CN103619356B - Peptide oligonucleotide conjugates - Google Patents

Peptide oligonucleotide conjugates Download PDF

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CN103619356B
CN103619356B CN201180071918.XA CN201180071918A CN103619356B CN 103619356 B CN103619356 B CN 103619356B CN 201180071918 A CN201180071918 A CN 201180071918A CN 103619356 B CN103619356 B CN 103619356B
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amino acid
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CN103619356A (en
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贡纳·J·汉森
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SA Leputa Medical Co
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SA Leputa Medical Co
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Priority claimed from US13/101,942 external-priority patent/US20110269665A1/en
Priority claimed from US13/107,528 external-priority patent/US9238042B2/en
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Abstract

There is provided the oligonucleotide analogs being conjugated in carrier peptides.The compound of the disclosure can be used for treating various diseases, for example, the disease of favourable therapeutic effect can be produced by wherein suppressing protein expression or correction exception mRNA splicing products.

Description

Peptide oligonucleotide conjugates
The cross reference of related application
The application No. 13/101942 U.S. according to filed in the 120th article of the chapter of United States Code No. 35 requires on May in 2011 5 The rights and interests of No. 13/107528 U.S. Patent application filed in state's patent application and 13 days Mays in 2011, these applications are by drawing In being fully incorporated herein.
Background
Technical field
This patent disclosure relates generally to can be used as the oligonucleotide compound (oligomer) of antisense compounds, relate more specifically to knot Close the oligomerization compounds on cell permeability peptide, and purposes of such oligomerization compounds in antisense application.
Description of Related Art
The actual utility of many medicines with potentially useful bioactivity, usually due to being difficult to arrive such medicine delivery Its target and be obstructed.Generally have to be delivered out to be delivered from the extracellular environment containing a large amount of water to intracellular compound Come, and then penetrate lipophilic cell membrane to enter cell.Except immaterial by specific transport mechanism is come active transport, many point Son, especially macromolecular, otherwise too lipophilic and can not actually dissolve, or it is too many hydrophilic and film can not be penetrated.
The HIV Tat protein fragments being made up of amino acid residue 49-57 (Tat49-57, with sequence RKKRRQRRR) It is used to peptide and protein delivery with bioactivity to intracellular (such as Barsoum et al., 1994, PCT Pubn.No.WO94/04686).Tat (49-60) is used to promote the delivering (Astriab- of phosphonothiolic acid oligonucleotides Fisher,Sergueev et al.2000;Astriab-Fisher,Sergueev et al.2002).Reverse Tat or rTat (57-49) (RRRQRRKKR) is had what is improved to deliver fluorescein to intracellular effectiveness by report compared to Tat (49-57) (Wender,Mitchell et al.2000;Rothbard,Kreider et al.2002).Rothbard and Wender are also public Other have been opened rich in arginic transport polymer (WO01/62297 PCT Publications；No. 6306993 United States Patent (USP)；The No. 2003/0032593 U.S. Patent Application Publication).
Oligonucleotides is the medical compounds of the potentially useful of a class, and it delivers the obstacle often treated and used. For this this, it has been found that be connected with the morpholino oligomers (PMOs of phosphoric acid diamides；See such as Summerton and Weller, 1997) have more prospect than the powered oligonucleotide analogs of such as D2EHDTPA.PMOs is water-soluble, neutral Or substantially without the antisense molecule of electric charge, it is by preventing splicing or the combination of body translation element or progress come suppressor Expression.Also show that PMOs can suppress or prevent virus replication (Stein, Skilling et al.2001;McCaffrey,Meuse et al.2003).They have highly resistant (Hudziak, Barofsky et al.1996) to enzymic digestion.Nothing in vitro (Stein, Foster et al.1997 in cell and cell culture model;Summerton and Weller1997), and in vivo Zebra fish, frog and sea urchin embryos in (Heasman, Kofron et al.2000;Nasevicius and Ekker2000), with And in adults model, such as rat, mouse, rabbit, dog and pig is (see such as 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), it is special that PMOs illustrates high antisense Property and effect.
Also show that antisense PMO oligomers can be ingested to intracellular, and compared to other widely used ASONs, More consistent validity and less nonspecific effect is (see such as P.Iversen, " Phosphoramidite in vivo Morpholino Oligomers",in Antisense Drug Technology,S.T.Crooke,ed.,Marcel Dekker,Inc.,New York,2001).Their cell can be increased and take the photograph by having shown that PMOs is attached on Arginine-rich peptide Take (see for example, No. 7468418 United States Patent (USP))；Waited however, the toxicity of the conjugate slow down them as feasible medicine Select the development of thing.
Although having been achieved for obvious progress, this area is still had to the antisense with raising or anti-gene performance The need for oligonucleotide conjugates.The antisense of such raising or anti-gene performance include：Hypotoxicity, to the stronger of DNA and RNA Affinity is without endangering sequence selectivity；The pharmacokinetics and Tissue distribution of raising；The cell of raising is delivered and reliable With controllable distribution in vivo.
Summary of the invention
The compound of the present invention can solve these problems, and provide the improvement for exceeding the existing antisense molecule in this area.It is logical Cross and cell permeability peptide is connected on the nucleic acid analog substantially without electric charge by glycine or amino proline acid, this hair Bright inventor solves the toxicity problem related to other peptide oligomer conjugates.In addition, being connected and/or end section between subunit To the modification of the combination at the 5 ' of oligonucleotide analogs and/or 3 ' ends, for example, morpholino oligonucleotide, can also improve this and sew The performance of compound.For example, in certain embodiments, compared to other oligonucleotide analogs, the conjugate of the disclosure has The toxicity of reduction and/or cell delivering, potency and/or the Tissue distribution of raising, and/or can be more effectively delivered to target device Official.These superior performances can produce favourable treatment index, the clinical dosage of reduction and lower merchandise cost.
Therefore, in one embodiment, the conjugate that the disclosure is provided is included：
(a) carrier peptides, it includes amino acid subunit；With
(b) nucleic acid analog, it includes substantially without the skeleton of electric charge and is attached to target nucleic acid for sequence-specific On target-seeking base sequence；
Wherein：
Two or more the amino acid subunit is positively charged amino acid, and the carrier peptides, which are included, is located at carrier The glycine (G) or proline (P) of the c-terminus of peptide, and the carrier peptides are covalently attached to the nucleic acid analog.Also provide Composition comprising above conjugate and pharmaceutically acceptable carrier.
In another embodiment, present disclose provides the method that albumen is produced is suppressed, this method includes should by coding The nucleic acid of albumen is exposed to the conjugate of the disclosure.
Another aspect of the present disclosure includes promoting to transport nucleic acid analog into intracellular method, and this method includes will power Profit requires that the carrier peptides described in 1 are attached on nucleic acid analog, and wherein relative to the nucleic acid analog of unconjugated form, promotes The nucleic acid analog transported intracellular.
In another embodiment, the method that the disclosure is directed to disease in treatment target body, this method is included pharmacy Conjugate is administered to object disclosed in effective dose.Additionally provide the method for preparing the conjugate, their application method and can use In the carrier peptides being attached on nucleic acid analog.
By reference to the following detailed description, the aspect of these and other of the invention can become apparent.For this Individual purpose, is set forth herein different bibliography, and some background informations, program, chemical combination is described in more detail in they Thing and/or composition, and each of which is all incorporated to by completely quoting herein.
Brief description of the drawings
Figure 1A shows the exemplary morpholino oligomers structure connected comprising phosphoric acid diamides.
Figure 1B is shown with reference to the morpholino oligomers in the end of carrier peptides 5 '.
Fig. 1 C show the morpholino oligomers for being attached to the end of carrier peptides 3 '.
Fig. 1 D-G show the repetition subunit fragments of exemplary morpholino oligonucleotide, are appointed as 1D to 1G.
Fig. 2, which is depicted, is connected to morpholino --- connected between the exemplary subunit on T parts.
Fig. 3 prepares the reaction scheme of the linking arm for synthesis in solid state for display.
Fig. 4 illustrates the preparation of the solid support (support) synthesized for oligomer.
Fig. 5 A, 5B and 5C are respectively illustrated, exemplary compared to known conjugate in mouse musculus quadriceps, barrier film and heart The exon skipping data of conjugate.
Fig. 6 A, 6B and 6C are respectively, exemplary conjugated compared to known conjugate in mouse musculus quadriceps, barrier film and heart The selective representative of the exon skipping data of thing.
Fig. 7 A and 7B respectively depict mouse blood urea nitrogen (BUN) level treated with different peptides-oligomer conjugate And survival rate.
Fig. 8 A and 8B respectively illustrate the injury of kidney mark of the mouse treated with different peptides-oligomer conjugate (KIM) data and lectin (Clu) data.
Fig. 9 A, 9B, 9C and 9D are respectively, compared to known conjugate, to compare the mouse treated with exemplary conjugate Internal exon skipping, BUN levels, the figure of % survival rates and KIM levels.
Figure 10 illustrates the KIM data of the mouse treated with different conjugates.
Figure 11 shows the BUN analysis results of the mouse treated with different conjugates.
Figure 12 is the figure of the concentration of different oligomers in display kidney of mouse.
Detailed description of the invention
I.Definition
In the following description, in order to provide fully understanding for different embodiments, some details be set forth.However, It will be appreciated by those skilled in the art that the present invention can be implemented in the case of without these details.In other examples, do not have There is display or well known structure is described in detail, to avoid unnecessarily obscuring the description of the embodiment.Unless context is another Require, this specification and appended claims in the whole text in, word " including (comprise) " and its deformation, e.g., " including (comprises) " and " including (comprising) " is to be interpreted as open, inclusive implication, that is, be construed to " including, but It is not limited to ".Moreover, title provided herein is merely for convenience, and scope of the claimed invention or implication are not explained.
This specification in the whole text in, refer to " embodiment " or " embodiment " refer to description with the embodiment party The relevant specific features of case, structure or characteristic are included at least one embodiment.Therefore, this specification in the whole text in, Phrase " in one embodiment " or " in embodiments " are not necessarily all referring to identical in the appearance of diverse location and implemented Scheme.Furthermore, it is possible in any suitable manner by specific features, structure or property combination in one or more embodiments In.It is singulative " one (a) ", " one (an) " and " described in addition, as used in this specification and appended claims (the) plural reference object " is included, unless the context.It should also be noted that term "or" generally with including Implication including "and/or" is used, unless the context.
As used herein, following term has following meanings, unless otherwise indicated：
" amino " refers to-NH2Base.
" cyano group " or " nitrile " refers to-CN bases.
" hydroxyl (Hydroxy) " or " hydroxyl (Hydroxyl) " refers to-OH bases.
" imino group " refers to=NH substituents.
" guanidine radicals " refers to-NHC (=NH) NH2Substituent.
" amidino groups " refers to-C (=NH) NH2Substituent.
" nitro " refers to-NO2Base.
" oxo " refers to=O substituents.
" thio " refers to=S substituents.
" cholate " refers to following structure：
" dexycholate " refers to following structure：
" alkyl " refers to the hydrocarbon chain base of straight or branched, and it is saturated or unsaturated (that is, containing one or more Double bond and/or three keys), with 1 to 30 carbon atom, and it is connected on the remainder of molecule by singly-bound.Including including 1 To the alkyl of 30 any number of carbon atoms.Alkyl comprising up to 30 carbon atoms is referred to as C1-C30Alkyl, similarly, example Such as, the alkyl comprising up to 12 carbon atoms is C1-C12Alkyl.It is analogously represented comprising other number of carbon atoms alkyl (and The other parts being defined herein).Alkyl includes, but not limited to C1-C30Alkyl, C1-C20Alkyl, C1-C15Alkyl, C1-C10Alkane Base, C1-C8Alkyl, C1-C6Alkyl, C1-C4Alkyl, C1-C3Alkyl, C1-C2Alkyl, C2-C8Alkyl, C3-C8Alkyl and C4-C8Alkane Base.Representational alkyl includes, but not limited to methyl, ethyl, n-propyl, 1- Methylethyls (isopropyl), normal-butyl, isobutyl Base, sec-butyl, n-pentyl, 1,1- dimethyl ethyls (tert-butyl group), 3- methylhexyls, 2- methylhexyls, vinyl, propyl- 1- alkene Base, but-1-ene base, amyl- 1- alkenyls, amyl- 1,4- dialkylenes, acetenyl, propinyl, butyl- 2- alkynyls, butyl- 3- alkynyls, pentyne Base, hexin base etc..Unless it is expressly stated otherwise in this manual, it can optionally replace alkyl by following description.
" alkylidene " or " alkylidene chain " refers to the straight or branched being connected to the remainder of molecule on group Bivalent hydrocarbon chain.Alkylidene can be saturated or unsaturated (that is, containing one or more double bonds and/or three keys).It is representative Alkylidene include, but not limited to C1-C12Alkylidene, C1-C8Alkylidene, C1-C6Alkylidene, C1-C4Alkylidene, C1-C3Alkylene Base, C1-C2Alkylidene, C1Alkylidene.Representational alkylidene is including, but not limited to methylene, ethylidene, propylidene, Asia just Butyl, ethenylidene, allylidene, sub- n-butene base, sub- propinyl, sub- positive butynyl etc..Alkylidene chain passes through singly-bound or double Key is connected on the remainder of molecule and is connected to by singly-bound or double bond on group.Alkylidene chain is connected to the residue of molecule Can be to pass through a carbon or any two carbon in the chain on part and to the connection site on group.Unless in this manual It is expressly stated otherwise, can be by following description optionally substituted alkylene chain.
" alkoxy " refers to formula-ORaBase, wherein RaFor alkyl as defined.Unless separately had in this manual clearly Illustrate, can be by following description optionally substituted alkoxy.
" alkoxyalkyl " refers to formula-RbORaBase, wherein RaFor alkyl as defined, and wherein RbSuch as to be defined Alkylidene.Unless it is expressly stated otherwise in this manual, can be by following description optionally substituted alkoxy alkyl.
" alkyl-carbonyl " refers to formula-C (=O) RaBase, wherein RaFor alkyl as defined above.Unless in this manual It is expressly stated otherwise, it can optionally replace alkyl-carbonyl by following description.
" alkoxy carbonyl " refers to formula-C (=O) ORaBase, wherein RaFor alkyl as defined.Unless in this specification In it is expressly stated otherwise, can be by following description optionally substituted alkoxy carbonyl.
" alkyl amino " refers to formula-NHRaBase or-NRaRaBase, wherein each RaAll it independently is alkane as defined above Base.Unless it is expressly stated otherwise in this manual, it can optionally replace alkyl amino by following description.
" amide groups " refers to formula-N (H) C (=O) RaBase, wherein RaFor alkyl or aryl as defined herein.Unless It is expressly stated otherwise in this specification, can be by following description optionally substituted amide group.
" Amidinylalkyl " refers to formula-Rb-C(=NH)NH2Base, wherein RbFor alkylidene as defined above.Unless in this theory It is expressly stated otherwise in bright book, it can optionally replace Amidinylalkyl by following description.
" Amidinylalkyl carbonyl " refers to formula-C (=O) Rb-C(=NH)NH2Base, wherein RbFor alkylidene as defined above.Remove It is non-expressly stated otherwise in this manual, it can optionally replace amidino groups alkyl-carbonyl by following description.
" aminoalkyl " refers to formula-Rb-NRaRaBase, wherein RbFor alkylidene as defined above, and each RaAll independently For hydrogen or alkyl.
" alkylthio " refers to formula-SRaBase, wherein RaFor alkyl as defined above.Unless separately had in this manual Clearly state, can optionally replace alkylthio.
" aryl " refers to coming the group of the hydrocarbon ring system of self-contained hydrogen, 6 to 30 carbon atoms and at least one aromatic ring.Aryl Can be monocyclic, two rings, three rings or Fourth Ring ring system, it can include fusion or bridge joint ring system.Aryl includes, but does not limit In the aryl from following hydrocarbon ring system：The luxuriant and rich with fragrance alkene of aceanthrylene, acenaphthylene, vinegar, anthracene, Azulene (az μ lene), benzene, bend (chrysene), it is glimmering Anthracene, fluorenes, asymmetric indacene (as-indacene), s-indacene (s-indacene), indane, indenes, naphthalene, that non-alkene (phenalene), phenanthrene, seven days of the week alkene (pleiadene), pyrene and benzophenanthrene.Unless expressly stated otherwise in this manual, term " aryl " or prefix " fragrant (ar) " (such as in " aralkyl (aralkyl) ") are intended to the aryl for including being optionally substituted.
" aralkyl " refers to formula-Rb-RcBase, wherein RbFor alkylidene chain as defined above, and RcTo be as defined above One or more aryl, for example, benzyl, diphenyl methyl, trityl etc..Unless separately had in this manual specifically Bright, aralkyl is optionally substituted.
" aryl carbonyl " refers to formula-C (=O) RcBase, wherein RcFor one or more aryl as defined above, for example, Phenyl.Unless expressly stated otherwise in this manual, aryl carbonyl is optionally substituted.
" aryloxycarbonyl " refers to formula-C (=O) ORcBase, wherein RcFor one or more aryl as defined above, example Such as, phenyl.Unless expressly stated otherwise in this manual, aryloxycarbonyl is optionally substituted.
" aromatic alkyl carbonyl " refers to formula-C (=O) Rb-RcBase, wherein RbFor alkylidene chain as defined above, and RcFor One or more aryl as defined above, for example, phenyl.Unless expressly stated otherwise in this manual, aromatic alkyl carbonyl Optionally it is substituted.
" aromatic alkoxy carbonyl " refers to Ji Shi-C (=O) ORb-Rc, wherein RbFor alkylidene chain as defined above, and Rc For one or more aryl as defined above, for example, phenyl.Unless expressly stated otherwise in this manual, aralkoxy Carbonyl is optionally substituted.
" aryloxy group " refers to formula-ORcBase, wherein RcFor one or more aryl as defined above, for example, phenyl.Remove Non- expressly stated otherwise in this manual, aryl carbonyl is optionally substituted.
" cycloalkyl " refers to stabilization, non-aromatic, monocyclic or polycyclic carbocyclic ring, and it can include fusion or bridge joint Ring system, it is saturated or unsaturated, and is connected to by singly-bound on the remainder of molecule.Representational cycloalkyl bag Include, but be not limited to, the cycloalkanes with 3 to 15 carbon atoms and 3 to 8 carbon atoms.Monocyclic cycloalkyl includes, for example, cyclopropyl, Cyclobutyl, cyclopenta, cyclohexyl, suberyl and cyclooctyl.Many ring groups include, for example, adamantyl, norborny, decahydronaphthalene Base and the ring of 7,7- dimethyl-two [2.2.1] heptane base.Unless expressly stated otherwise in this manual, cycloalkyl is optionally It is substituted.
" cycloalkyl-alkyl " refers to formula-RbRdBase, wherein RbFor alkylidene chain as defined above, and RdMore than such as The cycloalkyl of definition.Unless expressly stated otherwise in this manual, cycloalkyl-alkyl is optionally substituted.
" naphthene base carbonyl " refers to formula-C (=O) RdBase, wherein RdFor cycloalkyl as defined above.Unless in this explanation Expressly stated otherwise in book, naphthene base carbonyl is optionally substituted.
" cyclo alkoxy carbonyl " refers to formula-C (=O) ORdBase, wherein RdFor cycloalkyl as defined above.Unless at this Expressly stated otherwise in specification, cyclo alkoxy carbonyl is optionally substituted.
" fusion " refers to any cyclic structure described herein being fused in existing cyclic structure.Work as fusion Ring when being heterocyclic ring or heteroaryl ring, can use turns into the heterocyclic ring or thick of fusion in the existing cyclic structure of nitrogen-atoms substitution Any carbon atom of the heteroaryl ring part of conjunction.
" guanidine alkylation " refers to formula-Rb-NHC(=NH)NH2Base, wherein RbFor alkylidene as defined above.Unless at this It is expressly stated otherwise in specification, optionally it is substituted by following description guanidine alkylation.
" guanidine alkylation carbonyl " refers to formula-C (=O) Rb-NHC(=NH)NH2Base, wherein RbFor alkylidene as defined above. Unless it is expressly stated otherwise in this manual, optionally it is substituted by following description guanidine alkylation carbonyl.
" halo " or " halogen " refers to bromo, chloro, fluoro or iodo.
" haloalkyl " refers to the alkyl as defined above replaced by one or more halogens as defined above, example Such as, trifluoromethyl, difluoromethyl, methyl fluoride, trichloromethyl, 2,2,2- trifluoroethyls, the fluoro ethyls of 1,2- bis-, the bromo- 2- fluorine third of 3- Base, 1,2- dibromoethyls etc..Unless expressly stated otherwise in this manual, haloalkyl is optionally substituted.
" perhalogeno " or " perfluor " refers respectively to the part that wherein each hydrogen atom is replaced by halogen atom or fluorine atom.
" heterocyclic radical ", " heterocycle " or " ring of heterocycle " refer to 3 yuan to 24 yuan stable non-aromatic ring groups, and it includes 2 to 23 Individual carbon atom and selected from 1 to 8 following hetero atom：Nitrogen, oxygen, p and ses.Unless it is expressly stated otherwise in this manual, it is miscellaneous Ring group can be monocyclic, two rings, three rings or Fourth Ring ring system, and it can include the ring system of condense or bridge joint；And it is miscellaneous Nitrogen, carbon or sulphur atom in ring group are optionally oxidized；The nitrogen-atoms is optionally quaternized；And heterocyclic radical can be Partially or completely saturation.The example of such heterocyclic radical includes, but not limited to dioxolanyl (dioxolanyl), thienyl [1,3] dithiane base, Decahydroisoquinolinpreparation base, imidazolinyl, imidazolidinyl, isothiazole alkyl (isothiazolidinyl), different Oxazole alkyl, morpholinyl, octahydro indyl, octahydro isoindolyl, 2- oxygen piperazinyl, 2- Oxypertines base, 2- oxygen pyrrolidinyl, Evil Oxazolidinyl, piperidyl, piperazinyl, 4- piperidone bases, pyrrolidinyl, pyrazolidinyl, quinoline ring group, thiazolidinyl (thiazolidinyl), tetrahydrofuran base, trithiane base (trithianyl), THP trtrahydropyranyl, thio-morpholinyl, thia Quinoline base (thiamorpholinyl), 1- oxygen-thio-morpholinyl, 1,1- dioxido-thiomorpholines base, 12-crown-4,15- crown-s 5,18- Crown- 6,21- crown-s 7, azepine -18- crown-s 6, diaza -18- crown-s 6, azepine -21- crown-s 7 and diaza -21- crown-s 7.Unless Expressly stated otherwise in this specification, heterocyclic group is optionally substituted.
" heteroaryl " refer to comprising hydrogen atom, 1 to 13 carbon atom, selected from nitrogen, oxygen, p and ses 1 to 6 hetero atom With 5 to 14 yuan of ring system bases of at least one aromatic ring.For the purposes of the present invention, heteroaryl can be monocyclic, two rings, three rings Or Fourth Ring ring system, its can comprising fusion or bridge joint ring system；And nitrogen, carbon or the sulphur atom in heteroaryl are optionally It is oxidized；The nitrogen-atoms is optionally quaternized.Example includes but is not limited to, azepines base, acridinyl, benzimidazole Base, benzothiazolyl, benzindole base, benzodioxole group (benzodioxolyl), benzofuranyl, benzo Oxazolyl, benzothiazolyl, diazosulfide, benzo [b] [1,4] Dioxepane base (benzo [b] [1,4] Dioxepinyl), 1,4- benzodioxan bases (1,4-benzodioxanyl), benzo aphthofurans base, benzoxazolyl, benzene And dioxolyl (benzodioxolyl), benzodioxan base (benzodioxinyl), benzopyranyl, benzo Pyrans ketone group, benzofuranyl, benzofuran ketone group, benzothienyl (benzo thio-phenyl), BTA 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, different two Hydrogen indoles base, isoquinolyl, indolizine base (indolizinyl), isoxazolyls, naphthyridines base (naphthyridinyl), Evil bis- Oxazolyl (oxadiazolyl), 2- oxygen azepines Ji, oxazolyls, epoxy ethyl, 1- pyridine oxides base, 1- oxidations pyrimidine radicals, 1- oxygen Change pyrazinyl, 1- oxidations pyridazinyl, 1- phenyl -1H- pyrrole radicals, phenazinyl, phenothiazinyl, phenoxazine groups (phenoxazinyl), phthalazinyl, pteridyl, purine radicals, pyrrole radicals, pyrazolyl, pyridine radicals, pyrazinyl, pyrimidine radicals, pyridazine Base, quinazolyl, quinoxalinyl, quinolyl, quinoline ring group, isoquinolyl, tetrahydric quinoline group, thiazolyl, thiadiazoles, triazole Base, tetrazole radical, triazine radical and thio-phenyl (that is, thienyl).Unless expressly stated otherwise in this manual, heteroaryl can appoint Selection of land is substituted.
All of above group can be substitution or unsubstituted.As used herein, the term " substituted " refer to can be with By any of the above group (that is, alkyl, alkylidene, alkoxy, alkoxyalkyl, alkyl-carbonyl, alkoxy carbonyl, alkyl amino, Amide groups, Amidinylalkyl, Amidinylalkyl carbonyl, aminoalkyl, aryl, aralkyl, aryl carbonyl, aryloxycarbonyl, aralkyl carbonyl Base, aromatic alkoxy carbonyl, aryloxy group, cycloalkyl, cycloalkyl-alkyl, naphthene base carbonyl, cycloalkyl alkyl carbonyl, cycloalkyloxy carbonyl Base, guanidine alkylation, guanidine alkylation carbonyl, haloalkyl, heterocyclic radical and/or heteroaryl) further function dough, wherein at least 1 The key that individual hydrogen atom is connected on non-hydrogen atom substituent is replaced.Unless be expressly recited in this manual, substituent It can include being selected from following one or more substituents：Oxo (oxo) ,-CO2H, nitrile, nitro ,-CONH2, hydroxyl, sulphur oxygen (thiooxy), alkyl, alkylidene, alkoxy, 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- oxides, acid imide and enamine；The group of silicon atoms, such as trialkylsilkl, dialkyiarylsilyl, Allcyldiaryl silyl, diarye silyl, perfluoroalkyl or perfluoro alkoxy, for example, trifluoromethyl or fluoroform Epoxide." substituted " also refers to any of the above group, and wherein one or more hydrogen atoms are connected to such as in oxo, carbonyl, carboxylic Higher-order key on the hetero atom of oxygen in base and ester group and the nitrogen such as in imines, oxime, hydrazone and nitrile group is (for example, double Key or three keys) substitution.For example, " substituted " include any of the above group, wherein one or more hydrogen atoms are taken by following radicals Generation：-NRgC(=O)NRgRh、-NRgC(=O)ORh、-NRgSO2Rh、-OC(=O)NRgRh、-ORg、-SRg、-SORg、-SO2Rg、- OSO2Rg、-SO2ORg、=NSO2RgWith-SO2NRgRh." substituted " also refers to any of the above group, wherein one or more hydrogen atoms Replaced by following radicals：-C(=O)Rg、-C(=O)ORg、-CH2SO2Rg、-CH2SO2NRgRh、-SH、-SRgOr-SSRg.In foregoing base In group, RgAnd RhTo be same or different, and it independently is：Hydrogen, alkyl, alkoxy, alkyl amino, alkylthio, aryl, Aralkyl, cycloalkyl, cycloalkyl-alkyl, haloalkyl, heterocyclic radical, N- heterocyclic radicals, cycloheteroalkylalkyl, heteroaryl, N- heteroaryls And/or heteroaryl alkyl.In addition, each foregoing substituents can also optionally be replaced by one or more above-mentioned substituents.In addition, Any of the above group can be substituted with comprising one or more internal oxygen or sulphur atom.For example, alkyl can be by one or more interior Portion's oxygen atom replaces to form ether or polyether-based.Similarly, alkyl can be replaced to be formed by one or more internal sulphur atoms Thioether, disulphide etc..Acid amides base section can be replaced by up to 2 halogen atoms, and other above-mentioned groups can be one or more Halo atom replaces.Any of the above group can also be replaced by amino, alkyl monosubstituted amino, guanidine radicals or amidino groups (amidynyl).More than The optionally substituted base of any group also includes aryl phosphoryl, such as-RaP(Ar)3, wherein RaFor alkylidene, and ArFor aryl portion Point, such as phenyl.
Term " antisense scant polymer " or " antisense compounds " are used interchangeably, and refer to subunit sequence, each of which tool By carrying base on the skeleton subunit being made up of ribose or other pentoses or morpholino group, and wherein described backbone radical Connected by the connection between subunit, it allows the base in the compound to pass through Watson-Crick base pairings and nucleic acid Target sequence in (being usually RNA) hybridizes and forms nucleic acid：Oligomer heteroduplex in the target sequence.Oligomer can be with target The sequence of sequence formation exact complementarity or near-complementary.Such antisense scant polymer is designed to prevent or suppressed containing target sequence MRNA is translated, and can be said to be the sequence that " sensing " hybridizes therewith.
" morpholino oligomers " or " PMO " refer to having support can be with the base on Hydrogenbond to typical polynucleotide Skeleton polymerizable molecular, wherein the polymer lacks pentose skeleton part, and more specifically, its described skeleton is passes through phosphorus Acid diesters key (it typically is nucleotides and the phosphodiester bond of nucleosides, but containing the ring nitrogen of the combination by the ring nitrogen) connection Ribose backbone.Exemplary " morpholino " oligomer is included to be connected by (thio) phosphoramidate or (thio) di(2-ethylhexyl)phosphate amido link Morpholino subunit structures together, the morpholino nitrogen of a subunit is attached to outside the 5' rings of neighbouring subunit on carbon, each by it Subunit is all included can be effectively incorporated into the purine or pyrimidine base in polynucleotides in base by base specific Hydrogenbond Base mating section.For example, No. 5698685, No. 5217866, No. 5142047, No. 5034506, the 5166315th Number, No. 5185444, No. 5521063, No. 5506337 United States Patent (USP) and pending U.S. patent application 12/271036,12/ 271040 and WO/2009/064471 PCT Publications in, morpholino oligomers (including antisense scant polymer), institute are described in detail There are these files to be all fully incorporated herein by quoting.Representational PMOs includes being connected as connecting (A1) between wherein subunit PMOs。
" PMO+ " refers to that what is be previously described includes any number of (1- piperazines) phosphinylidene oxygen ((1- Piperazino) phosphinylideneoxy), (1- (4- (ω-guanidine radicals-silane alcohol base))-piperazine) phosphinylidene oxygen connection (A2 And A3) phosphoric acid diamides morpholino oligomers (see for example, PCT Publication WO/2008/036127, it is fully incorporated by quoting Herein).
" PMO-X " refers to disclosed herein comprising at least one (B) connection or at least one disclosed end modified Phosphoric acid diamides morpholino oligomers.
" phosphamide " base includes the phosphorus of the oxygen atoms with 3 connections and the nitrogen-atoms of 1 connection, and " phosphoric acid diamides " The phosphorus of oxygen atom of the base (see for example, Fig. 1 D-E) comprising 2 connections of tool and the nitrogen-atoms of 2 connections.Herein with the 61/th The neutral of oligomer described in No. 349783 and No. 11/801885 pending U.S. patent application or the Asia by modification In being connected between base, 1 nitrogen is always in side joint to (pendant to) skeletal chain.In the connection of phosphoric acid diamides, second nitrogen leads to It is often the ring nitrogen in morpholino cyclic structure.
" thioate " or " D2EHDTPA diamides ester " connection is respectively that phosphoramidate or di(2-ethylhexyl)phosphate carboxylic acid amide esters connect Connect, wherein 1 oxygen atom, usually the oxygen on side joint to skeleton, is replaced by sulphur.
" being connected between subunit " refers to connecting the connection of 2 morpholino subunits, such as structure (I).
As used herein, " powered ", " uncharged ", " cation " and " anion " is referred near neutral PH, the principal states of e.g., from about 6 to 8 times chemical parts.For example, this term can refer in physiological pH, i.e., about 7.4 times chemical parts Principal states.
" low alkyl group " refers to the alkyl of 1 to 6 carbon atom, such as with methyl, ethyl, normal-butyl, isobutyl group, tertiary fourth Base, isopentyl, n-pentyl and isopentyl are 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；That is methyl or ethyl.Similarly, " low-grade alkenyl " refers to It is 2 to 6, the alkenyl of preferably 3 or 4 carbon atoms, such as using acrylic and cyclobutenyl as example.
" non-interference " substituent is that the energy that its pre-determined target is put on will not be attached to antisense scant polymer as described in this article Power produces the substituent negatively affected.Such substituent includes small and/or relatively non-polar group, such as methyl, ethyl, Methoxyl group, ethyoxyl or fluoro base.
If oligomer is in physiological conditions with more than 37 DEG C, more than 45 DEG C, preferably at least 50 DEG C, and usually 60 DEG C- 80 DEG C or higher of TmWith target hybridization, then oligonucleotides or antisense scant polymer and target polynucleotide " specific hybrid ".Term " the T of oligomerm" for oligomer 50% with complementary polynucleotide hybridize when temperature.TmAt the standard conditions in physiological saline Measure, such as example, Miyada et al., Methods Enzymol.154:Described in 94-107 (1987).Such hybridization can Generation antisense scant polymer and target sequence " approximate " or " substantially " are complementary, and exact complementarity.
When being hybridized between two single stranded polynucleotides with anti-parallel arrangement, polynucleotides are described as each other " mutually Mend ".According to the base pairing rules generally received, based on the expected base ratio for forming hydrogen bond each other in relative chain, Complementary (1 polynucleotides degree complementary with another polynucleotides) can be quantified.
If polynucleotides its sequences for can be specifically bound to or can in physiological conditions with the second polynucleotide sequence The First ray of specific hybrid, then First ray is " antisense sequences " for the second sequence.
Term " targeting sequence " is complementary (in addition, the meaning is base with target sequence in rna gene group in oligonucleotide analogs It is complementary in sheet) sequence.The full sequence of analog compounds or only one part can be complementary with target sequence.For example, In analog with 20 bases, only 12-14 may be targeting sequence.Generally, targeting sequence is by continuous in analog Base composition, but be alternatively made up of discrete sequence, i.e., when these discrete sequences are put together, example Such as, from the other end of analog, the sequence across target sequence is constituted.
" skeleton " (for example, uncharged oligonucleotide analogs) of oligonucleotide analogs refer to that support base is matched somebody with somebody To the structure of part；For example, for morpholino oligomers, as described in this article, " skeleton ", which is included, passes through connection (example between subunit Such as, phosphorous connection) come the morpholino cyclic structure that connects." substantially without the skeleton of electric charge " refers to oligonucleotide analogs Skeleton, wherein it is powered to be connected between subunit less than 50% close under neutral pH.For example, substantially without the bone of electric charge Frame can be included less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or connected between even 0% subunit, It is being powered close under neutral pH.In some embodiments, substantially without the skeleton of electric charge, every 4 neutrals (at physiological ph) connection include at most 1 comprising connection between at most 1 electrically charged (at physiological ph) subunit, every 8 Or every 16 include at most 1 uncharged connection.In some embodiments, nucleic acid analog described herein is complete To be uncharged.
When hybridization occurs with anti-parallel arrangement, target and targeting sequence are described as " complementary " each other.Targeting sequence can With with complementary to target sequence " approximate " or " substantially ", and still worked with the purpose of presently described method, Remain as " complementary ".Preferably, the oligonucleotide analogs compound and target sequence used in presently described method, often 10 nucleotides have at most 1 mispairing, and have at most 1 mispairing in preferably 20.Or, antisense scant polymer used with such as Exemplary targeting sequence specified herein has at least 80%, at least 90% sequence homology or at least 95% sequence homology Property.In order to which complementation is attached in RNA target mark, and as described below, guanine base can be mutual with cytimidine or uracil RNA bases Mend.
" heteroduplex " refers to the double-strand formed between oligonucleotide analogs and target RNA complementary portion." resistance to nuclease Heteroduplex " refers to putting on the heteroduplex to be formed by the way that antisense scant polymer is attached into its complementary target, so that the hydridization is double Chain is substantially resistant to the internal degraded of such as RNase H intracellular and extracellular nucleases, and wherein nuclease can cut double-strand RNA/RNA or RNA/DNA compounds.
When medicament can enter cell by the mechanism in addition to passing through cell membrane except Passive diffusion, the medicament " is moved by lactation Thing cell is actively absorbed ".The medicament refers to transporting for example, by ATP dependences for example, can transport by " active transport " Mechanism is transported to transport medicament through mammalian cell membrane, or by " easyization transport ", is referred to by needing medicine Agent, which is attached to, can then promote the medicament that will be combined to be transported through the transport mechanism on the transport protein of film to carry out transport antisense medicine Agent passes through cell membrane.
Term " regulation expression " and/or " antisense activity " refer to expression or translation of the antisense scant polymer by RNA interfering To increase or more generally reduce the ability for the expression for giving albumen.In the situation of the protein expression of reduction, antisense oligonucleotide Thing can directly prevent the expression of given gene, or promote the RNA accelerated decomposition come from that genetic transcription.As retouched herein The morpholino oligomers stated by previous (steric occlusion) mechanism it is believed that worked.The preferred antisense traget of steric occlusion oligomer Including ATG initiation codon region, splicing site, the 5'- non-translational regions close to the region of splicing site and mRNA, although using Morpholino oligomers successfully regard other regions as target.
" amino acid subunit " is usually a-amino acid residue (- CO-CHR-NH-)；But can also be β-or other amino acid Residue is (for example ,-CO-CH2CHR-NH-), wherein R is amino acid side chain.
Term " naturally occurring amino acid " refers to the amino acid being present in the protein found in nature.Term " alpha-non-natural amino acid " refers to being not present in those amino acid in the protein that is found in nature；Example includes Beta-alanine (β-Ala) and 6-aminocaprolc acid (Ahx).
" effective dose " or " therapeutically effective amount " refers to being administered to the amount of the antisense scant polymer of mammalian object, no matter It is single dose or a series of part as dosage, it is generally by suppressing the translation of selected target nucleic acid sequence come effectively Produce desired therapeutic effect.
" treatment " of individual (such as mammal, such as people) or cell is for attempting the natural of the change individual or cell Any types intervention of process.Treatment includes, but not limited to give pharmaceutical composition, and can be used as prevention, or in pathology Event is carried out after being contacted with cause of disease medicament.
II.Carrier peptides
A.The performance of carrier peptides
As described above, this disclosure relates to the conjugate of carrier peptides and nucleic acid analog.The carrier peptides can be generally effectively increased The Premeabilisation of cells of nucleic acid analog.In addition, applicant have surprisingly discovered that glycine (G) or proline (P) subunit are included in into core (for example, in c-terminus or aminoterminal of carrier peptides) can reduce the poison of the conjugate between acid-like substance and the residue of carrier peptides Property, while relative to the conjugate with the different connections between carrier peptides and nucleic acid analog, effect keeps constant or obtained Improve.Therefore, when the conjugate of the disclosure has more preferable therapeutic window than other peptides-oligomer conjugate and is more promising Drug candidates.
Except the toxicity of reduction, the presence of glycine or proline subunit between nucleic acid analog and carrier peptides is it is believed that also Other advantage can be provided.For example, glycine is cheap, and it is readily incorporated into nucleic acid analog (or optional linking arm) On, and in the absence of any possibility of racemization.Similarly, proline is easily combined without occurring racemization, And additionally provide be not spiralization thing carrier peptides.The hydrophobicity of proline can also impart on carrier peptides and cytolipin Some advantages of bilayer interaction, and the carrier peptides (for example in certain embodiments) comprising multiple proline can be with The anti-chains of G- tetra- are formed.Finally, in certain embodiments, when proline moieties are adjacent to arginine subunit, the proline moieties Conjugate metabolic is assigned, because the Arg-Pro amido link can not be by conventional endopeptidase enzymatic lysis.
As described above, compared to other known conjugates, it is similar comprising nucleic acid is connected to by glycine or proline subunit The conjugate of carrier peptides on thing has relatively low toxicity and similar effect.Compared to other conjugates, this Shen of the support of progress Experiment please shows that the toxicity using the marker for nephrotoxicity of the conjugate of the disclosure is much lower (see for example, in embodiment 30 The injury of kidney mark (KIM) and blood urea nitrogen (BUN) data of description).Although being not intended to by theory constraint, present invention invention People believes that the toxicity of the reduction of disclosure conjugate may relate in the peptide moiety of nucleic acid analog is connected to (for example, carboxyl End) alpha-non-natural amino acid such as aminocaproic acid or Beta-alanine is not present.Because these alpha-non-natural amino acids are not cut in vivo, It is believed that the concentration of poison for not cutting peptide can accumulate and cause poisonous effect.
Glycine or proline moieties can be located at the aminoterminal or c-terminus of carrier peptides, and in some cases, carrier Peptide can be directly connected on nucleic acid analog by glycine or proline subunit, or carrier peptides can pass through optional company Arm is connect to be connected on nucleic acid analog.
In one embodiment, this disclosure relates to which conjugate, it is included：
(a) carrier peptides, include amino acid subunit；With
(b) nucleic acid analog, target nucleic acid is attached to comprising the skeleton substantially without electric charge and for sequence-specific Target-seeking base sequence；
Wherein：
Two or more amino acid subunits are positively charged amino acid, and the carrier peptides, which are included, is located at the carrier peptides C-terminus glycine (G) or proline (P) subunit, and the carrier peptides are covalently attached to nucleic acid analog.In some realities Apply in scheme, not more than 7 continuous amino acid subunits are arginine, and the continuous amino acid subunit of such as 6 or less is smart ammonia Acid.In some embodiments, the carrier peptides include the glycine subunit positioned at c-terminus.In other embodiments, institute State carrier peptides and include the proline subunit for being located at c-terminus.Further in other embodiments, the carrier peptides are included and are located at The single glycine or proline of c-terminus are (that is, not comprising the glycine positioned at c-terminus or proline dimer or trimer Deng).
In certain embodiments, when being attached to on the antisense scant polymer substantially without the skeleton of electric charge, phase For the antisense scant polymer of unconjugated form, carrier peptides can effectively facilitate antisense scant polymer and be attached to its target sequence, such as by with It is lower to be proved：
(i) when the translation initiation codon that antisense scant polymer is attached to the albumen that coding can be effectively prevented on its target sequence When, relative to the expression by unconjugated oligomer offer, the expression of the albumen of coding declines, or
(ii) it can effectively prevent Pre-mRNA when antisense scant polymer is attached to its target sequence (it encodes institute when correctly being spliced State albumen) in aberrant splicing site when, relative to the expression by unconjugated oligomer offer, the expression of the albumen of coding increases Plus.It is described further below the analysis for being adapted to measure these effects.In one embodiment, the conjugated of peptide is provided without thin The activity in born of the same parents' translation analysis, as described in this article.In some embodiments, activity be enhanced at least 2 times, at least 5 times or at least 10 times.
Optionally or additionally, relative to the analog of unconjugated form, carrier peptides can effectively facilitate nucleic acid analog transport To intracellular.In certain embodiments, transport is increased by least 2 times, at least 2 times, at least 5 times or at least 10 times.
In other embodiments, relative to conjugated comprising the carrier peptides for lacking terminal glycine or proline subunit Thing, carrier peptides are effectively reduced the toxicity (that is, increasing maximal tolerance dose) of conjugate.In certain embodiments, toxicity is subtracted At least 2 times, at least 2 times, at least 5 times or at least 10 times are lacked.
Other advantages of peptide transport section can stablize the double-strand between antisense scant polymer and its target nucleic acid sequence for its expection Ability.Although being not intended to by theory constraint, the ability of the stable double-strand may be by positively charged transport section and negatively charged Nucleic acid between electrostatic interaction cause.
The length of carrier peptides is not particularly limited, and is had differences in various embodiments.In some embodiments, Carrier peptides include 4 to 40 amino acid subunits.In other embodiments, carrier peptides comprising 6 to 30,6 to 20,8 to 25 Individual or 10 to 20 amino acid subunits.In some embodiments, carrier peptides are straight chain, and in other embodiments, its To there is side chain.
In some embodiments, carrier peptides are rich in positively charged amino acid subunit, such as arginine subunit.If extremely Few 10% amino acid subunit carries positive electricity, then the positively charged amino acid of carrier peptides " being rich in ".For example, in some embodiments In, 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% amino acid is sub- Base band has positive electricity.Even in other embodiments, all amino acid subunits in addition to glycine or proline subunit are carried Positive electricity.In other embodiments, all positively charged amino acid subunits are all arginine.
In other embodiments, the number of positively charged amino acid subunit is 1 to 20, such as 1 to 10 in carrier peptides It is individual or 1 to 6.In certain embodiments, in carrier peptides the number of positively charged amino acid for 1,2,3,4,5,6,7,8,9, 10th, 11,12,13,14,15,16,17,18,19 or 20.
Positively charged amino acid can be naturally occurring, non-naturally occurring, synthesis, modification or naturally occurring Amino acid analog.For example, can with specific designs with net positive charge modification amino acid be used for the present invention, such as with Lower more detailed description.A variety of different types of modifications to amino acid are well known in the art.In certain embodiments, band The amino acid of positive charge is histidine (H), lysine (K) or arginine (R).In other embodiments, carrier peptides are only included Natural amino acid subunit (that is, not comprising alpha-non-natural amino acid).In other embodiments, can be for example with acetyl group, benzene first Acyl group or stearyl part are capped to end amino acid, such as in N- ends.
H, K and/or R any number, combination and/or sequence may be present within carrier peptides.In some embodiments In, all amino acid subunits in addition to carboxyl terminal glycine or proline are all positively charged amino acid.In other implementations In scheme, the positively charged amino acid of at least one is arginine.For example, in some embodiments, all positively charged ammonia Base acid is all arginine, and even in other embodiments, carrier peptides are by arginine and carboxyl terminal glycine or proline Composition.Further in other embodiments, carrier peptides include not more than 7 continuous arginine, and such as not more than 6 continuous Arginine.
The positively charged amino acid of other types is contemplated.For example, in certain embodiments, at least one positively charged The amino acid of lotus is arginine analog.For example, the arginine analog can be to include RaN=C(NH2)RbSide-chain structure sun from Subtype a-amino acid, wherein RaFor H or Rc；RbFor Rc、NH2, NHR or N (Rc)2, wherein RcFor low alkyl group or low-grade alkenyl, and Optionally include oxygen or nitrogen, or RaAnd RbRing can be formed together；And wherein described side chain passes through RaOr RbIt is connected to amino acid On.Carrier peptides can include these any number of arginine analogs.
Positively charged amino acid can occur in any sequence in carrier peptides.For example, in some embodiments, Positively charged amino acid can be alternate or continuous.For example, carrier peptides can include sequence (Rd)m, wherein RdGoing out every time It is current independently all to be positively charged amino acid, and m is 2 to 12,2 to 10,2 to 8 or 2 to 6 integer.For example, some In embodiment, RdFor arginine, and carrier peptides include and are selected from following sequence：(R)4、(R)5、(R)6、(R)7(R)8, or choosing From：(R)4、(R)5、(R)6(R)7, for example in certain embodiments, carrier peptides include sequence (R)6, such as (R)6G or (R)6P。
In other embodiments, carrier peptides are by sequence (Rd)mConstituted with carboxyl terminal glycine or proline, wherein Rd Positively charged amino acid independently all is at each occurrence, and m is 2 to 12,2 to 10,2 to 8 or 2 to 6 integer.At certain In a little embodiments, RdArginine, histidine or lysine independently all are at each occurrence.For example, in some embodiment party In case, RdFor arginine, and carrier peptides selected from following sequence by constituting：(R)4、(R)5、(R)6、(R)7(R)8, and carboxyl Terminal glycine or proline.For example in certain embodiments, carrier peptides are by sequence (R)6G or (R)6P is constituted.
In some other embodiments, carrier peptides can include one or more hydrophobic amino acid subunits, described to dredge Aqueous amino acid subunit includes substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl or aralkyl side chain, wherein the alkane Base, alkenyl and every 6 carbon atoms of alkylyl side chain include at most 1 hetero atom.In some embodiments, hydrophobic amino acid is Phenylalanine (F).For example, carrier peptides can include two or more continuous hydrophobic amino acid such as phenylalanines (F), example Such as 2 continuous phenylalanine moieties.Hydrophobic amino acid can be located at any point in carrier peptide sequence.
In other embodiments, carrier peptides include sequence [(RdYbRd)x(RdRdYb)y]zOr [(RdRdYb)y(RdYbRd)x ]z, wherein RdAll independently be positively charged amino acid at each occurrence, x and y independently all be at each occurrence 0 or 1, condition is that x+y is 1 or 2, z are 1,2,3,4,5 or 6, and YbFor：
-C(O)-(CHRe)n-NH-
(Yb)
Wherein n is 2 to 7, and each ReHydrogen or methyl independently all are at each occurrence.The one of these embodiments In a little, RdArginine, histidine or lysine independently all are at each occurrence.In other embodiments, each RdAll it is Arginine.In other embodiments, n is 5, and YbFor amino-hexanoic acid moiety.In other embodiments, n is 2, and YbFor β- Alanine moiety.In other embodiments, ReFor hydrogen.
In foregoing some embodiments, x is that 1, y is 0, and carrier peptides include sequence (RdYbRd)z.In other embodiment party In case, n is 5, and YbFor amino-hexanoic acid moiety.In other embodiments, n is 2, and YbFor Beta-alanine part.Further exist In other embodiments, Re is hydrogen.
Further in other foregoing embodiments, x is that 0, y is 1, and carrier peptides include sequence (RdRdYb)z.At other In embodiment, n is 5, and YbFor amino-hexanoic acid moiety.In other embodiments, n is 2, and YbFor Beta-alanine part.Enter One step in other embodiments, ReFor hydrogen.
In other embodiments, carrier peptides include sequence (RdYb)p, wherein RdAnd YbAs defined above, and p be 2 to 8 Integer.In other embodiments, each RdAll it is arginine.In other embodiments, n is 5, and YbFor aminocaproic acid Part.In other embodiments, n is 2, and YbFor Beta-alanine part.Further in other embodiments, Re is hydrogen.
In other embodiments, carrier peptides include sequence ILFQY.Except any other sequence disclosed herein, institute ILFQY sequences can also be included by stating peptide.For example carrier peptides can include ILFQY and [(RdYbRd)x(RdRdYb)y]z、[(RdRdYb)y (RdYbRd)x]z、(RdYb)pOr combinations thereof, wherein Rd, x, y and YbAs defined above.[(the RdYbRd)x (RdRdYb)y]z、[(RdRdYb)y(RdYbRd)x]zOr (RdYb)pSequence can be located at the aminoterminals of ILFQY sequences, c-terminus or this Two ends.In certain embodiments, x is that 1, y is 0, and carrier peptides include and are connected to ILFQY sequences by optional Z linking arms On (RdYbRd)z。
In other related embodiments, carrier peptides include sequence ILFQ, IWFQ or ILIQ.Other embodiments include Include sequence PPMWS, PPMWT, PPMFS or PPMYS carrier peptides.Except any other sequence described herein, for example except Sequence [(RdYbRd)x(RdRdYb)y]z、[(RdRdYb)y(RdYbRd)x]zOr (RdYb)p, carrier peptides can also include these sequences, Wherein Rd, x, y and YbAs defined above.
Some embodiments of carrier peptides include the modification to naturally occurring amino acid subunit, for example, can modify amino End or carboxyl-terminus amino acid subunit.Such modification includes being capped to free amine group or free carboxy with hydrophobic group.Example Such as, it can be capped with acetyl group, benzoyl or stearyl part to aminoterminal.For example, any peptide sequence can in table 1 With such modification, it is not expressly recited in table.In these embodiments, the aminoterminal of carrier peptides can be following institute Description：
Further in other embodiments, carrier peptides include alanine, aspartic acid, cysteine, glutamine, sweet At least one in propylhomoserin, histidine, lysine, methionine, serine or threonine.
In some embodiments disclosed herein, carrier peptides are by the sequence and carboxyl terminal glycine or proline that annotate Subunit is constituted.
In some embodiments, carrier peptides are not made up of (amino terminal to carboxyl terminal) following sequence：R6G、R7G、 R8G、R5GR4G、R5F2R4G、Tat-G、rTat-G、(RXR2G2)2Or (RXR3X)2G.In other embodiments, carrier peptides not by R8G、R9G or R9F2G is constituted.Further in other embodiments, carrier peptides are not made up of following sequence：Tat-G、rTat-G、 R9F2G、R5F2R4、R4G、R5G、R6G、R7G、R8G、R9G、(RXR)4G、(RXR)5G、(RXRRBR)2G、(RAR)4F2Or (RGR)4F2。 In other embodiments, carrier peptides are not by " cell-penetrating peptide Penetratin " or " R6Pen " is constituted.
In another aspect, present disclose provides peptide-nucleic acid analog conjugate, it is included：
Nucleic acid analog, it has the skeleton and target-seeking base sequence substantially without electric charge, and
Be covalently attached to the peptide on nucleic acid analog, its include carboxyl terminal glycine or proline subunit, and by selected from Other other subunits composition of following 8 to 16：RdSubunit, Y subunits and optional Z subunits, include at least eight RdSubunit, extremely Few 2 Y subunits and at most 3 Z subunits, wherein>50% subunit is RdSubunit, and wherein：
(a) each RdSubunit all independently represents arginine or arginine analog, the arginine analog be comprising RaN=C(NH2)RbThe cationic a-amino acid of side-chain structure, wherein RaFor H or Rc；RbFor Rc、NH2, NHR or N (Rc)2, wherein RcFor low alkyl group or low-grade alkenyl, and optionally include oxygen or nitrogen, or RaAnd RbRing can be formed together；And it is wherein described Side chain passes through RaOr RbIt is connected on amino acid；
(b) at least two Y subunits are YaOr Yb, wherein：
(i) each YaAll it independently is the neutral a-amino acid subunit with the side chain independently selected from following group：Take Generation or unsubstituted alkyl, alkenyl, alkynyl, aryl and aralkyl, wherein the side chain, is elected to alkyl, the alkene from substitution It is every 2, preferably every 4 when base and alkynyl, and more preferably every 6 carbon atoms include at most 1 hetero atom, and wherein described Asia Base is the continuous or side positioned at linking arm part, and
(ii)YbFor：
-C(O)-(CHRe)n-NH-
(Yb)
Wherein n is 2 to 7, and each ReHydrogen or methyl independently all are at each occurrence；And
(c) Z represents to be selected from following amino acid subunit：Alanine, aspartic acid, cysteine, glutamine, sweet ammonia Acid, histidine, lysine, methionine, serine, threonine and the amino acid with side chain, the side is linked as naturally occurring side 1 carbon of chain or 2 carbon homologues, electronegative side chain (such as carboxylate side chains) under physiological pH is not included in.In some embodiment party In case, side chain is neutral.In other embodiments, Z side chains are the side chain of naturally occurring amino acid.In some embodiments Optional Z subunits are selected from：Alanine, glycine, methionine, serine and threonine.Carrier peptides can include 0,1,2 Or 3 Z subunits, and in some embodiments comprising at most 2 Z subunits.
In the embodiment selected, carrier peptides have type YaJust 2 Y subunits, it is continuous or positioned at half The side of cystine subunit.In some embodiments, 2 YaSubunit is continuous.In other embodiments, YaSubunit Side chain includes the side chain and its 1 carbon or 2 carbon homologues of naturally occurring amino acid, and side chain powered under physiological pH is not included in.Its His possible side chain is the side chain of naturally occurring amino acid.In a further embodiment, side chain is aryl or aralkyl side chain； For example, each YaCan be independently selected from：Phenylalanine, tyrosine, tryptophan, leucine, isoleucine and valine.
In the embodiment selected, each YaAll it is independently selected from phenylalanine and tyrosine；In other embodiments, Each YaAll it is phenylalanine.This includes, for example, by arginine subunit, phenylalanine subunit, glycine or proline subunit, Optional linking arm part and the conjugate of nucleic acid analog composition.A kind of such conjugate includes having formula Arg9Phe2Aa's Peptide, wherein aa are glycine or proline.
Aforementioned bearer peptide can also include ILFQY, ILFQ, IWFQ or ILIQ.Other embodiments include including sequence PPMWS, PPMWT, PPMFS or PPMYS aforementioned bearer peptide.
Peptide-oligomer conjugate of the present invention is more more effective than unconjugated oligomer in difference in functionality, including：In egg Suppress the expression of said target mrna, including cell free translation system in white matter expression system；Suppress the splicing of target Pre-mRNA；With pass through The cis-acting elements that viral nucleic acid duplication or mRNA are transcribed will be controlled as target, to suppress the duplication of virus.
Also include being conjugated for other pharmacological agents (that is, being not nucleic acid analog) and carrier peptides within the scope of the present invention Thing.Specifically, some embodiments provide conjugate, and it includes：
(a) carrier peptides of amino acid subunit are included；With
(b) pharmacological agent；
Wherein：
Two or more amino acid subunit is positively charged amino acid, and the carrier peptides, which are included, is located at the carrier The glycine (G) or proline (P) subunit of the c-terminus of peptide, and the carrier peptides are covalently attached to pharmacological agent.These realities It can be any carrier peptides described herein to apply the carrier peptides in scheme.Additionally provide by the way that pharmacological agent is conjugated into load The method that it is delivered on body peptide.
Pharmacological agent to be delivered can be bioactivator, such as therapeutic agent or diagnosticum, although it can be use In the compound of detection, such as fluorescent chemicals.Bioactivator includes being selected from following medicine：Biomolecule, such as peptide, albumen Matter, carbohydrate or nucleic acid, especially ASON, or " small molecule " organic or inorganic compound." small molecule " can be changed Compound is broadly defined as organic and inorganic or organo-metallic compound, and it is not biomolecule as described above.Generally, this Class compound has the molecular weight less than 1000, or, in one embodiment, less than 500.
In one embodiment, pharmacological agent to be delivered does not include single amino acids, dipeptides or tripeptides.Another In individual embodiment, it does not include short oligopeptides；That is, the oligopeptides having less than 6 amino acid subunits.In other embodiments In, it does not include longer oligopeptides；That is, the oligopeptides with 7 to 20 amino acid subunits.Further in other embodiments, It is not included with the oligopeptides or protein for being more than 20 amino acid subunits.
Relative to unconjugated form and/or the pharmacological agent with less toxicity, lack glycine relative to being conjugated to Or the pharmacological agent on the corresponding peptide of proline subunit, the carrier peptides can effectively facilitate pharmacological agent and transport cell It is interior.In some embodiments, transport is provided of at least 2 times, at least 5 times or at least 10 times.In other embodiments, it is malicious Property has been reduced at least 2 times, at least 5 times or at least 10 times of (that is, maximal tolerance dose is reduced).
B.Peptide linking arm
Can be can be used by those skilled in the art a variety of methods by carrier peptides be connected to medicament to be delivered (for example, Nucleic acid analog, pharmacological agent etc.) on.In some embodiments, carrier peptides are directly connected on nucleic acid analog, and Do not use intermediate link arm.Thus, on nucleic acid analog between end amino acid and the unhindered amina of free carboxy It may be useful to forming conjugate to form amido link.In certain embodiments, by carboxyl terminal glycine or proline Subunit is directly connected on 3 ' ends of nucleic acid analog, for example can by carboxyl terminal glycine or proline moieties and Amido link is formed between 3 ' morpholino ring nitrogen to connect carrier peptides (see for example, Fig. 1 C).
In some embodiments, by the way that nucleic acid analog is conjugated in carrier peptides selected from following linking arm part： YaOr YbSubunit, cysteine subunit and uncharged non-amino acid linking arm part.In other embodiments, by positioned at Nucleic acid analog is directly connected in carrier peptides by the glycine or proline moieties of the 5 ' of nucleic acid analog or 3 ' ends.One In a little embodiments, carrier peptides are directly connected to 3 ' ends of nucleic acid analog by glycine or proline subunit, for example, led to Superamide key is directly connected on 3 ' morpholino nitrogen.
In other embodiments, conjugate includes the coupling part between terminal glycine or proline subunit.At these In some of embodiment, linking arm is up to 18 atomic lengths, and it, which is included, is selected from following connection (bonds)：Alkyl, hydroxyl Base, alkoxy, alkyl amino, acid amides, ester, carbonyl, carbamate, phosphoric acid diamides, phosphoamide, phosphonothiolic acid and phosphoric acid Diester.In certain embodiments, linking arm includes phosphoric acid diamides and piperazine.For example, in some embodiments, linking arm With following structure (XXIX)：
Wherein R24To be not present, H or C1-C6Alkyl.In certain embodiments, R24For in the absence of, and other implementation In scheme, 5 ' ends of nucleic acid analog (for example, morpholino oligomers) are connected in carrier peptides (see example by structure (XXIX) Such as, Figure 1B).
In some embodiments, RdThe pendant moiety of subunit independently selected from：Guanidine radicals (HN=C (NH2) NH-), amidino groups (HN =C(NH2)C<), 2- amino dihydro-pyrimidins base, 2- amino tetrahydro-pyrimidine base, PA base and 2- aminopyrimidine bases.
If desired, multiple carrier peptides can be connected in single compound；Or, multiple compounds can be attached to On single transport thing.Linking arm between carrier peptides and nucleic acid analog can also be by natural or alpha-non-natural amino acid (for example, 6- ammonia Base caproic acid or Beta-alanine) composition.Linking arm can also include the c-terminus for transporting peptide and the amino or hydroxyl of nucleic acid analog Between the direct key that is formed (for example, at 3 ' morpholino nitrogen or 5 ' OH) by the condensation promoted by such as carbodiimides.
In general, linking arm can include any non-reacted part, its transport for not disturbing conjugate or function.Even It is not cleavable linking arm to connect arm and can be selected under the normal condition used, for example, containing ether, thioether, acid amides or amino Formic acid ester bond.In other embodiments, including in vivo cleavable carrier peptides with compound (for example, oligonucleotides is similar Thing, pharmacological agent etc.) between connection be desirable.Be known in the art in vivo for cleavable connection, and including for example, Carboxylate (it is enzymatically hydrolyzed) and disulphide (it leaves cut in glutathione).Pass through putting for application suitable wavelength Penetrate, the cleavable connection of photodissociation, such as O-Nitrophenylfluorone ether can be also cut in vivo.Further contain cleavable disulfide group The exemplary Heterobifunctional group bridging agent of group, including 3- [(4- azidos phenyl) two is thio] propionic acid-n-hydroxysuccinimide Ester and Vanin, E.F. and Ji, T.H., Biochemistry20:Other materials described in 6754-6760 (1981).
C.Exemplary carrier peptide
The table of exemplary carrier peptide sequence and oligonucleotide sequence is provided in table 1 below.In some embodiments, originally It is open to provide peptide oligomer conjugate, wherein the peptide is included or is made up of any one of table 1 peptide sequence.At another In embodiment, nucleic acid analog is included or is made up of any oligonucleotide sequence in table 1.Further in other embodiments In, present disclose provides peptide oligomer conjugate, wherein the peptide is included or is made up of any one of table 1 peptide sequence, and Nucleic acid analog is included or is made up of any oligonucleotide sequence in table 1.In other embodiments, present disclose provides bag The peptide for containing or being made up of any one of table 1 sequence.
The exemplary carrier peptide of table 1. and oligonucleotide sequence
Aa=glycine or proline；B=Beta-alanine；X=6-aminocaprolc acid；Tg=unmodified aminoterminal, or use acetyl Amino terminal (that is, acetyl group acid amides, benzoyl acid amides or stearyl acyl that base, benzoyl or stearyl are capped Amine), and YbFor：
-C(O)-(CHRe)n-NH-
Wherein n is 2-7, and each ReHydrogen or methyl independently all are at each occurrence.For the sake of simplicity, it is not all Sequence is all annotated with end tg groups；However, above sequence each of can comprising unmodified aminoterminal or with acetyl group, The aminoterminal that benzoyl or stearyl are capped.
III.Antisense scant polymer
Included in the present invention conjugate in nucleic acid analog for can base specific be attached to polynucleotides target sequence The synthesis oligomer substantially without electric charge of row, for example antisense oligonu-cleotides are like thing.Such analog includes, for example, first Base phosphonate ester, peptide nucleic acid, N3' → P5' phosphoramidates and morpholino oligomers substantially without electric charge.
The nucleic acid analog base sequence provided by the base pairing group of analog skeletal support can be any sequence, its Described in the base pairing group that supports include standard or A, T, C, G and U base by modification, or off-gauge creatinine And miscellaneous-G the bases of 7- denitrogenations (I).
In some embodiments, nucleic acid analog is morpholino oligomers, i.e. the morpholino subunit of form as shown in Figure 1 The oligonucleotide analogs of structure composition, wherein：(i) structure passes through phosphorous connection (1 to 3 atomic length, preferably 2 originals Sub- length) be joined together, the morpholino nitrogen of 1 subunit is attached to outside the 5' of neighbouring subunit in ring carbon, and (ii) Pi and Pj is that the purine or pyrimidine bases mating portion in polynucleotides in base can be effectively attached to by base specific Hydrogenbond Point.Purine or the pyrimidine bases mating section is usually adenine, cytimidine, guanine, uracil or thymidine.Below Further describe synthesis, structure and the binding characteristic of morpholino oligomers, and No. 5698685, No. 5217866, Carried out in No. 5142047, No. 5034506, No. 5166315, No. 5521063 and No. 5506337 United States Patent (USP)s in detail Thin description, it is all these to be all fully incorporated herein by quoting.
The desired chemical property of oligomer based on morpholino include with high TmComplementary base target nucleic acid include target RNA, is even as short as the energy in the ability of the oligomer selective cross of 8-14 base, active transport to mammalian cell Power, and oligomer：The ability of the resistance to RNase degraded of RNA heteroduplexs.
In preferred embodiments, morpholino oligomers are the length of about 8-40 subunit.More typically, oligomer is About 8-20, about 8-16, about 10-30 or the length of about 12-25 subunit.For some applications, such as antiseptic, short widow Polymers, the length of e.g., from about 8-12 subunit, it may be possible to particularly advantageous, especially when be connected to peptide as disclosed herein fortune When on defeated body (peptide transporter).
A.With the oligomer connected between the subunit by modification
One embodiment of the disclosure is related to comprising containing the nucleic acid analog (example connected between the subunit by modification Such as, morpholino oligomers) peptide-oligomer conjugate.In some embodiments, the conjugate compared to it is corresponding without The oligomer of modification has higher affinity to DNA and RNA affinity, and compared to the widow connected between other subunits Polymers shows cell delivering, potency and/or the Tissue distribution performance of raising.In one embodiment, conjugate includes one Or connected between multiple (A) type subunits as defined below.In other embodiments, conjugate is for example following fixed comprising at least one Connected between type (B) subunit of justice.Further in other embodiments, between subunit of the conjugate comprising (A) type and type (B) Connection.Further in other embodiments, conjugate includes morpholino oligomers as described in more detail below.Following The described in more detail architectural feature and performance of different connection types and oligomer in discussion.
1.Connect (A)
Applicant have discovered that by preparing with the oligomer connected between different subunits, can optimize antisense activity, The raising of bio distribution and/or other desired performances.For example, oligomer can be optionally sub- comprising one or more (A) types Connected between base, and in certain embodiments, oligomer is connected comprising at least one (A) type, such as each connection can be (A) type.In some other embodiments, each (A) type connection has identical structure.(A) type connection can be included altogether With the connection disclosed in No. 7943762 United States Patent (USP) possessed, it is fully incorporated herein by quoting herein.Connect (A) tool There are following structure (I) or the salt or isomers of the structure, wherein 3 ' and 5 ' indicate respectively morpholino ring (that is, knot discussed below Structure (i)) to the tie point of 3 ' and 5 ' ends：
Wherein：
W independently is S or O at each occurrence；
X independently is-N (CH at each occurrence3)2、-NR1R2、-OR3Or
Y independently is O or-NR at each occurrence2,
R1Hydrogen or methyl independently all are at each occurrence；
R2Hydrogen or-LNR independently all are at each occurrence4R5R7；
R3Hydrogen or C independently all are at each occurrence1-C6Alkyl；
R4Hydrogen, methyl ,-C (=NH) NH independently all are at each occurrence2、-Z-L-NHC(=NH)NH2Or-[C (=O) CHR'NH]mH, wherein Z are-C (=O)-or direct key, and R' is the side chain or its 1 carbon or 2 carbon homologues of naturally occurring amino acid, And m is 1 to 6；
R5Hydrogen, methyl or electronics pair independently all are at each occurrence；
R6Hydrogen or methyl independently all are at each occurrence；
R7Hydrogen, C independently all are at each occurrence1-C6Alkyl or C1-C6Alkoxyalkyl；And
L is the linking arm of up to 18 optional atomic lengths, and it includes alkyl, alkoxy or alkylamino, or they Combination.
In some instances, oligomer is connected comprising at least one (A) type.In some other embodiments, oligomer bag Containing the continuously coupled of at least two (A) type.In other embodiments, at least 5% connection is (A) type in oligomer；For example exist In some embodiments, 5% to 95%, 10% to 90%, 10% to 50% or 10% to 35% connection can be connection (A) type.One In a little specific embodiments, at least one (A) type is connected as-N (CH3)2.In other embodiments, each (A) type connection For-N (CH3)2, and even in other embodiments, each connection in oligomer is-N (CH3)2.In other embodiment party In case, at least one (A) type is connected as piperazine -1- bases, such as unsubstituted piperazine -1- bases (for example, A2 or A3).At other In embodiment, each (A) type connection is piperazine -1- bases, such as unsubstituted piperazine -1- bases.
In some embodiments, W independently is S or O at each occurrence, and in certain embodiments, W is O.
In some embodiments, X independently is-N (CH at each occurrence3)2、-NR1R2、-OR3.In some implementations In scheme, X is-N (CH3)2.In terms of others, X is-NR1R2, and in other examples, X is-OR3。
In some embodiments, R1Hydrogen or methyl independently all are at each occurrence.In some embodiments, R1 For hydrogen.In other embodiments, X is methyl.
In some embodiments, R2All it is hydrogen at each occurrence.In other embodiments, R2Occurring every time Shi Douwei-LNR4R5R7.In some embodiments, R3Hydrogen or C independently all are at each occurrence1-C6Alkyl.In other realities Apply in scheme, R3For methyl.In other embodiments, R3For ethyl.In some other embodiments, R3For n-propyl or Isopropyl.In some other embodiments, R3For C4Alkyl.In other embodiments, R3For C5Alkyl.In some implementations In scheme, R3For C6Alkyl.
In certain embodiments, R4Hydrogen independently all is at each occurrence.In other embodiments, R4For methyl. Further in other embodiments, R4For-C (=NH) NH2, and in other embodiments, R4For-Z-L-NHC (=NH) NH2。 Further in other embodiments, R4For-[C (=O) CHR'NH]mH.In one embodiment, Z be-C (=O)-, and another In one embodiment, Z is direct key.R' is the side chain of naturally occurring amino acid.In some embodiments, R ' is naturally to deposit In 1 carbon or 2 carbon homologues of the side chain of amino acid.
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, R5Hydrogen, methyl or electronics pair independently all are at each occurrence.In some embodiment party In case, R5For hydrogen.In other embodiments, R5For methyl.In other embodiments, R5For electronics pair.
In some embodiments, R6Hydrogen or methyl independently all are at each occurrence.In some embodiments, R6 For hydrogen.In other embodiments, R6For methyl.
In other embodiments, R7Hydrogen, C independently all are at each occurrence1-C6Alkyl or C2-C6Alkoxyalkyl. In some embodiments, R7For hydrogen.In other embodiments, R7For C1-C6Alkyl.Further in other embodiments, R7For C2-C6Alkoxyalkyl.In some embodiments, R7For methyl.In other embodiments, R7For ethyl.Further In other embodiments, R7For n-propyl or isopropyl.In some other embodiments, R7For C4Alkyl.In some realities Apply in scheme, R7For C5Alkyl.In some embodiments, R7For C6Alkyl.Further in other embodiments, R7For C2Alkane Epoxide alkyl.In some other embodiments, R7For C3Alkoxyalkyl.Further in other embodiments, R7For C4 Alkoxyalkyl.In some embodiments, R7For C5Alkoxyalkyl.In other embodiments, R7For C6Alkoxy alkane Base.
As described above, linking arm group L contains in its skeleton is selected from following connection：Alkyl (such as-CH2-CH2-)、 Alkoxy (for example ,-C-O-C-) and alkyl amino (such as-CH2- NH-), condition is L terminal atom (for example, neighbouring carbonyl Or the atom of nitrogen) it is carbon atom.Although may have the connection (such as-CH of tool side chain2-CHCH3-), but linking arm is usually without branch Chain.In one embodiment, linking arm is hydrocarbon linking arm.Such linking arm can have structure (CH2)n-, wherein n is 1- 12, preferably 2-8, and more preferably 2-6.
There is provided the oligomer connected with any number of (A) type.In some embodiments, oligomer does not have (A) Type is connected.In certain embodiments, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% connection is connection (A).In selected embodiment, 10% to 80%, 20% to 80%, 20% to 60%, 20% to 50%, 20% to 40% or 20% to 35% Connection for connection (A).In other embodiments, each connection is (A) type.
2.Connect (B)
In some embodiments, oligomer is connected comprising at least one (B) type.For example oligomer can comprising 1,2,3, 4th, 5,6 or more (B) type connection.(B) type connection can be neighbouring or can be dispersed in whole oligomer.(B) type connects Connect with following structure (I)：
Or the salt or isomers of the structure, wherein：
W independently is S or O at each occurrence；
X independently is-NR at each occurrence8R9Or-OR3；And
Y independently is O or-NR at each occurrence10,
R3Hydrogen or C independently all are at each occurrence1-C6Alkyl；
R8Hydrogen or C independently all are at each occurrence2-C12Alkyl；
R9Hydrogen, C independently all are at each occurrence1-C12Alkyl, C1-C12Aralkyl or aryl；
R10Hydrogen, C independently all are at each occurrence1-C12Alkyl or-LNR4R5R7；
Wherein R8And R9It may be combined to form the monocyclic or bicyclic heterocycles of 5-18 members, or R8、R9Or R3Can be with R10With reference to The heterocycle of 5-7 members is formed, and wherein when X is 4- piperazinyls, X has following structure (III)：
Wherein：
R11C independently all is at each occurrence2-C12Alkyl, C1-C12Aminoalkyl, C1-C12Alkyl-carbonyl, aryl, heteroaryl Base or heterocyclic radical；
R independently is electronics to, hydrogen or C at each occurrence1-C12Alkyl；And
R12Hydrogen, C independently all are at each occurrence1-C12Alkyl, C1-C12Aminoalkyl ,-NH2、-CONH2、-NR13R14、- NR13R14R15、C1-C12Alkyl-carbonyl, oxo (oxo) ,-CN, trifluoromethyl, amide groups, amidino groups, Amidinylalkyl, Amidinylalkyl carbonyl Base guanidine radicals, guanidine alkylation, guanidine alkylation carbonyl, cholate, dexycholate, aryl, heteroaryl, heterocycle ,-SR13Or C1-C12Alkane Epoxide, wherein R13、R14And R15C independently all is at each occurrence1-C12Alkyl.
In some instances, oligomer includes 1 (B) type connection.In some other embodiments, oligomer includes 2 Individual (B) type connection.In some other embodiments, oligomer includes 3 (B) type connections.In some other embodiments, Oligomer includes 4 (B) type connections.Further in other embodiments, (B) type is connected as continuously (that is, type (B) company Connect located adjacent one another).In other embodiments, at least 5% connection is type (B) in oligomer；For example in some embodiments In, 5% to 95%, 10% to 90%, 10% to 50% or 10% to 35% connection can be the connection of (B) type.
In other embodiments, R3Hydrogen or C independently all are at each occurrence1-C6Alkyl.In other embodiments In, R3It can be methyl.In some embodiments, R3It can be ethyl.In some other embodiments, R3Can be for just Propyl group or isopropyl.In other embodiments, R3Can be C4Alkyl.In some embodiments, R3Can be C5Alkyl. In some embodiments, R3Can be C6Alkyl.
In some embodiments, R8Hydrogen or C independently all are at each occurrence2-C12Alkyl.In some embodiments In, R8For hydrogen.In other embodiments, R8For ethyl.In some other embodiments, R8For n-propyl or isopropyl. In some embodiments, R8For C4Alkyl.In other embodiments, R8For C5Alkyl.In other embodiments, R8For C6Alkane Base.In some embodiments, R8For C7Alkyl.In other embodiments, R8For C8Alkyl.In other embodiments, R8 For C9Alkyl.In other embodiments, R8For C10Alkyl.In some other embodiments, R8For C11Alkyl.In other realities Apply in scheme, R8For C12Alkyl.In some other embodiments, R8For C2-C12Alkyl, and the C2-C12Alkyl includes one Individual or multiple double bonds (for example, alkene), three keys (for example, alkynes) or both.In some embodiments, R8To be unsubstituted C2-C12Alkyl.
In some embodiments, R9Hydrogen, C independently all are at each occurrence1-C12Alkyl, C1-C12Aralkyl or virtue Base.In some embodiments, R9For hydrogen.In other embodiments, R9For C1-C12Alkyl.In other embodiments, R9 For methyl.In other embodiments, R9For ethyl.In some other embodiments, R9For n-propyl or isopropyl.One In a little embodiments, R9For C4Alkyl.In some embodiments, R9For C5Alkyl.In other embodiments, R9For C6Alkane Base.In some other embodiments, R9For C7Alkyl.In some embodiments, R9For C8Alkyl.In some embodiments In, R9For C9Alkyl.In some other embodiments, R9For C10Alkyl.In some other embodiments, R9For C11Alkyl. In other embodiments, R9For C12Alkyl.
In some other embodiments, R9For C1-C12Aralkyl.For example, in some embodiments, R9For benzyl, And the benzyl can be optionally substituted on phenyl ring or benzyl carbon.Thus, substituent includes alkyl and alcoxyl Base, such as methyl or methoxy.In some embodiments, it instead of benzyl with methyl at benzyl carbon.For example, one In a little embodiments, R9With following structure (XIV)：
In other embodiments, R9For aryl.For example, in some embodiments, R9For phenyl, and the phenyl can To be optionally substituted.Thus, substituent includes alkyl and alkoxy grp, such as methyl or methoxy.In other implementations In scheme, R9For phenyl, and the phenyl includes crown ether moiety, the crown ether of such as 12-18 members.In one embodiment, it is preced with Ether is 18 yuan, and can further include other phenyl moiety.For example, in one embodiment, R9With following knot One kind in structure (XV or XVI)：
In some embodiments, R10Hydrogen, C independently all are at each occurrence1-C12Alkyl or LNR4R5R7, wherein R4、R5And R7Such as above with respect to the definition of connection (A).In other embodiments, R10For hydrogen.In other embodiments, R10 For C1-C12Alkyl, and in other embodiments, R10For-LNR4R5R7.In some embodiments, R10For methyl.At other In embodiment, R10For ethyl.In some embodiments, R10For C3Alkyl.In some embodiments, R10For C4Alkyl. In other embodiments, R10For C5Alkyl.In some other embodiments, R10For C6Alkyl.In other embodiments In, R10For C7Alkyl.In other embodiments, R10For C8Alkyl.In some embodiments, R10For C9Alkyl.At other In embodiment, R10For C10Alkyl.In other embodiments, R10For C11Alkyl.In some other embodiments, R10For C12Alkyl.
In some embodiments, R8And R9Combine to form the monocyclic or bicyclic heterocycles of 5-18 members.In some embodiments In, heterocycle is 5 or 6 yuan of monocyclic heterocycles.For example, in some embodiments, connection (B) has following structure (IV)：
Wherein Z represents 5 or 6 yuan of monocyclic heterocycles.
In other embodiments, heterocycle is two rings, such as 12 yuan of bicyclic heterocycles.Heterocycle can be piperazinyl.It is miscellaneous Ring can be morpholino.Heterocycle can be piperidyl.Heterocycle can be Decahydroisoquinolinpreparation.Representational heterocycle includes as follows：
In some embodiments, R11C independently all is at each occurrence2-C12Alkyl, C1-C12Aminoalkyl, aryl, Heteroaryl or heterocyclic radical.
In some embodiments, R11For C2-C12Alkyl.In some embodiments, R11For ethyl.In other implementations In scheme, R11For C3Alkyl.In other embodiments, R11For isopropyl.In some other embodiments, R11For C4Alkane Base.In other embodiments, R11For C5Alkyl.In some embodiments, R11For C6Alkyl.In other embodiments, R11For C7Alkyl.In some embodiments, R11For C8Alkyl.In other embodiments, R11For C9Alkyl.In other implementations In scheme, R11For C10Alkyl.In some other embodiments, R11For C11Alkyl.In some embodiments, R11For C12Alkane Base.
In other embodiments, R11For C1-C12Aminoalkyl.In some embodiments, R11For methylamino.One In a little embodiments, R11For ethylamino.In other embodiments, R11For C3Aminoalkyl.In other embodiments, R11 For C4Aminoalkyl.In some other embodiments, R11For C5Aminoalkyl.In other embodiments, R11For C6Aminoalkyl. In other embodiments, R11For C7Aminoalkyl.In some embodiments, R11For C8Aminoalkyl.In other embodiments, R11 For C9Aminoalkyl.In other embodiments, R11For C10Aminoalkyl.In some other embodiments, R11For C11Aminoalkyl. In other embodiments, R11For C12Aminoalkyl.
In other embodiments, R11For C1-C12Alkyl-carbonyl.In other embodiments, R11For C1Alkyl-carbonyl. In other embodiments, R11For C2Alkyl-carbonyl.In some embodiments, R11For C3Alkyl-carbonyl.In other embodiments In, R11For C4Alkyl-carbonyl.In some embodiments, R11For C5Alkyl-carbonyl.In some other embodiments, R11For C6 Alkyl-carbonyl.In other embodiments, R11For C7Alkyl-carbonyl.In other embodiments, R11For C8Alkyl-carbonyl.One In a little embodiments, R11For C9Alkyl-carbonyl.In other embodiments, R11For C10Alkyl-carbonyl.Implement some other In scheme, R11For C11Alkyl-carbonyl.In some embodiments, R11For C12Alkyl-carbonyl.In other embodiments, R11 For-C (=O) (CH2)nCO2H, wherein n are 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.At it In his embodiment, n is 6.
In other embodiments, R11For aryl.For example, in some embodiments, R11For phenyl.In some implementations In scheme, phenyl is for example replaced with nitro.
In other embodiments, R11For heteroaryl.For example, in some embodiments, R11For pyridine radicals.At other In embodiment, R11For pyrimidine radicals.
In other embodiments, R11For heterocyclic radical.For example, in some embodiments, R11For piperidyl, such as piperazine Pyridine -4- bases.
In some embodiments, R11For ethyl, isopropyl, piperidyl, pyrimidine radicals, cholate, dexycholate or-C (=O)(CH2)nCO2H, wherein n are 1 to 6.
In some embodiments, R is electronics pair.In other embodiments, R is hydrogen, and in other embodiments In, R is C1-C12Alkyl.In some embodiments, R is methyl.In some embodiments, R is ethyl.In other implementations In scheme, R is C3Alkyl.In other embodiments, R is isopropyl.In some other embodiments, R is C4Alkyl. In other embodiments, R is C5Alkyl.In some embodiments, R is C6Alkyl.In other embodiments, R is C7Alkane Base.In other embodiments, R is C8Alkyl.In other embodiments, R is C9Alkyl.In some embodiments, R For C10Alkyl.In other embodiments, R is C11Alkyl.In some embodiments, R is C12Alkyl.
In some embodiments, R12Hydrogen, C independently all are at each occurrence1-C12Alkyl, C1-C12Aminoalkyl ,- NH2、-CONH2、-NR13R14、-NR13R14R15, oxo (oxo) ,-CN, trifluoromethyl, amide groups, amidino groups, Amidinylalkyl, amidino groups Alkyl-carbonyl guanidine radicals, guanidine alkylation, guanidine alkylation carbonyl, cholate, dexycholate, aryl, heteroaryl, heterocycle ,-SR13Or C1-C12Alkoxy, wherein R13、R14And R15C independently all is at each occurrence1-C12Alkyl.
In some embodiments, R12For hydrogen.In some embodiments, R12For C1-C12Alkyl.In some embodiment party In case, R12For C1-C12Aminoalkyl.In some embodiments, R12For-NH2.In some embodiments, R12For-CONH2. In some embodiments, R12For-NR13R14.In some embodiments, R12For-NR13R14R15.In some embodiments, R12For C1-C12Alkyl-carbonyl.In some embodiments, R12For oxo.In some embodiments, R12For-CN.At some In embodiment, R12For trifluoromethyl.In some embodiments, R12For amide groups.In some embodiments, R12For amidine Base.In some embodiments, R12For Amidinylalkyl.In some embodiments, R12For Amidinylalkyl carbonyl.In some realities Apply in scheme, R12For guanidine radicals, such as monomethyl guanidine radicals or dimethyl guanidine radicals.In some embodiments, R12For guanidine alkylation. In some embodiments, R12For Amidinylalkyl carbonyl.In some embodiments, R12For cholate.In some embodiments In, R12For dexycholate.In some embodiments, R12For aryl.In some embodiments, R12For heteroaryl.One In a little embodiments, R12For heterocycle.In some embodiments, R12For-SR13.In some embodiments, R12For C1-C12Alkane Epoxide.In some embodiments, R12For dimethylamino.
In other embodiments, R12For methyl.In other embodiments, R12For ethyl.In some embodiments In, R12For C3Alkyl.In some embodiments, R12For isopropyl.In some embodiments, R12For C4Alkyl.At other In embodiment, R12For C5Alkyl.In other embodiments, R12For C6Alkyl.In some other embodiments, R12For C7 Alkyl.In some embodiments, R12For C8Alkyl.In other embodiments, R12For C9Alkyl.In some embodiments In, R12For C10Alkyl.In other embodiments, R12For C11Alkyl.In other embodiments, R12For C12Alkyl.At it In his embodiment, the moieties are substituted with one or more oxygen atoms and form ether moiety, for example methoxy methyl base portion Point.
In some embodiments, R12For methylamino.In other embodiments, R12For ethylamino.At other In embodiment, R12For C3Aminoalkyl.In some embodiments, R12For C4Aminoalkyl.In other embodiments, R12For C5 Aminoalkyl.In some other embodiments, R12For C6Aminoalkyl.In some embodiments, R12For C7Aminoalkyl.At some In embodiment, R12For C8Aminoalkyl.In other embodiments, R12For C9Aminoalkyl.In some other embodiments, R12 For C10Aminoalkyl.In other embodiments, R12For C11Aminoalkyl.In other embodiments, R12For C12Aminoalkyl.One In a little embodiments, aminoalkyl is dimethylamino.
In other embodiments, R12For acetyl group.In some other embodiments, R12For C2Alkyl-carbonyl.One In a little embodiments, R12For C3Alkyl-carbonyl.In other embodiments, R12For C4Alkyl-carbonyl.In some embodiments In, R12For C5Alkyl-carbonyl.In other embodiments, R12For C6Alkyl-carbonyl.In some other embodiments, R12For C7 Alkyl-carbonyl.In some embodiments, R12For C8Alkyl-carbonyl.In other embodiments, R12For C9Alkyl-carbonyl.One In other a little embodiments, R12For C10Alkyl-carbonyl.In some embodiments, R12For C11Alkyl-carbonyl.In other embodiment party In case, R12For C12Alkyl-carbonyl.Alkyl-carbonyl is replaced by carboxy moiety, and for example alkyl-carbonyl is substituted to form butanedioic acid portion Divide (that is, 3- Carboxyalkylcarbonyls).In other embodiments, alkyl-carbonyl is replaced by end-SH bases.
In some embodiments, R12For amide groups.In some embodiments, amide groups includes what is be further substituted Moieties, for example, replaced by-SH, carbamate or combinations thereof.In other embodiments, amide groups is by aryl portion Such as phenyl is divided to replace.In certain embodiments, R12There can be following structure (IX)：
Wherein R16Hydrogen, C independently all are at each occurrence1-C12Alkyl, C1-C12Alkoxy ,-CN, aryl or heteroaryl Base.
In some embodiments, R12For methoxyl group.In other embodiments, R12For ethyoxyl.In others implementation In scheme, R12For C3Alkoxy.In some embodiments, R12For C4Alkoxy.In some embodiments, R12For C5Alcoxyl Base.In some other embodiments, R12For C6Alkoxy.In other embodiments, R12For C7Alkoxy.At some its In his embodiment, R12For C8Alkoxy.In some embodiments, R12For C9Alkoxy.In other embodiments, R12For C10Alkoxy.In some embodiments, R12For C11Alkoxy.In other embodiments, R12For C12Alkoxy.
In certain embodiments, R12For pyrrolidinyl, such as pyrrolidin-1-yl.In other embodiments, R12For Piperidyl, such as piperidin-1-yl or piperidin-4-yl.In other embodiments, R12For morpholino, such as morpholine -4- bases. In other embodiments, R12For phenyl, and even in other embodiments, the phenyl is for example replaced with nitro.At it In his embodiment, R12For pyrimidine radicals, such as pyrimidine -2-base.
In other embodiments, R13、R14And R15C independently all is at each occurrence1-C12Alkyl.In some implementations In scheme, R13、R14Or R15For methyl.In other embodiments, R13、R14Or R15For ethyl.In other embodiments, R13、R14Or R15For C3Alkyl.In other embodiments, R13、R14Or R15For isopropyl.In other embodiments, R13、R14 Or R15For C4Alkyl.In some embodiments, R13、R14Or R15For C5Alkyl.In some other embodiments, R13、R14Or R15For C6Alkyl.In other embodiments, R13、R14Or R15For C7 alkyl.In other embodiments, R13、R14Or R15For C8Alkyl.In other embodiments, R13、R14Or R15For C9Alkyl.In some embodiments, R13、R14Or R15For C10Alkane Base.In some embodiments, R13、R14Or R15For C11Alkyl.In other embodiments, R13、R14Or R15For C12Alkyl.
As described above, in some embodiments, R12For the amide groups replaced by aryl moiety.Thus, R16It is every Secondary appearance can be same or different.These embodiments it is some in, R16For hydrogen.In other embodiments, R16For-CN.In other embodiments, R16For heteroaryl, such as tetrazole radical.In certain other embodiments, R16For methoxy Base.In other embodiments, R16For aryl, and the aryl is optionally substituted.Thus, optional substituent bag Include：C1-C12Alkyl, C1-C12Alkoxy, such as methoxyl group；Trifluoromethoxy；Halogen, such as chloro；And trifluoromethyl.
In other embodiments, R16For methyl.In other embodiments, R16For ethyl.In some embodiments In, R16For C3Alkyl.In some other embodiments, R16For isopropyl.In other embodiments, R16For C4Alkyl. In other embodiments, R16For C5Alkyl.In other embodiments, R16For C6Alkyl.In some other embodiments, R16 For C7Alkyl.In some embodiments, R16For C8Alkyl.In other embodiments, R16For C9Alkyl.In some other realities Apply in scheme, R16For C10Alkyl.In other embodiments, R16For C11Alkyl.In some other embodiments, R16For C12 Alkyl.
In some embodiments, R16For methoxyl group.In some embodiments, R16For ethyoxyl.In other embodiment party In case, R16For C3Alkoxy.In some other embodiments, R16For C4Alkoxy.In other embodiments, R16For C5Alkane Epoxide.In some other embodiments, R16For C6Alkoxy.In other embodiments, R16For C7Alkoxy.At some its In his embodiment, R16For C8Alkoxy.In other embodiments, R16For C9Alkoxy.In some other embodiments, R16For C10Alkoxy.In some embodiments, R16For C11Alkoxy.In some other embodiments, R16For C12Alcoxyl Base.
In some other embodiments, R8And R9Combine to form the crown ether of 12-18 members.For example, in some embodiments In, crown ether is 18 yuan, and in other embodiments, crown ether is 15 yuan.In certain embodiments, R8And R9With reference to Formed with heterocycle a kind of in following structure (X) or (XI)：
In some embodiments, R8、R9Or R3With R10Combine to form the heterocycle of 5-7 members.For example, in some embodiments In, R3With R10Combine to form the heterocycle of 5-7 members.In some embodiments, heterocycle is 5 yuan.In other embodiments, it is miscellaneous Ring is 6 yuan.In other embodiments, heterocycle is 7 yuan.In some embodiments, heterocycle can pass through following structure (XII) represent：
Wherein Z ' represents the heterocycle of 5-7 members.In some embodiments of structure (XI), R12All it is at each occurrence Hydrogen.For example, connection (B) can have one kind in following structure (B1), (B2) or (B3)：
In certain other embodiments, R12For C1-C12Alkyl-carbonyl or amide groups, it is by aryl non-phosphoryl moiety, example As triphenyl phosphorus acyl moiety is further substituted with.Connection example with the structure includes B56 and B55.
In certain embodiments, connection (B) is without any structure A1-A5.Table 2 shows the representative of (A) type and (B) Property connection.
Connected between the representational subunit of table 2.
In subsequent sequence and discussion, the title of above connection is often used.For example, including PMOapnThe alkali of connection Base is described asapnB, wherein B are base.Other connections can be named similarly.Further, it is possible to use abbreviation title, for example, can With in use above bracket abbreviation title (for example,aB, is referred toapnB).Other abbreviations easily recognized can also be used Word.
B.Oligomer with the end group by modification
Except carrier peptides, conjugate can also include the oligomer containing the end group by modification.Applicant is It was found that providing favourable curative properties (example after modifying 3 ' and/or 5 ' ends of oligomer with various chemical parts for conjugate Such as, cell delivering, potency and/or Tissue distribution of raising etc.).In various embodiments, the end group bag by modification Containing hydrophobic parts, and in other embodiments, the end group by modification includes hydrophilic segment.By the end of modification Group may have or in the absence of above-mentioned connection.For example, in some embodiments, the oligomer combined with carrier peptides is included One or more end groups and (A) type by modification is connected, and such as wherein X is-N (CH3)2Connection.In other embodiment party In case, oligomer comprising it is one or more by modification end groups and (B) type connect, such as wherein X be 4- amino piperidines- The connection of 1- bases (that is, APN).Further in other embodiments, oligomer includes one or more ends by modification Group and the mixing of connection (A) and (B).For example, oligomer can include one or more end group (examples by modification Such as, trityl or triphenyl acetyl group) and wherein X be-N (CH3)2Connection, and wherein X is 4- amino piperidine -1- base Connection.Other combinations of end group by modification and the connection by modification also provide favorably therapeutic for oligomer Energy.
In one embodiment, there is following structure (XVII) comprising end modified oligomer：
Or the salt or isomers of the structure, wherein X, W and Y be connected as described above defined in any one in (A) and (B), And：
R17All independently be at each occurrence be not present, hydrogen or C1-C6Alkyl；
R18And R19All independently be at each occurrence be not present, hydrogen, carrier peptides, natural or alpha-non-natural amino acid, C2- C30Alkyl-carbonyl ,-C (=O) OR21Or R20；
R20Guanidine radicals, heterocyclic radical, C independently all are at each occurrence1-C30Alkyl, C3-C8Cycloalkyl；C6-C30Aryl, C7-C30Aralkyl, C3-C30Alkyl-carbonyl, C3-C8Naphthene base carbonyl, C3-C8Cycloalkyl alkyl carbonyl, C7-C30Aryl carbonyl, C7- C30Aromatic alkyl carbonyl, C2-C30Alkoxy carbonyl, C3-C8Cyclo alkoxy carbonyl, C7-C30Aryloxycarbonyl, C8-C30Arylalkoxy Base carbonyl or-P (=O) (R22)2；
Pi independently is the part of base pairing at each occurrence；
L1For the linking arm of up to 18 optional atomic lengths, it, which is included, is selected from following connection：Alkyl, hydroxyl, alkane Epoxide, alkylamino, acid amides, ester, disulphide, carbonyl, carbamate, phosphoric acid diamides, phosphoamide, thiophosphate, Piperazine and di-phosphate ester；And
X is 0 or bigger integer；And wherein R18Or R19Middle at least one is R20；And
Wherein R18Or R19Middle at least one is R20, and condition is R17And R18Be in the absence of.
Oligomer with the end group by modification can be connected comprising any number of (A) type and (B) type.Example Such as, oligomer can only include the connection of (A) type.For example, the X in each connection can be-N (CH3)2.Or, oligomer can With only comprising connection (B).In certain embodiments, oligomer includes the mixing of connection (A) and (B), such as 1 to 4 (B) type Connection, and the remainder of the connection is (A) type.Thus, it is amino piperidine base that connection, which includes, but not limited to wherein X, (B) type connection and X connect for (A) type of dimethylamino.
In some embodiments, R17For in the absence of.In some embodiments, R17For hydrogen.In some embodiments In, R17For C1-C6Alkyl.In some embodiments, R17For methyl.In other embodiments, R17For ethyl.In some realities Apply in scheme, R17For C3Alkyl.In some other embodiments, R17For isopropyl.In other embodiments, R17For C4Alkane Base.Further in other embodiments, R17For C5Alkyl.In some other embodiments, R17For C6Alkyl.
In other embodiments, R18For in the absence of.In some embodiments, R18For hydrogen.In some embodiments In, R18For carrier peptides.In some embodiments, R18For natural or alpha-non-natural amino acid, such as trimethylglycine.One In a little embodiments, R18For R20。
In other embodiments, R19For in the absence of.In some embodiments, R19For hydrogen.In some embodiments In, R19For carrier peptides.In some embodiments, R19For natural or alpha-non-natural amino acid, such as trimethylglycine.One In a little embodiments, R19For-C (=O) OR17, such as R19There can be following structure：
In other embodiments, R18Or R19For C2-C30Alkyl-carbonyl, such as-C (=O) (CH2)nCO2H, wherein n are 1 To 6, such as 2.In other instances, R18Or R19For acetyl group.
In some embodiments, R20Guanidine radicals, heterocyclic radical, C independently all are at each occurrence1-C30Alkyl, C3-C8 Cycloalkyl；C6-C30Aryl, C7-C30Aralkyl, C3-C30Alkyl-carbonyl, C3-C8Naphthene base carbonyl, C3-C8Cycloalkyl-alkyl carbonyl Base, C6-C30Aryl carbonyl, C7-C30Aromatic alkyl carbonyl, C2-C30Alkoxy carbonyl, C3-C8Cyclo alkoxy carbonyl, C7-C30Aryloxy group Carbonyl, C8-C30Aryl-alkoxy carbonyl ,-C (=O) OR21Or-P (=O) (R22)2, wherein R21To include one or more oxygen or hydroxyl The C of base section or combinations thereof1-C30Alkyl, and each R22All it is C6-C12Aryloxy group.
In certain other embodiments, R19For-C (=O) OR21, and R18For hydrogen, guanidine radicals, heterocyclic radical, C1-C30Alkyl, C3-C8Cycloalkyl；C6-C30Aryl, C3-C30Alkyl-carbonyl, C3-C8Alkyl-carbonyl, C3-C8Cycloalkyl alkyl carbonyl, C7-C30Aryl Carbonyl, C7-C30Aromatic alkyl carbonyl, C2-C30Alkoxy carbonyl, C3-C8Cyclo alkoxy carbonyl, C7-C30Aryloxycarbonyl, C8-C30Virtue Base alkoxy carbonyl or-P (=O) (R22)2, wherein each R22All it is C6-C12Aryloxy group.
In other embodiments, R20Guanidine radicals, heterocyclic radical, C independently all are at each occurrence1-C30Alkyl, C3-C8 Cycloalkyl；C6-C30Aryl, C3-C30Alkyl-carbonyl, C3-C8Naphthene base carbonyl, C3-C8Cycloalkyl alkyl carbonyl, C7-C30Aryl carbonyl Base, C7-C30Aromatic alkyl carbonyl, C2-C30Alkoxy carbonyl, C3-C8Cyclo alkoxy carbonyl, C7-C30Aryloxycarbonyl, C8-C30Aryl Alkoxy carbonyl or-P (=O) (R22)2.And in other instances, R20All independently be at each occurrence guanidine radicals, heterocyclic radical, C1-C30Alkyl, C3-C8Cycloalkyl；C6-C30Aryl, C7-C30Aralkyl, C3-C8Naphthene base carbonyl, C3-C8Cycloalkyl-alkyl carbonyl Base, C7-C30Aryl carbonyl, C7-C30Aromatic alkyl carbonyl, C2-C30Alkoxy carbonyl, C3-C8Cyclo alkoxy carbonyl, C7-C30Aryloxy group Carbonyl, C8-C30Aryl-alkoxy carbonyl or-P (=O) (R22)2。
In some embodiments, R20For guanidine radicals, such as monomethyl guanidine radicals or dimethyl guanidine radicals.In other embodiments In, R20For heterocyclic radical.For example, in some embodiments, R20For piperidin-4-yl.In some embodiments, piperidin-4-yl Replaced by trityl or Boc bases.In other embodiments, R20For C3-C8Cycloalkyl.In other embodiments, R20For C6-C30Aryl.
In some embodiments, R20For C7-C30Aryl carbonyl.For example, in some embodiments, R20With following Structure (XVIII)：
Wherein R23Hydrogen, halogen, C independently all are at each occurrence1-C30Alkyl, C1-C30Alkoxy, C1-C30Alkoxy Carbonyl, C7-C30Aralkyl, aryl, heteroaryl, heterocyclic radical or Heterocyclylalkyl, and wherein 1 R23Can be with another R23With reference to shape Into heterocyclic ring.In some embodiments, at least one R23For hydrogen, for example, in some embodiments, each R23All it is Hydrogen.In other embodiments, at least one R23For C1-C30Alkoxy, for example in some embodiments, each R23All it is first Epoxide.In other embodiments, at least one R23For heteroaryl, such as in some embodiments, at least one R23With with It is a kind of in lower structure (XVIIIa) or (XVIIIb)：
In other embodiments, 1 R23With another R23Combine to form heterocyclic ring.For example, in an embodiment In, R20For CF.
In other embodiments, R20For C7-C30Aromatic alkyl carbonyl.For example, in various embodiments, R20With with It is a kind of in lower structure (XIX), (XX) or (XXI)：
Wherein R23Hydrogen, halogen, C independently all are at each occurrence1-C30Alkyl, C1-C30Alkoxy, C1-C30Alkoxy Carbonyl, C7-C30Aralkyl, aryl, heteroaryl, heterocyclic radical or Heterocyclylalkyl, wherein 1 R23Can be with another R23Combine to form Heterocyclic ring, X is-OH or halogen, and m is 0 to 6 integer.In some specific embodiments, m is 0.In other implementations In scheme, m is 1, and in other embodiments, m is 2.In other embodiments, at least one R23For hydrogen, such as at some In embodiment, each R23All it is hydrogen.In some embodiments, X is hydrogen.In other embodiments, X is-OH.At other In embodiment, X is Cl.In other embodiments, at least one R23For C1-C30Alkoxy, such as methoxyl group.
Further in other embodiments, R20For C7-C30Aralkyl, such as trityl.In other embodiments, R20For Methoxytrityl.In some embodiments, R20With following structure (XXII)：
Wherein R23Hydrogen, halogen, C independently all are at each occurrence1-C30Alkyl, C1-C30Alkoxy, C1-C30Alkoxy Carbonyl, C7-C30Aralkyl, aryl, heteroaryl, heterocyclic radical or Heterocyclylalkyl, and wherein 1 R23Can be with another R23With reference to shape Into heterocyclic ring.For example, in some embodiments, each R23All it is hydrogen.In other embodiments, at least one R23For C1- C30Alkoxy, such as methoxyl group.
In other embodiments, R20For C7-C30Aralkyl, and R20With following structure (XXIII)：
In some embodiments, at least one R23For halogen, such as chloro.In some other embodiments, 1 R23For positioned at the chloro of contraposition.
In other embodiments, R20For C1-C30Alkyl.For example, in some embodiments, R20For C4-C20Alkyl, And optionally include one or more double bonds.For example, in some embodiments, R20For comprising three keys, such as terminal triple link C4-10Alkyl.In some embodiments, R20For hexin -6- bases.In some embodiments, R20With following structure (XXIV), one kind in (XXV), (XXVI) or (XXVII)：
Further in other embodiments, R20For C3-C30Alkyl-carbonyl, such as C3-C10Alkyl-carbonyl.In some implementations In scheme, R20For-C (=O) (CH2)pSH or-C (=O) (CH2)pSSHet, wherein p are 1 to 6 integer, and Het is heteroaryl. For example, p can be 1, or p can be 2.In other instances, Het is pyridine radicals, for example pyridine -2- bases.In other implementations In scheme, C3-C30Alkyl-carbonyl is replaced by other oligomers, for example in some embodiments, and oligomer, which is included, is located at 3 ' positions The C put3-C30Alkyl-carbonyl, oligomer is connected to 3 ' positions of another oligomer by it.It is such end modified to be included in this public affairs In the range of opening.
In other embodiments, R20For the C being further substituted with by aryl non-phosphoryl moiety3-C30Alkyl-carbonyl, such as three Diphenylphosphoryl base.Such R20The example of group includes the structure 33 in table 3.
In other instances, R20For C3-C8Naphthene base carbonyl, such as C5-C7Alkyl-carbonyl.In these embodiments, R20 With following structure (XXVIII)：
Wherein R23Hydrogen, halo, C independently all are at each occurrence1-C30Alkyl, C1-C30Alkoxy, C1-C30Alkoxy Carbonyl, C7-C30Aralkyl, aryl, heteroaryl, heterocyclic radical or Heterocyclylalkyl, and wherein 1 R23Can be with another R23With reference to shape Into heterocyclic ring.In some embodiments, R23For cycloheteroalkylalkyl, for example in some embodiments.R23With following knot Structure：
In some other embodiments, R20For C3-C8Cycloalkyl alkyl carbonyl.In other embodiments, R20For C2- C30Alkoxy carbonyl.In other embodiments, R20For C3-C8Cyclo alkoxy carbonyl.In other embodiments, R20For C7- C30Aryloxycarbonyl.In other embodiments, R20For C8-C30Aryl-alkoxy carbonyl.In other embodiments, R20For- P(=O)(R22)2, wherein each R22All it is C6-C12Aryloxy group, for example in some embodiments, R20With following structure (C24)：
In other embodiments, R20Include one or more halogen atoms.For example, in some embodiments, R20Including Any above R20Partial perfluoro analog.In other embodiments, R20For p-trifluoromethyl phenyl, trifluoromethyl three Benzyl, perfluoropentyl or pentafluorophenyl group.
In some embodiments, 3 ' ends include modification, and in other embodiments, 5 ' ends include modification.At other In embodiment, 3 ' and 5 ' ends are all comprising modification.Therefore, in some embodiments, R18For in the absence of and R19For R20.At it In his embodiment, R19For in the absence of and R18For R20.Further in other embodiments, R18And R19Respectively R20。
In some embodiments, except 3 ' or 5 ' modifications, oligomer also includes cell permeability peptide.Therefore, at some In embodiment, R19For cell permeability peptide, and R18For R20.In other embodiments, R18For cell permeability peptide, and R19 For R20.In the further embodiment of aforementioned schemes, cell permeability peptide is Arginine-rich peptide.
In some embodiments, it may be present or lack 5 ' terminal groups (that is, R19) it is connected to the linking arm L of oligomer1。 The linking arm can include any number of functional group and length, and condition is that the linking arm remains with it and is connected to 5 ' terminal groups Ability on oligomer, and condition are that the linking arm does not disturb being attached to sequence-specific fashion on target sequence for oligomer Ability.In one embodiment, L is connected comprising phosphoric acid diamides and piperazine.For example, in some embodiments, L has Following structure (XXIX)：
Wherein R24To be not present, hydrogen or C1-C6Alkyl.In some embodiments, R24For in the absence of.In some embodiment party In case, R24For hydrogen.In some embodiments, R24For C1-C6Alkyl.In some embodiments, R24For methyl.In other realities Apply in scheme, R24For ethyl.Further in other embodiments, R24For C3Alkyl.In some other embodiments, R24 For isopropyl.In other embodiments, R24For C4Alkyl.In some embodiments, R24For C5Alkyl.In other embodiment party In case, R24For C6Alkyl.
Further in other embodiments, R20For C3-C30Alkyl-carbonyl, and R20With following structure (XXX)：
Wherein R25For hydrogen or-SR26, wherein R26For hydrogen, C1-C30Alkyl, heterocyclic radical, aryl or heteroaryl, and q is 0 to 6 Integer.
It is in office how on other embodiments, R23Hydrogen, halo, C independently all are at each occurrence1-C30Alkyl, C1-C30Alkoxy, aryl, heteroaryl, heterocyclic radical or Heterocyclylalkyl.
In some other embodiments, 3 ' ends of only oligomer are incorporated on 1 above-described group.At some In other embodiments, 5 ' ends of only oligomer are incorporated on 1 above-described group.In other embodiments, 3 ' With 5 ' ends all comprising 1 above-described group.Terminal groups can be illustrated in above-described any 1 group or table 3 Any special groups.
The representational terminal groups of table 3.
C. the performance of conjugate
As described above, this disclosure relates to the conjugate (that is, oligomer) of carrier peptides and oligonucleotide analogs.Oligomer can To include the various modifications for assigning the desired performance (for example, increased antisense activity) of oligomer.In certain embodiments, Oligomer is included to be connected 1 between the skeleton containing the morpholino ring-type structure sequence by connecting combination between subunit, the subunit 3 ' ends of individual morpholino cyclic structure are connected on 5 ' ends of neighbouring morpholino cyclic structure, wherein each morpholino ring Shape structure is all incorporated on the part of base pairing, so that oligomer can be attached on target nucleic acid with sequence-specific fashion. Morpholino cyclic structure can have following structure (i)：
Wherein Pi independently is the part of base pairing at each occurrence.
Each morpholino cyclic structure supports base pairing moiety (Pi), to form base pairing moiety sequence, and it leads to Often it is designed to the antisense traget selected in intracellular or receiving treatment subject to hybridize.Base pairing moiety can be The purine or pyrimidine found in n DNA or RNA (A, G, C, T or U) or the like, such as hypoxanthine (base of nucleoside inosine Component) or 5-methylcytosine.The analog base for assigning the binding affinity that oligomer is improved can also be utilized.With regard to this Speech, exemplary analog includes pyrimidine, 9- (amino ethoxy) phenoxazine (G- hair fasteners) that C5- propinyls are modified etc..
As described above, according to one aspect of the present invention, oligomer can be modified to include one or more (B) connection, Uncharged connection up to about 1 (B) connection for example per 2-5, generally every 10 uncharged to be connected as 3-5 (B) Connection.Some embodiments also include one or more (B) types and connected.In some embodiments, if up to about half Skeleton is connected as type (B), then, it is seen that to the optimal raising of antisense activity.When (B) with peanut such as 10-20% is connected, It is typically seen to arrive some but off-peak raising.
In one embodiment, (A) type and the connection of (B) type are spread along skeleton.In some embodiments, oligomer (A) and (B) connection without the strict alternate mode along its whole length.Except carrier peptides, oligomer can also be optional Ground includes as described above 5 ' and/or 3 ' modifications.
Also contemplate the oligomer with multiple (A) contiguous blocks He (B) contiguous block；For example, multiple (B) contiguous blocks can position In the side of center (A) contiguous block, or it is as the same in turn.In one embodiment, oligomer has approximate isometric 5 ', 3 ' End and center, and the percentage of (B) or (A) connection of center is greater than about 50%, or greater than about 70%.Applied for antisense Oligomer lengths generally range from about 10 to about 40 subunits, more preferably from about 15 to 25 subunits.For example, sub- with 19-20 The oligomer of the present invention of base (length useful to antisense scant polymer) can ideally have 2 to 7, such as 4 to 6 or 3 to 5 Individual (B) is connected, and remaining (A) connection.Oligomer with 14-15 subunit can ideally have 2 to 5, such as 3 Or 4 (B) connection, and remaining (A) connection.
Morpholino subunit can also be connected by being not based on connecting between the subunit of phosphorus, it is as described further below.
Other oligonucleotide analogs can also be used to connect, its be in the presence of unmodified state it is uncharged, But there can also be side amino-substituent.For example, the nitrogen-atoms of 5 ' in morpholino ring can be used in sulfonamide connection (or urine Element connection, wherein phosphorus is replaced by carbon or sulphur respectively).
In some embodiments that antisense is applied, oligomer can be complementary with nucleic acid target sequence 100%, or it can be with Comprising mispairing, for example, for accommodating variant, as long as the heteroduplex formed between oligomer and nucleic acid target sequence is sufficiently stable, And it is resistant to nucleus enzyme and the in vivo effect of possible other patterns degraded occurred.Mispairing (if present) makes heteroduplex The unstable possibility of stub area is less than intermediate region.According to well known duplex stability principle, it is allowed to mispairing number take G in length certainly in oligomer, double-strand:The position that mispairing occurs in the percentage and double-strand of C base-pairs.Although such antisense is few Polymers is not necessarily complementary with nucleic acid target sequence 100%, and it still effectively can stablize and be specifically binding on target sequence, so that The expression of the albumen of the bioactivity of nucleic acid target, such as coding is adjusted.
The duplex stability formed between oligomer and target sequence is to combine TmWith the double-strand to the sensitiveness of cell digestion Function.T of the antisense compounds on complementary series RNA can be measured by conventional methodm, such as Hames et al., Nucleic Acid Hybridization, IRL Press, the method described in 1985, pp.107-108, or such as Miyada C.G. and Wallace R.B.,1987,Oligonucleotide hybridization techniques,Methods Described in Enzymol.Vol.154pp.94-107.
In some embodiments, the combination T on complementary series RNA that each antisense scant polymer hasmMore than body temperature, Or in other embodiments, more than 50 DEG C.In other embodiments, TmFor 60-80 DEG C or bigger.According to well known original Then, by increasing C in double-strand:The ratio of the paired bases of G, and/or by increasing the length (in base-pair form) of heteroduplex, Oligomerization compounds can be increased on the T based on complementary RNA hybridizationm.Meanwhile, in order to optimize cellular uptake, limit oligomerization The size of thing is probably favourable.For this reason, it has been in a ratio of and has obtained high TmValue needs to be more than the compound of 20 bases, The compound of high Tm (50 DEG C or bigger) is generally preferably shown in 20 bases or less bases longs.For some applications, Longer oligomer, some advantages may be had by being for example longer than 20 bases.For example, in certain embodiments, longer widow Polymers is particularly useful for exon skipping or splicing regulation and control.
Targeting sequence base can be normal DNA base or its analog, for example, can be carried out with target sequence RNA bases The uracil and creatinine of Watson-Crick base pairings.
When target nucleotide is uracil residues, oligomer can also be incorporated to guanine base to replace adenine.Work as target When sequence has differences between different virus species and the variation of any given nucleotide residue is cytimidine or uracil, This is useful.By using guanine at the variant sites in target-seeking oligomer, it is possible to use well known guanine and urine Pyrimidine (is referred to as C/U:G base pairings) base pairing ability.By being incorporated to guanine in these positions, single oligomer can be with Effectively it regard the RNA target variability of wider range as target.
Compound (for example, connection, terminal groups between oligomer, subunit) can exist with different isomeric forms, for example, tie Structure isomers (for example, dynamic isomer).On stereoisomer, compound can have chiral centre, and can occur as Racemic modification, mixture, single enantiomer, mixture or the diastereomer or single diastereomer of enantiomer enrichment.Institute There are these isomeric forms to be all included in the present invention, include their mixture.Compound can also have axial chirality, and it can To cause the formation of atropisomer.In addition, some crystal formations of compound may have for polymorphic, it is included in the present invention It is interior.In addition, some compounds can also be with water or other organic solvents formation solvate.Such solvate also similarly by It is included within the scope of the invention.
Can be by method of the oligomer described herein for suppressing albumen generation or virus replication.Therefore, one In individual embodiment, the nucleic acid for encoding this albuminoid is exposed to oligomer as disclosed herein.In foregoing further reality Apply in scheme, antisense scant polymer includes the terminal groups or combinations thereof of 5 ' or 3 ' modifications, and as disclosed herein, and base is matched somebody with somebody Part B, which is formd, effectively to be hybridized effectively to suppress the sequence that albumen is produced with nucleic acid moiety at certain position. In one embodiment, the position is mRNA ATG initiation codon region, the splicing site of Pre-mRNA, or such as following institute The Virus target sequence stated.
In one embodiment, oligomer has greater than about 50 DEG C of the Tm on being attached on target sequence, and it can Absorbed by mammalian cell or bacterial cell.In another embodiment, oligomer can be incorporated into transport section example On such as Arginine-rich peptide, as described in this article, to promote such intake.In another embodiment, it is described herein It is end modified can be worked as transport section, with promote by mammal and/or bacterial cell intake.
Below with No. 5185444 United States Patent (USP) and WO/2009/064471, morpholino described in more detail The preparation of oligomer and performance, each of which are fully incorporated herein by quoting.
D.The preparation of conjugate and give
The disclosure additionally provides preparation and the delivering of disclosed conjugate.Therefore, in one embodiment, the disclosure is related to And the composition comprising peptide as disclosed herein-oligomer conjugate and pharmaceutically acceptable carrier.
Conjugate is effectively delivered to the importance that target nucleic acid is treatment.The approach bag of antisense scant polymer delivering Include, but be not limited to, various systemic routes, including oral and parenteral route, for example, intravenous, subcutaneous, intraperitoneal And intramuscular, and suction, percutaneous and local delivery.Suitable approach can be determined by those skilled in the art, its It is adapted to the situation for receiving the object for the treatment of.For example, the suitable pathways of delivering antisense scant polymer are in treatment skin virus infection Local delivery, and the delivering for the antisense scant polymer for being used to treating viral respiratory infection is by suction.Can also be by oligomerization Thing is delivered directly in viral sites of infection or blood flow.
Conjugate can physiologically and/or in pharmaceutically acceptable convenient carrier be given any.Such composition can With any in the pharmaceutically acceptable carrier of the multiple standards used including those of ordinary skill in the art.Example includes, But it is not limited to, salt solution, phosphate buffered saline (PBS) (PBS), water, such as ethanol water, oil/water emulsion or triglycerides emulsion Emulsion, tablet and capsule.Pattern is given according to what is selected, the selection of suitable physiologically acceptable carrier will change.
Generally the compound (for example, conjugate) of the present invention can be used as free acid or free alkali.Or, can be with acid Or the form of base addition salts uses the compound of the present invention.The free ammonia of the present invention can be prepared by methods known in the art The acid-addition salts of based compound, and it can form by organic and inorganic acid.Suitable organic acid includes maleic acid, anti-fourth Enedioic acid, benzoic acid, ascorbic acid, butanedioic acid, pyrovinic acid, acetic acid, trifluoroacetic acid, oxalic acid, propionic acid, tartaric acid, salicylic acid, Citric acid, gluconic acid, lactic acid, mandelic acid, cinnamic acid, aspartic acid, stearic acid, palmitic acid, glycolic, glutamic acid and benzene sulphur Acid.Suitable inorganic acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and nitric acid.Base addition salts include being formed by carboxylate anion Those salt, and including by such as selected from alkali and alkaline earth metal ions (for example, lithium, sodium, potassium, magnesium, barium and calcium) and ammonium ion The formation of organic and inorganic cation salt and derivative that they are substituted (for example, benzhydryl ammonium, benzyl ammonium, 2- hydroxyls Base ethyl ammonium etc.).Therefore, " the pharmaceutically acceptable salt " of nomenclature structure (I) is intended to include any and all acceptable salt shapes Formula.
In addition, prodrug is also included in the context of the present invention.Prodrug is can when such prodrug is administered to patient Any covalently bound carrier of structure (I) compound is discharged in vivo.Generally by modifying functional group in a certain way, so as to The modification can by routine operation or in vivo be cut and produce maternal compound to prepare prodrug.Prodrug includes, for example originally The compound of invention, wherein hydroxyl, amino or sulfydryl are incorporated into any its cleavable group when being administered to patient, from And form the hydroxyl, amino or sulfydryl.Therefore, the representative example of prodrug includes but is not limited to the second of structure (I) compound Acetate, formates and the benzoate derivatives of alcohol and amido functional group., can be with moreover, in the case of carboxylic acid (- COOH) Using ester, such as methyl ester, ethyl ester.
In some cases, liposome can be used for promoting ASON intake arriving intracellular.(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).Hydrogel can also be used as carrier to give antisense scant polymer, for example, as described in WO93/01286. Or, oligonucleotides can be given with microballoon or particulate form.(see, for example, Wu, G.Y.and Wu, C.H., J.Biol.Chem.262:4429-4432,1987).Or, it can be increased using the inflation microbubble being combined with antisense scant polymer To the delivering of target tissue, as described in No. 6245747 United States Patent (USP).Slow releasing composition can also be used.These can be wrapped Semi-permeable polymeric matrices containing shaped granule such as film or microencapsulation form.
In one embodiment, by making the cell that virus infects with can effectively suppress the antisense medicine that specific virus are replicated Agent is contacted, and Antisense Suppression is effective in the virus infection for the treatment of host animal.Can be by the antisense medicine in suitable pharmaceutical carrier Agent is administered to mammalian object, for example, give people or the domestic animal of virus infection.Host can be prevented by contemplating ASON The growth of internal RNA virus.It can quantitatively reduce RNA virus, or eliminate the virus and be allowed to normal growth to host Or development has seldom or no ill-effect.
In the one side of this method, the object is the experimenter, for example, being diagnosed as with topically or systemically virus sense The patient of dye.The situation of patient also can indicate that the preventative of antisense scant polymer of the present invention is given, such as in such situation Under, wherein patient (1) is immunologic inadequacy；(2) it is by burns victims；(3) there is inlying catheter；Or (4) will undergo or Operation is just lived through.In a preferred embodiment, oligomer is the di(2-ethylhexyl)phosphate in pharmaceutical acceptable carrier Acid amides morpholino oligomers, and oral delivery.In another preferred embodiment, oligomer is included in pharmaceutically acceptable Phosphoric acid diamides morpholino oligomers in carrier, and delivered through vein (i.v.).
In the another application of this method, object is domestic animal, for example, chicken, turkey, pig, milk cow or goat etc., and treatment is It is preventative or curative.Present invention additionally comprises livestock and poultry food composition, it contains, and to be supplemented with sub- therapeutic dose above-mentioned The food grain of the antiviral antisense compound of type.Also contemplate and combined with being supplemented with sub- treatment level antiviral oligonucleotides The method of the food grain stock raising and poultry of thing, wherein being improved to sub- therapeutic dose antiviral oligonucleotides group as described above Compound supplements food grain.
In one embodiment, with the amount for the peak value blood concentration that can effectively cause at least 200-400nM antisense scant polymers Conjugate is given with mode.Generally at certain intervals, i.e. during about 1 to 2 week, the antisense for giving one or more dosage is few Polymers.The preferred dose of orally administration is about 1-1000mg oligomers per 70kg.In some cases, more than 1000mg oligomerizations The dosage of thing/patient is probably necessary.Given for i.v., preferred dose is about 0.5-1000mg oligomers per 70kg.Can The short time gives conjugate in certain intervals, for example, giving two weeks by a definite date or shorter time daily.However, in some situations Under, conjugate is intermittently given in longer time section.Can after giving, or while giving, give antibiotic or other Treatment.Can be according to indicated by being checked based on immunoassay results, other biochemical tests and the physiology for receiving treatment target, regulation Therapeutic scheme (dosage, frequency, approach etc.).
According to duration, dosage, frequency and approach is given, and receive the situation for the treatment of target, sewed using the present invention Effective vivo treatment protocols of compound can change (that is, preventative give is given to response locality or systemic infection). Therefore, in order to obtain preferred treatment results, such interior therapeutic is usually needed by suitable for treated particular type virus The detection of infection is monitored the corresponding regulation with dosage or therapeutic scheme.For example, the general of disease and/or infection can be passed through Index, such as full blood count (CBC), nucleic acid detection method, immunodiagnosis test, the detection of viral cultures or heteroduplex are supervised Depending on treatment.
Can before and after, during the antisense scant polymer is given, from be derived from object biological sample (tissue, blood, Urine etc.) determine the antiviral conjugate for suppressing or eliminating one or more type RNA virus growths for giving the present invention in vivo Effect.The analysis of such sample includes：(1) program well known by persons skilled in the art, such as Electrophoretic gel mobility are used Analysis, monitoring and the existence or non-existence of target sequence and non-target sequences formation heteroduplex；(2) monitoring such as passes through such as ELISA Or the amount that the virus protein that measures of the standard technique of western blot is produced, or (3) measurement is to the effect of virus titer, example Such as, Spearman-Karber method is passed through.(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
Connection and the preparation of oligomer containing it can be such as embodiment and the between morpholino subunit, the subunit of modification Described in No. 5185444 and No. 7943762 United States Patent (USP), they are fully incorporated herein by quoting herein.Can according to General reaction scheme I prepares morpholino subunit down.
The preparation of the morpholino subunit of reaction scheme 1.
On reaction scheme 1, wherein B represents base pairing moiety, and PG represents protection group, it is as implied above can be from corresponding Ribonucleotide (1) prepares morpholino subunit.By being reacted with suitable protection based precursor such as trityl chloride, Ke Yiren Selection of land protection morpholino subunit (2).3 ' protection groups are generally removed during solid-state oligomer is synthesized, and such as following retouches in more detail State.Base pairing moiety can suitably be protected, for the synthesis of solid phase oligomer.The suitable guarantor of adenine and cytimidine Protecting base includes benzoyl, and the appropriate protection base of guanine includes phenylacetyl group, and hypoxanthine (I) appropriate protection Ji Bao Include pivaloyloxymethyl.Pivaloyloxymethyl can be incorporated on the N1 positions of hypoxanthine heterocyclic base.Although can adopt With unprotected hypoxanthine subunit, but the yield when the base is protected in priming reaction is much higher.Other are suitable Protection group includes the protection group disclosed in No. 12/271040 pending U. S. application, and it is fully incorporated herein by quoting herein.
The phosphorus compound 4 of 3 and activation is reacted and the morpholino substituent with desired coupling part (5) is produced.Can To use the compound of any number of method preparation structure 4 well known by persons skilled in the art.For example, can be by corresponding Amine and phosphoryl chloride phosphorus oxychloride reaction prepare such compound.Thus, any method known in the art can be used to prepare the amine Those methods described in parent material, such as embodiment and No. 7943762 United States Patent (USP).Although above scheme is described (B) preparation of type connection is (for example, X is-NR8R9), (A) type connection (for example, X is dimethyl amine) can be prepared in a similar fashion.
The compound of structure 5 can be used to solid phase to automate oligomer synthesis to prepare comprising the oligomerization connected between subunit Thing.Such method is known in the art.Briefly, can 5 ' end modified structures 5 compound so that solid phase carry Include linking arm on body.For example, can be by including L1And/or R19Linking arm compound 5 is connected on solid phase carrier. Illustrative methods are elaborated in figs. 3 and 4.By this way, complete oligomer synthesis and carrying the oligomer from solid phase After being cut down on body, oligomer can comprising 5 '-it is end modified.Once supported, 5 protection group (for example, trityl) meeting It is removed, and unhindered amina can react with the activation phosphorus part of the second compound of structure 5.Repeat the sequence and expect length until obtaining The oligomer of degree.If it is desire to obtaining 5 ' modifications, it can remove or retain the 5 ' terminus protecting groups positioned at terminal.It can use and appoint The method of what number, oligomer is removed from solid phase carrier, for example, handle to cut to the connection on solid phase carrier with base.
Can in the presence of suitable activator (for example, HATU), by by desired peptide (according to mark known in the art It is prepared by quasi- peptide symthesis method) combined to prepare peptide oligomerization with the oligomer comprising free NH (such as 3 ' NH of morpholino oligomers) Thing conjugate.Multiple technologies known in the art, such as SCX chromatography purified conjugation thing can be used.
The preparation of the morpholino subunit and peptide oligomer conjugate of modification described in more detail in embodiment.Can be with Using method described herein, methods known in the art and/or herein by the method for quoting description, prepare to contain and appoint The peptide oligomer conjugate of what connection of the number through modification.Also described in embodiment by being previously described (see for example, PCT Publication WO2008036127) the overall modification of the PMO+ morpholino oligomers prepared.
F.The antisense activity of oligomer
The disclosure, which is additionally provided, suppresses the method that protein is produced, and this method includes being exposed to the nucleic acid of encoding proteins matter Peptide as disclosed herein-oligomer conjugate.Therefore, in one embodiment, make the nucleic acid of this proteinoid of coding sudden and violent Conjugate is exposed to, as disclosed herein, the sequence of wherein base pairing moiety Pi formation is with can effectively suppress protein generation Position at nucleic acid moiety effectively hybridize.Oligomer can target ATG initiation codon region, precursor for example, mRNA MRNA splicing site or Virus target sequence as described below.
In another embodiment, present disclose provides improve comprising with the morpholino by connecting combination between subunit The method of subunit sequence, the antisense activity of the peptide oligomer conjugate of the oligonucleotide analogs of the part of support base pairing, This method includes carrier peptides as described in this article being attached on oligonucleotides.
In some embodiments, the raising of antisense activity can be proved by following：
(i) when the combination of antisense scant polymer and its target sequence can effectively prevent the translation initiation codon of the protein of coding When, the expression provided relative to corresponding unmodified oligomer, the protein expression of coding is reduced, or
(ii) code for said proteins when the combination of antisense scant polymer and its target sequence effectively can prevent correctly to be spliced Pre-mRNA in aberrant splicing site generation when, the expression provided relative to corresponding unmodified oligomer, coding Protein expression increase.It is described further below the analysis for being adapted to measure these effects.In one embodiment, exist Function animal model system is analyzed or is obtained from as described in this article in splicing correction translation in cell free translation analysis, cell culture In the splicing correction of system, modification provides the activity.In one embodiment, activity has been enhanced at least 2 times, at least 5 times Or at least 10 times.
The following describe the different exemplary applications of the conjugate of the present invention, including antiviral application, treatment neuromuscular Disease, bacterium infection, inflammation and POLYCYSTIC KIDNEY DISEASE.The description is not intended to limit the present invention in any manner, but for illustration The humans and animals disease states scope of conjugate processing described herein can be used.
G.The exemplary treatment purposes of conjugate
Be attached to oligomer in carrier peptides and include good effect and hypotoxicity, thus produce than with other oligomers or The treatment window that peptide-oligomer conjugate is obtained preferably treats window.Following description provides exemplary but nonrestrictive The example of the conjugate therapeutical uses.
1.Target the stem ring secondary structure of ssRNA viruses
One class exemplary antisense antiviral compound is morpholino oligomers as described in this article, and it has 12-40 Subunit sequence and the target-seeking with the stem ring secondary structure relevant range complementation in the base of 5' ends 40 of target viral plus RNA chains Sequence.(see for example, WO/2006/033933 PCT Publications or No. 20060269911 and No. 20050096291 U.S. Application is open, and it is fully incorporated herein by quoting).
Method includes：Virus target sequence is determined first --- the area in the base of 5' ends 40 of infectious virus positive-sense strand Domain, its sequence of virus therein can form internal stem ring secondary structure.Then by progressively synthesis in solid state, building has and energy shape Into the morpholino oligomers of the targeting sequence of at least 12 complementary subunits of the viral genomic region of internal double bonds structure, wherein The heteroduplex structure that the oligomer can be made up of with Virus target sequence formation viral plus chain and oligonucleotide compound, and It is characterized in that at least 45 DEG C of dissociation Tm and the destruction of such loop-stem structure.The oligomer is incorporated into carrier described herein On peptide.
By the way that the computer journey of secondary structure prediction can be performed based on the minimum free energy state of search input RNA sequence Sequence, can be by analyzing 5'- end sequences, and such as base of 5'- ends 40 identifies target sequence.
In related aspect, conjugate can be used to suppress that there is single-stranded, just gene in mammalian host cell The infectious RNA virus of group and the method selected from a kind of following viral duplication：Flaviviridae, Picornaviridae (Picornoviridae), Caliciviridae, Togaviridae, Arteriviridae, coronaviridae, Astroviridae or liver Scorching Viraceae.This method includes viral inhibitory amount conjugate as described herein being administered to infected host cell, institute State conjugate mutual with the region that the base interior energy of 5'- ends 40 with positive strand virus genome forms internal stem ring secondary structure The targeting sequence at least 12 subunits mended.When being administered to host cell, the conjugate can be effectively formed heteroduplex Structure, its (i) by virus positive-sense strand and oligonucleotide compound constitute, and (ii) be characterized as at least 45 DEG C dissociation Tm and this The destruction of class stem ring secondary structure.The conjugate can be administered to the lactation infected virus or there is viral infection risk Animal target.
The exemplary targeting sequence of the end loop-stem structure of dengue fever virus and japanese encephalitis virus will be targetted individually below It is listed as SEQ ID NOs:1 and 2.
Targeting ssRNA diseases can also be found in No. 11/801885 U. S. application and PCT Publication WO/2008/036127 Other exemplary targeting sequences of the end loop-stem structure of poison, it is incorporated herein by quoting.
2.Target the first ORFs of ssRNA viruses
The exemplary conjugate of Equations of The Second Kind is for suppressing picornavirus, calicivirus, togavirus, coronavirus With the conjugate of the viral growth of flaviviridae, there is virus therein single-stranded, just genome and coding less than 12kb to contain There is the first ORFs of the polyprotein of a variety of functional proteins.In certain embodiments, virus is from coronavirus The RNA virus of section or West Nile Virus, flavivirus or dengue fever virus from flaviviridae.Inhibition conjugate Comprising antisense scant polymer described herein, it has substantially rises with the AUG across the ORFs of viral genome first Beginning site the complementary target-seeking base sequence of Virus target sequence.In an embodiment of this method, conjugate is administered to The mammalian object of virus is infected.See, for example, WO/2005/007805 PCT Publications and No. 2003224353 U.S. State's application is open, and it is incorporated herein by reference.
It is preferred that target sequence be across the ORFs of viral genome first (ORF1) AUG initiation sites region. First ORF generally polyproteins of the coding containing such as non-structural protein of polymerase, unwindase and protease." cross over AUG Initiation site " refers to that target sequence includes at least three base in the side of AUG initiation sites, and includes at least two alkali in opposite side Base (at least eight base altogether).Preferably, it includes at least four base (at least 11 alkali altogether in every side of initiation site Base).
More generally, target site preferably includes the conservative target between a variety of virus isolates.Other favourable sites The site replicated including IRES (internal ribosome entry site), trans-activator binding site and starting.Compiled by targetting The host cell gene that code cell entry and host response virus are present, effectively can will can provide multiple redundancy genes Complicated and big viral genome is used as target.
A variety of virus genome sequences can be obtained from resource known to such as NCBI Genbank databases.Can also be in base ORF1 AUG initiation sites are identified in factor data bank or reference based on it, or can be by expected ORF1 start bits The sequence of AUG codons is searched in the region of point to find the site.
It following present respective general gene organization's form in 4 Viraceaes, and then obtained in each section The exemplary target sequence of selected member's (genus and species or strain).
3.Target influenza virus
The 3rd exemplary conjugate of class is used for the growth for suppressing orthomyxovirus coe virus and treatment virus infection.At one In embodiment, host cell is contacted with conjugate as described in this article, for example comprising can effectively with selected from following target The conjugate of the base sequence of area hybridization：1) base of 5 ' or 3 ' end 25 of negative-sense viral rna fragment；2) justice cRNA The base of end 25 at 5 ' or 3 ' ends；3) 45 bases around influenza virus mRNAs AUG initiation sites；With 4) experience choosing 50 bases around the influenza mRNAs of selecting property montage splicing donor or acceptor site.(see for example, WO/2006/ No. 047683 PCT Publication；No. 20070004661 U. S. application is disclosed；And No. 2010/056613 PCT application and the 12/th No. 945081 U. S. applications, it is incorporated herein by reference).
Thus, exemplary conjugate is included comprising containing SEQ ID NO:The conjugate of 3 oligomer.
Table 4. includes the influenza targeting sequence of connection or terminal groups between the subunit for passing through modification
**3 '-benzhydryl；*+ be connected as PMO+ junctions be acylated trimethylglycine；PMOm is represented in 3- nitrogen position Put the T bases with methyl.
The conjugate is used especially for treating the influenza infection of mammal.Conjugate can be administered to infection Influenza virus or the mammalian object that there is influenza infection risk.
4.Target the virus of Picornaviridae
The 4th exemplary conjugate of class is used for the growth for suppressing Picornaviridae virus and treatment virus infection. The conjugate is used especially for treating enterovirus and/or the rhinovirus infection of mammal.In this embodiment, conjugate Comprising the morpholino oligomers with 12-40 subunit of sequence, its include with virus 5 ' two 32 of non-translational region guard At least 12 subunits of the targeting sequence of the viral RNA sequence relevant range complementation in nucleotide region in one.(see, for example, No. WO/2007/030576 and WO/2007/030691 PCT Publications, or it is pending and the jointly owned 11/518058th Number and No. 11/517757 U. S. application, it is incorporated herein by reference).Exemplary targeting sequence is listed as SEQ following NO:6。
5.Target flaviviridae
The 5th exemplary conjugate of class is used for the duplication for suppressing flavivirus in zooblast.The exemplary of the type is conjugated Thing includes morpholino oligomers, and it is the length of 8-40 nucleotide base, and with including at least part normal chain Fo Lawei Virus (flaviviral) RNA 5 '-cyclization sequence (5'-CS) or the viral positive chain RNA genome area of 3 '-CS sequences are complementary At least eight base sequence.Highly preferred target is 3'-CS, the exemplary targeting sequence of dengue fever virus below by It is listed as SEQ ID NO:7.(see for example, (WO/2005/030800) number PCT Publication or pending and jointly owned No. 10/913996 U. S. application, it is incorporated herein by reference).
6.Target norovirus (Nidovirus) coe virus
The 6th exemplary conjugate of class be used to suppressing being infected by the virus the duplication of norovirus in zooblast.Such Exemplary conjugate includes morpholino oligomers, and it contains 8-25 nucleotide base, and has and can destroy in positive strand virus base Because the sequence of base pairing between the transcription regulating nucleotide sequence (TRS) in group 5' leaders and minus strand 3' subgenomes area is (see example Such as, WO/2005/065268 PCT Publications or No. 20070037763 U. S. application are disclosed, and it is incorporated by reference into this Text).
7.Target filamentous virus (Filoviruses)
In another embodiment, by making cell be contacted with conjugate as described in this article, for example with by The complementary target-seeking base sequence of the target sequence of at least 12 continuous base compositions sews in normal chain mRNA AUG initiation sites region Compound, one or more conjugates as described in this article can be used to suppress Ebola virus or Marburg virus in host In the method for intracellular duplication, it is as described further below described in.
Filamentous virus genome is the single stranded RNA of about 19000 bases, and it is ameristic, and is antisense orientation.The base Because of a group 7 kind protein of the coding from the monocistronic mRNA s complementary with vRNA.
Target sequence is AUG initiation codons downstream (25 alkali for crossing over or being located just at selected Ebola virus albumen In base) or upstream (in 100 bases) normal chain (justice) RNA sequence or the negative strand viruses RNA base of 3' ends 30.It is preferred that Protein target is virus polymerase subunits VP35 and VP24, although L, nucleoprotein NP and VP30 are also under consideration.In these, Early protein is more favored, for example, VP35 is more favored than the L polymerases of later expression.
In another embodiment, by make cell with as described in this article have with by filamentous virus mRNA sequence The complementary target-seeking base sequence of the target sequence of at least 12 continuous base compositions sews in normal chain mRNA AUG initiation sites region Compound is contacted, and can be used to suppress Ebola virus by one or more conjugates as described in this article or Marburg virus exists In the method replicated in host cell.(see for example, WO/2006/050414 PCT Publications or No. 7524829 and No. 7507196 United States Patent (USP)s, and No. 12/402455, No. 12/402461, No. 12/402464 and the 12/853180th The continuous application of number U. S. application, it is incorporated herein by reference).
8.Target arenavirus
In another embodiment, by a kind of species in Arenaviridae, it will can be conjugated as described in this article Thing is used to suppress in the method that mammalian cell inner virus infect.In one aspect, conjugate can be used to treat and infected The mammalian object of virus.(see, for example, WO/2007/103529 PCT Publications or No. 7582615 United States Patent (USP), It is incorporated herein by reference).
Table 5 is target viral inventory of the exemplary conjugate by the present invention as target, its by they old world or The classification of New World arenavirus carries out tissue.
The target arenavirus of table 5.
The genome of arenavirus is made up of two single stranded RNA fragments for being appointed as S (small) and L (big).In virion In, the S sections of mol ratios to L sections of RNA substantially 2:1.It has been determined that full S sections of RNA sequences of several arenavirus, and its scope For 3366-3535 nucleotides.Full L sections of RNA sequences of several arenavirus also measured were, and it is 7102 to 7279 nucleosides Acid.17 are identicals in last 19 nucleotides of 3 ' end sequences of S and L RNA fragments.In all known arenavirus In these end sequences be conservative.5 '-end 19 of each geneome RNA head end or 20 nucleotides and each corresponding 3 ' End is perfect complementary.Due to the complementarity, 3 ' and 5 ' ends are considered as that base pairing and panhandle spline structure (panhandle can be formed structures)。
Infectious virus particle or viral RNA (vRNA) replicate to form viral complementarity RNA (vcRNA) chain of anti-gene Occur infected intracellular.VRNA and vcRNA encode complementary mRNAs；Therefore, arenavirus is classified as ambisense RNA Virus, and non-negative justice or positive sense RNA virus.The ambisense of viral gene is oriented in L sections and S sections.NP and pol gene point Not Wei Yu S and L vRNA fragments 3 ' ends, and encoded that (that is, they are mutual by transcribing vRNA or genome with conventional antisense Benefit property mRNAs is expressed).Gene positioned at 5 ' ends of S and L vRNA fragments --- it is respectively GPC and Z, in mRNA modes It is encoded, but evidence suggests they are directly from genome vRNA translations.These genes are not by from anti-gene Group (that is, vcRNA), the genome vRNA total length complementary copies for playing replicative intermediate carry out the mRNAs of open gene group meaning And expressed.
The exemplary targeting sequence of arenavirus coe virus is listed as SEQ ID NO following:8.
9.Target respiratory syncytial virus (RSV)
Respiratory syncytial virus (RSV) (RSV) is most important a kind of respiratory pathogen in child's body.It is less than caused by RSV The lower respiratory tract illness of the children of 1 year old, such as capillary bronchitis and pneumonia, it is often necessary to hospitalization.Youngster with heart and lung diseases Virgin and preterm children is particularly susceptible for by serious disease caused by the infection.Rsv infection is also the important of old man and high-risk adult Disease, and it is the cause of disease (Falsey, the Hennessey et for the second most common determination for causing the elderly's viral pneumonia al.2005).World Health Organization estimation RSV worldwide causes 64,000,000 clinical infections and 160,000 death every year. Currently without the vaccine of available prevention rsv infection.Although it is many in the past few decades we to RSV biology, epidemic disease Learn, the understanding of Pathological Physiology and host immune response has occurred in that major progress, however it remains the baby on having infected RSV The very big arguement of child's optimum management.Ribavirin (Ribavirin) is the only approved antiviral agent for being used to treat rsv infection Thing, but it is using being only limitted to high-risk or grave illness baby.The application of Ribavirin is by its cost, variable efficiency and produces antiviral Limitation (the Marquardt1995 of trend;Prince2001).It is many institute's weeks the need for the current anti-RSV medicaments effective to other Know.
The PMO (PPMO) that known peptide is combined can effectively suppress in tissue cultures and in vivo animal model system RSV(Lai,Stein et al.2008).In the culture of two kinds of people's air flue cell line, test and be designed to targeting and include Two kinds of antisense PPMOs of RSV L mRNA 5 '-stub area and the sequence in translation initiation site region anti-RSV activity.It In one kind (RSV-AUG-2;SEQ ID NO10), reduce>2.0log10Virus titer.RSV- is used before RSV inoculations AUG-2PPMO intranasal (i.n.) handles BALB/c mouse, after infection when (p.i.) the 5th day, is generated in lung tissue 1.2log10The reduction of virus titer, and after infection the 7th when alleviate lung inflammation.These as shown by data RSV-AUG-2 is provided Effective anti-RSV activity, is worth as the candidate of potential treatment use further studying (Lai, Stein et al.2008).Despite the presence of RSV-AUG-2PPMO as described above success, solved using conjugate as disclosed herein The toxicity problem related to previous peptide conjugate is suitable.Therefore, in another embodiment of the present invention, by making Cell is contacted with conjugate as described in this article, for example with the AUG initiation sites region of the mRNA from RSV extremely The conjugate of the complementary target-seeking base sequence of the target sequences of few 12 continuous base compositions, can will one kind as described in this article Or multiple conjugates be used for suppress in the method that RSV is replicated in host cell, it is as further discussed below described in.
The key component of RSV L gene code virus RNA dependant RNA polymerase compounds.With RSV-AUG-2PPMO Form be directed to antisense PPMO across the sequences Design of the AUG translation initiation site codons of RSV L gene mRNAs with from presence " gene starting " sequence (GS) in L mRNA 5 ' ends arrives the sequence complementation for entering 13 nucleotides of coded sequence.Therefore, it is excellent The L gene targetings sequence of choosing upwardly extends 40 bases or entered and encodes as following with the 5 ' ends from L gene mRNAs in 3 ' sides SEQ ID NO are shown as in table 6:Any 12 continuous base complementrities of 22 bases of L genes of 9 sequence.Exemplary RSV L Gene targeting sequence is listed as SEQ ID NOs in following table 6:10-14.Described herein of the invention can be appointed Modify and be integrated into oligomer between what subunit, with the Intracellular delivery and/or organizing specific for providing increased antisense activity, improving Property is to improve therapeutic activity.The exemplary oligomer sequence connected between subunit containing the present invention is recited in table 6 below.
Table 6.RSV target sequences and targeting sequence
10.Neuromuscular disease
It is used to treat the neuromuscular disease with mammalian object there is provided treatment conjugate in another embodiment Sick related disease states.Antisense scant polymer is (for example, SEQ ID NO:16) shown in MDX mouse models to Du's Xing Shi fleshes battalion Support bad (DMD) active.The exemplary oligomer sequence for incorporating the connection used in some embodiments is recited in In table 7 below.In some embodiments, the conjugate, which is included, is selected from following oligomer：
(a) antisense scant polymer of people's myostatin is targetted, it has with passing through SEQ ID NO:The people of 18 identifications The base sequence of at least 12 continuous base complementrities in myostatin mRNA targeting regions, for treating muscular atrophy Illness, as described previously (see for example, No. 12/493140 U.S. Patent application, it is incorporated herein by reference；And PCT Open WO2006/086667).Exemplary mouse targeting sequence is listed as SEQ ID NOs:19-20；With
(b) antisense scant polymer, it can be such as with selected from SEQ ID NOs:The PMO of 22-35 sequence DMD albumen Exon skipping is produced in (dystrophin), to recover the amount of activated of dystrophin, for treating DMD, As described previously (see for example, No. WO/2010/048586 and WO/2006/000057 PCT Publications or US09/ No. 061960 U.S. Patent Publication, all these to be all incorporated herein by reference).
Several other neuromuscular diseases can be treated using the connection through modification and terminal groups of the present invention.Beg for below The exemplary compounds for treating myeloid muscular atrophy (SMA) and myotonia atrophica (DM) are discussed.
SMA is that, due to autosomal recessive disease caused by chronic loss α-motor neuron in spinal cord, and can influence Children and adult.The reduction expression of motor neuron existence (SMN) is (Hua, Sahashi et the reason for causing the disease al.2010).SMA mutation is caused to be located at SMN1 genes, but a kind of paralogous --- SMN2, if outer aobvious from lacking 7 (δ 7SMN2) of son alternative splicing form expression, can allow existence by compensating SMN1 loss.Have shown that The antisense compounds of targeting introne 6, exon 7 and introne 7 all can induce different degrees of exon 7 and include (inclusion).The antisense compounds of introne 7 are targetted to be preferred (see for example, No. WO/2010/148249, WO/ No. 2010/120820, WO/2007/002390 PCT Publications and No. 7838657 United States Patent (USP)).Target SMN2 precursors MRNA simultaneously induces the exemplary antisense sequence that the exon 7 of raising is included to be listed as SEQ ID NOs following:36-38.Phase Than performance known in the art, it is contemplated that using connection and terminal groups described herein through modification to these oligomer sequences The modification selected is by the performance with raising.Additionally, it is contemplated that the introne 7 of targeting SMN2 genes and integrating spy of the invention Any oligomer levied all has the potentiality that inducing exon 7 is included, and provides therapeutic effect for SMA patient.Myotonic is sought It is the central genetic caused by toxicity rna expression causes neuromuscular to be degenerated to support bad Class1 (DM1) and type 2 (DM2) Property disease.DM1 and DM2 respectively with transcription steinert's disease protein kinase (DMPK) and zinc finger protein 9 (ZNF9) 3 '- UTR repeats related (see for example, WO2008/036406) to the poly- CUG and poly- CCUG of the length in introne 1 region.Although normal There are individual up to 30 CTG to repeat, and DM1 patient carries 50 to thousands of more big figure repetitions.The seriousness and hair of the disease The sick age is related to the number repeated.The patient of adulthood morbidity shows lighter symptom and with 100 repetitions are less than, and green grass or young crops is few Term morbidity DM1 patient carries up to 500 repetitions, and congenital situation generally there are about 1000 CTG to repeat.It is increased to contain The transcript for having CUG to repeat is formed secondary structure, gathered in the form of core stove (nuclear foci) in core, and isolates RNA Associated proteins (RNA-BP).Several RNA-BP are related to the disease, including blind flesh sample (MBNL) albumen and CUG associated proteins (CUGBP).MBNL protein and the blind flesh of drosophila (Mbl) albumen homology necessary to light receptor and muscle differentiation.MBNL and CUGBP It is confirmed as influenceing the Antagonism splicing instrumentality of transcript in DM1, such as serum cardiac troponin T (cTNT), insulin receptor And muscle specific chloride channel (ClC-1) (IR).
The ASON of the increased repetition of targeting DMPK genes known in the art can take in DM1 animal model Isolate for RNA-BP and reverse myotonic reaction (WO2008/036406).Contemplating the oligomer comprising feature of present invention will be DM1 and DM2 patients provide the activity and treatment potentiality improved.Target the exemplary of poly- CUG described above and poly- CCUG repetitions Sequence is listed below as SEQ ID NOs:39-55, and be further described in No. 13/101942 U. S. application, it is whole simultaneously Enter herein.
Other embodiments that the present invention is used to treat neuromuscular disorder are contemplated, including are designed to treat other The oligomer of DNA repeat instability hereditary diseases.These diseases include Huntington's chorea (Huntington ' s disease), Spinocebellar ataxia (spino-cerebellar ataxia), X spinal cord and bulbar muscular atrophy and Spinocebellar ataxia Class1 0 (SCA10), as described in WO2008/018795.
Table 7. includes M23D sequences (the SEQ ID NO of connection and/or 3 ' and/or 5 ' terminal groups between the subunit for passing through modification: 15)
*Dimerization refer to the oligomer by connect two monomers 3 ' ends connection by dimerization.For example, described Connection can be-COCH2CH2-S-CH(CONH2)CH2-CO-NHCH2CH2CO- or any other suitable connection.EG3 refers to three Glycol tail (see for example, conjugate in embodiment 30 and 31).
11.Antibacterial applications
In another embodiment, the present invention includes anti-for treating including for mammalian hosts bacterial infections in vivo The conjugate of bacterium antisense scant polymer.In some embodiments, oligomer includes 10-20 base and the continuous base of at least ten Targeting sequence, the acyl carrier protein (acpP) of continuous base therein and infectious bacteria, gyrase A subunits (gyrA), FtsZ, ribosome protein S 10 (rpsJ), the mRNA of leuD, mgtC, pirG, pcaA and cma1 gene targeting regions complementation, its Described in targeting regions contain bacterium mRNA or positioned at translation initiation codon upstream (that is, 5 ') or downstream (that is, 3 ') direction 20 The translation initiation codon of sequence in individual base, and wherein described oligomer is joined to form heteroduplex on mRNA, from And suppress the duplication of the bacterium.
12.Adjust nuclear hormone receptor
In another embodiment, the present invention relates to for adjusting the core from nuclear hormone receptor superfamily (NHRSF) The composition and method of hormone receptor (NHR) expression, the mainly spelling by controlling or changing the Pre-mRNA for encoding this receptor Connect.Specific NHRs example includes：Glucocorticoid receptor (GR), PgR (PR) and androgen receptor (AR).Some In embodiment, conjugate described herein can cause the non-ligand dependent of this receptor or other selected form acceptors Expression increase, and the expression of their inactive form are reduced.
Embodiment of the present invention is included comprising oligomer, and such as extron or intron sequences with selected NHR is mutual The conjugate of the oligomer of benefit, extron or introne therein except other NHR domains described herein, in addition to " the ligand-binding extron " of NHRSF Pre-mRNAs and/or neighbouring introne.Term " ligand-binding extron " refers to It is to be present in wild type mRNA, but non-ligand dependent form is gone divided by prepared from primary transcript (" Pre-mRNA ") MRNA extron.In certain embodiments, complementarity can be based on the sequence in the Pre-mRNA sequence for crossing over splicing site Row, it includes, but not limited to the complementarity based on the sequence connected across exon: intron.In other embodiments, Complementarity can be based only upon the sequence of introne.In other embodiments, complementarity can be based only upon the sequence of extron. (see for example, No. 13/046356 U. S. application, it is incorporated herein by reference).
NHR instrumentalities can be used for the related diseases for the treatment of NHR, including with transcribing the gene of stimulation or suppression by NHRs Expression product relevant disease.For example, can suppress AP-1 and/or NF- κ B NHRs instrumentality can be used for treat it is inflammatory and Immunity disease and such as following illness：Osteoarthritis, rheumatic arthritis, multiple sclerosis, asthma, inflammatory bowel Disease, graft rejection and graft versus host disease(GVH disease), and other types described herein and known in the art.Antagonism is trans to swash Compound living can be used for treating the metabolic disease related to increased glucocorticoid levels, such as diabetes, osteoporosis Disease and glaucoma and other diseases.In addition, promoting the compound of (agonize) trans-activation to can be used for treatment and sugared cortical hormone Plain not enough related metabolic disease, such as Addison's disease (Addison ' s disease) and other diseases.
Embodiment of the present invention includes regulation intracellular nucleic NHR activity or the method for expression, including make cell with comprising The conjugate contact of carrier protein and antisense scant polymer, antisense scant polymer therein is by by the way that the morpholino nitrogen of a subunit is connected Connection constitutes come the morpholino subunit connected between the phosphorous subunit being connected on the outer carbon of the 5' rings of neighbouring subunit, wherein the few core Thuja acid contains the targeting sequence of 10-40 base and at least ten continuous base complementary with target sequence, wherein the target sequence For NHR Pre-mRNA transcript, so as to adjust NHR activity or expression.In certain embodiments, before oligomer can change The splicing of body mRNA transcripts, and increase the expression of NHR variant.In some embodiments, oligomer induction precursor The complete or partial exon skipping of one or more extrons of mRNA transcripts.In certain embodiments, it is one Or multiple exons coding at least a portion NHR ligand binding domains, and variant is NHR non-ligand dependent shape Formula.In certain embodiments, one or more of exons coding NHR at least a portion transactivation domain, and Variant has reduced transcriptional activation activity.In certain embodiments, one or more of exons coding NHR are extremely Few a part of DNA binding structural domains.In certain embodiments, at least one of one or more of exons coding NHR Divide the terminal activating domains of N-.In certain embodiments, one or more of exons coding NHR at least a portion carboxylic Base terminal domains.In certain embodiments, the variant is attached on NF-KB, AP-l or both, and reduces one Or the transcription of they multiple pro-inflammatory target genes.
In certain embodiments, oligomer promotes (agonize) NHR trans-activation transcriptional activity.In other implementations In scheme, oligomer antagonism NHR trans-activation transcriptional activity.In certain embodiments, oligomer promotes NHR trans suppression System activity.In other embodiments, oligomer antagonism NHR trans inhibitory activity.In certain embodiments, oligomer Antagonism NHR trans-activation transcriptional activity, and promote NHR trans inhibitory activity.(see for example, No. 61/313652 U.S. Application, it is incorporated herein by reference).
Embodiment
Unless otherwise indicated, all chemicals are all obtained from Sigma-Aldrich-Fluka.Benzoyl adenosine, benzene first Acyl group cytidine and phenylacetyl group guanosine are obtained from Britain Carbosynth Limited.
Use known in the art and No. 12/271036 and No. 12/271040 pending U. S. application and WO/ Method described in No. 2009/064471 PCT Publication completes PMO, PMO+, PPMO and connected as described in this article containing other The PMO of modification synthesis is connect, they are fully incorporated herein by quoting herein.
Substantially modified by having been synthesized described in WO/2009/064471 PCT Publications with 3 ' trityls PMO, in addition to trityl removal step is omitted.
Embodiment 1
4- (2,2,2- trifluoroacetyls amido) piperidines -1- t-butyl formates
Trifluoroacetic Acid Ethyl Ester (35.6mL, 0.300mol) is added dropwise to the 4- amino piperazines in DCM (250mL) under stirring In pyridine -1- t-butyl formates (48.7g, 0.243mol) and DIPEA (130mL, 0.749mol) suspension., will after 20 hours Solution is washed with citric acid solution (200mL x3, the 10%w/v aqueous solution) and sodium bicarbonate solution (200mL x3, concentrated aqueous solution) Wash, dry (MgSO4) and filtered by silica (24g).Wash silica with DCM, and by the elutriant part of merging Concentrate (100mL), and be directly used in next step.C12H19F3N2O3APCI/MS calculated values be 296.1, measured value m/z= 294.9(M-1)。
Embodiment 2
2,2,2- tri- fluoro- N- (piperidin-4-yl) acetamide hydrochloride
The hydrogen chloride solution (250mL, 1.0mol) that Isosorbide-5-Nitrae-dioxane (4M) will be dissolved in is added dropwise to the embodiment 1 of stirring and marked In the DCM solution (100mL) for inscribing compound.Persistently stir 6 hours, then filter suspension, and washed with diethyl ether (500mL) The solid provides the title compound (54.2g, 96% yield), and it is white solid.C7H11F3N2O APCI/MS calculated values For 196.1, measured value m/z=196.9 (M+1).
Embodiment 3
(4- (2,2,2- trifluoroacetyls amido) piperidin-1-yl) dichloro phosphoric acid
POCl3 (23.9mL, 0.256mol) and DIPEA (121.7mL, 0.699mol) are added dropwise in DCM In cooling (ice bath/water-bath) suspension of the title compound of embodiment 2 (54.2g, 0.233mol) in (250mL), and stir. After 15 minutes, withdraw cryostat and continue to stir mixture, to allow it to be warming up to environment temperature.After 1 hour, partial concentration (100mL) mixture, filters suspension and washs solid with diethyl ether to provide the title compound (43.8g, 60% production Rate), it is white solid.Partial concentration (100mL) elutriant, filtering gained suspension simultaneously washs solid to provide with diethyl ether Other title compounds (6.5g, 9% yield).1- (4- nitrobenzophenones) bridged piperazine derivatives C17H22ClF3N5O4P ESI/MS meters Calculation value 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- bases) -4- trityls Quinoline -2- bases) methyl (4- (2,2,2- trifluoroacetyls amido) piperidin-1-yl) chlorinated phosphonate
By Mo (Tr) T# (22.6g, 46.7mmol), 2,6- lutidines (21.7mL, 187mmol) and 4- in 10 minutes The DCM solution (100mL) of (dimethylamino) pyridine (1.14g, 9.33mmol) is added dropwise to the embodiment 3 for being dissolved in DCM (100mL) In the stirring of title compound (29.2g, 93.3mmol) and (ice bath/water-bath) solution of cooling.Cryostat is allowed to be warming up to environment Temperature.After 15 hours, solution is washed with citric acid solution (200mLx3, the 10%w/v aqueous solution), (MgSO is dried4), concentration, And raw oil is loaded directly on post.Concentration chromatography [SiO2Post (120g), hexane/EtOAc elutriants (gradient 1:1 to 0: 1) x3, is repeated] fraction (fraction) provides the title compound (27.2g, 77% yield), and it is white solid.1-(4- Nitrobenzophenone) bridged piperazine derivatives C46H50F3N8O8P ESI/MS calculated values are 930.3, measured value m/z=929.5 (M-1).
Embodiment 5
((2S, 6R)-6- (6- benzamido-9H- purine-9- bases)-4- trityls morpholine -2-yl) methyl (4- (2, 2,2- trifluoroacetyls amido) piperidin-1-yl) chlorinated phosphonate
To have synthesized the title compound similar to the method described in embodiment 4, to provide the title compound (15.4g, 66% yield), it is white solid.1- (4- nitrobenzophenones) bridged piperazine derivatives C53H53F3N11O7P ESI/MS is calculated It is worth for 1043.4, measured value m/z=1042.5 (M-1).
Embodiment 6
(R)-methyl (1- phenethyls) amino phosphinylidyne dichloro
By 2,6- lutidines (7.06mL, 60.6mmol) and (R)-(+)-N, a- dimethyl benzylamine under stirring The DCM solution of (3.73g, 27.6mmol) is continuously added dropwise to the POCl3 (2.83mL, 30.3mmol) that is dissolved in DCM (30mL) Cool down in (ice bath/water-bath) solution.After 5 minutes, cryostat is removed, and allow reactant mixture to be warming up to environment temperature.1 hour Afterwards, reaction solution is washed with citric acid solution (50mLx3, the 10%w/v aqueous solution), dries (MgSO4), pass through SiO2Filtering, and Concentrate to provide the title compound (3.80g), it is white foam.1- (4- nitrobenzophenones) bridged piperazine derivatives C19H25N4O4P ESI/MS calculated values be 404.2, measured value m/z=403.1 (M-1).
Embodiment 7
(S)-methyl (1- phenethyls) amino phosphinylidyne dichloro
To have synthesized the title compound similar to the method described in embodiment 6, to provide the title compound (3.95g), it is white foam.The C of 1- (4- nitrobenzophenones) bridged piperazine derivatives19H25N4O4P ESI/MS calculated values are 404.2, measured value m/z=403.1 (M-1).
Embodiment 8
((2S, 6R)-6- ((the 2H)-yl of 5- methyl-2,4- dioxy-3,4- dihydro-pyrimidins-1)-4- trityls morpholine -2- Base) methyl ((R) -1- phenethyls) chloro phosphoramidate
To have synthesized the title compound similar to the method described in embodiment 4, to provide the title compound amino Phosphoryl chloride phosphorus oxychloride (4.46g, 28% yield), it is white solid.C38H40ClN4O5P ESI/MS calculated values are 698.2, measured value m/z =697.3(M-1)。
Embodiment 9
((2S, 6R)-6- ((the 2H)-yl of 5- methyl-2,4- dioxy-3,4- dihydro-pyrimidins-1)-4- trityls morpholine -2- Base) methyl ((S) -1- phenethyls) chloro phosphoramidate
To have synthesized the title compound similar to the method described in embodiment 4, to provide the title compound amino Phosphoryl chloride phosphorus oxychloride (4.65g, 23% yield), it is white solid.C38H40ClN4O5P ESI/MS calculated values are 698.2, measured value m/z =697.3(M-1)。
Embodiment 10
(4- (pyrrolidin-1-yl) piperidin-1-yl) phosphinylidyne dichloro hydrochloride
By 2,6- lutidines (19.4mL, 167mmol) and 4- (1- pyrrolidinyls)-piperidines (8.58g, 55.6mmol) DCM solution (30mL) is added to the cooling (ice bath/water-bath) for the POCl3 (5.70mL, 55.6mmol) for being dissolved in DCM (30mL) In solution, and stir 1 hour.Suspension is filtered, and washs solid to provide the title pyrrolidines with excessive diethyl ether (17.7g, 91% yield), it is white solid.1- (4- nitrobenzophenones) bridged piperazine derivatives C19H30N5O4P ESI/MS calculated values For 423.2, measured value m/z=422.2 (M-1).
Embodiment 11
((2S, 6R)-6- ((the 2H)-yl of 5- methyl-2,4- dioxy-3,4- dihydro-pyrimidins-1)-4- trityls morpholine -2- Base) methyl (4- (pyrrolidin-1-yl) piperidin-1-yl) chlorinated phosphonate hydrochloride
By Mo (Tr) T# (24.5g, 50.6mmol), 2,6- lutidines (17.7mL, 152mmol) and 1- first in 10 minutes The DCM solution (100mL) of base imidazoles (0.401mL, 5.06mmol) is added dropwise to the dichlor-phosphoryl amine ester 8 for being dissolved in DCM (100mL) In the stirring of (17.7g, 50.6mmol) and (ice bath/water-bath) solution of cooling.Cryostat is allowed to be warming up in stirred suspension Environment temperature.After 6 hours, suspension is poured on diethyl ether (1L), stirred 15 minutes, filters and gained is washed with other ether Solid provides white solid (45.4g).Pass through chromatography [SiO2Post (120 grams), DCM/MeOH elutriants (gradient 1:0 to 6: 4)] purification of crude product, and the fraction of merging is poured on diethyl ether (2.5L), is stirred 15 minutes, is filtered and with other ether Wash gained solid to provide the title compound (23.1g, 60% yield), it is white solid.1- (4- nitrobenzophenones) piperazine Derivative C48H57N8O7P ESI/MS calculated values are 888.4, measured value m/z=887.6 (M-1).
Embodiment 12
3- (tert-butyl group disulfonyl base) -2- (isobutoxy carbonyl amino) propionic acid
H will be dissolved in2O (20mL) K2CO3(16.5g, 119.5mmol), which is added to, is dissolved in CH3CN (40mL) the S- tert-butyl groups In sulfydryl-Cys (10g, 47.8mmol).Stirring 15 minutes after, be slowly injected into isobutyl chlorocarbonate (9.4mL, 72mmol).Reaction is allowed to carry out 3 hours.Pass through Celite filter aid white solids；Filtrate is concentrated to remove CH3CN.Will Residue is dissolved in ethyl acetate (200mL), is washed with 1N HCl (40ml X3), bittern (40X1), uses Na2SO4Dry.Chromatography Desired product (2) is obtained after (5%MeOH/DCM).
Embodiment 13
4- (3- (tert-butyl group disulfonyl base) -2- (isobutoxy carbonyl amino) propionamido-) piperidines -1- t-butyl formates
HATU (8.58g, 22.6mmol) is added to be dissolved in DMF (50ml) acid (compound 2 from embodiment 12, 6.98g, 22.6mmol) in.After 30 minutes, by Hunig alkali (4.71ml, 27.1mmol) and 1-Boc-4- amino piperidines (5.43g, 27.1mmol) is added in mixture.Lower will react is stirred at room temperature persistently to carry out other 3 hours.Removed under high vacuum DMF, crude residue is dissolved in EtAc (300ml), H is used2O (50ml X3) is washed.After ISCO purifying (5%MeOH/DCM) To end-product (3).
Embodiment 14
3- (tert-butyl group disulfonyl base) -1- oxygen -1- (piperidin-4-yl amino) propyl- 2- aminocarbamic acid isobutyl esters
30ml 4M HCl/ dioxanes are added to prepared in embodiment 13 compound 3 (7.085g, 18.12mmol) in.Reaction is completed after 2 hours at room temperature.By the HCl salt (4) without be further purified be directly used in it is next Step.
Embodiment 15
3- (tert-butyl group disulfonyl base) -1- (1- (dichlor-phosphoryl) piperidin-4-yls amino) -1- oxo propyl- 2- base amino Iso-butyl formate
In -78 DEG C under an argon by POCl3(1.69ml, 18.12mmol) is slowly injected into what is prepared in embodiment 15 In the DCM solution (200ml) of compound 4 (7.746g, 18.12mmol), Et is subsequently added3N (7.58ml, 54.36mmol).Room The lower stirring reaction of temperature 5 hours, concentrates to remove excessive alkali and solvent.ISCO purifying (50%EtAc/ hexanes) obtains product afterwards (5), it is white solid.
Embodiment 16
(((((5- methyl -2,4- dioxy -3,4- dihydros are phonetic by (2S, 6R) -6- for chloro by 1- by 3- (tert-butyl group disulfonyl base) -1- Pyridine-1 (2H)-yl)-4- trityls morpholine -2-yl) methoxyl group) phosphoryl) piperidin-4-yl amino)-1- oxo propyl- 2- base ammonia Base iso-butyl formate
Lutidine (1.92ml, 16.47mmol) and DMAP (669mg, 5.5mmol) are added to 0 DEG C and are dissolved in DCM 1- ((2R, 6S)-6- (methylol)-4- trityls morpholine -2-the yl)-5- methylpyrimidines-2,4 (1H, 3H)-two of (100ml) In ketone (moT (Tr)) (5.576g, 10.98mmol), 4 (6.13g, 12.08mmol) are subsequently added.Reaction is stayed in and stirred at room temperature Mix 18 hours.Desired product (6) is obtained after ISCO purifying (50%EtAc/ hexanes).
Embodiment 17
((2S, 6R)-6- ((the 2H)-yl of 5- methyl-2,4- dioxy-3,4- dihydro-pyrimidins-1)-4- trityls morpholine -2- Base) methylhexyl (methyl) chloro phosphoramidate
In N2It is lower that DCM (80ml) solution of N- methylols amine (4.85ml, 32mmol) is cooled to -78 DEG C.It is slowly added to DCM (10ml) solution of phosphoryl chloride phosphorus oxychloride (2.98ml, 32mmol), is then slowly added to Et3N (4.46ml, 32mmol) DCM (10ml) solution.Persistently stirred when allowing reaction to carry out, to be warming up to room temperature overnight.Obtained after ISCO purifying (20%EtAc/ hexanes) Desired product (1) is obtained, it is edible vegetable oil.
Lutidine (3.68ml, 31.6mmol) and DMAP (642mg, 5.27mmol) are added to 0 DEG C and are dissolved in DCM In the moT (Tr) (5.10g, 10.54mmol) of (100ml), 1 (4.89g, 21.08mmol) is subsequently added.Reaction is stayed in into room temperature Lower stirring 18 hours.Desired product (2) is obtained after ISCO purifying (50%EtOAc/ hexanes).
Embodiment 18
((2S, 6R)-6- ((the 2H)-yl of 5- methyl-2,4- dioxy-3,4- dihydro-pyrimidins-1)-4- trityls morpholine -2- Base) methyl dodecyl (methyl) chloro phosphoramidate
General procedure according to described in embodiment 6 and embodiment 8 prepares the title compound.
Embodiment 19
((2S, 6R)-6- ((the 2H)-yl of 5- methyl-2,4- dioxy-3,4- dihydro-pyrimidins-1)-4- trityls morpholine -2- Base) methyl morpholine is for chlorinated phosphate ester
General procedure according to described in embodiment 6 and embodiment 8 prepares the title compound.
Embodiment 20
((2S, 6R)-6- ((the 2H)-yl of 5- methyl-2,4- dioxy-3,4- dihydro-pyrimidins-1)-4- trityls morpholine -2- Base) methyl (S) -2- (methoxy) pyrrolidin-1-yl chlorinated phosphate ester
General procedure according to described in embodiment 6 and embodiment 8 prepares the title compound.
Embodiment 21
((2S, 6R)-6- ((the 2H)-yl of 5- methyl-2,4- dioxy-3,4- dihydro-pyrimidins-1)-4- trityls morpholine -2- Base) methyl 4- (3,4,5- trimethoxy-benzamides base) piperidin-1-yl chlorinated phosphate ester
Hunig alkali (1.74ml, 10mmol) is added to the 1-Boc-4- piperidines (1g, 5mmol) for being dissolved in DCM (20ml) In, it is subsequently added 3,4,5- trimethoxy-benzoyl chlorides (1.38g, 6mmol).Reaction is carried out 3 hours at room temperature, concentrates to go Except solvent and excessive alkali.Dissolve the residue in EtAc (100ml), with 0.05N HCl (3X15ml), saturation NaHCO3 (2X15ml) is washed, and uses Na2SO4Dry.Product (1) is obtained after ISCO purifying (5%MeOH/DCM).
15ml 4N HCl/ dioxanes are added in 7, terminating reaction after 4 hours.Obtain as the 8 of white solid.
In N2It is lower that 8 (1.23g, 4.18mmol) DCM (20ml) solution is cooled to -78 DEG C.It is slowly added to phosphoryl chloride phosphorus oxychloride DCM (2ml) solution of (0.39ml, 4.18mmol), is then slowly added to Et3N (0.583ml, 4.18mmol) DCM (2ml) Solution.Persistently stirred when allowing reaction to carry out, to be warming up to room temperature overnight.Expected after ISCO purifying (50%EtAc/ hexanes) Product (9).
By lutidine (0.93ml, 8mmol) and DMAP (49mg, 0.4mmol) be added to 0 DEG C of moT (Tr) (1.933g, In DCM (20ml) solution 4.0mmol), 9 (1.647g, 4mmol) are subsequently added.Reaction is stayed and is stirred at room temperature 18 hours. Desired product (10) is obtained after ISCO purifying (50%EtAc/ hexanes).
Embodiment 22
Synthesis containing subunit (CPT endoxan)
MoT subunits (25g) are suspended in DCM (175ml), and add NMI (N- methylimidazoles, 5.94g, 1.4eq.) with Obtain settled solution.Paratoluensulfonyl chloride is added in reactant mixture, and monitors reaction process until completing by TLC (about 2 hours).Aqueous treatment is completed by using 0.5M citrate buffer solutions (pH=5) and subsequent brine.Separate organic layer And use Na2SO4Dry.Solvent is removed to obtain raw product with rotary evaporator, and it is used directly to without being further purified Next step.
MoT toluene fulfonates made above are mixed (1g/10ml) with Propanolamine.Reactant mixture is then placed in 45 DEG C baking oven in overnight, then with DCM (10ml) dilute.Washed by using 0.5M citrate buffer solutions (pH=5) and subsequent bittern Wash and complete aqueous treatment.Separation organic layer simultaneously uses Na2SO4Dry.Remove solvent to obtain raw product with rotary evaporator.It is logical Cross NMR and HPLC analyses and determine the raw product, it is standby as next step without being further purified.
The raw product is dissolved in DCM (2.5ml DCM/g, 1eq.), and mixed with DIEA (3eq.).With dry ice-the third Ketone cools down the solution, and POCl is added dropwise3(1.5eq.).Stirring gained mixture is stayed overnight at room temperature.By using 0.5M lemons Acid buffer (pH=5) and subsequent brine complete aqueous treatment.Separation organic layer simultaneously uses Na2SO4Dry.Use rotary evaporation Device removes solvent to obtain raw product, and it is slightly yellow solid.By silica gel column chromatography (raw product/silica=1 to 5 Ratio, gradient DCM to 50%EA/DCM) raw product is purified, and the fraction for merging elution is analyzed according to TLC.Remove solvent with Desired product is obtained, it is the mixture of diastereomer.This is analyzed by HPLC (NPP is quenched) and NMR (H-1 and P-31) pure The product of change.
Mixture of diastereomers is separated according to following procedure.The mixture (2.6g) is dissolved in DCM.The sample is loaded into On RediSepRf posts (80g positives, Teledyne Isco manufactures), and eluted 20 minutes with 10%EA/DCM to 50%EA/DCM. Collect elutriated fraction and analyzed by TLC.Elutriated fraction is merged according to TLC analyses, and removed at room temperature with rotary evaporator Solvent.The diastereomer ratio for merging fraction is determined by P-31NMR and NPP-TFA analyses.If it is desired, repeating procedure above Until diastereoisomer ratio reaches 97%.
Embodiment 23
PMO+ total cholic acid modification
Succinimide activated chlolic acid derivatives are prepared for according to following procedure.By cholic acid (12g, 29.4mmol), N- HOSu NHS (4.0g, 34.8mmol), EDCI (5.6g, 29.3mmol) and DMAP (1g, 8.2mmol) load round bottom and burnt In bottle.DCM (400ml) and THF (40ml) is added to dissolve.Stirring reaction mixture is stayed overnight at room temperature.Then by water (400ml) It is added in reactant mixture, separates organic layer and with water (2X400ml) and then use saturation NaHCO3(300ml) and bittern (300ml) is washed.Then use Na2SO4Dry organic layer.Remove solvent to obtain white solid with rotary evaporator.This is rough Product is dissolved in chloroform (100ml), and it is deposited in heptane (1000ml).Solid is collected by filtration, by HPLC and NMR is analyzed, not purified directly to use.
Weigh appropriate PMO+ (20mg, 2.8 μm of ol) to be encased in bottle (4ml), and be dissolved in DMSO (500 μ l).By every The cholate (13mg, 25 μm of ol) of activation is added in reactant mixture by the ratio of the equivalent active ester of decorating site 2, is then existed It is stirred overnight at room temperature.Reaction process is determined by MALDI and HPLC (C-18 or SAX).
(as the disappearance by originating PMO+ is determined) after the completion of reaction, once reaction completes to add 1ml concentration ammonia Enter into reactant mixture.Then reaction bottle is placed in baking oven (45 DEG C) overnight (18 hours), room temperature is then cooled to, And diluted with 1% ammonia (10ml) soluble in water.The sample is loaded on SPE posts (2cm), and rushed with 1% ammonia solution (2X2ml) Wash bottle.With 1% ammonia stripping being dissolved in water (3X6ml) the SPE posts, and with 45% acetonitrile being dissolved in 1% ammonia spirit (6ml) Elute the product.Elutriated fraction containing oligomer is identified by UV photo densitometries.Pass through desivac separation product.Pass through MALDI and HPLC (C-18 and/or SAX) determines purity and characteristic.
The identical program is applied to deoxycholic acid and activates and be attached to PMO+On.
Embodiment 24
PMO+'s is total guanidinated
Appropriate PMO+ (25mg, 2.8 μm of ol) is weighed to be encased in bottle (6ml).By 1H- pyrazoles -1- chlorination carbonamidines (15mg, 102 μm of ol) and potassium carbonate (20mg, 0.15mmol) are added in bottle.Water (500 μ l) is added, and is stirred at room temperature Reactant mixture is mixed to stay overnight (about 18 hours).Determine that reaction is completed by MALDI.
Once completing, i.e., with 1% ammonia diluting reaction thing being dissolved in water (10ml), and it is loaded on SPE posts (2cm).With 1% Ammonia solution (2X2ml) rinses bottle, and with 1% ammonia stripping SPE posts being dissolved in water (3X6ml).With being dissolved in 1% ammonia spirit The 45% acetonitrile eluted product of (6ml).Elutriated fraction containing oligomer is identified by UV photo densitometries.Pass through desivac point From product.Purity and characteristic are determined by MALDI and HPLC (C-18 and/or SAX).
Embodiment 25
PMO+ (M23D) total ethanethioyl modification
Weigh appropriate PMO+ (20mg, 2.3 μm of ol) to be encased in bottle (4ml), and be dissolved in DMSO (500 μ l).By N- Succinimido-S- acetylthioacetates (SATA) (7mg, 28 μm of ol) are added in reactant mixture, and are allowed in room It is stirred overnight under temperature.Reaction process is monitored by MALDI and HPLC.
Once completing, i.e., 1% ammonia soluble in water is added in reactant mixture, and it is small to be stirred for 2 at room temperature When.The solution is loaded on SPE posts (2cm).Bottle is rinsed with 1% ammonia solution (2X2ml), and with being dissolved in water (3X6ml) 1% ammonia stripping SPE posts.With 45% acetonitrile eluted product being dissolved in 1% ammonia spirit (6ml).Identified by UV photo densitometries Elutriated fraction containing oligomer.Pass through desivac separation product.Purity is determined by MALDI and HPLC (C-18 and/or SAX) And characteristic.
Embodiment 26
PMO+ total butanedioic acid modification
Weigh appropriate PMO+ (32mg, 3.7 μm of ol) to be encased in bottle (4ml), and be dissolved in DMSO (500 μ l).By N- Ethyl morpholine generation (12mg, 100 μm of ol) and succinic anhydride (10mg, 100 μm of ol) are added in reactant mixture, and are allowed in room It is stirred for staying overnight under temperature.Reaction process is monitored by MALDI and HPLC.
Once completing, 1% ammonia soluble in water is added in reactant mixture, and is stirred at room temperature 2 hours. The solution is loaded on SPE posts (2cm).Bottle is rinsed with 1% ammonia solution (2X2ml), and with 1% be dissolved in water (3X6ml) Ammonia stripping SPE posts.With 45% acetonitrile eluted product being dissolved in 1% ammonia spirit (6ml).Contained by the identification of UV photo densitometries There is the elutriated fraction of oligomer.Pass through desivac separation product.By MALDI and HPLC (C-18 and/or SAX) determine purity and Characteristic.
Procedure above is also applied for PMO+ glutaric acid (glutaric anhydride) and tetramethylene glutaric acid (tetramethylene glutaric acid Acid anhydride) modification.
Embodiment 27
Prepare the oligonucleotide analogs for including the terminal groups through modification
Farnesyl- bromide (1.75 μ l, 6.452 μm of ol) and diisopropylethylamine (2.24 μ L, 12.9 μm of ol) are added to In DMSO (300 μ L) solution of 25-mer PMO (27.7mg, 3.226 μm of ol) containing free 3 ' ends.It is stirred at room temperature Reactant mixture 5 hours.With 10mL 1% aqueous NH4OH dilutes crude reaction mixture, is then subsequently loaded into 2mL On Amberchrome CG300M posts.Then the post is rinsed with the water of 3 pillar volumes, and use 6mL1:1 acetonitrile and water (v/ V) eluted product.Subsequent Solutions in Freeze-drying is to obtain the title compound, and it is white solid.
Embodiment 28
Prepare morpholino oligomers
The preparation of tritylpiperazine carbanilate 35 (see Fig. 3)：The carbon in water (4mL/g potassium carbonate) will be dissolved in Sour potassium (3.2eq) solution, which is added to, to be dissolved in the cooling suspension of the compound 11 of dichloromethane (6mL/g11).It is slowly added to molten Phenyl chloroformate (1.03eq) solution (2g/g phenyl chloroformates) in dichloromethane is into the mixture of the two-phase.Will reaction Mixture is warming up to 20 DEG C.Once reaction completes (1-2 hours), different layers are isolated.It is washed with water organic layer, and with anhydrous Potassium carbonate is dried.By crystallizing separation product 35 from acetonitrile.Yield=80%.
The preparation of carbamate ethanol (carbamate alcohol) 36：Sodium hydride (1.2eq) is suspended in 1- first In base -2-Pyrrolidone (32mL/g sodium hydrides).Triethylene glycol (10.0eq) and compound 35 (1.0eq) are added to the suspension In.Gained slurries are heated to 95 DEG C.Once reaction completes (1-2 hours), the mixture is cooled to 20 DEG C.By 30% dichloro Methane/methyl tertiary butyl ether (v:V) it is added to water in the mixture.The NaOH aqueous solution, butanedioic acid aqueous solution and saturation are used successively Sodium-chloride water solution washs the product containing organic layer.Pass through the Crystallization Separation product from dichloromethane/methyl tertiary butyl ether/heptane 36.Yield=90%.
The preparation of tail acid 37：Succinic anhydride (2.0eq) and DMAP (0.5eq) are added to the compound for being dissolved in tetrahydrofuran In 36 solution (7mL/g36).Heat the mixture to 50 DEG C.Once reaction completes (5 hours), mixture is cooled to 20 DEG C, and use NaHCO3The aqueous solution is adjusted to pH8.5.Methyl tertiary butyl ether is added, and product is extracted into water layer.Add dichloromethane Alkane, and adjusted mixture to pH3 with aqueous citric acid solution.With the citrate buffer and saturated sodium-chloride water solution of pH=3 Mixture washing the product containing organic layer.37 dichloromethane solution is directly used for compound 38 without separation In preparation.
38 preparation：By N- hydroxyl -5- ENB -2,3- dicarboxylic acid imides (HONB) (1.02eq), 4- dimethyl Aminopyridine (DMAP) (0.34eq) is added in the solution of compound 37, is subsequently added 1- (3- dimethylaminopropyls)-N'- Ethyl-carbodiimide hydrochloride (EDC) (1.1eq).Heat the mixture to 55 DEG C., will once reaction completes (4-5 hours) Mixture is cooled to 20 DEG C, and successively with 1:1 0.2M citric acids/bittern and brine.Dichloromethane solution to acetone and Exchange of solvent then is carried out to DMF, and by being precipitated to saturation from acetone/DMF Sodium-chloride water solution in separate the product.In water by raw product it is repulped several times come remove remnants N, N- dimethyl methyls Acid amides and salt.From yield=70% of the preparation of compound 36 38.By the program for being incorporated to subunit during synthesis in solid state in NMP Middle " tail " for introduce activation is on disulphide grappling resin.
Preparation for the solid support (support) of synthesis of morpholino oligomer：In the jacket type peptide pipe of silanization The program is carried out in (customizing the ChemGlass companies in New Jersey), there is gross porosity (40-60 μm) glass to melt for it Block, overhead type stirrer and 3 lead to Teflon plug valves to allow N2Pass through the frit up bubbling or vacuum extraction.By following Temperature control is realized in ring water-bath in reaction vessel.
Resin treatment/washing step in following program is made up of two basic operations：Resin fluidized and solvent/solution Extraction.For resin fluidized, plug valve is placed in appropriate location to allow N2Flow upwardly through frit, by specified resin treatment/ Cleaning solution is added in reactor, and allows its infiltration and complete wetting resin.Then start mixing, and resin grout liquid is mixed The time specified.For solvent/solution extraction, stop mixing and N2Stream, and vavuum pump is opened, then plug valve is placed in suitably Position is to allow resin treatment/cleaning solution being expelled to reject chute (waste).All resin treatments/cleaning solution volume is all 15mL/g resins, unless otherwise indicated.
By 1-Methyl-2-Pyrrolidone (NMP;20ml/g resins) it is added to the amino first in the jacket type peptide pipe of silanization Base polystyrene resin (100-200 mesh；~1.0mmol/g N2Substituent；75g, 1eq, Polymer Labs, UK, part# In 1464-X799), and allow resin expansion under agitation 1-2 hours.Empty after expanded solvents, with dichloromethane, (2x1-2 divides Clock), 5% diisopropylethylamine (2x3-4 minutes) and dichloromethane (2x1-2 minutes) that are dissolved in 25% isopropanol/dichloromethane wash Wash resin.After final cleaning solution is emptied, with being dissolved in 1-Methyl-2-Pyrrolidone (0.17M；15mL/g resins ,~2.5eq) The solution fluidisation resin of disulphide deadman 34, and heat resin/reagent mixture 60 hours at 45 DEG C.Reacting Cheng Shi, stopping heating, spare anchor determines solution side by side, then with 1-Methyl-2-Pyrrolidone (4x3-4 minutes) and dichloromethane (6x1- 2 minutes) washing resin.With being dissolved in dichloromethane (16mL/g;2x5-6 minutes) 10% (v/v) pyrocarbonic acid diethyl ester solution at Resin is managed, is then washed with dichloromethane (6x1-2 minutes).In N2Flow down dry resin 39 (see Fig. 4) 1-3 hours, then exist Dried under vacuum to constant weight (± 2%).Yield：110-150% initial resin weight.
The load of aminomethylpolystyre.e-disulphide resin is determined：Pass through the trityl group (triphen of every gram of resin Methyl) number spectrometric analysis, determine resin load (potentially useful reaction site number).
The dry resin (25 ± 3mg) of known weight is transferred in the 25ml volume capacities bottle of silanization, and addition is dissolved in Dichloromethane~5mL 2% (v/v) trifluoroacetic acid.Content is mixed by gently rotating, then 30 minutes are stood.With molten In dichloromethane 2% other (v/v) trifluoroacetic acid by volume dilution to 25mL, and be thoroughly mixed content.Use positive discharge capacity Pipette, 1 part of solution (500 μ L) containing trityl is transferred in 10mL volumetric flasks, and with pyrovinic acid by volume dilution To 10mL.
By triphenylmethyl cation content of the UV absorbances in the whole solution of measurement at 431.7nm, and using suitable Volume, dilution factor, extinction coefficient (ε:41 μm of ol-1cm-1) and weight resin with every gram of resin of trityl (μm ol/g) calculate Resin load.Repeat the analysis with three, and calculate average load.
Resin load program in the embodiment will provide the resin with about 500 μm of ol/g loads.If at room temperature Disulphide deadman incorporation step is carried out 24 hours, then can obtain 300-400 μm of ol/g load.
Tail load：Set and volume using with preparing aminomethyl polystyrene-disulphide resin identical, can be by institute Tail is stated to be incorporated into molecule.For coupling step, use and be dissolved in 38 of the NMP containing 4- ethyl morpholines (NEM, 0.4M) The solution of (0.2M), rather than disulphide deadman solution.45 DEG C after 2 hours, with being dissolved in the 5% of 25% isopropanol/dichloromethane Diisopropylethylamine washing resin 39 twice, and washed once with DCM.By benzoyl oxide (0.4M) and NEM (0.4M) solution It is added in resin.After 25 minutes, reactor jacket is cooled to room temperature, and it is different be dissolved in 25% isopropanol/dichloromethane 5% 2 Propylethylamine washing resin twice, and is washed 8 times with DCM.Filtering and dry resin 40 under high vacuum.The load of resin 40 is simultaneously fixed Justice is the load of the initial ammonia methylated polystyrene-disulphide resin 39 used in tail load.
Synthesis in solid state：In 2mL Gilson polypropylene reaction columns in Gilson AMS-422 automated peptide synthesizers (Part#3980270) morpholino oligomers are prepared in.When they are located on synthesizer, the aluminium block with water stream channel is put Put around pillar.AMS-422 can alternately add reagent/wash solution, keep specifying the time, and use vacuum-evacuate post Son.
For the oligomer of up to about 25 subunit length, the aminomethyl of the load preferably with about 500 μm of ol/g resins Polystyrene-disulphide resin.For larger oligomer, the ammonia first of the load preferably with 300-400 μm of ol/g resin Base polystyrene-disulphide resin.If the molecule with 5 '-tail is desired, instruct to select using identical load Load has the resin of tail.
It is prepared for following reagent solution：
Trityl removal solution：It is dissolved in 4:10% cyanoacetic acid (w/v) of 1 dichloromethane/acetonitrile；Neutralize solution：It is dissolved in 3:5% diisopropylethylamine of 1 dichloromethane/isopropanol；Couple solution：Desired base and connection type 0.18M (or 0.24M grows to the oligomer for being longer than 20 subunits) activation morpholino subunit and 0.4M N-ethylmorpholines, be dissolved in 1,3- Methylimidazole alkanone.Dichloromethane (DCM) is used as separating the transitional cleaning solution of different solvents solution cleaning solution.
On synthesizer, block is set to 42 DEG C, 2mL 1-Methyl-2-Pyrrolidone is added to the ammonia containing 30mg In each pillar of methylated polystyrene-disulphide resin (or tail resin), and allow to keep at room temperature 30 minutes.With After 2mL dichloromethane is washed 2 times, following synthesis circulation is employed：
The sequence of single oligomer is programmed into synthesizer, so that each pillar is suitable with suitable sequential reception Couple solution (A, C, G, T, I).When in pillar oligomer complete mix its final subunit when, from block remove pillar, and with by The coupling solution of 4- methoxyl groups triphenylmethyl chloride (0.32M is dissolved in DMI) composition containing 0.89M4- ethyl morpholines is manual Finally circulated.
From resin cleavage and removal base and skeleton protection group：After Methoxytrityl, with 2mL1- methyl -2- pyrroles Pyrrolidone washing resin 8 times.Add 1mL by be dissolved in 1-Methyl-2-Pyrrolidone 0.1M1,4- dithiothreitol (DTT)s (DTT) and The cutting solution of 0.73M triethylamines composition, closes the lid, and allow it to stand 30 minutes at room temperature to pillar.That is for the moment Between after, make solution flow into Wheaton bottles of 12mL in.The resin significantly shunk is washed with 300 μ L cutting solution twice.Will The ammoniacal liquor (being stored in -20 DEG C) of 4.0mL concentrations is added in solution, firmly covers bottle (with the nut of Teflon tape strain line), And rotated mixture carrys out mixed solution.Bottle is placed in 45 DEG C of baking ovens 16-24 hours, to produce base and skeleton protection group Cutting.
Initial oligomer isolation：The ammonolysis solution for loading bottle is withdrawn from baking oven, and allows to cool to room temperature.Use 20mL 0.28% ammoniacal liquor dilute solution, and be passed to the 2.5x10cm posts containing Macroprep HQ resins (BioRad).Salt gradient (A:0.28% ammonia, B:1M sodium chloride in 0.28% ammonia；0-100%B, 60 minutes) it is used to elution and contains Methoxytrityl Peak.Merge the fraction of combination and further handled according to desired product.
The demethoxylation tritylation of morpholino oligomers：Use 1M H3PO4Handle the merging purified from Macroprep Fraction, 2.5 are down to by pH.After initial mixing, sample is set to stand 4 minutes at room temperature, now with 2.8% ammonia water by them With to pH10-11.Pass through SPE (SPE) purified product.
By Amberchrome CG-300M (Rohm and Haas;Philadelphia, PA) (3mL) put into 20mL tool In sieve aperture post (BioRad Econo-Pac chromatographic columns (732-1011)), and resin is rinsed with 3mL following material：0.28% NH4OH/80% acetonitriles；0.5M NaOH/20% ethanol；Water；50mM H3PO4/ 80% acetonitrile；Water；0.5NaOH/20% ethanol；Water； 0.28%NH4OH。
Future, the solution of autospasy Methoxytrityl was loaded on pillar, and with 3-6mL0.28% ammonia water rinse resins 3 times.Wheaton bottles (12mL) are placed under pillar, and washes twice and washes by using 2mL 45% acetonitriles for being dissolved in 0.28% ammoniacal liquor It is temporarily released from one's regular work thing.The solution is freezed in dry ice, and bottle is placed in freeze-dryer, to produce fluffy white powder. Sample is soluble in water, and by 0.22 micron filter, (Pall Life Sciences, Acrodisc25mm syringe type are filtered Device, with 0.2 micron of HT Tuffryn film) filtered using syringe, and optical density (OD) is measured on UV spectrophotometers, with The OD units that oligomer is presented are determined, and distribution sample is used to analyze.Then solution is put back in Wheaton bottles, for freezing It is dry.
The analysis of morpholino oligomers：The composition of the fraction in purifying is determined with MALDI-TOF mass spectral analyses, and The proof of the characteristic (molecular weight) of oligomer is provided.With 3,5- dimethoxy-4 's-hydroxycinnamic acid (sinapic acid), the hydroxyls of 3,4,5- tri- After the solution dilution of benzoylformaldoxime (THAP) or alpha-cyano -4- hydroxycinnamic acids (HCCA), upshift operation sample is used as.
Use the sodium acetate of the sodium acetate of 25mM pH=5,25% acetonitrile (buffer A) and 25mM pH=5,25% acetonitrile, 1.5M chlorine Change potassium (buffer B) (gradient 10-100%B, 15 minutes) or 25mMKH2PO425% acetonitrile, pH=3.5 (buffer A) and 25mM KH2PO425% 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.It is previous System is used for the positively charged oligomer adhered to without peptide, and the latter is used for peptide conjugate.
Morpholino oligomers are purified by cation-exchange chromatography：Sample is dissolved in 20mM sodium acetates, pH=4.5 (buffer solution A in), and be applied to Source30 cationic ion-exchange resins (GE Healthcare) pillar, and with 20mM sodium acetates and 0.5M NaCl gradients in 40% acetonitrile, pH=4.5 (buffer B) elution.The merging level containing product is neutralized with concentrated gas liquor Point, and it is applied to Amberchrome SPE posts.By as above elution, freeze and lyophilized products.
Embodiment 29
The preparation of exemplary conjugate
Peptide sequence AcR is prepared according to standard peptide synthesis methods known in the art6G.At room temperature by diisopropylethylamine (36 μ L, 5eq) it is added to PMO (NG-05-0225,3 '-H being dissolved in DMSO (3mL):M23D:5 '-EG3 are small for being attached to mdx Sequence on the exon 23 of mouse, 350mg, 1eq), AcR6In G (142mg, 2eq), HATU (31mg, 2eq) solution.1 hour Afterwards, start reaction and chromatographed by SCX and (eluted to Gradient：A:It is dissolved in 25% acetonitrile/H2O 20mM NaH2PO4, pH7.0； B:It is dissolved in 25% acetonitrile/H2O 1.5M guanidine HCl and 20mM NaH2PO4, pH7.0) and the desired peptide-oligomer conjugate of purifying.Make The fraction experience SPE of combination (1M NaCl then carry out water elution).Conjugate is obtained after lyophilized for white powder (257mg, 65.5% yield).
Embodiment 30
MDX mouse are handled with the exemplary conjugate of the present invention
MDX mouse are the animal model of Duchenne muscular dystrophy (DMD) generally acknowledging and fully levying surely, and it seeks in flesh Support in the exon 23 of bad GFP containing mutation.M23D antisense sequences (SEQ ID NO:15) extron is can induce known to 23 reparations skipped with the expression of feature dystrophin.Given with one kind in following conjugate by tail vein injection MDX mouse 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 " refer to Be following structure：
It is connected on 5 ' ends of oligomer by piperazine linking arm (that is, structure XXIX).
One week after is injected, MDX mouse is put to death and extracts RNA from different musculature.With terminal PCR (End-point PCR) determine the dystrophin mRNA containing exon 23 and lack outer aobvious due to the exon skipping that antisense is induced The mRNA of son 23 relative abundance.Percentage exon 23 is skipped as the measurement of internal antisense activity.Fig. 5 and Fig. 6 are shown respectively After processing 1 week 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) Result.Dose response between AVI-5225 and other conjugates is similar.In arginine series, R6G peptides are four There is highest effect in head flesh and barrier film, and it is similar with other effects of arginine series peptide in heart.
Embodiment 31
The BUN levels and survival rate of the mouse treated with exemplary conjugate
With described in embodiment 30 conjugate handle mouse, and according to described in example 3 below 2 and this area The general procedure known determines KIM-1 levels, BUN levels and survival rate.It is especially surprising that Fig. 7 A show that all glycine connect The conjugate connect has the notable low BUN levels of conjugate (AVI-5225) than XB connections.In addition, what is connected with glycine sews The treated mouse of compound, longer, wherein R of being survived under the higher dosage of the conjugate (Fig. 7 B) connected than XB8G conjugates are most Arginine polymer is resistant to less.Use R6The treated all mouse of G conjugates (NG-11-0009) all can be in up to 400mg/kg Dosage under survive (data are not shown).
The KIM-1 (Fig. 8 A) and lectin (Fig. 8 B) level for the mouse that the conjugate connected with glycine is treated are significantly low In the mouse treated with AVI-5225.The as shown by data, conjugate of the invention has the poison lower than previous conjugate Property, and as shown in above example 30, effect of the conjugate is not reduced.Therefore, conjugate of the present invention is than known to other Conjugate there is preferably treatment window, and for potential more preferable drug candidate.
Embodiment 32
The toxicity of exemplary conjugate
The toxicity of 4 kinds of exemplary conjugates of the present invention is tested in Mice Body.The 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 " refer to Be following structure：
It is connected on 5 ' ends of oligomer by piperazine linking arm (that is, structure XXIX).
Male mice (C57/BL6 8 weeks big is handled with the above conjugate for being configured to salt；Jackson Laboratories, 18-22 gram).Mouse is tamed minimum 5 days before experimental arrangement is started.
With every cage up to 3 in the micro- isolation cage of cleaning makrolon for proving qualified and irradiated contact bedding and padding Density group animal only.The cage observes Animal Welfare Law (including all amendments) and the state of the U.S. positioned at Washington Experimental animal nursing and instruction (Guide for the Care and Use of that academic press of family is published for 2010 Laboratory Animals, National Academy Press, Washington, D.C., 2010) the middle standard stated.
Animal is assigned randomly in treatment group again based on the cage illustrated in below table.The explanation in research record Component is matched somebody with somebody.
The toxicological study of table 8. is designed
That day that dosage is given in research is designated as research the 1st day.As slow push type pill (~5 seconds) via tail vein Give conjugate.All animals give dosage 2 days.It was that 1-8 groups give dosage at first day, and was that 9-16 groups are given at second day Give dosage.It is that treatment group (TG) 13-16 gives dosage according to upper table.Result from these TG does not influence other TG progress. The initial 2 TG dosage of every kind of conjugate is given according to upper table.If all animals in 100mg/kg groups are all dead, that The remaining TG of test article will not then give dosage, and research stops.If in 100mg/kg groups at least 1 animal to Give and being survived 2 hours after dosage, then then give 150mg/kg group dosage.If all animals in 150mg/kg groups are all dead, Then the remaining TG of that test article will not then give dosage, and research stops.If at least 1 is moved in 150mg/kg groups Thing is survived 2 hours after dosage is given, then then gives 200mg/kg group dosage.
The dying rate and the death rate of an animal are observed daily.According to Numira Biosciences S.O.P. people It is genuine to show danger and disaster sign, especially dead imminent any animal euthanasia.After arrival the same day, give dosage work as Body weight is recorded on the day of it and ptomatopsia.Detailed clinical observation is carried out, and 0 minute, 15 minutes and 2 hours after dosage is given Record, to assess the tolerance of injection.
Give after dosage 3 days, blood sample (largest body is obtained from all animals by cardiac puncture before ptomatopsia Product, about 1mL).Blood Sample Collection is interior to red top Microtainer pipes, and maintain at least 30 points at room temperature before centrifugation Clock but no longer than 60 minutes.Sample is centrifuged 15-20 minutes to obtain serum under about 1500-2500rpm.
The animal of unlikely survival to next predetermined observation is weighed and is euthanized.Animal to finding dead Weigh, and estimate the death time as closely as possible.Do not collect blood and tissue sample.
All animals humanity, is euthanized by the 3rd day (giving after dosage 2 days) with carbon dioxide.According to the acceptable U.S. Veterinary Medical Association (AVMA) is euthanized in June, 2007 on the guide of euthanasia.
Local gross necropsy includes inspection and the record of inspection situation.It has evaluated all outer surfaces and aperture.Completely retouch State and have recorded all abnormal conditions observed during collection tissue.Its hetero-organization is not gathered.
Acquire left kidney and right kidney.Tissue is gathered within 15 minutes or less the time that is euthanized.Changed between treatment group The all appts and instrument used.By all tissue IQFs and it is stored in as early as possible after collection<At -70 DEG C.
By injury of kidney mark number evidence is obtained as below.Use Quick gene Mini80Tissue Kit SII (Fuji Film the RNA from kidney of mouse) is purified.Briefly, about 40mg tissue is added in MagnaLyser Green In 0.5ml lysis buffers (5 μ l2- mercaptoethanols are dissolved in 0.5ml lysis buffers) in Bead bottles (Roche), and use MagNA Lyser (Roche) carry out homogeneity with 2 series 3x3800RPM and 3 series 1x6500RPM.In each low speed series Between between each high-speed cruising by sample in cooled on ice 3-4 minutes.400xg, at room temperature will homogenate centrifugation 5 minutes.Immediately Processing homogenate, for according to Quick gene Mini80 scheme purifying RNAs.Sample experienced using DNA enzymatic I's (Qiagen) Upper prop DNA digests 5 minutes.Use the quantitative total serum IgE of the spectrophotometers of NanoDrop 2000 (Thermo Scientific).
Use the reagent (One step RT-PCR) and prior designed primer/probe of Applied Biosystems companies Serial (ACTB, GAPDH, KIM-1, lectin-FAM reporters) carries out qRT-PCR.
Reagent Company Cat. no
One-step method PCR kit Applied Biosystems 4309169
GAPDH mouse primer/probe sets Applied Biosystems 4352932E
KIM-1 mouse primer/probe sets Applied Biosystems Mm00506686_m1
Each reaction contains following component (30 μ l altogether)：
By following operation qRT one-step method programs：
1.48 DEG C, 30 minutes
2.95 DEG C, 10 minutes
3.95 DEG C, 15 seconds
4.60 DEG C, 1 minute
5. repeat step 3-4 is counted 39 times, 40 circulations altogether
The Run sample in triplicate hole, and value is averaged for further analyzing.Carried out using Δ Δ Ct methods Analysis.Briefly, experiment Δ Ct [Ct (target)-Ct (reference)]-control Δ Ct [Ct (target)-Ct (reference)]=Δ Δ Ct. The multiple excursion calculated：2^- (Δ Δ Ct+SD) to 2^- (Δ Δ Ct-SD).The animal groups of control=vehicle treated (are closed And), target=KIM-1；With reference to=GAPDH；SD=Sqrt [(SD target ^2)+(SD refers to ^2)].
The result of KIM data is shown in Fig. 10.Conjugate comprising the carrier peptides with terminal glycine has low KIM Concentration, wherein R6G peptides have minimum KIM concentration.The presence of end G and alpha-non-natural amino acid (aminocaproic acid) seems all conjugated Played a role in the toxicity of thing.
The blood serum sample of freezing is delivered into IDEXX laboratories (West Sacramento, CA) on dry ice is used to handle. If necessary serum-dilution is carried out according to IDEXX S.O.P.s (SOPs).Analyze Blood chemistry results.Blood urea nitrogen level Display is in fig. 11.Again, the conjugate of G connections has low BUN levels, and end G and overall peptide sequence seem all sewing Played a role in the poisonous substance attribute of compound.
Accurate weighing nephridial tissue (the about 150mg) in the 2mL screw cap vials for be filled partially with ceramic bead.10U/mL will be contained 5 parts by volume tissue PE LB buffer solutions (G Biosciences) of Proteinase K (Sigma) are added in 1 part of tissue.Use Roche MagnaLyser (4x40 seconds@7000rpm, have cooling between operation) is incubated 30 minutes by sample homogeneity, and at 40 DEG C.Need When wanting, high sample concentration is reduced to calibration range with BSAsal (3mg/mL BSA+20mM NaCl) dilution tissue homogenate.
The BSA solution systems that the reinforcing of normative reference product is dissolved in 20mM NaCl 3mg/mL are suitably analyzed by using known quantity Standby calibration sample.Each sample preparation repetition series of 8 samples.Μ LOQ are 40 μ g/mL, and LLOQ is 0.065536 μ g/ mL.Internal standard compound (NG-07-0775) is added to outside being appointed as double blank (without medicine, without internal standard compound) blank sample except some All samples in.Pass through the μ L aliquots of vortex 100 and the methanol extraction sample of 3 times of volumes.
Centrifuge after (15 minutes, 14,000rpm), supernatant liquor is transferred in new pipe, and done in Speedvac It is dry.Dry sample is used and is dissolved in the appropriate of [10mM Tris pH8.0+1mM EDTA+100mM NaCl]-acetonitrile (75-25) FDNA (5 ' dFAM-ATTTCAGGTAAGCCGAGGTTTGGCC3 ') is reconstructed.
Using anion-exchange chromatography (Dionex DNAPac4x250mm posts) in Dionex Μ ltiMate3000HPLC Upper analysis sample.Injected slurry volume is 5 μ L.Mobile phase is by 20% acetonitrile and contains 25mM Tris pH8.0 and increases NaCl concentration ladder The 80% water composition of degree.Flow velocity is 1mL/ minutes, and run time is every sample 10 minutes.Fluorescence detector is arranged on EX494nm and EM520nm.Peak identification is based on retention time.Ratio of peak (analyte：Internal standard compound) it be used to quantify.Based on repetition Calibration sample (one operate in the batch starting when, another is at the end of the batch) average response coefficient calculate correction Curve.The linearity curve being fitted with 1/x weight factors is used.Blank sample has been used (not add the school of reference compound Quasi- sample) and double blank sample (not adding internal standard compound) ensure analysis 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.
Data above shows, compared to other conjugates, and conjugate of the invention has similar effect and improved toxicity. Fig. 9 A-D are outlined on R6These results of G conjugates (NG-11-0009).
Different embodiments described above can be combined to provide other embodiments.Referred in this specification and/ Or all United States Patent (USP)s, U.S. Patent Application Publication, U.S. Patent application, foreign patent, outer listed in the application tables of data State's patent application and non-patent publications are all fully incorporated herein by quoting.If it is necessary, the side of embodiment can be changed Face uses different patents, application and the concept of publication, to provide other embodiments.Can in detail it be retouched more than State and these and other changes are carried out to embodiment.In general, in the following claims, the term used should not be explained For claim to be restricted to the specific embodiment disclosed in specification and claims, and it should be interpreted that including with this A little claims enjoy all possible embodiment of the full breadth of the equivalent of scope.Therefore, the claim is not Limited by the disclosure.

Claims (59)

1. conjugate, it is included：
(a) carrier peptides of amino acid subunit are included, the carrier peptides include the glycine (G) of the c-terminus positioned at the carrier peptides Or proline (P) subunit；
(b) morpholino oligomers (PMO), target nucleic acid is attached to comprising the skeleton substantially without electric charge and for sequence-specific On target-seeking base sequence；With
(c) covalent attachment between the morpholino oligomers and the carrier peptides, the covalent attachment by with the c-terminus Glycine or the acid amides connection composition of proline connection；
Wherein：
Two or more of the amino acid subunit are positively charged amino acid, and not more than 7 continuous amino acid are sub- Base is arginine.
2. conjugate according to claim 1, wherein the carrier peptides include the glycine positioned at c-terminus.
3. conjugate according to claim 1, wherein the carrier peptides include the proline positioned at c-terminus.
4. conjugate according to claim 1, wherein the carrier peptides include 4 to 40 amino acid subunits.
5. conjugate according to claim 1, wherein the carrier peptides include 6 to 20 amino acid subunits.
6. conjugate according to claim 1, wherein the positively charged amino acid is histidine (H), lysine (K), arginine (R) or combinations thereof.
7. conjugate according to claim 1, the positively charged amino acid of wherein at least 1 is arginine.
8. conjugate according to claim 1, wherein in addition to the carboxyl terminal glycine or proline, each ammonia Base acid subunit is all positively charged amino acid.
9. conjugate according to claim 1, wherein each positively charged amino acid is arginine.
10. conjugate according to claim 1, the positively charged amino acid of wherein at least 1 is that arginine is similar Thing, the arginine analog is cationic a-amino acid, and it is R comprising structureaN=C (NH2)RbSide chain, wherein RaFor H or Rc；RbFor Rc、NH2, NHR or N (Rc)2, wherein RcInclude for low alkyl group or low-grade alkenyl and optionally oxygen or nitrogen, or Ra And RbRing can be formed together；And wherein described side chain passes through RaOr RbIt is connected on the amino acid.
11. conjugate according to claim 1, the amino acid subunit of wherein at least 20% is positively charged amino Acid.
12. conjugate according to claim 1, the amino acid subunit of wherein at least 50% is positively charged amino Acid.
13. conjugate according to claim 1, the amino acid subunit of wherein at least 80% is positively charged amino Acid.
14. conjugate according to claim 1, wherein the carrier peptides include sequence (Rd)m, wherein RdWhen occurring every time Positively charged amino acid independently all is, and m is 2 to 12 integer.
15. conjugate according to claim 14, wherein RdArginine, histidine independently all are when occurring every time or is relied Propylhomoserin.
16. conjugate according to claim 14, wherein each RdAll it is arginine.
17. conjugate according to claim 1, wherein in addition to the carboxyl terminal glycine or proline, each amino Sour subunit is all arginine.
18. conjugate according to claim 1, wherein the carrier peptides include at least one hydrophobic amino acid, it is described to dredge Aqueous amino acid includes substitution or unsubstituted alkyl, alkenyl, alkynyl, aryl or aralkyl side chain, wherein the alkyl, Alkenyl and every 6 carbon atoms of alkylyl side chain include at most 1 hetero atom.
19. conjugate according to claim 18, wherein the carrier peptides include two or more hydrophobic amino acids.
20. conjugate according to claim 18, wherein the carrier peptides include two or more continuous hydrophobicitys Amino acid.
21. conjugate according to claim 18,1 hydrophobic amino acid of wherein at least is phenylalanine.
22. conjugate according to claim 18, wherein each hydrophobic amino acid is phenylalanine.
23. conjugate according to claim 1, wherein the carrier peptides have following structure (Yb) comprising at least one Neutral amino acid：
-C(O)-(CHRe)n-NH-
(Yb)
Wherein n is 2 to 7, and each ReHydrogen or methyl independently all are at each occurrence.
24. conjugate according to claim 23, wherein the carrier peptides include sequence [(RdYbRd)x(RdRdYb)y]z, its Middle RdPositively charged amino acid independently all is at each occurrence, and x and y independently are 0 or 1 at each occurrence, Condition is that x+y is 1 or 2, and z is 1 to 6 integer.
25. conjugate according to claim 24, wherein RdAll independently be at each occurrence arginine, histidine or Lysine.
26. conjugate according to claim 24, wherein each RdAll it is arginine.
27. conjugate according to claim 24,1 Y of wherein at leastbFor 6-aminocaprolc acid or Beta-alanine.
28. conjugate according to claim 1, wherein the carrier peptides include sequence ILFQY.
29. conjugate according to claim 1, wherein the carrier peptides include sequence ILFQ, IWFQ or ILIQ.
30. conjugate according to claim 1, wherein the carrier peptides comprising sequence PPMWS, PPMWT, PPMFS or PPMYS。
31. conjugate according to claim 1, wherein, in addition to the covalent attachment, the carrier peptides also include third In propylhomoserin, aspartic acid, cysteine, glutamine, glycine, histidine, lysine, methionine, serine or threonine It is at least one kind of.
32. conjugate according to claim 1, wherein the carrier peptides include the acetyl positioned at the carrier peptides aminoterminal Base, benzoyl or stearyl part.
33. conjugate according to claim 1, wherein each amino acid subunit is natural amino acid.
34. conjugate according to claim 1, is combined wherein uncharged skeleton includes connection between passing through subunit Morpholino ring-type structure sequence, connect between the subunit 3 ' ends of a morpholino cyclic structure are attached to it is neighbouring Quinoline is on 5 ' ends of cyclic structure, wherein each morpholino cyclic structure is attached on the part of base pairing so that oligomerization Thing can be attached on target nucleic acid with sequence-specific fashion, wherein connection has following universal architecture (I) between the subunit：
Or connection (I) independently is connection (A) or connection (B) between its salt or isomers, and wherein each subunit：
Wherein for connection (A)：
W independently is S or O at each occurrence；
X independently is-N (CH at each occurrence3)2、-NR1R2、-OR3；Or
Y independently is O or-NR at each occurrence2,
R1Hydrogen or methyl independently all are at each occurrence；
R2Hydrogen or-LNR independently all are at each occurrence4R5R7；
R3Hydrogen or C independently all are at each occurrence1-C6Alkyl；
R4Hydrogen, methyl ,-C (=NH) NH independently all are at each occurrence2,-Z-L-NHC (=NH) NH2Or-[C (O) CHR' NH]mH, wherein Z are carbonyl (C (O)) or direct key, and R' is the side chain of naturally occurring amino acid or its 1 carbon or 2 carbon homologues, And m is 1 to 6；
R5Hydrogen, methyl or electronics pair independently all are at each occurrence；
R6Hydrogen or methyl independently all are at each occurrence；
R7Hydrogen C independently all is at each occurrence1-C6Alkyl or C1-C6Alkoxyalkyl；
L is the optional linking arm of up to 18 atomic lengths, including alkyl, alkoxy or alkylamino, or combinations thereof；And
Wherein for connection (B)：
W independently is S or O at each occurrence；
X independently is-NR at each occurrence8R9Or-OR3；And
Y independently is O or-NR at each occurrence10,
R8Hydrogen or C independently all are at each occurrence2-C12Alkyl；
R9Hydrogen, C independently all are at each occurrence1-C12Alkyl, C1-C12Aralkyl or aryl；
R10Hydrogen, C independently all are at each occurrence1-C12Alkyl or-LNR4R5R7；
Wherein R8And R9It may be combined to form the monocyclic or bicyclic heterocycles of 5-18 members, or R8、R9Or R3Can be with R10Combine to form The heterocycle of 5-7 members, and wherein when X is 4- piperazines, X has following structure (III)：
Wherein：
R11C independently all is at each occurrence2-C12Alkyl, C1-C12Aminoalkyl, C1-C12Alkyl-carbonyl, aryl, heteroaryl or Heterocyclic radical；And
R independently is electronics to, hydrogen or C at each occurrence1-C12Alkyl；And
R12Hydrogen, C independently all are at each occurrence1-C12Alkyl, C1-C12Aminoalkyl, NH2、CONH2、-NR13R14、- NR13R14R15、C1-C12Alkyl-carbonyl, oxo (oxo) ,-CN, trifluoromethyl, amide groups, amidino groups, Amidinylalkyl, Amidinylalkyl carbonyl Base guanidine radicals, guanidine alkylation, guanidine alkylation carbonyl, cholate, dexycholate, aryl, heteroaryl, heterocycle ,-SR13Or C1-C12Alkane Epoxide, wherein R13、R14And R15C independently all is at each occurrence1-C12Alkyl.
35. conjugate according to claim 34, a kind of the wherein at least morpholino cyclic structure has following structure (i)：
Wherein Pi independently is the part of base pairing at each occurrence.
36. it is connected as connecting (A) between conjugate according to claim 34,1 subunit of wherein at least.
37. conjugate according to claim 34, wherein being connected between each subunit as connection (A).
38. conjugate according to claim 34, wherein each connection (A) has identical knot at each occurrence Structure.
39. conjugate according to claim 34, wherein for the appearance of at least 1 time connection (A), X is-N (CH3)2。
40. conjugate according to claim 34, wherein X are all-N (CH connecting when (A) occurs every time3)2。
41. conjugate according to claim 34, wherein each connection is connection (A), and X is-N (CH3)2。
42. conjugate according to claim 34, wherein for the appearance of at least 1 time connection (A), X has following structure：
43. conjugate according to claim 34, wherein for the appearance of at least 1 time connection (B), X has following structure：
44. conjugate according to claim 34, wherein for the appearance of at least 1 time connection (B), X has following structure：
45. conjugate according to claim 34, wherein W and Y are all respectively O at each occurrence.
46. it is connected as connecting (B) between conjugate according to claim 34, the subunit of wherein at least 5%.
47. conjugate according to claim 34, wherein each connection (B) has identical knot at each occurrence Structure.
48. conjugate according to claim 34, wherein the nucleic acid analog has following structure (XVII)：
Or its salt or isomers, wherein：
R17All independently be at each occurrence be not present, hydrogen or C1-C6Alkyl；
R18And R19All independently be at each occurrence be not present, hydrogen, the carrier peptides, C2-C30Alkyl-carbonyl ,-C (=O) OR21Or R20；
R20Guanidine radicals, heterocyclic radical, C independently all are at each occurrence1-C30Alkyl, C3-C8Cycloalkyl；C6-C30Aryl, C7-C30 Aralkyl, C3-C30Alkyl-carbonyl, C3-C8Naphthene base carbonyl, C3-C8Cycloalkyl alkyl carbonyl, C7-C30Aryl carbonyl, C7-C30Virtue Alkyl-carbonyl, C2-C30Alkoxy carbonyl, C3-C8Cyclo alkoxy carbonyl, C7-C30Aryloxycarbonyl, C8-C30Aromatic alkoxy carbonyl Or-P (=O) (R22)2；
R21To include the C of one or more ehter bonds, hydroxylic moiety or combinations thereof1-C30Alkyl；
Each R22All it independently is C6-C12Aryloxy group；
Pi is the part of base pairing；
L1And L2The respectively linking arm of direct key or up to 18 atomic lengths, the linking arm, which is included, is selected from following connection： Alkyl, hydroxyl, alkoxy, alkyl amino, acid amides, ester, carbonyl, carbamate, phosphoric acid diamides, phosphoamide, thio phosphine Acid and di-phosphate ester；
Z is 0 or bigger integer；And
Wherein R17And R18It is not in the absence of and R18Or R19Middle at least one is the carrier peptides.
49. conjugate according to claim 48, wherein R18For the carrier peptides.
50. conjugate according to claim 48, wherein R19For the carrier peptides.
51. conjugate according to claim 48, wherein R18Or R19In at least one be R20。
52. conjugate according to claim 48, wherein R19For-C (=O) OR21。
53. conjugate according to claim 52, wherein R19For
54. conjugate according to claim 48, wherein L1Combined comprising phosphoric acid diamides and piperazine.
55. conjugate according to claim 54, wherein L1With following structure (XXIX)：
Wherein R24To be not present, H or C1-C6Alkyl.
56. conjugate according to claim 55, wherein R24For in the absence of.
57. conjugate according to claim 48, wherein R18For carrier peptides, L2For direct key, and the carrier peptides are in institute State and connection is formed between the c-terminus of carrier peptides and 3 ' terminal nitrogens of the nucleic acid analog.
58. composition, it includes the conjugate and pharmaceutically acceptable carrier described in claim 1.
59. the conjugate described in claim 1 is preparing the purposes in being used to treat the medicine of disease, wherein the disease is disease Malicious infection, neuromuscular disease, bacterium infection, inflammation or POLYCYSTIC KIDNEY DISEASE.
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