阅读说明：本技术 修饰核苷、核苷酸和核酸聚合物及其制备方法与应用 (Modified nucleoside, nucleotide and nucleic acid polymer as well as preparation method and application thereof ) 是由 王升启 何小羊 鲁丹丹 杨静 代玉 任晋 邓新秀 于 2019-09-26 设计创作，主要内容包括：本发明涉及修饰核苷、核苷酸和核酸聚合物及其制备方法与应用。所述修饰核苷选自具有如式(I)所示结构的化合物、其盐或其异构体；其中,R<Sub>1</Sub>选自取代或未取代的碱基或其盐；X代表O或S；R<Sub>2</Sub>选自氢或者取代或未取代的：C<Sub>1</Sub>～C<Sub>6</Sub>烷基、C<Sub>1</Sub>～C<Sub>6</Sub>杂烷基、C<Sub>2</Sub>～C<Sub>6</Sub>烯基、C<Sub>2</Sub>～C<Sub>6</Sub>炔基、芳基或杂芳基；R<Sub>3</Sub>和R<Sub>4</Sub>独立地选自氢或者取代或未取代的：C<Sub>1</Sub>～C<Sub>6</Sub>烷基、C<Sub>1</Sub>～C<Sub>6</Sub>杂烷基；W<Sub>1</Sub>和W<Sub>2</Sub>独立地选自H或保护基团。所述修饰核苷在6’位引入了腈基,并在此基础上进一步获得修饰核苷酸和核酸聚合物,极大地改善了它们的核酶耐受性,对靶RNA具有高度选择性和较强结合亲和力。<Image he="402" wi="664" file="DDA0002215769850000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention relates to modified nucleosides, nucleotides and nucleic acid polymers, and preparation methods and applications thereof. The modified nucleoside is selected from a compound with a structure shown in a formula (I), a salt or an isomer thereof; wherein R is 1 Selected from substituted or unsubstituted bases or salts thereof; x represents O or S; r 2 Selected from hydrogen or substituted or unsubstituted: c 1 ～C 6 Alkyl radical, C 1 ～C 6 Heteroalkyl group, C 2 ～C 6 An alkenyl group,C 2 ～C 6 Alkynyl, aryl or heteroaryl; r 3 And R 4 Independently selected from hydrogen or substituted or unsubstituted: c 1 ～C 6 Alkyl radical, C 1 ～C 6 A heteroalkyl group; w 1 And W 2 Independently selected from H or a protecting group. The nitrile group is introduced into the 6' position of the modified nucleoside, and the modified nucleotide and the nucleic acid polymer are further obtained on the basis, so that the ribozyme tolerance of the modified nucleoside is greatly improved, and the modified nucleoside has high selectivity and strong binding affinity to target RNA.)

1. A modified nucleoside selected from compounds having a structure represented by formula (I), salts thereof or isomers thereof:

wherein R is₁Selected from substituted or unsubstituted bases or salts thereof; x represents O or S;

R₂selected from hydrogen, or R₂Selected from substituted or unsubstituted: c₁～C₆Alkyl radical, C₁～C₆Heteroalkyl group, C₂～C₆Alkenyl radical, C₂～C₆Alkynyl, aryl or heteroaryl; said C is₁～C₆The heteroatom of heteroalkyl or said heteroaryl is selected from O, S, N, P or Si;

R₃and R₄Independently selected from hydrogen, or R₃And R₄Independently selected from substituted or unsubstituted: c₁～C₆Alkyl radical, C₁～C₆A heteroalkyl group;

W₁and W₂Independently selected from H or a protecting group.

2. The modified nucleoside of claim 1, wherein R is₁Selected from substituted or unsubstituted: a purine, pyrimidine or their respective salts;

preferably, said R is₁Selected from substituted or unsubstituted: adenine, guanine, thymine, cytosine, uracil, or their respective salts;

preferably, when said R is₁When selected from substituted cytosines, said R₁Is a 5 '-alkyl-substituted cytosine, and more preferably a 5' -methyl-substituted cytosine.

3. A nucleotide, wherein the nucleotide comprises a 3' -phosphoramidite derivative of a modified nucleoside of claim 1 or 2, or a salt thereof.

4. The nucleotide according to claim 3, wherein the nucleotide is selected from a compound having a structure represented by formula (II), a salt thereof, or an isomer thereof:

5. the nucleotide of claim 4, wherein W in the formula (II)₂Is 4, 4' -dimethoxytrityl.

6. A nucleic acid polymer, characterized in that it comprises at least one nucleotide according to any one of claims 3 to 5.

7. The nucleic acid polymer of claim 6, wherein the nucleic acid polymer comprises ribonucleic acid, deoxyribonucleic acid, or a copolymer of ribonucleic acid and deoxyribonucleic acid.

8. A method of preparing a modified nucleoside according to claim 1 or 2, comprising the steps of:

when R is₃And R₄Independently of one another is hydrogen, R₁In the case of thymine, the method comprises:

reacting the reaction substrate (I) with thymine in the presence of a protective agent to produce a compound (II); in the presence of a catalyst A, carrying out intramolecular cyclization reaction on the compound (II) to generate a compound (III); protecting group W of Compound (III)₂' removing and oxidizing in the presence of an oxidizing agent to form compound (IV); nucleophilic substitution of Compound (IV) with a nitrile Source in the Presence of catalyst BReacting to obtain a compound (V) and/or an isomer thereof; converting compound (V) and/or its isomer into compound (VI) and/or its isomer under basic conditions;

wherein the structural formula of each compound is shown as the following formula, W₁、W₂And W₂' is hydrogen or a protecting group:

9. the process according to claim 8, characterized in that the catalyst A is selected from 4-dimethylaminopyridine and/or trifluoromethanesulfonic anhydride;

preferably, the oxidizing agent is selected from at least one of 2-iodoxybenzoic acid, dess-martin reagent, dimethyl sulfoxide-oxalyl chloride, preferably 2-iodoxybenzoic acid;

preferably, the catalyst B is selected from AlCl₃、CeCl₃、ZnCl₂、TiCl₄Preferably AlCl₃；

Preferably, the nitrile source is selected from at least one of trimethylnitrile silane, sodium cyanide, potassium ferricyanate, preferably trimethylnitrile silane.

10. Use of the nucleic acid polymer according to claim 6 or 7 for the preparation of a nucleic acid diagnostic and/or therapeutic agent.

Technical Field

The invention relates to the fields of chemical modification of nucleotides and nucleic acid medicines, in particular to modified nucleosides, nucleotides and nucleic acid polymers as well as preparation methods and applications thereof.

Background

Compared with the traditional micromolecule and protein drugs, the nucleic acid drug has the advantages of rapid design, universal target, high specificity, capability of playing a role in cells, relatively rapid synthesis and preparation and the like, can break through the treatment of serious diseases of which the protein target is difficult to prepare, and has unique value particularly in emergency research and development of special cases, new emergent infectious diseases and the like.

However, natural oligonucleotide molecules also present a number of problems that must be overcome in order to be effective as therapeutic agents, such as: (1) the in vivo stability is poor, and the natural phosphodiester-linked oligonucleotides are easily and rapidly degraded by various ribozymes which are widely present in blood and cells in vivo; (2) low binding affinity and specificity with target genes, susceptibility to off-target effects, immunostimulation and toxic side effects, especially hepatotoxicity; (3) the bioavailability is low, oligonucleotides are usually polyvalent anionic macromolecules, and thus, target organs and tissues are difficult to enter, and lipophilic cell membranes are not easy to permeate into cells.

Chemical modification of nucleic acids is a key technology and bottleneck for the development of nucleic acid drugs. In view of the above problems, since 1970s, many successful oligonucleotide structure modification strategies have been developed by modifying phosphate backbone, sugar group, and base, such as phosphorothioate, 2 '-OMe modified at 2' -position, 2 '-OMOE, 2' -F, Morpholino, PNA, etc., which greatly improves the nuclease resistance of oligonucleotides, the affinity and specificity to target genes, reduces immune stimulation and toxic side effects, and promotes the development of nucleic acid drugs.

However, the currently approved nucleic acid drugs are all focused on the rare disease field, and further breakthrough is still expected in terms of major diseases, the problem of drug formation of the nucleic acid drug ADME-Tox and the like is still outstanding, and the need for a novel modified structure with high efficiency and low toxicity is very urgent, and particularly for the second-generation antisense nucleic acid drug gapmer, the development of C6' substituted LNA with high nuclease resistance and target RNA affinity is an important development direction of the new-generation nucleic acid chemical modification, and has important theoretical and practical significance.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a modified nucleoside, wherein a nitrile group is introduced into the position of a bridge ring C6' to improve the ribozyme tolerance of the modified nucleoside when the modified nucleoside is used for nucleic acid drugs.

Meanwhile, the invention further provides modified nucleotides and nucleic acid polymers on the basis of the modified nucleosides, wherein the modified nucleotides and the nucleic acid polymers have good ribozyme tolerance, high selectivity and strong binding affinity for target RNA, and are beneficial to reducing off-target effect.

The invention also provides the application of the nucleic acid polymer in preparing nucleic acid diagnostic agents and/or nucleic acid therapeutic agents.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a modified nucleoside selected from a compound having a structure represented by formula (I), a salt thereof, or an isomer thereof:

wherein R is₁Selected from substituted or unsubstituted bases or salts thereof; x represents O or S;

R₂selected from hydrogen, or R₂Selected from substituted or unsubstituted: c₁～C₆Alkyl radical, C₁～C₆Heteroalkyl group, C₂～C₆Alkenyl radical, C₂～C₆Alkynyl, aryl or heteroaryl; said C is₁～C₆The heteroatom of heteroalkyl or said heteroaryl is selected from O, S, N, P or Si;

R₃and R₄Independently selected from hydrogen, or R₃And R₄Independently selected from substituted or unsubstituted: c₁～C₆Alkyl radical, C₁～C₆A heteroalkyl group;

W₁and W₂Independently selected from H or a protecting group.

Alternatively, the protecting group may be selected from hydroxyl protecting groups conventional in the art, such as benzyl (Bn), 4' -dimethoxytrityl (DMTr), silicon protecting groups (e.g., t-butyldiphenylsilane, etc.), and the like.

The modified nucleoside provided by the invention is modified by introducing a nitrile group (-CN) at the site of bridge ring C6' to obtain the modified nucleoside with a novel structure, and can effectively enhance the tolerance of the modified nucleoside to nuclease.

After a polar group CN is introduced to the 6' position, the steric hindrance of a methylene bridge in a nucleoside structure can be further increased, and higher nuclease tolerance is facilitated to be obtained; the hydration of the polar group CN is stronger, the antisense activity and the tissue distribution can be further improved, more structures are provided, and the optimization space of modification is enlarged; the introduction of the polar group CN can greatly improve the cutting specificity of RNaseH enzyme on target RNA; meanwhile, the length of the antisense gapmer sequence is shortened, and the binding specificity to the target RNA is greatly improved, so that the off-target effect of antisense nucleic acid is greatly reduced, and the hepatotoxicity is reduced.

Alternatively, the R is₁Selected from substituted or unsubstituted: purine, pyrimidine orTheir respective salts.

Alternatively, the R is₁Selected from substituted or unsubstituted: adenine (a), guanine (G), thymine (T), cytosine (C), uracil (U), or their respective salts.

Alternatively, when said R is₁When selected from substituted cytosines, said R₁Is 5' -alkyl substituted cytosine.

Alternatively, the R is₁Is 5 '-methyl-substituted cytosine (5' -mC).

Alternatively, when R₁、R₂、R₃And R₄When substituted, the substituents may be independently selected from halogen, alkyl, heteroalkyl, alkenyl, alkynyl, hydroxy, mercapto, amino, amide, carbonyl, carboxyl, sulfonic, ester groups, and the like.

Alternatively, the R is₂Is hydrogen.

Alternatively, the R is₃And R₄Independently selected from hydrogen.

On the basis of any of the above nucleosides, the present invention also provides a nucleotide including a 3' -phosphoramidite derivative of any of the above modified nucleosides or a salt thereof.

Alternatively, the nucleotide is selected from a compound having a structure shown in formula (II), a salt thereof or an isomer thereof:

optionally, W in the formula (II)₂Is 4, 4' -dimethoxytrityl (DMTr).

Modified oligonucleotides can be obtained by incorporating the above nucleotides into oligonucleotide sequences using standard synthetic methods.

As an embodiment, the modified oligonucleotide is obtained by inserting the above-mentioned nucleotide into an oligonucleotide sequence by an automated synthesizer using a standard phosphoramidite method.

According to another aspect of the present invention, there is also provided a nucleic acid polymer.

The nucleic acid polymer comprises at least one nucleotide structure as described above.

Alternatively, the nucleic acid polymer contains a condensation product structure of the above-mentioned nucleotide.

Optionally, the nucleic acid polymer comprises ribonucleic acid, deoxyribonucleic acid, or a copolymer of ribonucleotides and deoxyribonucleotides.

The invention also provides a preparation method of the nucleoside. When R is₃And R₄Independently of one another is hydrogen, R₁In the case of thymine, the method comprises:

reacting the reaction substrate (I) with thymine in the presence of a protective agent to produce a compound (II);

in the presence of a catalyst A, carrying out intramolecular cyclization reaction on the compound (II) to generate a compound (III); protecting group W of Compound (III)₂' removing and oxidizing in the presence of an oxidizing agent to form compound (IV);

in the presence of a catalyst B, carrying out nucleophilic substitution reaction on the compound (IV) and a nitrile source to obtain a compound (V) and/or an isomer thereof;

converting compound (V) and/or its isomer into compound (VI) and/or its isomer under basic conditions;

wherein the structural formula of each compound is shown as the following formula, W₁、W₂And W₂' is hydrogen or a protecting group:

optionally, the protecting reagent is N, O-bis (trimethylsilyl) acetamide (BSA).

Alternatively, the catalyst A is selected from 4-Dimethylaminopyridine (DMAP) and/or trifluoromethanesulfonic anhydride (Tf)₂O)。

Optionally, the oxidizing agent is selected from at least one of 2-iodoxybenzoic acid, dess-martin reagent, dimethyl sulfoxide-oxalyl chloride, preferably 2-iodoxybenzoic acid.

Optionally, the catalyst B is selected from AlCl₃、CeCl₃、ZnCl₂、TiCl₄Preferably AlCl₃。

Optionally, the nitrile source is selected from at least one of Trimethylnitrilosilane (TMSCN), sodium cyanide, potassium ferricyanate, preferably trimethylnitrilosilane.

The invention also provides the application of the nucleic acid polymer in preparing nucleic acid diagnostic agents and/or nucleic acid therapeutic agents.

Compared with the prior art, the invention has the beneficial effects that:

according to the modified nucleoside provided by the invention, a novel 6'-CN modified structure is obtained by introducing-CN at the position of C6', and a nucleotide and a nucleic acid polymer are obtained by further modification, so that the ribozyme tolerance is greatly improved, the affinity to target RNA is high, the modified nucleoside has high RNA selectivity, good antisense property is shown, and the hepatotoxicity is low.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the results of the hydrolysis tolerance of Snake Venom Phosphodiesterase (SVPDE) comprising a sequence having a nitrile group at the 6' position substituted for the nucleotide structure and other alignment sequences in accordance with one embodiment of the present invention;

FIG. 2 shows the mass spectrum of the nucleotide sequence ON1 according to one embodiment of the present invention;

FIG. 3 shows the mass spectrum of the nucleotide sequence ON2 according to one embodiment of the present invention;

FIG. 4 shows the result of mass spectrometry of nucleotide sequence ON3 according to one embodiment of the present invention;

FIG. 5 shows the mass spectrum of the nucleotide sequence ON4 according to one embodiment of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

As used herein, the term "C₁～C₆"refers to a group having any integer value of carbon atoms in the backbone ranging from 1 to 6, for example, 1, 2, 3, 4,5, 6 carbon atoms. Similarly, the term "C₂～C₆"refers to a group having any integer value of carbon atoms in the backbone ranging from 2 to 6, for example 2, 3, 4,5, 6 carbon atoms.

As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon group having a straight chain or a branched chain; non-limiting examples thereof include methyl, ethyl, propyl, n-butyl, t-butyl, pentyl, hexyl and the like.

As used herein, the term "alkenyl" refers to a hydrocarbyl group having at least one carbon-carbon double bond at one or more positions along the carbon chain of the alkyl group; non-limiting examples thereof include ethenyl, propenyl, butenyl and the like.

As used herein, the term "alkynyl" refers to a hydrocarbon group having at least one carbon-carbon triple bond at one or more positions along the carbon chain of the alkyl group; non-limiting examples thereof include ethynyl, propynyl, and the like.

As used herein, the term "aryl" refers to a group comprising a carbocyclic aromatic system; non-limiting examples thereof include phenyl, naphthyl, anthryl, phenanthryl, pyrenyl, and the like; when the aryl group includes a plurality of rings, the respective rings may be fused to each other.

As used herein, the term "heteroaryl" refers to a group having a carbocyclic aromatic system containing at least one heteroatom selected from N, O, Si, P and S as a ring-forming atom; non-limiting examples thereof include pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, quinolyl, isoquinolyl and the like; when the heteroaryl group includes a plurality of rings, the respective rings may be fused to each other.

As used herein, the terms "heteroalkyl," "heteroaryl" refer to an alkyl group containing at least one heteroatom selected from N, O, Si, P and S.

The term "salt" as used herein refers to a corresponding salt, e.g., a pharmaceutically acceptable salt, of a modified nucleoside compound (or nucleotide compound) of the present invention which can be conveniently or desirably prepared, purified and/or processed. Unless otherwise indicated, reference to a particular compound in the present invention also includes its salt form.

As used herein, the term "nucleic acid polymer" may refer to any nucleic acid molecule, including but not limited to DNA, RNA, and hybrids thereof, including but not limited to single stranded, double stranded, and the like. The number of nucleotides polymerized to form the nucleic acid is 2, 3 or more, and may be an oligonucleotide having a number of nucleotides of 20 or less, or a polymer having a number of nucleotides of 20 or more.

As used herein, the portion between the wavy lines in the nucleic acid polymer is a structure embedded in the nucleic acid polymer sequence, and the portion outside the wavy lines represents other sequences in the nucleic acid polymer.

In one embodiment, the modified nucleoside of the present invention is selected from a compound having a structure represented by the following formula:

wherein R is₁Selected from substituted or unsubstituted: purine, pyrimidineOr their respective salts; r₂、R₃And R₄Are all hydrogen.

In one embodiment, the modified nucleoside of the present invention is selected from a compound having a structure represented by the following formula:

wherein R is₁Selected from substituted or unsubstituted: a purine, pyrimidine or their respective salts; r₂、R₃And R₄Are all hydrogen.

As an embodiment, the nucleotide can be obtained by subjecting the hydroxyl group at the 3' -position or the protecting group structure to a phosphating treatment on the basis of the modified nucleoside in the present invention, and the nucleotide is selected from a compound having a structure represented by the following formula:

in one embodiment, the nucleic acid polymer is obtained by condensing a compound containing the nucleotide to obtain a condensate.

As an embodiment, a polymer or a sequence comprising a condensate of the above-mentioned nucleotide also falls within the scope of the nucleic acid polymer of the present invention.

In one embodiment, the structure of the nucleic acid polymer of the present invention includes a structure in which at least one nucleotide is modified by being incorporated into an oligonucleotide sequence.