TW200838541A - Novel nucleoside analogues and methods for preparing the same - Google Patents

Abstract

New pyrrolidine derivatives are disclosed having the formula wherein R1 and R2 are protecting groups independently selected from R-C(=O), wherein R is straight or branched C1-C15 alkyl, C6-C15 aryl, substituted C5-C15 aryl including from 1 to 5 heteroatoms and R3 is a nucleobase selected from thymine I, cytosine II, adenine III, guanine IV, uracile V, xanthine VI and hypoxanthine VII, a derivative of said nucleobases I-VII protected at the reactive functionalities NH2 or imidic NH, a deazacarbocyclic derivative of said nucleobases I-VII optionally protected at the reactive functionality NH2. These pyrrolidine derivatives are nucleoside isosteres and can be employed for the synthesis of DNA chimeras or as termination sequences for diagnostic and therapeutic applications. Also disclosed are novel intermediates useful for the preparation of the aforementioned pyrrolidine derivatives, as well as to methods for preparing both the intermediates and the final pyrrolidine derivatives.

Description

200838541 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to novel nucleoside analogs for the preparation of DNA products for diagnostic and therapeutic applications. The present invention also relates to a novel process for the preparation of the above-described nucleoside analogs, a process for preparing an intermediate product and the like, and a novel oligomer, a synthesis thereof, and an oligomeric treatment thereof. Application of diagnostic or therapeutic agents. [Prior Art] Nucleic acids as carriers or transmitters of genetic messages are of considerable importance in natural life. Therefore, since its discovery, it has promoted a wide range of scientific concerns that led to the clarification of its structure and mechanism of action. Molecular biology The gradual understanding of these basic mechanisms has led to the creation of new compounds in recent years. This technology opens up new opportunities for medical diagnostics and treatment as well as plants. ® Therefore, some of the research in this field is about the development of anti-pathogens or treatments based on the development of nuclear compounds that interfere with the metabolism of bacteria, viruses or cancer. " Nucleoside analogs as inhibitors of viral RNA polymerase are disclosed in a number of prior publications, such as International Patent Application No. 2006/002231, which discloses that aza nucleoside can be inhibited by RNA polymerase Agent, the viral RNA polymerase such as (but similar to the inter-product and the middle-energy, in this group of cancer nucleosides, the WO virus is not limited to 5,385,384,41) hepatitis B virus, hepatitis C virus, ridge千久白暂, (Polio) Coxsackie A plastic and ® disease, < glass% Qin, (Rhino) Yike virus (Ech〇), smallpox virus, Yimao Maqin and West Nile Virus (West Nile) polymerase, while A 5,246,93 1 reveals that carbocyclic nucleoside analogs can act as a scrambler 1 碥 sharp compound' which fights against herpesvirus type 1 舆3 disease resistance Qin (Hs\ HSV -2) Human immunodeficiency virus (HIV), type A 犴4 and specialized U) Pathogens of non-A, non-B hepatitis viruses. On the other hand, in the field of nucleoside analogues

Part B The development of compounds for the detection of nucleic acids, which is about the primary structure of the ', the sex and its sequence'. The specific detectability of a nucleic acid is based on the nucleic acid in which the molecular heterogeneity is interacted with or hybridized with other nucleic acids in the form of a gas-slit (ie, providing an indicator group). Oblique) J uses the nucleic acid (target) of the square. 'Complementary enough to implement the primary structure (sequence) 7 to the state, which is the premise of the application of specific and specific applications of nucleic acid nucleic acids in molecular problems and methods. Specific hybrids between nucleic acids are also utilized in the final sequencing. The labeled nucleic acid fragments are also used for the above. Examples of nucleoside analogs for detecting nucleic acids are disclosed in many prior publications, such as U.S. Patent No. 6, 丨74,998, which describes pyrrole-[3,2-pyrimidine, pyrazolium-[4,3-d The sequence of pyrimidine (pyraz〇1〇-[4,3-d]pyrimidine) and the heterocyclic test group. This sequence of information is next to 200838541 Cancer 咬 咬 糖 ( (also known as nucleic acid. C-nucleoside) modified by a signal group for the detection of nucleic acid nucleoside analogs is also disclosed in the British application GB 2 309 969, the description of which is based on ten small bases... Biha labeled nucleoside analogs for labeling nucleic acids or for incorporation of nucleotides.

In this novel and challenging technical field, Heavy & Eight strives to design more effective nucleoside analogs for therapeutic or diagnostic purposes. In this regard, some studies have invested in the discovery of novel nucleoside analogs based on pyrrolidine derivatives, which are considered to mimic naturally occurring DNA or RN A, because the presence of pyrrolidine rings can be used as a natural hydrazine. An isostere of a furan ring of a nucleic acid.

Thus, for example, (3i?, 4i?)-3-trans-base-4 used to prepare a nuclear wise as a purine nucleoside phosphorylase (PNP) inhibitor (a compound called BCX-4208) - Synthesis of pyrrolidine, disclosed in Kotian et al. (Org. proc. Res. 2005, 9, 193) and U.S. Patent Application No. 2006128789 i. .methyl Dev·, 7 , which applies benzylamine according to the following reaction pathway:

200838541 However, according to this method, it is necessary to use very expensive thiol sultam trimethyl decyl methoxy to synthesize (3 i? 5 4 i?) - 3 - peroxy-4-hydroxypyrrolidine. Trimethylsilylmethoxymethy Ibenzylamine ij contains difficulties.

Karlsson et al. (Tetrahedron: Asymmetry, 2001, 197 7) also published an asymmetric 1,3-dipolar cyclization addition reaction (3i?, 4i?)-3-hydroxy-4-hydroxymethylpyrrolidine This is accomplished by using palmphorsultam and (iS)-phenethylamine as a chirality inducer according to the following reaction: base, while amine is trapped 12, synthetic pathway Induced

However, the synthesis of (3i?, 4i?)-3-hydroxy-4-hydroxymethylpyridine according to this method also utilizes very expensive reagents such as the above-mentioned mercaptosulfonyltrienylmethoxycarbonyl group. Trimethylsilylmethoxymethylphenyethylamine, and the yield has not exceeded 50%. (3J?, 4i?)-3-hydroxy-4. hydroxypyridyl pyrrolidine can also be obtained from International Patent Application No. WO2005 03 3 076, which is shown by Tyler et al. 200838541 CAL Novozyme 435 completes the racemization and the enzyme analysis of -1 ·#·benzyl-4-transmethoxy-3-ethoxycarbonyl-pyrrolidine, followed by reduction and hydrogenolysis reaction products according to the following reaction pathway: trans

However, the synthesis of (3i?, 4i?)-3-hydroxy-4-hydroxymethylpyrrolidine according to this method must utilize the kinetic analysis of the enzyme, and it has been described that the yield is not more than 50%. Pure mirror phase (3i?, 4i?)-3-hydroxy-4-hydroxymethyl fluorenidine can also be obtained by means of Galeazzi et al. (Tetrahedron: Asymmetry, 2004, 15, 3249) by inclusion ( Reaction route of the reaction of S)·phenethylamine with ethyl 2-silyloxy-3-methoxycarbonyl-3-butenoate:

R3SiOv ^COOMe .....>

COOMe

0; X to produce 3,4· and -3-3⁄4yl-4-oxo-oxygen ϋ 嗣 (3,4-irflfn*s-3-hydroxy-4-alkoxycarbonylpyrrolidin_2-one) 9 200838541 The intermediate product pyrrolidine-2-one is then converted into (3i?,4i?)-3-hydroxy-4-hydroxymethylpyrrolidine by a cumbersome process according to the following reaction route, which comprises LAH and chlorine chloroformate. Chioroalkyl chioroformate: R3SiOw vCOOMe

RsSiO^ ^COOMe

HO^ >CH2OH

H

However, according to this method, the synthesis of (3 i?, 4 i?) - 3 - via benzyl-4 - fluorenyl sigma acridine must utilize both metal hydride and chloroethyl chloro decanoate, thereby hindering the preparation of large amounts of Target compound. SUMMARY OF THE INVENTION In one aspect, the present invention provides novel nucleoside analogs that are compatible with DNA synthesis reagents and devices, such that they are suitable for the preparation of DNA chimeras and are selective for simultaneous migration under mild conditions. In addition to the protective group. In another aspect, the invention provides suitably protected nucleoside analogs to increase the efficiency of nucleic acid oligomer synthesis. In still another aspect, the invention provides nucleoside analogs that are compatible with commercially available products and equipment (e.g., automated synthesizers). In still other aspects, the invention provides novel intermediates for the preparation of the above-described nucleoside analogs, as well as convenient high yield synthetic methods for the preparation of intermediates and nucleoside analogs. In still other aspects, the present invention provides modified novel oligomers capable of forming novel palmitic DNA chimeras, methods for their synthesis, their use in the treatment of oligomer 10 200838541, and their use as diagnostic reagents application. According to its first aspect, the invention relates to the formula r2o

a 1-mercapto-3-hydroxy-4-hydroxymethylpyrrolidine derivative of D, wherein

Ri and R2 are respectively selected from fluorene and RC (= 0), wherein a linear or branched alkyl group, an aryl group, a substituted aryl group including 1 to 5 hetero atoms, and 1 to 3 hetero atoms are included. The heterocyclic group and R3 are the nucleobase. Advantageously, the nucleoside analog of formula D according to the invention is a compound which can be incorporated (by enzyme or chemical means) a nucleic acid (DNA or RNA) chain, and which is capable of binding to a nucleoside in the complementary strand The acid residues form base pairs or bases stacked in the appropriate nucleic acid strand. In a preferred embodiment, the 1-mercapto-3-trans-yl-4-methylpyrrolidine derivative of the present invention has the same electrons as the C-3 and C-4 carbon atoms of a naturally occurring deoxyribose ring. Isosteric Stereogenic Structure (3R, 4R) The nucleoside analogs of the invention can be advantageously used in both therapeutic and diagnostic methods, as will be described in more detail below. Further advantageously, the above-described nucleoside analogs of the present invention include sterically hindered-N-(C=0)-CH2- linkages to nucleobases in their structure, which are effectively enhanced thereby The resistance of the oligomer to the naturally occurring nuclease 11 200838541 with peptidase. Further advantageously, the nucleoside analogs of the invention are capable of approximately mimicking the nucleoside structure of naturally occurring RNA or DNA due to the above-described steric hindrance -1^-(€ = 0)-€112- The bond is linked to the presence of a pyrrolidine ring which constitutes the same electron alignment of the ribose or deoxyribose ring.

When the 1-indolyl-3-hydroxy-4-hydroxydecylpyrrolidinide derivative of the present invention is in its double-protected form, that is, when R! and R2 are RC (= 0), the modified nucleoside is similar. , which achieves both the appropriate protection of the 3 hydroxy and 4-hydroxy thiol functional groups and their subsequent selective removal of protection under mild conditions. The mild conditions used herein mean that the reaction is carried out at a temperature ranging from 0 ° C to 40 ° C and that there is no strong base which affects the stereogenic center structure on C-3 and C-4 or the cut ring. In a preferred embodiment, the groups R: and R2 are protective groups wherein the R-lined straight or branched Ci-C15 alkyl group, the C6-C15 aryl group, including 1 to 5 heteroatoms are substituted A C5-C15 aryl group, a C3-C5 heterocyclic group comprising from 1 to 3 heteroatoms. Further, in this method and in the following, the 1-mercapto-3-trans-yl-4-thiol-based derivative of the present invention can easily protect its 3-hydroxyl group and/or 4 during its synthesis. - hydroxymethyl functional groups, and at the same time at the most appropriate time during or after oligomer synthesis, under mild conditions, the protection of one or both functional groups is easily removed. In a preferred embodiment, R is selected from the group consisting of methyl, ethyl, propyl, isopropyl 12 200838541, t-butyl, 2-methylbutyl, allyl '3,3-dimethylene A linear or branched CpCu alkyl group of a propyl group, a 3-methylbutyl group, a 3-methyl-2-butenyl group, an octyl group or a fluorenyl group.

In this method, the 1-mercapto-3-hydroxy-4-hydroxymethylpyrrolidine derivative of the present invention can remove the protection of 3-hydroxyl and/or 4-hydroxydecyl functional groups under specific mild conditions, for example, An ion exchange resin in the form of a carbonate ion in methanol or a hydroxide in methanol without affecting other functional groups or affecting the stereoisomer center at 20QC. In another preferred embodiment, R is a substituted C5-C15 aryl group of 1 to 5 hetero atoms or a C3-C5 heterocyclic group including 1 to 3 hetero atoms, and the hetero atom is selected from oxygen. , atmosphere, sulfur, fluorine, chlorine, bromine or moth. The presence of an aryl group can be advantageous to obtain a more lipophilic product, so that the solubility in the organic solvent is increased and the purification is simpler. In another preferred embodiment, R is selected from the group consisting of C6H5, CH3C6H4, 2-furazyl, 3-indolyl, 4-oxooxyphenyl, 4-nitrophenyl, 4-chlorophenyl , 4-bromophenyl, 4·iodophenyl, 4-trifluoromethyl, 2,6-diphenylene, • 2,6-dioxaphenyl, 2,4-dinitrophenyl, 2 , 4-diphenyl, 2,4-dibromophenyl, 2,4-diiodophenyl, 2,4-dimethoxyphenyl aryl or heterocyclic group. These preferred aryl or heterocyclic groups may advantageously further increase the lipophilicity of the bite derivative and its solubility in an organic solvent to make its purification easier. According to the present invention, all of the above-mentioned advantages achieved by the novel nucleoside analogs can be achieved when R3 is a naturally occurring nucleobase and when R3 is a derivative thereof.

Thymus gland I, cell bit II, adenine III, bird 吟 IV, urinary vaginal V, jaundice VI and hypoxanthine VII, the above nucleobase I-VII reaction functional group ΝΗ 2 or An imidazole-protected derivative, the above-mentioned nucleobase I-VII reactive functional group NH2 is selectively protected by a deazacarbocyclic derivative.

In another preferred embodiment of the present invention, R3 may be a reactive functional group NH2 of nucleobase I-VII or a protected amine of NH imine NH or a reactive functional group NH2 of nucleobase I-VII described above may be selectively subjected to A protected azacarbon ring derivative wherein the above reactive functional group NH2 or imine NH is protected by a protective group P which is independently selected from the group consisting of a benzyl group, a benzinium group, 2,4-Dimethoxybenzyl, diphenylindenyl, allyl, 4-methoxybenzyl (PMB), 4-methoxybenzyloxycarbonyl, 4-methoxybenzyl, 4-nitrate Benzyl fluorenyl, 4-fluorobenzyl hydrazino, 4-bromobenzylidene, 4-iodobenzyl, phthalenyl, 14 200838541 methyl thiol, ethyl hydrazino, propyl fluorenyl, trimethyl acetyl Pi (piVal F al〇yi) v carbonyloxycarbonyl (Cbz), t-butoxy-based-benzhydryloxycarbonyl (Bhc), adamantyloxycarbonyl, ketone methoxycarbonyl ( Fluoryl methyloxy carbonyl, F mo c), allyloxycarbonyl (Alloc) carbonyl carbonyl nucleobase reactive functional group protection can be advantageously limited W & Γ nervous reduce

The side reaction of the final production. Furthermore, the use of a protective group can distort the oleophilicity of the same core, thus making it easier to separate the excitation (w 〇 r k - u ρ ) from the intermediate traits. Wherein a nucleobase, in a preferred embodiment of the invention, R3 is a hip hop derivative derivative derivative selected from the group consisting of:

among them? 1 is selected from the group consisting of benzyloxycarbonyl (cbz), 4-methoxybenzyloxycarbonyl, dibenzomethoxycarbonyl (Bhc), fluorenylmethoxycarbonyl (Fmoe), allyloxycarbonyl (AUoc), adamantane The protective group of oxycarbonyl and P2 is selected from the group consisting of a protective group of a benzyl group, a 4-oxobenzyl group (PMB), a 2,4-dioxaxybenzyl group, a benzhydryl group, and an allyl group. Also in this example, the protection of the reactive functional groups of the nucleobase derivative 15 200838541 can advantageously limit the side reactions which may reduce the final yield and increase the lipophilicity of the nucleobase, thus making the separation of the excitation and intermediate products easier. . In a preferred embodiment of the invention, r3 is a deazacarbacyclic derivative protected by the above nucleobase, which is selected from the group consisting of:

XII

XIV wherein P! is selected from the group consisting of benzyloxycarbonyl (Cbz), 4-methoxybenzyloxycarbonyl, dibenzyloxycarbonyl (Bhc), fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), A protective group of an adamantyloxycarbonyl group and P2 is a protective group selected from the group consisting of a benzyl group, a 4-oxobenzyl group (PMB), a 2,4-dimethoxybenzyl group, a diphenylmethyl group, and an allyl group. .

Also in this example, the protection of the reactive functional groups of the nucleobase derivative can advantageously limit side reactions that may reduce the final yield and increase the lipophilicity of the nucleobase, thus making the separation of the excitation and intermediate products easier. According to another aspect thereof, the present invention relates to

Ri〇-^\ /—FU 'R2〇

A-mercapto-3 of A is thiol-derivative derivative, 16 200838541 wherein R! and R2 are defined above and R4 is a leaving group. Most advantageously, the pyrrolidine derivatives of the above formula A of the present invention are directly obtained and are useful intermediates which can be used to prepare the nucleoside analogs of formula D of the present invention by causing N-mercapto groups. The method of the derivative is achieved and described in more detail later.

In a preferred embodiment, the 1-indolyl-3-hydroxy-4-hydroxymethylpyrrolidine derivative of formula A according to the present invention has a C-3 and C-4 carbon with a naturally occurring deoxyribose ring. The stereoisomeric structure (3R, 4R) of atoms arranged in the same electron. In a preferred embodiment, R and R2 may be preferred groups as defined above. In a preferred embodiment, R4 is a leaving group selected from C1, Βι*, I or OS02-Y, wherein Y is selected from the group consisting of methyl, isopropyl, phenyl, phenyl, 4-nitrobenzene. Base, 2,4-dinitrophenyl, 4-chlorophenyl, 4-bromophenyl and trifluoromethyl. The presence of both halogen and thiol-based derivatives advantageously allows the nucleophilic substitution reaction of the nuclear assay to proceed smoothly. According to another aspect thereof, the present invention relates to

1-alkyl-3-hydroxy '4-methylpyrrole derivative, wherein R! and R2 are defined above but R2 is not CH3, R5 is linear or branched alkane 17 200838541 and R6 is straight A chain or branched alkyl group, an aryl group or a substituted aryl group including 1 to 5 hetero atoms. In a preferred embodiment, the alkyl-3-hydroxy-4-hydroxymethylpyrrolidine derivative of formula B according to the present invention has a C-3 and C-4 carbon atom with a naturally occurring deoxyribose ring. Stereoisomeric structure (3R, 4R) arranged in the same electron.

Most advantageously, the formula B-1 • alkyl-3-trans-yl-4-methyl-haha bite derivative is a useful intermediate which can prepare 1-mercapto-3-hydroxy- of the above formula A The 4-hydroxymethylpyrrolidine intermediate product, the resulting intermediate product can then be used to prepare a nucleoside analog of formula D of the present invention. Further, the 1-alkyl-3-hydroxy-4. hydroxymethylpyrrolidine derivative of the above formula B in a double-protected form can be used to prepare the above-mentioned formula A of the hydrazin-3-hydroxy-4-hydroxyl group The pyrrolidine intermediate, which is described in more detail below by the method of inserting a thiol group into N-1. In a preferred embodiment, Ri and R2 may be preferred groups as defined above. In a preferred embodiment, in the 1·alkyl-3-hydroxy-4-hydroxydecylpyrrole derivative of formula B according to the present invention, the groups R5 and R6 are linear or branched C^-Cs, respectively. Alkyl groups, which contribute to the improvement of the lipophilicity of the product, making its purification easier. More preferably, R5 and R6 are each a linear or branched alkyl group selected from the group consisting of methyl, ethyl, propyl, isopropyl, t-butyl, 2-methylbutyl, Allyl, 3,3-didecylallyl, 3-nonylbutyl, 3-methyl-2-butanyl, octyl. In a preferred embodiment, the 1-alkyl-3-hydroxyl group of formula B according to the present invention is 18

200838541 In the 4-hydroxyindole derivative, the base ring R6 may be a C6-C8 aromatic to 5 hetero atom substituted C5-C8 aryl group, which is advantageous for lipophilicity and solubility in an organic solvent such that #易. In this regard, it is noted that the presence of an aryl group such as R6 can advantageously be accomplished by the ease of insertion of an acid group into N-1, effective, safe and low cost deuteration treatment, as will be described. More preferably, R6 is an aryl group or a substituted aromatic system S from C6H5, CH3C6H4, 4-methoxyphenyl, 4-nitrophenyl, 4-bromophenyl, 4-iodophenyl, 4- Trifluoromethylphenyl, 2,6 2,6-dimethoxyphenyl, 2,4.dinitrophenyl, 2,4-dichlorophenylphenyl, 2,4-diiodophenyl, 2 , 4-dioxaphenyl. According to a preferred aspect of the invention, all of the C-1' atoms defined above are stereogenic centers. In this method, a non-mirrored rapid separation of the alkyl-3-trans-yl-4-methyl-pyroline derivative of the formula G (hereinafter referred to) can be achieved, for example by conventional chromatographic techniques or fractions. The specific embodiment 3 of the section is described in more detail. Thanks to this feature, it is advantageous to carry out the preferred embodiment of the process for the intermediate product and to produce the most end product, all of which are pure to avoid the need for low yield, complex and time consuming racemate resolution steps to occur in organic synthesis techniques. in. The base, including 1 to enhance the electroforming of the product is more porous or substituted. This is based on a more detailed group, the base, 4-chlorobenzene-dimethylphenyl, ., 2,4-dibromo Derivatization of the double-protected 1-isomeric form of the crystal will be mirrored by all of the following inventions, whereby it is generally 19 200838541 according to another cancer sample thereof. The present invention relates to the inclusion of a plurality of nueleo monomers. The oligomer, wherein at least one of the above nuclear monomers is:

Wherein the X and Z lines are each selected from the group consisting of 〇 and s and the r3 line is defined by the nucleobase. Most advantageously, model studies have shown that the polymer of the present invention has a stereoisomeric structure (3R, 4R) aligned with the C-3 and C-4 carbon atoms of a naturally occurring deoxyribose ring, and is oriented Arranged by a binding competent conformation that improves the binding properties of the target nucleic acid.

As used herein, a binding competent construct means the spatial orientation of a heterocyclic tester required for the sequence-specific binding of a duplex or single strand of DNA or RNA in an oligomer. Thus, this feature makes the oligomers of the invention suitable for both therapeutic and diagnostic methods, as will be described in more detail below. The core monomer of the present invention is generally characterized by a group or a residue in which a homofuranose ring (i.e., a pyrrolidine ring) of a furanose ring is substituted for a furanose ring (generally found in a nucleotide). The discovery of these nuclear monomers and their properties is based on the model study 2008 200838, which (1) predicts that the above analogs are compatible with the binding of the competent oligomers and (2) defines the molecular characteristics when incorporated into the oligomers. It can be assumed that the above-mentioned nuclear precursor has not lost the binding ability. As used herein, the binding ability means pairing with Watson_Crick bases of single-stranded DNA or single-stranded RNA or Hoogsteen pairing with double-stranded nucleic acids (including double-stranded DNA or double-stranded RNA). °

Incorporation of the nucleomonomers described herein into oligomers may allow for the synthesis of improved compounds with respect to, for example, (i) enhanced lipophilicity by excluding the charge associated with phosphodiester linkages and (Π) pairs. Resistance to decomposition of enzymes such as nucleases and peptidases. Thus, oligomers containing these nucleoside analogs are quite suitable for hybridization to a target sequence or molecule. The invention provides a range of core monomers that can be incorporated into a competing oligomer. The oligomer of the present invention can be decomposed by nucleases and is stable under physiological conditions. Nuclease stability is an important consideration for the development of a polymer that is expected to act as a therapeutic agent by binding to specific DNA or RNA (mRNA, hnRNA, etc.) sequences. The combination of the above specific target sequences constitutes its therapeutic efficacy by interfering with the normal biological function of the nucleic acid sequence associated with pathological symptoms. According to the present invention, oligomers of any length, including l-mers (10-nuclear monomers), 20-mers, 5 〇-mers '1 00 mers, or longer oligomers, can be easily Produced by the nucleoside analog utilization technique of the present invention and the solid phase or liquid phase synthesis method known in the description, for example, Khydatov, YE; 21 200838541

Fields, H. A, Eds· Artificial DNA: methods and applications^ CRC Press, New York, 2002 Herdewijn, P. Oligonucleotide synthesis: methods and applications, Humana Press, New York, 2005 0 Contains 2 to 30 nucleomonomers Oligomers are preferred. In general, the oligomer of the present invention is preferably synthesized by a solid phase method in which a core monomer is sequentially added to a first monomer bonded to a solid support according to procedures and equipment known to those skilled in the art. unit. In an additional aspect thereof, the 1-formyl-3-hydroxy-3-hydroxyindole oxacyclidine derivative of the above formula D can be prepared by a stereoselective multi-step process, which must prepare the above formulas A and B. The intermediate of the new double protection. Therefore, in its additional aspect, the present invention relates to a preparation formula

A method of double-protected 1-alkyl-3-hydroxy-4-hydroxymethylpyrrolidine, wherein the R2 is independently selected from the group consisting of R_c(=〇), wherein r is a linear or branched alkane a aryl group, a substituted aryl group having 1 to 5 hetero atoms, a heterocyclic group including 1 to 3 hetero atoms; R5 is a linear or branched alkyl group: or a linear or branched alkyl group An aryl group or a substituted aryl group comprising 1 to 5 heteroatoms 22 200838541 which comprises the following steps: a)

M Q

The diprotected 1·alkyl-3-trialkylphosphonium-4-alkoxycarbonylpyrrolidin-2-one derivative is reacted with a reducing reagent capable of releasing a hydride ion (hydridei ο η), wherein Μ is SiR9RI0 And R7, R8, R9 and Rio are linear or branched alkyl groups, and R5 and R6 are defined above, to obtain

a 1-alkyl-3-hydroxy-4-hydroxymethylpyrrolidine derivative wherein R5 and R6 are defined above, b) a 1-bromo-3-light group of the formula obtained in the step a) -4-The fluorenyl sigma-pyridinium derivative is reacted with an anhydride of the formula (R6C0)20 or a fluorenyl derivative of the formula R6COZ wherein R6 is defined above and Z is hydrogen. This method facilitates the direct removal of both the lactam carbonyl and the oxycarbonyl group on C-4, as well as the protective group on C-3 23 200838541 (eg, 3_3 - butyl) Methyl methoxy group). The double protection of formula G · alkyl = ^ ^ ^ ^, - alkyl decyloxy - 4-alkoxycarbonyl decylamine is better than 'Yu (9) non-image isomer. Preferred implementation of this method In the case of 'R, a preferred group.

In a preferred embodiment of the method, 'R7 is selected from a linear or branched Ci_C8 alkyl group, preferably selected from the group consisting of ethyl, propyl, secondary-propyl, and butyl, and most preferably is butyl' to allow The chromatographic separation of the mirror image isomers is preferred. In a preferred embodiment of the method, R" is selected from a linear or branched Cl_C8 alkyl group, preferably a methyl group, to allow for better separation of the non-imagewise mixture. In a preferred embodiment of the method, R9 and R! are respectively selected from a straight bond or a branched c"c8 alkyl group, preferably selected from the group consisting of methyl, ethyl propyl, bis-propyl t-butyl I' and most preferably a fluorenyl group, so that A preferred chromatographic separation of the non-image isomers is permitted. The reducing agent used in step a) is preferably selected from the group consisting of bh3, LiAlH4, LiBH4, Bf3. triethyldecane, a1H3. Advantageously, the internal carbonyl group is advantageously removed to produce a pyrrolidine ring, and the alkoxycarbonyl group is converted into a ruthenium group and a trialkyl methoxy group by a simple and unambiguous reaction reaction. To provide a free hydroxyl group on C-3. — _ In the example of the filial father, 'the above reaction is carried out in an organic solvent capable of dissolving the reducing agent' and it is preferably selected from the group consisting of cyclic or dialkyl ethers, cyclic 24 200838541 or linear alkanes or a mixture of the above. In a preferred embodiment, the above reaction is carried out in hydrazine. 〇 to 9〇. The temperature of (: is compatible with the reducing agent. The above reaction is more preferably carried out at a temperature of from 20 ° C to 90 ° C, still more preferably 6 〇. (: to 80 ° C. In general, step a The reaction time is a function of the selected reaction temperature and is between 1 hour and 1.5 hours, and the yield of the reaction is preferably distributed between 60% and 70%.

Under these conditions, a high yield product can be advantageously obtained directly by one-pot treatment, which involves removal of both the indole amine carbonyl and the alkoxycarbonyl group on C-4, and the protection on C-3. a group (for example, 3-tris-butyldimethylmethoxy). The method of preparing a 1-alkyl-3-hydroxy-4-hydroxymethylpyrrolidine intermediate of the formula 较佳 preferably also includes one or more additional steps which are suitably prepared according to methods known to those skilled in the art. The treatment reagents and/or reaction products are preferably removed from any unwanted by-products such as those produced by the reducing agent. Thus, for example, when the reducing agent is ΒΗ3 or BF3-triethyl decane, the reaction product is first treated in one or more steps with an acid (eg, HCl) in an organic solvent (eg, decyl alcohol) so that Performing a transesterification reaction of a boron complex species formed on a 3-amino functional group, followed by treatment with a base (eg, NaOH) in a solvent (eg, decyl alcohol) to remove the anion of the acid and form a free form 3-hydroxy functional group. On the other hand, when the reducing agent is, for example, LiAlH4, LiBH4 or A1H3, firstly, in one or more steps, an organic solvent (for example, ethyl acetate) 25 200838541 is formed to form an A1 complex salt, and a salt (for example, tartaric acid) is formed. The acid (eg, HCl) treatment reaction product of Nazhong is treated with an organic weak acid or tartaric acid in a solvent (for example, water) to form two treatments of the homogenous reaction product. For example, ethyl acetate or dichlorohydrazine) the extraction & machine steps and appropriate reagents (for example,

The dry @ step performed by Na2S04 or MgS04) to obtain a pure product. This method is the above step b) Gabriel easily protects C · 3 and

The hydroxyl group and the hydroxymethyl group on C-4 thus avoid side reactions in further steps of the synthesis process. In a preferred embodiment of the method for preparing the double protection of formula B, ρ夂/ / each bite derivative, for use in step b); < thiol derivative r〇coz or •0-(C = 0) The halogen z of -R7-C0Z is F, C1 or . Further, in a preferred embodiment of the method for preparing a double-protected pyrrolidine derivative of the formula, the fluorenyl derivative 〇-((:: = 0 υ〇ζ υ〇ζ group R7 is a linear c4-c8 In a preferred embodiment of the method for preparing the double-protected pyrrolidine derivative of formula B, the above reaction step b) is carried out in a polar or non-polar solvent. The solvent is preferably selected from the group consisting of ethers and aromatic hydrocarbons. a class, a chlorinated hydrocarbon, dimethylformamide (DMF), dimethyl hydrazine (DMS0) or a mixture of the foregoing, which provides a clear solution thereby increasing the rate of reaction. In a preferred embodiment of the method of pyrrolidine derivative, the reaction step b) is carried out in a tertiary amine having a molecular weight equivalent to the above-mentioned anhydride (r〇c〇) 2〇 or the above-mentioned 26 200838541 mercapto derivative ROCOZ Execution in the presence of (R')3-N, wherein R' represents the same or different straight or branched C2_C8 alkyl group; or in the presence of a tertiary cyclic amine R'(R'')2-N, Wherein R'' is -(CH2)n- and η = 4-6 〇, most advantageously, the above-mentioned tertiary amines neutralize the acid formed by the reaction. Thereby increasing its output by up to about 90%.

In a preferred embodiment, the tertiary amine (R') 3-N is selected from the group consisting of triethylamine, tributylamine, diisopropylethylamine, triisopropylamine, and R'(R'')2-N It is selected from N-ethylpyrrolidine and N-ethylpiperidine, the salts of which are soluble in organic solvents; in addition, unreacted amines can be easily removed under reduced pressure due to low boiling point . In a preferred embodiment of the process for the preparation of the double protected pyrrolidine derivative of formula B, reaction step b) is carried out at a temperature between -10 ° C and 60 ° C, more preferably between 0 ° C and 40 ° C. The temperature between, and more preferably between 0 ° C and 20 ° C. In this process, an improved solubility product can be obtained which advantageously reduces or avoids separation problems. In general, the reaction time of step b) is a function of the selected reaction temperature and is between 0.5 hours and 2 hours, and the yield of the reaction is preferably distributed between 85% and 90%. In accordance with an additional aspect thereof and in the framework of the stereoselective multi-step process of the above-described 1-indolyl-3.hydroxy-4-hydroxymethylpyrrolidine derivative of formula D, the present invention relates to the preparation of formula 27 200838541

Ri

a double protected 1-mercapto-3-hydroxy-4-hydroxymethylpyrrolidine derivative of R4, wherein

Ri and R2 are each a protective group selected from R-C(=0), wherein R is defined above and R4 is a leaving group' which comprises the following steps:

Double protected 1·alkyl-3-hydroxy-4-hydroxydecyl pyrrolidine of B, wherein

Ri and R2 are defined as above; R5 is a linear or branched alkyl group and R6 is a linear or branched alkyl group, an aryl group or a substituted aryl group having 1 to 5 hetero atoms; and the formula 〇LA/R4 The mercapto halide reaction of c, wherein L is a halogen atom and R4 is defined above. 28 200838541 This method advantageously advantageously cleaves the N-1 of the pyrrolidine ring to avoid the preparation of the 3-methoxy-4-methoxymethylpyrrolidine derivative, which is difficult to decompose and provides a product of decomposition. Complex mixture. In a preferred embodiment of this process, R, R5, R6 and the leaving group R4 are preferred groups as defined above. In a preferred embodiment of this method, L is a halogen atom selected from the group consisting of F, C1 or Br. In particular, Br obtains a better yield of substituted product.

In a preferred embodiment, the above-described deuteration step is carried out in an organic solvent. The solvent is preferably selected from the group consisting of dichloromethane, chloroform, THF, diisopropyl ether, diisobutyl ether, dioxane, acetonitrile, dimethylguanamine or a mixture thereof. The preferred solvent is more preferably dichlorosilane, which provides a clear solution of the reaction mixture, which readily dissolves the two reaction products and thus increases the reaction rate. 0 In the preferred embodiment, the above reaction is carried out at 〇 ° C. The temperature between 50 ° C is preferably 0. (: to 40. (: between the temperature, and more preferably between 15 ° C and 25 ° C. In this method, the reaction rate is maintained at the optimum value while avoiding temperatures above 50 ° C An undesired side reaction that causes partial cleavage of the protective group on C-3 and C-4. Generally, the reaction time is a function of the selected reaction temperature and is between 6 hours and 12 hours. The yield of the reaction is preferably distributed between 50% and 72%. 29 200838541 According to one of the additional aspects, the invention relates to a preparation

Method for the double protection of 1 - mercapto-3 -hydroxy-4-hydroxymethylpyrrolidine derivative of D, wherein

R! and R2 are each a protective group selected from R-C(=0), wherein the R system is defined above and R3 is a nuclear test group, which comprises the following steps:

A

The double-protected 1 - mercapto-3 - thiol-4 - nucleobase reaction in a methyl quinone derivative with a non-aqueous solution (including a base capable of forming an anion of the above nucleobase R3), wherein 1^ and R2 are defined above and R4 is a leaving group. This process can advantageously be operated under mild conditions and a high yield of the final product can be obtained. In a preferred embodiment of the method, the base capable of forming an anion of the nucleobase R3 is selected from the group consisting of a base having a PKb of from 4 to 9, a lithium amine or a metal hydride. In the framework of the preferred embodiment, the above-mentioned bases are preferably selected from the group consisting of 30 200838541

LiiCOs, Na2C〇3' K2CO3, CS2CO3 or a mixture of the above. Furthermore, in the framework of the preferred embodiment, the lithium amine is preferably selected from the group consisting of lithium bis(t rim ethyl silyl), amide, LiHDMS, diisopropylamine. (LDA) or a mixture of the above. Finally, in the framework of the preferred embodiment, the metal hydride described above is preferably selected from the group consisting of NaH, KH or a mixture thereof.

In a preferred embodiment of this method, 'R1, R2, the nucleoside R 3 and the leaving group R4 may be preferred groups defined above. In a preferred embodiment, the above reaction step is carried out in an organic solvent. Preferably, the solvent is selected from the group consisting of DMF, DMSO, N.methylpyrrolidone, HMPA or a mixture of the foregoing. In a preferred embodiment, the reaction step is carried out at a temperature between 0 ° C and 10 ° C, preferably 0. (: a temperature between 60 ° C, and more preferably between i5 〇 c and 20 ° C. In this method, the reaction rate is maintained at an optimum value while strongly suppressing side reactions such as hydroxyl groups and Random cleavage of the protective group on both hydroxymethyl functional groups. In general, the reaction time is a function of the selected reaction temperature and is between 2 hours and 24 hours, and the yield of the reaction is preferred. Between 48% and 70%. According to one of the additional aspects, the invention relates to the preparation of the formula 31 200838541 HO-\ yT-'R4 Μ

A method of a fluorenyl derivative of HO Ε 1 · fluorenyl-3 -transyl-4-yl-methyl-pyrene-derivative, wherein R 4 is a leaving group, which comprises the following steps:

R2o

The double-protected 1-indolyl-3-hydroxy-4-hydroxydecylpyrrolidine derivative of A is equal to or less than the pKa in the presence of a carbonate ion or a polymer comprising a supporting 〇 or a CO group 1 8 one-stage alcohol reaction, wherein

R! and R2 are each a protective group selected from R-C(=0), wherein R is defined above and R4 is a leaving group. This method advantageously facilitates the cleavage of the Ri and R2 protecting groups to produce a deprotected 1-indolyl-3-hydroxy-4-yl-purine-based derivative of the formula E in a simple and suitable manner. According to one of its additional aspects, the invention relates to the preparation of the formula 32 200838541

A method of a 1-mercapto-3-hydroxy-4. hydroxypyridyl pyrrolidine derivative, wherein R3 is a nucleobase, which comprises the following steps:

The double-protected 1-indolyl-3-hydroxy-4-hydroxydecyl pyrrolidine derivative is present in the presence of a carbonate ion or a polymer comprising a supporting hydrazine or a C Of group with a pKa equal to or less than 18 A primary alcohol reaction wherein R! and R2 are each a protecting group selected from RC (=0), wherein R is defined above and R3 is a nuclear group. According to a preferred embodiment of the present invention, the selective removal protection step of the 3-hydroxy and 4-hydroxyindenyl groups is carried out in two processes in a non-aqueous solution, preferably comprising a pKa equal to or less than the above. 1 8 alkaloid In this method, one of the solvent of the deprotection step can be advantageously carried out as a solvent function for the reaction species of the simplified reaction, and the solid reagent and the R! and R2 alkyl groups are simply filtered off. The esters are then advantageously obtained directly from the reaction 33 200838541 in the vehicle as a reaction product of an alcoholic solution. Further, in this preferred embodiment, the R! and R2 protecting groups remain dissolved in the primary alcohol (e.g., methanol) and can be recovered in a simple manner after filtration and removal of the solvent under reduced pressure. In a preferred embodiment of these two methods, Ri, R2, nucleobase R3 and leaving group R4 may be preferred groups defined above.

In a particularly preferred embodiment of the method, the primary alcohol is selected from the group consisting of linear or branched C!-C5 alcohols, benzyl alcohols or mixtures thereof, and more preferably selected from the group consisting of decyl alcohol, ethanol, and propanol. , 2-methylbutan-1-ol, 3-mercaptobutan-1-ol or a mixture of the above. In this method and the above-mentioned mention, a reaction product like an alcohol solution in the reaction medium can be obtained after simply filtering off the solid reagent and the R! alkyl ester. In a preferred embodiment of this method, the carbonate ion is derived from a carbonate selected from the group consisting of Li2C03, K2C03, Na2C03, Cs2C03 or a mixture of the foregoing. Further, the polymer comprising a supporting hydrazine or a C〇r_ group is preferably a macroreticular ion exchange resin selected from the group consisting of hydrazine or CO. In a preferred embodiment, the selective removal of the 3-hydroxyl and 4-hydroxymethyl groups is carried out at a temperature of from 〇C to 50. The temperature between the crucibles is preferably from 0 ° C to 4-0. The temperature between C is more preferably 1 5 . Temperature between C and 2 5 ° C. In this method, the reaction rate is maintained at the optimum value while avoiding the undesired side reaction which causes partial cracking of the 1-mercapto group which is bonded to the Ν-l nitrogen atom when the temperature exceeds 50 °C. In general, the reaction times of the above two methods are a function of the selected reaction temperature and are between 1 hour and 3 hours, and the reaction yield is preferably between 70% and 90%.

Further, in general, all of the above methods also include the implementation of the extraction and purification steps of the initial reagent and/or the desired end product, which are described in accordance with the procedures known to those skilled in the art and examples thereof will be described below. In the example. The present invention relates to a 1-mercapto-3-hydroxy-4-hydroxymethylpyrrolidine derivative of the formula D for use in a therapeutic, prophylactic or diagnostic method, according to additional aspects thereof and matters already mentioned above. The oligomers defined above will be described in more detail below. According to additional aspects thereof, the present invention also relates to a 1-mercapto-3-hydroxy-4-hydroxydecyl-pyrrolidine derivative of formula F comprising a pharmaceutical carrier and a pharmaceutically effective amount, and/or a pharmaceutically effective amount of the above A pharmaceutical composition of one of the oligomers (disclosed above) will be described in more detail below. According to an additional aspect thereof, the present invention also relates to the use of a hydrazin-3-hydroxy-4-hydroxymethyl-pyrrolidine derivative of the formula D and/or an oligomer as disclosed above for the preparation of a therapeutic or prophylactic viral infection. The agent will be described in more detail below. According to an additional aspect thereof, the present invention relates to a diagnostic composition comprising the above oligomers and labels and to detecting or analyzing DNA or RNA sequences from an individual using the oligomers described above, as will be described in more detail below. . 35 200838541 According to additional aspects thereof, the present invention also relates to the use of the above oligomers for the preparation of diagnostic compositions for detecting or analyzing DN A or RN A sequences from an individual, preferably a mammal. Use, Treatment, and Diagnostic Applications of Nucleoside Analogs of the Invention In general, the nucleoside analogs disclosed herein can be used in the treatment of nucleoside metabolism, such as interfering with bacteria, viruses, or cancer, or with appropriate labeling (which can be readily familiar) As determined by the skilled person, it is used to detect the diagnosis of nucleic acids.

Furthermore, the novel nucleoside analogs disclosed herein can be used alone or as sequences to construct DN A analogs for controlling gene expression (and ultimately biological processes), thus providing gene functional PCR clamp studies and anti-infection development. New method. It is possible to insert coding information into a macromolecule (eg, a DNA analog) in a structural manner with the novel nucleoside analogs disclosed herein, with the intention of obtaining information at a later point in time so that new nucleoside analogs can be used to prepare the security marker. Nano barcode. Furthermore, the nucleoside analogs disclosed herein can be used in synthetic DNA chimeras or as termination sequences for diagnostic and therapeutic applications, as will be described in more detail below. Use, therapeutic and diagnostic applications of the oligomers of the invention Since the oligomers of the invention have significant single or double stranded target nucleic acid-binding activities to form stable binding of double strands, triplexes or other forms, These oligomers are useful for the diagnosis and treatment of diseases associated with the performance of one or more genes (eg, those associated with pathological symptoms). 36 200838541 Therapeutic applications can use oligos to specifically inhibit gene expression associated with the establishment or maintenance of pathological symptoms (or to inhibit translation of RNA sequences encoded by these genes). Exemplary genes that can be targeted or RNAs encoded by these genes include those encoding enzymes, hormones, serum proteins, transmembrane proteins, adhesion molecules (lfA-1, GPIIb/IIIa, ELAM-1, VACM-1, ICAM-l, E-selectin (seleetin, etc.),

Receptor molecules include cytokine receptors (IL-1 receptor, IL-2 receptor, etc.), cell mediators (IL-1, IL-2, IL-3, IL-4, IL-6, etc.) Etc., genes for oncogenes, growth factors and interleukin. The target gene or RN A line may be associated with any pathological condition, such as those associated with inflammatory conditions, cardiovascular abnormalities, immune responses, cancer, viral infections, bacterial infections, and the like. The oligomers of the invention are suitable for use in both in vivo (v/vo) and ex vivo (ex Wvo) therapeutic applications. Signs for ex vivo applications include treatment of bone marrow or peripheral blood in symptoms such as leukemia (chronic myeloid leukemia, acute lymphocytic leukemia) or viral infection. The target gene or the RNA encoded by these genes can be used as a target for cancer treatment including oncogenes or overexpression sequences and genetic translocations such as ras, k-ras, bcl-2, c-myb, bcr. , c-myc, c-abl·' and the overexpressed sequence such as mdm2, oncostatiir IL, IL-6 (Kaposi's sarcoma), HER_2, and the genetic translocation such as bcr/abl 〇37 200838541 the oligomer Can be used to inhibit the proliferation of DNA or RNA viruses, the disease

Toxic such as blister virus, papilloma virus (p api 11 〇ma viruse s) and so on. Herpesviruses such as CMV, HSV-1, HSV-2, retroviruses such as HTLV-1, HIV-1, HIV-2 or other DNA such as HBV, HPV, VZV, influenza virus, rhinovirus, etc. The viral gene sequences of the polymerase or reverse transcriptase genes of RNA viruses or the RNAs encoded by these genes are also suitable targets. The application of specific combinations of oligomers can be applied in conjunction with other therapeutic treatments. Other therapeutic indications of the oligomers of the invention include: (1) modulating the inflammatory response by modulating gene expression, which plays an important role in regulating the inflammatory response, such as IL-1 receptor, IL-1, ICAM- 1 or E-selectin, and (2) regulation of cell proliferation in, for example, arterial occlusion (revascular restenosis) symptoms after vascular repair, by modulating the following manifestations: (a) growth or cell division factors, such as Non-muscle myosin, myc, fos, PCNA, pdgf or FGF or its receptor, or (b) cell proliferation factor, such as c-myb. Other potentiating factors or signal transduction factors such as TGFa, TGFp, IL-6' ylNF, protein kinase C, glucosinolates (such as p210, pl90) may be targeted for the treatment of psoriasis or other symptoms. In addition, EGF receptor, TGFa or MHC dual genes can be targeted in autoimmune diseases. - The delivery of the oligomer of the invention into a cell can be effected by any suitable means, including calcium phosphate, DMSO, glycerol or dextran transfection, electroporation or by international publication number WO 90 /1 4074, W0 3δ 200838541

A cationic, anionic and/or neutral lipid composition or liposome method as described in U.S. Patent No. 4,897,355. The oligomer can be delivered to the cells by complexing with a cationic lipid such as DOTM A, which may or may not form a liposome, followed by contacting the complex with the cells. Suitable cationic lipids include, but are not limited to, N-(2,3-bis(9-(Z) octadecyloxy))-propan-1.yl-indole, hydrazine, hydrazine-trimethylammonium (N-( 2,3-di(9-(Z)octadecenyloxyl))-prop-l-yl-N,N,N-trimet hylammonium,DOTMA)and its salts, 1-0-oleyl- 2- 0-oleyl- 3-O-Isopropyl-2-0-oleyl-3-dimethylamino-propyl-p-hydroxyet hyl ammonium and its salts and 1,2-double (oil) Oxy)-3-(trimethylammonium) propane and its salts. Increasing the delivery of the oligomer of the present invention can also be achieved by using (i) viruses such as Sendai virus (Bartzatt, R., Biotechnol Appl Biochem (1 989), 11: 133-135) or adenovirus (Wagner, E. Etc., Proc Natl Acad Sci (1992) 89: 6099-6013); (ii) Polyamine or polycation conjugates utilizing, for example, polylysine, protamine or Ν1, Ν12·double (Ethyl) cetylamine compounds (Wagner, Ε. et al, Proc Natl Acad Sci (1 991) 88: 4255-4259; Zenke, M, et al, Proc Natl Acad Sci (1 990) 87:3 6 55 -3659; Chank, Β·Κ· et al, Biochem Biophys Res Commun (1 988) 1 57:264-270; US Patent 5,1 3 8,045); (iii) lipopolyamine complexes, For example, the compound of 39 200838541 lipid cetylamine is used (Behr, J.-Ρ· et al, Proc Natl Acad Sci (1989) 86: 6982-6986; Loeffler, JP et al. J Neurochem (1990) 54: 1 8 1 2- 1 8 1 5); (iv) Negative, neutral or pH-sensitive lipids, including, for example, phosphatidyl glycerol, cardiolipin, phosphatidic aci d) or phosphorus: a compound of a negatively charged fat of phosphatidylethanolamine (Lee, K, D. et al., Biochim Biophys Acta (1 992) 1 103: 185-197;

Cheddar, G. et al., Arch Biochem Biophys (1 992) 294:1 88-192; Yoshimura, T. et al., Biochem Int (1990) 2 0: 697-706); (v) conjugates, such as a compound of transferrin or biotin, or (vi) a conjugate having a compound such as serum protein (including albumin or antibody), prion protein or polymer (including polyethylene glycol), which can increase oligomers Pharmacodynamic properties in an individual. As used herein, "transfecti" refers to any method suitable for transporting a polymeric polymer into a cell. Any reagent that can be used in a transfection procedure, such as a lipid, or any reagent, such as a virus, is generally referred to herein as a "permeation enhancing agent." Transferring the oligo to the cell can be accomplished by co-transfection with other nucleic acids in step 70, /, such as a nucleic acid such as (1) a transcriptase or a fragment of a protein fragment, or (ii) encoding a conformational protein Or a translatable rna of a protein fragment. Thus, the oligomer and A e h 1 can be sweated into any suitable formulation that enhances the transport of the oligomer into the cell. Appropriate dropouts Formulations also include those materials that are administered by topical application of the compound to the cell or group and continue to be used in the application. Such as poly 40 200838541 ethylene glycol, π ^ propylene glycol, in % / , ^ month not (aζοne), early goods u Λ, oleic acid, Ι > 1 7 本 醇 醇 - 9 (nonoxonyl, 9), polymer The topical formulation of the oligomer of the present invention or the preparation of the reagent S0, polyamine or lipopolyamine polymer of the sputum can be used to contain the oligo of the present invention. Do not use it as a local formulation for Yongxiang as a researcher to suppress gene expression. A reagent that is intended to inhibit the expression of gene expression. What mechanisms are currently available to suppress the effective and specific use of the target gene Weifang F. Oligomers that have previously been shown to inhibit performance typically have the effect of not reducing the specificity of the poor and/or not showing the gene's performance to a very low 4〇%p madness (less than the degree of uninhibition) The oligomers described herein constitute an agent which can be used in an inhibition method for the expression of these proteins in an individual or a cell, or the expression of a protein, wherein the sequence is encoded and the translation is also performed, 1 includes a protein system. From the RNA sequence, the next step: the narrowing of the present invention into the 4 call and allows the oligomer to (10) A, ... "object into the cell; w "A or RNA to form three strands or form a double with DNA戍RNa In order to inhibit the expression of the protein or the proteins by the RNA method of the present invention. The method of the present invention and the characterization of the oligomer Q/4 are regulated in both prokaryotes and eukaryotes. Due to performance, such as bacteria, *P* contains about 8:: parasites, yeasts and mammalian cells. The target nucleic acid sequence is formed: as few as the oligomers can be made with Or a three-strand structure to effectively suppress the appearance of the target protein. The target 1 ^ is formed by double or triple strands and inhibits the target oligo 6 main linear oligomer #h protein base. M Gui has about 10 to a modified ridged ridge. The present invention contains fine, The two modified nuclear bitter oligomers can easily form a ring 41 200838541

Type, as in International Patent Application No. WO 92/1 9732; Κοοί, Ε·Τ· J Am Chem Soc (1991) 113:6265-6266; Prakash, G· et al. J Am Chem Soc (19 92) 114:3523 -3 5 27 as described in . The above oligomers are suitable for binding to single or double stranded nucleic acid targets. The cyclic oligomers can have different sizes. The above oligomers having a size ranging from about 22 to 50 core monomers can be easily prepared. The cyclic polymer has about three to six nuclear monomer residues in the cyclic region to separate the binding region of the oligomer (Prakash, G. ibid). The ruthenium polymer can inhibit transcription by regulating (Maher, LJ et al. Science (1 989) 245: 725-730) or the interaction of the translated nucleic acid binding proteins regulates the expression of the target gene. Therefore, the polymer is suitable as a sequence-specific reagent for competing nucleic acid binding proteins, including ribosomes, RNA polymerase, DNA polymerase, translation initiation factor, transcription factors that increase or decrease transcription, and protein-hormone transcription factors. and many more. Thus, a suitably designed oligomer permeable mechanism enhances the synthesis of a protein of interest, such as binding to a transcription factor to suppress the regulatory position of the expression or in the vicinity, or by inhibiting the expression of an inhibitory protein selected thereby. In therapeutic applications, oligomers are used in methods suitable for treating a variety of conditions by inhibiting the appropriate target surface of the target. For the above treatments, the oligomers can be formulated into a variety of different modes of administration, including systemic, topical or regional administration. Technology - Ageing - Tong. Changke. Found in Remingt〇n, s Pharmaceutical Sciences, Mack Publishing c〇.5 Easton, PA latest edition. The oligomer active ingredient is typically incorporated into a carrier such as a diluent or a formulation comprising a filler, an extender, a binder, a wetting agent, a disintegrating agent, a surfactant or a lubricant, This depends on the mode of administration and the nature of the dosage form. Typical dosage forms include tablets, powders, liquid formulations (including suspensions, emulsions and solutions), granules, capsules and suppositories, and liquid formulations for injection (including liposome preparations). For systemic administration, injections are preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous injections. For injection, the polymer of the present invention is formulated as a liquid solution, preferably a physiologically compatible buffer such as Hank's solution or Ringer's solution. In addition, the oligomers can be formulated in solid form and redissolved or suspended prior to use. Also included are freeze-dried forms. A preferred range of dosages that can be used for systemic administration is one or two doses between about 1 mg/kg to 5 mg/kg for mother's day. However, different dosage schedules can be used depending on (i) the potential of each oligomer to inhibit the activity of its target DNA or RNA, and (ii) the severity or extent of the pathological disease state associated with a known gene. , or (iii) the pharmacokinetic profile of known oligomers. Systemic administration can also be administered by crossing the mucosa or across the skin' or the compound can be administered orally. For the application through the mucosa or through the skin, apply a penetrating agent suitable for the barrier to be infiltrated. The above penetrants are well known in the art and include, for example, bile salts and fusidic acid derivatives for administration across the mucosa. In addition, 43 200838541 can use surfactants to accelerate penetration. Transmucosal administration can be accomplished by the use of, for example, nasal sprays or suppositories. For oral administration, the oligomers are formulated into conventional oral forms such as capsules, tablets and tonics. For topical application, the oligomers of the invention are formulated as ointments, ointments, gels or creams, as is conventional in the art. The formulations of the oligomers of the invention for use in ocular signs (e. g., viral infections) are based on standard compositions well known in the art.

In addition to use in therapy, the oligomers of the invention can be used in diagnostic reagents to detect the presence or absence of a target nucleic acid sequence to which they specifically bind. The increased binding affinity of the oligomers of the invention is an advantage of their use as primers and probes. Diagnostic tests can be performed by miscellaneous cooperation, which is formed by double-stranded or triple-stranded spirals and then detected by conventional methods. For example, radioactive, fluorescent or chromogenic labels can be used to label the oligomer and detect the presence of a label attached to the solid support. Alternatively, the presence of double-stranded or triple-stranded spirochetes can be detected by clearly identifying these forms of antibodies. A method of performing the assay using the above oligomer as a probe is conventional. It is advantageous to utilize the oligomer of the present invention as a diagnostic reagent by the formation of triple helix, since the triple helix is formed under mild conditions and thus the test can be carried out without the test specimen being subjected to severe conditions. Diagnostic tests based on the detection of RNA to distinguish bacterial, fungal or protozoan sequences usually require the separation of -RNA from the sample or in the laboratory - biologically isolated, which is laborious and time consuming because of the nucleic acid present in the RN A The enzyme is very sensitive. 44 200838541 The coupon I probe may also include additional modifications (eg, modified linkages) to make the oligomer particularly stable in nucleases and thus useful for cell or tissue extracts (usually containing nuclease activity) Exist in the test of implementation. A polymer comprising a terminal modification typically retains its ability to bind to complementary sequences without loss of specificity (Uhlmann et al. Chemical Reviews (1990) 90: 543-584). As noted above, the probes of the present invention may also comprise a linker that allows for specific binding to another DNA strand by incorporation of a linker that allows for such binding (Froehler, BC et al. Biochemistry (1992) 31 :1603- 1 609) ; Horne et al. J Am Chem Soe (1990) 112:2435-2437). Incorporating the oligomer of the present invention into a probe that also contains a covalent crosslinking reagent has the potential to increase sensitivity and reduce background values in diagnostic or detection assays. In addition, the use of cross-linking reagents will allow for the detection of new changes, such as (1) long-term use of cross-linking: two-probe recognition (2) incorporating denaturing wash steps to reduce background values, and (3) miscellaneous Hybridization and cross-linking are carried out at or near the melting temperature of the complex to reduce the secondary structure in the target and increase the specificity of the probe. The oligomers of the present invention are suitable for use in diagnostic assays which, as described, employ a method of attaching an oligomer or nucleic acid to be detected to a solid support (U.S. Patent No. 4,775, 619). PNA oligos are also suitable for use in diagnostic assays. The 辏 辏 辏 - 酶 酶 酶 酶 酶 酶 扩增 扩增 扩增 扩增 扩增 扩增 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( The oligomer of the present invention comprises a 3' end which can be used as an primer, which is compatible with the poly 45 200838541 synthase used in the polymerase chain reaction method, such as Taq or Vent8 (New England Biolabs) polymerase. Thus the PNA oligomers of the invention can be used as primers in the PCR protocol. The oligomer of the present invention can also be used as a primer for a discontinuous sequence or as a primer with a random sequence.

Random sequence primers are typically about 6, 7 or 8 nucleomonomers in length. The above primers can be used in different nucleic acid amplification procedures (PCR, ligase chain reaction, etc.) or in the selection process. The substitutional linkages of the present invention generally do not interfere with the ability of the polymer to act as an primer. The oligomer of the present invention has a 2' positional modification in addition to the 3, terminal residue, and other modifications which render the RNase oxime incapable of oligo or otherwise identifiable to the nuclease may advantageously be modified. Probe or primer for RNA or DNA sequences in cell extracts or other solutions containing nucleases. Thus, an oligomer can be used in a procedure for amplifying a nucleic acid in a sample by mixing the oligomer with a sample containing the target nucleic acid, followed by hybridizing the oligomer with the target nucleic acid and by PCR, LCR or other appropriate Method Amplification of a target nucleic acid. Oligomers derived from a chelating agent such as EDTA, DTPA or 1,2-diaminocyclohexane acetic acid analogs can be used in a variety of in vitro diagnostic tests (U.S. Patent Nos. 4,772,548, 4,707, 44 0 and 4,707,352). ). Or 'the polymer of the present invention may be derivatized with a crosslinking agent such as 5-(3-iodoethylamine-1-yl)-2'-deoxyuridine (5 - (3 - iodoacetamidoprop" - y 1 )- 2'-deoxyuridine) or 5-(3-(4-bromobutyramine)-prop-1-yl)-2'-deoxyuridine (5-(3-(4-bromobutyramido)-prop- L-yl)-2'-deoxyuridine), and 46 200838541 Various test methods or kits for description (International Publication No. WO 90/14353) In addition to the above applications, the ability of the polymer to inhibit gene expression can be It has been demonstrated in in vitro (in v/iro) systems that this is done by measuring the degree of expression in individual cells or recombinant systems by any suitable method (Grae ssmann, Μ· et al. Nucleic Acids Res (1991) 19:53 -59).

Finally, the polymer of the present invention can be used in structural nanotechnology to replace DNA with the goal of obtaining an object, lattice or device (Mao, C., et al. Nature (1 999), 397: 1 44). - 146) or tissue nanoparticle (Xiao, S. et al. J. Nanopart. Res. (2002) 4: 313-317). The additional objects, features and advantages of the present invention will become more apparent from the accompanying drawings. However, it is to be understood that the following examples and illustrations are only for the purpose of description and are not to be construed as limiting the scope of the invention. [Embodiment] Definition of Nucleosteroids The term "nucleomonomer" as used herein means a nucleoside analog comprising (1) - a nucleobase, covalently linked to (2) - selective Protected 3-amino-4-hydroxymethylpyrrolidine ring. The core monomer of the present invention lacks a sugar or furanium group such as ribose or deoxyribose, and can be joined to form a PNA oligomer which binds to the target or the complementary sequence of the nucleotide in the 2008 38, 481,451 nucleic acid in a sequence-specific manner. . As used herein, the term "nucleobase" includes those heterocyclic analogues or derivatives thereof that contain not only naturally occurring purine and pyrimidine heterocycles but also selective protection and tautomer. A group such as a selectively protected deaza carbocyclic derivative thereof.嘌呤 includes adenine, guanine, jaundice, and hypoxanthine, as well as analogs or derivatives thereof. Pyrimidines include thymine, uracil and cytosine as well as analogs or derivatives thereof. Nucleoside The term "nucleoside" as used herein means that the nucleoside is covalently linked to a selectively protected 3-amino-4-hydroxymethylpyrrolidine ring. The term "linkage" as used herein means that the invention to which an adjacent nucleomonomer is attached does not comprise a urethane linkage of phosphorus. The "oligomer (ο 1 i g 〇 m e r)" as defined herein is a covalently coupled two or more core monomers to each other by a bonding group defined above. Thus, the oligomer can have as few as two covalently bonded core monomers (dimers). The oligomer can be a single or double-stranded nucleic acid sequence that binds competently and is therefore base-matchable. Short oligomers (e.g., dimer-6 hexamers) can also be used in the treatments described herein. An example of the oligomer according to the present invention is the structure shown in Fig. 6. Protective basis is based on the "Protectinggroup" used here.

48

200838541 Includes a book that avoids reactions and chelating reactions on carboxylic acids, thiols, etc. - Transfection "Transfection" as used herein means a suitable method for the transfer of an element into a cell. Lithium body "Subject" as used herein means a plant or an animal, especially a human. Sequence Specificity As used herein, "sequence-specific binding" is used in its generally accepted definition to occur in combinations such as oligomers and DNA or RNA targets, which form a base of hydrogen bonds according to common rules. It is to be understood that the scope of the specification and the scope of the following claims are to be understood as otherwise, the meaning of the total number, the number, the percentage, etc. may be understood to be modified by the word "about" in all examples. Further ranges include any combination of the disclosed maximum and minimum values, which may or may not be explicitly recited. In addition, for the purpose of this specification and the following patent application, the meaning of a plurality of carbon atoms indicated by Cn-Cm (wherein η and πι are integers, for example, c 1 - C ! 5) is disclosed. It will be understood that including all of the explicit and non-limiting embodiments in which the disclosed isomers may or may not be explicitly referred to herein, the application will be applied*

Ac = CH3CO & He group. How to raise the fundraising, including the pair between the sexually integrated concepts. 'Unless the other number will be used, all and include any of the fixed values of the numerical values. The downsizing 49 200838541

Bz = CeHsCO DABCO = 1,4-dioxinbicyclo[2.2.2]octane DBU=1,8·diindole bicyclo [5.4·0] eleven-7-ene DCM = dichlorodecane DIPEA = diisopropyl Ethylethylamine DMAP = 4-diamine amine bite DMF = dimercaptoamine

DMS0 = disulfoxide

LiHDMS=Lithium bis(trimethyldecyl)guanamine lithium TBDMS = t-butyldimethylmethyl THF = four nitrogen bites σ South Detailed description of the preferred embodiment Examples 1-3 According to Figure 1 (Figure 1 Process Paths Described to Prepare Preferred 3-Trialkylsulfonyloxy-4-alkoxycarbonyl Indoleamines of Formula G·(37?,4 and,1'5)-3-Tris-Butyl Methyl dream lactyl-4-oxime acetophenethyl) hydrazine bismuth-2 - copper ((3i?54i??l,iS,)-3-i^ri-ButyldimethyIsilyloxy-4-methoxycarbo nyl- L-(l,-phenylethyl)pyrroli-din-2-one)3 and its (35,45,1'5〇-isomer 4. Example.1 — · - ..... - 1_B 4-mercapto-2-yl-3-methylidene succinate 1 50 200838541

Add in a mixture containing ethyl glyoxalate (13.3 g, 50% in benzene, 65 mmol) and methyl acrylate (4.5 mL, 50 mmol) dissolved in THF (30 mL) DMSO (7.5 mL), H2 (4.5 mL, 250 mmol) and DABCO (2·8 g, 25 mmol). After stirring for 12 hours at 20 ° C, the oil was purified by EtOAc (EtOAc:EtOAc (EtOAc:EtOAc) . 1 Η NMR (200 MHz, CDC13): δ 1.24 (t, J = 7.2, 3 Η), 3·51 (d, J = 6.2, ΟΗ), 3.76 (s, 3 Η), 4.22 (q, J = 7· 2, 2Η), 4·83 (d, J = 6.2, 1H), 5.92 (s, 1H), 6.34 (s, 1H). Example 2 L. Ethyl-4-methyl-2-tris-butyldimethylhydrazino-3 Methylene succinate 2

Add imidazole (3·) to a solution containing ester 1 (8.16 g, 43 mmol) dissolved in DCM (48 mL) and butyl dimethyl dimethyl decane (7·13 g, 47 3 mm 〇1) at 20 °C. 22 g' 47·3 mmol) and mix the mixture for 4 hours. After removing the "treat" under reduced pressure, the residue was dissolved in ethyl acetate (5.0 mL), brine (50 mL) and ethyl acetate (2 χ } 〇〇 mL). After drying (Na2S 〇 4), the solvent was removed in vacuo and the residue was purified eluting with EtOAc (EtOAc EtOAc EtOAc EtOAc Rate: 89%). 1H NMR (200 MHz, CDC13): δ 〇.〇6 (s,3Η), 0.10 (s,3Η), 0·87 (s,9Η),1·21 (t,3Η,J = 7· 1) 3.72 (s5 3H), 4·13 (q, 2H, J = 7.1), 5.03 (s, 1H), 6·〇(s, 1H), 6·31 (s, 1H). Example 3

Tertiary-T-based dimethyl helium-based 4-carbazinium phenethyl)pyrrolidin-2-one 3舆(3&4m)-isoindole 4

Add (iS)-phenethylamine (2.4 g, 2 〇mm〇1) to a solution containing thiol derivative 2 (6 〇g, 2 〇 mmol) dissolved in methanol (50 mL) at 2 〇〇c The mixture was stirred for 2 hours. After removing the solvent, the residue was dissolved in EtOAc (50 mL). The solvent was evaporated under reduced pressure and the residue was crystallised from ethyl acetate (5 mL) washed with 3M EtOAc. The solvent was dried (N EtOAc (m.). Further elution gave the isomer 4 (2.0 g, yield: 38%) as a yellow oil. 52 200838541 Compound 3: 4 NMR (200 MHz, CDC13): δ 0·14 (s, 3H) ), 0.18 (s, 3Η), 0.88 (s5 9Η), 1.56 (d5 3Η5 J = 7.2), 2·99 (dd, 1Η, J = 7.3. J = 9.4), 3.04 - 3.16 (m5 1H)? 3.87 (s, 3H)? 3.69 (dd, 1H, J = 7.6, J = 9.4), 4 · 4 5 (d, 1 H, J = 6.7), 5.44 (q, 1H, J = 7.2), 7.23-7.36 (m, 5 ArH).

Compound 4: 1H NMR (200 MHz, CDCh): δ 0·13 (s, 3H), 0.17 (s, 3H) 5 0.84 (s, 9H), 1·53 (d, 3H, J = 7.1), 3.14 - 3.26 (m, 1H), 3.30 (dd, 1H, J = 9.5, J = 9.6), 3.39 (dd, 1H, J = 6.8, J = 9.6), 3·63 (s, 3H), 4.47 (d , 1H, J = 6·4), 5.45 (q, 1H, J = 7.1), 7.25-7.35 (m, 5 ArH). EXAMPLES 4-6 Preferred double protected 1-alkyl-3.hydroxy-4-hydroxydecyl pyrrolidines of formula B according to the reaction scheme depicted in Figure 2 (Figure 2): (37?, 47? , 1' Magic-3-Ethyloxy-4·Ethyloxycarbonyl-1-(indolyl-phenyl)pyrrolidine 6, (3 and, 4i?, rS)-3-benzyloxy- 4-benzyloxymethylphenethyl) 吼L. Example 4 Hydroxy-4-hydroxymethyl-1·(Γ-stupyl)-pyrrolidine 5

The product 3 (0.38 g, 1 mmo 1) was refluxed for 1 hour in CH3OH (1 mL) containing 12 M EtOAc (1 mL). The solvent was then removed under reduced pressure and the reflux was repeated twice under the same conditions as phase 53 200838541. The residue was dissolved in dry THF (3 mL) and the solution was cooled to EtOAc. Lithium bis(trimethyldecyl) guanamine (LiHMDS) (3 mmo di dissolved in THF Φ, T 3 mL of M solution) was added and the mixture was stirred for 5 minutes. After adding 1 μηΓΊτ M HCl (3 mL), the mixture was extracted with Et EtOAc (5 〇 mL) and dried (Na2S 〇 4). The solvent was removed under reduced pressure and the residue was crystalljjjjjjjjjj At 7 baht. The solution was refluxed in argon for 1.5 hours and then cooled to 2 〇 DC. Add 15 mL)

With 12 M HCl (5 mL) and refluxing the mixture, the solvent was removed in 3 liters. The residue was dissolved again in methanol (5 mL) and 12 M HC1 V 5 m L) and refluxed at 70 ° C. mixture! hour. This step was repeated and after addition of a saturated aqueous solution of Na2C03 (1 mL), extraction was performed with EtOAe (2 χ 5 〇 mL). The organic layer was dried (Na 2 SO 4 ) and decomposed under reduced pressure to give the product 5 with a yield of 70% and a purity of more than 90%, which was directly applied without further purification. NMR (200 MHz, CDC13): δ·i, 4l (d 3 Η, J = 6.8), 2.15 - 2·27 (m, 1 Η), 2.46 (dd, 1 Η, J > 5 4 ; = 9.5), 2.62 (dd, 1H, J = 3·4, J = 10.3), 2.72 - 2·92 (m 2H + 2 OH), 3·30 (q, 1H, J = 6.8), 3·68 (d5 2H, J > 5 4) 4.18 - 4.26 (m, 1H), 7.21 - 7.37 (m, 5 ArH). Example 5 (3 and, 4 and 1, 1 - 3 - ethyl fluorenyl-4-ethyl fluorenylmethyl) 1 _ (Γ - stupid B $ 6 6 200838541

Compound 5 (0.63 g, 2.6 mmol) was dissolved in EtOAc (EtOAc) (EtOAc) After 2 hours, the mixture was poured into ice water and extracted with ethyl acetate (2×20 mL). Separate the organic layer and dry it (Na2S〇〇

The solvent is finally removed under reduced enthalpy. The residue was purified by EtOAc (EtOAc EtOAc (EtOAc: EtOAc) , CDC13) : δ 1.35 (d? 3H? j = 6 , 〇·>), 2.02 (s, 3H), 2·05 (s, 3H), 2.12-2.53 (m, 3H), 2.62-2 84 ( m, 2H), 3.21 (q, 1H, J = 6.5), 4.04 (dd, 1H, J = 7 〇; 11.0), 4.16 (dd, 1H, J = 6·3, J = 11.0), 4.93 (m , 1H) 7.18-7.34 (m, 5 ArH, Example 6, Magic-3-3⁄4, I.oxy-4-benzyloxycarbonyl)-pyrrolidine

Bz0, A —V: 2 5 (0.21 g, 1 mmol) was dissolved in DCM (10 mL) containing DMAP (〇36 g, 2.85 mmol) and was added to the argon (2 77 μ! After 45 minutes, the mixture was poured into ice water and treated with ethyl acetate 55 200838541

(3 χ lOmL) extraction. The organic layer was separated and dried (Na 2 SO 4 ) and finally solvent was removed under reduced pressure. The residue was purified by EtOAc EtOAc (EtOAc:EtOAc: 1 H NMR (200 MHz, CDC13): δ 1.41 (d, 3 Η, J = 6.8), 2·32 (dd, J = 6.7, J = 8.1), 2·74 (dd, 1 Η, J = 3 ·6, J = 8.2), 2.79 - 2.95 (m, 1H), 3.01 (dd, 1H, J = 6·4, J = 8.2), 3·15 (dd, 1H, J = 8·1, J = 8.1), 3.32 (q, 1H, J = 6.8), 4.59 (d, 2H, J = 6·6), 5.27 - 5·36 (m, 1H), 7.18 - 7.65 (m5 11 ArH), 7 ·97 - 8·07 (m, 4 ArH). EXAMPLES 7 - 8 Preferred double protected 1 mercapto-3-hydroxy-4-hydroxymethyl derivatives of formula A are prepared according to the reaction schemes depicted in Figures 3 and 4 (Figures 3 and 4). :(3i?,4i?)-3-acetoxy-4-ethenyloxymethyl-1-bromoacetylpyrrolidinium 8, (3i?,4i〇-3·benzyloxy-4- Benzyl oxime oxime bromide fluorenylpyridinium 9. Example 7 (3j?, 4i 〇-3. ethenyl-4-ethenyloxymethylpyrrolidine 8

Compound 6 (1 2 2 mg, ... 0 ·_4 mm ο 1) was dissolved in dry DC Μ (1.2 mL) and then bromine bromo bromide (1 〇 6 pL, 1-2) was added at 20 °C. Mm). After 30 minutes, pyridine (6 5 μ! 加入 was added and the mixture was stirred for 12 hours. Then EtOAc (5 mL) was added with water (2 mL), then the organic layer was separated, then sat. Na 2 C03 (2 mL), 3 M HC1 (2 work "with water (5 mL) washed and finally dried (NhSO4). After removing the solvent, the residue was chromatographed on silica gel (cyclohexane: Et 〇Ae 4:6 as eluent) To obtain a pure light colored oil 8 (90 mg, yield: 7 %). lH nmr (2 〇〇 MHz, CDC 13) · δ 2 · 〇 5 (s, 3H, 40%), 2.06 ( s, 3H, 6〇%), 2 〇7 (s, 3H, 60%), 2·〇8 (s, 3H, 4G%), 2 57 — 2 78 (4), ih), 3 4i a 3.62 (m , 2H), 3.71 - 3.89 (m, 1H), 3.77 (s, 2H, 60%), 3·80 (s, 2H, 4G%), 3.93 - 4" (m, 1H), 4.G3 - 4 · 09 (m, 2H), 5.12 - 5.19 (m, 1H). Example 8 Benzyloxymethyl n-bromopyridinyl pyrrolidine 9

Add bromine oxime bromide (106 μί, 1.2 mmol) to dry DCM (1·2 mL) i compound 7 (169 mg, 0.4 mm 〇1) at 20 ° C, followed by ▲ 30 min After joining. Pyridine (65, and stir the mixture for 12 hours. Then add 'Et〇AC (5 mL) and water (2 mL), separate the organic layer, then

Na2C〇3 saturated solution (2 mL), 3 M hci (2 mL) and water (5 mL) were washed and finally dried (Ns 〇 4). After the solvent was removed, the residue was crystalljjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj NMR (200 MHz, CDC13): §·2·89 · 3.14 57 200838541 (m,1H),3.59 -3.87 (m,2H),3.80 (s,2H,55%),3,84 (s, 2H, 45%), 3.91 - 4.21 (m, 2H), 4.22 (dd, 2H, J = 3.9, J = 6.5), 5.50 - 5.57 (m, 1H), 7.36 - 7.51 (m, 4 ArH), 7.53 -7.65 (m, 2 ArH), 7.97 - 8.07 (m, 4 ArH). Example 9-14

A preferred nucleoside analog of formula D is prepared according to the reaction schemes depicted in Figures 5 and 6 (Figures 5 and 6): (37?, 4i?)-3-acetoxy-4-acetone Oxymethyl-1-(1 thymine-pyridyl)pyrrolidine 11, (3i?, 4i〇-3-benzyloxy-4-pyloryloxyl-1-(1'-cell)密基基乙基)° ratio 11 each bite 12, (3i?, 4i?)-3-acetoxy-4-acetoxymethyl adenyl ethyl) pyrrolidine 13, (3i? , 4i〇-3 -acetoxy-4-acetoxymethyl·1-[(2-amino-6-benzyloxyindole-9-yl)ethenyl]pyrrolidine 14 and (3i? , 4i?)-3-benzyloxy-4-benzyloxymethyl-1-(indolyl-thymidineyl)-yl 11 each bite 15. Example 9 2-Amino-6-benzyl Oxonium 10

Benzyl alcohol (3 7 · 5 g, 3 4 7 -m rii ό 1) was mixed with sodium hydroxide (2 · 9 6 g, 7 4 m ο 1) and heated to 80 ° C to dissolve the sodium hydroxide. After cooling, 2-amino-6-chloropurine (6.0 g, 35 mmol) was added and the mixture was stirred under stirring 58 200838541

Heat to 90 ° C for up to 5 hours. After cooling, EtOAc (120 ml) was added to the mixture and mixture was extracted twice with 1% aqueous sodium hydroxide (70 mL). The basic liquid phase layer was combined, washed with EtOAc and then treated with 35% hydrochloric acid until pH 6-8. The expected crystals were collected by filtration and dried under reduced pressure to give white powder of 2-amino-6-ethoxy hydrazide ( 7.6 g, yield: 92%): lU NMR (200 MHz, CDC13) ): δ 5.46 (s5 2Η)5 6.24 (br s, 2H5 NH), 7.24 - 7.54 (m9 5 ArH)? 7.81 (s? 1H), 12.43 (br s, 1H, NH) 〇 Example 1 0 ϋ B醯 -4- 醯 醯 醯 醯 篡 】 】 】 】 】 】 】 】 】 】 】 】 11 11 11 11 11 11 11

Compound 8 (63 mg, 〇·19 mm〇l) was at 20. (: Dissolved in dry DMF (0.5 mL) followed by dry molecular sieves 4A (25 mg) with slow stirring. After 1 hour, add thymine (51 mg, 〇·39 mmol) and dry K2C〇3 (136, 〇98 _(4) and the mixture was stirred at 20 ° C for 2.5 hours _. Then dcm (10 mL) and water (3 mL) were added with stirring and the organic layer was washed with water. Again with DcM (5 mL) The aqueous layer was extracted, the organic phase was collected and dried (NkSO4), and the solvent was removed under reduced pressure of 59 200838541. The residue was purified by EtOAc (EtOAc) to give viscous oil.

Product 11 (44 mg, yield: 63%). NMR (200 MHz, CDC13) ·· δ 1·92 (s, 3H), 2.08 (s5 3H), 2·〇9 (s, 3H, 50%), 2.10 (s, 3Η, 50%), 2.54 - 2,68 (m,1Η,50%),2·68 - 2,74 (m,1Η,50%),3.43 - 3.65 (m,2Η),3,75 - 3,87 (m,1Η), 3.93 - 4.19 (m, 3H), 4.35 (dd, 1H, J = ίο·., j = 16.8), 4·47 (dd, 1H, J = 7.3, J = 16.8), 5.11 — 5.22 (m5 1H) , 7·01 (s, 1H, 50%), 7·03 (s, 1H, 50%), 8.42 (s, ih, welcome, 50%), 8.43 (s, 1H, NH, 50%) . Example 11 (3i?, 4ig)-3-benzyloxy-4-benzyloxymethyl-1-(1 cytosinyl)pyrrolidine 1 2

Compound 9 (84 mg, 0.99 mmol) was dissolved in dry DMF (0.5 mL) then dry molecular sieve 4A (50 mg) was then slowly stirred at 20 °C. After 1 hour, cytosine (50 mg, 0. 38 mmol) and dry K2CO3 (133 mg, 0.96 mmol) were added and the mixture was stirred at 20 ° C for an additional 2.5 hours. Then DCM (10 mL) and water (3 mL) were added and the organic layer was washed with water. The aqueous layer was again extracted with DCM (5 mL) and the organic phase was collected and collected.

After drying (Na.sub.2SO.sub.4), EtOAc (EtOAc) 4 NMR (2 00 MHz, DMS0-d6): δ 2·75 - 2.92 (m, 1 Η, 50%), 2.93 - 3·06 (m5 1 Η, 50%), 3.12 - 4·16 (m, 3H) ,4·01 - 4·20 (m,1H),4·22 - 4·43 (m,2H),4·53 (s,2H, 50%),4·59 (s,2Η, 50%) , 5,3 5 - 5.45 (m5 1Η, 50%), 5.45 -5·55 (m, 1H, 50%), 5·80 (d, 1H, J = 7·3, Het-H), 7· 01 (br s,1H,Het-H), 7.40 - 7.69 (m,6 ArH), 7.77 - 7.98 (m,4 ArH) 〇Example 1 2 (3 and, 4;)-3-acetamidine_oxygen Base-4 - ethyl hydrazine methyl-, - adenyl acetyl group) p 嘻唆 13

Compound 8 (40 mg, 0·12 mmol) was at 20. (: Dissolved in dry THF (2 mL) and then agitated (19 mg, 0.14 mmol) and NaH (6 mg, 〇14 _ 〇1) and stirred at 2 〇〇c for 24 hours. The EtOAc (5 mL) and water (2 mL) were evaporated. After removing the solvent under reduced pressure, the residue was purified by silica gel chromatography (DCM: Me:H 75:25) to give the product of s. %). NMR (200 MHz, CDC13): δ 2·07 (s, 2 χ 3H), 2.43 - 2.85 (m, 2H), 3·35 - 3·75 (m, 4H), 3.95 - 4·95 (m,3H),4.85 - 5·04 (m, 1Η),5·18 (br s,2Η,ΝΗ2),8·31 (s,1 Het-H),8.35 (s,1 Het -H). Example 1 3 _C3i?, 4i〇-3-acetoxime·4-acetamidinehydrol-1-K2-amino-6-helium-based hydrazine

呤-9-yl)ethinylpyrrolidine 1 4

Compound 8 (84 mg, 0·19 mmol) was dissolved in dry DMF (0.5 mL) at 20 ° C then dry molecular sieve 4A (50 mg) was then slowly stirred. After 1 hour, 2-amino-6-benzyloxyindole (50 mg, 0.38 mmol) and dry K.sub.2CO.sub.3 ( 133 mg, EtOAc. Then DCM (10 mL) and water (3 mL) were added and the organic layer was washed with water. The aqueous layer was extracted with EtOAc (5 mL). Product 14 of oil (41 mg, yield: 56%). iHNMR (200 MHz, CDC13): δ 2.06 (s, 2 χ 3H), 2.55 - 2·82 (m, 2H), 3·45 - 3·91 (m, 4Η), 3.99 - 4.22 (m, 3H) , 4.90 (brs, 2H5NH2), 5.12-5.20 (m, 1H), 5.56 (s, 2H), 7.21 - 7·52 (m, 5 ArH), 7·72 (s, 1 Het-H) 〇 Example 1 4

(3 and 47?)-3-benzylindole-4-yloxymethyl-1-(1'-thymidine)pyrrolidine 1 5

Compound 9 (84 mg, 0.19 mmol) was dissolved in dry DMF (0.5 mL) at 20 °C then dry molecular sieve 4A (50 mg). After 1 hour, thymus smear (50 mg, 0. 38 mmol) and dry K2C03 (1 33 mg, 〇·96 mmol) were added and the mixture was further stirred at 20 ° C for 2.5 hours. Then DCM (10 mL) and water (3 mL) were added and the organic layer was washed with water. The aqueous layer was again extracted with D CM (5 mL). The organic phase was collected and dried (Na.sub.2.sub.4) and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (EtO Ac) to obtain a viscous oil. Product 15 (yield: 58%). NMR (200 MHz, CDC13): δ 1.94 (s,3H), 2·54 - 2,68 (m,1Η,50%),2·68 - 2,74 (m,1Η,50%), 63 200838541 3·43 - 3,65 (m,2H),3·75 - 3.87 (m5 1H), 3.93 - 4.19 (m, 3H), 4.35 (dd,1H, J = 10.0, J = 16.8), 4.47 (dd , 1H, I = 7.3, J = 16.8), 5.11 - 5.22 (m, 1H), 7.03 (s, 1H, 50%), 7.07 (s5 1H, 50%), 8.42 (s5 1H, NH, 50%) , 8.43 (s, 1H, NH, 50%).

Additional examples (not numbered) of nucleoside analogs according to the present invention are shown in Figures 5 and 6, which can be produced using the same synthetic methods described in the above examples with corresponding nucleobases. Head Head "k ★ * Header The above description explains and describes the present invention. In addition, although the disclosure shows only the preferred embodiments of the invention, it will be understood that the invention can be used in various other combinations, modifications and environments, and can be embodied herein. Changes or modifications in the scope of the concept, the above doctrine and/or technology or related technology. The embodiments described above are intended to further illustrate the best mode for carrying out the present disclosure, and the various modifications of the present invention and the specific application or use of the present disclosure are disclosed in the above or other embodiments. Therefore, the description is not intended to limit the disclosure of the invention disclosed herein. Furthermore, it is intended that the scope of the appended claims should be understood to include alternative embodiments. All publications, patents, and patent applications listed in this specification are hereby incorporated by reference herein in its entirety herein in the the the the the the the 64 200838541 Incorporated by reference. In the case of inconsistency, this disclosure is preferred. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic illustration of a preferred tris-decyloxy-4-alkoxycarbonyl decylamine of the formula G, which is used in the preparation of the formula B according to the present invention. a preferred 1-alkyl-3-hydroxy-4-hydroxymethylpyrrolidine derivative;

Figure 2 is a preferred double-protected 1-alkyl-3-hydroxy-4-hydroxyl group starting from the preferred 3-trimethylcarbyloxy-4-indolylamine in the formula G. A schematic representation of a mercaptopyrrolidine derivative; Figures 3 and 4 are preferred starting from a preferred 1-alkyl-3.hydroxy-4-hydroxydecylpyrrolidine derivative of the formula. Double-protected 1 - mercapto - 3 - thiol-4 - via methyl 11 ratio σ each bite derivative of the outline diagram; Figure 5 and 6 are self-type 双 double protection of 1-anthrace 3. Amino-4'-hydroxymethylpyrrolidine derivatives A schematic representation of the preferred synthesis of nucleoside analogs of formula D of the present invention. [Main component symbol description] 65

Claims (1)

200838541 X. The scope of application for patents: 1. One type D-mercapto-3-trans-yl-4-methyl-pyrene derivative of D, wherein Ri and R2 are respectively selected from fluorene and RC (= 0), wherein R is a linear or branched alkyl group, an aryl group, a substituted aryl group including 1 to 5 hetero atoms, and includes 1 to 3 hetero atoms. A heterocyclic group and R3 is a nucleobase. 2. A pyrrolidine derivative according to claim 1, wherein R is a linear or branched Ci-Cu alkyl group, a C6-C15 aryl group, and a substituted C5-C15 comprising 1 to 5 hetero atoms. An aryl group, a C3-C5 heterocyclic group comprising from 1 to 3 heteroatoms. 3. A pyrrolidine derivative as described in claim 2, wherein R is selected from the group consisting of methyl, ethyl, propyl, isopropyl, t-butyl, 2-nonylbutyl, allyl, A straight or branched chain CrCu alkyl group of 3,3-dimethylallyl, 3-nonylbutyl, 3-methyl-2-butenyl, octyl or decyl. 4. The pyrrolidine derivative according to any one of claims 1 to 2, wherein the hetero atom is selected from the group consisting of oxygen, nitrogen, sulfur, fluorine, chlorine, bromine or iodine. 66. The pyrrolidine derivative according to claim 1, wherein R is selected from the group consisting of C6H5, CH3C6H4, 2-furyl, 3-furyl, 4-methoxyphenyl, 4-phenylene , 4-chlorophenyl, 4 -> odor phenyl, 4- phenyl, 4-trimethyl fluorenyl, 2,6-dimethylphenyl, 2,6-dimethoxyphenyl, 2,4- Dinitrophenyl, 2,4-dichlorophenyl, 2,4-dibromophenyl, 2,4-diiodophenyl, 2,4-dimethoxyphenyl-monoaryl or heterocyclic group . Thymus sputum I, sputum bite II, adenine III, guanine IV, scutellarin V, jaundice VI and hypoxanthine VII, the reactive group NH2 or VII of the above nucleobase I-VII The imidic NH is protected by one of the derivatives, and the reactive functional group NH2 of the above nucleobase I-VII is selectively protected from the nucleocapsid of a deazacarbocyclic derivative. 67. The method of claim 6, wherein the reactive functional group ΝΗ or ΝΗ 2 is protected by a protective group ,, the protective group is independently Selected from benzyl, benzindenyl, 2,4-dimethoxybenzyl, diphenylmethyl, dipropyl, 4-methoxybenzy 1, PMB, 4-methoxybenzyloxycarbonyl (4-methoxybenzyloxycarbonyl) ^ 4-methoxybenzylhydrazino, 4-nitrobenzylidene, 4-fluorobenzyl, 4-bromobenzyl, 4-iodobenzyl, 1-naphthylmethyl, A Sulfhydryl, ethyl fluorenyl, propyl fluorenyl, pivaloyl, benzyloxy carbonyl (Cbz), t-butoxycarbonyl 9 t-Boc, diphenyl hydrazine Benzhydryloxycarbonyl (B he), adamantyloxy carbonyl, fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc) 〇 8. The pyrrolidine derivative according to claim 6, wherein the nucleobase derivative is selected from the group consisting of: 68 200838541 wherein P! is selected from the group consisting of benzyloxycarbonyl (Cbz), 4-methoxybenzyloxycarbonyl, dibenzomethoxycarbonyl (Bhc), fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc a protective group of an adamantyloxycarbonyl group, and the P2 system is selected from the group consisting of a benzyl group, a 4-methoxybenzyl group (PMB), a 2,4-dimethoxybenzyl group, a diphenyl fluorenyl group, and an allyl group. Protective group. 9. The pyrrolidine derivative according to claim 6, wherein the deazacarbocyclic derivative of the nucleobase is selected from the group consisting of: XII XIV Wherein Pi is selected from the group consisting of benzyloxycarbonyl (Cbz), 4-methoxybenzyloxycarbonyl, diphenylphosphonium carbonyl (Bhc), methoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), King Kong a protective group of an alkoxycarbonyl group, and a P2 group is a protective group selected from the group consisting of a benzyl group, a 4-methoxybenzyl group (PMB), a 2,4-dioxabenzyl group, a diphenyl fluorenyl group, and an allyl group. group. 1 〇 · - type 69 200838541 R10^^ r2〇 A 1-mercapto-3-hydroxy-4-hydroxymethylpyrrolidine derivative, The Ri and R2 systems are respectively selected from Η and RC (= 0), wherein R is as defined in any one of claims 1 to 5 and the R4 is a leaving group 〇11 The pyrrolidine derivative according to claim 10, wherein R4 is a leaving group selected from the group consisting of Cl, Br, I or OS02-Y, wherein the Y group is selected from the group consisting of an anthracenyl group, an isopropyl group, a phenyl group, Tolyl, 4-nitrophenyl, 2,4. dinitrophenyl, 4-chlorophenyl, 4-bromophenyl and trifluoromethyl. 12. - Type A double protected 1-alkyl-3-hydroxy-4-hydroxydecyl pyrrolidine derivative of B, wherein the Ri and R2 lines are each selected from the group consisting of RC (= 0) protective groups, wherein R is straight 70 200838541 Or a bond C2-C15, a C6-Ci5 aryl group, a substituted C5-C15 aryl group comprising 1 to 5 heteroatoms, a C3-C5 heterocyclic group comprising 1 to 3 heteroatoms; A chain or branched alkyl group and R6 is a linear or branched alkyl group, an aryl group or a substituted aryl group having 1 to 5 hetero atoms. The pyrrolidine derivative according to claim 12, wherein the R system is selected from the group consisting of ethyl, propyl, isopropyl, t-butyl, 2-methylbutyl, allyl a linear or branched C2-C15 alkyl group of 3,3-didecylallyl, 3,nonylbutyl, 3-nonyl-2-butenyl, octyl or decyl. The pyrrolidine derivative according to claim 12, wherein the hetero atom is selected from the group consisting of oxygen, nitrogen, sulfur, fluorine, chlorine, bromine or broken. 1 5 · The pyrrolidine derivative according to claim 12, wherein R is one selected from the group consisting of C6H5, CH3C6H4, 2-furyl, 3-furyl, 4-methoxyphenyl, 4-nitrobenzene Base, 4-chlorophenyl, 4-bromophenyl, 4-iodophenyl, 4-trifluoromethyl, 2,6-diphenylphenyl, 2,6-dimethoxyphenyl, 2,4- An aryl or heterocyclic group of dinitrophenyl, 2,4-dichlorophenyl, 2,4-dibromophenyl, 2,4-diiodophenyl, 2,4-dimethoxyphenyl. 16. The pyrrolidine derivative according to claim 12, wherein R5 is a straight chain or a branched alkyl group. 71 200838541 1 7 · The σ piroxime derivative as described in claim 12, wherein R6 is a straight bond or a bond Ci-Cg alkyl, C6-C8 aryl, including 1 to 5 heteroatoms It is substituted for the C5-C8 aryl group. 18. The pyrrolidine derivative according to any one of claims 12, 16 or 17, wherein R5 and R6 are each selected from the group consisting of methyl, ethyl, propyl, isopropyl, t-butyl. , 2-methylbutyl, allyl, 3,3-dimethylallyl, 3-methylbutyl, 3-decyl-2-butenyl, straight or branched Ci of octyl -Cg alkyl group. 1 9. The pyrrolidine derivative according to claim 17, wherein the FU system is selected from the group consisting of C6H5, CH3C6H4, 4-methoxyphenyl, 4-nitrophenyl, 4-chlorophenyl, 4- Bromophenyl, 4·iodophenyl, 4-trifluoromethyl, 2,6-dimethylphenyl, 2,6-dioxaphenyl, 2,4-dinitrophenyl, 2,4-dichloro An aryl group of phenyl, 2,4-dibromophenyl, 2,4-diiodophenyl, 2,4-dioxaphenyl. The pyrrolidine derivative according to claim 1, wherein the C-1' atomic system is a stereogenic centre. 21. An oligomer comprising a plurality of nucleomonomers, wherein at least one of the core monomers is of the formula: 72 200838541 22. The oligomer of claim 21, wherein R3 is a nucleobase as defined in any one of claims 6-9. 23. A preparative formula A method of protecting a 1-alkyl-3-hydroxy-4-hydroxymethylpyrrolidine derivative, wherein the Ri and the lines are each selected from the protective cyclist of RC (= 0), wherein the R is as patented Included in any of items 1 - 5; R5 is a straight or branched alkyl group and R6 is a straight or branched alkyl group, an aryl group or a substituted aromatic group comprising 1 to 5 heteroatoms; Base, which includes the following steps: a) M G A pair of protected 1-alkyl-3-trialkylsulfoxy-4-alkoxycarbonylpyrrolidin-2-one derivatives, wherein the lanthanide series SiR9R10, R7, R8, R9 and R!o are linear Or a branched alkyl group, and R5 and R6 are defined above, and react with a reducing agent capable of releasing a hydride ion to obtain a formula a 1-alkyl-3-hydroxy-4-hydroxymethylpyrrolidine derivative wherein R5 and R6 are defined above, b) a 1-alkyl-3-hydroxy group of the formula ) obtained in step a) The 4-hydroxydecyl pyrrolidine derivative is reacted with an anhydride of the formula (R6C0)20 or a monohydrazine derivative of the formula R6COZ wherein R6 is defined above and Z is hydrogen. 74. The method of claim 23, wherein R5 and FU are as defined in any one of claims 16 to 19 of the patent application. 25. The method of claim 23, wherein R7, R8, R9 and Rig are each selected from a linear or branched C^-Cs alkyl group. 2. The method of claim 25, wherein R7 is selected from the group consisting of ethyl, propyl, isopropyl and t-butyl. 27. The method of claim 25, wherein R8 is a methyl group. 28. The method of claim 25, wherein R9 and R10 are each selected from the group consisting of a decyl group, an ethyl group, a propyl group, a secondary-propyl group, and a t-butyl group. 29. The method of claim 23, wherein the reducing agent is selected from the group consisting of BH3, LiAlH4, LiBH4, BF3-triethyldecane, A1H3. The method of claim 23, wherein the step a) is carried out in an organic solvent. H. The method of claim 30, wherein the solvent is selected from the group consisting of cyclic or dialkyl ethers, chlorinated hydrocarbons, cyclic and linear burns, or the like. mixture. The method of claim 23, wherein the step a) is carried out at a temperature between 0 QC and 90 QC and suitable for the reducing agent. 33. The method of claim 23, wherein the Z system is F, C1 or Br 〇 The method of claim 23, wherein the step b) is carried out in a polar or non-polar solvent. 3. The method of claim 4, wherein the solvent is selected from the group consisting of ethers, aromatic hydrocarbons, chlorinated hydrocarbons, dimethyl decylamine (DMF), dimethyl hydrazine (DMSO). Or a mixture of the above. 3. The method of claim 23, wherein the step b) is a tertiary amine having a molecular weight equivalent to the above-mentioned anhydride (ROCO0) 20 or the above-mentioned mercapto derivative ROCOZ (R' Wherein 3-N is present, wherein R' represents the same or different straight or branched C2-C8 alkyl group; or in the presence of a tertiary cyclic amine R'(R'')2-N, wherein R'' is -(CH2)n- and η = 4-6. 37. The method of claim 23, wherein the step b-) is carried out at -10 ° C to 60. (: between the temperatures. 76 200838541 38· a preparation s R2〇 A R4 A pair of protected 1-mercapto-3-hydroxy-4-hydroxymethylpyrroles 'R and R2 are respectively selected from the protective group of RC (= 0) and any one of the first to fifth patent claims A group as defined in the above, which comprises the step of formulating a double protected 1-alkyl-3-hydroxy-4-hydroxymethylpyrrole wherein the Ri and R2 systems are defined; Rs is a direct bond or a bond a branched alkyl group, an aryl group or a fluorene derivative comprising 1 to 5 heteroatoms, wherein R is, for example, R4 is a derivatized derivative, and R6 is a linear or substituted aryl group. , 77 200838541 A sulfhydryl halide reaction wherein L is a halogen atom and R4 is defined above. 3 9. The method of claim 38, wherein R5 and R6 are as defined in any one of claims 1 to 6-1 of the patent application. The method of claim 38, wherein R4 is the leaving group as defined in claim 11 of the patent application. The method of claim 3, wherein L is a halogen atom selected from the group consisting of F, C1 or Br. 42. - Preparation A pair of protected 1-mercapto-3-hydroxy-4-hydroxymethylpyrrolidine derivatives, wherein the Ri and R2 lines are each selected from the group consisting of RC (= 0) protective groups, wherein R is 78 200838541 R3 is a nucleobase as defined in any one of claims 1 to 5, and includes the following steps: R2o A a pair of protected 1-indolyl-3-hydroxy-4-hydroxymethylpyrrolidine derivatives, wherein Ri and R2 are defined above and R4 is a leaving group, and a nucleobase in a non-aqueous solution In the base reaction, the non-aqueous solution comprises a base capable of forming an anion of one of the above-mentioned verification groups R3. 43. The method of claim 42, wherein the base is selected from the group consisting of alkalis, lithium amines or metal hydrides having a pKb value between 4 and 9. 44. The method of claim 43, wherein the base is selected from the group consisting of Li2C03, Na2C03, K2C03, Cs2C03 or a mixture thereof. 45. The method of claim 43, wherein the lithium amine is selected from the group consisting of lithium bis(trimethylsilyl)amide 5 LiHDMS, diisopropylamine (LDA) or a mixture of the above. The method of claim 43, wherein the metal hydride is selected from the group consisting of NaH, KH or a mixture thereof. 47. The method of claim 42 wherein R3 is a nucleobase as defined in any one of claims 6-9. 48. The method of claim 42, wherein R4 is a leaving group as defined in claim 11 of the scope of the patent application. 49. The method of claim 42, wherein the reaction is carried out in a solution of an organic solvent. 50. The method of claim 49, wherein the organic solvent is selected from the group consisting of DMF, DMSO, N-methylpyrrolidone, HMPA, or a mixture thereof. The method of claim 42, wherein the reaction is carried out at a temperature between 20 ° and 100 ° C. 52. —Preparation 80 200838541 a method of one of HO E - 1 -mercapto-3 -transyl-4 -methyl sigma singly derivatives, wherein R 4 is a leaving group, which comprises the following steps, A pair of protected 1-indolyl-3-hydroxy-4-hydroxymethylpyrrolidine derivatives, wherein Ri and R2 are each selected from a protective group of RC (= 0), wherein R is as claimed R4 is defined above in any of items 1 - 5 and is defined above in the presence of a carbonate ion or a polymer comprising a supporting (suPp〇rted) Oir or C0 plant group with a pKa equal to Or less than 18 one-stage alcohols. 53. Preparations 81 200838541 A method of a 1-mercapto-3-hydroxy-4-hydroxyindole acridine derivative, wherein R3 is a nucleobase, which comprises the following steps: A pair of protected 1-indolyl-3-hydroxy-4-hydroxymethylpyrrolidine derivatives, wherein R! and R2 are each selected from a protective group of RC (= 0), wherein R is as patented R3 is defined above in any of the ranges 1-5, and is in the presence of a carbonate ion or a polymer comprising a supporting OH/ or CO broad group with a pKa equal to or less than 1 8 one-stage alcohol reaction. 54. The method of claim 52, wherein R4 is the leaving group as defined in item 11 of the patent application. 55. The method of claim 53, wherein R3 is a nucleobase as defined in any one of claims 6-9. The method of any one of claims 5 or 5, wherein the primary alcohol is selected from the group consisting of linear or branched CpCs alcohols, benzyl alcohols or mixtures thereof. 57. The method of claim 56, wherein the primary alcohol is selected from the group consisting of methanol, ethanol, propanol, 2-methylbutan-1-ol, 3-methylbutan-1-ol or the above a mixture. The method of any one of claims 5 or 5, wherein the carbonate ion is derived from a carbonate selected from the group consisting of Li2C〇3, K2CO3, Na2C03, Cs2C03 or a mixture thereof. The method of any one of claims 52 or 53 wherein the polymer comprising the support group or the CO plant group is selected from the group consisting of macroreticular ion exchange resins of the OH or CO form. . The method of any one of claims 52 or 53, wherein the reaction is carried out in a non-aqueous solvent. The method of claim 60, wherein the solvent comprises the above-mentioned alcohol having a pKa of equal to or less than 18. The method of any one of claim 52, wherein the reaction is carried out at a temperature between 〇 ° C and 50 ° C. 6. A pyrrolidine derivative according to claim 1, which is for use in a method of treatment, prevention or diagnosis. The oligomer of any one of claims 21 or 22, which is for use in a method of treatment, prevention or diagnosis. 6 5. A pharmaceutical composition comprising a pharmaceutical carrier and a pharmaceutically effective amount of one of the formula HO—\ y--Rs HO F 1 -mercapto-3-trans-yl-4-thiol-based σ An anthracene derivative, wherein R3 is a verification base as defined in any of claims 1 and 6-9. 66. Application of a pyrrolidine derivative of any one of claims 1 to 9 or a polymer of any one of claims 2 to 21 or 22 for preparing a therapeutic or prophylactic virus Infected agent. 84 200838541 67. A diagnostic composition comprising an oligomer and a label as in any one of claims 21 or 22. 68. An application of an oligomer according to any one of claims 21 or 22 for detecting or analyzing a DNA or RNA sequence from a single body. 69. The use of claim 68, wherein the system is a mammal. The use of an oligomer according to any one of claims 21 or 22 for the preparation of a diagnostic composition for detecting or analyzing DNA or RNA sequence 歹>J from a single body. 71. The use of claim 70, wherein the system is a mammal. 85