WO2008101514A1

WO2008101514A1 - Nucleoside analogues for the treatment of viral infections and methods for preparing them

Info

Publication number: WO2008101514A1
Application number: PCT/EP2007/001532
Authority: WO
Inventors: Fabio Biondi
Original assignee: NANODREAM Srl UNIPERSONALE
Current assignee: NANODREAM Srl UNIPERSONALE
Priority date: 2007-02-22
Filing date: 2007-02-22
Publication date: 2008-08-28
Anticipated expiration: 2009-08-22
Also published as: EP2173738A1; TW200838541A

Abstract

New pyrrolidine derivatives are disclosed having formula (I) 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

NUCLEOSIDE ANALOGUES FOR THE TREATMENT OF VIRAL INFECTIONS AND METHODS FOR PREPARING THEM

DESCRIPTION

Background of the invention

The present invention relates to novel nucleoside analogues useful for the preparation of DNA analogues for diagnostic and therapeutic applications.

The invention also pertains to novel intermediates useful for the preparation of the aforementioned nucleoside analogues, to methods for preparing both the intermediates and the analogues as well as to novel modified oligomers, the synthesis thereof, their use in oligomer-based therapies and their use as diagnostic or therapeutic reagents.

Related art

Nucleic acids are of central importance in living nature as carriers or transmitters of genetic information. Therefore since their discovery they have stimulated a broad scientific interest which has led to the elucidation of their function, structure and mechanism of action. The increasing knowledge of these basic mechanisms in molecular biology has made it possible in recent years to make new combinations of genes. This technology opens for example new opportunities in medical diagnosis and therapy and in plant breeding.

Thus, part of the research in this field is concerned with the development of treatments against pathogens or tumors based on the development of nucleoside compounds which interfere with the metabolism of nucleosides be it bacterial, viral or tumoral.

Examples of nucleoside analogs useful as inhibitors of viral RNA polymerases are disclosed in various prior publications, such as for example, International patent application WO 2006/002231 which discloses aza nucleosides that are useful as inhibitors of viral RNA polymerases such as, but not limited to, hepatitis B, hepatitis C, Polio, Coxsackie A and B, Rhino, Echo, small pox, Ebola, and West Nile virus polymerases, whereas US patent No. 5,246,931 discloses carbocyclic nucleoside analogs useful as antitumor or antiviral compounds for combating pathogens such as herpes viruses 1 and 2 (HSV-I and HSV-2), human immunodeficiency viruses (HIV), hepatitis A, B and non-A, non-B viruses.

On the other hand, another part of the research in the field of nucleoside analogs is concerned with the development of compounds useful for detecting nucleic acids with regard to their specific detection as well as with regard to their sequence, i.e. their primary structure.

The specific detectability of nucleic acids is based on the property of these molecules to interact or hybridize with other nucleic acids by forming base pairs via hydrogen bridges. Nucleic acids (probes) labelled in a suitable manner i.e. provided with indicator groups, can thus be used to detect complementary nucleic acids (target).

The determination of the primary structure (sequence), i.e. the sequence of the heterocyclic bases of a nucleic acid, is carried out by means of sequencing techniques. This knowledge of the sequence is in turn a prerequisite for a targeted and specific use of nucleic acids in problems and methods of molecular biology. In the end sequencing also utilizes the specific hybridization among nucleic acids. Labelled nucleic acid fragments are also used for this as mentioned above.

Examples of nucleoside analogs useful for the detection of nucleic acids are disclosed in various prior publications, such as for example, US patent No. 6,174,998 which describes pyrrolo-[3,2-d]pyrimidine, pyrazolo-[4,3-d]pyrimidine and pyrimidine- furanosides, otherwise known as C-nucleosides, modified with signal groups for the detection of nucleic acids.

Examples of nucleoside analogs useful for the detection of nucleic acids are also disclosed in British patent application GB 2 309 969 which describes labelled nucleoside analogs based on indol-1-yl, pyrrol- 1-yl useful for labelling nucleic acids or for incorporation in oligonucleotides.

Within this new and challenging technological area, important efforts are devoted to the design of ever more effective nucleosides analogues for therapeutic or diagnostic purposes.

In this respect, part of the research is devoted towards finding new nucleosides analogues based on pyrrolidine derivatives which are deemed to mimick to a larger extent the naturally occurring DNA or RNA due to the presence of the pyrrolidine ring which acts as an isostere of the furanose ring of the naturally occurring nucleic acids.

Thus, for example, the synthesis of (3i?,4i?)-3-hydroxy-4-hydroxymethylpyrrolidine useful for the preparation of a nucleoside analog (a compound known as BCX-4208) useful as purine nucleoside phosphorylase (PNP) inhibitor, was disclosed by Kotian et al. (Org. Proc. Res. Dev., 2005, 9, 193) and in US Patent application No. 2006128789 according to the following reaction path employing benzylamine:

The synthesis of (3i?,4i?)-3-hydroxy-4-hydroxymethylpyrrolidine according to this method, however, entails the use of the very expensive acyl sultam, whereas trimethylsilyhnethoxymethylbenzylamine involves synthesis problems.

The synthesis of (3i?,4/?)-3-hydroxy-4-hydroxymethylpyrrolidine was also reported by using asymmetric 1,3 -dipolar cycloaddition by Karlsson et al. (Tetrahedron: Asymmetry, 2001, 12, 1977) exploiting both a chiral camphorsultam and (S)- phenylethylamine as chirality inducers according to the following reaction path:

^Ph

Also the synthesis of (3i?,4i?)-3-hydroxy-4-hydroxymethylpyrrolidine according to this method, however, entails the use of very expensive reagents such as the aforementioned acyl sultam and chiral trimethylsilyhnethoxymethylphenyethylamine, while yields not exceeding 50% have been reported.

(3/?,4i?)-3-hydroxy-4-hydroxymethylpyrrolidine was also obtained by Tyler et al as disclosed in International patent application No. WO2005033076 by enzymatic - A - resolution of racemic trans- l-N-benzyl-4-hydroxy-3-eΛoxycarbonyl-pyrrolidine carried out with CAL Novozyme 435, followed by reduction and hydrogenolysis of the reaction product according to the following reaction path: trans

The synthesis of (3i?,4i?)-3-hydroxy-4-hydroxymethylpyrrolidine according to this method, however, entails the use of a kinetic resolution via en2yme, while yields not exceeding 50% have been reported.

Enantiomerically pure (3if,4i?)-3-hydroxy-4-hydroxymethylpyrrolidine was also obtained by Galeazzi et al. (Tetrahedron: Asymmetry, 2004, 15, 3249) by means of a reaction path involving the reaction of (5)-phenylethylamine with ethyl 2-silyloxy-3- methoxycarbonyl-3-butenoate:

to yield 3,4-trα«s-3-hydroxy-4-alkoxycarbonylpyrrolidin-2-ones and then the conversion of the intermediate pyrrolidin-2-ones into (3i?,4Λ)-3-hydroxy-4- hydroxymethylpyrrolidine by using a troublesome methods, involving LAH and chloroalkyl chloroformate, according to the following reaction path:

The synthesis of (3i?,4i?)-3-hydroxy-4-hydroxymethylpyrrolidine according to this method, however, entails the use of both a metal hydride and chloroethyl chloroformate, thus preventing the preparation of large amounts of the target compound.

Summary of the invention

In one aspect, this invention provides novel nucleoside analogues which can be compatible with DNA synthetic reagents and instrumentation, so that they can be suitable for preparation of DNA chimeras and optionally having at the same time protecting groups capable of removal under mild conditions.

In another aspect, this invention provides suitably protected nucleoside analogues for increased efficiency during nucleic acid oligomer synthesis.

In still another aspect, this invention provides nucleoside analogues that are compatible with commercially available products and instrumentation, such as automated synthesizers.

In still other aspects, this invention provides novel intermediates useful for the preparation of the aforementioned nucleoside analogues, as well as convenient high- yield synthetic methods for preparing both the intermediates and the nucleoside analogues.

In still other aspects, this invention provides novel modified oligomers capable to form new chiral DNA chimeras, the synthesis thereof, their use in oligomer-based therapies and their use as diagnostic reagents.

In accordance with a first aspect thereof, the invention relates to a l-acyl-3-hydroxy-4- hydroxymethylpyrrolidine derivative of the formula

wherein

R₁ and R₂ are independently selected from H and R-C(=O), wherein R is straight or branched alkyl, aryl, substituted aryl including from 1 to 5 heteroatoms, a heterocyclic group including from 1 to 3 heteroatoms and R₃ is a nucleobase. Advantageously, the nucleoside analogues of formula D according to the invention is a compound which is capable of being incorporated, by enzymatic or chemical means, in a nucleic acid (DNA or RNA) chain, and is there capable of base-pairing with a nucleotide residue in a complementary chain or base stacking in the appropriate nucleic acid chain.

In a preferred embodiment, the l-acyl-3-hydroxy-4-hydroxymethylpyrrolidine derivatives of the invention have a stereogenic configuration (3R,4R) isosteric with the naturally-occurring deoxyribose ring at the C-3 and C-4 carbon atoms.

The nucleoside analogues of the invention may be used to advantage in both therapeutic and diagnostic methods as will be illustrated in greater detail in the following.

Most advantageously, furthermore, the aforementioned nucleoside analogues of the invention comprise in their structure a sterically hindered -N-(C=O)-CH₂- linkage to the nucleobase which effectively enhances the resistance of the oligomer obtained therefrom against the attack of naturally occurring nucleases and peptidases.

Most advantageously, furthermore, the nucleoside analogues of the invention are capable to closely mimic the structure of naturally-occurring nucleosides of RNA or DNA thanks to the presence of the aforementioned sterically hindered -N-(C=O)-CH₂- linkage and of the pyrrolidine ring which constitute an isostere of the ribose or deoxyribose ring.

The l-acyl-3-hydroxy-4-hydroxymethylpyrrolidine derivatives of the invention in their di-protected form, i.e. when R₁ and R₂ are R-C(=O), constitute improved nucleoside analogues which allow to achieve both a suitable protection of the 3 -hydroxy and of the 4-hydroxymethyl functional groups and their subsequent selective deprotection under mild conditions.

Mild conditions, as used herein, refer to reactions carried out at temperatures ranging from 0°C to 40°C, in the absence of strong bases which can affect the configuration of the stereogenic centres at C-3 and C-4 or cleave the ring.

In a preferred embodiment, the groups Ri and R₂ are protecting groups wherein R is straight or branched Ci-Ci₅ alkyl, C₆-Ci₅ aryl, substituted C₅-Ci₅ aryl including from 1 to 5 heteroatoms, C₃-C₅ heterocyclic group including from 1 to 3 heteroatoms.

In this way and as will be better apparent in the following, the l-acyl-3-hydroxy-4- hydroxymethylpyrrolidine derivatives of the invention may be conveniently protected at their 3 -hydroxy and/or 4-hydroxymethyl functionality during their synthesis, and at the same time conveniently deprotected at one or both functionalities under mild conditions at the most appropriate time during or after the oligomer synthesis.

In a preferred embodiment, R is a straight or branched C₁-Ci₅ alkyl moiety selected from methyl, ethyl, propyl, isopropyl, t-butyl, 2-methylbutyl, allyl, 3,3-dimethylallyl, 3- methylbutyl, 3-methyl-2-butenyl, octyl, decyl.

In this way, the l-acyl-3-hydroxy-4-hydroxymethylpyrrolidine derivatives of the invention may be deprotected at the 3 -hydroxy and/or 4-hydroxymethyl functionality under particularly mild conditions, such as for example the use of carbonate ion in methanol or ion exchange resin in the hydroxide form in methanol at 20°C which do not affect the other functional groups nor the stereogenic centres.

In another preferred embodiment, R is substituted C₅-C ₁₅ aryl including from 1 to 5 heteroatoms or a C₃-C₅ heterocyclic group including from 1 to 3 heteroatoms, while the heteroatom is selected from oxygen, nitrogen, sulphur, fluorine, chlorine, bromine or iodine.

Advantageously, the presence of an aryl group allows to obtain a more lipophilic product, so that solubility in organic solvents is increased, and purification can be made easier.

In another preferred embodiment, R is an aryl or heterocyclic moiety selected from C₆H₅, CH₃C₆H₄, 2-furyl, 3-furyl, 4-methoxyphenyl, 4-nitrophenyl, 4-chlorophenyl, 4- bromophenyl, 4-iodophenyl, 4-trifluoromethyl, 2,6-dimethylphenyl, 2,6- dimethoxyphenyl, 2,4-dinitrophenyl, 2,4-dichlorophenyl, 2,4-dibromophenyl, 2,4- diiodophenyl, 2,4-dimethoxyphenyl.

Advantageously, these preferred aryl or heterocyclic moieties further enhance the lipophilicity of the pyrrolidine derivative and its solubility in organic solvents rendering its purification still easier.

According to the invention, all the advantages achieved by the new nucleoside analogues described above are achieved both when R₃ is a naturally occurring nucleobase and when R₃ is a derivative thereof.

In a preferred embodiment of the invention, therefore, R₃ is a nucleobase selected from thymine I, cytosine II, adenine III, guanine IV, uracil V, xanthine VI and hypoxanthine VII of the formula

III IV

Vl 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.

In another preferred embodiment of the invention, R₃ may be a derivative of the nucleobases I- VII protected at the reactive functionalities NH₂ or imidic NH or a deazacarbocyclic derivative of said nucleobases I-VII optionally protected at the reactive functionality NH₂, wherein said reactive functionalities NH₂ or imidic NH are protected by means of a protecting group P independently selected from benzyl, benzoyl, 2,4-methoxybenzyl, benzhydryl, allyl, 4-methoxybenzyl (PMB), 4- methoxybenzyloxycarbonyl, 4-methoxybenzoyl, 4-nitrobenzoyl, 4-fluorobenzoyl, 4- bromobenzoyl, 4-iodobenzoyl, 1-naphthoyl, formyl, acetyl, propionyl, pivaloyl, benzyloxycarbonyl (Cbz), t-butoxycarbonyl (t-Boc), benzhydryloxycarbonyl (Bhc), adamantyloxycarbonyl, fiuorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc).

Advantageously, the protection of reactive functionalities of the nucleobases limits possible side reactions which can decrease the final yield. Moreover, the use of protecting groups increases the lipophilicity of the same nucleobases, thus making easier work-up and separation of the intermediates.

In a preferred embodiment of the invention, R₃ is a pyrrolidine derivative wherein the nucleobase derivative is selected from:

VIII IX X Xl

wherein Pi is a protecting group selected from benzyloxycarbonyl (Cbz), A- methoxybenzyloxycarbonyl, benzhydryloxycarbonyl (Bhc), fluorenylmethyloxycar- bonyl (Fmoc), allyloxycarbonyl (Alloc), adamantyloxycarbonyl and P₂ is a protecting group selected from benzyl, 4-methoxybenzyl (PMB), 2,4-dimethoxybenzyl, benzhydryl, allyl.

Also in this case, the protection of reactive functionalities of the derivatives of nucleobases advantageously limits the possible side reactions which can decrease the final yield and increases the lipophilicity of the nucleobases, thus making easier work- up and separation of the intermediates.

In a preferred embodiment of the invention, R₃ is a protected deazacarbocyclic derivative of said nucleobases selected from:

XII XIII XIV

wherein Pi is a protecting group selected from benzyloxycarbonyl (Cbz), A- methoxybenzyloxycarbonyl, benzhydryloxycarbonyl (Bhc), fluorenylmethyloxy- carbonyl (Fmoc), allyloxycarbonyl (Alloc), adamantyloxycarbonyl and P₂ is a protecting group selected from benzyl, 4-methoxybenzyl (PMB), 2,4-methoxybenzyl, benzhydryl, allyl.

Also in this case, the protection of reactive functionalities of the derivatives of nucleobases advantageously limits the possible side reactions which can decrease the final yield and increases the lipophilicity of the nucleobases, thus making easier workup and separation of the intermediates.

In accordance with another aspect thereof, the invention relates to a l-acyl-3-hydroxy-4- hydroxymethylpyrrolidine derivative of the formula

wherein

Ri and R₂ are as defined above and R₄ is a leaving group.

Most advantageously, the aforementioned pyrrolidine derivatives of formula A of the invention have been obtained straightforwardly and are useful intermediates which allow to prepare the nucleoside analogues of formula D of the invention by means of a method which leads to N-acyl derivatives as it will be described in more detail hereinbelow.

In a preferred embodiment, the l-acyl-3-hydroxy-4-hydroxymethylpyrrolidine derivatives of formula A according to the invention have the stereogenic configuration (3R,4R) isosteric with the naturally-occurring deoxyribose ring at the C-3 and C-4 carbon atoms.

In a preferred embodiment, Ri and R₂ may be the preferred groups defined above.

In a preferred embodiment, R₄ is a leaving group selected from Cl, Br, I, or OSO₂-Y, wherein Y is selected from methyl, isopropyl, phenyl, tolyl, 4-nitrophenyl, 2,4- dinitrophenyl, 4-chlorophenyl, 4-bromophenyl and trifluoromethyl.

Advantageously, the presence of both halogen and sulphonyl derivative allows an easy nucleophilic substitution by a nucleobase.

In accordance with another aspect thereof, the invention relates to a 1 -alky 1-3 -hydroxy- 4-hydroxymethylpyrrolidine derivative of the formula

B

wherein

R₁ and R₂ are as defined above with the proviso that R₂ is not CH₃, R₅ is straight or branched alkyl and R₆ is straight or branched alkyl, aryl or substituted aryl including from 1 to 5 heteroatoms.

In a preferred embodiment, the l-alkyl-3-hydroxy-4-hydroxymethylpyrrolidine derivatives of formula B according to the invention have the stereogenic configuration (3R,4R) isosteric with the naturally-occurring deoxyribose ring at the C-3 and C-4 carbon atoms.

Most advantageously, the l-alkyl-3-hydroxy-4-hydroxymethylpyrrolidine derivatives of formula B are useful intermediates which allow to prepare the aforementioned l-acyl-3- hydroxy-4-hydroxymethylpyrrolidine intermediates of formula A which in turn allow to prepare the nucleoside analogues of formula D of the invention.

Advantageously, furthermore, the aforementioned l-alkyl-3-hydroxy-4- hydroxymethylpyrrolidine derivatives of formula B, in their di-protected form, allow to prepare the aforementioned l-acyl-3-hydroxy-4-hydroxymethylpyrrolidine intermediates of formula A by means of a method which allows the insertion of an acyl group at N-I as will be described in more detail hereinbelow.

In a preferred embodiment, R₁ and R₂ may be the preferred groups defined above.

In a preferred embodiment, in the l-alkyl-3-hydroxy-4-hydroxymethylpyrrolidine derivatives of formula B according to the invention the groups R₅ and R₆ are independently a straight or branched C₁-C₈ alkyl which advantageously allow to increase lipophilicity of the product rendering its purification easier.

Still more preferably, R₅ and R₆ are independently a straight or branched alkyl moiety selected from methyl, ethyl, propyl, isopropyl, t-butyl, 2-methylbutyl, allyl, 3,3- dimethylallyl, 3-methylbutyl, 3-methyl-2-butenyl, octyl. In a preferred embodiment, in the l-alkyl-3-hydroxy-4-hydroxymethyl derivatives of formula B according to the invention the group R₆ may be C₆-C₈ aryl, substituted C₅-C₈ aryl including from 1 to 5 heteroatoms, which advantageously allow to increase lipophilicity and solubility in organic solvents of the product rendering its purification easier.

In this regard, it is to be noted that the presence of an aryl or substituted aryl as R₆ advantageously allows a convenient insertion of an acyl group at N-I by means of an effective, safe and low-cost acylation process as will be described in more detail hereinbelow.

Still more preferably, R₆ is an aryl or substituted aryl moiety selected from C₆H₅, CH₃C₆H₄, 4-methoxyphenyl, 4-nitrophenyl, 4-chlorophenyl, 4-bromophenyl, 4- iodophenyl, 4-trifluoromethylphenyl, 2,6-dimethylphenyl, 2,6-dimethoxyphenyl, 2,4- dinitrophenyl, 2,4-dichlorophenyl, 2,4-dibromophenyl, 2,4-diiodophenyl, 2,4- dimethoxypheny 1.

hi accordance with a preferred aspect of the invention, the C-I' atom of all the above- identified pyrrolidine derivatives is a stereogenic centre.

In this way, a ready separation can be realised of the diastereomeric forms of the di- protected l-aUcyl-3-hydroxy-4-hydroxymethylpyrrolidine derivatives of formula G given hereinbelow, for example by means of conventional chromatographic techniques, or by fractional crystallization, as will be better explained in specific Example 3 which follows.

Thanks to this feature, all the methods of the invention may be advantageously carried out in preferred embodiments thereof on intermediates, and give rise to final products, which are all enantiomerically pure, thereby avoiding the need of low-yielding, complex and time consuming procedures of resolving racemates which frequently occur in the art of organic synthesis.

In accordance with another aspect thereof, the invention relates to an oligomer comprising a plurality of nucleomonomers wherein at least one of said nucleomonomers is of the formula:

wherein

X and Z are independently selected from O and S and R₃ is a nucleobase as defined above.

Most advantageously, modelling studies showed that the oligomers of the invention, which have the stereogenic configuration (3R,4R) isosteric with the naturally-occurring deoxyribose ring at the C-3 and C-4 carbon atoms, are ordered toward a binding competent conformation which has improved target nucleic acid binding properties.

A binding competent conformation, as used herein, refers to the spatial orientation of heterocyclic bases in an oligomer required for binding to duplex or single stranded DNA or RNA in a sequence-specific manner.

This feature thus renders the oligomers of the invention suitable for both therapeutic and diagnostic methods as will be illustrated in greater detail hereinbelow.

The nucleomonomers of the present invention are generally characterized as moieties or residues that replace the furanose ring that is normally found in nucleotides with an isostere thereof, i.e. the pyrrolidine ring. The discovery of these nucleomonomers and their characteristics is based on modelling studies that both (1) predicted such analogs are compatible with a binding competent oligomer and (2) defined the range of molecular characteristics that such nucleomonomers could assume without the loss of binding competence, when incorporated into oligomers. Binding competence, as used herein, refers either to Watson-Crick base pairing with single-stranded DNA or single- stranded RNA or to Hoogsteen pairing with duplex nucleic acids including duplex DNA or duplex RNA.

Incorporation of the nucleomonomers described herein into oligomers permits synthesis of improved compounds with respect to properties such as (i) increased lipophilicity which results from eliminating the charge associated with phosphodiester linkages and (ii) resistance to degradation by enzymes such as nucleases and peptidases.

Consequently, oligomers containing these nucleoside analogs are quite suitable for hybridization to target sequences or molecules.

The present invention provides a series of nucleomonomers that can be incorporated into binding competent oligomers. The invention oligomers are resistant to nuclease digestion, are stable under physiological conditions. Nuclease stability is an important consideration for the development of oligomers that are intended to be used as therapeutic agents that function by binding to specific DNA or RNA (mRNA, hnRNA, etc.) sequences. Such specific target sequence binding underlies their therapeutic efficacy by interfering with the normal biological function of nucleic acid sequences associated with pathological conditions.

According to the invention, oligomers of any length, including 10-mers (10 nucleomonomers), 20-mers, 50-mers, 100-mers, or oligomers of greater length, may be conveniently produced starting from the nucleoside analogues of the invention using solid phase or solution phase synthesis methods known in the art and described, for example, in Khydatov, Y.E.; Fields, H.A, Eds. Artificial DNA: methods and applications, CRC Press, New York, 2002, and Herdewijn, P. Oligonucleotide synthesis: methods and applications, Humana Press, New York, 2005.

Oligomers containing 2 to 30 nucleomonomers are preferred. In general, the invention oligomers will be preferably synthesized by solid phase methods which sequentially add nucleomonomers to a first monomer unit bound to a solid support according to protocols and apparatuses known to those skilled in the art.

In additional aspects thereof, the l-acyl-3-hydroxy-4-hydroxymethylpyrrolidine derivative of formula D described above may be prepared by means of a stereoselective multi-step method which entails the preparation of new di-protected intermediates of the aforementioned formulas A and B.

Accordingly, in an additional aspect thereof, the invention relates to a method for preparing a di-protected l-alkyl-3-hydroxy-4-hydroxymethylpyrrolidine of the formula

B

wherein

R₁ and R₂ are protecting groups independently selected from R-C(=O), wherein R is straight or branched alkyl, aryl, substituted aryl including from 1 to 5 heteroatoms, a heterocyclic group including from 1 to 3 heteroatoms; R₅ is straight or branched alkyl and R₆ is straight or branched alkyl, aryl or substituted aryl including from 1 to 5 heteroatoms,

comprising the steps of:

a) reacting a di-protected l-alkyl-3-trialkylsilyloxy-4-alkoxycarbonylpyrrolidin-2-one derivative of the formula

wherein M is SiR₉R₁₀, R₇, Rs, R₉ and Ri₀ are straight or branched alkyl, and R₅ and R₆ are as defined above,

with a reducing agent capable to release hydride ions so as to obtain a l-alkyl-3- hydroxy-4-hydroxymethylpyrrolidine derivative of the formula

H

wherein R₅ and R₆ are as defined above, b) reacting the 1 -alky 1-3 -hydroxy-4-hydroxymethylpyrrolidine derivative of formula H obtained in step a) with an anhydride of the formula (R₆CO)₂O or an acyl derivative of the formula R₆COZ, wherein R₆ is as defined above and Z is halogen.

Advantageously, this method allows to directly remove both the lactam carbonyl and the alkoxycarbonyl group at C-4, together with the protecting group (for example 3-tert- butyldimethylsilyloxy) at C-3.

Preferably, the di-protected l-alkyl-3-trialkylsilyloxy-4-alkoxycarbonyl lactam of formula G is the (3R,4R,1'S) diastereomer.

In preferred embodiments of this method, R, R₅ and R^ may be the preferred groups defined above.

In preferred embodiments of this method, R₇ is selected from straight or branched Ci-C₈ alkyl, preferably from ethyl, propyl, sec-propyl, t-butyl, and most preferably is t-butyl, in order to allow a better chromatographic separation of the diastereomers.

In preferred embodiments of this method, Rs is selected from straight or branched Ci-C₈ alkyl, preferably methyl, in order to allow a better separation of the diastereomeric mixture.

In preferred embodiments of this method, R₉ and Rj o are independently selected straight or branched Ci-C₈ alkyl, preferably from methyl, ethyl, propyl, sec-propyl, t-butyl, and most preferably are methyl, in order to allow a better chromatographic separation of the diastereomers.

Preferably, the reducing agent used in step a) is selected from BH₃, LiAlH₄, LiBH₄, BF₃-triethylsilane, AlH₃.

Advantageously, the lactam carbonyl is removed yielding a pyrrolidine ring, the alkoxycarbonyl moiety is converted into a hydroxymethyl group and the trialkylsilyloxy group gives a free hydroxy moiety at C-3 in a simple and straightforward one-pot reaction.

In a preferred embodiment, the aforementioned reaction is carried out in an organic solvent capable to solubilize the reducing agent and which is preferably selected from cyclic or dialkyl ethers, cyclic and straight alkanes, or mixtures thereof.

In a preferred embodiment, the aforementioned reaction is carried out at temperature of from 0°C to 9O⁰C and compatible with the reducing agent. More preferably, the aforementioned reaction is carried out at a temperature of from 20⁰C to 90⁰C and, still more preferably, of from 60⁰C to 8O⁰C.

Generally speaking, reaction times for step a) will vary as a function of the reaction temperatures chosen between 1 h and 1.5 h, while the yield of the reaction will preferably range between 60% and 70%.

Under these conditions, the product is advantageously obtained straightforwardly and in high yield by a one-pot process involving removal of both the lactam carbonyl and the alkoxycarbonyl group at C-4, together with the protecting group (for example 3-tert- butyldimethylsilyloxy) at C-3.

Preferably, the method for preparing the l-alkyl-3-hydroxy-4-hydroxymethylpyrrolidine intermediate of formula H also includes one or more additional steps of treating the reagents and/or the reaction products with suitable reagents in order to remove any undesired by-products, such as those generated by the reducing agent, in accordance with methodologies known to those skilled in the art.

Thus, for example, when the reducing agent is BH₃ or BF₃-triethylsilane the reaction products are treated first in one or more steps with an acid, such as for example HCl, in an organic solvent, such as for example methanol, in order to carry out a transesterification reaction of complex boron species formed at the 3 -amino functionality and then with a base, such as for example NaOH, in a solvent, such as for example methanol, in order to remove the anion of the acid and form the 3 -hydroxy functionality in its free from.

On the other hand, when the reducing agent is for example LiAlH₄, LiBH₄ or AlH₃, the reaction products are treated first in one or more steps with an acid, such as for example HCl, in an organic solvent, such as for example ethyl acetate, in order to form Al complex salts and then with a salt of a weak organic acid, such as for example Na or K tartrates in a solvent such as for example water, in order to form a homogeneous solution.

Both treatments of the reaction products preferably end with an extraction step carried out with a suitable reagent, such as ethyl acetate or dichloromethane and with a drying step, carried out with a suitable reagent, such as Na₂SO₄ or MgSO₄ in order to have a pure product. Advantageously, the aforementioned step b) of this method allows to easily protect the hydroxy and the hydroxymethyl moieties at C-3 and C-4, respectively, thus avoiding side reactions in further steps of the synthetic procedure.

In a preferred embodiment of the method for preparing the di-protected pyrrolidine derivatives of formula B, the halogen Z of the acyl derivatives ROCOZ or -0-(C=O)- R₇-COZ used in step b), is F, Cl or Br.

In a preferred embodiment of the method for preparing the di-protected pyrrolidine derivatives of formula B, furthermore, the group R₇ of the acyl derivative -0-(C=O)-R₇- COZ, is straight C₄-C₈ alkyl.

In a preferred embodiment of the method for preparing the di-protected pyrrolidine derivatives of formula B, the aforementioned reaction step b) is carried out in a polar or apolar solvent.

Preferably, the solvent is selected from ethers, aromatic hydrocarbons, chlorinated hydrocarbons, dimethylformamide (DMF), dimethylsulfoxide (DMSO), or mixtures thereof, which are able to give a clear solution thus increasing the reaction rate.

In a preferred embodiment of the method for preparing the di-protected pyrrolidine derivatives of formula B, the reaction step b) is carried out in the presence of a tertiary amine (R')₃-N wherein R' represents equal or different straight or branched C₂-C₈ alkyl, or of a tertiary cyclic amine R'(R")₂-N wherein R" is -(CH₂)_n- and n = 4-6, in an equimolar amount to said anhydride (ROCO)₂O or said acyl derivative ROCOZ.

Most advantageously, the aforementioned tertiary amines neutralize the acid evolved by the reaction thereby enhancing the yield thereof up to values of about 90%.

In a preferred embodiment, the tertiary amine (R')₃-N is selected from triethylamine, tributylamine, diisopropylethylamine, triisopropylamine, while R'(R")₂-N is selected from N-ethylpyrrolidine and N-ethylpiperidine, whose salts are soluble in the organic solvent; moreover, owing to the low boiling point, the unreacted amine can be easily removed under reduced pressure.

In a preferred embodiment of the method for preparing the di-protected pyrrolidine derivatives of formula B, the reaction step b) is carried out at a temperature of from - 10°C to 60°C, more preferably of from 0°C to 40°C and, still more preferably, of from 0°C to 20°C. In this way, an increased solubility of the product is attained, advantageously reducing or preventing separation problems.

Generally speaking, reaction times for step b) will vary as a function of the reaction temperatures chosen between 0.5 h and 2 h, while the yield of the reaction will preferably range between 85% and 90%.

In accordance with an additional aspect thereof and within the framework of the aforementioned stereoselective multi-step method for preparing the l-acyl-3-hydroxy-4- hydroxymethylpyrrolidine derivative of formula D, the invention relates to a method for preparing a di-protected l-acyl-3-hydroxy-4-hydroxymethylpyrrolidine derivative of the formula

wherein

R₁ and R₂ are protecting groups independently selected from R-C(=O), wherein R is as defined above and R₄ is a leaving group,

comprising the step of reacting a di-protected l-alkyl-3-hydroxy-4- hhyyddrrooxxyymmeetthhyyllppyyrrrroolliiddiinnee ooff tthhee ffoorrmmuullaa

B

wherein

Ri and R₂ are as defined above; R₅ is straight or branched alkyl and R₆ is straight or branched alkyl, aryl or substituted aryl including from 1 to 5 heteroatoms,

with an acyl halide of the formula

wherein L is halogen atom and R₄ is as defined above.

Advantageously, this method allows to directly acylate the N-I of the pyrrolidine ring avoiding the preparation of 3-acyloxy-4-acyloxymethylpyrrolidine derivatives whose acylation resulted difficult, and afforded complex mixtures of degradation products.

In preferred embodiments of this method, R, R₅, R₆ and the leaving group R₄ may be the preferred groups defined above.

In preferred embodiments of this method, L is a halogen atom selected from F, Cl or Br. In particular, Br allows to obtain the better yields of substituted product.

In a preferred embodiment, the aforementioned acylating step is carried out in an organic solvent.

Preferably, the solvent is selected from dichloromethane, chloroform, THF, diisopropyl ether, diisobutylether, 1,3-dioxane, acetonitrile, dimethylformamide or mixtures thereof.

Still more preferably, the preferred solvent is dichloromethane which allows to obtain a clear solution of the reaction mixture, easily dissolving both the reaction product thus increasing the reaction rate.

In a preferred embodiment, the aforementioned reaction is carried out at a temperature of from 0°C to 50⁰C, preferably at temperature of from 0°C to 40°C and, still more preferably at temperature of from 15°C to 25°C.

In this way, the reaction rate may be kept at optimal values while avoiding, at temperatures over 50°C the occurrence of undesired side reactions leading to partial cleavage of the protecting groups at C-3 and C-4.

Generally speaking, as a function of the reaction temperatures chosen, reaction times will vary between 6 h and 12 h, while the yield of the reaction will preferably range between 50% and 72% .

In accordance with an additional aspect thereof, the invention relates to a method for preparing a di-protected l-acyl-3-hydroxy-4-hydroxymeΛylpyrrolidine derivative of the formula

wherein

Ri and R₂ are protecting groups independently selected from R-C(=O), wherein R is as defined above and R₃ is a nucleobase,

comprising the step of reacting a di-protected l-acyl-3-hydroxy-4- hydroxymethylpyrrolidine derivative of the formula

wherein Ri and R₂ are as defined above and R₄ is a leaving group,

with a nucleobase in a non-aqueous solution comprising a base capable to form an anion of said nucleobase R₃.

Advantageously, this method allows to operate in mild conditions and to recover the final product with high yields.

In preferred embodiments of this method, the base capable to form an anion of the nucleobase R₃ is selected from bases having a pK_b between 4 and 9, lithium amides or metal hydrides.

Within the framework of this preferred embodiment, the aforementioned base is preferably selected from Li₂CO₃, Na₂CO₃, K₂CO₃, Cs₂CO₃, or mixtures thereof.

Within the framework of this preferred embodiment, furthermore, the aforementioned lithium amide is preferably selected from lithium bis(trimethylsilyl)amide (LiHDMS), lithium diisopylamide (LDA), or mixtures thereof.

Within the framework of this preferred embodiment, finally, the aforementioned metal hydride is preferably selected from NaH, KH, or mixtures thereof.

In preferred embodiments of this method, R₁, R₂ the nucleobase R₃ and the leaving group R₄ may be the preferred groups defined above.

In a preferred embodiment, the aforementioned reaction step is carried out in an organic solvent.

Preferably, the solvent is selected from DMF, DMSO, N-methylpyrrolidone, HMPA or mixtures thereof.

In a preferred embodiment, the reaction step is carried out at a temperature of from 0° to 100°C, preferably at temperature of from 0⁰C to 60°C and, still more preferably at temperature of from 15°C to 20°C.

In this way, the reaction rate may be kept at optimal values while the side reactions are strongly overcome, such as random cleavage of the protecting groups at both the hydroxy and the hydroxymethyl functionalities.

Generally speaking, as a function of the reaction temperatures chosen, reaction times will vary between 2 h and 24 h, while the yield of the reaction will preferably range between 48% and 70%.

In accordance with an additional aspect thereof, the invention relates to a method for preparing a l-acyl-3-hydroxy-4-hydroxymethylpyrrolidine derivative of the formula

wherein R₄ is a leaving group,

wherein

with a primary alcohol having a pK_a equal to or lower than 18 in the presence of a carbonate ion or a polymer comprising supported OH^" or CO₃ ^" groups.

Advantageously, this method allows to easily cleave the R₁ and R₂ protecting groups producing the deprotected l-acyl-S-hydroxy^-hydroxymethylpyrrolidine derivative of formula E in a simple and economically feasible manner.

wherein R₃ is a nucleobase,

ccoommppririssiinngg tthhee sstteepp ooff rreeaaccttiinngg a di-protected l-acyl-3-hydroxy-4- hydroxymethylpyrrolidine derivative of the formula

wherein

R₁ and R₂ are protecting groups independently selected from R-C(=O), wherein R is as defined above and R₃ is a nucleobase,

According to a preferred embodiment of the invention, the selective deprotection step of the 3 -hydroxy and of 4-hydroxymethyl groups is carried out in both these methods in a non-aqueous solvent which preferably comprises the aforementioned primary alcohol having a pK_a equal to or lower than 18.

In this way, one of the reagents of the deprotection step advantageously carries out also the function of acting as a solvent of the reacting species simplifying the reaction and advantageously allowing to directly obtain the reaction product as an alcoholic solution in the reaction medium after a simple filtering off of the solid reagent together with the Ri and R₂AIlCyI esters.

In this preferred embodiment, furthermore, the R₁ and R₂ protecting groups remain soluble in the primary alcohol (for example methanol) and may be recovered in a simple manner after filtration and removal of the solvent under reduced pressure.

In preferred embodiments of both these methods, Ri, R₂ the nucleobase R₃ and the leaving group R₄ may be the preferred groups defined above.

In a particularly preferred embodiment of this method, the primary alcohol is selected from straight or branched Ci-C₅ alcohol, benzyl alcohol, or mixtures thereof and still more preferably, from methyl alcohol, ethyl alcohol, propyl alcohol, 2-methylbutan-l- ol, 3-methylbutan-l-ol, or mixtures thereof.

In this way and as mentioned above, the reaction product may be obtained as an alcoholic solution in the reaction medium after a simple filtering off of the solid reagent together with the R₁AIlCyI ester.

In a preferred embodiment of this method, the carbonate ion is derived from a carbonate selected from Li₂CO₃, K₂CO₃, Na₂CO₃, Cs₂CO₃, or mixtures thereof.

Preferably, furthermore, the polymer comprising supported OH^" or CO₃ ^" groups is selected from macroreticular ion-exchange resins in the OH^" or CO₃ ^"" form. In a preferred embodiment, the selective deprotection step of the 3 -hydroxy and of the 4-hydroxymethyl groups is carried out at temperature of from 0°C to 50°C, preferably at temperature of from 0°C to 40⁰C and, still more preferably at temperature of from 15⁰C to 25⁰C.

In this way, the reaction rate may be kept at optimal values while avoiding, at temperatures over 50°C the occurrence of undesired side reactions leading to partial cleavage of 1-acyl group linked to the N-I nitrogen atom.

Generally speaking, as a function of the reaction temperatures chosen, reaction times of both the aforementioned methods will vary between 1 h and 3 h, while the yield of the reactions will preferably range between 70% and 90%.

Generally speaking, furthermore, all the methods described above will also include extraction and purification steps of the starting reagents and/or of the desired final products carried out according to procedures well known to those skilled in the art and examples of which will be illustrated in the Examples hereinbelow.

In accordance with additional aspects thereof and as already mentioned above, the invention relates to the l-acyl-3-hydroxy-4-hydroxymethyl-pyrrolidine derivative of the formula D and to an oligomer as defined above, for use in a method of treatment, prophylaxis or diagnosis, as will be described in more detail hereinbelow.

In accordance with additional aspects thereof, the invention also relates to pharmaceutical compositions comprising a pharmaceutical carrier and a pharmaceutically effective amount of a l-acyl-3-hydroxy-4-hydroxymethyl-pyrrolidine derivative of formula F and/or a pharmaceutically effective amount of one of said oligomers disclosed above, as will be described in more detail hereinbelow.

In accordance with additional aspects thereof, the invention also relates to the use of a l-acyl-3-hydroxy-4-hydroxymethyl-pyrrolidine derivative of formula D and/or of an oligomer as disclosed above, for the manufacture of a medicament for the treatment or prophylaxis of a viral infection, as will be described in more detail hereinbelow.

In accordance still additional aspects thereof, the invention relates to a diagnostic composition comprising an oligomer as disclosed above and a marker as well as to the use of such an oligomer for detecting or analysing a DNA or RNA sequence from a subject, as will be described in more detail hereinbelow. In accordance with additional aspects thereof, the invention also relates to the use of an oligomer as disclosed above for the manufacture of a diagnostic composition for detecting or analysing DNA or RNA sequence from a subject which preferably is a mammal.

Uses, therapeutic and diagnostic applications of the nucleoside analogues of the invention

Generally speaking, the nucleoside analogues disclosed herein may be employed as such in therapy for interfering with the metabolism of nucleosides be. it bacterial, viral or tumoral or in diagnosis for detecting nucleic acids in combination with a suitable marker which may be readily determined by those skilled in the art.

Furthermore, the new nucleoside analogues disclosed herein can be used alone or as sequences as to build up DNA analogues with the aim to control gene expression and eventually biological processes, thus providing a new approach to gene functional PCR clamping studies and anti-infectives development.

Possibly, coded information can be inserted into macromolecules such as DNA analogues in a structured way with the new nucleoside analogues disclosed herein, with the intention of recovering that information at a later date, so that the new nucleoside analogues can be used for preparation of nanobarcodes for security markers.

Also, the nucleoside analogues disclosed herein can be employed for the synthesis of DNA chimeras or as termination sequences for diagnostic and therapeutic applications as will be illustrated in greater detail hereinbelow.

Uses, therapeutic and diagnostic applications of the oligomers of the invention

As the oligomers of the invention are capable of significant single-stranded or double- stranded target nucleic acid binding activity to form duplexes, triplexes or other forms of stable association, these oligomers are useful in diagnosis and therapy of diseases that are associated with expression of one or more genes such as those associated with pathological conditions.

Therapeutic applications can employ the oligomers to specifically inhibit the expression of genes (or inhibit translation of RNA sequences encoded by those genes) that are associated with either the establishment or the maintenance of a pathological condition.

Exemplary genes or RNAs encoded by those genes that can be targeted include those that encode enzymes, hormones, serum proteins, transmembrane proteins, adhesion molecules (LFA-I, GPIIb/IIIa, ELAM-I, VACM-I, ICAM-I, E-selectin, and the like), receptor molecules including cytokine receptors (IL-I receptor, IL-2 receptor and the like), cytokines (IL-I, IL-2, IL-3, IL-4, IL-6 and the like), oncogenes, growth factors, and interleukins. Target genes or RNAs can be associated with any pathological condition such as those associated with inflammatory conditions, cardiovascular disorders, immune reactions, cancer, viral infections, bacterial infections and the like.

Oligomers of the present invention are suitable for use in both in vivo and ex vivo therapeutic applications.

Indications for ex vivo uses include treatment of cells such as bone marrow or peripheral blood in conditions such as leukemia (chrome myelogenous leukemia, acute lymphocytic leukemia) or viral infection.

Target genes or RNAs encoded by those genes that can serve as targets for cancer treatments include oncogenes, such as ras, k-ras, bcl-2, c-myb, bcr, c-myc, c-abl or overexpressed sequences such as mdm2, oncostatin M, IL-6 (Kaposi's sarcoma), HER-2 and translocations such as bcr/abl.

The oligomers may be used to inhibit proliferation of DNA or RNA viruses such as herpes viruses, papilloma viruses and the like. Viral gene sequences or RNAs encoded by those genes such as polymerase or reverse transcriptase genes of herpes viruses such as CMV, HSV-I, HSV-2, retroviruses such as HTLV-I, HIV-I, HIV-2, or other DNA or RNA viruses such as HBV, HPV, VZV, influenza virus, rhinovirus and the like are also suitable targets. Application of specifically binding oligomers can be used in conjunction with other therapeutic treatments.

Other therapeutic indications for the oligomers of the invention include: (1) modulation of inflammatory responses by modulating expression of genes such as IL-I receptor, IL- 1, ICAM-I or E-Selectin that play a role in mediating inflammation and (2) modulation of cellular proliferation in conditions such as arterial occlusion (restenosis) after angioplasty by modulating the expression of (a) growth or mitogenic factors such as non-muscle myosin, myc, fos, PCNA, PDGF or FGF or their receptors, or (b) cell proliferation factors such as c-myb. Other suitable proliferation factors or signal transduction factors such as TGFα, TGFβ, IL-6, γINF, protein kinase C, tyrosine kinases (such as p210, pi 90), may be targeted for treatment of psoriasis or other conditions, hi addition, EGF receptor, TGFα or MHC alleles may be targeted in auto immune diseases.

Delivery of the oligomers of the invention into cells can be enhanced by any suitable method including calcium phosphate, DMSO, glycerol or dextran transfection, electroporation or by the use of cationic anionic and/or neutral lipid compositions or liposomes by methods described in International Publication Nos. WO 90/14074, WO 91/16024, WO 91/17424 and U.S. Patent 4,897,355. The oligomers can be introduced into cells by complexation with cationic lipids such as DOTMA (which may or may not form liposomes) which complex is then contacted with the cells. Suitable cationic lipids include but are not limited to N-(2,3-di(9-(Z)octadecenyloxyl))-prop-l-yl-N,N,N- trimethylammonium (DOTMA) and its salts, l-O-oleyl-2-O-oleyl-3-dimethylamino- propyl-β-hydroxyethylammonium and its salts and 1, 2-bis (oleyloxy) -3- (trimethylammonium)propane and its salts.

Enhanced delivery of the oligomers of the invention can also be mediated by the use of (i) viruses such as Sendai virus (Bartzatt, R., Biotechnol Appl Biochem (1989), 11:133- 135) or adenovirus (Wagner, E., et al, Proc Natl Acad Sci (1992) 89:6099-6013); (ii) polyamine or polycation conjugates using compounds such as polylysine, protamine or Nl, N12-bis(ethyl)spermine (Wagner, E., et al, Proc Natl Acad Sci (1991) 88:4255- 4259; Zenke, M., et al, Proc Natl Acad Sci (1990) 87:3655-3659; Chank, B.K., et al, Biochem Biophys Res Commun (1988) 157:264-270; U.S. Patent 5,138,045); (iii) lipopolyamine complexes using compounds such as lipospermine (Behr, J.-P., et al, Proc Natl Acad Sci (1989) 86:6982-6986; Loeffler, J.P., et al J Neurochem (1990) 54:1812-1815); (iv) anionic, neutral or pH sensitive lipids using compounds including anionic phospholipids such as phosphatidyl glycerol, cardiolipin, phosphatidic acid or phosphatidylethanolamine (Lee, K.-D., et al, Biochim Biophys Acta (1992) 1103:185- 197; Cheddar, G., et al, Arch Biochem Biophys (1992) 294:188-192; Yoshimura, T., et al, Biochem Int (1990) 20:697-706); (v) conjugates with compounds such as transferrin or biotin or (vi) conjugates with compounds such as serum proteins (including albumin or antibodies), glycoproteins or polymers (including polyethylene glycol) that enhance pharmacokinetic properties of oligomers in a subject.

As used herein, "transfection" refers to any method that is suitable for delivery of oligomers into cells. Any reagent such as a lipid or any agent such as a virus that can be used in transfection protocols is collectively referred to herein as a "permeation enhancing agent".

Delivery of the oligomers into cells can be via cotransfection with other nucleic acids such as (i) expressible DNA fragments encoding a protein(s) or a protein fragment or (ii) translatable RNAs that encode a protein(s) or a protein fragment.

The oligomers can thus be incorporated into any suitable formulation that enhances delivery of the oligomers into cells. Suitable pharmaceutical formulations also include those commonly used in applications where compounds are delivered into cells or tissues by topical administration. Compounds such as polyethylene glycol, propylene glycol, azone, nonoxonyl-9, oleic acid, DMSO, polyamines or lipopolyamines can be used in topical preparations that contain the oligomers.

The oligomers of the invention can be conveniently used as reagents for research or production purposes where inhibition of gene expression is desired. There are currently very few reagents available that efficiently and specifically inhibit the expression of a target gene by any mechanism. Oligomers that have been previously reported to inhibit target gene expression frequently have non-specific effects and/or do not reduce target gene expression to very low levels (less than about 40% of uninhibited levels).

Thus, the oligomers as described herein constitute a reagent that can be used in methods of inhibiting expression of a selected protein or proteins in a subject or in cells wherein the proteins are encoded by DNA sequences and the proteins are translated from RNA sequences, comprising the steps of: introducing an oligomer of the invention into-the cells; and permitting the oligomer to form a triplex with the DNA or RNA or a duplex with the DNA or RNA whereby expression of the protein or proteins is inhibited. The methods and oligomers of the present invention are suitable for modulating gene expression in both procaryotic and eucaryotic cells such as bacterial, fungal parasite, yeast and mammalian cells.

Oligomers containing as few as about 8 modified nucleosides can be used to effect inhibition of target protein(s) expression by formation of duplex or triplex structures with target nucleic acid sequences. However, linear oligomers used to inhibit target protein expression via duplex or triplex formation will preferably have from about 10 to about 20 modified nucleoside residues.

Oligomers containing modified nucleosides of the invention can be conveniently circularized as described in International patent application No. WO 92/19732; Kool, E.T. J Am Chem Soc (1991) 113:6265-6266; Prakash, G., et al. J Am Chem Soc (1992) 114:3523-3527). Such oligomers are suitable for binding to single-stranded or double- stranded nucleic acid targets. Circular oligomers can be of various sizes. Such oligomers in a size range of about 22-50 nucleomonomers can be conveniently prepared. The circular oligomers can have from about three to about six nucleomonomer residues in the loop region that separate binding domains of the oligomer as described (Prakash, G. ibid).

The oligomers can be utilized to modulate target gene expression by inhibiting the interaction of nucleic acid binding proteins responsible for modulating transcription (Maher, L. J., et al, Science (1989) 245:725-730) or translation. The oligomers are thus suitable as sequence-specific agents that compete with nucleic acid binding proteins (including ribosomes, RNA polymerases, DNA polymerases, translational initiation factors, transcription factors that either increase or decrease transcription, protein- hormone transcription factors and the like). Appropriately designed oligomers can thus be used to increase target protein synthesis through mechanisms such as binding to or near a regulatory site that transcription factors use to repress expression or by inhibiting the expression of a selected repressor protein itself.

hi therapeutic applications, the oligomers are utilized in a manner appropriate for treatment of a variety of conditions by inhibiting expression of appropriate target genes. For such therapy, the oligomers can be formulated for a variety of modes of administration, including systemic, topical or localized administration. Techniques and formulations generally can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, latest edition. The oligomer active ingredient is generally combined with a carrier such as a diluent or excipient which can include fillers, extenders, binders, wetting agents, disintegrants, surface-active agents, or lubricants, depending on the nature of the mode of administration and dosage forms.

Typical dosage forms include tablets, powders, liquid preparations including suspensions, emulsions and solutions, granules, capsules and suppositories, as well as liquid preparations for injections, including liposome preparations.

For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the oligomers of the invention are formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the oligomers can be formulated in solid form and redissolved or suspended immediately prior to use.

Lyophilized forms are also included. Dosages that can be used for systemic administration preferably range from about 0.01 mg/Kg to 50 mg/Kg administered once or twice per day. However, different dosing schedules can be utilized depending on (i) the potency of an individual oligomer at inhibiting the activity of its target DNA or RNA, (ii) the severity or extent of a pathological disease state associated with a given target gene, or (iii) the pharmacokinetic behaviour of a given oligomer.

Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, bile salts and fusidic acid derivatives for transmucosal administration. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be through use of nasal sprays, for example, or suppositories. For oral administration, the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics.

For topical administration, the oligomers of the invention are formulated into ointments, salves, gels, or creams, as is generally known in the art.

Formulation of the invention oligomers for ocular indications such as viral infections would be based on standard compositions known in the art.

In addition to use in therapy, the oligomers of the invention can be used as diagnostic reagents to detect the presence or absence of the target nucleic acid sequences to which they specifically bind. The enhanced binding affinity of the invention oligomers is an advantage for their use as primers and probes.

Diagnostic tests can be conducted by hybridization through either double or triple helix formation which is then detected by conventional means. For example, the oligomers can be labelled using radioactive, fluorescent, or chromogenic labels and the presence of label bound to solid support detected. Alternatively, the presence of a double or triple helix can be detected by antibodies which specifically recognize these forms. Means for conducting assays using such oligomers as probes are generally known.

The use of the oligomers of the invention as diagnostic agents by triple helix formation is advantageous since triple helices form under mild conditions and the assays can thus be carried out without subjecting test specimens to harsh conditions.

Diagnostic assays based on detection of RNA for identification of bacteria, fungi or protozoa sequences often require isolation of RNA from samples or organisms grown in the laboratory, which is laborious and time consuming, as RNA is extremely sensitive to ubiquitous nucleases.

The oligomer probes can also incorporate additional modifications such as modified linkages that render the oligomer especially nuclease stable, and would thus be useful for assays conducted in the presence of cell or tissue extracts which normally contain nuclease activity. Oligomers containing terminal modifications often retain their capacity to bind to complementary sequences without loss of specificity (Uhlmann et al., Chemical Reviews (1990) 90:543-584). As set forth above, the invention probes can also contain linkers that permit specific binding to alternate DNA strands by incorporating a linker that permits such binding (Froehler, B.C., et al, Biochemistry

(1992) 31 :1603-1609); Home et al., J Am Chem Soc (1990) 112:2435-2437).

Incorporation of the oligomers of the present invention into probes that also contain covalent crosslinking agents has the potential to increase sensitivity and reduce background in diagnostic or detection assays. In addition, the use of crosslinking agents will permit novel assay modifications such as (1) the use of the crosslink to increase probe discrimination, (2) incorporation of a denaturing wash step to reduce background and (3) carrying out hybridization and cross linking at or near the melting temperature of the hybrid to reduce secondary structure in the target and to increase probe specificity.

The oligomers of the invention are suitable for use in diagnostic assays that employ methods wherein either the oligomer or nucleic acid to be detected are covalently attached to a solid support as described (U.S. Patent No. 4,775,619). The PNA oligomers are also suitable for use in diagnostic assays that rely on polymerase chain reaction techniques to amplify target sequences according to described methods (European Patent Publication No. 0 393 744). The oligomers of the invention containing a 3' terminus that can serve as a primer are compatible with polymerases used in polymerase chain reaction methods such as the Taq orVentδ (New England Biolabs) polymerase. The PNA oligomers of the invention can thus be utilized as primers in PCR protocols.

The oligomers of the invention are also useful as primers that are discrete sequences or as primers with a random sequence.

Random sequence primers can be generally about 6, 7, or 8 nucleomonomers in length. Such primers can be used in various nucleic acid amplification protocols (PCR, ligase chain reaction, etc) or in cloning protocols. The substitute linkages of the invention generally do not interfere with the capacity of the oligomer to function as a primer. Oligomers of the invention having 2' modifications at sites other than the 3¹ terminal residue, other modifications that render the oligomer RNase H incompetent or otherwise nuclease stable can be advantageously used as probes or primers for RNA or DNA sequences in cellular extracts or other solutions that contain nucleases. Thus, the oligomers can be used in protocols for amplifying nucleic acid in a sample by mixing the oligomer with a sample containing target nucleic acid, followed by hybridization of the oligomer with the target nucleic acid and amplifying the target nucleic acid by PCR, LCR or other suitable methods.

The oligomers derivatised to chelating agents such as EDTA, DTPA or analogs of 1,2- diaminocyclohexane acetic acid can be utilized in various in vitro diagnostic assays as described (U.S. Patent Nos. 4,772,548, 4,707,440 and 4,707,352). Alternatively, oligomers of the invention can be derivative with crosslinking agents such as 5-(3- iodoacetamidoprop-l-yl)-2'-deoxyuridine or 5-(3-(4-bromobutyramido)-prop-l-yl)-2'- deoxyuridine and used in various assay methods or kits as described (International Publication No. WO 90/14353).

In addition to the foregoing uses, the ability of the oligomers to inhibit gene expression can be verified in in vitro systems by measuring the levels of expression in subject cells or in recombinant systems, by any suitable method (Graessmann, M., et al., Nucleic Acids Res (1991) 19:53-59).

Eventually, the oligomers of the present invention can be employed for the use in structural nanotechnology in place of DNA, with the aim to obtain objects, lattices or devices (Mao, C, et al., Nature (1999), 397: 144-146) or for organizing nanogold particles (Xiao, S., et al., J. Nanopart. Res. (2002) 4: 313-317).

Additional objects, features and advantages of the present invention will become more readily apparent from the following non-limitative examples thereof, given hereinbelow for illustration purposes with reference to the accompanying drawing figures. It is to be understood, however, that the following examples and drawings are designed solely for the purpose of illustration and not as a definition of the limits of the invention, for which reference should be made to the appended claims.

Brief Description of the Drawings

Figure 1 is a schematic representation of the synthesis of a preferred 3-trialkylsilyloxy- 4-alkoxycarbonyl lactam of formula G useful for the preparation of preferred l-alkyl-3- hydroxy-4-hydroxymethylpyrrolidine derivatives of formula B according to the invention;

Figure 2 is a schematic representation of the synthesis starting from the preferred 3- trialkylsilyloxy-4-alkoxycarbonyl lactam of formula G of preferred di-protected 1-alkyl- 3-hydroxy-4-hydroxymethylpyrrolidine derivatives of formula B;

Figures 3 and 4 are schematic representations of the synthesis of preferred di-protected l-acyl-3-hydroxy-4-hydroxymethylpyrrolidine derivatives of formula A starting from preferred l-alkyl-3-hydroxy-4-hydroxymethylpyrrolidine derivatives of formula B;

Figures 5 and 6 are schematic representations of the synthesis of preferred nucleoside analogues of formula D of this invention starting from the di-protected l-acyl-3-amino- 4-hydroxymethylpyrrolidine derivatives of formula A.

Detailed description of the presently preferred embodiments

Definitions

Nucleomonomer. As used herein, the term "nucleomonomer" means a nucleoside analog including (1) a nucleobase covalently linked to (2) an optionally protected 3- amino-4-hydroxymethylpyrrolidine ring. The invention nucleomonomers lack a sugar or furanose moiety such as ribose or deoxyribose and can be linked to form PNA oligomers that bind to target or complementary base sequences in nucleic acids in a sequence specific manner.

Nucleobase. As used herein, the term "nucleobase" includes those moieties which contain not only the naturally occurring purine and pyrimidine heterocycles, but also optionally protected heterocycle analogs or derivatives and tautomers thereof, such as optionally protected deazacarbocyclic derivatives thereof. Purines include adenine, guanine, xanthine and hypoxanthine and analogs or derivatives thereof. Pyrimidines include thymin, uracil and cytosine and their analogs or derivatives.

Nucleoside. As used herein, "nucleoside" means a nucleobase covalently attached to optionally protected 3-amino-4-hydroxymethylpyrrolidine ring.

Linkage. As used herein, "linkage" means the nonphosphorous containing carbamate linkages of the invention that link adjacent nucleomonomers. Oligomers. "Oligomers" are defined herein as two or more nucleomonomers covalently coupled to each other by a linkage moiety as defined above. Thus, an oligomer can have as few as two convalently linked nucleomonomers (a dimer). Oligomers can be binding competent and, thus, can base pair with cognate single stranded or double-stranded nucleic acid sequences. Short oligomers (e.g. dimers - hexamers) are also useful for diagnostic or therapeutic purposes as described herein. An oligomer according to the invention is exemplified by the structure shown in Figure 6.

Protecting group. "Protecting group" as used herein includes any group capable of preventing the O-atom or N-atom to which it is attached from participating in a reaction or bonding. Such protecting groups for O- and N-atoms in nucleomonomers are described and methods for their introduction are conventionally known in the art. Protecting groups also include any group capable of preventing reactions and bonding at carboxylic acids, thiols and the like.

Transfection. "Transfection" as used herein refers to any suitable method that for enhanced delivery of oligomers into cells.

Subject. "Subject" as used herein means a plant or an animal, including a mammal, particularly a human.

Sequence-specific binding. "Sequence-specific binding" is used herein in its commonly accepted sense to define the binding which occurs between, for example, an oligomer and a DNA or RNA target sequence via pairs of bases which form hydrogen bonds according to conventional rules.

For the purposes of the present description and of the claims that follow, except where otherwise indicated, all numbers expressing amounts, quantities, percentages, and so forth, are to be understood as being modified in all instances by the term "about". Also, all ranges include any combination of the maximum and minimum points disclosed and include any intermediate ranges therein, which may or may not be specifically enumerated herein.

Additionally, for the purposes of the present description and of the claims that follow, any definition of moieties including a plurality of Carbon atoms by means of the notation C_n-C_1n disclosing only the end points, wherein n and m are integers of specified values, such as for example C₁-C₁₅, is to be understood as including all the isomers falling within and including the end points disclosed, which may or may not be specifically indicated herein. In the specific non-limitative examples which follow, the following acronyms will be employed:

Ac = CH₃CO

Bz = C₆H₅CO

DABCO = 1,4-Diazabicyclo[2.2.2]octane

DBU = l,8-Diazabicyclo[5.4.0]undec-7-ene

DCM = Dichloromethane

DIPEA = Diisopropylethylamine

DMAP = 4-Dimethylaminopyridine

DMF = Dimethylformamide

DMSO = Dimethylsulfoxide

LiHDMS = Lithium bis(trimethylsilyl)amide

TBDMS = t-butyldimethylsilyl

THF = Tetrahydrofuran

Detailed description of the preferred embodiments

EXAMPLES 1-3

Preparation of a preferred 3-trialkylsilyloxy-4-alkoxycarbonyl lactam of formula G: (3/f,4i?,riS)-3-fert-Butyldimethylsilyloxy-4-methoxycarbonyl-l-(r-phenylethyl)pyrroli- din-2-one, 3, and its (35,45,1'S)-IsOmCr, 4, according to the reaction path illustrated in Fig. 1 (chart 1).

EXAMPLE 1

1 -Ethyl-4-methyl-2-hvdroxy-3-methylenebutanedioate, 1

EtOO

To a mixture containing ethyl glyoxalate (13.3 g, 50% solution in toluene, 65 mmol) and methyl acrylate (4.5 mL, 50 mmol) in THF (30 mL), DMSO (7.5 mL) and H₂O (4.5 mL, 250 mmol), DABCO (2.8 g, 25 mmol) was added. After stirring for 12 h at 20°C, the oil was purified by silica gel chromatography (cyclohexane:EtOAc 8:2) to give 1 (8.2 g, yield: 87%) as a colourless oil. ¹H NMR (200 MHz, CDCl₃): δ 1.24 (t, J = 7.2, 3H), 3.51 (d, J = 6.2, OH), 3.76 (s, 3H), 4.22 (q, J = 7.2, 2H), 4.83 (d, J = 6.2, IH), 5.92 (s, IH), 6.34 (s, IH).

EXAMPLE 2

1 -Ethyl-4-methyl-2-/gr/-butyldimethylsilyloxy-3 -methylenebutanedioate, 2

To a solution containing the ester 1 (8.16 g, 43 mmol) and t-butyldimethylchlorosilane (7.13 g, 47.3 mmol) in DCM (48 mL) at 20°C, imidazole (3.22 g, 47.3 mmol) was added, and the mixture was stirred for 4 h. After removal of the solvent under reduced pressure, the residue was dissolved in ethyl acetate (50 mL), brine (50 mL) was added and the mixture was extracted with ethyl acetate (2 x 100 mL). After drying (Na₂SO₄), the solvent was removed in vacuo and the residue purified by silica gel chromatography (cyclohexane:EtOAc 9:1), to give the compound 2 (10.79 g, yield: 89%) as a colourless oil. ¹H NMR (200 MHz, CDCl₃): δ 0.06 (s, 3H), 0.10 (s, 3H), 0.87 (s, 9H), 1.21 (t, 3H, J = 7.1), 3.72 (s, 3H), 4.13 (q, 2H, J = 7.1), 5.03 (s, IH), 6.0 (s, IH), 6.31 (s, IH).

EXAMPLE 3

(3i?,4i?J'5^-3-/er?-Butyldimethylsilyloxy-4-methoxycarbonyl-l-π'-phenylethyl)pyrroli- din-2-one. 3. and its GSΛS.rSVisomer. 4

TBDMSO

COOMe

EtOOC

2 -

To a solution containing the silyl derivative 2 (6.0 g, 20 mmol) in methanol (50 mL),

(S)-phenylethylamine (2.4 g, 20 mmol) was added and the mixture was stirred for 2 h at 20°C. After removal of the solvent, the residue was dissolved in toluene (50 mL) and the solution was refluxed for 2h. The solvent was then evaporated under reduced pressure, and the residue was dissolved in ethyl acetate (50 mL) that was washed with

3M HCl. The solvent was dried (Na₂SO₄) and evaporated in vacuo and the residue was purified by silica gel chromatography (cyclohexane:EtOAc 9:1), to give compound 3 (2.1 g, yield: 40%) as a yellow oil. Further elution gave the isomer 4 (2.0 g, yield: 38%) as a yellow oil.

Compound 3: ¹H NMR (200 MHz, CDCl₃): δ 0.14 (s, 3H), 0.18 (s, 3H), 0.88 (s, 9H), 1.56 (d, 3H, J = 7.2), 2.99 (dd, IH, J = 7.3. J = 9.4), 3.04 - 3.16 (m, IH), 3.87 (s, 3H), 3.69 (dd, IH, J = 7.6, J = 9.4), 4.45 (d, IH, J = 6.7), 5.44 (q, IH, J = 7.2), 7.23-7.36 (m, 5 ArH).

Compound 4: ¹H NMR (200 MHz, CDCl₃): δ 0.13 (s, 3H), 0.17 (s, 3H), 0.84 (s, 9H), 1.53 (d, 3H, J = 7.1), 3.14 - 3.26 (m, IH), 3.30 (dd, IH, J = 9.5, J = 9.6), 3.39 (dd, IH, J = 6.8, J = 9.6), 3.63 (s, 3H), 4.47 (d, IH, J = 6.4), 5.45 (q, IH, J = 7.1), 7.25-7.35 (m, 5 ArH).

EXAMPLES 4-6

Preparation of preferred di-protected l-alkyl-3-hydroxy-4-hydroxymethylpyrrolidine of formula B: (3i?,4i?,r5)-3-Acetoxy-4-acetoxymethyl-l-(r-phenylethyl)pyrrolidine, 6, (3if,4i?,rS)-3-Benzoyloxy-4-benzoyloxymethyl-l-(r-phenylethyl)pyrrolidine, 7, according to the reaction paths illustrated in Fig. 2 (chart 2). EXAMPLE 4

(3RAR, 1 'S)-3-Hvdroxy-4-hvdroxymethyl- 1 -( 1 '-phenylethvQpyrrolidine. 5

The product 3 (0.38 g, 1 mmol) was refluxed for 1 h in CH₃OH (1 mL) containing 12 M HCl (1 mL). Then, the solvents were removed under reduced pressure and reflux was repeated twice under the same conditions. The residue was subsequently dissolved in dry THF (3 mL) and the solution was cooled to 0°C. Lithium bis(trimethylsilyl)amide (LiHMDS) (3 mmol, 3 mL of 1 M solution in THF) was added and the mixture was stirred for 5 min. After addition of 1 M HCl (3 mL), the mixture was extracted with EtOAc (50 mL) and dried (Na₂SO₄). The solvent was removed under reduced pressure and the residue was added to a solution of BH₃ in THF (1 M, 5 mL). The solution was refluxed at 70 °C for 1.5 h under argon and then cooled to 20⁰C. Methanol (5 mL) and 12 M HCl (5 mL) were added and the mixture was refluxed for 30 min. The solvents were eliminated in vacuo, the residue was again dissolved in methanol (5 mL) and 12 M HCl (5 mL) and the mixture was refluxed at 70 ⁰C for 1 h. This procedure was repeated twice and, after addition of a saturated Na₂CO₃ aqueous solution (10 mL), extraction was performed with EtOAc (2 x 50 mL). The organic layer was dried (Na₂SO₄) and solvents evaporated under reduced pressure to give the product 5 with a yield of 70% and a purity higher than 90%, which was directly used without further purification. ¹H NMR (200 MHz, CDCl₃): δ 1.41 (d, 3H, J = 6.8), 2.15 - 2.27 (m, IH), 2.46 (dd, IH, J = 5.4, J = 9.5), 2.62 (dd, IH, J = 3.4, J = 10.3), 2.72 - 2.92 (m, 2H + 2 OH), 3.30 (q, IH, J = 6.8), 3.68 (d, 2H, J = 5.4), 4.18 - 4.26 (m, IH), 7.21 - 7.37 (m, 5 ArH).

EXAMPLE 5

(3RAR, 1 '■S^-3-Acetoxy-4-acetoxymethyl-l -(I '-phenylethyDpyrrolidine, 6

The compound 5 (0.63 g, 2.6 mmol) was dissolved in DCM (10 mL) containing DMAP (0.95 g, 7.8 mmol), and acetyl chloride (0.46 mL, 6.5 mmol) was added at 20°C. After 2 h the mixture was poured in ice water and extracted with ethyl acetate (2 x 20 mL). The organic layer was separated and dried (Na₂SO₄) and solvents were eventually removed under reduced pressure. The residue was purified by silica gel chromatography (cyclohexane:ethyl acetate 50:50), to give 6 (0.54 g, yield: 90%) as a colourless oil. ¹H NMR (200 MHz, CDCl₃): δ 1.35 (d, 3H, J = 6.5), 2.02 (s, 3H), 2.05 (s, 3H), 2.12-2.53 (m, 3H), 2.62-2.84 (m, 2H), 3.21 (q, IH, J = 6.5), 4.04 (dd, IH, J = 7.0, J = 11.0), 4.16 (dd, IH, J = 6.3, J = 11.0), 4.93 (m, IH), 7.18-7.34 (m, 5 ArH).

EXAMPLE 6

(3RAR, 1 'SV3-Berizoyloxy-4-benzoyloxymethyl-l -(I '-phenylethvQpyrrolidine. 7

The compound 5 (0.21 g, 1 mmol) was dissolved in DCM (10 mL) containing DMAP (0.36 g, 2.85 mmol), and benzoyl chloride (277 μL) was added at 0⁰C. After 45 min the mixture was poured in ice water and extracted with ethyl acetate (3 x 10 mL). The organic layer was separated and dried (Na₂SO₄) and solvents were eventually removed under reduced pressure. The residue was purified by silica gel chromatography (cyclohexane: EtOAc 1 :1), to give 7 (0.36 g; yield: 85%) as a colourless oil. ¹H NMR (200 MHz, CDCl₃): δ 1.41 (d, 3H, J = 6.8), 2.32 (dd, IH, J = 6.7, J = 8.1), 2.74 (dd, IH, J = 3.6, J = 8.2), 2.79 - 2.95 (m, IH), 3.01 (dd, IH, J = 6.4, J = 8.2), 3.15 (dd, IH, J = 8.1, J = 8.1), 3.32 (q, IH, J - 6.8), 4.59 (d, 2H, J = 6.6), 5.27 - 5.36 (m, IH), 7.18 - 7.65 (m, 11 ArH), 7.97 - 8.07 (m, 4 ArH).

EXAMPLES 7-8

Preparation of preferred di-protected l-acyl-3-hydroxy-4-hydroxymethyl derivatives of formula A: (3/?,4i?)-3-Acetoxy-4-acetoxymethyl-l-bromoacetylpyrrolidine, 8, (3RAR)- 3-Benzoyloxy-4-benzoyloxymethyl-l-bromoacetylpyrrolidine, 9, according to the reaction paths illustrated in Figs. 3 and 4 (charts 3 and 4).

EXAMPLE 7 (3RAR)-3 - Acetoxy-4-acetoxymethyl- 1 -bromoacetylpyrrolidine, 8

The compound 6 (122 mg, 0.4 mmol) was dissolved in dry DCM (1.2 mL) and then bromoacetyl bromide (106 μL, 1.2 mmol) was dropped in at 20⁰C. After 30 min pyridine (65 μL) was added, and the mixture was stirred for 12 h. Then EtOAc (5 mL) and water (2 mL) were added, the organic layer was separated, subsequently washed with Na₂CO₃ saturated solution (2 mL), 3 M HCl (2 mL) and water (5 mL) and eventually dried (Na₂SO₄). After removal of the solvent, the residue was chromatographed on silica gel (cyclohexane: EtOAc 4:6 as eluant), to give pure 8 (90 mg, yield: 70%) as a pale yellow oil. ¹H NMR (200 MHz, CDCl₃): δ 2.05 (s, 3H, 40%), 2.06 (s, 3H, 60%), 2.07 (s, 3H, 60%), 2.08 (s, 3H, 40%), 2.57 - 2.78 (m, IH), 3.41 - 3.62 (m, 2H), 3.71 - 3.89 (m, IH), 3.77 (s, 2H, 60%), 3.80 (s, 2H, 40%), 3.93 - 4.0 (m, IH), 4.03 - 4.09 (m, 2H), 5.12 - 5.19 (m, IH).

EXAMPLE 8

(3i?,4i-)-3-Benzoyloxy-4-ben2oyloxymethyl- 1 -bromoacetylpyrrolidine, 9

To the compound 7 (169 mg, 0.4 mmol) dissolved in dry DCM (1.2 mL), bromoacetyl bromide (106 μL, 1.2 mmol) was added at 20⁰C, followed after 30 min by pyridine (65 μL), and the mixture was stirred for 12 h. Then EtOAc (5 mL) and water (2 mL) were added, the organic layer was separated, subsequently washed with Na₂CO₃ saturated solution (2 mL), 3 M HCl (2 mL) and water (5 mL) and eventually dried (Na₂SO₄). After removal of the solvent, the residue was chromatographed on silica gel (cyclohexane: EtOAc 6:4), to give pure 9 (126 mg, yield: 72%) as a yellow oil. ¹H NMR (200 MHz, CDCl₃): δ 2.89 - 3.14 (m, IH), 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, IH), 7.36 - 7.51 (m, 4 ArH), 7.53 - 7.65 (m, 2 AiH), 7.97 - 8.07 (m, 4 AxH).

EXAMPLES 9-14

Preparation of preferred nucleoside analogues of formula D: (3i?,4i?)-3-Acetoxy-4- acetoxymethyl- 1 -( 1' -thymidylacetyl)pyrrolidine, 11 , (3/?,4i?)-3 -Benzoyloxy-4- benzoyloxymethyl-l-(r-citosylacetyl)pyrrolidine, 12, (3i?,4i?)-3-Acetoxy-4- acetoxymethyl-l-(l '-adenylacetyl)pyrrolidine, 13, (3/?,4i?)-3-Acetoxy-4- acetoxymethyl-l-[(2-amino-6-benzyloxypurin-9-yl)acetyl]pyrrolidine, 14, and (3R,4R)- 3-Benzoyloxy-4-benzoyloxymethyl-l-(r-thymidylacetyl)pyrrolidine, 15, according to the reaction paths illustrated in Figs. 5 and 6 (charts 5 and 6).

EXAMPLE 9

2-Amino-6-benzyloxypurine, 10

Benzyl alcohol (37.5 g, 347 mmol) and sodium hydroxide (2.96 g, 74 mmol) were mixed and sodium hydroxide was dissolved on heating at 80°C. After cooling, 2-amino- 6-chloropurine (6.0 g, 35 mmol) was added, and the mixture was heated at 90⁰C for 5 h under stirring. After cooling, EtOAc (120 ml) was added to the reaction mixture, and the mixture was extracted twice with 1% aqueous sodium hydroxide solution (70 mL). The aqueous alkali layers were combined, washed with EtOAc and then treated with 35% hydrochloric acid until pH 6-8. The precipitated crystals were collected by filtration and dried under reduced pressure, to give 2-amino-6-benzyloxypurine 10 (7.6 g, yield: 92%) as a white powder: ¹H NMR (200 MHz, CDCl₃): δ 5.46 (s, 2H), 6.24 (br s, 2H, NH), 7.24 - 7.54 (m, 5 ArH), 7.81 (s, IH), 12.43 (br s, IH, NH).

EXAMPLE 10

(3RARY3 - Acetoxy-4-acetoxymethyl- 1 -( 1' -thyrnidylacetvPpyrrolidine. 11

The compound 8 (63 mg, 0.19 mmol) was dissolved in dry DMF (0.5 mL) at 20 °C and then dried molecular sieves 4A (25 mg) were added under slow stirring. After 1 h, thymine (51 mg, 0.39 mmol) and dry K₂CO₃ (136 mg, 0.98 mmol) were added and the mixture was stirred for 2.5 h at 20°C. Then DCM (10 mL) and water (3 mL) were added under stirring and the organic layer was washed with water. The aqueous layer was again extracted with DCM (5 mL), the organic phases were collected and dried (Na₂SO₄) and after removal of the solvent under reduced pressure, the residue was purified by silica gel chromatography (EtOAc), to give the product 11 (44 mg, yield: 63%) as a viscous oil. ¹H NMR (200 MHz, CDCl₃): δ 1.92 (s, 3H), 2.08 (s, 3H), 2.09 (s, 3H, 50%), 2.10 (s, 3H, 50%), 2.54 - 2,68 (m, IH, 50%), 2.68 - 2,74 (m, IH, 50%), 3.43 - 3.65 (m, 2H), 3.75 - 3.87 (m, IH), 3.93 - 4.19 (m, 3H), 4.35 (dd, IH, J = 10.0, J = 16.8), 4.47 (dd, IH, J = 7.3, J = 16.8), 5.11 - 5.22 (m, IH), 7.01 (s, IH, 50%), 7.03 (s, IH, 50%), 8.42 (s, IH, NH, 50%), 8.43 (s, IH, NH, 50%).

EXAMPLE I l

(3RARY3 -Benzoyloxy-4-benzoyloxymethyl- 1 -( 1 ' -citosylacety Opyrrolidine, 12

The compound 9 (84 mg, 0.19 mmol) was dissolved in dry DMF (0.5 mL) and then dried molecular sieves 4A (50 mg) were added at 20 ⁰C under slow stirring. After 1 h, cytosine (50 mg, 0.38 mmol) and dry K₂CO₃ (133 mg, 0.96 mmol) were added and the mixture was stirred for another 2.5 h at 20⁰C. Then DCM (10 mL) and water (3 mL) were added under stirring and the organic layer was washed with water. The aqueous layer was again extracted with DCM (5 mL), the organic phases were collected and dried (Na₂SO₄) and after removal of the solvent under reduced pressure, the residue was purified by silica gel chromatography (EtOAc), to give the product 12 (62 mg, yield: 70%) as a viscous oil. ¹H NMR (200 MHz, DMSO-(I₆): δ 2.75 - 2.92 (m, IH, 50%), 2.93 - 3.06 (m, IH, 50%), 3.12 - 4.16 (m, 3H), 4.01 - 4.20 (m, IH), 4.22 - 4.43 (m, 2H), 4.53 (s, 2H, 50%), 4.59 (s, 2H, 50%), 5.35 - 5.45 (m, IH, 50%), 5.45 - 5.55 (m, IH, 50%), 5.80 (d, IH, J = 7.3, Het-H), 7.01 (br s, IH, Het-H), 7.40 - 7.69 (m, 6 ArH), 7.77 - 7.98 (m, 4 ArH).

EXAMPLE 12

(3RARY3 - Acetoxy-4-acetoxymethyl- 1 -( 1' -adenylacetyDpyrrolidine. 13

The compound 8 (40 mg, 0.12 mmol) was dissolved in dry THF (2 mL) at 20 °C and then adenine (19 mg, 0.14 mmol) and NaH (6 mg, 0.14 mmol) were added under stirring and the mixture stirred for 24 h at 20⁰C. Then EtOAc (5 mL) and water (2 mL) were added under stirring and the organic layer was washed with water. The aqueous layer was extracted with EtOAc (5 mL) and then with DCM (2 x 5 mL). The organic phases were collected and dried (Na₂SO₄) and after removal of the solvent under reduced pressure, the residue was purified by silica gel chromatography (DCM:MeOH 75:25), to give the product 13 (23 mg, yield: 48%) as a viscous oil. ¹H NMR (200 MHz, CDCl₃): δ 2.07 (s, 2 x 3H), 2.43 - 2.85 (m, 2H), 3.35 - 3.75 (m, 4H), 3.95 - 4.95 (m, 3H), 4.85 - 5.04 (m, IH), 5.18 (br s, 2H, NH₂), 8.31 (s, 1 Het-H), 8.35 (s, 1 Het-H).

EXAMPLE 13

(3J?.4i?V3-Acetoxy-4-acetoxymethyl-l-|^"(2-amino-6-benzyloxypurin-9-vπacetyl]pyrro- lidine. 14

The compound 8 (84 mg, 0.19 mmol) was dissolved in dry DMF (0.5 mL) at 20 ⁰C and then dried molecular sieves 4A (50 mg) were added under slow stirring. After 1 h, 2- amino-6-benzyloxypurine (50 mg, 0.38 mmol) and dry K₂CO₃ (133 mg, 0.96 mmol) were added and the mixture was stirred for another 2.5 h at 20°C. Then DCM (10 mL) and water (3 mL) were added under stirring and the organic layer was washed with water. The aqueous layer was again extracted with DCM (5 mL), the organic phases were collected and dried (Na₂SO₄) and after removal of the solvent under reduced pressure, the residue was purified by silica gel chromatography (EtOAc), to give the product 14 (41 mg, yield: 56%) as a viscous oil. ¹H NMR (200 MHz, CDCl₃): δ 2.06 (s, 2 x 3H), 2.55 - 2.82 (m, 2H), 3.45 - 3.91 (m, 4H), 3.99 - 4.22 (m, 3H), 4.90 (br s, 2H, NH₂), 5.12 - 5.20 (m, IH), 5.56 (s, 2H), 7.21 - 7.52 (m, 5 ArH), 7.72 (s, 1 Het-H).

EXAMPLE 14

(3RAR)-3 -Benzoyloxy-4-benzoyloxymethyl- 1 -( 1 '-thvmidylacetvDp yrrolidine, 15

The compound 9 (84 mg, 0.19 mmol) was dissolved in dry DMF (0.5 mL) at 20 °C and then dried molecular sieves 4 A (50 mg) were added under slow stirring. After 1 h, thymine (50 mg, 0.38 mmol) and dry K₂CO₃ (133 mg, 0.96 mmol) were added and the mixture was stirred for another 2.5 h at 20°C. Then DCM (10 mL) and water (3 mL) were added under stirring and the organic layer was washed with water. The aqueous layer was again extracted with DCM (5 mL), the organic phases were collected and dried (Na₂SO₄) and after removal of the solvent under reduced pressure, the residue was purified by silica gel chromatography (EtOAc), to give the product 15 (yield: 58%) as a viscous oil. ¹H NMR (200 MHz, CDCl₃): δ 1.94 (s, 3H), 2.54 - 2,68 (m, IH, 50%), 2.68 - 2,74 (m, IH, 50%), 3.43 - 3.65 (m, 2H), 3.75 - 3.87 (m, IH), 3.93 - 4.19 (m, 3H), 4.35 (dd, IH, J = 10.0, J = 16.8), 4.47 (dd, IH, J = 7.3, J = 16.8), 5.11 - 5.22 (m, IH), 7.03 (s, IH, 50%), 7.07 (s, IH, 50%), 8.42 (s, IH, NH, 50%), 8.43 (s, IH, NH, 50%).

In drawing figures 5 and 6 additional examples of nucleoside analogues according to the invention (not numbered) are shown which may be produced using the same synthetic approach disclosed in the Examples given hereinabove and the corresponding nucleobases.

*** * ***

The foregoing description of the disclosure illustrates and describes the present invention. Additionally, the disclosure shows and describes only the preferred embodiments of the invention but, as mentioned above, it is to be understood that the invention is capable of use in various other combinations, modifications, and environments and is capable of changes or modifications within the scope of the inventive concept as expressed herein, commensurate with the above teachings and/or the skill or knowledge of the relevant art.

The embodiments described hereinabove are further intended to explain best modes known of practicing the disclosure and to enable others skilled in the art to utilize the disclosure in such, or other, embodiments and with the various modifications required by the particular applications or uses of the disclosure. Accordingly, the description is not intended to-limit the disclosure to the form disclosed herein. Also, it is intended that the appended claims be construed to include alternative embodiments.

All publications, patents and patent applications cited in this specification are herein incorporated by reference, and for any and all purposes, as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference. In the case of inconsistencies, the present disclosure will prevail.

Claims

1. A l-acyl-S-hydroxy^-hydroxymethylpyrrolidine derivative of the formula

wherein

Ri and R₂ are independently selected from H and R-C(=O), wherein R is straight or branched alkyl, aryl, substituted aryl including from 1 to 5 heteroatoms, a heterocyclic group including from 1 to 3 heteroatoms and R₃ is a nucleobase.

2. A pyrrolidine derivative according to claim 1, wherein R is straight or branched C₁- Ci₅ alkyl, C₆-C₁₅ aryl, substituted C₅-C₁₅ aryl including from 1 to 5 heteroatoms, C₃-C₅ heterocyclic group including from 1 to 3 heteroatoms.

3. A pyrrolidine derivative according to claim 2, R is a straight or branched C₁-C₁₅ alkyl moiety selected from methyl, ethyl, propyl, isopropyl, t-butyl, 2-methylbutyl, allyl, 3,3- dimethylallyl, 3-methylbutyl, 3-methyl-2-butenyl, octyl, decyl.

4. A pyrrolidine derivative according to anyone of claim 1 , wherein said heteroatom is selected from oxygen, nitrogen, sulphur, fluorine, chlorine, bromine or iodine.

5. A pyrrolidine derivative according to claim 1 or 4, wherein R is an aryl or heterocyclic moiety selected from C₆H₅, CH₃C₆H₄, 2-furyl, 3-furyl, 4-methoxyphenyl, 4-nitrophenyl, 4-chlorophenyl, 4-bromophenyl, 4-iodophenyl, 4-trifluoromethyl, 2,6- dimethylphenyl, 2,6-dimethoxyphenyl, 2,4-dinitrophenyl, 2,4-dichlorophenyl, 2,4- dibromophenyl, 2,4-diiodophenyl, 2,4-dimethoxyphenyl.

6. A pyrrolidine derivative according to claim 1, wherein R₃ is a nucleobase is selected from thymine I, cytosine II, adenine III, guanine FV, uracil V, xanthine VI and hypoxanthine VII of the formula

IV

Vl VII

7. A pyrrolidine derivative according to claim 6, wherein said reactive functionalities NH or NH₂ are protected by means of a protecting group P independently selected from benzyl, benzoyl, 2,4-methoxybenzyl, benzhydryl, allyl, 4-methoxybenzyl (PMB), 4- methoxybenzyloxycarbonyl, 4-methoxybenzoyl, 4-nitrobenzoyl, 4-fluorobenzoyl, 4- bromobenzoyl, 4-iodobenzoyl, 1-naphthoyl, formyl, acetyl, propionyl, pivaloyl, benzyloxycarbonyl (Cbz), t-butoxycarbonyl (t-Boc), benzhydryloxycarbonyl (Bhc), adamantyloxycarbonyl, fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc).

8. A pyrrolidine derivative according to claim 6, wherein said nucleobase derivative is selected from:

IX Xl wherein P₁ is a protecting group selected from benzyloxycarbonyl (Cbz), 4- methoxybenzyloxycarbonyl, benzhydryloxycarbonyl (Bhc), fluorenylmethyloxycarbo- nyl (Fmoc), allyloxycarbonyl (Alloc), adamantyloxycarbonyl and P₂ is a protecting group selected from benzyl, 4-methoxybenzyl (PMB), 2,4-dimethoxybenzyl, benzhydryl, allyl.

9. A pyrrolidine derivative according to claim 6, wherein said deazacarbocyclic derivative of said nucleobases is selected from:

XII XIII XIV

wherein P₁ is a protecting group selected from benzyloxycarbonyl (Cbz), 4- methoxybenzyloxycarbonyl, benzhydryloxycarbonyl (Bhc), fluorenylmethyloxycarbo- nyl (Fmoc) allyloxycarbonyl (Alloc), adamantyloxycarbonyl and P₂ is a protecting group selected from benzyl, 4-methoxybenzyl (PMB), 2,4-methoxybenzyl, benzhydryl, allyl.

10. A l-acyl-3-hydroxy-4-hydroxymethylpyrrolidine derivative of the formula

wherein

Ri and R₂ are independently selected from H and R-C(=O), wherein R is as defined in anyone of claims 1-5 and R₄ is a leaving group.

11. A pyrrolidine derivative according to claim 10, wherein R₄ is a leaving group selected from Cl, Br, I, or OSO₂-Y, wherein Y is selected from methyl, isopropyl, phenyl, tolyl, 4-nitrophenyl, 2,4-dinitrophenyl, 4-chlorophenyl, 4-bromophenyl and trifluoromethyl.

12. A di-protected 1 -alky 1-3 -hydroxy-4-hydroxymethylpyrrolidine derivative of the formula

B

wherein

Ri and R₂ are protecting groups independently selected from R-C(=O), wherein R is straight or branched C₂-C₁₅ alkyl, C₆-C₁₅ aryl, substituted C₅-C₁₅ aryl including from 1 to 5 heteroatoms, C₃-C₅ heterocyclic group including from 1 to 3 heteroatoms, R₅ is straight or branched alkyl and R₆ is straight or branched alkyl, aryl or substituted aryl including from 1 to 5 heteroatoms.

13. A pyrrolidine derivative according to claim 12, wherein R is a straight or branched C₂-Ci₅ alkyl moiety selected from ethyl, propyl, isopropyl, t-butyl, 2-methylbutyl, allyl, 3,3-dimethylallyl, 3-methylbutyl, 3-methyl-2-butenyl, octyl, decyl.

14. A pyrrolidine derivative according to claim 12, wherein said heteroatom is selected from oxygen, nitrogen, sulphur, fluorine, chlorine, bromine or iodine.

15. A pyrrolidine derivative according to claim 12, wherein R is an aryl or heterocyclic moiety selected from C₆H₅, CH₃C₆H^ 2-furyl, 3-furyl, 4-methoxyphenyl, 4-nitrophenyl, 4-chlorophenyl, 4-bromophenyl, 4-iodophenyl, 4-trifluoromethyl, 2,6-dimethylphenyl, 2,6-dimethoxyphenyl, 2,4-dinitrophenyl, 2,4-dichlorophenyl, 2,4-dibromophenyl, 2,4- diiodophenyl, 2,4-dimethoxyphenyl.

16. A pyrrolidine derivative according to claim 12, wherein R₅ is a straight or branched C₁-C₈ alkyl.

17. A pyrrolidine derivative according to claim 12, wherein R₆ is straight or branched Ci-C₈ alkyl, C₆-C₈ aryl, substituted C₅-C₈ aryl including from 1 to 5 heteroatoms.

18. A pyrrolidine derivative according to anyone of claims 12, 16 or 17, wherein R₅ and R₆ are independently a straight or branched C₁-C₈ alkyl moiety selected from methyl, ethyl, propyl, isopropyl, t-butyl, 2-methylbutyl, allyl, 3,3-dimethylallyl, 3-methylbutyl, 3-methyl-2-butenyl, octyl.

19. A pyrrolidine derivative according to claim 17, wherein R₆ is an aryl moiety selected from C₆H₅, CH₃C₆H₄, 4-methoxyphenyl, 4-nitrophenyl, 4-chlorophenyl, 4- bromophenyl, 4-iodophenyl, 4-trifluoromethyl, 2,6-dimethylphenyl, 2,6- dimethoxyphenyl, 2,4-dinitrophenyl, 2,4-dichlorophenyl, 2,4-dibromophenyl, 2,4- diiodophenyl, 2,4-dimethoxyphenyl.

20. A pyrrolidine derivative according to anyone of the preceding claims, wherein the C-I' atom thereof is a stereogenic centre.

21. An oligomer comprising a plurality of nucleomonomers wherein at least one of said nucleomonomers is of the formula:

wherein

X and Z are independently selected from O and S and R₃ is a nucleobase.

22. An oligomer according to claim 21, wherein R₃ is a nucleobase as defined in anyone of claims 6-9.

23. A method for preparing a di-protected l-alkyl-3-hydroxy-4- hydroxymethylpyrrolidine derivative of the formula

B wherein

Ri and R₂ are protecting groups independently selected from R-C(=O), wherein R is as defined in anyone of claims 1-5; R₅ is straight or branched alkyl and R₆ is straight or branched alkyl, aryl or substituted aryl including from 1 to 5 heteroatoms,

comprising the steps of:

wherein M is SiR₉R_1O, R₇, R₈, R₉ and R₁₀ are straight or branched alkyl, and R₅ and R₆ are as defined above,

H

wherein R₅ and R₆ are as defined above,

b) reacting the l-alkyl-3-hydroxy-4-hydroxymethylpyrrolidine of formula H obtained in step a) with an anhydride of the formula (R₆CO)₂O or an acyl derivative of the formula R₆COZ, wherein R₆ is as defined above and Z is halogen.

24. A method according to claim 23, wherein R₅ and R^ are as defined in anyone of claims 16-19.

25. A method according to claim 23, wherein R₇, R₈, R₉ and Rio are independently selected from straight or branched C₁-C₈ alkyl.

26. A method according to claim 25, wherein R₇ is selected from ethyl, propyl, isopropyl and t-butyl.

27. A method according to claim 25, wherein R₈ is methyl.

28. A method according to claim 25, wherein R₉ and R₁₀ are independently selected from methyl, ethyl, propyl, sec-propyl and t-butyl.

29. A method according to claim 23, wherein said reducing agent is selected from BH₃, LiAlH₄, LiBH₄, BF₃-triethylsilane, AlH₃.

30. A method according to claim 23, wherein said step a) is carried out in an organic solvent.

31. A method according to claim 30, wherein said solvent is selected from cyclic or dialkyl ethers, chlorinated hydrocarbons, cyclic and straight alkanes, or mixtures thereof.

32. A method according to claim 23, wherein said step a) is carried out at temperature of from 0°C to 90°C and compatible with the reducing agent.

33. A method according to claim 23, wherein Z is F, Cl or Br.

34. A method according to claim 23, wherein said step b) is carried out in a polar or apolar solvent.

35. A method according to claim 34, wherein said solvent is selected from ethers, aromatic hydrocarbons, chlorinated hydrocarbons, dimethylformamide (DMF), dimethylsulfoxide (DMSO), or mixtures thereof.

36. A method according claim 23, wherein said step b) is carried out in the presence of a tertiary amine (R')₃-N wherein R' represents equal or different straight or branched C₂- C₈ alkyl, or of a tertiary cyclic amine R'(R")₂-N wherein R" is -(CH₂),,- and n = 4-6, in an equimolar amount to said anhydride (ROCO)₂O or said acyl derivative ROCOZ.

37. A method according to claim 23, wherein said step b) is carried out at temperature

38. A method for preparing a di-protected l-acyl-3-hydroxy-4- hydroxymethylpyrrolidine derivative of the formula

wherein

R₁ and R₂ are protecting groups independently selected from R-C(=0), wherein R is as defined in anyone of claims 1-5 and R» is a leaving group,

comprising the step of reacting a di-protected l-alkyl-3-hydroxy-4- hydroxymethylpyrrolidine derivative of the formula

B

wherein

R₁ and R₂ are as defined above; R₅ is straight or branched alkyl and R₆ is straight or branched alkyl, aryl or substituted aryl including from 1 to 5 heteroatoms,

with an acyl halide of the formula

O

R₄

wherein L is halogen atom and R₄ is as defined above.

39. A method according to claim 38, wherein R₅ and R₆ are as defined in anyone of claims 16-19.

40. A method according to claim 38, wherein R₄ is a leaving group as defined in claim

11.

41. A method according to claim 38, wherein L is a halogen atom selected from F, Cl or Br.

42. A method for preparing a di-protected l-acyl-3-hydroxy-4- hydroxymethylpyrrolidine derivative of the formula

wherein

Ri and R₂ are protecting groups independently selected from R-C(=O), wherein R is as defined in anyone of claims 1-5 and R₃ is a nucleobase,

wherein R₁ and R₂ are as defined above and R₄ is a leaving group,

43. A method according to claim 42, wherein said base is selected from bases having a pK_b between 4 and 9, lithium amides or metal hydrides.

44. A method according to claim 43, wherein said base is selected from Li₂CO₃, Na₂CO₃, K₂CO₃, Cs₂CO₃, or mixtures thereof.

45. A method according to claim 43, wherein said lithium amide is selected from lithium bis(trirnethylsilyl)amide (LiHDMS), lithium diisopylamide (LDA), or mixtures thereof.

46. A method according to claim 43, wherein said metal hydride is selected from NaH, KH, or mixtures thereof.

47. A method according to claim 42, wherein R₃ is a nucleobase as defined in anyone of claims 6-9.

48. A method according to claim 42, wherein R₄ is a leaving group as defined in claim 11.

49. A method according to claim 42, wherein said reaction is carried out in a solution of an organic solvent.

50. A method according to claim 49, wherein said organic solvent is selected from DMF, DMSO, N-methylpyrrolidone, HMPA or mixtures thereof.

51. A method according to claim 42, wherein said reaction is carried out at a temperature of from 20° to 100°C.

52. A method for preparing a l-acyl-3-hydroxy-4-hydroxymethylpyrrolidine derivative of the formula

wherein R₄ is a leaving group,

wherein

Ri and R₂ are protecting groups independently selected from R-C(=O), wherein R is as defined in anyone of claims 1-5 and R₄ is as defined above,

53. A method for preparing a l-acyl-3-hydroxy-4-hydroxymethylpyrrolidine derivative of the formula

wherein R₃ is a nucleobase,

wherein

R₁ and R₂ are protecting groups independently selected from R-C(=0), wherein R is as defined in anyone of claims 1-5 and R₃ is as defined above,

with a primary alcohol having a pK_a equal to or lower than 18 in the presence of a carbonate ion or a polymer comprising supported OH^" or CO₃ ^"" groups.

54. A method according to claim 52, wherein R₄ is a leaving group as defined in claim 11.

55. A method according to claim 53, wherein R₃ is a nucleobase as defined in anyone of claims 6-9.

56. A method according to anyone of claims 52 or 53, wherein said primary alcohol is selected from straight or branched Ci-C₅ alcohol, benzyl alcohol, or mixtures thereof.

57. A method according to claim 56, wherein said primary alcohol is selected from methyl alcohol, ethyl alcohol, propyl alcohol, 2-methylbutan-l-ol, 3-methylbutan-l-ol, or mixtures thereof.

58. A method according to anyone of claims 52 or 53, wherein said carbonate ion is derived from a carbonate selected from Li₂CO₃, K₂CO₃, Na₂CO₃, Cs₂CO₃, or mixtures thereof.

59. A method according to anyone of claims 52 or 53, wherein said polymer comprising supported OH^" or CO₃ ^" groups is selected from macroreticular ion-exchange resins in the OH^" or CO₃ ^" form.

60. A method according to anyone of claims 52 or 53, wherein said reaction is carried out in a non-aqueous solvent.

61. A method according to claim 60, wherein said solvent comprises said primary alcohol having a pK_a equal to or lower than 18.

62. A method according to anyone of claims 52 or 53, wherein said reaction is carried out at temperature of from 0°C to 50°C.

63. A pyrrolidine derivative according to anyone of claims 1-9 for use in a method of treatment, prophylaxis or diagnosis.

64. An oligomer according to anyone of claims 21 or 22 for use in a method of treatment, prophylaxis or diagnosis.

65. A pharmaceutical composition comprising a pharmaceutical carrier and a pharmaceutically effective amount of a l-acyl-3-hydroxy-4-hydroxymethylpyrrolidine derivative of the formula

wherein R₃ is a nucleobase as defined in anyone of claims 1 and 6-9.

66. Use of pyrrolidine derivative according to any one of claims 1-9 or of an oligomer according to anyone of claims 21 or 22 for the manufacture of a medicament for the treatment or prophylaxis of a viral infection.

67. A diagnostic composition comprising an oligomer according to anyone of claims 21 or 22 and a marker.

68. Use of an oligomer according to anyone of claims 21 or 22 for detecting or analysing a DNA or RNA sequence from a subject.

69. Use of an oligomer according to anyone of claims 21 or 22 for the manufacture of a diagnostic composition for detecting or analysing a DNA or RNA sequence from a subject.

70. Use according to anyone of claims 68 or 69, wherein said subject is a mammal.