New process for the synthesis of 2',4'-BNA ncnucleoside analogues

Abstract

The present invention relates to a method for the synthesis of nucleoside analogues. More specifically, the present invention relates to a process for the preparation of a compound of formula (IV) which is an intermediate for producing the 2',4'-BNANC of formula A. The present invention relates to a process for the preparation of the 2',4'-BNANC of formula A.

Description

[0001] NEW PROCESS FOR THE SYNTHESIS OF 2', 4'-BNANCNUCLEOSIDE

[0002] ANALOGUES

[0003] TECHNICAL FIELD

[0004] The present invention relates to a method for the synthesis of nucleoside analogues.

[0005] More specifically, the present invention relates to a process for the preparation of a compound of formula (IV) which is an intermediate for producing the 2’,4’-BNANCof formula A.

[0006] The present invention relates to a process for the preparation of the 2’,4’-BNANCof formula A.

[0007] BACKGROUND ART

[0008] In recent years, nucleic acid-based therapies have emerged as promising approach to regulate diverse molecular pathways (De Serres-Berard, T. et al. Int. J. Mol. Sci. 2022, 23 (21), 1-28). Their ability to selectively function on previously undruggable proteins, transcripts and genes broadens therapeutic targets (Zhu, Y. et al. Cell Death Dis. 2022, 13 (7), 1-15). However, nucleic acid drugs are extremely delicate and susceptible to nuclease degradation. Recent advances in this field have relied on new chemical modifications to the nucleoside core, that often result in oligonucleotides with improved pharmacological properties (Hagedorn, P. et al. Drug Discov. Today 2018, 23 (1), 101-114).

[0009] Among the structural analogues and configurational isomers of BNA developed to date, 2'- O,4'-C-aminomethylene bridged nucleic acid (2',4'-BNANC) has become extremely useful in nucleic-acid-based technologies. The utility of this compound is due to its unprecedented hybridizing affinity for complementary strands (RNA and DNA), its sequence selectivity, its aqueous solubility, and its improved biostability compared to that of natural oligonucleotides. Because of its utility, 2',4'-BNANCis now commercially available, and 2',4'-BNA-modified antisense oligonucleotides are entering human clinical trials.

[0010] Currently, the synthesis of 2',4'-BNANCrelies on established method outlined in PCT publication W02005021570 (Examples) and Ab dur Rahman, S. et al. (J. Am. Chem. Soc. 2008, 130 (14), 4886-4896). However, this conventional synthetic pathway has posed a significant hurdle, primarily due to their dependence on natural carbohydrates as starting materials. Also, it is characterized by lengthy processes, low product yields, and high production costs.

[0011] Consequently, there is a need for a new, efficient, and cost-effective synthesis route.

[0012] To address these challenges, the applicant has developed an innovative and short synthesis of 2',4'-BNANC, utilizing simple achiral starting materials and allowing low atom economy because of the amount of protecting groups used, promising a breakthrough in the synthesis of these important nucleoside analogues.

[0013] DEFINITIONS

[0014] The definitions and explanations below are for the terms as used throughout the entire application, including both the specification and the claims.

[0015] When describing the compounds of the invention, the terms used are to be construed in accordance with the following definitions, unless indicated otherwise.

[0016] In the present invention, the following terms have the following meanings:

[0017] The term "alkyl" by itself or as part of another substituent refers to a hydrocarbyl radical of Formula CiJbn+i wherein n is a number greater than or equal to 1. Generally, alkyl groups of this invention comprise from 1 to 18 carbon atoms, preferably from 1 to 12 carbon atoms, more preferably from 1 to 6 carbon atoms, still more preferably 1 to 2 carbon atoms. Alkyl groups may be linear or branched and may be substituted as indicated herein. Suitable alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl and t-butyl, pentyl and its isomers (e.g. n-pentyl, iso-pentyl), hexyl and its isomers (e.g. n-hexyl, isohexyl), heptyl and its isomers (e.g. n-heptyl, iso-heptyl), octyl and its isomers (e.g. n-octyl, iso-octyl), nonyl and its isomers (e.g. n-nonyl, iso-nonyl), decyl and its isomers (e.g. n-decyl, iso-decyl), undecyl and its isomers, dodecyl and its isomers. Preferred alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl and n-decyl. Saturated branched alkyls include, without being limited to, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, 2- m ethylbutyl, 3 -methylbutyl, 2-m ethylpentyl,

[0018] 3 -methylpentyl, 4-m ethylpentyl, 2-m ethylhexyl, 3 -methylhexyl, 4-m ethylhexyl, 5 -methylhexyl, 2,3-dimethylbutyl, 2, 3 -dimethylpentyl, 2,4-dimethylpentyl, 2, 3 -dimethylhexyl, 2,4-dimethylhexyl, 2, 5 -dimethylhexyl, 2,2-dimethylpentyl,

[0019] 2,2-dimethylhexyl, 3,3-dimtheylpentyl, 3, 3 -dimethylhexyl, 4,4-dimethylhexyl,

[0020] 2-ethylpentyl, 3 -ethylpentyl, 2-ethylhexyl, 3 -ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3 -ethylpentyl, 2-methyl-4-ethylpentyl,

[0021] 2-methyl-2-ethylhexyl, 2-methyl-3 -ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-diethylpentyl, 3,3- diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl.

[0022] Cx-Cy-alkyl refers to alkyl groups which comprise x to y carbon atoms.

[0023] When the suffix "ene" ("alkylene") is used in conjunction with an alkyl group, this is intended to mean the alkyl group as defined herein having two single bonds as points of attachment to other groups. The term "alkylene" includes methylene, ethylene, methylmethylene, propylene, ethylethylene, and 1,2- dimethylethylene.

[0024] The term "alkenyl" as used herein refers to an unsaturated hydrocarbyl group, which may be linear or branched, comprising one or more carbon-carbon double bonds. Suitable alkenyl groups comprise between 2 and 18 carbon atoms, preferably between 2 and 8 carbon atoms, still more preferably between 2 and 6 carbon atoms. Examples of alkenyl groups are ethenyl, 2- propenyl, 2-butenyl, 3-butenyl, 2-pentenyl and its isomers, 2-hexenyl and its isomers, 2,4- pentadienyl and the like.

[0025] The term "alkynyl" as used herein refers to a class of monovalent unsaturated hydrocarbyl groups, wherein the unsaturation arises from the presence of one or more carbon-carbon triple bonds. Alkynyl groups typically, and preferably, have the same number of carbon atoms as described above in relation to alkenyl groups. Non limiting examples of alkynyl groups are ethynyl, 2- propynyl, 2-butynyl, 3-butynyl, 2-pentynyl and its isomers, 2 -hexynyl and its isomers-and the like.

[0026] The term "cycloalkyl" as used herein is a cyclic alkyl group, that is to say, a monovalent, saturated, or unsaturated hydrocarbyl group having 1 or 2 cyclic structures. Cycloalkyl includes monocyclic or bicyclic hydrocarbyl groups. Cycloalkyl groups may comprise 3 or more carbon atoms in the ring and generally, according to this invention comprise from 3 to 10, more preferably from 3 to 8 carbon atoms still more preferably from 3 to 6 carbon atoms. Examples of cycloalkyl groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, with cyclopropyl being particularly preferred. When a bicyclic ring structure is envisaged, the two rings can be: - fused, meaning they share a common bond; exemplary cycloalkyl bicyclic fused systems include but is not limited to naphthal enyl, bicyclofl.1.0]butanyl, octahydropental enyl, decahydronaphthalenyl, octahydro- IH-indenyl;

[0027] - linked via a bond between the two cyclic structures; exemplary cycloalkyl bicyclic linked systems include but is not limited to bi-phenyl, bi-cyclopropanyl, bi-cyclopentenyl, bi- cyclohexanyl, cyclopropylcyclohexanyl, cyclopropylcyclopentanyl;

[0028] - bridged meaning that the two rings share three or more atoms, separating the two bridgehead atoms by a bridge containing at least one atom; exemplary cycloalkyl bicyclic bridged systems include but is not limited to bicyclo[2.2.1]heptanyl, bicyclo[2.2.2]octanyl;

[0029] - or represent a spiro bicyclic ring system wherein the two rings are connected through a single atom; exemplary cycloalkyl spiro bicyclic systems include but is not limited to spiro[2.2]pentanyl, spiro[2.4]heptanyl, spiro[4.4]nonanyl, spiro[5.5]undecanyl.

[0030] The term "halo" or "halogen" means fluoro, chloro, bromo, or iodo. Preferred halo groups are fluoro and chloro.

[0031] The term "substituent" or "substituted" means that a hydrogen radical on a compound or group is replaced by any desired group which is substantially stable under the reaction conditions in an unprotected form or when protected by a protecting group. Examples of preferred substituents include, without being limited to, halogen (chloro, iodo, bromo, or fluoro); alkyl; alkenyl; alkynyl, as described above; hydroxy; alkoxy; nitro; thiol; thioether; imine; cyano; amido; phosphonato; phosphine; carboxyl; thiocarbonyl; sulfonyl; sulfonamide; ketone; aldehyde; ester; oxygen (-0); haloalkyl

[0032] (e.g., trifluoromethyl); cycloalkyl, which may be monocyclic or fused or non-fused polycyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl), or a heterocycloalkyl, which may be monocyclic or fused or non-fused polycyclic (e.g., pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, or thiazinyl), monocyclic or fused or non-fused polycyclic aryl or heteroaryl (e.g., phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothiophenyl, or benzofuranyl); amino (primary, secondary, or tertiary); CO2CH3; CONH2; OCH2CONH2; NH2; SO2NH2; OCHF2; CF3; OCF3; and such moieties may also be optionally substituted by a fused-ring structure or bridge, for example -OCH2O-. These substituents may optionally be further substituted with a substituent selected from such groups. In certain embodiments, the term "substituent" or the adjective "substituted" refers to a substituent selected from the group consisting of an alkyl, an alkenyl, an alkynyl, an cycloalkyl, an cycloalkenyl, a heterocycloalkyl, an aryl, a heteroaryl, an arylalkyl, a heteroarylalkyl, a haloalkyl, -C(O)NRi7Ri8, -NRi9C(0)R2o, a halo, -OR19, cyano, nitro, a haloalkoxy, -C(O)Ri9, -NR17R18, -SR19, -C(O)ORi9, -OC(O)Ri9, -NRi9C(O)NRi7Ri8, -OC(O)NRI7R18,

[0033] -NRI9C(O)OR20, -S(O)rRi9, -NRi9S(0)Rr2o, -OS(0)Rr2o, S(O)rNRi7Ri8, -O, -S, and -N-R19, wherein r is 1 or 2; R17 and Ri8, for each occurrence are, independently, H, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted arylalkyl, or an optionally substituted heteroarylalkyl; or R17 and Ri8 taken together with the nitrogen to which they are attached is optionally substituted heterocycloalkyl or optionally substituted heteroaryl; and R19 and R20 for each occurrence are, independently, H, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted arylalkyl, or an optionally substituted heteroarylalkyl. In certain embodiments, the term "substituent" or the adjective "substituted" refers to a solubilizing group.

[0034] As will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The term "protecting group" as used herein, refers to a labile chemical moiety which is known in the art to protect reactive groups including without limitation, hydroxyl, amino and thiol groups, against undesired reactions during synthetic procedures. Protecting groups are typically used selectively and / or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group as is or available for further reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, and references cited therein.

[0035] The term “hydroxyl protecting group” as used herein, refers to a group capable of stably protecting a hydroxyl group during synthetic procedure. Non limiting examples of hydroxy- protecting group include acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1- ethoxyethyl, l-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, 2,6- dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, bi s(2-acetoxy ethoxy )m ethyl (ACE), 2- trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, triisopropyl silyl (TIPS), [(triisopropylsilyl)oxy]methyl (TOM), 2- (trimethylsilyl)ethoxymethyl ether (SEM), tetrahydropyranyl (TUP) ether, acetate (Ac), pivalate (Piv), methoxymethyl or p-methoxybenzyl (PMB) mesylate, tosylate, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMT), trimethoxytrityl, l-(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9- phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). Where more preferred hydroxyl protecting groups include, but are not limited to, acetyl, benzyl, t- butyldimethyl silyl, t-butyldiphenylsilyl and dimethoxytrityl wherein a more preferred hydroxyl protecting group is 4,4'-dimethoxytrityl.

[0036] The term “amino-protecting group” as used herein, refers to a group capable of stably protecting an amino group during synthetic procedure. Non limiting examples of amino- protecting group include carbamate-protecting groups, such as 2-trimethylsilylethoxycarbonyl (Teoc), I-m ethyl- l-(4-biphenylyl)-ethoxy carbonyl (Bpoc), t-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9- fluorenylmethyloxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide-protecting groups, such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; and imine- and cyclic imide-protecting groups, such as phthalimido and dithiasuccinoyl.

[0037] The term “reactive phosphorus group” as used herein refers to phosphorus compounds useful for forming internucleoside linkages including for example phosphodiester and phosphorothioate internucleoside linkages. Such reactive phosphorus groups are known in the art and contain phosphorus atoms in P111or Pvvalence state including, but not limited to, phosphoramidite, H- phosphonate, phosphate triesters and phosphorus containing chiral auxiliaries. A more preferred reactive phosphorus group is diisopropylcyanoethoxy phosphoramidite. “Phosphoramidites” derived from protected nucleosides are referred to as nucleoside phosphoramidites and are widely used in chemical synthesis of DNA, RNA, and other nucleic acids and their analogs.

[0038] The bonds of an asymmetric carbon can be represented here using a solid triangle ( ) or a dashed triangle ( •'•HI). SUMMARY

[0039] In a first embodiment (El) the present invention provides a method for preparing the compound of formula (IV): wherein:

[0040] Ri and R2 each independently represent a hydrogen atom; a hydroxy protecting group; a reactive phosphorus group; or Ri and R2 together form a cyclic group;

[0041] R4 represent a hydrogen atom, a Cl to C6 alkyl group, a C2 to C6 alkenyl group or a C2 to C6 alkynyl group;

[0042] Rs represent an oxygen or a NR5’ group, wherein R5’ is a hydrogen or a suitable amino protecting group; and

[0043] PG represents an amino protecting group, comprising the step of subjecting a compound of formula (II): wherein Ri, R2, R4 and R5 are as defined above, to a reduction of its nitro moiety using a suitable reducing agent to obtain the corresponding N- hydroxyl amine compound of formula (III): wherein Ri, R2, R4 and R5 are as defined above, the hydroxylamine function of which compound of formula (III) is immediately protected through in situ acylation of the amine moiety, to obtain the compound of formula (IV).

[0044] Further enumerated embodiments (E) of the invention are described herein. It will be recognized that features specified in each embodiment may be combined with other specified features to provide further embodiments of the present invention.

[0045] E2. The method according to El, wherein the nitro moiety of the compound of formula (II) is reduced using a suitable reducing agent selected from activated zinc / acetic acid, activated zinc / ammonium chloride and an aluminum / mercury amalgam.

[0046] E3. The method according to E2, wherein the nitro moiety of the compound of formula (II) is reduced using an activated zinc / ammonium chloride.

[0047] E4. The method according to E3, wherein the amount of zinc used to conduct the reduction step is comprised between 10.0 to 30.0 equivalents, calculated from the molar quantity of the compound of formula (II) and the amount of ammonium chloride used to conduct the reduction step is comprised between 1.0 to 3.0 equivalents.

[0048] E5. The method according to any one of El to E4, wherein the reduction step is conducted in a mixture of an organic solvent and water.

[0049] E6. The method according to E5, wherein the reduction step is conducted in a mixture of tetrahydrofuran and water.

[0050] E7. The method according to E6, wherein the molar ratio between tetrahydrofuran and water is of 9:1 and the reduction step is conducted at a concentration between 0.05 M to 1.0 M, calculated from the amount of the compound of formula (II). E8. The method according to any one of El to E7, wherein the hydroxylamine function of compound of formula (III) is immediately protected through in situ acylation of the amine moiety using a t-butoxycarbonyl protecting group.

[0051] E9. The method according to E8, wherein the amount of di-tert-butyl dicarbonate used to protect the amine moiety is comprised between 0.5 to 3.0 equivalents, calculated from the molar quantity of the compound of formula (II).

[0052] E10. The method according to any one of El to E9, wherein the reduction-protection step is conducted at a temperature ranging from 15°C to 100°C, preferably from 15°C to 50°C, more preferably from 15°C to 25°C, for 0.5 to 12 hours, preferably 0.5 to 6 hours.

[0053] Ell. The method according to any one of El to E10, wherein the compound of formula (IV) is further subjected to a bridge formation to obtain the compound of formula (V): wherein Ri, R2, R4, Rs and PG are as defined in El.

[0054] Ell. The method according to El l, wherein the bridge formation step is conducted in the presence of an organic or inorganic base having a pKa from 8 to 40 in an aprotic solvent.

[0055] E13. The method according to El l or E12, wherein the amount of base used in the bridge formation step is comprised between 0.5 to 3.0 equivalents, calculated from the molar quantity of the compound of formula (IV).

[0056] E14. The method according to any one of El l to E13, wherein the bridge formation step is conducted in the presence of sodium hydride.

[0057] E15. The method according to any one of El l to E14, wherein the bridge formation step is conducted in dimethyl formamide. E16. The method according to any one of El l to El 5, wherein the bridge formation step is conducted at a temperature ranging from 15°C to 100°C, preferably from 15°C to 50°C, more preferably from 15°C to 25°C, for 0.5 to 6 hours, preferably 0,5 to 3 hours.

[0058] E17. The method according to any one of El l to E16, wherein the compound of formula (V) is further deprotected under suitable conditions to obtain the compound of formula (VI): wherein Ri, R2, R4 and R5 are as defined in claim 1, which compound of formula (VI) is subjected to a reductive amination to give the compound of formula A: wherein R3 represents a Cl to Cl 8 alkyl group, a C2 to C18 alkenyl group, a C2 to C18 alkynyl group, a C3 to CIO cycloalkyl group, an aryl group or an acyl group; and

[0059] Ri, R2, R4 and R5 are as defined in El.

[0060] E18. The method according to E17, wherein the amine moiety of compound of formula (V) is deprotected under strong acidic conditions.

[0061] E19. The method according to El 8, wherein the deprotection step is conducted with trifluoracetic acid.

[0062] E20. The method according to any one of E17 to E19, wherein the reductive amination step is conducted by reacting the amine function of compound of formula (VI) with an aldehyde, or a ketone in the presence of a reducing agent selected from sodium triacetoxyborohydride, sodium cyanoborohydride, sodium borohydride, lithium borohydride and lithium aluminum hydride. Ell. The method according to E20, wherein the amount of aldehyde or ketone and reducing agent used in the reductive amination step is comprised between 0.5 to 3.0 equivalents, calculated from the molar quantity of the compound of formula (VI).

[0063] Ell. The method according to any one of E17 to E21, wherein the reductive amination step is conducted in a suitable solvent at a concentration between 0.01 M to 1.0 M, at a temperature ranging from 15°C to 100°C, preferably from 15°C to 50°C, more preferably from 15°C to 25°C, for 0.5 to 12 hours, preferably 1 to 6 hours.

[0064] E23. A method for preparing the compound of formula A: wherein:

[0065] Ri and R2 each independently represent a hydrogen atom; a hydroxy protecting group; a reactive phosphorus group; or Ri and R2 together form a cyclic group;

[0066] R3 represents a Cl to Cl 8 alkyl group, a C2 to C18 alkenyl group, a C2 to Cl 8 alkynyl group, a C3 to CIO cycloalkyl group, an aryl group or an acyl group;

[0067] R4 represent a hydrogen atom, a Cl to C6 alkyl group, a C2 to C6 alkenyl group or a C2 to C6 alkynyl group; and

[0068] Rs represent an oxygen or a NR.5- group, wherein Rs is a hydrogen or a suitable amino protecting group, comprising the following steps:

[0069] 1) Subjecting the compound of formula (II) to a reduction of its nitro moiety using a suitable reducing agent to obtain the corresponding N-hydroxyl amine compound of formula (III): wherein Ri, R2, R4 and Rs are as defined above, the hydroxylamine function of which compound of formula (III) is immediately protected through in situ acylation of the amine moiety, to obtain the compound of formula (IV): wherein Ri, R2, R4 and R5 are as defined above and PG represents an amino protecting group,

[0070] 2) subjecting the compound of formula (IV) to a bridge formation to obtain the compound of formula (V): wherein Ri, R2, R4, Rs and PG are as defined above, which compound of formula (V) is subsequently deprotected under suitable conditions to obtain the compound of formula (VI): wherein Ri, R2, R4 and Rs are as defined above, which compound of formula (VI) is subjected to a reductive amination to give the compound of formula A.

[0071] E24. The method according to any one of El to E23, wherein the compound of formula (II) is obtained by subjecting a compound of formula (I): wherein Ri, R2, R4 and R5 are as defined in El, to dehydration in the presence of a dehydrating agent and a base, in an organic solvent.

[0072] E25. The method according to E24, wherein the dehydrating agent is a condensing agent selected from dialkyl and diaryl carbonates or a fluorinating agent selected from (diethylamino)sulfur trifluoride and trifluoromethanesulfonic anhydride.

[0073] E26. The method according to E25, wherein the dehydrating agent is (diethylamino)sulfur tri fluoride.

[0074] E27. The method according to any one of E24 to E26, wherein the dehydration step is conducted in the presence of a base selected from sodium carbonate, potassium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide and sodium methoxide.

[0075] E28. The method according to E27, wherein the base is sodium bicarbonate.

[0076] E29. The method according to any one of E24 to E28, wherein the dehydration step is conducted in the presence of an aprotic organic solvent selected from dichloromethane, acetonitrile, toluene, ethyl acetate, butyl acetate, isobutyl acetate, propyl acetate, isopropyl acetate, chlorobenzene, M -di methyl form am ide and pyridine.

[0077] E30. The method according to any one of E24 to E29, wherein the dehydration step is conducted at a temperature ranging from -78°C to 100°C, preferably from -40°C to 50°C, more preferably at 0°C, for 0.5 to 12 hours, preferably 2 to 6 hours.

[0078] E31. The method according to any one of El to E30, wherein Ri and R2 each independently represent a hydroxy protecting group; or Ri and R2 together form a cyclic group.

[0079] E32. The method according to any one of El to E30, wherein Ri and R2 together form a cyclic group.

[0080] E33. The method according to E32, wherein Ri and R2 together represent a group selected from -OC(CH3)2O-, - (CH2)3-, -CH2SCH2-, -CH2OCH2-, -OCH(C6H5)O-, -OC(C6HIO)0-, - OC(C4H8)O-, -Si(CH3)2OSi(CH3)2-, -Si(OCH3)2OSi(OCH3)2-, -Si(CH3)2CH2Si(CH3)2-, and - OSi(CH3)2O-.

[0081] E34. The method according to E33, wherein Ri and R2 together represent -OC(CH3)2O-.

[0082] E35. The method according to El 7, wherein R3is a Cl to C6 alkyl group.

[0083] E36. The method according to E35, wherein R3is a methyl group.

[0084] E37. The method according to any one of El to E36, wherein R4is a methyl group and R5 is an oxygen.

[0085] E38. The method according to any one of El to E37, wherein when Ri and R2 together form a cyclic group, then Ri and R2 are further hydrolyzed under strong acidic conditions such as hydrochloric acid or trifluoracetic acid.

[0086] DETAILED DESCRIPTION

[0087] The present invention relates to a method for preparing a compound of formula (IV):

[0088] (IV), wherein:

[0089] Ri and R2 each independently represent a hydrogen atom; a hydroxy protecting group; a reactive phosphorus group; or Ri and R2 together form a cyclic group;

[0090] R4 represent a hydrogen atom, a Cl to C6 alkyl group, a C2 to C6 alkenyl group or a C2 to C6 alkynyl group; Rs represent an oxygen or a NR.5- group, wherein R5’ is a hydrogen or a suitable amino protecting group; and

[0091] PG represents an amino protecting group, comprising the step of subjecting a compound of formula (II) wherein Ri, R2, R4 and R5 are as defined herein above, to a reduction of its nitro moiety using a suitable reducing agent to obtain the corresponding N-hydroxyl amine compound of formula (III): wherein Ri, R2, R4 and R5 are as defined above, the hydroxylamine function of which compound of formula (III) is immediately protected through in situ acylation of the amine moiety to obtain the compound of formula (IV).

[0092] The term “immediately protected in situ” as used herein, refers to the fact that the hydroxylamine function is trapped by the protecting group as soon as it is formed, in a single flask, without a distinct separation phase, isolation, or purification of intermediate compound of formula (III).

[0093] This step is crucial to prevent any undesired reactivity of the amine function, that can lead to potential side-products such as the N-hydroxide compound as represented below:

[0094] Examples of such suitable reducing agent that may be used according to the invention include, but are not limited to, activated zinc / acetic acid, activated zinc / ammonium chloride and aluminum / mercury amalgam. In one embodiment, the nitro moiety of the compound of formula (II) is reduced using a suitable reducing agent selected from activated zinc / acetic acid, activated zinc / ammonium chloride and an aluminum / mercury amalgam. In a preferred embodiment, the suitable reducing agent is an activated zinc / ammonium chloride.

[0095] In one embodiment, the amount of zinc used to conduct the reduction step is comprised between 10.0 to 30.0 equivalents calculated from the molar quantity of the compound of formula (II). In a preferred embodiment, the amount of zinc used to conduct the reduction step is 20.0 equivalents, calculated from the molar quantity of the compound of formula (II).

[0096] In one embodiment, the amount of ammonium chloride used to conduct the reduction step is comprised between 1.0 to 3.0 equivalents, calculated from the molar quantity of the compound of formula (II). In a preferred embodiment, the amount of ammonium chloride used to conduct the reduction step is 2.0 equivalents, calculated from the molar quantity of the compound of formula (II).

[0097] According to one embodiment, the reduction step is conducted in the presence of a suitable solvent. Examples of such solvents include, but are not limited to: ethers, such as diethyl ether, diisopropyl ether, tetrahydrofuran, MTBE, dioxane, and dimethoxyethane; alcohols, such as methanol, ethanol, ethylene glycol, 1 -propanol, 2-propanol, 2-methoxyethanol, 1 -butanol, 2- butanol, iso-butyl alcohol, t-butyl alcohol, glycerol, and C1-C6 alcohols; halogenated hydrocarbons, such as dichloro methane, chloroform, carbon tetrachloride, and chlorobenzene; aromatic hydrocarbons, such as toluene; aliphatic hydrocarbons, nitriles, esters and polar aprotic solvents such as DMF, DMSO, Dimethylacetamide (DMAc), water; any mixtures of two or more thereof. In a preferred embodiment, the reduction step is conducted in mixture of organic solvent and water. In a more preferred embodiment, the reduction step is conducted in a mixture of tetrahydrofuran and water. In one embodiment, the molar ratio between organic solvent and water is of 9: 1 and the reduction step is conducted at a concentration between 0.05 M to 1.0 M, calculated from the amount of the compound of formula (II). In a preferred embodiment, at a concentration between 0.05 M to 0.5 M. In a more preferred embodiment, at a concentration between 0.05 M to 0.1 M.

[0098] In a preferred embodiment, the hydroxylamine function of compound of formula (III) is immediately protected through in situ acylation of the amine moiety using a suitable aminoprotecting group, to obtain the compound of formula (IV).

[0099] Non limiting examples of amino-protecting group that may be used according to the invention include carbamate-protecting groups, such as 2-trimethylsilylethoxycarbonyl (Teoc), I-methyl- l-(4-biphenylyl)-ethoxy carbonyl (Bpoc), t-butoxy carbonyl (BOC), allyloxy carbonyl (Alloc), 9- fluorenylmethyloxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide-protecting groups, such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenyl acetyl; sulfonamide- protecting groups, such as 2-nitrobenzenesulfonyl; and imine- and cyclic imide-protecting groups, such as phthalimido and dithiasuccinoyl. In a preferred embodiment, the hydroxylamine function of compound of formula (III) is immediately protected through in situ acylation of the amine moiety using a t-butoxycarbonyl protecting group.

[0100] In one embodiment, the amount of the amino-protecting group used to protect the amine moiety is comprised between 0.5 to 3.0 equivalents calculated from the molar quantity of the compound of formula (II). In a preferred embodiment, the amount of the amino-protecting group used to protect the amine moiety is 1.2 equivalents, calculated from the molar quantity of the compound of formula (II).

[0101] According to one embodiment, the reduction-protection step is conducted at a temperature ranging from 15°C to 100°C. In a preferred embodiment at a temperature ranging from 15°C to 50°C. In a more preferred embodiment at a temperature ranging from 15°C to 25°C.

[0102] According to one embodiment, the reduction-protection step is conducted for a duration of between 0.5 to 12 hours. In a preferred embodiment, for a duration of 0.5 to 6 hours. In a more preferred embodiment, for a duration of 2 hours.

[0103] In one embodiment, the compound of formula (IV) may be isolated directly from the reaction mixture itself after the reaction is complete, by filtration or after conventional work up with techniques such as quenching with a suitable reagent, extraction, evaporation of solvent or the like.

[0104] According to one embodiment, the compound of formula (IV) is further subjecting to a bridge formation to obtain the compound of formula (V): wherein Ri, R2, R4, Rs and PG are as defined herein above.

[0105] According to one embodiment, the bridge formation step is conducted in the presence of an organic or inorganic base having a pka from 8 to 40.

[0106] Examples of such organic or inorganic base include, but are not limited to alkali hydroxide like alkali metal hydroxides, such as, for example, lithium hydroxide, sodium hydroxide, potassium hydroxide, and cesium hydroxide or the like; alkaline earth metal hydroxides, such as, for example, barium hydroxide, strontium hydroxide, magnesium hydroxide, calcium hydroxide, or the like; alkali metal carbonates, such as, for example, sodium carbonate, potassium carbonate, lithium carbonate, cesium carbonate, or the like; alkaline earth metal carbonates, such as, for example, magnesium carbonate, calcium carbonate, or the like; alkali metal bicarbonates, such as, for example, sodium bicarbonate, potassium bicarbonate, or the like; organolithium, such as, for example, butyl lithium, lithium diisopropylamide, lithium diethylamide, or the like ; alkali metal amides, such as, for examples, lithium amide, sodium amide, potassium amide, or the like; hydrides, such as, for example, sodium hydride, potassium hydride, or the like.

[0107] In a preferred embodiment, the bridge formation step is conducted in the presence of sodium hydride.

[0108] In one embodiment the amount of base required may vary depending on the nature of base, reaction conditions etc.

[0109] In one embodiment, the amount of base used in the bridge formation step is comprised between 0.5 to 3.0 equivalents calculated from the molar quantity of the compound of formula (IV). In a preferred embodiment, the amount of base used in the bridge formation step is 2.5 equivalents, calculated from the molar quantity of the compound of formula (IV).

[0110] According to one embodiment, the bridge formation step is conducted in an aprotic organic solvent selected from dichloromethane, acetonitrile, toluene, ethyl acetate, butyl acetate, isobutyl acetate, propyl acetate, isopropyl acetate, chlorobenzene, VA -di methyl form am ide and pyridine. In a preferred embodiment, the aprotic organic solvent is N, V-dimethylformamide.

[0111] According to one embodiment, the bridge formation step is conducted at a temperature ranging from 15°C to 100°C. In a preferred embodiment at a temperature ranging from 15°C to 50°C. In a more preferred embodiment at a temperature ranging from 15°C to 25°C.

[0112] According to one embodiment, the bridge formation step is conducted for a duration of between 0.5 to 6 hours. In a preferred embodiment, for a duration of 0.5 to 3 hours.

[0113] According to one embodiment, the compound of formula (V) is further deprotected under suitable conditions to obtain the compound of formula (VI): wherein Ri, R2, R4 and R5 are as defined herein above, which compound of formula (VI) is subjected to a reductive amination to give the compound of formula A: wherein R3 represents a Cl to Cl 8 alkyl group, a C2 to C18 alkenyl group, a C2 to C18 alkynyl group, a C3 to CIO cycloalkyl group, an aryl group or an acyl group; and

[0114] Ri, R2, R4 and Rs are as defined herein above. According to one embodiment, the amine moiety of compound of formula (V) is deprotected under strong acidic conditions.

[0115] Examples of such strong acid include, but are not limited to, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, oxalic acid, lactic acid, malic acid, citric acid, benzoic acid, carbonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, aminomethylphosphonic acid, trifluoro acetic acid (TFA), phosphonic acid, sulfuric acid, nitric acid, phosphoric acid, hydrochloric acid, ethane sulfonic acid (ESA), or any combination thereof. In one embodiment, the acid is acetic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, trifluoro acetic acid (TFA), sulfuric acid, or hydrochloric acid. In a preferred embodiment, the acid is trifluoro acetic acid (TFA) or hydrochloric acid. In a more preferred embodiment, the acid is trifluoro acetic acid (TFA).

[0116] In one embodiment, the amount of acid used to conduct the deprotection step is comprised between 10.0 to 30.0 equivalents calculated from the molar quantity of the compound of formula (V). In a preferred embodiment, the amount of acid used to conduct the deprotection step is 15.0 equivalents, calculated from the molar quantity of the compound of formula (V).

[0117] According to one embodiment, the deprotection step is conducted for a duration of between 10 to 72 hours. In a preferred embodiment, for a duration of between 20 to 48 hours.

[0118] According to one embodiment, the deprotection step is conducted in a suitable solvent. Examples of such solvents include, but are not limited to: ethers, such as diethyl ether, diisopropyl ether, tetrahydrofuran, MTBE, dioxane, and dimethoxy ethane; alcohols, such as methanol, ethanol, ethylene glycol, 1 -propanol, 2-propanol, 2- methoxy ethanol, 1 -butanol, 2- butanol, iso-butyl alcohol, t-butyl alcohol, glycerol, and C1-C6 alcohols; halogenated hydrocarbons, such as dichloromethane, chloroform, carbon tetrachloride, and chlorobenzene; aromatic hydrocarbons, such as toluene; aliphatic hydrocarbons, nitriles, esters and polar aprotic solvents such as DMF, DMSO, Dimethylacetamide (DMAc), water; any mixtures of two or more thereof. In a preferred embodiment, the deprotection step is conducted in dichloromethane or a mixture of dichloromethane and water.

[0119] According to one embodiment, the reductive amination step is conducted by reacting the amine function of compound of formula (VI) with an aldehyde or a ketone in the presence of a reducing agent. Examples of such reducing agent include, but are not limited to, sodium triacetoxyborohydride, sodium cyanoborohydride, sodium borohydride, lithium borohydride, lithium aluminum hydride or the like or reduction by catalytic hydrogenation using Raney nickel, palladium, platinum, rhodium catalyst. In a preferred embodiment, the reductive amination step is conducted by reacting the amine function of compound of formula (VI) in the presence of sodium cyanoborohydride.

[0120] In one embodiment, the amount of aldehyde or ketone used in the reductive amination step is comprised between 0.5 to 3.0 equivalents calculated from the molar quantity of the compound of formula (VI). In a preferred embodiment, the amount of aldehyde or ketone used in the reductive amination step is 2.5 equivalents, calculated from the molar quantity of the compound of formula (VI).

[0121] In one embodiment, the amount of reducing agent used in the reductive amination step is comprised between 0.5 to 3.0 equivalents calculated from the molar quantity of the compound of formula (VI). In a preferred embodiment, the amount of reducing agent used in the reductive amination step is 1.0 equivalents, calculated from the molar quantity of the compound of formula (VI).

[0122] According to one embodiment, the reductive amination step is conducted in a suitable solvent. Examples of such suitable solvents include, but are not limited to: alcohols such as methanol, ethanol, isopropyl alcohol, «-butyl alcohol, isobutyl alcohol, sec -butyl alcohol, tert-butyl alcohol, pentanol, octanol and the like; ethers such as dimethyl ether, diethyl ether, diisopropyl ether, tert-butyl methyl ether, tetrahydrofuran, dioxane and the like; esters such as methyl acetate, ethyl acetate, tert-butyl acetate and the like; dimethyl formamide; dimethyl sulfoxide; dimethyl acetamide; water or mixtures thereof. In a preferred embodiment, the solvent is methanol.

[0123] According to one embodiment, the reductive amination step is conducted at a concentration between 0.01 M to 1.0M, calculated from the amount of the compound of formula (VI). In a preferred embodiment, at a concentration between 0.01 M to 0.5 M. In a more preferred embodiment, at a concentration between 0.01 M to 0.1 M.

[0124] According to one embodiment, the reductive amination step is conducted at a temperature ranging from 15°C to 100°C. In a preferred embodiment at a temperature ranging from 15°C to 50°C. In a more preferred embodiment at a temperature ranging from 15°C to 25°C. According to one embodiment, the reductive amination step is conducted for a duration of between 0.5 to 12 hours. In a preferred embodiment, for a duration of 1 to 6 hours.

[0125] The present invention also relates to a method for preparing the compound of formula A: wherein:

[0126] Ri and R2 each independently represent a hydrogen atom; a hydroxy protecting group; a reactive phosphorus group; or Ri and R2 together form a cyclic group;

[0127] R3 represents a Cl to Cl 8 alkyl group, a C2 to C18 alkenyl group, a C2 to Cl 8 alkynyl group, a C3 to CIO cycloalkyl group, an aryl group or an acyl group;

[0128] R4 represent a hydrogen atom, a Cl to C6 alkyl group, a C2 to C6 alkenyl group or a C2 to C6 alkynyl group; and

[0129] Rs represent an oxygen or a NR5’ group, wherein Rs is a hydrogen or a suitable amino protecting group, comprising the following steps:

[0130] 1) subjecting the compound of formula (II): wherein Ri, R2, R4 and R5 are as defined above, to a reduction of its nitro moiety using a suitable reducing agent to obtain the corresponding billy droxyl amine compound of formula (III): wherein Ri, R2, R4 and Rs are as defined herein above, the hydroxylamine function of which compound of formula (III) is immediately protected through in situ acylation of the amine moiety, to obtain the compound of formula (IV): wherein Ri, R2, R4 and R5 are as defined above and PG represents an amino protecting group,

[0131] 2) subjecting the compound of formula (IV) to a bridge formation to obtain the compound of formula (V): wherein Ri, R2, R4, Rs and PG are as defined herein above, which compound of formula (V) is further deprotected under suitable conditions to obtain the compound of formula (VI): wherein Ri, R2, R4 and Rs are as defined herein above, which compound of formula (VI) is subjected to a reductive amination to give the compound of formula A.

[0132] According to one embodiment, the compound of formula (II) is obtained by subjecting a compound of formula (I): wherein Ri, R2, R4 and R5 are as defined herein above, to dehydration in the presence of a dehydrating agent and a base, in an organic solvent.

[0133] According to one embodiment, the dehydration step is conducted in the presence of a dehydrating agent and a base, in an organic solvent. In one embodiment, the dehydrating agent is a condensing agent selected from dialkyl and diaryl carbonates. In one embodiment, the dehydrating agent is a fluorinating agent selected from (diethylamino)sulfur trifluoride and trifluoromethanesulfonic anhydride. In a preferred embodiment, the dehydrating agent is (diethylamino)sulfur trifluoride.

[0134] In one embodiment, the amount of the dehydrating agent used to conduct the dehydration step is comprised between 2.0 to 10.0 equivalents calculated from the molar quantity of the compound of formula (I). In a preferred embodiment, the amount of the dehydrating agent used to conduct the dehydration step is 5.0 equivalents, calculated from the molar quantity of the compound of formula (I).

[0135] Non limiting examples of base that may be used according to the invention include alkali or alkaline earth metal hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide, or alkali or alkaline metal carbonates or bicarbonates thereof such as sodium carbonate, potassium carbonate, sodium bicarbonate. In a preferred embodiment, the base is sodium bicarbonate.

[0136] In one embodiment, the amount of the base used to conduct the dehydration step is comprised between 2.0 to 10.0 equivalents calculated from the molar quantity of the compound of formula (I). In a preferred embodiment, the amount of the base used to conduct the dehydration step is 5.0 equivalents, calculated from the molar quantity of the compound of formula (I).

[0137] According to one embodiment, the dehydration step is conducted in the presence of an aprotic organic solvent selected from dichloromethane, acetonitrile, toluene, ethyl acetate, butyl acetate, isobutyl acetate, propyl acetate, isopropyl acetate, chlorobenzene, N,N- dimethylformamide and pyridine. In a preferred embodiment, the aprotic organic solvent is di chi or om ethane .

[0138] According to one embodiment, the dehydration step is conducted at a temperature ranging from -78°C to 100°C. In a preferred embodiment, the dehydration step is conducted at a temperature ranging from -40°C to 50°C. In a more preferred embodiment, the dehydration step is conducted at a temperature ranging from -40°C to 0°C. In an even more preferred embodiment, the dehydration step is conducted at 0°C.

[0139] According to one embodiment, the dehydration step is conducted for a duration of between 0.5 to 12 hours. In a preferred embodiment, for a duration of 2 to 6 hours. In a more preferred embodiment, for a duration of 6 hours.

[0140] According to one embodiment, Ri and R2 each independently represent a hydroxy protecting group.

[0141] Non limiting examples of hydroxy -protecting group that may be used according to the invention include acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1 -ethoxy ethyl, 1- (2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, bi s(2-acetoxy ethoxy )m ethyl (ACE), 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, triisopropyl silyl (TIPS), [(triisopropylsilyl)oxy]methyl (TOM), 2-(trimethylsilyl)ethoxymethyl ether (SEM), tetrahydropyranyl (THP) ether, acetate (Ac), pivalate (Piv), methoxymethyl or p- methoxybenzyl (PMB) mesylate, tosylate, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMT), trimethoxytrityl, l-(2- fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9-phenylxanthine-9-yl (Pixyl) and 9-(p- methoxyphenyl)xanthine-9-yl (MOX). Where more preferred hydroxyl protecting groups include, but are not limited to, acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl and dimethoxytrityl wherein a more preferred hydroxyl protecting group is 4,4'-dimethoxytrityl. According to one embodiment, Ri and R2 together form a cyclic group. In one embodiment, Ri and R2 together represent a group selected from -OC(CH3)2O-, -(CH2)3-, -CH2SCH2-, - CH2OCH2-, -OCH(C6H5)O-, -OC(C6HIO)0-, -OC(C4H8)O-, -Si(CH3)2OSi(CH3)2-, - Si(OCH3)2OSi(OCH3)2-, -Si(CH3)2CH2Si(CH3)2-, and -OSi(CH3)2O-. In a preferred embodiment, Ri and R2 together represent -OC(CH3)2O-.

[0142] According to one embodiment, R3 represents a Cl to Cl 8 alkyl group. In a preferred embodiment, R3 represents a Cl to C6 alkyl group. In a more preferred embodiment, R3 represents a methyl group.

[0143] According to one embodiment, R4is a methyl group and R5 is an oxygen.

[0144] According to a preferred embodiment, Ri and R2 together represent -OC(CH3)2O-; R3 represents a methyl group; R4represent a methyl group and R5 represent an oxygen.

[0145] According to one embodiment, when Ri and R2 together form a cyclic group, then Ri and R2 are further hydrolyzed under strong acidic conditions to give a compound of formula A in which Ri and R2 represent a hydrogen.

[0146] Examples of such strong acid include, but are not limited to, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, oxalic acid, lactic acid, malic acid, citric acid, benzoic acid, carbonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, aminomethylphosphonic acid, trifluoro acetic acid (TFA), phosphonic acid, sulfuric acid, nitric acid, phosphoric acid, hydrochloric acid, ethane sulfonic acid (ESA), or any combination thereof. In one embodiment, the acid is acetic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, trifluoro acetic acid (TFA), sulfuric acid, or hydrochloric acid. In a preferred embodiment, the acid is trifluoro acetic acid (TFA) or hydrochloric acid. In a more preferred embodiment, the acid is trifluoro acetic acid (TFA).

[0147] According to one embodiment, the compound of formula A is: The present invention also relates to a method for preparing the compound of formula (I), comprising the step of subjecting a halohydrin ketone compound of formula (I’): wherein Ri and R2 each independently represent a hydroxy protecting group; or Ri and R2 together form a cyclic group;

[0148] R4 represent a hydrogen atom, a Cl to C6 alkyl group, a C2 to C6 alkenyl group or a C2 to C6 alkynyl group; and

[0149] Rs represent an oxygen or a NFC- group, wherein R5’ is a hydrogen or a suitable amino protecting group, to an addition / cyclisation reaction with a 4 ’-nitromethane adduct, in the presence of an inorganic base selected from sodium hydride, sodium hydroxide, cesium fluoride, cesium hydroxide, potassium phosphate, lithium carbonate, sodium carbonate, potassium carbonate, and cesium carbonate; in a polar aprotic organic solvent, to obtain the C4’- substituted nucleoside analogue of formula (I): wherein Ri and R2 each independently represent a hydroxy protecting group; or Ri and R2 together form a cyclic group;

[0150] R4 represent a hydrogen atom, a Cl to C6 alkyl group, a C2 to C6 alkenyl group or a C2 to C6 alkynyl group; and

[0151] Rs represent an oxygen or a NFC- group, wherein R5’ is a hydrogen or a suitable amino protecting group.

[0152] In a preferred embodiment, the addition / cyclisation reaction is conducted in the presence of a base selected from lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, and potassium phosphate. In a more preferred embodiment, the base is potassium phosphate.

[0153] According to one embodiment, the base is introduced in an amount of 0.5 to 5.0 equivalents calculated from the molar quantity of the halohydrin ketone of formula (I’). In a more preferred embodiment, the base is introduced in an amount of 3.0 equivalents, calculated from the molar quantity of the halohydrin ketone of formula (I’).

[0154] According to one embodiment, the addition / cyclisation reaction is conducted in acetonitrile at a concentration between 0.05M to 1.0M, calculated from the amount of the compound of formula (I’). In a preferred embodiment, at a concentration between 0.05 M to 0.5 M.

[0155] According to one embodiment, the molar ratio between nitromethane and acetonitrile is ranging from 1 :1 to 1 :5. In a more preferred embodiment, the molar ratio between nitromethane and acetonitrile is of 1 :3.

[0156] According to one embodiment, the addition / cyclisation reaction is conducted at a temperature ranging from 15°C to 50°C. In a more preferred embodiment, at a temperature ranging from 15°C to 25°C.

[0157] According to one embodiment, the addition / cyclisation reaction is conducted for a duration of between 1 to 72 hours. In a preferred embodiment, for a duration of between 12 to 48 hours.

[0158] According to one embodiment, Ri and R2 together form a cyclic group. In a preferred embodiment, Ri and R2 together represent a -OC(CH3)2O-, - (CH2)3-, -CH2SCH2-, -CH2OCH2- , -OCH(C6H5)O-, -OC(C6HIO)0-, -OC(C4H8)O-, -Si(CH3)2OSi(CH3)2-,

[0159] Si(OCH3)2OSi(OCH3)2-, -Si(CH3)2CH2Si(CH3)2-, and -OSi(CH3)2O-. In a more preferred embodiment, Ri and R2 together represent a -OC(CH3)2O-.

[0160] According to one embodiment, R4is a methyl group and R5 is an oxygen.

[0161] According to one embodiment, the compound of formula (I) is: According to one embodiment, the halohydrin ketone compound of formula (I’) is prepared according to the method described in the PCT publication W02021 / 191830, comprising the step of halogenating an heteroaryl acetaldehyde of formula (1): wherein,

[0162] R4 represent a hydrogen atom, a Cl to C6 alkyl group, a C2 to C6 alkenyl group or a C2 to C6 alkynyl group; and

[0163] Rs represent an oxygen or a NFC- group, wherein R5’ is a hydrogen or a suitable amino protecting group, by proline catalysis to obtain the a-fluoroaldehyde compound of formula (2): which compound is then coupled to the ketone of formula (3): wherein Ri and R2 each independently represent a hydroxy protecting group; or Ri and R2 together form a cyclic group, to obtain the halohydrin ketone compound of formula (I’).

[0164] In some embodiments, intermediate products made in any method herein may be used in the next step without purification. In other embodiments, intermediate products made in any method herein may be purified prior to being used in the next step. In any embodiment, one or more intermediate products and / or the final product may be purified by known techniques including filtration, centrifugation, chromatography, evaporation, liquid-liquid extraction, distillation (e.g., vacuum distillation), sublimation, crystallization, or a combination of two or more thereof. In any embodiment, the purifying may include distillation.

[0165] EXAMPLES

[0166] Some abbreviations are defined below:

[0167] DAST Diethylaminosulfur trifluoride

[0168] DMF Dimethylformamide

[0169] EtOAc Ethyl acetate

[0170] THF Tetrahydrofurane

[0171] TFA Trifluoroacetic acid

[0172] Preferably, the reactants are agitated during the reaction period using suitable mechanical agitators or stirrers. The reactions can be conducted from about 0.5 to about 24 hours or more, depending on the temperatures, dilution volumes, catalysts, concentrations and / or nature of the materials in the reaction mixtures. The term ‘about’ as used herein means + / - 5 %, in particular + / - 2 %, more particularly + / - 1 %.

[0173] The structures of the compounds described were confirmed by the usual spectroscopic techniques. For example,JH NMR data is in the form of delta values, given in part per million (ppm), using the residual peak of the solvent (7.24 ppm for CDCh or 2.49 ppm for DMSOd6 or 33.1 ppm for CD3OD) as internal standard. Splitting patterns are designated as: s (singlet), d (doublet), t (triplet), m (multiplet), br or brs (broad singlet).

[0174] The Preparations herein below illustrate the invention but do not limit it in any way.

[0175] EXAMPLE 1: Preparation of 2\4’-BNANC(Me)

[0176] STEP 1: Synthesis of nitromethane adduct 2

[0177] 1 2 The reaction was done with standard glassware.

[0178] Commercial nitromethane (ACS reagent, >95%) and acetonitrile (HPLC grade, >99.9%) were used as received without further drying or distillation.

[0179] A 500 mL round-bottom flask was charged with ketofluorohydrin 1 (8.0 g, 25.28 mmol), acetonitrile (180 mL), and nitromethane (60 mL). At room temperature, freshly grinded potassium phosphate tribasic, >98%, K3PO4 (13.40 g, 63.24 mmol, 3.0 equiv.) was added portion-wise. The reaction was vigorously stirred for 3 days.

[0180] At room temperature, the reaction was quenched with potassium phosphate monobasic, KH2PO4 (200 mL, 1.0 M solution), then transferred to a 1 L separating funnel and diluted with EtOAc (100 mL). The organic phase was separated and the aqueous layer was extracted with EtOAc (70 mL, x5). The combined organic layers were washed with brine (250 mL), dried over MgSO4, and the solvent removed under reduced pressure. The crude product was purified by silica gel column chromatography (30% hexanes in EtOAc) to afford nitromethane adduct 2 as a slightly yellow powder (4.49 g, 50% yield).

[0181] Rf = 0.27 (20% hexanes in EtOAc)

[0182] [a]D20= -82.2° (c 0.63 in CH3CN)

[0183] ’H NMR (400 MHz, (CD3)2CO) 5 10.20 (s, lH,H-3), 7.49 (d, J= 1.4 Hz, 1H, H-6), 6.03 (d, J = 1.4 Hz, 1H, H-L), 5.51 (dd, J= 12.3, 1.5 Hz, 1H, H-l "), 5.41 (d, J= 4.0 Hz, 1H, OH-2'), 5.11 (d, J= 12.3 Hz, 1H, H-l"), 4.71 (ddd, J = 5.7, 4.1, 1.4 Hz, 1H, H-2 ), 4.38 (d, J= 10.4 Hz, 1H, H-5 ), 4.35 (d, J= 6.1 Hz, 1H, H-3 ), 4.09 (dd, J = 10.4, 1.5 Hz, 1H, H-5 ), 1.82 (d, J = 1.3 Hz, 3H, H-7), 1.58 (s, 3H), 1.47 (s, 3H)

[0184] 13C NMR (101 MHz, (CD3)2CO) 5 164.3 C-4, 151.1 C-2, 138.3 C-6, 111.6 C-5, 103.1, 95.9 C- r, 77.2 C-4', 77.0 C-3 , 75.8 C-l", 73.2 C-2', 68.1 C-5', 29.3, 19.9, 12.2 C-7

[0185] HRMS (ESLTOF) m / z Calculated for [Ci4H2iN3O8]+358.1250; Found 358.1241

[0186] STEP 2: Synthesis of nitro-anhydro compound 3

[0187] The reaction was done using flame-dried glassware, and under nitrogen atmosphere. Dichloromethane, CH2Q2 was distilled out from Cath prior to use. Diethylaminosulfur trifluoride (DAST) was purchased from oakwood chemicals and used as received.

[0188] A 250 mL round-bottom flask was charged with nitro-anhydro compound 2 (2.84g, 7.95 mmol), NaHCCh (3.34 g, 39.78 mmol, 5.0 equiv.), and dry CH2CI2 (153 mL, c = 0.05 M). Then, it was cooled to -40 °C, and DAST (5.25 mL, 39.78 mmol, 5.0 equiv.) was added dropwise. After addition, the reaction was slowly warmed to 0 °C and stirred for 12 h at 0 °C.

[0189] At 0 °C, the reaction was quenched with the slow addition of a saturated aqueous solution of NaHCCL (50 mL, 1.0 M solution), then transferred to a 1 L separating funnel and diluted with CH2Q2 (100 mL). The organic phase was separated, and the aqueous layer was extracted with 10% of MeOH in CH2Q2 (70 mL, x3). The combined organic layers were washed with brine (250 mL), dried over MgSCU, and the solvent removed under reduced pressure. The crude product was purified by silica gel column chromatography using 80% EtOAc in hexanes (500 mL), {then gradually increased the polarity of eluent system} EtOAc (500 mL), and (,5% MeOH in EtOAc) to afford nitro-anhydro compound 3 as an off-white powder (1.34 g, 50% yield)

[0190] Rf =0.33 (20% acetone in EtOAc)

[0191] [a]D20= -121.7° (c 0.85 in CH3CN).

[0192] ’H NMR (500 MHz, CD3CN) 5 7.43 (d, J = 1.4 Hz, 1H, H-6), 6.18 (d, J = 6.4 Hz, 1H, H-L), 5.47 (dd, J= 7.4, 6.4 Hz, 1H, H-2 ), 5.11 (d, J= 12.6 Hz, 1H, H-l "), 4.52 (dd, J= 12.6, 1.7 Hz, 1H, H-l"), 4.35 (d, J= 10.5 Hz, 1H, H-5 ), 4.31 (d, J = 7.4 Hz, 1H, H-3 ), 3.98 (dd, J = 10.5, 1.7 Hz, 1H, H-5'), 1.87 (d, J= 1.3 Hz, 3H, H-7), 1.52 (s, 3H), 1.48 (s, 3H).

[0193] 13C NMR (126 MHz, CD3CN) 5 172.6 C-4, 160.5 C-2, 132.0 C-6, 119.4 C-5, 104.1, 88.8 C- L, 81.8 C-3 , 81.7 C-2', 76.6 C-4', 75.6 C-l", 67.1 C-5', 28.9, 19.8, 14.0 C-7. HRMS (ESLTOF) m / z Calculated for [Ci4Hi9N3O7]+340.1145; Found 340.1155

[0194] STEP 3: Nitro group reduction with N-Boc protection and cyclisation

[0195] Step 3.1: Nitro group reduction with N-Boc protection

[0196] The reaction was done with standard glassware.

[0197] A 250 mL round-bottom flask was charged with nitro-anhydro compound 3 (1.38 g, 4.06 mmol), ammonium chloride (0.435 g, 8.12 mmol, 2.0 equiv.), THF (73 mL), and water (8 mL). The THF:water ratio was set to 9:1 and to keep a 0.1 M concentration, relative to the starting material. Then, BOC2O, 95%, (1.06 g, 4.87 mmol, 1.2 equiv.) was added in one portion. At room temperature, zinc dust (2.65 g, 40.62 mmol, 10.0 equiv.) was added portion-wise with vigorous stirring. Then, the reaction was stirred at room temperature for 4 hours.

[0198] Once full consumption of starting material was observed by TLC (10% acetone in EtOAc), the reaction was filtered through celite to remove the solids, the reaction flask was rinsed with EtOAc (20 x3 mL) and the celite washed with EtOAc (30 mL) to wash all product. The solvent was removed under reduced pressure and the remaining water was removed via azeotropic, reduced pressure distillation with toluene. The crude solid was triturated with 15 mL of Et2O and hexanes in 1 : 1, was repeated 2 times, and azeotropically dried with toluene. The crude product was used in the next reaction without further purification.

[0199] N.B. It is essential to remove all of the unreacted BOC2O before setting up the next reaction.

[0200] An analytical sample of compound 4 was isolated to gather characterization data.

[0201] Characterization data for compound 4.

[0202] Rf =0.50 (30% acetone in EtOAc)

[0203] [a]D20= -101° (c 0.10 in MeOH) ’H NMR (500 MHz, CD3CN) 5 7.38 (d, J= 1.4 Hz, 1H, H-6), 7.23 (s, 1H, OH), 6.18 (d, J = 6.4 Hz, 1H, H-L), 5.42 (dd, J= 7.6, 6.4 Hz, 1H, H-2 ), 4.15 (d, J= 7.6 Hz, 1H, H-3 ), 4.11 (d, J = 9.9 Hz, 1H, H-5 ), 4.00 (d, J = 15.7 Hz, 1H, H-l"), 3.82 (dd, J= 9.9, 1.6 Hz, 1H, H-5 ), 3.59 (dd, J= 15.7, 1.6 Hz, 1H, H-l"), 1.88 (s, 3H, H-7), 1.48 (s, 12H), 1.46 (s, 3H).

[0204] 13C NMR (126 MHz, CD3CN) 5 172.9 C-4, 160.6 C-2, 157.5, 149.3, 132.1 C-6, 119.3 C-5, 103.4, 88.6 C-L, 82.2 C-2', 81.6 C-3 , 77.8 C-4', 68.5 C-5', 50.3 C-l", 29.2, 28.5, 28.3, 27.8, 19.9, 14.0 C-7.

[0205] HRMS (ESI-TOF) m / z Calculated for [CI9H28N3O8]+426.1876; Found 426.1866

[0206] Step 3.2: Cyclization

[0207] The reaction was done using flame-dried glassware, and under nitrogen atmosphere. DMF was dried over 4A molecular sieves. Sodium hydride was purchased as a 60% dispersion in mineral oil and used as received.

[0208] A 100 mL round-bottom flask was charged with crude product 4 (1.55 g), vacuumed and refilled with nitrogen (x3). Dry DMF (40 mL, c = 0.1 M) was added to dissolve the crude. Then, in an ice bath, NaH, 60% dispersion in mineral oil, (0.244g, 10.15 mmol, 2.5 equiv.) was added portion-wise. Hydrogen gas evolution was observed. The chilling ice bath was then removed, and the reaction stirred at room temperature for 3 h.

[0209] The reaction was quenched by the dropwise addition of acetic acid (50.0 mL, 10% w / w aqueous solution) and partitioned with EtOAC (50mL x3). The organic phase was separated and the aqueous layer extracted with EtOAc (X mL, x3). The combined organic extracts were washed with brine (250 mL), dried over MgSCU and the solvent removed under reduced pressure. Silica gel column chromatography (25% acetone in hexanes) afforded the desired, cyclized product as a white solid (0.73g, 42.2% yield over two steps). Characterization data for compound 5.

[0210] Rf = 0.50 (20% acetone in CH2CI2)

[0211] [a]D20= +8.8° (c 0.80 in MeOH) ’H NMR (400 MHz, CD3CN) 5 9.10 (s, 1H, H-3), 7.28 (d, J= 1.3 Hz, 1H, H-6), 6.04 (s, 1H, H-L), 4.66 (d, J= 3.2 Hz, 1H, H-3'), 4.30 (d, J= 10.7 Hz, 1H, H-5 ), 4.03 (d, J= 10.7 Hz, 1H, H-5 ), 3.98 (m, 2H, H-l "), 3.61 (d, J = 3.2 Hz, 1H, H-2 ), 1.88 (d, J= 1.2 Hz, 3H, H-7), 1.51 (s, 3H), 1.48 (s, 12H).

[0212] 13C NMR (101 MHz, CD3CN) 5 164.6 C-4, 155.5, 151.1 C-2, 135.1 C-6, 111.2 C-5, 102.8, 87.3 C-L, 82.8, 80.4 C-3 , 73.4 C-4', 68.7 C-2', 67.5 C-5', 48.3 C-l", 29.2, 28.5, 19.8, 12.6 C-7.

[0213] HRMS (ESI-TOF) m / z: Calculated for [C19H28N3O8]+426.1876; Found 426.1859

[0214] STEP 4: N-Boc deprotection

[0215] The reaction was done with standard glassware. Commercial dichloromethane (HPLC grade, >99.9%) was used as received without further drying or distillation Trifluoroacetic acid (TFA, >99%) was purchased from Sigma-Aldrich and used as received.

[0216] A 25 mL round-bottom flask was charged with compound 5 (0.78 g, 1.83 mmol) in CH2Q2 (17.5 mL) and water (0.87 mL) the CH2Q2: water ratio was set to 20:1 and kept to a 0.1 M concentration. Then, it was cooled to 0 °C, and trifluoroacetic acid (TFA) (2.12 mL, 27.5 mmol, 15.0 equiv.) was added dropwise. After the addition of TFA, the reaction was stirred at room temperature for 36 h. After the completion of the starting material, CH2Q2 and TFA were removed under reduced pressure. The crude product was purified by silica gel column chromatography (10% MeOH in EtOAc) to afford the desired compound 6 as the trifluoroacetate salt a colorless sticky semisolid (0.53 g, 72% yield).

[0217] [a]D20= +11° (c 0.20 in MeOH)

[0218] I have attached the NMR files, sam-01 -83 -TFA-CD3CN, sam-01 -83 -TFA-CD3CN

[0219] ’H NMR (400 MHz, MeOD) 5 8.15 (d, J = 1.2 Hz, 1H, H-6), 6.25 (s, 1H, H-l'), 4.23 (d, J = 3.5 Hz, 1H, H-2'), 4.09 (d, J= 3.5 Hz, 1H, H-3'), 3.75 (d, J= 12.5 Hz, 1H, H-5'), 3.68 (d, J= 12.5 Hz, 1H, H-5'), 3.55 (d, J = 13.0 Hz, 1H, H-l"), 2.51 (dd, J = 13.0, 1.4 Hz, 1H, H-l"), 1.88 (d, J = 1.2 Hz, 3H, H-7).

[0220] 13C NMR (151 MHz, MeOD) 5 163.4 C-4, 152.0 C-2, 137.4 C-6, 110.6 C-5, 86.6 C-l', 83.8 C-4', 83.1 C-3', 64.9 C-2', 60.8 C-5' 47.8 C-l", 12.6 C-7.

[0221] HRMS (ESI-TOF) m / z Calculated for [C11H15N3O3]+286.1039; Found 286. 1035.

[0222] STEP 5: Synthesis ofBNANC

[0223] The reaction was done with standard glassware.

[0224] A 25 mL round-bottom flask was charged with compound 6 (0.78 g, 1.53 mmol) in MeOH (15 mL, c = 0.1 M). Then, it was cooled to 0 °C, and formaldehyde (36.5 - 38.0 % aqueous solution, 0.88 mL, 3.84 mmol, 2.5 equiv.), NaBFFCN (96 mg, 1.53 mmol, 1.0 equiv.), and acetic acid (0.26 mL, 4.58 mmol, 3.0 equiv.) were added at 0 °C. The mixture was stirred at room temperature and the reaction progress was monitored by TLC. After 60 minutes, all of the starting material was consumed as determined by TLC (ethyl acetate: Methanol 9: 1), with only a single non-polar spot as compared to the SM. The solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (5% MeOH in EtOAc) to afford the desired compound as a white solid (0.43 g, 93% yield).

[0225] [a]D20= +19.9° (c 0.50 in MeOH)

[0226] ’H NMR (600 MHz, MeOD) 5 8.01 (d, J = 1.2 Hz, 1H, H-6), 6.22 (s, 1H, H-l'), 4.23 (d, J = 2.7 Hz, 1H, H-3 ), 3.94 (d, J= 3.2 Hz, 1H, H-2 ), 3.76 (d, J= 12.5 Hz, 1H, H-5 ), 3.70 (d, J= 12.5 Hz, 1H, H-5'), 2.88, 2.72 (d, J = 11.4 Hz, 2H, H-l"), 2.72 (s, 3H, NCH3), 1.88 (d, J = 1.2 Hz, 3H, H-7).

[0227] 13C NMR (151 MHz, MeOD) 5 166.7 C-4, 152.0 C-2, 137.6 C-6, 110.5 C-5, 86.9 C-l', 84.9 C-3 , 82.6 C-4', 65.0 C-2', 61.1 C-5', 58.5 C-l", 45.7, 12.6 C-7. HRMS (ESI-TOF) m / z Calculated for [C12H18N3O3]+300.1196; Found 300.1198.

[0228] EXAMPLE 2: Screening of conditions to prepare compound 2

[0229] The same general protocol as the one used in STEP 1 was used. The reaction was performed on 25 mg to 8 g scale in the presence of different bases and at different reaction time (refer to table 1). The acetonitrilemitromethane ratio was set to 3: 1 to keep a 0.1 M concentration calculated from the amount of ketofluorohydrin 1.

[0230] Table 1 entry scale base (equiv.) time yield dr

[0231] 1 25 mg Li2CO3(3.0) 24 h

[0232] 2 25 mg Na2CO3(3.0) 24 h

[0233] 3 25 mg K2CO3(3.0) 24 h 39% 5: 1

[0234] 4 25 mg SrCO3(3.0) 24 h

[0235] 5 25 mg Cs2CO3(3.0) 7 h 44% 20: 1

[0236] 6 25 mg Cs2CO3(3.0) 24 h 29% 8.5: 1

[0237] 7 25 mg Cs2CO3(3.0) 48 h 30% 20: 1

[0238] 8 350 mg Cs2CO3(3.0) 18 h 41% 10: 1

[0239] 9 750 mg Cs2CO3(3.0) 18 h 31% n.d.

[0240] 10 1.8 g Cs2CO3(3.0) 18 h 26% 12: 1

[0241] 11 150 mg K3PO4(3.0) 2.5 d 42% n.d.

[0242] 12 1.0 g K3PO4(1.0) 2.5 d 21% 5: 1

[0243] 13 1.0 g K3PO4(1.5) 2.5 d 36% 11 : 1

[0244] 14 1.0 g K3PO4(2.0) 2.5 d 48% 15: 1

[0245] 15 1.3 g K3PO4 (3.0) 2.5 d 50% n.d.

[0246] 16 1.0 g K3PO4(4.0) 2.5 d 42% n.d.

[0247] 17 2.6 g K3PO4(3.0) 2.5 d 42% n.d.

[0248] 18 5.2 g K3PO4(3.0) 2.5 d 48% n.d.

[0249] 19 8.0 g K3PO4 (3.0) 3.0 d 50% n.d.

Claims

CLAIMS1. A method for preparing the Compound of formula (IV):wherein:Ri and R2 each independently represent a hydrogen atom; a hydroxy protecting group; a reactive phosphorus group; or Ri and R2 together form a cyclic group;R4 represent a hydrogen atom, a Cl to C6 alkyl group, a C2 to C6 alkenyl group or a C2 to C6 alkynyl group;Rs represent an oxygen or NFC- group, wherein R5’ is a hydrogen or a suitable amino protecting group; andPG represents an amino protecting group, comprising the step of subjecting a compound of formula (II)wherein Ri, R2, R4 and R5 are as defined above, to a reduction of its nitro moiety using a suitable reducing agent to obtain the corresponding N-hydroxyl amine compound of formula (III):wherein Ri, R2, R4 and R5 are as defined above, the hydroxylamine function of which compound of formula (III) is immediately protected through in situ acylation of the amine moiety, to obtain the compound of formula (IV).

2. The method according to claim 1, wherein the nitro moiety of the compound of formula (II) is reduced using a suitable reducing agent selected from activated zinc / acetic acid, activated zinc / ammonium chloride and an aluminum / mercury amalgam.

3. The method according to claim 2, wherein the nitro moiety of the compound of formula (II) is reduced using an activated zinc / ammonium chloride.

4. The method according to claim 3, wherein the amount of zinc used to conduct the reduction step is comprised between 10.0 to 30.0 equivalents, calculated from the molar quantity of the compound of formula (II) and the amount of ammonium chloride used to conduct the reduction step is comprised between 1.0 to 3.0 equivalents.

5. The method according to any one of claims 1 to 4, wherein the reduction step is conducted in a mixture of an organic solvent and water.

6. The method according to claim 5, wherein the reduction step is conducted in a mixture of tetrahydrofuran and water.

7. The method according to claim 6, wherein the molar ratio between tetrahydrofuran and water is of 9:1 and the reduction step is conducted at a concentration between 0.05 M to 1.0 M, calculated from the amount of the compound of formula (II).

8. The method according to any one of claims 1 to 7, wherein the hydroxylamine function of compound of formula (III) is immediately protected through in situ acylation of the amine moiety using a t-butoxycarbonyl protecting group.

9. The method according to claim 8, wherein the amount of di-tert-butyl dicarbonate used to protect the amine moiety is comprised between 0.5 to 3.0 equivalents, calculated from the molar quantity of the compound of formula (II).

10. The method according to any one of claims 1 to 9, wherein the reduction-protection step is conducted at a temperature ranging from 15°C to 100°C, preferably from 15°C to 50°C, more preferably from 15°C to 25°C, for 0.5 to 12 hours, preferably 0.5 to 6 hours.

11. The method according to any one of claims 1 to 10, wherein the compound of formula (IV) is further subjected to a bridge formation to obtain the compound of formula (V):wherein Ri, R2, R4, Rs and PG are as defined in claim 1.

12. The method according to claim 11, wherein the bridge formation step is conducted in the presence of an organic or inorganic base having a pka from 8 to 40 in an aprotic solvent.

13. The method according to claim 11 or 12, wherein the amount of base used in the bridge formation step is comprised between 0.5 to 3.0 equivalents, calculated from the molar quantity of the compound of formula (IV).

14. The method according to any one of claims 11 to 13, wherein the bridge formation step is conducted in the presence of sodium hydride.

15. The method according to any one of claims 11 to 14, wherein the bridge formation step is conducted in dimethyl formamide.

16. The method according to any one of claims 11 to 15, wherein the bridge formation step is conducted at a temperature ranging from 15°C to 100°C, preferably from 15°C to 50°C, more preferably from 15°C to 25°C, for 0.5 to 6 hours, preferably 0,5 to 3 hours.

17. The method according to claim 11, wherein the compound of formula (V) is further deprotected under suitable conditions to obtain the compound of formula (VI):wherein Ri, R2, R4 and Rs are as defined in claim 1,which compound of formula (VI) is subjected to a reductive amination to give the compound of formula A:Wherein R3 represents a Cl to C18 alkyl group, a C2 to Cl 8 alkenyl group, a C2 to Cl 8 alkynyl group, a C3 to CIO cycloalkyl group, an aryl group or an acyl group; andRi, R2, R4 and R5 are as defined in claim 1.

18. The method according to claim 17, wherein the amine moiety of compound of formula (V) is deprotected under strong acidic conditions.

19. The method according to claim 18, wherein the deprotection step is conducted with trifluoracetic acid.

20. The method according to any one of claims 17 to 19, wherein the reductive amination step is conducted by reacting the amine function of compound of formula (VI) with an aldehyde, or a ketone in the presence of a reducing agent selected from sodium triacetoxyborohydride, sodium cyanoborohydride, sodium borohydride, lithium borohydride and lithium aluminum hydride.

21. The method according to claim 20, wherein the amount of an aldehyde or ketone and reducing agent used in the reductive amination step is comprised between 0.5 to 3.0 equivalents, calculated from the molar quantity of the compound of formula (VI).

22. The method according to any one of claims 17 to 21, wherein the reductive amination step is conducted in a suitable solvent at a concentration between 0.01 M to 1.0 M, at a temperature ranging from 15°C to 100°C, preferably from 15°C to 50°C, more preferably from 15°C to 25°C, for 0.5 to 12 hours, preferably 1 to 6 hours.

23. The method according to any one of claims 1 to 22, wherein the compound of formula (II) is obtained by subjecting a compound of formula (I):wherein Ri, R2, R4 and R5 are as defined in claim 1, to dehydration in the presence of a dehydrating agent and a base, in an organic solvent24. The method according to claim 23, wherein the dehydrating agent is a condensing agent selected from dialkyl and diaryl carbonates or a fluorinating agent selected from (diethylamino)sulfur trifluoride and trifluoromethanesulfonic anhydride.

25. The method according to claim 24, wherein the dehydrating agent is (diethylamino)sulfur trifluoride.

26. The method according to any one of claims 23 to 25, wherein the dehydration step is conducted in the presence of a base selected from sodium carbonate, potassium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide and sodium methoxide.

27. The method according to claim 26, wherein the base is sodium bicarbonate.

28. The method according to any one of claims 23 to 27, wherein the dehydration step is conducted in the presence of an aprotic organic solvent selected from dichloromethane, acetonitrile, toluene, ethyl acetate, butyl acetate, isobutyl acetate, propyl acetate, isopropyl acetate, chlorobenzene, -di methyl form am ide and pyridine.

29. The method according to any one of claims 23 to 28, wherein the dehydration step is conducted at a temperature ranging from -78°C to 100°C, preferably from -40°C to 50°C, more preferably at 0°C, for 0.5 to 12 hours, preferably 2 to 6 hours.

30. The method according to any one of claims 1 to 29, wherein Ri and R2 each independently represent a hydroxy protecting group; or Ri and R2 together form a cyclic group.

31. The method according to any one of claims 1 to 29, wherein Ri and R2 together form a cyclic group.

32. The method according to claim 31, wherein Ri and R2 together represent a group selectedfrom -OC(CH3)2O-, - (CH2)3-, -CH2SCH2-, -CH2OCH2-, -OCH(C6H5)O-, -OC(C6HIO)0-, - OC(C4H8)O-, -Si(CH3)2OSi(CH3)2-, -Si(OCH3)2OSi(OCH3)2-, -Si(CH3)2CH2Si(CH3)2-, and -OSi(CH3)2O-.

33. The method according to claim 32, wherein Ri and R2together represent -OC(CH3)2O-.

34. The method according to claim 17, wherein R3is a Cl to C6 alkyl group.

35. The method according to claim 34, wherein R3is a methyl group.

36. The method according to any one of claims 1 to 35, wherein R4is a methyl group and Rs is an oxygen.

37. The method according to any one of claims 1 to 36, wherein when Ri and R2together form a cyclic group, then Ri and R2are further hydrolyzed under strong acidic conditions such as hydrochloric acid or trifluoracetic acid.

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