KR20110099333A - Sulfurizing reagents and their use for oligonucleotides synthesis - Google Patents

Abstract

(I)
(식 중, A는 동일위치에 치환되는 알킬렌기, 바람직하게는 CH₂이고; X 및 Y는 S 및 0 중에서 독립적으로 선택되고; R₀은 임의 치환된 탄소-결합된 유기 잔기, 예를 들어 구체적으로는 임의 치환된 알킬 또는 아릴, SR_x, OR_x 및 NR_xR_y (R_x 및/또는 R_y는 H 및 유기 잔기 중에서 선택되며 적어도 R_x는 H 이외의 치환기임)로 이루어진 군에서 선택됨). 본 발명의 다른 목적은 올리고뉴클레오타이드 제조에 유용한 황화제 및 그 제조이다.The present invention is at least one internucleotide linkage comprising a PSR linkage and an oligonucleotide comprising at least two nucleosides, wherein R corresponds to formula (I):

(I)
Wherein A is an alkylene group substituted at the same position, preferably CH ₂ ; X and Y are independently selected from S and 0; R ₀ is an optionally substituted carbon-bonded organic moiety, for example Specifically, in the group consisting of optionally substituted alkyl or aryl, SR _x , OR _x and NR _x R _y (R _x and / or R _y is selected from H and organic moieties and at least R _x is a substituent other than H) Selected). Another object of the present invention is a sulfiding agent useful for preparing oligonucleotides and the preparation thereof.

Description

Sulfurized reagents and their use for oligonucleotide synthesis {SULFURIZING REAGENTS AND THEIR USE FOR OLIGONUCLEOTIDES SYNTHESIS}

This application claims the benefit of US patent application Ser. No. 61/140391, filed December 23, 2008, the entire contents of which are incorporated herein by reference.

The present invention relates to the preparation of the phosphorothioate oligonucleotides (phosphorothioate oligonucleotides), a novel sulfiding reagent, the sulfiding reagent and the preparation thereof.

Oligonucleotides belong to a class of herbal drugs with great potential for the treatment of various diseases (including several, for example, cancer, viral infections and inflammatory diseases). One important way to develop oligonucleotides as therapeutics is to modify the oligomer backbone, among other things to provide metabolic resistance and chemical stability and to enhance in vivo delivery to the site of action. Examples of modified backbone compounds include: peptide nucleic acids (PNAs) (see Nielsen, Methods Mol. Biol., 208: 3-26, 2002), locked nucleic acids (LNAs) (Petersen & Wengel, Trends Biotechnol., 21) (2): 74-81, 2003), phosphorothioate (see Eckstein, Antisense Nucleic Acid Drug Dev., 10 (2): 117-21, 2000), methylphosphonate (Thiviyanathan et al., Biochemistry, 41 (3): 827-38, 2002), phosphoramidate (Gryaznov, Biochem. Biophys. Acta, 1489 (1): 131-40, 1999; Pruzan et al., Nucleic Acids Res., 30 (2): 559-68, 2002), thiophosphoamidate (Gryaznov et al., Nucleosides Nucleotides Nucleic Acids, 20 (4-7): 401-10, 2001; Herbert et al., Oncogene, 21 (4): 638-42, 2002). Formation of phosphorothioates is one of the most useful modifications because the substitution of P═O with the P═S moiety makes the oligonucleotides resistant to nucleolysis, while in most cases the biological oligomers This is because the characteristics are maintained.

Phosphorothioates can be formed by oxidative sulfidation (see Oligonucleotide synthesis, methods and applications, P. Herdewijn Methods in Molecular Biology, volume 288, Chapter 4, 51-63). Depending on the nature of the phosphite ester used in this reaction and the nature of the expected product, phosphorothioate is prepared basically by two methods. One of them is, for example, elemental sulfur, dibenzoyl tetrasulfide, 3-H-1,2-benzodithiol-3-one 1,1-dioxide (also known as Beaucage reagent (Iyer et al., J. Org.Chem. 55, 4693-4699 (1990)), tetraethylthiuram disulfide (TETD), dimethylthiuram disulfide (DTD), phenylacetyl disulfide (PADS) and bis (O, O-disisopro The introduction of unsubstituted sulfur atoms into the phosphorus via a foxy phosphinothioyl) disulfide (known as Stec reagent), these reactions are mainly used for the automatic synthesis of oligonucleotides on a solid support by the phosphoramidite method, Oxidative sulfiding of the phosphorous acid triester formed during the extension of the oligomer.

The second method for preparing oligomeric phosphorothioates is to use the H-phosphonate method, which reacts a H-phosphonate diester with a sulfur transfer reagent that transfers sulfur atoms with aliphatic or aromatic substituents to phosphorus. . Auxiliary substituents on sulfur serve as protecting groups in the synthetic operation and are generally cleaved at the final stage of oligonucleotide preparation. This method is particularly suitable for the synthesis of oligonucleotides in solution.

In contrast to the wide choice of reagents available for introducing unsubstituted sulfur atoms into phosphite esters, the range of groups for sulfiding H-phosphonate esters with protected sulfur is limited (eg, Dreef, et al. Synlett, 481-483, 1990, see US Pat. No. 6,506,894. Indeed, only cyanoethylsulfide groups have been used extensively in this reaction during oligonucleotide solution synthesis, where chromatographic purification is performed at each step. An important problem in oligonucleotide solution synthesis relates to the need to obtain high substrate conversions with good specificity at each synthesis step, in particular providing high purity products in the form of avoiding chromatography and facilitating simple purification. Given the lack of methods to enable economical solution phase synthesis, solution phase techniques do not appear to be currently used for commercial scale oligonucleotide synthesis.

Accordingly, the present invention aims to provide a novel sulfided reagent, a method for preparing the same, and particularly the use of the sulfided reagent in the economical and convenient synthesis and purification of a solution phase phosphorothioate oligonucleotide.

The present invention specifically relates to the invention described in the appended claims. The invention also relates to the methods and reagents generally described herein, particularly in the Examples.

The present invention has many advantages over existing PS linkage formation methods, particularly in the synthesis of oligonucleotides which are preferably carried out via the H-phosphonate method. For example, residue R delivered to oligonucleotides using novel reagents may facilitate crystallization or precipitation of oligonucleotides, allowing simple purification of the product with minimal or no chromatography. . For oligonucleotides prepared in this manner and having from 2 to at least 16 nucleotide units, it has been found that there is no need to purify by chromatography until the required oligonucleotides are finally deprotected. Intermediate oligomers can be obtained with any deprotection at the 5'- and 3'- positions, and if desired pure enough to couple these crude deprotected materials with more oligonucleotides. In another advantage, the methods of the present invention provide access to a variety of sulfiding reagents that can be used to modify the properties of the oligonucleotides formed in connection with maximizing the efficiency of the simple but chromatographic purification. . Another advantage of the process according to the invention is that, for example, sulfur-protected acyloxymethylene groups RC (O) -OCH ₂ are subjected to, for example, primary or secondary or hindered conditions under ambient conditions. It can be easily cleaved using amines (eg n-propylamine or tert-butylamine). In optional treatment with amines, spontaneous cleavage occurs, including sulfur-methylene bonds, and P = S bonds are clearly formed. Cleavage products can be easily removed from the product using solvents or aqueous washes. The stability characteristics of acyloxymethylene groups, for example under basic non-nucleophilic conditions, allow selective deprotection along the synthetic route, thus allowing more flexibility in the synthesis process, for example by preventing cleavage of nucleobase protecting groups. do.

An important factor, especially in developing economical methods for the synthesis of solution phase oligonucleotides, is the purity of the conversion product at each stage of oligonucleotide chain extension. Although the process according to the invention ensures high yield and high purity of the product, each extension period generally comprises three steps, and it is advantageous to remove them, since even small amounts of impurities will accumulate along the way. Because there are many steps, using chromatographic methods at each step may not be economically feasible in actual large scale oligonucleotide synthesis. Accordingly, we also disclose a methodology that does not use chromatography for the purification of oligonucleotides formed in the course of chain extension.

It is a first object of the present invention to provide at least one internucleotide linkage comprising a P-S-R bond and an oligonucleotide comprising at least two nucleosides, wherein R corresponds to formula (I):

Wherein A is an alkylene group substituted at the same position, preferably CH ₂ ; X and Y are independently selected from S and 0; R ₀ is an optionally substituted carbon-bonded organic moiety, for example Specifically, optionally substituted alkyl or aryl, SR _x , OR _x and NR _x R _y (R _x and R _y are selected from H and organic moieties and at least R _x is a substituent other than H) .

Synthetic intermediates in which the oligonucleotides according to the invention are important for the synthesis of P-sulphide oligonucleotides having advantageous properties on soluble characteristics, allowing for efficient purification which can be effectively achieved, for example, by a combination of precipitation and extraction techniques. It turned out that In addition, the oligonucleotides according to the present invention are effectively regarded as prodrugs capable of releasing phosphorothioate oligonucleotides in vivo by cleaving R groups in the human or animal body.

The term "oligonucleotide" within the constitution of the present invention specifically refers to oligomers of nucleoside monomer units including sugar units linked to nucleobases, wherein the nucleoside monomer units are internucleotide bonds. Are connected by "Internucleotide linkage" specifically refers to a chemical linkage between two nucleoside moieties, eg, to phosphodiester bonds or synthetic nucleic acids and nucleic acid analogs that are commonly present in nucleic acids found in nature. Indicates other associations that exist. Such internucleotide bonds may include, for example, a phospho group or a phosphite group, wherein one or more oxygen atoms of the phospho group or phosphite group are modified with a substituent (eg, a sulfur atom, or a mono- or Bonds substituted with other atoms (such as nitrogen atoms of the di-alkyl amino groups) may be included. Common internucleotide linkages are diesters of phosphoric acid or derivatives thereof, for example phosphates, thiophosphates, dithiophosphates, phosphamidates, thiophosphamidates.

The term "nucleoside" specifically refers to a compound composed of nucleobases linked to sugars. Such sugars include furanose rings (eg ribose), 2'-deoxyribose and non-pentagonal rings (eg cyclohexenyl, anhydrohexitol, morpholino) It is not limited to this. The modifications, substitutions and positions indicated below with respect to the sugars contained in the nucleosides are described with reference to the pentagonal ring, although the same modifications and positions apply to similar positions of other sugar rings. Sugars can be further modified. Non-limiting examples of sugar modifications include, for example, hydrogen; Hydroxy; Alkoxy such as methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy, methoxyethyl, alkoxy, phenoxy; Azido; Amino; Alkylamino; Fluoro; Chloro; And modification at the 2'- or 3'-position (specifically 2'-position) of a pentagonal sugar ring comprising bromo; Particular mention may be made of 2'-4'- and 3'-4'-linked pentagonal sugar ring modifications, wherein the modifications in the pentagonal sugar ring are for example ring 4'-O to S, CH ₂ , NR, CHF Or substituted with CF ₂ .

The term "nucleobase" is understood to specifically refer to a nitrogen-containing heterocyclic moiety that can be paired with a complementary nucleobase or nucleobase analog. Typical nucleobases include naturally occurring nucleobases including the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U); As modified nucleobases comprising other synthetic and natural nucleobases, for example 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- Propynyl uracil and cytosine, and other alkynyl derivatives of the pyrimidine base, 6-azo uracil, cytosine and thymine, 5-urasecil, 4-thiouracil, 8-halo, 8-amino, 8-thiol , 8-thioalkyl, 8-hydroxyl and other 8-substituted adenine and guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine And 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azadenine, 7-diazaguine and 7-diazadenine, 3-diazaguinine and 3-diazadenine, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, for example phenoxazine cytidine (1H-pyrimido [5,4-b] [1,4] benzoxazine-2 (3H) -one) , Phenothiazine cytidine (1H-pyrimido [5,4-b] [1,4] benzothiazine-2 (3H) -one), G-clamps such as substituted phenoxazine cytidines (eg , 9- (2-aminoethoxy) -H-pyrimido [5,4-b] [1,4] benzoxazine-2 (3H) -one), carbazole cytidine (2H-pyrimido [4 , 5-b] indol-2-one), pyridoindole cytidine (H-pyrido [3 ', 2': 4,5] pyrrolo [2,3-d] pyrimidin-2-one) Include. Other potentially suitable bases include universal bases, hydrophobic bases, promiscuous bases, and size-enlarging bases.

"Oligonucleotide" generally refers to a nucleoside subunit polymer having from about 2 to about 50 contiguous subunits. Nucleoside subunits may be linked by various interunit linkages. In addition, “oligonucleotides” include variants known to those skilled in the art, such as variants for sugar backbones (eg, phosphoramidates, phosphorodithioates), variants for sugars (eg, 2′-F, 2 'substitutions, such as 2'-OMe), variants for bases, and variants for 3' and 5 'termini. Typically, oligonucleotides in the present invention comprise 2 to 30 nucleotides. According to another embodiment of the invention, the oligonucleotide is ribonucleoside, 2'-deoxyribonucleoside, 2'-substituted ribonucleoside, 2'-4'-fixed-ribonucleoside, 3 Nucleosides selected from '-amino-ribonucleosides, 3'-amino-2'-deoxyribonucleosides.

In some oligonucleotides of the invention, R is selected from methyleneacyloxy groups, methylenecarbonate groups and methylenecarbamate groups.

When R is a methyleneacyloxy group, it preferably corresponds to the formula -CH ₂ -OC (O) -R ₀ , wherein R ₀ is a saturated, unsaturated, heterocyclic or aromatic, hydrocarbon moiety of C 1 -C 20. . When R ₀ is a saturated hydrocarbon residue, it is preferably selected from linear, branched or cyclic alkyl residues. R ₀ may for example be selected from lower alkyl or cycloalkyl (C 1 -C 7) residues. Specifically, the saturated hydrocarbon residue is selected from among methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl cyclopentyl and cyclohexyl. Methyl group, ethyl group or n-propyl group is preferable. Ethyl groups are more particularly preferred. When R ₀ is an aromatic moiety, it is suitably selected from aromatic systems having 6 to 14 carbon atoms. Specifically, the aromatic moiety is, for example, a phenyl group and a nap that can be substituted with aryl or heteroaryl, alkyl, cycloalkyl, heterocycle or heterosubstituents (eg halogen, amine, ether, carboxylate, nitro, thiol, sulfonic acid and sulfone). It is selected from the group. Phenyl groups are preferred. When R ₀ is a heterocyclic moiety it is often selected from heterocycles containing one or more cyclic N, O or S atoms bonded to a carbonyl group via a cyclic carbon atom. Specific examples of such heterocyclic moieties include pyridine and furan.

According to certain aspects, the oligonucleotide comprises at least two internucleotide bonds and at least three nucleotides, including a P-S-R bond (R is a methyleneacyloxy group as described herein).

When R is a methylenecarbamate group, it preferably corresponds to the formula -CH ₂ -OC (O) -NR _x R _y , wherein R _x and R _y are independently selected from alkyl or (hetero) aryl. Preferably R _x and / or R _y is an alkyl group. In this case R _x and / or R _y may for example be selected from lower alkyl or cycloalkyl (C 1 -C 7) residues. Specifically, the alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert butyl, cyclopentyl and cyclohexyl. Methyl group, ethyl group or n-propyl group is preferable. According to a particularly preferred aspect both R _x and R _y in the methylene carbamate group are alkyl groups, specifically as previously described. More particular preference is given to N, N-dimethyl or N, N-diethyl groups. According to another aspect of this embodiment, R _x and R _y together form a 3 to 8 membered ring optionally containing an additional cyclic heteroatom selected from O, N and S. Specific examples include N-piperidyl or N-pyrrolidyl groups.

When R is a methylene carbonate group, it preferably corresponds to the formula -CH ₂ -OC (O) OR _x , wherein R _x is selected from optionally substituted alkyl, cycloalkyl and (hetero) aryl groups. Preferably, R _x is an alkyl group. In this case R _x can be selected from among lower alkyl or cycloalkyl (C 1 -C 7) residues, for example. Specifically, the alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert butyl, cyclopentyl and cyclohexyl. Methyl group, ethyl group or n-propyl group is preferable. Ethyl groups are more particularly preferred. When R _x is an aryl group, it is suitably selected from aromatic systems having 6 to 14 carbon atoms. Specifically, the aromatic residue is selected from phenyl group and naphthyl group. Phenyl groups are preferred. When R _x is a heterocyclic moiety it is often selected from heterocycles containing one or more cyclic N, O or S atoms bonded to an oxycarbonyl group via a cyclic carbon atom. Specific examples of such heterocyclic moieties include pyridine and furan.

For the case where R is selected from methyleneacyloxy group, methylenecarbonate group and methylenecarbamate group, the definitions and preferences of the substituents R _x , R _y and R ₀ provided above are such that X and / or Y in formula (I) Of course, the same applies to the corresponding thioanalogue, which is sulfur. It is of course also possible that the substituents mentioned are optionally substituted by, for example, halogen or alkoxy substituents or modified by, for example, inserting a chain heteroatom, in particular oxygen, into the alkyl chain.

A specific second object of the invention relates to the sulfiding agent of the formula R ″ -S-R, wherein R is as previously defined with respect to the oligonucleotide according to the invention and R ″ is a leaving group.

It has been found that the sulfiding agents according to the invention enable sulfur transfer, which is particularly efficient in forming S-protected phosphorothioate internucleotide bonds, specifically in oligonucleotides. The sulfiding agent according to the present invention introduces protected sulfur and can selectively and efficiently cleave the protecting group from the protected sulfur.

In the sulfiding agent according to the invention, the leaving group R ″ is generally an electrophilic group. Often, R ″ is a group containing an electrophilic nitrogen atom bonded to sulfur. Electrophilic nitrogen atoms are suitably substituted with one or more electron-withdrawing groups.

In one particular embodiment of the sulfiding agent of the invention, the sulfiding agent corresponds to formula (II):

Wherein R ^A and R ^B are the same or different from each other and R ^A and R ^B At least one of which is selected from a substituted sulfonyl group or acyl group, wherein R ^A and R ^B optionally together form a cyclic substituent).

R ^A and R ^B When at least one of, preferably one of them is substituted sulfonyl, it is generally selected from alkylsulfonyl groups and arylsulfonyl groups. Preferably, when at least one of R ^A and R ^B is an alkylsulfonyl group, its alkyl substituent is preferably selected from lower alkyl or cycloalkyl (C 1 -C 7) residues. Specifically, the alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, cyclopentyl and cyclohexyl groups. Methyl group, ethyl group or n-propyl group is preferable. Methyl groups are more particularly preferred. R ^A and R ^B When at least one of is an arylsulfonyl group, its aryl substituent is, for example, an optionally substituted phenyl group. R ^A and R ^B When at least one, preferably both sides are acyl groups, they are generally selected from alkylacyl groups and arylacyl groups. Preferably, R ^A and R ^B When at least one of is an alkylacyl group, its alkyl substituent is preferably selected from lower alkyl or cycloalkyl (C1-C7) residues. Specifically, the alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, cyclopentyl and cyclohexyl groups. Methyl group, ethyl group or n-propyl group is preferable. Methyl group, ethyl group or n-propyl group is preferable. Methyl groups are more particularly preferred. In a particularly preferred embodiment, R ^A and R ^B are together an acyl group which forms a cyclic substituent, preferably a 4 to 7 membered cyclic substituent.

In one particular embodiment, the sulfiding agent corresponds to formula (III):

Wherein R ₁ , R ₃ and R ₄ are independently C ₁ -C 20, any unsaturated or aromatic hydrocarbon moiety, preferably a linear or branched alkyl group or a cycloalkyl group.

In another specific embodiment of the sulfiding agent according to the invention, R ″ is dicarboxyamide. In one specific aspect of the invention, the sulfiding agent corresponds to formula (IV):

로 이루어진 군 중에서 선택되는 기이며, 바람직하게 Z는

기임).(Wherein Z is -CH ₂ -CH _2- , -CH = CH-, -CH ₂ -O-CH _2- ,

Group is selected from the group consisting of, preferably Z is

Term).

A specific third object of the invention relates to a process for the synthesis of sulfiding agents according to the invention, which process comprises: (a) a sulfuryl halide, preferably sulforyl chloride, of the formula RSC (O) -R ₂ R is as previously described, and R ₂ is an organic moiety which is preferably reacted with a thioacetal of C 1 -C 20 any unsaturated or aromatic hydrocarbon moiety, wherein the formula RSW (W is halogen, preferably C1), and (b) reacting the intermediate with an N-sulfonyl compound or N-acyl compound. According to a more specific embodiment of the invention, the formula R ₁ -C (O) -O-CH ₂ -SC (O) -R ₂ , wherein R ₁ and R ₂ are independently C ₁ -C 20 any unsaturated or Thioacetals of aromatic hydrocarbon moieties are reacted with sulfuryl chloride to formula R ₁ -C (O) -O-CH ₂ -S-Cl, wherein R ₁ is independently C1-C20, any unsaturated or aromatic Intermediates). According to another specific embodiment of the invention, the intermediate in step (b) is a compound of formula R ₃ -S (O) ₂ -NH-R ₄ Wherein R ₃ and R ₄ are independently organic moieties and are preferably reacted with N-sulfonyl compounds of C 1 -C 20 any unsaturated or aromatic hydrocarbon moiety.

In the process according to the invention for the synthesis of sulfiding agents, the reaction of step (a) is generally carried out in an aprotic polar organic solvent, for example in a halogenated hydrocarbon solvent (especially a chlorinated hydrocarbon solvent such as methylene chloride).

In the process according to the invention for the synthesis of sulfiding agents, the reaction of step (a) is generally carried out at temperatures of -80 ° C to 30 ° C.

In the process according to the invention for the sulfiding synthesis, the reaction of step (b) is generally carried out in an aprotic polar organic solvent, for example in halogenated hydrocarbon solvents (especially chlorinated hydrocarbon solvents such as methylene chloride).

In the process according to the invention for the synthesis of sulfiding agents, the reaction of step (b) is generally carried out at temperatures of -20 ° C to 50 ° C, preferably 0 ° C to 30 ° C.

A fourth specific object of the present invention relates to a method for preparing oligonucleotides using the sulfiding agent according to the present invention.

In general, the method according to the invention comprises at least (a) a coupling step of forming a phosphorescent internucleotide bond between two reactants selected from nucleotides and oligonucleotides, and (b) the phosphorescence using another sulfiding agent in the present invention. Sulfiding inter-nucleotide bonds. Steps (a) and (b) can be repeated after deprotection of 3 'or 5' of the sulfided oligonucleotide.

Preferably step (a) of the preparation process comprises coupling an H-phosphonate monoester salt with a protected nucleoside or an oligonucleotide having a free hydroxy group to link the H-phosphonate diester Operation to form a. This coupling operation is preferably carried out in solution.

Step (a) is preferably carried out in a polar aprotic organic solvent, for example a halogenated solvent or a nitrogen-containing solvent, more specifically an N-heterocyclic solvent or a chlorinated hydrocarbon, even more specifically acetonitrile and pyridine (preferably Preferably pyridine). The reaction to form the H-phosphonate diester is preferably activated by carboxylic acid halides, in particular pivaloyl chloride.

Step (a) is generally carried out at a temperature of -40 ° C to 30 ° C, preferably 0 ° C to 20 ° C.

In step (a) of the method according to the invention and in certain embodiments thereof, the liquid reaction medium generally contains at least 20% by weight of H-phosphonate oligonucleotide relative to the total weight of the reaction medium. Preferably this content is at least 20% by weight. The liquid reaction medium generally contains up to 50% by weight of H-phosphonate oligonucleotide relative to the total weight of the reaction medium.

The coupling product of step (a), in particular H-phosphonate, is isolated and then sulfided in step (b). Preferably it may be used without isolation operation in step (b). The sulfidation of the diesters formed may be carried out by in situ addition of sulfiding agents in an appropriately dissolved state in the appropriate solvent or may be carried out after the diesters formed from the reaction mixture have been previously purified.

Step (b) is preferably carried out in a polar aprotic organic solvent, for example a solvent including a halogenated hydrocarbon solvent, in particular a chlorinated hydrocarbon solvent such as methylene chloride. In certain aspects, step (b) is carried out in a mixture solvent containing a halogenated hydrocarbon solvent and a nitrogen-containing solvent, more specifically an N-heterocyclic solvent, preferably pyridine. More particularly, the pyridine / methylene chloride mixture is more particularly preferred when the coupling product of step (a) is sulfided without isolation operation.

Step (b) is generally carried out at a temperature of −40 ° C. to 30 ° C., preferably 0 ° C. to 20 ° C.

In step (b), the molar ratio of the sulfiding agent to the amount of internucleoside linkages to be sulfided is generally at least 1, often 1.5 to 4.0, preferably 2.0 to 3.0.

In step (b), the intermediate H-phosphonate diester is preferably activated by an active agent, in particular a base. Suitable bases include alkylamines, specifically tertiary alkylamines, with diisopropylethylamine being preferred.

In a fifth aspect, the present invention relates to a method for purifying oligonucleotides according to the present invention having one or more P-S-R bonds as described herein above. According to one embodiment of this aspect, the method comprises precipitating at least a second oligonucleotide. According to a more specific embodiment, the method further comprises extracting the second oligonucleotide with a solvent, in particular from the solid material recovered from the precipitation step. Suitable solvents for extraction include polar organic solvents.

It has been found that such purification can be effectively achieved by combining the precipitation and extraction techniques of the protected oligonucleotides obtained according to the process of the invention. The exact conditions for precipitation can be determined by considering the sequence and length of a given oligonucleotide. Precipitation generally comprises (a) dissolving oligonucleotides in a polar organic solvent, and (b) adding a nonpolar organic solvent until the solution becomes cloudy.

It has been found that oligonucleotides according to the invention can generally be isolated and purified via precipitation.

The solvent used to dissolve the oligonucleotides in step (a) is preferably a halogenated hydrocarbon such as methylene chloride and chloroform; Nitrogen-containing solvents such as acetonitrile and pyridine; And carbonyl-containing solvents such as acetone.

Generally, in step (a), in the range of about 0.5 (n + 1) mL to about 2.0 (n + 1) mL, preferably about 1.0 (n + 1) mL, where n is phosphorothioate triester Solvent volume) is used.

Until the solution of the second oligonucleotide becomes cloudy, the solution is preferably selected from hydrocarbons such as alkanes solvents such as hexanes, in particular ether solvents such as MTBE and mixtures thereof (eg preferably hexane / MTBE mixtures) Treated with nonpolar organic solvent. In another specific embodiment, the turbid solution thus obtained is subsequently treated with a precipitation aid.

In this case, the precipitation aid is generally selected from inert porous solids, preferably from celite, coal, wood cellulose, and chromatographic stationary phases such as silica or alumina.

In this case, the precipitation aid is generally in the range of about 0.25 (n + 1) g to about 1.5 (n + 1) g, preferably about 0.75 (n + 1) g (n is a phosphorothioate triester bond) In the millimolar number).

Preferably, after addition of the precipitation aid, the mixture is treated with a second fraction of nonpolar organic solvent as described above. The volume of the fraction generally falls in the range of about 1 (n + 1) mL to about 4 (n + 1) mL, preferably about 2.0 (n + 1) mL (n is a phosphorothioate triester bond) Millimolar number).

After precipitation, in particular when using a precipitation aid, the resulting mixture is generally subjected to a solid / liquid separation operation (preferably a filtration operation). Oligonucleotides are generally recovered from the solids recovered from the solid-liquid separation operation, in particular from the precipitation aid by extraction with a polar organic solvent, wherein the polar organic solvent is preferably a carbonyl solvent (eg acetone), nitrogen- Containing solvents such as acetonitrile and halogenated hydrocarbons such as methylene chloride and chloroform.

Oligonucleotides obtained from the precipitation treatment can be further purified by partitioning between the organic solvent and water. This step usually separates the polar impurities from the product that are dissolved in the aqueous solution layer. In this embodiment, the oligonucleotides are suitably in organic solvents, in particular polar organic solvents such as nitrogen-containing solvents, in particular formamides such as acetonitrile, DMF, N-heterocycles such as pyridine, carbonyl solvents such as acetone, Or in THF or DMSO.

The volume of organic solvent used is generally in the range of about 2.0 (n + 1) mL to about 8.0 (n + 1) mL, preferably about 4.0 (n + 1) mL (n is phosphorothioate tree Millimolar number of ester bonds). The solution is treated with an aqueous medium, specifically water. The volume of aqueous medium used is generally from about 0.5 equivalent volumes to about 1.5 equivalent volumes of organic solvent, usually about 0.7 equivalent volumes of organic solvent. At the end of the treatment with the aqueous medium, the oligonucleotide-containing layer is generally separated and, if appropriate, can be further processed to obtain purified oligonucleotides.

A sixth specific object of the present invention relates to a method for preparing a second oligonucleotide having at least one phosphothioate group, the method comprising the steps of (a) providing a first oligonucleotide according to the present invention, and (b Cutting at least one R group from the first oligonucleotide to produce the second oligonucleotide having at least one thiophosphate bond. In some specific embodiments of the invention, the R group is cleaved by reacting the solution phase first oligonucleotide with a base, wherein the base is preferably in alkyl, cycloalkyl and aromatic amine; More preferably primary amines, for example alkyl primary amines, or secondary alkylamines, wherein the alkyl group comprises the same or different substituents preferably selected from C1 to C8 linear or branched alkyl; Most preferably selected from n-propyl and tert-butyl amines, preferably the base is a hindered primary amine.

According to one particular embodiment, the cleavage step according to the sixth aspect of the invention is carried out in the presence of an sterically hindered base and an active agent which generally consists of an N-heteroaromatic base. Preferably the active agent is 1,2,4-triazole or other triazole and tetrazole derivatives, more preferably such active agent is used in combination with sterically hindered bases, in particular tert-butyl amine.

Deprotection of S-methylene-ester, -carbonate or -carbamate groups can be achieved, for example, by treating the protected nucleotides with sterically hindered bases such as t-butylamine. These bulk amines are particularly highly selective because they do not react with nucleobases, especially nucleobases protected at the carbonyl oxygen position. These amines actually limit or substantially prevent possible side-reaction with the nucleobase moiety. It has been found that the active agent can be appropriately added to improve the reactivity of the hindered amine with, for example, S-methylenepropanoate under standard conditions. Examples of suitable active agents include N-heterocyclic bases such as diazoles, triazoles, and derivatives thereof. This embodiment enables particularly rapid and efficient clean deprotection reactions.

In some embodiments of the invention, the deprotection method utilizes substituted anilines as bases, wherein the aryl groups of the anilines contain linear or branched alkyl or aryl substituents in the second and / or 6 positions (eg, 2,6). Dimethylaniline and 2,6-diethylaniline).

The deprotection process according to the sixth aspect is preferably carried out in an aprotic polar organic solvent, for example in a nitrogen-containing solvent, more specifically in an N-heterocyclic solvent (preferably pyridine).

The deprotection method according to the sixth aspect is generally carried out at a temperature of -10 ° C to 50 ° C, preferably 0 ° C to 30 ° C.

In a sixth aspect of the invention and certain embodiments thereof, the liquid reaction medium generally contains at least 20% by weight of the first oligonucleotide relative to the total weight of the reaction medium. Preferably this content is at least 50% by weight.

In a sixth aspect of the invention, the amount of base used is generally in the range of 5n mmol to 15n mmol, preferably about 10n mmol (n is the millimolar number of phosphorothioate triester bonds).

In the case of using the active agent in the sixth aspect of the present invention, the amount of active agent used generally falls in the range of 0.5n mmol to 3n mmol, preferably 1.5n mmol (n is millimolar of phosphorothioate triester bond) Commission).

The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.

The definitions of these examples and abbreviations used throughout this specification are as follows:

CH ₂ Cl ₂ is dichloromethane, KI is potassium iodide, Na ₂ S ₂ O ₃ is sodium thiosulfate, DME is dimethoxyethane, DIPEA is diisopropylethylamine, NaCl is sodium chloride, MTBE is methyl tert-butyl ether, EtOAc is ethyl acetate, HCl is hydrochloric acid, Na ₂ SO ₄ is sodium sulfate, N ₂ is dinitrogen, Br ₂ is bromine, SO ₂ Cl ₂ is thionyl chloride, NaHCO ₃ is sodium bicarbonate, CDCl ₃ is deuterated Chloroform, THF is tetrahydrofuran, DMSO is dimethylsulfoxide, and DMF is N, N-dimethylformamide.

Ap, Gp, Tp are the 2-deoxyribose nucleobases as described above linked to A, G and T nucleobases respectively as described above, wherein A, G and T are protected as follows:

Ap is a 2-dioxyribose nucleobase where A is N- (purin-6-yl) benzamide, and Gp is G-N- (6- (2,5-dichlorophenoxy) -purin-2-yl) isopart It is a 2-dioxyribose nucleobase which is a tyramide, and Tp is a nucleobase whose T is 5-methyl-4- phenoxypyrimidin-2-one.

Ap (S), Gp (S) and Tp (S) are 4'O-P-thiomethyl propionate corresponding to Ap, Gp and Tp, respectively, as described above. Ap (H), Gp (H) and Tp (H) are 4'O-P-H propionates corresponding to Ap, Gp and Tp, respectively, as described above.

DMTr is a bis para-methoxy trityl protecting group known to those skilled in the art and when bound to the corresponding oligonucleotide is bound to 5-O 'of the oligonucleotide as described above. Lev is a pentane 1,4-dione protecting group known to those skilled in the art and when bound to the corresponding oligonucleotide, is bound to 3-O 'of the oligonucleotide as described above.

The practice of using the abbreviations used in the specification of the present invention will be further explained through the following schemes.

Example  One: Vis - Chloromethyl Disulfide  synthesis

Anhydrous CH ₂ Cl ₂ (200 mL) and dimethyl disulfide (27.1 mL, 300 mmol) were added to a 1.0 L round bottom flask. The mixture was stirred and cooled to −78 ° C. under a nitrogen atmosphere, and bubbles of chlorine (Cl ₂ ) gas were slowly generated over the stirred mixture. The addition of chlorine gas was stopped when the mixture turned to a yellowish green slurry. The cold bath was removed and the mixture was allowed to warm naturally to room temperature. When the HCl evolution disappeared from the solution, a red solution formed. The mixture was first bubbled with nitrogen for 15 minutes and then rotary evaporated to remove volatile CH ₂ Cl ₂ . To the residue was added fresh CH ₂ Cl ₂ (300 mL). The solution thus obtained was stirred in an ice-water bath, and an aqueous solution of KI (139.4 g, 840 mmol) dissolved in water (200 mL) was slowly added over 15 minutes. The cold bath was removed and the mixture was stirred at room temperature for 2 hours. The organic layer was separated and stirred in an ice-water bath; Saturated aqueous Na ₂ S ₂ O ₃ solution was slowly added until the color of I ₂ disappeared. The organic layer was separated and washed with water (100 mL). The organic layer was dried over Na ₂ S0 _{4 and} concentrated to give the product (42.2 g) in the form of a red oil. Yield: 87.3%. The crude product thus obtained was used for next step without further purification.

Example  2: Of bis (propionyloxymethyl) disulfide  synthesis

To a 1.0 L round bottom flask was added sodium iodide (1.50 g, 10.0 mmol), bis-chloromethyl disulfide (16.2 g, 100 mmol) and anhydrous DME (150 mL). After stirring for 20 minutes at room temperature, the mixture was cooled in an ice-water bath and then DIPEA (42.0 mL, 240 mmol) was added followed by propionic acid (16.4 mL, 220 mmol). The cold bath was removed and the mixture was stirred for 18 hours at room temperature. Solvent bulk was removed by rotary evaporation. Ethyl acetate (400 mL) was added to the residue, and the mixture was washed with water (150 mL x 2), followed by brine (80 mL). The organic layer was concentrated and the residue was purified by silica gel chromatography to give the desired product (9.62 g) in the form of orange oil. Yield 40.4%.

Example  3: N- methyl Methanesulfonamide  synthesis

To the cooled and stirred aqueous methylamine (40% in water, 152 mL, 1.75 mol) in an ice-water bath was added dropwise over 15 minutes methanesulfonyl chloride (38.7 mL, 500 mmol). The internal temperature was maintained at 20-24 ° C. upon addition. At the end of the addition, the cold bath was removed and the mixture was stirred overnight at room temperature. NaCl (40 g) was added and the mixture was stirred for 30 minutes at room temperature. The mixture was extracted using CH ₂ Cl ₂ (150 mL, 100 mL × 2). After drying over Na ₂ SO ₄ , the solvent was evaporated to afford the desired product (48.7 g) as a colorless oil. Yield 89.2%.

Example  4: N- methyl -N- Propionyloxymethylsulfanyl Methanesulfonamide  synthesis

In a dried 100 mL round bottom flask, N-methyl methanesulfonamide (1.09 g, 10.0 mmol), pyridine (1.66 g, 21.0 mmol), bis (propionyloxymethyl) disulfide (1.2 g, 5.0 mmol) and anhydrous CH ₂ Cl ₂ (8 mL) was added. The mixture was stirred at room temperature under nitrogen and a solution of Br ₂ (0.882 g, 5.52 mmol) dissolved in 4 mL of CH ₂ Cl ₂ was added dropwise over 30 minutes. The resulting mixture was stirred for 2 hours at room temperature. MTBE (15 mL) was added and the resulting mixture was filtered. The solid was washed with a mixture of CH ₂ Cl ₂ (5 mL) and MTBE (5 mL). The filtrate was concentrated and purified by silica gel chromatography to give the desired product (1.62 g) in the form of a colorless oil. Yield: 71.3%.

Example  5: Chloromethyl Propionate  synthesis

Paraformaldehyde (90.1 g, 3000.0 mmol) and anhydrous zinc chloride (8.18 g, 60.0 mmol) were added to a dried 500 mL round bottom flask. The vessel was placed in an ice-water bath and propionyl chloride (260.6 mL, 3000.0 mmol) was added slowly over 1 hour. After the addition, the mixture was stirred at 50 ° C. for 18 hours under nitrogen. The mixture was distilled off to give the desired product (212.2 g) in the form of a colorless oil. Yield 58%.

Example  6: propionic acid Acetylsulfanylmethyl  Synthesis of Ester

In a dry 2000 mL three neck round bottom flask equipped with a mechanical stirrer, dropping funnel and nitrogen inlet, chloromethyl propionate (168.0 g, 1370.9 mmol), anhydrous CH ₂ Cl ₂ (1000 mL), and diisopropylethylamine ( 194.9 g, 1508.0 mmol) was added thereto. After the solution was stirred and cooled in an ice-water bath, thioacetic acid (98.0 mL, 1370.9 mmol) was slowly added over 30 minutes. After the addition, the mixture was stirred and slowly warmed up to room temperature and stirred overnight at room temperature. Most of CH ₂ Cl ₂ was evaporated. To the mixture thus obtained, a mixture of ethyl acetate (500 mL) and MTBE (500 mL) was added. The mixture was filtered and the solid was washed with a mixture of ethyl acetate (100 mL) and MTBE (100 mL). The filtrate was concentrated and the residue was distilled to afford the desired product (169.8 g) in the form of a yellow oil. Yield: 76.4%.

Example  7: N- methyl -N- Propionyloxymethylsulfanyl Methanesulfonamide  synthesis

Propionate acetylsulfanylmethyl ester (70.0 g, 431.5 mmol) and anhydrous CH ₂ Cl ₂ (600 mL) were added to a dry 1000 mL round bottom flask. The solution was stirred in an ice-water bath under nitrogen and sulfuryl chloride (34.6 mL, 431.5 mmol) was added. After the addition, the cold bath was removed and the mixture was stirred for 1.5 hours at room temperature. The mixture was rotovap to remove all volatiles. The residue was dissolved in 80 mL of anhydrous CH ₂ Cl ₂ to give solution A.

To another dry 1000 mL round bottom flask was added N-methyl methanesulfonamide (49.5 g, 453.1 mmol), molecular sieve (4 cc, activated, 5.0 g) and anhydrous CH ₂ Cl ₂ (200 mL). The solution was stirred in an ice-water bath under nitrogen and anhydrous pyridine (41.9 mL, 517.8 mmol) was added. After the addition, Solution A was added slowly over 15 minutes. The resulting mixture was stirred for 1.5 hours at room temperature. Hexane (200 mL) was added slowly and the resulting mixture was stirred for 10 minutes at room temperature. The mixture was filtered and the solid was washed with a mixture of ethyl acetate: hexanes = 1: 1 (80 mL). The filtrate was concentrated and the residue was washed with 800 g of silica on a column (hexane (2 liters), ethyl acetate / hexanes (1: 9, 3 liters), ethyl acetate / hexanes (2: 8, 4 liters) and ethyl) Purification using eluent acetate / hexanes (3: 7, 2 liters) sequence. The main impurities eluted in 20% ethyl acetate and the product eluted in 30% ethyl acetate. Yield: 74.4%.

Example  8: N- Methanesulfonamide Succinimide  synthesis

Into a 250 mL three neck round bottom flask equipped with a magnetic stirrer, a thermocouple, a nitrogen line, and a cooling ice bath, a solution of 9.803 g (60.43 mmol) of acetylsulfanylmethyl dissolved in 80 mL of anhydrous dichloromethane was added. The contents of the flask were cooled to about 0 ° C., and a total of 11.33 g (83.94 mmol, 1.38 equiv) of sulfuric dichloride was slowly added to the solution at a rate that maintained a temperature of 0-5 ° C. The cold bath was removed and the reaction mixture was stirred for an additional 1.5 h at room temperature. The resulting yellow solution was concentrated on a rotary evaporator to yield 8.88 g of crude acetylmethylsulphenyl chloride in the form of a malodorous viscous yellow oil. This material was used directly in the next step for the sulfenylation of succinimide.

6.12 g (61.76 mmol, 1.02 eq.) Of succinimide and 8.17 g (80.73) dissolved in 80 mL of anhydrous dichloromethane in a 250 mL three neck round bottom flask equipped with a magnetic stirrer, thermocouple, nitrogen line and cooling bath. mmol, 1.33 equiv) was added a mixture of triethylamine. The mixture was cooled to 0 ° C. and a crude acetylmethylsulphenyl chloride (8.88 g) solution dissolved in 20 mL of anhydrous dichloromethane was maintained at a temperature of 0-5 ° C. in a pre-cooled suspension of succinimide and triethylamine. It was added slowly at the rate which becomes. Once the addition was complete, the cold bath was removed and the resulting brown suspension was stirred for an additional 2 hours at room temperature. The reaction mixture was quenched with chilled water (300 mL) and saturated NaHCO ₃ (100 mL) and extracted with dichloromethane (3 × 80 mL). The combined organic layers were then dried over Na ₂ SO ₄ and concentrated on a rotary evaporator to yield 13 g of dark viscous oil. This material was purified using 0-30% etaacetate on a silica gel column (120 g) using an EtOAc-hexane mixture as eluent to afford 4.12 g of sulfenamide N-methanesulfonamide succinimide in the form of a white solid. It was. R _f = 0.16 in 30% EtOAc-hexanes. Yield 32%.

Example  9: ethyl Acetylsulfanylmethyl Carbonate  synthesis

Into a dry 100 mL round bottom flask was added chloromethyl chloroformate (12.9 g, 100.0 mmol) and anhydrous acetonitrile (300 mL). The solution was stirred in an ice-water bath, to which was slowly added a mixture of anhydrous ethanol (4.6 g, 100.0 mL) and anhydrous pyridine (23.7 g, 300 ml) over 20 minutes. After the addition, the mixture was stirred for 1 hour at room temperature. Sodium iodide (1.50 g, 10.0 mmol) was added to the reaction mixture. The mixture was stirred in an ice-water bath and thioacetic acid (7.6 g, 100 mmol) was added over 5 minutes. At the end of the addition, the cold bath was removed and the mixture was stirred overnight at room temperature. Hexane (600 mL) was added to the reaction mixture and filtered. The filtrate was concentrated and distilled to give the desired product.

Example  10: N- methyl -N- Ethoxycarbonyloxymethylsulfanyl Methanesulfonamide  synthesis

Ethyl acetylsulfanylmethyl carbonate (8.9 g, 50.0 mmol) and anhydrous CH ₂ Cl ₂ (200 mL) were added to a dry 500 mL round bottom flask. The solution was stirred in an ice-water bath under nitrogen and sulfuryl chloride (4.0 mL, 50.0 mmol) was added. After the addition, the cold bath was removed and the mixture was stirred for 1.5 hours at room temperature. The mixture was rotovap to remove all volatiles. The residue was dissolved in 50 mL of anhydrous CH ₂ Cl ₂ to give solution A.

To another dry 500 mL round bottom flask was added N-methyl methanesulfonamide (6.0 g, 55.0 mmol), molecular sieve (4 cc, activated, 3.0 g) and anhydrous CH ₂ Cl ₂ (150 mL). The mixture was stirred in an ice-water bath under nitrogen and anhydrous pyridine (5.3 mL, 65.0 mmol) was added. After the addition, the solution A was added slowly over 10 minutes. The resulting mixture was stirred for 1.5 hours at room temperature. Hexane (200 mL) was added slowly and the resulting mixture was stirred for 10 minutes at room temperature. The mixture was filtered and the solid was washed with a mixture of ethyl acetate: hexanes = 1: 1 (60 mL). The filtrate was concentrated and the residue was purified by silica gel column to give the desired product.

Example  11: Acetylsulfanylmethyl Of dimethyl carbamate  synthesis

To a dry 100 mL round bottom flask was added chloromethyl chloroformate (12.9 g, 100.0 mmol), dimethylamine hydrochloride (8.15 g, 100 mmol) and anhydrous acetonitrile (300 mL). The mixture was stirred in an ice-water bath, to which N, N -diisopropylethyl amine (43.5 mL, 250 mmol) was slowly added over 30 minutes. After the addition, the mixture was stirred for 1 hour at room temperature. Sodium iodide (1.50 g, 10.0 mmol) was added to the reaction mixture. The mixture was stirred in an ice-water bath and thioacetic acid (7.6 g, 100 mmol) was added over 5 minutes. At the end of the addition, the cold bath was removed and the mixture was stirred overnight at room temperature. Hexane (300 mL) was added to the reaction mixture and filtered. The filtrate was concentrated and distilled to give the desired product.

Example  12: N- methyl -N- Dimethylcarbamoyloxymethylsulfanyl Methanesulfonamide  synthesis

To a dry 500 mL round bottom flask was added ethyl acetylsulfanylmethyl dimethyl carbonate (8.9 g, 50.0 mmol) and anhydrous CH ₂ Cl ₂ (200 mL). The solution was stirred in an ice-water bath under nitrogen and sulfuryl chloride (4.0 mL, 50.0 mmol) was added. After the addition, the cold bath was removed and the mixture was stirred for 1.5 hours at room temperature. The mixture was rotovap to remove all volatiles. The residue was dissolved in 50 mL of anhydrous CH ₂ Cl ₂ to give solution A.

To another dry 500 mL round bottom flask was added N-methyl methanesulfonamide (6.0 g, 55.0 mmol), molecular sieve (4 cc, activated, 3.0 g) and anhydrous CH ₂ Cl ₂ (150 mL). The mixture was stirred in an ice-water bath under nitrogen and anhydrous pyridine (5.3 mL, 65.0 mmol) was added. After the addition, the solution A was added slowly over 10 minutes. The resulting mixture was stirred for 1.5 hours at room temperature. Hexane (100 mL) was added slowly and the resulting mixture was stirred for 10 minutes at room temperature. The mixture was filtered and the solid was washed with a mixture of ethyl acetate: hexanes = 1: 1 (60 mL). The filtrate was concentrated and the residue was purified by silica gel column to give the desired product.

Example  13: intermediate H- Phosphonate  Fully protected dinucleotide comprising an isolation step Phosphorothioate  synthesis

Example 13-1. A mixture of 1.86 g (2.62 mmol, 1.15 equiv) of H-phosphonate 1 and 0.78 g (2.28 mmol) of 3'-protected dioxy-thymidine 2 was coevaporated with anhydrous pyridine (3x25 mL). . The oily residue was dissolved in 10 mL of anhydrous pyridine and cooled to about 0 ° C. under argon atmosphere. A total of 0.56 g (4.66 mmol, 2 equivalents) of pivaloyl chloride was added dropwise through a syringe, and the resulting mixture was allowed to warm up to room temperature. The reaction was stirred for an additional 15 minutes and then quenched with 50 g of ice and 100 mL of diluted saturated NaHCO ₃ (80 mL of water and 20 mL of sodium bicarbonate). The organics were extracted with dichloromethane (2x80 mL) and the extract was washed with a mixture of chilled water (70 mL), saturated sodium bicarbonate (30 mL) and brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated on a rotary evaporator to yield 6.74 g of crude product 3 in the form of a clear oil. ³¹ P (162 MHz, CDCl ₃ , δ): 8.58 (s) and 7.06 (s).

A solution of crude H-phosphonate 3 (6.74 g) dissolved in 20 mL of anhydrous pyridine was cooled to about 0 ° C. under argon atmosphere. A total of 1.21 g (5.43 mmol, 2.3 equiv) of reagent 4 was added dropwise to the reaction and after 5 minutes 0.502 g (3.88 mmol, 1.7 equiv) of diisopropylethyl amine was also added to the flask. The reaction was allowed to warm up to room temperature, stirred for an additional 1 hour and then quenched using cold diluted sodium bicarbonate (100 mL) solution. The organic product was extracted with dichloromethane (2x80 mL), washed with water (100 mL) and dried over sodium sulfate. The organic layer was concentrated on a rotary evaporator to yield 5.02 g of crude product 5 (about 90% pure as measured by HPLC) in the form of a yellow oil. ³¹ P (162 MHz, CDCl ₃ , δ): 26.77 (s) and 26.67 (s).

Example 13-2. Similar to Method A, H-phosphonate 1 (1.49 g, 2.1 mmol, 1.08 equiv) and 3′-protected dioxythymidine 2 (0.66 g, 1.94 mmol) were converted to pival chloride in 20 mL of anhydrous pyridine. Intermediate H-phosphonate 3 was obtained by reacting in the presence of 0.49 g (4.06 mmol, 2 equivalents) per day. The reaction mixture was quenched with a mixture of chilled water / NaHCO ₃ aqueous solution / brine, and then intermediate 3 was isolated by extraction with dichloromethane (3 × 30 mL). The organic extract was washed with water (50 mL), aqueous NaHCO ₃ solution (20 mL) and brine (10 mL). After drying with Na ₂ SO ₄ (for about 1 minute), the organic layer is concentrated to about 1/4 of its original volume, cooled to 0 ° C., and then S-transferant reagent 4 (0.96 g, 4.3 mmol, 2.2 equiv) Was added to intermediate 3 and then diisopropylethylamine (0.45 g, 3.48 mmol, 1.8 equiv) was added. After stirring at room temperature for an additional 1 hour, the reaction was quenched as described in Method A. The organic layer was concentrated on a rotary evaporator to yield 3.55 g of crude product 5 in the form of a clear yellow oil with 91% purity as determined by HPLC.

Example  14: DMTr - Ap (s) T- Lev Manufacture

Triethylammonium 6- N- (benzoyl) -5'- O- (4,4'-dimethoxytrityl) -2'-dioxyadenosine-3'H-phosphonate (4.94 g, 6.0 mmol) and A mixture of 3′- O -levulinylthymidine (1.70 g, 5.0 mmol) was evaporated to pyridine to anhydrous, diluted with anhydrous pyridine (12.5 mL) and stirred at 0 ° C. under nitrogen. Subsequently, pivaloyl chloride (1.24 mL, 10.0 mmol) was added slowly over 2 minutes. The reaction mixture was stirred at 0 ° C. for 5 minutes and sequestered between methylene chloride (100 mL) and 1.25N acetate sodium-acetic acid buffer (2 × 100 mL). The buffer was prepared by mixing 190 mL of 1.25N acetic acid sodium solution with 10 mL of 1.25N acetic acid aqueous solution. The organic layer was dried (on Na ₂ SO ₄ ) and concentrated. The residue was co-evaporated with 50 mL of toluene, dissolved in anhydrous methylene chloride (25 mL) and then N -propionyloxymethylthio- N -methyl methanesulfonamide (2.05) dissolved in anhydrous methylene chloride (1.0 mL). g, 9.0 mmol) was treated under nitrogen at 0 ° C. and then N, N -diisopropylethylamine (0.87 mL, 5.0 mmol) was added. After stirring at room temperature for 30 minutes, solution A (MTBE: hexane = 1: 2, 37.5 mL) was added over 10 minutes, followed by celite (7.5 g). The mixture was stirred and added an additional portion of A (37.5 mL) over 30 minutes. Stirring continued for an additional 30 minutes and the mixture was filtered. The solid was washed with a solvent mixture (60 mL) consisting of solution A and CH ₂ Cl ₂ in a ratio of 5: 1. The solid was then extracted with methylene chloride (4 x 40 mL). The methylene chloride extract was concentrated. The residue was dissolved in acetonitrile (20 mL) and stirred in an ice-water bath, then cooling water (14 mL) was added over 20 minutes. The bottom layer was sequestered between methylene chloride (100 mL) and brine solution (1: 1) (60 mL). The organic layer was dried (on Na ₂ SO ₄ ) and concentrated to afford the product (5.7 g) in the form of a white solid. Yield: 97%. ³¹ P NMR (CDCl ₃ , 121.5 MHz): δ = 26.7, 26.3.

Example  15: DMTr - Gp (H) Preparation

Phosphoric acid (78.7 g, 960.0 mmol) made anhydrous by evaporation with pyridine (500 mL), 2'-dioxy-6- O- (2,5-dichlorophenyl) -5'- O- (4,4 '-Dimethoxytrityl) -2- N -isobutyrylguanosine (62.8 g, 80.0 mmol) was added and the mixture was evaporated to pyridine and brought back to dryness. The residue was treated with anhydrous pyridine (480 mL) and pivaloyl chloride (64.0 mL, 520.0 mmol) added at 10 ° C. over 30 minutes. The reaction mixture was stirred at rt for 6 h and concentrated. The residue was dissolved in 800 mL of methylene chloride and washed sequentially with cooling water (800 mL) and triethylammonium bicarbonate (2.0 N, 400 mL x 2). The organic layer was dried over (anhydrous Na ₂ SO ₄ ) and concentrated. The residue was dissolved in 80 mL of methylene chloride and solution A (MTBE: hexane = 1: 2, 360 mL) was added over 20 minutes under stirring, followed by Celite (80 g). Next, an additional solution A (360 mL) was added slowly over 30 minutes. The mixture was filtered and the solid was washed with MTBE (300 mL). Then the solid was extracted with methylene chloride (200 mL x 4). The methylene chloride filtrate was concentrated to give the product (70.4 g) in the form of a white foam. Yield 93%. ³¹ P NMR (CDCl ₃ , 121.5 MHz): δ = 2.71.

Example  16: DMTr - Gp (s) T- OH Manufacture

Triethylammonium 2'-dioxy-6- O- (2,5-dichlorophenyl) -5'- O- (4,4'-dimethoxytrityl) -2- N -isobutyrylguanosine-3 A mixture of 'H-phosphonate (54.9 g, 58.0 mmol) and 3'- 0 -levulinyl-4- O -phenylthymidine (20.1 g, 48.3 mmol) is evaporated to pyridine, anhydrous, anhydrous Dilution with pyridine (121 mL). The solution so obtained was treated with pivaloyl chloride (11.8 mL, 96.6 mmol) over 5 minutes at 0 ° C. under nitrogen. After further stirring for 5 minutes, a solution of N -propionyloxymethylthio- N -methyl methanesulfonamide (22.0 g, 96.6 mmol) dissolved in anhydrous methylene chloride (20 mL) followed by N, N -diiso Propylethylamine (8.4 mL, 48.3 mmol) was added. The reaction mixture was stirred for 30 minutes at room temperature and diluted with 600 mL of methylene chloride. The organic layer was washed sequentially with chilled water (600 mL) and saturated sodium bicarbonate (500 mL × 2), dried (on anhydrous Na ₂ SO ₄ ) and concentrated. The residue was dissolved in 97 mL of methylene chloride and solution A (MTBE: hexane = 1: 2, 194 mL) was added over 20 minutes under stirring, followed by addition of celite (73 g), followed by Additional portion (194 mL) was added over 30 minutes. After further stirring for 30 minutes, the mixture was filtered. The solid was washed with MTBE: hexane = 4: 1 (200 mL) and extracted with methylene chloride (150 mL x 4). The methylene chloride filtrate was concentrated to give the product (68.1 g) in the form of a yellow foam. ³¹ P NMR (CDCl ₃ , 121.5 MHz): δ = 26.9, 26.2. This product was used in the next step without further purification. The product (64 g) was dissolved in 117 mL of methylene chloride and stirred at 0 ° C. to a solution of pyridine: acetic acid: hydrazine monohydrate = 37.5 mL: 25.0 mL: 2.5 mL (51.6 mmol) was added. After stirring at 0 ° C. for 1 hour, the reaction mixture was diluted with methylene chloride (200 mL) and washed with cold water (500 mL). The aqueous layer was extracted again with methylene chloride (100 mL). The methylene chloride extracts were combined (on anhydrous Na ₂ S0 ₄ ) and concentrated. The residue was dissolved in 235 mL of acetonitrile, stirred in an ice-water bath and treated with chilled water (188 mL) added slowly over 30 minutes. The lower organic layer was separated, diluted with 200 mL of methylene chloride and dried (on anhydrous Na ₂ SO ₄ ). After concentration, the residue was dissolved in 94 mL of methylene chloride and added over 20 minutes, solution A (MTBE: hexane = 1: 2, 94 mL), celite (70 g) and again over 30 minutes. Treated sequentially with solution A (94 mL) under stirring. The mixture was filtered and the solid was washed with MTBE: hexane = 4: 1 (300 mL) and then extracted with methylene chloride (150 mL x 4). The methylene chloride filtrate was concentrated to give the product (52 g) in the form of a yellow foam. Yield 87%. This product was used directly in the next step. ³¹ P NMR (CDCl ₃ , 121.5 MHz): δ = 28.1, 25.3.

Example  17: HO - Gp (s) A- Lev Manufacture

Triethylammonium 2'-dioxy-6- O- (2,5-dichlorophenyl) -5'- O- (4,4'-dimethoxytrityl) -2- N -isobutyrylguanosine-3 A mixture of 'H-phosphonate (45.4 g, 48.0 mmol) and 2'-dioxy-3'- 0 -levulinyl-6- N -benzoyladenosine (18.1 g, 40.0 mmol) was evaporated to pyridine Made into a state. The residue was diluted with anhydrous pyridine (100 mL) and the resulting solution was treated with pivaloyl chloride (9.9 mL, 80.0 mmol) over 10 minutes at 0 ° C. under nitrogen. The reaction mixture was further stirred at 0 ° C. for 5 minutes and then a solution of N -propionyloxymethylthio- N -methyl methanesulfonamide (18.2 g, 80.0 mmol) dissolved in anhydrous methylene chloride (10 mL) followed by N Treated with , N -diisopropylethylamine (7.0 mL, 40.0 mmol). After stirring at room temperature for 30 minutes, the reaction mixture was diluted with 600 mL of methylene chloride and washed sequentially with cooling water (600 mL) and saturated sodium bicarbonate (300 mL x 2). The organic layer was dried over (anhydrous Na ₂ SO ₄ ) and concentrated. The residue was dissolved in 80 mL of methylene chloride and solution A (MTBE: hexane = 1: 2, 160 mL) over 20 minutes, celite (60 g), and an additional portion of Solution A over 160 minutes (160 mL) It was processed sequentially using. After further stirring for 30 minutes, the mixture was filtered. The solid was washed with MTBE: hexane = 4: 1 (200 mL) and extracted with methylene chloride (150 mL x 4). The methylene chloride filtrate was concentrated. The residue was dissolved in 160 mL of acetonitrile and stirred in an ice-water bath and then treated with chilled water (112 mL) over 30 minutes. The lower organic layer was separated and sequestered between methylene chloride (320 mL) and water (320 mL). The organic layer was dried (on anhydrous Na ₂ SO ₄ ) and concentrated to give the product (64.1 g) in the form of a yellow foam. This product was used in the next step without further purification. ³¹ P NMR (CDCl ₃ , 121.5 MHz): δ = 26.2, 26.0.

To the solution (64.0 g) was dissolved in 120 mL of methylene chloride and stirred at 0 ° C., pyrrole (13.9 mL, 200.0 mmol) was added followed by dichloroacetic acid (16.5 mL, 200.0 mmol) over 20 minutes. It was. After stirring at 0 ° C. for 1 hour, the reaction mixture was quenched with saturated sodium bicarbonate (200 mL). The aqueous layer was extracted with methylene chloride (60 mL × 2), the methylene chloride extracts were combined (on anhydrous Na ₂ SO ₄ ) and concentrated. The residue was dissolved in 80 mL of methylene chloride and added over 15 minutes solution A (MTBE: hexane = 1: 1, 100 mL), celite (60 g) and again added solution A over 30 minutes (100 mL) was treated sequentially. The mixture was filtered and the solid was washed with MTBE (150 mL x 2) and extracted with methylene chloride (200 mL x 4). The methylene chloride filtrate was concentrated, dissolved in 160 mL of acetonitrile and stirred in an ice-water bath and treated with cold water (144 mL) over 20 minutes. The lower organic layer was separated and segregated between methylene chloride (320 mL) and brine solution (1: 1) (320 mL). The organic layer was dried (on anhydrous Na ₂ SO ₄ ) and concentrated to give the product (40.5 g) in the form of off-white solid. Yield 92%. ³¹ P NMR (CDCl ₃ , 121.5 MHz): δ = 26.4, 26.2.

Example  18: DMTr - Gp (s) Tp (H) Preparation

Phosphoric acid (29.8 g, 364.0 mmol) was evaporated to pyridine (182 mL) to anhydrous. To the residue, DMTr- Gp (s) T- OH (33.0 g, 26.0 mmol) was added and the resulting mixture was evaporated to pyridine and brought back to dryness. The mixture was diluted with anhydrous pyridine (130 mL) and treated with pivaloyl chloride (24.0 mL, 195.0 mmol) added at 10 ° C. over 30 minutes. The mixture was stirred at rt for 16 h, concentrated and the residue was dissolved in 400 mL of methylene chloride. The solution thus obtained was washed sequentially with chilled water (400 mL) and triethylammonium bicarbonate (2.0 N, 200 mL x 3). The organic layer was dried (on anhydrous Na ₂ SO ₄ ) and concentrated. After co-evaporation with 150 mL of toluene, the residue was dissolved in 52 mL of methylene chloride, solution A (MTBE: hexane = 1: 1, 78 mL), celite (39 g), and over 20 min. Treatment was carried out sequentially using an additional portion of Solution A (78 mL) over 30 minutes. The mixture was filtered and the solid was washed with a solvent mixture (180 mL) consisting of solution A and CH ₂ Cl ₂ in a ratio of 5: 1. The solid was extracted with methylene chloride (150 mL x 4) and the filtrate was concentrated to give the product (33.1 g) in the form of an off white foam. Yield 89%. ³¹ P NMR (CDCl ₃ , 121.5 MHz): δ = 26.9, 25.4, 3.0, 2.9.

Example  19: DMTr - Gp (s) Tp (s) Gp (s) A- OH Manufacture

A mixture of triethylammonium salts of DMTr- Gp (s) T p (H) (28.6 g, 20.0 mmol) and HO- Gp (s) A -Lev (16.9 g, 15.4 mmol) was evaporated to pyridine to anhydrous. The residue was diluted with anhydrous pyridine (62.0 mL) and treated with pivaloyl chloride (4.8 mL, 38.5 mmol) over 5 minutes at 0 ° C. under nitrogen. After 10 min of further stirring at 0 ° C., a solution of N -propionyloxymethylthio- N -methyl methanesulfonamide (7.0 g, 30.8 mmol) dissolved in anhydrous methylene chloride (10 mL) was followed by N, N Treated with diisopropylethylamine (2.7 mL, 15.4 mmol). After stirring for 1 hour at room temperature, the reaction mixture was diluted with 450 mL of methylene chloride, and washed sequentially with cooling water (450 mL) and saturated sodium bicarbonate (300 mL x 2). The organic layer was dried (on anhydrous Na ₂ SO ₄ ) and concentrated. After co-evaporation with 100 mL of toluene, the residue was dissolved in 62 mL of methylene chloride, solution A (MTBE: hexane = 1: 1, 124 mL), celite (46.2 g), over 20 minutes, And an additional portion of Solution A (124 mL) over 30 minutes. After additional stirring for 30 minutes, the mixture was filtered and the solid was washed with solution A: CH ₂ Cl ₂ = 6: 1 (140 mL). The solid was then washed with methylene chloride (150 mL x 4). The methylene chloride filtrate was concentrated. The residue was dissolved in 123 mL of acetonitrile and treated with cold water (86 mL) over 20 minutes with stirring in an ice bath. The lower organic layer was separated and segregated between methylene chloride (300 mL) and brine solution (1: 1) (200 mL). The organic layer was dried (on anhydrous Na ₂ SO ₄ ) and concentrated to give the product DMTr- Gp (s) Tp (s) Gp (s) A -Lev (43.4 g) in the form of a yellow foam. This product was used in the next step without further purification. ³¹ P NMR (CDCl ₃ , 121.5 MHz): δ = 27.9-25.8 (m).

DMTr- Gp (s) Tp (s) Gp (s) A- Lev (38.0 g) was dissolved in 38.0 mL of methylene chloride and stirred at 0 ° C., pyridine: acetic acid: hydrazine monohydrate = 14.3 mL: 9.5 mL: 0.95 mL (19.5 mmol) of cooling mixture was added. After stirring for 40 minutes at 0 ° C., the reaction mixture was diluted with methylene chloride (450 mL) and washed with cold water (300 mL × 2) and brine (150 mL). The methylene chloride layer was dried (on Na ₂ SO ₄ ) and concentrated. After co-evaporation with 100 mL of toluene, the residue is dissolved in 60 mL of methylene chloride and solution A (MTBE: hexane = 1: 1, 90 mL) is added over 20 minutes under stirring followed by a cell Light (45 g) was added. The mixture was stirred and an additional solution A (90 mL) was added over 30 minutes. The mixture was filtered and the solid was washed with a solvent mixture (150 mL) consisting of solution A and CH ₂ Cl ₂ in a ratio of 5: 1. The solid was extracted with methylene chloride (150 mL x 4), the extract was concentrated and the residue was purified through a short silica column using a gradient of 0-80% acetonitrile in ethyl acetate. The product fractions were concentrated to give the product (26.9 g) in the form of a yellow foam. Yield: 82%, ³¹ P NMR (CDCl ₃ , 121.5 MHz): δ = 27.6-26.0 (m).

Example  20: HO - Gp (s) Tp (s) Gp (s) A - Lev Manufacture

DMTr- Gp (s) Tp (s) Gp (s) A -Lev (7.2 g, 2.84 mmol) was dissolved in 14 mL of methylene chloride and stirred at 0 ° C. followed by pyrrole (2.0 mL, 28.4 mmol) , Dichloroacetic acid (2.34 mL, 28.4 mmol) was added over 3 minutes. After stirring at 0 ° C. for 30 minutes, the reaction mixture was diluted with 14 mL of methylene chloride, and then saturated sodium bicarbonate (50 mL) was added slowly. The aqueous layer was extracted with methylene chloride (40 mL), the methylene chloride extracts were combined (on anhydrous Na ₂ SO ₄ ) and concentrated. The residue was dissolved in 30 mL of methylene chloride, using solution A (MTBE: hexane = 2: 1, 45.0 mL), celite (9.0 g) and a further solution A solution (45.0 mL) added over 30 minutes. And sequentially processed. The mixture was filtered and the solid was washed with MTBE (40 mL x 2) and extracted with methylene chloride (50 mL x 4). The methylene chloride extract was concentrated and the residue was dissolved in 28.4 mL of acetonitrile and treated with cold water (28.4 mL) over 20 minutes with stirring in an ice bath. The organic layer was diluted with methylene chloride (80 mL), dried (on anhydrous Na ₂ SO ₄ ) and concentrated to give the product (5.7 g) in the form of an off-white solid. Yield 95%. ³¹ P NMR (CDCl ₃ , 121.5 MHz): δ = 27.4-26.2 (m).

Example  21: DMTr - Gp (s) Tp (s) Gp (s) Ap (H) Preparation

Phosphoric acid (4.7 g, 57.3 mmol) was evaporated with pyridine (29 mL) and mixed with DMTr- Gp (s) Tp (s) Gp (s) A- OH (8.7 g, 3.58 mmol). The mixture was made anhydrous by adding pyridine to the mixture and evaporated with anhydrous pyridine (29.0 mL). The mixture was stirred and thereto was added pivaloyl chloride (3.75 mL, 30.4 mmol) at 10 ° C. over 5 minutes. The resulting mixture was stirred at rt for 6 h and concentrated. The residue was dissolved in 200 mL of methylene chloride and washed sequentially with cooling water (100 mL) and triethylammonium bicarbonate (2.0 N, 100 mL x 3). The organic layer was dried (on anhydrous Na ₂ SO ₄ ) and concentrated to give the product (9.15 g) in the form of an off white foam. Yield: 98%. ³¹ P NMR (CDCl ₃ , 121.5 MHz): δ = 27.5-25.8 (m), 2.9, 2.8.

Example  22: HO - Gp (s) Tp (s) Gp (s) Ap (s) Gp (s) Tp (s) Gp (s) A- Lev Manufacture

DMTr- Gp (s) Tp (s) Gp (s) Ap (H) (10.1 g, 3.9 mmol) and HO- Gp (s) Tp (s) Gp (s) A -Lev (6.7 g, 3.0 mmol) Triethylammonium salt mixture was evaporated to pyridine to anhydrous and diluted with anhydrous pyridine (15.0 mL). Pivaloyl chloride (1.1 mL, 9.0 mmol) was slowly added over 2 minutes while stirring the mixture thus obtained at 0 ° C. The mixture was stirred at room temperature for 30 minutes, cooled again at 0 ° C., and then dissolved in anhydrous methylene chloride (2.0 mL) of N -propionyloxymethylthio- N -methyl methanesulfonamide (1.70 g, 7.5 mmol). After the solution was added N, N -diisopropylethylamine (0.78 mL, 4.5 mmol). The reaction mixture was stirred at room temperature for 1 hour and diluted with 300 mL of methylene chloride. The organic layer was washed sequentially with chilled water (300 mL) and saturated sodium bicarbonate (200 mL), then dried (on anhydrous Na ₂ SO ₄ ) and concentrated. The residue was dissolved in methylene chloride (24 mL) and solution A (MTBE: hexane = 1: 1, 48 mL) over 10 minutes, celite (18 g), and an additional portion (48 mL) of Solution A over 30 minutes ) Were processed sequentially. After stirring for 30 minutes, the mixture was filtered and the solid was washed with MTBE: hexane = 2: 1 (60 mL) and extracted with methylene chloride (50 mL x 4). Methylene chloride extract is concentrated; The residue was dissolved in acetonitrile (48 mL) and treated with cold water (34 mL) over 20 minutes with stirring in an ice-water bath. The lower layer was separated and segregated between methylene chloride (150 mL) and brine solution (1: 1) (160 mL). The organic layer was dried (on anhydrous Na ₂ SO ₄ ) and concentrated to afford the product (11.7 g) in the form of a gray solid. ³¹ P NMR (CDCl ₃ , 121.5 MHz): δ = 27.7-25.8 (m). The product was used directly in the next step without further purification.

To this solution (11.2 g) was dissolved in 18 mL of methylene chloride and stirred at 0 ° C., pyrrole (2.85 mL, 41.1 mmol) was added followed by dichloroacetic acid (3.2 mL, 38.4 mmol) over 5 minutes. It was. After stirring for 40 min at 0 ° C., saturated sodium bicarbonate (40 mL) was added slowly to quench the reaction mixture. The mixture was diluted with 20 mL of methylene chloride, the aqueous layer was separated and extracted with methylene chloride (40 mL), the methylene chloride extracts were combined (on anhydrous Na ₂ SO ₄ ) and concentrated. The residue was dissolved in 28 mL of methylene chloride and added over 10 minutes solution A (MTBE: hexane = 1: 1, 28 mL), celite (16.8 g) and again added solution 20 over 20 minutes. Treated sequentially with additional portion (28 mL). The mixture is filtered; The solid was washed with solution A (40 mL) and MTBE (40 mL) and then extracted with methylene chloride (4 x 50 mL). The methylene chloride extract was concentrated and purified through a short silica gel column. The column was eluted with methylene chloride. After concentration, the product (8.7 g) was obtained in the form of a gray solid. Yield 66%. ³¹ P NMR (CDCl ₃ , 121.5 MHz): δ = 27.8-26.3 (m).

Example  23: HO - Gp (s) Tp (s) Gp (s) Ap (s) Gp (s) Tp (s) Gp (s) Ap (s) Gp (s) Tp (s) Gp (s) Preparation of A-Lev  ( SEQ . ID NO : One)

DMTr- Gp (s) Tp (s) Gp (s) A p (H) (6.85 g, 2.64 mmol) and HO- Gp (s) Tp (s) Gp (s) Ap (s) Gp (s) Tp The triethylammonium salt mixture of (s) Gp (s) A -Lev (8.2 g, 1.81 mmol) was evaporated to pyridine to anhydrous. Anhydrous pyridine (14 mL) was added to the residue, and the resulting solution was stirred at 0 ° C. under nitrogen and treated with pivaloyl chloride (0.8 mL, 6.48 mmol) added over 3 minutes. The cold bath was removed and the mixture was stirred at room temperature for 30 minutes. The mixture was cooled to 0 ° C. and a solution of N -propionyloxymethylthio- N -methyl methanesulfonamide (1.23 g, 5.40 mmol) dissolved in anhydrous methylene chloride (2.0 mL) followed by N, N -diisopropyl Ethylamine (0.56 mL, 3.24 mmol) was added. After stirring at room temperature for 1 hour, the reaction mixture was diluted with 250 mL of methylene chloride and washed with cold semi-saturated sodium bicarbonate (250 mL). The organic layer was dried (on anhydrous Na ₂ SO ₄ ) and concentrated. The residue was coevaporated with 50 mL of toluene, dissolved in 43 mL of methylene chloride, and then solution A (MTBE: hexane = 1: 1, 43 mL), celite (19.4 g), and 30 over 20 minutes. Treatment was carried out sequentially using an additional portion of Solution A over 43 minutes (43 mL). After stirring for 30 minutes, the mixture was filtered and the solid was washed with a solvent mixture (100 mL x 2) with Solution A and CH ₂ Cl ₂ in a ratio of 4: 1. The solid was extracted with methylene chloride (100 mL x 4) and the extract was concentrated. The residue was dissolved in 65 mL of acetonitrile and treated with cold water (46 mL) over 20 minutes. The lower organic layer was separated and segregated between methylene chloride (200 mL) and brine solution (1: 1) (200 mL). The organic layer was dried (on anhydrous Na ₂ SO ₄ ) and concentrated to give the product (12.8 g) in the form of a yellow foam. ³¹ P NMR (CDCl ₃ , 121.5 MHz): δ = 30.0-24.8 (m).

To this solution (12.8 g) was dissolved in 20 mL of methylene chloride and stirred at 0 ° C., pyrrole (2.64 mL, 38.0 mmol) was added followed by dichloroacetic acid (3.0 mL, 36.0 mmol). After stirring for 30 min at 0 ° C., the reaction mixture was diluted with 200 mL of methylene chloride and then saturated sodium bicarbonate (100 mL) was added slowly. The organic layer was separated, dried (on Na ₂ SO ₄ ) and concentrated. The residue was dissolved in 40 mL of methylene chloride and added over 10 minutes solution A (MTBE: hexane = 1: 1, 40 mL), celite (20 g) and added solution A addition over 20 minutes ( 80 mL). The mixture was filtered and the solid was washed with a solvent mixture (120 mL) consisting of solution A and CH ₂ Cl ₂ in a ratio of 5: 1. The solid was extracted with methylene chloride (100 mL x 4) and the extract was concentrated. The residue was dissolved in 70 mL of acetonitrile and treated with cold water (49 mL) over 30 minutes. The lower organic layer was separated and segregated between methylene chloride (150 mL) and brine solution (1: 1) (150 mL). The organic layer was dried (on Na ₂ SO ₄ ) and concentrated to give the product (9.2 g) in the form of an off-white solid. This product was used directly in the next step. Yield 75%. ³¹ P NMR (CDCl ₃ , 121.5 MHz): δ = 27.7-26.4 (m).

Example  24: HO- Gp (s) Tp (s) Gp (s) Ap (s) Gp (s) Tp (s) Gp (s) Ap (s) Gp (s) Tp (s) Gp (s) Ap (s) Gp (s) Tp (s) Gp (s) Preparation of A-Lev ( SEQ . ID NO : 2)

DMTr- Gp (s) Tp (s) Gp (s) A p (H) (3.64 g, 1.4 mmol) and HO- Gp (s) Tp (s) Gp (s) Ap (s) Gp (s) Tp The triethylammonium salt mixture of (s) Gp (s) Ap (s) Gp (s) Tp (s) Gp (s) A- Lev (6.1 g, 0.89 mmol) was evaporated to pyridine to anhydrous. The residue was diluted with anhydrous pyridine (10 mL) and treated with pivaloyl chloride (0.37 mL, 3.0 mmol) added slowly over 3 minutes at 0 ° C. under nitrogen. The cold bath was removed and the mixture was stirred at room temperature for 1 hour. The mixture was cooled to 0 ° C. and a solution of N -propionyloxymethylthio- N -methyl methanesulfonamide (568.3 mg, 2.5 mmol) dissolved in anhydrous methylene chloride (1.0 mL) followed by N, N -diisopropyl Ethylamine (0.26 mL, 1.5 mmol) was added. After stirring at room temperature for 1 hour, the reaction mixture was diluted with 120 mL of methylene chloride and washed sequentially with chilled water (120 mL) and half saturated sodium bicarbonate (120 mL). The organic layer was dried (on anhydrous Na ₂ SO ₄ ) and concentrated. The residue was dissolved in 32 mL of methylene chloride and then solution A (MTBE: hexane = 1: 1, 32 mL) added over 20 minutes, celite (16.0 g), and solution A added over 30 minutes Treatment was carried out sequentially using an additional portion of (64 mL). After stirring for 30 minutes, the mixture was filtered and the solid was washed with a solvent mixture (60 mL) consisting of solution A and CH ₂ Cl ₂ in a ratio of 5: 1. The solid was extracted with methylene chloride (80 mL x 4) and the extract was concentrated. The residue was dissolved in 40 mL of acetonitrile and treated with cooling water (28 mL) over 20 minutes with stirring. The lower organic layer was separated and segregated between methylene chloride (150 mL) and brine solution (1: 1) (150 mL). The organic layer was dried (on Na ₂ SO ₄ ) and concentrated to afford the product (6.92 g) in the form of a yellow solid. ³¹ P NMR (CDCl ₃ , 121.5 MHz): δ = 27.6-26.3 (m). The product was used directly in the next step without further purification.

To this solution (6.92 g) was dissolved in 10 mL of methylene chloride and stirred at 0 ° C., pyrrole (1.74 mL, 25.0 mmol) was added followed by dichloroacetic acid (2.0 mL, 24.0 mmol) over 2 minutes. It was. After further stirring at 0 ° C. for 30 minutes, the reaction mixture was diluted with 100 mL of methylene chloride and then saturated sodium bicarbonate (50 mL) was added slowly. The organic layer was separated and the aqueous layer was extracted with 50 mL of methylene chloride. The CH ₂ Cl ₂ extracts were combined (on anhydrous Na ₂ SO ₄ ), dried and concentrated. The residue was dissolved in 32 mL of methylene chloride and added over 10 minutes solution A (MTBE: hexane = 1: 1, 32 mL), celite (16 g) and additional solution A added over 30 minutes ( 64 mL). The mixture was filtered and the solid was washed with a solvent mixture (90 mL) consisting of solution A and CH ₂ Cl ₂ in a ratio of 5: 1. The solid was extracted with methylene chloride (80 mL x 4) and the extract was concentrated. The residue was dissolved in 40 mL of acetonitrile and the mixture was treated with chilled water (28 mL) added over 10 minutes with stirring in an ice bath. The lower organic layer was separated and segregated between methylene chloride (100 mL) and brine solution (1: 1) (100 mL). The organic layer was dried (on anhydrous Na ₂ SO ₄ ) and concentrated to give the product (5.8 g) in the form of a yellow solid. Yield 71%. ³¹ P NMR (CDCl ₃ , 121.5 MHz): δ = 27.7-26.3 (m).

Example  25: 5'- OH  Fully protected Oligonucleotides Phosphorothioate HO - Gp (s) Tp (s) Gp (s) A -Full of Lev Deprotection

The fully protected tetramer HO- Gp (s) Tp (s) Gp (s) A- Lev (1.38 g, 0.62 mmol) was evaporated with addition pyridine to anhydrous. To the residue, 1,2,4-triazole (192.7 mg, 2.79 mmol), 4 'molecular sieves (1.5 g) and anhydrous pyridine (6.0 mL) were added. The resulting mixture was stirred and cooled to 0 ° C. under nitrogen, to which tert -butylamine (1.95 mL, 18.6 mmol) was added. The resulting mixture was stirred for 4 hours at room temperature. The mixture was filtered and the molecular sieve washed with pyridine (5 mL x 2). The filtrates were combined and concentrated to dryness. To this residue was added syn -2-pyridine aldoxinsim (909 mg, 7.44 mmol) followed by anhydrous acetonitrile (10 mL). The mixture was stirred and cooled to 0 ° C., then 1,8-diazabicyclo [5.4.0] undec-7-ene (1.67 mL, 11.2 mmol) was added. After stirring for 15 hours at room temperature, MTBE (50 mL) was added slowly over 10 minutes. After stirring for a further 20 minutes, the upper clear solution was decanted and the residue was washed with ethyl acetate (20 mL). The remaining solvent was removed by evaporation of the residue and dissolved in a mixture of 28% aqueous ammonium solution (10.0 mL) and 2-mercaptoethanol (0.5 mL). The resulting mixture was heated at 55 ° C. for 15 hours. After cooling, the mixture was added dropwise over 10 minutes to a stirred mixture of isopropanol: THF = 1: 3 (80 mL). After stirring for an additional 20 minutes, the upper clear solution was decanted and the residue was washed with THF (20 mL). The residue was purified via reverse phase C18 chromatography. The product thus obtained was charged into a column (8 cm x 3 cm diameter) of Amberlite ^® IR-120 (plus) ion exchange resin (sodium form). The column was eluted with water and the desired fractions were collected and lyophilized to give 684 mg of the product as a white solid in the form of sodium of fully deprotected oligonucleotide phosphorothioate. Yield 83%. ³¹ P NMR (D ₂ O, 121.5 MHz): δ = 55.4-54.6 (m).

Example  26: fully protected, as shown in the following scheme Dinucleotide  Synthesis of Phosphorothioate

Triethylammonium 5'-O- (4,4'-dimethoxytrityl) -thymidine-3'-H-phosphonate (425,9 mg, 0.6 mmol), 3'-0-levulinyl thymi A solution of dine (170.2 mg, 0.5 mmol) and dry pyridine (10.0 mL) was evaporated to dryness. The residue was redissolved in 10 mL of pyridine and again evaporated to dryness. To the residue, molecular sieves (300 mg, activated, 3x) and anhydrous pyridine (5.0 mL) were added. The resulting mixture was stirred at room temperature under nitrogen, to which pivaloyl chloride (0.22 mL, 1.75 mmol) was added. After stirring at room temperature for 5 minutes, a solution of N-methyl-N-propionyloxymethylsulfanyl methanesulfonamide (284.1 mg, 1.25 mmol) dissolved in CH ₂ Cl ₂ (1.0 mL) was followed by DIPEA (0.17). mg, 2.0 mmol) was added. The resulting mixture was stirred for 30 minutes at room temperature. Ethyl acetate (30 mL) was added. The mixture was filtered and the filtrate was washed with water (15 mL), half-saturated aqueous sodium bicarbonate solution (15 mL x 2) and brine (15 mL). The organic layer was dried and evaporated to yield 1.21 g of pale yellow oil. This crude product was purified via silica gel chromatography (EtOAc / acetone) to give the product (389 mg) in the form of a colorless foam. Yield: 74.4%.

<110> Girindus America Inc <120> Sulfurizing reagents and their use for oligonucleotides synthesis <130> GIR 2008/02 <140> US 61 / 140,391 <141> 2008-12-23 <160> 2 <170> PatentIn version 3.3 <210> 1 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <220> <221> modified_base <222> (1) N- (6- (2,5-dichlorophenoxy) -purin-2-yl) isobutyramide <220> <221> misc_feature (222) (1) .. (11) 4'O-P-thiomethyl propionate <220> <221> modified_base <222> (2) <223> 5-methyl-4-phenoxypyrimidin-2-one <220> <221> modified_base <222> (3) N- (6- (2,5-dichlorophenoxy) -purin-2-yl) isobutyramide <220> <221> modified_base <222> (4) N- (purin-6-yl) benzamide <220> <221> modified_base <222> (5) N- (6- (2,5-dichlorophenoxy) -purin-2-yl) isobutyramide <220> <221> modified_base <222> (6) <223> 5-methyl-4-phenoxypyrimidin-2-one <220> <221> modified_base <222> (7) N- (6- (2,5-dichlorophenoxy) -purin-2-yl) isobutyramide <220> <221> modified_base <222> (8) N- (purin-6-yl) benzamide <220> <221> modified_base <222> (9) N- (6- (2,5-dichlorophenoxy) -purin-2-yl) isobutyramide <220> <221> modified_base <222> (10) <223> 5-methyl-4-phenoxypyrimidin-2-one <220> <221> modified_base <222> (11) N- (6- (2,5-dichlorophenoxy) -purin-2-yl) isobutyramide <220> <221> modified_base <222> (12) <223> 3O'-pentan 1,4-dione <220> <221> modified_base <222> (12) N- (purin-6-yl) benzamide <400> 1 gtgagtgagt ga 12 <210> 2 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> synthesis exemple <220> <221> modified_base <222> (1) N- (6- (2,5-dichlorophenoxy) -purin-2-yl) isobutyramide <220> <221> misc_structure (222) (1) .. (15) 4'O-P-thiomethyl propionate <220> <221> modified_base <222> (2) <223> 5-methyl-4-phenoxypyrimidin-2-one <220> <221> modified_base <222> (3) N- (6- (2,5-dichlorophenoxy) -purin-2-yl) isobutyramide <220> <221> modified_base <222> (4) N- (purin-6-yl) benzamide <220> <221> modified_base <222> (5) N- (6- (2,5-dichlorophenoxy) -purin-2-yl) isobutyramide <220> <221> modified_base <222> (6) <223> 5-methyl-4-phenoxypyrimidin-2-one <220> <221> modified_base <222> (7) N- (6- (2,5-dichlorophenoxy) -purin-2-yl) isobutyramide <220> <221> modified_base <222> (8) N- (purin-6-yl) benzamide <220> <221> modified_base <222> (9) N- (6- (2,5-dichlorophenoxy) -purin-2-yl) isobutyramide <220> <221> modified_base <222> (10) <223> 5-methyl-4-phenoxypyrimidin-2-one <220> <221> modified_base <222> (11) N- (6- (2,5-dichlorophenoxy) -purin-2-yl) isobutyramide <220> <221> modified_base <222> (12) N- (purin-6-yl) benzamide <220> <221> modified_base <222> (13) N- (6- (2,5-dichlorophenoxy) -purin-2-yl) isobutyramide <220> <221> modified_base <222> (14) <223> 5-methyl-4-phenoxypyrimidin-2-one <220> <221> modified_base <222> (15) N- (6- (2,5-dichlorophenoxy) -purin-2-yl) isobutyramide <220> <221> modified_base <222> (16) N- (purin-6-yl) benzamide <220> <221> misc_feature <222> (16) <223> 3O'-pentan 1,4-dione <400> 2 gtgagtgagt gagtga 16

Claims (49)

At least one internucleotide linkage comprising a PSR linkage and an oligonucleotide comprising at least two nucleosides, wherein R corresponds to formula (I)

Wherein A is an alkylene group substituted at the same position, preferably CH ₂ ; X and Y are independently selected from S and 0; R ₀ is an optionally substituted carbon-bonded organic moiety, for example Specifically, optionally substituted alkyl or aryl, SR _x , OR _x and NR _x R _y , wherein R _x and / or R _y are selected from H and organic moieties and at least R _x is a substituent other than H ). The oligonucleotide according to claim 1, wherein R is selected from methyleneacyloxy group, methylenecarbonate group and methylenecarbamate group. The compound of claim 1 or 2, wherein R is of the formula -CH ₂ -OC (O) -R ₀ Wherein R ₀ is a saturated, unsaturated, heterocyclic or aromatic, hydrocarbon moiety of C 1 -C 20. 3. The compound of claim 1, wherein R is of the formula —CH ₂ —OC (O) —NR _× R _y , wherein R _x and R _y are independently selected from alkyl or (hetero) aryl, and preferably R _x and R _y are alkyl, or R _x and R _y together form a 3-8 membered ring optionally containing an additional cyclic heteroatom selected from O, N and S) Phosphorus oligonucleotide. 3. The methylenecarbonate group of claim 1, wherein R is a group of the formula -CH ₂ -OC (O) OR _{x wherein} R _x is selected from optionally substituted alkyl, cycloalkyl, and (hetero) aryl groups Oligonucleotides that are applicable. 6. The oligonucleotide of claim 2, wherein R _x is selected from phenyl comprising lower alkyl or cycloalkyl (C 1 -C 7) residues, substituted phenyl groups, and naphthyl groups. 7. The oligonucleotide of claim 1, wherein the oligonucleotide comprises 2 to 30 nucleotides. The ribonucleoside, 2'-deoxyribonucleoside, 2'-substituted ribonucleoside, 2'-4'-fixed-ribonucleoside according to any one of claims 1 to 6 Oligonucleotide containing a nucleoside selected from 3'-amino-ribonucleoside, 3'-amino-2'-deoxyribonucleoside. The oligonucleotide of any one of claims 1 to 8 as prodrug. A method of preparing a second oligonucleotide having at least one thiophosphate bond, the method comprising the steps of: (a) providing a first oligonucleotide according to any one of claims 1 to 8, and (b) said first oligonucleotide Cleaving at least one R group from to produce said second oligonucleotide having at least one phosphorothioate linkage. The compound of claim 10, wherein the R group is preferably in alkyl, cycloalkyl and aromatic amine; More preferably in primary amine or secondary alkylamine; Most preferably, cleaved by reacting a solution selected from a base selected from n-propyl and tert-butyl amine with the first oligonucleotide. The method of claim 10, wherein the base is a hindered primary amine. 13. The method of any one of claims 10-12, wherein the cleavage step is carried out in the presence of an sterically hindered base and an active agent which generally consists of N-heteroaromatic bases. The method of claim 24, wherein the first base is an alkyl primary amine having the same or different substituents selected from linear or branched alkyl of C 1 to C 20. The method of claim 24, wherein the first base is an aryl primary amine wherein the aryl group comprises a linear or branched alkyl or aryl group in the second and / or 6 positions. The method of claim 24 or 25, wherein the active agent is 1,2,4-triazole or other triazole and tetrazole derivatives. 27. A method of deprotecting an oligonucleotide according to any of claims 24 to 26, wherein the hindered base is tert-butyl amine. The method of any one of claims 9 to 16, further comprising the step of purifying said second oligonucleotide having one or more thiophosphoate linkages. The method of claim 17 comprising at least precipitating a second oligonucleotide. 19. The method of claim 17 or 18, further comprising extracting the second oligonucleotide with a solvent. The method of claim 18 or 19 comprising dissolving the second oligonucleotide in a polar organic solvent preferably selected from methylene chloride, chloroform, acetonitrile, acetone and pyridine. 21. The method of claim 20, wherein in the range of about 0.5 (n + 1) mL to about 2.0 (n + 1) mL, preferably about 1.0 (n + 1) mL (n is millimoles of phosphorothioate triester bonds) Aqueous solvent volume) is used. 22. The process according to claim 20 or 21, wherein the solution is treated with a nonpolar organic solvent preferably selected from hydrocarbons, ether solvents and mixtures thereof until the solution of the second oligonucleotide becomes cloudy. 23. The method of claim 22, wherein in the range of about 1 (n + 1) mL to about 4 (n + 1) mL, preferably about 2.0 (n + 1) mL (n is the millimoles of the phosphorothioate triester bonds Aqueous solvent volume) is used. The method according to claim 22 or 23, wherein the turbid solution is treated with a precipitation aid. The method of claim 23, wherein the precipitation aid is selected from inert porous solids preferably selected from celite, coal, wood cellulose, and chromatographic stationary phases such as silica or alumina. 26. The precipitating aid according to claim 24 or 25, wherein the precipitation aid is in the range of about 0.25 (n + 1) g to about 1.5 (n + 1) g, preferably about 0.75 (n + 1) g (n is phosphoro). Used in the amount of millimolar of thioate triester bonds). 26. The process of claim 25, wherein after the addition of the precipitation aid the mixture is treated with a second fraction of a non-polar organic solvent, the volume of the fraction generally being from about 1 (n + 1) mL to about 4 (n + 1) mL And preferably about 2.0 (n + 1) mL (where n is the millimolar number of phosphorothioate triester bonds). 28. The method of any of claims 24 to 27, wherein the solid material obtained in the precipitation step is filtered and washed. 29. The method of any one of claims 18-28, wherein the oligonucleotide is extracted from the oligonucleotide-containing precipitate using a solvent. The solvent of claim 29 wherein the solvent comprises a mixture of a polar organic solvent preferably selected from acetonitrile, acetone, THF, DMF, DMSO and pyridine, and preferably an aqueous medium which is water; The volume ratio of polar organic solvent / aqueous medium is preferably about 0.5 to 1.5, more preferably about 0.7. The method according to claim 10, wherein the R group preferably selected from a methyleneacyloxy group, a methylene carbamate group or a methylene carbonate group is cleaved in the human body or the body of an animal. A sulfiding agent of formula R ″ -SR, wherein R corresponds to formula (I)

Wherein A is an alkylene group substituted at the same position, preferably CH ₂ ; X and Y are independently selected from S and 0; R ₀ is optionally substituted alkyl or aryl, SR _x , OR _x and NR _x R _y (R _x and R _y are selected from H and organic moieties and at least R _x is a substituent other than H). 33. The sulfiding agent of claim 32, wherein R is selected from methyleneacyloxy group, methylenecarbamate group or methylenecarbonate group and R " is a leaving group. 34. The sulfiding agent according to claim 32 or 33, wherein R is as described in any one of claims 1 to 6. 35. The sulfiding agent according to any one of claims 32 to 34, which corresponds to formula (II):

Wherein R ^A and R ^B are the same or different from each other and R ^A and R ^B At least one of which is selected from a substituted sulfonyl group or acyl group, wherein R ^A and R ^B optionally together form a cyclic substituent). 36. The sulfiding agent according to any one of claims 32 to 35, wherein R "is a sulfonamide group, preferably N-substituted-N-alkylsulfonyl. 37. The sulfiding agent of claim 36 corresponding to formula (III):

Wherein R ₁ , R ₃ and R ₄ are independently any unsaturated or aromatic hydrocarbon moiety of C ₁ -C 20, preferably a linear or branched alkyl group or a cycloalkyl group. 36. A sulfiding agent according to any one of claims 32 to 35 wherein R "is dicarboxyamide. The sulfiding agent according to claim 38, which corresponds to formula (IV):

(Wherein Z is -CH ₂ -CH _2- , -CH = CH-, -CH ₂ -O-CH _2- ,

Group is selected from the group consisting of, preferably Z is

Term). A method of synthesizing oligonucleotides, the method comprising using a sulfiding agent according to any one of claims 32 to 39 to sulfide one or more phosphorus internucleotide linkages of the precursor of the oligonucleotide. How to include. 41. The method of claim 40, wherein the phosphorescent internucleotide bond is an H-phosphonate diester bond. 42. The method of claim 41, wherein coupling the H-phosphonate monoester salt with a protected nucleoside or with an oligonucleotide having a free hydroxyl group to form an H-phosphonate diester bond How to include more. 43. The method of claim 41 or 42, wherein the coupling operation is carried out in solution. 38. A method of synthesizing a sulfiding agent as described in any one of claims 32-37, wherein (a) a sulfuryl halide, preferably sulforyl chloride, is represented by the formula RSC (O) -R ₂ As described in claims 1 to 6, R ₂ is an organic moiety, preferably reacted with a thioacetal of C 1 -C 20 any unsaturated or aromatic hydrocarbon moiety, of formula RSW wherein W is halogen , Preferably C1), and (b) reacting the intermediate with an N-sulfonyl compound or N-acyl compound. 45. The compound of claim 44, wherein formula R ₁ -C (O) -O-CH ₂ -SC (O) -R ₂ Wherein R ₁ and R ₂ are independently C ₁ -C 20 any unsaturated or aromatic hydrocarbon moiety, reacting thioacetal with sulfuryl chloride to give the formula R ₁ -C (O) -O-CH ₂ -S- A process for producing an intermediate of Cl wherein R ₁ is independently C ₁ -C 20, any unsaturated or aromatic hydrocarbon residue. 46. The process of claim 44 or 45, wherein the intermediate in step (b) is a compound of formula R ₃ -S (O) ₂ -NH-R ₄ wherein R ₃ and R ₄ are independently organic moieties, preferably Is an optionally unsaturated or aromatic hydrocarbon residue of C 1 -C 20. 46. The process of claim 44 or 45, wherein in step (b) the intermediate is reacted with an N-acyl compound of formula

(Wherein Z is -CH ₂ -CH-, -CH = CH-, -CH ₂ -O-CH _2- ,

Group is selected from the group consisting of, preferably Z is

Term). 48. The compound of any one of claims 45 to 47, wherein R ₁ is selected from phenyl comprising a lower alkyl or cycloalkyl (C1-C7) moiety, a substituted phenyl group and a naphthyl group, more preferably an ethyl group. How to be. 48. The compound of any of claims 44-47, wherein R _{2 ,} R ₃ and R ₄ are selected from among phenyl comprising lower alkyl or cycloalkyl (C 1 -C 7) residues, substituted phenyl groups and naphthyl groups, More preferably a methyl group.