EP3016964A1 - Verfahren zur herstellung von schützendem acetal und acetalestergruppen und dessen anwendung zum schutz einer hydroxylfunktion - Google Patents

Verfahren zur herstellung von schützendem acetal und acetalestergruppen und dessen anwendung zum schutz einer hydroxylfunktion

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Publication number
EP3016964A1
EP3016964A1 EP14723139.3A EP14723139A EP3016964A1 EP 3016964 A1 EP3016964 A1 EP 3016964A1 EP 14723139 A EP14723139 A EP 14723139A EP 3016964 A1 EP3016964 A1 EP 3016964A1
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European Patent Office
Prior art keywords
group
reaction
alkyl
acetal
hydroxyl
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EP14723139.3A
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English (en)
French (fr)
Inventor
Wojciech Markiewicz
Agnieszka TOS-MARCINIAK
Marcin Chmielewski
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Instytut Chemii Bioorganicznej of PAN
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Instytut Chemii Bioorganicznej of PAN
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/167—Purine radicals with ribosyl as the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/067—Pyrimidine radicals with ribosyl as the saccharide radical
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention is the method for incorporation of acetal and acetal ester groups for protection of hydroxyl function.
  • the method is applied in particular in the processes of RNA synthesis.
  • the method can be employed in the synthesis of nucleosides with acetal and acetal ester groups for the protection of hydroxyl functions.
  • protecting groups are used.
  • the incorporation of protecting groups must be selective and efficient.
  • the linkage between a protecting group and a blocked functional group must be stable in the conditions of successivereactions, and the unblocking must be selective and efficient, e.g. as a result of applying easily accessible and non-toxic reagents, and/or as a result of using physical factors, e.g. heating, irradiation.
  • RNA The chemical synthesis of RNA consists of the reaction of condensation of nucleotide units.
  • An activated nucleotide unit binds to the free 5 '-OH group of the growing RNA chain.
  • an internucleotide linkage is formed between position 3 ' of one nucleotide and position 5 ' of the other nucleotide.
  • the remaining reactive centres of the nucleotide are temporarily blocked by protecting groups.
  • An appropriate selection of protecting groups enables efficient formation of an internucleotide linkage.
  • An appropriate selection of protecting groups, particularly for 2'-hydroxyl functions, and a method for their incorporation and removal, are the main problems associated with the synthesis of RNA chains.
  • the unit thus prepared is subjected to a reaction with a phosphitylation reagent in order to incorporate phosphite in position 3 ' .
  • Blocking of amine functions of in pyrimidine and purine bases of nucleosides/nucleotides can be performed in optional order depending on a type of amine protection.
  • the protecting groupof the 2'-OH function must remain stable until the synthesis of the RNA chain is complete, and its unblocking - which constitutes the final stage of RNA synthesis - cannot adversely affect the synthesized RNA chain.
  • a protecting group in position 2' has a direct impact on the reactivity and efficiency of internucleotide linkage formation during the chemical synthesis of RNA fragments, and:
  • oligoribonucleotide synthesis An important factor in the processes of oligoribonucleotide synthesis is the stability of protection of hydroxyl and amine functions throughout the entire duration of the process of synthesis - from the stage of preparation of the nucleotide unit used in synthesis, through the successive stages of synthesis, isolation of the final product, and the processes of removal of protecting groups from the final product.
  • hydroxyl groups particularly in position 2'
  • a range of protecting groups the most common of which are ethers, silyl ethers, acetals and acetal esters.
  • Czernecki (3) applied a benzyl group to protect 2'-hydroxyl function.
  • the group ensures adequate protection during oligoribonucleotide synthesis, however it is removed by direct hydrogenolysis which may be accompanied by partial hydrogenation of double bonds
  • Ohtsuka (4) used a photolabile 2-nitrobenzyl group removable by UV irradiation, which is why the synthesis of the nucleotide unit and the RNA chain must be conducted without the access of light, and furthermore the removal may induce reactions of photodimerization or photodestruction of nucleobases.
  • Acetal protecting groups of hydroxyl functions have been known for a long time, however not all of them can be used for protecting the 2'-OH function in the chemical synthesis of RNA.
  • the acid-labile tetrahydropyran-l-yl (thp) and methoxytetrahydropyran-4-yl (mthp) groups have known applications as protecting groups of 2'-hydroxyl function.
  • the structure of the thp group has a centre of asymmetry at the acetal carbon atom.
  • the incorporation of thp group results in the formation of a mixture of diastereoisomers, which might cause necessity of otherwise difficult their separation operation.
  • the mthp group has no centre of chirahty. Nevertheless, neither thp nor mthp groups can be employed in the chemical synthesis of RNA if the 5 '-hydroxyl function is blocked with the commonly used DMTr group.
  • Reese et al. (8) proposed the application of l-[(2-chloro-4-methyl)phenyl]-4- methoxypiperidin-4-yl (ctmp) group, stable in the conditions of DMTr group removal.
  • Beijer et al. (9) used a l-(2-fluorophenyl)-4-methoxypiperidin-4-yl (fpmp) group which is stable in the conditions of DMTr group removal. All the achiral acetal groups, mthp, ctmp and fpmp, introduce a large steric hindrance, and have a quaternary carbon atom in the a position relative to the 2 '-oxygen atom. Thus, this higher order has an adverse effect on the efficiency of formation of the internucleotide linkage (2).
  • a method for protecting the hydroxyl function with acetal or acetal ester derivatives of formaldehyde has been known.
  • This protecting groups are sufficiently stable in the acidic environment, as opposed to other acetals.
  • These formaldehyde derived acetals exhibit sufficient stability in acidic conditions which are used for DMTr group removal.
  • the application of acetalderivatives of formaldehyde makes it possible to use various unblocking conditions depending on the characteristics of the formaldehyde derivative, e.g. their structure allows their removal in conditions other than acidic.
  • Acetal or acetal ester derivatives of formaldehyde do not introduce large steric hindrances since they contain a secondary carbon atom in the a-position relative to the 2' oxygen atom. On account of their mixed nature, the groups maintain stability during the chemical synthesis of RNA.
  • the patent US5986084 discloses the application of a triisopropylsilyloxymethyl (TOM) group in the synthesis of long RNA fragments.
  • the method of incorporating the protecting group in the 2' position in the nucleotide comprises two stages. At the first stage, a substrate with free 3 '- and 2'-hydroxyl groups is subjected to a reaction with dibutyltin(IV) chloride, producing reactive cyclic 5 '-0-(4,4'-dimethoxytrityl)-2',3'-0-dibutylstannateribonucleoside which, at the second stage, is subjected to a reaction with triisopropylsilyloxmethyl chloride.
  • the resulting product is a m i xture o f tw o n u c l e o s i d e i s o m e rs , n am e l y 5 '-0-(4,4'-dimethoxytrityl)-2'-0- (triisopropylsilyloxymethyl)nucleoside (5'-0-DMTr-2'-0-TOM-nucleoside) a n d 5 '-0-(4,4'- dimethoxytrityl)-3'-0-(triisopropyl silyloxymethyl)nucleo side (5 -0-DMTr-3'-0-TOM- nucleoside).
  • Ohgi et al. 10-10 used a cyanoethoxymethyl (CEM) group for blocking the 2'-OH function in a ribonucleoside.
  • CEM cyanoethoxymethyl
  • Oghi 9
  • the reaction gives rise to reactive cyclic 5'-0-(4,4'-dimethoxytrityl)-2',3'- O-dibutylstannate-ribonucleoside which, at the next stage, is subjected to a reaction with (2- cyanoethoxy)methyl chloride.
  • Yoshinobu (11) disclosed a method for incorporation of a 2-cyanoethoxymethyl group protecting the 2'-hydroxyl function of nucleoside, based on the reaction of 3 '- and 5'-protected nucleoside with 2-cyanoethyl methylthiomethyl ether at a very low temperature, in the presence of N-iodosuccinimide and trifluoromethanesulphonic acid.
  • the reactions are performed at a temperature of -45 C since higher temperatures may induce the alkylation of pyrimidine bases.
  • the method requires the use of expensive reagents.
  • the US patent US8536318 discloses the use of a pivaloyloxymethyl group (PivOM) for the protection of hydroxyl function.
  • the incorporation of pivaloyloxymethyl group into the hydroxyl function is a two-stage process. It consists of the reaction between a nucleoside protected in position 5'-0-(4,4'-dimethoxytrityl) group and free 3'- and 2'-hydroxyl groups with dibutyltin(IV) oxide, resulting in the formation of cyclic 2',3 '-dibutylstannate-ribonucleoside which, at the next stage, reacts with pivaloyloxymethyl chloride.
  • the reaction produces two isomers: 5'-0-(4,4'- dimethoxytrityl)-2'-0-pivaloyloxymethylnucleoside (5'-0-DMTr-2'-0-PivOM-nucleoside) and 5'- 0-(4,4'-dimethoxytrityl)-2'-0-p ivaloylo xym ethyl nuc l eo s i de ( 5 '-0-DMTr-3'-0-PivOM- nucleoside). Both isomers require chromatographic separation, as only 5'-0-DMTr-2'-0-PivOM- nucleosideis suitable for RNA synthesis.
  • the efficiency of obtaining isomer 5 -0-DMTr-2 -0- PivOM-nucleoside is 34-49%.
  • the group is compatible with other protecting groups used in the chemical synthesis of oligoribonucleotides, however the disclosed method for its incorporation into the nucleoside is inefficient and involves multiple stages.
  • Lackey (12) applied a levulinyloxymethyl (ALE) group for protecting the 2'-hydroxyl function during RNA synthesis.
  • ALE levulinyloxymethyl
  • the starting component is 5',3'-0- (tetraisopropyldisiloxane-l,3-diyl)nucleoside which, in a reaction with dimethyl sulphoxide, acetic acid and acetic anhydride, is converted into 5',3'-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- (methylthiomethyl)nucleoside which, following isolation and purification, is subjected to a reaction with sulphuryl chloride, forming another intermediate product i.e.
  • the method comprises several stages and is time consuming, with an overall duration exceeding 30 hours. Furthermore, column chromatography and expensive crown ethers markedly increase the costs of synthesis.
  • the purpose of the invention was development of a simple and effective method for incorporation of acetal and acetal ester groups protecting the hydroxyl function in order to use in particular thus blocked compounds in further chemical reactions and to protect the hydroxyl function of the 2' group in nucleosides.
  • the present invention is a method for incorporation of an acetal or acetal ester group to protect hydroxyl function.
  • the method according to the invention consists of the reaction of an organic compound containing at least one hydroxyl group, soluble in an aprotic solvent, with a compound of the general formula 1, Ri-S-CH 2 -0-R 2 (1) where
  • Ri represents a Ci_ 6 alkyl; unsubstituted or substituted benzyl or naphthyl, with substituents including a Ci_ 7 alkyl, halogen, aminoacyl; in particular Ri represents -CH 3 -Ph, -Ph(4-Cl), - Ph(4-CH 3 ), -CH 2 Ph
  • alkyl-aryl in which the alkyl chain contains Ci_ 5 , whereas aryl contains from 1 to 8 unsubstituted or substituted rings, with substituents including a Ci_ 7 alkyl, halogen, aminoacyl, tertiary amine group, cyano group;
  • R 3 represents:
  • R4, R 5 and R5 are different or the same, and represent a Ci_ 2 8 alkyl or aryl containing from 1 to 8 rings or trimethylsilyl, where the total number of carbon atoms in the group of formula 3 is no less than 6 and no more than 30,
  • the method according to the invention can be applied for compounds containing at least one hydroxyl group, and the compounds must be soluble in aprotic solvents.
  • the method is only suitable for modifying organic compounds that are soluble in aprotic solvents.
  • the method can also be applied for compounds that are insoluble in aprotic solvents, on condition that they are first converted into a form which is soluble in these solvents.
  • ribose is insoluble in aprotic solvents, however incorporation of a protecting group, e.g. a silyltert-butyldimethylsilylgroup, into one or two hydroxyl groups will permit ribose dissolution in aprotic solvents.
  • the solvents that can be used include halogen derivatives of alkanes, particularly carbon tetrachloride, chloroform, dichloromethane or 1,2-dichloroethane; aromatic solvents, particularly benzene, toluene; cyclic ethers, particularly tetrahydrofuran; nitrile compounds, particularly acetonitrile, or a mixture of these solvents. It is particularly advantageous to use 1,2- dichloroethane.
  • the reaction is conducted in anhydrous environments, whereby it is possible for the reaction to be conducted in an environment containing trace amounts of water, however if this is the case, an appropriate excess of SnCl 4 must be ensured because a part of it is bound by water.
  • the organic compound containing at least one hydroxyl group and an appropriate compound of the general formula 1 is dissolved in a solvent, and then SnCl 4 is added to the reaction mixture. It is advantageous to introduce SnCl 4 in the form of a solution in the same solvent as that is the reaction medium or in 1,2-dichloroethane.
  • SnCl 4 is used in an amount not smaller than 0.01 mole per one mole of hydroxyl groups which are intended to be exchanged. It is advantageous to use SnCl in an amount from 1 to 6 moles per one mole of hydroxyl groups which are intended to be exchanged most advantageously in an amount from 2.5 to 4.5 moles.
  • the ratio of the compound of formula 1 to hydroxyl groups can be 1 : 1, however it is advantageous for the compound of formula 1 to be used in excess ranging from 1 to 8.
  • An excess of the compound of formula 1 makes it possible to achieve the highest possible process efficiency within the shortest time.
  • the reaction can be conducted over a broad temperature range, from very low temperatures up to temperatures not exceeding the boiling temperature of the reaction mixture.
  • the reaction is conducted in low temperatures, however not lower than the solidification point of the solvent used.
  • the higher the temperature the lower the efficiency of obtaining the target product and the greater the quantity of by-products. It is beneficial to conduct the reaction in temperatures from the range from -80 C to 0 C, and most advantageously - below 15 C. Due to that fact, it is advantageous to use solvents with low solidification points.
  • the reaction of hydroxyl group blocking involves preparation of a solution of a compound containing a hydroxyl group, and the compound of formula 1, followed by cooling of the mixture to a low temperature. After the cooling procedure, SnCl 4 is introduced, whereupon the reaction is advantageously conducted at a low temperature until the completion of the process.
  • it is advantageous to monitor the course of the blocking reaction by thin-layer chromatography on silica gel plates, or by high-performance liquid chromatography (HPLC).
  • the duration of the reaction depends on the types of substrates and temperature, and generally ranges from 4 to 16 hours.
  • the product is isolated and purified using known methods. Prior to product isolation it is advantageous to neutralize SnCl 4 with a neutralizing agent in an amount of at least 4 molar equivalents of SnCl 4 used. Depending on product stability, neutralization can be performed with:
  • a product of hydroxyl function protectionwith an acetal or acetal ester protecting group is an original compound in which the site of the hydroxyl group is occupied by the group of formula 4
  • R 2 has the meaning defined above.
  • the present invention relates to a method for protecting the hydroxyl function, particularly in position 2' in nucleoside derivatives, consisting of the incorporation of an acetal or acetal ester group.
  • the method according to the invention consists of the reaction between the compound of the general formula 5
  • B represents radicals of nucleobases, particularly uracil, or appropriately protected residuesof adenine, guanine, cytosine, uracil and thymine,
  • Yi and Y 2 are the same or different, and represent groups protecting hydroxyl functions in positions 3' and 5 '; in particular, they are silyl groups: triethylsilyl, tert-butyl-dimethyl- silyl, isopropyl-dimethyl-silyl, tert-butyl-diphenyl-silyl, methyl-diisopropyl-silyl, triphenylsilyl, triisopropylsilyl, methyl-di-tert-butylsilyl
  • the blocking compound the compound of the general formula 1 (hereinafter referred to as the blocking compound),
  • - i represents a C w alkyl; unsubstituted or substituted benzyl or naphthyl, with substituents including a Ci_ 7 alkyl, halogen, aminoacyl; in particular Ri represents -CH 3> -Ph,
  • alkyl-aryl in which the alkyl chain contains Ci_ 5 , and aryl contains from 1 to 8 unsubstituted or substituted rings, with substituents including a Ci_ 7 alkyl, halogen, aminoacyl, tertiary amine group, cyano group;
  • R 3 represents
  • ketone group o unsubstituted or substituted phenyl, with substituents including a Ci_ 7 alkyl, halogen, aminoacyl, tertiary amine group, cyano group,
  • R4, R 5 and R ⁇ are different or the same, and represent a Ci_ 2 8 alkyl or aryl containing from 1 to 8 rings or trimethylsilyl, with the total number of carbon atoms in that group is no less than 6 and no more than 30,
  • the method for the protection of the hydroxyl function in nucleosides consists in particular of incorporation of groups of formulas 12 and 13 in position 2'
  • the solvents that can be used include halogen derivatives of alkanes, in particular carbon tetrachloride, chloroform, dichloromethane or 1,2-dichloroethane; aromatic solvents: particularly benzene, toluene; cyclic ethers, particularly tetrahydrofuran; nitrile compounds, particularly acetonitrile, or a mixture of these solvents. It is particularly advantageous to use 1,2- dichloroethane.
  • the reaction is conducted in anhydrous media but it is possible for the reaction to be conducted in an environment containing trace amounts of water, however if this is the case, an appropriate excess of SnCl 4 must be ensured because a part of it is bound by water.
  • the compound of the general formula 5 or 6, in which substituents have the meaning defined above, and an appropriate blocking compound are dissolved in a solvent, and then SnCl 4 is added to the reaction mixture. It is advantageous to introduce SnCl 4 in the form of a solution in the same solvent as that is the reaction medium or in 1 ,2-dichloroethane. SnCl 4 is used in an amount not smaller than 0.01 mole per one mole of hydroxyl groups which are intended to be protected. It is advantageous to use SnC in an amount from 1 to 6 moles per one mole of hydroxyl groups which are intended to be protected, most advantageously in an amount from 2.5 to 4.5 moles.
  • the ratio of the compound of formula 1 to hydroxyl groups can be 1 : 1, however it is advantageous for the compound to be used in excess ranging from 1 to 8. An excess of the compound of the formula 1 makes it possible to achieve the highest possible process efficiency withm the shortest time.
  • the reaction can be conducted over a broad temperature range, from very low temperatures up to temperatures not exceeding the boiling temperature of the reaction mixture, however the temperature of the reaction must not be higher than the temperature of de-protecting the hydroxyl function in positions 3 ' and 5 '. It is advantageous to conduct the reaction in low temperatures, however not lower than the solidification point of the solvent used. The higher the temperature, the lower the efficiency of the process and the greater the quantity of by-products. It is advantageous to conduct the reaction in the temperature range from -80 C to 0 C, and most advantageous - below 15 C. Due to that fact, it is advantageous to use solvents with low solidification points.
  • the reaction of hydroxyl group protection involves preparation of a solution of a compound containing a hydroxyl group which is to be protected, and a blocking compound, followed by the cooling of the mixture to a low temperature. After the cooling procedure, SnCl 4 is introduced, and then the reaction is advantageously conducted at a low temperature until the completion of the process. In order to determine the optimum duration of the process, it is advantageous to monitor the course of the reaction by thin-layer chromatography on silica gel plates, or by high- performance liquid chromatography (HPLC). The duration of the reaction depends on the types of substrates and temperature, and generally ranges from 4 to 16 hours.
  • the product is isolated and purified using known methods, following the same procedure as in the method according to the first aspect of the invention.
  • the product of the reaction is the compound of the general formula 14,
  • the protecting groups used in the method according to the invention are unblocked in different conditions, on a case-by-case basis, depending on the structure of the group.
  • a characteristic property of acetal and acetal ester groups protecting the hydroxyl function is high flexibility in unblocking methods.
  • all known chemical unblocking methods can be employed, however due to the specific properties of acetal and acetal ester groups stemming from the nature of these compounds, it is possible to select appropriate conditions which are advantageous for a particular application of the compounds protected with these groups.
  • the selection of the most advantageous method of unblocking the hydroxyl function protected with the method according to the invention depends not only on the chemical nature of the blocking group but also on the specific properties of the blockage used.
  • the removal of acetal and acetal ester groups can be performed with solutions of inorganic bases, e.g. NaOH, KOH, and organic bases, e.g. amines in organic or inorganic solvents, also in their mixtures. It is advantageous to use weak bases (e.g. aqueous ammonia solution, methanol/ammonia solution, ethanol/ammonia solution, methylamine in methanol, n-butylamine in methanol), which is why during the unblocking there is practically no hydrolysis of mtemucleotide linkages.
  • weak bases e.g. aqueous ammonia solution, methanol/ammonia solution, ethanol/ammonia solution, methylamine in methanol, n-butylamine in methanol
  • Acetal and acetal ester protecting groups can also be unblocked in acidic conditions, and they are stable in conditions required for the removal of the acid-labile dimethoxytrityl group (DMTr) from the 5 '-hydroxyl position.
  • the groups are stable, which is their advantage, towards weak acid solutions used for the unblocking of the 5 '-hydroxyl group blocked with the acid-labile dimethoxytrityl group (DMTr).
  • DMTr acid-labile dimethoxytrityl group
  • the acetal or acetal ester group protecting the 2'-hydroxyl function is stable, which is a significant factor for the chemical synthesis of the RNA chain.
  • Acetal and acetal ester protecting groups can also be removed in reactions that are specific to a particular blocking group, e.g. in a reaction between the carbonyl group of the ieto-ketoester radical (e.g. levulinyl, Lv, H 3 CC(0)CH 2 CH 2 C(0)-,) and the unblocking reagent i.e. hydrazine solution.
  • the carbonyl group of the ieto-ketoester radical e.g. levulinyl, Lv, H 3 CC(0)CH 2 CH 2 C(0)-,
  • the unblocking reagent i.e. hydrazine solution.
  • the subjects of the present invention are new monothioacetals of the general formula 1,
  • ⁇ R 2 represents o-toluyl, benzoyl, pivaloyl.
  • R 2 has the meanings defined above
  • the reaction is conducted in the following manner: 1 eqval of an appropriate compound of formula 17 is dissolved in diethyl ether and combined with 1 egual of an amine. The solution is cooled down to the temperature of 0 C and, on stirring, 1 egualof an appropriate compound of formula 16 is added. On completion of the reaction, the cooling is stopped and a saturated sodium hydrogen carbonate solution is added successively until carbon dioxide no longer evolves from the reaction mixture. The mixture is separated and the organic layer containing the reaction product is dried, following which the solvent is evaporated and the final product is crystallized.
  • the method according to the invention is universal and can be applied for the protection of hydroxyl functions with acetal and acetal ester groups.
  • the method can be applied for the protection of hydroxyl groups not only in nucleosides and their analogues, but also in alcohols and complex chemical compounds containing a hydroxyl group.
  • the method makes use of both known and new compounds, developed specifically for the invention, containing a thioacetal or thioacetal ester group.
  • the method according to the invention has a particularly advantageous application in the chemical synthesis of RNA and its analogues.
  • the blockage of the hydroxyl function using the method according to the invention is compatible with other protecting groups applied during RNA chain synthesis; for example, it is stable in the conditions of unblocking of the 3'- and 5' -hydroxyl positions with fluoride ions.
  • N 6 -phenoxacetyl-2'- benzoyloxymethyl-3',5'-(tetraisopropyldisiloxane-l,3-diyl)adenosine was obtained in the yield of 72%.
  • N 2 - tert-butylphenoxacetyl-3',5'-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- benzoyloxymethylguanosine was obtained in the yield of 74%.
  • a volume of 30 ⁇ (0.3 mmol) of dry benzyl alcohol was transferred into a round-bottom flask and then dissolved in 1 ml of dry 1,2-dichloroethane, then 0.166 g (0.6 mmol) of benzoyloxymethylthiobenzene was added in the presence of 4 A molecular sieves.
  • the flask was closed with a septum provided with an argon-filled balloon.
  • the mixture was cooled down to the temperature of -25°C and thereafter, on stirring, 0.6 ml of 0.8 M solution of tin(IV) chloride (0.48 mmol) in 1,2-dichloroethane was added.
  • the reaction was conducted in argon atmosphere.
  • the mixture was stirred magnetically at a temperature of -25°C for 5 hours. Thereafter, the reaction was completed by adding an aqueous solution of sodium hydrogen carbonate until carbon dioxide stopped evolving,then the cooling bath was removed. The white precipitatewas filtered off, and the filtrate was extracted three times with 1,2-dichloroethane (3x3 ml). The organic layers were collected and dried over anhydrous sodium (VI) sulphate. The solvent was evaporated.
  • the raw product was purified on a preparative PLC plate covered with silica gel 60 RP-18, F 25 4, 1 mm, from Merck, using hexane-dichloromethane 2:3 as the mobile phase. The product was extracted with dichloromethane (15 ml). In this manner, benzyloxymethylbenzoyl was obtained in the yield of 56%.
  • the product was extracted six times with 5 ml of methylene di chloride. The organic layers were dried over anhydrous sodium sulphate. The solvent was evaporated. The raw product of the reaction was purified in a chromatographic column packed with silica gel 60 (63-200 ⁇ ) from Merck, using methylene dichloride-methanol (95 :5) as eluents. In this manner, 2'-0-(pivaloyloxymethyl)uridine was obtained in the yield of 84%.
  • Example 20 A portion of 50 mg (0.14 mmol) of 2'-(9-(pivaloyloxymethyl)uridine obtained in Example 18 was dissolved in THF (2.5 ml). Thereafter, 2 M n-butylamine solution in methanol (2.5 ml) was added. On completion of the unblocking reaction (34 hours), the solvent and amine residue were evaporated from the reaction mixture. The post-reaction mixture was introduced into a chromatography column packed with silica gel 60 (63-200 ⁇ ) from Merck, using methylene dichloride-methanol (60:40) as eluents. Uridine was isolated as the product of unblocking. NRM analysis confirmed that the compound resulting from the removal of the protecting group is uridine.
  • Example 20 A portion of 50 mg (0.14 mmol) of 2'-(9-(pivaloyloxymethyl)uridine obtained in Example 18 was dissolved in THF (2.5 ml). Thereafter, 2 M n-butylamine solution
  • the raw product was introduced into a chromatography column packed with silica gel 60 (63-200 ⁇ ) from Merck, using methylene dichloride-methanol (98 :2) as eluents.
  • the isolated product of unblocking of the protecting group in position 2 ' is 3 ' ,5 '-0-(tetraisopropyldisiloxane-l,3- diyl)uridine.
  • Spectroscopic analysis confirmed the identification of the compound - cf. the spectrum in Example 20.
  • the isolated product of unblocking of the protecting group in position 2' is 3 ',5'-0-(tetraisopropyldisiloxane-l,3-diyl)uridine.
  • Spectroscopic analysis confirmed the identification of the compound - cf. the spectrum in Example 20.
  • the raw product was introduced into a chromatographic column packed with silica gel 60 (63-200 ⁇ ) from Merck, using methylene dichloride-methanol (60:40) as eluents.
  • the collected fraction was evaporated.
  • the isolated product of the unblocking reaction was uridine, which was confirmed by NMR analysis - cf. the spectrum in Example 17.

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EP14723139.3A 2013-03-21 2014-03-19 Verfahren zur herstellung von schützendem acetal und acetalestergruppen und dessen anwendung zum schutz einer hydroxylfunktion Withdrawn EP3016964A1 (de)

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PL403256A PL221806B1 (pl) 2013-03-21 2013-03-21 Sposób wprowadzania acetalowych i acetaloestrowych grup ochronnych oraz związki do realizacji tego sposobu
PCT/PL2014/050012 WO2014148928A1 (en) 2013-03-21 2014-03-19 Method for incorporating protecting acetal and acetal ester groups, and its application for the protection of hydroxyl function

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EP3016964A1 true EP3016964A1 (de) 2016-05-11

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EP4441066A1 (de) * 2021-11-29 2024-10-09 Hongene Biotech Corporation Synthese von 2' acetylester-geschützten nukleosiden

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CA1193598A (en) * 1982-05-06 1985-09-17 Rafael Foguet 2-amino-benzoic acid derivatives and processes for their production
US5986084A (en) 1997-08-18 1999-11-16 Pitsch; Stefan Ribonucleoside-derivative and method for preparing the same
AU771876B2 (en) * 1998-07-27 2004-04-01 Meiji Seika Kaisha Ltd. Novel carbapenem derivatives
FR2931824B1 (fr) * 2008-05-29 2014-11-28 Centre Nat Rech Scient Procede de synthese d'arn par voie chimique.

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WO2014148928A1 (en) 2014-09-25
PL221806B1 (pl) 2016-05-31

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