WO2020256084A1 - 架橋型ヌクレオシド中間体の結晶及びその製造方法、並びに架橋型ヌクレオシドアミダイトの製造方法 - Google Patents
架橋型ヌクレオシド中間体の結晶及びその製造方法、並びに架橋型ヌクレオシドアミダイトの製造方法 Download PDFInfo
- Publication number
- WO2020256084A1 WO2020256084A1 PCT/JP2020/024037 JP2020024037W WO2020256084A1 WO 2020256084 A1 WO2020256084 A1 WO 2020256084A1 JP 2020024037 W JP2020024037 W JP 2020024037W WO 2020256084 A1 WO2020256084 A1 WO 2020256084A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- formula
- compound represented
- compound
- group
- added
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H9/00—Compounds containing a hetero ring sharing at least two hetero atoms with a saccharide radical
- C07H9/02—Compounds containing a hetero ring sharing at least two hetero atoms with a saccharide radical the hetero ring containing only oxygen as ring hetero atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/18—Acyclic radicals, substituted by carbocyclic rings
-
- 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
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/13—Crystalline forms, e.g. polymorphs
-
- 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 disclosure relates to crystals of a crosslinked nucleoside intermediate and a method for producing the same, and a method for producing a crosslinked nucleoside amidite.
- antisense method antigene method, aptamer method, siRNA method, etc. as treatment methods for diseases by nucleic acid drugs.
- the furanose ring of the nucleoside sugar moiety has a distorted conformation called N-type or S-type instead of a planar structure, and is biased to a specific conformation due to a substituent on the ring.
- N-type conformation is dominant. Imanishi et al. Succeeded in forcibly fixing the nucleoside conformation to N-type by cross-linking the 4'- and 2'-hydroxyl groups of the nucleoside sugar moiety.
- LNA Locked Nucleic Acids
- Patent Document 1 a method using a nucleoside as a raw material
- Patent Document 2 a method using a sugar as a starting material
- Patent Document 2 a method using a sugar as a starting material
- Non-Patent Document 3 Among the existing methods for synthesizing crosslinked nucleosides, the following synthesis method has been proposed by Tohoku University as one of the most excellent methods (Non-Patent Document 3).
- the existing method for synthesizing a crosslinked nucleoside (for example, the methods described in Patent Documents 1 and 2 and Non-Patent Document 2) aims to synthesize a crosslinked nucleoside amidite by a series of steps. Therefore, the stability and storage of reaction intermediates (hereinafter sometimes referred to as "crosslinked nucleoside intermediates”) have not been studied.
- an object of the present disclosure is to provide a crystal of a compound that can be used as a crosslinked nucleoside intermediate that can be stably stored for a long period of time, and a method for producing the same.
- Another object of the present disclosure is to provide a method for producing a crosslinked nucleoside amidite using crystals of the compound.
- a compound having a specific substituent can be easily crystallized and can be stably stored as a crosslinked nucleoside intermediate in the form of the crystal.
- R 1 represents a hydroxyl-protecting group and R 2 represents a leaving group.
- Step 1 A step of protecting the hydroxyl group of the compound represented by the formula 1 to obtain the compound represented by the formula 2.
- Step 2 A step of converting the 4-position dimethyldioxolanyl group of the compound represented by the formula 2 into an aldehyde group to obtain a compound represented by the formula 3.
- Step 3 A step of reducing the compound represented by the formula 3 to make the 4-position aldehyde group a hydroxyl group to obtain the compound represented by the formula 4.
- Step 4 A step of converting the 4-position hydroxyl group of the compound represented by the formula 4 into a leaving group to obtain a compound represented by the formula 5.
- Step 5 A step of crystallizing the compound represented by the formula 5 from a crystallization solvent to obtain crystals of the compound represented by the formula 5.
- R 1 represents a hydroxyl-protecting group and R 2 represents a leaving group.
- Step 6 After suspending the crystals of the compound represented by the formula 5 obtained by the production method of any one of [5] to [7] in a solvent, the isopropylidene group of the compound is converted into an acetyl group.
- Step 7 A step of condensing the compound represented by the formula 6 with a silylated base to obtain a compound represented by the formula 7
- Step 8 A step of obtaining the compound represented by the formula 7 Step 8: Represented by the formula 7.
- Step 9 A step of obtaining a compound represented by the formula 8 by performing a cyclization reaction at the same time as removing the protective group of the compound
- Step 9 The hydroxyl group protecting group of the compound represented by the formula 8 is removed to obtain the compound represented by the formula 9.
- Step 10 A step of introducing a protective group into the amino group at the base of the compound represented by the formula 9 as necessary to obtain the compound represented by the formula 10.
- R 1 is a hydroxyl-protecting group
- R 2 is a leaving group
- R 3 is a hydrogen atom or amino-protecting group.
- FIG. 1 shows a photograph of crystals of the compound represented by the formula 5.
- FIG. 2 shows a powder X-ray diffraction spectrum of a crystal of a compound represented by the formula 5.
- FIG. 3 shows the results of thermogravimetric measurement / differential thermal analysis of the crystals of the compound represented by the formula 5.
- FIG. 4 shows the results of an accelerated test of crystals of the compound represented by the formula 5.
- FIG. 5A shows the TLC analysis result of the solution of the crystal of the compound represented by the formula 3 obtained in step 2.
- FIG. 5 (a) shows the TLC analysis result of the reaction solution of the compound represented by the formula c obtained under the conditions described in Non-Patent Document 3.
- the present disclosure relates to crystals of a compound represented by the following formula 5.
- R 1 represents a hydroxyl-protecting group and R 2 represents a leaving group.
- the crystals of the present disclosure show the appearance as shown in FIG. 1 when observed with a microscope as described in Examples described later.
- the crystals of the present disclosure have characteristic peaks in powder X-ray analysis.
- the powder X-ray diffraction in the present specification shall be performed under the following conditions.
- [Scanning range] 2 ⁇ 4.0 to 40 °
- the crystals of the present disclosure are analyzed by a powder X-ray diffractometer using the conditions described above, they are characteristic in the vicinity of the diffraction angle (2 ⁇ ) as shown in Table 1 below, as described in Examples described later. It shows a peak (see FIG. 2).
- the diffraction angle (2 ⁇ ) in powder X-ray diffraction may include an error range of less than 5%. Therefore, in addition to crystals in which the peak diffraction angles in powder X-ray diffraction completely match, crystals in which the peak diffraction angles match with an error of less than 5% are also included in the crystals of the present disclosure.
- the diffraction angle (2 ⁇ ) of a peak with a relative intensity of 10% or more is 5.9 ⁇ 0.3, 11.4 ⁇ 0.6, 11.8 ⁇ 0.6, 13.2.
- the crystals of the compound represented by the formula 5 of the present disclosure are analyzed by a thermogravimetric / differential thermal analysis (TG / DTA) apparatus (heating rate 5 ° C./min) as described in Examples described later. It has an endothermic peak at 124 ° C (see FIG. 3).
- TG / DTA thermogravimetric / differential thermal analysis
- thermogravimetric / differential thermal analysis in the present specification shall be performed under the following conditions.
- Thermal analyzer STA7200 (Hitachi High-Tech Science)
- Measurement conditions The temperature in the range of 30 to 190 ° C. is raised by 5 ° C. per minute, and the change in calorific value of the sample is measured.
- Aluminum oxide is used as a reference.
- the crystals of the compound represented by the formula 5 of the present disclosure can be stably stored at room temperature for a long period of time.
- the long-term stability of the compound can be evaluated by carrying out an accelerated test under the conditions as described in Examples described later.
- the crystals of the present disclosure show an HPLC purity of 90% or more even after the accelerated test, preferably 95% or more, more preferably 98% or more, still more preferably 99% or more, still more preferably 99.5. % Or more (see FIG. 4).
- the present disclosure also relates to a method for producing a crystal of a compound represented by the formula 5, which comprises the following steps 1 to 5.
- Step 1 A step of protecting the hydroxyl group of the compound represented by the formula 1 to obtain the compound represented by the formula 2.
- Step 2 A step of converting the 4-position dimethyldioxolanyl group of the compound represented by the formula 2 into an aldehyde group to obtain a compound represented by the formula 3.
- Step 3 A step of reducing the compound represented by the formula 3 to make the 4-position aldehyde group a hydroxyl group to obtain the compound represented by the formula 4.
- Step 4 A step of converting the 4-position hydroxyl group of the compound represented by the formula 4 into a leaving group to obtain a compound represented by the formula 5.
- Step 5 A step of crystallizing the compound represented by the formula 5 from a crystallization solvent to obtain crystals of the compound represented by the formula 5.
- R 1 represents a hydroxyl-protecting group and R 2 represents a leaving group.
- the compound represented by the formula 2 can be obtained by protecting the hydroxyl groups at the 3- and 5-positions of the compound represented by the formula 1 with a 4-substituted benzyl group.
- the compound represented by the formula 5 of the present disclosure exhibits good crystallinity. Therefore, 4-fluorobenzyl group, 4-chlorobenzyl group, 4-bromobenzyl group, 4-iodobenzyl group.
- 4-Halobenzyl groups such as groups and 4-nitrobenzyl groups are preferable.
- 4-chlorobenzyl group, 4-bromobenzyl group, 4-iodobenzyl group, and 4-nitrobenzyl group are preferable and available because of the high crystallinity of the compound represented by the formula 5 of the present disclosure.
- a 4-bromobenzyl group is preferable in terms of ease of use and cost.
- the 4-substituted benzyl group may be introduced into the compound represented by the formula 1 according to a known method. For example, in the case of introducing a 4-bromobenzyl group, treatment with NaH at 0 ° C. for about 15 minutes to 3 hours in DMF and then reacting with 4-bromobenzyl bromide at room temperature for 1 hour to 24 hours is possible. Good.
- the compound represented by the formula 1 is known, a commercially available compound can be used, or it can be synthesized by a known method (for example, J. Org. Chem. 2015, 80, 5337-5343).
- Step 2 is a step of converting the 4-position dimethyldioxolanyl group of the compound represented by the formula 2 into an aldehyde group to obtain the compound represented by the formula 3.
- a deprotecting agent is added in order to deprotect the dimethyldioxolanyl group at the 4-position to form a diol.
- the deprotecting agent to be used acetic acid, trifluoroacetic acid, hydrochloric acid, sulfuric acid, iodine and the like can be used. Among these, acetic acid is preferably used because of its availability and handleability.
- the amount of the deprotecting agent added can be appropriately set depending on the type of the deprotecting material, but in the case of acetic acid, for example, it is preferable to use the amount of the solvent. Further, as will be described later, it is preferable to use water as an auxiliary solvent because purification by crystallization is easy.
- the ratio of the deprotecting agent to the auxiliary solvent is preferably about 10: 1 to 1: 1 on a volume basis, and more preferably 2: 1.
- an oxidizing agent is used to oxidatively cleave the diol formed as a result of deprotecting the dimethyldioxolanyl group at the 4-position and convert it into an aldehyde group.
- Available oxidizing agents include periodate such as sodium periodate and potassium periodate, lead tetraacetate and the like. Among these, from the viewpoint of cost, toxicity and the like, it is preferable to use 1 to 10 molar equivalents of sodium periodate with respect to the diol. Further, the amount used is more preferably 1 to 2 molar equivalents.
- an additive may be used in order to increase the reactivity of the oxidizing agent.
- the additive include iodine.
- the reaction can be carried out at 0 to 100 ° C.
- the reaction temperature is more preferably about 50 to 80 ° C.
- the reaction may be carried out under the above conditions for 1 to 24 hours.
- excess iodine and the like do not exist in the reaction system, and the solvent can be easily discarded after the reaction, which is suitable for industrial production.
- the target compound represented by the formula 3 can be obtained in a high yield of 70 to 90% or more by crystallization without using a purification means such as chromatography. Can be done.
- Step 3 is a step of reducing the compound represented by the formula 3 to make the 4-position aldehyde group a hydroxyl group to obtain the compound represented by the formula 4.
- the type of reducing agent is not particularly limited, and for example, sodium borohydride, lithium aluminum hydride, borane derivative and the like can be used. Among these, sodium borohydride is preferably used from the viewpoint of cost and safety.
- the amount of the reducing agent added can be appropriately adjusted depending on the type of the reducing agent. For example, in the case of sodium borohydride, it is preferable to use 0.25 to 10 molar equivalents, and more preferably 0.25 to 2 molar equivalents, relative to the compound represented by the formula 3. Under the above conditions, the reaction can be carried out at ⁇ 30 to 50 ° C., more preferably ⁇ 10 to 25 ° C. for 10 to 120 minutes.
- the solvent in this step can be appropriately used according to the type of reducing agent or the like.
- water can be used as the aqueous solvent
- alcohol solvents such as methanol and ethanol as organic solvents
- tetrahydrofuran 2-methyltetrahydrofuran
- 4-methyltetrahydropyran and combinations thereof and water can be used.
- Step 4 is a step of converting the hydroxyl group at the 4-position of the compound represented by the formula 4 into a leaving group to obtain the compound represented by the formula 5.
- the leaving group introduced in this step is not particularly limited, but is preferably a substituent having high reactivity in the nucleophilic substitution reaction in order to efficiently carry out the cross-linking reaction.
- a substituent include a 4-toluenesulfonyloxy group, a methanesulfonyloxy group, a chloromethanesulfonyloxy group, a trifluoromethanesulfonyloxy group, a halogeno group and the like.
- the 4-toluenesulfonyloxy group is preferable because the leaving group-introducing reagent is easily available and inexpensive.
- an organic solvent or a combination of water and an organic solvent can be appropriately selected according to the type of leaving group to be introduced.
- an organic solvent a combination of a halogen-based solvent such as pyridine, dichloromethane, chloroform and an organic base such as triethylamine, and as an aqueous solvent, an organic base such as water-triethylamine, an aqueous sodium hydroxide solution, and an aqueous potassium hydroxide solution.
- a Schottten-Baumann-type condition in combination with a halogen-based solvent such as dichloromethane or chloroform and an ether-based solvent such as tetrahydrofuran or 2-methyl tetrahydrofuran can also be used.
- a halogen-based solvent such as dichloromethane or chloroform
- an ether-based solvent such as tetrahydrofuran or 2-methyl tetrahydrofuran
- the amount of the reagent for introducing the leaving group to be introduced can be appropriately selected depending on the type of the substituent and the solvent.
- the reaction may be carried out at ⁇ 10 to 100 ° C., more preferably 0 to 60 ° C. for 1 to 24 hours.
- Step 5 is a step of performing a post-treatment operation on the synthetic reaction solution of the compound represented by the formula 5 to obtain crystals of the compound represented by the formula 5.
- the crystallization solvent for the compound represented by the formula 5 include an alcohol solvent such as ethanol and a combination of various good solvents and poor solvents.
- good solvents include ethyl acetate, tetrahydrofuran, pyridine, chloroform, acetonitrile, acetone, and derivatives thereof.
- the poor solvent include hexane, alcohol, water, and derivatives thereof.
- the isolation / purification operation may be performed after the step is completed, but it may not be performed.
- the isolation / purification method include various chromatographies such as ion exchange and adsorption, and crystallization.
- the compound obtained by the synthetic method of the present disclosure has high crystallinity, it is preferable to perform crystallization.
- the compound represented by the formula 3 is preferably purified by crystallization. That is, it is preferable to further include a crystallization step between the steps 2 and 3.
- the solvent for the reaction is preferably acetic acid-water.
- crystals of the compound represented by the formula 3 are precipitated.
- the target compound is precipitated as crystals as the reaction progresses, it is possible to prevent the formation of by-products due to decomposition due to the excessive reaction, and the purification effect is high. Therefore, in step 2, by-production and contamination of impurities can be suppressed without performing a complicated purification operation by chromatography (see FIG. 5A).
- the crystals of the compound represented by the formula 5 of the present disclosure can be stably stored for a long period of time, they can be used as a crosslinked nucleoside intermediate. In addition, this crystal is suitable for industrial production because it does not require a purification step by chromatography.
- a crosslinked nucleoside can be synthesized from the crystals of the compound represented by the formula 5 of the present disclosure by the following steps 6 to 10 in accordance with a known description such as Non-Patent Document 2.
- Step 6 After suspending the crystals of the compound represented by the formula 5 in a solvent, the isopropylidene group of the compound is converted into an acetyl group to obtain the compound represented by the formula 6.
- Step 7 Formula 6
- a cyclization reaction is carried out at the same time as the removal of the protective group of the compound represented by the formula 7 to obtain the compound represented by the formula 7 by condensing the compound represented by the above formula with a silylated base.
- Step 9 A step of removing the hydroxyl group-protecting group of the compound represented by the formula 8 to obtain a compound represented by the formula 9
- Step 10 A step of obtaining the compound represented by the formula 9 If necessary, the compound represented by the formula 9
- R 1 is a hydroxyl-protecting group
- R 2 is a leaving group
- R 3 is a hydrogen atom or amino-protecting group.
- Base in the above formula can be a base such as 2-thiouracil, 5-propynyluracil, 5-propynylcytosine, 2,6-diaminopurine in addition to thymine, uracil, adenine, cytosine, 5-methylcytosine, and guanine.
- these bases can undergo further chemical reactions during the process to be converted to other bases.
- the amino group on these bases may be protected by an acyl group such as an acetyl group, a phenoxyacetyl group, a benzoyl group or an isobutyryl group or a protecting group such as a dimethylformamidino group.
- Non-Patent Document 2 can be converted into the corresponding cross-linked nucleoside amidite.
- the method for producing a crosslinked nucleoside of the present disclosure uses a crystal of a compound represented by the formula 5 that can be stably stored as a crosslinked nucleoside intermediate, various crosslinked nucleosides can be used as needed. Can be manufactured.
- sodium periodate 30.1 g, 30.1 g, 50 mL of a 0.141 mol
- aqueous solution (253 mL, dissolved by heating at 60 ° C.) was added.
- 30 mL and the rest were added over 5 minutes and stirred at the same temperature for 3 hours.
- the reaction mixture was cooled to 0 ° C., stirred for 45 minutes, and the precipitated solid (crystal) was collected by filtration.
- the residue was dissolved in pyridine (dehydrated, 119 mL), 4-toluenesulfonyl chloride (22.7 g, 0.119 mol) was added, and the mixture was stirred at 30 ° C. for 17 hours.
- deionized water (10 mL) and stirring the reaction solution was concentrated. Ethyl acetate was added to the residue, and the mixture was washed with saturated aqueous sodium hydrogen carbonate solution.
- the thermal weight measurement / differential thermal analysis (TG / DTA) machine used was a thermal analyzer STA7200 (manufactured by Hitachi High-Tech Science Co., Ltd.), and the analysis conditions were as follows. Analytical conditions: The temperature was raised by 5 ° C. per minute in the range of 30 to 190 ° C., and the change in calorific value of the sample was measured. Aluminum oxide was used as a reference.
- Step for Obtaining Compound Formula C from Compound Formula b A compound represented by formula c was produced from a compound represented by formula b according to the method described in Non-Patent Document 3.
- the TLC result of the crystal of the compound 3A is shown in FIG. 5 (a), and the TLC result of the solution of the compound represented by the formula c described in Non-Patent Document 3 is shown in FIG. 5 (a). It was confirmed that when compound 3A was synthesized by the above method, the target crystal was precipitated as the reaction proceeded, and a highly pure compound 3A could be obtained by simply filtering the crystal without performing a chromatography operation. .. On the other hand, in the reaction mixture of the compounds represented by the formula c synthesized under the conditions described in Non-Patent Document 3, production of by-products was observed in addition to the compounds represented by the formula c. Since the compound represented by the formula c is non-crystalline, it has become clear that a chromatographic operation is required when further purification is performed to remove this by-product.
- Example 2 Using the compound 5A produced in Example 1 as a raw material, various crosslinked nucleoside amidites of thymine, adenine, 5-methylcytosine, and guanine were produced.
- Example 2-1 Production of thymine-crosslinked nucleoside amidite A thymine-crosslinked nucleoside amidite 11T was synthesized from compound 5A.
- Acetonitrile (105 mL) was added to N, O-bis (trimethylsilyl) acetamide (33.9 mL, 0.139 mol) at .1 mmol), and the mixture was stirred at 85 ° C. for 1 hour.
- reaction mixture was cooled to 0 ° C., trimethylsilyl trifluoromethanesulfonate (9.89 mL, 54.7 mmol) was added, and the mixture was stirred at 85 ° C. for 4 hours.
- the reaction mixture was cooled to 0 ° C., saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was stirred.
- the precipitated solid was removed by filtration through Celite and then extracted with ethyl acetate (remaining timine can be removed by washing the ethyl acetate layer with a 1 M aqueous sodium hydroxide solution and then a saturated aqueous solution of ammonium chloride).
- the organic layer was dried over anhydrous magnesium sulfate and concentrated to give compound 7T.
- Compound 8T (7.13 g, 11.7 mmol), ammonium formate (7.56 g, 0.12 mol) in methanol (120 mL), It was dissolved in ethyl acetate (120 mL), 20% by mass of palladium hydroxide-activated carbon (20% by mass Pd, 50% by mass of water) (3.57 g) was added, and the mixture was stirred at 60 ° C. for 4 hours.
- Step to obtain compound 10T from compound 9T Compound 9T (4.23 g, 15.7 mmol) is dissolved in pyridine (dehydrated, 52.3 mL), dimethoxytrityl chloride (7.45 g, 22.0 mmol) is added, and 4 at room temperature. Stirred for hours. Methanol (5 mL) was added to the reaction mixture, and the mixture was stirred and concentrated. Ethyl acetate was added to the residue, washed with saturated aqueous sodium hydrogen carbonate solution, and the organic layer was dried over anhydrous magnesium sulfate and concentrated.
- N 6- Toluene 85 mL, 0.4 mol / L
- benzoyl adenine 12.1 g, 50.6 mmol
- N, O-bis (trimethylsilyl) acetamide 25 mL, 101 mmol
- ammonium formate (21.9 g, 348 mmol) and deionized water (37 mL) were added, and the mixture was stirred at 60 ° C. until dissolved.
- palladium hydroxide-activated carbon (2.9 g) was added at room temperature, and the mixture was stirred at 60 ° C. for 17 hours.
- Base (R 3 ) N 6 -benzoyladenine
- Step to Obtain Compound 11A from Compound 10A Pyridine (77 mL, 0.3 mol / L) was added to Compound 10A that had undergone azeotropic dehydration (pyridine 70 mL, 3 times), and then dimethoxytrityl chloride (dimethoxytrityl chloride) was stirred at room temperature. 9.6 g (28.2 mmol) was added, and the mixture was stirred at the same temperature for 1 hour. Subsequently, dimethoxytrityl chloride (11.8 g, 34.8 mmol) was added, and the mixture was stirred for 12 hours.
- Acetic anhydride (30.0 g, 42.1 mmol) was added to compound 5A.
- Sodium acetate (1.50 g, 18.3 mmol) was added to the reaction mixture, and the mixture was stirred until completely dissolved and then concentrated.
- Compound 6A was dissolved in acetonitrile (ultra-dehydrated) (105 mL) and N 4 -benzoyl-5-methylcytosine (12.7 g, 55.4 mmol), N, O-bis (trimethylsilyl) acetamide (29.8 mL, 0.122 mol). was added, and the mixture was heated and stirred at 85 ° C. for 1 hour.
- reaction mixture was ice-cooled, trimethylsilyl trifluoromethanesulfonate (11.4 mL, 63.1 mmol) was added, and the mixture was heated and stirred at 85 ° C. for 8 hours.
- the reaction mixture was ice-cooled, saturated aqueous sodium hydrogen carbonate solution (150 mL) was added, and the mixture was stirred until foaming subsided. Extraction was performed with ethyl acetate (150 mL), and the organic layer was dried over anhydrous magnesium sulfate and concentrated to obtain Compound 7C.
- Compound 7C was dissolved in tetrahydrofuran (21 mL), and methanol (211 mL) and water were added.
- Sodium oxide (8.42 g, 0.211 mol) was added, and the mixture was heated and stirred at 40 ° C. for 14 hours.
- the reaction mixture was concentrated to a small amount, diluted with ethyl acetate (200 mL), and washed with 1 mol / L hydrochloric acid (100 mL) and saturated aqueous sodium hydrogen carbonate solution (100 mL).
- Compound 8C (16.07 g, 26.46 mmol) was dissolved in tetrahydrofuran (142 mL) and methanol (142 mL) to dissolve formic acid. Ammonium (16.8 g, 0.266 mol) was added and dissolved. 1 mol / L hydrochloric acid (26.4 mL, 26.4 mmol) and palladium hydroxide-activated carbon (8.04 g) were added, and the mixture was heated and stirred at 60 ° C. for 23 hours.
- Compound 9C (9.15 g, 34.0 mmol) is pyridine (dehydrated) (dehydrated). It was dissolved in 118 mL), benzoic anhydride (15.4 g, 68.1 mmol) was added, and the mixture was stirred at room temperature for 3 days. Ethanol (116 mL) and a 2 mol / L sodium hydroxide aqueous solution (174 mL, 0.348 mol) were added to the reaction solution, and after stirring for 1 hour, acetic acid (23 mL) was added.
- Step to Obtain Compound 11C from Compound 10C Compound 10C (7.00 g, 18.7 mmol) was azeotroped with pyridine three times and then dissolved in pyridine (dehydrated) (62.3 mL). Dimethoxytrityl chloride (8.24 g, 24.3 mmol) was added, and the mixture was stirred at room temperature for 2 hours, and then methanol (5 mL) was added to the reaction solution for concentration. Ethyl acetate (200 mL) was added to the residue, and the mixture was washed with saturated aqueous sodium hydrogen carbonate solution (50 mL).
- Step to Obtain Compound 12C from Compound 11C Compound 11C (11.50 g, 17.02 mmol) was dissolved in dichloromethane (super-dehydrated) (85.1 mL), and N, N-diisopropylethylamine (6.02 mmol, 35.2 mmol), (2-Cyanoethyl) (N, N-diisopropyl) chlorophosphoroamidite (6.83 mL, 30.6 mmol) was added, and the mixture was stirred at room temperature for 2 hours.
- Acetic anhydride (30.0 g, 42.1 mmol) was added to compound 5A.
- Sodium acetate (1.50 g, 18.3 mmol) was added to the reaction mixture, and the mixture was stirred until completely dissolved and then concentrated.
- reaction mixture was ice-cooled, trimethylsilyl trifluoromethanesulfonate (15.2 mL, 84.1 mmol) was added, and the mixture was heated and stirred at 85 ° C. for 7 hours.
- the reaction mixture was ice-cooled, saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was stirred until foaming subsided.
- the mixture was extracted with ethyl acetate (150 mL), the organic layer was washed with 1 M aqueous sodium hydroxide solution, dried over anhydrous magnesium sulfate, and concentrated to obtain Compound 7G.
- Compound 7G was dissolved in tetrahydrofuran (105 mL) and methanol (105 mL) was dissolved in methanol (105 mL). 421 mL) and a 25% by volume sodium methoxide methanol solution (80.9 mL) were added, and the mixture was heated and stirred at room temperature for 24 hours. The reaction solution was neutralized with 2M hydrochloric acid and then concentrated. Ethyl acetate was added to the residue, and the mixture was washed with saturated aqueous sodium hydrogen carbonate solution.
- Compound 8G was dissolved in tetrahydrofuran (213 mL) and methanol (213 mL), and ammonium formate (26.9 g) was dissolved. , 0.427 mol) was added and dissolved.
- Palladium hydroxide-activated carbon (13.5 g) was added, and the mixture was heated and stirred at 60 ° C. for 30 hours. Palladium hydroxide-activated carbon was removed by Celite filtration and washed with methanol (100 mL ⁇ 5).
- Compound 9G was dissolved in 100 mM Tris-HCl (pH 7.5) (420 mL), and adenosine deaminase (37 ⁇ L, 42 units) was added. The mixture was stirred at 40 ° C. for 24 hours. Adenosine deaminase (37 ⁇ L, 42 units) was added and stirred at the same temperature for an additional 24 hours. The precipitated solid was collected by filtration, washed with water, and vacuum dried to obtain 10 G of compound (8.50 g, 28.8 mmol, 68.4%).
- the residue was dissolved in pyridine (8.0 mL), ice-cooled isobutyryl chloride (336 ⁇ L, 3.18 mmol) was added, and the mixture was stirred at room temperature for 20 hours. After adding deionized water and stirring, the reaction solution was concentrated. Ethyl acetate was added to the residue, washed with saturated aqueous sodium hydrogen carbonate, and the organic layer was dried over anhydrous magnesium sulfate and concentrated.
- the residue was dissolved in methanol (10.0 mL), acidic ammonium hydrogen fluoride (860 mg, 15.0 mmol) was added, and the mixture was heated and stirred at 60 ° C. for 17 hours.
- Step to Obtain Compound 12G from Compound 11G Compound 11G (1.87 g, 5.12 mmol) was azeotroped with pyridine three times and then dissolved in pyridine (dehydrated) (17.1 mL). Dimethoxytrityl chloride (2.43 g, 7.17 mmol) was added, and the mixture was stirred at room temperature for 2 hours, and then methanol (1 mL) was added to the reaction solution for concentration. Ethyl acetate (200 mL) was added to the residue, and the mixture was washed with saturated aqueous sodium hydrogen carbonate solution.
- Step to Obtain Compound 13G from Compound 12G Compound 12G (3.20 g, 4.79 mmol) was dissolved in dichloromethane (super-dehydrated) (24.0 mL), and N, N-diisopropylethylamine (2.03 mL, 11.9 mmol), (2-Cyanoethyl) (N, N-diisopropyl) chlorophosphoroamidite (2.35 mL, 10.5 mmol) was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution was washed with saturated aqueous sodium hydrogen carbonate solution. The organic layer was dried over anhydrous magnesium sulfate and then concentrated.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
今西らは、ヌクレオシド糖部の4’位と2’位水酸基を架橋することにより、ヌクレオシドのコンフォメーションを強制的にN型に固定することに成功した。結果として、この架橋型ヌクレオシドを含むLNA(Locked Nucleic Acids)は、相補的な配列の核酸と極めて安定な2重鎖を形成することが明らかとなった(特許文献1参照)。
上記の特性等から、近年核酸医薬品の素材として、LNAに対する期待が高まっている。
すなわち、本開示の課題は、長期間に渡って安定的に保管することができる架橋型ヌクレオシド中間体として用いることが可能な化合物の結晶及びその製造方法を提供することである。また、本開示の課題は、当該化合物の結晶を用いる架橋型ヌクレオシドアミダイトの製造方法を提供することである。
[1]以下の式5で表される化合物の結晶。
工程1:式1で表される化合物の水酸基を保護し、式2で表される化合物を得る工程、
工程2:式2で表される化合物の4位ジメチルジオキソラニル基をアルデヒド基へと変換し、式3で表される化合物を得る工程、
工程3:式3で表される化合物を還元して4位アルデヒド基を水酸基とし、式4で表される化合物を得る工程、
工程4:式4で表される化合物の4位水酸基を脱離基へと変換し、式5で表される化合物を得る工程、
工程5:式5で表される化合物を結晶化溶媒から結晶化し、式5で表される化合物の結晶を得る工程
工程6:[5]~[7]のいずれか1つの製造方法によって得られる式5で表される化合物の結晶を溶媒に懸濁させた後、該化合物のイソプロピリデン基をアセチル基へと変換し、式6で表される化合物を得る工程
工程7:式6で表される化合物をシリル化した塩基と縮合し、式7で表される化合物を得る工程
工程8:式7で表される化合物の保護基の除去と同時に環化反応を行い、式8で表される化合物を得る工程
工程9:式8で表される化合物の水酸基保護基を除去し、式9で表される化合物を得る工程
工程10:必要に応じて式9で表される化合物の塩基部のアミノ基に保護基を導入し、式10で表される化合物を得る工程
本明細書における粉末X線回折は、以下の条件で行うものとする。
[X線解析装置]X’Pert PRO MPD(スペクトリス)
[ターゲット]Cu
[X線管電流]40mA
[X線管電圧]45kV
[走査範囲]2θ=4.0~40°
[使用機器]熱分析装置STA7200(日立ハイテクサイエンス)
[測定条件]30~190℃の範囲を1分間あたり5℃ずつ昇温し、試料の熱量変化を測定する。リファレンスとして酸化アルミニウムを用いる。
このとき、本開示の結晶は、前記加速試験後にあってもHPLC純度90%以上を示し、好ましくは95%以上、更に好ましくは98%以上、更に好ましくは99%以上、更に好ましくは99.5%以上を示す(図4参照)。
工程1:式1で表される化合物の水酸基を保護し、式2で表される化合物を得る工程、
工程2:式2で表される化合物の4位ジメチルジオキソラニル基をアルデヒド基へと変換し、式3で表される化合物を得る工程、
工程3:式3で表される化合物を還元して4位アルデヒド基を水酸基とし、式4で表される化合物を得る工程、
工程4:式4で表される化合物の4位水酸基を脱離基へと変換し、式5で表される化合物を得る工程、
工程5:式5で表される化合物を結晶化溶媒から結晶化し、式5で表される化合物の結晶を得る工程
脱保護剤の添加量は脱保護材の種類によって適宜設定可能であるが、例えば酢酸の場合であれば溶媒量を用いることが好ましい。
また、後述するように、結晶化による精製が容易であることから、水を補助溶媒として用いることが好ましい。脱保護剤と補助溶媒との比率は体積基準で10:1~1:1程度が好ましく、2:1がより好ましい。
この条件を用いる場合、0~100℃で反応を行うことができる。反応温度は、特に50~80℃程度がより好ましい。上記条件にて、1~24時間反応させればよい。
当該組み合わせを用いることにより、過剰なヨウ素等が反応系に存在せず、反応後の溶媒の廃棄が容易である等、工業的な生産に適している。また、式1で表される化合物を用いることにより、クロマトグラフィー等の精製手段を用いることなく、結晶化により70~90%以上という高い収率で目的の式3で表される化合物を得ることができる。
このとき、反応の進行に伴って目的化合物が結晶として析出するので、過剰反応による分解で副生成物が生成するのを防ぐことができ、精製効果も高い。そのため、工程2においては、クロマトグラフィーによる煩雑な精製操作を行わずとも不純物の副生及び混入を抑制することができる(図5(ア)参照)。
一方で、非特許文献3に記載された方法によって、上記の式bで表される化合物から式cで表される化合物を合成した場合には、不純物がより多く副生してしまう(図5(イ)参照)。さらに、式cで表される化合物は非結晶性であるため、精製を行う場合にはクロマトグラフィーによる煩雑な精製操作が必要となる。
工程6:式5で表される化合物の結晶を溶媒に懸濁させた後、該化合物のイソプロピリデン基をアセチル基へと変換し、式6で表される化合物を得る工程
工程7:式6で表される化合物をシリル化した塩基と縮合し、式7で表される化合物を得る工程
工程8:式7で表される化合物の保護基の除去と同時に環化反応を行い、式8で表される化合物を得る工程
工程9:式8で表される化合物の水酸基保護基を除去し、式9で表される化合物を得る工程
工程10:必要に応じて式9で表される化合物の塩基部のアミノ基に保護基を導入し、式10で表される化合物を得る工程
これら塩基上のアミノ基はアセチル基、フェノキシアセチル基、ベンゾイル基、イソブチリル基、といったアシル基やジメチルホルムアミジノ基といった保護基で保護されていても良い。
<化合物5Aの結晶の製造>
化合物1Aを出発原料として、以下の工程によって化合物5Aの結晶を製造した。なお、式中、「BPMO」及び「OMPB」はともにBPM基が結合したO基を表す。また、「BPM」はブロモ(フェニル)メチル(4-ブロモベンジルとも称される)を表す。
化合物1A(20.43g、70.37mmol)をジメチルアセトアミド(脱水、352mL)に溶解し、0℃に冷却した。水素化ナトリウム(60質量%油性、7.04g、0.176mol)を加え、1時間撹拌後、臭化4-ブロモベンジル(44.0g、0.176mol)を加え、室温で17時間撹拌した。反応液にメタノール(10mL)を加え撹拌後、濃縮した。残渣に酢酸エチルを加え水洗後、有機層を無水硫酸マグネシウム上乾燥、濃縮し、化合物2Aを得た。
化合物2Aを酢酸(507mL)に60℃で加熱溶解後、過ヨウ素酸ナトリウム(30.1g、0.141mol)水溶液(253mL、60℃で加熱溶解)のうち50mLを添加した。5分後に30mL、さらに残りを5分間かけて添加し、同温度で3時間撹拌した。反応液を0℃に冷却して45分間撹拌後、析出した固体(結晶)をろ取した。酢酸:脱イオン水(体積比で2:1)、次いで脱イオン水で洗浄後、真空乾燥し、化合物3Aの結晶(33.10g、59.51mmol、84.57%)を得た。
1H-NMR(CDCl3,400MHz);δ9.89(1H,s),7.48-7.44(4H,m),7.20-7.09(4H,m),5.86(1H,d),4.66(1H,d),4.63(1H,t),4.52(1H,s),4.47(1H,d),4.41(1H,d),4.31(1H,d),3.66(1H,d),3.60(1H,d),1.60(3H,s),1.36(3H,s).
化合物3Aの結晶(33.10g、59.51mmol)をメタノール(298mL)、テトラヒドロフラン(298mL)に懸濁し、0℃に冷却した。水素化ホウ素ナトリウム(563mg、14.9mmol)を少量ずつ添加し、5分間撹拌後、さらに同量の水素化ホウ素ナトリウム(563mg、14.9mmol)を添加し、2時間撹拌した。反応液を少量にまで濃縮後、残渣に酢酸エチルを加え、飽和炭酸水素ナトリウム水溶液で洗浄した。有機層を無水硫酸マグネシウム上乾燥、濃縮し、化合物4Aを得た。
化合物4Aをピリジンで3回共沸後、残渣をピリジン(脱水、119mL)に溶解し、塩化4-トルエンスルホニル(22.7g、0.119mol)を加え、30℃で17時間撹拌した。脱イオン水(10mL)を加え撹拌後、反応液を濃縮した。残渣に酢酸エチルを加え、飽和炭酸水素ナトリウム水溶液で洗浄した。有機層を無水硫酸マグネシウム上乾燥、濃縮後、残渣をトルエンで3回共沸した。固化した残渣を酢酸エチル(50mL)に溶解し、ヘキサン(100mL)を少量ずつ添加した(結晶析出)。さらにヘキサンを(100mL)添加し、終夜静置した。析出した固体をろ取、ヘキサンで洗浄後、真空乾燥し、化合物5Aの結晶(32.22g、45.20mmol、75.95%)を得た。
1H-NMR(CDCl3,400MHz);δ7.77(2H,d),7.45(2H,d),7.44(2H,d),7.29(2H,d),7.13(2H,d),7.08(2H,d),5.68(1H,d),4.63(1H,d),4.54(1H,dd),4.44(1H,d),4.43(1H,d),4.38(1H,d),4.32(1H,d),4.14(1H,d),3.51(1H,d),3.48(1H,d),2.42(3H,s),1.29(3H,s),1.27(3H,s).
上記で得られた化合物5Aの結晶について下記(1)~(4)の物性を測定した。
化合物5Aの結晶の結晶形をデジタルマイクロスコープで観察した。使用したデジタルマイクロスコープはDino-Lite AD-4113ZT(AnMo Electronics Corporation社製)、観察倍率は230倍を用いた。
化合物5Aの結晶を、粉末X線分析に供した。使用した粉末X線分析機はスペクトリス株式会社製、分析条件は以下の通りであった。
[X線解析装置]X’Pert PRO MPD(スペクトリス株式会社)
[ターゲット]Cu
[X線管電流]40mA
[X線管電圧]45kV
[走査範囲]2θ=4.0~40°
化合物5Aは、図2に示す回折角(2θ)付近に特徴的なピークを示した。
化合物5Aの結晶を、熱重量測定/示差熱分析(TG/DTA)に供した。使用した熱重量測定/示差熱分析(TG/DTA)機は、熱分析装置STA7200(日立ハイテクサイエンス社製)、分析条件は以下の通りであった。
分析条件:30~190℃の範囲を1分間あたり5℃ずつ昇温し、試料の熱量変化を測定した。リファレンスとして酸化アルミニウムを用いた。
化合物5Aの結晶は、124℃に吸熱ピークを示した。
化合物5Aの結晶を、安定性の加速試験に供した。当該加速試験の条件は、以下の通りである。
化合物5Aの結晶50mgをガラス製サンプル瓶に取り、80℃で7日間加熱した。試料1mgを5mMトリエチルアンモニウムアセテート-70体積%アセトニトリル1mLに溶解し、高速液体クロマトグラフィーにより純度を測定した。
また、比較対象として、化合物5Aのアセトニトリル溶液を同条件にて加速試験に供した。化合物5Aの結晶10mgをアセトニトリル1mLに溶解し、80℃で7日間加熱した。試料溶液を5mMトリエチルアンモニウムアセテート-70体積%アセトニトリルで10倍に希釈し、高速液体クロマトグラフィーにより純度を測定した。
結果を図4に示す。
このことから、化合物5Aは、結晶状態で保管する限り、特別な管理を行わない場合であっても、安定的に長期間保管できることが分かった。一方で、化合物5Aであっても、既存の架橋型ヌクレオシド中間体の合成法と同じく、溶液状態では中間体を長期間安定的に保管することができないことがわかった。
上記の方法で得た化合物3Aの結晶と、非特許文献3に記載された条件で合成された式cで表される化合物の溶液の性状とを比較した。
非特許文献3に記載の方法に従い、式bで表される化合物から式cで表される化合物を製造した。
TLCプレート:Merck社製TLC Silica gel 60 F254
展開溶媒:ヘキサン:酢酸エチル=2:1(体積比)
上記の方法で化合物3Aを合成すると、反応の進行に伴い目的物結晶が析出し、これをろ過するのみで、クロマトグラフィー操作を行うことなく、純度の高い化合物3Aが得られることが確認された。
これに対し、非特許文献3に記載の条件にて合成された式cで表される化合物の反応混合液では、式cで表される化合物の他に副生成物の生成が見られた。式cで表される化合物は非結晶性のため、この副生成物を除去するため更なる精製を行う場合、クロマトグラフィー操作が必要であることが明らかとなった。
実施例1にて製造した化合物5Aを原料として用い、チミン、アデニン、5-メチルシトシン、グアニンの各種架橋型ヌクレオシドアミダイトを製造した。
化合物5Aからチミン架橋型ヌクレオシドアミダイト11Tを合成した。
化合物5A(33.10g、59.51mmol)を酢酸(366mL)、無水酢酸(45.0mL)に懸濁し、硫酸(0.393mL)の酢酸溶液(30.0mL)を加え、室温で2時間撹拌した。反応液に酢酸ナトリウム(1.50g)を添加、撹拌後、濃縮し、残渣をトルエンで5回共沸した。残渣に酢酸エチルを加え、飽和炭酸水素ナトリウム水溶液で洗浄した。有機層を無水硫酸マグネシウム上乾燥、濃縮し、化合物6Aを得た。
化合物6A、チミン(7.96g、63.1mmol)、N,O-ビス(トリメチルシリル)アセトアミド(33.9mL、0.139mol)にアセトニトリル(105mL)を加え、85℃で1時間撹拌した。反応液を0℃に冷却後、トリフルオロメタンスルホン酸トリメチルシリル(9.89mL、54.7mmol)を加え、85℃で4時間撹拌した。反応液を0℃に冷却後、飽和炭酸水素ナトリウム水溶液を加え撹拌した。析出した固体をセライトろ過により除去後、酢酸エチルで抽出した(酢酸エチル層を1M水酸化ナトリウム水溶液、次いで飽和塩化アンモニウム水溶液で洗浄すると、残存するチミンを除去できる)。有機層を無水硫酸マグネシウム上乾燥、濃縮し、化合物7Tを得た。
化合物7Tにメタノール(211mL)を加え、水酸化ナトリウム(8.42g、0.211mol)を加え、40℃で3時間撹拌した。反応液に飽和塩化アンモニウム水溶液を加え、酢酸エチルで抽出した。有機層を無水硫酸マグネシウム上乾燥、濃縮後、残渣にエタノール(150mL)を加え、溶解するまで酢酸エチルを添加した。減圧下、酢酸エチルを留去し、析出した固体をろ取、エタノールで洗浄した。得られた固体を真空乾燥し、化合物8T(20.00g、32.88mmol、78.1%)を得た。
1H-NMR(CDCl3,400MHz);δ8.39(1H,s),7.49(2H,d),7.46(2H,d),7.46(1H,d),7.18(2H,d),7.14(2H,d),5.64(1H,s),4.62-4.45(5H,m),4.02-3.78(5H,m),1.70(3H,d).
化合物8T(7.13g、11.7mmol)、ギ酸アンモニウム(7.56g、0.12mol)をメタノール(120mL)、酢酸エチル(120mL)に溶解し、20質量%水酸化パラジウム-活性炭素(20質量%Pd、50質量%含水)(3.57g)を加え、60℃で4時間撹拌した。触媒をセライトろ過により除去後、ろ液を濃縮し、シリカゲルカラムクロマトグラフィー(シリカゲル150mL、クロロホルム:メタノール=10:1(体積比))により精製し、化合物9T(3.22g、11.9mmol、102%)を得た。
1H-NMR(D2O,400MHz);δ7.66(1H,s),5.67(1H,s),4.49(1H,s),4.22(1H,s),4.05-4.03(3H,m),3.96(1H,d),1.91(3H,s).
化合物9T(4.23g、15.7mmol)をピリジン(脱水、52.3mL)に溶解し、塩化ジメトキシトリチル(7.45g、22.0mmol)を加え、室温で4時間撹拌した。反応液にメタノール(5mL)を加え撹拌後、濃縮した。残渣に酢酸エチルを加え、飽和炭酸水素ナトリウム水溶液で洗浄後、有機層を無水硫酸マグネシウム上乾燥、濃縮した。残渣をトルエンで3回共沸後、シリカゲルカラムクロマトグラフィー(シリカゲル250mL、ヘキサン:酢酸エチル=1:1~1:2(体積比)~酢酸エチル)により精製し、化合物10T(8.99g、15.7mmol、100%)を得た。
1H-NMR(CDCl3,400MHz);δ8.54(1H,s),7.65-6.84(14H,m),5.63(1H,s),4.43(1H,s),4.28(1H,d),3.88(2H,d),3.81(2H,d),3.80(6H,s),3.58(2H,d),3.47(2H,d),2.31(1H,d),1.70(3H,s).
化合物10T(4.50g、7.86mmol)、N,N-ジイソプロピルエチルアミン(3.86mL、17.3mmol)をジクロロメタン(脱水、39.3mL)に溶解し、0℃に冷却した。2-シアノエチルジイソプロピルクロロホスホロアミジド(3.86mL、17.3mmol)を加え、室温で1時間撹拌した。反応液を飽和炭酸水素ナトリウム水溶液で洗浄後、有機層を無水硫酸マグネシウム上乾燥、濃縮した。残渣をシリカゲルカラムクロマトグラフィー(シリカゲル200mL、ヘキサン:酢酸エチル=1:1(体積比))により精製し、化合物11T(6.00g、7.76mmol、98.7%)を得た。
31P-NMR(CDCl3,160MHz);δ149.94,149.80.
化合物5A(24.0g、33.7mmol)を酢酸(340mL、0.1mol/L)に懸濁し、室温で撹拌しながら無水酢酸(35mL、262mmol)、硫酸(0.32mL、6.07mmol)の酢酸溶液(371mL)を順次加え、同温で3時間撹拌した。反応終了後、同温で 酢酸ナトリウム(1.2g、14.5mmol)を加えた後、同温で5分撹拌後、濃縮した。残渣を、トルエン(340mL)で3回共沸した。得られた残渣を酢酸エチル(400mL)に溶解させた後、飽和重曹水(100mL)による洗浄を3回実施した。水層に対し、酢酸エチル(400mL)による抽出を1回行った後、得られたすべての有機層を合わせ、硫酸マグネシウムによる乾燥と溶媒の減圧留去を順次行い、化合物6Aを得た。
化合物6A、N6-ベンゾイルアデニン(12.1g、50.6mmol)にトルエン(85mL、0.4mol/L)を加えた後、室温で撹拌しながらN,O-ビス(トリメチルシリル)アセトアミド(25mL、101mmol)を加え、加熱還流下で1時間撹拌した。続いて、0℃に降温した後、同温でトリフルオロメタンスルホン酸トリメチルシリル(7.9mL、43.8mmol)を添加し、加熱還流下で2時間撹拌した。反応終了後、0℃で酢酸エチル(300mL)と1mol/L水酸化ナトリウム水溶液(100mL)を加え、同温で5分撹拌した。続いて、析出した固体をろ過により除去した後、有機層と水層の分離を行った。得られた有機層を、1mol/L水酸化ナトリウム水溶液(50mL)で3回洗浄後、硫酸マグネシウムによる乾燥と溶媒の減圧留去を順次行い、化合物7Aを得た。
化合物7Aに対し、メタノール:テトラヒドロフラン(9:1(体積比)、170mL)を加えた後、水酸化ナトリウム(6.7g、169mmol)を加え、40℃で1時間撹拌した。反応終了後、同温で飽和塩化アンモニウム水溶液(170mL)を加えた後、同温で5分撹拌した。続いて、酢酸エチル(200mL)を加え、撹拌した後、析出している固体をろ過により除去した。ろ液の濃縮後、残渣を酢酸エチル(400mL)に溶解させた後、有機層と水層の分離を行った。得られた有機層に対して脱イオン水(100mL)による洗浄を1回行った後、硫酸マグネシウムによる乾燥と溶媒の減圧留去を順次行った。得られた残渣に対し、メタノール(67mL、0.5mol/L)を加え、加熱還流下で撹拌することで残渣を完全に溶解させた。その後、室温に降温し、終夜撹拌することで析出した結晶を回収した。得られた結晶をメタノール(20mL)で2回洗浄し、化合物8A(14.3g、23.2mmol、68.7%)を得た。
1H-NMR(CDCl3, 400MHz); δ8.33(1H,s),7.99(1H,s), 7.47(2H,d),7.41(2H,d),7.18(2H,d),7.10(2H,d), 6.03(1H,s), 5.63(2H,brs), 4.87(1H,s),4.58(1H,d), 4.57(2H,dd), 4.48(1H,d), 4.26(1H,s), 4.11(1H,d),3.99(1H,d),3.81(2H,dd).
化合物8A(14.3g、23.2mmol)に対し、酢酸エチル:メタノール(1:3(体積比)、600mL)を加えた後、ギ酸アンモニウム(21.9g、348mmol)と脱イオン水(37mL)を加え、60℃で溶解するまで撹拌した。その後、室温にて水酸化パラジウム-活性炭素(2.9g)を加え、60℃で17時間撹拌した。続いて、ギ酸アンモニウム(7.3g、116mmol)と水酸化パラジウム-活性炭素(0.7g)を追加し、60℃で24時間撹拌した。反応終了後、セライトろ過により触媒を除去後、溶媒の減圧留去を行い、化合物9Aを得た。
共沸脱水(ピリジン 100mL、3回)を行った化合物9Aに対し、ピリジン(116mL、0.2mol/L)を加えた後、0℃でクロロトリメチルシラン(20.6mL、162mmol)を加え、同温で30分撹拌した。次に、塩化ベンゾイル(13.5mL、116mmol)を同温で添加し、21時間撹拌した。続いてメタノール(116mL)を添加した後、室温で5分撹拌し、その後、アンモニア水(34mL)を加え、同温で2.5時間撹拌した。反応終了後、析出していた固体をろ過により除去した後、溶媒を減圧留去した。その後、ピリジン(100mL)を添加し、5分撹拌した後、溶解しなかった固体をろ過により除去した。溶媒を減圧留去し、化合物10Aを得た。
共沸脱水(ピリジン70mL、3回)を行った化合物10Aに対し、ピリジン(77mL、0.3mol/L)を加えた後、室温で撹拌しながら塩化ジメトキシトリチル(9.6g、28.2mmol)を加え、同温で1時間撹拌した。続いて、塩化ジメトキシトリチル(11.8g、34.8mmol)を加え、12時間撹拌した。反応終了後、メタノール(80mL)を加え、同温で5分撹拌した後、溶媒の減圧留去を行った。得られた残渣を酢酸エチル(320mL)に溶解させた後、脱イオン水(80mL)による洗浄を1回実施した。硫酸マグネシウムによる乾燥と溶媒の減圧留去を順次行った後、中圧シリカゲルカラムクロマトグラフィー(SiO2100g、酢酸エチル:ヘキサン=66:34(体積比)~酢酸エチル)により精製し、化合物11A(9.41g、13.7mmol、59.1%)を得た。
1H-NMR(CDCl3, 400MHz); δ9.10(1H,s),8.77(1H,s),8.28(1H,s),8.02(2H,d),7.63-7.21(12H,m),6.85(4H,d),6.13(1H,s),4.44(1H,d),4.04(2H,s),3.61(1H,d),3.56(1H,d),2.65(1H,d).
共沸脱水(トルエン70mL、3回)を行った化合物11A(9.41g、13.7mmol)に対しジクロロメタン(70mL)とN,N-ジイソプロピルエチルアミン(6.0mL、34.3mmol)を順次加えた後、0℃で撹拌しながら(2-シアノエチル)(N,N-ジイソプロピル)クロロホスホロアミダイト(6.7mL、30.1mmol)を加え、室温で2.5時間撹拌した。反応終了後、0℃で飽和重曹水(50mL)を加え、同温で5分撹拌した後、有機層と水層の分離を行った。水層に対して酢酸エチル(70mL)による抽出を1回行った後、得られたすべての有機層を合わせ、硫酸マグネシウムによる乾燥と溶媒の減圧留去を順次行った。その後、中圧シリカゲルカラムクロマトグラフィー(SiO2100g、酢酸エチル:ヘキサン=69:31~90:10(体積比))により精製し、化合物12A(9.4g、10.6mmol、77.0%)を得た。
31P-NMR(MeCN-d3,160MHz);δ149.54,149.04.
化合物5Aから5-メチルシトシン架橋型ヌクレオシドアミダイト12Cを合成した。
化合物5A(30.0g、42.1mmol)を酢酸(366mL)、無水酢酸(45.0mL)に懸濁し、濃硫酸(396μL)の酢酸溶液(30.0mL)をゆっくりと滴下後、室温で2時間撹拌した。反応液に酢酸ナトリウム(1.50g、18.3mmol)を加え、完全に溶解するまで撹拌後、濃縮した。残渣をトルエン-酢酸エチル(体積比で2:1)で共沸(40mL×5)後、残渣に酢酸エチル(300mL)を加え、飽和炭酸水素ナトリウム水溶液(150mL)で洗浄した。有機層を無水硫酸マグネシウム上乾燥後、濃縮した。残渣をトルエンで共沸(20mL×3)し、化合物6Aを得た。
化合物6Aをアセトニトリル(超脱水)(105mL)に溶解し、N4-ベンゾイル-5-メチルシトシン(12.7g、55.4mmol)、N,O-ビス(トリメチルシリル)アセトアミド(29.8mL、0.122mol)を加え、85℃で1時間加熱撹拌した。反応液を氷冷後、トリフルオロメタンスルホン酸トリメチルシリル(11.4mL、63.1mmol)を加え、85℃で8時間加熱撹拌した。反応液を氷冷後、飽和炭酸水素ナトリウム水溶液(150mL)を加え、発泡が収まるまで撹拌した。酢酸エチル(150mL)で抽出し、有機層を無水硫酸マグネシウム上乾燥後、濃縮し、化合物7Cを得た。
化合物7Cをテトラヒドロフラン(21mL)に溶解し、メタノール(211mL)、水酸化ナトリウム(8.42g、0.211mol)を加え、40℃で14時間加熱撹拌した。反応液を少量にまで濃縮した後、酢酸エチル(200mL)で希釈し、1mol/L塩酸(100mL)、飽和炭酸水素ナトリウム水溶液(100mL)で洗浄した。有機層を無水硫酸マグネシウム上乾燥後、濃縮した。残渣をアセトン(30mL)に溶解し、脱イオン水(15mL)を添加した。溶液が透明になるまでアセトンを加えた後、析出した固体をろ取した。固体を50体積%エタノールで洗浄後、真空乾燥し、化合物8C(18.71g、30.81mmol、73.17%)を得た。
1H-NMR(CDCl3,400MHz);δ8.54(1H,s),7.50-7.42(5H,m),7,19-7.11(4H,m),5.71(1H,s),4.71(1H,s),4.60-4.52(3H,m),4.38(1H,d),4.00(1H,d),3.87-3.79(4H,m),3.71(2H,s),1.67(3H,d).
化合物8C(16.07g、26.46mmol)をテトラヒドロフラン(142mL)、メタノール(142mL)に溶解し、ギ酸アンモニウム(16.8g、0.266mol)を加え、溶解した。1mol/L塩酸(26.4mL、26.4mmol)、水酸化パラジウム-活性炭素(8.04g)を加え、60℃で23時間加熱撹拌した。脱イオン水(27mL)を加え、析出物を溶解した後、セライトろ過により水酸化パラジウム-活性炭素を除去し、50体積%メタノール(100mL×5)で洗浄した。ろ液、洗浄液を濃縮し、残渣を脱イオン水(100mL)に溶解し、Dowex50W×8(H+型)カラム(60mL)に吸着させた。カラムを水洗後、0.2-0.5mol/Lアンモニア水で溶出した。目的物を含むフラクションを濃縮後、真空乾燥し、化合物9C(6.83g、25.4mmol、96.0%)を得た。
1H-NMR(D2O,400MHz);δ7.62(1H,s),5.66(1H,s),4.46(1H,s),4.19(1H,s),4.04(1H,d),4.04(2H,s),3.96(1H,d),1.98(3H,s).
化合物9C(9.15g、34.0mmol)をピリジン(脱水)(118mL)に溶解し、無水安息香酸(15.4g、68.1mmol)を加え、室温で3日間撹拌した。反応液にエタノール(116 mL)、2mol/L水酸化ナトリウム水溶液(174mL、0.348mol)を加え、1時間撹拌後、酢酸(23mL)を添加した。反応液を濃縮後、残渣を脱イオン水で2回共沸した。残渣を脱イオン水(25mL)に溶解し、析出した固体をろ取、水洗した。得られた固体を脱イオン水から再結晶化し、化合物10C(10.19g、27.29mmol、80.3%)を得た。
1H-NMR(DMSO-d6,400MHz);δ8.15-7.45(6H,m),5.67(1H,d),5.46(1H,s),5.22(1H,t),4.17(1H,s),3.90(1H,d),3.83-3.63(4H,m),2.00(3H,s).
化合物10C(7.00g、18.7mmol)をピリジンで3回共沸後、ピリジン(脱水)(62.3mL)に溶解した。塩化ジメトキシトリチル(8.24g、24.3mmol)を加え、室温で2時間撹拌後、反応液にメタノール(5mL)を加え濃縮した。残渣に酢酸エチル(200mL)を加え、飽和炭酸水素ナトリウム水溶液(50mL)で洗浄した。有機層を無水硫酸マグネシウム上乾燥後、濃縮し、残渣をトルエン(20mL×3)で共沸した。残渣を以下の条件で中圧シリカゲルカラムクロマトグラフィー(SiO2100g、ヘキサン:酢酸エチル=2:1~1:1~1:3(体積比))により精製し、化合物11C(12.14g、18.00mmol、96.26%)を得た。
1H-NMR(CDCl3,400MHz);δ8.32(2H,d),7.83(1H,s),7.55-7.25(12H,m),6.89-6.85(4H,m),5.70(1H,s),4.47(1H,s),4.29(1H,d),3.85(2H,dd),3.81(6H,s),3.62(1H,d),3.48(1H,d),1.90(3H,s),1.86(1H,d).
化合物11C(11.50g、17.02mmol)をジクロロメタン(超脱水)(85.1mL)に溶解し、N,N-ジイソプロピルエチルアミン(6.02mmol、35.2mmol)、(2-シアノエチル)(N,N-ジイソプロピル)クロロホスホロアミダイト(6.83mL、30.6mmol)を加え、室温で2時間撹拌した。反応液をクロロホルム(100 mL)で希釈後、飽和炭酸水素ナトリウム水溶液-飽和塩化ナトリウム水溶液(50mL+50mL)で洗浄した。有機層を無水硫酸マグネシウム上乾燥後、濃縮した。残渣を以下の条件で中圧シリカゲルカラムクロマトグラフィー(SiO2100g、ヘキサン:酢酸エチル=3:1~2:1~1:1(体積比))により精製し、化合物12C(10.70g、12.22mmol、71.80%)を得た。
31P-NMR(CDCl3,160MHz);δ150.10,150.04.
化合物5Aからグアニン架橋型ヌクレオシドアミダイト13Gを合成した。
化合物5A(30.0g、42.1mmol)を酢酸(366mL)、無水酢酸(45.0mL)に懸濁し、濃硫酸(396μL)の酢酸溶液(30.0mL)をゆっくりと滴下後、室温で2時間撹拌した。反応液に酢酸ナトリウム(1.50g、18.3mmol)を加え、完全に溶解するまで撹拌後、濃縮した。残渣をトルエン-酢酸エチル(体積比で2:1)で共沸(40mL×5)後、残渣に酢酸エチル(300mL)を加え、飽和炭酸水素ナトリウム水溶液(150mL)で洗浄した。有機層を無水硫酸マグネシウム上乾燥後、濃縮した。残渣をトルエンで共沸(20mL×3)し、化合物6Aを得た。
化合物6Aをアセトニトリル(超脱水)(105mL)に溶解し、2-アミノ-6-クロロプリン(10.7g、63.1mmol)、N,O-ビス(トリメチルシリル)アセトアミド(33.9mL、0.137mol)を加え、85℃で1時間加熱撹拌した。反応液を氷冷後、トリフルオロメタンスルホン酸トリメチルシリル(15.2mL、84.1mmol)を加え、85℃で7時間加熱撹拌した。反応液を氷冷後、飽和炭酸水素ナトリウム水溶液を加え、発泡が収まるまで撹拌した。酢酸エチル(150mL)で抽出し、有機層を1M水酸化ナトリウム水溶液で洗浄後、無水硫酸マグネシウム上乾燥、濃縮し、化合物7Gを得た。
化合物7Gをテトラヒドロフラン(105mL)に溶解し、メタノール(421mL)、25体積%ナトリウムメトキシドメタノール溶液(80.9mL)を加え、室温で24時間加熱撹拌した。反応液を2M塩酸で中和後、濃縮した。残渣に酢酸エチルを加え、飽和炭酸水素ナトリウム水溶液で洗浄した。有機層を無水硫酸マグネシウム上乾燥後、濃縮した。残渣を酢酸エチルに溶解し、活性炭(1.0g)を加え、60℃で1時間加熱撹拌した。活性炭をセライトろ過により除去後、ろ液を濃縮し、化合物8Gを得た。
化合物8Gをテトラヒドロフラン(213mL)、メタノール(213mL)に溶解し、ギ酸アンモニウム(26.9g、0.427mol)を加え、溶解した。水酸化パラジウム-活性炭素(13.5g)を加え、60℃で30時間加熱撹拌した。セライトろ過により水酸化パラジウム-活性炭素を除去し、メタノール(100mL×5)で洗浄した。ろ液、洗浄液を濃縮後、残渣を脱イオン水(300mL)に溶解し、トルエンで洗浄した。溶液をIRA93(OH-型)カラム(150mL)に通過、水洗し後、濃縮し、化合物9Gを得た。
化合物9Gを100mM Tris-HCl(pH7.5)(420mL)に溶解し、アデノシンデアミナーゼ(37μL、42units)を加え、40℃で24時間撹拌した。アデノシンデアミナーゼ(37μL、42units)を追加し、同温度でさらに24時間撹拌した。析出した固体をろ取、水洗後、真空乾燥し、化合物10G(8.50g、28.8mmol、68.4%)を得た
1H-NMR(DMSO-d6,400MHz);δ10.63(1H,s),7.79(1H,s),6.56(2H,s),5.68-5.67(2H,m),5.03(1H,t),4.28(1H,s),4.14(1H,d),3.88(1H,d),3.77(2H,m),3.71(1H,d).
化合物10G(600mg、2.03mmol)をN,N-ジメチルホルムアミド(脱水)(4.1mL)に溶解し、tert-ブチルクロロジメチルシラン(1.22g、8.09mmol)を加え、室温で20時間撹拌した。反応液を酢酸エチルで希釈、水洗後、有機層を無水硫酸マグネシウム上で乾燥、濃縮した。残渣をシリカゲルカラムクロマトグラフィー(SiO2100mL、クロロホルム:メタノール=20:1(体積比))により精製した。
残渣をピリジン(8.0mL)に溶解し、氷冷化塩化イソブチリル(336μL、3.18mmol)を加え、室温で20時間撹拌した。脱イオン水を加え撹拌後、反応液を濃縮した。残渣に酢酸エチルを加え、飽和重曹水で洗浄後、有機層を無水硫酸マグネシウム上で乾燥、濃縮した。残渣をトルエンで3回共沸後、残渣をシリカゲルカラムクロマトグラフィー(SiO2 100mL、クロロホルム:メタノール=50:1(体積比))により精製した。
残渣をメタノール(10.0mL)に溶解し、酸性フッ化水素アンモニウム(860mg、15.0mmol)を加え、60℃で17時間加熱撹拌した。反応液にシリカゲルを加え溶媒を留去したのち、シリカゲルカラムクロマトグラフィー(SiO2100mL、クロロホルム:メタノール=10:1~5:1(体積比))により精製し、化合物11G(0.53g、1.5mmol、74%)を得た。
1H-NMR(DMSO-d6,400MHz);δ12.11(1H,s),11.77(1H,s),8.09(1H,s),5.80(1H,s),5.73(1H,d),5.06(1H,t),4.37(1H,s),4.15(1H,d),3.92-3.72(4H,m),2.77(1H,m),1.12(6H,d).
化合物11G(1.87g、5.12mmol)をピリジンで3回共沸後、ピリジン(脱水)(17.1mL)に溶解した。塩化ジメトキシトリチル(2.43g、7.17mmol)を加え、室温で2時間撹拌後、反応液にメタノール(1mL)を加え濃縮した。残渣に酢酸エチル(200mL)を加え、飽和炭酸水素ナトリウム水溶液で洗浄した。有機層を無水硫酸マグネシウム上乾燥後、濃縮し、残渣をトルエン(20mL×3)で共沸した。残渣をシリカゲルカラムクロマトグラフィー(SiO2150g、ヘキサン:酢酸エチル=1:1~1:3(体積比)~酢酸エチル)により精製し、化合物12G(3.27g、4.90mmol、95.7%)を得た。
1H-NMR(DMSO-d6,400MHz);δ12.05(1H,s),8.93(1H,s),7.88(1H,s),7.44-7.13(9H,m),6.79(4H,m),5.78(1H,s),4.60(1H,s),4.44(1H,s),4.05(1H,d),3.99(1H,d),3.74(3H,s),3.74(3H,s),3.61(1H,d),3.55(1H,d),2.64(1H,m),1.24(3H,d),1.23(3H,d).
化合物12G(3.20g、4.79mmol)をジクロロメタン(超脱水)(24.0mL)に溶解し、N,N-ジイソプロピルエチルアミン(2.03mL、11.9mmol)、(2-シアノエチル)(N,N-ジイソプロピル)クロロホスホロアミダイト(2.35mL、10.5mmol)を加え、室温で3時間撹拌した。反応液を飽和炭酸水素ナトリウム水溶液で洗浄した。有機層を無水硫酸マグネシウム上乾燥後、濃縮した。残渣をシリカゲルカラムクロマトグラフィー(SiO2200g、ヘキサン:酢酸エチル=1:1~1:2(体積比))により精製し、化合物13G(3.36g、3.87mmol、80.8%)を得た。
31P-NMR(MeCN-d3,160MHz);δ149.09,148.74.
Claims (8)
- 粉末X線分析において、回折角(2θ)として5.9±0.3、11.4±0.6、11.8±0.6、13.2±0.7、16.2±0.8、17.2±0.9、18.5±0.9、19.5±1.0、19.7±1.0、20.1±1.0、20.4±1.0、21.4±1.1、22.0±1.1、23.0±1.2、24.1±1.2、24.3±1.2、26.4±1.3、29.9±1.5(°)にピークを示す、請求項1に記載の結晶。
- 熱重量測定/示差熱分析(TG/DTA)装置により測定したとき、124℃に吸熱ピークを示す、請求項1または2に記載の結晶。
- 式5で表される前記化合物は架橋型ヌクレオシド中間体である、請求項1~3のいずれか1項に記載の結晶。
- 下記工程1から5を含む、式5で表される化合物の結晶の製造方法。
工程1:式1で表される化合物の水酸基を保護し、式2で表される化合物を得る工程、
工程2:式2で表される化合物の4位ジメチルジオキソラニル基をアルデヒド基へと変換し、式3で表される化合物を得る工程、
工程3:式3で表される化合物を還元して4位アルデヒド基を水酸基とし、式4で表される化合物を得る工程、
工程4:式4で表される化合物の4位水酸基を脱離基へと変換し、式5で表される化合物を得る工程、
工程5:式5で表される化合物を結晶化溶媒から結晶化し、式5で表される化合物の結晶を得る工程
- 工程2と工程3との間に、結晶化工程を更に含む、請求項5に記載の結晶の製造方法。
- 式5で表される前記化合物は架橋型ヌクレオシド中間体である、請求項5または6に記載の結晶の製造方法。
- 以下の工程を含む、架橋型ヌクレオシドアミダイトの製造方法。
工程6:請求項5~7のいずれか1項の製造方法によって得られる式5で表される化合物の結晶を溶媒に懸濁させた後、該化合物のイソプロピリデン基をアセチル基へと変換し、式6で表される化合物を得る工程
工程7:式6で表される化合物をシリル化した塩基と縮合し、式7で表される化合物を得る工程
工程8:式7で表される化合物の保護基の除去と同時に環化反応を行い、式8で表される化合物を得る工程
工程9:式8で表される化合物の水酸基保護基を除去し、式9で表される化合物を得る工程
工程10:必要に応じて式9で表される化合物の塩基部のアミノ基に保護基を導入し、式10で表される化合物を得る工程
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2021526889A JPWO2020256084A1 (ja) | 2019-06-19 | 2020-06-18 | |
EP20827632.9A EP3988558A4 (en) | 2019-06-19 | 2020-06-18 | CROSSLINKED NUCLEOSIDE INTERMEDIATE CRYSTAL AND METHOD FOR PRODUCTION THEREOF, AND METHOD FOR PRODUCTION OF CROSSLINKED NUCLEOSIDE AMIDITE |
CA3142593A CA3142593A1 (en) | 2019-06-19 | 2020-06-18 | Crosslinked nucleoside intermediate crystal and method for producing same, and method for producing crosslinked nucleoside amidite |
US17/608,780 US20220315617A1 (en) | 2019-06-19 | 2020-06-18 | Crosslinked Nucleoside Intermediate Crystal And Method For Producing Same, And Method For Producing Crosslinked Nucleoside Amidite |
CN202080040731.2A CN113924308A (zh) | 2019-06-19 | 2020-06-18 | 交联型核苷中间体的结晶及其制造方法以及交联型核苷亚磷酰胺的制造方法 |
KR1020227000752A KR20220024461A (ko) | 2019-06-19 | 2020-06-18 | 가교형 뉴클레오시드 중간체의 결정 및 그의 제조 방법, 그리고 가교형 뉴클레오시드 아미다이트의 제조 방법 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2019113372 | 2019-06-19 | ||
JP2019-113372 | 2019-06-19 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2020256084A1 true WO2020256084A1 (ja) | 2020-12-24 |
Family
ID=74040857
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2020/024037 WO2020256084A1 (ja) | 2019-06-19 | 2020-06-18 | 架橋型ヌクレオシド中間体の結晶及びその製造方法、並びに架橋型ヌクレオシドアミダイトの製造方法 |
Country Status (7)
Country | Link |
---|---|
US (1) | US20220315617A1 (ja) |
EP (1) | EP3988558A4 (ja) |
JP (1) | JPWO2020256084A1 (ja) |
KR (1) | KR20220024461A (ja) |
CN (1) | CN113924308A (ja) |
CA (1) | CA3142593A1 (ja) |
WO (1) | WO2020256084A1 (ja) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH10304889A (ja) | 1997-03-07 | 1998-11-17 | Takeshi Imanishi | 新規ビシクロヌクレオシド及びオリゴヌクレオチド類縁体 |
JP2002521310A (ja) | 1997-09-12 | 2002-07-16 | エクシコン エ/エス | オリゴヌクレオチド類似体 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
TR200604211T1 (tr) * | 1999-02-12 | 2007-02-21 | Daiichi Sankyo Company Limiteddaiichi Sankyo Company Limited | Yeni nükleosid ve oligonükleotid analoglarıYeni nükleosid ve oligonükleotid analogları |
-
2020
- 2020-06-18 US US17/608,780 patent/US20220315617A1/en active Pending
- 2020-06-18 KR KR1020227000752A patent/KR20220024461A/ko unknown
- 2020-06-18 WO PCT/JP2020/024037 patent/WO2020256084A1/ja unknown
- 2020-06-18 CN CN202080040731.2A patent/CN113924308A/zh active Pending
- 2020-06-18 JP JP2021526889A patent/JPWO2020256084A1/ja active Pending
- 2020-06-18 EP EP20827632.9A patent/EP3988558A4/en active Pending
- 2020-06-18 CA CA3142593A patent/CA3142593A1/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH10304889A (ja) | 1997-03-07 | 1998-11-17 | Takeshi Imanishi | 新規ビシクロヌクレオシド及びオリゴヌクレオチド類縁体 |
JP2002521310A (ja) | 1997-09-12 | 2002-07-16 | エクシコン エ/エス | オリゴヌクレオチド類似体 |
Non-Patent Citations (9)
Title |
---|
"Ph. D Thesis", 2016, TOHOKU UNIVERSITY |
FUKUYAMA, KEI: "Development of Efficient Synthetic Methods for C4- Tetrasubstituted Nucleoside Derivatives with Pharmacological Activities", TOHOKU UNIVERSITY DOCTORAL DISSERTATION, 25 March 2016 (2016-03-25), pages 1 - 102, XP055773874, Retrieved from the Internet <URL:http://hdl.handle.net/10097/64032> * |
J. ORG. CHEM., vol. 66, 2001, pages 8504 - 8512 |
J. ORG. CHEM., vol. 80, 2015, pages 5337 - 5343 |
KOSHIKIN, ALEXEI A. ET AL.: "A Simplified and Efficient Route to 2'-0, 4'- C-Methylene-Linked Bicyclic Ribonucleosides (Locked Nucleic Acid", J. ORG. CHEM., vol. 66, 2001, pages 8504 - 8512, XP055319074, DOI: 10.1021/jo010732p * |
KOSHIKIN, ALEXEI A. ET AL.: "LNA (Locked Nucleic Acids): Synthesis of the Adenine, Cytosine, Guanine, 5-Methylcytosine, Thymine and Uracil Bicyclonucleoside Monomers", OLIGOMERISATION, AND UNPRECEDENTED NUCLEIC ACID RECOGNITION, TETRAHEDRON, vol. 54, 1998, pages 3607 - 3630, XP002268966, DOI: 10.1016/S0040-4020(98)00094-5 * |
See also references of EP3988558A4 |
TETRAHEDRON, vol. 54, 1998, pages 3607 - 3630 |
WAGA, YOSHIAKI ET AL.: "Studies on sugar-modified nucleosides", PART I. SYNTHESIS OF 4'-C- METHYLNUCLEOSIDES , BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY, vol. 57, no. 9, 1993, pages 1433 - 1438, XP000917028 * |
Also Published As
Publication number | Publication date |
---|---|
KR20220024461A (ko) | 2022-03-03 |
EP3988558A1 (en) | 2022-04-27 |
CN113924308A (zh) | 2022-01-11 |
EP3988558A4 (en) | 2022-09-28 |
JPWO2020256084A1 (ja) | 2020-12-24 |
US20220315617A1 (en) | 2022-10-06 |
CA3142593A1 (en) | 2020-12-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1594882B1 (en) | Process for preparing branched ribonucleosides from 1, 2-anhydroribofuranose intermediates | |
WO2019208571A1 (ja) | アミダイト化合物及び該化合物を用いたポリヌクレオチドの製造方法 | |
JP3653292B2 (ja) | 5−メチルウリジンを用いる2’,3’−ジデヒドロ−3’−デオキシチミジン(d4T)の大量製造法 | |
JP2008515968A (ja) | 2’−デオキシ−2’,2’−ジフルオロシチジンの製造方法 | |
JP7025064B2 (ja) | 4’-置換ヌクレオシド誘導体の立体選択的合成法 | |
WO2020256084A1 (ja) | 架橋型ヌクレオシド中間体の結晶及びその製造方法、並びに架橋型ヌクレオシドアミダイトの製造方法 | |
JPH0797391A (ja) | ヌクレオシド誘導体とその製造方法 | |
JP3914279B2 (ja) | 5−メチルウリジンからd4Tを製造する方法 | |
EP1253154B1 (en) | Method for purifying 5'-protected 2'-deoxypurine nucleosides | |
JP2007513134A (ja) | 2−置換アデノシンの改善された合成 | |
EP1258489B1 (en) | Method for purifying 5'-protected thymidines | |
US20030236397A1 (en) | Process for preparing beta-L-2'deoxy-thymidine | |
JP4174895B2 (ja) | ヌクレオシド誘導体とその製法 | |
JP5192807B2 (ja) | シュードウリジン保護体の安定結晶 | |
JP2004175791A (ja) | 3’,5’−o−スルフィニルキシロシチジン誘導体、その製造方法及びシチジン化合物の製造方法 | |
JP2005538080A (ja) | 2−デオキシ−l−リボースの合成方法 | |
JP2002293792A (ja) | ヌクレオシド又は糖のフッ素化誘導体の製造方法 | |
JP2001226394A (ja) | 2’−デオキシ−β−グアノシンの製造法およびその中間体 | |
WO2010079813A1 (ja) | イノシン誘導体の製造方法 | |
WO1997006179A1 (fr) | Procede permettant de produire des derives d'azidonucleoside | |
JPH11246591A (ja) | 8−(グリコシルオキシ)−プリンヌクレオシド誘導体およびその製造方法 | |
JPH09110893A (ja) | 3’−アミノ−3’−デオキシヌクレオシドの製造方法 | |
JP2005132775A (ja) | フラノシルアデニン誘導体及びその製造方法、並びに3’−アミノ−3’−デオキシアデノシン化合物及びその製造方法。 | |
HUT61029A (en) | Process for producing nucleoside derivatives | |
WO2005021571A1 (ja) | N4−ベンゾイルシチジン誘導体の製造法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 20827632 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2021526889 Country of ref document: JP Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 3142593 Country of ref document: CA |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 20227000752 Country of ref document: KR Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2020827632 Country of ref document: EP Effective date: 20220119 |