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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/50—Pyridazines; Hydrogenated pyridazines
  • A61K31/501—Pyridazines; Hydrogenated pyridazines not condensed and containing further heterocyclic rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • 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

Definitions

• the present invention relates to an improved method for preparing l-(2'- deoxy-2'-fluoro-beta-L-arabinofuranosyl) thymine (hereinafter, referred to simply as "clevudine”) useful as an anti-HBV (hepatitis B virus) agent.
• clevudine l-(2'- deoxy-2'-fluoro-beta-L-arabinofuranosyl) thymine
• the present invention relates to an improved method for preparing clevudine comprising an improved purification process of 2-0- imidazolylsulfonyl-l,3,5-tri-O-benzoyl-alpha-L-ribofuranoside (hereinafter, referred to simply as "ITBR”) used as a key intermediate in the preparation of clevudine, and a final deprotection process using an alkaline metal salt, which is safe and easy to handle, as a deprotecting reagent of l-(3,5-di-O-benzoyl-2- deoxy-2-fluoro-beta-L-arabinofuranosyl) thymine (hereinafter, referred to simply as "DFAT”) so that productivity can be increased.
• ITBR imidazolylsulfonyl-l,3,5-tri-O-benzoyl-alpha-L-ribofuranoside
• DFAT deprotecting reagent of l-(3,5
• Clevudine useful as an anti-HBV agent, is represented by Formula I below:
• Korean Patent No. 247,505 discloses a method for preparing clevudine, as depicted in Reaction Scheme I below: Reaction Scheme I
• FIG. 1 shows an example of clevudine crystals obtained from ethanol, methanol, acetonitrile, acetone, water, and the like.
• the present inventors studied crystal polymorphism of clevudine by analyzing clevudine crystals obtained from ethanol, methanol, acetonitrile, acetone, water, and the like through differential scanning calorimetry, X-ray diffractometry, and infrared spectrophotometry. From the analysis results, it can be confirmed that all of the clevudine crystals have the same crystal form (Type I). This indicates that there is no crystal polymorph of clevudine.
• Single-crystal crystallography analysis results ascertained that clevudine has a ⁇ - and L-type structure.
• FIG. 1 shows an example of clevudine crystals obtained from ethanol, methanol, acetonitrile, acetone, water, and the like. From the analysis results,
• ORTEP structure of clevudine and crystal information is as follows:
• Reaction Scheme I above and an improved method for preparing clevudine using thionyl chloride disclosed in Korean Patent No. 10-0484567 are disadvantageous beta-L-arabinofuranosyl) thymine (hereinafter, referred to simply as "chloroclevudine”) represented by the following Formula II as a process impurity in terms of the reaction mechanism exists in an amount of equal to or higher than a qualification limit (i.e., 0.15 %).
• a qualification limit i.e. 0.15
• chloride ions derived from the sulfuryl chloride used as a reagent in the preparation process of ITBR represented by Formula III are present in a trace amount in the intermediate ITBR, and 2-deoxy-2-chloro- 1 ,3,5-tri-O-benzoyl-alpha-L-ribofuranoside (hereinafter, referred to simply as "CTBR") represented by Formula V is formed as a byproduct from the remaining chloride ions during the preparation process of 2- deoxy-2-fluoro-l ,3,5-tri-O-benzoyl-alpha-L-ribofuranoside (hereinafter, referred to simply as "FTBR”) represented by Formula IV and is related to the impurity, i.e., chloroclevudine which is carry-overed to the impurity in clevudine.
• CBR 2-deoxy-2-chloro- 1 ,3,5-tri-O-benzoyl-alpha-L-ribofuranoside
• FTBR 2-
• the present inventors elucidated the structure of CTBR prepared with b reference to the document (J. Org. Chem. 1981, 46, 1790-1792) and monitored the reaction behaviors during the fluorination process shown in Reaction Scheme II by HPLC to ascertain a retention time of CTBR produced by the reaction.
• the present inventors conducted LC-MS analysis of corresponding peaks to determine a molecular weight.
• FIG. 2 shows a HPLC chromatogram of 0 compounds obtained during Reaction Scheme II.
• FIG. 3 shows LC-MS analysis results of the peak corresponding to 17.4 min of a retention time in the HPLC chromatogram of FIG. 2.
• the present invention has been made in view of the afore-mentioned problems, and it is one aspect of the present invention to provide an improved method for preparing clevudine, comprising an improved purification process of 2-O-imidazolylsulfonyl- 1 ,3,5-tri-O-benzoyl-alpha-L-ribofuranoside (ITBR) of Formula III so that formation of a process impurity can be inhibited and an increased total yield of clevudine and reduced manufacturing costs can be thus realized, and a final deprotection process using an alkaline metal salt, which is safe and easy to handle, as a deprotecting reagent, instead of an ammonia gas conventionally used in the art, so that a reaction time can be greatly shortened, a reaction by-product can be readily removed and productivity can be thus increased.
• ITBR 2-O-imidazolylsulfonyl- 1 ,3,5-tri-O-benzoyl-alpha-L-rib
• a method for preparing clevudine comprising the step of deprotecting l-(3,5-di-O-benzoyl-2-deoxy-2-fluoro-beta-L- arabinofuranosyl) thymine (DFAT) by reacting with an alkaline metal salt in the presence of methanol.
• DFAT deprotecting l-(3,5-di-O-benzoyl-2-deoxy-2-fluoro-beta-L- arabinofuranosyl) thymine
• the DFAT may be prepared by reacting 1,3,5-tri-O-benzoyl-alpha-L- ribofuranoside (HTBR) with sulfuryl chloride and imidazole to prepare 2-O- imidazolylsulfonyl-l,3,5-tri-O-benzoyl-alpha-L-ribofuranoside (ITBR), which is treated with an anion exchange resin (hydroxide form); introducing fluorine into
• ITBR is prepared from 1,3,5-tri-O- benzoyl-alpha-L-ribofuranoside of the following Formula VI (hereinafter, referred to simply as "HTBR") and an anion exchange resin is used to purify the ITBR, so that chloride ions present in the ITBR compound can be readily and efficiently removed, when compared to washing several times with distilled water which is conventionally used in the art.
• Suitable anion exchange resins include DIAION WAlO, DIAION WA20, DIAION WA30 and DIAION WA21J (SAMYANG Co., Ltd.)
• the method according to the present invention is characterized in that an alkaline metal salt is used as a deprotecting reagent in the presence of a methanol solvent in the final deprotection process of l-(3,5-di-O-benzoyl-2- deoxy-2-fluoro-beta-L-arabinofuranosyl) thymine of Formula VII (hereinafter, referred to simply as "DFAT").
• suitable alkaline metal salts include: carbonate such as sodium hydrogen carbonate, potassium hydrogen carbonate, sodium carbonate and potassium carbonate; and sodium alkoxide such as sodium methoxide (NaOMe) and sodium ethoxide (NaOEt).
• examples of cation exchange resins that can be used in the purification process following the reaction include TRILITE SCR-B, TRILITE SCR04, TRILITE SCRlO and TRILITE SCRl 2.
• FIG. 5 is a HPLC chromatogram of compounds obtained during the deprotection process.
• FIG. 6 shows LC-MS analysis results of the peak corresponding to 14.9 min of a retention time in the HPLC chromatogram of FIG. 5. Since methyl benzoate is highly soluble in an organic solvent and sparingly soluble in an aqueous solution, it can be readily removed in the processing step, compared to benzoate. According to the method of the present invention, a reaction time can be reduced and a final target compound can be prepared as a high purity and yield.
• HTBR 1,3,5-tri-O-benzoyl- alpha-L-ribofuranoside
• STYR sulfuryl chloride
• imidazole in the presence of dichloromethane at -10 0 C to 0°C in accordance with a general method to prepare 2-O-imidazolylsulfonyl-l,3,5-tri-O-benzoyl- alpha-L-ribofuranoside (ITBR, Formula III).
• IBR 2-O-imidazolylsulfonyl-l,3,5-tri-O-benzoyl- alpha-L-ribofuranoside
• IBR 2-O-imidazolylsulfonyl-l,3,5-tri-O-benzoyl- alpha-L-ribofuranoside
• IBR 2-O-imidazolylsulfonyl-l,3,5-tri-O-benzoyl- alpha-L-ribofuranoside
• the anion exchange resin (hydroxide form) is used in an amount of 0.4 to 3.0 parts by weight, preferably 1.0 to 2.0 parts by weight, based on the weight of HTBR.
• the reaction solution is stirred at - 10 0 C to 4O 0 C, preferably at O 0 C to 30 0 C, for 1 to 6 hours, vacuum-filtered and distilled under reduced pressure to give ITBR.
• fluorine is introduced into the ITBR using triethylamine'trihydro fluoride to prepare 2-deoxy-2-fluoro-l,3,5-tri-O-benzoyl- alpha-L-ribofuranoside (FTBR, Formula IV).
• the FTBR obtained in the previous process is halogenated by treating with hydrobromic acid in accordance with a general method and subjected to condensation with thymine to yield l-(3,5-di-O-benzoyl-2-deoxy-2-fluoro-beta- L-arabinofuranosyl) thymine (DFAT, Formula VII).
• the DFAT is reacted with a deprotecting reagent in the presence of methanol at 40 0 C to 80 0 C for 12 to 18 hours to yield clevudine as a final target compound.
• the deprotecting reagent used herein is an alkaline metal salt and examples thereof include: carbonate such as sodium hydrogen carbonate, potassium hydrogen carbonate, sodium carbonate and potassium carbonate; and sodium alkoxide such as sodium methoxide and sodium ethoxide. Preferred is sodium hydrogen carbonate.
• the deprotecting reagent is used in an amount of 0.1 to 4.0 equivalents, preferably 0.5 to 2.0 equivalents, with respect to DFAT.
• the reaction is carried out at 40°C to 80 0 C, preferably at 80 0 C for 12 hours while stirring.
• a total yield of clevudine obtained from ITBR is 43.2%, which is increased by about 1 1%, compared to 38.9%, a total yield of an existing method. As a result, manufacturing costs of clevudine can be reduced.
• the reaction time is 12 hours, which is shorter than the reaction time (i.e , 72 hours) of a
• the deprotection method of the present invention is useful in the final deprotection of other nucleoside-based compounds. For example, as can be seen from Reaction scheme VII below, the deprotection method is useful for preparing l-(2-deoxy-2-fluoro-beta-L-arabinofuranosyl)-5- fluorouracil.
• FJG. 1 shows an ORTEP structure of clevudine by single crystal crystallography
• FIG. 2 is a HPLC chromatogram of compounds obtained during Reaction Scheme II;
• FIG. 3 shows LC-MS analysis results of the peak corresponding to 17.4 min of a retention time in the HPLC chromatogram of FIG. 2;
• FIG. 4 shows LC-MS analysis results of the peak corresponding to 22.1 min of a retention time in the HPLC chromatogram of FIG. 2;
• FIG. 5 is a HPLC chromatogram of compounds obtained during the deprotection process
• FIG. 6 shows LC-MS analysis results of the peak corresponding to 14.9 min of a retention time in the HPLC chromatogram of FIG. 5;
• FIGs. 7 to 11 are X-ray diffraction spectra of clevudine crystals, and more specifically, FIGs. 7, 8, 9, 10 and 1 1 are X-ray diffraction spectra of a FIGs. 12 to 16 are differential scanning calorimetry spectra of clevudine crystals, and more specifically, FIGs. 12, 13, 14, 15 and 16 are differential scanning calorimetry spectra of clevudine crystals grown in ethanol, methanol, acetone, acetonitrile and water, respectively; and FIGs. 17 to 21 are IR spectra of clevudine showing a wavelength and a contributing carbon corresponding thereto, and more specifically, FIGs. 17, 18, 19, 20 and 21 are IR spectra of clevudine crystals grown in ethanol, methanol, acetone, acetonitrile and water, respectively.
• 1,3,5-tri-O-benzoyl-alpha-L-ribofuranoside (5.26 g, 11.4 mmole) was dissolved in 70 mL of dichloromethane, sulfuryl chloride (1.7 mL, 21.2 mmole) was slowly added dropwise thereto at -10 0 C to O 0 C and stirred at ambient temperature.
• a solution of imidazole (5.0 g, 73.4 mmole) in 40 mL dichloromethane was added dropwise to the reaction solution and allowed to react for 12 hours.
• reaction solution was washed with 30 mL of distilled water, a separated organic layer was distilled under reduced pressure, 20 mL of anhydrous ethanol was added to the remaining solution and stirred at ambient temperature for one hour. The resulting crystal was filtered to yield 5.26 g of the title compound as a white crystal.
• Example 1 Preparation of 2-O-imidazolylsulfonyl- 1,3, 5-tri-O-benzoyl- alpha-L-ribofuranoside (ITBR) - method of the present invention
• 1,3,5-tri-O-benzoyl-alpha-L-ribofuranoside (5.26 g, 11.4 mmole) was dissolved in 70 mL of dichloromethane, sulfuryl chloride (1.7 mL, 21.2 mmole) dichloromethane was added dropwise to the reaction solution and allowed to react for 12 hours. After completion of the reaction, 5.26 g of DIAION WA30 as a weakly basic anion exchange resin was added to the reaction solution and stirred at ambient temperature for one hour. The anion exchange resin was removed by filtration and the filtrate was distilled under reduced pressure, 20 mL of anhydrous ethanol was added thereto and stirred at ambient temperature for one hour. The resulting crystal was filtered to yield 5.26 g of the title compound as a white crystal.
• the 2-deoxy-2-fluoro-l ,3,5-tri-O-benzoyl-alpha-L-ribofuranoside (3.06 g, 6.59 mmole) obtained in Example 2 was dissolved in 23 mL of methylene chloride.
• 3.8 g of hydrobromic acid/acetic acid (33%, w/v, 21.0 mmole) was added to the solution at 10 0 C to 5°C and stirred at ambient temperature for 24 hours. After completion of the reaction, the reaction solution was distilled under reduced pressure and 23 mL of methylene chloride was added thereto and extracted twice with 12 mL of distilled water.
• the separated organic layer was neutralized with 3.5 mL of a saturated aqueous sodium hydrogen carbonate solution, dried over magnesium sulfate and filtered. The filtrate was distilled under reduced pressure to yield the title compound as brown syrup, which was used in the following process without further separation or purification.
• Example 5 Preparation of 1 ⁇ (2-deoxy-2-fluoro-beta-L-arabinofuranosyl) thymine (clevudine) - method according to the present invention
• Example 7 Preparation of l-(2-deoxy-2-fluoro-beta-L-arabinofuranosyl) thymine (clevudine) - method according to the present invention l-(3,5-di-O-benzoyl-2-deoxy-2-fluoro-L-arabinofuranosyl) thymine (10.0 g, 21.3 mmole) was suspended in 100 mL of methanol, 1.6 g of potassium carbonate was added thereto and refluxed for 12 hours. After completion of the reaction, the reaction solution was cooled, 25 mL of methanol was added thereto, and the resulting solution was adjusted to pH 6.0 to 6.5 using a H + resin.
• thymine (clevudine) - method according to the present invention l-(3,5-di-O-benzoyl-2-deoxy-2-fluoro-L-arabinofuranosyl) thymine (10.0 g, 21.3 mmole) was suspended in 100 mL of methanol, 0.3 g of sodium methoxide was added thereto and refluxed for 12 hours. After completion of the reaction, the reaction solution was cooled, 25 mL of methanol was added thereto, and the resulting solution was adjusted to pH 6.0 to 6.5 using a H + resin. After stirring for one hour, the reaction solution was filtered to remove the resin and washed with 10 mL of methanol. The filtrate was distilled under reduced pressure and
• Example 9 Preparation of l-(2-deoxy-2-fluoro-beta-L-arabinofuranosyl) thymine (clevudine) - the method according to the present invention 1 -(3,5-di-O-benzoyl-2-deoxy-2-fluoro-L-arabinofuranosyl) thymine (10.0 g, 21.3 mmole) was suspended in 100 mL of methanol, 0.4 g of sodium ethoxide was added thereto and refluxed for 12 hours. After completion of the reaction, the reaction solution was cooled, 25 mL of methanol was added thereto, and the resulting solution was adjusted to pH 6.0 to 6.5 using a H + resin.
• X-ray diffractometry was carried out using an X-ray diffractometer (ROTAFLUX 12KW, Rigaku Co., Ltd., Japan) under the following common conditions: initial angle: 10.000 ° ; final angle: 80.000° ; scanning rate: 5.000 sec, sampling interval: 0.020.
• FIGs. 7 to 1 1 are X-ray diffraction spectra of clevudine crystals, and more specifically, FIGs. 7, 8, 9, 10 and 11 are X-ray diffraction spectra of clevudine crystals grown in ethanol, methanol, acetone, acetonitrile, and water, TABLE 1
• Example 11 Differential Scanning Calorimetry (DSC) Differential scanning calorimetry was carried out using a differential scanning calorimeter (Seiko Exstar 6000, Instruments, Inc., Japan) under the following common conditions: temperature range: 25° to 300°; scanning rate: 0.5 sec, 10°/min.
• DSC Differential Scanning Calorimetry
• FIGs. 12 to 16 show differential scanning calorimetry spectra of clevudine crystals
• FIGs. 12, 13, 14, 15 and 16 are differential scanning calorimetry spectra of clevudine crystals grown in ethanol, methanol, acetone, acetonitrile and water, respectively. In these spectra, one absorption peak is observed in a range of 191.5 ° to 193.3 ° .
• Example 12 Infrared Spectrophotometry (IR) Infrared spectrophotometry was carried out using an IR-470 spectrophotometer (available from Dong-il SHIMADZU Corp.) with a KBr powder.
• IR-470 spectrophotometer available from Dong-il SHIMADZU Corp.
• FIGs. 17 to 21 are IR spectra of clevudine showing a wavelength and a contributing carbon corresponding thereto, and more specifically, FIGs. 17, 18, 19, 20 and 21 are IR spectra of clevudine crystals grown in ethanol, methanol, acetone, acetonitrile and water, respectively.
• Table 2 shows the wavelength corresponding to respective stretching in IR spectra of a clevudine crystal form (Type I). TABLE 2
• an anion exchange resin (hydroxide form), which is easy to utilize in industrial applications, readily available and recyclable, is used to prepare and purify 2-O-imidazolylsulfonyl- 1,3,5-tri-O-benzoyl-alpha-L-ribofuranoside (ITBR).
• IBR 2-O-imidazolylsulfonyl- 1,3,5-tri-O-benzoyl-alpha-L-ribofuranoside
• an alkaline metal salt is used in the final deprotection process of DFAT, so that the process following the reaction can be readily conducted, a reaction time can be greatly shortened (i.e., reduction from 72 hours of a conventional method to 12 hours), and an increased productivity and reduced manufacturing costs can be thus realized.
• Table 3 shows comparison in preparation rate of process impurity, a total yield of clevudine and reaction time between the method of the present invention and the conventional method.

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• 2006
  • 2006-12-08 KR KR1020060124497A patent/KR100839322B1/ko active IP Right Grant
• 2007
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  • 2007-12-06 JO JO2007521A patent/JO2677B1/en active

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