TW389755B - Novel process for preparing n-acetyl (1) -4-cyanophenylalanine ac -(1) -phe (4-cn) -oh and n-acetyl -(1)-p-amidinophenylalanine-cyclohexylglycine-<beta>-(3-n-methylpyridinium)-alanine ac-(1)-paph-chg-palme(3)-nh2 - Google Patents

Description

Α7 Β7 Η.1 Avoiding rectification without supplementing. Five \ _ 发 呵 1¾ Ming (, vol. • So, there is a need to prepare a compound Ac — (L)-pAph-Chg — Pa IMe (3)-NH2 Method of isomers • The key intermediate for the preparation of this stereoisomer is the isomeric N-ethylglycosyl- (L) -4 monocyanophenylalanine. The choice of the target technology • Some examples are crystallisation or differential absorption (decantation) after formation of non-mirromeric isomers [eg £ 11811 '^ 01116 ^, 1 ^ 0611 &gt; 81 ^ 8,811 (111-esolutions, J. Jacques, A. Collet, and SH Vilen, Wiley (1981)] *], chromatographic separation on palm stationary phases * dynamic analysis, and enzyme analysis • enzyme analysis, especially hydrolase Or esterase, which has been proven to be used in the analysis of amino acids "such as in Che-mistry and Biochemistry of the amino acids, Chapman and Hall, New York, 1984, Chap. 10 pp. 338-353; in Applications of Biochemical Systems in Organi-c Chemistry, Part IJB Jones, CJ Sih, and D. Perlman, Wiley, New York, 1976 , Chap 4. pp. 107-401; and Chemistry of the Amino Acids, vol.l. Wiley, New York, 1961, Chap 9. pp 728-750 · Even after choosing a method, get used to The technician must still perform exhaustive experiments to find the correct solvent, co-solvent (if needed), temperature, time, and other conditions to come up with effective and efficient racemate analysis to easily recover the target compound. Resulting in high yield * 髙 Enantiomeric excess * and methods that are not too difficult to implement • The present invention can solve N-ethylammonium- (D, L) -4 4-cyanophenylalanine This paper is in accordance with Chinese national standards (CNS) A4 Specification (210 X 297 mm) (Jing Xian "Read the Note # on the back side and fill out this page>--— — — — — — — — — — — — — — — — Intellectual Property Bureau of the Ministry of Economic Affairs Printed by employee consumer cooperatives Printed by the Central Standards Bureau of the Ministry of Economy Printed by A7 __B7____ V. Description of invention (1) Description of the invention = Field of the invention The present invention relates to N-acetylammonium (L)-4 monocyanoamphetamine Novel preparation method, Includes resolution of the racemic compound N-ethylamyl- (D, L) — 4 monocyanophenylalanine ethyl ester and the stereoisomers of Ac-(L) —pAph-Chg — Pa IMe (3) — NH2 The novel preparation method comprises using an intermediate N-ethylamyl- (L) -4-cyanophenylalanine. BACKGROUND OF THE INVENTION The racemic compounds N-ethenyl- (D, L) -4-aminophenylalanine and Ac- (L) -pAph-Chg-Pa IMe (3) -NH2 are disclosed and described in 1995. Of 08 / 428,404, filed on May 25, it is a partial later case of U.S. Patent Application Serial No. 08/2 3 3, 0 54 filed on April 26, 1994, and its And in this article as a reference. Its end product Ac — (L) — pAph — Chg — Pa IMe (3) — NH2 can be used as an inhibitor of factor Xa. Because stereoisomers may be better than racemates, for example, they have better efficiency, fewer side effects, lower levels, or no toxicity. Therefore, stereoisomers are more suitable for development into drugs than racemates. Sometimes, The advantages of these stereoisomers over racemates are not known until the late stages of drug development or sometimes even after the drug has been marketed. Many government agencies that approve the listing of drugs also prefer the stereoisomers of the approved drugs rather than their racemates. The paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) (please read the back page first) Please fill in this page again for attention) Order line-4-Feng Zhengnian, Yue Yue Λ ,. —V. Description of Invention (g)

Step A COOH

Compound I Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs_ (Compound (I)) Racemate was resolved into a truncated N-ethenyl-4 monocyanophenylalanine (compound (IA)) L Stereo isomers and D_isomers of esters_ (Compound D — (I)) are not substantially converted to acids. This allows the desired acid to be separated from undesired esters. As a complete explanation, the acid obtained by the method is mainly the L isomer, as shown by its 髙 ee factor. A sufficient amount of the compound (I) used in combination to form the reaction medium is to be recovered as L-Lithosperylene by the method of the present invention. At least twice as much as the structure is placed in. The reaction medium contains a sufficient amount of acetonitrile and a sufficient amount of an aqueous solution. Those sufficient amounts are required to facilitate the interaction of the additives according to the present invention. As used herein ， Additive # means any substance added to the reaction medium. The sufficient amount of acetonitrile is the amount that can dissolve the substantially compound (I) in the reaction medium. The better is to dissolve the compound (I) first. _In acetonitrile, then slowly add water soluble Until the aqueous solution needs to be engraved. • A turbid solution indicates that a part of the compound (I) has precipitated out, so it may not be necessary to add an additional amount of acetonitrile to dissolve the precipitate. For example, • a sufficient amount of acetonitrile. Standard (CNS &gt; A4 size (210 X 297 mm) (please read the precautions on the back before filling this page) -II _1 1 1! ^ 1 · 1 ^ 1 ^ 1 ^ 1 &gt; 1 ammf II * -12-Α7 Β7 Η.1 Avoiding the Correction and Complementing Nothing. Five \ _ 发 咧 1¾ 明 (, 〇Volume • So, there is a need to prepare a compound Ac— (L) —pAph-Chg—Pa IMe (3)-NH2 Method for the preparation of stereoisomers • The key intermediate for the preparation of this stereoisomer is the stereoisomer N-ethylglycosyl- (L) -4 4-cyanophenylalanine. The choice of technology used for this purpose • Some examples are crystallisation or differential absorption (decantation) after formation of non-mirromeric isomers [eg £ 11811 '^ 01116 ^, 1 ^ 0611 &gt; 81 ^ 8,811 (111 -esolutions, J. Jacques, A. Collet, and SH Vilen, Wiley (1981)] *], chromatography on palm stationary phases * Dynamic analysis, and enzyme analysis • Enzymatic analysis, especially hydrolytic enzymes or esterases, are proven to be used in the analysis of amino acids "such as in Che-mistry and Biochemistry of the amino acids, Chapman and Hall, New York, 1984, Chap. 10 pp. 338-353; in Applications of Biochemical Systems in Organi-c Chemistry, Part IJB Jones, CJ Sih, and D. Perlman, Wiley, New York, 1976, Chap 4. pp. 107 -401; and Chemistry of the Amino Acids, vol.l. Wiley, New York, 1961, Chap 9. pp 728-750 · Even after choosing a method, those skilled in the art must still Extensive experiments to find the correct solvent, co-solvent (if needed), temperature, time and other conditions to propose effective and efficient racemate analysis to easily recover the target compound Enantiomeric excess * and methods that are not too difficult to implement • The present invention can solve N-acetylammonium (D, L) -4 4-cyanophenylalanine This paper is sized to the Chinese National Standard (CNS) A4 (210 X 297 mm) (Jing "Read the Note # item on the back side and then fill out this page"--— — — — — — — — — — — — — — ———————————————————————————————————————————————————————————————————————————————————————————————————————————————---------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------)) (-) (7) Printed by the Consumers Cooperative of the Intellectual Property Bureau of the Ministry of Economy The analysis of these problems is related to the subsequent use of intermediate 雔 to prepare A c. — (L) — pAph — Chg-Pa IMe (3) -N Η 2, compound (Π). Summary of the Invention The present invention proposes a method for preparing N-ethylenyl- (L) -4-airyl phenylalanine, compound (IA):

Compound (IA which includes the following steps: a) Combine a sufficient amount of compound (I): 0 (Please read the note on the back before filling out this page) Printed compound printed by the staff consumer cooperative of Central Bureau of Economic Affairs of the Ministry of Economic Affairs (3 〇 a sufficient amount of an aqueous solution, a sufficient amount of acetonitrile, and a sufficient amount of a subtilisin to react with a real amount of the compound (I) to form a reaction medium; and b) the reaction medium is adjusted to be appropriate when the subtilisin is added This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 6 A7 B7 V. Description of the invention (4) P Η and maintain the appropriate value of ρ η when the reaction occurs to produce the compound (IA). The invention further proposes a compound A c — (L)-pAph-Chg — Pa IMe (3) -NH2, compound (Π):

A method of a salt acceptable to peony, including the following steps: a) Combining a sufficient amount of compound (I): Printed by Shellfish Consumer Cooperative, Central Bureau of Standards, Ministry of Economic Affairs

A sufficient amount of aqueous solution, a sufficient amount of acetonitrile, and a sufficient amount of subtilisin to react with the actual mass of the compound (I) to form a reaction medium; and (Please read the note on the back before filling this page)

This paper size applies to Chinese National Standard (CNS) A4 (210X297 mm) -7-Printed by A7 Consumers Cooperatives, Central Standards Bureau, Ministry of Economic Affairs __B7_ V. Description of Invention (5) b) When adding subtilisin The reaction medium is adjusted to an appropriate P Η value and maintained at the appropriate p Η value when the reaction occurs to produce a compound (IA): CN compound (IA) c) Coupling compound (IA) with compound (3):

d) The compound (4) is obtained by converting the cyano group of the compound (4) into a fatty group and methylating the nitrogen of the pyridyl group. Detailed description of the lingjia embodiment This paper size applies to Chinese national standard (CNS> A4 size (210X297mm) (Please read the note on the back before filling in this page) Φ _ 8-NH; A7 _B7_______ V. Description of the invention (6) The following terms have the meanings described below: (a) or &quot; 乙醯 基 # refers to a functionality with the formula = 0

II CH3-C—; (b) The term “fatyl I” refers to the following formula: (Please read the notes on the back before filling this page) c) ^ pyridyl # It is a general term for all stereoisomers with the following formula: J0, the Ministry of Economic Affairs, Central Tungfeng Bureau, Shellfish Consumer Cooperative, which prints stereoisomers that are different only in the orientation of their atoms in space. This includes isomers (non-mirro isomers) of compounds that have more than one palm center but are not mirror images of each other β 'mirror isomers # The term refers to two stereoisomers that are non-overlapping mirror images of each other体 物。 Structure. The term '^ palmity center' refers to a carbon atom followed by four different radicals "L / D or R / S is based on IUPAC-IUB Joint Commissi 〇-n on Biochemical Nomenclature, Eur. J. Biochem. 1 38 : 9-37 (1 984). Palm substances may contain equal amounts of R and S isomers (or L and D isomers), which is then called racemic 'or' ^ racemate ^, or it may not contain the same amount of R And S (or L and D isomers) in this case are called 'optical ##' 'non-racemic -line-

This paper size applies to China National Standard (CNS) A4 (210X 297 mm) 9 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 _____B7_ V. Description of the invention (7) The word 'analysis' means to be racemic The mixture is separated into its optically active constituents. The term 'mirror excess' or * e refers to a mirror isomer, E1, the percentage of the excess of the two mirror isomers, E1 plus E2, ie (+) — the term refers to positive Mirror image isomers, (a) refers to negative image isomers "" 1 "symbol refers to the key protruding from the paper surface &quot; linn., .. 'symbol refers to the key protruding from the paper surface backward The term 'pharmaceutically acceptable salts' includes with acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric or phosphoric acid and organic carboxylic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, maleic acid, tartaric acid, lemon Acid addition salts derived from the reaction of acids, salicylic acid, 2-ethoxybenzoic acid or organic sulfonic acids such as methanesulfonic acid, 4-toluenesulfonic acid and fruit acid. Of course, it can also be used in medical technology. Of other acids. As used herein, the term 'amino acid' is intended to include naturally occurring amino acids which are derived from translation of the genetic code and which constitute the building block of a protein. Unless otherwise specified, the term amino acid is also intended to include (1 ^) monoamino acids; (D) —both amino acids, chemically modified amino acids such as amino acid analogs, and are not generally Naturally occurring amino acids incorporated in proteins. Table 1 lists the abbreviations of amino acids and amino acid analogs that fall within the scope of this manual. (Please read the precautions on the back before filling in this card). This paper size applies to China National Standard (CNS) A4 (210X297 mm) -10-

A B7 V. Description of the invention (8) Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs Table 1 Amine Acid Symbol Alanine Ala Phenylalanine Phe P-Cyanophenylalanine Phe (4-CN) P-Phenylphenylalanine pAphe Cyclohexylglycine Chg / 3-(3-N-methylpyridine) -Alanine PalMe (3)

Procedure 1: Analysis procedure of compound (1) Procedure 1: Analysis procedure of N-ethylenyl- (D, L) -4-cyanophenylalanine ethyl ester A This paper is in accordance with China National Standard (CNS) A4 specification (210 '〆297mm） (Please read the notes on the back before filling this page) Order-line! -11-Feng Zheng Year and month Λ, — — 5. Description of the invention (g)

Step A COOH

Compound I Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs_ (Compound (I)) Racemate was resolved into a truncated N-ethenyl-4 monocyanophenylalanine (compound (IA)) L Stereo isomers and D_isomers of esters_ (Compound D — (I)) are not substantially converted to acids. This allows the desired acid to be separated from undesired esters. As a complete explanation, the acid obtained by the method is mainly the L isomer, as shown by its 髙 ee factor. A sufficient amount of the compound (I) used in combination to form the reaction medium is to be recovered as L-Lithosperylene by the method of the present invention At least twice as much as the structure is placed in. The reaction medium contains a sufficient amount of acetonitrile and a sufficient amount of an aqueous solution. Those sufficient amounts are required to facilitate the interaction of the additives according to the present invention. As used herein ， Additive # means any substance added to the reaction medium. The sufficient amount of acetonitrile is the amount that can dissolve the substantially compound (I) in the reaction medium. The better is to dissolve the compound (I) first. _In acetonitrile, then slowly add water soluble Until the aqueous solution needs to be engraved. • A turbid solution indicates that a part of the compound (I) has precipitated out, so it may not be necessary to add an additional amount of acetonitrile to dissolve the precipitants. Standard (CNS &gt; A4 size (210 X 297 mm) (please read the precautions on the back before filling this page) -II _1 1 1! ^ 1 · 1 ^ 1 ^ 1 ^ 1 &gt; 1 ammf II * -12-Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 ____ B7 V. Description of the invention (10) is about 5 to 95% by volume of the reaction medium and more preferably about 50 to 60% by volume. Better The concentration of the compound (I) is about 20 g to 140 g / L of the reaction medium and more preferably about 35 g to 65 g / L. The sufficient amount of the aqueous solution is a real mass of the enzyme Bacillus subtilis The amount of protease dissolved in the reaction medium. As used herein, the aqueous solution * is a solution containing water, and more preferably, a solution containing water and other additives that help increase the yield or ee factor. The term solution Does not necessarily mean that any additives added to the aqueous solution are to be dissolved; it may also mean Additives are dispersed to form a suspension. For example, 'the aqueous solution may further contain a sufficient amount of inorganic salts such as potassium gas, sodium chloride, lithium chloride, etc. as additives. Preferably, the inorganic salt is potassium chloride It is believed that the effect of the inorganic salt is to stabilize the subtilisin. A sufficient amount of the inorganic salt is an amount sufficient to stabilize the subtilisin. It is about 0.02 mol to about 0.20 mol / liter aqueous solution, more preferably It is about 0.05 to 0.15 mole / liter. For example, a 1M potassium chloride solution is combined at about 10 to about 15% by volume of the reaction medium. A sufficient amount is combined with compound (I), acetonitrile, and an aqueous solution. Subtilisin. A sufficient amount of the subtilisin is an amount capable of reacting with the real mass (the actual total amount when possible) of the compound (I). It is about 0.5 to about 10 milliequivalents of the amount of the compound (I). When adding enzymes, measure the p Η value of the reaction medium and adjust it to the appropriate ρ Η value if necessary. The appropriate ρ Η is the ρ Η value that the enzyme can respond to. The preferred ρ Η is from about 5 to about 9, and more preferably from about 6.5 to about 7.5. For the paper size of compound (I) and subtilisin, the Chinese National Standard (CNS) A4 specification (210X297 mm) is applicable (Please read the precautions on the back before filling this page) -13-Central Bureau of Standards, Ministry of Economic Affairs Printed by the Industrial and Consumer Cooperatives B7 V. Invention Description (11) The appropriate P value should be maintained when a reaction occurs. The time is about 15 minutes to 4 hours. Preferably, stirring or other appropriate methods are used to help the reaction. Any suitable means can be used to maintain the proper pH, for example, by adding a sufficient amount of tincture such as sodium hydroxide, potassium hydroxide, lithium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, preferably a 1 M solution Add, or via addition of buffers such as ammonium acetate, ammonium bicarbonate, sodium bicarbonate or phosphate buffers such as ammonium hydrogen phosphate, sodium hydrogen phosphate. A PQ meter can be used to monitor the PQ value of the reaction medium. The compound (IA) can be collected by a method known in the art. For example, the solution can be diluted by adding a solution of an alkaline inorganic salt such as sodium bicarbonate. The inorganic phase is extracted with an organic solvent such as ether, a chlorinated solvent such as dichloromethane, chloroform * toluene, heptane or ethyl acetate. The aqueous solution is acidified with a concentrated acid such as concentrated hydrochloric acid to a pH of about 1 to 3, and then extracted with an organic solvent to obtain the compound (I a) β. Table 2 and Table 3 Table 2 and Table 3 list the aqueous solution and acetonitrile (Table 2) And comparing the reaction with an aqueous solution and dimethylaniline I instead of acetonitrile (Table 3). Column A is the amount of compound (I) used in the test in grams. Column B lists the amounts of dimethoate (DMS 0) or acetonitrile (CH3CN), water and 1M potassium chloride solution used in the preparation of the aqueous solution in milliliter (mj?) Units. Column D lists the time taken to conduct the experiment. Column E is the excess of the mirror image isomer of the obtained compound (IA). This paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) &quot; &quot; &quot; '-14-(Please read the precautions on the back before filling this page) Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Preparation A7 B7 V. Description of the Invention (12) Column F shows the yield of the obtained compound (IA). 1. Compound (I A) contains DMSO and is very difficult to remove. 2. Evaporate at 90 ° C / 8 torr, dissolve in ethyl acetate and wash with water (3X) to remove DMSO »3. Compound (I A) is separated into a solid. Table 2 In item 1, the reaction was performed in the presence of an inorganic salt (potassium chloride). In item 2, the reaction was performed without inorganic salts. (Please read the notes on the back before filling this page)

This paper size applies Chinese National Standard (CNS) A4 specification (210X297mm) -15-V. Description of invention (13) A7 B7 Printed to 1—i marriage ΠΠ Ο ϋ CT &gt; QQ · · · · &Gt; Η- ^ W t ™ 1 ^ N · · \-/ 1— * 〇9C 〇— o cn o «« \ oo CO CD H- SS W • ◦ \ cn Cd Λ 亍 o tSD 1 —1 half o (W ^ grid 03 to 100 (3 .: rod. White enzyme mg: when 〇tO CT> «4 PP w sun / bid just 1 — * CO h — OD ^ Μ φ ^ σ 〇» CD clothes , CO oo CO Ν—Η 〇 &gt;-w CD CO OO ◦ OO / -N Painting m a pd (B mm tender ^ (Please read the notes on the back before filling this page) Alignment This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm) -16-V. Description of the invention (14) A7 B7 Case specific enzymes of egg white bacterium. A large amount of sub-A / -V II I use C to make the compound 3-in-1 (Please read the precautions on the back before filling Page)

Printed on the paper of the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) -17-V. Description of the invention (15) A7 B7 ABCDEF item (1) (g) DMS0 / H20 / Bacillus time (IA) (ΙΑ) 1M KC1 (ml) Protease (min)% ee% Yield (mg) (millie equivalent) 1 2.0 32/18 / 6.4 1.5 30 95.4 70 (oil, in (1.5) DMSO) 1 2 7. 8 125/100/25 12 30 94 88 (oil, in (3.0) DMSO) 1 3 25 400/200/81 19 40 88 70 (Oil, in (1.5) DMSO) 1 4 100 1600/900/320 75 60 88 90 (oil, in (1.5) DMSO V 5 2.8 10/8/2 4.4 65 86 50 (solid (3. 0) ) 2 6 2.0 42/26 / 6.4 1.5 120 80 70 (oil, in (1.5). DMSO) 1 This paper size is applicable to the Chinese National Standard (CNS) A4 size (210X297 mm) Please read ft back it first Matter refill line-18-Printed by ABC _B7, Shellfish Consumer Cooperative, Central Bureau of Standards, Ministry of Economic Affairs-V. Description of Invention (16) These results Shows that the use of acetonitrile can lead to effective and efficient analysis, facilitate easy recovery of compound (IA), and have high yields and high mirror image isomerization excesses. "Program 2 Program 2 is a follow-up to Program 1 and shows a Method for preparing compound A c — (L)-pAph-Chg-Pa IMe (3)-NH2, compound (Π). In procedure 2, step A, compound (3) is compound (IA) is compounded into compound (4). Compound (3) can be coupled by the azide method. For example, compound (IA) is first dissolved in an inert atmosphere, such as nitrogen, and dissolved in a suitable anhydrous organic solvent such as anhydrous dimethylformamide or anhydrous diamine. Chloromethane. To this solution is added diphenylphosphonium azide: L to 4 equivalents of a suitable hydrazone such as diisopropylethylamine and at least one equivalent, dissolved in a suitable anhydrous organic solvent such as anhydrous dimethyl Protected amino acids in formamidine or anhydrous dichloromethane. The reaction is then stirred for about 1 to 15 hours. The coupling is then separated and purified by techniques well known in the art such as extraction techniques, precipitation, crystallization and flash chromatography. Product (4). For example, the solvent is evaporated off, the coupling product (4) is precipitated with ether, washed and collected. In the procedure 2, steps B and D, the cyano group of the compound (4) is converted into the fatty group of the compound (Π). This transformation followed the procedure given in Wagner et a-1., DDR Patent No. 155,954, issue 21, 1982; November 9, 1988 re-examination via the corresponding compound (5) methylthiofluorene Imide (via compound (this paper size applies Chinese National Standard (CNS) A4 specification (210X297 cm)) (please read the precautions on the back before filling this page)) -19-Central Bureau of Standards, Ministry of Economic Affairs Printed by the consumer cooperative A7 ______B7_ V. Description of the invention (17) 4) The cyano group reacts with hydrogen sulfide to form) and is completed by amination; this patent is incorporated herein by reference. For example, in the procedure 2, step B, the compound (4) is dissolved in an organic solvent such as dimethylsulfoxide. Organic hydrazones such as pyridine, triethylamine, diisopropylethylamine, 2,6-dimethylpyridine, and trimethyl oxidine are added. A hydrogenated hydrogen stream was passed through the solution at room temperature until the compound (4) was consumed. The reaction can be kept at room temperature for another 1 to 18 hours. The compound (5) is collected according to methods well known in the art such as precipitation and filtration. Then the precipitate is washed with an organic solvent such as diethyl ether and dried in air. (Please read the notes on the back before filling this page)

Program 2: Synthesis of Ac— (L) -pAph-Chg-PaIMe (3) — N Η 2

Compound ΑΑ) Compound ⑶ Compound (4 &gt;,? Τ -Φ This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) -20-r A7 B7 V. Description of the invention (18) Step B Thioacetamidine Amine formation

Step C Methylation Step D 'fluorenyl formation

Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling in this x)

This paper size is applicable to Chinese National Standard (CNS) A4 (210X297 gong)-21-Printed by Aigong Consumer Cooperative, Central Standards Bureau, Ministry of Economic Affairs, printed A7 B7 5. Invention Description (19) In Procedure 2, Step C, the system will The pyridyl group of compound (5) is methylated to obtain compound (6). For this methylation, it is more common to use methyl halides such as methyl iodide, methyl bromide or methyl chloride. You can also use methyl fluorosulfonate, dimethyl sulfate, toluene-P-methyl sulfonate and other methylating agents such as Zoltewicz and Deady, Adv. Heterocycl. Che-m. 22, 7 1-1 2 1 ( 1 978) and Duffin, Adv. Heterocyc 1. Ch-em. 3, 1-5 6 (1 9 64), both of which are incorporated herein by reference. More preferred is the use of excess methyl halide and more preferred is the use of excess methyl iodide. This reaction is carried out in a solvent such as an alcohol such as methanol and ethanol, acetone, chloroform, acetonitrile, nitrobenzene, and dimethylformamide. More preferably, the reaction is performed in an acetone / dimethylarsine mixture and stirred at room temperature for 1 to 24 hours. Compound (6) is isolated according to procedures well known in the art, for example, excess methyl iodide is distilled off, and compound (6) is precipitated by adding ether, collected by decantation, washed and dried. In step 2, step D, the compound (6) methylthiofluorenimide is further converted into its corresponding compound (Π) fatty group. Preferably, the compound (6) is dissolved in an organic solvent such as methanol, and more preferably in the presence of acetic acid. To this solution was added acetic acid. The worse one, heat the reaction at a temperature in the range of 40 ° C to 65 ° C, and more preferably in the range of 50 ° C to 60 ° C and keep it at this temperature for 2 to 3 hours. The well-known procedure isolates the compound (II) and can be isolated into a peony acceptable salt. For example, the solvent is first distilled off, the residue is dissolved in acetonitrile, trifluoroacetic acid is added to precipitate, filtered and dried under vacuum. The following examples show that the typical combined paper sizes described in Procedure 1 and Procedure 2 are applicable to the Chinese National Standard (CNS) A4 (210X297 mm) (please read the notes on the back ^^ before filling out this page) )

-Line-22-A7 B7 V. Description of the invention (20) These embodiments are to be understood by those who are not sexually motivated and are not intended to limit the scope of the invention in any way. E. The order of the 9 steps in the procedures is selected according to the situation. The starting materials are all available on the market or easily made by procedures well-known in the art. As used in this article, the following terms have the meaning.% I G g t% m 0% Μ mol 9% mm 0 9. mol mol; L • liter tm L liter milliliter 9 β L m liter microliter t 'm ρ surface melting point 9% ° c Μ Table degrees Celsius &gt;% τ LC 雔 TLC t T Μ Table Moore volume concentration: and% R f Table retention factor 〇 Yin Example 1 (D &gt; L), N-ethyl alcohol -4 monocyanopropyl propylamine Ac — (D 9 L) P he (4 N) 0 Η Step A; 4 __ Gasoylacetamidodimalonate COOEt Human NHAc NC- &lt; (COOEt (Please read the precautions on the back before filling this page) Printed by the Central Laboratories Bureau of the Ministry of Economic Affairs Suspend diethyl acetamidomalonate (44 g, 0.203 mol), alpha-bromotoluene (40 g, 0.204 mol), and potassium iodide (10 g) in suspension Dioxin (400 ml) and sodium ethoxide solution (4.6 g of sodium in 200 ml of absolute ethanol) was added. The mixture was heated at reflux for 3-4 hours and allowed to stand overnight. The paper was scaled Applicable to China National Standard (CNS) A4 (210X297 mm) -23-Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs A7 __ B7_ V. Description of the invention (21) The mixture is poured on ice (2 liters) and filtered The precipitate was washed with water and dried on a freeze dryer. It was recrystallized from methanol to give white crystals 4 -Cyanobenzylacetamidomalonic acid diethyl ester (61 g, 91%) β Step N — (D , L) Ethyl aceto-4-cyanophenylalanine ethyl acetate, Ac- (D, L) Phe (4 — CN) -0Et COOEt

Diethyl 4-cyanobenzylacetamidomalonate (41.97 g, 0.126 mol) was suspended in ethanol (0.6 liter). Sodium hydroxide (6M) was added at the following intervals: 10 ml (60 mmol) at 0 minutes, 10 ml (60 mmol) after 30 minutes, and 5 ml (30 mmol) after 3 hours. The reaction was stirred while adding sodium hydroxide (GM) until the starting material disappeared (TL C, ethyl acetate, R f = 0.6 3). The solution was diluted with water (100 ml) and P 浓 was adjusted to 3 by adding concentrated hydrochloric acid. The ethanol was distilled off and the semi-solid product was dried on a lyophilizer all night to obtain N-ethylfluorenyl-4 -aminofluorenylacetamidine aminomalonate monoethyl ester. The prepared N-ethylamidine-4 monocyanoacetamidineaminomalonate was suspended in anhydrous dioxolane (0.5 liter) and heated at reflux for 2.5 to 3 hours. The dioxin garden was distilled off, and the solid was suspended in ethyl acetate (250 ml), and extracted with a saturated aqueous sodium hydrogen carbonate solution (3 ×), water, hydrochloric acid (0.5M), and brine in this order. Ethyl acetate solution This paper uses faili standard 4 (CNS) Α4 specification (210X297 mm)-24 _ (Please read the precautions on the back before filling this page)

Printed by the Consumer Cooperatives of the Central Bureau of Prospecting, Ministry of Economic Affairs A7 B7__V. Description of the invention (22) Dehydrate with magnesium sulfate, filter and evaporate. The product was recrystallized from ethyl acetate / hexane to give 28.95 g (88%) of ethyl N- (L, D) -ethenyl-4-monocyanophenylalanine. Example 2 Enzyme analysis of Ac- (D, L) P he (4-CN)-0 E t (6: 4, D: L) and (L) — N —Ethyl — 4 Alanine, Ac — (L) Phe (4 — CN) — OH 1 COOH NC in Ac- (D, L) Phe (4-CN) -0Et (6: 4, D: L, 67 g) in To the solution in acetonitrile (1 liter) was added potassium chloride solution (1M, 241 ml) and water (600 ml). The pH of the solution was adjusted from 7.3 to 6.9 and a solution of the subtilisin protease Carlsberg (50 mg) in an aqueous potassium vaporization solution (8 ml, 0.1 M) was added. Titrate with sodium hydroxide (1M) to maintain the PΗ value of the solution. After one hour, a solution of subtilisin (50 mg) in aqueous potassium chloride solution (8 ml, 0.1 M) was added and titrated with sodium hydroxide (1 M) to maintain the pH of the solution. After 2.5 hours, sodium bicarbonate solution (800 ml) was added and extracted with ethyl acetate (4 x 800 ml). The ethyl acetate solution was dehydrated with magnesium sulfate and the mixture was filtered. The filtrate was concentrated to obtain a solid A c-(D The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) — -25-(Please read the precautions on the back before filling this page) ^ 1

Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Invention Description (23)) 卩 116 (4-〇1 ^)-〇 £ 1; (38 g, 9 5%, 90% e e). The aqueous layer was acidified to PHI with concentrated hydrochloric acid (56 ml). The aqueous layer was extracted with ethyl acetate (4 × 800 ml). The organic layer was dehydrated with magnesium sulfate and the mixture was filtered. The filtrate was concentrated to give solid A c-(L) Phe (4-CN) -OH (21 g, 88%) mp 124-126. . . 99.4% ee. Example 3 N-Ethyl- (L) -p-fluorenylphenylalanine, Ac (L) _pAph- (L) -Chg- (L) -Pa IMe (3) -N H 2

Step A: N — third butoxycarbonyl mono (L) — yS — (3 —pyridyl) —alanine, Boc — (L) —Pa 1 — NH2

Suspend Boc— (L) — Pal— 0H (1.34 g, 5 mmol) in dichloromethane (50 ml) and cool to —1 5 (Please read the precautions on the back before filling this page )

This paper size applies to Chinese National Standard (CNS) A4 (210X297 mm) -26-Printed by A7 B7, Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (24) ° C »Add diisopropylethylamine ( 963 microliters, 5.5 millimoles) and isobutyl chloroformate (715 microliters, 5.5 millimoles). The mixture was stirred at -15 ° C for 15 minutes. A vigorous stream of anhydrous ammonia (dehydrated with solid sodium hydroxide) was passed through the solution for about 3 minutes. The mixture was stirred at 15 ° C for 10 minutes, and then stirred at room temperature for 20 minutes. Dichloromethane was distilled off, ethyl acetate (70 ml) was added and the mixture was extracted with a saturated aqueous sodium hydrogen carbonate solution, dehydrated with magnesium sulfate and evaporated to dryness. Crystallization from ethyl acetate / hexane gave Boc- (L) -Pal-NH2 (0.98 g, 75%). Step B: (L) — / 3-(3-pyridyl) -alanine hydrochloride, H- (L) -Pal-NH2 .2HC1 0S ^ 0NH2 2HCI nh2 Suspend Boc_ (L) — Pal-NH2 (0.98 g) in dichloromethane (15 ml) under slight heating and add hydrochloric acid (4M, 10 ml) / dioxolane solution. In 30 minutes After distilling off the dichloromethane and dioxin. The solid was dissolved in methanol. 'Ether was added to precipitate and filtered to obtain Η- (L) —Pa 1 -NH2 · 2 HC1 (0.86 g, 98%). Step C: N —Third-butoxycarbonyl- (L) -cyclohexyl glycine-(--- ^ --- j --- ^ ---. Ψ_ | (Please read the notes on the back before filling in this Page) Φ This paper size is in accordance with Chinese National Standard (CNS) A4 specification (210X297 mm) A7 B7 V. Description of the invention (25) L) —々 一 (3-pyridyl) —propionamine amine B〇c— ( L). -Chg- (L) -Pal-NH2

Printed by the Shellfish Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs, H-(L)-Pa 1-ΝΗ2 · 2HC1 (432 mg, 1.82 mmol), Boc- (L) -Chg-OH (609 mg, 2. 37 Millimoles, 1.3 equivalents), dicyclohexylcarbodiimide (494 mg, 2.4 millimoles), iclobenzotriazole (324 mg, 2.4 millimoles) and diiso A mixture of propylethylamine (4 mmol) in dimethylformamide (15 ml) was stirred overnight. Dimethylformamide was distilled off, ethyl acetate was added and the mixture was stored at room temperature for one hour. The diisopropylcarbodiimide cyclohexyl urea formed was filtered off, and the solution (3 ×) was extracted with a saturated sodium bicarbonate solution, dehydrated with magnesium sulfate, and evaporated to dryness. Boc-(L)-Chg-(L)-Pa 1-NH2 (598 mg, 81%) was obtained by crystallization by adding hexane. Step D: (L) —Cyclohexylglycine mono (L) in case of (3-pyridyl) monoalanine ammonium hydrochloride H— (L) — Chg_ (L) -Pal-NH2 * 2HC1 ( (Please read the notes on the back before filling this page)

This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm) -28-V. Description of invention (26) A7 B7

0! Η CD 2HCI

B0 C — (L) — C hg one (L) — P a 1 — N Η 2 (1 * 2 7 grams 3 • 1 2 millimoles) are suspended In dichloromethane (50 ml) f and add hydrochloric acid (10 ml> 4 M 9 in dioxolane) 0 30 minutes later 9 evaporate the dioxin and dichloromethane f to dissolve the solid Diethyl ether (200 ml) was added in methanol to precipitate the product. 9 Filtration gave Η — (L) — C hg — (L) — P a 1 — Ν Η 2 • 2 Η C 1 (1 • 1 2 g 95%) Step E; Ν — ethyl. ,, Iί acid group — P —- cyanophenylalanine mono (L) -cyclohexyl glycine — (L) — β — (3-pyridyl) monoalanine Phenamine AC- (L) -P he (4 — CN) — (L) — C hg — (L) — ρ a 1 — N Η 2 will be (L) — C hg — (L) — P a 1 — N Η 2. »2 Η C 1 (1 • 1 3 g &gt; 3 millimoles) &gt; AC-(L) P he (4 — C Ν ) — 0 Η (0 f 8 4 g &gt; 3 • 6 mmol) »Diisopropylethylamine (10 mmol) and Diphenylphosphonium azide (8 0 3 µl 9 3 • 6 mmol) j in dimethylformamide (30 ml) was stirred overnight. The dimethylformamide was distilled off, precipitated with ether, filtered and washed with ether to obtain Ac— (L ) —Phe ((please read the note on the back of the page before filling in this page) The paper size of the booklet is applicable to the Chinese standard (CNS) A4 (210X297 mm) -29-A7 B7 V. Description of the invention (27 ) 4-CN)-(L)-Chg-(L)-Pa 1-NH2 (1.282 g, 82%). Ο

Η CHb

Step F: N-Ethyl- (L) -p-thiamine phenylalanine- (L) -cyclohexylglycine- (L) _ / S-(3-pyridyl) -alanine acetamide Ac — (L)-Phe (4 —Thioxamine)-(L) -Chg-(L)-Pa 1-NH2 nh2

(Please read the note on the back ^ &amp;# &gt; before filling in this page) Order *-Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs Ac — (L) — Phe. (4 — CN) — ( L)-Chg-(L)-Pa 1-NH2 (1.18 g, 2. 28 mmol) was dissolved in dimethyl asus (20-40 ml) and pyridine (40 ml) and triethylamine (14 Ml)> Hydrogen sulfide was passed through the solution at room temperature for 30 minutes. After keeping the solution at room temperature all night, it was evaporated to a small volume and the product was precipitated with ether. Placed on ice-paper scale, using Chinese national standard (CNS> A4 specification (210X297mm) -30-A7 B7 V. Description of the invention (28) In the box for several hours, filtering, washing with ether and vacuum drying to obtain a yellow solid Ac — (L) — Phe (4—thiothiamine) — (L) —Chg — (L) — P a 1 Ν Η (1.26 g) Step G: N —Ethylfluorenyl-p —methyl Thiosamine root phenylalanine monocyclohexyl glycine mono (L) — / 5 — (3-methylpyridine) _alanine hydroiodate Ac — (L) —Phe (4 — methyl thioaniline Root) — (L) — Chg — (L) — Pa 1 (Me) — NH2 * 2 Η sch3

Θ Please read the note

I ^ ^ IjC ¥ i Order Printed by Shellfish Consumer Cooperative, Central Bureau of Standards, Ministry of Economic Affairs

Chg-(L)-Pa 1-NH2 (1.2 g) was suspended in dimethanine (10-20 ml) and acetone (80 ml). Add methyl iodide (14 ml, 50 eq) to keep the reaction at room temperature all night, and distill off the acetone and excess methyl iodide. Decant with ether (0.5-1 liter). After being placed at 4 ° C for several hours, the ether was decanted or filtered and the semi-solid on the wall was washed with ether and ethyl acetate. The product was dried under vacuum to obtain Ac — (L) — Phe (4-methylthioamido) — (L)-Chg-(L)-Pal (Me)-Ν Η 2 (CNS) A4 specification (210X297 gong) -31-Printed by A7 B7, Shellfish Consumer Cooperative of Central Standards Bureau of the Ministry of Economic Affairs 5. Description of invention (29) 2 Η I (1.31 g). Step H: Ν-ethenyl- (L) -p-ethenylaminophenylalanine-cyclohexylglycine- (L)-^-(3-N-methylming) -alanine trifluoride Ac_ (L) -pAphe — _Chg-(L) — Pa IMe — NHzTFA Ac — (L) —Phe (4 —methylthiocarbamate) — (L)-Chg-(L) — Pa 1 (Me) — NH2 · 2HI (1.31 g) was dissolved in methanol (50 ml) and acetic acid (0.5 ml). Ammonium acetate (0.8 g) was added. The mixture was heated to 55 ° C for three hours and 'evaporated' dissolved in acetonitrile / water (0.1% trifluoroacetic acid) (1: 1) 'filtered and freeze-dried. This gives Ac — (L)-pAphe-(L)-Chg-(L) -Pa IMe-NHz.TFA (1.22 g). NH2

The present invention relates to the preparation which can be used to prepare Ac-(L)-pAph-Chg-Pa IMe (3)-NH2, the intermediate of compound (π), N-ethylamyl-L-phenylalanine (I A). This compound (Π) can be used in the inhibition of coagulation proteins ’and is more specific (please read the precautions on the back before filling this page)

This paper size applies the Chinese National Standard (CNS) M specification (210X297 mm) -32-Printed by the Central Bureau of Specimen Bureau of the Ministry of Economic Affairs, Shellfish Consumer Cooperative, A7, B7__V. Description of the invention (30) In conclusion, it is used for coagulation Among the inhibition of enzyme factors and X a 〃, as described more fully in US Patent Application Serial No. 08 / 428,404 filed on April 25, 1995, which is incorporated herein by reference. . As used herein, factor Xa activity # refers to the activity of factor Xa 'itself or in a subunit assembly called a prothrombinase complex that catalyzes the conversion reaction of prothrombin into thrombin "in When used in reference to factor Xa activity, the term inhibition includes the direct and indirect inhibition of factor Xa activity. Direct inhibition of factor Xa activity can be accomplished, for example, by binding a peptide to factor Xa or prothrombinase to prevent prothrombin from binding to the active site of the prothrombinase complex. As used herein, when used to refer to the inhibition of factor Xa activity, `` specific ''-the word means that the compound can inhibit factor Xa activity without substantially inhibiting the activity of other indicated proteases (using Under the same inhibitor concentration). Their other indicated proteases include, for example, proteases involved in the coagulation cascade such as thrombin, hemoglobin, trypsin and elastase. Although the formation of blood clots in the circulatory system is the source of the disease in some disease states, it is not appropriate to completely suppress the coagulation system because life-threatening bleeding may follow. The blood coagulation process is a complex procedure that includes a growing series of enzyme activation reactions in which plasma zymogens are sequentially activated via restricted proteolysis. In terms of mechanism, the coagulation level connection is divided into two ways. ^ The two paper sizes of internal and external are applicable to the Chinese National Standard (CNS) A4 (210X297 mm) (please read the back first) (Please note this page before filling in this page.) -33-Seal of the Central Standards Bureau of the Ministry of Economic Affairs, Shellfish Consumer Cooperatives, Seal A7, B7 V. Invention Description (31). These pathways intersect at the activation of factor X, followed by the production of thrombin via a single 'common pathway #'. Current evidence suggests that the endogenous pathway plays an important role in the maintenance and growth of fibrin formation, while the exogenous pathway is important in the initial stages of blood coagulation. It is now generally accepted that the blood coagulation system physically starts when the tissue factor / factor VlHa complex is formed. Once formed, this complex rapidly initiates coagulation via activating factors IX and X. The newly generated X a forms a one-to-one complex with the factor Via and phospholipids. The so-called prothrombinase is responsible for converting soluble fibrinogen into insoluble hemoglobin. Over time, the activity of the factor \ la / tissue factor complex (exogenous pathway) will be suppressed by the kunitz-type protease inhibitor protein, TFPI, which, when complexed to factor Xa, can directly inhibit the factor YU a / Tissue factor proteolytic activity. In order to maintain the coagulation program in the presence of a suppressed exogenous system, more factor X a is generated via the endogenous pathway activity of thrombin-mediated formation. Factor thrombin has a dual &gt; autocatalytic ^ 1 effect, leading to its own production and conversion of fibrinogen to fibrin. The autocatalytic nature of thrombin production is an important defense against uncontrolled bleeding. It can make blood coagulation to complete and promote in the presence of a predetermined low level of prothrombinase, for example, the termination of bleeding. It is therefore most desirable to develop agents that inhibit coagulation but do not directly inhibit thrombin. The compound A c — p A ph — Chg-Pa IMe (3)-NH2-a kind of peptide analogue-which is synthesized by the method of the present invention, that is, the Chinese paper standard (CNS) A4 (210X297 mm) is applied to this paper size ( Please read the note on the back before filling this page) Threading ~ 34-A7 _ B7_ V. Description of the Invention (32) is a useful inhibitor of factor X a activity and does not substantially inhibit other proteases involved in the coagulation process active. (Please read the precautions on the back before filling this page) Order-printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs -l ·-I--* 1 This paper size applies to Chinese national standards (CNS &gt; A4 specifications (210X297 Mm)-35-

Claims (1)

1988 8. U miserable 'Λ 9 4 —-Μ- 389? ^ VI. Annex 1 (A) of the scope of patent application: Patent application No. 8 5 1 1 5 5 9 No. 2 Chinese patent application scope amendment Amendment 1 of the Republic of China in August 88 1 — — Preparation N —ethenyl — (L) 4-Cyanophenylalanine, compound (IA) method: CN Compound (I A) It includes the following steps: a) Combine a sufficient amount of the compound Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A sufficient amount of an aqueous solution containing 10 to 15% of an inorganic salt (calculated based on 1 mole of water of the inorganic salt), the amount of which is 5 to 95% of acetonitrile in the reaction medium, and its amount For the compound (I), use centimeters; ^ 0.5 to 10 milliequivalents of subtilisin to react with the compound (j 彡 to form a reaction medium: and this paper size applies to China National Standard (CNS) A4 (210X297) (Centimeter) 88 years 8. U miserable 'Λ 9 4 —-Μ- 389? ^ VI. Annex 1 (A) of the scope of patent application: Patent application No. 8 5 1 1 5 5 9 No. 2 Chinese patent application scope amendment Amendment 1 of the Republic of China in August 88 1 — — Preparation N —ethenyl — (L) 4-Cyanophenylalanine, compound (IA) method: CN Compound (I A) It includes the following steps: a) Combine a sufficient amount of the compound Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A sufficient amount of an aqueous solution containing 10 to 15% of an inorganic salt (calculated based on 1 mole of water of the inorganic salt), the amount of which is 5 to 95% of acetonitrile in the reaction medium, and its amount For the compound (I), use centimeters; ^ 0.5 to 10 milliequivalents of subtilisin to react with the compound (j 彡 to form a reaction medium: and this paper size applies to China National Standard (CNS) A4 (210X297) C) A8, B8, C8, D8, 389,755. The scope of patent application. B) The minute to 2 will be divided into 3 potassium chloride, 4 sufficient amount of sodium chloride. 6 Adequate amount of 7 Phosphate 8 (please read the notes on the back before filling this page) The reaction medium is adjusted to an appropriate pH between 5 and 9, and the appropriate pH value is maintained during the reaction and the reaction medium is stirred. Compound (IA) is produced by reacting for 15 to 4 hours. The method according to item 1 of the patent application 'wherein the acetonitrile fills 50 to 60% of the volume of the aqueous solution °. The method according to item 1 of the patent application' where the inorganic salt is 0 The method of item 1 wherein the appropriate PΗ value of the reaction medium is adjusted and maintained through the added base. The method according to item 4 of the patent application, wherein the hydrogen is oxygen. The method according to item 1 of the patent application, where the buffer medium is added to adjust and maintain the appropriate value of the reaction medium. The method according to item 6 of the patent application, wherein the buffer is a solution. Printed by the staff of the Intellectual Property Bureau of the Ministry of Economic Affairs on anti-corruption cooperation. For example, the method of applying for the first item of the patent scope, wherein the pH of the reaction medium is adjusted and maintained at a pH of 6.5 to 7.5. The method of this item further includes a step of collecting the compound (IA) from the reaction medium. C hg · —A compound for preparation Ac — (L) — pAph PalMe (3) -NH2, compound (π): This paper is in ft scale and is used in China National Standards (CNS) A4 (210X297 mm) -2-Α8 Β8 C8 D8 389755 6. Scope of patent application Or a method of its pharmaceutically acceptable salt, which includes the following steps: a) Combine the fully compound (I): (Please read the notes on the back before filling this page) Order the consumer cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs to print a sufficient amount of an aqueous solution containing 10 to 15% of an inorganic salt (calculated based on the volume of the 1 mol aqueous solution of the inorganic salt). Its child is 5 to 9 5 of the volume of the reaction medium. % Acetonitrile, and its amount is 0.5 to 10 milliliters of subtilisin in the amount of the compound (I) to react with the compound (1) to form a reaction medium; and b) adjusting the reaction medium to 5 to The appropriate pH between 9 and when the reaction occurs to maintain the appropriate P 并 value and stir the reaction medium to react for 15 minutes to 4 hours to produce the compound (ΙΑ). CNS) 8 cases (210 ×: 297 mm) -3-Α8 Β8 C8 D8 Scope of patent application CN compound (Γ A) couples compound (Γ A) with compound (3) And the compound (4 ^ Order · ------. ^ .— /: &gt; (Please read the notes on the back before filling in this X) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs d) The cyanide of compound (4) The compound is converted to a fatty group and the pyridyl nitrogen is methylated to obtain a compound (Π). 11. The method according to item 10 of the scope of patent application, wherein the sufficient amount of the acetonitrile is 50 to 60% of the aqueous solution. 12. The method according to item 10 of the scope of patent application, wherein the inorganic salt is potassium chloride. — "I. Add the method of Zhui Li Di Di 1 Π, it is appropriate. China and Vietnam are based on the standard of this paper and used by the Chinese country (CNS &gt; Α4 size (210X297 mm) -4 382755. VI. Application The scope of the patent is to add a sufficient amount of base to adjust and maintain the appropriate P η value of the reaction medium. 1 4. The method according to item 13 of the scope of patent application, wherein the base is sodium hydroxide. 1 5 The method according to item 10, wherein the sufficient ρ 添加 value of the reaction medium is adjusted and maintained by adding a sufficient amount of buffer solution. 1 6 · The method according to item 15 of the scope of patent application, wherein the buffer solution is a phosphate solution 1 7. The method according to item 10 of the scope of patent application 'wherein the appropriate pH value of the reaction medium is adjusted and maintained at a pH value of 6.5 to 7.5. 1 8. As the item 10 in the scope of patent application The method 'also includes the step of collecting the compound (I Α) from the reaction medium. --- (Please read the notes on the back before filling this page) • Words-Printed by the staff of the Intellectual Property Bureau of the Ministry of Economic Affairs Paper size using China National Standards (CNS) 4 Wash cells (210Χ297 mm)