KR20210092883A - Method for preparing lysine-linked deoxycholic acid salt - Google Patents

Abstract

The present invention relates to a method for preparing a lysine-deoxycholic acid salt. The method for preparing a lysine-deoxycholic acid salt comprises the step of performing a coupling reaction of deoxycholic acid (DOCA) and lysine (Lys) salt in the presence of a phosphite-based compound or an acyl halide-based compound. The method for preparing a lysine-deoxycholic acid salt of the present invention can economically and efficiently prepare a high-purity lysine-deoxycholic acid salt.

Description

Lysine-deoxycholic acid salt production method {METHOD FOR PREPARING LYSINE-LINKED DEOXYCHOLIC ACID SALT}

The present invention relates to a method for preparing a lysine-deoxycholic acid salt that can be used as a bio-enhancer, and more particularly, a method for preparing a lysine-deoxycholic acid salt of high purity economically and efficiently is about

Lysine-deoxycholic acid (LysDOCA) or methyl 6-amino-2-(4-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl) ^{Pentanamido) hexanoate is an amphipathic substance having a positive charge in aqueous solution, and N α-} deoxychoyl-L-lysyl-methylester (DCK) chemically combined with L-lysine (Lys) and deoxycholic acid (bile acid, DOCA). )am.

Deoxycholic acid is a kind of bile acid, has a chemical formula of C ₂₄ H ₄₀ O ₄ , and a molecular weight of 393 g/mol, which may correspond to 3α,12α-dihydroxycholan.

Lysine (lysine) is one of the basic α- amino acids with the formula H ₂ N(CH ₂ ) ₄ (NH ₂ )COOH and has a molecular weight of 146.19 g/mol. It is abundantly present in animal protein, and has a small content in plant protein, is not synthesized in the body as an essential amino acid, and is also used in food processing.

Lysine-deoxycholic acid acts as an oral absorption enhancer, and as it forms a complex by ionic bonding with a negatively charged hydrophilic active ingredient in an aqueous solution, it increases the lipophilicity of the hydrophilic drug molecule and distributes it into the oil phase. can improve Deoxycholic acid is an amphiphilic molecule composed of a hydrophobic portion and a hydrophilic portion, which binds perpendicularly to the membrane surface of the cellular lipid bilayer and disrupts the acyl chains of the lipid matrix, thereby increasing the flexibility of the intestinal cell membrane and the solubility of fat-soluble drugs in the membrane. It can improve the membrane permeability of drugs.

In addition, the drug-bound bile acid derivative, deoxycholic acid, can be recognized by the bile acid reuptake transporter present on the intestinal membrane surface, which causes a high concentration gradient of the intestinal mucosal surface drug, resulting in passive diffusion of the drug into the intestinal membrane. to increase Therefore, the absorption enhancing effect of the drug by selective interaction with the bile acid reabsorption carrier is different from the existing absorption enhancers added simply by physical mixing, the absorption enhancers in the gastrointestinal tract after administration are diluted by the fluid present in the gastrointestinal tract. Since this decrease can be minimized, the gastrointestinal absorption rate of the hydrophilic drug can be increased with minimal use.

That is, lysine-deoxycholic acid is a bio-enhancer drug that binds to the drug and acts on the ASBT transporter in the upper small intestine, and is used in the form of lysine-deoxycholic acid hydrochloride, which has the structure of Formula 5 below.

[Formula 5]

Lysine-deoxycholic acid hydrochloride of Formula 5 passes through the intermediate of Formula 2 activated with ethyl carbonic anhydride through ethyl chloroformate under the conditions of N-methylmorpholine, as shown in Scheme 1 below, It is known that it can be prepared by deprotection with acetyl chloride after undergoing the preparation method of Chemical Formula 4 by performing an amide coupling reaction with the amine group-protected lysine (Formula 3).

[Scheme 1]

However, in the known synthesis method, anhydrous reaction conditions under perfectly controlled reaction conditions and a large amount of expensive tetrahydrofuran solvent must be used, and it is generated from N-methylmorpholine, a strong base used in the reaction, and ethyl chloroformate with strong reactivity. Not only the yield is reduced due to the side reactants, but also a tedious column process must be used to remove these side reactants, thereby increasing the manufacturing cost for the solvent and the production process.

In addition, according to the known process, after deprotection, the organic by-product is transferred to the organic solvent layer, the distilled water layer containing the desired compound 5 is recovered, and then the distilled water layer is subjected to a process of freeze-drying for 24 hours or more. Anhydrous conditions and column processes required for this reaction, and a mass freeze-drying process for removing the water layer, etc. may be possible for small-scale production of 10 g, but have a decisive disadvantage in that they are not advantageous for large-scale production of units of kg.

Korean Patent Registration No. 1796604 (Registration Date: November 6, 2017) Korean Patent Registration No. 1895237 (Registration Date: August 30, 2018) Korean Patent Publication No. 2019-0070653 (published date: June 21, 2019)

An object of the present invention is to provide a method for economically and efficiently preparing a high-purity lysine-deoxycholic acid salt.

Another object of the present invention is to provide a novel intermediate for the preparation of lysine-deoxycholic acid salt.

Another object of the present invention is to provide a novel purification process for the preparation of lysine-deoxycholic acid salt.

Lysine-deoxycholic acid (LysDOCA) salt preparation method according to an embodiment of the present invention in the presence of a phosphite-based compound represented by the following Chemical Formula 6, deoxycholic acid (DOCA) and lysine (lysine) , Lys) coupling the salt.

[Formula 6]

In Formula 6, R ⁶¹ to R ⁶³ are each independently any one selected from the group consisting of a hydrogen group, an alkyl group, and an aryl group.

The phosphite-based compound may be triphenyl phosphite.

The deoxycholic acid, the lysine salt and the phosphite-based compound may be reacted in a molar ratio of 1: 0.94 to 0.42: 1.49 to 2.24 (deoxycholic acid: lysine salt: phosphite-based compound).

The coupling reaction step may be accomplished through an intermediate represented by the following Chemical Formula 7.

[Formula 7]

In Formula 7, R ⁶¹ and R ⁶² are each independently any one selected from the group consisting of a hydrogen group, an alkyl group, and an aryl group.

Lysine-deoxycholic acid (LysDOCA) salt preparation method according to another embodiment of the present invention in the presence of an acyl halide-based compound represented by the following Chemical Formula 8, deoxycholic acid (DOCA) and lysine (lysine, Lys) may include a coupling reaction of the salt.

[Formula 8]

In Formula 8, R ⁸¹ to R ⁸³ are each independently any one substituent selected from the group consisting of a hydrogen group, an alkyl group, and an aryl group, and X ⁸¹ is fluoride (F), chloride (Cl), bromine ( Br) and any one halogen substituent selected from the group consisting of iodine (I).

The acyl halide-based compound may be pivaloyl chloride.

The deoxycholic acid, the lysine salt and the acyl halide compound may be reacted in a molar ratio of 1: 2.37 to 3.56: 1.20 to 1.80 (deoxycholic acid: lysine salt: acyl halide compound).

The coupling reaction is performed in the presence of any one base selected from the group consisting of triethylamine (TEA), pyridine, lutidine, dimethylamine, and mixtures thereof. can be done

The coupling reaction step may be performed through an intermediate represented by the following Chemical Formula 9.

[Formula 9]

In Formula 9, X ⁸¹ is any one substituent selected from the group consisting of fluoride (F), chloride (Cl), bromine (Br), and iodine (I).

The deoxycholic acid may be represented by Formula 1 below, the lysine salt may be represented by Formula 3 below, and the lysine-deoxycholic acid salt may be represented by Formula 5 below.

[Formula 1]

[Formula 3]

[Formula 5]

The coupling reaction is selected from the group consisting of dimethylformamide (dimethylformaide, DMF), tetrahydrofuran (THF), dichloromethane (DCM), chloroform (chloroform), methanol (MeOH), and mixtures thereof. It can be made in any one solvent.

The coupling reaction may be performed at a temperature of 5 °C to 60 °C.

The lysine salt is an amine group-protected lysine salt, and the lysine-deoxycholic acid salt preparation method may further include deprotecting the amine group protected from the prepared lysine-deoxycholic acid salt.

The method for preparing a lysine-deoxycholic acid salt of the present invention can economically and efficiently produce a high-purity lysine-deoxycholic acid salt, and provides a novel intermediate and a novel purification process for the production of lysine-deoxycholic acid salt. can

Hereinafter, the present invention will be described in more detail.

Unless otherwise specified herein, the alkyl group includes a primary alkyl group, a secondary alkyl group and a tertiary alkyl group.

In the present specification, unless otherwise specified, a halogen group means a fluoro group, a chloro group, a bromo group, or an iodo group.

Unless otherwise specified herein, all compounds or substituents may be substituted or unsubstituted. Here, substituted means that hydrogen is a halogen group, a hydroxyl group, a carboxy group, a cyano group, a nitro group, an amino group, a thio group, a methylthio group, an alkoxy group, a nitrile group, an aldehyde group, an epoxy group, an ether group, an ester group, a carbonyl group, An acetal group, a ketone group, an alkyl group, a perfluoroalkyl group, a cycloalkyl group, a heterocycloalkyl group, a benzyl group, an aryl group, a heteroaryl group, derivatives thereof, and combinations thereof means replaced with any one selected from the group consisting of .

Unless otherwise specified in the present specification, the alkyl group is a straight-chain or pulverized alkyl group having 1 to 10 carbon atoms, the halogenated alkyl group is a straight-chain or pulverized halogenated alkyl group having 1 to 10 carbon atoms, the aryl group is an aryl group having 6 to 30 carbon atoms, and the heteroaryl group is a carbon number A heteroaryl group having 2 to 30 and an alkoxy group means an alkoxy group having 1 to 10 carbon atoms.

Unless otherwise specified herein, the halogenated alkyl group means an alkyl group in which some hydrogen atoms or all hydrogen atoms are substituted with halogen atoms. For example, the halogenated alkyl group includes a perfluoroalkyl group.

Unless otherwise specified in the present specification, an aryl group means a chemical group obtained by removing one hydrogen atom from a monocyclic or polycyclic compound having 2 to 30 carbon atoms and derivatives thereof including one or more benzene rings, for example, benzene A monocyclic or polycyclic compound containing a ring is a benzene ring, toluene or xylene with an alkyl side chain attached to a benzene ring, biphenyl, etc. in which two or more benzene rings are bonded by a single bond, a benzene ring is a cycloalkyl group or a heterocycloalkyl group and fluorene, xanthene, or anthraquinone condensed with , naphthalene or anthracene in which two or more benzene rings are condensed.

In the present specification, unless otherwise specified, the prefix hetero means that 1 to 3 hetero atoms selected from the group consisting of -N-, -O-, -S- and -P- are substituted for carbon atoms. For example, it may be pyridine, pyrrole or carbazole containing a nitrogen atom as a hetero atom, furan or dibenzofuran containing an oxygen atom as a hetero atom, or the like, or dibenzothiophene, diphenylamine, and the like.

Lysine-deoxycholic acid (LysDOCA) salt preparation method according to an embodiment of the present invention in the presence of a phosphite-based compound represented by the following Chemical Formula 6, deoxycholic acid (DOCA) and lysine (lysine) , Lys) coupling the salt.

[Formula 6]

In Formula 6, R ⁶¹ to R ⁶³ may each independently be any one selected from the group consisting of a hydrogen group, an alkyl group, and an aryl group, for example, a phenyl group. Accordingly, the phosphite-based compound may be, for example, triphenyl phosphite.

Deoxycholic acid may be represented by Formula 1 below, lysine salt may be represented by Formula 3 below, and lysine-deoxycholic acid salt may be represented by Formula 5 below. As illustrated in the formula below, the lysine salt may be a lysine hydrochloride in which an amine group is protected with a t-butyloxycarbonyl (BOC), lysine-deoxycholic acid salt is lysine-deoxy cholic acid hydrochloride.

[Formula 1]

[Formula 3]

[Formula 5]

The coupling reaction step may be accomplished through a novel intermediate represented by the following formula (7).

[Formula 7]

In Formula 7, R ⁶¹ and R ⁶² are a substituent derived from a phosphite-based compound, and may be the same ^{as R 61} and R ^{62 defined in Formula 6.}

Deoxycholic acid, lysine salt and phosphite-based compound may be reacted in a molar ratio of 1: 0.94 to 0.42: 1.49 to 2.24 (deoxycholic acid: lysine salt: phosphite-based compound). If the molar ratio is out of the range, unreacted material may be generated, but may be removed during the purification process.

The coupling reaction is any one selected from the group consisting of dimethylformamide (dimethylformaide, DMF), tetrahydrofuran (THF), dichloromethane (DCM), chloroform, methanol (MeOH), and mixtures thereof. It can be carried out in one solvent, and the use of dimethylformamide has the fastest reaction rate.

The coupling reaction may be carried out at a temperature of 5 °C to 60 °C, specifically, at room temperature (25 °C). If the coupling reaction temperature is too high, other by-products are likely to be formed.

As such, a method for preparing a lysine-deoxycholic acid salt is described in detail with an example as follows. First, dimethylformamide (DMF) is added and dissolved in lysine hydrochloride in which deoxycholic acid represented by Formula 1 and an amine group represented by Formula 3 are protected with a t-butoxycarbonyl group. Here, imidazole and triphenylphosphite represented by Formula 6 are added and stirred to react. After completion of the reaction, ethyl acetate and purified water were added, stirred, and the layers were separated to remove the purified water layer. Aqueous hydrochloric acid solution was added, stirred, and the layers were separated to remove the water layer. Moisture is removed using magnesium sulfate on the ethyl acetate layer. After filtration, washing with ethyl acetate and concentration under reduced pressure, the oil-type material is purified by column chromatography under ethyl acetate/hexane conditions to obtain lysine-deoxycholic acid hydrochloride represented by compound 4 in a solid form.

On the other hand, the method for preparing the lysine-deoxycholic acid salt may further include the step of deprotecting the amine group protected in the prepared lysine-deoxycholic acid salt.

Specifically, the step of deprotecting the amine group is a step of adding acetyl chloride dropwise to an alcohol-based solvent such as methanol, then dissolving the prepared lysine-deoxycholic acid salt and reacting it, evaporating it under reduced pressure to remove the solvent, and After dissolving the oil-type mixture in an alcohol-based solvent such as methanol, it can be slowly added dropwise to ethyl acetate and dispersed to crystallize the solid material.

As such, the lysine-phosphite-based compound and its by-products used in the preparation method of the lysine-deoxycholic acid salt may interfere with the recrystallization process of the lysine-deoxycholic acid salt as an organic by-product, but may be removed through a relatively easy column process.

In addition, there is no by-product reacting with the lysine-deoxycholic acid salt prepared in the method for preparing the lysine-deoxycholic acid salt, and the lysine-deoxycholic acid salt can be obtained in a high yield of 70% or more.

In addition, in the preparation method of lysine-deoxycholic acid salt, due to the complete separation of by-products, a method of dissolving an oil-type mixture in methanol in the final step and then slowly adding it dropwise to ethyl acetate and dispersing it can be used to crystallize a solid material. there is.

Lysine-deoxycholic acid (LysDOCA) salt preparation method according to another embodiment of the present invention in the presence of an acyl halide-based compound represented by the following Chemical Formula 8, deoxycholic acid (DOCA) and lysine (lysine, Lys) comprising a coupling reaction of the salt.

[Formula 8]

In Formula 8, R ⁸¹ to R ⁸³ may each independently be any one substituent selected from the group consisting of a hydrogen group, an alkyl group, and an aryl group, and may be, for example, a methyl group, an ethyl group, or a butyl group. X ⁸¹ may be any one halogen substituent selected from the group consisting of fluoride (F), chloride (Cl), bromine (Br) and iodine (I), and may be, for example, chloride. Accordingly, the acyl halide-based compound may be, for example, pivaloyl chloride.

Deoxycholic acid may be represented by Formula 1 below, lysine salt may be represented by Formula 3 below, and lysine-deoxycholic acid salt may be represented by Formula 5 below. As illustrated in the formula below, the lysine salt may be a lysine hydrochloride in which an amine group is protected with a t-butyloxycarbonyl (BOC), lysine-deoxycholic acid salt is lysine-deoxy cholic acid hydrochloride.

[Formula 1]

[Formula 3]

[Formula 5]

The coupling reaction step may be accomplished through a novel intermediate represented by the following Chemical Formula 9.

[Formula 9]

In Formula 9, X ⁸¹ is a substituent derived from an acyl halide-based compound, and may be the same ^{as X 81 defined in Formula 8.}

Deoxycholic acid, lysine salt and acyl halide compound can be reacted in a molar ratio of 1:2.37 to 3.56: 1.20 to 1.80 (deoxycholic acid: lysine salt: acyl halide compound). If the molar ratio is out of the range, unreacted material may be generated, but may be removed during the purification process.

The coupling reaction is performed in the presence of any one base selected from the group consisting of triethylamine (TEA), pyridine, lutidine, dimethylamine, and mixtures thereof. and triethylamine may be used in terms of price or reactivity.

The coupling reaction is any one selected from the group consisting of dimethylformamide (dimethylformaide, DMF), tetrahydrofuran (THF), dichloromethane (DCM), chloroform, methanol (MeOH), and mixtures thereof. It can be carried out in one solvent, and the use of dimethylformamide has the fastest reaction rate.

The coupling reaction may be carried out at a temperature of 5 °C to 60 °C, specifically, at room temperature (25 °C). If the coupling reaction temperature is too high, other by-products are likely to be formed.

As such, a method for preparing a lysine-deoxycholic acid salt is described in detail with an example as follows. First, deoxycholic acid represented by Formula 1 is dissolved by adding dimethylformamide (DMF), and triethylamine is added thereto and stirred. After adding pivaloyl chloride represented by Chemical Formula 8 to the reaction mixture and stirring, triethylamine was added and stirred. Lysine hydrochloride in which the amine group represented by Formula 3 is protected with a t-butoxycarbonyl group is added and reacted.

After completion of the reaction, ethyl acetate was added, stirred, filtered, and washed with ethyl acetate. Water was added to the filtrate, stirred, and left to separate the ethyl acetate layer. A saturated aqueous sodium bicarbonate solution was added to the ethyl acetate layer, stirred, and then left to stand to collect the ethyl acetate layers and remove moisture using magnesium sulfate. After filtration, the mixture was washed with ethyl acetate and concentrated under reduced pressure to obtain lysine-deoxycholic acid hydrochloride represented by compound 4 in the form of a solid.

On the other hand, the method for preparing the lysine-deoxycholic acid salt may further include the step of deprotecting the amine group protected in the prepared lysine-deoxycholic acid salt.

Specifically, the step of deprotecting the amine group is a step of adding acetyl chloride dropwise to an alcohol-based solvent such as methanol, then dissolving the prepared lysine-deoxycholic acid salt and reacting it, evaporating it under reduced pressure to remove the solvent, and After dissolving the oil-type mixture in an alcohol-based solvent such as methanol, it can be slowly added dropwise to ethyl acetate and dispersed to crystallize the solid material.

As such, the by-product of the acyl halide compound used in the method for preparing the lysine-deoxycholic acid salt is easily separated and removed even by washing with a simple organic solvent and water, and only the desired coupling product, lysine-deoxycholic acid salt, is organic. Since it is transferred to a solvent, it can be synthesized in a high yield of 94%.

In addition, since the deprotection process also completely removes the by-products of the previous reaction, the lysine-deoxycholic acid salt can be obtained in a high yield of 90% only by the crystallization process, and the overall reaction yield is 76% with a high yield of 76%. can do.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein.

[Preparation Example: Preparation of lysine-deoxycholic acid salt]

(Comparative Example 1)

1) methyl 6-(tertiary-butoxycarbonylamino)-2-(4-(3,12-dihydroxyloxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17) - Synthesis of yl) pentanamido) hexanoate

[Scheme 2]

40 g of 4-(3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoic acid (or deoxycholic acid) of Formula 1 ( 101.89 mmol) was dissolved in 1.5 l of anhydrous tetrahydrofuran and then filled with nitrogen gas to meet anhydrous conditions. After cooling to 0 °C, 9.7 ml (101.89 mmol) of ethylchloroformate was added little by little, and 11.2 ml (101.89 mmol) of 4-methylmorpholine was added little by little.

Stirred at 0° C. for 30 minutes, converted to room temperature, and stirred for 2 hours to activate (ethylcarbonic) 4-(3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclo of Formula 2) After penta[a]phenanthren-17-yl)pentanoic anhydride was obtained during the reaction, 11.2 ml (101.89 mmol) of 4-methylmorpholine was added little by little again. 30.2 g (101.89 mmol) of methyl 2-amino-6-(tertiary-butoxycarbonylamino)hexanoate hydrochloride of Formula 3 was added as a solid powder to the reaction solution, and then filled with nitrogen gas. After refluxing for 2 hours, the reaction was stirred at room temperature for 12 hours. After completion of the reaction, it was filtered using Celite® 545 and distilled under reduced pressure. Purification by silica gel column (CHCl ₃ : MeOH = 20 : 1 (v/v%)) to obtain compound methyl 6-(tertiary-butoxycarbonylamino)-2-(4-(3,12-dihy) 41 g (yield: 60%) of cy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)hexanoate was obtained.

1H NMR (DMSO, 400 MHz) δ 8.15 (1H, brs), 6.76 (1H, brs), 4.52 (1H, brs), 4.51 (1H, s), 4.49 (1H, brs), 3.66 (3H, s), 3.34(2H, m), 2.99 (2H, m), 2.05(2H, dd), 1.80 to 1.42(39H, m), 0.89(3H, s), .088(3H, s), 0.51(3H, s) )

2) methyl 6-amino-2-(4-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)hexanoate synthesis of hydrochloride

[Scheme 3]

44.76 ml (4.941 g, 630.0 mmol) of acetyl chloride was added to 600 ml of methanol at 0°C. After stirring at 0° C. for 30 minutes, methyl 6-(tert-butoxycarbonylamino)-2-(4-(3,12-dihydroxyloxy-10,13-dimethylhexadecahydro-1H-cyclo of Formula 4) 40 g (63.0 mmol) of penta[a]phenanthren-17-yl)pentanamido)hexanoate were added. After slowly raising the temperature to room temperature, the mixture was stirred for 12 hours. After distillation under reduced pressure, it was dissolved in 400 ml of distilled water and washed 3 times with 400 ml of chloroform. The distilled water layer was recovered and freeze-dried for 24 hours, and the desired methyl 6-amino-2-(4-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenane of Formula 5) 28.79 g of tren-17-yl)pentanamido)hexanoate hydrochloride (yield 80%) was obtained.

1H NMR(DMSO, 400MHz) δ 7.89(1H, brs), 4.20(1H, m), 3.64~3.54(3H, m), 3.60(3H, s), 2.72(2H, t), 2.12(2H, m) ), 1.85 to 1.04 (33H, m), 0.84 (3H, s), .082 (3H, s), 0.51 (3H, s)

In Comparative Example 1, the overall yield was 48%, and a low yield was obtained. In addition, due to the reactivity of ethylchloroformate, many by-products are generated, a transesterification reaction is also involved, and due to the formation of by-products similar to the polarity of the desired formula (4), the crystallization process is hindered even in the final process.

(Example 1: using triphenyl phosphite)

1) methyl 6-(tertiary-butoxycarbonylamino)-2-(4-(3,12-dihydroxyloxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17) -yl) pentanamido) hexanoate synthesis

[Scheme 4]

50 g (0.127 mole) of 4-(3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoic acid of Formula 1 and Formula 3 35.9 g (0.120 mole) of methyl 2-amino-6-(tertiary-butoxycarbonylamino)hexanoate hydrochloride was dissolved in 50 ml of dimethylformamide. 13 g (0.190 mole) of imidazole and 50 ml (0.190 mole) of triphenylphosphite of Formula 6 were added and stirred at room temperature for 5 hours. After completion of the reaction, 300 ml of ethyl acetate and 300 ml of purified water were added, and after stirring for 30 minutes, the layers were separated to remove the purified water layer. 200 ml of 1 N aqueous hydrochloric acid solution was added, stirred, and then the layers were separated to remove the water layer.

Moisture was removed by using 10 g of magnesium sulfate on the ethyl acetate layer. After filtration, the mixture was washed with 50 ml of ethyl acetate, concentrated under reduced pressure, and the oil-type material was purified by column chromatography under the condition of ethyl acetate / hexane = 1:1. Methyl 6-(tertiary-butoxycarbonylamino)-2-(4-(3,12-dihydroxyloxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene- of compound 4) 17-yl) pentanamido) hexanoate 56.6 g (yield 70%) was obtained in the form of a solid.

1H NMR (DMSO, 400 MHz) δ 8.15 (1H, brs), 6.76 (1H, brs), 4.52 (1H, brs), 4.51 (1H, s), 4.49 (1H, brs), 3.66 (3H, s), 3.34 (2H, m), 2.99 (2H, m), 2.05 (2H, dd), 1.80 to 1.42 (39H, m), 0.89 (3H, s), .088 (3H, s), 0.51 (3H, s) )

2) methyl 6-amino-2-(4-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)hexanoate synthesis of hydrochloride

[Scheme 5]

850 ml of methanol was added to a 2000 ml reaction vessel and cooled to 0-5 °C for 1 hour. 63.4 ml (1.27 mole) of acetyl chloride was slowly added dropwise and stirred at 0-5 °C for 1 hour. Methyl 6-(tert-butoxycarbonylamino)-2-(4-(3,12-dihydroxyloxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-) of formula 4 17-yl) pentanamido) hexanoate 50.30 g (0.079 mole) was dissolved in 130 ml of methanol, cooled to 0-5 ° C for 1 hour, added, isothermal slowly to room temperature, and stirred at 25 ° C for 12 hours did it After completion of the reaction, methanol was removed by evaporation under reduced pressure, 100 ml of methanol was added, and then evaporated under reduced pressure once more. After dissolving the oil-type mixture in 120 ml of methanol, it was slowly added dropwise to 1,100 ml of ethyl acetate for 1 hour and dispersed to produce a solid material. For the crystallization process, after stirring at room temperature for 24 hours, stirring at 0-5 ° C. for 2 hours to produce white crystals, followed by filtration. The white crystals obtained after filtration were dried at 40° C. in a vacuum dryer for 24 hours to obtain the target compound, methyl 6-amino-2-(4-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[). a]Phenanthrene-17-yl)pentanamido)hexanoate hydrochloride was obtained in an amount of 48.3 g (95% yield). The overall overall yield was 63%, and high purity and high yield of the target compound could be obtained.

1H NMR(DMSO, 400MHz) δ 7.89(1H, brs), 4.20(1H, m), 3.64~3.54(3H, m), 3.60(3H, s), 2.72(2H, t), 2.12(2H, m) ), 1.85 to 1.04 (33H, m), 0.84 (3H, s), .082 (3H, s), 0.51 (3H, s)

(Example 2: Using pivaloyl chloride)

1) methyl 6-(tertiary-butoxycarbonylamino)-2-(4-(3,12-dihydroxyloxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17) -yl) pentanamido) hexanoate synthesis

[Scheme 6]

Dimethylformamide in 1,600 g (4.07 mole) of 4-(3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoic acid of Formula 1 12,000 ml was added and dissolved. 738 ml (536 g, 5.29 mole) of triethylamine was added and stirred at 5° C. for 2 hours. 602 ml (589 g, 4.88 mole) of pivaloyl chloride was added to the reaction mixture, and the mixture was slowly isothermal to 25° C. and stirred at room temperature for 3 hours. Triethylamine 1,704 ml (1,237 g, 12.22 mole) was added and stirred for 30 minutes. 1,330 g (4.48 mole) of methyl 2-amino-6-(tertiary-butoxycarbonylamino)hexanoate hydrochloride of Formula 3 was added and stirred at 25° C. for 5 hours.

After completion of the reaction, 20,000 ml of ethyl acetate was added, stirred for 30 minutes, filtered, and washed with 5,000 ml of ethyl acetate. 25,000 ml of water was added to the filtrate, stirred for 10 minutes, and left to stand to separate the ethyl acetate layer. 20,000 ml of a saturated aqueous sodium bicarbonate solution was added to the ethyl acetate layer, stirred for 10 minutes, and left to stand, the ethyl acetate layers were collected and moisture was removed using magnesium sulfate. After filtration, the mixture was washed with 5,000 ml of ethyl acetate and concentrated under reduced pressure. The compound 4 was methyl 6-(tert-butoxycarbonylamino)-2-(4-(3,12-dihydroxyloxy-10,13-dimethylhexadeca). 2,432 g (yield 94%) of hydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)hexanoate was obtained in the form of a solid.

1H NMR (DMSO, 400 MHz) δ 8.15 (1H, brs), 6.76 (1H, brs), 4.52 (1H, brs), 4.51 (1H, s), 4.49 (1H, brs), 3.66 (3H, s), 3.34(2H, m), 2.99 (2H, m), 2.05(2H, dd), 1.80 to 1.42(39H, m), 0.89(3H, s), .088(3H, s), 0.51(3H, s) )

2) methyl 6-amino-2-(4-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)hexanoate synthesis of hydrochloride

[Scheme 7]

24,300 ml of methanol was added to the reaction vessel and cooled to 0-5 °C for 1 hour. Acetyl chloride 1,361 ml (1,503 g, 19.14 mole) was slowly added dropwise and stirred at 0-5 °C for 1 hour. Methyl 6-(tert-butoxycarbonylamino)-2-(4-(3,12-dihydroxyloxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-) of formula 4 17-yl)pentanamido)hexanoate 2,432 g (3.83 mole) was dissolved in 2,430 ml of methanol, cooled to 0-5 ℃ for 1 hour, added, isothermal slowly to room temperature, and stirred at 25 ℃ for 12 hours did it After completion of the reaction, methanol was removed by evaporation under reduced pressure, 5,000 ml of methanol was added, and then evaporated under reduced pressure once more. After dissolving the oil-type mixture in 3,000 ml of methanol, it was slowly added dropwise to 30,000 ml of ethyl acetate for 1 hour and dispersed to produce a solid material. For the crystallization process, after stirring at room temperature for 24 hours, stirring at 0-5 ° C. for 2 hours to produce white crystals, followed by filtration. The white crystals obtained after filtration were dried at 40° C. in a vacuum dryer for 24 hours to obtain the target compound, methyl 6-amino-2-(4-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta). [a] phenanthrene-17-yl)pentanamido)hexanoate hydrochloride was obtained in an amount of 1,750 g (80 % yield).

The overall overall yield was 76%, and the target compound of high purity and high yield could be obtained through the crystallization process without chromatographic separation.

1H NMR(DMSO, 400MHz) δ 7.89(1H, brs), 4.20(1H, m), 3.64~3.54(3H, m), 3.60(3H, s), 2.72(2H, t), 2.12(2H, m) ), 1.85 to 1.04 (33H, m), 0.84 (3H, s), .082 (3H, s), 0.51 (3H, s)

Although the preferred embodiment of the present invention has been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims are also provided. is within the scope of the

Claims (13)

Lysine-deoxycholic acid (LysDOCA) comprising the step of coupling reaction between deoxycholic acid (DOCA) and a lysine (Lys) salt in the presence of a phosphite-based compound represented by the following formula (6) ) method for the preparation of salts.
[Formula 6]

(In Formula 6,
wherein R ⁶¹ to R ⁶³ are each independently any one selected from the group consisting of a hydrogen group, an alkyl group, and an aryl group) The method of claim 1,
The phosphite-based compound is triphenyl phosphite (triphenyl phosphite) lysine-deoxycholic acid salt production method. The method of claim 1,
The deoxycholic acid, the lysine salt and the phosphite-based compound are 1: 0.94 to 0.42: 1.49 to 2.24 (deoxycholic acid: lysine salt: phosphite-based compound) to react in a molar ratio of lysine-deoxycholic acid A method for preparing a salt. The method of claim 1,
The coupling reaction step is a method for producing a lysine-deoxycholic acid salt through the intermediate represented by the following formula (7).
[Formula 7]

(In Formula 7,
wherein R ⁶¹ and R ⁶² are each independently any one selected from the group consisting of a hydrogen group, an alkyl group, and an aryl group) Lysine-deoxycholic acid (deoxycholic acid, DOCA) and a lysine (Lys) salt comprising a coupling reaction in the presence of an acyl halide-based compound represented by the following formula (8) LysDOCA) salt preparation method.
[Formula 8]

(In Formula 8,
Wherein R ⁸¹ to R ⁸³ are each independently any one substituent selected from the group consisting of a hydrogen group, an alkyl group, and an aryl group,
X ⁸¹ is any one halogen substituent selected from the group consisting of fluoride (F), chloride (Cl), bromine (Br) and iodine (I)) 6. The method of claim 5,
The acyl halide-based compound is a lysine-deoxycholic acid salt of pivaloyl chloride (pivaloyl chloride) production method. 6. The method of claim 5,
The deoxycholic acid, the lysine salt and the acyl halide compound are reacted in a molar ratio of 1: 2.37 to 3.56: 1.20 to 1.80 (deoxycholic acid: lysine salt: acyl halide compound) Lysine-deoxycholic acid A method for preparing a salt. 6. The method of claim 5,
The coupling reaction is performed in the presence of any one base selected from the group consisting of triethylamine (TEA), pyridine, lutidine, dimethylamine, and mixtures thereof. A method for producing a lysine-deoxycholic acid salt consisting of. 6. The method of claim 5,
The coupling reaction step is
A method for producing a lysine-deoxycholic acid salt through an intermediate represented by the following formula (9).
[Formula 9]

(In Formula 9,
X ⁸¹ is any one substituent selected from the group consisting of fluoride (F), chloride (Cl), bromine (Br) and iodine (I)) 6. The method of claim 1 or 5,
The deoxycholic acid is represented by the following formula (1),
The lysine salt is represented by the following Chemical Formula 3,
The lysine-deoxycholic acid salt is a method for producing a lysine-deoxycholic acid salt represented by the following formula (5).
[Formula 1]

[Formula 3]

[Formula 5]

6. The method of claim 1 or 5,
The coupling reaction is selected from the group consisting of dimethylformamide (dimethylformaide, DMF), tetrahydrofuran (THF), dichloromethane (DCM), chloroform, methanol (MeOH), and mixtures thereof. A method for producing a lysine-deoxycholic acid salt made in any one solvent. 6. The method of claim 1 or 5,
The coupling reaction is made at a temperature of 5 ℃ to 60 ℃ lysine-deoxycholic acid salt production method. 6. The method of claim 1 or 5,
The lysine salt is a lysine salt protected with an amine group,
The method for preparing the lysine-deoxycholic acid salt further comprises the step of deprotecting the amine group protected in the prepared lysine-deoxycholic acid salt.