Sodium salt of acetylated hyaluronic acid and method for producing the same

Abstract

The present invention relates to a sodium salt of acetylated hyaluronic acid and a method for preparing the same. More specifically, the present invention relates to a novel form of a sodium salt of acetylated hyaluronic acid useful for reducing visible signs of aging and a method for preparing the same. According to the method for preparing a sodium salt of acetylated hyaluronic acid of the present invention, the sodium salt of acetylated hyaluronic acid can be produced in large quantities while improving color, recovery rate, and reproducibility.

Description

Technology Field

[0001] The following examples relate to a sodium salt of acetylated hyaluronic acid and a method for preparing the same. More specifically, the present invention relates to a novel form of a sodium salt of acetylated hyaluronic acid useful for reducing visible signs of aging and a method for preparing the same. Background Technology

[0002] Acetylhyaluronic acid, one of the derivatives of hyaluronic acid, is a compound in which an acetyl group is introduced to the hydroxyl group of hyaluronic acid. It is a material with high industrial utility as it exhibits improved physicochemical properties while maintaining the advantages of hyaluronic acid. Conventional methods for synthesizing acetylhyaluronic acid involved reacting hyaluronic acid with an acetylating agent and using a basic catalyst such as 4-dimethylaminopyridine (DMAP).

[0003] However, while the sodium salt derived from acetylated hyaluronic acid has improved bioavailability, stability, and skin penetration, it is unsuitable for skin care compositions due to its off-white to yellow color. Furthermore, there are issues with the complexity of the mass production process and poor reproducibility and recovery rates. Therefore, it is necessary to mass-produce the sodium salt of acetylated hyaluronic acid while improving its color, recovery rate, and reproducibility. Prior art literature

[0004] Republic of Korea Published Patent Application No. 10-2024-0064056 The problem to be solved

[0005] The following examples are devised to solve the problems described above. The present invention relates to a method for preparing a sodium salt of acetylated hyaluronic acid, and more specifically, aims to produce a sodium salt of acetylated hyaluronic acid in large quantities while improving color, recovery rate, and reproducibility. The problems of the present invention are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0006] To achieve the aforementioned objectives of the present invention, one embodiment of the present invention provides a method for preparing a sodium salt of acetylated hyaluronic acid, comprising the steps of: adding acetylated hyaluronic acid to a first organic solvent to obtain a suspension; adding a pH adjusting agent to the suspension to obtain a fluid suspension; adding a second organic solvent to a filtrate from which solids have been filtered and removed from the fluid suspension to obtain a solid compound; and purifying the solid compound to obtain a sodium salt of acetylated hyaluronic acid; wherein the pH adjusting agent is sodium bicarbonate, sodium carbonate, or a mixture thereof, and the equivalent weight of the pH adjusting agent is 1 to 2 based on the equivalent weight of the acetylated hyaluronic acid, and the first organic solvent and the second organic solvent are each independently one or more selected from the group consisting of methanol, ethanol, isopropanol, n-propanol, acetonitrile, dimethyl sulfoxide, tetrahydrofuran, and acetone. Effects of the invention

[0007] According to the method for preparing a sodium salt of acetylated hyaluronic acid of the present invention, a sodium salt of acetylated hyaluronic acid can be produced in large quantities while improving color, recovery rate, and reproducibility. Brief explanation of the drawing

[0008] FIGS. 1 to 5, FIGS. 8 to 17, FIGS. 20, FIG. 21, FIG. 23, FIG. 24 and FIGS. 26 to 28 are images showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention. FIGS. 6, FIGS. 7, FIGS. 18, and FIGS. 22 are images showing the progress of the reaction of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention. FIGS. 19, 25 and 29 illustrate a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention. 1 This is the H NMR spectrum. Specific details for implementing the invention

[0009] Hereinafter, embodiments are described in detail with reference to the attached drawings. However, various modifications may be made to the embodiments, and thus the scope of the patent application is not limited or restricted by these embodiments. It should be understood that all modifications, equivalents, and substitutions to the embodiments are included within the scope of the rights.

[0010] Specific structural or functional descriptions of the embodiments are disclosed for illustrative purposes only and may be modified and implemented in various forms. Accordingly, the embodiments are not limited to the specific disclosed forms, and the scope of this specification includes modifications, equivalents, or substitutions that fall within the technical concept.

[0011] Terms such as "first" or "second" may be used to describe various components, but these terms should be interpreted solely for the purpose of distinguishing one component from another. For example, the first component may be named the second component, and similarly, the second component may be named the first component.

[0012] When it is stated that a component is "connected" to another component, it should be understood that it may be directly connected to or coupled with that other component, or that there may be other components in between.

[0013] The terms used in the embodiments are for illustrative purposes only and should not be interpreted as intended to be limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprising" or "having" are intended to indicate the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0014] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the embodiments pertain. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0015] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0016] In the embodiments of the present invention, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in the embodiments of the present invention.

[0017] The shapes, sizes, ratios, angles, numbers, etc. disclosed in the drawings for explaining embodiments of the present invention are exemplary, and therefore the present invention is not limited to the depicted details. Furthermore, in describing the present invention, if it is determined that a detailed description of related known technology may unnecessarily obscure the essence of the present invention, such detailed description is omitted. Where terms such as "includes," "has," or "is made up" are used in this specification, other parts may be added unless "only" is used. Where a component is expressed in the singular, it includes cases where it includes the plural unless specifically stated otherwise.

[0018] In interpreting the components, they are interpreted to include a margin of error even in the absence of a separate explicit statement.

[0019] The size and thickness of each component shown in the drawings are illustrated for convenience of explanation, and the present invention is not necessarily limited to the size and thickness of the illustrated components.

[0020] The features of each of the various embodiments of the present invention may be combined or combined with one another, either partially or wholly, and as will be fully understood by those skilled in the art, various technical interlocking and operation are possible, and each embodiment may be implemented independently of one another or together in an interlocking relationship.

[0021] Sodium salt of acetylated hyaluronic acid and method for preparing the same

[0022] In one aspect, the present invention provides a method for preparing a sodium salt of acetylated hyaluronic acid, comprising the steps of: adding acetylated hyaluronic acid to a first organic solvent to obtain a suspension; adding a pH adjusting agent to the suspension to obtain a flowable suspension; adding a second organic solvent to a filtrate from which solids have been filtered and removed from the flowable suspension to obtain a solid compound; and purifying the solid compound to obtain a sodium salt of acetylated hyaluronic acid; wherein the pH adjusting agent is sodium bicarbonate, sodium carbonate, or a mixture thereof, and the equivalent amount of the pH adjusting agent is 1 to 2 based on the equivalent amount of the acetylated hyaluronic acid, and the first organic solvent and the second organic solvent are each independently one or more selected from the group consisting of methanol, ethanol, isopropanol, n-propanol, acetonitrile, dimethyl sulfoxide, tetrahydrofuran, and acetone.

[0023] In one aspect, purifying the solid compound involves washing the solid compound with a third organic solvent. The third organic solvent is one or more selected from the group consisting of methanol, ethanol, isopropanol, n-propanol, acetonitrile, dimethyl sulfoxide, tetrahydrofuran, and acetone, independently of the first organic solvent and the second organic solvent.

[0024] In one aspect, the hyaluronic acid is an ultra-low molecular weight hyaluronic acid with a weight-average molecular weight of 10 to 50 kDa, the content of the hyaluronic acid is 1 to 30 weight% with respect to the total weight of the composition, the viscosity is 80 to 400 cPS when measured with a Brookfield viscometer under conditions of 64 spindles and a speed of 2 to 12 rpm for 2 minutes, and can be discharged by pumping from within a pump container.

[0025] Hyaluronic acid (youngmoisture) is an anionic natural polysaccharide that exists in salt form and is an ingredient classified as EWG Grade 1. As one of the three major components of the skin, hyaluronic acid plays a role in eliminating wrinkles through its moisture retention capacity within the dermis and its anti-inflammatory effects. It is primarily produced and purified using microorganisms; it exists in the connective tissue and extracellular matrix of the human body and is widely used as a moisturizer due to its high water content resulting from its anionic functional groups. The molecular structure of hyaluronic acid can be represented by Chemical Formula 1 below. Here, n represents the number of monomers and is between 30 and 100.

[0026] [Chemical Formula 1]

[0027]

[0028] In one embodiment, the hyaluronic acid is acetylated hyaluronic acid represented by the following chemical formula 2. n is the number of monomers and is 10 to 50.

[0029] [Chemical Formula 2]

[0030]

[0031] In one embodiment, the equivalent amount of the pH adjuster is 1 to 2 based on the equivalent amount of the acetylated hyaluronic acid. Since the acetylated hyaluronic acid (AcHA, acid form) actually synthesized has an acetic acid odor, an equivalent amount of pH adjuster of 1 or more is required. However, since using an excessive amount of pH adjuster causes hydrolysis of the acetyl group, it is very important to find an appropriate amount of base. More specifically, the equivalent amount of the pH adjuster may be 1 or more, 1.1 or more, 1.2 or more, 1.3 or more; 2 or less, 1.9 or less, 1.8 or less, 1.7 or less, 1.6 or less, 1.5 or less, 1.4 or less, 1.3 or less, but is not limited thereto.

[0032] In one embodiment, the pH adjuster is sodium bicarbonate (NaHCO3). Sodium bicarbonate (NaHCO3) needs to be introduced because a sufficient base is required in the process of forming the sodium salt of acetylated hyaluronic acid. Using sodium bicarbonate for pH adjustment is particularly useful in mass production because it prevents the pH at a locally high point during pH adjustment from becoming a strong base pH of 10 or higher.

[0033] In one embodiment, the first solvent, the second solvent, and the third solvent are all isopropanol. In order to selectively remove the organic solvent under reduced pressure after alkali treatment with a pH adjuster during the process of forming a sodium salt of acetylated hyaluronic acid, it is preferable that the first solvent, the second solvent, and the third solvent are all isopropanol.

[0034] In one embodiment, the sodium salt of the acetylated hyaluronic acid is a white solid compound. The sodium salt of the acetylated hyaluronic acid according to the present invention is suitable for a skin care composition because it exhibits a white color while having excellent bioavailability, stability, and skin penetration power.

[0035] In one embodiment, the recovery rate of the sodium salt of the acetylated hyaluronic acid is 70% or more. More specifically, the recovery rate of the sodium salt of the acetylated hyaluronic acid may be 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more; 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 89% or less, 88% or less, 87% or less, 86% or less, 85% or less, 84% or less, 83% or less, 82% or less, 81% or less, 80% or less, 79% or less, 78% or less, 77% or less, 76% or less, but is not limited thereto.

[0036] In one embodiment, the degree of acetylation of the sodium salt of the acetylated hyaluronic acid is 3.8 or higher. Hyaluronic acid is a polysaccharide composed of repeating units containing N-acetylglucosamine and glucuronic acid. It may exist in the form of its free acid or sodium salt. In its acetylated form, the term "degree of acetylation" is understood as a unit of the number of hydroxyl groups on the acetylated polymer repeating unit. Each repeating unit of the natural hyaluronic acid / salt polymer contains four hydroxyl groups that can be acetylated, and the degree of acetylation is an indication of how many groups are substituted by acetyl groups. The sodium salt of the acetylated hyaluronic acid according to the present invention is characterized by having a very high degree of acetylation. More specifically, the degree of acetylation of the sodium salt of the acetylated hyaluronic acid is 3.8 or higher or 3.9 or higher, and is characterized in particular by an average degree of acetylation of 4.

[0037] In one embodiment, the acetylated hyaluronic acid has a degree of acetylation of 0.75 or higher and is prepared through the following steps: mixing hyaluronic acid with an organic acid, acetic anhydride, and a basic catalyst and heating to obtain a homogeneous mixture; acetylating the homogeneous mixture to obtain an acetylated hyaluronic acid solution; removing acetic acid and hyaluronic anhydride from the acetylated hyaluronic acid solution to obtain an acetylated hyaluronic acid slurry; and converting the acetylated hyaluronic acid slurry into an acetylated hyaluronic acid solid under acidic conditions.

[0038] In one embodiment, the organic acid is acetic acid, the carboxylic anhydride is acetic anhydride, the basic catalyst is 4-(dimethylamino)pyridine (DMAP), and the nonpolar organic solvent is hexane.

[0039] In one embodiment, the acetylation step is performed at 95-105°C for 40-56 hours, the basic aqueous solution is an aqueous sodium bicarbonate solution with a concentration of 15-25% (w / v), and the alcohol is isopropanol. In addition, the separation under acidic conditions is performed using a diluted aqueous hydrochloric acid solution (concentrated HCl : distilled water = 1 : 37.5 volume ratio), the non-polar organic solvent washing is performed for 8-16 hours, the yield of the acetylated hyaluronic acid obtained by the above method is 75-85%, and the final product is obtained as a milky white solid.

[0040] In addition, the acetylation step comprises: (a) mixing hyaluronic acid, acetic acid, acetic anhydride, and 4-(dimethylamino)pyridine in a molar ratio of 1:15:15:1 in a 4-liter round-bottom flask; (b) reacting the mixture at 100°C for 48 hours; (c) cooling the reaction mixture to room temperature and removing the solvent using a rotary evaporator; (d) adding a diluted aqueous hydrochloric acid solution (concentrated HCl : distilled water = 1 : 37.5 volume ratio) to obtain a solid product; and (e) washing the solid product with distilled water and then drying it. and (f) a step of filtering the dried solid after stirring with hexane for 12 hours; wherein a homogeneous reaction mixture is obtained using acetic acid as a co-solvent in step (a), and the yield of the product obtained after step (f) is 75-85% (w / w) and the degree of acetylation of the product is 3.8-4.0.

[0041] Also, at this time, the basic aqueous solution treatment and alcohol precipitation steps comprise: (g) a step of dissolving the acetylated hyaluronic acid in a 20% concentration sodium bicarbonate aqueous solution; and (h) a step of adding isopropanol to precipitate and filter the acetylated sodium hyaluronate; wherein, through steps (g) and (h), a pure acetylated sodium hyaluronate in which no acetic acid peak is detected upon gas chromatography analysis is obtained with a weight recovery rate of 98-100% relative to the acetylated hyaluronate added, and the pure acetylated sodium hyaluronate dissolves until no insoluble particles are visible upon visual observation when a 1% (w / v) aqueous solution is prepared at 20°C, the C Log P value of the monomer of the pure acetylated sodium hyaluronate is -1.12, and it is confirmed by NMR analysis that acetylation has occurred on all four -OH groups present in the hyaluronic acid starting material, and the above manufacturing method is on a 50g scale It can be performed with a yield of 79-83% and up to a scale of 100g.

[0042] In one embodiment, the acetylated hyaluronic acid has a degree of acetylation of 0.75 or higher. According to the measurement and calculation of the degree of acetylation, the degree of acetylation of the acetylated hyaluronic acid is 0.75 to 0.82. The degree of acetylation of the acetylated hyaluronic acid may be 0.75 or higher, 0.76 or higher, 0.77 or higher, 0.78 or higher, 0.79 or higher; 0.82 or lower, 0.81 or lower, 0.8 or lower, 0.79 or lower, but is not limited thereto.

[0043] In one embodiment, the acetylated hyaluronic acid (AcHA) has a structure in which a hydrophilic -OH group is substituted with an acetyl group having amphipathic properties. The acetylated hyaluronic acid can be synthesized by an acetylation reaction of hyaluronic acid, the key to this reaction is to obtain a homogeneous product by acetylating all -OH groups present in the hyaluronic acid molecule (a total of 4 -OH groups exist per hyaluronic acid monomer) or by site-selectively acetylating specific -OH groups (1 to 4). The acetylation reaction performed at this time can be carried out according to Reaction Scheme 1 below.

[0044] [Reaction Equation 1]

[0045]

[0046] In one aspect, n is the number of monomers and is 10 to 50. The present invention has an octanol-water partition coefficient (log P) of -1 to 5 and a distribution coefficient (log D @ pH7.4) (log D 7.4 The present invention provides a sodium salt of acetylated hyaluronic acid having a molecular weight of -2 to 5, a number average molecular weight (Mn) of 5000 to 7000 Da, and a weight average molecular weight (Mw) of 5000 to 30000 Da.

[0047] In one aspect, sodium salts of acetylated hyaluronic acid are prepared by the above-described preparation method.

[0048] In one embodiment, the sodium salt of the acetylated hyaluronic acid has an octanol-water partition coefficient (log P) of -1 to 5 and a distribution coefficient (log D @ pH7.4) of -2 to 5 (log D 7.4It is characterized by having a value.

[0049] {Example}

[0050] The present disclosure will be explained in detail below through examples. However, the following examples are merely illustrative to aid in the overall understanding of the present disclosure, and the content of the present disclosure is not limited to the following examples.

[0051] <Reference Example 1> Preparation of Acetylated Hyaluronic Acid

[0052] 50.0 g (0.131 mol, monomer basis) of hyaluronic acid was placed in a 4-liter round-bottom flask, followed by 750 mL of acetic acid, 750 mL of acetic anhydride, and 16.02 g (0.131 mol) of 4-(dimethylamino)pyridine (DMAP). The reaction mixture was heated to 100°C and stirred for 48 hours. Approximately 4.5 hours after the start of the reaction, a homogeneous solution was formed.

[0053] After the reaction was complete, the mixture was cooled to room temperature, and acetic acid and acetic anhydride were removed using a rotary evaporator. A diluted aqueous hydrochloric acid solution (40 mL of concentrated HCl diluted in 1500 mL of distilled water) was added to the residue and stirred for 20 minutes to obtain a solid product. This solid was filtered, washed with 500 mL of distilled water, and dried. 500 mL of hexane was added to the dried solid and stirred for 12 hours, after which it was filtered to obtain 57 g of acetylated hyaluronic acid (AcHA) (yield 79%).

[0054] <Reference Example 2> Setting of Candidate Organic Solvents

[0055] Candidate organic solvents that are soluble in water and have lower polarity than water were set as water (dielectric constant 80), acetonitrile (dielectric constant 37.5), methanol (dielectric constant 32.6), ethanol (dielectric constant 24.3), acetone (dielectric constant 20.7), and isopropanol (dielectric constant 18.3).

[0056] <Preparation Example 1> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 1)

[0057] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 24 mL (8 volumes) of methanol and stirred for 10 to 15 minutes. The acetylated hyaluronic acid (AcHA) did not dissolve and formed a sticky mass. 6 mL of an aqueous solution containing 0.92 g (10.92 mmol) of sodium bicarbonate was slowly added to the mixture over 10 minutes while stirring continued. The mixture initially formed a sticky mass, and after stirring for another 30 minutes, a solution or a milky white colloid was obtained.

[0058] 120 mL (40 volumes) of isopropanol was added to the above solution or milky white colloid and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered to obtain 2.56 g of sodium salt of acetylated hyaluronic acid (recovery rate 82%).

[0059] The product obtained in this way 1 When residual organic solvent was observed in the H NMR spectrum, hexane was added to the product and stirred for 2 to 3 hours, then filtered and dried to remove the residual organic solvent.

[0060] Figure 1 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0061] <Preparation Example 2> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 2)

[0062] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 24 mL (8 volumes) of acetone and stirred for 10 to 15 minutes. Most of the acetylated hyaluronic acid (AcHA) was dissolved. 6 mL of an aqueous solution containing 0.92 g (10.92 mmol) of sodium bicarbonate was added to the mixture, and stirring was continued. A white precipitate formed in the mixture.

[0063] The mixture was stirred for 10 to 15 minutes, 30 mL of acetone was added to the mixture, and the mixture was stirred for 30 minutes to obtain a solid compound. The solid compound was filtered to obtain 1.45 g of sodium salt of acetylated hyaluronic acid (recovery rate 46%).

[0064] Figure 2 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0065] <Preparation Example 3> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 3)

[0066] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 90 mL (30 volumes) of methanol and stirred for 10 to 15 minutes. The acetylated hyaluronic acid (AcHA) did not dissolve and formed a sticky mass. To the mixture, 6 mL of an aqueous solution containing 0.92 g (10.92 mmol) of sodium bicarbonate was slowly added over 15 minutes while stirring continued. The mixture initially formed a sticky mass, and after stirring for another 30 minutes, a solution or a milky white colloid was obtained.

[0067] 270 mL (90 volumes) of isopropanol was added to the above solution or milky white colloid and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered, washed with 30 mL (10 volumes) of isopropanol, and dried to obtain 2.71 g of sodium salt of acetylated hyaluronic acid, which is a solid compound (recovery rate 87%).

[0068] Figure 3 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0069] <Preparation Example 4> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 4)

[0070] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 40 mL (13.5 volumes) of acetone and stirred for 10 to 15 minutes. Most of the acetylated hyaluronic acid (AcHA) was dissolved. 10 mL of an aqueous solution containing 0.92 g (10.92 mmol) of sodium bicarbonate was added to the mixture, and stirring was continued. A white precipitate formed in the mixture.

[0071] The mixture was stirred for 10 to 15 minutes, 40 mL of acetone was added to the mixture, and the mixture was stirred for 30 minutes to obtain a solid compound. The solid compound was filtered to obtain 1.07 g of sodium salt of acetylated hyaluronic acid, which is an off-white fluid solid (recovery rate 34%).

[0072] Figure 4 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0073] <Preparation Example 5> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 5)

[0074] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 80 mL (26.7 volumes) of acetone and stirred for 10 to 15 minutes. Most of the acetylated hyaluronic acid (AcHA) dissolved. 100 mL of an aqueous solution containing 0.92 g (10.92 mmol) of sodium bicarbonate was added to the mixture, and stirring was continued. Most of the acetylated hyaluronic acid (AcHA) dissolved. After stirring for 10 to 15 minutes, 400 mL of acetone was added to the mixture and stirred for 30 minutes, but the sodium salt of the acetylated hyaluronic acid did not precipitate as a solid.

[0075] <Preparation Example 6> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 6)

[0076] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 55 mL (18.3 volumes) of isopropanol and stirred for 10 to 15 minutes to obtain a suspension. The acetylated hyaluronic acid (AcHA) did not dissolve. 70 mL of an aqueous solution containing 0.92 g (10.92 mmol) of sodium bicarbonate was added to the suspension while stirring was continued. The mixture initially formed a sticky mass, but the mixture gradually became transparent when about half of the solution was added.

[0077] 650 mL of isopropanol was added to the above mixture (50 mL aliquots) and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered, washed with 50 mL of isopropanol, and dried to obtain 0.82 g of sodium salt of acetylated hyaluronic acid, which is a solid compound (recovery rate 26%).

[0078] Figure 5 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0079] Figure 6 is an image showing the progress of the reaction of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0080] <Preparation Example 7> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 7)

[0081] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 55 mL (18.3 volumes) of acetone and stirred for 5 minutes. Most of the acetylated hyaluronic acid (AcHA) dissolved. 70 mL of an aqueous solution containing 0.92 g (10.92 mmol) of sodium bicarbonate was added to the mixture, and stirring was continued. No precipitate was formed in the mixture.

[0082] Stirred for 10 to 15 minutes, 400 mL of acetone was added to the mixture (50 mL a little) and stirred for 30 minutes. No precipitation occurred.

[0083] Figure 7 is an image showing the progress of the reaction of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0084] <Preparation Example 8> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 8)

[0085] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 60 mL (20 volumes) of acetone and stirred for 5 minutes. Most of the acetylated hyaluronic acid (AcHA) dissolved to form a thin gel solution with low viscosity. 12 mL of an aqueous solution containing 0.92 g (10.92 mmol) of sodium bicarbonate was added to the mixture while stirring was continued. A white precipitate formed in the mixture.

[0086] 240 mL of isopropanol was added to the above mixture (60 mL aliquots) and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered, washed with 60 mL of isopropanol, and dried to obtain 2.74 g of sodium salt of acetylated hyaluronic acid, which is a solid compound (recovery rate 87%).

[0087] FIG. 8 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0088] <Preparation Example 9> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 9)

[0089] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 60 mL (20 volumes) of isopropanol and stirred for 10 to 15 minutes to obtain a suspension. The acetylated hyaluronic acid (AcHA) did not dissolve, so coarse particles were mixed in a milky, dilute solution. 12 mL of an aqueous solution containing 0.92 g (10.92 mmol) of sodium bicarbonate was added to the suspension, and stirring was continued. This mixture initially forms a sticky mass, and further stirring yields a free-flowing suspension.

[0090] 180 mL of isopropanol was added to the above mixture (60 mL aliquots) and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered, washed with 60 mL of isopropanol, and dried to obtain 2.93 g of sodium salt of acetylated hyaluronic acid, which is a solid compound (recovery rate 93%).

[0091] FIG. 9 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0092] <Preparation Example 10> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 10)

[0093] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 60 mL of methanol and isopropanol (30 mL : 30 mL, 20 volumes) and stirred for 10 to 15 minutes to obtain a suspension. The acetylated hyaluronic acid (AcHA) did not dissolve, and coarse particles were mixed in a milky, dilute solution. 12 mL of an aqueous solution containing 0.92 g (10.92 mmol) of sodium bicarbonate was added to the suspension, and stirring was continued. This mixture initially formed a sticky mass, and further stirring resulted in a free-flowing suspension.

[0094] 240 mL of isopropanol was added to the above mixture (60 mL aliquots) and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered, washed with 60 mL of isopropanol, and dried to obtain 2.65 g of sodium salt of acetylated hyaluronic acid, which is a solid compound (recovery rate 85%).

[0095] FIG. 10 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0096] <Preparation Example 11> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 11)

[0097] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 60 mL of ethanol and isopropanol (30 mL : 30 mL, 20 volumes) and stirred for 10 to 15 minutes to obtain a suspension. The acetylated hyaluronic acid (AcHA) did not dissolve, so coarse particles were mixed in a milky, dilute solution. 12 mL of an aqueous solution containing 0.92 g (10.92 mmol) of sodium bicarbonate was added to the suspension, and stirring was continued. This mixture initially forms a sticky mass, and further stirring yields a free-flowing suspension.

[0098] 240 mL of isopropanol was added to the above mixture (60 mL aliquots) and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered, washed with 60 mL of isopropanol, and dried to obtain 2.41 g of sodium salt of acetylated hyaluronic acid, which is a solid compound (recovery rate 77%).

[0099] FIG. 11 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0100] <Preparation Example 12> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 12)

[0101] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 60 mL of acetonitrile and isopropanol (30 mL : 30 mL, 20 volumes) and stirred for 10 to 15 minutes. The acetylated hyaluronic acid (AcHA) was dissolved. 12 mL of an aqueous solution containing 0.92 g (10.92 mmol) of sodium bicarbonate was added to the mixture, and stirring was continued. The mixture initially formed a sticky mass, and upon further stirring, a free-flowing suspension was obtained.

[0102] 240 mL of isopropanol was added to the above mixture (60 mL aliquots) and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered, washed with 60 mL of isopropanol, and dried to obtain 2.54 g of sodium salt of acetylated hyaluronic acid, which is a solid compound (recovery rate 81%).

[0103] FIG. 12 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0104] <Preparation Example 13> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 13)

[0105] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 60 mL of acetone and isopropanol (30 mL : 30 mL, 20 volumes) and stirred for 10 to 15 minutes. Most of the acetylated hyaluronic acid (AcHA) dissolved to form a thin gel solution with low viscosity. 12 mL of an aqueous solution containing 0.92 g (10.92 mmol) of sodium bicarbonate was added to the mixture, and stirring was continued. The mixture initially formed a sticky mass, and further stirring yielded a free-flowing suspension.

[0106] 240 mL of isopropanol was added to the above mixture (60 mL aliquots) and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered, washed with 60 mL of isopropanol, and dried to obtain 2.61 g of sodium salt of acetylated hyaluronic acid, which is a solid compound (recovery rate 84%).

[0107] FIG. 13 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0108] <Preparation Example 14> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 14)

[0109] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 60 mL of tetrahydrofuran and isopropanol (30 mL : 30 mL, 20 volumes) and a suspension was obtained for 10 to 15 minutes. The acetylated hyaluronic acid (AcHA) did not dissolve, and coarse particles were mixed in a milky, dilute solution. 12 mL of an aqueous solution containing 0.92 g (10.92 mmol) of sodium bicarbonate was added to the suspension, and stirring was continued. This mixture initially formed a sticky mass, and upon further stirring, a free-flowing suspension was obtained.

[0110] 240 mL of isopropanol was added to the above mixture (60 mL aliquots) and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered, washed with 60 mL of isopropanol, and dried to obtain 2.83 g of sodium salt of acetylated hyaluronic acid, which is a solid compound (recovery rate 91%).

[0111] FIG. 14 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0112] <Preparation Example 15> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 15)

[0113] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 60 mL (20 volumes) of isopropanol and stirred for 10 to 15 minutes to obtain a suspension. The acetylated hyaluronic acid (AcHA) did not dissolve, so coarse particles were mixed in a milky, dilute solution. 12 mL of an aqueous solution containing 0.92 g (10.92 mmol) of sodium bicarbonate was added to the suspension, and stirring was continued. This mixture initially forms a sticky mass, and further stirring yields a free-flowing suspension.

[0114] 180 mL of isopropanol was added to the above mixture (60 mL aliquots) and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered, washed with 60 mL of isopropanol, and dried to obtain 3.04 g of sodium salt of acetylated hyaluronic acid, which is a solid compound (recovery rate 97%).

[0115] FIG. 15 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0116] <Preparation Example 16> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 16)

[0117] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 60 mL (20 volumes) of acetonitrile and stirred for 10 to 15 minutes. The acetylated hyaluronic acid (AcHA) was dissolved. 12 mL of an aqueous solution containing 0.92 g (10.92 mmol) of sodium bicarbonate was added to the mixture, and stirring was continued. The mixture initially forms a sticky mass, and upon further stirring, a free-flowing suspension is obtained.

[0118] 300 mL of isopropanol was added to the above mixture (60 mL aliquots) and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered, washed with 60 mL of isopropanol, and dried to obtain 2.37 g of sodium salt of acetylated hyaluronic acid, which is a solid compound (recovery rate 76%).

[0119] FIG. 16 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0120] The solvents and recovery rates of Entry 1 to 16 are shown in Table 1 below.

[0121] division menstruum Recovery rate (%) Entry 1 MeOh : Water = 24 : 6 (mL) 82 Entry 2 Acetone : Water = 24 : 6 (mL) 46 Entry 3 Meoh : Water = 90 : 10 (mL) 87 Entry 4 Acetone : Water = 40 : 10 (mL) 34 Entry 5 Acetone : Water = 80 : 100 (mL) - Entry 6 IPA : Water = 55 : 70 (mL) 26 Entry 7 Acetone : Water = 55 : 70 (mL) - Entry 8 Acetone : Water = 60 : 12 (mL) 87 Entry 9 IPA : Water = 60 : 12 (mL) 93 Entry 10 MeOh:IPA:water = 30:30:12 (mL) 85 Entry 11 EtOh : IPA : Water = 30 : 30 : 12 (mL) 77 Entry 12 IPA : Water = 30 : 30 : 12 (mL) 81 Entry 13 Acetone : IPA : Water = 30 : 30 : 12 (mL) 84 Entry 14 THF : IPA : Water = 30 : 30 : 12 (mL) 91 Entry 15 IPA : Water = 60 : 12 (mL) 97 Entry 16 ACN : Water = 60 : 12 (mL) 76

[0122] As a result of the experiment, it was confirmed that the cleanest and highest yield product was obtained in the case of Entry 15, in which 60:12 (mL) of isopropanol (IPA) and 180 mL of isopropanol were added.

[0123] <Preparation Example 17> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 17)

[0124] The process of Entry 15 above was expanded and reproduced on a scale ten times larger.

[0125] 30 g (54.6 mmol) of acetylated hyaluronic acid (AcHA) was added to 600 mL (20 volumes) of isopropanol and stirred for 10 to 15 minutes to obtain a suspension. The acetylated hyaluronic acid (AcHA) did not dissolve. 120 mL of an aqueous solution containing 9.2 g (109.2 mmol) of sodium bicarbonate was added to the suspension while stirring was continued. The reaction was carried out for 10 minutes using an addition funnel. The reaction temperature rose slightly (about 2 to 3°C) but did not change significantly. The mixture initially forms a sticky mass, and with further stirring, a free-flowing suspension is obtained within 15 to 20 minutes.

[0126] 1800 mL of isopropanol was added to the above mixture (600 mL aliquot) and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered (filtration time 30 minutes), washed with 600 mL of isopropanol, and dried (takes 10 minutes) to obtain 29.3 g of sodium salt of acetylated hyaluronic acid, which is a solid compound (recovery rate 94%).

[0127] FIG. 17 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0128] FIG. 18 is an image showing the progress of the reaction of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention. During the alkali treatment and precipitation process, the color of the reaction mixture was cloudy brown, which is very unusual for the product to be obtained as a white solid.

[0129] <Experimental Example 1> Measurement of the degree of acetylation of acetylated hyaluronic acid (Entry 17)

[0130] One of the critical issues in the process of generating sodium salts is whether partial hydrolysis of the acetyl group occurs after the alkali treatment step, and this is particularly important in scale-up. The degree of partial hydrolysis of the acetyl group was measured for the sodium salt of acetylated hyaluronic acid. Spectra were obtained using a Bruker Avance III 400 MHz NMR spectrometer. Chemical shift values ​​were corrected using tetramethylsilane (TMS) as an internal standard. Figure 19 shows the sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention. 1 This is the H NMR spectrum.

[0131] From Fig. 19, it can be seen that partial hydrolysis of the acetyl group occurs under the conditions of Entry 17 (degree of acetylation: 3.3~3.4 / total 4).

[0132] It was confirmed that sodium salt of acetylated hyaluronic acid (NaAcHA) with high yield and purity (color) was obtained through the use of isopropanol (IPA) as a co-solvent. However, it was also confirmed that partial hydrolysis of the acetyl group occurs, indicating that finding experimental conditions to prevent or at least minimize this is a very important task.

[0133] The reason for initially adding 2 equivalents of sodium bicarbonate was based on the observation that sufficient base is required for the formation of sodium salts, particularly when reintroducing hydrochloric acid during the acetylation step. In fact, since the synthesized acetylated hyaluronic acid (AcHA, acid form) emits an acetic acid odor, it was evident that more than 1 equivalent of base was required. However, because the use of an excessive amount of base can be one of the causes of hydrolysis of the acetyl group, it became very important to find the appropriate amount of base, and experiments for this purpose were conducted below.

[0134] <Preparation Example 18> Preparation of sodium salt of acetylated hyaluronic acid (Entry 19)

[0135] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 60 mL (20 volumes) of isopropanol and stirred for 10 to 15 minutes to obtain a suspension. The acetylated hyaluronic acid (AcHA) did not dissolve, so coarse particles were mixed in a milky, dilute solution. 12 mL of an aqueous solution containing 0.6 g (7.10 mmol) of sodium bicarbonate was added to the suspension while stirring was continued. This mixture initially forms a sticky mass, and further stirring yields a free-flowing suspension.

[0136] 180 mL of isopropanol was added to the above mixture (60 mL aliquots) and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered, washed with 60 mL of isopropanol, and dried to obtain 2.68 g of the sodium salt of acetylated hyaluronic acid, a solid compound (recovery rate 86%). Of the sodium salt of acetylated hyaluronic acid 1 In the H NMR spectrum, the peak area of ​​the acetyl group (δ 1.78-1.82 ppm) decreased from 15.01 to 14.32.

[0137] FIG. 20 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0138] <Preparation Example 19> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 20) (Perform secondary filtration)

[0139] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 60 mL (20 volumes) of isopropanol and stirred for 10 to 15 minutes to obtain a suspension. The acetylated hyaluronic acid (AcHA) did not dissolve, so coarse particles were mixed in a milky, dilute solution. 12 mL of an aqueous solution containing 0.6 g (7.10 mmol) of sodium bicarbonate was added to the suspension while stirring was continued. This mixture initially forms a sticky mass, and further stirring yields a free-flowing suspension.

[0140] The mixture was stirred for 30 minutes, and 0.3 g of solids were removed from the mixture by filtration (first filtration).

[0141] 180 mL of isopropanol was added to the filtrate (60 mL aliquots) and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered (secondary filtration), washed with 60 mL of isopropanol, and dried to obtain 2.14 g of the sodium salt of acetylated hyaluronic acid, a solid compound (recovery rate 69%). Of the sodium salt of acetylated hyaluronic acid 1 In the H NMR spectrum, the peak area of ​​the acetyl group (δ 1.78-1.82 ppm) decreased from 15.01 to 14.01.

[0142] Meanwhile, the characteristics of pKa (3.14; 4.52), Log P (1.10) and Log D (-1.39 @ pH7.4), number average molecular weight (Mn) 5804 Da, weight average molecular weight (Mw) 15864 Da, and maximum peak molecular weight (Mp) 7927 Da were observed.

[0143] FIG. 21 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0144] FIG. 22 is an image showing the progress of the reaction of a sodium salt of acetylated hyaluronic acid according to one embodiment of the invention. It can be seen that a much cleaner product can be obtained compared to the case of Entry 17.

[0145] Here, a gray solid was produced in the first filtration, and NMR analysis revealed that it contained the sodium salt (NaAcHa) of the desired acetylated hyaluronic acid along with impurities (which were later confirmed to be sodium bicarbonate through acid treatment). On the other hand, the material obtained from the second filtration was a very clean solid, although the yield was slightly reduced (69% recovery).

[0146] <Preparation Example 20> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Entry 21) (Addition of 2 equivalents of sodium bicarbonate)

[0147] 3.0 g (5.46 mmol) of acetylated hyaluronic acid (AcHA) was added to 60 mL (20 volumes) of isopropanol and stirred for 10 to 15 minutes to obtain a suspension. The acetylated hyaluronic acid (AcHA) did not dissolve, so coarse particles were mixed in a milky, dilute solution. 12 mL of an aqueous solution containing 0.92 g (10.92 mmol) of sodium bicarbonate was added to the suspension, and stirring was continued. This mixture initially forms a sticky mass, and further stirring yields a free-flowing suspension.

[0148] The mixture was stirred for 30 minutes, and 0.61 g of solids were removed from the mixture by filtration (first filtration).

[0149] 180 mL of isopropanol was added to the filtrate (60 mL aliquots) and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered (secondary filtration), washed with 60 mL of isopropanol, and dried to obtain 2.33 g of the sodium salt of acetylated hyaluronic acid, a solid compound (recovery rate 75%). Of the sodium salt of acetylated hyaluronic acid 1In the H NMR spectrum, the peak area of ​​the acetyl group (δ 1.78-1.82 ppm) decreased from 15.01 to 14.52.

[0150] FIG. 23 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0151] <Preparation Example 21> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Scale-Up 1)

[0152] We intended to identify potential issues that may arise during the mass production process by scaling up Entry 20, and specifically to verify the possibility of hydrolysis of acetyl groups by conducting artificial time-delay experiments considering the production process on a kg scale. Under the scale-up conditions of Entry 20, the remaining processes, such as sedimentation and filtration, were carried out after primary filtration and an artificial time-delay. This time-delay was artificially set to simulate situations that may occur when handling homogeneous mixtures during mass production.

[0153] 15 g (27.3 mmol) of acetylated hyaluronic acid (AcHA) was added to 300 mL (20 volumes) of isopropanol and stirred for 15 minutes to obtain a suspension. The acetylated hyaluronic acid (AcHA) did not dissolve. 60 mL of an aqueous solution containing 3.0 g (35.5 mmol) of sodium bicarbonate was added to the suspension while stirring was continued. The mixture was reacted for 10 minutes using an addition funnel. The mixture initially formed a sticky mass, and with further stirring, a free-flowing suspension was obtained within 15 to 20 minutes. The solids were removed from the mixture by filtration (2.3 g, filtration time 30 minutes).

[0154] After stirring the filtrate for 1.5 hours, 900 mL of isopropanol was added (300 mL aliquots) and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered, washed with 300 mL of isopropanol, and dried to obtain 10.7 g of the sodium salt of acetylated hyaluronic acid, a solid compound (recovery rate 34%). Of the sodium salt of acetylated hyaluronic acid 1 In the H NMR spectrum, the peak area of ​​the acetyl group (δ 1.78-1.82 ppm) decreased from 15.01 to 14.2.

[0155] The number average molecular weight (Mn) was 5560 Da, the weight average molecular weight (Mw) was 16457 Da, and the maximum peak molecular weight (Mp) was 7488 Da.

[0156] FIG. 26 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0157] <Experimental Example 2> Analysis of the degree of hydrolysis of acetyl groups (Scale-up 1)

[0158] FIG. 25 is a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention. 1 This is the H NMR spectrum. From Fig. 25, it can be confirmed that the hydrolysis reaction of the acetyl group was negligible (NMR integration margin of error ±5%).

[0159] <Preparation Example 22> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Scale-up 2) (Time-delay of 1 hour 30 minutes)

[0160] 20 g (54.6 mmol) of acetylated hyaluronic acid (AcHA) was added to 400 mL (20 volumes) of isopropanol and stirred for 15 minutes to obtain a suspension. The acetylated hyaluronic acid (AcHA) did not dissolve. 80 mL of an aqueous solution containing 4.0 g (47.3 mmol) of sodium bicarbonate was added to the suspension while stirring was continued. The mixture was reacted for 10 minutes using an addition funnel.

[0161] The mixture was stirred for 1 hour and 30 minutes, and 5.1 g of solids were removed from the mixture by filtration (filtration time 30 minutes). The mixture initially forms a sticky mass, and upon further stirring, a free-flowing suspension is obtained within 15 to 20 minutes.

[0162] 1200 mL of isopropanol was added to the filtrate (600 mL aliquot) and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered, washed with 200 mL of isopropanol, and dried to obtain 13.9 g of the sodium salt of acetylated hyaluronic acid, a solid compound (recovery rate 67%). Of the sodium salt of acetylated hyaluronic acid 1 In the H NMR spectrum, the peak area of ​​the acetyl group (δ 1.78-1.82 ppm) decreased from 15.01 to 14.08.

[0163] FIG. 27 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0164] <Preparation Example 23> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Scale-up 3) (Time-delay of 3 hours)

[0165] 20 g (54.6 mmol) of acetylated hyaluronic acid (AcHA) was added to 400 mL (20 volumes) of isopropanol and stirred for 15 minutes to obtain a suspension. The acetylated hyaluronic acid (AcHA) did not dissolve. 80 mL of an aqueous solution containing 4.0 g (47.3 mmol) of sodium bicarbonate was added to the suspension while stirring was continued. The mixture was reacted for 10 minutes using an addition funnel.

[0166] The mixture was stirred for 3 hours, and 5.1 g of solids were removed from the mixture by filtration (filtration time 30 minutes). The mixture initially forms a sticky mass, and upon further stirring, a free-flowing suspension is obtained within 15 to 20 minutes.

[0167] 1200 mL of isopropanol was added to the filtrate (600 mL aliquot) and stirred for 30 minutes to obtain a solid compound. The solid compound was filtered, washed with 200 mL of isopropanol, and dried to obtain 14.8 g of the sodium salt of acetylated hyaluronic acid, a solid compound (recovery rate 71%). Of the sodium salt of acetylated hyaluronic acid 1 In the H NMR spectrum, the peak area of ​​the acetyl group (δ 1.78-1.82 ppm) decreased from 15.01 to 14.4.

[0168] FIG. 28 is an image showing the appearance of a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention.

[0169] <Preparation Example 24> Preparation of Sodium Salt of Acetylated Hyaluronic Acid (Optimized Final Reaction Conditions)

[0170] 15.0 g (27.3 mmol) of acetylated hyaluronic acid (AcHA) was added to 300 mL (20 volumes) of isopropanol and stirred for 15 minutes to obtain a suspension. 60 mL of an aqueous solution containing 2.98 g (35.5 mmol) of sodium bicarbonate was slowly added to the suspension over 10 minutes while stirring continued. The mixture initially forms a sticky mass, and after stirring for another 30 minutes, a free-flowing suspension is obtained within 15 to 20 minutes.

[0171] The solid component was removed from the above mixture by filtration (2.3 g, filtration time 30 min), 900 mL of isopropanol was added to the filtrate (300 mL at 5-minute intervals) and stirred for 30 minutes to obtain a white solid compound. The white solid compound was filtered, washed with 150 mL of isopropanol, and dried to obtain 11.2 g of sodium salt of acetylated hyaluronic acid, which is a white solid compound (recovery rate 71%).

[0172] <Experimental Example 3> Analysis of the Degree of Hydrolysis of Acetyl Groups (Optimized Final Reaction Conditions)

[0173] FIG. 29 is a sodium salt of acetylated hyaluronic acid according to one embodiment of the present invention. 1This is the H NMR spectrum. From Fig. 29, it can be confirmed that the hydrolysis reaction of the acetyl group was negligible.

[0174] Although the present invention has been described in relation to the preferred embodiments mentioned above, various modifications and variations are possible without departing from the essence and scope of the invention. Accordingly, the appended claims will include such modifications and variations insofar as they fall within the essence of the invention.

Claims

Claim 1 A method for preparing a sodium salt of acetylated hyaluronic acid, comprising: a step of adding acetylated hyaluronic acid to a first organic solvent to obtain a suspension; a step of adding a pH adjusting agent to the suspension to obtain a flowable suspension; a step of adding a second organic solvent to a filtrate from which solids have been removed by filtering the flowable suspension to obtain a solid compound; and a step of purifying the solid compound to obtain a sodium salt of acetylated hyaluronic acid; wherein the pH adjusting agent is sodium bicarbonate, sodium carbonate, or a mixture thereof, and the equivalent weight of the pH adjusting agent is 1 to 2 based on the equivalent weight of the acetylated hyaluronic acid, and the first organic solvent and the second organic solvent are each independently one or more selected from the group consisting of methanol, ethanol, isopropanol, n-propanol, acetonitrile, dimethyl sulfoxide, tetrahydrofuran, and acetone. Claim 2 A method for preparing a sodium salt of acetylated hyaluronic acid according to claim 1, wherein the pH adjusting agent is sodium bicarbonate, the first organic solvent and the second organic solvent are both isopropanol, the sodium salt of acetylated hyaluronic acid is a white solid compound, the weight recovery rate of the sodium salt of acetylated hyaluronic acid is 70% or more, and the degree of acetylation of the sodium salt of acetylated hyaluronic acid is 3.8 or more. Claim 3 A method for preparing a sodium salt of acetylated hyaluronic acid according to claim 1, wherein the acetylated hyaluronic acid has a degree of acetylation of 0.75 or higher and is prepared through the following steps: a step of mixing hyaluronic acid with an organic acid, acetic anhydride, and a basic catalyst and heating to obtain a homogeneous mixture; a step of acetylating the homogeneous mixture to obtain an acetylated hyaluronic acid solution; a step of removing acetic acid and hyaluronic anhydride from the acetylated hyaluronic acid solution to obtain an acetylated hyaluronic acid slurry; and a step of converting the acetylated hyaluronic acid slurry into an acetylated hyaluronic acid solid under acidic conditions. Claim 4 A method for preparing a sodium salt of acetylated hyaluronic acid, wherein, in paragraph 3, the organic acid is acetic acid and the basic catalyst is 4-(dimethylamino)pyridine (DMAP). Claim 5 The octanol-water partition coefficient (log P) is -1 to 5, and the distribution coefficient (log D @ pH7.4) (log D 7.4 A sodium salt of acetylated hyaluronic acid prepared by the method of one of claims 1 to 4, wherein ) is -2 to 5, the number average molecular weight (Mn) is 5000 to 7000 Da, and the weight average molecular weight (Mw) is 5000 to 30000 Da.

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