Hyaluronic acid derivative, and preparation method therefor and use thereof

Abstract

The present invention relates to a hyaluronic acid derivative as shown in formula I, and a preparation method thereof and the use thereof in the preparation of an anti-infection drug. The structure of the compound or the pharmaceutically acceptable salt thereof of the present invention is significantly different from that of commercially available hyaluronic acid substances such as TCIsHA and R&D HA, and the compound or the pharmaceutically acceptable salt thereof can form a molecular structure recognized by HepSS-1. The molecule with the new structure can achieve an anti-infection effect by means of inhibiting the binding of SARS-Cov-2 spike protein to ACE2 of a host cell, and is less likely to cause serious adverse reactions clinically, such as bleeding and thrombocytopenia, as compared with heparin.

Description

Hyaluronic acid derivatives and their preparation methods and applications

[0001] This application claims priority to a prior application filed with the State Intellectual Property Office of China on June 29, 2023, with patent application number 202310778756.8, entitled “Hyaluronic acid derivatives, preparation methods and applications thereof.” The entire text of that application is incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to a hyaluronic acid derivative and a preparation method and application thereof. Background Art

[0003] Hyaluronic acid (HA) is a glycosaminoglycan composed of D-glucuronic acid and N-acetylglucosamine alternately linked by β-1,4-glycosidic bonds and β-1,3-glycosidic bonds. It is an important component of the natural extracellular matrix. Hyaluronic acid substances may have different pharmacological activities depending on their molecular weight and modified groups, and therefore can be widely used in cosmetics, food, and pharmaceuticals.

[0004] Hyaluronic acid's high water absorption capacity and high viscoelasticity give it unique properties in biomaterials and make it suitable for a variety of medical and pharmaceutical applications. We can find a variety of hyaluronic acid products in our daily lives. For example, because hyaluronic acid retains moisture, it is used in some cosmetics to maintain youthful and fresh skin. Although hyaluronic acid is abundant in the skin, its water-retention capacity decreases with age due to depolymerization. Therefore, maintaining an abundant supply of hyaluronic acid in the skin can help us maintain youthfulness.

[0005] Researchers have proposed many ideas for applying hyaluronic acid in areas other than cosmetics; however, there are few actual applications in medicine, primarily in the treatment of osteoarthritis and in ophthalmic medical devices.

[0006] Heparin is also a glycosaminoglycan, formed by alternating 1,4-glycosidic bonds between hexuronic acid and N-acetylglucosamine. It is widely used clinically as an anticoagulant, and some modified hyaluronic acid compounds have similar anticoagulant effects to heparin. However, due to the anticoagulant activity of heparin and its analogs, they can easily cause serious adverse reactions such as bleeding and thrombocytopenia.

[0007] Summary of the Invention

[0008] In order to improve the technical problems existing in the prior art, in a first aspect, the present invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein the structure of formula I is shown below:

[0009] In the formula I, R is the same or different and is independently selected from H or SO3H;

[0010] M is selected from a proton or a monovalent cation;

[0011] n is selected from 3-9.

[0012] According to an embodiment of the present invention, n is selected from 3, 4, 5, 6, 7, 8, and 9.

[0013] According to an embodiment of the present invention, the monovalent cation is selected from Na + or K + .

[0014] According to an embodiment of the present invention, the weight average molecular weight of Formula I is less than 10000. In some embodiments, the weight average molecular weight of Formula I is 2480-6590.

[0015] According to an embodiment of the present invention, in Formula I, at least some of R are selected from SO3H (i.e., not all R are H). In some embodiments, in Formula I, the degree of substitution by sulfate is less than or equal to 4.2; in some embodiments, in Formula I, the degree of substitution by sulfate is greater than 3.65, preferably, the degree of substitution by sulfate is greater than or equal to 3.75; in some embodiments, in Formula I, the degree of substitution by sulfate is less than or equal to 4.2 and greater than 3.65; in some embodiments, in Formula I, the degree of substitution by sulfate is less than or equal to 4.2 and greater than or equal to 3.75; in some embodiments, in Formula I, the degree of substitution by sulfate is less than or equal to 4.0 and greater than or equal to 3.75; for example, the degree of substitution by sulfate is selected from 3.70, 3.75, 3.80, 3.85, 3.90, 3.95, 4.00, 4.05, 4.10, 4.05, 4.10, 4.15 or 4.20.

[0016] According to an embodiment of the present invention, in the formula I, NHCOCH3 is not substituted by sulfate.

[0017] According to an embodiment of the present invention, the structure of formula I can be recognized by HepSS-1.

[0018] In a second aspect, the present invention provides a method for preparing the compound of formula I or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0019] (1) mixing water with hyaluronic acid or a water-soluble salt thereof;

[0020] (2) adding a sulfating agent and reacting;

[0021] (3) Add ethanol, mix and centrifuge;

[0022] (4) removing the supernatant and adding water, adding ethanol, mixing and centrifuging;

[0023] (5) Remove the supernatant and let stand for 4-24 hours;

[0024] (6) Add water, filter, and freeze-dry to obtain the product.

[0025] According to an embodiment of the present invention, the sulfating agent is selected from a complex of sulfur trioxide, preferably a pyridine complex of sulfur trioxide.

[0026] According to an embodiment of the present invention, the hyaluronic acid or a water-soluble salt thereof is selected from Hyalonano. Preferably, the hyaluronic acid or a water-soluble salt thereof is purchased from kewpie.

[0027] According to an embodiment of the present invention, in step (1), 0.5-3 g of hyaluronic acid or a water-soluble salt thereof is mixed with every 1 ml of water. Preferably, 0.8-1.2 g of hyaluronic acid or a water-soluble salt thereof is mixed with every 1 ml of water. For example, 1 g of hyaluronic acid or a water-soluble salt thereof is mixed with every 1 ml of water.

[0028] According to an embodiment of the present invention, the water-soluble salt is selected from sodium hyaluronate.

[0029] According to an embodiment of the present invention, in the step (2), the reaction temperature is 0-20°C, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20°C, preferably, 3-10°C; the reaction time is 24-100 hours, preferably, 36-90 hours, for example, 36, 48, 60, 72, 84 hours.

[0030] According to an embodiment of the present invention, the mass ratio of the sulfating agent to hyaluronic acid or a water-soluble salt thereof is 0.5-6:100, preferably 1-3:100, for example, 1.5:100, 2.0:100, 2.5:100, 3.0:100, 3.5:100, or 4.0:100.

[0031] According to an embodiment of the present invention, in step (3), the amount of ethanol added is: 10-50 ml of ethanol is added to every 1 g of hyaluronic acid or its water-soluble salt, preferably, 20-30 ml of ethanol is added, for example, 25 ml of ethanol is added.

[0032] According to an embodiment of the present invention, in step (4), the amount of water added is: per 1g of hyaluronic acid or its water-soluble salt, add 0.5-2ml of water, preferably, add 1ml of water; the amount of ethanol added is: per 1g of hyaluronic acid or its water-soluble salt, add 20-30ml of ethanol, preferably, add 25ml of ethanol;

[0033] According to an embodiment of the present invention, in step (3) and step (4), the centrifugation is carried out at 0-10°C, 2000-4000 rpm, and 5-30 minutes; preferably, the centrifugation temperature is 3-6°C, for example, 4°C; preferably, the centrifugation speed is 3000 rpm; preferably, the centrifugation time is 10-20 minutes, for example, 15 minutes.

[0034] According to an embodiment of the present invention, in step (5), the storage temperature is -20 to 5°C, preferably, -10 to 4°C; and the storage time is preferably 6-12 hours.

[0035] According to an embodiment of the present invention, in step (6), the amount of water added is: 0.5-2 ml of water is added for every 1 g of hyaluronic acid or its water-soluble salt, preferably, 1 ml of water is added.

[0036] According to an embodiment of the present invention, in step (6), a disc filter is used, preferably, a 0.45 μm disc filter is used.

[0037] In a third aspect, the present invention further provides a composition comprising the compound of formula I or a pharmaceutically acceptable salt thereof.

[0038] According to an embodiment of the present invention, the composition further comprises a pharmaceutically acceptable carrier.

[0039] In a fourth aspect, the present invention provides use of the compound of formula I or a pharmaceutically acceptable salt thereof or the composition in the preparation of anti-infective drugs.

[0040] According to an embodiment of the present invention, the infection is selected from infections caused by coronavirus (COVID-19); the coronavirus is selected from SARS-COV-2.

[0041] According to an embodiment of the present invention, the preparation of the medicine can be applied topically through the skin, for example, selected from creams, patches, ointments, cream preparations, gels and sprays, etc., and can also be applied orally (i.e., oral preparations), for example, in solid form, selected from tablets, granules, capsules, pills, dripping pills, etc. In some embodiments, the preparation of the medicine can also be selected from liquid or semisolid dosage forms (liquids, suspensions, solutions, dispersions, ointments, creams, emulsions, microemulsions, sprays, patches, drops). Injections, parenterals, inhalations, buccal administration, nasal preparations, etc. are also within the scope of the present invention.

[0042] The preparation further includes a physiologically acceptable carrier (e.g., a biocompatible material), and optionally includes a surfactant, an excipient, a moisturizer, an emulsifying promoter, a suspending agent, a salt or buffer for regulating the osmotic pressure, a colorant, a flavor, a stabilizer, a bactericide, a preservative or other conventional supplements.

[0043] In a fifth aspect, the present invention provides an anti-infection method, comprising administering to a patient a therapeutically effective amount of the compound of formula I or a pharmaceutically acceptable salt thereof or the composition of the present invention.

[0044] According to an embodiment of the present invention, the infection is selected from the infection caused by coronavirus (COVID-19).

[0045] Beneficial effects of the present invention

[0046] The present invention discovered that the compound of Formula I or a pharmaceutically acceptable salt thereof has a significantly different structure from commercially available hyaluronic acid substances such as TCIsHA and R&D HA, and can form a molecular structure recognized by HepSS-1. This novel molecule can inhibit the binding of the SARS-CoV-2 spike protein to the host cell's ACE2, thereby exerting an anti-infective effect. Compared with heparin, it is less likely to cause serious adverse reactions in clinical practice, such as bleeding and thrombocytopenia. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 shows the structure of sHA 1 H NMR spectrum;

[0048] Figure 2 shows the results of HepSS-1 recognition experiments;

[0049] Figure 3 shows the HPLC spectrum of sHA synthesized by the present invention, wherein the numbers represent the number of sugar residues, and the arrows represent the same number of disaccharide units (wherein the numbers 2, 4, 6, etc. represent the number of sugar residues; the solid arrows indicate that sulfation screening has been performed to a higher MV; the dotted arrows indicate that the maximum MW limit of sHA is almost the same as that of Fragmin);

[0050] FIG4 shows the results of binding experiments of the compounds of the present invention with the spike protein of SARS-COV-2;

[0051] FIG5 shows the prothrombin time of sHA and control substances. DETAILED DESCRIPTION

[0052] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0053] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0054] Example 1

[0055] Preparation of the compounds of the present invention

[0056] 1.1 Preparation method of compound sHA of formula I

[0057] 1.11 Preparation of Compound sHA-1 of Formula I

[0058] 20g hyaluronic acid (trade name Hyalonano, kewpie38399) was added and mixed in 20mL deionized water, and then 0.3g pyridine-sulfur trioxide complex (purchased from FUJIFILM Wako Pure Chemical Corporation, Fujifilm Wako Pure Chemical Co., Ltd.) was added and mixed at 4°C for 72 hours. 500mL ethanol was added, mixed and centrifuged (4°C, 3000rpm, 15 minutes). The supernatant was removed and 20mL deionized water was added, 500mL ethanol was added, mixed and centrifuged (4°C, 3000rpm, 15 minutes). The supernatant was removed and 20mL deionized water was added, 500mL ethanol was added, mixed and centrifuged (4°C, 3000rpm, 15 minutes). The supernatant was removed and stored in a refrigerator overnight. 20mL deionized water was added and applied to a disc filter (0.45μm: ADVANTEC), the solution was lyophilized, and 1.73g of product (i.e. sHA-1) was obtained.

[0059] 1.12 Referring to the process of 1.11, different mass ratios of pyridine-sulfur trioxide complex and hyaluronic acid were selected to prepare compounds of formula I sHA-2 to sHA-7.

[0060] 1.2 Structural Identification of Compound sHA of Formula I

[0061] 1.2.1 Detection method

[0062] (1) 1 H NMR

[0063] (2)HPLC

[0064] (3) HepSS-1 identification (based on ELISA)

[0065] The samples used are shown in the following table:

[0066] Among them, highly sulfated hyaluronic acid TCIs HA can also be prepared according to the literature [Highly sulfated hyaluronic acid maintains human induced pluripotent stem cells under feeder-free and bFGF-free conditions. Taichi Miuraa, Noriyuki Yuasac, Hayato Otab, Masato Habuc, Mitsuko Kawanoa, Fumiaki Nakayamaa, Shoko Nishihara. August 2019, Biochemical and Biophysical Research Communications 518(3)].

[0067] The steps are as follows:

[0068] Each sample (20 μg / 100 μL) was immobilized on a 96-well plate;

[0069] incubated with HepSS-1;

[0070] Incubate with HRP-anti mouse IgM;

[0071] incubation with 3,3'-diaminobenzidine (DAB) and 30% hydrogen peroxide;

[0072] iMark plate Reader wavelength: 405nm.

[0073] 1.2.2 Analysis of identification results

[0074] After identification, the structures of compounds sHA-1 to sHA-7 are shown below:

[0075] In the above structure, R is H or SO3H, GlcA: glucuronic acid; GlcNac: N-acetylglucosamine; and n=3.

[0076] (1) Sutra 1 H NMR and HPLC identification showed that in the structures of sHA-1 to sHA-7, n was 3 (8mers), the weight-average molecular weight (MW) was 2480, and the degree of sulfate substitution (i.e., [SO3 - ] groups) is less than or equal to 4.2 and greater than 3.65 (the degree of sulfate substitution of a disaccharide completely sulfated at both ends is 5). In TCIsHA, the degree of sulfate substitution is 3.65.

[0077] (2) 1 H NMR showed that in the structures of sHA-1 to sHA-7, the NHCOCH3 of GlcNac was not replaced by SO3-. However, these sHAs can be recognized by HepSS-1 (Figure 2). HepSS-1 recognizes N-sulfation of heparan sulfate, but not O-sulfation of heparan sulfate, heparin, or hyaluronic acid. Some O-SO3- is tightly bound to the NHCOCH3 in GlcNac in three dimensions, forming a molecular structure recognized by HepSS-1. The results of the HepSS-1 recognition experiment further showed that sHA can be recognized by HepSS-1, while TCIsHA and R&DHA cannot be recognized by HepSS-1 (see Figures 1-2).

[0078] 1.3 Preparation and identification of other compounds

[0079] Experimental verification shows that using hyaluronic acid with other molecular weights, for example, hyaluronic acid with n=3-9 (8-20mers) and weight average molecular weight (MW) of 2480-6590, other than hyaluronic acid with n=3, the above-mentioned products with similar sulfate substitution degree can also be prepared by referring to the process of 1.11 (see Figure 3). 1 H NMR analysis showed that HepSS-1 could recognize the saccharides when n was 3-9 (8-20mers), the weight-average molecular weight (MW) was 2480-6590, and the degree of sulfate substitution (i.e., the number of [SO3-] groups per disaccharide) was <4.2 and >3.65 (the degree of sulfate substitution of a disaccharide completely sulfated at both ends was 5).

[0080] Example 2

[0081] Physiological activities of the compounds of the present invention

[0082] 2.1 Anti-SARS-COV-2 infection test

[0083] The binding activity of the sHA of the present invention to the spike protein of SARS-COV-2 was evaluated using an ELISA-based immunoassay, and the experimental steps were as follows:

[0084] sHA-1 (20 μg / 100 μL, 20 μg / 100 μL) prepared according to Example 1 was added to a 96-well plate coated with SARS-Cov-2 spike protein;

[0085] Antibodies were incubated using HepSS-1 (anti- N -acetylheparan sulfate, mouse IgM), and anti-immunoglobulin was added twice;

[0086] incubation with 3,3'-diaminobenzidine (DAB);

[0087] iMark plate Reader wavelength: 405nm.

[0088] The experimental results show (see Figure 4) that the compounds of the present invention can inhibit the binding of SARS-Cov-2 spike protein to ACE2 of host cells, thereby exerting an anti-infection effect.

[0089] 2.2 Effects of the compounds of the present invention on blood coagulation

[0090] According to the reference [Sulakshan Sulakshana et al., Anesth Essays Res., 2021, 15(4): 341–347], the anticoagulant activity of the compound was evaluated by testing the prothrombin time.

[0091] The experimental methods and steps are as follows:

[0092] 1) Obtain approximately 2 mL of blood from the abdominal aorta of male SD rats (8 weeks old). 1 sample / rat, n = 3;

[0093] 2) Each substance is added to blood at a concentration of 27.8 μg / mL or 278 μg / mL and mixed (usually, 27.8 μg / mL is used for blood transfusion).

[0094] 3) To evaluate the anticoagulation effect of the samples, prothrombin time was measured using CoaguChek X (Sekisui Medical).

[0095] Control group:

[0096] 1) Normal saline;

[0097] 2) Unfractionated heparin (UFH), heparin sodium injection 5000 units / 5 mL, Mochida (average molecular weight 15000);

[0098] 3) Fragmin: low molecular weight heparin, average molecular weight 5000, 5000 units / 5mL;

[0099] 4) Arixtra: low molecular weight heparin, average molecular weight 1728 (5mers), 5000 units / 5 mL;

[0100] 5) No additives.

[0101] The test results are shown in Figure 5. At 27.8 μg / mL, no substance caused any change. At 278 μg / mL, the prothrombin time of UFH, Fragmin or Arixtra was significantly longer than that of the normal saline group and the no-addition group. However, sHA-1 prepared according to Example 1 had no effect. Among them, Fragmin and Arixtra are heparin drugs with low molecular weight, the purpose of which is to reduce anticoagulant activity, but according to existing studies, they have weak binding to the SARS-COV2 spike protein. sHA-1 did not show anticoagulant activity even at 10 times the clinical use concentration of heparin. Therefore, compared with heparin or even low molecular weight heparin, the sHA of the present invention is not easy to cause serious adverse reactions such as bleeding and thrombocytopenia in clinical practice.

[0102] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof, wherein: The structure of Formula I is shown below: In the formula I, R is the same or different and is independently selected from H or SO3H; M is selected from a proton or a monovalent cation; n is selected from 3 to 9; In the formula I, the degree of sulfation substitution is less than or equal to 4.2 and greater than 3.65; The structure of formula I can be recognized by HepSS-1.

2. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein In the formula I, NHCOCH3 is not substituted by sulfate; Preferably, n is selected from 3, 4, 5, 6, 7, 8, 9; Preferably, the monovalent cation is selected from Na + or K + .

3. A compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein: In the formula I, the degree of sulfation substitution is less than or equal to 4.2 and greater than or equal to 3.

75.

4. A method for preparing a compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, comprising the following steps: (1) mixing water with hyaluronic acid or a water-soluble salt thereof; (2) adding a sulfating agent and reacting; (3) adding ethanol, mixing and centrifuging; (4) removing the supernatant and adding water, adding ethanol, mixing and centrifuging; (5) Remove the supernatant and let stand for 4-24 hours; (6) Add water, filter, and freeze-dry to obtain the product.

5. The method according to claim 4, characterized in that: The sulfating agent is selected from the pyridine complex of sulfur trioxide.

6. The method according to claim 4, characterized in that: In the step (1), 0.5-3 g of hyaluronic acid or its water-soluble salt is mixed with every 1 ml of water.

7. The method according to any one of claims 4 to 6, characterized in that: In the step (2), the reaction temperature is 0-20°C; the reaction time is 24-100 hours; The mass ratio of the sulfating agent to hyaluronic acid or its water-soluble salt is 0.5-6:100; In the step (3), the amount of ethanol added is: 10-50 ml of ethanol is added for every 1 g of hyaluronic acid or its water-soluble salt; In the step (4), the amount of water added is: 0.5-2 ml of water is added for every 1 g of hyaluronic acid or its water-soluble salt.

8. A composition comprising the compound of formula I according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

9. The composition according to claim 8, characterized in that The composition also includes a pharmaceutically acceptable carrier.

10. Use of the compound of formula I according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof or the composition according to claim 8 in the preparation of an anti-infective drug.

11. The use according to claim 10, characterized in that: The infection is selected from infections caused by coronavirus (COVID-19).

12. An anti-infection method, comprising administering to a patient a therapeutically effective amount of the compound of formula I according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, or the composition according to claim 8.

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