Oral anticoagulant deoxycholic acid modified glucopyranose sulfate octasaccharide as well as preparation method and application thereof

Abstract

The invention discloses an oral anticoagulant deoxycholic acid modified glucopyranose sulfate octasaccharide as well as a preparation method and application thereof. The octaparin-DOCA modifier is synthesized by connecting amido bonds formed by condensation of sulphated glucogalactan octasaccharide and lipophilic DOCA molecules, and the octaparin-DOCA modifier is synthesized by connecting the amido bonds with the lipophilic DOCA molecules. The octaparin-DOCA modifier disclosed by the invention shows longer anticoagulation duration and higher bioavailability after oral administration, can effectively inhibit thrombosis, has a good application prospect in the aspect of preparing an oral anticoagulant, and is particularly suitable for chronic thrombotic disease patients needing long-term anticoagulation treatment.

Description

Technical Field

[0001] This invention belongs to the field of anticoagulant technology, and relates to an oral anticoagulant, deoxycholic acid modified glucosamine sulfate (octaparin-DOCA), its preparation method and application. Background Technology

[0002] Anticoagulation therapy plays a crucial role in the prevention and treatment of various vascular diseases, including venous thromboembolism (VTE), atrial fibrillation-related thrombosis, and stroke. Currently, widely used anticoagulants primarily include heparin and its derivatives, such as unclassified heparin (UFH), low molecular weight heparin (LMWH), and synthetic heparin analogs. While these drugs have excellent anticoagulant effects, almost all require subcutaneous or intravenous administration, significantly limiting their convenience and patient compliance in long-term anticoagulation therapy.

[0003] While oral anticoagulants (such as warfarin, dabigatran, and rivaroxaban) have been developed, they primarily target clotting factors or thrombin and cannot yet replace heparin-based drugs, which primarily work by inhibiting clotting enzymes. Heparin-based drugs are mostly highly polar, high-molecular-weight polysaccharides that cannot effectively cross the gastrointestinal epithelial barrier and are easily degraded by heparinases in the body, resulting in extremely low bioavailability and rendering them ineffective for anticoagulation after oral administration.

[0004] To overcome these challenges, several research groups have attempted to improve the oral absorption efficiency of heparin using various methods, including delivery systems such as liposomes, emulsions, nanoparticles, and enteric coatings, or by forming complexes with hydrophobic small molecules to improve its gastrointestinal adhesion and membrane permeability. Other studies have evaluated the effects of absorption enhancers, including EDTA, acidic buffers, sodium caprylate, and deoxycholic acid (DOCA) modification, on oral heparin absorption, providing preliminary validation of its anticoagulant activity in animals. Li et al. found that deoxycholic acid modification can effectively extend the duration of efficacy of low molecular weight heparin after oral administration, thereby enhancing its persistence and stability under oral administration (DYLee, Journal of Controlled Release 2007, 118, 310). However, these methods generally suffer from short duration of efficacy, unstable delivery systems, and low transport efficiency, and still cannot meet the clinical demand for safe, controllable, and sustained anticoagulation therapy.

[0005] The inventors previously reported a novel heparin analogue, riclinoctaose, synthesized from riclinoctaose via a one-pot sulfonation reaction. This analogue exhibits excellent anticoagulant properties and, compared to traditional heparin, shows a significantly reduced bleeding risk and fewer platelet-related adverse reactions, demonstrating higher safety (Yu N, Fang R, Ding Z, et al. Preparation and structural characterization of a sulfated octasaccharide with heparin-like anticoagulant activity[J]. Carbohydrate Polymers, 2025, 347:122782.). Summary of the Invention

[0006] To address the problems of low oral bioavailability, short anticoagulant time, and inconvenience for long-term use of existing heparin drugs, this invention provides an oral anticoagulant, deoxycholic acid-modified glucosamine sulfate, its preparation method, and its application.

[0007] The deoxycholic acid-modified glucosamine sulfate of the present invention has the following structural formula:

[0008]

[0009] The above-mentioned method for preparing deoxycholic acid-modified glucogalactocalose sulfate includes the following steps:

[0010] (1) Synthesis of succinimide DOCA: Deoxycholic acid (DOCA), dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were reacted with tetrahydrofuran (THF) as the reaction solvent to synthesize succinimide DOCA.

[0011] (2) Synthesis of amino-modified DOCA (DOCA-NH2): Amino-modified DOCA was synthesized by reacting succinimide DOCA with ethylenediamine using N,N-dimethylformamide (DMF) as the reaction solvent.

[0012] (3) Synthesis of octaparin-DOCA modified compound: using formamide as the reaction solvent, glucosamine sulfate and amino-modified DOCA were dehydrated in the presence of a condensing agent to synthesize octaparin-DOCA modified compound. The condensing agent was N,N'-dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC).

[0013] The above-mentioned application of deoxycholic acid-modified glucosamine sulfate in the preparation of oral anticoagulants.

[0014] The oral anticoagulant described in this invention is an oral preparation, including but not limited to tablets, capsules, liquid preparations, enteric-coated microparticles, etc.

[0015] The oral anticoagulant described in this invention can be administered to any animal that may have or has already developed thrombotic diseases such as venous thrombosis or pulmonary embolism. These animals include both human and non-human animals, such as pets or livestock.

[0016] The dosage of the oral anticoagulant described in this invention depends on the recipient's age, health, weight, treatment frequency, etc.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] (1) Based on the structure of glucogalactocalose sulfate, the present invention obtains octaparin-DOCA modified product by chemically reacting its carboxyl group with DOCA molecules through condensation and dehydration. This modified product significantly improves the lipophilicity and intestinal absorption capacity of glucogalactocalose sulfate. Animal experiments show that after oral administration of octaparin-DOCA modified product, a significant prolongation of whole blood clotting time can be observed. Its anticoagulant effect lasts for a significantly longer period than that of unmodified glucogalactocalose sulfate, and its bioavailability is significantly improved.

[0019] (2) The oral anticoagulant octaparin-DOCA modified of the present invention improves the adhesion and transmembrane transport capacity of galactostatus sulfate in the gastrointestinal tract by modifying galactostatus sulfate with DOCA, thereby enhancing its absorption efficiency through the intestine and achieving efficient delivery and oral anticoagulation in the intestine, thus expanding the use of heparin-like drugs. Compared with traditional injectable heparin, the oral anticoagulant octaparin-DOCA modified is more convenient to administer, significantly improving patient compliance and quality of life. In addition, the oral dosage form eliminates the need for injection, avoiding problems such as pain, skin damage, and local infection.

[0020] (3) The oral anticoagulant octaparin-DOCA modified of the present invention has good stability and sustained efficacy, and is suitable for patients requiring long-term anticoagulation therapy. It is especially suitable for continuous use in outpatient or home settings for patients undergoing long-term anticoagulation therapy, particularly for the prevention and control of chronic thrombotic diseases in outpatient management and home treatment scenarios. In addition, the oral anticoagulant octaparin-DOCA modified is expected to reduce dependence on medical resources and lower overall treatment costs. Attached Figure Description

[0021] Figure 1 This is a one-dimensional proton spectrum of the octaparin-DOCA modified compound.

[0022] Figure 2 Fourier transform infrared spectrum of octaparin-DOCA modified compound.

[0023] Figure 3 Figure 1 shows the in vivo anticoagulant effect of octaparin-DOCA modified mice. Detailed Implementation

[0024] Unless otherwise specified, the methods used in the following embodiments are conventionally used in the art. Unless otherwise specified, all raw materials used in the following embodiments are commercially available products. Unless otherwise defined, all technical and scientific terms used in this invention have the meanings commonly understood by one of ordinary skill in the art.

[0025] The glucogalactocaccharide sulfate described in this invention is prepared according to the reference [Yu N, Fang R, Ding Z, et al. Preparation and structural characterization of a sulfated octasaccharide with heparin-like anticoagulant activity[J]. Carbohydrate Polymers, 2025, 347:122782.].

[0026] Example 1

[0027] 1. Synthesis of Octaparin-DOCA modified compounds, including the following steps:

[0028] (1) Synthesis of succinimide DOCA

[0029] 5.0 g of deoxycholic acid, 4.2 g of dicyclohexylcarbodiimide, and 2.35 g (38.2 mmol) of N-hydroxysuccinimide were weighed and dissolved together in 50 mL of THF. The reaction was carried out at room temperature for 12 hours. After the reaction was completed, the white precipitate dicyclohexylurea (DCU) was removed by filtration. The filtrate was added dropwise to excess n-hexane for precipitation. The obtained succinimide DOCA precipitate was thoroughly washed with n-hexane and then vacuum dried at room temperature. The dried product was stored at -20 °C for later use.

[0030] (2) Synthesis of amination-modified DOCA (DOCA-NH2)

[0031] Weigh g of succinimide DOCA2, dissolve it in 10 mL of DMF, and slowly add it dropwise to 26.8 mL of ethylenediamine. React at room temperature for 6 hours. After the reaction, add the mixture dropwise to excess distilled water to precipitate the product. Collect the white solid DOCA-NH2, wash it three times with distilled water, and dry it under vacuum to obtain the target product DOCA-NH2.

[0032] (3) Synthesis of Octaparin-DOCA modified compounds

[0033] Weigh 0.2 g of glucogalactoctose sulfate and add it to 10 mL of formamide. Heat until dissolved. Add 0.274 g of EDAC at room temperature, followed by slowly adding 1.35 g of DOCA-NH2 solution. React at room temperature for 24 hours. After the reaction is complete, pour the mixture into excess pre-cooled acetone to precipitate. Collect the precipitate and wash it thoroughly three times with cold acetone to remove unreacted DOCA-NH2. Then dry it under vacuum. Dissolve the dried octaparin-DOCA modifier in deionized water and freeze-dry to obtain a white powdery octaparin-DOCA modifier.

[0034] 2. NMR spectral detection of octaparin-DOCA modified compounds

[0035] Using deuterated heavy water D2O / deuterated methanol CD3OD (1.5:1, v / v) as the solvent, the reaction was performed on a 300MHz nuclear magnetic resonance spectrometer. 1 ¹H NMR was used to verify the conjugated structure of octaparin and DOCA.

[0036] The structure of octaparin-DOCA modified compounds was determined by one-dimensional hydrogen nuclear magnetic resonance (NMR). 1 The H-NMR spectrum was analyzed, and the results are as follows: Figure 1 As shown in the spectrum, multiple characteristic peaks of DOCA appeared in the range of 0.65 to 2.30 ppm, mainly corresponding to the methyl and methylene proton signals in the DOCA molecule, indicating that DOCA was successfully covalently introduced into the structure of glucosamine sulfate. Furthermore, a new lower-field signal was detected at 8.04 ppm, attributed to the amino proton signal generated by the new amide bond formed between glucosamine sulfate and DOCA, further confirming the occurrence of the acylation reaction. 1 H-NMR analysis results strongly demonstrate that the structural modification of the octaparin-DOCA modifier has been successfully achieved.

[0037] 3. Fourier transform infrared (FTIR) detection of octaparin-DOCA modified compounds

[0038] To verify the covalent bond and structural modification between galactocalactose sulfate and DOCA, Fourier transform infrared spectroscopy was used to characterize and analyze the functional groups in the sample. 10 mg of the purified octaparin-DOCA modified sample was weighed and analyzed using an infrared spectrometer. The scanning wavelength range was 4000 cm⁻¹ in transmission mode. -1 Up to 400cm -1 The resolution is 0.48cm. -1 Record its absorption spectrum.

[0039] Figure 2 Fourier transform infrared (FTIR) spectra of glucogalactocataose sulfate and its octaparin-DOCA-modified form are presented to further verify the chemical bonding between the two. Compared with unmodified glucogalactocataose sulfate, the octaparin-DOCA-modified form exhibits higher spectral density at 1,665 cm⁻¹. -1 A distinct new absorption peak appears at 1200–1300 cm⁻¹, which can be attributed to the amide I band (C=O stretching vibration), indicating the formation of a new amide bond between the glucogalactocataose sulfate molecule and DOCA. This change demonstrates that DOCA has been successfully covalently linked to the glucogalactocataose sulfate molecule via acylation. Furthermore, octaparin-DOCA shows a peak at 1200–1300 cm⁻¹. -1 The presence of significantly enhanced COC and S=O related peaks suggests that its skeletal structure still retains the basic characteristics of sulfate groups.

[0040] In summary, the FT-IR results further demonstrate that galactocalose sulfate and DOCA are successfully coupled via amide bonds without disrupting the major functional group structure of the original galactocalose sulfate molecule, thus verifying its structural basis as an oral delivery modifier.

[0041] Example 2: Animal experiment on the oral absorption effect of Octaparin-DOCA modified compound

[0042] To evaluate the anticoagulant effect and duration of efficacy of the octaparin-DOCA modified formulation under oral administration, this study used unmodified octaparin as the control group. Male C57BL / 6J mice (weighing 25-30g) were used and fasted for 12 hours before the experiment, but allowed free access to water. All animal experiments were strictly conducted in accordance with the relevant provisions of the "Guidelines for the Care and Use of Laboratory Animals". Mice in the experimental group were administered a single dose of the octaparin-DOCA modified formulation orally via a stainless steel blunt-tipped gavage needle inserted into the stomach through the esophagus to ensure accurate delivery and minimize mechanical damage to tissues. The octaparin-DOCA modified formulation was administered in propylene glycol at a dose of 100 mg / kg (calculated based on the mass of the octaparin-DOCA modified formulation). The control group received an equal dose of the unmodified glucosamine sulfate formulation orally under the same conditions. Blood samples were collected once via the retroorbital venous plexus at multiple time points after administration to measure blood clotting time, comparing the differences in anticoagulant effect and duration of efficacy between the two groups under oral administration.

[0043] Figure 3 The graphs show the changes in coagulation time at different time points after a single oral administration of galactooctasaccharide sulfate (OP) and octaparin-DOCA modified form (OP-DOCA) to mice. The x-axis represents the detection time point, and the y-axis represents the coagulation time. The results showed that the coagulation time in the non-OP group changed little after gavage administration, increasing slightly only within 0.5 hours, and then rapidly returning to baseline levels within 1.5 hours, indicating extremely low oral absorption efficiency. In contrast, the OP-DOCA group showed a significant prolongation of coagulation time within 1-3 hours after administration, peaking at approximately 150 seconds, followed by a slow decline, remaining above the OP group level until 8 hours, and essentially returning to normal by 10 hours. These results indicate that DOCA modification significantly improves the oral bioavailability of galactooctasaccharide sulfate, giving it significant anticoagulant activity in the oral state and prolonging the duration of its effect.

[0044] It is noteworthy that, compared with the oral administration results of DOCA-modified low molecular weight heparin (DOCA-LMWH) reported in the literature (DYLee, Journal of Controlled Release 2007, 118, 310.), the OP-DOCA group exhibited significantly more sustained efficacy. In the literature, DOCA-LMWH reached its peak coagulation time in mice approximately 20 minutes after oral administration and returned to baseline within about 2 hours. In contrast, the OP-DOCA group in this invention maintained its peak coagulation time for 1-3 hours, and its efficacy lasted for more than 8 hours. This difference suggests that OP-DOCA has superior pharmacokinetic properties compared to DOCA-LMWH, providing a broader application prospect for non-invasive administration of heparin-like drugs.

Claims

1. A deoxycholic acid-modified glucosamine sulfate, characterized in that, The structural formula is: 。 2. The method for preparing deoxycholic acid-modified glucosamine sulfate according to claim 1, characterized in that, Includes the following steps: (1) Synthesis of succinimide DOCA: Deoxycholic acid, dicyclohexylcarbodiimide and N-hydroxysuccinimide were reacted with tetrahydrofuran as the reaction solvent to synthesize succinimide DOCA; (2) Synthesis of amino-modified DOCA: Amino-modified DOCA was synthesized by reacting succinimide DOCA with ethylenediamine using N,N-dimethylformamide as the reaction solvent; (3) Synthesis of octaparin-DOCA modified compound: using formamide as the reaction solvent, glucosamine sulfate and amino-modified DOCA undergo a dehydration reaction in the presence of a condensing agent to synthesize octaparin-DOCA modified compound. The condensing agent is N,N'-dicyclohexylcarbodiimide or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

3. The application of the deoxycholic acid-modified glucosamine sulfate according to claim 1 in the preparation of oral anticoagulants.

4. The application according to claim 3, characterized in that, Oral anticoagulants are available in tablet, capsule, liquid, or enteric-coated microparticle form.

5. The application according to claim 3, characterized in that, Oral anticoagulants are administered to animals that may have or have already developed thrombosis-related diseases.

6. The application according to claim 5, characterized in that, Thrombosis-related diseases include venous thrombosis or pulmonary embolism.

7. The application according to claim 5, characterized in that, The animals are either human or non-human.

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