Anticoagulant heparin-derived oligosaccharide biphenyl dimer, and preparation method therefor and use thereof

Abstract

The present invention relates to an anticoagulant heparin-derived oligosaccharide biphenyl dimer, and a preparation method therefor and a use thereof. The anticoagulant heparin-derived oligosaccharide biphenyl dimer has a structure as represented by general formula I, and a benzene ring can be replaced with substituted phenyl, an aromatic heterocyclic ring or a substituted aromatic heterocyclic ring. The heparin-derived oligosaccharide biphenyl dimer of the present invention has few synthesis steps and a significantly high overall yield, has antithrombin-dependent potent and specific anti-Xa factor activity and no obvious anti-IIa activity, anticoagulant activity being efficiently neutralized by a protamine, and has excellent pharmacokinetics characteristics. Therefore, the present invention is suitable for preparing novel anticoagulant and antithrombotic drugs having cost advantages, and high quality and safety.

Description

An anticoagulant heparin oligosaccharide phenyl dimer and its preparation method and application Technical Field

[0001] The present invention relates to an anticoagulant heparin oligosaccharide phenyl dimer and a preparation method and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Heparin, an ancient natural polysaccharide anticoagulant, remains widely used in clinical indications such as thromboembolic disease, myocardial infarction, cardiovascular surgery, cardiac catheterization, extracorporeal circulation, and hemodialysis. The global market has reached $8 billion and continues to grow. Currently commercialized natural heparin drugs primarily include unfractionated heparin (UFH), extracted from porcine small intestinal mucosa, and various low-molecular-weight heparins (LMWH) obtained through chemical or enzymatic partial depolymerization. LMWH drugs have gradually surpassed UFH as the preferred clinical anticoagulant due to their advantages such as long in vivo half-life, high bioavailability, minimal side effects, and safe use. However, their anticoagulant activity is generally lower than that of UFH. UFH and LMWH derived from animal tissues are not only subject to impurity contamination and fragile raw material supply chains, but also, as highly sulfated glycosaminoglycans, their variable sulfation and isomerization modifications are unevenly distributed along the sugar chain, resulting in a highly complex and heterogeneous fine structure, leading to insurmountable clinical limitations.

[0003] Studies have shown that the common structural basis for the anticoagulant effect of UFH and LMWH is the unique pentasaccharide sequence (abbreviated as: GlcNS / Ac6S-GlcA-GlcNS6S3S-IdoA2S-GlcNS6S) in the heparin chain that specifically binds to antithrombin (AT), and its methyl glycoside derivative, fondaparinux sodium (trade name ) was successfully launched in 2001 and is the only fully synthetic single heparin compound approved for marketing to date. However, despite its excellent clinical anticoagulant efficacy, fondaparinux sodium cannot be neutralized by protamine like animal-derived heparin. This prevents the termination of anticoagulant therapy based on the course of treatment and effectively prevents adverse reactions such as bleeding, significantly limiting its clinical application. Therefore, the development of new anticoagulant heparin molecules that can be neutralized by protamine has important clinical value.

[0004] However, despite the long history of protamine as a reversal agent for animal-derived heparin in clinical practice, its research is not systematic and in-depth. It is generally believed that the neutralization efficiency of protamine is determined by the length, charge density, and distribution of heparin. For example, Professor Jian Liu of the United States used a chemoenzymatic method to synthesize a heparin dodecasaccharide containing a single AT-binding pentasaccharide and four consecutive trisulfate disaccharides (IdoA2S-GlcNS6S). Its anticoagulant activity can be effectively neutralized by protamine, and it is widely believed that the anticoagulant heparin dodecasaccharide containing four trisulfate disaccharides is the smallest heparin molecule that can be neutralized by protamine. However, the synthesis of the above-mentioned neutralizing anticoagulant heparin dodecasaccharide requires 22 to 23 steps, is cumbersome, costly, and has a low overall yield. In addition, it has a short half-life in vivo and has potential adverse reactions such as HIT.

[0005] Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides an anticoagulant heparin oligosaccharide phenyl dimer and a preparation method and application thereof.

[0007] Terminology Notes:

[0008] AT: antithrombin

[0009] UDP-GlcNTFA: Uridine diphosphate-N-trifluoroacetylglucosamine

[0010] UDP-GlcNAc: Uridine diphosphate-N-acetylglucosamine

[0011] UDP-GlcA: uridine diphosphate-glucuronic acid

[0012] PAPS: 3'-phosphoadenosine-5'-phosphosulfate.

[0013] KfiA: Escherichia coli K5 N-acetylglucosaminyltransferase

[0014] PmHS2: Pasteurella multocida Heparosan synthase 2

[0015] NST: N-sulfatyltransferase

[0016] C5-epi: C5-isomerase

[0017] 2OST: 2-O-sulfatyltransferase

[0018] 6OST: 6-O-sulfatase

[0019] 3OST: 3-O-sulfotransferase

[0020] The technical solutions of the present invention are as follows:

[0021] The first object of the present invention is to provide an anticoagulant heparin oligosaccharide dimer.

[0022] The anticoagulant heparin oligosaccharide phenyl dimer, or a pharmaceutically acceptable salt thereof, has a structure shown in general formula I:

[0023] Wherein, R1 is a sulfonyl group (-SO3H) or an acetyl group (-COCH3); R2 and R3 are sulfonyl groups or hydrogen (-H); and n is an integer of 0-5.

[0024] According to the present invention, preferably, n=0, 1, or 2.

[0025] According to the preferred embodiment of the present invention, the benzene ring connected to the heparin oligosaccharide can be replaced by substituted phenyl, aromatic heterocycle, substituted aromatic heterocycle, C 1- C5 alkyl or cycloalkyl.

[0026] According to the preferred embodiment of the present invention, the substituents of the substituted phenyl and substituted aromatic heterocycle are halogen, hydroxyl, nitro, trifluoromethyl, C 1- C5 alkyl or cycloalkyl.

[0027] Further preferably, the substituted phenyl group is selected from one of the following:

[0028] More preferably, the substituted aromatic heterocycle is selected from one of the following:

[0029] Further improvement, C 1- The alkyl group of C5 is

[0030] More preferably, the cycloalkyl group is cyclohexane.

[0031] According to the present invention, the anticoagulant heparin oligosaccharide dimer is preferably one of the following compounds:

[0032] In formula I-1 to formula I-5, R1 is a sulfonyl group (-SO3H) or an acetyl group (-COCH3).

[0033] Preferably, according to the present invention, the anticoagulant heparin oligosaccharide phenyl dimer molecule has significant anti-factor Xa activity and no obvious anti-factor IIa activity; its anti-factor Xa activity can be effectively neutralized by protamine, with a neutralization rate of >70%; it has pharmacokinetic characteristics similar to those of short-chain heparin oligosaccharides, a long half-life in vivo, and high bioavailability; and it is not likely to cause HIT adverse reactions.

[0034] The second object of the present invention is to provide a method for preparing anticoagulant heparin oligosaccharide phenyl dimer, which adopts a chemical enzymatic synthesis strategy.

[0035] A method for preparing an anticoagulant heparin oligosaccharide phenyl dimer, the method using a chemically synthesized GlcA-containing dimer intermediate as a starting material, and combining four or five of the following steps: steps a and b of glycosyltransferase-catalyzed reactions repeated at least once, and steps c, d, e, f, and g of chemoenzymatic modification reactions;

[0036] Step a: Under the catalysis of N-acetylglucosaminyltransferase (KfiA) or Heparosan synthase 2 (PmHS2), UDP-GlcNTFA or UDP-GlcNAc is used as the glycosyl donor, and the GlcNTFA or GlcNAc residue is transferred to the two GlcAs at the non-reducing ends of the substrate phenyl di-O-glucoside or the oligosaccharide intermediate dimer with a non-reducing end of GlcA via an α-1,4 glycosidic bond to generate a dimeric intermediate product with an extended sugar chain;

[0037] Step b, under the catalysis of PmHS2 enzyme, UDP-GlcA is used as the glycosyl donor to react, and the GlcA residue is linked to the two glucosamines GlcNTFA or GlcNAc at the non-reducing end of the substrate via a β-1,4 glycosidic bond to generate an intermediate product of sugar chain extension;

[0038] Step c, the heparin oligosaccharide dimer intermediate is dissolved in an alkaline aqueous solution and placed on ice to detrifluoroacetylate all the GlcNTFA residues of the sugar chain to convert them into GlcNH2, and then converted into GlcNS under the catalysis of N-sulfotransferase (NST) to obtain an N-sulfated heparin oligosaccharide dimer intermediate;

[0039] Step d, under the co-catalysis of C5-isomerase (C5-epi) and 2-O-sulfotransferase (2OST), all GlcA residues located between two GlcNS in the dimer sugar chain are converted to 2-O-sulfated iduronic acid (IdoA2S), obtaining an IdoA2S-containing heparin oligosaccharide dimer intermediate;

[0040] In step e, all specific GlcA residues located between two GlcNS in the substrate are converted to 2-O-sulfated gluconic acid (GlcA2S) under the catalysis of 2OST alone;

[0041] Step f, under the joint catalysis of 6-O-sulfotransferase 1 and 6-O-sulfotransferase 3 (6OST1 and 6OST3), the 6-OH groups of all GlcNS or GlcNAc residues in the substrate sugar chain are sulfated to generate an intermediate product containing a 6-O-sulfated heparin oligosaccharide dimer;

[0042] Step g: Under the catalysis of 3-O-sulfotransferase 1 (3OST1), the 3-OH of GlcNS6S between GlcA and IdoA2S in the substrate sugar chain is sulfated (GlcNS6S3S) to obtain the anticoagulant heparin oligosaccharide dimer target compound.

[0043] According to the present invention, the preferred dimer intermediate containing GlcA is phenyl di-O-glucoside or substituted phenyl di-O-glucoside, aromatic heterocyclic di-O-glucoside, substituted aromatic heterocyclic di-O-glucoside, C 1- C5 alkyl bis O-gluconoside or cycloalkyl bis O-gluconoside.

[0044] According to the present invention, the starting material phenyl di-O-glucoside is preferably prepared by the following method:

[0045] Using fully acetylated glucose as the starting material, acetylhydrazine is used to remove the terminal acetyl group to obtain an intermediate. The exposed anomeric hydroxyl group reacts with trichloroacetonitrile (CNCCl3) to obtain glycosyl trichloroacetimidate, which is then glycosylated with p-diphenol in the presence of a promoter to generate an intermediate. Subsequently, alcoholysis removes the acetyl group to obtain phenyl di-O-glucoside. Finally, PhI(OAc)2 and Tempo are used to selectively oxidize C6-OH to obtain the target product.

[0046] The synthetic route is shown in the following formula II:

[0047] According to the preferred embodiment of the present invention, the reaction solvent for removing the terminal acetyl group of fully acetylated glucose 1 by using acetylhydrazine is anhydrous DMF, and the reaction is carried out at room temperature for 1 hour.

[0048] According to a preferred embodiment of the present invention, the exposed anomeric hydroxyl group of intermediate 2 is reacted with trichloroacetonitrile (CNCCl3) at a temperature below 0°C for more than 3 hours, and the reaction solvent is a mixture of DBU and anhydrous DCM. The mixing ratio of the two can be arbitrary.

[0049] According to a preferred embodiment of the present invention, the reaction of glycosyl trichloroacetimidate 3 with p-diphenol is carried out at -5°C for 1 hour using anhydrous DCM containing molecular sieves as a solvent and BF3-Et2O as a promoter. The reaction solution is extracted with EA, vacuum dried, and recrystallized with methanol to obtain intermediate 4. Intermediate 4 is treated with anhydrous MeOH-sodium methoxide at room temperature for more than 1 hour to remove the acetyl group. The solution is then neutralized with an acidic resin until neutral, filtered, vacuum dried, and crystallized to obtain intermediate 5. The dosage of each reagent is determined according to the prior art.

[0050] Preferably, according to the present invention, intermediate 5 is selectively oxidized to C6-OH using PhI(OAc)2 and Tempo, and the solvent used is t-BuOH, DCM and H2O in a volume ratio of 4:4:1. The reaction is carried out at room temperature overnight, and then recrystallized from acetonitrile to obtain the target product phenyl di-O-gluconoside 6.

[0051] The dosage of each reagent is carried out according to the existing technology in this field.

[0052] The preferred method for preparing the anticoagulant heparin oligosaccharide phenyl dimer of the present invention uses phenyl di-O-gluconoside 6 as a raw material and adopts the method of steps a, b and steps c, d, f, and g to prepare the target compound I-1. The synthetic route is shown in Formula III:

[0053] The chemoenzymatic synthesis route of I-1 can be simplified as: a→b→a→b→c→d→a→c or none→f→g.

[0054] According to the preferred embodiment of the present invention, the synthetic routes of formula I-2 to formula I-5 are as follows:

[0055] I-2: a→b→a→b→c→d→a→b→c→d→a→c or none→f→g;

[0056] I-3: a→b→a→b→c→e→a→b→c→d→a→c or none→f→g;

[0057] I-4: a→b→a→b→c→d→a→b→c→d→a→b→c→d→a→c or none→f→g;

[0058] I-5: a→b→a→b→c→d→f→a→b→a→b→c→d→a→c or none→f→g;

[0059] According to the preferred embodiment of the present invention, the enzymes KfiA and PmHS2 are respectively derived from Escherichia coli K5 and Pasteurella multocida, and are recombinantly expressed in Escherichia coli. The buffer used for the enzymatic sugar chain extension reaction is 50 mmol / L Tris-HCl, pH = 7.0 to 7.5, the reaction temperature is 20°C to 37°C, and the enzymatic reaction solution is purified by reverse phase C18 or anion exchange column chromatography to obtain the product.

[0060] The addition amount of enzyme and substrate and reaction time are carried out according to the existing technology.

[0061] According to the present invention, preferably, the heparinase modification enzymes NST, C5-epi, 2OST, 6OST1, 6OST3, and 3OST1 are obtained by recombinant expression in Escherichia coli, yeast, or insect cells, and the buffer for the catalytic reaction of each modification enzyme of NST, 2OST, 6OST1, 6OST3, and 3OST1 is 50 mmol / L 2-(N-morpholino)ethanesulfonic acid (MES), pH = 7.0 to 7.5, the reaction temperature is 20°C to 37°C, and 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is used as a sulfate donor. The enzymatic reaction solution is purified by anion exchange column chromatography to obtain the product.

[0062] The addition amount of enzyme and substrate and reaction time are carried out according to the existing technology.

[0063] The third object of the present invention is to provide an application of a heparin oligosaccharide dimer containing an AT binding sequence, which has specific anti-factor Xa activity, no obvious anti-IIa activity, and its anticoagulant activity can be neutralized by protamine, with better pharmacokinetics, for the preparation of a potent and safe anticoagulant and antithrombotic drug.

[0064] An anticoagulant and antithrombotic pharmaceutical composition comprises the above-mentioned heparin oligosaccharide phenyl dimer molecule containing an AT binding sequence and one or more pharmaceutically acceptable carriers or excipients.

[0065] The ratio of heparin oligosaccharide phenyl dimer to carrier or excipient is arbitrary.

[0066] The present invention is the first to use two molecules of glucuronic acid covalently linked to the same benzene ring as starting materials and successfully synthesize an anticoagulant heparin oligosaccharide dimer containing an AT binding sequence using a chemical enzymatic method. The dimer not only has potent anticoagulant activity, but most importantly, exhibits protamine neutralization properties similar to long-chain heparin molecules and ideal pharmacokinetic characteristics similar to short-chain molecules. In addition, it has a low risk of inducing adverse reactions such as HIT and requires only a few preparation steps, with the minimum being only nine. It can be developed into a new generation of safer and higher-quality innovative anticoagulant drugs with independent intellectual property rights, and has important application value.

[0067] The technical features and advantages of the present invention are as follows:

[0068] 1. This invention demonstrates for the first time that new dimer molecules of different heparin oligosaccharides (hexasaccharide, octasaccharide, and decasaccharide) containing AT binding sequences can be successfully prepared using phenyl di-O-gluconoside containing two GlcA groups as the starting material through 9 to 18 steps of chemoenzymatic reactions.

[0069] 2. The chromogenic substrate assay showed that the novel heparin oligosaccharide phenyl dimer containing an AT binding sequence of the present invention has significant anti-factor Xa activity but no significant anti-factor IIa activity. This is the first confirmation that the two AT binding sequences in the heparin oligosaccharide phenyl dimer can activate AT without interfering with each other. The half-maximal inhibitory molar concentration (IC50) for inhibiting factor Xa is 2.37kJ / mL. 50 ) was significantly lower than the positive control fondaparinux sodium and the long-chain heparin dodecasaccharide molecule reported by Jian Liu.

[0070] 3. The specific anti-factor Xa activity of the novel heparin oligosaccharide phenyl-linked dimer containing an AT binding sequence of the present invention can be efficiently neutralized by protamine, with a neutralization rate of >70%, which is comparable to that of long-chain unfractionated heparin UFH and reported heparin dodecasaccharides. The anti-factor Xa activity of the commercially available fondaparinux sodium is almost completely unneutralized by protamine, and the existing reported anticoagulant heparin hexasaccharides, octasaccharides, and decasaccharides are all incapable of neutralization by protamine. Therefore, the heparin oligosaccharide phenyl-linked dimer of the present invention has properties similar to those of long-chain heparin molecules of heparin dodecasaccharides and above, and can be neutralized by generating a high affinity interaction with protamine.

[0071] 4. The heparin oligosaccharide phenyl dimer of the present invention has ideal pharmacokinetic characteristics. For example, the in vivo half-life of compound I-1-1 is comparable to that of commercially available fondaparinux sodium, and is significantly superior to animal-derived heparin and the currently reported heparin dodecasaccharide molecules.

[0072] 5. The sugar chains and modified groups outside the AT binding sequence in the heparin oligosaccharide phenyl dimer structure of the present invention have little effect on the anti-Xa activity, but have a significant effect on the neutralization efficiency and pharmacokinetic characteristics of protamine.

[0073] 6. The heparin oligosaccharide phenyl dimer of the present invention can be used to prepare a cost-effective, higher-quality, safer, and potent anticoagulant and antithrombotic drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 is a diagram of phenyl bis-O-glucoside 6 prepared in Example 1 1 H NMR spectrum;

[0075] Figure 2 is a high performance liquid chromatography (A) and mass spectrum (B) of the heparin hexasaccharide phenyl dimer I-1-1 prepared in Example 5;

[0076] Figure 3 is a diagram of the heparin hexasaccharide phenyl dimer I-1-1 prepared in Example 5 1 H NMR (A) and HSQC (B) spectra;

[0077] FIG4 shows the in vitro anti-Xa factor activity of the heparin hexasaccharide phenyl dimer I-1-1 prepared in Example 5;

[0078] FIG5 shows the neutralization effect of protamine on the anticoagulant activity of the heparin hexasaccharide phenyl dimer I-1-1 of Example 5 in vitro.

[0079] FIG6 shows the in vivo pharmacokinetic characteristics of the heparin hexasaccharide phenyl dimer I-1-1 prepared in Example 5. DETAILED DESCRIPTION

[0080] The present invention is further described below with reference to specific examples, but the scope of protection of the present invention is not limited thereto. The target compounds in the following examples are numbered the same as in Table 1. Unless otherwise specified, the drugs and reagents involved in the examples are common commercial products.

[0081] Example 1: Chemical Synthesis of Phenyl Bis-O-Gluconoside 6

[0082] Compound 1 (1 g, 2.6 mmol) and AcOH / NH2-NH2 (1.4 equiv. 3.6 mmol, 330 mg) were dissolved in 5 ml of anhydrous DMF and reacted at room temperature for 1 h. After completion of the reaction, the reaction solution was diluted with EA, and the organic layer was washed with H2O and saturated brine, respectively. The residue was dried under vacuum and purified by silica gel column chromatography (PE:EA = 2:1) to obtain 752 mg of 2 as a colorless oil in an 84.3% yield.

[0083] Compound 2 (752 mg, 2.16 mmol) was dissolved in 5 ml of anhydrous DCM. CNCCl (3 equiv. 6.5 mmol, 0.65 ml) and DBU (0.2 equiv. 0.43 mmol) were added sequentially at low temperature and allowed to react for 3 h. After completion of the reaction, the reaction solution was directly vacuum-dried to obtain a residue, which was purified by silica gel column chromatography (PE:EA = 4:1) to afford 893 mg of 3 as a colorless oil in an 83.8% yield.

[0084] Compound 3 (893 mg, 1.8 mmol, 2.2 equiv.) and p-diphenol (0.82 mmol, 83 mg) were dissolved in 10 ml of anhydrous DCM containing an appropriate amount of molecular sieves. BF₃-Et₂O (4 equiv. 3.28 mmol, 0.4 ml) was added at -5°C, and the reaction was continued at this temperature for 1 h. After completion of the reaction, the reaction solution was diluted with EA, and the organic layer was washed sequentially with saturated sodium bicarbonate solution, water, and saturated brine. The washed organic layer was dried under vacuum, and the resulting residue was recrystallized from methanol to obtain 462 mg of compound 4 as a white solid in a 73% yield. 1H NMR(400MHz,Chloroform-d)δ6.91(d,J=4.6Hz,4H),5.30–5.11(m,6H),4.98(dd,J=7.4,3.6Hz,2H),4.26(dd,J=1 2.4, 5.2Hz, 2H), 4.15 (dt, J = 12.2, 2.8Hz, 2H), 3.80 (ddt, J = 10.0, 5.2, 2.4Hz, 2H), 2.04 (dt, J = 13.2, 4.8Hz, 24H).

[0085] Compound 4 (462 mg, 0.6 mmol) and sodium methoxide (0.1 equiv. 3 mg) were dissolved in 10 ml of anhydrous MeOH and reacted at room temperature for 1 h. After completion of the reaction, an acidic resin was added and stirred for 10 min. The reaction solution was neutralized to neutrality and filtered. After vacuum drying of most of the solvent, the remaining solution was refrigerated to precipitate, yielding 247 mg of compound 5 as a white solid (95% yield).

[0086] Compound 5 (247 mg, 0.57 mmol), PhI(OAc)2 (8 equiv. 4.6 mmol, 1.5 g), and Tempo (4 equiv. 2.3 mmol, 355 mg) were dissolved in 18 mL of a mixed solvent (t-BuOH:DCM:H2O = 4:4:1) and allowed to react overnight at room temperature. After completion of the reaction, the reaction solution was diluted with EA, the organic layer was washed with water, and the aqueous layer was spin-dried. The residue was vacuum-dried and recrystallized from acetonitrile to afford 162 mg of compound 6 as a white solid, in a 62% yield.

[0087] The synthetic route is shown in the following formula II:

[0088] Compound 6 1 The H NMR spectrum is shown in Figure 1.

[0089] Important 1 H NMR (400 MHz, D2O) data were δ 7.13 (dd, J = 2.8, 1.8 Hz, 4H), 5.16–5.06 (m, 2H), 4.10–4.00 (m, 2H), 3.72–3.56 (m, 6H).

[0090] Example 2: Enzymatic Synthesis of Heparin Pentasaccharide Backbone Dimer 8

[0091] 50 mg of phenyl di-O-glucoside 6 was weighed and dissolved in ~50 mL of 50 mmol / L Tris-HCl buffer (containing 6 mmol / L MnCl2, pH = 7.2). 2.2 equivalents of UDP-GlcNTFA and 2.5 mL of KfiA enzyme were added and stirred at room temperature overnight. The reaction was detected by PAMN-HPLC under the chromatographic conditions of 0→100% KH2PO4 gradient elution over 45 min at a flow rate of 0.5 mL / min and a detection wavelength of 280 nm. When the reaction yield was ≥99%, the reaction was terminated by adjusting the pH to 2-3 with trifluoroacetic acid (TFA) and C 18 Purification was performed on a 3×50 cm column, eluting with methanol-water containing 0.1% TFA to obtain the target fraction. The resulting product was placed in 50 mL of the same buffer as above, and 2.2 equivalents of UDP-GlcA and 3 mL of PmHS2 enzyme were added. The product was stirred at room temperature overnight. PAMN-HPLC analysis was performed to yield ≥99%, and intermediate 7 was purified using a C18 column. The KfiA and PmHS2 enzyme reactions described above were repeated to obtain heparin pentasaccharide backbone dimer 8, whose molecular weight, as determined by ESI, was consistent with theoretical value.

[0092] The synthetic route is shown in the following formula IV:

[0093] Example 4: Chemoenzymatic Synthesis of Heparin Pentasaccharide Dimer 10 Containing IdoA2S

[0094] Pentasaccharide backbone dimer 8 was dissolved in 100 mL of deionized water and placed on ice. 0.5 mol / L LiOH solution was added dropwise to a pH of 12, and the mixture was placed in an ice bath for 2 h. The reaction progress was monitored by PAMN-HPLC. After the reaction, the pH was adjusted to neutral with glacial acetic acid, and an appropriate amount of 1 mol / L MES solution (pH = 7.4) was added to a final concentration of 50 mmol / L. 4.4 equivalents of PAPS and 3 mL of NST enzyme were also added. The mixture was stirred at room temperature overnight and monitored by PAMN-HPLC. When the reaction yield was >95%, the pH was adjusted to 4-5 with acetic acid to terminate the reaction. The mixture was purified using a Q Sepharose column (1 × 20 cm) at a flow rate of 3 mL / min using a 0→100% gradient elution buffer containing 1 mol / L NaCl and 50 mmol / L NaAc (pH = 5). Detection wavelengths were 260 nm and 280 nm. The target fractions were collected, desalted, and dried to obtain the N-sulfated product 9. ESI-MS measured its molecular weight as 2130.22 Da.

[0095] The above product 9 was added to 50 mmol / L MES buffer at pH 7.0-7.4, 2 mmol / L CaCl2 and an appropriate amount of enzyme C5-epi were added, and the reaction volume was adjusted to 100 mL. The reaction was allowed to react in a 37°C water bath for 2 h. Approximately 2.5 equivalents of PAPS, additional C5-epi, and sufficient 2OST enzyme were then added, and the reaction was allowed to proceed overnight at room temperature. The reaction was monitored by PAMN-HPLC, and additional enzyme or PAPS was added as needed until the reaction was complete. The reaction solution was purified using a Q-Sepharose strong anion column (1×20 cm) to obtain heparin pentasaccharide dimer 10. Its molecular weight was determined by ESI-MS to be 2290.20 Da.

[0096] The synthetic route is shown in the following formula V:

[0097] Example 5: Chemoenzymatic Preparation of Heparin Hexasaccharide Dimer I-1-1

[0098] Pentasaccharide dimer 10 was used as a substrate, and the sugar chain was elongated using KfiA enzyme as described in Example 1. The reaction solution was purified using a Q-Sepharose strong anion column (1 cm × 20 cm). The resulting product was subjected to chemoenzymatic N-sulfation modification as described in Example 4 to obtain heparin hexasaccharide phenyl dimer 11. ESI-MS analysis determined its molecular weight to be 2772.41 Da, consistent with the theoretical value.

[0099] Hexasaccharide phenyl dimer 11 was placed in 50 mmol / L MES buffer at pH 7.0-7.5. Seven equivalents of PAPS, 4 mL of 6OST1, and 4 mL of 6OST3 enzyme were added to adjust the reaction volume to 140 mL and incubated at 37°C in a water bath overnight. Reaction progress was monitored by SAX-HPLC, and enzyme or PAPS was added as needed. Chromatographic conditions were: a flow rate of 1 mL / min, a gradient elution from 0 to 100% of eluent B (50 mmol / L NaAc + 2 mol / L NaCl, pH 5), and detection wavelengths of 260 nm and 280 nm. When the yield was >99%, the reaction was terminated with dilute acetic acid, adjusted to pH 4-5, and the enzyme was removed by freeze-thawing at -20°C without purification. The solution was adjusted to pH 7.0-7.5, and approximately 2.5 equivalents of PAPS and 5 mL of 3OST1 enzyme were added to adjust the reaction volume to 200 mL. The reaction was incubated at 37°C in a water bath overnight. PAMN-HPLC assay was performed until the substrate modification rate exceeded 99%. The reaction solution was adjusted to pH 4-5 with dilute acetic acid and then purified using a Q-Sepharose strong anion column (1 cm × 10 cm) to obtain the target heparin hexasaccharide phenyl dimer I-1-1 with a purity exceeding 92% (Figure 2A). ESI-MS analysis determined its molecular weight to be 3411.99 Da (Figure 2B), consistent with the theoretical value. 1 Its structure was characterized by H NMR and HSQC (Figure 3).

[0100] The synthetic route is shown in the following formula VI:

[0101] Test Example 1: Determination of the in vitro anticoagulant activity of heparin hexasaccharide phenyl dimer I-1-1

[0102] The IC value of the anti-FⅩa activity of the heparin hexasaccharide phenyl dimer I-1-1 prepared in Example 5 was measured by the chromogenic substrate method. 50 The value was 7.2nmol / L (28.15ng / mL). Under the same conditions, the IC values ​​of unfractionated heparin (UFH) and fondaparinux sodium were measured. 50 The values ​​were 378.3 ng / mL and 10.8 nmol / L (18.63 ng / mL), respectively, which are the IC values ​​of I-1-1 anti-FⅩa activity in terms of molar concentration. 50 The value is significantly lower than that of fondaparinux, indicating that it is a potent Xa inhibitor, as shown in Figure 4. At the same time, the target compound I-1-1 is similar to fondaparinux and has no significant anti-factor IIa activity (data omitted) as determined by the chromogenic substrate method. Therefore, the heparin hexasaccharide phenyl dimer I-1-1 prepared by the present invention is a specific inhibitor of factor Xa, and because the two AT binding sequences it contains can activate AT without interfering with each other, it shows superiority over fondaparinux containing a single AT binding sequence and the heparin dodecasaccharide reported by Jian Liu, with a half inhibition molar concentration (IC 50 ) was significantly lower than the positive control fondaparinux sodium and the long-chain heparin dodecasaccharide molecule reported by Jian Liu.

[0103] Experimental Example 2: Neutralization of the Anticoagulant Activity of Heparin Hexasaccharide Dimer I-1-1 by Protamine

[0104] The effect of adding different concentrations of protamine on the anti-FXa activity of the heparin hexaglycophenyl dimer I-1-1 from Example 5 was determined using a chromogenic substrate method. The results are shown in Figure 5 . As shown in Figure 5 , similar to UFH, the in vitro anti-FXa activity of the heparin hexaglycophenyl dimer I-1-1 was almost completely reversed by protamine. Therefore, the heparin hexaglycophenyl dimer I-1-1 prepared by the present invention is a novel heparin analog whose anticoagulant activity can be neutralized by protamine.

[0105] Experimental Example 3: In vivo pharmacokinetic characteristics of heparin hexaglycophenyl dimer I-1-1

[0106] A chromogenic substrate method was used to establish a standard curve using a low molecular weight heparin standard. The titers of fondaparinux and heparin hexaglycophenyl dimer I-1-1 were determined to be 1290.9 IU / mg and 1121.5 IU / mg, respectively. Both fondaparinux and heparin hexaglycophenyl dimer I-1-1 were subcutaneously injected at a dose of 300 IU / kg into Wister male rats weighing 200±20g (n≥3). Blood samples were collected from the jugular vein at 0, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 8, 12, and 24 hours (according to the actual time of blood collection). After centrifuging the blood sample at 5000 rpm for 15 minutes, the supernatant was collected and the anti-factor Xa activity was determined using the above method. The residual concentrations of fondaparinux and heparin hexaglycophenyl dimer I-1-1 in the rat body were calculated. The drug concentration-time curves were plotted, as shown in Figure 6. It can be seen that the curves of fondaparinux and heparin hexaglycophenyl dimer I-1-1 of the present invention are very similar. The data were further processed using DAS 2.0 to obtain the t of heparin hexaglycophenyl dimer I-1-1. 1 / 2 Up to 2.269h, slightly longer than the t of fondaparinux sodium 1 / 2 The half-life of the heparin hexaglycophenyl dimer I-1-1 was 2.166 h.

Claims

1. An anticoagulant heparin oligosaccharide phenyl dimer, or a pharmaceutically acceptable salt thereof, having the structure shown in general formula I: in, R1 is a sulfonyl group (-SO3H) or an acetyl group (-COCH3); R2 and R3 are a sulfonyl group or hydrogen (-H); and n is an integer of 0-5.

2. The anticoagulant heparin oligosaccharide phenyl dimer according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: n=0, 1, or 2. The benzene ring connected to the heparin oligosaccharide can be replaced by a substituted phenyl group, an aromatic heterocycle, a substituted aromatic heterocycle, a C1-C5 alkyl group, or a cycloalkyl group. The substituent of the substituted phenyl group or the substituted aromatic heterocycle is a halogen, a hydroxyl group, a nitro group, a trifluoromethyl group, a C1-C5 alkyl group, or a cycloalkyl group.

3. The anticoagulant heparin oligosaccharide phenyl dimer according to claim 2, or a pharmaceutically acceptable salt thereof, characterized in that: The substituted phenyl group is selected from the following: The substituted aromatic heterocycle is selected from one of the following: The C1-C5 alkyl group is Cycloalkyl is cyclohexane.

4. The anticoagulant heparin oligosaccharide phenyl dimer according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: The anticoagulant heparin oligosaccharide phenyl dimer is specifically one of the following compounds: In formula I-1 to formula I-5, R1 is a sulfonyl group (-SO3H) or an acetyl group (-COCH3).

5. The method for preparing the anticoagulant heparin oligosaccharide phenyl dimer according to claim 1, which method uses a chemically synthesized GlcA-containing dimer intermediate as a starting material, and comprises a combination of the following steps a and b: glycosyltransferase-catalyzed reactions repeated at least once, and four or five of the following steps c, d, e, f, and g: chemoenzymatic modification reactions; Step a: Under the catalysis of N-acetylglucosaminyltransferase (KfiA) or Heparosan synthase 2 (PmHS2), UDP-GlcNTFA or UDP-GlcNAc is used as the glycosyl donor, and the GlcNTFA or GlcNAc residue is transferred to the two GlcAs at the non-reducing ends of the substrate phenyl di-O-glucoside or the oligosaccharide intermediate dimer with a non-reducing end of GlcA via an α-1,4 glycosidic bond to generate a dimeric intermediate product with an extended sugar chain; Step b, under the catalysis of PmHS2 enzyme, UDP-GlcA is used as the glycosyl donor to react, and the GlcA residue is linked to the two glucosamines GlcNTFA or GlcNAc at the non-reducing end of the substrate via a β-1,4 glycosidic bond to generate an intermediate product of sugar chain extension; Step c, the heparin oligosaccharide dimer intermediate is dissolved in an alkaline aqueous solution and placed on ice to detrifluoroacetylate all the GlcNTFA residues of the sugar chain to convert them into GlcNH2, and then converted into GlcNS under the catalysis of N-sulfotransferase (NST) to obtain an N-sulfated heparin oligosaccharide dimer intermediate; Step d, under the joint catalysis of C5-isomerase (C5-epi) and 2-O-sulfotransferase (2OST), all GlcA residues located between two GlcNS in the dimer sugar chain are converted to 2-O-sulfated iduronic acid (IdoA2S), obtaining an IdoA2S-containing heparin oligosaccharide dimer intermediate; In step e, all specific GlcA residues located between two GlcNS in the substrate are converted to 2-O-sulfated gluconic acid (GlcA2S) under the catalysis of 2OST alone; Step f, under the joint catalysis of 6-O-sulfotransferase 1 and 6-O-sulfotransferase 3 (6OST1 and 6OST3), the 6-OH groups of all GlcNS or GlcNAc residues in the substrate sugar chain are sulfated to generate an intermediate product containing a 6-O-sulfated heparin oligosaccharide dimer; Step g: Under the catalysis of 3-O-sulfotransferase 1 (3OST1), the 3-OH of GlcNS6S between GlcA and IdoA2S in the substrate sugar chain is sulfated (GlcNS6S3S) to obtain the anticoagulant heparin oligosaccharide dimer target compound.

6. The preparation method according to claim 5, characterized in that The dimer intermediate containing GlcA is phenyl di-O-glucoside or substituted phenyl di-O-glucoside, aromatic heterocyclic di-O-glucoside, substituted aromatic heterocyclic di-O-glucoside, C 1- C5 alkyl bis-O-gluconoside or cycloalkyl bis-O-gluconoside; The starting material, phenyl di-O-gluconoside, was prepared as follows: Using fully acetylated glucose as the starting material, acetylhydrazine is used to remove the terminal acetyl group to obtain an intermediate. The naked anomeric hydroxyl group reacts with trichloroacetonitrile (CNCCl3) to obtain glycosyl trichloroacetimidate, which is then glycosylated with p-diphenol in the presence of a promoter to generate an intermediate. Subsequently, alcoholysis removes the acetyl group to obtain phenyl di-O-glucoside. Finally, PhI(OAc)2 and Tempo are used to selectively oxidize C6-OH to obtain the target product.

7. The preparation method according to claim 6, characterized in that The fully acetylated glucose 1 was treated with acetylhydrazine to remove the terminal acetyl group in anhydrous DMF at room temperature for 1 h. The exposed anomeric hydroxyl group of the intermediate is reacted with trichloroacetonitrile (CNCCl3) at less than 0°C for more than 3 hours, and the reaction solvent is a mixture of DBU and anhydrous DCM; The reaction of glycosyl trichloroacetimidate 3 with p-diphenol was carried out at -5°C for 1 h using anhydrous DCM containing molecular sieves as solvent and BF3-Et2O as a promoter. The reaction solution was extracted with EA, vacuum dried, and recrystallized with methanol to obtain intermediate 4. Intermediate 4 was treated with anhydrous MeOH-sodium methoxide at room temperature for more than 1 h to remove the acetyl group. The solution was then neutralized with an acidic resin until neutral, filtered, vacuum dried, and crystallized to obtain intermediate 5. Intermediate 5 was selectively oxidized to C6-OH using PhI(OAc)2 and Tempo. The solvent used was t-BuOH, DCM and H2O in a volume ratio of 4:4:

1. The reaction was carried out at room temperature overnight and then recrystallized from acetonitrile to obtain the target product phenyl di-O-gluconoside 6.

8. [Corrected 08.11.2023 according to Rule 91] The preparation method according to claim 7, characterized in that Using phenyl di-O-glucoside 6 as raw material, the target compound I-1 was prepared by the method of steps a, b and steps c, d, f, and g. The synthetic route is shown in Formula III:

9. [Corrected 08.11.2023 according to Rule 91] The preparation method according to claim 5, characterized in that The synthetic routes of formula I-2 to formula I-5 are as follows: I-2: a→b→a→b→c→d→a→b→c→d→a→c or none→f→g; I-3: a→b→a→b→c→e→a→b→c→d→a→c or none→f→g; I-4: a→b→a→b→c→d→a→b→c→d→a→b→c→d→a→c or none→f→g; I-5: a→b→a→b→c→d→f→a→b→a→b→c→d→a→c or none→f→g; 10. [Corrected 08.11.2023 according to Rule 91] The preparation method according to claim 5, characterized in that The enzymes KfiA and PmHS2 are derived from Escherichia coli K5 and Pasteurella multocida, respectively, and are recombinantly expressed in E. coli. The enzymatic sugar chain extension reaction uses a buffer of 50 mmol / L Tris-HCl, pH = 7.0-7.5, and a reaction temperature of 20°C-37°C. The enzymatic reaction solution is purified by reverse phase C18 or anion exchange column chromatography to obtain the product. The heparinase modification enzymes NST, C5-epi, 2OST, 6OST1, 6OST3, and 3OST1 are obtained by recombinant expression in Escherichia coli, yeast, or insect cells. The buffer for the catalytic reaction of each modification enzyme of NST, 2OST, 6OST1, 6OST3, and 3OST1 is 50 mmol / L 2-(N-morpholino)ethanesulfonic acid (MES), pH=7.0-7.5, and the reaction temperature is 20°C-37°C. 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is used as a sulfate donor. The enzymatic reaction solution is purified by anion exchange column chromatography to obtain a product.

11. [Corrected 08.11.2023 according to Rule 91] The use of a heparin oligosaccharide phenyl dimer containing an AT binding sequence as described in claim 1, which has specific anti-factor Xa activity, no obvious anti-factor IIa activity, and whose anticoagulant activity can be neutralized by protamine, has better pharmacokinetics, and is used to prepare a potent anticoagulant and antithrombotic drug.

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