Heparin oligosaccharyl-4-methylumbelliferone derivative as well as preparation method and application thereof

Abstract

The invention provides a heparin oligosaccharyl-4-methylumbelliferone derivative and a preparation method and application thereof. The heparin oligosaccharyl-4-methylumbelliferone derivative has a structure shown in a general formula I. The invention also relates to a chemoenzymatic preparation method of the heparin oligosaccharyl-4-methylumbelliferone derivative and application of the heparin oligosaccharyl-4-methylumbelliferone derivative as a heparanase inhibitor in preparation of anti-tumor and anti-inflammatory disease drugs and anti-wrinkle whitening products. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a heparin oligosaccharide-4-methylumbelliferone derivative and a preparation method and application thereof. Background Art

[0002] Heparan sulfate proteoglycan (HSPG) is a glycoconjugate widely distributed on the surface of mammalian cells and in the extracellular matrix. Its structure consists of a core protein and four heparan sulfate (HS) sugar chains, which are mainly distributed in the intima and media of large blood vessels and the basement membrane under the capillary endothelium. Heparanase (HPA) is the only known endogenous endoglucuronidase found in mammals. It can specifically recognize different HS chains on HSPG and degrade them into short sugar chains of 4kDa to 7kDa, affecting and reshaping the physical barriers of the extracellular matrix and basement membrane, thereby releasing a variety of cytokines such as growth factors, cytokines and enzymes that bind to HSPG.

[0003] Heparanase HPA is mainly distributed in the cell membrane or extracellular matrix of platelets, mast cells, placental basement membrane, keratinocytes, and leukocytes in the body. It has two natural substrates, heparin and heparan sulfate. Previous studies have found that HPA is overexpressed in a variety of cancers such as lung cancer, breast cancer, liver cancer, and intestinal cancer, and is closely related to the metastasis, angiogenesis, and lymphangiogenesis of cancer cells, suggesting that HPA is closely linked to the metastasis and invasiveness of cancer and is a promising target for new anti-tumor drugs (Antitumor activity and structure-activity relationship of heparanase inhibitors: Recent advances, Eur J Med Chem, 2020, 193: 112221). At the same time, HPA has also been shown to play a key role in the development of other diseases such as atherosclerosis, sepsis, diabetic nephropathy, and inflammatory glomerulonephritis. Therefore, inhibitors targeting HPA are expected to treat or improve the corresponding pathological processes or diseases. In addition, the use of HPA inhibitors through oral, injection and topical administration can control heparanase in the skin and can also very effectively prevent or improve skin wrinkles, especially fine lines.

[0004] As a special form of HS, the anticoagulant heparin can competitively inhibit the activity of HPA. Its chemically modified non-anticoagulant derivative SST0001 is a potent HPA inhibitor and has been approved for clinical trials for the treatment of advanced multiple myeloma with good tolerance. However, the disadvantage is that SST0001 is a multi-component mixture made from animal-derived multi-anticoagulant heparin, and it is impossible to conduct precise structure-activity relationship and drug metabolism studies, which restricts its drugability. In addition, there are a large number of reports on other oligosaccharides and polysaccharides, natural small molecules, and synthetic small molecule HPA inhibitors, all of which are in the early research stage (Research Progress of Heparan Sulfate Enzyme (HPSE) Inhibitors, Chinese Journal of Medicinal Chemistry, 2022, 32: 231-244). In short, the development of heparin and its derivative molecules with defined structures as HPA inhibitors for the treatment of tumors, inflammatory diseases and the relief of skin wrinkles is one of the hot directions in the current research and development of innovative drugs and cosmetics.

[0005] Patent document CN114957354A discloses a heparin pentasaccharide compound and a preparation method thereof, wherein the heparin pentasaccharide compound has HPA inhibitory activity and can be used to treat or prevent pathological physiological processes or diseases in which HPA participates, or diseases in which HPA overexpression or activity enhancement brings about pathological physiological changes, such as reducing tumor metastasis and invasion, prolonging the survival time of tumor patients, or treating kidney diseases, cardiovascular diseases, inflammatory diseases, pathological angiogenesis, etc. The heparin pentasaccharide compound is synthesized by chemical method, with as many as dozens of synthesis steps and a total yield of no more than 1%, which is low in yield and extremely high in cost; it has obvious anticoagulant activity (~120 IU / mg), and there may be a risk of bleeding when used in high doses. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a heparin oligosaccharide-4-methylumbelliferone derivative and a preparation method and application thereof.

[0007] Terminology Note:

[0008] UDP-GlcNTFA: uridine diphosphate-N-trifluoroacetylglucosamine;

[0009] UDP-GlcA: uridine diphosphate-glucuronic acid;

[0010] PAPS: 3'-phosphoadenosine-5'-phosphosulfate;

[0011] KfiA: Escherichia coli K5 N-acetylglucosaminyltransferase;

[0012] PmHS2: Pasteurella multocida heparosan synthase 2;

[0013] NST: N-sulfatase;

[0014] C 5 -epi:C 5 - Isomerase;

[0015] 2OST: 2-O-sulfotransferase;

[0016] 6OST: 6-O-sulfotransferase;

[0017] 3OST: 3-O-sulfotransferase.

[0018] The technical solution of the present invention is as follows:

[0019] The first object of the present invention is to provide a heparin oligosaccharide-4-methylumbelliferone derivative, which is an inhibitor of HPA and has significant HPA inhibitory activity.

[0020] The heparin oligosaccharide-4-methylumbelliferone derivative, or a pharmaceutically acceptable salt thereof, has a structure shown in the following general formula I:

[0021]

[0022]

[0023] Among them, R 1 , R 2 Sulfonyl (-SO 3 H) or hydrogen (-H); R 3 , R 4 is hydrogen (-H), halogen, nitro, trihalomethyl, cyano, hydroxyl or hydroxymethyl; and n is an integer.

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

[0025] According to the present invention, the heparin oligosaccharide-4-methylumbelliferone derivative is preferably one of the compounds having the following structures I-1 to I-9:

[0027]

[0028]

[0029] The heparin oligosaccharide-4-methylumbelliferone derivative of the present invention has significant HPA inhibitory activity.

[0030] The second object of the present invention is to provide a method for preparing the above-mentioned heparin oligosaccharide-4-methylumbelliferone derivative, which adopts a chemical enzymatic synthesis strategy.

[0031] The method for preparing the above-mentioned heparin oligosaccharide-4-methylumbelliferone derivative, which uses glucuronic acid with a reducing end connected to 4-methylumbelliferone or a derivative as a starting substrate, and is a method of combining the following steps a and b of glycosyltransferase catalysis reactions at least once with three or four of the steps c, d, e, f, and g of chemical enzymatic modification reactions;

[0032] Step a, under the catalysis of N-acetylglucosyltransferase (KfiA) or heparosan synthase 2 (PmHS2), using UDP-GlcNTFA or UDP-GlcNAc as a glycosyl donor, the GlcNTFA residue or GlcNAc residue of the glycosyl donor is transferred to the GlcA at the non-reducing end of the substrate via an α-1,4 glycosidic bond to obtain an intermediate compound;

[0033] Step b, under the catalysis of PmHS2 enzyme, using UDP-GlcA as a glycosyl donor, the GlcA residue of the glycosyl donor is connected to the glucosamine (GlcNTFA or GlcNAc) at the non-reducing end of the substrate through a β-1,4 glycosidic bond to obtain an intermediate compound;

[0034] Step c: The obtained heparin intermediate is placed on ice in a mild alkaline aqueous solution, and all the GlcNTFA residues of the sugar chain are detrifluoroacetylated (TFA) to be converted into GlcNH 2 , and then converted into GlcNS under the catalysis of N-sulfatase transferase (NST) to obtain an N-sulfated intermediate;

[0035] Step d, in C 5 -Isomerase (C 5 Under the joint catalysis of N-sulfated product sugar chain, the specific GlcA residue between two GlcNS or between GlcNS (non-reducing end) and GlcNAc is converted into 2-O-sulfated iduronic acid (IdoA2S) to obtain an intermediate containing IdoA2S residue.

[0036] Step e, under the catalysis of 2OST alone, a specific GlcA residue between two GlcNS or between the non-reducing end of GlcNS and GlcNAc of the substrate is converted into 2-O-sulfated glucuronic acid (GlcA2S), thereby obtaining an intermediate containing GlcA2S;

[0037] Step f, under the co-catalysis of 6-O-sulfotransferases 1 and 3 (6OST1, 6-OST3), the 6-OH of all GlcNS residues in the substrate sugar chain is sulfated to become GlcNS6S, thereby obtaining a 6-O-sulfated compound;

[0038] 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), and the target compound is finally obtained.

[0039] According to the present invention, preferably, the starting substrate is 4-methylumbelliferyl-β-D-glucuronide (GlcA-4MU), or a product formed by connecting glucuronic acid with a 4-methylumbelliferyl derivative via a β1→4 glycosidic bond.

[0040] Preferably according to the present invention, in steps a and b, N-acetylglucosamine transferase (KfiA) and Heparosan synthase 2 (PmHS2) are both recombinantly expressed in Escherichia coli, N-acetylglucosamine transferase (KfiA) is derived from Escherichia coli K5, and Heparosan synthase 2 (PmHS2) is derived from Pasteurella multocida.

[0041] According to the preferred embodiment of the present invention, in steps a and b, the buffer used for the enzyme catalytic reaction is 40-60 mmol / L Tris-HCl, and the Tris-HCl contains 6 mmol / L MnCl 2 , pH = 7.0-7.5, reaction temperature 20°C ~ 37°C, the amount of enzyme and substrate added and the reaction time are not limited; the obtained enzymatic reaction solution is purified by reverse phase C18 or anion exchange column chromatography to obtain an intermediate compound.

[0042] Preferably according to the present invention, in steps a and b, the amount of glycosyl donor added is 1.2 times or more of the equivalent of the substrate.

[0043] Preferably according to the present invention, in step c, the mild alkaline aqueous solution is a 0.1-0.5 mol / L LiOH solution.

[0044] Preferably, in steps c, d, e, f, and g, the heparinase modification enzymes NST, C 5 -epi, 2OST, 6OST1, 6-OST3 and 3OST1 are all obtained by recombinant expression in Escherichia coli, yeast or insect cells; heparin modification enzymes NST, 2OST, 6OST1, 6-OST3 and 3OST1 all use 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as a sulfate donor; the buffer in the catalytic reaction system of each modification enzyme is 40-60mmol / L 2-(N-morpholino)ethanesulfonic acid (MES), pH=7.0-7.5, the reaction temperature is 20℃-37℃, the added amount of the enzyme and the heparin intermediate substrate and the reaction time are not limited, and the obtained reaction solution is purified by anion exchange column chromatography to obtain the product.

[0045] Preferably according to the present invention, in steps c, d, e and f, the amount of sulfate donor added is 1.5-10 times the equivalent of the substrate.

[0046] The preparation method of the heparin oligosaccharide-4-methylumbelliferone derivative of the present invention is established by the inventors through a large number of repeated experiments to study the catalytic activity and substrate specificity of various glycosyltransferases and heparin modifying enzymes to different heparin intermediate molecules.

[0047] Specifically, the preparation method of the heparin oligosaccharide-4-methylumbelliferone derivative shown in formula I-1 or I-2 comprises the following steps:

[0048] 1) Under the catalysis of N-acetylglucosyltransferase (KfiA) or heparosan synthase 2 (PmHS2), UDP-GlcNTFA is used as the glycosyl donor, and the GlcNTFA residue of the glycosyl donor is transferred to the GlcA at the non-reducing end of the starting substrate via an α-1,4 glycosidic bond to obtain a disaccharide backbone intermediate;

[0049] 2) Under the catalysis of PmHS2 enzyme, UDP-GlcA is used as the glycosyl donor, and the GlcA residue of the glycosyl donor is connected to the GlcNTFA at the non-reducing end of the disaccharide backbone intermediate through a β-1,4 glycosidic bond to obtain a trisaccharide backbone intermediate;

[0050] 3) Repeating steps 1) and 2) to extend the sugar chain to obtain a pentasaccharide backbone intermediate;

[0051] 4) The pentasaccharide backbone intermediate is placed on ice in a mild alkaline aqueous solution, and all the GlcNTFA residues of the sugar chain are detrifluoroacetylated (TFA) to convert to GlcNH 2 , and then converted into GlcNS under the catalysis of N-sulfatase transferase (NST) to obtain an N-sulfated pentasaccharide intermediate;

[0052] 5) In C 5 -Isomerase (C 5 Under the joint catalysis of the enzymes 2-O-sulfotransferase (2OST), the specific GlcA residue between the two GlcNS in the sugar chain of the N-sulfated pentasaccharide intermediate is converted into 2-O-sulfated iduronic acid (IdoA2S), resulting in the heparin pentasaccharide containing one IdoA2S residue.

[0053] 6) Under the co-catalysis of 6-O-sulfotransferases 1 and 3 (6OST1, 6-OST3), the 6-OH of all GlcNS residues containing an IdoA2S residue is sulfated to become GlcNS6S, thereby obtaining the target compound I-1;

[0054] 7) Under the catalysis of 3-O-sulfotransferase 1 (3OST1), the 3-OH of GlcNS6S between GlcA and IdoA2S in the sugar chain of the substrate of compound I-1 is sulfated (GlcNS6S3S) to obtain the target compound I-2.

[0055] Specifically, the preparation method of the heparin oligosaccharide-4-methylumbelliferone derivative shown in formula I-3 comprises the following steps:

[0056] Using the N-sulfated pentasaccharide intermediate in step 4) of the preparation method of the heparin oligosaccharide-4-methylumbelliferyl derivative shown in formula I-1 or I-2 as a substrate, under the catalysis of 2OST alone, the GlcA residue between the two GlcNS at the reducing end of the sugar chain of the N-sulfated pentasaccharide intermediate is converted into 2-O-sulfated glucuronic acid (GlcA2S) to obtain a heparin pentasaccharide containing two GlcA2S residues; referring to step 6), all the GlcNS residues are modified to GlcNS6S to obtain the target compound I-3.

[0057] Preferably, according to the present invention, the preparation method is selected from one of the following synthetic routes:

[0058] I-1: The synthetic route is a→b→a→b→c→d→f;

[0059] I-2: The synthetic route is a→b→a→b→c→d→f→g;

[0060] I-3: The synthesis route is a→b→a→b→c→e→f.

[0061] I-4: The synthetic route is a→b→a→b→c→d→a→b→c→d→f;

[0062] I-5: The synthetic route is a→b→a→b→c→d→a→b→c→d→f→g;

[0063] I-6: The synthetic route is a→b→a→b→c→e→a→b→c→e→f;

[0064] I-7: The synthetic route is a→b→a→b→c→d→a→b→c→d→a→b→c→d→f;

[0065] I-8: The synthetic route is a→b→a→b→c→d→a→b→c→d→a→b→c→d→f→g;

[0066] I-9: The synthesis route is a→b→a→b→c→e→a→b→a→b→c→e→f.

[0067] The amount of enzyme and substrate added and the reaction time are carried out according to the prior art.

[0068] The amount of enzyme and substrate added and the reaction time are carried out according to the prior art.

[0069] The third object of the present invention is to provide the use of heparin oligosaccharide-4-methylumbelliferone derivatives in the preparation of drugs for inhibiting HPA activity.

[0070] Preferably according to the present invention, the drug that inhibits HPA activity is a cancer treatment drug, a kidney disease treatment drug, a cardiovascular disease treatment drug, an inflammatory disease treatment drug, a pathological blood vessel treatment drug or a wrinkle improving agent.

[0071] A pharmaceutical composition for treating cancer, kidney disease, cardiovascular disease, inflammatory disease, and pathological blood vessels, comprising the above-mentioned heparin oligosaccharide-4-methylumbelliferone derivative and one or more pharmaceutically acceptable carriers or excipients. The proportion of the heparin oligosaccharide-4-methylumbelliferone derivative in the pharmaceutical composition is not limited.

[0072] A wrinkle improving agent is a personal care product containing the above-mentioned heparin oligosaccharide-4-methylumbelliferone derivative.

[0073] The technical features and advantages of the present invention are as follows:

[0074] 1. The heparin oligosaccharide-4-methylumbelliferone derivatives represented by general formula I of the present invention are potent inhibitors of HPA, among which I-5 to I-9 have significantly better inhibitory activity than the heparin pentasaccharide disclosed in patent document CN114957354A.

[0075] 2. The heparin oligosaccharide-4-methylumbelliferone derivative shown in the general formula I of the present invention is different from animal-derived heparin, fondaparinux and the heparin pentasaccharide disclosed in the patent document CN114957354A. Its structure does not contain an anticoagulant pharmacophore, i.e., an antithrombin binding sequence, and is a non-anticoagulant heparin oligosaccharide product with low anticoagulant activity. There is no risk of bleeding even when used in high doses.

[0076] 3. The synthesis steps of the heparin oligosaccharide-4-methylumbelliferone derivative represented by the general formula I of the present invention are only 7 to 16 steps, and the total yield is 10% or more, with high yield and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 is the mass spectra of the heparin oligosaccharide-4-methylumbelliferone intermediates 6 (A) and 7 (B) prepared in Example 1;

[0078] Figure 2 1 is the mass spectra of the heparin pentasaccharide, 4-methylumbelliferone I-1 (A), I-2 (B) and the heparin heptasaccharide, 4-methylumbelliferone I-4 (C), I-5 (D) prepared in Example 2;

[0079] Figure 3It is the heparin heptasaccharide--4-methylumbelliferone I-5 prepared in Example 2 1 H NMR spectrum;

[0080] Figure 4 This is the relationship curve of the HPA inhibition of the heparin heptasaccharide - 4-methylumbelliferone I-4 (A) and I-5 (B) prepared in Example 2 at different concentrations. DETAILED DESCRIPTION

[0081] The present invention is further described below in conjunction with specific examples, but the protection scope of the present invention is not limited thereto. The target compounds in the following examples are numbered the same as in Table 1. The drugs and reagents involved in the examples are all common commercially available products unless otherwise specified.

[0082] Example 1: Chemoenzymatic Synthesis of Heparin Oligosaccharide-4-Methylumbelliferone Intermediates 6 and 7

[0083] 1) Weigh 4-methylumbelliferyl ketone-β-D-glucuronide (GlcA-4MU) 1 and dissolve it in 50mmol / L Tris-HCl buffer (containing 6mmol / L MnCl) at 2mg / mL. 2 , pH = 7.2), and substrate UDP-GlcNTFA (~1.2 equivalents) and 5 mL KfiA enzyme were added at the same time, and the reaction was stirred at room temperature. The yield was detected by PAMN-HPLC until ≥ 95%; the reaction solution was adjusted to pH 2-3 with trifluoroacetic acid (TFA) to terminate the reaction, and purified with a C18 chromatography column (3.0×50 cm), and the disaccharide backbone intermediate 2 was eluted with methanol-water containing 0.1% TFA and collected. Disaccharide intermediate 2 was placed in 50 mmol / L Tris-HCl buffer (containing 6 mmol / L MnCl) at 2 mg / mL. 2 , pH = 7.2), UDP-GlcA (~1.2 equivalents) and 5 mL PmHS2 enzyme were added at the same time, and the reaction was stirred at room temperature. The PAMN-HPLC detection reaction was carried out until the yield was ≥ 97%. The reaction solution was purified using a C18 chromatography column (3.0×50 cm) to obtain the trisaccharide skeleton intermediate 3. The PAMN-HPLC chromatography conditions were 0→100% KH 2 PO 4 Gradient elution, flow rate of 0.5 mL / min, detection wavelength of 310 nm. Repeat the above KfiA, PmHS2 reaction to obtain pentasaccharide skeleton intermediate 4, four-step total yield of 76.8%, ESI-MS measured its weight of 1218.32 Da, consistent with the theoretical value.

[0084] 2) 200 mg of pentasaccharide skeleton intermediate 4 was dissolved in 50 mL of deionized water, placed on ice, and 0.5 mol / L LiOH solution was added dropwise to pH = 12, and the mixture was placed in an ice bath for 2 h. The reaction progress was detected by PAMN-HPLC. After the reaction was completed, the pH was adjusted to neutral with glacial acetic acid, and 1 mol / L MES mother solution (pH = 7.5) was added to make the final concentration 50 mmol / L. PAPS (~2.5 times equivalent) and 3 mL NST enzyme were added at the same time. The reaction was stirred at room temperature overnight, and the reaction endpoint was detected by PAMN-HPLC. Acetic acid was added to adjust the pH to 4-5 to terminate the reaction. The reaction was purified using a Q Sepharose column (30×1.6 cm) at a flow rate of 3 mL / min, with a flow rate of 0→100% containing 1 mol / L NaCl and 50mmol / LNaAc buffer (pH=5) were used for gradient elution, and the detection wavelengths were 260nm and 310nm. The target components were collected, desalted and dried to obtain N-sulfated heparin pentasaccharide 5; its purity was measured by PAMN-HPLC to be 94%, and its component was measured by ESI-MS, which was consistent with the theoretical value.

[0085] 3) Take heparin pentasaccharide 5 and dissolve it in 50mmol / L MES buffer at pH=7.0-7.5, add 2mmol / L CaCl 2 and a small amount of enzyme C 5 -epi, adjust the reaction volume, and react in a 37°C water bath for 2 h. Then add PAPS (about 1.5 times equivalent), additional C 5 -epi and sufficient 2-OST enzyme were reacted at room temperature overnight, the reaction was detected by PAMN-HPLC and enzyme or PAPS was added as needed until the reaction rate reached 85%; the reaction solution was purified by Q-Sepharose strong anion column (30×1.6 cm) to obtain product 6. The purity of 6 measured by PAMN-HPLC was >96%, and its molecular weight was determined by ESI-MS to be 1266.22 Da, which was consistent with the theoretical value.

[0086] 4) Taking the pentasaccharide intermediate 6, sequentially subjecting it to KfiA and PmHS2 enzyme-catalyzed extension of the sugar chain to a heptasaccharide, the reaction conditions are the same as those in the above step 1), and purification is performed by Q Sepharose column chromatography; then chemical enzymatic N-sulfation modification is performed, and the reaction conditions and purification method are the same as those in the above step 2); finally, C 5 Under the catalysis of -epi and 2-OST enzymes, the newly introduced GlcA residue located between GlcNS is converted into IdoA2S. The reaction conditions and purification method are the same as those in step 3). The purity of heparin heptasaccharide 7 containing two IdoA2S residues was 97% by PAMN-HPLC, and its molecular weight was 1763.59 Da by ESI-MS, which is consistent with the theoretical value; the mass spectra of heparin oligosaccharide-4-methylumbelliferyl intermediates 6 and 7 are shown in Figure 1.

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

[0088]

[0089] Example 2: Enzymatic preparation of heparin oligosaccharide-4-methylumbelliferone

[0090] 1) 100 mg of the IdoA2S-containing heparin pentasaccharide-4-methylumbelliferyl intermediate 6 (or the heparin heptasaccharide 7 containing two IdoA2S residues) synthesized in Example 1 was placed in 50 mmol / L MES buffer (pH = 7.5), and PAPS (2 times the equivalent of GlcN residue) was added at the same time to adjust the pH to 7.5, and then 3 mL 6OST-1 and 3 mL 6OST-3 were added to adjust the reaction volume to 100 mL, and the reaction was allowed to react overnight at room temperature. The reaction progress was detected by PAMN-HPLC, and enzyme or PAPS was added as needed until the reaction yield was > 95%. Acetic acid was added to the reaction solution to adjust the pH to 4-5 to terminate the reaction, and then the target heparin pentasaccharide-4-methylumbelliferone I-1 (or heparin heptasaccharide-4-methylumbelliferone I-4) was obtained by gradient elution. The purities of I-1 and I-4 measured by HPLC were 95% and 99%, respectively. The molecular weights measured by ESI-MS were 1426.36 Da and 1989.43 Da, respectively, which were consistent with the theoretical values.

[0091] 2) The compound I-1 (I-4) was placed in an appropriate amount of 50 mmol / L MES buffer (pH = 7.5), and PAPS (2.5 equivalents) was added at the same time to adjust the pH to 7.5, and then 3 mL 3OST-1 was added to adjust the reaction volume, and the reaction was allowed to react at room temperature overnight. PAMN-HPLC was used to detect the progress of the reaction, and enzyme or PAPS was added as needed until the reaction yield was > 99%. Acetic acid was added to the reaction solution to adjust the pH to 4-5 to terminate the reaction, and then the target heparin pentasaccharide-4-methylumbelliferone I-2 (or heparin heptasaccharide-4-methylumbelliferone I-5) was obtained by gradient elution. The purity of I-2 and I-5 measured by HPLC was 97% and 99%, respectively, and the molecular weights measured by ESI-MS were 1502.55 Da and 2069.2 Da, respectively, which were consistent with the theoretical values; the total yields were 59.4% and 34.1%, respectively.

[0092] The mass spectra of heparin pentasaccharide, 4-methylumbelliferone I-1, I-2 and heparin heptasaccharide, 4-methylumbelliferone I-4, I-5 are shown in Figure 2 , heparin heptasaccharide-4-methylumbelliferone I-5 1 H NMR spectrum Figure 3 , indicating that the present invention successfully synthesized heparin oligosaccharide-4-methylumbelliferone derivatives.

[0093] The synthetic route is shown in the following formula III:

[0094]

[0095] Experimental Example: Determination of HPA Enzyme Inhibitory Activity of Heparin Heptasaccharide-4-Methylumbelliferone

[0096] HPA enzyme activity was assayed using a synthetic HS hexasaccharide containing a single cleavage site (GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6A-GlcA-PNP) as substrate.

[0097] The specific method is as follows: take 15 μL of the above HS hexasaccharide substrate (final concentration 40 μmol / L), add 7 μL NaAc (1 mol / L, pH = 5) and a series of different concentrations of heparin oligosaccharide-4-methylumbelliferone, add double distilled water to 165 μL, and finally add 10 μL of recombinant heparanase (0.0136 IU / μL, 1 IU = the amount of enzyme required to cleave the HS hexasaccharide substrate per minute to release 1 nmol of cleavage product), mix well, react in a 37°C water bath for 30 minutes, and boil for 3 minutes to terminate the reaction. After centrifugation, each reaction solution was filtered with a 0.22 μm filter head, and then detected by PAMN-HPLC, the method is: gradient elution with 0.8→1 mol / L KH2PO4 solution within 15 minutes, the detection wavelength is 310 nm, each sample is measured three times, the reaction rate is calculated, and the inhibition rate of the sample is calculated according to the following formula:

[0098] Inhibition rate = (reaction rate of blank group - reaction rate of experimental group) / reaction rate of blank group × 100%.

[0099] It was calculated that the HPA inhibition rate was 48.98% when the concentration of unfractionated heparin was 1 μg / mL, which is consistent with the literature report (Antitumor activity and structure-activity relationship of heparanase inhibitors: Recent advances, Eur J Med Chem, 2020, 193: 112221). The inhibition rate of each sample on HPA at different concentrations was determined, and the inhibition curve of each compound on HPA activity was drawn. The half-maximal inhibitory molar concentration (IC 50 ) were 5.512μmol / L and 1.922μmol / L, respectively, indicating that they are potent inhibitors of HPA.

Claims

1. A heparin oligosaccharide-4-methylumbelliferone derivative, or a pharmaceutically acceptable salt thereof, having the structure shown in the following general formula I: in, R1 and R2 are sulfonyl (-SO3H) or hydrogen (-H); R3 and R4 are hydrogen (-H), halogen, nitro, trihalomethyl, cyano, hydroxyl or hydroxymethyl; n is an integer.

2. The heparin oligosaccharide-4-methylumbelliferone derivative according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: n=0, 1 or 2; the heparin oligosaccharide-4-methylumbelliferone derivative is one of the compounds having the following structures I-1 to I-9:

3. A method for preparing a heparin oligosaccharide-4-methylumbelliferone derivative according to any one of claims 1 to 2, wherein the method uses glucuronic acid whose reducing end is connected to 4-methylumbelliferone or a derivative as a starting substrate, and is a method in which the glycosyltransferase catalyzed reaction of steps a and b is repeated at least once and three or four of the chemical enzymatic modification reactions of steps c, d, e, f, and g are combined; Step a, under the catalysis of N-acetylglucosyltransferase (KfiA) or heparosan synthase 2 (PmHS2), using UDP-GlcNTFA or UDP-GlcNAc as a glycosyl donor, the GlcNTFA residue or GlcNAc residue of the glycosyl donor is transferred to the GlcA at the non-reducing end of the substrate via an α-1,4 glycosidic bond to obtain an intermediate compound; Step b, under the catalysis of PmHS2 enzyme, using UDP-GlcA as a glycosyl donor, the GlcA residue of the glycosyl donor is connected to the glucosamine (GlcNTFA or GlcNAc) at the non-reducing end of the substrate through a β-1,4 glycosidic bond to obtain an intermediate compound; Step c, the obtained heparin intermediate is placed on ice in a mild alkaline aqueous solution, and all the GlcNTFA residues of the sugar chain are detrifluoroacetylated (TFA) to be converted into GlcNH2, and then converted into GlcNS under the catalysis of N-sulfotransferase (NST) to obtain an N-sulfated intermediate; Step d, under the co-catalysis of C5-isomerase (C5-epi) and 2-O-sulfotransferase (2OST), a specific GlcA residue between two GlcNS or between GlcNS (non-reducing end) and GlcNAc in the sugar chain of the N-sulfated product is converted into 2-O-sulfated iduronic acid (IdoA2S), thereby obtaining an intermediate containing an IdoA2S residue; Step e, under the catalysis of 2OST alone, a specific GlcA residue between two GlcNS or between the non-reducing end of GlcNS and GlcNAc of the substrate is converted into 2-O-sulfated glucuronic acid (GlcA2S), thereby obtaining an intermediate containing GlcA2S; Step f, under the co-catalysis of 6-O-sulfotransferases 1 and 3 (6OST1, 6-OST3), the 6-OH of all GlcNS residues in the substrate sugar chain is sulfated to become GlcNS6S, thereby obtaining a 6-O-sulfated compound; 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), and the target compound is finally obtained.

4. The preparation method according to claim 3, characterized in that: The starting substrate is 4-methylumbelliferyl-β-D-glucuronide (GlcA-4MU), or a product formed by connecting glucuronic acid with a 4-methylumbelliferyl derivative via a β1→4 glycosidic bond.

5. The preparation method according to claim 3, characterized in that: In steps a and b, N-acetylglucosaminyltransferase (KfiA) and Heparosan synthase 2 (PmHS2) are both recombinantly expressed in Escherichia coli, N-acetylglucosaminyltransferase (KfiA) is derived from Escherichia coli K5, and Heparosan synthase 2 (PmHS2) is derived from Pasteurella multocida.

6. The preparation method according to claim 3, characterized in that: In steps a and b, the buffer used for the enzyme catalytic reaction is 40-60mmol / L Tris-HCl, the Tris-HCl contains 6mmol / L MnCl2, the pH is 7.0-7.5, the reaction temperature is 20°C to 37°C, and the amount of enzyme and substrate added and the reaction time are not limited; the obtained enzymatic reaction solution is purified by reverse phase C18 or anion exchange column chromatography to obtain an intermediate compound, and in steps a and b, the amount of glycosyl donor added is 1.2 times the equivalent of the substrate or more.

7. The preparation method according to claim 3, characterized in that: In steps c, d, e, f, and g, the heparinase modification enzymes NST, C5-epi, 2OST, 6OST1, 6-OST3, and 3OST1 are all obtained by recombinant expression in Escherichia coli, yeast, or insect cells; the heparin modification enzymes NST, 2OST, 6OST1, 6-OST3, and 3OST1 all use 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as a sulfate donor; the buffer in each modification enzyme catalytic reaction system is 40-60mmol / L 2-(N-morpholino)ethanesulfonic acid (MES), pH=7.0-7.5, the reaction temperature is 20°C-37°C, the addition amount of the enzyme and the heparin intermediate substrate, and the reaction time are not limited, and the obtained reaction solution is purified by anion exchange column chromatography to obtain the product; In steps c, d, e and f, the amount of sulfate donor added is 1.5-10 times the equivalent of the substrate.

8. The preparation method according to claim 3, characterized in that: The preparation method is selected from one of the following synthetic routes: I-1: The synthetic route is a→b→a→b→c→d→f; I-2: The synthetic route is a→b→a→b→c→d→f→g; I-3: The synthetic route is a→b→a→b→c→e→f; I-4: The synthetic route is a→b→a→b→c→d→a→b→c→d→f; I-5: The synthetic route is a→b→a→b→c→d→a→b→c→d→f→g; I-6: The synthetic route is a→b→a→b→c→e→a→b→c→e→f; I-7: The synthetic route is a→b→a→b→c→d→a→b→c→d→a→b→c→d→f; I-8: The synthetic route is a→b→a→b→c→d→a→b→c→d→a→b→c→d→f→g; I-9: The synthesis route is a→b→a→b→c→e→a→b→a→b→c→e→f.

9. Use of the heparin oligosaccharide-4-methylumbelliferone derivative according to claim 1 or 2 in the preparation of a drug for inhibiting HPA activity, wherein the drug for inhibiting HPA activity is a drug for treating cancer, a drug for treating kidney disease, a drug for treating cardiovascular disease, a drug for treating inflammatory disease, a drug for treating pathological blood vessels, or a wrinkle improving agent.

10. A pharmaceutical composition for treating cancer, kidney disease, cardiovascular disease, inflammatory disease, and pathological blood vessels, comprising the above-mentioned heparin oligosaccharide-4-methylumbelliferone derivative and one or more pharmaceutically acceptable carriers or excipients, and the proportion of the heparin oligosaccharide-4-methylumbelliferone derivative in the pharmaceutical composition is not limited; A wrinkle improving agent is a personal care product containing the above-mentioned heparin oligosaccharide-4-methylumbelliferone derivative.

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