OLIGOSACCHARIDE COMPOUND FOR INHIBITING INTRINSIC COAGULATION FACTOR X-ENZYME COMPLEX, AND PREPARATION METHOD THEREFOR AND USES THEREOF

Abstract

A purified oligosaccharide compound having antithrombotic activity or a mixture of a homologous compound thereof and a pharmaceutically acceptable salt thereof, a preparation method for the mixture, a pharmaceutical composition containing the mixture, and uses thereof serving as an intrinsic factor X-enzyme (Xase) inhibitor in the preparation of drugs for preventing and/or treating thrombotic diseases.

Claims

1. An oligosaccharide compound or a pharmaceutically acceptable salt thereof, characterized in that, the oligosaccharide compound has antithrombotic activity, and has a general structure represented by Formula (I): ##STR00024## in the formula, R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are optionally and independently H or SO.sub.3H; R.sub.6 is optionally H, a substituted or unsubstituted C1-C6 hydrocarbon group or a C7-C12 aryl group; R.sub.7 is optionally H, SO.sub.3H, C2-C5 acyl; R.sub.8 is optionally a group represented by Formula (II), Formula (III) or Formula (IV): ##STR00025## in Formula (II), Formula (III) and Formula (IV), R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are defined as above; R.sub.9 and R.sub.10 are optionally H, a substituted or unsubstituted C1-C6 hydrocarbon group or a C7-C12 aryl group; R.sub.11 is optionally NHR.sub.12, OR.sub.13, wherein R.sub.12 and R.sub.13 are optionally H, a substituted or unsubstituted C1-C6 hydrocarbon group or a C7-C12 aryl group; and n is optionally 0 or a natural number of 18.

2. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, when R.sub.8 is the group represented by Formula (II), the oligosaccharide compound has a general structure represented by Formula (V): ##STR00026## wherein, R.sub.1H, R.sub.2R.sub.3R.sub.4R.sub.5SO.sub.3; or R.sub.1R.sub.3R.sub.4R.sub.5SO.sub.3; R.sub.2H.

3. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, when R.sub.8 is a group represented by Formula (III), the oligosaccharide compound has a general structure represented by Formula (VI): ##STR00027## wherein R.sub.1H, R.sub.2R.sub.3R.sub.4R.sub.5SO.sub.3; or R.sub.1R.sub.3R.sub.4R.sub.5SO.sub.3, R.sub.2H.

4. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, when R.sub.8 is the group represented by Formula (IV), the oligosaccharide compound has a general structure represented by Formula (VII): ##STR00028## wherein: R.sub.1H, R.sub.2R.sub.3R.sub.4R.sub.5SO.sub.3; or R.sub.1R.sub.3R.sub.4R.sub.5SO.sub.3, R.sub.2H.

5. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, in the general structure represented by Formula (I), n is optionally 1, 2, 3 or 4.

6. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, the pharmaceutically acceptable salt is optionally an alkali metal salt, an alkaline earth metal salt or an organic ammonium salt.

7. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that, the pharmaceutically acceptable salt is optionally a sodium salt, a potassium salt or a calcium salt.

8. An oligosaccharide mixture or a pharmaceutically acceptable salt thereof composed of the oligosaccharide compound of claim 1 and having antithrombotic activity, characterized in that, the oligosaccharide mixture is composed of homologues of the oligosaccharide compound of claim 1; and in the oligosaccharide compound of Formula (I) composing the oligosaccharide mixture, R.sub.8 is a group represented by Formula (II), Formula (III), or Formula (IV), and in the molar ratio, the proportion of the oligosaccharide compound of Formula (I) in which R.sub.8 is a group simultaneously represented by Formula (II), simultaneously represented by Formula (III) or simultaneously represented by Formula (IV) is not less than 95% in the mixture.

9. A preparation method of the oligosaccharide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: preparing a carboxylate of natural fucosylated glycosaminoglycan, followed by subjecting the fucosylated glycosaminoglycan to -elimination reaction and terminal reduction reaction, optionally in the presence of a strong base and a reducing agent, to obtain a mixture of the homologous oligosaccharide compounds; or subjecting the fucosylated glycosaminoglycan to -elimination reaction and terminal peeling reaction in the presence of a strong base to obtain a mixture of the homologous oligosaccharide compounds; then isolating and purifying, and optionally performing a substituent modification to obtain the desired purified oligosaccharide compound.

10. The preparation method according to claim 9, characterized in that: the oligosaccharide compound has the general structure represented by Formula (I) as defined in claim 1, and R.sub.8 is the group represented by Formula (II) as defined in claim 1; the method comprises: (a) converting fucosylated glycosaminoglycan into a quaternary ammonium salt form, followed by completely or partially converting the carboxyl group on hexuronic acid residue in the obtained fucosylated glycosaminoglycan quaternary ammonium salt into a carboxylate in an organic solvent; (b) in an anhydrous organic solvent in presence of a reducing agent, treating the fucosylated glycosaminoglycan carboxylate with a strong base to cause -elimination depolymerization and terminal reduction reaction, to obtain a mixture of homologous oligosaccharide compounds having D-acetylaminogalactitol at the reducing terminal; (c) converting the oligosaccharide mixture obtained in the step (b) into an alkali metal salt, followed by subjecting its carboxylate to basic hydrolysis in an aqueous solution, to obtain a mixture of the homologous oligosaccharide compounds containing a free carboxyl group; (d) isolating the oligosaccharide mixture obtained in the step (c) by chromatography, to obtain a purified oligosaccharide compound; (e) optionally, subjecting the purified oligosaccharide compound obtained in the step (d) to a substituent structural modification.

11. The preparation method according to claim 10, characterized in that, in the preparation steps: in the step (a), the fucosylated glycosaminoglycan quaternary ammonium salt is benzethonium salt (N,N-dimethyl-N-[2-[2-[4(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy]ethyl]benzylammonium salt); the organic solvent is DMF or a DMF-ethanol mixture; the carboxylate is a benzyl ester, and the complete or partial conversion into carboxylate means that the degree of carboxyl esterification of fucosylated glycosaminoglycan is in the range of from about 30% to about 100%; in the step (b), the organic solvent is DMF or a DMF-ethanol mixture; the reducing agent is sodium borohydride; the strong base is sodium ethoxide; in the step (c), the conversion of the oligosaccharide mixture to an alkali metal salt means that a saturated aqueous solution of sodium chloride is added to the reaction solution to convert the oligosaccharide mixture into a sodium salt form; the basic hydrolysis means that the carboxylate group of the homologous oligosaccharide compounds is hydrolyzed in NaOH aqueous solution with a concentration of 0.25 M1 M; in the step (d), the chromatography includes, but is not limited to, gel chromatography and/or ion exchange chromatography; in the step (e), the further substituent structural modification includes, but is not limited to, carboxyl esterification of D-glucuronic acid group and unsaturated hexenuronic acid group in the oligosaccharide compound; deacetylation and optional reacylation or resulfation of D-acetylgalactosamine; hydroxyalkylation at the C1 position of the reducing terminal -D-GalNAc-ol.

12. The preparation method according to claim 11, characterized in that, the oligosaccharide compound has a general structure represented by Formula (I) as defined in claim 1, and R.sub.8 is a group represented by Formula (III) or Formula (IV) as defined in claim 1, the method comprises the specific steps of: (a) converting fucosylated glycosaminoglycan into a quaternary ammonium salt form, followed by completely or partially converting the carboxyl group on hexuronic acid residue in the obtained fucosylated glycosaminoglycan quaternary ammonium salt into a carboxylate in an organic solvent; (b) in an anhydrous organic solvent, treating the fucosylated glycosaminoglycan carboxylate with a strong base to cause -elimination depolymerization, followed by subjecting the depolymerized product to peeling reaction by adding a small amount of aqueous solution of a strong base to lose the -D-GalNAc residue at the reducing terminal, thereby obtaining a mixture of homologous oligosaccharide compounds having glycosyl group -D-GlcA at the reducing terminal; (c) converting the oligosaccharide mixture obtained in the step (b) into an alkali metal salt, and subjecting the carboxylate of the homologous oligosaccharide compounds to basic hydrolysis in an aqueous solution, to obtain a mixture of the homologous oligosaccharide compounds containing a free carboxyl group; (d) isolating and purifying the oligosaccharide compounds in the oligosaccharide mixture obtained in the step (c) by chromatography; (e) optionally, subjecting the purified oligosaccharide compound obtained in the step (d) to a further substituent structural modification.

13. The preparation method according to claim 12, characterized in that, in the preparation steps: in the step (a), the quaternary ammonium salt is benzethonium salt; the organic solvent is DMF or a DMF-ethanol mixture; the carboxylate is benzyl ester, and the complete or partial conversion into carboxylate means that the degree of carboxyl esterification of fucosylated glycosaminoglycan is in the range of from about 30% to about 100%; in the step (b), the organic solvent is DMF or a DMF-ethanol mixture; the strong base is sodium ethoxide; the small amount of aqueous solution of a strong base means a 1 M2 M aqueous solution of NaOH that is equivalent to about to 1/10 of the total volume of the reaction solution; in the step (c), converting the oligosaccharide mixture into an alkali metal salt means that a saturated aqueous solution of sodium chloride is added to the reaction solution to convert the oligosaccharide mixture into a sodium salt form; the basic hydrolysis means that the carboxylate of the homologous oligosaccharide compounds is hydrolyzed in NaOH aqueous solution with a concentration of 0.05 M1 M; in the step (d), the chromatography includes, but is not limited to, gel chromatography and/or ion exchange chromatography; in the step (e), the further substituent structural modification includes, but is not limited to, carboxyl esterification of D-GlcA and UA in the oligosaccharide compound; deacetylation and optional reacylation or resulfation of D-GalNAc; alkylation, reduction, reductive amination or reductive alkylation of the hemiacetal at the C1 position of the reducing terminal -D-GlcA.

14. A pharmaceutical composition having antithrombotic activity, characterized in that, it comprises an anti-thrombotic effective amount of an active ingredient and a pharmaceutically acceptable excipient, the active ingredient is the oligosaccharide compound or the pharmaceutically acceptable salt thereof according to claim 1, or the oligosaccharide mixture or the pharmaceutically acceptable salt thereof according to claim 8.

15. The pharmaceutical composition according to claim 14, characterized in that, the preparation form of the pharmaceutical composition is an aqueous solution for injection or a lyophilized powder for injection, and its unit dosage form contains 20 mg100 mg of the active ingredient.

16. Use of the oligosaccharide compound or a pharmaceutically acceptable salt thereof according to claim 1, or the oligosaccharide mixture or a pharmaceutically acceptable salt thereof according to claim 8 in the preparation of a medicament for the treatment and/or prevention of thrombotic diseases, the thrombotic diseases are venous thrombosis, arterial thrombosis and/or ischemic cardiovascular and cerebrovascular diseases.

17. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to any one of claim 2, characterized in that, in the general structure represented by Formula (V), n is optionally 1, 2, 3 or 4.

18. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to any one of claim 3, characterized in that, in the general structure represented by Formula (VI), n is optionally 1, 2, 3 or 4.

19. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to any one of claim 4, characterized in that, in the general structure represented by Formula (VII), n is optionally 1, 2, 3 or 4.

Description

DESCRIPTION OF THE DRAWINGS

[0218] FIG. 1. HPGPC profiles of Compounds A1A4

[0219] FIG. 2. .sup.1H NMR spectrum and assignments for Compound A1

[0220] FIG. 3. .sup.13C NMR spectrum and assignments for Compound A2

[0221] FIG. 4. .sup.13C-.sup.1H HSQC spectrum and assignments for Compound A3

[0222] FIG. 5. Q-TOF MS spectrum and assignments for Compound A1

[0223] FIG. 6. .sup.1H NMR spectrum and assignments for Compound B1

[0224] FIG. 7. .sup.13C NMR spectrum and assignment for Compound B2

[0225] FIG. 8. .sup.13C-.sup.1H HSQC spectrum and assignments for Compound B3

[0226] FIG. 9. Q-TOF MS spectrum and assignments for Compound B2

[0227] FIG. 10. HPGPC profiles of Compound B6-B8

[0228] FIG. 11. .sup.13C NMR spectrum and assignments for Mixture C1

[0229] FIG. 12. HPLC profile of Mixture D1

[0230] FIG. 13. .sup.13C NMR spectrum and assignments for Mixture D1

[0231] FIG. 14. Effect of A2 and D1 on thrombosis of the inferior vena cava in

[0232] FIG. 15. Effect of A2 and D1 on the amount of bleeding loss in mice

DETAILED DESCRIPTION OF THE INVENTION

[0233] The following examples are intended to describe the contents of the present invention in detail, but do not limit the scope of the present invention.

Example 1

[0234] Preparation of Compounds A1, A2, A3, A4 and A5:

[0235] L-2,4-disulfated fucosyl-(1.fwdarw.3)-L-4-deoxy-threo-hex-4-enepyranosyluronic acid-(1.fwdarw.3)-{-D-N-acetyl-2-deoxy-2-amino-4,6-disulfated galactosyl-(1.fwdarw.4)-[L-2,4-disulfated fucosyl-(1.fwdarw.3)]-D-glucuronyl-(1.fwdarw.3)}.sub.(n+1)-D-N-acetyl-2-deoxy-2-amino-4,6-disulfated galactitol (n=0, 1, 2, 3 and 4; hexasaccharide, nonasaccharide, dodecasaccharide, pentadecasaccharide and octadecasaccharide)

[0236] 1.1 Materials

[0237] SvFG, Natural FG (sodium salt) from Stichopus variegatus, which was prepared according to the literature method (Zhao L Y et al., PNAS, 2015, 112: 8284-8289), with a purity of 98% (HPGPC, area normalization method) and a weight average molecular weight (Mw) of about 70 kDa.

[0238] The reagents used such as benzethonium chloride, benzyl chloride, DMF, sodium hydroxide, sodium chloride, and ethanol were all commercially available analytical reagents. Sephadex G10, medium (50-100 m), GE Healthcare; Bio-Gel P-6/P-2 gel, fine (45-90 m), Bio-Rad; Bio-Gel P-10 gel, medium (90-180 m), Bio-Rad; HPLC Chromatograph, Agilent 1200/1260 Series Chromatograph.

[0239] 1.2 Methods

[0240] (1) Quaternary ammonium salt conversion of SvFG: 2.0 g of SvFG was dissolved in 30 mL of deionized water; and 5.0 g of benzethonium chloride was dissolved in another 80 mL of deionized water. The SvFG solution was titrated with the benzethonium chloride solution with stirring to give a white precipitate. The obtained precipitate was washed three times with 55 mL of deionized water and dried under vacuum to give 5.34 g of SvFG quaternary ammonium salt.

[0241] (2) Carboxyl esterification of SvFG: The SvFG quaternary ammonium salt obtained in the step (1) was placed in a round bottom flask, dissolved in 26 mL of DMF, then added with 0.769 mL of benzyl chloride, reacted at 35 C. for 24 h with stirring; and allowed to stand and let the solution cool to room temperature (25 C.). The product sample was taken for .sup.1H NMR detection and the degree of carboxyl esterification of the FG was calculated to be about 41%.

[0242] (3) -elimination depolymerization in the presence of a reducing agent: a freshly prepared 8.9 mL of 0.08 M sodium ethoxide-ethanol solution (containing 0.4 M NaBH.sub.4) was added to the reaction solution of the step (2), and stirred for 30 min.

[0243] (4) Sodium salt conversion and carboxyl ester hydrolysis of the depolymerized product: 35 mL of a saturated NaCl solution and 284 mL of absolute ethanol were added to the reaction solution of the step (3), centrifuged at 4000 rpm10 min to obtain a precipitate. The obtained precipitate was dissolved in 90 mL of water, added with 1.5 mL of 6 M NaOH solution, and stirred at room temperature for 30 min, and then dropwise added with 6 M HCl to neutralize the reaction solution (pH 7.0). The reaction solution was filtered through a 0.45 m filter, and the obtained filtrate was desalted by a G10 gel column chromatography and lyophilized to obtain a total of 1.059 g of depolymerized product dSvFG (depolymerized SvFG) (yield 53%).

[0244] (5) Isolation and purification of Compounds A1A5: 1 g of dSvFG was dissolved in 10 mL of 0.2 M NaCl, loaded on a Bio-Gel P-10 gel column (2 cm, 1 200 cm), eluted with 0.2 M NaCl solution at a flow rate 10 mL/h, and the eluate fractions of 2.5 mL/tube were collected. The eluate fractions were monitored and the elution profiles were plotted by the cysteine-sulfuric acid method, and the eluate fractions having the same compositions were combined. Purity was determined by HPGPC method (TSK gel G2000SW XL, 7.8 mml 300 mm column). Unpurified samples were further purified on a Bio-Gel P-10 gel column. Purified oligosaccharides were desalted on a Sephadex G-10 or Bio-Gel P-2 gel column and then lyophilized.

[0245] (6) Spectral analysis: .sup.1H-/.sup.13C- and 2D-NMR were detected using Bruker DRX 800 MHz NMR spectrometer with a spectral width of 16025.6 Hz, an acquisition time of 2.0447 s, a pulse width of 9.5 s, a relaxation time of 1 s, and a scan of 32 times. The sample had a concentration of (10-15) g/L, and was repeatedly lyophilized three times with heavy water before the test; ESI-Q-TOF MS was analyzed by micrOTOF-QII ESI-MS (Bruker, Germany) mass spectrometer. The mass spectrometry conditions were: capillary voltage 2500 V, nebulizer voltage 0.6 bar, dry gas flow rate 4.0 L/min, dry gas temperature+180 C., m/z scan range 503000. Data were analyzed using Bruker Compass Data-Analysis 4.0 (Bruker-Daltonics, Germany) software.

[0246] 1.3 Results

[0247] (1) Compound A1 35 mg, A2 45 mg, A3 55 mg, A4 35 mg, and A5 20 mg were obtained by the method described, and the purity was determined to be about 99% by HPGPC method. The HPGPC patterns of oligosaccharide compounds A1A5 are shown in FIG. 1.

[0248] (2) Structural analysis of Compounds A1A5: The .sup.1H NMR spectrum and assignments for oligosaccharide compound A1 are shown in FIG. 2; the .sup.13C NMR spectrum and assignments for Compound A2 are shown in FIG. 3; the .sup.13C-.sup.1H HSQC spectrum and assignments for Compound A3 are shown in FIG. 4; the Q-TOF MS spectrum and assignments for Compound A1 are shown in FIG. 5; the .sup.1H/.sup.13C NMR signal assignments for Compounds A1A2 are shown in Tables 1 and 2, respectively.

[0249] According to .sup.1H-/.sup.13C-, 2D-NMR and Q-TOF MS analysis, the chemical structure of Compounds A1A5 is L-Fuc.sub.2S4S-(1,3)-UA-(1,3)-{D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)]-D-GlcA-(1,3)}.sub.n+1-D-GalNAc.sub.4S6S-ol, wherein n=1, 2, 3, 4 and 5, that is, Compounds A1A5 are hexasaccharide, nonasaccharide, dodecasaccharide, pentadecasaccharide and octadecasaccharide, respectively, having the chemical structural formula of:

##STR00014##

[0250] In A1, n=1, in A2, n=2; in A3, n=3; in A4, n=4; in A5, n=5.

TABLE-US-00002 TABLE 1 .sup.1H/.sup.13C NMR signal assignments and coupling constant of Compound A1 (ppm, Hz) rA U F A dU dF H-1 3.636 4.536 5.615 4.550 4.838 5.432 J.sub.1, 2 = 6.72 J.sub.1, 2 = 8.22 J.sub.1, 2 = 3.72 J.sub.1, 2 = 8.28 J.sub.1, 2 = 7.98 J.sub.1, 2 = 3.60 H-2 4.199 3.666 4.426 4.079 3.820 4.355 J.sub.2, 3 = 7.80 J.sub.2, 3 = 8.61 J.sub.2, 3 = 9.72 J.sub.2, 3 = 9.60 J.sub.2, 3 = 8.34 J.sub.2, 3 = 10.44 H-3 4.220 3.721 4.095 4.088 4.438 4.055 J.sub.3, 4 = 8.94 J.sub.3, 4 = 4.02 J.sub.3, 4 = 2.46 J.sub.3, 4 = 2.64 H-4 4.328 3.879 4.795 4.912 5.688 4.623 J.sub.4, 5 = 9.18 / H-5 4.343 3.713 4.892 3.989 4.282 J.sub.5, 6 = 6.36 J.sub.5, 6,6 = 7.08, 4.62 J.sub.5, 6 = 6.78 H-6 4.090 1.320 4.245/4.145 1.231 J.sub.6, 6 = 11.10 AcCH.sub.3 1.947 1.985 C-1 62.64 106.03 99.70 102.73 106.31 99.19 C-2 54.57 77.29 77.96 54.34 73.28 77.90 C-3 78.08 80.35 69.47 79.10 79.33 69.37 C-4 80.80 78.34 84.08 79.22 109.30 83.63 C-5 70.48 79.22 69.23 74.80 149.97 69.25 C-6 72.63 177.63 18.78 70.42 171.83 18.59 (Ac) CO 177.19 177.97 (Ac) CH.sub.3 25.02 25.46 Note: rA represents D-GalNAc-ol at the reducing terminal; dU and dF represent UA and L-Fuc linked to UA.

TABLE-US-00003 TABLE 2 .sup.1H/.sup.13C NMR signals assignments for Compound A2 (ppm, Hz) rA rU rF A U F dA dU dF H-1 3.626 4.520 5.615 4.512 4.395 5.628 4.530 4.832 5.435 H-2 4.189 3.661 4.412 4.010 3.579 4.412 4.047 3.830 4.348 H-3 4.211 3.737 4.093 3.956 3.688 4.093 4.073 4.446 4.060 H-4 4.403 3.958 4.803 4.711 3.880 4.803 4.908 5.686 4.623 H-5 4.327 3.706 4.863 3.919 3.600 4.863 3.971 4.288 H-6 4.079 1.299 4.206/4.123 1.311 4.230/4.122 1.231 AcCH.sub.3 1.947 1.980 1.988 C-1 62.62 105.92 99.58 102.71 106.92 99.38 102.68 106.02 99.10 C-2 54.53 77.23 77.89 54.19 76.76 77.90 54.20 73.22 77.84 C-3 78.06 80.15 69.41 78.14 79.93 69.41 78.88 79.23 69.32 C-4 80.76 78.33 83.96 79.18 78.33 83.96 79.09 109.26 83.54 C-5 70.43 79.10 69.24 74.69 79.69 69.20 74.65 149.95 69.20 C-6 72.59 177.62 18.86 70.27 177.88 18.86 70.21 171.78 18.59 (Ac) CO 177.15 177.88 177.94 (Ac) CH.sub.3 24.99 25.52 25.45 Note: in the table, rA, rU and rF represent GalNAc-ol at the reducing terminal, D-GlcA and L-Fuc glycosyl near the reducing terminal, respectively; dU, dA and dF represent AUA, D-GalNAc linked to AUA, and L-Fuc linked to AUA, respectively.

[Example 2] Preparation of Compounds B1, B2, B3, B4 and B5

[0251] L-3,4-disulfated fucosyl-(1,3)-L-4-deoxy-threo-hex-4-enepyranuronyl-(1,3)-{D-N-acetyl-2-deoxy-2-amino-4, 6-disulfated galactosyl-(1,4)-[L-3, 4-disulfated fucosyl-(1,3)-]D-glucuronyl-(1,3)}.sub.n-D-N-acetyl-2-deoxy-2-amino-4,6-disulfated galactose-[L-3,4-disulfated fucosyl-(1,3)-]-L-gulonic acid (n=0, 1, 2, 3 and 4, pentasaccharide, octasaccharide, hendecasaccharide, tetradecasaccharide, and heptadecasaccharide).

[0252] 2.1 Materials

[0253] HsFG Natural FG (sodium salt) from Holothuria fuscopunctata; which was prepared according to the literature method (Zhao L Y et al., PNAS, 2015, 112: 8284-8289), with a purity of 98% (HPGPC method), and a weight average molecular weight (Mw) of about 50 kDa.

[0254] The used reagents such as benzethonium chloride, benzyl chloride, DMF, sodium hydroxide, sodium chloride, and ethanol were all commercially available analytical reagents.

[0255] Sephadex G10/G25, medium (50-100 m), GE Healthcare; Bio-Gel P-2 gel, fine (45-90 m), Bio-Rad; Bio-Gel P-10 gel, medium (90-180 m), Bio-Rad; 1200/1260 Series HPLC Chromatograph, Agilent.

[0256] 2.2 Methods

[0257] (1) Quaternary ammonium salt conversion of HsFG: 3.5 g of HsFG was treated according to the method described in 1.2 (1) of Example 1, obtaining 10.3 g of HsFG quaternary ammonium salt.

[0258] (2) Carboxyl esterification of HsFG:HsFG quaternary ammonium salt was treated according to the method described in 1.2 (2) of Example 1 to obtain HsFG carboxylate, and the sample was taken for .sup.1H NMR detection, and the degree of carboxyl esterification of the obtained product was calculated to be about 44%;

[0259] (3) -elimination depolymerization and terminal peeling reaction of HsFG: To the reaction solution obtained in the step (2), a freshly prepared 16.7 mL of 0.08 M sodium ethoxide-ethanol solution was added, and stirred at room temperature for 30 min, and then 2.5 mL of 2 M NaOH solution was added, and stirred at 60 C. for 30 min.

[0260] (4) Sodium salt conversion, carboxylic ester hydrolysis and terminal reduction of depolymerized product: To the reaction solution in the step (3) was added sequentially 67 mL of saturated NaCl solution, 536 mL of absolute ethanol, centrifuged at 4000 rpm for 10 min; the obtained precipitate was dissolved in 120 mL of water, added with 1.0 mL of 6 M NaOH solution, and stirred at room temperature for 30 min; NaBH.sub.4 was added to a final concentration of about 0.1 M, and stirred at room temperature for 30 min, then 6 M HCl was added dropwise to neutralize the reaction solution (pH 7.0). The reaction solution was filtered through a 0.45 m filter, and the filtrate was ultrafiltered through a 30 kDa ultrafiltration membrane; the ultrafiltrate was concentrated and desalted by G25 gel column chromatography and lyophilized to obtain 1.53 g of depolymerized product dHsFG (yield 43.7%).

[0261] (5) Isolation and purification of Compounds B1B5: 1 g of dHsFG in the step (4) was dissolved in 10 mL of 0.2 M NaCl, loaded on a Bio-Gel P-10 gel column (2 cm, 1 200 cm), eluted with 0.2 M NaCl solution at a flow rate 15 mL/h, and the eluate fractions of 2.5 mL/tube were collected. UV spectrophotometry (max 234 nm) was used for monitoring. HPGPC (TSK G2000 SW column) was used to detect the sample purity and composition of the eluate fractions. The unpurified fractions were continued to be purified on a Bio-Gel P-10 gel column until the HPGPC spectrum of the product exhibited a single elution peak. The purified fractions were desalted on a Sephadex G-10 or Bio-Gel P-2 column and then lyophilized.

[0262] (6) Spectral analysis: the same as the method described in 1.2 (6) of Example 1, .sup.1H-/.sup.13C- and 2D-NMR was detected using Bruker DRX 800 MHz NMR spectrometer, ESI-Q-TOF MS was analyzed using microTOF-QII ESI-MS (Bruker, Germany) mass spectrometer. The detected data were analyzed using Bruker Compass Data-Analysis 4.0 (Bruker-Daltonics, Germany) software.

[0263] 2.3 Results

[0264] (1) Compound B1 54 mg, B2 177 mg, B3 154 mg, B4 86 mg, B5 57 mg were obtained by the method described above, and the purity was determined to be >99% by HPGPC method.

[0265] (2) Structure analysis of Compounds B1B5: The .sup.1H NMR spectrum of oligosaccharide Compound B1 is shown in FIG. 6; the .sup.13C NMR spectrum and assignments for Compound B2 are shown in FIG. 7; the .sup.13C-.sup.1H HSQC spectrum and assignments of Compound B3 are shown in FIG. 8; the Q-TOF MS spectrum and assignments for Compound B2 are shown in FIG. 9; the .sup.1H/.sup.13C NMR signal assignments for Compounds B1B2 are shown in Tables 3 and 4, respectively.

[0266] Combined with .sup.1H-/.sup.13C-/2D-NMR and Q-TOF MS analysis, the chemical structure of Compounds B1 B5 is L-Fuc.sub.3S4S-(1,3)-L-UA-(1,3)-{D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.3S4S-(1,3)]-D-GlcA-(1,3)}n-D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.3S4S-(1,3)]-D-GlcA-ol (wherein n=0, 1, 2, 3 and 4). That is, Compounds B1B5 are pentasaccharide, octasaccharide, hendecasaccharide, tetradecasaccharide, and heptadecasaccharide, respectively, having the chemical structural formula of:

##STR00015##

[0267] In B1, n=0; in B2, n=1; in B3, n=2; in B4, n=3; in B5, n=4.

TABLE-US-00004 TABLE 3 .sup.1H/.sup.13C NMR signal assignments and coupling constants for Compound B1 (ppm, Hz) rU rF rA dU dF H-1 3.743/3.700 5.039 4.639 4.859 5.201 J.sub.1, 1' = 11.82 J.sub.1, 2 = 3.96 J.sub.1, 2 = 8.52 J.sub.1, 2 = 8.64 J.sub.1, 2 = 3.78 H-2 4.060 3.886 4.043 3.822 3.886 J.sub.1/1', 2 = 3.96/6.78 J.sub.2, 3 = 10.38 J.sub.2, 3 = 8.70 J.sub.2, 3 =7.56 J.sub.2, 3 = 10.38 H-3 4.019 4.546 4.167 4.418 4.529 J.sub.3, 4 = 2.76 J.sub.3, 4 = 2.16 J.sub.3, 4 = 2.40 J.sub.3, 4 = 2.82 H-4 4.071 4.831 4.918 5.693 4.831 J.sub.4, 5 = 7.44 H-5 4.256 4.363 4.019 4.273 J.sub.5, 6 = 6.84 J.sub.5, 6/6' = 8.24, 3.52 J.sub.5, 6 = 6.48 H-6 1.240 4.268/4.146 1.231 J.sub.6, 6' = 10.68 (Ac)-CH.sub.3 1.986 C-1 65.28 104.26 104.31 105.82 101.04/175.8 C-2 72.59 69.39 54.35 72.90 69.27 C-3 82.44 77.93 78.56 79.12 77.96 C-4 75.17 81.70 78.94 109.52 81.72 C-5 75.17 69.55 74.64 149.51 69.02 C-6 180.08 18.85 70.64 171.65 18.56 (Ac)-C = O 177.65 (Ac)-CH.sub.3 25.19 Note: in the table, rU and dU represent D-GlcA-ol at the reducing terminal and UA at the non-reducing terminal, respectively; rF and dF represent L-Fuc glycosyl groups linked to the reducing terminal D-GlcA-ol and UA, respectively. A represents D-GalNAc.

TABLE-US-00005 TABLE 4 .sup.1H-/.sup.13C-NMR signal assignments for Compound B2 (ppm, Hz) rU rF rA U F A dU dF H-1 3.733/3.691 5.033 4.622 4.426 5.286 4.530 4.846 5.204 H-2 4.066 3.886 3.976 3.546 3.871 4.090 3.830 3.886 H-3 4.012 4.545 3.981 3.632 4.458 4.087 4.426 4.530 H-4 4.012 4.837 4.755 3.954 4.973 4.912 5.686 4.831 H-5 4.248 4.370 4.022 3.641 4.799 4.019 4.275 H-6 1.241 4.211/4.129 1.343 4.321/4.222 1.231 AcCH.sub.3 1.992 1.997 C-1 65.21 104.17 104.17 106.4 101.97 102.39 105.8 100.9 C-2 72.59 69.24 54.26 76.24 69.16 54.26 72.96 69.10 C-3 82.33 77.91 78.07 81.88 78.07 78.55 79.10 77.96 C-4 75.03 81.68 78.87 77.91 82.09 78.98 109.4 81.69 C-5 74.62 69.52 75.03 79.86 69.00 74.62 149.6 69.36 C-6 180.08 18.84 70.52 177.88 18.78 69.99 171.6 18.56 (Ac) CO 177.74 177.74 (Ac) CH.sub.3 25.28 25.26 Note: in the table, rA represent GalNAc near the reducing terminal, and rU and dU represent D-GlcA-ol at the reducing terminal and AUA at the non-reducing terminal, respectively; rF and dF represent L-Fuc glycosyl linked to D-GlcA-ol at the reducing terminal and AUA, respectively.

[Example 3] Preparation of Compounds B6, B7, B8

[0268] L-3,4-disulfated fucosyl-(1,3)-L-4-deoxy-threo-hex-4-enopyanosyluronyl-(1,3)-{D-N-acetyl-2-deoxy-2-amino-4,6-disulfated galactosyl-(1,4)-[L-3,4-disulfated fucosyl-(1,3)-]D-glucuronyl-(1,3)}.sub.n-D-N-acetyl-2-deoxy-2-amino-4,6-disulfated galactosyl-(1,4)-[L-3,4-disulfated fucosyl-(1,3)-]-D-glucuronic acid (n=0, 1 and 2)

[0269] 3.1 Materials:

[0270] HsFG, FG sodium salt derived from Holothuria fuscopunctata, derived from the same as described in 2.1 of Example 2.

[0271] The reagents used such as benzethonium chloride, benzyl chloride, DMF, sodium hydroxide, sodium chloride and ethanol were all commercially available analytical reagents.

[0272] 3.2 Methods:

[0273] (1) Quaternary ammonium salt conversion of HsFG: 9.55 g of HsFG quaternary ammonium salt was prepared from 3.5 g of HsFG by the method as described in 2.2(1) of Example 2.

[0274] (2) Carboxyl esterification of HsFG: carboxyl esterified HsFG was obtained by the method described in 2.2 (2) of Example 2, and the degree of carboxyl esterification was determined to be about 44% by .sup.1H NMR;

[0275] (3) -elimination depolymerization of HsFG: To the reaction solution obtained in the step (2), a freshly prepared 16.0 mL of 0.08 M sodium ethoxide-ethanol solution was added, and stirred at room temperature for 30 min.

[0276] (4) Sodium salt conversion and carboxylic ester hydrolysis of the depolymerized product: 67 mL of saturated sodium chloride solution and 536 mL of absolute ethanol were added to the reaction solution obtained in the step (3), centrifuged at 4000 rpm10 min; the obtained precipitate was dissolved in water (125 mL), 1.05 mL of 6 M NaOH solution was added, stirred at room temperature for 30 min, and then neutralized by dropwise addition of 6 M HCl (pH 7.0). The reaction solution was filtered through a 0.45 m filter, and the filtrate was ultrafiltered through a 30 kDa ultrafiltration membrane package. The ultrafiltrate was desalted by G25 gel column chromatography and lyophilized to obtain 1.623 g of depolymerized product dHsFG (yield 46.4%).

[0277] (5) Isolation and purification of Compounds B6B8: 1 g of depolymerized product dHsFG was dissolved in 10 mL of 0.2 M NaCl, loaded on a Bio-Gel P-10 gel column (2 cm, l 200 cm), and eluted with 0.2 M NaCl solution at a flow rate of 15 mL/h. The eluate fractions of 2.5 mL/tube were collected. Ultraviolet spectrophotometry (max 234 nm) was used for monitoring and the same eluate fractions were combined. HPGPC (TSK G2000 SW column) was used to detect the purity and composition of chromatographic samples. The unpurified samples were further purified by Bio-Gel P-10 column chromatography. The purified oligosaccharides were desalted on a Sephadex G-10 or Bio-Gel P-2 gel column and then lyophilized.

[0278] (6) Spectral analysis: By the same method described in 1.2 (6) of Example 1, .sup.1H-/.sup.13C- and 2D-NMR were detected using Bruker DRX 800 MHz NMR spectrometer, Q-TOF MS was analyzed using microTOF-QII ESI-MS (Bruker, Germany) mass spectrometer. The detected data were analyzed using Bruker Compass Data-Analysis 4.0 (Bruker-Daltonics, Germany) software.

[0279] 3.3 Results

[0280] (1) 47 mg of Compound B6, 55 mg of B7, 35 mg of B8 were obtained according to the treatment procedure described in 3.2. The purity was detected to be >99% by HPGPC method (area normalization method).

[0281] (2) Structural analysis of Compounds B6B8: The HPGPC profiles of Compounds B6 B8 are shown in FIG. 10. Combined with .sup.1H-/.sup.13C-/2D-NMR and Q-TOF MS analysis, Compounds B6, B7 and B8 are L-Fuc.sub.3S4S-(1,3)-L-UA-(1,3)-{D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.3S4S-(1,3)]-D-GlcA-(1,4)}.sub.n-D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.3S4S-(1,3)]-D-GlcA (wherein n=0, 1 and 2, namely pentasaccharide, octasaccharide and hendecasaccharide), having the chemical structural formula of:

##STR00016##

[0282] In B6, n=0; in B7, n=1; in B8, n=2.

[Example 4] Preparation of Compounds A6, A7 and A8

[0283] L-Fuc.sub.2S4S-(1,3)-[6-Me-UA-(1,3)]-{D-Gal-NAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)-]-D-6-Me-GlcA-(1,4)}.sub.2-D-GalNAc.sub.4S6S-ol, and L-Fuc.sub.2S4S-(1,3)-L-UA-(1,3)-{D-GalNS.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)-]-D-GlcA-(1,3)}.sub.2-D-GalNS.sub.4S6S-ol and L-Fuc.sub.2S4S-(1,3)-[6-Me-UA-(1,3)]-{D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)-]-D-6-Methyl-GlcA-(1,3)}.sub.2-D-1-Me-GalNAc.sub.4S6S-ol

[0284] 4.1 Materials

[0285] Compound A2, its preparation method and chemical structure were the same as described in Example 1.

[0286] Hydrazine sulfate, hydrazine hydrate, Et.sub.3N.SO.sub.3 ([Et.sub.3NSO.sub.3H]Cl, N,N-diethyl-N-sulfoethanammonium chloride) were all commercially available analytical reagents.

[0287] 4.2 Methods and Results

[0288] (1) Preparation of Compound A6: 10 mg of Compound A2 was dissolved in 0.5 mL of water, converted into H+ type by a Dowex 50X8 hydrogen-type cation exchange resin column, and the eluate was neutralized with tetrabutylammonium hydroxide and lyophilized to obtain 19 mg of A2 tetrabutylammonium salt. The obtained A2 tetrabutylammonium salt was dissolved in 1 mL of dimethyl sulfoxide (DMSO), 15 L of 2 M trimethylsilyldiazomethane (TMSD) was added and reacted for 60 min at room temperature, 15 L of acetic acid was added to remove the remaining TMSD, 4 mL of absolute ethanol was added at 4 C., and centrifuged at 4000 rpm30 min, and the obtained precipitate was dissolved in 1 mL of water, and converted into sodium-type by a Dowex/r50w8 50-100 (Na+ type) exchange resin. The obtained product was desalted on a Sephadex G-10 column and lyophilized to obtain 8.35 mg of A6. .sup.1H-/.sup.13C- and 2D-NMR were detected by the method described in 1.2 (6) of Example 1, and the structure of Compound A6 (the methyl ester group signal on UA was located at 3.70 ppm) was confirmed to be L-Fuc.sub.2S4S-(1,3)-[6-Methyl-UA-(1,3)]-{D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)-]-D-6-Methyl-GlcA-(1,4)}.sub.2-D-GalNAc.sub.4S6S-ol, having the structural formula of:

##STR00017##

[0289] (2) Preparation of compound A7: 10 mg of Compound A2 was added with 2.5 mg of hydrazine sulfate and 0.25 mL of hydrazine hydrate, and stirred under nitrogen atmosphere at 105 C. for 15 h; 0.5 mL of 16% NaCl solution and 3 mL of absolute ethanol were added to the reaction solution, and centrifuged at 4000 rpm20 min. The resulting precipitate was dissolved in 10 mL of water and dialyzed with a 500-1000 Da dialysis bag. The retentate was lyophilized to obtain 8 mg of deacetylated product. The NMR spectrum was detected by the method described in 1.2 (6) of Example 1, and the chemical structure of the deacetylated product (the Ac methyl signal at 2.0 ppm disappeared and the H signal at the 2-position of GalNH.sub.2 appeared at 3.0 ppm) was confirmed to be:

##STR00018##

[0290] The A2 deacetylated product was dissolved in 1 mL of water, 36 mg of Na.sub.2CO.sub.3 was added and heated to 55 C., and 15 mg of Et.sub.3N.SO.sub.3 was added at 0, 5 and 10 h after the start of the reaction, respectively. The reaction mixture was stirred at 55 C. for 15 h. Then 1 mL of 16% NaCl solution and 8 mL of absolute ethanol were added to the reaction solution, and centrifuged at 4000 rpm20 min; the precipitate was collected and dissolved in 10 mL of water, and then dialyzed with a 500-1000 Da dialysis bag. The dialysis retentate was lyophilized to obtain 6.8 mg of N-sulfated product A7.

[0291] .sup.1H-/.sup.13C- and 2D-NMR were detected according to method as described in 1.2(6) of Example 1, and the chemical structure of Compound A7 was confirmed to be L-Fuc.sub.2S4S-(1,3)-L-UA-(1,3)-{D-GalNS.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)-]-D-GlcA-(1,3)}.sub.2-D-Gal-NS.sub.4S6S-ol. The structural formula is:

##STR00019##

[0292] (3) Preparation of Compound A8: 20 mg of Compound A2 was added with Dowex 50X8 hydrogen type cation exchange resin as a catalyst, and then 5 mL of methanol solution was added, and heated to reflux under a nitrogen atmosphere overnight. The resin was removed by filtration, and the filtrate was evaporated to remove the solvent to obtain C1-hydroxyalkylated product of A2. The product was converted into H+ type by a Dowex 50X8 cation exchange resin column, and the eluate was neutralized with tetrabutylammonium hydroxide and lyophilized to obtain 37 mg of tetrabutylammonium salt of the C1 hydroxyalkylated product of A2. The obtained product was dissolved in 2 mL of DMSO, added with 30 L of 2 M TMSD, and reacted for 60 min at room temperature. Then 30 L of acetic acid was added to remove the remaining TMSD, and 2 mL of 16% NaCl solution and 8 mL of absolute ethanol were added at 4 C., centrifuged at rpm30 min, the obtained precipitate was dissolved in 2 mL of water and converted into sodium type by a Dowex/r50w8 50-100 (Na+ type) exchange resin column. The obtained product was desalted on a Sephadex G-10 column and lyophilized to obtain 13.5 mg of A8.

[0293] .sup.1H-/.sup.13C- and 2D-NMR were detected by the method as described in 1.2 (6) of Example 1, and the structure of compound A8 (the methyl signal of UA methyl ester was at 3.70 ppm, and the methyl signal at the reducing terminal was at 3.23 ppm) was confirmed to be L-Fuc.sub.2S4S-(1,3)-[6-Methyl-UA-(1,3)]-{D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)-]-D-6-Methyl-GlcA-(1,3)}.sub.2-D-1-Methyl-Gal-NAc.sub.4S6S-ol, having the structural formula of:

##STR00020##

[0294] Similarly, according to the method of the present example, an alcohol corresponding to a C2-C6 linear or branched alkane or an alkene may be selected to prepare the corresponding hydroxyalkylated product A8.

[Example 5] Preparation of Compounds B9, B10 and B11

[0295] L-Fuc.sub.2S4S-(1,3)-L-UA-(1,3)-D-Gal-NAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)]-D-GlcA-(1,3)-D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)]-D-1-Bn-GlcA, L-Fuc.sub.2S4S-(1,3)-L-6-Me-UA-(1,3)-D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)]-D-6-Me-UA-GlcA-(1,3)-D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)]-D-1-Bnz-6-Me-GlcA and L-Fuc.sub.2S4S-(1,3)-L-UA-(1,3)-{D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)]-D-GlcA-(1,3)}.sub.2-D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)]-D-1-deoxy-1-amino-GlcA-ol-N-4-benzoic ethyl ester

[0296] 5.1 Materials

[0297] Using SvFG as a starting material, hendecasaccharide (B3) was prepared according to the method described in Example 2, and octasaccharide (B7) was prepared according to the method described in Example 3.

[0298] Ethyl 4-aminobenzoate, tetrabutylammonium hydroxide, dichloromethane, pyridine, acetic anhydride, benzyl alcohol, boron trifluoride, ether, and so on were all commercially available analytical reagents.

[0299] 5.2 Methods and Results

[0300] (1) Preparation of Compound B9: 40 mg of Compound B7 was dissolved in 4 mL of water, converted into H.sup.+ type by a Dowex 50X8 hydrogen type cation exchange resin column, and the eluate was neutralized with tetrabutylammonium hydroxide and lyophilized to obtain 80 mg of B7 tetrabutylammonium salt. The obtained B7 tetrabutylammonium salt was added with 8 mL of pyridine and 8 mL of acetic anhydride, stirred at 100 C. for 30 min, and blowing-dried with nitrogen at room temperature. The residue was dissolved in 4 mL of dichloromethane, added with 128 L of benzyl alcohol, and then added dropwise with 20 L of boron trifluoride etherate (BF.sub.3OEt.sub.2) at 0 C., and heated under reflux for 36 h. The reaction was terminated by adding water. After shaking and standing, the CH.sub.2Cl.sub.2 layer was taken and evaporated to dryness to remove CH.sub.2Cl.sub.2. The residue was added with 4 mL of 0.02 M sodium methoxide-methanol solution at room temperature, stirred for 10 min to remove acetyl; evaporated to dryness to remove methanol, converted into H+ type by a Dowex 50X8 hydrogen-type cation exchange resin column. The eluate was neutralized with sodium hydroxide, and isolated and purified by Bio-gel P6, and the sugar-containing samples were combined, concentrated and desalted on a Sephadex G-10 column and lyophilized to obtain 24 mg of Compound B9.

[0301] .sup.1H-/.sup.13C- and 2D-NMR were detected by the same method as described in 1.2 (6) of Example 1, and the structure of Compound B9 (the benzyl-CH.sub.2 signal was at 4.6 ppm and the benzene ring signal was at 7.3 ppm) was confirmed to be L-Fuc.sub.2S4S-(1,3)-L-UA-(1,3)-D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)]-D-GlcA-(j 1,3)-D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)]-D-1-Benzyl-GlcA-ol, having the structural formula of:

##STR00021##

[0302] (2) Preparation of Compound B10: 10 mg of Compound B9 was dissolved in 0.5 mL of water, converted into H+ type by a Dowex 50X 8 hydrogen type cation exchange resin column, and the eluate was neutralized with tetrabutylammonium hydroxide and lyophilized to obtain 19 mg of B9 tetrabutylammonium salt. The obtained B9 tetrabutylammonium salt was dissolved in 1 mL of dimethyl sulfoxide (DMSO), added with 15 L of 2 M trimethylsilyldiazomethane (TMSD), and reacted at room temperature for 60 min, then added with 15 L of acetic acid to remove the remaining TMSD. 4 mL of absolute ethanol was added at 4 C., centrifuged at 4000 rpm30 min, and the obtained precipitate was dissolved in 1 mL of water and converted into sodium type by Dowex/r50w8 50-100 (Na+ type) exchange resin. The obtained product was purified with Bio-Gel P-6, desalted on a Sephadex G-10 column and lyophilized to obtain 8.35 mg of B10.

[0303] .sup.1H-/.sup.13C- and 2D-NMR were detected by the method as described in 1.2 (6) of Example 1, and the structure of compound B10 (methyl signal of carboxyl ester was at 3.7 ppm, CH.sub.2 signal of benzyl was at 4.6 ppm and the benzene ring signal was at 7.3 ppm) was confirmed to be L-Fuc.sub.2S4S-(1,3)-L-6-Methyl-UA-(1,3)-D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)]-D-6-Methyl-UA-GlcA-(1,3)-D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)]-D--1-Benzyl-6-Methyl-GlcA, having the structural formula of:

##STR00022##

[0304] (3) Preparation of compound B11 330 mg of ethyl 4-aminobenzoate was dissolved in 80 L of a mixture solution of glacial acetic acid and methanol (1:9), and 70 mg of sodium cyanoborohydride was added and dissolved. 20 mg of Compound B3 was dissolved in 2 mL of water, reacted with the mixed solution at 60 C. for 4 h in a constant temperature water bath, extracted with 2 mL of chloroform, and the aqueous phase was purified by Bio-gel P10 column chromatography, desalted by a Sephadex G-10 column and lyophilized to obtain about 14 mg of compound B11.

[0305] .sup.1H-/.sup.13C- and 2D-NMR were detected by the method as described in 1.2 (6) of Example 1, and the structure of compound B11 (the CH.sub.3 and CH.sub.2 signals of carbethoxy were located at 1.3 ppm and 4.3 ppm, respectively, and the benzene ring signals were divided into two groups at 6.78 ppm and 7.68 ppm, respectively) was confirmed to be L-Fuc.sub.2S4S-(1,3)-L-UA-(1,3)-{D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)]-D-GlcA-(1,3)}.sub.2-D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)]-D-1-deoxy-1-amino-GlcA-ol-N-4-benzoic ethyl ester, having the structural formula of:

##STR00023##

[0306] Similarly, when benzyl alcohol is replaced by a corresponding C8-C12 aromatic alcohol (for example, p-methylbenzyl alcohol, p-pentylbenzyl alcohol), a series of derivatives B9 having the corresponding C8-C12 aromatic hydrocarbon group were obtained according to the preparation method of B9 in this Example; when ethyl 4-aminobenzoate is replaced by 4-amino-aromatic (C8-C12) carboxylate (propyl 4-aminobenzoate, pentyl 4-aminobenzoate), a series of derivatives B11 having the corresponding C8-C12 aromatic hydrocarbon group were obtained according to the preparation method of B11 in this Example.

[Example 6] Preparation of Oligosaccharide Mixture C1

[0307] 6.1 Materials

[0308] SvFG, obtained as described in Example 1.

[0309] The reagents used such as benzethonium chloride, benzyl chloride, DMF, sodium hydroxide, sodium borohydride, sodium chloride, and ethanol are all commercially available analytical reagents. Sephadex G-50 (medium, 50-100 m), GE Healthcare product.

[0310] 6.2 Methods

[0311] (1) Quaternary ammonium salt conversion of SvFG: 70 g of SvFG was dissolved in 1 L of water; and 175 g of benzethonium chloride was dissolved in 2.8 L of water. The SvFG solution was titrated with a benzethonium chloride solution with stirring. After the completion of the titration, the mixture was centrifuged, and the precipitate was washed three times with deionized water and dried under vacuum to obtain 210 g of SvFG quaternary ammonium salt.

[0312] (2) Carboxyl esterification of SvFG: The SvFG quaternary ammonium salt obtained in the step (1) was dissolved in 1.020 L of DMF, added with 29 mL of benzyl chloride, and stirred at 35 C. for 24 h, and then the reaction solution was allowed to stand and cool down to room temperature (25 C.). Sample was taken for detecting .sup.1H NMR spectrum and the degree of carboxyl esterification of FG was calculated to be about 46%;

[0313] (3) -elimination depolymerization of SvFG in the presence of a reducing agent: To the reaction solution of the step (2), a freshly prepared 333 mL of 0.08 M sodium ethoxide-ethanol solution containing 0.4 M NaBH.sub.4 was added, and stirred at room temperature for 30 min.

[0314] (4) Post-treatment: To the reaction solution obtained in the step (3), 1.333 L of a saturated sodium chloride solution and 10.7 L of absolute ethanol were added, and centrifuged at 4000 rpm10 min, and the obtained precipitate was dissolved in 5 L of water, added with 40 mL of 6 M NaOH solution, and reacted for 30 min at room temperature. Then 6 M HCl was dropwise added to neutralize the reaction solution (pH 7.0). The obtained product was ultrafiltered through a 0.1 m.sup.2 10 kDa and 3 kDa ultrafiltration membrane pack (Millipore) to remove macromolecular and small molecular impurities, to obtain 35 g of oligosaccharide mixture C1.

[0315] (5) Spectral analysis: .sup.1H-/.sup.13C-/2D NMR spectra were detected according to the method described in 1.2 (6) of Example 1.

[0316] 6.3 Results

[0317] (1) 35 g of oligosaccharide mixture C1 was obtained according to the described method, with a yield of 50%;

[0318] (2) HPGPC analysis showed that C1 contained hexasaccharide, nonasaccharide, dodecasaccharide, pentadecasaccharide, octadecasaccharide and heneicosasaccharide, which were 14.2%, 23.1%, 4.1%, 16.0%, 8.9%, and 5.1%, respectively.

[0319] (3) The .sup.13C NMR spectrum and assignments for the oligosaccharide mixture C1 is shown in FIG. 11. In the .sup.1H NMR of the oligosaccharide mixture C1, three strong signal peaks were observed in the range of 5.45.7 ppm, wherein the signal peak at 5.77 ppm was H-4 position signal at the non-reducing terminal UA of the oligosaccharide mixture C1. The signals at 5.6 ppm and 5.43 ppm were a terminal hydrogen signal of L-Fuc.sub.2S4S in the sugar chain near the reducing terminal and a terminal hydrogen signal of L-Fuc.sub.2S4S attached to the non-reducing terminal UA, respectively.

[0320] By the signal analysis of the reducing terminal, in particular the carbon signal analysis of the C1 position (CH.sub.2) of -D-GalNAc.sub.4S6S-ol and -D-GlcA-ol, the content of the oligosaccharide compound having -D-GalNAc.sub.4S6S-ol at the reducing terminal structure was greater than 95%.

[0321] In combination with the .sup.13C-NMR and 2D-NMR analysis, C1 is composed of homologous oligosaccharide compounds, having the structure of L-Fuc.sub.2S4S-(1,3)-L-UA-(1,3)-{D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.2S4S-(1,3)]-D-GlcA-(1,3)}.sub.n-D-GalNAc.sub.4S6S-ol (n is a natural number), wherein the total content of the compounds of n=17 is about 95%.

[Example 7] Preparation of Oligosaccharide Mixtures D1 and D2

[0322] 7.1 Materials

[0323] HsFQG, derived as described in Example 2.

[0324] The reagents used such as benzethonium chloride, benzyl chloride, DMF, DMSO, TMSD, sodium hydroxide, sodium borohydride, sodium chloride, and ethanol were all commercially available analytical reagents. Ultrafiltration membrane (0.5 m.sup.2) with molecular weight cutoff of 30 kDa, 10 kDa, 3 kDa, Merk Millipore.

[0325] 7.2 Methods

[0326] (1) HsFG quaternary ammonium salt conversion: 330 g of HsFG was dissolved in 4.9 L of water; 825 g of benzethonium chloride was dissolved in another 13.2 L of water, the resulting solution was added to the HsFG solution under stirring, centrifuged at 4000 rpm for 10 min. The precipitate was washed three times with 9 L of deionized water and vacuum dried to obtain 804 g of HsFG quaternary ammonium salt.

[0327] (2) Carboxyl esterification of HsFG: The HsFG quaternary ammonium salt obtained in the step (1) was placed in a 30 L reactor, dissolved in 3.9 L of DMF. 97 mL of benzyl chloride was added at 35 C., stirred for 24 h, and then the reaction solution was allowed to stand and cool down to room temperature (25 C.). Sample was taken for .sup.1H NMR detection and the degree of carboxyl esterification was calculated to be about 46%;

[0328] (3) -elimination depolymerization and terminal peeling reaction of HsFG: a freshly prepared 1.3 L of 0.08 M sodium ethoxide-ethanol solution was added to the reaction solution of step (2), stirred at room temperature for 30 min, and then 2.5 mL of 2 M NaOH solution was added to the reaction solution and stirred at 60 C. for 90 min.

[0329] (4) Post-treatment: 5.36 L of saturated sodium chloride solution and 57 L of absolute ethanol were added to the reaction solution of the step (3), centrifuged at 4000 rpm10 min. The resulting precipitate was dissolved in 14.5 L of water, added with 122 mL of 6 M NaOH, stirred at room temperature for 30 min, and then added with 54.3 g of NaBH.sub.4, stirred at room temperature for another 30 min; and dropwise added with 6 M HCl to neutralize the reaction solution (pH 7.0). The obtained reaction solution was filtered through a 0.45 m membrance filter, and the filtrate was sequentially ultrafiltered with 0.5 m.sup.2 of 30 kDa (to obtain filtrate), 10 kDa (to obtain filtrate), and 3 kDa (to obtain retentate) ultrafiltration membrane package (Millipore product) and lyophilized, thereby obtaining an oligosaccharide mixture D1 (98.7 g). [Note: After the detection, the undepolymerized macromolecular compositions contained in the retentate obtained from ultrafiltration through a 30 kDa ultrafiltration membrane contains fucan and hexosamine-containing polysaccharides].

[0330] (5) D1 carboxymethylation: 20 g of oligosaccharide mixture D1 was dissolved in 300 mL of water, 800 mL of 6.25% benzethonium chloride solution was added with stirring, allowed to stand and then centrifuged at 4000 rpm10 min. The precipitate was washed three times with 300 mL of deionized water and vacuum dried to obtain 58 g of D1 quaternary ammonium salt. The obtained D1 quaternary ammonium salt was dissolved in 5.8 L of DMSO, added with 87 mL of 2 M TMSD, stirred for 60 min at room temperature, and then added with 87 mL of acetic acid to remove the remaining TMSD; 5.9 L of saturated sodium chloride solution and 63 L of 95% ethanol was sequentially added under stirring, centrifuged at 4000 rpm30 min. The resulting precipitate was dissolved in 2 L of deionized water, desalted by ultrafiltration through a 3 kDa ultrafiltration membrane, and the retentate was lyophilized to obtain D2 (16.3 g).

[0331] (6) Spectral analysis: .sup.1H-/.sup.13C- and 2D-NMR were detected according to the method described in 1.2 (6) of Example 1.

[0332] 7.3 Results

[0333] (1) Yield and Chemical Composition Analysis of Oligosaccharide Mixture D1

[0334] 98.7 g of oligosaccharide mixture D1 was obtained according to the method, with a yield of about 30%.

[0335] HPGPC analysis (FIG. 12) showed that D1 contained pentasaccharide, octasaccharide, hendecasaccharide, tetradecasaccharide, heptadecasaccharide, eicosasaccharide, which were 4.3%, 17.1%, 18.0%, 16.3%, 14.1%, and 11.1%, respectively. The total content of pentasaccharidenonacosasaccharide was about 96%.

[0336] The .sup.13C-NMR spectrum of the oligosaccharide mixture D1 is shown in FIG. 13. In the .sup.1H-NMR of D1, there was a strong signal at 5.685 ppm, which was from 4-position hydrogen of UA. There were three strong signal peaks at 5.05.6 ppm (5.283, 5.201 and 5.030 ppm), which were the a-anomeric proton signals of L-Fuc.sub.3S4S linked to D-GlcA, UA and D-GlcA-ol, respectively. By analyzing the terminal hydrocarbon signal of the reducing terminal glycosyl group, the content of the oligosaccharide compound having -D-GlcA-ol at the reducing terminal glycosyl group in D1 was more than 95%.

[0337] Combined with .sup.13C- and 2D-NMR analysis, it can be seen that the mixture D1 was a mixture of homologous oligosaccharide compounds having a structure of L-Fuc.sub.3S4S-(1,3)-L-U-(1,3)-{D-GalNAc.sub.4S6S-(3,4)-[L-Fuc.sub.3S4S-(1,3)]-D-GlcA-(1,3)}.sub.n-D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.3S4S-(a 1,3)]-D-GlcA-ol (n is a natural number).

[0338] (2) Analysis of Yield and Chemical Composition of Oligosaccharide Mixture D2

[0339] 16.3 g of oligosaccharide mixture D2 was obtained according to the method, with a yield of about 80%;

[0340] HPGPC analysis showed that it contained pentasaccharide, octasaccharide, hendecasaccharide, tetradecasaccharide, heptadecasaccharide, eicosasaccharide, which were 3.34%, 16.71%, 17.03%, 17.25%, 13.78%, and 12.25%, respectively.

[0341] Compared to the .sup.1H NMR spectrum of D1, new methyl (ester) group signals linked to hexuronic acid (-ol) appeared in the .sup.1H NMR of D2, which were at 3.7 ppm and 3.2 ppm, respectively. In combination with .sup.1H-/.sup.13C- and 2D-NMR analysis, it can be seen that the mixture D2 is a mixture of homologous oligosaccharide compounds having a structure of L-Fuc.sub.3S4S-(1,3)-L-6-Me-UA-(1,3)-{D-GalNAc.sub.4S6S-(1,4)-[L-Fuc.sub.3S4S-(1,3)]-D-6-Me-GlcA-(1,4)}.sub.n-D-GalNAc.sub.4S6S-(1,3)-[L-Fuc.sub.3S4S-(1,3)]-D-6-Me-GlcA-ol (n is a natural number). Wherein, the sum of the contents of the compounds of n=19 is about 96%.

[Example 8] Analysis of Anticoagulation and Coagulation Factor Inhibitory Activity

[0342] 8.1 Materials

[0343] Samples: oligosaccharide compounds A1A8, B1B11, oligosaccharide mixtures C1, D1, and D2, prepared according to the method described in Examples 17.

[0344] Control: Enoxaparin Sodium Injection (LMWH, Mw 35005500 Da, Sanofi-Aventis product);

[0345] Reagents: coagulation-controlled plasma (047B-D024A), activated partial thromboplastin time (APTT), prothrombin time (PT) assay kits, all of which were TECO GmbH company (Germany) products; Factor VIII test kit, Heparin Cofactor II (HCII), AT-dependent anti-factor IIa detection kit, AT-dependent anti-factor Xa detection kit, thrombin (factor IIa), thrombin substrate CS01 (38), KK substrate CS31 (02) were HYPHEN BioMed company (France) products; Factor VIII (FVIII), Bayer Healthcare LLC (Germany) product; ADP, Chronolog company (USA) product; sodium citrate, chloral hydrate, natural saline, were all commercial reagents.

[0346] Instruments: XS105 electronic balance, FE20 pH meter, METTLER TOLEDO products; HH-4 constant temperature water bath, Gongyi Yuhua company product, China; VOR76X-6 vortex oscillator, Hainan Qilin Bell product; Spectrafuge-24 D907386 centrifuge, Labnet product; MC-4000 blood coagulation instrument, TICO GmbH company (Germany) product; Microplate Reder ELx 808 microplate reader, Bio-Tek company product; Chronolog-700 platelet aggregation instrument, Chrono-log company (USA) product.

[0347] 8.2 Methods

[0348] (1) Preparation of sample solution: Oligosaccharide compounds A1A8, B1B11 and oligosaccharide mixtures C1, D1, D2 were all dissolved in Tris-HCl buffer and diluted to the desired series of solubility.

[0349] (2) Anticoagulant activity assay: 90 L of human-controlled plasma was added to the sample or 10 L of the control solution, and then the clotting time (APTT and PT) was detected by the MC-4000 coagulometer according to the method described in the APTT and PT kit instructions.

[0350] (3) Coagulation Factor Inhibitory Activity Analysis:

[0351] Xase inhibitory activity assay: Detection was performed according to kit instructions and literature methods by combining the Factor VIII and Factor VIII detection kits. Specifically, to each well of a 96-well plate, 30 L of test solution, control solution, or Tris-HCl buffer (negative control) was added, and 30 l of FVIII (2 IU/ml), 30 l of R.sub.2 (60 nM FIXa, containing FIIa, PC/PS, Ca.sup.2+) were sequentially added, mixed by shaking the plate, incubated at 37 C. for 2 min; and then 30 L of R.sub.1 (50 nM FX, containing direct thrombin inhibitor) was added, mixed by shaking the plate, incubated at 37 C. for 1 min; and then 30 L of R.sub.3 (FXa chromogenic substrate SXa-11, about 8.4 mM) was added. The absorbance at 405 nm (OD.sub.405) was detected with a microplate reader, continuously measuring for 7.5 min at a interval of 30 s. The Xase activity and IC.sub.50 value of Xase inhibition of the test sample were calculated based on the OD.sub.405 change value.

[0352] AT-dependent Xa inhibitory activity assay: Heparin Anti-FIIa kit was used for detection. To a 96-well plate, 30 L of sample, control solution or Tris-HCl buffer (negative control) was added, then 30 L of 1 IU/mL AT solution was added, mixed well and incubated at 37 C. for 1 min; and than 30 L of 8 g/mL FXa solution was added, mixed well and incubated at 37 C. for 1 min, then 30 L of pre-warmed 1.25 mM Xa chromogenic substrate SXa-11 was added. OD.sub.405 was detected by a microplate reader.

[0353] AT-dependent IIa inhibitory activity assay: Heparin Anti-FIIa kit was used for detection. To a 96-well plate, 30 L of sample, control solution or Tris-HCl buffer (negative control) was added, and then 30 L of 1 IU/mL AT solution was added, mixed well by shaking the plate and incubated at 37 C. for 2 min; 30 L of 24 IU/mL FIIa solution was added, mixed well by shaking the plate and incubated for 2 min at 37 C., and then 30 L of pre-warmed 1.25 mM FIIa specific chromogenic substrate CS-01 (38) was added, mixed well by shaking the plate. The OD.sub.405 was detected by a microplate reader and the IC.sub.50 value of FIIa inhibition of each sample was calculated.

[0354] HC-II-dependent IIa inhibitory activity assay: 30 L of sample, control solution or Tris-HCl buffer (negative control) was added, 30 L of 1 M HCII solution was added, and incubated at 37 C. for 2 min; and then 30 L of 20 NIH/mL FIIa was added, and incubated at 37 C. for 1 min; and finally 30 L of pre-warmed 4.5 mM FIIa chromogenic substrate CS-01 (38) was added. OD.sub.405 was detected by a microplate reader and the IC.sub.50 value of FIIa inhibition of each sample was calculated.

[0355] Data processing: The average value of OD.sub.405 detected by the duplicated well was used as the detection value of the test sample and the reference of each concentration, and the slope of the linear fit between the detected value to the time value (the change rate of the absorbance value OD.sub.405/min) indicated enzymatic activity of coagulation factor. Taking the clotting factor activity of the negative control well as 100%, coagulation factor activity (percentage) in the presence of the test sample was calculated. The coagulation factor activity in the presence of the test sample was plotted against the concentration of the test sample, and fitted according to the following formula, to calculate the IC.sub.50 value:

B=(IC.sub.50).sup.n/{(IC.sub.50).sup.n+[I].sup.n}

[0356] in the formula, B is the coagulation factor activity (percentage) in the presence of the test sample, [I] is the concentration of the test sample, IC.sub.50 is the half inhibitory concentration (concentration of the test sample required to inhibit 50% of the activity), and n is the Hill coefficient.

[0357] (4) Effect on Surface Activation and Platelet Activity:

[0358] FXII activation activity assay: To a 96-well plate was added 30 L of series concentration sample and reference solution, respectively, and then 30 L of human standard plasma that was diluted 4 times with a 0.02 M Tris-HCl (pH 7.4) buffer containing 0.15 M NaCl was added, and incubated at 37 C. for 2 min, and then 30 L of 6 mM kallikrein chromogenic substrate CS-31 (02) was added, and the OD.sub.405 value was detected by a microplate reader.

[0359] Platelet activation activity test: Anticoagulated blood was collected from healthy volunteers to prepare platelet-rich plasma (PRP) and platelet-poor plasma (PPP). Chronolog-700 platelet aggregation instrument and turbidimetry were used to detect platelet-induced aggregation activity of serial concentration solutions of the test sample, which were prepared by dissolving in natural saline.

[0360] 8.3 Results

[0361] Anticoagulation and coagulation factor inhibitory activity: The results are shown in Table 5. The oligosaccharide compounds and the mixture thereof according to the present invention have significant prolonged APTT activity, without affecting PT and TT, indicating that they can have significant anticoagulant activity against intrinsic coagulation pathway, and have no significant effect on extrinsic coagulation. The oligosaccharide compounds and the mixture thereof according to the present invention have significant inhibitory activity on factor Xase; in the presence or absence of antithrombin (AT), they have no significant effect on coagulation factors such as coagulation factors IIa, Xa, XIIa, but may have a certain intensity of heparin cofactor II (HC-II)-dependent IIa inhibitory activity.

[0362] An alcohol corresponding to a C2-C6 linear or branched alkane or alkene was selected to prepare the corresponding hydroxyalkylated product A8 according to Example 4. Studies on the activity of these series of derivatives show that they have similar activity to A8, that is, they have prolonged APTT activity (with the drug concentration for doubling the APTT clotting time being 7.0-10 g/mL), without affecting PT and TT; have significant selective inhibitory activity against factor Xase (IC.sub.50, 50-100 ng/mL), have no significant effect on coagulation factors such as factor IIa, Xa, XIIa, and have a certain intensity of heparin cofactor II (HC-II)-dependent IIa inhibitory activity.

[0363] According to the preparation method of Example B9, a series of derivatives B9 having a corresponding C8-C12 aromatic hydrocarbon group were obtained, and according to the preparation method of Example B11, a series of derivatives B11 having a corresponding C8-C12 aromatic hydrocarbon group were obtained. They have similar activities to B9 and B11, respectively; have drug concentration of doubling APTT clotting time of 6.0-9 g/mL, without affecting PT and TT; have significant selective inhibitory activity against factor Xase (IC.sub.50, 40-110 ng/mL), and have a certain intensity of heparin cofactor II (HC-II)-dependent IIa inhibitory activity.

TABLE-US-00006 TABLE 5 Anticoagulant and coagulation factor inhibitory activity of oligosaccharide compounds and oligosaccharide mixtures Drug concentration required for multiplication of Drug concentration required coagulation time to inhibit 50% of coagulation (g/mL) factor activity (IC.sub.50, ng/mL) APTT PT TT Xase Xa (AT) Ha (AT) Ha (HC-II) A1 60.1 # # ## ## ## 450 A2 7.2 # # 60.5 ## ## 323 A3 6.8 # # 39.8 ## ## 258 A4 5.3 # # 28.2 ## ## 231 A5 3.9 # # 23.6 ## ## 320 A6 7.5 # # 68.4 ## ## 385 A7 6.9 # # 58.9 ## ## 298 A8 8.2 # # 70.6 ## ## 335 B1 53.9 # # 850 ## ## 705 B2 7.5 # # 64.2 ## ## 753 B3 6.1 # # 31.1 ## ## 402 B4 4.5 # # 20.6 ## ## 408 B5 4.0 # # 19.8 ## ## 365 B6 50.2 # # 1670 ## ## 817 B7 7.3 # # 58.3 ## ## 432 B8 5.8 # # 29.6 ## ## 412 B9 7.6 # # 59.6 ## ## 706 B10 7.9 # # 61.5 ## ## 721 B11 6.3 # # 30.3 ## ## 375 C1 4.0 # # 22.3 ## ## 404 D1 4.3 # # 21.2 ## ## 230 D2 5.1 # # 23.6 ## ## 278 LMWH 7.8 64 4.0 120.0 16 36 431 Note: #, >128 g/mL; ##, >5000 ng/mL

[0364] (2) Effect on Surface Activation and Platelet Activity:

[0365] XII activation activity analysis: Within the concentration range of not more than 100 g/ml, all the oligosaccharide compounds and oligosaccharide mixtures have no significant XII activation activity;

[0366] Platelet activation activity assay: Within the concentration range of not more than 50 g/ml, all oligosaccharide compounds and oligosaccharide mixtures have no significant platelet activation activity.

[Example 9] Effect on Antithrombotic Activity and Bleeding

[0367] 9.1 Materials

[0368] The preparation of A2 was as shown in Example 1, and the preparation of D1 was as shown in Example 7.

[0369] Control: Low molecular weight heparin (LMWH), Sanofi-Aventis (France) product, batch number 4SH69.

[0370] Reagents: chloral hydrate (hydrated trichloroacetaldehyde), Sinopharm Chemical Reagent Co., Ltd.; natural saline, Kunming Nanjiang Pharmaceutical Co., Ltd.

[0371] Experimental animals: SD rats, weighing 250350 g, male, provided by Hunan Slack Jingda Experimental Animal Co., Ltd., license number SCXK (Xiang) 2011-0003; New Zealand rabbits provided by Kunming Medical University, SCXK (Dian) 2011-0004, used to make rabbit brain powder infusion.

[0372] 9.2 Methods

[0373] 9.2.1 Anti-Venous Thrombosis Experiment

[0374] Grouping and Administration: Rats were randomly divided into 8 groups with 8 animals in each group. The experimental groups and the dose of the animals in each group were (1) natural saline (NS) control group; (2) LMWH 4.0 mg/kg group; (3) A2 2.5 mg/kg group; (4) A2 group 5.0 mg/kg; (5) A2 10 mg/kg group; (6) D1 2.5 mg/kg group; (7) D1 5.0 mg/kg group; (8) D1 10 mg/kg group. The rats in each group were administered subcutaneously (sc.) into the back, and the administration volume was 1 mL/kg. The modeling experiment was performed 1 hour after administration.

[0375] Preparation of Rabbit Brain Powder Infusion:

[0376] A New Zealand rabbit was sacrificed, and the rabbit brain was taken out immediately. Rabbit brain powder infusion was prepared according to the literature method (Thromb Haemost, 2010, 103(5): 994-1004), and stored at 20 C. for use.

[0377] Induction of Inferior Vena Cava Thrombosis by Rabbit Brain Powder Infusion:

[0378] The rats were anesthetized by intraperitoneally injecting with 10% chloral hydrate (300 mg/kg), the abdominal wall was cut longitudinally along the midline of the abdomen, the viscera was removed, and the inferior vena cava and its branches were isolated. A ligature was passed through the lower margin of the left renal vein of the inferior vena cava, to ligate the inferior vena cava branches below the left renal vein. The femoral vein was injected with 2% rabbit brain powder infusion (1 mL/kg). After 20 seconds, the lower margin of the left renal vein was ligated. After the operation, the viscera was placed back into the abdominal cavity and covered with medical gauze (infiltrated with natural saline). After 20 minutes, the blood vessel was clamped at 2 cm below the ligature, and the blood vessel was longitudinally dissected to take out the thrombus. The length of the thrombus was measured, and the wet weight of the thrombus was weighed and then dry weight was weighed after drying at 50 C. for 24 h.

[0379] Data Processing and Statistics: The SPSS software was used to organize and analyze the data, and the measurement data were expressed as meanstandard deviation (xs). Data normality in different groups was tested using One-Sample KS test, variance homogeneity was tested using Levene test. If the data conformed to the normal distribution, and the variance was uniform, the significance was judged by One-Way ANOVA, otherwise, the significance was judged by Two-Independent-Samples Test.

[0380] 9.2.2 Bleeding Tendency Detection

[0381] Grouping and administration: Mice were randomly divided into 10 groups with 8 animals in each group. The experimental groups and the dose of the animals in each group were (1) natural saline (NS) control group; (2) LMWH 4.0 mg/kg group; (2) LMWH 20 mg/kg group; (3) LMWH 100 mg/Kg group; (4) A2 5 mg/kg group; (5) A2 25 mg/kg group; (6) A2 125 mg/kg group; (7) D1 5 mg/kg group; (8) D1 25 mg/Kg group; (10) D1 125 mg/kg group. The rats in each group were administered subcutaneously (sc.) into the back, and the dose volume was 10 mL/kg.

[0382] Test Methods:

[0383] After 60 min of subcutaneous administration in each experimental group, the mice were placed in a mouse holder, and the tail tip was cut by 5 mm by tail-clipping method, and the mouse tail was immersed in 40 mL of purified water (37 C.) in the beaker. Timing was started from the first drop of blood from the cut mouse tail, and stirring was continued. At 60 min, the beaker was placed for 60 min and then the absorbance of the solution (OD540) was detected by a UV spectrophotometer.

[0384] In addition, whole blood of healthy mice was taken, and the whole blood of different volumes of mice was added to 40 mL of purified water, stirred uniformly and allowed to stand for 60 min. The absorbance (OD.sub.540) of the solution was detected by the same method and the volume-absorbance curve was plotted and used as the standard curve for calculating the amount of bleeding. The amount of bleeding in each experimental group was calculated by the standard curve.

[0385] Data Processing and Statistics:

[0386] The SPSS software was used to organize and analyze the data, and the detected data was expressed as meanstandard deviation (xs). Data normality in different groups was tested using One-Sample KS test, variance homogeneity was tested using Levene test. If the data conformed to the normal distribution, and the variance was uniform, the significance was judged by One-Way ANOVA, otherwise, the significance was judged by Two-Independent-Samples Test.

[0387] 9.3 Results

[0388] (1) Antithrombotic activity: As shown in FIG. 14, the results show that both A2 and D1 have significant antithrombotic activity at the experimental dose, and the inhibition rate of thrombosis may reach above 70% at the dose of 5 mg/kg10 mg/kg.

[0389] (2) Bleeding tendency influence: As shown in FIG. 15, under high dose administration of equal multiple of the equivalent antithrombotic dose, the amount of bleeding in A2 and D1 administration groups is significantly lower than that in LMWH administration group.

[Example 10] Preparation of A3 Pharmaceutical Composition as Lyophilized Powder for Injection

[0390] 10.1 Materials

[0391] Compound A3, purified dodecasaccharide compound prepared according to the method described in Example 1.

[0392] NaCl, commercially available, pharmaceutical grade; Sterile water for injection; 2 mL medium borosilicate tube glass bottle for injection, Millipore Pellicon 2 ultrafiltration system (Merk Millipore); VirTis Ultra 35 EL lyophilizer.

[0393] 10.2 Formulation

TABLE-US-00007 Raw material (Excipient) Dosage A3 20 g NaCl 4 g H.sub.2O 500 mL Totally prepared into 1000 vials

[0394] 10.3 Preparation Process

[0395] (1) Process procedure: Twice the prescribed amount of A3 (40 g) and NaCl (8 g) were weighed and dissolved in 1.0 L of water for injection. After dissolved completely under stirring, it was treated by a Millipore ultrafiltration device having an ultrafiltration membrane package with a molecular weight cut-off of 10 kDa to remove the pyrogen. In a sterile environment, after 0.22 m membrane filtration and sterilization, the solution was filled in a 2 mL vial of 0.5 mE per vial while monitoring the filling process, partially stoppered, and placed in the drying box of the pilot lyophilizer (VirTis, US), lyophilized according to the programmed lyophilization process, stoppered, withdrawn from the lyophilizer, capped, and inspected.

[0396] (2) Lyophilization process:

[0397] Pre-cooling: The samples were placed in the lyophilizer; the temperature of shelves was dropped to 25 C., maintaining for 1 h, then dropped to 45 C., maintaining for 3 h; the temperature of cold trap was dropped to 50 C., and the vacuum degree was pumped to 40 Pa.

[0398] Sublimation: The temperature was increased uniformly to 30 C. within 1 h, maintaining for 2 h; increased uniformly to 20 C. within 2 h, maintaining for 6 h; the vacuum degree was maintained at 4030 Pa.

[0399] Drying: The temperature was increased to 5 C. within 2 h, maintaining for 2 h, and the vacuum was maintained at 3020 Pa; the temperature was increased to 10 C. within 0.5 h, maintaining for 3 h, and the vacuum degree was maintained at 3020 Pa; the temperature was increased to 40 C. within 0.5 h, maintaining for 4 h, and the vacuum degree was pumped to the lowest.

[0400] 10.4 Results

[0401] According to the preparation process, 1,960 vials of qualified products of A3 lyophilized preparation were obtained, and the qualified rate of the finished product was about 98%. After testing, the lyophilized cake had regular appearance; the sterility, pyrogen and insoluble particulate testing were all qualified; the moisture testing results showed that the water content was less than about 3%, and the loading testing results showed that the loading was within 95115% of the planned loading.

[Example 11] Preparation of D1 Pharmaceutical Composition as Lyophilized Powder for Injection

[0402] 11.1 Materials

[0403] Oligosaccharide mixture D1, prepared according to the method described in Example 7. NaCl, commercially available, pharmaceutical grade; sterile water for injection; 2 mL medium borosilicate tube glass bottle for injection, Millipore Pellicon 2 ultrafiltration system (Merk Millipore); Lyophilizer (LYO-20 m.sup.2), Shanghai Toffion Sci &Tech Co., Ltd.

[0404] 11.2 Formulation

TABLE-US-00008 Raw material (Excipient) Dosage D1 50 g NaCl 9 g H.sub.2O 1.0 L Totally prepared into 1000 vials

[0405] 11.3 Preparation Process

[0406] (1) Process procedure: 20 times the prescribed amount of D1 (1000 g) and NaCl (180 g) were weighed and dissolved in 20 L of water for injection. After dissolved completely under stirring, it was treated by a Millipore ultrafiltration device having an ultrafiltration membrane package with a molecular weight cut-off of 10 kDa to remove the pyrogen. In a sterile environment, after 0.22 m membrane filtration and sterilization, the solution was filled in a 2 mL vial of 0.5 mL per vial while monitoring the filling process, partially stoppered, and placed in the drying box of a production lyophilizer (VirTis, US), lyophilized according to the programmed lyophilization process, stoppered, withdrawn from the lyophilizer, capped, and inspected to be qualified, to obtain the final products.

[0407] (2) Lyophilization Process:

[0408] Pre-cooling: The samples were placed in the lyophilizer; the temperature of shelves was dropped to 25 C., maintaining for 1 h, then dropped to 45 C., maintaining for 3 h; the temperature of cold trap was dropped to 50 C., and the vacuum degree was pumped to 40 Pa.

[0409] Sublimation: The temperature was increased uniformly to 30 C. within 1 h, maintaining for 2 h; increased uniformly to 20 C. within 2 h, maintaining for 6 h; the vacuum degree was maintained at 4030 Pa.

[0410] Drying: The temperature was increased to 5 C. within 2 h, maintaining for 2 h, and the vacuum degree was maintained at 3020 Pa; the temperature was increased to 10 C. within 0.5 h, maintaining for 3 h, and the vacuum degree was maintained at 3020 Pa; the temperature was increased to 40 C. within 0.5 h, maintaining for 4 h, and the vacuum degree was pumped to the lowest.

[0411] 11.4 Results:

[0412] According to the preparation process, 17,600 vials of qualified samples of D1 lyophilized preparation were obtained, and the qualified rate of the finished product was about 88%.

[0413] Appearance/characteristic: This product was a white loose mass.

[0414] Loading testing: The gravimetric testing was in compliance with the regulations.

[0415] Sterility testing: An appropriate amount of this product was taken and tested according to law (1101, Volume IV, Chinese Pharmacopoeia Edition 2015). The test results showed that the batch of samples met the quality requirements of injection.

[0416] Pyrogen testing: The product was prepared into a solution containing 3.5 mg of D1 per 1 mL, and tested according to the law (1142, Volume IV, Chinese Pharmacopoeia Edition 2015), the results showed that this batch of samples met the quality requirements of pyrogen testing for injection.