WO2024061270A1

WO2024061270A1 - Heparin pentasaccharide-based low molecular weight heparin mimetic, preparation method therefor and use thereof

Info

Publication number: WO2024061270A1
Application number: PCT/CN2023/120054
Authority: WO
Inventors: 李铁海; 张良伟; 马文静
Original assignee: 中国科学院上海药物研究所
Priority date: 2022-09-21
Filing date: 2023-09-20
Publication date: 2024-03-28
Also published as: CN117777320A

Abstract

Disclosed in the present invention is a heparin pentasaccharide-based low molecular weight heparin mimetic, which is a heparin pentasaccharide dimer or heparin pentasaccharide trimer. Also disclosed in the present invention is a preparation method for a heparin pentasaccharide dimer or heparin pentasaccharide trimer. The heparin pentasaccharide dimer or heparin pentasaccharide trimer of the present invention has anticoagulant activity, and the heparin pentasaccharide trimer can be neutralized by protamine.

Description

Low molecular weight heparin mimetic based on heparin pentasaccharide and preparation method and use thereof

Technical field

The present invention relates to the field of medicine, and specifically to a uniform low molecular weight heparin mimetic based on heparin pentasaccharide and its preparation method and use. As an anticoagulant drug, it is mainly used to prevent and treat venous thrombosis.

Background technique

Heparin is a highly sulfated glycosaminoglycan composed of β-D-glucuronic acid (or α-L-iduronic acid) and α-D-glucosamine (or α-D-acetamide Glucose) is a mucopolysaccharide composed of repeating disaccharide units and has strong anticoagulant and antithrombotic properties. According to its molecular weight, it can be divided into unfractionated heparin (average molecular weight about 15,000), low molecular weight heparin (average molecular weight range 3,500-6,000) and synthetic ultra-low molecular weight heparin (molecular weight usually less than 3,000). Unfractionated heparin is extracted from animal livers, bovine lungs and pig intestines, and is mainly a complex mixture composed of different active polysaccharides; low molecular weight heparin is produced by chemical or enzymatic degradation of ordinary unfractionated heparin. Heparin has been used clinically for decades. The separated and extracted unfractionated heparin and low molecular weight heparin have problems such as the risk of contamination by pathogenic microorganisms, and the differences between different batches of animal tissues causing unstable quality. In 2008, in the large-scale recall of heparin sodium in the United States, allergic reactions and other side effects occurred due to poor quality control, and even led to the death of many people.

Compared with unfractionated heparin, low molecular weight heparin has the advantages of good injection absorption, long half-life, and high bioavailability. However, excessive use of low molecular weight heparin can cause bleeding complications. Although protamine can partially neutralize its anticoagulant activity, it cannot achieve the effect of complete neutralization and rapid prevention of bleeding. The isolated and extracted low molecular weight heparin has problems such as heterogeneity, the risk of pathogenic microbial contamination and bleeding risk, which seriously limit the widespread use of this drug. Synthesizing low molecular weight heparin polysaccharides with uniform structure can avoid the occurrence of related complications, but it is extremely difficult to synthesize linear heparin polysaccharides composed of pentasaccharide repeating units. Therefore, the preparation of sugar cluster compounds containing different numbers of heparin pentasaccharides can be used as low molecular weight heparin mimetics, and then screen out compounds that can be completely neutralized by protamine, which is expected to solve the problem of the difficulty in obtaining uniform and neutralizable low molecular weight heparin.

Contents of the invention

The object of the present invention is to provide a uniform low molecular weight heparin mimetic based on heparin pentasaccharide, preparation method and use.

A first aspect of the present invention provides a heparin pentasaccharide dimer having the following structure:

A second aspect of the present invention provides a heparin pentasaccharide trimer having the following structure:

A third aspect of the present invention provides a method for preparing the heparin pentasaccharide dimer described in the first aspect, the preparation method comprising the following steps:

After the heparin pentasaccharide derivative 4 reacts with the connecting arm modified with a bismaleimide group, dithiothreitol is added to react to obtain the heparin pentasaccharide dimer.

In another preferred embodiment, a large excess (20 equivalents) of heparin pentasaccharide derivative 4 is needed to promote complete reaction of the bismaleimide group-modified connecting arm and ensure that the two heparin pentasaccharide fragments are connected to obtain their dimerization. things.

In another preferred example, compound 4 is synthesized by the following steps:

After the reaction of heparin pentasaccharide 3 modified with the pentylamino connecting arm at the reducing end and 3,3'-dithiodipropionic acid bis(N-hydroxysuccinimide) ester, dithiothreitol was added to react to obtain heparin pentasaccharide 3. Sugar derivatives 4.

In another preferred embodiment, an excess amount (5 equivalents) of 3,3'-dithiodipropionic acid bis(N-hydroxysuccinimide) ester is required to ensure complete reaction of heparin pentasaccharide 3, and subsequently a large amount of Dithiothreitol (10 equivalents) reduces all disulfide bonds.

In another preferred example, the heparin pentasaccharide 3 modified with the reducing terminal pentamino linker arm is synthesized through the following steps:

i) Compound 5 reacts with PMe ₃ to completely convert the azide group into an amino group to obtain an amino-containing intermediate;

ii) The amino-containing intermediate reacts with NMe ₃ ^. SO ₃ to obtain an N-sulfonated intermediate;

iii) The N-sulfonated intermediate undergoes hydrogenation reaction to obtain heparin pentasaccharide intermediate 3.

In another preferred example, an excess of PMe ₃ (7.5 equivalents) is needed to ensure that all three azide groups are reduced to amino groups, and then an excess of NMe ₃ ·SO ₃ (15 equivalents) is added to ensure that the three amino groups are completely reduced. Sulfonation.

For compound 5, it can be synthesized by referring to the methods disclosed in ChemMedChem 2014, 9, 1071-1080 and J.Am.Chem.Soc. 2009, 131, 47, 17394-17405.

A fourth aspect of the present invention provides a method for preparing the heparin pentasaccharide trimer described in the second aspect, the preparation method comprising the following steps:

After the heparin pentasaccharide derivative 4 reacts with the connecting arm modified with a trimaleimide group, dithiothreitol is added to react to obtain the heparin pentasaccharide trimer.

In another preferred embodiment, a large excess of heparin pentasaccharide derivative 4 (30 units) is required to promote the complete reaction of the trimaleimide group-modified linking arm and ensure that the three heparin pentasaccharide fragments are connected to obtain the three heparin pentasaccharide fragments. Polymer.

In another preferred embodiment, compound 4 is synthesized by the following steps:

i) Compound 5 reacts with PMe ₃ to completely convert the azide group into an amino group to obtain an amino-containing intermediate; (Chemical For similar methods for the preparation of compound 5, please refer to published articles ChemMedChem 2014, 9, 1071-1080 and J.Am.Chem.Soc. 2009, 131, 47, 17394-17405)

ii) The amino-containing intermediate reacts with NMe ₃ ·SO ₃ to obtain an N-sulfonated intermediate;

A fifth aspect of the present invention provides a pharmaceutical composition, comprising:

The heparin pentasaccharide dimer as described in the first aspect or the heparin pentasaccharide trimer as described in the second aspect; and

A pharmaceutically acceptable carrier.

A sixth aspect of the present invention provides the use of the heparin pentasaccharide dimer as described in the first aspect or the heparin pentasaccharide trimer as described in the second aspect for preparing anticoagulant/antithrombotic drugs.

In another preferred embodiment, the heparin pentasaccharide dimer described in the first aspect or the heparin pentasaccharide trimer described in the second aspect is used to prepare drugs for preventing and treating venous thrombosis.

In another preferred embodiment, the heparin pentasaccharide dimer as described in the first aspect or the heparin pentasaccharide trimer as described in the second aspect is used to prepare a factor Xa inhibitor.

In another preferred embodiment, the anticoagulant activity of the heparin pentasaccharide dimer described in the first aspect or the heparin pentasaccharide trimer described in the second aspect, especially the heparin pentasaccharide trimer described in the second aspect, is The anticoagulant activity of the polymer is neutralized by protamine.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions. Each feature disclosed in the specification may be replaced by any alternative feature serving the same, equivalent or similar purpose. Due to space limitations, they will not be described one by one here.

Description of drawings

Figure 1 shows that heparin pentasaccharide trimer 1, dimer 2 and enoxaparin have similar inhibitory activities on factor Xa.

FIG2 shows the results of in vitro neutralization activity studies of heparin pentasaccharide trimer 1, dimer 2, enoxaparin and fondaparinux sodium.

Detailed ways

After extensive and in-depth research, the inventor of the present application has developed a neutralizable and uniform low-weight heparin mimetic to solve the problem of non-uniformity of current low-weight heparin as an anticoagulant, the risk of pathogenic microbial contamination and the risk of bleeding. And other issues.

Aiming at the problem of difficulty in synthesizing heparin pentasaccharide derivatives, the inventors developed a method for synthesizing heparin pentasaccharide derivatives with a connecting arm modified with a thiol functional group at the reducing end.

Heparin pentasaccharide derivatives modified with thiol functional group linking arm at reducing end

In order to solve the problem of difficulty in obtaining low-weight heparin, the inventor developed a method for preparing a sugar cluster compound composed of multiple pentasaccharide fragments as a uniform low-weight heparin mimetic.

Low molecular weight heparin mimetics based on heparin pentasaccharide dimerization

Low molecular weight heparin mimetics based on heparin pentasaccharide trimerization

Overuse of heparin drugs can cause bleeding in the body. Neutralizing drugs are antagonists that reverse the anticoagulant effect of heparin to reduce the risk of bleeding. In order to solve the problem of difficulty in obtaining uniform low molecular weight heparin that can be neutralized, a low molecular weight heparin mimetic composed of three heparin pentasaccharides was screened for biological activity and can be completely neutralized by protamine.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. Experimental methods without specifying specific conditions in the following examples are usually carried out according to conventional conditions (such as conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or according to manufacturing Conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as familiar to one skilled in the art. In addition, any methods and materials similar or equivalent to those described can be used in the method of the present invention. The preferred implementation methods and materials described in this article are for demonstration purposes only.

Example 1

First, 1 equivalent of compound 5 was dissolved in a mixed solvent of tetrahydrofuran and water (volume ratio 1:1), then 7.5 equivalents of PMe ₃ (1.0M in THF) was slowly added at room temperature, and the reaction was continued to stir until LC-MS analysis It shows that the starting material is completely converted (the azide group is completely converted into an amino group), and is subsequently purified by RP-C18 column chromatography (eluent: methanol and water) to obtain an amino-containing intermediate. Re-dissolve the intermediate with a mixed solvent of pyridine and triethylamine (volume ratio 1:1), add 15 equivalents of NMe ₃ ·SO ₃ at room temperature, and continue stirring for 5 hours until LC-MS analysis shows that the raw material has been completely converted. . The reaction mixture solution was concentrated under reduced pressure, exchanged with SCR732 sodium ion exchange resin (eluent: methanol), and purified by RP-C18 column chromatography (eluent: methanol and water) to obtain the N-sulfonated intermediate. Subsequently, the intermediate was dissolved in a mixed solvent of tert-butyl alcohol and water (volume ratio 1:1), palladium hydroxide/carbon was added, and the mixture was degassed and stirred at room temperature under a hydrogen atmosphere until mass spectrometry analysis showed that the hydrogenation reaction was complete. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to remove the solvent. The crude product obtained was purified by size exclusion chromatography, polyacrylamide P4 (Bio-gel P-4) (eluent: 0.1M NH ₄ HCO ₃ ). , the fractions containing the product were collected and freeze-dried, and finally the heparin pentasaccharide derivative 3 modified with the reducing terminal pentamino linker arm was obtained in a yield of 70%.

¹ H NMR (500MHz, D ₂ O): δ5.62 (d, J = 3.7 Hz, 1H, H-1-GlcN3), 5.57 (d, J = 3.5 Hz, 1H, H-1-GlcN2), 5.28 (d,J＝3.6Hz,1H,H-1-IdoA),5.18(d,J＝3.7Hz,1H,H-1-GlcN1),4.98(d,J＝3.0Hz,1H,H-5- IdoA),4.69(d,J＝7.9Hz,1H,H-1-GlcA),4.52-4.49(m,1H,H-6-GlcN),4.42-4.31(m,6H,H-3-GlcN2, H-2-IdoA,4 x H-6-GlcN),4.25-4.19(m,3H,including H-6-GlcN,H-4-IdoA),4.12-3.98(m,4H),3.94-3.84( m,3H),3.81-3.71(m,3H,including H-3-GlcN1,O CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH ₂ ),3.67-3.59(m,3H,including H-3-GlcN3, O CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH ₂ ),3.51-3.45(m,2H,H-2-GlcN2,H-2-GlcA),3.34-3.29(m,2H,H-2-GlcN3, H-2-GlcN1),3.09-3.06(m,2H,OCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH ₂ ),1.82-1.66(m,4H,OCH ₂ CH ₂ _CH ₂ CH 2 CH ₂ NH ₂ ) ,1.57-1.49(m,2H,OCH ₂ CH 2 CH ₂ _CH ₂ CH ₂ NH ₂ ).HRMS(ESI):[M-2H] ^2- m/z calcd for C ₃₅ H ₆₀ N ₄ O ₄₉ S ₈ ,788.0046; found,788.0048.

Example 2

Heparin pentasaccharide derivative 4 modified with reducing terminal thiol functional group linker arm

Dissolve the heparin pentasaccharide 3 modified with the reducing terminal pentylamino linker arm and five times the equivalent of 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide) ester in dry dimethyl sulfoxide (DMSO), add triethylamine to adjust the reaction solution to a slightly alkaline state (pH 7-9), and stir the reaction at room temperature under an argon atmosphere. After 30 minutes of reaction, measure the pH value of the reaction solution again to ensure that it is in a slightly alkaline state, and stir at room temperature for 8 hours. After mass spectrometry monitors the complete conversion of the raw materials, add 10 units of dithiothreitol (DTT) to the reaction solution, stir at room temperature for 3 hours, and then freeze-dry to obtain the crude product. The obtained crude product was separated and purified by size exclusion chromatography polyacrylamide P2 (Bio-gel P-2) (eluent: 0.1M NH ₄ HCO ₃ ). The fractions containing the product were collected and freeze-dried, with a yield of 98%. Heparin pentasaccharide derivative 4 modified with a connecting arm modified by a reducing terminal thiol functional group was obtained.

ESI-MS: [M-2H] ^2- C ₃₈ H ₆₄ N ₄ O ₅₀ S ₉ ^2- , theoretical value: m/z=832.01, actual value: 831.80.

Example 3

Low molecular weight heparin mimetic 2 of heparin pentasaccharide dimer

The heparin pentasaccharide derivative 4 (20 equivalents) modified with the connecting arm modified by the reducing terminal thiol functional group and the connecting arm modified with the bismaleimide group (1 equivalent) were dissolved in a degassed mixed solvent (100 mM PBS buffer, (The volume ratio of dioxane to acetonitrile is 4:1:1), add sodium hydroxide solution to adjust the pH to 7.5-9.0, and stir at room temperature under an argon atmosphere for 24 hours. Subsequently, 40 equivalents of dithiothreitol was added to the reaction solution, and after stirring at room temperature for 3 hours, it was purified by size exclusion chromatography polyacrylamide P4 (Bio-gel P-4) (eluent: 0.1M NH ₄ HCO ₃ ), the fractions containing the product were collected and lyophilized to obtain heparin pentasaccharide dimerized low molecular weight heparin analog 2 with a yield of 65%.

¹ H NMR (600MHz, D ₂ O): δ5.56 (d, J = 3.7 Hz, 2H, 2 x H-1-GlcN3), 5.52 (d, J = 2.6 Hz, 2H, 2 x H-1- GlcN2),5.16-5.11(m,4H,2 x H-1-GlcN1,2 x H-1-IdoA),4.94-4.89(m,2H,2 x H-5-IdoA),4.61(d,J ＝7.8Hz,2H,2 x H-1-GlcA),4.45(d,J＝10.8Hz,2H,2 x H-6-GlcN),4.42-4.20(m,15H),4.20-4.10(m, 6H),4.09-3.92(m,7H,include 2 x S CH CO),3.92-3.78(m,8H),3.77-3.48(m,13H),3.48-3.11(m,10H,2 x H-2 -GlcN3,2 x H-2-GlcN2,2 x H-2-GlcN1,2 x H-2-IdoA,2 x H-2-GlcA),3.09-2.74(m,6H),2.73-2.56 (m,5H),2.56-2.38(m,7H),1.72-1.57(m,4H),1.56-1.48(m,4H),1.47-1.33(m,4H).

Example 4

Heparin pentasaccharide trimerized low molecular weight heparin mimetic 1

Dissolve the heparin pentasaccharide derivative 4 (30 equivalents) modified with the reducing terminal thiol functional group linker arm and the trimaleimide group-modified linker arm (1 equivalent) in a degassed mixed solvent (100mM PBS buffer, (The volume ratio of dioxane to acetonitrile is 4:1:1), add sodium hydroxide solution to adjust the pH to 7.5-9.0, and stir at room temperature under an argon atmosphere for 24 hours. Subsequently, 60 equivalents of dithiothreitol was added to the reaction solution, and after stirring at room temperature for 3 hours, it was separated and purified by size exclusion chromatography polyacrylamide P6 (Bio-gel P-6) (eluent: 0.1M NH ₄ HCO ₃ ), the fractions containing the product were collected and lyophilized to obtain heparin pentasaccharide trimerized low molecular weight heparin mimetic 1 with a yield of 57%.

¹ H NMR (800MHz, D ₂ O): δ5.60-5.54(m,6H,3 x H-1-GlcN3,3 x H-1-GlcN2),5.25-5.10(m,6H,3 x H- 1-GlcN1,3 x H-1-IdoA),4.98-4.88(m,3H,3 x H-5-IdoA),4.68-4.59(m,3H,3 x H-1-GlcA),4.47-4.07 (m,31H),4.06-3.79(m,20H),3.78-3.50(m,29H),3.49-3.16(m,34H,including 3 x H-2-GlcN3,3 x H-2-GlcN2,3 x H-2-GlcN1,3 x H-2-IdoA,3 x H-2-GlcA),3.11-2.77(m,10H),2.73-2.66(m,4H),2.64-2.58(m,3H) ,2.58-2.53(m,3H),2.52-2.47(m,3H),2.46-2.42(m,3H),1.75-1.60(m,6H),1.58-1.50(m,6H),1.46-1.36( s,6H),0.85(s,3H,- CH ₃ ).

Example 5

Anti-factor Xa activity test

The synthesized dimers and trimers of heparin pentasaccharide and the reference low molecular weight heparin (enoxaparin) were diluted into a series of concentration gradients (0–1000ng/mL) with Tris-EDTA-NaCl-BSA (pH 8.4) buffer. )The solution. Add 40 μL of each of the above compound solutions to a 96-well plate, and add antithrombin ATIII (40 μL, 1IU/mL), mix and incubate at 37°C for 2 minutes. Subsequently, coagulation factor Xa (FXa, 40 μL, 1 IU/mL) was added and incubated at 37°C for 2 minutes. Then add the chromogenic substrate CS-11(65) of factor at Measure the absorbance and calculate the activity of the factor at different compound concentrations:
FXa (%) = (OD _{plus drug group} - OD _{plus blank} ) / (OD _{no medicine control} - OD _{plus blank} )

Excel draws the relationship curve between FXa activity and compound concentration, and analyzes it through the four-parameter fitting algorithm in nonlinear regression. The IC ₅₀ values of heparin pentasaccharide dimer and trimer and enoxaparin are obtained to evaluate the dimerized low molecular weight. Anti-factor Xa activity of heparin mimetics and trimeric low molecular weight heparin mimetics.

The concentration IC ₅₀ of the compound when the FXa activity is 50% was calculated through four-parameter algorithm fitting.

Figure 1 shows that the low-weight heparin mimetic of heparin pentasaccharide dimer (Compound 2) and trimer (Compound 1) is slightly better than the control group enoxaparin in anti-factor Xa activity.

Example 6

Protamine Neutralization Experimental Test

The synthetic low-weight heparin of heparin pentasaccharide dimer and trimer and the reference substances enoxaparin and fondaparinux were diluted into a 5000ng/mL solution with Tris-EDTA-NaCl-BSA (pH 8.4) buffer. , use Tris-EDTA-NaCl-BSA (pH 8.4) buffer to prepare a series of concentration gradients of protamine sulfate (0–200 μg/mL). Add 40 μL of each of the above compound solutions to a 96-well plate, and add 20 μL of protamine sulfate with the above concentration gradient, mix well, and incubate at 37°C for 10 minutes. Add antithrombin ATIII (40 μL, 1IU/mL), and mix. After homogenization, incubate at 37°C for 2 minutes. Coagulation factor Xa (40 μL, 1 IU/mL) was then added and incubated at 37°C for 2 minutes. Then add the chromogenic substrate CS-11(65) of factor Measure the absorbance and calculate the activity of factor Xa at different compound concentrations:
FXa (%) = (OD _{plus drug group} - OD _{plus blank} ) / (OD _{no medicine control} - OD _{plus blank} )

Excel draws the relationship curve between FXa activity and compound concentration to evaluate the effect of protamine neutralizing heparin pentasaccharide polymer on inhibiting FXa.

Figure 2 shows the results of the in vitro protamine neutralizing activity study of heparin pentasaccharide dimer and trimer. Compared with enoxaparin, heparin pentasaccharide dimer 2 exhibits a weaker neutralizing effect, while Heparin pentasaccharide trimer 1 is completely neutralized by protamine.

All documents mentioned in this application are incorporated by reference in this application to the same extent as if each individual document was individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

A heparin pentasaccharide dimer having the following structure:
A heparin pentasaccharide trimer with the following structure:
The preparation method of heparin pentasaccharide dimer as claimed in claim 1, characterized in that the preparation method includes the following steps:

After the heparin pentasaccharide derivative 4 reacts with the connecting arm modified with a bismaleimide group, dithiothreitol is added to react to obtain the heparin pentasaccharide dimer.
The preparation method of heparin pentasaccharide trimer as claimed in claim 2, characterized in that,

After the reaction of heparin pentasaccharide derivative 4 with the trimaleimide group-modified connecting arm, dithiothreitol was added The reaction yields the heparin pentasaccharide trimer.
The preparation method according to claim 3 or 4, characterized in that compound 4 is synthesized through the following steps:

After the reaction of heparin pentasaccharide 3 modified with the pentylamino connecting arm at the reducing end and 3,3'-dithiodipropionic acid bis(N-hydroxysuccinimide) ester, dithiothreitol was added to react to obtain heparin pentasaccharide 3. Sugar derivatives 4.
The preparation method according to claim 5, characterized in that the heparin pentasaccharide 3 modified by the reducing terminal pentamino linker arm is synthesized through the following steps:

i) Compound 5 reacts with PMe 3 to completely convert the azide group into an amino group to obtain an amino-containing intermediate;

ii) The amino-containing intermediate reacts with NMe 3 . SO 3 to obtain an N-sulfonated intermediate;

iii) The N-sulfonated intermediate undergoes hydrogenation to obtain the heparin pentasaccharide intermediate 3.
A pharmaceutical composition, characterized in that it contains:

The heparin pentasaccharide dimer according to claim 1 or the heparin pentasaccharide trimer according to claim 2; and a pharmaceutically acceptable carrier.
The use of the heparin pentasaccharide dimer as claimed in claim 1 or the heparin pentasaccharide trimer as claimed in claim 2 is used to prepare anticoagulant/antithrombotic drugs.
The use of the heparin pentasaccharide dimer according to claim 1 or the heparin pentasaccharide trimer according to claim 2 is characterized in that it is used to prepare drugs for preventing and treating venous thrombosis.
The use of the heparin pentasaccharide dimer according to claim 1 or the heparin pentasaccharide trimer according to claim 2 is used to prepare factor Xa inhibitors.