Preparation of new intermediate of heparin pentasaccharide and preparation method thereof

Abstract

The present invention relates to a process of a chemically synthetic drug, and in particular, to a new intermediate of a heparin pentasaccharide and a preparation method thereof. The process has high reaction efficiency, and an easy reaction operation. The reaction intermediate is easy to be purified, and is appropriate for an industrialization production.

Claims

1. A compound having the following formula I, which can be used as an intermediate for the preparation of heparin pentasaccharide, ##STR00018## wherein the configuration of monosaccharide units and the stereochemistry of the connecting bond among each of the monosaccharides is D-glucosyl--1,4-D-glucuronosyl--1,4-D-glucosyl--1,4-L-idoronosyl--1,4-D-glucose, wherein the definition of each substituent is as follows: R represents C.sub.1-C.sub.20 alkyl or substituted alkyl, wherein the alkyl can be either linear alkyl or branched or cyclic alkyl; R.sub.4 represents benzyl or substituted benzyl, and R.sub.4 in the same molecular formula represents either identical group or different group; R.sub.5 represents hydrogen ion or alkali metal ion.

2. The compound according to claim 1, characterized in that R is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, benzyl, or cyclohexyl.

3. The compound according to claim 1, characterized in that R is methyl; R.sub.4 is benzyl or substituted benzyl; and R.sub.5 is hydrogen ion or sodium ion.

4. A compound having the following formula II, which can be used as an intermediate for the preparation of heparin pentasaccharide ##STR00019## wherein the configuration of monosaccharide units and the stereochemistry of the connecting bond among each of the monosaccharides is D-glucosyl--1,4-D-glucuronosyl--1,4-D-glucosyl--1,4-L-idoronosyl--1,4-D-glucose, wherein the definition of each substituent is as follows: R represents C.sub.1-C.sub.20 alkyl or substituted alkyl, wherein the alkyl can be either linear alkyl or branched or cyclic alkyl; R.sub.4 represents benzyl or substituted benzyl, and R.sub.4 in the same molecular formula represents either identical group or different group.

5. The compound according to claim 4, characterized in that R is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, benzyl, or cyclohexyl.

6. The compound according to claim 4, characterized in that R is methyl; and R.sub.4 is benzyl or substituted benzyl.

7. A process for the preparation of the compound of formula I according to claim 1, characterized in that the process comprises the following steps: a) firstly, treating the fully protected pentasaccharide compound of the following formula III with sodium hydroxide, ##STR00020## such that the compound of formula III is subjected to ester hydrolysis, in order to obtain the compound having the following formula IV wherein R.sub.5 is sodium ion, alternatively, the compound is subjected to hydrolysis and subsequent acid neutralization, in order to obtain the compound having the following formula IV wherein R.sub.5 is hydrogen ion, ##STR00021## wherein R represents C.sub.1-C.sub.20 alkyl or substituted alkyl, wherein the alkyl can be either linear alkyl or branched or cyclic alkyl; R.sub.1 represents C.sub.1-C.sub.20 alkyl, substituted alkyl, hydrogen ion, sodium ion, wherein the alkyl can be either linear alkyl or branched or cyclic alkyl, R.sub.1 in the same molecular formula represents either identical group or different group; R.sub.2 represents linear or branched aliphatic acyl or aryl acyl, R.sub.2 in the same molecular formula represents either identical group or different group; R.sub.3 represents azido group; R.sub.4 represents benzyl or substituted benzyl, R.sub.4 in the same molecular formula represents either identical group or different group; R.sub.5 is hydrogen ion or sodium ion; b) secondly, treating the compound of formula IV with a reductant, i.e. the group R.sub.3 is reduced to amino group, in order to obtain the compound of formula I.

8. The process according to claim 7, characterized in that R.sub.2 in the compound of formula III is selected from acetyl, 4-oxovaleryl, and benzoyl; and wherein the acid is preferably hydrochloric acid.

9. The process according to claim 7, wherein the compound of formula IV is subjected to reduction after separation and purification.

10. A process for the preparation of the compound according to claim 1 having the following formula I: ##STR00022## comprising the following steps: a) firstly, treating the fully protected pentasaccharide compound of formula III with a reductant, ##STR00023## reducing R.sub.3 group into amino group, in order to obtain the compound of the following formula V, ##STR00024## wherein R represents C.sub.1-C.sub.20 alkyl or substituted alkyl, wherein the alkyl can be either linear alkyl or branched or cyclic alkyl; R.sub.1 represents C.sub.1-C.sub.20 alkyl, substituted alkyl, hydrogen ion, sodium ion, wherein the alkyl can be either linear alkyl or branched or cyclic alkyl, R.sub.1 in the same molecular formula represents either identical group or different group; R.sub.2 represents linear or branched aliphatic acyl or aryl acyl, R.sub.2 in the same molecular formula represents either identical group or different group; R.sub.4 represents benzyl or substituted benzyl, R.sub.4 in the same molecular formula represents either identical group or different group; b) secondly, treating the compound of formula V with sodium hydroxide, such that the compound of formula V is subjected to ester hydrolysis, in order to obtain the compound of formula I wherein R.sub.5 is sodium ion, alternatively, it is subjected to ester hydrolysis and subsequent acid neutralization, in order to obtain the compound of formula I wherein R.sub.5 is hydrogen ion.

11. The process according to claim 10, characterized in that R.sub.2 in the compound of formula V is selected from acetyl, 4-oxovaleryl, and benzoyl; and wherein the acid is preferably hydrochloric acid.

12. The process according to claim 7, characterized in that the reductant is selected from trimethylphosphine, triethylphosphine, tributylphosphine, triphenylphosphine, or hydrogen sulfide.

13. A process for the preparation of the compound of formula II: ##STR00025## characterized in that treating the compound having the following formula I: ##STR00026## with a sulfating reagent, and treating the obtained crude product with sodium ion exchange resin, in order to obtain the compound of formula II; wherein the sulfating reaction can be carried out in a single step or in several steps.

14. The process according to claim 13, characterized in that the sulfating agent is selected from sulfur trioxide-pyridine complex, sulfur trioxide-trimethylamine complex, sulfur trioxide-triethylamine complex, or gaseous sulfur trioxide.

15. A process for the preparation of heparin pentasaccharide compound of formula VI, characterized in that treating the compound according to claim 4 having the following formula II: ##STR00027## with a catalyst, palladium hydroxide or palladium on carbon, under hydrogen condition, such that all R.sub.4 groups of the compound of formula II are removed, in order to obtain heparin pentasaccharide compound according to formula VI ##STR00028##

16. The process according to claim 7, wherein the heparin pentasaccharide is fondaparinux sodium.

17. The process according to claim 10, characterized in that the reductant is selected from trimethylphosphine, triethylphosphine, tributylphosphine, triphenylphosphine, or hydrogen sulfide.

18. The process according to claim 13, wherein the heparin pentasaccharide is fondaparinux sodium.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 is the .sup.1H NMR of the compound of formula II-1 prepared according to Example 5.

(2) FIG. 2 is the HSQC NMR of the compound of formula II-1 prepared according to Example 5.

(3) FIG. 3 is the .sup.1H NMR of fondaparinux sodium prepared according to Example 7.

EXAMPLES

Example 1

Preparation of methyl O-(2-azido-3,4-di-O-benzyl-2-deoxy--D-glucopyranosyl)-(14)-O-(2,3-di-O-benzyl--D-glucopyranuronosyl)-(14)-O-(2-azido-2-deoxy--D-glucopyranosyl)-(14)-O-(3-O-benzyl--L-idopyranuronosyl)-(14)-2-azido-3-O-benzyl-2-deoxy--D-glucopyranoside (IV-1)

(4) ##STR00011##

(5) Fully protected pentascharride compound of formula III-1 (10 g, 5.4 mmol) was dissolved in tetrahydrofuran (220 mL). Aqueous solution of sodium hydroxide (110 mL, 1.0M, 20 eq., 110 mmol) was added dropwise. After the addition was completed, it was stirred overnight at room temperature, until the reaction was complete. It was then neutralized with 1 M hydrochloric acid and extracted with ethyl acetate. The organic phases were combined and were successively washed with water, 10% citric acid, saturated saline solution, and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to remove the solvent, in order to obtain 8.0 g compound of formula IV-1, in the form of foamy solid.

(6) ESMS: m/Z=1486 [M+1].sup.+, 1484 [M1].sup..

Example 2

Preparation of methyl O-(2-amino-3,4-di-O-benzyl-2-deoxy--D-glucopyranosyl)-(14)-O-(2,3-di-O-benzyl--D-glucopyranuronosyl)-(14)-O-(2-amino-2-deoxy--D-glucopyranosyl)-(14)-O-(3-O-benzyl--L-idopyranuronosyl)-(14)-2-amino-3-O-benzyl-2-deoxy--D-glucopyranoside (I-1)

(7) ##STR00012##

(8) Under the protection of nitrogen, the pentasccharide compound of formula IV-I (8.0 g) was dissolved in tetrahydrofuran (400 mL), aqueous solution of sodium hydroxide (32 mL, 1.0 M) was added, a solution of trimethylphosphine in tetrahydrofuran (54 mL, 1 M) was added dropwise in ice bath, and then the temperature was slowly raised to room temperature. It was stirred overnight, until the reaction is complete. Dilute hydrochloric acid was added for neutralization, until the pH reaches about 7. It was concentrated under reduced pressure to remove the solvent, in order to obtain 8.8 g compound of formula I-1, in the form of foamy solid. The crude product was subjected to the next reaction without further purification.

(9) HPLC purity: 90%.

(10) ESMS: m/z 1408 [M+1].sup.+, 1406 [M1].sup..

Example 3

Preparation of methyl O-(2-amino-3,4-di-O-benzyl-2-deoxy--D-glucopyranosyl)-(14)-O-(2,3-di-O-benzyl--D-glucopyranuronosyl)-(14)-O-(2-amino-2-deoxy--D-glucopyranosyl)-(14)-O-(3-O-benzyl--L-idopyranuronosyl)-(14)-2-amino-3-O-benzyl-2-deoxy--D-glucopyranoside (I-1)

(11) ##STR00013##

(12) Under the protection of nitrogen, the pentasccharide compound of formula IV-I (4.0 g) was dissolved in tetrahydrofuran (200 mL), aqueous solution of sodium hydroxide (16 mL, 1.0 M) was added, triphenylphosphine (7.1 g) was added in ice bath, and then the temperature was slowly raised to room temperature. It was stirred overnight, until the reaction is complete. Dilute hydrochloric acid was added for neutralization, until the pH reaches about 7. It was concentrated under reduced pressure to remove the solvent, in order to obtain 11.4 g crude compound of formula I-1, in the form of foamy solid. The crude product was subjected to the next reaction without further purification.

(13) ESMS: m/z 1408 [M+1].sup.+, 1406 [M1].sup..

Example 4

Preparation of methyl O-(2-amino-3,4-di-O-benzyl-2-deoxy--D-glucopyranosyl)-(14)-O-(2,3-di-O-benzyl--D-glucopyranuronosyl)-(14)-O-(2-amino-2-deoxy-D-glucopyranosyl)-(14)-O-(3-O-benzyl--L-idopyranuronosyl)-(14)-2-amino-3-O-benzyl-2-deoxy--D-glucopyranoside (I-1)

(14) ##STR00014##

(15) Under the protection of nitrogen, the pentasccharide compound of formula III-I (5.0 g, 2.7 mmol) was dissolved in tetrahydrofuran (200 mL), water (20 mL) was added, a solution of trimethylphosphine in tetrahydrofuran (27 mL, 1 M) was added dropwise in ice bath, and then the temperature was slowly raised to room temperature. It was stirred overnight, until the reaction is complete. Aqueous solution of sodium hydroxide (55 mL, 1.0 M) was added dropwise, and it was stirred at room temperature until the reaction was complete. Dilute hydrochloric acid was added for neutralization, until the pH reaches about 7. It was concentrated under reduced pressure to remove the solvent, in order to obtain 8.4 g crude compound of formula I-1, in the form of foamy solid. The crude product was subjected to the next reaction without further purification.

(16) HPLC purity: 87%.

(17) ESMS: m/z 1408 [M+1].sup.+, 1406 [M1].sup..

Example 5

Preparation of methyl O-(3,4-di-O-benzyl-2-deoxy-6-O-sulfo-2-sulfoamino--D-glucopyranosyl)-(14)-O-(2,3-di-O-benzyl--D-glucopyranuronosyl)-(14)-O-(2-deoxy-3,6-di-O-sulfo-2-sulfoamino--D-glucopyranosyl)-(14)-O-(3-O-benzyl-2-O-sulfo--L-idopyranuronosyl)-(14)-3-O-benzyl-2-deoxy-6-O-sulfo-2-sulfamino--D-glucopyranoside, decasodium salt (II-1)

(18) ##STR00015##

(19) Under the protection of nitrogen, the pentasccharide compound of formula I-I (8.8 g) was dissolved in the mixed solvent of pyridine (50 mL) and triethylamine (10 mL), sulfur trioxide-pyridine complex (10 g, 63 mmol) was added. It was stirred overnight at room temperature, until the reaction was complete. Saturated aqueous solution of sodium hydrogen carbonate was added in order to quench the reaction. It was concentrated under reduced pressure to remove the solvent, to obtain a red brown oily product. It was successively purified with ion exchange column and sephadex LH-20 column chromatography. The products were combined and the solvent was removed under reduced pressure, and then dried to obtain 8.2 g compound of formula II-1, in the form of foamy solid. Its .sup.1H NMR spectrum is as shown in FIG. 1, and HSQC NMR is as shown in FIG. 2.

(20) ESMS: m/z=1055.5 [M7Na+5H].sup.2/2, 688.9 [M9Na+6H].sup.3/3.

(21) .sup.1H NMR (400 MHz, D.sub.2O): (selected for anomeric H) 5.59 (d, J=2.8 Hz, 1H), 5.37 (s, 1H), 5.27 (d, J=7.6 Hz, 1H), 4.93 (according to HSQC NMR), 4.72 (according to HSQC NMR).

Example 6

Preparation of methyl O-(3,4-di-O-benzyl-2-deoxy-6-O-sulfo-2-sulfoamino--D-glucopyranosyl)-(14)-O-(2,3-di-O-benzyl--D-glucopyranuronosyl)-(14)-O-(2-deoxy-3,6-di-O-sulfo-2-sulfoamino--D-glucopyranosyl)-(14)-O-(3-O-benzyl-2-O-sulfo--L-idopyranuronosyl)-(14)-O-benzyl-2-deoxy-6-O-sulfo-2-sulfamino--D-glucopyranoside, decasodium salt (II-1)

(22) ##STR00016##

(23) Under the protection of nitrogen, the pentasccharide compound of formula I-I (3.0 g) was dissolved in N,N-Dimethylformamide (100 mL), sulfur trioxide-trimethylamine complex (7.2 g) was added. It was heated to 50 C., until the reaction was complete. Saturated aqueous solution of sodium hydrogen carbonate was added in order to quench the reaction. It was concentrated under reduced pressure to remove the solvent, to obtain a red brown oily product. It was successively purified with ion exchange column and sephadex LH-20 column chromatography. The parts containing sacchrides were combined and the solvent was removed under reduced pressure, and then dried to obtain 2.7 g foamy solid. The foamy solid was dissolved in the mixed solvent of pyridine (50 mL) and triethylamine (10 mL), and sulfur trioxide-pyridine complex (5.1 g) was added. It was stirred overnight at room temperature, until the reaction was complete. Saturated aqueous solution of sodium hydrogen carbonate was added in order to quench the reaction. It was concentrated under reduced pressure to remove the solvent, to obtain a red brown oily product. It was successively purified with ion exchange column and sephadex LH-20 column chromatography, and qualified samples were collected. They were combined and concentrated under reduced pressure to remove the solvent, and were dried to obtain 2.6 g compound of formula II-1, in the form of foamy solid.

Example 7

Preparation of methyl O-(2-deoxy-6-O-sulfo-2-sulfoamino--D-glucopyranosyl)-(14)-O-(-D-glucopyranuronosyl)-(14)-O-(2-deoxy-3,6-di-O-sulfo-2-sulfoamino--D-glucopyranosyl)-(14)-O-(2-O-sulfo--L-idopyranuronosyl)-(14)-2-deoxy-6-O-sulfo-2-sulfoamino--D-glucopyranoside, decasodium salt (VI-1, Fondaparinux sodium)

(24) ##STR00017##

(25) 8.2 g pentasaccharide of formula II-1 was dissolved in 150 ml mixed solvent of methanol and water (1:1, v/v), 3 g palladium on carbon (10%) was added. The reaction was carried out under hydrogen pressure of 70 psi, until the reaction was complete. The palladium on carbon catalyst was removed by filtration, and the solvent was removed under reduced pressure. It was successively purified with anion exchange column and sephadex G25, in order to obtain 6.4 g final product. Its .sup.1H NMR was as shown in FIG. 3.

(26) ESMS: m/z=1727.7 [M+H].sup.+, 1749.7 [M+Na].sup.+.

(27) .sup.1H NMR (400 MHz, D.sub.2O): (selected for anomeric H) 5.61 (d, J=3.2 Hz, 1H), 5.51 (d, J=2.4 Hz, 1H), 5.19 (d, J=2.4 Hz, 1H), 5.02 (d, J=3.2 Hz, 1H), 4.62 (d, J=7.6 Hz, 1H).

(28) Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.