COMPOSITION CONTAINING NITROGEN HETEROCYCLIC HEXAPEPTIDE PRECURSOR AND PREPARATION METHOD AND APPLICATION THEREOF

Abstract

Disclosed is a preparation method of a composition containing a nitrogen heterocyclic hexapeptide precursor, composition A and B containing a nitrogen heterocyclic hexapeptide precursor of a compound having formula I and formula II obtained by the method, and an application of the composition used for preparing a compound having formula III.

##STR00001##

Claims

1. A process for preparing a composition comprising a compound of formula I and a compound of formula II, comprising steps of: (a) dissolving the crude compound of formula I in an aqueous solution of an organic solvent (i); (b) precipitating solids from the solution by cooling and/or adding an organic solvent (ii) to obtain a suspension containing the compound of formula I; (C) obtaining composition A containing the compound of formula I and the compound of formula II by centrifugation or filtration; ##STR00057##

2. The process of claim 1, wherein, in step (a), the volume percentage of water in the aqueous solution of organic solvent (i) is from 8% to 30%, preferably from 10% to 25%, more preferably from 12% to 22%, and most preferably from 16% to 22%.

3. The process of claim 1, wherein, in step (a), the organic solvent (i) is selected from one or more of methanol, ethanol, n-propanol, isopropanol, isobutanol, n-butanol, and acetone.

4. The process of claim 1, wherein, in step (b), the organic solvent (ii) is selected from one or more of C.sub.3-7 ester, hexane, n-heptane, n-pentane, dichloromethane; and preferably from one or more of ethyl acetate, isopropyl acetate, n-hexane.

5. The process of claim 1, wherein steps (a)-(c) may be repeated one or more times.

6. A suspension containing the compound of formula I obtained by the process of any one of claims 1-4, wherein the diameter of the solid particles in the suspension is greater than or equal to 0.1 μm, preferably greater than or equal to 0.2 μm, more preferably greater than or equal to 0.3 μm.

7. Composition A comprising a compound of formula I and a compound of formula II obtained by the process of any one of claims 1-5, wherein the composition comprises from 1% to 40%, preferably 5% to 35%, more preferably 10-30% of an organic solvent (mass percent) and 1% to 15%, preferably 2% to 12%, more preferably 3% to 10% of water (mass percent); the organic solvent is selected from a group consisting of methanol, ethanol, n-propanol, isopropanol, isobutanol, n-butanol, acetone, ethyl acetate, isopropyl acetate, n-hexane, dichloromethane.

8. Composition A of claim 7, wherein, in composition A, the dry content of the compound of formula I is more than 95%, and the dry content of the compound of formula II is from 0.0001% to 2.0%; preferably from 0.0001% to 0.49%; more preferably from 0.0001% to 0.2%; and most preferably from 0.0001% to 0.1%.

9. Composition A of claim 8, wherein the dry content of each component in the composition is determined by HPLC assay.

10. Composition A of claim 9, wherein the HPLC assay is: Chromatographic Column: Symmtry C18 3.5 um 2.1×150 mm; Mobile phase: acetonitrile/water=39/61; Flow rate: 0.4 ml/min; Column temperature: 30° C.; Sample diluent: acetonitrile/water=39/61; Injection temperature: 5° C.; Detection wavelength: 205 nm.

11. The process of claim 1, wherein, after step (c), step (d) is further included: (d) composition A obtained in step (c) is further dried to obtain a dry composition B.

12. The process of claim 11, wherein the moisture content of composition B is less than or equal to 5%.

13. The process of claim 11, wherein, in the dry composition B, the dry content of the compound of formula I is more than 95%; and the dry content of the compound of formula II is from 0.0001% to 2.0%, preferably from 0.0001% to 0.49%, more preferably from 0.0001% to 0.2%, and most preferably from 0.0001% to 0.1%.

14. Composition B comprising a compound of formula I and a compound of formula II; ##STR00058## in the composition, the dry content of the compound of formula I is more than 95%; and in the composition, the dry content of the compound of formula II is from 0.0001% to 2.0%, preferably from 0.0001% to 0.49%, more preferably from 0.0001% to 0.2%, and most preferably from 0.0001% to 0.1%.

15. Composition B of claim 14, wherein the dry content of each component in the composition is determined by HPLC assay.

16. Composition B of claim 15, wherein the HPLC assay is: Chromatographic Column: Symmtry C18 3.5 um 2.1×150 mm; Mobile phase: acetonitrile/water=39/61; Flow rate: 0.4 ml/min; Column temperature: 30° C.; Sample diluent: acetonitrile/water=39/61; Injection temperature: 5° C.; Detection wavelength: 205 nm.

17. Use of composition B of any one of claims 14-16 for preparing a compound of formula X; TABLE-US-00005 Formula X Compound X R X (a = O, b = 2H) X1 a b X2 a b X3 a b X4 a b X5 a b X6 a b X7 a b X8 a b X9 a b X10 a b X11 a b X12 a b.

18. A composition comprising a compound of formula X and a compound of formula XI; TABLE-US-00006 Formula X Formula XI Compound X Compound XI R X (a = O, b = 2H) X1 XI1 a b X2 XI2 a b X3 XI3 a b X4 XI4 a b X5 XI5 a b X6 XI6 a b X7 XI7 a b X8 XI8 a b X9 XI9 a b X10 XI10 a b X11 XI11 a b X12 XI12 a b.

19. The composition of claim 18, wherein the content of the compound of formula X in the composition is more than 95%.

20. The composition of claim 18, wherein the HPLC content of the compound of formula XI in the composition is from 0.0001% to 2.0%, preferably from 0.0001% to 0.49%, more preferably from 0.0001% to 0.2%, most preferably from 0.0001% to 0.1%.

21. Use of the composition of any one of claims 18-20 for preparing a compound of formula III; ##STR00086##

22. A compound of formula III, wherein the compound of formula III is prepared from Composition A prepared by the process of claim 1 or from Composition B prepared by the process of claim 11 as a raw material; ##STR00087##

Description

DESCRIPTION OF DRAWINGS

[0084] FIG. 1 shows a HPLC chromatogram of the crude compound of formula I obtained in Example 1.

[0085] FIG. 2 shows a HPLC chromatogram of composition A10 obtained in Example 6.

[0086] FIG. 3 shows a HPLC chromatogram of the compound of formula X2a obtained in Example 10.

[0087] FIG. 4 shows a HPLC chromatogram of the compound of formula III obtained from Example 18 (caspofungin).

MODES FOR CARRYING OUT THE INVENTION

[0088] The invention will now be further described with reference to specific embodiments. It is to be understood that these examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods not specified in the following examples are generally prescribed in accordance with conventional conditions or in accordance with the conditions recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

[0089] Unless otherwise defined, all professional and scientific terms used herein are of the same meaning as those skilled in the art. In addition, any method and material similar to or equivalent to the described contents may be applied to the method of the present invention. The preferred embodiments and materials described herein are for exemplary purposes only.

[0090] The contents of Pneumocandin B0 and serine analogs are analyzed using the following HPLC assay:

[0091] Chromatographic Column: Waters Symmtry C18 3.5 um 2.1×150 mm;

[0092] Mobile phase: acetonitrile/water=39/61;

[0093] Flow rate: 0.4 ml/min;

[0094] Column temperature: 30° C.;

[0095] Sample diluent: acetonitrile/water=39/61;

[0096] Injection temperature: 5° C.;

[0097] Detection wavelength: 205 nm.

[0098] The contents of caspofungin intermediate and serine analogs thereof are analyzed using the following HPLC assay:

[0099] Chromatographic Column: YMC-Pack ODS-A 250×4.6 mm, 5 um;

[0100] Mobile phase A: 0.1% perchloric acid and 0.075% aqueous solution of sodium chloride;

[0101] Mobile phase B: acetonitrile;

[0102] Flow rate: about 1.5 ml/min;

[0103] Column temperature: 30° C.;

[0104] Detection wavelength: 220 nm;

[0105] Gradient procedure:

TABLE-US-00004 Time (min) % A % B 0 65.5 34.5 14.5 65.5 34.5 35 50 50 45 35 65 50 20 80 52 20 80 53 65.5 34.5 66 65.5 34.5

EXAMPLE 1

Preparation of Crude Compound of Formula I

[0106] According to WO2000/008197, a crude product containing the compound of formula I was obtained by the fermentation process of Glarea Lozoyensis (Zalerion arboricla).

[0107] According to WO2005026323, the above obtained crude product containing the compound of formula I was purified by chromatography and re-fined fractions were collected, so as to obtain 267 g of the compound of formula I. The content of the compound of formula II was determined as 3.1%.

EXAMPLE 2

Preparation of a Crude Product of the Compound of Formula I

[0108] According to “Pneumocandin B0 Production by Fermentation of the Fungus Glarea lozoyensis: Physiological and Engineering Factors Affecting Titer and Structural Analogue Formation”, the crude product of the compound of formula I was obtained by adding serine during fermentation.

[0109] According to WO2005026323, the above obtained crude product containing the compound of formula I was purified by chromatography and re-fined fractions were collected, so as to obtain 36 g of the compound of formula I. The content of the compound of formula II was determined as 8.0%.

EXAMPLE 3

Preparation of a Crude Product of Compound of Formula I

[0110] According to WO2005026323, threonine was added during fermentation to give a crude product of the compound of formula I. The above obtained crude product containing the compound of formula I was purified by chromatography and re-fined fractions were collected, so as to obtain 22 g of the compound of formula I. The content of the compound of formula II was determined as 0.5%.

EXAMPLE 4

[0111] 36 g of the crude product of the compound of formula I obtained in Example 1, in which the content of the compound of formula II was 3.1%, was dissolved in an isobutanol solution having a moisture content of 18% and a volume of 720 ml. 1440 ml of ethyl acetate was slowly added and stirred to obtain a suspension containing the compound of formula I. The diameter of solid particles of the compound of formula I in the suspension were determined as 0.3 um or higher using Malvern particle size meter 2600C. Composition A was obtained through solid-liquid separation by filtration, and the loss of the compound of formula I was 1.5%. The content of the compound of formula I in composition Al was determined as 98.6% and the content of compound of formula II was determined as 0.95% by HPLC.

[0112] The above-obtained composition Al (content of the compound of formula II was 0.95%) was divided into two portions. One portion was dried in vacuo to give 17.6 g of composition B1, the moisture content was determined as 4%, and the content of the compound of formula II was determined as 0.95%.

[0113] Another portion of composition Al was dissolved in an isobutanol solution having a moisture content of 12% in a volume of 240 ml. 460 ml of isopropyl acetate was slowly added and stirred to obtain a suspension containing the compound of formula I. The diameter of solid particles of the compound of formula I in the suspension were determined as 0.5 um or higher using Malvern particle size meter 2600C. Composition A2 was obtained through solid-liquid separation by filtration, and the loss of the compound of formula I in the liquid was 1.6%. The content of compound of formula II was determined as 0.38% by HPLC.

[0114] Composition A2 (in which the content of the compound of formula II was 0.38%) above-obtained by filtration was dissolved in an iso-butanol solution having a moisture content of 20% in a volume of 246 ml. 460 ml of isopropyl acetate was slowly added and stirred to obtain a suspension containing the compound of formula I. The diameter of solid particles of the compound of formula I in the suspension were determined as 0.2 um or higher using Malvern particle size meter 2600C. Composition A3 was obtained through solid-liquid separation by filtration, and the loss of the compound of formula I in the liquid was 1.2%. The content of compound of formula II was determined as 0.16% by HPLC.

[0115] Composition A3 (in which the content of the compound of formula II was 0.16%) above-obtained by filtration was dissolved in an iso-butanol solution having a moisture content of 15% in a volume of 206 ml. 410 ml of isopropyl acetate was slowly added and stirred to obtain a suspension containing the compound of formula I. The diameter of solid particles of the compound of formula I in the suspension were determined as 0.4 um or higher using Malvern particle size meter 2600C. Composition A4 was obtained through solid-liquid separation by filtration, and the loss of the compound of formula I in the liquid was 1.0%. The content of compound of formula II was determined as 0.09% by HPLC.

[0116] The above composition A4 was dried in vacuo to give 17 g of composition B2, the moisture content was determined as 2.4%, and the content of the compound of formula II was determined as 0.09%.

EXAMPLE 5

[0117] 6 g of the crude product of the compound of formula I obtained in Example 2, in which the content of the compound of formula II was 8%, was dissolved in an isobutanol solution having a moisture content of 8% and a volume of 150 ml. 250 ml of isopropyl acetate was slowly added and stirred to obtain a suspension containing the compound of formula I. The diameter of solid particles of the compound of formula I in the suspension were determined as 0.2 um or higher using Malvern particle size meter 2600C. Composition A5 was obtained through solid-liquid separation by filtration, and the loss of the compound of formula I in the liquid was 1.9%. The content of the compound of formula I in composition A5 was determined as 96.4% and the content of compound of formula II was determined as 3.1% by HPLC.

[0118] Composition A5 (in which the content of the compound of formula II was 3.1%) above-obtained by filtration was dissolved in an iso-butanol solution having a moisture content of 12% in a volume of 120 ml. 230 ml of isopropyl acetate was slowly added and stirred to obtain a suspension containing the compound of formula I. The diameter of solid particles of the compound of formula I in the suspension were determined as 0.3 um or higher using Malvern particle size meter 2600C. Composition A6 was obtained through solid-liquid separation by filtration, and the loss of the compound of formula I in the liquid was 1.6%. The content of compound of formula II was determined as 1.4% by HPLC.

[0119] Composition A6 (in which the content of the compound of formula II was 1.4%) above-obtained by filtration was dissolved in an iso-butanol solution having a moisture content of 20% in a volume of 123 ml. 230 ml of isopropyl acetate was slowly added and stirred to obtain a suspension containing the compound of formula I. The diameter of solid particles of the compound of formula I in the suspension were determined as 0.4 um or higher using Malvern particle size meter 2600C. Composition A7 was obtained through solid-liquid separation by filtration, and the loss of the compound of formula I in the liquid was 1.2%. The content of compound of formula II was determined as 0.6% by HPLC.

[0120] Composition A7 (in which the content of the compound of formula II was 0.6%) above-obtained by filtration was dissolved in an iso-butanol solution having a moisture content of 15% in a volume of 103 ml. 205 ml of isopropyl acetate was slowly added and stirred to obtain a suspension containing the compound of formula I. The diameter of solid particles of the compound of formula I in the suspension were determined as 0.6 um or higher using Malvern particle size meter 2600C. Composition A8 was obtained through solid-liquid separation by filtration, and the loss of the compound of formula I in the liquid was 1.0%. The content of compound of formula II was determined as 0.2% by HPLC.

[0121] Composition A8 (in which the content of the compound of formula II was 0.2%) above-obtained by filtration was dissolved in an iso-butanol solution having a moisture content of 15% in a volume of 80 ml. 190 ml of isopropyl acetate was slowly added and stirred to obtain a suspension containing the compound of formula I. The diameter of solid particles of the compound of formula I in the suspension were determined as 0.1 um or higher using Malvern particle size meter 2600C. Composition A9 was obtained through solid-liquid separation by filtration, and the loss of the compound of formula I in the liquid was 1.0%. The content of compound of formula II was determined as 0.08% by HPLC.

[0122] The above composition A9 was dried in vacuo to give 5.3 g of composition B3, the moisture content was determined as 4.9%, and the content of the compound of formula II was determined as 0.08%.

EXAMPLE 6

[0123] 9 g of the crude product of the compound of formula I obtained in Example 3, in which the content of the compound of formula II was 0.5%, was dissolved in an methanol-isobutanol solution (methanol:isobutanol=2:8 v/v) having a moisture content of 28% and a volume of 120 ml. 380 ml of ethyl acetate was slowly added and stirred to obtain a suspension containing the compound of formula I. The diameter of solid particles of the compound of formula I in the suspension were determined as 0.4 um or higher using Malvern particle size meter 2600C. Composition A10 was obtained through solid-liquid separation by filtration, and the loss of the compound of formula I in the liquid was 1.9%. The content of the compound of formula I in composition A10 was determined as 98.8% and the content of compound of formula II was determined as 0.2% by HPLC.

[0124] Composition A10 (in which the content of the compound of formula II was 0.2%) above-obtained by filtration was dissolved in an ethanol solution having a moisture content of 12% in a volume of 120 ml. 230 ml of dichloromethane was slowly added and stirred to obtain a suspension containing the compound of formula I. The diameter of solid particles of the compound of formula I in the suspension were determined as 0.2 um or higher using Malvern particle size meter 2600C. Composition A11 was obtained through solid-liquid separation by filtration, and the loss of the compound of formula I in the liquid was 1.6%. The content of compound of formula II was determined as 0.12% by HPLC.

[0125] Composition A11 (in which the content of the compound of formula II was 0.12%) above-obtained by filtration was dissolved in an isopropanol solution having a moisture content of 30% in a volume of 95 ml. 230 ml of n-hexane was slowly added and stirred to obtain a suspension containing the compound of formula I. The diameter of solid particles of the compound of formula I in the suspension were determined as 0.1 um or higher using Malvern particle size meter 2600C. Composition A12 was obtained through solid-liquid separation by filtration, and the loss of the compound of formula I in the liquid was 0.8%. The content of compound of formula II was determined as 0.06% by HPLC.

[0126] Composition A12 (in which the content of the compound of formula II was 0.06%) above-obtained by filtration was dissolved in an n-butanol solution having a moisture content of 15% in a volume of 103 ml. 205 ml of ethyl acetate was slowly added and stirred to obtain a suspension containing the compound of formula I. The diameter of solid particles of the compound of formula I in the suspension were determined as 0.2 um or higher using Malvern particle size meter 2600C. Composition A13 was obtained through solid-liquid separation by filtration, and the loss of the compound of formula I in the liquid was 1.0%. The content of compound of formula II was determined as 0.01% by HPLC.

[0127] The above composition A13 was dried in vacuo to give 8.6 g of composition B4, the moisture content was determined as 1.5%, and the content of the compound of formula II was determined as 0.01%.

Comparative Example 1

[0128] (When the Moisture Content is Controlled within 8%, the Compound of Formula I is in a Crystalline State without Impurity-Removing Effects)

[0129] 2.6 g of the crude product of the compound of formula I obtained in Example 1, in which the content of the compound of formula II was 3.1%, was dissolved in a methanol solution having a moisture content of 7% and a volume of 22 ml. 100 ml of isopropyl acetate was added slowly, and solids precipitated. The solids were filtered and analyzed by HPLC to determine the content of the compound of formula II as 3.1% without any removing effect. X-ray powder diffraction (XRPD) analysis showed that the above precipitated solids were in crystalline form.

Comparative Example 2

[0130] (When the Moisture Content is Controlled within 8%, the Compound of Formula I is in a Crystalline State without Impurity-Removing Effects)

[0131] 2.6 g of the crude product of the compound of formula I obtained in Example 1, in which the content of the compound of formula II was 3.1%, was dissolved in a n-propanol solution having a moisture content of 3% and a volume of 100 ml. 200 ml of ethyl acetate was added slowly, and solids precipitated. The solids were filtered and analyzed by HPLC to determine the content of the compound of formula II as 3.1% without any removing effect. X-ray powder diffraction (XRPD) analysis showed that the above precipitated solids were in crystalline form.

EXAMPLE 7

Preparing the Compound of Formula X1a from COMPOSITION B, wherein R is phenylthio

[0132] Under an atmosphere of nitrogen, acetonitrile (300 ml), composition B2 (10.0 g, wherein the content of the compound of formula II was 0.09%), phenylboronic acid (2.0 g) and thiophenol (3.6 g) were homogeneously stirred and cooled to −20˜−15° C. Trifluoromethanesulfonic acid (2.5 ml) was added dropwise, and upon addition, the reaction was performed at −20˜−15° C. for about 2.5 h. TLC showed that the reaction was completed. The reaction was quenched, and an aqueous NaOAc solution (2.3 g NaOAc dissolved in 5 ml of water) was slowly added. Upon addition, the temperature was raised to 20° C. and the reaction system was stirred for 2h. Large amount of solids precipitated, cooled to below 0° C., and filtered. The filter cake was washed with 125 ml of acetonitrile/water=9:1 (v/v) for three times and dried in vacuo for 5 h to obtain 9.0 g of sample containing the compound of formula X1a, and the content of the compound of formula XI1a was determined as 0.088% by HPLC.

EXAMPLE 8

Preparing the Compound of Formula X5a from Composition B, wherein R is methoxyphenylthio

[0133] Under an atmosphere of nitrogen, acetonitrile (30 ml), composition B1 (1.0 g, wherein the content of the compound of formula II was 0.95%), phenylboronic acid (0.20 g) and methoxythiophenol (0.38 g) were homogeneously stirred and cooled to −20˜−15° C. Trifluoromethanesulfonic acid (0.25 ml) was added dropwise, and upon addition, the reaction was performed at −20˜'15° C. for about 2.5 h. TLC showed that the reaction was completed. The reaction was quenched, and an aqueous NaOAc solution (0.23 g NaOAc dissolved in 5 ml of water) was slowly added. Upon addition, the temperature was raised to 20° C. and the reaction system was stirred for 2h. Large amount of solids precipitated, cooled to below 0° C., and filtered. The filter cake was washed with 12.5 ml of acetonitrile/water=9:1 (v/v) for three times and dried in vacuo for 5 h to obtain 0.94 g of sample containing the compound of formula X5a, and the content of the compound of formula XI5a was determined as 0.93% by HPLC.

EXAMPLE 9

Preparing the Compound of Formula X2a from Composition B, wherein R is m-hydroxyphenylthio

[0134] Under an atmosphere of nitrogen, acetonitrile (30 ml), composition B4 (1.0 g, wherein the content of the compound of formula II was 0.01%), phenylboronic acid (0.2 g) and hydroxythiophenol (0.36 g) were homogeneously stirred and cooled to −20˜−15° C. Trifluoromethanesulfonic acid (0.25 ml) was added dropwise, and upon addition, the reaction was performed at −20˜−15° C. for about 2.5 h. TLC showed that the reaction was completed. The reaction was quenched, and an aqueous NaOAc solution (0.23 g NaOAc dissolved in 0.5 ml of water) was slowly added. Upon addition, the temperature was raised to 20° C. and the reaction system was stirred for 2h. Large amount of solids precipitated, cooled to below 0° C., and filtered. The filter cake was washed with 1.25 ml of acetonitrile/water=9:1 (v/v) for three times and dried in vacuo for 5 h to obtain 0.93 g of sample containing the compound of formula X2a, and the content of the compound of formula XI2a was determined as 0.009% by HPLC.

EXAMPLE 10

Preparing the Compound of Formula X2a from Composition B, wherein R is m-hydroxyphenylthio

[0135] Under an atmosphere of nitrogen, acetonitrile (300 ml), composition B1 (10 g, wherein the content of the compound of formula II was 0.95%), phenylboronic acid (2.0 g) and m-hydroxythiophenol (3.6 g) were homogeneously stirred and cooled to −20˜−15° C. Trifluoromethanesulfonic acid (2.5 ml) was added dropwise, and upon addition, the reaction was performed at −20˜−15° C. for about 2.5 h. TLC showed that the reaction was completed. The reaction was quenched, and an aqueous NaOAc solution (2.3 g NaOAc dissolved in 5 ml of water) was slowly added. Upon addition, the temperature was raised to 20° C. and the reaction system was stirred for 2h. Large amount of solids precipitated, cooled to below 0° C., and filtered. The filter cake was washed with 12.5 ml of acetonitrile/water=9:1 (v/v) for three times and dried in vacuo for 5 h to obtain 9.3 g of sample containing the compound of formula X2a, and the content of the compound of formula XI2a was determined as 0.87% by HPLC.

EXAMPLE 11

Preparing the Compound of Formula X2a from Composition B, wherein R is m-hydroxyphenylthio

[0136] Under an atmosphere of nitrogen, acetonitrile (300 ml), composition B2 (10 g, wherein the content of the compound of formula II was 0.09%), phenylboronic acid (2.0 g) and m-hydroxythiophenol (3.6 g) were homogeneously stirred and cooled to −20˜−15° C. Trifluoromethanesulfonic acid (2.5 ml) was added dropwise, and upon addition, the reaction was performed at −20˜−15° C. for about 2.5 h. TLC showed that the reaction was completed. The reaction was quenched, and an aqueous NaOAc solution (2.3 g NaOAc dissolved in 5 ml of water) was slowly added. Upon addition, the temperature was raised to 20° C. and the reaction system was stirred for 2h. Large amount of solids precipitated, cooled to below 0° C., and filtered. The filter cake was washed with 12.5 ml of acetonitrile/water=9:1 (v/v) for three times and dried in vacuo for 5 h to obtain 9.2 g of sample containing the compound of formula X2a, and the content of the compound of formula XI2a was determined as 0.09% by HPLC.

EXAMPLE 12

Preparing the Compound of formula X9a from Composition B, wherein R is

[0137] ##STR00054##

[0138] Under an atmosphere of nitrogen, acetonitrile (30 ml), composition B3 (1.0 g, wherein the content of the compound of formula II was 0.08%), phenylboronic acid (0.20 g) and Tetrazole (0.27 g) were homogeneously stirred and cooled to −20˜−15° C. Trifluoromethanesulfonic acid (0.25 ml) was added dropwise, and upon addition, the reaction was performed at −20˜−15° C. for about 2.5 h. TLC showed that the reaction was completed. The reaction was quenched, and an aqueous NaOAc solution (0.23 g NaOAc dissolved in 5 ml of water) was slowly added. Upon addition, the temperature was raised to 20° C. and the reaction system was stirred for 2h. Large amount of solids precipitated, cooled to below 0° C., and filtered. The filter cake was washed with 12.5 ml of acetonitrile/water=9:1 (v/v) for three times and dried in vacuo for 5 h to obtain 0.91 g of sample containing the compound of formula X9, and the content of the compound of formula XI9a was determined as 0.079% by HPLC.

EXAMPLE 13

Preparing the Compound of formula X10 from Composition B, wherein R is

[0139] ##STR00055##

[0140] Under an atmosphere of nitrogen, acetonitrile (30 ml), composition B3 (1.0 g, wherein the content of the compound of formula II was 0.08%), phenylboronic acid (0.20 g) and pyridine (0.32 g) were homogeneously stirred and cooled to −20˜−15° C. Trifluoromethanesulfonic acid (0.25 ml) was added dropwise, and upon addition, the reaction was performed at −20˜−15° C. for about 2.5 h. TLC showed that the reaction was completed. The reaction was quenched, and an aqueous NaOAc solution (0.23 g NaOAc dissolved in 5 ml of water) was slowly added. Upon addition, the temperature was raised to 20° C. and the reaction system was stirred for 2h. Large amount of solids precipitated, cooled to below 0° C., and filtered. The filter cake was washed with 12.5 ml of acetonitrile/water=9:1 (v/v) for three times and dried in vacuo for 5 h to obtain 0.90 g of sample containing the compound of formula X10, and the content of the compound of formula XI10a was determined as 0.079% by HPLC.

EXAMPLE 14

Preparing the Compound of formula X11a from Composition B, wherein R is

[0141] ##STR00056##

[0142] Under an atmosphere of nitrogen, acetonitrile (30 ml), composition B4 (1.0 g, wherein the content of the compound of formula II was 0.01%), phenylboronic acid (0.20 g) and mercaptobenzothiazole (0.45 g) were homogeneously stirred and cooled to −20˜−15° C. Trifluoromethanesulfonic acid (0.25 ml) was added dropwise, and upon addition, the reaction was performed at −20˜−15° C. for about 2.5 h. TLC showed that the reaction was completed. The reaction was quenched, and an aqueous NaOAc solution (0.23 g NaOAc dissolved in 5 ml of water) was slowly added. Upon addition, the temperature was raised to 20° C. and the reaction system was stirred for 2h. Large amount of solids precipitated, cooled to below 0° C., and filtered. The filter cake was washed with 12.5 ml of acetonitrile/water=9:1 (v/v) for three times and dried in vacuo for 5 h to obtain 0.98 g of sample containing the compound of formula X11, and the content of the compound of formula XI11a was determined as 0.009% by HPLC.

EXAMPLE 15

Reduction Reaction of the Compound of Formula X1a

[0143] Under an atmosphere of nitrogen, the compound of formula X1a (2.0 g) obtained in Example 7, phenylboronic acid (0.28 g) and anhydrous tetrahydrofuran (80 ml) were heated under reflux for 30 min, and cooled to room temperature. BSTFA (2.1 ml) was added, stirred for 1h at room temperature, and cooled to −10˜−5° C. Borane-dimethyl sulfide complex (0.8 ml, 0.94%) was added dropwise, and upon addition, warmed to 10-15° C. for reacting for 3.5h. The reaction was monitored by HPLC and the conversion rate was 82%. Afterwards, hydrochloric acid (2 N, 4.8 ml) was added dropwise and water (160 ml) was added. The solvent was removed under reduced pressure and then stirred at room temperature for 24 h. The residue was loaded onto a preparative column, and eluted with 22% acetonitrile/water (0.15% acetic acid). Collections rich in products were pooled, diluted twice with water, loaded on a preparative column, and eluted with 90% acetonitrile/water (0.15% acetic acid). Fractions were collected and lyophilized to give 1.6 g of sample containing the compound of formula X1b. The content of the compound of formula XI1b was determined as 0.082% by HPLC.

EXAMPLE 16

Preparing the Compound of Formula III from the Compound of Formula X1b

[0144] Under an atmosphere of nitrogen, the compound of formula X1b (1.0 g) obtained in Example 15 was dissolved in methanol (4.2 ml), and cooled to −20 to −15° C. Ethylenediamine (4.2 ml) was added dropwise. Upon addition, the reaction was warmed to room temperature for 48 h, and the reaction conversion rate was determined as 99% by HPLC monitoring. The reaction system was added dropwise into a solution of glacial acetic acid solution (8.3 ml) in water (18.5 ml), then diluted twice with water, loaded onto a preparative column and eluted with 22% acetonitrile/water (0.15% acetic acid). Collections rich in products were pooled, diluted twice with water, loaded on a preparative column, and eluted with 90% acetonitrile/water (0.15% acetic acid). Fractions were collected and concentrated to dryness under reduced pressure. The above concentrate was dissolved in a solution (ethanol/water/acetic acid=210.0/19.7/1.0, v/v/v, 10 ml), and ethyl acetate (14 ml) was added dropwise at 5 to 20° C. for crystallization. White crystalline solids (caspofungin acetate, 0.71 g) were obtained by filtration, the purity of which was determined as 99.82% by HPLC, and the content of the compound of formula IV was 0.072%.

EXAMPLE 17

Reduction Reaction of the Compound of Formula X2a

[0145] Under an atmosphere of nitrogen, the compound of formula X2a (2.0 g) obtained in Example 11, phenylboronic acid (0.28 g) and anhydrous tetrahydrofuran (80 ml) were heated under reflux for 30 min, and cooled to room temperature. BSTFA (2.1 ml) was added, stirred for 1h at room temperature, and cooled to −10˜−5° C. Borane-dimethyl sulfide complex (0.8 ml, 0.94%) was added dropwise, and upon addition, warmed to 10-15° C. for reacting for 3.5h. The reaction was monitored by HPLC and the conversion rate was 82%. Afterwards, hydrochloric acid (2 N, 4.8 ml) was added dropwise and water (160 ml) was added. The solvent was removed under reduced pressure and then stirred at room temperature for 24 h. The residue was loaded onto a preparative column, and eluted with 22% acetonitrile/water (0.15% acetic acid). Collections rich in products were pooled, diluted twice with water, loaded on a preparative column, and eluted with 90% acetonitrile/water (0.15% acetic acid). Fractions were collected and diluted twice with water, loaded on a preparative column, and eluted with 90% acetonitrile/water (0.15% acetic acid). Fractions were collected and lyophilized to give 1.42 g of sample containing the compound of formula X2b. The content of the compound of formula XI2b was determined as 0.09% by HPLC.

EXAMPLE 18

Preparing the Compound of Formula III from the Compound of Formula XI2b

[0146] Under an atmosphere of nitrogen, the compound of formula X2b (1.0 g) obtained in Example 17 was dissolved in methanol (4.2 ml), and cooled to −10 to −5° C. Ethylenediamine (4.2 ml) was added dropwise. Upon addition, the reaction was warmed to room temperature for 48 h, and the reaction conversion rate was determined as 99% by HPLC monitoring. The reaction system was added dropwise into a solution of glacial acetic acid solution (8.3 ml) in water (18.5 ml), then diluted twice with water, loaded onto a preparative column and eluted with 22% acetonitrile/water (0.15% acetic acid). Collections rich in products were pooled, diluted twice with water, loaded on a preparative column, and eluted with 90% acetonitrile/water (0.15% acetic acid). Fractions were collected and concentrated to dryness under reduced pressure. The above concentrate was dissolved in a solution (ethanol/water/acetic acid=210.0/19.7/1.0, v/v/v, 10 ml), and ethyl acetate (14 ml) was added dropwise at 5 to 20° C. for crystallization. White crystalline solids (caspofungin acetate, 0.72 g) were obtained by filtration, the purity of which was determined as 99.76% by HPLC, and the content of the compound of formula IV was 0.06%.

Comparative Example 3

Preparation of Caspofungin Acetate without a Compound of Formula IV According to the Method of CN102070707A

[0147] Caspofungin acetate with less than 0.05% of the compound of formula IV was prepared according to the method disclosed in Examples of CN102070707A, and results showed that about 1 g of caspofungin acetate was prepared, which was purified through preparative HPLC (containing RP C-18 resin filler) with acetic acid and acetonitrile buffer, about 6 L of organic solvent, acetonitrile was used, and the used production equipment and resin filler are very expensive.

[0148] However, according to Examples 4, 5 and 6, it was found that about 0.3 L of solvent was used for preparing 1 g of the compound of the formula I in which the content of the impurity, the compound of formula II, was 0.1% or less, therefore, caspofungin acetate can be directly prepared in which the content of the compound of formula IV was less than 0.05%, and it is not necessary to use preparative HPLC for purification.

Comparative Example 4

Preparation of Caspofungin Acetate Intermediate without a Compound of Formula IV According to the Method of CN102947327A

[0149] Caspofungin acetate intermediate with low content of serine analog caspofungin intermediate was prepared according to methods disclosed in Examples of CN102947327A. When Caspofungin acetate intermediate with less than 0.1% of serine analog caspofungin intermediate was prepared according to methods described in CN102947327A, the yield was only 20% to 40%, and the value for industrial production was very small.

[0150] The foregoing is intended only as a preferred embodiment of the present invention and is not to limit the scope of the essential technical contents of the invention, the spirit of which is broadly defined within the scope of the claims, and any technical entity or method achieved by others, if it is identical with or an equivalent modification as that defined in the scope of the claims, is to be regarded as encompassed within the scope of the claims.