Method for isolating caspofungin

Abstract

The present invention relates to a method for isolating caspofungin and to a novel crystalline form of caspofungin diacetate thus obtained.

Claims

1. Method for isolating caspofungin diacetate comprising adding a liquid ester to a solution of caspofungin in an alcohol followed by isolation of the solid material thus obtained, wherein said adding is carried out in a stainless steel reactor.

2. Method according to claim 1 wherein said liquid ester is chosen from the group consisting of ethyl acetate, isopropyl acetate, methyl acetate and propyl acetate and said alcohol is chosen from the group consisting of ethanol, isopropanol, methanol and propanol.

3. Method according to claim 1 wherein the concentration of caspofungin in said alcohol is from 5 to 100 g.Math.L.sup.−1.

4. Method according to claim 3 wherein the volume of said solution of caspofungin in an alcohol is from 10 to 1000 L.

5. Method according to claim 1 wherein the amount of water in said solution of caspofungin in an alcohol is less than 10%.

6. Crystalline caspofungin diacetate, wherein the crystalline caspofungin diacetate has x-ray powder diffraction data selected from the group consisting of: (a) An XRD powder diffraction pattern with peaks at 2.91±0.2, 3.07±0.2, 5.09±0.2, 5.38±0.2, 5.78±0.2, 6.12±0.2, 8.96±0.2 and 10.19±0.2 degrees 2-theta; and (b) An XRD powder diffraction pattern as depicted in FIG. 1.

7. Crystalline caspofungin diacetate according to claim 6 wherein a ratio between the intensity of said peak at 3.07±0.2 degrees 2-theta and the intensity of any peak in the region 9.0±0.2 degrees 2-theta is at least 5.

8. Crystalline caspofungin diacetate according to claim 6 wherein the water content of the crystalline caspofungin diacetate is from 0.1% to 8% (w/w).

9. Crystalline caspofungin diacetate according to claim 6 wherein the water content of the crystalline caspofungin diacetate is from 0.1% to 10% (w/w).

10. Crystalline caspofungin diacetate according to claim 6 wherein the water content of the crystalline caspofungin diacetate is from 2% to 6% (w/w).

11. Crystalline caspofungin diacetate according to claim 6 wherein the crystalline caspofungin diacetate has a x-ray powder diffraction pattern with peaks at 2.91±0.2, 3.07±0.2, 5.09±0.2, 5.38±0.2, 5.78±0.2, 6.12±0.2, 8.96±0.2 and 10.19±0.2 degrees 2-theta.

12. The method for isolating caspofungin according to claim 1, wherein the isolated caspofungin diacetate has a x-ray powder diffraction pattern with peaks at 2.91±0.2, 3.07±0.2, 5.09±0.2, 5.38±0.2, 5.78±0.2, 6.12±0.2, 8.96±0.2 and 10.19±0.2 degrees 2-theta.

13. The method for isolating caspofungin according to claim 1, wherein the isolated caspofungin diacetate has a x-ray powder diffraction pattern as depicted in FIG. 1.

14. A method for isolating crystalline caspofungin diacetate comprising: adding a liquid ester to a solution of caspofungin in an alcohol in a stainless steel vessel; and isolating the solid material thus obtained as crystalline caspofungin diacetate, wherein the crystalline caspofungin diacetate has an XRD powder diffraction pattern with peaks at 2.91±0.2, 3.07±0.2, 5.09±0.2, 5.38±0.2, 5.78±0.2, 6.12±0.2, 8.96±0.2 and 10.19±0.2 degrees 2-theta.

15. The method according to claim 14, wherein said liquid ester is chosen from the group consisting of ethyl acetate, isopropyl acetate, methyl acetate and propyl acetate and said alcohol is chosen from the group consisting of ethanol, isopropanol, methanol and propanol.

16. The method according to claim 14, wherein the concentration of caspofungin in said alcohol is from 5 to 100 g.Math.L.sup.−1.

17. The method according to claim 16, wherein the volume of said solution of caspofungin in an alcohol is from 10 to 1000 L.

18. The method according to claim 14, wherein the amount of water in said solution of caspofungin in an alcohol is less than 10%.

19. The method according to claim 14, wherein a ratio between the intensity of the peak at 3.07±0.2 degrees 2-theta and the intensity of any peak in the region 9.0±0.2 degrees 2-theta is at least 5.

20. The method according to claim 14, wherein the water content of the crystalline caspofungin diacetate is from 0.1% to 8% (w/w).

Description

LEGEND TO THE FIGURES

(1) FIG. 1 is the XRD spectrum of caspofungin diacetate (Form A, Example 1). X-axis: 2-theta value (deg). Y-axis: intensity (cps). The zoomed view indicated by the arrow is the area where the two major peaks at 2.920 and 3.080 degrees can be clearly observed. The following distinct peaks can be discerned:

(2) TABLE-US-00001 Peak no. 2-Theta (deg) Flex width d-Value Intensity I/Io 1 2.920 0.118 30.2319 15599 86 2 3.080 0.118 28.6618 18341 100 3 5.100 0.165 17.3131 3823 21 4 5.400 0.141 16.3519 2055 12 5 5.800 0.165 15.2251 2495 14 6 6.140 0.212 14.3827 2884 16 7 9.000 0.282 9.8176 1839 11 8 10.200 0.235 8.6651 1844 11

(3) FIG. 2 is the XRD spectrum of caspofungin diacetate (Form A, Example 2). X-axis: 2-theta value (deg). Y-axis: intensity (cps). The zoomed view indicated by the arrow is the area where the two major peaks at 2.920 and 3.060 degrees can be clearly observed. The following distinct peaks can be discerned:

(4) TABLE-US-00002 Peak no. 2-Theta (deg) Flex width d-Value Intensity I/Io 1 2.920 0.118 30.4404 17334 83 2 3.060 0.118 28.8491 20913 100 3 5.080 0.165 17.3812 3947 19 4 5.360 0.118 16.4739 2098 11 5 5.760 0.165 15.3307 2715 13 6 6.100 0.188 14.4770 3261 16 7 8.920 0.306 9.9055 1665 8 8 10.180 0.141 8.6821 1829 9

(5) FIG. 3 is the XRD spectrum of caspofungin diacetate (Form B, Example 3). X-axis: 2-theta value (deg). Y-axis: intensity (cps). The zoomed view indicated by the arrow is the area where only a single peak at 2.980 degrees can be clearly observed. The following peaks can be discerned:

(6) TABLE-US-00003 Peak no. 2-Theta (deg) Flex width d-Value Intensity I/Io 1 2.980 0.165 29.6234 26979 100 2 5.120 0.165 17.2455 4287 16 3 6.000 0.353 14.7180 3246 13 4 6.660 0.118 13.2609 1340 5 5 7.900 0.094 11.1820 1305 5 6 9.060 0.212 9.7527 2035 8 7 10.200 0.212 8.6651 1814 7 8 10.420 0.094 8.4827 1722 7

EXAMPLES

General

(7) X-Ray Powder Diffraction analysis

(8) Samples were analyzed on an Ultima IV X-ray powder diffractometer from Rigaku.

(9) TABLE-US-00004 Source: X-ray tube Target: Cu Tube Voltage: 40 kV Tube Current: 40 mA Start Angle: 2 deg Stop Angle: 40 deg Scan Axis: −2 Theta/Theta Method: Continuous Counting Units: CPS (Counts Scan Speed: 2 deg/min per sec) Div slit: −2/3 deg DHL slit: −10 mm Scattering slit: −2/3 deg Rec slit: −0.3 mm
HPLC Analysis Injection volume: 5 μL Detection: UV (210 and 270 nm) Flow: 0.35 mL.Math.min.sup.−1. Column: Waters XBridge C18, 3.5 μm, 150 mm*2.1 mm (part no 186003023) Column temp: 40° C. Mobile phase A: 50 mM phosphate buffer pH 7 (700 mL)+acetonitrile (300 mL)” Mobile phase B: 75% acetonitrile Gradient:

(10) TABLE-US-00005 Time (min) 0 12 15 22 32 34 40 % A 100 84 84 0 0 100 100 % B 0 16 16 100 100 0 0
Solution of Caspofungin in Ethanol

(11) Solutions of caspofungin in ethanol were prepared as follows (see also WO 2012/041801).

(12) A solution of phenylthio pneumocandin B.sub.0 amine (1.6 L; 17.3 g phenylthio pneumocandin B.sub.0 amine; 15 mmol; 44 vol % water) was cooled to −10° C. Under stirring ethylenediamine (EDA; 320 mL; 4.7 mol) was added in 20 min between −10 and −3° C. Immediately a white precipitate was formed, which dissolved later on. The reaction mixture was stirred for 15 h at 21-22° C. Additional EDA (170 mL; 2.5 mol) was added at 10-20° C. and stirring at ambient temperature was continued for 31 h. Under stirring the reaction mixture and acetic acid (1430 mL; 24.7 mol) were simultaneously added to 1.5 L pre-cooled water of 0° C., keeping the temperature below 10° C. and the pH between 5.1 and 5.5 (quenching is exothermic). At 10° C. and pH 5.3 the mixture was extracted with heptane (2×300 mL). To improve phase separation water (500 mL) was added. The heptane phases were combined and the mix was back-extracted with water (2×250 mL). All aqueous phases were combined and diluted with water to 9.45 L in order to decrease the ethanol content to less than 10%.

(13) Silica gel 100 C.sub.18 (900 g) was suspended in 75% acetonitrile (2 L). The mixture was placed in a column (height 19.6 cm; internal diameter 10 cm). A bed-volume of 1540 mL was obtained. The column was first washed with three bed-volumes of acetonitrile (100%) and then equilibrated with three bed-volumes aqueous acetic acid (0.15%) with a flow of 80 mL.Math.min.sup.−1 at approximately 1 bar. The caspofungin solution (9.45 L) as prepared above was used as such for loading on the column. The flow rate was ˜50 mL.Math.min.sup.−1. The flow was adjusted to keep the pressure below 5 bar. The linear flow rate was ˜0.6 cm.Math.min.sup.−1. The loading capacity was 10 g caspofungin diacetate per L resin (12 g total caspofungins per L). Next, the column was washed with aqueous acetic acid (0.15%, 3.2 L) with a flow rate of 70 mL.Math.min.sup.−1 after which the column was eluted with different solvent compositions at the same flow rate: 10% Acetonitrile/0.15% acetic acid (15 L; 9.7 bed volumes) 13% Acetonitrile/0.15% acetic acid (6375 mL; 4.1 bed volumes) 20% Acetonitrile/0.15% acetic acid (7125 mL; 4.6 bed volumes)

(14) The column was regenerated by washing with 75% acetonitrile/0.15% acetic acid (5625 mL; 3.7 bed volumes). The eluate was monitored continuously with UV (280 nm and 254 nm) and fractions 37-69 were pooled, giving a caspofungin solution of 12.4 L with a yield of 94.7% and a concentration of caspofungin-diacetate of 1.2 g.Math.L.sup.1 (based on yield and input before column step). The mass balance of caspofungin over all fractions was 99.1%.

(15) Next, a column with a diameter of 10 cm was filled with Amberchrom XT30 resin giving a bed volume of 628 mL and a bed height of 8 cm. The column was equilibrated with 0.15% acetic acid. Loading, washing, and elution were carried out in two subsequent runs. After the first run the column was equilibrated with 0.15% acetic acid. The eluate was monitored continuously with UV (280 nm and 254 nm). The caspofungin solution (12.4 L) as prepared above was diluted to 30 L with 0.15% acetic acid (=feed). Loading 1: Caspofungin feed (21 L) was loaded up-flow on the column (16.7 g caspofungin per L resin) with a flow rate of 80 mL.Math.min.sup.−1). The linear flow rate was 1.0 cm.Math.min.sup.−1. The pressure did not exceed 2.5 bar. Washing 1: The column was washed in down-flow with 0.15% acetic acid (1660 mL). Elution 1: The column was eluted down-flow with ethanol/0.15% acetic acid (1000 mL). Fractions of 200 mL were collected starting from the point where the UV signal started to rise (after approximately 1 bed volume) and analyzed by HPLC and by Karl-Fischer for water analysis (Table 1).

(16) TABLE-US-00006 TABLE 1 Results of column run 1 Water Caspofungin Frac- Dilu- Volume % Total Yield Cum. Yield tion tion (mL) (v/v) Area Area (%) (%) Feed 1 21000 — 110.6 2323104 100 100 1 100 200 22.6  84.6 1691540 72.8 72.8 2 100 200 3.5 21.5 430940 18.6 91.4 3 20 200 0.3 9.7 38680 1.7 93.0 4 10 200 n.d. 15.2 30416 1.3 94.3 5 10 200 n.d. 13.6 27248 1.2 95.5

(17) The column was equilibrated with 0.15% acetic acid. Loading 2: Caspofungin feed (10.345 L) was loaded up-flow on the column (8 g caspofungin per L resin) with a flow rate of 70 mL.Math.min.sup.−1). The linear flow rate was 0.9 cm.Math.min.sup.−1. The pressure did not exceed 2.5 bar. Washing 2: The column was washed up-flow with 0.15% acetic acid (947 mL). Elution 2: The column was eluted down-flow with ethanol/0.15% acetic acid (735 mL). Fractions were collected starting from the point where the UV signal started to rise (after approximately 1 bed volume) and analyzed by HPLC and by Karl-Fischer for water analysis (Table 2).

(18) TABLE-US-00007 TABLE 2 Results of column run 2 Caspofungin Frac- Volume Water Total Yield Cum. Yield tion Dilution (mL) % (v/v) Area Area (%) (%) Feed 1 10345 — 77.0 796948 100 100 1 100 105 26.6 54.7 574833 72.1 72.1 2 20 100  2.7 55.9 111710 14.0 86.1 3 5 94 n.d. 102.4 48136 6.0 92.2 4 5 113 n.d. 58.9 33291 4.2 96.4 5 1 113 n.d. 86.3 9757 1.2 97.6 6 1 210 n.d. 18.0 3782 0.5 98.1

(19) Fractions 1 up to and including 5 of both runs were pooled, giving 1525 mL of a caspofungin solution with an estimated concentration of 9.5 g.Math.L.sup.1 (based on yield and input before both runs). The overall yield over both runs was 96.2%. The water concentration of the pooled fractions was analyzed by Karl-Fischer: 5.5%.

(20) The solution obtained above (see Table 3) was used as such for crystallization.

(21) TABLE-US-00008 TABLE 3 HPLC results of caspofungin solution after concentration and solvent switch Retention Peak time Height Area Rel. Area no. (min) Peak name (mAU) (mAU * min) (%) 1 2.21 1.193 0.254 0.23 2 9.43 0.003 0.020 0.02 3 10.23 0.001 0.028 0.03 4 10.99 0.001 0.018 0.02 5 12.17 0.579 0.160 0.14 6 12.68 Caspofungin 226.299 109.042 98.43 7 20.76 3.843 0.852 0.77 8 21.19 0.007 0.327 0.29 9 22.73 0.583 0.076 0.07

Example 1

Caspofungin Crystallization in Stainless Steel Reactor

(22) The crystallization was started with 29 L of a caspofungin solution in ethanol (13.6 g.Math.L.sup.−1; 394 g caspofungin) containing 6.1 w/w % water and 0.4% acetic acid, obtained as outlined in the General section. The caspofungin solution was charged into a stainless steel reactor of 350 L, equipped with an anchor type stirrer. Under stirring ethyl acetate (19 L) was slowly added in about 3 hours at about 20° C. and stirring was continued for 1 hour allowing crystals to form. Next ethyl acetate (38 L) was slowly added in 12 hours. After stirring at about 20° C. for another 2 hours, the crystals were successively filtered off by centrifugation, washed two times with ethyl acetate/ethanol/water (62.5/35.5/2 (v/v/v), 3.6 L), washed with ethyl acetate (two times 2 L), and dried under vacuum yielding 224 g caspofungin as white crystals (Form A, FIG. 1).

Example 2

Caspofungin Crystallization in Stainless Steel Reactor

(23) The crystallization was started with 32.5 L of a caspofungin solution in ethanol (12.9 g.Math.L.sup.−1; 419 g caspofungin) containing 7.6 w/w % water and 0.4% acetic acid, obtained as outlined in the General section. The caspofungin solution was charged into a stainless steel reactor of 350 L, equipped with an anchor type stirrer. Under stirring ethyl acetate (25 L) was slowly added in about 4.5 hours at about 20° C. Stirring was continued at about 20° C. for 1 hour allowing crystals to form. Next ethyl acetate (29 L) was slowly added in 12 hours. After stirring at about 20° C. for another 2 hours, the crystals were successively filtered off by centrifugation, washed two times with ethyl acetate/ethanol/water (62.5/35.5/2 (v/v/v), 4.4 L), washed with ethyl acetate (two times 2 L), and dried under vacuum yielding 303 g caspofungin as white crystals (Form A, FIG. 2).

Example 3

Caspofungin Crystallization in Glass Round Bottom Flask

(24) The crystallization was started with 400 mL of a caspofungin solution in ethanol (13.6 g.Math.L.sup.−1; 5.44 g caspofungin) containing 6.1 w/w % water and 0.4% acetic acid, obtained as outlined in the General section. The caspofungin solution was charged into a 2 L glass three necked round bottom flask equipped with a glass stirrer. Under stirring ethyl acetate (240 mL) was slowly added in about 3 hours at about 20° C. and stirring was continued for 1 hour allowing crystals to form. Next ethyl acetate (420 mL) was slowly added in 12 hours. After stirring at about 20° C. for another 2 hours, the crystals were successively filtered off, washed three times with ethyl acetate/ethanol/water (62.5/35.5/2 (v/v/v), 50 mL), washed with ethyl acetate (three times 50 mL) and dried under vacuum, yielding 4.0 g caspofungin as white crystals (Form B, FIG. 3).