PURIFICATION OF PLEUROMUTILIN

Abstract

The present invention relates to purification methods of pleuromutilin (I) by means of crystallisation and/or recrystallisation. The process according to the present invention is carried out in the presence of i-propylacetate.

##STR00001##

Claims

1. A method for purifying pleuromutilin ##STR00014## by means of crystallisation and/or recrystallisation in the presence of i-propylacetate.

2. A method according to claim 1 wherein crystallisation and/or recrystallisation is carried out in the presence of an organic anti-solvent.

3. A method according to claim 2, wherein said anti-solvent is selected from the group consisting of heptane, hexane, and mixtures thereof.

4. A method according to claim 1, wherein crystallisation and/or recrystallisation is carried out in the presence of a combination of i-propylacetate and an anti-solvent selected from the group consisting of: i-propylacetate in the presence of heptane, i-propylacetate in the presence of hexane, and mixtures thereof.

5. A method according to claim 2, wherein the ratio of i-propylacetate to anti-solvent is from 8 to 0.25.

6. A method according to claim 1, comprising the step of employing seed crystals to initiate the crystallisation and/or recrystallisation.

7. A method according to claim 2, wherein the ratio of i-propylacetate to anti-solvent is from 2 to 0.4.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0041] The trivial name mutilin refers to the IUPAC systematic name (1S, 2R, 3S, 4S, 6R, 7R, 8R, 14R)-3,6-dihydroxy-2,4,7,14-tetramethyl-4-vinyl-tricyclo[5.4.3.0.sup.1.8]tetradecan-9-one.

##STR00010##

[0042] In the following discussion the mutilin numbering system described by H. Berner (Berner, H.; Schulz, G.; Schneider H. Tetrahedron 1980, 36, 1807-1811) is used.

##STR00011##

[0043] The present invention relates to efficient purification processes/methods by means of (re)-crystallisation of pleuromutilin.

[0044] The present invention enables the purification of pleuromutilin and can be employed in the initial isolation/crystallisation after the product is extracted from the fermentation broth Alternatively, after the pleuromutilin has been isolated, a recrystallization can be performed using the solvents and solvent systems as specified above.

[0045] The pleuromutilin after isolating from fermentation has a reasonable high purity which can vary between about 85 to 95%. However, there is significant number of impurities present which are getting of concern if the pleuromutilin is used as starting material to produce semi-synthetic APIs for the treatment of animals or humans. In particular the presence of 2,3 pleuromutilin epoxide is of high concern.

[0046] In the tables below the purities of commercial (crude) pleuromutilin and purified pleuromutilin is presented.

TABLE-US-00003 Purified pleuromutilin batches after recrystallization using organic solvent/solvent systems Area % by HPLC Methyl- Ethyl- i-Propyl- n-Propyl- i-Butyl- t-Butyl- Commercial acetate acetate acetate acetate acetate acetate pleuromutilin (MeOAc) (EtOAc) (i-PrOAc)* (n-PrOAc) (i-BuOAc) (t-BuOAc) Impurity batch Procedure A Procedure B Procedure C Procedure D Procedure E Procedure F 1 0.47 0.32 0.34 0.16 0.14 0.16 0.23 2 0.31 0.27 0.27 0.14 0.18 0.21 0.26 3 0.37 n.d. n.d. n.d. n.d. n.d. n.d. 4 0.80 n.d. n.d. n.d. n.d. n.d. n.d. 5 5.56 1.73 2.02 1.99 1.78 1.92 2.81 6 0.30 n.d. n.d. n.d. n.d. n.d. n.d. 7 0.11 n.d. n.d. n.d. n.d. n.d. n.d. 8 0.25 n.d. n.d. n.d. n.d. n.d. n.d. 9 0.25 n.d. n.d. n.d. n.d. n.d. n.d. 10 0.12 n.d. n.d. n.d. n.d. n.d. n.d. 11 0.35 n.d. n.d. n.d. n.d. n.d. n.d. 12 1.68 n.d. n.d. n.d. n.d. n.d. n.d. 13 0.53 n.d. n.d. n.d. n.d. n.d. n.d. 14 0.51 n.d. n.d. n.d. n.d. n.d. n.d. 15 0.65 n.d. n.d. n.d. n.d. n.d. n.d. 16 0.20 n.d. n.d. n.d. n.d. n.d. n.d. 17 0.42 n.d. n.d. n.d. n.d. n.d. n.d. Sum of other 0.95 0.12 0.12 0.05 0.05 0.05 0.05 impurities below 0.10% area Pleuromutilin 86.17 97.56 97.25 97.66 97.85 97.66 97.56 purity *Example according to the present invention

TABLE-US-00004 Purified pleuromutilin batches after recrystallization using organic solvent/ anti solvent Area % by HPLC i-Propyl-acetate/ i-Propyl-acetate/ Commercial heptane (i-PrOAc/ heptane (i-PrOAc/ pleuro- heptane) isolated heptane) isolated mutilin at room temperature* at 0 to 5 C.* Impurity batch Procedure G Procedure H 1 0.47 0.21 0.21 2 0.31 0.16 0.17 3 0.37 n.d. n.d. 4 0.80 n.d. n.d. 5 5.56 1.87 2.11 6 0.30 n.d. n.d. 7 0.11 n.d. n.d. 8 0.25 n.d. n.d. 9 0.25 n.d. n.d. 10 0.12 n.d. n.d. 11 0.35 n.d. n.d. 12 1.68 n.d. n.d. 13 0.53 n.d. n.d. 14 0.51 n.d. n.d. 15 0.65 n.d. n.d. 16 0.20 n.d. n.d. 17 0.42 n.d. n.d. Sum of other 0.95 0.05 0.05 impurities below 0.10% area Pleuromutilin 86.17 97.71 97.46 purity n.d.: not detected *Examples according to the invention

[0047] The usage of hexane as anti-solvent, e.g. i-propylacetate/hexane leads to a comparable purification result.

[0048] As can be seen from the tables above with all purification methods applied, a significant purification is achieved.

[0049] After purification there are only three impurities remaining, with two of them being known impurities, namely 14-acetylmutilin and 2,3-pleuromutilin epoxide. The structure of 2,3-pleuromutilin epoxide has been elucidated by isolating the material from crude pleuromutilin and subjecting to analytical procedures like NMR and MS.

TABLE-US-00005 Relative Response Factor (RRF) to pleuromutilin at 210 nm detection wavelength in used Structure HPLC method Impurity 1 Unknown 1 Impurity 2 2,3-Pleuromutilin epoxide [00012] 0.32 Impurity 5 14-Acetylmutilin [00013] 0.96

[0050] The HPLC method to analyze the pleuromutilin batches is as follows:

TABLE-US-00006 HPLC column Phenomenex Kinetex C18 Detection 210 nm Eluent A 1 mL H.sub.3PO.sub.4 for HPLC + 1 L HPLC-Water Eluent B 1 mL H.sub.3PO.sub.4 for HPLC + 1 L Acetonitrile Injection volume 7 L

[0051] In order to determine the weight content of the impurities in the pleuromutilin, the area % values must be corrected with the Relative Response Factor (RRF) values for the impurities. Applying the response factor corrections leads to the following results for the two known impurities:

TABLE-US-00007 Impurity w/w % after response factor correction 2,3-Pleuromutilin epoxide 14-Acetylmutilin Commercial and purified reduction reduction pleuromutilin batches after compared to compared to recrystallization using organic commercial commercial solvent/solvent systems batch batch Commercial pleuromutilin 0.96 N/A 5.79 N/A batch Methyl-acetate (MeOAc) 0.84 12.5% 1.80 68.9% Ethyl-acetate (EtOAc) 0.84 12.5% 2.10 63.7% i-Propyl-acetate (i-PrOAc)* 0.44 54.1% 2.08 64.0% n-Propyl-acetate (n-PrOAc) 0.56 41.7% 1.85 68.0% i-Butyl-acetate (i-BuOAc) 0.66 31.2% 2.00 65.5% t-Butyl-acetate(t-BuOAc) 0.81 15.6% 2.93 49.4% i-Propyl-acetate/heptane 0.50 47.9% 1.95 66.3% (i-PrOAc/heptane) isolated at room temperature* i-Propyl-acetate/heptane 0.53 44.8% 2.20 62.0% (i-PrOAc/heptane) isolated at 0 to 5 C.* *Examples according to the invention

[0052] Surprisingly the reduction of the two known impurities is not the same for the series of acetic acid ester solvents used, optionally in the presence of an anti-solvent, to purify the commercial pleuromutilin batches. Most surprisingly the reduction of the critical 2,3-pleuromutilin epoxide is significantly enhanced when as crystallization solvent i-PrOAc, or a combination of i-PrOAc/heptane is used.

[0053] Especially, it is surprising that compared with n-PrOAc the reduction of the 2,3-pleuromutilin epoxide is still enhanced with i-ProAc in combination with an anti-solvent (heptane). This is surprising because while it is known that using an anti-solvent increases the yield of the process but tends to reduce the efficiency of the purification.

[0054] The above very surprising effects are very valuable in the further synthesis of semi-synthetic pleuromutilin derivatives. The reduction of 14-acetylmutilin is largely the same except for t-BuOAc where larger amounts are remaining in the pleuromutilin. However, as mentioned above, the impurity 14-acetylmutilin is not as important/critical as 2,3-pleuromutilin epoxide.

[0055] The impurity thresholds for APIs used in humans are very strict.

[0056] Drug substances for the human market have to fulfill the regulatory requirements defined in the corresponding ICH guidelines (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use). The ICH guideline on impurities in new drug substances (Q3A(R2)) includes the following thresholds:

TABLE-US-00008 Maximium daily Reporting Identification Qualification dose threshold threshold threshold <2 g 0.05% 0.10% 0.15% >2 g 0.03% 0.05% 0.05%

[0057] As can be seen from the ICH thresholds above it is desirable to have all individual unknown impurities below 0.10% area and the structure elucidated impurities below 0.15%, respectively. Pleuromutilin purification processes/methods provided according to the present invention supports to produce APIs (Active Pharmaceutical Ingredients) within the desired specifications and fulfilling ICH requirements.

[0058] It has been found that the (re)-crystallisation process according to the present invention is usable on large scale with high recoveries (in most cases >50%).

[0059] Of course, the (re-)crystallisation method according to the present invention can be repeated until the desired degree of purification is reached.

Abbreviations

[0060] API Active pharmaceutical ingredient [0061] EP European Pharmacopoeia [0062] g gram [0063] kg kilogram [0064] 1 liters [0065] M molar [0066] mmol millimolar [0067] min minutes [0068] ml milliliters [0069] RRF Relative Response Factor [0070] w/v weight/volume [0071] w/w weight/weight

Experimental SectionPleuromutilin Purification Methods

A) Solvent: Methyl Acetate

[0072] Pleuromutilin (10.02 g, 26.5 mmol) and methyl acetate (20 ml) were charged to a flask and heated to 65 C. The batch was stirred at this temperature for 30 min to ensure dissolution, then cooled to 0-5 C. over 4 h, and stirred at this temperature for 1 h. The batch was then filtered, and the solid washed with cooled methyl acetate (10 ml) and dried in a vacuum oven at 40 C.

[0073] Yield: 6.70 g

B) Solvent: Ethyl Acetate

[0074] Pleuromutilin (10.07 g, 26.6 mmol) and ethyl acetate (20 ml) were charged to a flask and heated to 80 C. The batch was stirred at this temperature for 30 min to ensure dissolution, then cooled to 0-5 C. over 4 h, and stirred at this temperature for 1 h. The batch was then filtered, and the solid washed with cooled ethyl acetate (10 ml) and dried in a vacuum oven at 40 C.

[0075] Yield: 7.06 g

C) Solvent: Isopropyl AcetateAccording to the Invention

[0076] Pleuromutilin (10.04 g, 26.5 mmol) and isopropyl acetate (20 ml) were charged to a flask and heated to 90 C. The batch was stirred at this temperature for 30 min to ensure dissolution, then cooled to 0-5 C. over 4 h, and stirred at this temperature for 1 h. The batch was then filtered, and the solid washed with cooled isopropyl acetate (10 ml) and dried in a vacuum oven at 40 C.

[0077] Yield: 5.83 g

D) Solvent: n-Propyl Acetate

[0078] Pleuromutilin (10.01 g, 26.4 mmol) and n-propyl acetate (20 ml) were charged to a flask and heated to 90 C. The batch was stirred at this temperature for 30 min to ensure dissolution, then cooled to 0-5 C. over 4 h, and stirred at this temperature for 1 h. The batch was then filtered, and the solid washed with cooled n-propyl acetate (10 ml) and dried in a vacuum oven at 40 C.

[0079] Yield: 4.64 g

E) Solvent: Isobutyl Acetate

[0080] Pleuromutilin (10.00 g, 26.4 mmol) and isobutyl acetate (20 ml) were charged to a flask and heated to 90 C. The batch was stirred at this temperature for 30 min to ensure dissolution, then cooled to 0-5 C. over 4 h, and stirred at this temperature for 1 h. The batch was then filtered, and the solid washed with cooled isobutyl acetate (10 ml) and dried in a vacuum oven at 40 C.

[0081] Yield: 6.43 g

F) Solvent: tert-Butyl Acetate

[0082] Pleuromutilin (10.00 g, 26.4 mmol) and tert-butyl acetate (20 ml) were charged to a flask and heated to 90 C. The batch was stirred at this temperature for 30 min but complete dissolution did not occur. The batch was cooled to 0-5 C. over 4 h, stirred at this temperature for 1 h, filtered, and the solid washed with cooled tert butyl acetate (10 ml). The solid product was then dried in a vacuum oven at 40 C.

[0083] Yield: 7.44 g

G) Solvent/Anti-solvent: Isopropyl Acetate/HeptaneMethod 1According to the Invention

[0084] Pleuromutilin (10.01 g, 26.4 mmol) and isopropyl acetate (20 ml) were charged to a flask and heated to reflux to ensure dissolution. The batch was then cooled to 40-45 C. over 2 h. Heptane (40 ml) was then added dropwise over approximately 1 h, maintaining the temperature at 40-45 C. The batch was then stirred for 1 h at 40-45 C., cooled to room temperature over 1 h and stirred at this temperature for 1 h. The batch was then filtered, and the solid washed with cooled isopropyl acetate-heptane (1:1, 210 ml). The resulting solid was dried in a vacuum oven at 40 C.

[0085] Yield: 7.69 g

H) Solvent/Anti-solvent: Isopropyl Acetate/HeptaneMethod 2According to the Invention

[0086] Pleuromutilin (10.01 g, 26.4 mmol) and isopropyl acetate (20 ml) were charged to a flask and heated to reflux to ensure dissolution. The batch was then cooled to 40-45 C. over 2 h. Heptane (40 ml) was then added dropwise over approximately 1 h, maintaining the temperature at 40-45 C. The batch was then stirred for 1 h at 40-45 C., cooled to room temperature over 1 h and stirred at this temperature for 1 h. The batch was then cooled to 0-5 C. over 1 h and stirred at this temperature for a further 1 h. The batch was then filtered, and the solid washed with cooled isopropyl acetate-heptane (1:1, 210 ml). The resulting solid was dried in a vacuum oven at 40 C.

[0087] Yield: 8.01 g

I) Pleuromutilin Tosylation

[0088] Pleuromutilin (10.03 g, 26.5 mmol) and acetonitrile (40 ml) were charged to a flask and a solution of sodium hydroxide (1.62 g, 40.5 mmol) in water (13.3 ml) was added, followed by acetonitrile (5 ml) as a line rinse. The batch was then cooled to 17 C. and p-toluenesulfonyl chloride (5.12 g, 26.9 mmol) was added, followed by acetonitrile (5 ml) as a line rinse. The batch was then stirred for 1.5 h at room temperature until complete by HPLC. Methyl tert-butyl ether (50 ml) and water (50 ml) were charged, stirred and allowed to settle. The lower aqueous layer was removed. The batch was then washed with 5% aqueous sodium chloride solution (250 ml). After separation and polish filtration, the batch was concentrated to approximately 6 vol, MTBE (100 ml) added and concentrated again to 6 vol. A further portion of MTBE (35 ml) was added and the batch concentrated to 6 vol. A mixture of diisopropyl ether (24 ml) and heptane (35 ml) was then added and the batch concentrated to 6 vol. A further mixture of diisopropyl ether (24 ml) and heptane (35 ml) was then added and the batch stirred for 30 mins. The batch was then heated to 57 C., stirred for 2 h and cooled to room temperature overnight before being filtered and washed with diisopropyl ether-heptane (2:3, 220 ml). The resulting solid was dried in a vacuum oven at 40 C.

[0089] Yield: 12.46 g

J) Solvent: Isopropyl Acetate with SeedingMethod 1According to the Invention

[0090] Pleuromutilin (200 g, 0.528 mol) and isopropyl acetate (400 ml) were charged to a flask and heated to reflux. The solution was stirred at reflux for 30 min. The batch was then cooled to 53 C. over 2 h, with pleuromutilin seeds (200 mg) added at 55 C. The batch was stirred for 2 h at 53 C., cooled to 20 C. over 2 h and stirred at this temperature for 2 h. The batch was then further cooled to 1 C. over 1 h and stirred at this temperature for a further 13 h. The batch was then filtered, and the solid washed with cooled isopropyl acetate (2200 ml) and dried in a vacuum oven at 40 C.

[0091] Yield: 124.04 g

K) Solvent: Isopropyl Acetate with SeedingMethod 2According to the Invention

[0092] Pleuromutilin (200 g, 0.528 mol) and isopropyl acetate (400 ml) were charged to a flask and heated to reflux. The batch was stirred at reflux for 30 min to ensure dissolution, then cooled to 18 C. over 4 h, with pleuromutilin seeds (200 mg) added at 55 C. The batch was stirred for 2 h at 18 C., filtered, and the solid washed with cooled isopropyl acetate (2200 ml) and dried in a vacuum oven at 40 C.

[0093] Yield: 106.59 g

L) Solvent/Anti-Solvent: Isopropyl Acetate/Hexane with SeedingAccording to the Invention

[0094] Pleuromutilin (200 g, 0.528 mol) and isopropyl acetate (400 ml) were charged to a flask and heated to reflux to ensure dissolution. The batch was then cooled to 45 C. over 3 h, with pleuromutilin seeds (60 mg) added after 2 h. The batch was then stirred for 1 h at 45 C., cooled to 17 C. over 2 h and stirred at this temperature for 12 h. The batch was then cooled to 5 C. over 1 h and stirred at this temperature for a further 2 h. Hexane (794 ml) was then added dropwise over approximately 4 h, maintaining the temperature at 5 C. The batch was then stirred at 5 C. for 2 h, filtered, and the solid washed with cooled isopropyl acetate (132 ml), followed by further cooled isopropyl acetate (66 ml), then cooled hexane (200 ml). The resulting solid was dried in a vacuum oven at 40 C.

[0095] Yield: 167.89 g

M) 2,3-Pleuromutilin Epoxide

[0096] 2,3-pleuromutilin epoxide is isolated from pleuromutilin via a series of prep-HPLC separations. An initial purification was performed, followed by a second prep-HPLC separation to purify further. Finally, a third purification was carried out by SFC (Supercritical Fluid Chromatography) to provide the final 2,3-pleuromutilin epoxide.

[0097] In total about 7 g of 2,3-pleuromutilin epoxide were isolated from about 5 kg of bulk (crude) pleuromutilin with a purity >90%.

[0098] .sup.1H-NMR (200 MHz, DMSO-d.sub.6) 6.13 (dd, J.sub.cis=10.8 Hz, J.sub.trans=18.0 Hz, 1H, H-19), 5.58 (d, J=8.8 Hz, H-14), 5.25 (t, 1H, 22-OH), 5.05 (d, J.sub.cis=13.2, 1H, H-20a), 5.01 (d, J.sub.trans=9.2 Hz, 1H, H-20b), 4.46 (d, J=6.0Hh, 1H, 11-OH), 3.88 (AB, 2H, H-22), 3.60 (d, J=3.2 Hz, 1H, H-3), 3.32 (d, J=3.2 Hz, 1H, H-2), 3.23 (dd, 1H, H-11), 2.28-1.15 (11H, series of multiplets related to H1, H4, H6, H7, H8, H10 and H13), 1.18 (s, 3H H-15), 1.05 (s, 3H, H-18), 0.74 (d, J=7.2 Hz, 3H, H-17), 0.63 (d, J=6.8 Hz, 3H, H-16)

[0099] Mass Spec:

[0100] m/z=396.3 [M+H.sub.2O]; m/z=423.5 [M+HCOO.sup.]