Synthesis of Delta 9,11 Steroids

Abstract

The present invention is directed to a process for the preparation of ?9,11 steroids by deoxygenation of 9,11-epoxy steroids using HI. The disclosed process selectively forms ?9,11 steroids such as Vamorolone and can also be used for steroid synthesis of ?9,11 steroids in pharmaceutical purity.

Claims

1. A method of making a ?9,11 steroid from a 9,11 epoxy steroid, comprising treating the 9,11 epoxy steroid with hydroiodic acid (HI).

2. The method of claim 1, wherein the ?9,11 steroid is a steroid of formula I, ##STR00031## wherein the dotted line is a single or a double bond; R.sup.1 is H or OH; one of R.sup.2 or R.sup.3 is CH.sub.3 and the other is H or both are H; and X is H, halo, OR, wherein R is H or C(O)R.sup.I, and R.sup.I is CF.sub.3, C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkoxy, C.sub.5-C.sub.12 cycloalkyl, aryl, heteroaryl or NR.sup.II, wherein NR.sup.II is a tertiary amine, wherein the tertiary amine is part of a linear or cyclic substructure.

3. The method of claim 1, wherein the ?9,11 steroid is a steroid of formula If, ##STR00032## wherein R.sup.3 is CH.sub.3 or H; and X is H, halo, OR, wherein R is H or C(O)R.sup.I, and R.sup.I is CF.sub.3, C.sub.1-C.sub.6 alkyl, C.sub.5-C.sub.12 cycloalkyl, aryl, heteroaryl or C.sub.1-C.sub.6 alkoxy, or NR.sup.II, wherein NR.sup.II is a tertiary amine, wherein the tertiary amine is part of a linear or cyclic substructure.

4. The method of claim 3, wherein the ?9,11 steroid is Vamorolone or Vamorolone Acetate, ##STR00033##

5. A process for preparing a ?9,11 steroids of formula I, ##STR00034## wherein the dotted line is a single or a double bond; R.sup.1 is H or OH; one of R.sup.2 or R.sup.3 is CH.sub.3 and the other is H or both are H; and X is H, halo, OR, wherein R is H or C(O)R.sup.I, and R.sup.I is CF.sub.3, C.sub.1-C.sub.6 alkyl, C.sub.5-C.sub.12 cycloalkyl, aryl, heteroaryl or C.sub.1-C.sub.6 alkoxy, or NR.sup.II, wherein NR.sup.II is a tertiary amine, wherein the tertiary amine is part of a linear or cyclic substructure, the process comprising treating an 9,11 epoxy steroid of formula II ##STR00035## wherein the dotted line is a single or a double bond; R.sup.1 is H or OH; one of R.sup.2 or R.sup.3 is CH.sub.3 and the other is H or both are H; and X is H, halo, OR, wherein R is H or C(O)R.sup.I, and R.sup.I is CF.sub.3, C.sub.1-C.sub.6 alkyl, C.sub.5-C.sub.12 cycloalkyl, aryl, heteroaryl or C.sub.1-C.sub.6 alkoxy, or NR.sup.II, wherein NR.sup.II is a tertiary amine, with hydroiodic acid, HI, to form the ?9,11 steroid of formula I.

6. The process of claim 5, wherein the ?9,11 steroid is a compound of formula Id, ##STR00036## wherein one of R.sup.2 or R.sup.3 is CH.sub.3 and the other is H or both are H; and X is H, halo, OR, wherein R is H or C(O)R.sup.I, and R.sup.I is CF.sub.3, C.sub.1-C.sub.6 alkyl, C.sub.5-C.sub.12 cycloalkyl, aryl, heteroaryl or C.sub.1-C.sub.6 alkoxy, or NR.sup.II, wherein NR.sup.II is a tertiary amine, wherein the tertiary amine is part of a linear or cyclic substructure, and wherein the 9,11 epoxy steroid is a compound of formula IId, ##STR00037## wherein one of R.sup.2 or R.sup.3 is CH.sub.3 and the other is H or both are H; and X is H, halo, OR, wherein R is H or C(O)R.sup.I, and R.sup.I is CF.sub.3, C.sub.1-C.sub.6 alkyl, C.sub.5-C.sub.12 cycloalkyl, aryl, heteroaryl or C.sub.1-C.sub.6 alkoxy, or NR.sup.II, wherein NR.sup.II is a tertiary amine, wherein the tertiary amine is part of a linear or cyclic substructure, with hydroiodic acid, HI, to form the compound of formula Id.

7. The process of claim 6, wherein the ?9,11 steroid is a compound selected from Vamorolone or Vamorolone Acetate ##STR00038## and the 9,11 epoxy steroid is a compound of 8-DM and 8-DM acetate, respectively. ##STR00039##

8. The process of claim 5, wherein the ?9,11 steroid is prepared using aqueous HI in an organic solvent and in the presence of an organic acid or without the addition of an organic acid and wherein treatment is carried out at a temperature of below 15? C.

9. The process of claim 8, wherein the organic solvent is MeCN, CH.sub.2Cl.sub.2, or Toluene; or a mixture of two or all three thereof, and wherein the organic acid is acetic acid.

10. The process of claim 5, wherein the ?9,11 steroid is prepared using aqueous HI, at a concentration of between 48 and 68 wt %, in toluene.

11. The process of claim 5, wherein if X is OH, the 9,11 epoxy steroid is acetylated before the treating of the 9,11 epoxy steroid with HI.

12. The process of claim 11, wherein the ?9,11 steroid obtained after treating the 9,11 epoxy steroid with HI, is de-acetylated.

13. The process of claim 5, further comprising a step of recrystallizing the ?9,11 steroid obtained, in iPrOH or a mixture of iPrOH and water, thereby preparing a pharmaceutically pure ?9,11 steroid.

14. A process of purifying Vamorolone comprising a step of re-crystallizing crude Vamorolone in isopropanol or in a mixture of water and isopropanol, thereby forming purified Vamorolone of 99.5 wt % purity or more.

15. A ?9,11 steroid produced by the process of claim 5.

16. A pharmaceutically pure ?9,11 steroid produced by the process of claim 13.

17. A pharmaceutical composition comprising the compound of claim 15.

18. A ?9,11 steroid produced by the method of claim 1.

19. a purified Vamorolone produced by the process of claim 14.

20. A pharmaceutical composition comprising the compound of claim 16.

Description

DETAILS OF THE INVENTION

[0051] As used herein, the term alkyl means a straight or branched unsaturated hydrocarbon chain; preferred are C.sub.1-C.sub.6 alkyl having from 1 to 6 carbon atoms. The definition of alkyl and C.sub.1-C.sub.6-alkyl includes for example the meanings methyl, ethyl, n-, isopropyl, n-, iso-, sec- and t-butyl, n-pentyl, n-hexyl, 1,3-dimethylbutyl, and 3,3-dimethylbutyl.

[0052] The term alkoxy refers to an alkyl (carbon and hydrogen chain) group singularly bonded to oxygen, RO; preferred are C.sub.1-C.sub.6 alkoxy groups having from 1 to 6 carbon atoms.

[0053] The term halo preferably means bromo, chloro or iodo.

[0054] Cycloalkyl groups in connection with the present invention are, unless defined otherwise, ring-shaped saturated hydrocarbon groups. Preferred are C.sub.5-C.sub.12 cycloalkyl having 5-12 ring C-atoms

[0055] Aryl in connection with the present invention is, unless defined otherwise, an aromatic hydrocarbon. Preferred is phenyl or substituted phenyl. The term heteroaryl defines an aryl, which can have one, two or more heteroatoms which are selected from O, N, P and S and can optionally be substituted by further groups. Aryl may be substituted or unsubstituted. Substituted aryl means aryl as defined above but containing one or more substituents. Preferred are substituted aryl containing one or more alkoxy substituents, for instance methoxy groups, and/or one or more halogens, such as Cl. Heteroaryl may be substituted or unsubstituted. Substituted heteroaryl means heteroaryl as defined above but containing one or more substituents. Preferred are substituted heteroaryl containing one or more alkoxy substituents, for instance methoxy groups, and/or one or more halogens, such as Cl.

[0056] The term tertiary amine denotes an amino group in which the nitrogen atom is bonded to three organic radicals, where two of these radicals may also together be part of a ring. In the present invention, NR.sup.II is a tertiary amine, wherein the tertiary amine is part of a linear or cyclic substructure. A preferred tertiary amine is a substituted or unsubstituted piperazin moiety, e.g.

##STR00019##

[0057] The phrase organic phase is abbreviated in this specification by OP, aqueous phase by AP.

[0058] The term pharmaceutical purity defines an at least 99 wt % pure compound, e.g. ?9,11 steroid, of the present invention, as determined by HPLC or other conventional methods. More preferably, the compound, e.g. ?9,11 steroid, of the present invention, is at least 99.5 wt % pure as determined by HPLC or other conventional methods. Most preferably, the compound, e.g. ?9,11 steroid, of the present invention, is at least 99.9 wt % pure as determined by HPLC or other conventional methods.

[0059] The following solvents and reagents employed in the process of the present invention are identified by the abbreviations indicated: ethyl acetate (EtOAc); acetic acid (HOAc); tetrahydrofuran (THF); dimethylsulfoxide (DMSO); triethylamine (Et.sub.3N); diisopropylethylamine (H?nigs base); methanol (MeOH); 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU); triphenylphosphine (PPh.sub.3); diisopropyl ether (iPr.sub.2O); dimethoxyethane (DME); t-butylmethyl ether (t-BuOMe); N,N-dimethylaminopyridine (DMAP); dimethylformamide (DMF); p-toluenesulfonyl chloride (TsCl); Bu.sub.3Sn.sub.2O (TBTO) Polymethylhydrosiloxane (PMHS).

[0060] The present invention comprises a process, designated Process A, deoxygenating the steroid of the formula II to form a ?.sup.9,11 steroid of the formula I, as shown in Reaction Scheme A.

##STR00020##

[0061] In Reaction Scheme A, a compound of formula II is treated with HI to form the compound of formula I. The present invention further comprises the process of Reaction Scheme A as shown above, wherein a compound of the formula IIa, II, IIc, IId or IIe is used instead of the compound of formula II.

[0062] As an example, Reaction Scheme A is shown. In Reaction Scheme A, a compound of formula IId is treated with HI to form the compound of formula Id.

##STR00021##

[0063] Most preferred is the synthesis of Vamorolone and Vamorolone Acetate (see Reaction Schemes B and B), respectively.

##STR00022##

[0064] In Reaction Scheme B, 8-DM Acetate is treated with HI to form Vamorolone Acetate.

##STR00023##

[0065] In Reaction Scheme B, 8-DM is treated with HI to form Vamorolone. Surprisingly, the primary alcohol (21-hydroxy in 8-DM) does not undergo transformation to a corresponding alkyl iodide (21-I).

[0066] According to the present invention, the deoxygenation reactions of the present invention with HI can take place in any organic solvent. The solvent choice, however, is limited in as far as the solvent needs to be stable towards strong acid (HI) and the potentially formed 12, and should also not interfere with any of the reaction intermediates (e.g. iodohydrin). This reduces the number of solvent choices.

[0067] Any aromatic solvent, any chlorinated solvent (CH.sub.2Cl.sub.2, chloroform, 1,2-DCE etc.) and any nitrile solvent or mixtures of any of these solvents can be used. Preferred aromatic solvents are toluene, xylene, benzene, PhCF.sub.3. Preferred chlorinated solvents are CH.sub.2Cl.sub.2, chloroform, 1,2-DCE. Preferred nitrile solvents are MeCN, propionitrile, butyronitrile

[0068] Three solvents are most preferred according to the present invention, i.e. toluene, MeCN and CH.sub.2Cl.sub.2. These solvents can be used alone or in mixtures of any of the three or a mixture of all tree solvents.

[0069] Alcoholic solvents, which are stable in the presence of HI, can be used as well. For example, CF.sub.3CH.sub.2OH and HFIP (hexafluoroisopropanol) may be used.

[0070] Etheral solvents, which are stable in the presence of HI, can be used as well. For example, DIPE (di-isopropylether) may be used.

[0071] The reaction can also be performed directly in an organic acid without any other solvent, e.g. directly in formic acid, acetic acid (i.e. AcOH), TFA etc. Examples of organic acids according to the present invention are acetic acid, TFA, formic acid, and propionic acid.

[0072] More preferably, the organic acid is AcOH. Organic acids, preferably AcOH, are good solvents for the reaction, as they facilitate elimination of the hydroxy-group at C11. However, in this embodiment, preferably an organic solvent (e.g. toluene, MeCN and CH.sub.2Cl.sub.2) needs to be added prior to quenching of the formed iodine with aqueous Na.sub.2SO.sub.3, to avoid by-product formation of insoluble residues.

[0073] Another solvent according to the present invention is H.sub.2O, wherein the reaction works as well. Preferably, H.sub.2O is used in a mixture with any of the organic acids as mentioned above. However, in H.sub.2O without organic acids, I.sub.2 will be formed and will precipitate from H.sub.2O. This creates the need of removing precipitated I.sub.2, quantitatively.

[0074] The reagent hydroiodic acid, HI, is preferably used in a high concentration aqueous form, more preferably in a concentration of 1%-70%, preferably 5%-70%, more preferably 10%-70%, even more preferably 30%-70% aqueous HI by mass, most preferably 48-57% aqueous HI or 64%-68% aqueous HI by mass.

[0075] The deoxygenation reaction with hydroiodic acid is performed at temperatures below room temperature RT (i.e. 25? C.), preferably below 15? C., more preferably below 10? C., most preferably below 5?, e.g. between 1 and 5? C.

[0076] To purify the compound of formula II, the final compound is recrystallized. Recrystallization may be performed from polar solvents such as water or alcohols and mixtures thereof and alternatively using Acetonitrile, Acetone, Methyl ethyl ketone (MEK), Methyl isopropyl ketone (MIK) and mixtures thereof. Preferably, re-crystallization is performed from isopropanol (iPr-OH) or a mixture of iPr-OH and water. More preferably recrystallization takes place in a mixture of iPr-OH: water between 60:40 wt % and 100:0 wt %, preferably between 80:20 wt % and 100:0 wt %

[0077] Typically the last crystallization of crude Vamorolone from isopropanol or isopropanol water mixtures is able to provide a purity upgrade of more than 1 wt %, e.g. from 98.5 wt % to between 99.6 wt % and 99.9 wt % while still providing good yields above 90%. This final polishing step by crystallization allows excellent control of the impurity profile of the final API.

[0078] In a further aspect of the present invention a protecting group for the primary hydroxy group on C-21 can be added in place of moiety X before the deoxygenation of formula II, IIa, IIb, IIc, IId, IIe or IIf, respectively.

[0079] The protecting group in place of X, is OR, wherein R is C(O)R.sup.I, wherein R.sup.I is CF.sub.3, C.sub.1-C.sub.6 alkyl, C.sub.5-C.sub.12 cycloalkyl, aryl, heteroaryl or C.sub.1-C.sub.6 alkoxy.

[0080] After the addition of the protecting group, the deoxygenation according to the present invention is performed, followed by a de-protection step, i.e. a step in which the protecting group is removed.

[0081] In this step moiety X, which is OR, wherein R is C(O)R.sup.I, and R.sup.I is CF.sub.3, C.sub.1-C.sub.6 alkyl, C.sub.5-C.sub.12 cycloalkyl, aryl, heteroaryl or C.sub.1-C.sub.6 alkoxy, is converted into X, which is OH. OH can furthermore be protected using methoxymethyl ether, tetrahydropyranyl ether, t-butyl ether, benzyl ether, dimethoxybenzyl ether, t-butyldimethylsilyl ether, t-butyldiphenylsilyl ether, acetic acid ester, pivalic acid ester, benzoic acid ester, acetonide or benzylidene acetal or any other conventional protecting groups.

[0082] In a preferred embodiment, 8-DM is acetylated before the deoxygenation with HI. The obtained 8-DM Acetate is deoxygenated with HI to Vamorolone Acetate and subsequently de-acetylated to form Vamorolone. If Vamorolone Acetate is partially deacetylated during the HI reaction, it is optional to re-acetylate the Vamorolone partially de-acetylated, again. Quantitative acetylation may be intended to quantitatively isolate Vamorolone Acetate and obtain a quantitative total synthesis of Vamorolone, if the synthesis involves Vamorolone Acetate formation by adding HI to 8-DM acetate.

[0083] The acetylation procedure can be performed using known reaction conditions specified in this patent in the experimental section or conditions known to the skilled person. However, particularly preferred is a reaction using Ac.sub.2O in an organic solvent, preferably in Acetonitrile. Most preferably, AC.sub.2O in Acetonitrile is used with catalytic amounts of DMAP. The 8-DM-Acetate may be obtained as a crystalline product after aqueous quenching. The acetylation may be performed at room temperature. Preferred temperature ranges are between 20 and 30? C., more preferably 22-25? C.

[0084] The de-acetylation hydrolysis procedure can be performed using reactions known in the art. However, particularly preferred is de-acetylation with K.sub.2CO.sub.3 in an alcohol or an alcohol/water-mixture. Most preferred is K.sub.2CO.sub.3 in MeOH/H.sub.2O. In a further embodiment, Vamorolone Acetate is de-acetylated with KOH in an alcohol or an alcohol/water-mixture. Most preferred is KOH in MeOH.

[0085] The temperature of the de-acetylation reaction is lower than RT, preferably lower than 15? C., more preferably lower than 10? C., most preferably between 0?-5? C.

[0086] In a preferred embodiment of the present invention, the ?9,11 steroid is prepared by deoxygenation of a respective 9,11-epoxy steroid using HI, wherein the ?9,11 steroid is prepared using aqueous HI at a concentration of between 48 and 68 wt %, in MeCN, CH.sub.2Cl.sub.2, or Toluene; or a mixture of two or all three thereof, in the presence or absence of an organic acid, wherein treatment with HI is carried out at a temperature of below 15? C.

[0087] In a more preferred embodiment of the present invention, the ?9,11 steroid is prepared by deoxygenation of a respective 9,11-epoxy steroid using HI, wherein the ?9,11 steroid is prepared using aqueous HI at a concentration of between 48 and 68 wt %, in MeCN, CH.sub.2Cl.sub.2, or Toluene; or a mixture of two or all three thereof, in the presence or absence of an organic acid, which is acetic acid, wherein treatment with HI is carried out at a temperature of below 15? C.

[0088] In an even more preferred embodiment of the present invention, the ?9,11 steroid is prepared by deoxygenation of a respective 9,11-epoxy steroid using HI, wherein the ?9,11 steroid is prepared using aqueous HI at a concentration of between 48 and 68 wt %, inToluene; in the presence or absence of an organic acid, which is acetic acid, wherein treatment with HI is carried out at a temperature of below 15? C.

[0089] In an even more preferred embodiment of the present invention, the ?9,11 steroid is prepared by deoxygenation of a respective 9,11-epoxy steroid using HI, wherein the ?9,11 steroid is prepared using aqueous HI at a concentration of between 48 and 68 wt %, in toluene; in the presence or absence of an organic acid, which is acetic acid, wherein treatment with HI is carried out at a temperature of below 15? C., wherein if X is OH, the 9,11 epoxy steroid is acetylated before the treating of the 9,11 epoxy steroid with HI. In this embodiment, the ?9,11 steroid obtained after treating the 9,11 epoxy steroid with HI, is preferably de-acetylated.

[0090] Most preferably, the processes of the present invention produce Vamorolone or Vamorolone Acetate, more preferably from the commercial 8-DM.

[0091] The ?9,11 steroids obtained by the processes of the present invention are ideally pharmaceutically pure ?9,11 steroids. Purification can be performed by recrystallization of any of the ?9,11 steroids obtained by any of the processes of the present invention, which are typically obtained as crude ?9,11 steroids, in iPrOH or a mixture of iPrOH and water.

EXAMPLES

Example 1: Unsuccessful Attempts

1.1 Unsuccessful Conditions

[0092] Deoxygenation was tried but found to be unsuccessful with the following reactions: [0093] a) Rhenium-catalysts: CH.sub.3ReO.sub.3, Re.sub.2O.sub.7 or perrhenic acid) in combination with Triphenylphosphite P(OPh) 3 in toluene at reflux, [0094] b) Electrochemical Reduction with aq. NH.sub.4Br/THF with Zn-electrodes. Constant Current, 30 mA, and [0095] c) PPh.sub.3 & I.sub.2 in MeCN.

[0096] None of these reactions known from the literature turned out to be successful.

1.2 Useful but Complex and Toxic Reaction with HBr

[0097] Alternative deoxygenation conditions using HBr followed by tin mediated PMHS reduction and subsequent water elimination involves large quantities of a tin reagent and liquid sulfur dioxide as the solvent for the elimination step. Neither the use of tin nor toxic gases as solvents or reagents is desirable for the downstream manufacturing of an API if it can be avoided.

[0098] Thus there is a need for a new process to circumvent the use of hazardous and toxic chemicals towards the end of the chemical sequence to Vamorolone.

[0099] Furthermore, the HBr route to Vamorolone has the potential to form PMIs (Potentially Mutagenic Impurities) such as allylic bromides of Vamorolone that could represent a patient risk. These impurities are hard to trace and must be controlled rigorously to very low contamination levels.

##STR00024## ##STR00025##

[0100] The steps of scheme 2 turned out to be possible, but unintended for several reasons: Debromination with Bu.sub.2Sn.sub.2O comprises the in situ generation of Bu.sub.3SnH, which is toxic. Bu.sub.3Sn.sub.2O (TBTO) used as biocid in ship paintings to reduce biofouling. TBT is highly toxic itself towards nontarget organisms. Toxic effects occur already at 1 nano-gram per liter of water. SO.sub.2 is toxic and it represents a safety hazard in a manufacturing plant and needs specific safety installations, controls and waste treatment facilities.

1.3 Useful but Imperfect Reaction with TMSI

[0101] Deoxygenation was tried, but found to be uncomfortable, by use of NaI (3.0 eq.) & TMSCl (1.5 eq.) in MeCN. After in situ formation of TMS-iodohydrin, and subsequent elimination the deoxygenation took place:

[0102] However, incomplete transformation of 8-DM-Acetate and a non-satisfactory resulting impurity profile required a new solution. Thus, the inventors sought to provide a new, even simpler and more straight forward and scalable synthesis.

Example 2: Synthesis of the Present Invention

[0103] ##STR00026##

[0104] Vamorolone was synthesized in three synthetic steps from commercially available 8-DM.

[0105] The synthetic route started with the acetylation of 8-DM using acetic anhydride and catalytic DMAP in THF, followed by crystallization of 8-DM Acetate after aqueous quench. Then, a deoxygenation reaction converted 8-DM Acetate directly into Vamorolone Acetate. This deoxygenation proceeded via initial formation of an iodohydrin with excess aq. HI, followed by simultaneous I.sub.2 and H.sub.2O-elimination to give Vamorolone Acetate. During the reaction, partial de-acetylation occurred (20-25%) and therefore re-acetylation with acetic anhydride was necessary. After completed re-acetylation, Vamorolone Acetate was directly crystallized by addition of H.sub.2O. Finally, the acetate group is cleaved under basic conditions to give crude Vamorolone, which was recrystallized from iPrOH to obtain the pure product.

##STR00027##

[0106] A 10 L glass dj (double jacketed reactor)-reactor was charged with 8-DM (490 g, 1.32 mol, 1.0 eq.) and DMAP (16.1 g, 0.132 mmol, 0.10 eq.). THF (1.25 L, 2.5 vol.) was added at IT=20-25? C. Then, Ac.sub.2O (201 g, 187 mL, 1.97 mol, 1.5 eq.) was added dropwise over 20-40 min, keeping IT below 30? C. during the addition. After complete addition, the reaction mixture was stirred at IT=20-25? C. for 30 min. IPC control by LC/MS indicated >99% conversion of 8-DM to 8-DM Acetate.

[0107] The reaction mixture was quenched by dropwise addition of H.sub.2O (4.9 L, 10 vol.) over 30-45 min, keeping IT below 25? C. The resulting aqueous suspension was aged at IT=20-25? C. for 1 h. The product was filtered off, washed with H.sub.2O (3?0.5 L), and dried on a rotary evaporator (900-10 mbar, 65? C. bath temperature) to provide 8-DM Acetate (539 g, 1.30 mol, 99% yield, >99% a/a, 98% w/w) as a white solid (cryst 1 #1).

Analytical Data:

[0108] LC/MS column: Zorbax RRHD SB-Aq, 2.1?50 mm, 1.8 ?m [0109] Program: G_005% B_TFA_0,800 ml_2.00 min [0110] Eluent A: Water/TFA 100:0.04, Eluent B: Acetonitrile [0111] IPC preparation for LC/MS [0112] 10 microliter in 1 mL H2O:MeCN 1:1 [0113] Conversion was determined with respect to consumption of 8-DM relative to formation of 8-DM Acetate. [0114] Detected mass: [M+1]=373.19 for 8-DM and [M+1]=415, 19 8-DM Acetate.

##STR00028##

[0115] A 10 L glass dj-reactor was charged with 8-DM Acetate (500 g, 1.21 mol, 1.0 eq.). Toluene (2.5 L, 5 vol.) was added. The suspension was cooled to IT=0-5? C. and then a solution of 57% aqueous HI (1.08 kg, 637 mL, 4.83 mol, 4.0 eq.) in AcOH (1.25 L, 2.5 vol.) was added via peristaltic pump over 45-60 min, keeping IT below 5? C. during the addition. The resulting dark purple to brown solution was stirred at IT=3-5? C. for 24 h. IPC control by LC/MS indicated >98% conversion of 8-DM Acetate/intermediate iodohydrin to Vamorolone Acetate/Vamorolone.

[0116] The reaction mixture was quenched by dropwise addition of 25% aq. Na.sub.2SO.sub.3 solution (2.0 L, 4 vol.) over 10-20 min, keeping IT below 15? C. After complete addition, EtOAc (1.0 L, 2 vol.) was added and the biphasic mixture was warmed to IT=15-20? C. Stirring was stopped and the phases were separated (Organic Phase 1 and aqueous Phase 1; goal pH of the aqueous Phase 1:2; aqueous Phase 1 disposed). 25% aq. Na.sub.2SO.sub.3 solution (1.25 L, 2.5 vol.) was added to Organic Phase 1 and the biphasic mixture was stirred at IT=15-20? C. for 5 min, stirring was stopped and phases separated (Organic Phase 1 and aqueous Phase 2; goal pH aqueous Phase 2:4-5; aqueous Phase 2 disposed). 25% aq. Na.sub.2SO.sub.3 solution (1.25 L, 2.5 vol.) was added to Organic Phase 1 and the biphasic mixture was stirred at IT=15-20? C. for 5 min, stirring was stopped and phases separated (Organic Phase 1 and aqueous Phase 3; goal pH aqueous Phase 3:5-6; aqueous Phase 3 disposed). H.sub.2O (0.5 L, 1.0 vol.) was added to Organic Phase 1 and the biphasic mixture was stirred at IT=15-20? C. for 5 min, stirring was stopped and phases separated (Organic Phase 1 and aqueous Phase 4; goal pH aqueous Phase 4:5-6; aqueous Phase 4 disposed).

[0117] A slight vacuum was applied to the double-jacketed reactor (100-150 mbar), containing Organic Phase 1, and toluene was distilled off at 70? C. jacket temperature (ET) from the reaction mixture with continuous addition of MeCN, and the distillation continued until target residual toluene value has been reached (goal: less than 5% toluene according to 1H-NMR of reaction mixture. Final volume in reactor after distillation: ca. 3.5 L (7.5 vol.).

[0118] Once toluene was removed, the vacuum was broken with N.sub.2 and resulting fine suspension cooled to IT=20-25? C. At this point, the amount of Vamorolone was assessed by IPC (typical ratio: Vamorolone Acetate to Vamorolone:75:25; x=25% a/a). DMAP (3.7 g, 0.0302 mol, 0.025 eq.) was added, followed by slow addition of Ac.sub.2O (61.6 g, 57 mL, 0.603 mol, 0.5 eq.) over 5-10 min at IT=20-25? C. After complete addition of Ac.sub.2O, the reaction mixture was stirred for 30 min at IT=20-25? C. IPC control by LC/MS indicated ?2% a/a Vamorolone (ratio: Vamorolone Acetate to Vamorolone:98.5:1.5).

[0119] The reaction mixture was quenched by slow addition of H.sub.2O (4.9 L, 10 vol.) over 15-30 min, keeping IT below 25? C. The resulting aqueous suspension was cooled to IT=0-5? C. and aged at this temperature for 2 h. The product was filtered off, washed with H.sub.2O/MeCN 4:1 (2?0.5 L), and dried on a rotary evaporator (900-10 mbar, 65? C. bath temperature) to provide Vamorolone Acetate (301 g, 0.76 mol, 63% yield, 98% a/a, 98% w/w) as an off-white solid (cryst 1 #1).

[0120] Over the course of the reaction, partial de-acetylation of Vamorolone Acetate to Vamorolone was observed (between 20-25% a/a). Therefore, after aq. workup and solvent switch to MeCN, the ratio of Vamorolone Acetate to Vamorolone was assessed by LC/MS (in % a/a), and the following amounts of DMAP and Ac2O were added: [0121] x=amount of Vamorolone in % a/a (e.g. x=20% a/a) [0122] DMAP eq.=(0.1.Math.x)/100 (e.g. 0.02 eq.) [0123] Ac2O eq.=(2.0.Math.x)/100 (e.g. 0.40 eq.)

Analytical Data

[0124] LC/MS column: Zorbax RRHD SB-Aq, 2.1?50 mm, 1.8 ?m [0125] Program: G_005% B_TFA_0,800 ml_2.00 min [0126] Eluent A: Water/TFA 100:0.04, Eluent B: Acetonitrile [0127] IPC preparation for LC/MS [0128] 10 microliter in 1 mL H.sub.2O:MeCN 1:1 [0129] Conversion was determined with respect to consumption of the sum of (8-DM Acetate+intermediate iodohydrin) relative to the sum of (Vamorolone Acetate+Vamorolone). [0130] Detected mass: [M+1]=415.19 for 8-DM Acetate, [M+1]=357.28 for Vamorolone, 399.20 for Vamorolone Acetate and 543.12 for intermediate lodohydrin

2.3 De-Acetylation

[0131] ##STR00029##

[0132] A 10 L glass dj-reactor was charged with Vamorolone Acetate (280 g, 0.703 mol, 1.0 eq.). MeOH (1.54 L, 5.5 vol.) was added. The suspension was cooled to IT=0-5? C. and then a solution of K.sub.2CO.sub.3 (107 g, 0.773 mol, 1.1 eq.) in H.sub.2O (0.7 L, 2.5 vol.) was added dropwise via peristaltic pump over 20-40 min, keeping IT below 10? C. during the addition. After complete addition, the reaction mixture was warmed IT=20-25? C. and stirred for 5 h. IPC control by LC/MS indicated 99.3% conversion of Vamorolone Acetate to Vamorolone.

[0133] The reaction mixture was cooled to IT=15-17? C. and quenched by dropwise addition of 1 M aq. HCl (950 mL, 0.95 mol, 1.35 eq.) over 20-40 min, keeping IT below 20? C. during the addition (goal pH: 5-6). The resulting aqueous suspension was aged at IT=15-20? C. for 12 h. The product was filtered off, washed with H.sub.2O/MeOH 2:1 (3?0.3 L), and dried on a rotary evaporator (900-10 mbar, 65? C. bath temperature) to provide Vamorolone (241.5 g, 0.68 mol, 96% yield, >99% a/a, 98% w/w) as a slightly yellow solid (crude 1 #1).

Analytical Data

[0134] LC/MS column: Zorbax RRHD SB-Aq, 2.1?50 mm, 1.8 ?m [0135] Program: G_005% B_TFA_0,800 ml_2.00 min [0136] Eluent A: Water/TFA 100:0.04, Eluent B: Acetonitrile [0137] IPC preparation for LC/MS [0138] 10 microliter in 1 mL H.sub.2O:MeCN 1:1 [0139] Conversion was determined with respect to consumption of Vamorolone Acetate relative to formation of Vamorolone.

2.4 Recrystallization

[0140] ##STR00030##

[0141] A 10 L glass dj-reactor was charged with Vamorolone (230 g, 0.645 mol, 1.0 eq.). iPrOH (5 L, 22 vol.) was added. The suspension was heated to reflux (jacket temperature ET=97? C.) and stirred until complete dissolution of Vamorolone occurred (10-15 min on this scale). After complete dissolution, the clear yellow solution was slowly cooled to IT=0-5? C. over the course of 12 h and then aged at IT=0-5? C. for 1 h. The recrystallized product was filtered off, washed with cold iPrOH (2?250 mL), and dried on a rotary evaporator (900-10 mbar, 65? C. bath temperature) to provide Vamorolone (201 g, 87% recovery, >99% a/a, 99% w/w) as an off white glimmery solid (cryst 1 #1).

[0142] Iso-propanol (iPrOH) was found to the best solvent for recrystallization with excellent purity upgrading properties (by rejection of impurities), although a high dilution is necessary to completely dissolve the crude Vamorolone at reflux temperature. Higher concentrations for the recrystallization satisfactory results are obtainable using mixtures of isopropanol and water. Maximum solubility of Vamorolone was determined to be at reflux of a 80:20 (isopropanol:water) mixture.

2.5 Improving Purity by Charcoal Treatment

[0143] To improve Vamorolone Acetate purity, a charcoal treatment was envisaged. Two options were found to be useful.

Option 1: Charcoal Treatment on Isolated Vamorolone Acetate

[0144] Vamorolone Acetate (10 g) from HI step (ELN293-1469.1) was suspended in MeCN (100 mL, 10 vol.) and H.sub.2O (10 mL, 1 vol.). The suspension was heated to IT=60-65? C. and stirred until Vamorolone Acetate was completely dissolved. Then, charcoal (1.0 g, 10% w/w) was added and stirred for 1 h at IT=60-65? C. The mixture was filtered hot through a Whatman glass microfiber filter into a 500 mL round-bottom flask. Additional H.sub.2O (90 mL, 9 vol.) was slowly added to induce crystallization of Vamorolone Acetate. The suspension was slowly cooled to 0? C. and aged for 1 h. The white solid was filtered off, washed with additional MeCN:H2O 1:4 (2?10 mL) and dried under reduced pressure on the rotavap (65? C.) for extended amount of time (9.0 g, 90% recovery).

Option 2: Charcoal Treatment During HI Step

[0145] A 1 L glass dj-reactor was charged with 8-DM Acetate (30.0 g, 72.4 mmol, 1.0 eq.). Toluene (150 mL, 5 vol.) was added. The suspension was cooled to IT=0-5? C. and then a solution of 57% aqueous HI (65.0 g, 38.2 mL, 290 mmol, 4.0 eq.) in AcOH (75 mL, 2.5 vol.) was added via peristaltic pump over 45-60 min, keeping IT below 5? C. during the addition. The resulting dark purple to brown solution was stirred at IT=3-5? C. for 24 h. IPC control by LC/MS indicated >98% conversion of 8-DM Acetate/intermediate iodohydrin to Vamorolone Acetate/Vamorolone.

[0146] The reaction mixture was quenched by dropwise addition of 25% aq. Na.sub.2SO.sub.3 solution (120 mL, 4 vol.) over 10-20 min, keeping IT below 15? C. After complete addition, EtOAc (60 mL, 2 vol.) was added and the biphasic mixture was warmed to IT=15-20? C. Stirring was stopped and the phases were separated (Organic Phase 1, i.e. OP1 and aqueous Phase 1, i.e. AP1; goal pH AP1:2; AP1 disposed). 25% aq. Na.sub.2SO.sub.3 solution (75 mL, 2.5 vol.) was added to OP1 and the biphasic mixture was stirred at IT=15-20? C. for 5 min, stirring was stopped and phases separated (OP1 and AP2; goal pH AP2:4-5; AP2 disposed). 25% aq. Na2SO3 solution (75 mL, 2.5 vol.) was added to OP1 and the biphasic mixture was stirred at IT=15-20? C. for 5 min, stirring was stopped and phases separated (OP1 and AP3; goal pH AP3:5-6; AP3 disposed). H2O (30 mL, 1.0 vol.) was added to OP1 and the biphasic mixture was stirred at IT=15-20? C. for 5 min, stirring was stopped and phases separated (OP1 and AP4; goal pH AP4:5-6; AP4 disposed).

[0147] A slight vacuum was applied to the double-jacketed reactor (100-150 mbar), containing OP1, and toluene was distilled off at 70? C. jacket temperature (ET) from the reaction mixture with continuous addition of MeCN, and the distillation continued until target residual toluene value has been reached (goal: less than 5% toluene according to 1H-NMR of reaction mixture. Final volume in reactor after distillation: ca. 225 mL (7.5 vol.))

[0148] Once toluene was removed, the vacuum was broken with N.sub.2 and resulting fine suspension cooled to IT=20-25? C. At this point, the amount of Vamorolone was assessed by IPC (ratio: Vamorolone Acetate to Vamorolone:75:25; x=25% a/a). DMAP (221 mg, 1.81 mmol, 0.025 eq.) was added, followed by slow addition of Ac.sub.2O (3.7 g, 3.4 mL, 36.2 mmol, 0.5 eq.) over 2-3 min at IT=20-25? C. After complete addition of Ac.sub.2O, the reaction mixture was stirred for 30 min at IT=20-25? C. IPC control by LC/MS indicated less than 1% a/a Vamorolone.

[0149] The reaction mixture was quenched by slow addition of H.sub.2O (30 mL, 1 vol.) over 5-10 min. The suspension was heated to IT=60-65? C. and stirred until complete dissolution has occurred. Then, charcoal (3.0 g, 10% w/w) was added and stirred for 1 h at IT=60-65? C. The mixture was filtered hot through a Whatman glass microfiber filter into a 1 L round-bottom flask. Additional H.sub.2O (195 mL, 6.5 vol.) was slowly added to induce crystallization of Vamorolone Acetate. The suspension was slowly cooled to 0? C. and aged for 1 h. The white solid was filtered off, washed with additional MeCN:H.sub.2O 1:4 (2?40 mL) and dried under reduced pressure at the rotavap (65? C.) for extended amount of time (18.7 g, 46.9 mmol, 65% yield, >99% a/a).