PROCESS FOR PREPARING INTERMEDIATES FOR THE SYNTHESIS OF OPTICALLY ACTIVE BETA-AMINO ALCOHOLS BY ENZYMATIC REDUCTION AND NOVEL SYNTHESIS INTERMEDIATES

Abstract

Subject-matter of the present invention is a process for preparing intermediates for the synthesis of optically active beta-amino alcohols by enzymatic reduction of the corresponding beta-amino ketones. Subject-matter of the invention are also said novel synthesis intermediates and the use thereof in the preparation of active pharmaceutical ingredients, among which vilanterol and the salts thereof.

Claims

1. A process for preparing an optically active compound of Formula (I) ##STR00021## wherein R.sub.1 and R.sub.2 are independently selected from hydrogen, OPr, CH.sub.2OPr, OH, CH.sub.2OH and C.sub.1-C.sub.4-alkoxy; wherein Pr is a hydroxy protective group and, when two Pr protective groups are in the compound of formula (I): said two Pr protective groups can be the same or different from one another; or said two Pr protective groups, together with the oxygen atoms to which they are bound, may form a cycle fused with the benzene; R.sub.3 and R.sub.4, together with the carboxamide group (HNCO) bonding them, constitute an imide, said process comprising the enzymatic reduction of the compound of Formula (II) ##STR00022## wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are as defined above.

2. The process according to claim 1, wherein said two Pr protective groups, together with the oxygen atoms to which they are bound, form a cycle fused with the benzene.

3. The process according to claim 2, wherein said fused cycle has the following formula ##STR00023##

4. The process according to claim 1, wherein said imide is succinimide.

5. The process according to claim 1, wherein said enzymatic reduction is carried out by at least one oxidoreductase enzyme.

6. The process according to claim 5, wherein said oxidoreductase enzyme has a sequence selected from the sequences SEQ ID No: 1 and SEQ ID No: 2 or has an amino acid sequence having at least 60% of the amino acids identical to the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO:2.

7. The process according to claim 6, wherein said oxidoreductase enzyme has the sequence SEQ ID No: 1.

8. The process according to claim 1, wherein said enzymatic reduction is carried out by at least one oxidoreductase enzyme, in the presence of at least one cofactor and at least one co-substrate regenerating said cofactor.

9. A compound selected from the compounds of formula B, C, Y, Z ##STR00024## and the salts and/or hydrates and/or solvates thereof.

10. (canceled)

11. The process according to claim 3, wherein said imide is succinimide.

12. The process according to claim 5, wherein said oxidoreductase enzyme has an amino acid sequence having at least 80% of the amino acids identical to the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO:2.

13. The process according to claim 5, wherein said oxidoreductase enzyme has an amino acid sequence having at least 90% of the amino acids identical to the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO:2.

Description

DESCRIPTION OF THE INVENTION

[0017] The Applicant has surprisingly found that, by using a beta-amino ketone wherein the amine group has lost its basicity, for example by transforming to an amide, imide or carbamate group, it is possible to carry out its enzymatic reduction and obtain optically active amino-alcohols, with high yields and purities.

[0018] Thus, according to one of its aspects, subject-matter of the invention is a process for preparing an optically active compound of Formula (I)

##STR00002##

[0019] wherein [0020] the asterisk indicates the chiral carbon is in the (S) form or (R) form; [0021] X represents an amide, imide or carbamate residue bound to the compound of formula (I) through the nitrogen atom; [0022] R.sub.1 and R.sub.2 are independently selected from hydrogen, OPr, CH.sub.2OPr, OH, CH.sub.2OH and C.sub.1-C.sub.4-alkoxy; wherein Pr is a hydroxy protective group and, when two Pr protective groups are in the compound of formula (I): [0023] said two Pr protective groups can be the same or different from one another; or [0024] said two Pr protective groups, together with the oxygen atoms to which they are bound, may form a cycle fused with the benzene;

[0025] said process comprising the enzymatic reduction of the compound of Formula (II)

##STR00003##

[0026] wherein X, R.sub.1 and R.sub.2 are as defined above.

[0027] According to a preferred embodiment, the compound of Formula (I) is a compound of Formula (I)

##STR00004##

[0028] wherein [0029] the asterisk, R.sub.1 and R.sub.2 are as defined above; [0030] R.sub.3 is selected from an hydrogen, alkyl, alkoxy, aryl, alkylaryl and arylalkyl; [0031] R.sub.4 is selected from hydrogen, alkyl, aryl, alkylaryl and arylalkyl;

[0032] or else [0033] R.sub.3 and R.sub.4, together with the carboxamide group (HNCO) bonding them, constitute an imide.

[0034] The expression chiral carbon is in the (S) form or in the (R) form herein means that at least 80%, preferably at least 90-95%, more preferably 98-99.9% of the compound of Formula (I) and (I) is in said configuration.

[0035] According to a preferred embodiment, the chiral carbon of the compound of Formula (I) and (I) is in the (R) configuration.

[0036] The term alkyl herein means a saturated, linear or branched, alkyl residue, preferably having 1 to 6 carbon atoms, advantageously 1 to 4 carbon atoms, for example the methyl or ethyl group.

[0037] The term alkoxy herein means a saturated, linear or branched, alkoxy residue, preferably having 1 to 6 carbon atoms, advantageously 1 to 4 carbon atoms, for example the methyl or ethyl group.

[0038] The term aryl herein means an aromatic hydrocarbon residue, preferably selected from phenyl and naphthyl.

[0039] The term alkylaryl herein means an aromatic hydrocarbon residue, preferably selected from phenyl and naphthyl, substituted with one or more alkyls, as defined above.

[0040] The term arylalkyl herein means a saturated, linear or branched, alkyl residue, preferably having 1 to 6 carbon atoms, advantageously 1 to 4 carbon atoms as defined above, substituted with one or more aryls, as defined above.

[0041] The expression hydroxy protective group means any protective group able to protect the hydroxy function without interfering with the reduction reaction. Such groups can be for example selected from those mentioned in T. W. Greene, John Wiley & Sons, Ltd, Protective Groups in Organic Synthesis, 5th edition, 2014. According to a preferred embodiment of the invention, the OPr groups, as defined above, include esters and acetonides.

[0042] Other protective groups according to the present invention include the silanes, for example the silyl group; alternatively, when there are two Pr groups, said silane protective groups, together with the two oxygen atoms to which they are bound, may form a cycle fused with the benzene.

[0043] According to another embodiment, when the groups R.sub.1 and R.sub.2 are on adjacent carbon atoms and there are two Pr groups, said two Pr groups represent a carbonyl group (CO) bound to the two oxygen atoms.

[0044] According to a preferred embodiment, R.sub.1 and R.sub.2 are both OH or OPr, wherein the Pr groups can be the same or different from one another, preferably the same.

[0045] According to another preferred embodiment, one of R.sub.1 and R.sub.2 is OH or OPr and the other is CH.sub.2OH or CH.sub.2OPr.

[0046] According to another preferred embodiment, R.sub.1 and R.sub.2 are different from hydrogen and are on adjacent positions on the benzene ring, preferably in the 3 and 4 positions of the benzene ring.

[0047] According to another preferred embodiment, when R.sub.1 and R.sub.2 are each independently OPr or CH.sub.2OPr, they form a cycle fused with the benzene ring; in this case, according to a more preferred embodiment, they are on adjacent positions of the benzene ring.

[0048] According to a preferred embodiment, R.sub.3 is hydrogen, saturated and linear C.sub.1-C.sub.6 alkyl, preferably methyl or ethyl and R.sub.4 is hydrogen or methyl.

[0049] According to another preferred embodiment, R.sub.3 and R.sub.4, together with the carboxamide group bonding them, represent a succinimide or a phthalimide possibly substituted, advantageously the succinimide or substituted succinimide, for example with one or more fluorine atoms.

[0050] Preferred compounds of Formula (II) are selected from the following:

##STR00005##

[0051] wherein X is a substituent selected from

##STR00006##

[0052] wherein ALK represents an alkyl as defined above, advantageously methyl or ethyl, said substituent X being bound to the molecule through the nitrogen atom, as depicted by the dashed line.

[0053] According to a particularly preferred embodiment, subject-matter of the invention is a process for preparing an optically active compound of Formula (I)

##STR00007##

[0054] wherein [0055] the asterisk, R.sub.1 and R.sub.2 are as defined above; [0056] R.sub.3 and R.sub.4, together with the carboxamide group (HNCO) bonding them, constitute an imide,

[0057] said process comprising the enzymatic reduction of the compound of Formula (II)

##STR00008##

[0058] wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are as defined above.

[0059] According to the last embodiment, preferably the Pr groups form a cycle fused with the benzene, advantageously having the following formula:

##STR00009##

[0060] Still according to the last embodiment, preferably said imide is succinimide.

[0061] The compounds of Formula (II) and (II) can be easily prepared according to the methods known in the art.

[0062] The process of the invention preferably uses at least one oxidoreductase enzyme, which is preferably a polypeptide originating from yeasts or bacteria, advantageously from bacteria.

[0063] The enantioselective enzymatic reduction can be carried out by using the oxidoreductase enzyme in suspension in a reaction mixture, or immobilized according to known techniques. The enzyme can be purified, only partially purified, or can be contained in cells. The cells themselves can be in a native state, in a permeabilized state or in a lysed state. Preferably, the enzyme is expressed in E. coli and used as a suspension of native cells.

[0064] According to a preferred embodiment, said enzymatic reduction is carried out with at least one oxidoreductase enzyme, in the presence of at least one cofactor and at least one co-substrate that regenerates said cofactor.

[0065] The process of enzymatic reduction of the compounds of Formula (II) or (II) can be carried out for example in a reaction mixture comprising said compound of Formula (II), an oxidoreductase enzyme, NADH or NADPH as cofactor, a co-substrate.

[0066] According to a preferred embodiment, said at least one oxidoreductase enzyme that is used in the process of the invention, has a sequence selected from the sequences SEQ ID No: 1, herein below also indicated as OX 56, described in EP1963516 (IEP) and SEQ ID N.sup.o: 2 herein below also indicated as OX 62, described in EP1929001 (BASF).

[0067] According to a more preferred embodiment, said at least one oxidoreductase enzyme that is used in the process of the invention has the sequence SEQ ID No: 1.

[0068] The sequences are described in the sequence listing herein attached and depicted below:

TABLE-US-00001 SEQIDNO.1 MetArgLeuLysGlyLysAlaAlaIleValThrGly GlyAlaSerGlyIleGlyArgAlaThrAlaIleArg PheAlaGluGluGlyAlaLysValAlaValSerAsp IleAsnGluGluGlyGlyGluGluThrValArgLeu IleArgGluLysGlyGlyGluAlaIlePheValGln ThrAspValAlaAspSerLysGlnValSerArgLeu ValGlnThrAlaValAspAlaPheGlyGlyLeuHis IleLeuPheAsnAsnAlaGlyIleGlyHisSerGlu ValArgSerThrAspLeuSerGluGluGluTrpAsp ArgValIleAsnValAsnLeuLysGlyValPheLeu GlyIleLysTyrAlaValProValMetLysGlnCys GlyGlyGlyAlaIleValAsnThrSerSerLeuLeu GlyIleLysGlyLysLysTyrGluSerAlaTyrAsn AlaSerLysAlaGlyValIleLeuLeuThrLysAsn AlaAlaLeuGluTyrGlyLysPheAsnIleArgVal AsnAlaIleAlaProGlyValIleAspThrAsnIle IleThrProTrpLysGlnAspGluArgLysTrpPro IleIleSerLysAlaAsnAlaLeuGlyArgIleGly ThrProGluGluValAlaAsnAlaValLeuPheLeu AlaSerAspGluAlaSerPheIleThrGlyAlaThr LeuSerValAspGlyGlyGlyLeuThrPhe SEQIDNo.2 MetThrThrThrSerAsnAlaLeuValThrGlyGly SerArgGlyIleGlyAlaAlaSerAlaIleLysLeu AlaGlnGluGlyTyrAsnValThrLeuAlaSerArg SerValAspLysLeuAsnGluValLysAlaLysLeu ProIleValGlnAspGlyGlnLysHisTyrIleTrp GluLeuAspLeuAlaAspValGluAlaAlaSerSer PheLysGlyAlaProLeuProAlaArgSerTyrAsp ValPheValSerAsnAlaGlyValAlaAlaPheSer ProThrAlaAspHisAspAspLysGluTrpGlnAsn LeuLeuAlaValAsnLeuSerSerProIleAlaLeu ThrLysAlaLeuLeuLysAspValSerGluArgPro ValAspLysProLeuGlnIleIleTyrIleSerSer ValAlaGlyLeuHisGlyAlaAlaGlnValAlaVal TyrSerAlaSerLysAlaGlyLeuAspGlyPheMet ArgSerValAlaArgGluValGlyProLysGlyIle HisValAsnSerIleAsnProGlyTyrThrLysThr GluMetThrAlaGlyIleGluAlaLeuProAspLeu ProIleLysGlyTrpIleGluProGluAlaIleAla AspAlaValLeuPheLeuAlaLysSerLysAsnIle ThrGlyThrAsnIleValValAspAsnGlyLeuIle Ala

[0069] It has been found that polypeptides that have an amino acid sequence having at least 60%, preferably at least 80%, advantageously at least 90% of the amino acids identical to the amino acid sequence of SEQ ID No: 1 and SEQ ID No: 2 lead to the reduction of the compound of Formula (II) or (II) in the (R) configuration with high yields and high enantiomeric selectivity. In fact, the obtained enantiomeric excess of the compound of Formula (I), (I) and (I) in the (R) configuration is at least about 90%, preferably at least about 95% and more preferably at least about 99%. According to an advantageous embodiment of the invention, the cofactor is selected from nicotinamide adenine dinucleotide phosphate (NADP) and nicotinamide adenine dinucleotide (NAD).

[0070] The cofactor is preferably in the reaction mixture at a concentration from about 0.01 mM to about 5 mM, advantageously from about 0.05 mM to about 0.5 mM. According to an advantageous embodiment of the invention the co-substrate is a secondary alcohol, preferably a secondary alcohol up to 10 carbon atoms, such as 2-propanol, 2-butanol, 2-pentanol, 4-methyl-2-pentanol, 2-heptanol and 2-octanol, preferably 2-propanol or 4-methyl-2-pentanol, more preferably 2-propanol. According to a preferred embodiment, the co-substrate is present in the reaction mixture from about 10% to about 80% (v/v), more preferably from about 10% to about 50%, advantageously at the rate of 15-25%.

[0071] Preferably the process of enzymatic reduction of the compounds of Formula (II) or (II) is carried out in a solvent, advantageously in the presence of a suitable buffer. The reaction of enzymatic reduction of the invention can be carried out in a mono-phase system or a bi-phase system of the kind of water/organic solvent, according to known techniques. In the latter case, an organic solvent that is not involved in the regeneration of the cofactor can be added to the reaction mixture. Examples of such solvents include diethyl ether, tert-butyl methyl ether, diisopropyl ether, dibutyl ether, ethyl acetate, butyl acetate, heptane, hexane or cyclohexane. Such a solvent can be present at a rate of about 1% to 50% in volume based on the volume of the reaction mixture.

[0072] When the enzyme is used as a suspension of native cells, the reaction mixture preferably contains from about 100 to 2000 g cells per kg of raw product that has been produced by the reduction.

[0073] The buffer used can, for example, be selected from potassium phosphate buffer or triethanolamine buffer, and can further comprise ions for stabilizing the enzyme, for example a source of magnesium ions. Other additives that can be present in the buffer for stabilizing the enzymes can include a polyol, such as glycerol, sorbitol and the like, sulfur compounds such as 1,4-DL-dithiothreitol, glutathione, cysteine or the like, amino acids and peptides or detergents, such as DMSO.

[0074] A preferred stabilizer for the enzyme is a polyol, in particular glycerol, that can be present at a rate of about 10-80%, preferably about 50% by weight based on the weight of the cell suspension.

[0075] The oxidized cofactor that is formed during the reduction of the compound of Formula (II) or (II) is regenerated by oxidation of the co-substrate and the oxidation can also be catalyzed by the oxidoreductase itself. Therefore, a particular practical and economical advantage of the present process is that the oxidoreductase affects both the reduction of the compound of Formula (I), (I) and (I) and the oxidation of the co-substrate, and thus it is not necessary to use other enzymes for regenerating the cofactor.

[0076] The pH of the reaction mixture, after the addition of all the components, will be between 5 and 10, preferably from 7 to 9 and optimally about 7.5. The enzymatic reduction according to the present invention is carried out at a temperature of about 10-45 C., preferably about 20-40 C., preferably about 35-40 C. The process of enantioselective reduction is convenient and eco-friendly, in addition to providing the alcohols of Formula (I), (I) and (I) with high yields and high enantiomeric selectivity.

[0077] The compound of Formula (I), (I) and (I) in the (R) configuration with high optical purity can be obtained in the presence of the oxidoreductase enzyme in the reaction conditions mentioned above in about 2 to 96 hours, preferably from about 4 to 24 hours. During the incubation, the pH of the mixture is preferably maintained within the ranges indicated above. The efficiency of the enantioselective enzymatic reduction can be expressed by the total turnover number (TTN) that is the moles of the chiral alcohol of Formula (I), (I) and (I) produced per mole of cofactor used. The TTN of the enantioselective enzymatic reduction is from about 102 to 105, preferably >103.

[0078] According to an embodiment, the at least one oxidoreductase enzyme that is used in the process of the invention, as described above, has a sequence selected from the sequences SEQ. ID No: 1 and SEQ. ID No: 2.

[0079] In addition to the sequences SEQ ID No: 1 and SEQ ID No: 2, it has been found that polypeptides that have an amino acid sequence having at least 60%, preferably at least 80%, advantageously at least 90%, identical to the amino acid sequence of SEQ ID No: 1 and SEQ ID No: 2 lead to the reduction of the compound of Formula (II) or (II) in the (R) configuration with high yields and high enantiomeric selectivity. In fact, the enantiomeric excess of the compound of Formula (I) or (I) is at least about 90%, preferably at least about 95% and more preferably at least about 99%. According to a preferred embodiment, subject-matter of the invention is a process of enantioselective reduction of the preferred compounds of Formula (II) and (II), as reported above, comprising the use of at least one enzyme selected from the sequences SEQ. ID No: 1 and SEQ. ID No: 2 or of polypeptides that have an amino acid sequence having at least 60%, preferably at least 80%, advantageously at least 90%, of the amino acids identical to the amino acid sequence of SEQ. ID No: 1 and SEQ. ID No: 2.

[0080] The compound of Formula (I), (I) and (I) can be easily transformed in the corresponding optically active beta-amino alcohol, for example by treating with bases or acids, according to the techniques well known to the person skilled in the art.

[0081] Some detailed examples of the reactions described above are reported in the following Experimental section.

[0082] Therefore it is understood that, in a completely unexpected way, it has been found that the enzymatic reduction of beta-amino ketones to give optically active beta-amino alcohols, can be carried out provided that the amine group is deprived of its basicity. This fact was not at all conceivable a priori and is an important technical progress in the field of stereoselective reductions.

[0083] The compounds of Formula (I), (I) and (I) are versatile synthesis intermediates and can be easily converted to active pharmaceutical ingredients, such as for example adrenalin, noradrenalin, vilanterol, R-salbutamol, R-colterol, R-isoproterenole, ()-arbutamine and the like.

[0084] According to another of its aspects, subject-matter of the invention is a compound of Formula (I) selected from the following compounds:

##STR00010##

[0085] and the salts and/or hydrates and/or solvates thereof.

[0086] According to another of its aspects, subject-matter of the invention is a compound of Formula (II) selected from the following compounds:

##STR00011##

[0087] and the salts and/or hydrates and/or solvates thereof.

[0088] According to another of its aspects, subject-matter of the invention is the use of at least one compound selected from the compounds of Formula (I), (I), (I), (II) or (II) for preparing a compound selected from adrenalin and noradrenalin or one of the salts thereof.

[0089] According to another of its aspects, subject-matter of the invention is the use of at least one compound selected from the compounds of Formula (I), (I), (I), (II) or (II) for preparing vilanterol or one of the salts thereof.

[0090] According to another of its aspects, subject-matter of the invention is the use of the compound having formula B, C

##STR00012##

[0091] and the salts and/or hydrates and/or solvates thereof, for preparing vilanterol.

[0092] According to another of its aspects, subject-matter of the invention is the use of the compound having formula Y, Z,

##STR00013##

[0093] and the salts and/or hydrates and/or solvates thereof, for preparing vilanterol. The examples described in the following Experimental section are provided purely by way of illustration and in no way as limiting.

EXPERIMENTAL SECTION

Abbreviations

[0094] DMF=dimethylformamide [0095] DCM=dichloromethane [0096] MTBE=methyl tert-butyl ether [0097] IPA=2-propanol [0098] MP=4-methyl-2-pentanol [0099] THF=tetrahydrofuran [0100] DMS=dimethyl sulfide

[0101] Analytic Methods [0102] UPLC purity method (Waters Acquity UPLC): [0103] Stationary phase: BEH SHIELD RP18 1.7 m 2.150 mm Column, Mobile phase: H.sub.2O+0.05% TFA, CH.sub.3CN+0.05% TFA, Gradient: 5% to 100% CH.sub.3CN. [0104] Chiral HPLC method (HPLC Agilent 1200): [0105] Stationary phase: CHIRALPAK AD-H 2504.6 mm-5*m, Mobile phase: heptane/MeOH/iPrOH 90:5:5+0.1% ethanolamine.

Preparation 1

Preparation of the Enzymatic Solution

[0106] Competent Escherichia coli cells Starb121 (DE3) (Invitrogen) have been transformed with the construct pET21a-MIX encoding for the oxidoreductases. The colonies of cells transformed with the constructs have been cultured in 200 ml LB medium (1% tryptone, 0.5% yeast extract and 1% NaCl) with 50 g/ml ampicillin or 40 g/ml kanamycin, respectively, until an optical density of 0.5 measured at 550 nm is achieved. The expression of the recombinant protein has been induced by adding isopropyl-thiogalactoside (IPTG) with a concentration of 0.1 mM. After 16 hours of induction at 25 C. and 220 revolutions/minute, the cells have been collected and frozen at 20 C. For the preparation of the enzymatic solutions, 30 g cells have been re-suspended in 150 ml triethanolamine buffer (100 mM, pH 7, 2 mM MgCl.sub.2, 10% glycerol) and homogenized by using a high pressure homogenizer. Subsequently, the enzymatic solution has been mixed with 150 ml glycerol and stored at 20 C.

Example 1

Preparation of a Compound of Formula (II) and (II)

[0107] ##STR00014##

[0108] Hal=halogen, preferably bromine or chlorine

[0109] 5 g compound A (wherein Hal=Cl) is dissolved in 50 ml DMF. 3.5 g succinimide potassium salt has been added to the solution. Upon complete reaction, 50 ml water and 100 ml DCM have been added. The organic phase has been dried over sodium sulphate, filtered and concentrated to a residue. 60 ml heptane and 10 ml DCM have been added, then the bulk was filtered, collecting 4.7 g of compound B that, in the dried form, provided 4.5 g of compound B, with HPLC purity higher than 99%.

Example 2

[0110] ##STR00015##

[0111] 1 g of compound B has been dissolved in 3 ml 2-propanol and 4 ml MTBE. 13 ml aqueous potassium buffer at pH 8, 1 mg NAD and 5 ml enzyme SEQ ID No: 1 (OX 56) have been added. The bulk has been stirred at about 35 C. until complete conversion of ketone to alcohol. The separated aqueous phase has been extracted twice with DCM, then the organic phases have been combined and concentrated to a solid. Yield 900 mg, HPLC purity>96%, S-alcohol not detectable (chiral HPLC analysis).

[0112] By operating as described in Example 2, the reactions on compound B and the following compounds have been carried out in the conditions and with the results reported in Table (I):

TABLE-US-00002 Compound Enzyme Cofactor Solvent ee (S) ee (R) B SEQ ID No.: 1 NAD MP nd >99.9% (ox56) D SEQ ID No.: 1 NAD IPA nd 100% (ox56) E SEQ ID No.: 1 NAD IPA 0.6% 99.4% (ox56) E SEQ ID No.: 1 NAD MP 0.7% 99.3% (ox56) F SEQ ID No.: 2 NAD IPA 2.4% 97.6% (ox62) F SEQ ID No.: 2 NAD MP 2.0% 98.0% (ox62) G SEQ ID No.: 1 NAD MP nd 100% (ox56) G SEQ ID No.: 1 NAD IPA nd 100% (ox56) [00016][00017][00018][00019]

Example 3

[0113] ##STR00020##

[0114] 350 mg of compound C has been dissolved in 11 ml ethanol and about 1 g of 20% caustic soda (20 g/100 ml). The bulk has been heated to reflux for about 1 hour. The bulk has been extracted at room temperature with DCM that has been then dried over sodium sulphate, filtered and concentrated to a residue, thus obtaining 210 mg of compound H.