EP2909327A1 - A process for preparing indoline derivatives - Google Patents

A process for preparing indoline derivatives

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Publication number
EP2909327A1
EP2909327A1 EP13780341.7A EP13780341A EP2909327A1 EP 2909327 A1 EP2909327 A1 EP 2909327A1 EP 13780341 A EP13780341 A EP 13780341A EP 2909327 A1 EP2909327 A1 EP 2909327A1
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EP
European Patent Office
Prior art keywords
transaminase
group
formula
compound
arthrobacter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP13780341.7A
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German (de)
French (fr)
Inventor
Sven NIERDINGER
Ferdinand Zepeck
Wolfgang Kroutil
Robert Simon
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Sandoz AG
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Sandoz AG
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Publication date
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Priority to EP13780341.7A priority Critical patent/EP2909327A1/en
Publication of EP2909327A1 publication Critical patent/EP2909327A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom

Definitions

  • the present invention relates in general to the field of organic chemistry, and in particular to the preparation of indoline derivatives. These indoline derivatives can be used as
  • silodosin (1 -(3-hydroxypropyl)-5-[(2R)-( ⁇ 2- [2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethyl ⁇ amino)propyl]indoline-7-carboxamide) is an ch-adrenoceptor antagonist with high uroselectivity, which is particularly suitable for the treatment of benign prostatic hyperplasia.
  • JP2001 -199956 discloses a process for preparation of silodosin, in which process
  • (fi)-5-(2-aminopropyl)-1 -(3-benzoyloxypropyl)-indoline-7-carbonitrile of formula is prepared via a three step reaction.
  • a first step 1 -(3-benzoyloxypropyl)-5-(2-oxo propyl)- indoline-7-carbonitrile and (R)-2-phenyl ethylamine are reacted under acid catalysis in order to obtain the corresponding imine.
  • the imine is reduced under a hydrogen atmosphere in the presence of platinum oxide catalyst, wherein the resulting reaction mixture is hydrogenated in the presence of Pd on charcoal in a third step.
  • the object of the present invention is to provide an improved process for preparing indoline derivatives representing valuable key intermediates for the preparation of pharmaceutically active agents such as silodosin or derivatives thereof.
  • CN is selected from the group consisting of CN, NH 2 , N0 2, halogen and a group convertible to carbamoyl,
  • hydroxy protecting group means any group known in the art which can be used for protecting a hydroxy group, with the proviso that the cleavage conditions for selectively removing said hydroxy protecting group will not adversely affect the structure of compound of formula I I or a derivative thereof.
  • amino protecting group means any group known in the art which can be used for protecting an amino group, with the proviso that the cleavage conditions for selectively removing said amino protecting group will not adversely affect the structure of compound of formula I I or a derivative thereof.
  • alkyl as used herein means straight, branched or cyclic hydrocarbons.
  • aryl as used herein means hydrocarbon aryls, preferably single or condensed six- membered rings, more preferably phenyl or naphthyl, in particular phenyl.
  • alkylaryl as used herein means that the aforementioned aryl moieties are incorporated into the aforementioned straight or branched alkyl moieties either at one of the proximal or distal ends of the alkyl chain or between the aforementioned alkyl chains.
  • proximal end means for e.g. adjacent to the indoline ring moiety of compound of formula I or I I, while distal means the terminal carbon of the alkyl moiety which is furthermost from said indoline ring moiety.
  • substituted as employed herein means that one or more, preferably 1-3 hydrogen atoms of a structural moiety are replaced independently from each other by the
  • substituents include, without being limited thereto, for example halogen, trifluoromethyl, cyano, nitro, -NR', -OR', -N(R')R" and R'", wherein each of R', R" and R'" are selected from the group consisting of linear or branched C1 - C6 alkyl.
  • substituent(s) are at positions where their introduction is/are chemically possible, that is positions being known or evident to the person skilled in the art to decide (either experimentally or theoretically) without inappropriate effort whether a particular substitution is possible.
  • substituents which may be unstable or may affect reactions disclosed herein may be omitted.
  • R 4 and R 5 are unsubstituted.
  • ⁇ -transaminase as used herein means an enzyme converting a keto compound into an amino compound and vice versa, ⁇ -transaminase belongs to the class of enzymes having the Enzyme Commission (EC) number EC 2.6.1.
  • amine donor means a compound which donates its amino group to a substrate in a transamination reaction, wherein the amino group of the amine donor is converted to a keto group.
  • ⁇ -transaminase is (R)-selective, preferably, the (R)-selective ⁇ -transaminase is derived from an organism selected from the group consisting of (R)-Arthrobacter sp., Hyphomonas neptunium,
  • Aspergillus terreus and Arthrobacter round 1 1 mutant preferably (R)-Arthrobacter sp. and Aspergillus terreus, more preferably (R)- Arthrobacter sp.
  • (R)-selective means ⁇ -transaminase enzymes which selectively or essentially selectively convert the prochiral carbonyl substrate of formula I to the respective amine compound of formula II in form of its (fi)-enantiomer.
  • Arthrobacter round 11 mutant means a mutant which was obtained and identified after the eleventh round of mutation in the process for preparing Arthrobacter mutant disclosed in C. K. Savile et al., Science 2010, 329, p. 305-309.
  • ⁇ -transaminase is derived from an organism selected from Chromobacterium violaceum, Hyphomonas neptunium, Bacillus megaterium, (R)- Arthrobacter sp. , Aspergillus terreus, Pseudomonas putida, Pseudomonas fluorescens, and Arthrobacter citreus, preferably Pseudomonas fluorescens and (R)- Arthrobacter sp.
  • the ⁇ -transaminase enzymes defined in items (2) and (3) above may be derived from the above indicated bacteria and fungi in their original, natural form, or in a modified, so-called mutant form.
  • mutant refers to mutants comprising a deletion(s), substitution(s), addition(s), or insertion(s) of one or more, preferably one or several, amino acids in the amino acid sequences or partial sequences thereof; or mutants showing about 80% or more, about 85% or more, preferably about 90% or more, more preferably about 95% or more, about 97% or more, about 98% or more, or about 99% or more identity with the amino acid sequences or partial sequences thereof.
  • mutants include homologs of bacterial or fungal species differing from natural bacteria or fungi and natural mutants thereof such as mutants based on polymorphic mutation among bacteria or fungi of the same species (e.g., race).
  • mutants refers to an integer of 10, 9, 8, 7, 6, 5, 4, 3, or 2.
  • mutants may be preferably selected from those disclosed in C. K. Savile et al., " Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture", Science 2010, 329, p. 305-309.
  • amine donor is selected from the group consisting of alanine, 1 -ethylamin, 1 -propylamine, 2- propylamine, 1 -indolamine, phenethylamine and others, preferably alanine and 2- propylamine, more preferably 2-propylamine.
  • optically active means enantiopure enantiomer or a mixture of enantiomers of the same compound, in which mixture the quantitative content of one enantiomer predominates.
  • optically active means that compound of formula II has an enantiomeric excess as defined in item (6) below.
  • enantiomeric excess means the difference between the percentage of one enantiomer of an optically active compound and the percentage of the other enantiomer of the same optically active compound. For example, an optically active compound which contains 75% R-enantiomer and 25% S-enantiomer will have an enantiomeric excess of 50% of R-enantiomer.
  • the ⁇ -transaminase is provided in the form of permeabilized E. coli cells overexpressing the ⁇ -transaminase or as a crude enzyme extract or a freeze-dried residue thereof or as a (partially) purified enzyme preparation or a freeze-dried residue thereof or as an immobilized preparation, preferably the ⁇ -transaminase is provided in the form of permeabilized E. coli cells overexpressing the ⁇ -transaminase, more preferably a freeze-dried residue of any enzyme solution.
  • overexpressing the ⁇ -transaminase in any form mentioned in item (10) to the molar amount of compound of formula I is 10 g/1 mol to 2000 g/1 mol.
  • reaction mixture further comprises alanine dehydrogenase (Ala- DH), formate dehydrogenase (FDH), nicotinamide adenine dinucleotide (NAD + ), pyridoxal-5 ' -phophat (PLP) and ammonium formate.
  • Al- DH alanine dehydrogenase
  • FDH formate dehydrogenase
  • NAD + nicotinamide adenine dinucleotide
  • PDP pyridoxal-5 ' -phophat
  • the reaction mixture further comprises alanine dehydrogenase (Ala- DH), glucose dehydrogenase (GDH), nicotinamide adenine dinucleotide (NAD + ), pyridoxal-5 ' -phophat (PLP) and glucose and ammonium salts, or alternatively, the reaction mixture further comprises alanine dehydrogenase (Ala-DH), phosphite dehydrogenase (PTDH), nicotinamide adenine dinucleotide (NAD + ), pyridoxal-5 ' - phophat (PLP) and ammonium phosphite.
  • R 3 is a hydroxy protecting group or H
  • R and R 5 are independently from each other selected from substituted or unsubstituted C1-C10 alkyl or substituted or unsubstituted C3-C10 aryl or substituted or unsubstituted C3-C10 alkylaryl, which process comprises the steps of:
  • R ⁇ R 2 , R 5 and m are defined as above, and
  • amine acceptor means a keto compound whereon an amino group of the substrate is transferred in transamination reaction, wherein the keto group of the amine acceptor is converted to an amine group.
  • first and second type of enantiomers means that in a mixture of (R)- and (S)- enantiomers of compound of formula II, one enantiomer, e.g. the (fi)-enantiomer represents the first type of enantiomer, while the (S)-enantiomer represents the second type of enantiomer, or vice versa.
  • the first type of enantiomer is the (S)-enantiomer of compound of formula II
  • the second type of enantiomer is the (fi)-enantiomer of compound of formula II.
  • ⁇ -transaminase is (S)-selective, preferably the (S)-selective ⁇ -transaminase is derived from an organism selected from the group consisting of Chromobacterium violaceum, Pseudomonas putida I or II, Bacillus megaterium, preferably Pseudomonas putida I and Bacillus megaterium.
  • (S)-selective means that the ⁇ -transaminase enzyme selectively converts an amine substrate in the form of the (S)-enantiomer into the respective carbonyl compound, while there is no or substantially no conversion of an amine substrate in the form of the (fi)-enantiomer.
  • Pseudomonas putida I means a Pseudomonas putida transaminase having a sequence corresponding to Seq. ID 20 disclosed in WO2010/089171 A2
  • Pseudomonas putida II means a Pseudomonas putida transaminase having a sequence corresponding to Seq. ID 22 disclosed in WO2010/089171 A2.
  • Arthrobacter sp. preferably Pseudomonas putida I or II, Aspergillus terreus, Bacillus megaterium and Arthrobacter sp.
  • ⁇ -transaminase enzymes defined in items (15) and (16) above may be derived from the above indicated bacteria and fungi in their original, natural form, or in a modified, so-called mutant form.
  • step i) is optically active.
  • the optically active mixture provided in step i) is prepared according to a process according to any one of items (1) to (13).
  • step i) is a racemic mixture of compound of formula II.
  • racemic mixture means a mixture wherein R- and S-enantiomers are present in equivalent amounts.
  • the racemic mixture is suitably obtained by achiral chemical synthesis.
  • immobilized preparation means that ⁇ -transaminase enzymes are chemically, physically or by means of genetic engineering methods confined at or localized at/in a chemically organic or inorganic support material, wherein catalytic activity of the enzyme is retained and the preparation can preferably be used repeatedly and continuously.
  • overexpressing the ⁇ -transaminase in any form mentioned in item (22) to molar amount of compound of formula II is 10 g/1 mol to 2000 g/1 mol.
  • reaction mixture further optionally comprises an aqueous buffer system selected from the group consisting of phosphate buffer, TRIS buffer, PIPES buffer and HEPES buffer, preferably the buffer system is a phosphate buffer, more preferably a potassium phosphate buffer; most preferably a buffer can be omitted.
  • an aqueous buffer system selected from the group consisting of phosphate buffer, TRIS buffer, PIPES buffer and HEPES buffer, preferably the buffer system is a phosphate buffer, more preferably a potassium phosphate buffer; most preferably a buffer can be omitted.
  • aqueous buffer system means an admixture of a weak acid and its conjugate base or a weak base and its conjugate acid, which admixture is dissolved in water and is capable to stabilize the pH value of an aqueous solution.
  • a "Tris buffer” as used herein means an admixture of e.g. tris(hydroxymethyl)aminomethane (TRIS) and a strong inorganic acid such as hydrochloric acid
  • phophate buffer as used herein means an admixture of e.g. K 2 HP0 4 and KH 2 P0 4
  • PIPES buffer means an admixture of e.g.
  • piperazine- N,N'-bis(2-ethanesulfonic acid) and an alkali hydroxide such as NaOH or KOH and HEPES buffer means an admixture of e.g. 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES) and an alkali hydroxide such as NaOH or KOH.
  • HEPES 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid
  • amine protective group selected from the group consisting of tert-butylcarbonyl (Boc), benzyloxycarbonyl (Z), 9-Fluorenylmethoxycarbonyl (Fmoc) and allyloxycarbonyl (alloc).
  • the hydroxy protective group R 3 is selected from the group consisting of benzoyl, tosyl, methoxymethyl, tetrahydropyranyl, t-butyl, allyl, benzyl (Bz), t-butylmethylsilyl, t-butyldiphenylsilyl, acetyl, pivaloyl.
  • R 2 ' is -(CH 2 ) 3 -0-R 3 ' or -(CH 2 ) 3 -0-CO-R 4 ', in which R 3 ' and R' 4 represent substituted or unsubstituted C1 -C10 alkyl or substituted or unsubstituted C3-C10 aryl or substituted or unsubstituted C3-C10 alkylaryl, prepared by a process according to any one of items (1 ) to (27), and b) converting the compound of formula II" of step a) to silodosin.
  • R 3 ' and R' 4 represent substituted or unsubstituted C1 -C10 alkyl or substituted or unsubstituted C3-C10 aryl or substituted or unsubstituted C3-C10 alkylaryl, prepared by a process according to any one of items (1 ) to (27), and b) converting the compound of formula II" of step a) to si
  • chiral indoline derivatives are synthesized by means of chemical synthesis affording the desired indoline derivative in the form of a racemat. Therefore, in order to obtain enantiomerically enriched or pure indoline derivative, in conventional processes, it is necessary to subject racemic indoline derivatives to resolution of racemates. Resolution of racemates is a laborious procedure, since it typically requires multiple subsequent resolution steps in order to obtain a satisfactory high degree of enantioenrichment. These multiple resolution steps in turn result in relatively poor yields of enantiomerically enriched or enantiopure product.
  • EP 0 987 332 A1 describes the use of a ⁇ -transaminase derived from Arthrobacter sp. for transamination of a substrate having the structural formula
  • 807-813 describes a screening of several putative (R)-selective amine transaminases for asymmetric synthesis of 2-aminohexane, 2-amino-4- phenylbutane, 1 -N-Boc-3-aminopyrrolidine and 1 -N-Boc-3-amopiperidine from the corresponding ketones.
  • R 3 is a hydroxy protecting group or H
  • R and R 5 are independently from each other selected from substituted or unsubstituted C1 -C10 alkyl or substituted or unsubstituted C3-C10 aryl or substituted or unsubstituted C3-C10 alkylaryl,
  • indoline compounds of formula I or II represent valuable (intermediate) compounds in the field of pharmacy and have been surprisingly found, albeit their complex structure, to be susceptible substrates of ⁇ -transaminase enzymes, and even further surprisingly with achieving enantiomeric excess combined with beneficial properties, such as purity and/or yield.
  • compound of formula II wherein
  • F is selected from the group consisting of CN, NH 2 , N0 2 and a group convertible to carbamoyl,
  • R 3 is a hydroxy protecting group
  • R and R 5 are independently from each other selected from substituted or unsubstituted C1 -C10 alkyl or substituted or
  • the procedural concept according to this aspect of the invention provides for a compound of formula II representing a highly valuable intermediate for the preparation of pharmaceutically active agents such as silodosin or derivatives thereof. It was surprisingly found by the present inventors that compound of formula I is converted via enzymatically catalysed reductive transamination reaction to compound of formula II, which conversion surprisingly can be effected, depending on further suitable conditions such as enzyme type selection, the procedural concept of reductive amination or kinetic resolution respectively provides for enantiomeric excess with a good combination of high yield and chemical purity.
  • the ⁇ -transaminase is (R)-selective, and preferably, the (R)-selective ⁇ -transaminase is derived from an organism selected from the group consisting of Arthrobacter sp. , Aspergillus terreus, Pseudomonas putida and Arthrobacter round 1 1 mutant, in particular the (R)-selective ⁇ -transaminase is (R)- Arthrobacter sp. and Aspergillus terreus, more preferably (R)-Arthrobacter sp. and Aspergillus terreus, and in particular (R)- Arthrobacter sp.
  • the ⁇ -transaminase is suitably selected in order to obtain the (fi)-enantiomer of compound of formula II.
  • (R)-selective ⁇ -transaminases enantioselectively convert the prochiral substrate compound of formula I into the (fi)-enantiomer of compound of formula II.
  • compound of formula II represents e.g. an intermediate for silodosin
  • the (R)- enantiomer is particularly preferred, since in case of silodosin, this enantiomer is approved e.g. by the Federal Drug Administration (FDA) of the United States as pharmaceutically active agent.
  • FDA Federal Drug Administration
  • the ⁇ -transaminase is derived from an organism selected from Chromobacterium violaceum, Hypophomonas neptunium, Bacillus megaterium, Arthrobacter sp., Aspergillus terreus, Pseudomonas putida, Pseudomonas fluoresceins, Arthrobacter citreus and Arthrobacter round 1 1 mutant, preferably Pseudomonas fluoresceins and (R)-Arthrobacter sp, preferably (R)-Arthrobacter sp.
  • the optically active form has an enantiomeric excess of at least 60-100%, more preferably 75-100%, even more preferably 85-100%, yet even more preferably 90-100%, still yet even more preferably 95-100% and in particular 99-100%.
  • compound of formula II is obtained in advantageous to exceptionally high enantiomeric excess, preferably in high enantiomeric excess.
  • the amine donor is selected from the group consisting of alanine, 1 -ethylamine, 1 -propylamine, 2-propylamine, 1 -indolamine, phenethylamine and others, preferably alanine and 2-propylamine, more preferably 2- propylamine.
  • the aforementioned compounds represent particularly suitable amine donor compounds.
  • compound of formula I is provided in a concentration of 1 to 200 mM in the reaction mixture, preferably 5 to 150 mM, more preferably 10 to 100 mM and in particular 15 to 50 mM. In this way, conversion rate is significantly improved.
  • ⁇ -transaminase is derived from an organism selected from the group consisting of Chromobacterium violaceum, Bacillus megaterium, Arthrobacter sp., Aspergillus terreus, Pseudomonas putida, Pseudomonas fluorescens and Arthrobacter citreus
  • a relatively low reaction temperature for carrying out the process of 16- 40°C, preferably 20-36°C, more preferably 24-34°C, even more preferably 26-32°C is particularly suitable.
  • the process is preferably carried out at a reaction temperature of 35-50°C, preferably 40-45°C whereby particularly advantageous conversion rates are obtained.
  • the reaction mixture further comprises alanine dehydrogenase (Ala-DH), formate dehydrogenase (FDH), nicotinamide adenine dinucleotide (NAD + ), pyridoxal-5 ' -phophat (PLP) and ammonium formate.
  • Al-DH alanine dehydrogenase
  • FDH formate dehydrogenase
  • NAD + nicotinamide adenine dinucleotide
  • PDP pyridoxal-5 ' -phophat
  • the reaction mixture further comprises alanine dehydrogenase (Ala-DH), glucose dehydrogenase (GDH), nicotinamide adenine dinucleotide (NAD + ), pyridoxal-5 ' - phophat (PLP), glucose and ammonium salts, or according to another alternative, the reaction mixture further comprises alanine dehydrogenase (Ala-DH), phosphite dehydrogenase (PTDH), nicotinamide adenine dinucleotide (NAD + ), pyridoxal-5 ' -phophat (PLP) and ammonium phosphite.
  • a particularly suitable alanine-dehydrogenase cofactor recycling system is provided.
  • R 3 is a hydroxy protecting group
  • R and R 5 are independently from each other selected from substituted or unsubstituted C1 -C10 alkyl or substituted or
  • R ⁇ R 2 , R 5 and m are defined as above, and
  • a further, alternative process for preparing a compound of formula II having enantiomeric excess and good chemical purity, preferably high enantiomeric excess is provided.
  • a mixture of enantiomers of compound of formula II e.g. a racemic mixture thereof, serves as a particularly suitable substrate for efficiently enantiomerically enriching compound of formula II by means of kinetic resolution using ⁇ -transaminase enzymes.
  • ⁇ -transaminase enzymes an individual one, e.g.
  • the undesired enantiomer of the compound of formula II is converted to compound of formula I which can be easily separated by physical and/or chemical methods from compound of formula II, while the other individual one, e.g. the desired enantiomer of compound of formula II is converted a lesser extend, preferably not or at least substantially not converted to compound of formula I.
  • the ⁇ -transaminase is derived from an organism selected from the group consisting of Hyphomonas neptunium, Chromobaterium violaceum, Pseudomonas putida I or II, Aspergillus terreus, Bacillus megaterium and Arthrobacter sp., preferably Pseudomonas putida, Aspergillus terreus, Bacillus megaterium and Arthrobacter sp.
  • the ⁇ -transaminase is (S)-selective, more preferably the (S)-selective ⁇ -transaminase is derived from an organism selected from the group consisting of Chromobacterium violaceum, Pseudomonas putida I or II, Bacillus megaterium, even more preferably Pseudomonas putida I and Bacillus megaterium.
  • the optically active chiral amine of compound of formula II obtained in step ii) has an enantiomeric excess of 70-100%, preferably 80- 100%, more preferably 90-100%, even more preferably 95-100%, yet even more preferably 99-100%.
  • compound of formula II is obtained in advantageous to exceptionally high enantiomeric excess, preferably in high enantiomeric excess.
  • the kinetic resolution process is carried out at a reaction temperature of 16-40°C, preferably 20-36°C, more preferably 24-34°C, even more preferably 26-32°C.
  • a molar ratio of compound of formula II to amine acceptor is 1 : 1 to 1 : 10, preferably 1 : 1.2 to 1 :8, more preferably 1 : 1.2 to 1 :4, most preferably 1 : 1.5 to 1 :2.5;
  • the ⁇ -transaminase overexpressing the ⁇ -transaminase or as a crude enzyme extract or a freeze-dried residue thereof or as a (partially) purified enzyme preparation or a freeze-dried residue thereof or as an immobilized preparation, preferably the ⁇ -transaminase is provided in the form of permeabilized E. coli cells overexpressing the ⁇ - transaminase, more preferably a freeze-dried residue of any enzyme solution;
  • the process is carried out in the presence of an organic solvent selected from the group consisting of DMF, DMSO, THF, MeCN, 1 ,2-dimethoxy ethane (DME), and C1 - C6-alcohols, preferably DMF, DMSO, THF, MeCN, DME and C1 -C3-alcohols, more preferably DMF, DMSO, TH F, MeCN, DME, in particular DMF and DMSO;
  • an organic solvent selected from the group consisting of DMF, DMSO, THF, MeCN, 1 ,2-dimethoxy ethane (DME), and C1 - C6-alcohols, preferably DMF, DMSO, THF, MeCN, DME and C1 -C3-alcohols, more preferably DMF, DMSO, TH F, MeCN, DME, in particular DMF and DMSO;
  • the reaction mixture further optionally comprises an aqueous buffer system selected from the group consisting of phosphate buffer, TRIS buffer, PI PES buffer and HEPES buffer, preferably the buffer system is a phosphate buffer, more preferably a potassium phosphate buffer; most preferably a buffer can be omitted;
  • an aqueous buffer system selected from the group consisting of phosphate buffer, TRIS buffer, PI PES buffer and HEPES buffer, preferably the buffer system is a phosphate buffer, more preferably a potassium phosphate buffer; most preferably a buffer can be omitted;
  • R 2 represents an amine protective group selected from the group consisting of tert- butylcarbonyl (Boc), benzyloxycarbonyl (Z), 9-Fluorenylmethoxycarbonyl (Fmoc) and allyloxycarbonyl (alloc);
  • the hydroxy protective group R 3 is selected from the group consisting of benzoyl, tosyl, methoxymethyl, tetrahydropyranyl, t-butyl, allyl, benzyl (Bz), t-butylmethylsilyl, t- butyldiphenylsilyl, acetyl, pivaloyl; and
  • any one of features (i) to (viii) represents particularly advantageous process conditions.
  • feature (iv) it was surprisingly found that the appropriate choice of solvent significantly improves conversion rate.
  • silodosin having the formula HO
  • R 2 ' is -(CH 2 ) 3 -0-R 3 ' or -(CH 2 ) 3 -0-CO-R 4 ', in which R 3 ' and R 4 ' represent substituted or unsubstituted C1 -C10 alkyl or substituted or unsubstituted C3-C10 aryl or substituted or unsubstituted C3-C10 alkylaryl, prepared by a process according to any one of items (1) to (27) indicated above, and
  • step b) converting the compound of formula II" of step a) to silodosin.
  • the above mentioned aspect provides a particularly advantageous process for preparing silodosin owing to the application of the present procedural aspects of reductive amination and/or kinetic resolution and the beneficial effects involved therewith.
  • a pharmaceutical formulation which comprises silodosin of formula
  • silodosin is entirely free of metal impurities.
  • metal impurities as used herein means impurities deriving from a catalyst in the form of elemental metal, covalent metal compounds or metal compounds in the form of a salt used in conventional chemical syntheses for preparing silodosin. More specifically, “metal impurities” are meant to be derived from metal catalysis conventionally applied in the chemical preparation of silodosin, in which typically e.g. Pd on charcoal and/or Pt0 2 on BaS0 4 is/are applied.
  • pharmaceutically acceptable excipient means any physiologically inert, pharmacologically inactive material known in the art being compatible with the physical and chemical characteristics of the active agent.
  • amino acid sequence of the below listed ⁇ -transaminases originates from the indicated literature, however, the present inventors employed codon optimized genes for improved expression of ⁇ -transaminase in E. coli.
  • Pseudomonas putida I corresponds to Seq. ID 20
  • P seudomonas putida II to Seq. ID 22.
  • ⁇ -transaminase derived from Arthrobacter citreus was taken from WO 2006 / 063336 (or we us US 7247460 B2).
  • Arthrobacter citreus corresponds to Sep. ID:02 in this patent.
  • All ⁇ -transaminases were expressed in Escherichia coli.
  • the ⁇ -transaminases are applied in form of lyophilized E. coli cell preparations.
  • n.d. means "not determined", and in this single case, deviation between the two determinations was > 10%; Pseudomonas putida listed under
  • the reductive amination process according to the present invention is particularly suitable to obtain the desired product in enantioenriched form.
  • the ⁇ -transaminases is derived from Pseudomonas fluorescens and (R)- Arthrobacter sp. providing both high enantiomeric excess and a high conversion rate. More beneficially, the ⁇ -transaminases is derived from (R)- Arthrobacter sp. providing the (fi)-enantiomer of compound of formula II' representing a highly desirably intermediate product for the preparation of silodosin in both high
  • Lyophilized cells of E. coli containing overexpressed ⁇ - ⁇ derived from Arthrobacter round 1 1 mutant (20 mg and 40 mg respectively) were rehydrated in a potassium phosphate buffer (pH 7.0, 100 mM) containing PLP (1 mM) at room temperature for 30 minutes.
  • the substrate was added afterwards (10 and 25 mM resectively) together with 10 vol% DMSO, and 2- propylamine (250 mM) as N-donor in amount determined by the ratio n(substrate)/n(donor), and the reaction was carried out at 45°C in an Eppendorf orbital shaker (700 rpm) for 24 h.
  • Arthrobacter round 11 mutant are obtained with the conditions according to entry 4 in terms of enantiomeric excess, and the conditions according to entry 1 in terms of conversion. At the conditions according to entry 2, preferably both enantiomeric excess and conversion are well balanced.
  • Exemplary preparative procedure for preparative scale batch Lyophilized cells of E. coli containing overexpressed ⁇ - ⁇ from (R)- Arthrobacter sp. (225 mg) were rehydrated in a KPi buffer (9 mL, pH 7.0, 100 mM) containing PLP (1.00 mM), NAD + (1.00 mM), ammonium formate (150 mM), FDH (1 1 U), Ala-DH (12 U) and D-alanine (500 mM) at 22°C for 30 minutes.
  • Pseudomonas putida I which provides the (fi)-enantiomer of compound of formula II' representing a highly desirably intermediate product for the preparation of silodosin in both relative high enantiomeric excess and relative high yield.

Abstract

The present invention relates in general to the field of organic chemistry, and in particular to the preparation of indoline derivatives. These indoline derivatives can be used as intermediates in the synthesis of pharmaceutically active agents such as silodosin or derivatives thereof.

Description

A process for preparing indoline derivatives
Field of the invention
The present invention relates in general to the field of organic chemistry, and in particular to the preparation of indoline derivatives. These indoline derivatives can be used as
intermediates in the synthesis of pharmaceutically active agents such as silodosin or derivatives thereof.
Background of the invention
Compounds comprising an indoline moiety are important intermediates for the preparation of active pharmaceutical active agents. For example, silodosin (1 -(3-hydroxypropyl)-5-[(2R)-({2- [2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethyl}amino)propyl]indoline-7-carboxamide) is an ch-adrenoceptor antagonist with high uroselectivity, which is particularly suitable for the treatment of benign prostatic hyperplasia.
EP 0 600 675 A1 and Sorbera L.A. et. al. in "Drugs of the future", Vol. 26, No 6, 2001 , p. 552 to 560, disclose a process for preparing silodosin respectively, in which process racemic 1 - acetyl-5-[2-[2-[2-(2,2,2-trifluoroethoxy)-phenoxy]ethylamino]propyl]indoline-7-carbonitrile of structural formula
is subjected to resolution of racemates by treating with (+)-mandelic acid in order to obtain the respective (fi)-enantiomer.
JP2001 -199956 discloses a process for preparation of silodosin, in which process
(fi)-5-(2-aminopropyl)-1 -(3-benzoyloxypropyl)-indoline-7-carbonitrile of formula is prepared via a three step reaction. In a first step, 1 -(3-benzoyloxypropyl)-5-(2-oxo propyl)- indoline-7-carbonitrile and (R)-2-phenyl ethylamine are reacted under acid catalysis in order to obtain the corresponding imine. In a second step, the imine is reduced under a hydrogen atmosphere in the presence of platinum oxide catalyst, wherein the resulting reaction mixture is hydrogenated in the presence of Pd on charcoal in a third step. The thus obtained crude product of racemic 1 -(3-benzoyloxypropyl)-5-(2-aminopropyl)-indoline-7-carbonitrile is subjected to resolution of racemates by treating with L-tartaric acid in order to obtain the (R)- enantiomer in an optical purity of 97.6 %ee.
The object of the present invention is to provide an improved process for preparing indoline derivatives representing valuable key intermediates for the preparation of pharmaceutically active agents such as silodosin or derivatives thereof.
Summary of the invention
Various aspects, advantageous features and preferred embodiments of the present invention as summarized in the following items, respectively alone or in combination, contribute to solving the object of the invention.
(1 ) pound of formula I I
, wherein m is 1 to 8,
is selected from the group consisting of CN, NH2, N02, halogen and a group convertible to carbamoyl,
R2 is selected from the group consisting of H, N-protecting group, -(CH2)n-0-R3 and -(CH2)n-0-CO-R4 in which n = 1 to 10, R3 is a hydroxy protecting group or H, and R4 and R5 are independently from each other selected from substituted or unsubstituted C1-C10 alkyl or substituted or unsubstituted C3-C10 aryl or substituted or unsubstituted C3-C10 alkylaryl, in which process a compound of formula I
, wherein R^ R2, R5 and m are defined as above,
is reacted with an amine donor in the presence of a ω-transaminase.
The term "hydroxy protecting group" as used herein means any group known in the art which can be used for protecting a hydroxy group, with the proviso that the cleavage conditions for selectively removing said hydroxy protecting group will not adversely affect the structure of compound of formula I I or a derivative thereof.
The term "amino protecting group" as used herein means any group known in the art which can be used for protecting an amino group, with the proviso that the cleavage conditions for selectively removing said amino protecting group will not adversely affect the structure of compound of formula I I or a derivative thereof.
The term "alkyl" as used herein means straight, branched or cyclic hydrocarbons.
The term "aryl" as used herein means hydrocarbon aryls, preferably single or condensed six- membered rings, more preferably phenyl or naphthyl, in particular phenyl.
The term "alkylaryl" as used herein means that the aforementioned aryl moieties are incorporated into the aforementioned straight or branched alkyl moieties either at one of the proximal or distal ends of the alkyl chain or between the aforementioned alkyl chains. For example, proximal end means for e.g. adjacent to the indoline ring moiety of compound of formula I or I I, while distal means the terminal carbon of the alkyl moiety which is furthermost from said indoline ring moiety.
The term "substituted" as employed herein means that one or more, preferably 1-3 hydrogen atoms of a structural moiety are replaced independently from each other by the
corresponding number of substituents. Typical substituents include, without being limited thereto, for example halogen, trifluoromethyl, cyano, nitro, -NR', -OR', -N(R')R" and R'", wherein each of R', R" and R'" are selected from the group consisting of linear or branched C1 - C6 alkyl. It will be understood that the substituent(s) are at positions where their introduction is/are chemically possible, that is positions being known or evident to the person skilled in the art to decide (either experimentally or theoretically) without inappropriate effort whether a particular substitution is possible. For example, substituents which may be unstable or may affect reactions disclosed herein may be omitted. Preferably, R4 and R5 are unsubstituted.
The term "ω-transaminase" as used herein means an enzyme converting a keto compound into an amino compound and vice versa, ω-transaminase belongs to the class of enzymes having the Enzyme Commission (EC) number EC 2.6.1.
The term "amine donor" as used herein means a compound which donates its amino group to a substrate in a transamination reaction, wherein the amino group of the amine donor is converted to a keto group.
(2) The process according to item (1), wherein the ω-transaminase is (R)-selective, preferably, the (R)-selective ω-transaminase is derived from an organism selected from the group consisting of (R)-Arthrobacter sp., Hyphomonas neptunium,
Aspergillus terreus and Arthrobacter round 1 1 mutant, preferably (R)-Arthrobacter sp. and Aspergillus terreus, more preferably (R)- Arthrobacter sp.
The term "(R)-selective" as used herein means ω-transaminase enzymes which selectively or essentially selectively convert the prochiral carbonyl substrate of formula I to the respective amine compound of formula II in form of its (fi)-enantiomer.
The term "enantiomer " as used herein means two stereoisomers which are mirror images of one another, but which are not superimposable. The designations "(R)" and "(S)" used herein indicate the absolute configuration at chiral center(s) of the respective compounds, determined by means of the Cahn Ingold Prelog convention (CIP-convention) known in the art (cf. e.g. M.B. Smith, J. March, "March's Advanced Organic Chemistry: Reactions, Mechanisms and Structure", 6th edition, John Wiley&Sons, Inc., p. 155-158). For example, compound of formula II' in (R)-configuration has the following structural formula
wherein the C-N bond in bold indicates that this bond is located above the plane of the paper according to the CIP-convention.
The term " Arthrobacter round 11 mutant' as used herein means a mutant which was obtained and identified after the eleventh round of mutation in the process for preparing Arthrobacter mutant disclosed in C. K. Savile et al., Science 2010, 329, p. 305-309.
(3) The process according to item (1) or (2), wherein the ω-transaminase is derived from an organism selected from Chromobacterium violaceum, Hyphomonas neptunium, Bacillus megaterium, (R)- Arthrobacter sp. , Aspergillus terreus, Pseudomonas putida, Pseudomonas fluorescens, and Arthrobacter citreus, preferably Pseudomonas fluorescens and (R)- Arthrobacter sp.
The ω-transaminase enzymes defined in items (2) and (3) above may be derived from the above indicated bacteria and fungi in their original, natural form, or in a modified, so-called mutant form.
The term "mutant" as used herein refers to mutants comprising a deletion(s), substitution(s), addition(s), or insertion(s) of one or more, preferably one or several, amino acids in the amino acid sequences or partial sequences thereof; or mutants showing about 80% or more, about 85% or more, preferably about 90% or more, more preferably about 95% or more, about 97% or more, about 98% or more, or about 99% or more identity with the amino acid sequences or partial sequences thereof. Examples of such mutants include homologs of bacterial or fungal species differing from natural bacteria or fungi and natural mutants thereof such as mutants based on polymorphic mutation among bacteria or fungi of the same species (e.g., race). The term "several" as used herein refers to an integer of 10, 9, 8, 7, 6, 5, 4, 3, or 2. For example, in case of ^rfftrobacter ω-transaminase, mutants may be preferably selected from those disclosed in C. K. Savile et al., " Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture", Science 2010, 329, p. 305-309.
(4) The process according to any one of items (1) to (3), wherein the amine donor is selected from the group consisting of alanine, 1 -ethylamin, 1 -propylamine, 2- propylamine, 1 -indolamine, phenethylamine and others, preferably alanine and 2- propylamine, more preferably 2-propylamine.
(5) The process according to any one of items (1) to (4), wherein the chiral amine
compound of formula II is optically active.
The term "optically active" as used herein means enantiopure enantiomer or a mixture of enantiomers of the same compound, in which mixture the quantitative content of one enantiomer predominates. Preferably, "optically active" means that compound of formula II has an enantiomeric excess as defined in item (6) below.
As regards the meaning of the term "enantiomer", reference is made to the explanations under item (2) above.
(6) The process according to item (5), wherein the optically active amine compound of formula II has an enantiomeric excess of at least 60-100%, preferably 75-100%, more preferably 85-100%, even more preferably 90-100%, yet even more preferably 95-100% and in particular 99-100%.
The term "enantiomeric excess" as used herein means the difference between the percentage of one enantiomer of an optically active compound and the percentage of the other enantiomer of the same optically active compound. For example, an optically active compound which contains 75% R-enantiomer and 25% S-enantiomer will have an enantiomeric excess of 50% of R-enantiomer.
(7) The process according to any one of items (1) to (6), wherein the process is carried out at a reaction temperature of 16-40°C, preferably 20-36°C, more preferably 24- 34°C, even more preferably 26-32°C; and /or
wherein in case the ω-transaminase is Arthrobacter round 1 1 mutant, the process is carried out at a reaction temperature of 35-50°C, preferably 40-45°C. As regards the meaning of the term "Arthrobacter round 1 1 mutant ", reference is made to the explanations under item (2) above.
(8) The process according to any one of items (1) to (7), wherein compound of formula I is provided in a concentration of 1 to 200mM in the reaction mixture, preferably 5 to 150 mM, more preferably 10 to 100 mM and in particular 15 to 50 mM.
(9) The process according to any one of items (1) to (8), wherein a molar ratio of compound of formula I to amine donor is 1 :5 to 1 :40, preferably 1 :8 to 1 : 30, more preferably 1 : 10 to 1 :26, most preferably 1 :20 to 1 :25.
(10) The process according to any one of items (1) to (9), wherein the ω-transaminase is provided in the form of permeabilized E. coli cells overexpressing the ω-transaminase or as a crude enzyme extract or a freeze-dried residue thereof or as a (partially) purified enzyme preparation or a freeze-dried residue thereof or as an immobilized preparation, preferably the ω-transaminase is provided in the form of permeabilized E. coli cells overexpressing the ω-transaminase, more preferably a freeze-dried residue of any enzyme solution.
(1 1 ) The process according to item (10), wherein the ratio of mass of E. coli cells
overexpressing the ω-transaminase in any form mentioned in item (10) to the molar amount of compound of formula I is 10 g/1 mol to 2000 g/1 mol.
(12) The process according to any one of items (1) to (1 1 ), wherein in case the amine donor is alanine, the reaction mixture further comprises alanine dehydrogenase (Ala- DH), formate dehydrogenase (FDH), nicotinamide adenine dinucleotide (NAD+), pyridoxal-5'-phophat (PLP) and ammonium formate.
(13) The process according to any one of items (1) to (11), wherein in case the amine donor is alanine, the reaction mixture further comprises alanine dehydrogenase (Ala- DH), glucose dehydrogenase (GDH), nicotinamide adenine dinucleotide (NAD+), pyridoxal-5'-phophat (PLP) and glucose and ammonium salts, or alternatively, the reaction mixture further comprises alanine dehydrogenase (Ala-DH), phosphite dehydrogenase (PTDH), nicotinamide adenine dinucleotide (NAD+), pyridoxal-5'- phophat (PLP) and ammonium phosphite. (14) A process for preparing an optically active chiral amine compound of formula II
wherein is selected from the group consisting of CN, NH2, N02 and a group convertible to carbamoyl,
R2 is selected from the group consisting of H, N-protecting group, -(CH2)n-0-R3 or - (CH2)n-0-CO-R4 in which n = 1 to 10, or H
R3 is a hydroxy protecting group or H, and R and R5 are independently from each other selected from substituted or unsubstituted C1-C10 alkyl or substituted or unsubstituted C3-C10 aryl or substituted or unsubstituted C3-C10 alkylaryl, which process comprises the steps of:
i) reacting a mixture of first and second type of enantiomers of compound of formula II
, wherein R^ R2, R5 and m are defined as above,
with an amine acceptor in the presence of a ω-transaminase for converting the first type of enantiomer of compound of formula II to a compound of formula I
, wherein R^ R2, R5 and m are defined as above, and
ii) separating the remaining, unreacted second type of enantiomer of compound of formula II from the reaction mixture.
As to the meaning of the terms "alkyl", "aryl, "alkylaryl", "substituted", "ω-transaminase" and "N-protecting group", reference is made to item (1) above. As to the meaning of the term "enantiomer", reference is made to item (2) above. As to the meaning of the term "optically active", reference is made to item (5) above. The term "amine acceptor" as used herein means a keto compound whereon an amino group of the substrate is transferred in transamination reaction, wherein the keto group of the amine acceptor is converted to an amine group.
The term "first and second type of enantiomers" means that in a mixture of (R)- and (S)- enantiomers of compound of formula II, one enantiomer, e.g. the (fi)-enantiomer represents the first type of enantiomer, while the (S)-enantiomer represents the second type of enantiomer, or vice versa. Preferably, the first type of enantiomer is the (S)-enantiomer of compound of formula II, while the second type of enantiomer is the (fi)-enantiomer of compound of formula II.
(15) The process according to item (14), wherein the ω-transaminase is (S)-selective, preferably the (S)-selective ω-transaminase is derived from an organism selected from the group consisting of Chromobacterium violaceum, Pseudomonas putida I or II, Bacillus megaterium, preferably Pseudomonas putida I and Bacillus megaterium.
The term "(S)-selective" as used herein means that the ω-transaminase enzyme selectively converts an amine substrate in the form of the (S)-enantiomer into the respective carbonyl compound, while there is no or substantially no conversion of an amine substrate in the form of the (fi)-enantiomer.
The term "Pseudomonas putida I" means a Pseudomonas putida transaminase having a sequence corresponding to Seq. ID 20 disclosed in WO2010/089171 A2, and the term "Pseudomonas putida II" means a Pseudomonas putida transaminase having a sequence corresponding to Seq. ID 22 disclosed in WO2010/089171 A2.
(16) The process according to item (14), wherein the ω-transaminase selected from the group consisting of Hyphomonas neptunium, Chromobaterium violaceum,
Pseudomonas putida I or II, Aspergillus terreus, Bacillus megaterium and
Arthrobacter sp. , preferably Pseudomonas putida I or II, Aspergillus terreus, Bacillus megaterium and Arthrobacter sp.
The ω-transaminase enzymes defined in items (15) and (16) above may be derived from the above indicated bacteria and fungi in their original, natural form, or in a modified, so-called mutant form.
As to the meaning of the term "mutant", reference is made to the explanations under item (3) above. (17) The process according to any one of items (14) to (15), wherein the amine acceptor is a C3-C8-ketone, a C2-C8-a-keto acid, or a salt of a C2-C8-a-keto acid, preferably acetone or pyruvate, more preferably pyruvate.
(18) The process according to any one of items (14) to (17), wherein the mixture of
enantiomers provided in step i) is optically active.
Preferably, the optically active mixture provided in step i) is prepared according to a process according to any one of items (1) to (13).
(19) The process according to any one of items (14) to (17), wherein the mixture of
enantiomers provided in step i) is a racemic mixture of compound of formula II.
The term "racemic mixture" as used herein means a mixture wherein R- and S-enantiomers are present in equivalent amounts.
In this embodiment, the racemic mixture is suitably obtained by achiral chemical synthesis.
(20) The process according to any one of items (14) to (19), wherein the optically active chiral amine of compound of formula II obtained in step ii) has an enantiomeric excess of 70-100%, preferably 80-100%, more preferably 90-100%, even more preferably 95-100%, yet even more preferably 99-100%.
(21 ) The process according to item (14) to (20), wherein a molar ratio of compound of formula II to amine acceptor is 1 :1 to 1 : 10, preferably 1 :1.2 to 1 :8, more preferably 1 : 1.2 to 1 :4, most preferably 1 :1 .5 to 1 :2.5.
(22) The process according to any one of items (14) to (21 ), wherein the ω-transaminase is provided in the form of permeabilized E. coli cells overexpressing the ω- transaminase or as a crude enzyme extract or a freeze-dried residue thereof or as a (partially) purified enzyme preparation or a freeze-dried residue thereof or as an immobilized preparation.
The term "immobilized preparation" as used herein means that ω-transaminase enzymes are chemically, physically or by means of genetic engineering methods confined at or localized at/in a chemically organic or inorganic support material, wherein catalytic activity of the enzyme is retained and the preparation can preferably be used repeatedly and continuously.
(23) The process according to item (22), wherein the ratio of mass of E. coli cells
overexpressing the ω-transaminase in any form mentioned in item (22) to molar amount of compound of formula II is 10 g/1 mol to 2000 g/1 mol.
(24) The process according to any one of items (14) to (24), wherein the process is carried out at a reaction temperature of 16-40°C, preferably 20-36°C, more preferably 24- 34°C, even more preferably 26-32°C.
(25) The process according to any one of items (1) to (24), wherein the process is carried out in the presence of an organic solvent selected from the group consisting of DMF, DMSO, THF, MeCN, 1 ,2-dimethoxy ethane (DME), and C1 -C6-alcohols, preferably DMF, DMSO, THF, MeCN, DME and C1 -C3-alcohols, more preferably DMF, DMSO, THF, MeCN, DME, in particular DMF and DMSO.
(26) The process according to any one of items (1) to (25), wherein the reaction mixture further optionally comprises an aqueous buffer system selected from the group consisting of phosphate buffer, TRIS buffer, PIPES buffer and HEPES buffer, preferably the buffer system is a phosphate buffer, more preferably a potassium phosphate buffer; most preferably a buffer can be omitted.
The term "aqueous buffer system" as used herein means an admixture of a weak acid and its conjugate base or a weak base and its conjugate acid, which admixture is dissolved in water and is capable to stabilize the pH value of an aqueous solution. In particular, a "Tris buffer" as used herein means an admixture of e.g. tris(hydroxymethyl)aminomethane (TRIS) and a strong inorganic acid such as hydrochloric acid, "phophate buffer" as used herein means an admixture of e.g. K2HP04 and KH2P04, PIPES buffer means an admixture of e.g. piperazine- N,N'-bis(2-ethanesulfonic acid) and an alkali hydroxide such as NaOH or KOH and HEPES buffer means an admixture of e.g. 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES) and an alkali hydroxide such as NaOH or KOH.
(27) The process according to any one of items (1) to (26), wherein R2 represents an
amine protective group selected from the group consisting of tert-butylcarbonyl (Boc), benzyloxycarbonyl (Z), 9-Fluorenylmethoxycarbonyl (Fmoc) and allyloxycarbonyl (alloc). The process according to any one of items (1) to (26), wherein the hydroxy protective group R3 is selected from the group consisting of benzoyl, tosyl, methoxymethyl, tetrahydropyranyl, t-butyl, allyl, benzyl (Bz), t-butylmethylsilyl, t-butyldiphenylsilyl, acetyl, pivaloyl.
The process according to any one of items (1) to (26), wherein in compounds of formulae I and II, m = 1 to 5, Ri is CN, R2 is -(CH2)n-0-CO-R4, wherein n = 1 to 5, R4 is linear or branched C1 -C4 alkyl or phenyl, and R5 is linear or branched C1 -C4 alkyl, preferably m = 1 to 3, Ri is CN, R2 is -(CH2)n-0-CO-R4, wherein n = 1 to 3 and R5 is methyl or ethyl, more preferably m = 1 , Ri is CN, R2 is -(CH2)3-0-CO-Ph and R5 is methyl.
(30) A process for preparing silodosin having the formula
, wherein R2' is -(CH2)3-0-R3' or -(CH2)3-0-CO-R4', in which R3' and R'4 represent substituted or unsubstituted C1 -C10 alkyl or substituted or unsubstituted C3-C10 aryl or substituted or unsubstituted C3-C10 alkylaryl, prepared by a process according to any one of items (1 ) to (27), and b) converting the compound of formula II" of step a) to silodosin. Detailed description of the invention
The present invention is now described in more detail by referring to further preferred and further advantageous embodiments and examples, which are however presented for illustrative purposes only and shall not be understood as limiting the scope of the present invention.
Conventionally, chiral indoline derivatives are synthesized by means of chemical synthesis affording the desired indoline derivative in the form of a racemat. Therefore, in order to obtain enantiomerically enriched or pure indoline derivative, in conventional processes, it is necessary to subject racemic indoline derivatives to resolution of racemates. Resolution of racemates is a laborious procedure, since it typically requires multiple subsequent resolution steps in order to obtain a satisfactory high degree of enantioenrichment. These multiple resolution steps in turn result in relatively poor yields of enantiomerically enriched or enantiopure product.
The general use of ω-transaminase enzymes for converting keto compounds into amine compounds (reductive amination) or kinetic resolution of an amine are known. For example, EP 0 987 332 A1 describes the use of a ω-transaminase derived from Arthrobacter sp. for transamination of a substrate having the structural formula
wherein p is 0 or 1 ; q is an integer of 0 to 8; r is an integer of 0 to 4; R is any one of as substituted or unsubstituted aryl group having 6 to 14 carbon atoms, a heteroaryl group having 4 to 12 carbon atoms, carboxyl group, an alkoxycarbonyl group having 1 to 6 carbon atoms, methyl group or hydrogen atom; and X is any one of hydroxyl group, carboxyl group, an alkoxycarbonyl group having 1 to 6 carbon atoms or hydrogen atom.
R. L. Hanson et al., "Preparation of (fi)-Amines from Racemic Amines with an (S)-Amine Transaminase from Bacillus meg ate um", Adv. Synth. Catal. 2008, 350, p. 1367-1375 describes the use of ω-transaminase derived from Bacillus megatehum for kinetic resolution of a substrate in the form of 1-cyclopropylethylamine and sec-butylamine respectively.
In WO 201 1/026556 A1 , identification and preparation of a ω-transaminase derived from Aspergillus terreus is described. The substrate specificity of said transaminase was tested by using a-methylbenzyl amine as model substrate. M. Hohne et al., "Rational assignment of key motifs for function guides in silico enzyme identification", Nat. Chem. Biol. 2010, 6, p. 807-813 describes a screening of several putative (R)-selective amine transaminases for asymmetric synthesis of 2-aminohexane, 2-amino-4- phenylbutane, 1 -N-Boc-3-aminopyrrolidine and 1 -N-Boc-3-amopiperidine from the corresponding ketones.
U. Kaulmann et al., "Substrate spectrum of ω-transaminase from Chromobacterium violaceum DSM30191 and its potential for biocatalysis", Enzyme Microb. Technol. 2007, 41, p. 628-637 describes the use of ω-transaminase derived from Chromobacterium violaceum for assessing conversion rate of kinetic resolution of different amine substrates and transamination of different ketoacid and aldehyde substrates, wherein no attention is paid to the enantiomeric excess of the obtained product.
However, in the above identified prior art documents, relatively simple substrates are used which chemical structure strongly differs from that of indoline compounds of formula I or II
, wherein m is 1 to 8,
is selected from the group consisting of CN, NH2, N02 and a group convertible to carbamoyl,
R2 is selected from the group consisting of H, N-protecting group, -(CH2)n-0-R3 and - (CH2)n-0-CO-R4 in which n = 1 to 10,
R3 is a hydroxy protecting group or H, and R and R5 are independently from each other selected from substituted or unsubstituted C1 -C10 alkyl or substituted or unsubstituted C3-C10 aryl or substituted or unsubstituted C3-C10 alkylaryl,
The above indicated indoline compounds of formula I or II represent valuable (intermediate) compounds in the field of pharmacy and have been surprisingly found, albeit their complex structure, to be susceptible substrates of ω-transaminase enzymes, and even further surprisingly with achieving enantiomeric excess combined with beneficial properties, such as purity and/or yield. For example, compound of formula II wherein
Ri = CN, R2 = -(CH2)3-0-CO-Ph and R5 is methyl, that is a compound having the structural formula represents a highly valuable precursor compound in the synthesis of silodosine having the formula
It was surprisingly found by the present inventors that the above indoline derivatives having the formula I and II represent particularly suitable substrates for ω-transaminase enzymes. Furthermore, by suitably selecting particular types of enzymes, both (S)- and (fi)-enantiomer of compound of formula (II) can be obtained in an advantageous high enantiomeric excess or even in enantiomerically pure state.
Accordi und of formula II
, wherein m is 1 to 8,
F is selected from the group consisting of CN, NH2, N02 and a group convertible to carbamoyl,
R2 is selected from the group consisting of H, N-protecting group, -(CH2)n-0-R3 or - (CH2)n-0-CO-R4 in which n = 1 to 10,
R3 is a hydroxy protecting group, and R and R5 are independently from each other selected from substituted or unsubstituted C1 -C10 alkyl or substituted or
unsubstituted C3-C10 aryl or substituted or unsubstituted C3-C10 alkylaryl, is prepared by a process wherein a compound of formula I
, wherein R2, R5 and m are defined as above,
is reacted with an amine donor in the presence of a ω-transaminase.
The procedural concept according to this aspect of the invention provides for a compound of formula II representing a highly valuable intermediate for the preparation of pharmaceutically active agents such as silodosin or derivatives thereof. It was surprisingly found by the present inventors that compound of formula I is converted via enzymatically catalysed reductive transamination reaction to compound of formula II, which conversion surprisingly can be effected, depending on further suitable conditions such as enzyme type selection, the procedural concept of reductive amination or kinetic resolution respectively provides for enantiomeric excess with a good combination of high yield and chemical purity.
According to a preferred embodiment, the ω-transaminase is (R)-selective, and preferably, the (R)-selective ω-transaminase is derived from an organism selected from the group consisting of Arthrobacter sp. , Aspergillus terreus, Pseudomonas putida and Arthrobacter round 1 1 mutant, in particular the (R)-selective ω-transaminase is (R)- Arthrobacter sp. and Aspergillus terreus, more preferably (R)-Arthrobacter sp. and Aspergillus terreus, and in particular (R)- Arthrobacter sp.
In this way, the ω-transaminase is suitably selected in order to obtain the (fi)-enantiomer of compound of formula II. It was surprisingly found by the present inventors that (R)-selective ω-transaminases enantioselectively convert the prochiral substrate compound of formula I into the (fi)-enantiomer of compound of formula II. For example, in a further preferred case wherein compound of formula II represents e.g. an intermediate for silodosin, the (R)- enantiomer is particularly preferred, since in case of silodosin, this enantiomer is approved e.g. by the Federal Drug Administration (FDA) of the United States as pharmaceutically active agent.
Alternatively, in case there is no specific preference for the kind of enantiomer, that is either the (R) or the (S)-enantiomer may be obtained, the ω-transaminase is derived from an organism selected from Chromobacterium violaceum, Hypophomonas neptunium, Bacillus megaterium, Arthrobacter sp., Aspergillus terreus, Pseudomonas putida, Pseudomonas fluoresceins, Arthrobacter citreus and Arthrobacter round 1 1 mutant, preferably Pseudomonas fluoresceins and (R)-Arthrobacter sp, preferably (R)-Arthrobacter sp.
Preferably, irrespective whether the (fi)-enantiomer or the (S)-enantiomer predominates in the optically active form of compound of formula II, the optically active form has an enantiomeric excess of at least 60-100%, more preferably 75-100%, even more preferably 85-100%, yet even more preferably 90-100%, still yet even more preferably 95-100% and in particular 99-100%. According to this embodiment of the invention, compound of formula II is obtained in advantageous to exceptionally high enantiomeric excess, preferably in high enantiomeric excess.
According to another preferred embodiment, the amine donor is selected from the group consisting of alanine, 1 -ethylamine, 1 -propylamine, 2-propylamine, 1 -indolamine, phenethylamine and others, preferably alanine and 2-propylamine, more preferably 2- propylamine. The aforementioned compounds represent particularly suitable amine donor compounds.
Preferably, compound of formula I is provided in a concentration of 1 to 200 mM in the reaction mixture, preferably 5 to 150 mM, more preferably 10 to 100 mM and in particular 15 to 50 mM. In this way, conversion rate is significantly improved.
It was surprisingly found that in case the ω-transaminase is derived from an organism selected from the group consisting of Chromobacterium violaceum, Bacillus megaterium, Arthrobacter sp., Aspergillus terreus, Pseudomonas putida, Pseudomonas fluorescens and Arthrobacter citreus, a relatively low reaction temperature for carrying out the process of 16- 40°C, preferably 20-36°C, more preferably 24-34°C, even more preferably 26-32°C is particularly suitable. On the other hand, in case the ω-transaminase is Arthrobacter round 11 mutant, the process is preferably carried out at a reaction temperature of 35-50°C, preferably 40-45°C whereby particularly advantageous conversion rates are obtained.
In case alanine is selected as amine donor, preferably, the reaction mixture further comprises alanine dehydrogenase (Ala-DH), formate dehydrogenase (FDH), nicotinamide adenine dinucleotide (NAD+), pyridoxal-5'-phophat (PLP) and ammonium formate.
Alternatively, the reaction mixture further comprises alanine dehydrogenase (Ala-DH), glucose dehydrogenase (GDH), nicotinamide adenine dinucleotide (NAD+), pyridoxal-5'- phophat (PLP), glucose and ammonium salts, or according to another alternative, the reaction mixture further comprises alanine dehydrogenase (Ala-DH), phosphite dehydrogenase (PTDH), nicotinamide adenine dinucleotide (NAD+), pyridoxal-5'-phophat (PLP) and ammonium phosphite. In this way, a particularly suitable alanine-dehydrogenase cofactor recycling system is provided.
According to another aspect of the invention, an optically active chiral amine compound of formula
wherein is selected from the group consisting of CN, NH2, N02 and a group convertible to carbamoyl,
R2 is selected from the group consisting of H, N-protecting group, -(CH2)n-0-R3 or - (CH2)n-0-CO-R4 in which n = 1 to 10,
R3 is a hydroxy protecting group ,and R and R5 are independently from each other selected from substituted or unsubstituted C1 -C10 alkyl or substituted or
unsubstituted C3-C10 aryl or substituted or unsubstituted C3-C10 alkylaryl, is provided by a process comprises the steps of:
of enantiomers of compound of formula II
, wherein R^ R2, R5 and m are defined as above,
with an amine acceptor in the presence of a ω-transaminase for converting the first ula II to a compound of formula I
, wherein R^ R2, R5 and m are defined as above, and
ii) separating the remaining, unreacted second type of enantiomer of compound of formula II from the reaction mixture.
According to this preferred aspect of the invention, a further, alternative process for preparing a compound of formula II having enantiomeric excess and good chemical purity, preferably high enantiomeric excess, is provided. It was surprisingly found by the present inventors that a mixture of enantiomers of compound of formula II, e.g. a racemic mixture thereof, serves as a particularly suitable substrate for efficiently enantiomerically enriching compound of formula II by means of kinetic resolution using ω-transaminase enzymes. In particular, in this kinetic resolution an individual one, e.g. the undesired enantiomer of the compound of formula II is converted to compound of formula I which can be easily separated by physical and/or chemical methods from compound of formula II, while the other individual one, e.g. the desired enantiomer of compound of formula II is converted a lesser extend, preferably not or at least substantially not converted to compound of formula I.
According to a preferred embodiment, in the kinetic resolution step i), the ω-transaminase is derived from an organism selected from the group consisting of Hyphomonas neptunium, Chromobaterium violaceum, Pseudomonas putida I or II, Aspergillus terreus, Bacillus megaterium and Arthrobacter sp., preferably Pseudomonas putida, Aspergillus terreus, Bacillus megaterium and Arthrobacter sp. Preferably, the ω-transaminase is (S)-selective, more preferably the (S)-selective ω-transaminase is derived from an organism selected from the group consisting of Chromobacterium violaceum, Pseudomonas putida I or II, Bacillus megaterium, even more preferably Pseudomonas putida I and Bacillus megaterium.
According to a further preferred embodiment, the optically active chiral amine of compound of formula II obtained in step ii) has an enantiomeric excess of 70-100%, preferably 80- 100%, more preferably 90-100%, even more preferably 95-100%, yet even more preferably 99-100%. According to this embodiment of the invention, compound of formula II is obtained in advantageous to exceptionally high enantiomeric excess, preferably in high enantiomeric excess.
Preferably, the kinetic resolution process is carried out at a reaction temperature of 16-40°C, preferably 20-36°C, more preferably 24-34°C, even more preferably 26-32°C.
The following procedural factors (i) to (viii) respectively represent particularly suitable process conditions for both procedural aspects, namely reductive amination and kinetic resolution, of the present invention:
(i) a molar ratio of compound of formula II to amine acceptor is 1 : 1 to 1 : 10, preferably 1 : 1.2 to 1 :8, more preferably 1 : 1.2 to 1 :4, most preferably 1 : 1.5 to 1 :2.5;
(ii) the ω-transaminase is provided in the form of permeabilized E. coli cells
overexpressing the ω-transaminase or as a crude enzyme extract or a freeze-dried residue thereof or as a (partially) purified enzyme preparation or a freeze-dried residue thereof or as an immobilized preparation, preferably the ω-transaminase is provided in the form of permeabilized E. coli cells overexpressing the ω- transaminase, more preferably a freeze-dried residue of any enzyme solution;
(iii) the ratio of mass of lyophilized E. coli cells overexpressing the ω-transaminase in any form mentioned in item (22) to molar amount of compound of formula II is 10 g/1 mol to 2000 g/1 mol;
(iv) the process is carried out in the presence of an organic solvent selected from the group consisting of DMF, DMSO, THF, MeCN, 1 ,2-dimethoxy ethane (DME), and C1 - C6-alcohols, preferably DMF, DMSO, THF, MeCN, DME and C1 -C3-alcohols, more preferably DMF, DMSO, TH F, MeCN, DME, in particular DMF and DMSO;
(v) the reaction mixture further optionally comprises an aqueous buffer system selected from the group consisting of phosphate buffer, TRIS buffer, PI PES buffer and HEPES buffer, preferably the buffer system is a phosphate buffer, more preferably a potassium phosphate buffer; most preferably a buffer can be omitted;
(vi) R2 represents an amine protective group selected from the group consisting of tert- butylcarbonyl (Boc), benzyloxycarbonyl (Z), 9-Fluorenylmethoxycarbonyl (Fmoc) and allyloxycarbonyl (alloc);
(vii) the hydroxy protective group R3 is selected from the group consisting of benzoyl, tosyl, methoxymethyl, tetrahydropyranyl, t-butyl, allyl, benzyl (Bz), t-butylmethylsilyl, t- butyldiphenylsilyl, acetyl, pivaloyl; and
(viii) in compounds of formulae I and II , m = 1 to 5, is CN , R2 is -(CH2)n-0-CO-R4, wherein n = 1 to 5, R is linear or branched C1 -C4 alkyl or phenyl, and R5 is linear or branched C1 -C4 alkyl, preferably m = 1 to 3, Ri is CN , R2 is -(CH2)n-0-CO-R , wherein n = 1 to 3 and R5 is methyl or ethyl, more preferably m =1 , is CN, R2 is - (CH2)3-0-CO-Ph and R5 is methyl.
Any one of features (i) to (viii) represents particularly advantageous process conditions. In particular, as regards feature (iv), it was surprisingly found that the appropriate choice of solvent significantly improves conversion rate.
According to another aspect of the invention, silodosin having the formula HO
is provided by a process comprising the steps of:
a) providing a compound of formula II
, wherein R2' is -(CH2)3-0-R3' or -(CH2)3-0-CO-R4', in which R3' and R4' represent substituted or unsubstituted C1 -C10 alkyl or substituted or unsubstituted C3-C10 aryl or substituted or unsubstituted C3-C10 alkylaryl, prepared by a process according to any one of items (1) to (27) indicated above, and
b) converting the compound of formula II" of step a) to silodosin.
The above mentioned aspect provides a particularly advantageous process for preparing silodosin owing to the application of the present procedural aspects of reductive amination and/or kinetic resolution and the beneficial effects involved therewith.
According to a still further aspect of the invention, a pharmaceutical formulation is provided which comprises silodosin of formula
as a pharmaceutically active ingredient and at least one pharmaceutically acceptable excipient, wherein silodosin is entirely free of metal impurities.
The following examples further illustrate the invention. They are provided for illustrative purposes only and are not intended to limit the invention in any way. The examples and modifications or other equivalents thereof will become apparent to those versed in the art in the light of the present entire disclosure.
The term "metal impurities" as used herein means impurities deriving from a catalyst in the form of elemental metal, covalent metal compounds or metal compounds in the form of a salt used in conventional chemical syntheses for preparing silodosin. More specifically, "metal impurities" are meant to be derived from metal catalysis conventionally applied in the chemical preparation of silodosin, in which typically e.g. Pd on charcoal and/or Pt02 on BaS04 is/are applied.
The term "pharmaceutically acceptable excipient" as used herein means any physiologically inert, pharmacologically inactive material known in the art being compatible with the physical and chemical characteristics of the active agent.
Examples Materials
General: All starting materials were obtained from commercial suppliers and used as received with exception of racemic 3-(7-cyano-5-(2-(amino)propyl)indolin-1 -yl)propyl benzoate (compound of formula ΙΓ) and 3-(7-cyano-5-(2-(oxo)propyl)indolin-1 -yl)propyl benzoate (compound of formula Γ). The reactions were carried out with standard Schlenk techniques under dry N2 atmosphere in oven-dried (120°C) glassware.
3-(7-cyano-5-(2-(2,2,2-trifluoroacetamido)propyl)indolin-1 -yl)propyl benzoate having the structural formula
was used as reference compound for the below described chiral analysis. This compound was prepared as follows:
To a stirred solution of 3-(7-cyano-5-(2-(amino)propyl)indolin-1 -yl)propyl benzoate (100 mg, 275 μηιοΙ) in 5 mL DME were added trifluoroacetic anhydride (TFAA) (72 mg, 49 μΙ_, 344 μηιοΙ) and DMAP (1 .7 mg, 14 μηιοΙ). The reaction was quenched after 12 hours by the addition of saturated NaHC03 and was then extracted three times with small amounts of CH2CI2. Combined organic layers were dried over MgS04, filtrated and concentrated under reduced pressure. The residue was purified via filter flash chromatography on silica (eluent: PE/EtOAc = 80/20) to afford the product as slightly yellow oil in 61 % yield (77 mg, 168 μηιοΙ). RF [(PE/EtOAc) 60/40] = 0.41. 1H-NMR (300 MHz, CDCI3): δΗ [ppm] = 1 .20 (d, = 6.6 Hz, 3 H, 3'-H), 2.17 = (tt, 3J2",i" = 6.5 Hz, 3J2",3- = 7.1 Hz, 2 H, 2"-H), 2.61 (dd, 3JrHa,2' = 7.4 Hz, 2Jra,rb = 13.8 Hz, 1 H, 1 '-Ha), 2.72 (dd, 3Jrb,2' = 5.9 Hz, 2Jrb,ra = 13.8 Hz, 1 H, 1 '-Hb), 2.97 (t, 3J3,2 = 8.7 Hz, 2 H, 3-Hind), 3.61 (t, 3J2,3 = 8.7 Hz, 2 H, 2-Hind), 3.76 (t, 3J2..,T = 7.2 Hz, 2 H, Γ- H), 4.15 (mc, 1 H, 2'-H), 4.47 (t, 3Jr,2- = 6.3 Hz, 2 H, Γ-Η) , 6.1 1 (d, 3JNH, = 7.9 Hz, 2 H, NH), 6.93 (mc, 2 H, arom-Hind), 7.44 (mc, 2 H, arom.-H), 7.55 (mc, 1 H, arom.-H), 8.06 (mc, 1 H, arom.-H). 13C-NMR (75 MHz, CDCI3): 5C [ppm] = 19.5 (C-3') , 27.3 (C-2"), 27.5 (C-3), 40.9 (C- 1 ') , 45.3 (C-1 ") , 47.5 (C-2') , 53.5 (C-2), 62.7 (C-3") , 88.0 (C-7), 1 19.4 (CF3), 125.5 (CN), 128.8 (arom.-CH), 129.5 (arom.-CHind), 129.9 (arom.-CH), 130.3 (arom.-C1pso), 132.0 (arom.- CHind), 133.2 (arom.-CipSo), 133.3 (arom.-C1pSo), 152.2 (COCF3), 166.8 (COO). 19F-NMR (285 MHz, CDCI3): δΗ [ppm] = -76.00 (s, 3 F, CF3).
Enzymes and Microorganisms
Formate dehydrogenase from Candida boidinii (aqueous buffer solution with glycerol, 215 U mL"1 [one unit will oxidise 1 .0 μηιοΐβ of formate to C02 per min at pH 7.6 at 37 °C], catalog no. FDH 002) and β-NAD free acid were purchased from Codexis. Lyophilised E. coli cells containing overexpressed ω-Transaminase (ω-ΤΑ) and purified recombinant L-alanine dehydrogenase were respectively prepared as reported in F. G. Mutti et. al., Adv. Synth. Catal. 201 1 , 353, p. 3227-3233.
The amino acid sequence of the below listed ω-transaminases originates from the indicated literature, however, the present inventors employed codon optimized genes for improved expression of ω-transaminase in E. coli.
The sequence of ω-transaminase derived from Arthrobacter sp. was taken from EP 0 987 332 A1.
The sequence for round 11 mutant of ω-transaminase derived from Arthrobacter was taken from C. K. Savile et al., "Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones applied to Sitagliptin Manufacture" Science, 2010, 329, p. 305-309. ω-transaminase derived from Aspergillus terreus was prepared as described in F. G. Mutti, Adv. Synth. Catal. 2010, 353, p. 3227-3233. ω-transaminase derived from Bacillus megaterium was prepared as described in R. L.
Hanson et al., "Preparation of (fi)-Amines from Racemic Amines with an (S)-Amine
Transaminase from Bacillus megaterium", Adv. Synth. Catal. 2008, 350, p. 1367-1375. ω-transaminase derived from Chromobacterium violaceum was prepared as described in U. Kaulmann et al., "Substrate spectrum of ω-transaminase from Chromobacterium violaceum DSM30191 and its potential for biocatalysis", Enzyme Microb. Technol. 2007, 41, p. 628- 637.
The sequence for ω-transaminase derived from P. fluorescens was taken from e.g. N. Ito et al., Biosci. Biotechnol. Biochem., 201 1 , 75, p. 2093-2095.
The sequences for ω-transaminase derived from P. putida are described in WO 2010/089171 A2. Pseudomonas putida I corresponds to Seq. ID 20, and P seudomonas putida II to Seq. ID 22.
The sequence for ω-transaminase derived from Arthrobacter citreus was taken from WO 2006 / 063336 (or we us US 7247460 B2). Arthrobacter citreus corresponds to Sep. ID:02 in this patent. All ω-transaminases were expressed in Escherichia coli. In particular, in the following examples, the ω-transaminases are applied in form of lyophilized E. coli cell preparations.
Analytical Methods
1H and 13C NMR spectra were recorded at 20°C on a 300 Bruker NMR unit; chemical shifts are given in ppm relative to Me4Si (1H: Me Si = 0.0 ppm) or relative to the resonance of the solvent (1H: CDCI3 = 7.26 ppm; 13C: CDCI3 = 77.0 ppm).
Determination of conversion in enzymatic kinetic resolution and reductive amination reaction: After the indicated reaction time, saturated Na2C03 solution was added (100 μΙ_) and the mixture was shaken vigorously. The aqueous phase was completely removed under reduced pressure via a SPEEDVAC-system and the residue resuspended with MeCN (500 μΙ_). After filtration through a plug of Celite 545, additional MeCN was added (500 μΙ_) and the process repeated. HPLC conditions: column = Luna RP-C18 (Phenomenex); length = 25 cm, internal diameter = 4.6 mm; isocratic flow with 1.0 mL/min using MeCN / H20 65:35 as eluent (+ 0.1 vol% TFA); ft, (II") = 2.51 min ft, (Γ) = 9.68 min.
Determination of enantiomeric excess (ee): after derivatisation of 3-(7-cyano-5-(2- (amino)propyl)indolin-1 -yl)propyl benzoate to the corresponding TFA-acetamide, a sample of MeCN-solution was withdrawn (500 μΙ_) and concentrated using a rotary evaporator. The residue was dissolved in CH2CI2 (200 μΙ_) and TFAA (ca. 3 eq.) was added. After shaking for 15 minutes at 30°C, the sample was concentrated again. The sample was ready for the measurement after reuptake in organic solvent (/7-heptane / 2-PrOH 50:50) and filtration through a plug of cotton. HPLC conditions: column = OD-H (Chiracel), length = 25 cm, internal diameter = 4.6 mm, isocratic flow with 1.5 mL/min; eluent: /7-heptane/2-PrOH 95:5. ft, [(ft)-N'-TFA-ester] = 19.35 min, ft, [(S)- ll'-TFA-ester] = 22.85 min.
Example 1 : Asymmetric Reductive Amination a) Pre-tests
General preparative procedure for microscale batch: Lyophilized cells of E. coli containing the corresponding overexpressed ω-ΤΑ (20 mg) were rehydrated in a potassium phosphate buffer (pH 7.0, 100 mM) containing PLP (1 mM), NAD+ (1 mM), ammonium formate (150 mM), FDH (1 1 U), Ala-DH (12 U) and D- respective L-alanine (500 mM) at room temperature for 30 minutes. The substrate was added afterwards (3.6 mg / 100 μί DMSO) and the reaction carried out at 30 °C in an Eppendorf orbital shaker (700 rpm) for 24 h. The experimental results are summarized in Table 1 below, in which the listed values represent average values obtained from double determination values (deviations between two determinations < 10%):
entry tji -transaminase conversion [%] ee [%]
1 Chromobacterium violaceum 22 88 (S)
2 Bacillus megaterium 28 95 (S)
3 Arthrobacter citreus 15 89 (S)
4 Pseudomonas fluorescens 84 >99 (S)
5 Pseudomonas putida n .d . 56 (S)*>
6 (R)-Arthrobacter sp. 98 >99 (R)
7 Aspergillus terreus 8 >99 (R)
*' entry 5: n.d. means "not determined", and in this single case, deviation between the two determinations was > 10%; Pseudomonas putida listed under
entry 5 is Pseudomonas putida I
Table 1. Asymmetric reductive amination of 3-(7-cyano-5-(2-(oxo)propyl)indolin-1 -yl)propyl using various ω-ΤΑ.
As becomes obvious from the experimental data listed in Table 1 above, the reductive amination process according to the present invention is particularly suitable to obtain the desired product in enantioenriched form. Preferably, the ω-transaminases is derived from Pseudomonas fluorescens and (R)- Arthrobacter sp. providing both high enantiomeric excess and a high conversion rate. More beneficially, the ω-transaminases is derived from (R)- Arthrobacter sp. providing the (fi)-enantiomer of compound of formula II' representing a highly desirably intermediate product for the preparation of silodosin in both high
enantiomeric excess and high yield. b) Testing of ω-ΤΑ derived from Arthrobacter round 11 mutant General preparative procedure for microscale batch:
Lyophilized cells of E. coli containing overexpressed ω-ΤΑ derived from Arthrobacter round 1 1 mutant (20 mg and 40 mg respectively) were rehydrated in a potassium phosphate buffer (pH 7.0, 100 mM) containing PLP (1 mM) at room temperature for 30 minutes. The substrate was added afterwards (10 and 25 mM resectively) together with 10 vol% DMSO, and 2- propylamine (250 mM) as N-donor in amount determined by the ratio n(substrate)/n(donor), and the reaction was carried out at 45°C in an Eppendorf orbital shaker (700 rpm) for 24 h.
The experimental results are summarized in Table 2 below:
Table 2. Asymmetric reductive amination of 3-(7-cyano-5-(2-(oxo)propyl)indolin-1 -yl)propyl using Arthrobacter round 11 mutant.
As becomes obvious from the experimental data listed in Table 2 above, properties of conversion rate and/or enantiomeric enrichment are achieved by the reductive amination process according to the present invention. The best results with ω-ΤΑ derived from
Arthrobacter round 11 mutant are obtained with the conditions according to entry 4 in terms of enantiomeric excess, and the conditions according to entry 1 in terms of conversion. At the conditions according to entry 2, preferably both enantiomeric excess and conversion are well balanced.
The above general preparative procedure was carried out applying 20 mg lyophilized cells of E. coli containing overexpressed ω-ΤΑ derived from Arthrobacter round 1 1 mutant, wherein reaction conditions were varied in that a decreased reaction temparature of 40°C was applied and various DMSO concentrations were tested. The experimental results are summarized in Table 3 below: Entry vol % (D SO) ee (amine) [%] conversion [%]
1 5 62 68
2 5 67 77
3 10 62 70
4 10 69 78
5 15 64 74
6 15 64 83
7 20| 64 88
8 20 60 77
Table 3. Asymmetric reductive amination of 3-(7-cyano-5-(2-(oxo)propyl)indolin-1 -yl)propyl using Arthrobacter round 11 mutant at decreased reaction tempature of 40°C and varying DMSO concentrations.
As becomes obvious from the experimental data listed in Table 3 above, properties of conversion rate and/or enantiomeric enrichment are achieved by the reductive amination process according to the present invention. At decreased reaction temperature of 40°C, enantiomeric excess was generally slightly increased compared to entry 3 of Table 2. On the other hand, beneficially, all conditions of entries 1 to 8 of Table 3 provide for a
advantageously high conversion rate. c) Testing of solvent and substrate concentration effects for ω-ΤΑ derived from Arthrobacter sp.
In order to evaluate if the substrate concentration of 3-(7-cyano-5-(2-(oxo)propyl)indolin-1 - yl)propyl could be further enhanced without affecting the stereochemical outcome, the conversion as function of organic co-solvent was investigated with the ω-ΤΑ from Arthrobacter sp. Notably, since the compound of formula Γ is almost insoluble in water and unpolar aprotic organic solvents (e.g. toluene, EtOAc, CH2CI2, TBME and Et20), the inventors focused onto the polar and water miscible solvents DMSO, DMF, THF and MeCN. All reactions were conducted at varying substrate concentration (5-25 mivi 3-(7-cyano-5-(2- (oxo)propyl)indolin-1 -yl)propyl) at 30°C for 24 hours. The experimental results are summarized in Scheme 1 below:
Scheme 1. Reductive amination of compound of formula Γ (3-(7-cyano-5-(2- (oxo)propyl)indolin-1 -yl)propyl) as a function of organic co-solvent and substrate concentration (5-25 mM) using the (R)-selective ω-ΤΑ from Arthrobacter sp.
As becomes obvious from the experimental data illustrated in Scheme 1 above, properties of conversion rate and/or enantiomeric enrichment are achieved by the reductive amination process according to the present invention. The solvents DMSO and DMF provide for a significantly improved conversion rate compared with THF and acetonitrile (MeCN).
Furthermore, small substrate concentration provide for a significantly improved conversion rate compared with relatively high substrate concentrations. d) Preparative scale batch
Exemplary preparative procedure for preparative scale batch: Lyophilized cells of E. coli containing overexpressed ω-ΤΑ from (R)- Arthrobacter sp. (225 mg) were rehydrated in a KPi buffer (9 mL, pH 7.0, 100 mM) containing PLP (1.00 mM), NAD+ (1.00 mM), ammonium formate (150 mM), FDH (1 1 U), Ala-DH (12 U) and D-alanine (500 mM) at 22°C for 30 minutes. Compound of formula Γ (3-(7-cyano-5-(2-(oxo)propyl)indolin-1 -yl)propyl) (54 mg, 0.15 mmol) dissolved in 1.0 mL DMF was added afterwards and the reaction was shaken for 26 hours at 30°C reaching 93% conversion. Saturated Na2C03 solution was added (1.00 mL) afterwards and the aqueous phase concentrated under reduced pressure. The residue was resuspended with MeCN (3 x 5 mL) and filtered through a plug of Celite to afford the optically pure amine (fi)-ll' in 88% yield after silica flash chromatography. 1H- MR (300 MHz; CD3CN): δΗ [ppm]: 0.97 (d, J = 6.3 Hz, 3 H), 1.43 (brs, 2 H, - H2), 2.10 (tt, Ji = 7.2 Hz, J2 = 6.2 Hz, 2 H), 2.31 (dd, Ji = 7.6 Hz, J2 = 13.3 Hz, 1 Ha), 2.43 (dd, Ji = 5.7 Hz, J = 13.3 Hz, 1 Hb), 2.87-2.99 (m, 1 H), 2.93 (t, 8.8 Hz, 2 H), 3.57 (t, 8.7 Hz, 2 H), 3.73 (t, 7.2 Hz, 2 H), 4.41 (t, 6.2 Hz, 2 H), 6.94 (s, 1 H), 7.01 (s, 1 H), 7.43-7.51 (m, 2 H), 7.60 (mc, 1 H), 8.00-8.07 (m, 2 H).
13C- MR (75 MHz; CD3CN): δΗ [ppm]: 23.8, 37.5, 27.9, 45.9, 46.1, 49.3, 53.9, 63.6, 88.2, 120.4, 129.4, 130.0, 130.3, 130.9, 131.3, 132.1, 134.0, 134.1, 152.5, 167.2.
Example 2: Kinetic Resolution
General preparative procedure for microscale batch: Lyophilised cells of E. coli containing the corresponding overexpressed ω-transaminase (20 mg) were rehydrated in a potassium phosphate buffer (pH 7.0, 100 mM) containing PLP (1 mM) and sodium pyruvate (1 equivalent) at room temperature for 30 minutes. The substrate was added afterwards (3.6 mg / 100 μΙ_ DMSO) and the reaction was carried out at 30°C in an Eppendorf orbital shaker (700 rpm) for 24 hours.
The experimental results are summarized in Table 4 below, in which the listed values represent average values obtained from double determination values (deviations between two determin
rac-ll' (R) / (S)-W
entry ω-transaminase conversion [%] ee [%] E-value
1 Chromobacteri urn violaceum 43 36 (R) 4
2 Bacillus megaterium 42 67 (R) 52
3 Pseudomonas putida I 45 78 (R) >100
4 Pseudomonas putida II 43 54 (R) 10
5 Arthrobacter citreus 25 34 (R) >100
6 Pseudomonas fluorescens 29 45 (R) 32
7 (R)-Arthrobacter sp. 48 96 (S) >200
8 Aspergillus terreus 44 83 (S) 96
9 Hyphomonas neptunium 35 26 (S) 90
Table 4. Kinetic resolution of racemic compound of formula II' (3-(7-cyano-5-(2- (amino)propyl)indolin-1 -yl)propyl) using various ω-transaminases (ω-ΤΑ). As becomes obvious from the experimental data listed in Table 4 above, properties of conversion rate and/or enantiomeric enrichment are achieved by the kinetic resolution process according to the present invention. Preferably, the ω-transaminases Pseudomonas putida I and II, Aspergillus terreus, Bacillus megaterium and Arthrobacter sp. are applied which provide for both high enantiomeric excess and a high conversion rate. More beneficially, Pseudomonas putida I is applied which provides the (fi)-enantiomer of compound of formula II' representing a highly desirably intermediate product for the preparation of silodosin in both relative high enantiomeric excess and relative high yield.

Claims

Claims pound of formula II
, wherein m is 1 to 8,
is selected from the group consisting of CN, NH2, N02 and a group convertible to carbamoyl,
R2 is selected from the group consisting of H, N-protecting group, -(CH2)n-0-R3 and -(CH2)n-0-CO-R4 in which n = 1 to 10,
R3 is a hydroxy protecting group, and R and R5 are independently from each other selected from substituted or unsubstituted C1 -C10 alkyl or substituted or
unsubstituted C3-C10 aryl or substituted or unsubstituted C3-C10 alkylaryl,
I
, wherein R^ R2, R5 and m are defined as above,
is reacted with an amine donor in the presence of a ω-transaminase.
The process according to claim 1 , wherein the ω-transaminase is derived from an organism selected from the group consisting of Chrome-bacterium violaceum, Hyphomonas neptunium, Bacillus megaterium, (R)- Arthrobacter sp., Aspergillus terreus, Pseudomonas putida, Arothobacter round 11 mutant, Pseudomonas fluorescens and Arthrobacter citreus, preferably Pseudomonas fluorescence and (R)- Arthrobacter sp; or
the ω-transaminase is a (R)-selective transaminase, preferably the (R)-selective ω- transaminase is derived from an organism selected from the group consisting of (R)- Arthrobacter sp., Hyphomonas neptunium, Aspergillus terreus and Arthrobacter round 1 1 mutant, more preferably (R)- Arthrobacter sp. and Aspergillus terreus, in particular (R)-Arthrobacter sp. The process according to claim 1 or 2, wherein the amine donor is selected from the group consisting of alanine, 1 -ethylamine, 1 -propylamine, 2-propylamine, 1 - indolamine, phenethylamine and others, preferably alanine and 2-propylamine, more preferably 2-propylamine.
The process according to any one of claims 1 to 3, wherein the chiral amine compound of formula II is optically active, preferably the optically active compound of formula II has an enantiomeric excess of at least 60-100%, preferably 75-100%, more preferably 85-100%, even more preferably 90-100%, yet even more preferably 95- 100% and in particular 99-100%.
The process according to any one of claims 1 to 4, wherein compound of formula I is provided in a concentration of 1 to 200 mM in the reaction mixture, preferably 5 to 150 mM, more preferably 10 to 100 mM and in particular 15 to 50 mM; and/or
the process is carried out at a reaction temperature of 16-40°C, preferably 20-36°C, more preferably 24-34°C, even more preferably 26-32°C; and/or
in case the ω-transaminase is Arthrobacter round 1 1 mutant, the process is carried out at a reaction temperature of 35-50°C, preferably 40-45°C.
The process according to any one of claims 1 to 5, wherein in case the amine donor is alanine, the reaction mixture further comprises alanine dehydrogenase (Ala-DH), formate dehydrogenase (FDH), nicotinamide adenine dinucleotide (NAD+), pyridoxal- 5'-phophat (PLP) and ammonium formate; or
the reaction mixture further comprises alanine dehydrogenase (Ala-DH), glucose dehydrogenase (GDH), nicotinamide adenine dinucleotide (NAD+), pyridoxal-5'- phophat (PLP), glucose and ammonium salts; or
the reaction mixture further comprises alanine dehydrogenase (Ala-DH), phosphite dehydrogenase (PTDH), nicotinamide adenine dinucleotide (NAD+), pyridoxal-5'- phophat (PLP) and ammonium phosphite.
7. hiral amine compound of formula II
wherein is selected from the group consisting of CN, NH2, N02 and a group convertible to carbamoyl, R2 is selected from the group consisting of H, N-protecting group, -(CH2)n-0-R3 or - (CH2)n-0-CO-R4 in which n = 1 to 10,
R3 is a hydroxy protecting group ,and R and R5 are independently from each other selected from substituted or unsubstituted C1 -C10 alkyl or substituted or
unsubstituted C3-C10 aryl or substituted or unsubstituted C3-C10 alkylaryl, which process comprises the steps of:
of enantiomers of compound of formula II
, wherein R^ R2, R5 and m are defined as above,
with an amine acceptor in the presence of a ω-transaminase for converting the first ula II to a compound of formula I
, wherein R^ R2, R5 and m are defined as above, and
ii) separating the remaining, unreacted second type of enantiomer of compound of formula II from the reaction mixture.
8. The process according to claim 7, wherein the ω-transaminase selected from the group consisting of Hyphomonas neptunium, Chromobaterium violaceum,
Pseudomonas putida I or II, Aspergillus terreus, Bacillus megaterium and
Arthrobacter sp., preferably Pseudomonas putida I, Aspergillus terreus, Bacillus megaterium and Arthrobacter sp; or
the ω-transaminase is (S)-selective, preferably the (S)-selective ω-transaminase is derived from an organism selected from the group consisting of Chromobacterium violaceum, Pseudomonas putida I or II, Bacillus megaterium, more preferably
Pseudomonas putida I and Bacillus megaterium.
9. The process according to claim 7 or 8, wherein the amine acceptor is pyruvate.
10. The process according to any one of claims 7 to 9, wherein the optically active chiral amine of compound of formula II obtained in step ii) has an enantiomeric excess of 70-100%, preferably 80-100%, more preferably 90-100%, even more preferably 95- 100%, yet even more preferably 99-100%.
1 1 . The process according to any one of claims 1 to 10, performed under any one, or a combination of the following procedural formula condition(s) (i) to (v):
(i) a molar ratio of compound of II to amine acceptor is 1 : 1 to 1 : 10, preferably 1 : 1 .2 to 1 :8, more preferably 1 : 1 .2 to 1 :4, most preferably 1 : 1.5 to 1 :2.5.
(ii) the ω-transaminase is provided in the form of permeabilized E. coli cells
overexpressing the ω-transaminase or as a crude enzyme extract or a freeze- dried residue thereof or as a (partially) purified enzyme preparation or a freeze- dried residue or a freeze-dried residue thereof or as
an immobilized preparation, preferably the ω-transaminase is provided in the form of permeabilized E. coli cells overexpressing the ω-transaminase, more preferably the freeze-dried residue of any enzyme solution;
(iii) the ratio of mass of lyophilized E. coli cells overexpressing the ω-transaminase to molar amount of compound of formula I I is 10 g/1 mol to 2000 g/1 mol;
(iv) the process is carried out in the presence of an organic solvent selected from the group consisting of DM F, DMSO, THF, MeCN, DM E, and C1 -C6-alcohols, preferably DM F, DMSO, THF, MeCN , DME and C1 -C3-alcohols, more preferably DMF, DMSO, THF, MeCN, DME and in particular DMF and DMSO;
(v) the reaction mixture further optionally comprises an aqueous buffer system
selected from the group consisting of phosphate buffer, TRIS buffer, PIPES buffer and HEPES buffer; preferably, a buffer is omitted.
12. The process according to any one of claims 1 to 1 1 , wherein compound of formulae I and I I are independently from each characterized by at least one structural features a), b) and/or c):
a) R2 represents an amine protective group selected from the group consisting of tert-butylcarbonyl (Boc), benzyloxycarbonyl (Z), 9-Fluorenylmethoxycarbonyl (Fmoc) and allyloxycarbonyl (alloc);
b) the hydroxy protective group R3 is selected from the group consisting of benzoyl, tosyl, methoxymethyl, tetrahydropyranyl, t-butyl, allyl, benzyl (Bz), t- butylmethylsilyl, t-butyldiphenylsilyl, acetyl, pivaloyl; and/or
c) m = 1 to 5, R is CN , R2 is -(CH2)n-0-CO-R4, wherein n = 1 to 5, R is linear or branched C1 -C4 alkyl or phenyl, and R5 is linear or branched C1 -C4 alkyl, preferably m = 1 to 3, Ri is CN, R2 is -(CH2)n-0-CO-R , wherein n = 1 to 3 and R5 is methyl or ethyl, more preferably m = 1 , Ri is CN, R2 is -(CH2)3-0-CO-Ph and R5 is methyl.
A process for preparing silodosin having the formula
, wherein R2' is -(CH2)3-0-R3' or -(CH2)3-0-CO-R4', in which R3' and R4' represent substituted or unsubstituted C1 -C10 alkyl or substituted or unsubstituted C3-C10 aryl or substituted or unsubstituted C3-C10 alkylaryl, prepared by a process according to any one of claims 1 to 12, and b) converting the compound of formula II" of step a) to silodosin.
EP13780341.7A 2012-10-18 2013-10-18 A process for preparing indoline derivatives Withdrawn EP2909327A1 (en)

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