Process for preparation of 7 alpha-carboxyl 9, 11-epoxy steroids and intermediates useful therein an

专利摘要:
Formula I Formula III Several novel schemes, new process steps and novel intermediates are provided for the synthesis of epoxymexrenone and other compounds of formula (I), where -AA- represents a group -CHR 4 -CHR 5 -or -CR 4 = CR 5- . R 3 , R 4 and R 5 are independently selected from the group consisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano, aryloxy, R 1 represents an α-oriented lower alkoxycarbonyl or hydroxycarbonyl radical, -BB- represents a group -CHR 6 -CHR 7 -or an α- or β-alignment group of formula III, and R 6 and R 7 Is independently selected from the group consisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, R 8 and R 9 is hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyal Independently selected from the group consisting of chel, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, or R 8 and R 9 together form a carbocyclic or heterocyclic ring structure, or R 8 or R 9 together with R 6 or R 7 form a carbocyclic or heterocyclic ring structure condensed into a 5-ring D ring.
公开号:KR20040071782A
申请号:KR10-2004-7011499
申请日:1996-12-11
公开日:2004-08-12
发明作者:엔지존에스.；와핑티.；바에즈줄리오에이.；리우친；앤더슨데니스케이.；로손존피.；에르프던하르트；위초릭조지프；무치아리엘로겐나로；반자넬라포르투나토；쿤다새스트리에이.；레턴더레오제이.；포조마크제이.；싱유엔룽엘.；요난에드워드이.
申请人:지.디. 썰 엘엘씨；
IPC主号:

专利说明:

TECHNICAL FOR PREPARATION OF 7 ALPHA-CARBOXYL 9, 11-EPOXY STEROIDS AND INTERMEDIATES USEFUL THEREIN AND A GENERAL PROCESS FOR THE EPOXIDATION OF OLIFINIC DOUBLE BONDS}
[7] The present invention relates to novel processes for the preparation of 9,11-epoxy steroid compounds, in particular the 20-spiroxane family and their analogues, novel intermediates useful for the preparation of steroid compounds, and methods for preparing such novel intermediates. Most specifically, the present invention relates to methyl hydrogen 9,11α-epoxy-17α-hydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone (eplerenone; epoxymexrenone The present invention relates to a novel and advantageous method of manufacturing.
[8] Methods for preparing 20-spiroxane based compounds are described in US Pat. No. 4,559,332. Compounds prepared according to the method of the '332 patent
[9] Formula IA
[10]
[11] [In the above formula
[12] -AA- is a group -CH ₂ -CH ₂ - or represents -CH = CH-,
[13] R ¹ represents an α-oriented lower alkoxycarbonyl or hydroxycarbonyl radical,
[14] -BB- represents a group -CH ₂ -CH ₂ -or an α- or β-alignment group of formula III
[15] Formula III
[16]
[17] R ⁶ and R ⁷ are hydrogen
[18] X represents two hydrogen atoms or oxo,
[19] Y ¹ and Y ² together represent an oxygen bridge bond -O-, or
[20] Y ¹ represents hydroxy, and
[21] Y ² represents hydroxy, lower alkoxy or lower alkanoyloxy when X is H ₂ ], the salts of such compounds wherein the open oxygen containing rings E and X are oxo, Y ² is hydroxy, ie the corresponding 17β-hydroxy Has -21-carboxylic acid.
[22] U. S. Patent No. 4,559, 332 describes various preparations of epoxymexrenone and related compounds of formula (IA). The approach of new and extended clinical use for epoxymexrenone requires an improved method of preparing several related steroids.
[1] 1 is a schematic flowchart of a process for bioconversion of a canrenone or canrenone derivative to a corresponding 11α-hydroxy compound.
[2] FIG. 2 is a schematic flowchart of a preferred process for bioconversion of canrenones and canrenone derivatives by 11α-hydroxylation.
[3] 3 is a schematic flowchart of a particularly preferred process for bioconversion of canrenones and canrenone derivatives by 11α-hydroxylation.
[4] FIG. 4 shows the particle size distribution of canrenon prepared according to the process of FIG. 2.
[5] FIG. 5 shows the particle size distribution of canrenone sterilized in a transformed fermenter according to the process of FIG. 3.
[6] Corresponding reference properties indicate corresponding parts throughout the drawings.
[23] Summary of the Invention
[24] It is a primary object of the present invention to provide improved methods for the preparation of epoxymexrenone, other 20-spiroxanes, and other steroids having conventional structural features. It is a specific object of the present invention to provide an improved process for producing products of formula (IA) and other related compounds in high yield; To provide such a method comprising a minimum separation step; It is to provide such a method that can satisfy a reasonable price cost and operate at a reasonable conversion cost.
[25] Accordingly, the present invention provides a series of synthetic schemes for epoxymexrenone, intermediates useful for the preparation of eplerenone; And the synthesis of such novel intermediates.
[26] The novel synthetic schemes are described in detail in the description of the preferred embodiments. The novel intermediates of the present invention are immediately later.
[27] Compounds of formula IV correspond to the following structures:
[28] Formula IV
[29]
[30] In the above formula:
[31] -AA- represents the group -CHR ⁴ -CHR ⁵ -or -CR ⁴ = CR ^5-
[32] R ³ , R ⁴ and R ⁵ are independently selected from the group consisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxy carbonyl, cyano, aryloxy,
[33] R ¹ represents an α-oriented lower alkoxycarbonyl or hydroxycarbonyl radical,
[34] R ² is an 11α-leaving group and its removal is effective to create a double bond between 9- and 11-carbon atoms;
[35] -BB- represents a group -CHR ⁶ -CHR ⁷ -or an α- or β-alignment group of formula III:
[36] Formula III
[37]
[38] R ⁶ and R ⁷ are independently selected from the group consisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy ,
[39] R ⁸ and R ⁹ are independently selected from the group consisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy Or R ⁸ and R ⁹ together form a carbocyclic or heterocyclic ring structure, or R ⁸ or R ⁹ together with R ⁶ or R ⁷ form a carbocyclic or heterocyclic ring structure condensed into a 5-ring D ring.
[40] Compounds of formula IVA correspond to formula IV, wherein R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure of formula XXXIV:
[41] (Formula XXXIV)
[42]
[43] X, Y ¹ , Y ² and C (17) in the above formula are as described above.
[44] Compounds of formula IVB correspond to formula IVA, wherein R ⁸ and R ⁹ together form the structure of formula XXXIII:
[45] (Formula XXXIII)
[46]
[47] Compounds of formula (IVC), formula (IVD) and formula (IVE) are each of -AA- and -BB-, -CH ₂ -CH _2- , R ³ is hydrogen, R ¹ is alkoxycarbonyl, preferably methoxycarbonyl Corresponds to any of IV, Formula IVA, or Formula IVB. Compounds within the range of formula IV can be prepared by reacting a lower alkylsulfonylation or acylation reagent or halide generating agent with a corresponding compound within the range of formula V.
[48] Compounds of formula V correspond to the following structures:
[49] Formula V
[50]
[51] -AA-, -BB-, R ¹ , R ³ , R ⁸ and R ⁹ in the above formula are as defined in formula IV.
[52] Compounds of formula VA correspond to formula V and R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure of formula XXXIV:
[53] (Formula XXXIV)
[54]
[55] X, Y ¹ , Y ² and C (17) in the above formula are as described above.
[56] Compounds of formula VB correspond to formula VA and R ⁸ and R ⁹ together form the structure of formula XXXIII:
[57] (Formula XXXIII)
[58]
[59] Compounds of formula (VC), formula (VD) and formula (VE) are: -AA- and -BB- are -CH ₂ -CH _2- , R ³ is hydrogen, R ¹ is alkoxycarbonyl, preferably methoxycarbonyl Corresponds to any of V, Formula VA, or Formula VB. Compounds within the range of formula (V) can be prepared by reacting alkali metal alkoxides with the corresponding compounds of formula (VI).
[60] Compounds of formula VI correspond to the following structures:
[61] Formula VI
[62]
[63] -AA-, -BB-, R ³ , R ⁸ and R ⁹ in the above formula are as defined in formula IV.
[64] Compounds of formula VIA correspond to formula VI and R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure of formula XXXIV:
[65] (Formula XXXIV)
[66]
[67] X, Y ¹ , Y ² and C (17) in the above formula are as described above.
[68] Compounds of formula VIB correspond to formula VIA, and R ⁸ and R ⁹ together form the structure of formula XXXIII:
[69] (Formula XXXIII)
[70]
[71] Compounds of formula VIC, formula VID and formula VIE correspond to any of formula VI, formula VIA or formula VIB, wherein -AA- and -BB- are -CH ₂ -CH _2- , R ³ is hydrogen. Compounds of formula VI, formula VIA, formula VIB, and formula VIC are prepared by hydrolyzing a compound corresponding to formula VII, formula VIIA, formula VIIB, or formula VIIC, respectively.
[72] Compounds of formula VII correspond to the following structures:
[73] Formula VII
[74]
[75] -AA-, -BB-, R ³ , R ⁸ and R ⁹ in the above formula are as defined in formula IV.
[76] Compounds of formula VIIA correspond to formula VII, and R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure of formula XXXIV:
[77] (Formula XXXIV)
[78]
[79] X, Y ¹ , Y ² and C (17) in the above formula are as described above.
[80] Compounds of formula VIIB correspond to formula VIIA, and R ⁸ and R ⁹ together form the structure of formula XXXIII:
[81] (Formula XXXIII)
[82]
[83] Compounds of formula VIIC, formula VIID and formula VIIE correspond to any of formula VII, formula VIIA or formula VIIB, wherein -AA- and -BB- are -CH ₂ -CH _2- , R ³ is hydrogen. Compounds in the range of formula (VII) can be prepared by cyanating compounds in the range of formula (VIII).
[84] Compounds of formula VIII correspond to the following structures:
[85] Formula VIII
[86]
[87] -AA-, -BB-, R ³ , R ⁸ and R ⁹ in the above formula are as defined in formula IV.
[88] Compounds of formula VIIIA correspond to formula VIII, and R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure of formula XXXIV:
[89] (Formula XXXIV)
[90]
[91] X, Y ¹ , Y ² and C (17) in the above formula are as described above.
[92] Compounds of formula VIIIB correspond to formula VIIIA, and R ⁸ and R ⁹ together form the structure of formula XXXIII:
[93] (Formula XXXIII)
[94]
[95] Compounds of formula (VIIIC), formula (VIIID) and formula (VIIIE) correspond to any of formulas (VIII), (VIIIA) or (VIIIB), wherein -AA- and -BB- are -CH ₂ -CH _2- , R ³ is hydrogen. Compounds within the range of formula (VIII) are prepared by oxidizing a substrate consisting of the compound of formula (XXX) as described below by fermentation effective to introduce 11-hydroxy groups into the α-oriented substrate.
[96] Compounds of formula XIV correspond to the following structures:
[97] Formula AXIV
[98]
[99] -AA-, -BB-, R ³ , R ⁸ and R ⁹ in the above formula are as defined in formula IV.
[100] Compounds of formula XIVA correspond to formula XIV, and R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure of formula XXXIV:
[101] (Formula XXXIV)
[102]
[103] X, Y ¹ , Y ² and C (17) in the above formula are as described above.
[104] Compounds of formula (XIV) correspond to formula (XIVA) and R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure of formula (XXXIII):
[105] (Formula XXXIII)
[106]
[107] Compounds of formula XIVC, formula XIVD and formula XIVE correspond to any of formulas XIV, XIVA or XIVB, wherein -AA- and -BB- are -CH ₂ -CH _2- , R ³ is hydrogen. Compounds in the range of formula (XIV) can be prepared by hydrolyzing the corresponding compounds in the range of formula (XV).
[108] Compounds of formula XV correspond to the following structures:
[109] Formula AXV
[110]
[111] -AA-, -BB-, R ³ , R ⁸ and R ⁹ in the above formula are as defined in formula IV.
[112] Compounds of formula XVA correspond to formula XV, and R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure of formula XXXIV:
[113] (Formula XXXIV)
[114]
[115] X, Y ¹ , Y ² and C (17) in the above formula are as described above.
[116] Compounds of formula XVB correspond to formula XVA, and R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure of formula XXXIII:
[117] (Formula XXXIII)
[118]
[119] Compounds of formula XVC, formula XVD and formula XVE correspond to any of formula XV, formula XVA or formula XVB wherein -AA- and -BB- are -CH ₂ -CH _2- , R ³ is hydrogen. Compounds in the range of formula (XV) can be prepared by cyanating the corresponding compounds in the range of formula (XVI).
[120] Compounds of formula XXI correspond to the following structures:
[121] Formula XXI
[122]
[123] -AA-, -BB-, R ³ , R ⁸ and R ⁹ in the above formula are as defined in formula IV.
[124] Compound of formula (XXIA) corresponds to formula (XXI) and R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure of formula (XXXIV):
[125] (Formula XXXIV)
[126]
[127] X, Y ¹ , Y ² and C (17) in the above formula are as described above.
[128] Compounds of formula (XXIB) correspond to formula (XXIA), and R ⁸ and R ⁹ together form the structure of formula (XXXIII):
[129] (Formula XXXIII)
[130]
[131] Compounds of formula (XXIC), formula (XXID) and formula (XXIE) correspond to any of formulas (XI), (XXIA) or (XXIB) wherein -AA- and -BB- are -CH ₂ -CH _2- , R ³ is hydrogen. Compounds within the range of Formula (XXI) can be prepared by hydrolyzing the corresponding compounds within the range of Formula (XXII).
[132] Compounds of formula XXII correspond to the following structures:
[133] Formula XXII
[134]
[135] -AA-, -BB-, R ³ , R ⁸ and R ⁹ in the above formula are as defined in formula IV.
[136] Compounds of formula XXIIA correspond to formula XXII and R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure of formula XXXIV:
[137] (Formula XXXIV)
[138]
[139] X, Y ¹ , Y ² and C (17) in the above formula are as described above.
[140] Compounds of formula XXIIB correspond to formula XXIIA, and R ⁸ and R ⁹ together form the structure of formula XXXIII:
[141] (Formula XXXIII)
[142]
[143] Compounds of formula XXIIC, formula XXIID and formula XXIIE correspond to any of formulas XXII, XXIIA or XXIIB, wherein -AA- and -BB- are -CH ₂ -CH _2- , R ³ is hydrogen. Compounds in the range of formula (XXII) can be prepared by cyanating the corresponding compounds in the range of formula (XXIII).
[144] Compounds of formula XXIII correspond to the following structures:
[145] Formula XXIII
[146]
[147] -AA-, -BB-, R ³ , R ⁸ and R ⁹ in the above formula are as defined in formula IV.
[148] Compounds of formula (XXIIIA) correspond to formula (XXIII), and R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure of formula (XXXIV):
[149] (Formula XXXIV)
[150]
[151] X, Y ¹ , Y ² and C (17) in the above formula are as described above.
[152] Compound of formula (XXIIIB) corresponds to formula (XXIIIA), and R ⁸ and R ⁹ together form the structure of formula (XXXIII):
[153] (Formula XXXIII)
[154]
[155] Compounds of formulas XXIIIC, XXIIID and XXIIIE correspond to any of formulas XXIII, XXIIIA or XXIIIB, wherein -AA- and -BB- are -CH ₂ -CH _2- , R ³ is hydrogen. Compounds within the range of formula (XXIII) can be prepared by oxidizing the compound of formula (XXIV).
[156] Compound of Formula 104 corresponds to the structure:
[157]
[158] In the above formula, -AA-, -BB-, R ³ is as defined in formula IV, and R ¹¹ is C ₁ to C ₄ alkyl.
[159] Compounds of formula 104A correspond to formula 104 wherein -AA- and -BB- are -CH ₂ -CH _2- , and R ³ is hydrogen. Compounds within the range of Formula 104 may be prepared by pyrolysing the compound of Formula 103.
[160] The compound of formula 103 corresponds to the structure:
[161]
[162] -AA-, -BB-, R ³ and R ¹¹ in the above formula are as defined in Formula 104.
[163] Compounds of formula 103A correspond to formula 103 wherein -AA- and -BB- are -CH ₂ -CH ₂ -and R ³ is hydrogen. Compounds within the range of Formula 103 may be prepared by reacting a dialkylmalonate with a corresponding compound of Formula 102 in the presence of a base such as an alkali metal alkoxide.
[164] Compounds of Formula 102 correspond to the following structures:
[165]
[166] -AA-, -BB-, R ³ and R ¹¹ in the above formula are as defined in Formula 104.
[167] Compounds of formula 102A correspond to formula 102 wherein -AA- and -BB- are -CH ₂ -CH ₂ -and R ³ is hydrogen. Compounds within the range of Formula 102 may be prepared by reacting a trialkylsulfonium compound with the corresponding compound of Formula 101 in the presence of a base.
[168] Compounds of Formula 101 correspond to the following structures:
[169]
[170] -AA-, -BB-, R ³ and R ¹¹ in the above formula are as defined in Formula 104.
[171] Compounds of formula 101A correspond to formula 101 wherein -AA- and -BB- are -CH ₂ -CH ₂ -and R ³ is hydrogen. Compounds within the range of Formula 101 may be prepared by reacting trialkylorthoformate with 11α-hydroxyandrosten-3,17-dione or other compounds of Formula XXXVI in the presence of an acid.
[172] Based on the disclosure of the specific schemes described later, it will be apparent that these compounds have maximum utility for the particular scheme. The use of the compounds of the present invention is useful as intermediates for epoxymexrenone and other steroids.
[173] Other objects and features will be in part apparent and in part pointed out later.
[174] According to the present invention, various novel methods for the preparation of epoxymexrenone and other compounds corresponding to formula (I) have been studied:
[175] Formula I
[176]
[177] In the above formula,
[178] -AA- represents the group -CHR ⁴ -CHR ⁵ -or -CR ⁴ = CR ^5-
[179] R ³ , R ⁴ and R ⁵ are independently selected from the group consisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano, aryloxy,
[180] R ¹ represents an α-oriented lower alkoxycarbonyl or hydroxyalkyl radical,
[181] -BB- represents a group -CHR ⁶ -CHR ⁷ -or an α- or β-alignment group of formula III:
[182] Formula III
[183]
[184] R ⁶ and R ⁷ are independently selected from the group consisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy ,
[185] R ⁸ and R ⁹ are independently selected from the group consisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy Or R ⁸ and R ⁹ together form a carbocyclic or heterocyclic ring structure, or together with R ⁶ or R ⁷ , R ⁸ or R ⁹ together form a carbocyclic or heterocyclic ring structure condensed into a 5-ring D ring.
[186] Unless stated otherwise, the term "lower" in this specification is said to contain 7 or fewer carbon atoms, preferably 1-4.
[187] Lower alkoxycarbonyl radicals are preferably derived from alkyl radicals of 1 to 4 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl; Especially preferred are methoxycarbonyl, ethoxycarbonyl and isopropoxycarbonyl. The lower alkoxy radical is preferably derived from the above-mentioned C ₁ -C ₄ alkyl radicals, especially those of primary C ₁ -C ₄ alkyl radicals; Especially preferably, it is methoxy. The lower alkanoyl radicals are preferably derived from straight chain alkyl of 1 to 7 carbon atoms; Particularly preferably formyl and acetyl.
[188] The methylene bridge at the 15,16-position is preferably β-orientation.
[189] A preferred class of compounds that can be prepared according to the process of the invention corresponds to the 20-spiroxane compound described in U.S. Patent No. 4,559,332, ie, the formula
[190] Formula IA
[191]
[192] In the above formula
[193] -AA- is a group -CH ₂ -CH ₂ - or represents -CH = CH-,
[194] -BB- represents a group -CH ₂ -CH ₂ -or an α- or β-alignment group of formula IIIA:
[195] Formula IIIA
[196]
[197] R ¹ represents an α-oriented lower alkoxycarbonyl or hydroxycarbonyl radical,
[198] X represents two hydrogen atoms, oxo or = S
[199] Y ¹ and Y ² together represent an oxygen bridge bond -O-, or
[200] Y ¹ represents hydroxy, and
[201] Y ² represents hydroxy, lower alkoxy or lower alkanoyloxy when X is H ₂ ,
[202] Preferably, the 20-spiroxane compound produced by the novel process of the present invention is of formula I wherein Y ¹ and Y ² together represent an oxygen bridge bond -O-.
[203] Particularly preferred compounds of formula I are those wherein X is oxo.
[204] Among the 20-spiroxane compounds of formula (IA) in which X is oxo, most preferably Y ¹ together with Y ² represents an oxygen bridge bond -O-.
[205] As mentioned above, 17β-hydroxy-21-carboxylic acid may be in the form of a salt thereof. In particular metals and ammonium salts, such as alkali metal and alkaline earth metal salts, for example sodium, calcium, magnesium and, preferably potassium, salts, and ammonia or ammonium salts derived from suitable, preferably physiologically resistant, organic nitrogen-containing bases This can be considered. Amines as bases such as lower alkylamines (eg triethylamine), hydroxy-lower alkylamines [eg 2-hydroxyethylamine, di- (2-hydroxyethyl) -amine or tri- (2 -Hydroxyethyl) -amine], cycloalkylamines (eg dicyclohexylamine) or benzylamines (eg benzylamine and N, N'-dibenzylethylenediamine), as well as nitrogen-containing heterocyclic compounds, for example Consideration also has to be made of aromatic properties (eg pyridine or quinoline) or at least partially saturated heterocyclic rings (eg N-ethylpiperidine, morpholine, piperazine or N, N'-dimethylpiperazine) Can be.
[206] Preferred compounds which are also included are alkali metal salts, in particular potassium salts, and in the compounds of formula (IA), R ¹ represents alkoxycarbonyl, X represents oxo, Y ¹ and Y ² each represent hydroxy.
[207] Particularly preferred compounds of formula (I) and formula (IA) are, for example:
[208] 9α, 11α-epoxy-7α-methoxycarbonyl-20-spirox-4-ene-3,21-dione,
[209] 9α, 11α-epoxy-7α-ethoxycarbonyl-20-spirox-4-ene-3,21-dione,
[210] 9α, 11α-epoxy-7α-isopropoxycarbonyl-20-spirox-4-ene-3,21-dione, and
[211] 1,2-dehydro analogs of each compound,
[212] 9α, 11α-epoxy-6α, 7α-methylene-20-spirox-4-ene-3,21-dione,
[213] 9α, 11α-epoxy-6β, 7β-methylene-20-spirox-4-ene-3,21-dione,
[214] 9α, 11α-epoxy-6β, 7β; 15β, 16β-bismethylene-20-spirox-4-ene-3,21-dione, and
[215] 1,2-dehydro analogs of each of these compounds,
[216] 9α, 11α-epoxy-7α-methoxycarbonyl-17β-hydroxy-3-oxo-pregin-4-ene-21-carboxylic acid,
[217] 9α, 11α-epoxy-7α-ethoxycarbonyl-17β-hydroxy-3-oxo-pregin-4-ene-21-carboxylic acid,
[218] 9α, 11α-epoxy-7α-isopropoxycarbonyl-17β-hydroxy-3-oxo-pregan-4-ene-21-carboxylic acid,
[219] 9α, 11α-epoxy-17β-hydroxy-6α, 7α-methylene-3-oxo-pregin-4-ene-21-carboxylic acid,
[220] 9α, 11α-epoxy-17β-hydroxy-6β, 7β-methylene-3-oxo-pregin-4-ene-21-carboxylic acid,
[221] 9α, 11α-epoxy-17β-hydroxy-6β, 7β; 15β, 16β-bismethylene-3-oxo-pregin-4-ene-21-carboxylic acid, and alkali metal salts of these respective acids, in particular potassium salts Or ammonium, and 1,2-dehydro analogs of each of the carboxylic acids or salts thereof mentioned.
[222] 9α, 11α-epoxy-15β, 16β-methylene-3,21-dioxo-20-spirox-4-ene-7α-carboxylic acid methyl ester, ethyl ester and isopropyl ester,
[223] 9α, 11α-epoxy-1565β, 16β-methylene-3,21-dioxo-20-spiroxa-1,4-diene-7α-carboxylic acid methyl ester, ethyl ester and isopropyl ester, and
[224] 9α, 11α-epoxy-3-oxo-20-spirox-4-ene-7α-carboxylic acid methyl ester, ethyl ester and isopropyl ester,
[225] 9α, 11α-epoxy-6β, 6β-methylene-20-spirox-4-en-3-one,
[226] 9α, 11α-epoxy-6β, 7β; 15β, 16β-bismethylene-20-spirox-4-en-3-one, and
[227] 9α, 11α-epoxy, 17β-hydroxy-17α (3-hydroxy-propyl) -3-oxo-androst-4-ene-7α-carboxylic acid methyl ester, ethyl ester and isopropyl ester,
[228] 9α, 11α-epoxy, 17β-hydroxy-17α- (3-hydroxypropyl) -6α, 7α-methylene-androst-4-en-3-one,
[229] 9α, 11α-epoxy, 17β-hydroxy-17α- (3-hydroxypropyl) -6β, 7β-methylene-androst-4-en-3-one,
[230] 9α, 11α-epoxy, 17β-hydroxy-17α- (3-hydroxypropyl) -6β, 7β; 15β, 16β-bismethylene-androst-4-en-3-one,
[231] 17α- (3-acetoxypropyl) and 17α- (3-formyloxypropyl) analogs of the androstane compounds mentioned,
[232] 1,2-dehydro analogs of all compounds mentioned in the androst-4-en-3-one and 20-spirox-4-en-3-one families.
[233] The chemical names of the compounds of the formulas (I) and (IA), and analogue compounds having structural properties of the same properties, are according to the conventional nomenclature of the following schemes: compounds in which Y ¹ together with Y ² represent -O- are derived from For example, a compound of formula (IA) wherein X is oxo, Y ² and Y ¹ represents —O— is derived at 20-spiroxane-21-one); Each of Y ¹ and Y ² represents hydroxy and X represents oxo is from 17β-hydroxy-17α-pregeneene-21-carboxylic acid; It is derived from 17β-hydroxy-17α- (3-hydroxypropyl) -androstan, wherein Y ¹ and Y ² each represent hydroxy and X represents two hydrogen atoms. Ring and open chain forms, i.e. lactones and 17β-hydroxy-21-carboxylic acids and their salts, respectively, are understood above and below because they are closely related to each other so that the latter can only be thought of as the former hydrated form. As specifically stated, unless otherwise stated, both the final product of formula (I) and the starting material and the intermediate of the analogous structure may both be in the form mentioned together in each case.
[234] According to the invention, several separate processes have been provided for the preparation of the compounds of formula I in high yields and at reasonable costs. Each synthetic scheme proceeds through a series of intermediate preparations. Many of these intermediates are novel compounds and the preparation of these intermediates is novel.
[235] Scheme 1 ( Canrenon Or start with the relevant substance)
[236] One preferred method for the preparation of compounds of formula (I) discloses canrenones or related starting materials corresponding to formula (XIII)
[237] Formula XIII
[238]
[239] In the above formula
[240] -AA- represents the group -CHR ⁴ -CHR ⁵ -or -CR ⁴ = CR ^5-
[241] R ³ , R ⁴ and R ⁵ are independently selected from the group consisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano, aryloxy,
[242] -BB- represents a group -CHR ⁶ -CHR ⁷ or an α- or β-alignment group of formula III:
[243] Formula III
[244]
[245] R ⁶ and R ⁷ are independently selected from the group consisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy , And
[246] R ⁸ and R ⁹ are independently selected from the group consisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy Or R ⁸ and R ⁹ together form a carbocyclic or heterocyclic ring structure, or together with R ⁶ or R ⁷ , R ⁸ and R ⁹ form a carbocyclic or heterocyclic ring structure condensed into a 5-ring D ring. Compounds of formula VIII are prepared by introducing an α-oriented 11-hydroxy group into a compound of formula XIII using a biotransformation process of the type shown in FIGS. 1 and 2:
[247] Formula VIII
[248]
[249] -AA-, -BB-, R ³ , R ⁸ and R ⁹ in the formula are as described above. Preferably, the compound of formula XIII has the structure
[250] (Formula XXXA)
[251]
[252] 11α-hydroxy product has the structure
[253] Formula VIIIA
[254]
[255] [In each chemical formula
[256] -AA- is a group -CH ₂ -CH ₂ - or represents -CH = CH-,
[257] -BB- represents a group -CH ₂ -CH ₂ -or an α- or β-alignment group of formula IIIA:
[258] Formula IIIA
[259]
[260] X represents two hydrogen atoms, oxo or = S,
[261] Y ¹ and Y ² together represent an oxygen bridge bond -O-, or
[262] Y ¹ represents hydroxy, and
[263] Y ² represents hydroxy, lower alkoxy or lower alkanoyloxy when X is H ₂ ] and a salt of a compound wherein X is oxo and Y ² is hydroxy, and the compound of formula VIII produced in the reaction is represented by formula VIIIA. Corresponds:
[264] Formula VIIIA
[265]
[266] -AA-, -BB-, Y ¹ , Y ² and X in the above formula are as defined in formula XXXA. More preferably, R ⁸ and R ⁹ together form a 20-spiroxane structure of the formula XXXIII:
[267] (Formula XXXIII)
[268]
[269] In the formula, -AA- and -BB- are each -CH ₂ -CH _2- , and R ³ is hydrogen.
[270] Preferred organisms that can be used in this hydroxylation step areAspergillus ochraceusNRRL 405,Aspergillus ochraceusATCC 18500,Aspergillus nigerATCC 16888 and ATCC 26693,Aspergillus nidulansATCC 11267,Rhizopus oryzaeATCC 11145,Rhizopus stoloniferATCC 6227b,Streptomyces fradiaeATCC 10745,Bacillus megateriumATCC 14945,Pseudomonas cruciviaeATCC 13262, andTrichothecium roseumATCC 12543. Other preferred organisms areFusarium oxysporum f.sp.cepaeATCC 11171 andRhizopus arrhizusATCC 11145.
[271] Other organisms that have shown activity for this reactionAbsidia coerulaATCC 6647,Absidia glaucaATCC 22752,Actinomucor elegansATCC 6476,Aspergillus flavipesATCC 1030,Aspergillus fumigatusATCC 26934,Beauveria bassianaATCC 7159 and ATCC 13144,Botryosphaeria obtusaIMI 038560,Calonectria decoraATCC 14767,Chaetomium cochliodesATCC 10195,Corynespora cassiicolaATCC 16718,Cunninghamella blakesleeanaATCC 8688a,Cunninghamella echinulataATCC 3655,Cunninghamella elegansATCC 9245,Curvularia clavataATCC 22921,Curvularia lunataATCC 12071,Cylindrocarpon radicicolaATCC 1011,Epicoccum humicolaATCC 12722,Gongronella butleriATCC 22822,Hypomyces chrysospermus,Mortierella isabellinaATCC 42613,Mucor mucedoATCC 4605,Mucor griseo -cyanusATCC 1207A,Myrothecium verrucariaATCC 9095,Nocardia corallina,Paecilomyces carneusATCC 46579,Penicillum patulumATCC 24550,Pithomyces atro-olivaceusIFO 6651,Pithomyces cynodontisATCC 26150,Pycnosporium sp.ATCC 12231,Saccharopolyspora erythraeATCC 11635,Sedonium chrysospermumATCC 13378,Stachylidium bicolorATCC 12672,Streptomyces hygroscopicusATCC 27438,Streptomyces purpurascensATCC 25489,Syncephalastrum racemosumATCC 18192,Thamnostylum piriformeATCC 8992,Thielavia terricolaATCC 13807, andVerticillium theobromaeATCC 12474.
[272] Additional organisms that can be expected to be active for 11α-hydroxylation includeCephalosporium aphidicola(Phytochemistry (1996), 42 (2), 411-415),Cochliobolus lunatas(J. Biotechnol. (1995), 42 (2), 145-150),Tieghemella orchidis(Khim.-Farm. Zh. (1986), 20 (7), 871-876),Tieghemella hyalospora(Khim.-Farm. Zh. (1986), 20 (7), 871-876),Monosporium olivaceum (Acta Microbiol. Pol. Ser. B. (1973), 5 (2), 103-110),Aspergillus ustus(Acta Microbiol. Pol., Ser. B. (1973), 5 (2), 103-110),Fusarium graminearum(Acta Microbiol. Pol., Ser. B. (1973), 5 (2), 103-110),Verticillium glaucum(Acta Microbiol. Pol., Ser. B. (1973), 5 (2), 103-110), andRhizopus nigricans(J. Steroid Biochem. (1987), 28 (2), 197-201).
[273] Prior to scale fermentation production for the hydroxylation of canrenone or other substrate of Formula XIII, the inoculum of cells is produced in a spawn fermentation tank, or in a spawn fermentation system consisting of a series of two or more spawn fermenters. The working spore suspension is introduced with the culture broth to the fermenter of the first strain for cell growth. If the volume of inoculum required or desired for production exceeds that produced in the first spawn fermenter, the inoculum volume can be serially progressively and geometrically amplified through the remaining fermenters in the spawn fermentation heat. Preferably, the inoculum produced in the spawn fermentation system is a sufficient volume of viable cells for rapid initiation of the reaction in production fermentation tanks, relatively short production batch circulation, and high production fermentation tank activity. Whatever the number of containers of the series of seed fermenters, the second and subsequent seed fermenters are preferably sized so that the degree of dilution is essentially the same in each step of this row. The initial dilution of the inoculum in each spawn fermentation tank may be approximately the same as the dilution in the production fermentation tank. Canrenone or other Formula XIII substrate is added to the production fermentation bath together with the inoculum and culture and the hydroxylation reaction is carried out here.
[274] The spore suspension in the spawn fermentation system is derived from a vial of a working spore suspension taken from a plurality of vials constituting a working storage cell bank which is stored under cryogenic conditions before use. The work storage cell bank is then derived from the main storage cell bank prepared in the following manner. Spore samples obtained from a suitable source such as ATCC are initially suspended in an aqueous medium such as saline solution, culture or surfactant solution (e.g., nonionic surfactant such as Tween 20 at a concentration of about 0.001% by weight). The suspension is dispensed into culture plates, each plate containing a solid culture mixture based on non-degradable polysaccharides, typically agar, in which spores grow. The solid culture mixture preferably comprises about 0.5% to about 5% glucose, about 0.05% to about 5% nitrogen source, such as peptone, about 0.05% to about 0.5% phosphorus source, For example, alkali metal phosphates or ammonium phosphates, such as dipotassium hydrogen phosphate, from about 0.25% to about 2.5% by weight yeast lysate or extract (or other amino acid source such as meat extract or brain heart culture), from about 1% by weight to It contains about 2% by weight agar or other non-degradable polysaccharides. Optionally, the solid culture mixture may further comprise or contain about 0.1% to about 5% malt extract by weight. The pH of the solid culture mixture is preferably about 5.0 to about 7.0 and adjusted as needed by alkali metal hydroxide or orthophosphoric acid. Useful solid growth media include:
[275] 1.Solid Medium # 1: 1% Glucose, 0.25% Yeast Extract, 0.3% K ₂ HPO ₄ and 2% Agar (Bacto); Adjust pH to 6.5 with 20% NaOH.
[276] 2. Solid medium # 2: 2% peptone (Bacto), 1% yeast extract (Bacto), 2% glucose and 2% agar (Bacto); Adjust pH to 5 with 10% H ₃ PO ₄ .
[277] 3. Solid Medium # 3: 0.1% Peptone (Bacto), 2% Malt Extract (Bacto), 2% Glucose and 2% Agar (Bacto); pH 5.3.
[278] 4. Liquid medium: 5% blackstrap molasses, 0.5% cornsteep liquor, 0.25% glucose, 0.25% NaCl and 0.5% KH ₂ PO ₄ , pH adjusted to 5.8.
[279] 5. Difco Mycological Agar (low pH).
[280] The number of agar plates used for the development of the primary storage cell bank can be chosen as needed for future primary storage, but typically about 15 to 30 plates are thus made. After a suitable growth period, for example 7 to 10 days, the plates are scraped for harvesting spores in the presence of an aqueous excipient, typically saline or buffer, and the resulting main storage suspension is obtained in small amounts, eg 1 ml, in each of a plurality of 1.5 ml vials. Distribute each time. In order to prepare a work spore suspension for use in research or production fermentation, the content of one or more of these second-generation main storage vials is dispensed on the agar plate in the manner described above for the production of the main storage spore suspension, Can be cultured. Given a certain manufacturing operation, as many as 100 to 400 plates can be used to generate the second generated work storage. Each plate was scraped into separate working storage vials and each vial was prepared containing typically 1 ml of inoculum. For permanent preservation, both the primary storage suspension and the second generation production inoculation are advantageously stored in the vapor space of cryogenic storage vessels containing liquid N ₂ or other cryogenic liquids.
[281] In the process shown in FIG. 1, an aqueous growth medium is prepared to include a peptone, yeast derivative or equivalent, a nitrogen source such as glucose and a phosphorus source such as phosphate. Spores of microorganisms are cultured in this medium in a spawn fermentation system. Preferred microorganisms are Aspergillus ochraceus NRRL 405 (ATCC 18500). The spawn storage thus prepared is then introduced into the production fermentation tank together with the substrate of formula XIII. The fermentation broth is stirred for a sufficient time to stir and react and terminate at the desired degree.
[282] The medium for the spawn fermenter is preferably about 0.5% to about 5% glucose, about 0.05% to 5% nitrogen source, for example peptone, about 0.05% to about 0.5% artificial Sources such as ammonium or alkali metal phosphates, such as ammonium phosphate monobasic or dipotassium hydrogen phosphate, from about 0.25% to about 2.5% by weight yeast acetate or extract (or other amino acid source that can be dissolved in a still) Consisting of an aqueous mixture containing from 1% to about 2% by weight agar or other non-degradable polysaccharides. Particularly preferred seed growth media are from about 0.05% to about 5% by weight of a nitrogen source, such as peptone, from about 0.25% to about 2.5% by weight of self-decomposed yeast or yeast extract, from about 0.5% to about 5% by weight. Glucose, and a phosphorus source such as about 0.05% to about 0.5% by weight of ammonium phosphate monobasic. Particularly economical process operations include from about 0.5% to about 5% by weight of cornstalk solution, from about 0.25% to about 2.5% by weight of autolyzed yeast or yeast extract, from about 0.5% to about 5% by weight of glucose and about Obtained using another preferred seed culture containing 0.05% to 0.5% by weight of ammonium phosphate monobasic. Constipates are particularly preferred economic sources of proteins, peptides, carbohydrates, organic acids, vitamins, metal ions, traces and phosphates. Meshes from other grains may be used in place of or in addition to corn tips. The pH of the medium is preferably adjusted within the range of about 5.0 to about 7.0 by addition of alkali metal hydroxide or orthophosphoric acid. If cornsip liquor is used as the nitrogen and carbon source, the pH is preferably adjusted within the range of about 6.2 to about 6.8. The medium consisting of peptone and glucose is preferably adjusted to a pH of about 5.4 to about 6.2. Useful growth media for use in spawn fermentation include:
[283] 1. Medium # 1: 2% peptone, 2% autolyzed yeast (or yeast extract) and 2% glucose; pH was adjusted to 5.8 with 20% NaOH.
[284] 2. Medium # 2: 3% corn steep solution, 1.5% yeast extract, 0.3% ammonium phosphate monobasic and 3% glucose; Adjust pH to 6.5 with 20% NaOH.
[285] Microbial spores are typically introduced into this medium from vials containing about 10 ⁹ spores per ml of suspension. Optimal productivity of spawning is achieved by diluting the growth medium without reducing spore density to about 10 ⁷ per ml at the start of spawning. Preferably, the spores are cultured in the spawn fermentation system until the fill mycelial volume (PMV) in the spawn fermentation tank is at least about 20%, preferably 35% to 45%. Since the circulation in the spawn fermentation vessel (or any of a plurality of vessels consisting of spawn fermentation heat) depends on the initial concentration of the vessel, it may be desirable to provide two or three spawn fermentation steps to accelerate the overall process. However, if the spawn fermentation is carried out through an excessive number of steps, it is preferable not to use a series of spawn fermenters significantly more than three because the activity may be lowered. Seed culture fermentation is carried out under stirring at a temperature in the range of about 23 ° C. to about 37 ° C., preferably in the range of about 24 ° C. to about 28 ° C.
[286] Cultures from the spawn fermentation system are introduced into the production fermenter along with the productive medium. In one embodiment of the invention, sterile canrenone or another substrate of formula (XIII) is used as the reaction substrate. Preferably, the substrate is added to the production fermentation bath in the form of a slurry of 10% to 30% by weight in the growth medium. In order to increase the effective surface area for the 11α-hydroxylation reaction, the particle size of the formula XIII substrate is reduced by passing the substrate through an offline micronizer before introducing it into the fermentor. Secondary sterile cultures containing yeast derivatives, such as glucose containing sterile culture storage and self-lyzed yeast (or comparable amino acid preparations based on other sources that can be dissolved in stills) are also introduced separately. The medium may contain about 0.5% to about 5% glucose, about 0.05% to about 5% nitrogen source, such as peptone, about 0.05% to about 0.5% phosphorus source, such as hydrogen phosphate Alkali metal phosphates or ammonium phosphates such as potassium, about 0.25% to about 2.5% yeast lysate or extract (or other amino acid source that can be dissolved in a still), about 1% to about 2% by weight agar or It consists of an aqueous mixture containing other non-degradable polysaccharides. Particularly preferred productivity medium is a nitrogen source such as about 0.05% to about 5% by weight peptone, about 0.25% to about 2.5% self-decomposed yeast or yeast extract, about 0.5% to about 5% glucose And from about 0.05% to about 0.5% by weight phosphorus source, such as ammonium phosphate monobasic. Other preferred production media include about 0.5% to 5% by weight of cornstalk solution, about 0.25% to about 2.5% by weight of self-decomposed yeast or yeast extract, about 0.5% to about 5% by weight of glucose and about 0.05% % To about 0.5% by weight of ammonium phosphate monobasic. The pH of the production fermentation medium is preferably adjusted in the manner described above for the seed fermentation medium in the same preferred range as the pH of the peptone / glucose medium and the cornsip liquor medium. Useful biotransformation growth media are:
[287] 1. Medium # 1: 2% peptone, 2% autolyzed yeast (or yeast extract) and 2% glucose; pH was adjusted to 5.8 with 20% NaOH.
[288] 2. Medium # 2: 1% peptone, 1% autolyzed yeast (or yeast extract) and 2% glucose; pH was adjusted to 5.8 with 20% NaOH.
[289] 3. Medium # 3: 0.5% peptone, 0.5% autolyzed yeast (or yeast extract) and 0.5% glucose; pH was adjusted to 5.8 with 20% NaOH.
[290] 4. Medium # 4: 3% corn steep solution, 1.5% yeast extract, 0.3% ammonium phosphate monobasic and 3% glucose; Adjust pH to 6.5 with 20% NaOH.
[291] 5. Medium # 5: 2.55% corn steep solution, 1.275% yeast extract, 0.255% ammonium phosphate monobasic and 3% glucose; Adjust pH to 6.5 with 20% NaOH.
[292] 6. Medium # 6: 2.1% corn steep solution, 1.05% yeast extract, 0.21% ammonium phosphate monobasic and 3% glucose; Adjust pH to 6.5 with 20% NaOH.
[293] Non-sterile canrenones and sterile cultures are fed continuously to the production fermentation tank with 5 to 20 parts, preferably 10 to 15 parts, preferably substantially the same part, for the production batch circulation. Advantageously, the substrate is initially introduced in an amount sufficient to establish a concentration of from about 0.1% to about 3%, preferably from about 0.5% to about 2% by weight before incubation with the spawn fermentation broth and then about It is easily added periodically every 8 to 24 hours at a cumulative rate of 1% to about 8% by weight. Additional substrates are added alternately every 8 hours, and the total addition may be slightly lower than if the substrates are added on a daily basis only, for example 0.25% to 2.5% by weight. In the latter example the cumulative canrenone addition may require a range of 2% to about 8% by weight. Supplementary culture mixtures supplied during the fermentation reaction are preferably sterilized from concentrates, for example from about 40% to about 60% by weight of sterile glucose, from about 16% to about 32% by weight of sterile yeast extract or other yeast derivatives. Mixtures containing a source (or other amino acid source). Since the substrate supplied to the production fermentation tank of Figure 1 is non-sterile, antibiotics are periodically added to the fermentation broth to control the growth of unwanted organisms. Antibiotics such as kanamycin, tetracycline and cephalexin can be added without adversely affecting growth and biotransformation. Preferably, the fermentation broth is at a concentration of, for example, from about 0.0004% to about 0.002%, based on the total amount of the culture, for example from about 0.0002% to about 0.0006% kanamycin sulfate, based on the total amount of the culture. It is introduced at a concentration consisting of 0.0002% to about 0.006% tetracycline HCl and / or about 0.001% to 0.003% cephalexin.
[294] Typically, the fermentation batch cycle is about 80-160 hours. Thus, the Formula XIII substrate and a portion of the culture are each added typically every 2 to 10 hours, preferably every 4 to 6 hours. Advantageously, antifoaming is also included in the spawn fermentation system and production fermentation bath.
[295] Preferably, in the process of FIG. 1, the inoculum charged to the production fermenter is from about 0.5 to about 7 volume percent, more preferably from about 1 to about 2 volume percent, based on the total mixture in the fermenter, and the glucose concentration is a total batch. It is preferably about 0.01% to about 1.0% by weight, preferably about 0.025% to about 0.5% by weight, and more preferably about 0.05% by weight, based on the filling, in periodic additions of about 0.05% to about 0.25% by weight. It is maintained at weight percent to about 0.25 weight percent. The fermentation temperature is easily controlled in the range of about 20 ° C. to about 37 ° C., preferably about 24 ° C. to about 28 ° C., but less than about 60%, more preferably less than about 50%, of the filled mycelial volume (PMV). It may be desirable to have a temperature, such as a 2 ° C. increment, that decreases step by step during the reaction to prevent the viscosity of the fermentation broth from interfering with sufficient mixing. Once biomass growth is extended over the liquid surface, the substrate retained in the biomass can be carried out in the reaction zone and becomes invalid for the hydroxylation reaction. For productivity, it is desirable to reach PMV in the range of 30-50%, preferably 35% -45% within the first 24 hours of the fermentation reaction, but preferably further growth within the limits described above Take some control. The pH of the fermentation broth during the reaction is controlled to about 5.0 to about 6.5, preferably about 5.2 to about 5.8, and the fermentor is stirred at a speed of about 400 to about 800 rpm. The dissolved oxygen level of at least about 10% saturation is achieved by venting the batch at about 0.2 to about 1.0 vvm and maintaining the pressure in the headspace of the fermenter near atmospheric pressure to about 1.0 bar gauge, most preferably near 0.7 bar gauge. Is achieved. The agitation rate can be increased as needed to maintain the minimum dissolved oxygen level. Advantageously, the dissolved oxygen level is kept at least 10%, in fact as high as 50%, to facilitate the conversion of the substrate. Keep the pH in the range of 5.5 ± 0.2 for optimal bioconversion. Foaming is controlled by adding a conventional antifoaming agent as necessary. After all the substrates have been added, the reaction is continued, preferably until the molar ratio of the product of formula VIII to the remaining unreacted formula XIII substrate is at least about 9 to 1. Such conversion can be achieved within the above batch cycle of 80-160 hours.
[296] It was found that high conversion was associated with reducing the initial culture level to the initial fill level by controlling the rate of aeration and agitation to prevent the substrate from jumping out of the liquid culture. In the process of Figure 1, the culture level is reduced to less than about 60% of the initial fill level, preferably to about 50%, and then maintained; On the other hand, in the process of Figures 2 and 3, the culture level was maintained after reducing it to about 80% or less, preferably about 70%, of the initial fill level. The aeration rate is preferably in the range of 1 vvm or less, more preferably about 0.5 vvm; On the other hand, the stirring speed is preferably 600 rpm or less.
[297] Particularly preferred methods for the preparation of compounds of formula VIII are shown in FIG. 2. Again, the preferred microorganismAspergillus ochraceusNRRL 405 (ATCC 18500). In this method, the growth medium is preferably about 0.5% to about 5% by weight of cornstalk solution, about 0.5% to about 5% by weight glucose, about 0.1% to about 3% by weight yeast extract, and about 0.05% to about 0.5% by weight of ammonium phosphate. However, other productivity badges described herein can also be used. The spawn culture is essentially produced in the manner described for the process of FIG. 1 using any spawn fermentation medium described herein. Undifferentiated canrenone or other substrate suspensions of formula (XIII) in growth media are preferably aseptically prepared in the blender at a relatively high concentration of from about 10% to about 30% by weight substrate. Preferably, the aseptic preparation may comprise sterilization or pasteurization of the suspension after mixing. The total amount of sterile substrate suspension required for the production batch is introduced into the production fermentation tank at the beginning of the batch or by periodic continuous feeding. The particle size of the substrate is reduced by wet grinding in an on-line sheer pump that carries the slurry to the production fermentation tank, thereby eliminating the need for the use of an offline micronizer. When the sterile conditions are pasteurized rather than sterilized, the degree of cohesion may not be important, but the use of sheer pumps may be desirable to provide reliable control of the particle size. Bactericidal growth medium and glucose solution are introduced into the production fermentation tank essentially in the same manner as above. Prior to introduction of all feed ingredients into the production fermentation tank, sterilization is eliminated.
[298] Preferably, in the process operation of FIG. 2, the inoculum is introduced into the production fermentation bath at a concentration of about 0.5% to about 7%, and the fermentation temperature is about 20 ° C to about 37 ° C, preferably about 24 ° C to about 28 ° C. And the pH is controlled from about 5.3 to about 5.5, preferably by introduction of gaseous ammonia, aqueous ammonium hydroxide, aqueous alkali metal hydroxide, or orthophosphoric acid. As in the method of FIG. 1, the temperature is preferably adjusted to control the growth of biomass so that the PMV does not exceed 55-60%. The initial glucose charge is preferably about 1% to about 4%, most preferably 2.5% to 3.5% by weight, but preferably deviates to less than about 1.0% by weight during fermentation. Supplementary glucose is periodically fed with a portion of from about 0.2% to about 1.0% by weight based on the total batch fill to within a range from about 0.1% to about 1.5%, preferably from about 0.25% to about 0.5% Maintain glucose concentration in the fermentation zone. Optionally, the nitrogen and phosphorus sources can be supplemented with glucose. However, since full canrenone filling is produced at the start of the batch cycle, an essential supply of nitrogen and phosphorus containing cultures can be introduced when using only supplemental glucose solution during the reaction. Agitation rates and properties can vary significantly. Moderately vigorous stirring facilitates mass transfer between the solid substrate and the water phase. However, low sheer impellers are used to prevent the microbial myelin sensitivity. The optimum stirring speed will vary within the range of 200 to 800 rpm depending on the culture viscosity, the oxygen concentration and the mixing conditions affected by the vessel, baffle and impeller configurations. Typically, the preferred stirring speed is in the range of 350-600 rpm. Preferably the stirring impeller provides a downward shaft pumping function to aid in good mixing of the fermentation biomass. The batch is preferably vented at a rate of about 0.3 to 1.0 vvm, preferably 0.4 to 0.8 vvm, and the pressure in the headspace of the fermenter is preferably about 0.5 to about 1.0 bar gauge. The temperature, agitation, venting and back pressure are preferably controlled to maintain the dissolved oxygen amount in the range of at least about 10 volume percent during bioconversion. The total batch circulation is typically about 100 to about 140 hours.
[299] Although the operating principle of the process of FIG. 2 substantially depends on the initial introduction of full canrenon filling, it will be appreciated that the growth of fermentation broth can be effected prior to filling most canrenones. Optionally, small amounts of canrenone may be added later to the batch. Generally, however, at least about 75% of sterile canrenone filling should be introduced into the transgenic fermenter within 48 hours after the start of fermentation. Furthermore, it is preferred to introduce at least about 25% by weight canrenone, or biotransformation enzyme (s) at least within the first 24 hours at the start of the fermentation.
[300] In another preferred process shown in Figure 3, the entire batch fill and culture are sterilized in a production fermentation vessel prior to introduction of the inoculum. Of these, not only the preferred one but also the culture medium which can be used are essentially the same as in the process of FIG. 2. In an embodiment of the invention, the substrate agglomerates do not become sheer of the stirrer impeller and others tend to form upon sterilization. It was found that the reaction proceeded sufficiently when the average particle diameter of the canrenone was less than about 200 mu and at least 75 wt% of the particles was smaller than 240 mu. At moderate speeds ranging from 200 to 800 rpm with a tip speed of at least about 400 cm / s, the use of a suitable impeller, for example a disk turbine impeller, maintains such particle size properties despite the agglomeration that tends to occur during sterilization in a production fermentation tank. It was found that it provides sufficient shear velocity. The in-process residual operation of FIG. 3 is essentially the same as in the process of FIG. The process of FIGS. 2 and 3 provides several distinct advantages over the process of FIG. 1. Particular advantages follow the use of inexpensive culture bases, such as corn steep solution. However, other benefits are realized by the elimination of antibiotics, the simplification of the feeding process and the batch sterilization of canrenone or other formula XIII substrates. Another particular advantage is the use of a simpler glucose solution than the supplemental complex culture in the reaction cycle.
[301] In the process shown in Figures 1-3, the product of Figure VIII is a crystalline solid with biomass that can be separated from the reaction liquid by filtration or low speed centrifugation. Alternatively, the product can be extracted from the total reaction solution with an organic solvent. The product of formula VIII is recovered by solvent extraction. For maximum recovery, both liquid filtrate and biomass filter or centrifugal cake are treated with extractant, but usually ≧ 95% of the product is associated with biomass. Typically, hydrocarbons, esters, chlorinated hydrocarbons and ketone solvents can be used for extraction. Preferred solvent is ethyl acetate. Typically other suitable solvents include toluene and methylisobutylketone. For extraction from the liquid phase, it may be easy to use a volume of solvent approximately equal to the volume of the reaction solution in contact. In order to recover the product from the biomass, the latter is suspended in large quantities for the initial charge of the solvent, preferably the substrate, for example 50-100 ml solvent per gram of the initial canrenone charge, and the suspension obtained is preferably biomass pore. And reflux for 20 minutes to several hours to deliver the product from the recess to the solvent phase. The biomass is then removed by filtration or centrifugation and the filter cake is preferably washed with both fresh solvent and deionized water. The aqueous and solvent washes are combined and the phases separated. The product of formula VIII is recovered by crystallization from solution. To maximize the yield, the hyphae are contacted twice with fresh solvent. The product is recovered from the solvent phase after standing to complete separation of the aqueous phase. Most preferably, the solvent is removed in vacuo until crystallization is initiated, and then the concentrated extract is 0 ° C. to 20 ° C., preferably about 10, for a time sufficient for crystal precipitation and growth, typically 8 to 12 hours. Cool to a temperature of about 15 ° C to about 15 ° C.
[302] Particularly preferred is the process of FIG. 2 and in particular the process of FIG. 3. These processes operate at low viscosity and follow close control of process scales such as pH, temperature and dissolved oxygen. Moreover, sterilization status is easily maintained without depending on antibiotics.
[303] Since the bioconversion process is exothermic, heat must be removed using a jacketed fermentation tank or cooling coil in the production fermentation tank. Alternatively, the reaction culture may be circulated through an external heat exchanger. At least about 5% by volume, preferably at least about enough to reliably convert glucose into CO ₂ and H ₂ O and provide energy to the reactants by controlling the rate of air entering the reactor to determine the oxygen potential in the culture The amount of dissolved oxygen is preferably maintained at a level of 10% by volume. The pH is preferably adjusted at about 4.5 to about 6.5.
[304] In each other method for 11-hydroxylation of a substrate of formula (XIII), productivity is limited by mass transfer to the phase interface at which an aqueous phase or reaction occurs in the solid substrate. As mentioned above, productivity is not significantly limited to the mass transfer rate as long as the average particle diameter of the substrate is reduced to less than about 300 mu and at least 75% by weight of the particle is less than 240 mu. However, the productivity of these processes can be further increased in any other embodiment that provides a substantial fermentation of canrenone or other Formula XIII substrates in an organic solvent to the production fermentation bath. According to one option, the substrate is dissolved in a water immiscible solvent and mixed with an aqueous growth medium inoculum and a surfactant. Useful water immiscible solvents include, for example, DMF, DMSO, C ₆ -C ₁₂ fatty acids, C ₆ -C ₁₂ n-alkanes, vegetable oils, sorbitan and surfactant aqueous solutions. Stirring of this charge results in an emulsion reaction system having an extended interfacial area for mass transfer of the substrate from the organic liquid phase to the reaction site.
[305] A second option is to initially dissolve in water miscible solvents such as acetone, methylethylketone, methanol, ethanol or glycerol at concentrations substantially greater than solubility in water. The solubility is increased by preparing the initial substrate solution at elevated temperature, thereby increasing the amount of solution to form the substrate introduced into the reactor and eventually increasing the reactor payload. The warm substrate solution is placed in a production fermentation tank reactor with a relatively cold water charge containing growth medium and inoculum. When the substrate solution is mixed with an aqueous medium, the substrate is precipitated. However, under conditions of substantial supersaturation and moderately vigorous stirring, nucleation leads to crystal growth and forms fine particles with high surface area. High surface area facilitates mass transfer between liquid and solid substrates. Moreover, the equilibrium concentration of the substrate in the aqueous liquid phase is increased in the presence of a water miscible solvent. Thus, productivity is accelerated.
[306] Although microorganisms may not necessarily be resistant to high concentrations of organic solvents in the water phase, concentrations of ethanol, such as from about 3% to about 5% by weight, may be advantageously used.
[307] A third option is to dissolve the substrate in aqueous cyclodextrin. Examples of cyclodextrins include hydroxypropyl-β-cyclodextrin and methyl-β-cyclodextrin. The molar ratio of substrate: cyclodextrin may be about 1: 1 to about 1: 1.5 substrate: cyclodextrin. The substrate: cyclodextrin mixture can then be aseptically added to the bioconversion reactor.
[308] 11α-hydroxykanrenone and other products (Formula VIII and Formula VIIIA) during the 11α-hydroxylation process are novel compounds, which can be isolated by filtering the reaction medium and extracting the product from the biomass collected on the filtration medium. have. Conventional organic solvents such as ethyl acetate, acetone, toluene, chlorinated hydrocarbons and methyl isobutyl ketone can be used for extraction. The product of formula VIII can then be recrystallized from organic solvents of the same type. Compounds of formula (VIII) have substantial value as intermediates for the preparation of compounds of formula (I), in particular formula (IA).
[309] Preferably, the compound of formula VIII corresponds to formula VIIIA, wherein -AA- and -BB- are -CH ₂ -CH ₂ , R ³ is hydrogen, lower alkyl or lower alkoxy, and R ⁸ and R ⁹ are Together constitute a 20-spiroxane ring of formula XXXIII:
[310] (Formula XXXIII)
[311]
[312] Also according to the process of Scheme 1, the compound of formula VIII is reacted with a source of cyanide ions under alkaline conditions to produce an enamine compound of formula VII:
[313] Formula VII
[314]
[315] -AA-, R ³ , -BB-, R ⁸ and R ⁹ in the formula are as described above.
[316] When the substrate corresponds to formula VIIIA, the product is of formula VIIA:
[317] Formula VIIA
[318]
[319] -AA-, -BB-, R ³ , Y ¹ , Y ² and X in the above formula are as defined in formula (XIII).
[320] Cyanation of the 11α-hydroxyl substrate of formula VIII can be effected by reacting with a cyanide ion source such as ketone cyanohydrin, most preferably acetone cyanohydrin in the presence of a base and an alkali metal salt, most preferably LiCl. . Alternatively, cyanation can be carried out without cyanohydrin by using alkali metal cyanide in the presence of an acid.
[321] In the ketone cyanohydrin process, the reaction is carried out using a solution, preferably an aprotic polar solvent such as dimethylformamide or dimethyl sulfoxide. Formation of enamines requires at least 2 moles of cyanide ion source per mole of substrate, preferably using a slightly excess cyanide source. The base is preferably a nitrogenous base such as dialkylamine, trialkylamine, alkanolamine, pyridine and the like. However, inorganic bases such as alkali metal carbonates or alkali metal hydroxides may also be used. Preferably the substrate of formula VIII is initially provided at a concentration of about 20% to about 50% by weight and the base is provided at a rate of 0.5 to 2 equivalents per equivalent of substrate. The reaction temperature is not limited but productivity is increased by operation at elevated temperatures. Thus, for example when using triethylamine as the base, the reaction is advantageously carried out at temperatures in the range of about 80 ° C to about 90 ° C. At such a temperature, it proceeds to complete the reaction in about 5 to about 20 hours. When diisopropylethylamine is used as the base and the reaction is carried out at 105 ° C., the reaction is completed at 8 hours. When the reaction time is complete, the solvent is removed in vacuo and the residual oil is dissolved in water and neutralized to pH 7 with dilute acid, preferably hydrochloric acid. The product is precipitated from this solution, then washed with dilute water and dried with air. Free HCN is stripped with inert gas and quenched with alkaline solution. The dry precipitate is taken up in chloroform or other suitable solvent and then extracted with concentrated acid, for example 6N HCl. The extract is neutralized to pH 7 by addition of an inorganic base, preferably alkali metal hydroxide, and cooled to a temperature in the range of 0 ° C. The precipitate obtained is washed and dried and then recrystallized in a suitable solvent such as acetone to give the product of formula VII suitable for use in the next step of the process.
[322] Alternatively, the reaction can be carried out in an aqueous solvent system consisting of a water miscible organic solvent such as methanol or in a two phase system consisting of water and an organic solvent such as ethyl acetate. Alternatively, the product can be recovered by diluting the reaction solution with water and extracting the product using an organic solvent such as methylene chloride or chloroform and then extracting it again from the organic extract using a concentrated inorganic acid such as 2N HCl. See US Pat. No. 3,200,113.
[323] According to another alternative, the reaction is carried out in a water miscible solvent such as dimethylformamide, dimethylacetamide, N-methyl, pyrrolidone or dimethyl sulfoxide and then the reaction product solution is diluted with water and alkaline by addition of alkali metal carbonate. This can be followed by cooling to 0-10 [deg.] C. to precipitate the product. Preferably, the system is quenched with hypohalite or other reagent that is effective to prevent evaporation of cyanide. After filtration and washing with water, the precipitated product is suitable for use in the next step of the process.
[324] According to another alternative, the enamine product of formula (VII) is formed of an excess of alkali metal cyanide, preferably NaCN, in an aqueous solvent consisting of an aprotic water miscible polar solvent such as dimethylformamide or dimethylacetamide. In the presence of a substrate of formula VIII. The proton source is preferably an inorganic acid or C ₁ to C ₅ carboxylic acid, with sulfuric acid being particularly preferred. In unusual cases, it is not necessary to add a separate proton source when the cyanide reagent is typically LiCN in DMF.
[325] Cyanide ions are placed in the reactor at a ratio of preferably about 2.05 to about 5 molar equivalents per equivalent of substrate. Inorganic acids or other proton sources are thought to be promoted by the addition of HCN through double bonds of 4,5 and 6,7 and are preferably provided in a ratio of at least 1 molar equivalent per molar equivalent of the substrate; However, the reaction system is made basic by maintaining an excess amount of alkali metal cyanide than the acid present. The reaction is preferably carried out at a temperature of at least about 75 ° C., typically 60 ° C. to 100 ° C., for about 1 to 8 hours, preferably about 1.5 to about 3 hours. When the reaction is complete, the reaction mixture is preferably cooled to near room temperature; The resulting enamine is precipitated by acidifying the reaction mixture and mixing near cold water, preferably near the ice bath temperature. Acidification is thought to approach 17-lactone, which tends to open under basic conditions that undergo cyanation. The reaction mixture is easily acidified with the same acid present in the reaction, preferably sulfuric acid. Water is preferably added at a rate of about 10 to about 50 molar equivalents per mole of product.
[326] Compounds of formula VII are novel compounds and have substantial value as intermediates for the preparation of compounds of formula I, in particular formula IA. Preferably, the compound of formula VII corresponds to formula VIIA, wherein -AA- and -BB- are CH ₂ -CH ₂ , R ³ is hydrogen, lower alkyl or lower alkoxy, and R ⁸ and R ⁹ together Formula 20 constitutes a 20-spiroxane ring of XXXIII:
[327] (Formula XXXIII)
[328]
[329] Most preferably the compound of formula VII is 5'R (5'α), 7'β-20'-aminohexadecahydro-11'β-hydroxy-10'α, 13'α-dimethyl-3 ', 5-dioxospyro [furan-2 (3H), 17'α (5'H)-[7,4] metheno [4H] cyclopenta [a] phenanthrene] -5'-carbonitrile.
[330] In the next step of the synthesis, the enamine of formula VII is hydrolyzed to yield the diketone compound of formula VI:
[331] Formula VI
[332]
[333] -AA-, R ³ , -BB-, R ⁸ and R ⁹ in the above formula are as defined in formula VIII. Any organic or inorganic acid can be used for hydrolysis. Hydrochloric acid is preferred. In order to increase productivity, water miscible organic solvents such as lower alkanols are preferably used as cosolvents. The acid should be provided in a proportion of at least one equivalent per equivalent of the substrate of formula VII. In aqueous systems, the enamine substrate VII can be substantially converted to the diketone of formula VII at about 80 ° C. for about 5 hours. Operation at elevated temperatures increases productivity, but the temperature is not limited. Suitable temperatures are chosen based on the solvent system and the volatility of the acid.
[334] Preferably, the enamine substrate of formula VII corresponds to formula VIIA
[335] Formula VIIA
[336]
[337] The diketone product corresponds to the formula VIA:
[338] Formula VIA
[339]
[340] -AA-, -BB-, Y ¹ , Y ² and X in each formula are as defined in formula VIIIA.
[341] At the end of the reaction, the solution is cooled to 0-25 ° C. to crystallize the product. The product crystals can be recrystallized from a suitable solvent such as isopropanol or methanol to obtain a product of formula VI suitable for use in the next step of the process, but recrystallization is usually not necessary. The product of formula VI is a novel compound, which has substantial value as an intermediate for the preparation of compounds of formula I, in particular formula IA. Preferably the compound of formula VI corresponds to formula VIA, wherein -AA- and -BB- are -CH ₂ -CH _2- , R ³ is hydrogen, lower alkyl or lower alkoxy, and R ⁸ and R ⁹ are Together constitute a 20-spiroxane ring of formula XXXIII:
[342] (Formula XXXIII)
[343]
[344] Most preferably, the compound of formula VI is 4'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β-dimethyl-3 ', 5,20'- Trioxosepyro [furan-2 (3H), 17'β- [4,7] methano [17H] cyclopenta [a] phenanthrene] -5'β (2'H) -carbonitrile.
[345] In a particularly preferred embodiment of the invention, the enamine product of formula VII is prepared from the compound of formula VIII in the manner described above and converted in situ to the diketone of formula VI. In an embodiment of the invention, the substrate of formula VIII is reacted with an excess of alkali metal cyanide in an aqueous solvent containing a proton source, or optionally with an excess of ketone cyanohydrin in the presence of a base and LiCl described above. However, instead of cooling the reaction mixture, water is added to the calculated concentration to acidify and cause precipitation of the enamine, and substantial cooling of the reaction mixture is preferably omitted. Water and an acid, preferably an inorganic acid such as sulfuric acid, are added to the mixture at the end of the cyanation reaction, the concentration of acid added being sufficient to neutralize excess alkali metal cyanide, and at least one molar equivalent acid per mole of the substrate of formula VIII, preferably Preferably, about 2 to about 5 molar equivalents per substrate equivalent is usually required. However, the temperature is kept sufficiently high and the dilution is sufficiently large to avoid substantial precipitation and to proceed the hydrolysis of enamine to diketone in the liquid phase. Therefore, the process of minimal interference and high productivity is advanced. Hydrolysis typically takes place at temperatures in the range of preferably at least 80 ° C., more preferably about 90 ° C. to 100 ° C. for about 1 to about 10 hours, more preferably about 2 to about 5 hours. The reaction mixture is then cooled to an ice bath temperature, preferably from about 0 ° C. to about 15 ° C., advantageously from about 5 ° C. to about 10 ° C. for the precipitation of the diketone product of Formula VI. The solid product can be recovered by filtration and impurities can be removed by washing with water.
[346] In the next step of the synthesis, the diketone compound of formula VI is reacted with a metal alkoxide to open the bond between positions 4 and 7 and break the bond between the carbonyl group and the 4-carbon to cleave the α-oriented alkane oil at position 7 Form a carbonyl substituent and remove the cyanide from 5-carbon. The product of this reaction is a hydroxyester compound corresponding to formula (V):
[347] Formula V
[348]
[349] In the above formula, -AA-, R ³ , -BB-, R ⁸ and R ⁹ are as defined in formula VIII, and R ¹ is lower alkoxycarbonyl or hydroxycarbonyl. The metal alkoxide used for the reaction corresponds to the formula R ¹⁰ OM in which M is an alkali metal and R ¹⁰ corresponds to an alkoxy substituent of R ¹ . The yield of this reaction is most satisfactory when the metal alkoxide is K or Na methoxide, but other lower alkoxides may be used. K alkoxides are particularly preferred. Phenoxides, other aryloxides as well as arylsulfides may also be used. The reaction is easily conducted in the presence of an alcohol R ¹⁰ corresponds to formula R ¹⁰ OH as described above. Other conventional solvents can be used. Preferably, Formula VI substrate is provided at a concentration of about 2% to about 12% by weight, more preferably at least about 6% by weight and R ¹⁰ OM is provided at a rate of about 0.5 to about 4 moles per mole of substrate. . The temperature is not limited but the elevated temperature increases productivity. The reaction time is typically about 4 to about 24 hours, preferably about 4 to 16 hours. Preferably, the reaction is carried out at ambient reflux temperature depending on the solvent used.
[350] In converting the diketone of formula VI to the hydroxyester of formula VI, the byproduct cyanide ion can be reacted with the product to form a 5-cyanoester. Since it is more preferable to equilibrate at low temperatures, it is preferred that the reaction be rather high in dilution, for example 40: 1, for reaction with Na methoxide. It has been found that significantly higher productivity can be realized with the use of K methoxide over Na methoxide, since dilution in the range of about 20: 1 when K methoxide is a reagent is usually sufficient to minimize reverse cyanation.
[351] In accordance with the present invention, it was further found that the reverse cyanation reaction can be suppressed by removing byproduct cyanide ions from the reaction zone by making appropriate chemical or physical measurements. Thus, in a further embodiment of the invention, the reaction of diketones with alkali metal alkoxides can be carried out in the presence of a precipitant for cyanide ions, such as salts consisting of cations forming insoluble cyanide compounds. Such salts include, for example, any halides, sulfates or other salts that require alkaline earth or transition metals that are more soluble than zinc iodide, ferric sulfate, or the corresponding cyanide. If zinc iodide is provided in a ratio in the range of about 1 equivalent per equivalent of diketone substrate, the productivity of the reaction is substantially increased compared to the process carried out in the absence of alkali metal halides.
[352] Even when a precipitant is used for the removal of cyanide ions, it is desirable to dilute considerably higher, but the use of a precipitant can significantly reduce the solvent to diketone substrate molar ratio compared to the reaction without such a precipitant. Recovery of the hydroxyester of formula V can be carried out according to any of the following extraction or non-extraction procedures.
[353] Preferably, the diketone substrate of Formula VI corresponds to Formula VIA
[354] Formula VIA
[355]
[356] The hydroxyester product corresponds to the formula VA:
[357] Formula VA
[358]
[359] In each formula, -AA-, -BB-, Y ¹ , Y ² and X are as defined in formula XIII and R ¹ is as defined in formula V.
[360] Compounds of formula V are novel compounds, which have substantial value as intermediates for the preparation of compounds of formula I, in particular formula IA. Preferably, the compound of formula V corresponds to formula VA, wherein -AA-, -BB- is -CH ₂ -CH _2- , R ³ is hydrogen, lower alkyl or lower alkoxy, R ⁸ and R ⁹ Together constitute a 20-spiroxane ring of formula XXXIII:
[361] (Formula XXXIII)
[362]
[363] Most preferably, the compound of formula V is methyl hydrogen 11α, 17α-dihydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.
[364] The compound of formula V can be separated by acidifying the reaction solution, for example with concentrated HCl, cooling to ambient temperature and extracting the product with an organic solvent such as methylene chloride or ethyl acetate. The extract is washed with an aqueous alkaline wash and filtered dry and then the solvent is removed. Alternatively, the product containing reaction solution of formula V can be quenched with concentrated acid. The product solution is concentrated, cooled to 0-25 [deg.] C. and the resulting solid is separated by filtration.
[365] According to a preferred mode of product recovery of formula V, methanol and HCN are removed by distillation of the reaction with water and acid added before or during the distillation followed by termination of the reaction. The addition of water before distillation simplifies the operation, while the progressive addition during distillation keeps the volume of the still still substantially constant. The product of formula V is crystallized at the bottom of the distiller as distillation proceeds. Recovery in this manner provides a high quality crystal product without any extraction operation.
[366] According to another alternative, the reaction solution containing the product of formula V is quenched with an inorganic acid such as 4N HCl and then the solvent is removed by distillation. Removal of the solvent is also effective to remove residual HCN from the reaction product. It has been found that complex solvent extraction for the purification of the compound of formula V is not necessary when the compound of formula V is used as an intermediate in the process for preparing epoxymexrenone described herein. In fact, such extraction can often be omitted completely. When solvent extraction is used for product purification, it is preferred to assist the solvent by washing with brine and caustic wash liquor. However, when omitting solvent extraction, brine and caustic washes are also omitted. The omission of extraction and washing significantly increases the productivity of the process without sacrificing yield or product quality and also eliminates the need for drying the washing solution with desiccant, such as sodium sulfate. The crude 11α-hydroxy-7α-alkanoyloxycarbonyl product is absorbed back into the solvent for the next reaction step of the process and the 11-hydroxy group is converted to a good leaving group at the 11 position to yield the compound of formula IV:
[367] Formula IV
[368]
[369] In the above formula, -AA-, R ³ , -BB-, R ⁸ and R ⁹ are as defined in formula VIII, R ¹ is as defined in formula V, and R ² is lower arylsulfonyloxy, alkyl Sulfonyloxy, acyloxy or halide. Preferably, 11α-hydroxyl is esterified by reaction with lower arylsulfonyl halides, acyl halides or acid anhydrides and added to the solution containing the intermediate product of formula (V). Lower alkylsulfonyl halides are preferred, in particular methanesulfonyl halides. Alternatively, the 11α-hydroxy group can be converted to a halide by reaction of a suitable reagent such as thionyl bromide, thionyl chloride, sulfuryl chloride or oxalyl chloride. Other reagents for forming the 11α-sulfonic acid esters include tosyl chloride, benzenesulfonyl chloride and trifluoromethanesulfonic anhydride. The reaction is carried out in a solvent containing a hydrogen halide scavenger such as triethylamine or pyridine. Inorganic bases such as K or Na carbonates can also be used. The initial concentration of the hydroxyesters of formula V is preferably from about 5% to about 50% by weight. The esterification reagent is preferably provided in slightly excess amount. Methylene chloride is a particularly suitable solvent for the reaction, but other solvents such as dichloroethane, pyridine, chloroform, methylethylketone, dimethoxyethane, methylisobutylketone, acetone, other ketones, ethers, acetonitrile, toluene and tetrahydrofuran Can be used. The reaction temperature is mainly determined by the volatility of the solvent. For methylene chloride, the reaction temperature is preferably in the range of about -10 ° C to about 10 ° C.
[370] Preferably, the hydroxyester substrate of formula V corresponds to formula VA
[371] Formula VA
[372]
[373] The product corresponds to the formula IVA:
[374] Formula IVA
[375]
[376] In each formula, -AA-, -BB-, Y ¹ , Y ² and X are as defined in formula (XIII), R ¹ is lower alkanoyloxycarbonyl or hydroxycarbonyl, and R ² is in formula (IV) As defined.
[377] The product of formula IV is a novel compound, which has substantial value as an intermediate for the preparation of compounds of formula I, in particular formula IA. Preferably, the compound of formula IV corresponds to formula VA, wherein -AA- and -BB- are -CH ₂ -CH _2- , R ³ is hydrogen, lower alkyl or lower alkoxy, and R ⁸ and R ⁹ Together constitute a 20-spiroxane ring of formula XXXIII:
[378] (Formula XXXIII)
[379]
[380] Most preferably, the compound of formula IV is methyl hydrogen 17α-hydroxy-11α- (methylsulfonyl) oxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.
[381] If desired, the compound of formula IV can be isolated by removal of the solvent. Preferably, the reaction solution is first washed with an alkaline washing aqueous solution, for example 0.5-2N NaoH, followed by an acid wash with 0.5-2N HCl. The product is recrystallized after removal of the reaction solvent, for example, the product is taken up in methylene chloride and then precipitated in crystalline form by addition of another solvent, such as ethyl ether, which degrades the solubility of the product of formula IV. In the recovery of the product of formula IV or in the preparation of a reaction solution for the conversion of the intermediate of formula IV to the intermediate of formula II, which is further described below, all extraction and / or washing steps are carried out, provided that the solution is It may be omitted if it is instead treated with an ion exchange resin for removal. The solution is first treated with anion exchange resin and then with cation exchange resin. Alternatively, the reaction solution may be treated with an inorganic adsorbent such as basic alumina or basic silica followed by a dilute acid wash. Basic silica or basic alumina can typically be mixed with the reaction solution at a rate of about 5 to about 50 g per kg of product, preferably about 15 to about 20 g per kg of product. If ion exchange resins or inorganic adsorbents are used, the treatment can be carried out by simply slurrying the reaction solution and the resin or inorganic adsorbent under stirring at ambient temperature and then removing the resin or inorganic adsorbent by filtration.
[382] In an alternative and preferred embodiment of the invention, the resulting compound of formula IV is recovered in crude form such as a concentrated solution by removal of some solvent. This concentrated solution is used directly in the next step of the process and the 11α-leaving group is removed from the compound of formula IV to produce the following ester of formula II:
[383] Formula II
[384]
[385] -AA-, R ³ , -BB-, R ⁸ and R ⁹ in the above formula are as defined in formula VIII, and R ¹ is as defined in formula V. For the purpose of this reaction, the R ² substituent in the compound of formula IV may be any leaving group and is considered effective to produce a double bond between 9- and 11-carbon. Preferably, the leaving group is a lower alkylsulfonyloxy or acyloxy substituent which is removed by reaction with acid and alkali metal salts. Inorganic acids may be used, but lower alkanoic acids are preferred. Advantageously, the alkali metal salt of alkanoic acid used as a reagent for reaction is further mentioned. The leaving group is mesyloxy and the removal reagents are formic acid and potassium formate and relatively high ratios of 9,11 to 11,12-olefins are observed. If free water is present during the removal of the leaving group, impurities tend to form, especially 7,9-lactone
[386]
[387] Silver is difficult to remove from the final product.
[388] Thus, acetic anhydride or other desiccant is used to remove the water present in the formic acid. The free water content in the reaction mixture prior to the reaction should be maintained at less than about 0.5% by weight, preferably less than about 0.1% by weight, as determined by Karl Fisher analysis of water based on the total reaction solution. While it is desirable to keep the reaction mixture as dry as possible, satisfactory results have been realized with 0.3% by weight water. Preferably, the reaction charge mixture contains from about 4% to about 50% by weight of the substrate of formula IV in alkanoic acid. About 4% to about 20% by weight of the alkali metal salt of the acid is preferably included. When acetic anhydride is used as the drying agent, it is preferably provided in a ratio of about 0.05 mole to about 0.2 mole per mole of alkanoic acid.
[389] The concentrations of by-products 7,9-lactone and 11,12-olefins in the reaction mixture were used as reagents for the removal of leaving groups and for the formation of esters (9,11-olefins). And relatively low when made up of a combination of potassium acetate. Trifluoroacetic anhydride is used as the desiccant and should be provided at a concentration of at least about 3% by weight, more preferably at least about 15% by weight, most preferably about 20% by weight, based on the trifluoroacetic acid remover.
[390] Alternatively, the ester of formula II can be prepared by removing the 11α-leaving group from the compound of formula IV and heating the solution of formula IV in an organic solvent such as DMSO, DMF, DMA.
[391] According to the invention, the compound of formula IV is initially reacted with an alkenyl alkanoate such as isopropenyl acetate in the presence of an anhydrous inorganic acid such as sulfuric acid or an acid such as toluene sulfonic acid to react the 3-enol ester of the compound of formula IV Forms:
[392] Formula IV (Z)
[393]
[394] Alternatively, 3enol esters can be formed by treatment with bases and acid anhydrides such as acetic acid and sodium acetate. Another alternative is the preparation of formula IV (Z) by treatment with ketene in the presence of an acid. The intermediate of formula IV (Z) is then reacted with alkali metal formate or acetate in the presence of formic acid or acetic acid to yield the Δ-9,11 enol acetate of formula IV (Y):
[395] Formula IV (Y)
[396]
[397] This may then be converted to the ester of formula II in an organic solvent, preferably an alcohol such as methanol, by either pyrolysis of the enol acetate or reaction with an alkali metal alkoxide. The elimination reaction has a higher selectivity for the esters of formula (II) than 11,12-olefins and 7,9-lactones, and this selectivity is maintained through the conversion of enol acetate to enons.
[398] Preferably, the substrate of formula IV corresponds to formula
[399] Formula IVA
[400]
[401] The ester product corresponds to the formula (IIA):
[402] Formula IIA
[403]
[404] In each formula, -AA-, -BB-, Y ¹ , Y ² and X are as defined in formula XIII and R ¹ is as defined in formula V.
[405] If desired, the compound of formula (II) can be separated by removing the solvent, absorbing the solid product in cold water and extracting with an organic solvent such as ethyl acetate. After appropriate washing and drying steps, the product is recovered by removing the extraction solvent. The ester is then dissolved in a solvent suitable for conversion to the product of formula (I). Alternatively, the ester can be separated by adding water to the concentrated product solution and filtering the solid product to preferentially remove 7,9-lactone. The conversion of the substrate of formula (II) to the product of formula (IA) can be carried out by the method described in US Pat. No. 4,559,332, which is incorporated herein by reference, or more preferably by a novel reaction using the haloacetamide promoter described below. .
[406] In another embodiment of the invention, the hydroxyesters of formula V can be converted to the esters of formula II without separating intermediate compounds of formula IV. In this method, the hydroxyester is absorbed into an organic solvent such as methylene chloride; An acylating agent such as methanesulfonyl chloride or a halogenated reagent such as sulfuryl chloride is added to the solution. The mixture is stirred and an HCl scavenger such as imidazole is added when the halogenation is involved. Mixing of the base and the solution is very exothermic and should be carried out at a controlled rate with complete cooling. After addition of the base, the resulting mixture is allowed to warm to a suitable temperature, for example 0 ° C. to room temperature or slightly higher and typically reacted for 1 to 4 hours. After completion of the reaction, the solvent is stripped under high pressure (e.g., 24 " to 28 " Hg) conditions, preferably at -10 [deg.] C. to +15 [deg.] C., more preferably at about 0 [deg.] C. to about 5 [deg.] C. to concentrate the solution and Remove the base. The substrate is then redissolved in an organic solvent, preferably a halogenated solvent, such as methylene chloride, for conversion to the esters.
[407] The leaving group remover is preferably prepared by mixing organic acids, organic acid salts and desiccants, preferably formic acid, alkali metal formate and acetic anhydride, respectively, in a drying reactor. The addition of acetic anhydride is exothermic to release CO, so the rate of addition must be controlled. In order to promote the removal of water, this reaction temperature is preferably maintained in the range of from 60 ° C. to 90 ° C., most preferably from about 65 ° C. to about 75 ° C. This reagent is then added to the product solution of the compound of formula IV to effect the removal reaction. After 4-8 hours, the reaction mixture is preferably heated to a temperature not exceeding at least about 85 ° C. or about 95 ° C. until all volatile distillates are removed and for an additional time, typically about 1-4 hours. Terminate the reaction. The esters can be recovered as desired by cooling the reaction mixture, recovering with standard extraction techniques and then evaporating the solvent.
[408] It was found that after the removal reaction, the ester of formula (II) can be recovered from the reaction solution in another way that does not require an extraction step, thus saving costs and providing an improvement in yield and / or productivity. In this method, the ester product is precipitated by dilution of the reaction mixture with water after removal of formic acid. The product is then separated by filtration. No extraction.
[409] According to another alternative for converting the hydroxyester of formula V to the ester of formula II without separating the compound of formula IV, the 11α-hydroxy group in the hydroxyester of formula V is substituted with halogen, and The ester is formed in situ by thermal dehalogenation. Substitution of the hydroxy group by halogen is effected by reaction with a sulfuryl halide, preferably sulfuryl chloride, upon cooling in the presence of a hydrogen halide scavenger such as imidazole. The hydroxyester is dissolved in a solvent such as tetrahydrofuran and cooled to 0 ° C to -70 ° C. Sulfuryl halide is added and the reaction mixture is allowed to warm to a suitable temperature, eg room temperature, for a time sufficient to terminate the reaction, typically 1 to 4 hours. The method of this embodiment combines the two steps into one, as well as halogenating reaction solvents; Acids (such as acetic acid); And no desiccant (acetic anhydride or sodium sulfate). Moreover, the reaction does not require reflux conditions and suppresses the generation of byproduct CO obtained when acetic acid is used as a desiccant.
[410] According to a particularly preferred embodiment of the invention, the diketone compound of formula (VI) can be converted to another compound of formula (I) or epoxymexrenone without separating any intermediate in purified form. According to this preferred method, the reaction solution containing hydroxyester is quenched with a strong acid solution, cooled to ambient temperature and then extracted with a suitable extraction solvent. Advantageously, an aqueous solution of an inorganic salt, such as a 10 wt% saline solution, is added to the reaction mixture before extraction. The extract is washed and dried by azeotropic distillation to remove residual methanol solvent from ketone cleavage.
[411] The resulting concentrated solution containing from about 5% to about 50% by weight of the compound of formula V is then cold contacted with an acylating agent or alkylsulfonylating agent to form a sulfonic acid ester or a dicarboxylic acid ester. After completion of the alkylsulfonation or carboxylation reaction, the reaction solution is passed through an acidic and basic exchange resin column to remove basic and acidic impurities. After each passage the column is washed with a suitable solvent, for example methylene chloride, to recover residual sulfonic acid or dicarboxylic acid ester therefrom. The combined eluate and wash fractions are combined and preferably reduced in vacuo to produce a concentrated solution containing the sulfonic acid ester or dicarboxylic acid ester of formula IV. This concentrated solution is then contacted with a desiccant consisting of a formulation effective for the removal of the 11α-ester leaving group and dehydrogenation to form 9,11 double bonds. Preferably, the reagent for removing the leaving group consists of the formic acid / alkali metal formate / acetic anhydride desiccant solution described above. After completion of the reaction, the reaction mixture is cooled and formic acid and / or other volatile components are removed in vacuo. The residue is cooled to ambient temperature, subjected to a suitable washing step and then dried to give a concentrated solution containing the ester of formula (II). This ester can then be converted to epoxymexrenone or another compound of formula (I) using the method described in the text or the method described in US Pat. No. 4,559,332.
[412] In a particularly preferred embodiment of the invention, the solvent is removed from the reaction solution under vacuum and the product of formula IV is partitioned between water and a suitable organic solvent, for example ethyl acetate. The aqueous layer is then extracted again with an organic solvent and the reextract is washed with an alkaline solution, preferably a solution of alkali metal hydroxides containing alkali metal halides. The organic phase is preferably concentrated in vacuo to give the ester product of formula II. The product of formula (II) is then absorbed into an organic solvent such as methylene chloride and further reacted by the method described in the '332 patent to produce the product of formula (I).
[413] When trihaloacetonitrile is used in the epoxidation reaction, it has been found that the choice of solvent is important because halogenated solvents are very preferred and methylene chloride is particularly preferred. A fairly satisfactory yield is obtained with solvents such as dichloroethane and chlorobenzene, but is usually better in methylene chloride reaction medium. Poor yields are usually obtained with solvents such as acetonitrile and ethyl acetate, while the reaction in solvents such as methanol or water / tetrahydrofuran results in little desired product.
[414] It has also been found that according to the present invention numerous improvements in the synthesis of epoxymexrenone can be realized using trihaloacetamide rather than trihaloacetonitrile as the peroxide activator for the epoxidation reaction. In a particularly preferred method, epoxidation is carried out by reacting hydrogen peroxide with a substrate of formula IIA in the presence of trichloroacetamide and a suitable buffer. Preferably, the reaction is conducted at a pH in the range of about 3 to about 7, most preferably about 5 to about 7. However, despite the subject of these considerations, successful reactions have been made outside the desired pH range.
[415] Particularly preferred results are a buffer consisting of a combination of potassium dihydrogen phosphate and dipotassium hydrogen phosphate and / or a buffer consisting of dipotassium hydrogen phosphate in a relative ratio ranging from about 1: 4 to about 2: 1, most preferably about 2: 3. Obtained. Borate buffers may also be used, but usually achieve lower conversions than dipotassium phosphate or K ₂ HPO ₄ or K ₂ HPO ₄ / KH ₂ PO ₄ mixtures. Whatever the configuration of the buffer, pH should be provided in the above ranges. Apart from the overall composition or exact pH of the buffer that may be provided, it has been observed that the reaction will proceed more effectively if at least some of the buffer consists of dihydrogen phosphate ions. It is believed that these ions can essentially precipitate as a homogeneous catalyst in the formation of complexes or adducts consisting of promoters and hydrogen peroxide ions, which in turn can be essential for the entire epoxidation reaction mechanism. Thus, the quantitative requirements for dihydrogen phosphate (preferably K ₂ HPO ₄ ) may be only low catalyst concentrations. In general, it is preferred that HPO ₄ is provided in a proportion of at least about 0.1 equivalents, such as from about 0.1 to about 0.3 equivalents per substrate equivalent.
[416] The reaction is carried out in a suitable solvent, preferably methylene chloride, but alternatively other halogenated solvents such as chlorobenzene or dichloroethane can also be used. Toluene and mixtures of toluene and acetonitrile were also found sufficient. Without conducting a specific theory, it is assumed that the reaction can proceed most effectively in a two-phase system that forms an intermediate hydrogen peroxide, is distributed in a low water content organic phase and reacts with a substrate in the organic phase. The preferred solvent is therefore low in water solubility. Effective recovery from toluene is facilitated by including other solvents such as acetonitrile.
[417] In converting the substrate of formula (II) to the product of formula (I), toluene offers the benefits of the process because the substrate is freely soluble in toluene and there is no product. Thus, a three phase mixture is prepared in which the product precipitates during the reaction when the conversion reaches 40-50% and the product can be easily separated by filtration. Methanol, ethyl acetate, acetonitrile alone, THF and THF / water did not prove to be as effective as the halogenated solvent or toluene in carrying out this stage of conversion in the process.
[418] Trichloroacetamide, on the other hand, is a very preferred reagent, and other trihaloacetamides such as trifluoroacetamide can also be used. Trihalomethylbenzamides and other compounds having an arylene moiety between the electron leaving trihalomethyl group and the carbonyl of the amide may also be useful. 3,3,3-trihalopropionamide may also be used, but the result is undesirable. In general, peroxide activators can correspond to the following formula:
[419] R ⁰ C (O) NH ₂
[420] Wherein R ⁰ is a group having an electron escape intensity (measured in sigma terms) at least as high as the monochloromethyl group. More specifically, the peroxide activator may correspond to the formula:
[421]
[422] Wherein X ¹ , X ² and X ³ are independently selected from halo, hydrogen, alkyl, haloalkyl and cyano and cyanoalkyl, and R ^P is selected from arylene and-(CX ⁴ X ⁵ ) n- And n is 0 or 1 and at least one of X ¹ , X ² , X ³ , X ⁴ and X ⁵ is halo or perhaloalkyl. When any of X ¹ , X ² , X ³ , X ⁴ or X ⁵ is not halo, it is preferably haloalkyl, most preferably perhaloalkyl. Particularly preferred activators include n is 0 and at least two of X ¹ , X ² and X ³ are halo; Or all X ¹ , X ² , X ³ , X ⁴ and X ⁵ are halo or perhaloalkyl. Each of X ¹ , X ² , X ³ , X ⁴ and X ⁵ may be suitable mixed halides which may be perchloralkyl or perbromoalkyl and combinations thereof, but is preferably Cl or F, most preferably Cl .
[423] Preferably, the peroxide activator is provided at a ratio of at least about 1 equivalent, more preferably about 1.5 to about 2 equivalents, per equivalent of substrate initially provided. Hydrogen peroxide should be added to the reaction at least in an appropriate excess or added continuously as the epoxidation reaction proceeds. Although the reaction consumes only 1 to 2 equivalents of hydrogen peroxide per mole of substrate, the hydrogen peroxide is preferably in a substantial excess relative to the substrate and the activator is initially provided. Without defining the present invention in any particular theory, the reaction mechanism involves the formation of an activator and an adduct of OOH ⁻ , an equilibrium reversible and advantageously substantial initial excess to react in the forward direction, favoring reverse reactions with the formation of this reactant. It is thought that hydrogen peroxide is required. The reaction temperature is not very limited and can be effectively carried out in the range of 0 ° C to 100 ° C. The optimum temperature depends on the solvent selection. Generally, the preferred temperature is about 20 ° C. to 30 ° C., but in certain solvents such as toluene the reaction is advantageously carried out in the range of 60 ° C.-70 ° C. At 25 ° C., the reaction typically requires less than 10 hours, typically 3 to 6 hours. If necessary, an activator and hydrogen peroxide can be added at the end of the reaction cycle to completely convert the substrate.
[424] At the end of the reaction cycle, the aqueous phase may be removed and preferably the organic reaction solution may be washed to remove the water soluble impurities and then the solvent may be removed to recover the product. Before removing the solvent, the reaction solution should be washed at least gently with a suitable alkaline washing solution, for example sodium carbonate. Preferably, the reaction mixture is a mild reducing solution such as a weak (eg 3% by weight) solution of sodium sulfite in water; Alkaline solutions such as NaOH or KOH (preferably about 0.5N); Acid solutions such as HCl (preferably about 1N); And a final neutral wash with water or brine, preferably saturated brine to minimize product loss. Prior to removing the reaction solvent, the product can be recovered by distillation and crystallization to remove an organic solvent, preferably another solvent such as ethanol, to remove the more volatile reaction solvent.
[425] The novel epoxidation process using trichloroacetamide or other new peroxide activators is in fact to produce epoxymexrenones which can be used to form epoxides through olefinic double bonds in a wide range of substrates reacted in the liquid phase. Understand that you are applying several schemes. The reaction is particularly effective in unsaturated compounds, where olefinic carbons are tetra- and trisubstituted, i.e. R ^a R ^b C = CR ^c R ^d and R ^a R ^b C = CR ^c RH (R ^a to R ^d are other than hydrogen Substituents). The reaction proceeds most rapidly and completely and the substrate in the formula is either a trisubstituted double ring compound, or a cyclic or acyclic compound with tetrasubstituted double bonds. Substrates for this reaction include Δ-9,11-canrenone, and
[426]
[427]
[428] Can be mentioned.
[429] Because of the faster and more complete reaction with tri- and tetra-substituted double bonds, it is particularly effective for selective epoxidation through such double bonds among compounds which may contain other double bonds in which the olefinic carbon is mono- or di-substituted.
[430] It is understood that this reaction can advantageously be used for epoxidation of mono- or di-substituted double bonds, such as 11,12-olefins in various steroid substrates. However, because of the preferential epoxidation of polysubstituted double bonds such as 9,11-olefins with high selectivity, the process of the present invention is particularly effective in achieving high yields and productivity in the epoxidation step of the various schemes described herein. Do.
[431] How to improve
[432]
[433] By epoxidation of formulas (IB) and (IC)
[434] Formula IB
[435]
[436] (Formula IC)
[437]
[438] It has been found that it is particularly advantageous to apply to the preparation.
[439] Trichloroacetamide instead of trichloroacetonitrile as the oxygen carrier in the epoxidation reaction has demonstrated several advantages over the process of the present invention. The trichloroacetamide reagent system provides strict regioregulation for epoxidation through the α, β-keto olefin and disubstituted trisubstituted double bonds in the same molecular structure. Thus, reaction yield, product profile and final purity increase substantially. It was further found that the substantial excess of oxygen evolution observed with the use of trihaloacetonitrile does not occur with trichloroacetamide, which gives improved stability to the epoxidation process. In addition, the trichloroacetamide reaction, compared to the trichloroacetonitrile promoted reaction, exhibits a minimal exothermic effect, thereby facilitating control of the reaction heat profile. The stirring effect is minimal and the reactor performance is more consistent and is observed with other advantages over the trichloroacetonitrile process. The reaction can be increased further than the trichloroacetonitrile promoted reaction. Product separation and purification is simple and as done, e.g. m-chloroperoxybenzoic acid or other peracids and the reagents are inexpensive, readily available and easily processed Bayer-Villager (Bayer) -Villager) No oxidation was observed.
[440] The novel epoxidation process of the present invention is very useful as the final step in the synthesis of Scheme 1. In a particularly preferred embodiment, the overall process of Scheme 1 proceeds as follows:
[441]
[442] Scheme 2
[443] The second novel scheme of the present invention (Scheme 2) discloses canrenones or other substrates corresponding to formula XIII:
[444] Formula XIII
[445]
[446] -AA-, R ³ , -BB-, R ⁸ and R ⁹ in the above formula are as defined in formula VIII. In the first step of this process, the substrate of formula (XIII) is converted to the product of formula (XII) using a cyanation scheme substantially the same as described above for the conversion of the substrate of formula (VIII) to the intermediate of formula (VII).
[447] Formula XII
[448]
[449] Preferably, the substrate of formula XIII corresponds to formula XIIIA
[450] Formula XIIIA
[451]
[452] Enamine products correspond to formula XIIA:
[453] Formula XIIA
[454]
[455] -AA-, -BB-, Y ¹ , Y ² and X in each formula are as defined in formula (XIII).
[456] In the second step of Scheme 2, the enamine of Formula XII is converted to Formula XI using a cyanation scheme that is substantially the same as described above for the conversion of the substrate of Formula VIII to the intermediate of Formula VII.
[457] Formula XI
[458]
[459] Hydrolyze to the intermediate diketone product of formula (wherein -AA-, R ³ , -BB-, R ⁸ and R ⁹ are as defined in formula VIII). Preferably the substrate of formula (XII) corresponds to formula (XIIA)
[460] Formula XIIA
[461]
[462] The diketone product corresponds to the formula XIA:
[463] Formula XIA
[464]
[465] In each formula, -AA-, -BB-, Y ¹ , Y ² and X are as defined in formula VIIIA.
[466] Also according to Scheme 2, the diketone of formula XI is reacted with an alkali metal alkoxide to form mexrenone or another product corresponding to formula X:
[467] Formula X
[468]
[469] In each formula, -AA-, R ³ , -BB-, R ⁸ and R ⁹ are as defined in formula VIII. R ¹ is as defined in formula (V). The method is carried out using the reaction scheme substantially the same as described above for the conversion of the compound of formula VI to that of formula V. Preferably, the substrate of formula XI corresponds to formula XIA
[470] Formula XIA
[471]
[472] The intermediate product corresponds to formula XA:
[473] Formula XA
[474]
[475] -AA-, -BB-, Y ¹ , Y ² and X in each formula are as defined in formula (XIII). R ¹ is as defined in formula (V).
[476] Canrenone or another compound of formula X is subjected to the following 9α-hydroxylation by a novel biotransformation process to yield the product of formula IX:
[477] Formula IX
[478]
[479] -A-A-, R in the above formula³, -B-B-, R⁸And R⁹Is as defined in formula VIII, R is^OneIs as defined in formula (V). The organisms that can be used in this hydroxylation step areNocardia conicruriaATCC 31548,Nocardia aurentiaATCC 12674,Corynespora cassiicolaATCC 16718,Streptomyces hydroscopicusATCC 27438,Mortierella isabellinaATCC 42613,Beauvria bassianaATCC 7519,Penicillumm purpurogenumATCC 46581,Hypomyces chrysospermusIMI 109891,Thamnostylum piriformeATCC 8992,Cunnignhamella blakesleeanaATCC 8688a,Cunningnhamella echinulataATCC 3655,Cunninghamella elegansATCC 9245,Trichothecium roseumATCC 12543,Epicoccum humicolaATCC 12722,Saccharopolyspora eythraeATCC 11635,Beauvria bassianaATCC 13144,Arthrobacter simplex,Bacterium cyclooxydansATCC 12673,Cylindrocarpon radicicolaATCC 11011,Nocardia aurentiaATCC 12674,Nocardia canicruria,Norcardia restrictusATCC 14887,Pseudomonas testosteroniATCC 11996,Rhodococcus equiATCC 21329,Mycobacterium fortuitumATCC-6842, andRhodococcus rhodochrousATCC 19150. The reaction is carried out substantially in the manner described above with respect to FIGS. 1 and 2. The method of FIG. 1 is particularly preferred.
[480] Growth media useful for biotransformation are preferably from about 0.05% to 5% effective nitrogen; About 0.5% to about 5% glucose; About 0.25% to about 2.5% yeast derivative; And about 0.05% by weight and about 0.5% by weight effective phosphorus. Particularly preferred growth media include:
[481] Soya flour: about 0.5% to about 3% glucose; About 0.1% to about 1% soybean flour; About 0.05% to about 0.5% by weight of the alkali metal halide; Yeast derivatives, such as from about 0.05% to about 560.5% by weight of autolyzed yeast or yeast extract; Phosphate, such as from about 0.05% to about 0.5% by weight K ₂ HPO ₄ ; pH = 7; Peptone-yeast extract-glucose: about 0.2% to about 2% peptone; About 0.05% to about 0.5% yeast extract; And from about 2% to about 5% glucose;
[482] Mueller-Hinton: from about 10% to about 40% by weight of beef culture; From about 0.35 wt% to about 8.75 wt% cassamino acid; From about 0.15% to about 0.7% starch.
[483] Fungi can be grown on soybean wheat or peptone nutrients, while radiation fungi and milk bacteria are soybean wheat (0.5% to 1% carboxylic acid such as Na formate for plus biotransformation) or Muller-Hinton culture Can grow in
[484] The preparation of ll [beta] -hydroxymexrenone from mexrenone by fermentation is discussed in Example 19.
[485] The product of formula (IX) is a novel compound which can be separated by filtration, washed with a suitable organic solvent such as ethyl acetate and recrystallized in the same or similar solvent. This has substantial value as an intermediate for the preparation of compounds of formula I, in particular formula IA. Preferably, the compound of formula IX corresponds to formula IX, wherein -AA- and -BB- are -CH ₂ -CH _2- , R ³ is hydrogen, lower alkyl or lower alkoxy, R ⁸ and R ⁹ together constitute a 20-spiroxane ring of formula XXXIII:
[486] (Formula XXXIII)
[487]
[488] In the next step of the synthesis scheme 2, the product of formula IX is reacted with a dehydrating agent to produce a compound of formula II
[489] Formula II
[490]
[491] In the above formula, -AA-, R ³ , -BB-, R ⁸ and R ⁹ are as defined in formula VIII, and R ¹ is as defined in formula V. When the substrate corresponds to formula IXA, the product is formula IIA
[492] Formula IXA
[493]
[494] Formula IIA
[495]
[496] In each formula, -AA-, -BB-, Y ¹ , Y ² and X are as defined in formula XIII and R ¹ is as defined in formula V.
[497] In the final step of this synthesis scheme, the product of formula (II) is converted into a compound of formula (I) by epoxidation according to the process described in US Pat. No. 4,559,332 or preferably by the novel epoxidation process of the invention described above. Let's do it.
[498] In a particularly preferred embodiment, the overall process of Scheme 2 proceeds as follows:
[499]
[500] Scheme 3
[501] Synthesis in this case starts with a substrate corresponding to formula (XX):
[502] Formula XX
[503]
[504] -AA- and R ³ in the above formula are as defined in formula (VIII), -BB- is defined in formula (VIII) except that both R ⁶ and R ⁷ are not ring moieties condensed into D ring at position 16,17 As is, R ²⁶ is lower allyl, preferably methyl. Reaction of the substrate of Formula (XX) with sulfoniumlides produces an epoxide intermediate corresponding to Formula (XIX):
[505] Formula XIX
[506]
[507] In the above formula, -AA-, R ³ , -BB- and R ²⁶ are as defined in formula (XX).
[508] In the next step of Synthesis Scheme 3, the intermediate of Formula (XIX) is further converted to the intermediate of Formula (XVIII):
[509] Formula XVIII
[510]
[511] In the formula, -AA-, R ³ and -BB- are as defined in formula (XX). In this step, the formula XIX substrate is converted to the formula XVIII intermediate by reaction with NaCH (COOEt) _{2 in} the presence of a base in a solvent. The compound of formula (XVIII) is exposed to hot water and an alkali halide to produce a decarboxylated intermediate compound corresponding to formula (XVII):
[512] Formula XVII
[513]
[514] In the formula, -AA-, R ³ and -BB- are as defined in formula (XX). The process of converting a compound of Formula (XX) to a compound of Formula (XVII) essentially corresponds to the methods described in US Pat. Nos. 3,897,417, 3,413,288 and 3,300,489, which are incorporated herein by reference. When the substrates are different, the reagents, mechanisms and conditions for the introduction of the 17-spirolactone moiety are essentially the same.
[515] Reaction of the intermediate of Formula XVII with a dehydrogenation reagent further provides an intermediate of Formula XVI:
[516] Formula XVI
[517]
[518] In the formula, -AA-, R ³ and -BB- are as described above.
[519] Typically useful dehydrogenation reagents include dichlorodicyanobenzoquinone (DDQ) and chloranyl (2,3,5,6-tetrachloro-p-benzoquinone). Alternatively, dehydrogenation could be achieved by halogenation followed by dehalogenation reactions in a series of carbon-6.
[520] The intermediate of formula XVI is converted to the enamine of formula XV:
[521] Formula XV
[522]
[523] In the formula, -AA-, R ³ and -BB- are as defined in formula (XX). The conversion is essentially cyanation in the manner described above for converting the 11α-hydroxy compound of formula VIII to the enamine of formula VII. Typically, the cyanide ion source may be an alkali metal cyanide. The base is preferably pyrrolidine and / or tetramethylguanidine. Methanol solvents can be used.
[524] The product of formula XV is a novel compound, which can be separated by chromatography. These and other novel compounds of formula AXV have substantial value as intermediates for the preparation of compounds of formula I, in particular formula IA. Compounds of formula AXV correspond to the following structures:
[525] Formula AXV
[526]
[527] In the formula, -AA-, R ³ and -BB- are as defined in formula (XX). In the most preferred compounds of formula XV, -AA- and -BB- are -CH ₂ -CH ₂ .
[528] According to the above hydrolysis to prepare the diketone compound of formula VI, the enamine of formula XV can be converted to the diketone of formula XIV:
[529] Formula XIV
[530]
[531] In the formula, -AA-, R ³ and -BB- are as defined in formula (XX). Particularly preferred for the synthesis of epoxymexrenone are the compounds of formula XIV which are within the scope of formula VIA.
[532] The product of formula XIV is a novel compound, which can be separated by precipitation. These and other novel compounds of formula AXIV have substantial value as intermediates for the preparation of compounds of formula I, in particular formula IA. Compounds of formula AXIV correspond to the following structures:
[533] Formula AXIV
[534]
[535] In the formula, -AA-, R ³ , R ⁸ and R ⁹ are as described above. In the most preferred compounds of formula AXIV and formula XIV, -AA- and -BB- are -CH ₂ -CH ₂ .
[536] Compounds of formula (XIV) are converted to compounds of formula (XXXI) using essentially the methods described above for the conversion of diketones of formula (VI) to hydroxyesters of formula (V). In this case, the formula XXXI
[537] (Formula XXXI)
[538]
[539] The intermediate of Formula (XXXII)
[540] (Formula XXXII)
[541]
[542] It is necessary to further convert to the product of formula (wherein -A-A- and -B-B- are as defined in formula (XX)). Preferred compounds of formula (XXXI) are those in formula (IA). Compounds of formula (XXXI) are converted to compounds of formula (XXXII) using the methods described or described above in US Pat. No. 4,559,332. In a particularly preferred embodiment, the overall process of Scheme 3 proceeds as follows:
[543]
[544] Scheme 4
[545] The first three steps of Scheme 4 are identical to that of Scheme 3, ie starting with a compound corresponding to Formula XX, to prepare an intermediate of Formula XVII.
[546] Next, the intermediate of formula XVII is epoxidized using the method of US Pat. No. 4,559,332 to produce the compound of formula XXIV:
[547] Formula XXIV
[548]
[549] -AA-, R ³ and -BB- in the above formula are as defined in formula (XX). However, in a particularly preferred embodiment of the invention, the substrate of formula (XVII) is converted to an amide type peroxide activator, most preferably Triclo, according to the method described above in Scheme 1 for converting the ester of formula (II) to the product of formula (I). It is epoxidized via a 9,11-double bond using an oxidizing agent consisting of loacetamide.
[550] The epoxidation of the substrate of formula (XVII) can also be carried out in very good yields using peracids such as m-chloroperoxybenzoic acid. However, trichloroacetamide reagents provide excellent results by minimizing Bayer-Villager oxide formation as a byproduct. The latter byproduct can be removed, but it must be triturated from a solvent such as ethyl acetate and then crystallized from another solvent such as methylene chloride. The epoxy compound of formula XXIV is dehydrogenated by reaction with a dehydrogenation (oxidation) agent such as DDQ or chloranyl, or a double bond between 6- and 7-carbons in the order of bromination / dehydrobromide (or other halogenated / dehalogenated) To form another novel intermediate of formula (XXIII):
[551] Formula XXIII
[552]
[553] In the above formula, -A-A- and -B-B- are as defined in formula (XX). Particularly preferred compounds of formula (XXIII) are those wherein -A-A- and -B-B- are as defined in formula (XIII).
[554] Direct oxidation is effective for product formation of Formula (XXIII), but yields are generally low. Thus, preferably, the oxidation is carried out in two steps, first by halogenating the substrate of formula (XXIV) at the C-6 position and then by dehalogenating the 6,7-olefin. Halogenation is preferably carried out with an N-halo organic reagent such as N-bromosuccinamide. Bromination is carried out in a suitable solvent such as acetonitrile in the presence of a halogenated promoter such as benzoylperoxide. The reaction proceeds effectively at temperatures ranging from about 50 ° C. to 100 ° C., easily at ambient reflux in solvents such as carbon tetrachloride, acetonitrile or mixtures thereof. However, 4 to 10 hours of reaction are typically required to complete the reaction. The reaction solvent is stripped off and the residue is taken up in a water immiscible solvent such as ethyl acetate. The resulting solution is washed successively with a mild alkaline solution (e.g. alkali metal bicarbonate) and water, or preferably saturated brine, to minimize product loss, after which the solvent is stripped and other residues suitable for dehydrohalogenation reaction ( Eg dimethylformamide).
[555] Suitable dehydrohalogenation reagents, such as 1,4-diazabicyclo [2,2,2] octane (DABCO), are added to the solution together with alkali metal halides such as LiBr and the solution is added at a suitable reaction temperature, for example 60 The reaction was heated to < RTI ID = 0.0 > C < / RTI > Additional dehydrobromination broth may be added as needed during the reaction cycle to complete the reaction. The product of formula XXIII is recovered by, for example, adding water to precipitate the product and then separating by filtration and preferably washing with an additional amount of water. The product is preferably recrystallized from dimethylformamide.
[556] Products of formula (XXIII), such as 9,11-epoxycanrenone, are novel compounds and can be separated by extraction / crystallization. This has substantial value as an intermediate for the preparation of compounds of formula I, in particular formula IA. For example, it can be used as a substrate for preparing a compound of formula XXII. In the most preferred compounds of formula (XXIII) and -AA- and -BB- are -CH ₂ -CH ₂ .
[557] Subsequently, the process described above for the preparation of the compound of formula VII is used to react the compound of formula XXIII with cyanide ions to produce a novel epoxyenamine compound corresponding to the corresponding formula XXII:
[558] Formula XXII
[559]
[560] In the formula, -AA-, R ³ and -BB- are as defined in formula (XX). Particularly preferred compounds of formula (XXII) are those wherein -AA- and -BB- are as defined in formula (XIII).
[561] The product of formula (XXII) is a novel compound and can be separated by precipitation and filtration. This has substantial value as an intermediate for the preparation of compounds of formula I, in particular formula IA. Most preferred compounds of formula (XXII) and -AA- and -BB- are -CH ₂ -CH ₂ .
[562] The epoxyeneamine compound of formula (XXII) is converted to the novel epoxydiketone compound of formula (XXI) using substantially the above described method for preparing the compound of formula (VI).
[563] The product of formula XXI is a novel compound and can be separated by precipitation and filtration. This has substantial value as an intermediate for the preparation of compounds of formula I, in particular formula IA. Particularly preferred compounds of formula (XXI) are those in which -AA- and -BB- are as defined in formula (XIII). Most preferred compounds of formula XXI and -AA- and -BB- are -CH ₂ -CH ₂ .
[564] Compounds of formula XXI are converted to compounds of formula XXXII using the method of US Pat. No. 4,559,332 or the epoxidation method described above. In a particularly preferred embodiment, the overall process of Scheme 4 proceeds as follows:
[565]
[566] Scheme 5
[567] The process of Scheme 5 starts with a substrate corresponding to Formula XXIX:
[568] Formula XXIX
[569]
[570] -A-A- and -B-B- in the formula are as defined in formula (XX). This substrate is reacted with trimethylorthoformate to convert to the product of formula XXVIII:
[571] Formula XXVIII
[572]
[573] -AA-, R ³ and -BB- in the above formula are as defined in formula (XX). After formation of Formula (XXVIII), the compound of Formula (XXIX) is converted to the compound of Formula (XXVII) using the method described above for the conversion of the substrate of Formula (XX) to Formula (XVII). The compound of formula XXVII has the structure
[574] Formula XXVII
[575]
[576] -AA- and -BB- in the above formula are as defined in formula (XX), and R ^X is any of the usual hydroxyl protecting groups.
[577] Using the method described above for the preparation of compounds of formula XVI, the compounds of formula XXVII are oxidized to yield novel compounds corresponding to formula XXVI:
[578] Formula XXVI
[579]
[580] -A-A- and -B-B- in the formula are as defined in formula (XX). Particularly preferred compounds of formula (XXIX), formula (XXVIII), formula (XXVII) and formula (XXVI) are those wherein -A-A- and -B-B- are as defined in formula (XIII).
[581] The product of formula (XXVI) is a novel compound and can be separated by precipitation / filtration. It has substantial value as an intermediate for the preparation of compounds of formula (I), in particular formula (IA). Particularly preferred compounds of formula (XXVI) are those in which -AA- and -BB- are as defined in formula (XIII). Most preferred compounds of formula (XXVI) and -AA- and -BB- are -CH ₂ -CH ₂ .
[582] The novel intermediate of formula (XXVI) is converted to the novel 9-hydroxyenamine intermediate of formula (XXV) using the method described above for cyanating the compound of formula (VIII):
[583] Formula XXV
[584]
[585] In the formula, -AA-, R ³ and -BB- are as defined in formula (XX).
[586] The product of formula XXV is a novel compound and can be separated by precipitation / filtration. This has substantial value as an intermediate for the preparation of compounds of formula I, in particular formula IA. Particularly preferred compounds of formula (XXV) are those in which -AA- and -BB- are as defined in formula (XIII). Most preferred compounds of formula (XXVI) and -AA- and -BB- are -CH ₂ -CH ₂ .
[587] The 9-hydroxyenamine intermediate of Formula XXV is converted to the diketone compound of Formula XIV using essentially the conditions described above for the preparation of the diketone compound of Formula VI. Note that in this case the reaction simultaneously hydrolyzes the enamine structure and introduces 9,11 double bonds by dehydration at the 9,11 position. The compound of formula (XIV) is converted into a compound of formula (XXXI) and then to the compound of formula (XIII) using the same steps described above in Scheme 3.
[588] In a particularly preferred embodiment, the overall process of Scheme 5 proceeds as follows:
[589]
[590] Scheme 6
[591] Scheme 6 is chemical formula XXXV
[592] (Formula XXXV)
[593]
[594] Formula XXXVI starting with 11α-hydroxylation of androstenedione or another compound of formula (-AA-, R ³ and -BB- is as defined in Formula XIII)
[595] (Formula XXXVI)
[596]
[597] Provided is an advantageous method for the preparation of epoxymexrenone and other compounds corresponding to formula (I) for preparing intermediates corresponding to (-AA-, R ³ and -BB- as defined in formula (XIII)). In addition to the selectivity of the substrate, the method for effecting 11α-hydroxylation is essentially as described above in Scheme 1. The following microorganisms can be subjected to 11α-hydroxylation of androstenedione or another compound of formula XXXV:
[598] Aspergillus ochraceusNRRL 405 (ATCC 18500);
[599] Aspergillus niger ATCC 11394;
[600] Aspergillus nidulansATCC 11267;
[601] Rhizopus oryzaeATCC 11145;
[602] Rhizopus stoloniferATCC 6227b;
[603] Trichothecium roseumATCC 12519 and ATCC 8685.
[604] 11α-hydroxyandrost-4-ene-3,17-dione, or another compound of formula XXXVI, is reacted with an etherifying agent such as trialkylorthoformate in the presence of an acid catalyst to form 11α-hydroxy-3 of formula 101 Convert to, 4-enol ether:
[605] Formula 101
[606]
[607] -AA-, R ³ and -BB- in the above formula are as defined in formula XIII and R ¹¹ is methyl or other lower alkyl (C ₁ to C ₄ ). To effect this conversion, the 11α-hydroxy substrate is acidified by mixing with an acid such as, for example, benzenesulfonic acid hydrate or toluenesulfonic acid hydrate, and dissolved in a lower alcohol solvent, preferably ethanol. Trialkylorthoformates, preferably triethylorthoformates, are introduced gradually over 5 to 40 minutes while maintaining the cooling mixture preferably at about 0 ° C to about 15 ° C. The mixture is warmed and the reaction is carried out at a temperature of 20 ° C to about 60 ° C. Preferably the reaction is carried out at 30 ° C. to 50 ° C. for 1 to 3 hours and then heated to reflux for an additional time, typically 2 to 6 hours to complete the reaction. The reaction mixture is preferably cooled to 0 ° C. to 15 ° C., preferably about 5 ° C. and the solvent is removed in vacuo.
[608] The 17-spirolactone moiety of Formula (XXXIII) is introduced into the compound of Formula (101) using the same scheme as described in Scheme 3 above to convert the compound of Formula (XX) to the compound of Formula (XVII). For example, a substrate of Formula 101 may be reacted with sulfoniumide in the presence of a base such as an alkali metal hydroxide in a suitable solvent such as DMSO to produce an intermediate corresponding to Formula 102:
[609]
[610] In the above formula, -AA-, R ³ , R ¹¹ and -BB- are as defined in Formula 101. The intermediate of Formula 102 is then reacted with the malonic acid diester in the presence of an alkali metal alkoxide to form a 5-membered spirolactone ring, to prepare the intermediate of Formula 103:
[611]
[612] In the above formula, -AA-, R ³ , R ¹¹ , R ¹² and -BB- are as defined in formula (XIII). Finally, the compound of formula 103 in a suitable solvent such as dimethylformamide is heated in the presence of an alkali metal halide, the alkoxycarbonyl moiety is separated and an intermediate of formula 104 is prepared:
[613] Formula 104
[614]
[615] In the above formula, -AA-, R ³ , R ¹¹ and -BB- are as defined in formula (XIII).
[616] The 3,4-enol ether compound 104 is then converted into a compound of the formula XXIII, ie R ⁸ and R ⁹ together with a compound of the formula VIII, which together form part of the formula XXXIII. This oxidation step is carried out in essentially the same manner as the oxidation step for converting the compound of formula (XXIV) to the intermediate of formula (XXIII) in Synthesis Scheme 4. Direct oxidation can be carried out using a reagent such as 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or tetrachlorobenzoquinone (chloranyl), or preferably both The step oxidation is first brominated with an N-halo bromination agent such as N-bromosuccinamide or 1,3-dibromo-5,5-dimethyl hydantoin (DBDMH) followed by a base such as in the presence of LiBr and heat. This is done by dehydrobromination with DABCO. When NBS is used for bromination, it can be used to convert acidity 3-enol ethers to enons. Ionic DBDMHs other than free radical bromination reagents are effective for the conversion of bromination itself and enol ethers to enons.
[617] The compound of formula VIII is then converted to epoxymexrenone or another compound of formula I by the steps described above for Scheme 1.
[618] Each of the intermediates of Formula (101), (102), (103) and (104) is a novel compound of substantial value as an intermediate for epoxymexrenone or other compounds of formula (IA) and formula (I). In each of the compounds of Formula 101, 102, 103 and 104, -AA- and -BB- are preferably -CH ₂ -CH ₂ -and R ³ is hydrogen, lower alkyl or lower alkoxy. Most preferably, the compound of formula 101 is 3-ethoxy-11α-hydroxyandrost-3,5-diene-17-one, and the compound of formula 102 is 3-ethoxyspiro [androst-3,5 -Diene-17β, 2'-oxirane] -11α-ol, the compound of formula 103 is ethyl hydrogen 3-ethoxy-11α-17α-dihydroxypregna-3,5-diene-21,21-dica Carboxylate, γ-lactone, and the compound of formula 104 is 3-ethoxy-11α-17α-dihydroxypregna-3,5-diene-21-carboxylic acid, γ-lactone.
[619] In a particularly preferred embodiment, the overall process of Scheme 6 proceeds as follows:
[620]
[621] Scheme 7
[622] Epoxymexrenone and other compounds of formula I are synthesized using a starting substrate consisting of β-sitosterol, cholesterol, stigmasterol or other compounds of formula XXXVII in Scheme 7:
[623] (Formula XXXVII)
[624]
[625] In the above formula, -AA-, R ³ and -BB- are as defined in formula (XIII), DD is -CH ₂ -CH ₂ -or -CH = CH-, R ¹³ , R ¹⁴ , R ¹⁵ and R Each of ¹⁶ is independently selected from hydrogen C ₁ to C ₄ alkyl.
[626] In the first step of the synthesis, 11α-hydroxyandrostenedione or another compound of formula XXXV is prepared by biotransformation of a compound of formula XXXVII. The biotransformation process is carried out substantially according to the method described above for 11α-hydroxylation of canrenone (or other substrate of formula XIII).
[627] In the 11α-hydroxyandrostenedione synthesis, 4-androsten-3,17-dione is initially prepared by biotransformation of the compound of formula XXXVII. This initial conversion can be effected by the method described in US Pat. No. 3,759,791, which is incorporated herein by reference. 4-Androsten-3,17-dione is then substantially converted to 11α-hydroxyandrostenedione according to the method described above for 11α-hydroxylation of canrenone (or other substrate of Formula XIII).
[628] The remainder of the synthesis scheme 7 is the same as in scheme 6. In a particularly preferred embodiment, the overall process of Scheme 7 proceeds as follows:
[629]
[630] The methods, processes and compositions of the present invention and the conditions and reagents used in the text are further described in the following examples.
[631] (Example)
[632] Example One
[633] Slant was prepared in the growth medium as shown in Table 1.
[634] Y P D A (medium for slants and plates) Yeast extract20 g peptone20 g Glucose20 g Agar20 g Sufficient distilled water-pH 6.7-H ₃ PO ₄ Adjusted to pH 5 with 10% w / v1000 ml Dispense-slant: 7.5 ml-plate (180 cm in Φ) to 180 x 18 mm tubes Sterilization-sterilization at 120 ° C. for 25 ml-20 minutes in 200 x 20 mm tubes pH: 5
[635] To produce a first developmental culture,Aspergillus ochraceusA colony of is suspended in distilled water (2 ml) in vitro; 0.15 ml aliquots of this suspension were applied to each slant prepared as described above. After incubating the slant at 25 ° C. for 7 days, the appearance of the surface culture was white cotton mycelia. The lower part of the reverse was colored orange and the upper part was colored orange.
[636] The first generation slant culture was suspended in a sterile solution (4 ml) containing a Tween 80 nonionic surfactant (3% by weight), and a 0.15 ml aliquot of the suspension was prepared in a second development sludge prepared as a growth medium as shown in Table 2. Used to inoculate the lances.
[637] (Second Generation and Routine Slant) Malt Extract20 g peptone1 g Glucose20 g Agar20 g Dispense into a sufficient amount of distilled water-pH 5.3-tube (180 x 18 mm) and sterilize at 120 ° C for 7.5 ml-20 minutes1000 ml
[638] The second embryonic slant was incubated at 25 ° C. for 10 days to produce a high mass of gold-colored spores; a brown-orange colored reverse.
[639] Protective media were prepared with the compositions in Table 3.
[640] Protection badge Skim milk10 g Distilled water100 ml Skim milk is added to a 250 ml flask containing 100 ml of distilled water at 50 ° C. Sterilize at 120 ° C. for 15 minutes. Cool to 33 ° C and use before day passes.
[641] Five second developmental slant cultures were suspended in protective solution (15 ml) in a 100 ml flask. This suspension was partitioned into aliquots (0.5 ml each) in 100 × 10 mm tubes for lyophilization. They were pre-frozen at -70 ° C to -80 ° C in acetone / dry ice bath for 20 minutes and then transferred directly to the drying chamber previously cooled to -40 ° C to -50 ° C. The previously frozen aliquots were lyophilized at a residual pressure of 50 μHg and ≦ 30 ° C. At the end of lyophilization, two to three granules of sterile silica gel were added to each tube with a moisture indicator and flame seal.
[642] In order to obtain a mother culture slant suitable for industrial scale fermentation, a single aliquot of the lyophilized culture prepared by the above-described method was suspended in distilled water (1 ml) and a suspension of 0.15 ml aliquot was grown in a medium having the composition shown in Table 2. Used to inoculate the provided slant. Parental slants were incubated at 25 ° C. for 7 days. At the end of the incubation, the cultures developed in the slant were stored at 4 ° C.
[643] To prepare a routine slan culture, the culture from the parent slant was suspended in sterile solution (4 ml) containing Tween 80 (3% by weight) and the resulting suspension was coated with the growth medium described in Table 2. In 0.15 ml aliquots. Routine slant cultures were used to inoculate primary seed flasks for experimental or industrial fermentation.
[644] To prepare primary seed flask cultures, the cultures from the routine slant prepared as described above were removed and suspended in a solution (10 ml) containing Tween 80 (3% by weight). 0.1 aliquots of the resulting suspension were introduced into a 500 ml baffle flask containing growth medium having the composition shown in Table 4.
[645] (Primary and transformed flask cultures and round bottom flasks) Glucose20 g peptone20 g Yeast autolysate20 g Sufficient distilled water-pH 5.2-20% adjusted to pH 5.8 with NaOH-100 ml dispensed into 500 ml baffle flask-500 ml dispensed into 2000 ml round bottom flask (3 baffles)-120 ° C x 20 min sterilization-pH approximately 5.7 after sterilization
[646] The seed flask was incubated on a rotary shaker (200 rpm, 5 cm shift) at 28 ° C. for 24 hours, thereby producing a culture in the form of pelleted hyphae with a diameter of 3-4 mm. Microscopically observed spawn cultures are synnematic growth (a term used to describe the long, twisted, rope-like arrangement of fungal cells), large mycelia and well-twisted pure cultures I knew that. The pH of the suspension was between 5.4 and 5.6. PMV was 5-8% as measured by centrifugation (3000 rpm x 5 minutes).
[647] Transformed flask cultures were prepared by inoculating biomass (1 ml) and growth medium (100 ml) having the composition shown in Table 4 in a second 500 ml shaker flask from the seed culture flask. The resulting mixture was incubated at 28 ° C. on a rotary shaker (200 rpm, 5 cm shift). The culture was examined and found to consist of pelleted hyphae with a diameter of 3-4 mm. Microscopic observation determined that the culture was synergistic and that the culture was pure with fibrous growth, where the apex cells were filled with cytoplasm and the globules had little fear. The pH of the culture suspension was between 5 and 5.2 and PMV was measured by centrifugation between 10% and 15%. Therefore, the culture was considered suitable for the transformation of canrenone into 11α-hydroxycanrenone.
[648] Canrenone (1 g) was micronized to about 5 μ and suspended in sterile water (20 ml). 40% (w / v) glucose sterilization solution in this suspension; Sterilizing solution of 16% (w / v) self-decomposed yeast; And antibiotic sterilization solution; All concentrations are shown in Table 5 for a 0 hour reaction time. Antibiotic solutions were prepared by dissolving kanamycin sulfate (40 mg), tetracycline HCl (40 mg) and cephalexin (200 mg) in water (100 ml). Steroid suspensions, glucose solutions and autolyzed yeast solutions were slowly added to the cultures contained in shaker flasks.
[649] Indication of steroids and solutions (additives and antibiotics) during the biotransformation of canrenones in shake flasks Reaction timeSteroid Suspension ml about mg.Glucose solution mlYeast autolysis solution ml.Antibiotic solution ml 0One50One0.5One 821002One242100One0.5One 3252502One482100One0.5One 5652502One723150One0.5One 90
[650] As the reaction proceeded, the reaction mixture was analyzed periodically to determine the glucose content and the conversion to 11α-hydroxycanrenone by thin layer chromatography. Additional canrenone substrate and nutrients were added to the fermentation reaction mixture during the reaction at a controlled rate to maintain the glucose content in the range of about 0.1% by weight. Addition schedules for steroid suspensions, glucose solutions, autolyzed yeast solutions and antibiotic solutions are listed in Table 5. The transformation reaction continued for 96 h at 25 ° C. on a rotary shaker (200 rpm and 5 cm shift). The pH range during fermentation was 4.5-6. Each time PMV rose to 60% or above, a 10 ml portion of the culture was recovered and replaced with 10 ml distilled water. The loss of canrenone and the appearance of 11α-hydroxycanrenone were sampled at intervals 4, 7, 23, 31, 47, 55, 71, 80 and 96 hours after the start of the fermentation cycle during the reaction, Samples were monitored by analyzing by TLC. The reaction progress measured by these samples is shown in Table 6.
[651] Biotransformation of Canrenon in Shake Flasks Time timeTransformed bicanenone Rf. 11α hydroxycanrenone RF. = 0.81 RF. = 0.29 01000.0 45050 72080 232080 313070 472080 553070 712575 801585 96To 10～ 90
[652] NOTE _Rf and RF are abbreviations for "solventfront" used in thin layer chromatography to describe the mobility of compounds relative to solvent fronts.
[653] Example 2
[654] Primary seed flask cultures were prepared by the method described in Example 1. The culture mixture was prepared with the composition shown in Table 7.
[655] 10 l Transformation Culture in Glass Fermentation Tankamountg / l Glucose80 g20 peptone80 g20 Self-decomposed yeast80 g20 Antifoam SAG 4710.5gSufficient deionized water-Sterilized empty fermenter for 30 minutes at 130 ° C-Filled with 3l of deionized water-Heated at 40 ° C-Add medium with stirring-Stir for 15 minutes, to a volume of 3.9 l-pH 5.1 Adjust to 5.8-Sterilize at 120 ℃ × 20 minutes- After sterilization pH 5.5-5.74l
[656] An initial charge of this nutrient mixture (4 L) was placed in a 10 L geometric volume transgenic fermenter. The fermenter was cylindrical in shape with a height to diameter ratio of 2.58. Two nos with six blades each It was equipped with a 400 rpm turbine stirrer with 2 disc wheels. The outer diameter of the impeller was 80mm, each blade was 25mm in radius, 30mm in height, the upper wheel was positioned 280mm below the top of the vessel, the lower wheel was 365mm below the top, the container baffle was 210mm in height and the vessel It extends radially inwardly 25 mm from the inner vertical wall of the wall.
[657] The spawn culture (40 ml) was mixed with nutrient filling in the fermentor, and the transformed culture was settled by incubating for 22 hours at 28 ° C. and aeration rate of 0.5 l / l-min at a pressure of 0.5 kg / cm ² . At 22 hours, the PMV of the culture was 20-25% and the pH was 5-5.2.
[658] The suspension was prepared with canrenone (80 g) in sterile water (400 ml) and a 10 ml portion was added to the mixture in the transformation fermenter. At the same time, 40% (w / v) glucose sterilization solution, 16% (w / v) autolyzed yeast sterilization solution and antibiotic sterilization solution were added at the ratio shown in Table 8 at 0 hour reaction time. Antibiotic solutions were prepared by the method described in Example 1.
[659] 10 l Addition of steroids and solutions (additives and antibiotics) during the biotransformation of canrenones in glass fermentation tanks Reaction timeSteroid suspension ml mg.Glucose solution mlYeast autolysis solution ml.Antibiotic solution ml 01042512.540 4 2512.581042512.512 2512.5161042512.520 2512.5241042512.540 281042512.53212.552512.53612.552512.54012.552512.54412.552512.54812.552512.540 5212.552512.55612.552512.56012.552512.56412.552512.56812.552512.57212.552512.540 7612.552512.5808488
[660] As the reaction proceeded, the reaction mixture was analyzed periodically to determine the glucose content and the conversion to 11α-hydroxycanrenone by thin layer chromatography. Based on the TLC analysis of the reaction culture sample described below, additional canrenones were added to the reaction mixture as long as the canrenone substrate was consumed. Glucose levels were monitored each time the glucose concentration dropped below about 0.05% by weight and the supplemental glucose solution was added to increase the concentration to about 0.25% by weight. Nutrients and antibiotics were also added at separate times during the reaction cycle. Addition schedules for steroid suspensions, glucose solutions, autolyzed yeast solutions and antibiotic solutions are listed in Table 8. The transformation reaction was continued for 90 hours at an aeration rate of 0.5 (vvm) of air volume per liquid volume per minute at a positive head pressure of 0.3 kg / cm ² . After maintaining the temperature at 28 ° C., the PMV reached 45%, then lowered to 26 ° C. and keeping the PMV at 45% to 60% at that temperature and then adjusting at 24 ° C. The initial stirring speed was 400 rpm and after 40 hours, the speed was increased to 700 rpm. The pH was maintained between 4.7 and 5.3 by addition of 2M orthophosphoric acid or 2M NaOH. Bubble expansion was controlled by adding a few drops of antifoam SAG 471 in bubble development. The loss of canrenone and the appearance of 11α-hydroxycanrenone were monitored at 4 hour intervals during the reaction by TLC analysis of the culture sample. When most of the canrenone was lost from the culture, additional increments were added.
[661] After all canrenone was added, the reaction was terminated when TLC analysis indicated that the concentration of canrenone substrate for the 11α-hydroxycanrenone product was reduced to about 5%.
[662] At the end of the reaction cycle, the fermentation broth was filtered through cotton for separation of mycelia from the liquid culture. The mycelium fraction was resuspended in ethyl acetate using about 65 volumes (5.2 liters) per gram of canlenone charged during the reaction. The suspension of mycelium in ethyl acetate was refluxed for 1 hour under stirring, cooled to about 20 ° C. and filtered in Buchner. The mycelia cake was washed successively with ethyl acetate (5 volumes per g charge of canlenone; 0.4 L) and deionized water (500 ml) to release the ethyl acetate extract from the cake. The filter cake was poured out. The rich extracts, solvent washes and water washes were collected in a separator and then left for 2 hours for phase separation.
[663] The aqueous phase was then decanted and the organic phase was concentrated in vacuo to 350 ml residual volume. The bottom of the distiller was cooled to 15 ° C. and stirred for about 1 hour. The resulting suspension was filtered to remove the crystalline product and the filter cake was washed with ethyl acetate (40 ml). After drying, the yield of 11α-hydroxykanrenone was determined to be 60 g.
[664] Example 3
[665] Spore suspensions were prepared from rutin slants by the method described in Example 1. In a 2000 ml baffle round bottom flask (3 baffles, 50 mm × 30 mm each), an aliquot (0.5 ml) of spore suspension was placed in a culture solution (500 ml) having the composition of Table 4. The resulting mixture was incubated in a flask for 24 hours at 25 ° C. in another shaker (120 strokes per minute; 5 cm shift) to produce a culture that appeared to be a pure culture with well-twisted mycelia when viewed under a microscope. The pH of the culture was about 5.3 to 5.5 and PMV (measured by centrifugation at 3000 rpm for 5 minutes) was 8 to 10%.
[666] Using this culture, spawn cultures were prepared in a vertical cylindrical stainless steel fermenter (height = 985 mm; diameter = 425 mm) having an aspect ratio of 2.31 and a geometric volume of 160L. The fermentor was equipped with a disk turbine type stirrer with two wheels, each wheel having six blades with an outer diameter of 240 mm, each blade having a radius of 80 mm and a height of 50 mm. The upper wheel was positioned at a depth of 780 mm from the top of the fermenter and a second at a depth of 995 mm. A vertical baffle 890 mm in height extended radially inwardly 40 mm from the inner vertical wall of the fermenter. The stirrer was operated at 170 rpm. A nutrient mixture (100 L) having the composition of Table 9 was introduced into the fermenter, followed by the introduction of some pre-inoculation sources (1 L), prepared as described above and having a pH of 5.7.
[667] Approximately 8L is required for spawn production fermenters for growth culture in 160L fermentersamountg / L Glucose2 kg20 peptone2 kg20 Self-decomposed yeast2 kg20 Antifoam SAG 4710.010kga very small amount Sufficient deionized water-empty fermentation tank sterilization for 1 hour at 130 ° C-filled with 6L of deionized water; heating at 40 ° C-adding medium with stirring-stirring for 15 minutes, volume of 95L-sterilization at 121 ° C for 30 minutes-sterilization PH 5.7- add sterilized deionized water to 100L100L
[668] The inoculation mixture was incubated for 22 hours at an aeration rate of 0.5 L / L-min at a head pressure of 0.5 kg / cm ² . After controlling the temperature to 28 ° C., PMV reached 25% and then lowered to 25 ° C. The pH was adjusted in the range of 5.1 to 5.3. Growth of mycelial volume is shown in Table 10 along with the dissolved oxygen content and pH profile of the seed culture.
[669] Mycelial Growth in Seed Culture Fermentation Fermentation timepHCharged mycelial volume (pmv)% (3000 rpm 5 minutes)Dissolved oxygen% 05.7 ± 0.1 100 45.7 ± 0.1 100 85.7 ± 0.112 ± 385 ± 5 125.7 ± 0.115 ± 372 ± 5 165.5 ± 0.125 ± 540 ± 5 205.4 ± 0.130 ± 535 ± 5 225.3 ± 0.133 ± 530 ± 5 245.2 ± 0.135 ± 525 ± 5
[670] Using the spawn culture thus produced, the transformation fermentation was carried out in a vertical cylindrical stainless steel fermenter with a diameter of 1.02m, a height of 1.5m, and a geometric volume of 1.4m ³ . The fermentor was equipped with two impellers, one of which was positioned 867 cm below the top of the reactor and the other was located 1435 cm below the top. Each wheel had six blades, each 95 cm in diameter and 75 cm in height. Vertical baffles 1440 cm high extended radially inwards from the inner vertical wall of the reactor. Nutrients mixtures were prepared with the compositions in Table 11.
[671] Biotransformation in 1000L Fermenteramountg / L Glucose16 kg23 peptone16 kg23 Self-decomposed yeast16 kg23 Antifoam SAG 4710.080kga very small amount Sufficient deionized water-Sterilized empty fermentation tank for 1 hour at 130 ° C-Filled with 600L of deionized water; Heat at 40 ° C-Add with stirring medium-Stir for 15 minutes, 650L volume-Sterilize at 121 ° C for 30 minutes-After sterilization pH 5.7- 700L with sterilized deionized water700L
[672] An initial charge (700 L) of this nutrient mixture (pH = 5.7) was placed in a fermenter followed by a seed inoculum (7 L) of this example prepared as described above.
[673] The nutrient mixture containing the inoculum was incubated for 24 hours at an aeration rate of 0.5 L / L-min at a head pressure of 0.5 kg / cm ² . The temperature was adjusted to 28 ° C. and the stirring speed was 110 rpm. The growth of mycelial volume is shown in Table 12 along with the dissolved oxygen content and pH profile of the spawn culture reaction.
[674] Mycelial Growth in Fermenter of Transgenic Cultures Fermentation timepHCharged mycelial volume (pmv)% (3000rpm × 5 minutes)Dissolved oxygen% 05.6 ± 0.2 100 45.5 ± 0.2 100 85.5 ± 0.212 ± 395 ± 5 12 15 ± 390 ± 5 165.4 ± 0.120 ± 575 ± 5 205.3 ± 0.125 ± 560 ± 5 225.2 ± 0.130 ± 540 ± 5
[675] At the end of the cultivation, pelletization of the hyphae was observed, but the pellets were usually small and relatively loosely packed. The dispersed mycelia were suspended in the culture. Final pH was 5.1 to 5.3.
[676] The thus-transformed culture was added with a suspension of canrenone (1.250 kg; micronized to 5 mu) in sterile water (5 L). Additive sterilization solution and antibiotic solution were added at the ratio indicated at reaction time 0 in Table 14. The composition of the additive solution is described in Table 13.
[677] Additive solution (transformation culture)amount Dextrose40 kg Yeast autolysate8 kg Antifoam SAG 4710.010kg Sufficient deionized water-150 l empty fermenter sterilized for 1 hour at 130 ° C-70 l filled with deionized water; heated at 40 ° C-added with stirring "component solution"-stirring for 30 minutes, volume of 95 l-pH 4.9- Sterilization at 120 ℃ × 20 minutes- pH after sterilization 5100 l
[678] Bioconversion was performed for about 96 hours with 0.5 L / L-min aeration at a head pressure of 0.5 kg / cm ² and pH ranging from 4.7 to 5.3 adjusted as needed by adding 7.5 M NaOH or 4 M H ₃ PO ₄ . It was. Stirring speed was initially 100rpm, increased to 165rpm at 40 hours, 250rpm at 64 hours. The initial temperature was 28 ° C., and decreased to 26 ° C. when PMV reached 45%, and decreased to 24 ° C. when PMV rose to 60%. Aesthetic SAG 471 was added as needed to control foaming. Glucose levels in fermentation were monitored at 4 hour intervals and an increase in additive sterilization solution (10 L) was added to the batch whenever the glucose concentration dropped below 1 gpl. The loss of canrenone and the appearance of 11α-hydroxycanrenone were also monitored by HPLC during the reaction. An increase of 1.250 kg of canrenone was added when at least 90% of the initial canrenone charge was converted to 11α-hydroxycanrenone. Another 1.250 kg increase was introduced, indicating that 90% of the canenone in the increase was converted. Using the same criteria, different increments (1.250 kg each) were applied to introduce a full reactor charge (20 kg). After conveying the full canrenone charge to the reactor, the reaction was terminated when the concentration of unreacted canrenone was 5% relative to the amount of prepared 11α-hydroxycanrenone. The schedules for addition of canrenone, additive sterilization solution and antibiotic solution are shown in Table 14.
[679] Addition of Steroids and Solutions (Additives and Antibiotics) during the Biotransformation of Canrenone in Fermenters Reaction timeCanrenon kg cumulative kg in suspensionAdditive Sterilization SolutionAntibiotic Solution lVolume liter 01.2501.25108700 4 10 81.2502.510 12 10 161.250 10 20 10 241.2505108800 281.250 10 321.250 10 361.250 10 401.250 10 441.250 10 481.25012.5108900 521.250 10 561.250 10 601.250 10 641.250 10 681.250 10 721.250201081050 76 0 80848892gun
[680] Upon completion of the biotransformation, the hyphae were separated from the culture by centrifugation in a basket centrifuge. The filtrate was removed by HPLC, containing only 2% of the total amount of 11α-hydroxykanrenone in the harvested culture. The mycelia were suspended in ethyl acetate (1000 L) in a 2 m ³ extraction tank. The suspension was heated for 1 h under stirring and ethyl acetate reflux conditions, then cooled and centrifuged in a basket centrifuge. Mycelia cake was washed with ethyl acetate (200L) and then discharged. The steroid rich solvent extracts were left for 1 hour to separate the aqueous phase. The aqueous phase was extracted with an additional amount of ethyl acetate solvent (200 L) and then discharged. The combined solvent phases were purified by centrifugation, placed in a concentrator (500 L geometric volume) and concentrated in vacuo to 100 L residual volume. When the evaporation took place, the initial charge of the combined extracts and washes into the concentrator was 100 L and this volume was kept constant with continuous or periodic addition of the combined solutions as the solvent was removed. After completion of the evaporation step, the bottom of the distiller was cooled to 20 ° C. and stirred for 2 h and then filtered on a Buchner filter. The concentrator pot was washed with ethyl acetate (20 L) and this wash was used to wash the cake on the filter. The product was dried under vacuum at 50 ° C. for 16 hours. The yield of 11α-hydroxycanrenone was 14 kg.
[681] Example 4
[682] Aspergillus ochraceusLyophilized spores of NRRL 405 were suspended in cornstalk liquid growth medium (2 ml) having the composition shown in Table 15.
[683] Corn tip liquid medium (growth medium for primary seed culture) Corn tip liquid30 g Yeast extract15 g Ammonium Phosphate Monobasic3 g Glucose (filled after sterilization) Sufficient amount of distilled water 1000mlpH: 4.6, adjusted to pH 6.5 with 20% NaOH 50ml dispensed into 250ml Erlenmeyer flask Sterilized at 121 ℃ for 20 minutes30 g
[684] The suspension obtained was used as inoculum for propagation of spores on agar plates. Ten agar plates were prepared, each containing a solid glucose / yeast extract / phosphate / agar growth medium having the composition shown in Table 16.
[685] GYPA (glucose / yeast extract / phosphate agar for plates) Glucose (filled after sterilization)10 g Yeast extract2.5g K ₂ HPO ₄ 3 g Sterilize at 121 ℃ for 30 minutes after adjusting to 1000mlpH 6.5 of agar20 g
[686] 0.2 ml aliquots of the suspension were transferred to the surface of each plate. After incubating the plates at 25 ° C. for 10 days, spores from all plates were harvested in sterile cryogenic protective medium having the composition shown in Table 17.
[687] GYP / Glycerol (Glucose / Yeast Extract / Phosphate / Glycerol Medium for Storage Vials) Glucose (filled after sterilization)10 g Yeast extract2.5g K ₂ HPO ₄ 3 g Sterilize at 121 ° C for 1000 mL of distilled water for 30 minutes20 g
[688] The resulting suspension was divided into 20 vials carrying 1 ml into each vial. These vials constitute the main cell bank, which can produce working cell banks for use in inoculation for the bioconversion of canrenone to 11α-hydroxycanrenone. The vial consisting of the main cell bank was stored at −130 ° C. in the vapor phase of a liquid nitrogen freezer.
[689] To prepare working cell banks, spores from a single main cell bank were resuspended in sterile growth medium (1 ml) having the composition shown in Table 15. This suspension was divided into 10 0.2 ml aliquots and each aliquot was used to inoculate agar plates containing solid growth media having the compositions in Table 16. These plates were incubated at 25 ° C. for 10 days. On the third day of culture, the lower surface of the growth medium was brown-orange. At the end of the culture, a lot of gold spores were produced. Spores from each plate were harvested by the method described above for the preparation of the main cell bank. A total of 100 vials were prepared and each contained 1 ml of suspension. These vials constituted a working cell bank. Working cell bank vials were stored and stored in the vapor phase of a liquid nitrogen freezer at -130 ° C.
[690] Growth medium (50 ml) having the composition in Table 15 was placed in a 250 ml Erlenmeyer flask. An aliquot (0.5 ml) of working cell suspension was placed in a flask and mixed with the growth medium. The inoculated mixture was incubated at 25 ° C. for 24 hours to produce a primary seed culture with a percent filled mycelial volume of about 45%. Observation by eye revealed that the culture consisted of pelleted hyphae of 1-2 mm diameter; Observation under a microscope revealed that the culture was pure.
[691] Cultivation of the secondary seed culture was initiated by inoculating the medium with a portion of the primary seed culture (10 ml) of this example prepared in the growth medium having the composition shown in Table 15 in a 2.8 L Fernbach flask prepared as described above. . The inoculation mixture was incubated at 25 ° C. for 24 hours on a rotary shaker (200 rpm, 5 cm shift). At the end of the culture, the culture exhibited the same characteristics as described above for the primary seed culture and was suitable for use in the transformation fermentation that bioconverts canrenone to 11α-hydroxycanrenone.
[692] Transformation was carried out in a Braun E Biostat fermenter consisting of:
[693] Capacity: 15 liter round bottom
[694] Height: 53 cm
[695] Diameter: 20cm
[696] H / D: 2.65
[697] Impellers: 7.46 cm diameter, 6 paddles each 2.2 × 1.4 cm
[698] Impeller spacing: 65.5, 14.5 and 25.5 cm from bottom of tank
[699] Baffles: 4 1.9 × 48cm
[700] Sparger: 10.1 cm diameter, 21 holes-1 mm diameter
[701] Temperature control: supplied as outer container jacket
[702] 20 g / L of canrenone was suspended in deionized water (4 L) and a portion of the growth medium (2 L) having the composition shown in Table 18 was added while stirring the mixture in the fermenter at 300 rpm.
[703] (Growth medium for biotransformation culture in 10L fermenter)amountQuantity / L Glucose (filled after sterilization)160 g20 g peptone160 g20 g Yeast extract160 g20 g Antifoam SAF4714.0ml0.5ml Depleted water of canrenone 7.5L sterilized at 121 ℃ for 30 minutes160 g20 g
[704] The resulting suspension was stirred for 15 minutes and then the volume was brought to 7.5 L with additional deionized water. At this point the pH of the suspension was adjusted to 5.2-6.5 by adding 20% by weight of NaOH solution, and then the suspension was sterilized by heating at 121 ° C. for 30 minutes in a Brown's fermenter. After sterilization the pH was 6.3 ± 0.2 and the final volume was 7.0L. The sterilization suspension was inoculated with a portion of the secondary seed culture (0.5 L) of the present Example prepared as described above, and the volume was adjusted to 8.0 L by adding 50% glucose sterilization solution. The fermentation was carried out at a temperature of 28 ° C. to reach 50% PMV, then to 26 ° C. and further down to 24 ° C. when PMV exceeded 50% to maintain a constant PMV of less than about 60%. Air was introduced through the sparger at a rate of 0.5vvm based on the initial liquid volume and the pressure in the fermenter was maintained at 700 millibar gauge. Agitation was initiated at 600 rpm and stepped up to 1000 rpm as needed to maintain the dissolved oxygen amount above 30 vol%. Glucose concentration was monitored. After lowering the initial glucose high concentration to less than 1% because it is consumed by the fermentation reaction, supplemental glucose was provided through 50% by weight of glucose sterilization solution to maintain the concentration in the range of 0.05% to 1% through the batch circulation residue. . The pH before inoculation was 6.3 ± 0.2. After the pH was lowered to about 5.3 during the initial fermentation, ammonium hydroxide was added to maintain in the range 5.5 ± 0.2 for the residue of the circulation. The bubbles were controlled by adding a polyethylene glycol antifoam agent sold under the trade name SAG 471 by OSISpecialties, Inc.
[705] Cultures were first grown during the first 24 hours of circulation, with PMV of about 40%, pH of about 5.6 and dissolved oxygen of about 50% by volume. Canrenone conversion was initiated as the culture grew. Concentrations of canrenone and 11α-hydroxycanrenone were monitored during biotransformation by analyzing samples daily. The sample was extracted with hot ethyl acetate and the sample solution obtained was analyzed by TLC and HPLC. Bioconversion was terminated when the residual canrenon concentration was about 10% of the initial concentration. Approximate conversion time was 110-130 hours.
[706] At the end of the biotransformation, the mycelial biomass was separated from the culture by centrifugation. The supernatant was extracted with the same volume of ethyl acetate and the aqueous layer was decanted. The mycelium fraction was resuspended in ethyl acetate using about 65 volumes per g of canlenone charged to the fermentation reactor. The mycelium suspension was refluxed for 1 hour under stirring, cooled to about 20 ° C. and filtered in a Buchner funnel. The mycelial filter cake was washed twice with 5 volumes of ethyl acetate per gram of canlenone in the fermenter, followed by washing with deionized water (1 L) to discharge the remaining ethyl acetate. Aqueous extracts, abundant solvents, solvent washes and water washes were combined. The residue from which the hyphae cake was removed was decanted or re-extracted depending on the analysis for residual steroids. The combined liquid phases were left for 2 hours. The aqueous phase was then separated and decanted and the organic phase was concentrated in vacuo to a residual volume of about 500 ml. The still bottle was cooled to about 15 ° C. with gentle stirring for about 1 hour. The crystalline product was recovered by filtration and washed with cold ethyl acetate (100 ml). The solvent was removed from the crystals by evaporation and the crystalline product was dried under vacuum at 50 ° C.
[707] Example 5
[708] Aspergillus ochaceusLyophilized spores of ATCC 18500 were suspended in cornstain liquid growth medium (2 ml) described in Example 4. Agar plates were prepared by the method of Example 4. Plates were incubated and harvested as described in Example 4 to obtain main cell banks. Vials containing the main cell banks were stored in the vapor phase of a liquid nitrogen freezer at -130 ° C. From the vials of the main cell bank, working cell banks were prepared as described in Example 4 and stored in a nitrogen freezer at -130 ° C.
[709] Growth medium (300 mL) having the composition described in Table 19 was placed in a 2 L baffle flask. An aliquot (3 mL) of working cell suspension was placed in a flask. The inoculation mixture was incubated on a rotary shaker (200 rpm, 5 cm shift) at 28 ° C. for 20-24 hours to produce a primary seed culture with a percent filled mycelial volume of about 45%. Observation by eye revealed that the culture consisted of pelleted mycelium of 1-2 mm; Observation under a microscope revealed that the culture was pure.
[710] Growth medium for primary and secondary seed culturesQuantity / L Glucose (filled after sterilization)20 g peptone20 g Yeast extract20 g Sufficient deionized water 1000mL sterilized at 121 ℃ for 30 minutes
[711] Cultivation of the secondary seed culture was initiated by introducing 8 L of growth medium having the composition shown in Table 19 into a 14 L glass fermenter. Fermenter is inoculated with 160 mL to 200 mL of the primary seed culture of this example. The preparation thereof is as described above.
[712] The inoculation mixture was incubated at 28 ° C. for 18-20 hours at 200 rpm stirring, 0.5vvm aeration rate. At the end of breeding the culture showed the same characteristics as described above for the primary seed.
[713] Transformation was substantially carried out in a 60L fermenter by the method described in Example 4 except that the growth medium had the composition shown in Table 20 and the initial fill of the secondary seed culture was 350 mL to 700 mL. The stirring speed was initially 200 rpm, but was increased to 500 rpm as necessary to maintain the dissolved oxygen amount at 10 vol% or more. The approximate bioconversion time for 20 g / L canrenones was 80 to 160 hours.
[714] Growth medium for biotransformation in 60L fermenteramountQuantity / L Glucose (filled after sterilization)17.5 g0.5g peptone17.5 g0.5g Yeast extract17.5 g0.5g Canrenone (filled as 20% slurry in sterile water)700 g20 g Sufficient deionized water 35L 30 minutes sterilization at 121 ℃
[715] Example 6
[716] Primary and secondary seed cultures were prepared substantially in the manner described in Example 4 using spore suspensions from working cell banks prepared according to the method described in Example 4. Using the secondary seed culture prepared in this way, two biotransformation operations were performed according to the modification method of the type shown in FIG. 1, and the two operations were made as the method shown in FIG. The transgenic growth medium, canrenone addition schedule, harvest time and conversion for these manipulations are listed in Table 21. Operation R2A uses the canrenone addition lumen based on the same principle as in Example 3, while operation R2C adds canrenone only two times, one at the start of the batch and the other after 24 hours. The lumen was modified. In operations R2B and R2D, the full canrenon filling is introduced at the start of the batch and the process is described in Example 4 except that the canlenone filling is sterilized in a separate container and added during the batch process of glucose before filling into the fermenter. It was usually carried out as. Waring blenders were used to reduce aggregates produced during sterilization. In operations R2A and R2B, canrenone was introduced into a batch in methanol solution, and these operations were different from those of Examples 3 and 4, respectively.
[717] Initial Canrenon Biotransformation Process Description Operation numberR2AR2BR2CR2D Medium (g / L) Cornstalk Liquid Yeast Extract NH ₄ H ₂ PO ₄ Glucose OSApHAdjusted to 6.0 with 30153150.5ml2.5NNaOHSame operation as R2A30153300.5ml2.5NNaOH to 6.5Same operation as R2C CanrenonAdd 10 g / 80 ml MEOH at 0, 18, 24, 30, 36, 42, 50, 56, 62 and 68 hoursAdd 80g / 640ml MEOH at 0h at the same timeSterilization and blending; 0 hours: 25 g 24 hours: 200 gSterilization and blending 0 hours: 200 g added Harvest time143 hours166 hours125 hours104 hours Biotransformation45.9%95.6%98.1%95.1%
[718] Note: OSA designates the antifoam SAG 471 manufactured by Sistersville OSI Specialty Company, West Virginia, USA.
[719] In the manipulations R2A and R2B, the methanol concentration accumulated at about 6.0% in the fermentation beer, which was found to inhibit the growth and biotransformation of the culture. However, based on the results of these operations, methanol or other water-miscible solvents can be effectively used at lower concentrations to increase canrenone filling and provide canrenones as particulate precipitates to provide large interfacial canrenones to the reaction. I knew you could.
[720] Canrenones are stable at sterilization temperature (121 ° C.) but aggregated into lumps. The Waring blender was used to break up the mass into fines which were successfully converted to the product.
[721] Example 7
[722] Primary and secondary seed cultures were prepared substantially by the method described in Example 4 using spore suspensions from working cell banks prepared according to the method described in Example 4. The description and results of Example 7 are shown in Table 22. Using a secondary seed culture produced in this way, one biotransformation (R3C) was carried out substantially as described in Example 3, and three bioconversions were normally carried out according to the method described in Example 5. In the latter three operations (R3A, R3B and R3D), canrenones were sterilized in a movable tank with growth media excluding glucose. Glucose was aseptically fed from another tank. Sterile canlenone suspensions were placed in fermenters either during the initial stages of biotransformation or before inoculation. In operation R3B, supplementary sterilization canrenone and growth medium were introduced at 46.5. A lump of canrenone formed during sterilization was pulverized through a Waring blender to prepare a fine particle suspension into a fermenter. Transformation growth medium, canrenone addition schedule, nutrient addition schedule, harvest time and conversion for these manipulations are shown in Tables 22 and 23.
[723] Canrenon Biotransformation Process Description Operation numberR3AR3BR3CR3D Medium (g / L) Cornstalk Liquid Yeast Extract NH ₄ H ₂ PO ₄ Glucose OSApH3015315 Adjusted to 6.5 with 0.5 ml 2.5 N NaOHSame operation as R3APeptone: 20 yeast extract: 20 glucose: 20 OSA: adjusted to 6.5 with 3 ml 2.5 N NaOHSame operation as R3A Canrenon chargingSterilize and blend canrenones. BI: 50g16.5hours: 110gOperation as in R3A BI: 50 g 16.5 hours: 110 g 46.5 hours: 80 gNon-sterile Canrenones: Fill according to the schedule listed in Table 23Operation as in R3A BI: 50 g 16.5 hours: 110 g supplySee Table 23See Table 23See Table 23See Table 23 Harvest time118.5 hours118.5 hours118.5 hours73.5 hours Biotransformation93.7%94.7%60.0%68.0%
[724] Supply Schedule for Canrenone, Glucose, and Growth Medium in Developmental Experiments Addition timeR3CR3AR3BR3D Canrenone 200g / 2L sterilization DIg50% glucose solution g20gg each of peptone and yeast extract in IL waterAntibiotic: 20 mg kanamycin 20 mg tetracycline 100 mg cephalexin in 50 mlCanrenon / Growth Badges 22 g / LCanrenon / Growth Badges 22 g / LCanrenon / Growth Badges 22 g / L 0----50g / 0.4L50g / 0.4L50g / 0.4L 14.5161002550 ml--- 16.5----110g / 1.2L110g / 1.2L110g / 1.2L 20.51614025---- 28.51614025---- 34.51615025---- 40.5161502550 ml--- 46.588013025--80g / 0.8L- 52.516012025---- 58.516015025---- 64.51601802550 ml--- 70.516014025----
[725] Fermenter cultures that were very viscous due to fibrous growth were present in all four operations of this example. In order to overcome the problems high viscosity caused for aeration, mixing, pH control and temperature control, the aeration rate and agitation rate were increased during these operations. The conversion proceeded sufficiently under more stringent conditions, but a dense cake formed on the liquid culture surface. Any unreacted canrenone was carried out of the culture by this cake.
[726] Example 8
[727] The description and results of Example 8 are summarized in Table 24. Four fermentations were performed, wherein 11α-hydroxycanrenone was prepared by bioconversion of canrenone. In two of these manipulations (R4A and R4D), the biotransformation was carried out in substantially the same manner as the manipulations R3A and R3D of Example 6. In operation R4C, canrenone was usually converted to 11α-hydroxycanrenone by the method described in Example 3. In operation R4B, the process is carried out substantially as described in Example 4, i.e. sterilization of growth medium and canrenone in the fermenter immediately before inoculation, all nitrogen and phosphorus nutrients are introduced at the start of the batch and contain only glucose Was fed to the fermenter to maintain glucose levels as the batch progressed. In the latter process (operation R4B), the glucose concentration was monitored every 6 hours and the glucose solution was added as directed to control the glucose level in the range of 0.5 to 1%. The schedule of addition of canrenones for these operations is in accordance with Table 25.
[728] Explain the development experiment process of the Kanrenon biotransformation Operation numberR4AR4BR4CR4D Medium (g / L) Cornstalk Liquid Yeast Extract NH ₄ H ₂ PO ₄ Glucose OSApHAdjusted to 6.5 with 30153150.5ml2.5NNaOHSame operation as R4APeptone: 20 Yeast Extract: 20 Glucose: 20OSA Adjusted to 6.5 with 3ml2.5NNaOHOperation same as R4A Canrenon chargingSterilize and blend canrenones.BI: 40g23.5 hours: 120g160g canrenon sterilized in fermenterNon-sterile Canrenones: Fill according to the schedule listed in Table 25Sterilize and blend canrenones.BI: 40g23.5 hours: 120g Badge chargingSee Table 25See Table 25See Table 25See Table 25 Harvest time122 hours122 hours122 hours122 hours Biotransformation95.6%97.6%95.4%96.7%
[729] Supply Schedule of Canrenone, Glucose and Growth Medium in the Developmental Experiment Addition timeR4CR4AR4BR4D Canrenone 200g / 2L sterilization water g50% solution of glucose20gg each of peptone and yeast extract in 1L waterAntibiotic: 20 mg kanamycin 20 mg tetracycline 100 mg cephalexin in 50 ml (added to canrenone slurry)See Growth Badges Table 24See Growth Badges Table 24See Growth Badges Table 24 146001352550 ml--- 20-100----- 23----120g / 1.2L-120g / 1.2L 26-10025---- 32-13525---- 385001202550 ml--- 44-10025---- 50-10025---- 56-15025---- 625001502550 ml--- 68-20025---- 74-30025---- 8--10025---- 86-12525---- 92-17525---- 98-150----- 104-175----- 110-175----- 116-200-----
[730] All fermenters were operated under high agitation and aeration during most fermentation cycles as the fermentation beer became very viscous within one day or after inoculation.
[731] Example 9
[732] Transformation growth medium, canrenone addition schedule, harvest time and conversion for the manipulation of this example are described in Table 26.
[733] Four biotransformation operations were carried out substantially by the method described in operation R4B of Example 8 except for the following. In operation R5B, a marine impeller pumping down a normal turbine disk impeller (mechanism consisting of blades attached to the hub concentrically attached to the stirring shaft of the fermenter) used for stirring in another operation. Boat propeller-like blades, allowing the liquid to flow in a specific direction, mainly up and down). The downward pumping action axially poured the culture into the center of the fermenter and reduced cake formation. Methanol (200 ml) was added immediately after inoculation on operation R5D. Because canrenone is sterilized in fermenters, all nutrients except glucose were added at the start of the batch, eliminating the need for a nitrogen source, a phosphorus source or a continuous supply of antibiotics.
[734] Process development experiment of 10L scale biotransformation Operation numberR5AR5BR5CR5D Medium (g / L) Cornstalk Liquid Yeast Extract NH ₄ H ₂ PO ₄ Glucose OSApHAdjusted to 6.5 with 30153150.5ml2.5NNaOHOperation same as R5APeptone: 20 Yeast Extract: 20 Glucose: 20OSA Adjusted to 6.5 with 3ml2.5NNaOHSame operation as R5A Canrenon charging160g canrenon sterilized in fermenter160g canrenon sterilized in fermenter160g canrenon sterilized in fermenter160g canrenon sterilized in fermenter Badge supplyGlucose supplyGlucose supplyGlucose supplyGlucose supply Harvest time119.5 hours119.5 hours106119.5 hours Biotransformation96%94.1%88.5%92.4%
[735] In order to maintain the solid phase immersion growing on the liquid surface, the growth medium (2 L) was added to each fermenter and the batch was started after 96 hours. Mixing problems were not overcome by either the addition of growth medium or the use of a downward pumped impeller (operation R5B), but the results of the operation demonstrated the feasibility and benefits of the process and that sufficient mixing could be provided according to conventional practice. Indicated.
[736] Example 10
[737] Three biotransformation operations were carried out substantially by the method described in Example 9. Transformation growth medium, canrenon addition schedule, harvest time and conversion for the manipulations of this example are listed in Table 27:
[738] 10L scale biotransformation process description Operation numberR6AR6BR6C Medium (g / L) Cornstalk Liquid Yeast Extract NH ₄ H ₂ PO ₄ Glucose OSApHAdjusted to 6.5 with 30153150.5ml2.5NNaOHOperation same as R6APeptone: 20 Yeast Extract: 20 Glucose: 20OSA 0.5 ml 2.5 NNAOH adjusted to 6.5 Canrenon charging160g canrenon sterilized in fermenter160g canrenon sterilized in fermenter160g canrenon sterilized in fermenter Badge supplyGlucose supply; 1.3L medium and 0.8L sterile water at 71 hoursGlucose supply; 0.5 L medium and 0.5 L sterile water at 95 hoursGlucose supply; no other additions Harvest time120 hours120 hours120 hours Biotransformation95%96%90% Mass balance59%54%80%
[739] Growth medium (1.3 L) and sterile water (0.8 L) were added after 71 hours in operation R6A and the mycelia cake grown on the surface of the liquid culture was immersed. For the same purpose, growth medium (0.5 L) and sterile water (0.5 L) were added after 95 hours in operation R6B. Material balance data indicated that a better mass balance could be measured when minimizing cake enhancement on the liquid surface.
[740] Example 11
[741] Fermentation was performed by comparing the growth medium and the sterilization of canrenone with the pre-sterilization of canrenone in the transformed fermenter. In operation R7A, the process was run as shown in FIG. 2 under conditions comparable to operations R2C, R2D, R3A, R3B, R3D, R4A and R4D. Operation R7B was as shown in FIG. 3 under conditions comparable to Examples 4, 9 and 10 and Operation R4B. Transformation growth medium, canrenone addition schedule, harvest time and conversion for the manipulations of this example are described in Table 28:
[742] 10L scale biotransformation process description Operation numberR7AR7B Medium (g / L) Cornstalk Liquid Yeast Extract NH ₄ H ₂ PO ₄ Glucose OSApHAdjusted to 6.5 with 30153150.5ml2.5NNaOHOperation same as R7A Canrenon chargingSterilize and mix 160g canrenone outside fermenter160g canrenon sterilized in fermenter Badge chargingGlucose supply; Kanrenon was added as a growth medium for 1.6LGlucose supply; no other additions Harvest time118.5 hours118.5 hours Biotransformation93%89%
[743] The mass balance based on the final sample taken from operation R7B was 89.5% with no significant substrate loss or biotransformation reduction. Mixing was determined appropriately for both operations.
[744] Residual glucose concentrations were above the desired 5-10 gpl control range during the initial 80 hours. Manipulation performance was not clearly affected by light cakes accumulated in the headspace of both fermenters.
[745] Example 12
[746] Extraction efficiency was measured by a series of 1L extraction operations summarized in Table 29. In each of these operations, steroids were extracted from mycelia using ethyl acetate (1 L / L fermentation volume). Two consecutive extractions were performed in each operation. About 80% of the total steroid was recovered in the first extraction based on RP-HPLC; Recovery was increased to 95% by the second extraction. An additional 3% steroid was recovered by the third extraction. The remaining 2% is lost in the aqueous phase of the supernatant. The extract was dried under vacuum but not washed with any additional solvent. Judging by an economical process, the solvent will be chased to improve the recovery from the initial extraction.
[747] Recovery of 11α-hydroxykanrenone from 1 liter extraction (% of total) Operation numberFirst extractionSecond extractionThird extractionSupernatant R5A79%16%2%2% R5A84%12%2%2% R4A72%20%4%4% R4A79%14%2%5% R4B76%19%4%One% R4B79%16%3%2% R4B82%15%2%One% Average79%16%3%2%
[748] Methylisobutylketone (MIBK) and toluene were evaluated as extraction / crystallization solvents for 11α-hydroxycanrenone at 1 L culture scale. Using the extraction protocol described above, two MIBK and toluene were compared with ethyl acetate in both extraction efficiency and crystallization performance.
[749] Example 13
[750] As part of the in-process evaluation of FIGS. 2 and 3, particle size studies were performed on canrenon substrates provided at the start of the fermentation circulation in each of these processes. As mentioned above, the canrenone supplied to the process of FIG. 1 was micronized and put into the fermenter. In this process canrenone is not sterilized and unwanted microbial growth is controlled by the addition of antibiotics. Canrenone is sterilized before reacting in the method of FIGS. 2 and 3. In the method of FIG. 2, this is done in a blender before canrenone is placed in the fermenter. In the method of FIG. 3, the suspension of canrenone in the growth medium is sterilized in a fermenter at the start of the batch. As mentioned above, sterilization tends to cause aggregation of canrenone particles. Due to the limited solubility of canrenones in aqueous growth media, the productivity of the process depends on mass transfer from the solid phase and thus can be expected to depend on the interfacial area provided by the solid particle substrate and depends on the particle size distribution. It is believed that they are initially used as obstacles to the method of FIGS. 2 and 3.
[751] However, the agitation in the blender of FIG. 2 and the fermentation tank of FIG. 3, together with the action of the shear pump used in the batch movement of FIG. 2, resulted in a reasonably close particle size range to the non-sterile and micronized canrenone supplied to the process of FIG. 1. Was found to reduce aggregates. This is illustrated by the particle size distribution for canrenone available in the contours of the reaction cycle in each of the three processes. See Table 30 and FIGS. 4 and 5.
[752] Particle Distribution of Three Different Canrenon Samples sample45-125μ<180 μAverage size μOperation #:% Biotransformation Canrenon charging75%95%-R3C: 95.1% (120 hours) R4C: 94.3% (120 hours) Formulated Sample31.2%77.2%139.5R3A: 94.6% (120 hours) R3B: 95.2% (120 hours) Sterile d sample24.7%65.1%157.4R4B: 97.6% (120 hours) R5B: 93.8% (120 hours)
[753] From the data in Table 30, it should be noted that the stirrer and sheer pump are effective to reduce the average particle diameter of sterile canrenone to the same size as the non-sterile substrate, but there is a significant difference in size on the non-sterile substrate. Despite this difference, the reaction performance data indicated that the presterilization process was at least as productive as in the process of FIG. 1. Further advantages can be realized in the process of FIG. 2 by pasteurization rather than by wet grinding and / or sterilization of certain steps for further reduction and control of the particle diameter, for example.
[754] Example 14
[755] Seed cultures were prepared by the method described in Example 5. At 20 hours, the mycelia in the inoculated fermenter were pulp of 40% PMV. The pH was 5.4 and 14.8 gpl glucose remained unused.
[756] Transformed growth medium (35L) was prepared with the composition shown in Table 20. In the preparation of the feed medium, the glucose and yeast extracts were sterilized separately and mixed in a single feed at an initial concentration of 30% glucose and 10% yeast extract. The feed pH was adjusted to 5.7.
[757] Using this medium (Table 20), two biotransformation operations were performed for the conversion of canrenone to 11α-hydroxycanrenone. Each operation was performed in a 60L fermenter with a stirrer consisting of one Rushton turbine impeller and two Lightning A315 impellers.
[758] The initial charge of the growth medium into the fermenter was 35 liters. Undifferentiated and non-sterile canrenones were added at an initial concentration of 0.5%. The medium in the fermenter was inoculated with a seed culture prepared by the method described in Example 5 at an initial inoculation ratio of 2.5%. Fermentation was carried out at a temperature of 28 ° C., agitation speed of 200 to 500 rpm, aeration rate of 0.5vvm and back pressure sufficient to maintain at least 20 volume percent dissolved oxygen level. Transgenic cultures developed during production were in the form of fine oval pellets (about 1-2 mm). Canrenone and supplemental nutrients were continuously fed to a fermenter, usually by the method described in Example 1. Nutrients were added every 4 hours at a rate of 3.4 g glucose and 0.6 g yeast extract per liter of culture in the fermentor.
[759] Listed in Table 31 are the prevailing aeration rate, stirring rate, dissolved oxygen amount, PMV and pH as well as glucose addition during the batch at the intervals mentioned during each operation of this example. Table 32 shows canrenone conversion profiles. Operation R11A was terminated after 46 hours; Operation R11B continued for 96 hours. In the latter operation, 93% conversion was at 81 hours; One or more feed additions were made at 84 hours; Next, the supply was terminated. Note that a significant change in viscosity occurs between the stop of the feed time and the end of the operation.
[760] Fermentation R11A timeAir (lpm)rpm% DOBack pressurePMV (%)pHGluc cc (g / l) 0.12020093026.175.8 72020085.1056.035.5 12.42030050.20 5.4321.82040025.50386.980 2920500170355.2230.22050018.810 5.0131205007910 4.81One 35.72050010010455.570 46.220500236455.8One Total glucose: 27.5 g / l Whole yeast extract: 8.75 g / l Fermentation R11B timeAir (lpm)rpm% DOBack pressurePMV (%)pHGluc cc (g / l) 0.12020092.9025.985.4 72020082.3055.95 12.42030049.50 5.4821.820400180407.120 292050036.80355.13 35.72050094.510 4.740 46.22050014.56455.322 552050016.710 5.310.5 58.62050019.415 5.32One 61.9205001315405.362 71.7205001315425.370 81.12050022.915 5.422.5 85.6205002215455.48One 97.52050010815456.470 117.720500 7.380 Total Glucose: 63g / l Whole Yeast Extract: 14.5g / l
[761] Fermentation R11A: Canrenon Conversion Concentration (g / l)Conversion rateCalc OH-canSwitching speed (g / l / h) sampletimeOH-canCanrenonall(%)(g / l)TheoryMeasure R11A-00.100.005.415.41 R11A-77.000.184.895.073.580.180.030.03 R11A-2221.802.022.124.1448.752.440.150.12 R11A-2929.003.674.147.8147.034.480.280.23 R11A-3635.706.681.448.1282.277.740.490.45 R11A-4646.207.090.417.5194.488.590.080.04 Fermentation R11B: Canrenon Conversion Concentration (g / l)Conversion rateCalc OH-canSwitching speed (g / l / h) sampletimeOH-canCanrenonall(%)(g / l)TheoryMeasure R11B-00.10.005.605.60 R11B-77.00.204.985.183.780.190.030.03 R11B-2221.82.512.464.9750.492.520.160.16 R11B-2929.04.4816.9921.4720.874.690.300.27 R11B-3635.78.1810.3518.5344.169.700.750.55 R11B-5555.017.0313.2030.2356.3319.500.320.36 R11B-5958.620.8011.7332.5363.9521.970.691.05 R11B-6261.922.198.6230.8172.0224.500.770.42 R11B-7271.726.623.6130.2388.0629.460.510.45 R11B-8181.127.132.0529.1892.9730.320.090.05 R11B-8685.626.872.0228.8893.0230.11-0.04-0.06 R11B-9797.523.951.7125.6693.3430.220.01-0.25 R11B-118117.724.101.6825.7993.4730.260.000.01
[762] Example 15
[763] In general, various cultures were tested for effectiveness in the bioconversion of canrenone to 11α-canrenone according to the method described above.
[764] Aspergillus nigerATCC 11394,Rhizopus arrhizusATCC 11145 andRhizopus stoloniferEach working cell bank in ATCC 6227b was prepared by the method described in Example 5. Growth medium (50 mL) with the composition described in Table 18 was inoculated with a spore suspension (1 mL) from a working cell bank and placed in the incubator. Seed cultures were prepared in the incubator by fermentation at 26 ° C. for about 20 hours. The incubator was stirred at a speed of 200 rpm.
[765] A spawn culture aliquot (2 ml) in each microorganism was used to inoculate the transformed flask containing the growth medium (30 ml) of Table 18. Each culture was used to inoculate two flasks, six in total. Canrenone (200 mg) was dissolved in methanol (4 ml) at 36 ° C. and 0.5 ml aliquots of this solution were placed in each flask. Bioconversion was usually performed under the conditions described in Example 5 with the addition of 50% by weight glucose solution (1 ml) daily. Observation after the first 72 hours showed that the hyphae developed in each transgenic fermentation flask:
[766] ATCC 11394-Good Growth
[767] ATCC 11145-good growth in the first 48 hours, but ball agglomerated mycelia; No growth seen in the last 24 hours;
[768] ATCC 6227b-good growth; Mycelial lumps form agglomerated balls.
[769] Culture samples were taken and analyzed for the degree of bioconversion. After 3 days, the fermentation using ATCC 11394 has a conversion of 11α-hydroxykanrenone at 80-90%; ATCC 11145 has a 50% conversion rate; ATCC 6227b had a conversion of 80-90%.
[770] Example 16
[771] Additional microorganisms were tested for effectiveness in the conversion of canrenone to 11α-hydroxycanrenone substantially using the method described in Example 15. The organisms were tested and the test results are shown in Table 33.
[772] Cultures Tested for Bioconversion of Canrenone to 11α-hydroxycanrenone CultureATTC #Badge ¹ resultApproximate conversion rate Rhizopus oryzae1145CSL+50%- Rhizopus stolonifer6227bCSL+80-90%- Aspergillus nidulans11267CSL+50%80% Aspergillus niger11394CSL+80-90%- Aspergillus ochraceusNRRL 405CSL+ 90% Aspergillus ochraceus18500CSL+ 90% Bacillus subtilis31028P & CSL-0%0% Bacillus subtilis31028CSL-0%0% Bacillus sp.31029P & CSL-0%0% Bacillus sp.31029CSL-0%* Bacillus megaterium14945P & CSL+5%80% * Bacillus megaterium14945CSL+5%10% * Trichothecium roseum12519CSL+80% *90% * Trichothecium roseum8685CSL+80% *90% * Streptomyces fradiae10745CSL+<5%<10% Streptomyces fradiae10745TSB-** Streptomyces lavendulae13664CSL-0%* Streptomyces lavendulae13664TSB-0%0% Nocardiodes simplex6946BP-0%0% Nocardiodes simplex13260BP-** Pseudomonas sp.14696BP-** Pseudomonas sp.14696CSL+<5%<10% Pseudomonas sp.14696TSB-0%* Pseudomonas sp.13261BP+*<10% Pseudomonas cruciviae13262BP #<10% Pseudomonas putida * other unidentified product formation15175BP-0%0% Medium ¹ : CSL-cornsip solution; TSB-Tryptic Soybean Culture; P & CSL-peptone and corn tip solution; BP-beef extract and peptone.
[773] Example 17
[774] Various microorganisms were tested for the effectiveness of the conversion of canrenone to 9α-hydroxycanrenone. Fermentation medium for the operation of this example was prepared as described in Table 34.
[775] Soybean Mill: Dextrose20 g Soybean Wheat5 g NaCl5 g Yeast extract5 g KH ₂ PO ₄ 5 g waterUp to 1L pH7.0 Peptone / Yeast Extract / Glucose: Glucose40 g Bactopeptone10 g Yeast extract5 g waterUp to 1L Müller-Hinton: Beef broth300 g Cassamino17.5 g Starch1.5 g waterUp to 1L
[776] The fungus is grown in soybean wheat medium and peptone yeast extract glucose; Radiation fungi and milk bacteria were grown in soybean wheat (0.9 wt.% Na formate for plus biotransformation) and Mueller-Hinton cultures.
[777] Starting cultures were inoculated with frozen spore storage (20 ml soybean wheat in 250 ml Erlenmeyer flasks). The flask was coated with a milk filter and biohazard shield. Starting cultures (after 24 or 48 hours) were used to inoculate metabolism cultures (20 ml in 250 ml Erlenmeyer flasks) with 10% to 15% crossing volume (volume of inoculation relative to the volume of medium to be inoculated). Were incubated for 24 to 48 hours before adding the steroid substrate for the transformation reaction.
[778] Canrenone was dissolved / suspended in methanol (20 mg / ml), the filter was sterilized and added to the culture at a final concentration of 0.1 mg / ml. All transgenic fermentation flasks were shaken at 250 rpm (2 ″ draw) at 26 ° C. controlled room temperature and 60% humidity.
[779] Biotransformation harvested from 5 to 48 hours, or 24 hours after addition of the substrate. Harvest was initiated by adding ethyl acetate (23 ml) or methylene chloride to the fermentation flask. The flasks were then shaken for 2 minutes and the contents of each flask poured into a 50 ml centrifuge tube. To separate the phases, the tube was centrifuged at 4000 rpm for 20 minutes in a room temperature unit. A device used to transport the organic layer from each tube into a 20 ml borosilicate glass vial and remove the solvent from a speed vac small sample, which typically applies a vacuum to the sample while centrifuging to promote solvent evaporation. Evaporated). The vial was capped and stored at -20 ° C.
[780] To obtain the structural measurement material, biotransformation was scaled up to 500 ml by increasing the number of fermentation shake flasks to 25. At harvest time (24 or 48 hours after substrate addition) ethyl acetate was added to each flask and the flask was capped and returned to the shaker for 20 minutes. The contents of the flask were then poured into polypropylene bottles and centrifuged to separate the phases or poured into a separatory funnel and the phases separated by gravity. The organic phase was dried to give a crude extract of the steroid contained in the reaction mixture.
[781] The reaction product was first analyzed by thin layer chromatography on a plate followed by fluorescent material (254 nm) and silica gel (250 μm). Ethyl acetate (500 μL) was added to each vial containing dry ethyl acetate extracted from the reaction mixture. Further analysis was performed by high performance liquid chromatography and mass spectrometry. Plates were developed in 95: 5 v / v chloroform / methanol medium.
[782] Further analysis was performed by high performance liquid chromatography and mass spectrometry. Watts HPLC with Millennium software, photodiode array detector, and autosampler was used. Reversed phase HPLC used Watts NovaPak C-18 (4 μm particle diameter) RadialPak 4 mm cartridge. The 25 minute linear solvent gradient started with a column starting with water: acetonitrile (75:25) and ending with water: acetonitrile (25:75). This was done with a 3-minute gradient and 100-minute equilibration wash with 100% acetonitrile and the column was regenerated at initial conditions.
[783] For LC / MS, ammonium acetate was added to both acetonitrile and the aqueous phase at a concentration of 2 nM. Chromatography was not significant. The eluate from the column was split into 22: 1 and most of the material was directed to the PDA detector. Residual 4.5% of the material was directed to the electrospray ionization chamber of the Sciex API III mass spectrometer. Mass spectrometry was performed in a positive mode (mass spectrometry more fully described as chemical ionization cation mode). Analog data lines from PDA detectors on HPLC were moved single wavelength chromatograms to mass spectrometer for co-analysis of UV and MS data.
[784] Fractional patterns of mass spectrometry proved useful for sorting among hydroxylated substrates. The two expected hydroxylated canrenones, 11α-hydroxy- and 9α-hydroxy, lose water at different frequencies in a consistent way that can be used for diagnostic purposes. In addition, 9α-hydroxykanrenone formed an ammonium adduct more easily than 11α-hydroxykanrenone. Listed in Table 35 is a summary of TLC, HPLC / UV and LC / MS data for canrenon fermentation and showed that the microorganisms tested were effective for bioconversion of canrenone to 9α-hydroxycanrenone. Among them, the preferred microorganism was Corynespora cassiicola ATCC 16718.
[785] TLC, HPLC / UV, and LC / MS Data Summary for Canrenon Fermentation9αOH-canrenon evidence CultureTLC spot at 9αQH-ADHPLC-peak at 9αOH-canrenone w / UVMS: 357 (M + H), 339 (-H ₂ O) & 375 (+ NH ₄ )Absidia coerulaATCC 6647nyy / nAbsidia glaucaATCC 22752n Actinomucor elegansATCC 6476trytrAsperqillus flavipesATCC 1030tr Asperqillus fumigatus ATCC 26934trynAsperqillus nidulansATCC 11267tryyAsperqillus niqerATCC 16888nyyAsperqillus niqerATCC 26693nynAsperqillus ochraceus ATCC 18500nynBacterium cyclo - oxydans (Searle) ATCC 12673ntrnBeauveria bassianaATCC 7159tryyBeauveria bassianaATCC 13144yyyBotryosphaeria obtusaIMI 038560ytrtrCalonectria decoraATCC 14767ntryChaetomium cochliodesATCC 10195trtry / nComomonas testosteroni(Searle) ATCC 11996trtrnCorynespora cassiicoalATCC 16718yyyCunninghamella blakesleanaATCC 8688ayyyCunninghamella echinulataATCC 3655yyyCunninghamella elegansATCC 9245yyyCurcularia clavataATCC 22921nyy / nCurvularia lunataATCC 12071ynnCylindrocarpon radicicola(Searle) ATCC 11011trnnEpicoccum humucolaATCC 12722yyyEpicoccum oryzaeATCC 12724trtrtrFusarium oxysporumATCC 7601tr Fusarium oxysporum f.sp.cepaeATCC 11171n Gibberella fujikuroiATCC 14842tryyGliocladium deliguescensATCC 10097ytrtrGonqronella butieriATCC 22822yy UV yHypomyces chrysospermus Tul .IMI 109891yyyLipomyces lipoferATCC 10792n Melanospora ornataATCC 26180trnnMortierella isabellinayATCC 42613yynMucor grisco - cyanusATCC 1207an Mucor mucedoATCC 4605tryyMycobacterium fortuitumnATCC 6842 Myrothecium verrucariaATCC 9095trtryNocardia aurentia(Searle) ATCC 12674ntrnNocardia cancicruria(Searle)yynNocardia corallinaATCC 19070n
[786] Paecilomyces carneusATCC 46579nynPenicillium chrysogenumATCC 9480n Penicillium patulumATCC 24550yyy / nPenicillium purpurogenumATCC 46581tryyPithomyces atro - olivaceusATCC 6651trytrPithomyces cynodontisATCC 26150ntrtr Phycomyces blakesleeanusyyy / nPycnosporium sp. ATCC 12231yyy / n Rhizopogon sp. Rhizopus arrhizusATCC 11145trynRhizopus stoloniferATCC 6227bn Rhodococcus equiATCC 14887ntrnRhodococcus equiATCC 21329trtrn Rhodococcus sp.nnnRhodococcus rhodochrousATCC 19150ntrnSaccharopolyspora erythaeaATCC 11635yyySedonium ampullosporumIMI 203033nnnSedonium chrysospermum ATCC 13378n Septomyxa affinisATCC 6737ny UV y / nStachylidium bicolor ATCC 12672yyy / nStreptomyces californicusATCC 15436n Streptomyces cinereocrocatusATCC 3443n Streptomyces coelicolorATCC 10147n Streptomyces flocculusATCC 25453 Streptomyces fradiaeATCC 10745n Streptomyces griseus subsp . griseusATCC 13968n Streptomyces griseusATCC 11984n Streptomyces hydrogenansATCC 19631n Streptomyces hygroscopicusATCC 27438yyyStreptomyces lavendulaePanlab 105n Streptomyces paucisporogenesATCC 25489n Streptomyces purpurascensATCC 25489ntrtrStreptomyces roseochromogenesATCC 13400 Streptomyces spectabilisATCC 27465n Stysanus microsporusATCC 2833 Syncephalastrum racemosumATCC 18192n Thamnidium elegansATCC 18191 Thamnostylum piriformeATCC 8992ytryThielavia terricolanATCC 13807 nTrichoderma virideATCC 26802n Trichothecium roseumATCC 12543tryy / nVerticillium theobromaeATCC 12474ytrtr
[787] Example 18
[788] Various cultures were tested for the effectiveness of the bioconversion of androstenedione to 11α-hydroxyandrostenedione according to the methods described above.
[789] Aspergillus ochraceusNRRL 405 (ATCC 18500);Aspergillus nigerATCC 11394;Aspergillus nidulansATCC 11267;Rhizopus oryzaeATCC 11145;Rhizopus stoloniferATCC 6227b;Trichothecium roseumEach working cell bank of ATCC 12519 and ATCC 8685 was prepared essentially by the method described in Example 4. Growth medium (50 ml) with the composition described in Table 18 was inoculated with spore suspension (1 ml) from the working cell bank and placed in the incubator. Seed cultures were produced in the incubator by fermentation at 26 ° C. for about 20 hours. The incubator was stirred at a speed of 200 rpm.
[790] An aliquot of the spawn culture of each microorganism (2 ml) was used to inoculate the transformed flask containing the growth medium (30 ml) of Table 15. Each culture was used to inoculate two flasks, a total of 16. Androstenedione (300 mg) was dissolved in methanol (6 ml) at 36 ° C. and 0.5 ml aliquots of this solution were placed in each flask. Bioconversion was usually performed under the conditions described in Example 6 for 48 hours. After 48 hours, the samples of the culture were pooled as in Example 17 and extracted with ethyl acetate. Ethyl acetate was concentrated by evaporation and the sample was analyzed by thin layer chromatography to determine whether a product with a chromatographic mobility similar to that of the 11α-hydroxy androstenedione standard (Sigma Chemical Co., St. Louis) was present. The results are shown in Table 36. Positive results are indicated by "+".
[791] Bioconversion of Androstenedione to 11α-hydroxy-Androstenedione CultureATTC #badgeTLC results Rhizopus oryzae11145CSL+ Rhizopus stolonifer6227bCSL+ Aspergillus nidulans11267CSL+ Aspergillus niger11394CSL+ Aspergillus ochraceusNRRL 405CSL+ Aspergillus ochraceus18500CSL+ Trichothecium roseum12519CSL+ Trichothecium roseum8685CSL+
[792] The data in Table 36 demonstrate that each of the listed cultures can produce compounds from androstenedione with the same Rf value as the 11α-hydroxyandrostenedione standard.
[793] Aspergillus ochraceusNRRL 405 (ATCC 18500) was retested in the same manner as above and the culture product was isolated and purified by conventional phase silica gel column chromatography using methanol as solvent. Fractions were analyzed by thin layer chromatography. The TLC plate was Whatman K6F silica gel 60 mm, 10 × 20 size, 250μ thick. The solvent system was methanol: chloroform, 5:95, v / v. Both the crystallized product and the 11α-hydroxyandrostenedione standard were analyzed by LC-MS and NMR spectroscopy. Both compounds obtained similar profiles and molecular weights.
[794] Example 19
[795] Various microorganisms were tested for effectiveness in the conversion of mexrenone to 11β-hydroxymexrenone. Fermentation medium for this example was prepared as described in Table 34.
[796] Fermentation conditions and analysis method were the same as in Example 17. TLC plate and solvent system were as described in Example 18. The ratios for the chromatographic analysis are as follows: 11α-hydroxymexrenone and 11α-hydroxycanrenone had the same chromatographic mobility. 11α-hydroxycanrenone and 9α-hydroxykanrenone showed the same mobility patterns as 11α-hydroxyandrostenedione and 11β-hydroxyandrostenedione. Therefore, 11β-hydroxymexrenone should have the same mobility as 9α-hydroxycanrenone. Therefore, the compound extracted from the growth medium was manipulated for 9α-hydroxykanrenone as a standard. The results are shown in Table 36.
[797] TLC data summary for 11β-hydroxymexrenone formation from mexrenone microbeBadge ¹ Spot characteristics ² Absidia coerulaATCC 6647M, SRiverAspergillus niger ATCC 16888S, PAbout (S) (P)Beauveria bassianaATCC 7159PRiverBeauveria bassianaATCC 13144S, P , Botryosphaeria obtusaIMI 038560 aboutCunninghamella blakesleeanaATCC 8688a echinulataATCC 3655 elegansATCC 9245S, PS, PS, PRiverCurvularia lunataATCC 12017SRiverGongronella butleriATCC 22822S, PRiverPenicillium patulumATCC 24550S, PRiverPenicillium purpurogenumATCC 46581S, PRiverPithomyces atro - olivaceusIFO 6651S, PaboutRhodococcus equiATCC 14887MaboutSaccharopolyspora erythaeaATCC 11635M, SFaboutStreptomyces hygroscopicusATCC 27438M, SFRiverStreptomyces purpurascens ATCC 25489M, SFaboutThamnidium elegansATCC 18191S, PaboutThamnostylum piriformeATCC 8992S, PaboutTrichothecium roseumATCC 12543P, SAbout (P) (S)¹ M = Müller-Hinton P = PYG (Peptone / Yeast Extract / Glucose) S = Soybean Wheat SF = Soybean Wheat Plus Formate ² = Probable Controls and Differences in Problem
[798] These data suggest that most organisms listed in this table produce products similar or identical to 11β-hydroxymexrenone from mexrenone.
[799] Example 20
[800] Scheme 1: Step 1: 5'R (5'α), 7'β-20'-aminohexadecahydro-11'β-hydroxy-10'a, 13'α-dimethyl-3 ', 5-di Preparation of Oxospyro [furan-2 (3H), 17'α (5'H)-[7,4] metheno [4H [cyclopenta [a] phenanthrene] -5'-carbonitrile.
[801] Into a 50 gallon glass line reactor was added 61.2 L (57.8 kg) of DMF followed by 23.5 kg of 11-hydroxycanrenone 1 with stirring. 7.1 kg of lithium chloride was added to the mixture. The mixture was stirred for 20 minutes, 16.9 kg of acetone cyanohydrin and 5.1 kg of triethylamine were added thereto. The mixture was heated to 85 ° C. and kept at this temperature for 13-18 hours. After the reaction, 353 L of water was added, followed by 5.6 kg of sodium bicarbonate. The mixture was cooled to 0 ° C. and 130 kg of 6.7% sodium hypochlorite solution was slowly quenched and transferred to a 200 gallon glass line reactor. The product was filtered off and washed with 3 × 40 L of some water to give 21.4 kg of product enamine.
[802]
[803] Example 21
[804] Scheme 1: Step 2: 4'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β-dimethyl-3 ', 5,20'-trioxospyro [ Preparation of furan-2 (3H), 17'β- [4,7] methano [17H] cyclopenta [a] phenanthrene] -5'β (2'H) -carbonitrile.
[805] 50 kg of Enamine 2 , about 445 L of 0.8 N diluted hydrochloric acid and 75 L of methanol were placed in a 200 gallon glass line reactor. The mixture was heated to 80 ° C. for 5 hours and cooled to 0 ° C. for 2 hours. The solid product was filtered to give 36.5 kg of dry product diketone.
[806]
[807] Example 22
[808] Scheme 1: Step 3A: Preparation of methyl hydrogen 11α, 17α-dihydroxy-3-oxopregin-4-ene-7α-21-dicarboxylate, γ-lactone
[809] The four-necked 5L bottom flask was equipped with a mechanical stirrer, an isostatic addition funnel with a nitrogen inlet tube, a thermometer and a chiller with a bubbler. The bubbler was connected to two 2 L traps via Tygon tubing, the first empty, and placed in the reaction vessel to prevent back-suction of material (1 L concentrated sodium hypochlorite solution) in the second trap. Diketone 3 (79.50 g; [uncorrected weight for purity, 85%]) was added to a flask containing 3 L methanol. 25% methanolic sodium methoxide solution (64.83 g) was placed on the funnel and added dropwise with stirring under nitrogen over 10 minutes. After the addition was complete, the orange reaction mixture was heated to reflux for 20 hours. Thereafter, 167 mL of 4N HCl was added dropwise through the addition funnel into the distillation (note: HCN evaporated at this time) and the reaction mixture was refluxed. The reaction mixture became pale golden orange in color. The cooler was then replaced with a take off head and 1.5 L of methanol was removed by distillation while at the same time 1.5 L of water was added through the funnel according to the rate of distillation. The reaction mixture was cooled to ambient temperature and extracted twice with 2.25 L aliquots of methylene chloride. The combined extracts were washed successively with 750 mL aliquots of cold NaCl solution, 1N NaOH, and again with saturated NaCl. The organic layer was dried over sodium sulfate overnight, filtered and reduced to ˜250 mL under vacuum. Toluene (300 mL) was added and the remaining methylene chloride was stripped under reduced pressure during which time the product began to form on the wall of the flask as a white solid. The flask was cooled overnight and the solids were removed by filtration. Wash with 250 mL of toluene, wash twice with 250 mL aliquots of ether and dry on a vacuum funnel to give 58.49 g of a white solid with a purity of 97.3% by HPLC. When the mother liquor was concentrated, an additional 6.76 g of 77.1% purity product was obtained. The overall yield adjusted for purity was 78%.
[810] Example 23
[811] Scheme 1: step 3B: methyl hydrogen 11α, 17α-dihydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone methyl hydrogen 17α-hydroxy-11α- (methyl Sulfonyl) oxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, conversion to γ-lactone.
[812] The trapping system was mounted as in the example above, except that the trapping system was installed under the bubbler. A large amount of 138.70 g of hydroxyester was added to the flask, followed by 1425 mL of methylene chloride while stirring under nitrogen. The reaction mixture was cooled to -5 ° C using salt / ice bath. Methanesulfonyl chloride (51.15 g, 0.447 mol) was added quickly, followed by the dropwise addition of triethylamine (54.37 g) in 225 mL of methylene chloride. The addition which took -30 minutes was adjusted so that reaction temperature might not become about 5 degreeC. After the addition, stirring was continued for 1 hour, and the reaction contents were transferred to a 12 L separatory funnel, to which 2100 mL of methylene chloride was added. The solution was washed successively with 700 mL aliquots of cold 1N HCl, 1N NaOH, and saturated aqueous NaCl solution, respectively. The aqueous washes were combined and extracted again with 3500 mL of methylene chloride. All organic washes were combined into a 9 L jug, to which 500 g neutral alumina, activity grade II and 500 g anhydrous sodium sulfate were added. The contents of the jug were thoroughly mixed for 30 minutes and filtered. The filtrate was dried under vacuum to give a sticky yellow bubble. This was dissolved in 350 mL of methylene chloride and added dropwise with stirring 1800 mL of ether. The rate of addition was adjusted so that the ether was about half over 30 minutes. After adding about 750 mL, the product began to separate as a crystalline solid. Residual ether was added within 10 minutes. The solid was removed by filtration and the filter cake was washed with 2 L of ether and dried overnight in a vacuum oven at 50 ° C. to give 144.61 g (88%) of an almost white solid, m.p. 149 ° C-150 ° C. Materials prepared in this way are typically 98-99% pure by HPLC (area%). In one operation, the material obtained had a melting point of 153 ° C.-153.5 ° C. and a purity of 99.5% as measured by HPLC area.
[813] Example 24
[814] Scheme 1: Step 3C: Method A: Preparation of methyl hydrogen 17α-hydroxy-3-oxopregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone.
[815] The 1 L four neck flask was mounted as in the second example. Formic acid (250 mL) and acetic anhydride (62 mL) were added to the flask with stirring under nitrogen. Potassium formate (6.17 g) was added and the reaction mixture was heated in an oil bath to an internal temperature of 40 ° C. for 16 hours (which is repeated later to get better results). After 16 hours, mesylate 5 was added and the internal temperature increased to 100 ° C. Heating and stirring were continued for 2 hours, after which the solvent was removed in vacuo on a rotary evaporator. The residue was stirred with 500 mL of ice water for 15 minutes, and then extracted twice with 500 mL portions of ethyl acetate. The organic phases were combined and washed successively with 250 mL aliquots of cold saturated sodium chloride solution (twice), 1N sodium hydroxide solution, and again saturated sodium chloride. The organic phase was then dried over sodium sulphate, filtered and dried in vacuo to yield an yellowish white bubble which was triturated with glass by tapping with a spatula. 14.65 g of the powder thus formed were analyzed with a mixture of 82.1% 6 7.4% 8 and 5.7% 9 (HPLC area%).
[816] Example 25
[817] Scheme 1: Step 3C: Method B: Preparation of methyl hydrogen 17α-hydroxy-3-oxopregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone.
[818] A 5 L four-necked flask was mounted as in the example above, and 228.26 g of acetic acid and 41.37 g of sodium acetate were added with stirring under nitrogen. The mixture was heated to an internal temperature of 100 ° C. using an oil bath. Mesylate (123.65 g) was added and heating continued for 30 minutes. At the end of this time, heating was stopped and 200 mL of ice water was added. The temperature was lowered to 40 ° C. and stirring was continued for 1 hour, after which the reaction mixture was slowly poured into 1.5 L of ice water in a 5 L stirred flask. The product was separated as sticky oil. The oil was dissolved in 1 L ethyl acetate and washed with 1 L of cold saturated sodium chloride solution, 1 N sodium hydroxide and finally saturated sodium chloride. The organic phase was dried over sodium sulphate and filtered. The filtrate was dried under vacuum to obtain a bubble collapsed into sticky oil. It was triturated with ether for some time and finally solidified. The solid was filtered and washed with more ether to give 79.59 g of an off white solid. This ^{consisted of the} desired Δ ^9,11 ester 6 70.4%, Δ ^11,12 ester 8 12.3%, 7α, 9-α-lactone 9 10.8% and unreacted 5 5.7%.
[819] Example 26
[820] Scheme 1: step 3D: synthesis of methyl hydrogen 9,11α-epoxy-17α-hydroxy-3-oxopregin-4-ene-7α, 21-dicarboxylate, γ-lactone.
[821] The four-neck jacketed 500 mL reactor was equipped with a mechanical stirrer, cooler / bubble, thermometer and addition funnel with nitrogen inlet tube. Into the reactor, 8.32 g of crude ester in 83 mL of methylene chloride was added while stirring under nitrogen. To this was added 4.02 g of potassium phosphate dibasic, followed by 12 mL of trichloroacetonitrile. External cooling water was passed through the reactor jacket and the reaction mixture was cooled to 8 ° C. 36 mL of 30% hydrogen peroxide was added to the addition funnel over 10 minutes. The initial pale yellow reaction mixture turned almost colorless after the addition was complete. The reaction mixture was added overnight and kept stirring (total 23 hours) and kept at 9 ± 1 ° C. Methylene chloride (150 mL) was added to the reaction mixture and the entire contents were added to ˜250 mL ice water. This was extracted three times with 150 mL aliquots of methylene chloride. The combined methylene chloride extracts were washed with 400 mL of cold 3% sodium sulfite solution to decompose the residual peroxide. This was washed with 330 mL of cold 1N sodium hydroxide, 400 mL of cold 1N hydrochloric acid, and finally 400 mL of brine. The organic phase was dried over sodium sulfate, filtered and the filter cake washed with 80 mL of methylene chloride. The solvent was removed in vacuo to yield 9.10 g of crude product as a pale yellow solid. This was recrystallized from 2-butanone-25 mL to obtain 5.52 g of almost white crystals. Finally, recrystallization from acetone (-50 mL) gave 3.16 g as long needle crystals, mp 241-243 ° C.
[822] Example 27
[823] Scheme 1: Step 3: Option 1: 4'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β-dimethyl-3 ', 5,20'-tree Oxosity [Furan-2 (3H), 17'β- [4,7] methano [17H] cyclopenta [a] phenanthrene] -5'β (2'H) -carbonitrile to methyl hydrogen 9,11α -Epoxy-17α-hydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.
[824] The diketone (20 g) was placed in a clean dried reactor and then 820 ml of MeOH and 17.6 ml of 25% NaOMe / MeOH solution were added. The reaction mixture was heated to reflux (˜67 ° C.) for 16-20 hours. The product was quenched with 40 mL 4N HCl. The solvent was removed at atmospheric pressure by distillation. 100 mL of toluene was added and residual methanol was removed by azeotropic distillation with toluene. After concentration, crude hydroxyester 4 was dissolved in 206 mL of methylene chloride and cooled to 0 ° C. Methanesulfonyl chloride (5 mL) was added followed by the slow addition of 10.8 ml of triethylamine. The product was stirred for 45 minutes. The solvent was removed by vacuum distillation to give crude mesylate 5 .
[825] To the separately dried reactor, 5.93 g of potassium formate, 240 mL of formic acid and then 118 mL of acetic anhydride were added. The mixture was heated to 70 ° C. for 4 hours.
[826] The formic acid mixture was added to the concentrated mesylate solution 5 prepared above. The mixture was heated to 95-105 ° C for 2 hours. The resulting mixture was cooled to 50 ° C and the volatile components were removed by vacuum distillation at 50 ° C. The product was partitioned between 275 mL of ethyl acetate and 275 mL of water. The aqueous layer was back extracted with 137 mL of ethyl acetate and washed with 240 ml of cold 1N sodium hydroxide solution and 120 ml of saturated NaCl. After phase separation, the organic layer was concentrated under vacuum distillation to afford crude ester.
[827] The product was dissolved in 180 mL of methylene chloride and cooled to 0-15 ° C. 8.68 g of dipotassium hydrogen phosphate was added followed by 2.9 mL of trichloroacetonitrile. 78 mL of 30% hydrogen peroxide solution was added to the mixture over 3 minutes. The reaction mixture was stirred at 0-15 ° C. for 6-24 hours. After the reaction, the biphasic mixture was separated. The organic layer was washed with 126 mL of 3% sodium sulfite solution, 126 mL of 0.5N sodium hydroxide solution, 126 mL of 1N hydrochloric acid and 126 mL of 10% brine. The product was dried over anhydrous sodium sulfate or filtered through celite and the solvent methylene chloride was distilled off at atmospheric pressure. The product was crystallized twice in methyl ethyl ketone to give 7.2 g of eplerenone.
[828]
[829] Example 28
[830] Scheme 1: Step 3: Option 2: 1'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β-dimethyl-3 ', 5,20'-tree Methyl hydrogen 9,11α of oxospyro [furan-2 (3H), 17'β- [4,7] methano [17H] cyclopenta [a] phenanthrene] -5'β (2'H) -carbonitrile Epoxy-17α-hydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone without conversion to intermediate.
[831] The four-necked 5L round bottom flask was equipped with a mechanical stirrer, an addition funnel with a nitrogen inlet tube, a thermometer, and a chiller with a bubbler attached to a sodium hypochlorite scrubber. Diketone (83.20 g) was added to a flask containing 3.05 L methanol. In the addition funnel was placed 67.85 g of a 25% (w: w) solution of sodium methoxide in methanol. While stirring under nitrogen, the methoxide was added dropwise to the flask over 15 minutes. Dark orange / yellow slurry was developed. The reaction mixture was heated to reflux for 20 hours and 175 mL of 4N hydrochloric acid was added dropwise while refluxing was continued. (Note, HCN evaporation during this operation.) The reflux condenser was replaced with a takeoff head and 1.6 L of methanol was removed by distillation while 1.6 L of 10% aqueous sodium chloride solution was added dropwise through a funnel at a rate satisfying the distillation rate. The reaction mixture was cooled to ambient temperature and extracted twice with 2.25 L aliquots of methylene chloride. The combined extracts were washed with cold 1N sodium hydroxide aliquots and 750 mL saturated sodium chloride solution. The organic layer was dried to a final volume of 1 L (0.5% of the total was removed for analysis) by azeotropic distillation of methanol at 1 atmosphere.
[832] The concentrated organic solution (hydroxyester) was added again to the usual reaction flask equipped as above, but there was no HCN trap. The flask was cooled to 0 ° C. and 30.7 g of methanesulfonyl chloride was added with stirring under nitrogen. 32.65 g of triethylamine was added to the addition funnel, which was added dropwise over 15 minutes while maintaining the temperature at 5 ° C. Stirring was continued for 2 hours while the reaction mixture was allowed to warm to ambient temperature. A column consisting of 250 g Dowex 50W × 8-100 acidic ion exchange resin was prepared and washed with 250 mL of water, 250 mL of methanol and 500 mL of methylene chloride before use. The reaction mixture was run on this column and collected. A new column was prepared and the process repeated. A third 250 g column consisting of Dowex 1 × 8-200 basic ion exchange resins was prepared and pretreated as in the acidic resin treatment described above. The reaction mixture was run on this column and collected. A fourth column of basic resin was prepared and the reaction mixture was again run on the column and collected. After passing through each column, the column was washed twice with 250 mL of methylene chloride and each took ~ 10 minutes to pass through. The solvent washes were combined with the reaction mixture and the volume was reduced to ˜500 mL in vacuo, of which 2% was removed for qc. The residue was further reduced to 150 mL final volume (crude mesylate solution).
[833] In a conventional 5 L reactor, 960 mL of formic acid, 472 mL of acetic anhydride and 23.70 g of potassium formate were charged. The mixture was heated with stirring at 70 ° C. under nitrogen for 16 h. The temperature was then increased to 100 ° C. and crude mesylate solution was added over 30 minutes through an addition funnel. The temperature was lowered to 85 ° C. and methylene chloride was distilled off from the reaction mixture. After all was removed, the temperature was increased back to 100 ° C. and held there for 2.5 hours. The reaction mixture was cooled to 40 ° C. and formic acid was removed under pressure to reach minimum stirring volume (˜150 mL). The residue was cooled to ambient temperature and 375 mL of methylene chloride was added. The diluted residue was washed with a cold 1 L portion of saturated sodium chloride solution, 1N sodium carbonate, and sodium chloride solution. The organic phase was dried over magnesium sulfate (150 g) and filtered to give a dark reddish brown solution (crude ester solution).
[834] The four-neck jacketed 1 L reactor was equipped with a mechanical stirrer, cooler / bubble, thermometer and addition funnel with nitrogen inlet tube. To the reactor was added a crude ester solution (measured at 60 g) in 600 mL of methylene chloride while stirring under nitrogen. To this was added 24.0 g of potassium phosphate dibasic followed by 87 mL of trichloroacetonitrile. External cooling water was passed through the reactor jacket and the reaction mixture was cooled to 10 ° C. 147 mL 30% hydrogen peroxide was added to the mixture over 30 minutes via an addition funnel. The initial dark reddish brown reaction mixture turned pale yellow after the addition was complete. The reaction mixture was added and stirred overnight to maintain at 10 ± 1 ° C. (total 23 hours). The phases were separated and the aqueous portion was extracted twice with 120 mL portions of methylene chloride. The combined organic phases were washed with 210 mL 3% sodium sulfite and added. This was repeated twice, after which both the organic and aqueous portions were negative for peroxide with starch / iodide test paper. The organic phase is washed successively with 210 mL aliquots of cold 1N sodium hydroxide, 1N hydrochloric acid and finally washed twice with brine. The organic phase was azeotropically dried at a volume of ˜100 mL, fresh solvent (250 mL) was added and azeotropically distilled to 100 mL in the same manner and the residual solvent was removed in vacuo to yield 57.05 g of crude product as a sticky yellow bubble. The portion (51.01 g) was further dried to a constant weight of 44.3 g and quantified by HPLC. Evaluated at 27.1% EPX.
[835] Example 29
[836] 11α-hydroxyandrostenedione (429.5 g) and toluene sulfonic acid hydrate (7.1) were placed in a reaction flask under nitrogen. Ethanol (2.58 L) was added to the reactor and the resulting solution was cooled to 5 ° C. Triethylorthoformate (334.5 g) was added to the solution over 15 minutes at 0 ° C to 15 ° C. After completion of the triethylorthoformate addition the reaction mixture was warmed to 40 ° C. and reacted at this temperature for 2 hours, after which the temperature was increased to reflux and the reaction continued under reflux for an additional 3 hours. The reaction mixture was cooled in vacuo and the solvent removed in vacuo to afford 3-ethoxyandrostar-3,5-diene-17-one.
[837] Example 30
[838] 11α- From hydroxycanrenone Enamine formation
[839]
[840] Sodium cyanide (1.72 g) was placed in a 25 mL three neck flask equipped with a mechanical stirrer. Water (2.1 mL) was added and the mixture was stirred while heating to dissolve the solid. Dimethylformamide (15 mL) was added followed by 11α-hydroxykanrenone (5.0 g). A mixture of water (0.4 mL) and sulfuric acid (1.49 g) was added to the mixture. The mixture was heated to 85 ° C. for 2.5 h at which time HPLC analysis indicated the conversion to product was complete. The reaction mixture was cooled to room temperature. Sulfuric acid (0.83 g) was added and the mixture was stirred for 1 hour and a half. The reaction mixture was added to 60 mL of water and cooled in an ice bath. The flask was washed with 3 mL DMF and 5 mL water. The slurry was stirred for 40 minutes and filtered. The filter cake was washed twice with 40 mL of water and dried in a vacuum oven at 60 ° C. overnight to give 11α-hydroxy enamine, 5′R (5′α), 7′β-20′-aminohexadecahydro-11 ' β-hydroxy-10'α, 13'α-dimethyl-3 ', 5-dioxospyro [furan-2 (3H), 17'α (5'H)-[7,4] metheno [4H] Cyclopenta [a] phenanthrene] -5'-carbonitrile (4.9 g) was obtained.
[841] Example 31
[842] 11α- Of hydroxycanrenone To diketone transform
[843]
[844] Sodium cyanide (1.03 g) was placed in a 50 mL three neck flask equipped with a mechanical stirrer. Water (1.26 mL) was added and the flask heated slightly to dissolve the solids. Dimethylacetamide [or dimethylformamide] (9 mL) was added followed by 11α-hydroxycanrenone (3.0 g). A mixture of water (0.25 mL) and sulfuric acid (0.47 mL) was added to the reaction flask with stirring. The mixture was heated to 95 ° C. for 2 hours. HPLC analysis indicated the reaction was complete. Sulfuric acid (0.27 mL) was added and the mixture was stirred for 30 minutes. Additional water (25 mL) and sulfuric acid (0.90 mL) were added and the reaction mixture was stirred for 16 h. The mixture was then cooled to 5-10 ° C. in an ice bath. The solid was separated by filtration through a sintered glass filter and then washed twice with water (20 mL). Solid diketone, ie 4'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β-dimethyl-3 ', 5,20-trioxospyro [furan- 2 (3H), 17'β- [4,7] methano [17H] cyclopenta [a] phenanthrene] -5'β (2'H) -carbonitrile was dried in a vacuum oven to give 3.0 g of solid. .
[845] Example 32
[846] A suspension of 5.0 g of diketone prepared by the method described in Example 31 in methanol (100 mL) was heated to reflux and a 25% solution of potassium methoxide in methanol (5.8 mL) was added over 1 minute. The mixture was homogeneous. After 15 minutes a precipitate formed. The mixture was heated to reflux and again homogeneous after about 4 hours. Heat reflux was continued for a total of 23.5 hours and 4.0N HCl (10 mL) was added. The entire 60 mL solution of hydrogen cyanide in methanol was removed by distillation. Water (57 mL) was added to the distillation residue over 15 minutes. The solution temperature was increased to 81.5 ° during water addition and an additional 4 mL of hydrogen cyanide / methanol solution was distilled off. After completion of the water addition, the mixture became cloudy and the heat source was removed. The mixture was stirred for 3.5 hours and the product slowly crystallized. The suspension was filtered and the collected solid was washed with water, dried with a funnel of air flow and dried at 92 ° (26 in. Hg) for 16 hours to give 2.98 g of an off-white solid. The solid was a hydroxyester, i.e., methyl hydrogen 11α, 17α-dihydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, 91.4 wt% of γ-lactone. The yield was 56.1%.
[847] Example 33
[848] The diketone prepared by the method described in Example 31 was placed in a three-necked reaction flask equipped with a thermometer, a Deanstark trap and a mechanical stirrer, which was cleanly dried. Methanol (24 mL) was added to the reactor at room temperature (22 ° C.) and the resulting slurry was stirred for 5 minutes. 25% by weight of a solution of sodium methoxide in methanol (52.8 mL) was placed in the reactor and the mixture was stirred at room temperature for 10 minutes during which time the reaction mixture turned into a light brown clear solution with slight exotherm (2-3 ° C.). . The rate of addition was controlled to prevent the pot temperature from exceeding 30 ° C. The mixture was then heated to reflux (about 67 ° C.) and continued at reflux for 16 h. Samples were then taken and the conversion analyzed by HPLC. The reaction was continued under reflux and the residual ketone was less than 3% of the diketone charge. During reflux, 4N HCl (120 mL) was placed in the reaction port and HCN was generated and quenched with a scrubber.
[849] After completion of the reaction, 90-95% methanol solvent was distilled off in the reaction mixture under atmospheric pressure. The head temperature during distillation was changed to 67-75 ° C. and treated with caustic bleach before treating the distillate containing HCN. After removing the methanol the reaction mixture was cooled to room temperature and the solid product began to precipitate as a mixture cooled in the range of 40-45 ° C. An aqueous solution optionally containing 5% by weight sodium bicarbonate (1200 mL) at 25 ° C. was placed in a cooling slurry and the resulting mixture was cooled to 0 ° C. in about 1 hour. Sodium bicarbonate treatment is effective to remove residual unreacted diketone from the reaction mixture. The slurry was stirred at 0 ° C. for 2 hours to complete precipitation and crystallization, after which the solid product was recovered by filtration and the filter cake was washed with water (100 mL). The product was dried to constant weight at 80-90 ° C. under 26 ″ mercury vacuum. The water content after drying was less than 0.25% by weight. The adjusted molar yield was about 77-80% by weight.
[850] Example 34
[851] The diketone (1 equiv) prepared according to Example 31 was reacted with sodium methoxide (4.8 equiv) in methanol solvent in the presence of zinc iodide (1 equiv). The reaction product can be manipulated according to any of the extraction or non-extraction processes described herein, and the methylene chloride extract, brine and caustic wash and sodium sulfate are removed in a drying step. Also in the non-extraction process, toluene was replaced with 5% by weight sodium bicarbonate solution.
[852] Example 35
[853] The hydroxyester (1.97 g) prepared in Example 34 was combined with tetrahydrofuran (20 mL) and the resulting mixture was cooled to -70 ° C. Sulfuryl chloride (0.8 mL) was added and the mixture was stirred for 30 minutes, after which imidazole (1.3 g) was added. The reaction mixture was allowed to warm to room temperature and stirred for an additional 2 hours. The mixture was then diluted with methylene chloride and extracted with water. The organic layer was concentrated to give crude ester (1.97 g). Small amounts of samples in the crude product were analyzed by HPLC. The analysis indicated that the ratio of 9,11-olefin: 11,12-olefin: 7,9-lactone was 75.5: 7.2: 17.3. If not different from the above, the reaction yielded a product having a 9,11-olefin: 11,12-olefin: 7,9-lactone distribution of 77.6: 6.7: 15.7. This method combines the introduction and removal of leaving groups in one step for the introduction of the 9,11-olefin structure of the esters, i.e., the reaction is carried out in which sulfuryl chloride is reacted by the halide of the 11α-hydroxy group of the hydroxy ester of Subsequent to substitution, the dehalogenated hydrogen gave a structure of Δ-9,11. Therefore, the formation of the ester is carried out without using a desiccant such as acetic anhydride or a strong acid (such as formic acid). It is also removed by reflux in other processes that produce carbon monoxide.
[854] Example 36
[855] The hydroxyester (20 g) and methylene chloride (400 mL) prepared in Example 34 were placed in a three-necked round bottom flask equipped with a mechanical stirrer, an addition funnel and a thermocouple, which was cleanly dried. The resulting mixture was stirred at ambient temperature to give a complete solution. The solution was cooled to 5 ° C. using an ice bath. Methanesulfonyl chloride (5 mL) was added to a hydroxyester containing CH ₂ Cl ₂ solution followed by rapid dropwise addition of triethylamine (10.8 mL). By adjusting the addition rate, the reaction temperature did not exceed 5 ° C. The reaction was very exothermic and required cooling. The reaction mixture was stirred at about 5 ° C. for 1 hour. At the end of the reaction (HPLC and TLC analysis), the mixture was concentrated at about 0 ° C. under 26 in Hg vacuum to give a thick slurry. The resulting slurry was diluted with CH ₂ Cl ₂ (160 mL) and the mixture was concentrated at about 0 ° C. under 26 in Hg vacuum to give a concentrate. Concentrate (the mesylate product of formula IV (R ³ = H, -AA- and -BB- are both -CH ₂ -CH _2- ), ie methyl hydrogen 11α, 17α-dihydroxy-3-oxopregne 4-ene-7α, 21-dicarboxylate, γ-lactone to methyl hydrogen 17α-hydroxy-11α- (methylsulfonyl) oxy-3-oxopregan-4-ene-7α, 21-dicar It was found that the purity of the carboxylate, γ-lactone) was 82% (HPLC area%). This material was used for the next reaction without separation.
[856] Potassium formate (4.7 g), formic acid (16 mL) and acetic anhydride (8 mL, 0.084 mol) were placed in a reactor equipped with a mechanical stirrer, a cooler, a thermocouple and a heating mantle cleanly dried. The resulting solution was heated to 70 ° C. and stirred for about 4-8 hours. The addition of acetic anhydride is exothermic and generates gas (CO), so the rate of addition was adjusted to control both temperature and gas evolution (pressure). The reaction time for preparing the deactivator was dependent on the amount of water present in the reactants (the formic acid and potassium formate each contained about 3-5% water). The removal reaction is sensitive to the amount of water present; If> 0.1% water (KF), the level of 7,9-lactone impurities can be increased. This by-product is difficult to remove from the final product. If KF showed <0.1% water, the deactivator was transferred to a concentrate of mesylate (0.070 mol) prepared in the previous step. The resulting solution was heated to 95 ° C. and the volatiles were distilled off and collected in a Dean Stark trap. When volatile evaporation was stopped, the Dean Stark trap was replaced with a cooler and the reaction mixture was heated at 95 ° C. for an additional hour. At the end (TLC and HPLC analysis; <0.1% starting material), the contents were cooled to 50 ° C. and vacuum distillation (26 in Hg / 50 ° C.) was started. The mixture was concentrated to a concentrated slurry and then cooled to ambient temperature. The resulting slurry was diluted with ethyl acetate (137 mL), the solution was stirred for 15 minutes and diluted with water (137 mL). The layers were separated and the lower aqueous layer was extracted again with ethyl acetate (70 mL). The combined ethyl acetate solution was washed once with brine solution (120 mL) and twice with ice cold 1N NaOH solution (120 mL each). The pH of the aqueous solution was measured and the organic layer was washed again if the pH of the wash solution used was <8. When the pH of the used washing solution was observed to be> 8, the ethyl acetate layer was washed once with brine solution (120 mL) and concentrated to dryness by rotary evaporation using a 50 ° C. water bath. 92 g (77% molar yield) of the obtained ester, solid product, ie, methyl hydrogen 17α-hydroxy-3-oxopregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone Weighed by.
[857] Example 37
[858] The hydroxyester (100 g; 0.22 mol) prepared in Example 34 was placed in a 2 L three neck round bottom flask equipped with a mechanical stirrer, addition funnel and thermocouple. A circulating cooling bath was used for thermostatic control. Since methanesulfonyl chloride is sensitive to water, the flask was dried prior to use in the reaction.
[859] Methylene chloride (1 L) was placed in a flask and hydroxyester was dissolved therein under stirring. The solution was cooled to 0 ° C. and methanesulfonyl chloride (25 mL; 0.32 mol) was added to the flask through an addition funnel. Triethylamine (50 mL; 0.59 mol) was added to the reactor through an addition funnel and the funnel was washed with additional methylene chloride (34 mL). The addition of triethylamine was very exothermic. The addition time was about 10 minutes under stirring and cooling. The fill mixture was cooled to 0 ° C. and maintained at this temperature under stirring for an additional 45 minutes during which the head space of the reaction flask was filled with nitrogen. A sample of the reaction mixture was then analyzed by thin layer chromatography and the reaction was checked by high performance liquid chromatography. The mixture was stirred for additional 30 min at 0 ° C. and the reaction was checked again. Analysis indicated that the reaction was substantially complete at this point; Solvent methylene chloride was stripped at 0 ° C. under 26 ″ mercury vacuum. Gas chromatographic analysis of the distillate showed the presence of both methanesulfonyl chloride and triethylamine. Methylene chloride (800 mL) was added to the reactor and the resulting mixture was stirred for 5 minutes at a temperature in the range of 0-15 ° C. The solvent was again stripped at 0-5 ° C. under 26 ″ mercury vacuum, where R ³ is H, -AA- and -BB- are -CH ₂ -CH ₂ -and R ¹ is methoxycarbonyl The rate was obtained. The purity of the product was about 90-95 area%.
[860] To prepare the removal reagent, potassium formate (23.5 g; 0.28 mol), formic acid (80 mL) and acetic anhydride (40 mL) were mixed in separate dried reactors. Formic acid and acetic anhydride were placed in the reactor and the temperature was kept below 40 ° C. during the addition of acetic anhydride. The reagent mixture was heated to 70 ° C. to drain water from the reaction system. This reaction was continued until the water content was less than 0.3% by weight as measured by Karl Fischer analysis. The removal reagent solution was then placed in a reactor containing the crude mesylate concentrated solution prepared as described above. The resulting mixture was heated to a maximum temperature of 95 ° C. and volatile distillates were collected until no more distillate formed. Distillation was stopped at about 90 ° C. After completion of the distillation, the reaction mixture was stirred for additional 2 hours at 95 ° C. and the reaction was checked by thin layer chromatography. At the end of the reaction, the reactor was cooled to 50 ° C. and formic acid and solvent were removed from the reaction mixture at 50 ° C. under 26 ″ mercury vacuum. The concentrate was cooled to room temperature, ethyl acetate (688 mL) was introduced and the mixture of ethyl acetate and concentrate was stirred for 15 minutes. At this point, 12% saline solution (688 mL) was introduced to remove water soluble impurities from the organic phase. The phases were then left to stand for 20 minutes. The aqueous layer was transferred to another vessel filled with an additional amount of ethyl acetate (350 mL). This back extraction of the aqueous layer was carried out for 30 minutes, after which the phases were allowed to stand and the ethyl acetate layers combined. Saturated sodium chloride solution (600mL) was added to the combined ethyl acetate layers and stirred for 30 minutes. The organic phase was separated from the second used wash. The organic phase was then washed with 1N sodium hydroxide (600 mL) under stirring for 30 minutes. The phases were left for 30 minutes to remove the aqueous layer. The pH of the aqueous layer was checked and found to be> 7. Further washes were performed with saturated sodium chloride (600 mL) for 15 minutes. Finally the organic phase was concentrated at 50 ° C. under 26 ″ mercury vacuum and the product was recovered by filtration. The final product was a bubbling brown solid when dried. Further drying at 45 ° C. under reduced pressure for 24 hours gave 95.4 g of ester product, which was estimated at 68.8%. The molar yield was 74.4% corrected for both starting hydroxyester and final ester.
[861] Example 38
[862] The complex washing step was repeated in Example 37 except that the reaction solution was omitted by treatment with an ion exchange resin. Basic alumina or basic silica. The conditions for treating with basic silica are shown in Table 38. Each of these treatments was found to be effective in removing impurities without the combined wash of Example 44.
[863] factorSet pointExperimental purposeKey result Basic alumina2g / 125g productTreatment of the reaction mixture with basic alumina removes Et ₃ N.HCl salt and 1N NaOH and 1N HCl wash solutionYield 93% Basic silica2g / 125g productThe reaction mixture was treated with lower cost basic silica to remove Et ₃ N.HCl salts and 1N NaOH and 1N HCl washesYield 95%
[864] Example 39
[865] Potassium acetate (4 g) and trifluoroacetic acid (42.5 mL) were mixed in a 100 mL reactor. Trifluoroacetic anhydride (9.5 mL) was added to the mixture at a controlled rate to maintain a temperature below 30 ° C. during the addition. The solution was then heated at 30 ° C. for 30 minutes to provide a removal reagent useful for converting the mesylate of formula IV to the ester of formula II.
[866] A preformed TFA / TFA anhydride removal reagent was added to the previously prepared mesylate solution of formula IV. The resulting mixture was heated at 40 ° C. for 4½ hours and the conversion was periodically checked by TLC or HPLC. At the end of the reaction, the mixture was placed in a 1-neck flask and concentrated to dryness at room temperature (22 ° C) under reduced pressure. Ethyl acetate (137 mL) was added to the mixture to completely dissolve the solid substance, and then the water / brine mixture (137 mL) was added and the resulting biphasic mixture was stirred for 10 minutes. The phases were separated for 20 minutes. Brine strength was 24% by weight. The aqueous phase was contacted with an additional amount of ethyl acetate (68 mL) and the biphasic mixture thus prepared was stirred for 10 minutes and then left for 15 minutes for phase separation. The ethyl acetate layers from the two extractions were combined and washed with 24 wt% brine (120 mL), another aliquot 24 wt% saline (60 mL), 1N sodium hydroxide solution (150 mL) and another aliquot saline (60 mL). After each aqueous phase addition, the mixture was stirred for 10 minutes and left for 15 minutes for separation. The resulting solution was concentrated to dryness under reduced pressure at 45 ° C. using a water aspirator. The solid product (8.09 g) was analyzed by HPLC and found to contain 83.4 area% of ester, 2.45 area% of 11,12-olefin, 1.5% of 7,9-lactone and 1.1% of unreacted mesylate.
[867] Example 40
[868] Mesylate (1.0 g), isopropenyl acetate (10 g) and p-toluenesulfonic acid (5 mg) having a structure prepared in Example 23 were placed in a 50 mL flask and heated to 90 ° C while stirring. After 5 hours the mixture was cooled to 25 ° C. and concentrated in vacuo at 10 mm Hg. The residue was dissolved in CH ₂ Cl ₂ (20 mL) and washed with 5% aqueous NaHCO ₃ . The CH ₂ Cl ₂ layer was concentrated in vacuo to give 1.47 g of a tan oil. This material was recrystallized in CH ₂ Cl ₂ / Et ₂ O to yield 0.50 g of enol acetate of formula IV (Z).
[869] This material was added to a mixture of acetic acid (2.0 ml) and sodium acetate (0.12 g) preheated to 100 ° C. with stirring. After 60 minutes, the mixture was cooled to 25 ° C. and diluted with CH ₂ Cl ₂ (20 ml). The solution was washed with water (20 ml) and dried over MgSO ₄ . The desiccant was removed by filtration and the filtrate was concentrated in vacuo to afford 0.4 g of the desired 9,11-olefin, IV (Y). The crude product contained less than 2% of 7,9-lactone impurities.
[870] Example 41
[871] DMSO Of Mesylate Heat removal
[872]
[873] A mixture of 2 g of mesylate and 5 ml of DMSO in the flask was heated at 80 ° C. for 22.4 h. HPLC analysis of the reaction mixture showed no starting material was detected. Water (10 ml) was added to the reaction and the precipitate was extracted three times with methylene chloride. The combined methylene chloride layers were washed with water, dried over magnesium sulfate and concentrated to give an ester.
[874] Example 42
[875] In a 50 mL flask, mixed with methylene chloride (15.0 mL) under stirring with an ester of formula (IA) (evaluated at 1.07 g 74.4%), trichloroacetamide (0.32 g) and dipotassium hydrogen phosphate (0.70 g) as a solid It was. A clear solution was obtained. Hydrogen peroxide (30% by weight; 5.0 mL) was added via pipette over 1 minute. The resulting mixture was stirred at room temperature for 6 hours at which time HPLC analysis indicated that the ratio of epoxymexrenone to esters in the reaction mixture was about 1: 1. Additional trichloroacetamide (0.32 g) was added to the reaction mixture and the reaction was continued under stirring for at least 8 hours, indicating that the residual ratio of esters was reduced to 10%. Additional trichloroacetamide (0.08 g) was added and the reaction mixture was left overnight at which time only 5% of unreacted ester remained in proportion to the epoxymexrenone in the mixture.
[876] Example 43
[877] The ester of formula (IA) (5.4 g, 74.4% ester evaluation) was placed in a 100 mL reactor. Both trichloroacetamide (4.9 g) and dipotassium hydrogen phosphate (3.5 g) were added to the ester in solid form, followed by methylene chloride (50 mL). The mixture was cooled to 15 ° C. and 30% hydrogen peroxide (25 g) was added over 10 minutes. The reaction mixture was brought to 20 ° C. and stirred at this temperature for 6 hours at which time the conversion was checked by HPLC. Residual ester was measured at less than 1% by weight.
[878] The reaction mixture was added to water (100 mL), the phases were separated and the methylene chloride layer was removed. Sodium hydroxide (0.5 N; 50 mL) was added to the methylene chloride layer. After 20 minutes the phases were separated and HCl (0.5N; 50 mL) was added to the methylene chloride layer, then the phases were separated and the organic phase was washed with saturated brine (50 mL). The methylene chloride layer was dried over anhydrous magnesium sulfate and the solvent was removed. White solid (5.7 g) was obtained. The sodium hydroxide aqueous layer was acidified, extracted and extracted to further give 0.2 g of product. The yield of epoxymexrenone was 90.2%.
[879] Example 44
[880] The esters of formula (IA) were prepared in Example 43 except that the following differences were made, except that the initial charge consisted of the esters (5.4 g 74.4% ester evaluation), trichloroacetamide (3.3 g) and dipotassium hydrogen phosphate (3.5 g). It was converted to epoxymexrenone according to the method described. Hydrogen peroxide solution (12.5 mL) was added. The reaction was run overnight at 20 ° C. after which HPLC showed 90% conversion of the esters to epoxymexrenone. Additional trichloroacetamide (3.3 g) and 30% hydrogen peroxide (5.0 mL) were added and the reaction was run for an additional 6 hours at which point the residual ester was only 2% based on the ester charge. After operating as described in Example 43, 5.71 g of epoxymexrenone was obtained.
[881] Example 45
[882] Esters of formula (IA) were converted to epoxymexrenone by the method normally described in Example 43. In the reaction of the present example, the ester charge was 5.4 g (74.4% ester evaluation), the trichloroacetamide charge was 4.9 g, the hydrogen peroxide charge was 25 g, and the dipotassium hydrogen phosphate charge was 3.5 g. The reaction was run at 20 ° C. for 18 hours. Residual ester was less than 2%. After operation, 5.71 g of epoxymexrenone was obtained.
[883] Example 46
[884] The ester of formula (IA) was converted to epoxymexrenone by the method described in Example 43 except that the reaction temperature in this example was 28 ° C. Substances charged to the reactor included ester (2.7 g), trichloroacetamide (2.5 g), dipotassium hydrogen phosphate (1.7 g), hydrogen peroxide (17.0 g) and methylene chloride (50 mL). After 4 hours of reaction, the unreacted ester was only 2% based on the ester charge. After operating as described in Example 43, 3.0 g of epoxymexrenone was obtained.
[885] Example 47
[886] The ester of formula (IA) (evaluated as 17 g, 72% ester) was dissolved in methylene chloride (150 mL) and trichloroacetamide (14.9 g) was added with gentle stirring. The temperature of the mixture was adjusted to 25 ° C. and a dipotassium hydrogen phosphate (10.6 g) solution in water (10.6 mL) was stirred in an ester substrate solution under 400 rpm stirring. Hydrogen peroxide (30 wt% solution; 69.4 mL) was added to the substrate / phosphate / trichloroacetamide solution over 3-5 minutes. No exothermic or oxygen evaporation was observed. The reaction mixture thus prepared was stirred at 25 ° C. and 400 rpm for 18.5 hours. No oxygen evaporation was observed during the reaction. The reaction mixture was diluted with water (69.4 mL) and the mixture was stirred at about 250 rpm for 15 minutes. No temperature control was necessary during this operation and essentially conducted at room temperature (acceptable at temperatures in the 5-25 ° C. range). The aqueous and organic layers were separated and the lower methylene chloride layer was removed.
[887] The aqueous layer was back extracted with methylene chloride (69.4 mL) under stirring at 250 rpm for 15 minutes. The layers were separated and the lower methylene chloride layer was removed. The aqueous layer (177 g; pH = 7) was measured for hydrogen peroxide. The product (12.2%) showed that only 0.0434 mol of hydrogen peroxide consumed in the reaction was 0.0307 mol of olefin. A small amount of back extraction of methylene chloride volume was sufficient to confirm that there was no loss of epoxymexrenone in the aqueous layer. This result confirmed that only trichloroacetamide was recovered using a large amount of second methylene chloride extraction.
[888] The combined methylene chloride solutions from the above extractions were combined and washed with 3% by weight sodium sulfite solution (122 mL) at about 250 rpm for at least 15 minutes. A negative starch iodide test (KI paper; no color observed; purple solution in the positive test indicates the presence of peroxide) was observed at the end of the agitation.
[889] The aqueous and organic layers were separated and the lower methylene chloride layer was removed. The aqueous layer (pH = 6) was decanted. Note that the addition of sodium sulfite solution causes some exotherm and such addition should be done under temperature control.
[890] The methylene chloride phase was washed with 0.5 N sodium hydroxide (61 mL) at a temperature in the range of 15-25 ° C. and about 250 rpm (pH = 12-13). Impurities derived from trichloroacetamide were removed by this process. The acidification of the alkaline water fraction followed by extraction of methylene chloride revealed that very little epoxymexrenone was lost in this operation.
[891] The methylene chloride phase was washed once with 0.1 N hydrochloric acid (61 mL) for 15 minutes under 250 rpm stirring at a temperature in the range of 15-25 ° C. The layers were then separated and the lower methylene chloride layer was removed and washed again with 10% by weight aqueous sodium chloride (61 mL) for 15 minutes at a temperature in the range of 15-25 ° C. and about 250 rpm. The layers were separated again and the organic layer was removed. The organic layer was filtered through a Solkafloc pad and evaporated to dryness under reduced pressure. Drying was completed at a water bath temperature of 65 ° C. An off-white solid (17.95 g) was obtained and analyzed by HPLC. Epoxymexrenone analysis was 66.05%. The molar yield adjusted for the reaction was 93.1%.
[892] The product was dissolved in hot methyl ethyl ketone (189 mL) and the resulting solution was distilled at atmospheric pressure and then 95 mL of ketone solvent was removed. The temperature was lowered to 50 ° C. and the product crystallized. Stirring was continued at 50 ° C. for 1 hour. The temperature was then lowered to 20-25 ° C. and stirring continued for an additional 2 hours. The solid was filtered off and washed with MEK (24 mL) and the solid was dried to a constant weight of 9.98 g, which contained 93.63% epoxymexrenone by HPLC analysis. This product was dissolved in hot MEK (106 mL) and the hot solution was filtered through a 10 micron line filter under pressure. An additional MEK 18 mL was washed and the filtered MEK solution was distilled at atmospheric pressure and 53 mL of solvent was removed. Lower the temperature to 50 ° C. and crystallize the product; Stirring was continued at 50 ° C. for 1 hour. The temperature was then lowered to 20-25 ° C. and this temperature was maintained while stirring was continued for an additional 2 hours. The solid product was filtered off and washed with MEK (18 mL). The solid product was dried to a constant weight of 8.32 g and contained 99.6% epoxymexrenone by quantitative HPLC analysis. The final loss upon drying was less than 1.0%. The total epoxymexrenone yield according to the operation and reaction of the present example was 65.8%. This overall yield reflected 93% reaction yield, 78.9% initial crystallization recovery and 89.5% recrystallization recovery.
[893] Example 48
[894] Chemical formula using toluene IIA of Epoxidation
[895] The ester of formula (IA) was converted to eplerenone by the method normally described in Example 46 except that tolene was used as the solvent. Materials charged to the reactor included esters (2.7 g), trichloroacetamide (2.5 g), dipotassium hydrogen phosphate (1.7 g), hydrogen peroxide (17.0 g) and tolene (50 mL). The reaction was exothermic to 28 ° C. and completed in 4 hours. The resulting three phase mixture was cooled to 15 ° C., filtered, washed with water and dried in vacuo to give 2.5 g of product.
[896] Example 49
[897] 9,11- Dienon Epoxidation
[898] Compound (VII.A) (40.67 g) in which -AA- and -BB- are both -CH ₂ -CH _2- (40.67 g) represented by Formula XVIIA is dissolved in methylene chloride (250 mL) in a 1 liter three-necked flask and the salts externally The mixture was cooled with ice. Dipotassium phosphate (22.5 g) and trichloroacetamide (83.5 g) were added and the mixture was cooled to 2 ° C. and 30% hydrogen peroxide (200 g) was added slowly over 1 hour. The reaction mixture was stirred at 12 ° C. for 8 hours and at room temperature for 14 hours. The organic layer was added dropwise and checked for any starting eon and found to be <0.5%. Water (400 mL) was added and stirred for 15 minutes and the layers were separated. The organic layer was washed successively with 200 mL of potassium iodide (10%), 200 mL of sodium thiosulfate (10%) and 100 mL of saturated sodium bicarbonate solution and the layers were separated each time. The organic layer was dried over anhydrous magnesium sulfate and concentrated to give crude epoxide (41 g). The product was crystallized from ethyl acetate: methylene chloride to give 14.9 g of pure material.
[899] Example 50
[900] m- Chloroperbenzoic acid Compound used XVIIA of Epoxidation
[901] Compound XVIIA (18.0 g) was dissolved in 250 mL of methylene chloride and cooled to 10 ° C. Under stirring solid m-chloroperbenzoic acid (50-60% purity, 21.86 g) was added for 15 minutes. No temperature rise was observed. The reaction mixture was stirred for 3 hours and checked for the presence of dienes. The reaction mixture was treated successively with sodium sulfite solution (10%), sodium hydroxide solution (0.5N), hydrochloric acid solution (5%) and finally with 50 mL saturated brine solution. After drying over anhydrous magnesium sulfate, evaporation gave 17.64 g of epoxide, which was used directly in the next step. The product was found to contain a Bayer-Billiger oxidation product which was triturated from ethyl acetate and then crystallized from methylene chloride. On the 500 g scale, the precipitated m-chlorobenzoic acid was filtered and then operated normally.
[902] Example 51
[903] Trichloroacetamide Compound used XVIIA of Epoxidation
[904] Compound XVIIA (2 g) was dissolved in 25 mL of methylene chloride. Trichloroacetamide (2 g) and dipotassium phosphate (2 g) were added. 30% hydrogen peroxide (10 mL) was added while stirring at room temperature, and stirring was continued for 18 hours to obtain an epoxide (1.63 g). No Bayer-Billiger product was formed.
[905] Example 52
[906] Potassium hydroxide (56.39 g; 1005.03 mmol; 3.00 equiv) was placed in a 2000 mL flask and slurried with dimethyl sulfoxide (750.0 mL) at ambient temperature. Trienone (100.00 g; 335.01 mmol; 1.00 equiv) corresponding to formula XX (R ³ is H and -AA- and -BB- are -CH ₂ -CH _2-, respectively) is combined with THF (956.0 mL). Put together in the flask. Trimethylsulfonium methyl sulfate (126.14 g; 670.02 mmol; 2.00 equiv) was added to the flask and the resulting mixture was heated to reflux at 80 to 85 ° C. for 1 hour. Conversion to 17-spirooxymethylene was checked by HPLC. About 1 L of THF was stripped from the reaction mixture under vacuum, then water (460 mL) was charged over 30 minutes and the reaction mixture was cooled to 15 ° C. The resulting mixture was filtered and the solid oxirane product was washed twice with 200 mL aliquots of water. The product was observed to be very crystalline and was easily filtered. The product was then dried at 40 ° C. under vacuum. 104.6 g of 3-methyl enol ether Δ-5,6,9,11, -17-oxirane steroid product was isolated.
[907] Example 53
[908] Sodium ethoxide (41.94 g; 616.25 mmol; 1.90 equiv) was placed in a dry 500 mL reactor under a blanket of nitrogen. Ethanol (270.9 mL) was placed in the reactor and sodium methoxide was slurried in ethanol. Diethylmalonate (103.90 g; 648.68 mmol; 2.00 equiv) was added to the slurry, followed by addition of the oxirane steroid (104.60 g; 324.34 mmol; 1.00 equiv) prepared in Example 52, followed by heating to reflux at 80-85 ° C. It was. The heating was continued for 4 hours and then the end of the reaction was checked by HPLC. Water (337.86 mL) was added to the reaction mixture over 30 minutes and the mixture was cooled to 15 ° C. Stirring was continued for 30 minutes and the reaction slurry was filtered to obtain a filler cake consisting of fine amorphous powder. The filter cake was washed twice with water (200 mL each) and then dried under vacuum at ambient temperature. 133.8 g of 3-methyl enolether-Δ5,6,9,11, -17-spirolactone-21-methoxycarbonyl intermediate were isolated.
[909] Example 54
[910] 3-methyl enolether-Δ5,6,9,11, -17-spirolactone-21-methoxycarbonyl intermediate prepared in Example 53 (Formula XVIII, R ³ is H and -AA- and -BB- is each -CH ₂ -CH ₂ - is; 133.80g; 313.68mmol; 1.00 eq.) of sodium chloride (27.50g; under 1.50 eq) and stirred into the reactor in dimethylformamide (709mL) and water (5mL) together; 470.52mmol Put into 2000 mL reactor. The resulting mixture was heated to reflux at 138 to 142 ° C. for 3 hours and then the reaction mixture was checked by HPLC for completion of the reaction. Water was then added to the mixture over 30 minutes and the mixture was cooled to 15 ° C. Stirring was continued for 30 minutes and then the reaction slurry was filtered and the amorphous solid reaction product was recovered as a filter cake. The filter cake was washed twice (200 mL aliquots of water) and then dried. The product 3-methylenolether-17-spirolactone was dried to give 91.6 g (82.3% yield; 96 area% evaluation).
[911] Example 55
[912] Enol ether (91.60 g; 258.36 mmol; 1.00 equiv), ethanol (250 mL), acetic acid (250 mL) and water (250 mL) prepared according to Example 54 were added to a 2000 mL reactor and the resulting slurry was heated to reflux for 2 hours. Water (600 mL) was added over 30 minutes and the reaction mixture was cooled to 15 ° C. The reaction slurry was filtered and the filter cake was washed twice with water (200 mL aliquot). The filter cake is then dried; 84.4 g of product 3-keto Δ4,5,9,11-17-spirolactone were isolated (compound of formula XVII, R ³ is H and -AA- and -BB- is -CH ₂ -CH _2- ; 95.9 % Yield).
[913] Example 56
[914] Compound XVIIA (1 kg; 2.81 mol) was placed in a 22 L four-necked flask with carbon tetrachloride (3.2 L). N-bromo-succinamide (538 g) was added to the mixture followed by acetonitrile (3.2 L). The resulting mixture was heated to reflux and maintained at reflux temperature of 68 ° C. for about 3 hours to obtain a clear orange solution. After heating for 5 hours, the solution turned dark. After 6 hours the heat was removed and the reaction mixture was sampled. The solvent was stripped under vacuum and ethyl acetate (6 L) was added to the residue at the bottom of the still. The resulting mixture was stirred and then 5% sodium bicarbonate solution (4 L) was added and the mixture was stirred for 15 minutes and the phases were then left to stand. The aqueous layer was removed and saturated brine solution (4 L) was introduced to the mixture and stirred for 15 minutes. The phases were separated again and the organic layer was stripped under vacuum to give a thick slurry. Dimethylformamide (4 L) was then added and stripping continued at a pot temperature of 55 ° C. The bottom of the distiller was left overnight and DABCO (330 g) and lithium bromide (243 g) were added. The mixture was then heated to 70 ° C. After heating for 1-1.5 hours, a liquid chromatography sample was taken and after 3.50 hours additional DABCO (40 g) was added. After heating for 4.5 hours, water (4 L) was added and the resulting mixture was cooled to 15 ° C. The slurry was filtered and the cake washed with water (3 L) and dried in filter overnight. The wet cake (978 g) was placed again in a 22 L flask and dimethylformamide (7 L) was added. The resulting mixture was heated to 105 ° C. at which point the cake was absorbed into the total solution. Heat was removed and the mixture in the flask was stirred and cooled. Ice water was added to the reactor jacket and the mixture in the reactor was cooled to 14 ° C. and maintained for 2 hours. The resulting slurry was filtered and washed twice with 2.5 L aliquots of water. The filter cake was dried under vacuum overnight. 510 g of a light brown solid product were obtained.
[915] Example 57
[916] In a 2 L four-necked flask, 9,11-epoxy canrenone (100.00 g; 282.1 mmol; 1.00 equiv), dimethylformamide (650.0 mL), lithium chloride (30.00 g; 707.7) prepared in Examples 49, 50, or 51 mmol; 2.51 equiv) and acetone cyanohydrin (72.04 g; 77.3 mL; 846.4 mmol; 3.00 equiv). The resulting suspension was mechanically stirred and treated with tetramethyl guanidine (45.49 g; 49.6 mL; 395.0 mmol; 1.40 equiv). The system was then filtered with water and cooled with a cooler and a dry ice cooler (filtered with dry ice in acetone) to prevent the escape of HCN. The discharge line from the dry ice cooler was passed through a scrubber filled with excess chlorine detergent. The mixture was heated to 80 ° C.
[917] After 18 hours a dark reddish brown solution was obtained which was cooled to room temperature with stirring. Nitrogen was dispersed in the solution during the cooling process to remove residual HCN while passing the discharge line through a detergent in a scrubber. After 2 hours the solution was treated with acetic acid (72 g) and stirred for 30 minutes. The crude mixture was poured into ice water (2 L) with stirring. The stirred suspension was further treated with 10% aqueous HCl (400 mL) and stirred for 1 hour. The mixture was then filtered to give a dark red brick solid (73 g). The filtrate was placed in a 4 L separatory funnel and extracted with methylene chloride (3 x 800 mL); The organic layers were combined and back extracted with water (2 × 2 L). The methylene chloride solution was concentrated in vacuo to give 61 g of a dark red oil.
[918] After leaving the aqueous wash fractions overnight, significant precipitation occurred. This precipitate was collected by filtration and the pure product enamine (14.8 g) was measured.
[919] After drying, the original red oil (73 g) was analyzed by HPLC and determined that the main component was 9,11-epoxyenamine. HPLC showed that enamine was the main component of the red oil obtained from the methylene chloride operation. The molar yield of the enamine was 46%.
[920] Example 58
[921] 9,11-Epoxyenamine (4.600 g; 0.011261 mol; 1.00 equiv) prepared according to Example 57 was placed in a 1000 mL round bottom flask. Methanol (300 mL) and 0.5 wt% aqueous HCl (192 mL) were added to the mixture and refluxed for 17 h. Methanol was then removed under vacuum to reduce the amount of material in the still pot to 50 mL to form a white precipitate. Water (100 mL) was added to the slurry and then filtered to give a white solid cake which was washed three times with water. The yield of solid 9,11-epoxydiketone product was 3.747 g (81.3%).
[922] Example 59
[923] Epoxydiketone (200 mg; 0.49 mmol) prepared according to Example 58 was suspended in methanol (3 mL) and 1,8-diazabicyclo [5.4.0] undes-7-ene (DBU) was added to the mixture. . Upon heating under reflux for 24 hours, the mixture became homogeneous. It was then concentrated to dryness at 30 ° C. on a rotary evaporator and the residue was partitioned between methylene chloride and 3.0 N HCl. The concentrate of the organic phase was a yellow solid (193 mg) which was measured by 22 wt% epoxymexrenone. Yield 20%.
[924]
[925] Example 60
[926] To 100 mg of diketone suspended in 1.5 mL of methanol was added 10 μl (0.18 equiv) of a 25% (w / w) solution of sodium methoxide in methanol. The solution was heated to reflux. There was no diketone remaining after 30 minutes and 5-cyanoester was present. To the mixture was added 46 μl of a solution of sodium methanol in 25% (w / w) methanol. The mixture was heated to reflux for 23 hours at which time the main product was determined as eplerenone by HPLC.
[927]
[928] Example 61
[929] 0.34 mL of triethylamine was added to 2 g of diketone suspended in 30 ml of dry methanol. The suspension was heated to reflux for 4.5 hours. The mixture was stirred at 25 ° C. for 16 h. The obtained suspension was filtered to obtain 1.3 g of 5-cyano ester as a white solid.
[930] 2.8 mL of triethylamine was added to 6.6 g of diketone suspended in 80 mL of methanol. The mixture was heated to reflux for 4 h and stirred at 25 × for 88 h during which time the product crystallized out of solution. After filtration, washing with methanol gave 5.8 g of cyanoester as a white powder. The material was recrystallized from chloroform / methanol to give 3.1 g of crystalline material, which was homogeneous by HPLC.
[931] As stated above, several objects of the present invention are achieved and other advantages are attained.
[932] As various changes could be made in the above compositions and methods without departing from the scope of the invention, it is intended that all materials contained in the above description and shown in the accompanying drawings be interpreted as illustrative and not in a limiting sense.
[933] According to the present invention, production is achieved through improved processes for the preparation of epoxymexrenone, other 20-spiroxanes, and other steroids having conventional structural features. According to the invention: the product of formula IA and other related compounds is prepared in high yield; Such a method is provided comprising a minimal separation step; Such a method is provided that can satisfy a reasonable price cost and operate at a reasonable conversion cost.
[934] Accordingly, the present invention provides a series of synthetic schemes for epoxymexrenone, intermediates useful for the preparation of eplerenone; And methods of synthesizing such novel intermediates.

权利要求:
Claims (11)
[1" claim-type="Currently amended] Formula V
Formula V

[In the above formula
-AA- represents the group -CHR ⁴ -CHR ⁵ -or -CR ⁴ = CR ^5-
R ³ , R ⁴ and R ⁵ are independently selected from the group consisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano, aryloxy,
R ¹ represents an α-oriented lower alkoxycarbonyl or hydroxycarbonyl radical,
-BB- represents a group -CHR ⁶ -CHR ⁷ -or an α- or β-alignment group of formula III:
Formula III

R ⁶ and R ⁷ are independently selected from the group consisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy ,
R ⁸ and R ⁹ are independently selected from the group consisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy Or R ⁸ and R ⁹ together form a carbocyclic or heterocyclic ring structure, or R ⁸ or R ⁹ together with R ⁶ or R ⁷ form a carbocyclic or heterocyclic ring structure condensed into a 5-ring D ring; In the preparation method of a compound of
Reacting an alkali metal alkoxide corresponding to the formula R ¹⁰ OM (M is an alkali metal and R ¹⁰ O- corresponds to an alkoxy substituent of R ¹ ) with a compound of formula VI having the structure Way.
Formula VI

In the above formula, -AA-, R ³ , -BB-, R ⁸ and R ⁹ are as described above.
[2" claim-type="Currently amended] The compound of claim 1, wherein the compound of Formula V
Formula VA

[In the above formula
-AA- is a group -CH ₂ -CH ₂ - or represents -CH = CH-,
R ¹ represents an α-oriented lower alkoxycarbonyl radical,
-BB- represents a group -CH ₂ -CH ₂ -or an α- or β-alignment group of formula IIIA:
Formula IIIA

X represents two hydrogen atoms or oxo
Y ¹ and Y ² together represent an oxygen bridge bond -O-, or
Y ¹ represents hydroxy, and
Y ² represents hydroxy, lower alkoxy or lower alkanoyloxy when X is H ₂ ] and X corresponds to a salt of a compound which represents oxo and Y ² represents hydroxy;
The method has an alkali metal alkoxide corresponding to the formula R ¹⁰ OM (M is an alkali metal and R ¹⁰ O- corresponds to an alkoxy substituent of R ¹ ) in the presence of an alcohol having the formula R ¹⁰ OH and Reacting a compound of formula VI.
Formula VIA

In the above formula, -AA-, -BB-, Y ¹ , Y ² and X are as defined in formula VA.
[3" claim-type="Currently amended] The compound of formula (V) according to claim 1, wherein the compound of formula (V) is methyl hydrogen 11α, 17α-dihydroxy-3-oxopregin-4-ene-7α, 21-dicarboxylate, γ-lactone, 4'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β-dimethyl-3 ', 5,20'-trioxospyro [furan-2 (3H) , 17'β- [4,7] methano [17H] cyclopenta [a] phenanthrene-5'β (2'H) -carbonitrile.
[4" claim-type="Currently amended] The method of claim 1, wherein cyanide ions are formed as reaction byproducts, and the method further comprises removing cyanide ions from the reaction zone during the reaction to reduce any degree of reaction of the cyanide ions with the product of formula V. How to feature.
[5" claim-type="Currently amended] 5. The process of claim 4 wherein the cyanide ions are precipitated with a precipitant and removed from the reactants.
[6" claim-type="Currently amended] 6. The process of claim 5 wherein said reaction is carried out in a solvent medium and said precipitant consists of a salt consisting of a cation forming a cyanide compound having a lower solubility in said medium than that of said precipitant in said medium.
[7" claim-type="Currently amended] The method of claim 6, wherein the cation is selected from the group consisting of alkaline earth metal ions and transition metal ions.
[8" claim-type="Currently amended] A compound of formula
Formula VI

In the above formula
-AA- represents the group -CHR ⁴ -CHR ⁵ -or -CR ⁴ = CR ^5- ,
R ³ , R ⁴ and R ⁵ are independently selected from the group consisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy,
-BB- represents a group -CHR ⁶ -CHR ⁷ -or an α- or β-alignment group of formula III:
Formula III

R ⁶ and R ⁷ are independently selected from the group consisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy ,
R ⁸ and R ⁹ are independently selected from the group consisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy Or R ⁸ and R ⁹ together form a carbocyclic or heterocyclic ring structure, or R ⁸ or R ⁹ together with R ⁶ or R ⁷ form a carbocyclic or heterocyclic ring structure condensed into a 5-ring D ring.
[9" claim-type="Currently amended] The compound of claim 8, wherein the compound is of formula VIA
Formula VIA

[In the above formula
-AA- is a group -CH ₂ -CH ₂ - or represents -CH = CH-,
-BB- represents a group -CH ₂ -CH ₂ -or an α- or β-alignment group of formula IIIA:
Formula IIIA

X represents two hydrogen atoms or oxo,
Y ¹ and Y ² together represent an oxygen bridge bond -O-, or
Y ¹ represents hydroxy, and
Y ² represents hydroxy, lower alkoxy or lower alkanoyloxy when X is H ₂ ] and X corresponds to a salt of a compound which represents oxo and Y ² represents hydroxy.
[10" claim-type="Currently amended] The compound of claim 8, wherein the compound is 4'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β-dimethyl-3 ', 5,20'-tree Formula VI, characterized in that it is oxisopyro [furan-2 (3H), 17'β- [4,7] methano [17H] cyclopenta [a] phenanthrene-5'β (2'H) -carbonitrile Of compounds.
[11" claim-type="Currently amended] Formula VIII
Formula VIII

[In the above formula
-AA- represents the group -CHR ⁴ -CHR ⁵ -or -CR ⁴ = CR ^5-
R ³ , R ⁴ and R ⁵ are independently selected from the group consisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano, aryloxy,
-BB- represents a group -CHR ⁶ -CHR ⁷ -or an α- or β-alignment group of formula III:
Formula III

R ⁶ and R ⁷ are independently selected from the group consisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy ,
R ⁸ and R ⁹ are independently selected from the group consisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy Or R ⁸ and R ⁹ together form a carbocyclic or heterocyclic ring structure, or R ⁸ or R ⁹ together with R ⁶ or R ⁷ form a carbocyclic or heterocyclic ring structure condensed into a 5-ring D ring; In the preparation method of a compound of
And wherein the substrate compound corresponding to formula (X) is oxidized by fermentation in the presence of a microorganism effective to introduce an 11-hydroxy group into said substrate of α-orientation corresponding to the formula:
Formula XIII

In the above formula, -AA-, R ¹ , R ³ , -BB-, R ⁸ and R ⁹ are as described above.

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公开号 | 公开日 | 专利标题

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同族专利:

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公开号 | 公开日

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HU9900493A2|1999-05-28|

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CA2347345A1|1997-06-19|

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HU222453B1|2003-07-28|

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KR100579527B1|2006-05-12|

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CA2347345C|2007-05-15|

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AU1355197A|1997-07-03|

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HU9900493A3|1999-11-29|

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KR100523146B1|2005-10-24|

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CN1903872A|2007-01-31|

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KR20050085950A|2005-08-29|

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AU719260B2|2000-05-04|

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KR19990076572A|1999-10-15|

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KR100523227B1|2005-12-21|

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CA2347346C|2007-01-02|

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CA2347346A1|1997-06-19|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1995-12-11|Priority to US845595P

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1995-12-11|Priority to US60/008,455

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1996-12-11|Application filed by 지.디. 썰 엘엘씨

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1996-12-11|Priority to PCT/US1996/020780

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2004-08-12|Publication of KR20040071782A

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2005-10-24|Application granted

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2005-10-24|Publication of KR100523146B1

优先权:

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申请号 | 申请日 | 专利标题

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US845595P| true| 1995-12-11|1995-12-11|

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US60/008,455|1995-12-11|

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PCT/US1996/020780|WO1997021720A2|1995-12-11|1996-12-11|Processes for preparation of 7 alpha-carboxyl 9,11-epoxy steroids and intermediates useful therein and a general process for the epoxidation of olifinic double bonds|