1,2-DEHYDROGENATION OF STEROIDAL 21-ESTERS WITH A. SIMPLEX

Abstract: The present invention involves processes for 1,2-dehydrogenation of DELTA <4>-3-keto C21-hemiester steroids of formula (I), with Arthrobacter simplex or Bacterium cyclooxydans to produce the corresponding DELTA <1,4>-3-keto C21 hemiester of formula (II), or free alcohol thereof with improved yields. The advantage is that the processes utilize higher substrate concentrations and give shorter reaction times than previously were known.

Description: 1.2-DEHYDROGENATIQ OF STEROIDAL 21-ESTER5 WITH .4. SIMPLEX BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention involves processes for 1, 2-dehydrogenatio of certain Δ -3-keto C2 ] _-hemiester steroids with Arthrobac ter simple or Bacterium cyclooxydans to produce the corresponding Δ 1 ' -3-ket &21 hemiester or free alcohol with improved yields, and shorte bioconversion time. 2. Description of the Related Art The 1,2-dehydrogenation of Δ -3-keto steroids to the cor responding A- 1 -' -3-keto steroids with Septomyxa af finis , A . simple and other microorganisms is well known in the art and has valuabl commercial significance. US Patent 2,837,464 claims a process for 1,2-dehydrogenation o Δ -3-ketopregnenes to Δ^'^-pregnadienes where the C21 hydroxyl grou is either free or esterified with a monocarboxylic acid by the use o Corynebacterium (Arthrobacter) simplex or Corynebac terium hoagii . US Patents 2,902,410 and 2,902,411 claim processes for the 1,2 dehydrogenation of 1,2-saturated steroids to 1,2-dehydro steroid with Septomyxa af finis . US Patent 3.360,439 discloses a process for the 1, 2-dehydrogena tion of Δ^-3-keto steroids to the corresponding A - • -3-keto steroid using A . simplex cells which had been previously treated with a lowe alkanol or alkanone. The process of the present invention does no require pretreatment of the cells and may be performed using viabl cells in a fermentation broth. Alternatively cells pretreated by ai or heat drying or by exposure to a lower alkanone such as acetone ma be used. The steroid-1-dehydrogenase of these organisms can also b used in a cell-free form. US Patent 3,770,586 claims an improved process for the produc¬ tion of 11/3,21-dihydroxypregna-l, , 17(20) -trien-3-one (Example 1) an 11/3, 21-dihydroxy-6 -methylpregna-l,4, 17(20) -trien-3-one (Example 5) whereby the alkali metal salts of the Δ^-3-keto 21-hemiester sub¬ strates thereof where the acyl radical is that of a dicarboxylic acid of 3 to 12 carbon atoms are 1,2-dehydrogenated by use of S . af fini s . Using shake flasks, 11/3, 21-dihydroxypregna-4, 17(20)-dien-3-one 21- hemisuccinate potassium salt at a concentration of 5.0 g/1 and medium 1, the desired Δ^'"*-product was produced in 100% yield after 5 days (Example 8). Example 1 uses larger stirred tanks and shows a lower yield. With medium 2, and 2.0-7.0 g/1 of 113,21-dihydroxypregna- 4,17(20) -dien-3-one 21-hemisuccinate potassium salt yields of 91.1- 96.6% were obtained in 6 days. US Patents 4,524,134 and 4,704,358 disclose improved microbial processes to produce a 1,2-dehydrogenated steroid from the correspon¬ ding 1,2-saturated Δ^-3-keto-steroid using air-dried or heat-dried cells of Arthrobacter simplex and Bacterium cyclooxydans respective- ly. The processes of the present invention provide Improved yields and at the same time • permit the use of higher substrate concentra¬ tions and reduced reaction times. SUMMARY OF THE INVENTION Disclosed Is a process for the production of a Δ 1, -3-keto 21- hemiester steroid (II) where (B-I) Rg is =CH 2 or α-R 6 . 1 :/3-H where R 6 ._ is -H, -F and -CH3, R7 (B-II) Rg is g_3 Rg. and R7 is R7_3:R7_ where one of Rg.3 and S-6-4- ta ken together with one of R7.3 and R-7.4 form a second bond between Cg and C , the other of Rg_3 and Rg.4 is -CH3 and the other of R7..3 and R7.4 is -H; (C-I) R_]_ is α-R^]_.-j_:^-R^.2 where one of Rχχ-1 and ]_]_.2 - s taken together with Rg to form a second bond between Cg and Ci-r , and the other of χχ-1 and χχ-2 ^ s ~^' (C-II) Rg is -H, -F, -Cl or Br and R χι Is =0 or α-R^. β - β'^H. where one of -3 and χχ-4 is -H and the other of R___3 and Rχi-4 Is -H or -OH; (C-III) R ] _ Is where β-0- is taken together with Rg to form an epoxide between Cg and C^ in the ?-configuration; (D-I) R £ is Ri6-i^Ri6-2 and R 17 ^ s R 17-l :R 17-2' w ere one of R_5_]_ and χg_2 ^- s "^ and t ^ ιe otrιer taken together with one of ] _7_ ] _ or χ7-2 f° rms a second bond between C ] _g and C-^ j and the other of R 17-l and R 17-2 is -C0-CH 2 -0-R2i where R 21 is -H, -CO-(X-j -C00R 2 - , where X ] _ is -CH=CH- and -(CH ) n ι- where n ] _ is 1-8 and where 2I-I is -H or a cation; (D-II) R 16 is α-H:3-H, and where 17 is where R is as defined above; (D-III) R 16 is =CH 2 or * α-R 1 g_ 5 :,9-R 1 g_g where ] _ -5 is -H, -OH o -CH3, i . is -H or -CH3 with the proviso that one of i .5 or Rχg. is -H, and where Rχ 7 is Q-R 17 _5:^-CO-CH 2 -0-R2 ] _ where 17-5 is -H or -OH, where R 2] _ is as defined above, which comprises contacting a Δ 3-keto 21-hemiester steroid (I) where (D-I) R_g is Rιg-l'Rlg-2 and R 17 is R 17-l :R 17-2> where one o • R 16-l and 16-2 ^- s "* and t ^ ιe otner ta ke together with one of Rχ7- or R 1 7. 9 for™ 3 a second bond between C^ and Q-^η and the other o R17-I and R_7_2 is -C0-CH -0-C0-(X]_) -C00R 2 ι_ι, wnere x l and R 21-l ar as defined above; (D-II) R 16 is α-H: / 9-H, and where R 17 is =CH-CH 2 -0-C0- (X j _) - C00R 2 _ _where Xη_ and R 2 l-i are as defined above; (D-III) R]_g is =CH 2 or α"R 16-5 : ^ "R 16-6 wher e R i6-5 is ~H > ~0H o -CH3, i .g is -H or -CH3 with the proviso that one of R]_6-5 or 16- is -H, and where 17 is Q-R17.5 :/3-CO-CH 2 -0-CO- (X ] _) -COOR 2 χ_ι wher R_7-5 i -H or -OH and where X ] _ and R τ__ ] _ a e as defined above, an where R , Rg and ] _χ are as defined above, with the steroid-1- dehydrogenase of Arthrobacter simplex or Bac terium cyclooxydans . DETAILED DESCRIPTION OF THE INVENTION The process of the present invention comprises contacting a Δ → - 3-keto 21-hemiester steroid of formula (I) with either the steroid- 1-dehydrogenase from A . simplex or B . cyclooxydans or with the cells of A . simplex or B . cyclooxydans containing steroid-1-dehydrogenase to produce the corresponding Δ-"-' -3-keto steroid (II) as the 21-hemi¬ ester and/or free 21-hydroxy compound. The Δ -3-keto steroidal 21-hemiester (I) starting materials are known to those skilled in the art or can be readily prepared from known compounds by methods known to those skilled in the art, see for ■ example US Patents 3,025,311, 3,209,000 and 3,770,586. The Δ -3-keto 21-hemiesters (I) can be made from either the corresponding Δ^-3- keto 21-halo or Δ -3-keto 21-hydroxy steroids by simple well known displacement reactions. The Δ -3-keto 21-halo, preferably the 21- chloro, steroid is reacted with a salt of the desired diacid to produce the Δ -3-keto steroidal 21-hemiester (I). Alternatively, a Δ -3-keto 21-hydroxy steroid is reacted with the appropriate an¬ hydride or acid halide of the desired diacid to produce the Δ^-3-keto steroidal 21-hemiester (I) . With the Δ 4 -3-keto 21-hemiesters (I) it is preferred that R .χ be -H, -F, -CH 3 and =CH 2 . It Is preferred that the C-ring be either Δ 9(ll) or ^ ave i]_ α -0H, llS-OH or have 2 hydrogen atoms at C ] _χ. With regard to the D-ring it Is preferred that the substitution at C-^- j be =CH-CH 2 -0-CO-(X 1 )-COO-R 21 . 1 and α-0H: -C0-CH 2 -0-C0-(X χ ) -COOR 21 . 1 . It is preferred that X ] _ is -CH=CH- or -(CH) n _- where _ is 2-4, more preferably 2. It is preferred that R 2 χ is succinate and fumarate. It Is preferred that the cation be monovalent and be selected from the group consisting of sodium or potassium, ammonium or -H + . The preferred steroidal portion of the hemiesters are selected from the group consisting of 17αr,21-dlhydroxypregna-4,9(11)-diene-3,20-dione, 11 / 9,21-dihydroxypregna-4,17(20)-dien-3-one, 11 / 9,17α,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione, 11 / 9,21-dihydroxy-6α-methylpregna-4,17(20) -dien-3-one, 11/9,17α,21-trihydroxypregn-4-ene-3,20-dione, 17α,21-dihydroxy-6α-methylpregna-4,9(11) -diene-3,20-dione, 17a,21-dihydroxy-16/3-methylpregna-4,9(11) -diene-3,20-dione, 6α-fluoro-21-hydroxyρregna-4,9(11) ,16-trIene-3 ,20-dione, 21-hydroxypregna-4,9(11) ,16-triene-3 ,20-dione, 17α,21-dlhydroxy-16α-methylpregna-4,9(11) -diene-3 ,20-dione. Most preferred Is 11 / 9,21-dIhydroxypregna-4,17(20) -dien-3-one as the 21-hemisuccinate. The microorganisms A. simplex and B . cyclooxydans are well known, see US Patents 2,837,464, 4,524,134 and 4,704,358. The process of the present Invention may be practiced using the sterold-l- dehydrogenase activity from either organism in any of several forms. The enzyme from either organism Is operable but It is preferred that A. simplex be used as the microbial source. The bioconversion may be conducted with either cell-free steroid-1-dehydrogenase or with cells of the microorganisms. Preferably, whole microbial cells in the fermentation broth are used in the practice of the Invention to avoid additional manufacturing steps. When using Isolated cells, these may be used after collection and concentration from the nutrient medium by conventional means such as centrifugation, flocculation and filtration, precipitation and filtration or ultrafiltration. The isolated cells can be used in a wet state or can be immobilized standard techniques, such as entrapment in collagen, alginate carrageenin as described in Methods in Enzymology, Vol.XLIV, p.1 317, (1976), Academic Press, Inc. , New York or can be dried treatment with a lower alcohol or alkanone such as acetone described in US Patent 3,360,439, by vacuum-drying with heat, freeze-drying, by air-drying with heat or by spray-drying as descr bed in US Patents 4,524,134 and 4,704,358. Steroid-1-dehydrogenas activity in a cell-free form can also be used in solution or as immobilized enzyme to catalyze steroid-1-dehydrogenation. It shoul be understood that the subject process covers the use of any form o steroid-1-dehydrogenase preparation from A . simplex and B . cyclo oxydans for the steroid bioconversion of Δ -3-keto 21-hemieste steroids (I) to the corresponding Δ- 1 -' -3-keto steroids (II). The bioconversion is accomplished by exposure of the steroi substrate, Δ -3-keto 21-hemiester steroids (I), to the preparatio containing steroid-1-dehydrogenase activity in a predominantl aqueous system with a pH range of 6 to 10, preferably 7 to 8. Th aqueous system can contain less than about five per cent of a water miscible organic solvent. Suitable water-miscible organic solvent include, for example, dimethyl sulfoxide, dimethylformamide, methan ol, ethanol, acetone and the like. Optionally, a water-immiscibl organic solvent can be added (provided that it is non-toxic to th steroid-1-dehydrogenase activity) in an amount of from about 2 t about 50%. Suitable water-immiscible organic solvents includ toluene, benzene, butyl acetate, heptane, methylene chloride and th like; preferred is toluene. The organic solvents can be used t dissolve or suspend the Δ -3-keto 21-hemiester steroid substrate (I or an exogenous electron carrier prior to its/their addition to th steroid-1-dehydrogenase preparation. It is preferred that th contacting of the steroid substrate take place in the presence of a exogenous electron carrier. It is not necessary to have the ex ogenous electron carrier present with all the Δ -3-keto 21-hemieste substrates (I) as is known to those skilled in the art, but it i preferred. It is particularly preferred when the bioconversion i performed cell free or with dried cells. The exogenous electro carrier can be added to stimulate steroid-1-dehydrogenation and/or t prevent other steroid-degrading activities in preparations where these activities have not been previously eliminated. It is prefer¬ red that the exogenous electron carrier be sele.cted from the group consisting of menadione, menadione bisulfite, 1,4-naphthoquinone, phenazine methosulfate, phenazine ethosulfate, dichlorophenol indophenol, and cytochrome c. It Is more preferred that the exo¬ genous electron carrier be menadione, menadione bisulfite or 1,4- naphthoquinone. The exogenous electron carrier Is used in catalytic amounts from about 0.01 g/1 In the manner described in US Patent 4,524,134, to about 3.0 g/1, provided hydrogen peroxide accumulation is minimized. It is preferred that about 0.10 to about 1.0 g/1 be used. During the biotransformation process the mixture preferably has access to molecular oxygen and is stirred. The temperature is maintained between about 5 and about 45° , preferably in the range of about 25 to ' about 35° . The type of bioconversion procedure to be used to dehydrogenate a specific Δ -3-keto 21-hemiester steroid substrate (I) will vary dependent on the structure and nature of the particular steroid. For example, the substrate discussed in Example 1 is bloconverted to completion using whole cells in a fermentation broth, whereas its conversion is substantially poorer using an equivalent amount of heat-dried cells of A. simplex. The optimum type and level of the enzyme preparation, optimum substrate concentration, time of sub¬ strate addition and duration of the bioconversion can be readily determined In each individual sterold-l-dehydrogenatlon reaction with routine preliminary experimentation within the expertise of one of ordinary skill in the art. Most of the Δ -3-keto 21-hemiester steroid substrates (I) bloconvert well only in a system which is about 95% or greater aqueous. Not all the Δ -3-keto 21-hemiester steroids (I) will work well under all of the above conditions. Some work better with only an aqueous system, some with a water-miscible or water-immiscible organic solvent; some work better with ferment¬ ation cells and others with dried cells, etc. Known steroid-1-dehydrogenation processes with A. simplex cells in fermentation broths use Δ -3-keto steroid substrates which at the C 2 χ position have 21-hydroxy or 21-monocarboxylic esters functiona¬ lity, preferably acetic acid esters. The substrate concentrations of these 21-hydroxy- or 21-acyloxy- Δ 4 -3-keto steroids in the fermenta tion media is from about 1.5 to about 5 g/liter, as described in U Patent 2,837,464. Known processes to 1-dehydrogenate Δ -3-ket steroid 21-hemiescers (I) use substrate concentration from about 2 t about 10 g/liter based on the 21-ester weight or from about 1.4 t about 7.1 g/liter based on the 21-hydroxy steroid parent, see U Patent 3,770,586. The process of the present invention using A . simplex permits the advantageous use of higher substrate levels i bioconversions performed with fermentation broths using 21-di- carboxylic acid ester Δ -3-keto 21-hemiester steroid substrates (I) and a complete reaction in a shorter time period. The process of the present invention permits the advantageous use of higher substrate concentrations with the advantageous resul of shorter reaction times. The process of the present inventio permits use of Δ -3-keto 21-hemiester steroid (I) concentrations o about 4 to about 30 g/1 based on the non-esterified Δ -3-keto steroid. Below about 4 g/1 is operable but not commercially impor¬ tant. Above about 30g/l the residuals increase to a point where it is preferable to perform the reaction at lower substrate levels. It is preferred tnat the Δ -3-keto steroid (I) concentration be equiv¬ alent to about 4 to about 12 g/1 based on the parent 21-hydroxy steroid. The process of the present invention provides the Δ-'- ' -3-keto steroids (II) in improved yields and in less time than previously required. The reaction producing the Δ 1 ' -3-keto steroids (II) is complete in about 4 to about 72 hr. Present steroid-1-dehydrogen¬ ation processes usually average about 120 hr using hemiesters with S . af finis , see US Patent 3,770,586. When the Δ -3-keto 21-hemiester steroid starting material has been converted to the corresponding A 1 ' -3-keto steroid (II) product it is isolated by means well known to those skilled in the art. The Δ- 1 -' -3-keto steroid (II) products are useful as inter¬ mediates in the production of steroid pharmaceuticals . DEFINITIONS AND CONVENTIONS The definitions and explanations below are for the terms as used throughout this entire document including both the specification and the claims . I. CONVENTIONS FOR FORMULAS AND DEFINITIONS OF VARIABLES The chemical formulas representing various compounds or molecu¬ lar fragments in the specification and claims may contain variable substituents In addition to expressly defined structural features. These variable substituents are identified by a letter or a letter followed by a numerical subscript, for example, "Z ] _" or "R^" where "i" is an integer. These variable substituents are either monovalent or bivalent, that Is, they represent a group attached to the formula by one or two chemical bonds. For example, a group Z- | _ would repres- ent a bivalent variable if attached to the formula CH3-C(=Z ] _)H. Groups R^ and R-- would represent monovalent variable substituents if attached to the formula CH3-CH 2 -C R j _) (R^)H 2 . hen chemical formulas are drawn in a linear fashion, such as those above, variable sub¬ stituents contained in parentheses are bonded to the atom immediately to the left of the variable substituent enclosed in parenthesis. When two or more consecutive variable substituents are enclosed in parentheses, each of th.e consecutive variable substituents is bonded to the immediately preceding atom to the left which is not enclosed in parentheses. Thus, in the formula above, both R^ and R,- are bonded to the preceding carbon atom. Also, for any molecule with an established system of carbon atom numbering, such as steroids, these carbon atoms are designated as C j _, where "i" is the integer cor¬ responding to the carbon atom number. For example, Cg represents the 6 position or carbon atom number In the steroid nucleus as tradition- ally designated by those skilled in the art of steroid chemistry. Likewise the term ,r Rg" represents a variable substituent (either monovalent or bivalent) at the Cg position. Chemical formulas or portions thereof drawn in a linear fashion represent atoms in a linear chain. The symbol "-' r in general represents a bond between two atoms in the chain. Thus CH3-0-CH 2 - CH(R ; r_ -CH3 represents a 2-substituted-l-methoxypropane compound. In a similar fashion, the symbol "=" represents a double bond, e.g., CH =C(Rχ) -O-CH3, and the symbol "≡" represents a triple bond, e.g., HC≡C-CH(R£) -CH 2 -CH.3. Carbonyl groups are represented in either one of two ways: -CO- or -C(=0)~, with the former being preferred for simplicity. Chemical formulas of cyclic (ring) compounds or molecular fragments can be represented in a linear fashion. Thus, the compoun 4-chloro-2-methylpyridine can be represented in linear fashion by N*=C(CH3> -CH=CC1-CH=C*H with the convention that the atoms marke with an asterisk (*) are bonded to each other resulting in th formation of a ring. Likewise, the cyclic molecular fragment, 4 (ethyl) -1-piperazinyl can be represented by -N - (CH ) 2 -N(C 2 H5) -CH 2 C*H 2 . A cyclic (ring) structure for any compound herein defines a orientation with respect to the plane of the ring for substituent attached to each carbon atom of the cyclic compound. In formula depicting such compounds , a substituent attached to a carbon ato below the plane of the ring is identified as being in the alpha (α configuration and is indicated by a broken, dashed or dotted lin attachment to the carbon atom, i.e., by the symbol "- - -" o "....". The corresponding substituent attached above the plane o the ring is identified as being in the beta (β) configuration. Whe a variable substituent is bivalent, the valences may be take together or separately or both in the definition of the variable. For example, a variable R^_ attached to a carbon atom as -C(=R. j _) might be bivalent and be defined as oxo or keto (thus forming carbonyl group (-CO-) or as two separately attached monovalen variable substituents -R j _.^ and β-R± -k - When a bivalent variable, R^, is defined to consist of two monovalent variable substituents, the convention used to define the bivalent variable is of the for " and .( -Rχ-k are attached- to the carbon atom to yield -C(α-R^ j) ( / S-R^.^)-. For example, when the bivalent variable R , -C(=Rg)-is defined .to consist of two monovalent variable substituents, two monovalent variable substituents are α-Rg_- ] _ : / S-Rg_ 2 α-Rg_g :9-Rg . 10 , etc, yielding -C(α-R . 1 ) ( / 9-R 6 . 2 ) - , .... -C(α-R 6 _ ) ( / 3-R . 10 ) - , etc. Likewise, for the bivalent variable Rι , -C(=Rιι)-, two monovalent variable substituents are Q - n-i'/9-Rn. 2 • For a ring substituent for. which separate a and β orientations do not exist (e.g. due to the presence of a carbon carbon double bond in the ring), and for a substituent bonded to a carbon atom which is not part of a ring the above convention is still used, but the α and β designations are omitted. Just as a bivalent variable may be defined as two separate monovalent variable substituents, two separate monovalent variable substituents may be defined to be taken together to form a bivalent variable. For example, in the formula -C_(R j _)H-C 2 (Rj)H- (C ] _ and C 2 define arbitrarily a first and second carbon atom, respectively) R j _ and R,- may be defined to be taken together to form (1) a second bond between C^ and C 2 or (2) a bivalent group such as oxa (-0-) and the formula thereby describes an epoxide. When R j _ - and RJ are taken together to form a more complex entity, such as the group -X-Y- , then the orientation of the entity is such that C ] _ in the above formula is bonded to X and C is bonded to Y. Thus, by convention the designa¬ tion "... R £ and Ri are taken together to form -CH 2 -CH 2 -0-CO- ..." means a lactone in which the carbonyl is bonded to C . However, when designated "... R j and R j _ are taken together to form -CH 2 -CH 2 -0-CO- the convention means a lactone in which the carbonyl is bonded to C ] _. The carbon atom content of variable substituents is indicated in one of two ways. The first method uses a prefix to the entire name of the variable such as "C_-C ή _" , where both "1" and "4" are Integers representing the minimum and maximum number of carbon atoms in the variable. The prefix is separated from the variable by a space. For example, "C- j _-C alkyl" represents alkyl of 1 through 4 carbon atoms, (Including Isomeric forms thereof unless an express indication to the contrary Is given) . Whenever this single prefix is given, the prefix indicates the entire carbon atom content of the variable being defined. Thus C 2 -C4 alkoxycarbonyl describes a group CH3-(CH 2 ) n -0- - CO- where n is zero, one or 2. By the second method the carbon atom content of only each portion of the definition is indicated separate¬ ly by enclosing the "Ci-Cj" designation in parentheses and placing it immediately (no intervening space) before the portion of the definl- tion being defined. By this optional convention (C1-C3)alkoxy¬ carbonyl has the same meaning as C 2 -C alkoxycarbonyl because the " c l' c 3" refers only to the carbon atom content of the alkoxy group. Similarly while both C 2 -Cg alkoxyalkyl and C ] _-C3)alkoxy C -C3 alkyl define alkoxyalkyl groups containing from 2 to 6 carbon atoms, the two definitions differ since the former definition allows either the alkoxy or alkyl portion alone to contain 4 or 5 carbon atoms while the latter definition limits either of these groups to 3 carbon atoms . When the claims contain a fairly complex (cyclic) substituent, at the end of the phrase naming/designating that particular sub¬ stituent will be a notation in (parentheses) which will correspond to the same name/designation in one of the CHARTS which will also set forth the chemical structural formula of that particular substituent. II. DEFINITIONS All temperatures are in degrees Centigrade. HPLC refers to high pressure liquid chromatography. DMF refers to dimethylformamide. hen solvent pairs are used, the ratios of solvents used are volume/volume (v/v) . A . simplex refers to Arthrobacter simplex . B . cyclooxydans refers to Bacterium cyclooxydans . The concentrations of Δ -3-keto 21-hemiester steroid substrates (I) are expressed as grams/liter, the weight (g) being expressed as the amount of the corresponding 21-hydroxy steroid. EXAMPLES Without further elaboration, it is believed that one skilled in the art can, using the preceding description, practice the present invention to its fullest extent. The following detailed examples describe how to prepare the various compounds and/or perform the various processes of the invention and are to be construed as merelv illustrative, and not limitations of the preceding disclosure in any way whatsoever. Those skilled in the art will promptly recognize appropriate variations from the procedures both as to reactants and as to reaction conditions and techniques . EXAMPLE 1 A . simplex Bioconversion of 11/3, 21-Dihydroxypregna- 4,17(20) -diene-3-one 21-Hemisuccinate Potassium Salt (I) A. Primary Microorganism Seed Development Stage Cells from a culture of Arthrobacter simplex (UC582, ATCC 6946), maintained oh nutrient agar slants, are resuspended in sterile water (5 ml) and used to inoculate 300 ml primary seed medium which is composed of nutrient broth (12 g/liter of Difco brand) , which had been sterilized at 121° for 30 minutes prior to inoculation. The mixture is incubated in 1 liter shake flasks at 28° on a rotary shaker (250 rpm, 2 inch throw) for two days. B. Secondary Seed Development Seed Stage The growth medium used for the secondary proliferation of the microorganism consists of an aqueous mixture of the following nutrient materials: Concentration of Ingredients Supplier Dried Corn steep liquor 20 g/1 Roquette Freres (Solulys A) Lard oil (No. 2) 5 g/1 Geo. Pfau & Son Silicone defoamer 0.1 ml/1 Union Carbide (SAG-471) Cortisone acetate 0.1 g/1 The Upjohn Company The pH of the medium Is adjusted to pH 7.0 with reagent grade sodium hydroxide prior to sterilization, as described above. Two shake flasks (300 ml) from the primary stage above are used to Inoculate a 250 liter secondary seed medium. The fermentor is run at about 28° , at an agitation rate of 280 rpm, an aeration rate of 100 standard liters per minute (slm) and at 5 psi back-pressure. The secondary seed stage Is incubated for two days. C. The Fermentation (Reaction) Stage of the Process of this Invention. An aqueous medium containing 20 g/1 of dried corn steep liquor (Solulys A), 20 g/1 of lard oil No. 2 , 0.1 g/1 of cortisone acetate and 0.2 ml/1 of a silicone defoamer (SAG-471 - Union Carbide) is adjusted to pH 6.0 with sodium hydroxide prior to sterilization of the mixture. The resulting medium is sterilized at 121° for 30 min in a fermenter having a working volume of 250 liters. The medium in the tank is stirred at a temperature of 28° , an agitation rate of 280 rpm, an aeration rate of 25 slm and with a 5 psi backpressure. The fermentor contents are inoculated with 5 percent by volume of the final fermented volume of a 48 hour growth (secondary seed medium from above) of Arthrobacter simplex (ATCC 6946) . The resulting culture mixture is allowed to grow for 36 hours at which time the pH was adjusted to 7.5 and maintained by an automatic addition of sulfuric acid or sodium hydroxide. Menadione is added after the pH adjustment, to a final concentration of 0.17 g/1. The Δ -3-keto 21- hemiester steroid substrate (I), 11 / , 21-dihydroxypregna-4, 17(20) - diene-3-one 21-hemisuccinate potassium salt (I) is added in DMF to give a final concentration of 11.35 g/1 (which is equivalent to 8 g/1 of the corresponding 21-hydroxy Δ -3-keto steroid) . This mixture is incubated for 48 hours. The completeness of the reaction is determined by an HPLC assay. The products, a mixture of 11 / 9,21-dihydroxypregna-1,4,17(20) -triene- 3-one (II) and 11 / 9,21-dihydroxypregna-l,4, 17(20) -triene-3-one 21- hemisuccinate (II) are hydrolyzed to 11 / ,21-dihydroxypregna- l,4,17(20)-triene-3-one (II) by base. This is followed by filtration of the mixture where the A-'-' -3-keto steroid (II) is retained in the filter cake and subsequently extracted with aqueous acetone and crystallized by procedures well known in the art. The products were identified by comparison with known samples by HPLC on a silica based C-8 column, 25 cm, eluting with a mobile phase of acetonitrile- /water/acetic acid (40/60/0.1). The retention time of the 21-hydroxy compound is 12.5 min. and of the 21-hemisuccinate is 28.5 min. EXAMPLES 2-20 Following the general procedure of EXAMPLE 1 and making non- critical variations but starting with the Δ -3-keto 21-hemiester steroids (I) of Column A below, the corresponding ^ • steroids (II) are obtained either as the free 21-alcohol or the 21-hemiester or a mixture thereof: EXAMPLE Column A 2 17α,21-dihydroxypregna-4,9(11) -diene-3,20-dione 21-hemi¬ succinate 3 11 / 9,17Q,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione 21- hemisuccinate potassium salt 4 11/9,21-dihydroxy-6α-methylpregna-4,17(20) -dien-3-one 21- hemisuccinate sodium salt 5 11 / 9,17α, 21-trihydroxypregn-4-ene-3,20-dione 21- hemisuccinate triethylamine salt 6 17α,21-dihydroxy-6α-methylpregna-4, 9(11) -diene-3,20-dione 21-hemisuccinate 7 17a,21-dihydroxy-16/3-methylpregna-4,9(11) -diene-3,20-dione 21-hemisuccinate sodium salt 8 6α-fluoro-21-hydroxypregna-4, 9(11) , 16-triene-3,20-dione 21- hemisucclnate potassium salt 9 21-hydroxypregna-4, 9(11) ,16- riene-3.20-dione 21-hemisuc- cinate 10 17α,21-dihydroxy-16α-methylpregna-4,9(11) -diene-3,20-dione 21-hemisuccinate 11 11 / 9,21-dihydroxypregna-4,17(20) -dieπ-3-one 21-hemifumarate 12 17α,21-dihydroxypregna-4,9(11) -diene-3,20-dione 21-hemioxy- late 13 ll^,17α,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione 21- . hemimaleate sodium salt 14 ll / 9,21-dihydroxy-6α-methylprεgna-4,17(20) -dien-3-one 21- hemiglutarate sodium salt 15 11/3,17α,21-trIhydroxypregn-4-ene-3,20-dione 21-hemiadipate triethyla lne salt 16 17α,21-dihydroxy-6α-methylpregna-4,9(11) -diene-3,20-dione 21-hemifumarate sodium salt 17 17α,21-dihydroxy-16^-methylpregna-4.9(ll) -diene-3,20-dione 21-hemimaleate 18 6α-fluoro-21-hydroxypregna-4,9(11) ,15-triene-3,20-dione 21- hemiglutarate 19 21-hydroxypregna-4,9(11) ,16-trlene-3.20-dione 21-hemi * oxy- late 20 17α,21-dihydroxy-16α-methylpregna- ..9(ll)-diene-3,20-dione 21-hemimalate EXAMPLE 21 A. simplex (dried cell) Bioconversion of 6α-fluoro-21- hydroxypregna-4,9(11) ,16(17) -triene-3,20-dione 21- hemisuccinate (I) Following the general procedures of US Patent 4,524,134, and making non-critical variations 1.0 gram of dried A. simplex cells are suspended in potassium phosphate buffer (.05 M, 100 ml, pH 7.5). Menadione (8.6 mg) is dissolved in ethanol (1 ml) and Is added to the cell suspension. The Δ -3-keto 21-hemiester steroid substrate (I), 6α-fluoro-21-hydroxypregna-4,9(11) ,16(17)-triene-3, 20-dione 21-hemi¬ succinate (I) is dissolved in DMF and added to the shake flask to a final steroid concentration of 4.0 g/1 (based on the corresponding 21-hydroxy steroid) and a final DMF concentration of about 5 percent. Replicate flasks are incubated on a rotary shaker at 31° for three days . Analysis of the bioconversion mixture by HPLC indicates tha 99 per cent of the Δ -3-keto steroid 21-hemiester substrate (I) i dehydrogenated to the corresponding Δ -1 - product and is present as mixture of the 21-hemisuccinate (II) and 21-hydroxy forms (II) . Th products were identified by comparison with known samples by HPLC, o a silica based C-8 column, eluting with acetonitrile/THF/water/aceti acid (9/31/60/0.3), retention time of the 21-hydroxy compound is 8.1 min, the 21-hemisuccinate is 12.51 and the substrate is 16.25 min. EXAMPLE 22 B . cyclooxydans Bioconversion of 11/9, 17α,21-trihydro xy-6α-methylpregna-4-ene-3, 20-dione 21 hemisuccinat succinate (I) Following the general procedure of US Patent 4,704,358 an making non-critical variations but starting with 11 / 9, 17α,21-trihydro- xy-6α-methylpregna-4-ene-3 ,20-dione 21-hemisuccinate (I) and using B . cyclooxydans, the title compound is obtained. The bioconversion is monitored by thin layer chromatography of butyl acetate extracts and known standards on a silica gel (fluorescence) plate in a chloroform/methyl-cellosolve/95% ethyl alcohol (87.5/5/7.5) solvent system. CHART A

Claims: CLAIMS 1. A process for the production of a Δ 1 • 4 -3- ' keto 21-hemiester steroid of formula (II) where (B-I) Rg is =CH 2 or α-Rg.-^-H where Rg. ] _ is -H, -F and -CH3, R7 is α-H: / 9-H; (B-II) Rg is Rg_3.-Rg_ and R7 is R _3:R7_ where one of R -3 and Rg_4 taken together with one of R7.3 and R7.4 form a second bond between Cg and C , the other of -3 and Rg.4 is -CH3 and the other of R 7-3 and R 7-4 is -H; (C-I) Rχ]_ is Q - li-1'/S-Rχι_2 where one of Rχχ.χ and Rχχ_2 is taken together with Rg to form a second bond between Cg and Oχχ, and the other of Rχχ.χ and Rχχ_2 is -H; (C-II) Rg is -H, -F, -Cl or Br and Rχχ " is =0 or Q- χχ-3 ' / 9- χχ-4 where one of Rχχ_3 and χχ-4 is -H and the other of Rχχ.3 and Rχχ.4 is -H or -OH; (C-III) R x is α-H:/3-0-, where β-O- is taken together with Rg to form an epoxide between Cg and 0χχ in the /3-configuration; (D-I) Rx is Rlg-l ' Rχg-2 and R-17 i 17-l : 17-2> where one of R ] _ _χ and Ri -2 i "H and the other taken together with one of 17.1 or Rχ7-2 forms a second bond between C^g and C1 and the other of R 17-l and R 17-2 is -CO-CH 2 -0-R 2 ι where R 21 is -H, -CO- (X-]_) -C00R 2 ι_ 1 , where X^ is -CH=CH- and -(CH 2 ) n ι- where ni is 1-8 and where R 2 i-i i -H or a cation; (D-II) Ri is α-H: / 3-H, and where R i7 is where R 2 χ is as defined above; (D-III) R 16 Is =CH 2 or α-R 16 . 5 :/9-R 16 .g where χg-5 is -H, -OH or -CH3, ig.g is -H or -CH3 with the proviso that one of Rι -5 or i . is -H, and where 7 is -Rι 7 .5: / 9-CO-CH 2 -0-R 2 where 17.5 is -H or -OH, where R 2 is as defined above, which comprises contacting a Δ - 3-keto 21-hemiester steroid of formula (I) where (D-I) Rτ_ Is i6_ι' ιg-2 and R 17 is l7-i * i7-2 ' where one of Rχg_l and Rχg. 2 i "H and the other taken together with one of Rχ7.χ or χ7_ 2 forms a second bond between 0 and C17 and the other of R 17-l and R 17-2 is -C0-CH 2 -0-C0-(X )-C00R 21 _ , where X-j_ and R _ are as defined above; (D-II) R 16 Is α-H: / 3-H, and where R 7 is =CH-CH 2 -0-CO-(∑χ)- C00R 2 _ι where Xi and 2 l-1 a e as defined above; (D-III) R 16 is =CH 2 or α-R 16 . 5 :/9-R_g.g where i .5 is -H, -OH or -CH3, Rχg.g is -H or -CH3 with the proviso that one of χg_5 or Rχg.g is -H, and where R i7 Is -Rι 7 . 5 :/3-CO-CH 2 -0-CO-(Xi) -C00 ι_ where R17.5 Is -H or -OH and where Xi and R 2 l-l are as defined above, and where R , Rg and R ] _ are as defined above, with the steroid Δ-*-- dehydrogenase of Arthrobacter simplex or Bacterium cyclooxydans . 2. A process for the production of a Δ ■ -3-keto steroid (II) according to claim 1 where the process is performed in the presence of an effective amount of an exogenous electron acceptor. 3. A process for the production of a A""- ' -3-keto steroid (II) according to claim 2 where the exogenous electron carrier Is selected from the group consisting of menadione. menadione bisulfite, 1,4 naphthoquinone, phenazine methosulfate , phenazine ethosulfate, dichlorophenol indophenol, cytochrome c. 4. A process for the production of a Δ 1, -3-keto steroid (II) according to claim 3 where the exogenous electron acceptor i menadione, menadione bisulfite and 1,4-naphthoquinone. 5. A process for the production of a ^ • -3-keto steroid (II) according to claim 1 where Rg_ι is -H, -F, -CH3 and =CH . 6. A process for the production of " a A- 1 -' -3-keto steroid (II) according to claim 1 where one of χi-3 and Rχι_ is -H and the other is -H or -OH. 7. A process for the production of a Δ^' -3-keto steroid (II) according to claim 1 where R]_]_ is <x-R\ . '- β - Rιl - 2 where one of n- and Rn-2 i s taken together with Rg to form a second bond between Cg and On, and the other of n-i and n_ is -H. 8. A process for the production of a Δ ■ -3-keto steroid (II) according to claim 1 where R i7 is =CH-CH 2 -0-C0- (X ] _) -COO-R 2 ι_ι. 9. A process for the production of a Δ 1 ' -3-keto steroid (II) according to claim 1 where Rχ 7 is α-OH: / 9-CO-CH 2 -0-CO- (Xi) -C00R 2 -i. 10. A process for the production of a Δ 1 ' -3-keto steroid (II) according to claim 1 where X is -CH=CH-. 11. A process for the production of a A^ ' -3-keto steroid (II) according to claim 1 where Xi is -(CH2) n χ-. 12. A process for the production of a Δ-*-' -3-keto steroid (II) according to claim 11 where n is 2-4. 13. A process for the production of a Δ^'^-3-keto steroid (II) according to claim 11 where n^ is 2. 14. A process for the production of a A - ' -3-keto steroid (II) according to claim 1 where the cation Is monovalent. 15. A process for the production of a -• -3-keto steroid (II) according to claim 14 where the monovalent cation is sodium or potassium, ammonium or -H . 16. A process according to claim 1 where the Δ -3-keto 21-hemiester steroid (I) Is selected from the group consisting of the hemiesters (-C0-X ] _-C00R 2 χ.ι) at C 2 χ of the following Δ 4 -3-keto steroids 17α,21-dihydroxypregna-4,9(11) -diene-3,20-dione, 11 / 9,21-dihydroxypregna-4,17(20)-dien-3-one, 11/3,17α,21-trlhydroxy-6α-methylpregn-4-ene-3,20-dione, 11 / 9, 21-dihydroxy-6α-methylpregna-4,17(20) -dien-3-one, 11/9, 17α,21-trihydroxypregn-4-ene-3,20-dione, 17α,21-dihydroxy-6α-methylpregna-4, (11) -diene-3,20-dione, 17α,21-dihydroxy-16 / 9-methylρregna-4,9(11) -diene-3,20-dione, 6α-fluoro-21-hydroxypregna-4,9(11) ,16- riene-3 ,20-dione, 21-hydroxypregna-4,9(ll) ,16-trlene-3 ,20-dione. 17α . 21-dihydroxy-16α-methylpregna-4 , 9 (11) - diene- 3 , 20-dione . 17 . A process for the production of a A-*- ' - 3 -keto steroid (II) according to claim 1 where the contacting Is performed with cells of A. simplex or B . cyclooxydans . 18 . A process for the production of a Δ^ ' - 3 -keto steroid (II) according to claim 2 where the contacting with A. simplex or B . cyclooxydans Is with dried cells of A. simplex or 3 . cyclooxydans . 19. A process for the production of a Δ-"-' -3-keto steroid (II) according to claim 18 where the dried cells are air or heat dried. 20. A process for the production of a Δ-*-' -3-keto steroid (II) according to claim 1 where the contacting with A. simplex or B . cyclooxydans is with viable reproducing cells under aerobic fermenta¬ tion conditions In an aqueous nutrient medium. 21. A process for the production of a A- < - 3 -keto steroid (II aaccccoorrddiinngg ttoo ccllaaiimm 22 wwhheerree tthhee steroid Δ -dehydrogenase is in th form of a cell free preparation.

Inventors: KOMINEK LEO A, STEIERT PAULA S, WOLF HOLLY JO, KOMINEK, LEO, A., STEIERT, PAULA, S., WOLF, HOLLY, JO
Applicants: UPJOHN CO, THE UPJOHN COMPANY
Application year: 1988
Main classification: C12P33
IPC codes: C12P33, C12R1