CA1172590A - Steroids of the pregnane series substituted in the 17- position, and their manufacture and use - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The present invention provides a microbiological process for the manufacture of 11.beta.-hydroxy-steroids of the pregnane series substituted in the 17.alpha.-position by an acetal group of the formula

Description

1~72590 The present invention relates to a microbiological pro-cess for the preparation of ll~-hydroxy-steroias of the preg-nane series.

This application is a divisional application of copending application No. 320,177 filed January 24, 1979 which latter application is directed to new corticoids, processes for their manufacture, their use and pharmaceutical preparations con-taining these corticoids.
It has been known for a long time that the topical activ-ity of anti-inflammatorally active 17a-hydroxy-corticoids can be increased by esterifying their 17-hydroxyl group. [In this connection see the general report of Thomas L. PoppPr and Arthur S. Watnick, "Anti-Inflammatory Steroids in Anti-Inflam-matory Agents" Volume 1, Academic Press, New York, San Fran-cisco, London (1974~, pages 268-271].

It has been found in accordance with the invention of 20 copending application No. 320,177 that the topical activity and/or the dissociation between the desired topical anti-inflammatory activity and the undesired systemic activity can be further increased by replacing the hydrogen atom of the 17~-hydroxy-groups of these corticoids, not by an ester group, but by an acetal group or a thioacetal group.

The invention of the copending application 320,177 accord-ingly provides compounds of the general formula I

- (~2~3 ~ oc~, O ..

in ~hich each o~ the bonds .~æ~ in the 1,2- and 6,7-positions represent~ a ~ingle carbon-to-carbo~ bond or 2 double carDon-to-ca~bon bond, represents a hydrogen atom, a ~lucr~e ~tom, ~ chlorina ~ atom or a methyl group~
Y represents a hydrogen atom and Z rep~esent~ a h~drogen atom, a fluorine atom or a chlorine atom or ~ 2nd Z together represent a carbon-t~-carbon bondS
V represents a ~-hydroxymethylene group, a ~-chloromethy-lene group or a carbo~yl group, W repre~ents a methylene group, an ethylidene group or a vInyl~dene group~
Q represents an oxygen atom or a sulphur atom~

Rl represents an alkyl group containing 1 to 8 carbon atoms which may~ be interrupted by an oxygen atom, or represents a benzyl group and R2 represents a hydrogen atom or an alkyl group containing 1 to 6 carbon atoms or Rl and R2 together represent a trimethylene group or a tetra-methylene group, and R3 represents a hydrogen ato,m, a fluorine atom, a chlorine atom or a free or esterified hydroxyl group.
The esterified hydroxyl group represented by R3 is under-stood herein to include a group derived not only from an or-ganic acid but also from an inorganic acid.

As compounds of the invention of the copending application there may be mentioned, for example, compounds of the general formula Ia ~72590 1~2R~5 R2 =o HO ~ .~ ~ ~9C~Q ~

~ (Ia) o . ~ ' ' '.
.

~ n ~hich W, Q, ~ ~ R2 and ~ h~ve the meanings gi~en above, r~presents a hydrogen atom, a ~luorine a~o~ or a ~ethyl group and Z~ represents a hydrogen atom, a ~luorine atom or a chlorl-te atom, compol~nds o~ the general formula Ib ~72590 l 2R3 2 C=O
~0~ aHQRl ~ (Ib) i~ which Zl ~ Ty~ Q, Rl, R2 a3~d R~ have the meanings give above ~ and compo~l~ds of the general ~ormula Ic - I 2R~ IR2 C~O
H0 ~ OCHQR L

~L~ (I c ) in ~Thich Xl, W~ Q, Rl, R2 and R3 h2ve the mea~ings g~ven aboveO

1~72590 The new corticoids o~ the general for~ula I
may contain as the substituent represented by ~ a straight chained or branched chain al~yl group containing 1 to 8, preferably 1 to 6, carbon atoms. Such alkyl groups are, for e~a~ple, the methyl group, the ethyl group, the propyl group, the isopropyl group, the butyl group, the isobutyl group, the tertO-butyl ~roup, the pentyl group, the isopentyl group, the hexyl group, the heptyl ~roup or the octyl group, Eowever, the alkyl group represe~ted by ~ may also be interrupted by an oxy~en atom. Such groups are, ~or e~ample, the 2-methoxyethyl group, the 3-metho~ypropyl group or the 2 etho~yethyl groupO
The symbol R2 in the corticoid~ of the general iormula I ~ay repr-esent an alkyl group containing 1 to 6, pre~erably 1 to 4, carbo~ atom~ and may represe~t~
for example, the methyl group, the ethyl gro~p, the propyl group or the butyi group. ~oteworthy are those corticoids o~ the general for~ula I in ~Ihich R2 repre-sents a hydro~en atom, as they are unable to fo~m diastereoiso~eric mixtures.
The ince~tion o~ act~on ~nd the duration of action oi the new corticoids and also their solubllity in physiologically tolerable solvents depend, as is the case with the known ~orticoids, especially on whether any 1~72590 -- 8 ~

h~droxyl group i~ the 21-position i~ estcrified and, i~
80, by what acid.
As e~terified 2i-hydroxy-gl-oups ~epre~ented by there come into con~ideration preierabl~ acyloxy group~
contai~ing 1 to 16 carbon atome in the acyl group, more prefer~bly 1 to 8 c~rbon atoms in the acyl group, sulphate groups or phosphate groups. ~uitable acylo~y groups are, ~or e~ample, those derived from straight chained or branched chained, saturated or unsatur~ted aliphatic mono- or di-carboxylic acids, which may be sub~tituted in ~he usual manner, ~or example by hydrogyl groups or amino groups or halogen atoms.
There are also suitable as acyloxy group~ those derived ~rom cycloaliphatic, aro~atic, ~i~ea aromatic-alipha~ic or heterocyclic acid6, unich may also be E~bstituted in the usu~l manner. ~ suitable acylo~y groups there may be mentioned, for example9 for~yloxy, acetoxy, propionylo~y, butyrylo~y, pentanoylo~y, he~anoyl-o~y, octanoylo~y, undecanoyloxy, dimet~yl~cetoxy~ tri-~et~yl2cetoxy, diethylaceto~, tert.-butylacetoxy, benzoylo~y, phenacetyloxy, cyclopentyl-propionyloxy, hydro~yaceto~y, monochloracetoxy, dichloracetoxy and trichloracetoxy groups and al~o di~ethyl~m~noacetoxy, trimethyl2minoaceto~y, diethylaminoaceto~y, piperidino-aceto~y, nicotinoylo~y, ~-carboxypropionyloxy and w-car~o~ypentanoylo~y groups.

1~72590 For the production of water-soluble active substances the 21-acyloxy-compounds containing a basic nitrogen group in the acyl group may be converted into the corresponding acid addition salts, lor example the hydrochlorides, hydrobromides, sulphates, phosphates, oxalates, tartrates or maleates.
Furthermore, the 21-d carboxylic acid monoesters, and also the sulphuric acid esters and phosphoric acid esters may be con-verted into their alkali salts, for example the sodium or potassium salts, in order to increase their solubility in water.

The new corticoids of the general formula I rnay be pre-pared according to one of the processes disclosed in the copending application as de~ined below.
15 .
The copending application thus provides a process for the manufacture of a compound of the general formula I, wherein a 17~-hydroxy-steroid of the general formula II

1 ~7 2 590 ' ' . C=O
~ 0~

0 ~ (II) in which the bonds ~ , Y, Z, ~ and W have the meaning6 given above and Rl3 represents a hydrogen atom, a ~luorlne atom, & chlori~e atom or an esterified hydroxyl group, i~ reacted, i~ desired after inter~ediate protection of any ll~-hydroxyl ~roup, ~) when Q represents an o~ygen atom, ~nth an acetal of the general formula III
.R ~C(ORl)2 (III) in which P~ and R2 have the meanings given above, or tb) ~en Q represents an oxygen atom, with an -halogen ether o~ the general ~or~ula IV

Hal-R2C~- ~ (IV) in ~hich ~ ~nd R2 have the meanings given above and ~1 represents a chlorine atom, a bromine atom or a~
iodine atom, or ~1 ~7~590 (c) ~hen Q represents an oxygen atom and R2 represents a hydrogen atom or an alkyl group containing 1 to 3 carbon atoms, with a vinyl ether of the g~neral ~orinula V

R 2~ g ~ ~ (V) in which ~ haB ths meaning given above and R~2 repre-sent~ a hydrogen atom or an alkyl group containing 1 ~o 3 carbon ato~s, or (d) when Q represents a sulphur atom, with a sulphox~de o~ ~he general formula Vl R2CH2SORl in ~ihich P.l c~d R2 have the meanings given above, and, i~ desired, in ~ny resulting steroid saturated in the 1,2-posit~on obta-ned by any one of the variants (a) to (d) dehydrogenation is carried out in the 1,2-position, and/or in any res~lting steroid an~
hydroxyl group is oxidized to form an o~o group, andlor any 21-ester group is hydrol~sed and/or any 21-hydroxyl group i8 esterified or exchanged for a fluorine atom or - a chlorine atomO
Th~s process may be carried out under conditions that are kno~m [Synthesi~
1975, 2786, J.Chem.SocO (66), 1974, 431, J.Amer. Chem.
SocO 14, (1952), 1239, United State~ Patent ~o~

~7~590 3,38~,~94 and AngewO Chemie~ ~, (1978)~ 289]o Thus, for e~ample, the steroids o~ the general iormula II may be reacted with an acetal ol the ~ene-~l formula III in the presence of acid catalysts, for e~ample perchloric acid or para-toluenesulphonic ac~d, or advantageously phosphorus pen-toxideO This reaction may be carried out in the absence of ~urther ~olvents or in the presence of inert solvents (for example chloroform, methyl~ne chloride, tetrachlorethane, tetrachloromethane~
toluene~ diethyl ether, tetrahydrofuran or dioxan)O
The reaction is usually carried out at a reaction tempera-~ure bet~een -20C to +50C~ and is sui~able especially lor the preparation of those steroids of the general formula I in which R2 represent~ a hydroge~ a~omO
On the other hand, the s~eroids of the general for~ula II may also be reacted wi~h a vinyl ether of the -general formula V. This reaction is prefer2bly carried out in one of the above-mentioned inert solvents with the additio~ of acid catalyst~ ~for e~ample perchloric acid, pàra-toluenesulphonic acid or methanesulphonic acid)O
The reaction is preferably carried out at a reaction temperatura o~ -20~ to 100Co ~ urthe~more, the steroids of the ~eneral formula II may also be reacted with an a-halogen-ether of the ~ener~l formula IVo This reaction may be carried out~
for ~xample, in an inert polar solvent, for example ~L~ 7~590 ( acetonitrile~ dimethylform~aide, ~-~eth~l-pyrrolidone, he~amethyl-phosphoric acid triamide or he~methyl-phosphoric acid tri~mide, with the additlon o~ baslc catalyst~, for eY.ample silver oxide, triethylamine or dii~opropylethylamine. The reaction i~ pre~erably carried out at a reaction temperature o~ -20C to ~100C~
In the above-~entioned reactions there are ~ormed corticoids o~ the general formula I in ~/hich Q represents an o~ygen atom. For the preparation of corticoids in which Q repr~sents a sulphur atom the corticoids of the general.formula II ~ay be reacted with ~ulphoxides of the ge~eral ~ormula VIo This reaction may be carried out, for example~ by reacti~g the sulphoxide and the steroid~
i~ desired in an inert solvent (for example methylene chloride, tetrachlorethane or tetrah~dro~uran), at approximately -20C to 100C with the addition of anhydrides (preferably acetic Pnhydride) and acid catalysts (for e~ample acetic acid or boron tri~luoride).
I~ for this process there are used as starting compounds ll~-h~droxy-corticoids o~ the general ~ormula II it i8 ad~antageous to protect the ll~-hydro~y group intermediately, in order to avoid its partial acetalisation. This can be achieved, for ~ample, by converting the ll~-hydro~y group be~ore the ^cetalisation into the corresponding nitrate, ~ormate, " ~72590 or tr;~logeno-acetate (especi~lly trlfluoracetate) and then splitting thi~ ester after carrying out the processO
The esterification of the ll~-hydroxy-corticoids of the general formula II with nitric acid may be brought about, for ex~mple, with acet~l nitrate, prepared by mixing fu~ing nitric acid with acetic anhydride. After the 17a-acetalisation the nitrates can be re-co~verted into the ll~-hydroxy-corticoids, ~or eY.ample, by reaction ~ith ~inc dust in acetic acid.
The esterification of the ll~-h~-droxy--corticoids of the general for~ula II with formic acid can be brought about, for example, by means o~ formic acid-acetic ~&~d anh~dride ~ith the use of 4-dimethylaminopyridine as catalystO After the 17a-acetalisation, the resulting for~ylo~y-corticoids can then be con~erted i~to the corrésponding ll~-hydrox~-steroidq by basic hydrolysis (for example by means of a solution o~ sodium methyla~e) or by enzym~tic hydrolysis.
The es~erification of the ll~-hydroxy-corticoids o~ the general form~la II with a trihalo~eno-acetic zcid, especially tri~luoracetic acld, ma~ be brou~ht about~ for example~ by reactin~ the 11~-hydro~y-corticoids with a trihalogeno-acetic anhydride in pyridine.
After ~he 17~-acetalisation, the trihalo~eno-acyl group can be split off by hydrolysis (for example in a lo~er alco~ol with the addition of ~eakly basic catalysts, for e~mple sodium acetate or tristhylamine).
The copending application further provides a process for the manufacture of a compound o~ the general ~o~mula I, wherein (a) a 9,11-dehydro-steroid of the general formula VII

.

2~ R
. C=O 12 ' ' 10 ~ W

tVII~ , 0~

in which the bond ,..~., X, W~ Q, Rl, R2 and ~ have the me~ings - given above, is additive~y comb1ned with chlorine or h~pochlorous acid, or . 20 (b) the epoxy-ring of a 9,11-epo}~-steroid oD the ; ~eneral ~ormula VIII

" i~7~59ll C~O l O~HQRL

(VIII ) 0~ . .

in ~Jhich the bonds ,~, :~, W, Q, :R~., R2 and R~; h~ve ~he me~n;ngs given above, is opened up with hydrogen fluoride or h~drogen chloride, or ~c) Z" or HZ" i8 9plit off ~rom a 9-haloge}lo-steroid of the gener 1 formula I~

~7 2 590 1 2~ 12 H0 ~ ~ ~ ~ ~

0~

in which the bonds ~, X, W, Q, ~ ~ R2 and ~ have the meal~Lngs given above and Z" represellts a chlorine atom or a bromine atom, a~d, i~ aesired, in any resulting sterQid saturated in the 1,2-position obtained by any one o~ the variants (a) to ~c) dehydrogenation is carried out in the 1,2-position, ~nd/or in any resulting steroid ~y ll~-hydroxyl group is oxidi~ed to form an o~o group, and/or any 21-ester group is h~d olysed and/or any 21-h~dro~yl group is esterified or exchanged for a fluorine atom or a chlorine atom.
This process may also be carried out under condition~ knolm per se (~nited State~
Patents NosO 3,678,034~ 3,718,671, 3,845,085 and 3,894, 063)o .

1~7~590 Thus, for eæample, the 9,11-deh~dro-steroida of the general ~ormula VII ~ay be reacted in an inert solvent (for example acetic acid, tetrahydrofuran, dioxan or acetonitrile) with reagents which in the presence of water and acids (for example sulphuric acid, phosphoric acid or perchloric acid) liberate hypochlorous acid during the reaction, thus especially reagents form~ ng halogen cations, ~or e~ample ~I-chloracylamides (especially ~-chloracetamide) or ~-chloracylimides (especially ~-chlorosuccinimide). In this reaction there are obtained as the m~in products 9-chloro~ -hydroxy-corticoids of the general formula I, and also frequently as b~-products the corresponding 9a,11~dichloro-corticoids o~ the general formula I~ ~he latter are obtained as m~in products if the reaction is carried out ~rith the exclusion of water in the presence of hydrogen chloride as acid.
On the other hand, the epo~y-r~ng of 9,11-epo~g-steroids of the general for~ula VIII can be opened up, for e~mple, with hydrogen chloride or hydrogen fluoride, by dissol~ing the compounds of the general ~or~ula VIII
in an inert sol~ent saturated with hydrogen chloride or hydrogen fluoride and, if desired, introducing into this solution additional hydrogen chloride gas. Suitable ~nert sol~ents are, for example, ether~ (for e~ample diethyl ether, diisopropyl ether, dioxan or tetrahydro-furan) or chlorinated hydrocarbons (for e}~ple methylenechloride, cbloro~orm, carbon tetracllloride or tetra-chloreth~ne). In this reaction there are ~ormed the 9~-fluoro- or 9a-chloro-11~-hydroxy-corticoids of the gener.~l ~or~ula I.
~ or splitting off a halogen from the 9-halogeno-steroids of the general fo~mula IX the latter may be reacted, for example, in an inert solvent in the presence oi rPd~cal former~ (~or example azodiisobutyronitrile, di-tertO-butyl peroxide or ultraviole~ light) with trialkyl tin hydrides (for e~ample triethyl tin hydride or tributyl ti~ hydride). ~uitable inert solven'Gs are, for exa3ple, ethers (~or example diethyl ether, glycol dimethyl ether, dio~an or tetrahydrofuran), hydrocarbons (for e~ample cyclohex&n~, ben~ene or toluene), alcohols (for e~a~ple meth~lol, ethanol or isopropanol) or n~triles (for example acetonitrile)0 In this raactio~
there are formed the ll~-hydro~y-corticoids of the general ~ormula I unsubstituted in the ga-positionO
The splitting off of a hydrogen h~lide from the 9-haloSeno-steroids ol t~e general formula IX may be carried out under the conditions which are customarily used in steroid chemistl~ for splittin~ o~ a hydroge~ halide from h~lohydrin~0 -` 13 7;~590 ~hus, for e~ample, the compounds of the ~e~eral .. formula IX may be heated u~der re~lu~ in a tertiary ~mine~
for eY.ample pyridine, lutidine or e~pecially collidine.
A further suitable method for splitting off hydrogen bromide i~, for e~ample, the reaction of these compounds with lithium salts (for e~ample lithium chloride) and/or calcium carbonate in dimethylforma~ide or dimethylacet-amide. In these reactions there are formed the ~-corticoids of the general fo~mula I.
~ he starting substances of the general formulae VII to IX required for th~s process o~ the pre~ent invention can be prepared by acetalising ~he correspond-ing 17a-hydro~-steroids under the conditions giren ~ above for the process of the present invention starting from compounds of the ~eneral forMula II, or from the 17-acetalised corticoids unsubstituted in the ~-position by dehydrat~ng the latter to form the 9,11-deh~dro-~teroids of the general Dormula VII, additivel~ combin-i~g HBr at the 9~ double bond, and converting th~
resulting 9-bro~o-steroids of the general formula IX b~
means of bases into thç epoxidçs of thç general formula ~IIIo ~7~590 The products obtained by each o~ the variants o~
the processes of the c~ding application, as de~ined above, may, if desired, be further converted by dehydrogenating in the 192-position corticoids tha'i are saturated in the 1,2-position, and/or o~idizing any ll~-hydro~yl group in these compounds to the ll-oxo group, and/or hydrolysing any 21-ester group and/or esteri~ying any 21 hydroxyl group or e~changing it for a fluorine atom or a chlorine atom~
The conditions under which corticoids saturated in the 1~2-posltion can be dehydroge~ated in the 1,2-posi-tion are described later on.
A preferred method of exchanging a 2l-hydroxyl group for a fluorine or chlorine atom con~ists o~ es~eri-iying the 21-hydroxyl grOllp with a sulphonic acid~
preferably with methanesulphonic acid or para-toluene-s-.~phonic acid~and then exchangin~ the sulphonic acid group for a halogen atom. ~he esterification o~ the 21-hydro~yl group is carried out, for example, by allowing a sulphonic acid chloride to act upon the 21-hydroxy-steroids in the presence of an organic base, for example pyridine, or in the presence of aqueous alkalisO The exchange of the sulphonic acid group for a halogen atom is preferably carried out by reacting the 21-sulphonic ac~d esters with an alkali halide, for example lithium chloride or pot SSil~ hydrogen fluoride, in the presence _ 22 --of a polar solvent, for e~ample dimethylform~mide, at a reaction t.emperature of 50C to 180Co The present invention provides a process for the manufacture o~ an ll~-hydrox~-steroid of the pregnane series substituted in the 17~-position by an acetal group of the general ~ormula X

--OC~-ORl (X) in ~hich -Rl and R2 have the-meanings given above, herein a corresponding ll-deso~y-steroid of the pregnane series or, when applicable, a 21-ester thereo~ is fermented with a culture of an ll~-hydro~lating micro-orgænis~. The ferment2tion is prefer2bly carried out with a fungal culture of a strain of the genus Curvl12r~a, for e~a~ple a fun~al culture of a strain of the species ~Y~ L~ lunata.
.~ preferred embodiment of this microbiological process is a process for the manufacture of an ll~-hydro~y-steroid of the general ior~l~a Id ~ 3~7~590 _ 2~ --~W
.. l .
~ (Id) 0~

in w'nich the bolld _, X, W, Q, Rl and ~2 have the meanings given above and R4 represents a hydrogen atom or a b~7dro~yl ~roup, wherein zn ll-deso~-steroid o~ the ~eneral ~ormula XI

~7~590 - 2~ -1 20R~4 R2 C~O I
OCXQ~

/ ~ (YI) 0~
X
in which X, W, Q, ~ and R2 have the meanings given above and R'4 represents a hydrogen atom, a hydroxyl group or an a~kanoyloxy group containing 1 to 6 carbon atoms, is fermented with a fungal culture of a strain o~ the genus Cur~ulPriQ~ and, if desired, the resulting compound of the ~eneral *ormula Id saturated in the 1~2-position is dehydrogenated in the 1,2-position.
The in~ention further provides the use o~
the ll~-hydro~y-steroids of the general formula Id as inter ediates for the production of steroids o~ the general formula ~II

C~2R4 ~o ~ ~1~ ~
(XII) 0~
t in ~Jhich the bond ....0~ X, W and R~ have the meanings given above.
It has been kno~n to prepare an ~ -infla~matorially ac~ive ll~-hydroxy-steroids (for example the corticoids hydrocortisoneS Prednisolone, De~methasone, ~e~amethasone 9 Preanylidenes, Triamcinolone, Fluocinolone and ~luran-drenolone) by means o~ a very expensive multi-stage partial synthesis from naturally occurring steroids (for example diosgenine), which have become more di~ficult tl~e to obtain in sul~ficient q~ ntities. Withinlmulti-st~ge synthesis of these compounds the microbiological introduction of the ll~-hydroxyl group into the steroid structure is generally the most expensi~e and most wasteful step of the synthesis.
In the year 1966 a process was developed by means of which the yield in the ll~-hydro~ylation of 11-a:
des~y-17-hydrox~--steroids of the pregnane series ca~

~i -` 1~72S9O

be considerably increæsed by e~terifying the 17a-hydroxyl group, then hydroxylating by mean~ o~ fungi of the genus Curvularia and hydrolysing the resulting ll~-hydroxy-17-acyloxy-steroids (German Patent Speci~ication ~o~
1~618~599)o However~ the acylation of the 17-hydro~yl group is qu~te expensive and the yields obtained in this manner are ofte~ unsatisfactory.
Tne hydrolysis of the ll~-hydroxy-17a-acylo~y-steroids is also dilDficult as by-products mostly form so that an e~pensive and wasteful purification olD the resultin~ products is necessary to make them lulfil the criteria of purity necessary for medicinally active - substances.
I~ the ~crobiological process of the present ~nvention there are used, on the other hand, starting steroids ~.hich can be obtained in good yields from the corresponding 17-hydro~y-steroids, and the resulting products o~ the process can be hydrolysed rapidly and quantitatively to the corresponding 11~,17a-diky2ro~y-steroids.
The steroids used ~n accordance with t~is proce~s may be substituted in the usual manner andJor contain double bonds.

1~L7Z590 The presence ol hydroxyl groups or acyloxy group~, ~or example in the 21-position, the presence o~ halogen atoms, preferably ~luorine atoms, methyl groups or ethylene groups, ~or example in the 6- and/or 16-position(s) have no ef~ect in the carrying out of this process o~ the present invention. ~or thi~ process of the present invention there are preferably used a~
starting compounds those steroi~s ~ich contain an oYo group in the 3-position and a double bond in the 4,5-positionO
~ part from the use of di~fere~t starting compounds, the microbiological process of the present invention is carried out under the conditions customarily used ~or the ll~-hydroy~ylation of steroids with fungi o~ the genu~
Curvularia.
~ ungi of the genus Curvularia suitable for the ~ydro~ylatlon are, for e~a~ple, Cu~ 2ria f~lcuta ~I-102 E, C~lrvularia entic~ata I~0 (6284), Cur~llaria l~t~ta ~URL 2380, I~UR~ 2434, ATCC 12017 or I~0 (6286) or _ur~ula~ia ~culans I~0 (6292).
It may be mentioned that ll~-hydro~ylating micro-organisms other than those of the ~enus Curvularia are also suitable for carrying out the microbiological process, but usually these microorganisms have no advant~ge~ over those o~ the genus C`ur~laria.

~7~590 Under the cultivation conditions customarily uGed ~or these microorganisms submerged cultures are gro~nn in a sultable nutrient medium with aeration. ~here i8 then added to the culture~ the substrate (dissolved in a suitable solvent or preferably in an emulsified form) and the fermentation is carried out until a maximum conversion o* substrate is achieved.
~ uitable substrate solvents are, for example, me~hanol, ethailol, glycol monomethyl ether, di ethyl-~ormamide and dimethyl sulphoxide. Tlle emulsification of t,he substrate may be carried out, for eYample, by injecting it i~ micronized form or dissolved in asolvent miscible with ~ater (for e~ample methanol, ethanol, acetone, glycol monomethyl ether, dinethylformamide or di eth~rl sulpho~,ide) under strong tul~bulence into (preferably decalcified) water ~Ihich contains the usual e~ulsification assist~nts. ~uitable emulsi~ication assistants are non-ionic emulsi~iers, for ex~mple eth~lene oxide adducts or fatty acid esters o~ poly-glycols. As suitable emulsifiers there m2y be mentioned by ~ay of example the usual commercial ~etting agents Te~in(R), ~a~attR), ~,een(R) and Span(R).
The emulsification of the substrate frequently e~ablos the throughput of substrate to be increased and thus the concentration o~ substratc to be increased.

1~7259C~

~o~Jever, it is ob-viou31y also possible in carrying out the microbiological process of the present invention t~
use such other methods for incre~6ing the throu~hput of substrate that are well known to the fe~entation expert.
The optimum concentration of substrate, the time o~
addition o~ the substrate a~d the duration of the fermen-tation depends on the structure of the substra-te used and the nature of the microorganisms used. These factor~
must, as is generally necessary in microbiological steroid con-rer~ionc~, be determined ~ n each particular case by preliminary tests of the type that are familiar to the expert.
~ As a subsequent optional step the dehydrogenation of ~4-steroids of the general for~ula I saturated in the 1-position may be carried out both by ~icrobiological methods and also purely chemicc~l methodsO Thus, for ex~mple, the ~4-steroids can be dehydrogenated in the 1-position under the usual conditions ~ith bacterial cultures of the genus ~acillus (for e~mple ~cillu3 ent.us or acillus ,s~haericv.s) or Arthrobacter (for example hrthrobacter sim~lex)O On the other hand, it i8 also possible to carry out the A -dehydrogenation by heating the a4-steroids with the oxidizing agents normally used for this reaction, for exc~mple selenium 1~72s90 dioxide or 2,3-dichloro-5,6-dicyænobenzoquinone in inert solventsO
The resulting products of the microbiological process can be split up in a simple manner to form the corresponding 11~,17a-dihydrox~-steroids.
This splitting is carried out under the conditions which are conventionally used for the hydrolysis or alcoholysis of acet~ls. Thus, for example, the compounds may be split up by reacting them in a lo~er alcohol, for e~a~ple methanol or ethanol, or in an aqueous or~anic solvent, for example glycol monomethyl ether, tetrahydro-furan, dio~an, d~methylformamide, dimethyl sulphoxide, hexamethyl-phosphoric zcid triamide or acetone, with a mineral acidj for e~ample hydrochloric acid, sulphuric acid, phosphoric acid or perchloric acidr æ sulphonic acid, *or e~àmple para-toluene sulphonic acid~ a strongly acidic carbo~ylic acid, for example formic acid, acetic acid or trifluoracetic acid, acid ion-exchan~ers or ~ith a I~wis acid, for e~ample boron tri*luoride, zinc c~oride, zinc bromide or tit~n~um te~rachloride.
The new corticoids of the copending application of the general formula I are distinguished, as already mentioned, in topical appiication by a ~ery good anti-inlla~matory activi~y and they possess a very favourable dissociation bet~een the desired topical activ1ty and the undesired systemic side effec~.

. .

. -The topical activity can be determined by means of the vasoconstriction test as follows.
The test is carried out on healthy test persons o~
both sexes 8 at a time, who have h~d no local cortico-steroid treatment in the previous tl~o weeks. Bfter removal of the Stratum corneum up to the Stratu~ lucidum on the backs of the test perso~s (20-40 Tesa film tears), there is appl~ed in each case Ool gm O~ the preparatlons on areas of 4 square cms without an occulusive dressing.
In order ~o avoid the ~ame preparations being applied at any time to identical skin areas, the applications are made i~ rotational successionO
The vasoconstriction is evaluated visually after 4 and 8 hours by the examiner in accordance with the follo~Jing de~rees of action: 1 = absolute paling. 2 - little residual erythema. ~ = medium degree of erythema, reddening intensity in the central region of the stripped, untreated and undamaged skin. 4 - el~thema with little clearingO 5 - no paling or intensification of the erythemaO
~ he individual evaluations are averaged.
In each of the series of tests there is used as reference substance diflucortolone-21-valerate (= 6a,9-difluoro~ hydroxy-16a-methyl-21-valeryloxy- ~'4-pregnadiene-~,20-dione = D~V~.

-In each case the di~ference ~ between the average degrees o~ action of DFV and the test substances in the individual series of investigations is determined.
Positive deviatiors ~ indicate a more favourable, and ne~ative deviations a less favourable, val~ation o~ the test substance as compared with DFVo In the follol7ing ~ables are given the observed test results, which are produced in the treatment o~
the test person~ with a preparation containing Ool ppm of the active substance.
The systemic activity of the compounds can be determined by t~e adjuvant-oedema test as follows.
~ or the production of a focus of inflammation SP~-rats weighing 1~0 to 150 gms are injected in the ri~ht _ rear pau with 0.1 ml o~ a suspension of 0O5% strength of ~cobacterium butyricvm (obtained lrom the American firm Difkoj. ~efore the in~ection the volumes of the paws of the rats are measuredO 24 ho~s after the in~ection the volume of the paws are again measured to determine the extent of the oedema. ~here are then administered to the rats orally or subcutaneously different quantities o~ the test substance dissolved in a mi~tura of 29% of ben3yl benzoate and 71~o of castor oil.
~fter a further 24 hours the paw volume~ are again measured.

,~

The control animals are treated in the same way but with the difference that the~ are injected with a benzyl benzoate-castor oil mixture free from test substance.
S ~rom the paw volu~es obtained there is determined in the usual manner the qu2ntity of test substance which is necessary to produce a reduction in the experimentally produced paw oedema of about 5O~o by volume.
In the following Tables are given the test results obtained, the compounds of the copending application being compared in each case with the most.structurally analogous kno-~ c.orticoids present in commercial preparations.

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- 1~72590 The new compounds of the copending application are suit-able in comhination with the carriers customarily used in, for example, galenical pharmacy for the local treatment of contact dermatitis, exzemas of a very wide variety of types, neurodermatoses, erythrodermia, burns, Pruritis vulvae et ani, Rosacea, Erythematodes cutaneus, psoriasis, Lichen ruber planus et verrucosus and similar skin diseases.

The copending application accordingly further provides the use of a compound of the general formula I as an anti-inflammatory agent.

The copending application further provides a pharmaceu-tical preparation which comprises a compound of the general formula I, in admixture or conjunction with a pharmaceutically suitable carrier. The preparation may, if desired, contain one, two or three compounds of the general formula I.

The pharmaceutical preparation may be in a form suitable, for example, for local tre~atment.

The manufacture of the pharmaceutical preparations may be carried out in the usual manner by converting the active substances with suitable additives into the desired form of application, for example solutions, lotions, salves, creams or plasters. In the pharmaceutical preparations so formulated the concentration of ~7~S90 active substance depends on the form of application~
In the case of lotion3 and s~lves there is prefer~blg used a concentratio~ of active sub3t~nce ~thin the range of ~rom 0.001~ to l~o by ~Jeight.
~ urthermore, the new compounds of the general for~ula I, if desired in combination with the usual carrier substances and au~iliary substances, are also well suited for the production of inhala~ preparations, which can be used for the therapy of allergic diseases of the respiratory system, for e~ample bronchial asthma or rhinitis.
. ~urthermore, the new compounds of the general formula I are also suitable for the production of capsules, tablets or dragées, l~hich preferably contain 10 to 200 mg of active substance and are applied orally, or for the production of suspensions which may be in unit dosage form preferably containing 100 to 500 mg of active substance per dosage unit and are applled rectally, and also for the treat~ent of allergic aisea~es o~ the intestinal tract, for e~ample Colitis ulcerosa and Colitis granulomatosa.

~7~590 .The iollowing ~ample~ illu~trate the inventiGn:
~x~Ple 1' a) 21063 gms o~ 3~921-diacetox~-17a-~ydro~.y--~5-pregne~-20-one we~e dissolved in 150 ml o~ anhydrous methylene chloride and 100 ml of anhydrous ~olmaldehyde-dimethyl-acet~ he solution was then cooled ~ith water and a mi~ture of 2106 g~s of phosphorus pentoxide and 43 gms o~ kieselguhr was poured into tlle solution, and the ~Jhole was stirred for one hour at room temperature. ~he reaction mi~ture was then ~iltered a~d the residue was washed with methylene cl~loride, triethylamine was added to the filtrate until the pE-value was 9 and the mixture was conce~trated in vacuo. ~he residue wa~ recryst~llized from methanol-~ethylene chloride and 22.68 ~ms of 3~,21-- diacetoxy-17a-metho~yme~G]lo~y-~5-pregne~-20-one melting at 182-184Q were obtained.
b) ~n Erlenmeyer flask o~ 2 litres capacity containiDg 1 litre o~ a sterile nutrient soluiion containing 0.3%
of yeast extract, 002~ of~corn steep li~uor and 002~o o~ glucose, adjusted to a p~-~alue o-~ 700, ~as inoculated with a dry culture ol l~vobacteritlm _eh~dro~ienans ATCC
1~,930 and the whole was agitated at 30C for two days at 175 revolutions per minute.
~ n Erlenmeyer flask o~ 500 ml oapacity containing ~5 ml of the same nutrient medium was inoculated with 10 mi of the lavobacterium dehydro~en~ns el~own culture 1~7;~590 and the ~ole was agitated at 30C for 7 hour~ at 175 revolutions per ~im~te. There were then added to the culture 5 ml of a sterile solution o~ 0.5 ~m of 3~,21-di~cetoxy-17a-~ethoxymethoxy-~5-pregnen-20-one in dimethyl-formamide and the whole was agitated at 30C for a ~urther 65 hours at 175 revolutions per minute. After the resulting fe~mentation the culture ~as extracted twice with 100 ml of ethylene chloride, the e~ract was con-centrat~d in vacuo? the residue was puri~ied by chlomato-gr~phy over aluminiu~ o~ide and 402 ~g of 21--hydro~y-17a-methoxymetho~y-~4-pregnene-3,20-dione melting at 152-153a were obtained.
c) ~n ~rlenmeyer flask of 2 litres capacity containing 1 litre o~ a sterile nutrient solution containing 2~, o~
gluco~e and 2% of corn steep liquor~ adjusted to a p~-value o~ 6O5~ was inoculated with a washing of a dry culture of Curvularia ~ RRL 2~80 and the ~,rhole was agitated at 30C for 60 hours at 175 revolutions per minute.
~ n Erlenmeyer flas~ of 500 ml capacity co taining 90 ml of a sterile nutrient solution containing 1~070 of corn steep liouor and 1~25~o of soya pow~er, adjusted to a p~-value of 6,2, was inoculated with 10 ml of the Cul~llaria lunata grow~ culture and ~he ~rhole was agitated at 30C ~or 7 hours at 175 revolutions per minute. There ~72590 was then added to the culture 006 ml of a sterile solu-tion of 30 mg of 21-hydroxy~17-methoxymetho~y-~4-pregnene-3,20-dione in dimet~ylformamide and the ~ole was fer~ented for a ~urther 65 hours under the conditions given.
~ he ~ermentation culture was worked up as described i~ Example l(b) and 27 mg o~ ,21-dihydro~y-17a-metho~ymethoxy-~4-pregnene-3,20-dione melting at 180-182a were obtainedO
d) 2.5 ~ms of 11~,21-dihyarox~-17a-methoxymethoxy-~4-pregnene-3,20-dione were dissolved in 40 ml of ~nhydrous methylene chloride, the solution ~as cooled to 0C and in the course of 15 min~tes under argon there ~-zs added a solution of 2.25 ml of titanium tetrachloride in 10 ml of methylene chloride. The reaction mixture uas stirred for 90 minutes at room temperature, there ~ere then added 150 ml o~ methylene chloride ~nd 100 ~1 of a saturated aqueous solution of sodium bicar~on2te~
and the mixture ~las stirred for 15 minutes, and the org&nic phase was separated off, washed until neutral, dried over sodium.sulphate and concentrated in ~
The residue was recrystallized from chloroform and 2019 ~ms of 11~,17~,21-trihydroxy-~4-pregnene-3,20-dione ~ecomposing at 215a were obtainedO

~7ZS~O
.

x~mple 2 a) To 50 gms of 3~,21-diaceto~-17a-~ydro~y-~5-pregnen-20-one were added 50 mg of anhydrous para-toluene-sulphonic acid and ~50 ml of anhydrous methylene chloride, the mixture was cooled to 0C and, after the addition of 10 gms of methyl vinyl ether, the mixture ~Jas stirred ~or 4 houra at 0C. Trie~hylamine was then added to the reaction mi~ture until the pH-value was 9 and the ~ixture was concentrated in vacuo. There were obtained 58 gms OI 3,Bf21-diaceto~y-17a~ -~et.hoxyethoxy)-~5-pregnen-20-one in the form of a diastereoisomeric ~ixture melt~lg at 80-118C~ (A test sample recrystallized from methanol melted at 132-134C)o b) ~nder the conditions given in ~xaQ~le l(b) a solution o~ 600 mg of a 3~,21-diacetoxy-17a-(1'-metho~y-etho~y)-~5-pregnen-20-one diastereoisomeric mixture ; n 5 ml of dimethylformamide ~as reacted with a culture of Fla~obacterium dehydro~enans ATCC 13,930, the mixture was worked up and there ~ere obtained 394 o~ 21-hydroxy-17-~ methoxyethoxy~-~4-pregnene-3,20-dione in the form o~
a diastereoisomeric mixture melting at 166-178C.
c) A solution o~ 100 mg o~ a 21-hydroxy-17~
methoxyethoxy)-~4-pregnene-3,20-dione diastereoisomeric mixtllre in 2 ml of dimethylformamide was fermented under the conditions described in ~xa~ple l(c) ~ith a culiure ~72590 of ~t~rLlaria lunata l~RL 2380, the mixture was ~iorked up and there were obtained 10~ mg o~ ,21-dihydroxy-17~ -~ethoxyetho~y)-~4-pregnene-3r20-dione in the form of an oily diastereoisomeric miY~ture.
~ o this mixture were added 3 ml of methanol and 0.5 ml of 2~-aqueous hydrochloric acid and the wllole was agitæted for 5 ho-~rs at room temperature. 4 ml of water were then added to the mixtur~, the mixtuxe ~as ~eutralized with a saturated agueous solution of ~odium bicarbonate, and e~tracted twice with 8 ml of ethylene chloride each time, the organic phase was evaporated in v~ , the residue was purified by chromatography over a colwnn of æluminium oxide and 69 mg o~ ,17,21-trihydro~y-~4-preg~ene-3,20-dione melting at 217-219C
-~ere obtained.
~ m~le ~
a) To lOo O ~mS of 21-acetoxy-17a-hydro~y-~4-pregnene-

3,20-dione were added 13 mg of anhydrous para-toluene-sulphonic acid and 130 ml of anllydrous methylene chloride, the mi~ture was cooled to 0C and, after the addition of 2.3 gms of methyl vinyl ether, the mi~ture was stirred for 7 hours at 0C. The reaction mixture ~as worked up as described in ~ample 2(a) and there were o~tained 1106 gm~ o:~ 21-acetoxy-17~-(l'-metho~cyetho~ry)-~4-pregnene- , 3,20-dione melting at 135-150C.

, ~ ~7~ 59 0 b) A solution of Ool gm of a 21-~ceJ.oxy-17~
metho~:yethoxy)-~4-pregnene-3,20-dione diastereoisomeric mixture i~ 2 ml of dimethylfor~amide wa~ fermented under the conditions described in Example l(c) with a culture of _u~llari~ RRL 2380 and wor~ed up. The 11~,21-dihydroxy-17a-(1~-methoxyetho~ 4-pregnene-3,20-dione so obtained was then hydrolysed under the conditions described in ~xample 2(c) and 78 mg of 11~,17a,21-trillydro~y- ~-pregnene-3,20-dione melting at 217-219~ were obtained.
E~ample 4 a) lOoO gms of 21-acetoxy-17a-hydro}y-~4-pregnene-3,20-dione were reacted under the conditions described in E~mple 3(a) with 2.50 gms of ethyl vinyl ether and worked up, and 12.5 gms of 21-aceto~y-17c-(1'-etho~y-etho~y)-~4-pregnene-3,20-dione ~ere obtained in the form of an oily diastereoisomeric mi~tureO
b) A solution of Ool gm of the 21-acetoxy-17a-(1~-e-tho~yethoxy)-~4-pregnene-3,20-dione mi~ture in 2 ml of di~ethylformamide ~-as hydroxylated l~der the conditions described in ~xa~ple l(c) with ~urvularia lunata ~URL
2380 and worked up, and there was obtained 17a~
ethoxyethox~)-11~,21-dihydrox~-~4-pregnene-3,20-dione, w~lch was hydrolysed under the conditions described in E~ample 2(c) to form 63 mg of 11~,17a,21-trihydro~y-~4-pregnene-3,20-dione melting at 216-21705C.

1J 7~590 Exam~le ~
a) ~nder the conditions described in ~xample 3(a) 10.0 gms of 21-acetoxy-17a-hydro~y-~4-pregnene-3,20-dione were reacted ~ith 400 gms of isobutyl vinyl ether and worlced up, and there were obtained 13b5 g~s of 21-acetoxy-17a-(1~-isobuto}yetho~y)-~4-pregnene-3 t 20-dione in the form of an oily diæstereoisomeric mi~ture.
b) Under the conditions described in ~x~mple l(o) a 601ution o~ 0.1 gm o~ ~he 21-aceto~y-17a~ isobuto~y-ethoxy)-~4-pl egnene-3 7 20-dione mixture in 2 ml of dimethyl-formamide was hydro~ylated with Curvularia u~ata I~RL
2380 and worked up, and the re~ulting li~,21-dihydro~y-17a-(l~-isobutoxyethoxy~-~4-pre~nene-3520-dione was hydrolysed under the conditions described in ~ample 2(c) to form 68 mg of llB,17a,21-trihydrox~--~4-pregnene-3,20-dione melting at 214-216C tWith decomposition).
le 6 a) ~o 1O95 gms of 21-acetox~-17a-~ydro~y-~4-pregnene-3,20-dione were added 5 mg of anhydrous para-~olucne-sulphonic acid, 25 ml of an~ydrous metllylene chloride and 3.5 ~1 of dihydropyran ~ld the whole ~as stirred for 13 hours at room temperature. The reaction mi~ture l~as worked up ~s described in ~ample 3(a) and there iere obtained 200 gms o~ 21-acetoxy-17-(2'-tetrahydropyranyl -oxy)-~4-pregnene-3,20-dione in the form of a diastereo-isomeriF mixture melting at 185-200Co _ 5~) --b) Under the conditions described in ~ample l(c) a solution of 0.1 ~m ;o~ thè 21-acetoxy-17-(2~-tetra-hydropyranyloxy)-~4-pregnene-3,20-dione miYture in 2 ml of dimethyl~orma~ide was hydroxylated with ~urvularia luna~a l~WL 2380 and worked up, and there was obtained ___ 11~,21-dihydro~ 17a-(2~-tetral~dropyranyloxy)-~4-pregnene-3,20-dione, which uas hydrolysed under the conditions described in E~ample 2(c) to form 72 mg o~
11~,17a,21-trihydroxy-~4-pregnene-3,20-dione melting at 215C (with decomposition)O
~amPle a) ~nder the conditions described in ~xample 3(a) 50 g~s of 21-acetoxy-17a-hydro~y-6a-methyl-~4-pregnene-3,20-dione ~ere reacted with l~o9 ~s of met.hyl vinyl e~her and worked up, and 59 ~ms of 21-acetox~T-17a-(l'-me~hoxyethoxy)-6a-methyl-~4-pregnene-3,20-dione were obtained in the form of an ~morphous mass.
b) Under the conditions described in Example l(c) a solution of 200 mg of 21-acetoxy-17a-(1l-methoxyetho~y)-6a-methyl - ~4-pre~nene-3~20-dione in 0.4 ml o~ dimethyl-.
for~amide was hydroxylated wi~h ~urvularia lunata ~
2~80 and worked up, and the result;ng 11~,21-dihydroxy-17a-(1'-methoxye~ho~y)-6a-metllyl-~4-pregn.elle-3,20-dione was hydrolysed under the condition~ described in ~xample 2(c) to form 15 mg of 11~,17a,21-trihydro~y-6-methyl-~4-1~7;~S90_ 51-pregnene-3,20-dione meltin~ at 189~192~o ~ - !
. j a) To 2.0 gms of 21-aceto~y-17-hydro~-16~-methyl-a4-pre~lene-3,20-dione ~ere added 5 mg o~ para-toluene-sulphonic acid (anhydrous) and 25 ml of anhydrous methylene chloride and the mixture was cooled to 0C.
There was then added to the mixture while stirring 0.5 gm of methyl vinyl ether, the mixture was stirred for 5 hours at 0C and ~or a further 12 hours at room ~emperature and then ~orked up as desoribed in EY.amP1e 3 ~2) and 2.1 gms of 21-aceto~.y-17a~ -metho~yetho~y)-16~-methyl- ~ -pregnene-3,20-dione were obtained in the form of an oily diastereoisomeric mi~ure.
b) ~nder the conditions described in EYample l(c) a solution of 50 mg o~ the 21-acetoxy-17a-(1'-methox~-ethoxy)-16~-methyl-~4-pregnene-~,20-dione miYture in 1 ml of dimethylformamide ~as hydroxylated with ll~ata ~URL 2380 and ~orked up and the res-~ting 11~,21-dihydro~y-17-(1'-methoxyethoxy)-16~-methyl-~4-pregnene-3,20-dione wa~ hyàrolysed under the conditions describe~
in ~xample 2(c) to form 32 mg of 11~,17~,21-trihydroxy-16~-methyl-~4-pregnene-3,20-dione melting at 204-207C.
~m~le 9 .
a) Under the conditions described in ~xample 3~a) 50 gms o~ 17a-hydrox~-~4-pregnene-~,20-dione were reacted 11725~90 r~J

with 15 gms of methyl vinyl ether and worked up, and there were obta~ned 51.2 gms o~ 17~ -methoxyetho~y)-~4-pregnene-3,20-dione melting at 115-152C.
b) Under the condition~ des~ribed in 3xample l(c) a Q~
solutionlO01 gm o~ 17a-(1~-metho~yethoxy)-~4-pregnene-3,20-dione in 1 ml of dimethylformamide was fermented with a culture of ~urvulari~ lunata l~RL 2380 and ~Jorked upO ~he resulting ll~-hydroxy-17a-(1~-~ethoxyetho~y)-~4-preg~ene-3,20-dione (melting at 85-103C) wa~
~ydrolysed under the condition~ described in Exa~ple 2(c) and there were obtained 63 mg of 11~,17a-dihydroxy-~4-pregnene-3,20-dione melting at 222-223c~5C.
le 10 a) Under the conditions described in ~xample l(a) - 50 gm ol 3,~ J 21-diacetox;5r-17~:-hydro~ 5-pre~ en-20-one in 150 ml o~ methylene chloride were reacted with 380 gm of formaldehyde bis-glycol monomethyl ether acetal, 50 gms o~ phosphoru~ pentoxide and 100 gm of kieselguhr d worked up, and there were obtained 4508 gms of 3~,21-~iaceto:~-17~x-t21-~aetho:~;yetno~y-metho~ 5-pregnen-20-one melting at 160-161C.
b) ~nder the conditions described in ~xample l(b) 0.5 ~m o~ 3~,21-diacetoxy-17-(2'-metho~etho~y-methoxy)-~5-pregnen-20-one wa~ reacted with a cul~ure of Fl~.vobact.erium dehvdro~ene~ns A~CC 13,930 and worked up, and there were obtained 390 mg of 2l-hydroxy-l7~(2~-1~7~590 ~ 53 -metho~yetho~y-methoxy)-~4-pregnene-3,20-dione in the form of a vitreous massO
c) ~nder the conditions described in Example l(c) 30 mg of 21-hydro};y-17~-(2~-methoxyetho~-methoxy)-~4-pregnene-3,20-dione were reacted with a culture of Cur~aria unata I~UR~ 2380 and worhed up5 and there were obtained 24 mg of ll~t21-dihydro~-17a-(2~-metho~y-ethoxy-methoxy)-~4-pregnene-3,20-dione melting at 143-147C.
~) Under the conditions described in ~zample l(d) -metho 10 mg of 11~,21-dihydroxy-17a-(2'-~etho~yetho~
pregnene-3~20-dione in 2 ml of methylene chloride and OoOl ml of titanium tetrachloride were reacted and worked up, - and there were obtained 8 mg of 11~,17,21-trihyaro~y-~-pregnene-3,20-dione decomposing at 213Co Ex.~nple 11 An ~rlenmeyer flask of 2.litres capacity containing 500 ml of a sterile nutrient solution containing Ool~O of yeast extrac`t 0O5~o of corn steep liquor Ool~O of glucose adjusted to a pH value of 7cO
was inoculated with a washing of a dry culture of Arthro-bacter im~le~ ATCC 6946 and the mixture was a~itated at 30C for 48.hours at 190 revolutions per minute.

~72590 "

An Erlenmeyer ~lask of 500 ml capacity containing 90 ml of the above described nutrient medium was inoculated ith 10 ml o~ the ~ a~ simvlex gro~rn culture and the w~lole wa~ agitated at 30C for 6 hours at 165 revolu-tions per minute~ There ~7as 1;hen added to the cultu~e 1 ml of a sterile solution of 50 mg of 11~,21-dihydrox~-17a-metho~ymetho~y-~4-pregnene-3,20-dione in dimethyl-form~mide and the mixture was fermented for a further 42 hours The fermentation culture was worked up as described in ~a~ple l(b) and there ~lere obtained 44.5 mg of 11~,21-dihy~ro~y-17a-methoxymetho~y-~1'4-pregn~diene-3,20-dione melting at 229/230-231C~
~xam~le 12 rlenmeyer flask o~ 2 litre capacity containi~
500 ml-of a sterile nutrient solution containin~
1~ of yeast extract (Difco) 0.45~0 of disodium hydrogen phosphate 0~34~o o~ potassium hydrogen phospllate - 0027to 0~ T-Y7een 80 adjusted to a pH-value of 6.7 was inoculated with a washing of a dry culture o~
~ocardia ~lober~a ~TCC 9356 and the mixture was agit~ted at 30C for 72 hours at 190 revolu~ions per minute.

-` ~3 7~59 ~ n ~rlenmeyer flask of 2 litres capacity containing 950 ml of a sterile nutrient ~olution cont~ining 200~o Of cornsteep liquor 003% of diammon1um hydrogen phosphate OG 25% of Tween 80 adjusted to a pH-~alue of 605 was inoculated ~i~h 50 mi of the Nocardia ~lober~la~
grown culture and the mixture was agitated at 30C for 24 hours at 190 revolutions per minute. ~here were then added to the cultlIre 5 ml o~ a sterile solution of 0~25 gm of 11~,21-dih~droxy-17a~ metho~yetho~y)-6-methyl-a4-pregnene-3,20-dio~e in dimethylformamide and the mi~tule ~as fermented for a further 72 hour~. The ~ermentation culture was ~Jorked up as described ~n Exa~ple l(b) and there ~as obtained 0021 gm of 11~,21-dihydro~r-17a-(1~-methoxyetho~y)-6~-methyl~ '4-pregna-diene-3,20-dione melting at 17~C.
~x~ le_13 ~) 50 gms of 21-aceto~y-17-hydroxy-~4-pregn ~e-3,20-dione were suspended with 600 ml of form21dehyde-die~hylacetal and 600 ml of methylene chloride and the mixture was cooled to -~0 to ~40CO There was then introduced while stirring a mixture of 75 gm3 Of phos-phorus pel~to~ide and 150 ~ms of kieselguhr and the mi~ture was stirred for ~0 hours at -~0C~ The solution was filtered and neutralized with triethylamine~ After distilling of~ the solvent the mixture was again distilled with methanol and the residue was recrystallized from methanol. There ~Jere obtained 35.9 gms of 21-acetoxy-17-ethox~methoxy-~4-pre~nene~3,20-dione which, after repeated recrystallization, melted at 137-139~C.
b) An ~rle~eyer flask of 2 litres capacit~ con~aining 1 litre of a sterile nutrient solution cGn~aining 1~ of corn steep liquor 1025% of soya powder ad~usted to a pH-value of 602 was inoculated with a washing of a dry culture of Curvularia l~nata ~R~ 2380 and agitated at 30C for 72 ho-~rs at 175 revolutions per minute.
A fermenter of 50 litres capacity containing 29 litres of a sterile nutrient medium as described above was inoculated with 1 litre of the Cur~laria lunata ~ro~m culture and cultivation was carried out for 24 hour~ at 30C ~ith aeration at the rate of 2 cubic metres per hour.
~ fermenter of 50 litres capacity containing 36 litres of a sterile nutrien~ solution as described above inoculated with 4 litres of the ~u~ laria lunata pre-fermenter culture and cultivated for 10 hours at 30C
~ith aeration at the rate of 2 ~ubic metres per hour and 1~7ZS~O

~tirring at 220 revolu~ion~ per minute. ~here were the~
added to the culture 10 gm9 of 21-acetoxy-17a-etho~.y-~ethoxy-Q4-p.e~nene-~,20-dione in 200 ml of ethylene glycol monomethyl ether. From the 10th hour the pH-value was mainta~ned ~et~een 605 and 7.00 ~ter a fur~her

4 hours there were added a further 10 gms of 21-acetoxy-17a-etho~ymetho~y-~4-pregnene-~,20-dione in 200 ml of ethylene glycol monoethyl ether and the fermentation , ~as continued for a further 2~ hours un~er the conditions g'ivenO
The fermentation cultu,re was extra.cted three time~
with 10 litres of ethylene chloride and was then ~urther worked up as described in E~mple l(b). There were obtained 1~.8 gms of 11~21-dihydroxy-17a-etllo~y~etho~y-~4-pregnene-3,2~-dione meltin~ at 153-154C.
Example 14 ~ ' a) 25 gms of 21-acetoxy-17-hydrox~--A4-pregnene-3, 20-dione ~ere suspended with 200 ml of formaldehyde-di~ropyl-acetal and ~20 ml of methylene chloride and cooled to -20C. h mixture of.490~ gms of phosphorus pentoxide and 97 gms of kieselguhr was introduced while stirring and stirring was continued for 22 hours at -20Co ~e solution ~as filtered and neutrali~ed with triethylamine~
The methylene chloride was distilled off in v.~~uo ~nd the formaldehyde-dipropylacetal phase was dec~lted from oil ~172590 - _ 58 --that had separated. After distilling off further solvcllt in vacuo 19 gms of 21-aceto~y-17a-propoxymethoxy-~4-. pregnene-3,20-dione ~elting at 145-147~ crystalli~edO
b) 10 gms of 21-acetoxy-17~-propoxymethoxy-~4-prcgnene-3,20-dione were ground with 1 gm of ~Jeen 80 and three ~imes the quantity of water in a Dyno-mill type ~D~
(~irm ~achofen, Basle)O ~his ground material was steri-lized with hydrogen pero~ide of l~o strength for at least 4 hours.
Curvularia l~lata I~3RR~ 2380 ~7as cultivated, a~
described in ~ample 13(b), in a shaking flask and pre-fermenter and the main fermenter was inoculated therewith.
This fermenter was also prepared as described in Example 13(b) and cultivated for 10 hours also in accorda~ce with the conditions described in ~xample 13(b). ~here w~s then added to the culture the ground material containing the 21-aceto~-17a-propoxymethoxy-~4-pregnelle-3,20-dione and fermentation was car~ied out for a further 44 hours, t.he pH-value being ~aintained bet~een 604 and 6070 ~he fer.mentation cultwre was wor~ed up as described in E~ample 13tb) and there ~lere obtained 6.5 gms of 11~,21-dihydro~y-17-propoxy~ethoxy-~4-pregnene-3,20-dione melting at 134/135~ C-.

~L~7~59 Fx~].e_15 a) 50 ~ms of 21-ace~oxy-17~-hydro~y-~4-pregnene-3 20-dione were suspended in 500 ml of formaldehyde-dibu~yl-acetal and 500 ml o~ me~hylene chloride and the whole waa cooled to -35C. ~ mixture of 74 ~ms of phosphorus pentoxide and 150 gms of ~ieselguhr ~as introduced ~hile stirring and the whole was st.irred for 30 hours at -35qC.
The solution was filtered and neutralized with triethyl-amine. rThe methylene chloride was distilled off in -vacuo and the formaldeh~Te-dibutylacetal phase was decanted from oil that had separated.- A~ter distilling off further Eolvent in ~acuo 38.7 gms OL 21-aceto~y-17-buto~ymetho~y-~4-pregnene-3,20-dione mel-ting at 123.5-124.5C crystal-- lizedO
,, 8 gm8 of 21-aceto~y-17a-buto~ymethoxy-~4-pregnene-3,20-dione were ground with 008 gm of ~Teen 80 as described in B~a~ple 14(b)o u _ ~aria _l~ata NRRL 2380 was cultivated and fer~ented in a shaking flask, pre-fermenter and main fermen'~;er as ~escribed in ~xample l~(b). At the 10th hour of the main fermenter the steroid substrate prepared as described above was added and fermentation ~as continued ~or a further 50 hours.
The fermentation culture was worked up as described in Bxample 13(b) and 3O7 gms o~ ,21-dihydro~17-butoxymethoxy--~4-pregnene-3,20-dione melting at 79-81C

117~590 were obtained~
~ ' a) 10.60 gms of 21-acetoxy-6~-fluoro-17a-hydroxy-~4-pregnene-3,20-dione were dissolved in 265 ml of methylene cbloride and 47.7 ml of formaldehyde-dimethylacetal.
~ixture of 7.95 gms o~ pllosphorus pentoxide and 15.9 gm9 of kieselguhr ~as added in portions and the mixture was stirred for 90 minutes under nitrogen at room temperature. The solu~ion was ~iltered and 201 ml of trietllylamine were added. ~he solvents were distilled off and the residue was recrystallized from methanolD
There were obtained 7.6 gms of 21-acetoxy-6-fluoro-17a-metho2ymet~loxy-~4-pregnene-~,20-dione melting 2t 161-167Co b) -rvularia lvnata ~LRL 2380 was cultivated in a sha~ing flask, pre-fermenter and main fermenter as described in Example l~(b). At ~he 10th ho~r of the rnain fe~men~er 5 gms of 21-aceto~-6~-fluoro-17a-methox~-methoxy-~4-pregnene-3,20-dione in 100 nl of ethylene ~lycol monome~hyl ether were added. From this moment the pX-value ~Jas m~in~ained between 605 and 7Ø ~t the 14th hour a fur~her 5 gms of 21-acetoxy-6a-fluoro-17a-metho~ymethox~-~4-pregnene-~,20-dione in 100 ml of ethylene glycol monomethyl ether were added and the mi2ture was fermented for a Iurther 26 hours.

~7~S90 The fermentation culture was worked up as described in Example 13~b) and there were obtained 4,2 gms of 11~,21-dihydroxy~6~-fluoro~17~-methoxymethoxy-~4~pregnene-3/20-dione melting at 190~ 192C.

. .
Example 17 a) 43 gms of 3~,21-diacetoxy-17~-hydroxy-16~-methyl-~ -pregnen-20-one were dissolved in 800 ml of formaldehyde-dimethylacetal and the mixture was cooled to -15C. A mix-ture of 43 gms of phosphorus pentoxide and 86 gms of kieselguhr was introduced in portions and the mixture was sitrred for 15 hours at about -15C. The solution was filtered, then neutrali-zed with triethylamine and the solvents were distilled off in vacuo. The residue was recrystallized from methanol and there were obtained 31.5 gms of 3~,21-dicetoxy-17~-methoxymethoxy-16~-methyl-a5-pregnen-20-one melting at 117-118C.

b) Flavobacterium dehydrogenans ATCC 13,930 was culti-vated and fermented as described in Example l(b). At the 7th hour there were added to the culture 4 ml of a sterile solution of 0.2 gm of 3~,21-diacetoxy-17~-methoxymethoxy-16~-methyl-a5-pregnen-20-one in dimethylformamide and the mixture was agitated for a further 65 hours.
After the fermentation the culture was worked up as described in Example l(b) and there were obtained 163 mg of 21-hydroxy-17~-methoxymethoxy-16~-methyl-~4-pregnene-3,20-dione melting at 126/128-129C.

~3~, - 6 1 1~7~S9O

c) ~ h~ nata hlU~L 2380 was gro-rm a~d ~ermented as described in ExaJmple l(c)o At the 7th hour there was added to the culture 1 ml of a sterile solution of 50 mg oi 21-hydro~y-17a-methoxymetho~y-16~-methyl-~4-pre~nene-3,20-dione ~n dimetnylformamide and fermentation was continued for 65 hours. The fermentation culture was worked up as described in Example l(b) and there were obtained 3405 mg of 11 ~,21-dihydroxy-17c-mel~hoxymetho~y-16~-methyl-~4-pregnene-3,20-dione melting at 204/205-206Co ExamP1e 18 ~ o 3 gms of 11~,21-dihydro~y-17a-methoxymetho2~-~4-pregnene-3,20-dione in 20 ml of pyridine were added 5 ml o~ acetic alhydride ænd the mixture was stirred for 5 hours at room temperature. After precipitation in 200 ml o* ice-~ter the mixture was filtered ~rith suction and there ~lere obtained 3.2~ ~ms of 21-acetoxy-11~-l~dro~y-17-methoxymetho~y-~4-pregnene-3,20-dione uhich melted at 172-177C after recrystallization from acetoneO
Ex?~le 1 To 4 gms of 11~,21-dihydroxy-17a-methoxymethoxy~
~4-pregnene-3,20-dione in 25 ml of pyridine were added 8 ml of propionic a~lydride and the m~ture was ~tirred for 105 minutes at room temperature. After precipitation in 500 ml of ice-water stirring was carried out until the ~7~590 anhydride had been split, fi].tration with suction ~JaS
then carried out and there were obt~ined 4~43 gms of 11~-hydro}~r-17~-~ethoxymethoxy-21-propionylox~T-~4-pregnene-3,20-dione which melted at 119-121C after recrystalliza-t~on from methanolO
Exam~le 20 To 4 g~s of llB,21-dihydro~y-17a-metho~ymethoY~-~4-pregnene-3,20-dione in 25 ml of pyridine were added 9 ml of butyric anhydride and the whole was stirred for 105 minutes at room temperature. After precipitation in ~00 ml of ice-vater the mixture was further stirred for 3 hour3, then filtered with suction ~nd there were obtained 4 . $5 gms of 21-butyryloxy-11~-hydroYy-17a-methoxymethoY~-~4-pregnene-3,20-dione which melted at 140-142C after recry~tallization from metnanolO

To 4 g~s of 11~,21-dihydro~y-17a-methoxymethox~-~4-pregnene-3,20-dione in 25 ml of pyridine were added 11 ml o~ trimethylacetic anhydride and 100 mg of 4-dimetl~l-amino-pv~idine. After SiY hours at room tempela+ul~e ice-~ater was added, the mixture was extracted with methylene chloride, the eY~tract was w2shed with aqueous acetic acid, a solution of sodiwm bicarbonate and water, then dried witk sodium sulphate and the solvent was distilled off in vacuo~ T~ere were obtained 4~8 ~ms of 11~-hydroxy-17-1~7~590-- 64 --met~lo~;~ethoxy-2l-trimethylacetoxy-~4-preglle~e-~, 20-dione which melted at 182-184C after recry~tallizatio~
~rom methanol.
Ex~mle 22 ~ o 5 gms of 11~,21-dihydro~y-17a-methoxymetho~y-al'4-pregnadiene-~920-dione in 30 ml of pyridine were added 8 ml of acetic anhydride and the mixture was stirred for 1D 5 hours at room temperæt,ureO Afte~ preci-pitation in 300 ml of ice-water the mixture was filtered with suction and there were obtained 5~08 gms of 21-acetox~T~ -hydroxy-17a-metllo~ymethoxy-~1'4-pre~nadiene-3,20-dione ~rhich melted at 214C after recrystallizat,ion from methanol and some methylene chlorideO
xam~le 2~
~ ~o 1,gm of 11~,21-dihydroxy-17~-metho~ymetho~y-~1'4-pre~ladiene-3,20~diolle in 6 ml of pyridine ~lere added 2 ml of butyric an~ydride and the mixture JaS
stirred for 1.5 hours at room tempera~ure. ~fter precipitatio~ in ice-~later the mixture was stirred lor 2 hours, then filtered with suction and there were obtained 1.11 ~ms of 21-butyr~loxy-11~-hydrox~--17a-~ethoxymethoxy-~1'4-pregnadiene-~,20-dione which melted at 182C af~er recrystalli~,ation from methanol with a small amount of methylene chlorideO

~7~S90 _ C: -~

Ex~l~le 24 ~ o 0.~2 g~ of pyridine chlorocllromate were added 2 ml of anhydrous meth~lene chloride and there w~s then added while stlrring a solution of 0.45 gm of 21-aceto2~r-~ hydroxy-17a-methoxymethoxy-~4-pregnene-3,20-dione in 7 ml of methylene chloride. ~fter 4 hours at 20C the mix~ure was filtered with suction over kiesel~uhr, then washed with methylene chloride/diethyl ether 1:1, a few drops of methanol were added and the solvent wæs distilled o~f in ~acllo. The residue ~'2S stirred with water and ~iltered off with ~uction. Recrystallization from methanol yielded 0.28 gm of 21-acetox~T-17a-methoxy--methoxy-~4-pregnene-3,11,20-trione melting at 160-161C.
x~m~le 25 - 6 gms of cortisone acetate were dissolved in 12Q ~;
ml of formaldehyde-dimethylacet~l and 120 ml of methylene ~ i chloride and, while cooling with ice, a mixture of 12 gms of phosphorus pentoxide and 24 gms of kieselg~ r was added. After stirring the mixture for 4~5 hours it ~a~
filtered, then neutralized with trietilyla~ine and the ~olvellt ~-as distilled o~f in V2CUO- The residue was chromatogr2phed o~er silica gel ~ith toluene-ethyl acetate mixtures and there were obta1ned 3085 ~s of 2l-acetoxy-l7a-methoxymetho~y-A4-pre~;ne~e-3,11,20-trione which melted at 160-161~ af~er recrys~allization from meth~nol.

:

-` 117~590 ExamPle_26 a) 2 gms of 21-acetoxy~ -hydrox~-17a-metho~ymethoxg-~4-pregnene-3,20-dione ~ere dissolved in 20 ml of pyridine ,.~
and 0.6 ml of thionyl chloride ~ adAed while cooling with ice. After 30 minutes precipitation in ice-water and filtration with suction were carried outO There were obtained 1.76 gms o~ 21-acetoxy-17a-methoxymet~loxy-~4'9(11)-pregnadiene-3,20-dione which melted at 194-196C
after recrystallization ~rom methanol ~th some methylene chloride.
b) 5 gms of 21-acetoxy-17a-methox~rmethoxy- ~'9(11)-pre~ladiene-3,20-dione were suspended i~ 50 ml of tetra-hydro~uran and at ~20C 20056 ml of l~-perchloric acid and 5014 gms of ~-bromosuccinimide were added. The mixture was then stîrred for 15 minutes. The reaction mixture lJas precipi~ated in a solution of 5.14 gms of sodium sulphite and 350 ~1 of ice-water. The crystalli-zate was filtered o.f with suction, washed until neutral with ~a~er and the still moist crystalli3ate w~s recl~stal-li~ed from J~ethanol/water. ~here were obt~ined 5~0 gms of 21-acetoxy-9a-bromo~ -hydroxy-17-~ethox~methoxy-~4-pregnene-3,20-~ione mel-ing at 130-131Co c) 5108 gms of 21-acetoxy-9-bromo-11~-'nydrox~-17a-metho~ymet~o~y-~4-pregnene-3,20-dione were suspended in 518 ml of ethanol and 45O3 gms of anhydrous potassium acetate were added. The mixture was refluxed for one hour and, after cooling to +20C, it w~s precipitated in 5180 ml of ice-uater. The crystalli7ate ~Jas filtered o~f with suction, washed with water and dried at ~20C.
There were obtained 41.75 gm~ of 21-acetox~-9~
epo~y-17a-methoxymethoxy-~4-pregnene-3,20-dione ~Jhich melted at 138-139~5a after recry~tallization from methanol.
d) 1.0 gm of 21-aceto~y-9~,]1~-epo~y-17a-methox~methoxy-~4-pregnene-3,20-dione was dissolved in 10 ml of methylene chloride and cooled with ice-water. Hydrogen chloride gas dried over sulphuric acid was introduced in a slow stream until no starting material ~as pre~ent in the th~n-layer chromatogram~ ~he reaction mixture was preci-pitated in 120 ml of a solution of 1,~ strength of sodiu~
bicarbonate. The methylene chloride phase was separated~
~ashed until neutral with ~ater, dried and concentrated to dryness. ~here were obtained 1.1 ~ms of 21-acetoxy-9a-clloro-11~-hydroxy-17a-methox~methoxy-~4-pregnene-~,20-dione ~ ich had a melting point of 194.5C after rec-lystalli'zation.
~x~m~le 27 500 gms of 21-acetoxy-17-metho~ymethox~-~'9(11)-pregnadie~e-~,20-dione were suspended in 50 ml of tetra-hydrofur~n and at ~20C 20.~6 ml of l~-perchloric acid and 2.78 gms o~ N-chlorosuccinimide were added. The mixture .

~7~590 was then ~tirred for 24 hours and a further 3052 ml o~
perchloric acid of 7 ~ strength were added. The reaction mixture was precipitated in a solution of 5.14 gm9 of ~odium sulphite and 350 ml of ice-water. The crystal-lizate ~Jas filtered o~f with suction, washed until neutral with water, and the moist crystallizate was recr~stallized from methanol/~aterO There were obtained 1.5 gms o~ 21-aceto~ 9a-chloro-ll,B-hydro~y-17a-methoxyMethoxy-~4-pregnene-3,20-dione which melted at 189-192C after cry~tallization from ethyl acetate and acetoneD
~x~m~le 28 10 gms o~ 9a-~luoro-hydrocortisone acetate_were dissolved in 67.4 ml of methylene c~loride and 13408 ml of methylal and the solution was cooled with an ice-methanol bath. Into this solution were introduced 10 gms of ~losphorus pento~ide mixed with 20 &ms of kiesel-~ihr and the mixture was then stirred at -15C for 5 '~
hours. The ~olution wa~ filtered and neutrali~ed with triethylamineO l~ter distilling off the solvent the mixture was further distilled with methanol and the residue was crystallized fro~ methanol. ~y chro~a~ography over silica gel with methylene chloride and 5~ of me~hanol the product was separated, and there were obtained 3.28 ~ms o~ 21-acetoxy-9a-fluoro-11~,17a-dimethoxymethoxy-~-pregnene-~,20-dione melting at 132-1~4C, 0~56 gm~

~ 1,17~590 of 21-aceto~y-9a-fluoroll~-hydroxy-17a-methoxymet~loxv-~4-pregnene-3,20-dione melting at 211-214C ~nd 0~79 gm of 21-acetoxy-9~-Iiuoro-17a-hydroxy-11~-methoxymethoxy-~4-pregnene-3;20-dione melti.ng at 196-199C. ..
Exam~le 29 500 g~s of 21-acetoxy-9-chloro-11~-hydroxy-17a-methoxyrne~hoxy-~4-pregnelle-~,20-dione were suspended in .
~0 ml of methylene chloride and 20 ml of methanol and the mixture was cooled to ~3C. Dur~ng the course of 5 minutes a solution of 0.31 gm of potassil~ hydroxide in 11 ml of methanol tJas ~dded dropwise and the mixture was then stirred for 80 nlinutes. ~he reaction mix~ure as neutralized with 0.~4 ml of glacial a.cetic acid and precipitated in 350 ml of waterO The cr~stælli3ate ~,as filtered off with suction and dried. There were ob~ai-ned 2032 gms of 9a-chloro-11~,21-dihy~roxy-17-methoxymethox~r-~4-pregnene-~,20-dione which ~d a melting point of 188-189C after recrystallization from methanol/methylelle chloride.
E~.am~le ~0 a) To a solution of 5.0 gms of prednisolone 21-acetate in 25 ml of pyridine were added drop~ise at -15C ~ml of trifluoracetic anh~rdride and the mixture was stirred ~or 10 minutes at -10C. The mixtl~e was introduced in~o a solution of ice-l~ater and sodium chloride and the ~ ' ~7~590 precipitate was filtered offO The residue was taken up in methylene chloride, washed until neutral and concentra-ted in vacuo after drying over sodium sl~phate. The yield was 6.3 gms of 21-acetos~-17a-hydroxy-11~-trifIuor-.
acetoxy-~l'4-pregnadiene-3,20-dioneO
b) ~.0 gms of the above crude product were stirred overnight in a mixture of 25 ml oi dimethyl sulhoxide, 15 ml of acetic anhydride and 408 ml of glacial acetic acid at room temperature. ~he reaction solution was added to a solution of lO~o strength of sodium carbonate and the precipitate was filtered of~. The residue was diæsolved in methylene chloride and worked up in the usual manner after being washed until neutralO A~ter chromatography over 350 mg of .silica gel with a methylene chloride-&cetone gradient (0-8~o of acetone) there ~-ere isolated 2.83 gms of 21-acetoxy-17-methylthio~etho2~r-11,6-trifluGraceto,.y-~1'4-pregnadiene-3J20-dione.
c) 1.5 gms o~ 21-acetoY.y-17~-methylthio~ethos.-y~
trifluoracetoxy~ pregnadiene-3,20-dione in 38 ml of methanol and lo9 ml of triethylamine ~ere stirred for 4 ho~s at room temperatureO The crude product was purified over ~00 gm~ of silica gel with a methylene chloride-acetone gradient (Q-S~o of acetone) and there were isolated lo 2 ~ns of 21-ace~os~y-11~-hydroxy-17a-methylthiomethoxy-~1'4-pregnadiene-~,20-dione melting at 155C.
.

--~ 13L72590 ~:xam~le ~
~. .
a) 20.0 ~ms of 21-acetoxy-9-fluoro~ ,17a-dihydroxy-~4-pre~lene-3,20-dione were reacted in a manner analogous to that described in Ex~mple 30(a) with tri~luoracetic anhydride to form 2306 gms of 21-aceto~y-9a-fluoro-17a-hydroxy-11,8-tri~luoraceto~ 4-pregnene-3,20-dioneO
b) 3- gms of the above crude product were treated in a manner analogous to thatidescribed in ~xample 30(b) ~ith dimethyl sul~hoxide, acetic anhydride and glacial acetic acid. The crude product was purified over 300 gms of silica gel with a methylene chloride-acetone gradient (0-~ of acetone)O The yield o~ 2O58 gms of ?l-acetoxy-9a-fluoro-17-methylthiomethoxy-11~- -trifluoracetoxy-~4-pre~lene-3,20-dione in 28 ml of-methanol was reacted with 104 ml of triethylamine in a manner analogous to that described in Exa~ple 30(c) and the crude product was purified over 100 gms of ~ilica gel with a methyl chloride-acetone gradient (0-12~o of acetone).
The yield was 914 mg of 21-acetoxy-9a-fluoro-11~-hydroxy-17a-3lethylthiomethoxy-~4-pregnene-3,20-dione melting at 193C.
x2m~1e ~2 a) To a ~uspension of 6.0 gms of 21-acetoxy-17-hydroxy-~l'4'9-pregnatriene-3,20-dione in 46 m~ of anhydrous acetonitrile were added 11~5 ml of methoxy-~72590 _ 72 --ethoxymethyl chloride and llo~ ml o~ diiso~ropylethylamineand the mixture was stirred ~or 7.5 hours at 30Co A~ter precipitation in ice-water the precipitate filtered o~
with suction was dissolved in methylene ci~loride, washed until neutral and concentrated after being dried. The reaction product was purified over 800 gms of silica gel with a hexane-ethyl acetate gradient (0-30% of eth~l acetate). The yield was 409 gms o~-21-acetoxy-17a-(1,~5,6-trioxaheptyl)-~1'4'9-pregnatriene-3,20-dione melting at 140Co b) To a solution of loO gm o~ 21-acetox~-17a-(1,3,6-trioxaheptyl)-~1'4'9-pregnatriene-3,20-dione in 10 ml of diox~l were added 900 mg of ~-chlorosuccinimide and 5 ml of perchloric acid of lO~o strengthD The mixture ~as stirred for 3O5 hours at room temperature and introduced into an ice-water-sodium chloride-sodi~lm hydrogen sulphate solution. r~he mixture was filtered ~nd the residue ~-as taken up in methylene chloride, washed until neutral and conce~trated after being dried over sodium sulphate.
The crude product was purified over 100 gms o~ silica gel with a methylene chloride-acetone gradient (0-15~o of acetone). The yield was 760 mg o~ 21-acetoxy-9a-chloro-ll~-hydrox~y-17a-(1,3,6-trioxaheptyl)-~1'4-pregna-diene-3,20-dione (meltin~ point 204C), and also 180 mg of 21-aceto~y-9~ -dichloro-17a-(1,3,6-trioxaheptyl3-~1'4-pre~nadiene-3,20-dione (melting polnt 148C)o ~172590 7, ---E~am~]e~
a) 4.0 gms of 21-acetoxy-17a-hydro~y-~1'4'9-pregpatri-ene-3,20-dione were dissolved in 28 ml of anhydrous methylene chloride and 18 ml of fornaldehyde-dimethyl-acetal and there was added ill portions a mixture of 600 gms o~ kiesel~uhr ~i 20 and 3.0 gms o~ phosphorus pentoxide.
The mi~ture was ~irred for 45 minutes at room temperature, then filtered with suction and the residue was eluted again uith methylene chloride which contained 3-5~0 of triethylamine. The crude product was purified over 750 gm~ of silica gel with a methylene c~loride-acetone gra-d~ent (0-12% of acetone). The yield uas 3.3 gms of 21-ace~ox~-17~-methoxymethoxy-~l'459-pregnatriene-3,20-dione melting at 160Co b) 1.6 gms of 21-aceto~y-17a-methc~methoxy-~1'4'~-~regnatriene-3,20-dione ~Jere dissolved in 16 ml of dioxan and 1.~ ~ms of N-chlorosuccinimide ~ere added. After the dropwise addition of 8 ml of ao~ueous perchloric acid of lO~o strength, the mixture was then stirred for 3 hours at room tcmperature and introduced into an ice-water-sodium chloride-sodium hydrogen sulphite solution. The product uas ~iltered off and l~orked up in a manner analogous to t~at described in E~:ample ~2(b)o The crude product was purified o~er 175 ~ms of silica gel with a metl~lene chloride-acetone gradient tO-12% of acetone). The yield l~as 1.1 gms o~ 21-aceto~y-9~-chloro-11~-hydroxy-1~7~5 17a-metho~ymethoxy-~1'4-pregnadiene-3,20-dione (melting poin~ 224C), and 250 mg o* 21-acetoxy 9a,11~-dichloro-17a-methoxymethox~y-~1'4-pregnadiene-3,20-dione (melting poi~t 162C).
~X~
a~ 1.8 gms of 21-acetoxy-17-(1,3,6-trioxaheptyl)~
~1'4'9-p~egnatriene-3,20-dione were dissolved in 18 ml of dioxan and lo 6 gms of IJ-bro~osuccinimide were added.
After the dropwise addition of 8.5 ml of aqueous per-chloric acid of lO~o strength the mixture was then stirred for 30 minutes at room temperature and introduced into an ice-water-sodivm chloride-sodium hydrogen sulphite solution. Working up was ca~ried out in a manner analogous to tnzt described in ~x~mple 32(b).
There were obtained 2.3 ~ms of cru~ 21-aceto~y-9~-bromo-ll~B-hydro~y-17G:-(1,3,6-.,rioxa~.eptyl)-~1'4-pregnadiene-3,20-dioneO
b) 2.0 gms of the above crude product were dissolved in 20 ml of he~amethyl-phosphoric acid tri~ide and stirred with 2.4 ~ms of lithium chloride for 0.5 hour at a bath temperature of 80C. After precipitation in ice-water-sodium chloride the product wzs filtered off and wor~ed up in the usual ma~ler. The crude vroduct was purified over 350 gms of silica gel with a meth~lene chloride-acetone gradient (0-15~o of acetone). The yield ~t7~590 was 570 mg of 21-acetoxy~ hydroxy-17-(1,~,6-trio~a-heptyl)-~l'4~8-pregna'Griene-3,20-dione melting at 170~o x.~m~le_3~
a) 3O2 gms of tristriphellyl-phosphine rhodium-(I) chloride ,~a~ldissolved in a mixture of 100 ml o~ methanol a~d 300 ml of ben~ene and prehydrogenation was carried out for 105 hours. After the addition o~ 400 gms of 21-acetoxy-17a-metho~yrnethoxy-~1'4'9-pregnatriene-3,20-dione hydrogenation ~as continued for 6.5 hours under atmospheric press~e. The solution was concentrated in a rotary e~aporator and the residue was purified over 400 gms of silica gel with a methylene chlorlde-acetone gradient (0-12~o of acetone). The yield ~as 201 gms of 21 -acetoYy-17~-me~hoxymetho~y-~4'9-pregnadiene-3,2Q-dione.
b) In a manner analogous to that described in ~xample 33(b) l.l gms of 21-acetoxy-17~-methoxymetho~ 4'9-pregnadiene-3,20-dione were treated with ~-chlorosucci~-imide and perchloric acid. After purification 430 mg o~ 21-acetoxy-9a-chloro~ -hydroxy-17~-~ethoxymethoxy-~4-pregnene-3,20-dione mel-ting at 195C were isolated.
Example 36 a) 17.5 ~ms o~ 21-chloro-17a-hydroxy-~4'9-pregnadiene-3,20-dione were re~cted and wor~ed up in a ma~er analo-gous to that described in Example 33(a) ~th 236 ml oi for~aldehyde-dimethylace~al. The cr~lde product was ., .

1~7;~S9O

pl~i~ied over 2025 kg o~ silica gel with a ~ethylene chloride-acetone gradient (0-4% of acetone). The yield was 7.6 gms of 21-chloro-17a-metho~metlloxy-~4'9-pre~la~
diene-3,20-dione meltin~ at 152a.
b) 1.8 gms of 21-chloro-17a-methoxymetho~y-~4'9-pregn~diene-3 r 20-dione were treated in a manner analogou3 to that described in ~Y,ample 3~(b) with l~-chlorosuccinimide and perchioric acid. '~he crude product ~as purified over 100 gms o~ silica gel with a methylene chloride-acetone gradient (0-lOC~ of acetone). ~here ~ere i~olated 126 rng of 9~,21-dichloro-11~-hydro~y-17~-metho~ymethoxy-~4-pre~nene-3,20-dione melting at 197C (with ~ecomposition).
~xar~ le ~7 a) 3~0 gms o~ 21-fluoro-17a-hydroxy-~1'4'9-pre~natriene-3,2G-dione were ~eacted in a manner analogous to that described in ~xample 33(a) with 14 ml of formaldehyde-dimethylacetal. l.~orking up was carried out l~der the conditions described in Ex~mple 40 '~he crude product was purified over 450 gms of silica gel with a metllyle~e c~loride-acetone gradient (0-8% of zcetone). The yield ~as 105 gms of 21-fluoro-17a-metho~ynethoxy-~1'4'9-pregnatliene-3,20-dione.
bj Under the conditions given in ~xæ~ple 33(k) 500 mg of 21-fluoro-17a--metnoxymetho~y-~1'4'9-pre~latriene-3,20-dione were reacted with ~-chlorosuccin~mide ~ld per-7~5~0 rl ~1 chloric acidO ~fter the working up procedure alreadydescribed and purification over silica gel there were isolated 420 mg o~ 9a-chloro-21-~luoro~ -hydroxy-17a-~ethox~methoxy-~1'4-pregnc.diene-3,20-dione melting at 245Co Ex2m~1e 38 a) loO gm of 17a-lydroxy-~1'4'9-pre~natriene-3,20-dione ~ ~ reacted under the conditions described in E~ample 3~(a) w~th forL1aldehyde- dimethylacetalO There were isolated 823 mg of 17a-methoxyme~hox~-~1'4'9-pregnatriene-3,20-dione as a crude productO
b) 823 mg o~ the above crude product ~lere treated in a manner analo~ous to that described in ~xample 33(b) ~th ~I-chlorosuccinimide and perchlo~ic acid and working up and purilication were carried out under the conditions described thereinO The yield was 410 mg of 9a-chloro-ll~-llydroxy-17a-methox~rmetho~y-~l'4-pregn2diene-3,20-dione meltin~ at 227Co ~x~ le ~
-a) In a manner analogous to that described in ~xample 34(a) 1.0 gm of 21-fluoro-17c-methoxymethoxy- ~'4'9 pregnatrien~-3,20-dione ~as treated with 900 mg of N-bromosucciniMide and S ml of aqueous perchloric acid of -stren~thO There ~iere i$olated lol ~ms of Sa-bromo-21-fluoro-11~-hydroxy-17a-methoxymethox~T-~1'4-pregnadiene-3~20-dione.

-1~7~S9O

b) . lol gms of the crude 9a-bromo-21-fluoro~ hydroxy-, 17a-methoxymetho-~y-~1'4-pregnadiene-~,20-dione were ,'~ 'reacted with, 104 ~ns of lithium chloriAe in a ~anner, analogous ~o that described in Example 34(b~ to form 21-fluoro~ -hydro~y-17u-methoxymethoxy-~1'4'8-pregnatriene-3,20-dione. The yield was 490 mg melting at 218C~
~x~m~le_~0 a) 3.3 ~s of 21-chloro-17a-hydro,-.y-~1'4'9-pregnatriene-3,20-diolle were reacted under the conditions given in Example 33(a) with formaldehyde-di~ethylacetal. There were isolated 204 gms o~ 21-chloro-17a-rnethoxymetho~y-~1'4'9-pre~latriene-3,20-dione.
b) 1-4 ~s of 21-chloro-17-metho~ymethoxy~ 4 ?9_ pre~n triene-3,20-dione were treated with N-bromosuccin-imide in a manner analogous to that described in ~xample 34(a~ and there weré ~solated 107 gms o~ 9a-bromo-21-cl~oro-ll~-hydroxy-17a-methoxymetho~-,y-~1'4-pregnadiene-3,20-dione in ~he lorm of a crude productO
c) Under the conditions gi~en in ~xample 34(b) 1.7 gms of 9-~ro~o-21-chloro-11~-hydroxy-17Q-mèthox~rmethoxy-'4-pre~nadiene-3,20-dione were reacted with 2~1 gms of lithium chlorideO The cr~de product was purified over 300 gms of silica gel with a methylene chloride-acetone gradient (0-8% of acetone). The yield was 530 mg of 21-chloro-11~-hydroxy-17a-methoxymethoxy-~1~4~8_ pre~natriene-3,20-dione melting at 166Co ~72S~O

le al a) 3.4 ~ms of 21-fluoro-17a-methoxymetho~y-~4~9-pregnadiene-3,20-dione were prepared in a manner analogous to that de~cri~ed in ~xample 33(a) from 7.6 gms of 21-fluoro-17a-hydroxy-~4'9-preenadiene-~,20-dione and 68 ml of formaldehyde-dimethylacet~10 b) Under the conditions described in ~ample 33(b) lo 4 gms of 21-fluoro-17~-metho~ymetho~4~9-pregnadiene-3,20-dione were treated with N-chlorosuccinimide and perchloric acidO The ~c~de product was purified over 100 gms of silica~gel with a metllylene chloride-acetone -gradient (0~1~' o-f acetone)O ~he yield wa~ ~0 mg of 9a-chloro-21-fluoro-11~-hydrox~-17~-metho~ymetho~y-a4-pregnene-3,20-dione melting at 214C (with deco.~position)O

E~a~nle 42 a) Into a mi~ture of 100 ml of pyridine and 12 ml o~
trifluoracetic anhydride were introduced at -10C 2000 gms of 9a-fiuoro-11~,17-dihydroxy-21-propionylox~ '4-pregnadiene-3,20-dione and the mixture was then s~irred for 10 minu~es at -10C. A~ter precipitation in ice-water the mixture was filtered and the residue was ta~en up in methylene chlorideO hfter being ~.~shed and dried, the organic solution was concentrated in vacuo, whereby 2200 ~ms of 9-fluoro-17~-hydroxy-21-propionyloxy-11~_ tr~fiuoracetoxy-~l'4-pre&nadiene-3,20-dione were isolated.

.

1~7~5~

b) 22,0 gms of the aboYe crude porduct were reacted under the conditions gi~en in Example 33~a) with 90 ml of formaldehyde~di-methylacetal~ and 9~fluoro~17~methoxymethoxy-21-propionyloxy~ trifluoracetoxy.~ 4~3,2C-dione was iso-lated as a crude product.
c) The crude 9~-fluoro-17~-methoxymethoxy-21-propionyl-oxy~ -trifluoracetoxy-~l'4-pregnadiene-3,20-dione was dis-solved in 500 ml of methanol and, after the addition of 25 ml of triethylamine, was then stirred for 30 minutes at room temperature, The reaction solution was concentrated to dry-ness in vacuo and the residue was chromatographed over 2.25 . _ kg of silica gel with a methylene chloride-acetone gradient (0-12% of acetone). The yield was 12.3 gms of 9~-fluoro-11~-hydroxy-17~-methoxymethoxy-21-propionyloxy-~1'4-pregnadiene-lS 3,20-dione melting at 241C.

Example 43 a) Under the conditions given in Example 42(a) 1.0 gram 20 of 21-butyryloxy-9~-fluoro~ 17-dihydroxy-~1'4-pregnadiene-3,20-dione was reacted with trifluoracetic anhydride and worked up. There was isolated 0.9 gm of 21-butyryloxy-9~-fluoro-17~-hydroxy-ll~-trifluoracetoxy-~l'4-pregnadiene-3,20 dione.

b) 800 mg of the above crude product were treated with 3.6 ml of formaldehyde-dimethylacetal in a manner analogous to that described in Example 33(a). After ~7~sgo worl~ing up there were obtained 1.1 ~m~ of crude 21-butyrylo~y-9a-fluoro-17~-methoxymethoxy~ -trifluor-aceto~y-~l'4-pregnadiene-3,20-dione~
c) 1~1 gms of crude 21-butyrylox.y-9~-fluoro-17a-.metho~etho~y-ll~-tri.fluoracetoxy- ~ '4-pregnadiene-3,20-dione were reacted with triethylamine in a manner analogous to that described in Exa~ple 42(c). The crude product wa~ o~rom~tographed over 75 gms of eilica gel with a ~ethylene chloride-acetone gradient (0-15 of acetone ? . The yield ~Jas 540 mg of 21-butyryl~o~y-9c~-fluoro-ll,B-llydroxy-17a-metho.xymetho~ 1'4-pregna-diene-3,20-dione melting at 247C.

~xæ~
200 gms of 21-butyryloxy~9a-fluoro-17a-hydrox~T-~1'4~pregnadielle-3,11,20-trione were reacted with 9 ml of formaldehyde-~imethylacetal and worked up in a manner analogous to that described in Example 33(a)0 The crude product ~as purified over 300 gms of ~ilica gel with a methylene chloride-acetone gradient (O-lO~o 0~ acetone).
The yield ~9 2.07 g~s of 21-butyryloxy-9~-fluoro-17~-methoxymethox~-~l'4-pregnadiene-3,11,20-trione melting at 192C.

xam~le 45 In a ma~ler analogous to that described in E~ample ~2~a) 700 mg of 21-butyryloxy-9-fluoro-17~-hyaroxy-~1'4-7;~590 pregnadiene-3,11,20-trione were reacted with 1054 ml o~
methoxyethoxymethyl chlorideO The crude product ~J2S
purified over 135 gms of ~ilica gel l~ith a methylene ~hloride-acetone gradient (0-5~o of acetone). The yield was 430 mg of 21-butyrylo~y-9a-fluoro-17a-(1,3,6-trioxa-heptyl)-~l'4-pregnadiene-3,]1,20-trione melting at 126Co ~xam~le ~6 a) 15.2 gms of 9a-fluoro-11~,17a-dihydroxy-16~-methyl-21-propionyloxy-Al'4-pregnadiene-3,20-dione, prepared ~rom 9~-fluoro~ ,17a,21-trihydroxy-16,~-methyl-~1~4-preg~adiene-3,20-dione and propionic a~lydride, were treated with 9~1 ml of trifluoracetic anhydride in a manner analogous to that described n 3~ample 42(a).
~here were obtained 15.4 gms of 9a-fluoro-17a-hydroxy-16,B-methyl-21-propionylo~ -ll,B-trifluoracetoxy-~1'4-pregnadiene-3,20-dioneO
b~ 15O4 gms of the above crude product ere reacted under the conditions gi~en in ~xample 33(a) with formal-dehyde-dimethylacetal to form 9a-fluoro-17-~ethoxy-methoxy-16~-methyl-21-propionyioxy-11~-trifluoracetoxy-~1'4-pregnadiene-3,20-dione. The yield ~as 16 o 9 gms of the crude product.
c) A solution of the crude 9a-fluoro-17a-methox~metho~y-16~-methyl-21-propionyioxy-11~-trifluoraceto~y-~1'4- -pregnadiene-3,20-dione in 250 ml of methanol ~as treated ~72590 .

with 30 ml of triethyl~nine in a manner analogous to that described in Exa~ple 42(c). After working up, the crude product was purified over 1.5 kg of silica gel with a methylene chlo~ide-acetone gradient (0-10% of acetone)0 The yield was 9.6 gm8 cf 9~-fluoro-11~-hydroxy-17a-metho~ymethox~r-16~-methyl-21-propionyloxy-~1'4-pregnadiene-3,20-dione melting at 169C.
~:Z
. . .
Under the conditions given in Example 35ta) 600 mg of 9a-fluoro-11~-hydro~y-17a-me~hoxy~etho~.y-16~-methyl-21-propionylo~y-~1'4-pregnadiene-3,20-dione were hydrogen-ated with 500 mg of ~ristriphenyl-pllosphine rhodium-(I) c~loride and wor~ed upO After chromatography of the crude product over 65 gms o~ silica gel with a methylene chloride-acetone gradient (0-10~ o~ acetone) there were isolated 347 mg of 9a-fluoro-ll~-hydrox-J-l7a-me metho~-y-16~-methyl-21-propionyloxy-~4-pre~nene-3,20-dione melting at 165C.
Ex~n~le a8 h suspension of 6.9 ~ms of 9a-fluolo-11~-hydro~y-17a-~qetllox~rmethoxy-16~-methyl-21-propionylox~-~1'4-pregnadiene-3,20-dione in 80 ml of a 0.2i~.T-methanolic solution of potassium hydro~ide was stirred for 45 mi~utes at 0C. ~eutrali~ation was carried out with acetic acid of 10% strength ~Id, after precipitation in ice-wa~er ~72590 -- &4 --and working up, there wa~ obtained a c~de p-roduct w~ich was puri~ied over 450 gm~ of ~ilica gel with a methylene chloride-acetone gradient (0-20~o of acetone). ~he yield wa~ 401 gms of 9a-fluoro~ ,21-dihydro~y-17a-methoxy-methox~-16~-methyl-~1'4-pregnadiene-3,20-dione melting at 220C.

.
~xam~le 49 a) A solution of 1~7 gms of 9-fluoro-11~,21 dihydroxy-17a-methoxymethoxy-16~-methyl-~1'4-pregnadiene-~,20-dione in 1'l ml of pyridine was stirred with 2.04 gm9 of ~osyl chloride for 1 hour at room temperature. After precipitation in ice-~Jater, the precipitate was taken up in methylene chloride and worked up in the usual manner.
The c~lde product W2S purified over 135 g~s of silica gel with a ~ethylene chloride-acetone gr2dient (O-lO~o of acetone). The yield was 876 mg of 9-fluoro~
hydroxy-17a-methoxymethoxy-16~-methyl-21-tosyloYy-~1'4-preenadiene-3,20-dioneO
b) 876 mg of the above product in 17 ml of hexar~ethyl-phosphoric acid triamide were s~irred with 880 rng of lithium chloride for 1 hour at 80C. The mixture ~as poured onto ice-water and the prec~pitate laS filtered off, After the usual working up, the crude~ product was recr~rstq-lized from hexane/acetone. The yield was 485 mg of 21-chloro-9a-fluoro-11~-hydroxy-17a-metho~rmetho~y-16~-metl~yl-~1'4-pregnadiene-~,20-dione melting at 204C.

~7Z590 Exa~le 50 a) ~ suspension of 1102 gms of 9a-fluoro~ -hydroY.y-17-~etho~ymethoxy~ propionyloY.y-~l'4-pregn2diene-3,20-dione in 129 ml o~ a 0.21~-methanolic solution o~ potas9ium hydroxide was prepare(l. The suspension was stirred for 1 hour at room temperature and worked up as described in Example 48. After chromatography over 105 kg of silica gel with a methylene chloride-acetone grædient (0-35~
of acetone) there were isolated 7O5 grns of 9a-fluoro-1]~, 21-dillydroY.~-17a-methoxymethoxy-~1'4-pregnadiene-3,20-dione.
b) In a manner analogou~ to that described in E~ample 49(2) loO glm of the above crude product was reacted with 2.0 gms of tosyl chloride. The crude product was ~uri-fied over 200 gms of silica gel ~7ith a methylene chloride-acetone gradient (0-105~ of acetone). The yield ~l2S 886 rng of 9~-fluoro-11~-hydroxy-17a-methoxymet'noxy-21-tosyloxy-'4-pregnadiene-3,20-dione~
c) 886 ~g of 9a-fluoro-11~-hydro~y-17a-methoxy~et)loxy-21-tosyloxy-~l'4-pl~e~r.adiene-3,20-dione ~7ere treated under the co~ditions given in Example 49(b) with lithiu~ chloride and worked up. Purification ~las carried out by recrystal-lization from acetone/hexaneO The yield was 392 mg of 21-chloro-9lx-fluoro-11,6-llydroxy-17~-methoxymetho~ '4- ,~
pregnadiene-3,20-dione melting at 225C. .;j ;

7~S9O

~ample 51 a) 2000 gm~ of 21-acetoxy-9a-fluoro-11~,17a-dihydroxy-~4-pregnene-3,20-dione were reacted in a manner analogous to that described in ~x~nple 42(a) ~ith 12 ml of trifluor-acetic anhydride to *orm 21-acetoxy-9~-fluoro-17a-hydroxy-ll~-trifluoracetoxy-~4-pre~nene-3,20-dione.
b) 5Q gms of the above crude product were converted in a manner analogous to that described ill Example 33(a) with 22.5 ml of formaldehyde-dimethylacetal into 21-aceto:Ry-9-fluoro-17a-methoxrymetho~-ll,B-trifluoracetoY.y-~4-pregnene-3,20-dioneO The yield was 5.3 gms~
c) 503 gms of 21-aceto~y-g~-fluoro-17~-methoxymetho~y-ll~-trifluoraceto~y-~4-pregnene-3,20-dione were treated with triethylamine in a manner analogous to th~t described in Exa~ple 42(c)o The crude product was puriIied over 500 gms of s;lica gel with a methylene c~tloride-acetone gradien~ (0-8~ of acetone)O The yield was 560 ~g of 21-acctoxy-9a-fluoro-11~-hydroxy-17a-metho~ymetho~y-~4-pregnene-3,20-dione melbing at 213~
Exam~le 52 Under the conditions given in Example 35(a) loO gm of 21-butyrylo:~--9-fluoro-ll,B-hydroxy-17Q-methoxymethoxy-94-pregnadiene-3,20-dione was hydrogel~ted with 800 mg ol tristriphenyl-phosphine rhodi~-(I:) chloride and worked up. After chromato~raphy of the crude product over ~7~590 100 gm o~ silica gel with a methylene chloride-acetone gr~dient (0-10~ of acetone) there were isolated 620 mg of 21-butyryloxy-9-fluoro-11~-hydroxy-17a-methoxymetho~y-~4-pregnene-3,20-aione melting at 1~3C.
am le 5~
a) In a manner analogous to that described in ~ample 48 28.0 gm6 of 21-acetoxy-9~-fluoro-11~-hydro~-17a-me~hoxymethoxy-~4-pregnene-~,20-dione were hydrolysed with 2 002~-methanolic solution of potassium hydroYide to fol~ 9~-fluoro-11~,21-dihyAroxy-17a-methoxymetho~y-~4-pregnene-3,20-dioneO
b) ~ solution of 500 mg of 9a-fluoro~ ,21-dihydro~.y-17a-~etho~ymetho~y-~4-pregnene-3,20-dione in 5 ml of pyridine was stirred with 7.5 ml of n-valeric anhydride ~or 1 hour at room temperature. AI^ter precipitation in ice-~Jater, the precipitate was fil~ered olf and wor`~ed up in tl~e usual malmer. The crude product was purified over 400 gms of silica gel with a hexane-et~yl ace~ate gradient (0-30~o of ethyl acetate). The yield was 235 mg of 9a-fluoro-11~-hydro~y-17a-methoxymetho~y-21-valeryloxy-~4-pregnene-3,20-dione melting at 181Co ~xample ~
a) 29O1 gms of 21-acetoxy-6-chloro-17a-hydro~y-~'6_ prenadiene-3,20-dione were dissolved in 730 ml of methvvlene chloride aIld l~loO ml of formaldehyde-dimethyi-~37'~590 acetal. A miYture o~ 22012 gms of phosphorus pentoxideand 44 ~s of kieselguhr l~as added in portions and the mixture tras stirred for 205 hours under nitrogen at room temperature. The solution was filtered and 5.8 ml of trietllylamine were added. The solvents were diætilled off and the residue was recrystalli~ed ~rom methanol ~ith the addition of acti~e carbon and l~o of triethyl-amine. There were obtained 15~6 gms of 21-aceto~g-6-chloro-17a-methoxymethoYy-~4'6-pregnadiene-3,20-dione meltin~ at 183-186Co b) ~urvularia lunata IDURL 2380 t~as grotm, as descrlbed in ~xample 13(b) r in a ~haking flask and pre- and main-fermenter. At the 10th hour of the main fermenter there ~ere added 3 gms of 21-aceto~-6-c~oro-17-metho~rmethoxy-~4'6-pre~nadiene-3,20-dione in 60 ml of ethylene g~l~Tcol monomethyl ether. From this moment the ~-value was maintained be~t~een 604 and 607 ~ld fermelltation ~;as continued for a further 20 hours. The fer~entation culture was ~orked up as de~cribed in Example 13(b) and there were obtained 1.8 gms of 11~,2~-dihydro~y-6-chloro-17-metho}.ymethox~-~41 -pregna~iene-3,20-~ione melting at 234/2~5-236Co .ample 5~
a) 38085 gms of 21-acetoxy-17~-hydro~ 4-pregnene-3,20-dione were stirred with 235 ml of formaldehyde-~3 7'~S9O
_ ~9 _ dii~opropyl~cetal ?~d 500 ml o~ methylene chlorifle andcooled to -20C. ~ mixture o~ 75 gms o~ phos~hol~s pentoxide and 150 gms of kieselguhr was introduced while stirring and the mixture was stirred for 20 hour~ at -20C.
Tho mixture ~;~as ~iltered, washed with methylene chloride and adjusted to a pH-value of 9 with triethyll~mine. The sol~rents were aistilled off in vacllo and the re~idue ~JaS
taken u~ in methylene chlorideO l'he resultin~ solution was washed ~Jith a semi-satvrated solution of sodium chloride,~dried with sodium sulphate, tre~ted with active carbon~ passed over'kiesel~ihr witll suction ~nd concentrated in vacuo. ~he residue uas chromatographed over silica gel ~r.th mi~tures of toluene and ethyl acetate.
There were obtained 3508 gms of 21-acetoxy-17a-isopropoxy-metho}y-h4-preg~eno-3,20-dione -l~hich, after crystalliza-tion with pentalle, melted at 111-117Co b) ~ -unata I~UR~ 2380 ~s ~rown, as described in Example 13(b), in a shaking flask and pre- and main-fermenter. ht the 10th hour of the main ferr~enter 12 gms of 21-acetox3-17a-isopropoxymetho~.y-~4-pregllene-3,20-dione in 240 ml of ethylene glycol monomethyl ether were added. ~rom this moment the pH-value was maintained between 605 and 7.0 ~d the ~ermentation was continued for a f~rther 15 hours. The fermentation culture ~a~
worked up as described in Example 13(b) and there were ob~àined 804 gms of 11~,21-dihydroxy-17a-isopropoxy-7~590 methoxy-~4-pregnene-3,20-dione melting at 71/73-78Co c) Arthrobacter s~m~le~ ATCC 6946 ~:as grown as described in Example 11 in a growth flask and a ~ermentation flask A~
~le. (~t h ~c.~r ~ere~ w~s c~ l. h t~ r~r~ sk:.
1 ml of a sterile solution of 50 mg o~ ,21-dihydroxy-17a-isopropoxymetho~ 4-pregnene-3,20-dione in ethylene glycol monomethyl ether and the fermentation was continued for a further 42 hour~.
The fermentation culture was worked up as described ln ~xample l(b) and there were obtained 32 mg of 11~,21-dihydro~y-17a-isopropoxymethox~T-~1'4-pIegnadiene-3,20-dione melting at ~8/63-65C. - `
~am~le 56 a) lOoO gms of prednisolone 21-acetate were dissolved with 40 gms o~ 4-dimethylaminopyriaine and 500 ml of ethylene chloride, and the miY.ture ~as cooled to -15C
and 25 ml of ace-tic anhydride ~7ere added. In the course of 10 minutes 10 ml of formic acid ~Jere added dropwise and the mixture was stirred ~or a further 135 minutes at -10 to -15C. The solution was extracted with water, ~drochloric acid o~ 4~o strength and a solution ol sodium bicarbonate, and the or~anic phase was dried ~ith sodium ~ulphate and concentrated in acuo. The resi2ue ~as recrystallized from met`nanol ~ith the addition of some metl~lene chloride and there were obtained ~O34 gm~ of 21-acetoxy-11~-formyloxy-17~-hydroxy-~1'4-pre~nadiene-3,20-dione melting at 221-223C.

~3 7~590 b) 5 gr~s o~ 21-acetoxy~ -forMyloxy-17a-hydro~y-~1'4-pregnadiene-3,20-dione were di~solved in 150 ml o~
methylene chloride and 30 ml of for~aldehyde-diethylacetal and the whole was cooled to 0C. A mixture of 5 gms of phosphorus pentoxide ~nd 10 gms of kieselg~hr was intro-duced while stirring and the miY~ture was s-tirred for 2.5 hours in an ice-bath. ~he mixture was filtered, washed with ~ethylene chloride and adjusted to a p~-value of 9 with triethylamineO After distilling off the solvents there were obtained 10.5 ~ms of crv.de 21-acetoxy-17a-etho~y~ethoxy~ ormylo~y-~l'4-pre~ladiene-3~20-dione in the form of a semi-solid massO
c) 0O34 gm of crude 21-acetoxy-17-ethoxymethoxy-11~-for~ylo~y-~l'4-pregnadiene-3,20-dione;~ re dissolved in 13 ml of ~e'~hanol and under argon the solution was added to a solution of 00126 gm of sodi~ bicarbonate ln 1032 ml of ~rater at 60Co ~he mixture was heated under reflux for 10 minutes, then cooled down, water was &dded and the mi~ture was extracted wi~h me~hylene chloride~ The methylene chloride solution was dried with sedium sulphate 9 then concentrated and the residue was chromato~r~phed over silica gel with mixtures of toluene and ethyl acetate.
There was obtained 0.1 gm of 17-ethoxymetho~y~ r 21-dihydroxy-~l'4-pregnadiene-3,20-dione melting at 149.5-152Co 1~7;~S90 Y.am~le 57 a) 20.0 ~ms of 11~,21-dihydro}~-17a-metho~.ymethoxy-~4-pre~ene-3,20-dione were dissolved in 200 ml of pyridine and 20 ml of-methallesulpllonic acid c~iloride ~Jere added drop~Yise while cooling~ The ~ixture tJas stirred for 30 minutes at room temperature and precipitated in 2000 ml of ice-~ater. After filtering with suction, washing and dryin~ there were obtained 23085 gms of 11~-hydro~-21-methanesulphonyloxy-17~-methoxynethoxy-~4-pregnene-3,20-dlone decomposin~ at 154-155Co b) To 20 gms of 11~-hydro~y-21-methanesul~honyloxy-17a-methoxymethoxy-~4-pre~ ene-3,20-dione were added 600 ml o~ acetone, a solution of 20 gms of sodium iodide in 520 ml o-f acetone was added and the mixture was he~ted wlàer reflux for 11 hours. After cooling the mi};tuL-e, ~he insoluble matter was filtered off, the acetone solu-tion was concentrated in vacuo and there las added to t~le residue w2ter and a s~all a~ovnt of a solution of soaium thiosulphate. The cl~lde p-oduct ~as filtered off ~th suction, washed with water and reclystalli~ed from ace~oneO ~here were obtained 1606 ~ms of 11~-hyaro}~-21-iodo-17~-methoxymethoxy-~4-pre~nene-3,20-dione decomposing at 123-126C.
c) 1 ~m of 11~-hydroxy-21-iodo-17a-metho~ymet~o~y-~4-pre~nene-3,20-dione ~as heated under argon for 30 minutes at ~C with 20 ml of toluene and 1~ ml of tributyl tin 1::L7;2S9O

hydride. The toluene uas then distilled off in .vacuo a~d the residue was treated wrth pentane. There ~as obtained zs a crystallizate 0.64 gm of 11~-hydroxy-17a-methoxymetho~ 4-pregnene-3,20-dione ~elti~g at 191-194C0 ~am~le 58 a) ~ gms of 21-acetoxy-17a-hydroxy-~4'9( )-pregna-diene-3,20-dione were dissolved with 72 ml of formaldelyde-diethylacetal and 280 ml of methylene chloride and the solution ~Jas cooled to 0CO A mixture o~ 9.0 gms of phosphorus pento~ide and 18 ~ms of kieselg~lr was introduced while stirring and the whole ~as stirred for 2.5 hours in an ice bath. The mi~ture was filtered and washed with methylene chloride~ The solution was adjusted to a p~--value o~ 9 with triethylamine, then concen~ra~ed and the residue was chrornatographed over silica gel with mi~tures of toluene and ethyl acetateO There were obtained 5.93 ~ms o~ 21-acetox~-17a-et'noxyn~etho~y-~4'9( pre~adiene-3,20 dione melting at 167 - 169Co b) 508 gms o~ 21-aceto2y-17~-ethoxymetho~y-~4'9(ll)-pregnadiene-3,20-dinne were reacted under the conàitions given in ~xa~ple 26(b), but without recrystalli~ation, and there were obtained 7O6 gms of crude 21-acetoxy-17a etllo~ymetho~y-9a-bromo-11~ ydrox~-~4-pre~lcne-3,20-dione in the form of a vitreous substance.

~ ` i 7~s~O

c) 7.6 ~s of crude 21-acet~xy~17~-ethoxymethoxy-9~
bromo~ hydrox~6 ~pregnene~3,20~dione were reacted under the conditions descri~ed in ~xample 26(c) and the ~ethanolic solution of the crude produc~ was filtered over silica gel, There were obtained 5.49 gms of crude 21-acetoxy-17~-ethoxy-:
methoxy-s~ epoxy-~4-pregnene-~20-dione as an amorphous substance.
~ - ' I
d) 5.4 gms o crude 21-acetoxy-17~-ethoxymethoxy-9~ -epoxy-~4-pregnene-3~20-dione were reacted under the conditions described in Example 26(d). The crude product was chromatographed over silica gel with mixtures of toluene and ethyl acetate and there were obtained 1.78 gms of 21-acetoxy-17a-ethoxymethoxy-9a-chloro~ -hydroxy-~4-pregnene-3,20-dione melting at 148-151C.

Example 59 - a) 6.0 gms of hydrocortisone 21-acetate-ll~formate were dissolved in 150 ml of methylene chloride and 63 gms of formaldehyde-dihexylacetal and the solution was cooled to 10C. A mixture of 6 gms of phosphorus pentoxide and 12 gms : of kieselguhr was added in portions under argon and the mix-ture was s~rred for one hour at 10C and one hour at 15 C.
The mixture was filtered, washed with methylene chloride and adjusted to a pH-value of 8 with triethylamine. The methylene chloride was evaporated ln vacuo and the residue was cooled in an ice bath. The solution was decanted from the oil that " .- ., ; . . . : . . ~..... " ~
:
:; , . . .

.

.

1~7~590 .

had precipi~at,ed and chromatographed over silica gel ~ith ~ixtures o~ toluene ~nd ethyl a_eiate. There were obtained 4.2 ~s of 21-acetoxy~ -formyloxy-17a-hexyloxymetnoxy-,Q4-pre~lene-3,20-dione which melted at 110C after crystalliz~tion ~ith pentane.
b) 0038 gm of 21-acetoxy-11~-formyloxy-17a-hexyloxy-metho}~-a4-pregnene-3,20-dione was reacted un~er the condit~ons'de~cribed in Example 56(c) ~nd Ool gm O~
,21-dihydroxy-17a-hexylo~ymetho}~-~4-pregnene-3,20-dione was obtainea.
ExaQ~le 60 a) 6.0 gms of h~drocortisone 21-acetate-11-~orm~te were dissolved in 150 ml of ~et~ylene chloride and 65 g~s ,ol ~ormaldehyde-dibenzylacetal and in a ~ater bath a~ room temperature there was added in por~ions while stirrin~ under argon a mixture of 6 gms of phosphorus pentoxide and 12 gms of kieselg~hrO After 4 hours the mixture was filtered, washed with methylene chloride and adjusted to a pH-value of 8 with triethylamine. ~he~
methylene chloride was evaporated in vacuo and the residue wa~ chrom~tographed over silica gel with mi~tures of toluene and ethyl acetate. ~here were obtained 3.0 gms of 21-acetoxy-17-benzyloxymethox~-11~-~ormyloxy-a4-pregnene-3,20-dione.

~ ~7'~590 b) 0038 gm of 21-aceto~y-17a-benæyloxymethoxy~
forMyloxy-~ -pregllene-3,20-dione was reacted under the conditions described in EYample 56(c) and there ~as obtained Ooll gm of 17~-benzylo~ymet~oxy~ ,21-dih~droxy-~4-pregnene-3,~0-dione.
~c~le 61 a) Flavobacteri1ua dehvdro-en~ns B~CC 13,930 ~JaS
cultivated and fermented as described in ~xa~ple l(b)o At the 24th hour of the main fermenter 19.5 gms of 16a-me~hylene-3~,21-~iacetoxy-17~-methoxymet}loxy-~5-pregnen-20-one in 500 ml o~ dimethylformamide were added and the fermentation was continued Por a further 28 hoursO
~ he fermentation culture was worked up as described in Example l(b) and there were obtained 15.5 gms oP 16-methylene-21-hydroxy-17a-metho~ymethoxy-~4-pregnene-3,20-dione melting at 147/150-151C.
b) Cllrvularia lunata ~RR~ 2380 ~las grown, as described in Example 13(b), in a shaking Plask and a pre- and main-fermenter. At the 10th hour of the main Permenter there were added 20 gms of 16-methylene-21-hydroxy-17a-metho~y-methoxy-~4-pregnene-3,20-dione in 400 ml of ethylene glycol monomethyl ether. From this moment the pH-value was maintained between 6.4 and 607 and the fermentation was continued for a fuL~ther 8 hours.

~7~59 The fermentation culture IJas ~orked up as described in EY.ample 13(b) ~nd -there ~7ere obtained 11 gms of 16-methylens-11~,21-dihydro}~-17a-methoY~ymethoxy-~4-pre~nene-3,20-dione melting at 205/206-208C.
le 62 ~ r sim~ AT~C 6946 W2S ~rol~n as described in Example 11 in a gro~7th flask and a fermentation ~lask.
At the 6th hour there ~as added to the fermentation flask 1 ml of a sterile solution of 50 mg of 11~521-dihydroxy-17a-propox~etho~-a4-pregnene-3,20-dione in ethylene glycol monomethyl ether and fermentation was continued -for a further 42 hours. The fermentation culture ~Jas worked up as described in ~ample l(b) and there ~Jere obtained ai m~ of ll,B,21-dihydrox~r-17a-propoxymethoxy-~1'4-pregnadiene-3520-dione melting at 121/125-127C.
Ex~l,;~le 63 300 gms of 11~,21-dihydroxy-17~-methoxymethoY.y-16-methylene-a4-pre~lene-3,20-diolle were dissolved in 12 ml of pyridine at ~20C and 2.37 ml of acetic ~lydride ~lere added. T~e reaction mixture was then stirred for 2 hours ~ l7as at ~20C~and/then precipitated in 144 ml of ice-water.
The whole was then stirred for one hour. The crystallizate was filtered off l~ith suction, ~ashed ~ith l~ater and dried.
After recryst~ ation from et~yl acetate there ~ere obtained 2.74 ~ms of 21-acetoxy-11~-hydroxy-17~-methoxy-methoxy-16-metllylene-~4-pregne~e-3~20-~ione meltin~ at 166-167Co

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLU-SIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the manufacture of an 11.beta.-hydroxy-steroid of the pregnane series substituted in the 17.alpha.-position by an acetal group of the general formula X

(X) in which R1 represents an alkyl group containing 1 to 8 carbon atoms which may be interrupted by an oxygen atom or represents a benzyl group and R2 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, wherein a corresponding 11-desoxy-steroid of the pregnane series or, when applicable, a 21-ester thereof is fermented with a fungal culture of a strain of the genus Curvularia.