AU621857B2 - Novel compounds, process for the preparation thereof and uses thereof - Google Patents

Description

Werner Waldegg Single Signature, by special power To: The Commissioner of Patents iocecnnoiogische Forschung mbH P Dr. oahim Klein Qe ut Zeittrager Wissenschaftlicher Administrativer Geschcftsfhrer Geschaftsftihrer 6.87 521 n L~-t I ill r IYI II I i P I 's.

AUSTRALIA

PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE 6' 9 1 5 71 Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority: Related Art: TO BE COMPLETED BY APPLICANT SName of Applicant: ressof Aplic Address of Applicant: Actual Inventors: Address for Service: CIBA-GEIGY AG and GESELLSCHAFT FUR BIOTECHNOLOGISCHE FORSCHUNG mbH Klybeckstrasse 141, 4002 Basle, Switzerland, and Mascheroder Weg 1, 3300 Braunschweig, Federal Republic of Germany 1) Prof. Dr. Hans Reichenbach, 2) Dr. Wolfram Trowitzsch-Kienast, 3) Dr. Klaus Gerth 4) Dr. Herbert Irschik Dr. Brigitte Kuze, 6) Dr. Hermann Auqustiniak, 7) Dr. Norbert Badorf, 8) Dr. Rolf Jansen, 9) Prof. Dr. Gerhard Hofle, Heinrich Steinmetz ARTHUR S. CAVE CO.

Patent Trade Mark Attorneys Level Barrack Street SYDNEY N.S.W. 2000

AUSTRALIA

Complete Specification for the invention entitled NOVEL COMPOUNDS, PROCESS FOR THE PREPARATION THEREOF AND USES THEREOF.

The following statement is a full description of this invention including the best method of performing it known to me:- 1 ASC 49 la The present invention relates to novel polycyclic alkaloids having ao o antibiotic properties, to a process for the preparation of these com- So" pounds by cyanidation and optional acylation or sulfonylation of the oo fermentation product of a microorganism of the species Myxococcus *oo* xanthus, to pharmaceutical compositions that contain the novel compounds, and to the use of said novel compounds as antibiotics and as tumour- S° inhibiting agents and for the preparation of pharmaceutical compositions.

o Specifically, the present invention relates to compounds of formula o 0 eCH 3

CH

3 fO 0 S"o A COCH3 "t o si

H

wherein R is hydrogen, acyl, sulfonyl, sulfa or phospho, and A is C-O or C-OH or acylated C-OH, and the dashed line denotes a C=C double bond at the site of the central bond when A is C=O or at the site of the two outer bonds when A is C-OH or acylatod C-OH, and to salts of such compounds, especially pharmaceutically acceptable salts.

2 The compounds of formula I are closely related to the compounds isolated from Streptomyces lavendulae and named saframycins. For example, the fermentative production of saframycins A, B, C, D and E is disclosed in US patent specification 4 248 863, and the production of saframycin S is disclosed in US patent specification 4 372 947. The structure of the known saframycins is cited in T. Arai, Antimicrobial Agents and Chemotherapy 28, 5 (1985). In contrast, the compounds of formula I of this invention differ from the saframycins disclosed in the prior art at least in the presence of the alanyl radical in the side-chain, in place of which the saframycins of the prior art contain the acyl radical of pyruvic acid or another acyl radical, and, in addition, in the presence #of the methoxy group in the bridged bicyclic nucleus. Saframycin deri- Svatives containing an alanyl radical in the side-chain are disclosed in European patent application 173 649, but they differ from the compounds of this invention in that they do not contain a metlhcvy group.

o European patent application 55 299 discloses the antibiotics Y-16482 a a and B, which are obtained by fermentation of a strain of Pseudomonas fluorescens. These compounds, also called safracins, are closely related Sto the saframycins according to Y. Ikeda at al., J. Antibiotics 36, 1284 (1983), and hence also to the compounds of formula I of this invention.

The safracins, however, differ from the compounds of this invention in °v o 6 the meaning of A (one of the groups A is C-H) and in the absence of a methoxy group in the bridged bicyclic nucleus.

Further closely related compounds can be isolated from the sponge Reniera sp, These compounds, named as renieramycins by J.M. Frinke and D.J. Faulkner, J. Am. Chem. Soc. 104, 265 (1982), differ from the compounds of formula I in at least the side-chain which, instead of alanylaminomethyl, is (Z)-2-methyl-2-butenoyloxymethyl (an angelic acid ester) in the renieramycins.

The configuration at the centres of chirality of the compounds of formula I is not known with certainty. However, pointers to the relative configuration at the different centres of chirality result from the nuclear Overhauser effect in proton nuclear resonance spectra and from -3the analysis of the alanine liberated during hydrolysis. In analogy to the known absolute configuration of saframycin C determined by X-ray structural analysis Arai et al., Tetrahedron Letters 1979, 2355) and of brominated safracin A Ueda at al., Acta Cryst. C 40, 1578 (1984)], it must be assumed that the compounds of this invention conform to the perspective formula

CH

CM

3

CC

3 3 ~JJZJ. Cfl (Ia).

OCH

CH

3 0

NHR

a *o

CHJ

I

In compounds of forotula I and Ia, R is hydrogen, acyl, sulfonyl, sulfo or phospho. An acyl group R is, for example, the acyl group of a carboxylic S acid, of a half-ester of carbonic acid, of carbamic acid or of a thiocarbamic acid having up to 2C0 carbon atoms, for example C-CzOalkanyl, C2-Cialkanoyl which is substituted by hydroxy, etherified or esterified hydroxy, amino, acylated amino, carboxy, amnidated or osterified carboxy, oxo and/or halogen, or aroyl, CQ-C 7 alkoxycarbonyl, Cl-C 7 alkylaminocarbonyl, Cl-C7alkylaminothiocarbonyl, arylaminocarbonyl or arylaminothiocarbonyl. R 2 as acyl may also be the radical of a polypeptide, for example of a polypeptide of naturally occurring amino acids, preferably of 2-200 amino acids, R as sulfonyl is, for example, the radical of a sulfonic acid having up to 20 carbon atoms, for example Ci-C 7 alkanesulfonyl or arylsulfonyl, or unsubstituted or substituted aminoasulfonyl. Sulfo is the sulfuric acid radical -503H. Phospho is the phosphoric acid radical -POjH.

-4-

CI-C

2 oAlkanoyl is preferably Cl-C 7 alkanoyl, for example formyl, acetyl, propionyl, n-butyryl, 2-methyipropionyl, n-pentanoyl, 2, 2-dimethylpropionyl, 2-methylbutyryl, 3-methylbutyryl or n-hexanoyl, or is straight-chain CB-C 2 ualkanoyl containing an even number of carbon atoms, for example n-octanoyl, n-decanoyl, n-dodecanoyl, n-tetradecanoyl, n-hexadecatioyl, n-octadecanoyl or n-icosanoyl.

Hydroxy-substituted CR-C 7 alkanoyl is, for example, glycolyl, glyceroyl or lactoyl, in which the hydroxy group may be etherified, for example with Cj-C 4 alkyl such as methyl, ethyl or isopropyl, or with arylalkyl containing 1 to 4 carbon atoms in the alkyl moiety, for example benzyl, or esterified, for example with C 1

-C

7 alkanoyl such as formyl, acetyl, propionyl or pivaloyl.

Amino-substituted CZ-C 7 alkanoyl is, for example, glycyl, alanyl, 0-alanyl, valyl, norvalyl, leucyl, isoleucyl, '-aminobutyryl, a,y'-diaminobutyryl, lysyl or ornithyl. R2 as acyl also comprises acyl radicals of other amino acids, for example acyl radicals of naturally occurring a-amino acids, such as prolyl, arginyl, cysteinyl, aspartyl, R-aspartyl, asparaginyl, glutamyl, 1-glutamyl, glutaminyl, phenylalanyl, tyrosyl, seryl, histidinyl, mothionyl, tryptophyl or threonyl, either in the natural L-form or in the unnatural D-form. The amino group or groups of the cited amino acids may be acylated, for example by one of the acyl groups mentioned above or hereinafter, for example by Cl-Ci~alkoxycarbonyl such as tert-butoxycarbonyl, or by arylallkoxycarbonyl containing 1 to 4 carbon atoms in the alkoxy group, for example benzyloxycarbontyl.

Carboxy-substituted Cz-C7alkanoyl ist, for example, malonyl, smccinyl, glutaryl or adipoyl, each containing a free carboxy group. This carboxy group can also be amidated or esterified, for example with a CI-C 7 alkanol such as methanol, ethanol or tert-butanol, or with an arylalkamol of I to 4 carb." z-iMS such as benzyl alcohol.

Oxo-substituted C2-C 7 alkanoyl is, for example, glyoxyloyl or pyruvoyl.

Halo-substituted C,'-0 7 alkanoyl is, for example, trifluorqacetyl or mono-, di- or trichloroacetyl.

Aroyl is, for example, benzoyl or 1- or 2-naphthoyl, wherein the phenyl or naphthyl ring may be substituted by nitro, amino, halogen, for example chloro or bromo, hydroxy, Cl-Ciialkoxy, for example inethoxy, carboxy, carbamoyl and/or esterified carboxy, for example CI-Ci~alkoxycarbonyl such as methoxycarbonyl, and is, for example, 4-nitrobenzoyl, benzoyl, anthraniloyl, 2,6-dichlorobenzoyl, salicylr-->L, galloyl, 3or 4-anisoyl, phthaloyl, terephthaloyl, 2-carbamoylbenzoyl or 2-methoxycarbonylbenzoyl.

CI-C

7 Alkoxycar~bonyl is, for example, methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl, isobutoxycarbonyl or tert-butoxycarbonyl.

:C1-C7Alkylaminocarbonyl and Ci-Cyalkylaminothiocarbonyl are, for example, methylaminocarbonyl or methylaminothiocarbonyl, ethylaminocarbonyl or ethylaminothiocarhonyl, n- or tert-butylaminocarbonyl or n- or tertbutylaminothiocarbonyl, or n-hexylaminocarbonyl or n-hexylaminothiocarbonyl. Arylaminocarbonyl and aryithiocarbonyl are, for example, phenylaminocarbonyl or phenylaminothiocarbonyl, 1- 2-naphthylaminocarbonyl or 1- or 2-naphthylaminothiocarbonyl, or phony lamino ca rbo nyl or phenylaminothiocarbonyl, whorein phenyl may be substituted by CI-Ci~alkyl, 00 for example methyl such as 2- or 4-methyl or 3,5-dimethyl, nitro, for 0 example 4-nitro, halogen, for example chloro or bromo such as 4-chloro, 4-bromo or 2,6-dichloro, Or CI-C~alkoxy, for example methoxy such as 2or 4-methoxy.

Ci-C 7 -Alkanesulfonyl is, for example, inethanesulfonyl, othanesulfonlyl or n-hutanesulfonyl. Arylsulfonyl is, for example, beozenesulfonyl, 1- or 2-naphtbalonesulfonyl or substituted benzenesulfonyl, wherein the substituent may be Cl-Cilalkyl such as methyl or isopropyl, nitro, halogen such as Qhloro or bromo, or Ci-Ci~alkoxy such as methoxy, for example as in p-toluenesulforiyl, p-nitrobenzenesulfonyl, 2, 4-dinitrobenzenesulforyl, p-bromobenzenesulfonyl or p-inethoxybenzenosulfonyl.

Substituted aminosulfonyl is, for example, substituted by one or two C1-C7alkyl groups and/or arylalkyl groups containing 1 to 4 carbon atoms in the alkyl moiety. in substituted aminosulfonyl, amino may also be part -4~4 -6of a 5- or 6-membered heterocycle containing one or two nitrogen atoms and, if desired, an oxygen or sulfur atom. Examples of substituted aminosulfonyl are methylaminosulfonyl, ethylaminosulfonyl, n- or tertbutylaminosulfonyl, diviethylaminosulfonyl, diethylaminosulfonyl, benzylamimosulfonyl, pyrrolidinosulfonyl, piperidinosulfonyl, 4-methylpyridazinylsulfonyl, morpholinosulfonyl or thiomorpholinosulfonyl.

Preferred acyl ra±dicals R are Cl-C 7 alkanoyl, for ei~ample formyl, acetyl, propionyl, n-butyryl, 2,2-dimethyipropionyl or n-hexanoyl, C2-C 7 alkanoyl S which is substituted by amino or acylated amino and/or free or esterified 0* carboxy, for example glycyl, alanyl, B-alanyl, valyl, y-aminobutyryl or 0.succinyl, glutaryl or adipoyl containing a free carboxy group, aspartyl, 0 -aspartyl, glutamyl or x-glutamyl, or one of the cited radicals wherein 0 the amino group is acylated by benzyloxycarbonyl anid/or wherein the carboxy group is esterified by methyl or benzyl, aroyl, for example 0 benzoyl, phthaloyl or 2-methoxycarbonylbenzoyl, arylaminocarbonylt for example phenyl aminooa rbony 1, or arylaminothiocarbonyl, for example e00 phenylaminothiocarbonyl. The preferred sulfonyl radical R is arylsulfon- 000 yl, for example benzenesulfonyl, p-toluenesulfonyl or p-nitrobenzenesulfonyl.

0 The acyl radical in the group A having the significance of acylatod C-Oil 0 0 has the same moaning as the acyl group The preferred acyl radical in the group A is, for example, CI-Cialkanoyl such as fornyl, acetyl, propionyl or 2,2-dimethylpropionyl, Or CZ-C7alkanoyl which is substituted by carboxy or esterifiod carboxy, for example succinyl or glutaryl, each free or esterified by methyl.

Preferred compounds of formula :I are those wherein R is hydraoen, the acyl group of a carboxylic acid, of a half-ester of carbonic acid, of a carbamic acid or of a thiocarbamic acid containing up to 20 carbon atoms, the acyl radical of a polypeptide from 2-200 naturally occurring amino acids, the radical of a sulfonic acid containing up to 20 carbon atoms, unsubstituted or substituted aminosulfonyl, sulfo or phospho, and A is C-0 icr C-OH or acylated C-OH, wherein C-Oi may be acylated by a carboxylic acid containing up to 20 carbon atoms, and the dashed line denotes a C=C double bond at the site of the central bond when A is C=O or at the site of the two outer bonds when A is C-OH or acylated C-OH, and salts of such compounds.

Particularly preferred compounds of formula I are those wherein R is hydrogen, Cl-Caoalkanoyl, C 2 -C~alkanoyl which is substituted by hydroxy, etherified or esterified hydroxy, amino, acylated amino, carboxy, amidated or esterified carboxy, o%,o and/or halogen, or is aroyl, Cl-C 7 alkoxycarbonyl, Cl-C7alkylaminocarbonyl, CI-C 7 alkYlaminothiocarbonyl, arylaminocarbonyl, arylaminothiocarbonyl, the acyl radical of a polypeptide from 2-200 naturally occurring amino acids, CI-Cvalkanesulfopyl, arylsulfonyl, aminosulfonyl, CjlCvalkylaminosulfonyl, dialkyl- B arinosulfonyl containing 1 to 7 carbon atoms in each of the alkyl moieties, arylalkylaminosulfonyl containing 1 to 4 carbon atoms in the alkyl moiety, cyclic 5- or 6-membered aminosulfonyl, wherein the ring contains one or two nitrogen atoms and, if desired, one oxygen or sulfur atom, sulfo or phospho, and A is C=O or C-Oil or acylated C-O11, wherein SC-OHf may be acylated by Cl-Czaallkanoyl, Ca-C 7 alkanoyl which is substituted by hydroxy, ethorified or esterified hydroxy, amino, acylated amino, arboxy, amidated or esterified carhoxy, oxo and/or halogen, or by V aroyl, and the dashed line has the meaning given above, and ialts of such compounds, More, particularly preferred compounds of formula I are those wherein R is hydrogen,, one of the acyl or sulfonyl groups mentioned above as preferred, sulfo or phospho, and A is 0=0 or C-Oil or acylated C-09, wherein C-Otj may be acylated by one of the acyl radicals cited above aa preferrod, and the dashed line has thle meaning given above, and pharmaceutically acceptable aalti of$ such compounds.

The invention relates in particular to compounda of formula, I wherein R is hydrogen, Cj-c 7 alkanoyl, for example formyl, ncotyl, 2,2-dimethylpropionyl or n-hexanoyl1, Ca-C7alkanoyl which insu~bstituted by Catboxy, Ci-Ct~all~xycarbonyl or bonzy1oxycarbonyl and/or amino or bonzyloxycarbomylamino, for example sucainy. or glutaryl containing a free patboxy group, 3-mothoxycarbonylpropionyl, 4-mothoxycarbonylbutyryl, 0-aspartyl or 3-bonzyloxycarbonyl-3-bonzyloxycirbonylaminopropienyl, phonylatnino- -8carbonyl, phenylaminothiocarbonyl, p-toluenesulfonyl or sulfo, and A is C=O or C-OH or acylated C-OH, and the dashed line has the meaning given above, and pharmaceutically acceptable salts of such compounds.

First and foremost, the invention relates to the compounds named in the Examples and the pharmaceutically acceptable salts thereof. Preferred compounds are 21-cyano-saframycin Mx 1 and N-formyl-21-cyano-saframycin Mx 1.

I. f The compounds of this invention are referred to hereinafter as 21-cyano- S saframycins Mx 1. Without affix, this nomenclature denotes compounds of o formula I in which A is C-OH. With the affix "BC" (bisquinone), this nomenclature is employed to denote compounds in which A is C=O.

00 0 o0 Salts of compounds of the formula I are especially pharmaceutically acceptable non-toxic acid addition salts. Examples of acid addition salts no° S with non-toxic, physiologically well tolerated acids are salts with 0o 6 inorganic acids, for example hydrochloric acid, sulfuric acid or phosphoric acid, or with organic carboxylic, sulfonic or sulfe acids, for 0 example formic acid, acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid, and also amino acids, for example a-amino acids, as well as methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid or naphthalene-2-sulfonic acid, or with other acidic organic compounds, such as ascorbic acid.

Compounds in which R is carboxy-substituted alkanoyl, or aroyl, sulfo or phospho, can also form inner salts or salts with organic bases. Preferred salts are salts with pharmaceutically acceptable non-toxic bases, for example with physiologically well tolerated organic amines, for example with tert-butylaminQ, hydroxyethylamine, cyclohexylamine, diethylamine, 1 S-9di-n-butylamine, bis(hydroxyethyl)amine, trimethylamine, triethylamine, tris(hydroxyethyl)amine or diethyIhydroxyethylamine, and also with organic ammonium salts such as tetramethylammonium or tetraethylammonium.

For isolation or purification it is also possible to use pharmaceutically unsuitabe salts,.

The compounds of formula I and pharmaceutically acceptable salts thereof have valuable pharmacological properties. For example, compounds of formula I are effective in low concentrations of less than 0.3 pg/ml against various bacteria, for example against Staphylococcus aureus, S Bacilus subtilis, Micrococcus lutous and Escherlchia coli. In addition to antibiotic properties, the compounds also have tumour-inhibiting properties, for example against the experimental tumours leukaemia L 1210/S2, human melanoma SK-MEL 109, human pulmonary carcinoma MBA 9812, colon adenocarcinoma 26 and teticulosarcoma M5076, The lifespan of mice with leukaemia L1210/S2 is significantly prolonged p by more than 70 by treatment with 21 -cyano-saframycin Mx 1, N-caproyl- S 21 .c yano-saframycin Mx 1, N-pivaloyl-21-cyano-saframycin Mx 1 or N-formyl-21-cyano-saframycin Mx 1 comprising administering intraperit~neal V injections on four successive days of 1,25, 2.5 or 5 mg/kg per day. The treatment with 21-cyano-aframycion Mx 1 (1.25 to 5,0 mg/lkg) cures 20 to of the mice with leukaemia 1210/S2 (survival time more than days). By injecting 1.25, 2.5 or 5 mg/kg of 21-cyano-saframycin Mx 1 per day on seven successive days, the weight of human melanoma SK-MEL 109 implanted in nude mice is reduced by 83 77 and 92 respectively, compared with that in untreated mice (determined after 3 weeks). Under identical conditions, a reduction of 35 and 66 respectively is achieved with 1.25 or 2.5 mg/kg of N-caproyl-21-cyano-saframycin Mx 1, a reduction of 46 and 69 respectively with 2.5 and 5 mg/kg of N-pivaloyl-21-cyano-saframycin Mx 1, and a reduction of 84 and 73 respectively with 2.5 and 5 mg/kg of N-formyl-21-cyano-saframycin Mx 1.

10 Processes The invention also relates to processes for the preparation of compounds of formula I. These compounds are obtained by converting, in a compound of formula II

CH

3 C3 wh n i3 i (ID V 21 C U 0 2

NH

4 44 wherein A is C=O or C-OH., in any order, the hydroxy group at the 21- 4 carbon atom into the cyano fgroup by cyanidation, and the NH group into the NHR group by acylation or sulfonation, where R is as defined for formula I, or. to prepare compounds of formula I, wherein R is hydrogen, converting the hydroxy group of a compound of formula II at the 21-carbon atom into the cyano group by cyanidation, and, if desired, converting a resultant compound into another compound of the invention and/or a resultant salt into the free compound or a resultant free compound into a salt, In the following description, acylation will be understood as meaning also the introduction of a sulfo group (acyl -SOH) or a phospho group (acyl w -POaH 2 The cyanidation is performed by reaction with an aqueous solution of a cyanide salt. Preferred cyanide salts are alkali metal cyanides, for example sodium cyanide or potassium cyanide, and also cyanide salts of dibasic metals, for example zinc cyanide, and ammonium cyanides, for example unsubstituted ammonium cyanide, trialkylammonium cyanides, for

~R

11 example triethylammonium cyanide, tetraalkylammonium cyanide, for example tetramethylammonium cyanide or tetrabutylammonium cyanide, or benzyltrialkylammonium cyanides, for example benzyltrimethylammonium cyanide. The reaction is carried out in aqueous or aqueous-organic solution, preferably in a buffer solution in the weakly acid range or close to the neutral point, for example in the pH range from 5 to 8, and in the temperature range from 00 to about 40 0 C, preferably at room temperature.

The reaction time is from a few minutes to several hours, for example from 10 to 30 minutes, depending on the temperature and the pH of the So* reaction mixture. The reaction is preferably carried out in an inert gas S atmosphere, for example under nitrogen. If atmospheric oxygen is not completely excluded, then under the reaction conditions a compound of formula I, wherein A is C-Ol, will be oxidised to the corresponding o quinone in which A is C=0.

The acylation or sulfonylation of a compound of formula II, or of a compound of formula I obtainable by cyanidation, wherein R is hydrogen, is effected by methods which are known per se, for example by reaction with an acid of formula R-OH or with a reactive functional derivative S thereof.

When using a free carboxylic acid for the acylation, the reaction is .O normally carried out in the presence of a suitable condensing agent, for example of a carbodiimide, for example dicyclohoxylcarbodiimide or, preferably, N-othyl-N'-3-dimethylaminopropylarbodiimide. The condensation is carried out in a buffered aqueous-organic solvent mixture or in an aqueous buffer close to the neutral point, for example in the pH range from 6 to 8, if appropriate with cooling or heating and in an inert gas atmosphere, for example under nitrogen, A reactive functional derivative of a carboxylic acid, of a half-stoer of carbonic acid or of a suifonic acid, is a suitable anhydride, for example a symmetrical anhydride, for example acetic anhydride, a mixed anhydride of the acid z-011 with an inorganic or organic acid, for example with a hydrohalic acid such as hydrochloric acid or hydrobromic acid, ie. for example a carbonyl chlori: or bromide, a chloreformate or a sulfonyl chloride, or a mixed anhydride of a carboxylic acid with another car- -12boxylic acid, for example formic-acetic anhydride, or with a half-ester of carbonic acid, for example the ethyl or isobutyl half-ester of carbonic acid. A fur !,er reactive functional derivative of a carboxylic acid or of a half-ester of carbonic acid is, for example, an activated ester, for exan~ple, a N-hydroxy ester such as the ester of N-hydroxyp~jaeridino, N-hydroxysuccinimide, N-hydroxyphthalimide or 1-hydroxybenztriazole. A reactive functional derivative of a carbamic acid or of a, thiocarbamic acid is the corresponding isocyanate or isothiocyanate.

The suitahily Rubsti~uted sulfamidyl chloride, for example unsubstituted sulfamidy'. chloride, dimethylsulfamidyl chloride, tert-butylsulfamidyl chloride o'r sulfopytrolidinyl chloride, is used to introduce art unsubstituted or subotituted aminosutlonyl group, Instead of the chloride it is also poasible to use an activai~ed ester, for example an ester of a phenol carrying olectrophiliq- stibstituents, for example pentafluorophenol or 2,4, 5-triohlorophen l.

The sulfo group is introduced with ohlorosulfoniQ acid or, preferably, a com ex of sulfur trioxide with an organia amine, for example with trimethylamine, triothylamine, benzyldimethylaniline, N-methylmorpholine or pytidino, The phoipho- group is nuitahly introduced with a reactive amide of phophoric acid, for example phosphoroimidazolidQ. 13stevs may also nultably be used, fo'- example enters of hydrochloric acid and mixed esters ofhydrochloric acid and a readily removable alcohol, for example phoaphoroXy chloride* bit(212*2-trichloroothyl) phoophorochloridite, 2,2,2-tribrornomet'hyl phosphorochloridito morpholide, 2,2 ,2-tribromomethyl phosphotodichloridite and 2,2, 2-trichloroothyl phosphorodichloridite.

When using an ester of hydrochloric acid or a suitable corresponding mixed ester, the acylatioo is followed by an ester hydrolysis or ester cleavae by methodsn Unown per ae in or-der to liberate the desired phospho groupq The a~'ylation Gi dulfqnylation reactions wiO% reactive functional dorivativoa of, R-Ofl are carried out in organic solution or, preferably, in aqueoo-4-organtc homogeneoup or two-phase solvent mixtures, if flesired with cooling or gentle boating, for example in the temperature range from 4bou~t 30 to~ +50"C, preferably at room tomperatate, and, if desired, in 13 13 an inert gas atmosphere, for example under nitrogen. An acid acceptor is added, for example suitable organic base, for example an amine such as trimethylamine, triethylamine, N-methylmorpholine, lo[4.3.0]non-5-ene, 1,8-diazabicyclo[5.4.0O-undec-7-ene, pyridine or dimethylaminopyridine, or an organic base, for example an alkali metal hydroxide or alkaline earth metal hydroxide, for example sodium, potassium or calcium hydroxide, an alkali metal carbonate or alkaline earth metal carbonate, for example sodium carbonate, sodium bicarbonate or calcium carbonate, or an alkali metal phosphate, for example sodium or potassium phosphate or sodium or potassium hydrogen phosphate. The o4 l, acylation is preferably carried out in an aqueous buffer close to the neutral point, for example in the pH range from 6 to 8, in which case the buffer base acts as acid acceptor. If desired, the pH is kept in the 4u o range from 6 to 8 by the continuous addition of an inorganic base, for example sodium hydroxide. A water-miscible organic solvent is preferably used as solubiliser, for example tetrahydrofuran, dioxan or acetonitrile, 6 000 When carrying out the reaction in a pure organic solvent, then pyridine U will preferably be used.

To inhibit the acylation of a phenolic hydroxy group A, an alcohol, for exampl' methanol or ethanol, is added to the aqueous-organic solvent mixture. However, if it is desired to acylate the phenolic hydroxy group A, then the reaction will be carried out with a suitably large excess of acylating agent.

The conversion of a compound of formula I into another compound of formula I is effected by methods which are known per se, for example by oxidation, reduction, acylation, sulfonylation, or by the per se known conversion of an acyl group, Conversions of hydroquinonos, i.e, compounds of formula I in which A is C-0H, into corresponding quinones, i.e.compounds of formula I in which A is CO4, by oxidation and conversion of the quinones into hydroquinones by reduction, can be cattied out, for example, by procedures analogous to those described in Mothodon der Organischen Chemis (Houben-Weyl), 4th edition, Vol, VII/3a, Thieme Verlag Stuttgart, 1977, Suitable mild oxidising agents are, for example, metal salts and metal oxides, for 3 a-: i 14example silver oxide in diethyl ether, benzene or toluene, in the presence of a drying agent, for example sodium sulfate, an iron (III) salt such as iron (III) chloride, in water or aqueous ethanol, a copper salt or thallium salt, for example thallium (III) trifluoroacetate. A suitable oxidising agent is also atmospheric oxygen in neutral solution, for example in a buffer solution of pH 7 to 8. Examples of suitable reducing agents are hydrogen in the presence of heterogeneous catalysts, for example platinum oxide or palladium on carbon, or homogeneous catalysts, for example tris(triphenylphosphine)rhodium(I) chloride, and also metal hydrides, for example borohydrides such as sodium borohydride, or aluminium hydrides such as lithium aluminium hydride, reducing salts, for example sodium dithionite, and also light in the presence of a hydrogen donor, for example ordinary daylight and methanol. Conversions S of quinones and hydroquinones into one another can also be carried out by electrochemical methods, for example electrolysis in weakly acid saltcontaining solutions, for example buffer solutions of pH 4 to 6.

Acylations or aulfonylations are carried out in the above described manner. If it is desired to acylate a compound of formula I, wherein R is acyl and A is C-OH, at the phenolic groups, then in this case a purely organic solvent in the presence of an organic base may also be used in place of the customary aqueous or aqueous-organic solvents. Examples of suitable solvents are ethers, for example tetrahydrofuran or dimethoxyethane, amides such as dimethyl formamide or dimethyl acetamide, or acetonitrile, in each case in the presence of one of the amine bases mentioned above. The reaction can also be carried out direct in pyridine.

Conversions of acyl groups R can be carried out in a manner known per se.

In these conversions, the reaction conditions must be so chosen that the other functional groups of the compounds of the invention are not chantged. It is possible, for example, in an acyl group R in which an amino group is protected by benzyloxycarbonyl and/or a carboxy group is protected by benzyl, to liberate said groups by mild hvdrngenolysis, for example with hydrogen in the presence of a noble metal catalyst, for example palladium on carbon.

15 If it is desired to prepare compounds of formula I, wherein R is acyl, sulfonyl, sulfo or phospho, it is possible first either to cyanidate the starting material of formula I and then to carry out acylation or sulfonylation, or first to acylate or sulfonylate said starting material and then to effect cyanidation. The resultant intermediates can either be isolated after carrying out the first step or subjected to the second step without being isolated.

i Salts of compounds of formula I can be prepared in a manner known per se, for example by reacting the free compound with preferably a stoichiometric amount, or with a small excess, of an acid that forms an acid addition salt or of the salt-forming organic amine.

j Acid addition salts can be converted into the free compounds or into salts with bases in conventional manner, for example by treatment with an equimolar amount of a free base, for example a hydroxide such as an )J Q alkali metal hydroxide, for example lithium, potassium or sodium hydrox- *u a ide, an alkaline earth metal hydroxide, for example calcium hydroxide or magnesium hydroxide, or ammonium hydroxide, for example unsubstituted a ammonium hydroxide or benzyltrimethylammonium hydroxide, or an organic tertiary amine, for example triethylamine. It must, however, be borne in mind that the free compound of formula I has only limited storage o stability and, for storage, must be converted into an acid addition salt.

Salts with organic amines are likewise converted in conventional manner into the free compounds or into acid addition salts, for example by treatment with an equimolar amount of a mineral acid, for example hydrochloric acid, sulfuric acid or phosphoric acid, or of an organic strong acid, for ^nample formic acid, trifluoroacetic acid or p-toluenesulfonic acid, Salts of compounds of formula I are converted in a manner known per se into other salts. The conve.sion of salts into other salts is preferably effected with ion exchangers which are charged with the desired anion or cation, or by adsorption chromatography in a solvent which contains an excess of the acid of the desired acid addition salt or the desired organic amine or ammonium ion.

16 16 The invention also relates to those embodiments of the process which comprise using an intermediate obtainable in any stage of the process as starting material and carrying out the remaining steps, discontinuing the process at any stage and/or further processing in situ a compound obtainable by the process of the invention.

Starting materials and intermediates Compounds of formula II, wherein A is C=O or C-OH, form the subject matter of European patent application 87 810 454.6, They are prepared, Ot'" for example, by culturing the strain Mx x48 of the species Myxococcus S* xanthus in a culture containing carbon and nitrogen compounds and essential inorganic salts in readily assimilable form, in the temperature range from 150 to 40 0 C and in the pH range from 5 to 9, under aerobic S0 0 0o a conditions, and isolating the compounds of formula I so obtained.

4 0 0 The microorganism Myxococcus xanthus, strain Mx x 48 is deposited with o"i: the Natinal Collection of Industrial and Marine Bacteria, Aberdeen, 0 0 1 Scotland, under the number NCIB 12 268.

The culture medium used for the growth must contain a source of carbon 4 04 and nitrogen and also essential inorganic salts. Suitable carbon and nitrogen sources are amino acids, peptides and proteins and also degradation products thereof, such as peptone or tryptone, and also meat extracts, cereal meal, for example of corn or wheat, beans, especially soya beans, fish meal, seeds, for example of cotton plants, distillation residues from the production of alcohol, yeast extracts and the like Essential inorganic salts contained in the nutrient solution may be, for example, chlorides, carbonates, sulfates, phosphates of alkali metals or alkaline earth metals, for example sodium, potassium, magnesium and calcium, and also salts of iron, zink, manganese, molybdenum and copper.

The growth is carried out preferably in liquid cultures, especially aqueous cultures.

17 A suitable liquid culture medium is especially MD 1 (peptone from casein, tryptically digested, 0.3 CaCl 2 *2H 2 0 0.05 MgSOi.-7H 2 0 0.2 Other suitable liquid culture media consist, for example, of peptone from casein 0.05 CaClz 2 2H 2 0 0.05 MgS047HIO 0.02 to which there may be added, as desired, 0.5 single cell protein, corn steep liquor, yeast extract, amino acid hydrolysate of casein, or protein from fish meal.

The culture is preferably grown in the temperature range from 250 to Suitable pH values are from 6.5 to Preferably, growth is carried out in serveral stages by first producing a Spreculture, for example in one of the cited culture media (inoculum), which is then, after about a one to two day fermentation, used to S inoculate a larger culture, for example in a dilution ratio of from 1:10 to 1:5000, This culture can in turn, after about a two to three day fermentation, be used to inoculate an even larger main culture, for example in a dilution ratio of from 1:10 to 1:1000.

The course of the fermentation can be followed analytically by sampling S during fermentation, for example by measuring the pH value of the culture which, during fpfrmentatior., rises from about pH 7.2 to pH 8.0, by measuring the optical density, which is a measure of the growth of the strain, gravimetrically by means of the dry weight of the biomass formed, by thin-layer chromatography, by reverse phase high-pressure liquid Schromatography, or by determining the antibiotic activity of the components contained in the culture filtrate.

To isolate the desired compounds from the crude fermentation liquor, the latter is stirred, preferably for several hours, with macroporous non-ionic adsorber resins. Suitable adsorber resins have a pore volume from about 0.5 to about 4.5 ml/g, a specific surface area of about 01001000 mg/g, and an average pore diameter from about 4 to about 130 nm, and are available, for example, under the trade name AMBERLITE® XAD ER 180 of' Rohin Haas.

18 After separating the fermentation liquor from the adsorber resin, the latter is washed with water and then eluted with isopropanol. The eluates are acidified and concentrated under vacuum.

The crude extracts are purified by chromatographic methods, for example by chromatography on an ion exchanger having carboxy groups, by adsorption or partition chromatography and/or high-pressure liquid chromatography on silica gel containing long-chain alkyl groups ("reversed phase").

Intermediates of formula oous Q f OH CH CH3

CH

S 1 So- NIHR a wherein R is acyl and A is C=O or C-OH or acylated C-OH, likewise form the subject matter of European patent application 87 810 454.6. They can be obtained from a compound of formula II by one of the acylation procedures described above.

19 Intermediates of formula CH3 CH3 S(Ilb), 02 /NHRb 4 o a 44 wherein R is acyl which is substituted by carboxy or esterified carboxy, or is sulfo or phospho, and A is C=O or C-OH or acylated C-OH, and the dashed line denotes a C=C double bond at the site of the central bond when A is C=O, or at the site of the two outer bonds when A is C-OH or acylated C-OH, and salts of such compounds, especially pharmaceutically acceptable salts, are novel and likewise constitute an object of the present invention.

Concerning the configurations at centres of chirality of the compounds of formulae II, Ila and lIb, the same applies as has been stated in respect of those of formula I.

In the compounds of formula Ilb, acyl, sulfo, phospho and A have the same meanings as given for formula I.

Acyl is, for example, substituted Ci-Caoalkanoyl, preferably C 1

-C

7 alkanoyl, C 1

-C

7 alkenoyl, aroyl containing up to 20 carbon atoms or heteroaroyl containing 1 to 20 carbon atoms and I or 2 hetero atoms, for example nitrogen, oxygen and/or sulfur atoms. In addition to carrying one or more carboxy and/or esterified carboxy groups, the cited acyl radicals may also carry other substituents, for example amino, acylamino, for example CI-C7alkanoylamino, C1-C7alkoxycarbonylamino or arylalkoxycarbonylamino containing 1 to 4 carbon atoms in the alk;oxy group, hydroxy, Ci-C7alkoxy C=O or C-OH or aizylated C-OH, wherein C-OH may be acylated by a carboxylic acid containing up to 20 carbon atoms, and the dashed line denotes a a-nd/or Cl-C 7 alkanoyloxy. The amino or hydroxy substituents can also be I protected by a protective group commonly employed in peptide or alkaloid chemistry.

Esterified carboxy is, for example, Cl-C 7 alkoxycarbonyl or arylalkoxycarbonyl containing 1 to 4 carbon atoms in the alkoxy group.

Sulfo is the sulfuric acid radical -SO 3 H. Phospho is the phosphoric acid radical -PO 3

H

2 is for example, one of the above mentioned acyl groups, preferably o~Cl-C 7 alkanoyl, carboxy-substituted Cl-C7alkanoyl, or CI-C 7 alkanoyl which s substituted by Cl-C 7 alkoxycarbonyl or arylalkoxycarbonyl containing to 4 carbon atoms in the alkoxy group.

Cl-CzoAlkanoyl is preferably C 1 -C7alkanoyl, for example formyl, acetyl, propionyl, n-butyryl, 2-methyipropionyl, n-pentanoyl, 2, 2-dimethyl- 0propionyl, 2-metbylbutyryl, 3-methylbtityryl or n-hexanoyl, or in straight- 00chain CO-C2oalkanoyl containing an even number or carbon atoms, for example n-octanoyl, n-decanoyl, n-dodecamoyl, n-tetradecanoyl, n-hexadecanoyl, n-octadecanoyl or n-icosanoyl,

C

1

-C

7 Alkenoyl is, for example, acryloyl, crotonoyl or Allylcarbonyl.

Aoyl is, for example, benzoyl, 1- of 2-naphthoyl, indape-'l- or -2carbonyl, indene-l-, or -5-carbonyl, 4-biphenylylcarbonyl or 9luriylcarbonyl.

Heteroaroyl is, for example, 2- or 3-furoyl, 2- or 3-thonoyl, pyrrole-2or -3-carbonyl, pyridine-2-, or -4-carbonyl, indole-3- or yl, quinoline-2-carbonyl or isoquinoline-1-carbonyl.

0 1

-C

7 Alkoxy is, for example, methoxy, ethoxy, propoxy, i~opropoxy, n-butoxy, tert-butoxy, tert-pentoxy or n-hexoxy. Arylalkoxy containing 1 to 4 carbon atoms in the alkoxy group is, for example, unsubstituted or substituted benzyloxy such as p-nitrobenzyloxy, 2-phenyl--2-propoxy or 2-phenylethoxy.

Examples of acyl radicals Rb of the compounds of formula Ilb are carboxyalkanoyl containing 1 to 7 carbon atoms, in the alkanoyl group, for example oxalyl, malonyl, succinyl, glutaryl or adipoyl having a free (,arboxy group, or suitably esterified radicals, for example rethoxalyl, ethoxalyl, methoxycarbonylacptyl, ethoxvcarbonylacotyl or benzyloxycarbonylacetyi, methoxycarbonyl-3-propionyl, ethoxyoarbonyl-3-propionyl *'or bnavloxycarbony]l-3-propionyl, methoxycarbonyl"4-butyryl or ethoxycarbonyl-4-butyryl or methorp.abonyl-5-pentanoyl or 4 pentanoyl, carboxyalkenoyl containing, 1 to 4 carbon atoms in the allkonoyl group, for example ftumaroyl, maleoyl, citraconoyl or mesaconoyl having a free carboxy group, or suitably esterified radicals, for example cis- or trans-me' inoxy- or -e hxcroy-0-acryloyl, carboxyaroyl, for example Sphthaloyl, isophthaloyl or terephthaloyl having a free carboxy group, 3-'carboxy-9-naphthoyl or 4'-carboxybiphenylytl-4-cnrbonyl, or suitably V esterified radicals, for example 3- or 4-methoxyaarbonylbonzoyl or 23- -ethoxycarbonylphaoyl, 3-me thoxy carbonyl- 2-naphtha loylI or 3-ohoxcarony-2-aphh~lylor 4'-methoxyaarbonylbiphenylyl-4-carbonyl or 4'-ethoxyearbonylbiphenylyl-4-carbonyl, carboxy-heteroatoyl, for k 0 example 3-carboxy-4-furoyl, 3-catboxy-4-thenoyl, 2-carboxynicotinoyl or 2- or 3-carboxyisonicotinoyl, or suitably esterified radicals, for example 3-methoxycarbonyl-4-furoyl or !-ethoxycarbonyl-4-furoylo or 3-metboxycarbonyl-4-tbenoyl or 3-otho~cycatbonyl-4-thonoyl, arinocarboxyalkanoyl containing 1 to 7 carbon atoms in the allanoyl group, for example or j-aspartyl or a- or 7-glutamyl, or suitably eotorified and/or acyJlated radicals* for excampl~e 3-benzyloxycarbonyl-2- or -3-benzyloXycarbopylaminopropionyl, 3-tort-butoxycarbonyl-2- or -3-tert-butoxycarbonylaminopropionyl, or 4-benzyloxycarbonyl-2- or -4-benzyloxycarbonylaminobutyryl, hydroxycarboxyallkanoyl, containing 1 to 7 carbon atoms in the alkanoyl group, for example maloyJ. or tartaroyl having a free carboxy group or corresponding radicals whichi are ontorified at the tarboxy group and/or astorifled or etherified at the hydroxy group, for -22 example 3-methoxycarbonyl-2--hydroxypropionyl, 3-methoxycarbonyl-2-methoxypropionyl or 3-methoxycarbonyl-2-acetoxypropionyl, or 3-methoxycarbonyl- 2, 3-dihydroxypropionyl, 3-methoxycarbonyl-2, 3-dime thoxypropionyl or 3-methoxy-2,3-diacetoxypropionyl, sulfo or phospho.

Examples of acyl radicals in the group A having the significance of acylated C-OH are acetyl, propionyl, pivaloyl or n-hexanoyl, and also 3-carboxypropionyl, 3-methoxycarbonyipropionyl or 3-ethoxycarbonylpropionyl, 2-carboxybenzoyl or 2-methoxycarbonylbenzoyl or 2-ethoxy- Preferred compounds of formula 11b are those wherein Rb is carboxyalkanoy! containing 1 to 7 carbon atoms in the alkanoyl group, for example oxalyl, malonyl, succinyl, glutaryl or adipoyl having a free carboxy Sgroup, alkoxycarbonylalkanoyl in which the alkoxy and alkanoyl group each Scontains 1 to 7 carbon atoms, for example methoxalyl, ethoxalyl, methoxycarbonylacetyl or ethoxycarbonylacetyl, 3-mo thoxycarbonylpropionyl, 3-ethoxycarbonylpropionyl or 3-tert-utox-,ycarbonylpropionyl, or 4-mothoy carbony],butyryl or 4-ethoxycarbonylbutyryl, arylalkoxycarbonylalkan- Soyl containing 1 to 4 carbon atoms -in the alktoxy group and 1 to 7 carbon atoms in the alkanoyl group, for example benzyloxycarbonylaaetyl, 3-benzyloxycarbonylpropionyl or 4-benzyloxycarbonylbutyryl, carboxyairoyl, for example 3- or 4-qarboxybenzoyl or 4'-carhoxybiphenylyl-4-carbon- Yl, CI-C 7 lkoxycarbonylaroyl, for example 3- or 4-mothoxycarbonylbenzoyl, 3- or 4-ethoxycorbonylbenzoyl or 4'-rnthoxycarbonylbiphenylyl-4-ca-rbonyl or~ 4'-ethoxycarbonylhiphenyl-yl-4-carbonyl, arinocarboxyalkanoyl containing 1 to 7 carbon atoms in the alkanoyl group, for example Q- or f-aspatyl, or or 1-gltutamyl, aaylamimoal1koxycarbanyl-, alkanoyl in which the alkoxy and aJlkanoyl group each contains I to 7 carbon atoms, for example 3-tort-bUtoycarbony2l-2- or -3-tert-butoxycarbonylaminopropionyl, acylaiinoarylalkox-ycarbonylaldtanoyl containing 1 to 4 carbon atoms in the alkoxKy group and I to 7 carbon atoms in the alkanoyl group, for e,4mple 3-benzyloxycarbonyl-2- or -3-ben. yloxycarbomylaminopropionyl, hydroxycaboxyalkaoy!l containing I to 7 tarbon atoms in the alkanoyl group, for example 3-carboxy-2-hydrotypropionyl or 3-carboxy-2 3-dihydtoxypropionyl, acyloXyAlkoxycarboiiylalkanoyl in which the olkoxy and alkanoyl group eaxch contains 1 to 7 carbon atoms, for -23example 3-metoxycarbonyl-2-acetoxypropionyl or 3-methoxycarbony1-2 ,3-diacetoxypropionyl, or 3-ethoxycarbonyl-2-acetoxypropionyl or 3-ethoxycarbonyl-2,3-diacetoxypropionyl, sulfo or phospho, and A is C=O or C-OH or acylated C-OH, wherein C-OH may be substituted by Cl-C 7 alkanoyl, for example acetyl, propionyl, pivaloyJ. or n-hexanoyl, by carboxyalk~anoyl containing 1 to 7 carbon atoms in the alkanoyl group, for example 3 -carboxypropionyl, by alkoxycarbonylalkanoyl in which the alkoxy and alkanoyl group each contains 1 to 7 carbon atoms, for example 3-methoxycarbonyipropionyl or 3-ethoxycarbonyipropionyl, by carboxyaroyl, for example 2-carboxybenzoyl, or by CI-C 7 alkoxycarbonylaroyl, for example 0 2 -methoxycarbonylbenzoyl or 2-ethoxycarbonylbenzoyl, and the dashed line Sdenotes a C=C double bond at the site of the central bond when A is C=0, or at the site of the two outer bonds when A is C-Oil or acylated C-OH1, 0and salts of such compounds.

Particularly, preferred compounds of formula Ilb are those wherein ibs O carboxyalkanoyl containing 1 to 7 carbon atoms in the alkanoyi. group, for example malonyl, succinyl or glutaryl having a f ree carboxy group, 000allkoxycarbonylalkanoyl in which the alkoxy and alkanoyl group each o ~contains 1 to 7 carbon atom-i, for excample nothoxycarbonylaqetyl or othoxycalrbonylacetyl, 3 -mo thoxycarbony].propionyl or 3-ethoxycarbonylt- Spropionyl, or 4-methoXycarbonybutyryl or: 4-ethoxycarbonylbutyryl, carboxyaroyl, for example carboxyaroyl, for example 3- or 4-carboxybonzoyl, 0-C7alkoxycarbonylaroyl, for example 3- or 4-methoxycarbonylbenzoyl or 3- or 4-ethoxcycarbonylbenzoyl, ttminocatrboxyalkanoyl containing 1 to 7 carbon atoms in the allonoyrl group, for example ai- or 0-asparty1. or Q- or y-glutamyl, sulfo or phospho, and A is C-0 or C-OH or acylated C-O11, w~herein C-Oil may be substituteod by 0i-C~alkanoyl, for example acetyl, propianyl or piValoyl, by carboxyalkanoyl containing 1 to 7 car~bon a~tomns in the alkanoyl groUpt for example 3-carbcuxypropionyl, or by alkoxycarbonylaJlkanoy]. In which the alkoxy And alkanoyl group each contains 1 to 7 carbon atoms, for, example 3-mothoxycarbonylprapionyi.

or 3-ethoxycArborylpropionylf and the dashed line has the given moanng and salts of said compounds.

-24 Most particularly, the present invention relates to compounds of formula IIb, wherein Rb is carboxyalkanoyl containing 1 to 7 carbon atoms in the alkanoyl group, for example succinyl or glutaryl having a free carboxy group, alkoxycarbonylalkanoyl. in which the alkoxy and alkanoyl group each contains 1 to 7 carbon atoms, for example 3-methoxycarbonylpropionyl or 4-methoxycarbonylbutyryl, carboxybenzoyl, for example 3or 4-carboxybenzoyl, or sulfo, and A is 0=0 or C-OH or acetylated C-OH, and the dashed tine has the above meaning, and pharmaceutically acceptable salts of said uompounds.

V First and foremost, the invention rolates to the compounds named in the Examples and the pharmaceutically acceptable salts thereof, Ii: ,,,The compounds of formula IIb are hereinafter referred to as8 N-acylsafra- *2.mycins Mx 1, Without affix, this nomenclature denotes compounds of aformula I in which A is C-OH, With the affix "DCO (hisquinono), this nomenclature denotes compounds in which A -is C=O, Salts of compounds of formula TIb are especially pharmaceutically acceptable non-toxic acid addition salts. EXamples of acid addition salts a aWith non-toXic physiologically Well tolerated acids are salts with Sinorganic ticids, for example hydrochloric acid, sulfuric acid or phosphoric acid, or With organic carboxylia, sulfomic or sulfo acids, for example formic acid, acetic acid, propionia acid, glycolic acid, succinio 'acid, naleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, citric acid, benzoic acid, cinnarsic acid, mandelic acid, oalicylic acid, 4-aminosalioylia acid, 2-phenoxybenzoia acid, 2-acetoxybenzoic acid, embonic acid, Aicotinic acid or isonicotinic, acid, and also amino acids, for example a-amino acids, as well as ,ethanesulfomic acid, ethanosulfcomic acid, 2-hydrox-yothanesulfonic Acid, ethane-l ,2-disulfonic acid, bazenesulfonic acid, 4-methytbonzeonulfonic acid or naphthalene-2-sulf'onia acid, or With other acidic organic compounds, such as ascorbic acid.

The compounds of formula M~ which carryf a carboxy, sultfo or phospho group can also form inner salts or salts with organit bases. l'ro1$rred salts are salta with pharmaceutically acceptable non-toxic bases, for 4 example with physiologically well tolerated organic amines, for example with tert-butylamine, hydroxyethylamine, cyclohexylamine, diethylamine, di-n-butylamine, bis(hydroxyethyl)amine, trimethylamine, triethylamine, tris(hydroxyethyl)amine or diethylydroxyethylamine, and also with organic ammonium salts, for example with tetramethylammonium or tetraethylammonium.

For isolCtion or purification it is also possible to use pharmaceutically unacceptable salts.

The compounds of formula IIb and their pharmaceutically acceptable salts have valuable pharmacological properties. For example, compounds of formula Ib are effective in low concentrations of less than 0,3 pig/lml Sagainst vatious bactoria, for example against Staphylococcus aureus, Bacillus subtilis, Ricrococcus lutous and Escherichia coli In addition to antibiotic proporties, the compounds also have tumour-inhibiting propertia, for example against the experimental tumours leukaemia L 121Q/S2, human melanoma SK-MEL 109, human pulmonary carcinoma MBA 9812, colon adenocarcinoma 26 and roticulosarcoma H5076.

ProcePsoe The prosnt invention also relates to processo for the preparation of compounds of formula I1b, Those compounds are obtained by acylating a compound of formula II, wheroein A is C-0 or C-01, and, if dosirted, converting a resultant compound into another compound of the invention and/or a resultant salt into the free compound or into another salt, or a resultant freo compound into a salt.

Acylation will alsoa be understood as meaning the introduction of a sulfo group (acyl -$0SQ13) or of a phpho group (acyl -PO 3 111).

The preparation of a compound of formula Ilb or a salt thereof is effected by methods whi.h are known per so as described for the proparation of compounds of formula 1, 26- The invention also relates to those embodiments of the process in which an intermediate obthinable in any stnge of the process is used as starting material and the remaining steps are carried out therewith, or the process is discontinued at ,y stage and/or a compound obtainable by the process of the invention is prepared under the conditions of the process and further processed in s.:u.

Pharmaceutical cormpition The compounds of the present invention and their pharmacologically acceptable salts can be used e.g, for the preparation of pharmaceutical compositions which contain an effective amount of the aocive ingredient, Spreferably in admixture with a significant amount of inorganic or organic, solid or liquid pha .naceutically acceptable excipients, The Sinventioa also relates to such pharmaceutical compositions and to the preparation and use thereof.

The pharmaceutical compoviticns of this invention are suitable for n* parenterali e.g, intravenous or intramuscular, adminiutration, and, as 4" circumtancn may require, also for oral administration or topical S application.

0 ci the cowpounds of formula I are used, for example, in the form of injectable compoaitions, e.g. for intravenous administration, or of infusion solutions. Such solutions are psaferably isotonic aqueous solutions or suspensions which can be preparod prior to use, e.g. from lyophilised preparations which contain the active ingredient alone or together with a carrier, e.g. dextra, moannitol or albumin. Pharmaceutical compositions for oral administration may be sterilised and can contain adjuvants, e.g, preservatives, stabilisors, wetting agents and/or emulsifier, solubilise*s, salts for regulating the osmotic pressure, and/or buffers. Also suitable are appropriate t.ily injection solutions in which the carriers are lipophilic solventO or vohiclIs, for example fatty oils such as sesame oil, triglycorlideo or syntheti fatty acid esters, for example ethyl oloato. The injectahle pharmaceutical compositions which, if desired, may contain further pharmacologically useful substancos, for 27 example active ingredients, contain about 0.1 to 100 preferably about 1 to 50 and, in the case of lyophilisates, up to 100 of the active ingredient.

Suitable carriers for oral compositions, for example dragees, tablets or film-coated tablets, are in particular fillers such as sugar, for example lactose, saccharose, mannitol or sorbitol, cellulose preparatons and/or calcium phosphates, e.g. tricalcium phosphate or calcium biphosphata, and also binders such as starch pastes, e.g. maize, corn, rice or potato starch, gelatin, tragacanth, methyl cellulose and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the above-mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate. Excipients are in particular glidants and lubricants, for example silica, talcum, stearic acid or salts thereof such as magnesium stearate or calcium S stearate, and/or polyethylene glycol. Drag6e cores can be provided with suitable coatings which are non-resistant or resistant to gastric juics.

Further pharmaceutical compositions for oral administration are drya filled capsules made of gelatin and also soft sealed capsules consisting of gelatin and a plasticiser such as glycerol or sorbitol.

Suitable pharmaceutical compositions for rectal administration are e.g. suppositories, which consist of a combination of the active ingredient with a suppository base. Compositions for oral and reactal administrr zion contain about 0.1 to 50 preferably about 1 to 10 of the active ingredient and, if desired, further pharmacologically useful compounds.

Pharmaceutical compositions for topical application are, in particular, creams, gels, ointments, pastes, foams, tinctures and solutions, which contain about 0.05 to 10 preferably about 0,5 to 5 of the active ingredient.

The pharmaceutical compositions are prepared in a manner known per se, e.g. by conventional diasolving or lyophilising methods described in textbooks of pharmacology.

I" -28-

N

The invention also relates to the use of the compounds of formula I or salts thereof as medicaments, for example in the form of pharmaceutical compositions, for the treatment of bacterial infections caused by gram-positive bacteria and for the treatment of tumours, especially lung and gastro-intestinal tumours and leukaemia, in warm-blooded animals, for example humans and mammals, by enteral, for example oral or, preferably, parenteral, administration of therapeutically effective doses.

Depending on the species, age, individual condition, severity of the disease and method of administration, the daily doses are from about 0.01 mg to about 10 mg, especially from about 0.1 mg to about 1 mg, S per kg of body weight, as considered appropriate by the doctor preso cribing the treatment.

(0 0, r o o f C. o The following Examples illustrate the invention but do not limit the scope thereof in any way.

o o oExample 1: Myxococcus xanthus Mx x48 bacterium Sa) Origin and availability of the production strain; The bacterium Myxococcus xanthus strain Mx x48 of the Myxococcaceae family of the order oo Myxobacteral' s was isolated in May 1980 from a soil sample from Gabes Tunisia. The Mx x48 strain was deposited with the National Collection of Industrial and Marine Bacteria, Aberdeen Scotland, on 26th May 1986 under the number NCIB 12 268 in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

b) Description of the production strain: Small vegetative rods, cylindrical with abruptly tapered ends, cigar-shaped, 0,8 x .5-5 pm, distinguished by crawling-gliding movement on surfaces. On suitable media, for example VY/2-agar (see below), the organism forms "fruiting bodies" 50-150 pm in diameter in the form of intensely red-orange to blue-grey soft-slimy drops, vesicles or capituli on the agar surface. Inside them are spherical, strongly refracting myxospores 1.8-1.9 Itm in diameter. The excess of the acid of the desired acid addition salt or the desired organic amine or ammonium ion.

h 4 I 29 colonies tend to spread like filmy "swarms" on the agar surface and can grow up to several centimeters in size. On some media they are intensely greenish-yellow to orange in colour.

c) Culture conditions: The organisms grows well on peptone agar, for example CY-agar (Casitone [Difco] 0.3 yeast extract [Difco] 0.1 CaClz 2 2HO 0.1 agar 1.5 pH 7.2) or on yeast agar, for example VY/2 agar (baker's yeast 0.5 based on fresh weight, CaC12*2H 2 0 0.1 agar 1.5 pH In liquid media Mx x48 grows in homogeneous cell suspension both in shaking bottles (at approximately 160 rpm) and in bioreactors. A suitable culture medium is, for example, MD1 1.m. (peptone Sfrom casein, tryptically digested [Merck, Darmstadt) 0.3 MgS04i7H20 0.2 CaClz2-H 2 0 0.05 pH Mx x48 is strictly aerobic. All cultures are maintained at 30°C. The generation time in MD1 l.m. is approximately 6.5 hours (p 0.15 per hour), The maximum optical density j m attained (623 nm, 1 cm path length) is from 1.3 to 1.5. The concentration of the peptone from casein can also be increased, for example to 0.5 or 1 The generation time does not vary in this case, but the optical s density increases to approximately 2 or 3.5. With increasing concentra- Stion of peptone, however, the production of antibiotic becomes lower j compared with the production in MD1 l,m. Further suitable culture media Sin which the strain grows with antibiotic formation are: 0.05 casitone, 0.2 MgSO,'7HzO and 0.05 CaClI.2HO, pH 7.2, containing either 0.5 Probion S (single cell protein, Hoechst) or 0.5 corn steep liquor or Protaminal (fish protein concentrate, Asta) or 0.5 yeast extract or 0.3 casamino acids (Difco).

d) Possible preservation methods: 1. By freezing vegetative cells of plate or liquid cultures in peptone solution at -80°C (viable for several years). 2. By drying fruiting bodies on filter paper (viable for several years at room temperature). 3. By drying fruiting bodies in milk, and storing under nitrogen at room temperature in ampoules sealed by melding (viable for several years). 4. By suspending vegetative cells of plates or liquid cultures in peptone solution and freezing in liquid nitrogen (viable for several years).

30 Example 2: Production of cell supernatants containing saframycin Mx 1 a) Preculture: 50 ml of MD1 l.m. are inoculated with 0.5 ml and maintained at 30 0 C and 160 rpm for 2 days.

b) Prefermentation: a 25 litre bioreactor available from Braun, Melsungen, having a flat blade agitator system is filled with MD1 l.m.

medium and inoculated with the preculture The temperature is maintained at 30°C. The speed of the agitator is 200 rpm and the aeration is 1 0.1 Nm 3 Without further modifications the fermentation is carried out over a period of 66 hours. After this time an OD (623 nm) of 1.1 and a pH of 7.9 are attained.

i c) Main fermentation: A bioreactor with a capacity of 700 litres, available from Giovanola, Monthey, Switzerland, and having a flat blade agitator system, is filled with MD1 l.m. medium. After inoculation with j t he prefermentation product b) the stirring speed is 150 rpm and the aeration is 1 Nm 3 By increasing the aeration and the speed the oxygen Ipartial pressure is maintained above 60 saturation during the course of S the cultivation. The fermentation is monitored by taking regular samples, working up the samples in accordance with Example 3 and analysis by HPLC, and discontinued as soon as the formation of saframycin Mx 1 and Mx 1 Bc j no longer increases. After 50 hours an 00 of 1.3 and a pH of 8 are attained. In the agar diffusion test against Staph. aureus, a still just visible ii..nibiting areola (approximately 7 mm in diameter) is obtained by diluting the culture supernatant in the ratio 1:64.

Example 3: Isolation and purification of saframycin Mx 1 and Mx 1 BC (1-BC) a) Working up a 700 litre fermentation: When the fermentation of Example 2c) is complete, 4.0 litres of XAD-resin ER-180® (sold by Rdhm Haas) are introduced into the culture liquor. The mixture is stirred slowly for 5 hours. The resin is separated from the cells in which there is no antibiotic and from the medium using a sieve. It is transferred to a chromatography column and eluted in one pass with 12 litres (1 litre/hour) of isopopanol. Over 90 of the antibiotic is eluted with the fist 6 litres of isopropanol. Immediately 0.05 of acetic acid is added to the fractions containing saframycins and the whole is I 31 concentrated to 1-2 litres (ph 4.2-4.5) at a maximum temperature of up to in a rotary evaporator in brown flasks. The concentrated XAD extract contains about 22 g of solids.

The saframycins are basic, light-sensitive and oxidation-sensitive hydroquinone/quinone derivatives that are stable only at pH values of less than 7. Purification is therefore carried out substantially with the exclusion of light and at pH values around 5. Temporary storage of the substance-containing fractions is in brown-tinted glass containers, and long-term storage is at b) Purification of the saframvcins: The crude extract is dissolved in S. 300 ml of 0.07 M phosphate buffer pH 5 and applied to an open column So filled with 1 litre of swelled CM-Sephadex® C-25 (sold by Pharmacia) in I 60 0.07 M phosphate buffer pH 5. First all non-absorbed material is eluted S with 2 litres of phosphate buffer. Then saframycin Mx 1 and saframycin 4 o Mx 1 BC are eluted from the column at a rate of flow of 4 litres/h 0.07 M phosphate buffer of pH 5 and a sodium chloride gradient which is carried by 0 to 0.6 M sodium chloride. A migrating yellowish band makes this S* elution visible. The eluate is monitored continuously in a by-pass with a 4 c photometer at 277 nm. A first fraction gives 1,2 g of pure saframycin Mx 1 and a second fraction gives 1.8 g of a 1:1 mixture of saframycin .o SM 1 and Mx 1 BC (bisquinon)., These fractions still contain residues of phosphate salt. The salt is removed by chromatography over a XAD-2® column, washing with 2 bed volumes of water and elution with methanol.

SChromatography can also be performed over a SephadexO L 20 column instead of XAD-2 and eluting with methanol and formic acid or acetic acid, phosphate being replaced by formate or acetate. The purified product is analysed by HPLC (Nucleosil® 0-18, 7.5 jm, methanol/water 60:40/0.005 M hoptanesulfonic acid or HD-Sil® 18, 7 fm, methanol/phosphate buffer of pt 6.5 60:40) and then contains only saframycin Mx 1 or a mixture of saframycin Mx 1 and the biscuinone derivative, saframycin Mx 1 BC.

Too long a wait before working up a fermentation broth (addition of the adsorber resin not until after 50 hours or longer) leads to the slow conversion of saframycin MX 1 and Mx 1 BC into saframycin Mx 2 and I I. _I 1 1

L

32 Mx 2 BC, i.e. into compounds of formula I in which R 1 and R 2 are hydrogen and A is C-OH and C=O respectively. The mixture of saframycins can be separated by semi-preparative HPLC over a RP-18 column (HD-Sil®, ex Organogen) with methanol/water 1:1 and 0.5 triethylammonium formate buffer of pH Example 4: 21-Cyano-saframycin Mx 1 and 21-cyano-saframycin Mx 1 BC (2-BC) mg of saframycin Mx 1 are dissolved in 2 ml of phosphate buffer of pH 5.5 (0,1 M) and to the solution are added 5 mg of sodium cyanide (dissolved in the same buffer). The mixture is stirred under nitrogen for S,,1 15 minutes at room temperature. The HPLC diagram under standard condi- S tions (HD-Sil® 18, 7 ipm, elution with methanol:phosphate buffer (0.06 M, S pH 6.5) 60:40, rate of flow 1.5 ml/min) shows even after this time dQ quantitative reaction (rentention times Rt(1) 3.03 min, S Rt 4.12 min, R 3.61 min, R (2-BC) 6.0 min), t tt The entire reaction mixture is applied to a XAD-2® column (2.5 cm x 10 cm) and the column is washed with two bed volumes of water to remove excess salts. Elution is then effected with one bed volume of a methanol and the eluate is lyophilised, affording 21-cyano-saframycin o MXI 1 as residue.

a" H-NMR in CD0D: 6 4.02 ppm 7-OMe), 3.73 17 OMe), 3.66 14-OMe), 2,40 N-Me), 2.20 16-Me), 1.92 6-Me), 0.74 If the reaction is not carried out with rigorous exclusion of atmospheric oxygen, then the corresponding bisquinone 2-BC is formed simultaneously.

A higher pH value (pH 7) and longer reaction times (45 instead of 15 min) favour the formation of the bisquinone 2-BC.

The ratio of 2 to its bisquinone 2-BC can thus be controlled as desired, The separation of both substances is effected by medium pressure chromatography on reversed phase silica gel (HD-Sil 18, eluant: methanol/ phosphate buffer of pH 6.5 70:30).

i 33- 21-Cyano-Saframycin Mx 1 BC UV: X 264 nm (1g E 4.31, in max -1 methanol). IR(film): v 1685, 1655, 1615 cm 1 3

C-NMR(CD

3 0D), formate instead of phosphate: 6 188.0 ppm C-15), 186.8 184.2 C-18), 182.7 172.0 C-23), 168.1 formate), 157.2 156.7 C-17), 143.0 142.5 C-19), 137.8 C-10), 136.7 C-20), 130.5 C-16), 129.3 117.9 71.0 C-14), 61.5 (2q, 7- und 17-OCH3), 61.3 C-21), 59.7 14-0CH 3 58.3 and 58.2 C-13 or 55.7 and 54.9 (2d, C-11 or 50.1 C-24), 42.8 C-22), 42.2 N-CH 3 26.7 C-4), 18.0 CH 3 8.8 (2q, 0-6 and C-16). 1H-NMR(CD30D): 6 4.70 ppm J 2.5 Hz [13-21], 1H, 21-H), 4.16 (dd, J =3 Hz [3-11], 4011 SJ 1.2 Hz [11-13], 1H, 11-H), 4.10 1H, 14-H), 4.04, 3H, 17-0CH 3 4.01 311, 7-0CH 3 3.95 1H, 1-H, crosspeaks in 2D-COSY with 22a-1, 22b-H and 4b-1), 3.77 J= 7.2 Hz 1H, 24-H), 3.60 1H, 22a-1, cross-peaks with 1-H and 22b-H), 3.60 1H, 13-H, cress-peak with 21-H and 11-H), 3.59 3H, S 14-OCH3), 3.45 (dd, J 3.5 Hz [1-22b], J 14.0 Hz [22a-22b], 1H, 22b-H), 2,.93 (ddd, J 12 Hz J 2 Hz [3-4aj, J 3 Hz [3-111], 111, 2.83 (dd, J 17 Hz [4a-4b], J -2 Hz IH, 4

-H)

S 2.50 3H, N-CH 3 2.00 311, 16-CH 3 1.95 3H, 6-CH3), 1.34 (m, 111, 4-Hb2 cross-peaks with 4-Hg, 3-11 und 1-11), 1.21 J 7.2 Hz, 3H, 24-0i). FAB-MS (pos.ions): m/z 598 (11+11), m/z -496 1M-CHNHCOCH(CHC )NHfl (loss of the side-chain at Hih resolution: found 598.2851, for C3O114oNs0 cal. 598.2877; found 496.073, for CgHadoN 3 0 7 cal. 496.2084.

Example 5t N-Acyl-21-cyano-saframycin Mx 1 derivatives General procedure: 50 mg of saframycin Mx 1 (11) are dissolved under nitrogen in 3 mi of phosphate buffer of pH 6.5. To this solution are added 10 mg of sodium cyanide and the mixture is stirred for 10 minutes at room temperature. The p1! is then adjusted to 7.5 v'ith 1N NaOH and the mixture is diluted with 3 ml of methanol. To this solution are added dropwise 1-2 molar equivalents of the appropriate acylating reagent in the form of the carboxylic anhydride dissolved in 5 ml of totrahydrofurano The reaction mixture is stirred for 30 minutes. Depending an the acylanting agent, IIPLC (under the standard conditions of Example 4) shaow that the starting Mx 1 is completely consumed after 5-20 minutes. The pH

I

34 of the reaction solution is monitored constantly and kept continuously at pH 7.5 with IN NaOH. Upon completion of the reaction, the entire reaction mixture is charged to a XAD-2® column (2.5 cm x 10 cm). The column is washed with two bed volumes of water and then the desired product is eluted with methanol. The methanol is removed by distillation under vacuum and the acylated saframycin is isolated from the aqueous residue by filtration or by lyophilisation.

If the solution is contacted with atmospheric oxygen, then N-acyl-21cyano-saframycin Mx BC (the bisquinone) is formed simultaneously in addition to N-acyl-21-cyano-saframycin Mx I in accordance with the procedure described in this Example. The two compounds are separated by semi-preparative HPLC on reversed phase silica gel (HD-Sil® 18).

9 9 ,,The carboxylic anhydrides used as acylating reagents are commercially S available or are prepared as follows: to 1 mmol of carboxylic acid in o 5 ml of tetrahydrofuran is added 0.5 mmol of dicyclohexylcarbodiimide and the mixture is stirred for 30 minutes at room temperature. The precipitated dicyclohexylurea is isolated by filtration. The filtrate, which contains the desired carboxylic anhydride, is added dropwise as solution 'o0 direct to saframycin Mx 1 in phosphate buffer.

4 0 0 Instead of the 21-cyano-saframycin Mx 1 can also be acylated.

The same procedure is followed, but the treatment with sodium cyanide is omitted.

The following compounds, for example, are prepared in accordance with this procedure: a) N-Acetyl-21-cyano-saframycin Mx 1 IH-NMR in CD 3

OD:

ppm 7-OMo), 3.72 17-0Me), 3.68 14-OMe), 2.42 N-Me), 2.20 1 6 1.93 N-acetyl), 1.91 6-Me), 0.72 25-Me), HPLC, standard conditions: R t 3.17 min.

b) N-Acetyl-21-cyan-arm f Mx 1 BC H-NMR in CD 3

OD:

4.08 pp 7 .6 s 1-fe,3.0(s 1-ye,2.0(s -M) 1.08 6-Me), 1.92 6-14e 1.750(s, acetyl) 1.13 FAB-MS: found 635.2588, for G 3 2

H

37 Nr0 9 cal. 635.2592, HPLC, standard conditions: t=5. 32 min.

c) N-Caproyl-21-cyano-safrarnvc:n lMx 1 IfH-NMR in CD 3 0D: 4.01 3. 71 17-011e) 3. 67 14-ome) 2.42 N-Me) 2.20 U(s, 16-Me), 2.20 caproyl-c-CH,,), 1.92 6-Me), 1.58 caproyl-R-CH 2 1.35 (nj, caproy-7',6-CHia), 0.95 caproyl-Me), 0.72 25-Me). FXB-MS:, found 694.3411, for C 3 6 1I4nN50 9 cal. 694.3452, U of the bishydroqluinone. UPLC, standard conditions: 0Rt 6.16 min.

d) N-Caproyl-21-cyano-saframycin M 1 BC 'H-NMR in CD 3 Ofl: 4.07 7-Moe), 4,04 17-QOe), 3.60 14-O~e), 2,50 N-4{e), S2,39 2H1, caproyl-c(-CH 1.69 caproyl-B-CHO2, 1.37 (at, captoyl-1,6-CH2), 1.10 25-Mo), 0.93 capL'oyl-Me), PC Sstandard conditions: Rt 15. 44 mnt e) N-Benzoyl1-21-cyano.saframycinE_ xl VAB-MS: found 697.2762, for f) N-Bonzoyl-21-cyino-aarfrainycin Mx I BC (5-7Bc): IH-NMP. in 7,4-7.8 (in, phenyl), 3.99 17-Qo)O, 3.97 7-ONe), 3.61 14-0me), 2.50 N-ge), 1,92 6-Me), 1.68 16-Me), 1.28 25-Me), HTPLC, standard conditionsi Rt 9.22 mint.

g) N-Pivaloyl,-21-cyano-s- af'vanwcin Mx 1 IH-NMR in CDCl3; 3.95 3.68 (,17-OMe), 3.60 14-O~e), 2.46 N-Ne), 2.15 16-Me), 1.82 6-Me), 1.07 pivalayl-Me), 0.61 FAB-NS: found 677.3057, -for, C 3 5H4 3

N

5 09 cal. 677.3061, of the bishydroquinone. IIPLCI, standard conditions: R,=4.54 min.

-36h)N-Pivaloyl-21-cyalo-saframycil Mx 1 BC 1 H-NMR in CD 3

OD:

3.99 17-O~e), 3.95 3.52 14-OMe), 2.35 N-Me), 1. 89 6- or 16-MQ) 1. 79 s, 6- or 16-Ne) 1 .10 1.07 pivaloyl-Me). HPLC, standard conditions: Rt 9.79 min.

i) N-(3-Carboxypropiolyl)-21-yalo-saframycil Mx 1 prepared with I! succinic anhydride: 'H-NMR in CD 3 OD: 4.03 3.71 17-O~e), 3.65 14-O~e), 2.7-2.3 (in, 4H, succinyl-CH2), 2.45, N-Me), I 2.34 16-Ne), 1.89 6-Me), 0.70 25-Me). FAI3-MS: found 693.2654, for C 3 i4U 3 9N 5 QII cal. 693.2646, of the bishydroquinone as triethylainmonium salt. HPLC, standard conditions: R' 2.06 min, t~ j) N-(3-Carboxypropionyl)-21-cyano-saframycin Mv 1 BC Il- MR in

CD

3 OD: 4.08 7-Ole), 4.06 17-014e), 3.60 14-Ollo), 2.50 N-Me), 2.36 (mn, 2H, succinyl-CH2), 2.25 2H, succinyl-CIZ), 4 61.97 16-Me), 1.90 6-Me), 1,14 25-Me). IiPLO, standard conditions: Rt =3,83 min.

k) N-(0-Benzy1-N-benzyloxycarbonyl-53-aspartyl)-21-cyano-saframycin IMx 1 3-bonzyloxycarbonyl-3-benzvloxycarbonylaininopropionyl 1-cyanosaframycinMx 11 1 U-NKR in CD3OD: 7.37 broad, 2 x~ phonyl), 5,19 benzyl-C1ia), 5.14 bonzyl-Cliz), 4.57 (dcl, aspattyl-a-CH), 3,98 7-WOe), 3.67, 17-O~e), 5320 14-OMo), 2.74 (dddd, aspartyl-o-ClHa), 2.41 N-Ne), 2.16 16-Me), 1.88 6-No), 0.70 25-Me), FAI3-MS: found 932.3592, for Ci~qH$ZN60 1 3 cal. 932.3592 HPLC, standard conditions; t 11.7 min.

N-(0-Benzyl-Nt-benzyloxycarbony-o-aspartyl) .2l-cyano-saframycin Mx 1 BC IH-NNR in CDIOD: 7k37 broad 2 x phonyl), 5.16 benzy3.-CHa), 5.13 benzyl-OtI 4.45 aspartyl-a-CH), 4.00 17-O~e), 3.99 3.57 14-O~e), 2.56 aspartyl-13-CIi7), 2.47 N-No), 1.91 16-Me), 1.86 6-Me), 1.10 25-Mo). UPLO, standard contitions: R t -35,9 min.

-37- Example 6: N-Formyl-21-cyano-saframycin Mx 1 (9) 160 mg of formic acid, 340 mg of acetic anhydride and 28 mg of p-N,N-dimethylaminopyridine (Dl4AP) are added in succession to a solution, cooled to -30'C, of 50 mg of 21-cyano-saframycin Mx 1 in 5 ml of pyridine.

The mixture is stirred for 30 minutes at the same temperature. The entire reaction solution is then concentrated under an oil pump vacuum and the residue is chromatographed by medium-pressure reversed phase chromatography with methanol /phosphate buffer of pH 6.5 -1:1 and at a rate of flow of 12.,5 ml/min. Removal of salt over XAD-2 gives the pure title compound 9.

1 11-NMR (CD 3 0D): 7.95 formyl-H), 4.0 7-Ofe), 3,72 17-O~e), 3.66 14-O~e), 2.41 N-Me), 2.18 (16-No), 1.91 6-Me), 0.8 25-Mec). FAB-MS: found 6.93.2580, for C31H37N509 cal. 623.2591, HPIAC, standard conditions; R t =2,79 min, HPIJC analysis of the N-formyl-21-cyano-saframycin Mx 1 MB, (9-nc) formed simultaneously and separated by chromatography shows a retention time of 4o38 minotes., Example 7; N-PhonylaminocarbonyJ- and N-pheny lthiocarbonvl1-21-cvano- 4 safra-mycin Mx 1 (10) and (11) mg of 2i-cyano-saframycin Xx 1 are treated under nitrogen in a, 1:1 mixture of phosphate buffer (piH 7,8) and totrahydrofur.n equivalents of phenyl isocyamate and phenyl isothiocyanate respectively.

The mixture is stirred at room temperature (phenyl isocyAnate) or at (phenyl isothiocyanate) for 30 minutes. The reaction is followed by means of HPLC (conditions as in Example The cooled solution is partitioned between ethyl acetate and water and chromatographed over reversed phase silica gel, The bisquinone obtained as by-product is separated simultaneously in this chromatography, a) N-Pheny lamino carbony 1-21 -cyano- saf ramvcin MxI1 111-NMR in CDC1 3 7.0-7.35 (in, phenyl), 6.63 Nft), 6.31 NI), 5.07 (do NHl), 3,98 17- or 3.96 17- or 3.51 14-O~e), 2.49 N-Me), 1.96 6-No), 1.76 16-Me), 1.10 FAII-bs: found 712.2850, for C3I14N0gO cal. 712,2856, UIPLC, standard conditions: R t-i 10.50 min.

-38b) N-Phenylaminothiocarbonyl-21-cYano-saframycin Mx 1 I1-NMR in

CD

3 OD: phenyl), 3,99 7-OMe), 3.73 17-OMe), 3.63 14-Ole), 2.38 N-Me), 2.13 6-Me), 1.89 16-Me), 0.84 25-Mo). FAB-MS: found 728.2616, for C 3 7MONGOaS cal, 728.2628, (N HPLC, standard conditions: Rt 4.75 min.

c) NPhenylaminothiocarbonyl21-cyano-saframycil Mx 1 BC (11-BC): IH-NMR in CD 3 OD: 7.2-7.5 phenyl), 4,06 17-OWe), 4,00 7-OMe), 3.59 14-Oe), 2,49 N-Me), 1.95 6-4le), 1.81 16-Me), 1.21 25-le), 1PLC, standard conditions: Rt 11.44 min.

Example 8: N,O,0-Triacetyl-21"-cano-safram'cin 1 (12) The phenolic 01 groups of the hydroquinone part are ticylated as follows: mg of N-acotyl-21-cyno-saframycin lx 1 are dissolved in I ml of pyridine and to the solution is added 0,5 ml of acetic anhydride, The mixture is stirred for 60 minutes at room temperature, Excess reagent and aolVont are removed tinder an oil pump vacuum, The residue is purified by medium-prssiUre chromatography (UD-Sil' 18, methanol/phosphate buffer of pH 6 5 60;4Q). In Acldition to the title compound 12, small amounts of s~l~ aframycin Mx 1 BC (1-BC) are isolated, SH-NMR in CD30D: 4,06 7-ONe), 3.78 17-OMe), 3.56 14-ONe), 1 2.49, (3 x P, N-Mo, 2 x 0-acotyl), 2,16 16-Me), 1.88 6-Ne), 1.72 (a N-acetyl) 1.02 25-lie). VAB-NS: found 721,2971, for C1 6

HI

3 NS011 cal, 721,2960, TPLO, standard conditions- Rt 3.5$ min, Example 5: N-p-Toludnesutfonyl-21-cyaflno-nfrlrflin Mx 1 (13) mg of 21-cyano-saframyain Xx 1 are dissolved in pyridino, the solutioia is cooled to 0'C and treated under nitrogen with an equimolar amount of toluenesulfonyl chloride, The reaction, mixture is allowed to warm to room temperature over 30 minuteo*3 then partitioned between ethyl acetate and water, The ethyl acetate phase is dried and the residue is chromatographod over reVersed phasIe silica gel with mthanol/buffer of p11 6.5 60:40. t

NONIE

-39- 'H-NMR in CD 3 OD): 7.64 (di, phenyl), 7.38 phenyl), 4.04 7-O~e), 3.69 17-Ole), 3.63 14-O~e), 2.48 tolyl-Me), 2.31 N-MC' 2.15 16-M1e), 1.92 0.69 25-Ne). FAB-bl,: found 750.2758, for C3 7 l1140NsS1o cal. 750.2808. HPLC, standard conditions: Rt 5.97 min.

In the above chromatography, N-p-toluienesulfonyl-1--cyano-saframycin Mx 1 BC (13-Bc) is separated as by-product, Rt .9.47 min under HPLO standard conditions.

Example 10: N-Sulfo-21-cyano-saframycin M% 1 and Mx 1 BC (14) mg of 21-cyano-saframycdn Mx 1 are dissolved in 3 ml of~ methanol and 3 ml of phosphate buffer of pHf 7.8 (0.12 it) and to the solu~tion is added half the equivalent amount of pyidine-sulfur trioxide complej, The pH is adjusted to the initial value of 7,8 with IN NaCH. Then the same amount of pyricline-sulfur trioxide comploex is again added, The reaction is complete after- 10 minutes, The solution is then concentrated under an oil pump vacuum, The title compounds are separated from each other hy reversed phase medium-pres sure chromatography and isolated as triothylammoiumsalts.

N-gulfo m can-aframvvin M\ (14) -NR(D0) 4.06 3,74 (vi 17-Ote), 3.64 (sj 14-WOe), 3.9.3 3x cu2, triothylammoiu-Cla), 2.46 N-4I), 2.27 16-Me), 1.88 Ca, 6-Me), 1.34 5X~ Cti3, triethYLaIMMonIUM-C113), 0.73 25-Mo). found 674.2101, for C~a1I3N~s$Qu cal. 674,2131, (lI).-HPLC, standard conditions: 9 1.82 mm.

b) N-Sulfo-21-cynno-saftanvein Mtx I BC 'U-NMR (CI30D):. 4,03 or 17-O1fe), 4.02 7- or~ 17-O~e), 3-61 Cs, 140NOH, 3.25 3x Oita, trio thylammonium-Clz) 2i49 N4-Me), 2.03 (on, 16-Ne), 1,91 6-MO), 1.34 Ct, 3- cff~t triothyl~mmonium-C1[3)s 1-09 254M()- HPI.G* standard conditions: 9 2.38 mm.,

BI

Example 11: N-Trifluotracetyl-21-cyan-saframycin Mx 1 mg of 21-cyano-saframycin Mx 1 are dissolved in 2 ml of pyridine and treated, under argon, with an excess of trifluoroacetic anhydride. The reaction mixture is stirred for 30 minutes, then poured into a cold solution of sodium bicarbonate and extracted with ethyl acetate. The ethyl acetate extrat is dried and concentrated by evaporation. The title compound is purified by reversed phase medium-pressure chromatograobhy with methanol/buffer of pV 6.5 55:45 as el- -t.

HPLC (column: RPI18 Nucleoasil, 5 pm, 10 cmia; cluant: methanol: buffer (pH 6.5) 7:45, rate of flow: 1.5 mll/in): R 5.57 min (safra- S myain Mx 3:3,12 min). 1H-NMR (CDC13 5 CD 3 D):6 3.91 ppm (a,7-oMe), 3.65 17-OMe), 3.57 14-oe), 2.32 N-Mo), 2,09 (s, a 6-Ne), 1.83 6-MNe), 0,63 J 6.9 Hz, S 130-NMR (QDC1 3 5 cD 3 0b): 6 157.77 J 37.6 tz, 0 C-CF 3 115.56 ppm J 2P6.0 Hz, CF 3 -C UV (methanol): a 266 nm (lg E 4.32), Example 12: N-(3-Carboxypropiony 1)-21-CN-saframycin Mx 1 (2) tg of N-(3-carboxypropianyl)laframycin Mx 1 (Example 13a) are taken up in 5 mi of phosphate buffer solution of pfI 6. The solution is treated 0 with a ats .11 excess of sodium cyanide and the mixture is stirred for minutes, The aqueous solution ia chargod to 4 small column of XAD-2 equilibrated wih water. The salt is temoved by washing the column with two column volumes of water, T h. title compound is eluted with methanol.

The phyh.aQochemical chara. tertistic are the same as in Example Example 13: N-Acyl-saframytin Mx 1 derivative General procedure Under nitrogen, 50 mg of saframycin Mx 1 are dissolved in 3 ml of phosphate buffee of pit 7,5 and the solution is diluted with 3 mi of methanol. A solution of 1-2 molar equivalents of the appropriate tacylating reagent as catrboxylic anhydride in 5 ml of totrahydrofuran is added dropwise to the above solution, and the reaction mixture is stirred for 30 minutes. Depending on the acylating agent, IIPLC (standard conditions! HID-SilO 18, 7 -im, elution with methanol/phosphate buffer 0.06 14, pit 6.5 60:40, rate of flow S ml/min) retention time t M 3.03 min) i, 1.

e.g. by conventional dissolving or lyophilising methods described in textbooks of pharmacology.

-41 shows that the starting material Mx 1 is completely consumed after 5-20 minutes. The pH of the reaction solution is constantly monitored and kept cuntinuously at p11 7.5 with 1N Na0H. Upon completion of the reaction, the entire reaction mixture is charged to a XAD-20 column cm x 10 cm). The column is washed with two bed volumes of water and the desired product is thereafter eluted with methanol. The methanol is removed by distillation under vacuum, and the acylated saframycin is isolated from the aqueous residue by filtration or by lyophilisation.

If the solution is contacted with atijiospheric oxygen, then N-acyl-saframycin Mx 1 BC (the bisquinone) is also formed simultaneously in accordance with this procedure. The two compounds are separated by semipreparative HPLC on reversed phase silica gel (HD-SilO 18).

The following compounds were prepared in accordance with the above Procedure.- a) N-3-Carboxypropionyl-saframycin Mx 1 prepared from succinic *1 anhydride: 1 11-NMR in CD 3 OD: 6 4.00 ppm, 3.72 Cs, 17-O~e), 3.66 14-O~e), 2o60-2.30 (,2x succinyl-CHa), 2.48 (s, N-Me), 2.25 16-Me), 1.89 6-Ne), 0.78 25-No). FAB-NS of the triethylammonium salt: found 668.2717, for C311i4ON 4 01 1 cal. 668.2693, (M-11a0-1)_ of the bishydroquinone. HPLC, standard conditions: R t =1.81 min.

b) N-(3-carboxvypropionyl)-saframycin Mx% 1 BC 1 H-NMR in 6=4.07 ppm Cs, 17-014e), 4.03 7-Qoe), 3.62 14-OMe), 2.45 (s, N-Ne), 2.33 Cm., succinyl-C1 2.27 succinyl-C12), 1.99 Cs, 16-Me), 1.90 Cs, 6-Nc), 1.18 Cd, 25-tRc). HVLC, standard conditions: Rt.2.24 min.

c) N-2-CarboxybenzoXI-saframycin Mx 1 prepared from phthalic anhydride, ilU-NMR in CDAQD 6 7.82 ppm Cm, 11W, aromatic compound), 7.47 (in, 211, aromatic compound), 7,39 1H1, airomatic compound), 4.02 (a, 3.72 17-O~e), 3.5 1 4 -OMe), 2.57 N-Me), 2.10 Ca, -42- 16-Me), 1.84 6-Me), 0.94 25-Nt -FAB-MS: found 716.2684, for

C

3 7 H~oNi,0 1 1 cal. 716.2693, (14-120) HPLC, standard conditions: Rt 1.75 min.

d) N-2-Carboxybenzoyl-saframycin Mx 1 BC IH-NMR in CD 3

OD:

6 7.68 ppm (in, Il, aromatic compound), 7.41 (in, 2H, aromatic compound), 7.34 (in, 1H, aromatic compound), 4.08 17-OMe), 3.98 7-OMe), 3.62 14-OMe), 2.45 N-He), 1.99 6-Me) 1 1.63 16-Me), 1.09 (d, HPLC, standard conditions: Rt =2.40 min.

e) N- 3-Me thoxvcarbony Iprop ionyl-s af ramycin Mx 1 prepared from monomethyl succinate and dicyclohexylcarbodiimide: 'I-NMR in CD 3

OD:

6 3.99 ppm 3.74 17-OMQ), 3.71 ester-Me), 3.69 (s, 14-OMe), 2.54 N-Mo), 2.54 (in, 2H, succiny1-CI12), 2.43 (in, 211, succinyl-CH,,), 2.23 16-Mo), 1.90 6-Me), 0.9 25-Me). -FAB-MS: found 682.2839, for Ca10412NI1011 cal. 682.2850, (M-11 2 UPLC, standard conditions; Rt =3.15 min.

Example 14: N-Sulfo-saframycin Mx 1 and Mx 1 BC and (4-BC) of saframycin Mx 1 arc- dissolved in 3 ml of methanol and 3 ml of phosphate buffer of pH 7.8 (0.12 M) and to this solution is added half the equivalent amount of pyridine-sulfur trioxide complex,., The pH is adjusted to the initiail value of 7.8 with 1N NaOIH. Subsequently the same amount of pyridine-sulfur trioxde complex is added once more., The reaction is complete after 10 minutes. The solution is concenttrated by evaporation under an oil pump vacuum. The title compounds are separated fromn each other by reversed phase medium-pressure chromatography and isolated in the fort, of the triethylammonium salts.

a) N-Sulfo-saframycinM~ 1 1 H-NMR (CD30D): 6 4.04 ppm 7-OMO), 3.76 17-O~e), 3.6$ 14-ome), 3.25 3% CH 2 triethylammonium-cUlz), 2.67 N-Mo), 2.31 16-Me), 1.89 6-Ne), 1.34 3x CH3, triethylammonium-113), 0.82 25-Mo). HPLO, standard conditions: llt 2.23 min.

-43b) N-Sulfo-saframycin Mx 1 BC IH-NMR (CD30D): 6 4.04 ppm 7-or 1 7 -OMe), 4.03 7- or 17-OMe), 3.62 14-OMe), 3.25 3x CHZ, triethylammonium-CH2), 2.50 N-Me), 2.04 16-Me), 1.91 6-Me), 1.35 3x CH 3 triethylammonium-CH3), 1.13 FAB-MS: found 798.2322, for C 3 6 Ho0N5S014 cal. 798.2292 (M nitrobenzyl alcohol from the matrix). HPLC, standard conditions: R t 3.19 min.

Example 15: Cytotoxicities of the derivatives against mouse fibroblasts L 929 Methanolic solutions of the tested compounds are diluted in titre plates with 96 dishes 3 times with DME medium (Dulbecco modification of the Eagle medium with 10 foetal calf serum) as follows: n 2 3 4 5 6 7 8 9 10 11 12 conc. 37 000 12 000 4 100 1 400 460 150 51 17 5.6 1.9 0.6 ng/ml To 60 p l of the dilutions were added 120 p1 of a cell suspension of mouse fibroblasts of the line L 929 obtained by trypsinisation, which suspension contains 25 000 cellsiml of DME medium (L 929: permanent cell culture growing as monolayer). The titre plates are incubated for 5 days at 37*C and under 10 C002 in a moistened incubator. Afterwards the growth of the mouse cells is assessed microscopically and the minimum inhibitory concentrations (MIC in ng/ml) of the compounds are determined (growth 50 Compounds minimum inhibitory concentration MIC (ng/ml) of formula I 4 days (comparison) 0.6 2-BC 51 3 150 3-BC 150 4+ 4-BC 150 6 6-BC 150 7 4100 7-BC 4100 ii

I

44 8 9 I1I 11-BC 12 13 1 3-BC 14 14-BC Compounds of formula I~b 1 1-BC 2 3 4100 51 150 150 150 4100 150 150 4100 4100 minimum inhibitory concentration MIC (ng/ml) after 4 days 4100 4100 4100 150 '4 a a a a' a a .4 4 4 4

R

3 0 H 3 CH3 OR'\ \6 /5\O/I\3>C113

CH

3

O

H

/NHR'

3 Compounds of formula I No.

1: RI-OH, R.

2 I, R 3 2: R1-CN, R 2 RI H f 3: RI-CN, R 2 =acetyl, R 3

H

4: R1-CN, R 2 -caproyl, R 3 if

IB

i 45

CN,

CN,

CN,

CN,

CN,

CN,

CN,

CN,

CN,

ON,

benzoyl, R 3

H

pivaloyl, R 3

H

succinyl, R 3 H H -aspartyl-N(Z)-0(Bn), R 3

H

formyl, R 3

H

-C(=O)-NHPhe, R 3

H

-C(=S)-NHPhe, R 3

H

acetyl, R3 acetyl tosyl, R3 H sulfa, R 3

H

Compounds of formula IIb No.

1: R' 01 2: RI= 01 3: 01 4: RI 01 3-carboxypropionyl 2-carboxybenzayl 3-mthoxycarbonylpropionyl sulfo Example 16: Pharmaceutical composition for parenteral administration ml of a sterilo aqueous solution of 1 of 21-cyano-saframycin Mx 1 (as PIua format) are filled into 5 ml ampoules or vials under aseptic conditions and lyophilised, The ampoules or vials are sealed under nitrogen and tested.

Solutions of the acylated and sulfonylated 21-cyano- and 21-hbdroxysaframycin Mx 1 and Mx 1 BC derivatives are processed in the same manner.

I--

Claims (1)

1. 46- The claims defining the invention are as follows: 1. A compound of formula CH 3 *CH3 *en i a C 30 ef.~ t a\ 4 fI (I) H 0 i 1 NHR wherein R is hydrogen, acyl, sulfonyl, Sulfo or phospho, and A is C=0 or C-OH or acylatd C-0H, and the dashed line denotes a C=C double bond atI the site ac the central bond when A is C=0 or at the site of the twoI outer bonds when A is C-OH or acylated C-OH, or a salt thereof. 2. A compound according to claim 1, which conforms to the petspective formula Ia 47 CH 3 CH3 CH3 H* O(Ia) C H3 1,H3 Ct CN C~aH /NHR 0 CH3 3. A compound of formula I according to either claim 1 or claim 2, wherein R is hydrogen, the acyl group of a carboxylic acid, of a half- ester of carbonic acid, of carbamic acid or of a thiocarbamic acid containing up to 20 carbon atoms, the acyl radical of a polypeptide from 2-200 naturally occurring amino acids, the radical of a sulfonic acid containing up to 20 carbon atoms, unsubstituted or substituted amino- sulfonyl, sulfo or phospho, and A is 0=0 or C-OH or acylated C-OH, wherein C-OH may be acylated by a carboxylic acid containing up to carbon atoms, and the dashed line denotes a C=C double bond at the site of the central bond when A is 0=0 or at the site of the two outer bonds when A is C-OH or acylated C-OH, or a salt thereof. 4, A compound of formula I according to either claim 1 or claim 2, wherein R is hydrogen, Cl-Czoalkanoyl, CZ-C 7 alkanoyl which is substituted by hydroxy, etherified or esterified hydroxy, amino, acylated amino, carboxy, amidated or esterified carboxy, oxo andor halogen, or is aroyl, C1 -C7alkoxycarbonyl, Cl-7alkylaminocarbonyl, CI-c 7 alkylaminothioqarbon- yl, arylaminocarbonyl, arylaminothiocarbonyl, the acyl radical of a polypeptide from 2-200 naturally occurring amino acids, C 1 -C7alkaneul- fonyli, arylsulfonyl, aminosulfonyl, Ci-C 7 alkylaminosulfonyl, dialkyl- aminosulfonyl containing 1 to 7 carbon atoms in each of the alkyl moieties, arylalkylaminosulfonyl containing 1 to 4 carbon atoms in the alkyl moiety, cyclic 5- or 6-membered aminosulfonyl, wherein the ring contains one or two nitrogen atoms and, if desired, one oxygen or ulfur 48 atom, or is sulfo or phospho, and A is C=O or C-OH or acylated C-OH, wherein C-OH may be acylated by CI-C20alkanoyl, by Cz-C 7 alkanoyl which is substituted by hydroxy, etherified or esterified hydroxy, amino, acylated amino, carboxy, amidated or esterified carboxy, oxo and/or halogen, or by aroyl, and the dashed line has the meaning given above, or a salt thereof. A compound of formula I according to either claim 1 or claim 2, ?i wherein R is hydrogen, Ci-C 7 alkanoyl, C-C 7 alkanoyl which is substituted by amino or acylated amino and/or esterified carboxy, or is aroyl, aryl- aminocarbonyl, arylaminothiocarbonyl, arylsulfonyl, sulfo or phospho, and A is C=O or C-OH or acylated C-OH, wherein C-OH may be acylated by SCi-C 7 alkanoyl or C2-C 7 alkanoyl which is substituted by carboxy or Sesterified carboxy, and the dashed line has the given meaning, or a S pharmaceutically acceptable salt thereof. 6. A compound of formula I according to either claim 1 or claim 2, S wherein R is hydrogen, Ci-C 7 alkanoyl, C2-C7alkanoyl which is substituted 4i by carboxy, CI-C4alkoxycarbonyl or benzyloxycarbonyl and/or amino or benzyloxycarbonylamino, or is phenylaminocarbonyl, phenylaminothio- carbonyl, p-toluenesulfonyl or sulfo, and A is C=O or C-OH or acetylated C-OH, and the dashed line has the given meaning, or a pharmaceutically acceptable salt thereof. 7. The compound of formula I according to either claim 1 or claim 2, wherein R is hydrogen and A is C-OH, and the dashed line has the given meaning, or a pharmaceutically acceptable salt thereof. 8. The compound of formula I according to either claim 1 or claim 2, wherein R is formyl and A is C-OH, and the dashed line has the given meaning, or a pharmaceutically acceptable salt thereof. 9. The compound of formula I according to either claim 1 or claim 2, wherein R is n-hexanoyl and A is C-OH, and the dashed line has the given meaningi or a pharmaceutically acceptable salt thereof. 49 The c6mpound of formula I according to either claim 1 or claim 2, wherein R is 2,2-dimethylpropionyl and A is C-OH, and the dashed line has the given meaning, or a pharmaceutically acceptable salt thereof. 11. A process for the preparation of a compound of formula I according to either claim 1 or claim 2, which comprises converting, in a compound of formula II CH 3 CH3 ".V O 0c, (i) 0 OCH3 S II 3 carbon atom into the cyano group by cyanidation and the 2 group into the IIHR group by acylation or sulfonation, where R is as defined for oformula I, or, to prepare a compound of formula X, wherein R is hydrogen, conwherin A is C=g or C-OH, in any order, the hydroxy group of a compound of formula II at the 21-carbon S carbon atom into the cyano group by cyandation and, if desired, convertoup in toa resultant compound into another compound of the invention andt'or a resultant salt into the freeHR group by acylaton or sulfonation, where is sultant free compounfined into a salt. 2. A pharmaceutical compare a compound of formula I, herein R is hydrogenr a pha convermaceuticallng the hydoxy group acceptableof a compound of formula II at eithe 21-carbon atom ino the cyano group by cyanidation, and, if desired, converting a a resultant compound into another compound of the invention and/or a| resultant salt into the free compound or a resultant free compound into a salt. 0 -t J2. A pharmaceutical composition containing a compound of formula I or a pharmaceutically acceptable salt thereof as claimed in either claim 1 or claim 2, in admixture with pharmaceutically acceptable carriers, §1; z;: 50 13. A method for the manufacture of pharmaceutical preparations which comprises admixing a compound as defined by any one of claims 1 or 2, or pharmaceutically acceptable salts thereof, with a pharmaceutically acceptable carrier. 14. A compound of formula CHO3 (IIb), r t k I t G, rl r o rr wherein Rb is acyl which is substituted by carboxy or esterified carboxy, or is sulfo or phospho, and A is C=O or C-OH or acylated C-OH, and the dashed line denotes a C=C double bond at the site of the central bond when A is C=O, or at the site of the two outer bonds when A is C-OH or acylated C-OH, or a salt thereof. A compound according to claim 14 which conforms to the perspective formula IIb' 4 44 I 1 I tt° -51- CH3H 3 A01 CO 0 3 16,A cmpundeiterof orulaUb ccrdig o cm1 roffruaUbcodn tocam 5 hrenR i 1 0 a~aol H 1 Caknyaolhvn pt 0cro atos r etrory hvig p o 0 arOn tm n otiig1 2htraos n 17. A compound either of formula Ilb according to claim 14 or of formula Tb' according to claim 15, wherein Rb is carboxalkanoyl contaUinoyhaing p to 0 carbonatminheaaoy 7 anatoms, aryl hng to2ycarbonaosn containing 1 o 4 carbon atoms h an~y goupa t oee 7rcaboatos inin sbtute byayrop carbo xyaoy, C-C 7 alkoxycnrbonyl arylamnocarboxyknyl containing 1 to 7 carbon atoms in the alknoy group nally- amino, aclkoxycrboydroyl i1whch e akoxy and the ikanoyly rou ei c contains 1 tos7 aon ataandnAi -OorC0-1 aylaoxyCnro-,aldathey codaid ing 1a te 4gcvebn masin the alkaly ghreoupad1tfabnaosi h~akny ruhdoyc tocam1,weenlbi aboxyalkanoyl containing 1 to 7 carbon atoms lin the alkanoylgrucyo-loy grukxcarboalkanoyl in which the alkoxy and alkanoyl group each contains 1 to 7cro 7aos, aslf oryloxyo, laandy Aoting is to o4 carbon orom acltdCOl hin thOe ay be susiutdb 1 7 laoyl, ario carboxyalkanoyl containing 1 to 7 carbon atoms in the lqo gupac- kny uak~carbonylalkanoyl in which the alkoxy and alkanoyl group each otigIt 7crn atmsif rpopo n sC0o -P rayae -U hri -Hmyb -,l -52- contains 1 to 7 carbon atoms, carboxyaroyl or C 1 -C 7 alkoxacarbonylaroyl, and the dashed line has the given meaning, or a salt thereof, 18, A compound either of formula nb according to claim 14 or of formula IIb' according to claim 15, wherein Rb is carboxyalkanoyl containing 1 to 7 carbon atoms in the alkanoyl group, alkoxycarbonylalkanoyl in which the alkoxy and alkanoyl group each contains 1 to 7 carbon atoms, carboxyaroyl, C 1 -C 7 alkoxycarbonylaroyl, amino-carboxy-alkanoyl in which the alkanoyl group contains 1 to 7 carbon atoms, sulfo or phospho, and A is C=O or I C-OH or acylated C-OH, wherein C-OH may be substituted by C 1 -C 7 alkanoyl, Scarboxyalkanoyl containing 1 to 7 carbon atoms in the alkanoyl group, alkoxycarbonylalkanoyl in which the alkoxy and alkanoyl group each contains 1 io 7 carbon atoms, and the dashed line has the given meaning, or a salt thereof. 19, A compound either of formula lib according to claim 14 or of formula IIb' according to claim 15, '-"ein Rb is carboxyalkanoyl containing 1 to 7 carbon atoms in the alkanoyl group, alkoxycarbonylalkanoyl in which the alkoxy and alkanoyl group each contains 1 to 7 carbon atoms, carboxybenzoyl or sulfo, and A is C=O or C-OH or acetylated C-OH, and the dashed line has the given meaning, or a pharmaceutically acceptable salt thereof. The compound either of formula IIb according to claim 14 or of formula IIb' according to claim 15, wherein Rb is 3-carboxyproptonyl and A is C-OH, and the dashed line has the given meaning, or a pharmaceutically acceptable salt thereof, 21. The compound either of formula lib according to claim 14 or of formula lIb' according to claim 15, wherein Rb is 2-carboxybenzoyl and A is C-OH, and the dashed line has the given meaning, or a pharmaceutically acceptable salt thereof. 22 A process for the preparation of a compound either of formula nib according to claim 14 or of formula JIb' according to claim 15, which comprises acylating a compound of formula I CH 3 0- OH 3 0 A OH CH 3 CH N CH. 3 0 OCH 3 (I 0 0 OH NH NH 2 0 OH 3 wherein A is (2=0 or C-OM' and, if desired, convertiniga resultant compound into another compound of the invention and/or a resultant salt into the free compound or into r nother salt, or a resultin8 free compound into a salt. 23. phrmacutial composition containing a compound of formula 11b or lIb', or a pharmaceutically acceptable salt thereof according to either claim 15 or claim 16, in admixture with pharmaceutically acceptable carriers, 24. A method for the manufacture of pharmaceutical preparations which comprises admixing a compound as defined by any one of claims 14 or 15 or pharmaceutically acceptable salts thereof, with a pharmaceutically acceptable carrier. A compound according to claim 1, 2, 14 or 15, or a process according to claim 11 or 22, or a compon*ItLion, according to claim 11 or 23, or a method for the manufacture of pharmaceutical preparations according to claim 13 or 24, substantially as herein described with reference to any one of the foregoing examples thereof, DAh.TED this 8th dahy ot jnutaryt 1992 CTBA-GEIGL AG and GESEISCIAt'T FUR t3-OTEC1INOLOGISCtIE FORSOUUNG bf i By TboiAr Patent Attorne-ys, DAVIES CObt 1 ISON OAVE (v