CZ2000633A3 - 4-Cyano-4-deformylsordaricin derivatives and a pharmaceutical and agrochemical composition comprising these derivatives - Google Patents

Abstract

4-Cyano-4-deformylsordaricin derivatives are fungicidal agents useful for the treatment and/or prevention of human and animal fungal infections and for the control of phytopathogenic fungi of agricultural crops.

Description

The invention relates to derivatives of 4-cyano-4-deformylsordaricin, * which are potent fungicidal agents with a broad spectrum of activity. The invention further relates to processes for the preparation of these derivatives, pharmaceutical and agricultural compositions containing these compounds and methods for the treatment of fungal infections in humans, animals and plant materials using these compounds.

State of the art

Sordarin is a fungicidal antibiotic isolated from the fungus Sordaria araneosa (see British patent GB 1162027 and Helvetica * Chimica Acta, 1971, 51, 119-120). Other fungicidal agents having the same structural basis as sordarin are known. Kokai * J62040292 describes a compound called zofimann, which was isolated from Zofiela marina sp.; Kokai J06157582 describes a compound called BE-31405, which was isolated from Penicillium sp.; and J. Antibiotics, 1995, 48, 1171-1172 describes a compound called SCH57404. Published PCT applications WO96/14326 and WO96/14327 describe semisynthetic derivatives of sordarin.

Sordaricin, which is the aglycone of sordarin, can be obtained by acid hydrolysis of sordarin, as described by Hauser and Sigg in Helvetica Chimica Acta, 1971, 51, 119-120; similarly, sordaricin methyl ester can be obtained from sordarin methyl ester. The total synthesis was described in Kato et al. J. Chem. Soc. Chem. Commun. 1993, 1002-1004, where O-methoxymethylsordaricin methyl ester is also described. 4-Deformyl-4-hydroxymethylsordaricin diacetate was described by Mander and Robinson in J. Org. Chem. 1991, 56(11), 3395-3601.

The biological activity of both sordaricin itself and its listed derivatives has never been described before. Nor have cyanoderivatives of the formyl group been described before.

The essence of the invention

The present invention relates to compounds of general formula (I):

COOR ¹⁰

CN

RO (I) where

R is (a) group C(=O)OR ¹ , (b) group C(=O)NR ² R ³ , (c) group C(=O)R ⁴ , (d) group CH(R ² )OR ⁵ , (e) group C (R ⁵ ) (R ⁷ ) (R ⁸ ) (f) group ^b (g) hydrogen atom;

, or

R ¹ is (a) an alkyl group containing 1 to 14 carbon atoms, • · (b) an alkenyl group containing 2 to carbon atoms, (c) an alkynyl group containing 2 to carbon atoms, (d) a cycloalkyl group containing 3 to 20 carbon atoms, (e) an aryl group, or (f) an aryl group substituted with

^R2 and ^R5

R ⁴ is

^R5 is

^R6 is

alkyl group 6 carbon atoms; containing 1 to are independent (a) hydrogen atom or (b) group R ¹ ; (a) hydrogen atom, (b) group R ¹ or (c) group - (CH?) _m NR ² R ³ ; (a) group R ¹ or (b) group - (CH ₂ ) _X O (CH ₂ ) _y H; (a) hydrogen atom, (b) alkyl group containing 1 to

carbon atoms, (c) an aryl group, (d) an aryl-substituted alkyl group containing 1 to carbon atoms, (e) a group - (CH ₂ ) _y CHR ⁹ (CH ₂ ) _Z H, (f) a group - (CH ₂ ) _y C=C (CH ₂ ) _Z H, (g) a group - (CH ₂ ) _y C (R ⁷ ) =CH (CH ₂ ) _Z H, • · · · • · (h) a group - (CHJ _y C=C (CHJ _m R ⁹ , (i) a group - (CH ₂ ) _y C (R ⁷ ) =CH (CH ₂ ) _m R ⁹ ;

R ⁷ and R ⁸ are independently (a) hydrogen or (b) an alkyl group containing 1 to 14 carbon atoms;

^R9 is

R ¹⁰ is (a) hydroxyl group or (c) NR ² RJ group (a) hydrogen atom, (b) -CťCgHs group, (c) -CH ₂ CH=CH ₂ group, (d) (e) (f) (g) (h) group group group group group

n is or 1;

m is 1 until 6; 4 X is 2 until 6; y is 0 until 6; from is 0 until 6; or

from pharmaceutical or salts of these compounds.

agriculturally acceptable

One embodiment of the invention relates to compounds of general formula (Ia)

where

R is (a) group C (=0) OR ¹ , (b) group C (=O)NR ² R ³ , (c) group C(=O)R ⁴ , (d) group _CH2OR5 ^, (e) group C(R ⁶ ) (R ⁷ ) (R ⁸ ! or (f) group AA , or

pharmaceutically acceptable salts of these compounds.

In one subgroup of compounds of general formula (Ia), R is a group C(=O)OR ¹ . In another subgroup, R is a group C(=O)NR 1 .

In another subgroup, R is a group C(=O)R ⁴ . In another subgroup, R is a group CH ₂ OR ⁵ . In another subgroup, R is a group C(R ^S ) (R ⁷ ) (R ^d ) .

In the next subgroup is the R group

O

A preferred embodiment of the present invention relates to compounds of general formula (Ia), wherein

R is the group CH(R ⁶ ) (R ⁷ ) ,

R ^b is (a) hydrogen, (b) an alkyl group containing 1 to 14 carbon atoms, (c) an aryl group, (d) an aryl-substituted alkyl group containing 1 to 6 carbon atoms, (e) a group - (CH ₂ ) _y CH (OH) (CH ₂ ) _Z H, (f) a group - (CH ₂ ) _y C (R ⁷ ) =CH (CH ₂ ) _Z H,

R' is a hydrogen atom or an alkyl group containing 1 to 6 carbon atoms.

Another preferred embodiment of the present invention is compounds of general formula (Ia), wherein

R is (a) a methyl group, (b) an ethyl group, (c) a -CH ₂ CH ₂ CH ₃ group, (d) a -CH ₂ CH ₂ CH ₂ CH ₃ group, (e) a -CH ₂ CH ₂ CH2CH ₂ CH ₃ group, (f) a -CH2CH2CH2CH2CH2CH2CH3 group, refers to • · (g) a -CH2CH2CH2CH2CH2CH2CH2CH2CH3 group, (h) a -CH2CH2CH2CH2CH2CH2CH2CH3 group, (i) a -CH2CH2CH2CH2CH2CH2CH2CH2CH3 group, (j) a -CH ₂ CH ₂ CH (CH ₃ ) z group, (k) a -CH ₂ C ₆ H5 group, (l) a -CH(CH ₃ ) ₂ group, (m) a -CH ₂ CH (CH ₃ ) ₂ group, (n) a -CH ₂ CH=CH ₂ group, (o) group -CH ₂ CH=CHCH ₃ , (p) group -CH ₂ CH=CHCH ₂ CH 3 , (q) group -CH 2 CH=CHCH ₂ CH ₂ CH 3 .

A more preferred embodiment of the present invention relates to

compounds of general formula (Ia), where R is (a) methyl group, (b) ethyl group, (c) group -CH ₂ CH ₂ CH ₃ , (d) group -CH2CH ₂ CH2CH _3/ (e) group -CH2CH2CH2CH2CH3, (f) group -CH ₂ CH (CH ₃ ) ₂ , (g) group -CH ₂ CH ₂ CH (CH ₃ ) 2, (h) group -CH ₂ CH=CHCH ₃ , (i) group -CH ₂ CH=CHCH ₂ CH ₃ , (j) the group -CH ₂ CH=CHCH ₂ CH ₂ CH ₃ .

Another aspect of the present invention is a pharmaceutical composition comprising a fungicidally effective amount of a compound of formula (I) and a pharmaceutically acceptable carrier. The invention also relates to a pharmaceutical composition prepared by combining a compound of formula (I) and a pharmaceutically acceptable carrier.

• ·

Another aspect of the present invention is also an agricultural composition comprising a fungicidally effective amount of a compound of formula (I) and an agriculturally acceptable carrier. The invention also relates to an agricultural composition which is made by combining a compound of formula (I) and an agriculturally acceptable carrier.

The present invention further relates to a method of treating a fungal infection in animals (including humans) which comprises administering a fungicidally effective amount of a compound of general formula (I) to those animals in need of such treatment.

Another aspect of the present invention is a method of controlling phytopathogenic fungi of plants, which comprises applying a fungicidally effective amount of a compound of general formula (I) to said plants.

Unless otherwise indicated, the following terms have the meanings set forth herein.

The term alkyl group used alone or as part of a group designation (e.g., aralkyl group), means a linear or branched alkyl chain containing the indicated number of carbon atoms, such as methyl group, ethyl group, n-propyl group, n-butyl group, isopropyl group, isobutyl group, isopentyl group, sec-butyl group, tert-butyl group, n-hexyl group, n-octyl group, decyl group, undecyl group, cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group, cyclobutylmethyl group, cyclopentylethyl group, cyclohexylmethyl group, and the like.

The term cycloalkyl group means a hydrocarbon containing one or more rings containing 3 to 12 carbon atoms, wherein the hydrocarbon contains a total of at most 20 carbon atoms. Examples of cycloalkyl groups include: • · cyclopropyl group, cyclopropylmethyl group, * cyclobutyl group, 2-cyclopentylethyl group, cyclopentyl group, cycloheptyl group, adamantyl group, cyclododecylmethyl group, 2-ethyl-1-bicyclo[4,4,0]decyl group, and the like.

The term "aryl group" used alone as part of a group designation includes a phenyl group or a heteroaryl group, both of which may be optionally substituted with one to three groups independently selected from the group consisting of a halogen atom, a hydroxyl group, an alkyl group containing 1 to 6 carbon atoms, an alkoxy group containing 1 to 6 carbon atoms or an alkoxy. carbonyl group containing 1 to 4 carbon atoms in the side chain. The heteroaromatic group may be a five- or six-membered heteroaromatic ring containing one or more heteroatoms selected from the group consisting of a nitrogen atom, an oxygen atom and a sulfur atom. Examples _of suitable heteroaromatic groups include a pyridyl group, a furyl group, a thienyl group and a pyrrolyl group.

The term alkenyl group means a linear or branched carbon chain containing at least one double bond between carbon atoms. Examples of such a group include vinyl, allyl, butenyl, isobutenyl, butadienyl, and the like.

The term "alkynyl group" means a linear or branched carbon chain containing at least one triple bond between carbon atoms. Examples of such a group include an acetylenyl group, a propargyl group, a butynyl group, a 1,3-pentadiinyl group, and the like.

The term "control" or "controlling" used in connection with phytopathogenic fungi includes prophylactic use (i.e. protection against infection) and curative use (i.e. eradication of infection).

The term "plants" includes whole living plants or parts thereof, leaves, flowers, seeds, fruits and other plant material. Due to the possibility of applying the active ingredient to the soil, the term also includes the roots of the plant.

The term "composition" means an agricultural or agrochemical composition which includes a product comprising the active ingredient(s), the inert ingredient(s) constituting the carrier and any product resulting, directly or indirectly, from the combination, complexation or grouping of any two or more ingredients or resulting from the dispersion of one or more ingredients or from other reactions or interactions of one or more ingredients.

The group of suitable salts of the compound of general formula (I) includes basic inorganic salts such as alkali salts (e.g. sodium or potassium salts), ammonium salts and organic basic salts. The group of suitable basic organic salts includes amine salts such as tetraalkylammonium salts (e.g. tetrabutylammonium or trimethylcetylammonium salts), trialkylamine (e.g. triethylamine), or dialkylamine salts (e.g. dicyclohexylamine), salts of optionally substituted benzylamine (e.g. phenylbenzylamine or p-bromobenzylamine), salts of ethanolamine, diethanolamine, N-methylglucosamine, N-methylpiperidine, pyridine and substituted pyridine (e.g. collidine, lutidine, 4-methylaminopyridine), salts of tri(hydroxymethyl)methylamine and salts of amino acids (e.g. lysine or arginine).

Compounds of general formula (I) can be prepared from sordarin (II) or its aglycone, sordaricin (III). Sordarin • · · · · · · * ' · · · · · · · · « • ····· · · · · · · is [IR-(la, 3aP, 4β, 4a3,7β, 7aa, 8ap)]-8a-[(6-deoxy-4-0methyl-^-D-altropyranosyloxy)methyl]-4-formyl4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4methano-s-indacene-3a(1H)-carboxylic acid of formula (II):

Sordarin can be obtained by culturing Sordaria araneosa NRRL 3196 (also deposited with the ATCC under the designation ATCC 36386) as described in British Patent GB 1162027 or in published PCT application WO96/14326. Sordarin can also be obtained by fermenting Rosellinia subiculata and an unidentified fungus designated ATCC 74387 as described below. Both cultures were deposited on August 27, 1996, in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA, under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

Sordaricin is [1R-(1α,3αΕ,4β,4α3,7β,7αα,8α0)]-4-formyl-8α-(hydroxymethyl)-4,4α,5,6,7,7α,8,8α-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene3a(1H)carboxylic acid of formula (III):

(III) » · · · · <

sordarin reaction As described in

Sordaricin can be prepared from concentrated hydrochloric acid, published in PCT application WO96/14326, sordaricin can also be obtained by fermentation of a mutant derived from Sordaria araneosa NRRL 3196 and biotransformation of sordarin by Koryneforms.

The compound of the present invention (compound of general formula I) can be prepared by the following methods. The conditions given are for illustrative purposes only and do not limit the scope of the present invention.

Scheme 1 shows the synthesis of 4-cyano-4-deformylsordaricin with a protected carboxyl group (IV) from sordarin. This sordarin derivative can be used as a starting compound in the synthesis of compounds of general formula (I). First, the hydroxyl group of sordarin is protected with a suitable protecting group (indicated by the letters PG in the schemes) and then the carboxyl group is protected with the same or another suitable protecting group. The formyl group is converted to an aldoxime group, which is further a suitable reagent, such as an inner salt \so-called.

Bur(methoxycarbonylsulfamoyl)triethylammonium hydroxide (gess reagent) is dehydrated to form a nitrile group (cyano group). Acid hydrolysis cleaves the protected saccharide and forms a compound of general formula (IV).

• · • · • · · · · · * · • ·· · · ··· · · · «····· · · · ·

PG and PG' may be (IV) the same or different

Scheme 2 shows the conversion of compound (IV) to sordaricin. After protection of the carboxyl and hydroxyl groups, the corresponding aldoxime is prepared. Subsequent dehydration with a suitable reagent, such as the inner salt of (methoxycarbonylsulfamoyl)triethylammonium hydroxide (so-called Burgess reagent) and removal of the hydroxyl protecting group gives the compound of formula (IV).

·· * ·· · ···· ·· · φ · · ··· · · ··

Scheme 2

+PG +PG'

Scheme 3 shows the possibility of preparing carbamates, esters and carbonic acid derivatives of 4-cyano-deformylsordaricin. Carbamates can be prepared by reacting a compound of general formula (IV) with an isocyanate (in the examples where R ³ is a hydrogen atom) or a carbamoyl halide or other activated reagent for preparing carbamates in an inert solvent.

Esters can be prepared in a similar manner by reacting a compound of formula (IV) with an activated carbonyl compound such as an acid chloride or mixed anhydride, preferably in the presence of an acylation catalyst such as N,N-dimethylaminopyridine and a base such as pyridine.

Carbonic acid derivatives can be prepared by reacting a compound of general formula (IV) with an activated carbonate such as chloroformate or pyrocarbonate. Preferably, the reaction • · · ·

an acylation catalyst such as N,N-dimethylaminopyridine and a base such as pyridine are used.

Scheme 3

PG is a carboxylic acid protecting group;

C(O)Y is an activated carbonyl group as an acid halide or anhydride.

Scheme 4 shows the synthesis of ether derivatives of 4-cyano4-deformylsordaricin. By reacting the compound of general formula (IV) with an α-haloether in basic medium or with a vinylether in • · • 9

9 9 9 • · in an acidic environment, substituted α-alkoxy ether derivatives are formed. Reaction of a compound of formula (IV) with a primary or secondary halide or sulfonate and a suitable base such as sodium hydride under conditions promoting S _N 2 addition gives the corresponding primary and secondary ether derivatives, while reaction of a compound of general formula (IV) with a tertiary alcohol, tertiary halide or tertiary sulfonate in the presence of a Lewis acid (including protic acids) under conditions promoting S _N 1 addition gives the corresponding tertiary ether derivatives.

• ·

Scheme 4

R ²

(R ² is not H or aryl) (R ⁵ , R ⁷ and R ^s are not H)

PG is a carboxylic acid protecting group;

X is a common leaving group such as halide or sulfonate.

Scheme 5 shows the preparation of cyclic acetals of the compound of formula (IV). Reaction of the compound of formula (IV) with a cyclic vinyl ether in the presence of an acid catalyst results in the formation of the corresponding cyclic acetal derivative of the compound of formula (IV).

9 99 9 9

9·· · e ««»· • · · · · · · · · * * 9 «····· Β · t r - · • · · · · » · « ♦ · »· ·· ·· »·· ·· ··

Scheme 5

PG is a carboxylic acid protecting group

Compounds of general formula (I) can also be prepared by prior derivatization of the hydroxyl group of sordaricin, with the sole exception that the hydroxyl group must not be converted to an ester group. This method is illustrated in Scheme 6. The starting carbamate, carbonic acid derivative, acetal, ether and cyclic acetal can be prepared by the general procedures illustrated in Schemes 3 to 5, with the exception that the starting compound in these cases is not 4-cyano-4-deformylsordaricin, but sordaricin with a protected carboxyl group. The aldoxime of the derivatized sordaricin can be prepared by reacting the corresponding aldehyde with hydroxylamine hydrochloride in a pyridine-containing alcohol. This aldoxime can be prepared using a suitable, for example, inner salt of ammonium hydroxide (so-called

dehydrating agent such as (methoxycarbonylsulfamoyl)triethylBurgess reagent) to the corresponding nitrile. Removal of the protecting group gives the compound of general formula (I).

Scheme β

COoH

(AND)

R'OH is a solvent which is a lower alcohol;

PG is a carboxylic acid protecting group.

Use

The compounds of general formula (I) are fungicidal agents which are useful for the manufacture of medicinal compositions intended for humans and other animals. The compounds of general formula (I) are also suitable as agents for the protection of agricultural crops.

The compounds of general formula (I) are very active fungicides useful in the control of fungal infections in • · · · · · tropicalis), Aspergillus fumigatus), coccidioides plasma (e.g.

animals, including humans. They can be used, for example, in the treatment of fungal infections caused by organisms such as various species of the Candida strain (e.g. Candida albicans, Candida glabrata, (Torulopsis glabrata), Candida tropicalis and Candida pseudoCryptococcus neoformans, Pneumocystis carinii, Sp. (e.g. Aspergillus flavus and Aspergillus Coccidioides (Coccidioides immitis), Para (e.g. Paracoccidicides brasiliensis), HistoHistoplasma capsulasum) or Blastomyces (e.g.

Blastomyces dermatitidis). These compounds may also be used for the treatment of other fungal infections caused by species of the genus Trichophyton, Microsporum or Epidermophyton (e.g. Trichophyton mentographites, Trichophyton rubrum, Microsporum canis or Epidermophyton floccosum) or for the treatment of mucosal infections caused by Candida albicans.

Compounds of general formula (Σ) can also be used for the treatment of other infections caused by various species of filamentous fungi such as Geotrichum (e.g. Geotrichum clavatum), Trichosporon (e.g. Trichosporon beigelii), Blastoschizomyces (e.g. Blastoschizomyces capizatus), Sporothrix (e.g. Sporothrix schenckií), Scedosporium (e.g. Scedosporíum apiosperum), Cladosporium (e.g. Cladosporíum carrionii) and Pityrosporum ovale.

The compounds of general formula (I) can also be used for the treatment of infections caused by protozoa such as Toxoplasma, Cryptosporidium, Leishmania, Trypanosoma, Giardia and Trichomonas.

The in vitro fungicidal activity of the compounds of the present invention was determined on liquid or solid fungicidal media by a two-fold serial dilution method to determine the minimum inhibitory concentration (MIC) of the fungicidal agent at which growth was inhibited after 24 to 48 hours of incubation at 35°C. In practice, the determination was carried out by inoculating a series of agar plates or microdilution panels of nutrient medium containing a two-fold dilution of the tested fungicidal agent with a standard culture of a clinically relevant pathogen such as Candida albicans. The agar plates or microdilution panels of nutrient medium were then tested for growth or not and the corresponding minimum inhibitory concentrations (MIC) were recorded. The live Almar Blue strain was used to visualize the endpoints.

The in vivo fungicidal activity of compounds of formula (I) can be determined by administering a series of different doses (e.g. subcutaneously, orally, intraperitoneally or intravenously) to mice that have been inoculated intravenously with Candida spp. Kidneys can be removed from the test animals and the viable Candida spp. content can be determined and the reduction in infection can be determined relative to an untreated control group of animals.

In terms of fungicidal activity, the compounds of general formula (I) are useful for the treatment and/or prevention of various fungal diseases in humans and other animals. Such infections include superficial, cutaneous, subcutaneous and systemic fungal infections such as respiratory tract infections, gastrointestinal infections, cardiovascular infections, urinary tract infections, central nervous system infections, candidiasis and chronic mucocandidiasis (for example, aphthous candidiasis and vaginal candidiasis) and fungal skin infections, cutaneous and mucocutaneous candidiasis, dermatophytosis including ringworm and tinea infections, tinea pedis, onychomycosis, pityriasis versicolor, erythrasma, impetigo, fungal exanthema, candidal vulvitis, candidal balanitis and otitis media. Furthermore, the compounds of general formula (I) can be used as prophylactic agents for the prevention of systemic and topical fungal infections. The use of these compounds as prophylactic agents may be useful, for example, in a diet for selective intestinal decontamination to prevent infection in immunocompromised patients (e.g., AIDS patients, cancer patients, or transplant patients). In certain disease syndromes or iatrogenic conditions, it may also be desirable to prevent fungal overgrowth during antibiotic therapy.

The compounds of general formula (I) also have a wide range of uses as agents for the protection of agricultural crops and are effective against a wide range of phytopathogenic fungi, in particular fungi from the group comprising deuteromycetes (e.g. Botritis spp., Septoria spp., Pyricularia spp., Stagnospora spp., Helminthosporium spp., Fusarium spp., Cercospora spp., Rynchosporium spp., Pseudocercosporella spp. and Alternaria spp.); basidiomycetes (e.g. Puccinia spp., Rhizoctonia spp. and Hemileia spp.); ascomycetes (e.g. Venturia spp., Podospharera spp., Erysiphe spp., Monilínia spp. and Uncinula spp.); and oomycetes (e.g. Phytophthora spp., Pemospora spp., Bremia spp., Pythium spp. and Plasmopara spp.). The foregoing list is illustrative of phytopathogenic fungi against which the compounds of the present invention exhibit fungicidal activity and is not intended to be limiting in any way. The compounds of the present invention have very advantageous curative and preventive fungicidal properties suitable for plant protection and can be used to inhibit or destroy microorganisms present on plants or plant parts (fruits, flowers, leaves, stems, tubers or roots) or on various crops of useful plants, while also protecting the later growing parts of the plants against such microorganisms.

• · · · • · ·♦· · · · · · » · * • · · · · · · · · » · · ·····♦ · ··· · · · · • · ·· · ·· · · · · · · · ·

The compounds of the present invention can also be used as an additive in the treatment of plant growth material, in particular seeds (fruits, tubers, grains) and plant cuttings (e.g. rice) to provide protection against fungal infections and against phytopathogenic fungi occurring in the soil. The compounds of the general formula (I) of the present invention are characterized by the fact that they are particularly well tolerated by plants and, in addition, are environmentally friendly.

The determination of the applicability of compounds of general formula (I) in agriculture can be carried out using the following tests:

1. Efficacy against Erypsiphe graminis on wheat

a) After one week of cultivation, wheat plants were treated with a spray containing 200 ppm of active ingredient, % acetone and 0.25% Triton X155, until it was consumed.

After 2 hours, the treated plants were infected with ascospores shaken from the inoculum plant. To determine the degree of protection provided by the test compound, fungal attack was evaluated after eight days of incubation at 22°C and 50% relative humidity.

b) After one week of cultivation, wheat plants were infected with ascospores shaken from the inoculum plant. After 24 hours, the wheat plants were treated with a spray containing 200 ppm of the active ingredient, 20% acetone and 0.25% Triton X155. To determine the therapeutic effect provided by the test compound, fungal attack was evaluated after eight days of incubation at 22°C and 50% relative humidity.

c) After one week of cultivation, wheat plants were infected with ascospores shaken from the inoculum plant. After 24 hours, the soil in which the plants were grown was watered with a solution containing 200 ppm of the active ingredient, 20% acetone and • · • ·

0.25% Triton X155. To determine the therapeutic effect provided by the test compound, fungal attack was evaluated after eight days of incubation at 22°C and 50% relative humidity.

2. Efficacy against Puccinia recondita on wheat

a) After one week of cultivation, wheat plants were treated with a spray containing 200 ppm of the active ingredient, 20% acetone and 0.25% Triton X155 until it was consumed. After 2 hours, the treated plants were infected with spores. To determine the degree of protection provided by the test compound, fungal attack was evaluated after one day of incubation at 20°C and 95-100% relative humidity followed by seven days of incubation at 25°C and 50% relative humidity.

b) After one week of cultivation, wheat plants were infected with a suspension containing spores. After 24 hours, the wheat plants were treated with a spray containing 200 ppm of the active ingredient, 20% acetone and 0.25% Triton X155 until it was consumed. To determine the therapeutic effect provided by the test compound, fungal attack was evaluated after one day of incubation at 20°C and 95-100% relative humidity followed by seven days of incubation at 25°C and 50% relative humidity.

c) After one week of cultivation, wheat plants were infected with a suspension containing spores. After 24 hours, the soil in which the plants were grown was watered with a solution containing 200 ppm of the active ingredient, 20% acetone and 0.25% Triton X155. To determine the therapeutic effect provided by the test compound, fungal attack was evaluated after one day of incubation at 20°C and 95-100% relative humidity followed by seven days of incubation at 25°C and 50% relative humidity.

Based on the range of effects, the compounds of the present invention can be used for the protection or treatment of plants against attack or already attacked by phytogenic fungi that attack various agricultural crops. The following list includes those plant species that are suitable for the applications of the compounds of the present invention described in the subject matter of the present invention: cereals (wheat, rye, oats, barley, rice, sorghum and related crops); beets (sugar beets or fodder beets); pome, stone and soft fruits (e.g. apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and blackberries); legumes (e.g. beans, peas, lentils and soybeans); oil plants (rapeseed, mustard, poppy, olives, sunflower, coconut, castor oil plants, cocoa beans and groundnuts); cucurbits (e.g. cucumber, pumpkin and melon); textile plants (e.g. cotton, flax, hemp and jute); citrus fruits (e.g. oranges, lemons, tangerines and grapefruits); vegetables (e.g. lettuce, cabbage, spinach, carrots, asparagus, peppers, onions, tomatoes and potatoes); plants from the Laurel family (avocado, cinnamon and camphor); or plants such as corn, tobacco, nuts, coffee, sugarcane, tea, grapevine, hops, banana and rubber as well as ornamental plants (flowers, shrubs, deciduous trees and evergreen plants such as conifers). However, all these listed plant species do not constitute a limiting list of plants with regard to the spectrum of action of the compounds according to the invention.

The compounds of general formula (I) are particularly suitable for the treatment of the following plant diseases:

Erysiphe graminis in cereals, Erysiphe cichoracearum and Sphaerotheca fuliginea in pumpkins, Podasphaera leucotricha in apples, Uncinula necator in grapevines, Puccinia sp. in cereals, Rhizoctonia solani in cotton, Ustilago sp. in cereals and sugarcane, Venturia inaequalis (scab) in apples, Helminthosporium sp. in cereals, Septoria nodorum in wheat, Botrytis cinerea (grey mold) in strawberries and grapes, Cercospora arachidicola in groundnuts, Pseudocercosporella herpotrichoides in wheat and barley, Pyricularia oryzae in rice, Phytophtora infestans in potatoes and tomatoes, Fusarium sp. and Verticillium sp. in various plants, Plasmopara viticola in grapes, Aiternaria sp. in fruits and vegetables. Compounds of general formula (I) can also be used for the protection of various materials (e.g. for the protection of wood raw material against Paecilomyces variotii).

Pharmaceuticals

Although it is in principle possible for the compounds of the present invention to be administered therapeutically alone, i.e. as raw material, it is preferred to administer the active ingredient in the form of a pharmaceutical composition. Accordingly, the invention further relates to a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt of a compound of formula (I) together with one or more pharmaceutically acceptable carriers for the compound of formula (I) and the therapeutic and/or prophylactic ingredients, which must be acceptable in the sense that they are compatible with the other ingredients of the said pharmaceutical composition and are not deleterious to the recipient thereof.

Pharmaceutical compositions according to the present invention include pharmaceutical compositions intended for oral, buccal, parenteral, implant, rectal, topical, ophthalmic or genito-urinary administration, or in a form suitable for administration in the form of implants or in a form suitable for inhalation or insufflation administration.

Tablets and capsules for oral administration may contain conventional excipients such as binders, for example syrup, acacia, gelatin, sorbitol, tragacanth, starch-based glue or polyvinylpyrrolidone; fillers, for example lactose, sugar, microcrystalline cellulose, corn starch, calcium phosphate or sorbitol; lubricants, for example magnesium stearate, stearic acid, talc, polyethylene glycol or silicon dioxide; disintegrating agents, for example potato starch or sodium starch glycolate or sodium croscarmellose; or wetting agents such as sodium lauryl sulfate. Tablets, which include chewable, soluble and effervescent tablets, may be coated according to well-known methods. Oral liquid preparations may take the form, for example, of aqueous or oily suspensions, solutions, emulsions, syrups or medicated drinks or may be in the form of a dry product for reconstitution with water or other suitable vehicle immediately before use. Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, for example sorbitol syrup, methylcellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hardened fats; emulsifying agents, for example lecithin, sorbate monooleate or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol; and preservatives, for example methyl or propyl p-hydroxybenzoates or sorbic acid.

The pharmaceutical composition of the present invention for buccal administration may take the form of tablets or lozenges formulated by conventional methods.

The pharmaceutical composition of the present invention for parenteral administration may be formulated for administration by injection or continuous infusion. Pharmaceutical compositions for injection may be in the form of single doses in ampoules or multi-dose storage bottles with an added preservative. These pharmaceutical compositions may be in the form of suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulating agents such as suspending agents, stabilizing agents and/or dispersing agents. Furthermore, the active ingredient may be in the form of a powder for constitution with a suitable vehicle, e.g. pyrogen-free water, just before use.

For inhalation administration, they are conveniently administered as an aerosol spray from pressurized containers using a suitable propellant gas, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases, or from a nebulizer. In the case of using a pressurized aerosol, it is possible to determine the individual dose by allowing the valve to deliver a metered amount of aerosol.

Another possibility for the inhalation administration of the pharmaceutical composition of the present invention is that the pharmaceutical composition is in the form of a dry powder composition, for example a powder mixture of a compound of the present invention and a suitable powder base such as lactose or starch, or a modified physical form of the drug substance itself. The powder composition may be supplied in unit dose form, e.g. in capsules or cartridges of e.g. gelatin or • · the vehicle included in blister packs, from which the powder can be administered by means of an inhaler or insufflator.

The pharmaceutical composition of the present invention may take the form of a suppository comprising, for example, a conventional suppository base, or the composition may take the form of a pessary comprising, for example, a conventional pessary base.

The pharmaceutical compositions of the invention may also be formulated for topical administration in the form of ointments, creams, gels, lotions, shampoos, powders (including spray powders), pessaries, tampons, sprays, medicated baths, aerosols, drops (e.g. eye drops, ear drops or nasal drops) or infusion solutions. Ointments and creams may be formulated, for example, using an aqueous or oily base with the addition of a suitable thickening or gelling agent. Ointments for administration to the eye may be manufactured under sterile conditions using sterile ingredients. Infusion solutions may be formulated, for example, for veterinary use in organic solvents containing oils, optionally with formulation agents such as stabilizing agents and solubilizers. Pessaries and tampons for insertion into the vagina may be formulated in conventional ways and, where appropriate, may contain effervescent Such pharmaceutical compositions may also contain other active ingredients such as suitable corticosteroids, antibiotics or antiparasitics.

Liquid compositions for intranasal use may be in the form of solutions or suspensions and may include conventional vehicles such as tonicity agents, for example sodium chloride, dextrose or mannitol; preservatives, for example benzalkonium chloride, thiomersal, phenylethyl alcohol; and other formulation agents such as suspending agents, buffering agents, stabilizing agents, dispersing agents and flavoring agents.

Transdermal administration can be effected by creating a suitable system that promotes absorption of the active compound through the skin and generally consists of a base composition enclosed in an adhesive patch comprising carrier films, membranes and release liners. Such systems may include absorption enhancing agents such as alcohols or may function on the basis of iontophoresis support.

The pharmaceutical composition of the present invention may also be formulated as a depot preparation. Such long-acting compositions may be administered by implantation (e.g., subcutaneously or intramuscularly) or intramuscular injection. The compound of the present invention may thus be formulated, for example, with suitable polymeric or hydrophobic materials (e.g., as an emulsion in a pharmaceutically acceptable oil) or ion exchange resins or as a nearly insoluble derivative, e.g., as a nearly insoluble salt.

When the pharmaceutical composition of the present invention comprises single doses, each dose preferably contains from 0.001 milligrams to 1000 milligrams, preferably from 0.01 milligrams to 400 milligrams, of the active ingredient in cases where the compound of the present invention is to be administered orally. The daily dose used for the treatment of adult humans is preferably from 0.001 milligrams to 5000 milligrams of the active ingredient, more preferably from 0.01 milligrams to 2000 milligrams of the active ingredient. This dose may be administered, for example, in 1 to 4 doses, depending on the route of administration and the condition of the patient and the disease to be treated.

The compound of the present invention may be administered, for example, by intravenous infusion at a dose of up to 50 milligrams of active ingredient.

components/kilogram/day. The duration of treatment is determined by the rate of response rather than a predetermined number of days.

The compounds of the invention may also be used in combination with other therapeutic agents and therefore a further aspect of the invention is a combination comprising a compound of the invention together with another therapeutically active agent.

The compounds of the present invention may thus be used, for example, in combination with one or more fungicides, such as a polyene derivative (e.g. amphotericin B, nystatin, lipid formulation of amphotericin B); an azole derivative (e.g. fluconazole, itraconazole, ketoconazole, miconazole, clotrimazole, ZD-08070, UK-109496, SCH56592), 5-fluorocytosine, a pneumocandin or echinocandin derivative such as cilofungin, LY-303366, L-733560,

L-743872 or other cell wall acting compounds such as nikkomycin Z and/or one or more immunosuppressive compounds such as interferon e.g. (IFN-), interleukin e.g. (IL-1, IL-2, IL-3 and IL-8) and cell stimulating agents, [(G)-CSF, (M)-CSF and (GM)-CSF] and defensins. Particularly suitable compounds for use with the compounds of the present invention are itraconazole, flucytosine, fluconazole or amphotericin B.

When the compounds of the present invention are administered in combination with another fungicidal compound, the compound of the present invention and the other fungicide may be administered at the recommended maximum clinical doses or at doses lower than these.

The above combinations may be intended for use in the form of pharmaceutical compositions and therefore pharmaceutical compositions comprising the above described combinations together with a pharmaceutically acceptable carrier for the combination are • fe • · ·· fefe fefe fefe fefe fefe a further aspect of the present invention. The individual components of such pharmaceutical composition may be administered either sequentially or simultaneously in separate or combined pharmaceutical compositions.

When a compound of the present invention is used in combination with a second drug for the same indication, the dosages of the individual compounds may differ from those when each compound is used alone. Suitable dosages will be readily determined by one skilled in the art.

Agrochemical compositions Compounds of the general formula (I) in unmodified form or preferably together with auxiliaries which are commonly used in the field of agrochemical compositions and are known from the point of view of their purpose of use mainly as: emulsifiable concentrates, spreadable pastes, directly sprayable or dilutable solutions, dilute solutions, suspensions (including aqueous, oily or other suspensions with a high percentage of water), dispersions, oily dispersions, spreading agents, wettable powders, soluble powders, dusts, granules and encapsulated compositions. The agrochemical compositions according to the invention are produced by known methods, e.g. by homogeneously mixing and/or grinding the active ingredients with additional components, e.g. solvents, solid carriers and, where appropriate, surfactants. Powders, dusts and spreading agents can be produced by mixing or grinding the active ingredients with a solid carrier. Granules, e.g. coated granules, impregnated granules or Homogeneous granules can be produced by binding active ingredients to solid carriers.

it is possible to use either • ·

The group of suitable solvents includes: aromatic hydrocarbons, preferably fractions containing 8 to 12 carbon atoms, such as mixtures of xylenes or substituted naphthalenes, chlorinated aromatics such as chlorobenzenes, phthalates such as dibutyl or dioctyl phthalate, aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and glycols and their ethers and esters such as ethanol, ethylene glycol, ethylene glycol monomethyl ether or ethylene glycol monoethyl ether, ketones such as cyclohexanone, amines such as ethanolamine, strongly polar solvents such as N-methyl-2-pyrrolidone, dimethyl sulfoxide or N,N-dimethylformamide and vegetable oils or epoxidized vegetable oils such as epoxidized coconut oil or soybean oil and water.

Examples of surfactants include: salts of aromatic sulfonic acids containing alkali metals, alkaline earth metals and ammonium cations, e.g. ligninsulfonic acid, phenolsulfonic acid, naphthalenesulfonic acid and dibutylnaphthalenesulfonic acid and the same salts of fatty acids, alkyl and alkylarylsulfonates and alkyllauryl ether and sulfates of fatty alcohols and salts of sulfonated hexadecanols, heptadecanols and octadecanols, salts of glycol ethers of fatty alcohols, condensation products of sulfonated naphthalene derivatives with formaldehyde, condensation products of naphthalene or naphthalenesulfonic acid derivatives with phenol and formaldehyde, polyoxyethyleneoctylphenol ethers, ethoxylated isooctylphenol, and ethoxylated nonylphenol, alkylphenol polyglycol ethers, tributylphenyl polyglycol ethers, alkylaryl polyether alcohols, isotridecyl alcohol, condensates of fatty alcohols with ethylene oxide, ethoxylated castor oil oil, polyoxyethylene alkyl ethers, ethoxylated polyoxypropylene, acetal • · • · · · · t» • · * <

• · · · · · · lauryl alcohol polyglycol ether, sorbitol esters, lignin sulfite waste liquor and methyl cellulose.

Examples of solid carriers include mineral earths such as silicic acids, silica gels, silicates, talc, kaolin, attapulgite, limestone, lime, chalk, stem, loess, clay, dolomite, diatomaceous earth, calcium aluminum sulfate, magnesium sulfate, magnesium oxide, ground plastics, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate and urea and its derivatives, and plant products such as whole grain flour, ground bark or ground wood and ground nut shells, cellulosic powders, etc.

The compounds of general formula (I) can be mixed and applied together with other active ingredients, for example herbicides, insecticides, bactericides, nematicides, molluscicide, growth regulators, micronutrients and fertilizers. The other ingredients can also include one or more fungicides belonging to, but not limited to, the following classes of fungicides: carboxamides, benzimidoazoles, triazoles, hydroxypyridines, dicarboxamides, phenylamides, thiadiazoles, carbamates, cyanooximes, cinnamic acid derivatives, morpholines, imidazoles, B-methoxyacrylates and pyridines/pyrimidines. Furthermore, these other active ingredients can be used as mixtures of several agrochemical compositions according to the present invention and, if desired, together with other adjuvants commonly used in the art to facilitate the application of the composition. Suitable carriers or excipients may be solid or liquid and correspond to substances generally used in technological processes for the formation of such compositions (e.g. natural or regenerated mineral compounds, solvents, dispersants and wetting agents) ···· · * · · • * » · · β · · · · · · • ····· · ··· «β ·

The following list of fungicides with which the compounds of general formula (I) can be combined is given only to illustrate the possible combinations without any limitation with respect to other possible combinations. As examples of fungicides which can be combined with the compounds of general formula (I) can be mentioned: sulfur, dithiocarbamates and their derivatives such as iron(III) dimethyldithiocarbamate, zinc dimethyldithiocarbamate, zinc ethylenebisdithiocarbamate, manganese containing ethylenebisdithiocarbamate, manganese and zinc containing ethylenebisdithiocarbamate, tetramethylthiuram disulfides, ammonium complex of zinc N,N'-ethylenebisdithiocarbamate, ammonium complex of zinc N,N'-propylenebisdithiocarbamate, zinc N,N'-propylenebisdithiocarbamate, N,N'-polypropylenebis(thiocarbamyl)disulfide; nitroderivatives such as dinitro(1-methylheptyl)phenyl crotonate, 2-sec-butyl-4,6-dinitrophenyl-3,3-dimethylacrylate, 2-sec-butyl-4,6-dinitrophenylisopropyl carbonate and diisopropyl-5-nitroisophthalate; heterocyclic compounds such as 2-heptadecylimidazol-2-yl acetate, 2,4-dichloro-6-(o-chloroanilino)-s-triazine, 0,O-diethyl phthalimidophosphonothioate, 5-amino-l[bis(dimethylamino)phosphinyl]-3-phenyl-1,2,4-triazole, 2,3-dicyano-1,4-dithioanthraquinone, 2-thio-l, 3-Dithio[4,5-b]quinoxaline, methyl 1-(butylcarbamoyl)-2-benzimidazole carbamate, 2-methoxycarbonylaminobenzimidazole, 2-(fur-2-yl)benzimidazole, 2-(thiazol-4-yl)benzimidazole, N-(1, 1,2,2-tetrachloroethylthio)tetrahydrophthalimide, N-trichloromethylthiotetrahydrophthalimide, N-dichlorofluoromethylthio-N', N'-dimethyl-N-phenylsulfondiamide, 5-ethoxy-3-trichloromethyl-1,2,3thiadiazole, 2-thiocyanomethylthiobenzothiazole, 1,4-dichloro-2,5dimethoxybenzene, 4-(2-chlorophenylhydrazono)-3-methyl-5-isoxazolone, 2-thiopyridine-1-oxide, 8-hydroxyquinoline and its copper-containing salt, 2,3-dihydro-5-carboxanilido-6-methyl-1,436 oxathiyne, 2,3-dihydro-5-carboxanilido-6-methyl-1,4-oxathiyne4,4-dioxide, 2-methyl-5,6-dihydro-4H-pyran-3-carboxanilide,

2-methylfuran-3-carboxanilide, 2,5-dimethylfuran-3-carboxanilide, 2, 4,5-trimethylfuran-3-carboxanilide, 2,5-dimethyl-N-cyclohexylfuran-3-carboxanilide, N-cyclohexyl-N-methoxy-2, 5-diethyl-furan-3-carboxanilide, 2-methylbenzanilide, 2-iodobenzanilide, N-formyl-N-morpholine-2,2,2-trichloroethyl acetal, piperazine-1,4-diylbis(1-(2,2,2-trichloroethyl)formamide),

1-(3,4-dichloroaniline)-1-formylamino-2,2,2,-trichloroethane,

2, 6-dimethyl-N-tridecylmorpholine and its salts, 2,6-dimethyl-Ncyclododecylmorpholine and its salts, N-[3-(p-tert.butylphenyl)-2methylpropyl]-cis-2,6-dimethyl-morpholine, N-[3-(p-tert.butylphenyl)-2-methyl-propyl]piperidine, 1-[2-(2,4-Dichlorophenyl)-4-ethyl-1,3-dioxolan-2-yl-ethyl]-1H-1,2,4-triazole, 1-[2-(2,4-dichlorophenyl)-4-n-propyl-1,3-dioxolan-2-yl-ethyl]-1H-1,2,4-triazole, N-(n-propyl)-N-(2,4,6-trichlorophenoxyethyl)-N-imidazole urea, 1-(4-Chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)butan-2-one, 1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H1,2,4-triazol-1-yl)butan-2-ol, α-(2-chlorophenyl)-α-(4-chlorophenyl)-5-pyrimidine-methanol, 5-butyl-(2-dimethylamino-4-hydroxy-6-methylpyrimidine, bis(p-chlorophenyl)-3-pyridinemethanol,

1,2-bis(3-ethoxycarbonyl-2-thioureido)benzene, 1,2-bis(3methoxycarbonyl-2-thioureido)benzene and various fungicides such as dodecylguanidine acetate, 3-[3-(3,5-dimethyl-2-oxycyclohexyl)-2hydroxyethyl]glutaramide, hexachlorobenzene, DL-methyl-N-(2,6-dimethylphenyl)-N-(fur-2-yl)alanate, methyl DL-N-(2,6-dimethylphenyl) -N-(2-methoxyacetyl)alanate, N-(2,6-dimethylphenyl)-Nchloroacetyl-DL-2-aminobutyrolactone, methyl DL-N-(2,6-dimethylphenyl) -N-(phenylacetyl)alanate, 5-methyl-5-vinyl-3-(3,5-dichlorophenyl) -2,4-dioxo-1,3-oxazolidine, 3-[3,5-dichlorophenyl]-5methyl-5-methoxymethyl-1,3-oxazolidine-2,4-dione, 3-(3,5-di37 chlorophenyl)-1-isopropylcarbamoylhydantoin, N-(3,5-dichlorophenyl)-1,2-dimethylcyclopropane-1,2-dicarboximide, 2-cyano-[N(ethylaminocarbonyl)-2-methoxyimino]acetamide, 1-[2-(2,4-dichlorophenyl )pentyl]-1H-1,2,4-triazole, 2,4-difluoro-α-(1H-1,2,4triazol-1-ylmethyl)benzhydryl alcohol, N-(3-chloro-2,6-dinitro4-trifluoromethylphenyl)-5-trifluoromethyl-3-chloro-2-aminopyridine and 1-((bis(4-fluorophenyl)methylsilyl)methyl-1H-1,2,4-triazole .

Depending on the nature of the agrochemical composition according to the present invention and on the objects to which the composition is to be applied and on the prevailing conditions, the method of application is selected, such as spraying, fine spraying, atomizing, scattering, coating, coating and pouring. One method of applying the active ingredient or the agrochemical composition containing at least one of the compounds mentioned is application to plants (i.e. foliar application). However, the active ingredient can penetrate the plant roots through the soil (i.e. soil application). This application can be carried out either by applying a liquid to the soil (drench application) or by applying granules containing the active ingredient(s).

The active ingredient of the present invention can also be applied to plant growth material, such as seeds (fruits, tubers or grains) or plant cuttings, either in liquid form (coating) or in solid form (coating). For example, the seeds can be coated before sowing. The compounds of the present invention can also be applied to grains, either by impregnating the grains with a liquid form or by coating the grains with a solid form of the agrochemical composition of the present invention. The agrochemical composition can also be applied directly to the place where the plants are grown, for example into the seed furrows during sowing.

• ·····» · · · « ’ β» · • · · ·· · ·«·· • · · · ·· ··· ·» ··

The usual preferred amount of the composition is from 10 grams to 50 kilograms of active ingredient per hectare, preferably 100 grams to 2 kilograms of active ingredient per hectare, more preferably 100 to 600 grams per hectare. The active ingredients of the compounds mentioned are generally used in the form of agrochemical compositions and can be applied to plants or parts of plants either simultaneously or sequentially with other active ingredients. These other active ingredients can be fertilizers, other micronutrients or other compounds affecting plant growth. However, they can also be selective herbicides, insecticides, bactericides, nematicides and molluscicide agents as well as other fungicides.

Preparation of starting material

Fermentative production of sordarin

For cultivation of Rosellinia subiculata (ATCC 74386) and

ATCC 74387 in the production of sordarin the following media were used:

Inoculation medium 1

Component

Yeast extract Malt extract Glucose Junlon gram/liter 4.0 8.0 4.0 1.5

The medium was prepared in distilled water, the pH was adjusted to 7.0 before sterilization, and then the medium was divided into 50 milliliter portions into uncovered Erlenmeyer flasks • · « · · 9 * · · · » · * ··»· • ·· · « * · · » « » « « ···*·« · · · ♦ -- » · • <· »· ·

Inoculation medium 2

Component gram/liter Corn extract (dried) 2.5 Tomato paste 40, 0 Oatmeal 10, 0 Glucose 10, 0 Trace element solution 10.0ml/1

Trace element solution

Folder gram/liter _{FeSO4.7H20 ​} 1.0 MnSO ₄ .4 H ₂ 0 1.0 CuC1 ₂ .2 H ₂ O 0.025 CaCl ₂ .H ₂ O 0.1 H3BO3 0.056 (NH ₄ ) ₆ Mo0 ₂₄ .4 H ₂ O 0.019 ZnSO ₄ .7 H ₂ O 0.2

The trace element solution of hydrochloric acid was prepared in

0, β N acid

The medium was prepared in distilled water, the pH was adjusted to 6.8 before sterilization, and then the medium was divided into 50 milliliter portions into uncovered Erlenmeyer flasks • * · · · » ···· » · * · » · · • « # · « ·«·· · * « t «····· · · · β » * β ·· · · t · ««»· • · · * »« ·»« ·· · · ο with a volume of 250 milliliters. The flasks were closed with cotton plugs and subsequent sterilization was carried out for 20 minutes at a temperature of 121 °C.

Solid production medium 1

1. Fixed part

675 milliliters of vermiculite were measured into a 2-liter cylindrical bottle. The bottle was closed with a latex stopper and placed in an autoclave for 60 minutes and then dried for 30 minutes.

2. Liquid part

220 milliliters of the following mixture was measured into a 500 milliliter bottle:

Folder gram/liter Glucose 150, 0 Glycerol 20, 0 Yeast extract 4.0 NaNO ₃ 1.0 Monosodium glutamate 3, 0 On ₂ HPO ₄ 0.5 _MgSCL.7H2O 1.0 K-elements 1.0ml/1 _CaCO3 8.0

K-elements Folder gram/liter FeCl ₃ .6 H ₂ O 5, 8 MnS0 ₄ .H ₂ 0 0.1

·· • ♦ « · · ( • · • · « · F · » • ···· * » β • · »»*'!&

» ♦ · · * < · • · · · · · 4¼

CoC1 ₂ .6 H ₂ O 0.02 CuSO ₄ .5 H ₂ O 0.015 Na ₂ Mo0 ₄ .2 H ₂ 0 0.012 ZnCl ₂ 0.02 SnCl?.2 H ₂ O 0.005 H3BO3 0.01 KCI 0.02 HCl (concentrated) 2.0ml/1

The medium was prepared in distilled water, the pH was adjusted to 7.0 before sterilization, the medium was divided into 500 milliliter bottles and then heat-treated in an autoclave for 15 minutes at a temperature of 121 °C. Glucose was heat-treated in an autoclave separately from the other components.

Liquid production medium 1

Folder gram/liter Glycerol 75, 0 Glucose 75, 0 Tomato paste 5.0 NZ amine type A 4.0 Ardamin PH 5.0 _{K2HPO4 ​} 0.5 MgSO ₄ .7 H ₂ 0 0.25 KCI 0.25 ZnSO ₄ .7 H ₂ O 0.5 _CaCO3 10.0

The medium was prepared in distilled water, the pH was adjusted to 7.0 before sterilization, the medium was divided into * · ·»·» • · • · ».B ¢-• » «· 9 i· · · · » i · * * · * ι.

9 ·. · c *» ·· I* ··« milliliters into uncovered 250 milliliter Erlenmeyer flasks. The flasks were closed with cotton plugs and subsequently sterilized for 20 minutes at a temperature of 121 °C.

Solid production medium 2

1. Fixed part

675 milliliters of vermiculite were measured into a 2-liter cylindrical bottle. The bottle was closed with a latex stopper and placed in an autoclave for 60 minutes and then dried for 30 minutes.

2. Liquid part

220 milliliters of the following mixture was measured into a 500 milliliter bottle:

Folder gram/liter Sucrose 60, 0 Glucose 80, 0 Glycerol 60, 0 Citric acid 15, 0 NZ amine type A 5.0 NaNO ₃ 1.0 KH ₂ PO ₄ 0.5 _MgSCL.7H2O 0.5 CaCCD 0.5 K-elements 1ml/1

• · · · ·

K-elements

Folder gram/liter FeCl ₃ .6 H ₂ O 5, 8 MnSO ₄ .H ₂ O 0.1 CoC1 ₂ .6 H ₂ O 0.02 CuSO ₄ .5 H _; 0 0.015 Na ₂ Mo0 ₄ .2 H ₂ O 0.012 ZnCl ₂ 0.02 _{SnCl2.2H2O ​} 0.005 h ₃ bo ₃ 0.01 KC1 0.02 HCl (concentrated) 2.0 ml/1

The medium was prepared in distilled water, the pH was adjusted to 7.0 before sterilization, the medium was divided into 220 milliliter bottles of 500 milliliters each and heat-treated in an autoclave for 15 minutes at a temperature of 121 °C.

Liquid production medium 2

The composition was identical to the liquid part of solid production medium 1. The medium was prepared in distilled water, the pH was adjusted to 7.0 before sterilization. The medium was divided into 50 milliliter portions into uncovered 250 milliliter Erlenmeyer flasks. The flasks were closed with cotton plugs and heat-treated in an autoclave at 121 °C for 15 minutes. Glucose was heat-treated in an autoclave separately from the other components.

Sordarin production by fermentation of Rosellina subiculata (MF6239,

ATCC 74386)

1. Culture:

A slant of the culture-containing agar was aseptically transferred to inoculum medium 1 (50 ml in an uncovered 250 ml flask). To obtain biomass, the mixture was incubated in a swinging conical shaker (cones diameter was approximately 5.1 cm (2 in)) for 5 days at 220 rpm, 25 °C, and 85% relative humidity. The biomass was divided, transferred to sterile vials containing glycerol, and frozen (hereafter referred to as frozen vegetative mycelium (FVM)). This mycelia, with a final glycerol concentration of 10-15%, was maintained at -75 °C. Secondary frozen vegetative mycelium (FVM) was prepared by transferring 1.0 milliliter of thawed primary frozen vegetative mycelium (FVM) to inoculation medium 2, incubating for 7 days at 25°C and 220 rpm, and then freezing as described above.

Inoculum:

The contents of the frozen ampoule (FVM) MF6239 were thawed and warmed to room temperature, used to inoculate the seed cultures in an amount of 1.0 milliliter per 50 milliliters of seed medium 2. The resulting mixture was grown for 7 days in a swinging conical shaker at a speed of 220 revolutions per minute, a temperature of 25 °C and a relative humidity of 85%.

• · « · · · · · * • ·· · · ··♦· • ······· · (

3. Production:

On a solid production medium

An aliquot (10-12 milliliters) of the inoculum was added to 220 milliliters of the liquid portion of solid production medium 1. The flask was shaken vigorously to disperse the biomass. The contents of each flask were poured into a 2-liter cylindrical culture vessel containing 675 milliliters of coarse-grained vermiculite. The contents of the culture vessel were shaken/mixed to ensure homogeneous inoculation and homogeneous coverage of the vermiculite. The culture vessel was incubated for 17 days in a horizontal position with rotation at a speed of approximately 4 revolutions per minute on a Wheaton apparatus at a temperature of 22 °C and a relative humidity of 70%.

In liquid production medium

Inoculation of the seed cultures was carried out as described above. Inoculation of 250 milliliters of production medium 1, each production flask containing 50 milliliters of liquid, was carried out with an aliquot (1.5 milliliters) of the inoculum. The flasks were incubated for 7-21 days in a swinging conical shaker at a speed of 220 rpm, a temperature of 25 °C and a relative humidity of 50-85%.

Sordarin production by fermentation of MF6232 (ATCC 74387)

1. Culture:

A slant of the agar containing MF6232 was aseptically transferred to inoculum medium 1 (50 ml in an uncovered 250 ml flask). To obtain biomass, the mixture was incubated in a swinging conical shaker for 3 days at 220 rpm, 25 °C and 85% relative humidity. The biomass was divided, transferred to sterile ampoules containing

• · · · · glycerol and frozen (hereinafter this mixture will be referred to as frozen vegetative mycelium (FVM)). This mycelia with a final glycerol concentration of 10-15% was maintained at -75 °C. Secondary frozen vegetative mycelium (FVM) was prepared by transferring 1.0 milliliter of thawed primary frozen vegetative mycelium (FVM) to inoculation medium 2, the composition of which is given below, incubating for 7 days at 25 °C and 220 rpm, and then freezing as described above.

Inoculum:

The contents of the frozen ampoule (FVM) MF6232 were used to inoculate the seed cultures after thawing and warming to room temperature in an amount of 1.0 milliliter per 50 milliliters of seed medium 2. The resulting mixture was grown for 7 days in a swinging conical shaker at a speed of 220 revolutions per minute, a temperature of 25 °C and a relative humidity of 85%.

3. Production:

On a solid production medium

An aliquot (10-12 milliliters) of the inoculum was added to 220 milliliters of the liquid portion of solid production medium 2. The flask was shaken vigorously to disperse the biomass. The contents of each flask were poured into a 2-liter cylindrical culture vessel containing 675 milliliters of coarse-grained vermiculite. The contents of the culture vessel were shaken/mixed to ensure homogeneous inoculation and homogeneous coverage of the vermiculite. The culture vessel was incubated for 17 days in a horizontal position with rotation at a speed of approximately 4 revolutions per minute on a Wheaton apparatus at a temperature of 22 °C and a relative humidity of 70%.

• · · ·

In liquid production medium

Inoculation of the seed cultures was carried out as described above. Inoculation of 250 milliliters of production medium 2, each production flask containing 50 milliliters of liquid, was carried out with an aliquot (1.5 milliliters) of the inoculum. The flasks were incubated for 7-21 days in a swinging conical shaker at a speed of 220 rpm, a temperature of 25 °C and a relative humidity of 50-85%.

Large-scale production of sordarin MF6232 (ATCC 74387)

The liquid portion of solid production medium 1 was used in both the seed fermenter and the production fermenter. Cerelose was added to both fermenters after sterilization, at a rate of 30 grams/liter in the seed fermenter and 150 grams/liter in the production fermenter. The seed fermenters were inoculated with 2 liters of the shake flask culture. The fermenters were then allowed to grow at 25°C for 30 hours until the oxygen uptake was approximately 3 millimoles/liter per hour. After 30 hours, 25 liters of the inoculum were transferred to the production fermenter.

Growth in the production fermenter reached an oxygen uptake of 8-10 millimoles/liter per hour after 50 hours and at the end of the cultivation this uptake had dropped to 5-7 millimoles/liter per hour. Oxygen dissolution was achieved by increased agitation of the mixture. The pH of the nutrient medium was not controlled and generally dropped to 5.3 after 200 hours. The process was carried out at a temperature of 25 °C.

After 280 hours of growth, the fermentation was terminated and isolation preparation began. The pH was adjusted to 12 with sodium hydroxide and the mixture was allowed to stand for 20 hours at the fermentation temperature. The pH was then adjusted to 6.0 with sulfuric acid before the mixture was transferred to cylinders for further processing.

Isolation of sordarin

Insulation I

The methyl ethyl ketone extract from the fermentation of culture MF6232 (ATCC 74387) corresponding to 64 milliliters of crude nutrient medium was evaporated to dryness in vacuo. The residue obtained, weighing 365 milligrams, was dissolved in such an amount of methanol/dichloromethane (2:98) that the volume of the solution was 4.6 milliliters. From this solution, 4.3 milliliters, i.e., a portion containing 341 milligrams of dissolved residue, were taken and applied to a flash chromatography column packed with 60 milliliters of silica gel 60 (0.040-0.0630 millimeters, 230-400 mesh, E. Merck) and equilibrated with a 2 percent methanol/dichloromethane solution. The column was eluted by step gradient elution and washed successively with 2, 5, 10 and 30 percent methanol in dichloromethane, each time using 240 milliliters of the respective solution, and finally the column was washed with 120 milliliters of methanol alone. For each system, 16 fractions of 15 milliliters were collected. By biological assay, it was determined that fractions 39-56 contained a large proportion of product.

The combined fractions were evaporated to dryness in vacuo to yield 103.1 milligrams of sample, from which a 34.4-milligram portion was taken and further purified by HPLC (a 9.4 x 250-millimeter column filled with Zorbax Rx-C8, 5 micrometer solid phase, which was • · 4 4 • · · · ·

4 4 4 4 · 4 eluted with a mobile phase consisting of 20% acetonitrile and 80% 0.01 molar aqueous K ₂ HPO ₄ solution, the pH of which was adjusted to 6.9 with concentrated H 3 PO 4 . The elution rate was set at 4 milliliters per minute at 40 °C. Detection was performed by diode array). 4 milliliter fractions were collected. Fractions 16-20, which contained a large proportion of product, were combined and concentrated in vacuo to approximately twenty-five percent of the original volume. The concentrate was extracted twice with an equal volume of ethyl acetate and the organic layers were combined, washed with an equal volume of brine, dried over anhydrous sodium sulfate and after evaporation of the solvents in vacuo, 3.7 milligrams of sordarin were obtained.

Insulation II

The methyl ethyl ketone extract from batch -004Y fermentation of culture MF6232 (ATCC 74387) corresponding to 980 milliliters of crude broth was evaporated to dryness in vacuo. The residue obtained, weighing 4.9 grams, was dissolved in such an amount of methanol/dichloromethane (1:9) that the volume of the solution was 21.5 milliliters. From this solution, 21 milliliters were taken, i.e. a portion containing 4.8 grams of dissolved residue, and this portion was applied to a chromatography column packed with 500 milliliters of silica gel 60 (0.040-0.0630 millimeters, 230400 mesh, E. Merck) and equilibrated with a 2 percent solution of methanol in dichloromethane. The column was eluted at a rate of 25 milliliters per minute by step gradient elution, first with 1 liter of a 2 percent methanol solution in dichloromethane, then with 1 liter of 5 percent methanol, and then with 2 liters of 15 percent methanol. Finally, the column was washed first with 1 liter of 30 percent methanol, and then with 2 liters of methanol alone. Twenty-five milliliter fractions were collected. By biological assay, fractions 75-85 and 111-121 contained a large proportion of product and, according to reverse-phase HPLC analysis, contained compound (I).

The combined fractions 75-85 and 111-121 were separately evaporated to dryness in vacuo to give samples weighing 69.3 milligrams and 95.3 milligrams, respectively. Two 34-milligram aliquots were taken from the sample obtained by evaporating the combined fractions 75-85 and were further purified by two identical HPLCs (a 21.2 x 250-millimetre column filled with Zorbax Rx-C8, 7 micrometre solid phase was used, which was eluted with a mobile phase consisting of 40% acetonitrile and 60% water with a total content _of 0.1% _H3PO4 . The elution rate was set at 20 millilitres per minute at 25°C. Detection was performed with a detector at a wavelength of 220 nanometres). 10 millilitre fractions were collected. Fractions 27-31 from both preparations, which contained a large proportion of the product, were combined and concentrated in vacuo to approximately forty percent of the original volume. The concentrate was extracted twice with an equal volume of ethyl acetate and the organic layers were combined, washed with an equal volume of brine, dried over anhydrous sodium sulfate and after evaporation of the solvents in vacuo, 27 milligrams of sordarin were obtained. Two 46-milligram portions were also purified under the same conditions, which were taken from the sample obtained by evaporation of the combined fractions 111-121. Fractions 25-28 from both preparations were combined and after treatment as described above, 17 milligrams of sordarin were obtained.

Preparation of sordaricin benzyl ester

To 2 milligrams of sordarin dissolved in 1 milliliter of acetone was added 0.2 milliliter of concentrated hydrochloric acid. The mixture was stirred for one day at room temperature. The solution was diluted with water, worked up aqueously (dichloromethane) and the organic portion was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The resulting mixture was dissolved in 2 milliliters of N,N-dimethylformamide (DMF) and to the resulting solution were gradually added 0.1 milliliter of benzyl bromide and an excess of solid NaHCO ₃ . The mixture was stirred overnight at room temperature and then concentrated in vacuo. Chloroform was added to the mixture and NaHCCn was filtered off. The filtrate was concentrated in vacuo and after purification by preparative thin layer chromatography (PTLC) 1.0 milligram of sordaricin benzyl ester was obtained.

¹ H NMR (CDCl ₃ ): δ 0.51 (3H, d, (1H, d, J=11.7), 5.31

7.31-7.40 (5H, m), 9.62

J=6.9), 0.82 (3H, d, J=6, 6) , 0.91 (1H, m) , 1.0 (3H, d, J=6, 6) , 1.18 (1H, d, J=12, 6), 1.50-2.00 (9H, m) , 2.24 (1H, m) , 2.51 (1H, m) , 3.48 (1H, d, J-11.0), 3.87 (1H, d, J=11.0), 5.11 (1H, d, J=11.7), 6.04 (1H, d, J=2.1), (1H, s) .

Preparation of p-methoxybenzyl ester of sordaricin

The procedure was similar to the preparation of the benzyl ester of sordaricin, except that 4-methoxybenzyl chloride was used instead of benzyl bromide. ² H NMR (CDCl3): δ 0.51 (3H, d, J=6.9), 0.82 (3H, d, J=6.9), 1.00 (3H, d, J=6.9), 0.90-2.00 (11H, m), 2.23 (1H, m), 2.49 (1H, t, J=3.8), 3.79 (3H, s),

4.61 (2H, s), 5.05 (1H, d, J=11.7), 5.26 (1H, d, J=11.7), 6.03 (1H, d, J=3.2), 6.88 (2H, d, J=8.7), 7.28 (2H, d, J=8.7), 9.60 (1H, s) .

• * • · « · * · *

Preparation of the allyl ester of sordaricin A similar procedure was followed for the benzyl ester of sordaricin, except that allyl bromide was used.

in the same way as in the preparation, except that instead of benzyl bromide,

Preparation of sordaricin

To a methanol solution of 0.6 milligrams of sordaricin benzyl ester was added Pearlman's catalyst. The mixture was stirred for 15 minutes under hydrogen pressure (pressure created by connecting a room filled with hydrogen). After filtration through cotton wool and concentration in vacuo, 0.4 milligrams of sordaricin was obtained. ¹ H NMR (CDCl ₃ ) : δ 0.82 (3H, d, J=6.8), 0.98 (3H, d, J=6.6),

1.01 (3H, d, J=6.9), 1.23 (1H, m) , 1.25 (1H, d, J=12, 6) , 1.582.10 (9H, m) , 2.34 (1H, m) , 2.41 (1H, t, J=3, 6) , 3.45 (1H, d, J=11.0), 4.14 (1H, d, J=11.0), 6.05 (1H, d, J=3.0), 9.75 (1H,

with) .

Following the present invention.

The examples of the invention are given for better illustration and do not limit the scope of this invention in any way.

Examples of embodiments of the invention

Example 1

Benzyl [IR-(Ια,3ββ,4β,4ββ,7β,7aa,8ηβ)]-8a-(hydroxymethyl)-4cyano-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl) 1,4-methano-s-indacene-3a(1H)-carboxylate

Method A

To a solution of 50 milligrams of sordarin in 3 milliliters of N,N-dimethylformamide were added 0.3 milliliters of benzyl bromide and 200 milligrams of sodium hydride (60% dispersion in mineral oil). The mixture was stirred overnight at room temperature. After aqueous workup (diethyl ether) and purification by preparative thin layer chromatography (PTLC), the benzyl ester of 2',3-'di-O-benzylsordarin was obtained.

To a solution of 1 equivalent of 2',3'-di-O-benzylsordarine benzyl ester, which was prepared as described above, in a mixture of ethanol/pyridine (2:1), an excess of hydroxylamine hydrochloride was added and the mixture was stirred for 2 hours at 70 °C. The mixture was then concentrated in vacuo and after aqueous work-up (dichloromethane) and subsequent purification by preparative thin layer chromatography (PTLC), 2',3'-di-O-benzyl4-aldoxime sordarine benzyl ester was obtained.

To a solution of 2',3'-di-O-benzyl4-aldoximesordarine benzyl ester, prepared as described above, in toluene was added an excess of the inner salt (methoxycarbonylsulfamoyl)triethylammonium hydroxide (Burgess reagent). The resulting mixture was stirred at 70 °C for 2 hours under nitrogen. After concentration in vacuo and purification by preparative thin layer chromatography (PTLC), 2',3'-di-O-benzyl-4-cyano-4-deformylsordarine benzyl ester was obtained.

A solution of 2',3'-di-O-benzyl-4-cyano-4-deformylsordarine benzyl ester in acetone was prepared and concentrated hydrochloric acid was added to this solution in an amount corresponding to 20% of the volume of the acetone solution. The mixture was stirred at room temperature for approximately one day or until analytical chromatography showed that the reaction had proceeded to a sufficient extent. Subsequently, the mixture was diluted with water and after aqueous work-up (dichloromethane), the organic fractions were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by preparative thin layer chromatography (PTLC) gave the final product.

Method B

To a solution of 161.2 milligrams of sordaricin benzyl ester in 6 milliliters of N,N-dimethylformamide was added one milliliter of p-methoxybenzyl chloride and 50 milligrams of a 60% dispersion of sodium hydride in mineral oil. The mixture was stirred overnight, diluted with ether, and carefully washed with water. The ether layer was dried over anhydrous sodium sulfate, and the volatiles were removed in vacuo. After purification of the residue by chromatography on silica gel, 192.5 milligrams (93%) of the corresponding p-methoxybenzyl ether were obtained.

150 milligrams of this ether were dissolved in 5 milliliters of dry ethanol and 3 milliliters of pyridine were added to the resulting solution. Then 96 milligrams of hydroxylamine hydrochlorides were added to the solution and the reaction mixture was heated for 3 hours at 70 °C. After cooling the mixture and concentrating in vacuo, the residue was dissolved in ether, washed with water and dried over anhydrous sodium sulfate. The residue obtained after removal of the ether in vacuo was purified by preparative thin layer chromatography (PTLC) and 143.4 milligrams (93%) of the desired aldoxime were obtained.

143 milligrams of this oxime were dissolved in 5 milliliters of toluene and 700 milligrams of the inner salt (methoxycarbonylsulfamoyl)triethylammonium hydroxide were added to the solution. The mixture was stirred for 1 hour at 70 °C. After concentration in vacuo, the residue was purified by preparative thin layer chromatography • · · · « « * · • · · · (PTLC) to give 116.6 milligrams (84%) of the desired nitrile.

67.5 milligrams of the nitrile thus obtained were dissolved in 5 milliliters of dichloromethane and 43 milligrams of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and 0.5 milliliters of water were added to the solution. The mixture was stirred for two hours at room temperature and after aqueous work-up and purification by preparative thin layer chromatography (PTLC) 47.6 milligrams (91%) of the final product were obtained.

Example 2

[1R-(1α,3αp,43,4α3,7β,7aa,8α3)]-8α-[(acetyloxy)methyl]-4-cyano-4,4α,5,6,7,7α,8,8α-octahydro-7-methyl-3-(1methyl ethyl)-1,4-methano-s-indacer.-3α (1H)-carboxylic acid

To a solution of 1.0 mg of 4-cyano-4-deformylsordaricin benzyl ester in 1 ml of pyridine and 1 ml of acetic anhydride was added a catalytic amount of 4-(N,N-dimethyl)aminopyridine (DMAP). The mixture was stirred at room temperature for approximately 60 minutes, concentrated in vacuo and purified by preparative thin layer chromatography (PTLC). The benzyl ester of the title compound was obtained, which was dissolved in 1 ml of methanol. Pearlman's catalyst was added to the solution and the mixture was stirred for 15 minutes under hydrogen pressure, which corresponded to the pressure in the inflated balloon. After filtration through cotton wool and concentration in vacuo, the final product was obtained.

• ·

Example 3

[IR-(10,3a β,4β,4a3,7β,7aa,8a3)]-8a[(undecanoyloxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8a-octahydro7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)carboxylic acid

To a tetrahydrofuran solution of 30 milligrams of undecanoic acid were added 34 microliters of triethylamine and 25 microliters of 2,4,6-trichlorobenzoyl chloride in sequence. The mixture was stirred for 15 minutes at room temperature, and then a solution of 1.0 milligrams of 4-cyano-4-deformylsordaricin benzyl ester in 1 milliliter of tetrahydrofuran (THF) and 20 milligrams of 4-(N,N-dimethyl)aminopyridine (DMAP) were added in sequence. The mixture was stirred for approximately 60 minutes at room temperature, and after purification by preparative thin layer chromatography (PTLC), the benzyl ester of the title compound was obtained. This undecanoate was dissolved in 1 milliliter of methanol. Pearlman's catalyst was added to the solution and the mixture was stirred for 15 minutes under hydrogen pressure, which corresponded to the pressure in the inflated balloon. After filtration through cotton wool and concentration in vacuo, the final product was obtained.

Example 4

[1R-(lα,3aβ,4β,4aβ,7β,7aoc,8aβ)]-8a[(propanoyloxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8a-octahydro-7methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H) carboxylic acid • · * ' « · # '· · * · · • · · · · ·»«· · * · · • »····· · · * · ^ř · · • · ·«· ·»►»· ·· · · ·· ··· ·· ··

To a solution of 0.5 milligrams of 4-cyano-4-deformylsordaricin benzyl ester in dichloromethane were added 0.2 milliliters of triethylamine and 0.1 milliliters of propionyl chloride. A catalytic amount of 4-(N,N-dimethyl)aminopyridine (DMAP) was added to the mixture and stirred for approximately 18 hours or until the reaction was complete by analytical chromatography. After purification by preparative thin layer chromatography (PTLC), the benzyl ester of the title compound was obtained, which was dissolved in 1 milliliter of methanol. Pearlman's catalyst was added to the solution and the mixture was stirred for 15 minutes under a hydrogen pressure corresponding to the pressure in the inflated balloon. After filtration through cotton wool and concentration in vacuo, the final product was obtained.

Example 5

[1R-(1a,3a3,43,4a3,7p,7aa,8a3)]-8a[(methoxycarbonyloxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8aoctahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene3a(1H)-carboxylic acid

By the same procedure as in Example 4, except that methyl chloroformate was used instead of propionyl chloride, the desired product was obtained.

Example 6

[1R-(1a,3a3,4p,4a3,7p,7aa,8a3)]-8a[(propylaminocarbonyloxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8aoctahydro-7-methyl-3-(1-methylethyl)-1, 4-methano-s-indacene3a(1H)-carboxylic acid

To a solution of 0.5 milligrams of 4-cyano-4-deformylsordaricin benzyl ester in chloroform was added 0.1 milliliters of n-propylisocyanate and a catalytic amount of 4-(N,N-dimethyl)aminopyridine (DMAP). The mixture was refluxed for 4 hours, concentrated in vacuo and purified by preparative thin layer chromatography (PTLC). The benzyl ester of the title compound was obtained, which was dissolved in 1 milliliter of methanol. Pearlman's catalyst was added to the solution and the mixture was stirred for 15 minutes under a hydrogen pressure corresponding to the pressure in the inflated balloon. After filtration through cotton wool, concentration in vacuo and purification by preparative thin layer chromatography (PTLC), the final product was obtained.

Example 7

[IR-(1α,3α3,4β,4ββ,7β,7aa,8α(3)]-8α[(methoxymethoxy)methyl]-4-cyano-4,4α,5,6,7,7α,8,8α-octahydro7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3α(1H)carboxylic acid

To a solution of 0.5 milligrams of 4-cyano-4-deformylsordaricin benzyl ester in dichloromethane were added 0.2 milliliters of diisopropylethylamine and 0.1 milliliters of methoxymethyl chloride (MOMC1), which was added at 0 °C. The mixture was stirred for approximately 18 hours at room temperature, concentrated in vacuo and purified by preparative thin layer chromatography (PTLC). The benzyl ester of the title compound was obtained, which was dissolved in methanol. Pearlman's catalyst was added to the solution and the mixture was stirred under a hydrogen pressure corresponding to the pressure in the inflated balloon until cleavage of the benzyl group was determined by thin layer chromatography (TLC). After concentration in vacuo and purification by preparative thin layer chromatography (PTLC), the final product was obtained.

Example 8

[IR-(Ia,3a3,43,4ap,73,7aa,8a3)]-8a[((methoxyethoxy)methoxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8aoctahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene3a(1H)-carboxylic acid

By the same procedure as in Example 7, except that methylethoxymethyl chloride was used instead of methoxymethyl chloride (MOMC1), the desired product was obtained.

Example 9

[IR-(Ia,3a3,43,4ap,7p,7aa,8a3)]-8a[(octyloxymethoxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8aoctahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene3a(1H)-carboxylic acid

By the same procedure as in Example 7, except that chloromethyl octyl ether was used instead of methoxymethyl chloride (MOMC1), the desired product was obtained.

• · · • · · · ·

Example 10

[IR-(1a,3a3,4β,4ββ,7β,7aa,8ββ)]-8a-[(methoxy)methyl]4-cyano-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylic acid

To the solution in milliliters 0.5 milliliters milligrams of sordaricin benzyl ester in

Methyl iodide and 100 milligrams of a 60% dispersion of sodium hydride in mineral oil were added sequentially to N,N-dimethylformamide. The mixture was stirred overnight at ambient temperature. After aqueous work-up (diethyl ether) and purification by preparative thin layer chromatography (PTLC), the corresponding ether derivative was obtained.

Milligrams of this ether were dissolved in 3 milliliters of ethanol and 1.5 milliliters of pyridine were added to the resulting solution. Then 100 milligrams of hydroxylamine hydrochloride were added to the solution and the reaction mixture was heated to 80 °C and stirred for 2 hours. After concentration in vacuo, aqueous work-up (dichloromethane) and purification by preparative thin layer chromatography (PTLC), the corresponding aldoxime was obtained.

Milligrams of this aldoxime were dissolved in 3 milliliters of toluene and the resulting solution was added with the salt (methoxycarbonylsulfamoyl). The mixture was stirred for 2 hours at 70 °C. After concentration in vacuo, the residue was purified by preparative thin layer chromatography (PTLC) to obtain the benzyl ester of the compound mentioned in the title example.

To a solution of 12 milligrams of this benzyl ester in 2 milliliters of methanol was added 20 milligrams of Pearlman's catalyst and the mixture was stirred for 15 minutes under hydrogen pressure,

200 milligrams of internal triethylammonium hydroxide.

• · » · • · ·· · « « · · · · • · · · · · · · · which corresponded to the pressure in the inflated balloon. After filtration through cotton wool and concentration in vacuo, the final product was obtained. MS (Cl): m/z=361 (M+NH ₄ ) ⁺

Example 11

[IR-(lot, 3a3, 4β, 4ββ, 7β, 7aoc, 8aP)] - 8a- [ (ethoxy) methyl] 4-cyano-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylic acid

The desired product was obtained by the same procedure as in Example 10, except that ethyl iodide was used instead of methyl iodide. MS (Cl): m/z=375 (M+NH ₄ ) ⁺

Example 12

[IR-(lot, 3a3,4β, 4a£, 7β, 7act, 8ap)]-8a[(propyloxy)methyl]-4-cyano-4, 4a, 5, 6, 7, 7a, 8, 8a-octahydro-7methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)carboxylic acid

The desired product was obtained by the same procedure as in Example 10, except that allyl bromide was used instead of methyl iodide. MS (Cl): m/z=389 (M+NH ₄ ) ⁺

Example 13

[IR-(Ια, 33β, 4β, 4a3,7β ,7act, 8a3)]-8a-[(2methylpropyloxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8a-octahydro7-methyl-3-(1-methylethyl)-1, 4-methano-s-indacene-3a(1H)carboxylic acid » * · · * « z o ·····*··· · o z ·«······ ··· • · · · · · · • · ·· « · · * · · ·

The desired product was obtained by the same procedure as in Example 10, except that 2-bromomethyl-1-propene was used instead of methyl iodide. MS (CI): m/z=403 (M+NH ₄ ) ⁺

Example 14

[IR-(Ια,3a3,4β,4a3,7β,7aa,8a3)]-8a[(butyloxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8a-octahydro-7methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)carboxylic acid

The desired product was obtained by the same procedure as in Example 10, except that butyl iodide was used instead of methyl iodide. MS (CI): m/z=403 (M+NH ₄ ) ⁺

Example 15

[IR-(1α,3α3,4β,4αβ,7β,7aa, 8α3)]-8α[(pentyloxy)methyl]-4-cyano-4,4α,5,6,7,7α,8,8α-octahydro-7methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3α(1H)carboxylic acid

The desired product was obtained by the same procedure as in Example 10, except that pentyl iodide was used instead of methyl iodide. MS (CI): m/z=417 (M+NH ₄ ) ⁺

Example 16

[1R-(1a,3a3,43,4a3,7p,7aa,8a3)]-8a[(hexyloxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8a-octahydro-763 • « · methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a (1H) carboxylic acid

The desired product was obtained by the same procedure as in Example 10, except that hexyl iodide was used instead of methyl iodide. MS (CI): m/z=431 (M+NHJ’

Example 17

[IR-(Ia,3a(3,43,4a3,7p,7aa,8a3)]-8a-[(S-2hydroxypropyloxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8a-octahydro7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a (1H)carboxylic acid

By the same procedure as in Example 10, except that (R)-glycidol benzenesulfonate was used instead of methyl iodide, the desired product was obtained.

Example 18

[IR-(10,3a3,4β,4aβ,7β,7a«,83β)]-8a-[(R-2hydroxypropyloxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8a-octahydro7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)carboxylic acid

By the same procedure as in Example 10, except that (S)-glycidol benzenesulfonate was used instead of methyl iodide, the desired product was obtained.

• 0 » · * · · · · · • · * · * · · • « · · · · í * · • · · » · ‘ »

Example 19

[1R-(1α,3α3,43,4α3,7β,7aa,8α3)]-8α-[(nheptyloxy)methyl]-4-cyano-4,4α,5,6,7,7α,8,8α-octahydro-7methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3α(1H)carboxylic acid

By the same procedure as in Example 10, except that hept-2-yn-1-ol benzenesulfonate was used instead of methyl iodide, the desired product was obtained.

Example 20

[1R-(1α,3αβ,43,4α3,73,7αα,8α)3)]-8α-[(noctyloxy)methyl]-4-cyano-4,4α,5,6,7,7α,8,8α-octahydro-7methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3α(1H)carboxylic acid

By the same procedure as in Example 10, except that oct-2-yn-1-ol benzenesulfonate was used instead of methyl iodide, the desired product was obtained.

Example 21

[1R-(lc<,3a3,43,4a3,73,7aa,8ap)]-8a-[(nnonyloxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8a-octahydro-7methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)carboxylic acid _e · · » · » · · · · « » · · * • « « · · · · · • ····« » ··· « · • · · * V « · · ·

By the same procedure as in Example 10, except that non-2-yn-1-ol benzenesulfonate was used instead of methyl iodide, the desired product was obtained.

Example 22

[1R-(1α,3αp,4β,4α3,7α,7aa,8α3)]-8α-[(ndecyloxy)methyl]-4-cyano-4,4α,5,6,7,7α,8,8α-octahydro-7methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3α(1H)carboxylic acid

By the same procedure as in Example 10, except that dec-2-yn-1-ol benzenesulfonate was used instead of methyl iodide, the desired product was obtained.

Example 23

[1R-(1a,3a3,43,4ap,73,7aa,8aP)]-8a-[(3-methylbut-loxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylic acid

The desired product was obtained by the same procedure as in Example 10, except that 1-iodo-3-methylbutane was used instead of methyl iodide. MS (CI): m/z=417 (M+NH ₄ ) ⁺ .

Example 24

[IR-(1α,3αp,43,4αp,7β,7αa,8αp) ]-8α-[(2propoxy)methyl]-4-cyano-4, 4α, 5,6,7,7α, 8, 8α-octahydro-7-methyl3-(1-methylethyl)-1,4-methano-s-indacene-3α(IH)-carboxylic acid • 9 ··· • * * · * · ·· · > * · 9 9 ·

S « · · · ι * » 9 · • · · · · * ·· ·· *··

By the same procedure as in Example 10, except that 2-iodopropane was used instead of methyl iodide, the desired product was obtained.

Example 25

[1R-(lc<,3a(3,4p,4a3,7p,7aa,8a8)]-8a-[2(tetrahydropyranyloxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8aoctahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene3a(1H)-carboxylic acid

To a solution of 10 milligrams of 4-cyano-4-deformylsordaricin benzyl ester in 3 milliliters of dichloromethane was added 50 microliters of 3,4-dihydro-2H-pyran and a catalytic amount of pyridinium-p-toluenesulfonate (PPTS). The mixture was stirred overnight at room temperature and then 1 milliliter of triethylamine was added. After concentration in vacuo and purification by preparative thin layer chromatography (PTLC), the benzyl ester of the title compound was obtained, which was dissolved in methanol.

Pearlman's catalyst was added to the solution and the mixture was stirred for 15 minutes under hydrogen pressure, which corresponded to the pressure in the inflated balloon. The mixture was filtered through cotton wool, concentrated in vacuo, purified by preparative thin layer chromatography (PTLC) to obtain the final product.

Example 26

[IR-(lot, 3ap, 4β, 4ββ, 7β, 7aa, 8ββ)] -8a- [ (1ethoxyethoxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8a-octahydro-76Ί methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)carboxylic acid

By the same procedure as in Example 25, except that ethyl vinyl ether was used instead of 3,4-dihydro-2H-pyran, the desired product was obtained.

Example 27 p-Methoxybenzyl [IR-(1a,3a3,4β,4πβ,7β,7aa,8a3)]-8a(hydroxymethyl)-4-cyano-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylate

By the same procedure as in Example 1, except that p-methoxybenzyl chloride was used instead of benzyl bromide, the desired product was obtained.

Example 28

[1Κ-(1α,33β,4β,4ηβ,7β,7aa,83β)]-8a[(benzyloxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8a-octahydro-7methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)carboxylic acid

To a solution of deformylsordaricin p-methoxybenzyl ester in N,N-dimethylformamide was added excess benzyl bromide and sodium hydride. The mixture was stirred overnight at room temperature and after aqueous work-up (ether) and purification by preparative thin layer chromatography (PTLC) the following was obtained:

4- methoxybenzyl ester of the compound mentioned in the title of the example. This ester was subsequently dissolved in excess acid ·· • 0 * • · * • 0 00 • · ·· ·· ·· ··

€0 · ·

0 0

000 formic acid and the resulting mixture was stirred for approximately three hours at room temperature. After concentration in vacuo and purification by preparative thin layer chromatography (PTLC), the desired product was obtained.

Example 29

[1R-(lo!,3a3,43,4aP,73,7aoc,8aš)]-8a-[(4bromobenzyloxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8a-octahydro-7methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)carboxylic acid

By the same procedure as in Example 28, except that 4-bromobenzyl bromide was used instead of benzyl bromide, the desired product was obtained.

Example 30

[IR-(Ια, 3ββ,.4β, 4ββ, 7β, 7aa, 83β)] - 8a- [ (l-but-2enyloxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylic acid

By the same procedure as in Example 28, except that crotyl chloride was used instead of benzyl bromide, the desired product was obtained.

Example 31

[1Κ-(1α,3Ηβ,4β,4ββ,7β,7βα,83β)]-83-[(1-pent-2enyloxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylic acid

By the same procedure as in Example 28, except that trans-1-bromo-2-pentene was used instead of benzyl bromide, the desired product was obtained.

Example 32

[1R-(1α,3α3,43,4αβ,7β,7αα,8α3)]-8α[(isobutenyloxy)methyl]-4-cyano-4, 4α, 5,6,7,7α, 8,8α-octahydro7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3α(1H)carboxylic acid

By the same procedure as in Example 28, except that 2-bromomethylpropene was used instead of benzyl bromide, the desired product was obtained.

Examples 33-51

Using the same procedure as in Example 4, the following esters could be prepared:

Acylating agent

The meaning of R in the product

CH ₃ (CH ₂ ) ₂ COC1 CH.3 (CHJ ₃ COC1 CH ₃ (CHJ ₄ COC1 CH ₃ (CH ₂ ) 5COCI

CH ₃ (CHJ ₂ C0 CH ₃ (CHJ ₃ CO CH ₃ (CH ₂ ) ₄ C0 CH ₃ (CHJ ₅ C0

CH ₃ (CH ₂ ) ₆ COC1 CH ₃ (CH ₂ ) 7COC1 CH ₃ (CH ₂ ) gCOCl (CH ₃ ) 3COCI (CH ₃ ) ₂ chcoci c ₆ h ₅ coci m-CH ₃ C ₆ H ₄ COCl o-FC _s H ₄ COC1 c ₆ h ₅ ch ₂ coci C _s H ₅ (CH ₂ ) 2 COCI

1- Naphthoyl chloride

2- naphthoyl chloride or cotinoyl chloride

2-pyrazinecarbonyl chloride

2-furoyl chloride

CH ₃ (CH ₂ ) ₅ COCH ₃ (CH ₂ ) 7COCH ₃ (CH ₂ ) ₈ CO(CH ₃ ) 3CO(CH ₃ ) ₂ chcoc ₆ h ₅ com-CH ₃ C _e H ₄ COo-FC ₆ H ₄ COc ₆ h ₅ ch ₂ coc ₆ h ₅ (ch ₂ ) ₂ co1-naphthoyl

2- naphthoyl nicotinoyl

2-pyrazinecarbonyl

2-furoyl

Examples 52-55

To a solution of 0.5 milligrams of 4-cyano-4-deformylsordarine p-methoxybenzyl ester in dichloromethane were added 0.2 milliliters of triethylamine, 10-100 milligrams of the appropriate acid chloride, and a catalytic amount of 4-(N,Ndimethyl)aminopyridine (DMAP). The mixture was stirred overnight at room temperature. After purification by preparative thin layer chromatography (PTLC), the p-methoxybenzyl esters of the products listed below were obtained. The ester obtained was always dissolved in excess formic acid and the mixture was stirred at room temperature for 3 hours or until the protecting group was cleaved. After concentration in vacuo and purification by preparative thin layer chromatography (PTLC), the following products were obtained:

• · • · • · every ₂ hours

Acylating agent

CH ₂ =CHCOC1 CH ₂ =CH(CH ₂ ) ₂ COC1 (CH ₃ ) ₂ c=chcoci p-ClC ₆ H ₄ COCl • · · · • · · · · • · · • · · ·

The meaning of R in the product ch ₂ =chcoch ₂ =ch (CH ₂ ) ₂ co(CH ₃ ) ₂ c=chcoP-C1C ₆ H ₄ COxamples 56-68

Using the same procedure as in Example 3, the following esters could be prepared:

Acylating agent

Meaning of R in the product cyclohexanecarboxylic acid cyclopentanecarboxylic acid cyclobutanecarboxylic acid p-CH ₃ C ₆ H ₄ COOH m-FC ₆ H ₄ COOH 4-biphenylcarboxylic acid cyclohexanecarbonyl cyclopentanecarbonyl cyclobutanecarbonyl p-CH ₃ C ₆ H ₄ COm-FC ₆ H ₄ CO4-biphenylcarbonyl acid 3-biphenylcarboxylic acid 4,4'terphenylcarboxylic acid 9-anthracenecarboxylic acid 2-pyrrolecarboxylic acid (CH ₃ ) ₂ CH (CH ₂ ) ₂ COOH HCOOH 2-thiophenecarboxylic acid

3-biphenylcarbonyl!

4,4'-terphenylcarbonyl 9-anthracenecarbonyl 2-pyrrolecarbonyl (CH ₃ ) ₂ CH (CH ₂ ) ₂ C0HCO2-thiophenecarbonyl

Examples 69-71

To each 0.16 millimol of 34 microliters of tetrahydrofuran solution containing the acids listed below were added triethylamine and 25 microliters of 2,4,6-trichlorobenzoyl chloride in sequence. The mixture was stirred for 15 minutes at room temperature, and then 1.0 milligram of sordarin p-methoxybenzyl ester dissolved in 1 milliliter of tetrahydrofuran (THF) and 20 milligrams of 4-(N,N-dimethyl)aminopyridine (DMAP) were added. The mixture was stirred for approximately one hour at room temperature. After purification by preparative thin layer chromatography (PTLC), the p-methoxybenzyl esters of the products listed below were obtained. The ester obtained was always dissolved in excess formic acid and the mixture was stirred at room temperature for 3 hours or until the protecting group was cleaved. After concentration in vacuo and purification by preparative thin layer chromatography (PTLC), the following products were obtained:

* *

Acylating agent

Meaning of R in the product o-C1C ₅ H ₄ COOH 3,5-dibromobenzoic acid cyclopropanecarboxylic acid o-C1C ₆ H ₄ CO3,5-dibromobenzoyl cyclopropanecarbonyl

Examples 72-82

Analogously to the procedure given in Example 5, the following carbonates were prepared:

were

what ₂ H Λ / RO 1 CN Ti ' 1 Me VP Acylating agent The meaning of R in a product ethyl chloroformate ethoxycarbonyl phenylchloroformate phenoxycarbonyl tert-butyl chloroformate tert-butoxycarbonyl cyclopropyl chloroformate cyclopropoxycarbonyl n-butyl chloroformate n-butoxycarbonyl (CH ₃ ) ₂ chch ₂ ococi (CH ₃ ) 2CHCH2OCO- sec-butyl chloroformate sec-butoxycarbonyl isopropyl chloroformate isopropoxycarbonyl _C6H5 ( _{CH2 ) 2OCOC1} C ₆ H ₅ (CH ₂ ) ₂ 0C0- n-propyl chloroformate n-propoxycarbonyl cyclohexyl chloroformate cyclohexylcarbonyl

• · • ·

Examples 83-89

To a dichloromethane solution of 0.5 milligrams of 4-cyano-4-deformylsordarine p-methoxybenzyl ester was gradually added

0.2 milliliters of aminopyridine triethylamine acyl chloride, 10-100 milligrams of the appropriate and a catalytic amount of 4-(N, N-dimethyl)(DMAP). The mixture was stirred for approximately 18 hours at room temperature. After purification by preparative thin layer chromatography (PTLC), the p-methoxybenzyl esters of the products listed below were obtained. The ester obtained was always dissolved in excess formic acid and the mixture was stirred at room temperature for 3 hours or until the protecting group was cleaved. After concentration in vacuo and purification by preparative chromatography, the following products were obtained:

on a thin layer of 'PTLC' were

Acylating agent

Meaning of R in the product benzyl chloroformate p-methylbenzyl chloroformate

1- Naphthyl chloroformate

2- naphthyl chloroformate allyl chloroformate crotyl chloroformate

CH?=CH (CH ₂ ) ₂ OCOC1 benzyloxycarbonyl p-methylbenzyloxycarbonyl

1- naphthyloxycarbonyl

2- naphthyloxycarbonyl allyloxycarbonyl crotyloxycarbonyl

CH?=CH (CH?) ₂ 0C0• ·

Examples 90-104

Analogously to the procedure given in Example 6, the following carbonates could be prepared:

Acylating agent

Meaning of R in the product methyl isocyanate phenyl isocyanate tert-butyl isocyanate cyclobutyl isocyanate (CH ₃ ) ₂ chch ₂ nco p-CH ₃ C ₆ H ₄ NCO isopropyl isocyanate cyclopentyl isocyanate 1-naphthyl isocyanate dimethylaminocarbonyl chloride N-methyl-N-butylaminocarbonyl chloride N-ethyl-Nbenzylaminocarbonyl chloride N-phenyl-N-2naphthylaminocarbonyl chloride N-methyl-Nphenylaminocarbonyl chloride methylaminocarbonyl anilinocarbonyl tert-butylaminocarbonyl cyclobutylaminocarbonyl (CH ₃ ) ₂ CHCH ₂ NHCOp-CH ₃ C ₆ H ₄ NHCOisopropylaminocarbonyl cyclopentylaminocarbonyl 1-naphthylaminocarbonyl dimethylaminocarbonyl

N-methyl-N-butylaminocarbonyl ethylaminocarbonyl

N-phenyl-N-2-naphthylaminocarbonyl

N-methyl-N-phenylaminocarbonyl • · ·

N-methyl-N-cyclopropylcarbonyl chloride N-methyl-N-cyclopropylcarbonyl

Examples 105-108

To a chloroform solution of 0.5 milligrams of p-methoxybenzyl ester of 4-cyano-4-deformylsordarine were added 10100 milligrams of the appropriate isocyanate and a catalytic amount of 4-(N,N-dimethyl)aminopyridine (DMAP). The mixture was refluxed for 4 hours or until the reaction was complete. After concentration in vacuo and purification by preparative thin layer chromatography (PTLC), the p-methoxybenzyl esters of the products listed below were obtained. The ester obtained was always dissolved in excess formic acid and the mixture was stirred at room temperature for 3 hours or until the protecting group was cleaved. After concentration in vacuo and purification by preparative thin layer chromatography (PTLC), the following products were obtained:

Acylating agent

Meaning of R in the product benzyl isocyanate allyl isocyanate o-iodphenyl isocyanate dibenzylaminocarbonyl chloride benzylaminocarbonyl allylaminocarbonyl o-iodophenylaminocarbonyl dibenzylaminocarbonyl • · • · « · · · · · · · · • · ···«· · ·

Examples 109-114

Analogously to the procedure given in Example 28, the following compounds could be prepared:

Alkylating agent 1-bromo-2-propyne 1-iodo-5-hexyne allyl bromide cis-1-bromo-2-butene cis-1-bromo-2-pentene cis-1-trimethylsiloxy-4-bromo-2-butene

Meaning of R in the product hc=cch ₂ hc^cch ₂ ch ₂ ch ₂ ch ₂ h ₂ c=chch ₂ cÍs-CH ₃ CH=CHCH ₂ cís-CH ₃ CH ₂ CH=CHCH ₂ cis-4-hydroxy-2-butenyl

Example 115

[IR-(1α,3αp,4β,4αp,7β,7aa,8αp)]-8α-[(nheptyloxy)methyl]-4-cyano-4,4α,5,6,7,7α,8,8α-octahydro-7methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3α(1H)carboxylic acid

The desired product was prepared by a similar procedure as in Example 10 using 1-benzenesulfonyloxy-2-heptine instead of methyl iodide. MS (Cl): m/z 445 (M+NH ₄ ) ⁺ • · ·· · · ··· · ·· ·· ·· • ··* ♦ · · · • · ·«· · · · · · · · · · · ·· · ·«· » · · • · · · · · · · · · · * · ·

Example 116

[1R-(1a,3aP,43,4aP,7p,7aoc _/ 8a3)]-8a-[(noctyloxy)methyl]-4-cyano-4,4a,5,6,7,7a,8,8a-octahydro-7methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)carboxylic acid

The desired product was prepared by a similar procedure to Example 10 using 1-benzenesulfonyloxy-2-octyne instead of methyl iodide. MS (Cl): m/z 459 (M+NH ₄ U

Example 117

[1R-(1a,3a3,43,4a3,7p,7aa,8a3)]-8a-[(nonyloxy)methyl]-4-cyano-4,4a,5,6,3,3a,8,8a-octahydro-7methyl-3-(1-methylethyl)-1,4-methane-s-indacene-3a(1H)carboxylic acid

By a similar procedure as in Example 10 using 1-benzenesulfonyloxy-2-nonine instead of methyl iodide, the desired product was prepared. MS (Cl): m/z 473 (M+NHJ*

Example 118

[IR-(1α,3α3,4β,4ββ,7β,7aa,8ββ)]-8α-(hydroxymethyl)-4cyano-4,4α,5,6,7,7α,8,8α-octahydro-7-methyl-3-(1-methylethyl)1,4-methane-s-indacene-3α(1H)-carboxylic acid

The benzyl ester of 4-cyano-4-deformylsordaricin prepared in Example 1 (Method B) was dissolved in 2 milliliters of methanol and to this solution was added approximately • · • · milligrams of palladium hydroxide on carbon (catalyst). The mixture was heated under an intense hydrogen atmosphere for 15 minutes, filtered through cotton wool and concentrated in vacuo.

5.0 milligrams (Pearlman's stirring in the filtrate was (94) of the desired product were obtained. MS (ESI): m/z 347 (M+NH ₄ )

Claims (16)

1. Compound PATENT CLAIMS of general formula (I): where R is (a) group C(=O) OR ¹ , (b) group C(=0) NR ² R ³ , (c) group C(=0) (d) group CH(R ^; ) OR ⁵ , (e) group C (R ⁶ ) (Rd (R ⁸ (f) (g) a hydrogen atom group; û)n or R ¹ is (a) an alkyl group containing 1 to 14 carbon atoms, (b) an alkenyl group containing 2 to 14 carbon atoms, (c) an alkynyl group containing 2 to 14 carbon atoms, (d) a cycloalkyl group containing 3 to 20 carbon atoms, (e) an aryl group or • · • · · · · · · · · · · · · · · * · ····· · (f) an aryl-substituted alkyl group containing 1 to 6 carbon atoms; R ² and R' ^: are independently (a) hydrogen or (b) R ¹ ; is (a) a hydrogen atom, (b) a group R ¹ or (d) a group - (CH ₂ ) _m NR ² R ³ ; R ⁵ is (a) a group R ¹ or (j) a group - (CH ₂ ) _x 0 (CH ₂ ) _y H; R ^s is (a) hydrogen, (b) an alkyl group containing 1 to 14 carbon atoms, (c) an aryl group, (d) an aryl-substituted alkyl group containing 1 to 6 carbon atoms, (e) a group - (CH ₂ ) _y CHR ⁹ (CH?) _Z H, (f) a group - (CH ₂ ) _y C=c (CH ₂ ) _Z H, (g) a group - (CH ₂ ) _Y C (R ⁷ ) =CH (CH ₂ ) _Z H, (h) a group - (CH?) _y C=C (CH ₂ ) _m R% (i) a group - (CH?) _y C (R ⁷ ) =CH (CH?) _m R ⁹ ; R ⁷ and R ² are independently (a) hydrogen or (b) an alkyl group containing 1 to 14 carbon atoms; 4 4 4 4 4 4 4 4 ···· 4 ♦ * 4 4 4 4 • * · 4 4 4 4 4 4 « β · · • 4 4 4 4 4« · ··« »· · • · 4 4 4 · 444 44 44 R is (a) (b) hydroxyl group or group NR ² R ^: ; ^R10 is (a) hydrogen atom, (b) group -CH ₂ CsH ₅ , (c) group -CH ₂ CH=CH ₂ , (d) group (e) group (f) group (g) group (h) group 0 or 1; 1 to 6; 2 to 6; 0 to 6; 0 to 6; or from a pharmaceutical or salt of these compounds. agriculturally acceptable • « · · · · • · « • · · · · • · · · 2. A compound according to claim 1 of general formula (Ia) wherein / l> . RO R is (a) group C (=0) OR ¹ , (b) group C (=O)NR ² R ³ , (c) group C(=O)R ⁴ , (d) group ch ₂ or ⁵ , (e) group C(R ⁶ ) (R ⁷ ) (R ^s ;f) the group š'' O ^{5 n} ; or pharmaceutically acceptable salts of these compounds. 3. A compound according to claim 2, wherein R is a group C(=O)OR ¹ . 4. A compound according to claim 2, wherein R is a group C(=O)NR ² R ³ ; or a pharmaceutically acceptable salt thereof. 5. A compound according to claim 2, wherein R is C(=O)R ⁴ ; or a pharmaceutically acceptable salt thereof. * « • ·····* · ··· « • · · · · · · · · · · · · 6. A compound according to claim 2, wherein R is CH(R ² )OR ⁵ ; or a pharmaceutically acceptable salt thereof. 7. A compound according to claim 2, wherein R is C( ^R5 )( ^R7 )( ^Ra ); or a pharmaceutically acceptable salt thereof. 8. A compound according to claim 2, wherein R is a group O^ ⁿ ; or pharmaceutically acceptable salts of these compounds. 9. Compound according to claim 2, wherein R is group CH(R ⁶ ) (R ⁷ ), ^R6 is (a) hydrogen atom, (b) alkyl group containing 1 to 14 atoms carbon, (c) aryl group (d) aryl-substituted alkyl a group containing 1 to 6 carbon atoms, (e) group - (CH ₂ ) _y CHR ⁹ (CH ₂ ) _Z H, (f) group - (CH?) _y C (R ⁷ ) =CH (CH ₂ ) _Z H, R ⁷ is a hydrogen atom or an alkyl group containing 1 to 6 carbon atoms. R ⁹ is a hydroxyl group; or pharmaceutically acceptable salts thereof. • β • · · · 10. The compound of claim 2, wherein R is (a) methyl . group, (b) ethyl group, (c) group - •ch ₂ ch ₂ ch ₃ , (d) group - CH2CH2CH2CH3, (e) group - _{CH2CH2CH2CH3 ,} (f) group - •CHoCHsCHzCHsCÍRCHs, (g) group - •CH2CH2CH2CH2CH2CH2CH3, (h) group - CH2CH2CH2CH2CH2CH2CH2CH3, (i) group - CHzCHzCHzCHzCHzCHzCHzCHzCHsCHs, from a pharmaceutical compound. 11. Compounds; R is (j) group -CH ₂ CH ₂ CH (CH ₃ ) 2, (to) group -CH ₂ C ₆ H5, (1) group -CH(CH ₃ ) 2/ (m) group -CH ₂ CH (CH ₃ ) ₂ , (n) group -CH ₂ CH=CH ₂ , (0) group -CH ₂ CH=CHCH ₃ , (P) group -CH ₂ CH=CHCH ₂ CH ₃ , (q) the group -CH ₂ CH=CHCH ₂ CH ₂ CH ₃ ; or it acceptable viewpoints salt according to claim 2, wherein (a) methyl group, (b) ethyl group, (c) group -CH ₂ CH ₂ CH ₃ , (d) group -CH ₂ CH ₂ CH ₂ CH ₃ , (e) group -CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ , (f) group -CH ₂ CH (CH ₃ ) ₂ , (g) group -CH ₂ CH ₂ CH (CH ₃ ) 2, of these group -CH ₂ CH=CHCH3, group -CH ₂ CH=CHCH ₂ CH ₃ , group -CH ₂ CH=CHCH ₂ CH ₂ CH ₃ ; or aspects of acceptable salts • · · · ·« « · · · · · e • · · · · · , « · » • · « · · »··· fe ··.- * · • ······ · · · * ·. i « • · · t · · · · » · M · · · · · · ♦ · · · · (h) (i) (j) from a pharmaceutical compound. these 12. A compound according to claim 1, wherein R is hydrogen, or a pharmaceutically acceptable salt thereof. to 13. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable carrier. and » 14. An agrochemical composition comprising a compound according to claim 1 and an agrochemically acceptable carrier. * 15. A method of treating or preventing fungal infections in animals, comprising administering to said animals a fungicidally effective amount of a compound according to claim 1. 16. A method of controlling phytopathogenic fungal infections, comprising treating plants in need of such control with a fungicidally effective amount of a compound according to claim 1.