RU2107687C1 - Derivatives of markfortine/paragerkwamide, method for suppressing of pests, insect and fungi nematodes - Google Patents

Abstract

FIELD: veterinary; agriculture. SUBSTANCE: synthetic derivatives having formula

Description

 Marcfortins are known compounds and are described by Polonsky et al. B. Journal of the Cemical Society Chemical Communications, 1980, 601-602 (Markfotin A) and Tetrahedron Letters, 1981, 22 1977-1980 (Markfotins B and C). The compounds are fungal metabolites of Penicillium rogneforti. Macrofortins are structurally related to paraherquamides, which are also known paragerquamide compounds disclosed by Yamazaki at al Tefraheolron Letters 1981 22 135-136, and Blanchflower et.al. Journal of Antibiotirs, 1991, 44, 492-497. US patent 4,866,060 and 4,923,867 describe the use of marcfortins A, B and C and several derivatives thereof as useful in the treatment and prevention of diseases caused by parasites in animals.

Макркфотрин A имеет следующую структуру:

Маркфортин B имеет следующую структуру:

Маркфортин C имеет следующую структуру:

WO 91/09961 (опубл. 11 июня 1991 г.) описывает различные производные маркфортина и парагерквамида и их (12) окисей также как и получение VW 29919 (парагерквамид) и VM 55596 (N(12) окись парагерквамида) (помимо прочего) из Penicillinm Sp. IMI 332995.Paragerquamide has the following structure:

Macrkhotrin A has the following structure:

Markfortin B has the following structure:

Markfortin C has the following structure:

WO 91/09961 (published June 11, 1991) describes various derivatives of marcfortine and paraherquamide and their (12) oxides as well as the preparation of VW 29919 (paraherquamide) and VM 55596 (N (12) paraherquamide oxide) (among other things) from Penicillinm Sp. IMI 332995.

 US Pat. No. 4,873,247 describes paraherkamide derivatives and Penicillinm charlessi strain MF 5123 (ATCC 20841) to produce paraherquamide. US 4,978,656 (as well as EP 390532-A, EP-301742-A) describes various synthetic derivatives, as well as the preparation of paraherquamide from Penicilli um charlelli MF 5123 (ATCC 20841).

 Paraherquamide is a compound produced under certain conditions by the fungal organism Penicillium paraherguei. WO 92/00300 (published January 9, 1992) discloses the syntheses of paraherquamide compounds derived from (produced) Penicillium paraherguei deposited at C. A. B. International Mycological Institute, Ferry Lane, Kew, London under the number CMI 68220 .

 This invention relates to the syntheses of C-18 thiomarcfortins A, B and C and their derivatives, C-18 thioparagerquamide and its derivatives, new N - I marcfortins, A, B and C and their derivatives, the new N - I paraherquamide and its derivatives, and the use of these compounds as anti-parasitic agents.

 The purpose of the invention is to describe these derivatives of thiomarkfortin, thioparaherquamide, marcfortine and paraherquamide.

 In addition, the purpose of this invention is to describe a method for producing these compounds.

 A further objective is to describe the use of these compounds as antiparasitic agents in the treatment and prevention of diseases caused by parasites.

 In addition, a further object is to describe compositions for treating diseases caused by parasites, containing the novel compounds of the invention as their active ingredient.

где
m - 0 или 1;
n = 1;
W = 5 или 0,
R_14a или R_14b - атом водорода;
R₂₄ и R₂₅ образуют двойную связь;
R_18a - атом водорода или C₁-C₇ - алкил;
R₁ - атом водорода;
-C(O)R¹ группа (где R¹ - цикло C₃-C₈ - алкил, феноксикарбонил, -C(O)-C₂-C₂₄-алкил, нитрофеноксикарбонил, 1,3-диоксо-2H-изоиндолил, динитробензолсульфенил, трихлорметилсульфенил, C₁-C₄-алкоксикарбонилсульфенил, бензолсульфонил, тетрагидрофуранил, 1,4-диокси-C₂-C₇-алкенил
или R₁ - группа -C(O)-NR₄R₅, где R₄ и R₅ вместе с атомом азота образуют насыщенное гетероциклическое кольцо: пирролидинил, морфолинил, пиперазинил, незамещенный или замещенных C₁-C₄-алкилом, C₁-C₄-алканоилом, C₁C₄-алкоксикарбонилом, 1,3- бензодиоксолил -C₁-C₄-алкилом, пиридинилом, галогенпиридинилом, фенилом (незамещенным или замещенным атомом галогена), хлоркарбонилом, трифтор C₁-C₄-алкоксикарбонилом, трифтор C₁-C₄-алкилтиадиазолинилом, или R₄ и R₅ образуют пиперидинил, незамещенный или замещенный фенилом или моно- или ди-замещенный C₁-C₄-алкилом,
или их фармацевтически приемлемые соли, или их гидраты.The compounds of this invention are represented by formula I:

Where
m is 0 or 1;
n is 1;
W = 5 or 0,
R _14a or R _14b is a hydrogen atom;
R ₂₄ and R ₂₅ form a double bond;
R _18a is a hydrogen atom or C ₁ -C ₇ is alkyl;
R ₁ is a hydrogen atom;
-C (O) R ¹ group (where R ¹ is cyclo C ₃ -C ₈ - alkyl, phenoxycarbonyl, -C (O) -C ₂ -C ₂₄ -alkyl, nitrophenoxycarbonyl, 1,3-dioxo-2H-isoindolyl, dinitrobenzenesulfenyl, trichloromethylsulphenyl, C ₁ -C ₄ alkoxycarbonylsulphenyl, benzenesulfonyl, tetrahydrofuranyl, 1,4-dioxo-C ₂ -C ₇ alkenyl
or R ₁ is a —C (O) —NR ₄ R ₅ group, where R ₄ and R ₅ together with a nitrogen atom form a saturated heterocyclic ring: pyrrolidinyl, morpholinyl, piperazinyl, unsubstituted or substituted with C ₁ -C ₄ alkyl, C ₁ -C ₄ -alkanoyl, C ₁ C ₄ -alkoxycarbonyl, 1,3-benzodioxolyl-C ₁ -C ₄ -alkyl, pyridinyl, halogen-pyridinyl, phenyl (unsubstituted or substituted by a halogen atom), chlorocarbonyl, C ₁ -C ₄ -alkoxycarbonyl trifluoro , trifluoro C ₁ -C ₄ -alkylthiadiazolinyl, or R ₄ and R ₅ form piperidinyl unsubstituted or substituted by phenyl or mono- or di-substituted by C ₁ -C ₄ -alkyl,
or their pharmaceutically acceptable salts, or their hydrates.

или N-1-маркфтортин IV

где заместитель имеют те же значения, что и для формулы I.Another aspect of the present invention is 18-thiomarkifluorin or derivatives of formula II

or N-1-marfluorotin IV

where the Deputy have the same meanings as for formula I.

, а

- насыщенный гетероцикл.Compounds I are preferred where R ₁ is

, a

- saturated heterocycle.

, являются
4-морфолин,
4-фенил-1-пиперазин,
4-(2-пиридинил)-1-пиперазин,
2,6-диметил-4-морфолин,
1-пирролидин,
4-метил-1-пиперазин,
1-пиперидин,
4-фенил-1-пиперидин,
2-метилпиперидин,
3-метилпиперидин,
4-метилпиперидин,
N-метилпиперазин,
1-метилгомопиперазин,
1-ацетилпиперазин
Предпочтительными соединениями изобретения являются
18-тиомаркфортин A (соединение 2),
18-тиомаркфортин B (соединение 2A),
1-пирролидинил-маркфортин A (соединение 12),
1-циклопропилкарбонил-маркфортин A (соединение 12A),
1-феноксикарбонил-маркфортин A (соединение 12B),
1-пальмитоил-маркфортин A (соединение 12C),
1-(4-нитрофеноксикарбонил)-маркфортин A (соединение 12D),
1-(1-пиперидинкарбонил)-маркфортин A (соединение 12F),
1-[[4-(этоксикарбонил)пиперазин-1-ил] карбонил]-маркфортин A (соединение 12G),
1-[[4-(бензил)пиперазин-1-ил]карбонил]-маркфортин A (соединение 12H),
1-[[4-(1,3-бензодиоксол-5-илметил)пиперазин-1-ил] карбонил]- -маркфортин A (соединение 12I),
1-[[4-(метил)пиперазин-1-ил]карбонил]-маркфортин A (соединение 12J),
1-(1,3-дигидро-1,3-диоксо-2H-изоиндол-2-ил)-маркфтортин A (соединение 12K),
1-[[4-(пиридин-2-ил)пиперазин-1-ил] карбонил] -маркфортин A (соединение 12L),
1-[[4-(фенил)пиперазин-1-ил]карбонил]-маркфортин A (соединение 12M),
1-[[4-(хлорокарбонил)пиперазин-1-ил] карбонил] -маркфортин A (соединение 12N),
10,10''-(1,4-дикарбонилпиперазин)бис(6', 7',8',9',10',10'a- гексагидро-1', 1', 4,4,12'-пентаметил)-[2'S-[2'альфа, 3'а.альфа, 9'а.альфа, 10/2'''R^*, 3'''aS^* 9'''aS, 10'''aR^*)10'a.бета]]-спиро[4H,8H-[1,4]диоксепино[2,3-g]индол- 8,2'(3'H)-[1H,4H-3a,9a](иминометано циклопента[b]хинолизин]-9,11'(10H)-дион (соединение 120),
1-[[4-(фенил)пиперидин-1-ил]карбонил]-маркфортин A (соединение 12P),
1-[[4-(диметил)пиперидин-1-ил]карбонил]-маркфортин A (соединение 12Q),
1-[[4-(5-хлорпиридазин-3-ил)пиперазин-1-ил] карбонил] -маркфортин A (соединение 12R),
1-[[4-(4-хлорфенил)пиперазин-1-ил] карбонил] -маркфортин A (соединение 12S),
10,10''-(1,4-диоксо-2-бутен)бис(6', 7',8',9',10',10'a-гексагидро- 1',1', 4,4,12'-пентаметил)-[2'S-[2'. альфа. , 3'a. альфа. , 9'a.альфа, 10(2'''R^*, 3'''aS^*, 9'''aS^*, 10'''aR^*) 10'a, бета]]-спиро[4H, 8H-[1,4]диоксепино[2,3-g] индол-8,2'(3'H)- [1H, 4H-3a, 9a] (иминометано)циклопента[b] хинолизин] -9,11'(10H)-дион (соединение 12T),
1-[[4-(трихлороэтоксикарбонил)пиперазин-1-ил] карбонил] - маркфортин A (соединение 12U),
1-[[4-(3-трифторометил-тиадиазол-1-ил)пиперазин-1-ил] карбонил] - маркфортин A (соединение 12V),
1-ацетоксиметил-маркфортин A (соединение 13)
1-(2,4-динитробензолсульфенил)-маркфортин A (соединение 14),
1-(4-морфолиносульфенил)-маркфортин A (соединение 14A),
1-(трихлорометилсульфенил)-маркфортин A (соединение 14B),
1-(метоксикарбонилсульфенил)-маркфортин A (соединение 14C),
1-(бензолсульфенил)-маркфортин A (соединение 14D),
1-(4-морфолинкарбонил)-маркфортина AN-окись (соединение 15),
1-(2-тетрагидрофуранил)-маркфортин A (соединение 16).Examples of heterocycles formed by —NR ₄ R ₅ ,

are
4-morpholine,
4-phenyl-1-piperazine,
4- (2-pyridinyl) -1-piperazine,
2,6-dimethyl-4-morpholine,
1-pyrrolidine,
4-methyl-1-piperazine,
1-piperidine,
4-phenyl-1-piperidine,
2-methylpiperidine,
3-methylpiperidine,
4-methylpiperidine,
N-methylpiperazine
1-methylhomopiperazine,
1-acetylpiperazine
Preferred compounds of the invention are
18-thiomarkfortin A (compound 2),
18-thiomarkfortin B (compound 2A),
1-pyrrolidinyl-marcfortine A (compound 12),
1-cyclopropylcarbonyl-marcfortine A (compound 12A),
1-phenoxycarbonyl-marcfortine A (compound 12B),
1-palmitoyl-marcfortine A (compound 12C),
1- (4-nitrophenoxycarbonyl) -markfortin A (compound 12D),
1- (1-piperidinecarbonyl) -markfortin A (compound 12F),
1 - [[4- (ethoxycarbonyl) piperazin-1-yl] carbonyl] -markfortin A (compound 12G),
1 - [[4- (benzyl) piperazin-1-yl] carbonyl] -markfortin A (compound 12H),
1 - [[4- (1,3-benzodioxol-5-ylmethyl) piperazin-1-yl] carbonyl] - α-marcfortine A (compound 12I),
1 - [[4- (methyl) piperazin-1-yl] carbonyl] -markfortin A (compound 12J),
1- (1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl) -markfluorotin A (compound 12K),
1 - [[4- (pyridin-2-yl) piperazin-1-yl] carbonyl] -markfortin A (compound 12L),
1 - [[4- (phenyl) piperazin-1-yl] carbonyl] -markfortin A (compound 12M),
1 - [[4- (chlorocarbonyl) piperazin-1-yl] carbonyl] -markfortin A (compound 12N),
10.10 '' - (1,4-dicarbonylpiperazine) bis (6 ', 7', 8 ', 9', 10 ', 10'a- hexahydro-1', 1 ', 4,4,12'-pentamethyl ) - [2'S- [2'alpha, 3'a.alpha, 9'a.alpha, 10/2 '''R ^* , 3''' aS ^* 9 '''aS,10''' aR ^* ) 10'a. Beta]] - spiro [4H, 8H- [1,4] dioxepino [2,3-g] indole-8,2 '(3'H) - [1H, 4H-3a, 9a] (iminomethane cyclopenta [b] quinolysin] -9.11 '(10H) -dione (compound 120),
1 - [[4- (phenyl) piperidin-1-yl] carbonyl] -markfortin A (compound 12P),
1 - [[4- (dimethyl) piperidin-1-yl] carbonyl] -markfortin A (compound 12Q),
1 - [[4- (5-chloropyridazin-3-yl) piperazin-1-yl] carbonyl] -markfortin A (compound 12R),
1 - [[4- (4-chlorophenyl) piperazin-1-yl] carbonyl] -markfortin A (compound 12S),
10.10 '' - (1,4-dioxo-2-butene) bis (6 ', 7', 8 ', 9', 10 ', 10'a-hexahydro-1', 1 ', 4.4, 12'-pentamethyl) - [2'S- [2 '. alpha. , 3'a. alpha. , 9'a. Alpha, 10 (2 '''R ^* , 3''' aS ^* , 9 '''aS ^* , 10''' aR ^* ) 10'a, beta]] - spiro [4H, 8H - [1,4] dioxepino [2,3-g] indole-8,2 '(3'H) - [1H, 4H-3a, 9a] (iminomethane) cyclopenta [b] quinolysin] -9,11' ( 10H) -dione (compound 12T),
1 - [[4- (trichloroethoxycarbonyl) piperazin-1-yl] carbonyl] - marcfortine A (compound 12U),
1 - [[4- (3-trifluoromethyl-thiadiazol-1-yl) piperazin-1-yl] carbonyl] - marcfortine A (compound 12V),
1-acetoxymethyl-marcfortine A (compound 13)
1- (2,4-dinitrobenzenesulfenyl) -markfortin A (compound 14),
1- (4-morpholinosulfenyl) -markfortin A (compound 14A),
1- (trichloromethylsulphenyl) -markfortin A (compound 14B),
1- (methoxycarbonylsulphenyl) -markfortin A (compound 14C),
1- (benzenesulfenyl) -markfortin A (compound 14D),
1- (4-morpholinecarbonyl) -marchfortin AN-oxide (compound 15),
1- (2-tetrahydrofuranyl) -markfortin A (compound 16).

 Pharmaceutically acceptable salts mean salts useful in administering the compounds of this invention as drugs, these salts include mesylate, hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, acetate, propionate, lactate, maleate, malate, succinate, tartrate and the like. These salts may be in hydrated form.

 The compound of formula II is prepared by the following methods.

Surprisingly, it was found that treatment of the Marcfortins A, B, and C or their substituted C-24, C-25, N-1, and N-18a derivatives with phosphorus pentasulfide or preferably 2,4-bis (methoxyphenyl) -1,3-dithia -2,4-diphosphetane-2,4-disulfide (Lawesson's reagent) selectively gives the corresponding 18-thio derivatives. Suitable C-24, C-25, N-1 and N-18a substituted derivatives of marcfortins A, B and C for this reaction are easily prepared by the methods described in [1], the description of which is incorporated herein by reference. The reactions are carried out in a suitable inert solvent, such as pyridine, collidine, toluene (preferably), xylene, dioxane, tetrahydrofuran and the like, at temperatures of 10 - 180 ^o C, preferably 80 - 140 ^o C. Or C-24, C -25 and N-1 derivatives can be prepared from 18-thiomarkfortins. For example, a large series of 18-thiomarkfortin analogs can be prepared by alkylation or acylation of N-1 and N-18a substituted 18-thiomarkfortins. Such derivatives can be easily prepared by sequentially treating a solution of 18-thiomarcfortin A, N-18a-substituted 18-thiomarcfortin B, or N-18a-substituted 18-thiomarcfortin C in a protic organic solvent such as tetrahydrofuran, diethyl ether, benzene and the like , an excess of a strong base, such as potassium hydride (preferably), sodium hydride, butyl lithium, potassium tert-butylate and the like, followed by treatment with a suitable alkylating or acylating agent at temperatures in the range 0-50 ^° C. from 0.25 to 48 h .

 Suitable alkylating agents include alkyl bromides, alkyl iodides, alkyl sulfonates, alkenyl iodides, alkynyl bromides, alkoxyalkyl chlorides and the like. Suitable acylating agents include acyl anhydrides, acyl chlorides, acyl bromides, substituted benzenesulfonyl chlorides, substituted benzenesulfonic anhydrides, sulfenyl chlorides, isocyanates, carbamoyl chlorides, chloroformates and the like.

Additional series of derivatives can be produced by modifying the C24-C25 double bond of the 18-thiomarcfortion A, B, and C. 24, 25 dihydro analogs are readily prepared by mixing a solution of a suitable 18-thiomarcfortin in an alcohol solvent such as methanol, ethanol, propanol and the like. with a catalyst such as palladium, platinum, tris (triphenylphosphine) - chlorodorium and the like in the presence of gaseous hydrogen. The product, which is an analogue of 24.25-dihydro-18-iomarkfortin, can be isolated and purified using methods known to those skilled in the art. It should be noted that the reactions described above to modify another part of the 18-thiomarkfortin structure can also be applied to 24, 25-dihydro 18-thiomarkfortin analogs to prepare the corresponding 24, 25-dihydro analogs. Additionally, C24-C25 double bond-modified analogues of 18-thiomarcfortins can be obtained via 24, 25 dibromide, which is easily prepared by treating a solution of 18-thiomarcfortins in a halogenated solvent such as dichloromethane, chloroform, carbon tetrachloride and the like with a molar equivalent of bromine temperature range from -20 to 25 ^o C for a time from 0.25 to 8 hours. This process affords the corresponding 24,25-dibromo derivative of 24,25-dihydro-18-tiomarkfortina which can be isolated and purified using techniques izves GOVERNMENTAL to those skilled in the art.

It should be noted that a 24.25 dichloro analogue can be prepared by substituting bromine for chlorine in the method described above. Analogs 24.25 - dibromo 24.25-dihydro-18-marcofortin described above are used as intermediates for the preparation of additional derivatives. Thus, the treatment of a solution of dibromide in an alcohol solvent, such as methanol, ethanol, propanol and the like, with a strong base, such as 1.8 diazabicyclo [5.4.0] undec-7-ene (DWI) at a temperature range of 0 - 30 ^o C for 0.25-24 hours gives analogues of 24-alkoxy-25 bromo-24, 25 dihydro-18-thiomarkfortin, which can be isolated and purified using methods known to those skilled in the art. These 24 alkoxy, 25-bromo derivatives can be debrominated by treating a solution of the compound in an aprotic organic solvent such as benzene, toluene, hexane and the like with a tin hydride reducing agent such as tri-butyl tin hydride, tri-pentyl tin hydride and the like with or without the addition of a radical initiator such as azobis-isobutyronitrile (AIBN) at a temperature range of 25-120 ^° C. for 0.5 to 48 hours. This method gives the corresponding derivatives of 24-alkoxy-18-thiomarkfortin (R ₂₄ = lower alkoc si in the general structure) that can be isolated and purified using methods known to those skilled in the art.

 Basic methods for preparing heteroaromatic N-oxides can be found in Chapter II of "Chemistry of the Heterocyclic N-Dxides" A.R. Kattitzky and T.M. Ladowski, pyb. 1971 Academic Press (Vol. 19 of ORGANIC CHEMISTRY - A Series of Monographs).

 Usually N - oxide is formed by the interaction of a with a percarboxylic acid in an aprotic solvent. It is most convenient to use aromatic peracid in a non-polar solvent, since the reaction can usually be carried out at room temperature. Suitable aromatic peracids include superbenzoic acid, chloronadobenzoic acid and perephthalic acid.

A compound of formula IV is prepared by alkylation or acylation of N-1 Marcfortine A and N-18a substituted Marcfortins B and C. These derivatives can easily be prepared by sequentially treating a solution of Marcfortine A or N-18a substituted Marcfortins B and C in an aprotic organic solvent such as tetrahydrofuran, diethyl ether, benzene and the like with an excess of a strong base such as potassium hydride (preferably), sodium hydride, butyllithium, potassium tert-butylate, and the like, followed by treatment with suitable alkyl a ligation or acylating agent at a temperature range of 0-50 ^° C. for 0.25 to 48 hours. Suitable alkylating or acylating agents include alkanoyloxyalkyl bromides, aminosulphenyl chlorides, phosphoryl chlorides, phosphonyl chlorides, acyl anhydrides, acyl chlorides, acyl benzyl bromides, acyl benzyl bromides, , carbamoyl chlorides, substituted phenoxychloroformates and the like. Treatment of Marcfortin A and N-18a substituted Marcfortins B and C and C24-C25 modified Marcfortins under these conditions gives 1-N-substituted analogs that can be isolated and purified using methods known to those skilled in the art.

Source material preparation
Parahervamide is isolated from Penicillium Sp. IMI 332995 and / or Penicillium charlessi MG 5123 (ATCC 20841) using standard enzymatic and separation techniques. The selection is described in detail in US patents 4973247 and 4978656, as well as EP 390532-A, EP-301742-A and WD91 / 09961 (all incorporated herein by reference).

 N-18a substituted marcfortins B and C and C24-C25 midfixed marcfortins are easily prepared by the methods described in [1].

 Markfortins A, B, and C are isolated, along with the previously known rockfortin, as fungal metabolites of Anicillinm roguerforti using standard enzymatic and separation methods. The isolation, as well as the analytical and structural characteristics of marcfortine A, are described in detail in Polonsky et al Journal of the Chemical Sosiiety Chemical Communisations 1980, 601-602. Isolation, as well as the analytical and structural characteristics of Marcfortins B and C, are described in detail in Polonsky et al Ketrahedron Letters 1981, 22, 1977-1980.

Alternatively and more preferably, Marcfortins A and C may be isolated from Penicillium sp. IP 7780 (strain number in the Culture Collection IP 7780, The Upjohn Company, Kalamazoo, MI). This strain was isolated from a soil sample taken from Illinois, deposited in the United States Department of Alricnetnre patent cnltnre collection in Peoria, IL and cataloged under the number NRRL 18887. To further characterize the fungus, a taxonomic study was performed according to the methods and materials described in I. John Pitt, The Genus Pinicillium, Acodemic Press, L ondon, (1979). Spores and hyphal surfaces were examined by electron scanning microscopy according to the methods of Dietz, A. and Matthews, J. Appl. Microlilogy 18: 694-696 (1969). Intact conidiophores were observed visually using light microscopy [AHS Onions et al, Smith's Introduction to Industrial Mycology, John Wiley and Sons, New York, pp 301-302 (1979)] after preparation of the microscopic preparation of the culture (s): a glass Petri dish containing glass beads, a microscopic preparation and a coverslip are sterilized. A small block of potato dextrose agar is placed on a microscopic preparation and inoculated on four sides with a fungal structure. A coverslip was placed on the inoculated agar block and sterile water was added to maintain moisture. The chamber is incubated for six days at 24 ^o C. A microscopic preparation is prepared by removing the coverslip and placing it on a drop of lactophenol cotton sienna.

 Characteristics of the Penicillium sp. IP 7780 (NRRL 18887) is as follows.

 Morphology - bimuth brush (two branch points between the conidia and peduncle). These branches (metula) support the supporting structures of phialids and conidia. Conidiophores (approximately 35 microns) end in whorls of 2-5 (10-14 microns) metulae. The phialids had the shape of cones (like an ancient Greek vessel for wine) in whorls from 2-5 (7 microns). The conidia were smooth and spheroidal (2 μm), usually forming a long gynostelium. The walls of the legs were smooth.

The culture was inoculated into three Petri dishes on Chapek’s yeast agar (SUA), one of which was 6 cm in diameter, the other contained malt extract agar (MEA) and agar with 25% glycerol nitrate (G 25 N). Inoculation was carried out from a semi-solid suspension (0.5 ml of 0.2% agar with 0.05% Tween 80). An inoculating loop with conidia was introduced into the tube and mixed. The suspension loop was inoculated into the sample in three places on each plate. A needle was used to introduce the inoculum into a 6 cm dish. Used incubation regimen: one CYA cup plus MEA and G 25 N cup at 24 ^o C, one CYA cup at 37 ^o C and 6 cm CYA cup at 5 ^o C. After seven days, the diameters, colors and other characteristics of the colonies were recorded and placed further in the table. 1. On potato dextrose PDA, Difco) agar, a dark red color appears on the bottom or on the back of the colony.

 No sexual stage was noted. These culture results (NRRL 18887) were determined by Penicillium. A determinant for defining a subculture. According to the Penicillium determinant, only the penicillin type determines the subgenus to which the species belongs. This species has some characteristics that distinguish it from the Biverticillium subgenus. For example, his brushes are whorled. The species reliably produces colonies of more than 10 mm in diameter in 7 days on agar with glycerol nitrate. Metulae appear longer than phialids and in whorls from 2-5. These characteristics allow this strain of Penicillium sp. (NRRL 18887) to the subgenus Furcatum.

 The above description illustrates the Penicillium strain IC 7780 (NRRL 18887), which can be used in the manufacture of Markfortin and its derivatives. In addition, the invention also includes mutants of the microorganism described above. For example, those mutants that are obtained by natural selection, or mutants obtained by mutating agents including ionizing radiation, such as ultraviolet radiation, or chemical mutagens, such as nitrosoguanidine, or the like, are also included in the scope of this invention.

 The subject and purpose of this invention is also the inclusion of mutual or internal genetic recombination processes using genetic techniques well known to those skilled in the art, such as, for example, conjugation, transultion and genetic engineering techniques.

 Penicillium sp. IP 7780 (NRRL 18887) can be cultivated under aerobic conditions by the method that is commonly used in the cultivation technique of a known strain of the genus Penicillium.

 As components of the medium can be used any well-known nutrients for Penicillium. For example, as an assimilable source, carbon, glucose, glycerin, maltose, dextrin, starch, lactose, sucrose, molasses, soybean oil, cottonseed oil, etc. preferably glucose and glycerin can also be used as an assimilable source of nitrogen, soy flour, peanut flour, cottonseed flour, fish meal, liquid corn soak, peptone, rice, bran, meat extract, yeast, yeast extract, sodium nitrate, nitrate ammonium, ammonium sulfate, etc. can be used. And inorganic salts such as sodium chloride, sodium phosphates, calcium carbonate, etc. can be added to the crop medium. If necessary, minor amounts of a metal salt may also be added. In addition, minor amounts of heavy metals can be added if necessary.

 In particular, in the cultivation of Penicillium sp. (NRRL 19997) under aerobic conditions, conventional aerobic cultivation methods, such as, for example, growing on a dry residue, growing under aeration and stirring (shaking), growing under shaking, etc., can be successfully used under aerobic conditions. Traces of metals, for example zinc, magnesium, manganese, cobalt, iron and the like, should be added to the enzymatic medium, since the level of reverse osmosis of water should be maintained.

 Anti-foaming agents, such as silicone oil, vegetable oils, surfactants, etc., can be successfully used in cultivation during aeration and agitation.

The pH of the medium can usually be in the range of pH 3-9, and preferably in or near the neutral region, and cultivation temperatures can usually be 20-30 ^° C, in particular about 21 ^° C.

 The cultivation can continue until Markfortin A accumulates significantly in the culture medium, usually from 20 to 240 hours, preferably 48 to 168 hours, and after cultivation, Marcfortin A can be isolated and isolated from the liquid culture medium (culture broth) by a suitable combination of various methods. For example, it may be extraction with an organic solvent, for example diethyl ether, ethyl acetate or chloroform; dissolving in a more polar solvent, for example acetone or alcohol; removal of impurities with a less solvent, for example, petroleum ether or hexane, absorption chromatography on activated carbon or silica gel; gel filtration through a Sephadex column (available from Pharmacia Co., Ltd. USA) and the like.

 Markfortin B can be prepared from Markfortin A by biotransformation using a microorganism in a water nutrient medium containing an assimilable source of nitrogen under aerobic conditions.

 The present invention also provides biologically pure cultures (organisms derived from a culture collection as apparently open organisms) of the genus Cunnirghamella characterized as species isolated from the group consisting of NRRL 1368, 1393 or 3655 and ATCC 8688 a. A method of using members of the genus Cunnirghamella, characterized as species isolated from the group consisting of NRRK 1368, 1393 or 3655 and ATCC 8688a, for the preparation of Marcfortin B, which involves the attachment of Macrfortin A to metabolizable cultures of these microorganisms.

 By "biotransformation" means the use of microorganisms or the isolation of partially purified enzymes to convert this substrate into a useful product. N.S. Davies, et al, "Biotransformations in Dreparative Organic Chemistrg", Akademic Press, N.Y., 1989, p.IX.

 By "metabolization" means the conduct of metabolic processes. A definition of metabolism can be found in A. L. Lehninger, "Principlles of Biochemistry", Worth Pnbl., New York, 1982, p. 333.

By using the new method of the invention, Markfortin B is prepared by adding Markfortin A to growing cultures of these organisms. The genus Cunnirghamella is preferred for performing this transformation, including Cunnirghamella echinulata sublp. elegans (-) NRRL 13b8, Cunninghamella blakesleeana (+) ATTC 8b88a Cunnirghamella echinufata subsp. elegans NRRL 1393, Cunnirghamella echinulata NRRL 3655;
in particular, strain NRRL 3655 is preferred.

 Subculture Cunnirghamella echinulata subsp. elegans (-) was deposited with the Budapest Treaty in the permanent collection of the Northem Region Research Center, ARS; U.S. Dept of Agriculture; Peoria, Illinois, USA.

 She was assigned the number NRRL 1368. Subculture Cunninghamella echinulata subsp. elegans (-) was also deposited of the Budapest Treaty in the permanent collection of the American Tupe Culture Collection, Rockville, MD, USA.

 She is assigned ATCC number 8688b.

 A subculture of Cunnirghamella blakesleeana (+) was deposited of the Budapest Treaty in the permanent collection of the American Tupe Culture Collection, Rockville, MD, USA.

 She is assigned ATCC number 8688a.

 Subculture Cunnirghamella echinulata subsp. elegans (-) deposited of the Budapest Treaty in the permanent collection of the Northem Region Research Center, ARS; U.S. Dept of Agriculture; Peoria, Illinois, USA.

 Its serial number is NRRL 1393. Subculture Cunnirghamella echinulata subsp. elegans (-) was deposited of the Budapest Treaty in the permanent collection of the American Tupe Culture Collection, Rockville, MD, USA.

 She is assigned ATCC serial number 10028b.

 The subculture Cunnirghamella echinulata was deposited of the Budapest Treaty in the permanent collection of the Nocthern Region Research Center, ARS; U.S. Dept of Agriculture; Peoria, Illinois, USA.

 She is assigned the number NRRL 3655.

 The use of these microorganisms in the method of the present invention is expressed in the preparation of Marcfortin B from Marcfortin A.

Markfortin B is obtained when Cunnirghamella echinulata subsp. elegans (-) NRRL 13b8, Cunnirghamella blakeslleana (+) ATTC 8b88a Cunnirghamella echinulata subsp. elegans NRRL 1393, Cunnirghamella echinulata NRRL 3b55
fermented in an aqueous nutrient medium under aerobic immersion in the presence of Markfortin A. Typically, a microorganism is fermented in a nutrient medium containing a carbon source and protein material. Preferred carbon sources include glucose, baked sugar, sucrose, glycerin, starch, corn starch, lactose, dextrin, molasses and the like. Preferred nitrogen sources include cotton flour (cottonseed flour), liquid corn impregnation, yeast, autolysed brewer's yeast with a dry milk substance, soy flour, corn flour, dry milk substance, pancreatic digested casein, animal-derived peptone extracts, meat extracts and bones and the like. Advantageously, combinations of these carbon and nitrogen sources can be used. There is no need to add traces of metals, for example, zinc, magnesium, manganese, cobalt, iron and the like, since tap water and raw ingredients are used as components of the medium.

The production of Marcfortin B can be induced at a certain temperature, contributing to a satisfactory growth of the microorganism between approximately 23 and 32 ^o C and preferably at about 28 ^o C.

 Typically, optimal formation of Markfortin B occurs approximately 1 to 4 days after the addition of Markfortin A to the growing culture, and preferably over a period of about 2 days. The fermentation broth usually remains slightly acidic to basic (pH 5.8-9.0) during fermentation. The final pH depends in part on the presence of a buffer, if present, and in part on the initial pH of the culture medium. It is advantageous to adjust the pH to about 6.5-7.5 and preferably 7.2 before sterilization.

 When growth is carried out in a shake flask or large vessel and collectors, it is preferable to use the vegetative form of the microorganism for inoculation, rather than using the spore form to avoid a pronounced delay in the production of Markfortin B and the associated inefficient use of equipment. Thus, it is desirable to obtain a vegetative inoculum in an aqueous nutrient medium by inoculating this medium with an aliquot of soil or culture on mowed agar. When a young, active vegetative seed is thus isolated, it is transferred aseptically into other shaken tubes or collectors. The environment in which the vegetative inoculum is obtained may be the same or different from that used to obtain Markfortin B, the duration of the process is adequate to the growth of the resulting microorganism.

 Various methods can be used to isolate and purify Markfortin A from the enzyme broth, for example by absorption chromatography followed by elution with a suitable solvent, column chromatography, partition chromatography and crystallization from solvents, as well as a combination of these methods.

 A preferred method for isolating Markfortin B is to extract from (untreated) whole beer. Column chromatography techniques, preferably on silica gel, are used to carry out the initial purification. Final purification of Markfortin B is achieved by chromatography and crystallization from organic solvents.

 Method A further illustrates the biotransformation method of the invention.

 These compounds of the present invention unexpectedly turned out to be potent antiparasitic agents against endo and ecto parasites, in particular helminths (parasitic worms) and arthropods, which lead to numerous diseases caused by parasites in humans, animals and plants.

 Diseases caused by parasites can be caused by both endoparasites and ectoparasites. Endoparasites are parasites that live inside the host’s body, either inside an organ (such as the stomach, lung, heart, intestines, etc.) or simply under the skin. Ectoparasites are parasites that live on the outer surface of the host, but still draw nutrients from the host.

 Diseases caused by endoparasites, commonly called helminth infections, are the result of infection of the host with parasitic worms, known as helminths. Helminthiasis is a widespread and serious global economic problem associated with infection of domestic animals such as pigs, horses, sheep, cattle, goats, dogs, cats and poultry. Many of these infections are caused by a group of worms called nematodes, which cause diseases of various organs in animals around the world. These diseases are often serious and can lead to the death of an infected animal. The most common genera of nematodes that infect the animals mentioned above include Haemonchus, Trichostrongylus, Ostertagia, Nematodirus, Cooperia, Ascaris, Bunostomum, Oesophagostomum, Chabertia, Trichuris, Strongylus, Trichonema, Dictyocaulus, Capillaria, Unicocida, Anetocoris, Anetocis, Anisocida, Anetocis, Anecida, Anis, Atomicis , Tokascaris, and Parascaris.

 Many parasites are species-specific (infect only one host) and most also have a preferred infection site for animals. So, Haemonchus and Ostertagia predominantly infect the stomach, while Nematodirus and Cooperia mainly attack the intestines. Other parasites prefer to linger in the heart, eyes, lungs, blood vessels and the like, while other immobile ones are subcutaneous parasites. Helminthiasis can lead to weakness, weight loss, anemia, intestinal damage, malnutrition and damage to other organs. If this disease is left untreated, it can lead to the death of the animal.

 Infection caused by ectoparasitic arthropods, such as ticks, mites, lice, autumn lighters, small cow lighters, carrion flies, fleas, etc. is also a serious problem. Infection with these parasites is expressed in loss of blood, skin lesions, can interfere with the normal absorption of food and lead to weight loss. These infections can also lead to the development of serious diseases, such as encephalitis, anaplasmosis, smallpox in pigs, etc., which can lead to a fatal outcome.

 Animals can be infected with several types of parasites at the same time, since infection with one parasite can weaken the animal and make it more susceptible to infection with a second type of parasite. Thus, a compound with a wide spectrum of activity has advantages in the treatment of such diseases. The compounds of this invention have unexpectedly high activity against these parasites and, in addition, are also active against Dirofilaria in dogs, Nematospiroides and Suphacia in rodents, stinging insects and migrating dipterous larvae such as Hypodorma sp. in cattle and Gastrophilus in horses.

 These compounds are also useful against endo and ecto-parasites, which lead to diseases caused by parasites in humans. Examples of such endoparasites that infect humans include gastrointestinal parasites of the genus Ancylostoma, Necator, Ascaris, Strongyloides, Trichinella, Capillaria, Trichuris, Enterobius, and the like. Other endoparasites that infect humans are found in the blood and other organs. Examples of such parasites are filamentous worms Wucheria, Brugia, Onchocerca and the like, as well as extraintestinal phases of the intestinal worms Strongylides and Trichinella. These are parasites that parasitize humans, including arthropods, such as ticks, fleas, ticks, lice, and the like, and as with pets, infection with these parasites can result in serious and even fatal diseases. The present compounds are active against these endo and ecto parasites and, in addition, are also active against stinging insects and other diptera parasites that bother a person. The current compounds, when administered orally or parenterally, are prescribed in doses ranging from about 0.05 to 20 mg / kg body weight of the animal. These compounds are also useful against indoor pests, such as Blatella sp (cockroaches), Tineola sp (clothes moth), Attagenus sp. (skin-eaters), Musca domestira (housefly) and against Solenopsis Invicta (Richter ant).

 In addition, the compounds are useful against agricultural pests such as aphids (Acyrthiosiphon sp.), Locusts and cotton weevil, as well as against harmful insects that corrode grains during storage, such as Tribolium sp. , and against insects at incomplete stages of development living in plant tissue. The compounds are also useful as a nematicide for controlling nematodes in the soil, which may be important for agriculture. When used as antiparasitic agents for animals, the compounds of this invention can be administered internally either orally, or by injection, or locally in the form of liquid irrigation or as a shampoo.

 For oral administration, the compounds may be administered in the form of capsules, tablets, drug infusion beads, or else they may be mixed with animal food. Capsules, tablets, and spheres contain the active ingredient in combination with an appropriate binder, such as starch, talc, magnesium stearate, or dicalcium phosphate. These unit dosage forms are prepared by intimately mixing the active ingredients with suitable solvents, excipients, disintegrating agents, suspending agents and / or binders, so that uniform solutions or suspensions are obtained. Inert ingredients are those that will not interact with these compounds and which are not toxic to animals to be treated. Suitable inert ingredients include starch, lactose, talc, magnesium stearate, vegetable gums and oils, and the like. These formulations may contain a wide variety of active and inactive ingredients, depending on numerous factors, such as the size and type of animal species and the type and severity of the infection. Active ingredients may also be administered as food additives by simply mixing the compound with the food or by applying the compound to the surface of the food. Otherwise, the active ingredient may be mixed with an inert carrier, and the resulting composition may then be either mixed with food or fed directly to the animal. Suitable inert carriers include corn flour, citrus flour, enzymatic residues, coarse soy flour, dried cereals, and the like. The active ingredients are intimately mixed with these inert carriers by grinding, shaking, crushing or tumbling, so that the final composition contains 0.001-5.0% by weight of the active ingredient.

 Otherwise, the compounds may be administered parenterally through injection of a composition containing the active ingredient dissolved in an inert liquid carrier. Injection can be carried out intramuscularly, intraruminally, intratracheally or subcutaneously. Formulations for injection contain the active ingredient mixed with an appropriate inert liquid carrier. Acceptable liquid carriers include vegetable oils, such as peanut oil, cottonseed oil, sesame oil and the like, as well as organic solvents such as solketal, glycerol formal and the like. Alternatively, aqueous parenteral formulations may also be used. Vegetable oils are the dissolution or suspension of the active ingredient in a liquid carrier, so that the final composition contains 0.005-20 wt.% The active ingredient.

 A topical application of these compounds is the possibility of using liquid sprinklers or shampoos containing the current compounds in the form of aqueous solutions or suspensions. These formulations typically contain a suspending agent, such as bentonite, and usually should also contain an anti-foaming agent. Acceptable are compositions containing 0.005-20 wt.% These compounds. The present compounds are mainly useful as antiparasitic agents for the treatment and / or prevention of helminthiases in domestic animals, such as cattle, sheep, horses, dogs, goats, pigs and poultry. They are also useful in the prevention and treatment of infections from parasites caused by ectoparasites in these animals, such as ticks, mites, lice, lighters and the like. They are also effective in the treatment of infections caused by parasites in humans. In the treatment of such infections, the compounds of this invention can be used individually or in combination with each other or with other unrelated antiparasitic agents. The dosages of the present compounds necessary for best results depend on several factors, such as the type and size of the animal, the type and severity of the infection, the treatment method and the compounds used. Oral treatment with these compounds at a dose level of 0.005-50 mg per 1 kg of animal body weight, either in a single dose or in several doses, carried out over several days, gives good results. A single dose of one of these compounds usually provides excellent control, however, repeated doses can be given to prevent reinfection or for species of parasites that occasionally remain. The technique for treating these animal compounds is known to those skilled in the veterinary field.

 The compounds of this invention can also be used to control agricultural pests that destroy the crop either in the field or during storage. The compounds are used for such purposes in the form of spraying liquids, dust preparations, emulsions and the like, either for growing plants or for harvested crops. Techniques for using these compounds for similar purposes are known to those working in the agricultural field.

 The following examples do not limit the scope of the present invention.

Example 1. Obtaining and isolation of Marcfortin A
The method of seed fermentation
Seed enzymes are inoculated using agar plugs from Penicillium sp. IC 7780 (NRRL 18887) stored in liquid nitrogen. Three plugs are thawed and used as an inoculum. GS-7 is made up of glucose and cotton flour (Pharmamedia brand "by Traders Protein, Procterk Gamble Oilseed Products Co., Memphis, TN, USA

Tap water is used without further purification to hydrate the components of the medium and the pH of the medium is adjusted to 7.2 with NH ₄ OH. The medium is dispensed into septum-free flasks representing a closed system, 300 ml per 1000 ml flask and sterilized by autoclaving at 121 ^° C. for 30 minutes. Each closed system flask containing 300 ml of GS-7 medium was inoculated with three agar plugs of Penicillium sp. 7780 (NRRL 18887) and shaken on a rotary vibrator at 250 rpm for 36 hours at 22 ^o C.

(0,35 кг/см²) и 250 об/мин в течение 36 ч.Method for secondary fermentation of seeds
Ripe seed cultures are used as inoculum for the secondary medium at 0.3% dose. The secondary medium is composed of glucose monohydrate (brand Cerelose by CPC International) 25 g, cottonseed flour (brand "Pharmamedia") 25 g MgCl ₂ • 6H ₂ O 329.8 mg; MnSO ₄ • H ₂ O 11.4 mg; FeSO ₄ • 7H ₂ O 3.2 mg; ; Na ₂ MoO ₄ • 2H ₂ O 1.8 mg; CaCl ₂ • 2H ₂ O 367.6 mg; NaCl 84.2 mg; KCl 5.8 mg; ZnSO ₄ • 7H ₂ O 0.1 mg; CoCl ₂ • 6H ₂ O 0.1 mg; CuSO ₄ • 5H ₂ O 3.1 mg and antifoam silicone (SAC - 471 Antifoam) 0.5 ml per 1 liter of water to provide reverse osmosis. The components of the medium are sufficient to produce 200 l of secondary medium for seeds hydrated in water with a reverse osmosis rate up to qs of 190 l in a 250-liter fermenter. After adjusting the pH, the medium was adjusted to 7.2 with NH ₄ OH and then the medium was sterilized at 121 ^° C for 30 minutes. Two flasks of a closed system with a culture of ripe initial seeds are used as an inoculum at a 0.3% dose of administration. Secondary seed culture incubated at 22 ^o C, with 125 slm backpressure aeration

(0.35 kg / cm ² ) and 250 rpm for 36 hours

The method of fermentation
The production medium consists of beet molasses 50 g of fish meal (Henhaden Select Fish Meal brand) 16 g, yeast extract (Fidro brand) 10 g; MgCl ₂ • 6H ₂ O 329.8 mg; MnSO ₄ • H ₂ O 11.4 mg; FeSO ₄ • 7H ₂ O 3.29 mg; Na ₂ MoO ₄ • 2H ₂ O 1.8 mg; CaCl ₂ • 2H ₂ O 367.6 mg; NaCl 84.2 mg; KCl 5.8 mg; ZnSO ₄ • 7H ₂ O 0.1 mg; CoCl ₂ • 6H ₂ O 0.1 mg CuSO ₄ • 5H ₂ O 3.1 mg and antifoam silicone (grade SAG - 471) 0.5 ml per 1 liter of water with a reverse osmosis index.

Media components sufficient for 5,000 L of medium are hydrated in water with a reverse osmosis index to a volume of 4,700 L in a 5,000 L fermenter. After adjusting the pH, the medium was adjusted to 7.0 with KOH, and then the medium was sterilized at 123 ^° C for 30 minutes. A ripe secondary seed culture is used as an inoculum at a 1.0% dose. The culture is incubated at 22 ^o C with 2,500 slm aeration, at 5 psig back pressure and 250 rpm for 96 hours

Isolation of Marcfortine A
Cells grown in culture are collected from 4900 L of fermented volume by passing through a finely chopped mixer into a cell collection vessel. After transfer, 4% w / v diatomaceous earth and 1/2 volume of methylene chloride are added. The harvested cell solution is then filtered using a filter press. The filtered precipitate is washed 2 times with 10% volume of methylene chloride.

 The resulting filtrate is decanted to remove the aqueous phase. The remaining methylene chloride phase enriched in the product is then concentrated to a volume of 44 l. The concentrate is then adjusted to the desired condition using 20% of the concentrate volume (9 L) of methylene chloride and diatomaceous earth on the filter.

 53 L of the conditioned concentrate is then purified to separate Marcfortin A from other components by silica gel chromatography and crystallization.

 Prior to chromatography, the conditioned concentrate is divided into four approximately equal aliquots. Each aliquot of the sample is chromatographed on a newly packed 9 '' diameter column filled with 25 kg of dry silica gel (filler volume 59 l). The loaded columns elute with 120 L of 10% acetone in methylene chloride, 120 L of 20% acetone in methylene chloride, 120 L of 30% acetone in methylene chloride, 160 L of 40% acetone in methylene chloride and 130 L of acetone, collecting 30 and 40% eluates in fractions 20 liters each Eluates were analyzed by TLC using, for example, a solvent system composed of 6% isopropanol and 0.3% ammonium hydroxide in methylene chloride to develop silica gel plates.

 The Marcfortin A fractions (containing a small amount of Marcfortin A, which chromatographically leaves with A) crystallize from acetone. The appropriate fractions (40-100 L) are concentrated under reduced pressure to approximately 5 L. The solution (or weak suspension) is then transferred to a rotary evaporator and continued to concentrate under reduced pressure. During the concentration, several 1 L portions of acetone are added until the methylene chloride is completely removed. The remaining acetone suspension (approximately 1 L volume) was cooled overnight, and Marcfortine A crystals were collected and washed with several small portions of chilled acetone and dried under reduced pressure. Such crystals may contain as an impurity a certain percentage of Marcfortine. Recrystallization from methylene / chloride / acetone (removal of methylene chloride as described) gives pure Marcfortine A.

Example 1A Obtaining and isolation of Marcfortins A and C
Primary seed fermentation method
Seed enzymes were inoculated using agar plugs from Penicillium sp. IC 7780 (NRRL 18887) stored in liquid nitrogen. Three plugs are thawed and used as inoculum for 100 ml of GS - 7 culture medium. GS-7 is made up of glucose and cottonseed flour (Pharmamedia by Traders Protein, Procter & Gamble Oilseed Products Co., Memphis, TN, USA), adding each component at a concentration of 25 g / l tap water. After setting the pH, GS-7 adjusted to 7.2 using NH ₄ OH. The medium is autoclaved in volumes of 100 ml in 500 ml of non-fermented fermentation flasks for 30 minutes, Sterile GS-7 is inoculated as described above, and shaken at 250 rpm for 35- 58 h, at 23 ^o C.

The method of fermentation (flask vibrator)
Ripe seed cultures are used as inoculum for the production medium at a 1% dose. The production medium consists of 45 g of glucose, 25 g of enzymatic digestion casein (Peptonized Milk Nutrient by Sheffield Products, Norwich, NYUSA 2.5 g of yeast extract (Bacto Yeast Extract Code: 0127 by Difco Laboratories, Detroit, MI) per 1 liter tap water. After adjusting the pH of the medium to be produced, adjust to 7.0 using potassium hydroxide. This medium is then autoclaved for 30 minutes in volumes of 100 ml contained in 500 ml of blocked fermentation flasks. The sterile production medium is inoculated as described above. and shake for 7-14 days at 250 rpm at 21 ^o C.

The method of fermentation (collections Labraferm tanks)
Ripe seed cultures are used as inoculum for a sterile production medium at a 0.5% dose. The production environment is described above. After adjusting the pH of the medium to 7.0 using KOH, 10 L of this medium was autoclaved for 90 minutes in 12 L Labroferm tanks (New Brunswier Scientific Co., Inc.). The collections were inoculated at a 0.5% dose and mixed at 500 rpm at 20 ^° C. for 5–9 days. The air flow rate was maintained between 10-15 l / min.

Isolation of Marcfortins A and C
West enzyme broth (35 L) was macerated at low speed in a large industrial mixer Waring'a and then mixed with an equal volume of methylene chloride. The mixture was allowed to stand overnight while cooling and then centrifuged to break the emulsion. The resulting clear methylene chloride layer was poured and evaporated under reduced pressure. A concentrated solution of the residue (37.4 g) in methylene chloride is applied to a column filled with a suspension of silica gel (1 kg) in methylene chloride. The column is eluted with increasing concentrations of acetone in methylene chloride (10%, 20%, 30%, 40% and 50% acetone). Fractions were analyzed by TLC and the appropriate fractions were evaporated and crystallized from acetone to give Marcfortin A and Marcfortin C.

 Example 1B Obtaining and isolation of Marcfortins A and C.

The method of seed fermentation
Seed enzymes are inoculated using agar plugs from Penicillium sp. IC 7780 (NRRL 18887) stored in liquid nitrogen. Three plugs are thawed and used as inoculum for 100 ml of GS - 7 seed medium. GS-7 is made from glucose from cottonseed flour (Pharmamedia by Traders Protein, Procter & Gamble Oilseed Products Co., Memphis, TN, USA), adding each component at a concentration of 25 g / l of tap water. After setting the pH, GS - 7 is adjusted to 7.2 using NH ₄ OH. The medium is autoclaved in volumes of 100 ml in non-blocked fermentation flasks for 30 minutes, Sterile GS-7 is inoculated as described above, and shaken at 250 rpm for 35 - 58 h at 23 ^o C.

Fermentation method (shake flask)
Ripe seed cultures are used as inoculum for the production medium at a 1% dose. The production medium consists of 20 g of glucose, 15 ml of glycerol from cotton seed flour (Pharmamedia brand by Traders Protein, Procter Gamble Oilseed Products (o., Memphis, TN, USA) 20 g of soy flour 10 g, K ₂ HPO ₄ 3 g per 1 liter of tap water. After adjusting the pH of the medium, the production was adjusted to 6.8 using potassium hydroxide. This medium was then autoclaved for 30 minutes in volumes of 100 ml in 500 ml of blocked fermentation flasks. The sterile production medium was inoculated as described above. and shake for 7-14 days at 250 rpm at 21 ^o C.

Fermentation Method (Labraferm Collections)
Ripe seed cultures are used as inoculum for a sterile production medium at a 0.5% dose. The production environment is described above. After adjusting the pH to 7.0 using KOH, 10 L of this medium was autoclaved for 90 min in 12 L volumes (Labroferm tanks (New Brunswick Scientific Co., Inc.). The volumes were inoculated at 0.5% dose and mixed at 500 rpm). / min at 20 ^o C for 5 to 9 days.The air flow rate is maintained between 10 - 15 l / min.

Isolation of Marcfortins A and C
All fermentation broth (35 L) is marced at low speed in a large industrial mixer Waring'a and then mixed with an equal volume of methylene chloride. The mixture was allowed to stand overnight while cooling and then centrifuged to break the emulsion. The resulting clear methylene chloride layer was poured and evaporated under reduced pressure. A concentrated solution of the residue (37.4 g) in methylene chloride is applied to a column filled with 91 kg silica gel suspension in methylene chloride. The column is eluted with increasing concentrations of acetone in methylene chloride (10%, 20%, 30%, 40% and 50% acetone). Fractions were analyzed by TCX and the appropriate fractions were evaporated and crystallized from acetone to give Marcfortin A and Marcfortin C.

Example 2. 18-thiomarkfortin A (Compound 2)
A solution of 30 g of marcfortine A and 22 mg of 2,4-bis- (methoxyphenyl) -1,3-dithia-2; β-diphosphetane-2,4-disulfide (Lawesson's reagent) in 5 ml of toluene is heated to boiling under reflux in a nitrogen atmosphere for 18 hours. The mixture is cooled and the solvent is distilled off under reduced pressure. The residue was subjected to preparative chromatography (TCX) on silica gel plates using 10% acetone in methylene chloride as an eluent to obtain 18-thiomarkfortin A.T. pl. 128 - 260 ^o C.

FAB-MS 494 (M ^{+ +} H)
According to the general method of Example 2, but using the appropriate 18-thiomarkfortin compounds below prepared using the appropriate marcfortine starting material:
1-methyl-18-thiomarkfortin A
1-benzyl-18-thioamarkfortin A
1-ethyl-18-thiomarkfortin B
1-benzyl-18-thiomarkfortin B
18a-ethyl-18-thiomarkfortin B
18a-benzyl-18-thiomarkfortin B
18a-methoxyethoxymethyl-18-thiomarkfortin B
18a-allyl-18-thiomarkfortin B
18a-propargyl-18-thiomarkfortin B
1,18a-bis-ethyl-18-thiomarkfortin B
1,18a-bis-benzyl-18-thiomarkfortin B
18a-ethyl-24-methoxy-18-thiomarkfortin B
18-thiomarkfortin B (Compound # 2A)
FAB-MS 480 (M ^{+ +} H)
18a-ethyl-24.25-dihydro-18-thiomarkfortin B
1-propyl-18-thiomarkfortin C
1-benzyl-18-thiomarkfortin C
18a-propyl-18-thiomarkfortin C
18a-benzyl-18-thiomarkfortin C
18a-methoxyethyl-18-thiomarkfortin C
18a-allyl-18-thiomarkfortin C
18a-propargyl-18-thiomarkfortin C
1,18a-bis-propyl-18-thiomarkfortin C
1,18a-bis-benzyl-18-thiomarkfortin C
18-thiomarkfortin C
1-palmitoyl-18-thiomarkfortin A
1-decanoyl-18-thiomarkfortin A
1-decanoyl-18-thiomarkfortin B
Example 3. 24, 25 dihydro-18-thiomarkfortin A
A mixture consisting of 15 mg of 5% palladium on carbon and 18-thiomarkfortin A (30 mg, 0.6 mmol) in 1 ml of methanol was vigorously stirred in a hydrogen atmosphere for 45 minutes. The reaction mixture was filtered through ^Celite® and the filtrate was evaporated under reduced pressure. Preparative thin layer chromatography of the residue on a 0.5 mm silica gel plate eluted with 3% methanol in methylene chloride affords 24, 25-dihydro 18-thiomarkfortin A.

Example 4. 24.25-dibromo-24.25-dihydro 18-thiomarkfortin A
A solution of bromine in chloroform (0.4 ml of a 12M solution, 0.048 mmol) is added dropwise to a chilled (ice bath) solution of 18-thiomarkfortin A (20 mg, 0.04 mmol) in 2 ml of chloroform. The resulting yellow solution was stirred at room temperature for 15 minutes and then evaporated in a stream of nitrogen. Preparative thin layer chromatography of the residue on a 0.5 mm silica gel plate eluted with 2% methanol in methylene chloride affords 24, 25 - dibromo-24.25-dihydro-18-thiomarkfortin A.

Example 5. 24-methoxy-24,25-dihydro 18-thiomarkfluorin B
A solution of bromine in chloroform (0.6 ml, 0.12 M solution, 0.072 mmol) is added dropwise to a cooled (ice bath) solution of 18-thiomarkfortin B (28 mg, 0.06 mmol) in 2 ml of chloroform. The resulting yellow solution was stirred at room temperature for 10 minutes, then at 0 ^° C. for 20 minutes, then evaporated in a stream of nitrogen. The yellow solid residue was dissolved in 2 ml of methanol, then 1,8-diazabicyclo [5.4.0] undec-7-ene (DWI, 0.015 ml, 0.10 mmol) was added. The solution was stirred at room temperature for 90 minutes, then evaporated under reduced pressure. Preparative thin layer chromatography of the residue on a 1.0 mm silica gel plate eluted with 2% methanol in methylene chloride affords 24-methoxy-25-bromo-24.25-dihydro-18-thiomarkfortin B. The 24-methoxy-25-bromo thus obtained -24.25-dihydro-18-thiomarkfortin B was dissolved in 2 ml of dry toluene, then tributylin hydride (1.2 ml, 0.45 mmol) was added. The solution was stirred at 100 ^° C. for 16 hours, then evaporated under reduced pressure. Preparative thin layer chromatography of the residue on a 2.0 ml silica gel plate eluted with 2% methanol in methylene chloride affords 24-methoxy-24.25-dihydro-18-thiomarkfluorin B.

Example 6. 24-propoxy 24,25-dihydro-18-thiomarkfluorin A
Substituting methanol for propanol in the method described above for 24-methoxy 24,25-dihydro-18-thiomarcfortin B (Example 5) and applying the modified procedure to 20 g of 18-thiomacrfortin A gives an oily residue. Preparative thin layer chromatography of the crude product on a 0.5 mm silica gel plate eluted with 2% methanol in methylene chloride gives 24-propoxy-24.25-dihydro-18-thiomarkfluorotin A.

Example 7. 1- (dimethylcarbamoyl) -18-thiomarkfluorotin A
Potassium hydride (50 mg of a 25% oil dispersion) is added to a solution of 18-thiomacrfortin A (15 ml, 0.030 mmol) in 1 ml of dry tetrahydrofuran. The solution was stirred at room temperature for 2 hours, then dimethylcarbamoyl chloride (0.028 ml, 0.30 mmol) was added. The mixture was stirred at room temperature for 18 hours, then partitioned between 5% aqueous sodium bicarbonate (1 ml) and methylene chloride (1 ml). The layers were separated and the aqueous layer was extracted with methylene chloride (2 ml). The combined extracts were dried with magnesium sulfate, filtered and evaporated under reduced pressure. Preparative thin layer chromatography of the residue on a silica gel plate eluted with 25% acetone in hexane gives 1- (dimethylcarbomoyl) -18-thiomarkfortin A.

According to example 7, but using the appropriate marcfortine starting material, the following other 18-thiomarkfortin compounds are prepared:
1-acetoxymethyl-18-thiomarkfortin A
1-diethoxyphosphoryl-18-thiomarkfortin A
1-cyclopropylcarbonyl-18-thiomarkfortin A
1-dimethylsulfamoyl-18-thiomarkfortin A
1- (1-piperidinyl) thiocarbonyl-18-thiomarkfortin A
1-succinoyl-18-thiomarkforoin A
1- (2,4-dinitrobenzenesulfenyl) -18-thiomarkfortin A
1- (4-morpholinosulfenyl-18-thiomarkfortin A
1- (p-toluenesulfonyl) -18-thiomarkfortin A
1-acetyl-18-thiomarkfortin A
1-methoxycarbonyl-18-thiomarkfortin A
1- (p-toluenesulfonyl) -18-thiomarkfortin B
1- (2,4-dinitrobenzene sulfonyl) -18a-ethyl-24-methoxy-24,25-dihydro
1- (p-bromobenzene sulfonyl) -18-thiomarkfortin C
1-propionyl-18-thiomarkfortin C
1- (4-carbetoxy-1,3-thiazolidin-3yl) carbonyl-18-thiomacrfortin A.

Example 8. 1-benzyl-18-thio-marfluorin A
Potassium hydride (75 ml of a 25% oil dispersion) is added to a solution of 18-thiomarkfortin A (15 mg, 0.030 mmol) in 1 ml of dry tetrahydrofuran. The solution was stirred at room temperature for 2 hours, then benzyl bromide (0.025 ml, 0.21 mmol) was added. The mixture was stirred at room temperature for 3 hours, then partitioned between water (1 ml) and methylene chloride (1 ml). The layers were separated and the aqueous layer was extracted with methylene chloride (2 ml). The combined extracts were dried with magnesium sulfate, filtered and evaporated under reduced pressure. Preparative thin-layer chromatography of the residue on a 1.0 mm silica gel plate eluted with 3% mathenol in methylene chloride afforded 1-benzyl-18-thiomarc fluorini A. According to Example 8, but using the following other mark-fluorotin starting materials, the following other compounds of 18-thiomarfluorotin were prepared:
1, 18a-bis-ethylene-18-thiomarkfortin B;
1-allyl-18-thiomarkfortin A;
1-methoxymethyl-18-thiomarkfortin A;
1-propargyl-18-thiomarkfortin A;
1-benzyl-18a-tyl-18-thiomarkfortin B;
Example 9. Halmonchus contortus / Trichostrongylus colubriformis / Tird Assay:
This in vivo (in vivo) test uses a gerbil infected with two important target parasites of ruminants, H.contortus (HC) and T. colubriformis (T. C) (helminth-sensitive or resistant worms can be used). Initially, activity was determined only against H. contortus, as described in GAConder et al, T. Parasitol, 76, 168-170 (1990), while in the subsequent stages, activity against both types of parasites was studied using the techniques described in GA Conderetal. , T. Parasitol. 77, 621-623 (1991). The activity is shown below in table. 2.

Example 10. According to example 2, but using the appropriate paraherquamide / thioparaherquamide starting preparation, the following other compounds of 18-thioparaherquamide are prepared:
18-thioparagerquamide
1-methyl-18-thioparaherquamide
1-ethyl-18-thioparaherquamide
1-benzyl-18-thioparagerquamide
1-palmitol-18-thioparagerquamide
1-decanoyl-18-thioparagerquamide
Example 10a According to example 7, but using the corresponding thioparaherkvamide starting preparation, the following other compounds of 18-thioparaherquamide are prepared:
1-acetoxymethyl-18-thiomarkfluorotin
1-diethoxyphosphoryl-18-thioparaherquamide
1-cyclopropylcarbonyl-18-thioparaherquamide
1-dimethiolsulfamoyl-18-thioparaherquamide
1- (1-piperidinyl) thiocarbonyl-18-thioparaherquamide
1-auctionil-18-thioparagerquamide
1- (2,4-dinitrobenzosulphenyl) -18-thioparaherquamide
1- (4-morpholinosulfenyl) -18-thioparaherquamide
1- (p-toluenesulfonyl) -18-type teraherquamide
1-acetyl-18-thioparaherquamide
1-methoxycarbonyl-18-thioparaherquamide
Example 11. According to example 8, but using the appropriate paraherquamide starting material, the following other compounds are prepared
18-thioparagerquamide:
1,18a-bis-ethyl-18-thioparagerquimide
1-allyl-18-thioparaherquamide;
1-methoxymethyl-18-thioparaherquamide;
1-propargyl-18-thioparaherquamide;
1-benzyl-18a-ethyl-18-thioparaherquamide.

 Example 12. 1-pyrrolidinyl-marcfortine A, comp. 12.

Potassium hydride (50 mg of a 25% oil dispersion) is added to a solution of marcfortine A (15 mg, 0.030 mmol) in 1 ml of dry tetrahydrofuran. The solution was stirred at room temperature for 2 hours, then pyrrolidinylcarbonyl (40 mg, 0.30 mmol) was added. The mixture was stirred at room temperature for 18 hours, then partitioned between 5% aqueous sodium bicarbonate (1 ml) and methylene chloride (1 ml). The layers were separated and the aqueous layer was extracted with methylene chloride (2 ml). The combined extracts were dried with magnesium sulfate, filtered and evaporated under reduced pressure. Preparative thin layer chromatography on a plate of silica gel eluted with 25% acetone in hexane gives 1-pyrrolidinyl carbonyl-marcfortine A
Hereinafter in the text:
1) in the spectra of ¹ H-NMR (NMR)
s - singlet - brs widened singlet
m - multiplet
d - doublet
t - triplet
dd - double doublet
2) Element. analysis:
calc - calculated
obsd - found
H ¹ NMR (CDCl ₃ ) δ 0.83 (s, 3H), 1.21 (s, 3H), 1.45 (s, 3H), 1.3-2.4 (m, 17H), 2, 38 (d, 1H), 2.6-2.8 (m, 2H), 2.84 (d, 1H), 3.00 (s, 3H), 3.4-3.6 (m, 4H) 3.72 (d, 1H); 4.85 (d, 1H); 6.41 (d, 1H); 6.84 (s, 2H)
According to the general method of example 12, but using the appropriate macrophortin starting material and carbonyl chloride, the following other macrophortin compounds are prepared:
1 - diethoxyphosphoryl - macrophortin A
1 - cyclopropylcarbonyl - marcfortine A, comp. # 12A.

¹ H NMR (CDCl ₃ ) δ 0.82 (s, 3H), 1.05 (s, 3H), 1.05 (s, 3H), 1.44 (2s, 6H), 0.9-1.7 ( m, 8H), 1.7-3.0 (m, 11H), 3.14 (s, 3H), 3.80 (d, 1H), 4.84 (d, 1H), 6.28 (d , 1H); 6.88 (s, 2H).

1- (1-piperidinyl) thiocarbonyl-macrophortin A
1-auctionil-macrofortin A
1-phenoxycarbonyl-marcfortine A Comp. # 12B.

¹ H NMR (CDCl ₃ ) δ 0.83 (s, 3H), 1.09 (s, 3H), 1.3-1.5 (m, 1H), 1.42 (s, 3H), 1, 46 (s, 3H), 1.5-3.0 (m, 13H), 3.14 (s, 3H), 3.70 (d, 1H), 4.83 (d, 1H), 6.27 (d, 1H); 6.90 (3, 2H); 7.2-7.5 (m, 5H).

1- (2,4-Dichlorophenoxycarbonyl) -18a-allyl-marcfortine B
1- (4-oxapentanoyl) -18a-methyl-marcfortine C
1-palmitoyl-marcfortine A, Comp. # 12C
FABS-MS 716 (M ^{+ +} H)
1- (4-nitrophenoxycarbonyl) -markfortin A, Comp. # 12D ¹ H NMR (CDCl ₃ ) δ 0.90 (s, 3H), 1.09 (s, 3H), 1.3-1.5 (m, 1H), 1.44 (s, 3H), 1.47 (s, 3H), 1.5-2.9 (m, 13H), 3.14 (s, 3H), 3.70 (d, 1H), 4.86 (d, 1H), 6 23 (d, 1H); 6.93 (ABg, 2H); 7.48 (d, 2H); 8.32 (d, 2H).

1- (1-piperidinecarbonyl) marcfortine A, comp. # 12F
FAB-MS 589 (M ^{+ +} H); H RNS m / z (M ⁺ + H, C ₃₄ H ₄₄ N ₄ O ₅ + H ₁ ) calc 589.3390, obsd 589.3398
1 - [[4- (ethoxycarbonyl) piperazin-1-yl] carbonyl] - marcfortine A, comp. # 12G
FAB-MS 662 (M ^{+ +} H); 684 (M ⁺ + Na); HRMS m / z (M ⁺ + H, C ₃₆ H ₄₇ N ₅ O ₇ + H ₁ ), calc 662.3553, obsd 662.3569
1 - [[4- (benzyl) piperazin-1-yl] carbonyl] - marcfortine A, comp. # 12H
¹ H NMR (300mHz, CDCl ₃ ): 0.80 (s, 3H), 1.26 (s, 3H), 1.41 (s, 3H), 1.45 (s, 3H), 1.2- 2.9 (m, H), 2.98 (s, 3H), 3.4-3.8 (m, 8H), 6.41 (d, 1H), 6.84 (s, 2H), 7 2-7.5 (m, 5H), FAB-MS 680 (M ⁺ + H); 702 (M ⁺ + Na)
1 - [[4- (1,3-benzodioxol-5-ylmethyl) piperazin-1-yl] carbonyl] - marcfortine A, comp. # 12I
¹ H NMR (300m Hz, CDCl ₃ ): 0.80 (s, 3H), 1.00-2.60 (m, 15H), 1.41 (s, 3H), 1.45 (s, 3H) 2.63 (t, 1H), 2.84 (d, 1H), 2.98 (s, 3H), 3.45-3.80 (m, 8H), 4.85 (d, 1H), 5.95 (3, 2H), 6.41 (d, 1H), 6.70-6.90 (m, 5H)
FAB-MS 724 (M ^{+ +} H)
1 - [[4- (methyl) piperazyl-1-carbonyl] -markfortin A, Comp. # 12J
¹ H NMR (300mHz, CDCl ³ ): 0.82 (s, 3H), 1.26 (s, 3H), 1.40 - 2.72 (m, 20H), 1.41 (s, 3H), 1.44 (s, 3H), 2.82 (d , 1H) 2.99 (s, 3H), 3.40-3.81 (m, 5H), 4.85 (d, 1H), 6.41 (d, 1H), 6.84 (s, 2H). FAB-MS 604 (M ⁺ + H) $ HRMS m / z (M ⁺ + H, C ₄ H ₄₅ N ₅ O ₅ + H ₁ ), Calc 604.30499, obsd 604.3506
1- (1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl) -markfortin A, Comp. # 12K FABS-MS 665 (V ^H )
1 - [[4-pyridin-2-yl) piperazin-1-yl] carbonyl] -markfortin A, Comp. 12 # L ¹ H NMR (300m Hz, CDCl ₃ ): 0.84 (s, 3H), 1.10-2.30 (m, 12H), 1.41 (s, 3H), 1.44 (s, 3H), 2.40 (d, 2H), 2.67 (t, 2H), 2.85 (d, 1H), 3.00 (s, 3H), 3.45-3.85 (m, 9H), 4.85 (d, 1H), 6.40 (d, 1H). 6.60-6.70 (m, 2H), 6.85 (s, 2H), 7.52 (t, 1H), 8.15-8.22 (m. 1H), FAB-MS 667 (M ⁺ + H)
1 - [[4- (phenyl) piperazin-1-yl] carbonyl] -markfortin A, Comp. # 12M ¹ H NMR (300 m Hz, CDCl ₃ ): 0.84 (s, 3H), 1.20-2.50 (m, 11H), 1.22 (s, 3H), 1.42 (s, 3H), 1.45 (s, 3H), 2.67 (t, 1H), 2.85 (d, 1H), 3.00 (s, 3H), 3.10-3.91 (m, 9H), 4.85 (d, 1H), 6.40 (d, 1H), 6.85 (s , 2H), 6.90-7.00 (m, 3H), 7.25-7.38 (m, 2H)
1 - [[4- (chlorocarbonyl) piperazin-1-yl] carbonyl] -markfortin A. Soed. # 12N
¹ H NMR (300m Hz, CDCl ₃ ): 0.74 (s, 3H), 1.18 (s, 3H), 1.30-2.65 (m. 20H), 2.74 (d, 1H), 2.81 (s, 3H), 3.48- 3.90 (m, 8H), 4.30 (d, 1H), 6.32 (d, 1H), 6.80 (s, 2H), FAB-MS 652 (M ⁺ + H)
10.10 '' - (1,4-dicarbonylpiperazine) bis (6 ', 7', 8 ', 9', 10 ', 10' ahexahydro-1 ', 1', 4,4,12 - pentamethyl) - [ 2'S - [2 'alpha, 3'a, alpha, 9'a alpha, 10/2''' R ^* , 3 '''a S ^* , 10''' a R ^* ), 10'a. beta]] - S pyro [4H, 8H - [1,4] dioxepino [2,3-g] indole - 8.2 '(3'n) - [1H, 4H - 3a, 9a] (iminomethane) cyclopenta [ b] quinolysin] - 9, II '(10H) - dione, Comp. # 12O
FAB-M ^{May 1093} (M ^{+ +} H)
1 - [[4- (phenyl) piperidin-1-yl] carbonyl] -macfortin A, Comp. # 12P ¹ H NMR (300m Hz, CDCl ₃ ); 0.76 (s, 3H) 1.10 - 2.30 (m, 13H), 1.14 (s, 3H), 1.35 (s, 3H), 1.38 (s 3H), 2.33 (d, 1H), 2.52-3.00 (m, 8H) , 3.00 - 3.20 (m, 1H), 3.50 - 3.75 (m, 2H), 4.05 (d, 1H), 4.33 (d, 1H), 4.79 (dd, 1H), 6.35 (dd, 1H), 6.78 (s , 2H), 7.10 - 7.30 (m, 5H). FAB-MS 665 (M ^{+ +} H)
1 - [[4- (dimethyl) piperidin-1-yl] carbonyl] - marcfortine A, Comp. 12Q ¹ H NMR (300m Hz, CDCl ₃ ); 0.75 (s, 3H), 0.90 - 1.95 (m, 13H), 0.92 (s, 3H), 0.96 (s 3H), 1.14 (s, 3H), 1.34 (s, 3H), 1.37 (s, 3H), 2.08 (d, 1H), 2.20 (brs, 1H), 2.33 (d, 1H), 2.60 (t, 2H), 2.77 (d, 1H), 2.92 (s, 3H), 3.45 - 3.75 (m, 4H) 4.77 (d 1H); 6.33 (d, 1H); 6.77 (s, 2H). FAB-MS 617 (M ^{+ +} H)
1 - [[4- (5-chloropyridazin-3-yl) piperazin-1-yl] carbonyl] - marcfortine A, Comp. 12 R ¹ H NMR (300m Hz, CDCl ₃ ): 0.83 (s, 3H), 1.20-2.30 (m, 11H), 1.22 (s 3H), 1.42 (s, 3H), 1.44 (s, 3H), 2.45 (d, 1H), 2.65 (t, 2H), 2.83 (s, 1H), 2.99 (s, 3H), 3.60-3.85 (m, 8H), 4.86 (d, 1H), 6.40 (d, 1H), 6.86 (s, 2H), 6.92 (d, 1H), 7.29 (d, 1H). FAB -MS 702 (M ⁺ + H).

1 - [[4-chlorophenyl) piperazin-1-yl] carbonyl] - marcfortine A, Comp. # 12S ¹ HNMR (300m Hz, CDCl ₃ ); 0.84 (s, 3H), 1.20 - 2.30 (m, 9H), 1.26 (s, 3H), 1.42 (s, 3H), 1.45 (s, 3H), 2.40 (d, 1H), 2.60 - 2.75 (m, 2H), 2.85 (d, 1H), 3.00 (s, 3H), 3.15 - 3.30 (m, 4H), 3.49 (d, 1H), 3.52 - 3.84 (m, 5H), 4.85 (d, 1H), 6.39 (d, 1H), 3.15 - 3.30 (m, 4H), 3.49 (d, 1H), 3.52 - 3.84 (m, 5H), 4.85 (d, 1H), 6.39 (d, 1H), 6.80 - 6.90 (m, 4H); 7.23 (d, 2H).

10'10 '' - (1,4-dioxo-2-butene) bis (6 ', 7', 8 ', 9', 10'a- hexahydro-1 ', 1', 4,4,12'- pentamethyl) - [2'S - 2'alpha, 3'a alpha, 3'a alpha, 9'a, alpha, 10 (2 '''R ^* , 3''' a S ^* 9 '''a S ^* , 10 '''a R ^* ), 10 "a beta]] - S-pyro [4H, 8H- [1,4] dioxepino [2,3-g] indole-8,2'(3'H) - [ 1H, 4H-3a, 9a] (iminomethane) cyclopenta [b] quinolizine] -9, 11 '(10H) -dione, compound # 12T
FAB - MS 1035 (M ^{+ +} H)
1 - [[4- (trichloroethoxycarbonyl) piperazin-1-yl] carbonyl] -markfortin A, compound # 12U
¹ H NMR (300m Hz, CDCL ₃ : 0.75 (s, 3H), 1.1-1.85 (m, 8H), 1.15 (s 3H), 1.34 (s, 3H), 1.34 (s, 3H), 1.92 (d, 1H), 2.00-2.40 (m, 2H), 2.34 (d, 1H), 2.55 (t, 24), 2.7b (d, 1H), 2.91 (s, 3H), 3.42-3.75 (m, 8H), 4.65-4.77 (m, 2H), 4.79 (d, 1H), 6.33 (d, 1H), 6.79 (s, 2H), FAB-MS 764 (M + H); HRMS m / z (M ⁺ + H, C ₆ H ₄₄ Cl ₃ N ₅ O ₇ + H ₁ ), sal 764.2384, obsd 764.2366
1 - [[4- (3-trifluoromethyl-thiadiazol-1yl) piperazin-1-yl] carbonyl] -markfortin A, compound # 12V
NMR (300m Hz, CDCl ₃ ): 0.83 (s, 3H), 1.26 (s, 34), 1.30-2.30 (m, 11H), 1.42 (s, 3H), 1.45 (S, 3H), 2.42 (d, 1H), 2.55-2.72 (m, 2H), 2.84 (d, 1H), 2.99 (S, 3H), 3.60-3.90 (m, 8H), 4.87 (d, 1H), 6.30 (d, 1H), 6.88 (s, 2H), FAB-MS742 (M ⁺ + H); HRMS m / z (M ⁺ + H, C ₃₆ H ₄₂ F ₃ N ₇ S ₁ + H ₁ ), calc 742.2998, obcd 742.3038
Example 13. 1-acetoxymethyl-marcfortine A, Comp. # 13
Sodium hydride (467 mg of a 60% oil dispersion) is added to a solution of marcfortine A (850 mg, 1.78 mmol) in 1 ml of tetrahydrofuran. The solution was stirred at room temperature for 4 hours and then methyl bromoacetate (280 mg 1.82 mmol) was added. The mixture was stirred at room temperature for 8 hours, then partitioned between 5% sodium bicarbonate (1 ml) and methylene chloride (1 ml). The layers were separated and the aqueous layer was extracted with methylene chloride (2 ml). The combined extracts were dried with magnesium sulfate, filtered and evaporated under reduced pressure. Chromatography on a silica gel plate, eluting initially with 5%, then 10% acetone in methylene chloride, gives 1 acetoxymethyl-marcfortine A as a colorless oil:
¹ H NMR (CDCl ₃ ) δ 0.75 (3H, s) 1.08 (3H, s), 1.43 (3H, s), 1.45 (3H, s), 2.05 (3H, s), 3.12 (3H, s), 4.86 (1H, d, J = 8), 5.85 (1H, d, J = 8), 5.96 (1H, d, J = 8), 6.26 (1H, d, J = 8), 6.77 (1H, d , J = 7), 6.86 (1H, d, J = 7).

According to example 13, but using the appropriate marcfortine starting material, the following other compounds of marcfortine are prepared:
1-t-butyryloxymethyl-marcfortine A
1-benzoyloesimethyl-marcfortine A
1-t-butyryloxymethyl-marcfortine A
1-acetoxymethyl-18a-benzyl marcfortine B
1-3,4-dichlorobenzoyloxy-marcfortine C
Example 14. 1- (2,4-Dinitrobenzenesulfenyl) -markfortin A, Comp. 14
Markfortin A (75 mg, o, 15 mmol) is added to a suspension of potassium hydride (180 mg, 1.50 mmol, 35 wt.% In vegetable oil) in THF (6 ml) at 5 ^o C followed by 2.4 -dinitrobenzenesulfenyl chloride (73 mg, 0.30 mmol). The reaction mixture was then stirred for 3 hours at 5 ^° C. After dilution with water and extraction with chloroform, the crude product was chromatographed on a 1 mm silica gel plate (TLC), eluting with 30% acetone in methylene chloride to give 1- (2,4-dinitrobenzenesulfenyl) -markfortin A (90 mg, 89.1%) as a yellow solid. Mp 155-160 ^° C (decomp.).

¹ H NMR (CDCl ₃ ); δ 0.86 - 0.97 (m, 3H), 1.13 - 1.45 (m, 15H), 1.56 - 1.78 (m, 5H), 1.86 (m, 1H), 2.00 (m, 1H), 2.15 (d, 1H), 2.30 (m, 1H), 2.45 (d, 1H), 2.67 (bd, 1H), 2.79 (d, 1H), 2.97 (m, 1H), 3.08 (s, 3H), 3.74 (d, 1H), 4.76 (bd, 1H), 5.85 (d, 1H), 6.87 (d, 1H), 6.97 (d, 1H), 7.28 (m, 0.5H), 7.62 (m, 0.5H), 8.36 (dd, 1H), 9.16 (d, 1H).

According to example 14, but using the appropriate marcfortine starting material, the following other compounds of marcfortine are prepared:
1- (4-morpholinosulfenyl) -markfortin A, Comp. # 14A
FAB-MS; m / e, 595 (m ⁺ + H)
1- (trichloromethylsulphenyl) -markfortin A, Comp. # 14B
FAB-MS; m / e, 626, 628, 630 (M ⁺ + H)
1- (methoxycarbonylsulphenyl) -markfortin A, Comp. # 14C)
FAB-MS; m / e, 568 (M ⁺ + H)
1- (benzenesulfenyl) -markfortin A, Comp. # 14D
FAB-MS; m / e, 586 (M ⁺ + H)
1-trichloromethylsulphenyl-18a-ethyl-marcfortine B
1-ethoxycarbonylsulphenyl-18a-methyl-marcfortine C
Example 15. 1- (4-Morpholinecarbonyl) -markfortin A N-oxide, Comp. # 15
1- (4-Morpholinocarbonyl) -markfortin A (Comp. # 12E, 15 mg) was treated with 3-chloronadbenzoic acid (15 mg) in methylene chloride (2 ml) at 0 ^° C for 10 minutes. The mixture was partitioned between 5% sodium bicarbonate (2 ml) and methylene chloride (2 ml). The layers were separated and the aqueous layer was extracted with methylene chloride (2 ml). The combined extracts were dried with magnesium sulfate, filtered and evaporated under reduced pressure. The residue was chromatographed on a 0.5 mm preparative thin layer silica gel plate, eluting with 10% methanol in methylene chloride to give the title compound (10 mg) as a solid. FAB - MS; 607 (M ⁺ + H).

Example 16 1- (2-Tetragylrofuranyl) Marcfortine A, Comp. # sixteen
Carbonylimidazole (50 mg) was added to a solution of marcfortine A (60 mg) in 6 ml of tetrahydrofuran. The mixture was heated to boiling under reflux and potassium hydride (120 mg) was added. The resulting mixture was stirred while continuing to boil under reflux for 1 h. The precipitate was filtered off and the filtrate was concentrated. The residue was chromatographed on a silica gel plate eluting with 25% acetone in methylene chloride to give the title compound (30 mg) as a solid. HRM S (FAB); m / e (M ⁺ + H), C ₃₂ H ₄₁ N ₃ O ₅ + H), calc. 548, 3124, obsd. 548, 3086.

Example 17. 1- (4-Morpholinecarbonyl) -markfortin A, Comp. # 12E
Markfortin A (1 g, 2.1 mmol) in THF (25 ml, purified by distillation over metallic sodium from benzophenone) and potassium hydride (35 wt.% 0.5 g, 4.4 mmol) are stirred for 0.5 h at room temperature in a nitrogen atmosphere. Morpholinecarbonyl chloride (1 ml, 8.4 mmol) is then added via syringe. After stirring at room temperature for 3 hours, the turbid reaction mixture was cooled in an ice bath and the reaction was quickly stopped by the addition of a cooled saturated sodium carbonate solution (5 ml) dropwise. The mixture was then diluted with water (75 ml) and extracted with CH ₂ Cl ₂ (175 ml). The organic layer was dried over magnesium sulfate, filtered and concentrated to obtain a crude product containing oil. Silica gel chromatography (2% to 5% MeOH / CH ₂ Cl ₂ ) gives 1-morpholincarbonyl-marcfortine A. (Silica gel chromatography on chromatotron (4% MeOH / CH ₂ Cl ₂ ) is the best purification method for 1-morpholincarbonyl-marcfortine A)
¹ H NMR (300 MHz, CDCl ₃ ); δ 0.81 (s, 3H), 1.21 (s, 3H), 1.19 - 1.94 (m, 8H), 1.41 (s, 3H), 1.45 (s, 3H), 1.98 (d, 1H), 2.08 - 2.28 (m , 2H), 2.40 (d, 1H), 2.62 (t, 2H), 2.82 (d, 1H), 2.99 (s, 3H), 3.50 - 3.86 (m, 8H), 4.86 (d, 1H), 6.41 ( d, 1H); 6.85 (s, 2H); FAB-MS 591 (M ⁺ + H); HRMS M / Z (M ⁺ + H, C ₃₃ H ₄₂ N ₄ O ₆ + H ₁ ), calc. 591.3182, obsd 591. 3200.

Example 18. According to example 12, but using the appropriate paraherquamide starting material, the following other paraherquamide compounds are prepared:
1-diethoxyphosphoryl-paraherquamide
1-cyclopropylcarbenyl-parageoquamide
1- (1-piperidinyl) thiocarbonyl-paraherquamide
1-succinoyl paraherquamide
1- (4-carbethoxy-1,3-thiazolidin-3-yl) carbonyl-paraherquamide
1-phenoxycarbonyl-paraherquamide
1-pyrrolidinyl-paraherquamide
1-piperidinylcarbonyl-paraherquamide
1- (4-morpholinecarbonyl) -paragerquamide
Example 19. According to example 13, but using the appropriate paraherquamide starting material, the following other paraherquamide compounds are prepared.

1-t-butyryloxymethyl-paraherquamide
1-benzoloxymethyl-paraherquamide
1-acetoxymethyl-18a-paraherquamide
1-3,4-dichlorobenzoidoxy-paraherquamide
Example 20. According to example 14, but using the appropriate paraherquamide starting material, the following other paraherquamide compounds are prepared:
1- (2,4-dinitrobenzosulphenyl) paragerquamide
1- (4-morpholinosulfenyl) paragerquamide
1-trichloromethylsulfenyl-18a-ethyl-paraherquamide
1-ethoxycarbonylsulphenyl-18a-methyl-paraherquamide
1-palmitol-paraherquamide
Method A
A. Seed enzyme Cunninghamtlla cchinulata subsp elegans (-) NRRL 1368, Cunningha-mella blakesleeana (+) Atcc SbSSa, Cunnirghamella echinulata subsp. sllgans (-) NRRL 1393, Cunnirghamella echinulata NRRL 3655.

Frozen agar plugs NRRL 1368 or 1393, ATTC 8688a or preferably NRRL 3655 (stored in liquid nitrogen "LN ₂ ") aseptically transfer 100 ml volume of GS-7 (sterile) medium contained in a 500 ml wide-neck shaking flask.

GS-7 is made up of (Cerelose) and Pharmamelig (Pharmaceuticals), each added at a concentration of 25 g / l of tap water. The pH of the medium is adjusted to 7.2 using ammonium hydroxide. The medium is sterilized by autoclaving for 30 minutes. The inoculated GS-7 is shaken at 150-300 (preferably 250 rpm) at 21-30 ^° C, preferably about 28 ^° C for 24-72 hours (preferably about 48-72 hours). This enzyme is used to inoculate the biotransformation process at about 1 to 5% dose (preferably about 5% dose).

B. Biotransformation Method
100 ml volumes of GS-7 contained in 500 ml wide-necked flasks were inoculated for the enzyme for 48-72 hours with seed enzyme at about 1-5% dose (preferably about 5% dose). Inoculated CS-7 is shaken at approximately 250 rpm at 28 ^° C. for 24-48 hours. During this time, Marcfortine A dissolved in dimethylformamide (DMF) is added to the enzyme in the range between 10-12 mg / flask, preferably at 10 mg per 0.4 ml DMF per 100 ml flask / enzyme. Fermentation in the presence of Markfortin A is continued, as previously indicated, for 24-72 hours.

The contents of 100 flasks, each containing 100 ml of enzyme, as described in the method for biotransformation, which was shaken at 28 ^o C for 24-72 hours, followed by the addition of Marcfortine A (0.010 g per flask, total amount 1 g) in DMF (0, 2 ml per flask, 20 ml), treated with CH ₂ Cl ₂ (100 ml) per flask. The contents are combined and mixed in a wacihg mixer for 5 minutes. The mixture is centrifuged and the resulting aqueous layer is decanted. The organic layer is collected, dried (sodium sulfate), filtered and concentrated. The components of the crude mixture are separated by a series of column chromatography using a silica gel-solid extract ratio of 50 - 125: 1. The columns are eluted with either 2-5% methanol in CH ₂ Cl ₂ or 15-60% acetone in CH ₂ Cl ₂ . Marcfortin B. HRMS (FAB) is separated from these chromatographic fractions; m / e found: 4642563; calculated: for C ₂₇ H ₃₃ N ₃ ) O ₄ + H, 4642549.

 If desired, Marcfortine B can be recrystallized from methylene chloride / acetone. For example, a solution of marcfortine B in methylene chloride is concentrated at a residual pressure on a rotary evaporator. The concentrate is diluted in portions of acetone during concentration until a suspension of marcfortine B in acetone remains.

Claims (7)

1. Derivatives of Marcfortine / Paraherquamide of Formula I

where m = 0 or 1;
n is 1;
W = S or O;
R _{1 4 a} and R _{1 4 b} - a hydrogen atom;
R _{2 4} and R _{2 5} form a double bond;
R _{1 8 a} is a hydrogen atom or C ₁ - C ₇ is alkyl;
R ₁ is a hydrogen atom, —C (O) R ¹ is a group (where R ¹ is cyclo C ₃ —C ₈ -alkyl), phenoxycarbonyl, —C (O) -C ₂ -C _{2 4} -alkyl, nitrophenoxycarbonyl, 1 , 3-dioxo-2H-isoindolyl, dinitrobenzenesulfenyl, trichloromethylsulphenyl, C ₁ - C ₄ -alkoxycarbonylsulphenyl, benzenesulfonyl, tetrahydrofuranyl, 1,4-dioxo-C ₂ -C ₇ alkenyl or R ₁ -group (C (O) NR ₄ R ₅ where R ₄ and R ₅ together with the nitrogen atom form a saturated heterocyclic ring selected from pyrrolidinyl, morpholinyl, piperazinyl, optionally substituted C ₁ - C ₄ alkyl, C ₁ -C ₄ -alkanoyl, C ₁ -C ₄ alkoxycarbonyl, 1,3-benzodioxolyl -C ₁ - C ₄ -alkyl, pyridinyl, halogen-pyridinyl, phenyl (optionally substituted by a halogen atom), chlorocarbonyl, trifluoro-C ₁ -C ₄ -alkoxycarbonyl, trifluoro-C ₁ -C ₄ -alkylthiadiazolinyl, or R ₄ and R ₅ form piperidinyl optionally substituted with phenyl or mono- or disubstituted with C ₁ -C ₄ alkyl,
or their pharmaceutically acceptable salts, or their hydrates. 2. The compound according to claim 1, characterized in that the group R ₁ represents -

and

- a saturated heterocyclic ring as defined in claim 1.  3. The compound according to claim 2, where the saturated heterocyclic ring is selected from 4-morpholine, 4-phenyl-1-piperazine, 4- (2-pyridinyl) -1-piperazine, 2,6-dimethyl-4-morpholine, 1- pyrrolidine, 4-methyl-1-piperazine, 1-piperidine, 4-phenyl-1-piperidine, 4-phenylpiperidine, 2-methylpiperidine, 3-methylpiperidine, 4-methylpiperidine, N-methylpiperidine, 1-methylhomopiperazine, 1-acetate.  4. Compounds according to claims 1 to 3, having anthelmintic and acaricidal properties.  5. A method of suppressing insects or nematodes - plant pests, comprising treating the affected cultures with a preparation consisting of an active component and a carrier, characterized in that the compound according to claims 1 to 3 is used as the active component.  6. The strain of the fungus Penicillium sp. NRRL 18887, producer of Marcfortine A and / or Marcfortine C. 7. The compound according to claim 1, characterized in that it represents
18-thiomarkfortin A (compound 2),
18-thiomarkfortin B (compound 2A),
1-pyrrolidinyl-marcfortine A (compound 12),
1-cyclopropylcarbonyl-marcfortine A (compound 12A),
1-phenoxycarbonyl-marcfortine A (compound 12V),
1-palmitoyl-marcfortine A (compound 12C),
1- (4-nitrophenoxycarbonyl) -markfortin A (compound 12D),
1- (1-piperidinecarbonyl) -markfortin A (compound 12F),
1 - [[4- (ethoxycarbonyl) piperazine 1-yl] carbonyl] -markfortin A (compound 12G),
1 - [[4- (benzyl) piperazin-1-yl] carbonyl] -markfortin A (compound 12H),
1 - [[4- (1,3-benzodioxol-5-ylmethyl) piperazin-1-yl] carbonyl] -markfortin A (compound 12I), 1 - [[4- (methyl) piperazin-1-yl] carbonyl] Marcfortin A (Compound 12J),
1- (1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl) -markfortin A (compound 12K),
1 - [[4- (pyridin-2-yl) piperazin-1-yl] carbonyl] -markfortin A (compound 12L),
1 - [[4- (phenyl) piperazin-1-yl] carbonyl] -markfortin A (compound 12M),
1 - [[4-chloro-carbonyl) piperazin-1-yl] carbonyl] -markfortin A (compound 12N),
10.10 '' - (1,4-dicarbonylpiperazine) bis (6 ', 7', 8 ', 9', 10 ', 10'a-hexahydro-1', 1 ', 4,4,12'-pentamethyl ) - [2'S- [2 '. alpha, 3'a.alpha, 9'a.alpha, 10] 2 '''R ^* , 3''' aS ^* 9 '''aS,10''' aR ^* ) -10'a. beta]] - spiro [4H, 8H- (1,4-dioxepino) - 2,3-g [indole-8,2 '- (3'H)] 1H, 4H-3a, 9a [(iminomethane cyclopenta [b ] quinolysin] -9.11 '- (10H) -dione (compound 12O), 1 - [[4- (phenyl) piperidin-1-yl] carbonyl] -markfortin A (compound 12P),
1 - [[4- (dimethyl) piperidin-1-yl] carbonyl] -markfortin A (compound 12Q),
1 - [[4- (5-chloropyridazin-3-yl) piperazin-1-yl] carbonyl] -markfortin A (compound 12R),
1 - [[4- (4-chlorophenyl) piperazin-1-yl] carbonyl] -markfortin A (compound 12S),
10.10 '' - (1,4-dioxo-2-butene) bis (6 ', 7', 8 ', 9', 10 ', 10a-hexahydro-1', 1 ', 4,4,12' -pentamethyl) - [2'S- [2'.alpha, 3'a.alpha, 9'a.alpha, 10 (2 '''R ^* , 3''' aS ^* 9 '''aS ^* , 10'''aR ^* ) 10'a. beta]] - spiro [4H, 8H- (1,4-dioxepino) -2,3-g [indole-8,2'(3'H) -1H, 4H-3a, 9a] (iminomethane) cyclopenta [b] quinolysin] -9.11 '- (10H) -dione (compound 12T),
1 - [[4- (trichloroethoxycarbonyl) piperazin-1-yl] carbonyl] -markfortin A (compound 12U),
1 - [[4- (3-trifluoromethyl-thiadiazol-1-yl) piperazine -1-yl] carbonyl) -markfortin A (compound 12V).

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