SI20073A - Novel polyketides, pyranone derivatives - Google Patents

Abstract

The invention refers to new polyketides, derivatives of pyranone, having the formula I, where A and B have meanings as described in the disclosure, each of new polyketide pyranone derivatives containing a carboxamide group. The new polyketides may be used either independently or as intermediates in the synthesis of new biologically active compounds exerting a potential therapeutical activity.

Description

Tehnični opisTechnical description

Predloženi izum se nanaša na poliketide, derivate piranona, in na postopek za njihovo pridobivanje, ki vključuje tehnike rekombinantne DNA in biosinteze na ta način dobljenih rekombinant. Novi poliketidi se lahko uporabljajo sami ali kot intermediati v proizvodnji novih biološko aktivnih spojin, ki imajo potencialno terapevtsko aktivnost. Posebno se predloženi izum nanaša na nove poliketide, derivate piranona, s formulo (I) kjer A pomeni in B pomemThe present invention relates to polyketides, pyranone derivatives, and a process for their preparation, which includes techniques for recombinant DNA and the biosynthesis of recombinants thus obtained. The new polyketides can be used alone or as intermediates in the production of novel biologically active compounds that have potential therapeutic activity. Specifically, the present invention relates to novel polyketides, pyranone derivatives of formula (I) wherein A is and B is

OH aliOH or

COOHCOOH

OOh

Η,Ν ali označene s tem, da ima vsak od novih poliketidnih derivatov piranona karboksamidno skupino.Η, Ν or characterized in that each of the new polyketide derivatives of pyranone has a carboxamide group.

Stanje tehnikeThe state of the art

Znano je, da so poliketidi velika in strukturno različna družina naravnih spojin s številnimi biološkimi in farmakološkimi lastnostmi. Nekateri primeri, vredni pozornosti, vključujejo antibiotike, kakor oksitetraciklin in eritromicin, zdravila za zdravljenje karcinoma, kot npr. tetracenomicin in doksorubicin, antiparazitno sredstvo avermektin, imunosupresivni rapamicin, zdravilo za zniževanje nivoja holesterola mevinolin in mnoge druge. Poliketidi se sintetizirajo s pomočjo encimskega kompleksa poliketid sintaze (PKS) na podoben biosintetski način kot maščobne kisline, sintetizirane s pomočjo encimskega kompleksa sintaze maščobnih kislin (FAS). PKS aromatskih poliketidov katalizirajo kondenzacijo enostavnih karboksilnih kislin, aktiviranih s koencimom A, z dekarboksilacijsko kondenzacijo. Velika raznolikost struktur naravnih poliketidov je posledica razlike v dolžini verige, izbire začetnih in nadaljevalnih enot in različnih redukcijskih ciklusov, kar vse vpliva na njihovo končno strukturo. Podobnosti v zaporedju amino kislin in mehanizmu delovanja med produkti PKS-genov so privedle do njihove klasifikacije na PKSsestave tipa I in tipa II, po sistemu klasifikacije, uporabljene za FAS (Hopwood in Sherman, Annu. Rev. Genet. 24:37, 1990). PKS modularnega tipa I so odgovorne za biosintezo kompleksnih poliketidov, kot so eritromicin, avermektin in rapamicin in so multifunkcionalni encimi, ki podpirajo številna edinstvena aktivna mesta, organizirana v obliki modula. V nasprotju s temi so PKS tipa II, kot so aromatski poliketidi aktinorodin, tetracenomicin, frenolicin, griseusin in pigment spor (produkt skupka vv/n-gena, katerega strukturo je še potrebno določiti), sestavljeni iz štirih do šestih mono- ali bifimkcionalnih proteinov, katerih aktivna mesta se uporabljajo zaporedno za zgodnjo sintezo in modifikacijo poliketidne verige. V kompleksih poliketidov so popolne skladnosti aktivnih mest in struktur biosinteznih produktov modularnih PKS spodbudile racionalno obliko novih kompleksnih poliketidov z zamenjavo PKS-genov z genetičnimi manipulacijami. Pot k racionalni obliki novih enostavnih aromatskih poliketidov pa ne more biti tako neposredna. Razlog za to so osnovne razlike v načinu, s katerim PKS-geni, odgovorni za biosintezo aromatskih in kompleksnih poliketidov, kontrolirajo strukturo proizvoda.Polyketides are known to be a large and structurally distinct family of natural compounds with many biological and pharmacological properties. Some noteworthy examples include antibiotics such as oxytetracycline and erythromycin, cancer medicines such as. tetracenomycin and doxorubicin, the antiparasitic agent avermectin, immunosuppressive rapamycin, cholesterol-lowering drug mevinolin and many others. The polyketides are synthesized using the polyketide synthase (PKS) enzyme complex in a similar biosynthetic manner to the fatty acids synthesized using the fatty acid synthase (FAS) enzyme complex. AKS of aromatic polyketides catalyze the condensation of simple carboxylic acids activated by coenzyme A with decarboxylation condensation. The great diversity of the structures of natural polyketides is due to the difference in chain length, the choice of initial and continuation units and different reduction cycles, all of which affect their final structure. Similarities in amino acid sequence and mechanism of action between PKS gene products have led to their classification into Type I and Type II PKS compositions by the FAS classification system (Hopwood and Sherman, Annu. Rev. Genet. 24:37, 1990) . Modular type I PKSs are responsible for the biosynthesis of complex polyketides, such as erythromycin, avermectin, and rapamycin, and are multifunctional enzymes that support many unique module-organized active sites. In contrast, type II PKCs, such as the aromatic polyketides of actinorodin, tetracenomycin, phrenolicin, griseusin, and pigment spores (a product of the vv / n-gene assembly whose structure remains to be determined), consist of four to six mono- or bifunctional proteins , whose active sites are used sequentially for the early synthesis and modification of the polyketide chain. In polyketide complexes, the complete consistency of the active sites and structures of the biosynthetic products of modular PKS promoted the rational design of novel complex polyketides by replacing the PKS genes by genetic manipulation. However, the path to the rational form of new simple aromatic polyketides cannot be so direct. This is due to the fundamental differences in the way in which the PKS genes responsible for biosynthesis of aromatic and complex polyketides control the structure of the product.

V nasprotju s popolno skladnostjo med aktivnimi mesti in strukturo produktov PKSgenov tipa I, pa so PKS-geni tipa II vsi strukturno zelo podobni. Glede na to pa na osnovi primarne strukture PKS-genov tipa II ni možno predpostaviti poliketidne strukture njihovih produktov. Zaradi tega je konstruiran poseben sestav vektor gostitelj za ekspresijo genetsko spremenjenih PKS-genov tipa II. Le-ta je sestavljen iz plazmida pRM5 in seva Streptomyces coelicolor CH999, iz katerega je odstranjen celoten skupek aktinorodin (act) genov. Pri poskusih kombiniranja PKS-genov, proizvajalcev različnih aromatskih poliketidov, je ta sestav uporabljen za sintezo mnogih novih aromatskih poliketidnih snovi in za razvoj strategije za njihovo racionalno obliko. Ti poskusi so pokazali, da je za dolžino ogljikove verige v aromatskih poliketidih odgovoren produkt c//gena (Khosla et al., WO 96/40968, 1996 in Hopwood, Chem. Rev. 97:2465, 1997).In contrast to the complete consistency between the active sites and the structure of type I PKSgen products, the type II PKS genes are all structurally very similar. In view of this, the polyketide structure of their products cannot be assumed on the basis of the primary structure of the Type II PKS genes. For this reason, a special host vector construct is constructed for the expression of genetically modified type II PKS genes. It consists of the plasmid pRM5 and the Streptomyces coelicolor CH999 strain, from which the entire set of actinorodin (act) genes is removed. In attempts to combine PKS genes, producers of different aromatic polyketides, this composition has been used to synthesize many new aromatic polyketide substances and to develop a strategy for their rational form. These experiments have shown that the carbon chain length in aromatic polyketides is responsible for the product of the c // gene (Khosla et al., WO 96/40968, 1996 and Hopwood, Chem. Rev. 97: 2465, 1997).

Vrsta S. rimosus, proizvajalec antibiotika oksitetraciklina (OTC), je ena od genetsko najbolje opisanih bakterij med industrijsko pomembnimi streptomicetami. Dva seva: M4018, preučevan v Veliki Britaniji, in R6, preučevan v Zagrebu, se temeljito preiskušata. Skupek genov za biosintezo OTC se klonira iz seva M15883, derivata seva M4018, in njegova restrikcijska mapa je izdelana [Hunter in Hill, Biotechnology of Antibiotics (2. izdaja), W.R. Strohl (ured.) Marcel Decker, New York, str. 21, 1997], Sekvenciranje DNA celotnega skupka je razkrilo funkcijo mnogih genov, za nekatere pa je le-to še potrebno določiti. Skupek o/c-genov seva R6 je prav tako kloniran. Njegova restrikcijska mapa se ne razlikuje od tiste za sev M15883 (Peric, magistrsko delo, Sveučilište v Zagrebu, 1995). Zato lahko podatke, dobljene za sev M15883 in sev R6 popolnoma primerjamo. Za produkt ofcDl-gena se verjame, da sodeluje pri prekrivanju, ciklizaciji in aromatizaciji hipotetičnega nonaketidnega intermediata biosinteze OTC [Hunter in Hill, Biotechnology of Antibiotics (2. izdaja), W.R. Strohl (ured.) Marcel Decker, New York, str. 21, 1997],S. rimosus, the manufacturer of the antibiotic oxytetracycline (OTC), is one of the most genetically described bacteria among the industrially important streptomycetes. Two strains: M4018 studied in the UK and R6 studied in Zagreb are being thoroughly tested. The set of OTC biosynthesis genes is cloned from strain M15883, a derivative of strain M4018, and its restriction map is produced [Hunter and Hill, Biotechnology of Antibiotics (2nd edition), W.R. Strohl (Ed.) Marcel Decker, New York, p. 21, 1997], DNA sequencing of the entire cluster has revealed the function of many genes, and some have yet to be determined. The cluster of o / c genes of strain R6 is also cloned. Its restriction map is no different from that for strain M15883 (Peric, M.Sc., University of Zagreb, 1995). Therefore, the data obtained for strain M15883 and strain R6 can be completely compared. The product ofcDl gene is believed to be involved in the overlap, cyclization, and aromatization of the hypothetical nonacetide intermediate of OTC biosynthesis [Hunter and Hill, Biotechnology of Antibiotics (2nd Edition), W.R. Strohl (Ed.) Marcel Decker, New York, p. 21, 1997],

Opis tehničnega problema s primeriDescription of a technical problem case

Ugotovili smo, da se z zgoraj opisanimi postopki izolirajo novi poliketidi, derivati piranona s formulo I, ki se dobijo iz genetično konstruiranega seva S. rimosus R6ZGL3 s fermentacijo, ekstrakcijo z organskim topilom, zgoščevanjem, čiščenjem z nizkotlačno kromatografijo na silikagelu in na koncu z gelsko filtracijo v koloni Sephadex LH-20, pri čemer se za eluiranje uporabi sistem topil kloroform-metanol.We have found that the above described procedures isolate new polyketides, pyranone derivatives of formula I, which are obtained from genetically engineered strain S. rimosus R6ZGL3 by fermentation, organic solvent extraction, concentration, purification by low-pressure chromatography on silica gel and finally by gel filtration in a Sephadex LH-20 column using a chloroform-methanol solvent system for elution.

Po podatkih, ki so nam dostopni, dobljene spojine do sedaj še niso bile opisane. Novi derivati piranona so označeni s tem, da vsi vsebujejo v strukturi karboksamidno skupino in različne dolžine ogljikovih verig, čeprav v genetično konstruiranem sevu (R6-ZGL3) c//-gen, odgovoren za dolžino ogljikove verige, ni spremenjen (Khosla et al., WO 96/40968, 1996).According to the information available to us, the compounds obtained have not yet been described. New pyranone derivatives are characterized by the fact that they all contain a carboxamide group and different carbon chain lengths in the structure, although in the genetically engineered strain (R6-ZGL3) the c // - gene responsible for the carbon chain length is unchanged (Khosla et al. , WO 96/40968, 1996).

Podroben opis izumaDETAILED DESCRIPTION OF THE INVENTION

V skladu s predloženim izumom so novi poliketidi, derivati piranona, s formulo IAccording to the present invention are novel polyketides, pyranone derivatives of formula I

označeni s tem, da ima vsak od novih poliketidnih derivatov piranona karboksamidno skupino, pripravimo pa jih lahko s postopki, opisanimi v nadaljevanju.characterized in that each of the new polyketide pyranone derivatives has a carboxamide group and can be prepared by the procedures described below.

Bakterijski sevi, plazmidi in pogoji za gojenjeBacterial strains, plasmids and growing conditions

Sev S. rimosus R6 (Hranueli et al., J. Gen. Microbiol. 114-295, 1979) uporabimo za inaktivacijo o/cDl-gena. Genetična konstrukcija seva S. rimosus R6-ZGL3 (otčDl::ermE) je opisana v nadaljevanju. Vse posege na DNA naredimo v celicah bakterije Escherichia coli DH5a (Sambrook et al., Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989). Plazmidno DNA razmnožimo v celicah bakterije E.coli NM544 (dem), da dobimo nemetilirano DNA pred transformacijo celic vrste S. rimosus (Marinus in Palmer, Gene 143:1. 1994).The S. rimosus R6 strain (Hranueli et al., J. Gen. Microbiol. 114-295, 1979) is used to inactivate the o / cDl gene. The genetic construction of S. rimosus strain R6-ZGL3 (otDl :: ermE) is described below. All DNA interventions are made in Escherichia coli DH5a cells (Sambrook et al., Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989). Plasmid DNA is propagated in E. coli NM544 (dem) cells to obtain unmethylated DNA before transformation of S. rimosus cells (Marinus and Palmer, Gene 143: 1. 1994).

Pla2mid vrste roda Streptomyces, pPFZlOl (Butler et al., Mol. Gen. Genet. 215:231, 1989), uporabimo kot izvir gena otcDl. Predhodno je že bilo dokazano, da fragment KpnI dolžine 6 kb (imenovan Kpnl₂6-Kpnl₃₃, na sliki 1) komplementira o/cD-mutanta vrste S. rimosus (Butler et al., Mol. Gen. Genet. 215:231, 1989). Plazmid bakterije E. coli, pIBI24 (Dente et al., Nucl. Acids. Res. j_l:1645, 1983), uporabimo za konstrukcijo inaktiviranega o/cDl-gena z vstavljanjem ermE-gena znotraj njegove strukture. Gen ermE, katerega produkt je odgovoren za odpornost proti antibiotiku eritromicinu (Bibb et al., Gene 38:215, 1985), dobimo iz plazmida pIJ4026 bakterije E. coli (M. Bibb, neobjavljeni rezultati), v katerem je er/uE-gen kloniran znotraj DNA člena vektorja (slika 1). Plazmid vrste roda Streptomyces, pIJ487 (Deng et al., Mol. Gen. Genet. 214:286. 1988), uporabimo za konstrukcijo dvojnega vektorja, ki se lahko razmnožuje v celicah bakterije E. coli, kot tudi v celicah vrste roda Streptomyces. Konstrukcija vseh plazmidov je opisana v nadaljevanju.A Pla2mid of the Streptomyces genus, pPFZlOl (Butler et al., Mol. Gen. Genet. 215: 231, 1989), was used as the source of the otcDl gene. It has previously been shown that a 6 kb long KpnI fragment (termed Kpnl ₂ 6-Kpnl ₃₃ , in Figure 1) complements an S. rimosus / cD mutant of the species (Butler et al., Mol. Gen. Genet. 215: 231 , 1989). An E. coli plasmid of pIBI24 (Dente et al., Nucl. Acids. Res. J_l: 1645, 1983) was used to construct an inactivated o / cDl gene by inserting an ermE gene within its structure. The ermE gene, whose product is responsible for antibiotic resistance of erythromycin (Bibb et al., Gene 38: 215, 1985), is derived from plasmid pIJ4026 from E. coli (M. Bibb, unpublished results), in which er / uE- gene cloned inside the DNA of a vector member (Figure 1). A plasmid of the Streptomyces genus, pIJ487 (Deng et al., Mol. Gen. Genet. 214: 286, 1988), is used to construct a double vector that can reproduce in E. coli cells as well as Streptomyces cells. The construction of all plasmids is described below.

Seve vrste E. coli vzgajamo in vzdržujemo pri standardnih pogojih (Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989). Kompletni tekoči (LM) in surovi (CM) hranljivi medij za vzgojo in vzdrževanje vrste S. rimosus (Hranueli et al., J. Gen.E. coli strains are grown and maintained under standard conditions (Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989). Complete liquid (LM) and crude (CM) nutrient medium for the cultivation and maintenance of S. rimosus (Hranueli et al., J. Gen.

Microbiol. £14:295, 1979) in fermentacijski medij (FM) za biosintezo poliketidov (Vešligaj et al., Appl. Environ. Microbiol. 41.:986, 1981) so opisani predhodno.Microbiol. £ 14: 295, 1979) and fermentation medium (FM) for polyketide biosynthesis (Vešligaj et al., Appl. Environ. Microbiol. 41.:986, 1981) have been described previously.

Manipulacija DNA in mikroorganizmovManipulation of DNA and microorganisms

Postopke za izolacijo in čiščenje kromosomskih in plazmidnih DNA, restrikcijsko cepljenje, elektroforezo v gelu agaroze, prenos DNA na membrano, označevanje DNA z digoksigeninom ter hibridizacijo in detekcijo opisujejo Sambrook et al. (Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).Methods for isolation and purification of chromosomal and plasmid DNAs, restriction vaccination, agarose gel electrophoresis, membrane transfer of DNA, digoxigenin DNA labeling, and hybridization and detection are described by Sambrook et al. (Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

Celice bakterije E. coli transformiramo s pomočjo DNA plazmida z uporabo naprave za elektroporacijo (Bio-Rad Pulsing apparatus) v skladu z navodili proizvajalca, medtem ko celice seva S. rimosus R6 transformiramo z uporabo enakega aparata po postopku, ki ga opisujeta Pigac in Schrempf (Appl. Environ. Microbiol. 61:352, 1995).E. coli cells were transformed using a DNA plasmid using an Bio-Rad Pulsing apparatus according to the manufacturer's instructions, while S. rimosus R6 cells were transformed using the same apparatus according to the procedure described by Pigac and Schrempf (Appl. Environ. Microbiol. 61: 352, 1995).

Konstrukcija plazmida, ki vsebuje inaktivirani o/cDl-genConstruction of a plasmid containing the inactivated o / cDl gene

Konstruiranje plazmid pGLW121, dvojni vektor, ki se lahko pomnožuje v celicah vrst roda Streptomyces in celicah bakterije E. coli, ki vsebuje inaktivirano kopijo o/cDlgena (o/cDl::erznE) iz vrste S. rimosus in del skupka o/c-gena navzgor in navzdol od o/cDl-gena. V prvi stopnji fragment 6 kb (Κ/?ηΙ₂6-Κ///7ΐ₃3, slika 1A) plazmida pPFZlOl, ki vsebuje o/cDl-gen in del skupka o/c-gena navzgor in navzdol od o/cDlgena, kloniramo v vektor pIBI24, da dobimo plazmid pGLW118. 5czcl₂9-restnkcijsko mesto, ki se nahaja znotraj o/cDl-gena izberemo kot mesto, v katerega bomo vstavili ermE-gen. Puščice vzdolž šrafiranih trakov kažejo smer transkripcije o/cDl- in o/cY2gena. Del DNA, ki vsebuje ermE-gen, modificiran tako, da ima dva iSocI-restrikcijska mesta z obeh strani gena, pripravimo takole: 1,7 kb Bg/II-fragment cm/E-gena iz plazmida pIJ4026 vstavimo v £cw?HI-restrikcijsko mesto plazmida pIBI24. Izolirani del DNA, ki vsebuje er/nE-gen, omejen z dvema Sod-restrikcijskima mestoma (eno iz DNA člena vektorja pIJ4026 in drugo iz DNA člena vektorja pIBI24), označimo kot plazmid pGLW119. Nato 5rid-fragment (ki vsebuje c m E-ge n) 1,7 kb iz plazmida pGLW119 vstavimo v restrikcijsko mesto Sacl₂₉ plazmida pGLW118. Vrh puščice iznad Sacl₂₉ kaze mesto vgradnje fragmenta SadermE, dobljenega iz plazmida pGLW119, da nastane pGLW114 (slika 1A). Končno konstrukcijo plazmida pGLW121 ustvarimo s povezovanjem DNA vektorja bakterije E. coli, pGLW114, linearizirane z restrikcijsko endonukleazo EčoRI, z DNA vektorja za vrste roda Streptomyces, pIJ487, ki je prav tako linearizirana z encimom EcoRI (slika IB). Restrikcijska mesta so označena s kraticami: A, ApaE, B, BamHI; Bc, Rc/I; Bg, Rg/II; C, C/αΙ; E, £coRI; EV, EcoRV; H, HindRl, K, Kpnl; P, Pstl; S, Sacl; Sa, Sa/GI; Srn, Smol; Sp, Sphl; St, Sstl; Xb, Xbal; Xh, Xhol; Xm, Xmal (sliki 1A in IB).Construction of plasmid pGLW121, a double vector that can be amplified in Streptomyces and E. coli cells, containing an inactivated copy of S. rimosus o / cDlgen (o / cDl :: erznE) and part of the o / c- gene up and down from the o / cDl gene. In the first stage, a 6 kb fragment (Κ /? ΗΙ ₂ 6-Κ /// 7ΐ ₃ 3, Fig. 1A) of the plasmid pPFZlOl containing the o / cDl gene and part of the o / c-gene cluster up and down from o / cDlgen , clone into pIBI24 vector to produce plasmid pGLW118. 5czcl _{2 The} 9-restriction site located inside the o / cDl gene is selected as the site where the ermE gene will be inserted. The arrows along the screwed bands indicate the transcription direction of the o / cDl- and o / cY2gens. The DNA portion containing the ermE gene modified to have two iSocI restriction sites on either side of the gene is prepared as follows: 1.7 kb Bg / II fragment cm / E gene from plasmid pIJ4026 is inserted into £ cw? HI - PIBI24 plasmid restriction site. The isolated DNA portion containing the er / nE gene bounded by two Sod restriction sites (one from the DNA member of the pIJ4026 vector and the other from the DNA member of the pIBI24 vector) is designated plasmid pGLW119. Then, a 5 kb fragment (containing cm E-ge n) of 1.7 kb from plasmid pGLW119 was inserted into the Sacl ₂₉ restriction site of plasmid pGLW118. The tip of the arrow above Sacl ₂₉ indicates the insertion site of the SadermE fragment obtained from plasmid pGLW119 to form pGLW114 (Fig. 1A). The final construct of plasmid pGLW121 is generated by linking the DNA vector of E. coli, pGLW114, linearized with restriction endonuclease EcoRI, to the vector vector for Streptomyces species, pIJ487, which is also linearized with the EcoRI enzyme (Figure IB). Restriction sites are indicated by the abbreviations: A, ApaE, B, BamHI; Bc, Rc / I; Bg, Rg / II; C, C / αΙ; E, £ coRI; EV, EcoRV; H, HindRl, K, Kpnl; P, Pstl; S, Sacl; Sa, Sa / GI; Srn, Smol; Sp, Sphl; St, Sstl; Xb, Xbal; Xh, Xhol; Xm, Xmal (Figures 1A and IB).

Konstrukcija seva 5. rimosus R6-ZGL3Construction of strain 5. rimosus R6-ZGL3

Inaktivacijo gena za predpostavljeno ciklazo/aromatazo (otcDl-gena) ustvarimo znotraj kromosoma seva S. rimosus R6. Inaktivirano kopijo o/cDl-gena (o/cDl::eraE, prisotna na plazmidu pGLW121) uvedemo v celice seva 5. rimosus R6 z elektroporacijo, transformante pa selekcioniramo na osnovi odpornosti proti antibiotikoma tiostreptonu in eritromicinu. Po rasti celic transformanta v tekočem mediju pri neselektivnih pogojih (v odsotnosti antibiotikov) v času, za katerega predpostavimo, da je prišlo do rekombinacije med DNA plazmida in DNA kromosomov, celice transformanta cepimo na CM-medij, ki prav tako ne vsebuje antibiotikov. V takem sestavu se plazmid pogosto izgubi v času delitve celic [segregacijska nestabilnost plazmida pIJ487 v odsotnosti selekcije, je dobro opisan pojav (Deng et al. Mol. Gen. Genet. 214:286, 1988)]. Do inaktivacije o/cDl-gena v kromosomu pride, kadar kopija otcDl::er/nE gena (prisoten na plazmidu pGLW121 zamenja izvirni o/<?Dl-gen v kromosomu ob stalni izgubi vektorja pIJ487 in pridruženih delov DNA. Da bi potrdili verodostojnost inaktivacije, celotno kromosomsko DNA, izolirano iz izbranih kolonij transformantov, razcepimo z velikim številom restrikcijskih endonukleaz, prenesemo na membrano (Sambrook et al. (Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press,Inactivation of the putative cyclase / aromatase gene (otcD1 gene) is generated within the chromosome of S. rimosus R6 strain. An inactivated copy of the o / cDl gene (o / cDl :: eraE present on plasmid pGLW121) is introduced into the cells of strain 5. rimosus R6 by electroporation, and the transformants are selected on the basis of antibiotic resistance thiostrepton and erythromycin. After the growth of the transformant cells in the liquid medium under non-selective conditions (in the absence of antibiotics) at the time that the recombination between the plasmid DNA and the chromosome DNA was assumed, the transformant cells were grafted onto CM medium that also did not contain antibiotics. In such an assembly, the plasmid is often lost during cell division [segregation instability of plasmid pIJ487 in the absence of selection is a well-described phenomenon (Deng et al. Mol. Gen. Genet. 214: 286, 1988)]. Inactivation of the o / cDl gene in the chromosome occurs when a copy of the otcDl :: er / nE gene (present on the plasmid pGLW121 replaces the original o / <? Dl gene in the chromosome with constant loss of the pIJ487 vector and associated DNA sections. inactivation, whole chromosomal DNA isolated from selected transformant colonies is cleaved by a large number of restriction endonucleases, transferred to the membrane (Sambrook et al. (Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press,

Cold Spring Harbor, Nev/ York, 1989) in hibridiziramo s sondo, ki vsebuje o/cDl-gen (ali fragment ΚρηΙχ-ΚρηΧη).Cold Spring Harbor, Nev / York, 1989) and hybridized with a probe containing the o / cDl-gene (or ΚρηΙχ-ΚρηΧη fragment).

Biosinteza novih poliketidovBiosynthesis of novel polyketides

Sev S. rimosus R6-ZGL3 (oicDl::erwE), hranjen v 40% glicerolu pri temperaturi -70 °C, cepimo v Petrijevih posodicah na surovi CM-medij, ki vsebuje eritromicin (50 pg/ml) [CM-medij na liter vsebuje: 10,0 g ekstrakta slada, 4,0 g ekstrakta kvasa, 4,0 g glukoze, 2% agarja (mas./vol.), destilirane vode do 1000 ml; pH, naravnan na 7,2 z 10% NaOH; sterilizacija: 40 min. pri 110 °C]. Petrijeve posodice inkubiramo v termostatu 5 do 7 dni (pri 25 do 30 °C), nato pa zrastejo posamezne kolonije streptomicet. Z eno kolonijo streptomicet cepimo 50 ml tekočega LM-medija, ki vsebuje eritromicin (50 pg/ml) [LM-medij na liter vsebuje: 40,0 g dekstrina, 16,0 g CSL (približno 50% trdne snovi), 7,0 g CaCO3, 2,0 g (NH₄)₂SO4, destilirane vode do 1000 ml; pH naravnan na 7,2 z 10% NaOH; sterilizacija: 30 min. pri 120 °C] in dobljeno suspenzijo streptomicet gojimo na rotacijskem stresalniku (150 - 300 vrt./min) 48 do 72 ur pri temperaturi od 25 do 30 °C. FM-medij za biosintezo poliketidov, ki vsebuje eritromicin (50 pg/ml), [FM-medij na liter vsebuje: 55 g koruznega škroba, 7,5 g CSS, 7,0 g CaCOi, 8,5 g (NH^hSCri, 2,0 g NHjCl, 0,14 g CoCl₂ x 6H₂O, 10,0 g olja sojinih semenk, vodovodne vode do 1000 ml, naravni pH; sterilizacija: 40 min. pri 120 °C] cepimo z 10% biomase streptomicet iz LM-medija. Biosintezo (fermentacijo) izvedemo na rotacijskem stresalniku (150 - 300 vrt./min.) v času od 5 do 7 dni pri temperaturi od 25 do 30 °C.S. rimosus strain R6-ZGL3 (oicDl :: erwE) stored in 40% glycerol at -70 ° C was vaccinated in Petri dishes on crude CM medium containing erythromycin (50 pg / ml) [CM medium on liter contains: 10.0 g malt extract, 4.0 g yeast extract, 4.0 g glucose, 2% agar (w / v), distilled water up to 1000 ml; pH adjusted to 7.2 with 10% NaOH; sterilization: 40 min. at 110 ° C]. Petri dishes are incubated in a thermostat for 5 to 7 days (at 25 to 30 ° C) and then individual streptomycet colonies are grown. Vaccine 50 ml of erythromycin-containing liquid LM medium (50 pg / ml) with one colony of streptomycetes [LM medium per liter contains: 40.0 g dextrin, 16.0 g CSL (approximately 50% solids), 7. 0 g of CaCO3, 2.0 g (NH _{4) 2} SO 4, distilled water to 1000 ml; pH adjusted to 7.2 with 10% NaOH; sterilization: 30 min. at 120 ° C] and the resulting streptomycet suspension was grown on a rotary shaker (150 - 300 rpm) for 48 to 72 hours at 25 to 30 ° C. FM erythromycin-containing polyketide biosynthesis medium (50 pg / ml), [FM medium per liter contains: 55 g corn starch, 7.5 g CSS, 7.0 g CaCOi, 8.5 g (NH ^ hSCri , 2.0 g NHjCl, 0.14 g CoCl ₂ x 6H ₂ O, 10.0 g soybean seed oil, tap water up to 1000 ml, natural pH; sterilization: 40 min at 120 ° C] is inoculated with 10% biomass streptomycet from LM medium Biosynthesis (fermentation) is performed on a rotary shaker (150 - 300 rpm) for 5 to 7 days at a temperature of 25 to 30 ° C.

Ekstrakcija, zgoščevanje in čiščenje novih poliketidovExtraction, thickening and purification of new polyketides

Fermentacijsko brozgo (1,5 1) seva S. rimosus R6-ZGL3 nasitimo z dodatkom anorganske soli, npr. natrijevega dihidrogenfosfata, in ekstrahiramo z organskim topilom, ki se slabo meša z vodo, npr. n-butanolom, n-oktanolom, kloroformom, etilacetatom, meti-izobutilketonom itd., najboljše z etil-acetatom. Topilo uparimo pri znižanem tlaku in temperaturi od 35 do 50 °C. Različne snovi, ki so v dobljeni surovini, ločimo v frakcije na silikagelu z nizkotlačno kromatografijo in z eluiranjem z zmesjo topil kloroforma-metanola. Frakcije, ki vsebujejo snovi: amid 4-(6-hidroksi-4okso-4H-piran-2-il)-3-okso-butan kislino (1), amid 2-[2,7-dihidroksi-2-(6-hidroksi-4okso-4H-piran-2-metil)-4-okso-kroman-5-il]-ocetno kislino (2), amid 2-[7-hidroksi-5(6-hidroksi-4-okso-4H-piran-2-ilmetil)-4-okso-4H-kromen-2-il]-ocetno kislino in 4-(2karbamoil-2,7-dihidroksi-4-okso-kroman-5-il)-3-hidroksibut-2-en kislino (4), dobljene z eluiranjem z zmesjo topil kloroforma-metanola (94:6), združimo in zgostimo z uparevanjem pri znižanem tlaku. Nato jih ločimo na koloni Sephadex LH-20 in ponovno eluiramo z zmesjo topil kloroforma-metanola. Frakcije, eluirane z zmesjo kloroforma-metanola (94:6), dajo tri posebne derivate piranona, spojine: 1 (15,2 mg), nato 2 (85 mg) in na koncu 3 (6,7 mg). Z nadaljnjim eluiranjem z zmesjo topil kloroforma-metanola (92:8) dobimo četrto spojino 4 (7,3 mg).The fermentation broth (1.5 l) of S. rimosus R6-ZGL3 strain was saturated with the addition of an inorganic salt, e.g. sodium dihydrogen phosphate, and extracted with an organic miscible water miscible, e.g. n-butanol, n-octanol, chloroform, ethyl acetate, meth-isobutyl ketone, etc., preferably with ethyl acetate. The solvent is evaporated under reduced pressure and at a temperature of 35 to 50 ° C. The various substances contained in the resulting feedstock were separated into silica gel fractions by low-pressure chromatography and eluted with a solvent mixture of chloroform-methanol. Fractions Containing Substances: 4- (6-Hydroxy-4-oxo-4H-pyran-2-yl) -3-oxo-butanoic acid amide (1), 2- [2,7-dihydroxy-2- (6- 2- [7-Hydroxy-5 (6-hydroxy-4-oxo-4H- hydroxy-4-oxo-4H-pyran-2-methyl) -4-oxo-chroman-5-yl] -acetic acid (2) pyran-2-ylmethyl) -4-oxo-4H-chromen-2-yl] -acetic acid and 4- (2carbamoyl-2,7-dihydroxy-4-oxo-chroman-5-yl) -3-hydroxybut-2 -en the acid (4) obtained by elution with a solvent mixture of chloroform-methanol (94: 6) is combined and concentrated by evaporation under reduced pressure. They were then separated on a Sephadex LH-20 column and eluted again with a solvent mixture of chloroform-methanol. The fractions eluted with a mixture of chloroform-methanol (94: 6) gave three specific pyranone derivatives, compounds: 1 (15.2 mg), then 2 (85 mg) and finally 3 (6.7 mg). Further elution with a solvent mixture of chloroform-methanol (92: 8) gave the fourth compound 4 (7.3 mg).

Ekstrakcijo, zgoščevanje in čiščenje poliketidov od 1 do 4 spremljamo s tankoplastno kromatografijo (TLC) na silikagelnih ploščah (Merck, kat.št. 15684) z uporabo zmesi topil kloroforma-metanola-vode (9:4:0,1) kot mobilne faze.The extraction, concentration and purification of polyketides 1 to 4 were monitored by thin layer chromatography (TLC) on silica gel plates (Merck, cat. No. 15684) using a solvent mixture of chloroform-methanol-water (9: 4: 0,1) as the mobile phase .

Kemijska karakterizacija novih poliketidovChemical characterization of new polyketides

Kemijsko strukturo dobljenih poliketidov določimo na osnovi 2D homonukleamih in heteronukleamih študij z uporabo COSY-45-tehnike za direktno določevanje H - H konjugacije, HC-COBI tehnike za določevanje H - C interakcije preko ene vezi (^!J) in HMBC-tehnike za določevanje interakcije preko dveh ( J) in treh vezi ( J) (sl. 2 - 5).The chemical structure of the obtained polyketides is determined on the basis of 2D homonucleam and heteronuclei studies using the COSY-45 technique for the direct determination of H - H conjugation, the HC-COBI technique for the determination of H - C interaction via a single bond ( ^! J) and the HMBC technique for determination interactions via two (J) and three bonds (J) (Figs 2 - 5).

IR-spekter razkriva številne absorpcijske trakove med 1716 in 1639 cm'¹, kar kaže na prisotnost karbonilov, značilnih za poliketide.The IR spectrum reveals a number of absorption bands between 1716 and 1639 cm &^lt; ^-1 &^gt;, indicating the presence of polyketide-specific carbonyls.

Priprava novih derivatov piranona je prikazana s spodnjimi Primeri, ki nikakor niso omejevalni faktor za izum.The preparation of novel pyranone derivatives is illustrated by the Examples below, which are by no means a limiting factor for the invention.

Primer 1Example 1

Amid 4-(6-hidroksi-4-okso-4H-piran-2-il)-3-okso-butanske kisline (1)4- (6-Hydroxy-4-oxo-4H-pyran-2-yl) -3-oxo-butanoic acid amide (1)

Z eno kolonijo seva S. rimosus R6-ZGL3 (otcDl::ermEj cepimo 50 ml LM-medija, ki vsebuje eritromicin (50 gg/ml). Dobljeno mikrobno suspenzijo gojimo na rotacijskem stresalniku (200 vrt./min.) 72 ur pri 28°C. Medij za biosintezo poliketidov (FM), ki vsebuje eritromicin (50 pg/ml), cepimo z 10% mikrobne biomase iz LM-medija. Biosintezo (fermentacijo) izvajamo na rotacijskem stresalniku (200 vrt./min.) 7 dni pri temperaturi 28°C. Fermentacij sko brozgo (1,5 1) nasitimo z dodatkom natrijevega dihidrogenfosfata (1,05 kg) in trikrat ekstrahiramo z enakim volumnom (1,5 1) etil acetata. Organske plasti združimo in uparimo pri znižanem tlaku pri 40°C do suhega ostanka. Suhi ostanek (1,5 g) frakcioniramo z nizkotlačno kromatografijo in eluiramo z zmesjo topil kloroforma - metanola (94 : 6). Frakcije, ki vsebujejo snovi 1, 2, 3 in 4, združimo, uparimo pri znižanem tlaku, porazdelimo na koloni Sephadex LH-20 in eluiramo z zmesjo topil kloroforma - metanola (94.6).One colony of S. rimosus R6-ZGL3 strain (otcDl :: ermEj was vaccinated with 50 ml of LM medium containing erythromycin (50 gg / ml). The resulting microbial suspension was grown on a rotary shaker (200 rpm) for 72 hours at 28 ° C. Polyketide biosynthesis medium (FM) containing erythromycin (50 pg / ml) is inoculated with 10% microbial biomass from LM medium Biosynthesis (fermentation) is performed on a rotary shaker (200 rpm) 7 days at 28 ° C. The fermentation slurry (1.5 l) was saturated with the addition of sodium dihydrogen phosphate (1.05 kg) and extracted three times with the same volume (1.5 l) of ethyl acetate. The organic layers were combined and evaporated under reduced pressure. at 40 [deg.] C. to the dry residue The dry residue (1.5 g) was fractionated by low-pressure chromatography and eluted with a solvent mixture of chloroform-methanol (94: 6). Fractions containing substances 1, 2, 3 and 4 were combined, evaporated under reduced pressure, distribute on a Sephadex LH-20 column and elute with a solvent mixture of chloroform-methanol (94.6).

Frakcije, eluirane z zmesjo topil kloroforma - metanola (94 : 6) iz kolone Sephadex LH-20, za katere je iz TLC razvidno, da vsebujejo spojino 1, zberemo in uparimo pri znižanem tlaku, da dobimo 15,2 mg spojine 1 v obliki belega praška.Fractions eluted with a mixture of chloroform-methanol solvents (94: 6) from a Sephadex LH-20 column, which are shown by TLC to contain compound 1, were collected and evaporated under reduced pressure to give 15.2 mg of compound 1 as white powder.

'H NMR (DMSOd-6); δ (ppm): 3,38 (2H, s, C₂); 3,83 (2H, s, C„); 6,05 (H, d, J=2,l C₆); 5,26 (H, d, J=2,1, C»); 7,52 in 7,11 (2H, NH₂); 11,80 (H, s, OH).1 H NMR (DMSOd-6); δ (ppm): 3.38 (2H, s, C ₂ ); 3.83 (2H, s, C '); 6.05 (H, d, J = 2, 1 C ₆ ); 5.26 (H, d, J = 2.1, C »); 7.52 and 7.11 (2H, NH ₂ ); 11.80 (H, s, OH).

¹³C NMR (DMSOd-6); δ (ppm): 167,5 (C_x); 50,1 (C₂); 199,6 (C₃); 46,8 (C₄); 159,4 (C₅); 102,8 (C₆); 170,2 (C₇); 89,0 (C₈); 163,7 (C₉). ¹³ C NMR (DMSOd-6); δ (ppm): 167.5 (C _x ); 50.1 (C ₂ ); 199.6 (C ₃ ); 46.8 (C ₄ ); 159.4 (C _5); 102.8 (C _6); 170.2 (C _7); 89.0 (C _8); 163.7 (C ₉ ).

Primer 2Example 2

Amid 2-(2,7-dihidroksi-2-(6-hidroksi-4-okso-4H-piran-2-metil)4-okso-kroman-5-il]-ocetne kisline (2)2- (2,7-Dihydroxy-2- (6-hydroxy-4-oxo-4H-pyran-2-methyl) 4-oxo-chroman-5-yl] -acetic acid amide (2)

Po fermentaciji in izolaciji, opisani v Primeru 1, dobimo z eluiranjem kolone Sephadex LH-20 z zmesjo topil kloroforma - metanola (94 : 6) frakcije (TLC), ki jih zberemo ter uparimo pri znižanem tlaku in tako dobimo spojino 2 (8,5 mg) v obliki belega praška.Following the fermentation and isolation described in Example 1, eluting with a Sephadex LH-20 column was obtained with a mixture of chloroform-methanol (94: 6) solvent fractions (TLC), which were collected and evaporated under reduced pressure to give compound 2 (8, 5 mg) as a white powder.

*Η NMR (DMSOd-6); δ (ppm): 6,73 in 7,09 (2H, NH₂); 3,82 in 3,70 (2H, dd, >15,2 C₂); 6,31 (H, d, C₄); 10,40 (H, s, Cs-OH); 6,21 (H, d, J=2,4, C₆); 2,55 in 2,97 (2H, dd, >16,0, Cio); 7,13 (H, s, Cn-OH); 2,99 in 3,07 (2H, dd, >14,4, C₁₂); 6,11 (H, d, >2,4, C₁₄); 5,27 (H, d, >2,4, C₁₆); 11,50 (H, s, C₁₇-OH).Η NMR (DMSOd-6); δ (ppm): 6.73 and 7.09 (2H, NH ₂ ); 3.82 and 3.70 (2H, dd, > 15.2 C ₂ ); 6.31 (H, d, C ₄ ); 10.40 (H, s, Cs-OH); 6.21 (H, d, J = 2.4, C _6); 2.55 and 2.97 (2H, dd, > 16.0, C10); 7.13 (H, s, Cn-OH); 2.99 and 3.07 (2H, dd,> 14.4 C _12); 6.11 (H, d,> 2.4, C _14); 5.27 (H, d,> 2.4, C _16); 11.50 (H, s, C ₁₇ -OH).

¹³C NMR (DMSOd-6); δ (ppm): 171,8 (Ci); 41,0 (C₂); 140,0 (C₃); 113,4 (C₄); 163,7 (C₅); 102,2 (C₆); 160,8 (C₇); 111,8 (C₈); 190,3 (C₉); 47,4 (C₁₀); 100,2 (C_n); 44,0 (C₁₂); 160,6 (C₁₃); 103,2 (C₁₄); 170,1 (C₁₅); 89,1 (C₁₆); 162,7 (C₁₇). ¹³ C NMR (DMSOd-6); δ (ppm): 171.8 (Ci); 41.0 (C ₂ ); 140.0 (C ₃ ); 113.4 (C ₄ ); 163.7 (C _5); 102.2 (C ₆ ); 160.8 (C _7); 111.8 (C _8); 190.3 (C ₉ ); 47.4 (C ₁₀ ); 100.2 (C _n ); 44.0 (C _12); 160.6 (C _13); 103.2 (C _14); 170.1 (C _15); 89.1 (C _16); 162.7 (C ₁₇ ).

Primer 3Example 3

Amid 2-(7-hidroksi-5-(6-hidroksi-4-okso-4H-piran-2-ilmetil)4-okso-4H-kromen-2-il]-ocetne kisline (3)2- (7-Hydroxy-5- (6-hydroxy-4-oxo-4H-pyran-2-ylmethyl) 4-oxo-4H-chromen-2-yl] -acetic acid amide (3)

Po fermentaciji in izolaciji, opisani v Primeru 1, in po ekstrakciji spojine 2, zberemo z nadaljnjim eluiranjem kolone Sephadex LH-20 z zmesjo topil kloroforma - metanola (94 : 6) frakcije (TLC), ki jih uparimo pri znižanem tlaku, da dobimo 7 mg spojine 3 v obliki belega praška.After fermentation and isolation as described in Example 1 and after extraction of compound 2, further eluting the Sephadex LH-20 column with a solvent mixture of chloroform-methanol (94: 6) fractions (TLC) was evaporated under reduced pressure to give 7 mg of compound 3 as a white powder.

*H NMR (DMSOd-6); δ (ppm): 7,17 in 7,63 (2H, NH₂); 3,47 (2H, C₂); 6,07 (H, C₄);1 H NMR (DMSOd-6); δ (ppm): 7.17 and 7.63 (2H, NH ₂ ); 3.47 (2H, C ₂ ); 6.07 (H, C _4);

6,76 (H, d, J=2,3, C₈); 10,86 (H, s, C₉-OH); 6,74 (H, d, J=2,3, C₁₀); 4,36 (2H, C_I2);6.76 (H, d, J = 2.3, C _8); 10.86 (H, s, C ₉ -OH); 6.74 (H, d, J = 2.3, C _10); 4.36 (2H, C _I2);

5,55 (H, d, J=2,1, C₁₄); 5,16 (H, d, J=2,1, C₁₆); 11,40 (H, s, C₁₇-OH).5.55 (H, d, J = 2.1, C _14); 5.16 (H, d, J = 2.1, C _16); 11.40 (H, s, C ₁₇ -OH).

¹³C NMR (DMSOd-6); δ (ppm): 168,4 (C,); 40,3 (C₂); 162,1 (C₃); 112,3 (C₄); 177,7 (C₅); 113,8 (C₆); 159,3 (C₇); 102,2 (C₈); 161,4 (C₉); 117,8 (C₁₀); 137,8 (C_lt); 37,4 (C₁₂); 165,7 (C₁₃); 99,6 (C₁₄); 170,6 (C₁₅); 88,2 (C₁₆); 163,9 (C₁₇). ¹³ C NMR (DMSOd-6); δ (ppm): 168.4 (C,); 40.3 (C ₂ ); 162.1 (C ₃ ); 112.3 (C _4); 177.7 (C _5); 113.8 (C ₆ ); 159.3 (C _7); 102.2 (C _8); 161.4 (C ₉ ); 117.8 (C ₁₀ ); 137.8 (C _lt ); 37.4 (C ₁₂ ); 165.7 (C _13); 99.6 (C _14); 170.6 (C _15); 88.2 (C _16); 163.9 (C ₁₇ ).

Primer 4Example 4

4-(2-karbamoil-2,7-dihidroksi-4-okso-kroman-5-il)-3-hidroksibut-2-en kislina (4)4- (2-Carbamoyl-2,7-dihydroxy-4-oxo-chroman-5-yl) -3-hydroxybut-2-ene acid (4)

Potem ko iz kolone Sephadex LH-20 eluiramo spojine 1, 2 in 3, dobimo s spremembo zmesi topil kloroforma - metanola (92 : 8) frakcije (TLC), ki jih zberemo in uparimo pri znižanem tlaku, da dobimo 7 mg spojine 4 v obliki belega praška.After eluting compounds 1, 2 and 3 from Sephadex LH-20 column, the fractions of chloroform-methanol (92: 8) fractions (TLC) were obtained by changing the solvent mixture, which was collected and evaporated under reduced pressure to give 7 mg of compound 4 v white powder form.

NMR (DMSOd-6); δ (ppm): 4,65 (H, C₂); 5,35 (H, C₃-OH); 4,06 in 3,96 (2H, dd, J=15,9, C₄); 6,29 (H, d, J=2,l, C₆); 7,49 (H, s, C₇-OH), 6,18 (H, d, J=2,1, C₈); 3,15 in 2,60 (2H, dd, J=16,0, C₁₂); 2,66 in 2,64 (2H, dd, J=14,5, C_H); 7,20 in 7,61 (2H, Cjr NH₂).NMR (DMSOd-6); δ (ppm): 4.65 (H, C ₂ ); 5.35 (H, C ₃ -OH); 4.06 and 3.96 (2H, dd, J = 15.9, C _4); 6.29 (H, d, J = 2, 1, C ₆ ); 7.49 (H, s, C ₇ -OH), 6.18 (H, d, J = 2.1, C _8); 3.15 and 2.60 (2H, dd, J = 16.0, C _12); 2.66 and 2.64 (2H, dd, J = 14.5, C _H ); 7.20 and 7.61 (2H, Cjr NH ₂ ).

¹³C NMR (DMSOd-6); δ (ppm); 165,2 (Cj); 86,8 (C₂); 163,0 (C₃); 37,4 (C₄); 139,7 (C₅); 113,0 (C₆); 162,8 (C₇); 102,8 (C₈); 161,3 (C₉); 111,3 (C₁₀); 190,6 (Cn); 48,0 (C₁₂); 100,6 (C₁₃); 43,8 (C₁₄); 171,3 (C₁₅). ¹³ C NMR (DMSOd-6); δ (ppm); 165.2 (Cj); 86.8 (C ₂ ); 163.0 (C ₃ ); 37.4 (C ₄ ); 139.7 (C _5); 113.0 (C _6); 162.8 (C _7); 102.8 (C _8); 161.3 (C ₉ ); 111.3 (C ₁₀ ); 190.6 (Cn); 48.0 (C _12); 100.6 (C _13); 43.8 (C _14); 171.3 (C ₁₅ ).

ZaFor

PLIVA farmaceutska, kemijska, prehrambena i kozmetička industrija, dioničko društvo:PLIVA pharmaceutical, chemical, food and cosmetic industry, joint stock company:

Claims (7)

PATENTNI ZAHTEVKIPATENT APPLICATIONS 1. Novi poliketidi, derivati piranona, s formulo I kjer A pomeni in B pomeni ali ali označeni s tem, da ima vsak od novih poliketidnih derivatov piranona karboksamidno skupino.Claims 1. New polyketides, pyranone derivatives of formula I wherein A is and B is or is characterized in that each of the new polyketide derivatives of pyranone has a carboxamide group. \/\ / OHOH 2. Novi poliketid s formulo I, označen s tem, da A pomeni2. A new polyketide of formula I, wherein A is 3. Novi poliketid s formulo I, označen s tem, da A pomeni \/ /3. A new polyketide of formula I, characterized in that A represents \ / / 4. Novi poliketid s formulo I, označen s tem, da A pomeniA new polyketide of formula I, wherein A is OHOH 5. Novi poliketid s formulo I, označen s tem, da A pomeniA new polyketide of formula I, wherein A is HOHO Η,ΝΗ, Ν OH in B pomeni .OH and B means. 6. Postopek za pridobivanje novih poliketidov, derivatov piranona, po zahtevku 1 s formulo I, označen s tem, da konstruiramo sev N rimosus R6-ZGL3 s pomočjo plazmidov pPFZlOl, pIBI24, pIJ4026 in pIJ487, nato sledi fermentacija 7 dni pri temperaturi 28 °C na rotacijskem stresalniku in nato izolacija zmesi spojin amid 4-(6hidroksi-4-okso-4H-piran-2-il)-3-okso-butanske kisline, amid 2-[2,7-dihidroksi-2-(6hidroksi-4-okso-4H-piran-2-metil)-4-okso-kroman-5-il]-ocetne kisline, amid 2-[7hidroksi-5-(6-hidroksi-4-okso-4H-piran-2-iknetil)-4-okso-4H-kromen-2-il]-ocetne kisline in 4-(2-karbamoil-2,7-dihidroksi-4-okso-kroman-5-il)-3-hidroksibut-2-en kisline z ekstrakcijo z organskim topilom in njihovim ločevanjem z nizkotlačno kromatografijo, nato pa sledi ločevanje na koloni Sephadex LH-20 z eluiranjem z zmesjo topil kloroforma - metanola in z uparevanjem frakcij, ki vsebujejo želeno spojino, pri znižanem tlaku do suhega.Process for the production of new polyketides, pyranone derivatives, according to claim 1 of formula I, characterized in that the N rimosus R6-ZGL3 strain is constructed using plasmids pPFZlOl, pIBI24, pIJ4026 and pIJ487, followed by fermentation for 7 days at 28 ° C on a rotary shaker and then isolation of a mixture of the compounds 4- (6-hydroxy-4-oxo-4H-pyran-2-yl) -3-oxo-butanoic acid amide, 2- [2,7-dihydroxy-2- (6-hydroxy- 2- [7-Hydroxy-5- (6-hydroxy-4-oxo-4H-pyran-2- 4-oxo-4H-pyran-2-methyl) -4-oxo-chroman-5-yl] -acetic acid iknetyl) -4-oxo-4H-chromen-2-yl] -acetic acid and 4- (2-carbamoyl-2,7-dihydroxy-4-oxo-chroman-5-yl) -3-hydroxybut-2-ene by extraction with an organic solvent and separation by low-pressure chromatography followed by separation on a Sephadex LH-20 column by eluting with a mixture of chloroform-methanol solvents and evaporating the fractions containing the desired compound under reduced pressure to dryness. 7. Novi poliketidi, derivati piranona, se lahko uporabljajo sami ali kot intermediati v proizvodnji novih biološko aktivnih spojin, ki imajo potencialno terapevtsko aktivnost.7. New polyketides, pyranone derivatives, may be used alone or as intermediates in the production of novel biologically active compounds that have potential therapeutic activity.