Microbial Pyran-2-ones and Dihydropyran-2-ones J. M. Dickinson Chemistry Department The University Stocker Road Exeter EX4 4QD" Selectively reviewing the literature published up until December 1991 1 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.9.1 2.9.2 2.9.3 2.9.4 2.10 2.10.1 2.10.2 2.10.3 2.10.4 2.10.5 2.10.6 2.10.7 2.10.8 2.10.9 2.10.10 3 3.1 3.2 3.2.1 3.2.2 3.2.3 3.3 3.4 3.5 3.5.1 3.5.2 3.5.3 3.6 3.6.1 3.6.2 4 Introduction Microbial Pyran-2-ones Simple Pyran-2-ones 6-A1 kylpyran-2-ones Citreoviridin and Derivatives The Aurovertins Asteltoxin and Citreomontanin The Pyrenocines and Macommelins The Styryl-Pyrones and Related Compounds Pyran-2-ones from the Gliding Bacteria The Phytotoxic Pyran-2-ones Radicinin and Related Compounds The Solanopyrones Poaefusarin and Sporofusarin Colletopyrone Other Pyran-2-ones Phacidin Elasnin Nectriapyrone and Related Compounds Vulgamycin Luteoreticulin Aszonapyrone A Coarctatin Taiwapyrone Stizolobic Acid and Stizolobinic Acid Muscaurin I1 Microbial Dihydropyran-2-ones Pestalotin and Related Compounds Aspyrone and Related Compounds Asp yrone Asperline Phomalactone and their Derivatives Astepyrone The Leptomycins Kazusamycins and Anguinomycins The Phoslactomycins and Related Compounds Other Polyketide-derived Dihydropyran-2-ones Alternaric Acid The Rubratoxins Phomopsolides A and B Non-polyketide Derived Dihydropyran-2-ones Fomannosin 23-Deoxyantheridiol References I Introduction The pyran-2-one moiety is found in a large number of natural products and is responsible for a wide range of biological effects e.g.antibiotic antifungal cytotoxic neurotoxic phytotoxic etc. Pyrones thus constitute an important class of compounds. Although certain aspects of individual naturally- occurring pyrones have been discussed within various reviews,' there does not appear to have been a recent review devoted entirely to these compounds. It is therefore the aim of this article to go some way towards redressing the balance. * Present address School of Chemistry University of Bristol Cantocks Close Bristol BS8 ITS. 0 OCHS I HO H OH (3) Pyrones have been isolated from an extensive range of natural sources such as plants (e.g.kawain (1) from Piper methysticum2) animals (e.g. bufalin (2) from Bufa vulgaris (t~ad)~), and marine organisms (e.g. diemensin A (3) from Siphonaria diemensis4). However in order to keep this review to a sensible length the present discussion will be restricted to pyrones that have been isolated from microbial sources. Literature coverage is by necessity not exhaustive but will highlight articles published up until the end of 1991. This review will be divided into two main sections the first will focus on the fully unsaturated pyran-2-ones whilst the second will look at the partially saturated dihydropyran-2- ones. Aspects of the isolation sturcture elucidation synthesis biosynthesis biological activity and mode of action will be discussed where possible.Fully saturated pyrones (6-lactones) fall outside the scope of this review. 2 Microbial Pyran-2-ones 2.1 Simple Pyran-Zones The simplest pyran-2-ones from both a structural and a biosynthetic viewpoint are the 4-hydroxypyrones triacetic lactone methyl triacetic lactone tetraacetic lactone and de hydroace tic acid. Methyl triacetic lactone (5) was the first of these to be isolated from a natural source Penicillium stipitatum,' and was later isolated from another strain of P. stipitatum along with triacetic lactone (4) and tetraacetic lactone (6).6 Triacetic lactone (4) had also been isolated from two unstipulated Penicillium species.'Dehydroacetic acid had been known as a synthetic compound long before its isolation from Ramaria apiculata.8.It has also been isolated from Hypocrea sulphurea. lo 71 NATURAL PRODUCT REPORTS 1993 OH -HF Scheme 1 0 OH 0 Tetraacetic lactone (6) was first synthesized at the end of the last century,l’ and was shown to be converted to triacetic lactone (4) on treatment with sulfuric acid.l2.I3 Compound (4) has also been synthesized by treatment of the disodio-derivative of acetylacetone with carbon dioxide followed by cyclization of the resultant acid with hydrogen fluoride to give the pyrone (Scheme l).14 Another route through to triacetic lactone (4) involved condensation of malonic acid or malonyl chloride with ethyl acetoacetate.Use of methylmalonyl chloride allowed the synthesis of methyl triacetic lactone (5).I53l6 The first synthesis of dehydroacetic acid (7) was also reported at the end of the last century and was achieved by passing ethyl acetoacetate vapour through a heated iron tube.17 Other routes include treatment of diketene with sodium phenoxide in benzene to give (7) and 2,6-dimethylpyran-4-one,ls and also base (sodium bicarbonate)-catalyzed condensation of ethyl aceto- acetate.lg The biosynthetic origins of the 4-hydroxypyran-2-ones were of considerable interest particularly because of their apparent relationship to the proposed ,!$polyketomethylene inter-mediates on the acetate-methylmalonate pathway. The bio- synthesis of triacetic lactone (4) methyl triacetic lactone (5) and tetraacetic lactone (6)was studied in cultures of Peniciffium stipitatum.2o This organism normally produces the tropolones stipitatonic acid (8) and stipitatic acid (9). However addition of ethionine to the culture medium resulted in accumulation of the pyran-2-ones -these were not encountered under normal fermentation conditions. Small amounts of orsellinic acid (10) and orcinol (1 1) were also isolated. [1-14C]Acetate was administered to cultures of ethionine- inhibited Peniciflium stipitatum and the radioactively-labelled pyrones were isolated. Degradation of tetraacetic lactone (6) gave the expected polyketide-derived labelling pattern (Scheme 2).20Although triacetic lactone and methyl triacetic lactone OH OH were not degraded their labelling pattern was assumed to be as shown.It had been reported that triacetic lactone (4) stimulated the formation of aromatic compounds by Peniciffiumurticae,21and increased stipitatic acid (9) formation in P. sti~itatum.~ Coupled with the fact that the pyrones accumulated under fermentation conditions where normal tropolone biosynthesis was inhibited it was suggested that the pyrones could be precursors of stipitatonic and stipitatic acids and other aromatic metabolites e.g. orsellinic acid orcino1.20 However when radioactively labelled triacetic lactone was incubated with P. stipitatum under normal growth conditions evidence was obtained which indicated that (4) had been broken down into acetate before incorporation into the tropolones and was not therefore a direct precursor.2o This was in agreement with other results which showed that [2-14C]triacetic lactone and [2-14C]tetraacetic acid were degraded to acetate in Penicilfium sp.before being incorporated into acetate-derived metabolites such as 6-methylsalicylic acid.’ The degradation of dehydroacetic acid (7) to triacetic acid via triacetic lactone (4) by a Pseudomonas species has been reported,22 as has the breakdown of triacetic lactone to acetoacetic acid and acetic acid via triacetic acid by rat liver and kidney and beef and rabbit liver h~mogenates.~~. 24 The NATURAL PRODUCT REPORTS 1993-5. M. DICKINSON CH3(CH2)4CH0+ NaCECH CH3(CH2)44H CECH I-OH ..... 11 111 1 CH3(CH2)4 (12) CH3(CH2)4Br+ NaCrCH t CH3( CH2)4C=CH v-vii CH3( C H2)4CECC HO viii / ix -CH3(CH2)400 (13) Reagents i Liq. NH,; 11 CrO,; 11 1 H,C(CO,Et), NaOEt; iv NaOH; v EtMgBr; vi CH(OEt),; vii H+;viii CH,CO,H AcOH; ix A Scheme 3 iii iv -20 (15) Reagents i Ac,O AcOH ; ii Pd/C A; iii NBS hv;iv LiCl Li,CO, DMF A Scheme 4 metabolism of these pyrones therefore appears to be similar in both fungal and animal systems. The biological activity of (4) (5) and (6) does not appear to have been reported. On the other hand the use of dehydroacetic acid (7) as a plasticizer fungicide and bactericide (in toothpaste^),^^ has led to a detailed toxicological study of this compound.26 It has an LD, (oral) of 1000 mg/kg in rats whilst its sodium salt has an LD, (oral) of 570 mg/kg.At higher dosage levels toxic effects include loss of appetite loss of body weight vomiting ataxia and convulsions. 2.2 6-Alkylpyran-Zones Other simple pyran-2-ones to have been isolated from microbial sources are the 6-alkyl- and 6-alkenyl-pyrones. 6-Pentylpyran-2-one (1 3) was the first of these to be identified as a fungal product of Trichoderma viride,*' although it had previously been reported to be a component of peach essence.28 It has since been isolated from strains of Trichoderma harzianum,29 31 Trichoderma ~iride,~~, 33 and an unidentified Aspergillus species,34 from peach35 and ne~tarine~~-~' essence and as an aroma component of roasted beef.39 In a study on pyrone production by cultures of Trichoderma viride the formation of (13) was found to be affected by the carbon source in the fermentation medium.32 6-Pentylpyran-2-one possesses a characteristic coconut odour.Such an odour was noted to be produced by isolates of Trichoderma viride T. koningii and T. hamatum during a study on volatile antibiotic production carried out by Dennis and Web~ter.~ Although the component responsible was not isolated it seems probable in view of the production of 6-pentylpyran-2-one by so many Trichoderma species that (13) was indeed being formed in this case. 6- Pentylpyran-2-one (1 3) has been produced by a strain of Trichoderma kaningii which was also reported -to produce 6- heptylpyran-2-one and a 'dehydro ' derivative of the latter.41 The only evidence presented for the latter two compounds however was the presence of the corresponding molecular ion peaks in the mass spectrum of partially-purified fungal extracts.6-Pentylpyran-2-one (1 3) is a co-metabolite with 6-pentenyl- pyran-2-one (16) in strains of Trichoderma harzianum.4z Compound (16) has also been isolated from a strain of Trichoderma ~iride,~~ and has been identified as a component of the queen pheromone of the red fire ant Solenopsis invicta (B~ren),~~ and of male mandibular gland secretions of the carpenter ants Camponotus pennsylvanicus C. herculeanus and C. noveboracensi~.~~ 6-Propenylpyran-2-one (1 9) named sibirinone has been isolated from Hypomyces sernitransl~cens.~~ The distinctive coconut aroma of (13) has attracted interest in its use as a possible flavourant in the food industry.As a result of this its synthesis was developed in order to ascertain its organoleptic properties before its first isolation from a natural source. The routes developed4' are outlined in Scheme 3. In both cases the key intermediate is the pyrone-acid (12) which is reported to undergo decarboxylation at 200 "C giving the required product. A synthesis of (13) has also been described in which it was an intermediate in the production of the pentenyl derivative (1 6) (Scheme 4).45 The keto-acid (14) (formed via the reaction of dipentylcadmium with ethyl 4-(chloroformy1)butyrate cyclized to give the dihydropyrone (1 5) which on dehydrogen- ation gave (13).4s The conversion of (13) to (16) was achieved via allylic bromination followed by dehydrobromination.A second synthesis of (1 6) has been reported by Rocca et al. NATURAL PRODUCT REPORTS 1993 (17) (18) (16) Reagents i Et,N; ii conc. H,SO ;iii SOCl,; iv H, Pd/BaSO, xylenes; v Butyl(tripheny1)phosphonium bromide base; vi I, benzene Scheme 5 (Scheme 5)., [2+41-Cycloaddition between the ketene derived from crotonyl chloride and trichloroacetyl chloride gave 6- trichloromethylpyran-2-one (17) which was converted to the aldehyde (1 8). Wittig methodology gave trans-6-pentenylpyran- 2-one (16). Sibirinone (19) has been synthesized by dimerization of the crotonyl-derived ketene (Scheme 6).,’ The antifungal properties of 6-pentylpyran-2-one (1 3) have been demonstrated on a number of occasions.It has been shown to significantly inhibit growth of Verticillium dahiiae V. fungicola Pyranoechaeta lysopersici Phomopsis scler~tioides,~~ Gaeumannomyces graminis (Take-all),31*42Chaetomium coch- lioides and C. ~pinusurn.~~ It is also partially effective against Rhizoctonia cerealis Fusarium oxysporum Aspergillus niger,, A. Jlavu~,~~,~~ and Botrytis ~inerea,~”~~ Ceratocystis ~lmi.~’ Pythium ultimum Sclerotinia sclerotiorum and Trichoderma harzianum showed negligible inhibiti~n,,~ and Bacillus subtiiis was not affected.,O Although growth of Sclerotinia sclerotiorum was not inhibited it was found that sclerotial development was significantly affected.This was also noted with Rhizoctonia cerealis.42 High atmospheric concentrations of 6-pentylpyran-2-one (13) affected germination of lettuce seedlings and subsequent de~eloprnent.~~ Compound (13) is reported to be non-toxic to greenhouse grown bean plants (Phaseolus vulgaris) corn plants (Zea mays) or tobacco (Nicotiana tabacum) and significantly inhibits growth of etiolated wheat coleoptile~.~~ Crude extracts of Trichoderma koningii which were reported to contain both 6-pentylpyan-2-one (1 3) and 6-heptylpyran-2- one were shown to be active against Gaeumannomyces graminis Rhizoctonia solani Phytophthora cinnamoni Pythium middle- toni Fusarium oxysporum and Bipolaris sor~kiniana.~~ It seems likely that this biological activity was due mainly to the presence of the 6-pentyl derivative (13) particularly as the length of the alkyl chain in 6-alklylpyran-2-ones has been shown to be crucial to the antifungal activity exhibited.48 A comparison between 6-pentenylpyran-2-one (16) and the 6-pentyl derivative (1 3) has shown that the two pyrones display similar antifungal properties (16) being as effective as (13).48 It has been reported that (16) was partially responsible for the 0 (CH,CH=CHCO)20 i-iii (19) Reagents i 550 “C 0.1 Torr; ii [4 +21 dimerization ; iii NaHCO or P-TsOH Scheme 6 (20) R’=CH3 R2=H (21) R’ = CH3 R2= CH3 Ho induction of oospore formation in Phytophthora ~innamoni.~~ Its biological role in combination with other pheromonal components has been demonstrated with red fire ants Solenopsis invicta (Buren),, but its role with respect to Carpenter ants (Camponotus sp.) from which it has also been isolated has not been e~tablished.,~ Sibirinone (19) appears to be biologically inert and only its inactivity against Staphylococcus aureus has been noted.46 2.3 Citreoviridin and Derivatives In the early part of this century the occurrence of cardiac beriberi in East Asia reached epidemic-like proportions.Mouldy rice was found to be responsible for the observed symptoms the principal mould being identified as Peniciliium citreoviride (P. toxic~rium).~~ The mould was found to produce a toxic principle named citreoviridin (28) which exhibited the bio- logical effects attributed to the micro-organism itself.51 Since then citreoviridin (28) has been found to occur in culture filtrates of Penicillium o~hrosalmone~m,~~~ P.53 P. pul~illorum,~~ pedemont~num,~~ P. ~harlesii,~~ P. ~itreoviride,~~ and Aspergillus terre~s.~’ This latter organism also produced a number of very similar metabolites to citreoviridin referred to as citreoviridins B C D E and F. The structures of citreoviridins C (20) and D (21) were assigned on the basis of spectroscopic data. Isocitreoviridin which is isomeric about the C-13,C- 14 double bond as compared to citreoviridin has also been isolated from culture filtrates of Peniciilium pulvillor~m,~~ but was shown to be an artefact after pure citreoviridin was found to be converted to a mixture of citreoviridin and isocitreoviridin under simulated fermentation and extraction conditions.In addition to citreoviridin (28) the related metabolites citreoviridinol (22) isocitreoviridinol (23) secocitreoviridin (30) and citreo- viral (29) have also been isolated from Penicillium ~pecies.~~-~O The synthesis of citreoviridin (28) has been the subject of a NATURAL PRODUCT REPORTS 1993-5. M. DICKINSON .. Reagents i HNEt,; ii MeI; iii H,O; iv polyphosphoric acid Scheme 7 Reagents i NaNH, NH,; ii CO, Ether; iii HF; iv DMSO K,CO, 2-butanone; v NBS peroxide CCl,; vi (CH,O),P toluene; vii (+)-(A) LDA THF HMPA Scheme 8 i-iv v-vii 0-glucose steps -6Bn viii ix vii x J (29) Reagents i Ac,O-BF,. Et,O 0 "C; ii 0.4 M NaOMe MeOH r.t.; iii NaBH, H,O MeOH (1 :2) r.t.; iv p-TsOH-Drierite acetone r.t.;v H, Pd/C MeOH r.t.; vi NaIO, MeOH :H,O (1 l) r.t.; vii Ph,=C(Me)CO,Me benzene; viii DIBAL-H toluene -78 "C; ix PDC DMF 0 "C; x Amberlite MeOH:H,O (2 I) r.t.; xi mCPBA CH,Cl, 0 "C; xii CsOH CH,Cl, r.t.; xiii MnO, CH,Cl, r.t.; xiv Ph,P=CHCO,Et benzene r.t.; xv (31) benzene Scheme 9 OCH3 OCH H3C (30) (31) Reagents i DMSO K,CO,; ii SeO, dioxan A; iii Ph,=CH-CHO; iv DIBAL-H THF; v MsC1 pyridine CH,Cl,; vi Ph,P benzene; vii NaH THF Scheme 10 number of papers and considerable success has been achieved. was reported in 1985,68 and is outlined in Scheme 9. The The synthesis of the pyrone moiety from y,i?-acetylenic-/?-oxo- absolute configurations of both citreoviridin (28) and citreoviral ester (24) via the enaminoester (25) has been reported (Scheme (29) were established based on D-glucose.7).61A second approach towards the synthesis of the pyrone Pyrone-phosphorane (3 l) widely used in the synthesis of moiety is outlined in Scheme 8.62 Methylacetylacetone (26) was citreoviridin (28) the aurovertins and related compounds was converted to 6-bromomethylpyrone (27) in five steps. Wittig prepared from secocitreoviridin (30) the total synthesis of methodology gave (-)-citreoviridin (28). The tetrahydrofuran which had already been reported.69 The route devised and the portion of the molecule was prepared from ( +)-citreoviral(29) subsequent conversion of (30) into (3l) are outlined in Scheme The for which a number of syntheses have been rep~rted.~~-~' 10.This synthesis enabled the structure of (30) to be established first total synthesis of citreoviridin (28) starting with D-glucose unequivocally -previously two possible structures had been NATURAL PRODUCT REPORTS 1993 H36-& H +A A tA HsCS-(CH2)2CH( NH2)COZH L H3 C -C -' 80H 18' 02 Scheme 11 68 The total synthesis of citreoviridinol (22) has also been reported. 70 Initial studies into the biosynthetic origins of citreoviridin (28) in Penicillium pulvillorum suggested that the molecule was derived from nine molecules of acetic acid and five molecules of methi~nine,'~ although degradation of the [14C]-labelled meta- bolite did not enable confirmation of all the positions of incorporation. Subsequent studies with [1-13C]- and [2-13C] acetate in Aspergillus terreus demonstrated the incorporation of nine acetate units as shown (Scheme 11).5i Oleic acid was suggested as a possible biosynthetic precursor of citreoviridin however feeding with [l-14C]oleic acid resulted in a low incorporation the distribution being consistent with degrada- tion of the acid to acetic acid.Labelling studies with [1-13C]- and [2-13C]acetate in PenicilZium puZvilZorum showed a similar incorporation pattern to that demonstrated in A. terre~s.~~ In addition to this the remaining methyl groups were shown to be methionine-derived and an acetate-starter unit was demonstrated by feeding experiments with [l-13C,2-2H,]acetate. Feeding experiments with [1-13C,180z]a~etate (P. pulvillorum) led to upfield isotope shifts for the C-2 C-4 and C- 6 resonances in the 13C NMR spectrum of (28) indicating that the corresponding carbon-oxygen bonds had remained intact throughout the biosynthetic pathway.Fermentation of cultures under an 1802 atmosphere and simultaneous addition of [l- 13C]acetate demonstrated the origin of the tetrahydrofuran ring oxygens from oxidative processes. i3 The discovery of citreoviridin (28) was as a result of the search for the cause of acute cardiac beriberi which was prevalent in rice-eating countries at the beginning of the century.50 Citreoviridin induced acute poisoning in cats and dogs (intraperitoneally (i.p.) subcutaneously (s.c.)) early symptoms of which were progressive paralysis of the hind legs vomiting and convulsions.Respiratory distress appeared gradually whilst cardiovascular disturbance and hypothermia were marked in the advanced stages. The final stage of toxicity was characterized by dyspnoea gasping and Cheyne-Sto kes respiration followed by respiratory arrest. Subacute ad-ministration in cats led to induced damage of the central nervous system. The LD, in male mice has been determined as 11 (s.c.) and 7.5 (i.p.) mg/kg whilst in female rats it is 3.6 mg/kg (s.c.). Pretreatment of test animals with vitamin B reduced acute intoxication whilst vitamin C led to an increase in the life-span of poisoned mice. Because of the similarity of the clinical manifestations of acute cardiac beriberi in humans with the toxicological effects of citreoviridin in poisoned animals the mycotoxin was presumed to be the cause of the cardiac beriberi that swept East Asia.The toxicity of citreoviridin (28) had been reported pre- vio~sly,~~ where it was noted that a single dose of 100 mg/kg (s.c.) was sufficient to kill all the experimental animals within two hours. Isocitreoviridin on the other hand had no effect at all. Citreoviridin monoacetate (32) is less potent than citreo- viridin (28) whilst the diacetate (33) is virtually ineffecti~e.~~. 75 Hydrogenation of the monoacetate reduced its potency considerably.75 The phytotoxic activity of citreoviridin (28) has been examined to a lesser degree. The compound was found to inhibit growth of wheat coleoptiles at concentrations of M OCH :5& ,.-CH 0 / / / /*--H3C H3C H (32)R' = Ac R2 = H (33) R' = R2 = Ac 3 (34) R1=H R2=CH3 R =R4 =COMe (35)R' = R2 = R4= H R3 = COMe (36) R'=OH R2=CH3 R3=COMe R4= H (37) R' = R3= R4= H R2 = CH3 and of corn seedlings with the inhibitory effects on the latter still in evidence two months after treatment at concentrations of lop2 M.On the other hand tobacco seedlings were not visibly affected by citreoviridin (28) indicating a degree of selectivity in its phytotoxic activity. 2.4 The Aurovertins The aurovertins are a group of mycotoxins related to citreoviridin (28). To date the structures of five aurovertins A (34) B (38) C (35) D (36) and E (37) have been reported. The first isolation of an aurovertin was from Calcarisporium arbu~cula'~-later evidence showed this to be aurovertin D (36).Aurovertin B (38) was also isolated from C.arb~sc~la," and its structure determined.78 At least nine aurovertins were reported in the mycelial extracts of this organism,77 although as mentioned structures have only been proposed for auro- vertins A through to E.i4 The total synthesis of aurovertin (38) has been rep~rted,'~ and the route is outlined in Scheme 12. As with citreoviridin D-glucose was used as starting material and so the absolute configuration of aurovertin B (38) was established on the basis of its derivation from this sugar. The biosynthetic origins of the aurovertins have been determined. Feeding experiments with [1-l3C]acetate in C.arbuscula demonstrated the incorporation of acetate units into aurovertin B (38) as shown in Scheme 13.80 Five units of [Me- 13C]methionine were also incorporated. Incorporation of [1,2-13C]acetate indicated that C(2) and C(3) of the ethyl side chain were derived from an intact acetate unit. These results thus NATURAL PRODUCT REPORTS 1993-5. M. DICKINSON OMe H OCOCH3 Me (38) Reagents i 80 % aq. AcOH; ii NaIO, MeOH-H,O; iii Ph,P=C(Me)CO,Me benzene; iv K,CO, MeOH; v CH,(OMe),-P,O, CHC1,; vi DIBAL-H THF; vii mCPBA CH,Cl,; viii (COCI), DMSO Et,N CH,Cl,; ix Ph,P=CHCO,Et benzene; x CF,CO,H CHC,; xi DIBAL- H THF; xii Trityl chloride pyridine CH,Cl,; xiii Ac,O pyridine r.t. ;xiv p-TsOH MeOH; xv BUtOOH Ti(OPtl), D-( -)-DET CH,Cl,; xvi H,/Pd black MeOH; xvii (COCI), DMSO Et,N CH,Cl,; xviii Ph,P=CHCHO benzene; xix CSA CH,Cl,; xx (31) THF Scheme 12 H&=EO,H +A A OMe t.r ~ H3CS-( CH&CH( NH,)C02H l 0a * H,CCH,CO,H ____t + + A '3 Medium derived Scheme 13 suggested that aurovertin B was derived from a C,,-polyketide precursor with the introduction of a methyl group from the C,- pool onto the methyl carbon atom of the chain-initiating acetate unit.However feeding experiments with [1-13C]pro-pionate demonstrated that this ethyl side chain could also be propionate-derived. Similar results were found for aurovertin D (36).,l Further labelling studies confirmed that aurovertins B (38) and D (36) were in fact derived from a C,,-polyketide which was methylated at C, of this precursor followed by the loss of the starter acetate unit through a retro-Claisen cleavage.*' This was shown by the incorporation of [2-13C]malonate with high enhancement at the positions also labelled by [2-13C]acetate -all atoms were labelled to a similar extent.The previous results with [l-13C]propionate still held and so the question was raised as to whether both pathways operated simultaneously. This was shown to be the case. It appeared that aurovertin C (35) was derived only via the single pathway using the acetate-polymalonateethionine route. Addition of [1-13C,1s02]acetate to cultures of C. arbuscula demonstrated that as in the case of citreoviridin (28) the pyrone oxygens of aurovertins B (38) and D (36) were acetate- derived.82 Fermentation of cultures under an "0 atmosphere and simultaneous addition of [1 -13C]acetate established the derivation of all the remaining oxygens by oxidative processes with the exception of the C(4)-oxygen atom which was presumably derived from the medium.With the above results in hand a detailed mechanism was proposed for the formation of the 2,6-dioxabicyclo[3.2. lloctane moiety from the postulated polyene precursor (39) (Scheme 14).82 The polyene is postulated to undergo monooxygenase- mediated epoxidation using molecular oxygen to give the (3R 4R 5R 6R 7R 8S)-triepoxide (40). Nucleophilic attack by water at C-4 leads to a series of ring closures to generate eventually the 2,6-dioxabicyclo[3.2. lloctane moiety of auro- vertin B (38).The biological activity and particularly the mode of action of the aurovertins has been studied in considerable detail. Whilst there is no apparent antibiotic activity against bacteria or pathogenic fungi toxicity against a number of animals has been noted. i4. i6 Pharmacological effects include brief stimu- lation followed by depression. Hypotension has also been noted along with a marked diuretic effect and enhanced secretion of Na' ions. The LD, for mice is 1.65 mg/kg. Intravenous injection of 1 mg/kg caused death in rabbits in less than 15 minutes and in dogs in less than 50 minutes.i6 Aurovertin does not appear to be significantly metabolized in the body.' Aurovertins B (38) and D (36) have been shown to inhibit oxidative phosphorylation 32Pi-ATP exchange and exchange of l80between PI and water in rat liver mitochondria.They also enhanced ATP hydrolysis induced by selenite selenate and deoxycholate. The hydrolysis of ATP induced by DNP and NATURAL PRODUCT REPORTS 1993 Me I Scheme 14 agents that uncouple oxidative phosphorylation were partially inhibited. It was ~onc1uded~~~~~ that aurovertin inhibited at a site located on the coupling factor F of ATPase. Enzymatic activity of highly purified mitochondrial ATPase from rat liver was found to be dependent on the anion of the buffer used. However aurovertins decreased enzymic activity to the same level regardless of the anion Aurovertins B (38) C (35) and D (36) were reported to have similar levels of activity whilst aurovertin E (37) was only a weak inhibitor of enzyme activity.Aurovertin A (34) was anomalous in its effects in that it was a powerful inhibitor of ADP-stimulated respiration but was impotent as an inhibitor of ATPase The inhibition of ATPase activity of soluble Escherichia coli coupling factor isolated from wild-type E. coli K-12 by aurovertin D (36) was found to be totally lost on acetylation or saponification of this metabolite.86 Aurovertin-resistant mutants of E. coli generated by nitroso- guanidine rnutagene~is,~’ were shown to be altered in the p-subunit suggesting that this was the position of the aurovertin binding site. Further evidence indicated that aurovertin B (38) and citreoviridin (28) bound to nonidentical subunits on the p-subunit of yeast F,-ATPase.88 The binding of citreoviridin was noncompetitive with respect to aurovertin and F,-ATPase obtained from aurovertin-resistant mutants was partly inhibited by citreoviridin.The ATPase activity and ATP-induced energization of photosynthetic membranes from Rhodopseudomonas capsulata a facultative photosynthetic bacterium were reported to be stimulated by phosphate -aurovertins completely inhibited the activity elicited by this anion.89 They also inhibited the energy transfer reactions of R. rubrum and the membrane-bound and soluble ATPases of this organism.go The R.rubrum coupling factor was suggested to be the site of action. Inhibitory action by aurovertins on chloroplast functions has not been found. 74 91 The aurovertins fluoresce weakly in solutions of methanol ethanol or alcohol-water mixtures when irradiated with light at 370 nm with an emission maximum at 470 nm.At lower temperatures or in glycerol solution an enhanced fluorescent intensity is ob~erved.’~ Aurovertin has found use as a fluorescent probe in following structural changes of membrane com-ponent~.~~ Thus when aurovertin was added to fragmented rat liver mitochondria the fluorescence was found to decrease on the addition of ADP. These changes were interpreted in terms of environmental constraints of the probe binding site. The formation of a complex between aurovertin and soluble mitochondrial ATPase (F,) was accompanied by a 55-fold enhancement of fluorescence. 93 This was partially quenched by ATP as previously noted for mitochondrial fragments and by Mg2+.Two binding sites were found on F in the presence of ATP and one site in the presence of ADP Mg2+ and dilute buffer. It was thus proposed that of the two binding sites for ;F1 -ATP ADP ATP Pi ADP Scheme 15 aurovertins only one participated in inhibition of ATPase activity. The fluorescence maxima was found to be during State-3 respiration and was partially quenched on anaerobiosis or on addition of respiratory inhibitor oligomycin or uncoupler suggesting that aurovertin bound cooperatively to State-3 mitoch~ndria.~~ Aurovertin thus induced conformational change in F binding sites in two ways firstly by directly acting as an allosteric effector of an oligomeric system ; and secondly indirectly by inhibiting State-3 respiration which changes the allosteric constant of the oligomeric system.Of the two binding sites for aurovertin one had high affinity binding and the other low affinity. A model was presented (Scheme 15) in which changes of the aurovertin fluorescence reflected conformational changes of the ATPase induced by its ligandsg5 The two conformations termed F and *F1 are in equilibrium. F contained one binding site per mole of enzyme which had a high affinity whilst *F exposed two binding sites one of high affinity and one of low affinity. Changes in the fluorescent properties of aurovertins were explained by a shift in equilibrium between the two conformations induced by binding of ligands to the enzyme.ATP and phosphate induce conformation *F, whilst ADP stabilizes conformation F,. Further evidence has been obtained in support of these conformational changes.96 ’’ The rate of change of fluorescence on addition of the various ligands was found to be pH dependent.98 2.5 Asteltoxin and Citreomontanin Asteltoxin (44) a mycotoxin structurally related to both citreoviridin (28) and the aurovertins was first isolated from toxic maize meal cultures of Aspergillus stellatus and its structure confirmed by single crystal X-ray cry~tallography.~~ NATURAL PRODUCT REPORTS 1993-5. M. DICKINSON Me. 0LeO MeOM' MewBz' PI BzlOwCHO i ii-iii 'c OH O B z H iViX I 1 xii -x xi Me H H Me (43) Reagents i hv;ii mCPBA NaHCO, CH,Cl,; iii THF; 3N HCl (3:1); iv Me,NNH, CH2Cl2 MgSO,; v EtMgBr THF; vi CSA acetone CuSO,; vii Li NH, Et,O; viii o-NO,C,H,SeCN Bu,P THF; ix H,O, THF; x 0,,CH,Cl, MeOH; xi CH,=CHMgBr THF; xii Ac,O Et,N DMAP Scheme 16 H3&-&2H t A H3CS-( CHp)BCH( NH2)C02H 16 II + H3C -C -I 'OH 18' 02 0 + Scheme 17 The polyene-pyrone citreomontanin (41) has been isolated the case of the aurovertins two biosynthetic pathways were in from Penicillium pedemontanum and also from a ci treoviridin- operation -the first in which C-14-3 was derived from producing strain of P.pulviIlorurn.lOO The structure of this methionine-malonate and the second in which these atoms compound has also been determined by means of X-ray were propionate-derived.crystallography and confirmed that the double bonds possessed In order to investigate the mechanism of the proposed 1,2-an all-E configuration. lol shift labelling studies with [I-13C,180,]acetate and with l80 The synthesis of the bistetrahydrofuran moiety of asteltoxin were carried Again as with both citreoviridin (28) and (44) has been reported and the route is illustrated in Scheme the aurovertins the pyrone ring oxygen atoms were found to be 16.1°2 Rearrangement of alcohol (42) gave the bistetra-acetate-derived. The remaining oxygen atoms with the ex-hydrofuran moiety of asteltoxin (43) with the correct relative ception of that attached to C(4) of the bistetrahydrofuran stereochemistry. system are 0 derived suggesting that this latter oxygen is Biosynthetic studies of the origin of asteltoxin in Aspergillus derived from the medium (Scheme 17).stellatus have demonstrated the incorporation of acetate with The mechanism proposed for the formation of asteltoxin (44) the position of label as shown in Scheme 17.1°3Feeding with in A. stellatus is outlined in Scheme 18.1°4 Thus the polyene [1,2-13C2]acetate proved the presence of eight intact acetate (45) was postulated to undergo monooxygenase-mediated units and also indicated the cleavage of a ninth unit via a 1,2-epoxidation to give the (3R 4R 5R 6R 7S 8S)-triepoxide bond migration. It was suggested that this cleavage occurred (46). Nucleophilic attack at C-4 by water would then initiate a via a pinacol or epoxide rearrangement to generate a branched series of reactions including the 1,2-shift resulting in the aldehyde which was subsequently utilized in the formation of formation of the bistetrahydrofuran moiety as shown.the tetrahydrofuran moiety. Both asteltoxin (44) and citreomontanin (41) were both led to incorporation tested for their effect on ATPase activity in Escherichia coli Feeding of (2S)-[methyl-13C]methionine of label at the C-4 and C-5 methyl groups and C-1 of the BF1.105 Whilst citreomontanin (41) was found to be completely bistetrahydrofuran moiety and at the two methyl groups of the inactive asteltoxin (44) was found to inhibit enzyme activity pyrone ring. [1-13C]Propionate was also incorporated with the with a potency intermediate between citreoviridin (28) and signal assigned to C-3 of the bistetrahydrofuran moiety in the aurovertin B (38).Asteltoxin (44) showed a large enhancement 13CNMR spectrum being enhanced. This suggested that as in of fluorescence upon interaction with BF, analogous to the NPR 10 NATURAL PRODUCT REPORTS 1993 1.2-ShiftI Me H Scheme 18 0 aurovertins and showed similar increase in intensity on treatment with ADP and was quenched on addition of Mg2+. However unlike the aurovertins asteltoxin did not enhance the binding affinity of BF for inorganic phosphate. 2.6 The Pyrenocines and Macommelins As well as citreoviridin Penicillium citreoviride has been the source of citreopyrone (58),lo6 and the novel dihydrothio- pyrone citreothiolactone (56).Io7 From a biogenetic point of view these may be derived from a common intermediate as will be discussed later.Citreopyrone (58) has also been isolated from Pyrenochaeta terrestris the causal agent of onion pink root disease and was named pyrenocine A. This latter name will be used in reference to structure (58) in the remainder of this review. The structurally-related pyrenocine B (55) was a co-metabolite.108~ log Pyrenocines A and B were originally erroneously assigned furan structures (47) and (48),loS re- spectively but were reassigned as (58) and (55) on the basis of an X-ray crystal structure determination of pyrenocine A.109 Pyrenocine C (49) has also been isolated from P. terrestris."' The macomellins (64) (59)-(61) which differ from the pyrenocines only with respect to the structure of the side chain at C-5 have been isolated from Macrophoma commelinae (fruit rot disease of apple and other plants)."' Rosellisin (1 14) was a co-metabolite of the macommelins ''I but had also previously been isolated from Hypomyces rosellus.112Its structure was originally misassigned as (50),but was later revised to (68) on the basis of biogenetic Rosellisin aldehyde (51) has also been isolated from Hypomyces ro~ellus."~ Compounds related to rosellisin have been reported.Macro- phin (53) has been obtained from Macrophoma commelinae along with macrophic acid (52).'14 Islandic acid (54) was isolated from Penicillium isiandicum.1'5 The biogenesis of pyrenocine A (58) and citreothiolactone (56) has been investigated in cultures of Penicillium citreo- H3CO OH CH20H p 0 CH3 / C02CH3 (49) (50) 0 (53)R= &CH3 0 OH (54) R = CH3 viride.l16 On addition of cysteine or methionine to the fermentation medium yields of the two metabolites decreased significantly.However addition of [1,2-13C,]acetate led to the isolation of enriched (58) and (56) as well as pyrenocine B (55) a metabolite not normally isolated from this organism. A possible biogenetic route was proposed (Scheme 19) in which all three intermediates were formed via a common intermediate (57). It was suggested that the sulfur atom in citreothiolactone was derived from enzyme-bound thiocrotonate although there is no evidence to support this. Feeding experiments with [1-13C]- and [1,2-13C2]acetate in Macrophoma commelinae resulted in formation of macom-melins (61) and (60) with a labelling pattern as shown (Scheme 20).l14Cultivation of the organism with diethyl [2-13C]malonate gave high enrichment at positions C-3 C-5 and C-7 of macommelinol (61) but only low incorporation at C-9.This result precluded the possibility that the macommelins were derived from a branched tetraketide chain,l16 but supported the 81 NATURAL PRODUCT REPORTS 1993-5. M. DICKINSON (55) 0 (57) Scheme 19 PH3CO HO H H * &OH+ OCH3 0 H3CmC02H -0 *o* o*o* Scheme 20 H3C0 H3C0 HO,H H3C0 OH * -hoH+ flH+ D&-COEH DD #CD3 0 * 0 * CH3 0 0 CH3 0 0 CH3 (59) (60) Scheme 21 theory proposed by Turner and Aldridgell' that an aromatic Compound (61) was only formed from (63) suggesting that this intermediate was formed with subsequent oxidative cleavage compound was at an early stage of the biosynthetic pathway.of the benzene ring followed by rearrangement. When [l-13C,2- [Methoxy-l4C] (59) was converted to radioactively-labelled 2H3]acetate was administered to M.commelinae deuterium was postulated intermediates (64) (67) (65) and (66). All were incorporated into the C-5 side chain of (59) (60) and (61) as transformed into (59) with the aldehyde (67) showing an shown in Scheme 21.'14 extremely high incorporation (91.7 Yo),suggesting that it is an A biogenetic pathway on the basis of the above results was immediate precursor. The incorporation of epoxide (66) into suggested to be as shown in Scheme 22.The epoxide (66) was (59) was low (2.5 %) with most of the radioactivity being taken postulated as intermediate in which a 1,2-hydride shift could into (60). The reduction of the alkene (65) to macommelin (64) occur thus accounting for the presence of a deuterium atom a was not detected by these experiments. Compound (64) to the pyrone ring in (59). [Methoxy-14C] (63) was prepared,l14 however was incorporated into (55). It was suggested that and shown to be incorporated into (59) (60) and (61). conversion of (65) to (64) was reversible. 6-2 OH HO HO*OH 'OH (70) The biosynthesis of rosellisin (68) a co-metabolite of the macommelins has been investigated in Hypomyces r0seZ1us.l~~ It was found that incorporation of [1-l3C]acetate and [2-I3C]acetate was as shown in Scheme 23.The branch carbons of the hydroxymethylene groups did not show any incorporation of label and so it was assumed that these were derived from the c pool. Both rosellisin (68) and rosellisin aldehyde (51) have been shown to be active against Staphyloccoccus aureus at low 113 The structurally related islandic acid (54) has exhibited cytotoxicity against Yoshida sarcoma cells in tissue culture and inhibited the transfection of BaciZZus phage M2.'15 Pyrenocines A (58) and B (55) were phytotoxic preventing germination of lettuce seeds and inhibiting root elongation in seedlings.lo8 Inhibition of root elongation was also demonstrated in rice and onion seedlings. Compound (58) showed the greater activity.Pyrenocine C (49) was reported to be only weakly phytotoxic.ll0 2.7 The Styryl-Pyrones and Related Compounds A number of conjugated arylpyrones e.g. yangonin (69) have been isolated from plant sources such as Piper Aniba Alpinia and Ranunculus. The first report of a styrylpyrone from a micro-organism however concerned the isolation of hispidin (79) from Polyporus hispidus,118. 119 a lignin-attacking white rot parasitic chiefly on ash (Fraxinus excelsior). Hispidin has since been isolated from a number of other species including Polyporus schweinitzii 12' Phellinus pomaceus where it is a co- metabolite with 3,14'-bishispidinyl (70),12' Phellinus igniarius,122 NATURAL PRODUCT REPORTS 1993 OH OH (72) X = H (73)X =OH 0 OH (74) X = H (75)X = OH OHI OH I and Gymnopilus species.123 Bisnoryangonin (7 1) was a co-metabolite in this latter organism and has also been reported to occur in Pholiota squarroso-adiposa 124 Gymnopilus spectabZis,'25 and G.decurrens.126A systematic study of the distribution of styrylpyrones and derivatives has been carried out among the Strophariuceue and related genera.12' Hispidin (79) and bisnoryangonin (71) were found to occur in Hypholoma Flammula Philiota and Gymnopilus species. Hypholomines A (72) and B (73) and fasciculines A (74) and B (75) were also found to be distributed amongst these same species. These compounds were also isolated from the fruiting bodies of Hypholoma fasciculare (Agaricales) (' Sulfur Tuft ').128 Other related compounds to have been isolated from micro-organisms were hymenoquinone (76) and leucohymeno- quinone (77) both metabolites of the fruiting bodies of Hyrnenochaete mougestii (Poriales).129 The synthesis of hispidin (79) has been reported on occasions and involves condensation of an aromatic aldehyde with the NATURAL PRODUCT REPORTS 1993-5.M. DICKINSON MOMO + MOMO OH i ii 1 OH OH - iii iv Reagents i EtOH KOH; ii H+; iii Ac,O; iv aq. H,SO, EtOH A Scheme 24 (80)R = n-C4Hg methyl ether of dehydroacetic acid to give a protected hispidin analogue (78). Deprotection followed by recyclization of the pyrone ring gave hispidin (79) (Scheme 24).l19* 130 Biosynthetic studies on the styrylpyrones have concentrated on the formation of hispidin (79) in cultures of Polyporus hispidus.Early studies demonstrated the incorporation of [U-14C]phenylalanine and [1-l*C]acetate and so suggested that hispidin was formed by condensation of a phenylpropanoid moiety with two acetate Conversion of phenylalanine to cinnamic acid in cultures of P.hispidus was also demonstrated and the presence of an enzyme capable of effecting hy- droxylation of cinnamic p-coumaric and benzoic acids was indicated. 132 p-Coumaric acid and caffeic acid were incor-porated efficiently into hispidin (79).133 The hydroxylase enzyme from P.hispidus was isolated and shown to catalyse hydroxylation of p-coumaric acid and even more readily hydroxylation of bisnoryangonin (7 1) to yield hispidin (79).NADH NADPH and ascorbate were found to serve as electron donor~.'~~,~~~ It was also noted that blue light (43k530 nm) stimulated pigment formation. The effect of light on growth pigmentation and enzymic activity in the biosynthesis of hispidin (79) was studied in more detail. It was found that conversion of cinnamic acid to p-coumaric acid was (81) R=H (82)R=CH3 enhanced whilst p-coumaric acid was converted to caffeic acid only in cultures exposed to light.134 Action spectrum studies showed that the system responded to light of 380 and 440 nm. Cycloheximide was found to block light-induced activity. 135 As the fruiting bodies of P. hispidus matured the amount of hispidin present decreased. Simultaneously the fruit- bodies became tougher and their fibrous 'woody' structure more pronounced.Oxidase enzymes isolated from cultures of P. hispidus were found to act on hispidin in vitro to bring about rapid oxidative polymerization,ll* and so a role for hispidin as a precursor of toughening polymers in Basidiomycetes had been suggested. No firm evidence either for or against this hypothesis has been reported although the presence of hispidin in other non-woody Basidiomycetes suggested that this might not be the case.133 2.8 Pyran-Zones from the Gliding Bacteria Gliding bacterium Myxococcus fulvus Mx f50 has been the source of the novel N-alkenylcarbamate pyrones myxo-pyronins A (84) and B (80),136whilst the structurally related corallopyronins A @I) B (82) and C (83) have been isolated from Corallococcus coralloides Cc c 127.13' NATURAL PRODUCT REPORTS 1993 s I 0 OH H36-C02H ___t A H3CS-( CH2)2CH( NH2)COZH Scheme 25 0 R' OyyR2 H 3H** C m H R20 (85) R' = CHO R2 = OCH3 (88)R' = CHO R2 = xylose (86) R' = CH20H R2 = OCH3 (89)R' = CH3 R2 = (glucose)eibose (87) R' = CHO R2 = -NHCH&H20H Biosynthetic studies on myxopyronin A have shown it to be derived from two polyketide chains with glycine being incorporated as a starter unit in one of the chains.138 Two of the remaining methyl groups and the carbamate methyl ester are incorporated from methionine with the third methyl group being derived from the C-2 of acetate (Scheme 25).Myxopyronins A (84) and B (80) were found to be active against a range of bacteria i.e.Corynebacterium mediolanum Arthrobacter simplex Bacillus megaterium B. subtilis Brevi- bacterium ammoniagenes Staphylococcus uureus Micrococcus luteus Acinetobacter calcoaceticus and Agrobacterium tume-faciens. Activity was marked for Gram-positive bacteria. However Gram-negative bacteria were hardly affected and yeasts and moulds were totally re~istant.'~~ Incorporation with Staphylococcus aureus sug-gested that the myxopyronins inhibited RNA synthesis and thus with a certain delay protein synthesis. This was confirmed when DNA-dependent RNA polymerase isolated from Escherichia coli was found to be strongly inhibited by the myxopyronins. Inhibition with both whole cells (S. aureus) or isolated enzyme (E.coli) was not however inhibited com- pletely. The inhibitory effect was not seen for wheat germ RNA polymerase 11 and so the biological activity appeared to be restricted to prokaryotic RNA polymerases. Myxopyronins had no acute toxicity for mice up to 100 mg/kg (s.c.). In general myxopyronin B (80) appeared to be more active than myxopyronin A (84). The corallopyronins (81)-(83) were found to exhibit similar biological activity to the myxopyronins,140 with the mechanism of action also being similar i.e. inhibition of bacterial RNA polymerases. However whereas the myxopyronins only par- tially inhibited RNA synthesis corallopyronin A (81) gave total inhibition both in whole cells and with the isolated enzyme. RNA synthesis was inhibited even after chain elongation had started.Once again eukaryotic cells were completely resistant. 2.9 The Phytotoxic Pyran-2-ones A number of pyran-2-ones have been isolated from micro- organisms which were pathogenic on plants and the pyrones themselves were found to exhibit some of the phytotoxic (92) (93) activity attributed to the organism. Such compounds include radicinin (94) the solanopyrones A (85) B (86) and C (87) poaefusarin (88) sporofusarin (89) and colletopyrone (90). These will be discussed in this section. 2.9.1 Radicinin and Related Compounds Radicinin (94) also named stemphylone was first reported in culture filtrates of Alternaria r~dicina,'~~ and has been isolated from Stemphylium radicinum. 142-'45 This latter organism has been shown to be identical with A.radi~ina.'*~ Radicinin (94) has also been found in culture filtrates of Curvularia lunata C. inequalis C. coisis and C. trij~lii,"~ and as a co-metabolite with the structurally related radicinol (91) in culture filtrates of Cochliobolus l~nata,~~' and Alternaria chrysanthemi.148 The X-ray crystal structure of radicinin (94) has been reported whilst the absolute configuration of the 4-O-p-bromobenzoyl ester of radicinin and by analogy of radicinin itself has been determined.138 Two other closely related metabolites deoxy- radicinin (92) and 3-epideoxyradicinol (93) have been isolated from Alternaria helianthi.'". lj0 NATURAL PRODUCT REPORTS 1993-5. M. DICKINSON Scheme 26 * H&-CO,H dH2(COZEt) (94) Scheme 27 The syntheses of (-t )-radicinin (94) and the dihydro analogue have been re~0rted.l~l-l~~ The route through to radicinin is outlined in Scheme 26.15' Construction of the second ring of radicinin was achieved via the Lewis acid-mediated con-densation of crotonyl chloride with the hydroxy-pyrone.Biosynthetic studies on radicinin have been carried out and its derivation from acetate established. Labelling studies with [ 1-'*C]acetate and [2-14C]malonate led to distribution of label as shown in Scheme 27 with evidence suggesting that radicinin (94) was formed by the condensation of two acetate-malonate- derived chains.'j4 Feeding studies with [ 1,2-13C,]acetate confirmed these results. Incorporation of [ 1-l3C]- and [2-l3C]acetate into deoxyradicinin (92) was shown to give a similar labelling pattern to that demonstrated for radicinin.150 Two pathways are biosynthetically possible for the formation of radicinin.The first would require the condensation of a C,-unit with a C,-unit whilst the second would require the condensation of two C,- units. Although the former is biosynthetically preferable no evidence has been obtained that favours one pathway over the other. Radicinol (91) was produced some time after the first production of radicinin (94) in cultures of Alternaria chrysanthemi and with concomitant decrease in concentration of the latter thus suggesting that radicinin was converted to radicinol by this Radicinin (94) has been shown to inhibit the germination of seeds of Lepidum sativum (cress) at concentrations of 5 x lop5M.At concentrations which permitted seed germi- nation considerable damage to root development was noted in that the roots were shorter than normal discoloured and devoid of root hairs. 142 Stems and seed-leaves were not affected. Radicinin has been found to be a constituent of necrotic lesions on chrysanthemum leaves that were artificially infected with Alternaria chrysanthemi. This suggested a causal role for the metabolite in foliar disease caused by this organism. Radicinol (91) has been shown to exhibit similar phytotoxic effects to that demonstrated for radicinin (94) and caused interveinal necrosis of cuttings of Canada thistle.lg8 3-Epideoxyradicinol (93) when applied over needle punctures on sunflower leaves caused small necrotic spots in less than 24 hours.These were enlarged after 24 hours and surrounded by chlorotic halos. These symptoms were also typical of the leaf spot phase of the disease caused by Alternaria helianthi. Although this suggested a causal role for 3-epideoxyradicinol in disease produced by the organism it has not been identified as a constituent of A. helianthi-induced sunflower leaf lesions. Deoxyradicinin (9 I) has been identified however. I5O (95) Radicinin has been reported to be inhibitory towards the growth of several Gram-positive bacteria such as Staphy-lococcus aureus Bacillus cereus and Clostridium species,142 146,148 and antagonistic towards Phytophthora erythroseptica.l'l In general however its antifungal properties are negligible.Experiments to determine the mode of action of radicinin (94) showed that it increased the rate of dehydrogenation of isopropyl alcohol by Fusarium lini. However there was no effect on the quantity or degree of desaturation of fatty acid synthesized.144 2.9.2 The Solanopyrones The solanopyrones A (85) B (86) and C (87) have been isolated from Alternaria solani the causal organism of early blight disease of tomato and potato. Solanopyrone A (85) induced a necrotic lesion on the leaf of potato at concentrations of 100 ,ug/,ul (rnethanol).'j6 2.9.3 Poaefusarin and Sporofusarin Poaefusarin (88) has been isolated from Fusarium poae and sporofusarin (89) from Fusariurn sporotrichiella.15' These Fusarium species have been identified as the causal organism of alimentary toxic aleukia. The toxins themselves have marked phytotoxic effects as well as exhibiting mammalian toxi~ity.'~' Phytotoxic symptoms include the death of branches of peas beans and tomatoes accompanied by extreme loss of turgor. The germination of pea beans and barley seeds was completely inhibited. In mammals the toxins caused temporary inflammation of the skin and oedematous haemorrhagic or leukocytorric reactions. The final stages of toxicity resulted in leucopenia and agranulocytosis necrotic angina haemorrhagic diathesis sepsis and necrosis of various parts of the alimentary tract especially the throat and exhaustion of the bone marrow.2.9.4 Colletopyrone Colletopyrone (90) has been isolated from Colletotrichum nicotianae a pathogenic fungus causative of tobacco anthra~n0se.l~~ It is related in structure to helipyrone (95) a dipyran-2-one that has been isolated from a higher plant Helichrysum italicurn. When solutions of colletopyrone (90) were placed on young tobacco leaves that had been pricked with a needle brown necrotic spots appeared within three days; the symptoms were analogous to those caused by the pathogenic fungus.lj8 NATURAL PRODUCT REPORTS 1993 0 00 0 (97) (99) (100) iii (CH,),SO, K,CO,; iv MeOCH,Cl TiCl,; v SeO Scheme 28 A Scheme 29 (98) Reagents i 2 BuLi; ii H,C(CH,),Br; 2.10 Other Pyran-Zones 2.10.1 Phacidin Phacidin (101) has been isolated from the canker fungus Potebniamyces balsamicola causal organism of bark disease in Abies grandis.160 161 Phacidin was originally assigned the pyran- 4-one structure (96),161 which was revised to that of structure (97) on the basis of spectroscopic evidence.162 However the actual structure was finally confirmed as the pyran-2-one (lOl) by an unambiguous total synthesis the route for which is outlined in Scheme 28.163 Thus pyrone (98) was 0-methylated under conditions that had been shown to produce only 4- methoxy-2H-pyran-2-ones to give methyl ether (99) which was then converted into phacidin via the formyl derivative (100). Labelling studies with [1-13C]-and [1 ,2-13C,]acetate demonstrated that phacidin (101) was derived from seven acetate units with two carbons derived from the C p00l.l~~ The distribution of label is shown in Scheme 29.Phacidin (101) showed potent antifungal properties and inhibited the growth of fungi in all the major groups. Fungi that were particularly affected were Phytophthora cactorum Xenomeris abietis Acremonium tsugae Sirococcus strobilinus Conisphora puteana Lenzites saepiariu Polyporus schweinitzii P. sulphureus Poria placenta P. subacida P. weirii and Serpula himuntioides.160 Although in most cases inhibition by the control antibiotic nystatin was greater phacidin was con- siderably more inhibitory against species of Pythium Phycomycetes and Phytophthora. The effect of phacidin (101) on fungi and yeasts that cause superficial and deep mycoses in man was also investigated.l'j4 It was found that amongst yeasts species of Cundida Sac- charomyces Trichosporon and Torulopsis were inhibited.Phacidin was also inhibitory against dermatophytes such as Epidermophyton floccosum Trichophyton mentagrophytes and T. rubrum its in vitro effectiveness being similar to that reported for griseofulvin. Its activity against systemic dimorphic fungi e.g. Histoplasma capsulatum Sporothrix schenckii Blastomyces dermatitidis and Coccidioides immitis was in general greater than that of the known antifungal agent 5-fluorocytosine. By comparison phacidin (101) was compara- tively ineffective against opportunistic fungi such as Aspergillus species. 2.10.2 Elasnin Elasnin (1 04) was isolated from Streptomyces norboritoensis.165 The synthesis of elasnin has been reported and the route followed is outlined in Scheme 30.166 Treatment of diketone (102) with a catalytic amount of acid and simultaneous azeotropic removal of water gave pyrone (103 R = H) which was then converted into elasnin (104). Original biosynthetic studies using [1 ,2-13C,]acetate as the labelled precursor suggested that elasnin (104) was derived from twelve molecules of acetate.167 This was confirmed in subsequent studies with the distribution of label as shown in SchemC 31.168 Elasnin (104) was originally isolated because of its inhibitory effect on human sputum (leukocyte) elastase an enzyme that has been implicated in many inflammatory disease states such as pulmonary emphysema acute arthritis and destruction of connective tissue.50 YOinhibition of this enzyme was achieved by elasnin at concentrations of 1.3 ,~g/ml.~~~* 169The compound appeared to be specific in its activity with considerably higher concentrations required in order to achieve 50 YOinhibition of pancreatic elastase. Chymotrypsin trypsin thermolysin and papain were unaffected. A number of analogues of elasnin have been prepared with the aim of developing more specific inhibitors of greater potency. The phenyl derivative (105) was as active as elasnin against human sputum elastase 170 and more effective against porcine pancreatic elastase or chymotrypsin whilst the octyl derivative (106) was thirty times more active than elasnir~.~'~ NATURAL PRODUCT REPORTS 1993-5.M. DICKINSON 0 CO2Me ... TCHO i ii 111 iv ___F C02Me -7 -1 (102) (103) v-viii J 0 (104) Reagents i Base condensation; ii DDQ dioxan; iii TsOH (cat.) toluene; iv Ac,O pyridine; v mCPBA ArSAr; vi 10% Pd-C EtOH; vii Jones reagent; viii conc. H,SO, 0 “C Scheme 30 0 ___t H3C -C02Na (104) Scheme 31 Scheme 32 Studies on the effects of substituents on biological activity have 2-10-3NectriaPYrone and Related Compounds also been carried 0~t.l’~ Nectriapyrone (107) has been isolated from Gyrostroma Elasnin (104) was reported to be of low toxicity with an missouriense the imperfect stage of Thyronectria rnissouri-and more recently from Gliocladium ~ermoesenii.”~ LD, in mice 290 mg/kg (i.p.) and > 1000 mg/kg rally).'^^ ensi~,~’~ Elasnin has no antibacterial or antifungal a~tivity.’~’ Vermopyrone (108) was a co-metabolite in this latter organism.NPR 10 D@-( C H&CH( NH2)COzH Scheme 33 A I IVI H3CS-( CH2)2CH( NH,)( (110) X=H (111) X=F Scheme 34 A closely related compound fusalanipyrone (109) has been isolated from Fusarium solani. li4 A route through to substituted 2-pyrones7 which involves the condensation of two molecules of @unsaturated acid chlorides with loss of two molecules of hydrogen chloride has been developed. Fusalanipyrone (109) was one of the pyrones synthesized by this method.175 When nectriapyrone (107) was first isolated it was reported to incorporate [2-14C]mevalonic acid and the conclusion was drawn that nectriapyrone was in fact a monoterpene.’i2 However there was no evidence from degradation studies to support this claim.Recent studies using [1,2-13C2]acetate and [Me-13C]methionine have demonstrated that this metabolite is acetate-derived with the extra methyl groups being introduced from methionine (Scheme 32).173 The labelling pattern for vermopyrone (108) suggested that the latter may have arisen through cleavage of nectriapyrone. The fact that vermopyrone was not isolated from shorter fermentations provided some support for this hypothesis. Fusalanipyrone (109) also appeared to follow the isoprene rule and the question was raised as to whether this might be a monoterpene. However feeding studies with [2H3C]methionine demonstrated that the C-3 and the C-l’methyl groups were methionine-derived and that presumably fusalanipyrone (1 09) was biosynthesized via a polyketide which was subsequently methylated (Scheme 33).176 Nectriapyrone (107) has been reported to display anti-bacterial activity against Staphylococcus aureus at a con-centration of 30 ppm.172 Fusalanipyrone was in contrast inactive against Staphylococcus and Escherichia coli although it displayed weak antibiotic activity against Candida albicans Mucor and Trichoderma k~ningii.l~~ 2.10.4 Vulgamycin Vulgamycin (1 lo) also named enterocin has been isolated from Streptomyces candidus var. enterostaticus S. virido-chromogenes,17i and S. hygroscopicus.178 An X-ray crystal structure determination of the m-bromobenzoyl ester of vulgamycin has been carried out.179 Biosynthetic feeding studies with [1-13C]- [2-13C]- and [1,2-13C2]acetate demonstrated that vulgamycin was derived from NATURAL PRODUCT REPORTS 1993 OCH3 I (113) R=H (114) R=CHs 0 0 Scheme 35 seven acetate units with the distribution of label as shown in Scheme 34.17* The methoxyl carbon of the pyrone ring was derived from methionine. [U-14C]Benzoate was incorporated specifically into the benzoyl portion of vulgamycin. It was thus proposed that vulgamycin was biosynthesized from methionine and seven acetate units with benzoate as the starter unit. Vulgamycin (1 10) was bacteriostatic against both Gram- positive and Gram-negative bacteria such as Escherichia coli and species of Proteus Sarcina Staphylococcus and Coryne- bacterium.Derivatives of vulgamycin were prepared in which the aromatic moiety was substituted with fluorine at various positions -these derivatives were formed by addition of the fluorinated benzoic acid to fermentations of Streptomyces hygroscopicus.180 Although p-fluorovulgamycin (1 11) showed stronger activity than the parent compound against Micro- coccus luteus the fluorinated derivatives displayed very little difference in their antimicrobial spectrum compared to vulgamycin itself. Vulgamycin had no activity against fungi and yeasts and its mammalian toxicity was 10w.l~’ 2.10.5 Luteoreticulin Luteoreticulin (1 12) has been isolated as a toxic metabolite of Streptomyces luteoreticuli.lsl NATURAL PRODUCT REPORTS 1993-5.M. DICKINSON HO t.r 0 Ho2C+co2H NHp l!, 0' -uu H CH2Br 7;O2H i ii iii __.F ___F -I Et02C00 H02C Reagents i NBS CCI, hv or peroxide; ii Na+ C(NHCOCH,)(CO,Et),; iii HCl/CH,CO,H 100 "C sealed tube Scheme 36 CO2H I 2.10.6 Aszonapyrone A Aszonapyrone A (1 13) has been isolated from Aspergillus zonatus,1s2and the crystal structure of the monomethyl ether (1 14) determined by X-ray analysis.183 It has been suggested that aszonapyrone A is biosynthesized by a combination of both the mevalonate-geranylgeranyl-pyrophosphate route and the acetate-polyketide route.lsz Aszonapyrone A showed antibacterial activity with an MIC of 6.3 ,ug/ml against Staphylococcus aureus.182 2.10.7 Coarctutin Coarctatin (1 16) has been isolated as an inactive metabolite of the fungus Chaetomium ~ourctatum'~~ and its structure was determined by spectroscopic means.Confirmation of the structure was obtained by X-ray crystallography studies on the dibromo-derivative (1 15).ls5 A number of possible biosynthetic pathways have been proposed for the formation of coarctatin. However feeding studies with [1,2-13C,]acetate demonstrated the incorporation of four intact acetate units into the structure (Scheme 35). The methyl carbon of [2-13C]acetate was also incorporated into the three remaining carbons as shown indicating that these were derived from the C 2.10.8 Tuiwapyrone Taiwapyrone (117) has been isolated from the fungus Cercospora taiwanensis a plant pathogen and its structure determined by spectroscopic means.lsi 2.10.9 Stizolobic Acid and Stizolobinic Acid Stizolobic acid (123) was first isolated from the cut surface of the epicotyl tips of etiolated seedlings of Stizolobium hassjoo (Velvet bean) and its structure reported to be that of the pyran- 4-one (1 18) on the basis of chemical degradations.ls8 It was also found to be present in other Stizolobium species and in Mucuna irukanda (Leguminoseae).The structure of stizolobic acid was later reassigned to that of the pyran-2-one (123) whilst the structure of a co-metabolite in S. hassjoo named stizolobinic acid was shown to be the related pyran-2-one (1 19).189 Both of these metabolites have since been isolated from the fungus Amanita pantherina a frequent cause of non-fatal mushroom poisoning in the Pacific Northwest.lgo The syntheses of stizolobic acid (123) and stizolobinic acid (1 19) have been re~0rted.l~~ The synthesis of the former is illustrated in Scheme 36. Thus ethyl 4-methylpyran-2-one-6- carboxylate (120) is treated with N-bromosuccinimide to give the bromomethyl derivative (12 I) which condenses with sodio diethyl acetamidomalonate to give pyrone (1 22). Hydrolysis of this compound gave the target diacid (123). Stizolobinic acid (1 19) was synthesized in an analogous manner taking ethyl 3-methylpyran-2-one-6-carboxylateas the starting pyran-2-one. Biosynthetic studies utilizing the plants S. hassjoo and Mucuna deeringiana demonstrated the derivation of both stizolobic acid (1 23) and stizolobinic acid (1 19) from tyrosine via DOPA with extradiol cleavage of the aromatic ring of DOPA being invoked in order to explain the formation of the heterocyclic rings of the pyrone-acid~.~~~-'~~ Th e enzymes responsible for this conversion have been isolated and purified.lg3.lg5 Feeding studies with [U-14C]DOPA utilizing Amanita pantherina demonstrated the incorporation of this amino acid into stizolobic acid thus indicating that a similar biosynthetic pathway was acting in the fungus as had been demonstrated for higher plants.lg6 2.10.10 Muscaurin II Muscaurin I1 (124) is one of a group of orange pigments to have been isolated from the caps of Amanita muscaria (Fly Agaric).lgi It is structurally related to stizolobic acid (123) and its synthesis from the latter has been reported.lg7 3 Microbial Dihydropyran-2-ones 3.1 Pestalotin and Related Compounds Pestalotin (also designated LL-P880a) (129) was originally isolated from the culture broth of Pestalotia cryptomeriaecola NATURAL PRODUCT REPORTS 1993 OCH OCH, I I OCH OCH, I I OH OH a fungus pathogen of Cryptomeriajaponica(Japanese Cedar).lg8 It has since been isolated from an unidentified Penicillium species,lg9 and an unidentified fungus which was believed to be neither a Pestalotia nor a Penicillium species. The keto- analogue (125) was a co-metabolite in this latter organism. Both pestalotin (129) and the keto analogue (125) have been isolated from another unidentified Penicillium species along with the fully unsaturated pyran-2-one dehydropestalotin (1 26).201 The dihydroxy analogues LL-P88Op (127) and LL-P880y (128) have also been isolated from an unidentified Penicillium species.2o2 The absolute configuration of pestalotin (129) at C-6 and C-1’ has been determined as (S).lg9 The structure of pestalotin (129) established by spectroscopic means,*03 has been confirmed by total synthesis,204 the route for which is illustrated in Scheme 37.The pyrone ring was constructed from ethyl acetoacetate whilst the side chain was synthesized from acrolein and butylmagnesium bromide. Reformatsky methodology yielded pestalotin (1 29) though in poor yield.The polyketide origin of pestalotin (129) has been determined from biosynthetic studies with labelled acetate. Thus addition of [1,2-13C,]acetate to fungal cultures led to isolation of pestalotin with the distribution of label as shown in Scheme 38.,05 Related metabolites are also presumably polyketide derived. Pestalotin (129) was originally isolated because of its role as a gibberellin synergist. Thus it was found that when pestalotin alone was applied to rice seedlings no effect was observed. However when it was applied in combination with gibberellic acid (GA,) the stimulative effect of this plant growth hormone was considerably enhanced. It was demonstrated that this enhancement was due to an increase in the promotive effect of GA on a-amylase synthesis.lg8 Pestalotin alone was also found to be capable of inducing sugar release although its activity was much lower than that of GA,.3.2 Aspyrone and Related Compounds 3.2.1 Aspyrone Aspyrone (1 30) was originally isolated from Aspergillus melleus,206and has since been isolated from A. elegans,”’ A. ochraceuS,208and another unidentified Aspergillus species.2o9 Its structure has been determined by spectroscopic means and by X-ray crystallographic studies.210 Considerable work has been carried out in elucidating the biosynthetic origins of aspyrone (130). Feeding studies with [I- 13C]- [2-13C]- and [1,2-13C,]acetate established the labelling pattern to be as shown in Scheme 39.211 It was proposed that the pyrone was formed via a pentaketide precursor which underwent a Favorskii-type rearrangement.Cleavage of an originally intact acetate unit would account for loss of 1,2-coupling in the 13C NMR spectrum as observed for the C-6 C H3COC H2C02E t i ii iii-v J J BrCH+=CHCO,Et CH,(CH2)3C?-CHO OCH OTHP vi vii I OCH, I OH ( 129) Reagents i CH(OCH,), H,SO (cat.); ii NBS CCl, A; iii acrolein; iv 0,;v Dihydropyran H+; vi Zn; vii H+ Scheme 37 OCH, I* ~~6-50,~ (1 29) Scheme 38 (130) Scheme 39 methyl group of [1 ,2-13C,]acetate enriched aspyrone.212-21* When a 13CNMR spectrum was obtained at 500 MHz a small coupling of 6.2 Hz between C-1 and C-8 was observed thus demonstrating that these carbons were originally derived from the same acetate unit.212 Incorporation of [2-14C]malonate into aspyrone demonstrated lower levels of label at C-10 thus identifying C-9-C-10 as the starter unit.Any possibility that aspyrone may have been derived from an aromatic intermediate such as mellein (1 3 1) (a co-metabolite) was discounted on the basis of feeding studies with [2-3H,]a~etate.215 Tritium was retained at C-7 whereas in the formation of mellein or other aromatic metabolites all three hydrogens would be removed in the formation of the aryl ring. Asperlactone (136) a co-metabolite of aspyrone (130) showed a similar incorporation pattern to that observed for the latter. This could be accounted for if both metabolites were derived from a common (post-Favorskii rearrangement) in- termediate.216 Feeding studies using 180-labelled precursors NATURAL PRODUCT REPORTS 1993-5.M. DICKINSON HOdMe t t +Me NAH’ -H20* Me t Scheme 40 HOh H3C4oAo \ H have allowed further elaboration of the biosynthetic pathway to aspyrone. When [l-13C,1s02]acetate was investigated as a precursor it was found that there were no 180-isotope induced shifts3n the 13C NMR spectrum indicating that none of the oxygen atoms of aspyrone were acetate derived. Growth of organisms under an atmosphere of 1802 gas and simultaneous addition of 13C-labelled acetate demonstrated that the epoxide and C-5-hydroxy oxygen atoms were derived from the atmosphere. In addition to this the C-2 and C-6 signals were also shifted but the intensity of their signals was approximately half that of the carbons attached to the hydroxyl and epoxide oxygen atoms.It was suggested that l80had been introduced onto C-2 from the atmosphere and that this was incorporated equally into both the carbonyl and ether oxygen of the pyrone ring. The remaining oxygen at C-2 would therefore be derived from the medium. Similar results were observed for asperlactone (I 36). A proposed biosynthetic pathway that takes into account all the above results has been postulated,216-220 and is illustrated in Scheme 40. Thus the epoxide (133) derived from the trienone intermediate (1 32) would undergo rearrangement to give the aldehyde (134). Further epoxidation and NAD+- mediated oxidation would then give the epoxycarboxylic acid (135).Ring closure to either end of the epoxide moiety would finally yield either aspyrone (130) or asperlactone (1 36). 3.2.2 Asperline Phomaluctone and their Derivatives Asperline (U-13,933) (140) was originally isolated from Aspergillus nidulans,221* 222 and has since been isolated from A. H36 -C02H (140) Scheme 41 * H,C-CO,H -H 0 3c~ (141) Scheme 42 carneus20i and A. caespito~u~.~~~ The epimer of asperline (1 37) was a co-metabolite in this latter organism as was the propenyl- derivative (1 38). Phomalactone (1 39) has been isolated from an unidentified Nigrospora species,224 and from an unidentified Phoma species.207 Feeding studies with [2-13C]acetate have demonstrated that asperline was polyketide-derived with the distribution of label as shown in Scheme 41.225 Asperline (140) displayed antibiotic activity against Staphy-lococcus aureus Proteus vulgaris Salmonella gallinarum Ba- cillus cereus Sarcina lutea Mycobacterium avium Salmonella pullorum Rhodopseudomonas spheroides and Chromobacterium violaceurn in vitro although it was inactive in P.vulgaris- infected mice when they were treated subcutaneously at the maximum tolerated dose.221,226 Asperline was also found to exhibit antifungal activity against Trichophyton violaceurn T. rubrum Homodendrum compactum Coccidioides immitis Blastomyces dermatitidis and Nocardia asteroides,226 but was inactive against Fusarium inoculijorm and Verticillium albo- ~trurn.~~~ Activity against Candida albicans was also noted.The related metabolites have been reported to exhibit a similar antimicrobial spectr~m.~~~~ 223 3.2.3 Astepyrone Astepyrone (141) has been isolated from cultures of Aspergillus terreus and has been shown to be a polyketide.22i The distribution of label derived from [1-l3C]acetate was as shown in Scheme 42. NATURAL PRODUCT REPORTS 1993 (142) R = CH3 R' = CH2CH3 R2 = CH20H (143) R = R' = CH3 R2 = CH20H (144) R = H R' = R2 = CH3 (145) R = H R' = CH2CH3 R2 = CH3 H3C-CO2H 0 OH HC ____t -C02H C02H (147) Scheme 43 Astepyrone was reported to exhibit antiulcerogenic activity in rat but also showed considerable toxicity. 3.3 The Leptomycins Kazusamycins and Anguinomycins A group of structurally-related cytotoxic dihydropyran-2-ones isolated from Streptomyces species are leptomycins A (146) and B (147) kazusamycins A (142) and B (143) and anguino- mycins A (144) and B (145).Leptomycin A (146) (also named PD 118,607) was isolated from Streptomyces 229 and from another Streptomyces species,23n with leptomycin B (147) as a co-metabolite. The latter compound which is identical to PD 114,720 and (3-940 has also been isolated from an unidentified Actinomycete. 231-232 Leptomycins A and B differ only in the nature of the alkyl group at C-3' of the polyene side chain. The kazusamycins are analogous to the leptomycins but bear a hydroxymethyl substituent at C-11' of the same side chain. Kazusamycin A (142) (identical to PD 114,721) has been isolated from the same unidentified Actinomycete as a co-metabolite of leptomycin and from a Streptomyces sp.233*234 B,231,232 Ka zusamycin B (143) (PD 124,895) was also isolated from this latter or-gani~m,~~~ as well as from another unspecified Streptomyces sp.236 Anguinomycins A (144) and B (145) isolated from a Streptomyces SP.,~~' differ from the leptomycins only with respect to the methyl substituent at C-5 of the dihydropyrone ring this group being absent in the former.Incorporation studies with labelled precursors ([1-13C]-acetate [1-13C]propionate and [1 -13C]butyrate) demonstrated that leptomycin A (146) was derived from four acetate units and eight propionate units with the distribution of label as shown in Scheme 43.Leptomycin B however was derived from four acetate units and seven propionate units with C-2' C-3' and the C-3' ethyl substituent being butyrate The biosynthetic origins of the kazusamycins and anguino- mycins have not been determined but are presumably analogous to the leptomycins As already mentioned all these compounds have been shown to be cytotoxic and as such their biological activity has been explored on a number of occasions. The leptomycins (146 and 147) have been shown to cause hyphal curling of Mucor racemosus M. rouxianus and Trichophyton mentagrophytes and to cause cell elongation of Schizosaccharornyces pombe.229.230 Their activity against other micro-organisms is however less well pronounced. They showed weak activity against Rhizopus and Rhodotorul~,~~~ whilst leptomycin B (147) was also active against Alcaligenes viscolactis Microcnccus luteus Staphylococcus aureus Streptococcus pj'ogenes S.pneumoniae and Bacillus With these exceptions most other fungi or yeasts were insensitive as were Gram-positive and Gram-negative bacteria e.g. species of Aerobacter Bacillus Mycobacterium Corynebacterium Staphylococcus Pseudo-monas Aspergillus Penicillium Paecilomyces and Candida."" Kazusamycin A (142) was active against fungi and some yeasts but was inactive against Gram-positive and Gram-negative bacteria.234 The antimicrobial spectrum of kazusamycin B (143) is similar to that of kazusamycin A.235 The mode of action of leptomycin B (147) with Schizo-saccharomyces pombe was examined.228 Low concentrations caused inhibition of cell division leading to the production of elongated cells with morphologically altered nuclei.High concentrations of leptomycin B inhibited nucleic acid synthesis. It appeared from the evidence obtained that the antibiotic inhibited a specific step possibly in the M phase just prior to nuclear division. Leptomycin A (146) was highly active against murine B16 melanoma in vivo and showed anticancer activity at very low dosages.239 Leptomycin B (147) was active in vitro against a number of human and mouse tumour lines and in vivo against murine experimental tumour systems such as P388 leukaemia L12 I0 leukaemia (IC50= 3 x M) Ridgeway osteogenic and M 5076 sarcomas and mammary adenocarcinoma 16/C.231.239 Growth of HeLa cells was also inhibited and the life-span of mice bearing Ehrlich ascite carcinoma or Lewis lung carcinoma was increased."O Kazusamycin A (142) was also strongly active against murine tumours in vi~o,~~~.241 as were the anguin~mycins.'~~ Anguinomycin B (145) was found to be more potent than anguinomycin A (144).237 NATURAL PRODUCT REPORTS 1993-5. M. DICKINSON HO CH (148) R’ = H R2 = OH (149) R’ = R2= H (150) R’ = R2 =OH (151) R = OC(O)CH(CH3)2 (152) R = H (1 53) R = OC(O)CH2CH(CH& (154) R = OC(O)CH2CH&H(CH& (155) R = OC(0)-(cyclohexyl) (1 56) R = OC(O)CH2CH2CH( CH3)CH2CH3 (157) R = OC(O)CH(CH3)CH2CH3 OH 0 Scheme 44 3.4 The Phoslactomycins and Related Compounds The phoslactomycins are a group of biologically active dihydropyran-2-ones containing a phosphate ester moiety.A number of structurally related compounds have also been isolated and these will be discussed together in this section. CI-920 (PD 110,161) (148) PD 113,270 (149) and PD 113,271 (150) were the first of this group of metabolites to be isolated in 1983 from Streptomyces pulveraceus subsp. fostreus.242 Their structures were determined by a combination of spectral and chemical The phoslactomycins A (151) B (152) C (153) D (154) E (155) and F (156) were isolated as a complex from culture broth of Streptomyces nigrescens.243Their structures have been shown to differ only with respect to the nature of the substituent on the cyclohexane ring.245 Phoslactomycin B (1 52) is identical to phospholine which has been isolated from Streptomyces hygroscopi~us,~~~- 247 whilst phoslactomycin C (153) is identical to phosphazomycin C, isolated from Streptomyces sp.248 Phosphazomycin C (1 57) was a co-metabolite in this latter organism,248 as was phosphazomycin A.249A structure has not been assigned to this metabolite although it appears to belong to the phoslactomycin class of antitumour antibiotics.CI-920 (148) has been shown to exhibit cytotoxic activity against murine P388 lymphocytic and L1210 lymphoid leu- kaemia as have PD 113,270 (149) and PD 113,271 (150) with the former being the most active.242 When CI-920 (148) was administered to mice (25 mg/kg i.p.) bearing approximately lo7 L1210 leukaemic cells it was found to be curative in about 10 % of the mice whilst the lifespan of mice that eventually died was increased typically in excess of 150%.The lactone and phosphate moieties were shown to be essential for anti-tumour activity whereas ring hydroxylation or removal of the terminal hydroxyl group had little effect. CI-920 (148) was inactive when given orally or subcutaneously and also failed to show activity against murine M5076 sarcoma B 16 melanoma or Ridgeway osteogenic CI-920 (148) PD 113,270 (149) and PD 113,271 (150) were all devoid of antimicrobial activity when tested against a range of micro-organisms including Bacillus subtilis Escherichia coli Penicillium avellaneum and Staphy-lococcus au~eus.~~~ The phoslactomycins (1 5 1)-( 156) showed strong antifungal activity against a range of organisms including Botrytis cinerea Alternaria sp.Chaetomium globosum Verticillium albo-atrum and Pseudocercosporella herpotrichoides.244 Antibacterial ac-tivity was weak although there was a slight inhibitory effect against some Gram-positive bacteria. A comparison of activity between phoslactomycins A to F showed that they had almost the same antimicrobial spectrum and so it was thought that the substituent on the cyclohexane ring was unimportant for On the other hand (21-920 (148) although struc- turally similar was devoid of antifungal activity suggesting that the aminomethyl and cyclohexadienyl moieties may be required for expression of antifungal activities of the phoslacto- mycins.Although the phoslactomycins have not been tested as a group for cytotoxic activity phoslactomycin B (1 52) has been shown to be active against L1210 P388 and EL-4 le~kaemias.,~~ Phosphazomycin C (1 57) along with phoslactomycin C (phosphazomycin C,) (153) has been shown to exhibit strong antifungal activity against Aspergillus sp. Trichophyton menta- grophytes Colletotrichum lagenarium Glomerella cingulata and Alternaria mali amongst others.248 Phosphazomycin A has also been shown to inhibit fungal growth being effective against the above named organisms and against yeasts (Saccharomyces cerevisiae Candida albi~ans).~~’ The inhibition of fungal growth by phosphazomycin A was found to be accompanied by swelling of the mycelia and in Saccharomyces cerevisiae p-1,3-glucan synthetase was inhibited at concen-trations of 100 ,ug/ml.Pot tests demonstrated that the metabolite prevented infection by cucumber grey mould disease and cucumber anthracnose at concentrations of 25 ppm. The LD, in mice was 19 mg/kg when administered orally. 3.5 Other Polyketide-derived Dihydropyran-Zones 3.5.1 Alternaric Acid Alternaric acid (158) has been isolated from the plant pathogen 252 Alternaria ~olani,~~’.and its structure elucidated by a combination of chemical degradation and spectroscopic studie~.~~~-~~’ Biosynthetic studies using [1-14C]-and [2-14C]acetate demonstrated the incorporation of nine acetate units into the molecule and thus established alternaric acid as a polyketide.258 The remaining carbons were derived from the C,-pool (Scheme 44).The possibility that alternaric acid may have been NATURAL PRODUCT REPORTS 1993 TOH 0 propionate-derived was precluded due to the low incorporation of [l-14C]propionate into the molecule. Alternaric acid did not display antibacterial activity but was shown to inhibit spore germination in a number of fungi (e.g. Absidia glauca Myrothecium verrucaria Mucor mucedo Thamnidium elegans) at concentrations of 1 ,ug/ml or less. Spore germination of other fungi was unaffected. However at high concentrations (100 ,ug/ml) the extension of germ-tubes after germination was retarded markedly in Botrytis allii Aspergillus tamari and Stemphylium species. 251 252 3.5.2 The Rubratoxins Rubratoxins A (159) and B (160) have been isolated from cultures of Penicillium rubrum and P.purpurogenum moulds responsible for diseases induced by infected corn. The isolation biosynthesis and biological activity of these metabolites has been reviewed,259 and so these aspects will only be summarized here. The effect of medium on toxin production has since been studied in P. rubrum.260s 261 Biosynthetic studies using 14C-labelled precursors have established that [l-14C]acetate [2-14C]malonate [l -14C]glucose [1,5-14C2]citrate,[l-14C]hexanoate and [U-14C]glucose were all incorporated into the rubratoxins although the position of label has not been established. The incorporation of both acetate and malonate provided evidence for the theory that these metabolites were polyketides.262 26‘3 The biological activity of the rubratoxins has been examined in Rats treated with these compounds developed within a short period anorexia diarrhoea and a porphyrin ‘OR (163) R = pBrC6H4CONHC0 (1 64) R = (-)-Camphanyl Scheme 45 discharge from the eyes ears and nose.Post-mortem exam- ination of these animals revealed extensive liver damage which consisted mainly of massive haemorrhagic necrosis with entire sections of the organ destroyed. The toxicity of the rubratoxins decreased when administered orally and rubratoxin B (160) displayed greater activity than any of its derivatives. The LD, of rubratoxin B in rats was 0.36 mg/kg (i.p.). Rubratoxin B was also found to exhibit antiprotozoal activity inhibiting growth of Tetrahymena pyrformis in vitro at concentrations of 25,4m1.264 3.5.3 Phomopsolides A and B The fungus Phomopsis oblonga has been shown to act as a biocontrol agent for the control of Scolytus scolytus the insect vector of Dutch elm disease.265 Phomopsolides A (161) and B (162) were isolated from this organism,266 and shown to act as boring/feeding deterrents against adult Scolytid beetles in vitr~.~~~ The structures of these metabolites were assigned on the basis of chemical and spectroscopic studies.3.6 Non-polyketide Derived Dihydropyran-Zones 3.6.1 Fomannosin Fomannosin (165) has been isolated from the wood-rotting fungus Fomes annosus and its structure determined on the basis of chemical and spectral data.268 An X-ray crystal structure of the p-bromobenzoylurethane derivative of dihydro- fomannosin (163) has been determined,269 as has that of the camphanate ester (1 6$).270 In direct contrast to other fungal pyrones fomannosin (165) has been shown to be a sesquiterpene derived from mevalona- lactone via trans,trans-farnesyl pyrophosphate and humulene.Feeding studies with [1,2-13C2]acetate have demonstrated the incorporation of label to be as shown (Scheme 45).2719272 The possibility that 1,2-hydride shifts may be occurring in the conversion of humulene to fomannosin was discounted on the basis of evidence obtained from feeding studies with [5,5-2H2]mevalonate.273 Fomannosin has been shown to be phytotoxic affecting seedlings of Pinus tadea and Chlorella pyrenoidosa thus NATURAL PRODUCT REPORTS 1993-J.M. DICKINSON 95 31 E. L. Ghisalberti M. J. Narbey M. M. Dewan and K. Sivasithamparam Plant Soil 1990 121 287 32 F. M. Yong and G. Lim MIRCEN J. Appl. Microbiol. Biotechnol. 1986 2 483 33 K. S. M. Sastry C. V. P. Rao and R. Manavalan Indian Perfum. 1985 29 193 (Chem. Abs. 1986 105 297840 34 T. Kikuchi T. Mimura K. Hariyama H. Yano T. Arimoto Y. Masada and T. Inoue Chem. Pharm. Bull. 1974 22 1946 35 A. J. Harvat G. W. Chapman Jr. J. A. Robertson F. I. Meredith R. Scorza A. M. Callahan and P. Morgens J. Agric. Food Chem. 1990 38 234 36 G. R. Takeoka R. A. Flath M. Guentert and W. Jennings J. Agric. Food Chem. 1988 36 553 37 K. H. Engel R.A. Flath R. G. Buttery T. R. Mon D. W. Ramming and R. Teranishi J. Agric. Food Chem. 1988 36 549 38 K. H. Engel D. W. Ramming R. A. Flath and R. Teranishi J. Agric. Food Chem. 1988 36 1003 39 C. M. Hsu R. J. Peterson Q. Z. Jin C. T. Ho and S. S. Chang, suggesting a role for this metabolite in diseases caused by J. Food Sci. 1982 47 2068 Fomes annosus. 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