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R E V I E W

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2-Pyrone natural products and mimetics: isolation, characterisation

and biological activity

Gerard P. McGlacken and Ian J. S. Fairlamb*

Department of Chemistry, University of York, Heslington, York, UK YO10 5DD.

E-mail: ijsf1@york.ac.uk; Fax: 44 1904 432516; Tel: 44 1904 434091

Received (in Cambridge, UK) 16th February 2005

First published as an Advance Article on the web 5th May 2005

Covering: December 1992 to December 2004

The review summarises natural products containing the 2-pyrone moiety. An emphasis has been placed upon the

biological activity associated with 2-pyrones, particularly with respect to potential therapeutic or anti-microbial

agents. Where appropriate, non-natural 2-pyrone analogues are discussed, particularly those derived from natural

product lead compounds.

1 Introduction

2 Naturally occurring 2-pyrones

2.1 Bufadienolides2.2 Fusapyrones and 4-hydroxy-2-pyrones

2.3 Gibepyrones

2.4 Herbarins A and B

2.5 6-Alkyl-2-pyrones

2.6 Peripyrones

2.7 Coumarins

2.8 Styrylpyrones

2.9 Others

3 Isolation and structure elucidation

4 Biological activity

4.1 HIV inhibitors

4.2 Treatment of Alzheimers disease

4.3 Treatment of high cholesterol

4.4 Treatment of cancer

4.5 Other uses

5 Future lead compounds and directions

6 References

Gerard P. McGlacken

Ian J. S. Fairlamb

Gerard McGlacken received his B.Sc. (Hons.) and Ph.D. from the National University of Ireland, Galway,

where he studied the asymmetric deprotonation of hydrazones under the supervision of Dr S. W. Breeden. Heis currently carrying out postdoctoral research with Dr I. J. S. Fairlamb at the University of York in England,studying asymmetricRu-catalysed cycloisomerisation reactionsof 1,6-enynesand 1,6-dienes. Further interests

include the synthesis of substituted 2-pyrones using Pd-catalysed coupling reactions, and the investigation ofunusual observations associated with this class of substrates.

Ian Fairlamb (born 1975, UK) was appointed to a lectureship in Organic Chemistry at York in late 2001,

following a successful Ph.D. under the guidance of Dr J. Dickinson investigating the rational design andsynthesis of squalene synthase inhibitors (1999), and a productive post-doctoral research stay with ProfessorG. C. Lloyd-Jones, studying the mechanisms of various Pd-catalysed processes (200001). At the age of 28,he was awarded the prestigious 2003 Meldola Medal and Prize by the Royal Society of Chemistry (awarded

in 2004) to recognise outstanding independent contributions in the application of transition metal catalysedreactions, particularly involving palladium, to the synthesis of medicinally relevant molecules and natural

products. He is a recipient of a Royal Society University Research Fellowship (started October 2004) for

Understanding, controlling and exploiting unusual observations in Pd-catalysed reactions. Research isbroadly at the interface between Organic, Inorganic and Medicinal/Biological Chemistry. Key areas involvetransition metal chemistry, catalyst design (chiral and achiral), mechanistic understanding, with strong linksto medicinal chemistry and drug discovery; utilisation of palladium chemistry in pharmaceutical design,

particularly in the preparation of novel heterocyclic compounds such as 2-pyrones and related thio-derivatives,and pyridines. The complexation of 2-pyrones and 2-pyridinones to a variety of transition metal moieties arestudied. Novel compounds are screened for interesting biological effects. The exploitation of medicinal lead

compounds possessing anti-cancer and carbon monoxide releasing properties is of interest to the researchgroup.

1 Introduction

2-Pyrone 1a is a six-membered cyclic unsaturated ester that

shares chemical and physical properties reminiscent of alkeneand aromatic compounds. It is highly abundant in bacteria,microbial, plant, insect and animal systems and takes part inmany different types of biological processes such as defenceagainst other organisms, as key biosynthetic intermediates, andas metabolites. Historically, simple 2-pyrones such as triaceticacid lactone 1b and tetraacetic acid lactone 1c, are used asprecursors for the synthesis of biologically important com-pounds such as pheromones,1 solanopyrones,2 a-chymotrypsin, 3

elastase,4 coumarins5 and analogues.6

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Some ten years ago, Dickinson reviewed the literatureon microbial 2-pyrones, dihydro-2-pyrones and secondarymetabolites belonging to fungi of several genera includingAlternaria, Aspergillus, Fusarium, Penicillium and Trichoderma.7

These exhibit a wide range of anti-fungal, cytotoxic, neu-rotoxic and phytotoxic properties. Investigations into theirplant growth-regulating,8,9 antitumour10,11 and HIV protease-inhibiting qualities1214 have demonstrated the medicinal impor-

tance of 2-pyrones. Given the recent flurry of reports in thisarea, a review of the literature since 1992 is required. Here weattempt to bring the reader up to date on this class of naturalproducts, focusing on investigations regarding the isolation ofnaturally occurring 2-pyrones, their mimetics and applicationas therapeutic and anti-microbial agents (dihydro-2-pyrones arenot covered here).

The phenomenal abundance of the 2-pyrone motif in natu-rally occurring molecules could justify several reviews. We donot claim to cite every natural 2-pyrone isolated in the lastthirteen years, nor all synthetic 2-pyrone analogues exhibitingbiological effects. It is intended to give an insight and broadoverview of 2-pyrones from an eclectic array of natural sources.Bioactivity will be discussed, along with the importance of 2-pyrones in the treatment of HIV, Alzheimers disease, cancer andotherhuman diseases. Naturalproducts basedon 2-pyrones, andrelated derivatives, form the basis of the material in this review.

2 Naturally occurring 2-pyrones

2.1 Bufadienolides

The bufadienolides are an important group of steroids contain-ing the 2-pyrone moiety.15 They are characterised by a 2-pyroneconnected at the five position by a steroid nucleus,e.g.bufalin2.

A thorough review by Steyn and van Heerden provides aninsight into the chemical findings of bufadienolides from 1977to 1997.15 This class of 2-pyrones can be found in severalfamilies of plants and animals and the biological activity of the

bufadienolides is diverse. The plant sources include the fam-ilies Crassulacceae, Hyacinthaceae, Iridaceae, Melianthaceae,Ranunculaceae and Santalaceae. Those from the family Cras-sulacceae can cause the symptoms of cardiac poisoning inanimals. Cytotoxic kalanchoside3was isolated fromKalanchoetomentosa, an ornamental plant from Madagascar.16 The animalsources of bufadienolides include the Photinus(fireflies),Rhab-dophis (snake) and Bufo or toad. Over eighty bufadienolideshave been isolated from the latter. The abundance of thesecompounds in some species is extraordinary. For example, ninenew polyhydroxylated bufadienolides were isolated from thenuchal glands ofRhabdophis tigrinus17,18 six of which are shown(4af). Resibufogenin 5 was isolated from the Chinese toadskin extract drug Chan Su, and found to significantly inhibit a

leukaemia cell line.19

2.2 Fusapyrones and 4-hydroxy-2-pyrones

While 4-hydroxy-2-pyrone derivatives are relatively abundant,little is known of their biosynthetic origin. One example ofa biosynthetic enzyme is 6-styryl-4-hydroxy-2-pyrone synthase(SPS), identified in gametophytes ofEquisetum arvense.20 Thesynthesis of the 4-hydroxy-2-pyrone skeleton may well involveenzymes belonging to the family of CHS-related proteins(CHS = chalcone synthases).21 Fusapyrone6and deoxyfusapy-rone 7 were originally isolated from the rice cultures ofFusariumsemitectum and structurally resolved by Evidente and co-workers.22 They were later tested for anti-microbial activity

in filamentous fungi and yeasts, including their zootoxic andphytotic activities.23 Both compounds exhibited considerableanti-fungal inhibitory activity and inhibitory activity towardagents of human mycoses. Deoxyfusapyrone 7 stimulated theroot elongation of tomato seedlings at doses of 10 and 100 lM.

The mortality of an assay of brine shrimp (Artemia salinaL.)larvae in a bioassay has proved a reliable and fast method totest anti-tumour and cytotoxic activity.24 This method has beenused to test the zootoxicities of6 and 7.23 Recently, structureactivity relationships of derivatives of 6 have been studied byAltomare and co-workers.25 The parent compounds, 6 and7, show considerable anti-fungal activity against moulds, lowzootoxicity and selective action, thus further investigations into

structural variants are warranted. Penta-acetylation of6to give8 and 9 resulted in an increase in toxicity. The structural changesto 6 causing an increase in toxicity in an A. salina bioassaywere attributable to lower hydrophobicity. YM-202204 10aandS39163/F-110b were isolated from the culture broth of marinefungus Phoma sp. Q60596.26 Compound 10a exhibited broadspectrum anti-fungal activity in vitro and showed retardationin fungal GPI-anchoring activity. GPI-anchoring represents amechanism involving the attachment of proteins to membranesthat areessential inyeastand fungi cells.27,28 Two new metabolites11a and 11b were isolated from the phytopathogenic fungusNeocosmospora vasinfecta NHL 2298, along with known 2-pyrones.29 Their structures were elucidated by spectroscopicmethods; the absolute stereochemistry by X-ray analysis andchemical synthesis.

The 2-pyrone 12 was isolatedby theMitsubishiPharmagroupfrom the culture broth of Epicoccum purpurascens. It shows

inhibitory propertiestowardtelomerase.30

Initial studies towardsits total synthesis include the construction and characterisationof the C-glycosidic moiety.31 The absolute stereochemistryof 12 is not known. 4-Hydroxy-2-pyrone derivatives can beformed after early termination,viatwo condensation reactions,

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of the valerophenone synthase (VPS) catalysed synthesis ofthe bitter acids in hops (Humulus lupulus).21 The pyronesynthase, 2PS, from Gerbera hybrida, synthesises 4-hydroxy-6-methyl-2-pyrone.32 Other more elaborate 4-hydroxy-2-pyronesare synthesised from 4-coumaroyl-CoA by CHS and relatedenzymes, including bisnoryangonin13 and 4-coumaroyltriaceticacid lactone14(CTAL).33 The leaf resin ofMimulus aurantiacusgave 3-geranyl-4-hydroxy-6-(2-hydroxypropyl)-2-pyrone15.34

The structure of the previously misidentified compound (withthe 2-hydroxypropyl and geranyl group alternatively positionedat the 3- and 2-positions, respectively) was unambiguously

assigned using spectroscopic methods. 6-Acetonyl-4-hydroxy-2-pyrone 16, synthesised from acetyl-CoA, was isolated fromstilbene synthase (from the rhizomes of Tatar rhubarb,RheumtataricumL.). Ascosalipyrone17was isolated from the fermen-tation broth ofA. salicorniae.35

As part of their study of non-pathogenic endophytes inplant protection, Furumai and co-workers isolated a 2-pyronedesignated fistupyrone 18 from Streptomyces sp. TP-A0569.36

Fistupyrone inhibited the in vivo infection of seedlings ofChinese cabbage by Alternaria brassiciolaTP-F0423, the causeof Alternaria leaf spot, without anyin vitro fungicidal activity.Both macroscopic and microscopic means were utilised inthe evaluation of 18 on the infection of Chinese cabbage byA. brassiciola TP-F0423. The disease effect was 12% of the

untreated plant at 100 ppm. On the untreated plants, sporessettled on the leaf and grew infectious hypha that penetratedthrough the cuticle into the host plant. In the treated plant, thespores did germinate but severely reduced numbers of sporesformed the infectious hypha. In fact, treatment of the leaves

with FP (100 ppm) resulted in an infection rate of 9% comparedwith 54% in the untreated leaves.

2.3 Gibepyrones

Gibepyrones are 6-substitued-3-methyl-2-pyrones. The firstmetabolite isolated fromGibberella fujikuroibearing a 2-pyronefunction,produced six novel compounds gibepyrones AF (19ad, 20 and 21).37 These were isolated from ICI 10% culture

medium, in which glucose had been replaced by calciumlactate as the carbon source. Gibepyrone A 19a and B 19bexhibited growth inhibitory activity against Bacillus subtilis,Staphylococcus aureus, Saccharomyces cerevisiae and Candidaalbicans.

2.4 Herbarins A and B

Two strains of the fungusCladosporium herbarumwere isolatedfrom the sponges Aplysina aerophoba and Callyspongia aerizusa,affording two 2-pyrones named herbarin A22and B23,38 alongwith the known citreoviridin A 24. Compounds 22 and 23exhibit inhibitory activity against Artemia salina. Compound24 exhibited growth inhibitory activity in an insecticidal assayagainstSpodoptera littoralis.

2.5 6-Alkyl-2-pyrones

The genus Trichoderma is an excellent source of biologicallyactive natural products. One of these is a metabolite, 6-pentyl-

2-pyrone (6PP). By optimising the time of harvest, temperature,light and spore inoculation, yields of 2 g of 6PP per kg of groundcorn have been achieved.39 Thein vitrobiological activity (anti-fungal and phytotoxic) was evaluated against various assaysincluding agar diffusion, agar dilution, air diffusion, wheat

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coleoptile and lettuce seed germination.40 At a concentrationof 0.1% (v/v) the 2-pyrone completely inhibited the outgrowthof B. cinerea. The same compound could prove effective inthe control ofB. cinerea rots in kiwi fruit. Fruit treated withthis food additive were less prone to accelerated ripening.41

The effect and metabolism of 6-pentyl-2-pyrone on Botrytiscinereahas been reported.42 This compound is toxic to manyfungal plant pathogens includingB. cinerea.43 Claydon reports

that unless fungal growth is totally suppressed, the fungi willoutgrow the 2-pyrone zones, suggesting that the 2-pyrone ismetabolised and that the metabolites are not toxic to the fungus.These results were confirmed by experiments on B. cinerea.Visible hyphal growth occurred after 7 days when the funguswas treated with 120lg mL1, whereas no growth was observedafter 30 days when 220 lg mL1 was administered. HPLCshowed the metabolism of 6PP and the appearance of threenew components corresponding to 26, 27and 28 (the structureof28 was tentatively assigned as either 28 or 29 based on MSdata). The presence of these compounds in the culture did notsuppress fungal growth, suggesting that these metabolites areless toxic, due to their decreased lipophilicity. The 2-pyrone isstable in 2M HCl and 2M CH3COONa and only degraded to2-heptanone on treatment with 1M NaOH. The transformationof25 to 26, 27and 28 could not be definitely attributed to acidor base hydrolysis. Cooney and co-workers isolated these three2-pyrones, along with a new metabolite 29 from B. cinerea.44

The same group later identified four new metabolites of25fromthree Pencillium isolates, a Sclerotinia isolate and a Fusariumisolate. Further oxidation to carboxylic acids was observed.45

Pinus radiata also metabolised 6-pentyl-2-pyrone in 7985%conversion to several isomers of25, the most abundant being 5-(2-pyron-6-yl)pentan-5-ol.46 6-Pentyl-2-pyrone is also producedbyTrichoderma harzianum, grown in liquid cultures containing14C labelled glucose. The study showed that ca. 1.2% of theglucose was converted to 6PP.47

2.6 Peripyrones

Aspergillus fumigatus FO-1289 was isolated from a soil sam-ple from Tokyo (Japan). Four biologically active compounds,pyripyropenes AD (30ad), sharing structural similaritiesnamely pyridine, 2-pyrone and sesquiterpene moieties, forminga steroid-like structure, were isolated from the culture brothof the producing strain.48,49 These demonstrated potent acyl-

CoA:cholesterol acyltransferase (ACAT) inhibitory activity.Pyripyropene C 30c was the most potent in screens with anenzyme assay system using rat liver microsomes (IC50 = 53 nM).The initial reports describing the isolation and biologicalevaluation of 30ad were followed by relative and absolute

structure elucidation,50 and finally the first total synthesis ofa potent member of the family, (+)-30a.51 The structurallysimilar arisugacin,52 and territrems53 serve as selective inhibitorsof AChE and have potential for the treatment of Alzheimersdisease. The structurally related GERI-BP001 31 was isolatedfrom a culture ofAspergillus fumigatus F37.54 It inhibits acyl-CoA:cholesterol acyltransferase (ACAT), the enzyme responsi-ble for the intercellular esterification of cholesterol. The total

synthesis was achieved in 1995.55

Phenylpyropene A 32a andB 32b were isolated from the fermentation broth of Pencil-lium griseofulvum F195956 along with the previously reportedphenylpyropene C 32c.57 These compounds are structurallysimilar to30a58 and pyripyropene E,59 sharing the 2-pyrone andsesquiterpene moieties, however these possess a phenyl ring inplace of pyridine. The level of inhibition is dependent on thenumber of acetoxy groups andon theexistence of an 11-hydroxylgroup32ais the most potent analogue.

2.7 Coumarins

Of the seventeen compounds isolated from the n-butanolfraction of the leaves ofPeucedanum japonicum, six containedthe coumarin moiety, e.g. 3335.60 All compounds were tested

for their radical scavenging capabilities. The coumarins wereless effective than other isolated compounds. Two new 6-acylcoumarins, racemosol36 and mammea A/AC cyclo F 37,were isolated from the leaf extract of Mesua racemosa. Theirstructures were solved by extensive spectroscopic analysis.61

Seven new coumarins, the achrocarpins AG, 3840, wereisolated62 from Ochrocarpos punctatus, along with five knowncoumarins.6366 These compounds exhibit broad spectrum cyto-toxicity against the A2780 human ovarian cancer cell line.

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2.8 Styrylpyrones

Naturally occurring styrylpyrones, without a 4-hydroxy moiety,include the previously known compounds,41aand41b, isolatedfrom the trunk wood and bark of an Anibaspecies and exotic 2-pyrone dimers (possibly photochemical artifacts resulting fromdimerisation). 67 The plant Miliusa balansae provides a newstyrylpyrone42.68 Dihydro-5,6-dehydrokawain43awas isolatedfrom Alpina speciosa leaves. It caused a 35% reduction in thehypocotyl length of lettuce seedlings, compared to a control.69

Several derivatives were synthesised, with43b the most potent,showing a 66% reduction in hypocotyl length.

2.9 Others

The carboxylic acid 44 was isolated from the aspen blue stainfungus Ophiostoma crassivaginata, along with fifteen other com-pounds. A series of biologically active secondary metabolites,

bearing the 2-pyrone moiety, are often detected in marinemicroorganisms such as fungi, bacteria and algae.7072 Thedecaturins A and B (45and 46, respectively) were isolated fromPencillium decaturense and Penicillium thiersii. Both compoundsfeature a new polycyclic ring system and 46 exhibits potentantiinsectan activity against the fall armyworm (Spodoptera

frugiperda).73

A recent analogue of luteoreticulin,74,75 griseulin 47 wasreported to show inhibitory activity against nematodes (P. redi-vivus,C. elegans, andH. glycines) and mosquito (A. eagypti).76

Lyga in his studies developed a convenient convergent synthesisto both natural products, allowing variation of both phenyl and2-pyrone rings.77

Davidson and co-workers isolated four 2-pyrones from aStreptomycete,named BD-26T(20), culturedfrom shallow watermarine sediment; Wailupemycins AC, 4850, and 3-epi-5-deoxyenterocin and derivatives, 51.78 Compounds weretested foranti-microbial activity against Bacillus subtilis, Staphylococcusaureus and Escherichia coli in vitro. Compound 48 showedinhibitory properties against E. coli, whereas compound 51aexhibited activity againstS. aureus.

Five novel cytotoxic compounds, NF00659 A152a, A252b, A352c, B152dand B252e, were isolated from a culture mycelium ofAspergillus sp.(Table1).10 Thesecompounds possess potent anti-tumour properties against A2780 human ovarian carcinoma

and SW480 human colorectal adenocarcinoma cells. An elegantstructure elucidation (without stereochemistry) of the noveltricyclic core is a feature of the study. 79

Lin-Chi is an oriental crude drug used in the treatment ofmild ailments and to promote good health.80 It consists of the

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Table 1 Novel 2-pyrones based on generic structure 52

NF00659 R1 R2

A1,52a I OHA2,52b II OHA3,52c Ac OHB1,52d I HB2,52e II H

fruit bodies of the fungus Ganoderma lucidum. G. lucidum canbecome infected with the mycoparacitic fungus, Cladobotryumvarium.

As part of a search for anti-fungal agents by Kikuchi andco-workers, this fungus was isolated from its culture brothand found to inhibit the mycelial growth ofG. lucidum.81 Sixnovel 2-pyrones, namely 53, 54ab, 54c, 54ef, were isolatedalong with known 6. Cladobotrins II 54a and III 54b, androsellisin aldehyde 54d, exhibited inhibitory activity against G.lucidum, indicating that a 5-formyl group might be necessary forinhibitory activity.

2-Pyrone-4,6-dicarboxylic acid was isolated from Potentillaanserine.82 While this compoundhas been isolatedfrom bacteria,e.g. Pseudomonas,83 this is the first time that it has been shownto be produced in plants. This was confirmed by the growthof seedlings under sterile conditions, eliminating the possibilitythat the 2-pyrone originated from bacteria or parasitic fungi.Daldiniapyrone 55 was isolated from the ethyl acetate extractof Daldinia concentrica along with known 2-pyrone, (+)-orthosporin.84 Novel fermentation products 11G219a,b,c andd of the 2-pyrone class were isolated from the fungal cultureLL-11G219.85 Isolation experiments were carried out by RP-HPLC. The structure of 56 was assigned by 13C, 1H, DEPT

and COSY NMR along with analysis of the UV spectra andHETCOR or HMQR measurements. The structures of57, 58and59 were assigned by comparison of their NMR shifts. Thescreening program was initiated in an effort to discover non-steroidal androgen receptor agonists or antagonists.

As can be seen from the overlay of 56 with testosterone(highlighted in bold), both compounds are well aligned andpossess more conformational freedom, and potentially could actas testosterone mimetics. The butyl group of56 is flexible andthus could fit into a variety of enzyme pockets. Compounds5658were shown to be attached in a membrane bound androgenreceptor assay. Epoxide59 exhibited anti-fungal activity. Someof the more structurally interesting biologically active 2-pyronesare the macrocyclic 2-pyrone derivatives 61 and 62. Thesecompounds were isolated as secondary metabolites from the

red algaPhacelocarpus labillardieri.8689

The crude extracts fromthese algae were shown to exhibit neuromuscular blockingactivity, which may be due to structures of this type. The useof ring closing alkyne methathesis,90,91 potentially allows thesecompounds to be prepared if the alkenyl regio- and stere-oselectivity can be controlled and preserved.92 Fujimoto andco-workers have isolated multiforisins AF.93,94 More recentlyisolated compounds by the same group include multiforins GI.95 These compounds are discussed in detail in the treatmentof Alzheimers in section 4.2. Two compounds, 63a and 63b,were isolated from the bark of Peruvian medicinal plant palode Sangue orBrosimum rubescens.96

Isoprenoid-substituted 3-arylcoumarins, glyasperinL 64 fromGylcyrrhiza aspera,97 and kanzonol W65, fromG. glabra98 were

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isolated. 4,4-Di-O-methylscandenin 66 has been isolated fromDerris scandens.99 The biosynthetic origin of some coumarinshave been studied. Examples include the linear psoralen 68,thought to be biosynthesised from umbelliferone 67.100 Relatedanalogues have been studied, employing labelled deuteratedmarmesina known intermediate. The biosynthesis of 7-acetoxy-4-methylcoumarin 69has been studied usingTrigonella

foenumgraecum.101 Examples of naturally occurring coumestans

include 4-hydroxycoumestrol 70102

and sigmoid K 71, isolatedfrom the Cameroonian medicinal plant Erythrina sigmoides.103

Both demonstrated anti-bacterial activity. Psoralidin 72 wasisolated from Psoralea corylifolia and exhibits cytotoxicityagainst stomach cancer celllines.104 Recently, a new 6-substituted2-pyrone 73, along with 6-pentyl-2-pyrone, was isolated from thecultural filtrate ofTrichoderma viride. Viridepyronone showedantagonistic activityin vitrotowardSclerotium rolfsii, which isthe cause of stem rot of artichoke.105 Pedras and co-workersanalysed a number of blackleg causing fungi.106 A new groupof metabolites has been identified able to attack the brownmustard plant (Brassica junceaL.), a plant previously thoughtto be resistant to blackleg disease. The known metabolitesphomapyrone A 74,107 phomenin B 79108 and infectopyrone80109 were isolated, along with new 2-pyrones phomapyronesDG (7578). The group has previously isolated a numberof similar compounds from Leptosphaeria maculans.110 Themethanol extract of whole plants ofHypericum japonicumgavea new 2-pyrone saropyrone 81.111 15-Deoxyoxalicine 82 wasisolated fromPenicillium decaturenseand is a member of a rarestructural class.73

The territrems A, B and C were isolated from rice cultures of

Aspergillus terreus (Hyphomycetes) by Yang and co-workers andwill be discussed later in more detail (section 4.2). 53,112,113 Crewsand co-workers isolated nectriapyrone A 83 and nectriapyroneB 84 from an unidentified fungus obtained from a Stylotellasp. sponge near Taveuni, Fiji.114 Aspergione B 85 was isolatedfrom an Aspergillus versicolor.115 Pyrenocines D 86 and E 87were isolated from fermentation ofSargassum ringgoldianum,the latter showing moderate toxicity against P388 leukaemiacells.116 Leptosphaerolide 88was isolated fromL. oraemaris.117

3 Isolation and structure elucidation

The exhaustive procedures involved in the successful isolationof naturally occurring 2-pyrones are exemplified in the isolationof compound 89 and its derivatives.118 Chaetomium quadran-

gulatum was cultivated on sterilized rice at 25

C for 33 days.The moldy rice was extracted with 30 L of EtOAc twice. Thisextract was partitioned betweenn-hexane and water. Each layerwas concentrated affording an n-hexane, EtOAc and aqueousfraction of 15.89, 16.07, and 7.60 g respectively. The layer

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showing greatest inhibition of monoamine oxidase (MAO) wasthe EtOAc at 29.6% (1.0 104 g mL1). The EtOAc fractionwas chromatographed on silica gel to give five fractions (IV). Fraction IV showed greatest inhibition and was furtherchromatographed to give six fractions (af). Fractions IVb andIVc were repeatedly chromatographed affording five chromones(983 mg) already described.119 Fraction IVd was further

chromatographed withn-hexaneacetone to give five fractionsIVd1-5; fraction IVd3 was treated with CHCl3 precipitating89(32 mg) as a white solid. Similar repetitions, crystallisation andHPLC separation afforded another five 2-pyrones to concludethe six new constituents from an Ascomycete. The structureelucidation of the 2-pyrone chaetoquadrin F, isolated from theEtOAc extract of an AscomyceteChaetomium quadrangulatum,has been reported.118 The optically active white powder showedinfrared data (mKBr cm1: 3109 (OH), 1678 (C=O), 1655, 1591(C=C), 1429, 1383, 1257, 1124, 1076 (CO)) suggesting an a,b-unsaturated carbonyl system. The 1H and 13C data showedthe presence of two methyl groups, two methines and fourquaternary carbons. Further study by COSY and HSQC (1H-detected single-bond heteronuclear correlation through multiple

quantum coherence) suggested either 89 (a 2-pyrone) or 90 (a4-pyrone). Methylation with CH2N2 gave a new derivative 91.NOE studies indicate that 90 is the precursor to 91. However,closer inspection of the UV and 13C NMR data show that theprecursor is 89. The characteristic differences seen in the UV(absorptions at 290 (4.02) and 263 (4.01), in nm (log e), for 89and 91, respectively) suggest that the precursor is a 2-pyrone andthat the methylated product is a 4-pyrone. Furthermore, the 13 CNMR signal at C-4 shifted to d 183.6 (+15.9 ppm) indicatingthat C-4, which has an aromatic carbon bearing a phenolicgroup, was now after treatment with CH2N2 a carbonyl carbon.On treatment with (R)- and (S)-MTPA acids, both hydroxylsubstituents were esterified. The modified Moshers method120

was applied and the absolute configuration assigned as (S).

Another example isG. heterospora74-T-542-1 which was cul-tivated on sterilized rice. The ethyl acetate layer was partitionedbetweenn-hexane and water. Evaporation of each solvent layerand testing of the crude products showed suppression of theCon A-induced proliferation of mouse splenic lymphocytes by

39, 100 and 59% at 50 lg ml1 in the case of the n-hexane, ethylacetate (defatted organic layer) and aqueous layer, respectively.Repeated chromatographic fractionation of the most active ethylacetate layer afforded compounds 92, 93 and 94 in 0.23, 0.19 and0.057%yields, respectively. The 1Hand 13C data of compound 92were similar to those of93.95 New NMR signals correspondingto the acetoxy group at d 2.08 (s, 3H), d 20.9 (q, 1C) andd 170.8(s, 1C), along with downfield shifts of C-11 and H-11, due to

added deshielding from the ester group, allowed the structure of92 to be assigned (atom numbering as in original paper). Thestructure was confirmed by acetylation of 2-pyrones 92 and 93.Both gave an identical doubly acetylated derivative 96. NOEcontacts were observed between H-7 and H-11 (9%) and H-7and H3-9 (5%).

In studies by Hua, tricyclic pyranopyrones were identified asa biologically important class of compounds.121,122 The synthesisof97 was achieved via the condensation of 4-amino-6-methyl-2-pyrone with 1-cyclohexenecarboxaldehyde, affording both97and98 in a 1 : 1.5 ratio.123 It was not possible to determine thestructures of these compounds by 1 H or13 C NMR spectroscopy.For example, H-10 of 97 resonates at d 8.15, the equivalentproton in98 is at d 8.5 (H-6); neither possesses a beacon signal

identifying the isomeric structure(atom numbering as in originalpaper). Recrystallisation of97 from ethanol and diethyl ethergave suitable crystals for X-ray diffraction studies (crystals of98were not suitable).

4 Biological activity

4.1 HIV inhibitors

Human immunodeficiency virus (HIV) protease124 is one ofthree essential viral enzymes in the replication of HIV. Inhi-bition of HIV protease (HIV PR) results in virus particles

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that are non-infectious towards other cells.125 The enormousresources dedicated to HIV research have identified inhibitionof HIV-1 protease as the successful target for the treatment ofAIDS.126,127 Despite these studies, many of the recent proteaseinhibitors suffer from serious restrictions. These include lowbioavailability,128 short half-lives,128 significant side-effects129,130

and the emergence of HIV strains resistant to these drugs. 131

Whilst inhibitors have been developed that inhibit HIV PR,132

it is important that the investigation of easily prepared, diverse,low molecular weight inhibitors continues, this considering theoccurrence of mutations and resulting inhibitor resistance. Agroup at Parke-Davis has focused its attention on synthetic,low molecular weight non-peptide molecules suitable for furtheroptimisation.133 A recent review by Hagen, Vara Prasad and Taitidentifies the need for such advances.133 In their investigations,approximately 150 000 chemical compounds were screenedviaahigh throughput scintillation proximity assay. The compoundswere initially screened as mixtures of ten at 100lM each. Ofthese, two lead compounds were chosen based on a number ofcriteria such as purity, selectivity, toxicityetc.The coumarin PD09956099and 2-pyrone PD 107067100exhibited IC50values of2.3 and 3.1 lM, respectively. The coumarin is similar in structureto warfarin101, which is a weak inhibitor of HIV protease6,134

and also interrupts HIV replication.135,136

Tummino in his studies found that 102 was the most potentwarfarin analogue. Coumarin99was the initial lead compound.X-Ray crystallographic studies illustrated that this compoundwas bound to HIV PR through two possible binding modes.In each mode, the 4-hydroxycoumarin ring of the inhibitordisplaced two water molecules. The fused phenyl ring wasorientated in the S1 site. In one mode, the flexible side chainorientated itself towards Arg 108 in the S 3

region, while in

the other it folded back towards the S2 region. Structuralmodification led to a marked improvement in inhibitory activity.For example, the IC50 value of103is 0.52lM.

137,138

4-Hydroxy-2-pyrones have become one of the most importantclasses of anti-HIV agents.135 In the late 1990s, a move todiscover cyclic non-peptide HIV protease inhibitors capable ofdisplacing the active site structural water molecule took place.139

The reorganization following water removal can lead to smallerand potentially more potent inhibitors.

High resolution X-ray diffraction studies on linear inhibitorswith HIV PR-1 have shown the presence of a tetra-coordinatedstructural water molecule linking the inhibitor to the flapsof the HIV PR dimer.140,141 The detailed relevance of this watermolecule is beyond the scope of this review.142,143 4-Hydroxy-2-

pyrones can replace the water molecule found in the active siteof the enzyme and the hydroxyl group forms hydrogen bondswith aspartates Asp 25 and Asp 125. Water is replaced, allowingthe lactone moiety to hydrogen bond with the flaps Ile 50 andIle 150.144

Several modifications to PD 107067 were carried out. Ex-

tension of the SPh region to SCH2Ph and SCH2CH2Phleads to improved activity.145 X-Ray diffraction studies showedimproved interaction between the SCH2Ph group and theS1

pocket. Systematic substitution of the phenyl group inSCH2Ph led to improved activity, with an isopropyl esteradjacent to the sulfur linkage being the most successful.146

Studies on the mode of action revealed a change in theoverall binding mode. The ester function resides in the S1

incontrast to that without the isopropyl ester group, where itoccupies the S2

pocket. Substitution of the phenyl ring with ahydroxyl at thepara-position, 3,4-benzodioxyl and 3,5-dimethylgroups, gave improved inhibition. Further studies suggested thatbranching at the 3-position might well achieve simultaneouspocket occupation at both the S1

and S2 pockets. Vara Prasad

et al.arrived at a new series of (4-hydroxy-6-phenyl-2-oxo-2H-pyran-3-yl)thiomethanes (Table 2).

The S-aliphatic series demonstrates, in general, increasedpotency over the aryl series. X-Ray crystallographic studies of104nand HIV-1 PR showed a unique mode of binding. In thiscase the lactone function only forms a hydrogen bond with theNH of Ile 50 (compared with previous examples where hydrogenbonding was observed with Ile 50 and Ile 150). The interactionwith Asp 125 and Asp 25 is also different. In this case, theenol function hydrogen bonds with Asp 125 and indirectly withAsp 25viaa bridging water molecule. The branching needed tooccupy the S1

and S2 pockets results in a chiral centre. The need

for this chiral centre could be removed with substitution of theSPh group, in which case the substituent and the phenyl group

itself could occupy both of these positions. A small library ofsuch compounds resulted in a highly inhibitory 2-pyrone 105c(IC50 = 0.037 lM).

147 X-Ray diffraction studies on 105a showthat the isopropyl group occupies the S 1

pocket, whereas the3-S-phenyl group partially occupies the S2

pocket.

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Table 2 C-3-S-aryl substituted 4-hydroxypyran-2-ones

Compound R1 R2 IC50/lM

104a Ph H 84.3104b Ph Ph 0.78104c Ph Cyclohexyl 2.44104d Ph Isobutyl 0.41104e Ph Isopentyl 0.39104f Benzyl Ph 0.48104g Benzyl Isobutyl 0.26104h Benzyl CH2cyclopropyl 0.084104i Cyclohexyl Ph 0.48104j Cyclohexyl Isobutyl 0.32104k Cyclohexyl CH2cyclopropyl 0.15104l Cyclohexyl Neopentyl 0.30104m Cyclopentyl Cyclopentyl 0.22104n Cyclopentyl Isobutyl 0.058104o Cyclopentyl CH2cyclopropyl 0.069

It was envisaged that the success to date with only threepockets occupied, could be further improved by habitation ofthe S3 pocket. Because this pocket is a spatial extension ofS1, substitution of the 6-phenyl ring could fill S3, furthermoreif these tethers extended beyond the active site opening theycould be used to alter the physical properties of the inhibitor.148

Compound 105c was the most potent with a IC50 value of0.019 lM. Some tricyclic 2-pyrones were also prepared, but onlyshowed moderate binding affinities.133 More recently the focushas shifted to 5,6-dihydro analogues.149

Compound 106 is the predominant constitutent of thealcoholic extract of Aspergillus terreus.150 This extract hasshown exceptional inhibitory activity towards HIV-1 at lowconcentration. 151,152 In was thus logical to examine the effectsof106 on eukaryotic cells and viruses.153 Infected H9 cells, anin vitromodel of chronic HIV infection, were treated with 106.The effective dose to reduce 50% viral replication (ED 50) was

12.5 lM, while the toxic dose for 50% of cells was 32.5 lM. Thisgives a TD50ED50ratio of 2.6. The activity of106on these cellsindicates that this molecule inhibits viral replication at post-integrational stages. The mycotoxin was also tested on Candidaalbicanswhich can complicate HIV infection.106decreased cell

proliferation and germ tube production in a dose-dependentmanner. This inhibitory effect correlated with a reduction incellular RNAand protein; therefore a decline in proteinsynthesisis most likely the mode of action. While this toxic effect mayoccur in other biological systems, it should be mentioned thatinhibition of human cell lines is less pronounced on HIV-1 andC. albicans.

A broad screening program identified phenprocoumon 107asa small molecule template for the inhibition of HIV protease.6

A favourable effect on activity was observed on replacement ofthe 4-hydroxycoumarin ring system with a 4-hydroxy-2-pyroneand with the introduction of carboxamide and sulfonamidegroups.6,154,155 Thus, modification of this compound based onstructureactivity relationships, identified 108, 109 and 110and ultimately a dihydropyrone, as next generation leads.

The final compound was dihydropyrone PNU-140690, whichsubsequently underwent clinical trials (not shown).156 In aparallel investigation, structureactivity relationship studies oninitial leads, replacing the carboxamide with a sulfonamide,led to the identification of another series with excellent invitro antiviral activity.157 The most active 2-pyrone 111 hasan IC50 value of 0.6 lM and represented a new directionfor the discovery of non-peptidic HIV protease inhibitors. In1996, Milne and co-workers identifiedfifteen non-peptide HIV-1proteaseinhibitors by pharmacophore searchingof the NCI DIS3D database.158 The most promising inhibitor identified againstHIV-1 protease was NSC 32180 112, a dimer of 4-hydroxycoumarin (ID50 = 0.32 lM). The tetra-coumarin derivative,bridged by a 1,4-benzene, NSC 158393 113, demonstrated

notable activity (ID50 = 1.7lM) and antiviral activity in HIV-1-infected CEM-SS cells (EC50 = 11.5lM).

4.2 Treatment of Alzheimers disease

Alzheimers disease is an age related neuro-degenerative dis-order characterised by progressive memory loss and globalloss of cognitive functions. The production and deposition of

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amyloid-b (Ab) peptides is thought to play a role in thepathogenic events leading to Alzheimers disease.159,160 Abpep-tides are derived from an amyloid precursor protein (APP). Theimpressive bioactivities of pyripyropeneA 30a49 and arisugacin52

in this area stemmed the synthesis and biological investigationof various analogues.121,161 Compound 30ahas been shown to bean acyl-coenzyme A:cholesterol O-acyltransferase (ACAT) in-hibitor, whereas arisugacin inhibits acetylcholinesterase. Hua inhisstudies identifiedactive molecules by mimicking theaction ofpyripyropene and synthesising tricyclic 2-pyrones with varyingfunctionalities.162 The common intermediate 114 isusedfor all ofthe 2-pyrones prepared. The successful candidates115 and 116protected from death MC65 cells that conditionally expressedwitha partial APP fusion protein. Surprisingly, tworegioisomerswere inactive, attributable to increased hydrophobicity.

Since approval by the United States Food and DrugAdministration (FDA) of E2020 (1-benzyl-4-[(5,6-dimethoxy-1-oxaindan-2-yl) methyl] piperidine) for the treatment of

Table 3 Inhibitory activities of tacrine, arisugacins and territremsagainst acetylcholinesterase and butyrylcholinesterase

Compound AChE (IC50/nM) BuChE (IC50/nM)

Tacrine, 117 200 12Arisugacin A,118a 1 >21000Arisugacin B,118b 25.8 >516000Territrem B,118c 7.6 >20000Territrem C,118d 6.8 >26000

Alzheimers disease,163 further attention has focused on the inhi-bition of acetylcholinesterase (AChE). The recently isolated nat-ural product arisugacin164 contains the 6-aryl-4-hydroxypyronemoiety and this function is necessary for biological activity.165

Arisugacin A118ahas shown huge potential in the treatment ofAlzheimers and other dementia diseases.166,167 In vitro potencygreater than existing anti-dementia therapeutics such as tacrine117,168 aricept169 and huperzine A, was reported.170,171 It issuggested172 that effective inhibitors should contain a nitrogenatom to mimic the binding action of the quaternary nitrogenof acetylcholine, a neurotransmitter responsible for memoryand other cognitive functions. A positively charged nitrogen is

believed to associate with Trp 84 situated near the anionicgorge of the active site.167

Interestingly, 118a bears no nitrogen atom, so it obviouslyfunctions by an alternative mechanism. Based on a modellingstudy by Omura and the known significance of the DE-ring,164 Hsung made the sensible assumption that binding withAChE may take the form of an electron-donatingelectron-withdrawing interaction.172 Electrondensity from the dimethoxygroup is coupled with the electron-withdrawing 2-pyrone ring.172

This prompted thegroup to synthesise a smalllibrary of6-aryl-4-hydroxy-pyrones that are analogues of118a. The original sourceof118aproduces only a small amount of the drug; an efficienttotal synthesis has been reported.173

Omura and co-workers isolated potent and selective in-

hibitors of AChE from a culture broth of Penicillium sp. FO-425952,164,174,175 along with 118a. Arisugacin B 118b, territremA 118c and territrem B 118d, selectively inhibit AChE (Ta-ble 3).174 Tacrine, an AChE inhibitor drug approved by the FDAdemonstrates improvementof cognitive function in patients withAD. This drug suffers from dose-limiting side effects thought tobe related to inhibition of butyrylcholonesterase (BuChE).176

A comparison of four of the isolated 2-pyrones with tacrine isshown in Table 3. The compounds show much greater selectivitythan tacrine, opening up the possibility for the treatment ofAD with fewer side effects. Compound118aand118bshow IC50valuesof 1 and26 nM, respectively, andalso protect mice againstamnesia induced by scopolamine.

The effect of functional group changes to the inhibitory

potencies on electric eel AChE has been reported.165

Saturationof the C-2 double bond, or reduction of C-1, caused a loss ofover 90% in activity. Epoxidation of the C-2 double bond hadlittle effect on activity. The 2-pyrone moiety proved crucial toactivity, as its disassembly was detrimental to activity.

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4.3 Treatment of high cholesterol

Hypercholesterolemia is a serious problem for our society.One approach to this is to inhibit cholesterol biosynthesis.Plant sterols such as compesterol and sitosterol are known toreduce plasma cholesterol by inhibiting absorption.177 Themodeof action seems to be a simple competition with cholesterolfor incorporation into micelles. Another method for reducingcholesterol absorption is the formation of complexes with

cholesterol that are excreted.178 New inhibitors of acyl-CoA-cholesterol acyltransferase have been found in the fermentationbroth ofPenicillium griseofulvum F1959.56 The ACAT inhibitoryactivity of phenylpyropene A (32a), B (32b) and the previouslyisolated C (32c) is thought to depend on the number of acetoxygroups and the existence of the hydroxyl group (as 32a is themost potent). A similar compound S1495 has been isolatedfrom aPenicilliumsp.179

Other 2-pyrone acyl-CoA-cholesterol inhibitors are the ter-reulactones AD (D is shown, 119, along with terreulactone A120, isolated fromAspergillus terreus).180

The absorption of dietary lipids into the body, followingdigestion, involves gastric lipase and three pancreatic enzymes:triglyceride lipase, phospholipase A2, and cholesterol esterase(CEase). CEase is thought to play a dual role. Firstly, itparticipates in the hydrolysis of triglycerides and secondly it

catalyses the hydrolysis of cholesterol esters.181 The mode ofaction of CEase is debatable. Rat studies suggest that rat CEasebinds to heparin on the cell membrane. This binding is thoughtto be associated with its 11 amino acid repeating unit near theC-terminal.182 Other studies (gene knockout), however do notsupport these ideas in human CEase.183 Inhibitors of pancreaticCEase have been shown to lower absorption of dietary choles-terol in animal studies.184186 3-Aryl-6-chloro-2-pyrones were firstdeveloped as irreversible inactivators of serine proteases thoughtto function as suicide inactivators.187,188 The success of recentlysynthesised 3-alkyl analogues may lie in their selectivity. Whilethe 3-alkyl versions inhibit chymotrypsin less successfully, theydo so reversibly and thus are promising as selective inhibitors. 3

Moreover, inhibitor 121d inhibits CEase in the presence and

absence of bile salts. In the absence of bile salts, CEase adopts aconformation in which theC-terminal hexapeptide is lodged intheactive site renderinga less competent active site.189,190 A seriesof 3-alkyl-6-chloro-2-pyrones with cyclohexane rings tetheredto the 3-position has been synthesised. The length of the alkylgroup affects the inhibition of cholesterol esterase. For example,in the case of121d, theKi is as low as 0.025lM.

181

To extend the previous study by Deck and co-workers,181 onthe selective inhibition of porcine CEase by 6-chloro-2-pyroneswith aliphatic groups at the 3-position, this group investigated 2-pyrones with a series of substituents at the 3- and 5-positions.191

Yeast CEase from Candida cylindracea (C. rugosaCRL 3) andCRL 1 were compared to porcine pancreatic cholesterol esterasefor inhibition by these series and related CRL 1 were compared

to CEase for inhibition. The activity of CRL 1 and CRL 3 wasmonitored in 25 mM Tris, pH 7, containing 6 mM taurocholate.Hydrolysis of p-nitrophenylbutyrate (1 mM) was followed at405 nm (25 C). Porcine CEase was monitored similarly. Kivalues were obtained by measuring rates with and without the

Table 4

Cpd. Structure

PorcineCEaseKi/lM

CRL 3Ki/lM

CRL 1Ki/lM

121a 0.04 58 960

121b 2.3 84 110

121c 2.2 12 120

121d 0.025 0.73 110

121e 0.13 96 120

121f 0.51 45 240

121g 0.80 16 >900

121h 0.50 53 225

addition of inhibitor. Small changes in the nature of the alkylgroup had profound effects on binding. Furthermore,no definitepattern emerged as regarding the preferential position of an alkylgroupat the3- or 5-position (Table 4).Porcine CEase hasalreadybeen demonstrated to function as a simple reversible competitiveinhibitor.181 Investigations into whether CRL 1 and CRL 3 actin a similar fashion were pursued by extraction of the assay andcomparative TLC. Unfortunately, no presumption can be made

as to the similarities between CRL 2 and mammalian CEase.For example, porcine CEase is 1450 times more sensitive thanCRL 3 to inhibition by 2-pyrone121a. Further conflict betweenporcine CEase and CRL 1 and CRL 3 is observed in their modeof action; CEase functionsas a reversible inhibitor. While CRL1significantly inhibited activity, removal of the inhibitor resultedin complete recovery of activity, suggesting that the 2-pyronesare not suicide inhibitors or irreversible inactivators of CRL 1.

Treatment of CRL 1 with an inhibitor, followed by analysis ofthe resulting products showed products consistent with hydroly-sis of the pyrone ring. In the case of CRL 3, recovery of activitytook several hours after removal of the inhibitor suggesting itfunctions as a pseudosubstrate. Possible mechanisms include(see scheme below): (1) suicide inhibition in which formation of

Bis followed by attack of an active site nucleophile, followed bydeacylation to C; (2)simple substrate inhibition in whichthe acylchlorideB hydrolizes to acylenzyme D followed by deacylationto regenerate active enzyme Ewith liberation of the substitutedglutaconic acid product; (3) simple reversible inhibition where

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an active site nucleophile attacks the 6-position on the 2-pyronewith release of chloride. Studies show that the 2-pyrone behavesas a pseudosubstrate inhibitor in which acylation is much fasterthan deacylation such that the acylenzyme accumulates andrearranges to the acid chloride, which hydrolyses while tetheredto serine followed by slow hydrolysis and release of product.

4.4 Treatment of cancerIn a study aimed at investigating the anti-cancer propertiesof tricyclic 2-pyrones with similar structures to pyripyropene(30ad) and arisugacins (118ab), novel TCPs were synthesisedand tested for their ability to prevent leukaemic cells fromsynthesising DNA and growingin vitro.192 The four pentahydro-3-aryl-1-oxopyrano-[4,3-b][1]benzopyrans122, 123aband 124all inhibit DNA synthesis by 7991% and tumour cell growth by93100%. Tricyclic compounds that lack the aryl group eithershow negligible or diminished anti-tumour activity, suggestingthat a greater conjugation is required for activity. Pyrone 122compared well with the known anti-cancer drug CPT; whileit proved less potent, it does not show any acute toxicityin vivo. A subsequent report introduced the novel 2-pyrone

125.193 Compounds 122 and 125 were tested for their abilityto disrupt microtubule (MT) dynamics; or to alter the mitoticindex and prevent murine EMT-6 mammary sarcoma cells fromsynthesising DNA and proliferating in vitro. Compound 124inhibits DNA synthesis, tubulin polymerisation and tumour cellgrowth, but to a lesser extent than 125. The former compoundmimics the effect of vincristine (VCR) but not that of paclitaxelon tubulin polymerisation. Further tests of these and related2-pyrones revealed that 122and 125 are more potent inhibitorsof DNA, RNA and protein synthesis than 124 at 1025lM.122

However,124is more effective at inhibiting the growth of L1210cells over a 4-day period at 525lM. This inconsistency may bedue to a difference in the molecular target in the inhibition ofmacromolecule synthesis and leukaemic cell growth.

Encouragingly, 124 is comparable with a number of anti-cancer drugs as an inhibitor of L1210 cell growth.

An array of N-substituted 4-aminocoumarin bicyclc andtricyclic pyrones (R1 and R2 = several moieties) was synthe-sised and tested for anti-proliferative properties.194 The anti-

proliferative and cytotoxic properties were evaluated by testingthe compounds for their inhibitory properties on DNA synthesis

in Ehrlich cells (an experimental tumour of the mouse) and forcytotoxicity by the MTT assay (HeLa cells, a tumour cell line ofhuman origin). The activity of the compounds depended on thetype of amino substituent.

In general, compounds showed increased anti-proliferativeactivity over cytotoxic activity. The most active compound was128 (NR1R2 = NHPh) with an IC50 value of 1.75 lM in theinhibition of DNA synthesis. Compound 126(NR1R2 =NHPh,Y = OCH2O, IC50 = 2.64 lM) shows good potential as ananti-proliferative agent due to its negligible cytotoxic activity.

As seen in many examples of naturally occurring 2-pyrones,variation of substituent at the 4-position is important. Novelmethods for the preparation of 4-substituted-6-methyl pyrones(130132) that allow the introduction of a plethora of alkyl,

aryl, alkenyl and alkynyl substituents have been developed.195

These involve cross-coupling of an electrophilic pyrone ringwith trialkylboranes, arylboronic acids, alkenylboronic acidsand alkynes. The synthesis of simpler yet variable 2-pyroneswas then carried out and they were screened for inhibitory

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activity against ovarian carcinoma (A2780) and human chronicmyelogenous leukaemia (K562) cell lines. The pyrones exhibitedgood cytotoxic activities. The 4-alkynyl-6-methyl-2-pyrones,130a and 130b, were most promising with IC50 values as lowas 4.0 and 1.8lM for K562 and A2720 cell lines, respectively.196

4.5 Other usesOver one third of all 2-pyrones screened by Fairlamb and co-workers showed prominent anti-proliferative activity.196 The 2-pyrones showed widespread antimicrobial activity against Bacil-lus subtilis, Escherichia coli, Staphylococcus aureus, Schizosac-charomyces pombeandBotrytis cinerea.

Selective cyclooxygenase-2 (COX-2) inhibitors have manyuseful applications. These include the treatment of neurode-generative disorders such as Alzheimers disease197 as previ-ously described, and inflammatory diseases such as rheumatoidarthritis.198 COX-2 inhibitors can also induce apoptosis in colon,stomach, prostate and breast cancer lines.198201 Diaryl heterocy-cles constitutes a major class of COX-2 inhibitors. Optimumpotency and selectivity have been shown (viastructural activity

relationship studies) to occur when a sulfonylmethyl group ispresent atthepara-positionof oneof thearyl rings anda fluorinesubstituent at thepara-position of the other.202

Etoricoxib 133202,203 bears a 6-membered pyridine ring, SC57666134,204 both an SO2Me and F group and Rolecoxib135,

205

a five membered lactone similar to a 2-pyrone function.Knaus and co-workers designed, synthesised and tested

various 2-pyrones with similar structural features to the above.206

136a, 136c and 138b showed an IC50 value of120 000). Insightsinto selectivity were gained using theoretical studies, whichreveal that the SO2Me substituent of 139 orients itself in thevicinity of the secondary pocket of COX-2. COX-1 possesses nosuch accessible pocket, due to the presence of the bulky Ile 523

residue.Investigations by the same group showed that 141 was a

potent and selective COX-2 inhibitor (IC50 = 0.02 lM).208 A

group of regioisomeric 3,4,6-triphenyl-2-pyrones with a MeSO2pharmacophore at the para-position of either a C-3 phenylor a C-4 phenyl substituent on the central 2-pyrone ring wasprepared and evaluated. COX-2 inhibitory potency is sensitiveto the substituent electronic properties at the para-position ofthe C-6 phenyl ring.An electron-withdrawing substituent (CF3)showed reduced selectivity and potency (IC50 >100 lM) towardsCOX-2.

5 Future lead compounds and directions

The review highlights the abundance of 2-pyrone naturalproducts possessing broad spectrum bioactivities, reported inthe last thirteen years. Since 2-pyrones represent a rich sourcefor isolation studies and lead discovery, here lies a wonderful

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opportunity to design, synthesise and identify novel synthetic2-pyrones and natural product 2-pyrone mimetics. The futureis bright for these compounds. The development of moreefficient syntheses to substituted 2-pyrones represents a syntheticchallenge. Here, cross-coupling (Sonogashira, Stille, Suzukietc.)209 and cyclisation strategies210 continue to be developed.It is quite apparent that simple natural and non-natural 2-pyrones possess important bioactivity, and although advanced

transformations are required for more elaborate structures, thereaders,particularly medicinalchemists, are encouraged to focuson simple analogues or relatives,e.g.2-pyridinones.

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