Bioactive pyrones and flavonoids from Cryptocarya ashersoniana seedlings

The bioassay directed fractionation of the EtOH extract from leaves of Cryptocarya ashersoniana seedlings led to the isolation of two flavonol glucosides: iso -quercitrin and hyperin, which exhibited free radical scavenging activity towards DPPH (IC 50 34.4 µ M and 32.7 µM, respectively) and were compared to standard compounds rutin (IC 50 27.0 µM) and catechin (IC 50 41.4 µM). Investigation of extracts from the seedlings roots and stems afforded one antifungal styrylpyrone: goniothalamine, and two dihydropyrones: 6-propyl-5,6-dihydro-2-pyrone and the new 6-[(4 ′ -ethyl-9 ′ -oxabicyclo[3.3.1]non-6 ′ -en-3 ′ -yl)methyl]-5,6-dihydro-2H-pyran-2-one, which had its structure determined by detailed analysis of MS and NMR data, including 2D experiments.


Introduction
Cryptocarya genus belongs to Lauraceae family and comprises more than 350 species which occur in South America, Asia and Australia.2][3][4] Alkaloids and pyrones are widely distributed in this genus in contrast to the chemical profile of Lauraceae family, in which lignans and neolignans are considered chemotaxonomical markers. 5ryptocarya species have been used as traditional medicines in South Africa for their antiinflammatory and other activities. 6,7Some of the pyrones and styrylpyrones showed larvicidal and antifertility activities, in addition to inhibition of breast cancer cell lines growth. 8-

Results and Discussion
5][16] They showed moderate free radical scavenging activity (IC 50 34.4µM and 32.7 µM), respectively when compared to standard compound rutin (IC 50 27.0µM) and higher activity than catechin (IC 50 41.4µM) (Figure 1), which is probably due to the α,β-unsaturated carbonyl moiety conjugated to ring B and the catechol group.Compounds 1 and 2 showed no antifungal activity as they did not inhibit Cladosporium cladosporioides and C. sphaerospermum growth on TLC plates.From the EtOH extract of stems and roots of C. ashersoniana seedlings, pyrones 3-5 were isolated and had their structures determination based on MS and NMR analysis, as well as comparison with literature data.
The 1 H NMR spectrum of compound 3 showed four signals at δ 5.65, δ 5.71, δ 5.95 and δ 6.80 suggesting the presence of two disubstituted double bonds, including one α,β-unsaturated carbonyl moiety; and three signals at δ 3.58, δ 3.93 and δ 4.30, assigned to oxymethine hydrogens.The 13 C NMR spectrum of compound 3 showed five signals for sp 2 carbons, including one α,β-unsaturated carbonyl moiety and 11 signals for sp 3 carbons, including three oxymethine carbons at δ 70.7, δ 76.0 and δ 77.8.Combined with DEPT 135 o data, which evidenced only five methylene carbons, and MS data, which showed the molecular ion peak at M +. 262, the formula C 16 H 22 O 3 was established, suggesting the structure of a pyrone ring attached to an unsaturated bicyclized C 11 aliphatic chain.One triplet at δ 0.86 (3H), correlating to a multiplet at δ 1.50 (2H) in the HOMOCOSY spectrum and to signals at δ 11.9 (C-11′) and δ 28.6 (C-10′), in the HMQC and HMBC spectra, respectively, evidenced an ethyl unit, which is linked to a methine carbon (δ C 45.4, δ H 1.75), assigned to C-4′, as shown by HMBC cross-peaks between H-11′ and C-4′, as well as by HOMOCOSY correlations of signals for H-4′ and H-10′.Two signals at δ 2.25 (m, 2H) and δ 4.30 (br tdd, 1H) in the 1 H NMR spectrum were assigned to H-5 and H-6 of the pyrone ring, respectively, and showed correlations with signals at δ 29.9 and δ 76.0 in the HMQC spectrum, which were then assigned to the methylene C-5 and oxymethine C-6, respectively.HMBC correlations of C-5 and C-6 to methylene hydrogens at δ 1.90 (H-7) and, from the latter to the signals at δ 36.9 (C-2′) and δ 37.5 (C-3′), established the linkage between the pyrone ring and the bicyclic moiety (Figure 2).One triplet at  δ 3.58 and one broad double-quadruplet at δ 3.93 for the two remaining oxymethine hydrogens, showing correlations with signals at δ 77.8 (C-5′) and δ 70.7 (C-1′) in the HMQC spectrum, suggested an unsaturated six-membered ring with an ether linkage, after observation of a cross-peak between the oxymethine hydrogens H-1′ (δ 3.93) and H-5′ (δ 3.58) in the HOMOCOSY and TOCSY spectra (Figure 3) as well as the HMBC correlation of H-5′ and C-1′ (Figure 2).The TOCSY spectrum showed correlations of signals at δ 1.75 (H-4′), δ 1.60 (H-3′) and δ 1.90 (H-7), which suggested an additional cyclization between C-3′ and C-4′, leading to a bicyclic moiety.This explains further correlations, e.g. from H-7 (δ 1.90) and H-3′ (δ 1.60) to C-4′ (δ 45.4), observed in the HMBC spectrum; and a long distance correlation (J 4 ) between H-7 and H-4′ in the HOMOCOSY spectrum.Additional HMBC and HOMOCOSY correlations confirmed the proposed structure for compound 3 (Table 1).The relative configuration of asymmetric centers C-1′, C-3′, C-4′ and C-5′ was established by analysis of coupling constants, e.g.J H-4′/H-5′ = 7.5 Hz, which evidenced the cis relationship of H-4′ and H-5′; and additionally by NOESY experiments, which showed correlations between H-3′/H-4′and H-4′/H-5′, and confirmed the cis orientation for these hydrogens.The cis relationship of H-1′/H-5′ was assumed due to high steric hindrance of the (H-1′/H-5′) trans isomer, as observed by molecular modeling and theoretical studies on steric energy minimization using the MM2 software and on heats of formation using the semiempirical PM3 and AM1 methods on SCF-MO-MOPAC-2000 software. 17A positive Cotton effect due to the carbonyl n→Π* transition of the α,β-unsaturated-δ-lactone moiety was observed at λ max 255 nm (∆ε +2.84) in the CD spectrum of 3.This observation indicated that the absolute configuration a C-6 was (S) on the basis of the rule first proposed by Snatzke 18 and later modified by Beecham, 19 considering the conformation of the α-pyrone ring, which was inferred due to steric factors. 20These observations led to the establishment of the new pyrone as 1′R*,3′S*,4′R*,5′S*,6S-6-[4′-ethyl-9′-oxabicycle[3.The biogenetic pathway leading to this novel pyrone was proposed, in which an acetate derived C 16 chain has gone through cyclization reactions to originate the pyrone ring via nucleophyllic attack followed by dehydration steps (Scheme 1).The subsequent attack of HO-5′ onto C-1′ with the ring closure via an ether linkage followed by dehydration and reduction steps might yield intermediate 8. Two additional dehydration steps would yield intermediate 9, which might go through a final cyclization, resulting from nucleophilic attack of C-4′ onto C-3′, and the subsequent formation of compound 3. Bicyclic pyrones resulting from cyclization of the sidechain onto the α,β-unsaturated carbonyl ring as cryptocaryolone (10) and goniopyrone (11) have been reported solely from other Cryptocarya (Lauraceae) and Goniothalamus (Annonnaceae) species, respectively 21,22 and led to structures in which the pyrone C-C double bond is lost.Compound 3 differs from those as the driving force for both cyclization steps do not involve the α,β-unsaturated carbonyl moiety of the pyrone ring.Conversely, nucleophylic attacks, dehydration and reduction reactions involving hydroxy groups and double bonds in the sidechain led to a bicyclic structure linked to an intact pyrone moiety.

ISSN 1424-6376
Page 133 © ARKAT USA, Inc Compound 4 was identified with goniothalamine, previously isolated from Goniothalamus macrophyllus (Annonnaceae), from the bark of Cryptocarya caloneura and C. moschata 23,24 and from leaves of C. ashersoniana. 25It showed CNS activity and induced fetal abnormalities in rats. 26,27Compound 5 was identified with 5,6-dihydro-6-propyl-2H-pyrane-2-one, which had been isolated previously from the fungus Lasiodiplodia theobromae, growing on fruits of Palmae species 28 and is now reported for the first time from a plant species.Compounds 3-5 were assayed for antifungal activity by bioautography with phytopathogen fungi Cladosporium cladosporioides and C. sphaerospermum. 29Compound 5 showed moderate activity on the TLC assay and had its MIC further determined (8 µg) and compared to standard fungicidal compounds nystatin (MIC 0.5 µg) and myconazol (MIC 0.5 µg).

Experimental Section
General Procedures.Silica gel (Merck, 230-400 and 70-230 Mesh), Sephadex LH-20 (Pharmacia) and polyvinylpolypyrrolidone (PVPP) were used for column chromatography unless otherwise stated and solvents were redistilled prior to use.NMR spectra were recorded on Bruker AC-200 or Varian INOVA 500 spectrometers at 200 or 500 MHz, respectively, for 1 H NMR, and at 50 or 125 MHz, respectively, for 13 C NMR, with TMS as internal standard and DMSO-d 6 or CDCl 3 as solvent.ES-MS were recorded on a VG Platform II spectrometer and elemental analysis was performed on a Perkin-Elmer Analyser model 2400CHN.Theoretical study was done using the semiempirical PM3 and AM1 methods on SCF-MO-MOPAC-2000 software.CD curves were obtained using a ISA Jobin Yvon CD6 dichrograph and optical rotation was measured on a Perkin-Elmer 241 polarimeter using a quartz cuvette (length 1 cm).
Bioautography with phytopathogen fungi on TLC plates. 25Each sample is submitted to TLC analysis and, after dry, the plate is sprayed with Cladosporium cladosporioides or C. sphaerospermum spore solution.The plates are kept at 25 o C in the dark for 2-3 days, when the fungi grow all over the plates, except for spots where antifungal compounds are present, which remain white.The inhibition zones are measured and compared to standard compounds nystatin and myconazol.
Bleaching experiments on β-carotene. 26Each extract (ca. 10 µg) was assayed for the presence of antioxidants using a TLC test and two eluents (CHCl 3 /MeOH 7:3 and hexane/EtOAc 1:1).After developing and drying, TLC plates were sprayed with a 0.02% solution of β-carotene (Aldrich) in CH 2 Cl 2 .Plates were placed under natural light until discoloration of background.The remaining yellow spots indicated the presence of antioxidant substances.Spectrophotometric assay on the reduction of 2,2-diphenyl-1-picrylhydrazyl radical. 27eaction mixtures containing test samples (dissolved in MeOH) at several concentrations and 0.004% DPPH methanolic solutions were incubated at 25 º C for 30 min.Absorbances of the resulting solutions were measured using a Milton Roy 20D spectrophotometer at 517 nm and the percent inhibition was determined by comparison with a MeOH treated control group.

Table 1 .
-NMR data for compound 3 a 13CDCl 3 , 500 MHz and 125 MHz for 1 H and13C NMR, respectively.Assignments were mostly based on HMQC and HOMOCOSY experiments.b multiplicities were obtained from DEPT 135 o experiment.c δ, multiplicity, J (Hz).