4-Alkyl- and 4-phenylcoumarins from Mesua ferrea as promising multidrug resistant antibacterials

PHYTOCHEMISTRY Phytochemistry 65 (2004) 2867–2879 www.elsevier.com/locate/phytochem

4-Alkyl- and 4-phenylcoumarins from Mesua ferrea as promising multidrug resistant antibacterials Luisella Verotta a,*, Erminio Lovaglio a, Giovanni Vidari b, Paola Vita Finzi b, Maria Grazia Neri c, Alessandro Raimondi c, Silvia Parapini c, Donatella Taramelli c, Antonella Riva d, Ezio Bombardelli d a

Dipartimento di Chimica Organica e Industriale, Universita` degli Studi di Milano, Via Venezian 21, 20133 Milano, Italy b Dipartimento di Chimica Organica, Universita` degli Studi di Pavia, Via Taramelli 10, 27100 Pavia, Italy c Istituto di Microbiologia, Universita` degli Studi di Milano, Via Pascal 36, 20133 Milano, Italy d Indena S.p.A., Viale Ortles 12, 20139 Milano, Italy Received in revised form 4 July 2004 Available online 31 July 2004 Dedicated to Kurt Hostettmann on the occasion of his 60th birthday

Abstract Supercritical CO2 selectively extracted a series of 4-alkyl and 4-phenyl 5,7-dihydroxycoumarins from Mesua ferrea blossoms. Chemical modifications of the isolated compounds allowed us to confirm the structures elucidated by spectroscopic means and to prepare new derivatives amenable to SAR studies and potential pharmaceutical development. Biological investigations towards the screening on a number of bacteria strains and Plasmodium falciparum, identified compounds 1–9 as weak antiprotozoal agents and potent antibacterials on resistant Gram-positive strains.
2004 Elsevier Ltd. All rights reserved. Keywords: Mesua ferrea; Guttiferae; 4-Alkyl and 4-phenyl 5,7-dihydroxycoumarins

1. Introduction In the course of our studies on plants belonging to the family Guttiferae, we have investigated the lipophilic extract of the aerial parts of Mesua ferrea L., a well-known tropical tree, used in folk medicine for the treatment of fever, dyspepsis and renal diseases (Dennis and Akshaya Kumar, 1998). Our interest in Guttiferae, which are prolific producers of metabolites, is related to the reported antibiotic, neuromodulator, antitumor, and antiviral *

Corresponding author. Tel.: +39-02-5031-4114; fax: +39-02-50314106. E-mail address: [email protected] (L. Verotta). 0031-9422/$ - see front matter
2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2004.07.001

activity of isolated phloroglucinols, xanthones, neoflavonoids, and coumarins (Verotta, 2003a,b). Different aerial parts of M. ferrea are traditionally used in the preparation of cosmetics and unguents. The pounded kernels of the seeds are applied externally for poulticing wounds and all forms of skin eruptions (Dennis and Akshaya Kumar, 1998; Perry and Metzger, 1980). Phytochemical investigation of different parts of the plant showed the occurrence of xanthones, coumarins, biflavones, cyclohexanedione derivatives, and an essential oil (Dennis and Akshaya Kumar, 1998). A preliminary investigation of a lipophilic extract of M. ferrea revealed a high antimicrobial activity against

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Gram-positive and anaerobic bacteria that led to antimicrobial or preservative formulations for pharmaceutical or cosmetic uses (Bombardelli and Morazzoni, 1998). Herein, we describe the complete composition of a lipophilic extract of M. ferrea, obtained under supercritical conditions, and the investigation of the bioactivity of the main components and related compounds on resistant bacteria strains. In addition, given the known antimalarial or insecticidal properties exhibited by most plants producing these coumarins (Dennis and Akshaya Kumar, 1998; Perry and Metzger, 1980; Neuwinger, 2000; Ito et al., 2000; Nagem and De A. e Silva, 1988; Bordoloi et al., 1997), the Plasmodium falciparum growth inhibitory activity of representative compounds was tested.

2. Results and discussion Multiple chromatographic separations of an extract of M. ferrea blossoms, obtained under supercritical conditions, allowed the identification of eighteen 4phenyl-5,7-dihydroxycoumarins, comprising four types of structures: (a) 6-acyl-8-prenyl derivatives [1 (mesuol) (Dennis and Akshaya Kumar, 1998; Chakraborty and Das, 1966; Chakraborty et al., 1985), 1a (mammea A/ AB) (Crombie et al., 1966, 1967), 1b (mammeisin, mammea A/AA) (Crombie et al., 1966, 1967), 2, 2a]; (b) 8-acyl-6-prenyl derivatives [3 (isomammeisin) (mammea A/BB) (Crombie et al., 1966, 1967), 3a (mammea A/BA) (Crombie et al., 1966, 1967), 4, 4a]; (c) 6-acyl-7,8-dihydrofurano derivatives [5 (mammea A/AD cycloF) (Crombie et al., 1972, 1987), 5a (mammea A/AB cycloF) (Crombie et al., 1972, 1987), 5b (mammea A/AA cycloF) (Crombie et al., 1972, 1987), 7, 7a]; (d) 6-acyl-7,8-pyrano derivatives [8 (mammea A/AD cycloD, mesuagin) (Chakraborty and Chatterji, 1969; Crombie et al., 1966, 1967, 1987; Carpenter et al., 1971), 8a (mammea A/AB cycloD, mammeigin) (Chakraborty and Chatterji, 1969; Crombie et al., 1966, 1967, 1987; Carpenter et al., 1971), 8b (mammea A/AA cycloD) (Crombie et al., 1972, 1987), 9, 9a]. In addition, two 4-(2 0 -hydroxypropyl)-5,7-dihydroxycoumarins [6 (assamene) (Bordoloi et al., 1997), 6a (surangin C) (Mahandru and Ravindran, 1986)], were isolated. The compounds were identified either in a pure state or as a mixture (labeled as MF) of chromatographically inseparable acyl homologues, through accurate analysis of high field NMR spectra recorded in two solvents (CDCl3 and acetone-d6). The majority of these data are reported in Section 3 to complete or update the literature. Compounds 2, 2a, 4, 4a, 7, 7a, 9 and 9a are new metabolites, while the other ones are known constituents of Mesua species.

The structures of isomeric coumarins 2 and 2a, and 4 and 4a were easily established by comparing their NMR spectra with those of the corresponding lower homologues 1a and 1b, and 3 and 3a, respectively. Indeed, the spectra resulted to be superimposable for the most part, with the notable difference of the characteristic signals of a geranyl (C10) chain replacing those of a C5 prenyl unit. In analogy, coumarins 9 and 9a resulted to be the geranyl homologues of 8 and 8a, respectively. Finally, the NMR signals of dihydrofurans 7 and 7a were superimposable to 5a and 5b, respectively, except for a 2-propenyl moiety replacing a 2-hydroxy-2-propyl group.

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Differentiation between each pair of regioisomeric 4phenyl-5,7-dihydroxycoumarins, i.e. between 2 and 4, was inferred by the chemical shifts of the corresponding phenolic hydroxyl groups, which, in case of compounds like 2 and 2a, resonated in the range of 10.5–10.3 ppm, both being hydrogen bonded to the ketone carbonyl group, while, in case of compounds like 4 and 4a, only one phenolic OH was shifted downfield (d 14.55), due to chelation with C-1000 . These assignments were definitely confirmed by routine HMBC spectra (Fig. 1). Structures 8 and 9 were further corroborated by submitting compounds 1 and 2, separately, to an intramolecular cyclization of the phenolic OH group onto the ortho-prenyl chain, which, in principle, can afford either dihydrofurano or pyrano substituted rings. Under suitable experimental conditions, we found that DDQ (Carpenter et al., 1971), or, more efficiently, Pd2+ catalyzed intramolecular cyclizations (Larock et al., 1998) of compounds 1 and 2, delivered the pyrano derivatives 8 and

Fig. 1. Most significant HMBC correlations in compounds 3 and 4.

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9, respectively, in good yields, and devoid of the corresponding dihydrofurano derivatives. Under the same conditions compound 3 yielded 3b, identical to ponnalide isolated from Calophyllum inophyllum (Murti et al., 1972). As expected, the cyclization of compound 3 occurred between the prenyl chain and the 5-OH substituent, since the 7-OH group was inhibited by the strong hydrogen bond with the C-1000 carbonyl group. Enantioselective total synthesis (Begley et al., 1987) proved the (S) configuration of the 2-methylbutanone side-chain of Mammea coumarins, most of which were isolated also from M. ferrea in the present study. This stereochemical assignment could not be confirmed by us, since all our efforts to establish the absolute stereochemistry of isolated compounds failed. Mixtures of isomeric coumarins (labeled as MF) or pure compounds, when available, possessing different prenyl chains (1 and 2, 3 and 4) or devoid of the alkyl chain (dihydrofurano and pyrano derivatives 5, 7, 8, 9) were tested as to their growth inhibitory activity on a series of bacteria strains (Tables 1 and 2) and on P. falciparum (Table 3). The in vitro susceptibility assays performed against strains of Escherichia coli, Enterobacter cloacae, Serratia marcescens, Pseudomonas aeruginosa, Acinetobacter spp., as well as against Candida albicans and Cryptococcus neoformans, did not prove an effective antimicrobial activity of Mesua coumarins against these Gram-negative bacteria and fungi. In fact, the minimal inhibitory concentrations (MICs) against the tested organisms were above 128 lg/ml. The activity of the Mesua derivatives was also assayed against Gram-positive organisms of the genera Staphylococcus, Enterococcus and a strain of Streptococcus durans. As shown in Tables 1a and 1b, the strains expressed different pattern of resistance to several known antibiotics. The choice of this group of strains aimed to challenge the isolated compounds against the most common and important resistance mechanisms encountered in Gram-positive bacteria. All the samples tested, except MF5, MF8 and MF9, showed an activity of potential interest either against the enterococci (MICs of 8–16 lg/ml) or against organisms of the genus Staphylococcus. It thus appears that none of the latter group of assayed strains is endowed with a specific mechanism of resistance to efficiently hinder the antimicrobial activity of Mesua coumarins. Such observation suggests that these new compounds may act by an uncommon inhibitory mechanism, possibly different from those endowed to other known antimicrobials. In addition, the anti-Staphylococcus activity of compounds 1–4 was compared with tetracycline, gentamicin, chloramphenicol, ciprofloxacin and erythromycin in an assay comprising a group of 42 different Staphylococcus strains either of the aureus or the non-aureus species. The results obtained with the individual samples are reported

h

g

f

e

d

c

Breakpoints are those of the National Committee for Clinical Laboratory Standards (1). S, susceptible; R, resistant. MIC, minimal inhibitory concentration. MET, methicillin; E, erythromycin; GM, gentamicin; CIP, ciprofloxacin; T/S, trimethoprim/sulfamethox; RIF, rifampicin; CL, clindamycin; TEICO, teicoplanin. 1a/1b: 4/1. 5a/5b/5: 5/1.5/1. 7/7a: 6/1. 8a/8b/8: 5/1.5/1. 9a/9: 4/1. b

S S – – – Strains Enterococcus faecalis 18292 Enterococcus faecalis 18250 Enterococcus faecalis 11268 Enterococcus faecium 5 Streptococcus durans 23

a

8 8 8 8 8 >128 >128 >128 – – >128 >128 – – – 16 16 – – – >128 >128 – – – 8 8 – – – 8 8 – – – 8 8 – – – S S – – –

– – – – –

R S – – –

– – – – –

S S – – –

R R – – –

R R – – –

8 8 – – –

Mesua extract MF9h MF8g MF7f MF5e 4 3 2 MF1d TET CIP

T/S

RIF

CL

MIC (lg/ml)b

GM E AMP

c

Resistancea Compound

Table 1a In vitro antibacterial activity of 10 Mesua derivatives against Gram-positive bacterial strains with defined resistance pattern

16 16 16 16 16

L. Verotta et al. / Phytochemistry 65 (2004) 2867–2879

Mesuol (1)

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in Table 2, in which data are expressed in terms of range of activity, concentration inhibiting 50% (MIC 50%) or 90% (MIC 90%) of the strains, and geometric mean. These results show that the reference antibiotics tetracycline, gentamicin and erythromycin were more effective than the Mesua derivatives against the most susceptible strains in the group. In fact, 50% of the strains were inhibited by the above antibiotics with a MIC of 0.25–0.5 lg/ml, whereas the Mesua derivatives exhibited a higher MIC 50% (2–4 lg/ml). Conversely, when the concentrations inhibiting 90% of the strains (MIC90%) were compared, some of the Mesua derivatives exhibited the best activity. Interestingly, this wider selection of strains included also organisms resistant to the reference antibiotics (MIC 90% =32 to >128), and all of them could, however, be inhibited by 4 lg/ml of MF1 and 4. Infections due to multidrug-resistant Gram-positive bacteria are a growing worldwide problem, in particular among seriously ill patients, and antibiotic options for patients with multidrug-resistant Staphylococcus aureus infections are severely limited. In this context, our findings confirm the interesting antibiotic properties of a few coumarins of this group. Table 3 shows the inhibitory properties of representative Mesua coumarins on the growth of P. falciparum, the protozoan responsible for the lethal malaria. The data point out a weak activity of all tested samples both on chloroquine-sensitive (D10) and resistant (W2) strains, at doses comprised between 1.2 lg/ml (W2) for 2 and 15.8 lg/ml (D10) for MF5.

3. Experimental 3.1. General Melting points were recorded employing a Bu¨chi SMP-20 apparatus or a Fisher–Johns hot-stage apparatus and are uncorrected. UV (in 95% EtOH) spectra were recorded on a Kontron UVIKON 941. Optical rotation values were recorded on a Perkin–Elmer 241 polarimeter. The 1H and 13C NMR spectra were determined by means of a Bruker CXP 300 spectrometer (300 and 75.47 MHz, respectively), or, alternatively, on a Bruker AMX 400 (400 and 100.61 MHz, respectively) and on a Bruker AC 200 (200 and 50 MHz, respectively). The chemical shifts for 1H and 13C NMR are referenced to CHCl3 at 7.26 ppm and CDCl3 at 77.0 ppm, respectively, or to CH3COCH3 at 2.1 ppm and CD3COCD3 at 28.8 ppm. The spectra were interpreted with the aid of standard COSY, HMBC and HMQC techniques. EI-MS (probe) 70 eV and CI-MS (iso-butane, probe), 200 eV spectra were taken on a VG 7070 EQ spectrometer. Column chromatography was performed using Silica Gel 60 (70–230 and 40–63 lm, Merck). The reactions were monitored by

Table 1b In vitro antibacterial activity of 10 Mesua derivatives against twelve strains of the Staphylococcus spp. with defined resistance pattern Compound

a b c d e f g h

MIC(lg/ml)b

AMPc

E

GM

CIP

T/S

RIF

CL

TET

MF1d

2

3

4

MF5e

MF7f

MF8g

MF9h

Mesua extract

Mesuol (1)

R R R R R R S S R R S R

S R R R S R R R R R R S

– R S R S S S S R S S R

R R R R R R S S R R S S

S S S S S S S S R S S S

S S S S S R S S R R S S

S S S R S S S S R S S S

S R S S S S S S S S S S

4 16 4 4 4 4 2 8 4 4 8 4

32 8 64 64 32 16 16 16 4 8 32 4

16 32 2 2 4 2 2 4 2 4 16 2

4 64 2 4 2 2 2 16 2 2 >128 2

>128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128

4 8 8 8 8 4 4 4 4 4 8 8

>128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128

>128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128

– 2 4 4 – 2 – – 2 – – 2

– 4 2 2 – 4 – – 2 – – 2

Breakpoints are those of the National Committee for Clinical Laboratory Standards (1). S, susceptible; R, resistant. MIC, minimal inhibitory concentration. MET, methicillin; E, erythromycin; GM, gentamicin; CIP, ciprofloxacin; T/S, trimethoprim/sulfamethox; RIF, rifampicin; CL, clindamycin; TEICO, teicoplanin. 1a/1b: 4/1. 5a/5b/5: 5/1.5/1. 7/7a: 6/1. 8a/8b/8: 5/1.5/1. 9a/9: 4/1.

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Strains S. aureus 18268 S. aureus 17380 S. aureus 17592 S. aureus 18110 S. aureus 17547 S. aureus 17728 S. aureus 3012 S. aureus 414 S. epidermidis 3112 S. epidermidis 2515 S. saprophyticus 3010 S. simulans 214

Resistancea

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Table 2 Comparative in vitro activities of four Mesua derivatives and other antimicrobial agents against 42 strains of Staphylococcus spp. Compound b

MF1 2 3 4 Tetracycline Gentamicin Erythromycin Chloramphenicol Ciprofloxacin

MIC (l g/ml)a Range

50%

90%

Mean

2 to 8 4 to 128 2 to 64 2 to >128 0.25 to >128 0.12 to >128 0.06 to >128 4 to 128 0.12 to >128

4 32 2 2 0:25 0:05 0:25 4 8

4 64 16 4 32 128 >128 64 64

4.13 23.51 4.00 2.67 0.70 3.16 0.83 6.56 3.22

a

50% and 90%: MICs for 50% and 90% of strains tested, respectively. The mean is the geometric mean. b Mixture 1a/1b: 4/1.

TLC on Merck 60 F254 (0.25 mm) plates, the spots were revealed under fluorescence (254 nm) and by spraying with ethanolic FeCl3 or with 0.5% vanillin in H2SO4/ EtOH (4:1) followed by heating. Commercially available reagents were used without prior purification. Solvent extracts of aqueous solutions were dried over anhydrous Na2SO4.

ether/CH2Cl2/EtOAc 25:25:1 (1 l), petroleum ether/ CH2Cl2/EtOAc 25:25:2 (2.5 l), CH2Cl2/EtOAc 25:1 (1.5 l), CH2Cl2/EtOAc 25:2 (1.3 l), CH2Cl2/EtOAc 10:1 (1.3 l) and CH2Cl2/MeOH 9:1 (1 l). Eluted material was collected in 10 · 400 ml and 6 · 1 l fractions. Identical fractions were combined to give six main fractions: A (fractions 3–4, 2.3 g), B (fractions 5–7, 3.8 g), C (fractions 8–10, 18.0 g), D (fractions 11–13, 1.8 g), E (fractions 14–15, 9.0 g), F (fraction 16, 3.5 g). Fraction A was subjected to flash column chromatography over Si gel (254 g, B = 6 cm, h = 18 cm) using petroleum ether/EtOAc 9:1 (1.5 l) and petroleum ether/EtOAc 85:15 (0.5 l) as eluent, to yield 3 main fractions: A1 (fractions 9–10, 160 mg) was further purified over Si gel (10 g) using petroleum ether/ CH2Cl2/EtOAc 10:10:0.1 (200 ml, 35 · 7 ml fractions) to afford, in fractions 14–33, 70 mg of MF9 (9a/9: 4/ 1); A2 (fractions 14–15, 650 mg) was rechromatographed by flash column chromatography over Si gel (87 g, B = 3.4 cm, h = 18 cm) using petroleum ether/ CH2Cl2/EtOAc 15:10:0.1 (1.5 l, 120 · 10 ml fractions). Fractions 48–86 yielded 460 mg of MF4 (4/4a: 4/1). 4 was purified from 4a by repeated crystallizations from hexane (275 mg); A3 (fractions 18–20, 360 mg) was crystallized from hexane (5 ml) to obtain 175 mg of MF3 (3/3a: 25/1).

3.2. Isolation A CO2 extract (51.7 g) of M. ferrea dried blossoms supplied by INDENA SpA (Bombardelli and Morazzoni, 1998) was fractionated by column chromatography over Si gel (600 g) with a stepwise gradient of petroleum ether/CH2Cl2/EtOAc, CH2Cl2/EtOAc and CH2Cl2/ MeOH: petroleum ether/CH2Cl2 1:1 (1.4 l), petroleum

Table 3 IC50 values of representative Mesua coumarins tested against D10 (CQ-S) and W2 (CQ-R) strains of P. falciparum Compounds MF1b Mesuol (1) 2 4 MF5c MF8d MF9e Chloroquine a

IC50 (lg/ml)a D10 (CQ-S)

W2 (CQ-R)

8.33 ± 1.06 10.75 ± 0.14 2.74 ± 0.45 11.21 ± 5.40 15.80 ± 3.25 9.19 ± 1.88 12.85 ± 2.69 0.011 ± 0.004

7.50 ± 1.24 8.91 ± 0.27 1.17 ± 0.61 8.38 ± 2.52 13.40 ± 4.25 7.53 ± 2.00 13.75 ± 5.15 0.229 ± 0.090

The antimalarial activity was assayed in vitro on P. falciparum strains using the pLDH assay. The results are expressed as IC50 ± SD of three different experiments each performed in triplicate. b Mixture 1a/1b: 4/1. c Mixture 5a/5b/5: 5/1.5/1. d Mixture 8a/8b/8: 5/1.5/1. e Mixture 9a/9: 4/1.

3.2.1. 5-Hydroxy-6-isobutyryl–8-methyl-8-(4-methylpent3-enyl)-4-phenyl-2H-pyrano[2,3-h]chromen-2-one (9) Yellow gum; Rf = 0.29 (petroleum ether/EtOAc 9:1, FeCl3: green; vanillin: dark violet); 1H NMR (200 MHz, CDCl3) d 14.60 (1H, s, 5-OH), 7.42–7.20 (5H, m, Ar), 6.95 (1H, J = 10 Hz, H-1000 ), 5.98 (1H, s, H-3), 5.58 (1H, d, J = 10 Hz, H-2000 ), 5.08 (1H, brt, J = 1.0 Hz, H-7000 ), 3.71 (1H, ept, J = 6.7 Hz, H-200 ), 2.20–1.75 (4H, m, H-5000 and H-6000 ), 1.71 and 1.62 (3H and 3H, two s, H-9000 and H-10000 ), 1.18 and 1.15 (1.5H and 1.5H, two s, H-4000 ), 1.19 (6H, d, J = 6.7 Hz, H-300 and H-400 ); EI-MS, m/z (rel. int.): 458 [M]+. 3.2.2. 5-Hydroxy-8-methyl-6-(2-methylbutanoyl)-8-(4methylpent-3-enyl)-4-phenyl-2H-pyrano[2,3-h]chromen2-one (9a) Yellow gum; Rf = 0.29 (petroleum ether/EtOAc 9:1, FeCl3: green; vanillin: dark violet); 1H NMR (200 MHz, CDCl3) d 14.60 (1H, s, 5-OH), 7.42–7.20 (5H, m, Ar), 6.95 (1H, J = 10 Hz, H-1000 ), 5.98 (1H, s, H-3), 5.58 (1H, d, J = 10 Hz, H-2000 ), 5.08 (1H, brt, J = 1.0 Hz, H-7000 ), 3.72 (1H, m, H-200 ), 2.20–1.75 (5H, m, H5000 , H-6000 and H-400 a), 1.71 and 1.62 (3H and 3H, two s, H-9000 and H-10000 ), 1.45 (1H, m, H-400 b), 1.18 and 1.15 (1.5H and 1.5H, two s, H-4000 ), 1.15 (3H, d, J = 6.7 Hz, H-300 ), 0.87 (3H, t, J = 7.4 Hz, H-500 ); EIMS, m/z (rel. int.): 472 [M]+ (23), 415 (12), 389 (100), 371 (19).

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3.2.3. 5,7-Dihydroxy-8-(2-methylbutanoyl)-6-[(E)-3,7dimethylocta-2,6-dienyl]-4-phenyl-2H-chromen-2-one (4) Colorless crystals; m.p. 90–92 C; Rf = 0.21 (petroleum ether/EtOAc 9:1, FeCl3: dark purple; vanillin: 25 light violet); – ½a
D ¼ 3:83 (CHCl3; c = 0.73); UV (EtOH) kmax 202, 226, 296, 333 nm; 1H NMR (400 MHz, CDCl3) d 14.55 (1H, s, 7-OH), 7.58–7.38 (5H, m, Ar), 6.01 (1H, s, H-3), 5.85 (1H, brs, 5-OH), 5.10 (1H, brt, J = 7.4 Hz, H-200 ), 5.00 (1H, brt, J = 6.5 Hz, H-700 ), 3.96 (1H, sext, J = 6.7 Hz, H-2000 ), 3.30 (2H, d, J = 7.4 Hz, H-100 ), 2.1–1.8 (4H, m, H-500 and H-600 ), 1.93 (1H, m, H-4000 b), 1.70 (3H, s, H-400 ), 1.60 and 1.52 (3H and 3H, two s, H-900 and H-1000 ), 1.47 (1H, m, H4000 a), 1.29 (3H, d, J = 6.7 Hz, H-3000 ), 1.02 (3H, t, J = 7.4 Hz, H-5000 ); 13C NMR (100 MHz, CDCl3): d 210.5 (s, C-1000 ), 166.8 (s, C-7), 158.6 (s, C-2), 157.0 (s, C-4), 155.4 (s, C-8a), 154.2 (s, C-5), 137.8 (s, C-1 0 ), 137.0 (s, C-300 ), 131.5 (s, C-800 ), 129.9 (d, C-4 0 ), 129.3 (d, C-3 0 and C-5 0 ), 127.4 (d, C-2 0 and C-6 0 ), 123.8 (d, C-700 ), 120.5 (d, C-200 ), 112.4 (s, C-6), 112.0 (d, C-3), 104.0 (s, C-8), 100.4 (s, C-4a), 46.8 (d, C-2000 ), 39.5 (t, C-500 ), 27.1 (t, C-4000 ), 26.4 (t, C-600 ), 25.5 (q, C-1000 ), 21.4 (t, C-100 ), 17.5 (q, C-900 ), 16.5 (q, C-3000 ), 16.1 (q, C4000 ), 11.7 (q, C-5000 ) EI-MS, m/z (rel. int.): 474 [M]+ (31), 417 (22), 405 (17), 389 (15), 351 (100), 333 (22), 293 (60). 3.2.4. 5,7-Dihydroxy-8-(3-methylbutanoyl)-6-[(E)-3,7dimethylocta-2,6-dienyl]-4-phenyl-2H-chromen-2-one (4a) 1 H NMR (400 MHz, CDCl3) d 14.55 (1H, s, 7-OH), 7.58–7.38 (5H, m, Ar), 6.01 (1H, s, H-3), 5.85 (1H, brs, 5-OH), 5.10 (1H, brt, J = 7.4 Hz, H-200 ), 5.00 (1H, brt, J = 1.0 Hz, H-700 ), 3.30 (2H, d, J = 7.4 Hz, H-100 ), 3.19 (2H, d, J = 6.7 Hz, H-2000 ), 2.10 (2H, m, H-3000 ), 2.1–1.8 (4H, m, H-500 and H-600 ), 1.70 (3H, s, H-400 ), 1.60 and 1.52 (3H and 3H, two s, H-900 and H-1000 ), 1.09 (6H, d, J = 6.7 Hz, H-4000 and H-5000 ). Fraction B (3.86 g) was fractionated by flash column chromatography over Si gel (250 g, B = 6.0 cm, h = 18 cm) using petroleum ether/EtOAc 85:15 (1 l) and petroleum ether/EtOAc 80:20 (1 l) to obtain two fractions. B1 (1.03 g) was purified by column chromatography over Si gel (flash 140 g, B = 4.0 cm, h = 18 cm) with petroleum ether/EtOAc 85:15 (1.5 l, 50 · 30 ml fractions); fractions 20–30 gave by crystallization from hexane, 370 mg of MF2 (2/2a: 9/1); 2 was purified from 2a by repeated crystallizations from hexane (296 mg). B2 (740 mg) was rechromatographed by flash column chromatography over Si gel (113 g, B = 4 cm, h = 18 cm) using petroleum ether/EtOAc 85:15 (1.6 l, 80 · 20 ml fractions); fractions 19–32 afforded, by crystallization from hexane, 327 mg of MF1 (1a/1b: 4/1). 3.2.5. 5,7-Dihydroxy-6-(2-methylbutanoyl)-8-[(E)-3,7dimethylocta-2,6-dienyl]-4-phenyl-2H-chromen-2-one (2) Yellow crystals; m.p. 89–90 C; Rf = 0.16 (petroleum ether/EtOAc 9:1, FeCl3: green; vanillin: dark violet); UV

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(EtOH) kmax 206, 285, 338, 431 nm; 1H NMR (400 MHz, CDCl3) d 10.52 (1H, s, 5-OH), 10.32 (1H, s, 7OH), 7.51–7.38 (5H, m, Ar), 5.96 (1H, s, H-3), 5.27 (1H, t, J = 6.9 Hz, H-2000 ), 5.06 (1H, brt, J = 6.5 Hz, H-7000 ), 3.62 (1H, m, H-200 ), 3.59 (2H, d, J = 6.9 Hz, H1000 ), 2.12–2.07 (4H, m, H-5000 and H-6000 ), 1.88 (3H, s, H-4000 ), 1.77 (1H, m, H-400 a), 1.66 (3H, brs, H-9000 ), 1.59 (3H, brs, H-10000 ), 1.36 (1H, m, H-400 b), 1.09 (3H, d, J = 6.7 Hz, H-300 ), 0.84 (3H, t, J = 7.4 Hz, H-500 ); 13C NMR (100 MHz, CDCl3) d 212.0 (s, C-100 ), 163.0 (s, C-7), 159.9 (s, C-2 and C-5), 156.8 (s, C-8a), 155.0 (s, C-4), 139.9 (s, C-1 0 ), 137.8 (s, C-3000 ), 132.1 (s, C-8000 ), 129.7 (d, C-4 0 ), 129.1 (d, C-3 0 and C-5 0 ), 127.6 (d, C-2 0 and C-6 0 ), 123.9 (d, C-7000 ), 120.6 (d, C-2000 ), 112.9 (d, C-3), 107.7 (s, C-8), 107.1 (s, C-6), 101.4 (s, C-4a), 47.0 (d, C-200 ), 40.0 (t, C-5000 ), 27.0 (t, C-400 ), 26.6 (t, C6000 ), 25.9 (q, C-10000 ), 21.9 (t, C-1000 ), 17.9 (q, C-9000 ), 16.8 (q, C-4000 ), 16.6 (q, C-300 ), 12.0 (q, C-500 ); EI-MS, m/z (rel. int.): 474 [M]+ (23), 417 (27), 405 (19), 389 (11), 351 (100), 333 (22), 293 (66). 3.2.6. 5,7-Dihydroxy-6-(3-methylbutanoyl)-8-[(E)-3,7dimethylocta-2,6-dienyl]-4-phenyl-2H-chromen-2-one (2a) 1 H NMR (400 MHz, CDCl3) d 10.52 (1H, s, 5-OH), 10.32 (1H, s, 7-OH), 7.51–7.38 (5H, m, Ar), 5.96 (1H, s, H-3), 5.27 (1H, t, J = 6.9 Hz, H-2000 ), 5.06 (1H, t, J = 6.5 Hz, H-7000 ), 3.01 (2H, d, J = 6.6 Hz, H-200 ), 3.59 (2H, d, J = 6.9 Hz, H-1000 ), 2.12–2.07 (4H, m, H-5000 and H-6000 ), 2.19 (1H, m, H-300 ), 1.88 (3H, s, H-4000 ), 1.66 (3H, s, H-9000 ), 1.59 (3H, s, H-10000 ), 0.90 (6H, d, J = 6.7 Hz, H-400 and H-500 ); 13C NMR (100 MHz, CDCl3) d 202.0 (s, C-100 ), 163.0 (s, C-7), 159.9 (s, C-2 and C-5), 156.8 (s, C-8a), 155.0 (s, C-4), 139.9 (s, C-1 0 ), 137.8 (s, C-3000 ), 132.1 (s, C-8000 ), 129.7 (d, C-4 0 ), 129.1 (d, C-3 0 and C-5 0 ), 127.6 (d, C-2 0 and C-6 0 ), 123.9 (d, C-7000 ), 120.6 (d, C-2000 ), 112.9 (d, C-3), 107.7 (s, C-8), 107.1 (s, C-6), 101.4 (s, C-4a), 53.5 (t, C-200 ), 40.0 (t, C-5000 ), 26.6 (t, C-6000 ), 25.9 (q, C-10000 ), 24.8 (d, C-300 ), 22.6 (q, C-400 and C-500 ), 21.9 (t, C-1000 ), 17.9 (q, C-9000 ), 16.8 (q, C-4000 ). Fraction C (18.0 g), was parcelled out in two main fractions (C1 and C2) by column chromatography over Si gel (500 g) using toluene/EtOAc 20:1 (2.4 l, 12 · 200 ml fractions) and EtOAc (0.8 l, 4 · 200 ml fractions). C1 (fractions 2–4, 5.3 g) was rechromatographed by column chromatography over Si gel (200 g), using petroleum ether/EtOAc 9.5:0.5 (0.3 l) and petroleum ether/EtOAc 9:1 (1.9 l) to obtain two fractions: C1a (fractions 41–63, 1.3 g) and C1b (fractions 64–76, 0.8 g). C1a was further purified by flash column chromatography over Si gel (220 g, B = 5 cm) using petroleum ether/t-BuOMe 9.5:0.5 (0.5 l, 20 · 25 ml fractions) and petroleum ether/t-BuOMe 9:1 (3.5 l, 140 · 25 ml fractions) to afford, in fractions 59–95, a mixture of 3 and 3a (0.63 g). The mixture was added to MF3 of A and purified by three crystallizations from hexane to give 120 mg of 3.

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C2 (fractions 5–10, 10.5 g) was rechromatographed by column chromatography over Si gel (600 g), using cyclohexane/EtOAc 9:1 (3.2 l), cyclohexane/EtOAc 8:2 (0.7 l) and EtOAc (2 l). Eluted material was collected in 30 · 100 ml and 5 · 400 ml fractions. Similar fractions were combined to give C2a (fractions 15–20, 1.6 g), C2b (fractions 21–23, 2.1 g) and C2c (fractions 24–35, 6.4 g). C2a was purified by column chromatography over Si gel (180 g) using hexane/CHCl3/EtOAc 10:10:0.2 (2.2 l) and hexane/CHCl3/EtOAc 10:10:0.25 (2.0 l). Fractions 4–8 were purified by crystallization from hexane (6 ml) to yield 504 mg of MF8 (8a/8ba/8: 5/1.5/1). C2b was purified by crystallization from hexane to give 660 mg of mesuol (1). C2c was purified by column chromatography over Si gel (350 g) using petroleum ether/ EtOAc 85:15 (15 · 100 ml fractions). Fractions 4–6 afforded by crystallization from hexane 0.92 g of MF1 (1a/1b: 4/1). C3 (fractions 11–12, 1.2 g) was added to D. 3.2.7. 5,7-Dihydroxy-8-(2-methylbutanoyl)-6-(3-methylbut-2-enyl)-4-phenyl-2H-chromen-2-one (mammea A/BB) (isomammeisin) (3) Colorless crystals; mp 124–125 C; Rf = 0.16 (petroleum ether/EtOAc 9:1, FeCl3: purple; vanillin: pink); 25 ½a
D ¼ 5:2 (CHCl3; c = 0.8); UV (EtOH) kmax 293, 331 nm; 1H NMR (400 MHz, CDCl3) d 14.58 (1H, s, 7-OH), 7.59–7.41 (5H, m, Ar), 6.01 (1H, s, H-3), 5.96 (1H, s, 5-OH), 5.10 (1H, tqq, J = 6.9, 1.1 Hz, H-200 ), 3.97 (1H, sext, J = 6.6 Hz, H-2000 ), 3.31 (2H, brd, J = 6.9 Hz, H-100 ), 1.95 (1H, ddq, J = 13.6, 7.3, 6.5 Hz, H-4000 a), 1.70 (3H, brs, H-400 ), 1.66 (3H, d, J = 1.1 Hz, H-500 ), 1.54 (1H, ddq, J = 13.6, 7.3, 6.5 Hz, H-4000 b), 1.30 (3H, d, J = 6.6 Hz, H-3000 ), 1.03 (3H, t, J = 7.3 Hz, H-5000 ); 13C NMR (100 MHz, CDCl3) d 210.5 (s, C-1000 ), 166.8 (s, C-7), 158.5 (s, C-2), 156.9 (s, C-4), 155.6 (s, C-8a), 154.1 (s, C-5), 136.7 (s, C-1 0 ), 134.0 (s, C-300 ), 130.1 (d, C-4 0 ), 129.5 (d, C-3 0 and C-5 0 ), 127.4 (d, C-2 0 and C-6 0 ), 120.7 (d, C-200 ), 112.6 (s, C-6), 112.1 (d, C-3), 104.1 (s, C-8), 100.4 (s, C-4a), 46.9 (d, C-2000 ), 27.1 (t, C-4000 ), 25.6 (q, C-500 ), 21.5 (t, C-100 ), 17.8 (q, C-400 ), 16.5 (q, C-3000 ), 11.7 (q, C-5000 ); EI-MS, m/z (rel. int.): 406 [M+] (28), 363 (12), 349 (60), 293 (100), 265 (5). 3.2.8. 5,7-Dihydroxy-8-(3-methylbutanoyl)-6-(3-methylbut-2-enyl)-4-phenyl-2H-chromen-2-one (mammea A/BA) (3a) 1 H NMR (400 MHz, CDCl3) d 14.58 (1H, s, 7-OH), 7.59–7.41 (5H, m, Ar), 6.01 (1H, s, H-3), 5.96 (1H, s, 5-OH), 5.10 (1H, tqq, J = 6.9, 1.1 Hz, H-200 ), 3.31 (2H, brd, J = 6.9 Hz, H-100 ), 3.19 (2H, d, J = 6.7 Hz, H-2000 ), 2.32 (1H, m, H-3000 ), 1.70 (3H, brs, H-400 ), 1.66 (3H, d, J = 1.1 Hz, H-500 ), 1.06 (6H, d, J = 6.6 Hz, H-4000 and H-5000 ); 13C NMR (100 MHz, CDCl3) d 206.5 (s, C1000 ), 166.8 (s, C-7), 158.5 (s, C-2), 156.9 (s, C-4), 155.6

(s, C-8a), 154.1 (s, C-5), 136.7 (s, C-1 0 ), 134.0 (s, C-300 ), 130.1 (d, C-4 0 ), 129.5 (d, C-3 0 and C-5 0 ), 127.4 (d, C-2 0 and C-6 0 ), 120.7 (d, C-200 ), 112.6 (s, C-6), 112.1 (d, C3), 104.1 (s, C-8), 100.4 (s, C-4a), 53.6 (t, C-2000 ), 26.4 (d, C-3000 ), 25.6 (q, C-500 ), 21.5 (t, C-100 ), 22.6 (q, C-4000 and C-5000 ), 17.8 (q, C-400 ); CI-MS, m/z (rel. int.): 407 [M+H]+ (100), 393 (20), 365 (13), 351 (25). 3.2.9. 5,7-Dihydroxy-6-(isobutyryl)-8-(3-methylbut-2enyl)-4-phenyl-2H-chromen-2-one (mesuol) (1) Spectral data were identical to those reported in the literature (Chakraborty et al., 1985). 3.2.10. 5,7-Dihydroxy-6-(2-methylbutanoyl)-8-(3-methylbut-2-enyl)-4-phenyl-2H-chromen-2-one (mammea A/AB) (1a) Rf = 0.11 (petroleum ether/EtOAc 9:1, FeCl3: pink; vanillin: blue); UV kmax (EtOH) 283, 337 nm; 1H NMR (400 MHz, CDCl3) d 12.15 (1H, brs, 5-OH), 10.70 (1H, brs, 7-OH), 7.48–7.45 (5H, m, Ar), 5.92 (1H, s, H-3), 5.21 (1H, tqq, J = 7.0, 1.2, 0.8 Hz, H2000 ), 3.82 (1H, sext, J = 6.7 Hz, H-200 ), 3.57 (2H, dqq, J = 7.0, 1.0 Hz, H-1000 ), 1.86 (3H, d, J = 0.8 Hz, H-4000 ), 1.80 (1H, ddq, J = 14.9, 7.4 Hz, H-400 a), 1.70 (3H, d, J = 1.2 Hz, H-5000 ), 1.38 (1H, ddq, J = 14.9, 7.4 Hz, H-400 b), 1.13 (3H, d, J = 6.7 Hz, H-300 ), 0.86 (3H, t, J = 7.4 Hz, H-500 ); 13C NMR (100 MHz, CDCl3) d 212.0 (s, C-100 ), 161.0 (s, C-7), 160.6 (s, C5), 158.5 (s, C-2), 157.4 (s, C-8a), 155.3 (s, C-4), 138.7 (s, C-1 0 ), 132.7 (s, C-3000 ), 128.4 (d, C-4 0 ), 127.4 (d, C-3 0 and C-5 0 ), 127.1 (d, C-2 0 and C-6 0 ), 121.0 (d, C-2000 ), 112.2 (d, C-3), 107.8 (s, C-8), 107.4 (s, C6), 101.8 (s, C-4a), 46.4 (d, C-200 ), 26.7 (t, C-400 ), 24.9 (q, C-5000 ), 22.0 (t, C-1000 ), 17.2 (q, C-4000 ), 15.8 (q, C-300 ), 11.1 (q, C-500 ); EI-MS, m/z (rel. int.): 406 [M]+ (34), 391 (4), 363 (8), 351 (15), 349 (42), 293 (100), 265 (9). 3.2.11. 5,7-Dihydroxy-6-(3-methylbutanoyl)-8-(3-methylbut-2-enyl)-4-phenyl-2H-chromen-2-one (mammea A/ AA) (mammeisin) (1b) 1 H NMR (400 MHz, CDCl3) d 12.15 (1H, brs, 5-OH), 10.70 (1H, brs, 7-OH), 7.48–7.45 (5H, m, Ar), 5.92 (1H, s, H-3), 5.21 (1H, tqq, J = 7.0, 1.2, 0.8 Hz, H-2000 ), 3.57 (2H, dqq, J = 7.0, 1.0 Hz, H-1000 ), 3.02 (2H, d, J = 6.7 Hz, H-200 ), 2.25 (1H, m, H-300 ), 1.86 (3H, d, J = 0.8 Hz, H-4000 ), 1.70 (3H, d, J = 1.2 Hz, H-5000 ), 0.92 (6H, d, J = 6.6 Hz, H-400 and H-500 ); 13C NMR (100 MHz, CDCl3) d 207.5 (s, C-100 ), 161.0 (s, C-7), 160.6 (s, C-5), 158.5 (s, C-2), 157.4 (s, C-8a), 155.3 (s, C-4), 138.7 (s, C-1 0 ), 132.7 (s, C-3000 ), 128.4 (s, C-4 0 ), 127.4 (d, C-3 0 and C-5 0 ), 127.1 (d, C-2 0 and C-6 0 ), 121.0 (d, C-2000 ), 112.2 (d, C-3), 107.8 (s, C-8), 107.4 (s, C-6), 101.8 (s, C-4a), 53.1 (t, C-200 ), 21.9 (q, C-400 and C-500 ), 24.9 (q, C-5000 ), 24.8 (d, C-300 ), 22.0 (t, C-1000 ), 17.2 (q, C-4000 ).

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3.2.12. 5-Hydroxy-6-isobutyryl-8,8-dimethyl-4-phenyl2H-pyrano[2,3-h]chromen-2-one (mammea A/AD cycloD) (mesuagin) (8) 1 H NMR (300 MHz, CD3COCD3) d 14.70 (1H, s, 5OH), 7.42–7.36 (5H, m, Ar), 6.82 (1H, d, J = 10.1 Hz, H1000 ), 5.92 (1H, s, H-3), 5.85 (1H, d, J = 10.1 Hz, H-2000 ), 3.97 (1H, ept, J = 6.7 Hz, H-200 ), 1.63 (3H, brs, H-4000 ), 1.62 (3H, brs, H-5000 ), 1.19 (6H, d, J = 6.7 Hz, H-300 and H-400 ); 13C NMR (75 MHz, CD3COCD3) d 211.7 (s, C-100 ), 164.3 (s, C-5), 158.2 (s, C-2), 157.7 (s, C-8a), 155.8 (s, C-4), 154.9 (s, C-7), 139.5 (s, C-1 0 ), 127.9 (d, C-2 0 , C-4 0 and C-6 0 ), 129.4 (d, C-3 0 and C-5 0 ), 127.1 (d, C-2000 ), 114.7 (d, C-1000 ), 112.4 (d, C-3), 106.6 (s, C-6), 102.0 (s, C-4a), 101.4 (s, C-8), 80.1 (s, C-3000 ), 39.6 (d, C-200 ), 28.0 (q, C-4000 and C-5000 ), 18.6 (q, C-300 and C-400 ). 3.2.13. 5-Hydroxy-8,8-dimethyl-6-(2-methylbutanoyl)-4phenyl-2H-pyrano[2,3-h]chromen-2-one (mammea A/AB cycloD) (mammeigin) (8a) Rf = 0.25 (petroleum ether/EtOAc 9:1, FeCl3: purple; vanillin: blue); 1H NMR (300 MHz, CD3COCD3) d 14.70 (1H, s, 5-OH), 7.42–7.36 (5H, m, Ar), 6.82 (1H, d, J = 10.1 Hz, H-1000 ), 5.92 (1H, s, H-3), 5.85 (1H, d, J = 10.1 Hz, H-2000 ), 3.86 (1H, sext, J = 6.7 Hz, H-200 ), 1.86 (1H, ddq, J = 13.4, 7.4 Hz, H-400 a), 1.63 (3H, brs, H-4000 ), 1.62 (3H, brs, H-5000 ), 1.43 (1H, ddq, J = 13.4, 7.4 Hz, H-400 b), 1.18 (3H, d, J = 6.7 Hz, H-300 ), 0.92 (3H, t, J = 7.4 Hz, H-500 ); 13C NMR (75 MHz, CD3COCD3) d 211.7 (s, C-100 ), 164.3 (s, C-5), 158.2 (s, C-2), 157.7 (s, C-8a), 155.8 (s, C-4), 154.9 (s, C-7), 139.5 (s, C-1 0 ), 127.9 (d, C-2 0 , C-4 0 and C-6 0 ), 129.4 (d, C-3 0 and C-5 0 ), 127.1 (d, C-2000 ), 114.7 (d, C-1000 ), 112.4 (d, C-3), 106.6 (s, C-6), 102.0 (s, C-4a), 101.4 (s, C-8), 80.1 (s, C-3000 ), 46.4 (d, C-200 ), 28.0 (q, C-4000 and C-5000 ), 27.3 (t, C-400 ), 16.1 (q, C-300 ), 11.1 (q, C-500 ); EI-MS, m/ z (rel. int.): 404 [M]+, (70), 389 (100), 371 (28), 347 (92). 3.2.14. 5-Hydroxy-8,8-dimethyl-6-(3-methylbutanoyl)-4phenyl-2H-pyrano[2,3-h]chromen-2-one (mammea A/ AA cycloD) (8b) 1 H NMR (300 MHz, CD3COCD3) d 14.70 (1H, s, 5OH), 7.42–7.36 (5H, m, Ar), 6.82 (1H, d, J = 10.1 Hz, H1000 ), 5.92 (1H, s, H-3), 5.85 (1H, d, J = 10.1 Hz, H-2000 ), 3.06 (2H, d, J = 6.7 Hz, H-200 ), 2.25 (1H, m, H-300 ), 1.63 (3H, brs, H-4000 ), 1.62 (3H, brs, H-5000 ), 0.98 (6H, d, J = 6.6 Hz, H-400 and H-500 ); 13C NMR (75 MHz, CD3COCD3) d 207.0 (s, C-100 ), 164.3 (s, C-5), 158.2 (s, C-2), 157.7 (s, C-8a), 155.8 (s, C-4), 154.9 (s, C-7), 139.5 (s, C-1 0 ), 127.9 (d, C-2 0 , C-4 0 and C-6 0 ), 129.4 (d, C-3 0 and C-5 0 ), 127.1 (d, C-2000 ), 114.7 (d, C-1000 ), 112.4 (d, C-3), 106.6 (s, C-6), 102.0 (s, C-4a), 101.4 (s, C-8), 80.1 (s, C-3000 ), 53.2 (t, C-200 ), 28.0 (q, C-4000 and C-5000 ), 24.7 (d, C-300 ), 21.9 (q, C-400 and C-500 ). Fraction D was chromatographed by flash column chromatography over Si gel (113 g, B = 4 cm, h = 18 cm) using petroleum ether/EtOAc 4:1 (1 l, 55 · 15 ml

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fractions). Fractions 21–45 (846 mg) were crystallized from MeOH yielding b-sitosterol. The mother liquors (560 mg) were purified by column chromatography over Si gel (50 g) with petroleum ether/CH2Cl2/EtOAc 10:10:0.4 (0.3 l), 10:10:0.5 (1 l) and 10:10:1 (1 l). 110 · 30 ml fractions were collected. Fractions 45–75 gave, by crystallization from hexane 254 mg of MF7 (7/7a: 6/1). 3.2.15. 8,9-Dihydro-5-hydroxy-6-(2-methylbutanoyl)-4phenyl-8-(prop-1-en-2-yl)furo[2,3-h]chromen-2-one (7) Rf = 0.10 (petroleum ether/EtOAc 8:2, FeCl3: green, vanillin: blue); 1H NMR (400 MHz, CDCl3) d 10.18 (1H, s, 5-OH), 7.40–7.26 (5H, m, Ar), 5.95 (1H, s, H3), 5.01 and 4.93 (1H and 1H, two brs, H-5000 ), 4.51 (1H, brt, H-2000 ), 3.83 (1H, m, H-200 ), 3.31 (2H, m, H1000 ), 1.93 (3H, s, H-4000 ), 1.82 (1H, m, H-400 a), 1.39 (1H, m, H-400 b), 1.14 (3H, d, J = 6.7 Hz, H-300 ), 0.89 (3H, t, J = 7.4 Hz, H-500 ); 13C NMR (100 MHz CDCl3) d 215.2 (s, C-100 ), 163.2 (s, C-7), 161.9 (s, C-5), 159.7 (s, C-2), 157.7 (s, C-8a), 156.5 (s, C-4), 145.9 (s, C-3000 ), 139.3 (s, C-1 0 ), 128.9 (d, C-4 0 ), 127.6 (d, C-3 0 and C5 0 ), 127.1 (d, C-2 0 and C-6 0 ), 112.1 (d, C-3), 110.9 (t, C-5000 ), 104.6 (s, C-6 and C-8), 102.5 (s, C-4a), 77.6 (d, C-2000 ), 46.5 (d, C-200 ), 28.6 (t, C-1000 ), 26.8 (t, C-400 ), 22.6 (q, C-4000 ), 16.5 (q, C-300 ), 11.7 (q, C-500 ); EI-MS, m/z (rel. int.): 404 [M]+ (31), 353 (100), 347 (75), 333 (60), 295 (50). 3.2.16. 8,9-Dihydro-5-hydroxy-6-(3-methylbutanoyl)-4phenyl-8-(prop-1-en-2-yl)furo[2,3-h]chromen-2-one (7a) Rf = 0.10 (petroleum ether/EtOAc 8:2, FeCl3: green, vanillin: blue); 1H NMR (400 MHz, CDCl3) d 10.17 (1H, s, 5-OH), 7.40–7.26 (5H, m, Ar), 5.95 (1H, s, H3), 5.01 and 4.93 (1H and 1H, two brs, H-5000 ), 4.51 (1H, brt, H-2000 ), 3.19 (2H, d, J = 6.8 Hz, H-200 ), 3.31 (2H, m, H-1000 ), 2.22 (1H, m, H-300 ), 1.93 (3H, s, H-4000 ), 1.06 (6H, d, J = 6.7 Hz, H-400 and H-500 ); 13C NMR (100 MHz, CDCl3) d 212.5 (s, C-100 ), 163.2 (s, C-7), 161.9 (s, C-5), 159.7 (s, C-2), 157.7 (s, C-8a), 156.5 (s, C-4), 145.9 (s, C-3000 ), 139.3 (s, C-1 0 ), 128.9 (d, C-4 0 ), 127.6 (d, C-3 0 and C-5 0 ), 127.1 (d, C-2 0 and C-6 0 ), 112.1 (d, C-3), 110.9 (t, C-5000 ), 104.6 (s, C-6 and C-8), 102.5 (s, C-4a), 77.6 (d, C-2000 ), 53.4 (t, C-200 ), 28.6 (t, C-1000 ), 26.8 (q, C-400 and C-500 ), 24.9 (d, C-300 ), 22.6 (q, C-4000 ). Fraction E was fractionated by column chromatography over Si gel (400 g) using petroleum ether/CHCl3/EtOAc 10:10:2 (0.5 l), CHCl3/EtOAc 10:1 (1.5 l), and CHCl3/EtOAc 1:1 (1 l) to yield three fractions: E1 (2.44 g), E2 (1.93 g) and E3 (2.76 g). E2 was purified by column chromatography over Si gel (flash 150 g, B = 4.0 cm, h = 18 cm) with petroleum ether/EtOAc 9:1 (1.5 l, 100 · 15 ml fractions). Fractions 65–75 yielded, by crystallization from hexane/EtOAc 9:1, 140 mg of 6; the mother liquors were further purified by silica gel

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(10 g). Elution with a gradient of hexane/EtOAc yielded 6a (80 mg). 3.2.17. 5,7-Dihydroxy-4-(1-hydroxypropyl)-8-(2-methylbutanoyl)-6-(3-methylbut-2-enyl)-2H-chromen-2-one (assamene) (6) Spectral data were identical to those reported in the literature (Bordoloi et al., 1997). 3.2.18. 5,7-Dihydroxy-4-(1-hydroxypropyl)-8-(2-methylbutanoyl)-6-[(E)-3,7-dimethylocta-2,6-dienyl]-2H-chromen-2-one (surangin C) (6a) Yellow crystals; mp 105–106 C; 1H NMR (200 MHz, CDCl3) d 14.20 (1H, s, 7-OH), 7.25 (1H, s, 5OH), 6.09 (1H, s, H-3), 5.20 (1H, brt, J = 7.0 Hz, H200 ), 5.05 (1H, tqq J = 7.0, 1.0 Hz, H-700 ), 4.72 (1H, t, J = 7.0 Hz, H-1 0 ), 3.72 (1H, m, H-2000 ), 3.39 (2H, d, J = 7.0 Hz, H-100 ), 2.05–1.95 (4H, m, H-500 and H-600 ), 1.95–1.25 (7H, m, H-2 0 , H-4000 and H-400 ), 1.63 and 1.56 (6H, two s, H-900 and H-1000 ), 1.20 (3H, d, J = 7.0 Hz, H-3000 ), 1.05 (3H, t, J = 7.0 Hz, H-5000 ), 0.87 (3H, t, J = 7.0 Hz, H-3 0 ); CI-MS, m/z (rel. int.) 456 [M+H]+ (70), 438 (18), 400 (15), 399 (51), 395 (11), 387 (14), 381 (17), 370 (10), 369 (36), 355 (16), 351 (18), 334 (22), 333 (100), 315 (75). 3.5 g of F were chromatographed by flash column chromatography over Si gel (255 g, B = 6 cm) using petroleum ether/EtOAc/MeOH 8:2:0.1 (2 l, 10 · 200 ml fractions) as an eluent. Fractions 7–8 (1.9 g) were rechromatographed (flash, 180 g, B = 5 cm) with petroleum ether/EtOAc 7:3 (900 ml, 30 · 30 ml fractions) to yield, by crystallization from hexane/EtOAc 9:1, 0.55 g of MF5 (5a/5b/5: 5/1.5/1). 3.2.19. 8,9-Dihydro-5-hydroxy-8-(2-hydroxypropan-2-yl)6-isobutyryl-4-phenylfuro[2,3-h]chromen-2-one (mammea a/AD cicloF) (5) and 8,9-dihydro-5-hydroxy-8-(2-hydroxypropan-2-yl)-6-(3-methylbutanoyl)-4-phenylfuro[2,3-h]chromen-2-one (mammea A/AA cicloF) (5b) NMR data of these compounds were identical to the literature (Crombie et al., 1972, 1987). 3.2.20. 8,9-Dihydro-5-hydroxy-8-(2-hydroxypropan-2-yl)6-(2-methylbutanoyl)-4-phenylfuro[2,3-h]chromen-2-one (mammea A/AB cycloF) (5a) Rf = 0.12 (petroleum ether/EtOAc 8.5:1.5, FeCl3: pink-purple, vanillin: blue); 1H NMR (400 MHz, CDCl3) d 14.59 (1H, s, 5-OH), 7.38–7.28 (5H, m, Ar), 5.92 (1H, s, H-3), 4.91 (1H, t, J = 8.9 Hz, H-2000 ), 3.61 (1H, sext, J = 6.7 Hz, H-200 ), 3.31 (2H, d, J = 8.9 Hz, H-1000 ), 1.80 (1H, m, H-400 a), 1.71 (1H, brs, 3000 -OH), 1.42 and 1.31 (3H and 3H, two s, H-4000 and H-5000 ), 1.41 (1H, m, H-400 b), 1.14 (3H, d, J = 6.6 Hz, H-300 ), 0.91 (3H, t, J = 7.4 Hz, H-500 ); 13C NMR (100 MHz, CDCl3) d 209.4 (s, C-100 ), 164.8 (s, C-7), 163.7 (s, C-5), 159.6 (s, C-2), 156.5 (s, C-4), 155.2 (s, C-8a), 139.1 (s,

C-1 0 ), 128.1 (d, C-4 0 ), 127.1 (d, C-3 0 and C-5 0 ), 127.5 (d, C-2 0 and C-6 0 ), 112.0 (d, C-3), 105.1 (s, C-8), 103.0 (s, C-6), 102.5 (s, C-4a), 92.6 (d, C-2000 ), 71.5 (s, C-3000 ), 45.6 (d, C-200 ), 26.7 (t, C-1000 ), 26.2 (t, C-400 ), 26.0 (q, C4000 ), 24.7 (q, C-5000 ), 16.4 (q, C-300 ), 11.6 (q, C-500 ); EIMS, m/z (rel. int.): 422 [M]+ (45), 408 (19), 365 (97), 347 (40), 307 (23), 293 (100). 3.3. Acylation of MF1 To a solution of 101 mg of MF1 (80% of compound 1a, 0.199 mmol) in 5 ml of dry CH3CN, p-Br-benzoylchloride (60 mg), and p-dimethylaminopyridine (0.82 mmol, 3.3 mol/eq) were added. After stirring 4 h, the reaction was worked up by dilution with 0.1 N HCl until pH 6 and extracted with Et2O (2 · 10 ml). The organic phase was washed with 0.1 N HCl, dried over Na2SO4 and evaporated to dryness. The residue (200 mg) was purified by flash column chromathography over Si gel (B = 1.5 cm) using hexane/EtOAc 9:1 (150 ml, 30 · 5 ml fractions) as eluent. Fractions 22–27 by crystallization from MeOH/H2O 9:1, afforded 95.2 mg of 1c (62%). 3.3.1. 5,7-Di(4-bromobenzoyloxy)-6-(2-methylbutanoyl)8-(3-methylbut-2-enyl)-4-phenyl-2H-chromen-2-one (1c) Yellow crystals; m.p. 136–137 C; 1H NMR (300 MHz, CDCl3) d 8.01 (2H, d, J = 10.1 Hz, Br–Ar), 7.69 (2H, d, J = 10.1 Hz, Br–Ar), 7.39 (4H, brs, Br–Ar), 7.25 (2H, d, J = 7.1 Hz, H-2 0 and H-6 0 ), 7.07 (2H, brt, J = 7.1 Hz, H-3 0 and H-5 0 ), 6.89 (1H, brd, J = 7.4 Hz, H-4 0 ), 6.25 (1H, s, H-3), 5.19 (1H, brt, J = 6.4 Hz, H2000 ), 3.68 (1H, sext, J = 6.9 Hz, H-200 ), 3.54 (2H, d, J = 6.5 Hz, H-1000 ), 1.64 (6H, s, H-4000 and H-5000 ), 1.57 (1H, m, H-400 a), 1.22 (1H, m, H-400 b), 0.91 (3H, d, J = 6.9 Hz, H-300 ), 0.63 (3H, t, J = 7.2 Hz, H-500 ); CIMS, m/z (rel. int.): 773 [M+H]+ (32), 589 (30), 573 (23), 407 (50), 391 (94), 337 (65), 201 (66), 185 (100). 3.4. Oxidation of MF1, coumarins 2, 3, 4, and mesuol (1) with PdCl2 Reaction of MF1 is described as an example. To a solution of 101 mg of MF1 (80% of compound 1a, 0.2 mmol) in DMSO/H2O 9:1 (15 ml), NaHCO3 (41 mg, 2 mol/eq) and PdCl2 (48 mg, 0.27 mmoli) were added. The reaction was stirred at room temperature for 8 h, then diluted with 0.1 N HCl until pH 6 and extracted with CH2Cl2 (7 · 5 ml). The organic phase was washed with water, dried with Na2SO4 and evaporated. The residue was purified by silica gel flash column chromatography (B = 1.5 cm, h = 15 cm) using petroleum ether/ AcOEt 9:1 as eluent (200 ml, 40 fractions) to afford, by crystallization from hexane, 68.8 mg of 8a (71%, fractions 30–40).

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The same reaction applied to compound 3 afforded 3b (22%) as major product, together with two more polar by-products 3c (17 mg, 16%) and 3d (10 mg, 9.5%). Compounds 4b, 8 and 9a were obtained, in a similar way, from 4 (11%), mesuol (1) (69%), and 2 (33%), respectively. 3.5. Oxidation of MF1 and coumarin 3 with DDQ To a solution of MF1 (25 mg, 0.05 mmol) in MeOH (2 ml), an excess of DDQ (43 mg, 0.19 mmol, 4 molar equivalents) was added. After stirring 6 h at room temperature, the reaction was worked up by dilution with water and extraction with CH2Cl2 (3 · 3 ml). The organic phase was washed with H2O, dried with Na2SO4 and concentrated. The residue was purified by silica gel (flash, B = 0.8 cm, h = 15 cm) using petroleum ether/ EtOAc 9:1 as eluent (100 ml, 20 fractions) to afford 5 mg of 8a (22%, fractions 9–15). The same reaction applied to compound 3 gave coumarin 3b (20%).

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3.5.3. 3,4-Dihydro-5-hydroxy-2,2-dimethyl–6-(2-methylbutanoyl)-4-oxo-10-phenyl-2H-pyrano[2,3-f]chromen-8one (3d) Colorless oil; 1H NMR (200 MHz, CDCl3) d 12.70 (1H, s, 7-OH), 7.44–7.22 (5H, m, Ar), 6.01 (1H, s, H3), 3.12 (1H, ddq, J = 6.6, 6.8 Hz, H-2000 ), 2.62 (2H, s, H-200 ), 1.92 (1H, m, H-4000 a), 1.53 (1H, m, H-4000 b), 1.26 (3H, d, J = 6.9 Hz, H-3000 ), 1.02 (6H, s, H-400 and H500 ), 1.02 (3H, t, J = 7.0 Hz,H-5000 ); 13C NMR (50 MHz, CDCl3) d 204.5 (s, C-1000 ), 197.4 (s, C-100 ), 162.3 (s, C-7), 159.6 (s, C-4), 158.6 (s, C-2), 155.3 (s, C-5 and C-8a), 139.7 (s, C-1 0 ), 128.5 (d, C-4 0 ), 128.2 (d, C3 0 and C-5 0 ), 127.2 (d, C-2 0 and C-6 0 ), 113.6 (d, C-3), 112.0 (s, C-6), 104.0 (s, C-8), 102.4 (s, C-4a), 81.3 (s, C-300 ), 49.4 (d, C-2000 ), 47.7 (t, C-200 ), 26.1 (q, C-400 and C-500 ), 25.7 (t, C-4000 ), 15.3 (q, C-3000 ), 12.0 (q, C-5000 ); CI-MS, m/z (rel. int.): [M+H]+ (100), 375 (20), 363 (45). 3.5.4. 5-Hydroxy-2-methyl-6-(2-methylbutanoyl)-2-(4methylpent-3-enyl)-10-phenyl-2H-pyrano[2,3-f]chromen8-one (4b) Colorless crystals; m.p. 95–96 C; 1H NMR (200 MHz, CDCl3) d 14.65 (1H, s, 7-OH), 7.40–7.24 (5H, m, Ar), 6.69 (1H, d, J = 10.3 Hz, H-100 ), 6.01 (1H, s, H-3), 5.35 (1H, d, J = 10.3 Hz, H-200 ), 4.89 (1H, brt, H-700 ), 3.97 (1H, sext, J = 6.5 Hz, H-2000 ), 1.93 (1H, m, H-4000 a), 1.65 (6H, brs, H-900 and H-1000 ), 1.59–1.44 (4H, m, H-500 and H-600 ), 1.47 (1H, m, H-4000 b), 1.30 (3H, d, J = 6.8 Hz, H-3000 ), 1.03 (3H, t, J = 7.4 Hz, H5000 ), 0.98 (3H, s, H-400 ); CI-MS, m/z (rel. int.): 473 [M+H]+ (100), 405 (12), 389 (46).

3.5.1. 5-Hydroxy-2,2-dimethyl-6-(2-methylbutanoyl)-10phenyl-2H-pyrano[2,3-f]chromen-8-one (3b)  Yellow crystals; m.p. 125–126 C; ½a
25 D ¼ 1:2 (CHCl3; c = 0.01); 1H NMR (300 MHz, CDCl3) d 14.59 (1H, s, 7-OH), 7.38–7.21 (5H, m, Ar), 6.62 (1H, d, J = 9.8 Hz, H-100 ), 5.99 (1H, s, H-3), 5.37 (1H, d, J = 9.8 Hz, H-200 ), 3.95 (1H, ddq, J = 6.4, 6.7 Hz, H2000 ), 1.95 (1H, ddq, J = 13.6, 7.4, 6.2 Hz, H-4000 a), 1.50 (1H, ddq, J = 13.6, 7.4, 6.2 Hz, H-4000 b), 1.28 (3H, d, J = 6.7 Hz, H-3000 ), 1.01 (3H, t, J = 7.4 Hz, H-5000 ), 0.95 (3H, s, H-400 ), 0.94 (3H, s, H-500 ); 13C NMR (75 MHz, CDCl3) d 210.4 (s, C-1000 ), 163.8 (s, C-7), 158.8 (s, C-2), 156.7 (s, C-4), 156.0 (s, C-5 and C-8a), 140.0 (s, C-1 0 ), 127.7 (d, C-4 0 ), 127.5 (d, C-3 0 and C-5 0 ), 127.0 (d, C-2 0 and C-6 0 ), 126.7 (d, C-200 ), 115.2 (d, C-100 ), 111.8 (d, C3), 105.8 (s, C-6), 103.6 (s, C-8), 102.1 (s, C-4a), 79.0 (s, C-300 ), 46.8 (d, C-2000 ), 27.3 (q, C-400 and C-500 ), 27.1 (t, C-4000 ), 16.5 (q, C-3000 ), 11.7 (q, C-5000 ); CI-MS, m/z (rel. int.): 405 [M+H]+ (100), 389 (34), 347 (20).

3.6.1. Bacterial strains The bacterial strains employed were recent clinical isolates from different hospital laboratories and were maintained in the culture collection of the Microbiology Institute of the University of Milano. Bacteria were stored frozen at 80 C in brain heart infusion (BHI) broth supplemented with 20% (v/v) glycerol (Sigma Chemical Co.).

3.5.2. 3,4-Dihydro-4,5-dihydroxy-2,2-dimethyl-6-(2-methylbutanoyl)-10-phenyl-2H-pyrano[2,3-f]chromen-8-one (3c) 25 Yellow crystals; m.p. 138–139 C; ½a
D ¼ 0; 1H NMR (300 MHz, CDCl3) d 15.39 (1H, s, 7-OH), 7.21–7.17 (5H, m, Ar), 6.02 (1H, s, H-3), 4.97 (1H, td, J = 6.5, 1.7 Hz, H-100 ), 3.95 (1H, ddq, J = 6.4, 6.7 Hz, H-2000 ), 3.43 (1H, s, 100 -OH), 1.93 (1H, ddq, J = 13.6, 7.4, 6.2 Hz, H-4000 a), 1.91 (2H, d, J = 6.5 Hz, H-200 ), 1.50 (1H, ddq, J = 13.6, 7.4, 6.2 Hz, H-4000 b), 1.29 (3H, d, J = 6.7 Hz, H-3000 ), 1.01 (3H, t, J = 7.4 Hz, H-5000 ), 0.81 (6H, s, H-400 and H-5000 );CI-MS, m/z (rel. int.): 423 [M+H]+ (15), 405 (100), 389 (37), 363 (17), 347 (14).

3.6.2. Susceptibility assay The minimal inhibitory concentration (MIC) was determined by an agar dilution method in Mueller–Hinton (M–H) agar according with the protocol of the National Committee for Clinical Laboratory Standards NCCLS, 2000. Stock solutions of the compounds were prepared by dissolving them in sterile 0.1 M potassium phosphate buffer (PB), pH 7.4 or in dimethyl sulfoxide (DMSO) (Sigma Chemical Co.) at 2560 lg/ml. Serial doubling dilutions of the stock solution were prepared in PB to obtain concentrations twenty times higher than those to be tested. One ml of these were mixed with 19 ml of melted M–H agar in a dish plate.

3.6. Biological evaluation

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The inocula were prepared by a direct saline suspension of isolated colonies from a 24 h M–H agar plate. The suspension was adjusted to obtain a turbidity comparable to that of the 0.5 McFarland standard and further on diluted 1:10 to obtain a bacterial concentration of approximately 107 CFU/ml. Few microliters of such dilution were deposited on the agar surface of the plates by a multipoint inoculating device (Denley-Tech Ltd., Billingshurst, UK) producing an inoculum of approximately 104–105 CFU per spot. The plates were incubated in air at 35 C for 24 h and the MIC was defined as the minimum concentration at which no visible grown was observed. 3.6.3. Parasite cultures RPMI 1640 medium was purchased from GibcoBRL, human A-positive red blood cells and plasma were kindly provided by the Blood Bank of the National Cancer Institute, Milano, Italy. Chloroquine diphosphate (CQ) (C-6628), and DMSO were all obtained from Sigma–Aldrich, Milan, Italy. P. falciparum cultures were carried out according to the method described by Trager and Jensen (1976). Briefly, the CQsensitive (CQ-S), moderately mefloquine-resistant clone D10 and the CQ-resistant (CQ-R), mefloquine-susceptible clone W2 were maintained at 5% haematocrit (human type A-positive red blood cells) in complete culture medium at 37 C. Complete medium contained RPMI 1640 medium (Gibco-BRL, NaHCO3 24 mM) with the addition of 10% heat-inactivated A-positive human plasma, 20 mM Hepes (Biological Industries, Kibbutz, Israel), 2 mM Glutammine (Biological Industries). All the cultures were maintained in a standard gas mixture consisting of 1% O2, 5% CO2, 94% N2. When parasitemia exceeded 5%, subcultures were taken; the culture medium was changed every second day. 3.6.4. Parasite growth and drug susceptibility assay Compounds were dissolved in either water (chloroquine), or DMSO (Mesua derivatives) and then diluted with medium to achieve the required concentrations (in all cases the final concentration contained <1% DMSO, which was found to be non-toxic to the parasite). Drugs were placed in 96-wells flat-bottom microplates (Costar #3596) and ten twofold dilutions were made starting from 20 lg/ml for all the compounds tested. Asynchronous cultures with parasitemia of 1– 1.5% and 1% final haematocrit were aliquoted into the plates and incubated for 72 h at 37 C. Parasite growth was determined spectrophotometrically by measuring the activity of the parasite lactate dehydrogenase (pLDH), in control and drug-treated cultures according to the method originally described by Makler and Hinrichs (1993). The pLDH activity is distinguishable from host LDH using the 3-acetyl pyridine adenine dinucle-

otide (APAD) as co-factor. Briefly, at the end of the incubation, the cultures are carefully resuspended, aliquots of 20 ll are removed and added to 0.1 ml of the Malstat reagent in a 96-well microtiter plate. The Malstat reagent is made with 0.125% Triton X-100, 130 mM L -lactic acid, 30 mM Tris buffer and 0.62 lM APAD. The spectrophotometric assessment of pLDH activity is facilitated by adding 25 ll of a solution of 1.9 lM nitro blue tetrazolium (NBT) and 0.24 lM phenazine ethosulfate (PES) to the Malstat reagent. As APADH is formed, the NBT is reduced and forms a blue formazan product that can be measured at 650 nm. The antimalarial activity of test compound was expressed as the 50% inhibitory concentration (IC50); each IC50 value is the mean and standard deviation of at least three separate experiments performed in triplicate. Statistical analysis was performed using the paired t test with Statview software.

Acknowledgement Work was in part supported by MIUR (funds PRIN) of Italy.

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