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Phytochemis

jo u rn al h om ep ag e: ww w.el1. Introduction

Mushrooms have proved to be a rich source of secondarymetabolites with unusual structures as well as interestingbiological activities (Hanson, 2008; Zaidman et al., 2005). Despitetheir potential for drug development, few bioactive metaboliteshave been reported from mushrooms as compared with higherplants and microbes.

In our screening project on bioactive metabolites of Basidio-mycetes we have investigated the metabolites produced by thefungus Clavicorona divaricata in MPG agar cultures and isolateddivaricatine A (1a) and B (1b) together with the nor-sesquiterpe-noids tsugicoline L and M (Arnone et al., 2003); successively, a stillliquid culture on MPGB medium gave rise to a complex mixture ofwhich tsugicoline A (2) was the main component although in pooryield. More interesting was the fermentation of the fungus indifferent conditions, 1 week still and 2 weeks shaken at 180 rpm. Inthis paper we describe the isolation and structure elucidation ofthe new aza-sesquiterpenoids divaricatine C (3a) and D (4a)obtained from shaking cultures in MPGB medium.

2. Results and discussion

The strain of C. divaricata (ATCC 22500) was grown on MPGBmedium in asks (see Section 3) and the metabolites wereextracted with EtOAc; four main metabolites, divaricatine C and D(3a and 4a), lentinellone (5), and tsugicoline A (2) were isolated bysilica gel chromatography (Fig. 1).

Divaricatine C (3a) was obtained as a cream powder, mp 112115 8C; a20D 17:6 (c 0.07, MeOH) and analyzed for C15H21NO3[M+, 263]. Treatment of 3a with pyridine/Ac2O gave the triacetate3b (see S.I.), indicating that the three oxygen atoms contained in 3awere part of hydroxy functions.

The 13C NMR spectrum of 3a (Table 1) (a non-systematic, butbiogenetically justied, numbering scheme deriving from theprotoilludane 2 is shown in the formula and it is used in theassignment of NMR spectral data; see Arnone et al., 2003)showed the presence of 15 signals attributable to 10 sp3- and 5sp2-hybridized carbon atoms. The signals attributable to sp3-hybridized carbon atoms were assigned to three methyl (C-8, -14and -15), two methylene (C-10 and -12), three methines (C-3, -9and -13, one of them hydroxy bearing), and two quaternary carbonatoms, one hydroxy bearing (C-7), while the signals attributable tosp2-hybridized carbon atoms were assigned to two methines (C-1and -4) and three quaternary (C-2, -5 and -6) carbon atoms. Theone-bond 1H13C coupling constants of 183 and 179 Hz observedfor C-1 and -4 (Stothers, 1973), together with the HMBCcorrelations presented by H-1 with C-4 and -6 and by H-4 withC-1, -5 and -6 (Table 1), indicated that the ve sp2 carbons were

Aza-sesquiterpenes

Protoilludane

Part 68 in the series Secondary mould metabolites for part 67, see Arnone et

al. (2009).

* Corresponding author. Tel.: +39 2 50316816; fax: +39 2 50316801.

E-mail address: loana.musso@unimi.it (L. Musso).

1874-3900/$ see front matter 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.doi:10.1016/j.phytol.2011.12.013Isolation and structure elucidation of aza-formed by shaken cultures of the fungu

Gianluca Nasini a, Alberto Arnone a, Adriana Bava a, a C.N.R., Istituto di Chimica del Riconoscimento Molecolare, Sezione Adolfo Quilico; Dip

via Mancinelli 7, 20131 Milano, ItalybDISMA, Dipartimento di Scienze Molecolari Agroalimentari, Universita` di Milano, via Ce

A R T I C L E I N F O

Article history:

Received 12 October 2011

Received in revised form 20 December 2011

Accepted 22 December 2011

Available online 5 January 2012

Keywords:

Clavicorona divaricata

Basidiomycetes

A B S T R A C T

Two novel sesquiterpenes o

from MPGB shaken cultures

elucidated by means of NM

against bacteria and inhib

mechanism of their format

2011 Phytoesquiterpenoids of protoilludane originClavicorona divaricata

oana Musso b,*

timento di Chimica, Materiali ed Ingegneria Chimica Giulio Natta del Politecnico,

ria 2, 20133 Milano, Italy

rotoilludane origin, the alkaloids divaricatine C and D, have been isolated

f the fungus Clavicorona divaricata (Basidiomycetes). Their structures were

studies and chemical correlations. The metabolites were weakly active

ed the germination of the water cress Lepidium sativum. A possible

n from the protoilludane tsugicoline A (2) is suggested.hemical Society of Europe. Published by Elsevier B.V. All rights reserved.

try Letters

s evier .c o m/lo c ate /p hyt ol

Fig. 1. Clavicorona divaricata metabolites (1a, 1b, 2, 3a, 4a and 5) and acetylated derivatives (3b and 4b).

G. Nasini et al. / Phytochemistry Letters 5 (2012) 224227 225part of the pyridine ring A in which C-1 and -4 were adjacent to thenitrogen atom. The upeld shifts exhibited by C-1 and C-4 withrespect to the corresponding carbons in pyridine indicated that theremaining hydroxy group must be linked to C-5.

The1H NMR analysis of 3a and the HMBC correlations showed thepresence of a cyclopentane ring substituted at position 11 with twomethyl groups, as demonstrated by their HMBC correlations with C-10, -11 and -12 (Table 1). Moreover the HMBC correlations observedbetween H-3 and C-1 and -12 and between H3-8 and C-6, -7 and -9allowed us to link C-2 to C-3 and C-7 to C-6 and C-9 to form the ring B,fused to the above described cyclopentane and the pyridine rings.

The relative conguration of the four stereogenic centres in 3awas determined by NOE difference spectra (Section 3). Specically,the fact that the 15-methyl group, assumed as b-oriented,Table 11H NMR (400 MHz) and 13C (100 MHz) spectroscopic data for 3a and 4a.

Position 3aa HMB

dC dH ppm (J, Hz)

1 137.5 d 8.25 dd (0.8,

G. Nasini et al. / Phytochemistry Letters 5 (2012) 2242272264a with those of 3a (Table 1) readily revealed that the twometabolites shared the same basic structure, the only relevantdifference being the presence in 4a of a vinyl group instead of atertiary alcohol on C-7. As expected, acetylation of 4a afforded thediacetate 4b (see S.I.).

The NMR and MS spectroscopic data of compound 5 were inagreement with those reported in literature for lentinellone(Wunder et al., 1996), a metabolite produced by Lentinelluscochleatus.

A putative biogenetic pathway of formation of divaricatine Cand D from tsugicoline A (2) (Arnone et al., 1995), isolated in thesame fermentation, can be proposed. The presence of a conjugatedcarbonyl function in the four-membered ring of tsugicoline A, isthe key to the reactivity of this interesting metabolite, which givesan easy opening of the 67 bond, with the formation of a largenumber of new nor- and sesquiterpenes (Arnone et al., 1997, 1998,2000).

Oxidation, amination and ring closure were assumably involvedin the formation of the pyridine moiety giving 3a, then convertedto 4a by water elimination.

The origin of the nitrogen source may be derived from a D-amino acid oxidase (DAAO), a FAD-dependent enzyme, thatcatalyzes the oxidative deamination of D-amino acids, to give a-keto acids and ammonia; this enzyme probably was formed in theaerobic fermentation of the fungus and the maltpeptone mediumis a rich source of amino acids (Pollegioni et al., 2007). The skeletonof divaricatine C and D has never been found among thesesquiterpenes of protoilludane origin, except for illudinine (Nairet al., 1969), a metabolite isolated from some strains of Clitocybeilludens.

Metabolites 3a, 4a, and 5 showed a weak antibacterial activityagainst Bacillus cereus and Sarcinea lutea (50 mg/disc), andinhibited the growth of Lepidum sativum. (After 48 h, the inhibitionof the root elongation was 85, 91 and 72%, respectively. For

Fig. 2. Selected NOE for compound 3a.experimental details see Arnone et al., 1990.)Compounds 3a and 4a were tested for their cytotoxicity against

the non-small cell lung tumor cell line H460, and showed noactivity [IC50 (mM) for 3a and 4a >100].

3. Experimental

3.1. General experimental procedures

Melting points were determined on a Koer apparatus andare uncorrected; the IR spectra were measured with a Perkin-Elmer 177 spectrophotometer; EI MS spectra with a BrukerEsquire 3000 Plus instrument, HRMS with a Bruker APEX-QZTICR. Optical rotations were measured on a JASCO-500 DIP-18polarimeter. NMR spectra were recorded on a Bruker ARX 400 orDMX 500 instrument at 305 K. The carbon signals were assignedfrom the heteronuclear correlation experiments via one-bond(HSQC) and long-range (HMBC) coupling constants. The nuclearOverhauser effects were determined by monodimensional NOEdifference spectra. Flash column chromatography was per-formed with Merck silica gel (0.040.063 mm), and TLC and PLCwith Merck HF254 silica gel.

3.2. Culture of C. divaricata, isolation and purication of metabolites

3a, 4a, and 5

A strain of C. divaricata (ATCC 22500), received from AmericanType Culture Collection, Rockville, was maintained on MPGA(malt, peptone, glucose, agar, 20:4:20:15 g L1) slants and amycelium suspension was inoculated into 40 Erlenmeyer asks(300 mL), each containing 100 mL of the sterile medium MPGB.Still cultures were incubated at 24 8C for a week and successivelyfor 2 weeks on a rotatory shaker at 180 rpm. After 3 weeks thecultures were extracted twice with EtOAc containing 1% MeOHand the organic phase was dried (Na2SO4) and evaporated to give0.6 g of the fraction containing a mixture of metabolites 2, 3a, 4aand 5. The extract was puried by ash column chromatographyon silica gel eluting with CH2Cl2MeOH at increasing polarity.Collected fractions were further puried by means of prep. TLCwith CH2Cl2MeOH 9:1 to give the pure metabolites in order ofdecreasing Rf value: tsugicoline A 2a (20 mg), compound 5(15 mg), divaricatine D (4a) (30 mg) and divaricatine C (3a)(35 mg).

3.2.1. Divaricatine C (3a)Cream powder, mp 112115 8C; a20D 17:6 (c 0.07, MeOH); UV

lmax (e) 203 (5070), 247sh (1370), 286 (1645) nm; IR (KBr) nmax3390 (br), 1655, 1561, 1438 cm1; EIMS, m/z 264 [MH]+(44%), 246[MH18]+(85), 245 [M18]+ (100), 230 (24), 212 (72); HREIMS:263.1529, calcd for C15H21NO3 263.1521; anal. C 68.35, H 8.00, N5.34%, calcd for C15H21NO3 C 68.41, H 8.03, N 5.32%;

1H and 13CNMR data are listed in Table 1. NOEs (acetoned6): {H-3} enhancedH3-8 (1.5%), H-12a (3.5%), {H-9} enhanced H-10b (3.5%), H-13(4%), H3-15 (1%), {H-13} enhanced H-9 (4.5%), H-12b (2.5%), H3-15(1%), {H3-14} enhanced H-10a (6%), H-10b (1%), H-12a (6%), H-12b (1%), {H3-15} enhanced H-9 (4.5%), H-10b (2.5%), H-12b(2.5%) and H-13 (5%).

3.2.2. Divaricatine D (4a)White powder, mp 110112 8C; a20D 105 (c 0.02, MeOH); UV:

lmax (e) 208 (6296), 249 (2110) 291 (1823) nm; IR (KBr) nmax 1675,1560 and 1458 cm1; EIMS m/z 246 [MH]+(100%), 245 [M]+(44),212 (24); HREIMS: 245.1419 (calcd for C15H19NO2 245.1415);

1Hand 13C NMR data are listed in Table 1.

Acknowledgements

We thank Dr. G. Pratesi (Istituto Nazionale dei Tumori, Milano)for the cytotoxicity tests and Professor L. Merlini for helpfuldiscussions.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.phytol.2011.12.013.

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G. Nasini et al. / Phytochemistry Letters 5 (2012) 224227 227

Isolation and structure elucidation of aza-sesquiterpenoids of protoilludane origin formed by shaken cultures of the fungus Clavicorona divaricataIntroductionResults and discussionExperimentalGeneral experimental proceduresCulture of C. divaricata, isolation and purification of metabolites 3a, 4a, and 5Divaricatine C (3a)Divaricatine D (4a)

AcknowledgementsSupplementary dataReferences