Antimicrobial Phenolics and Unusual Glycerides from Helichrysum

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Antimicrobial Phenolics and Unusual Glycerides from Helichrysum italicum subsp. microphyllum Orazio Taglialatela-Scafati,*,† Federica Pollastro,‡ Giuseppina Chianese,† Alberto Minassi,‡ Simon Gibbons,§ Warunya Arunotayanun,§ Blessing Mabebie,§ Mauro Ballero,⊥ and Giovanni Appendino*,‡ †

Dipartimento di Chimica delle Sostanze Naturali, Università di Napoli Federico II, Via Montesano 49, 80131 Napoli, Italy Dipartimento di Scienze Farmaceutiche, Università del Piemonte Orientale, Via Bovio 6, 28100, Novara, Italy § Department of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, U.K. ⊥ Dipartimento di Scienze Botaniche and Consorzio per lo Studio dei Metaboliti Secondari (COSMESE), Università di Cagliari, Viale S. Ignazio 13, 09123 Cagliari, Italy ‡

S Supporting Information *

ABSTRACT: During a large-scale isolation campaign for the heterodimeric phloroglucinyl pyrone arzanol (1a) from Helichrysum italicum subsp. microphyllum, several new phenolics as well as an unusual class of lipids named santinols (5a−c, 6−8) have been characterized. Santinols are angeloylated glycerides characterized by the presence of branched acyl- or keto-acyl chains and represent a hitherto unreported class of plant lipids. The antibacterial activity of arzanol and of a selection of Helichrysum phenolics that includes coumarates, benzofurans, pyrones, and heterodimeric phloroglucinols was evaluated, showing that only the heterodimers showed potent antibacterial action against multidrug-resistant Staphylococcus aureus isolates. These observations validate the topical use of Helichrysum extracts to prevent wound infections, a practice firmly established in the traditional medicine of the Mediterranean area.

variants of Staphylococcus aureus (MRSA), an important agent of nosocomial infections and a growing medical emergency.5

Helichrysum italicum L. (family Asteraceae), a plant endemic to the Mediterranean area, is known to contain a poorly characterized cortisone-like principle named helichrysine.1 The structure of helichrysine has long been elusive, but the identification of the heterodimeric phloroglucinyl pyrone arzanol (1a), a major constituent of H. italicum,2 as a potent dual inhibitor of pro-inflammatory transcription factors (NFκB)2 and inflammatory enzymes [(mPGES)-1,5-LO]3 suggests that helichrysine might consist of arzanol and/or related compounds. As a prelude to preclinical in vivo studies, a largescale isolation of arzanol from a Sardinian variety of H. italicum [H. italicum subsp. microphyllum (Willd.) Nyman] especially rich in this compound2 was carried out. In the course of this study, we have characterized several minor constituents of the plant, including an unprecedented class of lipids and several phenolics. The availability of these compounds provided the opportunity to study in a systematic way a second important property of helichrysine, namely, its antibacterial activity.1,4 Therefore, this report presents an account of the structure elucidation of the minor constituents of this plant and a study of the antibacterial action of the major phenolics from H. italicum against multidrug-resistant and methicillin-resistant © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Apart from arzanol (1a, ca. 0.32% on dried plant basis), the aerial parts of H. italicum subsp. microphyllum contain large amounts (ca. 0.40%) of ursolic acid (2) and phenolics belonging to three structural types: pyrones (both homo- and heterodimeric), benzofurans (bitalin esters), and prenylcoumarates. Ursolic acid could be obtained in high purity from crude extracts by the Passerini method, namely, a brief (10 min) reflux in acetone, cooling to obtain a crude triterpenoid mixture, and then recrystallization from methanol.6 The presence of large amounts of ursolic acid, a poorly soluble compound, complicated the fractionation of the extract, but it was found that, as an alternative to the Passerini method, this compound could also be largely removed by liquid/liquid Special Issue: Special Issue in Honor of Lester A. Mitscher Received: October 12, 2012

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dx.doi.org/10.1021/np3007149 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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partition of the extract between petroleum ether and aqueous methanol. Ursolic acid has a low solubility in both phases and remained undissolved during the partition of the crude extract between these two solvents. The two “de-triterpenated” phases were then independently fractionated by gravity column chromatography on silica gel to afford six major fractions (A−F).

Santinol A1 (5a) (C20H36O5, HR-ESIMS) was obtained as an optically active 1,3-diacylglycerol. The 1H NMR spectrum of 5a (CDCl3) showed a partially overlapped cluster of five resonances between δ 4.14 and 4.28, which, with the help of the COSY and HSQC spectra, could be deconvoluted and assigned to a 1,3-diacylated glycerol. The presence of an angelate group was evidenced by the appearance of typical 1H (δH 1.90, 3H, brs; 6.12, 3H, q; 1.99, 3H, d) and 13C NMR resonances, and the Z configuration of the stereogenic double bond was confirmed by the detection of a NOE correlation between the olefinic signal and the allylic methyl (H3-5′). Molecular formula considerations and the presence of a terminal branching identified the second acyl moiety as isolauric acid, an unusual C-12 fatty acid. The 1,3-acylation pattern was assessed by chemical shift considerations (the downfield resonance of the diastereotopic oxymethylene protons) and confirmed by the detection of HMBC crosspeaks between the methylene protons and the ester carbonyls (angelate carbonyl at δ 168.3; isolaurate carbonyl at δ 174.4). The presence of two markedly different acyl moieties provided a rationale for applying the modified Mosher’s method12 to assess the absolute configuration of 5a. To this aim, 5a was reacted with R- or S-MTPA chloride [MTPA = methoxy(trifluoromethyl)phenylacetic acid], obtaining the corresponding S (5d) and R (5e) monoesters. Analysis of Δδ (S − R) values for protons of the glycerol moiety (Figure 1) indicated,

The petroleum ether phase (fraction A) contained neryl acetate, the hallmark compound of the Helichrysum essential oils from the Tyrrhenian area,7 and the O-geranylated isomeric coumarates 3a,b,8 which could be separated by preparative HPLC on silica gel and could also be isolated from the aqueous methanol phase. Alkoxycinnamates have attracted considerable attention because of their action against drug-resistant Mycobacterium,9 but the concentration of these compounds in the extract (overall, ca. 35 ppm in the dried plant material) was too low to be associated with a specific activity. The remaining fractions were obtained by gravity column chromatography purification of the aqueous methanol phase. Fraction B was crystallized from ether to afford the dimeric pyrone helipyrone (4).10 The mother liquors afforded a series of angeloylated lipids (5a−c, 6−8) that have been named santinols in honor of Leonardo Santini, the pioneer in the study of the medicinal use of Helichrysum.1,11 By gravity column chromatography, a major glyceride was obtained apparently pure in terms of its 1H NMR spectrum, but ESIMS revealed a mixture of homologues, eventually resolved by RP-HPLC to afford pure santinols A1−A3 (5a−c).

Figure 1. Application of the modified Mosher’s method for the absolute configuration at C-2 of santinol A1 (5a). B



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according to Mosher’s model,12 a 2S configuration for the natural product. Santinols A2 (5b) and A3 (5c) differed from 4a only for the length of the saturated acyl moiety, an isomyristate and an isopalmitate, respectively. Since 5b and 5c share with 5a a positive optical rotation, it seemed reasonable to assume that they share the same S configuration at the stereogenic center. Santinol B (6, C13H20O5) was optically inactive, and its NMR spectra showed a 1,3-diangeloylated glycerol. Thus, the 1H NMR spectrum of 6 showed, apart from the signals of the glycerol protons, only the resonances of the angelate protons, while the 13C NMR spectrum showed only seven signals (two for the glycerol unit and five for the two angeloyl residues). Santinol C (7, C26H44O7) was found to be a triacyl glycerol. Analysis of its 1H and 13C NMR data identified, apart from the resonances of the glycerol backbone and of the angeloyl and lauryl moieties, also those of a 2-methyl-3-oxovaleroyl group. Thus, the COSY spectrum disclosed the presence, within this acyl moiety, of a methyl-bearing methine, in addition to the expected ω-methyl-bearing methylene, located between two carbonyl groups by analysis of the HMBC cross-peaks [correlation between the methyl doublet at δ 1.34 (H-3−6‴) and the ester carbonyl at δ 170.3 and with a ketone carbonyl resonating at δ 206.0]. The correlation of the methyl triplet H3-5‴ with C-3‴ indicated that an ethyl group bound to the ketone carbonyl completed the structure of this residue. 2Methyl-3-oxovaleric acid is an important marker for the diagnosis of propionic acidemia, a genetic disease caused by a defect in the biotin-dependent carboxylation of propionyl CoA,13 but has not been detected before in natural glycerides. The HMBC spectrum also defined the attachment of the acyl units, since the glyceryl methine (H-2, δ 5.35) showed a crosspeak with the ester carbonyl at δ 173.0 of the lauryl group. Santinol D (8) (C29H50O7) was found to be closely related to santinol C, differing only for the homologation of the α-methylbranched acyl moiety to 2,4-dimethyl-3-oxo-enanthate. In particular, the COSY spectrum of this acyl moiety disclosed two different spin systems (H-2‴−H3-8‴ and a C5 residue spanning from H3-9‴ to H3-7‴), for which the proton resonances were associated with those of the corresponding carbons by a HSQC experiment. Analysis of the HMBC crosspeak (H3-8‴ with both the ester carbonyl at δ 170.3 and a ketone carbonyl at δ 209.3 along with the cross-peak of H3-9‴ with the same ketone carbonyl) made it possible to combine the two spin systems into a final β-dicarbonyl acyl moiety, while the HMBC cross-peaks of H2-3 with C-1″ secured the attachment of this unit at O-3. Given the structural resemblances of santinols C and D with santinol A, especially in relation to the presence and location of the angeloyl moiety, it seems reasonable to assume that they share the same 2S configuration, while the configuration of the methyl-bearing acyl chain stereocenters of 6 and 7 could not be assigned. The branched acyl moiety of santinol D is unknown as a natural product and is structurally related to the pyrone moiety of arzanol (1a) and helipyrone (4), differing, however, by the location of the methyl groups. While a biogenetic polyketide derivation seems obvious for the fatty acyl moieties of santinols, their raison d’être is unclear. They could either represent aborted attempts to convert short ketide moieties into pyrones, the major constituents of the plant, or, otherwise, have a more precise and yet elusive role, with a general inclusion in cell membranes being ruled out by their low isolation yields (ca. 0.001%). Unusual lipids have been isolated from the

Mediterranean plant Thapsia garganica L.,14 suggesting that the distribution of this compound class deserves systematic studies. Apart from the santinols, fraction C also contained a mixture of medium- and long-chain tremetone derivatives. The oleyl and the nonanoyl esters of bitalin A (9a,b),15 both new compounds, could be obtained pure from this complex mixture. The isolation of an odd-number fatty acid ester was somewhat surprising and was confirmed by comparison with an authentic sample prepared by the esterification of bitalin A with nonanoic acid. Shorter chain esters of this benzofuran derivative were obtained from fractions D and E, along with the monomeric pyrone micropyrone (10).2 While the acetate (9c), the isobutyrate (9e), and the α-methylbutyrate (9f) ester are known,16 the propionate (9d) and the isocaproate (9g) are new. The structure elucidation of the new esters of bitalin A was straightforward. Consequently, the benzofuran core showed the signals (data for 9d as an example) of two ABX systems, one aromatic (δH 7.81, 7.80, and 6.82) and the other aliphatic (δH 5.39, 3.42, and 3.20), and of an oxymethylene (δH 4.70 and 4.63), an sp2-exomethylene (δH 5.37 and 5.39), and one acetyl (δH 2.53), while the acyl moieties were all common structural elements of phytochemicals. All tremetone esters showed a negative sign of optical rotation, suggesting the same configuration at the single stereogenic center of the dihydrobenzofuran core.

Fraction F contained heterodimeric pyrones. Arzanol (1a), the major constituent of the plant, could be crystallized from ether, and chromatographic purification of the mother liquors afforded the known less polar homologues 1b and 1c16 and the novel more polar derivative 11, which we have named heliarzanol. The heterodimer 1c shows a stereogenic center in its acyl moiety. Given the polyketide derivation of acylphloroglucinols, it seems reasonable to assume that 1c is assembled from an isoleucine-derived acyl starter. If so, the configuration of the stereogenic carbon on 1c should be S. C



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Figure 2. (a) Resonance-enhanced hydrogen bonding in ortho-hydroxylated phenones. (b) Possible rotameric and tautomeric processes involved in arzanol-type heterodimers (P = prenyl group; R = alkyl group).

Table 1. MIC (μg/mL) Values of a Selection of Phenolics from Helichrysum italicum subsp. microphyllum (ns > 128 μg/mL) compound

SA1199B

Xu212

ATCC25943

RN4220

EMRSA15

EMRSA16

arzanol (1a) micropyrone (10) helipyrone (4) 3a 3b 9a 9b 9c methylarzanol (12) cycloarzanol A (13) cycloarzanol B (14) 15a 15b 15c 15d 15e 15f norfloxacin

1 ns ns ns ns ns ns ns ns 128 ns 32 4 16 1 128 16 32

4 ns ns ns ns ns ns ns ns 128 ns 128 8 4 16 ns 16 8

1 ns 64 ns ns ns ns ns ns 32 ns 128 4 8 1 64 8 0.5

2 128 12 ns ns ns ns ns ns 32 ns 32 4 2 2 128 8 0.5

4 ns ns ns ns ns ns ns ns 128 ns 32 8 6 2 128 16 0.5

2 128 128 ns ns ns ns ns ns ns ns 128 8 8 2 128 8 128

isopropyl methine at δ 3.11 showed HMBC cross-peaks with a ketone carbonyl (δ 204.1) and with C-1 (δ 106.1), while crosspeaks of the oxymethine at δ 4.34 (H-17) with C-3, C-20, and the sp2 carbons C-18 and C-19 allowed the elucidation of the C-3 side chain and defined the full planar structure of heliarzanol as shown in 11. Some nonexchangeable signals in the 1H and 13C NMR spectra of arzanol and its analogues were rather broad at room temperature and were sharpened by heating to 60 °C, suggesting the presence of slow conformational interconversion between different rotameric and/or tautomeric forms. orthoHydroxylated phenones show resonance-enhanced hydrogen bonding (Figure 2a) that slows rotation around the bond linking the acyl moiety to the aromatic ring.17 As the phenolic hydroxy groups adjacent to the methylene bridge can form intramolecular hydrogen bonding with the oxygen function at C-2 and C-4 of the pyrone moiety, the rotameric equilibrium could, in principle, involve the interconversion of extensively hydrogen-bonded conformations around the bonds that link the phloroglucinyl core to its acyl and pyronylmethyl substituents (Figure 2b, rotamers A and B). Since the 4hydroxypyrone ring can, in turn, exist in two distinct tautomeric

Heliarzanol (11), C24H30O8 by HRESIMS, was identified as an arzanol-type compound on the basis of its NMR data. The 13C NMR spectrum of 11 showed 24 well-resolved signals and the resonances typical of the Helichrysum phloroglucinyl α-pyrones (from C-1 to C-15), with differences, however, on the prenyl moiety. Accordingly, the 1H NMR spectrum of 11 (CDCl3, 500 MHz) showed signals typical of the arzanol heterodimeric motif, including a deshielded methyl singlet (CH3-14, δ 1.95), an ethyl group, and a methylene broad singlet (H2-7, δ 3.70). The resonances of an additional methyl singlet (δ 1.86), a sp2 methylene group, and six multiplets, sorted as an isopropyl and a CH2CH−OH moiety by means of a 2D COSY spectrum, were also evident in the 1H NMR spectrum of heliarzanol (11). Association of all the proton resonances with those of the directly bound carbon atoms was obtained by the HSQC spectrum, and this allowed the correct interpretation of the HMBC spectrum. In addition to allowing the unambiguous location of methyl and ethyl groups on the pyrone moiety and confirming the presence of the methylene bridge (cross-peaks of H2-7 with C-4, C-5, C-6, C-8, C-9, C-15) to connect the two rings, the HMBC spectrum also allowed the identification of substituents at C-1 and C-3. In particular, the deshielded D



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forms, this scenario is further complicated by the involvement of different tautomeric forms of the pyrone moiety and rotation around the other bond of the pivotal methylene linker (cf., B and C in Figure 2b). These conformational issues are quite general for homo- and heterodimeric acylphloroglucinols,18 an important class of natural products, but have not yet been investigated systematically. Extracts of Helichrysum are used in folk medicine for the management of bacterial infection of the skin and the oral cavity, a use backed up by clinical observations1 and by the development in the former Soviet Union of arenarin (a mixture of phloroglucinols from Helichrysum arenarium L.) as a skin and eye antibacterial agent.4,19 It was therefore interesting to evaluate if, and to what extent, the phenolics from Helichrysum have significant activity against multidrug-resistant bacterial strains, a current medicinal urgency in both hospital and community settings especially for Staphylococcus aureus.5 Multidrug- and methicillin-resistant strains of S. aureus can efflux antibiotics from the cell by means of a pump in the cytoplasmic membrane. Some of these pumps, such as NorA and QacA, are able to efflux a range of antibiotics, antiseptics, and structurally unrelated compounds.20 Other pumps are more specific, for example TetK, which effluxes tetracycline. Simple, nonprenylated acylphloroglucinol derivatives show, in general, only modest antibacterial activity, enhanced, however, by C-prenylation.21 The very potent antibacterial action of arzanol demonstrated enhanced activity by the addition of a pyrone substituent. Arzanol had notable activities against S. aureus 1199B, which overexpresses the NorA pump (1 μg/L), EMRSA-16 (2 μg/L), and XU 212 (4 μg/L), all of which were lower than the MIC of the standard control antibiotic norfloxacin (Table 1). By analogy with the results of the antiinflammatory assays,2 the bitalin esters (9a−g) were inactive, as were the coumarates and the simple pyrone micropyrone (10), while the pyrone homodimer helipyrone (4) was only marginally active. The chimeric structure of arzanol, combining two distinct polyketide elements with prenylation and heterodimerization, seems therefore optimized for bioactivity, in terms of both antibacterial and anti-inflammatory action. Having validated arzanol (1a) as a lead structure for antibacterial activity, we also investigated the activity of some derivatives of the natural product and synthetic analogues available from a study on the total synthesis of arzanol.22 Remarkably, both methylation of the pyrone moiety (12) and cyclization of the prenyl moiety (13, 14) led to loss of activity (Table 1), suggesting that hydroxylation of the ortho-position of the phloracetophene core and of the pyrone moiety were critical for the antibacterial activity of arzanol, again in perfect analogy with the results of anti-inflammatory evaluation.3 The development of a total synthesis of the natural product made it possible to also evaluate the effect of the introduction of a substituent on the methylene linker and of the simplification of the pyrone substitution pattern. The introduction of a substituent on the methylene linker [methyl (15a), hexyl (15b), phenyl (15c)] led to a modest decrease in their antibacterial activity and not to an increase of potency, as seen in the MICs value ranges (1−128 μg/L). On the other hand, the replacement of the pyrone moiety of the natural product with dehydroacetic acid led to a substantial retention of activity both in the natural product series (15d) and in its substituted analogues 15e and 15f. Taken together, these observations show that the antibacterial action of arzanol is critically sensitive to changes involving the hydroxy groups, while the pyrone

substitution pattern can be simplified and the introduction of a substituent on the methylene linker may provide no advantage. The potent activity of arzanol and its strict structure−activity relationships suggested that a specific target underlies its antibacterial activity. Arzanol is a powerful inhibitor of the transition-metal (iron, copper)-promoted oxidative degradation of lipids (linoleic acid, cholesterol)23 and inhibits proinflammatory targets involving redox reactions [(mPGES)1,5-LO].3 It is therefore tempting to speculate that it might interfere with a bacterial target that uses a transition metal redox system. The anti-inflammatory and antibacterial action of helichrysine was investigated clinically in the 1940s, at the onset of research on corticosteroids and antibiotics.1 The extraordinary success of these agents made interest in helichrysine fade quickly, despite its excellent clinical performance1 and the observation that its clinical profile summarized the action of both classes of drugs. Seventy years later, we believe that arzanol (1a) has the potential to play a role in addressing the major limitations of antibiotics and corticosteroids, namely, the selection of resistant strains for the former and the development of side-effects for the latter.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations (CHCl3) were measured at 589 nm on a JASCO P2000 polarimeter, and IR spectra on a FT-IR Thermo Nicolet apparatus. 1H (500 MHz) and 13C

E



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(125 MHz) NMR spectra were measured on a Varian INOVA spectrometer. Chemical shifts were referenced to the residual solvent signal (CDCl3: δH = 7.26, δC = 77.0, CD3OD: δH = 3.34, δC = 49.0). Homonuclear 1H connectivities were determined by the COSY experiment. One-bond heteronuclear 1H−13C connectivities were determined with the HSQC experiment. Through-space 1 H connectivities were evidenced using a ROESY experiment with a mixing time of 250 ms. Two- and three-bond 1H−13C connectivities were determined by gradient 2D HMBC experiments optimized for a 2,3 J = 9 Hz. Low- and high-resolution ESIMS were obtained on a LTQ OrbitrapXL (Thermo Scientific) mass spectrometer. Silica gel 60 (70− 230 mesh) and RP-18 used for gravity column chromatography were purchased from Macherey-Nagel. Reactions were monitored by TLC on Merck 60 F254 (0.25 mm) plates, which were visualized by UV inspection and/or staining with 5% H2SO4 in ethanol and heating. Organic phases were dried with Na2SO4 before evaporation. HPLC separations were achieved on a Knauer apparatus equipped with a refractive index detector. The Knauer HPLC apparatus was used to purify all final products. LUNA (normal-phase, SI60, or reversed-phase RP-18, 250 × 4 mm) (Phenomenex) columns were used, with 0.7 mL/min as flow rate. Plant Material. Flowered aerial parts of H. italicum subsp. microphyllum were collected around Arzana (Sardinia) at the beginning of July 2010. The plant material was identified by M.B., and a voucher specimen (number 729/10) is kept at the General Herbarium of the Botany Department of the University of Cagliari. Extraction and Isolation. Powdered nonwoody aerial parts (flowers and leaves, 5 kg) of H. italicum subsp. microphyllum were extracted at room temperature with acetone (2 × 15 L). Evaporation of the solvent left a black, gummy residue (202 g, 4.0%), part of which (50 g) was partitioned between petroleum ether (1.5 L) and aqueous methanol (1:9, 1.5 L). A 5 L round-bottom flask was used, connected to a rotatory evaporator stirring motor. After stirring for 1 h in a water bath at 30 °C, the two phases were separated to afford, after evaporation, 10.3 g of petroleum ether phase and 29.4 g of aqueous methanol phase. The semisolid material resisting partition (8.2 g) was purified by refluxing in acetone (200 mL) for 10 min. Cooling at room temperature and then in the refrigerator at 4 °C afforded 5.1 g (0.40%) of crude ursolic acid, which was further purified from impurities of oleanolic acid by recrystallization from methanol. The petroleum ether phase (11.3 g) was dissolved in methanol (300 mL) and then cooled at 4 °C overnight. After filtration of the copious precipitate of waxes and triglycerides, the filtrate (7 g) was purified by filtration over neutral alumina and further fractionated by gravity column chromatography on silica gel (200 g, petroleum ether−EtOAc, 9:1, as eluant) to afford neryl acetate (290 mg), ω-oleoyloxylinalol,2 and a mixture of the coumarates 3a,b (42 mg, ca. 35 ppm in the dry plant material), which could be separated by preparative HPLC (petroleum ether−EtOAc, 9:1, as eluant). The aqueous methanol phase (24.7 g) was fractionated coarsely by gravity column chromatography on silica gel (400 g, petroleum ether−EtOAc gradient) into five fractions (B−F), and, in turns, these were variously purified. Fraction B (3.4 g) was crystallized from ether to afford helipyrone (4, 320 mg, 256 ppm from the dry plant material). The mother liquors were further purified by gravity column chromatography on silica gel (150 g, petroleum ether−EtOAc, 7:3) to afford the bitalin A ester 9a (21 mg, 17 ppm in the dried plant material) and 9b (35 mg, 28 ppm in the dried plant material) and a mixture of santinol diesters and triesters. This was further fractionated by HPLC on normal phase (hexane−EtOAc, 8:2) to afford two subfractions, which eventually yielded pure compounds by further chromatographic separation. The mixture of santinols A1−A3 (ca. 200 mg, 160 ppm in the dried plant material) was resolved using MeOH−H2O, 93:7, as eluant, affording santinol A1 (5a, 15.3 mg, 12.2 ppm in the dried plant material), santinol A2 (5b, 8.6 mg, 70 ppm in the dried plant material), and santinol A3 (5c, 7.1 mg, ca. 60 ppm in dried plant material). The second, less polar santinol subfraction (180 mg) was subjected to chromatography over a silica gel column (230−400 mesh) eluting with a solvent gradient of increasing polarity from n-hexane to EtOAc. Fractions eluted with n-hexane−EtOAc (9:1) were rechromato-

graphed by HPLC (n-hexane−EtOAc, 9:1) to give santinols D (8, 3.7 mg, ca. 3 ppm in dried plant material) and C (7, 2.5 mg, ca. 2 ppm in the dried plant material). Fractions eluted with n-hexane−EtOAc, 8:2, were subjected to repeated HPLC chromatography (n-hexane− EtOAc, 85:15), affording santinol B (6, 1.5 mg, ca. 1 ppm in the dried plant material). Fractions D (1.6 g) and E (1.1 g) contained, along with micropyrone (10, overall yield 130 ppm on dried plant material after trituration with ether of a combination of two subfractions), a complex mixture of esters of bitalin A, from which pure compounds were obtained using a combination of chromatographic purifications on silica gel. Thus, a mixture of 9d and 9g (126 mg) obtained from the purification of fractions D and E by gravity column chromatography on silica gel was subjected to HPLC (n-hexane−EtOAc, 8:2) to yield 9d (2.0 mg) and 9g (1.1 mg). Fraction F was crystallized from ether to afford arzanol (3.1 g) as a yellow powder. The mother liquors (2.9 g) were fractionated by gravity colum chromatography to afford a further 600 mg of arzanol (overall yield 3.0%) and a mixture of analogues (250 mg), which was resolved by preparative HPLC on normal-phase silica gel (hexane−EtOAc, 55:45), affording pure heliarzanol (11, 4.5 mg, 3.6 ppm in dried plant material), 1b (12 mg, 10 ppm in dried plant material), and 1c (31 mg, 25 mg in dried plant material). Santinol A1 (5a): colorless oil; [α]D +3.5 (c 0.2, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 6.12 (1H, dq, J = 7.4, 2.0 Hz, H-3′), 4.28 (1H, overlapped, H-1a), 4.23 (1H, overlapped, H-3a), 4.22 (1H, overlapped, H-1b), 4.16 (1H, overlapped, H-3b), 4.14 (1H, m, H-2), 2.35 (2H, t, J = 7.8 Hz, H2-2″), 1.99 (3H, d, J = 7.4 Hz, H3-4′), 1.90 (3H, brs, H3-5′), 1.63 (1H, m, H-10″), 1.60 (2H, overlapped, H2-3″), 1.25 (10H, m, H2-4″ to H2-8″), 1.15 (2H, m, H2-9″), 0.87 (6H, d, J = 6.7 Hz, H3-11″ and H3-12″); 13C NMR (CDCl3, 125 MHz) δ 174.4 (s, C1″), 168.3 (s, C-1′), 139.6 (d, C-3′), 127.4 (s, C-2′), 68.6 (d, C-2), 65.4 (t, C-3), 65.2 (t, C-1), 39.4 (t, C-9″), 34.4 (t, C-2″), 29.6 (t, C-4″ to C-8″), 28.0 (d, C-10″), 25.3 (t, C-3″), 22.9 (q, C-11″ and C-12″), 20.9 (q, C-5′), 16.2 (q, C-4′); (+) ESIMS m/z 379 [M + Na]+; HRESIMS m/z 379.2456 (calcd for C20H36O5Na, 379.2460). Santinol A2 (5b): colorless oil; [α]D +3.2 (c 0.2, CHCl3); 1H NMR spectrum (CDCl3, 500 MHz) was identical to that of 5a, except for the integration of the signal at δH 1.25 (14H); (+) ESIMS m/z 407 [M + Na]+; HRESIMS m/z 407.2777 (calcd for C22H40O5Na, 407.2773). Santinol A3 (5c): colorless oil; [α]D +2.7 (c 0.2, CHCl3); 1H NMR spectrum (CDCl3, 500 MHz) was identical to that of 5a except for the integration of the signal at δH 1.25 (18H); (+) ESIMS m/z 435 [M + Na]+; HRESIMS m/z 435.3090 (calcd for C24H44O5Na, 435.3086). Mosher Ester Analysis of Santinol A1. Santinol A1 (5a, 2.0 mg) was treated with R-MTPA chloride (30 μL) in 400 μL of dry pyridine with a catalytic amount of DMAP overnight at room temperature. The solvent was then removed, and the product was purified by HPLC (nhexane−EtOAc, 85:15) to obtain the S-MTPA ester 5d (1.8 mg). When santinol A1 (5a, 2.0 mg) was treated with S-MTPA chloride, following the same procedure, 1.7 mg of the R-MTPA ester 5e was obtained. Santinol A1 (S)-MTPA ester 5d: 1H NMR (500 MHz, CDCl3) δ (selected values) 5.58 (1H, m, H-2), 4.44 (1H, dd, J = 12.3, 3.5 Hz, H3a), 4.37 (1H, dd, J = 12.3, 4.0 Hz, H-1a), 4.30 (1H, dd, J = 12.3, 6.0 Hz, H-1b), 4.14 (1H, dd, J = 12.3, 7.2 Hz, H-3b); ESIMS (positive ion) m/z 595 [M + Na]+. Santinol A1 (R)-MTPA ester 5e: 1H NMR (500 MHz, CDCl3) δ (selected values) 5.58 (1H, m, H-2), 4.46 (1H, dd, J = 12.3, 3.5 Hz, H3a), 4.37 (1H, dd, J = 12.3, 4.0 Hz, H-1a), 4.25 (1H, dd, J = 12.3, 6.0 Hz, H-1b), 4.21 (1H, dd, J = 12.3, 7.2 Hz, H-3b); ESIMS (positive ion) m/z 595 [M + Na]+. Santinol B (6): colorless oil; 1H NMR (CDCl3, 500 MHz) δ 6.15 (2H, dq, J = 7.4, 2.0 Hz, H-3′ = H-3″), 4.24−4.22 (4H, m, H2-1 and H2-3), 4.20 (1H, m, H-2), 1.99 (6H, d, J = 7.4 Hz, H-4′ = H-4″), 1.88 (6H, brs, H3-5′ and H3-5″); 13C NMR (CDCl3, 125 MHz) δ 167.4 (s, C-1′ = C-1″), 139.7 (d, C-3′ = C-3″), 127.7 (s, C-2′ = C-2″), 68.5 (d, C-2), 65.0 (t, C-1 = C-3), 20.9 (q, C-5′ = C-5″), 16.1 (q, C-4′ = C4″); (+) ESIMS m/z 279 [M + Na]+; HRESIMS m/z 279.1211 (calcd for C13H20O5Na, 279.1208). Santinol C (7): colorless oil; [α]D −8.5 (c 0.1 in CHCl3); 1H NMR (CDCl3, 500 MHz) δ 6.12 (1H, dq, J = 7.0, 2.0 Hz, H-3′), 5.35 (1H, F



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m, H-2), 4.37 (1H, dd, J = 12.3, 3.5 Hz, H-3a), 4.35 (1H, dd, J = 12.3, 4.0 Hz, H-1a), 4.21 (1H, overlapped, H-3b), 4.20 (1H, overlapped, H1b), 3.55 (1H, q, J = 7.1 Hz, H-2‴), 2.60 (1H, m, H-4‴a,), 2.54 (1H, m, H-4‴b), 2.30 (2H, t, J = 7.5 Hz, H2-2″), 1.99 (3H, d, J = 7.0 Hz, H3-4′), 1.88 (3H, brs, H3-5′), 1.60 (2H, m, H2-3″), 1.34 (3H, d, J = 7.1 Hz, H3-6‴), 1.26 (16H, m, from H2-4″ to H2-11″), 1.06 (3H, t, J = 7.2 Hz, H3-5‴), 0.88 (3H, t, J = 6.9 Hz, H3-12″); 13C NMR (CDCl3, 125 MHz) δ 206.1 (s, C-3‴), 173.0 (s, C-1″), 170.3 (s, C-1‴), 167.4 (s, C1′), 139.7 (d, C-3′), 127.7 (s, C-2′), 68.6 (d, C-2), 63.3 (t, C-3), 62.0 (t, C-1), 52.6 (d, C-2‴), 35.1 (t, C-4‴), 34.4 (t, C-2″), 29.9 (t, C-4″ to C-11″), 25.8 (t, C-3″), 20.8 (q, C-5′), 16.1 (q, C-4′), 14.7 (q, C-12″), 13.1 (q, C-6‴), 8.8 (q, C-5‴); (+) ESIMS m/z 491 [M + Na]+; HRESIMS m/z 491.2993 (calcd for C26H44O7Na, 491.2985). Santinol D (8): colorless oil; [α]D +24.9 (c 0.4 in CHCl3); 1H NMR (CDCl3, 500 MHz) δ 6.13 (1H, dq, J = 7.0, 2.0 Hz, H-3′), 5.35 (1H, m, H-2), 4.37 (1H, dd, J = 12.3, 3.5 Hz, H-3a), 4.35 (1H, dd, J = 12.3, 4.0 Hz, H-1a), 4.21 (1H, overlapped, H-3b), 4.20 (1H, overlapped, H1b), 3.76 (1H, q, J = 7.1 Hz, H-2‴), 2.80 (1H, m, H-4‴), 2.30 (2H, t, J = 7.5 Hz, H2-2″), 1.99 (3H, d, J = 7.0 Hz, H3-4′), 1.87 (3H, brs, H35′), 1.60 (2H, m, H2-3″), 1.55 (2H, m, H-5‴), 1.34 (3H, d, J = 7.1 Hz, H3-8‴), 1.26 (16H, m, from H2-4″ to H2-11″), 1.25 (2H, m, H2-6‴), 1.13 (3H, d, J = 7.1 Hz, H-9‴), 0.88 (3H, t, J = 6.9 Hz, H3-12″), 0.87 (3H, t, J = 7.0 Hz, H-7‴); 13C NMR (CDCl3, 125 MHz) δ 209.3 (s, C-3‴), 173.0 (s, C-1″), 170.3 (s, C-1″), 167.5 (s, C-1′), 139.7 (d, C3′), 127.6 (s, C-2′), 68.6 (d, C-2), 63.3 (t, C-3), 62.0 (t, C-1), 52.9 (d, C-2‴), 43.5 (d, C-4‴), 34.4 (t, C-2″), 33.5 (t, C-5‴), 29.9 (from C-4″ to C-11″), 25.8 (t, C-3″), 20.5 (t, C-6‴), 20.5 (q, C-5′), 16.3 (q, C-4′), 15.5 (q, C-9‴), 14.7 (q, C-12″), 14.1 (q, C-7‴), 13.2 (q, C-8‴); (+) ESIMS m/z 533 [M + Na]+; HRESIMS m/z 533.3457 (calcd for C29H50O7Na, 533.3454). Oleoylbitalin A (9a): waxy gum; [α]D −75 (c 0.2, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 7.82 (1H, s, H-4), 7.79 (1H, d, J = 6.2 Hz, H-6) 6.82 (1H, d, J = 6.2 Hz, H-7), 5.40 (1H, t, J = 8.0 Hz, H-2), 5.38 (2H, brs, H-9′ and H-10′), 5.36 (1H, brs, H-11a), 5.32 (1H, brs, H11b), 4.71 (1H, d, J = 13.2 Hz, H-12a), 4.66 (1H, d, J = 13.2 Hz, H12b), 3.44 (1H, dd, J = 15.0, 8.0 Hz, H-3a,), 3.20 (1H, dd, J = 15.0, 8.0 Hz, H-3b), 2.53 (3H, s, H3-9), 2.28 (2H, t, J = 7.2 Hz, H2-2′), 2.00 (6H, brs, H2-3′, H2-8′ and H2-11′), 1.28 (22H, brs), 0.86 (3H, t, J = 7.2 Hz, H3-18′); 13C NMR (CDCl3, 125 MHz) δ 196.6 (s, C-8), 173.1 (s, C-1′), 163.2 (s, C-7a), 142.7 (s, C-10), 131.1 (s, C-5), 130.6 (d, C9′ and C-10′), 130.5 (d, C-4), 127.2 (s, C-3a), 124.8 (d, C-6), 114.6 (t, C-11), 109.1 (d, C-7), 84.0 (d, C-2), 64.1 (t, C-12), 34.8 (t, C-3), 33.9, 33.8, 33.6 (t, C-2′, C-8′, and C-11′), 31.7 (t, C-16′), 29.8, 29.7, 29.5, 29.4, 29.3 (t, C-4′, C-5′, C-6′, C-7′, C-12′, C-13′, C-14′, and C-15′), 26.7 (q, C-9), 25.3 (t, C-3′), 22.5 (t, C-17′), 14.1 (q, C-18′); (+) ESIMS m/z 505 [M + Na]+; HRESIMS m/z 505.3294 (calcd for C31H46O4Na, 505.3294). Nonanoylbitalin A (9b): waxy gum; [α]D −65 (c 0.2, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 7.82 (1H, s, H-4), 7.79 (1H, d, J = 6.2 Hz, H-6), 6.82 (1H, d, J = 6.2 Hz, H-7), 5.39 (1H, t, J = 8.0 Hz, H-2), 5.36 (1H, brs, H-11a), 5.27 (1H, brs, H-11b), 4.73 (1H, d, J = 13.2 Hz, H12a), 4.67 (1H, d, J = 13.2 Hz, H-12b), 3.48 (1H, dd, J = 15.0, 8.0 Hz, H-3a,), 3.20 (1H, dd, J = 15.0, 8.0 Hz, H-3b), 2.53 (3H, s, H3-9), 2.27 (2H, t, J = 7.2 Hz, H2-2′), 1.58 (4H, m, H2-3′ and H2-4′), 1.28 (8H, brs, H2-5′- H2-8′), 0.86 (3H, t, J = 7.2 Hz, H3-3′); 13C NMR (CDCl3, 125 MHz) δ 196.0 (s, C-8), 172.9 (s, C-1′), 162.7 (s, C-7a), 142.3 (s, C-10), 131.4 (s, C-5), 130.2 (d, C-4), 127.2 (s, C-3a), 125.7 (d, C-6), 114.7 (t, C-11), 109.4 (d, C-7), 84.0 (d, C-2), 64.2 (t, C-12), 34.9 (t, C-3), 34.1 (t, C-2′), 31.8 (t. C-7′), 29.4, 29.3 (t, C-6′ and C-5′), 28.7 (t, C-4′), 26.7 (q, C-9), 25.0 (t, C-3′), 22.5 (t, C-8′), 13.8 (q, C-9′); (+) ESIMS m/z 381 [M + Na]+; HRESIMS m/z 381.2056 (calcd for C22H30O4Na, 381.2042). Propanoylbitalin A (9d): colorless, amorphous solid; [α]D −52.8 (c 0.2, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 7.81 (1H, s, H-4), 7.80 (1H, d, J = 6.2 Hz, H-6) 6.82 (1H, d, J = 6.2 Hz, H-7), 5.39 (1H, t, J = 8.0 Hz, H-2), 5.37 (1H, brs, H-11a), 5.29 (1H, brs, H-11b), 4.70 (1H, d, J = 13.2 Hz, H-12a), 4.63 (1H, d, J = 13.2 Hz, H-12b), 3.42 (1H, dd, J = 15.0, 8.0 Hz, H-3a,), 3.20 (1H, dd, J = 15.0, 8.0 Hz, H-3b), 2.53 (3H, s, H3-9), 2.37 (2H, q, J = 7.2 Hz, H2-2′), 1.16 (3H, t, J = 7.2 Hz, H3-3′); 13C NMR (CDCl3, 125 MHz) 196.7 (s, C-8), 171.5 (s, C-1′),

163.7 (s, C-7a), 142.5 (s, C-10), 131.0 (s, C-5), 130.5 (d, C-4), 127.0 (s, C-3a), 125.7 (d, C-6), 114.9 (t, C-11), 109.2 (d, C-7), 84.2 (d, C2), 64.1 (t, C-12), 34.6 (t, C-3), 28.1 (t, C-2′), 26.4 (q, C-9), 10.1 (q, C-3′); (+) ESIMS m/z 297 [M + Na]+; HRESIMS m/z 297.1105 (calcd for C16H18O4Na, 297.1103). Isocaproylbitalin A (9g): colorless, amorphous solid; [α]D −43.1 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 7.81 (1H, s, H-4), 7.80 (1H, d, J = 6.2 Hz, H-6) 6.82 (1H, d, J = 6.2 Hz, H-7), 5.39 (1H, t, J = 8.0 Hz, H-2), 5.37 (1H, brs, H-11a), 5.29 (1H, brs, H-11b), 4.70 (1H, d, J = 13.2 Hz, H-12a), 4.63 (1H, d, J = 13.2 Hz, H-12b), 3.42 (1H, dd, J = 15.0, 8.0 Hz, H-3a), 3.20 (1H, dd, J = 15.0, 8.0 Hz, H-3b), 2.53 (3H, s, H3-9), 2.25 (2H, t, J = 7.0 Hz, H2-2′), 1.70 (1H, m, H-3′), 1.58 (2H, m, H2-3′), 0.95 (6H, d, J = 7.0 Hz, H3-5′ and H3-6′); 13C NMR (CDCl3, 125 MHz) δ 196.7 (s, C-8), 171.3 (s, C-1′), 163.7 (s, C-7a), 142.5 (s, C-10), 131.0 (s, C-5), 130.5 (d, C-4), 127.0 (s, C-3a), 125.7 (d, C-6), 114.9 (t, C-11), 109.2 (d, C-7), 84.2 (d, C-2), 64.1 (t, C-12), 35.5 (t, C-3′), 34.6 (t, C-3), 31.8 (t, C-2′), 28.2 (d, C-4′), 26.4 (q, C9), 23.0 (q, C-5′ and C-6′); (+) ESIMS m/z 339 [M + Na]+; HRESIMS m/z 339.1582 (calcd for C19H24O4Na, 339.1572). Heliarzanol (11): pale yellow, amorphous solid; [α]D +17.4 (c 0.15, CH3OH); IR (KBr) νmax 3331, 3000, 1675, 1625, 1570, 1321, 1175 cm−1; 1H NMR (CDCl3, 500 MHz) δ 4.89 (2H, brs, H2-19), 4.34 (1H, bt, J = 7.3 Hz, H-17), 3.70 (2H, brs, H2-7), 3.22 (1H, dd, J = 11.5, 7.3 Hz, H-16a), 3.11 (1H, hept, J = 6.9 Hz, H-22), 2.85 (1H, dd, J = 11.5, 7.3 Hz, H-16b), 2.55 (2H, q, J = 6.9 Hz, H2-12), 1.95 (3H, s, H3-14), 1.86 (3H, brs, H3-20), 1.20 (6H, d, J = 6.9 Hz, H3-23 and H324), 1.19 (3H, q, J = 6.9 Hz, H3-13); 13C NMR (CDCl3, 125 MHz) δ 204.1 (s, C-21), 169.4 (s, C-15), 168.7 (s, C-9), 161.9 (s, C-4), 161.4 (s, C-11), 161.1 (s, C-6), 159.5 (s, C-2), 144.2 (s, C-18), 111.6 (t, C19), 109.0 (s, C-10), 107.9 (s, C-3), 107.0 (s, C-5), 106.1 (s, C-1), 103.5 (s, C-8), 79.6 (d, C-17), 32.2 (t, C-16), 30.1 (d, C-22), 24.9 (t, C-12), 20.0 (q, C-23 and C-24), 19.2 (t, C-7), 18.1 (q, C-20), 11.9 (q, C-13), 10.0 (q, C-14); (−) ESIMS m/z 445 [M − H]−; HRESIMS m/ z 445.1866 (calcd for C24H29O8, 445.1862). Synthesis of Nonanoylbitalin A (9b). A solution of bitalin A (obtained by hydrolysis with methanolic KOH of a mixture of its esters, 104 mg, 0.45 mmol) in CH2Cl2 (3 mL) was treated sequentially with nonanoic acid (66 mg, 4.95 mmol, 1.1 molar equiv), DCC (108 mg, 4.95 mmol, 1.1 molar equiv), and DMAP (47 mg, 0.8 molar equiv). After stirring 12 h at room temperature, the reaction was diluted with ether (ca. 6 mL) and filtered over neutral alumina and then silica gel to obtain 119 mg (60%) of 9b as a colorless, waxy solid, identical (1H and 13C NMR and MS) with the compound obtained by isolation. Biological Assays. Bacterial Strains. Strain XU212, which possesses the TetK tetracycline efflux pump and is also resistant to methicillin and erythromycin, was a hospital isolate. XU212 and standard strain ATCC 25923 were obtained from Dr. Edet Udo.24 Strain SA-1199B overexpresses the NorA MDR efflux pump and was provided by Prof. Glenn Kaatz.25 RN4220, which has the MsrA macrolide efflux protein, was provided by J. Cove.26 The epidemic methicillin-resistant strains EMRSA-15 and EMRSA-16 are hospital isolates27,28 and were obtained from Dr. Paul Stapleton. All strains were cultured on nutrient agar (Oxoid) and incubated overnight at 37 °C prior to assay. Minimum Inhibitory Concentration (MIC) Determination. Sterile Mueller-Hinton broth (MHB; Oxoid) (100 μL) containing 20 and 10 mg/L of Ca2+ and Mg2+, respectively, was dispensed into 11 of the wells of a 96-well microliter plate (Nunc, 0.3 mL volume per well). All antibacterial agents apart from norfloxacin were dissolved in dimethylsulfoxide and diluted in MHB to give a stock solution. Then, 100 μL of the antibacterial agent stock solution (concentration 512 mg/L) was serially diluted into each well, and 100 μL of the bacterial innoculum was added to each well to give a final concentration range of 128−0.25 μg/L in the wells. All procedures were performed in duplicate, and the plates incubated for 18 h at 37 °C. A 20 μL aliquot of a 5 mg/L methanolic solution of 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma) was added to each well and incubated for 30 min. A blue coloration G



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subspecies,7 and we have found that several commercial samples of the plant are totally devoid of pyrone derivatives. It is therefore unsurprising that, in the lack of a proper standardization, the early Helichyrsum pharmaceutical products were doomed to failure. (12) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 4092−4096. (13) Lehnert, W.; Junker, A. Clin. Chim. Acta 1980, 104, 47−51. (14) Liu, H.; Olsen, C.; Christensen, S. B. J. Nat. Prod. 2004, 67, 1439−1440. (15) Bowen, D. M.; DeGraw, J. I.; Shah, V. R.; Bonner, W. A. J. Org. Chem. 1963, 6, 315−319. (16) (a) Bohlmann, F.; Zdero, C. Tetrahedron Lett. 1970, 41, 3575− 3576. (b) Haensel, R.; Cybulski, B.; Mericli, A. H.; Bohlmann, F.; Zdero, C. Phytochemistry 1980, 19, 639−644. (17) Emsley, J. Chem. Soc. Rev. 1980, 9, 91−124. (18) Crassipin A ( Socolsky, C.; Rates, S. M. K.; Stein, A. C.; Asakawa, Y.; Bardon, A. J. Nat. Prod. 2012, 75, 1007−1017. ) and myrtucommulone A ( Appendino, G.; Bianchi, F.; Minassi, A.; Sterner, O.; Ballero, M.; Gibbons, S. J. Nat. Prod. 2002, 65, 334−338 ) exemplify phloroglucinyl heterodimers showing, respectively, two distinct sets of signals or very broad spectra in their NMR spectra at room temperature. (19) Smirnov, V. V.; Fasil’ev, V. N.; Preobrazhenskaia, N. E.; Zhmura, L. V. Mikrobiol. Zh. 1990, 52, 56−59. (20) Marshall, N. J; Piddock, L. J. V. Microbiologia 1997, 13, 285− 300. (21) Shiu, W. K. D.; Gibbons, S. Phytochemistry 2006, 67, 2568− 2572. (22) Minassi, A.; Cicione, L.; Koeberle, A.; Bauer, J.; Laufer, S.; Werz, O.; Appendino, G.; Appendino, G. Eur. J. Org. Chem. 2012, 772−779. (23) Rosa, A.; Pollastro, F.; Atzeri, A.; Appendino, G.; Melis, M. P.; Deiana, M.; Incani, A.; Loru, D.; Dessi, M. A. Chem. Phys. Lipids 2011, 164, 24−32. (24) Gibbons, S.; Udo, E. E. Phytother. Res. 2000, 14, 139−140. (25) Kaatz, G. W.; Seo, S. M.; Ruble, C. A. Antimicrob. Agents Chemother. 1993, 37, 1086−1094. (26) Ross, J. I.; Farrell, A. M.; Eady, E. A; Cove, J. H.; Cunliffe, W. J. J. Antimicrob. Chemother. 1989, 24, 851−862. (27) Richardson, J. F.; Reith, S. J. Hosp. Infect. 1993, 25, 45−52. (28) Cox, R. A.; Conquest, C.; Mallaghan, C.; Marples, R. R. J. Hosp. Infect. 1995, 29, 87−106.

indicated bacterial growth. The MIC was recorded as the lowest concentration at which no visible growth was observed.

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ASSOCIATED CONTENT

* Supporting Information S

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: + 39 081 678509 (O.T.-S.); +39 0321 373744 (G.A.). Fax: + 39 081 678552 (O.T.-S.); +39 0321 0.375621 (G.A.). Email: [email protected] (O.T.-S.); [email protected] (G.A.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to MURST (Italy) for financial support (Project 2009RMW3Z5: Metodologie sintetiche per la generazione di diversità molecolare di rilevanza biologica). Mass and NMR spectra were recorded at “Centro di Servizio Interdipartimentale di Analisi Strumentale”, Università di Napoli “Federico II”.

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DEDICATION Dedicated to Dr. Lester A. Mitscher, of the University of Kansas, for his pioneering work on the discovery of bioactive natural products and their derivatives

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REFERENCES

(1) Santini, L. Minerva Med. 1952, 43, 714−719. (2) Appendino, G.; Ottino, M.; Marquez, N.; Bianchi, F.; Giana, A.; Ballero, M.; Sterner, O.; Fiebich, B. L.; Muñoz, E. J. Nat. Prod. 2007, 70, 608−612. (3) Bauer, J.; Koeberle, A.; Dehm, F.; Pollastro, F.; Appendino, G.; Northoff, H.; Rossi, A.; Sautebin, L.; Werz, O. Biochem. Pharmacol. 2011, 81, 259−268. (4) Negrash, A. K.; Pertsev, I. M.; Khristenko, L. A.; Salo, D. P. Khim.-Farm. Zh. 1977, 11, 107−110. (5) Gibbons, S. Nat. Prod. Rep. 2004, 21, 263−277. (6) Passerini, M.; Ridi, M.; Papini, P. Ann. Chim. 1954, 74, 783−786. (7) Angioni, A.; Barra, A.; Arlorio, M.; Coisson, J. D.; Russo, M. T.; Pirisi, F. M.; Satta, M.; Cabras, P. J. Agric. Food Chem. 2003, 51, 1030− 1034. (8) Weyerstahl, P.; Marschall, H.; Bork, W.-R.; Reiner, S.; Wahlburg, H.-C. Flav. Fragr. J. 1995, 10, 297−311. (9) (a) Zapesochnaya, G. G.; Dzyadevich, T. V.; Karasartov, B. S. Khim Prir. Soedin 1990, 409−410. (b) Lakshmanan, D.; Werngren, J.; Jose, L.; Suja, K. P.; Nair, M. S.; Varma, R. L.; Mandayoor, S.; Hoffner, S.; Kumar, R. A. Fitoterapia 2011, 82, 757−761. (10) Haensel, R.; Cybulski, E. M.; Cubukcu, B.; Mericli, A. H.; Bohlmann, F.; Zdero, C. Phytochemistry 1980, 19, 639−644. (11) Leonardo Santini (1904−1983), an Italian physician, carried out the first systematic studies on the clinical anti-inflammatory properties of H. italicum ( Santini, L. Atti Soc. Lomb. Sci. Med. Biol. 1949, 5, 18 ). Santini summarized the results of almost two decades of clinical observation on H. italicum in a review article that appeared in 1952.1 On the basis of these studies, extracts of various Helichrysum species were introduced to the Italian pharmaceutical market for the treatment of cough. The quality and origin of these extracts were questioned by L. Santini (personal communication of his son, Maurizio Santini, to G.A.), and they were eventually removed from the pharmaceutical market in the 1970s. H. italicum shows a remarkable infraspecific variation in its phytochemical profile, even without its various H


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