Antimycobacterial Coumarins from the Sardinian Giant Fennel (Ferula communis) Giovanni Appendino,* ,† Enrico Mercalli, ‡ Nicola Fuzzati, ‡ Lolita Arnoldi, ‡ Michael Stavri, § Simon Gibbons,* ,§ Mauro Ballero, ⊥ and Andrea Maxia ⊥ Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universita ` del Piemonte Orientale, Viale Ferrucci 33, 28100 Novara, Italy, Indena S.p.A., Via Don Minzoni 6, 20090 Settala (MI), Italy, Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, U.K., and Dipartimento di Scienze Botaniche, Universita ` di Cagliari, Viale Fra Ignazio 13, 09124 Cagliari, Italy Received September 4, 2004 The structure of a new prenylated coumarin (E-ω-benzoyloxyferulenol, 1b) from the Sardinian giant fennel (Ferula communis) has been confirmed by synthesis. The parent compound ferulenol (1a) showed sub- micromolar antimycobacterial activity, which was partly retained in 1b and in the simplified synthetic analogue 3, but diminished in its ω-hydroxy and ω-acetoxy derivatives (1c and 1d, respectively). The outstanding activity of 1a, its low toxicity, and the evidence for definite structure-activity relationships make this prenylated 4-hydroxycoumarin an interesting antibacterial chemotype worth further investiga- tion. The presence of two distinct chemotypes of giant fennel (Ferula communis L.; Apiaceae) has complicated the study of ferulosis, a hemorrhagic and often lethal intoxication of livestock pasturing in areas of Sardinia infested with this invasive species. 1 On the other hand, this chemical poly- morphism has given phytochemists a unique opportunity to obtain large amounts of structurally unrelated bioactive compounds from the same species. Thus, while the poison- ous chemotype is an excellent source of ferulenol (1a), 2 a hemorrhagic prenylated coumarin that shows also pacli- taxel (Taxol) mimicry, 3 the potent phytoestrogen ferutinin (2) can be obtained in large amounts from the nonpoisonous chemotype. 4 Remarkably, the two chemotypes occupy distinct enclaves, and an extensive investigation failed to detect mixed populations. 5 The basis of this behavior remains unknown, but preliminary studies have shown distinct genetic diversities between the two chemotypes, which are otherwise morphologically indistinguishable. 6 During the development of an expeditious HPLC proce- dure to fingerprint the two chemotypes, we noticed the presence of a minor compound having chromatographic behavior and a UV spectrum similar to those of ferulenol but with a higher molecular weight (+104 amu), corre- sponding to the introduction of a benzoyloxy group. From the mass fragmentation pattern, this moiety was located on one of the two ω-carbons of the prenyl group, but the low natural abundance and the close chromatographic behavior compared to ferulenol (1a), a major constituent of the extract, prevented its direct isolation. 7 To solve this matter, we planned to synthesize the E-isomer of the alleged structure (1b) from E-ω-hydroxyferulenol (1c), a compound easily available by isolation, 2 and compare the chromatographic and spectroscopic properties (UV, MS) of the synthetic compound with those of the unknown chro- matographic peak. Diastereomerically pure E-ω-hydroxyferulenol (1c) was obtained, in 1.05% yield, along with ferulenol (1a, 0.55%) and its ω-acetoxy derivative (1d, 0.20%), 8 from a sample of giant fennel collected in southern Sardinia. The presence of E,Z-mixtures of ω-oxygenated ferulenols has been previ- ously reported, 8 but in the sample under investigation, both ω-acetoxy and ω-hydroxyferulenol were obtained in a diastereomerically pure E-form. To prepare a compound with the alleged formula of the unknown peak, compound 1c was treated with an excess of benzoyl chloride to afford the dibenzoate 1e, which was then subjected to chemose- lective deprotection. Opening of the lactone ring and decarboxylation to a prenylacetophenone 9 were not ob- served when a transamidation rather than a hydrolysis reaction was employed. Pyrrolidine is the standard base for the chemoselective transamidation of phenolic esters, but in our case, the two reaction products (1b and N- * To whom correspondence should be addressed. Tel: +39 0321 375744 (G.A.); +44 207 753 5913 (S.G.). Fax: +39 0321 375631 (G.A.); +44 207 753 5909 (S.G.). E-mail: appendino@pharm.unipmn.it (G.A.); simon.gibbons@ulsop.ac.uk (S.G.). † Universita ` del Piemonte Orientale. ‡ Indena S.p.A. § University of London. ⊥ Universita ` di Cagliari. 2108 J. Nat. Prod. 2004, 67, 2108-2110 10.1021/np049706n CCC: $27.50 © 2004 American Chemical Society and American Society of Pharmacognosy Published on Web 11/25/2004