Sesquiterpenes from Oplopanax horridus Taichi Inui, †,‡,⊥ Yuehong Wang, ‡ Dejan Nikolic, † David C. Smith, § Scott G. Franzblau, ‡ and Guido F. Pauli* ,†,‡ Department of Medicinal Chemistry and Pharmacognosy and Institute for Tuberculosis Research, College of Pharmacy, UniVersity of Illinois at Chicago, Chicago, Illinois 60612, and Alaska Green Gold, Anchorage, Alaska 99510 ReceiVed October 21, 2009 From the anti-TB active fractions of the inner stem bark of Oplopanax horridus, two new heterocyclic nerolidol derivatives, 3,10-epoxy-3,7,11-trimethyldodeca-1,6-dien-11-ol, named neroplomacrol (1), and rel-(3S,6R,7S,10R)-7,10-epoxy-3,7,11- trimethyldodec-1-ene-3,6,11-triol, named neroplofurol (2), were isolated together with oplopandiol (3), falcarindiol (4), and sesamin (5). Extensive spectroscopic analysis revealed that 1 possesses a novel 3,10-oxanonacyclic ring system. Computer-iterated full spin system analysis led to the generation of 1 H NMR fingerprints that will facilitate future dereplication of analogues by providing characteristic spin-spin coupling patterns. The full spin analysis of 5 revealed asymmetric coupling patterns among the chemically equivalent spins, thus confirming the magnetic asymmetry of 5. It was further demonstrated that 1 H NMR fingerprints and MS data enable dereplication of isolates at a submilligram levels including their relative configuration. Countercurrent concentration of the anti-TB activity of the ethnobotanical O. horridus versus the Mycobacterium tuberculosis Erdman strain led to polyynes 3 and 4 as main anti-TB active principles. Their synergistic behavior is linked to a complex fraction containing the new nerolidiol sesquiterpenes, 1 and 2, as phytochemical marker compounds. Oplopanax horridus (Sm.) Miq. (Araliaceae) (syn. Fatsia horrida (Sm.) Benth. & Hook.; Echinopanax horridus (Sm.) Decne. & Planch; Panax horridum (Sm.)), commonly known as devil’s club, is an abundant deciduous shrub found along the Northern Pacific coast of North America. It is one of the most important plants among native tribes in the Alaskan region due to the fact that it has been used extensively in shamanistic rituals, as well as a treatment for various ailments, including tuberculosis. 1-3 There is a consistency in the applications of O. horridus as a medicinal plant even among tribes too far apart for direct communication, and at least eight distinctive cultural groups use O. horridus bark for tuberculosis treatment. 4 The crude extract of O. horridus was found to be active against Mycobacterium tuberculosis Erdman and M. aVium by disk diffusion assay. 5 Two independent GC-MS analyses of O. horridus identified 36 sesquiterpenes and oxygenated sesquiterpenes, including trans-nerolidol. 6,7 So far, the structures of five polyynes have been reported from the inner stem bark of the plant. 8 The absolute configuration of oplopandiol acetate, one of the five reported polyynes, has been determined as (11S, 16S) by the modified Mosher method and was confirmed by total synthesis. 9 The five polyynes from the inner stem bark have shown moderate antimy- cobacterial activities among the known constituents of O. horridus. 8,10 Recent investigations involving absorption-free countercurrent chromatography have proven the presence of anti-TB synergistic interactions among fractions of O. horridus. 11 When synergy is present, chemical standardization of ethnobotanical preparations requires marker compounds for the active extracts/fractions regard- less of the activities of markers as individual compounds. Dereplication, i.e., the process of rapid identification of already known natural products, can facilitate the standardization of ethnobotanicals with synergistic activity, as structurally similar secondary metabolites often have similar but distinct spectroscopic properties. Even subtle differences in the 1 H spin systems of two structures can give rise to second- or higher-order spin-spin coupling effects, which often produce significant differences in the splitting patterns of the 1 H NMR signals. Hence, a computer-iterated full spin system analysis of the isolated compounds will serve to facilitate the process of dereplication by providing more accurate spectral interpretation as well as characteristic 1 H NMR fingerprints for later comparison. Not only are observed complex peak patterns unique for each compound, they can also be fully reproduced by spectral simulation from the tabulated spin system parameters even if future spectra are measured at different magnetic field strengths. Therefore, a complete 1 H NMR spectral analysis facilitates unam- biguous, yet rapid structure dereplication through comparison of NMR fingerprints and represents complex but unique splitting patterns that are present in the majority of the high-resolution 1 H signals. 12,13 Results and Discussion The inner stem bark of O. horridus was consecutively extracted with CH 2 Cl 2 , MeOH, and 50% aqueous MeOH. In order to remove the anti-TB inactive lipophilic and hydrophilic constituents, the combined extracts were partitioned between n-hexane- EtOAc-MeOH-water (HEMWat) solvent systems (SSs), first HEMWat -8 then HEMWat 0, respectively. 14 The anti-TB activity was concentrated in the upper phase of HEMWat 0, which was further separated by countercurrent chromatography (CCC) involv- ing a sequence of finely tuned two-phase SSs, i.e., gradient-array CCC. 15 Further MPLC separation of the two most active fractions * To whom correspondence should be addressed. Tel: 312-355-1949. Fax: 312-996-2693. E-mail: gfp@uic.edu. † Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago. ‡ Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago. § Alaska Green Gold, Anchorage, Alaska. ⊥ Current address: Wm. Wrigley Jr. Company, Chicago, Illinois 60642. J. Nat. Prod. 2010, 73, 563–567 563 10.1021/np900674d  2010 American Chemical Society and American Society of Pharmacognosy Published on Web 03/10/2010