Isolation and Structure Elucidation of Parthenolide from Tanacetum

LABORATORY EXPERIMENT pubs.acs.org/jchemeduc

Nature’s Migraine Treatment: Isolation and Structure Elucidation of Parthenolide from Tanacetum parthenium Emma L. Walsh, Siobhan Ashe, and John J. Walsh* School of Pharmacy and Pharmaceutical Sciences, University of Dublin, Trinity College, Dublin 2, Ireland

bS Supporting Information ABSTRACT: The purpose of this experiment is to provide students with the essential skills and knowledge required to perform the extraction, isolation, and structural elucidation of parthenolide from Tanacetum parthenium or feverfew. Students are introduced to a background of the traditional medicinal uses of parthenolide, while more modern applications of parthenolide are also presented. Clinical data supporting the use of feverfew in the treatment of migraine is presented. Methods outlining the accurate extraction and isolation of parthenolide from the powdered, dried flowering tops of feverfew are described. The experiment allows students to acquire and use such skills as extraction, flash column chromatography, and thin-layer chromatography. Structural elucidation is carried out on parthenolide using techniques such as infrared (IR) spectroscopy, highresolution mass spectrometry (HRMS), and nuclear magnetic resonance (NMR) spectroscopy. Nuclear Overhauser enhancement spectroscopy (NOESY) and X-ray crystallography are employed to establish the three-dimensional conformation of the structure. The student can isolate parthenolide with an approximate yield of 0.2% and the experiment can be completed over two 3-h laboratory sessions. Finally, questions are provided in the student handout, requiring that students engage further in topics associated with the context of this practical. KEYWORDS: Graduate Education/Research, Upper-Division Undergraduate, Laboratory Instruction, Organic Chemistry, HandsOn Learning/Manipulatives, Medicinal Chemistry, NMR Spectroscopy, Natural Products, Thin Layer Chromatography, X-ray Crystallography

’ BACKGROUND Feverfew is a member of the Asteraceae (Compositae) family and has been classified as T. parthenium, Chrysanthemum parthenium, or Leucanthemum parthenium, with T. parthenium being the most favored classification.8 Feverfew has long been referred to as “a medieval aspirin”9 and was used to a treat a broad spectrum of disorders including fever, headache and migraine, menstrual problems, and childbirth difficulties as well as stomach ache, toothache, and insect bites.10 A surge in scientific interest in this plant during the 1980s led to the undertaking of a number of clinical trials assessing the effects of T. parthenium in the prophylaxis of migraine.11 14 More recently, it has been shown that parthenolide exhibits antiinflammatory properties15,16 and antileishmanial activity,17 as well as chemopreventative properties18 and antitumor activity.19

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any natural plant products have been used in the treatment of a wide variety of ailments and disease since ancient times. This experiment imparts a valuable learning experience to the students in the use of Tanacetum parthenium (Figure 1) as a herbal medicine and the application of important analytical techniques commonly used in the laboratory for extraction and isolation purposes. The principal isolation technique employed in this practical is flash column chromatography. Widespread use of flash column chromatography in research fields such as antiinflammatory,1 anti-allergic,2 and antimalarial3 drug design programs underpins its status as a valuable analytical technique. This experiment represents an effective educational tool in column chromatography, offering students the opportunity to perform and thoroughly master the skills required to achieve precise isolation of a substance of interest from a complex mixture. Students also utilize high-resolution mass spectrometry (HRMS), infrared (IR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy as a means to performing a complete structural elucidation study on parthenolide. Work on this project complements our previous developments in phytochemical isolation, including valtrate from Centranthus ruber,4 galantamine from Leucojum aestivum,5 as well as other laboratory-based experiments such as betulin from birch bark6 and monoterpenes from spearmint.7 Copyright r 2011 American Chemical Society and Division of Chemical Education, Inc.

’ EXPERIMENTAL OVERVIEW This experiment is aimed at fourth-year undergraduate or firstyear postgraduate students who have a comprehensive understanding of the spectroscopic techniques used in structure Published: October 31, 2011 134

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(6 mL/1 mL), 5:1 (5 mL/1 mL), and 4:1 (12 mL/3 mL). Fractions of approximately 0.5 mL in volume were obtained. The first 10 mL of mobile phase was run through the column and the eluent was collected. For the remaining 10 mL of solvent, 20 fractions were obtained in numbered glass sample vials. Fractions containing pure parthenolide were identified by TLC by comparison with a parthenolide standard, using hexane/ethyl acetate (3:1) as the solvent system. The appropriate fractions were combined and solvent was removed using a rotary evaporator. The yield of pure parthenolide was recorded. Crystals suitable for X-ray analysis were slowly grown from a methanol solution after pooling the isolated compound samples from five students.

Figure 1. Photograph of T. parthenium taken from author’s (J.J.W.) garden.

elucidation of organic compounds. It has been completed with consistency and reproducibility by students in a masters-level pharmaceutical analysis course. The plant material is readily available and was collected from the author’s (J.J.W.) garden. The experiment affords the student the opportunity to follow the entire procedure of parthenolide isolation, from its extraction from the flowering tops of T. parthenium to elucidation of its structure using spectroscopy. It places particular emphasis on the importance of flash column chromatography for the isolation of phytochemicals in pure form from complex mixture and the use of modern spectroscopic techniques for the identification of natural products.20 The students are not informed of the structure of the compound, which is of moderate complexity, therefore, providing a challenging exercise for an upper undergraduate or graduate student. A variety of one- and two-dimensional NMR spectra, as well as IR and mass spectra, are provided in the Supporting Information. The NMR data obtained on parthenolide include 1H NMR and 13C NMR spectra, in addition to proton proton correlated spectroscopy (HH COSY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond coherence (HMBC). IR and MS spectral evidence support the information gained from these spectra. Students also utilize X-ray crystallography and nuclear Overhauser effect spectroscopy (NOESY) to identify the exact stereochemical conformation of parthenolide.

’ EXPERIMENTAL DETAILS This laboratory was divided into three sections: (i) crude product extraction and isolation, (ii) chromatography (flash column and TLC), and (iii) spectroscopy (MS, IR, and NMR). In week one of the practical, approximately 1 mg of parthenolide was generated from 0.5 g of the powdered flowering tops of T. parthenium. The process involved mixing the finely powdered plant material in 5 mL of dichloromethane for 20 min using a magnetic stirrer. The mixture was filtered and reduced in volume to allow for easy transfer onto the flash column. A standard Pasteur pipet of internal diameter of 5 mm was used to prepare the column (see student handout in Supporting Information for precise details of column preparation). The solvent system of choice for the flash chromatography was hexane/ethyl acetate, for which the following gradient system was used to achieve optimal resolution; hexane/ethyl acetate 9:1 (9 mL/1 mL), 6:1

’ HAZARDS This experiment involves the use of some potentially toxic substances including dichloromethane, hexane/ethyl acetate, silica gel, and vanillin spray reagent. Dichloromethane is a carcinogen, is toxic by inhalation, and causes irritation and burning pain on prolonged contact. Hexane is a flammable liquid and vapor, is harmful or fatal if swallowed, and causes irritation to skin, eyes, and respiratory tract. Ethyl acetate is flammable and harmful if inhaled and slightly hazardous in case or skin or eye contact. Exercise great care when handling these substances and use in a fume hood. Laboratory coats and suitable eye protection must be worn while undertaking this experiment. Parthenolide is a known skin irritant. Protective gloves must be worn throughout the practical. ’ RESULTS AND DISCUSSION IR, NMR, and HRMS spectra were obtained on the substance isolated to fully elucidate its structure. A strong absorption band on the IR spectrum at 1756 cm 1 is indicative of an ester group, whereas the band at 940 cm 1 may suggest the presence of an epoxide. The weak absorption band at 1654 cm 1 indicates alkene functionality. HRMS provides a molecular ion peak with an m/z value of 271.1306. Taking into account the addition of a sodium adduct, a possible molecular weight of approximately 248 (271.1306 23) for the structure is a reasonable assumption. A peak representing an ion with an m/z value of 248.1646 justifies this. Referring to the 13C NMR spectrum, 15 carbon signals are evident, perhaps representative of a sesquiterpene-like compound, whereas a total proton count of 20 is provided by the 1 H NMR spectrum. Further analysis of the 13C and DEPT spectra supplies evidence of two CH3, five CH2, and four CH signals in addition to four quaternary carbon atoms. It may be deduced that, of the 15 signals present in the 13C NMR spectrum, one represents the carbonyl carbon of a conjugated ester, four are indicative of olefinic carbons, and three peaks signify carbons with a carbon oxygen single bond. Knowing the molecular weight of the compound to be 248.1646 and having proven the existence of 15 carbons and 20 hydrogens, three oxygen atoms must be present, therefore, deriving an empirical formula of C15H20O3. Further evidence to substantiate this formula is provided with data accompanying the mass spectrum, which generates a range of molecular compositions with a possible molecular weight of approximately 248. Of the 10 possible options, only one molecular formula contains the 15 required carbon atoms that correspond with the suggested empirical formula (see instructor’s notes in the Supporting Information). Calculation of the index of hydrogen deficiency yields an unsaturation index of 6. Already established is the presence of 135

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detected between H5 and H14, suggesting that H5, resonating as an overlapping double doublet with identical J values (9.0 Hz) is α oriented. The large coupling constant observed for H5 suggests that it is antiperiplanar to H4 and H6. The configuration of the C10 C11 double bond is established due to lacking evidence indicating correlation of H10 with H15. This suggests that these protons are located on opposite planes of the double bond, thus, representing H10 as β oriented and an E configuration for the double bond. Strong correlation between H10 and H6 establishes that H6 resides below the plane of the ring.

Figure 2. Parthenolide.

’ ASSOCIATED CONTENT

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Supporting Information Student handout; instructor’s notes; spectra. This material is available via the Internet at http://pubs.acs.org.

Figure 3. Ortep presentation of parthenolide generated by X-ray crystallography The X-ray crystal structure obtained was identical to that previously published.21

two CdC double bonds and one carbonyl group, suggesting ring formations to account for the remaining three unsaturation equivalents. In the IR spectrum, the CdO stretching vibrations at 1756 cm 1 is strongly indicative of an α, β-unsaturated γlactone functionality. In addition, the mass spectrum illustrates fragmentation of the structure with a loss of CO2, resulting in a daughter ion peak with an m/z value of 227.1404. Consideration of these features, coupled with application of the unsaturation equivalent rule, leads to an assumption that a lactone ring is present. Resonances for two carbon atoms in the chemical shift region of the 13C NMR spectrum consistent with a C O single bond, together with one unassigned oxygen atom, suggests ether or possibly epoxide functionality. IR spectral evidence of a ring deformation band at 984 cm 1 is consistent with the presence of an epoxide. Combining this evidence with an unsaturation equivalent establishes with high probability the presence of an epoxide. The one remaining unsaturation index must be due to an additional ring formation. Therefore, the suggestion of a sesquiterpene lactone with epoxide functionality as a possible structure is acceptable. Imperative to complete elucidation of the structure is detailed use of 1-D and 2-D NMR spectroscopic data. Using HH COSY, HMQC, and HMBC spectra, in conjunction with 1H NMR and 13 C NMR spectra, the C H framework of the molecule is assigned. One approach to the elucidation of the structure, shown in Figure 2, is outlined in the Supporting Information. With assignment of the structure complete, the conformation of the molecule requires clarification. A three-dimensional physical model of the structure was constructed. Manipulation of the model yielded two possible structural configurations, differing in the arrangement of the two CH3 (C14 and C15) groups above and one each above and below the plane. A conformation whereby both methyl groups are pointing upward was deduced. NOE spectral studies and X-ray crystallographic analysis (Figure 3) indicate that the molecular framework is restricted to a single conformation. Strong contours indicating through-space coupling of the two methyl group protons (H14 and H15) establish that both groups are situated above the plane of the ring. Very strong, through space, coupling is observed between H5 and H15 with medium coupling

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT We would like to thank John O’Brien, Brian Talbot, and Tom McCabe for recording the NMR, HRMS, and X-ray data, respectively, Justin Perry, University of Northumbria, Newcastle, for helpful suggestions with regard to analysis of parthenolide by X-ray crystallography. ’ REFERENCES (1) Barlow, J. W.; Walsh, J. J. Eur. J. Med. Chem. 2008, 43 (12), 2891–2900. (2) Barlow, J. W.; Walsh, J. J. Eur. J. Med. Chem. 2010, 45 (1), 25–37. (3) Walsh, J. J.; Coughlan, D.; Heneghan, N.; Gaynor, C.; Bell, A. Bioorg. Med. Chem. Lett. 2007, 17 (13), 3599–3602. (4) Doyle, A. M.; Reilly, J.; Murphy, N.; Kavanagh, P. V.; O’Brien, J. E.; Walsh, M. S.; Walsh, J. J. J. Chem. Educ. 2004, 81, 1486–1487. (5) Halpin, C. M.; Reilly, C.; Walsh, J. J. J. Chem. Educ. 2010, 87 (11), 1242–1243. (6) Green, B.; Bentley, M. D.; Chung, B. Y.; Lynch, N. G.; Jensen, B. L. J. Chem. Educ. 2007, 84, 1985–1987. (7) Davies, D. R.; Johnson, T. M. J. Chem. Educ. 2007, 84, 318–320. (8) Dewick, P. M.Medicinal Natural Products—A Biosynthetic Approach, 3rd ed.; John Wiley & Sons: Chichester, U.K., 2009; pp 214. (9) Knight, D. W. Nat. Prod. Rep. 1995, 12, 271–276. (10) Heptinstall, S. J. R. Soc. Med. 1988, 81, 373–374. (11) Johnson, E. S.; Kadam, N. P.; Hylands, D. M.; Hylands, P. J. Br. Med. J. 1985, 291, 569–573. (12) Murphy, J. J.; Heptinstall, S.; Mitchell, J. R. Lancet 1988, 23, 189–192. (13) Palevitch, D.; Earon, G.; Carasso, R. Phytother. Res. 1997, 11, 508–511. (14) Diener, H. C.; Pfaffenrath, V.; Schnitker, J.; Friede, M.; Henneicke-von Zepelin, H. H. Cephalalgia 2005, 25 (11), 1031–1041. (15) Kwok, B.; Koh, B.; Ndubuisi, M. I.; Elofsson, M.; Crews, C. M. Chem. Biol. 2001, 8, 759–766. (16) Saadane, A.; Master, S.; DiDonato, J.; Li, J.; Berger, M. Am. J. Respir. Cell Mol. Biol. 2007, 36, 728–736. (17) Tiuman, T. S.; Ueda-Nakamura, T.; Garcia Cortez, D. A.; Dias Filho, B. P.; Morgado-Díaz, J. A.; de Souza, W.; Nakamura, C. V. Antimicrob. Agents Chemother. 2005, 36, 176–182. (18) Won, Y.; Ong, C.; Shi, X.; Shen, H. Carcinogenesis 2004, 25 (8), 1449–1458. 136

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(19) Guzman, M.l.; Rossi, R. M.; Karnischky, L.; Li, X.; Peterson, D. R.; Howard, D. S.; Jordan, C. T. Blood 2005, 105 (11), 4163–4169. (20) Fischer, N. H.; Isman, M. B.; Stafford, H. A.Modern Phytochemical Methods; Plenum Press: New York, 1991; pp 271 317. (21) Quick, A.; Rogers, D. J. Chem. Soc., Perkin Trans. 2 1976, 5, 465–469.

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