Semi-Automated Separation of the Epimeric

Research Article

Received: 30 December 2013, Revised: 29 January 2014, Accepted: 2 February 2014 Published online in Wiley Online Library: 9 May 2014

(wileyonlinelibrary.com) DOI 10.1002/pca.2511 Semi-automated Separation of the Epimeric Dehydropyrrolizidine Alkaloids Lycopsamine and Intermedine: Preparation of their N-oxides and NMR Comparison with Diastereoisomeric Rinderine and Echinatine

Steven M. Colegate,a* Dale R. Gardner,a Joseph M. Betzb and Kip E. Pantera

ABSTRACT: Introduction – The diversity of structure and, particularly, stereochemical variation of the dehydropyrrolizidine alkaloids can present challenges for analysis and the isolation of pure compounds for the preparation of analytical standards and for toxicology studies. Objective – To investigate methods for the separation of gram-scale quantities of the epimeric dehydropyrrolizidine alkaloids lycopsamine and intermedine and to compare their NMR spectroscopic data with those of their heliotridine-based analogues echinatine and rinderine. Methods – Lycopsamine and intermedine were extracted, predominantly as their N-oxides and along with their acetylated derivatives, from commercial samples of comfrey (Symphytum officinale) root. Alkaloid enrichment involved liquid–liquid partitioning of the crude methanol extract between dilute aqueous acid and n-butanol, reduction of N-oxides and subsequent continuous liquid–liquid extraction of free base alkaloids into CHCl3. The alkaloid-rich fraction was further subjected to semi- automated flash chromatography using boronated soda glass beads or boronated quartz sand. Results – Boronated soda glass beads (or quartz sand) chromatography adapted to a Biotage Isolera Flash Chromatography System enabled large-scale separation (at least up to 1–2 g quantities) of lycopsamine and intermedine. The structures were confirmed using one- and two-dimensional 1H- and 13C-NMR spectroscopy. Examination of the NMR data for lycopsamine, intermedine and their heliotridine-based analogues echinatine and rinderine allowed for some amendments of literature data and provided useful comparisons for determining relative configurations in monoester dehydropyrrolizidine alkaloids. A similar NMR comparison of lycopsamine and intermedine with their N-oxides showed the effects of N-oxidation on some key chemical shifts. A levorotatory shift in specificrotationfrom+3.29°toÀ1.5° was observed for lycopsamine when dissolved in ethanol or methanol respectively. Conclusion – A semi-automated flash chromatographic process using boronated soda glass beads was standardised and confirmed as a useful, larger scale preparative approach for separating the epimers lycopsamine and intermedine. The useful NMR correlations to stereochemical arrangements within this specific class of dehydropyrrolizidine alkaloid cannot be confidently extrapolated to other similar dehydropyrrolizidine alkaloids. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Supporting information can be found in the online version of this article.

Keywords: Boronated soda glass chromatography; HPLC–ESI/MS; NMR; optical rotation; echinatine; dehydropyrrolizidine alkaloids; intermedine; lycopsamine; rinderine; comfrey; Symphytum ofﬁcinale

Introduction The C9 monoester DHPAs lycopsamine (1) and intermedine (2) are epimeric about C13 (Fig. 1) (Mackay et al., 1983) and are In vivo oxidative metabolites of the plant-derived esters of components in many DHPA-producing plants (Bull et al., 1968). dehydropyrrolizidine alkaloids (DHPAs) and their N-oxides are potentially acutely and chronically toxic to livestock and humans (Edgar et al., 2011; Molyneux et al., 2011). Consequently, to enable monitoring of animal feed, the human food supply, dietary * Correspondence to: Steven M. Colegate, Poisonous Plant Research Laboratory, Agriculture Research Service, US Department of Agriculture, 1150 East 1400 supplements and herbal medicines for the presence of the DHPAs, North, Logan, Utah 84341, USA. and to deﬁne the actual risk to health presented by individual E-mail: [email protected] DHPAs, pure alkaloids are required for analytical standards and, a on a larger scale, for toxicological studies. However, the sometimes Poisonous Plant Research Laboratory, Agriculture Research Service, US subtle diversities of structure and stereochemistry (relative and Department of Agriculture, 1150 East 1400 North, Logan, Utah, 84341, USA absolute) of the several hundreds of DHPAs known can present a b Ofﬁce of Dietary Supplements, National Institutes of Health, 6100 Executive challenge for analysis and for the isolation of pure alkaloids. Blvd., Room 3B01, Bethesda, Maryland, 20892, USA 429

Phytochem. Anal. 2014, 25, 429–438 Published 2014. This article is a U.S. Government work and is in the public domain in the USA. S. M. Colegate et al.

Lycopsamine (1) and intermedine (2) in extracts of Amsinckia intermedia have been previously separated from each other by taking advantage of their different affinities for the boronate anion (Frahn et al., 1980). Two methods were described: (i) an affinity-type column where 1 and 2 were selectively partitioned between aqueous borax-coated powdered soda glass and the CHCl3 elution solvent; and (ii) an ion exchange process in which the anionic nature of the respective boronate complexes differentially hindered retention on a cation exchange resin. Only one of these approaches, that is, the cation exchange, seems to subsequently have been utilised successfully, albeit only once and with mannitol as the lycopsamine/intermedine displacement ligand instead of glucose (Roitman, 1983). In the present study, both methods were re-examined for their ability to separate lycopsamine, intermedine and related alkaloids derived from Symphytum officinale roots with the intent of standardising an approach with modern availability of reagents, substrates and equipment.

Experimental

Chemicals and reagents Dry, powdered comfrey (S. officinale) root (Frontier Natural Products Co-op) was purchased from Take Herb (Alhambra, CA, USA). The Figure 1. Structures of dehydropyrrolizidine alkaloids. following were reagent ACS/USP/NF grade (Pharmaco Products, Brookfield, CT, USA): methanol, for extractions; petroleum ether (b.p. – fl Each can be biosynthesised relatively exclusively of the other, 35 60°C) for column packing; and CHCl3,for ashcolumnchromatog- such as the lycopsamine-N-oxide chemotype of Amsinckia raphy. All other reagents, that is, zinc powder, n-butanol, ammonia solution and concentrated sulphuric acid were of analytical grade intermedia (Colegate et al., 2013) and intermedine-N-oxide from and generally sourced. Acetonitrile was HPLC-certified solvent Cerinthe glabra (Luber et al., 2012), but more often they co-occur (Honeywell Burdick and Jackson, Muskegon, MI, USA) and water was at various relative levels as the free bases and/or their N-oxides Milli-Q-purified (18.2 MΩ/cm) (Millipore, Bedford, MA, USA). Formic (Kelley and Seiber 1992; Williams et al., 2011). acid was ‘For Analysis’ grade (> 99%; Acros Organics/Thermo Fisher In some cases a fortuitous combination of solubility properties Scientific, Bridgewater, NJ, USA). Ammoniated methanol was prepared and a lack of other DHPA co-metabolites has allowed for a by bubbling dry ammonia gas through cooled (ice bath) methanol. The relatively simple extraction, purification and precipitation resultant saturated ammoniated methanol solution was diluted 1:9 process to result in large quantities of relatively pure DHPA. Such with methanol for use in strong cation exchange, solid phase was the case, for example, in the isolation of the macrocyclic extraction (SCX SPE). The HPLC columns, with their guard columns, μ diester DHPAs riddelliine (Adams et al., 1942) and senecionine bulk SCX packing material (Sepra SCX, 50 m, 65A) and SCX SPE cartridges (Strata SCX 55 μm, 70 A; 5 g/20 mL Giga tube), were sourced (Culvenor, 1962). However, when the DHPAs are less tractable, from Phenomenex (Torrence, CA, USA). The indigo-carmine-based methods of separation include thin-layer chromatography redox resin was prepared and used as previously described (Colegate (TLC), column chromatography, centrifugal planar chromatogra- et al., 2005; Williams et al., 2011). Echinatine (3), rinderine (4), heliotrine phy, semi-preparative and preparative scale high-pressure liquid (5) (Fig. 1) and lasiocarpine (all > 99% pure based on HPLC–ESI(+)/MS chromatography (HPLC), rotation locular counter current and NMR analysis) were sourced from the stocks of extracted and chromatography (RLCCC), droplet counter current chromatogra- purified pyrrolizidine alkaloids kept by the USDA/ARS Poisonous phy (DCCC) and high-speed counter current chromatography Plant Research Laboratory. One- and two-dimensional (COSY, HSQC) 1 13 (HSCCC). Because of the time, effort and higher levels of losses H- (300 MHz) and C- (75 MHz) NMR data were acquired using a JEOL through degradation or adsorption-related effects usually Eclipse NMR spectrometer using solutions in deuterochloroform involved with necessary repetitive application of the solid (Sigma-Aldrich, St Louis, MO, USA) and the residual proton in the CHCl3 as the chemical shift reference. Optical activity was measured using an stationary phase approaches, they have mostly been used to iso- Autopol IV Automatic Polarimeter (Rudolph Research Analytical, late relatively small quantities of pure alkaloid for NMR-based Hackettstown, NJ, USA). structural studies (e.g. Roeder et al., 1991; Colegate et al., 2012). High-speed CCC, a hydrodynamic equilibration system, has been applied to up to 800 mg of sample for the larger scale HPLC–ESI(+)/MS separation of DHPAs from Amsinckia tessellata, Symphytum spp., μ Trichodesma incanum and Senecio douglasii var. longilobus (Cooper Analytical samples were prepared by adding an aliquot (10 L) to a lasiocarpine-spiked solution of 0.1% formic acid in water and methanol et al., 1996). However, while the resolution reported was sufficient (1:1, v/v) (100 μL). The lasiocarpine was included as an internal standard to to separate the open chain diesters (symphytine and related normalise all responses. An aliquot (5 μL) of an analytical sample was subse- isomers) from the monoesters (lycopsamine, intermedine and their quently injected (Agilent 1260 Infinity HPLC System, Agilent Technologies, acetylated derivatives) extracted from Symphytum spp., it was not CA, USA) onto a Synergi Hydro RP column (150 × 2 mm, 4 μ) fitted with a sufficient to separate the individual diesters or monoesters (such guard column of similar adsorbent (AC C18, 4 mm diameter × 2 mm) 430 as the epimeric lycopsamine and intermedine) from each other. (Security Guard Cartridge system). Sample components were eluted from

wileyonlinelibrary.com/journal/pca Published 2014. This article is a U.S. Government work and is in Phytochem. Anal. 2014, 25,429–438 the public domain in the USA. Isolation of Lycopsamine and Intermedine the column using a gradient ﬂow (400 μL/min) of 0.1% formic acid in water Isolera 1 ﬂash chromatography system (Biotage, Charlotte, NC, USA)

(mobile phase A) and acetonitrile (mobile phase B). Thus, mobile phase B and washed with CHCl3. At this stage the column effluent bypassed (3%) was held for 2 min before linearly increasing to 70% by 10 min. After the fraction-collection module of the system to avoid unnecessary holding at 70% for another 5 min the column was re-equilibrated to 3% mo- contamination of the UV detector cell and collection lines. Visual bile phase B over 2 min and held for a further 7 min before the next injection. inspection of the column effluent showed when any more excess water The column effluent was directed into a Velos Pro LTQ mass was flushed from the column and when the petroleum ether was spectrometer (Thermo Scientific, USA) equipped with a heated replaced completely by CHCl3. electrospray ionisation (HESI) source. Mass spectra were acquired using During typical use the packed and conditioned boronated soda glass alternating scans in the positive ion mode over the 15 min chromato- bead column was disconnected from the Isolera system and the column graphic run. The first full scan (m/z 200 – 800) was followed by a data end cap removed. The headspace CHCl3 was drained down to the sur- dependent, collision-induced dissociation (CID) scan using a generic face of the packed glass bead column and a solution of reduced extract

CID energy of 32%, Activation Q of 0.25 and an Activation Time of 10.0 of S. officinale (ca. 4 g) in CHCl3 (15 mL) was added slowly to the head of ms. The ionisation spray voltage was set at 3.45 kV, the source heater the column. The slow addition ensured effective exposure of the temperature at 305°C and the sheath gas flow was 40 units with an alkaloids to the aqueous borax film coating the glass beads such that auxiliary flow of 5 units. The capillary temperature was set at 275°C. the orange–red band occupied the first 1.5–3 cm of the column by the end of the addition. Chloroform (ca. 20 mL) was added to the head of the column, the top end cap was refitted and the column was connected

Extraction and isolation of crude alkaloid mixtures from to the Isolera system. The column was washed with CHCl3 at 2 mL/min. Symphytum officinale The first 100 mL was collected separately followed by up to 140 × 22 mL fractions using the Isolera 1 fraction collector. The flow rate was then Dry comfrey (Symphytum officinale) root powder (ca. 5 kg) was Soxhlet- increased to 10 mL/min and ca. 10 fractions of 900 mL were collected extracted with methanol (15 L) for 4 × 20 h and 1 × 60 h. After each 20 separately. The separation was monitored using HPLC–ESI(+)/MS. h extraction, the extract-loaded solvent was exchanged for fresh solvent Decreased separation efficiency after repeated use indicated a need for to decrease any degradation of DHPAs in the boiling methanol. The progress of the extraction was monitored using HPLC–ESI(+)/MS. cleaning and regeneration of the column. To regenerate the column, the glass beads were removed and efficiently cleaned by washing with metha- Each extract was separately evaporated to near dryness, affording a nol. Once thoroughly dried of all methanol, the clean beads were re-coated red oil–solid mixture that was partitioned between n-butanol (500 mL) with aqueous borax and repacked into the column as described. and 0.05 M sulphuric acid (250 mL). The pale orange aqueous acid layer was separated from the deep red n-butanol layer. The n-butanol layer was back-washed with 0.05 M sulphuric acid (2 × 250 mL). The Preparation of N-oxides partitioning and washes were monitored using HPLC–ESI(+)/MS. The combined aqueous acid fraction was made more acidic (to ca. 0.5 M A sample of lycopsamine (ca. 450 mg, ca. 1.5 mmol) and a sample of sulphuric acid) and stirred with zinc powder. The reduction was intermedine (ca. 700 mg, 2.3 mmol) were each separately dissolved in monitored as usual with HPLC–MS and took about 24 h to be complete. ethanol (10 mL). An aliquot of 30% hydrogen peroxide (5 mL, ca. 44 The filtered reduction product was made basic with ammonium hydrox- mmol) was added to the filtered ethanolic solutions and left to stand ide, until the copious precipitate of zinc hydroxide just redissolved, and at room temperature. The reaction solutions became slightly cloudy with fi was then continuously extracted with CHCl3 using a 2 L downward a ne white precipitate or suspension. There was a slight pressure build- displacement liquid–liquid extractor unit (Sigma-Aldrich). The extracting up that was released every hour or so for the first 4–5 h and then every – solvent was replaced after 20 h. The resultant, pale yellow CHCl3 extract 24 h. The progress of the reactions was monitored using HPLC ESI(+)/MS solution was dried over anhydrous sodium sulphate and evaporated to and MS/MS. dryness to afford a red–orange gum (ca. 16 g combined yield from the After about a week, the slightly cloudy solutions were evaporated un- repeat extractions) that was redissolved in CHCl3 (60 mL) and stored at der reduced pressure to remove most of the ethanol. The aqueous resi- 4°C until used. due from each reaction was diluted with 0.05 M sulphuric acid (ca. 10 mL) and each applied to a separate, conditioned SCX SPE cartridge. The loaded columns were treated as usual (Colegate et al., 2012) by washing Semi-automated flash column chromatography of alkaloidal with water and methanol before eluting the components using 10% fractions saturated ammoniated methanol. All column manipulations were monitored using HPLC–ESI(+)/MS. Soda glass beads (0.09–0.135 mm, ca. 1 kg) (Thomas Scientific, fi Swedesboro, NJ, USA) in a screw-cap media storage bottle (2 L) were The rst basic methanol fraction (ca. 20 mL) from each column was treated with 5% w/v aqueous borax (300 mL) by roller-mixing for 20 h immediately evaporated to dryness to afford lycopsamine-N-oxide (402 to ensure effective coating of the beads. The moistened beads were mg, 85% yield) and intermedine-N-oxide (622 mg, 84% yield), each as a added in small aliquots to an ACE Large Chromatography column white glassy foam. The foam structure was readily broken using a spatula (600 × 50 mm; Supelco/Sigma-Aldrich, Bellefonte, PA, USA) with a to yield white solids for each alkaloid N-oxide. bottom-end fitting that included a flow valve (adapter, filter, ptfe, #50 ace-thred to 1/4in-28unf, flow valve, 100 micron polyethylene filter, fetfe o-ring; ACE Glass, Vineland, NJ, USA) and an appropriate top-end fitting Results and discussion (adapter, filter, ptfe, #50 ace-thred to 1/4in-28unf, 100 micron polyethylene filter, fetfe o-ring; ACE Glass) and containing petroleum ether. After Extraction and concentration of comfrey root DHPAs each addition, the clumped aliquot of coated beads was carefully In contrast to an earlier study of S. officinale that reported the compressed using a plunger made by screwing a rubber stopper (slightly isolation of the open chain diester DHPAs symlandine, symphytine smaller diameter than the column) to a long wooden handle (60 cm). and echimidine (Kim et al., 2001), the present HPLC–ESI(+)/MS anal- This packing process was continued until all the beads were used, ysis of the crude methanol extract of S. officinale root powder producing the final packed column (550 × 45 mm). The column was washed with petroleum ether until no more water was visible in the showed the major presence of the N-oxides and free base forms column effluent, at which stage the petroleum ether was drained to of the monoester DHPAs lycopsamine, intermedine and their acet- the surface of the column and the headspace then topped up with ylated derivatives. There was a minor presence of the symlandine-

CHCl3. After ﬁtting the column top-end cap, the column was connected like isomers and their N-oxides (Fig. 2, Table 1). The MS/MS data to a ﬂow- (composition and rate) and fraction-collection-programmable (Table 1) for the acetylated derivatives that, under these 431

Phytochem. Anal. 2014, 25, 429–438 Published 2014. This article is a U.S. Government work and is in wileyonlinelibrary.com/journal/pca the public domain in the USA. S. M. Colegate et al.

100 4 90 80 A 6 70 3 * 60 50 40 30 20 5 10 1 2 8 0 2 90 80 B 1 70 5 60 50 40 * 30 20 3 10 4 7 0 1 2 3 4 5 6 7 8 9 10 11 12 13

Figure 2.TheHPLC–ESI(+)/MS base ion (m/z 200–800) chromatograms for (A) the crude methanol extract and (B) the zinc/acid-reduced extract of dried, powdered roots of Symphytum ofﬁcinale. Peaks labelled with ‘*’ are the internal standard, lasiocarpine. Peak identities are: 1, intermedine;2, lycopsamine; 3, intermedine-N-oxide; 4, lycopsamine-N-oxide; 5, acetylated lycopsamine/intermedine; 6, acetylated lycopsamine/intermedine-N-oxides; 7, symlandine- like isomers; 8, symlandine-like N-oxide isomers. The MS and MS/MS data are shown in Table 1.

Table 1. MS and MS/MS data for dehydropyrrolizidine alkaloids and N-oxides in a methanol extract of dried comfrey (Symphytum ofﬁcinale) root

Alkaloid MH+ m/z MS/MS m/z (% abundance) Symlandine isomers 382 382 (1.6), 338 (1.6), 300 (1), 238 (2), 220 (15.9), 138 (1), 121 (1.3), 120 (100), 118 (2) Symlandine isomers N-oxides 398 (100)/795(20) 398 (100), 380 (7), 354 (32.5), 308 (11.4), 298 (2.8), 292 (1.9), 282 (1.5), 254 (68.6), 237 (3), 220 (7.8), 218 (1.4), 154 (1.8), 137 (2.6), 136 (1.9), 120 (1.7) Acetylated lycopsamine/intermedine 342 198 (2), 180 (76.6), 162 (6.3), 124 (1), 120 (100), 118 (1.4), 103 (1.7) Acetylated lycopsamine/intermedine N-oxides 358(40)/715(100) 358 (62.4), 340 (9.6), 315 (3.4), 314 (31.3), 298 (2.5), 296 (1.8), 268 (14.5), 252 (4), 242 (2.2), 214 (100), 197 (4), 180 (6.5), 178 (5.7), 154 (1.9), 137 (4.7), 136 (2.9), 120 ( 2.9) Lycopsamine/intermedine 300 300 (2.7), 256 (2.8), 210 (1.3), 156 (6.8), 138 (100), 120 (14.8), 94 (27.9) Lycopsamine/intermedine N-oxides 316 (30)/ 631 (100) 316 (55.3), 298 (4.7), 272 (22.8), 254 (1.8), 226 (18.3), 210 (3), 172 (100), 156 (1.7), 155 (6.9), 154 (4.8), 138 (15), 137 (4.6), 136 (7.3), 111 (1.6), 94 (3.4)

chromatographic conditions, effectively co-eluted, indicated that dilute aqueous acid. The resultant red–brown aqueous suspen- the acetylation was at the C7 hydroxyl (rather than the C13 hy- sion–mixture was quite intractable with respect to further droxyl) by virtue of the strong ion at m/z 180 (Colegate et al., 2005). filtration, and therefore, after trialling several solvents, it was The major problem associated with the Soxhlet methanol partitioned with water-saturated n-butanol. The deep-red extraction of bulk, powdered root material from S. officinale is pigments, along with some of the DHPAs, partitioned into the the presence of large amounts of white solid and deep red n-butanol. Monitoring using HPLC-ESI/MS showed that a subse- pigmented co-extractives. Some of this solid was able to be quent wash of the n-butanol fraction with 0.05 M sulphuric acid removed by filtration as the bulk methanol extract was concen- recovered the DHPAs. Two approaches were investigated for trated in vacuo. Further concentration of the deep red filtrate the recovery of DHPAs from the combined aqueous acid 432 afforded a red oily residue that was subsequently extracted with fractions: (i) strong cation exchange (SCX) capture of DHPAs

wileyonlinelibrary.com/journal/pca Published 2014. This article is a U.S. Government work and is in Phytochem. Anal. 2014, 25,429–438 the public domain in the USA. Isolation of Lycopsamine and Intermedine and N-oxides followed by indigo-carmine-based redox resin-medi- was possible using the more usual Biotage column cartridges. ated reduction of the N-oxides; (ii) zinc/acid reduction of the N-ox- Maintaining a narrow band of applied DHPA extract and ides followed by liquid–liquid partitioning of the free base DHPAs. subsequent slow elution flow rates (2–5 mL/min) was essential The SCX-based capture of DHPAs and their N-oxides directly for efficient, differential partitioning of the various DHPAs along from the aqueous acid fraction was a scaled-up version of similar the column length. Consequently the chromatographic runs analytical applications (Betteridge et al., 2005) and required often exceeded 24 h duration. pumping of large volumes of aqueous acid solution through The first eluate collection of 100 mL, every fifth fraction large columns (70 × 160 mm) of the silica-based, bulk SCX resin. collector tube, and each of the 900 mL final wash fractions were Issues associated with monitoring column overloading compli- monitored using HPLC–ESI/MS (Fig. 3). The bulk of the colour cated this approach with uncertainty. However, once the eluted first, overlapping with elution of the symlandine-like columns were at capacity, they were washed and the DHPAs isomers and the acetylated derivatives of 1 and 2. Then, and N-oxides eluted as usual with ammoniated methanol. intermedine (2) eluted over a relatively tight band compared Evaporation of the solvent and reconstitution of the residue in with the subsequent elution of lycopsamine (1). Fractions were methanol was followed by treatment with indigo-carmine-based pooled based upon their constituents. Thus, evaporation of redox resin to effect reduction of the N-oxides. However, and fraction collector tubes 5–28 afforded a mixture of acetylated despite high-efficiency recovery of DHPAs with this method, derivatives, the symlandine isomers and intermedine as a red the slow rate of application combined with the limited capacity oil (0.8 g). Fractions 29–75 yielded intermedine as a pale yellow of the column and the consequent need for regular monitoring oil (0.87 g). Fractions 76–129 yielded a mixture of intermedine of the column effluent to prevent overloading, rendered the and lycopsamine as a yellow oil (0.6 g) and fractions 130–140 whole process quite slow for a large-scale preparation. plus the first six of the 900 mL fractions afforded lycopsamine The simplest approach, requiring less ‘hands-on’ and less time, as a white foamy glass (1.4 g) (Fig. 3). The very slow rate of was indeed the conventional liquid–liquid partitioning of the elution of lycopsamine with respect to intermedine presumably aqueous acid-solubles after treatment with zinc dust to reduce reflects a stronger boronate complex of the former. the N-oxides. Implementation of a continuous liquid–liquid Those fractions containing both 1 and 2 were variously extractor instead of the repeated extractions using a separating combined and re-chromatographed. The column is remarkably funnel expedited this approach. An overnight extraction of the stable and reusable. In one instance a column was prepared, basified solution of reduction products with CHCl3 using the used once then allowed to stand for 4 months before it was then liquid–liquid extractor was sufficient to recover most if not all reused another eight times over 5 months. The column does the DHPAs without any significant degradation of extracted retain some colour and, with repeated use, the efficiency of DHPAs in the boiling CHCl3 for this time. The HPLC–ESI(+)/MS separation of intermedine from lycopsamine reduces, indicating analysis showed that the reduced comfrey root extract that the column requires cleaning and regeneration. comprised mainly intermedine and lycopsamine (MH+ m/z 300) It was found that boronated quartz sand, treated in the same along with their acetylated derivatives (MH+ m/z 342) and minor way as the soda glass beads, performed just as well as the amounts of the open chain diester symlandine-like isomers boronated soda glass beads and so provides an alternative (MH+ m/z 382) (Fig. 1). matrix. However, most of the data and observations relating to stability, cleaning, regeneration and reuse of the column reported herein are derived from the soda glass columns. Semi-automated purification of lycopsamine (1) and intermedine (2) NMR comparison of lycopsamine stereoisomers 1–4 After a careful consideration of many options for separating lycopsamine (1) and intermedine (2) from co-extracted DHPAs Examination of the one- (1H and 13C) and two-dimensional and from each other, it was decided to reinvestigate the use of (COSY and/or HSQC) NMR spectroscopy data for lycopsamine boronated complexes. Despite the lack of literature reports (1), intermedine (2), echinatine (3) and rinderine (4) enabled describing its use following the initial description by Frahn unambiguous confirmation of structures and assignment of et al. (1980), their differential partitioning approach using resonance signals (Tables 2 and 3) (see online Supporting crushed soda glass, rather than their ion exchange approach, information for copies of the NMR spectra). Although most of was considered the most straightforward and more likely to the assignments were consistent with previous literature reports be amenable to semi-automated, larger scale (ca. ≥ 1g) (Jones et al., 1982; Zalkow et al., 1985; Asibal et al., 1989; purifications using a standardised treatment of a standardised Wiedenfeld and Roeder, 1991), the current two-dimensional matrix (soda glass beads or quartz sand). NMR experiments enabled an unambiguous reassignment for Commercial flash chromatography systems, such as the the methyl carbons 14, 16 and 17 of 1–4 relative to published Biotage Isolera 1 used in this study, offer the ability to pump data (Jones et al., 1982; Asibal et al., 1989; Wiedenfeld and various solvents in controlled isocratic or gradient elution modes Roeder, 1991). through packed chromatographic columns. Eluant fractions can The structures and absolute configurations of 1–4, in particu- then be automatically collected based on the most effective lar those of the esterifying viridifloric and trachelanthic acids, parameters. As the DHPAs lack a strong chromophore that have been confirmed using X-ray diffraction crystallography or would allow for sensitive detection in real time, equivolume by synthesis (Mackay et al., 1983; Zalkow et al., 1985; Asibal fractions were collected and then analysed post-run using et al., 1989). A comparison of the 1H- (Fig. 4) and 13C- (Fig. 5) HPLC–ESI(+)/MS, usually in overnight, automated analytical NMR spectra of the four diastereoisomers highlights some sequences. potentially diagnostic chemical shift differences within the two The use of the large glass column, adapted to the Biotage pairs of C13 epimers (i.e. 1/2 and 3/4) and the two pairs of C7 Isolera 1 system, allowed for a higher scale of separation than epimers (i.e. 1/3 and 2/4). The former relates to the difference 433

Phytochem. Anal. 2014, 25, 429–438 Published 2014. This article is a U.S. Government work and is in wileyonlinelibrary.com/journal/pca the public domain in the USA. S. M. Colegate et al.

Figure 3. The HPLC–ESI(+)/MS ion chromatograms (left column) of pooled fractions from semi-automated separation of dehydropyrrolizidine alkaloids on boronated soda glass beads. In the ion chromatograms, the peak marked with an ‘*’ is the internal standard, lasiocarpine. Also shown are the MS/MS data (right column) for the major components (see also Table 1).

Table 2. 1H-NMR spectroscopy data for the diastereoisomeric monoester dehydropyrrolizidine alkaloids lycopsamine (1), intermedine (2), echinatine (3) and rinderine (4)a

Hb Lycopsamine Intermedine Echinatine Rinderine 2 5.92, s 5.93, s 5.65, s 5.67, s 3d 3.96, sm 3.92, bd, J = 15.5 3.91, bdm 3.86, sm 3u 3.33, m 3.26, m 3.29, bdm 3.29, bdm 5d 3.44, dm 3.41, dm 3.21, dt, Jd = 10.6, Jt = 6.3 ca. 3.2, m 5u 2.77, dm 2.72, dm 2.59, bm 2.58, dm 6d ca. 2.0, sm 1.96, sm 1.91, m 1.94, sm 6u ca. 2.0, sm 1.96, sm 1.80, m 1.80, m 7 4.32, bs 4.25, bm 4.11, td, Jd = 3.7, Jt = 5.8 4.10, sm 8 4.17, bs 4.16, bm ca. 3.96, sm 3.87, sm 9d 4.86, d, J = 13.6 4.83, d, J = 12.7 5.01, d, J = 13.2 4.91, d, J = 13.0 9u 4.76, d, J = 14.2 4.75, d, J = 12.7 4.78, d, J = 13.5 4.84, d, J = 13.0 13 ca. 4.0, sm 4.08, q, J = 6.4 ca. 3.95, sm 4.06, sm 14 1.25, d, J = ca. 9 1.18, d, J = 6.4 1.29, d, J = 6.6 1.18, d, J = 6.4 15 2.16, sept, J = 6.8 2.02, sept, J = 6.9 2.16, sept, J = 6.8 2.02,sm 16 0.93, d, J = 6.8 0.92, d, J = 6.9 0.90, d, J = 6.8 0.92, d, J = 6.9 17 0.88, d, J = 6.8 0.91, d, J = 6.9 0.85, d, J = 6.7 bd = broad doublet; bdm = broad doublet of multiplets; bm = broad multiplet; bs = broad singlet; d = doublet; dm = doublet of multiplets; dt = doublet of triplets; m = multiplet; q = quartet; s = singlet; sept = septet; sm = superimposed multiplet; td = triplet of doublets. aAll assignments were supported by two-dimensional COSY and/or HSQC experiments and the spectra are included online as Supporting information. b‘ ’ ‘ ’ ﬁ ﬁ 434 u and d represent the up eld and down eld protons of a geminal pair.

Table 3. 13C-NMR spectroscopy data for the diastereoiso- in the esterifying acid, whereas the latter is the difference meric monoester dehydropyrrolizidine alkaloids lycopsamine between the heliotridine and retronecine-based DHPA (1), intermedine (2), echinatine (3) and rinderine (4)a monoesters. From the 1H-NMR data, an R configuration about C13 is C Lycopsamine Intermedine Echinatine Rinderine indicated by a slight upfield chemical shift of the C14 methyl protons of 2 (δ 1.18) and 4 (δ 1.18) relative to those with an S 1 132.74 132.69 136.23 136.13 configuration, that is, 1 (δ 1.24) and 3 (δ 1.29). Additionally, the 2 130.08 131.02 125.88 126.73 C17 methyl protons on the iso-propyl group are shifted slightly 3 62.85 62.88 62.12 61.86 downfield in 2 (δ 0.91) and 4 (δ 0.92) relative to those with an 5 53.94 53.95 54.32 54.2 S configuration, that is, 1 (δ 0.88) and 3 (δ 0.85) such that the 6 36.3 36.32 33.74 33.9 signal is superimposed (i.e. 4) or barely resolved (i.e. 2) from their 7 71.11 70.87 74.46 74.87 geminal C16 methyl protons. It has been reported that a 8 78.52 78.78 79.81 79.88 difference in chemical shift (Δδ) of the C9 protons of 0–0.2 9 62.65 62.56 62.0 62.45 ppm indicates a C12 S configuration, whereas a Δδ of ≥ 0.25 11 174.54 175.41 174.09 175.37 ppm indicates a C12 R configuration (Wiedenfeld and Roeder, 12 83.71 83.16 84.18 83.49 1991). This general rule is supported by the H9 Δδ observed 13 71.06 69.3 71.67 69.33 for 1, 2 and 4 but, with a Δδ of 0.23 ppm, is ambiguous for 3 14 17.31 17.33 17.34 17.25 (Table 2). Although the Δδ of the C9 protons between the C13 15 32.46 33.09 32.33 33.08 S and R epimers is very pronounced for the heliotridine-based b 16 16.04 17.21 15.82 17.19 epimeric pair 3 and 4, it is not so for the retronecine-based b 17 17.80 17.02 17.93 17.07 C13 epimers 1 and 2 (Fig. 4) and thus should be considered aAll assignments were supported by two-dimensional COSY cautiously and only in concert with other NMR stereochemical and/or HSQC experiments and the spectra are included indicators. A clear resolution of the C14, C16 and C17 methyl carbons in online as Supporting information. 13 bAssignments based on comparison with intermedine but the C-NMR spectrum was observed with the C13 S epimers 1 can be interchanged. and 3 compared with the C13 R epimers 2 and 4 (Fig. 5). This improved resolution is due to a significant downfield shift of

A heliotridine C13 S 14 16, 17

2 9d 9u 7 8 5u 15 6d,u

14 16, 17 B heliotridine C13 R 2 9d, 9u 7 8 5u 15 6d,u

14 16, 17 C retronecine C13 S 2 9d, 9u 6d,u 7 8 5u 15

14 16, 17 D retronecine C13 R 9d, 9u 2 7 8 5u 15 6d,u

E heliotridine C13 R

6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 f1 (ppm)

Figure 4. The 1H-NMR spectra for: (A) echinatine (3), (B) rinderine (4), (C) lycopsamine (1), (D) intermedine (2) and (E) heliotrine (5). For assignments see Table 2, but some key assignments described in the text are annotated on the spectra. Also annotated for each spectrum are the necine base and C13 absolute stereochemistry for ease of comparison. 435

Phytochem. Anal. 2014, 25, 429–438 Published 2014. This article is a U.S. Government work and is in wileyonlinelibrary.com/journal/pca the public domain in the USA. S. M. Colegate et al.

A heliotridine C13 S 1 2 8 7 17, 14, 16 13 6

heliotridine C13 R 1 B 8 14, 16, 17 2 7 13 6

13, 7 17, 14, 16 8 6 C retronecine C13 S 1 2

retronecine C13 R 14, 16, 17 1 D 8 7 13 6 2

E heliotridine C13 R

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 f1 (ppm)

Figure 5. The 13C-NMR spectra for: (A) echinatine (3), (B) rinderine (4), (C) lycopsamine (1), (D) intermedine (2) and (E) heliotrine (5). For assignments see Table 3, but some key assignments described in the text are annotated on the spectra. Also annotated for each spectrum are the necine base and C13 absolute stereochemistry for ease of comparison.

C17 and a significant upfield shift of C16, while the chemical (δ ca. 132.72, 78.65, 71, 130.5 and 36.3 respectively) (Fig. 5, shift of C14 remained fairly similar for all of the diastereoisomers Table 3), may also be diagnostic for the heliotridine-based (Table 3). Additionally, the 13C-NMR chemical shift of C13 is alkaloids including, as shown in this study, heliotrine. diagnostically downfield for the C13 S-configured lycopsamine (1) and echinatine (3), that is, δ 71.06 and 71.67 respectively, NMR comparison of the N-oxides of lycopsamine and compared with the C13 R-configured intermedine (2) and intermedine rinderine (4), that is, δ 69.3 and 69.33 respectively. The applicability of these observations is limited to the Despite the increased water solubility imparted to a DHPA by N- esterified viridifloric and trachelanthic acid epimers, because oxidation, the N-oxides of both lycopsamine and intermedine 1 13 extrapolation to, for example, the H- (Fig. 4) and C- (Fig 5) were remarkably soluble in CHCl3. This meant that the more NMR spectra of heliotrine (5) (Fig. 1) would result in incorrect usual approach to synthesising these N-oxides, that is, using a conclusions. Heliotrine has been shown to be an ester of solution of ethanol and CHCl3 from which the newly formed N- heliotric acid (methylated trachelanthic acid), that is, with a oxide precipitates (Molyneux et al., 1991), was not applicable. C12 S/C13 R configuration (Kochetkov et al., 1969). However, Therefore, the reaction solutions were processed using SCX isolated from other considerations, the large Δδ (ca. 0.44 ppm) SPE from the aqueous acid-soluble fraction of the reaction of the C9 protons would indicate an incorrect C12 R configura- products. The 1H- and 13C-NMR data for lycopsamine-N-oxide tion. Furthermore, the downfield shift of C13 (δ ca. 78) and the (Table 4), acquired using a solution in CDCl3, are similar to clear resolution of the C16 and C17 methyl protons might published data acquired using D2O (Roeder and Bourauel, indicate a C13 S configuration rather than the established R 1992) and 10% D2O in CDCl3 (Nishida et al., 1991). Apart from configuration. differences in some chemical shifts (up to about 2.7 ppm in Potentially diagnostic for the configuration of the necine base the 13C data and about 0.24 ppm in the 1H data), presumably are the chemical shifts of H2, H7, H8 and H6u that in the related to solvent effects, the major apparent discrepancies heliotridine-based 3 and 4 are all significantly upfield of their involve the current observation of a singlet and a broad retronecine counterparts in 1 and 2 (Fig. 4, Table 2). Further- multiplet for the geminal protons on C3 and C5, respectively, more, significant downfield shifts of C1(δ ca. 136), C8 (δ ca. compared with two doublets when recorded in D2O and a 79.85) and C7 (δ ca. 74–75), and upfield shifts of C2 (δ ca. doublet (C3 protons) and broad multiplet (C5 protons) when 436 126.3) and C6 (δ ca. 33.8), relative to their retronecine analogues recorded in 10% D2O in CDCl3. In addition, when acquired in

Table 4. 1H- and 13C-NMR spectroscopy data for the N-oxides of the epimeric monoester dehydropyrrolizidine alkaloids lycopsamine (1) and intermedine (2)a

Carbonb Lycopsamine-N-oxide Intermedine-N-oxide 1H 13C 1H 13C 1 NA 132.89 NA 132.95 2 5.79 122.30 5.80 (s) 122.25 3d 4.43 (s, 2H) 78.21 4.42 (s, 2H) 78.14 3u 5d 3.77 (bm, 2H) 69.51 3.72 (m, 2H) 69.38 5u 6d 2.58 (bm) 34.72 2.55 (m) 34.79 6u 2.01 (bm) 1.98 (bd, J6u,6d = 14) 7 4.70 (sbs) 69.89 4.66 (bs) 69.62 8 4.77 (sbs) 96.43 4.76 (bs) 96.0 9d 4.86 (d, J9d.9u = ca. 12) 61.50 4.84 (s, 2H) 61.4 9u 4.76 (sd, J9u.9d = ca. 12) 11 NA 174.21 NA 174.93 12 NA 84.70 NA 84.22 13 3.86 (q, J13,14 = 6.6) 72.87 4.11 (q, J13,14 = 6.3) 70.08 14 1.24 (d, J14,13 = 6.5) 17.31 1.17 (d, J14,13 = 6.4) 16.80 15 2.14 (sept, J15,16/17 = 6-7) 32.33 1.85 (sept, J15,16/17 = 6-7) 33.92 16 0.89 (d, J16,15 = 6.7) 15.65 0.97 (d, J16,15 = 7) 17.25 17 0.84 (d, J17,15 = 6.7) 18.18 0.91 (d, J17,15 = 6.8) 17.75 NA = not applicable; bd = broad doublet; bm = broad multiplet; bs = broad singlet; d = doublet; m = multiplet; q = quartet; s = singlet; sbs = superimposed broad singlet; sd = superimposed doublet; sept = septet. aAll assignments were supported by two-dimensional COSY and/or HSQC experiments and the spectra are included online as Supporting information. b‘u’ and ‘d’ represent the upﬁeld and downﬁeld protons of a geminal pair.

22 10% D2O in CDCl3 the C7 and C8 protons are reported as a single (Frahn et al., 1980). Luber et al. (2012) report [α]D = +12.0° peak. However, despite these differences, the current assign- (c = 0.01, methanol) for intermedine-N-oxide, but there do not ments based on the two-dimensional homonuclear (COSY) and seem to be values reported for lycopsamine-N-oxide. Further- heteronuclear (HSQC) correlation experiments were in accord more, Souza et al. (2005) isolated lycopsamine from Heliotropium 20 with previous data and confirmed the structure. indicum and reported [α]D = +0.12° (c = 0.05, chloroform). 22 There seems to be only two reports of NMR data for In the present study the [α]D for lycopsamine, intermedine, intermedine-N-oxide, one of a synthetic intermedine-N-oxide lycopsamine-N-oxide and intermedine-N-oxide were: À1.4° (Nishimura et al., 1987) and one of a natural compound isolated (c = 0.98, methanol), +3.7° (c = 0.81, methanol), +10.6° (c = 1.04, from Cerinthe glabra (Luber et al., 2012). Using two-dimensional methanol), +7.3° (c = 0.98, methanol) respectively. Obviously, heteronuclear NMR experiments, including HMQC, Luber et al. despite the MS and NMR evidence for lycopsamine herein, the (2012) were able to correct some of the earlier assignments by levorotatory value for the lycopsamine solution was a concern Nishimura et al. (1987), however, they could not unequivocally because all reported values have been dextrorotatory, even the assign C7 and C13. The current NMR data (Table 4) were similar very small value determined for the chloroform solution of to those of Luber et al. (2012) except that the C9 protons lycopsamine (Souza et al., 2005). Therefore, to investigate poten- resonated as a single peak (evident in both the 1H and HSQC tial levorotatory contamination of the lycopsamine sample, spectra) at 4.84 ppm in contrast to the doublet and a multiplet specific rotations were determined for another two samples of at 4.84 and 4.97 ppm, respectively, observed by Luber et al. lycopsamine isolated using the boronated soda glass bead (2012). However, the use of the HSQC experiment for determin- column and for lycopsamine isolated from a lycopsamine ing direct C–H coupling allowed for a higher resolving power in chemotype of Amsinckia intermedia (Colegate et al., 2013) that the 13C direction than the HMQC experiment used by Luber et al. had not been exposed to the borax solutions. In each case the 22 (2012) (Mandal and Majumdar, 2004) and, thereby, confirmed [α]D was À1.56° (c = 2.5, methanol), À1.5° (c = 1.4, methanol) their tentative assignment of C3 and C13. and À1.8° (c = 1.6, methanol), respectively, thereby confirming the levorotatory optical rotation of a methanolic lycopsamine solution. Optical rotation observations Subsequently, to better compare with literature reports, the Reported measurements of specific rotation ([α]) for ethanolic lycopsamine and intermedine were dissolved in ethanol. solutions of lycopsamine vary from +1.2° (Zalkow et al., 1985) Somewhat surprisingly, despite the recognition that the magni- to +5.7° (Frahn et al., 1980). For intermedine, also in ethanolic so- tude, and sometimes the sign, of the optical rotation is affected lution, they vary from +4.8° (Culvenor and Smith, 1966) to +9.8° by both solvent and temperature (Gottarelli et al., 1991), there 437

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was a significant dextrorotatory effect on the optical rotations Jones AJ, Culvenor CCJ, Smith LW. 1982. Pyrrolizidine alkaloids – a measured and, consequently, the specific rotations calculated carbon-13 N.M.R. study. Aust J Chem 35: 1173–1184. Kelley RB, Seiber JN. 1992. Pyrrolizidine alkaloid chemosystematics in when changing the solvent from methanol to ethanol. Thus for Amsinckia. Phytochemistry 31: 2369–2387. α 22 lycopsamine [ ]D = +3.75° (c = 2.4, ethanol) and for intermedine Kim N-C, Oberlies NH, Brine DR, Handy RW, Wani MC, Wall ME. 2001. 22 [α]D = +6.27° (c = 0.75, ethanol). The change in sign of the optical Isolation of symlandine from the roots of common comfrey rotation for lycopsamine was specifically confirmed by evaporat- (Symphytum officinale) using countercurrent chromatography. J Nat – ing the ethanolic solution to dryness under a stream of nitrogen, Prod 64: 251 253. Kochetkov NK, Likhosherstov AM, Kulakov VN. 1969. The total synthesis and redissolving the residue in methanol for measurement of its of some pyrrolizidine alkaloids and their absolute configuration. optical rotation. Tetrahedron 25: 2313–2323. Luber M, Musa A, Kadry HA, Bracher F. 2012. Isolation of the pyrrolizidine Acknowledgements alkaloid intermedine-N-oxide from Cerinthe glabra and ab initio calculation of its 13C NMR shifts. Z Naturforschung 67B: 411–416. The authors thank the anonymous reviewers for their comments, Mackay MF, Sadek M, Culvenor CCJ. 1983. Lycopsamine and intermedine, suggestions and constructive critique. C15H25NO5: Diastereoisomeric pyrrolizidine alkaloids. Acta Cryst C39: 785–788. Mandal PK, Majumdar A. 2004. A comprehensive discussion of HSQC and References HMQC pulse sequences. Concepts Magn Resonan A 20A:1–23. Molyneux RJ, Johnson AE, Olsen JD, Baker DC. 1991. Toxicity of Adams R, Hamlin Jr KE, Jalinek CF, Phillips RF. 1942. The structure of pyrrolizidine alkaloids from Riddell groundsel (Senecio riddellii)to riddelliine, the alkaloid in Senecio riddellii.I.J Am Chem Soc 64: cattle. Amer J Vet Res 52: 146–151. – 2760 2763. Molyneux RJ, Gardner DR, Colegate SM, Edgar JA. 2011. Pyrrolizidine Asibal CF, Glinski JA, Gelbaum LT, Zalkow LH. 1989. Pyrrolizidine alkaloids alkaloid toxicity in livestock: a paradigm for human poisoning? J Food from Cynoglossum creticum. Synthesis of the pyrrolizidine alkaloids Additiv Contamin 28: 293–307. – echinatine, rinderine, and analogues. J Nat Prod 52: 109 118. Nishida R, Kim C-S, Fukami H, Irie R. 1991. Ideamine N-oxides: Betteridge K, Cao Y, Colegate SM. 2005. An improved method for extrac- Pyrrolizidine alkaloids sequestered by the Danaine butterfly, Idea tion and LCMS analysis of pyrrolizidine alkaloids and their N-oxides in leuconoe. Agric Biol Chem 55: 1787–1792. honey: Application to Echium vulgare honeys. J Agric Food Chem 53: Nishimura Y, Kondo S, Takeuchi T, Umezawa H. 1987. Total syntheses of – 1894 1902. indicine N-oxide, intermedine N-oxide, and their enantiomers using Bull LB, Culvenor CCJ, Dick AT. 1968. In The Pyrrolizidine Alkaloids: Their carbohydrate as a chiral educt. Bull Chem Soc Jpn 60: 4107–4113. Chemistry, Pathogenicity and other Biological Properties. North-Hol- Roeder E, Bourauel T. 1992. Pyrrolizidine alkaloids from Neatostema land: Amsterdam. apulum. Phytochemistry 31: 3613–3615. Colegate SM, Edgar JA, Knill AM, Lee ST. 2005. Solid phase extraction and Roeder E, Breitmaier E, Birecka H, Frohlich MW, Badzies-Crombach A. – fi LC MS pro ling of pyrrolizidine alkaloids and their N-oxides: a case 1991. Pyrrolizidine alkaloids of Heliotropium spathulatum. Phytochem- – study of Echium plantagineum. Phytochem Anal 16: 108 119. istry 30: 1703–1706. Colegate SM, Gardner DR, Joy RJ, Betz JM, Panter KE. 2012. Roitman JN. 1983. The pyrrolizidine alkaloids of Amsinckia menziesii. Aust Dehydropyrrolizidine alkaloids, including monoesters with an J Chem 36: 769–778. unusual esterifying acid, from cultivated Crotalaria juncea (Sunn Souza JSN, Machado LL, Pessoa ODL, Braz-Filho R, Overk CR, Yao P, ‘ ’ – Hemp cv. Tropic Sun ). J Agric Food Chem 60: 3541 3550. Cordell GA, Lemos TLG. 2005. Pyrrolizidine alkaloids from Colegate SM, Gardner DR, Davis TZ, Welsh SL, Betz JM, Panter KE. 2013. Heliotropium indicum. J Braz Chem Soc 16: 1410–1414. fi Identi cation of a lycopsamine-N-oxide chemotype of Amsinckia Wiedenfeld H, Roeder E. 1991. Pyrrolizidine alkaloids from Ageratum – intermedia. Biochem Syst Ecol 48: 132 135. conyzoides. Planta Med 57: 578–579. Cooper RA, Bowers RJ, Beckham CJ, Huxtable RJ. 1996. Preparative Williams MT, Warnock BJ, Betz JM, Beck JJ, Gardner DR, Lee ST, Molyneux separation of pyrrolizidine alkaloids by high-speed counter-current RJ, Colegate SM. 2011. Detection of high levels of pyrrolizidine-N-ox- – chromatography. J Chromat A 732:43 50. ides in the endangered plant Cryptantha crassipes (Terlingua Creek fi Culvenor CCJ. 1962. Senecio magni cus F. Muell., a source of senecionine. Cat’s-eye) using HPLC–ESI-MS. Phytochem Anal 22: 532–540. – Aust J Chem 15: 158 159. Zalkow LH, Glinski JA, Gelbaum LT, Fleischmann TJ, McGowan LS, Gordon Culvenor CCJ, Smith LW. 1966. The alkaloids of Amsinckia species: A. MM. 1985. Synthesis of pyrrolizidine alkaloids indicine, intermedine, intermedia Fisch. & Mey., A.hispida (Ruiz. & Pav.) Johnst. and A. lycopsamine, and analogues and their N-oxides. Potential antitumor – lycopsoides Lehm. Aust J Chem 19: 1955 1964. agents. J Med Chem 28: 687–694. Edgar JA, Colegate SM, Boppré M, Molyneux RJ. 2011. Pyrrolizidine alkaloids in food: a spectrum of potential health consequences. J Food Additiv Contamin 28: 308–324. Frahn JL, Culvenor CCJ, Mills JA. 1980. Preparative separation of the Supporting information pyrrolizidine alkaloids, intermedine and lycopsamine, as their borate complexes. J Chromatogr 195: 379–383. Gottarelli G, Osipov MA, Spadat GP. 1991. A study of solvent effect on the Supporting information can be found in the online version of optical rotation of chiral biaryls. J Phys Chem 95: 3879–3884. this article. 438

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