Minor Diterpene Glycosides from the Leaves of Stevia rebaudiana

Apr 23, 2014 - (2) S. rebaudiana is a perennial bush indigenous to the mountainous regions of Paraguay and Brazil. The indigenous people of Paraguay a...
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Minor Diterpene Glycosides from the Leaves of Stevia rebaudiana Mohamed A. Ibrahim,†,‡ Douglas L. Rodenburg,† Kamilla Alves,† Frank R. Fronczek,§ James D. McChesney,*,†,⊥ Chongming Wu,⊥ Brian J. Nettles,⊥ Sylesh K. Venkataraman,⊥ and Frank Jaksch⊥ †

Ironstone Separations, Inc., Etta, Mississippi 38627, United States Department of Chemistry of Natural Compounds, National Research Center, Dokki 12622, Cairo, Egypt § Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, United States ⊥ ChromaDex, Inc., Suite G, Irvine, California 92614, United States J. Nat. Prod. 2014.77:1231-1235. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/19/19. For personal use only.

‡

S Supporting Information *

ABSTRACT: Two new diterpene glycosides in addition to five known glycosides have been isolated from a commercial extract of the leaves of Stevia rebaudiana. Compound 1 (rebaudioside KA) was shown to be 13-[(O-β-D-glucopyranosyl)oxy]ent-kaur-16-en19-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester and compound 2, 12-α-[(2-O-β-D-glucopyranosyl-β-Dglucopyranosyl)oxy]ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester. Five additional known compounds were identified, rebaudioside E, rebaudioside M, rebaudioside N, rebaudioside O, and stevioside, respectively. Enzymatic hydrolysis of stevioside afforded the known ent-kaurane aglycone 13-hydroxy-ent-kaur-16en-19-oic acid (steviol) (3). The isolated metabolite 1 possesses the ent-kaurane aglycone steviol (3), while compound 2 represents the first example of the isomeric diterpene 12-α-hydroxy-ent-kaur-16-en-19-oic acid existing as a glycoside in S. rebaudiana. The structures of the isolated metabolites 1 and 2 were determined based on comprehensive 1D- and 2D-NMR (COSY, HSQC, and HMBC) studies. A high-quality crystal of compound 3 has formed, which allowed the acquisition of X-ray diffraction data that confirmed its structure. The structural similarities between the new metabolites and the commercially available stevioside sweeteners suggest the newly isolated metabolites should be examined for their organoleptic properties. Accordingly rebaudiosides E, M, N, O, and KA have been isolated in greater than gram quantities.

company reported that the global market for S. rebaudiana sweetener had reached $500 million and could reach $10 billion in just a few years. The world demand for S. rebaudiana leaves is expected to exceed 6−8 million metric tonnes in the next 10 years.8 Stevioside and rebaudioside A are the major glycoside sweeteners from S. rebaudiana that have been commercialized for human consumption in countries throughout Asia, Europe, and North and South America.9,10 S. rebaudiana and its cultivars have been reported to produce as many as 33 other glycosides containing the same aglycone, steviol.11−17 Examination of both crude and partially processed commercial extracts of S. rebaudiana leaves has shown the presence of numerous currently unidentified metabolites that are very similar in chemical and physical properties and are often present as lowlevel impurities in commercial grade rebaudioside A and stevioside. Their availability as reference standards in process quality control is important. A second significant goal of the present effort was to prepare these minor components in

Stevia rebaudiana (Bertoni) Bertoni (Asteraceae) is a plant well known to produce a mixture of high-potency sweet compounds that have been on the market in Japan since the 1970s as lowcalorie sucrose substitutes.1 According to a recent publication by Ibrahim et al., the family Asteraceae represents a potential source for biologically active compound leads.2 S. rebaudiana is a perennial bush indigenous to the mountainous regions of Paraguay and Brazil. The indigenous people of Paraguay and Brazil have used S. rebaudiana leaves safely for sweetening for centuries.1 S. rebaudiana was first identified and described in 1905 by the botanist Moisés Santiago de Bertoni.3 Cultivars of S. rebaudiana have been selected for enhanced production of selected glycosides and are now grown commercially in mainland China, Japan, Korea, India, and elsewhere.4 The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has established a monograph for steviol glycosides, with the latest version issued in 2010.5 The United States Food and Drug Administration granted generally recognized as safe (GRAS) regulatory acceptance of rebaudioside A, commonly known as “rebiana”, in 2008 and of steviol glycosides in 2010.6 The European Union approved steviol glycosides for marketing in November 2011.7 In 2008, a leading market research © 2014 American Chemical Society and American Society of Pharmacognosy

Received: November 22, 2013 Published: April 23, 2014 1231

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sufficient quantities to make them available for biological evaluation. Minor components of complex natural mixtures such as the glycoside extract of S. rebaudiana leaves are frequently isolated and purified only in sufficient quantities for structure elucidation, which with contemporary instrumental methods usually means 1−3 mg in total. Rather, our strategy has been to isolate these compounds in sufficient quantity to fully characterize them and provide significant residual material for biological evaluation as well as for use as impurity standards. Our initial effort to isolate minor components from a commercial S. rebaudiana leaf extract has yielded two new glycosides (1 and 2) and the previously reported glycosides rebaudioside E, rebaudioside M, rebaudioside N, rebaudioside O, and stevioside.

Figure 1. X-ray structure of compound 3, including the methanol adduct molecule, with ellipsoids at the 50% level (Mercury).34

Table 1. 1H NMR and 13C NMR Spectroscopic Data (pyridine-d5) for Compounds 1 and 2 1a position

1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

13

The H and C NMR data of the isolated compounds 1 and the five known compounds showed a high degree of similarity especially for the signals representing the diterpene core, confirming the presence of the common diterpene ent-kaur-13 hydroxy-16-en-19-oic acid (3). Comparison of the 1H and 13C NMR data of the five known compounds to the previously reported ent-kaurane glycosides established them to be rebaudioside E, rebaudioside M, rebaudioside N, rebaudioside O, and stevioside. Many trials have been conducted to form high-quality crystals of this group of metabolites for X-ray diffraction processing. In the present investigation, the X-ray data of a crystal of compound 3 was used to confirm its structure (Figure 1). A low-temperature structure demonstrated good agreement with the previous room-temperature X-ray determinations18,19 including the absolute configuration, which was previously deduced.19 The 1H and 13C NMR data for compound 1 (Table 1), as well as the high-resolution mass spectrum (m/z 803.3881 for [M − H]−), suggested the molecular formula of C38H60O18 with the presence of three glucose units. The positions of attachment of the three sugars were established based on HMBC data. 3J HMBC correlations between H-1′ (5.12 ppm) and C-13 (87.0 ppm) in compound 1 were used to establish the attachment of the first glucose unit at C-13. The 3J HMBC correlations between H-1″ (6.23 ppm) and C-19 (176.1 ppm) confirmed the attachment of the second sugar at C-19. The position of attachment of the third glucose was established

R1 Glc′-1 Glc′2-1 R2 Glc-1 R3 Glc-1 Glc2-1

δH (J in Hz) 0.76 m, 1.75 m 1.70 m, 2.17 m 1.82 m, 2.14 m

2a δC, type

0.99 s

40.8, 20.2, 38.9, 44.5, 57.6, 22.2, 41.9, 42.2, 54.2, 39.8, 20.7, 38.0, 87.2, 44.8, 48.6, 153.8, 105.5, 29.4, 176.1, 16.5,

6.23 bs 5.10 m

93.8, CH 105.8, CH

5.12 m

99.7, CH

0.99 m 1.91 m, 2.20 m 1.31 m, 1.51 m 0.93 m 1.48 m 2.75 m, 1.10 m 1.94 m, 2.45 m 2.10 m 5.10 m, 5.64 s 1.42 s

CH2 CH2 CH2 C CH CH2 CH2 C CH C CH2 CH2 C CH2 CH2 C CH2 CH3 CO CH3

δH (J in Hz) 0.79 1.38 m, 2.13 m 2.36 m 1.03 m 1.84 m, 2.41 m 2.78 m 1.01 m 1.59 4.11 2.94 1.42 1.91

m bs bs m, 1.05 m m, 2.23 m

4.90 s, 5.24 m 1.26 s 1.04 s

6.18 (d, 8 Hz)

5.01 (d, 7.6 Hz) 5.24 m

δC, type 40.2, 19.7, 39.1, 44.6, 57.9, 21.1, 39.6, 34.9, 51.7, 44.6, 27.3, 78.4, 42.0, 39.9, 49.0, 148.4, 109.5, 29.1, 177.4, 13.3,

CH2 CH2 CH2 C CH CH2 CH2 C CH C CH2 CH CH CH2 CH2 C CH2 CH3 CO CH3

96.4, CH

103.0, CH 106.4, CH

a

Measured at 100 MHz for 13C in methanol-d4, (s) singlet; (d) doublet; (bs) broad singlet; (m) multiplet.

through the 3J HMBC correlation between C-1‴ (105.8 ppm) and H-2″ (4.43 ppm), where the position of H-2‴ was confirmed through the COSY correlation between H-2‴ (4.43 ppm) and H-1″ (6.23 ppm) (Figures S4 and S6, Supporting 1232

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Included were the fungi Candida albicans ATCC 90028, Cryptococcus neoformans ATCC 90113, and Aspergillus f umigatus ATCC 90906 and the bacteria methicillin-resistant Staphylococcus aureus ATCC 43300 (MRS), Escherichi coli ATCC 35218, Pseudomonas aeruginosa ATCC27853, and Mycobacterium intracellulare ATCC 23068. However, the two new compounds and the five known compounds isolated as well as the other known glycosides, rebaudiosides A−D, rebaudioside F, rubusoside, and dulcoside A, were inactive in both the antimalarial and antimicrobial assays used.

Information). This, in turn, confirmed the attachment of the third glucose unit at C-2″. The glucose units were determined to belong to the D-series through the comparison with a standard using an HPLC method reported by Tanaka et al.20 It is proposed to name compound 1 as rebudioside KA, and the structure was fully elucidated as shown. The 1H and 13C NMR data for compound 2 (Table 1), as well as the high-resolution mass (m/z 827.3626 for [M + Na]+), suggested a molecular formula of C38H60O18 with the presence of three glucose units. The closely comparable HMQC and HMBC spectra of compounds 2 and 1 showed a high degree of similarity except in the region from C-8 to C-15, which clearly indicated the presence of a different diterpene aglycone (Figure 2). Key HMBC correlations were observed to

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using an Autopol IV instrument at ambient temperature. NMR spectra were acquired on a Bruker AV NMR spectrometer (Bruker Biospin, Bruker Inc.) operating at 400 (1H) and 100 MHz (13C), and the chemical shift values are expressed in ppm. Multiplicity determinations (DEPT) and 2D-NMR spectra (COSY, HMQC, HMBC, NOESY) were obtained using standard Bruker pulse programs. The mass spectrometric data were obtained on an Agilent series 1100 SL mass spectrometer. HRMS were obtained by direct injection using a Bruker Bioapex-FTMS with electrospray ionization. HPLC was performed with a Hewlett-Packard Agilent 1100 with a single-wavelength detector at 205 nm. Plant Material. The starting material was a partially processed commercially available extract of Stevia rebaudiana leaves kindly provided by Cargill Inc., Minneapolis, MN, USA. HPLC comparison of that extract with several other S. rebaudiana extracts purchased from various sources showed high similarities, differing only in the relative concentrations of specific glycosides but not their presence or absence. Extraction and Isolation. The isolated compounds obtained were byproducts from the isolations and purification of rebaudioside C and rebaudioside D. Approximately 1.5 kg of commercially available S. rebaudiana leaf extract was processed. The composition of the extract was approximately 42% rebaudioside A, 31% stevioside, and 10% rebaudioside C, with the remaining 17% of the extract being other minor steviol glycoside components such as rebaudioside D, dulcoside A, rebaudioside F, rubusoside, rebaudioside B, steviolbioside, and the other four known compounds rebaudiosides E, M, N, and O and additional components with ≪1% compounds 1 and 2. Many different compound purification approaches were evaluated, and those used are described generally here. The commercial extract was dissolved in methanol or 10% aqueous methanol at about 200 mg/mL and allowed to crystallize. The crystalline products were rebaudioside A and stevioside, which accounted for approximately 50% of the starting mass. The enriched mother liquors were chromatographed by normalphase chromatography. There were five large-scale column runs. The loads were nominally 150 g of mother liquor dried onto 450 g of Celite 545 and packed into a 3 in. by 14 in. load column. The normalphase column was 6 in. i.d. × 54 in. length packed with 10 kg of 37−63 μm flash silica gel. The mobile phase was 100:18:14:0.1 v/v ethyl acetate−methanol−water−acetic acid. The columns were run with 16 L of mobile phase collected as a forerun followed by 70 1 L fractions. The fractions were analyzed by HPLC and pools produced. The pools for rebaudioside C were nominally fractions 40−60. The rebaudioside C pools were dried and dissolved in methanol, and stevioside was allowed to crystallize. The crystals of stevioside were filtered, and the mother liquors were dried and dissolved in water, with rebaudioside C allowed to crystallize. The rebaudioside C solids were collected, and the rebaudioside C mother liquors were then chromatographed again by three normal-phase chromatographic separations on a highperformance 7.5 cm i.d. × 50 cm long column packed with 1 kg of 10 μm spherical silica gel. The mobile phase was the same as used for the large column. Pools were selected of rebaudioside C and rebaudioside F. The rebaudioside C pool was dissolved in water, and another crop of rebaudioside C crystals was harvested. The rebaudioside C mother liquor and the rebaudioside F pool were then chromatographed in six reversed-phase column runs. The column was

Figure 2. Selected HMBC correlations for compound 2.

support this overall similarity, where H-18 (1.26 ppm) showed a 3J HMBC correlation to C-5 (57.9 ppm), H-20 (1.04 ppm) showed 3J HMBC correlations to both C-5 (57.9) and C-9 (51.7), and H-9 (1.01 ppm) showed a 3J HMBC correlation to C-15 (49.0 ppm). The 3J HMBC correlations between H2-17 (4.90 and 5.24 ppm) and both C-13 (42.0 ppm) and C-15 (49.0 ppm), in addition to the appearance of C-13 in the 135° DEPT spectrum as a CH signal, confirmed that compound 2 is not based on the expected ent-kaur-13-hydroxy-16-en-19-oic (steviol) acid aglycone. On comparing the NMR data of the aglycone of 2 to the literature, the structure could be proposed as ent-kaur-12-α-hydroxy-16-en-19-oic acid.21 The 3J HMBC correlation between H-12 (4.11 ppm) and C-1′ of Glc-1 (103.3 pm) was used to establish the attachment of the first glucose unit at C-12 rather than C-13, and this together with the correlations noted above suggested the diterpene aglycone to be ent-kaur-12-α-hydroxy-16-en-19-oic acid, the first report of this kaurenoic acid aglycone among the glycosides of S. rebaudiana.21−23 The 3J HMBC correlation between C-1″ of Glc-2 (106.4 ppm) and H-2′ of Glc-1 (4.12 ppm) established the attachment of the second glucose unit at C-2′ of Glc-1, and the position of H-2′ was confirmed through a COSY correlation between H-2′ (4.12 ppm) and H-1′ (5.01 ppm) (Figures S11 and S13, Supporting Information). The 3J HMBC correlation between H-1‴ of Glc′-1 (6.18 ppm) and C-19 (177.4 ppm) established the position of attachment of the third glucose unit at C-19. The glucose units were determined to be D-sugars through comparison with a standard using the previously mentioned HPLC method. Accordingly, compound 2 was assigned structurally as shown. The very high concentrations of glycosides in the leaves of S. rebaudiana suggest that they may have protective functions in the plant. To test this possibility, the isolated metabolites were evaluated for in vitro antimalarial and antimicrobial activity.24−29 Antimalarial activity was determined against chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum. Antimicrobial activity was determined against a panel of human pathogenic fungi and bacteria. 1233

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for 1 h at 60−70 °C, followed by the addition of phenylisothiocyanate with extended reflux for an additional 1 h at 60−70 °C to form the thiazoline derivative that was detected by UV spectroscopy. X-ray Crystallography of 3. The crystal structure and absolute configuration of 3 methanol solvate were determined, using data collected at T = 90 K with Cu Kα radiation on a Bruker APEX-II DUO CCD diffractometer, equipped with a microfocus source and an Oxford Cryostream cooler. The structure was solved using the program SHELXS-9731 and refined anisotropically by full-matrix leastsquares on F2 using SHELXL-97.31 H atoms were visible in difference maps, but were placed in idealized positions for the refinement, except for those of OH groups, which were refined. The absolute configuration was determined by refinement of the Flack32 parameter based on resonant scattering of the light atoms and computation of the Hooft parameter,33 which yielded a probability of 1.000 that the reported configuration with C4(R), C5(S), C8(R), C9(R), C10(S), and C13(S) is correct.19 Crystal data: (4α)-13-hydroxykaur-16-en-18olic acid methanol solvate, C20H30O3: CH3OH, Mr = 350.48, orthorhombic space group P212121, a = 7.3774(13) Å, b = 14.4459(17) Å, c = 17.666(3) Å, V = 1882.7(5) Å3, Z = 4, Dx = 1.236 Mg m−3, θmax = 67.6°, R = 0.031 for all 3214 unique data and 239 refined parameters. The Flack parameter is 0.09(17) and the Hooft parameter is 0.06(7) for 1327 Bijvoet pairs. These X-ray crystallographic data have been deposited with the Cambridge Crystallographic Data Centre under the deposition number CCDC 955759.

6 cm i.d. × 77 cm packed with 1.4 kg of 15 μm spherical C18 gel. The mobile phase was 65:35:0.1 v/v methanol−water−acetic acid. A pool of 1 was collected from each of these runs and combined and dried, yielding 655 mg. A second pool was collected from each of these runs and combined and dried, yielding 296 mg of 2. Compound 1 was then crystallized from methanol and yielded 309 mg at >98% purity by HPLC area. The pool of 2 did not crystallize from either water or methanol, and 180 mg was then chromatographed again by reversedphase chromatography. This column was 2.5 cm × 45 cm packed with 10 μm spherical C18 gel. The mobile phase was 72:28:1 v/v methanol−water−acetic acid. This pool yielded 82 mg of 2 at >95% purity by HPLC area. Following each of the five original large-scale 10 kg column runs, the column was regenerated using a solvent composition of 38:57:5:0.05 v/v ethyl acetate−methanol−water−acetic acid. The regeneration solvent pools contained rebaudioside D, rebaudioside N, and several other polar glycosides. A 5 g portion of one of these regeneration pools was dried onto 40 g of Celite 545, packed into a 3.2 cm i.d. × 13.5 cm long load column, and then chromatographed by normal-phase separation. The column was the 7.5 cm × 50 cm highperformance normal-phase column, and the mobile phase was 61.7:20.3:17.9:0.5 v/v ethyl acetate−methanol−water−acetic acid. A 2 L forerun and 50 125 mL fractions were collected. Rebaudioside M crystallized from fractions 2 and 3; two crops were taken from each of these two fractions. The first and second crops from fraction 2 and the first crop from fraction 3 when combined weighed 246 mg at 90% purity rebaudioside M by HPLC weighted average area and were then digested in methanol, yielding 109 mg at >97% purity by HPLC area. Rebaudioside E was the second crop from fraction 3, weighing 61.5 mg at 75% purity by HPLC area and was recrystallized from methanol, yielding 33.2 mg at 95% purity by HPLC area. A second of the largescale normal-phase chromatography regeneration pools after solvent removal was digested by exposure to hot 95:5 ethanol−water, and the insoluble rebaudioside D was filtered. The filtrate was then dried onto 45 g of Celite 545 and packed into a 3.2 cm i.d. × 13.5 cm long load column and chromatographed by normal-phase chromatography (7.5 cm × 50 cm high-performance normal-phase column). The mobile phase was 63.1:19.7:17.2:0.7 v/v ethyl acetate−methanol−water− acetic acid. A forerun of 2 L and 30 125 mL fractions were collected. A rebaudioside N pool was selected and concentrated, where rebaudioside N crystallized from the residual water, yielding an initial crop of 118 mg and following further concentration a subsequent crop of 105 mg. Both crops, 223 mg in total, represented >97% purity by HPLC area. A rebaudioside O pool was selected and dried, yielding 253 mg at 85% purity by HPLC area. Rebaudioside O did not crystallize from either methanol or water. Reversed-phase chromatography of rebaudioside O with mobile phase 72:28:0.1 v/v water−acetonitrile− acetic acid gave material of >96% purity. Greater than gram quantities of rebaudioside KA (1) (1.25 g), rebaudioside E (2.5 g), rebaudioside M (8.8 g), rebaudioside N (5.2 g), and rebaudioside O (2.7 g) were prepared in the present investigation. Stevioside, collected during the initial crystallizations of the commercial extract, was recrystallized from methanol to >99% purity by HPLC area. The stevioside was hydrolyzed enzymatically following the procedure described by Mosettig et al.,30 except ethyl acetate was used rather than ether for the partitioning. The enzymatic hydrolysis yielded 640 mg of steviol (3) at >99% purity by HPLC area from 6 g of stevioside. Compound 1: amorphous, off-white solid; [α]25D −27 (c 0.1, MeOH); 1H and 13C NMR (Table 1); HRMS m/z 803.3881 (calcd for C38H59O18, 803.3701). Compound 2: amorphous, off-white solid; [α]25D −38 (c 0.1, MeOH); 1H and 13C NMR (Table 1); HRMS m/z 827.3626 (calcd for C38H60O18Na, 827.3671). Sugar Unit Determination. The absolute configuration determination of the carbohydrate units of 1 and 2 was completed using an HPLC method reported by Tanaka et al.20 Each compound was hydrolyzed via refluxing in 1.0 N HCl for 2−3 h followed by extraction with ethyl acetate. The aqueous layer was then neutralized with silver carbonate, centrifuged to remove the insoluble precipitate, and dried. The residue was then refluxed with L-cysteine methyl ester in pyridine

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ASSOCIATED CONTENT

S Supporting Information *

Spectroscopic data and mass analysis of compounds 1 and 2, and the absolute configuration analysis of the carbohydrate units are available free of charge via the Internet at http://pubs. acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 303-808-4104. E-mail: jdmcchesney@ ironstoneseparations.com. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Dr. B. Avula for her help in obtaining HRESMS data. We are very grateful to Drs. S. I. Khan and M. Jacob for biological evaluations. This work was funded through Ironstone Separations, Inc., Etta, Mississippi, USA.

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REFERENCES

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Chicago, IL, 2008. (b) McQuate, R. S. U.S. Food and Drug Administration, Agency Response Letter GRAS Notice No. GRN 000304, CFSAN/Office of Food Additive Safety. GRAS Associates, LLC: Bend, OR, 2010. (7) The European Commission. Off. J. Eur. Union 2011, L 295−211. (8) Mitchell, W. Stevia World, Reassuring Numbers on Stevia’s Market Potential; Shared by William Mitchell, CFO of PureCircles, 2nd Stevia World Meeting: Shanghai, 2009. (9) Prakash, I.; Upreti, M. U.S. Pat. 8030481 B2, 2011. (10) DuBois, G. E.; Clos, J. F.; Wilkens, K. l.; Fosdick, L. E. Food Chem. Toxicol. 2008, 46, 575−582. (11) Rieck, U. W. J. Agric. Food Chem. 2012, 60, 886−895. (12) Kohda, H.; Kasai, R.; Yamasaki, K.; Murakami, K.; Tanaka, O. Phytochemistry 1976, 15, 982−983. (13) Sakamoto, I.; Yanaski, K.; Tanaka, O. Chem. Pharm. Bull. 1977, 25, 844−846. (14) Sakamoto, I.; Yanaski, K.; Tanaka, O. Chem. Pharm. Bull. 1977, 25, 3437−3439. (15) Starratt, A. N.; Kirby, C. W.; Pocs, R.; Brandle, J. E. Phytochemistry 2002, 59, 367−370. (16) Ohta, M.; Sasa, S.; Inoue, A.; Tamai, T.; Fujita, I.; Morita, K.; Matsuura, F. Japn. Soc. Appl. Glycosci. 2010, 57, 199−209. (17) Ceunen, S.; Geuns, M. C. J. J. Nat. Prod. 2013, 76, 1201−1228. (18) Dias Rodrigues, A. M. G.; de Almeida Santos, R. H.; LeChat, J. R. Acta Crystallogr. 1993, C49, 729−731. (19) Al’fonsov, V. A.; Bakaleinik, G. A.; Gubaidullin, A. T.; Kataev, V. E.; Kovylyaeva, G. I.; Konovalov, A. I.; Litvinov, I. A.; Strobykina, I. Yu.; Strobykin, S. I. Russ. J. Gen. Chem. 1998, 68, 1735−1743. (20) Tanaka, T.; Naskashima, T.; Ueda, T.; Kouno, I. Chem. Pharm. Bull. 2007, 55, 899−901. (21) Ortega, A.; Morales, F. J.; Salmon, M. Phytochemistry 1985, 24, 1850−1852. (22) Harinantenaina, L.; Kasai, R.; Yamasaki, K. Chem. Pharm. Bull. 2002, 50, 268−271. (23) Harinantenaina, L.; Kasai, R.; Yamasaki, K. Phytochemistry 2002, 61, 367−372. (24) Makler, M. T.; Hinrichs, D. J. Am. J. Trop. Med. Hyg. 1993, 48, 205−210. (25) NCCLS.. NCCLS Document M27-A2 2002, 22, 1−51. (26) NCCLS.. NCCLS Document M27-A7 2006, 22, 1−64. (27) NCCLS.. NCCLS Document M38-A 2002, 22, 1−51. (28) NCCLS.. NCCLS Document M24-A 2003, 22, 1−84. (29) Franzblau, S. G.; Witzig, R. S.; McLaughlin, J. C.; Torres, P.; Madico, G.; Hernandez, A.; Degnan, M. T.; Cook, M. B.; Quenzer, V. K.; Ferguson, R. M.; Gilman, R. H. J. Clin. Microbial. 1998, 36, 362− 366. (30) Mosettig, E.; Nes, W. R. J. Org. Chem. 1955, 20, 884−899. (31) Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112−122. (32) Flack, H. D. Acta Crystallogr. 1983, A39, 876−881. (33) Hooft, R. W. W.; Straver, L. H.; Spek, A. L. J. Appl. Crystallogr. 2008, 41, 96−103. (34) Macrae, C. F.; Edgington, P. R.; McCabe, P.; Pidcock, E.; Shields, G. P.; Taylor, R.; Towler, M.; van de Streek, J. J. Appl. Crystallogr. 2006, 39, 453−457.

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NOTE ADDED AFTER ASAP PUBLICATION This paper was published ASAP on April 23, 2014, with an error in the name of compound 2 in the abstract. The corrected version was reposted May 2, 2014.

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