Withanolide Artifacts Formed in Methanol - American Chemical Society

Article pubs.acs.org/jnp

Withanolide Artifacts Formed in Methanol Cong-Mei Cao, Huaping Zhang, Robert J. Gallagher, and Barbara N. Timmermann* Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045, United States S Supporting Information *

ABSTRACT: Methanol solutions of the main withanolides (6−8) naturally present in Physalis longifolia yielded five artificial withanolides (1−5), including three new compounds (1−3). Withanolides 1 and 2 were identified as intramolecular Michael addition derivatives, while withanolides 3−5 were the result of intermolecular Michael addition. A comprehensive literature investigation was conducted to identify potential withanolide Michael addition artifacts isolated from Solanaceous species to date.

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methanol at room temperature while highlighting the possible formation of such artifacts in previous investigations.

ithanolides are a group of modified C28 ergostane-type steroids with a C-22, C-26 δ-lactone side chain. They are distributed primarily within 25 genera of the Solanaceae, including Datura, Jaborosa, Physalis, and Withania.1 Recently, our group reported the isolation of 23 withanolides2,3 from Physalis longifolia Nutt., a native species in continental North America.4 Among them, the three major withanolides present in this species, withalongolides A (6) and B (7) as well as withaferin A (8), exhibited potent antiproliferative activity.2 Furthermore, a withanolide-based structure−activity relationship analysis confirmed that the presence of a Δ2-1-oxofunctionality in ring A is essential for the mediation of potent antiproliferative activity.1 Saturation of this functionality, such as in the case of 2,3-dihydro-3β-methoxywithaferin A (4), reduces cytotoxic potency compared to its unsaturated precursor 8.1 Previously, this saturation was achieved through Michael addition by adding the parent withanolide to either a refluxing methanol solution or a methanol sodium acetate solution.5,6 As part of an ongoing investigation related to the discovery of new bioactive withanolides, we sought to determine the stability of 6−8 in methanol in the absence of either a catalyst or heat. HPLC analysis of this solution displayed the emergence of five additional peaks. Each peak was isolated and identified as an artificial product (1−5) of the corresponding natural withanolide (6−8). Among them, 1−3 are new compounds, while 4 and 5 were identified as 2,3-dihydro-3β-methoxywithaferin A and withalongolide D, respectively.2 Two deuterated artificial withanolides (2a and 3a) were also isolated from a deuterated methanol solution of 7 to obtain additional information concerning the role of methanol in the formation of withanolide artifacts. These findings are supported by an exhaustive literature search for potential withanolide Michael addition artifacts reported to date from Solanaceous species. The combined data described herein confirm the instability of withanolides with a Δ2-1-oxo-functionality in ring A in pure © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

A methanol solution of 6−8 was examined at three time intervals (zero, three, and 10 days). HPLC analysis of the solution displayed the emergence of five additional peaks in a time-dependent manner (Figure 1). Among the five artifacts produced, 1 and 5 originated from 6, and 2 and 3 originated from 7, whereas 4 was produced from 8. In order to readily visualize the parent withanolides and their artificial products, a TLC plate of the purified artificial products 1−5, the purified compounds 6−8, and an unpurified solution of 6−8 with their artifacts was run (Figure S1, Supporting Information). Compound 1 exhibited the molecular formula C28H38O7, as determined from the HRESIMS and NMR data, the same as that of its precursor, 6. The NMR data of 1 (Table 1) closely resembled those of 6, except in the ring-A moiety, where the conjugated double bond signals observed in 6 were absent in 1. Instead, the 1H−1H COSY spectrum showed evidence for the partial structure of −CH2−CH(OR)− in 1 [δH 2.98 (dd, J = 18.8, 3.2 Hz, H-2β), 2.50 (dd, J = 18.8, 2.4 Hz, H-2α), 4.29 (dd, J = 3.2, 2.4 Hz, H-3)], which replaced the conjugated doublebond fragment −CHCH− in ring A of 6. This was confirmed by the HMBC correlations of H-3 to C-1 (δC 208.0), H-2β to C-4 (δC 69.7), and H-3 to C-5 (δC 60.8). These observations suggested that 1 contains an additional ring to satisfy the 10 degrees of unsaturation. The absence of the coupling between H-3 and H-4 in 1 suggested a dihedral angle of approximately 90° between H-3 and H-4. This indicated that a change at C-3 restricted the flexibility and confirmation of ring A in 1 as compared to 6. The chemical shifts of C-19 at δC 63.5, H-19 at δH 4.18 (d, J = 9.0 Hz), 3.86 (d, J = 9.0 Hz), and Received: April 8, 2013

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dx.doi.org/10.1021/np400296s | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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amount of DCl (Figure 2, type Ia).7 To further explore this observation and understand the role of methanol in this change, 7 was dissolved in deuterated methanol to afford the deuterated artifact 2a. The 1H NMR and 13C NMR data of 2a and 2 were indistinguishable except for the following observations: (1) the two protons at C-2 in 2 [δH 2.99 (H-2β) and 2.49 (H-2α)] were absent in 2a; (2) H-3 appeared as a double doublet at δH 4.29 (J = 3.3, 2.2 Hz) in 2, while it appeared as a broad singlet at δH 4.29 in 2a; (3) the carbon signal at δC 42.8 (C-2) in 2 was absent in 2a. This indicated that both C-2 hydrogens present in 2 were deuterated in 2a. Hence, H-2α and H-2β and their coupling with H-3 were absent in the 1H NMR spectrum of 2a. The 13C NMR data indicated that the strong methylene signal of C-2 in 2 was converted into a weak quaternary carbon signal in 2a. Furthermore, the coupling between the two deuterium atoms and C-2 resulted in a quintet. Hence, the C-2 signal in 2a could not be detected. Analysis of the ESIMS of 2a yielded a molecular ion at m/z 473 [M + H]+, confirming the presence of the two deuterium atoms in 2a. The polar protic deuterated methanol provided the deuterium necessary for keto−enol tautomerization resulting in H/D exchange to form 2a (Figure 3). This suggests strongly that withanolide intramolecular artifacts could form in the presence of any polar protic solvent. However, further experimentation with other solvents will be required to confirm this hypothesis. Similarly, as observed for the 19β-hydroxylated withanolides, withanolides containing a 6α-hydroxy group in close proximity to the β-carbon of their α,β-unsaturated carbonyl in ring A can undergo a similar transformation (Figure 2, type Ib). Therefore, it is reasonable to assume that a withanolide with a C-3−O−C6 bridge could be an intramolecular Michael addition artifact formed from a C-6-hydroxylated withanolide with the Δ2-1oxo-functionality (Table 2). For example, 5β,27-dihydroxy3α,6α-epoxy-1-oxowitha-24-enolide may result from an intramolecular Michael addition of jaborosalactone D (5β,6α,27trihydroxy-1-oxowitha-2,24-dienolide).8 In a similar manner, three withanolides with a C-3−O−C-6 bridge have been hypothesized to be intramolecular artifacts arising from the cyclization of 6α-hydroxywithanolides,9 namely, 3α,6α-epoxy4β,5β,27-trihydroxy-1-oxowitha-24-enolide,10 withaperuvin D,11 and withaperuvin F,12 arising from 4β,5β,6α,27-tetrahydroxy-1-oxowitha-2,24-dienolide, withaperuvin, and withaperuvin B, respectively. Compound 3 was isolated as colorless powder by semipreparative HPLC. Its molecular formula of C29H42O7 was deduced from its HRESIMS and NMR data. The NMR spectra of 3 (Table 1) resembled the 2,3-dihydro-3β-methoxy moiety of 4 and 5.2 The presence of a −OCH3 group at C-3 in 3 was confirmed by a 1H−1H COSY fragment [−CH2−CH(OCH3)− CH(OH) in ring A] and HMBC correlations (OCH3/C-3 and H-3/OCH3). Therefore, compound 3 was identified as 2,3dihydro-3β-methoxywithalongolide B (5β,6β-epoxy-4β,19β-dihydroxy-3-methoxy-1-oxowitha-24-en-22,26-olide). Compound 3 may be conjectured to be the result of an intermolecular Michael addition of 7 and methanol. This was confirmed when 7 in deuterated methanol produced the corresponding deuterated artifact, 3a (Figure 3). The ESIMS of 3a showed a molecular ion at m/z 529 [M + H]+, suggesting that four hydrogen atoms were replaced by four deuterium atoms. By comparing their NMR spectra, it was evident that the four hydrogen atoms at OMe-3 and H-2α in 3 were replaced by four deuterium atoms in 3a. The 1H NMR and 13C NMR

H-3 at δH 4.29 suggested an oxygen bridge between C-3 and C19 in 1. This deduction was confirmed by observed HMBC correlations [C-19 (δC 63.5)/H-3 (δH 4.29) and C-3 (δC 74.3)/ H-19α (δH 4.18)]. The structure of 1 was elucidated as shown, and this compound has been named isowithalongolide A (3β,19β:5β,6β-diepoxy-4β,19β,27-trihydroxy-1-oxowitha-24-en22,26-olide). The NMR spectroscopic assignments of 1 are summarized in Table 1. Compound 2 was isolated as a colorless powder and found to possess the same molecular formula, C28H38O6, determined by HRESIMS, as the precursor, 7. The NMR data of 2 were closely comparable to those observed for withanolide 1. The only difference observed corresponded to the signals at C-27, where a methylene (δH 4.39, 4.34; δC 57.6) was evident for 1, while a methyl group (δH 1.88; δC 12.6) was observed for 2. This information suggested that compound 2 is the 27dehydroxy derivative of 1, which was confirmed by the HMBC correlations (CH3-27/C-24, 25, and 26) in 2 and superimposable NMR signals of the side chain between 2 and 7. Compound 2 was identified as an isomer of 7, with a C-3−O− C-19 bridge, and named isowithalongolide B (3β,19β:5β,6βdiepoxy-4β,19β-dihydroxy-1-oxowitha-24-en-22,26-olide). In the artifactual formation of 1 and 2, a C-3−O−C-19 bridge between the 19-hydroxy group and the β-carbon of the α,β-unsaturated carbonyl in ring A of 6 and 7, respectively, was established. Isocinerolide is the only 19-hydroxylated withanolide-derived intramolecular Michael addition artifact reported in the literature, and it was obtained during the NMR acquisition of cinerolide in CDCl3, containing a trace B



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Figure 1. Time-dependent emergence of artifacts 1−5 in a methanol solution of 6−8.

1−5 occurred in a time-dependent manner; that is, the 10-day solution contained larger amounts of artificial products as compared to those formed in the three-day solution. As the Δ21-oxo-functionality is common in withanolides (Figure 2, type II), a detailed literature review was conducted in order to highlight possible artifacts formed through this reaction (Table 2). Among the reported ca. 800 withanolides,1 the only confirmed intermolecular Michael addition artifacts are 2,3dihydro-3-methoxywithaferin A derived from withaferin A,13−15 2,3-dihydro-3-methoxywithacnistine derived from withacnistine,6,15 and 2,3-dihydro-3β-methoxyixocarpalactones A and B derived from ixocarpalactones A and B, respectively.16 There are, however, several 2,3-dihydro-3-methoxy withanolides reported in the literature that were hypothesized to be Michael addition artifacts formed during their isolation. These include withalongolide E (2,3-dihydro-3-methoxywithalongolide C),2 2,3-dihydro-3-methoxywithanolide D and 18-acetoxy-2,3-dihydro-3-methoxywithanolide D,17 tubocapsenolide B (2,3-dihydro-3-methoxytubocapsenolide A), tubocapsanolide B (2,3-

(APT) data of 3 and 3a were indistinguishable except for the following observations: (1) the methoxy group signals at δH 3.37 (OMe-3) and δC 57.6 (OMe-3) in 3 were absent in 3a; (2) the double doublet of H-2β at δH 2.88 (J = 16.4, 8.3 Hz) in 3 changed to a broad doublet at δH 2.86 (J = 8.3 Hz) in 3a; (3) H-3 appeared at δH 3.71 (ddd, J = 8.3, 3.4, 2.6 Hz) in 3, while it appeared at δH 3.72 (brdd, J = 8.3, 3.4 Hz) in 3a; (4) the strong singlet (C-2, CH2, δC 40.5) of 3 appeared as a weak triplet (C2, CHD, δC 40.2) in 3a. These observed differences confirmed that OMe-3 and H-2α of 3 are deuterated in 3a and are formed through an intermolecular Michael addition with deuterated methanol acting as a nucleophile. Theoretically, other nucleophilic solvents such as ethanol and water could produce similar artifactual withanolides. Unlike in previous studies, this intermolecular Michael addition proceeded without heating or use of a catalyst. Subjecting withalongolide A (6), withalongolide B (7), and withaferin A (8) to methanol at room temperature resulted in the formation of 1−5. As shown in Figure 1, the formation of C



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Table 1. NMR Data of Withanolides 1−3, 2a, and 3a in CDCl3a 1 position 1 2

δC

δH (J in HZ)

208.0 42.8

3

74.3

4 5 6 7

69.7 60.8 56.9 29.8

8 9 10 11

28.7 36.8 50.0 22.1

12

39.3

13 14 15

43.2 54.8 24.2

16

27.3

17 18 19

52.2 11.8 63.5

20 21 22 23

38.8 13.5 78.8 29.9

24 25 26 27

153.0 125.8 167.1 57.6

28 OCH3 OH-4 a1

20.2

2.98 dd (18.8, 3.2) 2.50 dd (18.8, 2.4) 4.29 dd (3.2, 2.4) 3.69 d (7.1) 3.37 d (3.5) 2.13 dt (14.5, 3.5) 1.27 dd (14.5, 11.0) 1.62 m 1.90 m 1.18 m 0.95 dd (13.0, 3.8) 1.87 m 1.21 m 1.04 m 1.64 m 1.17 m 1.66 m 1.35 m 1.12 m 0.66 s 4.18 d (9.0) 3.86 d (9.0) 1.99 m 0.98 d (6.7) 4.42 dt (13.4, 3.5) 2.49 brdd (17.0, 13.4) 1.98 m

4.39 d (12.5) 4.34 d (12.5) 2.04 s 3.16 d (7.0)

2 δC 208.0 42.8

74.3 69.7 60.8 56.9 29.8

28.8 36.9 50.1 22.1

39.3 43.2 54.8 24.2 27.3 52.3 11.8 63.5 38.9 13.5 78.4 29.7

149.1 122.1 167.2 12.6 20.7

δH (J in HZ)

2a δC

δH (J in HZ)

208.0 2.99 dd (18.8, 3.3) 2.49 dd (18.8, 2.2) 4.29 dd (3.3, 2.2) 3.69 d (9.4) 3.37 d (3.2) 2.14 dt (14.6, 3.5) 1.29 dd (14.6, 11.3) 1.62 m 1.90 m 1.18 m 0.94 dd (13.2, 4.0) 1.88 m 1.22 m 1.05 m 1.64 m 1.17 m 1.68 m 1.36 m 1.12 m 0.66 s 4.19 d (9.0) 3.86 d (9.0) 1.98 m 0.97 d (6.7) 4.35 dt (13.3, 3.5) 2.42 brdd (17.0, 13.3) 1.92 m

3

3a

δC

δH (J in HZ)

δC

208.0 40.5

2.88 dd (16.4, 8.3)

208.0 40.2b

δH (J in HZ) 2.86 brd (8.3)

2.58 dd (16.4, 2.6) 74.3

4.29 brs

77.7

69.7 60.8 56.9 29.8

3.69 d (9.4)

72.7 62.4 57.5 31.0

28.8 36.9 50.1 22.1

39.3 43.2 54.8 24.2 27.3 52.3 11.8 63.5 38.9 13.5 78.4 29.7

3.37 d (3.2) 2.14 dt (14.6, 3.5) 1.29 dd (14.6, 11.3) 1.62 m 1.90 m 1.18 m 0.94 dd (13.2, 4.0) 1.88 m 1.22 m 1.05 m 1.64 m 1.17 m 1.68 m 1.36 m 1.12 m 0.66 s 4.19 d (9.0) 3.86 d (9.0) 1.98 m 0.97 d (6.7) 4.35 dt (13.3, 3.5) 2.42 brdd (17.0, 13.3) 1.92 m

1.88 s

149.1 122.1 167.2 12.6

1.88 s

1.94 s

20.7

1.94 s

3.16 d (9.4)

30.0 42.1 56.0 21.5

39.2 42.7 56.6 24.2 27.4 52.0 11.6 60.2 38.9 13.6 78.3 29.7

3.71 ddd (8.3, 3.4, 2.6) 3.43 d (3.4) 3.23 brs 2.27 dd (9.7, 2.0) 1.43 m 1.44 m 1.15 m 1.27m 1.18 m 1.89 m 1.01 m 0.96 m 1.67 m 1.13 m 1.68 m 1.37 m 1.08 m 0.63 s 4.39 d (10.1) 3.61 d (10.1) 1.95 m 0.96 d (6.7) 4.33 dt (13.5, 3.5) 2.41 brdd (17.0, 13.5) 1.90 m

149.1 122.1 167.2 12.6

1.86 s

20.7 57.6

1.93 s 3.37 s

77.5 72.7 62.4 57.5 31.0

30.0 42.1 56.0 21.5

39.2 42.7 56.6 24.2 27.4 52.0 11.6 60.2 38.9 13.6 78.3 29.7

3.72 brdd (8.3, 3.4) 3.43 d (3.4) 3.23 brs 2.27 dd (9.7, 2.0) 1.43 m 1.44 m 1.15 m 1.27m 1.18 m 1.89 m 1.01 m 0.96 m 1.67 m 1.13 m 1.68 m 1.37 m 1.08 m 0.63 s 4.39 d (10.1) 3.61 d (10.1) 1.95 m 0.96 d (6.7) 4.33 dt (13.5, 3.5) 2.41 brdd (17.0, 13.5) 1.90 m

149.1 122.1 167.2 12.6

1.86 s

20.7

1.93 s

3.16 d (9.4)

H NMR and C NMR data were recorded at 500 and 125 MHz, respectively. 13C NMR data were obtained from the APT spectrum. bThe strong singlet of 3 was changed to a weak triplet in 3a. 13

artifacts of 25,27-dihydro-4,7-didehydro-7-deoxyneophysalin A; physagulin N (2,3-dihydro-3-methoxyphysagulin A) is a physagulin A artifact;21 and physalin U (5,6:14,17:14,27triepoxy-13,20,22-trihydroxy-3α-methoxy-1,15-dioxo-γ-lactone δ-lactone) is an artifact of physalin F.22,23 5β,6β-Epoxy-4β,20βdihydroxy-3β-methoxy-1-oxowithanolide24 may be proposed as being a withanolide D artifact; 2,3-dihydro-3-methoxywithaphysacarpin may be proposed as being a withaphysacarpin artifact;25 and 5β,6β-epoxy-4β-hydroxy-3β-methoxywitha-24enolide26 may be proposed as being the artifact of 27desoxywithaferin A. Moreover, 2,3-dihydro-3-methoxywithacnistine6,15 and 2,3-dihydro-3-methoxyiochromolide seem to be 2,3-dihydro-3-methoxy artifacts of iochromolide.15 Although

dihydro-3-methoxytubocapsanolide A), tubocapsanolide G (2,3-dihydro-3-methoxytubocapsanolide F), and 20-hydroxytubocapsanolide G (2,3-dihydro-17α-hydroxy-3-methoxywithanolide D).18 On the basis of the data obtained herein for compounds 3−5, it is reasonable to assume that all 2,3-dihydro3-methoxy withanolides reported in the literature are most likely Δ2-1-oxo withanolide artifacts (Figure 2, type II). Therefore, it is highly likely that withaperuvin K (5β,6βepoxy-4β,14α,17β,20,28-pentahydroxy-3β-methoxy-1-oxowitha24-en-26,22-olide)19 is an artifact of visconolide; physalins I (2,3,25,27-tetrahydro-3α-methoxy-4,7-didehydro-7-deoxyneophysalin A) and II (2,3,25,27-tetrahydro-3β-methoxy-4,7didehydro-7-deoxyneophysalin A)20 are 2,3-dihydro-3-methoxy D



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are susceptible to methanol degradation when left at room temperature and without the addition of heat or catalyzers. This study emphasizes the risks associated with using methanol as a solvent in withanolide research, especially for compounds containing α,β-unsaturated carbonyl structural units that are of interest for their biological activities. It is our opinion that the use of methanol and other polar protic solvent nucleophiles should be avoided during the isolation and especially during the final purification stages of withanolides. In general, polar aprotic and nonpolar solvents should be preferred for handling withanolides bearing 19β-hydroxy and 6α-hydroxy groups, although intramolecular Michael addition could still occur, albeit at a much slower rate. Regardless of solvent type, it is advisable to reduce withanolide solvent exposure time whenever possible to minimize the potential formation of such artifacts.

Figure 2. Two major types of withanolide artifacts reported in Solanaceous plants.

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other 2,3-dihydro-3-methoxy withanolides have been isolated, the corresponding Δ2-1-oxo withanolides have yet to be reported from the same plant source, such as in the case of 2,3dihydro-3-methoxy-4,7-didehydrophysalin B;27 withangulatin H (2,3-dihydro-16-hydroxy-3-methoxywithanolide E);28 physalin W (2,3,5α,6-tetrahydro-3-methoxyphysalin B);28 withaphysalin Q (5,6-epoxy-3,18-dimethoxy-1-oxowith-24-enolide);29 tubocapsanolide H (4β,20β-dihydroxy-5β,6β-epoxy-3β-methoxy-1oxo-16-en-withanolide);18 bracteosin A (5β,6β:22,26-diepoxy4β,28-dihydroxy-3β-methoxyergost-24-ene-1,26-dione), bracteosin B (5β,6β:22,26-diepoxy-4β,28-dihydroxy-3β-methoxy1,26-dioxoergost-24-en-19-oic acid), and bracteosin C (22,26epoxy-4β,6β,27-trihydroxy-3β-methoxyergost-24-ene-1,26dione);30 2,3-dihydro-3-methoxy-27-hydroxywithacnistine, 2,3dihydro-3-methoxy-27-hydroxyiochromolide, and 2,3-dihydro3-methoxy-16α-hydroxywithacnistine;15 and physalactone.31 The formation of 1−5 and the comprehensive literature search performed herein both serve to highlight that withanolides containing α,β-unsaturated carbonyl structural units

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a Rudolph RS Autopol IV automatic polarimeter. UV spectra were scanned on Varian Cary 50 UV−visible spectrophotometer. IR data were obtained with a Thermo Nicolet Avatar 380 FTIR spectrometer. NMR spectra were recorded with a Bruker AV-400 or AV-500 instrument with a cryoprobe for 1H NMR, APT, COSY, HSQC, HMBC, and NOESY/ROESY experiments. Chemical shift values are given in δ (ppm) using the peak signals of the solvent CDCl3 (δH 7.26 and δC 77.23) as references, and coupling constants are reported in Hz. ESIMS data were measured with an Agilent 1200 Series LC coupling with an ion trap 6310 mass spectrometer. HRESIMS data were collected with a LCT Premier time-of-flight mass spectrometer (Waters Corp., Milford, MA, USA). Normal-phase silica gel G TLC plates (UV 254) were used for fraction detection. The spots were visualized using UV light at 254 nm and 10% EtOH− sulfuric acid spray reagent. Semipreparative HPLC was performed on an Agilent 1200 unit equipped with a DAD detector, utilizing a Phenomenex Luna RP-18 column (250 × 10 mm, 5 μm). Isolation and Purification. Compounds 6−8 were isolated from previous work as major withanolides from P. longifolia.2 Compounds 1 (8 mg) and 5 (20 mg) were obtained by leaving compound 6 (50 mg)

Figure 3. Proposed reaction mechanisms leading to 2a and 3a from 7. E



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Table 2. Possible Withanolide Artifacts Isolated from Solanaceous Species in the Literature compound name isocinerolide (3β,19-epoxy-14α,17α-dihydroxy-1-oxowitha-5,24-dien-22,26-olide) 3α,6α-epoxy-5β,27-dihydroxy-1-oxowitha-24-enolide 3α,6α-epoxy-4β,5β,27-trihydroxy-1-oxowitha-24-enolide withaperuvin D (3α,6α-epoxy-4β,5β,14α,17β,20α-pentahydroxy-1-oxoergosta-24-en22,26-olide) withaperuvin F (3α,6α-epoxy-4β,5β,17β,20α-tetrahydroxy-1-oxoergosta-14,24-dien22,26-olide) 2,3-dihydro-3β-methoxywithaferin A (5β,6β-epoxy-4β,27-dihydroxy-3β-methoxywitha24-en-22,26-olide) 2,3-dihydro-3-methoxyiochromolide 2,3-dihydro-3-methoxywithacnistine 2,3-dihydro-3β-methoxyixocarpalactone A 2,3-dihydro-3β-methoxyixocarpalactone B withalongolide E (5β,6β-epoxy-4β,11β,27-trihydroxy-3β-methoxy-1-oxowitha-24-en22,26-olide) 2,3-dihydro-3-methoxywithanolide D (iso-tubocapsanolide G) 18-acetoxy-2,3-dihydro-3-methoxy-withanolide D tubocapsenolide B (5β,6β-epoxy-4β,16α-dihydroxy-3β-methoxy-1-oxowitha-13,24-dien22,26-olide) tubocapsanolide B (5β,6β:16α,17α-diepoxy-3β-methoxy-4β-hydroxy-1-oxowitha-24-en22,26-olide) tubocapsanolide G (5β,6β-epoxy-4β,17β-dihydroxy-3β-methoxy-1-oxowitha-24-en22,26-olide) 20-hydroxytubocapsanolide G, (5β,6β-epoxy-4β,17β,20-trihydroxy-3β-methoxy-1oxowitha-24-en-22,26-olide) withaperuvin K (5β,6β-epoxy-4β,14α,17β,20,28-pentahydroxy-3β-methoxy-1-oxowitha24-en-22,26-olide) physalin I (2,3,25,27-tetrahydro-3α-methoxy-4,7-didehydro-7-deoxyneophysalin A) physalin II (2,3,25,27-tetrahydro-3β-methoxy-4,7-didehydro-7-deoxyneophysalin A) physagulin N (15α-acetoxy-5β,6β-epoxy-14α-hydroxy-3β-methoxy-1-oxowitha-16,24dien-22,26-olide) physalin U (16,24-cyclo-13,14-secoergosta-18,26-dioic acid; 5,6:14,17:14,27-triepoxy13,20,22-trihydroxy-3α-methoxy-1,15-dioxo-γ-lactone-δ-lactone) 5β,6β-epoxy-4β,20β-dihydroxy-3β-methoxy-1-oxo-withanolide 2,3-dihydro-3-methoxywithaphysacarpin 5β,6β-epoxy-4β-hydroxy-3β-methoxy-1-oxo-witha-24-enolide 2,3-dihydro-3-methoxy-4,7-didehydrophysalin B withangulatin H (5β,6β-epoxy-14α,16β,17β,20β-hydroxy-3β-methoxy-1-oxowitha-24en-22,26-olide) physalin W withaphysalin Q (5,6-epoxy-3,18-dimethoxy-1-oxowitha-24-en-22,26-olide) tubocapsanolide H (5β,6β-epoxy-4β,20β-dihydroxy-3β-methoxy-1-oxowitha-16-en22,26-olide) bracteosin A (5β,6β:22,26-diepoxy-4β,28-dihydroxy-3β-methoxyergost-24-ene-1,26dione) bracteosin B (5β,6β:22,26-diepoxy-4β,28-dihydroxy-3β-methoxy-1,26-dioxoergost-24en-19-oic acid) bracteosin C (22,26-epoxy-4β,6β,27-trihydroxy-3β-methoxyergost-24-ene-1,26-dione) 2,3-dihydro-3-methoxy-27-hydroxywithacnistine 2,3-dihydro-3-methoxy-27-hydroxyiochromolide 2,3-dihydro-3-methoxy-16α-hydroxywithacnistine physalactone (5β,6β-epoxy-4β,17β,20-trihydroxy-3β-methoxy-1-oxowitha-8,24-dien22,26-olide)

precursor

type

cinerolide (14α,17α-dihydroxy-1-oxowitha-2,5,24-trien22,26-olide) jaborosalactone D (5β,6α,27-trihydroxy-1-oxowitha-2,24dienolide) 4β,5β,6α,27-tetrahydroxy-1-oxowitha-2,24-dienolide withaperuvin (4β,5β,6α,14α,17β,20-hexahydroxy-1oxoergosta-2,24-dien-22,26-olide) withaperuvin B (4β,5β,6α,17β,20α-pentahydroxy-1oxoergosta-2,14,24-trien-22,26-olide) withaferin A

Ia

7

Ib

8

Ib Ib

10 11

Ib

12

II

13−15

II II II II II

15 6, 15 16 16 2

II

17, 18

II II

17 18

II

18

II

18

II

18

II

19

II II II

20 20 21

II

22, 23

24,25-dihydrowithanolide D withaphysacarpin 27-desoxywithaferin A no report no report

II II II II II

24 25 26 27 28

no report no report no report

II II II

28 29 18

no report

II

30

no report

II

30

no no no no no

II II II II II

30 15 15 15 31

iochromolide withacnistine ixocarpalactone A ixocarpalactone B withalongolide C (5β,6β-epoxy-4β,11β,27-trihydroxy-1oxowitha-2,24-dien-22,26-olide) withanolide D (5β,6β-epoxy-4β,20-dihydroxy-1oxowitha-2,24-dien-22,26-olide) 18-acetoxywithanolide D tubocapsenolide A (5β,6β-epoxy-4β,16α-dihydroxy-1oxowitha-2,13,24-trien-22,26-olide) tubocapsanolide A (5β,6β:16α,17α-diepoxy-4β-hydroxy1-oxowitha-2,24-dien-22,26-olide) tubocapsanolide F (5β,6β-epoxy-4β,17β-dihydroxy-1oxowitha-2,24-dien-22,26-olide) 17α-hydroxywithanolide D (5β,6β-epoxy-4β,17β,20trihydroxy-1-oxowitha-2,24-dien-22,26-olide) visconolide (5β,6β-epoxy-4β,14α,17β,20,28pentahydroxy-1-oxowitha-2,24-dienolide) 25,27-dihydro-4,7-didehydro-7-deoxyneophysalin A 25,27-dihydro-4,7-didehydro-7-deoxyneophysalin A physagulin A (15α-acetoxy-5β,6β-epoxy-14α-hydroxy-1oxowitha-2,16,24-trien-22,26-olide) physalin F

report report report report report

ref

Isowithalongolide A (3β,19β:5β,6β-diepoxy-4β,19β,27-trihydroxy-1-oxowitha-24-en-22,26-olide) (1): colorless, amorphous solid; [α]24 D +0.9 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 230 (3.96) nm; IR (film) νmax 3347 (br), 2941 (br), 2832, 1687, 1399, 1135, 1022 cm−1; 1H NMR and 13C NMR, see Table 1; ESIMS (+) m/z 487 [M + H]+ (100); HRESIMS (+) m/z 509.2515 [M + Na]+ (calcd for C28H38O7Na, 509.2522). Isowithalongolide B (3β,19β:5β,6β-diepoxy-4β,19β-dihydroxy-1oxowitha-24-en-22,26-olide) (2): colorless, amorphous solid; [α]24 D +1.1 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 230 (3.77) nm; IR

dissolved in 10 mL of methanol at room temperature for three days and purified by semipreparative HPLC. Compound 7 (49 mg) was dissolved in 10 mL of methanol and left to stand at room temperature for three days. This solution was subjected to semipreparative HPLC to yield compounds 2 (6 mg) and 3 (12 mg). To get a higher yield, compound 4 (6 mg) was obtained by leaving compound 8 (11 mg) dissolved in methanol at room temperature for 10 days and purified by semipreparative HPLC. F



Article

(film) νmax 3491, 2927 (br), 1734, 1697, 1396, 1132, 1026 cm−1; 1H NMR and 13C NMR, see Table 1; ESIMS (+) m/z 471 [M + H]+ (100), 453 [M+ H − H2O]+ (15); HRESIMS (+) m/z 493.2546 [M + Na]+ (calcd for C28H38O6Na, 493.2566). 2,3-Dihydro-3β-methoxywithalongolide B (5β,6β-epoxy-4β,19βdihydroxy-3-methoxy-1-oxowitha-24-en-22,26-olide) (3): colorless, amorphous solid; [α]24 D −1.0 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 230 (4.03) nm; IR (neat) νmax 3346 (br), 2928 (br), 1705, 1396, 1132, 1097 cm−1; 1H NMR and 13C NMR, see Table 1; ESIMS (+) m/z 503 [M + H]+ (13), 485 [M+ H − H2O]+ (100), 467 [M + H − 2H2O]+ (85); HRESIMS (+) m/z 525.2802 [M + Na]+ (calcd for C29H42O7Na, 525.2828).

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(16) Gu, J. Q.; Li, W.; Kang, Y. H.; Su, B. N.; Fong, H. H. S.; van Breemen, R. B.; Pezzuto, J. M.; Kinghorn, A. D. Chem. Pharm. Bull. 2003, 51, 530−539. (17) Raffauf, R. F.; Shemluck, M. J.; Le Quesne, P. W.. J. Nat. Prod. 1991, 54, 1601−1606. (18) Hsieh, P. W.; Huang, Z. Y.; Chen, J. H.; Chang, F. R.; Wu, C. C.; Yang, Y. L.; Chiang, M. Y.; Yen, M. H.; Chen, S. L.; Yen, H. F.; Lübken, T.; Hung, W. C.; Wu, Y. C. J. Nat. Prod. 2007, 70, 747−753. (19) Fang, S. T.; Liu, J. K.; Li, B. Steroids 2012, 77, 36−44. (20) Qiu, L.; Zhao, F.; Jiang, Z. H.; Chen, L. X.; Zhao, Q.; Liu, H. X.; Yao, X. S.; Qiu, F. J. Nat. Prod. 2008, 71, 642−646. (21) Abe, F.; Nagafuji, S.; Okawa, M.; Kinjo, J. Chem. Pharm. Bull. 2006, 54, 1226−1228. (22) Kuo, P. C.; Kuo, T. H.; Damu, A. G.; Su, C. R.; Lee, E. J.; Wu, T. S.; Shu, R.; Chen, C. M.; Bastow, K. F.; Chen, T. H.; Lee, K. H. Org. Lett. 2006, 8, 2953−2956. (23) Choudhary, M. I.; Yousaf, S.; Ahmed, S.; Samreen; Yasmeen, K.; Atta-ur-Rahman. Chem. Biodiversity 2005, 2, 1164−1173. (24) Kuroyanagi, M.; Shibata, K.; Umehara, K. Chem. Pharm. Bull. 1999, 47, 1646−1649. (25) Kennelly, E. J.; Gerhäuser, C.; Song, L. L.; Graham, J. G.; Beecher, C. W. W.; Pezzuto, J. M.; Kinghorn, A. D. J. Agric. Food Chem. 1997, 45, 3771−3777. (26) Jamal, S. A.; Qureshi, S.; Ali, S. N.; Choudhary, M. I.; Atta-urRahman. Chem. Heterocycl. Compd. 1995, 31, 1047−1059. (27) Qiu, L.; Jiang, Z. H.; Liu, H. X.; Chen, L. X.; Yao, X. S.; Qiu, F. J. Asian Nat. Prod. Res. 2008, 10, 881−885. (28) Damu, A. G.; Kuo, P. C.; Su, C. R.; Kuo, T. H.; Chen, T. H.; Bastow, K. F.; Lee, K. H.; Wu, T. S. J. Nat. Prod. 2007, 70, 1146−1152. (29) Ma, L.; Gan, X. W.; He, Q. P.; Bai, H. Y.; Arfan, M.; Lou, F. C.; Hu, L. H. Helv. Chim. Acta 2007, 90, 1406−1419. (30) Riaz, N.; Malik, A.; Aziz-ur-Rehman; Nawaz, S. A.; Muhammad, P.; Choudhary, M. I. Chem. Biodiversity 2004, 1, 1289−1295. (31) Maslennikova, V. A.; Tursunova, R. N.; Abubakirov, N. K. Chem. Nat. Compd. 1977, 13, 443−446.

ASSOCIATED CONTENT

S Supporting Information *

TLC of compounds 6−8 and respective artifacts 1−5; 1H NMR, 13C NMR, 1H−1H COSY, HSQC, HMBC, and ROESY spectra for 1−3; and 1H NMR and 13C NMR spectra for 2a and 3a are available. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +01-785-864-4844. Fax: +01-785-864-5326. E-mail: [email protected] (B.N.T.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by grant IND 0061464 (awarded to B.N.T.) from the Kansas Biosciences Authority (KBA) and Center for Heartland Plant Innovations (HPI).

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REFERENCES

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Withanolide Artifacts Formed in Methanol - American Chemical Society

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