17β-Hydroxy-18-acetoxywithanolides from ... - ACS Publications

Apr 12, 2016 - Ya-ming Xu, Daniel P. Bunting, Manping X. Liu, Hema A. Bandaranayake,. † and A. A. Leslie Gunatilaka*. Natural Products Center, Schoo...
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17β-Hydroxy-18-acetoxywithanolides from Aeroponically Grown Physalis crassifolia and Their Potent and Selective Cytotoxicity for Prostate Cancer Cells Ya-ming Xu, Daniel P. Bunting, Manping X. Liu, Hema A. Bandaranayake,† and A. A. Leslie Gunatilaka* Natural Products Center, School of Natural Resources and the Environment, College of Agriculture and Life Sciences, University of Arizona, 250 E. Valencia Road, Tucson, Arizona 85706, United States S Supporting Information *

ABSTRACT: When cultivated under aeroponic growth conditions, Physalis crassifolia produced 11 new withanolides (1−11) and seven known withanolides (12−18) including those obtained from the wild-crafted plant. The structures of the new withanolides were elucidated by the application of spectroscopic techniques, and the known withanolides were identified by comparison of their spectroscopic data with those reported. Withanolides 1−11 and 16 were evaluated for their potential anticancer activity using five tumor cell lines. Of these, the 17β-hydroxy-18-acetoxywithanolides 1, 2, 6, 7, and 16 showed potent antiproliferative activity, with some having selectivity for prostate adenocarcinoma (LNCaP and PC-3M) compared to the breast adenocarcinoma (MCF-7), non-small-cell lung cancer (NCI-H460), and CNS glioma (SF-268) cell lines used. The cytotoxicity data obtained for 12−15, 17, and 19 have provided additional structure−activity relationship information for the 17β-hydroxy-18-acetoxywithanolides. side chain, suggesting that withanolides bearing α- and βoriented side chains may target different biological sites in exerting their biological/pharmacological activities.5 It is noteworthy that about half of the number of 17βhydroxywithanolides known to date have been found to occur in Physalis species, but these withanolides have also been encountered in Jaborosa and Withania species and in Ajuga parvifolia of the plant family Lamiaceae.2 The genus Physalis contains over 75 species,6,7 of which some are employed in traditional medicines throughout the world, and the pharmacological activities of these have been attributed to their constituent withanolides.8 Some significant studies on biologically active withanolides from Physalis species include those on P. alkekengi,9 P. angulata,10 P. chenopodifolia,11 P. cinerascens,12 P. coztomatl,13 P. hispida,14 P. longifolia,15 P. minima,16 P. peruviana,17 P. philadelphica,18 and P. pubescens.19 Of these, the occurrence of 17β-hydroxywithanolides has been reported from P. angulata,10 P. chenopodifolia,11 P. cinerascens,12 and P. coztomatl.13 In a continuing search for natural-productbased anticancer agents,20 an extract of P. crassifolia Benth. was recently identified as the most promising lead among 18 000 natural product extracts/fractions screened in a highthroughput gene-expression assay for potential agents to treat castration-resistant prostate cancer (PC).5 Bioactivity-guided fractionation of this extract led to the isolation and identification of several 17β-hydroxy-18-acetoxywithanolides as active constituents with selective toxicity to PC cell lines (LNCaP and PC-3M) and also with in vivo anti-PC activity.5

W

ithanolides constitute a class of polyoxygenated steroids based on a C28 ergostane skeleton, of which many contain a modified side chain at C-17 derived from 22hydroxyergosta-26-oic acid.1 Over 650 natural withanolides have been encountered to date, mainly from 23 genera of the plant family Solanaceae including Acnistus, Datura, Jaborosa, Physalis, and Withania.2 Structurally, withanolides may be classified based on the nature and orientation of the side chain. Most of the withanolides contain δ-lactone or δ-lactol side chains, although some with γ-lactone side chains are also known. In many withanolides bearing a side chain at C-17 containing a δ-lactone moiety, this side chain has a βorientation, as exemplified by withaferin A (22) (Figure 1), the most extensively studied member of this class of compounds and a constituent of Withania somnifera (L.) Dunal employed in Indian Ayurvedic medicine.3 Withaferin A has been reported to have a variety of biological/pharmacological activities, and these have been attributed to the presence of the 2(3)-en-1-one moiety in ring A, the 5β,6β-epoxy moiety in ring B, and the unsaturated δ-lactone moiety in ring E, while the presence of other oxygen-containing substituents has been known to modulate these activities.4 Structural variations of withanolides consist mainly of combinations of oxygenated functions (hydroxy or acetoxy groups) at different positions of the steroid nucleus (C-14, C-15, C-16, C-17, and C-18) and the side chain (C-20, C-21, C-26, and C-27). The hydroxy group at C-17 may occur in either an α- or β-orientation. Among over 650 natural withanolides, only ca. 50 are known to contain a hydroxy substituent at C-17 with β-orientation.2 In contrast to withaferin A (22) and other withanolides bearing a 17αhydroxy group, 17β-hydroxywithanolides contain an α-oriented © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 13, 2015

A

DOI: 10.1021/acs.jnatprod.5b00911 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Structures of withanolides 1−18 from aeroponically grown Physalis crassifolia, 15α,18-diacetoxy-17-epi-withanolide K (19), withanolide A (20), hyoscyamilactol (21), and withaferin A (22).

The difficulty in collecting large quantities of P. crassifolia in the wild prompted an investigation of the application of aeroponic techniques for the cultivation of this plant as done previously for another plant of the family Solanaceae, W. somnifera, for the production and structural diversification of withaferin A (22).21,22 Aeroponically grown P. crassifolia afforded 18 withanolides, in contrast to the wild-crafted plant, from which only five withanolides were obtained.5 Eleven of the withanolides isolated from aeroponically grown P. crassifolia are new, and these were identified as 15α-hydroxyphysachenolide D (1), 27-hydroxyphysachenolide D (2), 15α-acetoxy-27hydroxyphysachenolide D (3), 15α-acetoxy-27-O-β-glucopyranosylphysachenolide D (4), 15α-acetoxy-28-O-β-glucopyranosylphysachenolide D (5), 15α-acetoxyphysachenolide C (6), 15α-acetoxy-2,3-dihydrophysachenolide D-3β-O-sulfate (7), 15α,18-diacetoxy-28-hydroxy-17-epi-withanolide K (8), 23βhydroxyphysacoztolide E (9), 14α-hydroxywithanolide A (10), and 16-oxo-17(20)-dehydrohyoscyamilactol (11). Comparison of spectroscopic data with those reported led to the identification of several previously known withanolides as physachenolide D (12),5,11 15α-acetoxyphysachenolide D (13),5,13b 15α-acetoxy-28-hydroxyphysachenolide D (14),5 physachenolide C (15),5,11 2,3-dihydrophysachenolide D 3βO-sulfate (2,3-dihydro-3-O-sulfonylphysachenolide D) (16),13b 18-acetoxy-17-epi-withanolide K (17),5 and physacoztolide E (18).13a Of these, we have previously isolated 12−14 from wild-crafted P. crassifolia and obtained 15 by epoxidation of 12.5 Intriguingly, despite the occurrence of a number of structurally diversified withanolides including physachenolide C (15) in aeroponically grown P. crassifolia, 15α,18-diacetoxy-17-epiwithanolide K (19) isolated from wild-crafted P. crassifolia was not encountered when it was grown under aeroponic conditions. Reported herein are descriptions of the structure

elucidation of withanolides 1−11 and the cytotoxic activity of the 17β-hydroxy-18-acetoxywithanolides.



RESULTS AND DISCUSSION When grown under aeroponic conditions, P. crassifolia produced flowers in ca. 2 months and reached maturity and produced fruits in ca. 5 months. Aerial parts of 2-month-old and 5-month-old aeroponically grown plants were therefore harvested and processed separately for their constituent withanolides. Aerial parts of 2-month-old P. crassifolia were found to contain eight withanolides (1−3, 12−14, 17, and 18) compared to 14 (2 and 4−16) produced by 5-month-old (mature) plants. The molecular formula of 1 (C30H40O9) and its 1H and 13C NMR data indicated that it is a deacetyl analogue of 15αacetoxyphysachenolide D (13), which has been encountered previously in wild-crafted P. crassifolia5 and P. coztomatl.13b Comparison of the 1H and 13C NMR data of 1 (Tables 1 and 3) with those of 135 suggested that in 1 the OAc-15 group is replaced with an OH group. This was confirmed by the expected upfield shifts for H-15 (Δ = −1.10 ppm) and C-15 (Δ = −1.7 ppm) and the downfield shift for C-16 (Δ = +4.5 ppm) of 1, when compared to 13. The orientation of this OH group at C-15 in 1 was determined to be α, the same as that of the OAc group in 13 from the NOE observed between H-15 and H-18 (Figure 2 and Figure S50, Supporting Information). The absolute configuration of C-22 was established as R through the positive Cotton effect at 250 nm in the CD spectrum.16 Acetylation (Ac2O/pyridine) of 1 afforded 13, the structure of which has been determined by single-crystal X-ray analysis.13b The foregoing evidence together with its HMBC data (Figure 2) helped establish the structure of 1 as 15α-hydroxyphysachenolide D [(20S,22R)-18-acetoxy-14α,15α,17β,20-tetrahydroxy1-oxowitha-2,5,24-trienolide]. B

DOI: 10.1021/acs.jnatprod.5b00911 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H NMR Data (400 MHz, δ, Hz) for Withanolides 1−5 in CDCl3a position 2 3 4

2

dd (10.0, 2.0) ddd (10.0, 4.9, 2.5) dd (22.0, 3.2) dd (22.0, 4.8) brd (5.6) m

19 21 22 23

1.83 2.33 1.41 2.32 2.18 2.43 4.06 2.01 2.35 4.56 4.24 1.18 1.36 4.87 2.46

m m m m m m dd (7.9, 7.9) m m d (11.6) d (11.6) s s dd (12.1, 3.9) m

27

1.84 s

28 OAc-15 OAc-18 Glc-1′ Glc-2′ Glc-3′ Glc-4′ Glc-5″ Glc-6

1.89 s

4.33 d (12.4) 4.28 d (12.4) 2.00 s

2.07 s

2.03 s

6 7 8 9 11 12 15 16 18

a

1 5.80 6.72 3.21 2.77 5.53 2.16

5.84 6.76 3.25 2.80 5.55 1.85 2.00 1.90 2.14 1.32 2.17 2.11 2.24 1.77 1.49 2.57 4.20

dd (10.0, 2.0) ddd (10.0, 4.9, 2.5) brd (21.2) dd (21.2, 4.8) brd (5.9) m m m m m m m m m m m brs

1.23 1.42 4.59 2.68

s s brd (12.2) m

3 5.82 6.73 3.23 2.78 5.50 1.78 2.26 1.82 2.37 1.37 2.35 1.70 2.49 5.13 2.11 2.50 4.68 4.22 1.18 1.38 4.94 2.60

dd (10.0, 1.9) ddd (10.0, 4.9, 2.5) br d (21.6) dd (21.6, 4.8) brd (5.5) m m m m m m m m dd (8.8) m m d (11.5) d (12.5) s s dd (12.4, 3.6) m

4.34 brs 2.03 s 2.10 s 2.06 s

4 5.78 6.73 3.21 2.76 5.48 1.77 2.25 1.80 2.33 1.35 2.30 1.67 2.43 5.01 2.05 2.43 4.54 4.21 1.14 1.28 4.89 2.53 2.70 4.54 4.37 2.01 2.02 2.04 4.31 3.26 3.26 3.40 3.41 3.69 3.81

brd (10.0) brd (10.0) m m brd (6.0) m m m m m m m m dd (8.4, 8.4) m m m d (11.2) s s brd (12.5) m m m d (11.1) s s s d (7.8) m m m m dd (12.0, 4.8) dd (12.0, 3.2)

5 5.78 6.73 3.21 2.76 5.48 1.78 2.24 1.78 2.33 1.38 2.28 1.70 2.44 5.11 2.07 2.41 4.51 4.26 1.15 1.29 4.83 2.42 2.88 1.84

dd (10.0, 2.0) ddd (10.0, 4.8, 2.4) m m brd (5.3) m m m m m m m m dd (8.8, 8.8) m m d (11.6) d (11.6) s s dd (13.2, 2.4) m brd (8.8) s

4.38 2.02 2.08 4.20 3.22 3.23 3.39 3.37 3.71 3.78

m s s d (7.7) m m m m dd (12.0, 4.4) dd (12.0, 3.2)

Assignments based on DEPT, HSQC, and HMBC data.

The HREIMS and 13C NMR data of 2 were consistent with the molecular formula, C30H40O9, indicating that it is an isomer of 1. The 1H and 13C NMR data of 2 (Tables 1 and 3) showed very close similarities to those of physachenolide D (12),5,11 except that one of the allylic CH3 groups in 12 is replaced by a CH2OH [δH 4.33 and 4.28 (2H, d, J = 12.4 Hz); δC 56.8] in 2. The position of this oxygenated methylene was determined to be C-27 on the basis of the strong cross-peaks observed for this CH2 with C-26 (δC 166.2) and C-24 (δC 155.5) in its HMBC spectrum (Figures S10 and S51, Supporting Information). The configuration at C-22 was determined as R based on the positive Cotton effect observed at 253 nm in its CD spectrum.16 Withanolide 2 was thus identified as 27hydroxyphysachenolide D [(20S,22R)-18-acetoxy14α,17β,20,27-tetrahydroxy-1-oxowitha-2,5,24-trienolide]. The molecular formula of 3 (C32H42O11) together with its 1H and 13C NMR data (Tables 1 and 3) suggested that it is a mono-oxygenated analogue of 15α-acetoxyphysachenolide D (13),5 in which one of the allylic CH3 groups has undergone oxidation to a CH2OH. The position of this oxygenated methylene was determined to be C-27 on the basis of strong cross-peaks observed for this CH2OH (δH 4.34) with C-26 (δC 166.0) and C-24 (δC 154.4) in its HMBC spectrum (Figures

S14 and S51, Supporting Information). The positive Cotton effect at 254 nm in the CD spectrum suggested the 22R configuration.16 Thus, the structure of 3 was established as 15αacetoxy-27-hydroxyphysachenolide D [(20S,22R)-15α,18-diacetoxy-14α,17β,20,27-tetrahydroxy-1-oxowitha-2,5,24-trienolide]. Withanolides 4 and 5 were determined to have the same molecular formula (C38H52O16) by their HREIMS and 13C NMR analyses. Comparison of the 1H and 13C NMR spectroscopic data of 4 with those of 15α-acetoxy-27hydroxyphysachenolide D (3) (Tables 1 and 3) indicated that, in addition to the signals due to the withanolide moiety of 3, compound 4 contained signals due to six additional carbons, of which all were oxygenated [five CH (δC 102.2, 76.4, 75.9, 73.2, 70.1) and a CH2 (δC 61.9)], and seven protons [δH 4.31 (1H) and 3.26−3.81 (6H)] characteristic of a β-glucopyranosyl moiety.13b The β configuration of the glucose moiety was further supported by the observed large coupling constant of the anomeric proton H-1′ [δH 4.31 (1H, d, J = 7.8 Hz)]. The attachment of the β-glucose moiety to C-27 was confirmed by the HMBC correlations of H-1′ (Glc)/C-27 (δC 62.4), H-27 (δH 4.37 and 4.54)/C-1′ (Glc), H-27/C-26 (δC 166.3), and H27/C-24 (δC 158.8) (Figure 2). The absolute configuration of C

DOI: 10.1021/acs.jnatprod.5b00911 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 1H NMR Data (400 MHz, δ, Hz) for Withanolides 6−11 in CDCl3a position

6

2

6.00 dd (10.1, 2.7)

3 4 6 7 8 9 11 12 14 15 16 17 18 19 21 22 23

7

8

9

6.81 ddd (9.9, 6.3, 2.3) 1.84 m 2.93 m 3.12 brs 1.69 m 2.00 m 2.49 m 1.95 m 1.41 m 2.09 m 2.06 m 2.33 m

2.67 m 2.75 m 4.47 ddd (14.0, 9.6, 6.9) 2.43 m 2.69 m 5.54 brs 1.81 m 2.26 m 1.83 m 2.12 m 1.21 m 1.64 m 2.03 m 2.33 m

3.24 2.71 5.57 6.00

d (19.8) dd (19.8, 4.4) m d (10.0)

5.60 1.94 2.38 1.97 2.53 1.23 2.01 1.73 2.49

m m m m m m m m m

5.03 dd (8.8, 8.8)

5.00 dd (9.2, 8.4)

5.18 dd (8.9, 8.9)

2.16 m 2.48 m

2.04 m 2.40 m

2.16 m 2.53 m

4.55 4.24 1.21 1.36 4.84 2.50

4.50 4.14 1.14 1.25 4.76 2.39 2.57

4.61 4.34 1.35 1.41 4.88 2.92 2.46

d (11.6) d (11.6) s s brd (11.5) d (18.3) m

1.87 4.44 4.28 2.08 2.14

s d (14.1) d (14.1) s s

d (11.7) d (11.7) s s dd (8.3, 8.3) m

d (11.6) d (11.6) s s dd (13.6, 3.2) m m

10 5.84 dd (10.1, 2.1)

5.85 ddd (10,0, 2.8, 0.8)

6.77 ddd (10.0, 4.9, 2.5) 3.26 brd (21.2) 2.82 dd (21.2, 4.8) 5.56 brd (6.0) 1.87 m 2.15 m 1.83 m 2.05 m 1.42 m 2.27 m 1.53 m 2.14 m

6.57 ddd (10.1, 5.1, 2.2) 2.67 m 2.52 dd (18.8, 5.2) 2.99 d (4.0) 3.43 dd (4.0, 1.8)

6.59 ddd (10.0, 5.2, 2.2) 2.68 brd (18.8) 2.53 dd (18.8, 5.2) 3.05 d (3.6) 3.25 dd (3.6, 2.0)

2.16 2.26 1.35 2.70 1.79 1.67

m m m m m m

2.16 1.93 1.46 1.63 2.66 4.37 4.05 1.20 1.41 4.13 4.37

1.72 1.94 1.68 2.08 2.18 1.06

m m m m m s

1.91 1.75 1.45 2.91 1.70 2.33 1.78 2.20 2.38

1.18 1.28 4.20 2.20 2.37

s s dd (13.3, 3.7) m brdd (17.2, 12.8)

m m m m dd (9.3,9.3) d (11.4) d (11.4) s s d (8.9) d (8.9)

26

a

27 28

1.87 s 1.91 s

1.78 s 1.86 s

OAc-15 OAc-18

2.07 s 2.10 s

2.01 s 2.02 s

11

5.85 dd (10.0, 2.0)

1.86 s 1.95 s

1.87 s 1.94 s

m m m m m m m m m

1.10 s 1.18 1.89 5.83 1.66 1.80 5.02 3.82 1.42 1.37

s s dd (11.2, 2.8) m m d (11.2) d (11.2) (OH) s s

2.07 s

Assignments based on DEPT, HSQC, and HMBC data.

OAc [δH 5.03 (1H, dd, J = 8.8, 8.8 Hz) and 2.07 (3H, s); δC 75.9 (CH), 170.9 (C), and 21.4 (CH3)] moiety present in 6. The HMBC data of 6 (Figures S28 and S50, Supporting Information) suggested that this OAc is attached to C-15, and the NOESY data (Figures S29 and S51, Supporting Information) helped to determine its orientation as α. Compound 6 was shown to be identical with the major product obtained by the epoxidation (m-CPBA/CH2Cl2) of 15α-acetoxyphysachenolide D (13), the structure of which has been determined by single-crystal X-ray analysis.13b Thus, the structure of 6 was established as 15α-acetoxyphysachenolide C [(20S,22R)-15α,18-diacetoxy-5β,6β-epoxy-14α,17β,20-trihydroxy-1-oxowitha-2,24-dienolide]. The HRESIMS and 13C NMR data of 7 were consistent with the molecular formula C32H44O14S, indicating that it is a sulfated withanolide similar to 2,3-dihydrowithaferin A 3β-Osulfate21 and 2,3-dihydrophysachenolide D 3β-O-sulfate (2,3dihydro-3β-O-sulfonylphysachenolide) (16).13b The 1H and 13 C NMR spectroscopic data of 7 (Tables 2 and 3), assigned with the help of the HSQC and HMBC spectra, were similar to those of 15α-acetoxyphysachenolide D (13),5 except for the absence of signals due to the 2(3)-en-1-one moiety in ring A of 7. Instead, the 1H and 13C NMR spectra exhibited signals due to a 1-oxo-3-O-sulfate moiety [δC 212.3 (C-1), δH 4.47 ddd (J =

C-22 was determined as R from the positive Cotton effect at 254 nm.16 On the basis of the foregoing evidence, the structure of 4 was tentatively established as 15α-acetoxy-27-O-βglucopyranosylphysachenolide D [(20S,22R)-15α,18-diacetoxy-27-O-β-glucopyranosyl-14α,17β,20-trihydroxy-1-oxowitha2,5,24-trienolide]. Comparison of its 1H and 13C NMR spectroscopic data with those of 4 (Tables 1 and 3) suggested that 5 also contains an O-β-glucopyranosyl moiety [δH 4.20 (1H, d, J = 7.7 Hz) and 3.22−3.78 (6H); δC 102.1, 76.4, 75.8, 73.3, 69.8, and 61.6] attached to C-28 [δH 4.38 (2H, m); δC 67.3]. This was confirmed by the HMBC correlations of H-1′ (Glc)/C-28, H-28/C-1′ (Glc), H-28/C-25 (δC 123.6), and H28/C-23 (δC 29.2) (Figures S23 and S51, Supporting Information). The positive Cotton effect at 256 nm in the CD spectrum suggested the 22R configuration.16 Thus, the structure of 5 was established as 15α-acetoxy-28-O-βglucopyranosylphysachenolide D [(20S,22R)-15α,18-diacetoxy-28-O-β-glucopyranosyl-14α,17β,20-trihydroxy-1-oxowitha2,5,24-trienolide]. The molecular formula of 6 (C32H42O11) and comparison of its 1H and 13C NMR data (Tables 2 and 3) with those of physachenolide C (15)5,11 indicated that the only difference between 6 and 15 is that one of the carbocyclic ring CH2 groups of 15 has undergone acetoxylation, leading to a CHD

DOI: 10.1021/acs.jnatprod.5b00911 J. Nat. Prod. XXXX, XXX, XXX−XXX

E

a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 OAc-18

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

2

204.3 C 127.7 CH 145.6 CH 33.4 CH2 135.4 C 124.2 CH 24.7 CH2 36.8 CH 36.0 CH 50.8 C 22.1b CH2 22.0b CH2 53.9 C 85.4 C 33.1 CH2 32.8 CH2 87.5 C 62.8 CH2 18.1 CH3 76.9 C 18.9 CH3 81.5 CH 33.6 CH2 155.5 C 124.7 C 166.2 C 56.8 CH2 20.0 CH3 21.1 CH3 171.1 C

3 203.8 127.9 145.2 33.3 134.7 124.9 25.6 37.7 35.7 50.5 22.7 25.9 57.3 79.8 75.8 43.5 85.1 64.8 18.7 78.8 19.3 80.3 34.0 154.4 125.4 166.0 57.0 20.2 21.4 170.2 21.3 171.3

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

4 204.5 127.7 145.8 33.2 134.5 125.0 25.5 37.6 35.7 50.4 22.8 25.7 57.0 79.4 76.1 42.7 84.6 64.5 18.7 78.4 18.4 80.8 34.1 158.8 122.2 166.3 62.4 20.5 21.1 170.6 21.2 171.6 102.2 73.2 75.9 70.1 76.3 61.9

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

5 204.6 127.7 145.8 33.2 134.5 125.0 25.5 37.6 35.7 50.5 22.6 25.6 57.1 79.4 76.1 42.9 84.7 64.8 18.6 78.5 18.9 81.2 29.2 148.8 123.6 166.6 12.0 67.3 21.3 171.3 21.1 171.6 102.1 73.3 75.8 69.8 76.4 61.6

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

Assignments based on DEPT, HSQC, and HMBC data. bAssignments are interchangeable.

Glc-1′ Glc-2′ Glc-3′ Glc-4′ Glc-5′ Glc-6′

OAc-15

1

204.1 127.8 145.4 33.3 134.3 125.4 25.9 37.8 35.8 50.6 22.8 26.4 57.1 80.1 74.1 48.2 85.1 65.0 18.8 78.9 19.1 80.1 33.8 150.2 121.6 166.3 12.3 20.6 21.3 170.3

position

Table 3. 13C NMR Data (100 MHz) of Withanolides 1−11 in CDCl3a 6 202.7 129.6 143.9 32.8 61.8 63.8 25.7 34.6 36.8 48.4 22.8 26.0 57.3 79.7 75.9 43.8 84.7 64.8 14.7 79.0 19.4 79.3 33.9 149.9 121.8 165.4 12.4 20.6 21.3 169.8 21.4 170.9

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

7 212.3 44.5 74.4 37.7 133.9 126.2 25.7 36.2 35.7 52.7 21.7 25.3 57.1 79.3 76.0 42.6 84.6 64.3 17.7 78.3 18.4 80.7 33.8 151.2 121.3 167.3 12.1 20.5 21.7 170.7 21.3 171.7

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

8 210.4 39.7 121.5 129.2 140.1 127.1 25.8 36.5 34.1 52.2 21.5 25.5 57.7 79.8 75.6 43.5 85.5 64.8 20.1 79.2 20.1 80.6 28.1 151.2 121.9 165.5 11.9 61.4 21.1 170.4 21.4 171.1

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

9 204.3 127.8 145.6 33.4 135.1 124.6 25.3 36.2 36.2 50.6 22.3 27.4 50.7 83.3 21.2 32.2 49.3 63.0 18.9 76.1 15.8 85.6 67.0 152.3 121.7 164.4 12.8 23.7 21.2 170.8

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

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

10 202.8 129.0 139.4 36.7 73.0 54.5 56.5 37.5 28.8 51.0 20.7 32.9 47.7 84.6 33.0 20.8 48.5 17.9 14.4 75.2 21.2 81.3 31.8 149.1 121.9 166.1 12.5 20.5

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

11 202.8 128.9 139.8 36.7 73.2 56.2 56.8 34.6 35.4 51.0 22.0 37.2 45.0 45.7 39.1 204.5 141.0 17.1 14.7 149.1 12.7 62.0 34.9 65.0 63.1 91.9 16.5 18.6

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The molecular formula of 9 (C30H40O8) and its 1H and 13C NMR data (Tables 2 and 3) suggested that it is a monooxygenated analogue of physacoztolide E (18), a withanolide found to co-occur in this extract and previously encountered in P. coztomatl.13a Comparison of their 1H and 13C NMR data suggested that in 9 the CH2-23 group of 18 underwent oxidation to a CHOH moiety [δH 4.37 (1H, d, J = 8.9 Hz); δC 67.0]. The HMBC correlations [CH3-28 (δH 1.95)/C-23 and H-23/C-25 (δC 121.7)] (Figure S41, Supporting Information) confirmed the presence of a hydroxy group at C-23 of 9. The large coupling constant observed for H-22/H-23 (J = 8.9 Hz) was consistent with the relevant dihedral angle of ca. 45° for the favorable conformation adopted by ring E with a β-OH at C-23, as suggested by the energy-minimized Chembio3D model of 9. The positive Cotton effect at 261 nm in the CD spectrum helped to establish the absolute configuration of C-22 as R.16 On the basis of the foregoing evidence, the structure of 9 was determined as 23β-hydroxyphysacoztolide E [(20S,22R)-18acetoxy-14α,20,23β -trihydroxy-1-oxowitha-2,5,24-trienolide]. The HRESIMS and 13C NMR data of 10 were consistent with the molecular formula, C28H38O7. The 1H and 13C NMR spectra of 10 (Tables 2 and 3), assigned with the help of HSQC and HMBC data (Figures S44 and S45, Supporting Information) and its molecular formula, suggested that 10 is a mono-oxygenated analogue of withanolide A (20).24 Comparison of the 13C NMR spectroscopic data of 10 with those of 20 indicated significant differences in the chemical shifts for C-14 (δC 84.6 in 10; 51.9 in 20), suggesting the presence of a hydroxy group at C-14. The HMBC correlations of CH3-18 (δH 1.94) to C-14 and C-17 (δC 48.5) further supported the presence of a OH-14 substituent. The chemical shift difference of C-9 and C-12 (δC 28.8 and 32.9 in 10; δC 35.5 and 40.3 in 20, respectively) induced by the gauche-effect, as well as the chemical shift of C-17 (δC 48.5 in 10; δC 54.4 in 20) due to a similar γ-interaction, implied that OH-14 is αoriented.25 The positive Cotton effect at 252 nm in the CD spectrum established the absolute configuration of C-22 as R.16 Thus, the structure of 10 was determined as 14αhydroxywithanolide A [(20S,22R)-6α,7α-epoxy-5α,14α,20-trihydroxy-1-oxowitha-2,24-dienolide]. The molecular formula of 11 (C28H36O7) and its 1H and 13C NMR data indicated that it is an analogue of hyoscyamilactol (21), previously encountered in Hyoscyamus niger.26 The 1H and 13C NMR data of 11 (Tables 2 and 3) suggested that when compared with 21 it has an additional enone moiety. The presence of this 17(20)-en-16-one moiety in 11 was determined using the HMBC spectrum, which showed correlations for H-21/C-16, H-22/C-17, H-23/C-20, and H21/C-17 (Figure 2). The configuration of the C-17(20) double bond was determined to be Z by the strong NOE correlation observed for CH3-18 and CH3-21 (Figure 2). Comparison of the 13C NMR data of 11 with those reported for the MnO2 oxidation product of the withanolide, exodeconolide A, suggested that these have almost the same 13C chemical shifts for C1−C21.27 The absolute configuration of C-22 was determined as R from the positive Cotton effect at 261 nm in its CD spectrum.16 On the basis of the foregoing evidence, the structure of 11 was established as 16-oxo-17(20)dehydrohyoscyamilactol [(20S,22R)-6α(7α),24α(25α)-diepoxy-5α-hydroxy-1,16-dioxowitha-2(3),17(20)-dienolide]. Withanolides 1−11 and 16 were evaluated for their cytotoxic activity in a panel of tumor cell lines consisting of LNCaP (androgen-sensitive prostate adenocarcinoma), PC-3M (an-

Figure 2. Key HMBC and NOE correlations for withanolides 1, 4, 7, and 11.

6.9, 9.6, and 14.0 Hz); δC 74.4 (CH-3)]. The presence of this 1oxo-3-O-sulfate moiety in 7 was confirmed by the presence of cross-peaks for H-3/C-1 and H-19 (δH 1.14)/C-1 in its HMBC spectrum (Figure 2). The orientation of the 3-O-sulfate moiety was determined as β from the large J(ax,ax) coupling pattern of H-3 (ddd, J = 6.9, 9.6, 14.0 Hz),23 whereas the orientation of the OAc-15 group in 7 was shown to be α from its NOE data (Figure 2). It has been shown previously that 2,3dihydrowithaferin A 3β-O-sulfate can be converted readily to withaferin A (22) in a cell-culture medium.21 Thus, when 7 was incubated in this manner (DMEM supplemented with 10% fetal bovine serum) for 24 h at 37 °C and monitored by HPLC,21 elimination of HOSO2OH proceeded smoothly, affording 15α-acetoxyphysachenolide D (13) as the only detectable product. The absolute configuration of C-22 was established as R based on the positive Cotton effect at 249 nm in the CD spectrum.16 On the basis of the foregoing evidence, the structure of 7 was determined as 15α-acetoxy-2,3dihydrophysachenolide D 3β-O-sulfate [(20S,22R)-15α,18diacetoxy-14α,17β,20-trihydroxy-1-oxowitha-5,24-dienolide-3βO-sulfate]. The HRESIMS and 13C NMR data of 8 were consistent with the molecular formula, C32H42O11, indicating that it is an isomer of 15α-acetoxy-28-hydroxyphysachenolide D (14).5 The 1 H and 13C NMR spectra of 8 (Tables 2 and 3), assigned with the aid of the HSQC spectrum (Figure S37, Supporting Information), were almost identical with those of 14,5 except for the absence of signals due to the 2(3)-en-1-one moiety in ring A of 8. Instead, the 1H and 13C NMR spectra exhibited signals due to a tetrasubstituted 3(4),5(6)-conjugated diene moiety [δH 5.57 (m), 5.60 (m), and 6.00 (1H, d, J = 10.0 Hz); δC 121.5 (CH), 129.2 (CH), 140.1 (C), 127.1 (CH)], similar to that of 18-acetoxy-epi-withanolide K (19).5 The positive Cotton effect at 248 nm in the CD spectrum suggested a 22R configuration.16 On the basis of the foregoing evidence, the structure of 8 was determined as 15α,18-diacetoxy-28-hydroxy17-epi-withanolide K [(20S,22R)-15α,18-diacetoxy14α,17β,20,27-tetrahydroxy-1-oxowitha-3,5,24-trienolide]. F

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Table 4. Cytotoxicity Data of 17β-Hydroxy-18-acetoxywithanolides and Withaferin A (22) against a Panel of Selected Tumor Cell Lines and Normal Cellsa cell lineb compound

LNCaP

PC-3M

MCF-7

NCI-H460

SF-268

HFF

1 2 6 7 12c 13c 14c 15c 16 17c 19c 22 doxorubicin

0.19 ± 0.01 3.7 ± 1.4 1.2 ± 0.00 0.12 ± 0.02 0.08 ± 0.01 0.44 ± 0.14 3.3 ± 0.3 0.02 ± 0.01 0.21 ± 0.05 1.1 ± 0.1 2.0 ± 0.3 0.87 ± 0.10 NT

0.60 ± 0.11 >5.0 1.8 ± 0.1 0.43 ± 0.08 0.13 ± 0.01 2.0 ± 0.4 >5.0 0.09 ± 0.01 0.56 ± 0.11 3.1 ± 0.2 >5.0 0.41 ± 0.01 0.25 ± 0.02

>2.5 >5.0 >2.5 >5.0 1.9 ± 0.4 >5.0 >5.0 0.21 ± 0.16 2.2 ± 0.3 >5.0 >5.0 0.57 ± 0.03 0.32 ± 0.09

>2.5 >5.0 >2.5 2.5 ± 0.3 1.1 ± 0.1 2.0 ± 0.5 >5.0 0.80 ± 0.20 3.8 ± 0.7 3.9 ± 1.1 >5.0 2.5 >5.0 >2.5 2.8 ± 0.2 3.4 ± 0.3 >5.0 >5.0 2.5 ± 0.4 >5.0 >5.0 >5.0 3.1 >2.8 NT >3.1 >5.0 >5.0 >5.0 >6.8 NT

Results are expressed as IC50 values in μM. Doxorubicin and DMSO were used as positive and negative controls. NT = not tested. Withanolides 3− 5, 8−11, and 18 had no activity up to 5.0 μM. bKey: LNCaP = androgen-sensitive human prostate adenocarcinoma; PC-3M = metastatic human prostate adenocarcinoma; MCF-7 = human breast cancer; NCI-H460 = human non-small-cell lung cancer; SF-268 = human CNS cancer (glioma); HFF = human foreskin fibroblast. cPreviously reported data.5 a

spectrophotometer. CD spectra were measured with a JASCO J-810 spectropolarimeter using MeOH as the solvent. 1D and 2D NMR spectra were recorded in CDCl3 using residual solvent as the internal standard on a Bruker Avance III 400 spectrometer at 400 MHz for 1H NMR and 100 MHz for 13C NMR, respectively. The chemical shift values (δ) are given in parts per million (ppm), and the coupling constants (J values) are in Hz. Low-resolution MS were recorded on a Shimadzu LCMS-DQ8000α and high-resolution MS on a JEOL HX110A or a Shimadzu LCMS-IT-TOF (225-07100-34) mass spectrometer. Analytical thin-layer chromatography (TLC) was carried out on silica gel 60 F254 aluminum-backed TLC plates (Merck). Preparative TLC was performed on Analtech silica gel 500 μm glass plates. Compounds were visualized with short-wavelength UV, by spraying with anisaldehyde−sulfuric acid spray reagent and heating until the spots appeared. Silica gel flash chromatography was accomplished using 230−400 mesh silica gel. Sephadex LH-20 for gel-permeation chromatography was obtained from Amersham Biosciences. HPLC purifications were carried out using a 10 × 250 mm Phenomenex Luna 5 μm C18 (2) column for reversed-phase (RP) chromatography and a 10 × 250 mm Econosil Si (10 μm) column for normal-phase (NP) chromatography, with a Waters Delta Prep system consisting of a PDA 996 detector. When required, MM2 energy minimizations of all possible conformers were carried out using Chembio3D Ultra 14.0 from CambridgeSoft Corp. Aeroponic Cultivation and Harvesting of P. crassifolia. The fruits of P. crassifolia were collected in the summer of 2009 in Yuma County, Arizona. Seeds obtained from dried fruits were germinated in 1.0 in. Grodan rock-wool cubes in a Barnstead Lab-line growth chamber kept at 28 °C with 16 h of fluorescent lighting and maintaining 25−50% humidity. After ca. 4 weeks in the growth chamber, seedlings with an aerial length of ca. 5.0 cm were transplanted to aeroponic culture boxes for further growth, as described previously for Withania somnifera.22 A few seedlings were transplanted from the grown chamber into pots containing soil, and the identity of the mature plants obtained was confirmed by comparison with a previous herbarium sample of P. crassifolia (accession number SPM7420) deposited at the University of Arizona Herbarium (ARIZ). Aerial parts of areoponically grown plants were harvested when they started to produce flowers (ca. 2 months under aeroponic growth conditions) and when fruits were almost mature (ca. 5 months under aeroponic growth conditions) and were processed separately. Harvested plant materials were dried in the shade, powdered, and stored at 5 °C prior to extraction. Extraction and Isolation of Withanolides from 2-Month-Old P. crassifolia. Dried and powdered plant material (50.0 g) was

drogen-insensitive metastatic prostate adenocarcinoma), MCF7 (breast adenocarcinoma), NCI-H460 (non-small-cell lung cancer), and SF-268 (CNS glioma) cells. Of these, only the 17β-hydroxy-18-acetoxywithanolides 1, 2, 6, 7, and 16 were found to be active, with many showing selectivity to the LNCaP and PC-3M cell lines. The data depicted in Table 4 together with our previously obtained data for 17β-hydroxy-18acetoxywithanolides (12−15, 17, and 19) and withaferin A (22)5 confirmed that in addition to the ring A 2(3)-ene-1-one and ring B 5β,6β-epoxide moieties the orientation of the side chain and/or the presence of a β-OH at C-17 was partly responsible for the potent and selective activity (IC50 values of 0.02−0.44 μM) to PC cell lines, especially LNCaP, when compared to other cancer cell lines. In addition, the absence of cytotoxic activity for 3−5, 8, and 9 at the highest concentration tested (5.0 μM) provided additional structure−activity relationship (SAR) information for the 17β-hydroxy-18-acetoxywithanolides, including decreases in activity due to (i) substitution at C-3 (7 vs 13 and 16 vs 12); (ii) isomerization of the 2(3) double bond to the 3(4) position (17 vs 12 and 19 vs 13); (iii) α-hydroxylation at C-15 (1 vs 12); (iv) α-acetoxylation at C-15 (7 vs 16, 19 vs 17, and 13 vs 12); (v) hydroxylation at C-27 (2 vs 12) and C-28 (14 vs 13), and (vi) oxyglucosylation at C-27 (4 vs 13) and C-28 (5 vs 13). These preliminary SAR data for substituents in rings C and D and the side chain of 17βhydroxy-18-acetoxywithanolides bearing a 17α side chain differed from those reported for withanolides with a 17β side chain without an OH at C-17,4c further suggesting that the orientation of the side chain and/or the presence of a β-OH at C-17 of withanolides may have a significant influence on their cytotoxic potency and selectivity to certain cancer cell lines. Further work on these two classes of withanolides to understand the effects of these and other structural features on their antiproliferative and other biological activities is currently in progress.



EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO Dip-370 polarimeter using MeOH as the solvent. UV spectra were recorded with a Shimadzu UV 2601 G

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extracted (3×) with MeOH (300 and 2 × 200 mL) in an ultrasonic bath at 25 °C for 1 h and filtered, and the combined filtrates were concentrated in vacuo to afford the crude extract (6.7 g). This extract was subjected to solvent−solvent partitioning using 80% MeOH(aq) (50 mL) and hexanes (3 × 50 mL). The 80% MeOH(aq) solution thus obtained was diluted with water to 50% MeOH(aq) and extracted with CHCl3 (3 × 30 mL). Combined CHCl3 extracts were concentrated, and the resulting CHCl3 fraction (755.0 mg) was subjected to column chromatography over RP silica gel (20.0 g) and eluted with 100 mL each of 70%, 80%, and 90% MeOH(aq) followed by MeOH. The fraction eluted with 80% MeOH(aq), which was found to contain most of the withanolides, was concentrated and further fractionated by RP HPLC with a solvent gradient of MeOH and water (flow rate, 3 mL/min; 0−15 min, 50% MeOH(aq); 16−60 min, 60% MeOH with UV detection at 250 nm). On the basis of the HPLC trace obtained, 10 fractions designated A−J [A (6.8 mg) at tR = 26.5 min; B (2.1 mg) at tR = 27.4 min; C (3.9 mg) at tR = 28.1 min; D (11.1 mg) at tR = 29.2 min; E (7.1 mg) at tR = 31.1 min; F (10.0 mg) at tR = 34.4 min; G (55.0 mg) at tR = 38.2 min; H (6.5 mg) at tR = 44.3 min; I (180.4 mg) at tR = 46.9 min; and J (8.3 mg) at tR = 58.1 min] were collected. Fractions A, D, and F−J, which contained withanolides, were further purified by silica gel NP HPLC using mixtures of CHCl3/ MeOH as eluants (3 mL/min, UV detection at 254 nm). Fraction A afforded 14 [6.1 mg, tR = 17.9 min, CHCl3/MeOH (97:3)]; D afforded 3 [6.5 mg, tR = 11.2 min, CHCl3/MeOH (97:3)]; F afforded 2 [6.8 mg, tR = 8.7 min, CHCl3/MeOH (97:3)]; G afforded 1 [48.9 mg, tR = 14.4 min, CHCl3/MeOH (97:3)]; H afforded 18 [6.0 mg, tR = 5.5 min, CHCl3/MeOH (97:3)]; I afforded 12 [168 mg, tR = 8.8 min, CHCl3/MeOH (97:3)] and 13 [14.2 mg, tR = 19.3 min, CHCl3/ MeOH (99:1); and fraction J afforded 17 [4.2 mg, tR = 8.0 min, CHCl3/MeOH (97:3)]. Extraction and Isolation of Withanolides from 5-Month-Old P. crassifolia. Dried, powdered aerial parts of P. crassifolia (1.0 kg) were extracted (3×) for 24 h each time with MeOH (1400, 800, and 800 mL) in a shaker at 25 °C and filtered. The resulting filtrates were combined and concentrated in vacuo to afford a crude extract (150.0 g). A portion (50.0 g) of this extract was subjected to solvent−solvent partitioning using 80% MeOH(aq) (200 mL) and hexanes (3 × 100 mL). The resulting 80% MeOH(aq) fraction was diluted with water to 50% MeOH(aq) and extracted with CHCl3 (3 × 100 mL). The combined CHCl3 extracts were concentrated under reduced pressure to afford a CHCl3 fraction (5.47 g). This fraction was subjected to column chromatography on RP C18 (100 g) and eluted with 200 mL each of 60%, 70%, 80%, and 90% MeOH(aq) and finally with MeOH to afford five fractions, A−E: A (698.0 mg) eluted with 60% MeOH(aq); B (608.0 mg) with 70% MeOH(aq); C (522.0 mg) with 80% MeOH(aq); D (1.63 g) with 90% MeOH(aq); and E (2.18 g) with MeOH. Further purification of fraction A (500.0 mg) by RP HPLC using a gradient solvent system (increasing MeOH concentration from 35% MeOH(aq) to 60% MeOH(aq) in 40 min) afforded subfractions A1 and A2. Subfraction A1 (72.8 mg) collected at tR = 15.4 min was separated by column chromatography over silica gel (10.0 g) and eluted with CHCl3/MeOH (8:2) to afford 6 (16.9 mg) and 7 (35.6 mg). Further purification of subfraction A2 (26.9 mg) collected at t R = 40.0 min by silica gel (25.0 g) column chromatography and elution with CHCl3/MeOH (85:15) afforded 5 (9.3 mg) and 16 (6.6 mg). Fraction B (608.0 mg) obtained above was subjected to further purification by RP HPLC using a gradient solvent system (increasing methanol concentration from 45% MeOH(aq) to 70% MeOH(aq) in 50 min) yielding eight subfractions (B1−B8) with retention times (tR values) of 20, 25, 27, 33, 35, 37, 40, and 42 min, respectively. TLC analysis of these indicated that only subfractions B5−B8 contained withanolides. Further purification of subfraction B5 (53.5 mg) by column chromatography over silica gel (20 g) and elution with CHCl3/MeOH (96:4) afforded 4 (24.8 mg) and 10 (5.5 mg). Similar purification of subfraction B6 (133.3 mg) gave 14 (124.0 mg) and 11 (5.4 mg). Subfraction B7 (32.2 mg) on further purification by silica gel (5.0 g) column chromatography and elution with CHCl3/ MeOH (95:5) afforded 2 (9.0 mg). Subfraction B8 (24.1 mg) on silica gel (20.0 g) column chromatography and elution with CHCl3/MeOH

(96:4) gave 9 (5.6 mg), 8 (4.8 mg), and 15 (2.4 mg). Fraction C (522.0 mg) resulting from the first column chromatographic separation was subjected to gel filtration chromatography on Sephadex LH-20 (100.0 g) eluted with CH2Cl2/hexanes (4:1). Fractions obtained were combined based on their TLC profiles to afford four subfractions, C1−C4. TLC investigation of these indicated that only C2 and C3 contained withanolides. Subfraction C2 (272.7 mg) on further purification by silica gel NP HPLC [CHCl3/MeOH (95:5), 3 mL/ min, UV detection at 254 nm) afforded 13 (95.0 mg, tR = 6.5 min) and 12 (105.0 mg, tR = 7.5 min). Subfraction C3 (28.2 mg) on further purification by RP HPLC (65% MeOH(aq), 3.0 mL/min, UV detection at 230 nm) gave 12 (15.5 mg, tR = 24.6 min). 15α-Hydroxyphysachenolide D (1): amorphous, colorless powder; [α]25 D +28 (c 0.16, MeOH); UV (MeOH) λmax (log ε) 222 (3.81) nm; CD (MeOH) [θ] +7976 (250 nm); 1H and 13C NMR data, see Tables 1 and 3, respectively; positive HRESIMS m/z 567.2570 [M + Na]+ (calcd for C30H40O9Na, 567.2570). 27-Hydroxyphysachenolide D (2): amorphous, colorless powder; [α]25 D +91 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 224 (4.19) nm; CD (MeOH) [θ] +17 131 (253 nm); 1H and 13C NMR data, see Tables 1 and 3, respectively; positive HRESIMS m/z 567.2570 [M + Na]+ (calcd for C30H40O9Na, 567.2570). 15α-Acetoxy-27-hydroxyphysachenolide D (3): amorphous, colorless powder; [α]25 D +65 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 222 (4.07) nm; CD (MeOH) [θ] +13 134 (254 nm); 1H and 13C NMR data, see Tables 1 and 3, respectively; positive HRESIMS m/z 603.2802 [M + H]+ (calcd for C32H43O11, 603.2805). 15α-Acetoxy-27-O-β-glucopyranosylphysachenolide D (4): amorphous, colorless powder; [α]25 D +49 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 221 (4.10) nm; CD (MeOH) [θ] +15 278 (254 nm); 1H and 13C NMR data, see Tables 1 and 3, respectively; positive HRESIMS m/z 765.3322 [M + H]+ (calcd for C38H53O16, 765.3334). 15α-Acetoxy-28-O-β-glucopyranosylphysachenolide D (5): amorphous, colorless powder; [α]25 D +43 (c 0.18, MeOH); UV (MeOH) λmax (log ε) 222 (4.07) nm; CD (MeOH) [θ] +13 037 (256 nm); 1H and 13C NMR data, see Tables 1 and 3, respectively; positive HRESIMS m/z 787.3131 [M + Na]+ (calcd for C38H52NaO16, 787.3153). 15α-Acetoxyphysachenolide C (6): amorphous, colorless powder; [α]25 D +80 (c 0.47, MeOH); UV (MeOH) λmax (log ε) 225 (3.94) nm; 1 H and 13C NMR data, see Tables 2 and 3, respectively; positive HRESIMS m/z 603.2805 [M + H]+ (calcd for C32H43O11, 603.2805). 15α-Acetoxy-2,3-dihydrophysachenolide D 3β-O-sulfate (7): amorphous, colorless powder; [α]25 D +81 (c 0.33, MeOH); UV (MeOH) λmax (log ε) 222 (3.83) nm; CD (MeOH) [θ] +15 527 (249 nm); 1H and 13C NMR data, see Tables 2 and 3, respectively; positive HRESIMS m/z 685.2525 [M + H]+ (calcd for C32H45O14S, 685.2530). 15α,18-Diacetoxy-28-hydroxy-17-epi-withanolide K (8): amorphous, colorless powder; [α]25 D +65 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 228 (3.97) nm; CD (MeOH) [θ] +19 361 (248 nm); 1H and 13C NMR data, see Tables 2 and 3, respectively; positive HRESIMS m/z 603.2792 [M + H]+ (calcd for C32H43O11, 603.2805). 23β-Hydroxyphysacoztolide E (9): amorphous, colorless powder; [α]25 D −57 (c 0.21, MeOH); UV (MeOH) λmax (log ε) 218 (4.04) nm; CD (MeOH) [θ] +2502 (261 nm); 1H and 13C NMR data, see Tables 2 and 3, respectively; positive HRESIMS m/z 529.2791 [M + H]+ (calcd for C30H41O8, 529.2801). 14α-Hydroxywithanolide A (10): amorphous, colorless powder; [α]25 D +62 (c 0.22, MeOH); UV (MeOH) λmax (log ε) 225 (4.03) nm; CD (MeOH) [θ] +9512 (252 nm); 1H and 13C NMR data, see Tables 2 and 3, respectively; positive HRESIMS m/z 509.2508 [M + Na]+ (calcd for C28H38O7Na, 509.2515). 16-Oxo-17(20)-dehydrohyoscyamilactol (11): amorphous, colorless powder; [α]25 D −19 (c 0.39, MeOH); UV (MeOH) λmax (log ε) 228 (3.80) nm; CD (MeOH) [θ] +3560 (261 nm); 1H and 13C NMR data, see Tables 2 and 3, respectively; positive HRESIMS m/z 507.2349 [M + Na]+ (calcd for C28H36O7Na, 507.2359). Acetylation of 15α-hydroxyphysachenolide D (1). To a solution of 1 (2.0 mg) in pyridine (1.0 mL) was added Ac2O (0.5 mL), and the reaction mixture was stirred at 25 °C for 5 h (TLC control). It was H

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(3) (a) Lavie, D.; Glotter, E.; Shvo, Y. J. Chem. Soc. 1965, 30, 7517− 7531. (b) Kupchan, S. M.; Doskotch, R. W.; Bollinger, P.; McPhail, A. T.; Sim, G. A.; Renauld, J. A. S. J. Am. Chem. Soc. 1965, 87, 5805− 5806. (c) Gupta, G. L.; Rana, A. C. Pharmacog. Rev. 2007, 1, 129−136. (4) (a) Yousuf, S. K.; Majeed, R.; Ahmad, M.; Sangwan, P. I.; Purnima, B.; Saxena, A. K.; Suri, K. A.; Mukherjee, D.; Taneja, S. C. Steroids 2011, 76, 1213−1222. (b) Nakano, D.; Ishitsuka, K.; Hatsuse, T.; Tsuchihashi, R.; Okawa, M.; Okabe, H.; Tamura, K.; Kinjo, J. J. Nat. Med. 2011, 65, 559−567. (c) Zhang, H.; Samadi, A. K.; Cohen, M. S.; Timmermann, B. N. Pure Appl. Chem. 2012, 84, 1353−1367. (d) Wijeratne, E. M. K.; Xu, Y.; Scherz-Shouval, R.; Marron, M. T.; Rocha, D. D.; Liu, M. X.; Costa-Lotufo, L. V.; Santagata, S.; Lindquist, S.; Whitesell, L.; Gunatilaka, A. A. L. J. Med. Chem. 2014, 57, 2851− 2863. (5) Xu, Y. M.; Liu, M. X.; Grunow, N.; Wijeratne, E. M. K.; PaineMurrieta, G.; Felder, S.; Kris, R. M.; Gunatilaka, A. A. L. J. Med. Chem. 2015, 58, 6984−6993. (6) Whitson, M.; Manos, P. S. Syst. Bot. 2005, 30, 216−230. (7) Kindscher, K.; Long, Q.; Corbett, S.; Bosnak, K.; Loring, H.; Cohen, M.; Timmermann, B. N. Econ. Bot. 2012, 66, 298−310. (8) Chen, L. X. I.; He, H.; Qiu, F. Nat. Prod. Rep. 2011, 28, 705−740. (9) 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. (10) 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. (11) Maldonado, E.; Torres, F. R.; Martinez, M.; Pérez-Castorena, A. L. Planta Med. 2004, 70, 59−64. (12) Maldonado, E.; Alvarado, V. E.; Torres, F. R.; Martinez, M.; Pérez-Castorena, A. L. Planta Med. 2005, 71, 548−553. (13) (a) Pérez-Castorena, A.-L.; Oropeza, R. F.; Vazquez, A. R.; Martinez, M.; Maldonado, E. J. Nat. Prod. 2006, 69, 1029−1033. (b) Zhang, H.; Cao, C. M.; Gallagher, R. J.; Day, V. W.; Kindscher, K.; Timmermann, B. N. Phytochemistry 2015, 109, 147−153. (14) Cao, C. M.; Zhang, H.; Gallagher, R. J.; Day, V. W.; Kindscher, K.; Grogan, P.; Cohen, M. S.; Timmermann, B. N. J. Nat. Prod. 2014, 77, 631−639. (15) Zhang, H.; Samadi, A. K.; Gallagher, R. J.; Araya, J. J.; Tong, X.; Day, V. W.; Cohen, M. S.; Kindscher, K.; Gollapudi, R.; Timmermann, B. N. J. Nat. Prod. 2011, 74, 2532−2544. (16) Guan, Y. Z.; Shan, S. M.; Zhang, W.; Luo, J. G.; Kong, L. Y. Steroids 2014, 82, 38−43. (17) Lan, Y. H.; Chang, F. R.; Pan, M. J.; Wu, S. J.; Chen, S. L.; Wang, S. S.; Wu, M. J.; Wu, Y. C. Food Chem. 2009, 116, 462−469. (18) Su, B.-N.; Misico, R.; Park, E. J.; Santarsiero, B. D.; Measecar, A. D.; Fong, H. H. S.; Pezzzuto, J. M.; Kinghorn, A. D. Tetrahedron 2002, 58, 3453−3466. (19) Glotter, E.; Sahai, M.; Kirson, I.; Gottlieb, H. E. J. Chem. Soc., Perkin Trans. 1 1985, 11, 2241−2245. (20) (a) Santagata, S.; Xu, Y. M.; Wijeratne, E. M. K.; Kotnik, R.; Rooney, C.; Perley, C. C.; Kwon, H.; Clardy, J.; Kesari, S.; Whitesell, L.; Lindquist, S.; Gunatilaka, A. A. L. ACS Chem. Biol. 2012, 7, 340− 349. (b) Tao, S.; Tillotson, J.; Wijeratne, E. M. K.; Xu, Y.; Kang, M.; Wu, T.; Lau, E. C.; Mesa, C.; Mason, D. J.; Brown, R. V.; La Clair, J. J.; Gunatilaka, A. A. L.; Zhang, D. D.; Chapman, E. ACS Chem. Biol. 2015, 10, 1916−1924. (21) Xu, Y. M.; Marron, M. T.; Seddon, E.; McLaughlin, S. P.; Ray, D. T.; Whitesell, L.; Gunatilaka, A. A. L. Bioorg. Med. Chem. 2009, 17, 2210−2214. (22) Xu, Y. M.; Gao, S.; Bunting, D. P.; Gunatilaka, A. A. L. Phytochemistry 2011, 72, 518−522. (23) Kasal, A.; Budesinsky, M.; Griffiths, W. J. In Steroid Analysis; Makin, H. L., Gower, D. B., Eds.; Springer Science: New York, 2010; Chapter 2, pp 99−102. (24) Liffert, R.; Hoecker, J.; Jana, C. K.; Woods, T. M.; Burch, P.; Jessen, H. J.; Neuburger, M.; Gademann, K. Chem. Sci. 2013, 4, 2851− 2857. (25) Gottlieb, H. E.; Kirson, I. Org. Magn. Reson. 1981, 16, 20−25. (26) Ma, C.-Y.; Williams, I. D.; Che, C.-T. J. Nat. Prod. 1999, 62, 1445−1447.

then poured into ice water (10.0 mL), and the resulting suspension was passed through a column of RP C18 silica gel (1.0 g). The column was washed with water (5.0 mL) followed by MeOH (5.0 mL). Evaporation of MeOH provided the crude product, which on column chromatography over silica gel (1.0 g) and elution with CHCl3/ MeOH (98:2) afforded a colorless, amorphous powder (1.9 mg). The 1 H NMR and APCI-MS data of the product were identical with those of 13.5,13b Epoxidation of 15α-acetoxyphysachenolide D (13). To a stirred solution of 13 (10.0 mg) in CH2Cl2 (2.0 mL) was added mchloroperbenzoic acid (5.0 mg), and the mixture was stirred at 25 °C. After 3 h (TLC control), the reaction mixture was partitioned between CHCl3 (10.0 mL) and water (20.0 mL). The CHCl3 layer was washed with water (20.0 mL), dried (Na2SO4), and subjected to purification by silica gel preparative TLC [CHCl3/MeOH (95:5)] to afford the major β epoxidation product as a pale, amorphous powder (4.0 mg). The 1H and 13C NMR and APCI-MS data of this product were identical with those of 6 obtained above. Cytotoxicity Assay. A resazurin-based colorometric (AlamarBlue) assay28 was used for evaluating the cytotoxicity of withanolides 1−11 against androgen-sensitive human prostate adenocarcinoma (LNCaP), metastatic human prostate adenocarcinoma (PC-3M), human breast (MCF-7), human non-small-cell lung (NCI-H460), and human CNS glioma (SF-268) cancer cell lines. Doxorubicin and DMSO were used as positive and negative controls, respectively.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00911. 1 H, 13C, and 2D NMR spectra of withanolides 1−11, key HMBC and NOE correlations for 2, 3, 5, 6, 9, and 10, and CD spectra of withanolides 1−5 and 7−11 (PDF)



AUTHOR INFORMATION

Corresponding Author

*Tel: (520) 621-9932. Fax: (520) 621-8378. E-mail: leslieg1@ email.arizona.edu. Present Address †

Biology Department, Pima Community College West Campus, 2202 W. Anklam Road, Tucson, Arizona 85709, United States.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS Financial support for this work was provided by grants R01 CA090265 (A.A.L.G.) and 5K12 GM000708-14 (H.A.B.) funded by NCI, NIH, and NIGMS, NIH, respectively. We thank Drs. K. Wijeratne, Natural Products Center, and H.-J. Kang, Department of Pharmacology and Toxicology, University of Arizona, for obtaining the CD spectra, and Prof. M. C. F. ́ Oliveira of Departamento de Quimica Orgânica e Inorgânica, Universidade Federal do Ceará, Brazil, for obtaining HRESIMS data for some of the withanolides.



REFERENCES

(1) Glotter, E. Nat. Prod. Rep. 1991, 8, 415−440. (2) Misico, R. I.; Nicotra, V. E.; Oberti, J. C.; Barboza, G.; Gil, R. R.; Burton, G. In Progress in the Chemistry of Organic Natural Products; Kinghorn, A. D.; Falk, H.; Kobayashi, J., Eds.; 2011; Vol 94, pp 127− 22910.1007/978-3-7091-0748-5_3. I

DOI: 10.1021/acs.jnatprod.5b00911 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

(27) Gil, R. R.; Misico, R. I.; Sotes, I. R.; Oberti, J. C. J. Nat. Prod. 1997, 60, 568−572. (28) Wijeratne, E. M. K.; Bashyal, B. P.; Liu, M. X.; Rocha, D. D.; Gunaherath, G. M. K. B.; U'Ren, J. M.; Gunatilaka, M. K.; Arnold, A. E.; Whitesell, L.; Gunatilaka, A. A. L. J. Nat. Prod. 2012, 75, 361−369.

J

DOI: 10.1021/acs.jnatprod.5b00911 J. Nat. Prod. XXXX, XXX, XXX−XXX