Antiproliferative Withanolides from Datura wrightii

Abstract

A new withanolide (1) named withawrightolide, and four known withanolides (2–5) were isolated from the aerial parts of Datura wrightii (Solanaceae). The structure of compound 1 was elucidated through 2D NMR and other spectroscopic techniques. In addition, the structure of withametelin L (2) was confirmed by X-ray crystallographic analysis. Using MTS viability assays, withanolides 1–5 showed antiproliferative activities against human glioblastoma (U251 and U87), head and neck squamous cell carcinoma (MDA-1986), and normal fetal lung fibroblast (MRC-5) cells with IC₅₀ values in the range between 0.56 and 5.6 μM.

Withanolides are a group of modified, highly-oxygenated C₂₈ ergostane-type steroids, present primarily in several genera of the Solanaceae which include Acnistus, Datura, Dunalia, Jaborosa, Physalis, and Withania. In recent years these compounds have gained significant scientific interest due to their structural and biological diversity, in conjunction with their antitumor capacities.^1–3 Recently we reported, as part of an ongoing study, the isolation and antiproliferative activities of a series of withanolides from Physalis longifolia, Vassobia brevifolia, and Withania somnifera with oxygenation at C-1, 3, 4, 5, 6, 7, 11, 17, 19, 20, 22, 27 and 28.^4–7

The Datura genus is a rich source of oxygen substituted C-21 withanolides² yet there have been limited biological activity studies. We therefore chose to investigate the native Kansas plant Datura wrightii Regel to continue our Solanaceae derived withanolide work, and to further probe withanolide structure-activity relationships.^3,4

We report herein the first phytochemical and bioactivity study of withanolides from D. wrightii including all details pertaining to the isolation, structure elucidation, and cytotoxicity (using MTS viability assays) of the new withanolide withawrightolide (1) and four known withanolides (2–5).

Compounds 1–5 were isolated from a CH₂Cl₂–MeOH (1:1) extract of the aerial parts of the title plant (see Experimental Section). The molecular formula of the minor component 1 was determined to be C₂₈H₃₈O₅ by HRESIMS and NMR experiments, equating to ten double-bond equivalents. The IR absorptions of 1 indicated the presence of OH (3435 cm⁻¹), keto and ester (1740 and 1705 cm⁻¹) groups. The ¹H NMR spectrum (Table 1) showed signals of three methyl groups at δ 0.72 (3H, s), 1.12 (3H, s), 1.42 (3H, s); four protons attached to oxygenated carbons at δ 3.31 (1H, t, J = 2.9 Hz), 3.71 (1H, dd, J = 3.3, 13.3 Hz), 3.87 (1H, d, J = 13.3 Hz), 4.64 (1H, brs); and two olefinic methine groups at δ 6.00 (1H, s) and 6.75 (1H, s). The ¹³C NMR (APT) and HSQC spectra for 1 (Table 1) displayed 28 carbon signals differentiated as three CH₃, ten CH₂ (including an olefinic at δ 130.3 and an oxygenated at δ 60.7), eight CH (including two oxygenated at δ 75.7 and 73.1), and seven C (including a keto carbonyl at δ 217.7, an ester carbonyl at δ 165.4, an olefinic at δ 139.0, and an oxygenated at δ 69.5), corresponding to C₂₈H₃₇. The remaining hydrogen atom was therefore assigned as an OH group, indicating that seven rings must be present in the structure.

Table 1.

¹H (500 MHz) and ¹³C (125 MHz) NMR Data for Withawrightolide 1 in CDCl₃

Position	δc, type	δH (J in Hz)	Position	δc, type	δH (J in Hz)
1	217.7, C		15	24.1, CH2	1.73, m; 1.26, m
2	39.6, CH2	2.81, ddd (1.7, 5.4, 18.1) 2.14, d (18.1)	16	26.7, CH2	1.74, m; 1.42, m
3	16.0, CH	1.39, m	17	47.7, CH	1.75, m
4	17.2, CH2	0.065, dd (3.8, 5.8) 0.78, ddd (1.7, 5.8, 5.9)	18	13.0, CH3	0.72, s
5	36.0, C		19	15.0, CH3	1.12, s
6	73.1, CH	3.31, t (2.9)	20	39.8, CH	1.84, m
7	37.4, CH2	1.96, m; 1.27, m	21	60.7, CH2	3.87, d (13.3); 3.71, dd (3.3, 13.3)
8	29.3, CH	1.86, m	22	75.7, CH	4.64, brs
9	47.4, CH	0.97, ddd (3.4, 10.7, 12.2)	23	33.5, CH2	2.0, dd (1.7, 9.0); 1.90, m
10	52.6, C		24	69.5, C
11	22.2, CH2	1.42, m; 1.34, m	25	139.0, C
12	39.5, CH2	1.89, m; 1.31, m	26	165.4, C
13	43.3, C		27	130.3, CH2	6.75, s; 6.00, s
14	55.8, CH	1.15, m	28	25.9. CH3	1.42, s

The NMR data of 1 were closely related to, a major isolate of our investigation, the six-ringed withanolide withametelin L (2) [(20R,22R,24R)-21,24-epoxy-12β-hydroxy-1-oxowitha-2,25,25(27)-trienolide].⁸ The structure of 2 was confirmed by X-ray crystallographic studies as shown in Figure 1. Compounds 1 and 2 were found to contain identical bicyclic side chain moieties; an exocyclic double bond [an olefinic methylene C-27 at δ_C 130.3 and two singlet olefinic protons at δ_H 6.00 and 6.75 (each 1H, s, H₂-27)] conjugated with the lactone carbonyl (C-26: δ_C 165.4); characteristic signals for oxygenated C-21 [a methylene with δ_C 60.7 and two protons at δ_H 3.87 (1H, d, J = 13.3 Hz), 3.71 (1H, dd, J = 13.3, 3.3 Hz)] and C-22 [a methine with δ_C 75.7 and δ_H 4.64 (1H, brs)], and a tertiary methyl group C-28 with δ_C 25.9 and δ_H 1.42 (3H, s) adjacent to the oxygenated quaternary C-24 with δ_C 69.5.

Figure 1.

X-ray ORTEP drawing of withametelin L (2)

The differences between 1 and 2 were observed within the steroid nucleus moieties. Compound 1 showed unusual NMR signals for a keto group (δ 217.7) and a methylene group [δ_C 17.2, δ_H 0.78 (1H, ddd, J = 1.7, 5.8, 5.9 Hz) and 0.065 (1H, dd, J = 3.8, 5.8 Hz)], implying the presence of a five-membered keto ring (the chemical shift value of a keto group in a six-membered-ring is less than 210 ppm) and a cyclopropane ring (when considering the markedly low chemical shift value at δ_H 0.065 and small geminal coupling constant 5.8 Hz), respectively. The presence of 1-oxo-3,5-cyclo-6-hydroxy in the A/B rings of the steroid nucleus was deduced by HSQC and ¹H-¹H COSY fragment of -C(2)H₂-C(3)H-C(4)H₂-; HMBC correlations between H₃-19 (δ 1.12, 3H, s) and C-1 (δ 217.7), C-5 (δ 36.0), C-9 (δ 47.4), and C-10 (δ 52.6); between H-3 (δ 1.39, 1H, m) and C-1, C-5, and C-6 (δ 73.1). The presence of a three-membered ring formed by C-3, C-4, C-5 was also supported by the chemical shift values of C-3 (CH, δ 16.0) C-4 (CH₂, δ 17.2), and C-5 (C, δ 36.0). Thus, the planar structure of 1 was represented as shown.

As for the stereochemistry, β orientation of the H-3 proton was assigned based on the ROESY correlation between H-3 and H₃-19. Axial β orientation of the OH group at C-6 was established by the small coupling constant between H-6 (δ 3.31, t, J = 2.9 Hz) and H₂-7, and the ROESY correlation between H-6 and H-4α (δ 0.78, 1H, ddd, J = 1.7, 5.8, 5.9 Hz). These NMR data and assignments were in good agreement with those withanolides reported in the literature with a 1-oxo-3,5-cyclo-6-hydroxy functionality in the A/B rings of the steroid nucleus.^9,10 Accordingly, the structure of 1 was elucidated as (20R,22R,24R)-21,24-epoxy-3α,5α-cyclo-1-oxowitha-25(27)-enolide on the basis of biogenetic grounds, and subsequently named withawrightolide. Detailed ¹H and ¹³C NMR spectrometric analysis utilizing 2D correlational techniques were undertaken and summarized in Table 1.

The four other withanolides were identified through data comparisons with those published in the literature, as withametelin L (2),⁸ withametelin also known as daturilin (3),^11,12 withametelin O (4),⁸ and withametelin F (5).¹³

Withawrightolide 1 contains a cyclopropane ring, a rarity in withanolides. A literature investigation showed that only five of the 820 withanolides reported to date contain a cyclopropane ring: physalin S isolated from Physalis alkekengi var. francheti,⁹ cilistols p, pm, p1 and u from Solanum cilistum (Solanaceae).¹⁰ This is the first report of such a structural type³ present in Datura species.

All the five withanolides (1–5) isolated were tested for cytotoxicity against human glioblastoma (U251 and U87), head and neck squamous cell carcinoma (MDA-1986), and normal fetal lung fibroblast (MRC-5) cells, the results of which were summarized in Table 2. Withanolides 1–5 demonstrated cytotoxicity with IC₅₀ values ranging from 0.56–3.6 μM in the cancer cells and 3.3–5.6 μM in the normal fibroblasts. Specifically, concentrations required to obtain cytotoxicity in the normal MRC-5 cells were 3.8–7.4 fold higher than those in U87 cells. Our previously published work on a commonly known withanolide, withaferin A,^4,13 is provided as a control for comparison. The potencies of 1–5 are on average slightly lower than withaferin A, suggesting that oxygenation of C-21 did not contribute toward the observed antiproliferative activities. Furthermore, the comparison of withanolides 2 (12β-hydroxy 3) and 3 revealed that oxygenation of C-12 is also not a contributor for antiproliferative activities. These observations are in good agreement to those reported in the literature.³ On the other hand, withanolides 1–5 appear to have greater selectivity than withaferin A for cancer cells (especially in the glioma and head and neck squamous cancer cells tested) compared to normal cells such as fibroblasts with average selectivity of between 4 and 7 fold. Such a level of selectivity may provide for a larger therapeutic window for treatment in vivo.

Table 2.

Cytotoxicity (IC₅₀) of Isolated Withanolides (μM) against Four Cell Lines^a

Compound	U87	U251	MDA-1986	MRC-5	MRC-5:U87
1	1.5	3.6	3.1	5.6	3.8
2	0.57	1.3	2.3	4.2	7.4
3	1.0	2.2	2.7	5.1	5.1
4	1.1	2.8	3.0	4.6	4.1
5	0.56	1.4	1.5	3.3	6.0
Withaferin Ab	1.1	0.69	0.80	0.20	0.19

^aFor cell lines used, see text.

^bWithaferin A was used as control.

Due to the observed proliferation inhibition of the withanolides 1–5, further U251cell-based western blot analysis was performed. Total cell expression of heat shock protein 70 (HSP70) and HSP32, proteins associated with cellular stress and withaferin A activity,¹³ were monitored 24 hours post-treatment (Figure 2). Post-treatment HSP70 upregulation was observed for 5 μM concentrations of withanolides 2, 3, and 5. Withanolides, 1–5, elevated HSP32 levels in a concentration-dependent manner.

Figure 2.

Western blot analysis of glioblastoma cell line U251 following treatment with withanolides 1–5. Total levels of HSP32 and HSP70, biomarkers of cellular stress and withanolide activity, were screened at 0, 0.1, 0.5, 1, and 5 μM concentrations of each compound for 24h. HSP70 was markedly upregulated at 5 μM with withanolides 2, 3, and 5, while HSP32 levels were elevated in a concentration-dependent manner with all compounds 1–5 tested. The β-actin protein was used as a loading control marker.

In this report, the heat shock effect and oxidative stress effect was abrogated following administration of N-acetyl-cysteine to the cells. This stress response effect was only observed with compounds 2, 3, and 5. It is likely that the epoxide at C5–6 in compound 5 may lead to free radical or reactive-oxygen species formation that could explain an oxidative stress on the cells. Additionally the double bond at C5–6 (compounds 2 and 3) may also convey some of the biologic activity seen with these molecules as compounds 1 and 4 lack either an epoxy group or a double bond at C5–6 and do not exhibit this stress response following treatment. Further studies, however, are warranted to validate this observation and more completely evaluate the mechanism of antiproferative action of these withanolides.

EXPERIMENTAL SECTION

General Experimental Procedures

Optical rotations were measured with a Rudolph RS Autopol IV automatic polarimeter. IR data were obtained with a Thermo Nicolet Avatar 360 FT-IR spectrometer. NMR spectra were recorded with a Bruker AV-400 or AV-500 instrument with a cryoprobe for ¹H, APT, COSY/DQF-COSY, HSQC, HMBC, and NOESY/ROESY. Chemical shift values are given in δ (ppm) using the peak signals of the solvent CDCl₃ (δ_H 7.26 and δ_C 77.23) as references and coupling constants were reported in Hz. ESIMS data were measured with an Agilent 1200 Series LC-MS/MS ion trap 6300 mass spectrometer. HRESIMS data were collected with a LCT Premier time of flight mass spectrometer (Waters Corp., Milford, MA). Column chromatography was performed on silica gel (particle size 12–25 μm) (Sorbent Technologies, Atlanta, GA), or MCI CHP20P (particle size 75–150 μm) (Sigma-Aldrich, St. Louis, MO), or Sephadex LH-20 (GE Healthcare, Piscataway, NJ), or C₁₈ reversed-phase silica gel (particle size 40–65 μm) (Sigma-Aldrich, St. Louis, MO). Normal-phase silica gel G TLC plates (w/UV 254) and reversed-phase C₁₈ TLC plates (w/UV 254) (Sorbent Technologies, Atlanta, GA) were used for fraction and compound detection. The spots were visualized using UV light at 254 nm and spraying with 10% EtOH-sulfuric acid reagent. Semi-preparative HPLC was performed on an Agilent 1200 unit equipped with a DAD detector, utilizing a Lichrospher RP-18 column (250 × 10 mm, 5 μm).

Plant Material

The fresh aerial parts of D. wrightii were collected in July 2010 from the Quivira National Wildlife Refuge (latitude: 38.10165°; longitude: 98.45496°), Stafford County, Kansas and authenticated by Dr. Kelly Kindscher of the Kansas Biological Survey, University of Kansas, Lawrence, Kansas, United States. A voucher specimen, 4091, was deposited in the R.L. McGregor Herbarium of the University of Kansas.

Extraction and Isolation

The collected biomass was air dried, ground to a coarse powder (2.1 Kg), and extracted three times with CH₂Cl₂–MeOH (50:50, 6.0 L) at room temperature. After removing the solvents under vacuum, the extract (220 g) was suspended in 400 mL H₂O, followed by successive partitions with n-hexane, ethyl acetate and n-butanol (3 × 500 mL). The resulting n-BuOH fraction (52 g) collected was applied to a MCI CHP20P column (2.0 Kg) and eluted subsequently with mixtures of H₂O–MeOH (100:0, 80:20, 60:40, 40:60, 85:15, 0:100), in order of increasing concentrations of MeOH. The 85% MeOH fraction (12.0 g) was subjected to silica gel CC, eluted with CH₂Cl₂-CH₃COCH₃ with increasing amounts of acetone to afford compounds withametelin L 2 (340 mg), withametelin also known as daturilin 3 (150 mg), withametelin O 4 (12 mg), and withametelin F 5 (7 mg). The fractions eluted by CH₂Cl₂-CH₃COCH₃ (6:1) were subjected to semi-preparative HPLC, with the mobile phase CH₃CN-H₂O (44:56), to afford 1 (8 mg).

Withawrightolide (1): amorphous powder; [α]²⁵_D +19.2 (c 0.18, CHCl₃); UV (MeOH) λ_max (log ε) 226 (3.05) nm; IR (neat) ν_max 3435 (br), 2922, 1740, 1705 cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1; ESIMS (positive-ion mode) m/z 455 [M + H]⁺; HRESIMS m/z 477.2610 [M + Na]⁺ (calcd for C₂₈H₃₈O₅Na, 477.2617).

Single-Crystal X-ray Structure Determination of withametelin L (2)

Crystal analysis was performed with a colorless cubic crystal (dimensions 0.48 × 0.12 × 0.05 mm³) obtained from CH₂Cl₂–CH₃CN (1:1) using Cu Kα radiation (λ = 1.54178 Å) on a Bruker APEX2 diffractometer equipped with a Bruker MicroStar microfocus rotating anode X-ray source and Helios multilayer optics. Crystal data for 2: C₂₈H₃₆O₅ (formula weight 452.57), monoclinic, space group P2₁, T = 100(2) K, crystal cell parameters a = 11.7590(3) Å, b = 6.5766(2) Å, c = 15.0921(4) Å, β = 96.0790(10)°, V = 1160.57(10) ų, D_c = 1.295 Mg/m³, Z = 2, F(000) = 488, absorption coefficient μ = 0.700 mm⁻¹. A total of 10166 reflections were collected in the range 2.94 < θ < 69.26°, with 2971 independent reflections [R_(int) = 0.0155], completeness to θ = 66° was 96.5%. Multi-scan absorption correction applied; full-matrix least-squares refinement on F², the number of data/restraints/parameters were 2971/1/443; goodness-of-fit on F² = 1.060; final R indices [I > 2σ(I)], R₁ = 0.0249, ωR₂ = 0.0646; R indices (all data), R₁ = 0.0249, ωR₂ = 0.0646; largest difference peak and hole, 0.169 and −0.126 e/Å⁻³.

Cell Culture and Cell Proliferation and Viability Assay

Two human glioblastoma cell lines, U251 and U87, were generously provided by Dr. Jann Sarkaria (Mayo Clinic, Rochester, MN). The head and neck squamous cell carcinoma (HNSCC) cell line MDA-1986 was a gift from Dr. Jeffrey Myers (University of Texas, M.D. Anderson Cancer Center, Houston, TX). The fetal lung fibroblast cell line MRC-5 was obtained from ATCC. Cell maintenance, experimental procedures, and data presentation were as similar as previously described.^4,14 The U87 and U251 cell lines were grown in DMEM (#6429; Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO) and 1% penicillin/streptomycin (100 IU/mL/100 μg/mL; Sigma-Aldrich). The MDA-1986 and MRC-5 cell lines were grown in similar media but also supplemented with 1% L-glutamine (200 mM; Sigma-Aldrich), 1% MEM-vitamin (100x; Hyclone, Logan, UT), and 1% MEM-nonessential amino acids (Sigma Aldrich). The cells were grown in a monolayer and were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ until they reached 75–90% confluence level. The viability of cells after treatment with withanolides (1–5) were determined using an MTS assay. Cells were seeded in 96-well plates at 2,500 cells/well in 90 μL of media. Following an approximate 6 h incubation period, 10 μL of drug-containing media in various concentrations was added to each well, and the cells were incubated for an additional 72 h. The number of viable cells was quantified by the colorometric CellTiter96 Aqueous MTS assay (Promega, Fitchburg, WI) at 490 nm on a BioTek Synergy 2 plate reader (BioTek, Winooski, VT) as per the manufacturer’s instructions. All experiments were carried out in triplicate on two separate occasions, and GraphPad (GraphPad Inc., San Diego, CA) was used to generate best-fit sigmoidal dose response curves for IC₅₀ determination.

Western Blotting Analysis

Cells were plated at an amount previously determined, based on the growth characteristics of the cells, to have a high enough yield without achieving complete confluency upon harvest and were allowed to grow overnight. Cells were treated with appropriate concentrations of the withanolides. Upon completion of treatment, proteins were collected, quantified, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and electrotransfered onto a Hybond nitrocellulose membrane as previously described.⁴ Actin levels were assessed to ensure equal loading and transfer of proteins. Western analysis was completed in the human U251 cell line. Primary antibodies were utilized against HSP70 (Enzo Life Sciences, Farmingdale, NY; ADI-SPA-810; 1:1000), HSP32/heme oxygenase 1 (Life Sciences, Farmingdale, NY; SPA-894; 1:1000), and total actin (EMD Millipore, Billerica, MA; MAB1501; 1:50,000). Donkey anti-rabbit IgG HRP (sc-2313; 1:5000) and goat anti-mouse IgG HRP (sc-2005; 1:5000) secondary antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, CA).