Withanolides from the stems and leaves of Physalis pubescens and their cytotoxic activity

Steroids 115 (2016) 136–146

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Withanolides from the stems and leaves of Physalis pubescens and their cytotoxic activity Guiyang Xia a,b, Yang Li a, Jiawen Sun a, Liqing Wang a, Xiaolong Tang a, Bin Lin c, Ning Kang b, Jian Huang a, Lixia Chen a,⇑, Feng Qiu a,b,⇑ a Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China b Tianjin State Key Laboratory of Modern Chinese Medicine and School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, People’s Republic of China c Department of Medicinal Chemistry, School of Pharmaceutical Engineering, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 4 July 2016 Received in revised form 17 August 2016 Accepted 7 September 2016 Available online 10 September 2016 Keywords: Physalis pubescens Withanolides Cytotoxic activity

a b s t r a c t A phytochemical study of Physalis pubescens L. afforded twelve compounds, including six new withanolides (1, 4, and 6i–9), four new withanolide glucosides (2, 3, 5, and 6), and two known withanolides (10 and 11). Their structures were established via extensive spectroscopic analysis. The absolute configuration of 3 was assigned using X-ray crystallography, and the absolute configurations of the 1,2-diol moiety in 1 were determined using the in situ dimolybdenum electronic circular dichroism method. Compounds 7, 9, and 10 exhibited significant cytotoxicity against human prostate cancer cells (C4-2B and 22Rvl), human renal carcinoma cells (786-O, A-498, Caki-2, and ACHN), human melanoma cells (A375 and A375-S2), and human normal hepatic cell line (L02) with IC50 values in the range of 0.17–5.30 lM. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction The genus Physalis (family Solanaceae) includes approximately 120 species worldwide, which are mainly distributed in tropical and temperate regions of America and in temperate regions of Europe and Asia. Five species and two varieties are found in China [1]. This genus has been reported to be a rich source of withanolides, a group of naturally occurring C28 steroids that are built on an ergostane skeleton and functionalized at C-1, C-22, and C-26 [2–4]. In recent years, withanolides have attracted researchers’ interest mainly due to their structural variety and significant biological activities, such as cytotoxic [5–7], antiinflammatory [8,9], and immunosuppressive [10] activities. Physalis pubescens L., which is also known as Deng-Long-Cao in China, has been used as a traditional folk medicine to treat sore ⇑ Corresponding authors at: Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China (L. Chen). School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, People’s Republic of China (F. Qiu). E-mail addresses: [email protected] (L. Chen), [email protected] (F. Qiu). http://dx.doi.org/10.1016/j.steroids.2016.09.002 0039-128X/Ó 2016 Elsevier Inc. All rights reserved.

throat, cough, urethritis, hematuria, and orchitis [11,12]. Earlier phytochemical investigations on this species have characterized several withanolides [13,14], some of which contain an a, b-unsaturated ketone in ring A, a 5b,6b-epoxy group in ring B, or a ninecarbon side chain with an a, b-unsaturated d-lactone group. These structural moieties were reported to be effective for increasing the cytotoxic activity of withanolides [4,6]. Our recent findings also suggest that withanolides could be potentially useful in the treatment of human tumors [15–18]. In our continuing search for the antiproliferative constituents of this plant, herein are reported the isolation and structure elucidation of six new withanolides (1, 4, and 6i–9), four new withanolide glucosides (2, 3, 5, and 6), and two known withanolides (10 and 11) (Fig. 1), from the crude extract of the stems and leaves of P. pubescens. X-ray crystallographic analysis was applied to determine the absolute configuration of 3. The in situ dimolybdenum electronic circular dichroism (ECD) method was used to assign the absolute configuration of 1. The isolates were tested for their cytotoxicity against eight human tumor cell lines (C4-2B, 22Rvl, 786-O, A-498, Caki-2, ACHN, A375, and A375-S2) and human normal hepatic cell line (L02). In this paper, we describe our results of the isolation, structural elucidation, and cytotoxic effect of these isolated withanolides.

G. Xia et al. / Steroids 115 (2016) 136–146

137

Fig. 1. Structures of compounds 1–11.

2. Experimental

2.2. Plant material

2.1. General methods

Physalis pubescens L. were collected from Shenyang, Liaoning Province, China, and identified by Professor Jincai Lu, Department of Pharmaceutical Botany, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University. A voucher specimen (CP-X20110829) has been deposited in the herbarium of the Department of Natural Products Chemistry, Shenyang Pharmaceutical University.

ECD spectra were determined on a Bio-Logic Science MOS-450 spectrometer. Optical rotations were measured with a PerkinElmer 241 polarimeter. Melting points were determined on an X-4 digital display micromelting point apparatus. UV spectra were recorded on a Shimadzu UV 2201 spectrophotometer, and IR (4000– 400 cm
1) spectra (KBr pellets) were recorded on a Bruker IFS 55 spectrometer. NMR experiments were performed on Bruker ARX300, AV-400, and AV-600 spectrometers. Chemical shifts given in d (ppm) using the peak signals of the solvents pyridine-d5 (dH 8.74, 7.58, and 7.22; and dC 150.35, 135.91, and 123.87), CDCl3 (dH 7.24 and dC 77.23) or CD3OD (dH 3.34 and dC 49.86) as references, and coupling constants are reported in Hz. HRESIMS were obtained on an Agilent 6210 TOF mass spectrometer. GC was carried out on an Agilent GC-series system and performed with an HP-5 column (30 m  0.25 mm  0.25 lm, Agilent, Santa Clara, CA). Silica gel GF254 prepared for TLC and silica gel (200–300 mesh) for column chromatography (CC) were obtained from Qingdao Marine Chemical Factory (Qingdao, People’s Republic of China). Sephadex LH-20 was a product of Pharmacia. Octadecyl silica gel was purchased from Merck Chemical Company Ltd (Germany). HPLC analyses were performed on a Waters HPLC instrument (Waters, USA) equipped with a Waters 996 photodiode array detector. RP-HPLC separations were conducted using an LC-6AD (Shimadzu, Kyoto, Japan) liquid chromatograph with the YMC Pack ODS-A columns (250  20 mm, S-10 lm, 12 nm; 250  20 mm, S5 lm, 12 nm) and SPD-10A VP UV/vis detector or RID-10A detector. All reagents were HPLC or analytical grade and were purchased from Tianjin Damao Chemical Company. Spots were detected on TLC plates under UV light or by heating after spraying with anisaldehyde/H2SO4 reagent.

2.3. Extraction and isolation The air-dried stems and leaves of P. pubescens L. (9.3 kg) were cut into approximately 2 cm pieces and extracted with EtOH/H2O (75:25 v/v) (2  100 L). The resulting extract (1.2 kg) was concentrated in vacuum, suspended in H2O (3.0 L), and partitioned successively with petroleum ether (PE) (3  3.0 L), EtOAc (3  3.0 L), and n-BuOH (3  3.0 L) to give EtOAc (99 g) and n-BuOH (100 g) fractions. The n-BuOH fraction (100 g) was subjected to silica gel CC (10  60 cm) and eluted with CH2Cl2/MeOH (100:1, 40:1, 20:1, 10:1, 4:1, 2:1, 1:1, and 0:1 v/v) to obtain nine combined subfractions (B1–B9). Fraction B2 (7.5 g) was subjected to a silica gel column (6  60 cm) and eluted with CH2Cl2/MeOH (from 30:1 to 0:1 v/v) to produce three subfractions (B21–B23). B22 (340 mg) was separated using preparative TLC (CH2Cl2/MeOH) and further purified via preparative HPLC (MeOH/H2O, 70:30 v/v) to obtain 1 (23.0 mg). Fraction B3 (15.2 g) was separated using a silica gel column (6  80 cm) with a gradient of MeOH in CH2Cl2 that ranged from 2% to 100% to afford six fractions (B31–B36). B35 (1.6 g) was subjected to RP-C18 CC (2.5  30 cm) that was eluted with MeOH/H2O (from 10:90 to 90:10 v/v) to produce eight subfractions (B351–B358). B356 (206 mg) was further purified on a Sephadex LH-20 column (2.5  80 cm) via elution with MeOH to produce 2

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(101 mg). Fraction B6 (12 g) was subjected to purification via a silica gel column (5.0  60 cm) eluted with isocratic CH2Cl2/MeOH (6:1 v/v) to produce three fractions (B61–B63). B63 (2.3 g) was chromatographed over RP-C18 CC (2.5  30 cm) that was eluted with MeOH/H2O (from 20:80 to 80:20 v/v) with increasing amounts of MeOH and further purified via preparative HPLC (MeOH/H2O, 70:30 v/v) to yield 6 (75.0 mg). Fraction B7 (30.0 g) was separated via silica gel CC (10  50 cm) and eluted with CH2Cl2/MeOH (from 8:1 to 3:1 v/v) to produce four subfractions (B71–B74). B74 (2.3 g) was subjected to a polyamide column (3.0  40 cm) and eluted with H2O/EtOH (from 100:0 to 0:100 v/v) to produce B741 and B742. B742 (220 mg) was chromatographed on a Sephadex LH-20 column (2.5  80 cm) that was eluted with MeOH and further separated via preparative HPLC (MeOH/H2O, 50:50 v/v) to yield 3 (43.0 mg). Fraction B8 (5.0 g) was subjected to silica gel CC (4.0  60 cm) eluting with isocratic CH2Cl2/MeOH (5:1 v/v) to produce three fractions (B81–B83). B81 (3  0.7 g) was applied to a Sephadex LH-20 column (3.0  80 cm) that was eluted with MeOH and further separated via preparative HPLC (MeOH/H2O, 60:40 v/v) to yield 5a/5b (320.0 mg). The EtOAc fraction (99 g) was subjected to a silica gel CC (10  50 cm) that was eluted with CH2Cl2/MeOH (100:1, 40:1, 20:1, 10:1, 4:1, 2:1, 1:1, and 0:1 v/v) to obtain nine combined sub-

fractions (E1–E9). Fraction E3 (19.0 g) was subjected to a silica gel CC (6  80 cm) and eluted with CH2Cl2/acetone (from 100:1 to 0:1 v/v) to produce eight subfractions (E31–E38). E35 (56.0 mg) was chromatographed on a Sephadex LH-20 column (2.5  80 cm) that was eluted with CH2Cl2/MeOH (1:1 v/v) and further purified via preparative HPLC (MeOH/H2O, 70:30 v/v) to yield 8 (8.9 mg). E36 (536.0 mg) was applied to a Sephadex LH-20 column (2.5  80 cm) that was eluted with CH2Cl2/MeOH (1:1 v/v) to give chlorophyll and E361(380.0 mg). E361 was further separated on a silica gel CC (2.5  30 cm) that was eluted with cyclohexane/EtOAc (10:1, 5:1, 2:1, 1:1, and 0:1 v/v) to yield E3611 and E3612. Subfraction E3612 (150.0 mg) was separated using preparative HPLC (MeOH/H2O, 60:40 v/v) to produce 7 (19.6 mg). E38 (3  1.3 g) was fractionated on a Sephadex LH-20 column (5.0  80 cm) that was eluted with CH2Cl2/MeOH (1:1 v/v) and was further purified via preparative HPLC (MeOH/H2O, 60:40 v/v) to yield 11 (2.2 g). Fraction E4 (15.0 g) was chromatographed on a silica gel column (6.0  80 cm) that was eluted with cyclohexane/EtOAc (from 100:1 to 0:1 v/v) to yield eight subfractions (E41–E48). E44 (620.0 mg) was chromatographed over Sephadex LH-20 (3.0  80 cm) that was eluted with CH2Cl2/MeOH (1:1 v/v) to give E441. E441 (220.0 mg) was subjected to a RP-C18 silica gel CC (2.5  30 cm) that was eluted with MeOH/H2O (10:90 to 0:100 v/ v) to yield E4411, and then it was purified using preparative HPLC

Table 1 C NMR Spectroscopic Data (150 MHz, in Pyridine-d5) of Compounds 1–6i, d in ppm.

13

Position

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 10 20 30 40 50 60 100 200 300 400 500 600 CH3O-24 CH3O-26 a

1a

2

3

4a

4b

5a

5b

6

6i

dC, type

dC, type

dC, type

dC, type

dC, type

dC, type

dC, type

dC, type

dC, type

73.2, CH 38.5, CH2 66.6, CH 41.6, CH2 137.5, C 125.8, CH 31.9, CH2 32.1, CH 41.8, CH 41.9, C 20.5, CH2 39.6, CH2 43.0, C 56.4, CH 24.5, CH2 27.3, CH2 52.1, CH 11.9, CH3 19.6, CH3 38.9, CH 12.9, CH3 80.9, CH 31.9, CH2 76.2, C 72.7, C 179.1, C 24.5, CH3 23.3, CH3

72.7, CH 38.2, CH2 74.2, CH 39.5, CH2 139.6, C 124.4, CH 32.6, CH2 32.5, CH 41.9, CH 42.5, C 20.9, CH2 40.1, CH2 43.2, C 56.7, CH 24.9, CH2 27.6, CH2 52.6, CH 12.2, CH3 20.0, CH3 39.6, CH 13.3, CH3 79.8, CH 33.6, CH2 77.4, C 73.6, C 179.2, C 25.3, CH3 23.7, CH3 103.2, CH 75.7, CH 78.9, CH 71.8, CH 78.6, CH 62.9, CH2

72.8, CH 38.3, CH2 74.8, CH 39.5, CH2 139.6, C 124.4, CH 32.6, CH2 32.5, CH 41.8, CH 42.4, C 20.9, CH2 40.1, CH2 43.2, C 56.7, CH 24.9, CH2 27.6, CH2 52.6, CH 12.1, CH3 19.9, CH3 39.6, CH 13.2, CH3 79.9, CH 33.6, CH2 77.4, C 73.6, C 179.2, C 25.3, CH3 23.7, CH3 103.6, CH 75.5, CH 78.8, CH 71.8, CH 77.4, CH 70.1, CH2 105.9, CH 75.6, CH 78.8, CH 72.0, CH 78.6, CH 63.0, CH2

73.1, CH 40.7, CH2 66.4, CH 43.8, CH2 140.6, C 123.9, CH 32.7, CH2 32.7, CH 42.1, CH 42.5, C 21.0, CH2 40.1, CH2 43.1, C 56.9, CH 25.0, CH2 27.8, CH2 53.7, CH 12.3, CH3 20.2, CH3 40.2, CH 14.1, CH3 73.6, CH 37.5, CH2 77.8, C 74.4, C 98.8, CH 16.5, CH3 24.0, CH3

73.1, CH 40.7, CH2 66.4, CH 43.8, CH2 140.6, C 123.9, CH 32.7, CH2 32.7, CH 42.1, CH 42.5, C 21.0, CH2 40.1, CH2 43.1, C 56.9, CH 25.0, CH2 28.0, CH2 53.9, CH 12.3, CH3 20.2, CH3 40.0, CH 13.9, CH3 69.4, CH 37.8, CH2 75.8, C 73.4, C 99.6, CH 22.9, CH3 25.8, CH3

72.8, CH 38.3, CH2 74.8, CH 39.5, CH2 139.6, C 124.4, CH 32.6, CH2 32.5, CH 41.8, CH 42.4, C 20.9, CH2 40.1, CH2 43.1, C 56.7, CH 25.0, CH2 27.8, CH2 53.6, CH 12.3, CH3 19.9, CH3 40.1, CH 14.0, CH3 73.6, CH 37.4, CH2 77.8, C 74.4, C 98.7, CH 16.4, CH3 24.0, CH3 103.6, CH 75.5, CH 78.8, CH 72.0, CH 77.3, CH 70.1, CH2 105.8, CH 75.6, CH 78.8, CH 71.8, CH 78.6, CH 63.0, CH2

72.8, CH 38.3, CH2 74.8, CH 39.5, CH2 139.6, C 124.4, CH 32.6, CH2 32.5, CH 41.8, CH 42.4, C 20.9, CH2 40.1, CH2 43.1, C 56.7, CH 25.0, CH2 28.0, CH2 53.9, CH 12.3, CH3 19.9, CH3 39.9, CH 13.9, CH3 69.3, CH 37.7, CH2 75.8, C 73.4, C 99.6, CH 22.9, CH3 25.8, CH3 103.6, CH 75.5, CH 78.8, CH 72.0, CH 77.3, CH 70.1, CH2 105.8, CH 75.6, CH 78.8, CH 71.8, CH 78.6, CH 63.0, CH2

72.8, CH 38.4, CH2 74.8, CH 39.6, CH2 139.7, C 124.4, CH 32.7, CH2 32.6, CH 41.8, CH 42.4, C 20.9, CH2 40.1, CH2 43.1, C 56.8, CH 25.1, CH2 28.3, CH2 53.6, CH 12.3, CH3 19.9, CH3 40.0, CH 13.8, CH3 69.2, CH 35.4, CH2 77.5, C 76.1, C 107.0, CH 22.9, CH3 19.0, CH3 103.7, CH 75.5, CH 78.8, CH 71.8, CH 77.4, CH 70.2, CH2 105.9, CH 75.6, CH 78.9, CH 72.0, CH 78.7, CH 63.1, CH2 50.7, CH3 55.7, CH3

73.1, CH 40.8, CH2 66.5, CH 43.8, CH2 140.7, C 124.1, CH 32.8, CH2 32.8, CH 42.2, CH 42.6, C 21.1, CH2 40.2, CH2 43.2, C 57.0, CH 25.1, CH2 28.3, CH2 53.7, CH 12.3, CH3 20.3, CH3 40.1, CH 13.9, CH3 69.3, CH 35.5, CH2 77.5, C 76.2, C 107.1, CH 23.0, CH3 19.0, CH3

Spectrum was obtained in CDCl3.

50.8, CH3 55.8, CH3

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2.3.2. Physapubside A (2) White powder; [a]25 D
54.3 (c 0.07, MeOH):UV, end absorption; IR (KBr) mmax 3448, 2938, 1728, 1385, 1068, 1021 cm
1; 1H NMR (600 MHz, Pyridine-d5) and 13C NMR (150 MHz, Pyridine-d5) data, see Tables 1 and 2; HRESIMS m/z 661.3560 [M+Na]+ (calcd for C34H54O11Na, 661.3564).

(MeOH/H2O, 70:30 v/v) to produce 10 (75.2 mg) and 1 (20.0 mg). E46 (825.0 mg) was subjected to a Sephadex LH-20 column (2.5  80 cm) that was eluted with CH2Cl2/MeOH (1:1 v/v), and it was further separated via preparative HPLC (MeOH/H2O, 60:40 v/v) to yield 9 (76.5 mg). Fraction E7 (2.2 g) was subjected to a silica gel CC (2  50 cm) that was eluted with CH2Cl2/acetone (15:1, 10:1, 5:1, 2:1, and 1:1 v/v) to obtain (E71–E78). E74 (0.8 g) was chromatographed over a RP-C18 CC silica gel (2.5  30 cm) that was eluted with MeOH/H2O (20:80 to 80:20 v/v) with increasing amounts of MeOH to produce E741. 4a/4b (77.0 mg) was obtained from preparative HPLC of E471, which was eluted by isocratic MeOH/H2O (67:33 v/v).

2.3.3. Physapubside B (3) Colorless flaky crystals, mp 241 °C; [a]25 D
53.3 (c 0.06, MeOH); UV, end absorption; IR (KBr) mmax 3458, 2972, 2939, 1718, 1376, 1062, 1048 cm
1; 1H NMR (600 MHz, Pyridine-d5) and 13C NMR (150 MHz, Pyridine-d5) data, see Tables 1 and 2; HRESIMS m/z 818.4527 [M+NH4]+ (calcd for C40H64O16NH4, 818.4538).

2.3.1. Physapubescin E (1) White powder; [a]25 D
25.7 (c 0.07, MeOH); UV, end absorption; IR (KBr) mmax 3458, 2942, 2904, 1721, 1384, 1262, 1143, cm
1; 1H NMR (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) data, see Tables 1 and 2; HRESIMS m/z 499.3035 [M+Na]+ (calcd for C28H44O6Na, 499.3036).

2.3.4. 26S-Physapubescin F (4a) and 26R-physapubescin F (4b) White powder; [a]25 D +30.0 (c 0.10, MeOH); UV, end absorption; IR (KBr) mmax 3422, 2972, 2938, 1641, 1384, 1079, 1050 cm
1; 1H NMR (600 MHz, Pyridine-d5) and 13C NMR (150 MHz, Pyridine-

Table 2 H NMR (600 MHz) Spectroscopic Data of Compounds 1–4 in Pyridine-d5, d in ppm.

1

Position

1 2 3 4 6 7 8 9 11 12 14 15 16 17 18 19 20 21 22 23 26 27 28 10 20 30 40 50 60

1a

2

3

4a

dH, (J in Hz)

dH, (J in Hz)

dH, (J in Hz)

dH, (J in Hz)

dH, (J in Hz)

3.83, 2.07, 1.72, 3.96, 2.36, 2.28, 5.57, 1.96, 1.56, 1.45, 1.58, 1.58, 1.46, 1.98, 1.20, 1.02, 1.62, 1.13, 1.74, 1.36, 1.05, 0.70, 1.02, 2.02, 0.96, 4.77, 1.91, 1.76,

4.07, 2.69, 2.13, 4.88, 2.91, 2.66, 5.58, 1.91, 1.66, 1.44, 2.22, 1.75, 1.45, 1.96, 1.17, 0.99, 1.47, 0.99, 1.65, 1.22, 1.05, 0.64, 1.02, 2.06, 1.06, 5.12, 2.09, 1.94,

brs brd (11.2) t (11.8) m dd (13.4, 3.5) t (13.9) brd (4.4) brd (16.2) m m td (11.7, 4.1) m m m td (12.8, 3.5) o m o m dd (22.3, 10.0) dd (22.3, 10.0) s s m d (6.5) dt (12.0, 3.5) t (13.5) dd (14.4, 3.5)

3.83, 2.83, 2.15, 4.80, 2.84, 2.65, 5.60, 1.90, 1.67, 1.42, 2.22, 1.70, 1.41, 1.93, 1.14, 0.98, 1.45, 0.98, 1.66, 1.22, 1.05, 0.63, 1.02, 2.07, 1.07, 5.12, 2.12, 1.95,

brs brd (15.1) t (12.4) m dd (12.1, 4.4) t (12.1) brd (3.6) brd (16.8) m m td (12.1, 4.1) m m brd (12.8) td (12.8, 2.7) m m m m dd (20.8, 9.7) dd (20.8, 9.7) s s m d (6.3) dt (12.1, 3.1) t (12.9) dd (14.4, 3.1)

1.64, 1.63, 5.05, 4.06, 4.27, 4.35, 3.87, 4.44, 4.41,

s s d (7.7) t (8.5) t (8.9) t (9.3) dt (9.3, 3.5) brd (10.9) dd (10.9,3.5)

1.65, 1.63, 4.94, 3.99, 4.18, 4.24, 3.95, 4.71, 4.37, 5.15, 4.06, 3.95, 4.24, 4.25, 4.52, 4.39,

s s d (7.7) t (8.3) t (8.9) t (6.4) o d (11.3) t (4.6) d (7.8) t (7.6) o t (6.4) t (6.6) d (11.5) t (4.9)

4.16, 2.63, 2.20, 4.77, 2.82, 2.81, 5.67, 2.01, 1.73, 1.52, 2.26, 1.75, 1.49, 1.95, 1.09, 1.02, 1.55, 1.05, 1.70, 1.30, 1.11, 0.70, 1.16, 2.08, 1.19, 3.92, 2.19, 1.94, 5.45, 2.00, 1.91,

4.16, 2.63, 2.20, 4.77, 2.82, 2.81, 5.67, 2.01, 1.73, 1.52, 2.26, 1.75, 1.49, 1.95, 1.09, 1.02, 1.55, 1.05, 1.80, 1.38, 1.17, 0.68, 1.15, 2.01, 1.18, 4.58, 2.13, 1.86, 5.56, 1.86, 2.11,

brs brd (14.0) t (11.9) m dd (13.4, 4.7) t (12.3) brd (3.7) brd (15.9) m m td (11.5, 4.6) m m brd (12.3) td (12.3, 4.4) m m m m dd (20.4, 10.5) dd (20.4, 10.5) s s m d (6.5) dt (12.0, 3.2) t (13.0) dd (13.5, 3.2)

1.39, s 1.30, s

100 200 300 400 500 600 a

Spectrum was obtained in CDCl3.

4b

brs brd (13.0) m m m m d (3.5) brd (16.4) m m td (12.1, 4.1) m m brd (12.9) t (12.1) m m m m dd (21.5, 10.7) dd (21.5, 10.7) s s m d (6.4) dt (12.2, 2.8) t (12.5) brd (13.0) d (4.1) s s

brs brd (13.0) m m m m d (3.5) brd (16.4) m m td (12.1, 4.1) m m brd (12.9) t (12.1) m m m m o o s s m d (6.4) dt (12.3, 2.7) o o d (4.1) s s

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d5) data, see Tables 1 and 2; HRESIMS m/z 501.3184 [M+Na]+ (calcd for C28H46O6Na, 501.3192).

2.3.5. 26S-Physapubside C (5a) and 26R-physapubside C (5b) White powder; [a]25 D
31.4 (c 0.07, MeOH); UV, end absorption; IR (KBr) mmax 3396, 2940, 2897, 1374, 1076, 1044 cm
1; 1H NMR (600 MHz, Pyridine-d5) and 13C NMR (150 MHz, Pyridine-d5) data, see Tables 1 and 3; HRESIMS m/z 825.4243 [M+Na]+ (calcd for C40H66O16Na, 825.4249).

2.3.6. (20S,22R,24R,25S,26R)-22,26-epoxy-24,26-dimethoxy1a,3b,25-trihydroxyergost-5-ene 3-O-[b-D-glucopyranosyl(1?6)]-bD-glucopyranoside (6) White powder; [a]25 D
63.3 (c 0.06, MeOH); UV, end absorption; IR (KBr) mmax 3406, 2941, 2896, 1375, 1051 cm
1; 1H NMR (600 MHz, Pyridine-d5) and 13C NMR (150 MHz, Pyridine-d5) data, see Tables 1 and 3; HRESIMS m/z 853.4538 [M+Na]+ (calcd for C42H70O16Na, 853.4562).

2.3.7. (20S,22R,24R,25S,26R)-22,26-epoxy-24,26-dimethoxy1a,3b,25-trihydroxyergost-5-ene (6i) White powder; [a]25 D
21.3 (c 0.15, MeOH); UV, end absorption; IR (KBr) mmax 3422, 2918, 2847, 1598, 1384, 1055, 1032, 1015 cm
1; 1H NMR (400 MHz, Pyridine-d5) and 13C NMR (150 MHz, Pyridine-d5) data, see Tables 1 and 3; HRESIMS m/z 529.3501 [M+Na]+ (calcd for C30H50O6Na, 529.3505). 2.3.8. Physapubescin G (7) Colorless needles; mp 118 °C; [a]25 D +70.7 (c 0.14, MeOH); UV (MeOH) kmax (log e) 214 (3.73); IR (KBr) mmax 3455, 2934, 1716, 1678, 1460, 1373, 1248, 1186, 1038 cm
1; 1H NMR (300 MHz, CD3OD) and 13C NMR (75 MHz, CD3OD) data, see Tables 4 and 5; HRESIMS m/z 553.2778 [M+Na]+ (calcd for C30H42O8Na, 553.2777). 2.3.9. Physapubescin H (8) White powder; [a]25 D +78.0 (c 0.10, MeOH); UV (MeOH) kmax (log e) 222 (3.77) nm; IR (KBr) mmax 3451, 2925, 1733, 1675, 1462, 1383, 1252, 1040 cm
1; 1H NMR (600 MHz, CDCl3) and 13C NMR

Table 3 H NMR (600 MHz) Spectroscopic Data of Compounds 5–6i in Pyridine-d5, d in ppm.

1

Position

1 2 3 4 6 7 8 9 11 12 14 15 16 17 18 19 20 21 22 23 26 27 28 10 20 30 40 50 60 100 200 300 400 500 600

5a

5b

dH, (J in Hz)

dH, (J in Hz)

dH, (J in Hz)

dH, (J in Hz)

4.08, 2.81, 2.15, 4.81, 2.85, 2.65, 5.60, 1.95, 1.75, 1.45, 2.21, 1.68, 1.40, 1.90, 1.07, 0.99, 1.53, 1.03, 1.70, 1.29, 1.11, 0.67, 1.00, 2.07, 1.19, 3.92, 2.19, 1.75, 5.45, 2.00, 1.91, 4.95, 4.00, 4.19, 4.25, 3.96, 4.72, 4.37, 5.16, 4.07, 3.95, 4.25, 4.26, 4.53, 4.39,

4.08, 2.81, 2.15, 4.81, 2.85, 2.65, 5.60, 1.95, 1.75, 1.45, 2.21, 1.68, 1.40, 1.90, 1.07, 0.99, 1.53, 1.03, 1.80, 1.37, 1.16, 0.66, 1.00, 2.01, 1.18, 4.58, 2.16, 1.86, 5.57, 1.85, 2.11, 4.95, 4.00, 4.19, 4.25, 3.96, 4.72, 4.37, 5.16, 4.07, 3.95, 4.25, 4.26, 4.53, 4.39,

4.08, 2.81, 2.15, 4.81, 2.84, 2.65, 5.61, 1.94, 1.72, 1.45, 2.22, 1.67, 1.41, 1.92, 1.07, 1.01, 1.58, 1.10, 1.74, 1.36, 1.10, 0.69, 1.02, 1.94, 1.11, 3.99, 1.78, 1.67, 4.63, 1.63, 1.75, 4.94, 3.99, 4.19, 4.25, 3.95, 4.45, 4.37, 5.16, 4.06, 3.95, 4.34, 4.25, 4.52, 4.37, 3.57, 3.37,

4.17, 2.63, 2.21, 4.77, 2.82, 2.81, 5.68, 1.97, 1.72, 1.51, 2.27, 1.71, 1.38, 1.94, 1.09, 1.02, 1.54, 1.13, 1.77, 1.37, 1.10, 0.71, 1.16, 1.94, 1.10, 4.00, 1.78, 1.68, 4.63, 1.63, 1.75,

brs brd (15.7) t (12.7) m dd (11.7, 4.6) t (11.7) brd (4.7) brd (16.8) m m td (11.0, 4.4) m m m td (12.1, 2.6) o m m m ddd (20.1, 9.4, 2.1) dd (20.1, 9.4) s s m d (6.4) dt (12.7, 2.8) t (12.9) dd (13.1, 1.8) s s s d (7.7) t (8.5) t (8.9) o m dd (11.6, 1.9) m d (7.8) t (7.9) o o o dd (11.8, 2.4) t (4.9)

MeO-24 MeO-26 a

6ia

6

Spectrum was measured at 400 MHz.

brs brd (15.7) t (12.7) m dd (11.7, 4.6) t (11.7) brd (4.7) brd (16.8) m m td (11.0, 4.4) m m m td (12.1, 2.6) o m m m o o s s m d (6.4) dt (12.2, 2.7) o o s s s d (7.7) t (8.5) t (8.9) o m dd (11.6, 1.9) m d (7.8) t (7.9) o o o dd (11.8, 2.4) t (4.9)

brs brd (15.1) t (11.9) m dd (11.5, 4.6) t (11.5) brd (4.8) brd (16.8) m m td (11.9, 4.5) brd (13.1) dd (13.1, 4.5) m td (11.9, 3.9) o m m m dd (21.2, 10.1) dd (21.2, 10.1) s s m d (6.6) dt (12.3, 3.3) t (12.8) brd (13.3) s s s d (7.7) t (8.5) t (8.9) o o d (10.2) m d (7.8) t (8.6) o t (8.8) o dd (11.8, 2.0) m s s

brs brd (12.8) t (11.1) m m m brd (5.0) m m m td (12.0, 4.7) m m m t (11.2) m m m m m m s s m d (6.7) dt (12.2, 3.2) t (12.8) br d (13.6) s s s

3.57, s 3.37, s

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(75 MHz, CDCl3) data, see Tables 4 and 5; HRESIMS m/z 537.2817 [M+Na]+ (calcd for C30H42O7Na+, 537.2828). 2.3.10. Physapubescin I (9) White powder; [a]25 D +16.4 (c 0.33, MeOH); UV (MeOH) kmax (log e) 216 (3.71) nm; IR (KBr) mmax 3466, 2965, 1746, 1679, 1460, 1377, 1260, 1039 cm
1; 1H NMR (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) data, see Tables 4 and 5; HRESIMS m/z 611.2823 [M+Na]+ (calcd for C32H44O10Na, 611.2832). 2.4. The in situ dimolybdenum ECD assays Dimolybdenum tetraacetate was purchased from SigmaAldrich. HPLC grade DMSO was dried with 4 Å molecular sieves. According to the published procedure, a mixture of the diol/Mo2(OAc)4 (1:1) of 1 was subjected to CD measurement at a concentration of 0.51 mg/mL. The first CD spectrum was recorded immediately after mixing, and its time evolution was monitored until stationary (approximately 15 min after mixing). The inherent CD spectrum was subtracted. The observed sign of the diagnostic band at approximately 305 nm in the induced CD spectrum was correlated to the absolute configuration of the 24,25-diol moiety. 2.5. X-ray crystallographic analysis of 3 Upon crystallization from MeOH using the vapor diffusion method, the colorless flaky crystals of 3 were obtained. The data was collected using a Sapphire CCD with a graphite monochromated Cu Ka radiation of k = 1.54184 Å at 173.00 (10) K. Crystal Table 4 C NMR Spectroscopic Data of Compounds 7–9a, d in ppm.

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 AcO-15 AcO-16

2.6. Enzymatic hydrolysis of the withanosides Cellulase (400 u/mg) was purchased from Treechem. A solution of the withanosides (8.0 mg of 2, 3, 5a/5b or 6) in acetate buffer (pH 4.8, 5.0 mL) was treated with cellulase (8.0 mg), and the entire mixture was stirred at 45 °C for 2 days. Then, the reaction mixtures were extracted with EtOAc. The EtOAc extracts were concentrated and purified using normal-phase silica gel CC to produce the

Table 5 H NMR Spectroscopic Data of Compounds 7–9a, d in ppm.

13

Position

data: 2 (C40H64O16), 2 (H2O), M = 1637.86, space group triclinic, P 1; unit cell dimensions were determined to be a = 6.8580 (2) Å, b = 11.1678 (4) Å, c = 26.7116 (8) Å, a = 89.405 (3)°, b = 89.537 (2)°, c = 86.193 (3)°, V = 2041.12 (11) Å3, Z = 1, Dx = 1.332 mg/m3, F(0 0 0) = 884, l (Cu Ka) = 0.863 mm
1. In total, 32,924 reflections were collected to hmax = 62.76° in which 11,152 reflections were observed [F2 > 4r (F2)]. The structure was solved via direct methods using the SHELXS-97 program and refined by the program, SHELXL-97, and using full-matrix least-squares calculations. In the structure refinements, non-hydrogen atoms were placed on the geometrically ideal positions using the ‘‘ride on” method. The hydrogen atoms bonded to oxygen were located by the structure factors with isotropic temperature factors. The final refinement gave R = 0.0361, RW = 0.0909, S = 1.051, and Flack = 0.02 (9). The crystallographic data for the structure of 3 were provided to the Cambridge Crystallographic Data Centre under reference number CCDC 1417179. Copies of the data can be obtained free of charge upon application to the Director at CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax:+44 (0)1223 336033, or e-mail: deposit@ ccdc.cam.ac.uk).

1

7

8

9

dC, type

dC, type

dC, type

202.8, C 131.7, CH 143.9, CH 69.7, CH 63.2, C 59.9, CH 30.6, CH2 29.3, CH 43.8, CH 47.5, C 20.9, CH2 39.2, CH2 42.8, C 58.7, CH 76.1, CH 36.4, CH2 50.2, CH 11.7, CH3 15.7, CH3 39.2, CH 11.5, CH3 73.6, CH 29.8, CH2 42.6, CH 213.2, C 27.7, CH3 16.8, CH3 161.4, C 171.4, C 20.0, CH3

203.6, C 129.2, CH 144.5, CH 33.0, CH2 61.8, C 63.3, CH 30.4, CH2 29.4, CH 44.4, CH 48.4, C 23.8, CH2 39.9, CH2 43.2, C 58.9, CH 75.9, CH 37.2, CH2 50.3, CH 12.9, CH3 15.4, CH3 38.6, CH 12.6, CH3 64.8, CH 29.5, CH2 65.0, C 63.9, C 91.7, CH 16.6, CH3 19.0, CH3 170.7, C 21.4, CH3

202.4, C 131.9, CH 142.6, CH 69.5, CH 63.5, C 62.0, CH 29.7, CH2 29.0, CH 43.5, CH 47.5, C 21.7, CH2 39.7, CH2 39.6, C 56.7, CH 71.7, CH 74.8, CH 55.9, CH 14.5, CH3 17.5, CH3 37.4, CH 13.0, CH3 64.8, CH 29.9, CH2 64.6, C 63.8, C 91.8, CH 16.5, CH3 18.9, CH3 169.6, C 20.9, CH3 170.1, C 20.6, CH3

a Spectrum of 7 was obtained in CD3OD, spectra of 8 and 9 were obtained in CDCl3. Spectra of 7 and 8 were measured at 75 MHz, spectrum of 9 was measured at 150 MHz.

Position

7

8

9

dH, (J in Hz)

dH, (J in Hz)

dH, (J in Hz)

2 3

6.18, d (9.9) 7.07, dd (9.9, 6.3)

6.16, d (10.0) 6.93, dd (10.0, 5.8)

4

3.66, d (6.3)

6 7

3.17, m 2.07, dd (15.1, 4.1) 1.51, m 1.68, m 0.98, td (11.7, 4.1) 1.71, m 1.18, m 1.72, dt (12.5, 3.5) 1.23, m 1.36, m 4.80, o 2.08, m 1.82, m 1.49, m 0.75, s 1.39, s 1.94, m 0.96, d (6.7) 4.81, m 1.88, m 1.47, dd (14.0, 9.9) 2.68, m 2.12, s 1.10, d (7.2) 8.07, s 2.06, s

6.02, dd (10.0, 2.8) 6.85, ddd (10.0, 6.1,2.3) 2.99, dt (19.1, 2.3) 1.92, dd (19.1, 6.1) 3.11, d (2.7) 1.99, dt (14.5, 2.8) 1.46, m 1.26, m 1.22, td (12.0, 4.2) 2.08, m 1.47, m 1.93, dt (12.0, 3.6) 1.28, m 1.26, m 4.77, td (9.5, 3.0) 2.09, m 1.51, m 1.31, m 0.74, s 1.26, s 1.73, m 0.89, d (6.7) 3,62, dt (11.4, 3.0) 1.77, m 1.66, brd (14.0) 5.00, 1.42, 1.41, 2.02,

4.94, 1.37, 1.36, 1.96, 2.07,

8 9 11 12 14 15 16 17 18 19 20 21 22 23 24 26 27 28 AcO-15 AcO-16

s s s s

3.74, d (5.8) 3.18, 2.02, 1.40, 1.75, 1.05, 1.84, 1.42, 1.93, 1.05, 1.34, 5.09, 5.11,

brs dd (15.0, 4.1) m m td (11.4, 4.1) m m dt (12.3, 3.5) m m t (8.0) t (7.9)

1.44, m 0.80, s 1.38, s 1.87, m 0.89, d (6.7) 3.51, dt (11.4, 2.6) 1.58, dd (13.9, 11.7) 1.81, brd (13.7) s s s s s

a Spectrum of 7 was obtained in CD3OD, spectra of 8 and 9 were obtained in CDCl3. Spectrum of 8 was measured at 300 MHz, spectra of 8 and 9 were measured at 600 MHz.

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aglycones. The aqueous solutions were deproteinated using the Sevag method [19]. The residual water layers were desalted with Amberlite MB-3 and dried in vacuum to produce the sugars of the withanosides. 2.7. Determination of the sugar components Approximately 1.0 mg of the sugars obtained from the enzymatic hydrolysis of each withanoside were dissolved in anhydrous pyridine (0.5 mL). Then, a pyridine solution (0.5 mL) of L-cysteine methyl ester hydrochloride (1.5 mg) was added to each of the sugar solutions. The mixtures were stirred at 60 °C for 2.0 h. After drying the solutions in vacuo, trimethylsilyl imidazole (150 lL) was added to each of the residues and stirred at 60 °C for another 1.0 h. After partition between cyclohexane (0.5 mL) and H2O (0.5 mL), the cyclohexane extracts were analyzed using gas chromatography (GC) with an HP-5 column. The temperatures of the injector and detector were 250 and 280 °C, respectively. A temperature gradient system was used for the oven starting at 100 °C and up to 140 °C at a rate of 4 °C/min, and then increased to 170 °C for 8 min at a rate of 13 °C/min; finally, increased to 200 °C at a rate of 5 °C/min. The peaks of authentic samples of standard D-glucose and L-glucose after treatment using the same method were detected at tR 26.35 and 27.03 min, respectively. 2.8. Cytotoxicity bioassay The C4-2B cell line was from Urocor Inc. (Oklahoma City, OK), and the 22Rvl, 786-O, A-498, Caki-2, ACHN, A375, A375-S2, and L02 cell lines were obtained from ATCC (Manassas, VA, USA). The C4-2B, 22Rvl, and 786-O cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS). The A-498, ACHN, A375, and A375-S2 cells were cultured in Eagle’s minimum essential medium (EMEM) with 10% FBS. The Caki-2 cells were cultured in McCoy’s medium with 10% FBS. The L02 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% FBS. All of these cells were cultured at 37 °C in a humidified atmosphere with 5% CO2. The cell viability was assessed by colorimetric measurement of the amount of insoluble formazan formed in the living cells based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-

trazolium bromide (MTT). Briefly, 100 lL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, whereas the suspended cells were seeded just before drug addition with an initial density of 1  105 cells/mL. Each cancer cell line was exposed to the test samples at various concentrations in triplicate for 48 h, using 5-fluorouracil as the positive control. After incubation, MTT was added to a final concentration of 1 mg/mL, and the incubation was maintained for 3 h at 37 °C. The cells were lysed with 100 lL DMSO after removal of the medium. The optical density (OD) of the lysate was measured at 490 nm. The IC50 values of the isolated compounds were derived from the mean OD values of the triplicate tests versus the drug concentration curves. 3. Results and discussion Compound 1 was isolated as a white powder. Its molecular formula was determined to be C28H44O6 based on positive HRESIMS, which indicated seven degrees of unsaturation. The 1H NMR spectrum of 1 displayed one olefinic proton [dH 5.57 (1H, brd, J = 3.7 Hz)], three oxymethine protons [dH 3.83 (1H, brs), 3.96 (1H, m), 4.77 (1H, dt, J = 12.0, 3.2 Hz)], and five methyl groups [dH 0.70 (3H, s), 0.96 (3H, d, J = 6.5 Hz), 1.02 (3H, s), 1.30 (3H, s), 1.39 (3H, s)]. The 13C NMR spectrum showed 28 carbon signals, including one lactone carbonyl carbon (dC 179.1), two olefinic carbons (dC 125.8 and 137.5), five oxygen-bearing carbons (dC 66.6, 72.7, 73.2, 76.2, and 80.9), and five methyl carbons (dC 11.9, 12.9, 19.6, 23.3, and 24.5). All of these features suggested a typical withanolide skeleton. The 1H and 13C NMR data (Tables 1 and 2) of 1 were similar to those of (20S,22R)-1a,3b-dihydroxy-witha-5,24dienolide obtained previously from the roots of Dunalia australis [20]. The major difference between their NMR spectra was the absence of two olefinic carbons, whereas two new oxygenated carbons were detected in 1. The degrees of unsaturation and the chemical shifts of C-24 (dC 76.2) and C-25 (dC 72.7) suggested that the D24,25 double bond was hydroxylated and both C-24 and C-25 were linked with a hydroxy group. This deduction was supported by the HMBC correlations from H3-27 to C-26, C-25, and C-24, H3-28 to C-25, C-24, and C-23, H3-21 to C-22, C-20, and C-17, and H-22 to C-21. The NOESY correlations of H3-19/H-1, H3-19/

Fig. 2. Key HMBC (A), selected NOESY (B) correlations and Newman projections for C-22/C-20 rotamer (C) and C-23/C-22 rotamer (D) of compound 1.

G. Xia et al. / Steroids 115 (2016) 136–146

H-2b, H3-19/H-4b, H-3/H-2a, and H-3/H-4a suggested a b-orientation for H-1 and an a-orientation for H-3 (Fig. 2). The two methyls in the lactone ring of 1 were found to be trans to H-22 due to the lack of NOESY correlations of H-22/H3-27 and H-22/H3-28. The 20S and 22R configurations of 1 were determined based on the characteristic coupling pattern of H-22: dH 4.77 (1H, dt, J = 12.0, 3.2 Hz) [21]. As illustrated in Fig. 2, the small coupling constant (3.2 Hz) between H-20 and H-22 suggested the gauche conformation for these protons, which was supported by the diagnostic NOESY correlations of H3-21/H-23ax, H-17/H-23eq, H-16a/H-22, H-16b/H-22, and H-22/H-20 (Fig. 2). The absolute configurations of the 24,25-diol were assigned using the in situ dimolybdenum ECD method developed by Snatzke and Frelek [22–24]. The positive Cotton effect (CE) at approximately 305 nm (see Supporting information, Fig. S8) permitted the assignment of 24S and 25S for 1 based on the empirical rule proposed by Snatzke [23]. A comparison of the NMR spectroscopic data of 2 and 3 with those of 1 indicated that they differ only in the sugar moieties. The enzymatic hydrolysis of 2 and 3 with cellulase afforded the aglycone (1) and the sugar moiety. The D-glucose was identified via GC analysis of the trimethylsilyl thiazolidine derivatives [25]. Additionally, the absolute configuration of 3 was determined based on the singlecrystal X-ray diffraction (Fig. 3). Consequently, compound 1, 2, and 3 were established as (20S,22R,24S,25S)-1a,3b,24,25-tetrahydroxywitha-5-en-22,26-olide (physapubescin E), (20S,22R, 24S,25S)-1a,3b,24,25-tetrahydroxywitha-5-en-22,26-olide 3-O-b(physapubside A), and (20S,22R,24S,25S)-1a, 3b,24,25-tetrahydroxywitha-5-en-22,26-olide 3-O-[b-D-glucopyranosyl(1?6)]-b-D-glucopyranoside (physapubside B), respectively. Compound 4 was isolated as a white powder. The molecular formula was determined to be C28H46O6 based on the positive HRESIMS data. A comprehensive study of the 1D and 2D NMR spectra showed that 4 was actually a mixture of two interconvertible epimeric isomers at C-26, which is similar to cilistol g, a reported withanolide mixture with a 24,25,26-trihydroxy-22,26-olide side chain [26]. The 1H and 13C NMR chemical shifts of rings A–D in 4 and 1 were almost identical. The major difference was observed in the side chain, where the lactone carbonyl carbon (dC 179.1) in 1 was modified into a hemiacetal carbon (dC 98.8/99.6) in 4. The proportion of the major (4a) and minor (4b) stereoisomers could be determined from the relative integration of the respective resonances of H-26 (dH 5.45) and H-26. (dH 5.56). The relative configurations of C-24, C-25, and C-26 in 4a were determined by the NOESY correlations of H-26 (dH 5.45)/H-22 (dH 3.92), H-22/H3-28 (dH 1.91), and H-26/H3-28. The absolute configurations of C-20 and C-22 were elucidated to be S and R, respectively, based on the characteristic chemical shift, coupling constants, and the NOESY correlations as described previously. The stereo configurations of 24R, 25S, and 26R for 4b were also assigned using the D-glucopyranoside

143

NOESY correlations of H-26. (dH 5.56)/H3-27. (dH 1.86), and H22. (dH 4.58)/H3-28. (dH 2.11). Therefore, the structures of 4a and 4b were determined to be (20S,22R,24R,25S,26S)-22, 26-epoxy-1a,3b,24,25,26-pentahydroxyergost-5-ene (26S-physapubescin F) and (20S,22R,24R,25S,26R)-22,26-epoxy-1a,3b, 24,25,26-pentahydroxyergost-5-ene (26R-physapubescin F), respectively. Compounds 5a and 5b were isolated as white powders, and they were also a pair of interconvertible epimeric isomers. Their molecular formulas were determined to be C40H66O16 based on the HRESIMS and 13C NMR spectroscopic data (Table 1). Their 1H and 13C NMR spectra were similar to those of 4a and 4b except for the signals representing the sugar moiety. The enzymatic hydrolysis of 5a and 5b liberated the D-glucose, which was detected using GC analysis of the sugar derivative [25]. The composition of the sugar moiety was identified as a gentiobiose (b-D-glucopyranosyl-(1?6)-b-D-glucopyranoside) by detailed analysis of the HMBC and 1H–1H COSY spectra. The location of the gentiobiose moiety was assigned to C-3 via the HMBC correlation from H-10 (dH 4.95) to C-3 (dC 74.8). Therefore, 5a and 5b were identified as (20S,22R,24R,25S,26S)-22,26-epoxy-1a,3b,24,25,26-pentahydroxyergost-5-ene 3-O-[b-D-glucopyranosyl(1?6)]-b-D-glucopyranoside (26S-physapubside C) and (20S,22R,24R,25S,26R)-22,26-epoxy1a,3b,24,25,26-pentahydroxyergost-5-ene 3-O-[b-D-glucopyranosyl(1?6)]-b-D-glucopyranoside (26R-physapubside C), respectively. The HRESIMS analysis of compound 6 provided the molecular formula of C42H70O16, which is appropriate for eight degrees of unsaturation. The NMR spectroscopic data of rings A-D for 6 closely resembled those of 5, except for the side chain. The 1H and 13C NMR spectra of 6 showed the presence of two methoxy groups [dH 3.37 (3H, s), dC 55.7; dH 3.57 (3H, s), dC 50.7]. Analysis of its HMBC spectrum revealed that the two OCH3 groups (dC 55.7 and 50.7) were connected at C-26 and C-24, respectively. The enzymatic hydrolysis of 6 liberated 6i and glucoses. The observed H22/H3-28, H-26/H3-27, CH3O-26/H-22, and CH3O-26/H3-28 NOESY correlations implied that OCH3-24, H-26, and CH3-27 were trans to H-22. The absolute configurations of C-20 and C-22 were elucidated to be S and R, respectively, based on the characteristic coupling constants and the NOESY correlations. Consequently, the side chain moiety in 6 was determined as depicted in Fig. 1. It is possible that 6 is an artifact because methanol was one of the solvents used during the isolation and purification procedure. Compound 7 was isolated as colorless needles. Its molecular formula was determined to be C30H42O8 with 10 degrees of unsaturation according to the HRESIMS and NMR spectroscopic data (Tables 4 and 5). The NMR spectra of 7 resembled those of physapubescin (11) [14,27]. In contrast to 11, the epoxy-d-lactol system with b,c-dimethyl groups in the side chain was absent in 7. Exten-

Fig. 3. X-ray ORTEP drawing of compound 3.

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sive analysis of the 1H, 13C, and 2D NMR spectra suggested the characteristic signals for the side chain [28]: three methyl groups [dH 0.96 (3H, d, J = 6.7 Hz), dC-21 11.5; dH 1.10 (3H, d, J = 7.2 Hz), dC-27 16.8; dH 2.12 (3H, s), dC-26 27.7], one methylene (dC-23 29.8), three methines (dC-20 39.2, dC-22 73.6, and dC-24 42.6), two carbonyl carbons (dC-25 213.2 and dC-28 161.4). The HMBC spectrum showed key correlations from H-28 to C-22, H3-26 to C-25 and C-24, and H3-27 to C-23, C-24, and C-25, confirming the structure of the side chain, as shown in Fig. 1. The configurations of the side chain moiety of 7 remain to be determined. Accordingly, the structure of 7 was elucidated as 15a-acetoxy-5,6b-epoxy-4b-hydroxy-1,25dioxo-25,26-secowitha-2-enolide, and it was named physapubescin G. Compound 8 was isolated as a white powder. The molecular formula was determined to be C30H42O7 based on the HRESIMS and 13 C NMR spectroscopic data (Table 4). A detailed comparison of the 1H and 13C NMR spectra of 8 with those of 11 and 2D NMR experiments indicated that 8 has the same substituent patterns in rings B-D and the side chain as 11, except for the absence of one oxygenated carbon resonance at dC 69.8 in 11, and the presence of one methylene carbon signal at dC 33.0 in 8. This evidence suggested the absence of the 4-hydroxy group in 8, which was confirmed by the HMBC correlations from H-4 to C-2, C-3, C-5, C-6, and C-10. The NOESY experiment showed correlations of H-26/ H3-27, H3-28/H-23ax, and H-23eq/H-22, confirming that H-26, CH3-27, and CH3-28 were trans to H-22 (Fig. 4). Based on all of the above evidence, the structure of 8 (physapubescin H) was determined to be (20S,22R,24S,25S,26R)-15a-acetoxy-5,6b:22, 26:24,25-triepoxy-26-hydroxyergost-2-en-1-one(see Table 6). The molecular formula of 9 was established as C32H44O10 via the HRESIMS ion at m/z 611.2823 [M+Na]+ (calcd for C32H44O10Na,

611.2827). The 1H and 13C NMR data of 9 exhibited characteristic signals for a withanolide skeleton. The almost identical 13C NMR data for rings A-C and the nine-carbon side chain of 9 and 11 indicated that the structural differences between them are restricted to the substituents in ring D. The methylene of C-16 (dC 37.2) was absent, instead, signals corresponding to an additional acetoxy group and a new oxygenated methine of C-16 (dC 74.8) were observed in 9. The new acetoxy group is determined to be attached to C-16 by the HMBC correlation of the signal at dH 5.11 (H-16) to the carbonyl carbon (dC 170.1). Both the 15-acetoxy and 16-acetoxy groups were deduced to be a-oriented based on the NOESY correlations of H-15/H3-18, H-16/H3-18, H-15/H-20, and H-16/H20. Therefore, the structure of 9 was identified as (20S,22R, 24S,25S,26R)-15a,16a-diacetoxy-5,6b:22,26:24,25-triepoxy-4b,26dihydroxyergost-2-en-1-one, and given the trivial name physapubescin K. The known compounds 10 and 11 were identified as (20S,22R,24S,25S,26R)-15a-acetoxy-5,6b:22,26:24,25-triepoxy-26methoxy-4b-hydroxyergost-2-en-1-one [16] and physapubescin [14,27], respectively, on the basis of detailed spectroscopic analysis and comparison with the reported data. The NOESY spectrum showed correlations of H-26/H3-27, H3-28/H-23ax, and H-23eq/ H-22, confirming that H-26, CH3-27, and CH3-28 were trans to H-22. Accordingly, the structure of 11 was elucidated as shown in Fig. 1. As methanol was one of the solvents used for the isolation of the materials, the possibility of 10 being an artifact cannot be excluded. The cytotoxic effects of the isolated compounds 1–11 were tested against human prostate cancer cells (C4-2B, 22Rvl), human renal carcinoma cells (786-O, A-498, Caki-2, and ACHN), and human melanoma cells (A375, A375-S2) using the MTT method

Fig. 4. Selected NOESY correlations of Compound 8.

Table 6 Cytotoxicitya of Compounds from Physalis pubescens L.

a b c d

Compoundsb

C4-2B

CWR22Rvl

786-O

A-498

Caki-2

ACHN

A375

A375-S2

L02

3 7 8 9 10 11 5-Fluorouracilc

6.31 ± 1.1 0.36 ± 0.11 >10 0.19 ± 0.02 0.25 ± 0.09 1.16 ± 0.03 5.64 ± 0.45

>10 0.38 ± 0.06 7.92 ± 1.31 0.31 ± 0.07 0.33 ± 0.06 3.97 ± 0.12 3.86 ± 0.36

>10 0.43 ± 0.09 7.75 ± 0.81 0.17 ± 0.03 0.29 ± 0.04 1.12 ± 0.09 >10

>10 0.97 ± 0.65 >10 0.34 ± 0.02 1.22 ± 0.12 0.71 ± 0.11 8.83 ± 0.88

>10 0.47 ± 0.03 9.64 ± 1.83 0.55 ± 0.01 0.38 ± 0.17 >10 9.47 ± 1.02

>10 0.43 ± 0.05 >10 0.39 ± 0.03 0.30 ± 0.06 >10 2.72 ± 0.79

>10 0.77 ± 0.07 8.44 ± 0.91 1.22 ± 0.09 1.02 ± 0.17 5.04 ± 0.23 >10

>10 2.60 ± 0.64 >10 5.30 ± 0.15 2.83 ± 0.52 >10 1.92 ± 0.54

–d 0.65 ± 0.10 – 1.00 ± 0.14 1.38 ± 0.33 9.22 ± 0.22 4.90 ± 0.50

Results for the compounds and positive control are expressed as IC50 values in lM. Compounds 1, 2, and 4–6i were inactive for all cell lines used (IC50 > 10 lM). Positive control. Not tested.

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[29,30]. Compounds 7, 9, and 10 containing the 4b-hydroxy-2-en1-one group in ring A and the 5b,6b-epoxy group in ring B, showed potent cytotoxicity against the eight cancer cell lines with IC50 values in the range of 0.17–5.30 lM. However, compounds 1–6i and 8 had a partial or total absence of these functional groups, showing weak or no cytotoxicity against the tested cancer cell lines. These results are in agreement with previous structure– activity relationship reports [6,31–34]. The potently cytotoxic isolates 7 and 9–11 were also tested against the L02 human normal hepatic cell line, and they were found to be active. Among these cytotoxic compounds, 9–11 showed less cytotoxicity against the normal hepatic L02 cell line compared with C4-2B and 786-O cancer cell lines. This preliminary selectivity testing result provides a favorable in vitro selectivity profile for any further development of these active withanolides. Additionally, compared to the activities of withanolides with those of other steroids previously isolated from this plant, such as alkesterol A, alkesterol B, and b-sitosterol [35], withanolides with functional groups (4b-hydroxy-2-en-1one in ring A and 5b,6b-epoxy in ring B) have certain advantages that make them active constituents of this plant. 4. Conclusion In conclusion, herein we have reported six new withanolides (1, 4, and 6i–9), four new withanolide glucosides (2, 3, 5, and 6), along with two known withanolides (10 and 11) from the stems and leaves of P. pubescens L. Structure analysis including X-ray crystallography and the in situ dimolybdenum ECD method allowed the determination of the absolute stereochemistry. All the isolated compounds were evaluated for their cytotoxic effect against eight human tumor cell lines. Compounds 7, 9, and 10 showed prominent cytotoxic effect with IC50 values in the range of 0.17–5.30 lM. This study demonstrated that P. pubescens L. may be a good source of bioactive substances, especially for withanolides. Acknowledgements This work was financially supported by grants from the National Natural Science Foundation of China (NSFC) [grant numbers 31270399, 21472138], Key Projects of the National Science and Technology Pillar Program [grant number 2012BAI30B02], Fund of the Educational Department of Liaoning Province [grant number L2011177], Liaoning Baiqianwan Talents Program [grant number 2013921043], Scientific Research Foundation for the Returned Overseas Chinese Scholars of Shenyang Pharmaceutical University [grant number GGJJ2015103], Liaoning Province Natural Science Foundation [grant number 201602689] and 2015 Career Development Program for Young and Middle-aged Teachers of Shenyang Pharmaceutical University [grant number ZQN2015015]. We gratefully acknowledge Prof. Xiaolin Zi (Department of Urology, University of California, Irvine, Orange, CA, USA) for the assistance with the evaluation of cytotoxicity activities against human tumor cell lines. We are grateful to Prof. Hao Gao (Jinan University, Guangzhou, China) for the X-ray diffraction analysis of compound 3. We thank Paul Owusu Donkor (University of Ghana, Accra, Ghana) for the language check and for the editorial assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.steroids.2016.09. 002.

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