Antiproliferative and Anti-inflammatory Withanolides from Physalis

Article pubs.acs.org/jnp

Antiproliferative and Anti-inflammatory Withanolides from Physalis angulata Cheng-Peng Sun,† Chong-Yue Qiu,† Ting Yuan,† Xiu-Fang Nie,† Hong-Xin Sun,† Qian Zhang,§ Hui-Xiang Li,§ Li-Qin Ding,‡ Feng Zhao,§ Li-Xia Chen,*,† and Feng Qiu*,†,‡ †

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 ‡ School of Chinese Materia Medica and Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, People’s Republic of China § School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, 264005, People’s Republic of China S Supporting Information *

ABSTRACT: Sixteen new withanolides, physangulatins A−N (1−14) and withaphysalins Y and Z (15 and 16), as well as 12 known analogues, were isolated from the stems and leaves of Physalis angulata L. Their structures were established using extensive spectroscopic data analyses. The absolute configurations of 1 and 9 were assigned via X-ray crystallography. The isolated compounds were tested for their antiproliferative effects against human prostate cancer cells (C4-2B and 22Rvl), human renal carcinoma cells (786-O, A-498, and ACHN), and human melanoma cells (A375-S2), as well as inhibitory effects on NO production induced by LPS in macrophages. Compounds 9, 17, 20, 21, 25, and 27 showed antiproliferative effects against all tested cancer cells, with IC50 values of 0.18−7.43 μM. Compounds 3−5, 9− 11, 17, 20−22, 24, 25, and 27 displayed inhibitory effects against NO production, with IC50 values of 1.36−11.59 μM. ithanolides are a class of natural C28 steroidal δ- or γlactones built on an ergostane skeleton derived from a parent 22-hydroxy-26-oic or 23-hydroxy-26-oic analogue. Withaferin A, as the first withanolide-type constituent, was reported from Withania somnifera in 1965.1 Over the last 51 years, investigations dedicated to the chemical constituents of the Solanaceae family have led to the isolation of approximately 750 withanolides.2 These withanolides can be divided into 22 types, such as normal withanolides, physalins, jaborols, nicandrenones, acnistins, withajardins, neophysalins, taccalonoides, withaphysalins, and 4-, 18-, and 19-nor-withanolides, based on the structural differences.3,4 Recent pharmacological studies have indicated that withanolides possess anti-inflammatory,5−8 antitumor,9−12 immunomodulatory,13 and antinociceptive14 activities. The genus Physalis (Solanaceae), a rich source of withanolides, comprises approximately 120 species mainly distributed in subtropical and tropical regions. Physalis angulata L. (Chinese name “Kuzhi”)15 has been used as a traditional folk medicine to treat various illnesses in China,16 such as dermatitis, trachitis, impaludism, rheumatism, and hepatitis, and it is also utilized to treat analogous conditions in other countries,7,17 including Indonesia, Peru, Mexico, and Brazil. Various bioactive withanolides, including physagulins A−Q, physangulidines A−C, withangulatins A−I, physalins B, D, F, G, and H, and withaminimin, have been isolated from this

W

© XXXX American Chemical Society and American Society of Pharmacognosy

plant, and some of them showed antiproliferative18 and antimicrobial activities,19 as well as inhibition of nitric oxide (NO) production.5 As a part of continuous research on the genus Physalis to identify a series of potential anticancer and anti-inflammatory drugs,5,6,9,10 the EtOH extracts of the dried stems and leaves of P. angulata were investigated and afforded 14 new unmodified withanolides (1−14), two rare withaphysalins (15 and 16), and 12 known analogues. In this paper, the structural elucidation of the withanolides was conducted using 1D and 2D NMR, electronic circular dichroism (ECD), and X-ray crystallographic analyses. The isolated compounds were assayed for antiproliferative activities against human prostate cancer cells (C4-2B and 22Rvl), human renal carcinoma cells (786-O, A-498, and ACHN), and human melanoma cells (A375-S2), as well as inhibitory effects on NO production induced by lipopolysaccharides (LPS) in macrophages.

■

RESULTS AND DISCUSSION The investigation of the constituents of the stems and leaves of P. angulata led to the isolation of 16 new withanolides, physangulatins A−N (1−14) and withaphysalins Y and Z (15 Received: February 2, 2016

A

DOI: 10.1021/acs.jnatprod.6b00094 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Chemical constituents of P. angulata.

the 16,17-epoxy [δH 3.73 (1H, s, H-16); δC 76.7 (C-17) and 59.5 (C-16)] and 15-acetoxy [δH 2.25 (3H, s); δC 170.3 and 21.1] moieties in 26 were absent, and signals of a double bond [δH 6.13 (1H, d, J = 2.6 Hz, H-16); δC 158.1 (C-17) and 127.5 (C-16)] in 1 were present, suggesting the presence of a Δ16,17 olefinic moiety and a hydroxy group at C-15. This conclusion was confirmed by the HMBC correlations from Me-18 to C12/C-13/C-14/C-17, H-15 to C-13/C-14/C-16/C-17, and H16 to C-13/C-14/C-15/C-17/C-20 (Figure 2). The NOESY correlation between H-15 and Me-18 (Figure 3) suggested a βorientation of H-15 and an α-orientation of OH-15. The ECD spectrum exhibited a positive Cotton effect at 252 nm, requiring a (22R)-configuration.27 Single-crystal X-ray crystallography data were used to unambiguously assign the absolute configuration of 1 (Figure 4). Therefore, the structure of physangulatin A (1) was defined as (5R,6R,8R,9S,10R,13R,14S,15S,20S,22R)-5,6,14,15-tetrahydroxy-1-oxowitha2,16,24-trienolide. Physangulatin B (2) has the same molecular formula as 1 based on HRESIMS (m/z 509.2515 [M + Na]+, calcd for C28H38O7Na, 509.2515) and 13C NMR data. The UV, IR, and 1 H and 13C NMR spectroscopic data were similar to those of 1, indicating that 2 possessed the same 2D structure as 1. HSQC and HMBC data confirmed this conclusion. The NOESY

and 16), and the known physagulins A (20),20 B (21),20 F (26),21 H (24),22 I (25),22 J (18),22 K (28),22 and N (22),23 physagulides I (27)24 and J (17),24 withaminimin (19),25 and withaminimin acetonide (23)25 (Figure 1). Physangulatin A (1) was shown to have the molecular formula C28H38O7 by HRESIMS (m/z 509.2516 [M + Na]+, calcd for C28H38O7Na, 509.2515) and 13C NMR data. The IR spectrum showed the presence of hydroxy (3398 cm−1), carbonyl (1676 cm−1), and olefinic (1648 cm−1) functionalities. The 1H NMR data of 1 displayed the characteristic signals of a 1-ox-2-ene-5α,6β-dihydroxywithanolide at δH 6.67 (1H, ddd, J = 10.0, 5.0, 2.0 Hz, H-3), 6.15 (1H, dd, J = 10.1, 2.4 Hz, H-2), and 4.32 (1H, br s, H-6). The deduction was supported by the 13 C NMR data [δC 205.6 (C-1), 142.7 (C-3), 129.5 (C-2), 78.1 (C-5), and 75.9 (C-6)]. The orientations of OH-5 and OH-6 were further confirmed by a negative Cotton effect at 331 nm in the ECD spectrum and the small coupling constant between H-6 and H-7a/H-7b.26,27 The side chain in 1 was established as a 3,4-dimethyl-5,6-dihydro-2H-pyran-2-one group using NMR data [δH 4.43 (1H, dt, J = 13.2, 3.8 Hz, H-22), 1.85 (3H, s, Me27), and 1.52 (3H, s, Me-28); δC 166.8 (C-26), 150.1 (C-24), 122.0 (C-25), 79.7 (C-22), and 32.9 (C-23)]. The 1H and 13C NMR data of 1 were consistent with those of physagulin F (26) isolated from P. angulata,21 with the exception that signals of B



Article

14 [δH 3.56 (1H, s)] with Me-18 [δH 0.99 (3H, s)], indicating a β-orientation of OH-14. Accordingly, the structure of physangulatin B (2) was defined as (5R,6R,8R,9S,10R,13R,14R,15R,20S,22R)-5,6,14,15-tetrahydroxy-1-oxowitha-2,16,24trienolide. Physangulatin C (3) was assigned the molecular formula C30H40O7 by HRESIMS (m/z 535.2667 [M + Na]+, calcd for C30H40O7Na, 535.2672) and 13C NMR data. The IR spectrum of 3 showed absorption bands of hydroxy (3395 cm−1) and olefinic (1646 cm−1) functionalities. The 1H and 13C NMR data of 3 indicated the presence of a 5α,6β-dihydroxy-2-en-1-one withanolide skeleton similar to 1 and 2. The 1H and 13C NMR data of 3 closely resembled those of 1, except for the presence of a methyl group at δH 1.34 (3H, d, J = 4.7 Hz, Me-2′) and a methine proton at δH 5.34 (1H, q, J = 4.7 Hz, H-1′) in 3 and the downfield shifts of C-14 and C-15 from δC 82.4 and 83.3 in 1 to δC 93.8 and 84.7 in 3, respectively, indicating the presence of an acetal moiety at C-14 and C-15. The HMBC correlations from H-1′ to C-2′/C-15, Me-2′ to C-1′, H-15 to C-1′/C-8/C14/C-16/C-17, H-16 to C-13/C-14/C-15/C-17/C-20, and Me-18 to C-12/C-13/C14/C-17 confirmed the presence of this functional group. The β-orientation of the methyl group at C-1′ and an α-orientation of the 14,15-acetal moiety were based on the NOESY correlations between H-1′ and H-16, Me-2′ and H9/H-15, and H-15 and Me-18. The ECD spectrum exhibited a positive Cotton effect at 252 nm, suggesting a (22R)configuration.27 Based on the above-mentioned data, the structure of physangulatin C (3) was defined as (1′R,5R,6R,8R,9S,10R,13R,14R,15S,20S,22R)-14,15-acetal-5,6-dihydroxy-1-oxowitha-2,16,24-trienolide. Physangulatin D (4) was isolated as an amorphous powder. Its molecular formula was established as C30H40O8 by HRESIMS (m/z 551.2614 [M + Na]+, calcd for C30H40O8Na, 551.2621) and 13C NMR data. The NMR data for rings A−E of 4 were similar to those of physagulin Q isolated from P. angulata L. var. villosa Bonati,28 with the exception that the C-6 signal was deshielded from δC 66.8 in physagulin Q to δC 71.2 in 4, suggesting a C-6 hydroxy group rather than a C-6 chloro group in 4. This conclusion was confirmed by the HMBC correlations from H-6 to C-4/C-5 and OH-6 to C-5/C-6/C-7 and the absence of ions containing Cl-isotopes in the HRESIMS spectrum. The chemical shift value of the C-19 methyl group was important for deducing the stereochemistry of the A/B ring junction in 5,6-dihydroxy- or 4,5,6-trihydroxywithanolides.21 For a cis-junction, the chemical shift appears at around δC 10, and in the trans-isomer, the methyl carbon resonates near δC 15. The chemical shift value of the C-19 methyl group (δC 7.8) in 4 indicated cis-fusion for rings A and

Figure 2. Selected HMBC correlations of compounds 1, 9, 13, and 15.

Figure 3. Selected NOESY correlations of compounds 1, 9, 13, and 15.

correlations between Me-19 and H-4a/H-8, H-6 and H-4b, and H-15 and H-9 indicated that OH-6 and OH-15 were both βoriented. The ECD spectrum suggested a (22R)-configuration and an α-orientation of OH-5 due to the positive and negative Cotton effects at 252 and 330 nm, respectively.27 The NOESY spectrum of 2 was also recorded in DMSO-d6 and showed correlations of H-8 [δH 1.82 (1H, br t, J = 13.5 Hz)] and OH-

Figure 4. ORTEP drawing of compound 1. C



Article

Figure 5. ORTEP drawing of compound 9.

B, which was supported by the NOESY correlation between OH-5 and Me-19. The NOESY correlations from H-6 to Me19, OH-14 to Me-18, and H-15 to Me-18 indicated a βorientation of OH-14 and the α-orientations of OH-6 and OAc15. The (R)-configuration of C-22 was determined based on the characteristic coupling pattern of H-22, δH 4.29 (1H, dt, J = 12.8, 3.6 Hz), and biosynthetic considerations.29 Therefore, the structure of physangulatin D (4) was defined as (5S,6S,8R,9S,10R,13R,14S,15S,20S,22R)-15-acetoxy-5,6,14-trihydroxy-1-oxowitha-2,16,24-trienolide. Physangulatins E (5) and F (6) possessed the same molecular formula, C29H40O7, according to HRESIMS and 13 C NMR data. The UV, IR, and 1H and 13C NMR spectra of 5 and 6 suggested that they had the same 2D structure. This conclusion was supported by HSQC and HMBC data. Comparison of the NMR data of 5 and 6 with those of 1 and 2 suggested that 5 and 6 were O-methylated derivatives of 1 and 2 because the C-15 signal was deshielded from δC 83.3 in 1 and 76.5 in 2 to δC 92.2 in 5 and 86.1 in 6, respectively. The HMBC correlations from OMe-15 [δH 3.25 (3H, s) for 5 and 3.28 (3H, s) for 6] to C-15 [δC 92.2 for 5 and 86.1 for 6] confirmed that conclusion. The orientations of OMe-15 in 5 and 6 were established by the NOESY correlations from H-9 to H-7a, H-8 to Me-19, H-15 to H-7b, and OMe-15 to H-7a in 5 and from H-15 to H-9 and OMe-15 to Me-18 in 6, indicating that OMe-15 was α-oriented in 5 and β-oriented in 6. The NOESY spectra of 5 and 6 were also recorded in DMSO-d6. The key correlations of H-8 [δH 1.84 (1H, td, J = 12.6, 2.7 Hz)] with OH-14 [δH 3.68 (1H, s)] and Me-19 [δH 1.10 (3H, s)] in 5 and of H-8 [δH 1.82 (1H, m)] with OH-14 [δH 3.41 (1H, s)] and Me-19 [δH 1.14 (3H, s)] in 6 established the β-orientations of OH-14 in 5 and 6. Thus, the structures of physangulatins E (5) and F (6) were defined as (5R,6R,8R,9S,10R,13R,14S,15S,20S,22R)-5,6,14-trihydroxy-15-methoxy-1-oxowitha2,16,24-trienolide and (5R,6R,8R,9S,10R,13R,14S,15R,20S,22R)-5,6,14-trihydroxy-15-methoxy-1-oxowitha-2,16,24-trienolide, respectively. Physangulatin G (7) was assigned a molecular formula of C30H42O9 based on HRESIMS and 13C NMR data. IR absorption bands suggested the presence of hydroxy (3396 cm−1), α,β-unsaturated δ-lactone (1711 cm−1), and olefinic (1646 cm−1) functionalities. Comparison of the NMR data of 7 and 1 indicated that signals of a methylene [δH 3.34 (1H, br dd, J = 14.3, 5.3 Hz, H-2a) and 2.76 (1H, br d, J = 14.2 Hz, H-2b); δC 47.2 (C-2)], an oxygenated methine [δH 4.85 (1H, br s, H3); δC 70.5 (C-3)], and an acetoxy group [δH 2.20 (3H, s); δC

171.2 and 21.9] in 7 replaced the signals of an olefinic moiety [δH 6.15 (1H, dd, J = 10.1, 2.4 Hz, H-2) and 6.67 (1H, ddd, J = 10.0, 5.0, 2.0 Hz, H-3); δC 129.5 (C-2) and 142.7 (C-3)] in 1. These features were reminiscent of the hydration of the Δ2,3 double bond and acetylation of OH-15 in 1 to afford 7. The HMBC cross-peaks from H-2a to C-1/C-3, H-2b to C-1/C-3/ C-4, H-4a to C-3, H-4b to C-3/C-5/C-10, and H-15 to OAc-15 confirmed the above assumption. The NOESY cross-peaks of OAc-15 with H-9 and H-8 with Me-18 and the small coupling constant between H-3 and H-2a/H-2b/H-4a/H-4b [δH 4.85 (1H, br s, H-3)]25 showed that OAc-15 and OH-3 were α- and β-oriented, respectively. The ECD spectrum indicated a (22R)configuration via the positive Cotton effect at 242 nm.27 Therefore, the structure of physangulatin G (7) was defined as (3R,5R,6R,8R,9S,10R,13R,14S,15S,20S,22R)-15-acetoxy3,5,6,14-tetrahydroxy-1-oxowitha-16,24-dienolide. HRESIMS and 13C NMR data of physangulatin H (8) provided the molecular formula C31H44O8. IR absorption bands indicated the presence of hydroxy (3395 cm−1), α,βunsaturated δ-lactone (1711 cm−1), carbonyl (1686 cm−1), and olefinic (1647 cm−1) functionalities. The NMR spectroscopic data of rings B−E in 8 closely resembled those of 7. The 1 H and 13C NMR data of 8 displayed the presence of an enoyl moiety [δH 6.57 (1H, ddd, J = 10.0, 5.3, 2.0 Hz, H-3) and 6.06 (1H, dd, J = 10.0, 2.7 Hz, H-2); δC 204.7 (C-1), 141.0 (C-3), and 129.7 (C-2)] and a methoxy group [δH 2.99 (3H, s); δC 49.9]. The HMBC correlation from OMe-5 to C-5 confirmed the location of the methoxy group. The positive and negative Cotton effects at 252 and 330 nm, respectively, in the ECD spectrum indicated a (22R)-configuration and an α-orientation of OMe-5.27 Thus, the structure of physangulatin H (8) was defined as (5R,6R,8R,9S,10R,13R,14S,15S,20S,22R)-15-acetoxy6,14-dihydroxy-5-methoxy-1-oxowitha-2,16,24-trienolide. The molecular formula of physangulatin I (9) was assigned as C31H42O9 based on HRESIMS (m/z 581.2726 [M + Na]+, calcd for C31H42O9Na, 581.2727) and 13C NMR data. The 1H and 13C NMR data of 9 were similar to those of physagulin H (24) isolated from P. angulata,22 except for the presence of a methoxy group [δH 3.20 (3H, s); δC 56.1], a methylene [δH 2.92 (1H, dd, J = 14.3, 4.9 Hz, H-2a) and 2.80 (1H, ddd, J = 14.3, 4.9, 1.4 Hz, H-2b); δC 43.3 (C-2)], and an oxygenated methine [δH 3.70 (1H, m, H-3); δC 73.9 (C-3)] in 9 and the absence of an olefinic unit [δH 6.70 (1H, ddd, J = 10.0, 6.0, 2.0 Hz, H-3) and 6.14 (1H, dd, J = 10.2, 2.0 Hz, H-2); δC 145.9 (C-3) and 128.5 (C-2)] in 24, suggesting the methoxylation of the Δ2,3 double bond at C-3. This finding was confirmed D



Article

The 1H and 13C NMR for rings A−C and E of 13 closely resembled those of 12. The differences were that signals of two methines [δH 1.68 (1H, d, J = 10.9 Hz, H-14) and 1.39 (1H, br d, J = 8.7 Hz, H-17); δC 59.4 (C-14) and 50.5 (C-17)], a methylene [δH 2.09 (1H, dd, J = 10.2, 4.6 Hz, H-16a) and 1.65 (1H, m, H-16b); δC 37.8 (C-16)], and an acetoxy group [δH 1.92 (3H, s); δC 171.0 and 21.6] in 13 replaced signals of the 16,17-epoxy group [δH 3.80 (1H, br s, H-16); δC 77.2 (C-17) and 62.9 (C-16)] and one oxygenated tertiary carbon [δC 82.9 (C-14)] in 12. The HMBC cross-peaks from H-14 to C-13/C15, H-15 to OAc-15, H-16a to C-13/C-15/C-17, H-16b to C14/C-15/C-20, and H-17 to C-15/C-16/C-18/C-20 (Figure 2) confirmed the above conclusion. The NOESY spectrum showed correlations from H-14 to H-9/H-17 and Me-18 to H-15/H-20 (Figure 3), indicating a β-orientation of the side chain at C-17 and the α-orientations of H-14, OAc-15, and H17. The positive Cotton effect at 252 nm in the ECD spectrum established a (22R)-configuration.27 Thus, the structure of physangulatin M (13) was defined as (5R,6R,8R,9S,10R,13R,14S,15S,17R,20S,22R)-15-acetoxy-5,6-dihydroxy-1-oxowitha-2,24-dienolide. Physangulatin N (14) was shown to have the molecular formula C28H40O7 based on HRESIMS (m/z 511.2671 [M + Na]+, calcd for C28H40O7Na, 511.2672) and 13C NMR data. IR absorption bands were observed at 3395, 1760, and 1647 cm−1, corresponding to hydroxy, α,β-unsaturated carbonyl, and olefinic functionalities, respectively. A comparison of the NMR data of 14 and 13 showed that differences involved the chemical shift of C-20 [δC 75.3 for 14 and 39.3 for 13] and the absence of the C-15 O-acetyl resonances [δH 1.92 (3H, s) for 13; δC 171.0 and 21.6 for 13], indicating the presence of hydroxy groups at C-15 and C-20 in 14. The HMBC correlations of H-14 with C-13/C-15/C-16/C-18, H-17 with C-13/C-20, and Me-21 with C-20 confirmed this conclusion. The NOESY spectrum showed a correlation between H-15 and Me-18, requiring an α-orientation of OH-15. The (20R,22R) absolute configurations were elucidated via biosynthetic considerations and the positive Cotton effect at 256 nm in the ECD spectrum.27 Thus, the structure of physangulatin N (14) was defined as (5R,6R,8R,9S,10R,13S,14S,15S,17S,20R,22R)-5,6,15,20-tetrahydroxy-1-oxowitha-2,24-dienolide. Withaphysalin Y (15) was assigned a molecular formula of C28H34O8 based on HRESIMS (m/z 521.2146 [M + Na]+, calcd for C28H34O8Na, 521.2151) and 13C NMR data. The 1H and 13C NMR data of 15 exhibited a 1-oxo-2,5-diene moiety [δH 6.62 (1H, ddd, J = 9.9, 4.9, 2.4 Hz, H-3), 5.88 (1H, dd, J = 10.0, 2.4 Hz, H-2), and 5.58 (1H, br d, J = 6.0 Hz, H-6); δC 204.1 (C-1), 146.3 (C-3), 135.9 (C-5), 128.1 (C-2), and 125.7 (C-6)]. Comparison of the 13C NMR data of 15 and 18βacetoxywithaphysalin C30 indicated differences at rings D and E. The proton signal at δH 5.27 (1H, t, J = 10.1 Hz) was correlated with C-15 (δC 76.8) via HSQC data and suggested the location of a hydroxy group at C-15. This finding was supported by the HMBC correlations from H-15 to C-14/C17, H-16a to C-17, and H-17 to C-13/C-16/C-18/C-20 (Figure 2). The NOESY correlations of H-11a with H-15/Me19 (Figure 3) suggested an α-orientation of OH-15. Ring E was deduced to be a γ-lactone ring linked between C-18 and C-20 using IR data (1769 cm−1), the chemical shift values of C-18 (δC 177.3) and C-20 (δC 85.3), and the HMBC cross-peaks from H-17 to C-13/C-18/C-20 and Me-21 to C-17/C-20/C22. The ECD spectrum suggested a (22R)-configuration due to

through an HMBC experiment showing a correlation from OMe-3 to C-3 (Figure 2). The NOESY correlation between H3 and Me-19 (Figure 3) indicated that OMe-3 was α-oriented. The ECD spectrum showed positive and negative Cotton effects at 247 and 294 nm, confirming a (22R)-configuration and a β-orientation of the 5,6-epoxy moiety, respectively.27 The absolute configuration of 9 was established by single-crystal Xray crystallography analysis (Figure 5). Therefore, the structure of physangulatin I (9) was defined as (3S,5S,6R,8R,9S,10R,13S,14S,15S,16S,17R,20R,22R)-15-acetoxy-5(6),16(17)diepoxy-14-hydroxy-3-methoxy-1-oxowith-24-enolide. Physangulatin J (10) had the molecular formula C31H42O9 based on HRESIMS (m/z 581.2727 [M + Na]+, calcd for C31H42O9Na, 581.2727) and 13C NMR data. The similarity of the NMR data of 10 and 8 suggested that the difference between 10 and 8 involved ring D. The HMBC correlations from H-15 to C-13/C-14/C-16/C-17, H-16 to C-13/C-14/C15, and Me-18 to C-12/C-13/C-14/C-17 suggested the presence of a 16,17-epoxy group [δC 77.0 (C-17) and 59.9 (C-16)]. Its β-orientation was established by the NOESY crosspeaks of H-12a with H-9/Me-21, H-16 with H-12a/H-20/Me21, and Me-19 with H-8. The ECD spectrum indicated a (22R)-configuration due to the positive Cotton effect at 254 nm.27 Therefore, the structure of physangulatin J (10) was defined as (5R,6R,8R,9S,10R,13S,14S,15S,16S,17R,20R,22R)15-acetoxy-16,17-epoxy-6,14-dihydroxy-5-methoxy-1-oxowitha2,24-dienolide. Physangulatin K (11) was assigned the molecular formula C31H44O9 on the basis of HRESIMS (m/z 583.2885 [M + Na]+, calcd for C31H44O9Na, 583.2883) and 13C NMR data. Comparison of the NMR data of 11 and physagulin K (28)22 suggested that 11 was an O-methylated derivative of 28 because the C-5 signal was deshielded from δC 77.3 in 28 to δC 83.1 in 11, and the signals of a methoxy group [δH 2.98 (3H, s); δC 50.0] were present in 11. The HMBC correlation from OMe-5 to C-5 confirmed this conclusion. The ECD spectrum showed positive and negative Cotton effects at 255 and 331 nm, requiring a (22R)-configuration and an α-orientation of OMe5, respectively.27 On the basis of the above evidence, the structure of physangulatin K (11) was defined as (5R,6R,8R,9S,10R,13R,14S,15S,17R,20R,22R)-15-acetoxy-6,14,17-trihydroxy5-methoxy-1-oxowitha-2,24-dienolide. Physangulatin L (12) was shown to have the molecular formula C28H38O8 by HRESIMS (m/z 525.2461 [M + Na]+, calcd for C28H38O8Na, 525.2464) and 13C NMR data. The 1H and 13C NMR data of 12 were analogous to those of 1, except for those of ring D. The 13C NMR spectrum of 12 indicated the presence of a 16β,17β-epoxy group [δC 77.2 (C-17) and 62.9 (C-16)] confirmed by the HMBC correlations from Me-18 to C-12/C-13/C-14/C-17, H-15 to C-13/C-14/C-16, H-16 to C13/C-14/C-15/C-17/C-20, and H-20 to C-13/C-16/C-17 and the NOESY correlations of H-9 with H-12a, H-16 with H-12a/ H-20/Me-21, and Me-19 with H-8. The ECD spectrum showed a positive Cotton effect at 252 nm, indicating a (22R)configuration.27 Thus, the structure of physangulatin L (12) was defined as (5R,6R,8R,9S,10R,13S,14S,15S,16S,17R,20R,22R)-16,17-epoxy-5,6,14,15-tetrahydroxy-1-oxowitha-2,24-dienolide. Physangulatin M (13) was shown to have the molecular formula C30H42O7 by HRESIMS (m/z 537.2830 [M + Na]+, calcd for C30H42O7Na, 537.2828) and 13C NMR data. The IR spectrum showed the presence of hydroxy (3398 cm−1), carbonyl (1681 cm−1), and olefinic (1647 cm−1) functionalities. E



Article

Table 1. 1H NMR Data of Compounds 1−4 position 2 3 4 6 7 8 9 11 12 15 16 18 19 20 21 22 23 27 28 OAc-15 H-1′ Me-2′ a

1a

2a

2b

3a

4c

4a

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

6.15 dd (10.1, 2.4) 6.67 ddd (10.0, 5.0, 2.0) 3.77 dt (19.8, 2.5) 2.45 dd (19.8, 5.2) 4.32 br s 3.47 td (13.2, 2.5) 2.82 m 2.86 td (12.5, 3.1) 3.79 td (12.5, 3.0) 2.82 m 1.64 br d (12.5) 2.34 td (13.2, 2.2) 1.94 dt (13.0, 3.0) 5.10 d (2.6) 6.13 d (2.6) 1.43 s 1.75 s 2.64 qd (11.3, 4.4) 1.26 d (7.1) 4.43 dt (13.2, 3.8) 2.47 br t (13.2) 2.07 dd (17.6, 2.7) 1.85 s 1.52 s

6.18 dd (10.1, 2.4) 6.69 ddd (10.1, 4.9, 2.0) 3.76 dt (19.7, 2.4) 2.44 dd (19.7, 5.0) 4.26 br s 2.94 td (13.4, 2.7) 2.65 dt (13.4, 3.0) 2.73 td (12.7, 3.0) 2.86 td (12.7, 2.7) 2.57 m 1.45 m 1.84 m 1.41 dt (13.0, 2.8) 5.25 br s 5.71 br s 1.39 s 1.73 s 2.58 m 1.20 d (7.1) 4.43 dt (13.1, 3.7) 2.52 m 2.06 dd (17.6, 2.7) 1.85 s 1.52 s

5.68 dd (9.8, 1.7) 6.62 ddd (9.8, 4.8, 1.9) 3.10 1.97 3.45 1.88 1.66 1.82 1.92 1.87 1.03 1.70 1.02 4.61 5.27 0.99 1.14 2.42 1.07 4.31 2.46 2.16 1.76 1.90

br d (19.9) dd (19.7, 4.5) br s m br d (13.2) br t (13.5) m m m m m d (5.3) br s s s m d (6.9) dt (12.8, 3.3) m br d (17.0) s s

6.18 dd (10.1, 2.5) 6.71 ddd (10.1, 5.0, 2.0) 3.78 dt (19.7, 2.5) 2.46 dd (19.7, 5.1) 4.31 br s 2.54 m 2.50 m 2.69 m 2.70 m 2.26 m 1.57 m 1.83 td (13.3, 3.0) 1.33 m 5.22 d (1.4) 5.44 d (1.4) 1.34 s 1.73 s 2.51 m 1.08 d (7.1) 4.33 m 2.34 br t (15.6) 2.14 dd (17.7, 2.8) 1.90 s 1.72 s

5.85 br d (10.4) 6.80 ddd (10.0, 5.0, 2.1) 2.58 br d (20.7) 2.44 m 3.58 m 2.14 m 1.27 m 1.73 m 1.98 td (12.2, 3.0) 1.16 m 0.87 dd (13.2, 2.5) 1.69 dd (13.0, 4.3) 1.21 d (13.0) 5.10 d (2.1) 5.57 d (1.9) 0.94 s 0.97 s 2.46 m 1.06 d (7.1) 4.29 dt (12.8, 3.6) 2.43 m 2.17 m 1.75 s 1.89 s 1.94 s

6.38 dd (10.1, 2.0) 6.91 ddd (9.9, 4.9, 2.1) 3.24 dt (20.5, 2.3) 2.42 m 4.37 m 3.07 m 2.58 m 2.18 br t (12.5) 2.40 m 2.36 dd (13.1, 4.3) 1.65 dd (15.0, 5.3) 1.78 dd (10.3, 3.6) 1.99 m 5.91 d (2.6) 6.16 d (2.5) 1.36 s 1.62 s 2.60 m 1.21 d (7.0) 4.37 m 2.46 m 2.10 m 1.84 s 1.51 s 2.08 s

5.34 q (4.7) 1.34 d (4.7)

Run at 400 MHz, pyridine-d5. bRun at 600 MHz, DMSO-d6. cRun at 400 MHz, DMSO-d6.

the positive Cotton effect at 250 nm.27 Thus, the structure of withaphysalin Y (15) was defined as (8R,9S,10R,13R,14R,15S,17R,20R,22R)-15-hydroxy-1-oxo-13,14-secowitha2,5,24-triene-18(20),26(22)-diolide. HRESIMS and 13C NMR data of withaphysalin Z (16) indicated the molecular formula C28H34O9. The 1H and 13C NMR spectra displayed the presence of a 4-hydroxymethyl-3methyl-5,6-dihydro-2H-pyran-2-one moiety [δH 4.60 (1H, dd, J = 12.8, 2.8 Hz, H-22), 4.24 (1H, dd, J = 14.9, 5.5 Hz, H-28a), 4.15 (1H, dd, J = 14.9, 5.4 Hz, H-28b), and 1.77 (3H, s, Me27); δC 165.6 (C-26), 152.9 (C-24), 119.8 (C-25), 78.5 (C-22), 59.7 (C-28), 26.1 (C-23), and 11.6 (C-27)], which was supported by the HMBC cross-peaks from H-22 to C-24, Me27 to C-24/C-25/C-26, and H-28a/H-28b to C-23/C-24/C25. Comparison of the NMR data of rings A−E in 16 and withaphysalin A31 indicated the presence of hydroxy groups at C-12 and C-15 in 16. The HMBC cross-peaks from H-8 to C14/C-15, H-9 to C-1/C-8/C-10/C-14, H-11b to C-12, H-12 to C-14/C-17/C-18, and H-17 to C-12/C-13/C-16/C-20 confirmed this conclusion. The NOESY cross-peaks of H-12 with H-9/H-17/Me-21, H-8 with Me-19, and H-15 with H-9/H-17 confirmed the β-orientations of OH-12 and OH-15. The ECD spectrum established a (22R)-configuration due to the positive Cotton effect at 255 nm.27 Thus, the structure of withaphysalin Z (16) was defined as (8R,9S,10R,12R,13R,14R,15R,17S,20R,22R)-12,14,15,28-tetrahydroxy-1-oxowitha-2,5,24-triene-18(20),26(22)-diolide. All compounds were evaluated for antiproliferative effects against human prostate cancer cells (C4-2B and 22Rvl), human

renal carcinoma cells (786-O, A-498, and ACHN), and human melanoma cells (A375-S2). As shown in Table 7, compounds 9, 17, 20−22, 25, and 27 showed significant antiproliferative effects against human prostate cancer cells (C4-2B and 22Rvl) and human renal carcinoma cells (786-O) with IC50 values of 0.18−2.43 μM, while compounds 17, 20, 21, 25, and 27 exhibited significant antiproliferative effects against human renal carcinoma cells (A-498 and ACHN) and human melanoma cells (A375-S2), with IC50 values of 0.42−7.43 μM. These results indicated that withanolides with a 5β,6βepoxy or 5α-Cl/6β-OH moieties possessed significant antiproliferative effects against all tested cancer cells. Nitric oxide (NO) is a well-known cellular signaling molecule and is considered an important regulator in many physiological mechanisms. Pharmacological studies have indicated that inflammation is related to overproduction of NO.32 The inhibitory effects of all compounds on NO production were evaluated, and the results are listed in Table 8. Hydrocortisone was used as a positive control and had an IC50 value of 58.79 μM. Compounds 9, 17, 20−22, 24, 25, and 27 displayed significant inhibitory effects against NO production (IC50 values from 1.36 to 5.56 μM), as expected for 5β,6β-epoxy or 5α-Cl,6β-OH withanolides. Other withanolides (3 and 5) without the above-mentioned moieties also exhibited significant inhibitory effects on NO production. A comparison of the inhibitory efficiency of withanolides indicated that the methoxy group was of pivotal importance. Compounds 10 and 11 showed significant inhibitory activities due to the methylation of OH-5. An analogous case was also observed for 5α,6βF



Article

Table 2. 1H NMR Data of Compounds 5−8 position

5a

5b

6a

6b

7a

8a

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

2

6.13 dd (10.1, 2.5)

5.64 dd (10.1, 2.3)

6.17 dd (10.1, 2.5)

5.68 dd (9.8, 2.0)

3 4

6.66 ddd (10.0, 5.0, 2.0) 3.77 dt (19.6, 2.5) 2.43 dd (19.6, 5.0) 4.30 br s 3.01 td (12.7, 2.7) 2.72 dt (12.7, 2.7) 2.76 td (12.1, 3.1) 3.50 td (12.1, 2.9) 2.75 dd (13.0, 3.0) 1.57 td (13.0, 2.8) 2.09 td (13.0, 2.7) 1.83 dt (13.0, 2.8) 4.34 d (2.2) 6.17 d (2.1) 1.37 s 1.71 s 2.60 qd (7.2, 4.3) 1.27 d (7.1) 4.41 dt (13.1, 3.8) 2.49 br t (15.2) 2.06 dd (15.2, 2.6) 1.86 s 1.54 s

6.59 ddd (9.9, 4.9, 2.1) 3.10 dt (19.8, 2.2) 1.92 dd (19.8, 4.9) 3.49 dd (7.0, 3.0) 2.16 td (12.6, 2.5) 1.79 dt (12.7, 2.5) 1.84 td (12.6, 2.7) 2.50 m 1.98 dd (12.8, 2.8) 1.02 br d (12.4) 1.58 td (12.8, 2.9) 1.46 dt (13.0, 2.5) 3.78 d (2.1) 5.81 d (1.8) 0.93 s 1.10 s 2.45 m 1.13 d (7.0) 4.30 dt (13.0, 3.7) 2.47 m 2.18 dd (14.7, 2.4) 1.76 s 1.90 s

6.69 ddd (10.0, 5.0, 2.1) 3.76 dt (19.6, 2.5) 2.43 dd (19.6, 5.0) 4.24 br s 2.48 m 1.30 m 2.61 m 2.74 td (12.0, 2.6) 2.52 m 1.43 td (12.0, 2.6) 1.83 m 1.33 m 4.43 br s 5.79 br s 1.33 s 1.70 s 2.59 m 1.25 d (7.1) 4.45 dt (13.1, 3.6) 2.53 m 2.05 dd (17.6, 2.5) 1.84 s 1.52 s

6.62 ddd (9.8, 5.1, 2.0) 3.10 dt (19.7, 2.5) 1.95 dd (19.9, 5.2) 3.47 d (2.8) 1.72 m 1.21 m 1.82 m 1.95 m 1.88 m 1.02 m 1.72 m 1.01 br d (9.5) 4.26 d (2.1) 5.59 br s 0.98 s 1.14 s 2.46 m 1.10 d (6.8) 4.34 dt (12.9, 3.5) 2.50 m 2.17 dd (17.9, 2.1) 1.76 s 1.91 s

3.25 s

3.16 s

3.28 s

3.36 s

6 7 8 9 11 12 15 16 18 19 20 21 22 23 27 28 OAc-15 OMe-5 OMe-15 a

3.34 br dd (14.3, 5.3) 2.76 br d (14.2) 4.85 br s 3.32 dd (14.5, 3.4) 2.14 br d (14.5) 4.21 br s 2.60 td (13.1, 2.6) 2.55 td (13.1, 2.6) 2.73 td (12.5, 3.5) 3.54 td (12.5, 2.2) 2.29 dd (12.1, 2.8) 1.42 br t (12.0) 2.03 m 1.90 dt (13.0, 3.2) 6.18 d (2.6) 6.01 d (2.6) 1.42 s 1.72 s 2.67 qd (7.1, 4.4) 1.26 d (7.1) 4.41 dt (12.8, 3.7) 2.43 br t (15.2) 2.04 m 1.85 s 1.50 s 2.20 s

6.06 dd (10.0, 2.7) 6.57 ddd (10.0, 5.3, 2.0) 3.36 dt (20.2, 2.4) 2.48 dd (20.2, 5.2) 4.24 br s 2.58 td (13.2, 2.8) 2.13 dt (13.2, 2.5) 2.70 td (12.0, 2.7) 3.19 td (12.0, 2.6) 2.69 dd (12.9, 3.2) 1.54 br t (13.0) 1.96 dd (13.0, 2.3) 1.87 dt (13.0, 3.0) 6.07 d (2.5) 6.02 d (2.5) 1.39 s 1.62 s 2.63 qd (7.1, 4.5) 1.25 d (7.1) 4.39 dt (12.9, 3.7) 2.42 br t (15.0) 2.01 dd (17.4, 2.3) 1.85 s 1.51 s 2.15 s 2.99 s

b

Run at 400 MHz, pyridine-d5. Run at 600 MHz, DMSO-d6. the herbarium of the Department of Natural Products Chemistry, Shenyang Pharmaceutical University. Extraction and Isolation. The dried stems and leaves of P. angulata (9.5 kg) were extracted with 75% EtOH (2 × 2 h × 110 L) to afford a residue after solvent removal in vacuo. The residue (1.3 kg) was suspended in H2O (5 L) and extracted with petroleum ether (3 × 5 L), EtOAc (3 × 5 L), and n-BuOH (3 × 5 L), successively. The EtOAc extracts (116 g) were subjected to silica gel CC (10 × 80 cm) eluted with CH2Cl2−MeOH (100:1, 80:1, 60:1, 40:1, 20:1, 10:1, 8:1, 5:1, 3:1, 1:1, and 0:1, v/v) to afford six fractions (E1−E6). Fraction E3 (35 g) was separated by silica gel CC (6 × 80 cm) with a gradient of acetone in petroleum ether ranging from 10% to 100% to produce seven subfractions (E31−E37). Subfraction E31 (5 g) was separated by silica gel CC (5 × 70 cm) eluted with petroleum ether−EtOAc (100:1 to 1:1) and preparative TLC (CHCl3−acetone, 4:1) to afford compounds 19 (10 mg) and 24 (75 mg). Subfraction E34 (4.2 g) was subjected to Sephadex LH-20 CC (3 × 80 cm) using CH2Cl2−MeOH (1:1) to yield two subfractions (E341 and E342). Purification of subfraction E342 (600 mg) by an ODS column (3 × 50 cm), with MeOH−H2O as solvent (1:9 to 1:0), yielded compound 20 (220 mg) and four subfractions (E3421−E3424). Separation of subfraction E3422 (150 mg) through preparative TLC (CH2Cl2−acetone, 4:1) and preparative HPLC (MeOH−H2O, 7:3) afforded compounds 22 (8 mg, tR = 35 min) and 27 (53 mg, tR = 27 min). Compound 25 (21 mg, tR = 19 min) was separated from subfraction E3423 (70 mg) by preparative HPLC (MeOH−H2O, 7:3). Subfraction E3424 (500 mg) was purified by preparative TLC (CH2Cl2−acetone, 4:1) to afford compounds 9 (17 mg), 17 (9 mg), 21 (37 mg), and 26 (250 mg) and an impure subfraction, which was further purified by preparative HPLC (MeOH−H2O, 7:3) to afford compounds 11 (8 mg, tR = 62 min), 15 (10 mg, tR = 42 min), and 18 (10 mg, tR = 20 min). Fraction E4 (15 g) was subjected to silica gel CC (5 × 70 cm), with CHCl3−

dihydroxywithanolides, compounds 5 and 6 showed significant inhibitory activities, whereas IC50 values of compounds 1 and 2 were only 36.47 and 38.23 μM, respectively, indicating that OMe-15 could significantly increase inhibitory activity on NO production.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a PerkinElmer 241 polarimeter. UV spectra were measured on a Shimadzu UV 2201 spectrophotometer. ECD spectra were recorded on a Bio-Logic Science MOS-450 spectrometer. IR spectra were recorded on a Bruker IFS 55 spectrometer. Bruker AV400 and AV-600 spectrometers were used in the NMR experiments. Chemical shift values are expressed in δ (ppm) using the peak signals of the solvent pyridine-d5 (δH 7.58 and δC 135.9) or DMSO-d6 (δH 2.50 and δC 39.5) as references, and coupling constants (J in Hz) are given in parentheses. HRESIMS data were acquired on an Agilent 6210 TOF mass spectrometer. Silica gel GF254 prepared for TLC was purchased from Qingdao Marine Chemical Factory (Qingdao, China). Silica gel (200−300 mesh, Qingdao Marine Chemical Factory), Sephadex LH-20 (Pharmacia, USA), and octadecyl silica gel (Merck Chemical Company Ltd., Germany) were used for column chromatography (CC). RP-HPLC was equipped with an LC-6AD liquid chromatograph, SPD-20A UV detector (Shimadzu, Kyoto, Japan), and RP-C18 column (250 × 20 mm, 120 Å, 5 μm, YMC Co. Ltd.). Spots were detected on TLC plates under UV light or by heating after spraying with anisaldehyde−H2SO4 reagent. Plant Material. The stems and leaves of P. angulata were collected from Nanning, Guangxi Province, China, in July 2013, and identified by Pharmacist Jia-Fu Wei, Guangxi Institute for Food and Drug Control. A voucher specimen (PA-20130826) has been deposited in G



Article

Table 3. 1H NMR Data of Compounds 9−12 9a position 2

3

δH (J in Hz)

δH (J in Hz)

6.05 dd (10.1, 2.5)

6.14 dd (10.0, 2.5)

3.32 br s 2.76 td (14.5, 2.7) 1.67 m

11

1.98 td (12.5, 3.7) 2.29 td (12.5, 3.6) 1.78 dd (13.0, 3.7) 1.30 m

6.56 ddd (10.0, 5.0, 2.1) 3.32 dt (20.3, 2.4) 2.44 dd (20.3, 5.0) 4.17 br s 2.47 m

6.56 ddd (9.9, 4.9, 1.9) 3.34 dt (19.7, 2.5) 2.44 dd (20.1, 5.0) 4.21 br s 2.46 m

1.88 m

2.05 m

2.43 td (13.0, 3.0) 2.94 td (13.0, 3.0) 2.60 m

2.68 m

1.55 br d (12.6) 1.81 m

12

1.68 m

15

1.56 dt (13.0, 3.2) 5.33 s

16

3.77 s

3.74 br s

18 19 20 21 22

1.27 s 1.31 s 2.52 m 0.97 d (7.1) 4.47 ddd (12.7, 5.7, 3.6) 2.29 m 2.11 dd (17.6, 2.8) 1.90 s 1.73 s 2.17 s 3.20 s

1.37 s 1.54 s 2.59 m 1.00 d (7.1) 4.48 ddd (12.7, 5.5, 3.7) 2.33 br t (15.2) 2.11 dd (17.5, 2.9) 1.91 s 1.73 s 2.21 s

27 28 OAc-15 OMe-3 OMe-5

14a

15a

16b

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

6.16 dd (10.1, 2.4) 6.68 ddd (10.0, 5.0, 2.1) 3.78 dt (19.6, 2.5) 2.46 dd (19.6, 5.1) 4.21 br s

6.18 dd (10.1, 2.6) 6.69 ddd (10.1, 5.0, 2.0) 3.81 dt (19.7, 2.5) 2.45 dd (20.0, 4.6) 4.27 br s

5.88 dd (10.0, 2.4) 6.62 ddd (9.9, 4.9, 2.4)

5.83 dd (10.1, 2.2) 6.95 ddd (10.0, 4.9, 2.5) 3.29 dd (21.6, 2.0) 2.88 dd (21.6, 4.9) 5.61 d (6.0)

2.51 m 2.01 dt (13.4, 2.9) 2.41 td (11.9, 2.8) 2.55 td (11.9, 2.9) 2.77 dd (13.2, 3.3) 1.54 td (13.2, 3.3) 1.93 m 1.46 dd (13.0, 3.2) 1.68 d (10.9) 5.02 td (9.7, 3.0) 2.09 dd (10.2, 4.6) 1.65 m 1.39 br d (8.7)

2.80 m 1.95 m

0.69 s 1.73 s 1.89 m 0.97 d (7.0) 4.26 dt (13.2, 3.4) 2.24 br t (14.1) 1.86 m

1.32 s 1.80 s 1.46 s 4.40 dd (13.3, 3.6) 2.40 br t (14.3) 2.18 m

27 28

1.94 s 1.67 s

1.88 s 1.65 s

OAc-15

1.92 s

position 2 3

1.54 br d (13.0) 1.95 td (13.3, 2.7) 1.66 dt (13.1, 2.8) 5.57 br s

23

a

δH (J in Hz) 6.04 dd (10.0, 2.4)

6 7

13a

12a

δH (J in Hz)

2.38 dd (14.5, 3.1) 1.67 m

9

11a

2.92 dd (14.3, 4.9) 2.80 ddd (14.3, 4.9, 1.4) 3.70 m

4

8

10a

Table 4. 1H NMR Data of Compounds 13−16

2.96 s

3.31 td (12.7, 3.4) 2.70 m

1.65 m 5.81 dd (8.6, 3.5) 2.95 dd (15.8, 8.7) 2.08 m 1.44 s 1.62 s 2.39 m 1.25 d (6.9) 5.05 dt (12.7, 3.1) 2.68 m 2.08 m 1.96 s 1.73 s 2.21 s

6.65 ddd (10.0, 5.0, 2.1) 3.76 dt (19.8, 2.5) 2.42 dd (19.8, 5.0) 4.26 br s 3.14 td (13.1, 2.6) 2.75 dt (13.1, 3.0) 2.61 td (12.9, 3.1) 3.71 td (12.3, 3.0) 2.82 dd (12.8, 3.3) 1.55 dd (12.8, 2.3) 2.38 td (13.3, 2.3) 1.70 dt (13.3, 3.2) 4.59 br s

4

6 7

8 9 11

12

14 15

3.80 br s

16

1.43 s 1.70 s 2.71 m 1.06 d (7.1) 4.57 ddd (12.7, 4.9, 3.7) 2.40 m 2.17 dt (17.6, 2.7) 1.93 s 1.74 s

17 18 19 20 21 22 23

2.98 s

Run at 400 MHz, pyridine-d5.

acetone (80:1 to 1:1) as solvent, to afford five subfractions (E41− E45). Subfraction E42 (2 g) was separated by Sephadex LH-20 CC (3 × 80 cm, MeOH), preparative TLC (CH2Cl2−acetone, 4:1), and preparative HPLC (MeOH−H2O, 7:3) to give compounds 8 (22 mg, tR = 42 min) and 13 (19 mg, tR = 33 min). Purification of subfraction E44 (512 mg) by ODS CC (3 × 50 cm) eluted with MeOH−H2O (1:9 to 1:0), preparative TLC (CH2Cl2−acetone, 2:1), and preparative HPLC (MeOH−H2O, 65:35) afforded compound 14 (4 mg, tR = 15 min). Subfraction E45 (4 g) was separated by an ODS column (3 × 50 cm) eluted with MeOH−H2O (1:9 to 1:0) to afford four subfractions (E451−E454). Subfraction E452 (800 mg) was purified by preparative TLC (CH2Cl2−acetone, 2:1), yielding compounds 1 (10 mg), 4 (10 mg), 6 (21 mg), and 28 (65 mg) and an impure subfraction, which was further separated by preparative HPLC (MeOH−H2O, 65:35) to yield compounds 5 (4 mg, tR = 26 min), 7 (3 mg, tR = 16 min), 10 (19 mg, tR = 12 min), 12 (6 mg, tR = 17 min), and 16 (6 mg, tR = 20 min). Compounds 2 (31 mg) and 23 (8 mg) were separated from subfraction E453 (200 mg) by preparative TLC (CH2Cl2−acetone,

a

2.64 m 2.64 m 2.80 m 1.69 td (12.8, 2.1) 2.18 m 1.70 m 1.75 t (9.9) 4.41 m 3.07 dt (13.7, 10.1) 1.98 m 2.08 d (10.1)

3.21 br d (21.0) 2.67 br d (21.0) 5.58 br d (6.0) 2.72 m 2.39 m

2.31 m 2.10 m

2.11 td (12.2, 5.0) 2.63 td (12.7, 3.0) 3.27 td (12.9, 3.0) 2.30 m

1.94 td (11.9, 4.8) 1.84 m

2.38 m 2.28 m

3.48 m

5.27 t (10.1)

3.98 dd (17.5, 7.8) 2.06 m

3.10 br d (13.8) 1.89 m 3.03 dd (13.8, 3.5)

2.12 m 2.05 m

1.80 m 2.54 m

1.64 s

1.16 s

1.64 s 4.69 dd (12.8, 3.5) 2.69 m

1.40 s 4.60 dd (12.8, 2.8) 2.58 m

2.25 dd (17.5, 3.2) 1.89 s 1.72 s

2.29 m 1.77 s 4.24 dd (14.9, 5.5) 4.15 dd (14.9, 5.4)

Run at 400 MHz, pyridine-d5. bRun at 400 MHz, DMSO-d6.

2:1). Finally, purification of subfraction E454 (150 mg) by preparative TLC (CH2Cl2−acetone, 2:1) and preparative HPLC (MeOH−H2O, 65:35) yielded compound 3 (10 mg, tR = 25 min). Physangulatin A (1): amorphous powder; [α]25 D +124 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 212 (4.2) nm; IR (KBr) νmax 3398, 2921, 2851, 1676, 1648, 1467, 1384, 1133 cm−1; ECD (MeOH) nm (Δε) 252 (+3.9), 331 (−1.7); 1H (400 MHz, pyridine-d5) and 13C NMR (100 MHz, pyridine-d5) data, see Tables 1 and 5; HRESIMS m/ z 509.2516 [M + Na]+ (calcd for C28H38O7Na, 509.2515). X-ray Crystal Structure Determination of Physangulatin A (1). The data were collected on an Xcalibur, Eos, Gemini diffractometer using monochromatized Cu Kα radiation. The structure was solved by direct methods using SHELXL. All H atoms were refined using the riding model. All non-H atoms were refined anisotropically. Crystallographic data have been deposited in the Cambridge Crystallographic H



Article

Table 5. 13C NMR Data of Compounds 1−6 1a 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 OAc-15 OMe-5 OMe-15 C-1′ C-2′ a

δC, type 205.6, 129.5, 142.7, 37.1, 78.1, 75.9, 29.0, 37.7, 37.4, 53.5, 24.8, 39.8, 53.1, 82.4, 83.3, 127.5, 158.1, 17.9, 16.0, 35.5, 18.4, 79.7, 32.9, 150.1, 122.0, 166.8, 13.0, 20.2,

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

2a δC, type 205.4, 129.4, 142.9, 37.0, 77.7, 75.4, 30.8, 37.7, 38.2, 53.2, 23.9, 40.4, 53.3, 84.0, 76.5, 127.2, 154.6, 17.4, 16.7, 35.1, 18.1, 79.4, 32.7, 150.0, 122.0, 166.8, 13.0, 20.2,


2b δC, type 204.1, 127.5, 142.8, 35.3, 76.0, 73.1, 28.8, 35.6, 36.4, 51.1, 22.3, 39.1, 51.9, 82.6, 74.9, 125.9, 153.1, 16.4, 15.4, 33.8, 17.4, 78.3, 31.5, 150.3, 120.4, 165.9, 12.3, 20.2,


3a δC, type 205.2, 129.3, 143.0, 36.9, 77.8, 75.5, 30.9, 34.1, 38.0, 54.1, 23.6, 39.3, 53.2, 93.8, 84.7, 121.0, 160.5, 19.9, 15.8, 36.0, 18.2, 79.9, 33.6, 149.5, 122.3, 166.5, 13.0, 20.4,


4c

4a

δC, type 201.7, 126.7, 144.4, 30.1, 77.3, 71.2, 30.6, 38.8, 39.5, 54.6, 22.0, 37.7, 52.0, 80.2, 83.2, 121.3, 160.8, 15.7, 7.8, 34.1, 17.4, 78.0, 31.5, 150.3, 120.3, 165.7, 12.2, 20.1, 169.2, 21.0,

δC, type

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

202.9, 128.7, 144.7, 31.7, 79.1, 72.8, 32.5, 40.7, 41.0, 56.5, 23.5, 39.1, 53.6, 82.0, 84.9, 122.9, 162.5, 16.9, 9.1, 35.7, 18.2, 79.2, 32.8, 149.8, 122.1, 166.6, 13.0, 20.2, 170.2, 21.6,


5a δC, type 205.5, 129.5, 142.7, 37.3, 78.0, 75.8, 29.0, 37.7, 37.2, 53.4, 24.3, 39.2, 53.2, 82.7, 92.2, 123.6, 160.6, 17.6, 15.9, 35.6, 18.7, 79.6, 33.1, 150.2, 122.0, 166.8, 13.0, 20.2,


57.0, CH3

5b δC, type 204.4, 127.7, 142.4, 35.5, 76.2, 73.4, 26.8, 35.6, 35.2, 51.6, 22.6, 37.6, 51.3, 80.9, 90.7, 122.5, 158.6, 16.2, 14.5, 34.2, 17.4, 78.2, 31.3, 150.5, 120.3, 165.9, 12.3, 20.2,


56.4, CH3

6a δC, type 205.4, 129.3, 142.9, 37.2, 77.6, 75.3, 30.4, 37.5, 38.0, 53.1, 23.8, 40.2, 53.3, 84.4, 86.1, 121.8, 156.2, 17.0, 16.7, 35.2, 18.1, 79.2, 32.7, 150.0, 122.0, 166.8, 13.0, 20.2,


57.0, CH3

6b δC, type 204.1, 127.4, 142.8, 35.3, 76.1, 73.0, 28.5, 35.5, 36.3, 51.1, 22.3, 38.9, 52.0, 83.2, 84.9, 120.9, 154.6, 16.1, 15.4, 34.0, 17.3, 78.2, 31.5, 150.4, 120.3, 165.8, 12.2, 20.1,


56.5, CH3

99.3, CH 19.6, CH3

Run at 100 MHz, pyridine-d5. bRun at 150 MHz, DMSO-d6. cRun at 100 MHz, DMSO-d6. 2920, 2849, 1647, 1469, 1384, 1130 cm−1; 1H (400 MHz, DMSO-d6; 400 MHz, pyridine-d5) and 13C NMR (100 MHz, DMSO-d6; 100 MHz, pyridine-d5) data, see Tables 1 and 5; HRESIMS m/z 551.2614 [M + Na]+ (calcd for C30H40O8Na, 551.2621). Physangulatin E (5): amorphous powder; [α]25 D +160 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 216 (4.2) nm; IR (KBr) νmax 3397, 2921, 2850, 1677, 1646, 1467, 1384, 1133 cm−1; ECD (MeOH) nm (Δε) 252 (+4.4), 330 (−1.7); 1H (400 MHz, pyridine-d5; 600 MHz, DMSO-d6) and 13C NMR (100 MHz, pyridine-d5; 150 MHz, DMSOd6) data, see Tables 2 and 5; HRESIMS m/z 523.2670 [M + Na]+ (calcd for C29H40O7Na, 523.2672). Physangulatin F (6): amorphous powder; [α]25 D +67 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 218 (4.1) nm; IR (KBr) νmax 3399, 2920, 2850, 1681, 1646, 1467, 1384, 1129 cm−1; ECD (MeOH) nm (Δε) 250 (+4.3), 332 (−1.2); 1H (400 MHz, pyridine-d5; 600 MHz, DMSO-d6) and 13C NMR (100 MHz, pyridine-d5; 150 MHz, DMSO-d6) data, see Tables 2 and 5; HRESIMS m/z 523.2672 [M + Na]+ (calcd for C29H40O7Na, 523.2672). Physangulatin G (7): amorphous powder; [α]25 D +69 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 220 (3.8) nm; IR (KBr) νmax 3396, 2921, 2850, 1711, 1646, 1469, 1384, 1261, 1121 cm−1; ECD (MeOH) nm (Δε) 242 (+1.9); 1H (400 MHz, pyridine-d5) and 13C NMR (100 MHz, pyridine-d5) data, see Tables 2 and 6; HRESIMS m/z 569.2732 [M + Na]+ (calcd for C30H42O9Na, 569.2727). Physangulatin H (8): amorphous powder; [α]25 D +164 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 216 (4.2) nm; IR (KBr) νmax 3395, 2921, 2850, 1732, 1711, 1686, 1647, 1469, 1383, 1242, 1131 cm−1;

Data Centre (CCDC number 1444697). Copies of the data can be obtained, free of charge, from the CCDC Web site (www.ccdc.cam.ac. uk). Crystal data: C28H40O8, M = 504.60, monoclinic (MeOH), size 0.25 × 0.18 × 0.09 mm3, a = 10.3465(3) Å, b = 12.0928(4) Å, c = 11.1061(4) Å, α = 90°, β = 112.619(4)°, γ = 90°, V = 1282.70(8) Å3, T = 104.0 K, space group P21 (no. 4), Z = 2, μ(Cu Kα) = 0.776, completeness θmax = 99.8%, F(000) = 544, 2θ range for data collection from 8.626° to 142.276°, 8683 reflections measured, 4582 unique (Rint = 0.0252), which were used in all calculations. The final wR(F2) was 0.1018 (all data). The Flack and Hooft parameters were 0.01(14) and 0.06(8), respectively. The largest difference peak and hole were 0.278 and −0.236 e Å−3. Physangulatin B (2): amorphous powder; [α]25 D +83 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 214 (4.3) nm; IR (KBr) νmax 3427, 2921, 2851, 1676, 1467, 1384, 1132 cm−1; ECD (MeOH) nm (Δε) 252 (+4.7), 330 (−2.3); 1H (400 MHz, pyridine-d5; 600 MHz, DMSO-d6) and 13C NMR (100 MHz, pyridine-d5; 150 MHz, DMSOd6) data, see Tables 1 and 5; HRESIMS m/z 509.2515 [M + Na]+ (calcd for C28H38O7Na, 509.2515). Physangulatin C (3): amorphous powder; [α]25 D +40 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 216 (3.9) nm; IR (KBr) νmax 3395, 2921, 2849, 1646, 1469, 1384, 1119 cm−1; ECD (MeOH) nm (Δε) 252 (+1.7), 331 (−1.1); 1H (400 MHz, pyridine-d5) and 13C NMR (100 MHz, pyridine-d5) data, see Tables 1 and 5; HRESIMS m/z 535.2667 [M + Na]+ (calcd for C30H40O7Na, 535.2672). Physangulatin D (4): amorphous powder; [α]25 D +66 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 218 (3.8) nm; IR (KBr) νmax 3395, I



Article

Table 6. 13C NMR Data of Compounds 7−16 7a δC, type

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 OAc-15

212.5, 47.2, 70.5, 38.0, 80.8, 75.9, 28.4, 37.2, 36.4, 56.8, 23.8, 39.4, 53.8, 82.3, 83.8, 122.7, 162.3, 17.4, 17.3, 35.6, 18.1, 79.2, 32.6, 149.6, 122.0, 166.7, 12.9, 20.1, 171.2, 21.9,

OMe-3 OMe-5 a

8a

9a

δC, type

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

204.7, 129.7, 141.0, 28.5, 83.0, 68.3, 28.8, 37.2, 36.8, 53.5, 24.7, 39.4, 53.6, 82.2, 84.0, 122.6, 162.6, 17.5, 16.2, 35.6, 18.3, 79.3, 32.8, 149.8, 122.0, 166.6, 13.0, 20.1, 170.8, 21.7,


10a

δC, type 211.2, 43.3, 73.9, 36.9, 62.3, 63.1, 26.6, 35.5, 37.9, 52.3, 22.1, 32.1, 47.1, 81.7, 78.7, 59.4, 76.7, 15.8, 15.4, 34.5, 14.2, 77.3, 33.2, 149.7, 122.2, 166.3, 13.0, 20.4, 170.1, 21.1, 56.1,

δC, type

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

204.6, 129.6, 141.1, 28.3, 82.8, 68.2, 29.4, 36.1, 36.3, 53.3, 23.4, 33.5, 47.4, 82.5, 77.7, 59.9, 77.0, 16.3, 16.1, 34.0, 14.4, 77.3, 33.2, 149.8, 122.2, 166.4, 13.0, 20.4, 170.2, 21.3,

49.9, CH3


49.9, CH3

11a

12a

δC, type 204.4, 129.8, 140.8, 28.5, 83.1, 68.2, 28.4, 36.1, 35.6, 53.5, 23.7, 31.9, 51.7, 87.9, 79.9, 48.8, 86.6, 16.3, 15.9, 42.8, 10.4, 77.7, 32.7, 150.9, 122.0, 167.2, 13.0, 20.5, 170.4, 21.7,

13a

δC, type

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

205.5, 129.5, 142.7, 37.1, 77.8, 75.9, 29.9, 36.9, 36.7, 53.3, 23.2, 33.5, 47.1, 82.9, 77.2, 62.9, 77.2, 16.5, 15.9, 33.6, 14.5, 77.7, 33.2, 149.9, 122.2, 166.6, 13.0, 20.4,

δC, type


205.2, 129.4, 142.7, 37.1, 77.8, 75.1, 34.3, 30.9, 42.3, 52.9, 24.1, 41.1, 44.2, 59.4, 76.5, 37.8, 50.5, 13.7, 16.7, 39.3, 13.7, 78.4, 29.8, 149.9, 122.2, 166.9, 13.1, 20.4, 171.0, 21.6,

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

14a

15a

δC, type 205.6, 129.6, 142.6, 37.2, 78.0, 75.4, 35.3, 30.9, 42.8, 53.1, 24.3, 42.1, 45.5, 64.1, 73.2, 36.1, 53.8, 16.4, 16.8, 75.3, 21.8, 82.4, 32.3, 149.9, 122.1, 166.9, 13.0, 20.4,

16b

δC, type

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

204.1, 128.1, 146.3, 33.7, 135.9, 125.7, 28.0, 41.1, 36.7, 52.1, 26.9, 46.5, 83.1, 100.4, 76.8, 22.1, 60.0, 177.3, 20.6, 85.3, 26.6, 77.4, 32.2, 149.7, 121.8, 165.3, 12.9, 20.4,

δC, type

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

203.2, 126.7, 147.2, 32.7, 133.8, 125.0, 24.1, 38.6, 35.3, 50.1, 32.0, 71.8, 64.3, 80.5, 74.1, 33.7, 47.3, 173.9, 19.0, 83.2, 23.3, 78.5, 26.1, 152.9, 119.8, 165.6, 11.6, 59.7,

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

50.0, CH3

b

Run at 100 MHz, pyridine-d5. Run at 100 MHz, DMSO-d6.

Table 7. IC50 Valuesa of Compounds 1−28 against Human Tumor Cell Lines compound

b

9 13 17 20 21 22 25 27 5-fluorouracild

C4-2B

22Rv1

786-O

A-498

ACHN

A375-S2

(mean ± SD, μM)

(mean ± SD, μM)

(mean ± SD, μM)

(mean ± SD, μM)

(mean ± SD, μM)

(mean ± SD, μM)

2.12 − 0.49 0.18 0.51 1.15 1.07 0.27 5.64

± 0.47 ± ± ± ± ± ± ±

0.15 0.02 0.12 0.31 0.26 0.03 0.45

1.17 11.58 0.90 0.63 0.48 2.23 2.28 2.38 3.83

± ± ± ± ± ± ± ± ±

0.24 1.35 0.27 0.13 0.09 0.26 0.12 0.13 0.16

2.20 − 0.39 0.66 0.42 2.43 0.69 0.52 −

± 0.07 ± ± ± ± ± ±

0.09 0.06 0.02 0.09 0.04 0.02

−c − 0.71 0.66 0.42 4.70 0.52 0.53 8.83

± ± ± ± ± ± ±

0.04 0.7 0.04 0.27 0.09 0.10 0.88

− − 0.73 1.08 4.80 7.50 0.78 0.40 2.73

± ± ± ± ± ± ±

0.07 0.12 0.26 0.57 0.08 0.02 0.79

− − 7.43 3.39 3.37 >10 5.00 3.85 1.91

± 1.15 ± 0.47 ± 0.42 ± 1.62 ± 0.66 ± 0.54

Results are expressed as IC50 values in μM. bCompounds 1−8, 10−12, 14, 18, 19, 23, 24, 26, and 28 were inactive for all cell lines used (IC50 > 10 μM). Compounds 15 and 16 were not tested for antiproliferative activities against all cell lines used. cInactive for the cell line used (IC50 > 10 μM). d Positive control. a

ECD (MeOH) nm (Δε) 252 (+2.9), 330 (−2.1); 1H (400 MHz, pyridine-d5) and 13C NMR (100 MHz, pyridine-d5) data, see Tables 2 and 6; HRESIMS m/z 565.2783 [M + Na]+ (calcd for C31H42O8Na, 565.2777). Physangulatin I (9): amorphous powder; [α]25 D +80 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (1.4), 220 (4.0) nm; IR (KBr) νmax 3395, 2921, 2850, 1740, 1712, 1647, 1467, 1382, 1228, 1134 cm−1; ECD (MeOH) nm (Δε) 247 (+5.9), 294 (−1.1); 1H (400 MHz, pyridine-d5) and 13C NMR (100 MHz, pyridine-d5) data, see Tables 3

and 6; HRESIMS m/z 581.2726 [M + Na]+ (calcd for C31H42O9Na, 581.2727). X-ray Crystal Structure Determination of Physangulatin I (9). The data were collected on an Xcalibur, Eos, Gemini diffractometer using monochromatized Cu Kα radiation. The structure was solved by direct methods using SHELXL. All H atoms were refined using the riding model. All non-H atoms were refined anisotropically. Crystallographic data have been deposited in the Cambridge Crystallographic Data Centre (CCDC number 1444698). Copies of the data can be obtained, free of charge, from the CCDC Web site (www.ccdc.cam.ac. J



Article

HRESIMS m/z 521.2146 [M + Na]+ (calcd for C28H34O8Na, 521.2151). Withaphysalin Z (16): amorphous powder; [α]25 D +7 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 220 (3.4) nm; IR (KBr) νmax 3395, 2920, 2849, 1742, 1646, 1469, 1421, 1384, 1120 cm−1; ECD (MeOH) nm (Δε) 255 (+0.9), 334 (−0.1); 1H (400 MHz, DMSO-d6) and 13C NMR (100 MHz, DMSO-d6) data, see Tables 4 and 6; HRESIMS m/z 537.2103 [M + Na]+ (calcd for C28H34O9Na, 537.2101). Antiproliferative Assays. Compounds were evaluated by the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method for antiproliferative activities against human prostate cancer cells (C4-2B and 22Rvl), human renal carcinoma cells (786-O, A-498, and ACHN), and human melanoma cells (A375-S2).33 The cells were incubated in RPMI-1640 or EMEM medium with 10% fetal bovine serum in a humidified atmosphere (5% CO2, 37 °C). Cells (1 × 104 cells/well) were added into the 96-well plates for 12 h before drug addition. The test compounds with various concentrations were added into the 96-well plates, then incubated for 48 h. 5-Fluorouracil was used as the positive control, and every assay was repeated three times. Cell viability was evaluated by the MTT reduction assay. NO Production Bioassay. All compounds were assayed for the inhibition of NO production according to the Griess method.5,34,35 A total of 1 × 106 cells/well of RAW 264.7 cells were added into the 96well plates and incubated at 37 °C for 24 h by the stimulation of LPS (1 μg/mL) with or without test compounds. After the addition of Griess reagent [0.1% N-(1-naphthyl)ethylenediamine (50 μL); 1% sulfanilamide in 5% H3PO4 (50 μL)], absorbance (540 nm) was recorded by using a microplate reader. The standard curve was used to calculate the NO concentrations and inhibitory rates.

Table 8. Inhibitory Effects of Compounds 1−28 on NO Production Induced by LPS in Macrophages compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a

IC50 (mean ± SD, μM) 36.47 38.23 11.15 11.59 11.36 18.74 >100 44.56 3.51 6.36 3.53 >100 17.62 71.69 25.91

± ± ± ± ± ±

2.50 2.41 0.92 0.88 0.87 1.33

± ± ± ±

3.02 0.17 0.39 0.25

± 1.12 ± 4.27 ± 1.53

compound 16 17 18 19 20 21 22 23 24 25 26 27 28 hydrocortisonea

IC50 (mean ± SD, μM) 17.53 1.36 43.75 64.06 3.02 3.38 5.56 55.92 2.54 1.42 59.51 3.42 >100 58.79

± ± ± ± ± ± ± ± ± ± ± ±

1.05 0.05 2.89 4.01 1.32 0.13 2.17 3.26 1.12 0.06 3.92 1.54

± 3.32

Positive control.

uk). Crystal data: C31H42O9.09464, M = 560.16, orthorhombic (CHCl3− MeOH), size 0.14 × 0.12 × 0.08 mm3, a = 7.21271(16) Å, b = 15.4107(4) Å, c = 25.7325(7) Å, α = 90°, β = 90°, γ = 90°, V = 2860.24(12) Å3, T = 104.0 K, space group P212121 (no. 19), Z = 4, μ(Cu Kα) = 0.780, completeness θmax = 99.9%, F(000) = 1203, 2θ range for data collection from 6.686° to 142.174°, 9629 reflections measured, 5413 unique (Rint = 0.0259), which were used in all calculations. The final wR(F2) was 0.0961 (all data). The Flack and Hooft parameters were 0.06(10) and −0.02(9), respectively. The largest difference peak and hole were 0.231 and −0.166 e Å−3. Physangulatin J (10): amorphous powder; [α]25 D +55 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 218 (4.0) nm; IR (KBr) νmax 3396, 2921, 2851, 1740, 1711, 1683, 1647, 1467, 1384, 1229 cm−1; ECD (MeOH) nm (Δε) 254 (+1.7), 329 (−0.9); 1H (400 MHz, pyridined5) and 13C NMR (100 MHz, pyridine-d5) data, see Tables 3 and 6; HRESIMS m/z 581.2727 [M + Na]+ (calcd for C31H42O9Na, 581.2727). Physangulatin K (11): amorphous powder; [α]25 D +40 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 223 (3.7) nm; IR (KBr) νmax 3395, 2920, 2849, 1646, 1469, 1384, 1121 cm−1; ECD (MeOH) nm (Δε) 255 (+1.5), 331 (−0.1); 1H (400 MHz, pyridine-d5) and 13C NMR (100 MHz, pyridine-d5) data, see Tables 3 and 6; HRESIMS m/z 583.2885 [M + Na]+ (calcd for C31H44O9Na, 583.2883). Physangulatin L (12): amorphous powder; [α]25 D +44 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 218 (3.9) nm; IR (KBr) νmax 3398, 2920, 2850, 1760, 1655, 1467, 1385, 1133 cm−1; ECD (MeOH) nm (Δε) 252 (+1.7), 333 (−1.0); 1H (400 MHz, pyridine-d5) and 13C NMR (100 MHz, pyridine-d5) data, see Tables 3 and 6; HRESIMS m/ z 525.2461 [M + Na]+ (calcd for C28H38O8Na, 525.2464). Physangulatin M (13): amorphous powder; [α]25 D +104 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 216 (4.2) nm; IR (KBr) νmax 3398, 2921, 2850, 1681, 1647, 1467, 1384, 1129 cm−1; ECD (MeOH) nm (Δε) 252 (+4.0), 332 (−1.9); 1H (400 MHz, pyridine-d5) and 13C NMR (100 MHz, pyridine-d5) data, see Tables 4 and 6; HRESIMS m/ z 537.2830 [M + Na]+ (calcd for C30H42O7Na, 537.2828). Physangulatin N (14): amorphous powder; [α]25 D +40 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 224 (4.0) nm; IR (KBr) νmax 3395, 2921, 2850, 1760, 1647, 1467, 1384, 1133 cm−1; ECD (MeOH) nm (Δε) 256 (+1.4), 333 (−0.4); 1H (400 MHz, pyridine-d5) and 13C NMR (100 MHz, pyridine-d5) data, see Tables 4 and 6; HRESIMS m/ z 511.2671 [M + Na]+ (calcd for C28H40O7Na, 511.2672). Withaphysalin Y (15): amorphous powder; [α]25 D +12 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 224 (4.0) nm; IR (KBr) νmax 3395, 2921, 2850, 1769, 1714, 1646, 1469, 1384, 1247, 1139 cm−1; ECD (MeOH) nm (Δε) 250 (+3.0), 335 (−1.4); 1H (400 MHz, pyridined5) and 13C NMR (100 MHz, pyridine-d5) data, see Tables 4 and 6;

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00094. 1D and 2D NMR, HRESIMS, UV, and ECD (except for 4) spectra for new compounds 1−16 (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-24-23986515. E-mail: [email protected] (L.-X. Chen). *Tel: +86-22-59596223. E-mail: [email protected] (F. Qiu). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was financially supported by grants from the National Natural Science Foundation of China (NSFC) (Grant Nos. 21472138 and 31270399), Key Projects of the National Science and Technology Pillar Program (Grant No. 2012BAI30B02), Fund of the Educational Department of Liaoning Province (Grant No. L2011177), Liaoning Baiqianwan Talents Program (Grant No. 2013921043), the Project of Innovation Team (LT2015027) of Liaoning and Scientific Research Foundation for the Returned Overseas Chinese Scholars of Shenyang Pharmaceutical University (Grant No. GGJJ2015103), and 2015 Career Development Program for Young and Middle-aged Teachers of Shenyang Pharmaceutical University (ZQN2015015).

■

REFERENCES

(1) Lavie, D.; Glotter, E.; Shvo, Y. J. Org. Chem. 1965, 30, 1774− 1778. K



Article

(2) Zhang, H. P.; Samadi, A. K.; Cohen, M. S.; Timmermann, B. N. Pure Appl. Chem. 2012, 84, 1353−1367. (3) Misico, R. I.; Nicotra, V. E.; Oberti, J. C.; Barboza, G.; Gil, R. R.; Burton, G. Prog. Chem. Org. Nat. Prod. 2011, 94, 127−229. (4) Chen, L. X.; He, H.; Qiu, F. Nat. Prod. Rep. 2011, 28, 705−740. (5) 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. (6) He, H.; Feng, Y. S.; Zang, L. H.; Liu, W. W.; Ding, L. Q.; Chen, L. X.; Kang, N.; Hayashi, T.; Tashiro, S.; Onodera, S.; Qiu, F.; Ikejima, T. Food Chem. Toxicol. 2014, 71, 128−135. (7) Choi, E. M.; Hwang, J. K. J. Ethnopharmacol. 2003, 89, 171−175. (8) Bastos, G. N. T.; Silveira, A. J. A.; Salgado, C. G.; Picanço-Diniz, D. L. W.; Do Nascimento, J. L. M. J. Ethnopharmacol. 2008, 118, 246− 251. (9) He, H.; Zang, L. H.; Feng, Y. S.; Wang, J.; Liu, W. W.; Chen, L. X.; Kang, N.; Tashiro, S.; Onodera, S.; Qiu, F.; Ikejima, T. J. Nat. Prod. 2013, 76, 880−888. (10) He, H.; Zang, L. H.; Feng, Y. S.; Chen, L. X.; Kang, N.; Tashiro, S.; Onodera, S.; Qiu, F.; Ikejima, T. J. Ethnopharmacol. 2013, 148, 544−555. (11) Hsieh, W. T.; Huang, K. Y.; Lin, H. Y.; Chung, J. G. Food Chem. Toxicol. 2006, 44, 974−983. (12) Hseu, Y. C.; Wu, C. R.; Chang, H. W.; Kumar, K. J. S.; Lin, M. K.; Chen, C. S.; Cho, H. J.; Huang, C. Y.; Lee, H. Z.; Hsieh, W. T.; Chung, J. G.; Wang, H. M.; Yang, H. L. J. Ethnopharmacol. 2011, 135, 762−771. (13) Kusumaningtyas, R. W.; Laily, N.; Limandha, P. Procedia Chem. 2015, 14, 367−372. (14) Bastos, G. N. T.; Santos, A. R. S.; Ferreira, V. M. M.; Costa, A. M. R.; Bispo, C. I.; Silveira, A. J. A.; Do Nascimento, J. L. M. J. Ethnopharmacol. 2006, 103, 241−245. (15) Institute of Botany of the Chinese Academy of Sciences, and Kunming Institute of Botany of the Chinese Academy of Sciences. Chinese Flora (Zhongguo Zhiwu Zhi); Science Press: Beijing, 1978; Vol. 67, pp 50−59. (16) Di Stasi, L. C.; Santos, E. M. G.; Moreira dos Santos, C.; Hiruma, C. A. In Plantas Medicinais da Amazonia; Editora Unesp: Sao Paulo, 1989; pp 45−46. (17) Branch, L. C.; Silva, M. F. D. Acta Amazonica 1983, 13, 737− 797. (18) Jin, Z.; Mashuta, M. S.; Stolowich, N. J.; Vaisberg, A. J.; Stivers, N. S.; Bates, P. J.; Lewis, W. H.; Hammond, G. B. Org. Lett. 2012, 14, 1230−1233. (19) Januario, A. H.; Rodrigues Filho, E.; Pietro, R. C. L. R.; Kashima, S.; Sato, D. N.; Franca, S. C. Phytother. Res. 2002, 16, 445− 448. (20) Shingu, K.; Yahara, S.; Nohara, T.; Okabe, H. Chem. Pharm. Bull. 1992, 40, 2088−2091. (21) Shingu, K.; Yahara, S.; Okabe, H.; Nohara, T. Chem. Pharm. Bull. 1992, 40, 2448−2451. (22) Nagafuji, S.; Okabe, H.; Akahane, H.; Abe, F. Biol. Pharm. Bull. 2004, 27, 193−197. (23) Abe, F.; Nagafuji, S.; Okawa, M.; Kinjo, J. Chem. Pharm. Bull. 2006, 54, 1226−1228. (24) Kong, L. Y.; Luo, J. G.; Zhang, W. N. CN Patent, CN103,396,470, 2013. (25) Gottlieb, H. E.; Cojocaru, M.; Sinha, S. C.; Saha, M.; Bagchi, A.; Ali, A.; Ray, A. B. Phytochemistry 1987, 26, 1801−1804. (26) Hsieh, P. W.; Huang, Z. Y.; Chen, J. H.; Chang, F. R.; Wu, C. C.; Yang, Y. L.; Chiang, M. Y.; Yen, M. H.; Chen, S. L.; Yen, H. F.; Lübken, T.; Hung, W. C.; Wu, Y. C. J. Nat. Prod. 2007, 70, 747−753. (27) Kuroyanagi, M.; Shibata, K.; Umehara, K. Chem. Pharm. Bull. 1999, 47, 1646−1649. (28) Ding, H.; Hua, Z. J.; Yu, L. Y.; Ma, Z. J.; Ma, X. Q.; Chen, Z.; Wang, D.; Zhao, X. F. Steroids 2014, 86, 32−38. (29) Vasina, O. E.; Abdullaev, N. D.; Abubakirov, N. K. Khim. Prir. Soedin. 1990, 3, 366−371. (30) Ma, L.; Gan, X. W.; He, Q. P.; Bai, H. Y.; Arfan, M.; Lou, F. C.; Hu, L. H. Helv. Chim. Acta 2007, 90, 1406−1419.

(31) Glotter, E.; Kirson, I.; Abraham, A.; Sethi, P. D.; Subramanian, S. S. J. Chem. Soc., Perkin Trans. 1 1975, 14, 1370−1374. (32) Mulligan, M. S.; Hevel, J. M.; Marletta, M. A.; Ward, P. A. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 6338−6342. (33) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. R. Cancer Res. 1988, 48, 589−601. (34) Li, J.; Zhao, F.; Li, M. Z.; Chen, L. X.; Qiu, F. J. Nat. Prod. 2010, 73, 1667−1671. (35) Dirsch, V. M.; Stuppner, H.; Vollmar, A. M. Planta Med. 1998, 64, 423−426.

L


Antiproliferative and Anti-inflammatory Withanolides from Physalis

Recommend Documents