Discovery of Polyoxypregnane Derivatives From Aspidopterys obcordata With Their Potential Antitumor Activity.

The bioassay-guided phytochemical study of an ethnic medicinal plant Aspidopterys obcorda ta Hemsl. var. obcordata results in the isolation of eight new polyoxypregnane derivatives, named aspidatasides A-H (1-8), along with ten known analogs (9-18). The series polyoxypregnane derivatives were screened for their cytoxic activity against HL-60 cells, and compound 2 showed the highest potency with an IC50 8.03 μM. Preliminary structure-activity relationship studies displayed that the sugar chain and double bond could notably impact their biological activity.

1 Introduction

Natural products, as major chemical resources, make up significant agents in modern drug discovery, and play an important role in treating and preventing diseases with novel molecular mechanisms over the last 200 years (Hanson, 2017). The latest report shows that half of the new drugs in the market come from natural products or structural modifications based on a natural chemical framework (Newman and Cragg, 2020).

The polyoxypregnanes and their derivatives as valuable therapeutic agents are naturally occurring C21 steroidal skeleton or substituted by oligosaccharide chain, benzoyl, acylated tigloyl ester groups, as well as an extra epoxide ring (Bai et al., 2007; Zheng et al., 2014), which exhibited diverse biological and medicinal activities, such as anti-inflammatory effect, immune-suppressive effect; chondro-protective effect; and antifungal, antioxidant, antifertility, anti-AchE, and anti-HIV activities (Abe et al., 2000; Niranjan et al., 2002; Plaza et al., 2004; Deng et al., 2005; Li et al., 2007; Sanyacharernkul et al., 2009; Ni and Ye, 2010; Wang et al., 2010; Itthiarbha et al., 2012; Pang et al., 2015; Zhang et al., 2015; Gu and Hao, 2016; Wang et al., 2016; Pang et al., 2017; Zhan et al., 2019). In particular, many of them have shown prominent anticarcinogenic or cancer inhibitory activities with great research and development potential, and received widespread attention by pharmacologists (Plaza et al., 2005; Panda et al., 2006; Bai et al., 2009; Shen and Zhang, 2010; Zhang et al., 2010; Xue et al., 2012; Sun et al., 2014; Yao et al., 2014; Zhang et al., 2015; Liu et al., 2018; Song et al., 2018). Natural monomeric polyoxypregnanes have been recognized as characteristic constituents in Asclepiadaceae plants, and there have been effective reports on the use of Marsdenia tenacissima (Asclepiadaceae) as a raw material in the treatment of leukemia, gastric carcinoma, liver cancer, prostate cancer, and lung cancer in China, notably (Zhao et al., 2007; Tong et al., 2015; Ge et al., 2019; Wang et al., 2019; Yu, 2019; Pu et al., 2020; Zhu et al., 2020). However, polyoxypregnanes are rarely isolated from the other families. Meanwhile, their antitumor mechanism and the structure–activity relationship of polyoxypregnane derivatives from the plant are not clearly demonstrated.

A. obcordata is a woody liana herb, mainly distributed in sunny and rainfall areas in Xishuangbanna, Yunnan Province. As a “Dai Medicine,” A. obcordata has been used frequently and commonly in the Dai Minority Hospital of Xishuangbanna, Southwest China, for the treatment of various diseases, such as cystitis, chronic nephritis, urinary tract infections, rheumatic bone pain, and postpartum body deficiency (Chen and Funston, 1997; Li et al., 2011). Although the effectiveness of the medicinal plant has been verified in clinic, there are few related studies about its material basis (Li et al., 2016; Li et al., 2019). In a continuous attempt to search for structurally diverse cytotoxicity, interesting C21 steroids from A. obcordata, eight new polyoxypregnane derivatives, named aspidatasides A–H (1–8), together with ten known analogs (9–18) (Figure 1) were obtained from the 95% EtOH extract of plant’s vines, which exhibited moderate antitumor effects, especially its antihuman myeloid leukemia (HL-60) activity. Herein, the isolated procedures, structural elucidation of the new compounds, as well as their inhibitory activity evaluation against HL-60 were reported in order to understand the structure–activity relationship (SAR).

FIGURE 1

Chemical structures of compounds 1–18.

2 Results and Discussion

2.1 Isolation and Structure Elucidation

Compound 1 was isolated as a white amorphous powder. Its IR spectrum exhibited absorptions for the groups of hydroxyl (3,382 cm−1) and carbonyl (1,717 cm−1). The molecular formula was established as C42H64O15 based on the molecular ion peak at m/z [M + Na]+ 831.4139 in the HR–ESI–MS (calculated for 831.4143 C42H64O15Na). In the 1H NMR spectrum (Table 1), signals for three angular methyl protons at δH 1.29 (3H, s, CH3-18), 1.32 (3H, s, CH3-19), and 2.16 (3H, s, CH3-21), and one olefinic proton signal at δH 5.36 (H-6) in conjunction with three carbons at δC 216.5, 140.6, and 119.5 (Table 2) suggested the basic skeleton of pregen-5-en-20-one (Wang et al., 2010; Zhang et al., 2015; Wang et al., 2019). The proton signals at δH 1.81 (3H, s, 11-OAc) and 6.73 (1H, q, J = 7.2 Hz, Tig-H-3), 1.57 (1H, d, J = 7.2 Hz, Tig-H-4), and 1.43 (3H, s, Tig-H-5), together with carbon resonances at δC 21.0, 172.9 (11-OAc), and δC 168.4, 129.9, 140.7, 14.8, and 12.3 (Tig-C), suggested the presence of acetoxyl function and tigloyl unit in the molecular structure. Two anomeric signals at δH 4.65 (1H, d, J = 9.6 Hz) and 4.72 (1H, d, J = 7.8 Hz) indicated the existence of two sugar moieties in compound 1. Two methoxyls at δH 3.39 (s) and 3.60 (s) were attributed to the sugar moiety. The IR and 1H and 13C-NMR spectra data identified that compound 1 is a polyoxypregnane glycoside derivative.

TABLE 1

1H NMR spectroscopic data (600 MHz, 1 in MeOD and 2–8 in DMSO) for compounds 1–8 (δH in ppm, J in Hz).

Position	1	2	3	4	5	6	7	8
1	1.19, m	1.22, m	1.18, m	1.26, m	1.20, m	1.21, m	1.20, m	1.22, m
	1.83, m	1.81, m	1.85, m	1.82, m	1.83, m	1.83, m	1.88, m	1.90, m
2	1.95, m	1.72, m	1.69, m	1.72, m	1.74, m	1.73, m	1.71, m	1.67, m
	1.72, m	1.91, m	1.97, m	1.97, m	1.99, m	1.98, m	1.91, m	1.98, m
3	3.69, m	3.61, m	3.47, m	3.74, m	3.60, m	3.71, m	3.47, m	3.59, m
4	2.27, m	2.21, m	2.27, m	2.27, m	2.21, m	2.27, m	2.12, m	2.17, m
	2.41, m	2.40, m	2.38, m	2.38, m	2.49, m	2.41, m	1.44, m	1.44, m
5	—	—	—	3.40, m	3.40, m	3.40, m	1.17, m	1.18, m
6	5.36, d (5.4)	5.27, d (4.2)	5.46, d (2.4)	—	6.20, dd (16.2)	5.46, dd (16.2)	1.04, m	1.04, m
	—	—	—	6.50, dd (10.2)	—	—	1.57, m	1.57, m
7	1.89, m	1.86, m	1.87, m	6.6, dd (10.2)	6.28, dd (16.2)	6.52, dd (16.2)	1.37, m	1.37, m
	2.27, m	2.25, m	2.29, m	—	—	—	1.66, m	1.67, m
8	—	—	—	—	—	1.92, m	—	—
9	1.95, d (10.8)	1.96, d (10.2)	2.29, d (10.2)	1.66, d (10.8)	1.83, d (10.8)	1.76, m	2.00, d (10.2)	2.17, d (10.2)
10	—	—	—	—	—	—	—	—
11	5.81, t (10.8)	5.70, t (10.8)	5.53, t (10.2)	5.80, t (10.8)	5.98, t (10.8)	5.28, t (10.2)	5.42, t (10.2)	5.61, t (10.2)
12	5.15, d (10.8)	4.89, d (10.8)	5.23, d (10.2)	4.92, d (10.8)	5.04, d (10.8)	5.44, d (10.2)	5.18, d (10.2)	5.17, d (10.2)
13	—	—	—	—	—	—	—	—
14	—	—	1.64, m	—	—	—	1.64, m	1.64, m
15	1.51, m	1.52, m	1.48, m	1.60, m	1.52, m	1.54, m	1.47, m	1.47, m
	1.53, m	1.58, m	1.37, m	1.54, m	1.61, m	1.56, m	1.53, m	1.53, m
16	2.20, m	1.77, m	1.81, m	1.78, m	1.75, m	1.77, m	1.71, m	1.71, m
	1.78, m	2.12, m	2.38, m	2.38, m	2.12, m	2.14, m	2.11, m	2.17, m
17	3.17, m	3.17, m	3.16, m	3.16, m	3.15, m	3.15, m	3.16, m	3.14, m
18	1.29, s	1.27, s	1.18, s	1.26, s	1.23, s	1.25, s	1.17, s	1.12, s
19	1.32, s	1.31, s	1.25, s	1.29, s	1.31, s	1.27, s	1.39, s	1.37, s
20	—	—	—	—	—	—	—	—
21	2.16, s	2.11, s	1.91, s	2.06, s	2.12, s	2.06, s	2.07, s	2.06, s
11-O	Ac	Tig	Tig	Tig	Bz	Tig	Tig	Bz
2	1.81, s	—	—	—	—	—	—	—
3	—	6.70, q (7.2)	6.52, q (7.2)	6.60, q (5.4)	7.81, dd (7.2.1.2)	6.59, q (5.4)	6.50, q (7.2)	7.70, qq (7.2.1.2)
4	—	1.70, d (7.2)	1.46, d (7.2)	1.55, d (5.4)	7.78, t (7.2)	1.57, d (5.4)	1.45, d (7.2)	7.34, t (7.2)
5	—	1.48, s	1.48, s	1.60, s	7.50, t (7.2)	1.45, s	1.37, s	7.50, t (7.2)
6	—	—	—	—	7.78, t (7.2)	—	—	7.34, t (7.2)
7	—	—	—	—	7.81, dd (7.2.1.2)	—	—	7.70, qq (7.2.1.2)
12-O	Tig	Bz	Bz	Bz	Bz	Bz	Bz	Bz
3	6.73, q (7.2)	7.80, dd (7.2.1.2)	7.81, dd (7.2.1.2)	7.75, dd (7.8.2.4)	7.49, dd (7.2.1.2)	7.43, dd (7.8.2.4)	7.74, dd (7.2.1.2)	7.65, dd (7.2.1.2)
4	1.57, d (7.2)	7.43, t (7.2)	7.49, t (7.2)	7.43, t (7.8)	7.47, t (7.2)	7.36, t (7.8)	7.48, t (7.2)	7.31, t (7.2)
5	1.43, s	7.58, t (7.2)	7.63, t (7.2)	7.43, t (7.8)	7.35, t (7.2)	7.57, t (7.8)	7.63, t (7.2)	7.63, t (7.2)
6	—	7.43, t (7.2)	7.49, t (7.2)	7.43, t (7.8)	7.47, t (7.2)	7.36, t (7.8)	7.48, t (7.2)	7.31, t (7.2)
7	—	7.80, dd (7.2.1.2)	7.81, dd (7.2.1.2)	7.75, dd (7.8.2.4)	7.49, dd (7.2.1.2)	7.43, dd (7.8.2.4)	7.74, dd (7.2.1.2)	7.65 dd (7.2.1.2)
Ole-1	4.65, d (9.6)	4.59, d (9.0)	4.62, d (9.6)	4.92, d (10.2)	4.55, d (8.4)	4.92, d (10.2)	4.60, d (9.6)	4.57, d (9.6)
2	1.93, m	1.91, m	1.91, m	1.15, m	1.99, m	1.98, m	1.91, m	1.93, m
	2.24, m	2.21, m	2.23, m	2.06, m	2.21, m	2.21, m	2.25, m	2.21, m
3	3.51, m	3.52, m	3.51, m	3.26, m	3.46, m	3.46, m	3.34, m	3.47, m
4	3.19, m	3.16, m	3.16, m	3.02, m	3.18, m	3.15, m	3.27, m	3.21, m
5	3.36, m	3.35, m	3.35, m	3.14, m	3.35, m	3.34, m	3.35, m	3.34, m
6	1.23, d (6.6)	1.09, d (6.0)	1.24, d (6.0)	1.24, d (6.0)	1.14, d (6.6)	1.23, d (6.0)	1.09, d (6.6)	1.08, d (6.0)
3-OCH3	3.39, m	3.39, s	3.45, s	3.26, m	3.35, s	3.37, s	3.45, s	3.44, s
Allo-1	4.72, d (7.8)	4.56, d (8.4)	4.62, d (8.4)	4.75, d (7.2)	4.74, d (10.8)	4.55, d (7.2)	4.62, d (8.4)	4.74, d (7.2)
2	3.48, m	3.47, m	3.47, m	3.47, m	3.44, m	3.47, m	3.47, m	3.47, m
3	3.66, m	3.62, m	3.80, m	3.45, m	3.61, m	3.61, m	3.61, m	3.61, m
4	3.39, m	3.37, m	3.35, m	3.04, m	3.34, m	3.27, m	3.34, m	3.34, m
5	3.68, m	3.62, m	3.53, m	3.19, m	3.66, m	3.71, m	3.66, m	3.60, m
6	1.32, d (6.6)	1.24, d (6.0)	1.46, d (6.0)	1.09, d (6.0)	1.20, d (6.6)	1.20, d (6.0)	1.24, d (6.6)	1.20, d (6.0)
3-OCH3	3.60, s	3.50, s	3.80, s	3.45, m	3.35, s	3.90, s	3.51, s	3.51, s

TABLE 2

13C NMR spectroscopic data (150 MHz, 1 in MeOD, and 2–8 in DMSO) for compounds 1–8.

Position	1	2	3	4	5	6	7	8
1	40.3	40.5	40.6	40.3	39.9	40.5	40.5	40.5
2	30.5	29.0	31.5	29.3	28.9	29.3	31.2	28.8
3	79.3	76.7	74.1	79.0	73.0	73.0	74.4	74.4
4	41.3	41.2	41.2	41.3	41.0	41.3	36.6	36.5
5	140.6	138.3	140.1	55.2	54.4	52.2	56.2	58.7
6	119.5	118.3	119.3	131.1	133.2	145.1	26.3	26.3
7	37.6	38.0	38.6	129.4	130.5	117.3	36.6	34.3
8	76.8	75.0	70.1	77.8	77.4	30.8	65.9	71.2
9	50.0	47.6	56.3	50.2	49.8	48.9	50.5	50.5
10	39.9	40.0	40.1	40.0	39.2	40.0	38.4	38.5
11	72.7	70.6	70.5	73.5	73.0	70.8	68.1	70.5
12	79.4	77.6	78.5	79.0	75.1	75.1	74.9	78.5
13	56.2	54.5	45.4	55.1	54.8	53.9	45.3	45.4
14	86.5	84.5	48.0	84.9	84.4	82.8	42.7	42.7
15	36.2	36.5	36.5	35.2	36.5	36.6	34.3	36.8
16	25.1	29.0	26.4	24.1	24.3	23.0	28.8	26.4
17	60.1	54.5	56.3	59.4	61.3	61.3	58.6	56.2
18	13.5	11.6	11.4	13.0	12.6	11.5	11.4	12.5
19	18.4	18.4	18.5	18.4	13.4	18.4	18.3	18.3
20	216.5	211.6	210.1	212.0	211.5	211.1	210.1	210.1
21	31.0	30.6	30.2	31.2	30.8	30.8	30.1	31.2
11-O	Ac	Tig	Tig	Tig	Bz	Tig	Tig	Bz
1	172.9	166.9	166.2	166.7	166.1	166.1	166.4	165.4
2	21.0	127.5	127.6	128.4	133.6	127.9	127.7	129.3
3	—	138.0	138.3	138.5	128.8	138.1	137.9	128.9
4	—	14.2	16.1	14.6	128.5	11.8	13.9	128.5
5	—	12.8	14.0	12.1	130.5	11.7	12.4	133.3
6	—	—	—	—	128.5	—	—	128.5
7	—	—	—	—	128.8	—	—	128.9
12-O	Tig	Bz	Bz	Bz	Bz	Bz	Bz	Bz
1	168.4	165.9	165.1	166.5	164.9	166.4	165.1	165.1
2	129.9	128.0	128.9	134.3	129.6	133.8	128.9	128.5
3	140.7	129.2	129.1	129.4	128.2	128.5	129.0	128.8
4	14.8	129.0	128.5	128.8	129.1	128.4	128.5	128.4
5	12.3	129.3	133.5	131.1	133.2	130.7	133.4	133.2
6	—	129.0	128.5	128.8	129.1	128.4	128.5	128.4
7	—	129.2	129.1	129.4	128.2	128.5	129.0	128.8
Ole-1	98.9	96.7	96.6	96.7	96.2	96.3	96.3	96.3
2	38.0	38.7	37.9	37.7	37.2	37.0	34.3	38.2
3	80.7	78.6	75.9	79.1	78.5	78.6	78.5	74.9
4	84.2	82.8	82.8	83.2	82.8	82.3	82.7	82.8
5	72.4	70.5	69.3	72.0	70.5	70.5	70.5	69.3
6	19.0	18.0	18.3	18.8	18.3	17.9	17.9	17.9
3-OCH3	57.6	56.3	58.8	56.7	56.2	56.3	56.2	56.2
Allo-1	102.4	100.8	100.8	101.2	100.7	100.8	100.7	100.7
2	73.8	71.6	71.6	73.5	71.1	70.8	71.1	71.5
3	84.1	82.3	82.2	82.7	82.2	82.2	82.2	82.2
4	75.2	73.0	73.0	75.6	71.5	71.6	73.0	73.0
5	71.4	69.4	68.3	71.0	69.3	69.3	69.3	68.8
6	18.4	17.2	17.9	18.4	17.9	14.1	16.3	16.4
3-OCH3	62.7	61.4	61.3	61.8	61.3	61.3	61.3	61.3

All assignments of proton signals were assigned by its HSQC spectrum (Supporting Information, Supplementary Figure S3). The connectivities of compound 1 were deduced mainly by 1H-1H COSY and HMBC spectra (Figure 2). The correlations among H-1/H-2/H-3/H-4, H-6/H-7, H-9/H-11/H-12, and H-15/H-16/H-17 were found in the 1H-1H COSY spectrum. The position of acetoxyl and tigloyl groups was determined at C-11 and C-12, respectively, as the HMBC correlations between δH5.81 (1H, t, J = 10.8 Hz, H-11) and δC 172.9 (11-OAc), δH5.15 (1H, d, J = 10.8 Hz, H-12), and δC 168.4 (Tig-C-1). Two hydroxyl substituents were located at C-8 and C-14, respectively, based on their chemical shifts (δC 76.8 and 86.5) together with the molecular formula above. The existence of D-oleandrose (Ole) and 6-deoxy-3-O-methyl-D-allopyranose (Allo) units was confirmed by co-TLC and further followed by gas chromatography in comparison with standard monosaccharides. The sugar sequence and linkage position were established by the HMBC correlations of δH 4.72 (H-1 of Allo) to δC 84.2 (C-4 of Ole) and δH 4.65 (H-1 of Ole) to δC 79.3 (C-3 of aglycone), as shown in Figure 2.

FIGURE 2

Key HMBC and 1H-1H COSY correlations of compound 1.

Meanwhile, the NOE correlations of H-3/H-1α, H3-19/H-1β, H-12/H-9, and H-12/H-17 indicated the α-orientations of H-3/12/17. The 3 J coupling constant (J = 10.8 Hz) between H-11 and H-12 also confirmed their anti-periplanar relationship. Meanwhile, the β configurations of the two sugars were established based on the large coupling constants (each 3 J 1, 2 > 7.0 Hz). Thus, the structure of 1 was established and named aspidataside A.

Compound 2 was obtained as a white amorphous powder. Its molecular formula was established as C47H66O15 according to the HR–ESI–MS spectrum at m/z [M + Na]+ 893.4305 (calculated for C47H66O15Na, 893.4299). An analysis of the 1H and 13CNMR data (Table 1; Table 2) displayed that the structure of 2 was similar to that of 1, except for the OAc group in 1 was absent in 2, extra signals of a benzoyl group at δH7.80 (2H, dd, J = 7.2, 1.2 Hz), 7.43 (2H, t, J = 7.2 Hz), 7.58 (1H, t, J = 7.2 Hz), and δC 165.9, 128.0, 129.2, 129.0, and 129.3 were found in 2. In the HMBC spectrum, the correlations from δH 5.70 (H-11) to the carbonyl signal at δC 166.9 (Tig-1), and from δH 4.89 (H-12) to δC 165.9 indicated that the acetoxyl group at C-11 in 1 was replaced by the tigloyl group in 2, while the tigloyl group at C-12 was changed to the benzoyl group. The absolute configuration of 2 was established by the analysis of the NOESY and CD spectra. Therefore, compound 2 was identified and named aspidataside B.

Compound 3 was suggested to possess the molecular formula C47H66O14, based on the m/z 877.4348 [M + Na]+ (calculated for C47H66O14Na, 877.4350), in the HR–ESI–MS, which was 16 amu less than that of 2. In the NMR spectra of 3 and 2 (Table 1; Table 2), the structure of 3 corresponded to 2, except a hydroxyl group absent at C-14. The up-field chemical shift of C-14 at δC 48.0 in 3 compared with C-14 (δC 84.5) in 2 together with the molecular weight of 3 in the HR–ESI–MS exhibited the disappearance of hydroxyl function at C-14 in 3. The difference was fully confirmed by the 2D NMR experiments. With the analysis of NOESY data (Supporting Information, Supplementary Figure S23), compound 3 was determined as shown and named aspidataside C.

Compound 4, with the molecular formula of C47H66O15 determined by the HR–ESI–MS ion at m/z [M + H]+ 871.4461 (calculated for C47H66O15Na, 871.4480), was an isomer of 2. NMR data analysis between 4 and 2 suggested that they had almost identical structures except for the location of double bond in the B-ring portion. The double bond located at C-6/C-7 was confirmed by the correlations from δH 3.40 (m, H-5) to δC 55.2 (C-5), δH 6.50 (dd, J = 10.2 Hz, H-6) to δC 131.1 (C-7), and δH 6.60 (dd, J = 10.2 Hz, H-7) to δC 129.4 (C-7) in the HSQC spectrum, and correlations from H-6 to C-5 and C-8 in the HMBC experiment. By the combined use of 1H-1H COSY and NOESY experiments (Supporting Information, Supplementary Figures S31, S32), the structure of 4 was finally elucidated and named aspidataside D.

Compound 5 with the molecular formula of C49H64O15 was determined by the HR–ESI–MS (m/z 893.4305, calcd for C49H64O15Na [M + H]+, 893.4323) and had the similar structure as 4 based on comparison of their NMR data (Table 1; Table 2). The differences displayed that the tigloyl group in 4 was replaced by a benzoyl unit in 5. In the 13C NMR data, the typical signals at δC 166.1, 133.6, 128.8, 128.5, 130.5, 164.9, 129.6, 128.2, 129.1, and 133.2, and the absence signals at δC 14.6 and 12.1 supported the differences above. The additional benzoyl function was linked to C-12, based on the correlations of the HMBC spectrum. As a result, the structure of 5 was illustrated and named aspidataside E.

Compound 6 exhibited its molecular ion peak at m/z 877.3334 [M + Na]+, corresponding to the molecular formula of C47H66O14. The 1H NMR and 13C NMR (APT) data were strongly related to those of 4 (Table 1; Table 2). The difference was that the signal at C-8 (δC 77.8) in 4 was changed to δC 30.8 in 6. Considering the loosening of 16 units in molecular weight of 6, it was deduced that the hydroxyl group at C-8 in 4 was missed in 6, which was fully supported by the 2D NMR spectra (Supporting Information, Supplementary Figures S47–S50). Taken together with the NOESY data, compound 6 was established and named aspidataside F.

Compound 7 was given a molecular formula as C47H68O14 based on the HR–ESI–MS ion peak at m/z 875.4196 (calcd for C47H68O14Na [M + Na]+, 875.4207). Its NMR data were almost identical to those of 3, except for one less degree of unsaturation, which suggested the disappearance of one double bond. In the 13CNMR data (Table 2), the carbon signals at δC 74.4 (C-3), 36.6 (C-4), 56.2 (C-5), 26.3 (C-6), 36.6(C-7), and 18.3 (C-19) indicated that there was no double bond between C-5 and C-6 in 7. In the HMBC spectrum, the correlations from δH 1.17 (1H, m, H-5) to δC 26.3 (C-6) and 36.6(C-7) confirmed the difference. As a result, compound 7 was a ductile product of compound 3. Therefore, compound 7 was finally established and named aspidataside G.

Compound 8 was determined as C49H66O14 according to the HR–ESI–MS at m/z 905.4664 (calcd for C49H66O14Na [M + Na]+, 905.4750). The 1H NMR and 13C APT spectra displayed that 8 was analogous to 7 (Table 1; Table 2), except for the tigloyl group at C-11 in 7 was substituted by a benzoyl group (δC 165.4, 129.3, 128.9, 128.5, and 133.3) in 8. The 2D NMR of HMBC experiments fully confirmed the difference (Supporting Information, Supplementary Figures S65–S68). Together with its NOESY data, the structure of compound 8 was fully identified and named aspidataside H.

So far, the skeleton connections of A, B, C, and D rings in all pregnanes were trans/trans/cis types, and the CH3-18, CH3-19, 8-H, and 14-H configurations in all the structures were β-oriented (Liu et al., 2017; Pang et al., 2017; Li et al., 2019). Considering the identical biosyntenic relationship, and the comparison of the experimental and calculated CD data (Supporting Information, Supplementary Figures S49–S56), the absolute configurations of the chiral carbons in the pregnane skeleton were determined to be 3S, 8S, 9S, 10R, 11S, 12S, 13S, 14R, and 17S.

The known polyoxypregnanes were identified as sinomarinoside E (9) (Chen et al., 1999), deacylmetaplexigein (10) (Chen et al., 1999), incisagenin (11) (Chen et al., 1999), drevogenin P (12) (Niranjan et al., 2002), obcordata G (13) (Li et al., 2019), obcordata H (14) (Li et al., 2019), obcordata I (15) (Li et al., 2019), sarcostin (16) (Ma et al., 2017), caretroside A (17) (Halim and Khalil, 1996), and 3-O-[6-deoxy-3-O-methyl-β-allopyanosyl (1→4)-β-oleandropyranosyl]-5,6-dihydro gen-11α,12β-di-O-tigloyl-17β-marsdenin (18) (Liu et al., 2017), based on the comparison of the observed spectroscopic data with those reported in the literature.

2.2 Inhibitory Effects of Compounds 1–18 on Human HL-60 Cell Line

The cytotoxic activity of the isolates (1–18) against HL-60 human promyelocytic acute leukemia cell in vitro was evaluated using the MTT method with the positive control doxorubicin IC50 3.87 μM, and the results are shown in Table 3. Compounds 2 and 9 exhibited potent activities with IC50 values of 8.03 ± 0.35 and 9.25 ± 0.45 μM, respectively. Compounds 1 and 3 displayed moderate activity with IC50 values ranging from 10.53 to 13.27 μM, and approximately two-fold more active than the compounds 7–8, 13–15, 17, and 18, and about three-fold more active comparing with the compounds 4–6, 10, and 11. Compounds 12 and 16 showed week cytotoxic activity against anti–HL-60 cell (IC50 > 50 μM).

TABLE 3

In vitro cytotoxic activity of the compounds (1–18) against HL-60 cell.

Compound	IC50 (μM)	Compound	IC50 (μM)
1	13.27±0.64 a	10	41.56±4.12
2	8.03±0.35	11	40.85±3.67
3	10.53±0.92	12	57.33±4.62
4	30.72±2.15	13	21.68±1.83
5	32.80±1.80	14	24.86±2.35
6	32.40±1.28	15	27.50±2.24
7	29.13±2.02	16	55.62±4.83
8	24.94±2.55	17	29.45±2.50
9	9.25±0.45	18	26.36±2.48
Doxorubicin b	3.87±0.28	Doxorubicin	3.87±0.28

Value presents mean ± SD of triplicate experiments.

Positive control substance.

2.3 Structure–Activity Relationships

The structure–activity relations of polyoxypregnane derivatives were described, as shown in Figure 3. It was found that all the isolates exhibited moderate inhibitory activity against HL-60 cell lines, which may be due to their unique pregen-5-en-20-one skeleton. It was reported that pregnane, as a component of steroids, plays an important role in regulating the metabolism of life. However, the presence of sugar moieties in the side chain also influenced the cytotoxic activity. Compounds 10–12, and 16 without sugar chain on the hydroxyl at C-3 displayed weak activity (39.0 < IC50 < 60.0 μM) when comparing with possessing oligosaccharides located at C-3, such as compounds 1–8, 9, 13–15, 17, and 18 with IC50 values ranging from 8.03 to 32.80 μM. These results indicated that the presence of sugar moiety at C-3 maybe improving the solubility of structures and could significantly enhance the cytotoxic activity, and played important roles in the structure.

FIGURE 3

Graphical explanation of the general SAR for cytotoxic activity of 2 and its derivatives.

Four (1–3, and 9, 8.03 < IC50 < 13.27 μM) compounds with a double bond between C-5 and C-6 showed the best potency among the 14 isolated constituents with sugar side chain. Compounds 7–8, 13–15, and 17–18 (21.68 < IC50 < 29.45 μM, vs. 1–3, and 9) characterizing without a double bond in skeleton exhibited moderate cytotoxic activity. In addition, the cytotoxic activities were obviously decreased due to a double bond at C-6/C-7, such as compounds 4–6 with the IC50 values from 30.72 to 32.80 μM comparing with 1–3 and 9. Thus, it is likely that the presence and location of double bond was important to the activity in this type of compounds. Furthermore, substituent groups, including O-Bz, O-Ac, and O-Tig, located at C-11 and C-12 were related with antitumor activity. More diverse modifications at C-11 and C-12 are warranted. These findings suggested that the sugar chain, the location of double bond, and polyester substitution in the C-11 and C-12 can substantially contribute to improve their cytotoxic activity.

In summary, the most promising compound, that is, compound 2, exerted significant cytotoxicity against the tested cancerous cell, and might be considered as a potential candidate for drug development and worthy of more detailed studies.

3 Conclusion

In conclusion, chemical investigation of the stems of A. obcordata has led to the discovery of eight new polyoxypregnanes (1–8) and ten known derivatives (9–18). In the in vitro assays, compounds 4–8 and 10–18 showed weak activity against HL-60 cells, while compounds 1–3 and 9 displayed strong inhibitory effects toward HL-60. Especially, compound 2 exhibited the best inhibition of HL-60 with an IC50 value of 8.03 μM, which was likely due to its hydrophilic sugar chains and bulky hydrophobic aliphatic chains linked with the complex conjugated system by SAR analysis. Nevertheless, the antitumor mechanism of action of compound 2 as anti–HL-60 agent is necessary for further studies.

4 Experimental

4.1 General Experimental Procedures

Optical rotations were measured with a Perkin-Elmer 341 digital polarimeter (PerkinElmer, Norwalk, CT, United States). UV and IR spectra were obtained on Shimadzu UV2550 and FTIR-8400S spectrometers (Shimadzu, Kyoto, Japan), respectively. CD spectra were obtained using a JASCO J-815 spectropolarimeter (Tokyo, Japan). NMR spectra were obtained using a Bruker AV III 600 NMR spectrometer with chemical shift values presented as δ values with TMS as the internal standard (Bruker, Billerica, German). HR–ESI–MS were performed on an LTQ-Orbitrap XL spectrometer (Thermo Fisher Scientific, Boston, MA, United States). Column chromatography (CC) was performed using silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical Plant, Qingdao, China). TLC analyses were carried out on silica gel GF254 precoated plates (Zhi Fu Huang Wu PilotPlant of Silica Gel Development, Yantai, China) with detection accomplished by spraying with 5% H2SO4 followed by heating at 100°C. Preparative HPLC was performed on a Lumtech K-1001 analytic LC equipped with two K-501 pumps, a K-2600 UV detector, and a YMC Pack C18 semi-preparative column (250 mm × 10 mm, i. d., 5 μM, YMC Co. Ltd., Japan), and eluted with CH3OH–H2O at a flow rate of 2 ml/min. All solvents used were of analytical grade (Beijing Chemical Works).

4.2 Plant Material

The vines of A. obcordata were collected in September 2016 from Jinghong, Yunnan Province, People’s Republic of China and identified by Prof. Dai-Gui Zhang, at the School of Biological Resources and Environmental Science, Jishou University, where a voucher specimen (JS161248) was deposited.

4.3 Extraction and Isolation

The dried and powdered vines of A. obcordata (4.0 kg) were extracted with 95% EtOH (20 L) three times (each time for 1 h). The solvents were filtrated and evaporated under reduced pressure to give the total extract (249.0 g), and dissolved successively with water (1.5 L), in the order, petroleum ether (MSO), dichloromethane (DCM), ethyl acetate (EtOAc), and n-butanol (NBA) to re-extract the water solution of the crude extract, and then have yielded different fractions. EtOAc fraction was selected for further separation due to its better inhibitory effect against HL-60 cells.

The EtOAc fraction (112.0 g) was subjected to CC over silica gel (100–200 mesh, 10 × 150 cm), eluting with a gradient of CH2Cl2–MeOH (from 1:0 to 0:1), and obtained twelve fractions, A–L. Fraction A (7.8 g) was subjected to chromatography using ODS MPLC elution with MeOH–H2O (30:70, 70:30, 90:10, and 100:0; v/v), to yield four fractions (Fr. A1–4), and Fr. A2 (0.5280 g) was separated through semi-preparative HPLC using a mobile phase of MeOH–H2O (70:30, v/v) to afford 12 (14.6 mg, R t 23.5 min) and 16 (13.8 mg, R t 28.8 min). Compounds 10 (17.2 mg, R t 20.4) and 11 (13.2 mg, R t 25.6) were isolated from Fr. A3 (0.2468 g) purified with MeOH–H2O (75:25, v/v). Fraction D (10.4 g) was subjected over ODS column chromatography elution with MeOH–H2O in a gradient manner (30:70, 50:50, 70:30, 80:20, and 100:0; v/v) to give five sub-fractions (Fr. D1–D5). Fr. D2 (0.348 g) was purified by HPLC with an isocratic of 55% MeOH–H2O on a YMC C18 column to get compounds 1 (8.5 mg, R t 13.1 min), 2 (9.8 mg, R t 16.3 min), 6 (10.4 mg, R t 21.6 min), and 9 (7.5 mg, R t 28.4 min). Compounds 3 (8.6 mg, R t 18.2 min), 4 (7.5 mg, R t 22.4 min), and 5 (12.2 mg, R t 27.8 min) were yielded from Fr. D3 (0.41 g) separated by semi-preparative HPLC with MeOH–H2O (50:50, v/v). Similarly, Fr. E (5.8 g) was isolated through ODS MPLC elution with MeOH–H2O (30:70, 50:50, 70:30, 90:10; 100:0, v/v) to give Fr. E1–5, and Fr. E2 (0.55 g) was prepared by semi-preparative HPLC elution with MeOH–H2O (55:45, v/v), to obtain compounds 7 (14.5 mg, R t 18.2 min), 8 (10.6 mg, R t 22.3 min), 13 (8.2 mg, R t 28.5 min), and 18 (14.8 mg, R t 31.5 min). Compounds 14 (15.4 mg, R t 17.5 min), 15 (7.8 mg, R t 21.5 min), and 17 (13.5 mg, R t 27.8 min) were yielded from Fr. E3 (0.3550 g) separated by semi-preparative HPLC with MeOH–H2O (50:50, v/v).

4.4 New Compounds

4.4.1 Aspidataside A

C42H64O15, white amorphous powder; [α]20 D+35o (c 0.15, MeOH); UV λ max (MeOH) nm (log ε): 225 and 272; IR (KBr) νmax cm−1: 3,423, 1,660; HR–ESI–MS m/z 831.4139 (calculated for 831.4143 C42H64NaO15). 1H-NMR (MeOD, 600 MHz) and 13C-NMR (MeOD, 150 MHz) (see Table 1; Table 2).

4.4.2 Aspidataside B

C47H66O15, white amorphous powder; [α]20 D+42.5o (c 0.12, MeOH); UV λ max (MeOH) nm (log ε): 272; IR (KBr) νmax cm−1: 3,350, 1,665, 1,605, 1,525; HR–ESI–MS m/z [M + Na]+ 893.4305 (calculated for C47H66NaO15, 893.4299). 1H-NMR (MeOD, 600 MHz) and 13C-NMR (DMSO, 150 MHz) (see Table 1; Table 2).

4.4.3 Aspidataside C

C47H66O14, white amorphous powder; [α]20 D+40.2o (c 0.15, MeOH); UV λ max (MeOH) nm (log ε): 275; IR (KBr) νmax cm−1: 3,318, 1,680, 1,600, 1,524; HR–ESI–MS m/z 877.4348 [M + Na]+ (calculated for C47H66NaO14, 877.4350). 1H-NMR (MeOD, 600 MHz) and 13C-NMR (DMSO, 150 MHz) (see Table 1 and Table 2).

4.4.4 Aspidataside D

C47H66O15, white amorphous powder; [α]20 D+52.5o (c 0.12, MeOH); UV λ max (MeOH) nm (log ε): 274; IR (KBr) νmax cm−1: 3,320, 1,675, 1,604, 1,525; HR–ESI–MS m/z 871.4461 [M + H]+ (calculated for C47H66NaO15, 871.4480). 1H-NMR (MeOD, 600 MHz) and 13C-NMR (DMSO, 150 MHz) see Table 1 and Table 2.

4.4.5 Aspidataside E

C49H64O15, white amorphous powder; [α]20 D+44.8o (c 0.10, MeOH); UV λ max (MeOH) nm (log ε): 275; IR (KBr) νmax cm−1: 3,315, 1,678, 1,605, 1,520; HR–ESI–MS m/z 893.4305 [M + H]+ (calculated for C49H64NaO15, 893.4323). 1H-NMR (MeOD, 600 MHz) and 13C-NMR (DMSO, 150 MHz) see Table 1 and Table 2.

4.4.6 Aspidataside F

C47H66O14, white amorphous powder; [α]20 D+50.3o (c 0.15, MeOH); UV λ max (MeOH) nm (log ε): 272; IR (KBr) νmax cm−1: 3,324, 1,692, 1,600, 1,524; HR–ESI–MS m/z877.3334 [M + Na]+ (calculated for C47H66NaO14, 877.3350). 1H-NMR (MeOD, 600 MHz) and 13C-NMR (DMSO, 150 MHz) (see Table 1 and Table 2).

4.4.7 Aspidataside G

C47H68O14, white amorphous powder; [α]20 D+60.4o (c 0.15, MeOH); UV λ max (MeOH) nm (log ε): 275; IR (KBr) νmax cm−1: 3,384, 1,689, 1,610, 1,532; HR–ESI–MS m/z 875.4196 [M + Na]+ (calculated for C47H68NaO14, 875.4207). 1H-NMR (MeOD, 600 MHz) and 13C-NMR (DMSO, 150 MHz) (see Table 1 and Table 2).

4.4.8 Aspidataside H

C49H66O14, white amorphous powder; [α]20 D+45.2o (c 0.15, MeOH); UV λ max (MeOH) nm (log ε): 275; IR (KBr) νmax cm−1: 3,372, 1,674, 1,538; HR–ESI–MS m/z 905.4664 [M + Na]+ (calculated for C49H66NaO14, 905.4750). 1H-NMR (MeOD, 600 MHz) and 13C-NMR (DMSO, 150 MHz) (see Table 1 and Table 2).

Acid hydrolysis of compounds was accomplished by the procedure described previously (Wang et al., 2019).

4.6 In vitro Cytotoxicity Bioassay

Compounds 1–18 were assessed for cytotoxicity against human cancer cell line HL-60. This used the MTT method as described in the previously published literature (Halim and Khalil, 1996). Briefly, cells were grown in DMEM supplied with 10% fetal bovine serum and cultured at a density of 1.1 × 105 cells/ml per well in a 96-well plate at 37°C in a 5% CO2 incubator overnight. Each concentration was tested in triplicate. Then, the cells were incubated with 10 µL of MTT (5 mg/ml) for additional 4 h. The residual liquid was removed, and 200 µL DMSO was added. The absorbance was recorded on a microplate reader at a wavelength of 570 nm. The experiments were conducted a minimum of three times.

4.7 Statistical Analysis

Each experiment was repeated for at least three times. Results were expressed as mean ± SD. The statistical significance of differences between groups was evaluated by the unpaired Student’s t-test and indicated with (**) p < 0.01, (*) p < 0.05.

Statistical software SPSS version 15.0 was used for statistical analysis. ANOVA analysis was used with Fisher’s LSD multiple comparison test for multiple comparisons. All p-values < 0.05 were considered statistically significant.