*2.1. Structure Elucidation of Compounds*

Compound **1** showed a quasi-molecular ion peak [M + H]+ at *m*/*z* 461.3731 (calculated to be 461.3738) in the HR-ESI-MS (spectrum showed in Supplementary material), which corresponds to the molecular formula C28H48N2O3. The IR spectrum showed absorption bands at 3424 (hydroxyl group), 1662 (amide carbonyl group), and 1623 (double bond) cm–1. The 1H and 13C NMR (DEPT) spectra (Table 1) displayed 28 carbon resonances due to four quaternary carbons, 10 methines, seven methylenes, and seven methyl groups, which revealed one amide carbonyl group and one double bond. The presence of five methyl signals [*δ*<sup>H</sup> 0.67 (3H, s, H-18), 1.21 (3H, s, Me-19), 0.92 (3H, d, *J* = 6.4 Hz, Me-21), 2.23 (6H, s, *N*,*N*-dimethyl)] and one nitric proton signal [*δ*<sup>H</sup> 6.09 (1H, d, *J* = 5.1 Hz, NH-3)] in the 1H NMR (CDCl3) spectrum in combination with 2D NMR data suggested that compound **1** belongs to the 20*α*-dimethylamino-3-amino-5*α*-pregnane type steroidal alkaloids [21].


**Table 1.** 1H (600 MHz) and 13C (150 MHz) NMR data of compounds **<sup>1</sup>**−**<sup>3</sup>** in CDCl3.

The presence of two methyl [*δ*<sup>H</sup> 1.76 (3H, d, *J* = 6.9 Hz, H-4 ), 1.84 (3H, s, H-5 )] and an olefinic proton [*δ*<sup>H</sup> 6.45 (1H, q, *J* = 6.9 Hz, H-3 )] signals in the 1H NMR spectrum together with two methyl [*δ*<sup>C</sup> 14.3 (C-4 ), 12.6 (C-5 )], one double bond [*δ*<sup>C</sup> 131.4 (C-2 ), 132.1 (C-3 )], and one carbonyl [*δ*<sup>C</sup> 170.6 (C-1 )] signals in the 13C NMR spectrum led to the deduction of a tigloyl moiety, which was supported by the 1H–1H COSY-correlated signal of H-4 /H-3 and the HMBC correlations of H-5 /C-3 , C-1 and H-3 /C-1 (Figure 2). Furthermore, the HMBC correlations of NH/C-2 (Figure 2) proposed that the location of the tigloyl group was at N-3. In addition, the two hydroxyl groups assigned at the C-2 (*δ*<sup>C</sup> 69.0) and C-4 (*δ*<sup>C</sup> 76.3) positions were deduced from the 1H NMR [*δ*<sup>H</sup> 2.84 (1H, d, *J* = 7.2 Hz, OH-2), 4.40 (1H, d, *J* = 3.0 Hz, OH-4)] and 13C NMR [*δ*<sup>C</sup> 69.0 (C-2), 76.3 (C-4)] data in combination with the 1H–1H COSY [OH-2 /H-2 (*δ*<sup>H</sup> 3.88) and OH-4 /H-4 (*δ*<sup>H</sup> 3.78)] and HMBC [OH-2/C-1 (*δ*<sup>C</sup> 44.2), C-3 (*δ*<sup>C</sup> 58.4) and OH-4/C-3, C-5 (*δ*<sup>C</sup> 45.5)] experiments. Therefore, the planar structure of compound **1** was constructed.

The 13C NMR data of the ring A**,** coupling constants of H-2, H-3 and H-4 and NOESY data clearly indicated that compound **1** differs from the previously reported compounds of hookerianamide M [10]

and sarcovagine A [18] with respect to the stereochemistry at C-2, C-3, and C-4 positions. In the ROESY spectrum (Figure 2), the correlations of H-19 [*δ*<sup>H</sup> 1.21 (3H, s)] with OH-2 and OH-4 indicated the *β* orientations of the two hydroxyl groups. Furthermore, the obvious ROESY correlations (Figure 2) of HN with H-2, H-4 and H-5 [*δ*<sup>H</sup> 1.26 (1H, m)] implied the *α* orientation of the tigloylamide group. The ring A of compound **1** may exist mainly as a stable boat conformation due to the substitution of 3*α* tigloylamide group [17]. Consequently, the structure and relative configuration of compound **1** was determined as (20*S*)-20-*N*,*N*-dimethylamino-2*β*,4*β*-dihydroxyl-3*α*-tigloylamino-5*α*-pregnane, which was named sarchookloide A (Figure 1).

**Figure 1.** Structures of compounds **1**−**7**.

Compound **2** had a molecular formula of C28H48N2O2, which was determined by HR-ESI-MS (*m*/*z* 445.3775 [M + H]+), suggesting six degrees of unsaturation. The IR spectrum displayed absorptions indicating a hydroxyl group (3443 cm–1), amide carbonyl group (1664 cm−1) and double bond (1629 cm−1). In the 13C NMR and DEPT spectra (Table 1), 28 carbon signals were observed, including 23 carbon resonances assigned to a 20*α*-dimethylamino-3-amino-5*α*-pregnane type steroidal alkaloid skeleton and 5 carbon resonances for a tigloyl group [21]. The 1H–1H COSY correlated signal of H-4 [*δ*<sup>H</sup> 1.75 (3H, d, *J* = 6.9 Hz)]/H-3 [*δ*<sup>H</sup> 6.37 (1H, q, *J* = 6.9 Hz)] and the HMBC correlations of H-5 [*δ*<sup>H</sup> 1.84 (1H, s)]/C-3 [*δ*<sup>C</sup> 130.8], C-1 [*δ*<sup>C</sup> 169.2], H-3 /C-1 and HN [*δ*<sup>H</sup> 5.82 (1H, d, *J* = 7.4 Hz)]/C-1 (Figure 2) suggested that the tigloyl group were attached to N-3. The COSY correlations of H-2 [*δ*<sup>H</sup> 3.97 (1H, brs)]/H-1 [*δ*<sup>H</sup> 1.16, 1.87 (2H, m)], H-3 [*δ*<sup>H</sup> 4.03 (1H, m)] proposed that the location of the hydroxyl group was at C-2.

The similarity of the NMR data of compounds **2** and 20*α*-dimethylamino-2*α*-hydroxyl-3*β*-tigloylamino-5*α*-pregnane [16] suggested that they possessed the same planar structure. The ROESY correlations (Figure 2) of HN with H-2 and H-5 [*δ*<sup>H</sup> 1.07 (1H, m)] implied the *α* orientation of the tigloylamide group and the *β* orientation of the hydroxyl group. This was also supported by the discrepant W1/2 (14.8) of H-2 [10] and the 13C NMR data of the ring A in **2** compared with the data of reported compounds [16]. The substitution of 3*α* tigloylamide group led to the main boat conformation of the ring A in compound **2**. Consequently, the structure and relative configuration of compound **2** was determined as (20*S*)-20-*N*,*N*-dimethylamino-2*β*-hydroxyl-3*α*-tigloylamino-5*α*-pregnane, which was named sarchookloide B (Figure 1).

Compound **3** was given the molecular formula C28H48N2O according to its HR-ESI-MS data at *m*/*z* 429.3829 [M + H]<sup>+</sup> (calculated as 429.3839), corresponding to six degrees of unsaturation. The IR spectrum of compound **3** included the absorption bands for amide carbonyl group (1665 cm−1) and double bond (1625 cm<sup>−</sup>1). The 13C NMR and DEPT spectra (Table 1) of compound **3** exhibited 28 carbon signals corresponding to four quaternary carbons, eight methines, nine methylene, and seven methyl groups, of which 23 carbon resonances were ascribed to 20*α*-dimethylamino-3-amino-5*α*-pregnane type steroidal alkaloid skeleton and five carbon resonances were attributed to a tigloyl group [21].

Compound **3** and pachysamine G [19] exhibited the same planar structure, which was supported by the similar NMR data found for both of them. The *α*-orientations of the tigloylamide group was assigned by the ROESY correlations (Figure 2) of HN [*δ*<sup>H</sup> 5.93 (1H, d, *J* = 6.6 Hz)] with H-5 [*δ*<sup>H</sup> 1.11 (1H, m)] in combination with the different shift of the ring A in compound **3** compared with the data of pachysamine G in 13C NMR spectrum. Similar to compounds **1** and **2**, the boat conformation of the ring A in compound **3** was the main conformation [17]. Therefore, the structure and relative configuration of compound **3** was determined as (20*S*)-20-*N*,*N*-dimethylamino-3*α*-tigloylamino-5*α*-pregnane, which was named sarchookloide C (Figure 1).

The known compounds **4**–**7** were identified as pachysamine G (**4**) [19], pachysamine H (**5**) [19], sarcovagine B (**6**) [18], and pachyaximine A (**7**) [20] through a comparison of their spectroscopic data with those reported in the literature.

**Figure 2.** Key 1H−1H COSY (-), HMBC (*→*) and ROESY (*↔*) correlations of compounds **1**–**3**.

#### *2.2. Results of the Cytotoxicity Test*

All compounds were evaluated using a MTT cytotoxicity assay against human cervical cancer cell line Hela, lung adenocarcinoma cell line A549, breast cancer cell line MCF-7, colon cancer cell line SW480, and leukemia CEM cells (adriamycin was used as the positive control). The IC50 values of all compounds against the indicated cancer cells are summarized in Table 2. Compound **5** had the greatest cytotoxicity to all cells, as its range of IC50 values was approximately 1.05–2.23 μM. All three amide-substituted compounds, except for pachyaximine A, exhibited significant cytotoxic activity on all cells, which suggests that the amide group of these compounds was the necessary group for the cytotoxicity. In addition, Hela and A549 were the more sensitive cell lines to these types of compounds compared to all tested cancer cells because the IC50 values of all compounds were less than 10 μM. Furthermore, all active compounds showed effects that were comparable to the chemotherapeutic drug adriamycin in inhibiting the growth of all cancer cells, which suggests that three amide-substituted pregnane-type steroidal alkaloids might have the potential to be anticancer agents.

**Table 2.** Cytotoxicity of compounds **<sup>1</sup>**−**<sup>7</sup>** <sup>a</sup> against Hela, A549, MCF-7, SW480, and CEM cells in vitro (IC50 b, μM).


<sup>a</sup> All results are expressed as mean ± SD; *<sup>n</sup>* = 3 for all groups. <sup>b</sup> IC50: 50% inhibitory concentration. <sup>c</sup> Adriamycin was the positive control.

#### **3. Materials and Methods**

#### *3.1. General Experimental Procedures*

Optical rotations were obtained on a JASCO model 1020 polarimeter (Horiba, Tokyo, Japan). UV spectra were measured on a Shimadzu UV-2401PC spectrophotometer (Shimadzu, Kyoto, Japan). IR (KBr) spectra were measured on a Bio-Rad FTS-135 spectrometer (Bio-Rad, Hercules, CA, USA). The 1D and 2D NMR spectra were recorded on Bruker AVANCE III-600 spectrometers with TMS used as an internal standard (Bruker, Bremerhaven, Germany). The mass spectra were obtained on a Waters AutoSpec Premier P776 (Waters, NY, USA). The silica gel (200–300 mesh) for column chromatography and the TLC plates (GF254) were obtained from Qingdao Marine Chemical Factory (Qingdao, Shandong, China). The Sephadex LH-20 (20–150 μm) used for chromatography was purchased from Pharmacia Fine Chemical Co. Ltd. (Pharmacia, Uppsala, Sweden). Fractions were visualized by heating silica gel plates sprayed with Dragendorff's reagent. The cell lines Hela, A549, MCF-7, SW480, and CEM were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), while MTT were obtained from Sigma Company.
