**1. Introduction**

The *Sarcococca* genus (Buxaceae) consists of about 20 species, widely distributed in the southwestern region of China and other south Asian countries [1]. The members of *Sarcococca* plants are used as TCM and traditional folk medicine for the treatment of stomach pain, rheumatism, swollen sore throat and traumatic injury [2–4]. Previous investigations on several species of this genus indicated that steroidal alkaloids are the major chemical components with a broad spectrum of biological activities, such as cholinesterase inhibition [5–7], antitumor [8], antibacterial [9], antileishamanial [10], antidiabetic [11] and estrogen biosynthesis-promoting [12].

*Sarcococca hookeriana*, one of *Sarcococca* plants, is usually confusedly used by ethnic minorities in China. Although dozens of steroidal alkaloids have been discovered from *S. hookeriana* of Nepal [13–17], there were few phytochemical or biological studies on this species which grows in China. Enlightened by the diverse bioactivities of steroidal alkaloids and the use of *Sarcococca* plants as folk medicine, *S. hookeriana* was chosen for searching antitumor agent by our research group and several cytotoxic steroidal alkaloids have been reported [18]. In continuation of our ongoing study on this plant, two new steroidal alkaloids, named hookerianine A (**1**) and hookerianine B (**2**), together with two known ones, sarcorucinine G (**3**) [19] and epipachysamine D (**4**) [20] (Figure 1), were characterized and their cytotoxicity were evaluated in vitro with a CCK-8 assay. Herein, we describe the isolation, structure elucidation and cytotoxicity of the isolates.

**Figure 1.** Structures of compounds **1**–**4**.

#### **2. Results and Discussion**

#### *2.1. Elucidation of the Chemical Structure of Compounds*

Hookerianine A (**1**) was obtained as white amorphous powder, positive to Dragendorff's reagent. The molecular formula of C31H48N2O was determined by HR-ESI-MS (*m*/*z* 465.3833, [M + H]+). The IR absorption at 3294, 1637, 1539, 1496 and 760 cm−<sup>1</sup> indicated the presence of a secondary amine, amide carbonyl and aromatic ring, respectively. The 1H-NMR spectra (Table 1) exhibited signals of five aromatic protons (*δ*<sup>H</sup> 7.37, 7.33 and 7.28) and five methyls (*δ*<sup>H</sup> 2.17, 0.87, 0.74 and 0.63). The 13C-NMR (Table 1) signals at (*δ*<sup>C</sup> 135.5, 129.4, 129.1 and 127.4) were characteristic for a monosubstituted aromatic ring, whereas the signal at *δ*<sup>C</sup> 170.1 was due to the carbonyl carbon. The NMR data of compound **1** was similar to epipachysamine D (**4**), having one more methylene. HMBCs (Figure 2) from H-C(2 ) to C(1 ), C(3 ) and C(4 ) indicated that the additional methylene was placed between C(1 ) and C(3 ). Thus, compound **1** possessed a novel phenylacetyl group instead of benzoyl group located at C(3). The relative configuration of C(3) was assigned as *α*-orientation by correlations of N-H with H*α*-C(1), H*α*-C(5), and H*β*-C(1) with H-C(19) in ROESY (Figure 2). Therefore, compound **1** was characterized as (20*S*)-20-(*N*,*N*-dimethylamino)-3*α*-phenylacetylamino -5*α*-pregnane, to which we give the trivial name hookerianine A.


**Table 1.** 1H- (500 MHz) and 13C- (125 MHz) NMR data of compounds **1**–**2** in CDCl3.


**Table 1.** *Cont.*

**Figure 2.** Key 1H,1H-COSY ( ), HMBC (H→C), and ROESY ( ) correlations of compound **<sup>1</sup>**.

Hookerianine B (**2**) was obtained as white amorphous powder also reacts positively with Dragendorff's reagent. The molecular formula of C30H44N2O2 was determined by HR-ESI-MS (*m/z* 465.3471, [M + H]+). The IR absorption at 3402, 1644, 1603, 1521, 1488 and 718 cm−<sup>1</sup> indicated the presence of a secondary amine, amide carbonyl and aromatic ring, respectively. The 1H-NMR spectra (Table 1) exhibited signals of five aromatic protons (*δ*<sup>H</sup> 7.74, 7.47 and 7.41) and five methyls (*δ*<sup>H</sup> 2.24, 1.14, 0.87 and 0.83). The 13C-NMR spectra (Table 1) displayed 30 carbon signals including one carbonyl carbon at (*δ*<sup>C</sup> 166.7) and six carbons of an aromatic ring (*δ*<sup>C</sup> 135.0, 131.2, 128.5 and 126.8), respectively. whereas the signals at (*δ*<sup>C</sup> 73.6 and 60.9) were due to two oxygenated carbons. The NMR data of compound **2** was similar to epipachysamine D (**4**), and the difference was the downfield chemical shift of C(16) and C(17) at *δ*(C) 73.6 and 60.9, which suggested that compound **2** possessed an epoxy group at C(16) and C(17), confirmed by HMBCs (Figure 3) from H-C(16) to C(14) and C(15), from H-C(18) and H-C(21) to C(15). The ROESY correlations (Figure 3) of N-H with H*α*-C(5), and H*β*-C(16) with N-Me suggested that the substituent at C(3) and the epoxy group at C(16) and C(17) all had *α*-orientations. Thus, compound **2** was characterized as (20*S*)-20-(*N*,*N*-dimethylamino)-16*α*,17*α*-epoxy-3*α*-benzoylamino-5*α*-pregnane, to which we give the trivial name hookerianine B.

**Figure 3.** Key 1H,1H-COSY ( ), HMBC (H→C), and ROESY ( ) correlations of compound **<sup>2</sup>**.

The structures of known compounds **3**–**4** were determined by comparing their spectral data with literature data. To further confirm the chemical structure of compound **4**, a colorless crystal was obtained from CH2Cl2, and X-ray crystallography analysis with Mo *K*α radiation was performed. Through structural refinement by direct method SHELX-2014 [21,22], the chemical structure of **4** was identified as shown in Figure 4.

**Figure 4.** ORTEP drawing of compound **4.**

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

The IC50 values of four compounds against five human cancer cell lines: SW480, SMMC-7721, PC3, MCF-7 and K562 are summarized in Table 2 (DDP and 5-FU was used as the positive control). The compound **2**, a new steroidal alkaloid, exhibited moderate cytotoxic activities to all cell lines with IC50 values in the range of 5.97–19.44 μM. Compared to the positive control 5-FU with IC50 values of 7.65 and 4.78 μM against SW480 and K562 cell lines, the compound **3** was the most effective one against these cell lines with IC50 values of 5.77 and 6.29 μM, respectively. The structure-activity relationships of compound **1** and **4** showed that steroidal alkaloids possessed a novel phenylacetyl group instead of benzoyl group located at C-3 can increase the cytotoxicity to human cancer cell lines: SW480, SMMC-7721, PC3 and K562. Interestingly, the cytotoxicity of compound **3** is stronger than compound **4**, which indicated that the presence of double bond between C-16 and C-17 can increase the cytotoxicity. Meanwhile, compared to compound **4**, compound **2** possessed an epoxy group at C-16 and C-17 also showed better cytotoxicity. The results suggested that C-16 and C-17 of steroidal alkaloids play an important role in anticancer potential.


**Table 2.** Cytotoxicity of compounds **1**–**4** against SW480, SMMC-7721, PC3, MCF-7 and K562 cells in vitro.

<sup>a</sup> Values of IC50 expressed as mean ± SD, *<sup>n</sup>* = 3 for all groups. <sup>b</sup> DDP, the abbreviation of cisplatin, used as reference drug. <sup>c</sup> 5-FU, the abbreviation of 5-fluorouracil, used as reference drug.

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

#### *3.1. General Experimental Procedures*

Optical rotations were measured with a Rudolph Autopol I automatic polarimeter (Rudolph, Hackettstown, NJ, USA). UV spectra were obtained on a Shimadzu UV-2401PC spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were measured with a Bruker TENSOR-27 spectrophotometer

(Bruker, Bremerhaven, Germany) using KBr pellets. The 1D and 2D NMR spectra were recorded on JEOL ECX 500 MHz spectrometers (JEOL Ltd, Kyoto, Japan) with TMS as an internal standard. Chemical shifts (*δ*) were expressed in ppm with reference to solvent signals. High-Resolution Electrospray Ionization Mass Spectrometry (HR-ESI-MS) was recorded on a Bruker Daltonics micrOTOF-Q II spectrometer (Bruker, Bremerhaven, Germany). Column chromatography (CC) was performed on Silica gel (200–300 and 300–400 mesh, Qingdao Marine Chemical Ltd., Qingdao, China). Fractions were monitored by TLC (GF 254, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), and spots were visualized by Dragendorff's reagent. Solvents were distilled prior to use for extraction and isolation.
