Four New Sesquiterpenoids from the Roots of Diarthron Tianschanica with Their Antineoplastic Activity

Abstract

Four new sesquiterpenoids, known as diarthronchas A–D (1–4), and one known daphnauranol B (5) were isolated from the methanol extract of the roots of Diarthron tianschanica. The compounds structures were determined on the basis of spectroscopic data. All of the isolated compounds were profiled for their antineoplastic activity.

1. Introduction

Diarthron tianschanica (Pobed.) Kit Tanis, a member of the genus Diarthron (Thymelaeaceae), is only observed in the Zhaosu County of Xinjiang Uygur Autonomous Region, China [1]. The roots of D. tianschanica have been used in folk medicine to cure a wide variety of ailments, including coughs; asthma [2]; bronchitis [3]; and tuberculosis of the skin, bone, and epididymis [4]. Previously, our group examined the chemical constituents of D. tianschanica, resulting in the identification of lignans, coumarins, and diarylpentanols [5,6]. As part of our ongoing phytochemical study, we further investigated the chemical constituents from the roots of D. tianschanica and obtained four new sesquiterpenoids, known as diarthronchas A–D (1–4), and one known daphnauranol B (5) [7] (Figure 1). In this paper, we elucidate the structure of these new compounds and their antineoplastic activity.

2. Results and Discussion

2.1. Purification of Compounds 1–4

The roots of D. tianschanica (10 kg) were soaked with MeOH at room temperature and extracted three times under reflux. The sesquiterpenoids were isolated and purified via silica gel chromatography, Sephadex LH-20 gel chromatography, and semi-preparative high-performance liquid chromatography (HPLC).

2.2. Structure Elucidation of Compounds 1–4

Compound 1 was obtained as a white powder. This compound’s molecular formula was established to be C₁₅H₂₂O₃, based on the HRESIMS quasimolecular ion at m/z 273.1485 [M + Na]⁺ (Calcd for. 273.1467 C₁₅H₂₂O₃Na). The infrared (IR) and ultraviolet (UV) spectra revealed absorptions for the hydroxyl (3365 cm⁻¹), and an α,β-unsaturated ketone moiety (216 nm; 1724 cm⁻¹) [8]. The ¹H-NMR spectrum (

Table 1. ¹H (600 MHz) and ¹³C-NMR (150 MHz) spectral data for compounds 1 and 2 (in DMSO-d₆).

Position	1		Position	2
	δc Type	δH (J in Hz)		δc Type	δH (J in Hz)
1	172.1		1	162.6
2	27.6	1.50 (1H, dd, 10.8, 9.6) 1.52 (1H, dd, 10.8, 2.4)	2	39.9	2.26 (2H, m)
3	39.5	1.99 (1H, m) 2.48 (1H, m)	3	34.0	2.48 (2H, m)
4	73.9		4	145.5
5	83.3		5	144.9
6	34.7	2.53 (1H, m) 2.65 (1H, m)	6	39.6	2.48 (2H, m)
7	42.2	2.67 (1H, m)	7	41.5	3.28 (1H, m)
8	47.9	2.16 (1H, dd, 12.0, 1.8) 2.60 (1H, dd, 12.0, 10.8)	8	49.6	2.81 (2H, m)
9	205.3		9	199.0
10	136.8		10	134.1
11	151.0		11	147.3
12	108.7	4.65 (1H, d, 1.8) 4.71 (1H, d, 1.8)	12	110.8	4.77 (1H, d, 1.8) 4.79 (1H, d, 1.8)
13	20.1	1.72 (3H, s)	13	20.1	1.75 (3H, s)
14	7.9	1.54 (3H, s)	14	2.3	1.80 (3H, s)
15 OH	27.5	1.19 (3H, s) 4.31 (br s) 5.27 (br s)	15 OH	58.8	4.15 (1H, dd, 11.5, 5.4)

) showed three tertiary methyl groups as singlets at δ_H 1.19 (3H, s, H₃-15), 1.54 (3H, s, H₃-14), and 1.72 (3H, s, H₃-13), and an exo-methylene as two broad doublets at δ_H 4.65 (1H, d, J = 1.8 Hz, H-12a) and 4.71 (1H, d, J = 1.8 Hz, H-12b). The ¹³C-APT NMR spectrum revealed 15 carbon signals due to three methyls, five methylenes, one methine, and six quaternary carbons. Among these signals, the carbon signals at δ_C 136.8 (C-10), 172.1 (C-1), and 205.3 (C-9) indicated the presence of an α,β-unsaturated ketone group, and the carbon signals at δ_C 20.1 (C-13), 108.7 (C-12), and 151.0 (C-11) revealed the existence of an anisopropenyl group [8]. The results described above suggested that compound 1 contained a guaiane-type skeleton [8]. All the signals and functional groups were confirmed by HSQC, HMBC, and ¹H-¹H COSY spectra. In the HMBC spectrum (Figure 2A), the protons of δ_H 1.54 (s, H₃-14) were correlated with δ_C 136.8 (C-10), 172.1 (C-1), and 205.3 (C-9), indicating that the connection of H₃-14 is at C-10 and the presence of an α,β-unsaturated ketone group. Moreover, the proton signal at δ_H 1.19 (s, H₃-15) had long-range correlations with carbon signals at δ_C 39.5 (C-3), 73.9 (C-4), and 83.3 (C-5), suggesting that the methyl group of H₃-15 was located at the quaternary carbon C-4. The proton signal at δ_H 2.67 (1H, m, H-7) was interrelated with δ_C 151.0 (C-11) and 108.7 (C-12). The double bond protons at δ_H 4.65 (1H, d, J = 1.8 Hz, H-12a) and 4.71 (1H, d, J = 1.8 Hz, H-12b) and the methyl protons at δ_H 1.72 (3H, s, H-13) were connected with δ_C 42.2 (C-7) and 151.0 (C-11), respectively, suggesting the presence of an isopropenyl group at C-7. Furthermore, the HMBC correlations from δ_H 4.31 (1H, br s) to δ_C 73.9 (C-4) and δ_H 5.27 (1H, br s) to δ_C 83.3 (C-5) indicated that two OH groups were attached to C-4 and C-5, respectively. The relative stereochemistry of compound 1 was determined by the Nuclear Overhauser Effect Spectroscopy (NOESY) experiment (Figure 2B), in which correlations were observed between the following protons (OH-4/OH-5, H-7/OH-5). Finally, because of the n–π electron transition effect of the α,β-unsaturated ketone group, the CD spectra of compound 1 showed the cotton effects at 325 nm (Δε −0.7) and 250 nm (Δε + 2.0); thus, the C-5 is in the S configuration [9]. Based on these results, compound 1 was elucidated as described and given the trivial name of diarthroncha A.

Compound 2 yielded the molecular formula of C₁₅H₂₀O₂, based on the HRESIMS analysis of m/z 255.1348 [M + Na]⁺ (Calcd for. 255.1361 C₁₅H₂₀O₂Na). Its ¹H and ¹³C-NMR (Table 1) data were very similar to 1, except for the additional double bond and hydroxymethyl groups in 2. In contrast to 1, the signal for C-4 and C-5 of compound 2 showed strong downfield chemical shifts to δ_C 145.5 (C-4) and 144.9 (C-5), indicating a sp² double bond between C-4 and C-5 in 2. Additionally, in the HMBC spectrum, the methylene protons at δ_H 4.15 (H-15a) and 4.24 (H-15b) had direct correlations with δ_C 145.5 (C-4), indicating that the methyl group at C-4 in 1 was oxidized to a hydroxymethyl group in 2. Thus, the structure of 2 was determined as shown and named diarthroncha B.

Compound 3 was obtained as a white powder with the molecular formula of C₁₅H₂₀O₂, in agreement with the positive HRESIMS ion peak at m/z 255.1349 [M + Na]⁺ (Calcd for. 255.1361 C₁₅H₂₀O₂Na). Comparison of this compound’s ¹H and ¹³C-NMR data (

Table 2. ¹H (600 MHz) and ¹³C-NMR (150 MHz) spectral data for compounds 3 and 4 (in DMSO-d₆).

Position	3		Position	4
	δc Type	δH (J in Hz)		δc Type	δH (J in Hz)
1	138.2		1	47.6	2.33 (1H, m)
2	40.4	2.90 (1H, m) 3.20 (1H, m)	2	29.1	1.09 (1H, m) 1.23 (1H, m)
3	202.8		3	30.9	1.43 (1H, m) 1.85 (1H, m)
4	137.3		4	34.7	2.56 (1H, m)
5	165.5		5	78.4
6	35.3	2.63 (1H, m) 2.80 (1H, m)	6	35.5	1.76 (2H, m)
7	37.2	2.65 (1H, m)	7	154.3
8	41.0	1.93 (1H, m) 1.89 (1H, m)	8	124.5	5.63 (1H, d, 1.2)
9	70.0	4.33 (1H, m)	9	202.1
10	130.4		10	84.3
11	149.8		11	146.3
12	110.1	4.71 (1H, s) 4.76 (1H, s)	12	125.6	6.09 (1H, d, 1.2) 6.12 (1H, d, 0.6)
13	20.2	1.75 (1H, s)	13	165.8
14	17.0	1.87 (1H, s)	14	15.9	1.92 (3H, s)
15	8.2	1.66 (1H, s)	15 MeO	22.2 51.7	0.90 (3H, d, 7.2) 3.62 (3H, s)

) revealed that its structure was similar to that of oleodaphnoic acid [10], except for the additional hydroxyl group at C-9 and methyl group at C-10 in 3. In the HMBC spectrum, the correlations from δ_H 4.33 (1H, m, H-9) to δ_C 41.0 (C-8) and 130.4 (C-10), together with the signal for C-9, revealed a powerful downfield shift to δ 70.0 (+ 44.4 ppm), confirming the presence of a hydroxy group at C-9. Additionally, the HMBC correlations of δ_H 1.87 (H₃-14) with δ_C 138.2 (C-1), 70.0 (C-9), and 130.4 (C-10) suggested that the carboxyl group at C-10 in the oleodaphnoic acid was reduced to a methyl group in 3. In the NOESY spectrum, the cross-peaks of H-7 and H-9 indicated the 9-OH was in a,β-orientation. Therefore, the structure of 3 was established as shown and named diarthroncha C.

Compound 4 was obtained as a white powder. The compound’s molecular formula of C₁₆H₂₂O₅ was determined via HRESIMS analysis of m/z 317.1348 [M + Na]⁺ (Calcd for. 317.1365 C₁₆H₂₂O₅Na). The ¹H-NMR spectrum (Table 2) showed two methyl groups as signals at δ_H 1.92 (3H, s, H₃-14) and 0.90 (3H, d, J = 7.2 Hz, H₃-15), an exo-methylene resonance as two broad doublets at δ_H 6.09 (1H, d, J = 1.2 Hz, H-12a) and 6.12 (1H, d, J = 1.2 Hz, H-12b), an olefinic proton signal at δ_H 5.63 (1H, s, H-8), and a methoxy signal at δ_H 3.62 (3H, s, 13-OMe). Aside from the single methoxy group, the ¹³C-NMR spectrum exhibited 15 carbon resonances, including two methyls, four methylenes, three methines, and six quaternary carbons (one ketone, one carboxymethyl, two olefinic carbons, and two oxygenated carbons).The ¹H and ¹³C-NMR data of 4 were characteristic of the guaiane-type skeleton with an α,β-unsaturated ketone (δc 124.5, 154.3, 202.1) and an acrylic ester group (δc 125.6, 146.3, 165.8). In the HMBC spectrum, the correlations of δ_H 6.09 (1H, d, J = 1.2 Hz, H-12a) and 6.12 (1H, d, J = 1.2 Hz, H-12b) with δc 146.3 (C-11), 154.3 (C-7), and 165.8 (C-13) suggested that the isopropenyl group at C-7 in the reported guaiane-type skeletons had been oxidized to an acrylic ester group in 4 [8]. Moreover, the proton of δ_H 5.63 (1H, s, H-8) was interrelated with δc 154.3 (C-7), and 202.1 (C-9) revealed the presence of an α,β-unsaturated ketone moiety at C-7/8/9. Furthermore, the ¹H-¹³C long-range signals from δ_H 3.62(-OCH₃) to δc 165.8 (C-13) and from δ_H 1.92 (H₃-14) to δc 84.3 (C-10) placed the methoxy group at C-13 and the methyl group at C-10. The downfield chemical shifts of C-5 (δc 78.4) and C-10 (δc 84.3), together with the molecular formula above, indicated the presence of OH groups at C-5 and C-10. The CD spectrum of 4 displayed strong cotton effects at 334 nm (Δε −1.2) and 287 nm (Δε + 2.8), which corresponded to the n → π* and π → π* transitions of the unsaturated dienone. On the basis of the CD excitation chirality method for the unsaturated dienone, C-10 possessed the S absolute configuration. Taken together with the NOESY spectrum, the structure of 4 was defined as shown and given the trivial name diarthroncha D.

2.3. Antineoplastic Activity of Compounds 1–5

All of the isolated compounds were tested in vitro for their cytotoxic activity against HepG-2, MCF-7, and HeLa human cancer cell lines, with paclitaxel serving as a positive control. The results (

Table 3. IC₅₀ values of compounds 1–5 against HepG-2, MCF-7, and HeLa.

Compound	HepG-2	MCF-7	HeLa
	IC50 (μM) *
Paclitaxel	1.80 ± 0.26	3.80 ± 0.31	4.25 ± 0.52
1	18.9 ± 0.02	48.7 ± 0.39	>50
2	41.3 ± 0.13	>50	39.6 ± 0.53
3	22.5 ± 0.09	>50	>50
4	>50	>50	>50
5	20.3 ± 0.24	>50	29.6 ± 0.61

* IC₅₀ = inhibitory concentration 50%.

) showed that compounds 1, 3, and 5 were moderately cytotoxic against HepG-2 cells with inhibitory concentration 50% (IC₅₀) values at 18.9, 22.5, and 20.3 μM, respectively, while compound 2 was weakly cytotoxic with an IC₅₀ value of 41.3 μM. Furthermore, compounds 2 and 5 exhibited weak cytotoxicity against HeLa cells with IC₅₀ values of 39.6 and 29.6 μM, respectively. None of the compounds had activity against MCF-7 cell lines.

3. Materials and Methods

3.1. General Experimental Procedures

Optical rotation data were obtained using a Perkin-Elmer 341 digital polarimeter (PerkinElmer, Norwalk, CT, USA). CD spectra were obtained using a JASCO J-815 spectropolarimeter (JASCO, Easton, Md., USA). UV and IR spectra were obtained using Shimadzu UV2550 and FTIR-8400S spectrometers (Shimadzu, Kyoto, Japan), respectively. NMR spectra were obtained using a Bruker AV III 600 NMR spectrometer (Bruker, Billerica, German) with chemical shift values presented as δ values and TMS (Tetramethylsilane) as the internal standard. HRESIMS was performed using an LTQ-Orbitrap XL spectrometer (Thermo Fisher Scientific, Boston, MA, USA). Column-chromatography (CC) was performed using silica gel (100–200 mesh, Qingdao Marine Chemical Plant, Qingdao, China) and Sephadex LH-20 (Pharmacia, Uppsala, Sweden). Precoated silica gel GF254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, China) were used for TLC. All of the solvents used were of analytical grade (Beijing Chemical Plant, Beijing, China).

3.2. Plant Material

The roots of Diarthron tianschanica were collected in September 2013 from Zhaosu city, Xinjiang Autonomous Region, China, and were identified by Prof Xiao-Guang Jia, Department of Pharmaceutical Chemistry, Xinjiang Institute of Chinese and Ethnic Medicine. A voucher specimen (NO. 13094) was deposited at the Xinjiang Institute of Chinese and Ethnic Medicine.

3.3. Isolation and Purification of Compounds 1–5

The roots of D. tianschanica (10 kg) were soaked with MeOH at room temperature (3 × 40 L, 3 h each) and were extracted three times under reflux. Removal of the MeOH under reduced pressure yielded a methanol extract (2189 g). The residue was dissolved in water and extracted with petroleum ether (3 × 1000 mL), chloroform (3 × 1000 mL), ethyl acetate (3 × 1000 mL), and n-butanol (3 × 1000 mL), successively. The petroleum ether fraction (108 g) was subjected to CC (column-chromatography) over a silica gel (100–200 mesh, 15 × 60 cm) eluting with a stepwise gradient of CH₂Cl₂-MeOH (from 1:0 to 0:1, that is, 100:0, 100:1, 80:1, 50:1, 30:1, 20:1, 15:1, 0:1, v/v) to yield fractions A–H. Fr. B was prepared using a Sephadex LH-20 column with MeOH to remove pigments, and purified by semi-preparative HPLC of MeOH-H₂O (60:40, v/v) as the mobile phase to yield compounds 1 (5.8 mg, t_R = 18.8 min) and 2 (2.5 mg, t_R = 31.2 min). Fr. C was purified by semi-preparative HPLC with MeOH-H₂O (70:30, v/v) as the mobile phase to yield compounds 3 (3.1 mg, t_R = 16.7 min), 4 (4.2 mg, t_R = 28.3 min), and 5 (3.5 mg, t_R = 37.5 min).

3.4. Characterization of Compounds 1–4

Diarthroncha A (1): white powder (MeOH); [α${]}_{\mathrm{D}}^{20}$ + 11.6 (c 0.1, MeOH); UV (MeOH) λmax (logε): 216 (3.62) nm, CD (MeOH) 325 nm (Δε −0.7) and 250 nm (Δε + 2.0); IR (film) ν_max: 3365, 2923, 2845, 1724, 1782 cm^–1; ¹H and ¹³C-NMR data (DMSO-d₆), (see Table 1); HR-ESI-MS m/z 273.1485 [M + Na]⁺.(Calcd for. 273.1467 C₁₅H₂₂O₃Na).

Diarthroncha B (2): white powder (MeOH); [α${]}_{\mathrm{D}}^{20}$ + 16.4 (c 0.1, MeOH); UV (MeOH) λmax (logε): 214 (4.83) nm; IR (film) ν_max: 3372, 2930, 2848, 1721, 1776 cm^–1; ¹H and ¹³C-NMR data (DMSO-d₆), (see Table 1); HR-ESI-MS m/z 255.1348 [M + Na]⁺ (Calcd for. 255.1361 C₁₅H₂₀O₂Na).

Diarthroncha C (3): white powder (MeOH); [α${]}_{\mathrm{D}}^{20}$ + 14.2 (c 0.1, MeOH); UV (MeOH) λmax (logε): 212 (3.71) nm; IR (film) ν_max: 3368, 2932, 2846, 1729, 1786 cm^–1; ¹H and ¹³C-NMR data (DMSO-d₆), (see Table 2); HR-ESI-MS m/z 255.1349 [M + Na]⁺ (Calcd for. 255.1361 C₁₅H₂₀O₂Na).

Diarthroncha D (4): white powder (MeOH); [α${]}_{\mathrm{D}}^{20}$ + 17.5 (c 0.1, MeOH); UV (MeOH) λmax (logε): 210 (4.56) nm; CD (DMSO-d₆) 334 nm (Δε –1.2) and 287 nm (Δε + 2.8); IR (film) ν_max 3364, 2927, 2839, 1736, 1783, 1233 cm^–1; ¹H and ¹³C-NMR data (DMSO-d₆), (see Table 2); HR-ESI-MS m/z 317.1348 [M + Na]⁺ (Calcd for. 317.1365 C₁₆H₂₂O₅Na).

3.5. Cytotoxicity Assay of Compounds 1–5

The tested human cancer cell lines were seeded in 96-well plates (10⁷ cells/well), and the compounds were added at various concentrations (2.5, 5, 10, 25, and 50μM, respectively). Keeping the treatment on for 48 h, MTT (0.5 mg/mL) solution was added to each well, which were incubated for a further 4 h at 37 °C. The supernatant was removed, and the formazan crystals were dissolved in DMSO (150 mL) with gentle shaking at room temperature. Finally, the optical density of each well was measured at 570 nm with a microplate reader.

4. Conclusions

In conclusion, five sesquiterpenoids were isolated and characterised by spectrometric analysis (1 and 2D-NMR, HRESIMS). Among the isolated compounds, compounds 1, 3, and 5 showed moderate cytotoxicity against HepG cells with IC₅₀ values at 18.9, 20.3, and 22.5 μM, respectively. Therefore, we believed that this plant was an important source for the diverse structure of sesquiterpenoids and should be further investigated for biological activities.