Abietane Diterpenoids With Potent Cytotoxic Activities From the Resins of Populus euphratica

Abstract

A new abietane norditerpenoid, 5-(6-isopropyl-2-methylnaphthalen-1-yl)pentan-2-one (1), with a rare 4,5-seco-19-norabietane skeleton possessing a rearranged angular methyl group at C-5, 2 new naturally occurring compounds, 19-norabieta-4,6,8,11,13-penttaen-3-one (2) and 14-hydroxy-7-oxo ester (3), along with 10 known analogs (4−13) were isolated from the resins of Populus euphratica. The new structures were elucidated by spectroscopic analysis (one-/two-dimensional nuclear magnetic resonance and high-reolution electrospray ionization mass spectrometry). Biological evaluation showed that compounds 1 and 2 display low cytotoxicity against normal cells and appreciable cytotoxicity in cancer cells, with 2 to be more sensitive in HepG2 cells with a half-maximal inhibitory concentration value of 27.0 μM.

Populus euphratica (Salicaceae) is mainly distributed in western Inner Mongolia, Gansu, Xinjiang and other places in China. ¹ P. euphratica, also known as the desert polar, could survive in the desert ecosystem characterized by salt stress, extreme temperatures, and drought. ² P. euphratica Scbrenk, the resins from P. euphratica, has been historically used as a traditional Chinese medicine and applied in Chinese folk remedy for the treatment of swelling and pain in throat, tuberculosed adenitis, and toothache. ³ Previous chemical studies on P. euphratica revealed that it contains salicin derivatives, volatile oils, and some phenolic compounds. ^4-6 Besides, anti-inflammatory activity of the resin extract was also reported. ¹ As a part of our studies on plant resins, the title material was investigated, resulting in the isolation of a new rearranged abietane norditerpenoid (1), 2 new naturally occurring compounds (2 and 3), and 10 known but first isolated abietane diterpenoids (4−13) from this species. Subsequently, the cytotoxicity of compounds 1-3 in human cancer cells was assayed.

Compound 1, a colorless gum, has the molecular formula C₁₉H₂₄O based on analysis of its high-resolution electrospray ionization mass spectrometry (HRESIMS) m/z 269.1902 [M + H]⁺ (calculated for C₁₉H₂₄O, 269.1900), ¹³C nuclear magnetic resonance (NMR), and distortion enhancement by polarization transfer (DEPT) spectra, indicating 8 degrees of unsaturation. The ¹H NMR data (Table 1) of 1 shows an ABX system [δ _H 7.99 (d, J = 8.8 Hz), H-11, 7.59 (d, J = 1.8 Hz), H-14, 7.41 (dd, J = 8.8 Hz, 1.8 Hz), H-12], a double bond [δ _H 7.25 (d, J = 8.1 Hz), H-6, 7.57 (d, J = 8.1 Hz), H-7], indicating the presence of one 1,2,3,4-tetrasubstituted and one 1,2,4-trisubstituted benzene rings. In addition, 4 methyl groups [δ _H 2.48 (s), 2.16 (s), 1.34 (d, J = 6.9 Hz, H₃-17), and 1.33 (d, J = 6.9 Hz, H₃-16)] are also observed. The ¹³C NMR and DEPT spectra (Table 1) display 19 carbons including 4 methyl, 3 methylene, 6 methine groups (5 olefinic and 1 aliphatic), and 6 carbons (1 keto-carbonyl and 5 olefinic). These data remind that 1 might be a diterpenoid derivative. Actually, the structural architecture of 1 was mainly assembled by using two-dimensional (2D) NMR. The ¹H−¹H COSY spectrum of 1 shows the correlations of H₂-1/H₂-2/H₂-3, H-6/H-7, H-11/H-12 and H₃-16/H-15/H₃-17 (Figure 1). The heteronuclear multiple bond correlations (HMBCs) of H-6/C-8, C-10, C-20, H-7/C-5, C-9, C-14, H-11/C-8, C-10, C-13 and H-12/C-9, C-14, C-15, H-14/C-9, C-15 indicate the presence of a substituted naphthalene ring. The HMBCs of H₂-1/C-5, C-9, C-10, H₂-2, H₂-3/C-4 (δ _C 208.9) and H₃-18/C-3, C-4 indicate the substituted group (4-ketone-1-pentane group) at C-10. An isopropyl group is connected to C-13 via C-15 supported by the HMBCs of H₃-16/C-13, C-15, C-17 and H₃-17/C-13, C-15 (Figure 1). Thus, the structure of compound 1, named 5-(6-isopropyl-2-methylnaphthalen-1-yl)pentan-2-one, was identified. Compound 1 is a rare 4,5-seco-19-norabietane skeleton diterpenoid bearing a rearranged angular methyl group at C-5. To the best of our knowledge, before only 3,4-dihydro-6-isopropyl-2-methyl-1-(4′-oxopentyl)naphthalene as a similar structure was synthesized. ⁷

Table 1

¹H (600 MHz) and ¹³C NMR (150 MHz) Data of 1-3 (δ in ppm, J in Hz, CD₃OD).

No.	1	δ C	No.	2	δ C	No.	3	δ C
	δ H (J in Hz)			δ H (J in Hz)			δ H (J in Hz)
1	3.04 (overlap)	28.0, t	1	2.62 (overlap); 2.24 (m)	33.3, t	1	2.29 (m); 1.63 (m)	37.2, t
2	1.91 (m)	24.1, t	2	2.73 (m); 2.62 (overlap)	34.2, t	2	1.77 (overlap)	18.3, t
3	2.58 (t, 7.0)	43.6, t	3		198.0, s	3	1.78 (overlap); 1.71 (m)	36.6, t
4		208.9, s	4		129.7, s	4		46.7, s
5		132.3, s	5		156.3, s	5	2.67 (dd, 14.0, 3.2)	43.5, d
6	7.25 (d, 8.4)	129.3, d	6	6.66 (d, 9.8)	123.7, d	6	2.76 (dd, 17.8, 14.0) 2.35 (dd, 17.8, 3.2)	37.9, t
7	7.57 (d, 8.4)	126.1, d	7	6.76 (d, 9.8)	133.5, d	7		205.3, s
8		132.9, s	8		130.8, s	8		114.8, s
9		130.9, s	9		142.9, s	9		153.8, s
10		134.9, s	10		38.8, s	10		37..6, s
11	7.99 (d, 8.8)	132.8, d	11	7.30 (d, 8.0)	123.9, d	11	6.77 (d, 8.0)	113.3, d
12	7.41 (dd, 8.8, 1.8)	125.9, d	12	7.15 (dd, 8.0, 1.9)	127.2, d	12	7.36 (d, 8.0)	133.6, d
13		145.0, s	13		147.5, s	13		134.9, s
14	7.59 (d, 1.8)	124.0, d	14	7.09 (d, 1.9)	126.5, d	14		160.9, s
15	3.04 (overlap)	34.0, d	15	2.89 (m)	33.7, d	15	3.32 (m)	26.2, d
16	1.34 (d, 6.9)	24.0, q	16	1.26 (d, 7.0)	24.0, q	16	1.22 (d, 6.9)	22.5, q
17	1.33 (d, 6.9)	24.0, q	17	1.25 (d, 7.0)	24.0, q	17	1.21 (d, 6.9)	22.3, q
18	2.16 (s)	30.2, q	18	1.90 (s)	10.7, q	18	1.33 (s)	16.7, q
19			19			19		177.9, s
20	2.48 (s)	20.2, q	20	1.39 (s)	30.1, q	20	1.24 (s)	22.3, q
						21	3.66 (s)	52.4, q

Figure 1

Key COSY and HMBCs of 1.

Compound 2, obtained as a yellow gum, has a molecular formula C₁₉H₂₂O deduced from its HRESIMS with a pseudo-molecular ion peak at m/z 267.1741 [M + H]⁺ (calculated for C₁₉H₂₂O, 267.1743), ¹³C NMR, and DEPT spectra. The ¹H NMR spectrum of 2 (Table 1) shows signals assignable to 4 methyl groups [δ _H 1.90 (s), 1.39 (s) 1.26 (d, J = 7.0 Hz, H₃-16) , and 1.25 (d, J = 7.0 Hz, H₃-17)] and 5 olefinic methines [δ _H 7.30 (d, J = 8.0 Hz), 7.15 (dd, J = 8.0, 1.9 Hz), 7.09 (d, J = 1.9 Hz), 6.76 (d, J = 9.8 Hz), 6.66 (d, J = 9.8 Hz)]. The ¹³C NMR and DEPT spectra (Table 1) display 19 carbons including 4 methyl, 2 methylene, 6 methine groups (5 olefinic and 1 aliphatic), and 7 carbons (1 keto-carbonyls, 5 olefinic, and 1 quaternary). Analyses of NMR data of 2 and 12 reveal that 2 is an analog of 12, based on the close similarity of their NMR data with the exception of the carboncarbon single bond (δ _C 27.0, C-6, δ _C 27.5, C-7) in 12 replacing the carboncarbon double bond (δ_C 123.7, C-6, δ _C 133.5, C-7) in 2. This conclusion is supported by ¹H-¹H COSY correlation of H-6 (δ _H 6.66)/H-7 (δ _H 6.76) and HMBCs of H-6, H-7/C-8 (δ _C 130.8). Thus, the structure of 2 was determined as depicted (Figure 2).

Figure 2

Chemical structures of compounds 1 to 13.

Compound 3 was obtained as a pale yellow solid. Its molecular formula was determined as C₂₁H₂₈O₄ on the basis of its HRESIMS m/z 345.2057 [M + H]⁺ (calcd for C₂₁H₂₈O₄, 345.2060), ¹³C NMR, and DEPT spectra. The ¹H NMR spectrum of 3 (Table 1) gives signals for 5 methyl groups [δ _H 3.66 (s), 1.33 (s), 1.24 (s), 1.22 (d, J = 6.9 Hz, H₃-16) and 1.21 (d, J = 6.9 Hz, H₃-17)] and two olefinic methine [δ _H 7.36 (d, J = 8.0 Hz) and 6.77 (d, J = 8.0 Hz)]. The ¹³C NMR and DEPT spectra (Table 1) display 21 carbons including 5 methyl, 4 methylene, 4 methine groups (2 olefinic and 2 aliphatic), and 8 carbons (2 keto-carbonyls, 4 olefinic, and 2 quaternary). Analyses of NMR data of 3 and 11 reveal that 3 is an analog of 11. The difference is that an additional hydroxyl at C-14 (δ_C 160.9) in 3 is observed, which is supported by the HMBCs of H-11, H-12/C-14 (δ _C 160.9). The structure of compound 3 was thus deduced.

Of note, both 2 and 3 were previously synthesized. ^8,9 However, they, as new naturally occurring products, were isolated from natural origins for the first time. Their NMR data were also unambiguously assigned by 2D NMR experiments for the first time. In addition, 10 known analogs were identified as 18-norabieta-8,11,13-4-ol (4), ¹⁰ 6,8,11,13-abiteratrien-18-oic acid (5), ¹¹ 19-nor-abieta-4(18),8,11,13-tetraen-7-one (6), ¹² abietane ester (7), ¹³ dehydroabietic acid (8), ¹⁴ 7-oxodehydroabietic acid (9), ¹¹ 7α-hydroxydehydroabietic acid (10), ¹⁵ 7-oxoabieta-8,11,13-trien-18-oate (11), ¹⁶ 4,4a,9,10-tetrahydro-1,4a-dimethyl-7-isopropyl-2(3H)-phenanthrone (12), ¹⁷ and methyl-13-acetyl-podocarpa-8,11,13-trien-18-oate (13), ¹⁸ respectively, by comparison their spectroscopic data with the literature. All the known compounds were isolated from the resins of P. euphratica for the first time.

In this study, compounds 1 and 2 were examined for their cytotoxic activities against human cancer cells including Kyse30, HepG2, A549, BGC-823, and MDA-MB231. The results showed that compounds 1 and 2 and 5-fluorouracil (5-FU) significantly inhibit the growth of 5 human cancer cell lines in a dose-dependent manner (Figure S1Figure S1). We found that the cytotoxicity of compound 2 is much better than that of 5-FU in all cancer cell lines (Table S1). On the other hand, the IC₅₀ values, which are higher in normal human umbilical vein endothelial cells than in the cancer cells, suggesting that these compounds exert acceptable cytotoxicity in normal cells and high selectivity in cancer cells.

Experimental

General

UV spectrum was recorded on a UV/VIS spectrophotometer. NMR spectra were recorded on a Bruker Avance III 600 MHz spectrometer, with TMS as an internal standard. ESIMS and HRESIMS were measured on an Agilent 6230 TOF MS spectrometer. C-18 silica gel (40-60 µm; Daiso Co., Japan), MCI gel CHP 20P (75-150 µm, Mitsubishi Chemical Industries, Tokyo, Japan), Sephadex LH-20 (Amersham Pharmacia, Uppsala, Sweden), and silica gel (200-300 mesh; Qingdao Marine Chemical Inc., China) were used for column chromatography. Semipreparative HPLC was carried out using an Agilent 1200 liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA). The column used was a YMC-Pack ODS-A 250 mm × 10 mm, i.d., 5 µm.

Plant Resins

The medicinal materials of P. diversifolia were obtained from Bayin, Xinjiang Autonomous Region, in November, 2011, where the trees of P. euphratica were grown. The material was secreted from P. euphratica and a voucher specimen (CHYX0573) is deposited at School of Pharmaceutical Sciences, Shenzhen University, China.

Extraction and Isolation

The dried resins (50 kg) were soaked with 95% EtOH (300 L × 3 × 24 h) to give a crude extract, which was extracted with EtOAc to afford an EtOAc soluble extract (12.0 kg). This extract was divided into eight fractions (Fr.A-Fr.H) by using a silica gel column eluted with petroleum ether–acetone (50:1, 35:1, 20:1, 15:1, 10:1, 7:1, 3:1, 1:1). Fr.A (640.0 g) was separated via MCI gel CHP 20P eluted with aqueous MeOH (50%-100%) to provide nine portions (Fr.A.1-Fr.A.9). Among them, Fr.A.3 (30.0 g) was subjected to a RP-18 column eluted with aqueous MeOH (50%-100%) to provide three portions (Fr.A.3.1-Fr.A.3.3). Fr.A.3.2 (26.0 g) was further separated via MCI gel CHP 20P eluted with aqueous MeOH (50%-100%) to yield nine fractions (Fr.A.3.2.1-Fr.A.3.2.9). Fr.A.3.2.6 (2.6 g) was passed through Sephadex LH-20 (MeOH) to yield two fractions (Fr.A.3.2.6.1 and Fr.A.3.2.6.2). Fr.A.3.2.6.1 (1.1 g) was divided into 5 fractions (Fr.A.3.2.6.1.1- Fr.A.3.2.6.1.5) by using silica gel chromatography (petroleum ether–acetone, 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 1:1). Fr.A.3.2.6.1.3 (245.7 mg) was purified by semipreparative HPLC (aqueous MeOH, 90%) to give 4 portions. Fr.A.3.2.6.1.3.1 (8.0 mg) was separated by RP-HPLC to produce compound 4 (1.2 mg, t _R = 11.3 min, flow rate: 3 mL/min). Fr.A.3.2.8 (1.1 g) was submitted to Sephadex LH-20 (MeOH) to provide three portions (Fr.A.3.2.8.1-Fr.A.3.2.8.3). Fr.A.3.2.8.3 (580.0 mg) was purified by semipreparative HPLC (aqueous MeOH, 90%) to give two portions. Compound 5 (2.8 mg, t _R = 17.0 min, flow rate: 3 mL/min) was purified from Fr.A.3.2.8.3.1 (3.2 mg) by semipreparative HPLC (aqueous MeCN, 80%). Fr.A.7 (60.0 g) was separated via an RP-18 column eluted with gradient aqueous MeOH (60%-100%) to yield 13 fractions (Fr.A.7.1-Fr.A.7.13). Fr.A.7.7 (1.2 g) was passed through Sephadex LH-20 (MeOH) to give 4 portions. Fr.A.7.7.3 (191.0 mg) was subjected to preparative TLC developed with petroleum ether–acetone (15:1) to afford eight portions (Fr.A.7.7.3.1-Fr.A.7.7.3.8). From Fr.A.7.7.3.2 (44.7 mg), compound 6 (5.2 mg, t _R = 19.5 min, flow rate: 3 mL/min) was obtained by HPLC separation (aqueous MeOH, 88%). Further purification of Fr.A.7.7.3.4 (26.7 mg) by semipreparative HPLC eluted with aqueous MeOH (88%) afforded compound 3 (7.2 mg, t _R = 22.0 min, flow rate: 3 mL/min). Compound 1 (2.6 mg, t _R = 18.6 min) was obtained from Fr.A.7.7.3.6 (15.8 mg) by HPLC separation (aqueous MeCN, 80%, flow rate: 3 mL/min). Fr.A.7.9 (393.0 mg) was submitted to Sephadex LH-20 (MeOH) to yield two fractions (Fr.A.7.9.1 and Fr.A.7.9.2), of which, Fr.A.7.9.2 (31.7 mg) was purified by semipreparative HPLC (aqueous MeCN, 93%) to yield compound 7 (27.2 mg, t _R = 17.1 min, flow rate: 3 mL/min). Fr.A.4 (180.0 g) was separated via RP-18 eluted with aqueous MeOH (65%-100%) to provide nine portions (Fr.A.4.1-Fr.A.4.9). Compound 8 (3.0 g) was obtained from Fr.A.4.3 (10.1 g) via MCI gel CHP 20P eluted with aqueous MeOH (50%-100%). Fr.A.4.4 (11.8 g) was subjected to an RP-18 column washed with aqueous MeOH (60%-100%) to provide 4 portions (Fr.A.4.4.1-Fr.A.4.4.4). Fr.A.4.4.1 (221.0 mg) was separated by semipreparative HPLC (aqueous MeOH, 80%) to give 5 portions. Fr.A.4.4.1.5 (50.0 mg) was further separated by HPLC (aqueous MeCN, 65%) to yield 9 (4.5 mg, t _R = 18.1 min, flow rate: 3 mL/min) and 10 (24.0 mg, t _R = 16.0 min). Separation of Fr.A.4.4.1.6 (11.0 mg) by HPLC (aqueous MeCN, 80%) afforded 11 (4.7 mg, t _R = 17.9 min, flow rate: 3 mL/min). Fr.A.4.1 (4.8 g) was submitted to Sephadex LH-20 (MeOH) to yield three fractions (Fr.A.4.1.1-Fr.A.4.1.3). Fr.A.4.1.3 (2.6 g) was further divided into 4 parts (Fr.A.4.1.3.1-Fr.A.4.1.3.4) by a silica gel column washed with increasing acetone in petroleum ether (100:1, 60:1, 30:1, 15:1, 5:1 1:1). HPLC separation of Fr.A.4.1.3.3 (30 mg) by aqueous MeOH (82%) yielded 13 (5.3 mg, t _R = 16.8 min, flow rate: 3 mL/min). Fr.A.5 (136.0 g) was subjected to an RP-18 column washed with aqueous MeOH (70%-100%) to provide nine portions (Fr.A.5.1-Fr.A.5.9). Fr.A.5.2 (1.5 g) was passed through Sephadex LH-20 (MeOH) to yield 4 fractions (Fr.A.5.2.1-Fr.A.5.2.4). Further purification of Fr.A.5.2.2 (415.5 mg) by semipreparative HPLC eluted with aqueous MeCN (72%) afforded compounds 2 (10.4 mg, t _R = 24.7 min, flow rate: 3 mL/min) and 12 (4.2 mg, t _R = 25.6 min, flow rate: 3 mL/min).

Compound 1

Colorless gum.

UV (MeOH) λ _max (log ε): 230 (5.03), 280 (3.91), 325 (3.34) nm.

¹H and ¹³C NMR: Table 1.

ESIMS m/z: 269 [M + H]⁺.

HRESIMS m/z 269.1902 [M + H]⁺ (calculated for C₁₉H₂₄O, 269.1900).

Biological Evaluation

Compounds 1 and 2 were evaluated for their inhibition on cancer cell proliferation. In this assay, human cancer cells (Kyse30, HepG2, A549, BGC-823, and MDA-MB231) derived from the stomach, liver, breast, lung, and esophagus were used. All cells were exposed to various concentrations of compounds (0-80 µM) for 48 hours, and cell viability was quantified by CCK-8 assay.