kaurane Diterpenoids from the Aerial Parts of - ACS Publications

Article Cite This: J. Nat. Prod. 2018, 81, 106−116

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7α,20-Epoxy-ent-kaurane Diterpenoids from the Aerial Parts of Isodon pharicus Zheng-Xi Hu,†,‡ Miao Liu,† Wei-Guang Wang,† Xiao-Nian Li,† Kun Hu,† Xing-Ren Li,† Xue Du,† Yong-Hui Zhang,*,‡ Pema-Tenzin Puno,*,† and Han-Dong Sun† †

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China ‡ Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China S Supporting Information *

ABSTRACT: A phytochemical investigation of an ethyl acetate extract of the aerial parts of Isodon pharicus led to the isolation of 21 new 7α,20-epoxy-ent-kaurane diterpenoids, pharicins C−W (1−21), and 29 known (22−50) analogues. The structural characterization of 1−21 and assignment of their relative configurations were accomplished by spectroscopic data interpretation, while the structures of 1 and 16 were confirmed by X-ray crystallography. The absolute stereostructure of 1 was confirmed by electronic circular dichroism data analysis. Twenty-five of the diterpenoids were screened for their cytotoxic activities against a panel of tumor cell lines, including HL-60, SMMC-7721, A-549, MCF-7, and SW-480. Compounds 11, 16, 38, and 48 exhibited inhibitory activities against these tumor cell lines with IC50 values ranging from 1.01 to 9.62 μM, while 2, 15, 29, and 47 exhibited moderate cytotoxic potency.

A

high elevation of about 4200 m was performed. As a result, a total of 50 7α,20-epoxy-ent-kaurane diterpenoids, including 21 new (1−21) and 29 known analogues, namely, oridonin (22),5 lasiokaurin (23),6 effusanin A (24),7 parvifoline I (25),8 parvifoline H (26),8 effusanin C (27),7 sodoponin (28),9 taibaihenryiin A (29),10 effusanin E (30),7 longikaurin C (31),11 lasiodonin (32),8 enmenol (33),12 rosthorin A (34),13 longikaurin B (35),14 longikaurin A (36),15 effusanin B (37),7 effusanin D (38),7 maoecrystal E (39),16 rubescensin C (40),17 isoadenolin E (41),18 xerophilusin XII (42),19 isoadenolin G (43),18 rabdoternin E (44),20 parvifoline J (45),8 adenolin E (46),21 nervosanin A (47),22 rabdocoetsin A (48),23 rabdoternin A (49),24 and rabdoternin B (50),24 were isolated from an EtOAc-soluble extract. In this report, the isolation, structure characterization, and bioactivity evaluation of these compounds are discussed.

s one of the largest genera in the Labiatae family, Isodon has approximately 150 species and is distributed throughout the world.1 During the past four decades, a series of phytochemical investigations on this genus have demonstrated that ent-kauranoids are the characteristic and main secondary metabolites exhibiting a broad spectrum of bioactivities, such as antibacterial, anti-inflammatory, and antitumor properties.1 Isodon pharicus (Prain) Hara, a perennial herb that is primarily distributed throughout the southern district of the Tibetan Region and the northwest district of Sichuan Province in China, has been commonly applied in folk medicine for deinsectization and treatment of inflammation of the eyes.2a Previously, our research group has performed a systematic chemical investigation on this plant growing in the Lhasa area of the Tibetan Autonomous Region, China, and several entkaurane diterpenoids with remarkable pharmacological effects were discovered.2 For instance, pharicin A affected the mitotic progression of several leukemia and solid-tumor-derived cells and induced mitotic arrest (G2/M) and apoptosis;3 pharicin B stabilized the RAR-α protein and enhanced all-trans retinoic acid for the differentiation of some acute myelocytic leukemia cell lines, particularly several primary leukemic cells in acute promyelocytic leukemia patients.4 In order to identify additional biologically active diterpenoids with extraordinary structural features, a chemical investigation of the aerial parts of I. pharicus distributed at a © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Pharicin C (1), obtained as colorless needles from MeOH, had a molecular formula of C22H32O7, according to the HRESIMS analysis (m/z 453.2131, [M + COOH]−, calcd for 453.2130) and 13C NMR data, which was indicative of seven indices of hydrogen deficiency. Its 1H NMR data (Table 1) showed signals of two methyl groups [δH 1.35 (H3-18) and 1.97 Received: August 23, 2017 Published: December 29, 2017 106

DOI: 10.1021/acs.jnatprod.7b00723 J. Nat. Prod. 2018, 81, 106−116

Journal of Natural Products

Article

Chart 1

group was located at C-19 (δC 67.2) based on the HMBC correlations from H2-19 (δH 4.50 and 4.70) to C-3 (δC 36.4), C-4 (δC 38.0), C-5 (δC 59.5), C-18 (δC 27.7), and the acetoxy carbonyl (δC 171.3). Thus, the 2D structure of 1 was determined. By repeated crystallization from MeOH at room temperature, 1 afforded a crystal suitable for X-ray diffraction crystallographic analysis (Figure 2). However, the crystallographic data [Flack parameter of 0.2(5)] only enabled us to determine its relative configuration. To define its absolute configuration, the DFT-calculated electronic circular dichroism (ECD) spectrum of (4R,5S,6S,7S,8S,9S,10R,12S,13R,15R)-1 obtained at the B3LYP/6-31G(d,p) level was compared with the experimental ECD spectrum (Figure 3) and confirmed the (4R, 5S, 6S, 7S, 8S, 9S, 10R, 12S, 13R, 15R) absolute configuration. Thus, the structure of pharicin C (1) was defined as 19-acetoxy-6β,7β,12α,15β-tetrahydroxy-7α,20epoxy-ent-kaur-16-ene.

(OAc)], two olefinic protons [δH 5.32 and 5.64 (both br s, H217)], and three oxymethine protons [δH 4.44 (br d, J = 5.7 Hz, H-6), 4.30 (m, H-12), and 5.20 (s, H-15)]. Except for one acetoxy group (δC 171.3 and 21.2), the remaining 20 carbon resonances were attributed to one methyl, eight methylenes (including two oxygen-bearing and one olefinic carbon), six methines (including three oxygen-bearing carbons), and five nonprotonated carbons (including one olefinic and one oxygen-bearing carbon). The aforementioned data suggested that 1 was assigned as a 7α,20-epoxy-ent-kaurane diterpenoid with the characteristic exocyclic double bond and an acetoxy group. Carbons 6, 7, 12, and 15 were substituted with hydroxy groups, as supported by the 2D NMR data (Figure 1), i.e., the 1 H−1H COSY correlations of H-5 (δH 1.85)/H-6 (δH 4.44) and H-9 (δH 2.35)/H2-11 (δH 1.85 and 2.07)/H-12 (δH 4.30) and HMBC correlations from H2-20 (δH 4.08 and 4.21) to C-7 (δC 97.9) and from H-15 (δH 5.20) to C-7, C-8 (δC 52.9), C-9 (δC 38.9), C-16 (δC 157.7), and C-17 (δC 109.7). An acetoxy 107



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Table 1. 1H NMR Spectroscopic Data of Compounds 1−7 (δ in ppm, J in Hz) no.

1a,b

2a,b

3a,b

4a,b

5a,b

6a,b

7a,b

3.76, m 1.96, m; 2.10, m 1.29, m; 2.38, m

1.14, m; 1.31, m 1.91, m; 1.97, m 3.75, m

1.00, m; 1.34, m 1.37, m; 1.50, m 1.05, m; 2.34, m

1.72, m 4.58, d (6.4, 9.9)

1.26, 1.56, 3.20, 5.15,

1.71, d (6.7) 4.61, dd (6.7, 10.0) 1.75, dd (6.4, 13.1) 2.04, m; 2.10, m 4.30, m 3.60, br d (17.9) 5.57, s

1 2 3

1.03, m; 1.41, m 1.33, m; 1.38, m 1.01, m; 1.87, m

1.11, m; 1.29, m 1.72, m; 1.80, m 3.67, m

3.73, m 1.94, m 1.27, m; 1.89, m

5 6

1.85, m 4.44, br d (5.7)

1.73, d (5.6) 4.69, dd (6.1, 10.2)

1.74, d (7.0) 4.45, m

1.12, m; 1.27, m 1.72, m; 1.80, m 3.66, dd (3.8, 11.9) 1.71, m 4.69, d (6.4, 9.9)

9

2.35, dd (5.7, 13.1)

1.55, m

1.98, m

1.72, m

1.79, d (7.3) 4.61, dd (7.4, 10.6) 2.03, m

11 12 13 14

1.85, m; 2.07, m 4.30, m 3.07, br d (4.5) 2.28, dd (4.6, 12.2); 2.42, br d (12.2) 5.20, s 5.32, br s; 5.64, br s

1.23, m; 1.58, m 1.33, m; 2.16, m 2.92, dd (4.2, 9.5) 2.29, br d (12.3); 2.48, dd (6.5, 12.3)

1.98, 1.58, 3.22, 5.32,

1.24, 1.55, 3.20, 5.15,

2.03, 1.65, 3.24, 5.35,

5.32, br s; 6.01, br s

1.35, s 4.50, d (11.1); 4.70, d (11.1) 4.08, d (9.9); 4.21, d (9.9) 1.97, s

1.78, s 4.79, d (11.8); 5.06, d (11.8) 3.99, d (10.2); 4.20, d (10.2) 1.75, s

5.53, br s; 6.30, br s 1.46, s 4.49, d (11.6); 4.87, d (11.6) 4.43, m; 4.85, m

15 17 18 19 20 OAc

m; 2.48, m m; 2.44, m br d (9.3) br s

1.98, s

m; 1.68, m m; 2.37, m br d (9.6) s

5.53, br s; 6.30, br s 1.77, s 4.82, d (11.8); 5.05, d (11.8) 4.03, d (10.1); 4.31, d (10.1) 1.81, s

m; 2.49, m m; 2.49, m br d (8.6) s

5.57, br s; 6.33, br s 1.67, s 4.13, d (10.4); 4.44, d (10.4) 4.57, d (10.2); 4.87, d (10.2)

1.73, m m; 1.68, m m; 2.39, m br d (9.6) s

5.54, br s; 6.30, br s 1.91 s 4.28, d (11.0); 4.74, d (11.0) 4.05, d (10.0); 4.36, d (10.0)

5.60, br s; 6.41, br s 1.63, s 4.09, m; 4.28, m 4.12, d (10.1); 4.33, d (10.1)

Recorded at 500 MHz in pyridine-d5. b“m” means overlapped or multiplet with other signals.

a

Pharicin D (2), purified as a white, amorphous powder, had a molecular formula of C22H30O7, on the basis of its HRESIMS data at m/z 429.1883 ([M + Na]+, calcd for 429.1884). The 1D NMR data (Tables 1 and 4) showed close similarities to those of the known compound longikaurin C (31), except for an sp3 methylene in 31 being replaced by an oxymethine (δC 77.8) in 2. A hydroxy group was located at C-3, based on the HMBC correlations from H3-18 (δH 1.78) and H2-19 (δH 4.79 and 5.06) to C-3 (δC 77.8), C-4 (δC 43.9), and C-5 (δC 62.5). The ROESY interactions (Figure 4) of H-3 (δH 3.67), H-5β (δH 1.73), and H3-18 (δH 1.78) and correlation between H-19 (δH 4.79) and H-20 (δH 4.20) suggested that C-3 hydroxy group was α-oriented. The similar ROESY data of 2 and 31 showed that both compounds had the same relative configurations. Thus, the structure of pharicin D (2) was defined as 19-acetoxy-3α,6β,7β-trihydroxy-7α,20-epoxy-entkaur-16-en-15-one. Pharicin E (3) was also purified as a white, amorphous powder. The HRESIMS analysis displayed a sodium adduct ion at m/z 445.1833 [M + Na]+ (calcd for 445.1833), suggesting a molecular formula of C22H30O8. The 1D NMR data (Tables 1 and 4) of 3 resembled those of the known compound longikaurin B (35), with the only distinction being

Figure 2. ORTEP drawing of compound 1.

the presence of an oxymethine carbon (δC 73.3) in 3. The H−1H COSY correlations of H-1 (δH 3.73)/H-2 (δH 1.94)/ H-3 (δH 1.27) and the HMBC correlation between H-1 and C20 (δC 65.1) showed the presence of a hydroxy group at C-1. A key ROESY correlation between H-1 and H-9β (δH 1.98) confirmed that the C-1 hydroxy group of 3 was α-oriented. Therefore, the structure of pharicin E (3) was defined as 191

Figure 1. Selected 1H−1H COSY and HMBC correlations of compounds 1, 8, and 13. 108



Article

sp3 methylene (δC 27.1, C-14) in 2 was replaced by an oxymethine (δC 74.0, C-14) in 4, which gave rise to deshielding of C-13 from δC 35.5 in 2 to δC 44.2 in 4, as supported by the HMBC correlations from H-14 (δH 5.15) to C-12 (δC 30.6), C-15 (δC 209.3), and C-16 (δC 153.4). Based upon the ROESY correlation between H-14 and H-20 (δH 4.03), the hydroxy group at C-14 was determined to be βoriented. Hence, the structure of pharicin F (4) was defined as 19-acetoxy-3α,6β,7β,14β-tetrahydroxy-7α,20-epoxy-ent-kaur16-en-15-one. Pharicin G (5) gave a molecular formula of C20H28O7, as assigned by a deprotonated molecule at m/z 379.1764 ([M − H]−, calcd for 379.1762) in the (−)-HRESIMS analysis, which was 42 mass units less than that of 3. Comparison of its 1D NMR data (Tables 1 and 4) with those of 3 indicated the replacement of a C-19 hydroxy group in 5 by a C-19 acetoxy group in 3, as supported by the key HMBC correlations between H3-18 (δH 1.67) and C-3 (δC 34.2), C-4 (δC 39.5), C5 (δC 61.9), and C-19 (δC 64.9). Similar ROESY data showed that 5 possessed the same relative configuration as 3. Therefore, the structure of pharicin G (5) was defined as 1α,6β,7β,14β,19-pentahydroxy-7α,20-epoxy-ent-kaur-16-en-15one. Pharicins H (6) and I (7) both had the molecular formula C20H28O7, based upon the HRESIMS analysis as well as the 13 C NMR and DEPT data. The 1D NMR data (Tables 1 and 4) of 6 showed a close structural resemblance to those of 4, defining that C-19 (δC 64.7) was deacetylated in 6, as supported by the HMBC cross-peaks of H-19 (δH 4.74) with

Figure 3. Comparison between experimental and calculated ECD spectra of 1.

acetoxy-1α,6β,7β,14β-tetrahydroxy-7α,20-epoxy-ent-kaur-16en-15-one. The HRESIMS ion at m/z 421.1872 ([M − H]−, calcd for 421.1868), together with the 13C NMR and DEPT data for pharicin F (4), revealed the molecular formula C22H30O8. Comparison of the 1D NMR data (Tables 1 and 4) of 4 with those of 2 suggested that these two substances featured the same carbon frameworks, with the only difference being that an

Table 2. 1H NMR Spectroscopic Data of Compounds 8−14 (δ in ppm, J in Hz) 8a,b

no.

9a,b

1

3.80, m

1.14, m; 1.28, m

2 3

1.91, m; 2.00, m 1.24, m; 1.88, m

5 6 9 11 12

1.93, d (6.3) 4.48, m 2.99, dd (6.0, 12.0) 1.92, m; 2.38, m 1.78, m; 2.44, m

1.68, m; 2.06, m 4.95, dd (3.8, 12.2) 1.80, m 5.06, m 1.73, m

13 14

2.90, br d (8.9) 5.15, d (2.1)

15 17

5.77, m 5.38, br s; 5.70, br s 1.40, s 4.54, d (11.2); 4.98, d (11.2) 4.45, d (10.1); 4.92, d (10.1)

18 19 20 1-OAc 3-OAc 6-OAc 11-OAc 19-OAc OCH2CH3 15-OH

10a,b

11a,b

12a,b

13a,b

14a,b

5.56, dd (5.2, 11.7) 1.67, m; 2.30, m 1.24, m; 1.35, m

1.70, m; 2.39, m

1.70, m; 2.41, m

1.53, m; 2.12, m

1.63, m; 2.36, m

1.39, m; 1.57, m 1.03, m; 1.74, m

1.42, m; 1.61, m 1.06, m; 1.84, m

1.39, m; 1.51, m 1.18, m; 1.42, m

1.40, m; 1.54, m 1.20, m; 1.41, m

1.69, d (4.6) 4.26, m 2.95, m

1.88, d (5.9) 5.99, d (5.9) 3.31, br d (8.5)

1.90, m 4.43, m 3.36, br d (8.3)

1.63, d (5.2) 4.18, m 3.22, br d (8.2)

1.68, d (4.0) 4.21, d (4.0) 3.29, br d (8.5)

1.24, m; 1.69, m 1.59, m; 2.37, m

4.59, m 1.89, m; 2.94, m

6.55, m 1.73, m; 3.17, m

6.60, m 1.76, m; 3.26, m

3.19, br d (9.5) 5.19, s

2.77, dd (4.4, 9.3) 2.04, m; 2.21, dd (4.6, 12.2) 5.34, s 5.18, br s; 5.41, br s 1.09, s 1.15, s

2.85, br d (8.7) 5.90, br s

2.89, br d (7.8) 5.92, br s

6.19, m 1.73, dd (6.3, 14.0); 3.18, m 2.91, br d (7.8) 5.38, s

6.58, m 1.74, dd (6.2, 13.7); 3.24, m 2.89, br d (7.9) 5.89, s

5.66, m 5.43, br s; 5.71, br s 1.06, s 4.45, d (11.2); 4.65, d (11.2) 6.22, d (2.3)

5.90, m 5.43, br s; 5.76, br s 1.35, s 4.43, d (11.1); 4.65, d (11.1) 6.18, d (2.3)

5.83, s 5.42, br s; 5.74, br s 1.18, s 1.06, s

5.88, m 5.42, br s; 5.75, br s 1.20, s 1.04, s

5.48, s

6.16, s

2.26, s 2.10, s 1.99, s

2.09, s 1.92, s

2.04, s

2.08, s

5.55, br s; 6.33, br s 1.55, s 4.27, d (11.6); 4.40, d (11.6) 3.97, d (9.5); 4.89, 4.44, d (10.0); d (9.5) 4.71, d (10.0) 2.14, s 2.04, s

1.97, s

3.49, m; 3.97, m 1.12, t (7.0) 6.81, s

Recorded at 500 MHz in pyridine-d5. b“m” means overlapped or multiplet with other signals.

a

109



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Table 3. 1H NMR Spectroscopic Data of Compounds 15−21 (δ in ppm, J in Hz) 15a,c

no.

16b,c

17b,c

18b,c 0.99, m; 1.34, m 1.35, m; 1.48, m 1.02, m; 2.32, m 1.68, m 4.59, dd (7.1, 9.7) 1.82, dd (5.6, 13.5) 2.02, m; 2.15, m 4.40, m 3.05, m

3.76, m

3.78, m

1.95, m; 2.09, m

1.95, m; 2.11, m

1.27, m; 2.37, m

1.24, m; 2.39, m

1.78, d (7.7) 4.62, dd (8.1, 10.5)

1.79, d (6.6) 4.57, m

2.05, m

2.06, m

2.14, m; 2.41, m

1.92, m; 2.55, m

1.66, m; 2.42, m 2.76, br d (8.2)

1.86, m; 2.07, m 2.91, m

5.55, s

5.27, s

5.44, s

2.93, m 3.98, m

3.76, m 3.82, m; 3.87, m

1

3.77, m

4.85, dd (5.4, 11.5)

4.85, dd (5.4, 11.5)

2

1.92, m; 1.99, m

1.53, m; 1.83, m

1.53, m; 1.81, m

3

1.24, m; 1.89, m

1.31, m; 1.34, m

1.33, m; 1.34, m

5 6

1.72, d (5.8) 4.38, m

1.49, d (5.4) 4.14, dd (5.7, 11.2)

1.51, m 4.19, dd (6.3, 10.6)

9

1.75, dd (6.0, 12.1)

1.66, dd (5.8, 12.3)

1.68, dd (5.7, 12.3)

11

1.86, m; 2.49, m

1.21, m; 1.99, m

1.28, m; 2.04, m

12 13

1.48, m; 1.86, m 2.71, m

1.37, m; 1.78, m 2.70, m

1.17, m; 2.13, m 2.55, m

14

2.52, br s

16 17

2.96, m 3.68, dd (9.5, 10.3); 3.78, dd (4.6, 10.3) 1.39, s 4.45, d (11.2); 4.87, d (11.2) 4.36, d (10.5); 4.77, d (10.5) 1.95, s 3.26, s

2.40, br d (12.4); 2.49, dd (3.3, 12.4) 2.92, m 3.64, m; 3.76, dd (4.7, 10.3) 1.18, s 1.06, s

2.34, br d (12.8); 2.76, dd (4.0, 12.8) 2.56, m 3.49, dd (5.0, 9.5); 3.55, m 1.22, s 1.06, s

18 19 20 OAc OCH3

19b,c

3.07, m 3.96, m; 4.02, dd (4.8, 9.1) 1.62, s 4.08, m; 4.26, d (10.9) 4.26, d (10.2); 4.43, 4.27, d (10.1); 4.47, 4.07, m; 4.33, d (10.2) d (10.1) d (9.9) 2.08, s 2.08, s 3.25, s 3.17, s 3.23, s

20b,c

21b,c 0.96, m; 1.32, m 1.33, m; 1.35, m 1.01, m; 1.85, m 1.62, d (6.6) 4.38, m 1.80, dd (5.9, 13.4) 2.00, m; 2.13, m 4.40, m 3.01, dd (4.8, 9.6) 5.51, s

3.06, br s 3.94, m; 4.01, dd (4.9, 9.1) 1.67, s 1.65, s 1.40, s 4.13, dd (4.7, 10.4); 4.14, dd (5.0, 10.5); 4.44, d (11.1); 4.42, dd (4.3, 10.4) 4.47, dd (4.7, 10.5) 4.65, d (11.1) 4.57, d (10.0); 4.86, d 4.57, d (10.1); 4.86, d 4.10, d (10.3); (10.0) (10.1) 4.15, d (10.3) 1.98, s 3.28, s 3.29, s 3.23, s

Recorded at 600 MHz in pyridine-d5. bRecorded at 500 MHz in pyridine-d5. c“m” means overlapped or multiplet with other signals.

a

COSY cross-peaks of H-9 (δH 1.75)/H2-11 (δH 2.04 and 2.10)/H-12 (δH 4.30) and HMBC correlations of H-12 with C-16 (δC 149.3). The ROESY correlations (Figure 4) of H-20 (δH 4.12)/H-11α (δH 2.04)/H-14 (δH 5.57) and H-9/H-11β (δH 2.10)/H-12 indicated that the 14-hydroxy group was β-

C-3 (δC 80.1), C-4 (δC 43.7), C-5 (δC 62.2), and C-18 (δC 24.4). Comparison of the 1H, 13C, and 2D NMR data of 6 and 7 suggested that 6 had a C-3 hydroxy group, as supported by the HMBC correlations of H3-18 (δH 1.91) with C-3 (δC 80.1), while 7 had a C-12 hydroxy group, as supported by the 1H−1H Table 4.

13

C NMR Spectroscopic Data of Compounds 1−10 (δ in ppm)

no.

1a

2a

3a

4a

5a

6a

7a

8a

9a

10a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OAc

31.1, CH2 19.1, CH2 36.4, CH2 38.0, C 59.5, CH 73.9, CH 97.9, C 52.9, C 38.9, CH 36.7, C 28.1, CH2 76.8, CH 49.1, CH 25.4, CH2 76.0, CH 157.7, C 109.7, CH2 27.7, CH3 67.2, CH2 66.8, CH2 171.3, C 21.2, CH3

29.0, CH2 28.6, CH2 77.8, CH 43.9, C 62.5, CH 74.6, CH 96.6, C 60.6, C 50.5, CH 36.9, C 17.3, CH2 30.0, CH2 35.5, CH 27.1, CH2 210.9, C 154.4, C 117.0, CH2 24.4, CH3 65.6, CH2 66.5, CH2 171.3, C 21.2, CH3

73.3, CH 30.5, CH2 34.3, CH2 38.1, C 61.8, CH 74.2, CH 99.0, C 63.3, C 54.4, CH 42.3, C 20.9, CH2 31.3, CH2 44.4, CH 74.0, CH 209.8, C 153.7, C 119.8, CH2 27.9, CH3 67.0, CH2 65.1, CH2 171.4, C 21.2, CH3

29.5, CH2 28.6, CH2 77.6, CH 43.8, C 61.7, CH 74.5, CH 99.0, C 63.0, C 52.5, CH 36.8, C 17.3, CH2 30.6, CH2 44.2, CH 74.0, CH 209.3, C 153.4, C 120.2, CH2 24.6, CH3 65.6, CH2 66.8, CH2 171.3, C 21.3, CH3

73.9, CH 30.9, CH2 34.2, CH2 39.5, C 61.9, CH 74.0, CH 99.2, C 63.5, C 55.0, CH 42.5, C 21.1, CH2 31.5, CH2 44.5, CH 74.1, CH 209.8, C 153.9, C 119.9, CH2 28.6, CH3 64.9, CH2 65.3, CH2

29.8, CH2 29.1, CH2 80.1, CH 43.7, C 62.2, CH 73.8, CH 99.0, C 63.1, C 52.6, CH 36.9, C 17.5, CH2 30.6, CH2 44.3, CH 74.1, CH 209.2, C 153.4, C 120.1, CH2 24.4, CH3 64.7, CH2 67.4, CH2

31.5, CH2 19.4, CH2 36.3, CH2 39.4, C 62.0, CH 73.6, CH 99.0, C 63.2, C 49.8, CH 37.1, C 29.0, CH2 74.7, CH 56.4, CH 72.6, CH 208.6, C 149.3, C 122.0, CH2 28.7, CH3 65.0, CH2 67.6, CH2

73.6, CH 30.7, CH2 34.1, CH2 38.1, C 58.8, CH 73.1, CH 100.4, C 54.0, C 45.7, CH 41.8, C 19.0, CH2 33.6, CH2 46.9, CH 76.6, CH 73.6, CH 161.8, C 109.5, CH2 27.4, CH3 66.9, CH2 64.7, CH2 171.3, C 21.1, CH3

29.7, CH2 25.1, CH2 80.6, CH 43.4, C 61.7, CH 74.1, CH 98.9, C 63.1, C 53.0, CH 36.6, C 17.6, CH2 30.7, CH2 44.2, CH 73.8, CH 209.0, C 153.5, C 120.2, CH2 25.0, CH3 63.0, CH2 67.1, CH2 171.1, C 21.5, CH3

77.3, CH 25.2, CH2 39.1, CH2 34.4, C 59.5, CH 74.9, CH 97.7, C 53.7, C 50.8, CH 41.1, C 62.8, CH 44.4, CH2 38.3, CH 28.7, CH2 75.4, CH 162.3, C 106.9, CH2 33.0, CH3 22.7, CH3 64.7, CH2 170.8, C 22.3, CH3

a

Recorded at 125 MHz in pyridine-d5. 110



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Figure 4. Selected ROESY correlations of compounds 2, 7, and 17.

Table 5.

13

C NMR Spectroscopic Data of Compounds 11−21 (δ in ppm) 11b

12b

13b

14b

15a

16b

17b

18b

19b

20b

21b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1-OAc

30.1, CH2 18.6, CH2 36.2, CH2 38.0, C 56.1, CH 73.7, CH 99.9, C 54.2, C 47.9, CH 40.9, C 68.8, CH 40.4, CH2 46.4, CH 77.0, CH 73.4, CH 160.1, C 111.0, CH2 27.1, CH3 67.3, CH2 96.4, CH




73.1, CH 30.4, CH2 34.2, CH2 38.2, C 63.2, CH 74.2, CH 96.2, C 61.6, C 52.3, CH 42.0, C 20.4, CH2 20.5, CH2 30.1, CH 29.3, CH2 225.0, C 57.9, CH 69.4, CH2 27.2, CH3 66.5, CH2 65.2, CH2

76.2, CH 25.8, CH2 38.9, CH2 34.3, C 62.5, CH 74.8, CH 96.0, C 61.2, C 51.3, CH 40.2, C 18.5, CH2 20.3, CH2 30.0, CH 29.0, CH2 224.6, C 57.9, CH 69.3, CH2 33.1, CH3 22.1, CH3 63.9, CH2 170.5, C 21.9, CH3

76.4, CH 25.9, CH2 39.0, CH2 34.3, C 61.9, CH 75.0, CH 96.0, C 61.0, C 51.0, CH 40.3, C 19.0, CH2 29.8, CH2 30.6, CH 27.2, CH2 224.7, C 60.7, CH 71.9, CH2 33.3, CH3 22.3, CH3 63.8, CH2 170.5, C 21.9, CH3

31.8, CH2 19.4, CH2 36.4, CH2 39.3, C 61.4, CH 73.6, CH 98.7, C 63.6, C 49.2, CH 36.9, C 28.6, CH2 74.1, CH 53.2, CH 74.4, CH 220.5, C 50.8, CH 75.0, CH2 28.9, CH3 65.1, CH2 67.6, CH2

74.0, CH 30.9, CH2 34.3, CH2 39.2, C 61.3, CH 74.0, CH 98.9, C 64.1, C 54.1, CH 42.3, C 20.9, CH2 31.2, CH2 39.4, CH 76.0, CH 222.0, C 58.2, CH 75.5, CH2 28.9, CH3 65.0, CH2 65.1, CH2

73.5, CH 30.8, CH2 34.0, CH2 39.6, C 62.5, CH 73.8, CH 98.7, C 63.4, C 54.0, CH 42.1, C 20.0, CH2 21.1, CH2 38.3, CH 74.8, CH 222.7, C 52.5, CH 69.4, CH2 28.1, CH3 64.4, CH2 65.3, CH2

31.2, CH2 19.0, CH2 36.4, CH2 37.9, C 61.4, CH 74.0, CH 98.6, C 63.5, C 48.7, CH 36.8, C 28.5, CH2 73.8, CH 53.2, CH 74.4, CH 220.6, C 50.8, CH 74.9, CH2 28.1, CH3 67.2, CH2 67.2, CH2

6-OAc

169.6, C 21.8, CH3 170.7, C 22.0, CH3 171.1, C 21.1, CH3

170.8, C 22.1, CH3 171.2, C 21.1, CH3

170.7, C 22.0, CH3

170.9, C 22.1, CH3

59.1, CH3

171.2, C 21.1, CH3 58.7, CH3

no.

11-OAc 19-OAc OCH3 OCH2CH3

171.3, C 21.2, CH3 59.1, CH3

59.1, CH3

59.0, CH3

58.7, CH3

58.7, CH3

64.4, CH2 15.8, CH3

a

Recorded at 150 MHz in pyridine-d5. bRecorded at 125 MHz in pyridine-d5.

oriented, whereas the hydroxy group at C-12 was α-oriented in 7. Thus, the structures of compounds 6 and 7 were defined as 3α,6β,7β,14β,19-pentahydroxy-7α,20-epoxy-ent-kaur-16-en-15one and 6β,7β,12α,14β,19-pentahydroxy-7α,20-epoxy-entkaur-16-en-15-one, respectively. Pharicin J (8) was obtained as a white, amorphous powder, and its molecular formula C22H32O8 (seven indices of hydrogen deficiency) was deduced based upon the HRESIMS analysis (m/z 469.2064, [M + COOH]−, calcd for 469.2079) and 13C NMR data. Comparison of the 1H and 13C NMR data (Tables 2 and 4) of 8 with those of the known enmenol (33) showed the presence of an acetoxy group and an oxymethylene (δC 66.9) in 8 and the presence of a methyl group in 33. In the

HMBC spectrum, correlations (Figure 1) from H2-19 (δH 4.54 and 4.98) to C-3 (δC 34.1), C-4 (δC 38.1), C-18 (δC 27.4), and the acetoxy carbonyl (δC 171.3), coupled with a ROESY correlation between H-19 (δH 4.54) and H-20 (δH 4.45), suggested an acetoxy group at C-19. Hence, the structure of pharicin J (8) was defined as 19-acetoxy-1α,6β,7β,14β,15βpentahydroxy-7α,20-epoxy-ent-kaur-16-ene. Pharicin K (9), purified as a white, amorphous powder, gave a molecular formula of C22H30O8, as deduced from a deprotonated molecule at m/z 421.1861 ([M − H]−, calcd for 421.1868) in the HRESIMS analysis, which was 42 mass units more than that of 6. The fact that its 1D NMR data (Tables 2 and 4) were comparable to those of 6 revealed that 111



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Pharicin P (14), obtained as a white, amorphous powder, gave a molecular formula of C22H32O8, via its (+)-HRESIMS data (m/z 447.1987, [M + Na]+, calcd for 447.1989). The 1H and 13C NMR spectra of 14 were similar to those of 12, except for the absence of a C-19 acetoxy group in 14, as inferred by the HMBC correlations from H3-19 (δH 1.04) to C-3 (δC 42.0), C-4 (δC 34.7), C-5 (δC 58.7), and C-18 (δC 34.3). Hence, the structure of pharicin P (14) was defined as 20(S*)11β-acetoxy-6β,7β,14β,15β,20-pentahydroxy-7α,20-epoxy-entkaur-16-ene. The molecular formula of pharicin Q (15) was assigned as C23H34O8, based upon the (+)-HRESIMS sodium adduct ion at m/z 461.2144 ([M + Na]+, calcd for 461.2146). The 1D and 2D NMR data (Tables 3 and 5) showed that 15 was structurally similar to enanderinanin G,25 except for the opposite C-1 configuration. A ROESY correlation between H-1 (δH 3.77) and H-5β (δH 1.72) verified the β-orientation of H-1 in 15. Thus, the structure of pharicin Q (15) was defined as 16(S*)-19-acetoxy-1α,6β,7β-trihydroxy-17-methoxy-7α,20epoxy-ent-kaur-15-one. Pharicins R (16) and S (17) showed identical molecular formulas (C23H34O7) via their HRESIMS and 13C NMR data. Their similar 1H and 13C NMR data revealed that these two compounds were a pair of C-16 epimers. Analysis of the 1D NMR data (Tables 3 and 5) suggested that 16 was a 7α,20epoxy-ent-kaurane diterpenoid possessing an acetoxy and a methoxy group and was structurally similar to adenolin E (46), except for the presence of an sp3 C-11 methylene (δC 18.5) in 16 rather than an oxymethine in 46, as inferred by the 1H−1H COSY correlations of H-9 (δH 1.66)/H-11 (δH 1.99)/H-12 (δH 1.37). To support the proposed structure, a single-crystal X-ray diffraction analysis with Cu Kα radiation (Figure 5) of

C-3 was acetylated in 9, as confirmed by the HMBC correlations between H2-19 (δH 4.27 and 4.40) and C-3 (δC 80.6), C-4 (δC 43.4), C-5 (δC 61.7), C-18 (δC 25.0), and an acetoxy carbonyl carbon (δC 171.1). The ROESY data of 9 and 6 suggested that these two compounds possessed identical relative configurations. Hence, the structure of pharicin K (9) was defined as 3α-acetoxy-6β,7β,14β,19-tetrahydroxy-7α,20epoxy-ent-kaur-16-en-15-one. Pharicin L (10), obtained as a white, amorphous powder, gave the molecular formula C22H32O7, on the basis of a sodium adduct ion at m/z 431.2047 [M + Na]+ (calcd for 431.2040) from the HRESIMS analysis. Comparison of the 1H and 13C NMR data (Tables 2 and 4) of 10 with the known parvifoline I (25) revealed that they are structural analogues, except for the replacement of a carbonyl group in 25 by an oxymethine at C15 (δC 75.4) in 10. This was corroborated by the HMBC correlations between H-15 (δH 5.34) and C-7 (δC 97.7), C-9 (δC 50.8), C-16 (δC 162.3), and C-17 (δC 106.9). Based upon the ROESY correlation between H-9 (δH 2.95) and HO-15 (δH 6.81), the HO-15 group was deduced as β-oriented. Hence, the structure of pharicin L (10) was defined as 1α-acetoxy6β,7β,11β,15β-tetrahydroxy-7α,20-epoxy-ent-kaur-16-ene. Pharicin M (11) was isolated as a white, amorphous powder with a molecular formula of C26H36O11, as established via its (+)-HRESIMS data (m/z 547.2148, [M + Na]+, calcd for 547.2150). Its 1D NMR data (Tables 2 and 5) were closely related to those of the known isoadenolin G (43), except that signals of a methoxy group were absent and two acetoxy groups were evident in 11. In the HMBC spectrum, H-6 (δH 5.99) and H2-19 (δH 4.45 and 4.65) correlated to two acetoxy carbonyls (δC 169.6 and 171.1), suggesting that acetoxy groups should be located at C-6 and C-19. The HMBC correlations of H-20 (δH 6.22) with C-7 (δC 99.9) and C-9 (δC 47.9) indicated the presence of a hydroxy group at C-20 (δC 96.4). The ROESY correlation of H-19 (δH 4.65) with H-20 indicated a (20S*) configuration. Hence, the structure of pharicin M (11) was defined as 20(S*)-6β,11β,19-triacetoxy7β,14β,15β,20-tetrahydroxy-7α,20-epoxy-ent-kaur-16-ene. Pharicin N (12) was isolated as a white, amorphous powder, displaying an [M + Na]+ ion at m/z 505.2043 (calcd for 505.2044) in the HRESIMS data, correlating to a molecular formula of C24H34O10, 42 mass units less than 11. The difference between the NMR data of 12 and 11 was that an acetoxy group in 11 was replaced by a C-6 hydroxy group in 12, as supported by the HMBC correlations between H-11 (δH 6.60) and H2-19 (δH 4.43 and 4.65) to the acetoxy carbonyls (δC 170.8 and 171.2), respectively, together with a key 1H−1H COSY correlation between H-5 (δH 1.90) and H-6 (δH 4.43). The similarities of their ROESY data suggested that 11 and 12 possessed identical relative configurations. Hence, the structure of pharicin N (12) was defined as 20(S*)-11β,19-diacetoxy6β,7β,14β,15β,20-pentahydroxy-7α,20-epoxy-ent-kaur-16-ene. Pharicin O (13) was also purified as a white, amorphous powder, giving the molecular formula C24H36O8, based upon the 13C NMR and HRESIMS data (m/z 475.2298, [M + Na]+, calcd for 475.2302). Compound 13 differed from 12 in that the C-20 hydroxy group was replaced by an ethoxy group as well as the absence of a C-19 acetoxy group, as supported by the 2D NMR analysis (Figure 1). The close resemblance of the ROESY data of 12 and 13 suggested their identical relative configurations. Hence, the structure of pharicin O (13) was defined as 20(S*)-11β-acetoxy-6β,7β,14β,15β-tetrahydroxy20-ethoxy-7α,20-epoxy-ent-kaur-16-ene.

Figure 5. ORTEP drawing of compound 16.

16 was obtained, and the Flack parameter of 0.2(5) enabled definition of its relative configuration. Furthermore, key ROESY correlations of H-12β (δH 2.13)/H-16 (δH 2.56) and H-14β (δH 2.76)/H-17 (δH 3.55) suggested the β-orientation of H-16 in 17 (Figure 4). Accordingly, the structures of 16 and 17 were defined as 16(S*)-1α-acetoxy-6β,7β-dihydroxy-17methoxy-7α,20-epoxy-ent-kaur-15-one and 16(R*)-1α-acetoxy6β,7β-dihydroxy-17-methoxy-7α,20-epoxy-ent-kaur-15-one, respectively. Pharicins T (18) and U (19) were determined to be a pair of isomers with the molecular formula C21H32O8, which was deduced from the HRESIMS analysis as well as comparison of their 1D and 2D NMR spectra. The only distinction was that 18 had a C-12 hydroxy group, while 19 had a C-1 hydroxy 112



Article

group. The 1H and 13C NMR data (Tables 3 and 5) of 18 and 7 suggested that their differences involved the reduction of the exocyclic Δ16(17) double bond and the presence of a methoxy group at C-17 in 18, as verified by HMBC cross-peaks of H-17 (δH 3.96) with C-13 (δC 53.2), C-15 (δC 220.5), C-16 (δC 50.8), and the methoxy group (δC 58.7). ROESY correlations of H-9β (δH 1.82)/H-11β (δH 2.15)/H-12 (δH 4.40)/H-16 (δH 3.07) indicated that the C-12 hydroxy group was αoriented, while H-16 was β-oriented in 18. The 1H−1H COSY spectrum showing cross-peaks of H-1 (δH 3.76)/H2-2 (δH 1.95 and 2.09)/H2-3 (δH 1.27 and 2.37) suggested the presence of a C-1 hydroxy group in 19. The ROESY correlation between H1 (δH 3.76) and H-5β (δH 1.78) suggested that the C-1 hydroxy group was α-oriented in 19. Accordingly, the structures of compounds 18 and 19 were defined as 16(R*)6β,7β,12α,14β,19-pentahydroxy-17-methoxy-7α,20-epoxy-entkaur-15-one and 16(R*)-1α,6β,7β,14β,19-pentahydroxy-17methoxy-7α,20-epoxy-ent-kaur-15-one, respectively. Pharicin V (20), also purified as a white, amorphous powder, had a molecular formula of C21H32O8, on the basis of its HRESIMS and 13C NMR data. The similarities of the 1D NMR data (Tables 3 and 5) of 19 and 20 revealed that these two compounds were a pair of C-16 epimers. The chemical shifts (δC 21.1, C-12; 69.4, C-17) in 20 were similar to those (δC 20.5, C-12; 69.4, C-17) in 15 and (δC 20.3, C-12; 69.3, C17) in 16, suggesting the α-orientation of H-16 in 20, as supported via a ROESY correlation between H-12β (δH 2.07) and H-17 (δH 3.87). Thus, the structure of pharicin V (20) was defined as 16(S*)-1α,6β,7β,14β,19-pentahydroxy-17-methoxy7α,20-epoxy-ent-kaur-15-one. The molecular formula C23H34O9 of compound 21 revealed 42 mass units more than that of 18, according to a deprotonated molecule in the HRESIMS data (m/z 453.2115, [M − H]−, calcd for 453.2130). The 1D NMR data (Tables 3 and 5) of 21 closely resembled those of 18, except for an acetoxy group replacing the C-19 hydroxy group, as supported by the HMBC correlations of H-19 (δH 4.65) to C-3 (δC 36.4), C-4 (δC 37.9), C-5 (δC 61.4), C-18 (δC 28.1), and an acetoxy carbonyl (δC 171.2). Thus, the structure of pharicin W (21) was defined as 16(R*)-19-acetoxy6β,7β,12α,14β-tetrahydroxy-17-methoxy-7α,20-epoxy-entkaur-15-one. Compounds 1−50 were isolated from an EtOAc extract of I. pharicus, which was collected in Gongbo’gyamda County of the Tibetan Autonomous Region. Structurally, all the isolates were characterized as 7α,20-epoxy-ent-kaurane diterpenoids, which were distinguished from the C-20 deoxy-ent-kaurane diterpenoids obtained from this plant in the Lhasa area, Tibetan Autonomous Region.2 These findings suggested that different ecological environments might greatly influence the chemical diversity of the Isodon species. Preliminary structure−activity relationship studies of selected diterpenoids were evaluated in terms of their in vitro cytotoxic activities against a small panel of human tumor cell lines, including HL-60, SMMC-7721, A-549, MCF-7, and SW-480, with the MTS viability assay approach.26 Compounds 11, 16, 38, and 48 (Table 6) displayed inhibitory activities with IC50 values in the range of 1.01−9.62 μM, while compounds 2, 15, 29, and 47 exhibited moderate cytotoxic potency. The remaining 17 compounds (1, 3−10, 12−14, and 17−21) were inactive (IC50 > 10 μM) against the tumor cell lines. These pharmacological data indicated that an α,βunsaturated carbonyl group was the cytotoxic pharmacophore,

Table 6. Cytotoxic Activities of Selected Compounds against Five Human Tumor Cell Linesa compound

HL-60

SMMC-7721

A-549

MCF-7

SW-480

2 11 15 16 29 38 47 48 cisplatinb paclitaxelb

12.48 5.53 18.04 3.77 5.36 2.43 17.20 4.24 2.06

kaurane Diterpenoids from the Aerial Parts of - ACS Publications

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