Five New ent-Kaurane Diterpenoids from Isodon pharicus

Chinese Journal of Natural Medicines 2009, 7(6): 04090413

Chinese Journal of Natural Medicines

Five New ent-Kaurane Diterpenoids from Isodon pharicus ZHAO Yong1,2, PU Jian-Xin 1, Li Li-Mei 1, XIAO Wei-Lie1, YANG Li-Bin1, SUN Han-Dong1* 1

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204; 2 School of Chemistry and Engineering, Yunnan Normal University, Kunming 650092, China Available online 20 Nov. 2009

[ABSTRACT] AIM: To study the chemical constituents of the aerial parts of Isodon pharicus. METHOD: Silica gel, C18 reversed phase silica gel and HPLC were used. The structures were elucidated by extensive spectroscopic methods (IR, UV, MS and NMR). RESULTS: Five ent-kaurane diterpenoids, pharicunins N–R (1–5), were isolated and identified. CONCLUSION: All of the compounds are new. [KEY WORDS] Labiatae; Isodon pharicus; Diterpenoids [CLC Number] R284.1

1

[Document code] A

[Article ID]1672-3651(2009)06-0409-05

Introduction

Isodon species (Labiatae) have proved to be a prolific source of new diterpenoids, especially the highly oxygenated ent-kaurane diterpenoids with diverse biological activity[1, 2]. Isodon pharicus (Prain) Hara, mainly distributed in the northwest of Sichuan Province and the southern district of Tibetan region, China, has been used for the deinsectization and treatment of inflammation of the eyes[3]. In our previous investigations, some ent-kuaranoids have been reported from this plant[4, 5]. In the course of searching for more biologically active ent-kauranoids, further investigation on its AcOEt extract resulted in the isolation of five 1-oxygenated new ent-kaurane diterpenoids, pharicunins N-R (1-5) (Fig. 1). The current paper describes the isolation and structural elucidation of these new compounds.

2

Results and Discussion Pharicunin N (1) was isolated as a white amorphous

[Received on] 17-Apr.-2009 [Foundation Item]This project was supported financially by the NSFC (No. 30772637), the NSFC-joint Foundation of Yunnan Province (No. U0832602), the Major State Basic Research Development Program of China (No. 2009CB522300), the Natural Science Foundation of Yunnan Province (No. 2008CD162) and the Key Project of Knowledge Innovation Project of CAS (No. KSCX2-YW-R-25). [ Corresponding author] SUN Han-Dong: Prof., Tel: 86-871-5223 251, Fax: 86-871-5216343, E-mail: [email protected] 2009 ᑈ 11 ᳜

㄀7ो

㄀6ᳳ

Fig. 1

Structures of 1–5

powder, [D]20.5 D –23.8 (c 0.11, MeOH). A molecular formula of C24H34O8 was determined as 1 from its HRESI-MS at m/z 473.215 0 [M + Na]+ (calcd. for C24H34O8Na, 473.215 1). IR absorptions at 3 428, 1 729, and 1 648 cm1 implied the presence of hydroxyl, carbonyl, and Į, ȕ-unsaturated ketone. Except for two acetyl moieties (įH 2.08, 6H, s; įC 171.1 and 170.3, s, 21.2 and 20.9, q), the 1H NMR spectrum of 1 (Table 1) displayed characteristic signals for an exocyclic methylene signals at įH 5.66 and 5.30 (each 1H, s), three methine protons at įH 3.09 (1H, br s, H-13Į), 1.63 (1H, br s, H-9ȕ), and 1.54 (1H, d, J = 11.6 Hz, H-5ȕ), two tertiary methyl singlets at įH 1.20 (3H, s, Me-20) and 0.98 (3H, s, Me-18), and an oxygenated methylene doublet at įH 4.21 (1H, d, J = 11.4 Hz, H-19a) and 3.93 (1H, d, J = 11.4 Hz, H-19b). The 13C and DEPT NMR spectral data (Table 2) indicated that 1 contains 20 carbons, including two methyls, six methylenes (including a olefinic carbon and an oxygenated), seven methines (of which four were oxygenated), and five Chin J Nat Med Nov. 2009

Vol. 7 No. 6

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ZHAO Yong, et al. /Chinese Journal of Natural Medicines 2009, 7(6): 409413 Table 1

1

H NMR data of compounds 1–5 (į, J in Hz) 1a,d

2b,e

3b,d

1ȕ

3.63 (1H, m)

4.03 (1H, dd, 11.4, 4.3)

2Į

1.84-1.89 (2H, overlap)

2.44 (1H, m)

4.96 (1H, t, 3.0)

5ȕ

1.54 (1H, d, 11.6)

1.92 (1H, m)

6Į

1.86 (1H, overlap)

6ȕ

1.98 (1H, m)

H

4c,d

5c,d

3.70-3.66 (1H, m)

3.43 (1H, m)

3.70 (1H, overlap)

1.99 (1H, m)

1.80 (1H, overlap)

1.97 (1H, m)

2.22 (1H, overlap)

1.80 (1H, overlap)

1.63 (1H, overlap)

1.88 (1H, m )

5.38 (1H, overlap)

4.92 (1H, t, 3.1)

4.60 (1H, br s)

4.81 (1H, t, 2.9)

1.67 (1H, br d, 12.6)

1.35 (1H, br d, 11.7)

1.53 (1H, d, 11.0)

1.96 (1H, m)

2.05 (1H, m)

1.81 (1H, overlap)

2.10 (1H, m)

0.85 (1H, m)

1.81 (1H, overlap)

1.62 (1H, overlap)

1.83 (1H, m)

2.27 (1H, br d, 12.4)

1.62 (1H, m)

1Į

2ȕ 3Į 3ȕ

7Į 7ȕ

4.30 (1H, dd, 12.4, 4.0)

4.92 (1H, m)

4.38 (1H, m)

4.13 (1H, m)

4.40 (1H, m)

9ȕ

1.63 (1H, br s)

2.23 (1H, overlap)

1.78 (1H, br d, 9.8)

1.76 (1H, overlap)

1.80 (1H, d, 10.2)

11Į

1.42 (1H, m)

2.33 (1H, m)

2.28 (1H, m)

2.35 (1H, m)

2.42 (1H, m)

11ȕ

2.87 (1H, dd, 15.8, 5.2)

3.93 (1H, d, 16.5)

3.93 (1H, d, 17.5)

3.70 (1H, overlap)

4.02 (1H, d, 18.1)

12Į

1.73 (1H, m)

5.18 (1H, br s)

12ȕ

2.07 (1H, overlap)

4.40 (1H, 4.4)

13Į

3.09 (1H, br s)

3.66 (1H, d, 3.6)

3.54 (1H, br s)

3.27 (1H, br s)

2.61 (1H, br s)

14Į

4.88 (1H, br s)

5.95 (1H, s)

5.05 (1H, br s)

4.99 (1H, br s)

5.05 (1H, br d, 7.6)

15Į

4.40 (1H, s)

4.09 (1H, m)

16Į

3.15 (1H, overlap)

3.91 (1H, m)

16ȕ

3.23 (1H, s)

17a

6.17 (1H, s)

6.30 (1H, br s)

6.06 (1H, s)

17b

5.42 (1H, s)

5.39 (1H, br s)

5.49 (1H, s)

3.53-3.70 (2H, overlap)

18

0.98 (3H, s)

1.09 (3H, s)

1.00 (3H, s)

0.78 (3H, s)

0.98 (3H, s)

19a

4.21 (1H, 11.4)

4.60 (1H, 11.5)

4.25 (1H, 11.5)

0.83 (3H, s)

1.01 (3H, s)

19b

3.93 (1H, 11.4)

4.25 (1H, 11.5)

3.99 (1H, 11.5)

20

1.20 (3H, s)

1.99 (3H, s)

1.02 (3H, s)

0.88 (3H, s)

1.10 (3H, s)

2.09 (3H, s)

2.16 (3H, s)

3.16 (3H, s)

3.51 (3H, s)

OAc

2.08 (3H, s)

2.03 (3H, s)

2.08 (3H, s)

2.08 (3H, s)

1.93 (3H, s)

2.02 (3H, s)

OMe a

4.09 (1H, dd, 9.4, 3.5) 3.70 (1H, dd, 9.4, 4.1)

Recorded in (CD3)2CO.

b

c

d

e

Recorded in C5D5N. Recorded in CDCl3. Recorded at 400 MHz. Recorded at 500 MHz.

quaternary carbons (including one sp2 carbon and one carbonyl). All evidence suggested that compound 1 was an ent-kaur-16-en-15-one[6]. The positions of the two acetoxyl groups were determined on the basis of the observed HMBC correlations from H-3 (įH 4.96) and H2-19 (įH 4.21, 3.93) to OAc (įC 170.3) and OAc (įC 171.1), respectively. The correlations in the HMBC spectra from H-1 (įH 3.63) to C-5, C-9, and C-20, from H-3 (įH 4.96) to C-5, C-18, and C-19, from H-7 (įH 4.30) to C-5, C-8, C-14, and C-15, and from H-14 (įH 4.88) to C-7, C-15, and C-16 established the locations of the oxymethines as Figure 2. This revealed that the gross structure of 1 as 1, 7, 14-trihydroxy-3, 19diacetoxy-ent-kaur-16-en-15-one.

In the ROESY spectrum of 1, H-1 correlated to H-5 and H-9, H-3 correlated to H-19, H-7 correlated to H-5 and H-9, and H-14 correlated to H-6Į and Me-20, indicating that HO-1, AcO-3, HO-7, and HO-14 adopt Į-, ȕ-, Į-, and ȕ-orientations, respectively. A computer-modeled structure of 1 (CS Chem 3D Pro Version 8.0 using MM2 force field calculations for energy minimization), in which the ROESY correlations were depicted (Fig. 2), further supported the stereochemistry assignments. Therefore, compound 1 was assigned as 1Į, 7Į, 14ȕ-trihydroxy-3ȕ, 19-diacetoxy-16-ent-kaur-15-one. Pharicunin O (2) exhibited the molecular formula C24H34O9 as determined by the positive HRESI-MS (m/z 489.210 5 ([M + Na]+ (calcd. for C24H34O9Na [M + Na]+,

ZHAO Yong, et al. /Chinese Journal of Natural Medicines 2009, 7(6): 409413 Table 2

13

C NMR Data of Compounds 1–5 (į)

Carbon

1a,d

2b,e

3b,d

4c,d

5c,d

1

75.5 d

75.9 d

75.8 d

75.4 d

76.0 d

2

33.7 t

34.2 t

33.9 t

32.8 t

33.8 t

3

75.1 d

75.0 d

73.9 d

73.8 d

77.6 d

4

40.5 s

41.2 s

41.5 s

36.4 s

36.7 s

5

46.4 d

47.4 d

46.8 d

45.7 d

45.8 d

6

27.4 t

29.5 t

28.9 t

27.5 t

28.5 t

7

74.8 d

74.2 d

75.7 d

73.8 d

73.7 d

8

62.2 s

62.3 s

62.1 s

60.8 s

63.3 s

9

55.1 d

58.7 d

51.3 d

51.3 d

49.4 d

10

44.9 s

44.3 s

45.2 s

43.9 s

44.8 s

11

19.6 t

28.7 t

37.6 t

37.7 t

36.5 t

12

31.1 t

73.0 d

207.4 s

211.9 s

208.9 s

13

46.5 d

56.0 d

64.7 d

51.8 d

52.1 d

14

73.2 d

71.6 d

74.0 d

72.1 d

74.3 d

15

208.3 s

209.7 s

204.9 s

215.9 s

217.1 s

16

147.7 s

148.3 s

146.0 s

58.4 d

58.7 d

17

118.0 t

116.7 t

118.7 t

67.8 t

71.1 t

18

22.3 q

22.3 q

22.4 q

27.4 q

27.7 q

19

66.0 t

66.6 t

64.7 t

21.3 q

21.5 q

20

14.4 q

13.8 q

13.7 q

12.9 q

13.0 q

OAc

171.1 s

170.9 s

171.0 s

170.8 s

170.4 s

170.3 s

170.4 s

170.4 s

21.2 q

21.0 q

20.9 q

20.9 q

21.2 q

20.9 q

20.6 q

20.7 q 58.9 q

59.5 q

OMe a d

b

c

Recorded in (CD3)2CO. Recorded in C5D5N. Recorded in CDCl3. Recorded at 100 MHz. e Recorded at 125 MHz.

HMBC : H

Fig. 2

C

ROESY : H

cidated as 1Į, 7Į, 12Į, 14ȕ-tetrahydroxy-3ȕ, 19- diacetoxy-16-ent-kaur-15-one. The HR-ESI-MS of pharicunin P (3) showed a pseudomolecular ion peak at m/z 487.193 7, consistent with the molecular formula C24H32O9Na (calcd. 487.194 4) revealing one more degree of unsaturation compared with 2. A careful analysis of its 2D NMR spectra data and comparison with 2 revealed that a carbonyl group at C-12 in 3 replaced a hydroxyl group at the same position in 2, which was proven by the HMBC correlations from H-9, H-11, H-13, and H-17 to C-12 (įC 207.4). In addition, in the 13C NMR spectrum, the downfield shift for C-11 from įC 28.7 in 2 to įC 37.6 in 3 and for C-13 from įC 56.0 in 2 to įC 64.7 in 3 confirmed this conclusion. Moreover, the correlations observed in the ROESY spectrum of 3 indicated that the configurations of the substituent groups in 3 are the same as those of 2. Eventually, compound 3 was established as 1Į, 7Į, 14ȕ-trihydroxy-3ȕ, 19-diacetoxy-16-ent-kaur-12, 15-dione. The molecular formula of pharicunin Q (4) (C23H34O8) was determined by the HR-ESI-MS at m/z 461.215 2 (calcd. 461.215 1), with seven degrees of unsaturation. The NMR data of 4 resembled those of pseurata F[7], a known compound also isolated from this plant, except for the signals due to an oxygenated methylene at C-17 in 4 rather than an olefinic methylene at the same position in pseurata F, which was verified by the HMBC correlations of H-17 (įH 3.533.70) with C-13, C-15, C-16, and OMe. A ȕ-configuration of the oxygenated methylene (C-17) was concluded based on the 1 H-1H correlations of H-11ȕ with H-17a observed in the ROESY experiment of 4 (Fig. 3). The observed significant upfield shift in the 13C NMR spectrum for C-13 from įC 64.8 in pseurata F to įC 51.8 in 4, caused by the Ȗ-steric compression effect between MeO-17 and H-13Į confirmed this conclusion. Therefore, compound 4 was identified as 1Į, 7Į, 14ȕ-trihydroxy-3ȕ-acetoxy-16ȕ-methoxymethyl-ent-kaur-12, 15-dione. Detailed comparison of NMR data of 5 with those of 4 revealed that they were a pair of isomer concerning the configuration of H-16. The NOE signal of H-13Į with H-17

H

Key HMBC and ROESY correlations of 1

489.210 0). Comparison of the 1H and 13C NMR data of 2 with those of 1 indicated that these two compounds were closely similar, and the only difference was that there is one more hydroxyl in 2. The correlations of H-12 (įH 4.40) with C-9, C-13, and C-14 in the HMBC experiment suggested that the hydroxyl group was attached to C-12. The relative configuration of HO-12 was deduced to be Į-oriented according to the ROESY correlations of H-12/H-17b. Thus, 2 was elu-

Fig. 3

Key HMBC and ROESY correlations of 4

ZHAO Yong, et al. /Chinese Journal of Natural Medicines 2009, 7(6): 409413

observed as well as the NOE signal of H-11ȕ with H-17 was not observed in 5 confirmed the relative configuration of H-16 to be ȕ-orientation. Thus, compound 5, named pharicunin R, was characterized as 1Į, 7Į, 14ȕ-trihydroxy-3ȕ-acetoxy-16Į-methoxymethyl-ent-kaur-12, 15-dione.

3

Experimental

3.1

General Optical rotations were measured using a Perkin-Elmer model 241 polarimeter. IR spectra were recorded on a Bio-Rad FTS-135 spectrometer with KBr pellets. 1D and 2D NMR spectra were measured on a Bruker DRX-400 and a Bruker DRX-500 instrument with TMS as internal standard. Mass spectra were obtained on a VG Auto Spec-3000 spectrometer or on a Finnigan MAT 90 instrument. Semipreparative HPLC was performed on an Agilent 1100 liquid chromatograph with a Zorbax SB-C18, 9.4 mm u 25 cm, column. Column chromatography was performed on silica gel (200–300 mesh; Qingdao Marine Chemical Inc., Qingdao, China), Lichroprep RP-18 gel (40–63 ȝm, Merck, Darmstadt, Germany), and MCI-gel CHP 20P (75–150 ȝm, Mitsubishi Chemical Corp., Tokyo, Japan). Thin-layer chromatography (TLC) was carried out on silica gel 60 F254 on glass plates (Qingdao Marine Chemical Inc.) using various solvent systems. 3.2 Plant Material The aerial parts of I. pharicus were collected in Lhasa area, Tibet Autonomous region, China, in October 2005. Voucher specimens (KIB 20051006) were deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, and were identified by Prof. LI Xi-Wen.

4

Extraction and Isolation

The milled aerial parts of I. pharicus (2.0 kg) were extracted with 70% aqueous acetone (3 u 10 L) at room temperature overnight. The extract was partitioned between EtOAc and H2O. The EtOAc extract (120g) was chromatographed on MCI gel CHP 20P (90% CH3OH–H2O, then 100% CH3OH). The 90% CH3OH fraction (92 g) was chromatographed over silica gel (200–300 mesh, 1.5 kg), eluted in a step gradient manner with CHCl3–CH3COCH3 (1ĩ0 to 0ĩ1) to afford fractions A–F. Fraction B (12 g) was submitted to repeated chromatography over silica gel (petroleum ether–acetone, from 40ĩ1 to 0ĩ1) to obtain fractions B1–B4. Compound 1 (3 mg) was obtained by RP-18 column chromatography (30%o60% MeOH–H 2 O) and semipreparative HPLC (40% MeOH–H2O). Compound 2 (2 mg) was purified by RP-18 column chromatography (37% MeOH–H 2 O) from fraction B3. Fraction C (7 g) was subjected to silica gel column chromatography, eluted with petroleum ether-acetone (9ĩ1o1ĩ1), to yield fractions C1-C5. Compound 3 (6 mg) was purified by semipreparative

HPLC (32% MeOH–H2O) from fraction C2. Fraction C5 was further chromatographed over RP-18 column (37% MeOH–H2O) followed by semi-preparative HPLC (32% MeOH–H2O) to give compounds 4 (4 mg) and 5 (5 mg). Pharicunin N (1): white amorphous powder; [D]20.5 D –23.8 (c 0.11, MeOH); UV (MeOH) Omax (log H): 231 (3.57) nm; IR (KBr) Qmax 3 428, 2 939, 2 878, 1 729, 1 648, 1 452, 1 377, 1 250, 1 185, 1 077, 1 034, 993, 982, 603 cm–1; ESI-MS m/z 473 [M + Na]+, 923 [2M + Na]+; HR-ESI-MS [M + Na]+ m/z 473.215 0 (calcd. for C24H34O8Na [M + Na]+, 473.215 1); 1 H NMR (CDCl3, 400 MHz) data see Table 1; 13C NMR (CDCl3, 100 MHz) data see Table 2. –8.8 (c 0.25, Pharicunin O (2): white powder; [D] 20.8 D MeOH); UV (MeOH) Omax (log H): 230 (3.38) nm; IR (KBr) Qmax 3 413, 2 982, 2 944, 2 910, 2 880, 1 722, 1 649, 1 446, 1 378, 1 259, 1 182, 1 092, 1 078, 1 034, 1 025, 995, 936, 819, 692, 607, 564 cm–1; ESI-MS m/z 489 [M + Na]+; HR-ESI-MS [M + Na]+ m/z 489.210 5 (calcd. for C24H34O9Na [M + Na]+, 489.210 0); 1H NMR (C5D5N, 500 MHz), 1H NMR (CDCl3, 400 MHz) data see Table 1; 13C NMR (CDCl3, 100 MHz) data see Table 2. Pharicunin P (3): white amorphous powder; [D]20.1 D +49.9 (c 0.56, MeOH); UV (MeOH) Omax (log H): 202 (3.40) nm; IR (KBr) Qmax 3 417, 2 981, 1 720, 1 645, 1 373, 1 248, 1 179, 1 077, 1 031, 981, 916 cm–1; positive ESI-MS m/z 487 [M + Na]+; HR-ESI-MS [M + Na]+ m/z 487.193 7 (calcd. for C24H32O9Na [M + Na]+, 487.194 4); 1H NMR (CDCl3, 400 MHz) data see Table 1; 13C NMR (CDCl3, 100 MHz) data see Table 2. Pharicunin Q (4): white amorphous powder; [D]19.1 D + 44.6 (c 0.10, MeOH); UV (MeOH) Omax (log H): 202 (3.38) nm; IR (KBr) Qmax 3 426, 2 961, 2 924, 1 712, 1 628, 1 449, 1 376, 1 247, 1 179, 1 091, 1 077, 1 038, 1 041, 991, 968, 606, 536 cm–1; ESI-MS m/z 461 [M + Na]+; HR-ESI-MS [M + Na]+ m/z 461.215 2 (calcd. for C23H34O8Na [M + Na] +, 461.215 1); 1 H NMR (CDCl3, 400 MHz) data see Table 1; 13C NMR (CDCl3, 100 MHz) data see Table 2. Pharicunin R (5): white amorphous powder; [D]20.0 D +54.8 (c 0.07, MeOH); UV (MeOH) Omax (log H): 203 (3.10) nm; IR (KBr) Qmax 3 436, 2 966, 1 718, 1 638, 1 461, 1 448, 1 377, 1 260, 1 195, 1 180, 1 103, 1 077, 1 041, 995, 959, 606 cm–1; ESI-MS m/z 461 [M + Na]+; HR-ESI-MS [M + Na]+ m/z 461.212 6 (calcd. for C23H34O8Na [M + Na]+, 461.215 1); 1 H NMR (CDCl3, 400 MHz) data see Table 1; 13C NMR (CDCl3, 100 MHz) data see Table 2.

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