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Planta Med 2004; 70(6): 540-550
DOI: 10.1055/s-2004-827155
Original Paper
Natural Product Chemistry
© Georg Thieme Verlag KG Stuttgart · New York

Structure of Kaurane-Type Diterpenes from Parinari sprucei and their Potential Anticancer Activity

Alessandra Braca1 , Annahil Armenise1 , Ivano Morelli1 , Jeannette Mendez2 , Qiuwen Mi3 , Hee-Byung Chai3 , Steven M. Swanson3 , A. Douglas Kinghorn3 , Nunziatina  De Tommasi4
  • 1Dipartimento di Chimica Bioorganica e Biofarmacia, Università di Pisa, Pisa, Italy
  • 2Escuela de Quimica, Universidad Central de Venezuela, Caracas, Venezuela
  • 3Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL, USA
  • 4Dipartimento di Scienze Farmaceutiche, Università di Salerno, Fisciano (SA), Italy
This work was supported in part by CNR (Consiglio Nazionale delle Ricerche, Roma) and by FONACIT (Fondo Nacional de Ciencia Tecnologia e Innovacion), and grant U19 CA 52 956 from the National Cancer Institute, NIH, Bethesda, MD, USA
Further Information

Prof. Nunziatina De Tommasi

Dipartimento di Scienze Farmaceutiche

Università di Salerno

Via Ponte Don Melillo

84084 Fisciano (SA)

Italy

Fax: +39-89-962828

Email: detommasi@unisa.it

Publication History

Received: October 27, 2003

Accepted: March 7, 2004

Publication Date:
01 July 2004 (online)

Also available at
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Table of Contents #

Abstract

Twenty-three kaurane-type diterpenes 1 - 23, including twenty new natural products 1 - 20, have been isolated from the leaves of Parinari sprucei and their structures elucidated by spectroscopic and chemical analysis. The isolated compounds were tested for their cytotoxic activity towards a panel of cancer cell lines. Compounds 9 and 10 showed activity against all cell lines with ED50 values in the range of 10 - 20 μg/mL. The previously known 13-hydroxy-15-oxozoapatlin 21 was evaluated in an in vivo hollow fiber test, and found to be active with KB and LNCaP cells at the concentrations used.

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Key words

Parinari sprucei - Chrysobalanaceae - leaves - kaurane diterpenes - cytotoxicity - hollow fiber test

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Introduction

Parinari sprucei Hook. f. (Chrysobalanaceae) is a tree growing in the Amazon forest of Venezuela, whose fruits are edible and constitute part of the diet of indigenous populations [1]. Plants of the genus Parinari have been used in several countries to treat different diseases [2]. Only few species of the genus Parinari have been studied, leading to the identification of several nor-kaurene and ent-kaurene diterpenes [2], [3], [4]. In the present study, twenty new (1 - 20) and three known kaurane-type diterpenes (21 - 23) have been isolated from the leaves of P. sprucei (Fig. [1]). The structures of compounds 1 - 20 were elucidated on the basis of their spectral properties and by chemical analysis. Since 13-hydroxy-15-oxozoapatlin (21) has shown activity in a tumour cell-line cytotoxicity evaluation [2], for the isolated kaurane diterpenes a biological evaluation of their cytotoxicity was carried out against six cell lines, while the potential anticancer activity of compound 21 was studied in vivo using a hollow fiber test.

Zoom Image

Fig. 1 Structures of compounds 1 - 23 isolated from Parinari sprucei leaves.

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Materials and Methods

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General experimental procedures

The instrumentation used in this work is described in our previous paper [5].

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Plant material

The leaves of P. sprucei Hook. f. were collected in the Amazonian region of Venezuela in August 1999. The plant material was identified by Prof. Anibal Castillo, Universidad Central de Venezuela, Caracas, where a voucher sample (number VEN 295627Y) is deposited.

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Extraction and isolation

The air-dried powdered leaves of P. sprucei (1 kg) were defatted with n-hexane and successively extracted for 48 h with CHCl3, CHCl3-MeOH (9 : 1), and MeOH, by exhaustive maceration (3 × 2 L), to give 5.0 g, 12.7 g, 13.7 g, and 31.0 g of the respective residues. Part of the chloroform extract (4.0 g) was submitted to silica gel column chromatography (4 × 50 cm, 50 g of silica gel for 1 g of crude extract), eluting with chloroform followed by increasing concentrations of MeOH (between 1 % and 50 %) in CHCl3; the following volumes of solvents were used: 7.30 L of CHCl3, 8 L of CHCl3-MeOH (99 : 1), 4 L of CHCl3-MeOH (98 : 2), 3.25 L of CHCl3-MeOH (95 : 5), 1.6 L of CHCl3-MeOH (9 : 1), 1.40 L of CHCl3-MeOH (1 : 1), 0.8 L of MeOH. Fractions of 25 mL were collected, analyzed by TLC (silica gel plates, in CHCl3 or mixtures of CHCl3-MeOH, 99 : 1, 98 : 2, 97 : 3, 9 : 1, 8 : 2), and grouped into 16 fractions. Fraction 3 (173.3 mg) was subjected to RP-HPLC on a C18 μ-Bondapak column (30 cm × 7.8 mm, flow rate 2.0 mL min-1) with MeOH-H2O (7 : 3) to yield pure compound 21 (100.0 mg, t R = 10 min). Fractions 4 (42.0 mg), 6 (49.3 mg), 8 (44.0 mg), and 12 (86.7 mg) were separately purified by RP-HPLC on a C18 μ-Bondapak column (30 cm × 7.8 mm, flow rate 2.0 mL min-1) with MeOH-H2O (3 : 2) to afford pure compound 9 (12.0 mg, t R = 7 min) from fraction 4, compound 13 (7.0 mg, t R = 12 min) from fraction 6, compound 14 (3.2 mg, t R = 9 min) from fraction 8, and pure compounds 8 (5.0 mg, t R = 15 min) and 18 (6.0 mg, t R = 20 min) from fraction 12. Fractions 7 (56.0 mg), 9 (37.0 mg), 10 (142.0 mg), 11 (56.7 mg), and 13 (32.0 mg) were separately chromatographed over RP-HPLC on a C18 μ-Bondapak column (30 cm × 7.8 mm, flow rate 2.0 mL min-1) with MeOH-H2O (1 : 1) to give pure compound 5 (7.3 mg, t R = 15 min) from fraction 7, pure compounds 20 (6.3 mg, t R = 8 min), 11 (1.0 mg, t R = 16 min), and 10 (2.0 mg, t R = 19 min) from fraction 9, compounds 1 (15.0 mg, t R = 15 min) and 17 (3.5 mg, t R = 20 min) from fraction 10, compound 7 (6.5 mg, t R = 20 min) from fraction 11, and compound 19 (3.7 mg, t R = 18 min) from fraction 13. Finally, fractions 14 (106.0 mg) and 15 (86.5 mg) were separately submitted to RP-HPLC on a C18 μ-Bondapak column (30 cm × 7.8 mm, flow rate 2.0 mL min-1), with MeOH-H2O (3.5 : 6.5), to yield pure compounds 6 (3.8 mg, t R = 11 min) and 3 (2.5 mg, t R = 15 min) from fraction 14 and compound 2 (7.7 mg, t R = 22 min) from fraction 15. Part of the chloroform-methanol residue (5.0 g) was chromatographed on Sephadex LH-20, using MeOH as eluent; fractions of 8 mL were collected and grouped into 12 major fractions by TLC on silica 60 F254 gel-coated glass sheets with n-BuOH-AcOH-H2O (60 : 15 : 25) and CHCl3-MeOH-H2O (40 : 9:1). Fraction 4 (283.5 mg) was chromatographed over RP-HPLC on a C18 μ-Bondapak column (30 cm × 7.8 mm, flow rate 2.0 mL min-1) with MeOH-H2O (8 : 2) to give pure compounds 15 (4.2 mg, t R = 21 min) and 16 (5.5 mg, t R = 23 min). Fraction 5 (255.5 mg) was purified by RP-HPLC on a C18 μ-Bondapak column (30 cm × 7.8 mm, flow rate 2.0 mL min-1) with MeOH-H2O (1 : 1) to afford pure compounds 4 (8.0 mg, t R = 9 min) and 22 (4.7 mg, t R = 24 min). Finally, fraction 6 (150.0 mg) was submitted to RP-HPLC on a C18 μ-Bondapak column (30 cm × 7.8 mm, flow rate 2.0 mL min-1) with MeOH-H2O (7 : 3) to yield pure compounds 12 (6.0 mg, t R = 13 min) and 23 (6.5 mg, t R = 17 min).

Compound 1 : Yellow crystals; m. p. 88 - 93 °C; [α]D 25: + 24.3° (c 0.1, MeOH); UV(MeOH): λmax = 274, 226 (sh) nm; ESI-MS (pos.): m/z = 365 [M + H]+, 347 [(M + H) - 18]+, 329 [(M + H) - 36]+, 319, 301, 283, 241; anal. found: C 65.88 %, H 7.75 %; calcd. for C20H28O6: C 65.92 %, H 7.74 %; NMR data, see Table [1].

Compound 2 : White needles; m. p. 100 - 103 °C; [α]D 25: + 15.0° (c 0.1, MeOH); UV (MeOH): λmax = 277, 226 (sh) nm; ESI-MS (pos.): m/z = 381 [M + H]+, 363 [(M + H) - 18]+, 337, 310, 306, 289, 277, 260; anal. found: C 63.08 %, H 7.45 %; calcd. for C20H28O7: C 63.14 %, H 7.42 %; NMR data, see Table [1].

Compound 3 : White crystals; m. p. 85 - 90 °C; [α]D 25: + 5.3° (c 0.1, MeOH); UV (MeOH): λmax = 271, 225 (sh) nm; ESI-MS (pos.): m/z = 381 [M + H]+, 363 [(M + H) - 18]+; anal. found: C 63.16 %, H 7.39 %, O 29.45 %; calcd. for C20H28O7: C 63.14 %, H 7.42 %, O 29.44 %; NMR data, see Table [1].

Compound 4 : White amorphous solid; m. p. 150 °C dec; [α]D 25: + 16.0° (c 0.1, MeOH); UV (MeOH): λmax = 276, 230 (sh) nm; ESI-MS (pos.): m/z = 397 [M + H]+, 379 [(M + H) - 18]+, 361 [(M + H) - 36]+; anal. found: C 60.54 %, H 7.15 %; calcd. for C20H28O8: C 60.59 %, H 7.12 %; NMR data, see Table [1].

Compound 5: White crystals; m. p. 95 °C; [α]D 25: + 16.0° (c 0.1, MeOH); UV (MeOH): λmax = 272, 215 (sh) nm; ESI-MS (pos.): m/z = 349 [M + H]+, 331 [(M + H) - 18]+; anal. found: C 68.90 %, H 8.15 %; calcd. for C20H28O5: C 68.94 %, H 8.10 %; NMR data, see Table [2].

Compound 6: Pale-yellow crystals; m. p. 152 - 160 °C; [α]D 25: + 20.0° (c 0.1, MeOH); UV (MeOH): λmax = 275, 230 (sh) nm; ESIMS (pos.): m/z = 365 [M + H]+, 347 [(M + H) - 18]+, 329 [(M + H) - 36]+; anal. found: C 65.90 %, H 7.70 %; calcd. for C20H28O6: C 65.92 %, H 7.74 %; NMR data, see Table [2].

Compound 7: White amorphous solid; m. p. 187 - 200 °C; [α]D 25: + 23.0° (c 0.1, MeOH); UV (MeOH): λmax = 275, 235 (sh) nm; ESI-MS (pos.): m/z = 349 [M + H]+, 331 [(M + H) - 18]+, 313 [(M + H) - 36]+; anal. found: C 68.92 %, H 8.13 %; calcd. for C20H28O5: C 68.94 %, H 8.10 %; NMR data, see Table [2].

Compound 8: White amorphous solid; m. p. 167 - 170 °C; [α]D 25: + 18.0° (c 0.1, MeOH); UV (MeOH): λmax = 277, 232 (sh) nm; ESI-MS (pos.): m/z = 349 [M + H]+, 331 [(M + H) - 18]+, 313 [(M + H) - 36]+; anal. found: C 68.88 %, H 8.12 %; calcd. for C20H28O5 : C 68.94 %, H 8.10 %; NMR data, see Table [2].

Compound 9: Pale-yellow crystals; m. p. 85 - 88 °C; [α]D 25: -68.0° (c 0.1, MeOH); UV (MeOH): λmax = 226 (sh) nm; ESI-MS (pos.): m/z = 333 [M + H]+, 318 [(M + H) - 15]+, 315 [(M + H) - 18]+, 287, 269, 241, 211; anal. found: C 72.30 %, H 8.51 %; calcd. for C20H28O4: C 72.26 %, H 8.49 %; NMR data, see Table [3].

Compound 10: White amorphous solid; m. p. 193 - 200 °C dec; [α]D 25: + 24.0° (c 0.1, MeOH); UV (MeOH): λmax = 218 (sh) nm; ESI-MS (pos.): m/z = 349 [M + H]+, 331 [(M + H) - 18]+; anal. found: C 68.90 %, H 8.11 %; calcd. for C20H28O5: C 68.94 %, H 8.10 %. NMR data, see Table [3].

Compound 11: White solid; m. p. 200 °C dec; [α]D 25: + 18.0° (c 0.1, MeOH); UV (MeOH): λmax = 220 (sh) nm; ESIMS (pos.): m/z = 365 [M + H]+, 347 [(M + H) - 18]+, 329 [(M + H) - 36]+; anal. found: C 65.88 %, H 7.77 %; calcd. for C20H28O6: C 65.92 %, H 7.74 %; NMR data, see Table [3].

Compound 12: Brownish solid; m. p. 137 - 142 °C; [α]D 25: + 5.0° (c 0.1, MeOH); UV (MeOH): λmax = 276, 232 (sh) nm; ESI-MS (pos.): m/z = 351 [M + H]+, 333 [(M + H) - 18]+; anal. found: C 68.45 %, H 8.65 %; calcd. for C20H30O5: C 68.55 %, H 8.63 %; NMR data, see Table [3].

Compound 13: White solid; m. p. 187 - 193 °C dec; [α]D 25: + 27.0° (c 0.1, MeOH); UV (MeOH): λmax = 280, 252 (sh), 217 nm; ESI-MS (pos.): m/z 365 = [M + H]+, 347 [(M + H) - 18]+; anal. found: C 69.18 %, H 8.87 %; calcd. for C21H32O5: C 69.20 %, H 8.85 %; NMR data, see Table [4].

Compound 14: White solid; m. p. 103 - 108 °C; [α]D 25: + 23.0° (c 0.1, MeOH); UV (MeOH): λmax = 218 (sh) nm; ESI-MS (pos.): m/z = 381 [M + H]+, 363 [(M + H) - 18]+; anal. found: C 66.25 %, H 8.50 %; calcd. for C21H32O6: C 66.29 %, H 8.48 %; NMR data, see Table [4].

Compound 15: White amorphous solid; m. p. 270 °C dec; [α]D 25: + 9.0° (c 0.1, MeOH); UV (MeOH): λmax = 273, 228 (sh) nm; ESI-MS (neg.): m/z = 483 [M - H]-, 465 [(M - H) - 18]-, 321 [(M - H) - 162]-; anal. found: C 64.37 %, H 9.18 %; calcd. for C26H44O8: C 64.44 %, H 9.15 %; NMR data, see Table [4].

Compound 16: White solid; m. p. 182 °C; [α]D 25: + 6.0° (c 0.1, MeOH); UV (MeOH): λmax = 235 (sh) nm; ESI-MS (pos.): m/z = 323 [M + H]+, 305 [(M + H) - 18]+; anal. found: C 74.46 %, H 10.64 %; calcd. for C20H34O3: C 74.49 %, H 10.63 %; NMR data, see Table [5].

Compound 17: Pale-yellow solid; m. p. 100 - 103 °C; [α]D 25: + 23.3° (c 0.1, MeOH); UV (MeOH): λmax = 220 nm; ESI-MS (pos.): m/z = 321 [M + H]+, 303 [(M + H) - 18]+; anal. found: C 71.19 %, H 8.80 %; calcd. for C19H28O4: C 71.22 %, H 8.81 %; NMR data, see Table [5] .

Compound 18: White crystals; m. p. 100 - 104 °C; [α]D 25: + 11.3° (c 0.1, MeOH); UV (MeOH): λmax = 223 nm; ESI-MS (pos.): m/z = 697 [M + H]+; anal. found: C 68.87 %, H 8.13 %; calcd. for C40H56O10: C 68.94 %, H 8.10 %; NMR data, see Table [6].

Compound 19: White amorphous solid; m. p. 175 - 180 °C; [α]D 25: + 28.3° (c 0.1, MeOH); UV (MeOH): λmax = 276, 230 (sh) nm; ESI-MS (pos.): m/z = 733 [M + Na]+; anal. found: C 67.62 %, H 7.68 %; calcd. for C40H54O11: C 67.59 %, H 7.66 %; NMR data, see Table [6].

Compound 20: White crystals; m. p. 100 - 105 °C; [α]D 25: + 35.0° (c 0.1, MeOH); UV (MeOH): λmax = 270, 225 (sh) nm; ESIMS (pos.): m/z = 711 [M + H]+, 363, 349; anal. found: C 69.34 %, H 8.20 %; calcd. for C41H58O10: C 69.26 %, H 8.23 %; NMR data, see Table [6].

Table 1 1H- and 13C-NMR data for compounds 1 - 4 (600 MHz, CD3OD)a
Position 1 2 3 4
δH δC δH δC δH δC δH δC
1a
1b
2a
2b
3a
3b
4
5
6a
6b
7a
7b
8
9
10
11a
11b
12a
12b
13
14a
14b
15
16
17a
17b
18
19
20		 1.94 dt (12.7, 6.0)
1.30 m
1.83 m
1.54 m
1.60 br dd (12.0, 6.0)
1.57 m

2.64 dd (14.5, 4.0)
1.67 m
1.26 m
1.77 m
1.48 dd (14.0, 8.8)



2.05 dd (9.5, 3.0)
1.40 m
2.07 dt (14.0, 3.3)
1.86 dt (12.0, 5.0)

2.27 d (12.7)
2.02 d (12.7)


3.81 d (11.5)
3.67 d (11.5)
1.08 s

1.28 s		 32.1

21.1

36.8

48.7
53.4
19.0

26.5

54.4
44.0
89.6
32.0

33.1

77.7
42.8

223.8
81.2
65.3

17.8
182.8
19.1		 2.14 br d (14.0)
1.61 dd (14.0, 4.5)
4.18 br dd (4.5, 3.5)

1.99 dd (12.5, 5.0)
1.87 dd (12.5, 3.5)

2.67 dd (14.0, 4.0)
1.67 m
1.26 m
1.76 m
1.50 dd (14.0, 8.2)



2.06 dd (10.0, 3.0)
1.48 m
2.11 dd (14.0, 2.5)
1.87 dt (11.5, 5.0)

2.27 d (12.6)
2.02 d (12.6)


3.81 d (11.3)
3.67 d (11.3)
1.07 s

1.29 s		 39.7

65.7

46.3

45.4
53.4
18.5

26.6

55.0
44.6
89.0
32.5

33.4

78.0
43.0

223.6
81.0
65.4

17.4
183.0
18.4		 1.98 dt (13.0, 5.5)
1.40 m
2.12 dt (12.0, 5.0)
1.41 m
3.62 dd (10.0, 5.0)


2.45 dd (14.0, 4.0)
1.67 m
1.26 m
1.77 m
1.50 dd (14.0, 8.7)



2.04 dd (10.0, 2.6)
1.43 m
2.10 dd (14.0, 3.0)
1.86 dt (12.0, 5.0)

2.28 d (12.7)
2.01 d (12.7)


3.81 d (11.5)
3.67 d (11.5)
1.15 s

1.28 s		 32.0

30.5

73.4

54.3
51.0
19.0

26.4

55.0
44.0
88.8
32.7

33.3

77.8
43.0

223.8
81.0
65.3

12.7
180.5
18.9		 2.18 br d (14.0)
1.61 dd (14.0, 5.0)
4.12 br dd (4.0, 3.0)

3.65 d (9.5)


2.62 dd (14.0, 4.0)
1.67 m
1.26 m
1.72 m
1.50 dd (14.0, 8.0)



2.02 dd (10.0, 2.5)
1.48 m
2.16 br d (12.0)
1.83 dt (12.0, 4.5)

2.27 d (12.7)
2.03 d (12.7)


3.81 d (11.5)
3.68 d (11.5)
1.15 s

1.29 s		 38.0

68.5

77.0

53.0
52.0
18.5

26.7

55.0
44.0
89.0
32.0

33.0

78.0
43.0

223.7
81.0
65.4

13.0
180.5
19.0
a J values are in parentheses and reported in Hz; chemical shifts are given in ppm.
Table 2 1H- and 13C-NMR data for compounds 5 - 8 (600 MHz, CD3OD)a
Position 5 6 7 8
δH δC δH δC δH δC δH δC
1a
1b
2a
2b
3a
3b
4
5
6a
6b
7a
7b
8
9
10
11a
11b
12a
12b
13
14a
14b
15
16
17a
17b
18
19
20		 1.97 dd (13.7, 7.5)
1.36 m
1.81 m
1.54 m
1.65 br dd (12.0, 6.0)
1.60 m

2.63 dd (14.5, 4.0)
1.67 m
1.31 m
1.68 m
1.47 dd (14.5, 8.5)



2.10 dt (9.5, 3.0)
1.32 m
1.94 m
1.87 dt (12.0, 6.0)
2.41 m
2.28 dd (12.5, 4.5)
1.65 dd (12.5, 3.0)


3.58 d (11.5)
3.46 d (11.5)
1.08 s

1.29 s		 31.8

21.1

36.8

48.8
53.4
19.0

27.1

53.0
45.0
90.2
32.5

25.5

38.3
36.9

224.0
82.0
66.7

17.0
182.0
19.0		 3.62 dd (9.5, 7.0)

2.09 m
1.61 m
1.68 br dd (12.0, 5.5)
1.60 m

2.99 dd (13.4, 6.0)
1.71 ddd (13.0, 11.0, 5.0)
1.30 m
3.80 dd (5.7, 2.0)




2.26 dt (10.0, 3.0)
1.52 m
1.88 dd (13.0, 5.0)
1.81 dt (12.5, 6.0)

2.28 d (12.0)
2.21 dd (12.0, 3.0)

2.26 m
1.08 d (6.0)

1.10 s

1.23 s		 71.9

35.0

34.4

47.4
48.7
30.0

68.4

63.6
46.0
92.3
32.5

31.6

74.8
46.2

221.0
57.0
6.92

17.4
182.6
18.4		 3.57 dd (8.0, 7.0)

2.26 m
1.51 m
1.64 br dd (12.0, 6.0)
1.62 m

2.58 dd (14.0, 5.0)
1.66 m
1.31 m
1.78 m
1.55 dd (15.0, 8.5)



2.02 dt (6.5, 5.0, 2.0)
1.76 m
1.90 dd (14.0, 5.0)
1.82 dt (12.0, 6.0)

2.35 d (11.0)
1.65 d (11.0)

2.23 m
1.13 d (6.0)

1.09 s

1.26 s		 72.1

32.7

34.7

46.7
51.2
19.3

27.3

57.5
45.6
91.6
35.0

31.4

75.0
49.0

224.0
55.0
6.74

17.4
182.0
19.2		 1.89 dt (13.5, 7.0)
1.32 m
1.81 m
1.57 m
1.71 br dd (12.0, 6.0)
1.62 m

2.59 d (10.0)
3.81 ddd (14.0, 10.0, 5.0)

1.97 dd (14.0, 5.0)
1.62 dd (14.0, 9.0)



2.03 dt (10.0, 3.0)
1.46 m
2.05 dd (14.0, 5.0)
1.88 dt (12.0, 6.5)

2.34 d (12.0)
1.62 d (12.0)

2.23 m
1.08 (6.0)

1.30 s

1.29 s		 32.4

21.2

38.1

49.0
59.7
64.7

39.0

58.0
43.1
90.3
32.5

31.0

75.0
48.0

223.2
55.0
7.30

20.1
182.6
19.1
a J values are in parentheses and reported in Hz; chemical shifts are given in ppm.
Table 3 1H- and 13C-NMR data for compounds 9 - 12 (600 MHz, CD3OD)a
Position 9 10 11 12
δH δC δH δC δH δC δH δC
1a
1b
2a
2b
3a
3b
4
5
6a
6b
7a
7b
8
9
10
11a
11b
12a
12b
13
14a
14b
15
16
17a
17b
18
19
20		 1.92 dt (13.2, 5.7)
1.24 m
1.80 m
1.55 m
1.64 br dd (12.0, 6.0)
1.60 m

2.64 dd (14.4, 4.0)
1.67 m
1.26 m
1.80 m
1.54 dd (14.0, 8.3)



1.94 dd (10.0, 3.0)
1.46 m
1.96 dd (14.0, 5.0)
1.83 dt (12.0, 6.0)

2.33 d (12.0)
1.62 d (12.0)

2.20 m
1.10 d (6.2)

1.07 s

1.26 s		 31.9

21.1

36.6

49.0
53.4
19.3

26.6

57.0
43.3
89.6
31.3

32.0

75.6
48.0

223.8
55.0
7.9

17.2
182.9
19.0		 1.99 dt (12.7, 5.7)
1.29 m
1.87 m
1.81 m
1.65 br dd (12.0, 5.5)
1.60 m

2.62 dd (14.0, 4.0)
1.66 m
1.27 m
1.83 m
1.56 dd (14.5, 8.0)



1.67 dd (9.5, 3.0)
1.47 m
1.94 dd (14.5, 3.0)
1.80 dt (13.0, 6.5)

2.33 d (11.0)
1.65 d (11.0)

2.43 t (6.0)
3.90 dd (11.5, 6.0)
3.85 dd (11.5, 6.0)
1.08 s

1.26 s		 31.7

21.0

36.3

49.0
53.0
19.0

27.0

57.0
43.9
90.0
32.6

32.0

75.6
48.3

221.0
62.0
58.3

17.0
182.7
19.0		 2.12 br d (14.0)
1.60 dd (14.0, 4.5)
4.15 br dd (4.5, 3.0)

2.00 dd (12.5, 5.0)
1.89 dd (12.5, 3.5)

2.63 dd (14.0, 4.0)
1.67 m
1.26 m
1.76 m
1.50 dd (14.0, 8.2)



1.67 dd (10.0, 3.5)
1.47 m
1.95 dd (14.0, 4.0)
1.80 dt (13.0, 6.0)

2.33 d (11.0)
1.63 d (11.0)

2.43 t (6.5)
3.90 dd (11.5, 6.0)
3.85 dd (11.5, 6.5)
1.09 s

1.28 s		 39.7

66.0

46.3

45.4
53.5
18.5

26.6

57.0
43.9
89.0
32.6

32.0

75.6
48.5

221.1
62.0
58.3

17.4
183.0
19.0		 1.89 m
0.85 dt (12.0, 3.2)
1.66 m
1.48 m
2.16 dd (13.5, 2.0)
1.06 m

1.16 dd (12.0, 2.0)
1.91 ddd (14.0, 3.0, 2.5)
1.70 ddd (14.9, 11.0, 4.0)
1.90 m
1.61 dd (14.0, 8.2)

1.16 dd (11.0, 9.5)

1.90 m
1.71 m
1.77 m
1.37 dt (13.0, 4.0)
2.50 m
2.43 d (13.0)
1.61 d (13.0)


3.60 d (11.0)
3.45 d (11.0)
1.23 s

1.08 s		 41.1

19.7

39.0

44.9
57.6
21.0

27.0

53.2
54.5
41.4
19.2

34.7

38.3
34.1

224.3
83.0
65.3

29.3
182.4
16.0
a J values are in parentheses and reported in Hz; chemical shifts are given in ppm.
Table 4 1H- and 13C-NMR data for compounds 13 - 16 (600 MHz, CD3OD)a
Position 13 14 15 16
δH δC δH δC δH δC δH δC
1a
1b
2a
2b
3a
3b
4
5
6a
6b
7a
7b
8
9
10
11a
11b
12a
12b
13
14a
14b
15a
15b
16
17a
17b
18
19
20
OCH3
1′
2′
3′
4′
5′
6′a
6′b		 1.88 m
0.86 dt (12.0, 3.5)
1.90 m
1.66 m
2.14 dd (13.0, 2.0)
1.09 m

1.20 dd (11.5, 2.5)
1.92 ddd (13.5, 3.5, 2.5)
1.73 ddd (14.5, 11.0, 4.0)
1.90 m
1.61 dd (14.0, 8.5)

1.17 dd (11.0, 9.0)

1.89 m
1.70 m
1.75 m
1.34 dt (13.0, 4.0)
2.50 m
2.41 d (14.0)
1.61 d (14.0)



3.59 d (11.0)
3.46 d (11.0)
1.21 s

0.96 s
3.67 s		 41.1

20.0

38.8

45.0
57.6
21.0

27.0

53.2
54.3
41.2
19.3

34.8

38.4
34.8

223.5

83.0
65.6

28.9
180.0
15.8
51.0		 2.14 dt (12.5, 6.0)
1.09 m
1.90 m
1.86 m
1.63 br dd (14.0, 6.0)
1.60 m

1.83 dd (12.5, 2.0)
1.66 m
1.30 m
1.77 m
1.07 dd (14.0, 8.8)



1.80 dd (9.5. 3.0)
1.48 m
2.15 m
1.43 dt (12.0, 5.0)
2.52 m
2.66 dd (14.0, 4.5)
1.77 dd (14.0, 3.0)



3.59 d (11.5)
3.48 d (11.5)
1.10 s

1.21 s
3.67 s		 38.6

20.0

36.5

46.0
50.3
19.0

28.8

53.6
45.0
78.2
32.3

30.0

37.7
35.2

222.6

82.0
66.2

17.6
182.5
29.2
51.7		 3.73 br m

1.96 m
1.47 m
1.66 dt (14.0, 4.0)
1.12 m

1.28 dd (12.0, 2.5)
1.56 m
1.28 m
1.46 m
1.40 dd (14.0, 8.0)

1.32 dd (11.0, 8.0)

2.23 m
1.46 m
2.10 m
1.53 dt (12.5, 4.5)
1.79 m
2.02 dd (14.0, 4.0)
1.04 dd (14.0, 3.5)
2.00 d (10.0)
1.13 d (10.0)

3.58 d (13.0)
3.35 d (13.0)
0.90 s
0.85 s
1.08 s

4.47 d (8.0)
3.24 dd (9.5, 8.0)
3.40 br t (9.5)
3.23 br t (9.5)
3.29 m
3.67 dd (12.0, 5.5)
3.87 (12.0, 2.5)		 71.8

27.2

35.8

34.0
49.5
20.8

42.5

43.4
42.6
47.0
18.0

26.3

48.6
38.6

49.7

86.0
66.5

33.8
21.9
18.7

100.0
75.6
78.4
71.6
77.9
62.6
 3.73 br m

2.04 m
1.51 m
1.66 dd (14.0, 4.0)
1.14 m

1.30 dd (12.0, 2.0)
1.56 m
1.40 m
2.06 m
1.46 dd (13.7, 8.0)

1.41 dd (11.0, 8.0)

1.56 m
1.12 m
1.85 m
1.54 dt (12.5, 4.5)
1.84 m
2.00 dd (13.6, 3.5)
1.10 dd (13.6, 3.0)
1.49 d (10.0)
1.47 d (10.0)

3.43 d (11.0)
3.33 d (11.0)
0.92 s
0.90 s
1.09 s		 71.6

26.4

35.7

34.0
49.5
20.8

42.2

44.7
42.6
44.3
18.7

27.8

48.5
39.0

53.6

80.8
70.6

33.8
22.0
18.7
a J values are in parentheses and reported in Hz; chemical shifts are given in ppm.
Table 5 1H- and 13C-NMR data for compounds 16 and 17 (600 MHz, CD3OD)a
Position 16 17
δH δC δH δC
1a
1b
2a
2b
3a
3b
4
5
6a
6b
7a
7b
8
9
10
11a
11b
12a
12b
13
14a
14b
15a
15b
16
17a
17b
18
19
20		 3.73 br m

2.04 m
1.51 m
1.66 dd (14.0, 4.0)
1.14 m

1.30 dd (12.0, 2.0)
1.56 m
1.40 m
2.06 m
1.46 dd (13.7, 8.0)

1.41 dd (11.0, 8.0)

1.56 m
1.12 m
1.85 m
1.54 dt (12.5, 4.5)
1.84 m
2.00 dd (13.6, 3.5)
1.10 dd (13.6, 3.0)
1.49 d (10.0)
1.47 d (10.0)

3.43 d (11.0)
3.33 d (11.0)
0.92 s
0.90 s
1.09 s		 71.6

26.4

35.7

34.0
49.5
20.8

42.2

44.7
42.6
44.3
18.7

27.8

48.5
39.0

53.6

80.8
70.6

33.8
22.0
18.7		 1.65 m
1.34 m
1.70 m
1.35 m
1.97 m
1.82 m


2.53m
2.53 m
1.46 m
1.46 m


2.24 dd (10.5, 6.0)
1.50 m
1.44 m
2.09 m
1.72 dt (12.5, 6.0)

2.01 d (11.0)
1.86 dd (11.0, 3.5)



3.88 d (11.0)
3.79 d (11.0)
1.66 s
0.93 s
 25.0

23.5

33.9

128.0
130.0
26.6

29.0

56.5
42.2
44.4
34.6

34.0

79.0
41.6

222.6

82.3
65.2

19.0
14.6
a J values are in parentheses and reported in Hz; chemical shifts are given in ppm.
Table 6 1H- and 13C-NMR data for compounds 18 - 20 (600 MHz, CD3OD)a
Position 18 19 20
δH δC δH δC δH δC
1a
1b
2a
2b
3a
3b
4
5
6a
6b
7a
7b
8
9
10
11a
11b
12a
12b
13
14a
14b
15
16
17a
17b
18
19
20
OCH3
 1′a
1′b
2′a
2′b
3′a
3′b
4′
5′
6′a
6′b
7′a
7′b
8′
9′
10′
11′a
11′b
12′a
12′b
13′
14′a
14′b
15′
16′
17′a
17′b
18′
19′
20′		 1.94 dt (12.6, 5.5)
1.33 m
1.93 m
1.59 m
1.65 dd (12.0, 6.0)
1.60 m

2.64 dd (14.0, 4.0)
1.65 m
1.29 m
1.81 m
1.66 dd (14.5, 8.5)



2.04 dd (10.0, 3.0)
1.39 m
2.12 dt (14.0, 3.5)
1.89 dt (12.0, 5.0)

2.31 d (13.5)
2.03 d (13.5)


4.46 d (11.5)
4.12 d (11.5)
1.09 s

1.29 s

1.88 m
0.86 dt (12.0, 3.2)
1.60 m
1.59 m
2.24 dd (13.0, 2.5)
1.11 m

1.16 dd (12.0, 2.0)
1.94 ddd (14.0, 3.0, 2.5)
1.88 ddd (14.9, 11.0, 4.0)
1.92 m
1.55 dd (14.0, 8.2)

1.17 dd (11.0, 9.5)

1.90 m
1.68 m
1.70 m
1.41 dt (13.0, 4.0)
2.51 m
2.45 d (13.5)
1.63 d (13.5)


3.47 d (11.5)
3.16 d (11.5)
1.24 s

0.93 s		 32.0

20.9

36.4

49.0
53.4
19.0

27.0

54.6
44.5
90.0
32.5

33.4

77.3
42.8

221.0
80.5
67.2

17.0
183.6
19.0

41.1

19.1

38.6

45.0
57.9
21.0

27.0

44.0
54.4
41.4
19.3

34.9

38.5
34.1

221.1
83.0
65.6

28.9
178.6
16.0		 2.06 dt (12.8, 5.0)
1.40 m
1.83 m
1.47 m
1.64 dd (12.0, 5.5)
1.59 m

2.62 dd (14.0, 4.5)
1.67 m
1.29 m
1.81 m
1.56 dd (14.2, 8.0)



1.93 dd (9.5, 3.0)
1.33 m
2.10 dt (14.0,4.0)
1.88 dt (12.0, 5.0)

2.30 d (12.6)
2.04 d (12.6)


4.48 d (11.5)
4.06 d (11.5)
1.08 s

1.30 s

1.93 m
1.28 dt (12.0, 3.0)
1.80 m
1.40 m
2.23 dd (13.0, 2.0)
1.05 m

1.81 dd (11.5, 2.0)
2.13 ddd (14.0, 3.0, 2.0)
2.10 ddd (14.8, 11.0, 4.0)
1.83 m
1.67 dd (14.0, 8.0)



5.77 br s

1.97 dd (12.0, 5.3)
1.80 dt (12.0, 1.5)
2.77 m
4.71 dd (5.0, 2.5)



3.58 d (11.4)
3.48 d (11.4)
1.27 s

0.99 s		 32.6

21.2

36.3

49.0
53.6
19.0

27.0

55.2
44.0
89.8
32.0

33.2

77.0
42.8

221.5
80.3
67.8

17.3
183.3
18.9

42.2

21.2

38.8

45.6
48.4
19.3

26.5

51.0
151.4
40.7
123.7

40.2

41.3
76.0

215.0
84.4
66.0

28.8
178.3
23.4		 2.19 dt (12.7, 5.0)
1.05 m
1.88 m
1.42 m
1.60 br dd (14.0, 6.0)
1.59 m

1.85 dd (12.0, 3.0)
1.65 m
1.30 m
1.87 m
1.65 dd (14.5, 8.0)



1.93 dd (9.5, 3.0)
1.48 m
2.14 dd (14.0, 4.5)
1.76 dt (12.0, 5.0)
2.52 m
2.66 dd (14.5, 13.0)
1.69 dd (13.0, 3.0)


1.07 s

1.08 s

1.20 s
3.66 s
1.92 m
1.29 dt (12.0, 2.5)
1.88 m
1.40 m
2.19 dd (13.0, 2.0)
1.11 m

1.83 dd (12.0, 2.0)
2.07 ddd (14.0, 3.0, 2.5)
1.80 ddd (14.9, 11.0, 4.0)
1.88 m
1.66 dd (13.7, 8.0)



5.72 br s

1.97 dd (13.0, 5.7)
1.81 dt (13.0, 1.5)
2.77 m
4.72 dd (5.0, 2.5)



3.58 d (11.5)
3.46 d (11.5)
1.24 s

1.00 s		 39.0

21.0

36.2

46.0
50.2
19.0

26.8

53.0
46.0
78.5
32.6

30.0

38.0
35.0

223.2
84.0
17.0

17.4
182.5
29.1
51.6
42.3

21.1

39.0

45.4
48.4
19.3

26.3

52.3
151.0
43.0
124.0

40.2

41.3
74.0

217.0
85.0
66.2

28.6
180.0
22.8
a J values are in parentheses and reported in Hz; chemical shifts are given in ppm.
#

Preparation of benzoyl ester and acetonide of 1

Compound 1 was condensed with benzoyl chloride by the standard procedure (Et3N, 4-N,N-dimethylaminopyridine) and the intermediate monobenzoyl ester 1a was converted to the acetonide derivative by a reported procedure [6].

#

Biological assays

Evaluation for cytotoxicity was carried out against the following six cell lines: Lu1 (human lung cancer), Col2 (human colon cancer), KB (human oral epidermoid carcinoma), LNCaP (hormone-dependent human prostate cancer), hTERT-RPE1 (human telomerase reverse transcriptase-retinal pigment epithelial cells), and HUVEC (human umbilical vein endothelial cells). These evaluations were performed by published procedures [7], [8]. The hollow fiber assay was conducted using Lu1, KB, and LNCaP cells, as described previously [9], [10]. In the treatment protocols, the compound was co-precipitated with PVP (polyvinylpyrrolidone; MW 360.000; Sigma, St. Louis, MU, USA) to increase solubility [11]. Paclitaxel (taxol) was used as a standard cytotoxic agent [10].

#

Results and Discussion

Compound 1 was assigned the molecular formula, C20H28O6, as established by ESI-MS ([M + H]+ at m/z = 365), 13C-, 13C DEPT NMR, and elemental analysis. The 1H-NMR spectrum (Table [1]) of 1 displayed signals due to two tertiary methyl groups at δ = 1.08 and 1.28 and one hydroxymethylene group at δ = 3.67 and 3.81. The 13C-NMR and 13C-DEPT experiments (Table [1]) revealed the presence of 20 carbon signals. A comparison of these carbon resonances with those of the related nor-kaurane γ-lactone diterpenes suggested that compound 1 possesses the same skeleton [3]. Analysis of the DQF-COSY and HSQC spectra of 1 indicated the presence of the same partial structure for rings A, B, and C as those of 13-hydroxy-15-oxozoapatlin (21), as shown in Fig. [1] [3], but indicated that ring D was the point of difference between the two molecules due to the presence of the 16,17-diol group in 1 instead of the double bond in 21. This was confirmed by the chemical shift of the keto function at C-15 at δ = 223.8 in 1 versus δ = 209.8 in 21 and the hydroxy group at C-13 at δ = 77.7 in 1 versus δ = 75.0 in 21. The structure of ring D was deduced based on the HMBC correlations of H2 - 17, H2 - 14, and H2 - 12 with C-16. The location of the keto carbon was established to be at C-15 from the correlations between C-15 and H-14a, H2 - 17, and H-7a, and the location of the 16,17-diol group was determined based on the HMBC correlations between H2 - 14, H2 - 12, and C-16 and between H2 - 17 and C-13 and C-16. The relative stereochemistry of 1 was assigned on the basis of a ROESY experiment and from the J data. The ROESY correlations between H-5/H-3ax, H-5/H2 - 17, H-5/Me-18, and between H2 - 17/H-11ax and Me-20/H-12ax, Me-20/H-14ax indicated a β orientation for H-5, H2 - 17 and an α-orientation for Me-20. The relative stereochemistry at C-13 and C-16 was determined as follows. The hydroxymethylene group at C-17 was esterified with a benzoyl group. The product obtained (1a) was converted to the acetonide 1b. The acetonide methyl groups of 1b appeared in the 1H-NMR spectrum at δ = 1.32 and 1.43 as two singlets; these data demonstrated that the 1,2-diol has the erythro configuration [12]. Thus, the structure of 1 was determined to be 10α,13α,16α,17-tetrahydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid γ-lactone.

Compound 2 (C20H28O7) exhibited in the ESI-MS an [M + H]+ ion at m/z = 381. Its NMR spectral data (Table [1]) suggested that the structure of 2 resembled that of 1, but differed in ring A. Analysis of the DQF-COSY and 1D-TOCSY spectra led to the determination of the spin systems of ring A (Table [1]). This was confirmed from HSQC and HMBC data. The α-orientation of the hydroxy group at C-2 was derived from the coupling constant values of the H-2 equatorial signal at δ = 4.18 (br dd, J = 4.5 and 3.5 Hz). On the basis of ROESY data, the relative stereochemistry of 2 was verified to be the same as that of 1. Thus, these spectral data corroborated 2α,10α,13α,16α,17-pentahydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid (19,10)-lactone as the structure of 2.

Compound 3 had a molecular formula of C20H28O7, as determined by 13C-, 13C-DEPT NMR, and ESI-MS ([M + H]+ at m/z = 381) experiments, the same as compound 2. The NMR data of 3 (Table [1]) indicated the same skeleton as compound 2; comparative analysis of the 1H-NMR spectra showed that the chemical shifts and the J values of the hydroxymethine group (Table [1]) were the points of difference. From ROESY data (rOe effect between H-3/H-5) the relative stereochemistry of OH group at C-3 was established as 3α. Consequently, compound 3 was established as 3α,10α,13α,16α,17-pentahydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid γ-lactone.

The ESI-MS of compound 4 (C20H28O8) showed a molecular ion peak at m/z = 397 that differed from that of compounds 2 and 3 by 16 amu. The proton coupling network within each ring system was traced out by DQF-COSY, 1D-TOCSY and HSQC experiments that indicated the presence of two hydroxy groups in ring A. The 1H-NMR data of 4 (Table [1]) showed signals at δ = 4.12 (br dd, J = 4.0 and 3.0 Hz) and 3.65 (d, J = 9.5 Hz) ascribable to the 2β- and 3β-protons, respectively. Analysis of 1D-ROESY experiments showed an rOe effect between H-3 and H-5, while no effects were observed when irradiating H-2. So, compound 4 was identified as 2α,3α,10α,13α,16α,17-hexahydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid (19,10)-lactone.

The 1H- and 13C-NMR spectra (Table [2]) of compound 5 (C20H28O5) were similar to those of 1 except for the presence of an additional methine group (δ = 2.41 and 38.3 ppm) instead of a quaternary hydroxy function (77.7 ppm). Comparison of chemical shifts with those of 1 suggested the absence of the 13-hydroxy group in 5. In the ROESY spectrum of 5, a correlation between H2 - 17/H-5 and H2 - 17/H-11ax confirmed that the stereochemistry of OH-16 was α; correlations between Me-20/H-12ax, Me-20/H-14ax permitted the α orientation of Me-20 to be established. Therefore, 5 was characterized as 10α,16α,17-trihydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid γ-lactone.

The 1H-NMR spectrum (Table [2]) of 6 (C20H28O6) combined with observations from the 1D-TOCSY and DQF-COSY experiments, suggested the sequences C-1 - C-3, C-5 - C-7, C-11 - C-12, and C-14 - C-17. The locations of the functional groups were confirmed by key correlations observed in the HMBC experiment. Thus, the signal of Me-17 correlated with C-13, C-16, and C-15, and that of H-14b with C-15, C-13, C-16, C-9, and C-7 permitting the keto function to be located at C-15 and the quaternary hydroxy group to be placed at C-13. The signal of H-1 correlated with C-3, C-5, C-9, and C-19 and that of Me-18 with C-5, C-19, and C-3 allowing a C-1 hydroxy group and a C-10/C-19 γ-lactone function to be established. The analysis of the J of the H-1 proton showed an axial orientation (dd, J = 9.5, 7.0 Hz), indicating therefore the α-configuration of the C-1 OH. 1D-ROESY measurements supported the proposed structure and proved the relative stereochemistry at C-5, C-7, C-9, and C-16. Thus, the irradiation of H-7 affected the Me-20 and H-14ax signals, while irradiation of H-5 interacted with Me-18 and H-1ax. The β-orientation of the hydroxy group at C-7 was confirmed by observation of a 1,3-diaxial interaction between H-5 (δ = 2.55) and OH-7. Consequently, 6 was established as 1α,7β,10α,13α-tetrahydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid γ-lactone.

The ESI-MS of compound 7 (C20H28O6) showed a protonated molecular ion peak at m/z = 349 [M + H]+. Analysis of the NMR data of compound 7 and comparison with those of 6 exhibited that 7 differed from 6 only by the absence of the OH group at C-7 (Table [2]). Therefore, 7 was identified as 1α,10α,13α-trihydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid γ-lactone.

Compound 8 (C20H28O5,) showed an [M + H]+ ion at m/z = 349. Its 1H-NMR spectrum (Table [2]), when compared to that of 7, showed the absence of the signal at δ = 3.57 (H-1) and the occurrence of a signal at δ = 3.81 ascribable to a hydroxymethine group; a further feature was the downfield shift exhibited by Me-18. Analysis of the DQF-COSY spectrum of 8 identified two different spin system for ring A (CH2-CH2-CH2) and B (CH-CHOH-CH2) when compared with analogous signals of compound 7. The β-orientation of OH-6 was evident from the chemical shift and the large J value of H-6 (δ = 3.81, ddd, J = 14.0, 10.0, 5.0 Hz) and H-5 (δ 2.59, d, J = 10.0 Hz) characteristic of two axial protons. Thus, compound 8 was identified as 6β,10α,13α-trihydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid (19,10)-lactone.

The analysis of the spectral data of compound 9 (C20H28O4) and comparison with those of 8 showed 9 to differ from 8 only in the absence of the hydroxy group at C-6 (Table [3]). Therefore, 9 was concluded to be 10α,13α-dihydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid γ-lactone.

Compound 10 (C20H28O5) exhibited spectral features characteristic of 9-methyl-15-oxo-20-norkauran-19-oic acid γ-lactone. NMR signals (Table [3]) for rings A, B, and C of 10 were close to those of 9 but differed in ring D, leading to a downfield shift of the resonances of H-16 and C-16 and an upfield shift of the C-15 signal. These changes could be explained by the presence of a hydroxymethylene at C-16 in 10 instead of a methyl group in 9. To determine the relative configuration at C-13 and C-16, the acetonide was prepared. The acetonide methyl groups of 10 displayed similar 1H-NMR chemical shifts at δ = 1.35 and 1.37 within the range characteristic of an anti-1,3-diol acetonide [6]. This was confirmed by the observed rOe correlation between the acetonide methyl group and H-11ax. In the ROESY spectrum of 10 a correlation between H2 - 17 and H-11ax led to the assignments of a β-configuration of the CH2OH group and an α-configuration of the OH at C-13. So, the structure of 10 was determined to be 10α,13α,17-trihydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid γ-lactone.

The NMR spectral data of 11 (C20H28O6) (Table [3]) suggested that the structure of 11 resembled that of 10, but differed in ring A. Analysis of NMR data led to the determination of the spin systems of ring A. The relative stereochemistry of H-2 was obtained from the J values of the signal at δ = 4.15 (br dd, J = 4.5 and 3.0 Hz). Therefore, the structure of 11 was defined as 2α,10α,13α,17-tetrahydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid (19,10)-lactone.

The ESI-MS spectrum of 12 (C20H30O5) showed a protonated molecular peak at m/z = 351 ([M + H]+). Its 1H-NMR spectrum showed two methyl singlets at δ = 1.08 and 1.23 and a hydroxymethylene group at δ = 3.45 and 3.60 (both d, J = 11.0 Hz) adjacent to a quaternary carbon, and the signal ascribable to H-13 of an ent-kaurane (δ = 2.50, m) [13]. The HSQC spectrum confirmed the presence of a CH2OH group and revealed the occurrence of a tertiary alcoholic carbon by the resonance at δ = 83.0 ppm (C-16) (Table [3]). The α (axial) disposition of the -COOH substituent followed from the downfield shift exhibited by C-4 (44.9 ppm) and the upfield shift experienced by C-3 (39.0 ppm), when compared to the corresponding signals of ent-kaur-16(17)-ene [13]. The carbon and proton chemical shifts of ring C and D could be explained by the presence of the 15-keto group (δ = 224.3). In the ROESY spectrum of 12 a correlation between H2 - 17/H-12ax and a correlation between H-5/H-9 and H-9/H2 - 17 confirmed that the stereochemistry of C-16 OH was α. Therefore, 12 was 16α,17-dihydroxy-15-oxo-ent-kaur-19-oic acid.

NMR spectral data of 13 (C21H32O5) compared with those of 12 showed that the only difference was the presence of a -COOCH3 group in 13 instead of a -COOH group in 12 (Table [4]) [14]. Hence, 13 was established as 16α,17-dihydroxy-15-oxo-ent-kaur-19-oic acid methyl ester.

The NMR spectrum of compound 14 (Table [4]) showed 21 carbon resonances. From the DQF-COSY, 1D-TOCSY, and HSQC spectral data, the spin systems C-1 - C-3, C-5 - C-7, C-12 - C-14 were established. The proton signal of H-5 correlated in the HMBC spectrum with Me-18, C-19, C-10, C-3, and C-7; the proton signal of Me-20 correlated with C-5, C-8, C-1, and C-11; the Me-18 signal correlated with C-4, C-3, C-10, and C-19; the signal of H-13 correlated with C-8, C-15, C-17, and C-11, and that of H-14 correlated with C-9, C-16, and C-7, while the signals of H2 - 17 correlated with C-15, C-8, and C-13. The relative stereochemistry at C-5, C-9, C-13, and C-16 was in agreement with that determined for compound 5 by using ROESY spectral data. Therefore, compound 14 was assigned as 10α,16α,17-trihydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid methyl ester.

Compound 15 (C26H44O8) showed an [M - H]- peak at m/z = 483 and a prominent fragment at m/z = 321 [M - H - 162]- due to the loss of a hexose unit. Its 13C-NMR spectrum (Table [4]) showed twenty-six signals of which twenty were assigned to a diterpenoid moiety and six to a saccharide portion. A HSQC experiment allowed the assignment of the substitution sites [14]. Key correlation peaks were observed in the HMBC experiment between H-1 and C-5, C-3; Me-20 and C-5, C-9, C-1; Me-18 and C-5; H-9 and C-20, C-5, C-7, C-14; H-14 and C-9, C-15, C-12; H-17 and C-15, C-13; H-1′ and C-16. The relative stereochemistry of H-1 was obtained from the coupling constant values of the signal at δ = 3.73 (1H, br m), which indicated its equatorial orientation. In the 1D-ROESY spectrum, significant correlations between H-5 and H-9, between H-9 and H2 - 17, and between Me-20 and Me-19 confirmed the relative stereochemistry at C-5, C-9, C-10, and C-16. Thus, the structure of 15 was determined to be 1β,16α,17-trihydroxy-ent-kaur-17-O-β-D-glucopyranoside.

The ESI-MS of compound 16 (C20H34O3) showed the [M + H]+ ion at m/z = 323. Analysis of the NMR data of compound 16 and comparison with those of 15 showed 16 to differ from 15 only in the absence of a glucopyranosyl unit linked at C-16 (Tables [4] and [5]). Therefore, the structure 1β,16α,17-trihydroxy-ent-kaurane was assigned to 16.

Analysis of the DQF-COSY and HSQC experiments of 17 (C19H28O4) indicated the presence of the same partial structure in rings C and D as that of 1, but showed that rings A and B were different. A double bond occurring in ring A (Δ4,5) was deduced from the signal due to a methyl linked at an sp 2 carbon at δ = 1.66 and from the subspectrum obtained by a 1D-TOCSY experiment. Irradiation of the signal at δ = 2.24 (H-10) showed connectivities with chemical shifts at δ = 1.65 and 1.34 (H2 - 1), 1.70 and 1.35 (H2 - 2), and 1.97 and 1.82 (H2 - 3) and 1.66 (Me-18). These observations were substantiated by the HMBC correlations of Me-19 (δ = 0.93) and C-10, C-8, and C-11; Me-18 (δ = 1.66) and C-3, C-5, and C-10; H-10 (δ = 2.24) and C-2, C-4, and C-8. The relative stereochemistry at C-9, C-13, and C-16 was elucidated from the ROESY spectrum to be identical to that of 1. Therefore, the structure of 17 was defined as 13α,16α,17-trihydroxy-9α-methyl-19,20-di-nor-kauran-4-en-15-one.

Compound 18 (C40H56O10) showed spectral features characteristic of a dimeric diterpenoid. Its 13C-NMR spectrum (Table [6]) showed 40 signals ascribable to a 10α,13,16α,17-tetrahydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid γ-lactone unit (compound 1) linked through a ester linkage at a 16α,17-dihydroxy-15-oxo-ent-kaur-19-oic acid unit (compound 12). The linkage was proposed to be between C-17 of the nor-kauran-19-oic acid γ-lactone unit and C-19′ of the ent-kaurane moiety, on the basis of the resonances of H2 - 17 and C-17 and of C-19′ (Table [5]). The HMBC experiment confirmed this inference through the observed correlation between H2 - 17 and C-19′. Thus, the structure of 18 was determined to be 10α,13α,16α-trihydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid γ-lactone-17-yl-16′α,17′-dihydroxy-15′-oxo-ent-kaur-19′-oate.

Compound 19 was assigned the molecular formula C40H54O11 by ESI-MS ([M + H]+ at m/z = 711) and NMR spectral data (Table [6]). NMR data indicated that 19 is a dimeric diterpenoid. On the analysis of 2D-NMR spectra, moiety I could be defined as the isolated compound 1, while moiety II was a new diterpene. The chemical shifts of protons of A and B ring spin systems exhibited a close similarity to that of compound 12. The spin system of ring C was obtained starting from proton at δ = 5.77 (H-11′) which correlated with signals at δ = 1.80 and 1.97 (H2 - 12′), 2.77 (H-13′), and 4.71 (H-14′). Thus, ring C had the structure -C=CH-CH2-CH-CHOH-C-. Moreover, rings A and B were connected with rings C and D by HMBC correlations: H-5′/C-19′, C-20′, C-1′; Me-20′/C-1′, C-5′, C-9′; H-11′/C-10′, C-8′, C-20′, C-13′, H-13/C-11′, C-8′, C-15′; H-14′/C-12′, C-9′, C-15′, C-16′; H2 - 17′/C-13′, C-15′, C-16′. Analysis of these data suggested that compound 19 is a novel dimeric ent-kaurane diterpenoid. The HMBC experiment was used to establish the linkage between the two moieties through the observed correlation between H2 - 17 and C-19′. Analysis of 1D-ROESY experiments showed rOe effect between H-14′ax and H-7ax. Thus, the structure of 19 was defined to be 10α,13α,16α-trihydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid γ-lactone-17-yl-14′α,16′α,17′-trihydroxy-15′-oxo-ent-kaur-11′-en-19′-oate.

Compound 20 was assigned the molecular formula C41H58O10 (ESI-MS, m/z = 711 [M + H]+). Its 13C-NMR data (Table [6]) indicated that 20 was a dimeric diterpenoid. Analysis of its NMR data revealed signals due to one moiety which matched very closely those obtained for moiety II of compound 19. Comparative analysis of the NMR data of this moiety with diterpenes 1 - 17 showed close similarities between this unit and compound 14; a point of difference was the absence of the CH2OH group at C-16 replaced in moiety I of 20 by a methyl group. The connectivity between moieties I and II was obtained from a HMBC experiment which showed correlations between Me-17 and C-19′, C-16, and C-13 and between Me-18 and C-5, C-4, and C-19. Compound 20 was established as 10α-hydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid methyl ester-16α-yl-14′α,16′α,17′-trihydroxy-15′-oxo-ent-kaur-11′-en-19′-oate.

To the best of our knowledge, diterpenes 1 - 20 are new natural products. The known compounds 21 - 23 were identified, by analysis of their spectroscopic data and comparison with those reported in the literature, as 13-hydoxy-15-oxozoapatlin (21) [3], 16α,17-dihydroxy-ent-kauran-19-oic acid (22) [15], and ent-kaur-16-en-19-oic acid (23) [16].

Compounds 1 - 7, 9 - 10, 12 - 20, 22 - 23 were tested for their cytotoxic activity towards six cell lines: Lu1, Col2, KB, LNCaP, hTERT-RPE1, and HUVEC [7], [8]. Paclitaxel (taxol) was used as a standard cytotoxic agent, and exhibited the following ED50 data (μg/mL) using these cell lines: Lu1 (0.002), Col2 (0.003), KB (0.0005), LNCaP (0.001), hTERT-RPE1 (0.004), and HUVEC (0.008). The results indicated that only compounds 9 and 10 showed weak activity for these cell lines, with ED50 values in the range 10 - 20 μg/mL for all the cell lines in the panel, with the exception of compound 9 in the HUVEC cell line (ED50 > 20 μg/mL). Compounds 8 and 11 were not tested because they were isolated in too small quantities for this purpose. Compound 21 was not evaluated against the cell panel because it is already known to be a cytotoxic agent [2]. 13-Hydroxy-15-oxozoapatlin (21) was evaluated in an in vivo hollow fiber model [9], [10] at doses of 25, 50, 75, and 100 mg/kg, after it was shown to be cytotoxic towards cultured KB, LNCaP, and Lu1 cells with ED50 values of 1.2, 1.5, and 5.2 μg/mL, respectively. It showed more than 50 % growth inhibition (Fig. [2]) with KB (up to 68.7 %) (A) and LNCaP (up to 87.7 %) (B) cells at the i. p. site, but no significant growth inhibition was observed with Lu1 cells (C). Relative to the PBS control, there were no significant growth inhibitory effects observed at the s. c. site with three tested cell lines. No significant weight loss was observed at each dose in any of the experiments. In conclusion, compound 21 was active with KB and LNCaP cells at the i. p. site in the hollow fiber test at the concentrations used. Mechanistically, this compound has been established by Rundle et al. [17] to be a G2 checkpoint inhibitor and an antimitotic agent.

Zoom Image

Fig. 2 The effect of 13-hydroxy-15-oxozoapatlin (21) on the growth of KB (Panel A), LNCaP (Panel B), and Lu1 (Panel C) cells implanted at the i. p. (solid column) and the s. c. (open column) compartments of NCr nu/nu mice. Results are shown as the average percentage cell growth relative to control, ± SE (bars). Changes in mouse body weight at the end of the experiment are listed at the bottom of the figure. ***, and **, treatment groups were significantly different from the control group (p < 0.0001, p < 0.001) using Student’s t-test, with n = 6 for the control group and n = 3 for treatment groups. Paclitaxel (taxol) was used as a standard cytotoxic agent [10].

#

Acknowledgements

The authors wish to thank Prof. Anibal Castillo for collecting and identifying the plant material.

#

References

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    Search in Google Scholar
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    CrossrefPubMedSearch in Google Scholar
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    CrossrefPubMedSearch in Google Scholar
  • 17 Rundle N T, Xu L, Andersen R J, Roberge M. G2 DNA damage checkpoint inhibition and antimitotic activity of 13-hydroxy-15-oxozoapatlin.  J Biol Chem. 2001;  276 48 231-6
    PubMedSearch in Google Scholar

Prof. Nunziatina De Tommasi

Dipartimento di Scienze Farmaceutiche

Università di Salerno

Via Ponte Don Melillo

84084 Fisciano (SA)

Italy

Fax: +39-89-962828

Email: detommasi@unisa.it

#

References

  • 1 Toledo C L, Kubitzki K, Prance G H. Flora de Venezuela. Vol. IV Ediciones Fundación Educación Ambiental Caracas; 1982: 411
    Search in Google Scholar
  • 2 Lee I S, Shamon L A, Chai H B, Chagwedera T E, Besterman J M, Farnsworth N R. et al . Cell-cycle specific cytotoxicity mediated by rearranged ent-kaurene diterpenoids isolated from Parinari curatellifolia .  Chem-Biol Interact. 1996;  99 193-204
    PubMedSearch in Google Scholar
  • 3 Garo E, Maillard M, Hostettmann K, Stoeckli-Evans H, Mavi SS. Absolute configuration of a diterpene from Parinari capensis .  Helv Chim Acta. 1997;  80 538-44
    CrossrefPubMedSearch in Google Scholar
  • 4 Uys A CU, Malan S F, van Dyk S, van Zyl R L. Antimalarial compounds from Parinari capensis .  Bioorg Med Chem Lett. 2002;  12 2167-9
    CrossrefPubMedSearch in Google Scholar
  • 5 Braca A, Bader A, Morelli I, Scarpato R, Turchi G, Pizza C. et al . New pregnane glycosides from Caralluma negevensis .  Tetrahedron. 2002;  58 5837-48
    CrossrefPubMedSearch in Google Scholar
  • 6 Boger D L, Hikota M, Lewis B M. Determination of the relative and absolute stereochemistry of fostriecin (CI-920).  J Org Chem. 1997;  62 1748-53
    CrossrefPubMedSearch in Google Scholar
  • 7 Likhitwitayawuid K, Angerhofer C K, Cordell G A, Pezzuto J M. Traditional medicinal plants of Thailand. XX. Cytotoxic and antimalarial bisbenzylisoquinoline alkaloids from Stephania erecta .  J Nat Prod. 1993;  56 30-8
    PubMedSearch in Google Scholar
  • 8 Seo E K, Kim N C, Mi Q, Chai H, Wall M E, Wani M C. et al . Macharistol, a new cytotoxic cinnamylphenol from the stems of Machaerium aristulatum .  J Nat Prod. 2001;  64 1483-5
    CrossrefPubMedSearch in Google Scholar
  • 9 Hollingshead M G, Alley M C, Camalier R F, Abbott B J, Mayo J G, Malspeis L. et al . In vivo cultivation of tumor cells in hollow fibers.  Life Sci. 1995;  57 131-41
    CrossrefPubMedSearch in Google Scholar
  • 10 Mi QQ, Lantvit DD, Reyes-Lim EE, Chai HH, Zhao WW, Lee I S. et al . Evaluation of the potential cancer chemotherapeutic efficacy of natural product isolates employing in vivo hollow fiber test.  J Nat Prod. 2002;  65 842-50
    CrossrefPubMedSearch in Google Scholar
  • 11 Waller D P, Zaneveld L JD, Fong H HS. In vitro spermicidal activity of gossypol.  Contraception. 1980;  22 183-7
    CrossrefPubMedSearch in Google Scholar
  • 12 Gu Z M, Fang X P, Zeng L, Kozlowski J F, McLaughlin J L. Novel cytotoxic annonaceous acetogenins: (2,4-cis and trans)-bulladecinones from Annona bullata (Annonaceae).  Bioorg Med Chem Lett. 1994;  4 473-8
    CrossrefPubMedSearch in Google Scholar
  • 13 Yamasaki K, Kohda H, Kobayashi T, Kasai R, Tanaka O. Structures of Stevia diterpene-glucosides: application of 13C NMR. Tetrahedron Lett 1976: 1005-7
    Search in Google Scholar
  • 14 Chen C Y, Chang F R, Cho C P, Wu Y C. Ent-kaurane diterpenoids from Annona glabra .  J Nat Prod. 2000;  63 1000-3
    CrossrefPubMedSearch in Google Scholar
  • 15 Wu Y C, Hung Y C, Chang F R, Cosentino M, Wang H K, Lee K H. Identification of ent-16β,17-dihydroxykauran-19-oic acid as an anti-HIV principle and isolation of the new diterpenoids annosquamosins A and B from Annona squamosa .  J Nat Prod. 1996;  59 635-7
    CrossrefPubMedSearch in Google Scholar
  • 16 Grande M, Moran J R, Macias M J, Macheño B. Carbon-13 nuclear magnetic resonance spectra of some tetracyclic diterpenoids isolated from Elaeselinum species.  Phytochem Anal. 1993;  4 19-24
    CrossrefPubMedSearch in Google Scholar
  • 17 Rundle N T, Xu L, Andersen R J, Roberge M. G2 DNA damage checkpoint inhibition and antimitotic activity of 13-hydroxy-15-oxozoapatlin.  J Biol Chem. 2001;  276 48 231-6
    PubMedSearch in Google Scholar

Prof. Nunziatina De Tommasi

Dipartimento di Scienze Farmaceutiche

Università di Salerno

Via Ponte Don Melillo

84084 Fisciano (SA)

Italy

Fax: +39-89-962828

Email: detommasi@unisa.it

   Permissions and Reprints
Zoom Image

Fig. 1 Structures of compounds 1 - 23 isolated from Parinari sprucei leaves.

Zoom Image

Fig. 2 The effect of 13-hydroxy-15-oxozoapatlin (21) on the growth of KB (Panel A), LNCaP (Panel B), and Lu1 (Panel C) cells implanted at the i. p. (solid column) and the s. c. (open column) compartments of NCr nu/nu mice. Results are shown as the average percentage cell growth relative to control, ± SE (bars). Changes in mouse body weight at the end of the experiment are listed at the bottom of the figure. ***, and **, treatment groups were significantly different from the control group (p < 0.0001, p < 0.001) using Student’s t-test, with n = 6 for the control group and n = 3 for treatment groups. Paclitaxel (taxol) was used as a standard cytotoxic agent [10].