Planta Medica Letters 2015; 2(01): e48-e51
DOI: 10.1055/s-0035-1557861
Letter
Georg Thieme Verlag KG Stuttgart · New York

Semi-Synthesis of Kaurenoic Acid Derivatives and Their In Vitro Cytotoxic Activities

Lei Zhang
1  Key Laboratory of Natural Drug Pharmacology in Fujian Province, School of Pharmacy, Fujian Medical University, Fuzhou, P. R. China
,
Fei Wang
1  Key Laboratory of Natural Drug Pharmacology in Fujian Province, School of Pharmacy, Fujian Medical University, Fuzhou, P. R. China
,
Xin-hua Ma
1  Key Laboratory of Natural Drug Pharmacology in Fujian Province, School of Pharmacy, Fujian Medical University, Fuzhou, P. R. China
,
Fang Zhou
1  Key Laboratory of Natural Drug Pharmacology in Fujian Province, School of Pharmacy, Fujian Medical University, Fuzhou, P. R. China
,
Sheng-mei Wen
1  Key Laboratory of Natural Drug Pharmacology in Fujian Province, School of Pharmacy, Fujian Medical University, Fuzhou, P. R. China
,
Tian-hua Zhong
2  Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen, P. R. China
,
Quan-yu Liu
1  Key Laboratory of Natural Drug Pharmacology in Fujian Province, School of Pharmacy, Fujian Medical University, Fuzhou, P. R. China
,
Shi-wu Chen
3  School of Pharmacy, Lanzhou University, Lanzhou, P. R. China
,
Yong-hong Zhang
1  Key Laboratory of Natural Drug Pharmacology in Fujian Province, School of Pharmacy, Fujian Medical University, Fuzhou, P. R. China
› Author Affiliations
Further Information

Correspondence

Yong-hong Zhang
Key Laboratory of Natural Drug Pharmacology in Fujian Province
School of Pharmacy
Fujian Medical University
88 Jiao Tong Road
Fuzhou 350004
P. R. China
Phone: +86 5 91 22 86 20 16   
Fax: +86 5 91 22 86 20 16   

Publication History

received 11 November 2014
revised 22 June 2015

accepted 09 July 2015

Publication Date:
06 November 2015 (online)

 

Abstract

The cytotoxic activities of the diterpene kaurenoic acid (1) and its 15 semi-synthesis derivatives were assessed on human cell cultures. The human tumor cells used comprised colon (SW620 and SW480), pancreatic (PANC-1 and BxPC-3), stomach (SGC-7901), esophageal (Eca-109), and leukemia (K562 and HL-60). Kaurenoic acid was inactive against the tumor cell lines; however, its derivatives which contain α,β-unsaturated ketone rendered compounds with cytotoxic activity. Compounds 514, 1719, and 24 with a substitution at the C-4 position showed significant inhibitory activity against the tested cell lines, while compound 3, without a substitution at the C-4 position, was slightly less active in these cell lines. The SW620 colon cancer cell was highly susceptible to all of the tested derivatives.


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Abbreviations

BrR: alkyl bromide
Br-R-Br: dibromoalkanes
DCC: dicyclohexylcarbodiimide
DMF: dimethylformamide
DMSO: dimethylsulfoxide
EI-MS: electron impact mass spectrometry
EtBr: bromine ethane
EtOH: ethanol
HOBt: hydroxybenzotriazole
HR-MS: high resolution mass spectrometer
IR: infrared spectrum
KI: potassium iodide
MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide
OD: optical density
PDC: pyridinium dichromate
t-BuOOH: peroxide tert-butyl alcohol
THF: tetrahydrofuran
TMS: tetramethylsilane
TLC: thin layer chromatography

Kaurenoic acid (1) [1], [2] is one of the active constituents in Wedelia prostrata (Hook. et Arn.) Hemsl. (Asteraceae) [3], a traditional Chinese herbal medicine [4]. It is an ent-kaurane diterpenoid [5], which is claimed to have important biological activities, mainly antimicrobial [6], antibacterial [7], cytotoxic [8], [9], anti-inflammatory [10], and anticonvulsant properties [11]. In the present paper, we describe the semi-synthesis of kaurenoic acid derivatives and their preliminary cytotoxic activities. A conversion of the 15-hydroxy group of kaurenoic acid to a ketone is made in order to incorporate an α,β-unsaturated ketone into the ent-kaurane skeleton. It is well know that a main structural determinant for cytotoxicity is present in an α,β-unsaturated ketone system [12], [13], which likely serves as an alkylating center and can be part of an ester, ketone, or lactone moiety. We also report several transformations on the carboxylic acid group at the C-4 position.

Compound 2 was obtained from 1 after treatment with SeO2/t-BuOOH in THF. Oxidation of the 15-hydroxy group of compound 2 using PDC produced α,β-unsaturated ketone 3 (Fig. 1 S, Supporting Information). Compound 2 was amidated with RNH2/DCC/HOBt in THF/DMF to yield 4a4d and oxidized with PDC to yield the corresponding amide derivatives 58. Amide derivatives 910 were synthesized from 2 under treatment with pyridine/DCC and then oxidized to ketone. Ester derivatives of compounds 1114 were obtained directly from 3 with BrR/K2CO3/KI in DMF. Treatment of 2 with K2CO3 and Br-R-Br in DMF formed 15a15c, which subsequently converted into compounds 16a-16c under treatment with piperidine and K2CO3 in THF. The oxidation of 16a16c with PDC yielded 1719. Compound 1 was esterfied with EtBr to give 20, which was reduced with LiAlH4 to form the alcohol 21. The reaction of 21 with Ac2O formed the corresponding ethyl ester 22. The reaction of 22 with SeO2 and t-BuOOH obtained compound 23 and the oxidation of 23 yielded 24 ([Fig. 1]). Compound 3 has been reported previously [14], while the 14 derivatives (514, 1719, 24) were reported here for the first time. The structures of the derivatives were confirmed by 1H-NMR, 13C-NMR, IR, HR-MS, EI-MS, and ESI-MS data (see Supporting Information).

Zoom Image
Fig. 1 Structure of kaurenoic acid (1) and its derivatives.

The cytotoxic activities of compound 1 and its 15 semi-synthesis derivatives were assessed on eight human cell lines ([Table 1]). The results of the cytotoxicity assays indicated that compound 1, without the α,β-unsaturated ketone, was inactive, while the 15 derivatives, which do contain this moiety, were active against all or some of the cell lines. Thus, as proposed in the literature [12], [13], the α,β-unsaturated ketone is the active center, possibly acting as an alkylation site. Compound 3 without a substitution at the C-4 position had moderate activities to SGC-7901 and K562 with IC50 values ranging from 7.37 to 7.53 µM, while compounds 514, 1719, and 24 with a substitution at the C-4 position showed stronger inhibitory activity than compound 3 to those tumor cell lines with IC50 values ranging from 0.25 to 2.47 µM. This indicated that the substitution of the acid moiety at the C-4 position led to significant changes in the cytotoxic activity. Compounds 57 with amide groups at C-4 displayed more potent cytotoxicity than compounds 1114 with ester groups at C-4 against the K562 cell line but were less potent to the SGC-7901 cell line. Meanwhile, compounds 1114 with only ester groups at C-4 showed higher cytotoxic activity than compounds 1719 with piperidine groups conjunct to them at C-4 to SGC-7901. Therefore, different kinds of substituted groups would cause different effects to different cell lines. The cytotoxicity of compounds 1113 with ethyl, propyl, and butyl ester groups at C-4 implied that the elongated aliphatic chain length did not influence their activities. This was yet again evidenced by the cytotoxicity of compounds 1719 with piperdinethyl, piperdinepropyl, and piperdinebutyl ester groups at the C-4 position. The activity of compounds 11 and 24 indicated that inversion of the ester bond had little affect on the activity. The SW620 colon cancer cell was highly susceptible to all tested derivatives, and the standard deviation of their average IC50 from compound 3 to compound 24 was 0.16 µM.

Table 1In vitro cytotoxicitives of 1, 3, 514, 1719, and 24 against selected tumor cell lines as IC50 (µM).

Compound

SW620

SW480

PANC-1

BxPC-3

SGC-7901

Eca-109

K562

HL-60

α Cell line: SW620 = colon; SW480 = colon; PANC-1 = pancreatic; BxPC-3 = pancreatic; SGC-7901 = stomach; Eca-109 = esophageal; K562 = leukemia; HL-60 = leukemia; NT: not tested

1

> 100

NT

> 100

NT

> 100

> 100

> 100

> 100

3

1.01

NT

5.60

NT

7.37

NT

7.53

7.91

5

0.98

NT

3.36

3.28

4.51

1.85

0.53

1.68

6

0.81

NT

0.90

1.46

2.46

1.23

0.25

1.40

7

0.97

NT

1.64

1.48

2.53

1.24

0.38

1.40

8

0.99

1.16

1.97

NT

1.95

1.26

2.47

0.42

9

0.73

NT

NT

NT

1.15

NT

1.09

NT

10

0.58

NT

NT

NT

2.65

NT

0.93

NT

11

0.70

1.13

1.22

NT

1.63

1.92

2.09

0.93

12

1.06

NT

NT

NT

1.28

NT

0.98

NT

13

0.89

NT

NT

NT

1.59

NT

1.21

NT

14

0.87

NT

NT

NT

1.23

NT

0.96

NT

17

0.96

1.48

1.64

NT

3.14

2.46

1.64

1.48

18

0.73

1.29

1.45

NT

2.59

1.93

1.29

1.09

19

0.70

1.14

1.45

NT

2.19

1.91

1.60

1.25

24

1.10

NT

NT

NT

2.47

NT

1.77

NT

Cisplatin

13.26

15.58

10.78

5.17

8.92

2.76

4.98

1.92

This semi-synthesis of kaurenoic acid derivatives has revealed several compounds with increased cytotoxic activity. Further studies are required to lower toxicity against normal cells and enhance the effect against cancer cell lines.

Materials and Methods

Isolation: The staring material kaurenoic acid [ent-kaur-16-en-19-oic acid, (1; [Fig. 1])] was isolated from the mangrove-associated plant of W. prostrata as previously described [3].

General: IR spectra were measured on a Nicolet FT-IR spectrometer with KBr pellets. 1H-NMR and 13C-NMR spectra were recorded on a Bruker AVANCE III spectrometer using TMS as the internal standard and CDCl3 as the solvent, reported in Supporting Information. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals. Column chromatography was performed on silica gel (100–200 mesh and 200–300 mesh; Qingdao Marine Chemical Group Corporation), ESI-MS data were obtained using a Bruker APEX II FT-MS, and EI-MS data was obtained with a ThermoFinnigan DECAX-30000 mass spectrometer. HR-ESI-MS data were obtained with a Bruker APEX II mass spectrometer. Optical rotations were obtained using a JASCO-20 polarimeter. TLC was carried out on silica gel GF254 on glass plates (Qingdao Marine Chemical, Inc.) using various solvent systems. The spots were visualized under UV light or by spraying with 5 % H2SO4 in EtOH followed by heating. All other reagents were purchased from Aladdin Reagent Company in analytic grade.

Cytotoxic activities against human tumor cell lines including SW620, SW480, PANC-1, BxPC-3, SGC-7901, Eca-109, K562, and HL-60 were evaluated with the MTT assay method [15].

15-Oxo-kaurenoic acid (3) [14]: amorphous solid (36 % yield); m. p. 179.8–180.9 °C; IR (KBr): ν max = 3106, 2926, 1682, 1731 cm−1; ESI-MS: m/z (rel. int.) = 316 [M]+ (95), 317 (26), 301 (27), 148 (60), 91 (53); anal. C 75.94, H 8.86, calcd for C20H28O3, C 75.91, H 8.92.

15-Oxo-kaurenoic acid propanamide (5): white foamy solid (19 % yield); m. p. 156.4–157.6 °C; [α]D 24: − 13.5 (CHCl3, c 0.10); IR (KBr): ν max = 3380, 2926, 2860, 1720, 1638, 1517, 1463 cm−1; HR-ESI-MS: [M + Na]+ m/z = 380.2568 (calcd. for C23H35NO2Na: 380.2566); EI-MS: m/z (rel. int.) = 357 [M]+ (90), 358 (23), 329 (95), 228 (76).

15-Oxo-kaurenoic acid isopropyl amide (6): white foamy solid (24 % yield); [α]D 24: − 12.9 (CHCl3, c 0.10); IR (KBr): ν max = 3418, 2958, 2860, 1724, 1632, 1518, 1448 cm−1; HR-ESI-MS: [M + Na]+ m/z = 380.2568 (calcd. for C23H35NO2Na: 380.2566); ESI-MS: m/z (rel. int.) = 357 [M]+ (100), 358 (26), 329 (97) 228 (48).

15-Oxo-kaurenoic acid butyramide (7): white foamy solid (21 % yield); [α]D 24: − 13.9 (CHCl3, c 0.10); IR (KBr): ν max = 3375, 2915, 2854, 1726, 1632, 1512, 1452 cm-1; HR-ESI-MS: [M + Na]+ m/z = 394.2722 (calcd. for C24H37NO2Na: 394.2723); EI-MS: m/z (rel. int.) = 371 [M]+ (56), 343 (60), 228 (45), 209 (48) 142 (100).

15-Oxo-kaurenoic acid benzoylamide (8): white foamy solid (20 % yield); [α]D 24: − 14.9 (CHCl3, c 0.10); m. p. 210.2–211.6 °C; IR (KBr): ν max = 3386, 2920, 2849, 1720, 1638, 1517, 1452, 1249 cm−1; HR-ESI-MS: [M + Na]+ m/z = 428.2567 (calcd. for C27H35NO2Na: 428.2566); ESI-MS: m/z (rel. int.) = 405 [M]+ (52), 377 (34), 243 (38), 228 (41).

15-Oxo-kaurenoic acid phenylalanine methyl ester amide (9): white amorphous solid (20 % yield); [α]D 24: − 11.7 (CHCl3, c 0.10); IR (KBr): ν max = 3408, 2942, 2855, 1726, 1643, 1517 cm−1; HR-ESI-MS: [M + Na]+ m/z = 500.2771 (calcd. for C30H39NO4Na: 500.2777); ESI-MS: m/z (rel. int.) = 477 [M]+ (100), 423 (5), 380 (6).

15-Oxo-kaurenoic acid valine methyl ester amide (10): white amorphous solid (22 % yield); [α]D 24: − 15.2 (CHCl3, c 0.10); IR (KBr): ν max = 3413, 2936, 2866, 1736, 1720, 1660, 1506, 1194 cm−1; HR-ESI-MS: [M + Na]+ m/z = 452.2783 (calcd. for C26H39NO4Na: 452.2777); ESI-MS: m/z (rel. int.) = 429 [M]+ (100), 398 (8), 380 (6).

15-Oxo-kaurenoic acid ethyl ester (11): amorphous solid (27 % yield), m. p. 148.5–149.6 °C; [α]D 24: − 14.5 (CHCl3, c 0.10); IR: (KBr) ν max = 3426, 2950, 2926, 1728, 1692, 1452, 1245 cm−1; HR-ESI-MS: [M + Na]+ m/z = 367.2253 (calcd. for C22H32O3Na: 367.2249); EI-MS: m/z (rel. int.) = 344 [M]+ (100), 329 (11), 271 (48), 91 (37).

15-Oxo-kaurenoic acid propyl ester (12): amorphous solid (23 % yield); [α]D 24: − 6.4 (CHCl3, c 0.10); IR: (KBr) ν max = 3426, 2955, 2926, 1727, 1692, 1456, 1245 cm−1; HR-ESI-MS: [M + Na]+ m/z = 382.2400 (calcd. for C23H34O3Na: 381.2406); EI-MS: m/z (rel. int.) = 359 [M + H]+ (100), 343 (16), 318 (5).

15-Oxo-kaurenoic acid butyl ester (13): amorphous solid (25 % yield); [α]D 24: − 14.1 (CHCl3, c 0.10); IR (KBr): ν max = 3428, 2955, 2926, 1729, 1692, 1456, 1244 cm−1; HR-ESI-MS: [M + Na]+ m/z = 395.2565 (calcd. for C24H36O3Na: 395.2562); EI-MS: m/z (rel. int.) = 373 [M + H]+ (100), 357 (6), 318 (5).

15-Oxo-kaurenoic acid benzyl ester (14): amorphous solid (31 % yield); [α]D 24: − 17.8 (CHCl3, c 0.10); IR (KBr): ν max = 3418, 2942, 2855, 1721, 1638, 1522, 1241 cm−1; HR-ESI-MS: [M + Na]+ m/z = 429.2403 (calcd. for C27H34O3Na: 429.2406); EI-MS: m/z (rel. int.) = 407 [M + H]+ (100), 389 (13), 338 (33), 315 (50).

15-Oxo-kaurenoic acid piperdinethyl ester (17): yellow oily liquid (17 % yield); [α]D 24: − 11.9 (CHCl3 , c 0.10); IR (KBr): ν max = 2931, 2854, 1721, 1638, 1457, 1221, 1162 cm−1; HR-ESI-MS: [M + Na]+ m/z = 450.2979 (calcd. for C27H41NO3Na: 450.2984); ESI-MS: m/z (rel. int.) = 427 [M]+ (5), 111 (34), 98 (100).

15-Oxo-kaurenoic acid piperdinepropyl ester (18): yellow oily liquid (18 % yield); [α]D 24: − 12.2 (CHCl3, c 0.10); IR (KBr): ν max = 2931, 2849, 1721, 1643, 1457, 1222, 1156 cm−1; HR-ESI-MS: [M + H]+ m/z = 442.3322 (calcd. for C28H44NO3: 442.3321); ESI-MS: m/z (rel. int.) = 441 [M]+ (8), 413 (4), 149 (15), 98 (100).

15-Oxo-kaurenoic acid piperdinebutyl ester (19): yellow oily liquid (21 % yield); [α]D 24: − 12.7 (CHCl3, c 0.10); IR (KBr): ν max = 2926, 2849, 1720, 1671, 1469, 1436, 1227, 1151 cm−1; HR-ESI-MS: [M + Na]+ m/z = 478.3293 (calcd. for C29H45NO3Na: 478.3297); EI-MS: m/z (rel. int.) = 455 [M]+ (8), 156 (10), 98 (100).

ent-15-Oxo-kaur-16-en-19-acetoxy (24): white crystals (19 % yield); m. p. 156.4–157.6 °C; [α]D 24: − 12.0 (CHCl3, c 0.10); IR (KBr): ν max = 2932, 2834, 1732, 1638, 1446, 1391, 1238, 1024 cm−1; HR-ESI-MS: [M + Na]+ m/z = 367.2244 (calcd. for C22H32O3Na: 367.2249); EI-MS: m/z (rel. int.) = 344 [M]+ (100), 318 (83), 304 (42).

The MTT assay was performed in 96-well plates. Test cells at the log phase of their growth cycle (3 × 104 cell/mL) were added to each well (100 µL/well), then treated in three replicates at various concentrations of the samples (0.1–100 µg/mL), and incubated for 24 h at 37 °C in a humidified atmosphere of 5 % CO2. After 48 h, 20 µL of MTT solution (5 mg/mL) per well were added to each cultured medium, which were then incubated for a further 4 h. Then, DMSO was added to each well (150 µL/well). After 15 min at room temperature, the OD of each well was measured on a microplate reader at a wavelength of 570 nm. IC50 values were obtained by a linear regression analysis of percent absorbance versus log drug concentration.

Supporting information

The 1H-NMR, 13C-NMR, EI-MS, ESI-MS, and HR-MS data of compounds 3, 514, 1719, and 24, and the general procedures for the synthesis of them are available as Supporting Information.


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Acknowledgements

This work was sponsored by the Natural Sciences Foundation of Fujian Province (No. 2010J01179), the Science and Technology Project of Fujian Province (No. 2012Y0035), and the Open Project of National Marine Bureau Key Laboratory of Marine Biogenetic Resources (HY201506).


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Conflict of Interest

The authors declare no conflict of interest.

Supporting Information


Correspondence

Yong-hong Zhang
Key Laboratory of Natural Drug Pharmacology in Fujian Province
School of Pharmacy
Fujian Medical University
88 Jiao Tong Road
Fuzhou 350004
P. R. China
Phone: +86 5 91 22 86 20 16   
Fax: +86 5 91 22 86 20 16   


Zoom Image
Fig. 1 Structure of kaurenoic acid (1) and its derivatives.