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

Key words

Wedelia prostrata - Asteraceae - kaurenoic acid derivatives - semi-synthesis - cytotoxic activity - cancer cell lines

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 4a–4d and oxidized with PDC to yield the corresponding amide derivatives 5–8. Amide derivatives 9–10 were synthesized from 2 under treatment with pyridine/DCC and then oxidized to ketone. Ester derivatives of compounds 11–14 were obtained directly from 3 with BrR/K₂CO₃/KI in DMF. Treatment of 2 with K₂CO₃ and Br-R-Br in DMF formed 15a–15c, which subsequently converted into compounds 16a-16c under treatment with piperidine and K₂CO₃ in THF. The oxidation of 16a–16c with PDC yielded 17–19. 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 (5–14, 17–19, 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).

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 5–14, 17–19, 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 5–7 with amide groups at C-4 displayed more potent cytotoxicity than compounds 11–14 with ester groups at C-4 against the K562 cell line but were less potent to the SGC-7901 cell line. Meanwhile, compounds 11–14 with only ester groups at C-4 showed higher cytotoxic activity than compounds 17–19 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 11–13 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 17–19 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 1  In vitro cytotoxicitives of 1, 3, 5–14, 17–19, 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, 5–14, 17–19, and 24, and the general procedures for the synthesis of them are available as Supporting Information.

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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