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Planta Medica Journal of Medicinal Plant and Natural Product Research
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Luc Pieters, Antwerp, Belgium
Senior Editor
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Rudolf Bauer, Graz, Austria Veronika Butterweck, Muttenz, Switzerland Thomas Efferth, Mainz, Germany Irmgard Merfort, Freiburg, Germany Hermann Stuppner, Innsbruck, Austria Yang-Chang Wu, Taichung, Taiwan
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© Georg Thieme Verlag KG Stuttgart · New York
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362
Original Papers
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Authors
Verónica Rachel Olate 1, Mariano Walter Pertino 1, Cristina Theoduloz 2, Erdem Yesilada 3, Francisco Monsalve 2, Paulo González 2, Daniel Droguett 2, Pascal Richomme 4, A. Hamid A. Hadi 5, Guillermo Schmeda-Hirschmann 1
Affiliations
The affiliations are listed at the end of the article
Key words " (4S,9R,10R) methyl 18‑carl boxy‑labda‑8,13(E)‑diene‑ 15‑oate amide derivatives " labdane diterpenes l " gastroprotective effect l " basal cytotoxicity l " Polyalthia macropoda l " Annonaceae l
Abstract !
Starting from the diterpene (4S,9R,10R) methyl 18-carboxy-labda-8,13(E)-dien-15-oate (PMD) and its 8(9)-en isomer [PMD 8(9)-en], 11 amides were prepared and assessed for a gastroprotective effect in the ethanol/HCl-induced gastric lesions model in mice. Basal cytotoxicity of the compounds was determined on the following human cell lines: normal lung fibroblasts (MRC-5), gastric epithelial adenocarcinoma (AGS), and hepatocellular carcinoma (Hep G2). All compounds are described for the first time. At the single oral dose of 0.1 mg/kg, compounds 1, 10, and 11 presented a strong gastroprotective effect, at least comparable with that of the reference compound lanso-
Introduction !
received revised accepted
June 9, 2011 Nov. 18, 2011 Nov. 27, 2011
Bibliography DOI http://dx.doi.org/ 10.1055/s-0031-1280453 Published online January 16, 2012 Planta Med 2012; 78: 362–367 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence G. Schmeda-Hirschmann Laboratorio de Química de Productos Naturales Instituto de Química de Recursos Naturales Universidad de Talca Casilla 747 Talca, Región del Maule Chile Phone: + 56 71 20 02 88 Fax: + 56 71 20 15 73
[email protected]
Terpenes ranging from sesquiterpenes to triterpenes have been isolated and identified as gastroprotective agents of several South American crude drugs, presenting activity both in animal and in vitro models [1–7]. Some natural products belonging to the terpenes such as glycyrrhetic acid derivatives and ecabet sodium have been developed into therapeutic agents [8–12]. The effect of the Mapuche crude drug Araucaria araucana resin on gastric lesions has been reported [6]. Several studies were undertaken to disclose the structure-activity relationships of labdane diterpenes from the resin as gastroprotective agents [13–16]. The effect of diterpene amide derivatives on the gastroprotective effect was reported for labdane [13, 15–16] and abietane [17] diterpenes. While the COOH function at C-4 in Araucaria labdanes is β-configurated, the carboxylic acid function of Polyalthia diterpenes is α-configurated. To disclose the relevance of this structural feature on the gastroprotective effect, the diterpene (4S,9R, 10R) methyl 18-carboxy-labda-8,13(E)-diene-15-
Olate VR et al. New Gastroprotective Labdeneamides … Planta Med 2012; 78: 362–367
prazole at 1 mg/kg, reducing gastric lesions by 76.7, 67.7, and 77.2 %, respectively. The leucyl amide methyl ester 3, tryptophanyl amide methyl ester 5, and benzyl amide 6 of PMD presented a selective basal cytotoxicity on Hep G2 cells with IC50 values of 136.8, 105.3, and 94.2 µM, respectively, while the IC50 values towards AGS cells were 439.5, 928.0, and 937.3 µM, respectively. The three compounds did not affect fibroblast viability with IC50 values > 1000 µM. Compounds 7, 8, 10, and 11 showed no toxic effect against the three selected cell lines. Supporting information available online at http://www.thieme-connect.de/ejournals/toc/ plantamedica
oate (PMD) isolated from the stem bark of Polyalthia macropoda (Annonaceae) [18] was used as starting material to prepare new amide derivatives. The relevance of the double bond either at 8(17) or 8(9) for both activities was investigated. PMD also differs from the Araucaria labdanes by the presence of an additional double bond at Δ13 (14). Diterpene amides from labdanes with aromatic amines [13] have shown good gastroprotective activity with lower basal cytotoxicity than the parent terpenes. The effect was higher than that elicited by amides with C-protected aminoacids [15, 16]. According to Halle and Spielmann, there is a significant correlation between cytotoxicity of mammalian cell culture systems and acute oral toxicity (LD50) in animals [19]. Basal cytotoxicity of the new compounds on human cell lines can be determined as a valid model to predict starting doses for in vivo lethality assays in rodents. Following our studies on structure-activity relationships of labdane diterpenes, we now report the synthesis, gastroprotective effect, and basal cytotoxicity of new amides derived from PMD.
This is a copy of the authorʼs personal reprint
This is a copy of the authorʼs personal reprint
New Gastroprotective Labdeneamides from (4S,9R,10R) Methyl 18-carboxy-labda-8,13(E)diene-15-oate
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Materials and Methods !
Equipment and general procedure
This is a copy of the authorʼs personal reprint
Optical rotations were obtained for solutions in CHCl3 (concentrations expressed in g/100 mL) on a Jasco DIP 370 polarimeter. IR spectra were recorded on a Nicolet Nexus FT‑IR instrument. 1 H‑NMR spectra were recorded at 400 MHz, and 13C‑NMR data were obtained at 100 MHz on a Bruker Avance spectrometer (and expressed in δ). Mass spectra were measured with an EBE trisector VG Autospec Micromass spectrometer operating at 70 eV and are presented as m/z (rel. int. %). Silica gel 60 (Merck, 63–200 µm particle size) was used for column chromatography; precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) were used for thin-layer chromatography (TLC) analysis. Gel permeation was performed on Sephadex LH-20 (Pharmacia), eluting with a petroleum ether (PE):DCM : MeOH 1 : 1 : 1 mixture. Reversed-phase silica gel 100 C8 (Fluka) was used to purify the amides. TLC spots were visualized by spraying the chromatograms with p-anisaldehyde-ethanol-acetic acid-H2SO4 (2 : 170 : 20 : 10 v/v) and heating at 110 °C for 3 min. All reactions were carried out under an inert dry nitrogen atmosphere.
Preparation of derivatives
The starting compound, (4S,9R,10R) methyl 18-carboxy-labda8,13(E)-dien-15-oate (PMD) was isolated from Polyalthia macropoda [18]. The 8(9)-en derivative was obtained by treating the diterpene with HBr in acetic acid. The amides were prepared according to the literature [13, 15, 16]. To obtain the amino acid methyl ester amides (compounds 1–5) and the amides with aromatic amines (compounds 6–11), the diterpene (1 mmol) was dissolved in dry CH2Cl2 (DCM, 5 mL) and ice cooled under nitrogen flow. To this solution, oxalyl chloride (40 mmol) in dry DCM was added dropwise with stirring. The mixture was stirred at room temperature overnight, then the DCM was evaporated, and the residue vacuum dried. The dry residue was dissolved in dry DCM (10 mL). To obtain the amino acid methyl ester amides (compounds 1–5), the C-protected amino acid (2 mmol, as hydrochloride) was added as well as 3 mmol triethylamine (TEA). The reaction mixture was left at room temperature under stirring and an inert atmosphere for two days. Then, the mixture was washed with water, and the aqueous phase extracted with DCM to afford the crude reaction mixture. To prepare the amides 6–11, the aromatic amines were added to the diterpene acid chloride, and after 48 h, the mixture was worked-up as described in the previous paragraph. Purification was undertaken using a combination of gel permeation in Sephadex LH-20 [column length: 58 cm, internal diameter: 5 cm; petroleum ether (PE) : DCM : MeOH 1 : 1 : 1; flow rate 2 mL/min, fraction size: 2 mL], silica gel column chromatography (column length: 53 cm, internal diameter: 9 cm; stepwise gradient of PE, PE : EtOAc 90 : 10, 80 : 20, 70 : 30, 50 : 50, and EtOAc, fraction size: 5 mL), and reversedphase silica gel chromatography (column length: 90 cm, internal diameter: 3 cm; MeOH : H2O 80 : 20 to MeOH in 1 h 30 min; flow rate 2.5 mL/min, fraction size: 5 mL) to afford the amides.
Ethanol/HCl-induced ulcer model in mice
The gastroprotective activity of the compounds was assessed in the ethanol/HCl-induced lesion model [1, 6, 14]. The purity of the tested compounds was higher than 95 % by NMR analysis. Male Swiss albino mice weighing 30 ± 3 g were used. Animals were fed a certified Champion diet with free access to water under standard conditions of 12-h dark-light period, 50 % relative
humidity, and 22 °C room temperature. The antisecretory drug lansoprazole (2-[[[3-methyl-4-(2,2,2-trifluroethoxy)-2-pyridyl] methyl]sulfinyl]benzoimidazole) at 5 mg/kg was used as the reference compound. The tensioactive compound Tween 80 (12 %, 10 mL/kg) was used as the vehicle to suspend the terpenes. To keep the animal number to a minimum, dose-response studies were performed with PMD at doses of 10, 5, and 1 mg/kg to set the conditions for single-dose comparison of a gastroprotective effect. The products 1–8 were assessed at a single oral dose of 1 mg/kg and compounds 1, 2, 4, 5, 7–11 were tested at 0.1 mg/ kg. Mice were randomly distributed into groups of eight animals each and fasted for 24 h with free access to water previous to the experiment. The diterpenes and their derivatives, as well as lansoprazole and the vehicle, were administered to fasted animals by gavage in a volume of 0.2 mL. 50 min after the administration of the substances, all groups of animals received orally 0.2 mL of a solution containing 60 % ethanol/0.3 M HCl (ethanol/HCl) for gastric lesion induction. After 60 min of lesion induction, animals were sacrificed by cervical dislocation. The stomachs were excised and filled with 1 mL of 5% formalin for fixation. After 30 min, the stomachs were opened along the greater curvature. The length (mm) of each lesion was measured, and the lesion index expressed as the sum of the length of all lesions [20, 21]. The protocols were approved by the Universidad de Talca Institutional Animal Care and Use Committee which follows the recommendations of the Canadian Council on Animal Care under the reference FONDECYT No. 1085306 from March, 2008. Tween 80 and lansoprazole (> 98 % purity by HPLC) were purchased from Sigma-Aldrich Co.
MRC-5 and Hep G2 cell culture
Human normal lung fibroblasts (MRC-5) (ATCC CCL-171) and hepatocellular carcinoma cells (Hep G2) (ATCC HB-8065) were grown as monolayers in minimum essential Eagleʼs medium (MEM), with Earleʼs salts, 2.0 mM L-glutamine (Sigma-Aldrich Co.), and 2.2 g/L sodium bicarbonate (Sigma-Aldrich Co.) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 µg/mL streptomycin in a humidified incubator with 5 % CO2 in air at 37 °C. Cell passage was maintained between 10 and 16 for MRC-5 and between 79 and 82 for Hep G2 cells. The medium was changed every 2 days. Culture media, antibiotics, and FBS were obtained from Life Technologies Corp.
AGS cell culture
Human gastric epithelial adenocarcinoma cells (AGS) (ATCC CRL1739) were grown as monolayers in Ham F-12 medium containing 1.0 mM L-glutamine and 1.5 g/L sodium bicarbonate, supplemented with 10% heat-inactivated FBS, 100 IU/mL penicillin, and 100 µg/mL streptomycin in a humidified incubator with 5 % CO2 in air at 37 °C. Cell passage was maintained between 42 and 48. The medium was changed every 2 days.
Cytotoxicity assay
Confluent cell cultures were treated with medium containing the compounds at the following concentrations: 0, 2, 4, 6, 12, 25, 50, 100, 200, 400, 600, 800, and 1000 µM. The products were first dissolved in DMSO and then in the corresponding culture medium supplemented with 2% FBS. The final content of DMSO in the test medium and controls was 1 %. Cells were exposed for 24 h to the assayed compounds. Untreated cells served as controls. Each concentration was tested in quadruplicate together
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Original Papers
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Results were expressed as the mean ± SD. In all experiments, statistical differences between several treatments and their respective control were determined by one-way ANOVA with StudentNewman-Keuls post hoc test. The level of significance was set at p < 0.05.
1453, 1433, 1389, 1358, 1218, 1146, 772 cm−1; HR‑MS (EI): m/z = 438.2535 [M + H]+ (calcd. for C28H39NO3: 437.6142). (4S,10R) methyl 18-carboxy-labda-8(9),13(E)-dien-15-oate p-toluidylamide (10): Colorless resin; Rf 0.54 (PE : EtOAc 8 : 2); [α]20 D : − 2.15 (c 1.18, CHCl3); IR (film): vmax = 3354, 2924, 1716, 1645, 1597, 1517, 1433, 1386, 1314, 1226, 1146, 756 cm−1; HR‑MS (EI): m/z = 438.2940 [M + H]+ (calcd. for C28H39NO3: 437.293). (4S,10R) methyl 18-carboxy-labda-8(9),13(E)-dien-15-oate 3-bromoanilylamide (11): Colorless resin; Rf 0.53 (PE : EtOAc 8 : 2); [α]20 D : − 2.13 (c 1.02, CHCl3); IR (film): vmax = 3358, 2936, 1716, 1653, 1581, 1517, 1473, 1429, 1409, 1386, 1222, 1146, 756 cm−1; HR‑MS (EI): m/z = 502.2133 [M + H]+ (calcd. for C27H36BrNO3: 501.1879).
Compounds
Supporting information
(4S,9R,10R) methyl 18-carboxy-labda-8,13(E)-dien-15-oate glycyl amide methyl ester (1): Colorless resin; Rf 0.26 (PE : EtOAc 7 : 3); [α]20 D : − 282 (c 0.07, CHCl3); IR (film): vmax = 3384, 2932, 2858, 1750, 1717, 1643, 1520, 1429, 1220, 1146, 772 cm−1; HR‑MS (EI): m/z = 442.2550 [M + Na]+ (calcd. for C24H37NO5Na: 442.2570). (4S,9R,10R) methyl 18-carboxy-labda-8,13(E)-dien-15-oate valyl amide methyl ester (2): Colorless resin; Rf 0.62 (PE : EtOAc 7 : 3); [α]20 D : − 215 (c 0.15, CHCl3); IR (film): vmax = 3454, 3384, 2945, 2928, 2863, 1746, 1717, 1647, 1503, 1438, 1228, 1146, 746 cm−1; HR‑MS (EI): m/z = 462.0527 [M + H]+ (calcd. for C27H43NO5: 461.3141). (4S,9R,10R) methyl 18-carboxy-labda-8,13(E)-dien-15-oate leucyl amide methyl ester (3): Colorless resin; Rf 0.43 (PE : EtOAc 8 : 2); [α]20 D : − 327 (c 0.23, CHCl3); IR (film): vmax = 3374, 2948, 2864, 1748, 1716, 1641, 1517, 1437, 1226, 1146, 772 cm−1; HR‑MS (EI): m/z = 484.3039 [M + Na]+ (calcd. for C27H43NO5: 461.3141). (4S,9R,10R) methyl 18-carboxy-labda-8,13(E)-dien-15-oate prolyl amide methyl ester (4): Colorless resin; Rf 0.42 (PE : EtOAc 7 : 3); [α]20 D : − 650 (c 0.16, CHCl3); IR (film): vmax = 3418, 2952, 1744, 1712, 1641, 1617, 1358, 1226, 1146, 746 cm−1; HR‑MS (EI): m/z = 460.7040 [M + H]+ (calcd. for C27H41NO5 459.2985). (4S,9R,10R) methyl 18-carboxy-labda-8,13(E)-dien-15-oate tryptophanyl amide methyl ester (5): Colorless resin; Rf 0.29 (PE : EtOAc 7 : 3); [α]20 D : + 64 (c 0.13, CHCl3); IR (film): vmax = 3387, 2930, 1748, 1717, 1639, 1507, 1438, 1216, 1150, 768, 740 cm−1; HR‑MS (EI): m/z = 549.7119 [M + H]+ (calcd. for C33H44 N2O5: 548.325). (4S,9R,10R) methyl 18-carboxy-labda-8,13(E)-dien-15-oate benzyl amide (6): Colorless resin; Rf 0.31 (PE : EtOAc 8 : 2); [α]20 D : − 37 (c 0.635, CHCl3); IR (film): vmax = 3557, 3364, 2932, 1717, 1643, 1520, 1429, 1384, 1224, 1146 cm−1; HR‑MS (EI): m/z = 438.0471 [M + H]+ (calcd. for C28H39NO3: 437.6142). (4S,9R,10R) methyl 18-carboxy-labda-8,13(E)-dien-15-oate p-toluidylamide (7): Colorless resin; Rf 0.49 (PE : EtOAc 8 : 2); [α]20 D : − 12 (c 0.283, CHCl3); IR (film): vmax = 3357, 2924, 1714, 1645, 1593, 1516, 1435, 1403, 1386, 1216, 1148, 771 cm−1; HR‑MS (EI): m/z = 438.0370 [M + H]+ (calcd. for C28H39NO3: 437.294). (4S,9R,10R) methyl 18-carboxy-labda-8,13(E)-dien-15-oate 3-bromoanilylamide (8): Colorless resin; Rf 0.56 (PE : EtOAc 8 : 2); [α]20 D : − 9 (c 0.38, CHCl3); IR (film): vmax = 3368, 2924, 1717, 1643, 1586, 1516, 1479, 1384, 1220, 1187, 1138, 772 cm−1; HR‑MS (EI): m/z = 501.9320 (calcd. for C27H36BrNO3: 501.1879). (4S,10R) methyl 18-carboxy-labda-8(9),13(E)-dien-15-oate benzyl amide (9): Colorless resin; Rf 0.41 (PE : EtOAc 8 : 2); [α]20 D : − 2.98 (c 1.49, CHCl3); IR (film): vmax = 3350, 2932, 1716, 1645, 1525,
The 1H NMR data of compounds 1–11 are presented in Tables 1S and 2S, and the 13C NMR data are shown in Table 3S.
with the control and repeated three times in separate experiments. At the end of the incubation, the neutral red uptake (NRU) assay was carried out to determine cell viability [7, 22]. Results were transformed to percentage of controls to calculate the IC50 values and graphically obtained from the dose-response curves.
This is a copy of the authorʼs personal reprint
Statistical analysis
Results !
Starting from PMD and PMD 8(9)-en, 11 amides with amino acid " Fig. 1). The methyl esters and aromatic amines were prepared (l percent w/w yields of the obtained compound was as follows: 1: 37; 2: 79; 3: 78; 4: 62; 5: 41; 6: 85; 7: 79; 8: 50; 9: 82; 10: 62; 11: 47. The spectroscopic data of the compounds are in agreement with the proposed structures. All compounds are described for the first time. When assessed at the single oral dose of 1, 5, and 10 mg/kg, PMD showed a strong gastroprotective effect reducing the lesion index by 68.3, 72.5, and 85.4 %, respectively. This fact suggests a strong activity but limited solubility of the compound. As the activity of PMD was strong at 1 mg/kg, the gastroprotective effect of the semisynthetic compounds 1–8 was first assessed at a single oral dose of 1 mg/kg in mice. As compounds 1–8 at 1 mg/kg reduced the lesion index in the range of 39.9–80.6 %, the derivatives 1, 2, 4, 5, and 7–11 were evaluated for gastric ulcer prevention at the " Table 1. dose of 0.1 mg/kg. The results are summarized in l While dose-response was not observed at the doses of 1 and 0.1 mg/kg for the PMD amino acid methyl ester amides 1, 2, and 4, the best gastroprotective effect at the lowest dose was observed for the glycyl, p-toluidyl, and 3-bromoanilyl amides 1, 10, and 11, reducing lesions by 76.7, 67.7, and 77.2 %, respectively at 0.1 mg/kg. When comparing the gastroprotective effect of PMD amides with the amides prepared with PMD 8(9)-en, the p-toluidyl and 3-bromoanilyl amides 10 and 11 were more active than compounds 7 and 8, suggesting that the position of the double bond either at 8(17) or 8(9) is relevant for the biological effect of this group of diterpenes. The diterpenes PMD and PMD 8(9)-en as well as the 11 semisynthetic derivatives were assessed for basal cytotoxicity in lung fibroblasts, gastric epithelial cells, and hepatocytes. The results are " Table 2. The compounds 7, 8, 10, and 11 were depresented in l void of toxicity in the three cell lines with IC50 values ≥ 1000 µM. Compound 4 was the most toxic and less selective product with IC50 values of 38.5, 28.6, and 34.9 µM against fibroblasts, AGS, and Hep G2 cells, respectively. PMD 8(9)-en was about three times less cytotoxic than the diterpene bearing the exomethylene group towards fibroblasts and AGS cells, but more toxic against Hep G2 cells. Compounds 3, 5, and 6 presented a selective effect on Hep G2 cells with IC50 values of 136.8, 105.3, and 94.2 µM, respectively; while IC50 values towards AGS cells were 439.5, 928.0,
Olate VR et al. New Gastroprotective Labdeneamides … Planta Med 2012; 78: 362–367
This is a copy of the authorʼs personal reprint
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Compound
PMD 1 mg/kg 5 mg/kg 10 mg/kg Lansoprazole 1 mg/kg Untreated control Compound 1 2 3 4 5 6 7 8 9 10 11 Untreated control
Lesion index
% Lesion
Lesion index
% Lesion
mean ± SD (mm)
reduction
mean ± SD (mm)
reduction
20.4 ± 12.5** 17.7 ± 15.6** 9.4 ± 9.1** 13.8 ± 11.2** 64.4 ± 57.7 Dose: 1 mg/kg 11.3 ± 10.0** 16.4 ± 18.9* 8.4 ± 7.6** 12.9 ± 9.9** 26.1 ± 31.2 17.9 ± 13.3* 18.8 ± 17.8* 14.5 ± 11.8* ND ND ND 43.4 ± 33.6
68.3 72.5 85.4 78.6 0.0
Dose: 0.1 mg/kg 10.4 ± 12.7** 18.7 ± 13.6* ND 16.5 ± 13.3* 39.1 ± 22.9 ND 17.8 ± 13.7* 24.3 ± 23.0 34.0 ± 17.5* 20.8 ± 28.2* 14.7 ± 18.1**
74.0 62.2 80.6 70.3 39.9 58.7 56.7 66.6
Table 1 Dose-response gastroprotective effect of PMD and gastroprotective effect of the compounds 1–11 on the ethanol/HCl induced gastric lesions in mice at the single oral dose of 1 mg/kg (compounds 1–8) and 0.1 mg/kg (compounds 1, 2, 4, 5, and 7–11).
76.7 56.9
62.0 9.9
59.0 44.0 47.2 67.7 77.2
ND: not determined; * p < 0.05 different from control; ** p < 0.01 different from control one-way ANOVA with Student-Newman-Keuls post hoc test; n = 8
and 937.3 µM, respectively. The three compounds were devoid of toxicity against fibroblasts with an IC50 value > 1000 µM. Several of the new derivatives were less cytotoxic than the reference compound lansoprazole. Comparing the effect of the PMD derivatives with PMD 8(9)-en derivatives, similar cytotoxicities were observed for the pairs 7 and 10 (p-toluidyl) as well as 8 and 11 (3-bromoanilyl), but relevant differences were observed for Hep G2 cells, where the IC50 values of 6 and 9 (benzyl) were 94.2 and 332.1 µM, respectively. Compared to fibroblasts and AGS cells, compounds 5 and 6 showed a 10-fold increase in basal cytotoxicity on hepatocytes, suggesting some selectivity against this cell line.
Discussion !
The gastroprotective activity of the new PMD amides was compared with other semisynthetic diterpene amides at single oral doses. At 1 and 0.1 mg/kg, the effect of the new compounds was higher than at 100 mg/kg of 15-hydroxy or 15-acetoxyimbricatolic acid derivatives [6], 25 mg/kg of labdeneamides with aromatic amines [13], 50 mg/kg amino acid methyl esters amides [15], or 25 mg/kg of amino acid ester monoamides and diamides [16]. Using structural modifications of natural products to disclose the critical positions for their biological effect as well as to find more active compounds and selective activity is an approach that has been successfully used for other terpenes [23–26]. PMD differs from the Araucaria labdanes in the stereochemistry of the
Olate VR et al. New Gastroprotective Labdeneamides …
Planta Med 2012; 78: 362–367
This is a copy of the authorʼs personal reprint
This is a copy of the authorʼs personal reprint
Fig. 1 Structure of (4S,9R,10R) methyl 18-carboxy-labda-8,13(E)-dien-15-oate (PMD) and its 8(9) en isomer and the semisynthetic compounds 1–11. Reagents and conditions: a appropriate amino acid or amine, DCC, DMAP, CH2Cl2; b CH3COOH/HBr, rt, 24 h; c appropriate amine, DCC, DMAP, CH2Cl2. Abbreviations: Gly: glycine methyl ester; Val: valine methyl ester; Leu: leucine methyl ester; Pro: proline methyl ester; Trypt: tryptophane methyl ester.
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Compound
Cytotoxicity (IC50 ± SEM, µM) MRC-5
PMD PMD 8(9)-en 1 2 3 4 5 6 7 8 9 10 11 Lansoprazole
122.0 ± 7.2 312.3 ± 15.6 153.5 ± 9.2 169.0 ± 10.1 > 1000 38.5 ± 2.3 > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 306 ± 11
AGS
Hep G2
95.1 ± 4.3 271.1 ± 13.6 75.5 ± 4.5 145.9 ± 10.2 439.5 ± 26.4 28.6 ± 1.7 928.0 ± 55.7 937.3 ± 56.2 > 1000 > 1000 > 1000 > 1000 > 1000 162 ± 6
> 1000 200.7 ± 10.0 73.8 ± 3.7 64.2 ± 3.2 136.8 ± 8.2 34.9 ± 2.1 105.3 ± 6.3 94.2 ± 5.7 > 1000 > 1000 332.1 ± 16.6 1000 > 1000 221 ± 9
Table 2 Cytotoxicity of PMD, PMD 8(9)-en, and the compounds 1–11 expressed as IC50 values (µM) towards normal lung fibroblasts (MRC-5), gastric adenocarcinoma cells (AGS), and hepatocellular carcinoma cells (Hep G2).
Confluent cultures were treated with culture medium containing the compounds at concentrations ranging between 0 and 1000 µM for 24 h. Cell viability was determined by the neutral red uptake assay. Data are expressed as arithmetic mean values of three different ex-
COOH function at C-18 and the Δ13(14) double bond. Therefore, new studies including other diterpenes from the same group are required to obtain a better insight into the structure-activity relationships of labdane diterpenes. The Polyalthia diterpene used to prepare the new amides was shown to display a strong leishmanicidal effect against the promastigote form of Leishmania donovani donovani [18, 27] and is structurally related to agathic acid, occurring in gymnosperms. Thus, new biological activities should be expected for the novel compounds. Diterpenes related to PMD with antimicrobial effect have been isolated from Copaifera paupera oleoresin [28]. The new diterpenes described in this report proved to be very active gastroprotective agents. Using the same gastric lesion induction model (ethanol/HCl) to assess the gastroprotective effect of the diterpenes, the new compounds presented a better effect than naturally occurring and semisynthetic labdanes [6, 13–16], dehydroabietic acid derivatives [17], abietanes [6, 13–16], ferruginol [21], and solidagenone derivatives [1, 4, 5]. Several of the new compounds showed a strong gastroprotective effect without a cytotoxic effect, suggesting potential as new antiulcerogenic agents. Further research work is required to disclose the mechanisms of action involved in the gastroprotective effect of the new compounds.
Acknowledgements !
This work was supported by FONDECYT, Project No. 1085306 and Programa de Investigación en Productos Bioactivos, Universidad de Talca. VRO thanks the Universidad de Talca for a Doctoral grant, and MWP thanks the Programa Bicentenario de Ciencia y Tecnologia PSD-50 for a postdoctoral fellowship. The skilful technical work of Irene Manriquez is gratefully acknowledged.
Conflict of Interest !
The authors state no conflict of interest.
Affiliations 1 2 3 4 5
Instituto de Química de Recursos Naturales, Universidad de Talca, Talca, Chile Facultad de Ciencias de la Salud, Universidad de Talca, Talca, Chile Faculty of Pharmacy, Yeditepe University, Istanbul, Turkey SONAS – IFR QUASAV, UFR des Sciences Pharmaceutiques, Angers, France Department of Chemistry, University of Malaya, Kuala Lumpur, Malaysia
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