Synthesis, Gastroprotective Effect and Cytotoxicity of New Amino Acid Diterpene Monoamides and Diamides †

Following our studies on the gastroprotective effect and cytotoxicity of terpene derivatives, new amides were prepared from the diterpene 8(17)-labden-15,19-dioic acid (junicedric acid) and its 8(9)-en isomer with C-protected amino acids (amino acid esters). The new compounds were evaluated for their gastroprotective effect in the ethanol/HCl-induced gastric lesions model in mice, as well as for cytotoxicity using the following human cell lines: normal lung fibroblasts (MRC-5), gastric adenocarcinoma cells (AGS) and liver hepatocellular carcinoma (Hep G2). A dose-response experiment showed that at 25 mg/kg the C-15 leucyl and C-15,19-dileucylester amides of junicedric acid reduced gastric lesions by about 65.6 and 49.6%, respectively, with an effect comparable to lansoprazole at 20 mg/kg (79.3% lesion reduction). The comparison of the gastroprotective effect of 18 new amino acid ester amides was carried out at a single oral dose of 25 mg/kg. Several compounds presented a strong gastroprotective effect, reducing gastric lesions in the 70.9-87.8% range. The diprolyl derivative of junicedric acid, the most active product of this study (87.8% lesion reduction at 25 mg/kg) presented a cytotoxicity value comparable with that of the reference compound lansoprazole. The structure-activity relationships are discussed.

It has been established that chronic gastric ulcers might lead to gastric and pancreatic cancer [18,19]. The resin of the tree Araucaria araucana is used to relieve gastric pain and the gastroprotective effect of the crude drug and main diterpene constituents has been reported [15]. The study started an investigation on the structure-activity relationships of labdane diterpenes from the resin for gastroprotective as well as for cytotoxic effect on normal and cancer cell lines [14,15,20]. Amide derivatives from labdane diterpenes with increased gastroprotective effect and low cytotoxicity were prepared [20]. However, the aromatic amines, used to prepare the amides, do not correspond to usual mammalian metabolites. Therefore, we selected amino acids esters to prepare new amides. A previous study on the synthesis of C-19 amino acid ester monoamides was carried out using imbricatolic acid and its acetate, as well as the 8(9)-en isomers, as starting compounds (Figure 1). The new C-19 monoamides showed gastroprotective effects but the activity was lower than that found for amides of aromatic amines [21]. There is, however, no information on the gastroprotective effect of labdane diterpene amides bearing the amide at C-15, nor is the effect of C-15, 19-diamides on preventing gastric lesions or modulating cytotoxicity known.
The aim of the present work was to synthesize new diterpene amides starting from the labdane diterpene imbricatolic acid. The starting compound was oxidized to obtain 8(17)-labden-15,19-dioic acid (junicedric acid) and then isomerized to the 8(9)-en derivative to prepare the new compounds. The new amides were assessed for gastroprotective effects in the ethanol/HCl-induced gastric lesions model in mice, as well as for cytotoxicity on normal human lung fibroblasts and the tumor cell lines AGS (gastric adenocarcinoma) and HepG2 (hepatocytes). The amino acids used to prepare the amides were C-methyl or ethyl esters of glycine, valine, leucine, proline and tryptophan.

Results and Discussion
Starting from junicedric acid and its 8(9)-en isomer, 20 new diterpene amides were synthesized from amino acids esters and 18 from them were investigated for gastroprotective effects and cytotoxicity ( Figure 2).
To select the dose for comparing the gastroprotective effect of the new compounds, junicedric acid C-15 leucyl methyl ester amide (compound 13) and C-15,19 dileucyl methyl ester amide (compound 18) (amino acid COOH as methyl ester) were assessed as gastroprotective agents at three oral doses: 25, 50 and 100 mg/kg. In the dose-response study, the higher doses of both compounds reduced gastric lesions by 79-89% and did not differ statistically from each other. At 25 mg/kg, compound 13 reduced gastric lesions by 65.6% (lesion index in mm: 15.8 ± 6.2) and compound 18 by 49.6% (lesion index in mm: 23.2 ± 12.9). Under the same experimental conditions, the lesion index of untreated animals (in mm) was 46.0 ± 5.7 and the standard compound lansoprazole at 20 mg/kg reduced lesions by 79.3% (lesion index: 9.5 ± 2.5). Based on the results, the efficacy of the compounds preventing gastric lesions in mice was compared at the single oral dose of 25 mg/kg (Table 1). For the junicedric acid 8(9)-en isomers, when the COOH function at C-19 was free, a higher gastroprotective activity was observed for the glycyl (1, 73.3%), leucyl (3,72.6%) and prolyl ester (4, 70.9%) amides, which were more effective as gastroprotective agents than the corresponding C-15,19diamides. However, for the tryptophanyl ester derivatives, better gastroprotection was found for the diamide 10 (61.7%) than for the monoamide 5 (25.4%), with lower cytotoxicity seen for compound 10. For the diamides with glycine, proline and tryptophan ester, better gastroprotection was observed for the derivatives with the exo methylene group.
When considering cytotoxicity (Table 2), significant differences can be observed between the compounds. Lower cytotoxicity with IC 50 values > 1,000 µM towards all cell lines was observed for the tryptophan ester monoamide 15 and the diamides 10 and 20, making these derivatives the most interesting compounds when looking for gastroprotective products with low cell toxicity. Compound 9 proved to be the more cytotoxic derivative, with IC 50 values of 119, 82 and 67 µM against human fibroblasts, AGS cells and Hep G2 hepatocytes, respectively. None of the new compounds presented selectivity against the cell panel used, but higher gastroprotective effect with lower toxicity was found for compounds 15 and 20. Compound 19, the most active product of this study, presented a cytotoxicity comparable with that of the reference compound lansoprazole, but higher than that of compounds 15 and 20, which presented IC 50 values > 1,000 µM.

General
Optical rotations were obtained for solutions in CHCl 3 (concentrations expressed in g/100 mL) on a Jasco DIP 370 polarimeter. Infrared spectra were recorded on a Nicolet Nexus FT-IR instrument (Thermo Electron Corporation). NMR spectra were recorded at room temperature in CDCl 3 using a Bruker Avance 400 instrument (Bruker, Germany) operating at 400 MHz for 1 H and 100 MHz for 13 C spectra. All chemical shifts values are reported relative to residual CHCl 3 .Chemical shifts (δ) are given in ppm and coupling constants (J) are reported in Hertz. 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 F 254 , 0.25 mm) were used for thin layer chromatography (TLC) analysis. Reversed-phase silica gel 100 C 8 (Fluka, Germany) was used to purify the the amides. TLC spots were visualized by spraying the chromatograms with p-anisaldehyde-ethanol-acetic acid-H 2 SO 4 (2:170:20:10 v/v) and heating at 110 ºC for 3 min.

Synthesis of the diterpene amides 1-20
The diterpene used as starting compound for the synthesis was obtained from the resin of Araucaria araucana as described in previous work [14,15]. 15-Hydroxyimbricatolic acid was treated with Jones reagent (CrO 3 /H 2 SO 4 /H 2 O) to afford after purification by column chromatography on silica gel the diacid diterpene junicedric acid. The 8(9)-en junicedric acid isomer was prepared treating junicedric acid with HBr in acetic acid. The amides described herein for the first time were prepared under an inert (N 2 ) atmosphere. Briefly, the diterpene was dissolved in dry dichloromethane (CH 2 Cl 2 , DCM) and ice cooled under nitrogen flow. To this solution, oxalyl chloride in dry DCM was added dropwise with stirring. The volume of dry DCM used to dissolve the diterpene was about 15-20 mL. Oxalyl chloride was dissolved in dry DCM in a 1:5 ratio The diterpene:oxalyl chloride molar ratio was 1:40. 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 and the C-protected amino acid ester (AAE) (as hydrochloride) was added as well as triethylamine (TEA) under constant N 2 flow. The diterpene:AAE:TEA molar ratio was 1:3:3. The reaction mixture was left at room temperature (18-20 ºC) under stirring and inert atmosphere for two days (48 h). Then, the mixture was washed two times with water, and the aqueous phase extracted with DCM to obtain the crude reaction mixture [21,22]. To prepare the C-15 monoamides, the diterpene dissolved in dry DCM was treated with N,N′dicyclohexylcarbodiimide (DCC)/dimethylaminopyridine (DMAP) and the amino acid ester.HCl. The reaction time was 1-3 h and the process was monitored by TLC. Purification was undertaken using a combination of gel permeation in Sephadex LH-20, eluting with an petroleum ether (PE):DCM:MeOH 1:1:1 mixture, silica gel column chromatography (CC) and reversed-phase silica gel chromatography (RP-8) to afford the amides. The reagents and conditions used are summarized in Figure 2. The structure of the compounds was determined mainly by NMR spectroscopy and by comparing the spectroscopic data with those of similar compounds already reported and by IR and mass spectra.

Ethanol/HCl-induced ulcer model in mice
The gastroprotective activity of the compounds was determined in the ethanol/HCl-induced lesion model in mice [14,15,20,21]. The purity of the tested compounds was higher than 95% by NMR

Cytotoxicity assay
Confluent cultures of MRC-5, AGS and Hep G2 cells were treated with medium containing the compounds at concentrations ranging from 0 up to 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 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 [20,21]. To calculate the IC 50 values (concentration that produces a 50% inhibitory effect on the evaluated parameter) the results were transformed to percentage of controls and the IC 50 value was graphically obtained from the dose-response curves.

Statistical analysis
Results were expressed as the mean ± SD. In all experiments, statistical differences between several treatments and their respective control were determined by one-way analysis of variance (ANOVA) and when the F value was significant, post hoc differences were determined by the Dunnett's multiple comparison test. The level of significance was set at P < 0.05. All statistical analyses were performed using the software Statistica 5.1 (StatSoft, Inc.) and Statistical Package S-Plus 2000.

Conclusions
In summary, the new compounds prepared were more active as gastroprotective agents than the C-19 monoamides prepared from imbricatolic acid, 15-acetoxyimbricatolic acid and the corresponding 8(9)-en isomers [21]. Relevant gastric lesion-preventing effects with very low cytotoxicity were found at a single oral dose of 25 mg/kg for some of the compounds. Further studies are needed to fully reveal the potential of labdane diterpenes as templates for the synthesis of new gastroprotective drugs.