Synthesis and Pharmacological Activity of Diterpenylnaphthoquinone Derivatives

New diterpenylquinones, combining a diterpene diacid and a naphthoquinone, were prepared from junicedric acid and lapachol. The new derivatives were assessed as gastroprotective agents by the HCl-EtOH-induced gastric lesions model in mice as well as for basal cytotoxicity on the following human cell lines: Normal lung fibroblasts (MRC-5), gastric epithelial adenocarcinoma (AGS), and hepatocellular carcinoma (Hep G2). Several of the new compounds were significantly active as antiulcer agents and showed selective cytotoxicity against AGS cells.


Introduction
In the year 2002 stomach cancer and peptic ulcer disease represented 1.5 and 0.5% of the total causes of death worldwide, respectively [1]. Chronic gastric ulcers can lead to gastric and pancreatic cancer [2,3]. Several natural products have been shown to display significant gastroprotective effects OPEN ACCESS in animal models of induced gastric ulcers. These agents include diterpenes with different structural skeletons [4][5][6][7][8][9][10][11]. The labdane diterpenes from Araucaria araucana (Molina) Koch (Araucariaceae) and their semisynthetic derivatives present gastroprotective activity with a wide range of basal cytotoxicity on human cell lines [9,10]. According to Halle and Spielmann there is a significant correlation between cytotoxicity of mammalian cell culture systems and acute oral toxicity (LD 50 ) in animals [12]. Basal cytotoxicity is a valid model to predict starting doses for in vivo lethality assays in rodents.
A new approach to drug design is to link two molecules with individual intrinsic effect into a single compound, named a "hybrid compound" [20]. A classification of "hybrid" molecules includes conjugates, cleavage conjugates, fused hybrid molecules, and merged hybrids [20][21][22][23]. The use of lapachol to obtain lapachol hybrid derivatives with diterpenes has not been explored previously, but changes in the gastroprotective effect and basal cytotoxicity of the resulting products should be expected.
In this paper we describe the preparation of junicedric acid and lapachol derivatives and examine the structure-activity relationships/trends of the new compounds as gastroprotective agents in mice. The basal cytotoxicity of the new compounds was also assessed towards the following human cell lines: normal lung fibroblasts (MRC-5), epithelial adenocarcinoma (AGS), and hepatocellular carcinoma (Hep G2).
Derivatives 6 and 8 were significantly active, protecting 3/8 and 5/9 animals, respectively, from lesions or stomach bleeding. An important gastroprotective effect was seen with derivatives 3, 4, 9 and 10 compared to untreated controls. However, for compounds 3, 4 and 9 all the stomachs showed at least some lesions or bleeding. Compound 10 was a very active gastroprotective agent. Compounds 8 and 10 are methyl esters of II and IV, the quinone moiety bearing either the prenyl side chain or the reduced prenyl side chain. Compound 6 was built from I and VI. It showed very low basal cytotoxicity (>1 mM) and proved to be one of the best gastroprotective products found in this study. The most active compounds are C19 methyl esters of the parent compounds. This fact shows that changes in the structure of any of the moieties will lead to changes in activity of the new compounds.
The diterpenylnaphthoquinones as well as I, II and IV were evaluated for basal cytotoxicity using MRC-5 fibroblasts, AGS and Hep G2 cells (Table 2). No significant differences in basal cytotoxicity were found between derivatives 1 and 7, where the diterpene moiety presents the double bond either at 8(17)-en-(compound 1) or at 8(9)-en-(compound 7). Cytotoxicity strongly decreases after methylation, showing the relevance of the COOH function at C-19 for this effect. In a broad sense, all compounds displayed lower basal cytotoxicity against fibroblasts than against AGS or Hep G2 cells. Some selectivity was seen for compounds 1, 5, 7 and 11 towards Hep G2 cells. The most cytotoxic compound of this series (5) was built with I and the hydrogenation product of lapachol VI. The IC 50 values of this compound towards fibroblasts, AGS and Hep G2 cells were 69, 40 and 27 µM, respectively. The increase in cytotoxicity was associated with poor selectivity. The effect is completely lost after methylation of the C-19 COOH function of the diterpene.
A similar approach, but starting from myrcecommunic acid and p-benzoquinone was used to prepare new diterpenylnaphthoquinones through a Diels-Alder cycloaddition [25]. Several of the diterpenylnaphthoquinones presented higher cytotoxicity than the quinone moiety itself, the naphthoquinone (NQ) system showing better effect than benzoquinone or anthraquinone derivatives [25,26]. The most promising compounds present variations in the decaline part of the naphthohydroquinone (NHQ) of the NQ system and show GI 50 values (growth inhibition values that reduce cell growth by 50%) in the 0.12-0.50 µM range towards the tumour cell lines A-549 (lung carcinoma), HT-29 (colon carcinoma), and MEL-28 (malignant melanoma) [25]. The main difference with this report is that [25] formed C-C bonds by cycloaddition, while we prepared esters of dicarboxylic diterpenes and napththoquinones.

Plant Material
Lapachol was isolated from the wood of Tabebuia heptaphylla as described previously [13] and purified by successive silica gel column chromatography, followed by crystallization. The diterpene junicedric acid (I) was obtained by saponification and oxidation of a mixture of labdane diterpenes from Araucaria araucana resin. The resin was collected from healthy trees in Conguillío, Araucanía Region, Chile. Voucher specimens have been deposited at the Herbarium of the Universidad de Talca.

General Procedure for the Synthesis of Compounds I-III and V-VI
The crude Araucaria araucana resin was worked-up as described previously [9,10] to obtain a mixture of diterpenes bearing an alcohol, aldehyde, acid or ester (acetate) function at C-15 and/or C-19. After saponification (KOH, methanol), the diterpene mixture was oxidized with CrO 3 to yield I. Isomerization of the double bond of I to II was carried out by treating I in acetic acid with 47% HBr, stirring constantly for 24 h. The reaction product was purified by silica gel column chromatography (86% w/w yield). Reduction of the double bond of I and IV was carried out by catalytic hydrogenation of the terpene dissolved in ethyl acetate with 10% Pd/C in a 1:10 molar ratio with respect to the diterpene, stirring constantly for 24 h to yield III and V, respectively. Compound VI was obtained by high pressure hydrogenation of compound IV.
Δ8(9) Junicedric acid (II). HBr (3 mL) was added to a solution of junicedric acid (I) (2.51 g, 7.47 mmol) in acetic acid (HOAc, 20 mL). The mixture was stirred at room temperature for 24 h, cooled in an ice bath, and after addition of water the aqueous phase was extracted with EtOAc (3 × 20 mL). The extract was dried over anhydrous Na 2 SO 4 , and taken to dryness under reduced pressure. The residue was purified by silica gel column chromatography, eluting with hexane/EtOAc (8:2), yielding 2.15 g (86%) of compound II.
Lapachoyl junicedrate (1). Junicedric acid (I) (170 mg, 0.506 mmol), DCC (104 mg, 0.506 mmol), a catalytic amount of DMAP and lapachol (IV) (120 mg, 0.506 mmol), were stirred at room temperature in dry CH 2 Cl 2 (20 mL) for 2-4 h. The reaction mixture was cooled in an ice bath. After addition of water, the aqueous phase was extracted with EtOAc (3 × 20 mL). The extract was dried over anhydrous Na 2 SO 4 and taken to dryness under reduced pressure. The residue was purified by silica gel column chromatography, eluting with hexane/EtOAc     Dihydroprenyl lapachoyl Δ8(9) junicedrate (9). Compound 9 was synthesized as described for compound 1, using terpene II and quinone V, yielding 91 mg (52%) of 9: pale yellow oil;   [7][8][9][10]27] The gastroprotective activity of the compounds was assessed in the EtOH-HCl-induced gastric lesion model. Male Swiss albino mice weighing 30 ± 3 g were used. The animals were fed on 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 mice were randomly distributed into groups of 7-14 animals each and fasted for 24 h with free access to water before the oral administration of test samples by gavage. The purity of the tested compounds was higher than 95% by NMR analysis. To keep the animal numbers to a minimum, dose-response studies were performed with lapachol at 5, 10 and 50 mg/kg to set the conditions for single-dose comparison of gastroprotective effect. The tested compounds, lansoprazole (20 mg/kg) or the vehicle (12% Tween 80, 10 mL/kg) were administered orally by gavage. After 50 min, all groups were orally treated with 0.2 mL of a solution containing 60% EtOH-0.3 M HCl for gastric lesion induction. Animals were sacrificed by cervical dislocation 1 h after the administration of EtOH-HCl, and the stomachs excised and inflated by injection of 5% formalin (1 mL). The ulcerated stomachs were fixed in 5% formalin for 30 min and 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. Based on previous studies on the parent diterpenes, comparison of the new compounds 1-12 was carried out at a single oral dose of 5 mg/kg. 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. Tween 80 and lansoprazole (>98% purity by HPLC) were purchased from Sigma-Aldrich. [28] The human cell lines MRC-5 normal lung fibroblasts (ATCC CCL-171), AGS gastric adenocarcinoma cells (ATCC CRL-1739), and Hep G2 hepatocellular carcinoma cells (ATCC HB-8065) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were grown as monolayers in the following media: MRC-5 and Hep G2 in minimum essential Eagle's medium (MEM), with Earle's salts, 2.0 mM L-glutamine (Sigma Chemical Co.) and 2.2 g/L sodium bicarbonate (Sigma Chemical 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% CO 2 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 Invitrogen Corp. Human gastric adenocarcinoma cells AGS (ATCC CRL-1739) 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% CO 2 in air at 37 °C. Cell passage was maintained between 42 and 48. The medium was changed every 2 days.

Cytotoxicity Assay
Confluent cultures of the different cell lines were treated with medium containing the compounds at concentrations ranging from 1 up to 1,000 µM. The antisecretory drug lansoprazole was used as reference compound. The substances were first dissolved in DMSO and then in the corresponding culture medium supplemented with 2% FBS. The final concentration of DMSO in the test medium and controls was 1%. The cells were exposed for 24 h to test medium with or without the compound (control). 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 assay was performed.

Statistical Analysis
Results were expressed as the mean ± SEM. In all experiments, statistical differences between several treatments and their respective control were determined by one-way ANOVA with Student-Newman-Keuls post-hoc test. The level of significance was set at P < 0.05.

Conclusions
The aim of this work was to synthesize diterpenylnaphthoquinones combining diterpene and quinone moieties. To determine structure-activity relationships/trends, 12 esters were prepared. The diterpenes used as building blocks comprised the diacids I, II and III. The naphthoquinone moieties included lapachol, dihydroprenyl lapachol, and dihydroprenyl-5,6,7,8-tetrahydrolapachol. Compounds 6, 8 and 10 were significantly active as antiulcer agents, protecting against induced gastric lesions in mice by 74, 84 and 76%, respectively. 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). Some compounds showed selective cytotoxicity against AGS cells. Derivative 5 was the most cytotoxic product, with IC 50 values of 69, 40 and 27 µM for fibroblasts, AGS and Hep G2 cells, respectively. Derivative 6, with strong gastroprotective activity, was devoid of cytotoxic effect towards the selected cell lines. The new compounds had different biological effects than the building blocks and some of them were less toxic than the starting molecules.