Dimeric Labdane Diterpenes: Synthesis and Antiproliferative Effects

Several diterpenes with the labdane skeleton show biological activity, including antiproliferative effects. Most of the research work on bioactive labdanes has been carried out on naturally occurring diterpenes and semisynthetic derivatives, but much less is known on the effects of diterpene dimers. The aim of the present work was to synthesize dimeric diterpenes from the labdane imbricatolic acid using esters, ethers and the triazole ring as linkers. Some 18 new derivatives were prepared and the compounds were evaluated for antiproliferative activity on human normal fibroblasts (MRC-5) and the following human tumor cell lines: AGS, SK-MES-1, J82 and HL-60. The diethers 8–10, differing in the number of CH2 units in the linker, presented better antiproliferative activity with a maximum effect for the derivative 9. The best antiproliferative effect against HL-60 cells was found for compounds 3 and 17, with IC50 values of 22.3 and 23.2 µM, lower than that found for the reference compound etoposide (2.23 µM). The compounds 9, 17 and 11 were the most active derivatives towards AGS cells with IC50 values of 17.8, 23.4 and 26.1 µM. A free carboxylic acid function seems relevant for the effect as several of the compounds showed less antiproliferative effect after methylation.


Introduction
Natural product monomers occur in all living organisms and many can also form polymeric structures, including rubber, cellulose or lignin.Less common are dimeric compounds formed by coupling two units by C-C, ester, ether, C-N or N-N bonds.
It has been reported that dimeric compounds can be employed as potential anticancer agents because they might interact with two different binding sites on a receptor or on two separate monomers of a dimeric protein [1].In some cases, it has been observed that dimers not only maintained the activity shown by its monomer, but could also increase it.The dimer montamine from Centaurea montana presented two times higher activity on in vitro colon cancer than its monomer [2].Monomeric ergolines presented weak antiplasmodial effect, while dimerized ergoline derivatives, prepared using different aliphatic or arylalkyl spacers, showed significantly increased activity [3].
Different biological activities have been reported for labdanes [15][16][17][18][19][20] including antiproliferative effects [21].Several labdane derivatives were prepared and assessed for gastroprotective and cytotoxic effects looking for structure-activity relationships.The semisynthetic labdanes included esters, ethers, amides with aromatic amines [17], amino acids [22], as well as hybrid molecules formed with naphthoquinones [23].This paper describes the synthesis of some new dimeric diterpenes including esters, ethers and dimers fused by triazole rings, starting from the naturally occurring labdane imbricatolic acid.The new compounds were evaluated for antiproliferative activity on human normal lung fibroblasts and selected cancer cell lines.
Other studies on the synthesis and cell toxicity of dimeric compounds include the work on dimeric epothilone A derivatives, prepared by using diacyl spacers.The new compounds were evaluated on tubulin polymerization and the cytotoxicity was determined against cancer cell lines [24].All the dimers were less cytotoxic than epothilone, however, several dimeric compounds inhibit endothelial cell differentiation and endothelial cell migration.Looking for new compounds with antimalarial and anticancer activity, C-10 non-acetal dimers of 10β-(2-hydroxyethyl)deoxoartemisinin were prepared and evaluated on cell lines [25].All the artemisinin dimers showed potent antimalarial activity in the nM range.One of the dimers evaluated in vitro was fifty times more potent than artemisinin.The cytotoxicity of the new products was determined on the National Cancer Institute (NCI) tumour cell panel, most of the compounds, except the phosphate dimer were not cytotoxic.The synthesis, cytotoxicity, in vivo anticancer and antiprotozoal effect of twelve artemisinin acetal dimers was reported [26].Several of the new dimers were more active than the corresponding monomers on the cancer cell lines tested with GI 50 values for the dimers between 8.7 and 0.019 μM.Hybrid compounds combining the artemisinin and a quinoline moiety were synthesized [27] and evaluated against Plasmodium falciparum.Two of the new compounds showed excellent activity against the protozoa in vitro.

Results and Discussion
Starting from the naturally occurring diterpene imbricatolic acid, fourteen dimers were synthesized using different linkers.The diterpene imbricatolic acid, used as starting compound for the synthesis was isolated from the resin of Araucaria araucana.Imbricatolic acid was methylated with diazomethane to form compound 1. 15-Hydroxyimbricatolic acid was treated with the Jones reagent (CrO 3 /H 2 SO 4 /H 2 O) to afford the diacid junicedric acid (JA, 2).Compound 1 was tosylated and then treated with NaN 3 in DMF to form the azide 3. Compounds 4-6 were prepared by "click chemistry" of 3 with different alkynes using CuSO  To prepare the ether 7, compound 1 was treated with NaH in DMF under constant stirring and then the tosylated compound 1 was added to the solution.The ethers 8-10 were prepared from 1 with NaH in DMF and the corresponding dibromoalkanes.To prepare the ester 11, the diterpene JA (2) dissolved in dry CH 2 Cl 2 was treated with N,N'-dicyclohexylcarbodiimide (DCC)/dimethylaminopyridine (DMAP) and compound 1.Compounds 13 and 14 were prepared from compound 1 with DCC/DMAP in dry CH 2 Cl 2 and succinic acid and phthalic acid, respectively (Scheme 2).Scheme 2. Preparation of derivatives 7-14 from imbricatolic acid methyl ester (1).Reagents and conditions: (a) (i) appropriate alkyne alcohol, DCC, DMAP, CH The dimers 15, 17 and 19 were prepared by "click chemistry".To prepare the dimers 15 and 17, JA (2) was treated with DCC/DMAP in dry CH 2 Cl 2 and different alkyne alcohols (propargyl alcohol and 3-butyn-1-ol), to afford the corresponding alkynes esters.These esters were treated whit the azide 3 and CuSO 4 •5H 2 O/sodium ascorbate in t-BuOH/H 2 O to yield the desired products.In the same way 19 was obtained treated imbricatolic acid with DCC/DMAP in dry CH 2 Cl 2 and 4-pentynoic acid, the ester obtained was then treated with the azide 3 (Scheme 3).All the C-19 methyl esters were prepared by reaction with a diazomethane solution (compounds 12, 16, 18 and 20).In all, some 19 compounds were prepared starting from the diterpenes labd-8(17)-en-15-hydroxy-19-oic acid (imbricatolic acid) and labd-8(17)-en-15,19-dioic acid (junicedric acid, compound 2).The new compounds include ethers and esters with different "linkers" (spacers) as well as 1,2,3-triazole-substituted derivatives prepared by click chemistry.
The purity of all derivatives was over 98% as assessed by 1 H-NMR spectroscopy.The syntheses are summarized in Schemes 1-3.All of the compounds were characterized by spectroscopic means and compounds 3-20 are described for the first time.The dimeric compounds prepared can be classified into symmetric dimers (compounds 7-10, 13-14) and asymmetric dimers (compounds 11-12, 15-20).Unlike the symmetric dimers, asymmetric dimers showed small variations in many of their 13 C-NMR signals (see Tables 1 and 2).1D and 2D-NMR methods were used for a better H and C assignation.Selected NMR spectra are presented as Supporting Information.28.83 q * 29.04 q * 28.80 q 28.80 q 28.80 q * 29.01 q * 28.81 q 28.81 q 28.82 q * 29.05 q * 28.86 q 28.86 q 28.84 q 28.84 q 28.78 q 28.78 q 12.58 q * 12.76 q * 12.53 q 12.53 q 12.55 q * 12.73 q * 12.54 q; 12.54 q 12.58 q * 12.79 q * 12.60 q 12.60 q 12.58 q * 12.79 q * 12.52 q 12.52 q OMe OMe' 51.10 q -51.11 q 51.11 q 51.11 q -51.13 q 51.13 q 51.16 q -51.17 q 51.17 q 51.17 q -51.17 q 51.17The compounds 1-20 were assessed for antiproliferative effect towards human lung fibroblasts (MRC-5) and the following human tumor cell lines: gastric adenocarcinoma (AGS), lung cancer cells (SK-MES-1), bladder carcinoma (J82) and promyelocytic leukemia (HL-60).IC 50 values >100 µM were considered as inactive.The results are summarized in Table 3.The new compounds as well as the diterpenes used to assemble the dimers were evaluated for antiproliferative effects using five different human cell lines.From the starting diterpenes, compounds 2 and 6 showed no antiproliferative effect on any of the cell lines.From the dimeric derivatives containing an ether function, compound 7 showed no antiproliferative effect.The diethers 8-10, differing in the number of CH 2 units of the linker, presented better antiproliferative activity with maximum effect for the derivative 9.The dimer formed by the esterification of compounds 1 and 2 (compound 11) and its corresponding methyl ester (compound 12) show the importance of a free carboxylic acid function in the antiproliferative effect.Compound 11 showed activity against MRC-5, AGS and HL-60 cell lines but not against SK-MES-1 and J82 cells.Compound 12 showed effect against SK-MES-1 and J82 cells but was devoid of activity against MRC-5, AGS and HL-60 cell lines.
The antiproliferative activity of both diesters with succinic and phthalic acid (compounds 13 and 14) was similar.
For compounds 15-18, methylation of the carboxylic acid function at C-19 reduced the antiproliferative effect, as can be seen comparing the pairs 15-16 and 17-18, respectively.Some selectivity against AGS and HL-60 cells was found for the dimers 15 and 17, with better effect for compound 17, differing from 15 in one CH 2 unit in the triazole linker.On the other hand, the compounds 19 and 20 showed no antiproliferative effect on any of the cell lines, except on HL-60 for compound 19.

Antiproliferative Assay
All human cell lines used in this work were purchased from the American Type Culture Collection (ATCC, Manasas, VA, USA).Normal lung MRC-5 fibroblasts (CCL-171), SK-MES-1 lung cancer cells (HTB-58) and J82 bladder carcinoma cells (HTB-1) were grown as monolayers in minimum essential Eagle medium (MEM) with Earles's salts, 2 mM L-glutamine and 1.5 g/L sodium bicarbonate.Gastric adenocarcinoma AGS cells (CRL-1739) were grown as monolayers in Ham F-12 medium containing 1 mM L-glutamine and 1.5 g/L sodium bicarbonate.Promyelocytic leukemia HL-60 cells (CCL-240) were grown in suspension in RPM1 medium containing 1 mM sodium pyruvate and 2.0 g/L sodium bicarbonate.All media were supplemented with 10% heat-inactivated FBS, 100 IU/mL penicillin and 100 µg/mL streptomycin.Cells were grown in a humidified incubator with 5% CO 2 in air at 37 °C.For the antiproliferative assay, adherent cells were plated at a density of 5 × 10 4 cells/mL and HL-60 cells at 30 × 10 4 cells/mL.Cells were seeded in 96-well plates (100 µL/well).One day after seeding, cells were treated with medium containing the compounds at concentrations ranging from 0 up to 100 µM during 3 days.The compounds were dissolved in DMSO (1% final concentration) and complete medium.Untreated cells (medium containing 1% DMSO) were used as 100% viability controls.Etoposide (98% purity, Sigma-Aldrich, St. Louis, MO, USA) was used as reference

Table 3 .
Antiproliferative activity of compounds 1-20 against MRC-5 normal fibroblasts and selected tumor cell lines.a a For cell lines used, see text.b Results are expressed as mean values ± SD.Each concentration was tested in sextuplicate together with the control and repeated two times in separate experiments.c Reference compound.