Design, Synthesis and Evaluation of New Marine Alkaloid-Derived Pentacyclic Structures with Anti-Tumoral Potency

This work describes the synthesis and biological evaluation of a new heterocyclic hybrid derived from the ellipticine and the marine alkaloid makaluvamine A. Pyridoquinoxalinedione 12 was obtained in seven steps with 6.5% overall yield. 12 and its intermediates 1–11 were evaluated for their in vitro cytotoxic activity against different cancer cell lines and tested for their inhibitory activity against the human DNA topoisomerase II. The analysis by electrophoresis shows that the pentacycle 12 inhibits the topoisomerase II like doxorubicine at 100 µM. Compound 9 was found to have an interesting profile, having a cytotoxicity of 15, 15, 15 and 10 μM against Caco-2, HCT-116, Pc-3 and NCI cell lines respectively, without any noticeable toxicity against human fibroblast.


Introduction
Research leading to the characterization of naturally occurring secondary metabolites continues to offer one of the most significant pathways contributing to drug discovery [1]. New leads for the preclinical development of therapeutic agents are often inspired by natural products, which engage initial interest owing to reports of their biological activity [2]. Indeed, the unique molecular architecture of these natural substances may serve as primary stimulus for innovation and creativity to provide precisely engineered chemical solutions for complex biological phenomena. Among secondary metabolites is found a class of compounds called alkaloids [3][4][5]. This concept, introduced by Meissner [6] from the latin alkali (basic) regroups many chemical structures that are important sources of bioactive compounds, such as ellipticine [7], neoamphimedine, isolated from xestospongia sponge [8] or makaluvamine A [9] (Figure 1), extracted from another marine sponge. In fact, marine biodiversity holds an exceptional number of species. So far, more than 300,000 marine species have now been discovered; some specialists agree that over than a million are still to be discovered [10,11]. Such diversity leaves a space wide-open for new discoveries, presenting the marine world as a great reservoir for new active drugs. Between 2001 and 2010, most of the compounds isolated from marine sources were alkaloids or macrocycles, and they have been isolated from sponges [12,13]. From the Latrunculiidae sponge family have been extracted some pyrroloiminoquinones [14][15][16], an important class of bioactive alkaloids. This class of metabolites includes discorhadbines and makaluvamines isolated primarily from sponges of the Zyzzya genus. They have been reported to express significant cytotoxic activity towards several tumor cells, as well as a potential activity against the topoisomerase II [17].
Our goal is to study the synthesis and the biological evaluation of new pyridoquinoxalinediones as potential intermediates for the synthesis of new analogues of makaluvamine A, along with pentacyclic structures as potential anti-topoisomerase II agents.

Analogue Design and Chemistry
As previously reported by our research group, the 10 steps of the synthesis of compound 3 were described (Scheme 1), starting from 2,5-dimethoxyaniline 1 [18].
In order to rapidly access new analogues, we have developed a new approach that could lead to the same compound in only seven steps, starting from 1,2-benzenediamine 4 (Scheme 2). Dihydroquinoxalinone 5 was obtained with 66% yield after condensation of dimethylacryloylchloride [19]. The tricyclic intermediate 7 was then synthesized in two steps with a 65% yield, after acylation conducted in pyridine and cyclisation using aluminum chloride in dichloromethane. The nitration of the tricyclic compound was achieved using fuming nitric acid in dichloromethane in a 42% yield, which could be easily reduced by catalytic hydrogenation leading to the amino intermediate 8 with a 70% yield. Unfortunately, oxidation to the desired iminoquinone using Fremy salts or cerium ammonium nitrate was never obtained. Reduction of pyridoquinoxalinedione 7 using boran/tetrahydrofuran reagent yielded compound 9, which could not be oxidized in order to reach the desired pyridoiminoquinone 3. We have recently described the synthesis of a pentacyclic hybrid molecule 10 (Scheme 3) of makaluvamine A and ellipticine [20]. Scheme 3. Synthesis of pentacycle 10.
As we had great interest in these polycylic structures with a topoisomerase II inhibition potential, we have developed a two-step pathway to the pentacyclic analogue compound in this series from intermediate 8 (Scheme 4). The Buchwald-Hartwig coupling between 8 and the 2-bromoiodobenzene led to compound 11 with 71% yield according to our previously reported procedure [20]. The cyclisation led to the desired pentacyclic structure 12 through the Heck reaction, using palladium acetate and potassium carbonate in dimethylacetamide in a sealed tube. All synthesized compounds were then biologically evaluated in order to determine their potential against different cancer cell lines, as well as their topoisomerase II inhibition efficiency.

Bioactivities of the New Synthesized Analogues
Synthesized molecules were tested against different cancer cell lines in order to evaluate their antitumoral potency. In a second stage we will describe the anti-topoisomerase II efficiency in order to determine the mechanism of action of these new synthetic analogues of marine alkaloids.

In Vitro Cytotoxicity
All synthesized compounds were evaluated for their in vitro cytotoxic activity against different human cancer cell lines, Caco-2, HCT-116, HUH-7, MDA-MB-231, Pc-3 and NCI. A human fibroblast cell line was used as control for the overall toxicity. Compounds were tested up to 25 μM (as shown in Table 1) for compounds with any activity at <25 μM. All other tested compounds were found to be inactive against these cell lines at 25 μM.
Cells were harvested during the logarithmic growth phase and seeded in 96-well plates at a density of 1 Å~10 4 cells/mL, and cultured at 37 °C in a humidified incubator (5% CO2) for 24 h, followed by exposure to various concentrations of compounds tested for 48 h. The microtitration colorimetric method of MTT reduction was used in order to identify surviving cells at the end of the treatment period. Subsequently, 20 μL of MTT solution (5 mg/mL) was added to each well and mixed, after which the cells were incubated for an additional 4 h. The solution in each well containing media, unmetabolized MTT and dead cells was removed by suction and 100 μL of DMSO was added. Cell growth inhibition was determined by measuring the absorbance (Abs) at λ = 570 nm using a microplate reader and then calculated. The half maximal inhibitory concentrations (IC50) were obtained from liner regression analysis of the concentration-response curves plotted for each tested compound. Stock solutions (5 mg/mL) were prepared by dissolving pure compounds in DMSO and storing them at 4 °C . Serial dilutions with culture media were prepared just prior to the addition to test plates. Doxorubicine was used as the positive control and vehicle (DMSO + Media) as Blank.

Topoisomerase II Decatenation Assay
The kinetoplast-DNA (kDNA) assay was performed according to the protocol of Inspiralis (Norwich, UK). The enzymatic reaction was performed in a total volume of 30 µL containing 200 ng kDNA, test compound (doxorubicine as control or synthesized compounds in DMSO), and 1 unit of topoisomerase II in assay buffer (5 mM Tris.HCl pH 7.5, 12.5 mM NaCl, 1 mM MgCl2, 0.5 mM DTT, 10 μg/mL albumin and 1 mM ATP). The mixture was incubated at 37 °C for 30 min and the reaction was terminated by the addition of 5 µL of stop buffer (5% sarkosyl (n-lauroylsarcosine, sodium salt), 0.125% bromophenol blue, 50% glycerol). DNA products were analyzed by electrophoresis through 1% agarose gels containing ethidium bromide. kDNA form extremely large networks of high molecular weight that fail to enter an agarose gel. Decatenation of kDNA by topoisomerase II results in minicircular DNAs moving in the gel due to their small size.

Conclusions
In short, we have developed the synthesis of a new series of marine alkaloids analogues, such as makaluvamine A or neoamphimedine, and have evaluated their potential as antitumor agents. Compounds 9, 11 and 12 showed activity against different cancer cell lines such as Pc-3 or HCT-116, with no apparent selectivity toward human fibroblast for compounds 11 and 12. Compound 9 was found to have an interesting profile, having a cytotoxicity of 15, 15, 15 and 10 μM against Caco-2, HCT-116, Pc-3 and NCI cell lines respectively, without toxicity against Human fibroblast. Although, pentacycle 12 was found to have cytotoxic effects on different cancer cell lines (HCT-116, Pc-3 and NCI), it did not express any real selectivity. Nevertheless, its DNA binding capacity showing its potential as a Topoisomerase II inhibitor will allow us to undertake new SAR studies by synthesizing analogues of compound 12 in order to search for more potent activity and better selectivity.