Synthesis and Antitumor Evaluation of 6-Aryl-substituted benzo[j]phenanthridine- and Benzo[g]pyrimido[4,5-c]isoquinolinequinones

A variety of novel 6-arylsubstituted benzo[j]phenanthridine- and benzo[g]-pyrimido[4,5-c]isoquinolinequinones were synthesized from 1,4-naphthoquinone, aryl-aldehydes and enaminones via a two-step synthetic approach. The cytotoxic activity of the aminoquinone derivatives was evaluated in vitro against one normal cell line (MRC-5 lung fibroblasts) and three human cancer cell lines (AGS human gastric adenocarcinoma; SK-MES-1 human lung cancer cells, and J82 human bladder carcinoma) in 72-h drug exposure assays using the MTT colorimetric method. Structure–activity relationships within the series of angular quinones reveal that the insertion of pyrrol-2-yl and furan-2-yl groups at the 6-position is more significant for the increase of the potency and selectivity index of the pharmacophores.


Introduction
The quinone nucleus is common to many natural and synthetic products associated with anticancer and antibacterial activities [1]. Among these compounds the polycyclic members are typically DNA-intercalating agents due to the ability of their large and planar structure to bind strongly between the base pairs through hydrogen bonds and -stacking interactions [2,3]. They usually have side chains or sugar substituents and basic nitrogens, which upon protonation further strengthen the DNA binding. Examples of quinone derivatives with antitumor activity include mitoxantrone [4], doxorubicin [4], mitomycin [5], streptonigrin [6] and actinomycin D (AMD) [2]. All known quinonic DNA intercalators have the potential to disrupt the normal function of DNA, leading to cell death [7]. This DNA damage can be caused either by the parent form or by its metabolic conversion to electrophilic or radical species [8].
Aza-and diaza-anthraquinones such as those depicted in Figure 1, represent an important class of antitumor agents that exhibit promising in vitro and in vivo activity on tumor cell lines [9][10][11][12][13]. The antitumor activity of these agents seem to be mediated by DNA intercalation and redox cycling processes that are improved by the basic and electron-withdrawing properties of the N-heterocyclic ring [12]. As part of a research program on the synthesis and antitumor evaluation of N-heterocyclic quinones we have described the synthesis and antitumor properties of benzo[j]phenanthridine-and benzo[g]pyrimido [4,5-c]isoquinolinequinone derivatives [14]. Recent evidences demonstrate that some members of these series have potential anti-cancer activity through inhibition of TOP1 catalytic activity and induction of apoptosis [15]. The synthesis of these tetracyclic quinones, such as 1 and 2 ( Figure 2), was accomplished by means a strategy involving the heterocyclization of acyl-1,4benzoquinones with enaminones to construct the ABC-ring systems followed by a Diels-Alder reaction to assemble the D-benzene nucleus. In this context, we envisaged an alternative synthetic approach to the tetracyclic frameworks, in which the assembly of the ABCD-ring system would be accomplished through a one-step process by heterocyclization of 2-acyl-1,4-naphthoquinones with enaminones. Taking advantage of recently published preliminary results on the synthesis of heteroaroylnaphthohydroquinones by solar-chemical photo-Friedel-Crafts heteroacylation of 1,4-quinones [16], we decided to explore the mentioned strategy. Herein, we wish to report the synthesis and the in vitro antitumor evaluation of a variety of 6-aryl-substituted benzo[j]phenanthridine-and benzo[g]pyrimido [4,5-c]isoquinolinequinones. In the light of the results, structure-activity relationships (SARs) were examined, using 1 and 2 as lead compounds, assessing the effects on antitumor activity of structural changes made to the pyridine fused ring B by insertion of aryl-and heteroaryl groups.

Chemistry
Acylnaphthohydroquinones 4a-f together the enaminones 3-aminocyclohex-2-en-1-one (5) and 5-amino-1,3-dimethyluracil (6) were selected as precursors to explore the synthesis of the designed N-heterocyclic quinones. The synthesis of the required hydroquinones 4a-f was performed by solar photo-Friedel-Crafts reaction of 1,4-naphthoquinone (3) with the corresponding aryl-and heteroarylcarbaldehydes, according to our recently reported procedure [16]. In all the examined cases acylhydroquinones 4 were obtained in good to excellent yields (Scheme 1). The structure of compounds 4a and 4c-f were confirmed by comparison of their spectral data to those of authentic samples [16]. In the case of the previously unreported compound 4b, its structure was established by 1 H-, 13 C-NMR, and HRMS data.

Scheme 1. Preparation of acylnaphthohydroquinones 4a-f.
The synthesis of the target members of the series of benzo[j]phenanthridine-and benzo[g]pyrimido[4,5-c]isoquinolinequinones 7 and 8, containing phenyl and heteroaryl substituents at 6-position, was accomplished by reaction of acylnaphthohydroquinones 4, enaminones 5/6 and silver (I) oxide in dichloromethane (Scheme 2) using a previously reported one-pot procedure [14]. The broad application of this synthetic pathway allowed us to synthesize derivatives with different aryl-substituents in position 6 of the heterocyclic ring systems. The results are summarized in Table 1.

Cytotoxic Activities
Compounds 7a, 7c-f, 8a-f were evaluated for in vitro anticancer activity against normal human lung fibroblasts MRC-5 and three human tumor cell lines: AGS gastric adenocarcinoma, SK-MES-1 lung, and J82 bladder carcinoma, in 72-h drug exposure assays. The cytotoxicity of the new compounds was measured using a conventional microculture tetrazolium reduction assay [17][18][19]. The broad variety of the synthesized compounds was designed in order to gain insight upon the influence on the biological activity of phenyl, 3,4,5-trimethoxyphenyl, furan-2-yl, thiophen-2-yl, thiophen-3-yl, and pyrrol-2-yl groups at the 6-position of the benzo[j]phenanthridine-and benzo[g]pyrimido [4,5c]isoquinolinequinone pharmacophores. The cytotoxic activities of compounds 7 and 8 are collected in Table 2. The cytotoxic activity of analogues 1 and 2 [14] is also included in Table 2 in order to compare the effect of the insertion of aromatic groups at 6-position of the corresponding chromophores. As indicated in Table 2, the tested compounds showed moderate to good activity in the in vitro antitumor screening expressed by the IC 50 values. The novel angular quinones are less cytotoxic than the anti-cancer agent etoposide used as reference.  Comparison of the cytotoxic potency of the previously reported benzo[j]phenanthridine-and benzo[g]pyrimido[4,5-c]isoquinolinequinones 1 and 2 with the 6-aryl-substituted analogs 7a-f/8a-f, evaluated in this study, clearly indicates that the biological activity on normal and human cancer cells depends on the nature of the substituent located at the 6-position.
The initial structure-activity relationship (SAR) was focused on the effects of insertion of aryl-substituents at the 6-position of the benzo[j]phenanthridinequinone chromophore (compound 1). According to the IC 50 values it can be deduced that the insertion of a phenyl group in the 6-position of 1, as in quinone 7a, does not induce a significant change on the cytotoxic activity and selectivity index (SI = IC 50 fibroblasts/IC 50 cancer cells) on human gastric adenocarcinoma cell line. However, the substitution strongly decreases the biological effect, respect to 1, on human lung cancer and bladder carcinoma cell lines.
Comparison of the cytotoxic potency between the furan-2-yl-substituted quinone 7b and 1, reveals that insertion of the furyl group at the 6-position results in a decreasing effect on the potency on human gastric adenocarcinoma cells and the suppression of the cytotoxic activity on normal lung fibroblasts, lung cancer and bladder carcinoma cell lines. The insertion of a thiophen-2-yl or thiophen-3-yl substituent, bonded at the 6-position of the pharmacophore, as in compounds 7c and 7d, promotes an increasing effect on normal lung fibroblasts and no significant changes on the cytotoxicy on the tested cancer cell lines.
The data of Table 2 reveal that on AGS cells analogue 7e shows higher antitumor potency but a similar selectivity index to compound 1. Also observed is that the insertion of the pyrrol-2-yl substituent suppresses the cytotoxic activity on lung cancer and bladder carcinoma cell lines.
We can conclude that among the evaluated 6-arylbenzo[j]phenanthridinequinones, the pyrrolylderivative 7e could be of interest for the design of new analogues with antitumor activity on human gastric adenocarcinoma cells.
Next, the SAR analysis was focused on the effects of the insertion of aryl groups at the 6-position of the benzo[g]pyrimido [4,5-c]isoquinolinequinone scaffold (compound 2). The data in Table 2 reveal that the insertion of a phenyl group in the 6-position of 2, as in quinone 8a, has a remarkable effect on the antitumor potency and selectivity index of all the tested cancer cell lines. It can be seen that the effect of such insertion on the cytotoxic activity on gastric and bladder cancer cells, reaches values nearly 18-and 16-times higher than the reference compound 2. Compound 8b was included in the cytotoxic evaluation on the basis of the biological activity of 8a and precedent structure-activity relationships of benzo[c]phenanthridines, which reveal that methoxyphenyl substituents lead to derivatives with enhanced antiproliferative activity on cancer cells [20] respect to the unsubstituted phenyl-derivative. According to the biological evaluation of 8b, the expected enhanced effect respect to compound 8a was not observed. On the contrary, compound 8b displayed significant less cytotoxic activity on the tested cell lines, compared to 8a.
Next we examined the influence on the insertion of heterocyclic groups in the 6-position of the benzo[g]pyrimido [4,5-c]isoquinolinequinone chromophore. The substitution of a furan-2-yl group, as in 8c, resulted in the suppression of the antitumor activity on lung cancer and bladder carcinoma cell lines but in a 25-fold increase in potency on gastric cell line compare to compound 2, with a high selectivity index (9.6). The insertion a thiophenyl substituent, bonded through the 2-or 3-position to the 6-position of the chromophore, as in compounds 8d and 8e, does not induce significant changes on the cytotoxicy of the tested cancer cell lines. Concerning the effect of the pyrrol-2-yl group, as in 8f, a strong decreasing effect on the cytotoxic activity of all the tested cell lines was observed.

General
All reagents were commercially available reagent grade and were used without further purification. Melting points were determined on a Stuart Scientific SMP3 apparatus and are uncorrected. The IR spectra were recorded on an FT Bruker spectrophotometer using KBr disks, and the wave numbers are given in cm −1 . 1 H-NMR spectra were run on Bruker AM-200 and AM-400 instruments in deuterochloroform (CDCl 3 ). Chemical shifts are expressed in ppm downfield relative to tetramethylsilane (TMS,  scale), and the coupling constants (J) are reported in Hertz. 13 C-NMR spectra were obtained in CDCl 3 at 50 and 100 MHz. Chemical shifts are reported in  ppm downfield from TMS, and J-values are given in Hertz. HRMS were obtained on a Thermo Finnigan spectrometer, model MAT 95XP. Silica gel Merck 60 (70-230 mesh) was used for preparative column chromatography and TLC aluminum foil 60F 254 for analytical TLC. Acylnaphthohydroquinones 4a-f were prepared according to a recently reported solar photoacylation procedure [16]. The structure of 4a, 4c-f were confirmed by comparison of their spectral properties with those of authentic samples.