Preparation of Novel Homodimers Derived from Cytotoxic Isoquinolinequinones. A Twin Drug Approach

The synthesis of five novel homodimers is reported based on the anilinoisoquinolinequinone scaffold. In these twin-drug derivatives, two units of the anilinoquinone pharmacophores are linked through a methylene spacer. The formation of dimers was achieved by reaction of isoquinolinequinones with 4, 4′-diaminodiphenylmethane via a sequence of two oxidative amination reactions. A preliminary in vitro screening of the homodimers reveals moderate to high cytotoxic activities against MDA-MB-21 breast adenocarcinoma and B16-F10 murine metastatic melanoma cell lines. The asymmetrical homodimer 15 stands out due to its cytotoxic potencies at submicromolar concentrations and high selectivity index (mean IC50 = 0.37 μM; SI = 6.97) compared to those of etoposide (mean IC50 = 3.67; SI = 0.32) and taxol (mean IC50 = 0.35; SI = 0.91) employed as reference anticancer drugs.


Introduction
The quinone nucleus is the common feature of many drugs used clinically in the therapy of solid cancers, such as daunorubicin, mitomycin, mitoxantrone, and saintopin. The most remarkable characteristics of quinonoid compounds are their ability to acts as DNA intercalators, reductive alkylators of biomolecules, and/or generators of reactive oxygen species (ROS), which can damage tumor cells [1][2][3][4][5][6]. Since many of the currently available anticancer drugs are incapable of differentiating between normal and neoplastic cells, there is a pressing need for new anticancer agents with high potency and less toxicity to noncancerous cells. Among the broad variety of synthetic quinonoid compounds, a group of donor-acceptor members derived from 2-anilino-1,4naphoquinone and 7-anilinoisoquinoline-5,8-quinone analogues such as compounds A, B and C (Figure 1), have a wide range of remarkable in vitro cytotoxic activity against a variety of cancer cell lines [7][8][9][10][11]. We have recently reported preliminary synthetic efforts aimed at the construction of homodimers of 7-anilinoisoquinolinequinones such as B and C, to produce twin drugs of these cytotoxic pharmacophores [12]. The linkage of two identical pharmacophoric entities, generating an "identical twin drug" or homodimer derivative, is a classical strategy used in medicinal chemistry to produce more potent and/or selective drugs compared to the single entities [13][14][15][16]. Our efforts to prepare homodimers from isoquinolinequinones and symmetric aryldiamines such as pphenylendiamine, benzidine, and dapsone were unsuccessful, since the diamines act as mononucleophiles to produce the corresponding arylaminoisoquinolinequinones [12]. The lack of reactivity of these amination products to undergo a further amination reaction with the isoquinolinequinones to give the corresponding homodimers is probably due to significant donoracceptor interactions between the quinone and the arylamino components [17], thus decreasing the nucleophilic character of the NH2 groups. Here, we report successful results on the access to five homodimers based on two identical anilinoisoquinolinequinone fragments linked via a methylene spacer, from isoquinolinequinones and 4, 4'-diaminodiphenylmethane. The preparation of monoamination compounds as valuable precursors of heterodimers is also described. A preliminary in vitro evaluation of the new homodimers and their corresponding arylaminoisoquinolinequinones intermediates is reported.

Chemistry
The isoquinolinequinones 1-4 and the commercially available symmetrical diamine 5, were selected as starting materials to prepare the target identical twin-drug homodimer types containing the anilinoisoquinolinequinone pharmacophores B and C ( Figure 2). Quinones 1-4 were synthesized by a previously reported method [9,10]. We first examined the reaction of quinone 1 with diamine 5 in a 2:1 mole ratio in the presence of catalytic amounts of CeCl3×7H2O in ethanol at room temperature. Work-up of the reaction mixture followed by column chromatography yielded product 6 and the symmetric homodimer 11 in 8 and 41% yields, respectively (Scheme 1). The structures of compounds 6 and 11, in which the quinoid moiety is bonded to the diamine through the 7-position, were fully characterized by infrared spectroscopy (IR), 1 H-and 13 C-nuclear magnetic resonance (NMR), bidimensional nuclear magnetic resonance (2D-NMR), and high resolution mass spectroscopy (HRMS).
It is evident that the reaction of quinone 1 with diamine 5 under aerobic conditions proceeds to give product 6, which, by a further oxidative amination with electrophile 1, yields homodimer 11. The results show that the amino group of compound 6 is as nucleophilic as those of diamine 5 to react with electrophile 1, to give 11. It can therefore be concluded that there are no significant electronic interactions between the amino group and the donor-acceptor anilinoisoquinolinquinone fragment of compound 6.
Based on this preliminary assay (Scheme 1), we focused on the selective access to the target symmetric homodimer 11 by performing the reaction of 1 with amine 5 in a 4:1 mole ratio under the above-mentioned conditions. Surprisingly, the reaction produced the expected dimer 11 in nearly quantitative yield (98%).
Taking into account the behavior of 6 to react with isoquinolinequinone 1 and its potential application in the synthesis of new heterodimers by reaction with different cytotoxic carbo-and heterocyclic quinones, we investigated the experimental conditions to allow selective access to compound 6. After several trials, we found that 6 is formed in 74% yield by reaction of 1 with amine 5 in a 1:2 mole ratio under the standard conditions (Table 1). Based on the optimal experimental conditions to prepare compounds 6 and 11, the synthesis of monoamination and homodimer products from isoquinolinequinones 2-4 and diamine 5 was attempted. The reactions of quinones 2, 3, and 4 with 5 in a 2:1 mole ratio produced the expected monoamination products 7, 8, and 9 in 55, 57 and 32% yield, respectively. From the reaction of quinone 3 with 5, the monoamination compound 10 was isolated along with 8, albeit in poor yield (6%) (Scheme 2; Table 2). The reaction of quinones 2, 3 and 4 with 5 in a 4:1 mole ratio produced the expected homodimers 12, 13, and 14 in 95, 20, and 58% yield, respectively. From the reaction of quinone 3 with 5, the asymmetrical homodimer 15 was isolated in 50% yield, along the symmetrical homodimer 13, ( Table 2). The findings on the reactions of quinone 3 with diamine 5 suggest that competitive nucleophilic attacks of compounds 8/10 to the 6-and 7-positions of quinone 3 are involved.   It should be noted that the oxidative amination reaction of quinone 1 with diamine 5 produced homodimer 11 as the unique regioisomer; however, two isomeric homodimers, 13 and 15, were generated in the reaction of quinone 3 with diamine 5. The differences in the regiochemistry of the oxidative amination reaction of quinones 1 and 3 with amine 5, are in agreement with previous studies on the oxidative amination of isoquinolinequinones with amines. For instance, it was determined that quinone 1 reacts with alkyl-and arylamines in a regiospecific manner to furnish the respective 7-substituted regioisomers [9,11]. In the case of quinone 3, the amination reactions take place with regioselective preferences to give the 7-substituted regioisomers, as the main products, along with the 6-substituted regioisomers [18]. The regiochemical control of the substitution reactions of quinones 1 and 3 can be explained assuming stereoelectronic interactions between the substituents at C-1 (CH3 and H) and the carbonyl group at C-8. These factors probably affect the electrophilicity of the C-7 and the preference of the nitrogen nucleophiles to attack this electrophilic centre. The structures of the arylaminoquinones and homodimers were determined by IR, 1 H-, 13 C-NMR, 2D-NMR and HRMS. The symmetrical homodimers 11-14 showed magnetically equivalent spectroscopic signal patterns, indicating their C2-symmetric molecular feature in solution. Heteronuclear multiple bond correlation (HMBC) experiments were used to establish the structure of symmetrical and asymmetrical homodimers 13 and 15 ( Figure 4).

Biological Results
The prepared homodimers 11-15 were evaluated for their in vitro cytotoxic activity using a conventional fluorescence assay (Cyquant direct cell proliferation assays) [19], against primary mouse embryo fibroblast cell line MEF and two cancer cells lines: MDA-MB-21 human breast adenocarcinoma and B16-F10 murine metastatic melanoma cells, in 72 h drug exposure assays. Monoamination products 6-10 were included in these preliminary assays. Table 2 shows the IC50 values, and selectivity indexes of the new compounds. Etoposide and taxol, used clinically as anticancer agents, were taken as positive controls. According to the data in Table 2, compounds 14 and 15 appeared as the most potent members of the synthesized homodimers. Compound 15 stands out due to its cytotoxic activity at submicromolar concentrations and high selectivity index (mean IC50 = 0.37 μM; SI = 6.97) compared to those of etoposide (mean IC50 = 3.67; SI = 0.32) and taxol (mean IC50 = 0.35; SI = 0.91) used as reference anticancer drugs.
In view of the high incidence of undesirable side effects induced by the majority of current anticancer drugs and considering the selective indexes of homodimers 14 and 15, they appear as promising and interesting leads, having potential anticancer activity.

Conclusions
In conclusion, we have prepared new homodimers and monoamination compounds derived from cytotoxic isoquinolinequinones and the symmetrical 4.4'-diaminodiphenylmethane. Selective access to these compounds was achieved through a one-step procedure, using appropriate reactant ratios. The high cytotoxic potencies of the usymmetrical homodimer 15 and the potential application of the monoamination compounds to prepare new heterodimers by combining different cytotoxic anilinoquinones, opens the possibility of constructing new twin drug scaffolds as more active and selective anticancer agents.
Supplementary Materials: The 1 H-NMR, 13 C-NMR, 2D-NMR spectra of compounds 6-15 are available as supporting data. Supplementary materials are available online.