Design, Synthesis, and Evaluation of Monoamine Oxidase A Inhibitors–Indocyanine Dyes Conjugates as Targeted Antitumor Agents

Monoamine oxidase A (MAOA) is an important mitochondria-bound enzyme that catalyzes the oxidative deamination of monoamine neurotransmitters. Accumulating evidence suggests a significant association of increased MAOA expression and advanced high-grade prostate cancer (PCa) progression and metastasis. Herein, a series of novel conjugates combining the MAOA inhibitor isoniazid (INH) and tumor-targeting near-infrared (NIR) heptamethine cyanine dyes were designed and synthesized. The synthesized compounds G1–G13 were evaluated in vitro for their cytotoxicity against PC-3 cells using the MTT assay, and molecular docking studies were performed. Results showed that most tested compounds exhibited improved antitumor efficacy compared with INH. Moreover, conjugates G10 and G11 showed potent anticancer activity with IC50 values (0.85 and 0.4 μM respectively) comparable to that of doxorubicin (DOX). This may be attributable to the preferential accumulation of these conjugates in tumor cells. G10, G11, and G12 also demonstrated moderate MAOA inhibitory activities. This result and the results of molecular docking studies were consistent with their cytotoxicity activities. Taken together, these data suggest that a combination of the MAOA inhibitor INH with tumor-targeting heptamethine cyanine dyes may prove to be a highly promising tool for the treatment of advanced prostate cancer.


Introduction
Global prostate cancer (PCa) incidence and mortality have substantially increased, compounded by an increase in the proportion of the elderly population and in the frequency of diagnosis. Although the majority of cases with metastatic PCa respond to the available therapeutic modalities, hormone-refractory PCa and advanced metastatic PCa remain inevitable [1][2][3][4]. Therefore, novel and highly effective treatments with high tumor-targeting specificity, fewer side effects, and improved efficacy are desirable for hormone-refractory PCa and metastatic PCa.

Chemistry
The synthetic route for the type I compounds is outlined in Scheme 1. Intermediates 1 and 2 were both synthesized by electrophilic substitution of 5-substitutional 3H-indole. The synthesis of key intermediate 4 was achieved according to procedures described in the

Chemistry
The synthetic route for the type I compounds is outlined in Scheme 1. Intermediates 1 and 2 were both synthesized by electrophilic substitution of 5-substitutional 3H-indole. The synthesis of key intermediate 4 was achieved according to procedures described in the literature [26]. Briefly, both intermediates 1 and 2 condensed with 3 via base-catalyzed (AcONa) aldol condensation reaction to give intermediate 4.

Chemistry
The synthetic route for the type I compounds is outlined in Scheme 1. Intermediates 1 and 2 were both synthesized by electrophilic substitution of 5-substitutional 3H-indole. The synthesis of key intermediate 4 was achieved according to procedures described in the literature [26]. Briefly, both intermediates 1 and 2 condensed with 3 via base-catalyzed (AcONa) aldol condensation reaction to give As illustrated in Scheme 2, symmetric heptamethine cyanine dye intermediate 6 was synthesized by one-step base-catalyzed (Et3N) aldol reaction of intermediates 1 and 2. However, asymmetric heptamethine cyanine dye intermediate 6 was afforded by the two-step aldol condensation reaction of intermediates 1, 2, and 5 in Ac2O with pyridine as catalyst. Finally, the desired compounds G4-G12 were generated by the coupling of 6 with INH in anhydrous DCM with DCC and DMAP acting as coupling agents. Meanwhile, the type III compound G13 was obtained in the sequence of steps outlined in Scheme 3. As illustrated in Scheme 2, symmetric heptamethine cyanine dye intermediate 6 was synthesized by one-step base-catalyzed (Et 3 N) aldol reaction of intermediates 1 and 2. However, asymmetric heptamethine cyanine dye intermediate 6 was afforded by the two-step aldol condensation reaction of intermediates 1, 2, and 5 in Ac 2 O with pyridine as catalyst. Finally, the desired compounds G4-G12 were generated by the coupling of 6 with INH in anhydrous DCM with DCC and DMAP acting as coupling agents. Meanwhile, the type III compound G13 was obtained in the sequence of steps outlined in Scheme 3.
MTT assay results revealed that nearly all tested compounds showed improved antitumor activity when compared with their parent compound, the MAOA inhibitor INH. The moderate cytotoxicity of these conjugates may be due to their preferential uptake by PC-3 cells mediated by OATPs and subsequent accumulation in the mitochondria of PC-3 cells, disrupting mitochondrial activities. The fact that INH exhibited negligible cytotoxicity may be attributed to its poor passive diffusion into PC-3 cells. G10, G11 (IC 50 values of 0.85 and 0.40 µM, respectively) possessed potent cytotoxicity comparable to that of doxorubicin (DOX) (IC 50 = 0.21 µM). G10 and G11 have very similar chemical structures except for the different chain length of carboxyalkyl connected to the N atom in indole moieties. Their structural similarity may explain their similar antitumor efficacy. These results indicated that the conjugation with INH was conducive to the enhancement of anti-tumor activity against PC-3 in vitro.

MAOA Inhibitory Activity of G10, G11, and G12
Because the prostate cancer cell line LNCaP expressed a relatively high level of MAOA [17,40], we tested the inhibitory effect of compounds G10, G11, and G12 on MAOA using LNCaP cells (Table 2). Our data showed that three synthesized novel compounds displayed an enhanced inhibitory action on MAOA levels as compared with the parent compound INH, while clorgyline-another MAOA inhibitor-exhibited the most potent inhibitory effect. The fact that heptamethine dye-INH conjugates possessed higher MAOA inhibition efficacy than INH may be attributed to the modification of the terminal hydrazine group in INH. This assay result is consistent with that of the cell viability assay, suggesting that the tested compounds inhibited cell growth by suppressing MAOA activity.

Materials
All reagents and solvents were obtained from commercial sources and were used as received unless otherwise stated. Phenylhydrazine hydrochloride, 4-bromophenylhydrazine

Molecular Docking
The crystal structure of MAOA with a resolution of 3 Å was retrieved from the Protein Data Bank (PDBID: 2BXR). AutoDock 4.2 suite, an automated docking tool that uses a Lamarckian genetic algorithm (LGA), was used to perform the molecular docking simulations [42]. Briefly, all the bound water molecules, ligands, and co-factors were eliminated from the protein, and the polar hydrogen was added to the proteins. Gasteiger charges were computed in AutoDockTools 4.2 [43]. 2D structures of G11 were constructed using ChemBioDraw ultra12.0 and were transformed to 3D. Subsequently, 3D structures of the aforementioned compounds were also deduced and then the structure was energetically minimized by using an MMFF94 force field.
In the docking process the grid point maps were calculated using AutoGrid 4.2. The program AutoGrid was used to generate the grid maps. Each grid was centered at the structure of the corresponding receptor. The grid dimensions for the grid maps were fixed at a spacing of 0.375 Å with the grid box of size 90 × 70 × 60 (number of points in x-, y-, and z-axes for both proteins). For each docking experiment, default parameters for docking run included number of final conformations, 100; population size, 150; maximum number of energy evaluations, 2.5 × 10 6 , and maximum number of generations, 27,000. Some low-binding-energy docked poses were investigated in detail for their binding interactions with the binding cavity of the target protein, using Discovery Studio Visualizer (DSV). The docked compound complexes were built using the low-free-energy binding positions.

Cytotoxicity Evaluation
The in vitro cytotoxicity activity of G1-G13 were measured by the colorimetric MTT assay. Briefly, PC-3 cells were incubated in 96-well plates at a density of 3 × 10 3 cells/well for 24 h. Then, the cells were exposed to various concentrations of tested compounds, INH and DOX, respectively for 96 h. After incubation, 10 µL of MTT (5 mg/mL) solution was added and the plates were incubated for 4 h at 37 • C. Next, the medium was replaced with 200 µL of DMSO to dissolve the formed formazan crystals. The absorbance was measured at 570 nm on a microplate reader (Synergy-HT, BioTek Instruments, Winooski, VT, USA).

MAOA Inhibition Activity Assay
In a 10-mm dish, 6 × 10 5 LNCaP cells were plated in medium supplemented with 10% FBS. After 24 h, cells were treated with various concentrations (10 pM, 1 nM, 100 nM, 10 µM) of the tested compounds G10, G11, G12, isoniazid, and clorgyline for 48 h. The reaction products were extracted and MAOA activities were measured as described in the introduction for the Cell MAOA Assay Kit (Shanghai Chengong Biotechnology, CHN, Lot No: 1-201838-10). The MAOA activity was assessed based on the substrate p-tyramine level after treatment with MAOB inhibitor pargyline.

Conclusions
In this study, we developed a series of novel near-infrared heptamethine dye-INH conjugates. Evaluation of IC 50 values indicated that nearly all conjugates showed moderate antitumor efficacy. Among them, conjugates G10 and G11 exhibited strong cytotoxicity comparable to doxorubicin in inhibiting PC-3 cell growth while INH showed negligible in vitro antitumor activity. Furthermore, MAOA inhibitory activities of G10, G11, and G12 and molecular docking analysis were consistent with the cell viability results, suggesting that the enhanced antitumor activity of these conjugates may be attributed to the preferential accumulation in cancer cells mediated by OATPs and the increased MAOA inhibitory activity derived from INH N-acylation. All these above results suggested that a combination of the MAOA inhibitor INH and tumor-specific targeting heptamethine cyanine dyes may provide a promising tool for the treatment of advanced prostate cancer.