Design of DNA Intercalators Based on 4-Carboranyl-1,8-Naphthalimides: Investigation of Their DNA-Binding Ability and Anticancer Activity

Abstract

In the present study, we continue our work related to the synthesis of 1,8-naphthalimide and carborane conjugates and the investigation of their anticancer activity and DNA-binding ability. For this purpose, a series of 4-carboranyl-1,8-naphthalimide derivatives, mitonafide, and pinafide analogs were synthesized using click chemistry, reductive amination, amidation, and Mitsunobu reactions. The calf thymus DNA (ct-DNA)-binding properties of the synthesized compounds were investigated by circular dichroism (CD), UV–vis spectroscopy, and thermal denaturation experiments. Conjugates 54–61 interacted very strongly with ct-DNA (∆Tm = 7.67–12.33 °C), suggesting their intercalation with DNA. They were also investigated for their in vitro effects on cytotoxicity, cell migration, cell death, cell cycle, and production of reactive oxygen species (ROS) in a HepG2 cancer cell line as well as inhibition of topoisomerase IIα activity (Topo II). The cytotoxicity of these eight conjugates was in the range of 3.12–30.87 µM, with the lowest IC50 value determined for compound 57. The analyses showed that most of the conjugates could induce cell cycle arrest in the G0/G1 phase, inhibit cell migration, and promote apoptosis. Two conjugates, namely 60 and 61, induced ROS production, which was proven by the increased level of 2′-deoxy-8-oxoguanosine in DNA. They were specifically located in lysosomes, and because of their excellent fluorescent properties, they could be easily detected within the cells. They were also found to be weak Topo II inhibitors.

Carboranyl-1,8-naphthalimide intercalators induce lysosomal membrane permeabilization and ferroptosis in cancer cell lines

“…In comparison with unmodified mitonafide and pinafide (Δ T m = 5.17 and 6.50 °C, respectively) 12 , this may indicate an intercalative interaction with DNA as the dominant binding mode. In addition, conjugates 35 and 37 stabilised ct-DNA most strongly among all 1,8-naphthalimides containing carborane clusters obtained in our laboratory 12 , 13 : the Δ T m values of selected 4-carboranyl-1,8-naphthalimides increased within the range of 81.67–86.33 °C (Δ T m = 7.67–12.33 °C), also suggesting their intercalation with DNA 13 .…”

“…The same compounds were the most cytotoxic against HepG2 cells among all the modified conjugated tested. 13 In the current work, these compounds were selected to be lead structures in the synthesis of new derivatives. Furthermore, studies revealed that specific substituent types on the naphthalic ring also might increased the cytotoxicity; e.g., 3-amino-, 3-nitro-, 3-methoxy groups gave the best results 9 .…”

“…The structure, purity, and homogeneity of these compounds were confirmed using 1 H-NMR, 13 C-NMR, 11 B-NMR, FT-IR, MS, RP-HPLC ( Figures S6–S61 , ESI ), and TLC. 3-Carboranyl-1,8-naphthalimide derivatives were synthesised with good yields using the same methodology 12 , whereas 4-carboranyl-1,8-naphthalimide derivatives 13 were synthesised using appropriate active esters bearing carborane clusters 16 . The methodology using active esters was unsuccessful for the synthesis of 12 – 19 .…”

“…A comparison of the activity of the 1,8-naphthalimides in the series showed that the conjugates obtained via amidation reaction 12 – 19 were slightly less cytotoxic than the modified 1,8-naphthalimides obtained using the reductive amination reaction 30 – 37 . Recently, we have reported that 4-carboranyl-1,8-naphthalimides synthesised using the reductive amination reaction were more cytotoxic against the HepG2 cell line than the conjugates obtained via the “click reaction,” Mitsunobu reaction, or amidation reaction 13 . Among compounds 12 – 19 and 30 – 37 , the conjugates containing a shorter linker (CH 2 ) 2 between the heteroaromatic system and carborane modification ( 12 – 15 and 30 – 33 ) were less cytotoxic than those containing a longer linker (CH 2 ) 6 between the heteroaromatic system and carborane ( 16 – 19 and 34 – 37 ).…”

“…In our previous studies, the methods used to synthesise 1,8-naphthalimides modified with a carborane group or mitonafide and pinafide analogs, through modification of imide 11 , bearing the carborane group at position 3 12 or 4 of the ring 13 , have been discussed ( Figure 1 ). It was observed that the attachment site of carborane to the 1,8-naphthalimide moiety and the structure of the linker between the carborane cluster and the heteroaromatic ring system are the factors influencing various physicochemical (interaction with DNA) and biological (cytotoxicity, cell death, cell cycle, ROS production) properties of a HepG2 cancer cell line.…”