Evaluation of New Naphthalimides as Potential Anticancer Agents against Breast Cancer MCF-7, Pancreatic Cancer BxPC-3 and Colon Cancer HCT- 15 Cell Lines

Copyright: © 2015 Noro J, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Evaluation of New Naphthalimides as Potential Anticancer Agents against Breast Cancer MCF-7, Pancreatic Cancer BxPC-3 and Colon Cancer HCT15 Cell Lines


Introduction
Naphthalimide and bisnaphthalimide are groups of aromatic compounds that have generated intense interests for a number of years by scientists around the world and this is due to their diverse applications in the medical and environmental sciences [1][2][3]. For example due to their inherent intense UV and fluorescence properties, the naphthalimido derivatives had been developed as highly selective fluoride ion sensors [4], molecular probes [5] and dyes [6]. However, the main application to date for the naphthalimides, is their potential as therapeutic agents [7] especially in the area of cancer therapy. The flat aromatic structural of the naphthalimido ring and its ability to bind to DNA by intercalation are attractive features that had attracted much attention in the development of new mono naphthalimides [8][9] and bisnaphthalimido [10][11] derivatives for enhanced anticancer activities and aqueous solubility. Among the mononaphthalimido derivatives, Mitonafide and analogues had reached clinical trial but failed to progress due to unpredicted neurological toxicity side effects [12]. The first generation of mononaphthalmides developed by Brana et al. had a short linker chain from the naphthalimido ring with a tertiary amino group [1]. Since then other workers have made many changes to the naphthalimido rings especially with substitution at positions 5 and 6. More recently Qian et al. have been working on 6 substituted naphthalimido derivatives and demonstrated that these compounds exhibited their toxicities due to a multi targets approach that involve inhibition of topoisomerase II and induction lysosomal membrane permeabilization leading eventually to apoptosis and cell death [13]. Here in this work we have focused our attention in the length of linker chains and the terminal groups. The linker alkyl chain was modified with 2, 3 and 4 carbons and the terminal groups include amino, imino, pyrrole, nitrobenzene, ureas. These compounds were screened against breast cancer MCF-7, colon cancer HCT-15 and pancreatic cancer BxPC-3 cell lines.

Results and Discussion
Chemistry 1,8-Naphtalimide compounds bearing 2,3 and 4 carbon chains, and different terminal functional groups were synthesized by methodologies based on well-established chemistry. Scheme 1 depicts the synthesis of naphthlimido alkylamine and alkyl alcohol products 1a-f in excellent yields. This was achieved by the reaction of naphthalic anhydride with corresponding diamines and amino alcohols in excess [14].
1.6 % cell growth inhibition. Within the three types of tumor cell lines tested, BxPC-3 turned out to be the most sensitive to the compound 4a. Interestingly a different trend was observed with the ureas, in particular compound bearing a p-nitrophenyl group. Urea 6f bearing a 4 carbon atoms chain showed to be the most active compound against MCF-7 cell line, displaying 84.5 ± 3.2 % inhibition of cellular growth. Shorter carbon chain resulted in the loss of activity particularly on MCF-7 cell line; this is evident when comparing the inhibition activity of compounds 6f with 6e (n=3; 28.3 ± 4.1 %), and 6d (n=2; 14.0 ± 5.9 %). The nature of terminal groups in the urea compounds was also found to be important. This was evident when comparing the inhibitory activity of the three ureas 6f (84.5 ± 3.2 %), 6i (10.8 ± 0.6 %) and 6l (8.6 ± 3.1 %), all possessing 4-carbon atoms chains. Certainly, compound 6i incorporates an extra methylene within the benzyl, extending the length of the compound, but as in the benzyl series (6g, 6h, 6i) the length of the carbon chain does not appear to have a relationship between activity and the structure of the molecules. However the relevant structural feature in terms of activity appears to be related with the presence of the nitro group attached to the phenyl group. a GI 50 , concentration of compound required to cause 50% cell growth inhibition after a continuous exposure for 48h. Results are presented as means ± SE of at least three independent experiments performed in duplicate. ND: not determined. Further analysis of the results obtained from the heterocylic imine compounds revealed that the 2c compound bearing a thiophene unit together with a 2 atom carbon chain presented better activity than its furan and pyrrole counterparts on , and HCT-15 (46.2 ± 9.3 % inhibition) cell lines. BxPC3 cell line was more sensitive to the furan compound 2b (33. 5 ± 7.8 % inhibition). Compound 2a bearing a pyrrole ring was the least active among the 2 carbon atoms chain imine compounds. The amino compounds in its hydrochloride salts (3f and 3g), obtained from the heterocyclic imines 2f and 2g, showed no activity on any of the analyzed tumor cell lines. For the MCF-7 cell the best inhibitor compound obtained was the compound 6f with a percentage of cell growth inhibition of 84.5% at 5 µM and a GI 50 of 2.44 µM, close to the one found for the reference drug, doxorubicin, with 99.5% and a GI 50 of 0.024 µM. HCT-15 was more susceptible to the compound 9d with 64.9% of cell growth inhibition at 5 µM, and a GI 50 of 3.6 µM, similar to doxorubicin which presented 73.6% and a GI 50 of 1.07 µM. These two compounds can be proposed as leaders for modification by medical chemistry.

Toxicity of the compounds to non-tumor cells:
The toxicity of the synthesized compounds was evaluated on THP-1 differentiated macrophages and compounds 1a, 2c, 6f and 9d were further tested on mouse bone marrow-derived macrophages (BMMØ) by the MTT assay. No toxicity was observed for a 10 µM concentration from most of the compounds on THP1 differentiated macrophages after 72h incubation (data not shown). The exception occurred with compound 6f, which presented 61.1 ± 3.0 % of cell viability, value below the minimum considered as non-toxic in the screening of new compounds (70 %). However, toxicity assays performed on BMMØ with 5 µM and 50 µM compound concentrations revealed no toxicity of compound 6f for any of the concentrations tested (Table 3). Results are presented as means ± SE of two independent experiments performed in triplicate. This contradictory result may be related with the fact that THP1 differentiated macrophages derived from an immortalized cell line, which although not tumorigenic is derived from the peripheral blood of a patient with acute monocytic leukemia in contrast with the primary non-tumor BMMØ. Also, no toxicity was observed on BMMØ for compounds 1a, 2c and 9d at a concentration of 5 µM. It is noteworthy to mention that at 50 µM concentration, compound 1a induced a decrease in cell viability to 46.2 ± 12.9 % and compound 2c to 67.3 ±

Compound
Inhibition of cell growth (%) 16.6 %. These values suggest some toxicity of these two compounds only at a concentration tenfold higher than the concentration used in our assays (5 µM).

Conclusions
Several naphthalimides were synthetized in good yields and showed very good GI 50 values towards MCF-7, HCT-15, and BxPC-3 cancer cell lines. By changing the alkyl chain length between the naphthalimido group and the functionality at the end of the chain, it was possible to find the best structural features from 1,8-naphthalimido derivatives to achieve enhanced anticancer activity, either generically speaking either against each type of cancer cell line. From these results new perspectives can be drawn to improve the activity of naphthalimides as anticancer agents.

Experimental Chemistry
General: All starting materials were purchased from Sigma-Aldrich, were of research-grade quality and were used without further purification. Compounds were purified by dry flash chromatography, using silica 60 <0.063 mm and water pump vacuum. TLC plates (silica gel 60 F 254 , Macherey-Nagel) were visualized either at UV lamp or with I 2 . 1 H NMR and 13 C NMR were carried out on a Varian Unity Plus 300 (300 MHz) and Brucker Avance III 400 (400 MHz) spectrometers. Infrared spectra were recorded on a Bomem MB 104. Samples were run as nujol mulls and oils as thin films. MS spectra were recorded on a VG Autospec M. spectrometer. Microanalyses were performed in a LECO-CHNS-932 analyzer. Melting points (m.p.) were determined on a Gallenkamp block and are uncorrected. The synthesis of compounds 1a-c were described before. 9a The yields were improved by increasing the number of equivalents of diamine, and by diminishing the coupling reaction time. Alcohols 1d-f were prepared by analogy to the synthesis reported for N- (3-propanol The ammonium chloride salts were obtained by dissolving the amines in dichloromethane and treatment with the solution with HCl gas, produced by heating a concentrated solution of HCl, and passing the gas through H 2 SO 4 .

Synthesis of ureas 6a-l
General procedure: To a solution of compounds 1a-c (0.15-0.26 g, 0.56-0.83 mmol) in dry toluene (8-16 mL). Kept stirring under nitrogen atmosphere in an ice/water bath, was added dropwise the isocyanate (1.0-2.5 equiv.). After addition is complete the reaction mixture was stirred for 1 h at rt, and then refluxed at 130°C in an oil bath for 4-6 h. The reaction mixture was concentrated in the rotary evaporator, refrigerated at -20°C for 20 h to give white, yellow solids as products, which are eventually purified by column chromatography (silica; solvent), 6a-l (55-98 %).