Novel 1,8-Naphthyridine Derivatives: Design, Synthesis and in vitro screening of their cytotoxic activity against MCF7 cell line

Abstract A series of new 2-phenyl-7-methyl-1,8-naphthyridine derivatives with variable substituents at C3 were synthesized for an in vitro evaluation of their anticancer activity against human breast cancer cell line (MCF7). On one hand, compounds 3f, 6f, 8c, and 10b showed IC50 values (6.53, 7.88, 7.89, 7.79 μM, respectively) compared to that of the mentioned drug staurosparine (IC50 = 4.51 μM). On the other hand, derivatives 10c, 8d, 4d, 10f and 8b displayed better activity than staurosporin with IC50 values (1.47, 1.62, 1.68, 2.30, 3.19 μM, respectively).


Introduction
Cancer is one of the deadliest diseases affecting public health around the world. The occurrence and death statistics represent that it is on the rise in the developed and economically developing countries [1,2]. Cancer related deaths in females is caused primarily by breast cancer. It constitutes 23% of the total cancer cases, out of which 14% lead to death, the incidence that rises significantly with age [1,2]. On a cellular level, cancer develops as a result of failed cell division and deregulation of signaling cascades. What follows is an uncontrolled cell division, increased cell survival and the development of resistance to anticancer therapies [3].
Even though an early discovery of the disease improves the survival rates through recognized therapeutic routes, metastatic breast malignancy is still the leading cause of breast cancer-related deaths [4]. Noticeably, about 30% of the primarily detected localized malignant masses will progress to an advanced or metastatic disease in 5 years [5,6]. Naphthyridine derivatives are broadly spread in naturally occurring products, principally as tricyclic benzo[f] [1,7] naphthyridines and benzo [c] naphthyridines [2,7]. These derivatives play a vital role in therapeutic products [7][8][9]. Naphthyridines derivatives have a wide spectrum of biological effects, for example anti-inflammatory, antimalarial, antifungal, and antibacterial [10][11][12]. Moreover, naphthyridine derivatives displayed good HIV-1 integrase inhibitor profiles and cytotoxicity [13,14]. Certain 1,8-naphthyridine derivatives are considered good DNA intercalators. They bind with double stranded DNA (ds-DNA) by intercalating between adjacent base pairs, thus changing the DNA conformation and inhibit DNA duplication or transcription leading to suppression of cancer cell growth [15][16][17]. Due to their wide range of biological effects, 1,8-naphthyridine derivatives have been considered promising antitumor agents [18][19][20]. Voreloxin (AG-7352, SPC-595, SNS 595, voreloxin) is a naphthyridine analog. It intercalates DNA in the presence of topoisomerase II, resulting in selective, replicationdependent DNA damage, irreversible G2 arrest and rapid apoptosis [21]  In addition, a novel class of 1,8-naphthyridine-3carboxamide carrying cyclic and open chain amino acids that were linked at C-3 position had been synthesized. These chemicals were tested for anticancer activity, and the results obtained revealed a promising in vitro cytotoxic activity profile against different human cancer cell lines such as breast, colon, oral [22][23][24][25][26]. As a continuation of our previous efforts [27][28][29], herein, we described the synthetic preparation of a new class of compounds bearing 4-hydroxy-7-methyl-2-phenyl-1,8-naphthyridine scaffold. This new class was hybridized with different heterocylic ring systems known to exhibit anticancer activity such as substituted pyrazole, pyridine and pyrimidine rings [30,31] (Figure 2). The new compounds were evaluated as cytotoxic agents against breast cancer cell lines (MCF7). Some of these compounds displayed a noticeable effect on cancer cells, with IC 50 values comparable with the used reference drug.

Materials and Methods
Informed consent has been obtained from all individuals included in this study.
Ethical approval: The conducted research is not related to either human or animals use.

General Information
Commercially available solvents and reagents were purified according to the standard procedures. All melting points were measured on a Barnstead international 1002 melting point apparatus and were calibrated. Thin layer chromatography (TLC) was performed on aluminum silica gel 60 F 254 (E-Merk). The spots were detected by iodine and UV light absorption. IR spectra were recorded for the compounds on a FTIR, Perkin Elmer SP 100 spectrometer. 1 H NMR and 13 C NMR spectra were recorded on Bruker WM 300 and 850 MHz spectrometers using TMS (0.00 ppm) or the signal of the deuterated solvent were used as internal standard. Chemical shift (δ) is given in ppm relative to the signal for TMS as standard, and coupling constant in expressed in Hz. Microanalysis was performed by Perkin Elmer elemental analyzer at the Faculty of Science, King Abdul Aziz University [32].
Ethical approval: The conducted research is not related to either human or animal use.

Chemistry
Synthetic methodology to attain target compounds is clarified in Scheme 1 and 2. Formylation of 7-methyl-2-phenyl-1,8-naphthyridin-4-ol 1 via Vilsmeier-Haack reaction, afforded the vital starting compound in our study: the 4-hydroxy-7-methyl-2-phenyl-1,8-naphthyridine-3-carbaldehyde 2 [35]. The structure of aldehyde 2 was identified based on microanalysis and spectral data. The IR spectra showed the absorption band at ν = 1661 cm −1 due to aldehydic carbonyl group. Its 1 H NMR spectrum showed the disappearance of C 3 -H at δ = 6.30 ppm and a characteristic singlet signal at δ = 9.56 ppm for -CHO group proton. 13 C NMR spectrum for 2 displayed the presence of carbon of a carbonyl group at δ = 190.05 ppm. The formed aldehyde derivative 2 was treated with different acetyl compounds in the presence of MgAl-hydrotalcite (HT) [36] in refluxing ethanol and it produced only one isolable product (as examined by TLC) identified as the chalcones derivatives 3a-g (Scheme 1). As a descriptive example, the 1 H NMR spectrum of 3a displays a characteristic signal at δ = 8.38 and 8.58 ppm as a pair of doublet referring to the two olefinic protons. 13 C NMR spectrum of 3e shows the existence of the carbon of methoxide group at δ = 55.9 ppm. The signals for the two olefinic carbons appeared at δ = 128 and 143 ppm, respectively. Treatment of chalcone 3 with different binuclophilic agents, namely phenylhydrazine, urea, and thiourea afforded in each case one isolable product (examined by TLC) corresponding to pyrazoline 4d,f,g, dihydropyrimidinone 5d,f,g, and dihydropyrimidinethione 6d,f,g derivatives (Scheme 1).

Biological Evaluation
The antitumor activity of compounds 3b, f, g, 4d, g, 5f, 6f, 7, 8a-d, and 10a-f was examined and compared with staurosporin as a standardized medicine antagonizing human breast cancer cell lines (MCF-7) using standard MTT assay process [35]. Cancer cells were either isolated In summary, breast malignant tumors MCF-7 cell lines were viciously inhibited by 1,8-naphthyridine scaffold showing its significant antitumor activity. Variation in the types of heterocyclic rings conjugated with it considerably affected the observed activity. In general, 1,8-napthyridine conjugated with pyridine and pyrazoline, specially 4-bromophenyl pyridine, 4-tolyl pyridine and 3-tolylpyrazoline, introduced novel candidates which could serve as key templates to fight against antitumor effects on breast cancer cells.
Supplementary Materials: Supplementary materials are available online.