Design, Synthesis, and In Vitro Evaluation of Novel 8-Amino-Quinoline Combined with Natural Antioxidant Acids

Overproduction of reactive oxygen species (ROS) and alterations in metallostasis are common and related hallmarks in several neurodegenerative diseases (NDDs). Nature-based derivatives always represent an attractive tool in MTDL drug design, especially against ROS in NDDs. On this notion, we designed a new series of 8-quinoline-N-substituted derivatives with a natural antioxidant portion (i.e., lipoic, caffeic, and ferulic acids). These compounds were shown to chelate copper, a metal involved in ROS-induced degeneration, and scavenger oxygen radicals in DPPH assay. Then, selected compounds 4 and 5 were evaluated in an in vitro model of oxidative stress and shown to possess cytoprotective effects in 661W photoreceptor-like cells. The obtained results may represent a starting point for the application of the proposed class of compounds in retinal neurodegenerative diseases such as retinitis pigmentosa (RP), comprising a group of hereditary rod–cone dystrophies that represent a major cause of blindness in patients of working age, where the progression of the disease is a multifactorial event, with oxidative stress contributing predominantly.


Introduction
Oxidative stress (OS) is a complex network of malfunctioning pathways resulting from an imbalance of oxidant and antioxidant processes occurring within cells and tissues. Balanced levels of reactive oxygen species (ROS) and nitrogen species (RNS) are deputed to physiological processes and are finely regulated for cell differentiation and migration, immune response, and apoptosis [1]. When this equilibrium is altered, ROS induce a progressive loss of tissues' function, thus contributing to the onset and/or progression of several neurodegenerative disorders [2]. Despite heterogeneity, OS represents a common hallmark in the pathogenesis of uncurable neurodegenerations including Parkinson (PD), Alzheimer (AD), Huntington (HD), Batten (BD) diseases, and amyotrophic lateral sclerosis (ALS) [3][4][5]. In this context, retinal neurodegenerations are a group of different disorders associated with retinal tissue. For example, age-related macular degeneration (AMD) is a multifactorial disease in which OS drives a series of events such as protein misfolding and deposition. In addition, retinitis pigmentosa (RP) is an orphan genetic disease (ORPHA:791) in which the primary death of rods carrying the mutation causes an increase in retinal oxygen levels. This event triggers the increment in ROS production and supports the progression of the disease, leading to secondary neurodegeneration of the cones. Several studies also reported that antioxidant molecules are effective in slowing cone death in Taking this into account, a new series of 8-quinoline-N-substituted derivatives 1-8 have been designed and synthetized by linking the 8-aminoquinoline scaffold, as chelating scaffold, with natural-based antioxidant compounds including ferulic (FA), caffeic (CA), and lipoic (LA) acids [33] (Figure 1), which have been widely investigated for their antioxidant behavior and have been proven to directly neutralize free radicals [34][35][36][37][38].
Compounds 1-8 were tested for their ROS-scavenging and copper-chelating properties. Finally, an in vitro photoreceptor-like model (661W cells) of H 2 O 2 -induced oxidative damage was used to evaluate cytoprotective and antioxidant activity by assessing cell ROS+ and levels of acrolein produced, a biomarker of oxidative stress.

Chemistry
Compounds 1-8 were synthetized, as depicted in Scheme 1. Briefly, protected amines 10a-c were obtained by the reaction between the amino acid 9a-c (i.e., glycine, β-alanine, or γ-aminobutyric acid) and di-tert-butyl dicarbonate. Then, the condensation of 10a-c with 8-aminoquinoline provided the amides 11a-c. N-Boc adducts 11a-c were then deprotected with TFA, affording the free amines 12a-c, which were condensed with lipoic, caffeic, and ferulic acids, yielding the final products 1-8. The amide-coupling reaction with lipoic acid and 12a-c was performed with TBTU/DIPEA as coupling reagents, whereas the same reaction with caffeic and ferulic acids was performed replacing TBTU/DIPEA with DCC/DMAP.

Copper-Chelating Activity Test
Compounds 1-8 were preliminarily tested for their capacity to chelate copper ions by UV-Vis spectroscopy. The specific absorbance peaks of compounds 1-8 were monitored in the range of 270-500 nm ( Figure 2). The co-incubation of 10 µM of the tested compound together with increasing amounts of CuCl 2 induced an isosbestic shift of the absorption for compounds 1, 4, 5, 6, and 7, indicating the formation of Cu 2+ -compound complexes. On the contrary, derivatives 2, 3, and 8 were found to be devoid of chelating activity (data not shown). We also evaluated the molar ratio between chelating ligands and copper ions. The stoichiometry of the Cu 2+ -compound complex was determined by using the molar ratio method ( Figure 2). The maximum variations of absorbance were recorded with a 10 µM solution of the tested compounds and an increased concentration of CuCl 2 (0-50 µM). As shown, the absorbance initially increased until a breakpoint, and the intersection of the two straight lines indicates the mole fraction of Cu 2+ -ligand complexes. From the analyzed data, we obtained that lipoic derivative 1, caffeic compounds 4 and 5, and ferulic ligand 7 gave a mole fraction of 2, indicating a 2:1 stoichiometry for the Cu 2+ -ligand complex, whereas the Cu 2+ -6 complex showed a 2.5:1 stoichiometry.

Radical-Scavenging Activity
The radical-scavenging properties of derivatives 1-8 were evaluated using a spectrophotometric in vitro assay with 1,1-diphenyl-2-picryl-hydrazyl (DPPH). Compounds 1-8 and their relative phytonutrient precursors (LA, CA, and FA) were initially tested at three different concentrations (200, 100, and 50 µM) and incubated for 45 min with a 500 µM DPPH methanolic solution. The antioxidant activity is depicted in Table 1 as the mean values ± standard deviations. Caffeic derivatives 4-6 were good antioxidants, showing a percentage of radical scavenging higher than caffeic acid (63.9%). Hence, the IC 50 s (defined as the concentration resulting in a 50% scavenging activity) were determined ( Figure 3). Although the FA derivatives showed the same scavenging capacity as the parent compound (e.g., FA) at all concentrations tested, the lipoic acid derivative 2 possessed only a modest scavenging capacity, at 200 µM, whereas 1 and 3 proved to be devoid of antioxidant activity.

Biological Evaluation of Compound in H 2 O 2 -Induced Cell Death
To further evaluate whether compounds 1-8 protect against OS-induced cell death, we assessed their efficacy in preserving the H 2 O 2 -induced 661W cell line death at five different concentrations (1-100 µM). The range of concentrations was chosen according to the antioxidant activity of the compounds observed through the DPPH assay. The administration of H 2 O 2 (500 µM for 3 h) to the cells led to an approximately 90% decrease in cell viability. All compounds tested were unable to protect the cells (see Figure S1 in Supporting Materials) except for compounds 4 and 5, which induced a significant reduction in H 2 O 2 -induced damage, preserving cell viability ( Figure 4). Based on the results, despite an increase in cell viability being found at concentrations of 50 and 75 µM, after the H 2 O 2induced cell death, 100 µM was considered the most suitable concentration of 4 and 5 to be used for further experiments.

Compounds 4 and 5 Protect 661W Photoreceptors from H 2 O 2 -Induced Cell Death by Reducing Intracellular ROS Generation
We then verified whether derivatives 4 and 5 attenuate H 2 O 2 -mediated cell death by suppressing intracellular ROS generation. Flow cytometry data showed that treatment with H 2 O 2 resulted in an increase in intracellular ROS generation in the 661W cell line compared to the control group ( Figure 5A). The bar graph in Figure 5B shows the percentage of the ROS-positive (ROS+) cell population in the control (black bar), stressed cells (gray bar), and the cells pre-treated with the selected compounds 4 and 5 (purple and light-blue bar, respectively). From the graph, it is possible to observe that the pre-treatment was able to significantly reduce the percentage of ROS+ cells. To validate the protective function of the two compounds against OS, we assessed the expression of acrolein, which is a marker of lipid peroxidation induced by oxidative stress. In the panel in Figure 6A, representative images, acquired by confocal microscopy, showing the presence of acrolein adducts (red staining) following damage with H 2 O 2 in the 661W cells, in green show a cell-specific marker (cone arrestin) and, in blue, the nuclei. The results obtained by the acquisition of the confocal image were confirmed by the quantification of acrolein cell fluorescence (CTCF), summarized in Figure 6B. Acrolein expression increased considerably in the 661W cells after exposure to H 2 O 2 ; however, pre-treatment with 4 or 5 seemed to significantly reduce the intensity of fluorescence relative to the acrolein, validating the antioxidant effect of the tested molecules. Overall, these results suggested that 4 and 5 reduce the H 2 O 2 -induced intracellular ROS generation in the 661W photoreceptor-like cells, leading to a decrease in oxidative stress-mediated cell death.

Discussion
In our previous studies, we demonstrated that the introduction of phytonutrients including lipoic, caffeic, and ferulic acids into the chemical scaffold of acetylcholinesterase inhibitors such as rivastigmine [32] and tacrine [33][34][35] leads to new molecules, which retain the cholinesterase inhibitory activity and show antioxidant and chelating properties, thus proving to be valuable disease-modifying candidates to treat AD. Following the same strategy and aiming at identifying new compounds with good antioxidant and metal-chelating profiles, herein we described the synthesis and the preliminary evaluation of a small series of new compounds obtained by linking LA, FA, and CA with the 8-amminoquinoline scaffold, known to possess chelating properties [36,37]. The choice of nature-based acids relies on the solid knowledge of their safe profiles and, moreover, on additional effects beyond their direct antioxidant activity. The activation of antioxidant enzymes such as SOD (superoxide dismutase) or CAT (catalase), antioxidant proteins such as GSH (glutathione), and autophagic machinery stimulation represents attractive aspects that support their use in anti-AD drug discovery [39][40][41]. The novel 8-amminoquinoline derivatives were then evaluated for their capability to chelate copper by UV-Vis spectroscopy. Results indicated that five out eight compounds were able to complex Cu 2+ with a stoichiometry of 2:1, whereas lipoic acid derivatives 2 and 3 were unable to chelate Cu 2+ . Further, we performed a DPPH assay to evaluate the radical-scavenging ability. As expected, the newly synthetized molecules retained the scavenging properties of the parent compounds, at least at higher concentrations. Of note, derivatives 4-6 showed the best antiradical activity, while 7-8 resulted to be two-thirds fold less active than the caffeic analogues (4-6), indicating the importance of the catecholic portion for the antioxidant potential. In order to evaluate the new molecules as potential tools to fight OS, we assessed the cytoprotective effects in the H 2 O 2 -induced damage of a photoreceptor-like cell line. Photoreceptors are prone to oxidative stress, mainly from a high metabolic rate, and OS alteration may lead to retinal dysfunction and progressive neurodegeneration of cone photoreceptors, which can be worsened by the disruption of metal ion homeostasis. Considering the key role of both ROS and metal ions in OS-induced damage, compounds 1-8 were assessed for protection against oxidative stress. Within the compounds synthetized, 4 and 5 proved to protect the 661W photoreceptors from H 2 O 2 -induced cell death by reducing intracellular ROS generation at concentrations in the range of 50-100 µM. Moreover, both compounds were able to decrease the expression of acrolein, a marker of lipid peroxidation induced by oxidative stress. Additionally, further investigation will be needed to elucidate whether the cytoprotective effect induced by compounds 4 and 5 is essentially due to the scavenger and chelating properties or is the result of a more complex multifunctional activity due to a polypharmacological profile.

Synthesis and Characterization
General Material and Methods. Commercial-grade anhydrous solvents were used without further drying. Commercially available chemicals were purchased from Sigma-Aldrich, Fluorochem, or Alfa Aesar and used without further purification. Evaporation was performed in a vacuum (rotating evaporator). Anhydrous Na 2 SO 4 was always used as the drying agent. Flash chromatography was performed on Merck 60 Å high-purity grade silica gel (0.40-63 µm). Reactions were followed by TLC, performed on Merck aluminum silica gel (60 F254) sheets. Spots were viewed under a UV lamp (254 nm) or with the aid of 10% phosphomolybdic acid in EtOH. Celite ® 545 was used as a filter agent. All melting points were determined on a Barnstead/Electrothermal MeL-Temp Model 1101D and were uncorrected. The 1 H and 13 C NMR spectra were obtained using a Bruker TopSpin 3.2 400 MHz Spectrometer and were recorded at 400 and 101, respectively. Chemical shifts are reported in parts per million (ppm) δ values, downfield from the internal reference tetramethylsilane (TMS), and referenced from solvent resonance as the internal standard: deuterochloroform (δ 7.26 (1H spectra), δ 77.16 (13C spectra)); deuterodimethylsulfoxide (δ 2.50 (1H spectra), δ 39.52 (13C spectra)); deuteromethanol (δ 3.31 (1H spectra)). Coupling constants J are reported in Hertz (Hz). The 13 C NMR spectra were 1 H decoupled. Signal patterns are indicated as follows: singlet (s), doublet (d), triplet (t), double-doublet (dd), double-triplet (dt), multiplet (m), broad singlet (br s), broad doublet (br d), broad triplet (br t), and broad multiplet (br m). High-resolution mass spectra were recorded on a ABSciex 3200 QTRAP using electrospray positive ionization. HPLC purity determination was performed on a Varian Pro Star 330PDA detector, a ternary HPLC pump Varian 9012, and a Rheodyne injector with 20 µL loop. An RP column ThermoScientific TM Hypersil TM C18 ODS (5 µm, 250 × 4.6 mmID) HPLC was used for all analyses (detection at 240 nm). Stock solutions of compounds 1-8 were prepared in a mixture of MeOH/ACN (2:3) and stored at 4 • C. The mobile phase was acetonitrile/water, and the elution gradient varied, according to the method depicted in the Supplementary section. HPLC analysis confirmed the ≥95% purity of all compounds, 1-8.

General Procedure for the Synthesis of Compounds 1-3
To a solution of commercial lipoic acid (30 mg, 0.14 mmol), in dry DMF, under N 2 atmosphere, and cooled to 0 • C, TBTU (46 mg, 0.14 mmol) and DIPEA (0.05 mL, 0.29 mmol) were added. After 30 min at 0 • C, the intermediate 12a-c (0.14 mmol) was added, and the temperature was kept at 0 • C for an additional 30 min. Later, the mixture was slowly warmed to rt and left under stirring overnight. Then, the organic solvent was reduced under vacuum, and the crude mixture was treated with H 2 O. The resulting precipitate was collected by filtration, dried, and further purified by flash chromatography.  To a solution of commercial caffeic acid (89 mg, 0.49 mmol) in DCM/DMF 1:1, DCC (111 mg, 0.54 mmol), a catalytic amount of DMAP, and amine 12a-c (0.45 mmol) were added. The mixture was stirred for 12 h at rt. Then, the organic solvent was evaporated under vacuum, and the crude product was purified by flash chromatography to afford the final compounds.     171.73, 166.52, 148.30, 147.30, 146.67,  140.77, 138.37, 136.41, 134.37, 128.01, 127.45, 127.30, 122.22, 121.77, 121.73, 118.41, 116.55, 114.65,  109.48, 55.95, 39.47, 35.65, and 25.06 ppm.

4-((tert-butoxycarbonyl)amino)butanoic acid (10c)
To a solution of commercial γ-aminobutyric acid (1.00 g, 11.22 mmol) in NaOH 1M/iPrOH 4:3 at 0 • C, Boc 2 O (2.45 g, 11.22 mmol) was added. The procedure followed was the same as that described for derivative 10a. The crude product (1.32 g, 6.51 mmol, 58% yield) was used for the next step, without further purification. Tert-butyl (3-oxo-3-(quinolin-8-ylamino)propyl)carbamate (11b) To a solution of compound 10b (400 mg, 2.11 mmol), in dry DMF, under N 2 atmosphere, and cooled to 0 • C, TBTU (677 mg, 2.11 mmol) and DIPEA (0.7 mL, 4.22 mmol) were added. After 30 min at 0 • C, commercial 8-aminoquinoline (306 mg, 2.11 mmol) was added, and the temperature was kept at 0 • C for an additional 30 min. Later, the mixture was slowly warmed to rt and left under stirring at rt for 72 h. Then, the organic solvent was reduced under vacuum, and the crude residue was diluted with DCM and washed with H 2 O and a solution of HCl 10%. The organic layer was dried over Na 2 SO 4 , and the solvent was evaporated, affording the intermediate 11b that was used for the next step, without further purification (313 mg, 0.99 mmol, 47% yield). Tert-butyl (4-oxo-4-(quinolin-8-ylamino)butyl)carbamate (11c) To a solution of compound 10c (400 mg, 1.97 mmol), in dry DMF, under N 2 atmosphere, and cooled to 0 • C, TBTU (632 mg, 1.97 mmol) and DIPEA (0.7 mL, 3.94 mmol) were added. After 30 min at 0 • C, commercial 8-aminoquinoline (285 mg, 1.97 mmol) was added, and the temperature was kept at 0 • C for an additional 30 min. Later, the mixture was slowly warmed to rt and left under stirring at rt for 72 h. Then, the organic solvent was reduced under vacuum, and the crude residue was treated with H 2 O. The resulting precipitate was collected by filtration and dried, affording the intermediate 11c that was used for the next step, without further purification (552 mg, 1.67 mmol, 85% yield).

Metal-Chelating Study
The copper-chelating activity was performed as previously reported and monitored spectrophotometrically using a UV-Vis PerkinElmer EnSpire 2300 spectrophotometer [27]. CuCl 2 was dissolved in absolute ethanol to make a concentration of 400 µM. Then, 100 µL of the CuCl 2 solution was added to make a final concentration of compounds of 10 µM, which were incubated at room temperature for 30 min. Then, the absorption spectrum was recorded with a UV-Vis spectrophotometer.

DPPH Assay
DPPH was dissolved in methanol to make a concentration of 1 mM, and compounds 1-8 were dissolved in methanol and diluted in a 96-well plate at several concentrations (200, 100, and 50 µM) [27]. Then, in each well, the DPPH solution was added (final concentration of 500 nM) and incubated at room temperature in the dark for 30 min. The absorbance was read at 531 nm with a PerkinElmer EnSpire 2300 multiplate reader. The capacity of scavenging free radicals was calculated as followed: capacity = (A DDPH − A compound /A DPPH ) × 100, where A DPPH and A compound are, respectively, the absorbances of the control and the tested compounds. Instead, to detect the IC 50 of the selected compounds, 300 µM methanolic solution of compounds was properly diluted to 150, 75, 37.5, 18.8, 9.4, 4.7, and 0 µM (duplicated), following the procedure previously described.

Cell Culture
The 661W photoreceptor cells were supplied by Dr. Muayyad Al-Ubaidi (University of Oklahoma Health Sciences Center). These cells were cultured in Dulbecco's Modified Eagle's medium, high glucose, (DMEM) with 10% (v/v) fetal bovine serum and 1% penicillin-streptomycin solution and maintained at 37 • C in a humidified incubator with 5% CO 2 . The material used for the cell cultures was purchased from Sigma-Aldrich (Merck, Darmstadt, Germany).

Drug Stock Preparation
The hydrogen peroxide solution (H 2 O 2 ) was purchased from Sigma-Aldrich and diluted to a working concentration in DMEM. The compounds were synthesized as described above, and stock solutions (10 mM) were prepared in DMSO and then diluted to working concentrations in DMEM.

Cell Viability
Cell vitality was tested by using CellTiter 96 AQueous One Solution Reagent (Promega, WI USA). The cells were seeded in a 96-well plate at a density of 5 × 10 3 cells per well and incubated overnight at 37 • C in 5% CO 2 . The cells were then pre-treated with the compounds (1-100 µM) for 24 h. The next day, H 2 O 2 was added at the concentration of 500 µM and, after 3 h, the cells were treated with the One Solution Reagent and further incubated for 2 h at 37 • C, 5% CO 2 . The absorbance was measured at 490 nm with a 96-well plate reader. The quantity of the formazan product, as measured by the amount of 490 nm absorbance, was directly proportional to the number of living cells in the culture. The percentage of cell viability was normalized to the control group (no compounds or H 2 O 2 exposure).

Intracellular Reactive Oxygen Species' (ROS) Determination
The cells undergoing oxidative stress, defined by the presence of ROS, namely, superoxide, were determined by a Muse ® Oxidative Stress Kit. Briefly, after culturing, in a 24-well plate at a density of 5 × 10 4 cells per well, the cells were pre-treated with compounds 4 and 5 at the chosen concentration of 100 µM. The next day, H 2 O 2 was added at the concentration of 500 µM, and, after 3 h, the cells were re-suspended at a concentration of 1 × 10 6 cells per mL in 1X assay buffer (Muse ® Oxidative Stress Kit). After that, the samples were incubated for 30 min at 37 • C, and then the ROS-positive cells were examined using the Muse ® Cell Analyzer.

Immunofluorescence
The cells were seeded onto an eight-well chamber slide at a density of 10 4 cells/well. The cells were pre-treated, with compounds 4 and 5, as indicated, fixed in 2% paraformaldehyde for 15 min, permeabilized with 2.5% bovine serum albumin (BSA) and 0.3% Triton X-100 for 10 min, blocked in 2.5% BSA for 1 h, then incubated overnight at 4 • C with primary antibodies against cone arrestin (Millipore, ab15282, 1:1000) and acrolein (Abcam, ab48501, 1:1000). The next day, the cells were incubated with Anti-Rabbit Alexa Flour-488 and Anti-Mouse Alexa Flour-568 for 2 h at room temperature. Finally, the cells were counterstained with DAPI and washed three times with PBS, and images were acquired by using a Nikon Ni-E confocal microscope using a 20X objective. For each treatment group, a total of 50 cells, distributed in three different fields, were measured using a 20X objective. The acrolein signal was calculated as "correct total cell fluorescence" (CTFC), based on the signal intensity and area of each individual cell, to normalize the signal distribution within the cells with different sizes. Each cell was contoured to the cell membrane using brightfield images. Then, each cell's area and integrated density, as well as five measurements of the surrounding background, were calculated using ImageJ software. CTCF was calculated as follows: CTCF = integrated density-(selected cell area × average background density). Values were subsequently normalized with the H 2 O 2 500 µM group.

Statistical Analysis in Vitro Assay
The Origin Lab 8.0 program (MicroCal, Northampton, MA, USA) was used for data analysis and graphical presentation. All data are presented as the means ± SE. Statistical analyses were performed by using a one-way ANOVA test followed by Levene's post-test, as indicated in each graphic. The p-value < 0.05 was considered to be statistically significant.

Conclusions
Neurodegenerative and orphan diseases include a wide range of pathologies, which share a very complex etiology, resulting in a lack of effective treatments to solve or at least relieve patients' symptomatology. In the last decades, accumulating evidence indicated a key role for ROS-dependent cellular damage in many cellular dysfunctions, also linked to abnormal metal accumulation. Our findings indicate that 8-aminoquinoline derivatives 4 and 5 significantly reduce oxidative stress in a photoreceptor-like cell line, supporting that the synthesis of molecules with dual antioxidant and chelating properties may be promising MTDL candidates to fight OS. This activity is particularly important in relation to neurodegenerative diseases, such as RP, where the progression of the pathology is driven by multifactorial events including the increase of OS leading to secondary degeneration and the death of cones, the photoreceptors essential for daytime vision in humans, which is a priority to protect in order to preserve a residual visual function in patients. Our work supports the rationale that opportunely designed pleiotropic agents, synthesized following the MTDL approach, could offer new opportunities for the identification of innovative and effective therapies against OS.
Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/ph15060688/s1. Figure S1. Copper chelating study; DPPH assay. Cell viability was analyzed by CellTiter 96 AQueous One Solution Reagent. Cells were pre-treated for 24 h with compounds at various concentrations (1-100 µM) and then exposed for 3 h with H 2 O 2 500 µM. The dashed line indicates the reference value of Ctrl (control group, no compounds or H 2 O 2 exposure). Values in the graph indicate % viability as the mean ± SE obtained from n = 3 of independent experiments; 1 H-NMR and 13 C-NMR spectra of final products 1-8; HPLC spectra of final compounds 1-8.