Synthesis, Crystal Structure, Cell Viability and in vitro Antiviral Activities Over Arboviruses of Novel Adamantane-Derived Schiff BasesAbstract
Arboviral diseases such as Zika, Mayaro and chikungunya fevers are neglected infections transmitted by arthropod vectors. The search for alternatives to circumvent the effects of arboviruses by modifying molecules previously known for their antiviral activities can be considered a good strategy to obtain new antiviral drugs. Here, we describe the synthesis of novel adamantane derived Schiff bases and their antiviral activities against Zika (ZIKV), Mayaro (MAYV) and chikungunya (CHIKV) viruses. Amantadine Schiff base was prepared by reaction of amantadine and 2-thiophenecarboxyaldehyde (atdTiof), while rimantadine Schiff base was prepared by reaction of rimantadine and 5-fluoro-2-hydroxybenzaldehyde (rtdFhba). The compositions of the Schiff bases were determined by elemental and mass spectrometric analyses as C15H19NS and C19H24FNO for atdTiof and rtdFhba, respectively. Spectroscopic characterizations added to single crystal X-ray diffraction permitted proposing the structures of Schiff bases. Cell viability and antiviral activities of the compounds were evaluated against ZIKV, MAYV and CHIKV viruses. The atdTiof and rtdFhba species exhibited the best inhibition rates of 58.5% over ZIKV and 69.5% over CHIKV, respectively. The results obtained in the in vitro studies open perspectives for future in vivo investigations of the synthesized Schiff bases in the search for alternatives for the treatment of arboviruses.
Keywords:
arboviruses; aminoadamantanes; Schiff bases; crystal structures; antiviral activity
Introduction
The World Health Organization (WHO) defines neglected diseases as illnesses that affect mostly populations with higher socioeconomical vulnerability, typically in the global south.1,2 Some of these illnesses are caused by arboviruses, which are transmitted by the bite of some species of mosquitoes, mainly the ones that belong to the genus Aedes. Although arboviral diseases primarily affect tropical and subtropical countries, climate change has caused global temperature to rise, thus allowing the mosquitoes to migrate and reproduce in previously unaffected colder areas.3 There are still no vaccines or medications targeted specifically towards arboviral infections, aside from the recently approved vaccine against dengue and chikungunya viruses.3-8 Zika, chikungunya and Mayaro fever are other arboviruses caused by the Zika (ZIKV), chikungunya (CHIKV) and Mayaro (MAYV) viruses, respectively, that remain without available specific treatment. The clinical symptoms of arboviruses vary from mild fevers and headaches to long-lasting joint pain and neurological impairment.7,9,10
In the search of new medications to treat arboviruses, aminoadamantanes have been studied as a potential class of antiviral drugs. Amantadine and rimantadine are examples of such class of antiviral agents that have been used in the clinics for the treatment and prevention of influenza A infections, acting as inhibitors of the uncoating process, which leads to the release of the viral genome and its subsequente replication.3,11 The rigid cage structure of the aminoadamantanes is associated with drug stability and its further distribution in blood plasma, as it protects adjacent functional groups from metabolic cleavage.12 Moreover, the rigid and three-dimensional structure of the adamantane allows the molecule to be positioned with precision, thus interacting more effectively with drug targets.13 Recent studies demonstrated the potential in vitro of aminoadamantanes as antiviral agents towards Zika,11 chikungunya14,15 and Mayaro.16 It was recently reported17,18 that amantadine and rimantadine succesfully inhibited CHIKV replication with half maximal effective concentration (EC50) of 63.37 and 71.64 µmol L-1, respectively. Both aminoadamantanes were also active against different ZIKV strains, with 50% cytotoxicity concentration (CC50) > 100 µg mL-1.11
Besides, the modification of bioactive molecules with known pharmacological properties is a useful strategy in drug development.4,19,20 In this scenario, Schiff bases derived from aminoadamantanes are considered promising candidates for testing as antiviral agents over arboviruses. Schiff bases are obtained from the condensation of an amine and a carbonyl group, typically an aldehyde, which generates the characteristic imine group.21 The biological activity observed in many Schiff bases can be associated with the proteolytic or hydrolytic cleavage of the C=N bond.22
The aldehyde moiety incorporated into Schiff bases also plays a significant role in determining their behavior in biological environments. Thiophene-based scaffolds have been explored in drug discovery in the treatment of several illnesses due to its antibacterial, anticancer, antidiabetic, antifungal and antiviral properties.23,24 They are present in compounds targeting viral capsid proteins and replication processes.25 The incorporation of sulfur into molecular frameworks significantly enhances their chemical and biological properties. Owing to its larger atomic radius, lower electronegativity and greater polarizability relative to lighter heteroatoms, sulfur can establish characteristic interactions with biomolecules that may influence cellular function.26 In combination with nitrogen donor atoms within the same molecule, sulfur also broadens the coordination behavior of these ligands by providing mixed hard-soft donor groups, thereby enabling the formation of complexes with diverse metal ions through a variety of binding modes.27
Furthermore, fluorinated hydroxybenzaldehyde derivatives have been used to synthesize biologically active compounds including metal complexes with antimicrobial, anticancer, antioxidant, and diagnostic applications, highlighting their therapeutic potential.28 Phenolic and halogenated aromatic compounds, including fluorinated derivatives, have shown antiviral effects through interference with viral entry and replication.29,30 The presence of aromatic and heteroaromatic rings enables these compounds to engage in π-π stacking interactions with proteins and nucleic acids.31,32 Such interactions may modulate the structure and function of these biomacromolecules, thereby contributing to the drug-like properties of the compounds32 and supporting their potential application in drug delivery systems.33
Schiff bases have a well-established profile of activity against cancer, bacteria, fungus, diabetes and other illnesses, and have been considered as promising candidates to antiviral drugs.21 Moreover, metal complexes with Schiff bases have also lead to promising results when evaluated for their activities over several illnesses.34,35 In a recent study,36 computational calculations pointed the drug-like properties of a CuII complex with a Schiff base ligand and its affinity towards key proteins associated with severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) and human papillomavirus (HPV). The in vitro inhibitory properties of metal complexes with Schiff bases over SARS-CoV-2, hepatitis B (HBV), herpes simplex (HSV) and human immunodeficiency (HIV) viruses were also reported,37-39 which highlights the antiviral potential of this class of compounds.
Aminoadamantane Schiff bases are particularly envisaged for drug development, constituting a total of 224 structures in the Cambridge Structural Database,12 with applications varying from the treatment of neurodegenerative diseases to viral infections.12,22 In special, amantadine and rimantadine-derived Schiff bases have been reported for their antidiabetic, antibacterial and antifungal properties,40-42 and their biological activities have been linked to their interaction with biomolecules such as deoxyribonucleic acid (DNA), peptides and enzymes.41-43 The coordination of aminoadamantane Schiff bases to several transition metals has also been explored as a drug development strategy, leading to complexes with biological activities against diabetes, Alzheimer’s disease, bacterial infections, Parkinson’s disease and some types of tumor.12,22,44,45 The use of adamantane-derived Schiff bases as antivirals is a promising but little explored strategy.
Within the present context, this work presents two novel Schiff bases obtained by the reaction of amantadine with 2-thiophenecarboxaldehyde (atdTiof) and rimantadine with 5-fluoro-2-hydroxybenzaldeyde (rtdFhba) and the study of antiviral activities of the Schiff bases against ZIKV, CHIKV and MAYV.
Experimental
Material and methods
Reagents and equipment
Amantadine (atdH) and rimantadine (rtdH) hydro-chlorides were purchased from Thermo Scientific and Sigma-Aldrich laboratories, respectively. Potassium hydroxide, 2-thiophenecarboxaldehyde and 5-fluoro-2 hydroxybenzaldeyde were purchased from Sigma-Aldrich laboratories and used as received.
Elemental analyses were performed using a Perkin Elmer 2400 Series II CHNS/O elemental analyzer. Infrared absorption spectra (IR) were obtained using an Agilent Cary 630 spectrophotometer in attenuated total reflectance (ATR) mode in the range 4000-400 cm-1 and with a resolution of 4 cm-1. The spectra were plotted using OriginLab software, version 8.1. Nuclear magnetic resonance (NMR) analyses were performed using Bruker Avance 600 MHz and 500 MHz spectrometers. Samples were evaluated in deuterated dimethylsulfoxide (DMSO-d6) solutions at 298 K and chemical shifts were reference to tetramethylsilane (TMS). The data were processed in the program TopSpin, version 4.5.0. High-resolution electrospray ionization mass spectra in positive-ion mode (ESI(+)-HRMS) of the complexes were obtained using an Orbitrap Thermo Q-Exactive mass spectrometer. A sample of atdTiof was prepared by dissolving 1.0 mg of the compound in 1.0 mL of DMSO, followed by filtration through a 0.22 μm filter. Subsequently, 50 µL of the resulting solution was diluted to a final volume 1.0 mL with a mixture of acetonitrile:water (1:1). The solvent used in the system was acetonitrile:water (1:1) with 0.1% formic acid. For rtdFhba, a sample was prepared by dissolving 1.0 mg of the compound in 1.0 mL of methanol, followed by filtration through a 0.22 μm filter. Subsequently, 50 µL of the resulting solution was diluted to a final volume of 1.0 mL with methanol:water (1:1) mixture. A solution of methanol:water (1:1) with 0.1% formic acid was used as the solvent system. Samples were infused into the ESI source at a flow rate of 200 μL min-1. The acquired data were processed using Thermo Scientific FreeStyle software, version 1.8.
Synthesis of the Schiff bases
The Schiff bases were obtained from a two-step process, starting with the neutralization of the amantadine and rimantadine hydrochlorides followed by the reaction with the 2-thiophenecarboxldeyde and 5-fluoro-2-hydroxybenzaldehyde, respectively.
Amantadine-Schiff base (atdTiof)
Initially, 5.0 mmol of amantadine hydrochloride and 5.5 mmol of KOH were dissolved in 50 mL of water and maintained under constant stirring. After 1 h, the white precipitate obtained was collected by filtration, rinsed with distilled water and dried in a desiccator over P2O5. The solid obtained corresponds to free amantadine (atd). Then, 2.5 mmol of atd and 2.75 mmol of 2-thiophenecarboxaldehyde were dissolved in 40 mL of methanol and refluxed in a round-bottom flask under constant stirring for 4 h (Scheme 1). The solvent was removed using a rotary evaporator, leading to the formation of a white precipitate. The solid was washed with distilled water and dried in a desiccator over P2O5. The yield of the synthesis was 59%. Elemental analysis led to the molecular formula C15H19NS. Anal. calcd. for C15H19NS (%): C 73.42, H 7.80, N, 5.71, found: C 73.26, H 7.46, N 5.53. The solid was redissolved in methanol and left to slow evaporation of the solvent, leading to the formation of crystals suitable for structural determination by single crystal X-ray diffraction technique.
Rimantadine Schiff base (rtdFhba)
The synthesis of rimantadine Schiff base followed a similar procedure used to synthesize the amantadine Schiff base. Free rimantadine was firstly isolated following procedure described in the literature.46 Briefly, 1.0 mmol of rimantadine hydrochloride was dissolved in 12 mL of deionized water and then 8.0 mL of an aqueous solution containing 5% KOH and sodium chloride was added. A liquid-liquid extraction was conducted using dichloromethane as organic solvent. A small amount of anhydrous sodium carbonate was added to the organic phase, followed by filtration through filter paper into a round-bottom flask and removal of the solvent using a rotary evaporator. A white powder was formed, corresponding to the neutral species rtd.
The rtdFhba Schiff base was synthesized by the reaction of 0.82 mmol of rtd and 0.90 mmol of 5-fluoro-2-hydroxybenzaldehyde in a round-bottom flask with 20 mL of methanol under reflux and constant stirring for 4 h (Scheme 2). The solvent was removed using a rotary evaporator, forming a yellow oil. After standing in a desiccator over P2O5, a bright-yellow powder was formed. The solid was washed with cold water, collected by filtration and left to dry in a desiccator. The yield of the synthesis was 62%. Elemental analysis led to the molecular formula C19H24FNO. Anal. calcd. for C19H24FNO (%): C 75.70, H 8.04, N 4.65, found: C 75.67, H 7.59, N 4.40. The compound was redissolved in DMSO and left to slow evaporation of the solvent, leading to the formation of crystals suitable for structural determination by single crystal X-ray diffraction technique.
Crystallographic characterization
The atdTiof and rtdFhba crystal structures were determined by single crystal X-ray diffraction technique. Data collection was carried out at 120 K on a Bruker Apex II Duo CCD diffractometer, using a fine focus sealed tube of Mo Kα (λ = 0.71073 Å) as the radiation source. The data collection strategy consisted of sets of ϕ and ω scans.47 Data reduction and cell refinement were performed using SAINT,47 while SADABS47 was used for absorption correction by the multi-scan method for atdTiof and by numerical method for rtdFhba. The crystal structures were solved by direct methods using SHELXS9748 and refined by full-matrix least square on F2 using SHELXL2014/749 and SHELXL tool (Qt version 6.4.0).50
For both structures, the CH and CH2 hydrogen atoms of the adamantane cage were assigned with C-H distances of 1.0000 and 0.9900 Å, respectively, and with Uiso(H) = 1.2Ueq(C) The N-C-H and aromatic hydrogen atoms were assigned with C-H distance of 0.9500 Å and with Uiso(H) = 1.2Ueq(C). In rtdFhba, the methylene hydrogen atoms were assigned with C-H distance of 0.9800 Å and Uiso(H) = 1.5Ueq(C), while the hydroxyl hydrogen atom was freely refined with an O-H distance of 0.90(2) and Uiso(H) = 1.5Ueq (O).
Additional information about the crystals, data collection and structure refinement is summarized in Table 1. The tables and figures were generated using the programs publCIF51 and Mercury.52
Biological studies
Cell viability
To determine the cytotoxicity of each molecule at different concentrations, Vero E6 cells (kidney tissue-derived cells from an African green monkey, ATCC) were seeded at 5 × 103 cells per well in 96-well microplates and incubated at 37 °C for 24 h under 5% CO2. Subsequently, the culture medium was replaced with Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1% W/S (penicillin-streptomycin), 1% NEAA (non-essential amino acids), 5% FBS (fetal bovine serum) containing the compounds at different concentrations (50, 10 and 2.0 μM). The cells were incubated for 24 or 72 h, to evaluate MAYV and CHIKV or ZIKV assays, respectively. The supernatant was removed, and 100 μL per well of a solution containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich) at 1.0 mg mL-1, diluted in non-supplemented culture medium was added. The plate was incubated at 37 °C for 30 min. Subsequently, the solution was removed and replaced with 100 μL per well of DMSO, and the absorbance was measured at 560 nm. Cell viability was determined as the ratio between the mean absorbance of treated wells multiplied by 100 and the mean absorbance of untreated control wells.39
Antiviral activity
The viruses used for antiviral assays were: (i) CHIKV expressing a nanoluciferase reporter (CHIKV-nanoluc), based on the CHIKV LR2006OPY1 strain (East/Central/South African genotype);53 (ii) MAYV expressing a nanoluciferase reporter (MAYV-nanoluc), based on the MAYV BeAr20290 strain (L genotype), isolated from Haemagogus mosquitoes in Brazil in 1960;16 (iii) a wild type ZIKV isolate obtained from a clinical sample of a patient in Brazil (ZIKVPE243).53
Antiviral assays were performed as previously described.16,54,55 Briefly, Vero E6 cells were seeded in microplates 48-well microplates at a concentration of 5 × 104 cells per well or in 96-well microplates at a concentration of 5 × 103 cells per well and incubated at 37 °C and 5% CO2 for 24 h. Each compound was added at the maximum non-cytotoxic concentration in the presence of the virus in a multiplicity of infection (MOI) of 0.1 for MAYV-nanoluc and CHIKV-nanoluc, and 0.005 for ZIKVPE243, diluted in DMEM 1% W/S, 1% NEAA, 2% FBS. DMSO was used as untreated control. For MAYV and CHIKV assays, the supernatant was collected after 24 h of incubation and processed according to the Renilla-luciferase Assay System (Promega) kit protocol, and subsequently submitted to a luminescence analysis on the GloMax (Promega) plate reader. For ZIKV assays, cells were fixed with 4% (v/v) formaldehyde after 72 h of incubation, washed with PBS, and treated with blocking buffer (BB) for the immunofluorescence assay. Focus-forming units were measured using a EVOS cell fluorescence microscopy (Thermo Fisher Scientific). The results obtained were normalized according to the mean luminescence or FFU (focus of infection) obtained from the DMSO control and multiplied by 100 to calculate the percentages.
Statistical analysis
Data were analyzed for normal distribution to demonstrate the applicability of parametric or nonparametric test. Then, two-way analysis of variance (ANOVA) test was employed to compare the treatment of each compound with the DMSO control, considering p < 0.05 as statistically significant.
SDS-PAGE gel electrophoresis
Solutions of the Schiff bases atdTiof and rtdFhba (200 and 100 µmol L-1) were prepared in phosphate buffer (HPO42-/H2PO4-, pH = 7.4) with 1% v/v DMSO. An aliquot of 5 or 2.5 µL was added to a microtube containing 10 µL of PBS and 5 µL of bovine serum albumin solution (100 µmol L-1 in PBS) resulting in final concentrations of 25.0 µmol L-1 of the investigated protein and 12.5, 25.0, and 50.0 µmol L-1 of each studied compound. The solutions were incubated at 37 °C for 18 h, centrifuged and 50 µL of loading buffer (62.5 mM Tris/HCl pH 6.8, 2.0% SDS, 0.01% bromophenol blue and 25.0% glycerol) were added to each solution. Samples were heated at 96 °C for 5 min, centrifuged, and 10 µL of each sample was loaded onto the SDS-polyacrylamide gels (precast 8-16% gradient gels). The electrophoretic run was performed at 100 V for 90 min. After electrophoresis, the gels were fixed (30% methanol, 10% acetic acid) to improve dye binding, washed with distilled water, dyed with a Coomassie blue solution (0.1% Coomassie blue, 18% methanol 2% and acetic acid), destained (45% methanol, 10% acetic acid) and photodocumented. In addition, Precision Plus Protein™ Dual Color standards (Bio-Rad) was employed to monitor protein separation and to estimate molecular weight. Samples containing only the protein in phosphate buffer were used as controls.
DNA gel electrophoresis assay
The influence of atdTiof and rtdFhba on plasmid DNA structure was investigated by agarose gel electrophoresis. Stock solutions of the compounds were prepared using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (80 mmol L-1 HEPES, 25 mmol L-1 NaCl, pH 7.4) with 1% DMSO and diluted to result in concentrations values varying from 1.0 to 100 µmol L-1. These samples were incubated with 200 ng of plasmid DNA pUC19 (Thermo Fischer Scientific) at 37 °C for 24 h. Subsequently, 6 × loading dye sample buffer was added to each sample, which was then loaded onto a 1.2% agarose gel. The agarose gel was prepared using 1 × Tris acetate-ethylenediaminetetraacetic acid (EDTA) buffer and stained with SYBR Green. The electrophoretic run was conducted at 70 V for 90 min using a Bio-Rad horizontal tank connected to a PowerPac Basic 300 V Power Supply Flier. The gel was visualized using a transilluminator coupled to a photodocumentation system (UVDoc, Delpho Instruments) and photographed with a digital camera.
Results and Discussion
Structural determinations of atdTiof and rtdFhba
Single crystal X-ray diffraction results showed that atdTiof, or (adamantan-1-yl)-1-(thiophen-2-yl)methanimine, and rtdFhba, or 2-{[1-(adamantan-l-yl)ethyl]iminomethyl}-4-fluorophenol Schiff bases crystallized in monoclinic P21 and triclinic P-1 space groups, respectively. In both cases, the asymmetric units of the crystals present only one molecule, as can be seen in Figure 1a (atdTiof) and Figure 1b (rtdFhba).
The asymmetric units of (a) atdTiof and (b) rtdFhba at 120 K, with displacement ellipsoids at 50% probability level.
Despite the structural and compositional differences between the Tiof and Fhba fragments bound to the adamantane cage portions, both fragments possess an aromaticity that imposes a planarity on a part of the structures. For atdTiof, the mean plane deviation of N1-C5-C4-C3-C2-C1-S shows a root mean square deviation (RMSD) of 0.016 Å, whereas the mean plane defined by N1-C7-C1-C2-C3-C4-C5-C6 in rtdFhba shows an RMSD of 0.011 Å. As expected, the bond lengths and dihedral angles between the atoms belonging to the atd and rtd cages, considering the uncertainties, are equal. These data, along with all the geometric parameters of atdTiof and rtdFhba, are provided in Table S1 (Supplementary Information (SI) section).
In the crystal of atdTiof, the molecules are connected in a head-to-head fashion, via non-classical, long intermolecular hydrogen bonds (C2-H2···S1i, see Table S2, SI section), forming chains that extend along the b-axis direction (Figure 2a). In rtdFhba, the molecules are stabilized by intramolecular hydrogen bond (O1-H1···N1, see Table S2, SI section) and interact with each other in head-to-tail manner, forming chains along the a-axis (Figure 2b), via non-classical hydrogen bond (C2-H2···O1i). Simplified molecular input line entry specification (SMILES) strings for the new molecular structures presented in this manuscript are also provided in the SI section.
Interactions of (a) atdTiof molecules, viewed along the a-axis and (b) rtdFhba molecules, viewed along the c-axis. In both Schiff bases, molecules are connected via non-classical intermolecular H-bond (green dashed lines).
Spectroscopic analysis
Both Schiff bases (atdTiof and rtdFhba) were further characterized by infrared and nuclear magnetic resonance spectroscopies. In the infrared spectra (Figure S1, SI section), it is possible to observe two sharp bands with the maxima at 2890 and 2846 cm-1 for atdTiof and at 2895 and 2844 cm-1 for rtdFhba. Both bands are attributed to the stretching modes ν(CH) and ν(CH2) of the aminoadamantane cage.22,43 The bands with maxima at 1625 cm-1 for atdTiof and 1631 cm-1 for rtdFhba, on the other hand, are associated with the stretching of the C=N bond, which indicates the formation of the imine group, characteristic of Schiff bases.22,43 Besides, the infrared spectra of both Schiff bases synthesized present C=C stretching absorption bands in the region of 1493 1428 cm 1,56,57 in addition to characteristic absorption bands, specifically a strong C-S stretching band at 725 cm-1 for atdTiof56 and C-O and C-F absorption bands at 1273 and 1251 cm-1, respectively, for rtdFhba.58,59
The 1H NMR (Figure 3) and 13C NMR spectra (Figure S2 and Table S3, SI section) further confirmed the proposed structures of the Schiff bases. In the 1H NMR spectra, the signals with chemical shifts up to 3 ppm relative to TMS are assigned to the aminoadamantane moiety, while those ones above 6 ppm correspond to the aldehyde unit. In the 13C NMR spectra, the chemical shifts associated with C11 in atdTiof and with C12 in rtdFhba (both over 150 ppm) reinforce the formation of the imine (C=N). The hydrogen atom numbering and corresponding 1H NMR assignments are also presented in Table 2.
1H NMR spectra of the Schiff bases atdTiof (500 MHz, DMSO-d6) and rtdFhba (600 MHz, DMSO-d6).
Mass spectrometric measurements
The composition of the Schiff bases was also confirmed by ESI(+)-HRMS analyses. The full rtdFhba and atdTiof spectra, presented in Figures 4a and 5a, respectively, show the presence of the monoprotonated ions [C19H25FNO]+ and [C15H20NS]+, respectively. The experimental isotopic patterns for [C19H25FNO]+ and [C15H20NS]+ (Figures 4b and 5b, respectively) were compared to the expected isotopic patterns based on the proposed compositions (Figures 4c and 5c). The agreement between calculated and experimental values was confirmed, with an error of -1.6 ppm (calcd. m/z 302.19147, exp. m/z 302.1910) for rtdFhba and an error of +1.2 ppm (calcd. m/z 246.13110, exp. m/z 246.1314) for atdTiof.
Mass spectra of the rtdFhba Schiff base. (a) Full experimental spectrum in the m/z range of 50-750; (b) zoom of the signal corresponding to the monoprotonated ion [C19H25FNO]+, and (c) simulated isotopic pattern.
Mass spectra of the atdTiof Schiff base. (a) Full experimental spectrum in the m/z range of 200-1000; (b) zoom of the signal corresponding to the monoprotonated ion [C15H20NS]+, and (c) simulated isotopic pattern.
Biological studies
Aiming to assess the cytotoxicity of the compounds, Vero E6 cells were treated with atdTiof and rtdFhba at concentrations of 50, 10 and 2.0 µM. Cell viability was subsequently evaluated by the MTT assay after 24 or 72 h of incubation. DMSO was used as untreated control. Analyzing the effects on viability, we found that cell viability > 90% was observed for the treatment with both compounds at 50 µM, with exception of the treatment with rtdFhba for 72 h, which maintained cell viability above 90% at 10 µM (Table 3).
In the sequence, we investigated the antiviral activity of atdTiof and rtdFhba employing Vero E6 cells infected with ZIKVPE243 at a MOI of 0.005 for 72 h, and CHIKV nanoluc or MAYV-nanoluc at a MOI of 0.1 for 24 h. The results demonstrated that atdTiof at 50 µM inhibited 58.5% of ZIKV infection, 23% of CHIKV infection and 35% of MAYV infection. Additionally, rtdFhba at 50 µM reduced CHIKV and MAYV replication in 69.5 and 34%, respectively. Alternatively, rtdFhba at 10 µM did not affect ZIKV infection (Table 3 and Figure 6).
Effects of atdTiof and rtdFhba on cell viability and replication rates of ZIKV (a), MAYV (b), and CHIKV (c).
The antiviral activity of aminoadamantanes, such as amantadine and rimantadine, has been investigated recently and reported in literature.60-63 Their mechanisms of action on flaviand alphaviruses are not fully elucidated, however, it is hypothesized that these compounds are able to target viroporin ion channels,9,64 similarly to how amantadine interacts with the M2 protein of influenza A virus.65 Notably, atdTiof showed its strongest antiviral effect against ZIKV, which is consistent with a previous study9 that demonstrated the suppression of this virus replication following the treatment with atdH. In contrast, rtdFhba exhibited a higher inhibition of CHIKV replication, in line with previous18 results from our group demonstrating the antiviral activity of rtdH against this virus.
It was possible to observe that the synthesis of adamantane-derived Schiff bases was a successful strategy to obtain compounds with improved antiviral activities in vitro than their aminoadamantane precursors. In a previous work, the antiviral activity of free atdH over ZIKV and CHIKV was determined as low (22% inhibition rate) or negligible, respectively, which contrasts to the 58.5 and 23% inhibition rates determined for atdTiof over the same viruses. The obtained results are in accordance with the previously discussed properties of several Schiff bases, which includes anticancer, antiviral, antifungal, and antibacterial activities.66,67 The incorporation of the imine group may contribute to antiviral activity by increasing molecular flexibility and potential interactions with viral or host targets.19,68 Furthermore, the aldehyde moieties incorporated into the Schiff bases atdTiof and rtdFHBA significantly increases their biochemical versatility. The presence of the sulfur-containing tiophene group in addition to the nitrogen-containing imine allows interaction with both hard and soft compounds,26 while halogenated molecules and aromatic compounds are reported to interfere in viral entry and replication processes.29-32
These findings highlight aminoadamantane-Schiff base derivatives as promising scaffolds for the design of novel antiviral agents. Nevertheless, additional studies need to be conducted to elucidate the mechanism of action of both compounds.
SDS-PAGE gel electrophoresis
Since proteins are potential targets for drugs and may be associated with their efficacy, it is essential to study drug-protein binding.69 For this purpose, serum albumin, abundant protein in plasma, was chosen. Serum albumins bind strongly to small molecules, acting as delivery systems for many drugs, including some antiviral ones. Bovine serum albumin (BSA) was used due to its structural similarity to human serum albumin (HSA), besides its greater availability and lower cost compared to HSA.69-71 Polyacrylamide gel electrophoresis was employed to assess compound-protein interaction. The compounds (atdTiof and rtdFhba) were evaluated at three concentrations, but no interaction was identified, since the same pattern of electrophoretic mobility was observed for the free protein and for the protein treated with the compounds (Figure S3, SI section). These findings suggest that proteins are not the primary targets of these compounds, at least initially.
DNA binding by gel electrophoresis
The investigation about interaction of drug candidates with nucleic acids (ribonucleic acid (RNA) or DNA) seeking for potential biomolecular targets is an important step in drug development, which includes antiviral agents. This perspective has also been applied even to drugs already used in clinical practice.72,73 Although Zika, chikungunya and Mayaro are RNA viruses, studies about nucleic acid interaction with the Schiff bases rtdTiof and rtdFhba were performed considering plasmid DNA as nucleic acid model. Since no changes were observed in the electrophoretic mobility of DNA in the presence or absence of the compounds (Figure S4, SI section), it can be concluded that the compounds were unable to induce significant structural alterations in DNA. Therefore, nucleic acids may not represent the primary target of the Schiff bases. Additional studies may be carried out in the future to confirm this hypothesis.
Conclusions
The Schiff bases atdTiof and rtdFhba were synthesized and characterized. Elemental analysis confirmed the composition C15H19NS for atdTiof and C19H24FNO for rtdFhba, while mass spectrometric data permitted identifying the monoprotonated species [C15H20NS]+ and [C19H25FNO]+, respectively. Mass errors were lower than 2 ppm, thereby confirming unequivocal elementary formulas. Infrared spectroscopic analyses confirmed the formation of the imine group from the reaction between the aminoadamantane and the respective aldehyde, with C=N stretching bands observed at 1625 cm-1 for atdTiof and 1631 cm-1 for rtdFhba. The structures of the Schiff bases were confirmed by single crystal X-ray diffraction analysis. The atdTiof crystallized in monoclinic P21 space group, while rtdFhba crystallized in triclinic P-1 space group. In the crystal of atdTiof, the molecules are connected in a head-to-head fashion, via non-classical, long intermolecular hydrogen bonds, while for rtdFhba, the molecules are stabilized by intramolecular hydrogen bonds.
Both componds were evaluated for their antiviral activity over Zika, Mayaro and chikungunya viruses, in their highest non-cytotoxic concentration. Results showed that atdTiof significantly inhibited ZIKV, MAYV and CHIKV replications with best inhibition rate of 58.5% over ZIKV. On the other hand, rtdFhba was able to reduce MAYV and CHIKV replication with the best inhibition rate of 69.5% over CHIKV, while no significant antiviral activity against ZIKV infection was detected. In addition, the compounds did not show interaction with BSA, ruling out this protein as a target. DNA binding studies by gel electrophoresis indicated that the compounds do not interfere with the conformational structures of DNA, which was used as a nucleic acid model. Such data suggest that atdTiof and rtdFhba may not target the genetic material of the viruses. Nevertheless, RNA may be specifically evaluated in future works to confirm the absence of interaction. Collectively, the results obtained open up prospects for future in vivo applications of Schiff bases in the search for new therapies for the treatment of arboviruses.
Supplementary Information
Supplementary data is available free of charge at http://jbcs.sbq.org.br as PDF file.
Supplementary PDF
Crystallographic data for the structures in this work were deposited in the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 2453708 (atdTiof) and 2453699 (rtdFhba). Copies of the data can be obtained, free of charge, via https://www.ccdc.cam.ac.uk/structures/.
Acknowledgments
This work was supported by grants from FAPESP (2025/07048 6, 2024/16650-9 and 2021/10265-8 Cancer Theranostics Innovation Center CancerThera-CEPID) and CNPq (304661/2024-4 and 150579/2024-1). The authors thank LIRMN (RRID:SCR_027247), LIEM (RRID:SCR_027240), LIRX (RRID:SCR_027392) and LISpec (RRID:SCR_027391) from CEMUIQ-UNICAMP for technical support. A. C. G. J. is grateful to FAPEMIG (APQ-01487-22 and APQ-04686-22), CNPq (409187/2023-2 and 310736/2022-6), and to CAPES (Prevention and Combat of Outbreaks, Endemics, Epidemics and Pandemics - Finance Codes No. 88881.506794/2020-01 and 001).
Data Availability Statement
The data presented in this study are available upon request from the corresponding author.
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Edited by
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Editor handled this article:
Izaura Cirino Nogueira Diógenes (Executive)
