Synthesis and Discovery of Schiff Base Bearing Furopyrimidinone for Selective Recognition of Zn²⁺ and its Applications in Cell Imaging and Detection of Cu²⁺

Introduction

Metal ions have pivotal functions for the growth and development process of organisms, and it is of great significance to identify and monitor metal ions in the environment and in organisms (Domingo 1994; Schmidt et al., 2009; Garza-Lombo et al., 2018). Fluorescence analysis technology has received increased attention in view of its utility for selective recognition of metal ion owing to its high selectivity, low toxicity, real-time detection, convenient and simple operation, and relatively friendly environment (Ma et al., 2015; Chae et al., 2020; Nsanzamahoro et al., 2020). In the past few years, many researchers have been devoted to finding and developing of some site-specific small-molecule fluorescence probes for highly selective recognition of metal ions (Liang et al., 2017; Bae et al., 2018; Yang et al., 2019) and for analyzing different metal ions, which are widely distributed in organisms and environments worldwide (Wang et al., 2019; Kim et al., 2021; Klenner et al., 2021; Sannigrahi et al., 2021). Many diseases, such as Alzheimer’s disease (Isaev et al., 2020), neuron disease (Marchetti 2014), Parkinson’s disease (Park et al., 2015), ischemia (Yin et al., 2019), epilepsy, and certain types of cancer and so on (Hildebrand et al., 2015; Hershfinkel 2018; Wang et al., 2020), are caused by the excessive or insufficient intake of Zn²⁺ and Cu²⁺ ions.

Recently, we have focused on the synthesis of nitrogenous heterocyclic compounds via aza-Wittig reaction, attempting to apply and evaluate their biological activities (Hu et al., 2010; Hu et al., 2014; Wang et al., 2016a; Wang et al., 2016b; Li et al., 2016; Liu M.-G. et al., 2019; Liu N. et al., 2019; Gao et al., 2019; Liu et al., 2021). Herein, we designed a Schiff base bearing furopyrimidinone scaffold (FPS) synthesized from 2-hydroxy-benzaldehyde with furo [2,3-d]-pyrimidine-5-carbohydrazide (Scheme 1), and fluorescence analysis showed that FPS displayed highly selective recognition Zn²⁺ with no apparent interference from other metal ions in MeOH solution and its applications in cell imaging and detection Cu²⁺ ions.

SCHEME 1. (A) The route of synthesis for FPS, (B) Dual-Response of FPS to Zn²⁺ and Cu²⁺, schematic of FPS applicated in cell imaging and detection Cu²⁺ ions.

Experimental Sections

Materials and General Methods

Unless otherwise stated, starting materials were commercially available and analytically pure, and the solvent was dried before use. The water used was redistilled water. The UV absorption and fluorescence emission spectra were recorded on a U-2550 Double-beam UV-Vis spectrophotometer (Japan) and a F-7000 fluorescence spectrometer (Japan), respectively. Melting points were recorded using an uncorrected X-4digital melting point apparatus. NMR were recorded on a Bruker Avance 400 MHz spectrometer (CDCl₃ and DMSO-d₆) with resonances relative to tetramethyl-silane (TMS) as an internal standard. Mass spectra (ESI) were recorded on a Waters XEVO G2-XSmass spectrometer. Fluorescence images of cells were analyzed by Dual photon confocal microscope (Olympus FV1000 MPE).

Synthesis

Synthesis of 2-Ethyl-3,4-dihydro-6-methyl-4-oxo-2-(propylamino)-3-p-tolyl-furo[2,3-d]pyrimidine-5-carboxylate 3

Preparation of ethyl-3,4-dihydro-6-methyl-4-oxo-2-(propylamino)-3-p-tolyl-furo-[2,3-d]pyrimidine-5-carboxylate 2. As described in methods previously (Hu et al., 2012). A mixture of 1 (2.5 g, 5 mmol) and p-tolyl isocyanate (5 mmol) in anhydrous methylene dichloride 24 h at 0–5°C under N₂, and then n-propylamine (5.2 mmol) was added, after the mixture was stirred for 1 h at room temperature. The solution was removed under reduced pressure and anhydrous EtOH (10 ml) with five drops of EtONa (10%) in EtOH was added. The mixture was stirred for 4 h at room temperature. The precipitated solid was collected and washed with ethanol to give ethyl 3,4-dihydro-6-methyl-4-oxo-2-(propyl-amino)-3-p-tolyl-furo [2,3-d]pyrimidine-5-carboxylate 2, which was used directly without further purification. A solution of 2 (5 mmol) and hydrazine hydrate (1 ml, 80%) in EtOH were stirred at 60–65°C for 15 h, after the solution was concentrated under reduced pressure and the residue recrystallized from CH₂Cl₂/EtOH (v:v = 4:1, 20 ml) to give 3,4-dihydro-6-methyl-4-oxo-2-(propylamino)-3-p-tolyl-furo [2,3-d]pyrimidine-5-carbohydrazide 3, white solid, m. p.: 260–262°C; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.8 (t, J = 8.0 Hz, 3H, CH₃), 1.46–1.51 (m, 2H, CH₂), 2.43 (s, 3H, CH₃), 2.65 (s, 3H, CH₃), 3.16–3.2 (m, 2H, NCH₂), 4.44 (s, 2H, NH₂), 6.45 (s, 1H, NH), 7.23–7.42 (m, 4H, ArH), 10.88 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 165.45, 161.63, 160.70, 153.22, 153.00, 139.52, 132.11, 131.25, 129.24, 110.52, 93.38, 43.52, 22.11, 21.36, 13.48, 11.58; MS (70 eV) m/z (100%): Anal. calcd for C₁₈H₂₁N₅O₃ (M, 355.16), found [M + H⁺, 356.17].

Synthesis of N'-(2-hydroxybenzylidene)-3,4-dihydro-6-methyl-4-Oxo-2-(propylamino)-3-P-tolylfuro[2,3-d]pyrimidine-5-carbohydrazide FPS

A mixture of 3 (1.1 g, 3 mmol) and salicylaldehyde (3 mmol) in 25 ml ethanol was stirred for 10 h at 70–75°C, after the solution was concentrated under reduced pressure and the solid was collected and recrystallized from CH₂Cl₂/EtOH to give FPS (1.1 g, 84%). m. p.: 216–218°C. ¹H NMR (400 MHz, DMSO-d₆): δ = 13.55 (bs, 1H, ArOH), 8.35 (s, 1H, N = CH), 7.58–6.89 (m, 8H, Ar-H), 6.51 (bs, 1H, NH), 3.23 (q, J = 8.0, 2H, CH₂), 2.72 (s, 3H, CH₃), 2.45 (s, 3H, CH₃), 0.82 (t, J = 8.0, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ = 11.1, 13.3, 20.9, 21.6, 43.1, 92.6, 109.8, 116.3, 118.6, 119.3, 128.8, 129.7, 131.0, 131.4, 131.5, 139.3, 147.4, 152.9, 155.2, 157.4, 158.0, 160.7, 165.1. HRESI-MS m/z anal. calcd for C₂₅H₂₅N₅O₄ (M, 459.1985), found [M + H⁺, 460.1982].

Spectroscopic Study

FPS was formulated into 1.0 mmol/L solution in DMSO and then diluted to definite concentration with methanol before the spectral experiment. The salts used in standard stock solutions of metal ions were MnSO₄, Ca(NO₃)₂, CuSO₄, Al(NO₃)₃, Ba(NO₃)₂, AgNO₃, CoCl₂, ZnSO₄, FeSO₄, NaCl, KCl, Pb(NO₃)₂, MgSO₄, Fe(NH₄) (SO₄)₂ in distilled water to prepare 0.050 mol/L. The spectral changes of the mixed solutions of FPS with various metal ions were studied by UV-Vis absorption and fluorescence spectroscopy at room temperature. The fluorescence emission of FPS were recorded with excitation at 480 nm.

CCK8 Assay

The cytotoxicity of FPS was researched by CCK8 assay according to reported methods (Hou et al., 2019). Hela cells were cultured in DMEM medium containing 10% fetal bovine serum cell culture medium with 5% CO₂ atmosphere at 37°C. The cells were transferred into 24-well plates and incubated for 24 h at 37°C. FPS, diluted to the desired concentrations (50–1,000 μmol/L) in culture medium, was added to the well. Then the original medium was removed after 24 h, and 10 μl of CCK-8 solution (5 mg/ml stock) was added to the Well and incubated for 2 h at 37°C. Absorbance at 450 nm was recorded with an enzyme-linked immunosorbent assay (ELISA) reader (Bio-Tek). The results showed that FPS exhibited low cytotoxicity against Hela cell lines with IC₅₀ more than 500 μmol/L.

Result and Discussion

Fluorescence and UV-Vis Properties of FPS

FPS showed low fluorescence emission could be because of the photoinduced electron transfer (PET) process between the imine group and the benzene ring. To research the diverse solvent effect on FPS and FPS-Zn²⁺, the emission and excitation spectra of FPS and FPS-Zn²⁺ were recorded in different solvents [MeOH, EtOH, DMSO, and DMF (Figure 1)], respectively. These results showed that FPS and FPS-Zn²⁺have higher fluorescence enhancement in MeOH than the other solvents.

FIGURE 1. Fluorescence spectra of FPS (A) and FPS-Zn²⁺ (B) in different solvents.

Selectivity of FPS

The fluorescence properties of FPS with various metal ions (10.0 equiv. of Na⁺, K⁺, Ba²⁺, Zn²⁺, Mn²⁺, Mg²⁺, Ca²⁺, Ag⁺, Co²⁺, Ni⁺, Fe³⁺, Cd²⁺, Pb²⁺, Cu²⁺, Al³⁺) were investigated, respectively. Results showed that Zn²⁺ caused significant fluorescent enhancement at 480 nm with the color change from colorless to the light blue. These phenomena indicated that FPS could be used as fluorescent sensors for Zn²⁺ recognition (Figure 2A). The ability was also explored of FPS to detect Zn²⁺ in the presence of other metal ions. A competitive test was carried out, in which 10 equivalent other metal ions were added to the solution of FPS and Zn²⁺ ion (Figure 2B), respectively. Figure 2B shows that the fluorescence quenching of FPS-Zn²⁺ undergoes a significant change with the addition of Cu²⁺ and Fe²⁺, and there is a little interference with the addition of Al³⁺, Fe³⁺, Co²⁺ and Ni²⁺ ions.

FIGURE 2. (A) Left: Fluorescent spectra of FPS (5 μmol/L) with various metal ions (10.0 equiv.) in methanol; Right: Dense bars indicate the fluorescence intensity (λ_ex = 300nm, λ_em = 480 nm) (B) Left: Fluorescent spectra of FPS (5 μmol/L) + 10.0 equiv. Zn²⁺ with various metal ions (10.0 equiv.) in methanol; Right: Dense bar portion indicates the fluorescence intensity (λ_ex = 300 nm, λ_em = 480 nm) in methanol solution, respectively.

Job’s Plot Measurements

Figure 3A is the Job’s plot of the fluorescence signal for FPS and Zn²⁺ solutions. The binding stoichiometry can be obtained from the plot. It revealed that a 1:1 binding was obtained between FPS and Zn²⁺ in methanol solution. Then the fluorescence characteristics of FPS to Zn²⁺ were further studied by fluorescence titration experiments (Figure 3B). The stability constant of FPS and Zn²⁺ was calculated to be 4.45×10⁴ (r² = 0.9626) from the nonlinear least squares fitting of the data, according to the Benesi-Hildebrand equation (Scheme 2). As shown in Figure 3B, with gradual addition of 0–20 μmol/L Zn²⁺ into the methanol solution of FPS (20.0 μmol/L), the fluorescence emission at 480 nm was increased gradually. Moreover, the detection limit of FPS to Zn²⁺ was calculated (LOD = 3σ/slope) to be 1.19 × 10^–8 mol/L (r² = 0.9959).

FIGURE 3. (A) Job’s plot for the stoichiometry determination of FPS and Zn²⁺ in the complexation and the fluorescence was performed as a function of the molar ratio [Zn²⁺]/([Zn²⁺] + [FPS]) (B) The fluorescence characteristics of FPS (20.0 μmol/L) with gradual addition of 0–20 μmol/L Zn²⁺ (λ_ex = 300 nm, λ_em = 480 nm).

Concentration Effect of Cu²⁺and Fe²⁺ on Complex FPS-Zn²⁺

Based on the results in Figure 2B, to evaluate further the effect of Cu²⁺ and Fe²⁺ concentration on the probe FPS for recognition Zn²⁺, respectively, the fluorescence properties of complex FPS-Zn²⁺ were studied in methanol solution (Figure 4). For Cu²⁺ ion, at low concentrations (c_Cu2+ ≤ 5 μmol/L), the fluorescence emission was continuously quenched with the increase of Cu²⁺ and there was a good linear relationship. However, for Fe²⁺, there were no similar phenomena, the fluorescence emission at 480 nm was quenched completely, at that moment, with the addition of Fe²⁺ even in small amounts. Then to investigate the time-dependent of fluorescence quenching for Cu²⁺, as shown in Figure 4, the florescence intensity tended to be stable after adding the Cu²⁺ ion 15 seconds. In addition, the detection limit of complex FPS-Zn²⁺ to Cu²⁺ was calculated (LOD = 3σ/slope) to be 2.25 × 10⁻⁷ mol/L (r² = 0.9987). These results show that a fluorescent probe composed of complex FPS-Zn²⁺ could be used as fluorescent sensors for Cu²⁺ detection real-time and for Fe²⁺ qualitative determination.

FIGURE 4. (A) The relationship between fluorescence intensity of 10.0 μmol/L FPS and the time (B) Fluorescence spectra (λ_ex = 300 nm) of 10.0 μmol/L FPS and 10 μmol/L Zn²⁺ in the presence of Cu²⁺ ion with various concentrations (from 0 to 5.0 μmol/L) in methanol solution.

pH Tolerance of Complex FPS-Zn²⁺

As shown in Figure 5, the fluorescent spectra of FPS (5 μmol/L) + 1.0 equiv. Zn²⁺ with pH (1–12) in DMSO/H₂O be studied, in the neutral solution, the fluorescence intensity of FPS + Zn²⁺ complex is strongest. At the same time, when the pH lower than five or higher than 10, the fluorescent of FPS + Zn²⁺ complex is quenching.

FIGURE 5. Fluorescent spectra of FPS (5 μmol/L) + 1.0 equiv. Zn²⁺ with pH (1–12) in DMSO/H₂O.

¹H NMR Experiment

The binding ability of FPS with Zn²⁺ was evaluated using ¹H NMR. As shown in Figure 6, when Zn²⁺ was added to FPS, the protons on the phenolic hydroxyl and amide of the FPS Ha and Hb were nearly despaired, respectively. This indicated that phenolic hydroxyl and amide coordinated to Zn²⁺ and the FPS-Zn²⁺ complex was formed.

FIGURE 6. ¹H NMR spectra (400 MHz, DMSO-d6, 298 K) of (A) FPS (6 mm) + Zn²⁺ (6 mm) (B) free FPS (6 mm). This indicated that phenolic hydroxyl and amide play important roles of FPS-Zn²⁺ complex.

Theoretical Calculations

Based on the experimental data and Job’s plot, to further elucidate the influence of the structure on the electronic properties, DFT calculations are performed for FPS, FPS-X (X = Zn²⁺, Fe²⁺, Cu²⁺). As shown in Figure 7, for complex FPS-X (X = Zn²⁺, Fe²⁺, Cu²⁺), the deprotonated O atom of phenolic hydroxyl, O atoms of carbonyl, imine and the O atom of methanol were coordinated with metal ions. The calculated distributions of molecular orbitals (HOMO, highest occupied molecular orbital; SOMO, single electron occupied molecular orbital; and LUMO, lowest unoccupied molecular orbital) are shown in Figure 7. The HOMO and LUMO of FPS are predominately determined by the phenol moiety, the bride including acyl hydrazine moiety, furan moiety, and sectionally listed in pyridine moiety, respectively. Once FPS coordinated to metal ions (Zn²⁺, Fe²⁺, Cu²⁺), the HOMO/SOMO and LUMO in FPS-X (X = Zn²⁺, Fe²⁺, Cu²⁺) were conversely localized on the pyridine moiety and metal ions one, respectively. Apparently, this phenomenon was attributed to the enlargement of the conjugated system due to the complexation of the FPS and metal ions. The lower energy gap between the HOMO and LUMO level of FPS-X (X = Zn²⁺, Fe²⁺, Cu²⁺) compared with 4.21 eV of FPS was in good agreement with the red shift of the experimental fluorescence spectra (Figure 2B). Furthermore, the calculational binding energy E_bind (Supplementary Table S1) of FPS-X (X = Zn²⁺,Fe²⁺, Cu²⁺) show that the minimal values of FPS-Zn²⁺ and the maximum values of FPS-Cu²⁺ which cause the greater complexation of Cu²⁺compared with Zn²⁺ and Fe²⁺, probably further leading to the fluorescence quenching of FPS-Cu²⁺.

FIGURE 7. The optimized structures and Frontier molecular orbitals of FPS and FPS-X (X = Zn²⁺, Fe²⁺, Cu²⁺).

Fluorescence calculations of FPS-X (X = Zn²⁺, Fe²⁺, Cu²⁺) (Supplementary Tables S2–S4) was in good agreement with the experimental fluorescence data.

Fluorescence Imaging in Living Cells

Hela cells were cultured in DMEM medium containing 10% fetal bovine serum cell culture medium with 5% CO₂ atmosphere at 37°C. The cells were transferred into 24-well plates and incubated for 24 h at 37°C. The first group Hela cells treated in a culture medium (DMSO: DMEM = 1: 99, v/v) alone were used as a control (Figure 8A). In group 2 and 3, Hela cells were cultured with probe FPS solution (5.0 μmol/L) for 25 min (Figure 8B). In groups 4 and 5, Hela cells were cultivated successively with probe FPS (5.0 μmol/L and 10.0 μmol/L) for 25 min, after being washed with PBS, and further incubated with Zn²⁺ (10.0 μmol/L and 20.0 μmol/L) for 5 min, respectively (Figure 8C).

FIGURE 8. CLSM images of Hela cells treated under various conditions: (A) Hela cells were incubated with culture medium as a control; (B) Hela cells were cultured with probe FPS solution (5.0 μmol/L) for 25 min; (C) Hela cells were cultured with probe FPS solution (5.0 μmol/L) for 25 min, and after being washed with PBS and further incubated with Zn²⁺ (10.0 μmol/L) were then incubated for 5 min.

SCHEME 2. Benesi-Hildebrand equation. Where F_max and F_min are the fluorescence intensity of FPS in the presence and absence of zinc ions, respectively. F represents fluorescent intensities (at 480 nm) of FPS as a function of Zn²⁺ concentration. [FPS] = 20.0 μmol/L and [Zn²⁺] = 0–20 μmol/L.

Conclusion

This work was to synthesize and discover a specific “Dual-Response” to Zn²⁺ and Cu²⁺ probe based on a Schiff base bearing furopyrimidinone scaffold. The probe FPS with excellent linear relationship for the Zn²⁺ detection showed good and weak potential in imaging the exogenous and endogenous Zn²⁺, respectively, which can lead to the exploitation of growingly specific probes, particularly fluorescent for detection of Zn²⁺, Cu²⁺ and diagnosis of Zn²⁺, Cu²⁺ related diseases. In addition, the DFT calculations results showed how the structure affects the fluorescent behavior of FPS, which may help us to understand the essence of metal ions regulating effect in nature, and even give valuable reference to extend the real application of cell imaging (Li et al., 2021), imaging-guided (Sun et al., 2019), stimuli-responsive bioimaging (Min et al., 2021).

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Author Contributions

YH: Conceptualization, Writing—original draft. CL, JL and JF: Living cells experiments. LG: Formal analysis, Investigation. XH: Project administration. JM: Conceptualization, Resources, Writing—review and editing. LL: Software, Methodology, Writing—review and editing.

Funding

This work was financially supported by the National Natural Science Foundation of China (No. 81773746), the Natural Science Foundation of Hubei Provincial Department of Education (No. D20192102), the Open Project of Hubei Key Laboratory of Wudang Local Chinese Medicine Research (Hubei University of Medicine) (No. WDCM2018001, WDCM2020006), the Hubei Provincial Innovation and Entrepreneurship Training Program for College Students (No. S201910929016).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2021.774090/full#supplementary-material

References