Synthesis, Photophysical Properties, Theoretical Studies, and Living Cancer Cell Imaging Applications of New 7-(Diethylamino)quinolone Chalcones

Abstract

In this article, three unsymmetrical 7-(diethylamino)quinolone chalcones with D-π–A-D and D-π–A-π-D type push–pull molecular arrangements were synthesized via a Claisen–Schmidt reaction. Using 7-(diethylamino)quinolone and vanillin as electron donor (D) moieties, these were linked together through the α,β-unsaturated carbonyl system acting as a linker and an electron acceptor (A). The photophysical properties were studied, revealing significant Stokes shifts and strong solvatofluorochromism caused by the ICT and TICT behavior produced by the push–pull effect. Moreover, quenching caused by the population of the TICT state in THF–H₂O mixtures was observed, and the emission in the solid state evidenced a red shift compared to the emission in solution. These findings were corroborated by density functional theory (DFT) calculations employing the wb97xd/6-311G(d,p) method. The cytotoxic activity of the synthesized compounds was assessed on BHK-21, PC3, and LNCaP cell lines, revealing moderate activity across all compounds. Notably, compound 5b exhibited the highest activity against LNCaP cells, with an LC₅₀ value of 10.89 μM. Furthermore, the compounds were evaluated for their potential as imaging agents in living prostate cells. The results demonstrated their favorable cell permeability and strong emission at 488 nm, positioning them as promising candidates for cancer cell imaging applications.

1. Introduction

In recent decades, organic small molecular dyes have gained great research value due to their low cost of production as well as their ease of synthesis from commercial materials and their possibility of modifications. Moreover, these molecules usually display a broad spectrum of applications, such as optoelectronic devices,¹ dye-sensitized solar cells,² nonlinear optical properties (NLO), molecular probes,³ biological properties,⁴ and sensors.⁵ Furthermore, fluorescent compounds are important tools in biomedical imaging due to their ability to selectively bind to specific targets within biological systems and emit light upon excitation.⁶ This property allows researchers to track the movement of molecules, monitor cellular activity, and visualize the progression of diseases. Currently, advances in fluorescence imaging technology have led to significant improvements in the sensitivity and resolution of biological imaging,⁷ enabling researchers to study biological systems with unprecedented detail. Therefore, the development of new and improved fluorescent compounds is crucial for the continued advancement of biomedical imaging, as well as the diagnosis and treatment of various diseases.⁸

In this regard, great effort has been made in developing new fluorophores with improved properties from classical scaffolds, the majority of these are created through structural alterations of a small number of classical “core” dyes,⁹ including naphthalimide,¹⁰ coumarin,¹¹ BODIPY,⁶ fluorescein,¹² and rhodamine.¹³ However, the creation of new “core” fluorophores could provide greater versatility and flexibility for developing novel fluorescent probes with unique properties. In this context, quinolones have been of great interest due to their excellent photochemical stability (laser irradiation), favorable biocompatibility,¹⁴ excellent mechanical properties,¹⁵ synthetic versatility,¹⁶ and good thermal stability.¹⁷ Even more the relevance of the incorporation of dialkylamino groups in the quinolone moiety has been assessed on its key effect on the photophysical properties,¹⁸ such as UV–vis spectra (causing a red shift), Stokes shift, and fluorescence quantum yield, generating high efficient materials in the electron transport, high photoluminescence efficiencies, and biological imaging properties.¹⁹ In addition, quinolones have exhibited interesting biological properties, such as antimicrobial,²⁰ antifungal,²¹ antiviral,²² antimalarial,²³ antituberculosis,²⁴ antioxidant,²⁵ and anticancer.²⁶

On the other hand, chalcones are a class of organic compounds that contain a central α,β-unsaturated carbonyl system, which has attracted significant attention,²⁷ due to their diverse applications in the fields of medicine,²⁶ material science, and optics.²⁸ Moreover, the chalcones due to the π-conjugated system and the two aromatic rings that act as donors and acceptors provide a reasonable fluorescence quantum yield,²⁷ efficient stabilization of charge-transfer processes in the excited state,²⁸ long-wavelength emission (λ_em), and large Stokes shifts,²⁹ which means that they have potential applications in fluorescence microscopy or bioimaging applications as they can improve image sensitivity by reducing the interference caused by self-absorption or autofluorescence.³⁰⁻³²

This study focuses on the design, synthesis, and investigation of the photophysical properties of three unsymmetrical 7-(diethylamino)quinolone chalcones. These chalcones contain electron-rich 7-(diethylamino)quinolone and vanillin moieties acting as electron donors (D), linked together through an α,β-unsaturated carbonyl system acting as a linker and an acceptor (A), resulting in D-π–A-D and D-π–A-π-D type push–pull molecular arrangements. Additionally, we evaluated the cell viability and toxicity in a normal cell line (BHK-21) and two prostate cancer cell lines (LNCaP and PC3). Furthermore, fluorescence imaging was carried out to evaluate their potential in bioimaging applications.

2. Results and Discussion

2.1. Synthesis

The detailed synthetic routes for 7-(diethylamino)quinolone chalcones 4a and 5a–b derivatives are outlined in Scheme 1. Target chromophores were readily synthesized following the literature method through a one-step Claisen–Schmidt condensation reaction between equimolar amounts of 7-(diethylamino)-2-oxo-quinolinaldehyde 1 and ketones 2 and 3a–b.³³ The reaction was carried out in methanol and KOH at room temperature for 36 h; after completion, it was neutralized with HCl, and the resulting precipitate was collected by filtration and washed with MeOH/H₂O, leading to the isolation of the expected target compounds 4a and 5a–b with yields between 58 and 73%. The noncommercial 7-(diethylamino)-2-oxo-quinolinaldehyde 1 was prepared in two steps. Initially, by a Meth–Cohn reaction from N-(3-(diethylamino)phenyl)acetamide, 2-chloro-7-(diethylamino)quinoline-3-carbaldehyde was obtained, followed by hydrolysis with acetic acid (70%). The structures of the new chromophores, 7-(diethylamino)quinolone chalcones 4a and 5a–b, were ascertained by FTIR, ¹H NMR, and ¹³C NMR (in DMSO-d₆), and mass spectrometric analysis is summarized in the Experimental Section. The ¹H NMR spectrum of the compound 5b showed four doublets at 7.74, 7.68 ppm (J = 15.7 Hz) and 7.62, 7.21 (J = 15.9 Hz) assigned to protons of the α,β-unsaturated moiety, confirming the condensation between 1 and 3b, as well as E configuration of the new double bonds formed. In the ¹³C NMR spectrum, the total expected signals (i.e., 24) for compound 5b were observed (see the Supporting Information).

Scheme 1. Synthesis of New 7-(diethylamino)quinolone Chalcones.

2.2. Photophysical Properties

The optical properties of 4, 5a, and 5b were determined by recording the UV–vis absorption and fluorescence emission spectra in seven different solvents (THF, DCM, acetone, DMF, DMSO, ACN, and EtOH) as outlined in Table 1. The UV–vis spectra (Figure 1) showed an intense absorption maximum centered in the region of 434–465 nm, which could be assigned to an intramolecular charge transfer (ICT) process from the diethylamino and vanillin moieties to the acceptor segments.^15,34 The nature of the solvent had a considerable effect on the UV–vis absorption properties of the three compounds, suggesting that the difference in the dipole moment between the equilibrium ground state and the Franck–Condon excited state is large.³⁵ For example, the maximum absorption of 5a shifted from 434 nm in THF to 463 nm in DMSO (Table 1). The intense red shift of the λ_max values indicates electronic transitions with ICT character, which can also be verified by the molar absorption coefficient magnitude for all of the dyes³⁶ (ε = 1.7 × 10⁴–6.9 × 10⁴ M^–1 cm^–1). Theoretical calculations were performed at the time-dependent wb97xd/6-311g (d,p) level in THF to determine the nature of the electronic transitions that gave rise to the experimental absorption bands. The intense absorption band experimentally registered in the 434–465 nm region is due to the transition to the lowest-lying singlet excited state S₁ predicted with a considerable oscillator strength (f = 1.63, 2.01, and 1.92 for 4, 5a, and 5b, respectively). This transition is depicted by the HOMO → LUMO monoexcitation in all of the dyes.

Table 1. Spectroscopic Properties of Fluorophores 4, 5a, and 5b at 5.0 μM.

compounds	4			5a			5b
solvent	Abs, nm (ε, M–1cm–1)	Emb, nm (φ)a	Stokes shift, cm–1	Abs, nm (ε, M–1cm–1)	Em, nm (φ)a	Stokes shift, cm–1	Abs, nm (ε, M–1cm–1)	Em, nm (φ)a	Stokes shift, cm–1
THF	437 (38 080)	502	2963	434 (69 500)	523 (0.33)	3921	443 (28 840)	525 (0.49)	3226
DCM	443 (23 820)	521 (0.66)	3380	452 (35 160)	552 (0.63)	4008	457 (39 480)	552 (0.63)	3766
acetone	440 (39 020)	521 (0.73)	3533	452 (21 000)	550	3942	445 (37 900)	552 (0.35)	4356
DMF	448 (21 740)	534 (0.31)	3489	456 (32 680)	564 (0.46)	4199	459 (32 340)	569 (0.42)	4212
DMSO	454 (32 580)	543 (0.53)	3610	463 (32 180)	576 (0.44)	4237	465 (17 420)	581 (0.37)	4294
ACN	441 (60 960)	539 (0.79)	4123	448 (26 380)	577 (0.47)	4990	455 (44 600)	579 (0.33)	4611
EtOH	443 (46 000)	565 (0.43)	4811	459 (34 880)	606 (0.14)	5284	462 (31 060)	604 (0.10)	5089

^aRelative quantum yield using Rhodamine B as standard.

^bThe measures were made at 2.0 μM.

Figure 1.

Absorption spectra of (a) 4, (b) 5a, and (c) 5b (5.0 μM) in different solvents.

The fluorescence emission spectra of compounds 4, 5a, and 5b (Figure 2) exhibited various emission profiles, which have a stronger dependence on the solvent environment than the absorption spectra, presenting structure bands with maximum emission in the 502–606 nm region. Interestingly, these compounds exhibited broad Stokes shifts (4 2963–4811, 5a 3921–5284, and 5b 3226–5089 cm^–1) in different solvents, indicating a fast relaxation from the excited state to lower energy vibrational states, which is beneficial for fluorescence imaging.³⁷ As the solvent polarity increased from THF to ethanol, the emission peaks of 4, 5a, and 5b displayed a red-shifted tendency from 502 to 563 nm, 523 to 606 nm, and 525 to 604 nm, respectively, showing a considerable bathochromic effect. These observations indicate that the excited state is highly polar compared to the ground state, resulting in a stronger interaction of polar solvents in the excited state. The decrease in fluorescence intensity, along with the change in emission maximum, can be attributed to the significant impact of the twisted intramolecular charge transfer (TICT) effect in polar solvents.^38,39 The observed shift toward red in the emission intensity of fluorophores is attributed to the TICT process due to its susceptibility to different nonradiative decay processes. Unexpectedly, all of the compounds showed a dual emission in DCM, which could be attributed to an excited state proton transfer (ESPT) from DCM to the amide group of the chromophores caused by the photodegradation of DCM in the presence of UV–vis radiation.^40,41 Moreover, the optical band gap (E^0-0) was estimated from the interception of the absorption and emission bands for all of the compounds, resulting in values of 2.67, 2.56, and 2.55 eV for 4, 5a, and 5b, respectively, exhibiting the same tendency as the calculated energy gap in THF.

Figure 2.

Emission spectra of (a) 4, (b) 5a, and (c) 5b in different solvents. Pictures of solutions under UV light (365 nm lamp) in solvents with increasing polarity.

2.3. Solvatofluorochromism

Preliminary studies to view the solvatofluorochromism effects were performed by checking the color change of 4, 5a, and 5b in solutions of solvents with different polarities. All compounds displayed color changes from green in a moderate- polar solvent (THF) to red in a high polar solvent (EtOH; Figure 3). The significant difference in Stokes shift observed for compounds 4, 5a, and 5b motivated us to investigate the charge transfer interaction more extensively. This was accomplished by examining the emission characteristics of the fluorophores in solvents of different polarities. The emission spectra showed positive solvatochromism properties with increasing solvent polarity because the highly polar TICT state is specially stabilized concerning a planar local excited (LE) state in polar solvents.^38,42 This positive solvatochromic behavior was quantitatively determined using different solvent polarity plots, such as the plot of the maximum emission band regarding the empirical polarity scale E_T(30)⁴³⁻⁴⁵ as well as the plot of the relationship between Stokes shifts vs orientation polarizability (Lippert–Mataga plot).^29,46 The plot of emission maximum vs E_T(30) (Figure 3d) displays a linear tendency of 4, 5a, and 5b with negative slopes of −206.0 cm^–1 (R² = 0.63), – 214.5 cm^–1 (R² = 0.96), and −222.6 cm^–1 (R² = 0.95), respectively (Figure 3d). Nevertheless, compounds 5a and 5b showed many sheer slopes with linear coefficients better than those of 4, evidencing a more significant variation of the solvatofluorochromism effect with the variation of solvent properties. These features confirm the assignment of this band to an ICT and TICT process produced by the push–pull effect into the D-π–A-D and D-π–A-π-D structures.⁴⁷ On the other hand, the Lippert–Mataga plot (Figure S2a) shows a low linearity tendency; this behavior can be due to specific solvent effects like preferential solvation, hydrogen bonding, and charge-transfer interactions, which are produced by one or a few neighboring molecules and are determined by the specific chemical properties of both the fluorophore and the solvent.⁴⁸ The spectral offsets caused by the formation of ICT and TICT states are not explained by the Lippert–Mataga equation, because the feature of the ICT and TICT emission depends on the electron-donating and electron-accepting properties of groups within or linked to the fluorophore instead of polarity. In order to establish a connection between the observed solvatochromic shift in a positive direction and the twisted intramolecular charge transfer (TICT) phenomenon, we studied the Rettig plots (Figure S2b). The Rettig plots for compounds 4, 5a, and 5b revealed a linear relationship between the Stokes shift and the solvent polarity parameter Δf, indicating that the primary state in which TICT occurs is the initial single state. Consequently, the linear association observed in the Rettig plots suggests the coexistence of both TICT and ICT within these fluorophores.^49,50

Figure 3.

Emission spectra of fluorophores (a) 4, (b) 5a, and (c) 5b in different solvents at 25 °C. Pictures of solutions under UV light (365 nm lamp) in solvents of increasing polarity. (d) Plot of emission maximum vs E_T(30) in aprotic solvents.

2.4. Solid-State Emission in Powder

The solid-state fluorescence spectra were registered at room temperature and exhibited emission bands centered at 624, 603, and 648 nm for 4, 4a, and 5b, respectively (Figure 4). Compound 5a showed a blue shift compared to 4 and 5b by the −OH group in the 4-position π-extended vanillin moiety, indicating the existence of a twisted arrangement in the solid form.⁵¹ In addition, the insertion of the electron-donating group −OCH₃ (5b) instead of −OH (5a) caused an increase in the donor ability of vanillin, leading to enhanced D–A interactions. However, compound 5a exhibits a broad and intense band caused by the highly conjugated system.⁵² On the other hand, the solid-state emission spectra are similar to those acquired in a highly polar solvent such as ethanol. Conversely, an opposite behavior was found in 4 and 5b, showing a red shift compared to those in solution, indicating the existence of intermolecular π–π interactions in the solid state.^52,53 The results suggest that changing the molecular structure makes it possible to control the fluorescence properties, such as fluorescence intensity, emission band offset, and intermolecular interaction in the solid state.

Figure 4.

Solid-state emission spectra of compounds 4, 5a, and 5b.

2.5. Acid–Base Responsive Emission

To investigate the acid–base response on the emission behavior of 4, 5a, and 5b, emission spectra in THF with TFA and TEA equivalents were recorded (Figure S3). In all cases, the emission spectra show a blue shift (negative acidocromism) with a gradually decreasing emission peak, which could be attributed to the protonation of nitrogen at the N,N-diethylamino terminal group.⁵⁴ On the other hand, the addition of TEA caused similar results to the TFA titration with a negative acidocromism, and the emission intensity decreased due to the deprotonation of the −OH group of the phenol moiety. However, compound 5b does not show this trend due to the absence of this group, which is replaced by a methoxy group.

2.6. Aggregation Properties

To investigate the effects of aggregation on the emission performance, emission spectra of 4, 5a, and 5b were recorded in different THF–water mixtures (from 0 to 90%; Figure 5). In all dyes, the emission displayed a fluorescence quenching with a red shift upon increasing amounts of water (f_W) attributed to the influence of the TICT effect. In the TICT state, an N,N-diethylamino donor undergoes a torsional motion of around 90° when exposed to light, creating a chemically active species that does not emit light.⁵⁵⁻⁵⁷ Molecules exhibiting TICT characteristics are consistently identified by their substantial molecular dipoles and a distinct uneven distribution of electrons in emission-related frontier molecular orbitals. These characteristics are confirmed by theoretical calculation results. Figure 6 shows that in the HOMO level, the electron cloud is primarily concentrated on the N,N-diethylamino unit, while in the LUMO level, it is predominantly situated on the quinolone unit.⁵⁸ On the other hand, all compounds could be candidates for determining water content in organic solvents acting as fluorescent sensors.⁵⁹

Figure 5.

Emission spectra of (a) 4, (b) 5a, and (c) 5b in THF/water and (d)–(f) plot between emission intensity vs fraction of water (f_W) in THF/water.

Figure 6.

Optimized structures, SEP, and frontier orbitals for 5a, 5b, and 4 obtained at the Wb97xd/6-311g(d,p) level of theory.

2.7. Theoretical Calculations

The minimum energy structures of all of the compounds were confirmed through frequency calculations, ensuring that no imaginary frequencies were generated. Figure 6 displays the structures with minimum energy; additionally, the uppermost part of this figure shows a 2D structure based on the studied compound with specific atoms arbitrarily labeled to facilitate comprehension of the electron flux within the D-π–A-π-D system. In this sense, a discussion of the geometry obtained through optimization is initially performed, followed by a discussion of how frontier orbitals affect the absorption and emission processes.

Table 2 shows the variation of some important bonds as well as the dipolar momentum (μ) in both the ground and the first excited state in THF solvent. Notably, except for the bonds between C₉–O₁₀ and C₁₁–O₁₂, all selected bonds exhibit a shorter length in the excited state than in the ground state. This observation supports the hypothesis mentioned above that the studied compounds demonstrate an intricate charge transfer toward the carbonyl moiety, coupled with the π–π* transition serving as the initial transition; in other words, this result suggests that an efficient electronic conjugation between D and A takes place. Conversely, the bonds denoted as C₉–O₁₀ and C₁₁–O₁₂ exhibit a contrary trend as they display a greater length in the excited state. This finding strongly indicates that, in both the O–H and O–CH₃ groups, the inductive effect predominates over the resonance effect, and they have not increased the electronic density over the aromatic ring. In addition, a close inspection of both bonds shows the probability of hydrogen bond formation due to the proximity of these two bonds; consequently, the potential formation of a hydrogen bond could reduce the electron density of the oxygen atoms and the donor effect via resonance.

Table 2. Variation of Some Specific Bonds in the Ground and First Excited States in THF Using the wb97xd/6-311g(d,p) Level of Theory and CPCM Approximation.

compounds	N1-C2	C3-C4	C4-C5	C5-C6	C6-O7	C9-O10	C11-O12	μ(D)
5a	1.37667	1.45386	1.33958	1.48395	1.21983	1.36463	1.34961	6.9220
5aa	1.3745	1.44542	1.36261	1.42571	1.29330	1.36539	1.35172	6.9968
5b	1.3741	1.45358	1.33973|	1.48313	1.21900	1.36561	1.36854	7.7461
5ba	1.37607	1.44560	1.36154	1.42706	1.29189	1.36750	1.36901	8.1422
4	1.37421	1.45471	1.33943	1.48329	1.21952	1.36463	1.34826	7.6802
4a	1.37003	1.41086	1.37636	1.45583	1.234387	1.36531	1.3497	9.1999

^aindicates first excitation state. Bond longitude in Anstrong.

Another essential factor that can be observed in Table 2 is the increase of the dipole momentum on going from the ground state to the first excited state. These findings are consistent with the experimental bathochromic shift observed in the absorption spectra of all studied compounds as the polarity of the solvent increases. In such scenarios, the relaxed excited state S1 is more energetically stable than the ground state S0, leading to a significant red shift in the absorption spectra.⁶⁰

The shape of its frontier orbitals can influence the UV absorption spectrum of a molecule. The HOMO of a molecule acts as an electron donor, while the LUMO accepts electrons. The spatial distribution and shape of these orbitals play a crucial role in determining the strength and orientation of electronic transitions that occur when the molecule absorbs UV radiation.⁶¹

The compounds studied are depicted in Figure 6, which displays the frontier orbitals and electrostatic potential maps. 5a and 4 exhibit similar behavior, with boundary orbitals dispersed throughout the molecular structure. Conversely, 5b displays a highly localized HOMO in the amino group, while its LUMO exhibits a distribution similar to that of the other compounds. Notably, the presence of dimethoxy groups significantly impacts electron fluxes, as indicated by the shape of the frontier orbitals. The absence of dimethoxy groups in the HOMO and LUMO orbitals suggests that they function as attractors rather than acceptors in the electron push–pull systems, confirming the explanation above based on bond distances. In addition, molecules with a HOMO localized in a donor group and a LUMO mainly localized in an acceptor group will have a strong absorption band at a wavelength corresponding to the energy difference between these orbitals. This happens because the transition from the HOMO to the LUMO is favorable and can be easily excited by UV radiation.⁶²⁻⁶⁴

Conversely, when a molecule’s HOMO and LUMO are distributed throughout the molecule, the energy gap between them may be narrow, leading to a wide absorption spectrum, as shown in Figure 1. Moreover, as the solvent polarity increases, the emission spectra broaden. This is due to the stabilization of HOMO–LUMO transitions by the solvent’s polarity, which promotes electronic transitions.⁶⁵

Table 3 shows the absorption excitation energy, wavelengths of the lowest electronic transitions, and oscillator strengths for all compounds in THF. In all cases, the maximum oscillation strength corresponds to the transition between the HOMO–LUMO orbitals, followed by the transition HOMO(−2)–LUMO(+2); these transitions are allowed due to the close in the shape of the orbital. Moreover, the occupied orbital was mainly localized in the aromatic ring. In contrast, the uncoupled orbital was centered on both the carbonyl and π moiety, strongly suggesting an ICT process (carbonyl group) and, therefore, a π–π* transition. However, the presence of the dimethoxy group seems to break the donor process; computational calculations in this work suggest that the presence of two methoxy groups in the ring prevents resonance to the electron-accepting group. This behavior of the methoxy group has been reported before.⁶⁶

Table 3. Excitation Energy, Wavelength, and Oscillator Strength Obtained from DFT Calculations in THF.

molecule	excited state	transition	excitation energy (eV)	wavelength	oscillator strength
5a	1	HOMO–LUMO	3.2458	381.99	2.0051
	2	HOMO(−1)–LUMO	3.9252	315.87	0.2447
	3	HOMO(−2)–LUMO(+2)	5.7780	214.58	0.7702
5b	1	HOMO–LUMO	3.2479	381.74	1.9161
	2	HOMO(−1)–LUMO	4.1031	302.17	0.3836
	3	HOMO(−2)–LUMO(+2)	5.7673	214.98	0.7751
4	1	HOMO–LUMO	3.3366	371.59	1.6313
	2	HOMO(−1)–LUMO	4.2715	290.26	0.1114
	3	HOMO(−2)–LUMO(+2)	5.8139	213.26	0.9813

The fluorescence efficiency is affected by the nature of a molecule’s frontier orbitals. For example, Figure 5 shows that molecules with highly delocalized orbitals display stronger fluorescence due to the greater likelihood of the excited state electron returning to its ground state via radiative decay rather than nonradiative decay through alternative pathways like heat. Moreover, the shape of the frontier orbitals explains the behavior of the Stokes shift, which is the energy difference between a molecule’s maximum absorption and maximum emission. Molecules with highly delocalized HOMO and LUMO experience a larger Stokes shift, resulting in a larger energy gap between excitation and emission. Nevertheless, a larger Stokes shift typically corresponds with a smaller quantum yield, as illustrated in Table 1.

2.8. Electrochemical Studies

Electrochemical redox behaviors (reduction and oxidation) of compounds 4, 5a, and 5b and their precursors (1, 2, 3a, and 3b) were investigated by cyclic voltammetry studies. All measures were obtained after finding the appropriate experimental conditions, such as applied potential range, scan rate (ν), and the number of cycles (n). Figure 7 shows the voltammograms obtained at 100 mV/s for each compound of interest.

Figure 7.

Cyclic voltammograms of the compounds (a) 4, (b) 5a, (c) 5b and their precursors (1, 2, 3a, and 3b) (1.2 × 10^–2 M) in CH₃CN with 1.2 × 10^–2 M TBAPF₆, ν = 100 mV/s, n = 5.

Compound 4 (see Figure 7a) shows two oxidation peaks, one at 0.819 V and the second at 1.068 V; possibly, these peaks are due to the influence of precursors 2 and 1, respectively. Precursor 2 shows an oxidation peak at 0.739 V, which could result from the oxidation of the OH attached to the aromatic ring,⁶⁷ while in 1, it is observed at 1.068 V, which originated from the oxidation of the quinolone structure, the latter being an irreversible signal in terms of current obtained and peak potential difference.

Compounds 5a and 5b (see Figures 7b,7c) show much more marked and defined oxidation and reduction peaks than compound 4. In Table 4, the peak potential values of each compound are detailed. Considering the voltammograms obtained for both compounds (5a and 5b), it is possible to infer that the oxidation process is linked to the redox reaction of the segment coming from compound 1; in both cases, it is observed that this signal is shown at lower peak potentials than in compound 4, that is, less energy is required for the charge transfer to occur, which could indicate electrocatalysis of the reaction. The reduction processes observed at approximately −0.046 and −0.191 V for compounds 5a and 5b, respectively, are irreversible and may be linked to the reduction reaction of the segment of the molecule coming from the precursors 3a and 3b, respectively.

Table 4. Electrochemical Parameters Acquired from Voltammograms.

compounds	Eonset,Oxi/V	Eonset,red/V	Ep, oxi/V	Ep, red/V	HOMO	LUMO	Eg/eV
4(1)	0.740		0.819		5.140
4(2)	0.980	1.106	1.068	0.991	5.380	5.506	0.126
5a	0.638	0.975	0.778	0.864	5.038	5.375	0.337
5a2		0.240		–0.046
5b	0.760	0.895	0.851	0.815	5.160	5.295	0.135
5b2		–0.018		–0.191

E_onset,_Oxi: potential where the oxidation peak is initiated, E_onset,red: potential where the reduction peak is initiated, Ep: peak potential.

In certain investigations, the initiation of oxidation or reduction signals (E_onset,Oxi; E_onset,red) is utilized to estimate the energy levels of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), respectively. This approach provides a means to quantify the electronic properties of molecules.⁶⁸ The results of these and other electrochemical parameters are listed in Table 4. The electrochemical Eg values obtained are much lower than those estimated by spectroscopy; possibly, this difference is due to the influence of the solvent and the supporting electrolyte in the charge transfer process determined by cyclic voltammetry.⁶⁹

2.9. Cell Viability

To determine the toxic effects of these compounds, the MTT assay was carried out using two prostate-cancer-derived cells and a normal cell line. The cellular viability of the compounds 4, 5a, and 5b was determined at different concentrations (60.0, 30.0, 15.0, 7.5, 3.8, 1.9 μM) to determine their 50% lethal concentration (LC₅₀) (Table 5 and Figure 8). Compound 5a exhibited higher toxicity to tumor cells compared to the results obtained in BHK-21 cells, being 4.6 times more toxic to the LNCaP tumor line than normal cells. This is notably an advancement in the search for antitumor compounds or compounds useful in detecting cancer cells.

Table 5. In Vitro Antiproliferative Activity of Compounds 4 and 5a–b against a Normal Cell Line (BHK-21) and Two Prostate Cancer Cell Lines (LNCap and PC3).

	compounds
	5a	5b	4
cell line	LC50 (μM)
BHK-21	49.70	36.00	8.10
PC3	33.03	65.30	>60
LNCaP	10.89	44.90	>60

Figure 8.

Cell viability of compounds 4 and 5a–b against two human prostate cancer cells (LNCaP and PC3) and BHk-21 cell.

Motivated by the extraordinary fluorescence properties of compounds 4 and 5a–b, we investigated their practical utility in live cell bioimaging and their efficient internalization in two human prostate cancer cells (LNCaP and PC3) and the normal cell BHk-21 (Figures 9–11). The results indicated the favorable cell permeability of compounds 4 and 5b, along with their significant green fluorescence emission after excitation at 450–490 nm. Moreover, these compounds exhibited low-intensity emission when excited at 365 nm, and no fluorescence emission was detected when cell cultures were excited at 575–625 nm and 460 nm. However, it is noteworthy that compounds 5b and 4 uniquely emitted light at a wavelength of 520 nm exclusively in PC3 cells (Figure 11). This observation can be attributed to the distinctive traits of tumor cells. PC3, a prostate cancer cell line, is characterized by being poorly differentiated and androgen-independent, making it a highly aggressive form of cancer. This finding opens up possibilities for further exploration, as it could potentially serve as a marker for identifying tumor cells exhibiting aggressive phenotypes.

Figure 9.

Microscopy fluorescence images of BHK-21 cells loaded with compound 4 (I–L) and compounds 5a (A–D) and 5b (E–H) excited at different wavelengths.

Figure 11.

Microscopy fluorescence images of PC3 cells loaded with compound 4 (I–L) and compounds 5a (A–D) and 5b (E–H) excited at different wavelengths.

Figure 10.

Microscopy fluorescence images of LNCaP cells loaded with compound 4 (I–L) and compounds 5a (A–D) and 5b (E–H) excited at different wavelengths.

In general, prostate cancer cells, such as PC3 and LNCap, are characterized by high vesicular expression. This leads to the presence of characteristic multivesicular bodies, from which exosomes bud.⁷⁰ This characteristic is primarily observed in PC3 in Figure 11(A,E,I). Figure 11(B, F,J) demonstrates that the studied compounds can associate with these cellular structures because they exhibit a fluorescence with greater intensity, primarily composed of cytoplasmic proteins, such as actin, tubulin, and genetic material, including various RNA molecules.⁷¹ It is noteworthy that both the nucleus and nucleolus, in all assays with different cell lines, exhibit much fainter fluorescence compared to the cytoplasm (Figures 9–11), suggesting that these compounds may not show affinity for nucleic acids but rather for various cytoplasmic proteins and different types of lipids. Additionally, when cells were excited at 450–490 nm, strong fluorescence was around the nuclei, suggesting an accumulation of the compound in the endoplasmic reticulum.^72,73 In this endomembrane system, lipid synthesis and modifications, as well as some protein synthesis, take place.⁷⁴ The potential association of the analyzed compounds with multivesicular bodies and the endoplasmic reticulum could be related to the slightly hydrophobic nature of these compounds, providing them with a certain selectivity when interacting with lipid or peptide cellular components. Future research on these compounds should focus on the mechanisms of action, cellular targets, and other potential effects, such as apoptosis activation, generation of reactive oxygen species, and genotoxic effects, among others.

3. Conclusions

In summary, we have synthesized three unsymmetrical 7-(diethylamino)quinolone chalcones with D-π–A-D and D-π–A-π-D type push–pull molecular arrangements via a Claisen–Schmidt reaction. The photophysical properties of compounds 4, 5a, and 5b were studied. These dyes exhibited absorptions around 434–465 nm and emissions in the 502–606 nm range with considerable Stokes shifts caused by the ICT and TICT behavior. The general solvent effects in solvatofluorochromism were determined by the Reichardt ET, Lipper–Mataga, and Rettig plots that confirm the ICT and TICT process produced by the push–pull effect into D-π–A-D and D-π–A-π-D. The solid-state emission evidenced a red shift compared to the solution emission caused by the intermolecular π–π interactions. The acid–base response shows the possible sites of protonation and deprotonation inside the molecules 4, 5a, and 5b. Finally, all of these compounds exhibited a quenching behavior with the increase of f_W caused by the typical TICT effect. The DFT results suggest that the compounds exhibit an intricate charge transfer within the D-π–A-D and D- π–A- π-D system, where the bonds involved in the charge transfer exhibit shorter lengths in the excited state, indicating an efficient electronic conjugation between the donor (D) and acceptor (A) groups. The obtained compounds showed moderate toxicity. However, compound 5a exhibited 4.6 times higher toxicity on LNCaP than the normal cell line BHK-2. Furthermore, these fluorophores hold promising potential for utilization as cancer biomarkers.

4. Experimental Section

4.1. Chemistry

4.1.1. General

All organic chemicals and solvents were procured from Sigma-Aldrich, Fluka, AK Scientific, and Merck (analytical grade reagent) and used without further purification. IR spectra were recorded on a Shimadzu FTIR 8400 ATR spectrophotometer. Melting points were measured using a Stuart SMP3 melting point apparatus and are uncorrected. The 1H and 13C NMR spectra (400 MHz for proton and 100 MHz for carbon) were recorded on a Bruker Avance II 400 MHz NMR spectrometer, using DMSO-d6 (deuteration degree min. 99.95% for NMR spectroscopy MagniSolv) as the solvent and tetramethylsilane as an internal standard. Mass spectra were recorded on a SHIMADZU-GCMS 2010-DI-2010 spectrometer (equipped with a direct inlet probe) operating at 70 eV. TLC analyses were performed on silica gel aluminum plates (Merck 60 F254) and spots were visualized with ultraviolet irradiation.

4.1.2. General Procedure for the Synthesis of bis 7-(Diethylamino)quinolone Chalcones

7-(diethylamino)-2-oxo-quinolinaldehyde 1 (1.0 mmol) was first dissolved in methanol (23 mL) and 5 mL of 10% KOH, and then ketones 2 and 3a–b (1.0 mmol) were added. The reaction mixture was stirred for 36 h at room temperature. When the reaction finished, it was neutralized with HCl, and the resulting precipitate was collected by filtration, washed with MeOH/H₂O, and finally recrystallized from ethanol.

4.1.2.1. 7-(Diethylamino)-3-((1E,4E)-5-(4-hydroxy-3-methoxyphenyl)-3-oxopenta-1,4-dien-1yl)quinolin-2(1H)-one (5a)

Red solid, mp 102–104 °C. FTIR (ATR) ν = [3363, 3168] (OH, NH), [1666, 1610] (C=O) cm^–1, ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 11.47 (s, 1H), 8.19 (s, 1H), 7.67 (dd, J = 11.3 Hz, 2H), 7.60 (d, J = 15.7 Hz, 1H), 7.48 (d, J = 9.0 Hz, 1H), 7.41 (s, 1H), 7.21 (d, J = 8.0 Hz, 1H), 7.13 (d, J = 15.8 Hz, 1H), 6.85 (d, J = 8.1 Hz, 1H), 6.71 (d, J = 9.2 Hz, 1H), 6.50 (s, 1H), 3.87 (s, 3H), 3.44 (q, 6.8 Hz, 4H), 1.16 (t, J = 6.9 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ ppm: 188.3, 161.5, 150.3, 149.5, 148.0, 142.4, 141.7, 141.6, 138.8, 130.1, 126.3, 124.7, 123.5, 123.4, 119.0, 115.6, 111.3, 110.0, 109.0, 99.5, 93.8, 55.7, 44.2, 12.5. EI MS (70 eV): m/z (%): 419 (M⁺: 5), 418 (17), 241 (100), 216 (9), 197 (13).

4.1.2.2. 7-(Diethylamino)-3-((1E,4E)-5-(3,4-dimethoxyphenyl)-3-oxopenta-1,4-dien-1-yl)quinolin-2(1H)-one (5b)

Red solid, mp 83–85 °Cν = [3165] (NH), [1661] (C=O) cm^–1, ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 8.15 (s, 1H), 7.74 (d, J = 15.7 Hz, 1H), 7.68 (d, J = 15.7 Hz, 1H), 7.62 (d, J = 15.9 Hz, 1H), 7.45 (d, J = 8.9 Hz, 1H), 7.44 (s, 1H), 7.32 (d, J = 8.3 Hz, 1H), 7.21 (d, J = 15.9 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.67 (dd, J = 9.0, 1.7 Hz, 1H), 6.50 (s, 1H), 3.86 (s, 3H), 3.83 (s, 3H), 3.42 (q, J = 6.8 Hz, 4H), 1.15 (t, J = 6.9 Hz, 6H). ¹³C NMR (101 MHz, DMSO) δ ppm: 188.9, 162.5, 151.5, 150.7, 149.5, 143.0, 142.4, 142.0, 139.9, 130.6, 128.2, 125.0, 124.9, 123.7, 119.4, 112.1, 110.9, 110.7, 109.4, 94.8, 56.1, 56.1, 44.6, 13.0. EI MS (70 eV): m/z (%): 432 (M+: 9), 255 (67), 241 (100), 229 (21), 211 (15), 191 (23)

4.1.2.3. (E)-7-(diethylamino)-3-(3-(4-hydroxy-3-methoxyphenyl)-3-oxoprop-1-en-1-yl)quinolin-2(1H)-one (4)

Orange solid, mp 100–101 °C. FTIR (ATR) ν = [3415, 3290] (OH, NH), [1647, 1610] (C=O) cm^–1, ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 11.44 (s, 1H), 8.32 (s, 1H), 8.17 (d, J = 15.4 Hz, 1H), 7.77 (d, J = 15.4 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.58 (s, 1H), 7.48 (d, J = 9.0 Hz, 1H), 6.95 (d, J = 8.2 Hz, 1H), 6.71 (d, J = 8.6 Hz, 1H), 6.51 (s, 1H), 3.88 (s, 3H), 3.43 (q, J = 6.2 Hz, 4H), 1.16 (t, J = 6.8 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ ppm: 187.3, 161.6, 151.6, 150.3, 147.8, 141.7, 141.2, 139.3, 130.1, 130.1, 123.1, 120.1, 119.1, 115.0, 111.4, 110.0, 109.0, 93.9, 55.7, 44.2, 12.5, 12.4. EI MS (70 eV): m/z (%): 392 (M+: 17), 241 (100), 197 (15) 151 (11),

4.2. Computational Details

The minimum energy surfaces of the compounds 4, 5a, and 5b were determined using density functional theory (DFT) at the wb97xd/6-311g (d,p) level of theory.^75,76 The minimum energy structures were validated through frequency calculations, which ensured that all force constant values were greater than zero and verified that the minimum energy surface existed in all directions. Subsequently, electrostatic surface potential (ESP), frontier orbitals (HOMO and LUMO), and the excited state were computed by applying time-dependent density functional theory.⁷⁷

4.2.1. Theoretical Solvation Models

The impact of the solvent on the behavior of the compounds described in the study was analyzed by using a polarized continuum solvation model based on density. The solvation model, which was implemented using G09 for Linux, was used to investigate the influence of the solvent on the compounds by incorporating the effect of solvent polarization into the calculations. The solvation model utilized the principles of the joint solvation model and the polarized continuum model, as described in the literature.^78,79 The goal of this study was to gain a deeper understanding of how the properties of the solvent impact the behavior of the compounds and to develop a more accurate and comprehensive model for predicting the behavior of similar compounds in different solvents. The results of this study are expected to provide important information for the design and optimization of new chemical compounds and to advance our understanding of the complex interactions between solvents and solutes.

4.3. Electrochemical Characterization

Electrochemical experiments were carried out on an INTERFACE 1010E Gamry potentiostat–galvanostat and its respective Gamry Instruments Framework electrochemical research software suitable for computer control and recording of current–potential (I–E) curves. All experiments were carried out at room temperature and in an anchor-type electrochemical cell using a homemade Ag/AgCl electrode referenced with the saturated calomel electrode (SCE) as the reference electrode and a spiral-shaped platinum wire as the auxiliary electrode and as the working electrode, and in all cases, a platinum disk of 2 mm diameter was used. The electrolyte solution was prepared using tetrabutylammonium hexafluorophosphate for electrochemical analysis ≥99.0% as the supporting electrolyte and acetonitrile grade HPLC (Merck) as the solvent.

4.4. Cytotoxicity Assays and Fluorescence Studies

To analyze the behavior of compounds in cells, cytotoxicity assays and fluorescence studies were conducted in cell culture. Normal cells (BHK-21) and prostate cancer cells (PC3 and LNCaP) were used. All assays were carried out in RPMI 1640 medium, with 10% FBS and 2% penicillin–streptomycin at 37 °C with 5% CO₂ and complete humidity.^80,81

To evaluate the cytotoxic effect of the compounds, the cell viability assay using the MTT reductase assay was employed, and 1 × 10⁴ cells were seeded in each well of a 96-well plate and incubated for 24 h in RPMI 1640 medium with 10% FBS and 2% penicillin–streptomycin. After the cell adherence period, the wells were treated with different concentrations (60.0, 30.0, 15.0, 7.5, 3.8, 1.9 μM) of compounds (4 and 5a, 5b). They were incubated for 24 h at 37 °C, 5% CO₂. Once the incubation time was completed, the medium was removed, and the wells were washed with PBS (pH: 7.4). A solution of MTT (0.5 mg/mL) diluted in serum-free RPMI was used. It was incubated again for 2–3 h to allow formazan formation. The medium was removed, and 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan. Once it was completely dissolved, the absorbance was measured at a wavelength of 570 nm using a microplate reader (FLUOstar Omega). Data analysis was performed considering the negative control.^82,83 The fluorescence study was conducted using a motorized, inverted fluorescence microscope (Zeiss Axio-observer Z1) to visualize cells at 40× magnification, and images were captured using an Axiocam HRm. 5 ×10⁴ cells were seeded in 24-well plates. Fifty thousand cells per well were grown in RPMI medium supplemented with 10% FBS and 2% penicillin–streptomycin at 37 °C and 5% CO₂. Subsequently, cell cultures were treated with the respective compound at 10 μM. Cells were imaged with a Zeiss Axio Observer Z1 motorized inverted fluorescence microscope at 40× magnification using the following filter cubes: FITC (set 10: excitation bandpass: 450–490 nm; emission bandpass: 515–565 nm), rhodamine (set 20: excitation bandpass: 546 ± 6 nm; emission bandpass: 575–640 nm), Cy5 (set 26: excitation bandpass: 575–625 nm; emission bandpass: 660–710 nm), DAPI (set 49: excitation: G365 nm; emission bandpass: 445 ± 25 nm), fields were selected, and images were taken for 5 and 15 min using a 40x objective and excitation channels at 488, 460, 520, and 660 nm.^84,85

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.3c07242.

• Absorption and emission normalized spectra of all compounds in a THF solution (S1), Lippert–Mataga and Rettig plots (S2). Emission spectra of 4, 5a, and 5b in THF with TFA (S3) and TEA and ¹H NMR and ¹³C NMR spectra of all compounds (Figures S4–S6) (PDF)

The authors declare no competing financial interest.