Naphth[1,2-d]imidazoles Bioactive from β-Lapachone: Fluorescent Probes and Cytotoxic Agents to Cancer Cells

Theranostics combines therapeutic and imaging diagnostic techniques that are extremely dependent on the action of imaging agent, transporter of therapeutic molecules, and specific target ligand, in which fluorescent probes can act as diagnostic agents. In particular, naphthoimidazoles are potential bioactive heterocycle compounds to be used in several biomedical applications. With this aim, a group of seven naphth[1,2-d]imidazole compounds were synthesized from β-lapachone. Their optical properties and their cytotoxic activity against cancer cells and their compounds were evaluated and confirmed promising values for molar absorptivity coefficients (on the order of 103 to 104), intense fluorescence emissions in the blue region, and large Stokes shifts (20–103 nm). Furthermore, the probes were also selective for analyzed cancer cells (leukemic cells (HL-60). The naphth[1,2-d]imidazoles showed IC50 between 8.71 and 29.92 μM against HL-60 cells. For HCT-116 cells, values for IC50 between 21.12 and 62.11 μM were observed. The selective cytotoxicity towards cancer cells and the fluorescence of the synthesized naphth[1,2-d]imidazoles are promising responses that make possible the application of these components in antitumor theranostic systems.


Introduction
The multifunctionality of theranostic agents introduces several advantages for medicine, overcoming pharmacokinetic and selectivity issues of conventional therapy and diagnostic agents [1], while providing the image monitoring of pathology progression as well as the pharmacokinetic profile of the drug in the body [2].
The design of a theranostic agent requires a combination of different areas, such as chemistry, physics, nanotechnology, biochemistry, and engineering, with the aim of obtaining a multifunctional platform capable of performing non-invasive therapy and diagnosis of a pathological condition [3]. Typically, a theranostic agent is composed of (i) an imaging agent, (ii) a therapeutic molecule, (iii) a target-specific ligand, and (iv) a carrier. The diagnostic agent is a fundamental part of a theranostic system. It favors the non-invasive visualization of cellular and subcellular processes of a pathological condition Herein, it is reported the synthesis of different naphth [1,2-d]imidazoles, with modification of substituents at the C2 position of the naphthoimidazole ring, and the following evaluation of their photophysical and anticancer activity as a part of a strategy to provide a new class of materials with promising biomedical applicability.

Results and Discussion
Naphth [1,2-d]imidazoles IM1-IM7 were prepared in two steps (Scheme 1) from lapachol 1, in which the natural 1,4-naphthoquinone was extracted from the heartwood of trees of the genus Tabebuia. In the first step, β-lapachone (β-Lap 2) was obtained from the acid-catalyzed cyclization of lapachol 1 using sulfuric acid (H 2 SO 4 ). In the following step, the compounds IM1-IM7 were synthesized through the Debus-Radziszewski reaction, in a one-pot process between β-Lap 2 and the corresponding aldehyde, using ammonium acetate as a source of ammonia (Scheme 1). The reactions were established at 70 • C in acetic acid with a reaction time range of 0.5-4.0 h. The crude reactions were treated with sodium bisulfite (NaHSO 3 ), and the products were purified using column chromatography or recrystallization. The naphthoimidazoles returned yields in the range of 9.9 to 52.0%, and their structures were scrutinized by analyzing the 1D and 2D Nuclear Magnetic Resonance (NMR) spectra, mass spectroscopy, and Fourier Transform Infrared (FTIR).

Optical Properties-Studies of Absorption and Fluorescence Spectra
Fluorescent molecules with the ability to absorb ultraviolet radiation and to emit in a range of wavelengths greater than that absorbed, that is, in the visible region, are extremely important to biomedical applications [21]. The characteristic time for the fluorescence process (on the order of 10 −9 s) depends on the interaction of molecules with the surrounding environment, being attractive for the evaluation of several phenomena from the biophysical properties of molecules [21]. Thus, the photophysical study of the naphth [1,2-d]imidazoles IM1-IM7 synthesized was carried out to verify their potential to emission of molecules for potential use in theranostic systems. The data for Ultravioletvisible (UV-vis) absorption and fluorescence spectroscopy from naphth [1,2-d]imidazoles IM1-IM7 are summarized in Table 1.

Solvatochromism Study
The dispersed molecules in a specific solvent can interact with other molecules of a fluorophore, affecting their emissive properties [22]. This phenomenon is called solvatochromism and depends on factors such as the polarity of the solvent, hydrogen bonding ability, pH, and viscosity of the solvent [22,23]. Considering the application of fluorescent probes in living cells and tissues, the solvatochromism study can evaluate the sensitivity and selectivity of the new compounds [24].
The influence of the solvent on optical characteristics of the synthesized naphth [1,2d]imidazoles IM1-IM7 was evaluated from the solvatochromism study that considered four solvents: hexane, dichloromethane (CH2Cl2), dimethyl sulfoxide (DMSO), and methanol (CH3OH) ( Table S1). From the UV-vis absorption spectra of the naphtha [1,2d]imidazoles (for different solvents), it was possible to determine the most suitable solvents and wavelengths for further photophysical studies. The criteria considered solvents for naphth [1,2-d]imidazoles that presented positive solvatochromism, i.e., redshift with increasing polarity of the solvent.

Optical Properties-Studies of Absorption and Fluorescence Spectra
Fluorescent molecules with the ability to absorb ultraviolet radiation and to emit in a range of wavelengths greater than that absorbed, that is, in the visible region, are extremely important to biomedical applications [21]. The characteristic time for the fluorescence process (on the order of 10 −9 s) depends on the interaction of molecules with the surrounding environment, being attractive for the evaluation of several phenomena from the biophysical properties of molecules [21]. Thus, the photophysical study of the naphth[1,2-d]imidazoles IM1-IM7 synthesized was carried out to verify their potential to emission of molecules for potential use in theranostic systems. The data for Ultraviolet-visible (UV-vis) absorption and fluorescence spectroscopy from naphth [1,2-d]imidazoles IM1-IM7 are summarized in Table 1.

Solvatochromism Study
The dispersed molecules in a specific solvent can interact with other molecules of a fluorophore, affecting their emissive properties [22]. This phenomenon is called solvatochromism and depends on factors such as the polarity of the solvent, hydrogen bonding ability, pH, and viscosity of the solvent [22,23]. Considering the application of fluorescent probes in living cells and tissues, the solvatochromism study can evaluate the sensitivity and selectivity of the new compounds [24].
The influence of the solvent on optical characteristics of the synthesized naphth[1,2d]imidazoles IM1-IM7 was evaluated from the solvatochromism study that considered four solvents: hexane, dichloromethane (CH 2 Cl 2 ), dimethyl sulfoxide (DMSO), and methanol (CH 3 OH) (Table S1). From the UV-vis absorption spectra of the naphtha [1,2-d]imidazoles (for different solvents), it was possible to determine the most suitable solvents and wavelengths for further photophysical studies. The criteria considered solvents for naphth [1,2d]imidazoles that presented positive solvatochromism, i.e., redshift with increasing polarity of the solvent.
The naphth [1,2-d]imidazoles IM2 and IM6 showed a higher bathochromic shift for polar solvents, a strong influence of the increasing solvent polarity on the absorption spectrum of these derivatives. On the other hand, IM7 showed a positive bathochromic effect in comparison to hexane, a nonpolar solvent. The other naphthoimidazoles (IM1, IM3, IM4, and IM5) exhibited an increasing redshift in CH 2 Cl 2 (Table S1).

Fluorescence Spectroscopy Experiments
Considering the UV-vis absorption spectra of the naphth[1,2-d]imidazoles IM1-IM7, the excitation wavelength of 345 nm was chosen to obtain the fluorescence spectra. Analyzing the fluorescence emission spectra of compounds IM1-IM7, the emission was observed in the UV-vis region, between the λ emis values of 366 and 457 nm ( Figure 2). IM3 (λ emis 457 nm) and IM5 (λ emis 422 nm), emitted at lower energy wavelengths, shifted more to the blue region. On the other hand, IM1, emitted at a higher energy wavelength, shifted toward the violet region ( Figure 2).

Fluorescence Spectroscopy Experiments
Considering the UV-vis absorption spectra of the naphth[1,2-d]imidazoles IM1-IM7, the excitation wavelength of 345 nm was chosen to obtain the fluorescence spectra. Analyzing the fluorescence emission spectra of compounds IM1-IM7, the emission was observed in the UV-vis region, between the λemis values of 366 and 457 nm ( Figure 2). IM3 (λemis 457 nm) and IM5 (λemis 422 nm), emitted at lower energy wavelengths, shifted more to the blue region. On the other hand, IM1, emitted at a higher energy wavelength, shifted toward the violet region ( Figure 2).
In addition, IM2 (2.49 × 10 6 au) and IM3 (1.41 × 10 6 au) showed fluorescence emission intensities that were thirty-six and twenty-three times greater, respectively, than IM1 (6.90 × 10 4 au), indicating that the substitution at the carbon C2 of the naphthoimidazole ring favored the fluorescence emission of naphth [1,2-d]imidazoles IM1-IM7 ( Figure 2). The increase in the fluorescence evaluated by the substitution of the carbon C2 position is due to the increase in the conjugation of double bonds, providing more-effective intramolecular displacement of electrons [25,33].  In addition, IM2 (2.49 × 10 6 au) and IM3 (1.41 × 10 6 au) showed fluorescence emission intensities that were thirty-six and twenty-three times greater, respectively, than IM1 Molecules 2023, 28, 3008 5 of 13 (6.90 × 10 4 au), indicating that the substitution at the carbon C2 of the naphthoimidazole ring favored the fluorescence emission of naphth [1,2-d]imidazoles IM1-IM7 ( Figure 2). The increase in the fluorescence evaluated by the substitution of the carbon C2 position is due to the increase in the conjugation of double bonds, providing more-effective intramolecular displacement of electrons [25,33].
By comparison between the fluorescence of IM4, IM5, and IM6, a positive influence on fluorescence was observed in compounds containing electron-donor substituents in the aromatic ring located at C2 of the naphth [1,2-d]imidazoles with IM4 and IM5 substituted with 4-hydroxyphenyl and 4-dimethylaminophenyl, respectively, showing higher fluorescence intensity than IM6, substituted with 4-nitrophenyl. The higher fluorescence of IM4 and IM5, if compared with other fluorophores, is attributed to a possible intramolecular charge transfer (ICT) [34].
DAPI exhibits photophysical characteristics of absorption and emission (λ abs 340 nm and λ emis 453-461 nm) similar to the synthesized naphth [1,2-d]imidazoles. By comparison of the fluorescence emission of DAPI (1.13 × 10 5 au) to that of IM2 (2.49 × 10 6 au), IM3 (1.41 × 10 6 au), IM4 (1.36 × 10 6 au), and IM5 (7.04 × 10 5 au), it was observed that IM compounds also present more intense fluorescence in the blue region than DAPI. This may be due to the extension of the conjugated double-bond system of the synthesized naphth [1,2-d]imidazoles enhanced by the presence of the naphthalene system associated with the 2-substituted imidazole ( Figure 3).
Molecules 2023, 28, x FOR PEER REVIEW 5 of 13 By comparison between the fluorescence of IM4, IM5, and IM6, a positive influence on fluorescence was observed in compounds containing electron-donor substituents in the aromatic ring located at C2 of the naphth [1,2-d]imidazoles with IM4 and IM5 substituted with 4-hydroxyphenyl and 4-dimethylaminophenyl, respectively, showing higher fluorescence intensity than IM6, substituted with 4-nitrophenyl. The higher fluorescence of IM4 and IM5, if compared with other fluorophores, is attributed to a possible intramolecular charge transfer (ICT) [34].
DAPI exhibits photophysical characteristics of absorption and emission (λabs 340 nm and λemis 453-461 nm) similar to the synthesized naphth [1,2-d]imidazoles. By comparison of the fluorescence emission of DAPI (1.13 × 10 5 au) to that of IM2 (2.49 × 10 6 au), IM3 (1.41 × 10 6 au), IM4 (1.36 × 10 6 au), and IM5 (7.04 × 10 5 au), it was observed that IM compounds also present more intense fluorescence in the blue region than DAPI. This may be due to the extension of the conjugated double-bond system of the synthesized naphth[1,2-d]imidazoles enhanced by the presence of the naphthalene system associated with the 2-substituted imidazole ( Figure 3).

Stokes Shift
Fluorophores with large Stokes shiftsΔST) are considered promising fluorescent probes for application in vivo cell-imaging studies, since they could minimize the background fluorescence in live tissues [36][37][38][39][40]. The naphth[1,2-d]imidazoles IM1-IM7 presented ΔST between 20 and 103 nm ( Table 2). If compared with the structure and ΔST of naphth[1,2-d]imidazoles IM1-IM7, it was observed that the substituents at C2 affect the ability of this compound to be a fluorophore. IM1 has no substituents on C2 and was the one with the smallest ΔST, as well as the lowest fluorescence emission (6.90 × 10 4 au). IM3 presented the largest displacement, which is substituted at C2 with a naphthyl group. It was also observed that the introduction of electron-withdrawing substituents, such as nitrophenyl, produced naphthoimidazoles with narrow ΔST, as shown for IM6 (ΔST 20 nm) and IM7 (ΔST 48 nm).

Stokes Shift
Fluorophores with large Stokes shifts (∆ ST ) are considered promising fluorescent probes for application in vivo cell-imaging studies, since they could minimize the background fluorescence in live tissues [36][37][38][39][40]. The naphth[1,2-d]imidazoles IM1-IM7 presented ∆ ST between 20 and 103 nm ( Table 2). If compared with the structure and ∆ ST of naphth[1,2-d]imidazoles IM1-IM7, it was observed that the substituents at C2 affect the ability of this compound to be a fluorophore. IM1 has no substituents on C2 and was the one with the smallest ∆ ST , as well as the lowest fluorescence emission (6.90 × 10 4 au). IM3 presented the largest displacement, which is substituted at C2 with a naphthyl group. It was also observed that the introduction of electron-withdrawing substituents, such as nitrophenyl, produced naphthoimidazoles with narrow ∆ ST , as shown for IM6 (∆ ST 20 nm) and IM7 (∆ ST 48 nm).

Cytotoxicity Assay
The cytotoxic activity of the naphth[1,2-d]imidazoles IM1-IM7 was assessed through colorimetric MTT assay [41,42]. The ability of these compounds to inhibit cell growth against human glioblastoma (SNB-19), human colorectal carcinoma (HCT-116), and human promyelocytic leukemia (HL-60) cell lines was evaluated. The IC 50 was determined for those compounds, returning a percentage inhibition of cell growth above 75% in at least two tested cell lines. Thus, from all seven naphth [1,2-d]imidazoles tested (IM1-IM7), only IM3 displayed low growth inhibition against all cell lines and did not have the IC 50 calculated due to low cytotoxic activity. Doxorubicin was used as the positive control, and Molecules 2023, 28, 3008 6 of 13 cytotoxic activities were expressed as IC 50 for all the naphth [1,2-d]imidazoles in Table 2. The substances that displayed significant results against the cancer cell lines were also investigated against a nontumor cell line of murine fibroblast (L929) to evaluate their selectivity index (SI) ( Table 2). As shown in Table 2, most of the naphth [1,2-d]imidazoles are characterized by a certain degree of cytotoxicity against at least one of the malignant cell lines tested. The IC 50 data showed that IM1 was the least potent imidazole of the series, demonstrating the importance of the substitution at the C2 carbon of the naphth[1,2-d]imidazole ring for the cytotoxic activity against the tested cancer cell lines. If considering the comparison of the influence of substituents at the C2 position, a significant decrease in cell growth inhibition was observed for naphthoimidazole with a naphthyl ring at C2 (IM3), suggesting that the phenyl substituent at the C2 position of the naphthoimidazole ring is relevant for the evaluated activity.
Against glioblastoma cells (SNB-19), the most cytotoxic compound was IM5 (IC 50 21.05 µM). As for HCT-116 cells and leukemia cells (HL-60), the most active naphth [1,2d]imidazoles were IM6 and IM4, with IC 50 of 21.12 and 8.71 µM, respectively. Comparing the three most cytotoxic imidazoles (IM4, IM5, and IM6) for each cancer cell line tested, it was observed that all of them presented as a substituent at the C2 position a 4-substituted phenyl ring with an electronegative group: -OH, -N(CH 3 ) 2, and NO 2 , respectively. Thus, it is possible to suggest that the substituted phenyl group located at the C2 carbon of the naphthoimidazole ring improves cytotoxicity activity and promotes selectivity.
In addition, for the cytotoxic activity of IM6 and IM7 (Table 2) for each cancer cell line tested, it can be seen that the 2-nitrophenyl substituent group at the C2 carbon of the naphthoimidazole ring makes the compound less cytotoxic to the evaluated tumor cell lines. Comparing the cytotoxic activity against three cancer cell lines, the tested naphthoimidazoles showed to be more active against the leukemia cell line (HL-60), presenting IC 50 between 8.71 and 29.92 µM. Among them, IM4 was the most active compound, with an emphasis on its high selectivity for leukemic cells (SI > 41.67).
By considering that high ε Abs values combined with large ∆ ST are desirable for fluorescent probes [43] and the photophysical and cytotoxic properties of each naphth [1,2-d]imidazole, one can infer that IM3 (fluorescence intensity 1.41 × 10 6 au, ε Abs 1.57 × 10 4 M −1 cm −1 and ∆ ST 103 nm) fits as a good fluorescent probe; however, it showed low cytotoxicity on the cell lines tested. One can also infer that IM2 (fluorescence intensity 2.49 × 10 6 au; ε Abs 2.43 × 10 4 M −1 cm −1 and ∆ ST 60 nm) has photophysical properties that qualify it as a fluorescent probe, and it has cytotoxicity against HCT-116 (IC 50 21.69 µM) and selectivity (SI 4.83).

Materials
All chemicals were purchased from commercial suppliers and used without further purification. Melting points were determined through a PFM-II (Instrumentation MS Tecnopon ® ) melting-point apparatus. The purity of the compounds synthesized was determined by thin-layer chromatography (TLC) using several solvent systems of different polarities. Purification of these compounds was done by column chromatography. Infrared (IR) spectra were recorded on a PerkinElmer (model 10.4.00) spectrophotometer equipped with an Attenuated Total Reflectance ATR sampling unit. NMR spectra were recorded on a Bruker Ascend 400 spectrometer, operating at 400 MHz for 1 H NMR and 100 MHz for 13 C NMR. CDCl 3 and DMSO-d 6 were used as solvents with tetramethylsilane (TMS) as the internal standard; chemical shifts (δ) are given in ppm and coupling constants (J) in Hz. Mass spectra were recorded with a Bruker Daltonics (TOF-Q-II) spectrometer using electrospray ionization. UV-vis absorption spectra were obtained using a Hach/Lange spectrophotometer (model DR 5000). Fluorescence emission spectra were obtained using the ISS spectrofluorometer (model PC1).

Synthesis of Naphth
Lapachol 1 was extracted from the wood of a plant of the genus Tabebuia and used after purification and identification, as described previously [44]. The access was registered in the National System of Genetic Heritage and Associated Traditional Knowledge (SISGEN) under the A5FDA89. Yield: 1.5% (m/m). Yellow solid, mp: 138.3-140.3 • C (Lit 139.0-141.0 • C) [45].
In a 25 mL reaction flask, the lapachol (484 mg, 2 mmol) was weighed and incorporated into a concentrated sulfuric acid (H 2 SO 4 ) solution (5 mL). The reaction mixture was stirred at room temperature for 1.0 h, then poured into 400 mL of ice-cold deionized water. The solid obtained was vacuum filtered and allowed to dry at room temperature [44], which resulted in a yield of 95%. Orange solid, mp: 155 • C (Lit 154-155 • C) [46]. 1

General Synthesis of the Naphth[1,2-d]imidazoles
The solution of β-Lap 2 (242 mg; 1.0 mmol) was prepared in glacial acetic acid (6 mL), and was added aldehyde adequate (2.5 mmol). The reaction mixture was placed at a temperature of 70 • C and added to ammonium acetate (1.27 g; 16.5 mmol) that was divided into three parts and remained at this temperature under stirring until the end of the reaction [10]. The reactions were followed by Thin Layer Chromatography (TLC), and the reaction times varied in the range of 30 min to 4 h. In experiments using 4-dimethylaminobenzaldehyde and 4-nitrobenzaldehyde, there was a precipitate formation in the reaction. However, in experiments employing formaldehyde, benzaldehyde, 1-naphthaldehyde, 4-hydroxybenzaldehyde, and 2-nitrobenzaldehyde, there was no precipitate formation in the reaction. Then, after the reaction time, the reaction mixture was poured into a cold solution of 5.0% (m/v) of NaHSO 3 for precipitate formation. The solid was filtered and washed with a solution of 5.0% (m/v) of NaHCO 3 , and water was deionized at neutral pH and dried at room temperature.  3435, 3088, 2971, 2925, 2841, 1608, 1538, 1484, 1453, 1362, 1252, 1164, 1122, 1054, , and 770, Figure S1. thesis of 4,5-dihydro-6,6-dimethyl-6H-2-(phenyl)-pyran [b-4,3] Figure S3.

Obtaining Visible Ultraviolet Absorption Spectra
A stock solution in dichloromethane (CH 2 Cl 2 ) of each compound was prepared at a concentration of 4000 µM. From the stock solution, solutions were prepared at a concentration of 20 µM of each compound in four different solvents: hexane, CH 2 Cl 2 , dimethyl sulfoxide (DMSO), and methanol (CH 3 OH). Then, measurement in the range of 190 to 800 nm was performed, with the wavelengths of maximum absorption (λ max ) of the compounds in the different solvents shown in Table S1 and Figure S8.

Molar Absorptivity Coefficient
The molar absorptivity coefficient (ε Abs ) was determined using an equation applied by Lambert-Beer (ε Abs = A/LxC, where A-maximum absorbance; L-the optical path of the cuvette used (1 cm); and C-concentration of the analyzed sample in M).

Fluorescence Emission Spectrum and Stokes Shift
Stock solutions used for each compound, at a concentration of 4000 µM, in the solvents in which the sample showed better resolution of the maximum absorption band, were DMSO for IM2, CH 3 OH for IM6, hexane for IM7, and CH 2 Cl 2 for the others. The stock solutions were diluted to a concentration of 20 µM, and readings used the excitation wavelength of 345 nm for all compounds. The Stokes shift (∆ ST ) was calculated from the difference between the absorbance and excitation wavelengths (λ Abs -λ Emis ), Figure S8.

Assessment of In Vitro Anticancer Activity
Cytotoxic potential of the naphth[1,2-d]imidazoles IM1-IM7 was assessed after 72 h of exposure to the tumor cell lines of human SNB-19, HCT-116, HL-60, and normal cell line L929. Cells were plated in 96-well plates (0.7 × 10 5 cells/well for SNB-19, 0.3 × 10 6 cells/well for HCT-116, and 0.3 × 10 6 cells/well for HL-60). Compounds were dissolved with DMSO at concentrations in the 0.078-10 µg.mL −1 range. Doxorubicin (0.001-1.10 µM) was used as the positive control, and negative control groups received the same amount of vehicle (DMSO). The cell viability was determined by the reduction of the yellow dye 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a blue formazan product [35]. At the end of the incubation time (69 h), the plates were centrifuged, and the medium was replaced with fresh medium (200 µL) containing 0.5 mg/mL of MTT. Three hours later, the MTT formazan product was dissolved in DMSO (150 µL), and the absorbance was measured using a multi-plate reader (Spectra Count, Packard, ON, Canada). The drug effect was quantified as the percentage of control absorbance of the reduced dye at 550 nm. All experiments were performed in three independent assays, and the half maximal inhibitory concentration (IC 50 ) and their 95% confidence intervals were achieved by nonlinear regression.

Conclusions
Naphth[1,2-d]imidazoles IM1-IM7 showed high levels of cytotoxic activity and selectivity against the tested cancer cells and promising optical properties. The cytotoxicity results and photophysical properties presented by naphth [1,2-d]imidazoles IM2, IM3, IM4, and IM5 qualify them for further studies in the development of fluorescent anticancer probes using this scaffold, making possible the use of naphth [1,2-d]imidazoles as fluorescent probes/therapeutic molecules in theranostic systems for cancer treatment/diagnosis.