Synthesis and Characterization of Sulfonamide-Containing Naphthalimides as Fluorescent Probes

A tumor-targeting fluorescent probe has attracted increasing interest in fluorescent imaging for the noninvasive detection of cancers in recent years. Sulfonamide-containing naphthalimide derivatives (SN-2NI, SD-NI) were synthesized by the incorporation of N-butyl-4-ethyldiamino-1,8-naphthalene imide (NI) into sulfonamide (SN) and sulfadiazine (SD) as the tumor-targeting groups, respectively. These derivatives were further characterized by mass spectrometry (MS), nuclear magnetic resonance spectroscopy (1H NMR), Fourier transform infrared spectroscopy (FT-IR), ultraviolet–visible spectroscopy (UV), and a fluorescence assay. In vitro properties, including cell cytotoxicity and the cell uptake of tumor cells, were also evaluated. Sulfonamide-containing naphthalimide derivatives possessed low cell cytotoxicity to B16F10 melanoma cells. Moreover, SN-2NI and SD-NI can be taken up highly by B16F10 cells and then achieve good green fluorescent images in B16F10 cells. Therefore, sulfonamide-containing naphthalimide derivatives can be considered to be the potential probes used to target fluorescent imaging in tumors.


Introduction
In recent years, fluorescent imaging has attracted increasing interest in a potential medical diagnostics modality for noninvasive detection in cancers [1][2][3].Although fluorescent imaging in thick tissue layers is hampered by low spatial resolution induced by strong light scattering and the absorption of tissues, the recent advances in instrumentation and fluorescent probes have led to the application of fluorescent imaging to be extended from preclinical research to the clinical detection of cancers [4][5][6].
Some compounds, such as indocyanine green (ICG), 5-aminolevulinic acid (5-ALA), and 1,8-Naphthalimide, are evaluated as potential fluorescent probes in FI to improve imaging contrast and sensitivity [7][8][9].1,8-Naphthalimide is a fluorescent compound generally used as an organic dye, luminophore, and anticancer agent [10].However, these widely used fluorescent probes with a low molecular weight are excreted from the body quickly and usually demonstrate no tumor-targeting biodistribution, which possess significant restrictions for the imaging of tumors.So, a tumor-targeting fluorescent probe is an ideal solution to demonstrate a favorable signal-to-background ratio and yield quantitative and spatially and temporally indexed information on both normal tissue and tumors [11].
Sulfonamide (SN) and sulfadiazine (SD) derivatives have been investigated as tumortargeting groups due to their potent and highly selective carbonic anhydrase (hCA IX) inhibitors, which are well-known transmembrane CA isoforms.These CA isoforms are highly expressed in different tumor types and present a rather limited expression in most normal cells [20,21].Therefore, SN and SD derivatives were reported as the active ingredients in treating cancer and suppressing metastasis.SD derivatives were concentrated into Walker carcinoma or Yoshida sarcoma with 2-3 times as large as the concentration in the liver [21][22][23].Gadolinium diethylenetriaminepentaacetic acid derivatives containing SD and SN groups were investigated as the potential tumor-targeting contrast agents and possessed high uptake by Hepatoma and Ehrlich ascites carcinoma in mice [24].Polyaspartamide gadolinium complexes containing SD groups also had specific uptake by Hepatoma and greatly enhanced the contrast of magnetic resonance images of Hepatoma in mice [25].
In this work, SD and SN were chosen as the tumor-targeting groups and then incorporated to the N-butyl-4-ethyldiamino-1,8-naphthalimide (NI) [26,27] fluorescent molecule to synthesize tumor-targeting fluorescent probes (Scheme 1).These fluorescent probes were further characterized, and their properties in vitro were also evaluated to find a potential to achieve highly sensitive imaging in tumor cells with high contrast enhancement.
Sulfonamide (SN) and sulfadiazine (SD) derivatives have been investigated as tumor-targeting groups due to their potent and highly selective carbonic anhydrase (hCA IX) inhibitors, which are well-known transmembrane CA isoforms.These CA isoforms are highly expressed in different tumor types and present a rather limited expression in most normal cells [20,21].Therefore, SN and SD derivatives were reported as the active ingredients in treating cancer and suppressing metastasis.SD derivatives were concentrated into Walker carcinoma or Yoshida sarcoma with 2-3 times as large as the concentration in the liver [21][22][23].Gadolinium diethylenetriaminepentaacetic acid derivatives containing SD and SN groups were investigated as the potential tumor-targeting contrast agents and possessed high uptake by Hepatoma and Ehrlich ascites carcinoma in mice [24].Polyaspartamide gadolinium complexes containing SD groups also had specific uptake by Hepatoma and greatly enhanced the contrast of magnetic resonance images of Hepatoma in mice [25].
In this work, SD and SN were chosen as the tumor-targeting groups and then incorporated to the N-butyl-4-ethyldiamino-1,8-naphthalimide (NI) [26,27] fluorescent molecule to synthesize tumor-targeting fluorescent probes (Scheme 1).These fluorescent probes were further characterized, and their properties in vitro were also evaluated to find a potential to achieve highly sensitive imaging in tumor cells with high contrast enhancement.

Synthesis
Experimental data, such as FT-IR, 1 H NMR, UV, MS, and a fluorescent assay, provided evidence for the formation ofsulfonamide-containing naphthalimides (SN-2NI and SD-NI) (Figures 1-5), which were synthesized by the incorporation of a NI fluorescent molecule to SN and SD as the tumor-targeting groups (Scheme 1).The MS spectra of SN-2NI displayed the correct [M+H] + peak of 875.03 in accordance with the molecular weight of SN-2NI (MW 874) (Figure 1a).The MS spectra of SD-NI also indicated the correct peak of [M+H] + (602.39), which was consistent with the molecular weight of SD-NI (MW 601) (Figure 1b).

Synthesis
Experimental data, such as FT-IR, 1 H NMR, UV, MS, and a fluorescent assay, provided evidence for the formation ofsulfonamide-containing naphthalimides (SN-2NI and SD-NI) (Figures 1-5), which were synthesized by the incorporation of a NI fluorescent molecule to SN and SD as the tumor-targeting groups (Scheme 1).The MS spectra of SN-2NI displayed the correct [M+H] + peak of 875.03 in accordance with the molecular weight of SN-2NI (MW 874) (Figure 1a).The MS spectra of SD-NI also indicated the correct peak of [M+H] + (602.39), which was consistent with the molecular weight of SD-NI (MW 601) (Figure 1b).fore, SN-2NI and SD-NI are expected to retain the same photophysical properties their predecessors (NI) to make good fluorescent imaging.Moreover, they should p sess a good tumor-targeting property to achieve highly sensitive imaging in tumor c with high contrast enhancement.

Cell Cytotoxicity
The effect of sulfonamide-containing naphthalimide derivatives (SN-2 to B16F10 cell growth and metabolism is shown in Figure 6.At aconcentrat of SN-2NI and SD-NI in growth medium, the viabilities of B16F10 cells in SN-2NI and SD-NI retained 69.7% and 53.0%, respectively, relative to the c concentration (100 µg/mL) of sulfonamide-containing naphthalimides in medium, the viabilities of B16F10 cells incubated with SN-2NI and SD-NI r and 45.2%, respectively.This illustrated that SN-2NI and SD-NI possessed city to B16F10 cells and SN-2NI displayed slightly higher cytotoxicity to B16

Cell Cytotoxicity
The effect of sulfonamide-containing naphthalimide derivatives (SN-2NI and SD-NI) to B16F10 cell growth and metabolism is shown in Figure 6.At aconcentration (1 µg/mL) of SN-2NI and SD-NI in growth medium, the viabilities of B16F10 cells incubated with SN-2NI and SD-NI retained 69.7% and 53.0%, respectively, relative to the control.At the concentration (100 µg/mL) of sulfonamide-containing naphthalimides in the growth medium, the viabilities of B16F10 cells incubated with SN-2NI and SD-NI retained 57.3% and 45.2%, respectively.This illustrated that SN-2NI and SD-NI possessed low cytotoxicity to B16F10 cells and SN-2NI displayed slightly higher cytotoxicity to B16F10 cells than that of SD-NI.
SD and SN are the antibacterial agents and anti-inflammatory drugs used in clinics.Moreover, SD and SN possess good tumor-targeting properties, and then SN-2NI and SD-NI can be selectively taken up by the tumor cells.So, SN-2NI and SD-NI selectively accumulated into B16F10 cells and exhibited obviously high anticancer efficiencies to B16F10 cells when the incubation concentration increased.The 1 H NMR spectra of SN-2NI and SD-NI indicated the typical peaks of naphthalimide ring and methyl and benzyl groups, which appeared at 8.9-8.0 ppm, 1.65-0.8ppm, and 7.7-7.4ppm, respectively (Figure 2a,b), indicating that NI was covalently bound to SN and SD, respectively.The IR spectra of SN-2NI and SD-NI showed the characteristic absorption peaks of amide bond (CONH)with 1640-1570 cm −1 , amino group (NH) at 3450-3420 cm −1 , and benzyl groups varying from 1150 to 660 cm −1 , respectively (Figure 3a,b), which indicated that both SN and SD were covalently bound to NI.
SN-2NI and SD-NI displayed similar UV and fluorescent properties as well as NI.The UV spectra of SN-2NI and SD-NI showed the characteristic UV absorption peaks of SD, SN, and NI structures, appearing at 265-267 and 417-435 nm (Figure 4), whist the fluorescent spectra of SN-2NI and SD-NI showed the same characteristic fluorescent property as NI.Meanwhile, SN-2NI and SD-NI had a maximum excitation wavelength at 437 nm and a maximum emission wavelength at 525 nm (Figure 5), respectively.It appears that the typical fluorescent emission wavelength was similar to that of NI.Therefore, SN-2NI and SD-NI are expected to retain the same photophysical properties as their predecessors (NI) to make good fluorescent imaging.Moreover, they should possess a good tumor-targeting property to achieve highly sensitive imaging in tumor cells with high contrast enhancement.

Cell Cytotoxicity
The effect of sulfonamide-containing naphthalimide derivatives (SN-2NI and SD-NI) to B16F10 cell growth and metabolism is shown in Figure 6.At a concentration (1 µg/mL) of SN-2NI and SD-NI in growth medium, the viabilities of B16F10 cells incubated with SN-2NI and SD-NI retained 69.7% and 53.0%, respectively, relative to the control.At the concentration (100 µg/mL) of sulfonamide-containing naphthalimides in the growth medium, the viabilities of B16F10 cells incubated with SN-2NI and SD-NI retained 57.3% and 45.2%, respectively.This illustrated that SN-2NI and SD-NI possessed low cytotoxicity to B16F10 cells and SN-2NI displayed slightly higher cytotoxicity to B16F10 cells than that of SD-NI.
Molecules 2024, 29, x FOR PEER REVIEW Figure 6.In vitro cytotoxicity assay of SN-2NI and SD-NI to B16F10 cells.

Fluorescent Imaging
The fluorescent imaging of B16F10 cells cultured with SN-2NI, SD-NI, an growth medium wasinvestigated, respectively,when excited by white light (com excitation wavelength: 400-750 nm, visible light) and blue light (excitation wave 450 nm) (Figure 7).The cells cultured with SN-2NI and SD-NI showed obviousl green fluorescent images when excited by blue light.However, cells culture growth medium indicated no fluorescent image in the same condition.Therefore, S and SD-NI can be taken highly upby B16F10 cells due to the tumor-targeting prop SN and SD groups.SD and SN are the antibacterial agents and anti-inflammatory drugs used in clinics.Moreover, SD and SN possess good tumor-targeting properties, and then SN-2NI and SD-NI can be selectively taken up by the tumor cells.So, SN-2NI and SD-NI selectively accumulated into B16F10 cells and exhibited obviously high anticancer efficiencies to B16F10 cells when the incubation concentration increased.

Fluorescent Imaging
The fluorescent imaging of B16F10 cells cultured with SN-2NI, SD-NI, and pure growth medium was investigated, respectively, when excited by white light (composite excitation wavelength: 400-750 nm, visible light) and blue light (excitation wavelength: 450 nm) (Figure 7).The cells cultured with SN-2NI and SD-NI showed obviously good green fluorescent images when excited by blue light.However, cells cultured with growth medium indicated no fluorescent image in the same condition.Therefore, SN-2NI and SD-NI can be taken highly upby B16F10 cells due to the tumor-targeting property of SN and SD groups.

Cell Uptake and Fluorescent Imaging
The cell uptake assay and fluorescent imaging of SN-2NI and SD-NI were evaluated to B16F10 cells excited by white light (composite excitation wavelength: 400-750 nm, visible light) and blue light (excitation wavelength: 450 nm) (Figures 8 and 9).Good green fluorescent images can be observed obviously in cells incubated with SN-2NI and SD-NI excited by blue light (Figures 8B2 and 9B5).However, cells incubated with pure growth medium displayedno fluorescent imaging in the same condition (Figures 8B1 and 9B4).
B16F10 cells indicateda significantly lower intensity of green fluorescent images than that of the original cell uptake assaywhen they were incubated previously by SN or SD solution for 1 h, respectively, and by SN-2NI or SD-NI solutions (0.25 µmol/L) later.It is likely that SN or SDcovered up earlysome receptor-binding affinity ofSN-2NI and SD-NI in tumor cells.After that, SN-2NI or SD-NI cannot be taken up more or be effectively internalized again by cells.Therefore, SN-2NI and SD-NI possessed good tumor-targeting and characteristic green fluorescent imaging in B16F10 cells via the tumor-targeting property of the SN and SD groups.

Cell Uptake and Fluorescent Imaging
The cell uptake assay and fluorescent imaging of SN-2NI and SD-NI were evaluated to B16F10 cells excited by white light (composite excitation wavelength: 400-750 nm, visible light) and blue light (excitation wavelength: 450 nm) (Figures 8 and 9).Good green fluorescent images can be observed obviously in cells incubated with SN-2NI and SD-NI excited by blue light (Figures 8B2 and 9B5).However, cells incubated with pure growth medium displayed no fluorescent imaging in the same condition (Figures 8B1 and 9B4).
B16F10 cells indicated a significantly lower intensity of green fluorescent images than that of the original cell uptake assaywhen they were incubated previously by SN or SD solution for 1 h, respectively, and by SN-2NI or SD-NI solutions (0.25 µmol/L) later.It is likely that SN or SD covered up earlysome receptor-binding affinity ofSN-2NI and SD-NI in tumor cells.After that, SN-2NI or SD-NI cannot be taken up more or be effectively internalized again by cells.Therefore, SN-2NI and SD-NI possessed good tumor-targeting and characteristic green fluorescent imaging in B16F10 cells via the tumortargeting property of the SN and SD groups.

Materials
N-Butyl-4-bromoacetyl ethyldiamino-1,8-naphthalimide (NI) [27], sodium sulfadiazine (SDNa), and sodium sulfonamide (SNNa2) [25] were synthesized by the methods cited in the literature.B16F10 mouse melanoma cells were provided by the China Center for Type Culture Collection of Wuhan University, China, and raised according to the method described in the literature [28].

Fluorescent Spectroscopy
Sulfonamide-containing naphthalimide derivatives (SN-2NI, SD-NI) were first dissolved in a minimum volume of CHCl 3 and then diluted with methanol to the desired concentration.Fluorescent spectra were recorded on a Varian Cary Eclipse fluorescence spectrophotometer (Varian, Palo Alto, CA, USA).

Figure 6 .
Figure 6.In vitro cytotoxicity assay of SN-2NI and SD-NI to B16F10 cells.

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Figure 7.In vitro fluorescent imaging of SN-2NI and SD-NI to B16F10 melanoma cells.

Figure 7 .
Figure 7.In vitro fluorescent imaging of SN-2NI and SD-NI to B16F10 melanoma cells.