Design and Synthesis of New Acridone-Based Nitric Oxide Fluorescent Probe

Nitric oxide (NO) is an important signaling molecule involved in a wide range of physiological and pathological processes. Fluorescent imaging is a useful tool for monitoring NO concentration, which could be essential in various biological and biochemical studies. Here, we report the design of a novel small-molecule fluorescent probe based on 9(10H)acridone moiety for nitric oxide sensing. 7,8-Diamino-4-carboxy-10-methyl-9(10H)acridone reacts with NO in aqueous media in the presence of O2, yielding a corresponding triazole derivative with fivefold increased fluorescence intensity. The probe was shown to be capable of nitric oxide sensing in living Jurkat cells.


Introduction
Being a unique molecule, nitric oxide (II) plays a vital role in many physiological processes. Nowadays, nitric oxide has been shown to take part in neurotransmission, vasodilatation, smooth muscle tone regulation, immune system reaction, and so forth [1][2][3][4][5]. It means that lack or excess of it will inevitably lead to pathological states, such as cardiovascular disease, neurodegeneration, or even the formation of tumors [6]. Hence, monitoring the level and transformation pathways of nitric oxide is crucial for normal body functioning and for the further understanding of nitric oxide's biological role.
The fluorescent microscopy technique, involving fluorescent probes specifically reacting with NO, allows one to achieve the abovementioned tasks. The design of the majority of such fluorescent probes is based on the conjugation of fluorophore with phenylene-1,2-diamine moiety, which undergoes oxygen-promoted reaction with NO forming the triazole ring. Such transformation causes significant growth of fluorescence quantum yield by blocking the photoinduced electron transfer [7,8]. To this day, a wide range of NO probes utilizing the abovementioned principle have been reported with BOD-IPY [9], coumarin [10,11], fluorescein [12], rhodamine [13,14], and other fluorophores. Several commercially available substances, such as DAN, DAR, or DAF (Figure 1), have found wide applications for the detection and imaging of NO in living cells [15]. 1,2-Diaminoanthraquinone (DAQ) is also a commercially available and cheap sensor that gives significant fluorescence intensity enhancement upon reaction with NO [16,17]. However, DAQ has several disadvantages, such as poor solubility in water and most organic solvents of a parent molecule and the triazole derivative and difficulties of modification. In order to expand the present range of fluorescent probes, we paid attention to acridone scaffold as the close analog of anthraquinone. Recently, several NO probes based on the acridine core has been reported [18,19]. Acridones are commonly available by intramolecular condensation of N-aryl-ortho-aminobenzoic acids [20] and could be easily modified at the N(10) atom [21,22]. We hypothesized that the incorporation of the o-diamine fragment along with the carboxylic group would improve water solubility.

Spectral Properties and NO Trapping
Both diaminoacridones 6 and 13 were characterized by UV-VIS and fluorescence spectroscopy, and their ability of nitric oxide sensing was studied. Figure 2a shows the absorption spectra of 6 in aqueous solution with 1% DMSO before and after reaction with the excess of diethylamine NONOate diethylammonium salt (NONOate) for 10 min. Acridone 6 has low absorbance in the visible region, and spectral changes upon reaction with NONOate were insignificant. The changes in fluorescence maximum and intensity were also subtle ( Figure 2b). Moreover, our attempts to obtain triazole by the diazotation of 6 were unsuccessful, and only complex reaction mixtures were obtained. Compound 13 has an absorption maximum at 447 nm (Table 1), which falls within the most widespread excitation ranges of modern fluorescent microscopes (450-490 nm). The absorbance curve after the addition of NONOate significantly changed, indicating that structural changes occurred. As shown in Figure 3b, fluorescence intensity increased by a factor of 5 after reacting with nitric oxide, with the fluorescence maximum shifted to 495 nm from 564 nm. Triazole 14a, which was obtained by the diazotation of 13 (Scheme 3), has weaker absorption in a visible region and a 7.6-fold increased fluorescence quantum yield compared with 13. Unfortunately, molecular ion [14a] +· was not observed by HRMS probably due to fast decomposition by the ejection of a N 2 molecule, so we prepared the methylated analog 14b, which has been well characterized by HRMS.

NO Imaging in Jurkat Cells
To demonstrate the applicability of 13 for the imaging of NO in living cells, we used the Jurkat cell line. A solution of 10 µM of 13 in water + 0.1% DMSO was added to the culture dish. After 15 min of incubation, cells were placed under a fluorescent microscope for observation. The standard filter set was used (excitation: 450-490 nm; emission: >515 nm). Figure 4A shows an image of Jurkat cells with bright fluorescence, which is the sign of 13 inside cells. The distribution of fluorescence in cells were inhomogeneous, as can be seen in Figure 4B. After the addition of 6 mM of NONOate, the fluorescence increase was visible. Figure 4C shows the same cell after 3 min. It justifies that 13 reacts with NO inside cells as well.

General Remarks
All chemicals were purchased from commercial sources and used without additional purification, unless otherwise noted. The progress of reactions was monitored by thinlayer chromatography (TLC) on Silica gel 60 F254 aluminum sheets. NMR spectra were recorded on Bruker Avance-300 (300.13 MHz for 1 H) and Avance-400 (400.13 MHz for 1 H, 100.62 MHz for 13 C) spectrometers; chemical shifts of 1 H and 13 C{ 1 H} are given in ppm, with solvent signals serving as the internal standard ( 1 H = 7.24 ppm and 13 C = 77.16 ppm for CDCl 3 ; 1 H = 2.50 ppm and 13 C = 39.52 ppm for DMSO-d 6 ). UV-VIS spectra were registered on a Shimadzu UV-1900 spectrophotometer, and fluorescence spectra were obtained on a Shimadzu RF-6000 fluorometer for 10 −4 M solutions in 1% DMSO in H 2 O. Quantum yields were determined according the standard procedure with rhodamine 6G (10 −6 M solution in H 2 O) as the standard. Masses of molecular ions were determined by high-resolution mass spectrometry (HRMS) by means of a DFS Thermo Scientific instrument (EI, 70 eV) or by using a Bruker micrOTOF-Q for ESI experiments. An inverted fluorescent microscope Carl Zeiss Axio Vert.A1 equipped with an Axiocam 503 mono high-sensitive camera was used in this study for the observation of Jurkat cells. The cells were grown in standard conditions: IMDM basic medium supplemented with L-glutamine, 25 mM HEPES, fetal bovine serum, and gentamicin in an incubator at 37 • C and 5% CO 2 . 9-Acridone and 4-carboxy-9(10H)acridone were synthesized according to the literature methods [23,24]. The spectra for synthesized compounds can be found in the Supplementary Materials. Synthesis of 10-(carboxymethyl)-9(10H)acridone ethyl ester (1). To a stirred solution of 1.87 g of 9-acridone (9.57 mmol) in 10 mL of DMF at 60 • C, 0.57 g (14.4 mmol) of NaH was added. The mixture was stirred for additional 15 min, and then 2.07 g (12.5 mmol) of ethyl bromoacetate was added. The reaction mixture was stirred for 3 h, cooled to room temperature, and poured in water. The obtained precipitate was filtered off and washed a few times with water. The obtained solid was recrystallized using ethanol as a solvent. Pale yellow solid. Yield, 1.48 g (55%). 1  ). An NMR spectrum is in good agreement with a literature one [25].
Synthesis of 2-nitro-10-(carboxymethyl)-9(10H)acridone ethyl ester (2). An amount of 0.670 mL (15 mmol) of nitric acid was added to a mixture of 1.4 g (5 mmol) of compound 1, followed by 3.3 mL (35 mmol) of acetic anhydride in 15 mL of acetic acid. The mixture was stirred for 1.5 h at 60 • C. The resulting mixture was cooled to room temperature and neutralized with 10% NaOH solution. The formed precipitate was filtered off and washed a few times with water. Yellow solid. Yield  Synthesis of 2-acetamide-1-nitro-10-(carboxymethyl)-9(10H)acridone ethyl ester (4). An amount of 0.34 g of compound 3 (1.27 mmol) was added to a mixture of 10 mL of acetic acid and 0.6 mL of acetic anhydride. The reaction mixture was stirred for 1.5 h at 60 • C. After the reaction completion, the mixture was cooled to room temperature and diluted with water until the precipitate was formed. The formed precipitate was filtered off and washed with water. Yellow solid, used without purification. Yield, 0.237 g (55%). 1  A mixture of the obtained 0.2 g (0.59 mmol) of acetamide, 0.3 mL (3 mmol) of acetic anhydride, and 0.06 mL (1.18 mmol) of nitric acid in 5 mL of acetic acid was stirred at 60 • C until reaction completion by TLC. The resulting mixture was cooled to room temperature, poured into water, and neutralized with sodium hydroxide solution. The formed precipitate was filtered off and washed a few times with water. The obtained solid was recrystallized using ethanol. Yellow solid. Yield, 0.097 g (40%). 1  Synthesis of 1,2-diamino-10-(carboxymethyl)-9(10H)-acridone (6). A mixture of 0.040 g (0.13 mmol) of compound 5, 0.072 mL (1.92 mmol) of formic acid, 0.230 mL of TEA, and 0.02 g of Pd/C in 10 mL of ethanol was refluxed for 2 h. The resulting mixture was filtered off while hot, and Pd/C was washed with hot ethanol. The solvent was evaporated at low pressure. The residue was diluted with 5% hydrochloric solution and neutralized with 5% NaHCO 3 solution. The formed precipitate was filtered off and washed with water. Dark solid. Yield, 0.021 g (60%). 1  Synthesis of methyl 10-methyl-9-oxo-9,10-dihydroacridine-4-carboxylate (8). An amount of 2.3 g (9.6 mmol) of 4-carboxy-(9,10H)acridone was suspended in 30 mL of DMF. Then 1.52 g (28.8 mmol) of NaH was slowly added with continuous stirring. After the addition of NaH, 4.1 g (28.8 mmol) of MeI was added dropwise to the mixture. The reaction mixture was stirred for 2 h at room temperature until completion by TLC. The reaction mixture was diluted with water until the residue was formed. The obtained residue was filtrated and washed a few times with water. If the purity of the product was not satisfying, it could be recrystallized from ethanol. Green solid. Yield, 2.1 g (82%). M.p. 164.8 • C. 1  Synthesis of methyl 10-methyl-7-nitro-9-oxo-9,10-dihydroacridine-4-carboxylate (9). An amount of 2 g (7.5 mmol) of compound 8 was suspended in 45 mL of glacial acetic acid. The mixture was heated to 60 • C, and a mixture of 6.67 mL of nitric acid and 6.67 mL of acetic acid was added. The reaction was stirred until completion by TLC. The reaction mixture was poured in water with ice. The obtained precipitate was filtrated and thoroughly washed with water. The obtained solid contained 2-nitro isomer and traces of 5-nitro isomer. Yellow solid, used without purification. Yield of all isomers, 1.68 g (72%). 1  Synthesis of methyl 7-amino-10-methyl-9-oxo-9,10-dihydroacridine-4-carboxylate (10). A mixture of 1.25 g (0.004 mol) of 9, 0.43 g of Pd/C, and 2 mL (0.04 mol) of N 2 H 4 ·H 2 O in 75 mL EtOH was refluxed with stirring for 1 h. Then the reaction mixture was filtrated while hot, and Pd/C was washed a few times with hot ethanol. The filtrate was evaporated in vacuo, and the obtained precipitate was dried on air. Orange solid, which contained impurities (approximately 20%), used without purification. Yield, 0.88 g (78%). 1  Synthesis of methyl 7-acetamido-10-methyl-8-nitro-9-oxo-9,10-dihydroacridine-4-carboxylate (11). To a solution of 0.6 g (2.13 mmol) of 10 in 12 mL of glacial acetic acid, 1 mL of Ac 2 O was added. The reaction mixture was stirred at 60 • C for 1 h, and then a mixture of 1.9 mL of nitric acid and 1.9 mL of glacial acetic acid was added. The reaction was monitored by TLC, until initial the 10 disappeared. After completion, the reaction mixture was poured in water with ice, and the formed precipitate was filtrated and thoroughly washed with water. Yellow solid with impurities (approximately 20%), used without purification. Yield, 0.58 g (74%). 1  Synthesis of 7-amino-10-methyl-8-nitro-9-oxo-9,10-dihydroacridine-4-carboxylic acid (12). An amount of 0.560 g of 11 was added to 37.5 mL of 4N NaOH solution and refluxed until complete dissolution of the initial precipitate. The mixture was cooled to room temperature and acidified with HCl to pH~7. The formed precipitate was filtrated and thoroughly washed with water. Dark brown solid, obtained as pure. Yield, 0.347 g (73%). Decomposes without melting at >150 • C. 1

Conclusions
In summary, a new fluorescent probe, 7,8-diamino-4-carboxy-10-methyl-9(10H)acridone, for nitric oxide sensing based on acridone moiety has been reported. Its spectroscopic properties fall within the most widespread excitation ranges of modern fluorescent microscopes. The probe has shown the ability to trap nitric oxide with a fluorescence increase. Furthermore, the probe has been shown to be capable of sensing nitric oxide in Jurkat living cells. In contrast to a commercial DAQ probe, the described diaminoacridone demonstrates solubility in water media. Furthermore, the suggested synthetic protocol allows simple modification at the N(10) atom.