Design and Synthesis of an Azo Reductase Responsive Flavonol–Indomethacin Hybrid Used for the Diagnosis and Treatment of Colitis

Human intestinal bacteria are the primary producers of azo reductase, and the content of azo reductase is closely associated with various intestinal diseases, including ulcerative colitis (UC). The rapid detection of changes in azo reductase levels is crucial for diagnosing and promptly intervening in UC. In this study, a therapeutic agent, FAI, specifically targeting UC, was designed and synthesized. This agent was developed by linking the anti-inflammatory drug indomethacin to flavonols with antioxidant activity via an azo bond (off–on). Breakage of the azo bond breaks results in the release of both fluorophores and drugs, achieving targeted tracing and integrated treatment effects. In vivo and in vitro fluorescence imaging experiments were used to demonstrate the potential of FAI in the diagnosis of UC, together with synergistic therapeutic effects through the release of both fluorophores and anti-inflammatory agents. Therefore, this diagnostic agent shows promise as a potential tool for diagnosing and treating UC.


Introduction
Ulcerative colitis (UC) is a chronic inflammatory bowel disease with an unknown pathogenesis, and its incidence is rising worldwide.UC has a prolonged course, is challenging to treat, and may progress to more severe conditions, such as colorectal cancer, posing a significant economic and medical burden [1][2][3].Currently, endoscopic biopsy is the only definitive diagnostic method for UC [4].Therefore, early detection and timely intervention are of great clinical importance.
Azo reductase is a flavin-dependent enzyme expressed in various prokaryotic and eukaryotic organisms, and it is commonly referred to as flavin mononucleotide reductase [5].This enzyme efficiently reduces arylazo (N=N) compounds to aromatic amines.It is found primarily in tumors and the intestines, linking it closely to diseases, such as cancer and UC [6][7][8][9][10][11]. Azo reductase is widely utilized in disease diagnosis and drug delivery.Azo groups are frequently incorporated to create off-on probes that specifically recognize azo-reducing enzymes for diagnostic purposes.Furthermore, azo groups can function as targeting moieties for colitis, where drug molecules are released in situ upon reduction by azo-reducing enzymes at the colon site.This mechanism allows for the highly effective targeted and controlled release of drugs [12][13][14][15][16][17][18], such as sulfasalazine (SASP), olsalazine, and sulphasalazine, which contain azo-targeting groups in their molecular structures.Although some tumor diagnostic and therapeutic agents based on azo bond design have been reported [19][20][21], there have been few investigations into their applications in the diagnosis and treatment of UC.
To facilitate the early diagnosis and prompt treatment of UC, the present study focused on the synthesis and biological assessment of a novel prodrug termed FAI, which is responsive to azo reductase.This therapeutic compound incorporates a flavanol component and indomethacin (INDO), functioning synergistically, to enhance the diagnosis and treatment of UC (Scheme 1).Flavonols are important natural compounds characterized by the chemical structure 3-hydroxy-2-phenyl-1-benzopyran-4-one.They are commonly found in flowers, fruit, vegetables, and traditional Chinese medicine and exhibit various pharmacological activities, including antioxidant [4], anti-inflammatory [22,23], antibacterial [24,25], antiallergic [26], anticancer [27,28], and antiviral [29] activities.Moreover, flavonols are also used as natural plant dyes due to their intrinsic excited-state intramolecular proton transfer (ESIPT) properties, associated with excellent photostability and high fluorescence quantum yields.As a result, flavonols are regarded as promising fluorescent agents with potential applications in biological fields.
To facilitate the early diagnosis and prompt treatment of UC, the present study focused on the synthesis and biological assessment of a novel prodrug termed FAI, which is responsive to azo reductase.This therapeutic compound incorporates a flavanol component and indomethacin (INDO), functioning synergistically, to enhance the diagnosis and treatment of UC (Scheme 1).Scheme 1. Schematic diagram of releasing indomethacin and azo bond cleavage from diagnosis and treatment agents (FAI) for fluorescence emission "turn on".

Chemistry
The synthesis of the compound FAI is outlined in Scheme 2. Chalcone (3) was synthesized using the Claisen-Schmidt condensation method.Briefly, acetophenone (1) and an aromatic aldehyde (2) were condensed and dehydrated in the presence of a dilute solution of aqueous sodium hydroxide, yielding chalcone 3 with a yield of 68%.Chalcone 3 then underwent cyclization through the Algar-Flynn-Yamada reaction under alkaline conditions in the presence of H2O2, resulting in Compound 4 with a moderate yield of 52% due to concurrent side reactions.Compound 7 was synthesized in a similar manner, resulting in a yield of 92%, through the azo coupling reaction between 4-aminobenzyl alcohol and phenol using NaNO2 under mildly acidic conditions.The phenolic hydroxyl group of azo Compound 7 then reacted with dibromoethane in the presence of K2CO3, resulting in the formation of Compound 8 (82% yield).The carboxylic group of indomethacin was esterified with Compound 8 in the presence of K2CO3, resulting in the formation of Compound 9 (85% yield).The alcohol hydroxy group of Compound 9 was then transformed into a bromo-hydrocarbon through the Appel reaction, catalyzed by CBr4 and PPh3, yielding Compound 10 with a 73% yield.Subsequently, flavonoidol (4) reacted with the indomethacin-azo complex (10) in the presence of K2CO3, leading to the synthesis of the target compound FAI with a yield of 79%.

Chemistry
The synthesis of the compound FAI is outlined in Scheme 2. Chalcone (3) was synthesized using the Claisen-Schmidt condensation method.Briefly, acetophenone (1) and an aromatic aldehyde (2) were condensed and dehydrated in the presence of a dilute solution of aqueous sodium hydroxide, yielding chalcone 3 with a yield of 68%.Chalcone 3 then underwent cyclization through the Algar-Flynn-Yamada reaction under alkaline conditions in the presence of H 2 O 2 , resulting in Compound 4 with a moderate yield of 52% due to concurrent side reactions.Compound 7 was synthesized in a similar manner, resulting in a yield of 92%, through the azo coupling reaction between 4-aminobenzyl alcohol and phenol using NaNO 2 under mildly acidic conditions.The phenolic hydroxyl group of azo Compound 7 then reacted with dibromoethane in the presence of K 2 CO 3 , resulting in the formation of Compound 8 (82% yield).The carboxylic group of indomethacin was esterified with Compound 8 in the presence of K 2 CO 3 , resulting in the formation of Compound 9 (85% yield).The alcohol hydroxy group of Compound 9 was then transformed into a bromo-hydrocarbon through the Appel reaction, catalyzed by CBr 4 and PPh 3 , yielding Compound 10 with a 73% yield.Subsequently, flavonoidol (4) reacted with the indomethacin-azo complex (10) in the presence of K 2 CO 3 , leading to the synthesis of the target compound FAI with a yield of 79%.

Photophysical Properties of Compound 4
To explore the photophysical characteristics of Compound 4, UV-VIS absorption and fluorescence spectra were first acquired and analyzed using various solvents.The concentration of probe 4 was maintained at 20 µmo/L using solvents, including dichloromethane (DCM), dimethyl sulfoxide (DMSO), ethyl acetate (EA), and tetrahydrofuran (THF).As depicted in Figure 1A, probe 4 exhibited an absorption peak around 400 nm (approximately 450 nm in DMSO), attributed to an intramolecular charge transfer transition.In the fluorescence emission spectra (Figure 1B), the excitation wavelength corresponded to the maximum absorption wavelength of each solvent, with probe 4 displaying a consistent maximum emission wavelength of approximately 570 nm across all four solvents, indicating a Stokes shift of 170 nm.The molar absorption coefficient of Compound 4 at 450 nm in the DMSO solution was 27,400 M −1 cm −1 , and the absolute fluorescence quantum yield was 13.6% (Figure S11 and Table S1).The emission spectra of the probes were further investigated in various volume fractions (fw) of DMSO/H 2 O mixtures (Figure 1C).Initially, as the moisture content increased, the emission intensity remained relatively stable.However, when the concentration was raised to above 80%, the fluorescence intensity increased gradually (Figure 1D), confirming the presence of the aggregation-induced emission (AIE) characteristics of this probe.

Photophysical Properties of Compound 4
To explore the photophysical characteristics of Compound 4, UV-VIS absorption and fluorescence spectra were first acquired and analyzed using various solvents.The concentration of probe 4 was maintained at 20 µmo/L using solvents, including dichloromethane (DCM), dimethyl sulfoxide (DMSO), ethyl acetate (EA), and tetrahydrofuran (THF).As depicted in Figure 1A, probe 4 exhibited an absorption peak around 400 nm (approximately 450 nm in DMSO), attributed to an intramolecular charge transfer transition.In the fluorescence emission spectra (Figure 1B), the excitation wavelength corresponded to the maximum absorption wavelength of each solvent, with probe 4 displaying a consistent maximum emission wavelength of approximately 570 nm across all four solvents, indicating a Stokes shift of 170 nm.The molar absorption coefficient of Compound 4 at 450 nm in the DMSO solution was 27,400 M −1 cm −1 , and the absolute fluorescence quantum yield was 13.6% (Figure S11 and Table S1).The emission spectra of the probes were further investigated in various volume fractions (fw) of DMSO/H2O mixtures (Figure 1C).Initially, as the moisture content increased, the emission intensity remained relatively stable.However, when the concentration was raised to above 80%, the fluorescence intensity increased gradually (Figure 1D), confirming the presence of the aggregation-induced emission (AIE) characteristics of this probe.

Release Properties of Compound FAI
Given the exceptional optical properties of FAI, an additional investigation was carried out to explore the effects of its fluorescence "off-on" behavior and drug-release characteristics on catalysis by azo reductase.
Initially, the absorption spectra of FAI were investigated in a buffer solution (pH 7.4, 1% DMSO) in the presence and absence of azo reductase and NADH.As depicted in Figure 2A, FAI exhibited a maximum absorption peak centered around 360 nm.This peak decreased with the addition of azo reductase, and a new peak emerged at 450 nm, indicating changes in the molecular structure.The fluorescence spectrum, illustrated in Figure 2B, demonstrated that the ESPIT effect of flavonol was disrupted by the azobenzene group, significantly reducing the fluorescence emission from the probe.In the presence of azo reductase, the fluorescence intensity at 570 nm increased in a concentration-dependent manner (Figure 2C), reaching a maximum at 0.45 µg/mL.A comparison with the fluorescence spectrum of Compound 4 showed that the fluorescence recovered to the original 90.6% after cleavage.Next, the incubation of FAI with different physiologically relevant substances, including K + , Na + , Mg 2+ , ClO − , Ca 2+ , GSH, Zn 2+ , Fe 2+ , alkaline phosphatase (ALP), Cys, OH − , VcNa, Cu 2+ , NADPH, glucose, H 2 O 2 , and azo reductase, was used to evaluate its selectivity for azo reductase.As shown in Figure 2D, the fluorescence intensity of FAI changed following the treatment with azo reductase.In contrast, the incubation of FAI with other biologically relevant species resulted in only a minimal increase in fluorescence.This finding suggests that FAI responds specifically to azo reductase, leading to drug release.

Release Properties of Compound FAI
Given the exceptional optical properties of FAI, an additional investigation was carried out to explore the effects of its fluorescence "off-on" behavior and drug-release characteristics on catalysis by azo reductase.
Initially, the absorption spectra of FAI were investigated in a buffer solution (pH 7.4, 1% DMSO) in the presence and absence of azo reductase and NADH.As depicted in Figure 2A, FAI exhibited a maximum absorption peak centered around 360 nm.This peak decreased with the addition of azo reductase, and a new peak emerged at 450 nm, indicating changes in the molecular structure.The fluorescence spectrum, illustrated in Figure 2B, demonstrated that the ESPIT effect of flavonol was disrupted by the azobenzene group, significantly reducing the fluorescence emission from the probe.In the presence of azo reductase, the fluorescence intensity at 570 nm increased in a concentration-dependent manner (Figure 2C), reaching a maximum at 0.45 µg/mL.A comparison with the fluorescence spectrum of Compound 4 showed that the fluorescence recovered to the original 90.6% after cleavage.Next, the incubation of FAI with different physiologically relevant substances, including K + , Na + , Mg 2+ , ClO − , Ca 2+ , GSH, Zn 2+ , Fe 2+ , alkaline phosphatase (ALP), Cys, OH − , VcNa, Cu 2+ , NADPH, glucose, H2O2, and azo reductase, was used to evaluate its selectivity for azo reductase.As shown in Figure 2D, the fluorescence intensity of FAI changed following the treatment with azo reductase.In contrast, the incubation of FAI with other biologically relevant species resulted in only a minimal increase in fluorescence.This finding suggests that FAI responds specifically to azo reductase, leading to drug release.
Moreover, the kinetics of Compound 4 and indomethacin release from FAI when exposed to azo reductase were investigated over 24 h.The HPLC chromatogram (Figure 2E) shows the initial appearance of a retention peak for FAI at 7.5 min.Two additional retention peaks subsequently emerged at 2.5 min (indomethacin) and 6.4 min (Compound 4), indicating the enzyme-mediated release of both compounds from FAI.After 24 h of incubation, the complete release of both Compound 4 and indomethacin was observed.Moreover, the kinetics of Compound 4 and indomethacin release from FAI when exposed to azo reductase were investigated over 24 h.The HPLC chromatogram (Figure 2E) shows the initial appearance of a retention peak for FAI at 7.5 min.Two additional retention peaks subsequently emerged at 2.5 min (indomethacin) and 6.4 min (Compound 4), indicating the enzyme-mediated release of both compounds from FAI.After 24 h of incubation, the complete release of both Compound 4 and indomethacin was observed.These findings suggest that FAI releases both Compound 4 and indomethacin upon exposure to azo reductase, thereby facilitating synergistic therapeutic effects.
Scheme 2 proposes a possible response mechanism.The non-fluorescent nature of the probe itself is attributed to the disruption of the ESPIT effect between the 3-hydroxy and 4-carbonyl groups of Compound 4 by the azobenzene group.When exposed to azo reductase, the reduction of the azobenzene group leads to the cleavage of Compound 4 and indomethacin, facilitating the ESPIT process and resulting in the generation of a strong fluorescent signal.To gain deeper insight into this mechanism, density functional theory (DFT) calculations were performed using Gaussian 09 software.The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of Compound 4 and FAI are shown in Figure 2F.The LUMO energy level of FAI indicates that the charge distribution primarily resides within the azobenzene group, confirming its role in disrupting the ESPIT process.
To investigate the binding interactions between FAI and azo reductase and COX-2, molecular docking was performed using Schrödinger-Maestro.The results show that the azo group of FAI fit snugly within the active pocket of azo reductase.Meanwhile, indomethacin was shown to enter the active pocket of COX-2 and form hydrogen bonds with amino acid residues Ser-530 and Arg-120.These experimental findings support the proposed response mechanism.
Compound 4 and FAI are shown in Figure 2F.The LUMO energy level of FAI indicates that the charge distribution primarily resides within the azobenzene group, confirming its role in disrupting the ESPIT process.
To investigate the binding interactions between FAI and azo reductase and COX-2, molecular docking was performed using Schrödinger-Maestro.The results show that the azo group of FAI fit snugly within the active pocket of azo reductase.Meanwhile, indomethacin was shown to enter the active pocket of COX-2 and form hydrogen bonds with amino acid residues Ser-530 and Arg-120.These experimental findings support the proposed response mechanism.

In Vitro Anti-Inflammatory Activity
Treatment with LPS activates macrophages, resulting in the generation of substantial amounts of nitric oxide (NO), which can exacerbate the progression of inflammatory diseases.Hence, NO production in LPS-treated RAW 264.7 mouse macrophages was evaluated to assess the inhibitory effects of Compound 4, indomethacin, Compound 4 + indomethacin, and FAI.The results are shown in Table 1, indicating that FAI demonstrated significant anti-inflammatory activity, with an IC 50 value of 5.1 ± 0.48 µM.This efficacy surpassed that of Compound 4 and indomethacin when used individually and was comparable to their combined activity.Notably, analysis of the cytotoxicity revealed that FAI significantly mitigated the toxicity associated with indomethacin when used alone.
The production of the inflammatory cytokines TNF-α and IL-6 by macrophages after FAI treatment was measured using ELISA.As seen in Figure 3A,B, treatment with all compounds at 1 µM resulted in significantly reduced inflammatory cytokine secretion.This inhibition of inflammatory cytokine secretion was most marked at an FAI concentration of 5 µM, underscoring its pronounced anti-inflammatory efficacy.The production of the inflammatory cytokines TNF-α and IL-6 by macrophages after FAI treatment was measured using ELISA.As seen in Figure 3A,B, treatment with all compounds at 1 µM resulted in significantly reduced inflammatory cytokine secretion.This inhibition of inflammatory cytokine secretion was most marked at an FAI concentration of 5 µM, underscoring its pronounced anti-inflammatory efficacy.

Antioxidant and Anti-Mitochondrial Damage
Recent research shows a strong association between mitochondrial oxidative stress and UC [30,31].Figure 4A illustrates the impact of FAI on the levels of reactive oxygen species (ROS) in LPS-induced RAW264.7 cells.Cells in the model group showed markedly increased intracellular ROS levels in comparison to the blank control group.Compound 4 significantly reduced the intracellular ROS levels, in contrast to indomethacin, which showed limited efficacy.Remarkably, FAI was more effective in reducing ROS levels compared to either drug alone and comparable to their combined use.Subsequently, the

Antioxidant and Anti-Mitochondrial Damage
Recent research shows a strong association between mitochondrial oxidative stress and UC [30,31].Figure 4A illustrates the impact of FAI on the levels of reactive oxygen species (ROS) in LPS-induced RAW264.7 cells.Cells in the model group showed markedly increased intracellular ROS levels in comparison to the blank control group.Compound 4 significantly reduced the intracellular ROS levels, in contrast to indomethacin, which showed limited efficacy.Remarkably, FAI was more effective in reducing ROS levels compared to either drug alone and comparable to their combined use.Subsequently, the ability of FAI to mitigate mitochondrial oxidative damage was assessed by monitoring changes in the mitochondrial membrane potential (MMP).Reduced membrane potentials prevent the accumulation of JC-1 dye in the mitochondrial matrix, resulting in green fluorescence, whereas higher membrane potentials are represented by the presence of red fluorescence associated with the polymer.It was found that FAI significantly reduced the levels of mitochondrial oxidative stress, outperforming the effects of the individual drugs and slightly surpassing their combined efficacy (Figure 4B).

The Protective Influence of FAI on LPS-Treated RAW264.7 Cells
As illustrated in Figure 5, the results of the live/dead cell staining of RAW264.7 cells indicate a significant reduction in the number of yellow-green Calcein-AM-labeled live cells and a significant increase in red-stained dead cells in the LPS-treated group compared to the blank group.However, the separate administration of indomethacin or Compound 4 did not result in any substantial improvement.Interestingly, the simultaneous administration of both drugs resulted in a more pronounced treatment effect.Particularly noteworthy is the marked protective effect exhibited by the prodrug FAI against LPS-induced RAW264.7 cells.
ability of FAI to mitigate mitochondrial oxidative damage was assessed by monitoring changes in the mitochondrial membrane potential (MMP).Reduced membrane potentials prevent the accumulation of JC-1 dye in the mitochondrial matrix, resulting in green fluorescence, whereas higher membrane potentials are represented by the presence of red fluorescence associated with the polymer.It was found that FAI significantly reduced the levels of mitochondrial oxidative stress, outperforming the effects of the individual drugs and slightly surpassing their combined efficacy (Figure 4B).

The Protective Influence of FAI on LPS-Treated RAW264.7 Cells
As illustrated in Figure 5, the results of the live/dead cell staining of RAW264.7 cells indicate a significant reduction in the number of yellow-green Calcein-AM-labeled live cells and a significant increase in red-stained dead cells in the LPS-treated group compared to the blank group.However, the separate administration of indomethacin or Compound 4 did not result in any substantial improvement.Interestingly, the simultaneous administration of both drugs resulted in a more pronounced treatment effect.Particularly noteworthy is the marked protective effect exhibited by the prodrug FAI against LPS-induced RAW264.7 cells.

Activity and Imaging of FAI In Vivo
The protective effects of FAI against DSS-induced acute colitis were also evaluated in vivo.C57BL/6 mice were randomly assigned to six groups.The normal group was treated with water, while the other groups received a 4% solution of DSS for 7 days to establish a model of acute colitis (Figure 6A).Subsequently, PBS, Compound 4, indomethacin, Compound 4 + indomethacin, and FAI were injected intraperitoneally on days 0, 2, 4, 6, 8, and 10, respectively, and the mice were sacrificed and imaged on days 0-2 and 11.During

Activity and Imaging of FAI In Vivo
The protective effects of FAI against DSS-induced acute colitis were also evaluated in vivo.C57BL/6 mice were randomly assigned to six groups.The normal group was treated with water, while the other groups received a 4% solution of DSS for 7 days to establish a model of acute colitis (Figure 6A).Subsequently, PBS, Compound 4, indomethacin, Compound 4 + indomethacin, and FAI were injected intraperitoneally on days 0, 2, 4, 6, 8, and 10, respectively, and the mice were sacrificed and imaged on days 0-2 and 11.During the course of treatment, the daily weight fluctuations of the mice were recorded, and the fecal characteristics and presence of occult blood were observed to evaluate the severity of the colitis.As shown in Figure 6B,C, the weight of the mice in each group increased in comparison to that in the control group, with the most significant increase observed in the FAI group.These results indicate that the intestinal absorption in the colitis mice recovered gradually after FAI treatment; this was confirmed by measurements of colon length, in which the mean length of the colon in the FAI-treated group was 6.90 ± 0.34 cm, significantly longer than that observed in the model group (4.50 ± 0.22 cm) and closer to that of the normal group.Furthermore, relative to the control group, the model group showed a significantly increased expression of IL-6, TNF-α, and calprotectin in the colon tissues (Figure 6D-F), while the expression of IL-6, TNF-α, and calprotectin in the colon tissues of the treated mice was reduced in comparison to the model group, with the reduction being most significant in the FAI group and the combined treatment group, where they approached normal levels.In vivo imaging of FAI was undertaken by intraperitoneal injection of all mice with FAI, followed by fluorescence imaging at various time points.The model group showed a significant increase in fluorescence compared to the normal group, reaching a peak at 8 h with a fluorescence intensity approximately five times higher than that of the normal group, indicating the ability of the probe to visualize azo reductase upregulation in colitis (Figure 6G,H).Subsequently, the colon, heart, liver, spleen, lungs, and kidneys of the mice in the FAI group were harvested and imaged using fluorescence imaging on days 0-2 (Figure 6I).No obvious fluorescence signal was detected in other organs apart from the colon tissue (Figure 6J), suggesting that azo reductase was mainly distributed in the inflammatory sites of the colon, and that FAI could be used as an indicator for studying colitis and similar diseases.On day 11, dissection of the colon tissues from each group showed that the fluorescence in the FAI group was markedly lower than that in the Compound 4 group, indicating that the FAI treatment was more effective than that of Compound 4, and led to decreased azo reductase levels (Figure 6K).

H&E and iNOS Immunofluorescence Staining
Furthermore, colonic tissues were assessed using hematoxylin and eosin (H&E) staining in each group.As depicted in Figure 7A, the mucosa of the colonic tissue in the control group appeared intact, with cells arranged in a regular pattern.Conversely, in the acute UC group, the mucosa of the colonic tissue was damaged, with the visible presence of inflammatory cells in the subepithelial layer.In contrast, both the FAI group and the combined treatment group showed a significant reduction in the number of inflammatory cells, with the mucosa of the colonic tissue appearing intact.Furthermore, the therapeutic efficacy of FAI was further verified through iNOS immunofluorescence staining.The results revealed a significant reduction in green fluorescence, marked by iNOS, nearly returning to normal levels.In summary, FAI demonstrates promising potential as a potent drug for the treatment of acute UC.

In Vivo Biosafety Evaluation of FAI
Assessing the safety of drugs is crucial for their clinical application.To evaluate the biocompatibility of FAI, mice were given oral doses of high concentrations of FAI (500 mg/kg), and then euthanized, and their hearts, kidneys, lungs, livers, and spleens were collected for H&E staining.As illustrated in Figure 8, H&E staining revealed no histopathological or pathological damage in either the PBS group or the FAI group, affirming the low toxicity and superior safety profile of FAI.
spleen, lungs, and kidneys of the mice in the FAI group were harvested and imaged using fluorescence imaging on days 0-2 (Figure 6I).No obvious fluorescence signal was detected in other organs apart from the colon tissue (Figure 6J), suggesting that azo reductase was mainly distributed in the inflammatory sites of the colon, and that FAI could be used as an indicator for studying colitis and similar diseases.On day 11, dissection of the colon tissues from each group showed that the fluorescence in the FAI group was markedly lower than that in the Compound 4 group, indicating that the FAI treatment was more effective than that of Compound 4, and led to decreased azo reductase levels (Figure 6K).combined treatment group showed a significant reduction in the number of inflammatory cells, with the mucosa of the colonic tissue appearing intact.Furthermore, the therapeutic efficacy of FAI was further verified through iNOS immunofluorescence staining.The results revealed a significant reduction in green fluorescence, marked by iNOS, nearly returning to normal levels.In summary, FAI demonstrates promising potential as a potent drug for the treatment of acute UC.

In Vivo Biosafety Evaluation of FAI
Assessing the safety of drugs is crucial for their clinical application.To evaluate the biocompatibility of FAI, mice were given oral doses of high concentrations of FAI (500 mg/kg), and then euthanized, and their hearts, kidneys, lungs, livers, and spleens were collected for H&E staining.As illustrated in Figure 8, H&E staining revealed no histopathological or pathological damage in either the PBS group or the FAI group, affirming the low toxicity and superior safety profile of FAI.

In Vivo Biosafety Evaluation of FAI
Assessing the safety of drugs is crucial for their clinical application.To evaluate the biocompatibility of FAI, mice were given oral doses of high concentrations of FAI (500 mg/kg), and then euthanized, and their hearts, kidneys, lungs, livers, and spleens were collected for H&E staining.As illustrated in Figure 8, H&E staining revealed no histopathological or pathological damage in either the PBS group or the FAI group, affirming the low toxicity and superior safety profile of FAI.

Experimental Procedures
All chemical reagents utilized in this study were commercially sourced.The solvents employed were of analytical purity, with common solvents procured from Guoyu Chemical Reagents Co., Ltd.(Shanghai, China).The 1 H NMR spectra were recorded on a 300 MHz Bruker AV spectrometer, while the 13 C NMR spectra were recorded on a 600 MHz Bruker AV spectrometer (Bruker Co., Billerica, MA, USA), using deuterated chloroform (CDCl 3 ) or deuterated dimethyl sulfoxide (DMSO) as solvents, with tetramethylsilane (TMS) serving as the internal standard.The fluorescence spectra were measured using an RF-5301PC fluorescence spectrophotometer (Shimadzu, Kyoto, Japan), while the UV-visible absorption spectra were measured using a TU-1900 UV-visible absorption spectrometer (Beijing Puxi General Instrument Co., Ltd., Beijing, China).An LSM710 confocal laser scanning microscope (Zeiss, Oberkochen, Germany) was used for fluorescence imaging.High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed on an Agilent 6500 Q-TOF LC/MS (Agilent Technologies, Santa Clara, CA, USA) using an ESI ion source.Column chromatography was carried out using 200-300 mesh silica gel (Qingdao Ocean Chemical Co., Ltd., Qingdao, China).The reaction was monitored using TLC glass plates (Yantai Huayang New Material Technology Co., Ltd., Yantai, China).Purification of all the tested compounds was carried out to achieve a purity of at least 95% by high-performance liquid chromatography (HPLC) using a Waters 1525 binary high-performance liquid chromatography pump equipped with a Waters 2998 photodiode array detector, and XBridge-C18 analytical column (5 µm, 4.6 mm × 150 mm).

UV and Fluorescence Spectra
Compound 4 was dissolved separately in different solvents (DCM, DMSO, EA, and THF) to prepare a 1 mmol/L compound stock solution.Compound 4 was then diluted to 20 µM, and its UV absorption spectrum was measured in each solvent.The fluorescence emission spectrum was then recorded under the excitation wavelength equivalent to the maximum absorption wavelength of different solvents.The emission wavelength was recorded between 500 and 600 nm.Different volumes of DMSO were diluted with water to prepare DMSO/water (DMSO/H 2 O) solutions of varying concentrations, and the AIE effect of the probe was determined.The excitation slit and emission slit were both adjusted to 5 nm.

Computational Methods of HOMO and LUMO
ChemDraw was employed for modeling all molecular conformations, which were initially optimized using Chem3D with the MM2 force field, and then saved in the mol2 format.Further structure optimization and frequency calculations were performed using Gaussian 09 software at the B3LYP/6-311G(d,p) level with GD3BJ dispersion correction.Excited state structures and fluorescence spectra were calculated using time-dependent density functional theory (TDDFT), setting the state to 20 and using the B3LYP/def2-TZVP level with GD3BJ dispersion correction.

Molecular Docking
ChemDraw was used to draw the molecular structures of the selected compounds, which were then saved in the .molformat.Using Schrödinger, the azo reductase and COX 2 protein receptor files were prepared by extracting the ligands, removing the surfacecrystallization water, hydrogenating the protein, and repairing the side chains.Compound 4 and FAI were then docked with the prepared azo reductase (PDB code: 2Z9C) and COX-2 protein (PDB code: 4OTJ) structures.The conformation with the lowest binding energy was selected as optimal.PyMOL was then used to determine the interaction mode between the target compounds and the proteins.

Cell Culture
RAW 264.7 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (100 U/mL each of penicillin and streptomycin, Solarbio) at 37 • C and 5% CO 2 .The cells were subsequently transferred to 35 mm cell culture dishes and incubated for 24 h to allow them to adhere to the surface.

Cell Cytotoxicity
Cell toxicity was assessed using MTT assays.RAW 264.7 cells were seeded in 96-well plates at a density of 10 5 cells/mL and incubated at 37 • C with 5% CO 2 for 12 h.Subsequently, 10 µL of various concentrations (20,40,80,160,320, and 640 µM) of Compound 4, indomethacin, a combination of 4 + indomethacin, and FAI solutions were added to the respective wells, and incubation was continued for an additional 24 h.Following this, 20 µL of MTT solution (at a final concentration of 0.5 mg/mL) was added to the individual wells.The MTT solution was removed after a 4 h incubation period, and 150 µL of DMSO was added to dissolve the formazan crystals.The absorbance of individual wells was then measured at 490 nm using a microplate reader.

NO Assay
The culture supernatants were collected, and the NO content was measured using a NO assay kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), according to the manufacturer's instructions.Absorbances were read at 540 nm in a microplate reader.The NO concentrations were calculated based on the standard curve of sodium nitrite.

In Vitro ROS-Scavenging Capacity of FAI
The ability of FAI to clear ROS in RAW 264.7 cells was evaluated.The cells (2 × 10 5 cells/well) were seeded into glass culture dishes and then incubated with LPS (10 µg/mL) at 37 • C for 12 h.All cells were then washed twice with PBS and incubated with DMEM containing 5 µM indomethacin, Compound 4, a combination of indomethacin and Compound 4, and FAI for 4 h.Following the removal of the media, the cells were washed once again with a fresh medium.Then, 1 mL of fresh medium with DCFH-DA (50 µM) was added to the cells.The cells were subsequently incubated at 37 • C for 30 min, followed by PBS washing (thrice), and the fluorescence images from DCF were observed under a laser confocal microscope.

Evaluation of Mitochondrial Membrane Potential
In brief, RAW 264.7 cells (2 × 10 5 /mL) were seeded into cell culture dishes and incubated with LPS (10 µg/mL) overnight.Then, the cells were incubated with 5 µM indomethacin, Compound 4, a combination of indomethacin and Compound 4, or FAI at 37 • C for another 24 h.JC-1 solution (10 µg/mL in DMEM) was used to replace the old medium, and the cells were incubated at 37 • C for 20 min.After incubation, the cells were separated and washed with PBS three times.A laser confocal microscope was used for evaluation and imaging.

Live/Dead Cell Staining
RAW 264.7 cells (2 × 10 5 /mL) were seeded into cell culture dishes and incubated with LPS (10 µg/mL) overnight.Then, the cells were incubated with 5 µM indomethacin, Compound 4, a combination of indomethacin and Compound 4, or FAI at 37 • C for another 24 h.After the treatment, 250 µL of AM-PI staining solution was added to each group and incubated for 20 min.The specimens were washed three times with PBS, and images were captured using a laser confocal microscope.

In Vivo Fluorescence Imaging
BALB/c mice (18-20 g, 6-8 weeks old) were purchased from the Experimental Animal Center of Nantong University.The mice were divided into 6 groups (n = 3).The experimental group mice received 4% DSS solution for 7 days, while the control group received distilled water.On day 7, all mice received intraperitoneal injections of indomethacin, Compound 4, a combination of indomethacin and Compound 4, or FAI (20 mg/kg).At specified time intervals, the mice were euthanized, and their hearts, livers, spleens, lungs, kidneys, and intestines were promptly collected and imaged using the IVIS Lumina system in vivo.

Levels of IL-6, TNF-α, and Calprotectin
Colonic tissues from all groups underwent enzyme-linked immunosorbent assay (ELISA) to determine the levels of TNF-α, IL-6, and calprotectin.Briefly, frozen tissue samples from the distal colon were homogenized in a phosphate potassium buffer (pH 6.0) containing a cocktail of protease inhibitors, followed by centrifugation at 2500 rpm and 4 • C for 5 min.The supernatants were subsequently centrifuged at 10,000 rpm and 4 • C for 10 min and the concentrations of TNF-α, IL-6, and calprotectin were measured by ELISA according to manufacturer's guidelines.The total protein concentrations were measured using a BCA kit (Thermo Fisher, Waltham, MA, USA).

Scheme 1 .
Scheme 1. Schematic diagram of releasing indomethacin and azo bond cleavage from diagnosis and treatment agents (FAI) for fluorescence emission "turn on".

a
Concentration of compound that can inhibit 50% of cell viability.b The half-maximal inhibitory concentration (IC50) indicates the concentration of the compound required to inhibit NO production by 50% relative to the LPS group, with the NO release in the LPS group being 60.01 ± 0.24 µM.

Figure 4 .
Figure 4. (A) The influence of FAI on ROS levels resulting from the LPS treatment of RAW264.7 cells.The ROS levels were determined using DCFH-DA in cells treated with indomethacin, Compound 4, a combination of indomethacin and Compound 4, and FAI, as shown by laser confocal microscopy.λex = 488 nm; λem = 500-550 nm; scale bar = 100 µm.(B) The influence of FAI on the mitochondrial membrane potential in LPS-treated RAW264.7 cells.Changes in the mitochondrial membrane potential were determined by JC-1 accumulation in cells treated with indomethacin, Compound 4, a combination of indomethacin and Compound 4, and FAI, as shown by laser confocal microscopy.Green channel: λex = 488 nm, λem = 510-550 nm; red channel: λex = 535 nm, λem = 590-610 nm; scale bar = 100 µm.

Figure 4 . 17 Figure 5 .
Figure 4. (A) The influence of FAI on ROS levels resulting from the LPS treatment of RAW264.7 cells.The ROS levels were determined using DCFH-DA in cells treated with indomethacin, Compound 4, a combination of indomethacin and Compound 4, and FAI, as shown by laser confocal microscopy.λex = 488 nm; λem = 500-550 nm; scale bar = 100 µm.(B) The influence of FAI on the mitochondrial membrane potential in LPS-treated RAW264.7 cells.Changes in the mitochondrial membrane potential were determined by JC-1 accumulation in cells treated with indomethacin, Compound 4, a combination of indomethacin and Compound 4, and FAI, as shown by laser confocal microscopy.Green channel: λex = 488 nm, λem = 510-550 nm; red channel: λex = 535 nm, λem = 590-610 nm; scale bar = 100 µm.Molecules 2024, 29, x FOR PEER REVIEW 8 of 17

Figure 6 .
Figure 6.(A) Schematic diagram showing the timing and frequency of administration of FAI.(B) The daily weight changes in the mice in each group after drug administration.(C) Corresponding changes in the lengths of the colon.(D) The influence of each treatment on the expressions of IL-6 (D), TNF-α (E), and calprotectin (F) proteins in the colon tissue of UC mice.(G) Fluorescence imaging of mice from the normal and UC groups after intraperitoneal injection of FAI (20 mg/kg) in terms of time point (1, 4, 8, 12, and 24 h).(H) Quantification of the average fluorescence intensity of (G).(I) Fluorescence images of ex vivo organs from colitis mouse models following FAI administration.(J) Quantification of the average fluorescence intensity of major organs according to (I).(K) Ex vivo fluorescence imaging of mice treated with Compound 4 and FAI after intraperitoneal injection.## p < 0.01 compared to the control group; * p < 0.05 and ** p < 0.01 compared to the PBS group.

Figure 6 .
Figure 6.(A) Schematic diagram showing the timing and frequency of administration of FAI.(B) The daily weight changes in the mice in each group after drug administration.(C) Corresponding changes in the lengths of the colon.(D) The influence of each treatment on the expressions of IL-6 (D), TNF-α (E), and calprotectin (F) proteins in the colon tissue of UC mice.(G) Fluorescence imaging of mice from the normal and UC groups after intraperitoneal injection of FAI (20 mg/kg) in terms of time point (1, 4, 8, 12, and 24 h).(H) Quantification of the average fluorescence intensity of (G).(I) Fluorescence images of ex vivo organs from colitis mouse models following FAI administration.(J) Quantification of the average fluorescence intensity of major organs according to (I).(K) Ex vivo fluorescence imaging of mice treated with Compound 4 and FAI after intraperitoneal injection.## p < 0.01 compared to the control group; * p < 0.05 and ** p < 0.01 compared to the PBS group.

Figure 7 .
Figure 7. (A) H&E-stained sections of colon tissues from the different groups.(B) The expression of iNOS in colonic tissue was detected by immunofluorescence staining.Scale bar = 100 µm.

Figure 7 .
Figure 7. (A) H&E-stained sections of colon tissues from the different groups.(B) The expression of iNOS in colonic tissue was detected by immunofluorescence staining.Scale bar = 100 µm.

Figure 7 .
Figure 7. (A) H&E-stained sections of colon tissues from the different groups.(B) The expression of iNOS in colonic tissue was detected by immunofluorescence staining.Scale bar = 100 µm.

Figure 8 .
Figure 8. H&E staining of ex vivo tissue sections (heart, kidney, lung, liver, and spleen) from the FAI group.

Table 1 .
Cytotoxic effects in RAW 264.7 cells and IC 50 values of FAI against NO production in LPS-stimulated RAW264.7 cells.The half-maximal inhibitory concentration (IC 50 ) indicates the concentration of the compound required to inhibit NO production by 50% relative to the LPS group, with the NO release in the LPS group being 60.01 ± 0.24 µM.
a Concentration of compound that can inhibit 50% of cell viability.b

Table 1 .
Cytotoxic effects in RAW 264.7 cells and IC50 values of FAI against NO production in LPSstimulated RAW264.7 cells.