Mitochondrial targeting near-infrared fluorescent probe, preparation method thereof and application of mitochondrial targeting near-infrared fluorescent probe in dynamic monitoring of hypoxic pulmonary hypertension

Abstract

The invention discloses a mitochondrial targeting near-infrared fluorescent probe and a preparation method and application thereof, and belongs to the field of medical living body fluorescence imaging, and the mitochondrial targeting near-infrared fluorescent probe designed and synthesized by the invention has high sensitivity (18.6 nM), quick response (5 minutes) and mitochondrial targeting characteristics (co-localization coefficient is 0.925). And an ideal tool is provided for dynamically monitoring the mitochondrial oxidation stress state in the hypoxic pulmonary hypertension model.

Description

Technical Field

[0001] The present invention relates to the field of medical fluorescent probes, and in particular to a mitochondria-targeted near-infrared fluorescent probe, a preparation method thereof, and an application thereof in dynamic monitoring of hypoxic pulmonary hypertension. Background Art

[0002] Hypoxia-induced pulmonary hypertension (HPH) is a common complication of hypoxic diseases such as chronic obstructive pulmonary disease (COPD), interstitial lung disease, and high-altitude heart disease. Its prevalence is increasing significantly worldwide with increasing air pollution and an aging population. Epidemiological data show that the incidence of HPH in COPD patients is as high as 30%-50%, and the 5-year survival rate for patients with HPH is less than 40%. Although existing treatments (such as endothelin receptor antagonists and phosphodiesterase-5 inhibitors) can temporarily improve hemodynamic parameters, they are unable to reverse the progression of pulmonary vascular remodeling, and the long-term prognosis for patients remains poor. Current research on HPH faces two major bottlenecks: 1. The complexity of the pathological mechanism: The vicious cycle of oxidative stress and inflammation drives the progression of HPH, but the spatiotemporal correlation between the dynamic changes of mitochondrial reactive oxygen species (ROS) and vascular remodeling remains unclear; 2. The limitations of diagnostic and therapeutic technologies: Traditional ROS probes suffer from poor tissue penetration and susceptibility to photobleaching, making it impossible to distinguish between mitochondrial and cytosolic ROS sources. The existing probes rely on the passive diffusion of hydrophobic triphenylphosphine (TPP), and their mitochondrial colocalization coefficient is ≤0.72. On the other hand, the sensitivity of the existing probes is limited and the sensitivity to O2 ·- The detection limit is ≥50nM. Therefore, the development of high-resolution in vivo imaging probes has become a key breakthrough in achieving early diagnosis of HPH. Summary of the Invention

[0003] In view of this, the present invention provides a mitochondria-targeted near-infrared fluorescent probe to solve the problem of low sensitivity of existing probes leading to low resolution of in vivo imaging.

[0004] In one aspect, the present invention uses a mitochondrial-targeted near-infrared fluorescent probe, the structural formula of which is as follows:

[0005]

[0006] Another aspect of the present invention provides an application of a mitochondria-targeted near-infrared fluorescent probe in the preparation of a dynamic monitoring tool for hypoxic pulmonary hypertension.

[0007] Preferably, the probe can monitor the level of reactive oxygen species in mice.

[0008] Preferably, the probe can dynamically monitor in vivo how aucubin regulates the PI3K / Akt / Nrf2 axis to improve hypoxic pulmonary hypertension.

[0009] On the other hand, the present invention provides a method for preparing a mitochondrial-targeted near-infrared fluorescent probe, which comprises a condensation reaction between 2,3,3-trimethylindoline quaternary ammonium salt and 5-chloro-2,4-dialdehyde phenol to obtain a heptamethine cyanine near-infrared fluorescent skeleton structure, and then introducing an active oxygen ion recognition site based on a cyano-phenol group through 4-chlororesorcinol. Finally, the probe is activated by trifluoromethanesulfonic anhydride to obtain a mitochondrial-targeted near-infrared fluorescent probe containing a trifluoromethanesulfonate group.

[0010] The mitochondrial-targeted near-infrared fluorescent probe M550 provided by the present invention, with its high sensitivity (18.6 nM), rapid response (5 minutes) and mitochondrial-targeting properties (colocalization coefficient 0.925), provides an ideal tool for real-time monitoring of the dynamics of mitochondrial oxidative stress in the HPH model.

[0011] Furthermore, the preparation method of the present invention adopts a distributed coupling strategy, has mild synthesis conditions, and is easy to promote.

[0012] Furthermore, the mitochondrial-targeted design and near-infrared imaging capabilities of the M550 probe enabled the first in vivo dynamic imaging of ROS in an HPH mouse model, providing a new tool for the early non-invasive diagnosis of HPH oxidative stress and filling the gap in clinical dynamic monitoring technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The spectrum test results of the active oxygen probe M550, where A represents the chemical structure of M550 and O2 ·- Sensing schematic diagram; B represents M550 (10 μM) in PBS buffer (10 mM, pH 7.4, 2% DMSO, v / v) in the absence or presence of O2 ·- C represents the absorption spectra of M550 (10 μM) in PBS buffer (10 mM, pH 7.4, 2% DMSO, v / v) in the presence of different concentrations of O2 ·- (0-90μM) fluorescence spectrum. D represents the fluorescence intensity and O2 ·- The linear relationship of concentration; E represents the effect of M550 (10μM) on O2 ·- (10 μM) and selectivity experiments with other relevant analytes (10 equiv.);

[0014] Figure 2 The superoxide anion (O2 ·- ) test results; A represents the fluorescence intensity and O2 ·- Concentration relationship diagram; B represents the addition of O2 ·- (10 μM) after the time-dependent fluorescence changes of M550 (10 μM); C represents the fluorescence intensity in the presence or absence of O2·- D represents the stability of M559 (10 μM) in PBS buffer (10 mM, pH 7.4, 2% DMSO, v / v) at different pH values ​​under O2 ·- Fluorescence intensity in the presence of

[0015] Figure 3 The cell survival rate of PASMCs after treatment with different concentrations of M550 (0, 5, 10, 15, 20, 25, 30, 35, 40 μM) for 24 h;

[0016] Figure 4 The results of fluorescence co-localization analysis of probe M550 in mitochondria and lysosomes in PASMCs;

[0017] Figure 5 The graph (A) and quantitative analysis (B) of the reactive oxygen species levels of PASMCs in each group detected by probe M550;

[0018] Figure 6 The M550 probe detects the level of reactive oxygen species in the lungs of mice in each group (A) and quantitative analysis (B);

[0019] Figure 7 Schematic diagram of probe M550 monitoring the improvement of HPH by regulating PI3K / Akt / Nrf2 axis by aucubin. DETAILED DESCRIPTION

[0020] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0021] In the following examples, male C57BL / 6J mice, weighing 25 g ± 5 g, and 8 weeks old, meeting SPF standards, were used as experimental materials. All experimental animals were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd. PASMCs were primary cultured cells (can be passaged up to three generations).

[0022] Example 1: Mitochondrial-targeted near-infrared fluorescent probe M550, whose structural formula is as shown in Formula 1:

[0023]

[0024] Example 2: Preparation of mitochondria-targeted near-infrared fluorescent probe M550. The synthesis route is shown in Formula 2 and includes the following steps:

[0025] (1) Synthesis of M174: 4.4 g of iodomethane (31 mmol, equivalent to 5 equivalents) was added to an acetonitrile solution containing 1 g of 2,3,3-trimethylindole (6.2 mmol, 1 equivalent) and the reaction was continued under reflux for 15 hours; after the reaction mixture was cooled to room temperature, it was concentrated under reduced pressure to obtain a solid residue. The obtained solid residue was washed several times with ether and then recrystallized in acetone to obtain a white powder of 2,3,3-trimethylindole quaternary ammonium salt M174 (yield 78.5%). HR-MS: Calc. for C 12 H 16 N + :174.1278,found:174.1274. 1 H NMR (400MHz, CDCl3): δ8.10(d,J=13.4Hz,1H),7.41(s,1H),7.30–7.24(m,3H),7.11–7.04(m,1H),6.86(d,J=7.8Hz,1H), 6.71(s,1H),5.61(d,J=13.4Hz,1H),3.37(s,3H),2.72–2.66(m,2H),2.65–2.57(m,2H),1.96–1.85(m,2H),1.67(s,6H). 13 C NMR (101MHz, CDCl3): δ174.76,166.82,160.42,157.87,143.86,139.77,139.60,133.44,132.07,128.31,127. 38,122.68,122.22,117.06,116.28,115.63,108.08,103.92,94.84,47.67,30.28,28.73,28.12,24.59,21.39.

[0026] (2) Synthesis of M172: Dimethylformamide (10 ml, 129 mmol, 5.4 eq) was added dropwise to a cooled solution of 10 ml of dichloromethane (CH2Cl2) in an ice-water bath, and phosphorus trioxide (POCl3, 10 ml, 107 mmol, 4.5 eq) was added. After 15 minutes of reaction, cyclohexanone (2.5 ml, 24 mmol, 1 eq) was added, and the resulting mixture was vigorously stirred and refluxed at 80°C for 8 hours. The reaction mixture was then poured into ice water and stored in a refrigerator for 24 hours to obtain 5-chloro-2,4-dialdehyde phenol M172 (3.8 g, 91% yield) as a yellow solid. The obtained product was used directly in the subsequent experimental steps without further purification. The product was stored at -20°C. M172 can also be purchased directly from the market.

[0027] (3) Synthesis of M483: M174 (2.09 g, 12 mmol, 2.4 eq) and M172 (0.86 g, 5 mmol) were dissolved in a mixture of 1-butanol (35 mL) and toluene (15 mL), and the mixture was heated under reflux for 4 h. The solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography using dichloromethane / methanol (CH2Cl2 / MeOH, 100:1, volume ratio) as the eluent to obtain M483 as a green solid.

[0028] HR-MS:Calc.for C 32 H 36 C l N2 + :483.2562,found:483.2554. 1 H NMR (400MHz, CDCl3): δ8.35 (d, J = 14.1Hz, 2H), 7.44–7.34 (m, 4H), 7.27–7.17 (m, 4H), 6.2 4(d,J=14.1Hz,2H),3.77(s,6H),2.76(t,J=6.2Hz,4H),2.04–1.93(m,2H),1.73(s,12H). 13 C NMR (101MHz, CDCl3): δ172.94,150.67,144.41,142.91,141.03,128.94,12 7.97,125.43,122.24,110.91,101.88,49.33,32.76,28.20,26.92,20.80.

[0029] (4) Synthesis of M418: Under nitrogen (N2) protection, 4-chlororesorcinol (173.47 mg, 1.2 mmol, 1.2 eq) and potassium carbonate (K2CO3, 414.63 mg, 3 mmol, 3 eq) were dissolved in 10 ml of acetonitrile (CH3CN), and the mixture was stirred at 80°C until the solution turned brown. Subsequently, M483 (483.26 mg, 1 mmol, 1 eq) was added to the reaction system. The reaction was stirred at 80°C for 4 hours, then cooled to room temperature, condensed under reduced pressure, and the residue was purified by silica gel column chromatography using dichloromethane / methanol (CH2Cl2 / MeOH, 45:1) as eluent to obtain blue solid M418. HR-MS: Calc. for C 26 H 25 ClNO2 + :418.1569,found:418.1559. 1H NMR (400MHz, DMSO): δ7.95–7.88(m,1H),7.87–7.79(m,1H),7.68–7.58(m,2H),3.98(d,J=1.0Hz,3H),2.78(d,J=1.0Hz,3H),1.53(s,7H). 13 C NMR (101MHz, DMSO): δ195.97,142.07,141.57,129.28,128.78,123.27,115.10,53.90,34.72,21.68,14.17.

[0030] (5) Synthesis of active probe M550: M418 (41.82 mg, 0.1 mmol) was dissolved in dichloromethane (6.0 ml) and triethylamine (100 μl, 1.32 mmol) was added under stirring. After cooling to 0 degrees Celsius, trifluoromethanesulfonic anhydride (Tf2O, 18.7 μl, 0.11 mmol) was added dropwise. The reaction mixture was stirred for 30 minutes and then stirred for 35 minutes after the temperature returned to room temperature. The reaction mixture was diluted with 20 ml of water and then 30 ml of dichloromethane was added for layering. After the layering was completed, the aqueous phase was extracted twice with 30 ml of dichloromethane. The obtained organic layers were combined and concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography with dichloromethane and methanol (volume ratio 60:1) as eluent. The target compound M550 was finally obtained as a purple solid. HR-MS: Calc.for C 27 H 24 ClF3NO4S + :550.1062,found:550.1043. 1 H NMR (500MHz, CDCl3): δ8.56 (d, J = 15.2Hz, 1H), 7.57–7.47 (m, 4H), 7.44 (s, 1H), 7.18 (s, 1H), 6.92–6. 83(m,2H),4.10(s,3H),2.80(t,J=6.0Hz,2H),2.71(t,J=6.1Hz,2H),1.98–1.88(m,2H),1.81(s,6H). 13C NMR (126MHz, CDCl3): δ180.1,157.2,151.2,146.3,145.5,142.4,142.0,133.6,129.7,128.9,128.4,126.8,123.1,123 .0,122.5,121.0(q,J=321.3Hz),118.7(q,J=322.6Hz),116.9,114.0,110.9,109.2,51.6,34.2,29.8,27.8,24.2,20.1.

[0031]

[0032] Example 3: Study on the chemical properties of probe M550

[0033] (1) Study the spectral characteristics of probe M550, such as Figure 1 , M550 at O2 ·- When present, its absorption spectrum is weakened in the range of 500 to 625 nm ( Figure 1 B), and enhanced around 700nm. Under 680nm excitation, M550 in the absence of O2 ·- It shows almost no fluorescence under the condition of O2 ·- With the increase of concentration, the fluorescence intensity at 725nm increased significantly ( Figure 1 C). Further linear analysis showed that the fluorescence intensity of M550 was closely related to O2 ·- There is a good linear relationship between the concentrations ( Figure 1 D)(F725nm=18.484[O2 ·- ]μM+26.82, R2=0.996), the detection limit was 18.6nM O2 ·- In addition, selectivity experiments confirmed that M550 is sensitive to O2 ·- Highly specific ( Figure 1 E). Time-dependent kinetic evaluation showed that M550 provided an adequate response within 5 minutes and remained stable over 24 hours.

[0034] (2) The probe M550 reacts with superoxide anion (O2 ·- )’s detection characteristics are studied, such as Figure 2 Figure A shows that in the 680-725nm spectral range, the probe fluorescence intensity is related to O2 ·- The concentration (0-10 μM) showed a linear positive correlation (R 2 =0.993), the detection limit was 18.6nM (3σ / S), and the probe had no significant cross-reaction with ROS such as H2O2 / ·OH (ΔFI < 5%), showing excellent sensitivity and specificity. Figure 2B / C reveals the dynamic response characteristics of the probe: at 10μM O2 ·- Under stimulation, the fluorescence intensity reached a plateau within 5 minutes (response time t90 = 4.2 ± 0.3 minutes), and the signal decay rate within 24 hours was <3% (RSD = 1.8%), indicating that the probe has temporal stability. Figure 2 The results showed that the probe maintained stable detection efficiency in the pH range of 6.0-8.0, with the optimal working pH being 7.4 (FI = 235 ± 8 a.u.), which is highly compatible with the physiological environment. Extreme pH conditions (<5 or >9) resulted in a signal attenuation of >50%, suggesting the advantage of the probe in a neutral microenvironment. The rapid response characteristics of the M550 probe (t90 <5 min) are related to mitochondrial O2 ·- Its pH stability window (6.0-8.0) perfectly covers the mitochondrial matrix pH (7.0-7.4), which provides an ideal tool for real-time monitoring of mitochondrial oxidative stress dynamics in the HPH model.

[0035] (3) The cytotoxicity of M550 was evaluated and tested using the MTT assay system. Figure 3 The results showed that M550 had low toxicity to PASMCs at concentrations below 20 μM. At low concentrations, M550 had no significant effect on cell viability, confirming its safety as a biological probe.

[0036] (4) Fluorescence co-localization analysis of intracellular mitochondria and lysosomes by probe M550. Specifically, primary PASMCs were cultured in a 25mm glass-bottomed culture dish and incubated with 10μM M550. After incubation at 37°C for 10 minutes, MitoTracker Green (200nM) and Lyso-Tracker-Green (1μM) dyes were added and incubated for 10 minutes. The cells were then washed with PBS and treated with fresh culture medium for imaging. Fluorescence imaging experiments were performed using an OLYMPUS FV3000 fluorescence microscope (Japan). Red channel (Ex=680nm, Em=700-750nm); Mito / Lyso-Tracker green channel (Ex=488nm, Em=500-545nm). The results are shown in Figure 5. Figure 4The upper row of images shows mitochondria labeled with Mito-Tracker Green, while the lower row of images shows lysosomes labeled with Lyso-Tracker Green. Fluorescence microscopy reveals green fluorescence from mitochondria and red fluorescence from lysosomes. Yellow areas in the merged image indicate colocalization of mitochondria and lysosomes, while isolated areas of green or red fluorescence indicate their independent distribution. Colocalization analysis revealed a correlation coefficient (Rr) of 0.925 between Mito-Tracker and the Red Channel, indicating high colocalization; the Rr between Lyso-Tracker and the Red Channel was 0.437, indicating low colocalization. This indicates that the near-infrared fluorescent probe M550 can target mitochondria.

[0037] Example 4: Application of a mitochondrial-targeted near-infrared fluorescent probe in dynamic monitoring of hypoxic pulmonary hypertension

[0038] (1) M550 probe can monitor the reactive oxygen species levels of PASMCs in vitro

[0039] The in vitro PASMCs model and groupings were as follows: 1. Normal group: Cultured in a standard cell culture incubator (37°C, 20% O2, 5% CO2). 2. HPH group: Cultured in a hypoxic cell culture incubator (37°C, 1% O2, 5% CO2). 3. HPH+AU group: Cultured under hypoxic conditions with the addition of 400 μmol / L of aucubin. 4. HPH+LTOT group: Cultured under hypoxic conditions with 12 hours of oxygen-enriched incubator (37°C, 30% O2, 5% CO2) daily.

[0040] The concentration of M550 was selected as 10 μM. After incubation for 15 minutes, the cell images were taken using a confocal microscope under the same conditions. The changes in the fluorescence intensity of the probe M550 in different groups were observed using a confocal microscope. Figure 5 The results showed that compared with the normal control group, in the hypoxia-induced HPH group, M550 detected a large amount of reactive oxygen species, which emitted bright fluorescence under confocal microscopy. Both AU and LTOT-treated PASMCs reduced oxidative stress levels in their corresponding PASMCs, with the AU group showing a more significant effect. This suggests that the M550 probe can be used to detect reactive oxygen species levels in PASMCs across various groups, providing an auxiliary tool for dynamic monitoring of hypoxic pulmonary hypertension.

[0041] (2) The M550 probe can monitor the level of reactive oxygen species in each group of mice

[0042] This study used 8-week-old male C57BL / 6J mice weighing 25 ± 5 g. After 4 weeks of normal culture and hypoxia, followed by 2 weeks of intraperitoneal treatment with aucubin (AU) and low-flow oxygen therapy, mice were randomly divided into four groups: Normal, HPH, HPH+AU, and HPH+LTOT. Three mice were randomly selected from each group. Each mouse underwent a brief 10-15 second gas anesthesia and was suspended by its dentition from a sling. Under monitoring by a small animal tracheal titration display system, 50 μl / mouse of M550 working solution was instilled into the mouse trachea using a 100 μl pipette. The mice were gently patted on the back. Fluorescence imaging was performed. After the drug had fully taken effect, the mice were imaged using a small animal fluorescence imaging system to detect the fluorescence enhancement of the probe M550 upon binding to reactive oxygen species.

[0043] The results are as follows Figure 6 As shown, the fluorescence intensity of the Normal group mice was low, indicating that their reactive oxygen levels were normal. The fluorescence intensity of the HPH group mice was significantly enhanced, indicating that the reactive oxygen levels in the pulmonary hypertension model group were significantly increased. The fluorescence intensities of the HPH+AU group and the HPH+LTOT group were both lower than those of the HPH group, indicating that both the AU group and the LTOT group could reduce the reactive oxygen levels in the HPH mice, among which the fluorescence intensity of the HPH+AU group was the lowest, indicating a significant therapeutic effect; although the fluorescence of the lungs of the HPH+LTOT group mice was reduced, it was not much different from that of the HPH group, indicating that its therapeutic effect was poor. The results of fluorescence changes using the M550 probe showed that both the AU group and the LTOT group could significantly reduce the reactive oxygen levels in the HPH model mice, among which the therapeutic effect of the AU group was better than that of the LTOT group. This shows that the M550 probe can monitor the reactive oxygen levels in each group of mice and can be used as a non-invasive tool for dynamic monitoring of hypoxic pulmonary hypertension. Figure 7 The diagram further shows that the probe M550 can dynamically monitor in vivo the regulation of PI3K / Akt / Nrf2 axis by aucubin to improve hypoxic pulmonary hypertension.

[0044] In summary, the novel superoxide anion fluorescent probe M550 provided by the present invention is prepared by a step-by-step coupling strategy, specifically, M174 is generated by iodomethane quaternization reaction (yield 78.5%), M172 is synthesized by Vilsmeier-Hack reaction (69.2%), M174 and M172 are condensed in a mixed solvent system to obtain M483 (62.4%), 4-chlororesorcinol group is further introduced to generate M418 (55.7%), and finally the target compound M550 (48.9%) is obtained by activation with trifluoromethanesulfonic anhydride.

[0045] Furthermore, spectroscopic studies have shown that the probe M550 undergoes significant photophysical changes under the action of O2·-: the absorbance at 500-625nm decreases by 42%, and a characteristic absorption peak appears at 700nm (ΔA=0.38).

[0046] Furthermore, fluorescence detection showed that its emission intensity was similar to that of O2 ·- The concentration was linearly correlated (R 2 =0.996), with a detection limit of 18.6 nM, which is superior to similar probes reported in the literature (50 nM). In addition, the probe still maintains a response specificity of >95% in the presence of 100-fold concentration of interfering substances.

[0047] The M550 probe developed in this study provides a new tool for the noninvasive detection of HPH. Its high sensitivity (18.6 nM), rapid response (5 minutes), and mitochondrial targeting (colocalization coefficient 0.925) make it suitable for in vivo imaging. Therefore, the M550 probe's mitochondrial targeting design and near-infrared imaging capabilities enable the first in vivo dynamic imaging of ROS in an HPH mouse model, providing a new tool for the early noninvasive diagnosis of oxidative stress in HPH and filling a gap in clinical dynamic monitoring technology.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A mitochondrial-targeted near-infrared fluorescent probe, characterized in that: Its structural formula is as follows:

2. Use of the mitochondria-targeted near-infrared fluorescent probe according to claim 1 in the preparation of a dynamic monitoring tool for hypoxic pulmonary hypertension.

3. The use according to claim 2, characterized in that The probe can monitor the level of reactive oxygen species in mice.

4. The use according to claim 2, characterized in that The probe can dynamically monitor in vivo how aucubin regulates the PI3K / Akt / Nrf2 axis to improve hypoxic pulmonary hypertension.

5. The method for preparing a mitochondrial-targeted near-infrared fluorescent probe according to claim 1, wherein: A near-infrared fluorescent skeleton structure of heptamethine cyanine was obtained by condensation reaction of 2,3,3-trimethylindoline quaternary ammonium salt and 5-chloro-2,4-dialdehyde phenol. Then, an active oxygen ion recognition site based on a cyano-phenol group was introduced through 4-chlororesorcinol. Finally, a mitochondrial-targeted near-infrared fluorescent probe containing a trifluoromethanesulfonate group was obtained by activation with trifluoromethanesulfonic anhydride.

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