Specific fluorescent probe of human monoamine oxidase B and preparation method thereof

Abstract

The invention provides a specific fluorescent probe for human monoamine oxidase B. The invention also provides a preparation method of the specific fluorescent probe of the human monoamine oxidase B. According to the specific fluorescent probe of the human monoamine oxidase B, high-precision imaging of living cell mitochondria is achieved through a double-targeting mechanism of enzyme aiming and charge driving, the selectivity of hMAO-B is obviously higher than that of hMAO-A, and the specific fluorescent probe has high specificity and can be used for early diagnosis and curative effect monitoring of neurodegenerative diseases (such as Parkinson's disease and Alzheimer's disease).

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe technology, and in particular to a specific fluorescent probe for human monoamine oxidase B and its preparation method. Background Technology

[0002] Monoamine oxidase (MAO) is a naturally occurring enzyme in the human body that catalyzes the metabolism of monoamines such as dopamine, playing a crucial role in neurotransmitter metabolism and oxidative homeostasis. MAO includes two closely related subtypes: monoamine oxidase A (hMAO-A) and monoamine oxidase B (hMAO-B), which differ in substrate specificity and tissue distribution. hMAO-A primarily metabolizes serotonin, norepinephrine, and dopamine, and its dysregulation is associated with mental disorders such as depression and schizophrenia. hMAO-B, on the other hand, primarily metabolizes phenylethylamine and dopamine, and its abnormal activity is directly related to neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD). Therefore, hMAO-B is an important biomarker for neurodegenerative diseases.

[0003] Neurodegenerative diseases impose a tremendous physical, emotional, and economic burden on patients and their families by gradually eroding cognitive abilities, motor function, and independence, profoundly disrupting daily life. Due to the aforementioned differences between the two subtypes, specific testing for hMAO-B and hMAO-A is crucial for elucidating disease mechanisms, achieving early diagnosis, and monitoring treatment effectiveness.

[0004] Enzyme-responsive (ER) fluorescent probes, activated by highly specific enzyme catalysis, are suitable tools for detecting human monoamine oxidase (hMAO) activity. They offer superior advantages in sensitivity, spatiotemporal resolution, and in-situ analysis, and can be used for real-time imaging of hMAO activity and disease diagnosis. However, the development of existing ER fluorescent probes has mainly focused on the selective detection of hMAO-A or both isomers. Specific detection of hMAO-B remains insufficient. Summary of the Invention

[0005] There are still technical limitations in the specific detection of hMAO-B. This invention first provides a specific fluorescent probe for human monoamine oxidase B; this invention also provides a method for preparing the aforementioned specific fluorescent probe for human monoamine oxidase B. The specific fluorescent probe for human monoamine oxidase B provided in this application achieves high-precision imaging of mitochondria in live cells through a dual-targeting mechanism of enzyme targeting and charge-driven targeting. It exhibits significantly higher selectivity for hMAO-B than hMAO-A, demonstrating high specificity, and can be used for the early diagnosis and treatment monitoring of neurodegenerative diseases (such as Parkinson's disease and Alzheimer's disease).

[0006] A specific fluorescent probe for human monoamine oxidase B has the structure shown in Formula I:

[0007] Formula I.

[0008] The method for preparing the above-mentioned specific fluorescent probe for human monoamine oxidase B includes: The reaction precursor of Formula II was reacted with 1-methyl-1,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester to give the compound of Formula I;

[0009] Formula II.

[0010] Preferably, it includes the following steps: Under an inert atmosphere, the reaction precursor of formula II, 1-methyl-1,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester, reaction aid and catalyst are added to the solvent, and the reaction is carried out at a temperature of 56~76℃ for 12~14h to obtain the product.

[0011] Preferably, the molar ratio of the reaction precursor of Formula II, 1-methyl-1,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester, reaction aid and catalyst is 10:(8-20):(40-100):(0.3-2).

[0012] Preferably, the inert atmosphere is any one of nitrogen, helium, or argon; The solvent is any one of tetrahydrofuran aqueous solution, dioxane aqueous solution, methyltetrahydrofuran aqueous solution, and dimethyl ethylene diether aqueous solution; The reaction aid is either potassium carbonate or sodium carbonate. The catalyst is Pd(PPh3)4.

[0013] Preferably, in the solvent, the volume ratio of tetrahydrofuran or dioxane, or methyltetrahydrofuran, or dimethyl ethylene ether to water is (10~1):1.

[0014] Preferably, after the reaction is complete, the product is cooled to room temperature, the organic layer is extracted with dichloromethane, dried and the solvent is removed, and separated by silica gel column chromatography using a mobile phase of petroleum ether / ethyl acetate = (10~2):1 to obtain the compound of formula I.

[0015] Preferably, anhydrous sodium sulfate is used for drying, and then the solvent is removed by rotary evaporation under reduced pressure.

[0016] The compounds of Formula I provided in this application use a substituted amine as the identification moiety. Substituted amines, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), are converted to MPP under hMAO-B catalysis.+ It simultaneously forms an optimized D-Π-A framework and acquires a positive charge. These properties synergistically enhance signal strength and mitochondrial binding capacity.

[0017] In existing technologies, a few fluorescent probes use amines as leaving groups, which have problems such as limited signal enhancement (usually <50-fold), lack of cell sub-localization ability, lack of "wash-free" operation design, and affecting the accuracy of live cell dynamic imaging. In contrast, this application designs a novel hMAO-B selective fluorescent probe THT-MTP by using N-methyltetrahydropyridine (MTP), a substituted amine, as the recognition part.

[0018] This application mimics the metabolic pathway of the natural hMAO-B substrate MPTP (→MPP) + The recognition component MTP used is catalytically oxidized by hMAO-B to form MPy. + An optimized D-Π-A framework was constructed, which enhances the electron-donating effect, significantly narrows the HOMO-LUMO band gap, promotes electronic transitions, thereby generating fluorophores and inducing a significant fluorescence-on response. The fluorescent probe provided in this application can achieve hMAO-B specific activation (without hMAO-A cross-reactivity), can be activated in situ within cells, and exhibits >100-fold fluorescence enhancement after enzyme activation, improving selectivity and signal amplification capabilities; it precisely locates mitochondria based on an enzyme-targeting + charge-driven dual-targeting mechanism, and supports wash-free live-cell imaging without additional washing steps.

[0019] This application represents a significant breakthrough in specificity, sensitivity, and applicability to neurological disease research. It provides a simple method for rapid and highly selective monitoring of hMAO-B activity and has great potential for the early diagnosis of hMAO-B-related pathologies. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a synthesis route diagram of the probe THT-MTP in Example 1 of the present invention.

[0022] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the probe THT-MTP in Example 1 of the present invention.

[0023] Figure 3This is the carbon NMR spectrum of the probe THT-MTP in Example 1 of this invention.

[0024] Figure 4 THT-MPy in Embodiment 2 of the present invention + The synthesis route map.

[0025] Figure 5 The concentrations of 5 μM THT-MTP (pink) and THT-MPy in Example 3 of this invention are... + (Blue) UV-Vis absorption spectrum.

[0026] Figure 6 The THT-MTP (pink, 5 μM, λ) in Example 3 of this invention ex =355 nm) and THT-MPy + (Blue, 5 μM, λ) ex Fluorescence emission spectrum (413 nm)

[0027] Figure 7 The fluorescence emission spectrum of THT-MTP (5 μM) and hMAO-B (blue) / hMAO-B+ inhibitor safenamide (pink) in Example 4 of this invention, after incubation at 37°C for 2 h.

[0028] Figure 8 The fluorescence emission spectra of THT-MTP in Example 4 of this invention, after incubation with different concentrations of hMAO-B (0, 1, 2, 5, 10 and 20 μg / mL) at 37°C for 2 h.

[0029] Figure 9 The fluorescence intensity of THT-MTP (5 μM) changes over time after adding hMAO-A or hMAO-B (20 μg / mL) to PBS in Example 5 of this invention.

[0030] Figure 10 This is a molecular docking diagram of the crystal structures of THT-MTP with hMAO-A (left) and hMAO-B (right) in Example 6 of the present invention.

[0031] Figure 11 This is a comparison of cell viability after treating various cells (SH-SY5Y, HepG2, and NIH-3T3) with different concentrations of THT-MTP (0, 1, 4, and 8 μM) in Example 7 of this invention.

[0032] Figure 12 This is a comparison of fluorescence intensity after treating various cells (SH-SY5Y, HepG2, and NIH-3T3) with different concentrations of THT-MTP (0, 1, 4, and 8 μM) in Example 7 of this invention.

[0033] Figure 13 The images show fluorescence images of different cells after incubation with THT-MTP (5 μM) for 0-120 min in Example 7 of this invention.

[0034] Figure 14 This is a fluorescence image of THT-MTP (5 μM) and a cell HepG2 / HepG2+ inhibitor incubated at 37°C for 2 h in Example 7 of this invention.

[0035] Figure 15 This is a co-localization image of HepG2 and NIH-3T3 with THT-MTP and Mito Tracker Red in Embodiment 8 of the present invention. Detailed Implementation

[0036] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0037] In the following experiments, the compound of formula I is abbreviated as THT-MTP, and the reaction precursor of formula II is abbreviated as THT-Br.

[0038] Example 1: Synthesis of the probe THT-MTP Under nitrogen (N2) conditions, the precursors THT-Br (81 mg, 0.25 mmol), 1-methyl-1,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester (72 mg, 0.32 mmol), K2CO3 (345 mg, 2.5 mmol), and Pd(PPh3)4 (29 mg, 0.025 mmol) were added to a tetrahydrofuran (THF) / water (H2O) (7.5 / 2.5 mL) solution. The mixture was refluxed at 66 °C for 12 h, cooled to room temperature, and the organic layer was extracted with dichloromethane (DCM) and dried over anhydrous sodium sulfate. The solvent was then evaporated under reduced pressure, and the mixture was separated by silica gel column chromatography (PE / EA = 2 / 1, i.e., petroleum ether / ethyl acetate = 2 / 1) to obtain a yellow solid product (yield 89%).

[0039] Characterization data: 1 H NMR (500 MHz, CDCl3) δ 8.68 (d, J= 8.0 Hz, 1H), 8.20 (s,1H), 7.68 – 7.55 (m, 3H), 7.41 (d, J = 4.0 Hz, 1H), 6.97 (d, J = 4.0 Hz, 1H), 6.20 (s, 1H), 3.19-3.15 (m, 2H), 2.15-2.68 (m, 2H), 2.68-2.62 (m, 2H), 2.44(s, 3H). 13 C NMR (126 MHz, CDCl3) δ 177.2, 146.7, 136.2, 135.0, 132.1, 131.8,131.3, 129.6, 129.5, 129.3, 127.9, 126.5, 125.1, 121.4, 120.8, 54.6, 51.9, 45.6, 28.0.

[0040] HRMS (ESI) m / z calculated C 19 H 17 NOS2[M+H] + 340.0216, found 340.0219. The reaction route is as follows: Figure 1 As shown. The 1H and 1C NMR spectra of THT-MTP are as follows. Figure 2 and Figure 3 As shown.

[0041] To mimic the metabolic pathway of the natural substrate hMAO-B, MPTP (→MPP) + Design the following experiment to synthesize THT-MPy + : Example 2 THT-MPy + Synthesis Step 1, Preparation of THT-Py: Under nitrogen (N2) conditions, a mixture of the precursors THT-Br (100 mg, 0.31 mmol), pinacol 4-pyridineborate (80 mg, 0.39 mmol), K2CO3 (427 mg, 3.1 mmol), and Pd(PPh3)4 (36 mg, 0.03 mmol) was added to a tetrahydrofuran (THF) / water (H2O) (7.5 / 2.5 mL) solution. The mixture was refluxed at 66 °C and reacted for 12 h. After cooling the mixture to room temperature, the organic layer was extracted with dichloromethane (DCM) and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the mixture was separated by silica gel column chromatography (PE / DCM / EA = 3 / 1 / 1 to 1 / 1 / 1) to obtain 90 g of yellow solid product (yield 89%).

[0042] Characterization data: 1 H NMR (500 MHz, Chloroform-d) δ 8.69 (d, J = 7.0 Hz, 1H), 8.58 (d, J = 6.5 Hz, 2H), 8.33 (s, 1H), 7.65 (d, J = 4.0 Hz, 2H), 7.63-7.58(m, 1H), 7.56 – 7.53 (m, 2H), 7.50 – 7.47 (m, 2H). 13C NMR (126 MHz, CDCl3) δ177.08, 150.39, 142.41, 141.75, 138.58, 136.23, 133.11, 131.98, 131.65,129.72, 128.60, 128.30, 126.66, 125.52, 124.65, 119.80.

[0043] HRMS (ESI) m / z calculated C18H11NOS2 [M+H]+: 322.0286, found322.0291.

[0044] Step 2, THT-MPy + Preparation: Iodomethane (75 μL, 1.2 mmol) was slowly added to a 10 mL solution of THT-Py (64 mg, 0.2 mmol) synthesized in step one in N,N-dimethylformamide (DMF), and the mixture was stirred overnight at room temperature in the dark. After the reaction was complete, anhydrous diethyl ether was added to precipitate the compound, and the purified product yielded a yellow solid compound (50 mg), with a yield of 54%.

[0045] Characterization data: 1 H NMR (500 MHz, DMSO- d 6) δ 9.45 (s, 1H), 8.84 (d, J = 6.5 Hz, 2H), 8.56 (d, J = 8.0 Hz, 1H), 8.38-8.34 (m, 2H), 8.29-8.25 (m, 1H), 8.07 (d, J =8.0 Hz, 1H), 7.97-7.94 (m, 1H), 7.86 (t, J = 7.5 Hz, 1H), 7.76 (t, J= 7.5 Hz, 1H), 4.25 (s, 3H). 13 C NMR (126 MHz, DMSO- d 6) δ 176.4, 148.5, 145.8, 143.7,138.9, 137.7, 136.5, 132.7, 131.4, 131.2, 129.2, 129.1, 128.0, 127.2, 126.5,122.0, 47.2.

[0046] HRMS (ESI) m / z calculated C 19 H 14 NOS2[M] + : 336.0501, found 336.0510.

[0047] The reaction route is as follows Figure 4 As shown.

[0048] Example 3 Chemical properties of the probe Prepare 5 μM THT-MTP and THT-MPy + The UV-Vis spectra of the compound were recorded using a UV-Vis spectrometer in a DMSO solution. The results are as follows: Figure 5 As shown in the figure. It can be seen that, compared to THT-MTP, THT-MPy... + The maximum absorption peak shifted from 355 nm to 413 nm, and the absorption intensity increased by more than two times.

[0049] Prepare 5 μM THT-MTP and THT-MPy + The fluorescence spectra of the compound were recorded using DMSO solution excited at 355 nm and 413 nm, respectively. The results are as follows: Figure 6 As shown, under 355 nm excitation, the maximum value of the THT-MTP fluorescence spectrum is at 518 nm, and the signal intensity is negligible, while THT-MPy... + Upon excitation at 413 nm, the signal intensity at a maximum of 515 nm showed an increase of over 100-fold. This significant difference in photophysical behavior confirms the enzyme activation "on" mechanism of the probe.

[0050] Example 4 Selective Verification At 37°C, the probe (5 μM) was incubated with the corresponding enzyme (human monoamine oxidase B, hMAO-B, 20 μg / ml) and the h-MAO inhibitor safenamide in PBS buffer (pH 7.4) for 2 h, and fluorescence spectra were obtained. The results are as follows: Figure 7As shown, the solution treated with hMAO-B elicited a significant fluorescence-on response at 515 nm, which was completely suppressed after pretreatment with safenamide, confirming the probe's specific selectivity for hMAO-B. Importantly, increasing the hMAO-B concentration (0-20 μg / mL) induces THT-MPy. + The concentration-dependent increase in fluorescence intensity indicates a strong enzyme activation response (e.g., Figure 8 (As shown).

[0051] Example 5: Rapid Response After adding 20 μg / mL hMAO-A or hMAO-B to a PBS solution of THT-MTP (5 μM), the THT-MTP (λ) level was monitored using a microplate reader. ex / em The fluorescence intensity change over time at 413 / 515 nm. Figure 9 Kinetic analysis showed that the fluorescence intensity of hMAO-B increased rapidly, with a signal enhancement of >90% within 40 minutes, while the hMAO-A system showed very little change.

[0052] Example 6: Molecular docking verification of selectivity Autodock was used to test the binding of THT-MTP to hMAO-A and hMAO-B proteins. Results are as follows: Figure 10 As shown, THT-MTP can bind well to the active pocket of hMAO-B. The thiophene group exhibits good hydrogen bonding interactions with Tyr326 (3.5 Å) and Cys172 (3.7 Å), while the thiophene ring forms a strong hydrogen bond with Tyr36 (2.6 Å). Docking results show that the binding energy of THT-MTP to hMAO-B (-11.9 kcal / mol) is significantly lower than that of hMAO-A (-10.9 kcal / mol), indicating that THT-MTP has good selectivity for hMAO-B.

[0053] Example 7: Detection of intracellular monoamine oxidase B activity Cell Culture: It is well known in the field that the SH-SY5Y cell line is a neuroblastoma subclonal cell derived from human bone marrow, possessing morphological and biological characteristics similar to human neurons, and is widely used in research on the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD). HepG2 cells are human liver cancer cell lines, and NIH-3T3 are mouse embryonic fibroblasts. The MAO concentration in these three cell lines decreases sequentially, and the activity and selectivity of THT-MTP can be verified through differences in signal intensity.

[0054] Logarithmically growing SH-SY5Y, HepG-2, and NIH-3T3 cells were digested with trypsin to prepare single-cell suspensions. The cell suspensions were seeded in 100 μL DMEM 96-well plates containing 10% fetal bovine serum or 10% NBCS and cultured at 37°C in a 5% CO2 incubator for 24 hours.

[0055] Cytotoxicity assay: After cell culture as described above, the old culture medium was aspirated and discarded. Then, 100 μL of DMEM containing different concentrations of the probe THT-MTP (8, 4, 1, and 0 μM) was added and the cells were incubated for 24 hours. Next, 20 μL of MTT was added to each well, and after incubation for 4 hours, the supernatant was discarded. 150 μL of DMSO was added to dissolve the crystals for 10 minutes until completely dissolved. The OD value was then measured at 490 nm using a microplate reader. Results are as follows: Figure 11 As shown, cells treated with graded probe concentrations (0-8 μM) for 24 hours exhibited a survival rate of >90% in all cell lines.

[0056] Cell imaging: Cells were cultured as described above. After cell attachment, the culture medium was removed, washed three times with PBS, and then culture medium containing 5 μM THT-MTP was added. After incubation at 37°C for different times (0, 0.5, 1, and 2 hours), the plates were washed three times with PBS. A control group was also set up; cells cultured in the same CO2 incubator were incubated with THT-MTP and the h-MAO inhibitor safinamide for 3 hours, with all other treatments being the same. Cell fluorescence imaging was performed using a cell imaging system, and the results are as follows: Figure 12-13 As shown, HepG-2 cells exhibited rapid fluorescence enhancement (50% signal intensity at 30 min) compared to SH-SY5Y cells (40% signal intensity at 60 min), while NIH-3T3 cells showed no significant fluorescence emission, consistent with the endogenous hMAO-B expression level. Furthermore, as... Figure 14 As shown, HepG-2 cells pretreated with inhibitors do not exhibit fluorescence.

[0057] Example 8: Mitochondrial colocalization and wash-free imaging HepG-2 and NIH-3T3 cells in logarithmic growth phase were seeded in cell culture dishes and cultured at 37°C in a 5% CO2 incubator for 24 h. After cell attachment, the culture medium was removed, and the cells were washed three times with PBS. Then, 5 μM THT-MTP and MitoTracker Red (mitochondrial red fluorescent probe) were added, and the cells were cultured for 2 h, followed by three washes with PBS. Cell fluorescence imaging was performed using a cell imaging system. (THT-MTP: λ) ex =488 nm, λ em=500-550 nm; Mito Tracker Red: λ ex =488 nm, λ em =600-650 nm). The results are as follows: Figure 15 As shown, after hMAO-B catalytic activation, the generated metabolite THT-MPy⁺ acquires a positive charge due to the mitochondrial membrane potential-dependent electrophoretic gradient, synergistically driving mitochondrial accumulation. Therefore, the commercial reagent Mito Tracker Red was used to quantify mitochondrial colocalization in HepG2 cells. A high Pearson correlation (R=0.81) indicates preferential accumulation of the probe in mitochondria. The intensity of hMAO-B expression in cells is proportional to the enzyme level. This suggests that the THT-MTP probe is a wash-free, dual-targeting mitochondrial probe with excellent cell permeability.

[0058] The above description is merely an embodiment of the present invention. It should be noted that those skilled in the art can make improvements without departing from the inventive concept of the present invention, but these improvements all fall within the protection scope of the present invention.

Claims

1. A specific fluorescent probe for human monoamine oxidase B, characterized in that, It has the structure shown in Equation I: Formula I.

2. The method for preparing the specific fluorescent probe for human monoamine oxidase B according to claim 1, characterized in that, include: The reaction precursor of Formula II was reacted with 1-methyl-1,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester to give the compound of Formula I; Formula II.

3. The preparation method according to claim 2, characterized in that, Includes the following steps: Under an inert atmosphere, the reaction precursor of formula II, 1-methyl-1,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester, reaction aid and catalyst are added to the solvent, and the reaction is carried out at a temperature of 56~76℃ for 12~14h to obtain the product.

4. The preparation method according to claim 3, characterized in that, The molar ratio of the reaction precursor of Formula II, 1-methyl-1,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester, reaction aid and catalyst is 10:(8~20):(40~100):(0.3~2).

5. The preparation method according to claim 3, characterized in that, The inert atmosphere is any one of nitrogen, helium, or argon. The solvent is any one of tetrahydrofuran aqueous solution, dioxane aqueous solution, methyltetrahydrofuran aqueous solution, and dimethyl ethylene diether aqueous solution; The reaction aid is either potassium carbonate or sodium carbonate; The catalyst is Pd(PPh3)4.

6. The preparation method according to claim 5, characterized in that, In the solvent, the volume ratio of tetrahydrofuran or dioxane, or methyltetrahydrofuran, or dimethyl ethylene ether to water is (10~1):

1.

7. The preparation method according to claim 3, characterized in that, After the reaction was complete, the product was cooled to room temperature, the organic layer was extracted with dichloromethane, dried and the solvent was removed, and the product was separated by silica gel column chromatography using a mobile phase of petroleum ether / ethyl acetate = (10~2):1 to obtain the compound of formula I.

8. The preparation method according to claim 7, characterized in that, The solvent was removed by drying with anhydrous sodium sulfate and then by rotary evaporation under reduced pressure.

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