A pet molecular probe targeting AQP-4 and a preparation method and application thereof

Abstract

The application discloses a PET molecular probe targeting AQP-4 and a preparation method and application thereof. Based on the unsuccessful copper salt catalyzed aromatic ring 18F-CF3 labeling strategy, a new labeling precursor and molecular probe are prepared 18 F]AEF-EF, the PET molecular probe is prepared through the labeling of a new labeling precursor AER-OTs by a radionuclide 18 F, the probe has high radiochemical purity and good chemical stability, can be specifically combined with a target molecule AQP-4, and can target AQP-4 imaging through animal in-vivo Micro-PET scanning; therefore, the application provides intuitive in-vivo evolution molecular imaging for early diagnosis, curative effect monitoring and treatment research of AD related lymphoid lesions, and lays a foundation for further research and development of new drugs for delaying or preventing AD development.

Description

Technical Field

[0001] This invention relates to a PET molecular probe targeting AQP-4, its preparation method, and its application, belonging to the field of bioengineering technology. Background Technology

[0002] With the increasing aging of the population, Alzheimer's disease (AD) has become one of the most important public health problems seriously affecting the health and quality of life of the global population. The latest data from 2022 predicts that the growth rate of AD in my country will be higher than the global average. However, there is currently no effective method to fundamentally prevent or slow the occurrence, development, or reduction of the lethality of AD by stopping the destruction and degeneration of neurons. One of the main reasons for this situation is the late intervention in treatment. Increasing research suggests that effective intervention goals can be achieved in the early reversible stage of AD. However, early diagnosis and treatment of AD remain very challenging, mainly due to the lack of ideal early diagnostic biomarkers that are well-correlated with cognitive function outcomes, which is also a pressing need. AD is characterized by the abnormal accumulation of β-amyloid (Aβ) and Tau protein, but the exact pathogenesis of AD, especially the pathological mechanisms in the early reversible stage, still requires further in-depth research.

[0003] Positron emission tomography (PET) neuroimaging biomarkers have advantages such as high diagnostic sensitivity and specificity, ease of operation, and non-invasiveness. Moreover, they can specifically and intuitively assess functional changes, making them one of the hot topics and important trends in AD research.

[0004] Recent domestic and international studies have suggested the existence of a glial-meningeal lymphatic system within the central nervous system. Similar in function to the peripheral lymphatic system in clearing metabolic waste, and possessing a characteristic dependent on astrocyte aquaporin 4 (AQP4), it is termed a "glymphatic system." Animal studies indicate that abnormalities in the glymphatic system are an early pathological change in Alzheimer's disease (AD) and are reversible, providing new possibilities for the early diagnosis and treatment of AD. AQP4 is polarly expressed and distributed in the terminal appendages of astrocytes. Moreover, the formation of astrocyte terminal appendages and the polar expression of AQP4 are temporally consistent with the formation of the glymphatic system, suggesting that AQP4 may be related to the operation of the glymphatic system. The glymphatic system promotes the exchange and circulation of cerebrospinal fluid (CSF) and interstitial fluid (ISF) through AQP4 channels. Simultaneously, the perivascular pathway network of the glymphatic system also promotes the clearance of interstitial solutes such as Aβ through AQP4. Summary of the Invention

[0005] The purpose of this invention is to address the current technical limitations of MRI, which mainly uses intrathecal gadolinium contrast agent to reflect lymphoid function, but lacks an effective PET imaging agent for assessing lymphoid function. Furthermore, MRI is invasive and its clinical application is limited, and it suffers from poor specificity. This invention provides a PET molecular probe targeting AQP-4 (hereinafter referred to as the AQP-4 molecular probe), its preparation method, and its application. This PET molecular probe can target functional metabolic changes associated with AD-related lymphoid alterations, enabling non-invasive, specific, and intuitive dynamic assessment and prediction of brain function changes.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a PET molecular probe targeting AQP-4, the chemical structural formula of which is shown below:

[0008]

[0009] Secondly, the present invention provides a method for preparing the above-mentioned PET molecular probe, wherein the PET molecular probe is prepared by radioactive nuclide from the precursor compound AER-OTs. 18 The marker for F is obtained, and the marked route is shown below:

[0010]

[0011] Preferably, the synthetic route for the precursor compound AER-OTs is as follows:

[0012]

[0013] Preferably, the synthetic route for the precursor compound AER-OTs is as follows:

[0014]

[0015] Thirdly, the present invention provides the application of the PET molecular probe described in the first aspect in the preparation of early diagnosis of AD-related lymphatic lesions.

[0016] Fourthly, the present invention provides the use of the PET molecular probe described in the first aspect in the preparation of reagents or kits for monitoring the progression of AD-related lymphoid lesions or the efficacy of treatment.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) This invention prepares PET molecular probes through an optimized and improved scheme. 18F]AER-EF, the PET molecular probe has high radiochemical purity and good chemical stability, can specifically bind to the target molecule AQP-4, and can be visualized in vivo.

[0019] (2) The molecular probe targeted AQP-4 type lymphocyte PET imaging provided by this invention provides intuitive in vivo evolution molecular images for early diagnosis, efficacy monitoring and treatment research of AD-related type lymphocyte damage, and lays the foundation for further development of new drugs to delay or prevent the development of AD. Attached Figure Description

[0020] Figure 1 The 1H NMR spectrum of the AER-270 cold product in Example 1;

[0021] Figure 2 The carbon NMR spectrum of the AER-270 cold product in Example 1;

[0022] Figure 3 The mass spectrum of the cold product of AER-270 in Example 1;

[0023] Figure 4 The 1H NMR spectrum of the AER-270-I intermediate in Example 1;

[0024] Figure 5 The carbon NMR spectrum of the AER-270-I intermediate in Example 1;

[0025] Figure 6 This is the mass spectrum of the AER-270-I intermediate in Example 1;

[0026] Figure 7 The 1H NMR spectrum of the precursor AER-270-Pre in Example 1;

[0027] Figure 8 The carbon NMR spectrum of the precursor AER-270-Pre in Example 1;

[0028] Figure 9 This is the mass spectrum of the precursor AER-270-Pre in Example 1;

[0029] Figure 10 The HPLC analysis chromatogram of the AER-270 molecular probe in Example 1;

[0030] Figure 11 The HPLC chromatogram of the precursor compound AER-270-Pre in Example 1 is shown below.

[0031] Figure 12 This is the HPLC chromatogram of the preparation and separation of the precursor compound AER-270-Pre and the labeled product AER-270 by HPLC in Example 1;

[0032] Figure 13 The 1H NMR spectrum of the intermediate compound AER-OTs in Example 2;

[0033] Figure 14 The carbon NMR spectrum of the intermediate compound AER-OTs in Example 2;

[0034] Figure 15 The mass spectrum of the intermediate compound AER-OTs in Example 2;

[0035] Figure 16 The 1H NMR spectrum of the precursor compound AER-EF in Example 2;

[0036] Figure 17 The carbon NMR spectrum of the precursor compound AER-EF in Example 2;

[0037] Figure 18 The mass spectrum of the precursor compound AER-EF in Example 2;

[0038] Figure 19 The liquid phase diagram is for AER-EF molecular probe standards;

[0039] Figure 20 This is the HPLC quality control chromatogram of the AER-EF molecular probe.

[0040] Figure 21 AQP-4 coupled sensor diagram;

[0041] Figure 22 The results show the interaction between AQP4 and the AER-270 molecular probe standard.

[0042] Figure 23 The results show the interaction between AQP4 and the AER-EF molecular probe standard.

[0043] Figure 24 The Time-Activity Curve (TAC) of the molecular probe in different brain regions at 90 minutes shows that the drug is rapidly taken up by the brain after injection, then washed off, and reaches equilibrium in about 40 minutes.

[0044] Figure 25 Images show the Micro-PET scan results of AQP-4KO model mice and normal controls; where A: PET imaging of AQP-4KO model mice; B: Quantitative analysis of each brain region of AQP-4KO model mice; C: PET imaging of corresponding WT controls; D: Quantitative analysis of each brain region of corresponding WT controls. Detailed Implementation

[0045] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0047] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0048] Example 1: Synthesis of AER-270, a PET molecular probe targeting AQP-4, and its precursor

[0049] 1. Synthesis of AER-270 molecular probe precursor

[0050]

[0051] 1) Synthesis of cold products from AER-270

[0052] 206 mg of phosphorus trichloride (1.5 mmol) was added to 10 mL of toluene solution containing 229 mg (1 mmol) of 3,5-di(trifluoromethyl)aniline and 207 mg (1.2 mmol) of 5-chlorosalicylic acid. The mixture was heated and stirred at 100 °C for 8 hours. After the reaction was completed and cooled to room temperature, the solution was diluted with water, and sodium bicarbonate solution was added dropwise to adjust the pH to 6. The solution was extracted three times with ethyl acetate, and the combined organic phases were concentrated to obtain a crude product. Further purification by silica gel column chromatography yielded 104 mg of the white target product (yield 27%). 1 H NMR (400MHz, CDCl3) δ11.35(s,1H),8.20(s,1H),8.14(s,2H),7.72(s,1H),7 .56(d,J=2.4Hz,1H),7.45(dd,J=8.9,2.4Hz,1H),7.03(d,J=8.9Hz,1H).13C NMR (101MHz, CDCl3) δ167.4,160.2,138.1,135.3,132.9,132.5,125.3,124.2,120.7,114.9.ESI-MS m / z:[MH] - Calculated for C 15 H7ClF6NO2: 382.01, found: 382.24. Its characterization spectrum is as follows: Figures 1-3 As shown.

[0053] 2) Synthesis of intermediate AER-270-I

[0054] The procedure was the same as above. 206 mg of phosphorus trichloride (1.5 mmol) was added to 10 mL of toluene solution containing 287 mg (1 mmol) of 3-iodo-5-trifluoromethylaniline and 207 mg (1.2 mmol) of 5-chlorosalicylic acid. The mixture was heated and stirred at 100 °C for 8 hours. After the reaction was complete and cooled to room temperature, the solution was diluted with water, and sodium bicarbonate solution was added dropwise to adjust the pH to 6. The solution was extracted three times with ethyl acetate, and the combined organic phases were concentrated to obtain the crude product. Further purification by silica gel column chromatography yielded 163 mg of a white solid (yield 37%). 1 HNMR (400MHz, CDCl3): δ11.43(s,1H),8.22(s,1H),8.05(s,1H),7.88(s,1H),7.79 (s,1H),7.52(d,J=2.4Hz,1H),7.42(dd,J=8.9,2.4Hz,1H),7.01(d,J=8.9Hz,1H). 13 C NMR (101MHz, CDCl3): δ167.2,160.2,138.0,135.2,132.7,130.9,125.2,124.1,120.7,117.1,115.0,94.0.ESI-MS m / z:[MH] - Calculated for C 14 H7ClF3INO2: 439.92, found: 440.18. Its characterization spectrum is as follows: Figures 4-6 As shown.

[0055] 3) Synthesis of the precursor AER-270-Pre

[0056] 135 mg of AER-270-I (305 μmol) and 80 μL of diisopropylethylamine (460 μmol) were dissolved in 20 mL of dichloromethane. 50 mg of bromomethyl methyl ether (400 μmol) was added dropwise under an ice-water bath. The ice-water bath was removed, and the reaction was continued at a lower temperature for 4 hours. After the reaction was complete, 20 mL of ammonium chloride aqueous solution was added to quench the reaction. The organic layer was concentrated and precipitated by column chromatography to give 91 mg of a white solid product (yield 61%). 1 H NMR (400MHz, CDCl3): δ9.81(s,1H),8.25(t,J=1.8Hz,1H),8.22(d,J=2.7Hz,1H),7.90(s,1H ),7.72(s,1H),7.45(dd,J=8.9,2.7Hz,1H),7.20(d,J=8.9Hz,1H),5.43(s,2H),3.59(s,3H). 13C NMR (101MHz, CDCl3): δ162.1,153.5,139.5,133.5,132.1,131.9,129.9,128.5,123.2,116.9,116.3,96.2,94.0,57.2.ESI-MS m / z:[MH] - Calculated for C 16 H 11 ClF3INO3: 483.94, found: 484.21. Its characterization spectrum is as follows: Figure 7-9 As shown.

[0057] 2. Synthesis of AER-270 molecular probe standards:

[0058] 1) Cold labeling condition screening

[0059]

[0060] Reaction steps: The catalyst (CuI) was added to KF / K2.2.2 acetonitrile solution and dried twice under N2 flow at 100℃; the precursor AER-270-Pre (2mg), TMEDA, and ClCF2COOMe were dissolved in anhydrous DMF (0.5mL); the two were mixed and heated to react for 20min; after cooling, 0.5mL of HCl solution (6M in dioxane) was added to the reaction solution and reacted at 100℃ for 15min; after cooling, 50μL of the reaction solution was taken and diluted to 500μL for liquid phase detection.

[0061]

[0062]

[0063] In the table, Ratio represents the molar ratio of AER-270-Pre:KF / K2.2.2:ClCF2COOMe:CuI:TMEDA.

[0064] Catalyst screening

[0065]

[0066] Catalyst screening: CuI, CuOTf (AER-270-Pre:KF / K2.2.2(Kryptofix 222):ClCF2COOMe=1:2:2)

[0067] Entry Catalyst Temp.℃ Yield 1 CuI 3eq 150 No detected 2 CuOTf 3eq 150 No detected 3 CuI / phenanthroline 3eq 150 11％ 4 CuI / proline 3eq 150 16％

[0068] Based on the above screening results, the labeling scheme is: AER-270-Pre:ClCF2COOMe:CuI:TMEDA = 1:2:3:3, reacted by heating at 150℃. The screened labeling scheme will be used as the conditions for the following thermal labeling.

[0069] 2) Thermal labeling

[0070]

[0071] No product was obtained from the thermal labeling reaction.

[0072] 3) Screening of HPLC analytical conditions:

[0073] A. HPLC analysis conditions:

[0074] Mobile phase A: water (0.1% TFA), mobile phase B: methanol; elution gradient: 0–6 min, 75–95% B; 6–15 min, 95% B; chromatographic column: 0.5 μm × 4.5 mm × 150 mm ODS C18 column.

[0075] Under these analytical conditions, the HPLC chromatograms of the precursor AER-270-Pre and the AER-270 molecular probe standard are as follows: Figure 10-11 As shown, the retention times of the two are similar, making them unsuitable as HPLC analysis conditions.

[0076] B. HPLC analysis conditions:

[0077] Mobile phase A = water (0.1% TFA), mobile phase B = acetonitrile; elution gradient: 0–15 min, 75–95% B; 15–20 min, 95% B; chromatographic column: 0.5 μm × 10 mm × 250 mm ODS C18 column.

[0078] Under these conditions, the HPLC analysis results are as follows: Figure 12 As shown, this condition can distinguish between the precursor AER-270-Pre and the AER-270 molecular probe standard; therefore, this condition is used as the HPLC analysis condition.

[0079] Example 2: Synthesis of AER-EF molecular probe

[0080] Because in Example 1, radionuclide labeling was prepared using the precursor AER-270-Pre compound [ 18 The labeling reaction of F]AER-270 was unsuccessful. In this embodiment, a new precursor was designed and synthesized, and then... 18 Preparation of PET molecular probes by F radionuclide labeling [ 18 F]AER-EF:

[0081] 1. Synthesis of AER-EF molecular probe standards

[0082]

[0083] 1) Synthesis of AER-OTs

[0084] AER-270 (384 mg, 1 mmol), ethylene glycol di-p-toluenesulfonate (408 mg, 1.1 mmol), and potassium carbonate (166 mg, 1.2 mmol) were added to a round-bottom flask containing 25 mL of acetonitrile. The mixture was heated and stirred at 80 °C for 4–6 hours. After the reaction was completed and cooled, crude silica gel was added, the mixture was concentrated, and further purified by silica gel column chromatography to obtain 460 mg of the white target product (yield 79%). 1 H NMR(400MHz, CDCl3)δ9.82(s,1H),8.26–8.18(m,3H),7.70(d,J=8.3Hz,2H),7.63(s,1H),7.46(dd,J=8.8,2 .8Hz,1H),7.24(d,J=8.3Hz,2H),6.91(d,J=8.8Hz,1H),4.59–4.52(m,2H),4.51–4.43(m,2H),2.40(s,3H). 13 C NMR (101MHz, CDCl3) δ162.3,154.1,145.8,139.5,133.5,132.8,132.4,132.4,1 32.1,130.1,128.3,127.8,123.0,120.7,117.8,114.0,67.6,67.1,21.7.ESI-MS m / z:[M+H] + Calculated for C 24 H 19 ClF6NO5S: 582.06, found: 582.30. Its characterization spectrum is as follows: Figure 13-15 As shown.

[0085] 2) Synthesis of AER-EF molecular probe standards

[0086] 291 mg of AER-OTs was dissolved in anhydrous acetonitrile, and 1 mL of tetrabutylammonium fluoride solution (1 M in THF) was added. The mixture was heated and stirred at 80 °C for 2 hours under nitrogen protection. After the reaction was completed and cooled, crude silica gel was added, the mixture was concentrated, and further purified by silica gel column chromatography to obtain 154 mg of the white target product (yield 72%). 1H NMR (400MHz, CDCl3) δ10.17(s,1H),8.26(d,J=2.7Hz,1H),8.22(s,2H),7.62(s,1H),7.48(dd,J=8.8,2.8H z,1H),6.98(d,J=8.8Hz,1H),5.08–4.97(m,1H),4.94–4.87(m,1H),4.54–4.47(m,1H),4.45–4.39(m,1H). 13 C NMR (101MHz, CDCl3) δ162.1,154.4,139.7,133.6,132.6,132.2,128.5,128.3,122.9,119.8,117.5,114.3,82.2,80.5,68.6,68.4.ESI-MS m / z:[MH] - Calculated for C 17 H 10 ClF7NO2: 428.03, found: 428.23. Its characterization spectrum is as follows: Figure 16-18 As shown.

[0087] 2. AER-OTs are performed 18 Labeling (preparation) of F radionuclides 18 F]AER-EF molecular probe)

[0088]

[0089] The cyclotron produced 18 F-fluoride ions were enriched on a QMA column and elute with 1.5 mL of eluent (containing 13 mg K2.2.2 and 3 mg potassium carbonate, acetonitrile / water = 9 / 1). 18 F-fluoride ions were eluted from the QMA column into the reaction tube. The mixture was heated to azeotropic drying at 100°C under high-purity nitrogen, and then 1 mL of anhydrous acetonitrile was added and azeotropically dried again. 5 mg of the precursor AER-OTs was dissolved in 1.5 mL of anhydrous acetonitrile and transferred to the reaction tube. The reaction was carried out at 115°C under sealed conditions for 15 minutes. After cooling, the reaction tube was diluted with 8 mL of pure water and loaded onto a C18 Sep-pak column. Impurities were then washed with 20 mL of pure water, followed by elution with 4 mL of ethanol. The eluent was collected by semi-preparative liquid chromatography and diluted with sterile physiological saline for later use.

[0090] 3. Purity testing and quality control

[0091] AER-EF molecular probe standard liquid phase diagram as follows: Figure 19 As shown, the HPLC analysis conditions are as follows:

[0092] Mobile phase: 83% ethanol, 4 ml / mL; Column: 0.5 μm × 10 mm × 250 mm ODS C18 column.

[0093] [ 18 F]AER-EF molecular probe quality control diagram as shown Figure 20 As shown, the HPLC analysis conditions are as follows:

[0094] Mobile phase: 83% ethanol, 4 ml / mL; Column: 0.5 μm × 10 mm × 250 mm ODS C18 column.

[0095] Example 3: In vitro stability test

[0096] Experimental method: The AQP-4 molecular probe ([ 18 The radiochemical purity of the F]AER-EF) was measured by TLC at room temperature over multiple time periods to observe its stability.

[0097] The results of the radiochemical purity measured in the stability test over 6 hours are shown in the table below:

[0098] Time point Radiochemical purity 1h 98.3％ 2h 98.1％ 3h 97.9％ 4h 97.6％ 5h 97.3％ 6h 96.8％

[0099] Example 4: Affinity Detection of AER-EF Molecular Probe Standards

[0100] AQP-4 coupling:

[0101] An interaction model between AQP-4 protein and AER-EF molecular probe standards was established using surface plasmon resonance (SPR). This model was used to detect the coupling between AQP-4 protein and AER-EF molecular probe standards. The results showed that the total AQP4 coupling amount was 7180.4 RU, as illustrated in the coupling sensing diagram below. Figure 21 As shown in the figure, the horizontal axis represents time (time, s), the vertical axis represents the coupling quantity (Response, unit: RU), and the curve represents the coupling channel activation coupling curve, which reflects the coupling process.

[0102] Affinity test:

[0103] AER-270 and AER-EF were measured according to their dissolution concentrations. The kinetic and steady-state fitting results for AER-270 and AER-EF are shown below. The fitted concentration gradients were 250, 125, 62.50, 31.25, 15.63, 7.81, and 0 μM. The interaction results between AQP4 and the AER-270 molecular probe standard are as follows. Figure 22As shown; the results of the interaction determination between AQP4 and the AER-EF molecular probe standard are as follows. Figure 23 As shown.

[0104] The interaction affinity between AER-270 molecular probe standard, AER-EF molecular probe standard and AQP4 was determined using Biacore. The protein-coupled assay was performed using a CM5 chip. The results are shown in the table below:

[0105] ligands Analytes KD(M) AQP4 AER-270 Molecular Probe Standard <![CDATA[2.137*10 -4 ]]> AQP4 AER-EF molecular probe standards <![CDATA[4.236*10 -4 ]]>

[0106] KD: dissociation constant, which reflects the affinity of the analyte for the target. The smaller the value, the stronger the affinity.

[0107] Example 5: PET scan imaging of animal models

[0108] Dosing to mice:

[0109] Each mouse was weighed before administration and injected once via tail vein with the synthetic [method] synthesized in Example 2 of this invention. 18 F]AER-EF molecular probe (approximately 0.37 MBq / g body weight, 0.1-0.2 mL).

[0110] Micro-PET scan:

[0111] Normal mice were anesthetized with isoflurane via indwelling needles and then immediately placed on the PET scanner for 90 minutes of dynamic imaging. The raw PET data was then divided into 30 frames (90 minutes). Dynamic time-to-radioactivity curves for each brain region were analyzed using PMOD. Based on the dynamic data, a static time point of 40 minutes post-administration was selected for brain imaging. AQP-4KO / 5xFAD and AQP-4KO model mice and corresponding controls were included. PMOD images of each brain region were analyzed, and statistical analysis was used to compare the differences and correlations between AQP-4KO / 5xFAD and AQP-4KO model mice and their corresponding control mice.

[0112] Scan results:

[0113] 90-minute brain dynamics TAC curves for each brain region are as follows: Figure 24 As shown, the Micro-PET scan results of the AQP-4KO model mouse and normal controls are as follows: Figure 25 As shown. The PET molecular probe synthesized in this invention showed reduced uptake in AQP-4KO model mice, which differed from that in normal control mice. Moreover, the SPR detection in Example 4 indicated that the PET molecular probe synthesized in this invention, which targets AQP-4, specifically binds to AQP-4, making it a potential AQP-4 probe that may be used for the early diagnosis of lymphoid lesions.

[0114] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A PET molecular probe targeting AQP-4, characterized in that, The chemical structural formula of the PET molecular probe is shown below:

2. The method for preparing the PET molecular probe according to claim 1, characterized in that, The PET molecular probe is derived from the precursor compound AER-OTs via radionuclide... 18 The marker for F is obtained, and the marked route is shown below:

3. The preparation method according to claim 2, characterized in that, The synthetic route for the precursor compound AER-OTs is shown below:

4. The application of the PET molecular probe according to claim 1 in the preparation of early diagnostic reagents for AD-related lymphatic lesions.

5. The use of the PET molecular probe of claim 1 in the preparation of reagents or kits for monitoring the progression of AD-related lymphoid lesions or the efficacy of treatment.

Images