A multimodal molecular imaging probe NIR-[ 68 Ga] and its preparation methods and applications

Abstract

The application discloses a kind of near-infrared fluorescence / positron emission tomography bimodal molecular imaging probe NIR-[ 68 Ga] and preparation method and application thereof, and the application is by the specific response of alzheimer disease beta amyloid monomer, oligomer and aggregate near-infrared fluorescent small molecule probe on radionuclide 68 Ga 3+ Labeling, obtain near-infrared fluorescence / positron emission tomography bimodal molecular imaging probe NIR-[ 68 Ga], to be applied to the bimodal imaging detection of AD A beta protein, especially early soluble A beta protein monomer and oligomer, to improve the sensitivity and accuracy of A beta protein on living body detection, to realize the early accurate diagnosis of AD.

Description

Technical Field

[0001] This invention relates to molecular imaging, specifically to a multimodal molecular imaging probe NIR-[ 68 Ga] and its preparation methods and applications. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive and irreversible cognitive loss, and is one of the most common types of dementia. According to the amyloid cascade hypothesis, one of the histopathological features of AD is the presence of β-amyloid (Aβ) plaques in the brain. In the chronic pathological process of AD, overexpressed Aβ monomers aggregate via intermediate transition states (dimers, oligomers, and Aβ protein fibrils, etc.) to form insoluble Aβ plaques. Besides the neurotoxic insoluble Aβ plaques, recent evidence suggests that both soluble oligomeric Aβ proteins and the final insoluble Aβ plaques are highly neurotoxic and contribute to the development of AD clinical symptoms. Therefore, these soluble and insoluble Aβ proteins in the AD brain can serve as biomarkers for AD. Based on this, imaging detection of these proteins is beneficial for the early and accurate diagnosis of AD and for evaluating the efficacy of anti-AD Aβ protein drug therapy.

[0003] Over the past few decades, numerous molecular imaging probes for detecting Aβ protein, including fluorescence (FL) probes, positron emission tomography (PET) probes, and magnetic resonance imaging (MRI) probes, have been developed for the in vivo visualization of Aβ protein. In particular, multimodal molecular imaging probes can combine the advantages of different imaging modalities to improve the sensitivity and accuracy of Aβ protein detection in the brain, and achieve more accurate diagnosis of AD through mutual verification of their respective imaging information. These offer significant advantages over single-modal imaging. For example, dual-modal imaging probes based on FL / MRI, FL / PET, and FL / SPECT, while designed for high-sensitivity and high-resolution imaging of Aβ protein, sometimes fail to capture fluorescence emission wavelengths in the near-infrared (NIR) emission band (λ) after binding to Aβ protein. emThe nanometer size (<650nm) is unfavorable for imaging and detecting Aβ proteins located in the brain. Furthermore, MRI imaging, due to its large dosage, also has low detection sensitivity. In addition, most of these multimodal imaging probes target late-stage Aβ plaque responses, failing to provide adequate early diagnosis of AD. Therefore, it is essential to develop a multimodal molecular imaging probe with NIR fluorescence emission that can target both soluble and insoluble Aβ proteins to improve the sensitivity and accuracy of Aβ protein detection, thereby achieving accurate early diagnosis of AD. Summary of the Invention

[0004] Objective of the Invention: The technical problem to be solved by the present invention is to address the shortcomings of existing technologies by providing a dual-modal molecular imaging probe for NIR-[NIR-FL / PET]. 68 Ga].

[0005] Another technical problem to be solved by this invention is to provide the above-mentioned multimodal molecular imaging probe NIR-[ 68 Preparation method of Ga].

[0006] A further technical problem to be solved by the present invention is to provide the above-mentioned multimodal molecular imaging probe NIR-[ 68 Applications of Ga].

[0007] Invention Concept: This invention labels radionuclides capable of PET imaging onto NIR fluorescent small molecule probes that specifically respond to Aβ protein monomers, oligomers, and aggregates. It utilizes the deep tissue penetration depth and high sensitivity of NIR fluorescence and PET imaging technologies to achieve highly sensitive and specific imaging detection of Aβ protein species and early accurate diagnosis of AD.

[0008] To solve the first technical problem mentioned above, the present invention discloses a curcumin derivative as shown in Formula I;

[0009]

[0010] The present invention also discloses a molecular imaging probe NIR-[Ga] containing the structure shown in Formula I above;

[0011]

[0012] This invention also discloses a multimodal molecular imaging probe NIR-[ containing the structure shown in Formula I above.] 68 Ga], which comprises the following components: (1) NIR fluorescent small molecule probes that respond to Aβ protein monomers, oligomers and aggregates; (2) NODA- for PET imaging. 68 Ga chelate; (3) intermediate alkyl linker chain used to balance lipophilicity and hydrophilicity; shown as a multimodal molecular imaging probe NIR-[ 68The specific structural formula of Ga is as follows:

[0013]

[0014] To address the second technical problem mentioned above, this invention discloses the aforementioned molecular imaging probe NIR-[ 68 The preparation methods of Ga] and NIR-[Ga] include the following steps:

[0015] Step a: Compound F1 converts the intracyclic double bond to an excyclic double bond under basic conditions to obtain intermediate F1-a;

[0016] Step b: Compound F1-a is reacted with Vilsmeier-Haack to give compound F2;

[0017] Step c: Compound F2 undergoes a condensation reaction with CRANAD-54 to obtain compound F3;

[0018] Step d: Compound F3 undergoes a Click reaction with 6-azido-hexylamine to give compound F4;

[0019] Step e: Compound F4 undergoes an acylation reaction with NODA-GA-NHS ester to give compound F5;

[0020] Step f: Compound F5 and radionuclide 68 Ga 3+ A coordination reaction was performed to obtain the dual-modal molecular imaging probe NIR-[ 68 Ga]; or compound F5 and Ga 3+ A coordination reaction was carried out to obtain the "cold" probe NIR-[Ga];

[0021]

[0022] Where M is selected from 68 Ga or Ga.

[0023] In step a, the reaction is carried out by reacting compound F1 in a mixed solvent of alkaline aqueous solution and diethyl ether to obtain compound F1-a.

[0024] The volume ratio of the alkaline aqueous solution to the diethyl ether is 2–3:1, preferably 2:1.

[0025] The alkaline aqueous solution includes, but is not limited to, potassium hydroxide aqueous solution; the concentration of the potassium hydroxide aqueous solution is 0.5–1.5N, preferably 1N.

[0026] The reaction temperature is 30–50°C, preferably 40°C.

[0027] The reaction is carried out under stirring.

[0028] The reaction time is 1 hour or more, preferably 1–4 hours, and more preferably 2 hours.

[0029] After the reaction is completed, the reaction solution containing F1-a is rotary evaporated, extracted, and dried to obtain F1-a.

[0030] In step b, the reaction is a Vilsmeier-Haack reaction between compound F1-a and Vilsmeier reagent (chloroimine salt) to obtain compound F2. Specifically, phosphorus oxychloride reacts with anhydrous N,N-dimethylformamide (DMF) to form Vilsmeier reagent, and the obtained Vilsmeier reagent reacts with F1-a to obtain compound F2.

[0031] The reaction is carried out under the protection of an inert gas.

[0032] The Vilsmeier reagent is obtained by reacting phosphorus oxychloride with anhydrous N,N-dimethylformamide under stirring at 0°C beforehand; the reaction time is 0.5h–2.0h, preferably 1.0h.

[0033] The molar ratio of phosphorus oxychloride to compound F1-a is 1:1–3, preferably 1:2.

[0034] Among them, F1-a and Vilsmeier reagent are reacted at 0℃ for 0.5h–2.0h, preferably 1.0h; (2) at room temperature–55℃ for 0.5h–2.0h, preferably at room temperature for 10min, and then at 40℃ for 1.0h; (3) quenched with an ice-water mixture, adjusted to pH=11 with a saturated sodium hydroxide aqueous solution, and then reacted at 50–80℃ for 1–10min, preferably at 60℃ for 3min.

[0035] After the reaction is completed, the reaction solution containing compound F2 is extracted, dried, and recrystallized to obtain compound F2; wherein the solvent for recrystallization includes, but is not limited to, anhydrous diethyl ether.

[0036] In step c, the reaction is a condensation reaction between compound F2 and CRANAD-54 (J.Am.Chem.Soc.2013,135,16397-16409) in a solvent to obtain compound F3.

[0037] The solvent includes, but is not limited to, acetic anhydride, and is preferably acetic anhydride.

[0038] The reaction temperature is 45–70°C, preferably 60°C.

[0039] The reaction is carried out under stirring.

[0040] The reaction time is 1 hour or more, preferably an overnight reaction.

[0041] After the reaction is completed, the reaction solution containing compound F3 is rotary evaporated and purified by silica gel column chromatography to obtain compound F3.

[0042] In step d, the reaction is a click chemical reaction between compound F3 and 6-azido-hexylamine in a mixed aqueous solution of CuSO4 and sodium vitamin C to obtain compound F4.

[0043] The solvents include, but are not limited to, dimethyl sulfoxide (DMSO).

[0044] The reaction temperature is 20–30°C, preferably room temperature.

[0045] The reaction is carried out under stirring.

[0046] The reaction time is 10–120 min, preferably 30 min.

[0047] After the reaction is completed, the reaction solution containing F4 is separated and purified by reversed-phase preparative liquid chromatography and then freeze-dried to obtain compound F4.

[0048] In step e, the reaction involves dissolving compound F4, diisopropylethylamine (DIPEA), and NODA-GA-NHS ester in a solvent and reacting them to obtain a reaction solution containing compound F5.

[0049] The molar ratio of compound F4, DIPEA and NODA-GA-NHS ester is 1:3.0–10:0.5–2.5, preferably 1:5:1.

[0050] The solvents include, but are not limited to, DMF.

[0051] The reaction temperature is 20–30°C, preferably room temperature.

[0052] The reaction is carried out under stirring.

[0053] The reaction time is 1 hour or more, preferably 1–7 hours.

[0054] After the reaction is completed, the reaction solution containing F5 is separated and purified by reversed-phase preparative liquid chromatography and then freeze-dried to obtain compound F5.

[0055] In step f, the reaction is between compound F5 and a radionuclide. 68 Ga 3+Marking, obtaining NIR-[ 68 Ga].

[0056] To address the third technical problem mentioned above, this invention discloses the aforementioned NIR fluorescence / PET dual-modal molecular imaging probe NIR-[ 68 Applications of Ga] and the corresponding "cold" probe NIR-[Ga].

[0057] The application is in the preparation of Aβ protein species detection reagents.

[0058] The application is in the detection of Aβ protein species.

[0059] The application is in the preparation of fluorescence imaging products for diagnosing Alzheimer's disease; wherein, when the probe is NIR-[ 68 When Ga] is used, the fluorescence imaging is NIR fluorescence / PET dual-modal imaging; the product includes a contrast agent.

[0060] This invention also discloses a reagent for detecting Aβ protein species, comprising the aforementioned molecular imaging probe NIR-[ 68 Ga] and NIR-[Ga].

[0061] This invention also discloses a fluorescence imaging product for diagnosing Alzheimer's disease, comprising the aforementioned molecular imaging probe NIR-[ 68 [Ga] and NIR-[Ga]; the products include contrast agents.

[0062] like Figure 1 As shown, before the bimodal probe binds to Aβ protein, the molecule's conformation in solution is freely rotating, with non-radiative energy release dominating and weak fluorescence. After binding to Aβ protein, the molecule's free rotation is hindered, and its molecular structure becomes planar. When the molecule is excited by excitation light, fluorescent radiation energy release dominates, and the fluorescence is activated. Simultaneously, the bimodal probe, after binding to Aβ protein, remains in the brain to a certain extent compared to the unbound probe, thus generating a stronger PET signal, which can distinguish transgenic AD model mice from control wild-type mice. Therefore, on the one hand, after the bimodal probe is injected into mice via the tail vein, the probe circulates through the blood-brain barrier (BBB) ​​and enters the brain parenchyma, where it binds to the highly expressed Aβ protein in the brain of AD mice, producing an "activation" fluorescent signal. On the other hand, after binding to Aβ protein, the bimodal probe remains in the brain to a certain extent, while unbound probes can re-enter the bloodstream through free diffusion and be cleared, thus generating an enhanced PET signal.

[0063] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0064] This invention provides a dual-modal molecular imaging probe, NIR-[, that responds to Aβ protein-mediated NIR fluorescence / PET] 68 [Ga] Utilizing the high sensitivity and deep tissue penetration of NIR fluorescence and PET, it is possible to perform highly sensitive and specific imaging detection of soluble and insoluble Aβ proteins highly expressed in the brains of AD model mice, thereby achieving accurate diagnosis of AD. Attached Figure Description

[0065] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0066] Figure 1 (A) Dual-modal molecular imaging probe for NIR fluorescence / PET 68 (A) Schematic diagram of the mechanism of Ga for Aβ protein detection; (B) NIR-[ 68 A schematic diagram of Ga's response to Aβ protein species in vivo.

[0067] Figure 2 (A) Dual-modal molecular imaging probe NIR-[ 68 Synthesis of Ga] and its “cold” probe NIR-[Ga]: (a)(i) 1NKOH / H2O-Et2O, 40℃; (ii) POCl3 / DMF, 0℃–rt, 89.5%; (b) Ac2O, 60℃, overnight, 60.1%; (c) NH2-(CH2)6-N3, CuSO4 / VitC, DMSO:H2O=10:1; (d) NODA-GA-NHS ester, DIPEA, DMF; (e) GaCl3, HCl, pH 2–3, rt, 12h; (f) 68 GaCl3, pH=4, 30℃, 15min. (B)NIR-[Ga] and NIR-[ 68 HPLC (monitored at 600 nm wavelength) and radiometric HPLC spectra of Ga[ (monitored by radiometric gamma signal). (C, D) NIR-[ 68 Radiometric HPLC spectra of Ga after incubation in PBS buffer (C) and plasma (D) for 1 h and 2 h.

[0068] Figure 3(AC) Fluorescence spectra of 50 nM NIR-[Ga] in PBS buffer with 2.5 μM Aβ42 protein monomers (A), oligomers (B), and aggregates (C). (DF) Fluorescence intensity of NIR-[Ga] in PBS buffer with 2.5 μM Aβ42 protein monomers (D), oligomers (E), and aggregates (F) as a function of probe concentration. (G) Fluorescence intensity of 50 nM NIR-[Ga] after incubation with 2.5 μM Aβ42 protein monomers, oligomers, and aggregates for 0–180 s. (H) Fluorescence intensity of 50 nM NIR-[Ga] with different concentrations of Aβ42 protein monomers, oligomers, and aggregates. (I) Fluorescence intensity of 50 nM NIR-[Ga] after incubation with 10 μg / ml Aβ42 protein species and other endogenous biological substances in PBS buffer (1: PBS, 2: OH·(200 μM Fe) 2+ +1mM H2O2),3: 1 O2(1mM H2O2+1mM ClO - ),4:O2 ·- (100 μM xanthine + 22 mU xanthine oxidase), 5: H2O2 (1 mM H2O2), 6: hMAO-A, 7: β-galactosidase, 8: AChE, 9: BuChE, 10: L-cysteine, 11: GSH, 12: Cytochrome C, 13: Vitamin C, 14: Amylin, 15: BSA, 16: Aβ 42 Monomer, 17: Aβ 42 Oligomer, 18: Aβ 42 (aggregates).

[0069] Figure 4 HPLC chromatograms of NIR-[Ga] incubated in PBS buffer (pH=7.4) (5 μM) for different number of days.

[0070] Figure 5 Cell viability of U87MG cells (A) and PC-12 cells (B) after incubation with different concentrations of NIR-[Ga] (0, 0.25, 0.5, 1.0, 2.5, 5.0 and 10.0 μM) for 24 h.

[0071] Figure 6 (A)NIR-[ 68 Ga] PET imaging of the brains of 9-month-old APP / PS1 transgenic AD model mice and control wild-type mice at 5, 10, 30, and 60 min, respectively. (B) NIR-[ 68PET signal intensity of [Ga] in the brains of 9-month-old AD model mice and wild-type mice over time. (C, D) NIR fluorescence imaging (C) and fluorescence signal intensity (D) of NIR-[Ga] (1.0 mg / kg) in the brains of 9-month-old AD model mice and wild-type mice at 5, 10, 30, 60, 120, 240, and 360 min. Values ​​are mean ± standard deviation. (*p<0.05, **p<0.01, n=3). Note: Circles in (A) indicate mouse brain regions.

[0072] Figure 7 (A)NIR-[ 68 Ga] was uptaken in specific brain regions of 9-month-old APP / PS1 mice and wild-type mice, respectively. (B) NIR-[ 68 [Ga] autoradiographic imaging of ex vivo brain tissue sections from 9-month-old APP / PS1 mice and wild-type mice, respectively. (C) NIR-[Ga] fluorescence imaging of ex vivo brain tissue sections from 10-month-old APP / PS1 mice. Aβ plaques were further validated by Thioflavin-T (ThT) staining. Note: Cortex (Cor), Hippocampus (H), Thalamus (T), Superior Colliculi (SC), Midbrain (M), Amygdala (A), Hypothalamus (HT).

[0073] Figure 8 Fluorescent staining of ex vivo brain tissue sections from wild-type mice (10 months old) 10 min after tail vein injection of NIR-[Ga] (2.0 mg / kg). The same sections were then co-stained with ThT to confirm Aβ protein plaques. Detailed Implementation

[0074] Specific embodiments are described below to further illustrate the present invention. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0075] Unless otherwise specified, all "%" in the following embodiments refers to weight percentage.

[0076] Reagents and Instruments: All chemical reagents and solvents were purchased from Sigma-Aldrich, Energie Chemicals, and TCI (Shanghai) Chemical Industry Development Co., Ltd. Analytical solvents and reagents were of chromatographic grade, and routine reagents were of analytical grade without further purification. Aβ 42Protein monomers were purchased from Nanjing Peptide Biotechnology Co., Ltd. (Nanjing, China). High-glucose Dulbecoo's Modified Eagle Medium (DMEM), high-glucose RPMI 1640 medium, fetal bovine serum (FBS), and trypsin were purchased from Thermo Fisher Scientific (Shanghai, China). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay kit was purchased from Jiangsu Kaiji Biotechnology Co., Ltd. (Nanjing, China). U87MG cells and PC-12 cells were purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China).

[0077] The compound was prepared using normal-phase rapid preparative liquid chromatography (HPLC). The equipment used includes a machine U200 (Sante Technology Changzhou Co., Ltd.) and a reversed-phase preparative high-performance liquid chromatograph (Thermo Scientific Dionex Ultimate 3000). The eluent for the preparative liquid chromatography is CH3CN / H2O containing 0.1% CF3COOH. 1 H-NMR and 13C-NMR spectra were obtained using a 400 MHz Bruker Avance III 400 NMR spectrometer. Chemical shifts (δ) are expressed in ppm. Singlets, doublets, triplets, quartets, dd (doublet of doublets) peaks, multiplets, and broad peaks are represented by s, d, t, q, dd, m, and br, respectively. Coupling constants (J) are expressed in Hz. The number of hydrogen atoms was determined by the integral value of the spectrum and labeled as nH. High-performance liquid chromatography (HPLC) was performed using a Thermo Scientific Dionex Ultimate 3000 with CH3CN / H2O (containing 0.1% CF3COOH) as the eluent. Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was performed using an AB SCIEX 4800Plus MALDI TOF / TOF™ mass spectrometer. Fluorescence spectra were measured using a HORIBA Jobin Yvon Fluoromax-4 fluorometer. MTT was measured using a microplate reader (Tcan). In vivo fluorescence images were acquired using the IVIS LuminaXR III system, and fluorescence intensity was quantified by measuring the signal in the circled region of interest using Living Image software (PerkinElmer, USA). PET imaging was performed on a small animal PET imaging system (SIEMENS INVEON). The small animal PET imaging results were further processed using PMOD software (Pmod Technologies, Zurich, Switzerland).

[0078] Example 1: Probe design, synthesis and characterization ( Figure 2 A)

[0079] Route S1 shows the probe NIR-[ 68 The structural design of Ga].

[0080]

[0081] Reaction conditions: Dual-mode probe NIR-[ 68 Synthetic route of Ga]. Reaction conditions: (a) 1N KOH / H2O, 40℃; (b) POCl3 / DMF, 0℃–rt, 89.5%; (c) Ac2O, 60℃, Overnight, 60.1%; (d) NH2-(CH2)6-N3, CuSO4 / VitC, DMSO:H2O=10:1; (e) NODA-GA-NHS ester, DIPEA, DMF; (f) 68 Ga 3+ pH 4, 37℃, 15min.

[0082] Synthesis of compound F2: F1 (2.0 g, 5.66 mmol) was dissolved in a mixed solution of 6 mL 1N KOH / H2O and 3 mL diethyl ether, and the mixture was stirred at 40 °C for 2 h. The reaction solution was extracted three times with diethyl ether, the ether layers were combined, and then washed three times with saturated NaCl solution. The mixture was dried over anhydrous Na2SO4, and the solvent was evaporated to obtain a yellow-brown oily substance F1-a. This intermediate did not require further purification and was used directly in the next reaction. Separately, under a nitrogen atmosphere, 1.5 mL POCl3 was added dropwise to anhydrous DMF that had been pre-cooled to 0 °C. After the addition was complete, the reaction mixture was allowed to react at 0 °C for another 1 h. Then, compound F1-a was dissolved in 2.0 mL of anhydrous DMF solution and added dropwise to a mixture of POCl3 and anhydrous DMF at 0 °C. After the addition was complete, the reaction mixture was allowed to react at 0 °C for another 1 h (under a nitrogen atmosphere). Finally, the reaction mixture was allowed to react at room temperature for 10 min, and then at 40 °C for 1 h. The reaction solution was poured into an ice-water mixture, and the pH was adjusted to 11 with saturated NaOH solution. The reaction was carried out at 60°C for 3 min. The reaction solution was extracted with ethyl acetate, washed three times with saturated NaCl solution, dried over anhydrous Na2SO4, filtered, and the solvent was evaporated to obtain a reddish-brown compound. The compound was then recrystallized from diethyl ether to give approximately 1.28 g of a reddish-brown solid, yield: 89.5%. 1 H-NMR (400MHz, CDCl3) δ10.03(d,J=8.9Hz,1H),7.31-7.24(m,2H),7.07(td,J=7.5,0.8Hz,1H),6.94(d,J=7.9Hz,1H),5. 46(d,J=8.9Hz,1H),3.84(t,J=7.6Hz,2H),2.30(td,J=6.7,2.6Hz,2H),2.11(t,J=2.6Hz,1H),1.92(m,2H),1.67(s,6H). 13 C-NMR (101MHz, CDCl3) δ186.80,173.03,143.12,139.52,128.25,122.65,122.02 ,108.30,99.10,82.74,70.13,47.65,41.79,29.82,24.92,16.22.MS:calcd.for C 17 H 20 NO + [M+H] + :254.1539; MALDI-TOF-MS see m / z 254.1388.

[0083] Synthesis of compound F3: CRANAD-54 in this reaction was synthesized according to the literature (J. Am. Chem. Soc. 2013, 135, 16397-16409). F2 (121 mg, 0.43 mmol) and CRANAD-54 (100 mg, 0.39 mmol) were dissolved in 2.0 mL of acetic anhydride, and the reaction solution was stirred overnight at 60 °C. The reaction solvent was then evaporated to dryness, and the remaining solid was purified by rapid liquid chromatography (silica gel column) (petroleum ether:ethyl acetate:dichloromethane = 4:1:1) to give approximately 122 mg of black solid, yield: 60.1%. 1 H-NMR (400MHz, CDCl3) δ8.32(t,J=13.4Hz,1H),7.82(d,J=15.4Hz,1H),7.46(d,J=8.8Hz,2H),7 .28-7.24(m,2H),7.07(t,J=7.4Hz,1H),6.92(d,J=8.0Hz,1H),6.68(d,J=8.7Hz,2H),6.42(d,J= 15.3Hz,1H),5.80(d,J=13.6Hz,1H),5.75(d,J=13.2Hz,1H),5.69(s,1H),3.90(t,J=7.3Hz,2H), 3.05(s,6H),2.32(td,J=6.6,2.6Hz,2H),2.14(t,J=2.6Hz,1H),1.99–1.89(m,2H),1.65(s,6H). 13 C-NMR(101MHz, CDCl3)δ177.24(s),174.52(s),168.38(s),152.11(s),144.97(s),143 .82(s),143.07(s),140.19(s),130.74(s),128.27(s),122.93(s),122.22(s),116.17 (s),113.97(s),112.11(s),108.40(s),100.55(s),97.38(s),82.86(s),70.21(s),48 .03(s),41.77(s),40.31(s),29.83(s),28.90(s),25.52(s),16.24(s).MS:calcd.for C 31 H 33 BF2N2NaO2 + [M+Na] + :537.2495; HRMS(ESI) see m / z 537.2534.

[0084] Synthesis of compound F4: F3 (60 mg, 0.12 mmol) was dissolved in 2.0 mL of DMSO, followed by the addition of 6-azidohexylamine (18 mg, 0.12 mmol), and then 200 μL of a deionized aqueous solution of CuSO4 (19 mg, 0.12 mmol) and sodium ascorbate (24 mg, 0.12 mmol). The mixture was stirred at room temperature for 30 min, purified by preparative HPLC, and lyophilized to give approximately 66 mg of a black solid, with a yield of 84%. 1 H-NMR (400MHz, DMSO-d6) δ8.18(t,J=13.40Hz,1H),7.90(s,1H),7.58(d,J=8.80Hz,2H),7.57(d,J=15.73Hz,1H),7 .51(d,J=7.30Hz,1H),7.32(t,J=8.20Hz,1H),7.21(d,J=7.90Hz,1H),7.13(t,J=7.40Hz,1H),6.75(d,J=9.08Hz,2 H),6.71(d,J=15.70Hz,1H),6.05(d,J=13.20Hz,1H),5.90(d,J=13.40Hz,1H),4.30(t,J=7.04Hz,2H),4.08-4.01( m,4H),3.02(s,6H),2.76-2.71(m,6H),2.01(m,J=7.44Hz,2H),1.80-1.76(m,2H),1.60(s,6H),1.53-1.45(m,4H). 13 C-NMR(101MHz,DMSO-d6)δ175.47,172.10,169.47,158.54,158.21,157.88,151.9 9,145.83,144.99,142.55,141.74,139.99,130.55,128.24,123.22,122.25,121. 91,116.06,111.97,109.67,98.23,79.30,78.97,78.64,49.05,47.75,42.19,40. 44,38.70,29.47,29.01,28.17,26.76,26.16,25.32,25.13,22.23.MS:calcd.for C 37 H 48 BF2N6O2 + [M+H] + :657.3894; HRMS (ESI) see m / z657.3891.MS:calcd.for C 37 H 47BF2N6NaO2 + [M+Na] + :679.3714; HRMS(ESI) see m / z 679.3706.

[0085] Synthesis of compound F5: Intermediate F4 (14 mg, 0.021 mmol) was dissolved in 0.7 mL of anhydrous DMF, and 5 equivalents of diisopropylethylamine (DIPEA) (18 μL, 0.106 mmol) were added. Then, 0.3 mL of anhydrous DMF solution of NODA-GA-NHS ester (10 mg, 0.021 mmol) was rapidly added to the reaction mixture. The reaction mixture was stirred at room temperature for 1 h, and HPLC monitoring showed that the reaction was complete. The reaction mixture was purified by preparative high-performance liquid chromatography and lyophilized to give approximately 9.1 mg of a black solid, with a yield of 42.3%. 1 H-NMR (400MHz, DMSO-d6) δ8.18(t,J=13.4Hz,1H),7.89(s,1H),7.74(t,J=5. 5Hz,1H),7.58(d,J=8.8Hz,2H),7.57(d,J=16.96Hz,1H),7.51(d,J=7.5Hz,1H ),7.32(t,J=8.2Hz,1H),7.19(d,J=7.9Hz,1H),7.13(t,J=7.4Hz,1H),6.75( d,J=9.1Hz,2H),6.71(d,J=15.6Hz,1H),6.05(d,J=13.3Hz,1H),5.90(d,J=13 .4Hz,1H),4.29(t,J=7.0Hz,2H),4.03(t,J=6.9Hz,2H),3.89(d,J=4.1Hz,2H ),3.90-3.88(m,2H),3.49(t,J=7.3Hz,1H),3.15-3.04(m,6H),3.02(s,6H),3 .00-2.88(m,8H),2.74(t,J=7.44Hz,2H),2.18(t,J=6.8Hz,2H),2.06-1.96( m,2H),1.95-1.83(m,2H),1.83-1.72(m,3H),1.60(s,6H),1.40-1.31(m,2H). 13C-NMR(101MHz,DMSO-d6)δ175.48,173.43,172.08,171.51,171.27,169.91,169.51,167.30,158.73,158.37,158.01,157.65,153.97 ,151.98,145.80,145.04,142.54,141.73,139.98,138.95,138.56,137.18,130.56,129.63,128.25,123.22,122.29,122.22,121.91 ,120.10,117.20,116.08,114.29,112.00,111.39,109.68,98.26,88.53,81.76,62.93,54.46,54.14,50.62,49.85,49.29,49.16,47 .98,47.76,46.70,44.78,42.20,40.43,38.38,31.99,29.66,28.95,28.31,28.17,26.13,25.80,25.59,25.21,22.22.MS:calcd.for C 52 H 70 BF2N9NaO9 + [M+Na] + :1036.5250; MALDI-TOF-MS see m / z 1036.7166.

[0086] Dual-mode probe NIR-[ 68 Synthesis of Ga: Ga was synthesized using a one-step labeled synthesis method, as follows: 5.0 mL of HCl (0.05 mol / L) was injected... 68 Ga 3+ The generator was used to take out 0.5, 0.5, 0.5, 1.4, 0.7, and 1.4 mL of solutions containing... 68 Ga 3+ The solution. After testing the isotope dosage, the solutions obtained from the fourth and fifth tests were transferred to the hot chamber using lead containers. Then 1.4 mL containing 68 Ga 3+ The solution was accurately transferred to a new centrifuge tube, and the pH was adjusted to 4.0 with 1M sodium acetate solution. Next, 400 μg of intermediate F5 was added to the mixture, and the mixture was incubated at 37°C for 15 min, with shaking every 5 min. A small amount of the solution was used for radioactive HPLC analysis; the remaining solutions were used directly.

[0087] Synthesis of the “cold” probe NIR-[Ga]: Compound F5 (10 mg, 0.01 mmol) was dissolved in 0.5 mL of anhydrous DMF, and 0.5 mL of HCl (1 M) solution was added. After mixing thoroughly, 0.2 mL of deionized water solution of GaCl3 (18 mg, 0.1 mmol) was added to the reaction solution (pH = 2–3). The reaction was continued at room temperature in the dark with stirring for 12 h. HPLC monitoring showed that the reaction was complete. The reaction solution was purified by preparative high performance liquid chromatography, and after lyophilization, approximately 8.8 mg of black solid was obtained, with a yield of 82%. MS: calcd.for C 52 H 67 BF2GaN9NaO9 + [M+Na] + :1102.4271; MALDI-TOF-MS see m / z 1102.5259.MS:calcd.for C 52 H 67 BF2GaKN9O9 + [M+K] + :1118.4010; MALDI-TOF-MS see m / z 1118.5046.

[0088] Example 2: Probe Performance Testing

[0089] 1. Dual-mode probe NIR-[ 68 Structural confirmation of Ga]

[0090] To confirm the dual-mode probe NIR-[ 68 The successful synthesis of Ga was first achieved by high-performance liquid chromatography (HPLC). Figure 2 B) and high-resolution mass spectrometry confirmed the successful synthesis of the "cold" probe NIR-[Ga]. Then, the labeled NIR-[Ga] was... 68 Ga was monitored using a radioactive high-performance liquid chromatograph at a wavelength of 600 nm and for radioactive gamma signals, respectively. Figure 2 As shown in B, the dual-mode probe NIR-[ 68 The retention time of [Ga] is consistent with that of the "cold" probe NIR-[Ga], indicating that NIR-[ 68 Successful synthesis of Ga].

[0091] 2. Dual-modal probe NIR-[ 68 Stability study of Ga]

[0092] NIR probe-[ 68[Ga] was added to 500 μL of PBS buffer (pH = 7.4) and plasma, respectively, and incubated at 37 °C. Equal volumes of solution were taken at 0 min, 60 min, and 120 min for radioactive HPLC analysis to investigate the stability of the compound under different incubation conditions. Figure 2 C(PBS buffer) and Figure 2 As shown in D (plasma), the dual-modal probe exhibits good stability in the physiological environment.

[0093] 3. In vitro performance testing of the probe after its response to Aβ protein

[0094] (1) Fluorescence spectra of the probe before and after binding to Aβ protein: NIR-[Ga] was added to 2.5 μM PBS buffer (pH = 7.4) for Aβ protein monomers, oligomers, and aggregates, respectively, to make the final concentration of the probe 50 nM. Figure 3 As shown in AC, the fluorescence intensity of the probe significantly increased after binding to Aβ protein monomers, oligomers, and aggregates, with enhancement factors of ~75-fold, ~38-fold, and ~36-fold, respectively. Simultaneously, the fluorescence emission wavelength of the probe underwent a blue shift, from 755 nm to 689 nm, 683 nm, and 689 nm, respectively. These results indicate that NIR-[Ga] has a strong interaction with Aβ protein monomers, oligomers, and aggregates, producing "activated" NIR fluorescence.

[0095] (2) Affinity test between the probe and Aβ protein: Different concentrations of NIR-[Ga] were added to 2.5 μM PBS buffer (pH = 7.4) containing Aβ protein monomers, oligomers, and aggregates, respectively, and the fluorescence intensity of the probe was nonlinearly fitted to the concentration gradient. Figure 3 As shown in DF, the dissociation constants (Ki) of the probe for Aβ protein monomers, oligomers, and aggregates are... d The values ​​were 18.22±0.41 nM, 21.31±1.13 nM, and 17.91±1.09 nM, respectively, all in the nanomolar range, indicating that the probe has a strong affinity for Aβ protein.

[0096] (3) Kinetics of probe binding to Aβ protein: 50 nM NIR-[Ga] was added to 2.5 μM PBS buffer (pH = 7.4) for Aβ protein monomers, oligomers, and aggregates, respectively. The fluorescence intensity of the probe over time (0–180 s) was immediately measured on a 96-well plate using a microplate reader. Figure 3 G, the fluorescence intensity of the probe after binding to Aβ protein monomers and oligomers reached its maximum at 10 s, while the fluorescence intensity after binding to aggregates reached its peak at 120 s. This result indicates that the probe has a relatively rapid binding kinetic process with Aβ protein.

[0097] (4) Detection limit test of the probe for Aβ protein: Different concentrations of Aβ protein monomers, oligomers, and aggregates were added to 50 nM NIR-[Ga] PBS buffer (pH = 7.4), and the fluorescence intensity of the probe was measured at different protein concentrations. Linear fitting of fluorescence intensity as a function of concentration was then performed on Aβ protein monomers (50 nM–3.2 μM), oligomers (50 nM–10 μM), and aggregates (50 nM–6.4 μM) within the corresponding concentration ranges. Figure 3 As shown in H, the limits of detection (LOD, 3σ / k) of the probe for Aβ protein monomers, oligomers, and aggregates are ~10.9, ~25.8, and ~20.2 nM, respectively.

[0098] (5) Selectivity test of probe for Aβ protein: 50 nM NIR-[Ga] and 10 μg / ml Aβ were measured in PBS buffer (pH = 7.4). 42 Fluorescence intensity resulting from interactions between protein species (monomers, oligomers, and aggregates) and other endogenous biological substances. These endogenous substances primarily include monoamine oxidases, β-galactosidase, acetylcholinesterase, butyrylcholinesterase, L-cysteine, GSH, cytochrome C, amylin, bovine serum albumin, and reactive oxygen species (such as OH·). 1 O2, O2 ·- And H2O2), etc. For example... Figure 3 I. When the probe binds to Aβ protein monomers, oligomers, and aggregates, the fluorescence intensity is significantly enhanced; however, when the probe is mixed with other interfering substances, no obvious fluorescence enhancement is observed, indicating that NIR-[Ga] has good selectivity for Aβ protein.

[0099] These results indicate that the "cold" probe NIR-[Ga] exhibits a strong fluorescence response and affinity for Aβ protein, and a rapid binding kinetics with Aβ protein; at the same time, it demonstrates high detection sensitivity and good selectivity for Aβ protein.

[0100] 4. Probe stability test

[0101] Next, the stability of the probe in PBS buffer (pH = 7.4) was investigated. NIR-[Ga] was prepared into a 5 μM solution in PBS buffer and incubated at room temperature. The probe was then analyzed by HPLC on days 1, 2, and 3. Figure 4 As shown, no obvious impurity peaks were observed after the probe was incubated in PBS for 3 days, and the retention time of the probe was consistent with that on day 1, indicating that the probe has good stability in the physiological environment of PBS.

[0102] 5. Cell culture and probe-based cell viability assay (MTT method)

[0103] Human glioma cells (U87MG cells) and normal neural cells (PC-12 cells) were purchased from the Shanghai Stem Cell Institute, Chinese Academy of Sciences, and cultured in DMEM (Dulbecco modified Eagle medium) and high-glucose RPMI 1640 medium, respectively. 10% (v / v) fetal bovine serum (FBS) was added to the medium, along with 100 units of penicillin and 100 units of streptomycin per milliliter of medium. All cells were cultured in a humidified environment (5% CO2) at 37°C.

[0104] Cultured neural cells were seeded in flat-bottomed 96-well plates (5000 cells / well, 100 μL / well) and incubated overnight at 37°C. The culture medium was aspirated from each well, and NIR-[Ga] solutions of different concentrations (0, 0.25, 0.5, 1.0, 2.5, 5.0, and 10 μM) were prepared and added to each well at 100 μL. After incubation for 24 hours, 50 μL of 1×MTT solution was added to each well. Incubation continued at 37°C for 4 hours, then the solution in each well was carefully removed. Next, 150 μL of DMSO solution was added to each well to dissolve the resulting purple crystals. The absorbance (OD) of each well solution at 490 nm was measured using a microplate reader (Tcan). The absorbance of blank cells (OD control) was used as a control. The percentage of viable cells per well was determined by the OD / OD ratio. control The result is calculated as ×100%, with each experiment repeated three times. For example... Figure 5 As shown, the cell survival rate after incubation of different concentrations of probe with the two cell lines for 24 hours was over 90%, proving that the probe had no obvious toxicity to the two cell lines.

[0105] 6. In vivo PET and NIR fluorescence imaging of APP / PS1 mice

[0106] The dual-mode probe NIR-[ 68 Ga (240–290 μCi) was injected via the tail vein into 9-month-old APP / PS1 transgenic AD model mice and control wild-type mice. Mice were dynamically scanned for 60 minutes using a micro-PET scanner (SIEMENS INVEON). Throughout the experiment, mice were anesthetized using a 2% isoflurane / oxygen system at a flow rate of 1.5 L / min. Real-time and dynamic images were processed using specialized image processing software (ASIPro 6.7.1.1, SIEMENS), and regions of interest (ROIs) were further analyzed and normalized according to the injected dose. Tissue uptake values ​​are expressed as a percentage of the injected dose per cubic centimeter of tissue (%ID / mL).

[0107] Figure 6Figures A and 6B show that APP / PS1-induced AD model mice exhibited higher PET signals in the brains of APP / PS1 mice than wild-type mice at 5, 10, and 30 minutes after probe injection. Specifically, at 5 minutes, the radioactive uptake signal in the brains of APP / PS1 mice reached ~1.34 ± 0.17% ID / g, which was ~1.5 times that of wild-type mice. As the probe was metabolized and cleared in vivo, the radioactive uptake in the brains of both APP / PS1 and wild-type mice gradually decreased, and at 10 minutes, the radioactive uptake in the brains of APP / PS1 mice was ~1.9 times that of wild-type mice. This may be because the probe interacts with Aβ protein in the brains of APP / PS1 mice, while in wild-type mice the probe is rapidly metabolized and cleared, thus gradually increasing the difference in probe activity between the AD model mice and wild-type mice.

[0108] Simultaneously, the "cold" probe NIR-[Ga] was injected intravenously into 9-month-old APP / PS1 transgenic AD model mice and control wild-type mice (1.0 mg / kg, 20% DMSO + 20% polyoxyethylene ether castor oil + 60% PBS buffer). Fluorescence signals in the mouse brain were collected at 5, 10, 30, 60, 120, 240, and 360 mins after administration using the IVIS Lumina XRⅢ small animal imaging system (Caliper LifeSciences, PerkinElmer). The target region of interest (ROI) was then delineated using Living Image software (4.5.2, PerkinElmer, MA, USA), and the data were quantified to analyze the data and evaluate the imaging effect. Figure 6 As shown in C and 6D, the fluorescence signal of the probe in the brain of APP / PS1 mice quickly reached its maximum (5–10 min) and was significantly higher than that in wild-type mice. These results indicate that the bimodal probe can rapidly penetrate the BBB and bind to the Aβ protein in the brain, thereby generating enhanced PET and NIR fluorescence signals, effectively distinguishing APP / PS1 mice from control wild-type mice.

[0109] 7. NIR-[ 68 Distribution of Ga in the brain of APP / PS1 mice

[0110] First, the in vivo PET imaging results were further analyzed using PMOD image analysis software (3.8, Pmod Technologies, Zurich, Switzerland). For example... Figure 7As shown in Figure A, the PET signals in the cerebral cortex (Cor, blue line) and hippocampus (H, dark green) of APP / PS1 mice were higher than those in wild-type mice, while there was no significant difference in PET signals in the thalamus (T) of the brains of APP / PS1 mice and wild-type mice. Autoradiography of ex vivo brain tissue sections ( Figure 7 B) also confirmed that the probe was mainly distributed in the cortex and hippocampus. Furthermore, the "cold" probe NIR-[Ga] was injected intravenously into 10-month-old APP / PS1 transgenic AD model mice and control wild-type mice (2.0 mg / kg, 20% DMSO + 20% polyoxyethylene ether castor oil + 60% PBS buffer). Ten minutes later, the mice were sacrificed under deep anesthesia, and brain tissue was extracted for in vitro brain tissue section staining. Co-staining with the positive probe Thioflavin-T (ThT) confirmed the positive result. Figure 7 As shown in Figure C, Aβ protein in the cortex and hippocampus of the APP / PS1 mouse brain was "lit up," showing good co-localization with the positive control probe ThT for Aβ plaques. In contrast, no obvious fluorescent signal of Aβ protein was observed in brain tissue sections from wild-type mice. Figure 8 These results further demonstrate that the dual-modal probe can effectively penetrate the BBB and non-invasively image Aβ patches via enhanced PET and NIR fluorescence signals. Note: Cortex (Cor), Hippocampus (H), Thalamus (T).

[0111] This invention utilizes near-infrared (NIR) small-molecule fluorescent probes that specifically respond to monomers, oligomers, and aggregates of β-amyloid (Aβ) protein in Alzheimer's disease (AD) for radionuclide detection. 68 Ga 3+ The labeling enables dual-modal imaging detection of Aβ protein in AD, especially early soluble Aβ protein monomers and oligomers, using NIR fluorescence and positron emission tomography (PET), thereby improving the sensitivity and accuracy of in vivo Aβ protein detection and achieving early and accurate diagnosis of AD.

[0112] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A molecular imaging probe NIR-[Ga]; NIR-[Ga].

2. A multimodal molecular imaging probe NIR-[ 68 Ga]; NIR-[ 68 Ga].

3. The method for preparing the molecular imaging probe according to claim 1 or 2, comprising the following steps: Step a: Compound F1 converts the intracyclic double bond to an excyclic double bond under basic conditions to obtain intermediate F1-a; Step b: Compound F1-a is reacted with Vilsmeier-Haack to give compound F2; Step c: Compound F2 undergoes a condensation reaction with CRANAD-54 to obtain compound F3; Step d: Compound F3 undergoes a Click reaction with 6-azido-hexylamine to give compound F4; Step e: Compound F4 undergoes an acylation reaction with NODA-GA-NHS ester to give compound F5; Step f: Compound F5 and radionuclide 68 Ga 3+ A coordination reaction was performed to obtain the molecular imaging probe NIR-[ 68 Ga]; or compound F5 and Ga 3+ A coordination reaction was carried out to obtain the multimodal molecular imaging probe NIR-[Ga]; Where M is selected from 68 Ga or Ga.

4. The use of the molecular imaging probe according to claim 1 or 2 in the preparation of Aβ protein species detection reagents.

5. A reagent for detecting Aβ protein species, characterized in that, Includes the molecular imaging probe described in claim 1 or 2.

6. The use of the molecular imaging probe of claim 1 or 2 in the preparation of fluorescence imaging products for diagnosing Alzheimer's disease.

7. The application according to claim 6, characterized in that, The product includes contrast agents.

8. A product for diagnosing Alzheimer's disease using fluorescence imaging, characterized in that, Includes the molecular imaging probe described in claim 1 or 2.

9. The product according to claim 8, characterized in that, The product includes contrast agents.

10. The application according to claim 6 or 7 or the product according to claim 8, characterized in that, The fluorescence imaging is NIR fluorescence / PET dual-modal imaging.

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