Mitochondria-targeting probe, preparation method therefor, and application thereof

Abstract

A mitochondria-targeting probe, a preparation method therefor, and an application thereof. The mitochondria-targeting probe has a structure represented by formula (I). The probe compound has the characteristics of being simple to prepare, having good chemical stability, and having high radiochemical purity, and can be used as a radionuclide or fluorescent probe in the field of myocardial perfusion.

Description

A type of mitochondrial-targeted probe and its preparation method and application Technical Field

[0001] The present invention relates to the field of radiopharmaceutical chemistry and fluorescent molecular imaging technology, and in particular to a class of mitochondria-targeted small molecule probes, a preparation method and application thereof. Background Art

[0002] Mitochondria are crucial sites in the body for energy metabolism, maintaining ion homeostasis, and transmitting apoptosis signals. They are targets for many drugs and toxins. Organs and tissues with high metabolic activity, such as the heart and tumor tissue, contain large numbers of mitochondria. Numerous studies have linked mitochondrial abnormalities to a variety of diseases, including heart failure, cancer, and neurodegenerative disorders. Therefore, the development of mitochondrial-targeted probes could provide a basis for studying mitochondrial function and disease.

[0003] Because the tissue metabolism of the heart and tumor is active, they have a higher mitochondrial membrane potential and a larger membrane potential difference. Delocalized lipophilic cationic compounds (DLCs) will be enriched in the mitochondrial matrix of cells with a large membrane potential difference. Currently, mitochondrial-targeted lipophilic cations are widely used in the fields of anti-tumor drugs, molecular imaging probes, and myocardial perfusion imaging. However, there are relatively few studies on lipophilic cationic radionuclide probes. At present, the research and development is mainly based on basic structures such as triphenylphosphonium salts (TPP) and rhodamine derivatives. For example 18 F-FBnTP(Nature 2019, 575(7782), 380-384), 18 F-FPEGBnTP(J Labeled Comp Radiopharm 2016, 59(3), 117-123), 18 F-FTPMP (Eur.J.Med.Chem.2016, 118, 90-97), 18 F-FMBTP (Mol. Pharm. 2014, 11(11), 3823-3831) and 18 F-FPTP (ACS Med. Chem. Lett. 2014, 5 (10), 1124-1128), etc., and other structures such as rhodamine derivatives as representatives 18 F-FERhB (Nucl. Med. Biol. 2010, 37(3), 365-370), 18 F-Rhodamin 6G (Medchemcomm 2017, 8(10), 1891-1896), etc.

[0004] However, there are few reports on radioactive probes of indolevinylquinolate F16 compounds (Cancer Cell 2002, 2(1), 29-42; Chem. Commun. (Camb.) 2014, 50(64), 8919-8922; Chemical Science 2019, 10(34), 7946-7951; J Med Chem. 2022, 65(1): 497-506), which are widely used for tumor mitochondrial targeting, and there are no related studies on chelation with radioactive metal nuclides. F16 compounds have the characteristics of lipophilic delocalized cations and can be highly enriched in tumor cells and cardiomyocytes. At the same time, F16 compounds themselves have fluorescent properties, which facilitates fluorescence imaging screening and clinical fluorescence imaging surgical navigation. Therefore, the development of radioactive nuclide probes of indolevinylquinolate F16 compounds complexed with radioactive metal nuclides is of great significance for broadening the field of myocardial perfusion and tumor PET / SPECT molecular probes. Summary of the Invention

[0005] One object of the present invention is to provide a class of compounds, which are indole vinyl pyridinium salt F16 compounds that can be used as chelated radionuclides or fluorescent probes for mitochondrial targeting.

[0006] Another object of the present invention is to provide a method for preparing the compound.

[0007] Another object of the present invention is to provide a use of the compound as a mitochondrial-targeted radionuclide or fluorescent probe.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] According to one aspect of the present invention, there is provided a compound of the following formula I:

[0010] In formula I, the linker group is selected from C1-C6 alkylene or -(CH2CH2O) m -Any one of, wherein m is an integer from 2 to 30;

[0011] n = 1 or 2;

[0012] X - is an anion in any form, preferably I - , Br - , Cl - , BF4 - or ClO4 - ;

[0013] R1 is independently selected from H, C1-C10 alkyl, substituted C1-C10 alkyl, C2-C10 alkenyl, substituted C2-C10 alkenyl, C6-C10 aryl, substituted C6-C10 aryl, C5-C10 heteroaryl, and substituted C5-C10 heteroaryl;

[0014] R2 is independently selected from H, C1-C10 alkyl, substituted C1-C10 alkyl, C2-C10 alkenyl, substituted C2-C10 alkenyl, C6-C10 aryl, substituted C6-C10 aryl, C5-C10 heteroaryl, substituted C5-C10 heteroaryl, halogen, nitro and cyano;

[0015] R3 and R4 are each independently selected from H, C1-C10 alkyl, substituted C1-C10 alkyl, C2-C10 alkenyl, substituted C2-C10 alkenyl, C1-C10 alkoxy, C6-C10 aryl, substituted C6-C10 aryl, C5-C10 heteroaryl, substituted C5-C10 heteroaryl, -NR7R8, halogen, nitro and cyano, wherein R7 and R8 are each independently C1-C6 alkyl, or R7 and R8 together with the carbon atom to which they are attached form a 5-6 membered nitrogen-containing heterocyclic ring;

[0016] R5 and R6 are each independently selected from H, C1-C10 alkyl, substituted C1-C10 alkyl, C2-C10 alkenyl, substituted C2-C10 alkenyl, C6-C10 aryl, C1-C10 alkoxy, substituted C6-C10 aryl, C5-C10 heteroaryl, substituted C5-C10 heteroaryl, halogen, nitro and cyano; or R5 and R6 together with the carbon atom to which they are attached form C6-C10 aryl, substituted C6-C10 aryl, C5-C10 heteroaryl or substituted C5-C10 heteroaryl,

[0017] Wherein, the substituted refers to being substituted by a substituent selected from halogen, C1-C10 alkyl, halogen-substituted C1-C10 alkyl, C1-C10 alkoxy, and halogen-substituted C1-C10 alkoxy;

[0018] R is a monovalent or divalent group,

[0019] The monovalent or divalent group is derived from one of a colored luminophore, an organic fluorophore, an inorganic luminophore, a light absorbing compound, a light reflecting compound, a light scattering compound, and a bioluminescent molecule, preferably, it is derived from a near-infrared fluorescent dye, more preferably, it is derived from ICG; or

[0020] The monovalent or divalent group is derived from a bifunctional chelating agent that chelates with a radioactive nuclide or a metal element capable of generating a nuclear magnetic resonance signal, and the bifunctional chelating agent is selected from 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA), 1,4,7-triazacyclononane, 1-pentanedioic acid-4,7-acetic acid (NODAGA), 2,2′,2″,2″-(5 2 , 13 2 -Dihydroxy-5- 5 , 13 5 -dimethyl-3,7,11,15-tetraaza-1,9(2,6)-dipyridine-5,13(1,3)-dibenzocyclohexanedione-3,7,1,11,15-tetrayl)tetraacetic acid (Dar), dimethyltriaminepentaacetic acid (DTPA), deferoxamine (DFO), hydrazinonicotinamide (HYNIC), mercaptoacetyl triglycine (MAG3), 1,4,7,10-tetraazacyclododecane-1-pentanedioic acid-4,7,10-triacetic acid (DOTAGA), 1,4,7-triazacyclononane, 1-pentanedioic acid-4,7-acetic acid (NODAGA).

[0021] The radionuclides include diagnostic radionuclides and therapeutic radionuclides:

[0022] The diagnostic radionuclide is selected from: 86 Y、Al[ 18 F]、 51 Mn, 52m Mn, 52g Mn, 64 Cu, 67 Ga, 68 Ga, 89 Zr, 99m Tc, 111 In, 123 I. 124 I. 125 Any one of I, preferably 86 Y、Al[ 18 F]、 64 Cu, 68 Ga, 89 Zr, 99m Tc, 124 Any one of I;

[0023] The therapeutic radionuclide is selected from: 67 Cu, 90 Y. 125 I. 131 I. 153 Sm,166 Ho, 177 Lu, 186 Re、 188 Re、 211 At 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227 Any one of Th; preferably 67 Cu, 90 Y. 125 I. 131 I. 177 Lu, 223 Ra, 225 Ac, 211 Any of At;

[0024] The metal element capable of generating a nuclear magnetic resonance signal is selected from any one of Gd, Fe, Eu, Mn, Cu, Si, and Nd.

[0025] In a specific embodiment, in Formula I,

[0026] Linker is C2-C6 alkylene;

[0027] X - Br - ;

[0028] R1 is selected from H, C1-C10 alkyl substituted by halogen, and C1-C10 alkyl substituted by halogenated C1-C10 alkoxy;

[0029] R2, R3, R5 and R6 are H;

[0030] R4 is selected from: H, halogen, cyano, nitro, C1-C6 alkoxy,

[0031] R is a group derived from DOTA, NOTA and ICG, and the radionuclide chelated to DOTA or NOTA is 68 Ga.

[0032] Specifically, the compound of formula I according to the present invention is selected from the following compounds:

[0033] According to another aspect of the present invention, a method for preparing the compound of formula I is provided, and the preparation route thereof is as follows:

[0034] The method is carried out by the following method 1 or method 2,

[0035] Method 1

[0036] The steps include:

[0037] (a) 4-methylpyridine 1 and a compound of the general formula X-Linker-NH2 wherein X is a halogen undergo a nucleophilic substitution reaction to give compound 2;

[0038] (b) Compound 2 undergoes nitrogen acylation reaction with di-tert-butyl dicarbonate to obtain compound 3;

[0039] (c) Compound 3 undergoes Knoevenagel condensation reaction with substituted or unsubstituted indole-3-carboxaldehyde to obtain compound 4;

[0040] (d) removing the tert-butyloxycarbonyl protecting group of compound 4 in a hydrochloric acid-methanol solution to obtain compound 5;

[0041] (e) Compound 5 undergoes condensation reaction with the NHS ester of a bifunctional chelating agent to obtain compound 6;

[0042] (f) Compound 6 undergoes a chelation reaction with a radioactive nuclide to obtain the final product 7.

[0043] Method 2

[0044] The steps include:

[0045] (a) 4-methylpyridine 1 and a compound of the general formula X-Linker-NH2 wherein X is a halogen undergo a nucleophilic substitution reaction to give compound 2;

[0046] (b) Compound 2 undergoes nitrogen acylation reaction with di-tert-butyl dicarbonate to obtain compound 3;

[0047] (c) Compound 3 undergoes Knoevenagel condensation reaction with substituted or unsubstituted indole-3-carboxaldehyde to obtain compound 4;

[0048] (d) removing the tert-butyloxycarbonyl protecting group of compound 4 in a hydrochloric acid-methanol solution to obtain compound 5;

[0049] (g) Compound 5 undergoes a condensation reaction with a near-infrared fluorescent dye having a carboxyl group (e.g., a carboxylic acid derivative of ICG) to obtain compound 8;

[0050] Among them R1, R2, R3, R4, R5, R6, R, X - , Linker are defined as above.

[0051] In a specific embodiment, in step (a) of method 1 and method 2, 4-methylpyridine 1 and a compound of the general formula wherein X is a halogen are added to methanol, and heated with stirring under reflux overnight.

[0052] In a specific embodiment, in step (b) of method 1 and method 2, the solvent is 1,4-dioxane, and the reaction temperature is room temperature.

[0053] In a specific embodiment, in step (c) of method 1 and method 2, compound 3, 1 equivalent of substituted or unsubstituted indole-3-carboxaldehyde and 0.2 equivalent of piperidine are added to anhydrous methanol and heated under reflux overnight.

[0054] In a specific embodiment, in step (e) of method 1 and step (g) of method 2, N,N-diisopropylethylamine and N,N-dimethylformamide are added to react.

[0055] In a specific embodiment, in step (g) of method 2, HATU is further added to carry out condensation reaction.

[0056] According to another aspect of the present invention, the present invention also provides use of the compound of formula I as a mitochondrial-targeted radionuclide or fluorescent probe.

[0057] In a specific embodiment, the mitochondria-targeted radionuclide or fluorescent probe is a myocardial perfusion PET imaging agent or a myocardial perfusion fluorescence imaging agent, respectively. Beneficial effects

[0058] The radioactive metal nuclide chelated F16 compound of the present invention has simple preparation, good chemical stability, high radiochemical purity, and can be used as a radionuclide probe in the field of myocardial perfusion.

[0059] The F16 compounds containing near-infrared fluorescent dye groups according to the present invention have the advantages of simple preparation, good chemical stability, and fluorescence, and can be used as fluorescent probes in in vivo imaging, such as in the field of myocardial perfusion.

[0060] In summary, this type of compound has the advantages of simple preparation, good chemical stability, high radiochemical purity, and fluorescence. After chelation with radioactive metal nuclides, PET / CT imaging results show that it has a high uptake in the target tissue heart. It is expected to be used in clinical practice and developed into a new type of myocardial perfusion PET imaging agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG1 is a mass spectrum of Ga-DOTA-F16 prepared in Example 3.

[0062] FIG2 is a mass spectrum of ICG-F16 prepared in Example 5.

[0063] FIG3 is a mass spectrum of ICG-2F16 prepared in Example 6.

[0064] FIG4 is a fluorescence confocal localization image of Ga-DOTA-F16 in cardiomyocytes in Experimental Example 4.

[0065] Figure 5 shows the results of Experiment 5. 68 Micro-PET / CT imaging effect of Ga-DOTA-F16 in normal Balb / c mice after 30 minutes of myocardial perfusion. DETAILED DESCRIPTION

[0066] The technical solutions of the present invention are further described in detail below with reference to specific embodiments, but the scope of protection and implementation of the present invention are not limited thereto. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0067] Furthermore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0068] In this application, C as a prefix of a group i -C j It means that the group contains ij carbon atoms, for example, C1-C10 alkyl refers to an alkyl group containing 1-10 carbon atoms, that is, an alkyl group containing any integer number (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) of carbon atoms in the range of 1 to 10. Other similar expressions should be understood similarly.

[0069] In the present application, the term "heteroaryl" refers to an aromatic group containing at least one heteroatom selected from N, O and S in the aromatic ring system.

[0070] Unless otherwise specified, the raw materials, reagents, experimental animals, etc. used in this application are conventional raw materials, reagents, experimental animals in this field and are commercially available. The animal experiments comply with the ethical requirements for experimental animals, and the equipment and methods used are conventional equipment and methods in this field.

[0071] Example 1: Preparation of DOTA-F16

[0072] Step 1: Preparation of intermediate 2

[0073] 4-Methylpyridine (2.27 g, 24.37 mmol) and 2-bromoethylamine hydrobromide (5 g, 24.4 mmol) were added to 30 ml of methanol and heated with stirring under reflux overnight. After the reaction was complete, most of the solvent was evaporated under reduced pressure. The reaction solution was cooled in a refrigerator at 4°C, with the formation of a precipitate. The precipitate was collected by filtration and washed with cold methanol to obtain 23.626 g of the product as a white solid, with a yield of 49.9%. 1 H NMR (600MHz, CD3OD) δ 8.92 (d, 2H), 8.03 (d, 2H), 4.95 (t, 1H), 3.69 (t, 2H), 2.72 (s, 3H). 13 C NMR (101MHz, CD3OD) δ161.37, 144.11, 129.03, 56.97, 39.19, 20.88.

[0074] Step 2: Preparation of Intermediate 3

[0075] Intermediate 2 (200 mg, 0.67 mmol) obtained in the previous step and sodium carbonate (166 mg, 1.57 mmol) were dissolved in 5 ml of water. A solution of di-tert-butyl dicarbonate (161 mg, 0.74 mmol) in 5 ml of 1,4-dioxane was added dropwise to the reaction solution. The mixture was allowed to react overnight at room temperature. After completion of the reaction, the solvent was removed by rotary evaporation under reduced pressure. An appropriate amount of methanol was added, and the mixture was filtered, concentrated, and purified by column chromatography to obtain 3, a white solid (186.4 mg) with a yield of 87.6%. 1 H NMR (600MHz, CD3OD) δ 8.77 (d, 2H), 7.95 (d, 2H), 4.63 (t, 1H), 3.62 (t, 1H), 2.68 (s, 1H), 1.32 (s, 9H). 13 C NMR (101MHz, CD3OD) δ161.29, 157.94, 145.32, 129.67, 80.55, 62.02, 41.77, 28.52, 22.06.

[0076] Step 3: Preparation of intermediate 4

[0077] Intermediate 3 (330 mg, 1.04 mmol) obtained in the previous step was added to 1.0 equivalent of indole-3-carboxaldehyde (151 mg, 1.04 mmol) and 0.2 equivalent of piperidine (20 μL, 0.2 mmol) in 20 mL of anhydrous methanol. The reaction solution was heated under reflux overnight and monitored by TLC. After completion of the reaction, the product was concentrated by rotary evaporation under reduced pressure and purified by column chromatography to yield 4 as an orange-yellow solid (235 mg, 50.8% yield). 1H NMR (400MHz, CD3OD) δ8.24 (d, 2H), 8.04-7.91 (m, 2H), 7.84 (s, 1H), 7.72 (d, 2H), 7.56-7.46 ( m, 1H), 7.28 (ddd, 2H), 6.98 (d, 1H), 4.33 (t, 2H), 3.55 (t, 2H), 3.41 (s, 1H), 1.34 (s, 9H).13C NMR (101MHz, CD3OD) δ157.89, 156.43, 143.85, 138.92, 138.01, 133.42, 126.26 , 124.29, 122.72, 121.58, 117.81, 115.43, 113.42, 80.52, 60.59, 41.67, 28.56.

[0078] Step 4: Preparation of Intermediate 5

[0079] The intermediate 4 obtained in the previous step (235 mg, 0.53 mmol) was added to 5 ml of 4 mol / L HCl-MeOH solution and reacted at room temperature for 4 h. After the reaction, the reaction solution was cooled in a -20°C refrigerator, accompanied by the formation of a precipitate. The precipitate was collected by filtration and washed with cold methanol. The precipitate was then washed two to three times with anhydrous ether to obtain an orange-yellow solid product 5, 182 mg, with a yield of 96%. 1 H NMR (500MHz, DMSO-d6) δ12.19 (d, 2H), 8.80 (d, 2H), 8.25 (d, 1H), 8.09 (dd, 3H), 7.91 (d, 1H) ,7.45-7.42(m,1H),7.22(d,1H),7.14(pd,2H),4.74(dd,2H),3.06(s,2H),2.41(p,2H).13C NMR (126MHz, DMSO) δ155.23, 143.90, 137.60, 132.62, 124.95, 122.93, 122.03, 121.20, 120.53, 116.82, 113.67, 112.72, 55.81, 48.59, 40.02, 39.86, 39.69, 39.52, 39.36, 39.19, 39.02, 38.93.

[0080] Step 5: Preparation of DOTA-F16

[0081] Intermediate 5 (2 mg, 0.006 mmol) obtained in the previous step, N,N-diisopropylethylamine (8.8 μL, 0.024 mmol), and tetraazacyclododecane tetraacetic acid succinimidyl ester (8.9 mg, 0.012 mmol) were dissolved in 2 mL of N,N-dimethylformamide and allowed to react overnight at room temperature. After completion of the reaction, semi-preparative liquid chromatography was used to obtain the labeled precursor compound DOTA-F16 as an orange solid (1.67 mg, 39.4% yield). LRMS (ESI) m / z: [M-Br] + Calculation: C 33 H 44 N7O7 + : 650.33, measured: 650.69.

[0082] Example 2: Preparation of DOTA-3C-F16

[0083] The synthesis method was the same as in Example 1, except that 3-bromopropylamine hydrobromide was used instead of 2-bromoethylamine hydrobromide. HPLC separation yielded 1.3 mg of DOTA-3C-F16 with a yield of 28.3%. LRMS (ESI) m / z: [M-Br] + Calculation: C 34 H 46 N7O7 + : 664.35, measured: 664.52.

[0084] Example 3: Preparation of Ga-DOTA-F16

[0085] The compound prepared in Example 1 was dissolved in a pH 4.5 sodium acetate buffer solution, and an excess of solid gallium chloride was added. The reaction was allowed to proceed at 90°C for half an hour. After completion of the reaction, the target peak was collected by semi-preparative liquid phase separation and lyophilized to obtain the target compound Ga-DOTA-F16. LRMS (ESI) m / z: [M-Br] + Calculation: C 33 H 42 GaN7O7 + : 716.24, measured: 716.62. The mass spectrum results are shown in Figure 1.

[0086] Example 4: 68 Radiochemical labeling of Ga-DOTA-F16

[0087] 40 μL of sodium acetate solution (1.5 M) was added to a glass reaction bottle, and 10 μg of DOTA-F16 prepared in Example 1 was added. Then, 5 mCi 68 GaCl3 (~2 mL) was heated in a 95°C metal bath for 10 min, cooled to room temperature after the reaction was completed, and separated and purified by HPLC to obtain a radioactive probe68 Ga-DOTA-F16.

[0088] Example 5: Preparation of ICG-F16

[0089] ICG-2COOH (53.7 mg, 0.068 mmol) and 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) (103.4 mg, 0.272 mmol) were dissolved in 5 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (40.8 μL, 0.272 mmol) was added and the mixture was allowed to react for 30 minutes. Intermediate 5 synthesized in Example 1 (93.62 mg, 0.272 mmol) was then added and the reaction continued at room temperature overnight. After completion of the reaction, the product was spin-dried and purified by HPLC to obtain 27.9 mg of ICG-F16 in a 36.8% yield. LRMS (ESI) m / z: [M-2Br] 2+ Calculation: C 64 H 69 N5O3 2+ : 477.77, measured: 477.80. The mass spectrum results are shown in Figure 2.

[0090] Example 6: Preparation of ICG-2F16

[0091] ICG-2COOH (53.7 mg, 0.068 mmol) and 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) (103.4 mg, 0.272 mmol) were dissolved in 5 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (40.8 μL, 0.272 mmol) was added and the mixture was allowed to react for 30 minutes. Intermediate 5 synthesized in Example 1 (93.62 mg, 0.272 mmol) was then added and the reaction continued at room temperature overnight. After completion of the reaction, the product was spin-dried and purified by HPLC to obtain 25.7 mg of ICG-2F16 in a 26.2% yield. LRMS (ESI) m / z: [M-3Br] 3+ Calculation: C 81 H 85 N8O2 3+ : 400.56, measured: 401.07. The mass spectrum results are shown in Figure 3.

[0092] Test Example 1: Optical Properties of DOTA-F16

[0093] The final compound prepared in Example 1 above was dissolved in PBS solution, the solution was diluted to a concentration of 10 μmol / L, and placed in a quartz cuvette. The absorption spectrum was measured using a Shimadzu UV-2600i spectrophotometer, and the maximum absorption wavelength was calculated. The sample with the measured absorption spectrum was placed in a Hitachi F-4500 fluorescence spectrometer, and its fluorescence emission spectrum was measured using its maximum absorption wavelength as the excitation wavelength to obtain the maximum emission wavelength. The maximum absorption wavelength and maximum emission wavelength of DOTA-F16 were found to be 429 nm and 540 nm, respectively.

[0094] Experimental Example 2: Optical Properties of ICG-F16

[0095] The compound prepared in Example 5 above was dissolved in methanol solution, the solution was diluted to a concentration of 10 μmol / L and placed in a quartz cuvette. The absorption spectrum was measured using a Shimadzu UV-2600i spectrophotometer, and the maximum absorption wavelength was calculated. The sample with the measured absorption spectrum was placed in a Horiba Scientific IHR320 model fluorescence spectrometer, and the emission spectrum was measured with an excitation wavelength of 808 nm, and the maximum emission wavelength was calculated. The maximum absorption wavelength and maximum emission wavelength of ICG-F16 were found to be 787 nm and 933 nm, respectively.

[0096] Experimental Example 3: Optical Properties of ICG-2F16

[0097] The compound prepared in Example 6 above was dissolved in methanol solution, the solution was diluted to a concentration of 10 μmol / L and placed in a quartz cuvette. The absorption spectrum was measured using a Shimadzu UV-2600i spectrophotometer, and the maximum absorption wavelength was calculated. The sample with the measured absorption spectrum was placed in a Horiba Scientific IHR320 model fluorescence spectrometer, and the emission spectrum was measured with an excitation wavelength of 808 nm, and the maximum emission wavelength was calculated. The maximum absorption wavelength and maximum emission wavelength of ICG-2F16 were 787 nm and 907 nm, respectively.

[0098] Experimental Example 4: Localization of Ga-DOTA-F16 in Subcellular Organelles of Cardiomyocytes

[0099] AC16 cells were passaged into the logarithmic growth phase and digested with 0.25% trypsin. The cells were collected and centrifuged. The supernatant was discarded and resuspended in 1 ml of DMEM high glucose medium. After counting with a hemocytometer, the cells were inoculated into a twelve-well plate with a cell slide placed in advance, with 1 ml per well, so that the number of cells in each well was 5 × 10 4Individual. Culture at 37℃ 5% CO2 saturated humidity for 24 hours. After the cells adhere to the wall, discard the DMEM high-glucose medium in the wells, add the Ga-DOTA-F16 solution prepared in Example 3 at a concentration of 2μM, and incubate at 37℃ 5% CO2 saturated humidity for 1 hour, then discard the liquid in the wells and rinse with PBS three times. Add Mito-Tracker Deep Red at a concentration of 500nM, and incubate at 37℃ 5% CO2 saturated humidity for 0.5 hour, then discard the liquid in the wells and rinse with PBS three times. Add 4% paraformaldehyde to fix for 15 minutes, discard the liquid in the wells and rinse with PBS three times. Add DAPI and discard it after 2 minutes, then rinse three times with PBS. Remove the slide and carefully seal it on a glass slide with a sealing agent. Observe the cells and photograph them under a laser confocal microscope. The results are shown in Figure 4. Where DAPI: λ ex =405nm,λ em =415-487nm.Ga-DOTA-F16:λ ex =488nm,λem=498-542nm.Mito-Tracker Deep Red:λ ex =633nm,λ em =643-750 nm. As can be seen from the results in FIG4 , the target compound overlaps with the commercial MitoTracker dye (red), thus, the Ga-DOTA-F16 prepared in this application has mitochondrial targeting.

[0100] Test Example 5: 68 Application of Ga-DOTA-F16 in myocardial perfusion micro-PET / C imaging in mice

[0101] Inject approximately 150 μCi / 200 μL into the tail vein of normal commercially available SPF-grade Balb / c mice. 68 Ga-DOTA-F16 (prepared in Preparation Example 4) was injected, and PET / CT imaging was performed at 0.5, 1, and 2 hours after injection. The distribution of the probe in the mouse body and its accumulation in the cardiac region were observed. The imaging results are shown in Figure 5. As can be seen from the figure, the probe clearly accumulates in the myocardium from 0.5 hours onward, with clear edge contours, and the probe remains in the myocardium until 2 hours.

[0102] Test Example 6: 68 Biodistribution of Ga-DOTA-F16 in mice

[0103] Sixteen normal commercially available SPF grade Balb / c mice were randomly divided into four groups and injected via the tail vein with 150 μCi / 200 μL of the drug prepared in Example 4. 68The Ga-DOTA-F16 probe was injected. 10, 30, 60, and 120 minutes after injection, the animals were sacrificed and their organs were harvested. The weights were measured using a CPM counter and the %ID / g was calculated. The results are shown in Table 1.

[0104] Table 1: 68 Uptake, distribution and ratio of Ga-DOTA-F16 in normal mice

[0105] As shown in Figure 5 and Table 1: 68 The biodistribution results of Ga-DOTA-F16 in normal mice showed a high initial uptake value and good retention in the myocardium. 10 minutes after injection, the myocardial uptake value was 9.12±1.69%ID / g. 120 minutes after injection, the myocardial uptake value was still 5.97±1.61%ID / g. The heart was clearly visible in PET / CT imaging. The probe had high uptake values ​​in both the liver and kidneys, indicating that 68 Ga-DOTA-F16 is primarily excreted through the liver and kidneys in mice. The ratio of heart to muscle is high, at 11.18 ± 2.01 at 10 minutes, which improves imaging contrast and leads to better diagnostic results.

[0106] The above description is only a preferred embodiment of the present invention, which is only illustrative but not restrictive of the present invention. A person skilled in the art can understand that many modifications or equivalent changes can be made within the spirit and scope defined by the claims of the present invention, but all of them fall within the scope of protection of the present invention.

Claims

1. A compound of the following formula I: In formula I, the linker group is selected from C1-C6 alkylene or -(CH2CH2O) m - any one of, wherein, m is an integer from 2 to 30; n = 1 or 2; X - is any form of anion, preferably I - ,Br - , Cl - , BF4 - or ClO4 - ; R1 is independently selected from H, C1-C10 alkyl, substituted C1-C10 alkyl, C2-C10 alkenyl, substituted C2-C10 alkenyl, C6-C10 aryl, substituted C6-C10 aryl, C5-C10 heteroaryl, and substituted C5-C10 heteroaryl; R2 is independently selected from H, C1-C10 alkyl, substituted C1-C10 alkyl, C2-C10 alkenyl, substituted C2-C10 alkenyl, C6-C10 aryl, substituted C6-C10 aryl, C5-C10 heteroaryl, substituted C5-C10 heteroaryl, halogen, nitro and cyano; R3 and R4 are each independently selected from H, C1-C10 alkyl, substituted C1-C10 alkyl, C2-C10 alkenyl, substituted C2-C10 alkenyl, C1-C10 alkoxy, C6-C10 aryl, substituted C6-C10 aryl, C5-C10 heteroaryl, substituted C5-C10 heteroaryl, -NR7R8, halogen, nitro and cyano, wherein R7 and R8 are each independently C1-C6 alkyl, or R7 and R8 together with the carbon atom to which they are attached form a 5-6 membered nitrogen-containing heterocyclic ring; R5 and R6 are each independently selected from H, C1-C10 alkyl, substituted C1-C10 alkyl, C2-C10 alkenyl, substituted C2-C10 alkenyl, C6-C10 aryl, C1-C10 alkoxy, substituted C6-C10 aryl, C5-C10 heteroaryl, substituted C5-C10 heteroaryl, halogen, nitro and cyano; or R5 and R6 together with the carbon atom to which they are attached form C6-C10 aryl, substituted C6-C10 aryl, C5-C10 heteroaryl or substituted C5-C10 heteroaryl, Wherein, the substitution refers to substitution by a substituent selected from halogen, C1-C10 alkyl, halogen-substituted C1-C10 alkyl, C1-C10 alkoxy, halogen-substituted C1-C10 alkoxy; R is a monovalent or divalent group, the monovalent or divalent group is derived from: (i) one of a colored luminophore, an organic fluorophore, an inorganic luminophore, a light absorbing compound, a light reflecting compound, a light scattering compound, and a bioluminescent molecule, preferably, it is derived from a near-infrared fluorescent dye, more preferably, it is derived from ICG; or (ii) a bifunctional chelating agent chelated with a radioactive nuclide or a metal element capable of generating a nuclear magnetic resonance signal, wherein the bifunctional chelating agent is selected from 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA), 1,4,7-triazacyclononane, 1-pentanedioic acid-4,7-acetic acid (NODAGA), 2,2′,2″,2″-(5 2 , 13 2 -Dihydroxy-5 5 , 13 5 - any one of dimethyl-3,7,11,15-tetraaza-1,9(2,6)-bipyridine-5,13(1,3)-dibenzocyclohexanedione-3,7,1,11,15-tetrayl)tetraacetic acid (Dar), dimethyltriaminepentaacetic acid (DTPA), deferoxamine (DFO), hydrazinonicotinamide (HYNIC), mercaptoacetyltriglycine (MAG3), 1,4,7,10-tetraazacyclododecane-1-pentanedioic acid-4,7,10-triacetic acid (DOTAGA), 1,4,7-triazacyclononane, 1-pentanedioic acid-4,7-acetic acid (NODAGA); The radionuclides include diagnostic radionuclides and therapeutic radionuclides; The diagnostic radionuclide is selected from: 86 Y、Al[ 18 F], 51 Mn, 52m Mn, 52g Mn, 64 Cu, 67 Ga, 68 Ga, 89 Zr, 99m Tc, 111 In, 123 I. 124 I. 125 Any one of I, preferably 86 Y、Al[ 18 F], 64 Cu, 68 Ga, 89 Zr, 99m Tc, 124 Any one of I; The therapeutic radionuclide is selected from: 67 Cu, 90 Y. 125 I. 131 I. 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 211 At 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227 Any one of Th; preferably 67 Cu, 90 Y. 125 I. 131 I. 177 Lu, 223 Ra, 225 Ac, 211 Any of At; The metal element capable of generating a nuclear magnetic resonance signal is selected from any one of Gd, Fe, Eu, Mn, Cu, Si, and Nd.

2. The compound according to claim 1, wherein In Formula I, Linker is C2-C6 alkylene; X - Br - ; R1 is selected from H, C1-C10 alkyl substituted by halogen, and C1-C10 alkyl substituted by halogenated C1-C10 alkoxy; R2, R3, R5 and R6 are H; R4 is selected from: H, halogen, cyano, nitro, C1-C6 alkoxy, R is a group derived from DOTA, NOTA and ICG, and the radionuclide chelated with DOTA or NOTA is 68 Ga.

3. The compound according to claim 1, wherein The compound of formula I is selected from the following compounds:

4. A method for preparing a compound of formula I, wherein the preparation route is as follows: In the above routes, R1, R2, R3, R4, R5, R6, R, X - , Linker, n are respectively as defined in claim 1, The method is carried out by the following method 1 or method 2, Method 1: When the substituent R in formula I is the case of (ii) in claim 1, The steps include: (a) 4-methylpyridine 1 and a compound of the general formula X-Linker-NH2 wherein X is a halogen undergo a nucleophilic substitution reaction to obtain compound 2; (b) Compound 2 undergoes nitrogen acylation reaction with di-tert-butyl dicarbonate to obtain compound 3; (c) Compound 3 undergoes Knoevenagel condensation reaction with substituted or unsubstituted indole-3-carboxaldehyde to obtain compound 4; (d) Compound 4 is subjected to the step of removing the tert-butyloxycarbonyl protecting group in a hydrochloric acid-methanol solution to obtain compound 5; (e) Compound 5 undergoes condensation reaction with the NHS ester of a bifunctional chelating agent to obtain compound 6; (f) Compound 6 undergoes a chelation reaction with a radioactive nuclide to obtain the final product 7, Method 2: When the substituent R in formula I is the case of (i) in claim 1, The steps include: (a) 4-methylpyridine 1 and a compound of the general formula X-Linker-NH2 wherein X is a halogen undergo a nucleophilic substitution reaction to obtain compound 2; (b) Compound 2 undergoes nitrogen acylation reaction with di-tert-butyl dicarbonate to obtain compound 3; (c) Compound 3 undergoes Knoevenagel condensation reaction with substituted or unsubstituted indole-3-carboxaldehyde to obtain compound 4; (d) Compound 4 is subjected to the step of removing the tert-butyloxycarbonyl protecting group in a hydrochloric acid-methanol solution to obtain compound 5; (g) Compound 5 undergoes a condensation reaction with a near-infrared fluorescent dye having a carboxyl group (such as the carboxylic acid of ICG) to obtain compound 8.

5. The method according to claim 4, wherein: In step (a) of method 1 and method 2, 4-methylpyridine 1 and a compound of the general formula wherein X is a halogen are added to methanol, and heated, stirred and refluxed overnight; and / or In step (b) of method 1 and method 2, the solvent is 1,4-dioxane and the reaction temperature is room temperature; and / or In step (c) of method 1 and method 2, compound 3, 1 equivalent of substituted or unsubstituted indole-3-carboxaldehyde and 0.2 equivalent of piperidine are added to anhydrous methanol and heated under reflux overnight; and / or In step (e) of method 1 and step (g) of method 2, N,N-diisopropylethylamine and N,N-dimethylformamide are added to react; and / or In step (g) of method 2, HATU is further added to carry out a condensation reaction.

6. Use of the compound of formula I as claimed in claim 1 as a mitochondrial targeted radionuclide or fluorescent probe.

7. The use according to claim 6, wherein: The mitochondria-targeted radionuclide or fluorescent probe is a myocardial perfusion PET imaging agent or a myocardial perfusion fluorescent imaging agent.

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