Mitochondrial targeting probe as well as preparation method and application thereof

Abstract

The invention relates to a mitochondrial targeting probe as well as a preparation method and application thereof. The mitochondrial targeting probe has a structure as shown in a formula I. The probe compound provided by the invention has the characteristics of simple preparation, good chemical stability and high radiochemical purity, and can be applied to the field of myocardial perfusion as a radionuclide or a fluorescent probe. # imgabs0 #

Description

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 mitochondrial-targeted small molecule probes, and a preparation method and application thereof. Background Art

[0002] Mitochondria are an important place in the body for energy metabolism, maintaining ion homeostasis, and transmitting apoptosis signals. They are the targets of many drugs and poisons. Organs and tissues with vigorous metabolism, such as the heart and tumor tissues, contain a large number of mitochondria in their cells. A large number of studies have shown that mitochondrial abnormalities are associated with a variety of diseases, such as heart failure, tumors, and neurodegenerative diseases. Therefore, the development of mitochondrial-targeted probes can provide a certain detection basis for studying mitochondrial functions and diseases.

[0003] Because the tissue metabolism of the heart and tumor is active, they have higher mitochondrial membrane potential and larger membrane potential difference. Delocalized lipophilic cationic compounds (DLCs) will be enriched in the mitochondrial matrix of cells with 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., other structures such as rhodamine derivatives 18 F-FERhB(Nucl.Med.Biol.2010,37(3),365-370)、 18 F-Rhodamin 6G (Medchemcomm2017, 8(10), 1891-1896), etc.

[0004] However, there are few reports on the radioactive probe research of indole vinyl quinoline salt 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) widely used in tumor mitochondrial targeting, and there is no relevant research 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 fluorescence characteristics, which are convenient for fluorescence imaging screening and clinical fluorescence imaging surgical navigation. Therefore, the development of radioactive nuclide probes of indole vinyl quinoline salt F16 compounds combined 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 chelates for radioactive nuclides or fluorescent probes for mitochondrial targeting.

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

[0007] Another object of the present invention is to provide the application of the compound as a radioactive nuclide or fluorescent probe for mitochondrial targeting.

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

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

[0010]

[0011] In formula I, the linking group Linker is selected from any one of C1 - C6 alkylene or -(CH 2 CH 2 O) m -, where m is an integer from 2 to 30;

[0012] n = 1 or 2;

[0013] X - is an anion in any form, preferably I - , Br - , Cl - , BF 4 - or ClO 4 -;

[0014] R 1 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;

[0015] R 2 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;

[0016] R 3 and R 4 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, -NR 7 R 8 , halogen, nitro and cyano, wherein, R 7 and R 8 are each independently C1-C6 alkyl, or, R 7 and R 8 together with the carbon atom to which they are attached form a 5- to 6-membered nitrogen-containing heterocycle;

[0017] R 5 and R 6 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 R 5 and R 6 together with the carbon atom to which they are attached form a C6-C10 aryl, substituted C6-C10 aryl, C5-C10 heteroaryl or substituted C5-C10 heteroaryl,

[0018] wherein, the substitution means being substituted by a substituent selected from halogen, C1-C10 alkyl, halogen-substituted C1-C10 alkyl, C1-C10 alkoxy, halogen-substituted C1-C10 alkoxy;

[0019] R is a monovalent or divalent group,

[0020] The monovalent or divalent group is derived from one of a chromogenic lumophore, an organic fluorophore, an inorganic lumophore, 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, and more preferably, it is derived from ICG; or

[0021] The monovalent or divalent group is derived from a bifunctional chelating agent that chelates with a radionuclide or a metal element capable of generating a nuclear magnetic resonance signal. 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-diacetic 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)-dipyridyl-5,13(1,3)-dibenzocyclohexanedione-3,7,1,11,15-tetrayl)tetraacetic acid (Dar), dimethyltriaminepentaacetic acid (DTPA), desferrioxamine (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-diacetic acid (NODAGA).

[0022] The radionuclides include diagnostic radionuclides and therapeutic radionuclides:

[0023] The diagnostic radionuclides are 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 I, any one of them, preferably 86 Y, Al 18 F], 64 Cu, 68 Ga, 89 Zr, 99m Tc, 124Any one of those in I;

[0024] 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 Th; preferably any one of 67 Cu, 90 Y, 125 I, 131 I, 177 Lu, 223 Ra, 225 Ac, 211 At;

[0025] The metal element capable of generating nuclear magnetic resonance signals is selected from any one of Gd, Fe, Eu, Mn, Cu, Si, Nd.

[0026] In a specific embodiment, in Formula I,

[0027] Linker is C2-C6 alkylene;

[0028] X - is Br - ;

[0029] R 1 is selected from H, C1-C10 alkyl substituted by halogen, and C1-C10 alkyl substituted by halo C1-C10 alkoxy;

[0030] R 2 , R 3 , R 5 and R 6 are H;

[0031] R 4 is selected from: H, halogen, cyano, nitro, C1-C6 alkoxy, and

[0032] R is a group derived from DOTA, NOTA and ICG, and the radionuclide chelated with DOTA or NOTA is68 Ga。

[0033] Specifically, the compound of formula I according to the present invention is selected from the following compounds:

[0034]

[0035]

[0036]

[0037] According to another aspect of the present invention, there is provided a method for preparing the compound of formula I, and the preparation route is as follows:

[0038]

[0039] The method is carried out by the following method 1 or method 2,

[0040] Method 1

[0041] Comprising the following steps:

[0042] (a) 4-Methylpyridine 1 and a general formula compound X-Linker-NH in which X is a halogen 2 Undergo a nucleophilic substitution reaction to obtain compound 2;

[0043] (b) Compound 2 and di-tert-butyl dicarbonate undergo a nitrogen acylation reaction to obtain compound 3;

[0044] (c) Compound 3 and substituted or unsubstituted indole-3-carbaldehyde undergo a Knoevenagel condensation reaction to obtain compound 4;

[0045] (d) Compound 4 removes the tert-butoxycarbonyl protecting group in a hydrochloric acid-methanol solution to obtain compound 5;

[0046] (e) Compound 5 and the NHS ester of a bifunctional chelating agent undergo a condensation reaction to obtain compound 6;

[0047] (f) Compound 6 and a radionuclide undergo a chelation reaction to obtain the final product 7.

[0048] Method 2

[0049] Comprising the following steps:

[0050] (a) 4-Methylpyridine 1 and a general formula compound X-Linker-NH in which X is a halogen 2 Undergo a nucleophilic substitution reaction to obtain compound 2;

[0051] (b) Compound 2 and di-tert-butyl dicarbonate undergo a nitrogen acylation reaction to obtain compound 3;

[0052] (c) Compound 3 undergoes a Knoevenagel condensation reaction with substituted or unsubstituted indole-3-carbaldehyde to obtain Compound 4;

[0053] (d) The tert-butoxycarbonyl protecting group of Compound 4 is removed in hydrochloric acid-methanol solution to obtain Compound 5;

[0054] (g) Compound 5 undergoes a condensation reaction with a near-infrared fluorescent dye with a carboxyl group (such as a carboxylic acid derivative of ICG) to obtain Compound 8;

[0055] wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R, X - , Linker are respectively defined as above.

[0056] In a specific embodiment, in step (a) of Method 1 and Method 2, 4-methylpyridine 1 and a general formula compound wherein X is a halogen are added to methanol, and the mixture is heated with stirring and refluxed overnight.

[0057] 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.

[0058] In a specific embodiment, in step (c) of Method 1 and Method 2, Compound 3, 1 equivalent of substituted or unsubstituted indole-3-carbaldehyde, and 0.2 equivalent of piperidine are added to anhydrous methanol, and the mixture is refluxed and heated overnight.

[0059] 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 for reaction.

[0060] In a specific embodiment, in step (g) of Method 2, HATU is further added for condensation reaction.

[0061] According to another aspect of the present invention, the present invention also provides the use of the compound of formula I as a mitochondrion-targeted radionuclide or fluorescent probe.

[0062] In a specific embodiment, the mitochondrion-targeted radionuclide or fluorescent probe is a myocardial perfusion PET imaging agent or a myocardial perfusion fluorescent imaging agent, respectively.

[0063] Beneficial effects

[0064] The F16-class compound chelated with a radioactive metal nuclide according to the present invention is simple to prepare, has good chemical stability, high radiochemical purity, and can be used as a radionuclide probe in the field of myocardial perfusion.

[0065] The F16-class compound containing a near-infrared fluorescent dye group according to the present invention is simple to prepare, has good chemical stability, and has advantages such as fluorescence, and can be used as a fluorescent probe in in vivo imaging, for example, in the field of myocardial perfusion.

[0066] In summary, this class of compounds has the advantages of simple preparation, good chemical stability, high radiochemical purity, and fluorescence. After chelation with a radioactive metal nuclide, the PET / CT imaging results show that the uptake is relatively high in the target tissue, the heart, and it is expected to be applied clinically and developed into a new type of myocardial perfusion PET imaging agent. Description of the Drawings

[0067] Figure 1 It is the mass spectrum of Ga-DOTA-F16 prepared in Example 3.

[0068] Figure 2 It is the mass spectrum of ICG-F16 prepared in Example 5.

[0069] Figure 3 It is the mass spectrum of ICG-2F16 prepared in Example 6.

[0070] Figure 4 It is the fluorescence confocal localization map of Ga-DOTA-F16 in cardiomyocytes in Test Example 4.

[0071] Figure 5 For Test Example 5 68 The micro-PET / CT imaging effect diagram of Ga-DOTA-F16 after 30 minutes of myocardial perfusion in normal Balb / c mice in vivo. Detailed Embodiments

[0072] The following further describes the technical solutions of the present invention in detail with specific embodiments, but the protection scope and implementation manners 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.

[0073] And, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0074] In this application, C as a prefix of a certain group i -C j means that the group contains i - j 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 within the range of 1 - 10. Other similar expressions should be understood similarly.

[0075] In this application, the term "heteroaryl" refers to an aryl group containing at least heteroatoms selected from N, O, and S in the aromatic ring system.

[0076] Unless otherwise specified, the raw materials, reagents, test animals, etc. used in this application are all conventional raw materials, reagents, and test animals in the art and are commercially available. Animal experiments comply with the ethical requirements of experimental animals. The equipment and methods used are all conventional equipment and methods in the art.

[0077] Example 1: Preparation of DOTA - F16

[0078] Step 1: Preparation of Intermediate 2

[0079]

[0080] 4 - Methylpyridine (2.27 g, 24.37 mmol) and 2 - bromoethylamine hydrobromide (5 g, 24.4 mmol) were added to 30 ml of methanol, heated and stirred under reflux overnight. After the reaction was completed, most of the solvent was evaporated under reduced pressure. The reaction solution was placed in a refrigerator at 4 °C to cool, and a precipitate was formed. The precipitate was collected by filtration, washed with cold methanol, and finally 3.626 g of a white solid product 2 was obtained, with a yield of 49.9%. 1 H NMR(600MHz,CD3OD)δ8.92(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.

[0081] Step 2: Preparation of Intermediate 3

[0082]

[0083] The 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, and the reaction was carried out overnight at room temperature. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure. An appropriate amount of methanol was added, filtered, and concentrated, and then purified by column chromatography to obtain 186.4 mg of white solid 3, with a yield of 87.6%. 1 H NMR (600 MHz, 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 (101 MHz, CD3OD) δ 161.29, 157.94, 145.32, 129.67, 80.55, 62.02, 41.77, 28.52, 22.06.

[0084] Step 3: Preparation of intermediate 4

[0085]

[0086] The intermediate 3 (330 mg, 1.04 mmol) obtained in the previous step, 1.0 equivalent of indole-3-carbaldehyde (151 mg, 1.04 mmol), and 0.2 equivalent of piperidine (20 μL, 0.2 mmol) were added to 20 ml of anhydrous methanol. The reaction solution was refluxed and heated overnight, and the reaction was monitored by TLC. After the reaction was completed, it was concentrated by rotary evaporation under reduced pressure and purified by column chromatography to obtain 235 mg of orange-yellow solid 4, with a yield of 50.8%. 1 H NMR (400 MHz, CD3OD) δ 8.24 (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 (101 MHz, CD3OD) δ 157.89, 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.

[0087] Step 4: Preparation of intermediate 5

[0088]

[0089] The intermediate 4 (235 mg, 0.53 mmol) obtained in the previous step was added to 5 ml of 4 mol / L HCl-MeOH solution, and the reaction was carried out at room temperature for 4 h. After the reaction was completed, the reaction solution was placed in a refrigerator at -20 °C to cool, and a precipitate was formed. The precipitate was filtered and washed with cold methanol, and the collected precipitate was washed with anhydrous ether two to three times to obtain an orange-yellow solid product 5, 182 mg, with a yield of 96%. 1 H NMR (500 MHz, DMSO-d6) δ 12.19 (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 (126 MHz, DMSO) δ 155.23, 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.

[0090] Step 5: Preparation of DOTA-F16

[0091]

[0092] The intermediate 5 (2 mg, 0.006 mmol), N,N-diisopropylethylamine (8.8 μL, 0.024 mmol) and tetraazacyclododecane tetraacetic acid-N-hydroxysuccinimide ester (8.9 mg, 0.012 mmol) obtained in the previous step were dissolved in 2 ml of N,N-dimethylformamide, and the reaction was carried out overnight at room temperature. After the reaction was completed, semi-preparative liquid chromatography was used for separation to obtain the labeled precursor compound orange-yellow solid DOTA-F16, 1.67 mg, with a yield of 39.4%. LRMS (ESI) m / z: [M-Br] + Calculated: C 33 H 44 N 7 O 7 + : 650.33, found: 650.69.

[0093] Example 2: Preparation of DOTA-3C-F16

[0094] The synthesis method was the same as that in Example 1, except that 3-bromopropylamine hydrobromide was used instead of 2-bromoethylamine hydrobromide. After separation by HPLC, 1.3 mg of the product DOTA-3C-F16 was obtained with a yield of 28.3%. LRMS(ESI) m / z: [M - Br] + Calculated: C 34 H 46 N 7 O 7 + : 664.35, found: 664.52.

[0095] Example 3: Preparation of Ga-DOTA-F16

[0096] An appropriate amount of the compound prepared in Example 1 above was dissolved in a sodium acetate buffer solution with a pH of 4.5, and an excessive amount of gallium chloride solid was added. The reaction was carried out at 90 °C for half an hour. After the reaction was completed, it was separated by semi-preparative liquid phase, and the target peak was collected and freeze-dried to obtain the target compound Ga-DOTA-F16. LRMS(ESI) m / z: [M - Br] + Calculated: C 33 H 42 GaN 7 O 7 + : 716.24, found: 716.62. The mass spectrometry results are shown in Figure 1 .

[0097] Example 4: 68 Radiochemical labeling of Ga-DOTA-F16

[0098] 40 μL of sodium acetate solution (1.5 M) was added to a glass reaction flask, and then 10 μg of DOTA-F16 prepared in Example 1 was added. Then, 5 mCi 68 GaCl 3 (~2 mL) was added and heated in a metal bath at 95 °C for 10 min. After the reaction was completed, it was cooled to room temperature, and the radioactive probe 68 Ga-DOTA-F16 was prepared by separation and purification through HPLC.

[0099] Example 5: Preparation of ICG-F16

[0100]

[0101] ICG-2COOH (53.7 mg, 0.068 mmol) and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATU (103.4 mg, 0.272 mmol) were dissolved in 5 ml of N,N-dimethylformamide. After adding N,N-diisopropylethylamine (40.8 μL, 0.272 mmol) and reacting for 30 min, the intermediate 5 synthesized in Example 1 above (93.62 mg, 0.272 mmol) was added and the reaction was continued overnight at room temperature. After the reaction was completed, the solvent was evaporated, and the product ICG-F16 27.9 mg was obtained by separation and purification by HPLC, with a yield of 36.8%. LRMS (ESI) m / z: [M-2Br] 2+ Calculated: C 64 H 69 N 5 O 3 2+ : 477.77, found: 477.80. The mass spectrometry results are shown in Figure 2 。

[0102] Example 6: Preparation of ICG-2F16

[0103]

[0104] ICG-2COOH (53.7 mg, 0.068 mmol) and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATU (103.4 mg, 0.272 mmol) were dissolved in 5 ml of N,N-dimethylformamide. After adding N,N-diisopropylethylamine (40.8 μL, 0.272 mmol) and reacting for 30 min, the intermediate 5 synthesized in Example 1 above (93.62 mg, 0.272 mmol) was added and the reaction was continued overnight at room temperature. After the reaction was completed, the solvent was evaporated, and the product ICG-2F16 25.7 mg was obtained by separation and purification by HPLC, with a yield of 26.2%. LRMS (ESI) m / z: [M-3Br] 3+ Calculated: C 81 H 85 N 8 O 2 3+ : 400.56, found: 401.07. The mass spectrometry results are shown in Figure 3 。

[0105] Test Example 1: Optical properties of DOTA-F16

[0106] The final compound prepared in Example 1 above was dissolved in PBS solution, and the solution was diluted to a concentration of 10 μmol / L, filled in a quartz cuvette, and its absorption spectrum was measured using a Shimadzu UV-2600i spectrophotometer to calculate the maximum absorption wavelength; the sample with the measured absorption spectrum above was placed in a Hitachi F-4500 fluorescence spectrometer, and its fluorescence emission spectrum was measured with 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 obtained as 429 nm and 540 nm, respectively.

[0107] Test Example 2: Optical properties of ICG-F16

[0108] The compound prepared in Example 5 above was dissolved in methanol solution, and the solution was diluted to a concentration of 10 μmol / L, filled in a quartz cuvette, and its absorption spectrum was measured using a Shimadzu UV-2600i spectrophotometer to calculate the maximum absorption wavelength; the sample with the measured absorption spectrum above was placed in a fluorescence spectrometer of model IHR320 of Horiba Scientific, and its emission spectrum was measured with 808 nm as the excitation wavelength to calculate the maximum emission wavelength; the maximum absorption wavelength and maximum emission wavelength of ICG-F16 were obtained as 787 nm and 933 nm, respectively.

[0109] Test Example 3: Optical properties of ICG-2F16

[0110] The compound prepared in Example 6 above was dissolved in methanol solution, and the solution was diluted to a concentration of 10 μmol / L, filled in a quartz cuvette, and its absorption spectrum was measured using a Shimadzu UV-2600i spectrophotometer to calculate the maximum absorption wavelength; the sample with the measured absorption spectrum above was placed in a fluorescence spectrometer of model IHR320 of Horiba Scientific, and its emission spectrum was measured with 808 nm as the excitation wavelength to calculate the maximum emission wavelength; the maximum absorption wavelength and maximum emission wavelength of ICG-2F16 were obtained as 787 nm and 907 nm, respectively.

[0111] Test Example 4: Subcellular localization of Ga-DOTA-F16 in cardiomyocytes

[0112] The AC16 cells were cultured to the logarithmic growth phase, digested with 0.25% trypsin, collected, centrifuged, and after discarding the supernatant, 1 ml of high-glucose DMEM medium was added to resuspend the cells and counted with a hemocytometer. Then the cells were inoculated into a 12-well plate with cell slides placed in advance, 1 ml per well, so that the number of cells per well was 5×10 4 cells. At 37 °C and 5% CO 2Cultivate for 24 h under saturated humidity. After the cells adhered to the wall, aspirate and discard the high-glucose DMEM medium in the wells, add the Ga-DOTA-F16 solution prepared in Example 3 with a concentration of 2 μM, and incubate at 37 °C in 5% CO 2 For 1 h under saturated humidity. Then aspirate and discard the liquid in the wells, and wash 3 times with PBS. Add Mito-Tracker Deep Red with a concentration of 500 nM, and incubate at 37 °C in 5% CO Figure 4 . Among them, DAPI: λ ex = 405 nm, λ em = 415 - 487 nm. Ga-DOTA-F16: λ ex = 488 nm, λ em = 498 - 542 nm. Mito-Tracker Deep Red: λ ex = 633 nm, λ em = 643 - 750 nm. It can be seen from the Figure 4 results that the target compound overlaps with the commercial MitoTracker dye (red). Therefore, the Ga-DOTA-F16 prepared in this application has mitochondrial targeting.

[0113] Test Example 5: 68 Application of Ga-DOTA-F16 in myocardial perfusion micro-PET / C imaging in mice

[0114] Intravenously inject about 150 μCi / 200 μL 68 Ga-DOTA-F16 (prepared in Preparation Example 4) into the tail vein of normal commercially available SPF-grade Balb / c mice, and perform PET / CT imaging at 0.5, 1, and 2 h after injection. Observe the distribution of the probe in the mice and its enrichment in the heart region. The imaging results are as Figure 5 shown. It can be seen from the figure that the probe starts to accumulate significantly in the myocardium from 0.5 h, with a clear edge contour, and the probe still remains in the myocardium until 2 h.

[0115] Test Example 6: 68 Bio-distribution of Ga-DOTA-F16 in mice

[0116] Take 16 normal commercially available SPF-grade Balb / c mice and randomly divide them into four groups. Intravenous injection of 150 μCi / 200 μL dose of the product prepared in Example 4 68The Ga-DOTA-F16 probe was used. The animals were sacrificed at 10 min, 30 min, 60 min, and 120 min after injection, and each organ was excised, weighed, and the CPM value was measured using a CPM counter. The %ID / g was calculated. The results are shown in Table 1.

[0117] Table 1: 68 Uptake distribution and ratio of Ga-DOTA-F16 in normal mice

[0118]

[0119] As Figure 5 shown in and Table 1: 68 The biodistribution results of Ga-DOTA-F16 in normal mice showed that there was a relatively high initial uptake value and good retention in the myocardium. At 10 min after injection, the myocardial uptake value was 9.12 ± 1.69 %ID / g, and at 120 min after injection, the myocardial uptake value was still 5.97 ± 1.61 %ID / g. The heart was clearly visible in the PET / CT imaging. The probe had relatively high uptake values in both the liver and kidneys, indicating that 68 Ga-DOTA-F16 was mainly excreted through the liver and kidneys in mice. The ratio to non-target tissues such as muscle was relatively high. At 10 min, the heart / muscle ratio was 11.18 ± 2.01, which was beneficial to improving the imaging contrast and obtaining better diagnostic effects.

[0120] The above are only preferred embodiments of the present invention, which are illustrative but not restrictive to the present invention. Those of ordinary skill in the relevant 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 belong to the protection scope of the present invention.

Claims

1. A compound of formula I: In Formula I, the linking group Linker is selected from any one of C1-C6 alkylene or -(CH 2 CH 2 O) m -, Wherein, m is an integer from 2 to 30; n = 1 or 2; X - is an anion in any form, preferably I - , Br - , Cl - , BF 4 - or ClO 4 - ; R 1 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; R 2 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; R 3 and R 4 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, -NR 7 R 8 , halogen, nitro and cyano, wherein R 7 and R 8 each independently is C1-C6 alkyl, or R 7 and R 8 together with the carbon atom to which they are attached form a 5- to 6-membered nitrogen-containing heterocycle; R 5 and R 6 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 R 5 and R 6 together with the carbon atom to which it is attached form a C6-C10 aryl, substituted C6-C10 aryl, C5-C10 heteroaryl or substituted C5-C10 heteroaryl, Wherein, the substitution means being substituted 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, and the monovalent or divalent group is derived from: (i) One of a colored lumophore, an organic fluorophore, an inorganic lumophore, a light absorption compound, a light reflection compound, a light scattering compound, 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 radionuclide 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-diacetic 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)-dipyridyl-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-diacetic acid (NODAGA); The radionuclide includes a diagnostic radionuclide and a therapeutic radionuclide; The diagnostic radionuclides are 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 radionuclides are 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 Th; preferably 67 Cu, 90 Y, 125 I, 131 I, 177 Lu, 223 Ra, 225 Ac, 211 At; The metal element capable of generating a nuclear magnetic resonance signal is selected from any one of Gd, Fe, Eu, Mn, Cu, Si, Nd.

2. The compound according to claim 1, Wherein, In formula I, Linker is C2-C6 alkylene; X - is Br - ; R 1 selected from H, halogen-substituted C1-C10 alkyl, and C1-C10 alkyl substituted with halo C1-C10 alkoxy; R 2 、R 3 、R 5 and R 6 are H; R 4 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 the compound of formula I, and its preparation route is as follows: In the above route, R 1 and R 2 and R 3 and R 4 and R 5 and R 6 and R, X - and Linker and n are respectively defined as 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, It includes the following steps: (a) 4-methylpyridine 1 and a general formula compound X-Linker-NH where X is a halogen 2 A nucleophilic substitution reaction occurs to obtain compound 2; (b) Compound 2 undergoes a nitrogen acylation reaction with di-tert-butyl dicarbonate to obtain compound 3; (c) Compound 3 undergoes a Knoevenagel condensation reaction with substituted or unsubstituted indole-3-carbaldehyde to obtain compound 4; (d) Compound 4 removes the tert-butoxycarbonyl protecting group in a hydrochloric acid-methanol solution to obtain compound 5; (e) Compound 5 undergoes a condensation reaction with the NHS ester of a bifunctional chelating agent to obtain compound 6; (f) Compound 6 undergoes a chelation reaction with a radionuclide to obtain the final product 7, Method 2: When the substituent R in formula I is the case of (i) in claim 1, It includes the following steps: (a) 4-Methylpyridine 1 and a general formula compound X-Linker-NH where X is a halogen 2 undergo a nucleophilic substitution reaction to obtain compound 2; (b) Compound 2 undergoes a nitrogen acylation reaction with di-tert-butyl dicarbonate to obtain compound 3; (c) Compound 3 undergoes a Knoevenagel condensation reaction with substituted or unsubstituted indole-3-carbaldehyde to obtain compound 4; (d) Compound 4 removes the tert-butoxycarbonyl 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 with 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 the general formula compound where X is halogen are added to methanol, heated with stirring 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-carbaldehyde and 0.2 equivalent of piperidine are added to anhydrous methanol and refluxed and heated overnight; and / or In step (e) of Method 1 and step (g) of Method 2, N,N-diisopropylethylamine and N,N-dimethylformamide are added for reaction; and / or In step (g) of Method 2, HATU is further added for condensation reaction.

6. Use of the compound of formula I as defined in claim 1 as a mitochondrion-targeted radionuclide or fluorescent probe.

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

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