PARP-targeted single-photon tracer agent as well as labeling precursor, preparation method and application of PARP-targeted single-photon tracer agent

Abstract

The invention relates to a PARP (poly ADP-ribose polymerase)-targeted single-photon tracer, a labeling precursor thereof, a preparation method and application, the tracer takes Olaparib as a lead compound, develops HYNIC-Olaparib, and adopts hydrophilic triglycine and EDDA as auxiliary ligands to successfully prepare the PARP-targeted single-photon tracer with high stability and purity (gt; the < 99mTc >-labeled PARP-targeting tracer agent with the molecular weight of 9mTc (9mTc, 90%) has good hydrophilicity, effectively reduces radiation accumulation in the abdominal cavity, can specifically target PARP-1 in MDA-MB-453 cells, clearly displays the MDA-MB-453 tumor focus through Micro SPECT / CT imaging, has obvious tumor / background contrast, can be efficiently removed from non-target organs, and has huge potential of becoming a novel PARP imaging radioactive tracer agent.

Description

Technical Field

[0001] This application belongs to the technical field of tracers for radioactive imaging, and in particular relates to a single-photon tracer targeting PARP, its labeling precursor, preparation method and application. Background Art

[0002] Poly(ADP-ribose) polymerase (PARP) is a family of enzymes with multiple cellular functions, including DNA repair, maintenance of genomic stability, and regulation of cell death. Among them, PARP-1 is the most widely studied one, and its expression level in tumor tissues is often higher than that in normal tissues. This enzyme plays a key role in the process of DNA damage repair, especially in cells carrying BRCA1 / 2 mutations or defects. Currently, several PARP inhibitors have been approved for the treatment of various cancers, such as talazoparib, niraparib, olaparib, and rucaparib, with indications covering pancreatic cancer, breast cancer, prostate cancer, and ovarian cancer. Monitoring the dynamic activity and expression level of PARP is crucial for patients who benefit from PARP inhibitor therapy.

[0003] To detect whether PARP is overexpressed in organs and tissues, common clinical methods include immunohistochemistry (IHC) staining and fluorescence in situ hybridization (FISH). However, the PARP expression at specific sampling sites often cannot accurately reflect the expression levels of the whole tumor and metastatic lesions. In addition, the invasiveness of pathological examinations and possible sampling errors reduce the reliability and reproducibility of these methods in efficacy evaluation.

[0004] In recent years, a number of studies have reported PARP radioactive tracers for PET imaging, most of which are 18 18F-labeled PARP inhibitor analogs structurally related to rucaparib or olaparib. Although these compounds show potential in targeting pancreatic cancer and breast cancer, their high lipophilicity and the characteristic of mainly undergoing hepatobiliary metabolism and excretion lead to significant radioactive accumulation in the abdomen. Given that the abdomen is a common metastatic site for prostate cancer and ovarian cancer, this physiological distribution may seriously affect the detection of lesions. In addition, the labeling efficiency of 18F-labeled compounds is usually low and further purification by semi-preparative high-performance liquid chromatography is required. Therefore, it is crucial to explore ligands that can bind to easily labeled metal radionuclides (such as 99m 99mTc, 68 68Ga, and 177 177Lu) to avoid cumbersome purification processes and improve the labeling efficiency. In addition, due to the high operation and maintenance costs, PET equipment is difficult to popularize in developing countries and underdeveloped regions. There is an urgent need to develop PARP radioactive tracers suitable for single-photon emission computed tomography (SPECT) imaging. Summary of the Invention

[0005] The technical problem to be solved by the present invention is: to solve the above-mentioned deficiencies in the prior art, and thus provide a single-photon tracer targeting PARP, its labeling precursor, preparation method and application.

[0006] The technical solution adopted by the present invention to solve its technical problems is:

[0007] In the first aspect of the present invention, a labeling precursor of a single-photon tracer targeting PARP is provided, and its chemical structure is:

[0008] In the second aspect of the present invention, a preparation method of the above-mentioned labeling precursor of a single-photon tracer targeting PARP is provided, including the following steps:

[0009] S1: Remove the R1 protecting group of to obtain The R1 is one of a Boc group, a Cbz group, and an Fmoc group, and the X is a halogen group, preferably one of chlorine (Cl), bromine (Br), and iodine (I);

[0010] S2: React in the presence of a condensation reagent and an organic base to generate

[0011] S3: Heat and react with hydrazine hydrate to obtain a labeling precursor of a single-photon tracer targeting PARP

[0012] Preferably, the Boc group in step S1 is removed by a strong acid, and the strong acid is preferably at least one of trifluoroacetic acid (TFA), hydrochloric acid, formic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, sulfuric acid, and phosphoric acid; the Cbz group is preferably removed by a palladium-carbon catalyst; the Fmoc group is preferably removed by a base, and the base is preferably concentrated ammonia water.

[0013] Preferably, the is obtained by reacting R1-piperazine in the presence of a condensation reagent and an organic base; after the reaction for preparing is preferably completed, a strong acid solution is added to neutralize the excess organic base and remove the excess R1-piperazine, the strong acid is preferably hydrochloric acid and / or citric acid, and the strong acid solution is further preferably a 5-10% hydrochloric acid solution or a saturated citric acid solution; for preparing The condensation reagent used is preferably at least one of HBTU, HATU, propylphosphonic anhydride (T3P), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), 1,1'-carbonyldiimidazole (CDI); the preparation The organic base used is preferably at least one of triethylamine (TEA), N-methylmorpholine (NMM), N,N-diisopropylethylamine (DIPEA), pyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), N-methylimidazole (NMI); the preparation The reaction is carried out in an organic solvent, and the organic solvent is preferably at least one of dichloromethane (DCM), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc).

[0014] Preferably, the reactions in steps S1 and S2 are carried out in an organic solvent, and the organic solvent is preferably at least one of dichloromethane (DCM), methanol, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc).

[0015] Preferably, the condensation reagent in step S2 is at least one of HBTU, HATU, propylphosphonic anhydride (T3P), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), 1,1'-carbonyldiimidazole (CDI).

[0016] Preferably, the organic base in step S2 is at least one of triethylamine (TEA), N-methylmorpholine (NMM), N,N-diisopropylethylamine (DIPEA), pyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), N-methylimidazole (NMI).

[0017] Preferably, the temperature of the heating reaction in step S3 is 50-120 °C, preferably 80 °C.

[0018] Preferably, the reaction in step S3 is carried out in an organic solvent, and the organic solvent is preferably at least one of methanol, ethanol, n-propanol, isopropanol, 1,2-dichloroethane, toluene.

[0019] The third aspect of the present invention provides a preparation method of a single-photon tracer targeting PARP, using EDDA and tricine as co-ligands, adding a reducing agent, and adding the labeling precursor of the single-photon tracer targeting PARP and 99mTc-pertechnetate for a heating reaction to obtain the single-photon tracer targeting PARP.

[0020] Preferably, the reducing agent solution is first added to the solution of DDA and tricine, and then the mixed solution is adjusted to neutral. The solution of DDA and tricine is preferably obtained by dissolving EDDA and tricine in an aqueous salt solution. The reducing agent is preferably stannous chloride and / or stannous chloride dihydrate (SnCl2·2H2O). The reducing agent solution is preferably obtained by dissolving the reducing agent in a strong acid aqueous solution.

[0021] Preferably, the temperature of the heating reaction is 50-120 °C, and the time is preferably 5-30 min.

[0022] The fourth aspect of the present invention provides a single-photon tracer targeting PARP prepared by the above method.

[0023] The fifth aspect of the present invention provides an application of a single-photon tracer targeting PARP, and the single-photon tracer is used to detect whether PARP is overexpressed in humans or animals.

[0024] The beneficial effects of the present invention are as follows: The present invention has established an alternative detection method for PARP expression that is more economical and accessible than positron emission tomography (PET). Using olaparib as a lead compound, HYNIC-Olaparib was developed and hydrophilic tricine and EDDA (diethylamine-N,N'-diacetic acid) were used as auxiliary ligands to successfully prepare 99m Tc][Tc-HYNIC / EDDA]-Olaparib with high stability and purity (>90%). The introduction of specific groups not only simplifies 99m the synthesis of the

[0025] 99m Tc][Tc-HYNIC / EDDA]-Olaparib can specifically target PARP-1 in MDA-MB-453 cells. In in vivo experiments, the MDA-MB-453 tumor lesions were clearly demonstrated by Micro SPECT / CT imaging, with a significant tumor / background contrast and efficient clearance from non-target organs. The research confirmed 99m Tc][Tc-HYNIC / EDDA]-Olaparib has great potential to become a new type of PARP imaging radioactive tracer. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The technical solutions of the present application will be further described below with reference to the drawings and embodiments.

[0027] Figure 1 For the compound 2 prepared in Example 1 of the present application​1 1H NMR spectrum;

[0028] Figure 2 ESI-MS spectrum of Compound 2 prepared in Example 1 of this application;

[0029] Figure 3 ESI-MS spectrum of Compound 3 prepared in Example 1 of this application;

[0030] Figure 4 1H NMR spectrum of Compound 6 prepared in Example 1 of this application 1 1H NMR spectrum;

[0031] Figure 5 ESI-MS spectrum of Compound 6 prepared in Example 1 of this application;

[0032] Figure 6 1H NMR spectrum of Compound 7 prepared in Example 1 of this application 1 1H NMR spectrum;

[0033] Figure 7 ESI-MS spectrum of Compound 7 prepared in Example 1 of this application;

[0034] Figure 8 1H NMR spectrum of Compound 8 prepared in Example 1 of this application 1 1H NMR spectrum;

[0035] Figure 9 ESI-MS spectrum of Compound 8 prepared in Example 1 of this application;

[0036] Figure 10 1H NMR spectrum of Compound HYNIC-Olaparib prepared in Example 1 of this application 1 1H NMR spectrum;

[0037] Figure 11 13C NMR spectrum of Compound HYNIC-Olaparib prepared in Example 1 of this application 13 13C NMR spectrum;

[0038] Figure 12 ESI-MS spectrum of Compound HYNIC-Olaparib prepared in Example 1 of this application;

[0039] Figure 13 Radiochromatogram of 99m [99mTc][Tc-HYNIC / EDDA]-Olaparib and 99m NaTcO4, where Figure (A) is the radiochromatogram of 99m [99mTc][Tc-HYNIC / EDDA]-Olaparib, and Figure (B) is99m Radiochromatogram of NaTcO4;

[0040] Figure 14 For 99m Stability analysis of [99mTc][Tc-HYNIC / EDDA]-Olaparib in mouse plasma (A) and saline (B) at 0, 2, and 4 hours;

[0041] Figure 15 Binding model of 5DS3 and [99mTc][Tc-HYNIC / EDDA]-Olaparib. (A) Protein is shown in cartoon mode, and small molecule is shown in ball-and-stick model; (B) Protein is shown in surface mode; detailed display of the interaction between 5DS3 and [99mTc][Tc-HYNIC / EDDA]-Olaparib is shown in (C) and (D): where (C) is a 3D interaction diagram, where small molecule is shown in stick model and protein is shown in cartoon mode; (D) is a 2D interaction diagram;

[0042] Figure 16 (A) is 99m Uptake of [99mTc][Tc-HYNIC / EDDA]-Olaparib in MDA-MB-453 cells and MDA-MB-453 cells blocked by Olaparib, n = 3, ***p < 0.001, ****p < 0.0001; (B) is 99m Time-activity curve and pharmacokinetic parameters of [99mTc][Tc-HYNIC / EDDA]-Olaparib in healthy BALB / c nude mice (n = 3, within 4 hours);

[0043] Figure 17 Miniature SPECT / CT images of [99mTc][Tc-HYNIC / EDDA]-Olaparib in tumor-bearing mice with MDA-MB-453 cells 1 hour after injection of radioactive tracer; (A) Maximum intensity projection (MIP) of unblocked mice; (B) Fusion image of unblocked mice; (C) Fusion image of blocked mice; the blocked group was injected with 500-fold dose of Olaparib 30 minutes before injection of 99m [99mTc][Tc-HYNIC / EDDA]-Olaparib;

[0044] Figure 18 (A) is the biodistribution of [99mTc][Tc-HYNIC / EDDA]-Olaparib in the MDA-MB-453 model, and (B) is the ratio of tumor to non-target tissue. Detailed implementation methods

[0045] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments may be combined with each other.

[0046] The technical solutions of the present application will be described in detail below with reference to the accompanying drawings and in combination with embodiments.

[0047] The reagents, chemicals, cancer cells, nude mice, etc. involved in the following embodiments are as follows:

[0048] The following compounds were provided by Shanghai Bide Pharmaceutical Technology Co., Ltd.: dimethyl (3-oxo-1,3-dihydroisobenzofuran-1-yl) phosphate (purity 98%), 2-fluoro-5-formylbenzonitrile (purity 98%), tert-butyl piperazine-1-carboxylate (purity 98%), 6-chloronicotinic acid (purity 98%); MDA-MB-453 breast cancer cells were provided by Wuhan Boster Biological Engineering Co., Ltd.; BALB / c nude mice (female, 18 - 20 g) were provided by Changzhou Cavens Experimental Animal Co., Ltd.; [99mTc]NaTcO4 was obtained by eluting a 99Mo / 99mTc commercial generator purchased from Jiangyuan Hospital (Wuxi, China).

[0049] The experimental methods involved in the following embodiments are as follows:

[0050] Electrospray ionization mass spectrometry (ESI-MS) analysis was completed using a Waters ZMD4000 LC / MS quadrupole tandem mass spectrometer from the United States; nuclear magnetic resonance spectroscopy (NMR) data were collected by a Bruker 400 MHz nuclear magnetic resonance spectrometer from Germany; high performance liquid chromatography (HPLC) and radioactive HPLC analysis were performed using a Waters pump system from the United States, equipped with a Phenomenex C18 chromatographic column (10 μm, 250×4.6 mm), and connected to a radioactive detector and a dual-wavelength ultraviolet absorption detector; SPECT / CT imaging was performed by a Bruker Albira Si micro SPECT / CT system from Germany; the results of cell uptake experiments, pharmacokinetics, and biodistribution studies were detected by a 2470Wizard γ radioimmunoassay counter from Perkin Elmer, Inc. in the United States.

[0051] The molecular docking simulations involved in the following embodiments are as follows:

[0052] The crystal structure of PARP-1 protein (PDB ID: 5DS3) was obtained from the Protein Data Bank (PDB). Using the MOE 2022 software from Chemical Computing Group in Canada, olaparib analogs were docked to the PARP-1 binding site. The specific parameters were as follows: ligand positioning was performed using the triangle matcher method, MMFF94x force field optimization, and 27dG energy rescoring. Finally, through the United States The Maestro v13.5.128 software of the company evaluates the three-dimensional binding mode of the analog with PARP-1.

[0053] Example 1

[0054] This example provides a preparation method for a labeling precursor (HYNIC-Olaparib) of a single-photon tracer targeting PARP, and the reaction route is as follows:

[0055]

[0056] (a) Boc-piperazine, HBTU, TEA, DCM, rt; (b) DCM, TFA, rt; (c) TEA, THF, rt; (d) ⅰ. NaOH, THF, 100 °C; ⅱ. NH2NH2·H2O, 70 °C; (e) Compound 3, HBTU, TEA, DCM, rt; (f) NH2NH2·H2O, EtOH, 80 °C.

[0057] The specific reaction steps include:

[0058] Synthesis of Compound 2

[0059] Dissolve 6-chloronicotinic acid (4.71 g, 30 mmol), Boc-piperazine (6.70 g, 36 mmol), HBTU (17.10 g, 45 mmol) and triethylamine (9.10 g, 90 mmol) in 90 mL of dichloromethane (DCM), and stir at room temperature overnight. Subsequently, add 10% HCl solution (90 mL) and continue stirring for 30 minutes. After the organic layer is washed with brine and water and dehydrated with anhydrous sodium sulfate, it is concentrated and dried to obtain white solid Compound 2 (8.10 g, yield 83%). 1 1H-NMR (600 MHz, DMSO-d6) δ 8.49 (dd, J = 2.4, 0.7 Hz, 1H), 7.93 (dd, J = 8.2, 2.4 Hz, 1H), 7.62 (dd, J = 8.3, 0.7 Hz, 1H), 3.60 (s, 2H), 3.34 (s, 2H), 1.41 (s, 9H). ESI-MS (m / z): 326.48 [M+H] + .

[0060] Synthesis of Compound 3

[0061] Compound 2 (3.26 g, 10 mmol) was dissolved in 32 mL of DCM, and 16 mL of trifluoroacetic acid (TFA) was added. After stirring for 30 minutes, the mixture was concentrated under reduced pressure. The residue was triturated with n-hexane / DCM, filtered, and dried in vacuo to give compound 3 (2.10 g, yield 93.3%). Since the chemical structures of compound 3 and 2 are similar (except for the removal of the Boc group), its 1 1H-NMR was not measured. ESI-MS (m / z): 226.25 [M+H] + .

[0062] Synthesis of compound 6

[0063] Compound 4 (35.00 g, 0.14 mol) and compound 5 (20.90 g, 0.14 mol) were dissolved in 330 mL of tetrahydrofuran (THF). Triethylamine (14 mL, 0.14 mol) was slowly added dropwise at ≤15 °C within 25 minutes. The reaction mixture was warmed to room temperature within 60 minutes and then concentrated under reduced pressure. The residue was stirred with 250 mL of water for 30 minutes, filtered, and washed successively with water (2×30 mL), n-hexane (2×30 mL), and diethyl ether (2×30 mL), and then dried to give compound 6 (37.2 g, yield 96%). 1 1H-NMR (600 MHz, DMSO-d6) δ 8.15 (dd, J = 19.1, 7.1, 6.7, 3.5 Hz, 2H), 8.08–8.05 (m, 1H), 7.99–7.97 (m, 1H), 7.91 (td, J = 7.6, 1.0 Hz, 1H), 7.75–7.62 (m, 3H), 6.96 (s, 1H). ESI-MS (m / z): 266.33 [M+H] + .

[0064] Synthesis of compound 7

[0065] Compound 6 (37.2 g, 0.14 mol) was dissolved in 200 mL of tetrahydrofuran. 50 mL of 13N aqueous NaOH was added, and the mixture was heated at 100 °C for 1 hour. After cooling to 70 °C, hydrazine hydrate (100 mL, 2 mol) was added, and the mixture was stirred at 70 °C for 18 hours. The pH was adjusted to 4 with 8N HCl (80 mL) at room temperature, stirred for 10 minutes, filtered, and washed successively with water (2×60 mL) and diethyl ether (3×50 mL), and then dried to give white powdery compound 7 (30.1 g, yield 77%). 1H-NMR (400 MHz, DMSO-d6) δ 13.22 (s, 1H), 12.60 (s, 1H), 8.27 (dd, J = 7.8, 1.5 Hz, 1H), 8.01–7.95 (m, 1H), 7.90 (ddd, J = 8.1, 7.2, 1.5 Hz, 1H), 7.83 (ddd, J = 9.7, 7.4, 1.8 Hz, 2H), 7.58 (ddd, J = 8.5, 4.6, 2.5 Hz, 1H), 7.24 (dd, J = 10.8, 8.5 Hz, 1H), 4.36 (s, 2H). ESI-MS (m / z): 299.34 [M+H] + 。

[0066] Synthesis of Compound 8

[0067] Compound 7 (2.98 g, 10 mmol), Compound 3 (2.7 g, 12 mmol), HBTU (5.7 g, 15.0 mmol) and triethylamine (3.1 g, 30.0 mmol) were dissolved in 70 mL of DCM and stirred at room temperature overnight. 100 mL of water was added and stirred for 30 minutes. After filtration, the residue was triturated with methanol, filtered and dried in vacuo to give Compound 8 (2.67 g, yield 53%). 1 H-NMR (600 MHz, DMSO-d6) δ 12.60 (s, 1H), 8.50 (s, 1H), 8.26 (d, J = 7.9 Hz, 1H), 7.89 (d, J = 64.8 Hz, 4H), 7.63 (s, 1H), 7.41 (d, J = 50.4 Hz, 2H), 7.24 (s, 1H), 4.34 (d, J = 6.4 Hz, 2H), 3.85–3.50 (m, 4H), 3.42 (s, 1H), 3.26 (t, J = 23.9 Hz, 3H). ESI-MS (m / z): 506.53 [M+H] + 。

[0068] Synthesis of HYNIC-Olaparib

[0069] Compound 8 (1.5 g, 3 mmol), ethanol (30 mL) and 80% hydrazine hydrate (30 mL) were heated under reflux at 80 °C for 6 - 8 hours. After removing the excess hydrazine by distillation under reduced pressure, the reaction solution was diluted with 30 mL of water and extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography (eluent: 1% - 3% methanol in DCM) to give off-white solid HYNIC-Olaparib (916 mg, yield 61%), HPLC purity > 98%. 11H-NMR (400 MHz, DMSO-d6) δ 12.61 (s, 1H), 8.27 (dd, J = 7.8, 1.5 Hz, 1H), 8.11 (d, J = 2.3 Hz, 1H), 7.99–7.93 (m, 2H), 7.85 (dtd, J = 25.4, 7.3, 1.4 Hz, 2H), 7.53 (dd, J = 8.8, 2.3 Hz, 1H), 7.45 (ddd, J = 8.2, 5.0, 2.2 Hz, 1H), 7.37 (dd, J = 6.5, 2.3 Hz, 1H), 7.24 (t, J = 9.0 Hz, 1H), 6.71 (d, J = 8.7 Hz, 1H), 4.34 (s, 2H), 4.25 (s, 2H), 3.72–3.63 (m, 2H), 3.57 (t, J = 5.2 Hz, 2H), 3.43 (d, J = 5.3 Hz, 2H), 3.25 (d, J = 5.3 Hz, 2H). 13 13C NMR (101 MHz, DMSO) δ 168.79, 164.53, 162.84, 159.87, 158.07, 155.63, 148.26, 145.32, 137.23, 135.30, 133.96, 132.27, 132.19, 132.03, 129.55, 129.39, 128.35, 126.55, 125.93, 124.10, 123.92, 119.26, 116.53, 116.31, 105.35, 46.94, 41.90, 36.91. ESI-MS (m / z): 502.57 [M+H] + .

[0070] In summary, the synthesis of Hynic-Olaparib was initiated by the condensation reaction of 6-chloronicotinic acid (1) with tert-butyl 1-piperazinecarboxylate to synthesize compound 2, followed by Boc deprotection to obtain compound 3. Intermediate 4 was coupled with 5 in the presence of triethylamine to form 6. The resulting mixture of Z and E isomers was directly reacted with hydrazine hydrate without further purification to form the phthalazinone core. Subsequently, base hydrolysis of the nitrile group generated 7. The condensation reaction of 7 with 3 produced the key intermediate 8. Finally, 8 reacted with hydrazine hydrate to generate Hynic-Olaparib.

[0071] Example 2

[0072] This example provides a preparation method for a labeling precursor (HYNIC-Olaparib) of a single-photon tracer targeting PARP. The reaction route is as follows:

[0073]

[0074] (a-1) Cbz-piperazine, T3P, TEA, DMF, rt; (b-1) CH3OH, Pd / C 10%, rt; (e-1)

[0075] HATU, TEA, DCM, rt; (f-1) NH2NH2·H2O, EtOH, 80 °C.

[0076] The specific reaction steps include:

[0077] Synthesis of compound 2-1

[0078] Dissolve 6-bromonicotinic acid (2.02 g, 10 mmol), Cbz-piperazine (3.10 g, 12 mmol), propylphosphonic anhydride (T3P) (4.77 g, 15 mmol) and triethylamine (3.00 g, 30 mmol) in 30 mL of DMF and stir at room temperature overnight. Subsequently, add 10% HCl solution (30 mL) and continue stirring for 30 minutes. After the organic layer is washed with brine and water and dehydrated with anhydrous sodium sulfate, it is concentrated and dried to obtain white solid compound 2-1 (3.35 g, yield 83%). ESI-MS (m / z): 404.33 [M+H] + .

[0079] Synthesis of compound 3-1

[0080] Dissolve compound 2-1 (2.02 g, 5 mmol) in 20 mL of CH3OH, add 100 mg of Pd / C 10%, introduce hydrogen and stir the reaction overnight, concentrate under reduced pressure, and dry in vacuo to obtain compound 3-1 (1.21 g, yield 90%). ESI-MS (m / z): 270.55 [M+H] + .

[0081] Synthesis of compound 8-1

[0082] Dissolve compound 7 (1.49 g, 5 mmol) prepared in Example 1, compound 3-1 (1.61 g, 6 mmol), HATU (2.85 g, 7.5 mmol) and triethylamine (1.55 g, 15 mmol) in 35 mL of DCM and stir at room temperature overnight. Add 50 mL of water and stir for 30 minutes. After filtration, the residue is ground with methanol, filtered and dried in vacuo to obtain compound 8-1 (1.51 g, yield 55%). ESI-MS (m / z): 550.25 [M+H] + .

[0083] Synthesis of HYNIC-Olaparib

[0084] Compound 8-1 (1.5 g, 3 mmol), toluene (30 mL) and 80% hydrazine hydrate (30 mL) were heated under reflux at 50 °C for 1 - 3 hours. After removing the excess hydrazine by distillation under reduced pressure, the reaction solution was diluted with 30 mL of water and extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated, and then purified by silica gel column chromatography (eluent: 1% - 3% methanol in DCM) to obtain a white solid, HYNIC-Olaparib (944 mg, yield 61%), with HPLC purity > 98%.

[0085] Example 3

[0086] This example provides a method for preparing a labeling precursor (HYNIC-Olaparib) of a single-photon tracer targeting PARP. The reaction route is as follows:

[0087]

[0088] (a-2) Fmoc-piperazine, HATU, NMM, DMF, rt; (b-2) DMF, concentrated ammonia water, rt; (e-2) T3P, NMM, DCM, rt; (f-2) NH2NH2·H2O, EtOH, 80 °C.

[0089] The specific reaction steps include:

[0090] Synthesis of Compound 2-2

[0091] 6-Iodonicotinic acid (4.98 g, 20 mmol), Fmoc-piperazine (7.39 g, 24 mmol), HATU (11.41 g, 30 mmol) and N-methylmorpholine (NMM) (6.07 g, 60 mmol) were dissolved in 60 mL of DMF and stirred at room temperature overnight. Subsequently, saturated citric acid solution (60 mL) was added and stirring was continued for 30 minutes. The organic layer was washed with brine and water, dried over anhydrous sodium sulfate, and then concentrated and dried to obtain a white solid, Compound 2-2 (9.16 g, yield 85%). ESI-MS (m / z): 540.13 [M + H] + .

[0092] Synthesis of Compound 3-2

[0093] Compound 2-2 (8.09 g, 15 mmol) was dissolved in 60 mL of DMF, 60 mL of concentrated ammonia water was added, and the reaction was stirred overnight. The reaction mixture was poured into water, and the aqueous phase was acidified with concentrated HCl aqueous solution. The precipitated product was filtered, washed with Et2O and dried in vacuo to obtain Compound 2-2, and then dried in vacuo to obtain Compound 3-2 (4.33 g, yield 91%). ESI-MS (m / z): 318.29 [M + H]+ 。

[0094] Synthesis of Compound 8-2

[0095] Dissolve Compound 7 (2.98 g, 10 mmol) prepared in Example 1, Compound 3-2 (3.80 g, 12 mmol), propylphosphonic anhydride (T3P) (4.77 g, 15.0 mmol) and N-methylmorpholine (NMM) (1.21 g, 30.0 mmol) in 70 mL of DCM, and stir at room temperature overnight. Add 100 mL of water and stir for 30 minutes. After filtration, the residue is triturated with methanol, filtered and dried in vacuo to obtain Compound 8-2 (3.34 g, yield 56%). ESI-MS (m / z): 598.49 [M+H] +

[0096] Synthesis of HYNIC-Olaparib

[0097] Heat Compound 8-2 (1.8 g, 3 mmol), 1,2-dichloroethane (30 mL) and 80% hydrazine hydrate (30 mL) under reflux at 120 °C for 0.5 - 1.5 hours. After removing the excess hydrazine by distillation under reduced pressure, the reaction solution is diluted with 30 mL of water and extracted with DCM (3 × 50 mL). The combined organic layers are washed with brine, dried over anhydrous sodium sulfate and concentrated, and then purified by silica gel column chromatography (eluent: 1% - 3% methanol in DCM) to obtain off-white solid HYNIC-Olaparib (977 mg, yield 63%), HPLC purity > 98%.

[0098] Example 4 99m Tc Labeling

[0099] This example provides a preparation method of a single photon tracer targeting PARP ( 99m Tc][Tc-HYNIC / EDDA]-olaparib), using EDDA and tricine as co-ligands and stannous chloride dihydrate (SnCl2·2H2O) as a reducing agent for the radiochemical synthesis of [99mTc][Tc-HYNIC / EDDA]-olaparib. The specific steps are as follows:

[0100] Preparation of ligand solution: Dissolve EDDA (5 mg) and tricine (15 mg) in 0.5 mL of normal saline in a sterile penicillin vial;

[0101] Addition of reducing agent: Add freshly prepared SnCl2·2H2O solution (20 μL, 2 mg / mL, dissolved in 0.1 N HCl), and adjust the pH to 7;

[0102] Precursor and radionuclide labeling: HYNIC-olaparib (20 μL, 1 mg / mL acetonitrile solution) and freshly eluted 99m Tc]NaTcO4 solution (350 μL, 74 - 259 MBq) were added sequentially, and heated at 95 °C for 15 minutes under nitrogen protection;

[0103] Quality control: After cooling to room temperature, radiochemical purity (RCP) and radiochemical yield (RCY) were analyzed by radio-HPLC. Chromatographic conditions: Mobile phase: Phase A (0.1% trifluoroacetic acid aqueous solution), Phase B (0.1% trifluoroacetic acid acetonitrile solution); Gradient program: 0 - 3 minutes (5% B), 3 - 30 minutes (linearly increased to 95% B), 30 - 35 minutes (decreased to 5% B); Flow rate: 1 mL / min.

[0104] Effect Example 1 Radiochemical Analysis

[0105] As Figure 13 shown, 99m the retention time of 99m Tc changed from 3.9 minutes to 16.5 minutes. The retention times of the complex 99m Tc][Tc-HYNIC / EDDA]-Olapari prepared in Example 4 and 99m NaTcO4 were significantly different ( 99m Tc][Tc-HYNIC / EDDA]-Olaparib was 16.5 minutes, 99m NaTcO4 was 3.9 minutes). The radiochemical purity (RCP) of the complex exceeded 90%, so it could be used for subsequent in vivo and in vitro experiments without further purification.

[0106] Effect Example 2 Stability Analysis

[0107] The in vitro stability of 99m Tc][Tc-HYNIC / EDDA]-Olaparib prepared in Example 4 was evaluated in saline and serum. Saline stability experiment: 99m Tc complex was incubated in saline for 0, 2, and 4 hours respectively, and stability analysis was carried out by HPLC. Serum stability experiment: 100 μL 99m Tc complex was incubated with 100 μL mouse serum at 37 °C for 0, 2, and 4 hours, then 200 μL acetonitrile was added to precipitate proteins, the supernatant was collected after centrifugation, filtered through a 0.22 μm filter membrane, and then analyzed by HPLC. The radiochemical purity (RCP) was calculated by measuring the HPLC peak integration of 99m Tc complex and its degradation products.

[0108] As Figure 14 shown, 99m[[99mTc]][Tc-HYNIC / EDDA]-olaparib showed good stability in both saline and serum, with the retention time at 16.5 minutes showing no significant change within 4 hours of incubation, and the radiochemical purity (RCP) of the complex still exceeding 90%.

[0109] Effect Example 3 Determination of Partition Coefficient

[0110] Approximately 74 - 148 kBq of 99m [[99mTc]]-labeled complex prepared in Example 4 was mixed with 2 mL of a mixed solvent (water / n-octanol = 1:1, v / v). After 5 minutes of vortex oscillation, the organic and aqueous phases were separated by centrifugation at 15,000 g. Subsequently, 100 μL samples were taken separately, and the radioactivity was quantified using a gamma-counter. This experiment was repeated five times (n = 5). As a result, the log P value of the partition coefficient of [[99mTc]][Tc-HYNIC / EDDA]-Olaparib prepared in Example 4 was 0.63 ± 0.25, indicating its hydrophilicity.

[0111] Effect Example 4 99m [[99mTc]][Tc-HYNIC / EDDA]-Olaparib Binding Mode Analysis with PARP-1

[0112] To study the 99m possible interaction between [[[99mTc]][Tc-HYNIC / EDDA]-Olaparib and PARP-1 protein, a three-dimensional structure was constructed using Maestro 13.5.128. As Figure 15 shown, [[99mTc]][Tc-HYNIC / EDDA]-Olaparib interacted with multiple amino acid residues in the 5DS3 structure ((in Figure (C), hydrogen bonds are represented by purple dashed lines, π-π interactions by green dashed lines, and halogen bonds by brown dashed lines; in Figure (D), hydrogen bonds are represented by purple arrows, π-π interactions by green solid lines, and halogen bonds by brown arrows)). Specifically, the analogue was able to form hydrogen bonds with SER904, GLY863, ARG878, and ASN868. In addition, π-π interactions occurred with TYR907 and HIS862, while ARG878 and SER864 were involved in the formation of halogen bonds. Hydrophobic interactions with ILE895, TYR896, and PHE897 (highlighted in green in Figure 15 Figures C and D) further stabilized the complex. Calculated by the London dG scoring function in MOE, the binding energy between the ligand and the receptor was -9.2 kcal / mol, indicating a strong binding affinity between the two.

[0113] Effect Example 5 In Vitro Cellular Uptake Experiment

[0114] To evaluate 99m the targeting efficiency of [Tc][Tc-HYNIC / EDDA]-Olaparib on PARP-1 positive tumor cells, its uptake was studied in MDA-MB-453 tumor cells. Human breast adenocarcinoma MDA-MB-453 cells in the logarithmic growth phase were harvested, resuspended in DMEM medium (5×105 cells / mL), and seeded into 12-well plates (1 mL per well), and cultured overnight at 37 °C under 5% CO2 conditions. After culture, the cells were washed with 1 mL PBS, and 99m the [Tc] complex solution (37 kBq / tube) prepared with glucose-free DMEM medium was added. The cells were incubated at 37 °C for 30 minutes, 1 hour, 2 hours, and 4 hours respectively to evaluate the cellular uptake of the radioactive complex. In the blocking experiment, the cells were pre-incubated with 50 μM Olaparib for 15 minutes before incubation to block specific binding sites, and then the 99m [Tc] complex (37 kBq / tube) was added under the same conditions. After the incubation, the medium was removed, the cells were washed twice with cold PBS (0.5 mL), and then the cells were lysed with 1 mL NaOH (0.1 M), the lysate was collected, and the radioactivity (CPM, counts per minute) was measured with a gamma-counter, and time decay correction was performed. The experiment was repeated independently three times.

[0115] 99m The highest uptake of [Tc][Tc-HYNIC / EDDA]-Olaparib in MDA-MB-453 cells occurred at 60 minutes (3.69 ± 0.13% AD), and further incubation slightly decreased the uptake to 3.59 ± 0.25% AD, and finally reached a plateau at about 3.38 ± 0.17% AD ( Figure 16 A). In contrast, when there was an excess of Olaparib, the uptake decreased to 1.09 ± 0.12% AD at 60 minutes and to 0.88 ± 0.13% AD at 240 minutes, indicating that the radioactive complex was specific for PARP-1.

[0116] Effect Example 6 Pharmacokinetics

[0117] Due to the introduction of the HYNIC group and hydrophilic ligands Tricine and EDDA, 99m ​[[Tc][Tc-HYNIC / EDDA]]-Olaparib has increased hydrophilicity compared to Olaparib, which is verified by its decreased log P value (0.63 ± 0.25, the log P of Olaparib is 0.8). This modification may lead to accelerated metabolism in vivo.

[0118] 99m [[[Tc][Tc-HYNIC / EDDA]]-Olaparib's pharmacokinetic characteristics were evaluated in healthy mice. 99m The [[Tc]] complex was administered by intravenous injection at 4.55–6.99 MBq / mouse (200 μL). Subsequently, blood samples were collected via the tail vein at 2, 5, 10, 30, 60, 90, 120, 180, and 240 minutes after administration, and their radioactivity was measured using a gamma counter, corrected to the activity value at the time of injection. The experimental data were expressed as %ID / g (radioactive uptake percentage normalized to per gram of blood), that is, the measured CPM value divided by the mass of the blood sample in the capillary. Subsequently, pharmacokinetic analysis was performed using GraphPad Prism v2.1.DAS. The time-activity curve and main pharmacokinetic parameters of [[99mTc][Tc-HYNIC / EDDA]]-Olaparib in the blood of healthy female mice are as Figure 16 shown in Figure B. The distribution half-life was approximately 2.6 minutes, and the elimination half-life was approximately 20.7 minutes. In addition, the apparent volume of distribution of the radiotracer was 0.955 L / kg.

[0119] Effect Example 7 SPECT / CT Imaging

[0120] MDA-MB-453 breast cancer cells were cultured in L-15 medium containing 15% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and the culture conditions were 37 °C and 5% CO2. A tumor-bearing mouse model was constructed by subcutaneous inoculation of approximately 1 × 10 7 cells (100 μL saline suspension) into the right groin of nude mice. After 6 - 8 weeks, when the tumor grew to 9 - 10 mm, biodistribution and small animal SPECT / CT imaging studies were performed.

[0121] After subcutaneous transplantation of MDA-MB-453 breast cancer cells to form 9 - 10 mm tumors in BALB / c nude mice (n = 3), 99m [[[Tc][Tc-HYNIC / EDDA]]-Olaparib (12.9 MBq, 200 μL saline) was injected intravenously under anesthesia, and SPECT / CT imaging was performed after injection. In the blocking experiment, the mice were injected 99m ​[[Tc][Tc-HYNIC / EDDA]-Olaparib (12.9 MBq), 10 minutes ago, 10 mg / kg olaparib was intravenously injected, and then SPECT / CT imaging was performed after the injection of 99m [[Tc][Tc-HYNIC / EDDA]-Olaparib (n = 3). The imaging parameters were as follows: SPECT acquisition: using a multi-pinhole collimator (MPH collimator); field of view (FOV): 80 mm; acquisition time: 30 seconds; CT scan: voltage: 45 kV; current: 0.4 mA; field of view (FOV): 64 mm; scan time: 15 minutes; SPECT images were reconstructed using the filtered back-projection (FBP) method, and CT images were processed using the ordered subsets expectation maximization (OSEM) algorithm (two iterations). Finally, the reconstructed images were analyzed using PMOD 4.3 software.

[0122] As Figure 17 shown, the radioactive signal mainly accumulated in the kidneys and bladder of the mice, indicating that the radioactivity was mainly excreted through the urinary system. 99m [[Tc][Tc-HYNIC / EDDA]-Olaparib uptake in tumors was approximately 3.45 ± 0.17% ID / g (1 hour). After blocking with Olaparib (500 times the standard therapeutic dose), 99m [[Tc][Tc-HYNIC / EDDA]-Olaparib tumor uptake at 1 hour after injection decreased from 3.45 ± 0.17% ID / g to 0.69 ± 0.13% ID / g. These results verified 99m [[Tc][Tc-HYNIC / EDDA]-Olaparib specificity for PARP-1.

[0123] Effect Example 8 Biodistribution Study

[0124] Biodistribution analysis was performed in a MDA-MB-453 breast cancer-bearing mouse model (10 - 12 weeks old, n = 3). The experimental mice were intravenously injected with 99m [[Tc][Tc-HYNIC / EDDA]-Olaparib (5.58–6.84 MBq, 200 μL normal saline). At 1 or 2 hours after injection, the experimental mice were decapitated, and then samples of different tissues and organs (liver, heart, lung, spleen, stomach, kidney, muscle, small intestine, bone, pancreas, thyroid, blood, tumor, and ovary) were collected, and their radioactivity was measured using a gamma counter. Finally, it was quantified as % ID / g (radioactive uptake percentage normalized to per gram of tissue).

[0125] To verify the specific uptake of olaparib in tumor tissues, a blocking experiment was conducted: mice in the experimental group were pre-injected with 100 μg olaparib 30 minutes before intravenous injection of 99m Tc][Tc-HYNIC / EDDA]-Olaparib (6.84 MBq). Thereafter, the mice were sacrificed 2 hours after intravenous injection to evaluate the blocking effect.

[0126] 99m The biodistribution of [Tc][Tc-HYNIC / EDDA]-Olaparib was consistent with the SPECT / CT imaging results, as shown in Figure 18 (A). The highest uptake was observed in the kidneys at 60 minutes after injection, which was 7.11 ± 0.79% ID / g, and decreased to 3.05 ± 0.76% ID / g after 2 hours. The uptake in the liver was 1.38 ± 0.26% ID / g and 0.80 ± 0.21% ID / g at 1 hour and 2 hours, respectively, indicating that the radioactive complex was mainly excreted through the urinary system. The initial uptake of the radioactive complex in the blood was relatively high, which was 2.25 ± 0.39% ID / g at 60 minutes, but decreased significantly to 0.28 ± 0.15% ID / g after 2 hours. The uptake in the tumor was 3.36 ± 0.26% ID / g at 60 minutes after injection and decreased to 2.37 ± 0.14% ID / g after 2 hours. Based on the highest tumor uptake, 1 hour was selected as the optimal time for imaging. In addition, the biodistribution study showed that the tumor-to-muscle ratios were 7.72 ± 1.05 and 22.30 ± 8.23 at 1 hour and 2 hours, respectively ( Figure 18 (B)).

[0127] In summary, the improvement of water solubility and the modification of the molecular structure affected the metabolism and distribution of the radioactive tracer in vivo. Compared with the reported 18F-labeled probe, 99m the uptake of [Tc][Tc-HYNIC / EDDA]-Olaparib in the kidneys was much higher than that in the liver. The high radioactive level in the kidneys at 1 hour after injection (7.11 ± 0.79% ID / g) indicated its preference for water-soluble compounds. The renal radioactivity decreased rapidly to a lower level within 2 hours (3.05 ± 0.76% ID / g), indicating its rapid clearance from the blood, and the radioactive complex was mainly excreted through the renal pathway. The compound was efficiently cleared from non-target organs, and the tumor-to-non-target organ ratios were significantly increased at 1 hour after injection (tumor / liver: 2.46 ± 0.31, tumor / muscle: 7.98 ± 0.17). Further in vivo experiments, including micro-SPECT / CT imaging, verified the previous speculation. 99m ​Micro-SPECT / CT imaging of [[Tc][Tc-HYNIC / EDDA]-Olaparib showed higher uptake in the kidneys and bladder than in the liver. Additionally, when blocked with excess Olaparib, 99m the high uptake of [[Tc][Tc-HYNIC / EDDA]-Olaparib in tumors decreased significantly, indicating that its uptake was specifically mediated by PARP-1.

[0128] In summary, the novel SPECT radiotracer of the present application 99m [[[Tc][Tc-HYNIC / EDDA]-olaparib, is used for PARP-1 imaging in vitro and in vivo. Micro-SPECT / CT imaging and biodistribution studies showed that 99m [[[Tc][Tc-HYNIC / EDDA]-olaparib was able to accurately measure PARP-1 expression in vivo, showing rapid clearance and enhanced imaging contrast. This provides a valuable tool for evaluating the PARP-1 expression level in patients and monitoring treatment efficacy.

[0129] Taking the above ideal embodiments of the present application as inspiration, through the above description, relevant staff can completely make various changes and modifications without departing from the technical idea of this application. The technical scope of this application is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A labeling precursor of a single-photon tracer targeting PARP, characterized in that, Its chemical structure is as follows:

2. A method for preparing a labeling precursor of a single-photon tracer targeting PARP according to claim 1, characterized in that, It includes the following steps: S1: Remove the R1 protecting group of to obtain The R1 is one of Boc group, Cbz group, and Fmoc group, and the X is a halogen group, preferably one of chlorine (Cl), bromine (Br), and iodine (I); S2: Add to react in the presence of a condensing agent and an organic base to form S3: Heat hydrazine hydrate to carry out a reaction to obtain a labeling precursor of a single-photon tracer targeting PARP ​ 3. The preparation method of the labeling precursor of the single-photon tracer targeting PARP according to claim 2, wherein The Boc group in step S1 is removed by a strong acid, and the strong acid is preferably at least one of trifluoroacetic acid (TFA), hydrochloric acid, formic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, sulfuric acid, and phosphoric acid; the Cbz group is preferably removed by a palladium-carbon catalyst; the Fmoc group is preferably removed by a base, and the base is preferably concentrated ammonia water.

4. The preparation method of the labeling precursor of the single-photon tracer targeting PARP according to claim 2 or 3, characterized in that, The is obtained by reacting R1-piperazine in the presence of a condensing agent and an organic base; after the reaction for preparing is preferably completed by adding a strong acid solution to neutralize the excess organic base and remove the excess R1-piperazine. The strong acid is preferably hydrochloric acid and / or citric acid, and the strong acid solution is further preferably a 5-10% hydrochloric acid solution or a saturated citric acid solution; for the preparation of the condensing agent used is preferably at least one of HBTU, HATU, propylphosphonic anhydride (T3P), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), 1,1'-carbonyldiimidazole (CDI); for the preparation of the organic base used is preferably at least one of triethylamine (TEA), N-methylmorpholine (NMM), N,N-diisopropylethylamine (DIPEA), pyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), N-methylimidazole (NMI); the reaction for preparing is carried out in an organic solvent, and the organic solvent is preferably at least one of dichloromethane (DCM), methanol, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc).

5. The preparation method of the labeling precursor of the single-photon tracer targeting PARP according to any one of claims 2-4, characterized in that, The reactions in steps S1 and S2 are carried out in an organic solvent, and the organic solvent is preferably at least one of dichloromethane (DCM), N,N-dimethylformamide (DMF), and N,N-dimethylacetamide (DMAc); the condensation reagent in step S2 is preferably at least one of HBTU, HATU, propylphosphonic anhydride (T3P), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), and 1,1'-carbonyldiimidazole (CDI); the organic base in step S2 is preferably at least one of triethylamine (TEA), N-methylmorpholine (NMM), N,N-diisopropylethylamine (DIPEA), pyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and N-methylimidazole (NMI).

6. The preparation method of the labeling precursor of the single-photon tracer targeting PARP according to any one of claims 2-5, characterized in that, The temperature of the heating reaction in step S3 is 50-120°C, preferably 80°C; the reaction in step S3 is carried out in an organic solvent, and the organic solvent is preferably at least one of methanol, ethanol, n-propanol, isopropanol, 1,2-dichloroethane, and toluene.

7. A preparation method of a single-photon tracer targeting PARP, characterized in that, Using EDDA and tricine as co-ligands, adding a reducing agent, and adding the labeling precursor of the single-photon tracer targeting PARP described in claim 1 and 99mTc-pertechnetate for a heating reaction to obtain the single-photon tracer targeting PARP.

8. The preparation method of the single-photon tracer targeting PARP according to claim 7, characterized in that, First, add the reducing agent solution to the solution of EDDA and tricine, and then adjust the mixed solution to neutral. The solution of EDDA and tricine is preferably obtained by dissolving EDDA and tricine in a saline solution. The reducing agent is preferably stannous chloride and / or stannous chloride dihydrate (SnCl2·2H2O). The reducing agent solution is preferably obtained by dissolving the reducing agent in a strong acid aqueous solution; the temperature of the heating reaction is preferably 50-120°C, and the time is preferably 5-30 min.

9. A single-photon tracer targeting PARP prepared by the method according to any one of claims 7-9.

10. An application of the single-photon tracer targeting PARP described in claim 9, wherein the single-photon tracer is used to detect whether PARP in a human or an animal is overexpressed.

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