Nuclide-labeled PARP-1 targeting probe and preparation method thereof

Abstract

The invention relates to the technical field of radionuclide imaging and therapeutic agents, in particular to a nuclide-labeled PARP-1 targeting probe and a preparation method thereof. The targeting probe based on PARP 1 inhibitor olaparib is used as a targeting molecular marker radionuclide, and the obtained specific targeting radionuclide marker can achieve accurate diagnosis and treatment effects and has good clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of radionuclide imaging and therapeutic agents, and in particular to a radionuclide-labeled PARP-1 targeting probe and its preparation method. Background Technology

[0002] Poly[ADP-ribose] polymerase 1 (PARP-1) is a key enzyme in eukaryotic cells responsible for DNA damage repair. When a single-strand break occurs in cellular DNA, PARP-1 can rapidly recognize and bind to the damage site, using its substrate nicotinamide adenine dinucleotide (NAD). + PARP-1 acts as a donor, catalyzing the synthesis of poly(ADP-ribose) (PAR) chains. This process not only performs post-translational modifications on histones and other nucleoproteins, altering chromatin structure, but also recruits other DNA repair protein complexes, thereby efficiently completing damage repair and maintaining genome stability. However, under pathological conditions, continuous oxidative stress or genotoxic stress leads to the continuous accumulation of DNA damage, resulting in the overactivation of PARP-1. This abnormal activation excessively consumes intracellular NAD+. + It and ATP lead to an energy crisis and promote the abnormal accumulation of PAR polymers, ultimately participating in the process of various diseases, including neurodegenerative diseases, ischemia-reperfusion injury, and malignant tumors, by inducing programmed cell death or inflammatory responses.

[0003] In recent years, research on PARP-1 in the field of oncology has been particularly in-depth. Numerous studies have shown that PARP-1 is significantly overexpressed in various solid tumors, such as breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, and glioblastoma, and its activity is closely related to tumor proliferation, invasion, metastasis, and treatment resistance. This biological characteristic makes PARP-1 a highly attractive target for anti-tumor therapy. PARP inhibitors (PARPi, such as olaparib, niraparib, and tapazolidone) developed based on the principle of "synthetic lethality" have been successfully applied clinically, showing remarkable efficacy, especially in tumor patients with BRCA1 / 2 and other homologous recombination repair deficiencies. PARPi inhibits the enzymatic activity of PARP-1, "capturing" it at DNA damage sites, hindering the progression of replication forks, and leading to the accumulation of DNA double-strand breaks, thus selectively killing tumor cells with specific DNA repair deficiencies.

[0004] Despite the revolutionary success of PARPi in some patients, its clinical application still faces significant challenges, most notably primary and acquired resistance. Not all theoretically sensitive patients respond to PARPi, and even those initially effective may eventually experience disease progression. Resistance mechanisms are complex and diverse, including increased expression of drug efflux pumps, restoration of homologous recombination repair function, and decreased inhibitor binding affinity due to PARP-1 gene mutations. This situation directly leads to a dilemma in clinical practice: physicians lack effective means to accurately predict patient response to PARPi treatment before treatment. Currently, BRCA gene testing, which relies on tumor tissue biopsy, is only one indicator for screening potential beneficiaries and cannot comprehensively and dynamically reflect the actual expression level, activity status, and spatial heterogeneity of PARP-1 within tumors. Therefore, developing a non-invasive, real-time, and visually assessable technology for evaluating PARP-1 expression and distribution in living tumor tissue is of crucial clinical significance for achieving personalized precision PARPi administration, dynamic monitoring of efficacy, early detection of resistance, and screening of new applicable populations.

[0005] Molecular imaging techniques, particularly positron emission tomography (PET), offer a potential solution to these problems. By designing and synthesizing radiolabeled probes that specifically target PARP-1, quantitative imaging of the PARP-1 protein in vivo can be achieved. Currently, research in this field mainly focuses on using the chemical structures of classic PARP1s (such as olaparib and rucapabani) as the core, and performing radioisotope (e.g., 18 F, 64 Cu、 89 Zr labeling. Although some early probes have entered preclinical and even clinical research stages, many limitations remain. For example, some probes are too lipophilic, which may lead to nonspecific binding and high hepatic uptake, affecting the targeting signal-to-noise ratio; some probes have insufficient metabolic stability in vivo, and dehalogenation or demetallization may interfere with the accuracy of quantitative analysis; in addition, how to balance the targeting affinity of probes with pharmacokinetic properties (such as rapid blood clearance and efficient tumor uptake) remains a core challenge in design. More importantly, the existing probe structures have limited diversity, and there is an urgent need to explore novel labeling compounds based on different pharmacophore or linker strategies to optimize imaging performance and meet more demanding clinical diagnostic needs.

[0006] In summary, developing novel, efficient, and stable radionuclide-labeled PARP-1 targeting probes, constructing corresponding standardized preparation and quality control methods, and ultimately applying them to the precise diagnosis and efficacy evaluation of tumors is a clear and urgent research direction in the interdisciplinary field of nuclear medicine and tumor targeted therapy. Summary of the Invention

[0007] The purpose of this invention is to provide a radionuclide-labeled PARP-1 targeting probe, its preparation method, and its application. Using a targeting probe based on the PARP-1 inhibitor olaparib as a target molecular marker for radionuclides, the resulting specific targeted radionuclide marker can achieve precise diagnosis and treatment, and has good clinical application prospects.

[0008] To achieve the above objectives, in the basic technical solution, the present invention provides a radionuclide-labeled PARP-1 targeting probe. The structure includes olaparib, a PARP 1 inhibitor, a hydrophilic structure, and a chelating agent, which are sequentially linked by covalent bonds; while the chelating agent and the radionuclide are linked by coordination bonds. The targeting probe is based on the PARP-1 inhibitor olaparib, the hydrophilic structure, and the chelating agent linked by an amide bond, as shown in Formula I.

[0009] The radionuclide includes any one of 68Ga, 177Lu, 64Cu, 86Y, 89Zr, 90Y, 111In, 123I or 124I, preferably 68Ga.

[0010] Wherein, the L group is selected from NH, and n is selected from positive integers between 0 and 10; the Y group is... ; The second technical solution provided by this invention is a method for preparing the radionuclide-labeled PARP-1 targeting probe. When the radioactive element is 68Ga, the preparation method includes the following steps: (1) Compound 1, olaparib, and compound PG-L-PEGn-COOH undergo a condensation reaction to give compound 3; the reaction formula is shown below: Wherein, PG represents the protecting group of the amino group, the L group is selected from NH, and n is selected from positive integers between 0 and 10; (2) Compound 3 was deprotected to obtain compound 4; the reaction formula is shown below: Wherein, PG represents the protecting group of the amino group, the L group is selected from NH, the L' group is selected from NH2, and n is selected from positive integers between 0 and 10; (3) Compound 4 and the chelating agent undergo a condensation reaction to obtain the compound shown in Formula I; wherein the reaction formula is as follows: Wherein, the chelating agent is selected from DOTA-tri(t-butyl ester); the L group is selected from NH, n is selected from positive integers between 0 and 10; the Y group is... ; (4) Radiolabel the compound represented by Formula I with a radionuclide to obtain the radionuclide-labeled PARP-1 targeting probe.

[0011] Preferably, in step (1), the molar ratio of olaparib to compound PG-L-PEGn-COOH is 1:(1~2); Preferably, the PG is a protecting group of an amino group, and the protecting group of the amino group is selected from Boc; Preferably, in step (1), the temperature of the condensation reaction is 20~40℃, and the time of the condensation reaction is 12~24 h; Preferably, in step (1), the condensation reaction is carried out in a solvent selected from dichloromethane or N,N-dimethylformamide; Preferably, in step (1), the condensation reaction is carried out in the presence of a condensing agent selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; Preferably, in step (2), the reagent used for the deprotection treatment is trifluoroacetic acid; Preferably, in step (2), the temperature of the deprotection treatment is 20~40℃, and the time of the deprotection treatment is 1~3 h.

[0012] Preferably, in step (3), the molar ratio of compound 4 to chelating agent is 1:(1~2); Preferably, in step (3), the temperature of the condensation reaction is 20~40℃, and the time of the condensation reaction is 12~24 h; Preferably, in step (3), the condensation reaction is carried out in a solvent selected from N,N-dimethylformamide; Preferably, in step (3), the condensation reaction is carried out in the presence of a condensing agent selected from N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU); Preferably, in step (4), the reaction temperature is 85~95℃ and the reaction time is 5~20 min.

[0013] The third technical solution provided by this invention is the application of the aforementioned radionuclide-labeled targeting probe in the preparation of precise tumor-targeting agents.

[0014] The fourth technical solution provided by the present invention is the application of the radionuclide-labeled targeting probe in the preparation of a contrast agent for early tumor diagnosis.

[0015] The fifth technical solution provided by the present invention is the application of the aforementioned radionuclide-labeled targeting probe in the preparation of tumor nuclear medicine imaging agents.

[0016] The sixth technical solution provided by the present invention is the application of the aforementioned radionuclide-labeled targeting probe in the preparation of radiotherapy agents.

[0017] The technical solution adopted in this invention can achieve the following beneficial effects: The radionuclide-labeled targeting probe provided by this invention integrates the PARP 1 inhibitor olaparib, a hydrophilic structure, and a specific chelating agent sequentially linked by amide bonds. 68 This invention utilizes a Ga radionuclide, achieving structural innovation and performance breakthroughs. The probe exhibits excellent radiochemical stability and targeting specificity in vivo, significantly improving the signal-to-noise ratio of tumor imaging. It also possesses the potential for "therapeutic integration"—enabling both high-sensitivity PET / SPECT imaging and providing a precise dosage basis for subsequent targeted internal irradiation therapy. Compared to existing technologies, this invention maintains high targeting accuracy while optimizing drug metabolism kinetics by introducing a controllable hydrophilic segment and selecting readily available and clinically suitable materials. 68 Ga nuclides have comprehensive advantages such as simple preparation process, controllable cost, and easy large-scale production, and have broad prospects for clinical application. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, forming part of the present invention. The illustrative embodiments of the present invention and their descriptions explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings: Figure 1 SPECT CT imaging results of the 68Ga-labeled targeting probes prepared in Examples 1 and 2. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. In the description of this invention, it should be noted that the term "or" is generally used to include the meaning of "and / or," unless otherwise expressly indicated.

[0020] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0021] The inventors, through research, discovered that PARP-1 expression levels in drug-resistant breast cancer cell lines are significantly lower than in sensitive cells, suggesting an intrinsic correlation between its expression level and tumor response to PARP inhibitors. Multiple clinical dosimetry studies further demonstrate that the abundance of PARP-1 expression in tumor tissue may directly correlate with the therapeutic efficacy of PARP inhibitors (PARPi). For example, existing literature reports that in triple-negative breast cancer (TNBC) patients, those with high PARP-1 expression may be more suitable for PARPi treatment than those with low expression. Based on this, developing radionuclide-labeled PARP-1 targeting probes capable of imaging PARP-1 expression levels holds promise for precise screening of PARPi-suitable populations, thereby improving treatment success rates and providing an important tool for predicting efficacy and dynamically assessing treatment response. Currently, research on radionuclide-labeled PARP-1 targeting probes in the field of tumor treatment is still in its early stages, and its therapeutic potential requires further systematic exploration and validation.

[0022] Unless otherwise specified, all raw materials and reagents used in this embodiment are commercially available products or prepared by methods well known to those skilled in the art. Compound 1 used in the examples was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0023] Example 1 This embodiment provides a PARP-1 targeting probe, which is a DOTA-PEG4-PARPi labeled with the radioactive nuclide 68Ga. The structural formula of the DOTA-PEG4-PARPi is shown below: The synthetic route for the radiolabeled DOTA-PEGn-PARPi (n = 4, 6) using the radionuclide 68Ga is shown below: (1) Synthesis of intermediate 3b Compound 2b (200 mg, 0.57 mmol) and 20 mL of anhydrous dichloromethane were added to a 100 mL round-bottom flask. EDCI (189 mg, 0.99 mmol), HOBT (133 mg, 0.99 mmol), and DIPEA (295 mg, 2.28 mmol) were added at room temperature. After reacting for 30 min at room temperature, compound 1, olaparib (229 mg, 0.63 mmol), was added, and the reaction was continued overnight at room temperature. The reaction was stopped when the starting material disappeared as detected by TLC. The reaction mixture was poured into water and extracted with dichloromethane (100 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain the crude product. The crude product was separated by column chromatography (PE:EA = 3:1) to give 273 mg of a white solid, with a yield of 78%. 1H-NMR (300 MHz, DMSO-d6) δ 8.45 - 8.37 (m, 1H),7.96 - 7.82 (m, 1H), 7.77 - 7.92 (m, 3H), 7.39 - 7.32 (m, 1H), 7.30 - 7.23(m, 1H), 5.82 (t, J = 8.7 Hz, 1H), 4.40 - 4.17 (m, 2H), 4.05 (s, 2H), 3.73 -3.56 (m, 22H), 3.39 - 3.10 (m, 2H), 1.40 (s, 9H).

[0024] (2) Synthesis of intermediate 4b Compound 3b (200 mg, 0.29 mmol) and 20 mL of anhydrous dichloromethane were added to a 100 mL round-bottom flask. The flask was placed in an ice bath, and 2 mL of trifluoroacetic acid was added dropwise. The reaction was carried out in an ice bath for 1 h, followed by a reaction at room temperature for 1.5 h. The reaction was stopped when the starting material disappeared as detected by TLC. The reaction solution was concentrated under reduced pressure to give a crude product, which was then slurried with n-hexane to give a yellow solid 4b (130 mg), with a yield of 78%.

[0025] (3) Synthesis of intermediate 5b In a 100 mL round-bottom flask, compound DOTA-tri (t-butyl ester) (100 mg, 0.17 mmol) and 20 mL of anhydrous DMF were added. HATU (86 mg, 0.23 mmol) and DIPEA (68 mg, 0.52 mmol) were added separately at room temperature. After stirring at room temperature for 1 h, intermediate 4b (98 mg, 0.19 mmol) was added, and the reaction was continued at room temperature for 14 h. The reaction was stopped when the starting material disappeared as detected by TLC. The reaction solution was poured into water and extracted with ethyl acetate (100 mL × 3). The organic phases were combined and washed with saturated NaCl aqueous solution (100 mL × 3). The mixture was dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to obtain the crude intermediate 5b. This crude product was used directly in the next step without further purification.

[0026] (4) Synthesis of DOTA-PEG4-PARPi Add intermediate 5b and 20 mL of anhydrous dichloromethane to a 100 mL round-bottom flask, then add 2 mL of 5 NHCl at room temperature. Stir the reaction mixture overnight at room temperature. Stop the reaction when the starting material disappears as detected by TLC. Concentrate the reaction solution under reduced pressure to obtain a crude product, which is then separated by column chromatography (DCM:CH3OH = 50:1) to give a white solid DOTA-PEG4-PARPi (58 mg), HPLC >95%, with a yield of 34% for the last two steps. MS (ESI+), m / z: 986.63 [M+H]+; 1H NMR (300MHz, DMSO-d6) δ 8.33 (t, J = 4.3 Hz, 1H), 8.02 - 7.91 (m, 1H), 7.89 - 7.78(m, 2H), 7.72 (d, J = 14.6 Hz, 1H), 7.52 - 7.43 (m, 1H), 7.42 - 7.35 (m, 1H), 4.32 (d, J = 11.9 Hz, 2H), 4.18 - 3.93 (m, 2H), 3.63 (d, J = 14.7 Hz, 18H),3.55 - 3.30 (m, 6H), 3.18 (t, J = 7.6 Hz, 10H), 3.11 - 2.88 (m, 12H), 2.68 (s, 2H).

[0027] (5) Radionuclide labeling The sodium acetate buffer containing the DOTA-PEG4-PARPi was mixed with a solution containing 68Ga, and the mixture was reacted in a metal bath at 95°C for 15 min to obtain the radionuclide-labeled targeting probe. Example 2 This embodiment provides a PARP-1 targeting nuclide probe, which is a radioactive 68Ga-labeled DOTA-PEG6-PARPi. The structural formula of the DOTA-PEG6-PARPi is shown below: (1) Synthesis of intermediate 3c Using compound 2c (200 mg, 0.46 mmol) and compound 1 (183 mg, 0.50 mmol) as starting materials, the preparation process was similar to that of intermediate 3b. The resulting filtrate was distilled under reduced pressure to obtain the crude product. The crude product was separated by column chromatography (PE:EA = 3:1) to give 210 mg of white solid, with a yield of 75%. 1H-NMR (300 MHz, DMSO-d6) δ 8.33 - 8.26 (m,1H), 7.80 - 7.66 (m, 3H), 7.43 - 7.32 (m, 2H), 7.30 - 7.23 (m, 1H), 5.87 (t,J = 7.7 Hz, 1H), 4.22 - 4.15 (m, 2H), 4.06 (s, 2H), 3.73 - 3.60 (m, 26H), 3.57 - 3.40 (m, 4H), 3.38 - 3.29 (m, 2H), 1.38 (s, 9H). (2) Synthesis of DOTA-PEG6-PARPi Using compound 3c (200 mg, 0.25 mmol) and compound DOTA-tri(t-butyl ester) (100 mg, 0.17 mmol) as starting materials, the preparation process was similar to that of DOTA-PEG4-PARPi. The resulting filtrate was distilled under reduced pressure to obtain the crude product. The crude product was separated by column chromatography (DCM:CH3OH = 50:1) to obtain a white solid DOTA-PEG6-PARPi (56 mg), HPLC >95%, with a yield of 30% for the last two steps. MS (ESI+), m / z: 1074.61 [M+H]+; 1H NMR (300 MHz, DMSO-d6) δ 8.26 - 8.14 (m, 1H), 7.98 (d, J = 7.2 Hz, 1H), 7.89 - 7.81 (m,1H), 7.75 (d, J = 26.3 Hz, 1H), 7.53 - 7.44 (m, 1H), 7.44 - 7.25 (m, 1H), 7.22 - 7.11 (m, 1H), 4.36 - 4.20 (m, 2H), 4.19 - 3.97 (m, 2H), 3.70 - 3.56(m, 10H), 3.49 (d, J = 10.5 Hz, 16H), 3.39 - 3.23 (m, 6H), 3.20 - 3.06 (m,12H), 2.99 (t, J = 7.5 Hz, 10H), 2.83 - 2.65 (m, 2H).

[0028] (3) Radionuclide labeling The sodium acetate buffer containing the DOTA-PEG6-PARPi and the solution containing 68Ga were mixed and reacted in a metal bath at 95°C for 15 min to obtain the radionuclide-labeled targeting probe. Application Example 1 The present application example does not have any particular limitation on the preparation or use method of the tumor precision targeting agent, tumor early diagnosis contrast agent, tumor nuclear medicine imaging agent and radiotherapy agent. Any method known to those skilled in the art can be used. For example, the specific targeting radionuclide marker can be directly used as a tumor precision targeting agent, tumor early diagnosis contrast agent, tumor nuclear medicine imaging agent or radiotherapy agent.

[0029] The effectiveness of the 68Ga-labeled targeting probes prepared in Examples 1 and 2 was verified: (1) 3.7 MBq of 68Ga-DOTA-PEG4-PARPi and 68Ga-DOTA-PEG6-PARPi were injected into one PSN-1 tumor-bearing mouse via the tail vein, and Micro-SPECT / CT imaging was performed 1 hour later.

[0030] (2) PSN-1 tumor-bearing rats were placed in a small animal gas anesthesia machine and anesthetized by inhalation with isoflurane.

[0031] (3) After anesthesia, fix the tumor-bearing mouse in a prone position on the scanning table of Micro SPECT / CT, set the corresponding parameters, and then acquire SPECT and CT images respectively.

[0032] Figure 1 SPECT CT imaging results of the 68Ga-labeled targeting probes prepared in Examples 1 and 2. As can be seen from the figures, 68Ga-DOTA-PEG4-PARPi and 68Ga-DOTA-PEG6-PARPi showed significant uptake in the liver and kidneys, and also showed high uptake at the tumor site.

[0033] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of the present invention.

Claims

1. A radionuclide-labeled PARP-1 targeting probe, characterized in that, The structure includes olaparib, a PARP 1 inhibitor, a hydrophilic structure, and a chelating agent, which are sequentially linked by covalent bonds; while the chelating agent and the radionuclide are linked by coordination bonds. The targeting probe is based on the PARP-1 inhibitor olaparib, the hydrophilic structure and the chelating agent are linked by an amide bond, as shown in Formula I; The L group is selected from NH, and n is selected from positive integers between 0 and 10; the Y group is... .

2. The radionuclide-labeled PARP-1 targeting probe according to claim 1, characterized in that, The radionuclide includes any one of 68Ga, 177Lu, 64Cu, 86Y, 89Zr, 90Y, 111In, 123I or 124I, preferably 68Ga.

3. A method for preparing a radionuclide-labeled PARP-1 targeting probe according to claim 2, characterized in that, When the radioactive element is 68Ga, the preparation method includes the following steps: (1) Compound 1, olaparib, and compound PG-L-PEGn-COOH were condensed to give compound 3; ； Wherein, PG represents the protecting group of the amino group, the L group is selected from NH, and n is selected from positive integers between 0 and 10; (2) Compound 3 was deprotected to obtain compound 4; ； Wherein, PG represents the protecting group of the amino group, the L group is selected from NH, the L' group is selected from NH2, and n is selected from positive integers between 0 and 10; (3) Compound 4 and the chelating agent undergo a condensation reaction to obtain the compound shown in Formula I; ； Wherein, the chelating agent is selected from DOTA-tri(t-butyl ester); the L group is selected from NH, n is selected from positive integers between 0 and 10; the Y group is... ; (4) Radiolabel the compound represented by Formula I with a radionuclide to obtain the radionuclide-labeled PARP-1 targeting probe.

4. The preparation method according to claim 3, characterized in that, The molar ratio of olaparib to compound PG-L-PEGn-COOH is 1:(1~2).

5. The preparation method according to claim 3, characterized in that, The protecting group of the amino group is selected from Boc.

6. The preparation method according to claim 3, characterized in that, The condensation reaction is carried out at a temperature of 20-40°C for 12-24 hours.

7. The use of a radionuclide-labeled targeting probe as described in claim 1 or 2 in the preparation of a precise tumor-targeting agent.

8. The use of a radionuclide-labeled targeting probe as described in claim 1 or 2 in the preparation of a contrast agent for early tumor diagnosis.

9. The use of a radionuclide-labeled targeting probe as described in claim 1 or 2 in the preparation of a tumor nuclear medicine imaging agent.

10. The use of a radionuclide-labeled targeting probe as described in claim 1 or 2 in the preparation of radiotherapy formulations.

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