PARP1-targeting small-molecule probe precursor as well as radiofluorination method and application of PARP1-targeting small-molecule probe precursor

Abstract

The invention belongs to the technical field of organic chemistry, radiopharmaceutical chemistry and clinical nuclear medicine, and more specifically relates to a PARP1-targeting micromolecular probe precursor, and a radiofluorination method and application thereof. The PARP1 targeting probe precursor disclosed by the invention has high computational chemical affinity, a radioactive small-molecule probe formed by chelating the PARP1 targeting probe precursor with radioactive diagnosis nuclide < 18 > F <-> has good in-vivo / in-vitro stability, PARP1 targeting and pharmacokinetic properties, and a tumor-bearing mouse PET imaging effect is good; the probe is expected to become a PARP1 imaging probe with a good application prospect and can guide particle radiotherapy injury effect evaluation from a molecular level.

Description

Technical Field

[0001] This invention belongs to the fields of organic chemistry, radiopharmaceutical chemistry and clinical nuclear medicine, and more specifically, relates to a small molecule probe precursor targeting PARP1 and its radiofluorination method and application. Background Technology

[0002] In the field of clinical cancer treatment, the significant value of particle radiotherapy (protons, carbon ions, etc.) is becoming increasingly apparent. Compared to traditional photon radiotherapy, high-energy particle beams, upon entering the body, can create a dose deposition peak (i.e., the Bragg peak) at a specific depth, while the radiation dose generated before reaching the tumor and after passing through the target area is relatively low. This greatly improves the precision and safety of radiotherapy. Particle radiotherapy primarily works by effectively disrupting the DNA structure of tumor cells, thereby inhibiting their proliferation and division capabilities, thus suppressing tumor growth and spread, and improving local control rates.

[0003] Poly(ADP-ribose) polymerase I (PARP1) is a key protein involved in DNA damage repair processes. When DNA is damaged by radiation, PARP1 immediately binds to the damaged area and is activated. Once PARP1 recognizes and binds to the damaged DNA site, it begins to play several important roles in promoting the DNA repair process. Firstly, PARP1 undergoes rapid modification, primarily through poly(ADP-ribose) glycosylation. In this process, PARP1 converts nicotinamide adenine dinucleotide (NAD) into N, while N, N, and N, ... + Using ADP as a substrate, the ADP-ribose group is added to itself and some related protein molecules in the surrounding environment. This modification may seem like just a chemical change to itself and other proteins, but it is actually very significant. It can alter the charge state, conformation, and interaction ability of these proteins with other molecules, thus creating favorable conditions for subsequent repair work.

[0004] Secondly, PARP1 acts as a "recruiter." Utilizing signals generated after its own modification and its interaction network with other proteins, it actively recruits a series of key factors involved in DNA repair. For example, it can recruit DNA repair enzymes such as DNA ligases and DNA polymerases, which play a central role in repairing broken DNA strands. Simultaneously, it can recruit auxiliary proteins, such as histones that help stabilize DNA structure. By concentrating these repair factors with different functions at the DNA damage site, PARP1 effectively organizes a "repair army," enabling each factor to work synergistically to repair damaged DNA.

[0005] Furthermore, PARP1 also plays a certain "regulatory" role in the repair process. It can control the progress and efficiency of DNA repair by regulating its own activity and its interaction with other regulatory factors. For example, when the repair work progresses to a certain stage, PARP1 can adjust its own modification level in a timely manner according to the actual situation of DNA repair, thereby affecting the activity of recruited repair factors, ensuring that the repair work can proceed in an orderly manner, neither leading to over-repair due to excessively high activity of repair factors, nor to incomplete repair due to excessively low activity.

[0006] Currently, although several approved PARP1 inhibitors are available for cancer treatment or combination therapy, neither targeted inhibition nor radiation damage can directly reflect the extent of DNA damage in tumor cells. Therefore, if radionuclide tracing could be used to "illuminate" PARP1 and then nuclear medicine functional imaging techniques could be employed to assess DNA damage in the tumor target area before and after radiotherapy or targeted inhibition, in order to further evaluate the precise killing effect of cancer cells and guide subsequent treatment, it would have significant scientific and clinical implications.

[0007] However, there is currently a lack of such small molecule imaging probes with clinical application value in China. Summary of the Invention

[0008] The purpose of this invention is to provide a small molecule probe precursor structure targeting PARP1, and to further prepare a radionuclide-labeled probe with the precursor structure as a ligand and a radiopharmaceutical containing the radionuclide-labeled probe.

[0009] The chemical structure of the small molecule probe precursor targeting PARP1 is as follows:

[0010]

[0011] For ease of description, the above structure is named PARPi-1.

[0012] PARPi-1 is derived from the therapeutic PARP1 inhibitor olaparib. PARPi-1 is obtained by replacing the cyclopropyl group (-C3H5) on the outer side of olaparib with the bifunctional chelator NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid).

[0013] This application further discloses a [Al] 18 The F-labeled radioactive small molecule probe is characterized in that the radioactive probe uses PARPi-1 as a ligand.

[0014] This application further discloses a radiodiagnostic drug, characterized in that the active ingredient of the drug is [Al] as described above. 18F]-labeled small molecule probes.

[0015] The preparation method of the radiodiagnostic drug is as follows:

[0016] accelerator 18 O(p,n) 18 F nuclear reaction produces 18 F - Passing through a QMA anion exchange column under helium gas propulsion. 18 F - Adsorbed onto the QMA column;

[0017] Rinse with physiological saline / acetic acid solution 18 F - Then, remove the contents. 18 F - The eluent was poured into a reaction tube pre-filled with PARPi-1 and AlCl3, and the reaction was heated.

[0018] After the reaction system is cooled to room temperature, it is diluted with water for injection. The product is loaded onto a C18 solid-phase extraction column and then rinsed with water for injection. The product is eluted with ethanol / physiological saline solution, dried under constant temperature N2, and then prepared into a formulation with ethanol / physiological saline solution and filtered through a sterile filter membrane for later use.

[0019] The above preparation method is further disclosed as follows:

[0020] accelerator 18 O(p,n) 18 F nuclear reaction produces 18 F - Passing through a QMA anion exchange column under helium gas propulsion. 18 F - Adsorbed onto a QMA column; eluted with 0.5 mL of physiological saline / acetic acid (pH 4.0) solution. 18 F - Then, transfer 80 μL containing 18 F - The eluent was added to a reaction tube containing 0.1 mL of 1 mg / mL PARPi-1 and 14 μL of 10 mM AlCl3, and reacted at 105 °C for 15 min. After the reaction system cooled to room temperature, 10 mL of water for injection was added for dilution. The product was loaded onto a C18 solid-phase extraction column and then washed with 5 mL of water for injection. The product was eluted with 1 mL of 70% ethanol / physiological saline solution, dried under N2 at 25 °C, and then prepared into a formulation with 5% ethanol / physiological saline solution and filtered through a 0.22 μm sterile filter membrane for later use. The radiochemical purity (RCP) and labeling rate (RCY) of the product were determined by radio-HPLC.

[0021] The radiodiagnostic drug RCP is not less than 99%, and RCY is not less than 25%.

[0022] This application finally discloses the above-mentioned content containing [Al] 18 F] The use of radioactive small molecule probes in the preparation of radiodiagnostic drugs.

[0023] The radiological diagnosis is PET imaging, or a combination of PET and CT or MRI imaging.

[0024] The beneficial effects of this invention are:

[0025] The invented PARP1 targeting probe has high computational chemical affinity activity. The molecular probe formed by its chelation with radiodiagnostic nuclides has good in vivo / in vitro stability, PARP1 targeting and pharmacokinetic properties, as well as PET imaging effect and in vivo targeting. It is expected to become a PARP1 imaging probe with good application prospects and is expected to guide the evaluation of particle radiotherapy damage effects at the molecular level.

[0026] Instruction manual illustrations:

[0027] Appendix Figure 1 :[Al 18 F]PARPi-1 radio-HPLC mass analysis chromatogram

[0028] Appendix Figure 2 :[Al 18 F]PARPi-1 in vitro stability analysis diagram

[0029] Appendix Figure 3 In vitro autoradiography of frozen sections of tumor tissue (A&B, experimental group; C&D, inhibition group)

[0030] Appendix Figure 4 :[Al 18 F]PARPi-1 pharmacokinetic curve

[0031] Appendix Figure 5 :[Al 18 F]PARPi-1 tumor-bearing mouse PET imaging MIP image

[0032] Appendix Figure 6 Autoradiography (A&B, ex vivo) and fluorescent staining (C) of tumor tissue from tumor-bearing mice (control)

[0033] Explanation of abbreviations:

[0034] RCP: Radio-chemical Purity

[0035] RCY: Radio-chemical Yield, radiochemical yield / labeling rate

[0036] QMA: Quaternary Methyl Ammonium salt

[0037] OCT adhesive: short for Optimal Cutting Temperature Compound. Detailed Implementation

[0038] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0039] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0040] Example 1: Preparation of PARPi-1

[0041]

[0042] Step 1: Piperazine N-alkylation reaction:

[0043] 2-Fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid (5.0 mmol) and piperazine (6.0 mmol) were dissolved in 30 mL of anhydrous acetonitrile, and potassium carbonate (12 mmol) and potassium iodide (0.5 mmol) were added. The mixture was refluxed at 80 °C and stirred for 12 hours under nitrogen protection. After cooling, the mixture was filtered, the filtrate was concentrated, and subjected to silica gel column chromatography (ethyl acetate / petroleum ether = 1:3). The product phase was subjected to rotary evaporation to remove the solvent, yielding a white solid.

[0044] Step 2: Acid-amine condensation reaction

[0045] 4.0 mmol of 2,2',2”-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid was dissolved in 20 mL of anhydrous dimethylformamide. Then, 8 mmol of N,N-diisopropylethylamine, 4.8 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 4.8 mmol of 1-hydroxybenzotriazole were added sequentially, and the mixture was activated in an ice bath for 30 minutes. Subsequently, a dimethylformamide solution containing 4.0 mmol of the product from step one was added dropwise, and the mixture was stirred overnight at room temperature. The reaction solution was precipitated with water, filtered, and dried to obtain a pale yellow solid.

[0046] Step 3: Purification

[0047] The crude product (3.0 mmol) obtained in step two was dissolved in 15 mL of dichloromethane, and 10 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature for 4 hours. The solvent was removed under reduced pressure, and the residue was adjusted to pH 8-9 with saturated NaHCO3 solution. After extraction with ethyl acetate, the residue was dried and concentrated. The final product was purified by preparative HPLC (acetonitrile / water + 0.1% TFA).

[0048] The data for the final product are as follows:

[0049] 1 H-NMR (400MHz, DMSO-d6) δ12.60(s,1H),8.27(d,J=8.4Hz,1H),7.96(d,J=7.8Hz,1H),7.90(t,J=7.7Hz,1H),7.84(t,J=7.5Hz,1H),7.4 7(s,1H),7.35(t,J=7.2Hz,1H),7.25(t,J=8.9Hz,1H),4.34(s,2H),4.16(s,1H),4.06(s,1H),3.80–3.50(m,12H),3.26–2.89(m,14H).

[0050] MS: [M+H] + =652.5.

[0051] Example 2: Simulation of the affinity of PARPi-1 for PARP1

[0052] This embodiment illustrates the affinity of the small molecule PARPi-1 for PARP1 provided by this invention. The affinity of PARPi-1 for PARP1 was obtained through molecular docking simulation and calculation of the binding energy between PARPi-1 and the active PARP1 structure 5DS3 (PDB ID: 5DS3). The crystal structure file of the receptor 5DS3 protein was downloaded directly from the RCSB PDB database. The 5DS3 crystal structure was preprocessed using the PyMol program to remove heteroatoms (solvents, ions, small molecules, etc.) from the protein and repair incomplete structures to obtain the structure of the protein to be docked. Based on the .cdxml file of the small molecule PARPi-1, the 3D conformation was generated using the RDKit program.

[0053] Using the AutoDock Vina 1.2.5 program, the region containing the olaparib (09L) molecule in the 5DS3 crystal structure was selected as the binding site. The treated 5DS3 protein was docked with 09L and PARPi-1 molecules, respectively. After each docking was completed, the conformation with the best score in each docking result was selected for analysis.

[0054] The results are shown in Table 1. PARPi-1 exhibits high computational chemical activity.

[0055] receptor ligands* Binding energy (kcal / mol) 5DS3 Olapalli (09L) -9.570 5DS3 PARPi-1 -10.910

[0056] Example 3: 18 Preparation of F-labeled PARPi-1 formulations

[0057] This embodiment is for illustration. 18 Preparation and analytical methods of F-labeled PARPi-1 formulations.

[0058] accelerator 18 O(p,n) 18 F nuclear reaction produces 18 F - Passing through a QMA anion exchange column under helium gas propulsion. 18 F - Adsorbed onto a QMA column; washed with 0.5 mL of physiological saline / acetic acid (pH 4.0) solution. 18 F - Then, transfer 80 μL containing 18 F - The eluent was added to a reaction tube containing 0.1 mL of 1 mg / mL PARPi-1 and 14 μL of 10 mM AlCl3, and reacted at 105 °C for 15 min. After the reaction system cooled to room temperature, it was diluted with 10 mL of water for injection. The product was loaded onto a C18 solid-phase extraction column and washed with 5 mL of water for injection. The product was eluted with 1 mL of 70% ethanol / physiological saline, dried under N2 at 25 °C, and then prepared into a formulation with 5% ethanol / physiological saline and filtered through a 0.22 μm sterile filter membrane for later use. The radiochemical purity (RCP) and labeling rate (RCY) of the product were determined by radio-HPLC.

[0059] The radio-HPLC analysis conditions are as follows:

[0060] Chromatography equipment: Agilent 1260 Infinity liquid chromatograph equipped with Gabi Star radiometric detector

[0061] Column: WondaSil C18-WR, 5μm, 4.6×150mm;

[0062] Injection volume: 20 μL; Detection wavelength: 250 nm

[0063] Mobile phase gradient: Pump-A, H2O; Pump-B, acetonitrile; 0-2 min, 5% B; 2-10 min, 5% B to 95% B.

[0064] Flow rate: 1 mL / min;

[0065] According to the test results, the formulation product [Al]18 F]PARPi-1 retention time (RT) is 7.28 min, RCP is not less than 99% (see attached). Figure 1 As shown), RCY is not less than 25%.

[0066] Example 4: Probe [Al] 18 In vitro stability of F]PARPi-1

[0067] [Al] with an activity of 14.8 MBq (0.4 mCi) was transferred. 18 F]PARPi-1 formulation was added to a 1.5 mL centrifuge tube, diluted with physiological saline or 5% fetal bovine serum (FBS) to 0.4 mL, mixed well, and incubated at 37°C. Radio-HPLC analysis was performed at 30 min, 60 min, 120 min, and 240 min (conditions as in Example 3), with two injections analyzed at each time point. The final data were normalized and the images were reconstructed, as shown in the attached figure. Figure 2 As shown.

[0068] The results show that [Al] 18 The F]PARPi-1 formulation retained over 98% of its RCP value after incubation at 37°C for 4 hours in physiological saline or 5% FBS, indicating that the probe has good in vitro stability.

[0069] Example 5: Probe [Al] 18 F]PARPi-1 PARP1 in vitro targeting specificity

[0070] This embodiment is divided into an experimental group and an inhibition group: the experimental group will use [Al] 18 F]PARPi-1 formulation was diluted to a physiological saline solution of 370 kBq / mL (10 μCi / mL) (Solution E). The inhibition group consisted of [Al] containing a final concentration of 1 μM inhibitor olaparib at a concentration of 370 kBq / mL. 18 F]PARPi-1 physiological saline solution (I solution).

[0071] Frozen sections of tumor tissue from tumor-bearing mice (22Rv1, 10μm) pre-frozen at -80℃ were thawed to room temperature and then washed twice with physiological saline to remove the embedding gel. Subsequently, the experimental group sections (n=2) were incubated in 10mL of solution E, while the inhibition group sections (n=2) were incubated in an equal volume of solution I. After 1 hour, the incubation solution was discarded, and the sections were immersed in physiological saline three times for 5 minutes each time. After removal, they were dried, exposed to a phosphorus storage screen for 30 minutes, and then scanned using an imaging system (see attached image). Figure 3 ).

[0072] The results showed that the experimental group slices had obvious concentrations of radioactive signals, while the signal in the inhibition group was almost completely suppressed, indicating that the probe [Al] was effective.18 F]PARPi-1 exhibits good in vitro targeting specificity for PARP1.

[0073] Example 6: [Al] 18 Pharmacokinetic analysis of F]PARPi-1 in normal BALB / c mice

[0074] [Al] 18 F]PARPi-1 was diluted to a 74 MBq / mL (2 mCi / mL) saline solution. Each mouse was injected with 7.4 MBq via the tail vein (n=3). Blood samples (2 μL / sample) were collected from the tail tip at 2 min, 5 min, 10 min, 15 min, 30 min, 45 min, 60 min, and 120 min after administration. The radioactivity counts of the blood samples were determined using a gamma counter. Separately, samples of [Al] at known concentrations were collected. 18 F]PARPi-1 injection solution was used as a standard and was simultaneously measured and counted with blood samples. After decay correction (referring to the injection time), the counts of each group were converted to obtain the radioactivity concentration values ​​(kBq / mL) of blood samples at different times. GraphPadPrism 6 software was used for data fitting and processing to plot concentration-time curves.

[0075] As attached Figure 4 As shown, according to calculations, [Al] 18 The distribution and elimination half-lives of F]PARPi-1 in normal mice were 4.47 min and 35.20 min, respectively, demonstrating that the imaging probe has good pharmacokinetic properties.

[0076] Example 7: Probe [Al] 18 PET imaging of tumor-bearing mice with F]PARPi-1

[0077] After anesthetizing pre-prepared PARP1-positive tumor-bearing mice (male, 20±2g, tumor diameter 0.8–1cm, n=3), 3.7MBq [Al] was injected via the tail vein. 18 F]PARPi-1 formulation (37 MBq / mL): Mice were fixed in a prone position on a self-made PET scanning bed, and imaging data were acquired at 10 min, 20 min, 30 min, 45 min and 60 min after drug administration in static scanning mode. Images were reconstructed using the ordered subsets expectation maximization (OSEM) algorithm, and image analysis and processing were performed using an Inveon Research Workplace 4.2 workstation.

[0078] The specific results are attached. Figure 5 As shown, probe [Al]18 PET images of F]PARPi-1 within 1 hour in PARP1-positive model mice showed that the probe had good stability and pharmacokinetic properties in small animals. The systemic background signal decreased rapidly over time, while the tumor area gradually showed obvious radioactive concentration signal.

[0079] Example 8: Probe [Al] 18 F]PARPi-1 PARP1 in vivo targeting specificity

[0080] Prepare PARP1-positive tumor-bearing mouse models (male, 20±2g, tumor diameter 0.8–1cm, n=2), and add [Al] 18 The PARPi-1 formulation was diluted to a 74 MBq / mL saline solution. Each mouse was injected with 7.4 MBq via the tail vein (n=2). One hour after administration, the mice were euthanized by cervical dislocation. Tumor and muscle tissue were dissected, rinsed with saline, dried with absorbent paper, embedded in OCT gel, and frozen. Sections were cut to a thickness of 10 μm, mounted on absorbent glass slides, and allowed to air dry. The slides were then exposed to a phosphorus storage screen for 3.5 hours, followed by scanning imaging. Tissue sections were stored at -20°C and thawed after 48 hours. The sections were then washed with saline to remove the gel, followed by staining with the PARP1-specific commercial fluorescent dye PARPi-FL for 15 minutes, during which the staining was observed under a fluorescence microscope. After staining, whole tissue sections were acquired using a laser confocal microscope (Zeiss, LSM800, ×10) Tiles module.

[0081] The specific results are attached. Figure 6 As shown, the autoradiography of ex vivo tumor tissue and the staining results with commercial fluorescent dyes are consistent, indicating that the probe [Al]... 18 F]PARPi-1 exhibits good in vivo targeting specificity.

[0082] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A small molecule probe precursor PARPi-1 targeting PARP1, characterized in that, The chemical structure is as follows:

2. A [Al] 18 A radioactive probe labeled with F, characterized in that, The radioactive probe uses PARPi-1 as a radiolabeled precursor as described in claim 1.

3. A radiodiagnostic drug, characterized in that, Containing the [Al] as described in claim 2 18 F] labeled radioactive probes.

4. The radiodiagnostic drug as described in claim 3, characterized in that, The diagnostic drug preparation method is as follows: accelerator 18 O(p,n) 18 F nuclear reaction produces 18 F - Passing through a QMA anion exchange column under helium gas propulsion. 18 F - Adsorbed onto the QMA column; Rinse with physiological saline / acetic acid solution 18 F - Afterwards, the transfer contained 18 F - The eluent was poured into a reaction tube containing the precursors PARPi-1 and AlCl3, and the mixture was heated to allow the reaction to proceed. After the reaction system is cooled to room temperature, it is diluted with water for injection. The product is loaded onto a C18 solid-phase extraction column and then rinsed with water for injection. The product is eluted with ethanol / physiological saline solution, dried under constant temperature N2, and then prepared into a formulation with ethanol / physiological saline solution and filtered through a sterile filter membrane for later use.

5. The radiodiagnostic drug as described in claim 4, characterized in that, Its preparation method is as follows: accelerator 18 O(p,n) 18 F nuclear reaction produces 18 F - Passing through a QMA anion exchange column under helium gas propulsion. 18 F - Adsorbed onto a QMA column; eluted with 0.5 mL of physiological saline / acetic acid (pH 4.0) solution. 18 F - Then, transfer 80 μL containing 18 F - The eluent was added to a reaction tube containing 0.1 mL of 1 mg / mL precursor PARPi-1 and 14 μL of 10 mM AlCl3, and reacted at 105 °C for 15 min. After the reaction system cooled to room temperature, it was diluted with 10 mL of water for injection. The product was loaded onto a C18 solid-phase extraction column and then rinsed with 5 mL of water for injection. The product was eluted with 1 mL of 70% ethanol / physiological saline solution. After drying with N2 at 25 °C, it was prepared into a formulation with 5% ethanol / physiological saline and filtered through a 0.22 μm sterile filter membrane for later use.

6. The radiodiagnostic drug as described in any one of claims 4 or 5, characterized in that, The radiochemical purity of the radiodiagnostic drug is not less than 99%, and the radiochemical yield is not less than 25%.

7. Use of the radioactive probe as described in any one of claims 4 or 5 for the preparation of radiodiagnostic drugs.

8. The radiodiagnostic drug as described in claim 7, characterized in that, The radiological diagnosis is PET imaging.

9. The radiodiagnostic drug as described in claim 7, characterized in that, The radiological diagnosis is a combination of PET and CT or MRI imaging.

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