PET (Polyethylene Terephthalate) imaging agent for targeting CSF1 receptor as well as labeled precursor, preparation method and application of PET imaging agent

Abstract

The invention relates to the technical field of medical imaging diagnosis, in particular to a CSF1 receptor-targeted PET (positron emission tomography) developer as well as a labeled precursor, a preparation method and application thereof. The structural formula of a labeled precursor compound pre-FPPA of the PET imaging agent for targeting the CSF1 receptor provided by the invention is as shown in a formula I, and the structural formula of the PET imaging agent 18F-FPPA for targeting the CSF1 receptor provided by the invention is as shown in a formula II. According to the invention, a 6-Aryl group is used as a pharmacophore, the structure of the 6-Aryl group is completely consistent with that of an original drug in a CSF1 receptor antagonist (DFFPA), only isotope difference exists, and the consistency of stability, affinity and specificity of the 6-Aryl group can be ensured to the greatest extent. The prepared PET imaging agent 18F-FPPA has good stability and pharmacokinetic characteristics, and a foundation is laid for scientific research and clinical application of an F-18 labeled targeted CSF1 receptor PET imaging agent.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical image diagnosis, in particular to a PET imaging agent targeting CSF1 receptor, a labeling precursor thereof, a preparation method and use thereof. BACKGROUND

[0002] CSF1R is a tyrosine kinase transmembrane receptor, belonging to the growth factor CSF-1 / platelet-derived growth factor (PDGF) receptor family. In the intracranial, CSF1R is specifically expressed in microglia cells; in the extracranial, CSF1R is mainly expressed in monocyte / macrophage cells. The ligands of CSF1R are colony stimulating factor 1 (CSF1) and IL-34. After CSF1R binds to CSF1 or IL-34, CSF1R needs to be dimerized and bind to ATP to activate the downstream signaling pathway, and regulate the polarization of microglia / macrophage cells. The unique physiological function of CSF1 receptor makes it a therapeutic target for Alzheimer's disease (AD), Parkinson's disease (PD), epilepsy, tumor and other diseases. Therefore, some scholars have proposed that CSF1 can be used as a potential target for microglia activation and inflammation imaging.

[0003] At present, most of the small molecule probes targeting CSF1R are 11 C-labeled. 11 The half-life of C is short (20.4 minutes), which seriously limits its application in the clinic, and one synthesis often only meets the diagnosis and treatment of 1 patient. The first reported 18 F-labeled CSF1R PET imaging agent is 18 F-FOMPyD, which is modified from CSF1R inhibitor GW2580, however 18 F-FOMPyD shows high non-specific binding in autoradiography of rat and human brain tissues. Subsequently, An X et al. synthesized 18 F-4 with high specificity and in vitro stability, but its metabolic products in plasma / cerebrospinal fluid are unknown, and it has not been evaluated in animal models using PET. 18 F-1 is an F-18 imaging agent based on CPPC modification, although it has good specific binding in lipopolysaccharide-induced acute neuroinflammation model mice, however 18 F-1 has certain off-target binding. Therefore, 18 F-labeled small molecule probes targeting CSF1R still have a large room for improvement. SUMMARY

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a PET imaging agent targeting the CSF1 receptor, its labeling precursor, preparation method, and application, in order to solve the problems in the prior art.

[0005] To achieve the above and other related objectives, the present invention first provides a labeled precursor compound (pre-FPPA) for a PET imaging agent targeting the CSF1 receptor, having the structure shown in Formula I:

[0006]

[0007] The present invention also provides a method for preparing the pre-FPPA of the PET imaging agent targeting the CSF1 receptor, comprising the following steps: subjecting compound 1 and compound 2 to a nucleophilic substitution reaction under alkaline conditions to obtain the pre-FPPA of the PET imaging agent targeting the CSF1 receptor.

[0008] This invention also provides a PET imaging agent targeting the CSF1 receptor. 18 F-FPPA), having the structure shown in Formula II:

[0009]

[0010] This invention also provides a PET imaging agent targeting the CSF1 receptor. 18 A method for preparing F-FPPA, the method comprising the following steps:

[0011] 1) The precursor compound pre-FPPA shown in Formula I is subjected to a nucleophilic fluorination reaction with a fluorinating agent to obtain an intermediate;

[0012] 2) After the reaction is complete, acid is added to the reaction system in step 1) for hydrolysis, followed by neutralization with alkali to obtain a PET imaging agent targeting the CSF1 receptor. 18 F-FPPA.

[0013] The present invention also provides the use of the above-mentioned PET imaging agent targeting the CSF1 receptor in the preparation of tumor diagnostic products or inflammation diagnostic products.

[0014] As described above, the PET imaging agent targeting the CSF1 receptor of the present invention, its labeling precursor, preparation method, and uses have the following beneficial effects:

[0015] Invented synthetic compounds 18F-FPPA has good stability and pharmacokinetic characteristics, can specifically bind to CSF1 receptor, has relatively appropriate biochemical properties, can be used as a radioactive imaging agent for detecting CSF1R in vivo to non-invasively monitor inflammation and identify the expression of CSF1 receptor in tumors, and lays a foundation for scientific research and clinical application of F-18 labeled CSF1 receptor targeting PET imaging agents. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 HPLC chromatogram of F-FPPA shown in Example 2 of the present application 18 HPLC chromatogram of F-FPPA shown in Example 2 of the present application

[0017] Figure 2 HPLC chromatogram of F-FPPA shown in Example 2 of the present application 18 HPLC chromatogram of F-FPPA shown in Example 2 of the present application

[0018] Figure 3 PET / CT imaging of a neuroinflammation model mouse shown in Example 6 of the present application. The crosshairs indicate the inflammatory tissue that has taken up the imaging agent. No significant imaging agent uptake is observed in the brain of the blocking group.

[0019] Figure 4 PET / CT imaging of a breast cancer model mouse shown in Example 6 of the present application. The tumor tissue is shown in the white oval box. DETAILED DESCRIPTION

[0020] The present application provides a pre-labeling compound (pre-FPPA) of a PET imaging agent targeting CSF1 receptor, having the structure shown in Formula I:

[0021]

[0022] The present application also provides a preparation method of the pre-labeling compound (pre-FPPA) of the PET imaging agent targeting CSF1 receptor, comprising the following steps:

[0023] Compound 1 and compound 2 undergo a nucleophilic substitution reaction under alkaline conditions to obtain the pre-labeling compound of the PET imaging agent targeting CSF1 receptor.

[0024]

[0025] In some embodiments of the present application, the equivalent ratio of compound 1, compound 2 and base is 1:(0.5-1.5):(1.5-2.5). The equivalent ratio of compound 1, compound 2 and base is selected from any one of the following ranges: 1:(0.5-0.7):(1.5-1.7), 1:(0.7-1.0):(1.7-2.0), 1:(1.0-1.3):(2.0-2.3), 1:(1.3-1.5):(2.3-2.5).

[0026] In a preferred embodiment of the present application, the equivalent ratio of compound 1, compound 2 and base is 1:1:2.

[0027] In some embodiments of the present application, the basic condition is provided by an organic base or an inorganic base.

[0028] In a preferred embodiment of the present application, the inorganic base is selected from potassium carbonate or sodium hydroxide.

[0029] In some embodiments of the present application, the reaction is carried out in an organic solvent.

[0030] In a preferred embodiment of the present application, the organic solvent is N,N-dimethylformamide (DMF).

[0031] In some embodiments of the present application, the temperature of the nucleophilic substitution reaction is 45-55℃. Preferably, the temperature of the nucleophilic substitution reaction is 50℃.

[0032] In some embodiments of the present application, the time of the nucleophilic substitution reaction is 10-20h. The time of the nucleophilic substitution reaction is selected from any one of the following ranges: 10-12h, 12-14h, 14-16h, 16-18h, 18-20h.

[0033] In a specific embodiment of the present application, the reaction progress is detected by liquid chromatography-mass spectrometry.

[0034] In some embodiments of the present application, it further comprises column purification of the system after the reaction is completed to obtain the labeled precursor of the PET imaging agent targeting CSF1 receptor with the structure of formula I.

[0035] The elution phase of the column purification is ethyl acetate and petroleum ether with a volume ratio of 1:1.

[0036] In the present application, the preparation steps of compound 1 refer to the published literature: J. Med. Chem. 2023, 66, 6959-6980.

[0037] The present application also provides a PET imaging agent targeting CSF1 receptor with the structure of formula I. 18F-FPPA), having a structure shown in Formula II:

[0038]

[0039] The present application also provides a preparation method of the PET imaging agent targeting CSF1 receptor (F-FPPA), comprising the following steps: 18 The preparation method of the PET imaging agent targeting CSF1 receptor (F-FPPA) comprises the following steps:

[0040] 1) carrying out nucleophilic fluorination reaction on the precursor compound pre-FPPA shown in Formula I with a fluorination reagent to obtain an intermediate;

[0041] 2) after the reaction is completed, adding acid hydrolysis to the reaction system in step 1) to remove the SEM protecting group, and then adding base neutralization to obtain the PET imaging agent targeting CSF1 receptor (F-FPPA). 18 F-FPPA.

[0042]

[0043] In some embodiments of the present application, in step 1), the fluorination reagent is [18F]KF / K 18 F]KF / K 222 complex.

[0044] In some embodiments of the present application, in step 1), the concentration of the precursor compound pre-FPPA shown in Formula I is 1.3-6.6 mmol / L.

[0045] In a preferred embodiment of the present application, in step 1), the concentration of the precursor compound pre-FPPA shown in Formula I is 5 mg / mL.

[0046] Further, the solvent for dissolving the precursor compound pre-FPPA is ultradry DMSO.

[0047] In the present application, the ultradry DMSO refers to DMSO with extremely low water content, and the water content is generally controlled to be ≤30 ppm.

[0048] In some embodiments of the present application, in step 1), the reaction temperature is 90-140°C.

[0049] In a preferred embodiment of the present application, in step 1), the reaction temperature is 120°C.

[0050] In some embodiments of the present application, in step 1), the reaction time is 8-20 min.

[0051] In a preferred embodiment of the present application, in step 1), the reaction time is 10 min.

[0052] In some embodiments of the present application, in step 2), the acid is HC1.

[0053] In some embodiments of the present application, in step 2), the molar equivalent of the base is equal to the molar equivalent of the acid.

[0054] In some embodiments of the present application, in step 2), the base is NaOH.

[0055] In some embodiments of the present application, further comprising column purification of the system after the reaction is completed to obtain the PET imaging agent targeting CSF1 receptor having the structure of Formula II 18 F-FPPA.

[0056] The elution phase of the column purification is acetonitrile and water with a volume ratio of 55:45.

[0057] The present application also provides the use of the PET imaging agent targeting CSF1 receptor described above in the preparation of a tumor diagnosis product or an inflammation diagnosis product.

[0058] In some embodiments of the present application, the tumor is selected from one or more of lung cancer, prostate cancer, breast cancer, esophageal cancer, liver cancer, bile duct cancer, gastric cancer, colorectal cancer, fibrosarcoma, pancreatic cancer, and glioma.

[0059] The inflammation is selected from neurological inflammation or cardiovascular inflammation. The neurological inflammation is accompanied by or derived from neurodegenerative disease, infectious disease, autoimmune disease, ion channel disease, or demyelinating disease.

[0060] The neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis. The infectious disease is selected from viral encephalitis, acute disseminated encephalomyelitis, brain abscess. The autoimmune disease is autoimmune encephalitis. The ion channel disease is epilepsy. The demyelinating disease is multiple sclerosis.

[0061] The cardiovascular inflammation is accompanied by or derived from atherosclerotic disease, Takayasu's arteritis, cardiac sarcoidosis, myocarditis, etc.

[0062] The embodiments of the present application are described below by way of specific examples, and other advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure herein. The present application can also be implemented or applied in other different specific embodiments, and various modifications or changes can be made to the details herein based on different perspectives and applications without departing from the spirit of the present application.

[0063] Before further description of the present application specific embodiments, it is understood that the present application is not limited in scope to the specific embodiments described herein; it is also understood that the terminology used in the following description is for the purpose of describing the particular embodiments only and is not intended to limit the present application as further described herein; in the description of the present application and in the claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0064] When a numerical range is given in the embodiments, it is understood that, unless otherwise stated by the present application, both ends of each numerical range and any number between the two ends can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, devices, materials used in the embodiments, any method, device and material of the prior art similar or equivalent to the method, device and material described in the embodiments of the present application can also be used to implement the present application according to the mastery of the prior art by those skilled in the art and the description of the present application.

[0065] Embodiment 1

[0066] Synthesis of precursor pre-FPPA having the structure shown in Formula I:

[0067]

[0068] Compound 1 (0.46 g, 1.0 mmol) and compound 2 (0.46 g, 1.0 mmol) were dissolved in DMF (5 mL), and then potassium carbonate (0.27 g, 2.0 mmol) was added. The mixture was stirred at 50°C until the reaction was complete (monitored by liquid chromatography-mass spectrometry). Subsequently, the mixture was cooled to room temperature, and the solvent was removed by evaporation under reduced pressure. The resulting solid product was purified by silica gel column chromatography (ethyl acetate / petroleum ether: 1 / 1) to obtain the product pre-FPPA. The pre-FPPA obtained by preparation was a colorless oil (45 mg, 0.060 mmol, yield 60%). The mass spectrometry, spectroscopy data of pre-FPPA are as follows:

[0069] ESI-MS: 761.1 [M+H] + ;

[0070] 1H NMR (500MHz, DMSO) δ8.23 (s, 1H), 7.81–7.76 (m, 3H), 7.64 (d, J = 8.5Hz, 2H), 7.47 (m, 3H),7.21(t,J=7.5Hz,1H),7.05(m,3H),6.71(s,1H),5.52(s,2H),5.01(s,2H),4.1 5–4.09(m,4H),3.76–3.71(m,2H),3.65–3.58(m,4H),3.56–3.53(m,2H),3.52–3.47 (m,2H),3.35(s,3H),2.40(s,3H),2.27(s,3H),0.86(t,J=7.5Hz,2H),0.08(s,9H).

[0071] Example 2

[0072]

[0073] 1) Dissolve [ 18 F]KF / K 222 Add the MeCN / water solution to the bottle, heat under N2 protection to evaporate to dryness to remove the solvent, then add ultra-dry MeCN, heat under N2 protection to remove the solvent, repeat this process three times to remove all H2O from the system;

[0074] 2) Then, add a mixed solution of the precursor pre-FPPA (3 mg) dissolved in ultra-dry DMSO (500 μL); react at 120 °C for 10 minutes;

[0075] 3) After the reaction is complete, add 500 μL of 4M HCl, hydrolyze for 10 min, then add 500 μL of 4M NaOH to obtain the reaction product. 18 F-FPPA.

[0076] After the reaction system was cooled to room temperature, it was separated by HPLC (YMC-Pack ODS-AM AM12S05-2510WT) (mobile phase: H2O:MeCN = 45:55), and the product was obtained. 18 The HPLC chromatogram of F-FPPA is as follows: Figure 1 As shown. The radiochemical purity of the product is greater than 97%. The total reaction time, starting from the first step, is 40 minutes. After decay correction, the product... 18 The radiochemical yield of F-FPPA is approximately 4–7%.

[0077] Example 3 18 In vitro stability test of F-FPPA

[0078] Take the product obtained in Example 2 18Approximately 10 μCi of F-FPPA was placed in 100 μL of 0.9% physiological saline and 0.1% BSA, respectively, and thoroughly mixed before being stored at 37°C. Samples were taken at 1 h, 2 h, 4 h, and 6 h, and the purity changes were examined using analytical HPLC. The results are shown in Table 1.

[0079] Table 1. Compounds 18 HPLC detection results of F-FPPA in physiological saline and BSA

[0080]

[0081] As shown in Table 1, the compounds 18 F-FPPA is very stable, with no significant decomposition observed within 6 hours.

[0082] Example 4 18 Pharmacokinetics of F-FPPA

[0083] Take the product obtained in Example 2 18 F-FPPA (120 MBq / kg) was injected intravenously into five 8-week-old male ICR mice. Tails were docked at 1, 3, 5, 10, 20, 30, 45, 60, 90, and 120 minutes post-injection. Approximately 5 μL of blood was collected via capillary tube and placed at the bottom of a counting tube for counting. Blood drug concentration-time curves were plotted. Results are as follows: Figure 2 As shown, this indicates that the probe has a relatively fast distribution half-life and a suitable clearance half-life.

[0084] Example 5 18 Biodistribution experiment of F-FPPA

[0085] The product obtained in Example 2 18 Approximately 100 μCi of F-FPPA was injected intravenously into 16 male nude mice at 8 weeks of age. Under anesthesia, blood was collected by enucleation. Four mice were sacrificed at 15, 30, 60, and 90 minutes. Tissue samples, including blood, brain, heart, lung, liver, spleen, kidney, stomach, colon, bone, and muscle, were collected, weighed, and their radioactivity counts were performed. After decay correction, the counts of each tissue sample were compared with standard counts. The results are expressed as %ID / g (the percentage of radioactivity per gram of sample tissue relative to the injected dose), representing the radioactivity of each organ. 18 The relative absorbance values ​​of F-FPPA are shown in Table 2.

[0086] Table 2 Compounds 18 F-FPPA biodistribution detection results

[0087]

[0088] As shown in Table 2, the compounds 18F-FPPA can be excreted through the liver and kidneys, and its uptake in the muscles, brain, and bones of normal mice is low.

[0089] Example 6 18 F-FPPA in vivo imaging experiment

[0090] 1) Construction of the model mouse:

[0091] Neuroinflammatory model mice: 300g male SD rats were used to induce neuroinflammatory reactions by stereotactic injection of lipopolysaccharide (LPS) into the right striatum.

[0092] Breast cancer model mice: Eight-week-old female nude mice were used to construct a breast cancer model by subcutaneously transfecting human CSF1R breast tumor cells in their right forelimbs.

[0093] 2) Experimental grouping: After successful construction, the experiment was divided into two groups.

[0094] Group 1: The product obtained in Example 2 was administered via direct injection through the tail vein. 18 The F-FPPA dosage for mice with neuroinflammatory models is 74 MBq / kg; the dosage for mice with breast cancer models is approximately 140 MBq / kg.

[0095] Group 2: An antidote was administered via tail vein injection approximately 30 minutes prior to the procedure. 19 After F-FPPA (4 mg / kg), injection 18 F-FPPA (rat: 74 MBq / kg, 140 MBq / kg) was used in different rat models for PET / CT imaging under continuous anesthesia.

[0096] The results are as follows Figure 3 , 4 As shown, the study found that in PET / CT images, the imaging agent... 18 F-FPPA can concentrate in the inflamed tissue of the right striatum in mice with inflammatory models, and is also observed in mice with breast tumor models. 18 F-FPPA imaging agent uptake was observed, and both showed reduced imaging agent uptake after injection of the blocking agent, demonstrating... 18 F-FPPA can specifically target CSF1R imaging in mouse models of neuroinflammation and tumors.

[0097] The above experiments show that the compounds synthesized in this invention... 18 F-FPPA exhibits strong stability, specifically binds to the CSF1 receptor, and possesses suitable biochemical properties. It can be used as a radioactive imaging agent to detect CSF1R in vivo for non-invasive monitoring of inflammation and identification of CSF1 receptor expression in tumors via PET / CT, laying the foundation for scientific research and clinical application of F-18-labeled PET imaging agents targeting the CSF1 receptor.

[0098] The foregoing examples are to be construed as merely illustrative of the presently disclosed embodiments, and not a limitation of the present application in any way. Furthermore, various modifications of the application, as well as the variations of the method of the application, will be apparent to those skilled in the art, without departing from the scope and spirit of the application. Although the present application has been described in detail with reference to various preferred embodiments, it should be understood that the application is not limited to such details. Indeed, various modifications of the application, as well as variations of the method of the application, will be apparent to those skilled in the art, without departing from the scope and spirit of the application.

Claims

1. A pre-labeling precursor compound pre-FPPA of a PET imaging agent targeting the CSF1 receptor, characterized in that, having a structure shown in Formula I:

2. Process for the preparation of the pre-labeling precursor compound pre-FPPA of the PET imaging agent targeting the CSF1 receptor according to claim 1, characterized in that, comprising the following steps: undergoing a nucleophilic substitution reaction of compound 1, compound 2 under alkaline conditions to obtain a labeling precursor of a PET imaging agent targeting CSF1 receptor.

3. The preparation method according to claim 2, characterized in that, comprising one or more of the following features: a. the equivalent ratio of compound 1, compound 2 and base is 1:(0.5-1.5):(1.5-2.5); b. the alkaline condition is provided by an organic base or an inorganic base; preferably, the inorganic base is selected from potassium carbonate or sodium hydroxide; c. the reaction is carried out in an organic solvent; preferably, the organic solvent is selected from N,N-dimethylformamide; d. the temperature of the nucleophilic substitution reaction is 45-55℃; e. the time of the nucleophilic substitution reaction is 10-20h.

4. A PET imaging agent targeting the CSF1 receptor 18 F-FPPA characterized in that, having a structure shown in Formula II:

5. The PET imaging agent targeted to the CSF1 receptor as described in claim 4 18 Process for the preparation of F-FPPA, characterized in that, comprising the following steps: 1) carrying out a nucleophilic fluorination reaction of precursor compound pre-FPPA shown in Formula I with a fluorination reagent to obtain an intermediate; 2) After the reaction is completed, add acid hydrolysis to the reaction system in step 1) to remove the SEM protecting group, and then add base to neutralize it to obtain the PET imaging agent targeting the CSF1 receptor 18 F-FPPA.

6. The method of claim 5, wherein the step of forming the first and second layers is performed by a method comprising: In step 1), comprising one or more of the following features: 11) the fluorinating reagent is [F]KF / K222 complex; 18 F]KF / K222 complex; 12) the concentration of precursor compound pre-FPPA shown in Formula I is 1.3-6.6mmol / L; 13) the reaction temperature is 90-140℃; 14) the reaction time is 8-20min.

7. The preparation method according to claim 5, characterized in that, In step 2), comprising one or more of the following features: 21) the acid is HCl; 22) the molar equivalent of the base is equal to the molar equivalent of the acid; 23) the base is NaOH.

8. The PET imaging agent targeted to the CSF1 receptor as described in claim 4 18 Use of F-FPPA in the manufacture of a diagnostic product for tumors or a diagnostic product for inflammation.

9. Use according to claim 8, characterized in that, The tumor is selected from one or several of lung cancer, prostate cancer, breast cancer, esophageal cancer, liver cancer, bile duct cancer, gastric cancer, colorectal cancer, fibrosarcoma, pancreatic cancer and glioma.

10. Use according to claim 8, characterised in that, The inflammation is selected from neural inflammation or cardiovascular inflammation.

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