Cxcr2-targeted compounds and nuclide-labeled probes, methods of making and using the same, pharmaceutically acceptable forms of existence, pharmaceutical compositions, and lyophilized kits and uses thereof

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a CXCR2 targeting compound and a nuclide-labeled probe, a preparation method and application thereof, a pharmaceutically acceptable form, a pharmaceutical composition, a freeze-drying kit and application thereof. The present application introduces a molecular chain with adjustable length between a targeting group and a coordination structure, so as to adjust the distance between the targeting group and the coordination structure and reduce mutual influence. Meanwhile, the structure is connected with different types of nuclide chelating groups to obtain different polar types of labels. In addition, the change in polarity of the small molecule can also improve the pharmacokinetic properties of the labeled complex, accelerate the clearance speed of the tracer in non-target tissues, increase the target / non-target ratio, enhance the affinity between the ligand and the receptor, improve the lesion uptake of the labeled complex, make the imaging clearer, and achieve better diagnosis and treatment effect by improving the lesion uptake.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to CXCR2 targeting compounds and radionuclide-labeled probes, their preparation methods and applications, pharmaceutically acceptable forms, pharmaceutical compositions and lyophilized kits and their applications. Background Technology

[0002] CXC motif chemokine receptor 2 (CXCR2) is a protein-coding gene involved in the G protein-coupled receptor (GPCR) signaling pathway. CXCR2 is specifically overexpressed on the surface of many immune and inflammatory cells and is involved in the development and progression of various inflammatory diseases and cancers. Activation of the chemokine receptor CXCR2 is a key event in neutrophil mobilization in the bone marrow, while bone marrow preservation is regulated by CXCR4. The diurnal regulation of CXCR2 and CXCR4 forms the basis of the circadian rhythm of homeostatic neutrophil flux. Neutrophils are polymorphonuclear neutrophils of the innate immune system, serving as the first line of defense against infection and maintaining tissue homeostasis. They differentiate in the bone marrow (BM), producing short-lived cytotoxic cells. CXCR2 plays multiple roles in pathophysiology, from combating bacterial and viral infections to its role in development, vascular stenosis, diseases of multiple organs including the heart, lungs, liver, and brain, and some cancer etiologies. Research on its main functions has focused primarily on its pro-angiogenic effects and its crucial role in neutrophil chemotaxis.

[0003] While neutrophils play a major role in protecting the host organism in the early stages of bacterial and fungal invasion, recent studies have highlighted their crucial role in inflammation-induced disease progression. Neutrophils play a key role in preventing infection and activating and regulating innate and adaptive immunity. Tumor-associated neutrophils (TANs) play a vital role in the tumor inflammatory process, with mechanisms involving promoting angiogenesis, extracellular matrix remodeling, metastasis, and immunosuppression. Simultaneously, neutrophils also demonstrate significant effects in anti-tumor processes, directly eliminating tumor cells and actively participating in the construction of anti-tumor cell networks.

[0004] The dual functions of TANs in the tumor microenvironment profoundly reveal the diversity and high plasticity of neutrophils. Bone marrow checkpoints and the specific context of tumors and tissues jointly shape the functional performance of neutrophils in response to conventional and immunotherapies. Based on this finding, we hypothesize that neutrophils have the potential to serve as an effective tool to optimize and modulate existing immunotherapy strategies. This will help open up new therapies centered on bone marrow cells, further complementing and enhancing existing treatments, thereby advancing the field of cancer therapy. Neutrophils exhibit high levels of expression of chemokine receptors CXCR1 and CXCR2, which play a crucial role in guiding neutrophil migration into the tumor microenvironment (TME). Furthermore, ligands of CXC chemokines expressed by tumor cells, tumor-infiltrating leukocytes, endothelial cells, and fibroblasts (including but not limited to CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8, also known as IL-8) further enhance the aggregation effect of neutrophils in this region.

[0005] Given that neutrophils serve as the primary line of defense against infection and tissue damage, they can rapidly respond and migrate to the site of inflammation in the early stages, maintaining the body's health by phagocytosing and killing pathogens. In the tumor environment, neutrophils release substances such as vascular endothelial growth factor (VEGF) to promote tumor angiogenesis, providing essential nutrients and oxygen for tumor cell growth and spread. Considering the complex and variable nature of tumor immune targets, molecular imaging-guided tumor diagnosis and treatment are increasingly attracting widespread attention in the medical community. Therefore, developing specific probes targeting CXCR2, a key neutrophil immune target, to provide personalized treatment plans for patients has become a current research focus and hot topic.

[0006] Despite the complex and diverse roles neutrophils play in inflammation and tumors, the importance of constructing molecular probes targeting CXCR2 is further highlighted. However, current research on the distribution of CXCR2 in systemic and lesion-specific models of inflammation or tumors using molecular imaging techniques targeting CXCR2 molecular probes is still limited, and there are even fewer reports of combining CXCR2 expression data with small molecule inhibitors for treatment. Combining the high dynamic monitoring, high resolution, and high sensitivity of positron emission tomography (PET) and single-photon emission computed tomography (SPECT), with small molecule probes specifically targeting CXCR2, holds promise for accelerating in-depth research into the immune mechanisms of CXCR2 in diseases, enhancing understanding of its target, and thus providing new therapeutic strategies for clinical immunotherapy. Summary of the Invention

[0007] The purpose of this invention is to provide CXCR2-targeting compounds and radionuclide-labeled probes, their preparation methods and applications, pharmaceutically acceptable forms, pharmaceutical compositions, and lyophilized kits and their applications. This invention achieves high lesion uptake through functionalized fragment modification, not only filling the gap in the visual diagnostic and therapeutic toolkit for this target at the technical level, but also representing significant progress in compound design and translational applications. It is an innovation that propels the integration of CXCR2-targeted diagnosis and therapy from "lack" to "availability," and from "usable" to "optimal use."

[0008] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a CXCR2 targeting compound, comprising the compound with the structure shown in Formula I or Formula II: Formula I; Formula II; Among them, X1, X2, and X3 independently include CH or N; R1 includes any of the following structures: ; R2 and R5 are either present or absent independently; when R2 or R5 exists, it independently includes any of the following structures: a and b are integers from 0 to 10; R3 includes any of the following structures: -H, -CH3, -CH2CH3, -CH(CH3)2, -CH(CH2CH3)2, -CH(CH2CH2CH3)2. , , , ; R4 includes any of the following structures: Where G is -H or -CH3; x and y are integers from 0 to 10; t and f are integers from 0 to 5; Z is the group to be labeled, including any of the following chelate structures: X and Y independently include -OH or -NH2.

[0009] This invention also provides a method for preparing the CXCR2-targeting compound described in the above technical solution. The preparation method of the compound with the structure shown in Formula I includes the following steps: Step 1: Reaction of aminophenol compounds with diethyl squaric acid yields monosubstituted intermediate 1 of squaric acid; Step 2: React the monosubstituted squaric acid intermediate 1 with an R1 linker compound containing an amino group to obtain squaric amide intermediate 2 with a linker introduced. Step 3: Deprotect the squaramide intermediate 2 to obtain the free amine intermediate 3; When R2 is present, step 4: the free amine intermediate 3 is coupled with an elongation reagent containing R2 and the protecting group is removed to obtain squaric acid intermediate 4; the squaric acid intermediate 4 is substituted with the Z active compound to obtain the compound with the structure shown in Formula I. When R2 is absent, step 4: the free amine intermediate 3 is subjected to a substitution reaction with the Z active compound to obtain the compound with the structure shown in Formula I; The structural formula of the aminophenolic compound is: ; The structural formula of the compound containing the amino group and its R1 linker arm is as follows: ; The structural formula of the R2-containing elongation reagent is: ; The preparation method of the compound with the structure shown in Formula II includes the following steps: Step a: Reaction of Boc-terminated aminophenolic compounds with diethyl squaric acid yields monosubstituted squaric acid intermediate 5; When R5 is not present, step b: react the monosubstituted squaric acid intermediate 5 with an R3 linker compound containing an amino group, and remove the protecting group to obtain intermediate 6. When R5 is present, step b1: remove the protecting group from the monosubstituted squaric acid intermediate 5 and react it with the R5 linker with the protecting group to obtain intermediate 7; step b2: react the intermediate 7 with the R3 linker compound containing an amino group and remove the protecting group to obtain intermediate 8. Step c: The intermediate 6 or the intermediate 8 is subjected to a substitution reaction with the Z active compound to obtain the compound with the structure shown in Formula II; The structural formula of the aminophenolic compound protected by the terminal Boc group is: ; The structural formula of the compound containing the amino group and its R3 linker arm is as follows: ; The structural formula of the R5 binder with a protective group is: ; R1, R2, R3, R4, R5, X1, X2, X3 in each raw material and intermediate are the same as R1, R2, R3, R4, R5, X1, X2, X3 in the compound with the structure shown in Formula I and Formula II.

[0010] The present invention also provides a radionuclide labeling probe, comprising a compound with the structure shown in Formula III or a compound with the structure shown in Formula IV; Formula III; Formula IV; Wherein, R1, R2, R3, R4, R5, X1, X2, X3 are the same as R1, R2, R3, R4, R5, X1, X2, X3 in the structural compound of Formula I and the structural compound of Formula II; Q is a radionuclide labeling group, obtained by coordinating the Z labeling group in the CXCR2 targeting compound prepared by the above-described technical solution or the preparation method described in the above-described technical solution with a radionuclide.

[0011] Preferably, the radionuclide includes 18 F, 43 Sc、 44 Sc、 47 Sc、 51 Cr 55 Co、 57 Co、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 72 As、 72 Se、 89 Zr、 86 Y、 89 Sr、 90 Y、 97 Ru、 99m Tc, 105 Rh、 101m Rh、 103 Pd, 109 Pd, 111 In、 119 Sb、 128 Ba、 139 La、 140 La、 142 Pr、 149 Pm, 149 Tb, 151 Tb, 151 Eu、 153 Eu、 169 Eu、 153 Sm、 152 Gd, 153 Gd, 157 Gd, 159 Gd, 161 Tb, 165 Dy、 166 Ho、 169 Er、 175 Yb、 177 Lu、 186 Re、188 Re、 197 Hg, 198 Au、 201 Tl、 203 Pb, 211 At、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 227 Th and 225 At least one of Ac.

[0012] This invention also provides a method for preparing the radionuclide-labeled probe described in the above technical solution, comprising the following steps: The CXCR2 targeting compound described in the above technical solution or the CXCR2 targeting compound prepared by the preparation method described in the above technical solution and the radionuclide are prepared by wet labeling method or freeze-drying labeling method.

[0013] The present invention also provides a pharmaceutically acceptable form of existence, including the pharmaceutically acceptable form of the CXCR2 targeting compound described in the above technical solutions and / or the pharmaceutically acceptable form of the radionuclide-labeled probe described in the above technical solutions, wherein the pharmaceutically acceptable form of existence includes one or more of salts, tautomers, stereoisomers, racemates, hydrates and solvates.

[0014] The present invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients; the active ingredient comprising one or more of the CXCR2 targeting compound described in the above-described technical solutions, the radionuclide-labeled probe described in the above-described technical solutions, and the pharmaceutically acceptable forms described in the above-described technical solutions.

[0015] The present invention also provides a lyophilized kit comprising a precursor and pharmaceutically acceptable excipients, wherein the precursor comprises one or more of the CXCR2-targeting compounds described in the above-described technical solutions and pharmaceutically acceptable forms of the CXCR2-targeting compounds described in the above-described technical solutions.

[0016] This invention also provides the use of the CXCR2 targeting compound described in the above-described technical solutions, the radionuclide-labeled probe described in the above-described technical solutions, the pharmaceutically acceptable form described in the above-described technical solutions, the pharmaceutical composition described in the above-described technical solutions, or the lyophilized kit described in the above-described technical solutions in the preparation of therapeutic agents, therapeutic drugs, diagnostic reagents, or diagnostic drugs for CXCR2 protein-mediated diseases.

[0017] Preferably, the diseases mediated by the CXCR2 protein include tumors or immune-related diseases; The tumors include breast cancer, lung cancer, colorectal cancer, prostate cancer, bone and connective tissue sarcoma, renal cell carcinoma, gastric cancer, pancreatic cancer, nasopharyngeal carcinoma, head and neck cancer, neuroendocrine tumors, or skin melanoma. The immune-related diseases include lupus, atherosclerosis, rheumatoid arthritis, kidney damage, diabetes, hepatitis, autoimmune encephalomyelitis, scleroderma, psoriasis, Sjögren's syndrome, polyarteritis nodosa, or asthma.

[0018] Compared with the prior art, the outstanding beneficial effects of the present invention are specifically described as follows: (1) This invention develops a class of CXCR2-targeting radioactive probes with high affinity and high specificity. The ligands selected by the radiotracer have the characteristics of strong labeling ability, short labeling time and high labeling yield, which is more conducive to the commercial application and clinical promotion of the labeled substances. (2) This invention introduces a molecular chain with adjustable chain length between the target group and the coordination structure, which can adjust the distance between the target group and the coordination structure and reduce mutual interference. At the same time, this structure can obtain markers with different polarities by connecting with different types of radionuclide chelating groups. In addition, the small molecule polarity change can also improve the pharmacokinetic properties of the labeled complex, accelerate the clearance rate of the tracer in non-target tissues, increase the target / non-target ratio, enhance the affinity between the ligand and the receptor, improve the lesion uptake of the labeled complex, make the imaging clearer, and achieve better diagnostic and therapeutic effects by improving lesion uptake.

[0019] (3) The CXCR2 protein-targeting compounds and their radionuclide markers described in this invention possess excellent in vivo biological properties. They exhibit high specific uptake in lesions with high CXCR2 protein expression, a high target-to-non-target ratio, and low non-specific background. These characteristics can reduce unnecessary radiation damage to normal tissues and organs, making them highly suitable for the diagnosis and treatment of tumors. Furthermore, these probes can also be used in combination with other immune checkpoint inhibitors (such as PD-1 / PD-L1 monoclonal antibodies) for combined therapy.

[0020] (4) The probe described in this invention uses nuclear medicine imaging to visualize and quantitatively analyze the dynamic expression of CXCR2 in the tumor microenvironment, overcoming the shortcomings of traditional detection methods such as sampling errors and poor accuracy, and providing a new research method for exploring the role of CXCR2 in tumor occurrence and development. Attached Figure Description

[0021] Figure 1 This is the mass spectrum of compound PD in this invention; Figure 2 This is the HPLC spectrum of compound PD in this invention; Figure 3This is the mass spectrum of compound PP2D in this invention; Figure 4 This is the HPLC spectrum of compound PP2D in this invention; Figure 5 This is the mass spectrum of compound PP2N in this invention; Figure 6 This is the HPLC spectrum of compound PP2N in this invention; Figure 7 This is the mass spectrum of compound PP2SD in this invention; Figure 8 This is the HPLC spectrum of compound PP2SD in this invention; Figure 9 This is the mass spectrum of compound PP4D in this invention; Figure 10 This is the HPLC spectrum of compound PP4D in this invention; Figure 11 This is the mass spectrum of compound PP4SD in this invention; Figure 12 This is the HPLC spectrum of compound PP4SD in this invention; Figure 13 This is the mass spectrum of compound PP4SN in this invention; Figure 14 This is the HPLC spectrum of compound PP4SN in this invention; Figure 15 This is the mass spectrum of compound Amyl-PP4D in this invention; Figure 16 This is the HPLC spectrum of compound Amyl-PP4D in this invention; Figure 17 This is the mass spectrum of compound Py-PP4D in this invention; Figure 18 This is the HPLC spectrum of compound Py-PP4D in this invention; Figure 19 The compound in this invention [ 68 Ga]Ga-PD,[ 68 Ga]Ga-PP2D,[ 68 Ga]Ga-PP4D, [ 68 Ga]Ga-PP2SD and [ 68 HPLC chromatogram of Ga]Ga-PP4SD labeled quality control; Figure 20 The compound of this invention [ 68 Ga]Ga-Amyl-PP4D, [ 68 Ga]Ga-Py-PP4D, [ 18 F]AlF-PP2N and [18 HPLC chromatogram of labeled AlF-PP4SN; Figure 21 The compound in this invention [ 68 Ga]Ga-PP4SD and [ 68 Stability of Ga]Ga-PP4D in physiological saline and serum; Figure 22 The compound in this invention [ 68 Ga]Ga-PP4SD(A) and [ 68 Ga]Ga-PP4D (B) Micro-PET imaging in normal mice for 0.5–1 hour; Figure 23 The compound in this invention [ 68 Ga]Ga-PP2D (Picture A), [ 68 Ga]Ga-PP2SD (Picture B), [ 68 Ga]Ga-PP4D (Picture C), [ 68 Ga]Ga-PP4SD (D image) Micro-PET imaging in mouse B16F10 model mice for 0.5–2 hours; Figure 24 The compound in this invention [ 68 Ga]Ga-PP2D (Picture A), [ 68 Ga]Ga-PP2SD (Picture B), [ 68 Ga]Ga-PP4D (Picture C), [ 68 Ga]Ga-PP4SD (D figure) Radioligand uptake values ​​of important tissues and organs in rodent B16F10 model mice from 0.5 to 2 hours; Figure 25 The compound in this invention [ 68 Ga]Ga-PP2D (Picture A), [ 68 Ga]Ga-PP2SD (Picture B), [ 68 Ga]Ga-PP4D (Picture C), [ 68 Ga]Ga-PP4SD (D figure) Target to non-target ratio of important tissues and organs in mouse B16F10 model mice at 0.5-2 hours; Figure 26 The compound in this invention [ 68 Ga]Ga-PP4SD Micro-PET imaging in PANC-02 mouse model mice for 0.5~2 hours; Figure 27 The compound in this invention [ 68Ga]Ga-PP4SD uptake of radioligands in important tissues and organs in mouse PANC-02 model mice from 0.5 to 2 hours (Figure A) and target to non-target ratio (Figure B). Figure 28 The compound in this invention [ 18 Micro-PET imaging of F]AlF-PP4SN in mouse-derived PANC-02 model mice for 0.5–2 hours; Figure 29 The compound in this invention [ 18 [F]AlF-PP4SN radioligand uptake in important tissues and organs of rat PANC-02 model mice from 0.5 to 2 hours (Figure A) and target to non-target ratio (Figure B). Figure 30 The compound in this invention [ 68 Ga-Py-PP4D micro-PET imaging in human A549 model mice for 0.5–1 hour; Figure 31 The compound in this invention [ 68 Ga-Py-PP4D uptake of radioligands in important tissues and organs in human A549 model mice from 0.5 to 1 hour (Figure A) and target to non-target ratio (Figure B). Figure 32 The compound in this invention [ 68 Ga-Amyl-PP4D micro-PET imaging in MC38 mouse model mice for 0.5–2 hours; Figure 33 The compound in this invention [ 68 Ga]Ga-Amyl-PP4D uptake of radioligands in important tissues and organs in MC38 rat model mice from 0.5 to 2 hours (Figure A) and target to non-target ratio (Figure B). Figure 34 The compound in this invention [ 68 Micro-PET imaging of Ga]Ga-PP4SD in mice with doxorubicin (DOX)-induced myocarditis and healthy control mice; Figure 35 The compound in this invention [ 68 Ga]Ga-PP4SD in the uptake of radioligands in important tissues and organs of doxorubicin (DOX)-induced myocarditis model mice and healthy control mice (Figure A) and myocardial ratio (Figure B). Figure 36 The compound in this invention [ 68 Ga]Ga-PP2D, [ 68 Ga]Ga-PP2SD, [ 68 Ga]Ga-PP4D, [68 Ga]Ga-PP4SD Micro-PET imaging in a mouse model of gouty ankle joint at 0.5 hours; Figure 37 The compound in this invention [ 68 Ga]Ga-PP2D, [ 68 Ga]Ga-PP2SD, [ 68 Ga]Ga-PP4D, [ 68 Radioligand uptake values ​​of Ga]Ga-PP4SD in important tissues and organs at 0.5 hours in a mouse model of gouty ankle joint; Figure 38 In this invention [ 68 Ga]Ga-PP4SD (Figure A) and [ 68 Ga]Ga-PP4D (Figure B) Micro-PET imaging at 0.5-1 hours in a mouse model of acute kidney injury due to ischemia-reperfusion. Figure 39 The compound in this invention [ 68 Ga]Ga-PP4SD (Figure A) and [ 68 Ga]Ga-PP4D (Figure B) uptake values ​​of the left and right kidneys and the ratio of the right kidney to the left kidney in a mouse model of acute kidney injury due to ischemia-reperfusion (Figure C). Figure 40 In this invention [ 68 Ga]Ga-PP4SD micro-PET imaging at 0.5 hours in mice with acute kidney injury model induced by different cisplatin doses; Figure 41 In this invention [ 68 Ga]Ga-PP4SD uptake values ​​in the heart (Figure A), liver (Figure B), kidney (Figure C), and muscle (Figure D) of mice with acute kidney injury induced by different cisplatin doses; Figure 42 In this invention [ 68 Micro-PET imaging of Ga]Ga-PP4SD uptake and blockade in a mouse model of cisplatin-induced acute kidney injury; Figure 43 In this invention [ 68 Quantitative renal uptake values ​​of Ga]Ga-PP4SD in a mouse model of cisplatin-induced acute kidney injury; Figure 44 In this invention [ 68 Autoradiography of heart, liver and kidney in cisplatin-induced acute kidney injury model mice using Ga]Ga-PP4SD; Figure 45 In this invention [ 68Quantitative autoradiography of heart, liver and kidney in a mouse model of cisplatin-induced acute kidney injury using Ga]Ga-PP4SD. Figure 46 In this invention [ 68 Quantitative biodistribution of Ga]Ga-PP4SD in a cisplatin-induced acute kidney injury model mouse. Detailed Implementation

[0022] This invention provides a CXCR2 targeting compound, comprising a compound with the structure shown in Formula I or a compound with the structure shown in Formula II: Formula I; Formula II; Where X1, X2, and X3 independently include CH or N; R1 includes any of the following structures: ; R2 and R5 may or may not exist independently; when R2 or R5 exists, it independently includes any of the following structures: a and b are integers from 0 to 10; R3 includes any of the following structures: -H, -CH3, -CH2CH3, -CH(CH3)2, -CH(CH2CH3)2, -CH(CH2CH2CH3)2. , , , ; R4 includes any of the following structures: Where G is -H or -CH3; x and y are integers from 0 to 10; t and f are integers from 0 to 5; Z is the group to be labeled, including any of the following chelate structures: X and Y independently include -OH or -NH2; In this invention, R1 has an active amine end that is connected to the -CO- at one end of the R2 fragment to form an amide bond, and the other end is connected to the parent structure; Z is connected to the -NH- at the other end of the R2 fragment in the form of an amide bond or a thiourea bond.

[0023] In this invention, R4 and the parent structure are connected by an amide bond; the other end of R4 is then connected to -CO- at one end of the R5 fragment to form an amide bond; Z is connected to -NH- at the other end of the R5 fragment in the form of an amide bond or a thiourea bond.

[0024] In this invention, when R2 or R5 is absent, the active amine at the other end of R1 or R4 is directly connected to Z via an amide bond or a thiourea bond. In specific embodiments, it is not restricted for either end of the active amine at both ends of R4 to be connected to the parent compound.

[0025] In this invention, the CXCR2 targeting compound preferably has any one of the following structures: When X1, X2, and X3 in equation (I) are all CH, R1 adopts R2 does not exist, Z adopts The structure of the CXCR2-targeting compound is shown below: Formula (PD); When X1, X2, and X3 in equation (I) are all CH, R1 adopts R2 adopts (a=2 or a=4), Z adopts or or or The structures of the CXCR2-targeting compounds are shown below: Formula (PP2D); Formula (PP2N); Formula (PP2SD); Formula (PP4D); Formula (PP4SD); Formula (PP4SN); When X1 is N, X2 and X3 are CH, and R1 adopts R2 adopts (a=4), Z adopts The structure of the CXCR2-targeting compound is shown below: Formula (Py-PP4D); When in equation (II), R3 adopts R4 is (x=1, G is CH3), R5 adopts (a=4), Y adopts The structure of the CXCR2-targeting compound is shown below: Formula (Amyl-PP4D).

[0026] The present invention also provides a method for preparing the CXCR2-targeting compound described in the above technical solution.

[0027] In this invention, R1, R2, R3, R4, R5, X1, X2, X3 in each raw material and intermediate are the same as R1, R2, R3, R4, R5, X1, X2, X3 in the compound with structure shown in Formula I and the compound with structure shown in Formula II.

[0028] In this invention, the preparation method of the compound with the structure shown in Formula I includes the following steps: Step 1: Reaction of aminophenol compounds with diethyl squaric acid yields monosubstituted intermediate 1 of squaric acid; Step 2: React the monosubstituted squaric acid intermediate 1 with an R1 linker compound containing an amino group to obtain squaric amide intermediate 2 with a linker introduced. Step 3: Deprotect the squaramide intermediate 2 to obtain the free amine intermediate 3; When R2 is present, step 4: the free amine intermediate 3 is coupled with an elongation reagent containing R2 and the protecting group is removed to obtain squaric acid intermediate 4; the squaric acid intermediate 4 is substituted with the Z active compound to obtain the compound with the structure shown in Formula I. When R2 is absent, step 4: the free amine intermediate 3 is subjected to a substitution reaction with the Z active compound to obtain the compound with the structure shown in Formula I; The structural formula of the aminophenolic compound is: ; The structural formula of the compound containing the amino group and its R1 linker arm is as follows: ; The structural formula of the R2-containing elongation reagent is: .

[0029] In this invention, a schematic flowchart of the preparation method of the compound with the structure shown in Formula I is shown below: .

[0030] Step 1 of this invention involves reacting an aminophenol compound with diethyl squaric acid to obtain a monosubstituted intermediate 1 of squaric acid.

[0031] In this invention, the molar ratio of the aminophenolic compound to diethyl squaric acid is preferably 1:1 to 20, specifically 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:10, 1:12, 1:15, 1:16, 1:18, or 1:20. The reaction is preferably carried out in an alcohol solvent, which preferably includes one or more of methanol, ethanol, isopropanol, and n-butanol, more preferably ethanol. The reaction temperature is preferably 0 to 60°C, specifically 0°C, 15°C, or 25°C. The reaction time is preferably 1 to 48 hours, specifically 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, or 48 hours. After the reaction, this invention also preferably includes post-treatment, which preferably includes: filtering the precipitated solid, washing it three times with petroleum ether, collecting the filter cake, and drying it at 45°C.

[0032] In obtaining the squaric acid monosubstituted intermediate 1, step 2 of the present invention involves reacting the squaric acid monosubstituted intermediate 1 with an R1 linker compound containing an amino group to obtain a squaric amide intermediate 2 with an introduced linker arm.

[0033] In this invention, the molar ratio of the monosubstituted squaric acid intermediate 1 to the amino-containing R1 linker compound is preferably 1:1 to 20, specifically 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:10, 1:12, 1:15, 1:16, 1:18, or 1:20; the solvent used preferably includes one or more of methanol, ethanol, isopropanol, n-butanol, tetrahydrofuran (THF), acetonitrile, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and N-methylpyrrolidone (NMP); the reaction temperature is preferably 25 to 50°C, more preferably room temperature, and the reaction time is preferably 4 to 8 hours (specifically 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours). In this invention, the reaction is preferably followed by a post-treatment, which preferably includes: removing the solvent using a rotary evaporator, then extracting three times with water and ethyl acetate, collecting the organic phase, washing with saturated brine, and drying with anhydrous sodium sulfate; filtering, concentrating the filtrate under reduced pressure; and performing column chromatography (dichloromethane:methanol = 1:0-50:1) on the concentrated crude product.

[0034] After obtaining the square amide intermediate 2, step 3 of the present invention is to deprotect the square amide intermediate 2 to obtain the free amine intermediate 3.

[0035] In this invention, the deprotection is preferably carried out under acidic conditions. The acidic reagent used is preferably trifluoroacetic acid (TFA) or hydrochloric acid, more preferably an organic solution of hydrochloric acid. The concentration of hydrochloric acid in the organic solution is preferably 1~12M, specifically 1M, 2M, 3M, 4M, 6M, 8M, 10M or 12M; more preferably an ethyl acetate solution of 4M hydrochloric acid. The deprotection temperature is preferably 0~50℃, more preferably room temperature, and the time is preferably 0.5~24h (specifically 0.5h, 1h, 5h, 10h, 12h, 16h, 20h, 24h). The solvent used for deprotection preferably includes one or more of ethyl acetate, 1,4-dioxane, methanol, ethanol, isopropanol, tetrahydrofuran (THF), acetonitrile and dichloromethane (DCM). In this invention, after the deprotection, post-treatment is preferably included, which preferably includes: filtering the solid, washing it three times each with ethyl acetate and dichloromethane; collecting the filter cake and drying it at 45°C.

[0036] After obtaining the free amine intermediate 3, when R2 is present, step 4 of the present invention is to couple the free amine intermediate 3 with an elongation reagent containing R2 and remove the protecting group to obtain squaric acid intermediate 4; and to perform a substitution reaction between the squaric acid intermediate 4 and the Z active compound to obtain the compound with the structure shown in Formula I.

[0037] In this invention, when R2 is present, the molar ratio of the free amine intermediate 3 to the elongating agent containing R2 is preferably 1:1 to 10; the coupling reaction is preferably carried out in the presence of a condensing agent and a basic reagent, wherein the condensing agent preferably includes one or more of HATU, PyBOP, HBTU, EDCI, and DIC; the basic reagent preferably includes one or more of DIPEA, TEA, and NMM; the molar ratio of the free amine intermediate 3 to the condensing agent is preferably 1:1 to 10 (specifically, it can be 1:1, 1:2, 1:3, 1:4, 1:1...). The molar ratio of the free amine intermediate 3 to the basic reagent is preferably 1:1 to 10 (specifically, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, or 1:10). The reaction solvent used in the coupling reaction preferably includes one or more of DMF, NMP, DMSO, acetonitrile, and DCM. The temperature of the coupling reaction is preferably 25 to 50°C, more preferably room temperature, and the time is preferably 4 to 12 hours (specifically, it can be 4 hours, 6 hours, 8 hours, 10 hours, or 12 hours). In this invention, after the coupling reaction, a post-treatment is also preferred, which preferably includes: adding ultrapure water to the system to quench the reaction and removing the reaction solvent using a rotary evaporator; extracting three times with ethyl acetate and collecting the organic phase; drying with anhydrous sodium sulfate; filtering; concentrating the filtrate under reduced pressure; and separating the concentrated crude product by column chromatography (dichloromethane:methanol = 1:0-20:1).

[0038] In this invention, the condition parameters for removing the protecting group are preferably the same as those for removing the protecting group in step 3, and will not be repeated here.

[0039] In this invention, the Z-active compound preferably comprises a Z-active compound with an activated ester group or an isothiocyanate group; the activated ester group preferably comprises an N-hydroxysuccinimide (NHS) activating group, an acid anhydride, tetrafluorophenyl ester, or pentafluorophenyl ester; the Z-active compound preferably comprises any one of the following structures: .

[0040] In this invention, the molar ratio of the squaric acid intermediate 4 to the Z active compound is preferably 1:1 to 10 (specifically, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, or 1:10); the solvent for the substitution reaction preferably includes one or more of DMF, NMP, DMSO, acetonitrile, and DCM; the substitution reaction is preferably carried out in the presence of an alkaline reagent, which preferably includes one or more of DIPEA, TEA, and NMM, with DIPEA being preferred; the temperature of the substitution reaction is preferably 25 to 50°C, more preferably room temperature, and the time is preferably 4 to 12 hours (specifically, it can be 4 hours, 6 hours, 8 hours, 10 hours, or 12 hours). In this invention, after the substitution reaction, a post-treatment is also preferred, which preferably includes: separation of the crude product by high-performance liquid chromatography (HPLC) column (column type: C18 reversed-phase column, 250 × 10 mm). 2 Thermo Scientific; Mobile phase: A: ultrapure water (containing 0.1% trifluoroacetic acid), D: acetonitrile (containing 0.1% trifluoroacetic acid); Mobile phase conditions: 5-95% D, 20 min, 3 mL / min); The separated product was lyophilized.

[0041] In this invention, when R2 is absent, the molar ratio of the free amine intermediate 3 to the Z active compound is preferably 1:1 to 10 (specifically, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, or 1:10); the solvent for the substitution reaction preferably includes one or more of DMF, NMP, DMSO, acetonitrile, and DCM; the substitution reaction is preferably carried out under alkaline conditions, and the alkaline reagent preferably includes one or more of DIPEA, TEA, and NMM, with DIPEA being preferred; the temperature of the substitution reaction is preferably 25 to 50°C, more preferably room temperature; the time is preferably 4 to 12 hours (specifically, it can be 4 hours, 6 hours, 8 hours, 10 hours, or 12 hours). In this invention, after the substitution reaction, a post-treatment is also preferably included, and the post-treatment is preferably the same as the post-treatment process of the substitution reaction in the above technical solution, which will not be repeated here.

[0042] In this invention, the preparation method of the compound with the structure shown in Formula II includes the following steps: Step a: Reaction of Boc-terminated aminophenolic compounds with diethyl squaric acid yields monosubstituted squaric acid intermediate 5; When R5 is not present, step b: react the monosubstituted squaric acid intermediate 5 with an R3 linker compound containing an amino group, and remove the protecting group to obtain intermediate 6. When R5 is present, step b1: remove the protecting group from the monosubstituted squaric acid intermediate 5 and react it with the R5 linker with the protecting group to obtain intermediate 7; step b2: react the intermediate 7 with the R3 linker compound containing an amino group and remove the protecting group to obtain intermediate 8. Step c: The intermediate 6 or the intermediate 8 is subjected to a substitution reaction with the Z active compound to obtain the compound with the structure shown in Formula II; The structural formula of the aminophenolic compound protected by the terminal Boc group is: ; The structural formula of the compound containing the amino group and its R3 linker arm is as follows: ; The structural formula of the R5 binder with a protective group is: ; In this invention, the flowchart of the preparation method of the compound with the structure shown in Formula II is shown below: .

[0043] Step a of the present invention is to react an aminophenol compound with a Boc-terminated end group with diethyl squaric acid to obtain a monosubstituted intermediate 5 of squaric acid.

[0044] In this invention, the molar ratio of the Boc-terminated aminophenol compound to diethyl squaric acid is preferably 1:1 to 20, more preferably 1:1 to 10, and specifically can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8 or 1:10; the solvent used in the reaction preferably includes one or more of methanol, ethanol, isopropanol, n-butanol, tetrahydrofuran (THF), acetonitrile and DMF; the reaction temperature is preferably 0 to 60°C, more preferably room temperature, and the reaction time is preferably 1 to 48 h (specifically 1 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, 48 h). In this invention, after the reaction, it is preferred to perform a post-treatment, which preferably includes: removing the solvent with a rotary evaporator, then extracting three times with ethyl acetate and water, taking the organic phase, washing once with saturated brine, and drying with anhydrous sodium sulfate; filtering, concentrating the filtrate under reduced pressure; and separating the concentrated crude product by column chromatography (dichloromethane:methanol = 1:0-50:1).

[0045] After obtaining the monosubstituted squaric acid intermediate 5, when R5 is not present, step b of the present invention is to react the monosubstituted squaric acid intermediate 5 with a compound containing an amino group and an R3 linker, and remove the protecting group to obtain intermediate 6.

[0046] In this invention, the molar ratio of the monosubstituted squaric acid intermediate 5 to the amino-containing R3 linker compound is preferably 1:1 to 20, more preferably 1:1 to 10, and specifically can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8 or 1:10; the solvent used in the reaction preferably includes one or more of methanol, ethanol, isopropanol, n-butanol, THF, acetonitrile, DMF, DMSO and NMP; the reaction temperature is preferably 25 to 50°C, and the reaction time is preferably 4 to 8 hours.

[0047] In this invention, the removal of the protecting group is preferably carried out under acidic conditions. The acidic reagent used is preferably TFA or hydrochloric acid, more preferably an organic solution of hydrochloric acid. The concentration of hydrochloric acid in the organic solution is preferably 1-12M, and in specific embodiments it can be 1M, 2M, 3M, 4M, 6M, 8M, 10M or 12M, more preferably a 4M ethyl acetate solution of hydrochloric acid. The reaction solvent used preferably includes one or more of ethyl acetate, 1,4-dioxane, methanol, ethanol, isopropanol, THF, acetonitrile and DCM. The temperature for removing the protecting group is preferably 0-50°C, more preferably room temperature, and the time is preferably 0.5-24h (specifically 0.5h, 1h, 5h, 10h, 12h, 16h, 20h, 24h). In this invention, after the removal of the protecting group, a post-treatment is also preferred. The post-treatment preferably includes: filtering the solid, washing it three times each with ethyl acetate and dichloromethane; collecting the filter cake, and drying it at 45°C.

[0048] After obtaining the monosubstituted squaric acid intermediate 5, when R5 is present, step b1 of the present invention is to remove the protecting group from the monosubstituted squaric acid intermediate 5 and react it with the R5 linker with the protecting group to obtain intermediate 7; step b2 is to react the intermediate 7 with the R3 linker compound containing an amino group and remove the protecting group to obtain intermediate 8.

[0049] In this invention, the condition parameters for removing the protecting group in step b1 are preferably the same as those for removing the protecting group in step b, and will not be repeated here.

[0050] In this invention, in step b1, the molar ratio of the deprotected free amine intermediate 5 to the protected R5 linker is preferably 1:1 to 10 (specifically, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, or 1:10); the reaction is preferably a coupling reaction, which is preferably carried out in the presence of a condensing agent and a basic reagent. The condensing agent preferably includes one or more of HATU, PyBOP, HBTU, EDCI, and DIC; the basic reagent preferably includes one or more of DIPEA, TEA, and NMM; the molar ratio of the deprotected free amine intermediate 5 to the condensing agent is preferably... The molar ratio of the free amine intermediate 5 (without the protecting group) to the basic reagent is preferably 1:1 to 10 (specifically, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, or 1:10); the molar ratio of the free amine intermediate 5 (without the protecting group) to the basic reagent is preferably 1:1 to 10 (specifically, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, or 1:10); the reaction solvent used in the coupling reaction preferably includes one or more of DMF, NMP, DMSO, acetonitrile, and DCM; the temperature of the coupling reaction is preferably 25 to 50°C, more preferably room temperature, and the time is preferably 4 to 12 hours (specifically, it can be 4 hours, 8 hours, 10 hours, or 12 hours). In this invention, the reaction is preferably followed by a post-treatment, which preferably includes: adding ultrapure water to the system to quench the reaction and removing the reaction solvent using a rotary evaporator; extracting three times with ethyl acetate and collecting the organic phase; drying with anhydrous sodium sulfate; filtering and concentrating the filtrate under reduced pressure; and separating the concentrated crude product by column chromatography (dichloromethane:methanol = 1:0~20:1).

[0051] In this invention, in step b2, the molar ratio of the intermediate 7 to the amino-containing R3 linker compound is preferably 1:1 to 20, more preferably 1:1 to 10, specifically 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, or 1:10; the reaction is preferably carried out in the presence of an alkaline reagent, which preferably includes one or more of DIPEA, TEA, and NMM, with DIPEA being preferred; the solvent used in the reaction preferably includes one or more of methanol, ethanol, isopropanol, n-butanol, THF, acetonitrile, DMF, DMSO, and NMP; the reaction temperature is preferably 25 to 50°C, more preferably room temperature, and the reaction time is preferably 8 to 24 hours (specifically 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, or 24 hours). In this invention, the reaction is preferably followed by a post-treatment, which preferably includes: removing the solvent using a rotary evaporator, then extracting three times with water and ethyl acetate, collecting the organic phase, washing with saturated brine, and drying with anhydrous sodium sulfate; filtering, concentrating the filtrate under reduced pressure; and performing column chromatography (dichloromethane:methanol = 1:0-50:1) on the concentrated crude product.

[0052] In this invention, the condition parameters for removing the protecting group in step b2 are preferably the same as those for removing the protecting group in step b, and will not be repeated here.

[0053] After obtaining intermediate 6 or intermediate 8, step c of the present invention is to perform a substitution reaction between intermediate 6 or intermediate 8 and the Z active compound to obtain the compound with the structure shown in Formula II.

[0054] In this invention, the type of the Z active compound is preferably as defined in the above-described technical solution, and will not be repeated here. In this invention, the molar ratio of intermediate 6 or intermediate 8 to the Z active compound is preferably 1:1 to 10, specifically 1:1, 1:1.2, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, or 1:10; the reaction solvent preferably includes one or more of DMF, NMP, DMSO, acetonitrile, and DCM; the substitution reaction is preferably carried out under alkaline conditions, and the alkaline reagent preferably includes one or more of DIPEA, TEA, and NMM, preferably DIPEA; the temperature of the substitution reaction is preferably 25 to 50°C, more preferably room temperature, and the time is preferably 4 to 12 hours (specifically 4 hours, 6 hours, 8 hours, 10 hours, or 12 hours). In this invention, after the substitution reaction, post-treatment is also preferred, which preferably includes: separation of the crude product by high-performance liquid chromatography (HPLC) column (C18 reversed-phase column, 250 × 10 mm). 2Thermo Scientific; Mobile phase: A: ultrapure water (containing 0.1% trifluoroacetic acid), D: acetonitrile (containing 0.1% trifluoroacetic acid); Mobile phase conditions: 5-95% D, 20 min, 3 mL / min); The separated product was lyophilized.

[0055] The present invention also provides a radionuclide labeling probe, comprising a compound with the structure shown in Formula III or a compound with the structure shown in Formula IV; Formula III; Formula IV; Wherein, R1, R2, R3, R4, R5, X1, X2, X3 are the same as R1, R2, R3, R4, R5, X1, X2, X3 in the structural compound of Formula I and the structural compound of Formula II; Q is a radionuclide labeling group, obtained by coordinating the Z labeling group in the CXCR2 targeting compound prepared by the above-described technical solution or the preparation method described in the above-described technical solution with a radionuclide.

[0056] In this invention, the radionuclide preferably includes 18 F, 43 Sc、 44 Sc、 47 Sc、 51 Cr 55 Co、 57 Co、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 72 As、 72 Se、 89 Zr、 86 Y、 89 Sr、 90 Y、 97 Ru、 99m Tc, 105 Rh、 101m Rh、 103 Pd, 109 Pd, 111 In、 119 Sb、 128 Ba、 139 La、 140 La、 142 Pr、 149 Pm, 149 Tb, 151 Tb, 151 Eu、 153 Eu、 169Eu、 153 Sm、 152 Gd, 153 Gd, 157 Gd, 159 Gd, 161 Tb, 165 Dy、 166 Ho、 169 Er、 175 Yb、 177 Lu、 186 Re、 188 Re、 197 Hg, 198 Au、 201 Tl、 203 Pb, 211 At、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 227 Th and 225 At least one of Ac.

[0057] This invention also provides a method for preparing the radionuclide-labeled probe described in the above technical solution, comprising the following steps: The CXCR2 targeting compound described in the above technical solution or the CXCR2 targeting compound prepared by the preparation method described in the above technical solution and the radionuclide are prepared by wet labeling method or freeze-drying labeling method.

[0058] In this invention, the wet labeling method preferably includes: mixing a solution containing a precursor compound with a radionuclide solution, performing a coordination reaction, and then diluting to obtain a reaction solution containing a radionuclide-labeled probe; the precursor compound is the CXCR2-targeting compound described in the above technical solution or a pharmaceutically acceptable form of the CXCR2-targeting compound described in the above technical solution.

[0059] The present invention does not specifically limit the solvent in the solution containing the precursor compound; any solvent well known to those skilled in the art can be used, such as a buffer solution, water, and one or more organic solvents. The buffer solution preferably includes PBS, acetate-acetate solution, or aluminum chloride-acetate solution; the organic solvent preferably includes one or more of acetonitrile, ethanol, DMSO, and DMF. In the present invention, when the nuclide is... 18 When F, the solvent preferably includes an acetic acid-acetate solution or an aluminum chloride-acetate solution; when the nuclide is 68 Ga or 177In the present invention, the solvent preferably comprises an acetic acid-acetate solution. In this invention, the concentration of the solution containing the precursor compound is preferably 0.001~1000 mg / mL, more preferably 0.01~1 mg / mL. In this invention, the mass ratio of the precursor compound to the radioactivity of the radionuclide in the radionuclide solution is preferably 20~400 µg: 1 kBq~1000 GBq, more preferably 20~400 µg: 0.037~74000 MBq, and even more preferably 20~200 µg: 0.037~7400 MBq. This invention does not have a particular limitation on the radionuclide solution; any radionuclide solution well known to those skilled in the art can be used, specifically as follows: 68 GaCl3 hydrochloric acid solution, 18 F-solution, 177 LuCl3 solution or Na 99m TcO4 eluent, the 68 The GaCl3 hydrochloric acid solution is preferably obtained by rinsing from a germanium-gallium generator, wherein the Na 99m The TcO4 eluent is preferably obtained by rinsing from a molybdenum technetium generator.

[0060] In this invention, the temperature of the coordination reaction is preferably 25-100°C, more preferably 80-100°C, and the time of the coordination reaction is preferably 10-60 min, more preferably 15-30 min. In this invention, when the temperature of the coordination reaction is higher than room temperature, after the coordination reaction, it is preferable to further cool the obtained coordination reaction system to room temperature, which can be diluted according to actual needs, and then filtered through a sterile membrane to obtain a radionuclide-labeled probe solution. This invention does not have any special limitations on the cooling; any cooling method well known to those skilled in the art can be used, such as natural cooling. In this invention, the dilution is preferably performed using physiological saline or water for injection. In this invention, the concentration of the radionuclide-labeled probe solution is preferably 0.037-3700 MBq / mL.

[0061] In this invention, when the nuclide is 18 In case F, the coordination reaction is preferably carried out in a mixed medium containing co-coordinate ions, a buffer solution, and an organic solvent; the co-coordinate ions are preferably metal ions, more preferably aluminum ions; the buffer solution is preferably an aluminum chloride-acetate solution; and the organic solvent is preferably acetonitrile.

[0062] In this invention, the lyophilization labeling method preferably includes the following steps: lyophilizing a solution containing the CXCR2 targeting compound and then sealing it to obtain a lyophilized kit; adding a solvent to the lyophilized kit for dissolution, then adding a radionuclide solution for coordination reaction and dilution to obtain a radionuclide-labeled probe solution. In this invention, the lyophilization preferably includes dispensing the solution containing the CXCR2 targeting compound into lyophilization containers before lyophilization; this invention does not specifically limit the lyophilization conditions, and lyophilization conditions well known to those skilled in the art can be used. In this invention, the relevant raw materials and condition parameters are preferably the same as those used in the wet labeling method, and will not be repeated here.

[0063] Preferably, the present invention adds pharmaceutically acceptable excipients to the injection solution containing the radionuclide-labeled probe obtained by the wet labeling method or the lyophilized medicine box obtained by the lyophilization labeling method as needed; the pharmaceutically acceptable excipients preferably include at least one of excipients, antioxidants, binders, buffers, colorants, diluents, disintegrants, emulsifiers, flavoring agents, flow aids, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents; the present invention does not have a special limitation on the types of the above-mentioned pharmaceutically acceptable excipients, and pharmaceutically acceptable excipients well known to those skilled in the art can be used.

[0064] In this invention, when the radiochemical purity of the radionuclide-labeled probe solution prepared by the wet labeling method or the lyophilization method is less than 95%, it is preferable to further purify the radionuclide-labeled probe solution. The purification preferably includes column separation purification, and the chromatographic column used for column separation purification preferably includes a Sep-Pak C18 separation column. The Sep-Pak C18 separation column is preferably activated and eluted sequentially with anhydrous ethanol and water before use. In this invention, the eluent used for column separation purification is preferably water and anhydrous ethanol sequentially. The eluent of anhydrous ethanol is collected and the solvent is removed, and then diluted to obtain a high-purity radionuclide-labeled probe solution. In this invention, the dilution is preferably performed using physiological saline or water for injection. In this invention, the radioactivity concentration of the high-purity radionuclide-labeled probe solution is preferably 0.037~3700 MBq / mL.

[0065] The preparation method provided by this invention has the advantages of simple and readily available labeling, good stability of the obtained radionuclide-labeled probe, and high tumor uptake, making it suitable for industrial production and clinical application.

[0066] The present invention also provides a pharmaceutically acceptable form of existence, including the pharmaceutically acceptable form of the CXCR2 targeting compound described in the above technical solutions and / or the pharmaceutically acceptable form of the radionuclide-labeled probe described in the above technical solutions, wherein the pharmaceutically acceptable form of existence includes one or more of salts, tautomers, stereoisomers, racemates, hydrates and solvates.

[0067] In this invention, the salt preferably includes trifluoroacetate, phosphate, formate, acetate, potassium salt, or sodium salt. This invention does not impose any particular limitation on the tautomer, racemate, hydrate, or solvate; any tautomer, racemate, hydrate, or solvate known to those skilled in the art can be used. This invention also does not impose any particular limitation on the preparation method of the tautomer, racemate, hydrate, solvate, or salt; any preparation method known to those skilled in the art can be used.

[0068] This invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients; the active ingredient comprises one or more of the CXCR2-targeting compounds described in the above-described technical solutions, the radionuclide-labeled probes described in the above-described technical solutions, and the pharmaceutically acceptable forms described in the above-described technical solutions. In this invention, the optional types of pharmaceutically acceptable excipients are the same as those described above, and will not be repeated here. In this invention, when the active ingredient comprises a radionuclide-labeled probe and / or a pharmaceutically acceptable form of a radionuclide-labeled probe, the dosage form of the pharmaceutical composition is preferably an injection, preferably administered intravenously; when the active ingredient does not contain a radionuclide-labeled probe and / or a pharmaceutically acceptable form of a radionuclide-labeled probe, this invention does not have special limitations on the dosage form and administration method of the pharmaceutical composition, and dosage forms and administration methods well known to those skilled in the art can be used.

[0069] The present invention also provides a lyophilized kit comprising a precursor and pharmaceutically acceptable excipients, wherein the precursor comprises one or more of the CXCR2-targeting compounds described in the above-described technical solutions and pharmaceutically acceptable forms of the CXCR2-targeting compounds described in the above-described technical solutions.

[0070] This invention also provides the use of the CXCR2-targeting compounds, radionuclide-labeled probes, pharmaceutically acceptable forms, pharmaceutical compositions, or lyophilized kits described in the above-described technical solutions in the preparation of therapeutic agents, therapeutic drugs, diagnostic reagents, or diagnostic drugs for CXCR2 protein-mediated diseases. In this invention, diagnostic methods preferably include single-photon emission computed tomography (SPECT) and positron emission tomography (PET); therapeutic methods preferably include radionuclide-targeted therapy, immunotherapy, and combinations thereof.

[0071] In this invention, the diseases mediated by the CXCR2 protein preferably include tumors or immune-related diseases; the tumors preferably include breast cancer, lung cancer, colorectal cancer, prostate cancer, bone and connective tissue sarcoma, renal cell carcinoma, gastric cancer, pancreatic cancer, nasopharyngeal carcinoma, head and neck cancer, neuroendocrine tumors, or skin melanoma; the immune-related diseases preferably include lupus erythematosus, atherosclerosis, rheumatoid arthritis, kidney injury, diabetes, hepatitis, autoimmune encephalomyelitis, scleroderma, psoriasis, Sjögren's syndrome, polyarteritis nodosa, or asthma.

[0072] The CXCR2-targeting compounds, pharmaceutically acceptable forms of the CXCR2-targeting compounds, and the radionuclide-labeled probes and pharmaceutically acceptable forms provided by this invention all possess excellent in vivo biological properties. They exhibit superior lesion uptake and long retention time in lesions with high CXCR2 expression, while being rapidly cleared and exhibiting low signal background in non-target organs such as normal tissues. Therefore, they have a high target / non-target ratio and have the potential for widespread clinical application.

[0073] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0074] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0075] Preparation Example 1 Synthesis of PD: 1) Synthesis of Compound 2 Diethyl squaric acid (starter 1, 1.89 g, 11.10 mmol) was dissolved in ethanol (EtOH, 7 mL). A solution of 3-amino-2-hydroxy-N,N-dimethylbenzamide (compound 1, 1 g, 5.55 mmol) (previously dissolved in 7 mL ethanol) was slowly added under ice bath conditions. The reaction was carried out at room temperature for 12 h until compound 1 was completely reacted. A solid precipitated at this point. The precipitated solid was filtered, washed three times with petroleum ether, and the filter cake was collected and dried at 45 °C to give crude compound 2 (1.2 g, 3.94 mmol). 1 H NMR (400 MHz, Chloroform- d ) δ 7.88 (s, 1H), 7.81 (s, 1H), 7.13 (d, J = 8.0 Hz, 1H), 6.91 (t, J = 8.3 Hz, 1H), 4.89 (q, J = 7.3 Hz, 2H), 3.21 (d, J = 2.2 Hz, 6H), 1.54 (dd, J =14.3, 2.2 Hz, 3H).

[0076] 2) Synthesis of compound 3 Compound 2 (1.2 g, 3.94 mmol) obtained from the reaction and starting material 2 (tert-butyl 4-aminopiperidine-1-carboxylate, 789.80 mg, 3.94 mmol) were dissolved in ethanol and reacted at room temperature for 6 hours until the reaction was complete. The ethanol was removed by rotary evaporation. The mixture was extracted three times with water and ethyl acetate, and the organic phase was collected, washed with saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The concentrated crude product was subjected to column chromatography (dichloromethane:methanol = 1:0-50:1) to give compound 3 (1 g, 2.18 mmol). 1 H NMR (400 MHz, Chloroform- d ) δ 7.88 (s, 1H), 7.81 (s, 1H), 7.13(d, J = 8.0 Hz, 1H), 6.91 (t, J = 8.3 Hz, 1H), 4.89 (q, J = 7.3 Hz, 2H), 3.21 (d, J =2.2 Hz, 6H), 1.54 (dd, J = 14.3, 2.2 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d ) δ183.85, 181.00, 171.35, 169.20, 163.62, 154.92, 147.79, 128.30, 122.88 (d, J =19.2 Hz), 119.15, 117.83, 80.13.

[0077] 3) Synthesis of compound 4 The obtained compound 3 (1 g, 2.18 mmol) was dissolved in 2 mL of ethyl acetate, and then ethyl acetate hydrochloride solution (4 M HCl) was added. . The reaction was carried out at room temperature for 2 hours with 1.5 mL of ethyl acetate (6 mmol) until complete. The solid was filtered and washed three times each with ethyl acetate and dichloromethane. The filter cake was collected and dried at 45 °C to give crude compound 4 (700 mg, 1.95 mmol). 1 H NMR (400 MHz, DMSO- d 6) δ 9.61 (s, 1H), 9.16 – 9.02 (m, 1H), 8.95 (d, J = 11.1 Hz, 1H), 8.84(d, J = 7.8 Hz, 1H), 7.81 – 7.68 (m, 1H), 6.95 – 6.82 (m, 2H), 4.21 (ddt, J =13.6, 9.2, 4.9 Hz, 1H), 3.39 – 3.27 (m, 2H), 3.06 (q, J = 11.0 Hz, 2H), 2.95(s, 6H), 2.19 – 2.08 (m, 2H), 1.91 – 1.78 (m, 2H).

[0078] 4) Synthesis of PD Compound 4 (3 mg, 8.37 μmol) was dissolved in 400 μL of dimethyl sulfoxide (DMSO), and DIPEA (5.41 mg, 41.85 μmol) and DOTA-activated ester (starting material 3, 8.40 mg, 16.74 μmol) were added sequentially. The reaction was carried out at room temperature for 12 hours. The crude product was separated by high performance liquid chromatography (HPLC) (C18 reversed-phase column, 250 × 10 mm). 2Thermo Scientific; Mobile phase: A: ultrapure water (containing 0.1% trifluoroacetic acid), D: acetonitrile (containing 0.1% trifluoroacetic acid); Mobile phase conditions: 5-95% D, 20 min, 3 mL / min). The separated product was lyophilized to obtain PD white solid powder (2.5 mg, 3.36 μmol). MS (ESI) m / z: calcd for C 34 H 49 N8O 11 + [M + H] + , 745.3516; found, 745.3475, see Figure 1 and Figure 2 .

[0079] Preparation Example 2 Synthesis of PP2D: Specifically, the following steps are included: 1) Synthesis of compound 5 Starting material 4 (2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatetradecane-14-acid, 46 mg, 167.41 μmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 64 mg, 167.41 μmol), and N,N-diisopropylethylamine (DIPEA, 54 mg, 418 μmol) were dissolved in anhydrous acetonitrile (ACN, 3 mL). After stirring at room temperature for 0.5 hours, compound 4 (50 mg, 139.51 μmol) was added, and the reaction was continued at room temperature for 4 hours. After the reaction was complete, the reaction was quenched with ultrapure water, and the acetonitrile was removed by rotary evaporation. The mixture was extracted three times with ethyl acetate, and the organic phase was collected. The solution was dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The concentrated crude product was subjected to column chromatography (dichloromethane:methanol = 1:0-20:1) to obtain compound 5 (60 mg, 97.13 μmol). 1 H NMR (400 MHz, Chloroform- d ) δ 8.65 (s, 1H), 8.05 (d, J = 8.3 Hz, 1H), 8.00 (dd, J = 8.2, 1.4 Hz, 1H), 7.00 (dd, J = 7.9, 1.5 Hz, 1H), 6.89 (t, J = 8.0 Hz, 1H), 4.41 (dd, J= 62.8, 11.4 Hz, 2H), 3.95 – 3.80 (m,2H), 3.72 (dt, J = 9.4, 5.1 Hz, 1H), 3.57 (h, J = 5.0, 4.6 Hz, 5H), 3.46 (t, J =5.3 Hz, 2H), 3.22 (d, J = 13.2 Hz, 4H), 3.14 (s, 6H), 2.86 – 2.72 (m, 2H), 2.55(dt, J = 15.6, 5.1 Hz, 1H), 2.16 – 1.91 (m, 2H), 1.56 (d, J = 12.6 Hz, 1H), 1.44(s, 1H), 1.42 (s, 10H), 1.37 – 1.30 (m, 1H), 1.26 (d, J = 12.4 Hz, 2H).

[0080] 2) Synthesis of Compound 6 The obtained compound 5 (60 mg, 97.13 μmol) was dissolved in 300 μL of ethyl acetate, and then ethyl acetate hydrochloride solution (4 M HCl) was added. . The reaction was carried out at room temperature for 2 hours with 0.5 mL of ethyl acetate (2 mmol) until complete. The solid was filtered and washed three times each with ethyl acetate and dichloromethane. The filter cake was collected and dried at 45 °C to give crude compound 6 (40 mg, 77.3 μmol). 1 H NMR (600MHz, DMSO- d 6) δ 10.02 (s, 1H), 9.30 (s, 1H), 8.45 (d, J = 7.6 Hz, 1H), 7.84 (s,2H), 7.79 – 7.74 (m, 1H), 6.94 – 6.81 (m, 2H), 4.25 (dt, J = 13.9, 4.5 Hz, 1H),4.12 (ddt, J = 14.4, 10.8, 5.4 Hz, 1H), 3.87 (dt, J = 14.4, 4.6 Hz, 1H), 3.65(td, J = 6.5, 4.5 Hz, 2H), 3.59 (t, J= 5.3 Hz, 2H), 3.56 (tt, J = 4.7, 2.6 Hz,4H), 3.21 – 3.14 (m, 1H), 2.99 (t, J = 5.4 Hz, 2H), 2.95 (d, J = 15.5 Hz, 6H), 2.84 (t, J = 12.2 Hz, 1H), 2.62 (td, J = 6.6, 2.8 Hz, 2H), 1.98 (ddd, J = 26.4,13.1, 4.3 Hz, 2H), 1.49 (qd, J = 11.1, 4.1 Hz, 1H), 1.37 (qd, J = 11.2, 4.2 Hz, 1H).

[0081] 3) Synthesis of compound PP2D Compound 6 (2 mg, 3.86 μmol) was dissolved in 400 μL of dimethyl sulfoxide (DMSO), and DIPEA (2.5 mg, 19.32 μmol) and DOTA-activated ester (starting material 3, 3.88 mg, 7.73 μmol) were added sequentially. The reaction was carried out at room temperature for 12 hours. The crude product was separated by high-performance liquid chromatography (HPLC) (column type: C18 reversed-phase column, 250 × 10 mm). 2 Thermo Scientific; Mobile phase: A: ultrapure water (containing 0.1% trifluoroacetic acid), D: acetonitrile (containing 0.1% trifluoroacetic acid); Mobile phase conditions: 5-95% D, 20 min, 3 mL / min). The separated product was lyophilized to obtain PP2D white solid powder (1.5 mg, 1.66 μmol). MS (ESI) m / z: calcd for C 41 H 62 N9O 14 + [M + H] + , 904.4411; found, 904.4348; calcd forC 41 H 61 N9O 14 Na + [M + Na] + , 926.4230; found, 926.4162, see Figure 3 and Figure 4 .

[0082] Preparation Example 3 Synthesis of PP2N: Compound 6 (2 mg, 3.86 μmol) was dissolved in 400 μL of dimethyl sulfoxide (DMSO), and DIPEA (2.5 mg, 19.32 μmol) and activated Nota ester (starting material 5, 3.09 mg, 7.73 μmol) were added sequentially. The reaction was carried out at room temperature for 12 hours. The crude product was separated by high performance liquid chromatography (HPLC) (C18 reversed-phase column, 250 × 10 mm). 2 Thermo Scientific; Mobile phase: A: ultrapure water (containing 0.1% trifluoroacetic acid), D: acetonitrile (containing 0.1% trifluoroacetic acid); Mobile phase conditions: 5-95% D, 20 min, 3 mL / min). The separated product was lyophilized to obtain PP2N white solid powder (2.1 mg, 2.32 μmol). MS (ESI) m / z: calcd for C 37 H 55 N8O 12 + [M + H] + , 803.3934; found, 803.3997, see Figure 5 and Figure 6 .

[0083] Preparation Example 4 Synthesis of PP2SD Specifically, the following steps are included: Compound 6 (4 mg, 7.73 μmol) was dissolved in dimethyl sulfoxide (DMSO, 500 μL), and then SCN-DOTA activated ester (starting material 6, 9.62 mg, 15.46 μmol) and N,N-diisopropylethylamine (DIPEA, 5 mg, 38.64 μmol) were added sequentially. The mixture was stirred at room temperature for 12 hours. The crude product was separated by high performance liquid chromatography (HPLC) (C18 reversed-phase column, 250 × 10 mm). 2 Thermo Scientific; Mobile phase: A: ultrapure water (containing 0.1% trifluoroacetic acid), D: acetonitrile (containing 0.1% trifluoroacetic acid); Mobile phase conditions: 5-95% D, 20 min, 3 mL / min). The separated product was lyophilized to obtain PP2SD white solid powder (2.5 mg, 2.19 μmol). MS (ESI) m / z: calcd for C 52 H 74 N 11 O 16 S + [M + H]+ , 1140.5030; found, 1140.4982, see Figure 7 and Figure 8 .

[0084] Preparation Example 5 Synthesis of PP4D: Specifically, the following steps are included: 1) Synthesis of compound 7 2,2-Dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaeicosano-20-acid (starting material 7, 123 mg, 334.82 μmol), HATU (127.31 mg, 334.82 μmol), and DIPEA (108 mg, 837.06 μmol) were dissolved in 3 mL of acetonitrile. After stirring at room temperature for 0.5 hours, compound 4 (100 mg, 279.02 μmol) was added, and the reaction was continued for 6 hours. After the reaction was completed, water was added to quench the reaction. Acetonitrile was evaporated using a rotary evaporator. The mixture was extracted three times with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was purified by rotary evaporation under reduced pressure and column chromatography (DCM:MeOH = 1:0-20:1) to give compound 7 (100 mg, 141.68 μmol). 1 H NMR (400 MHz, Chloroform- d δ 8.54 (s, 1H), 8.10 (dd, J = 8.1, 1.4 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.05 (dd, J = 7.9, 1.4 Hz, 1H), 6.93 (t, J = 8.0 Hz, 1H), 4.61 (d, J = 13.4 Hz, 1H), 4.36 (d, J = 8.9 Hz, 1H), 4.01 (d, J = 13.7 Hz, 1H), 3.88 (td, J = 9.2, 4.1 Hz, 1H), 3.76 (d, J = 5.4 Hz, 1H), 3.72 (dq, J = 5.7,3.2, 2.8 Hz, 2H), 3.70 – 3.67 (m, 2H), 3.66 (s, 1H), 3.65 (s, 2H), 3.64 (d,J =3.6 Hz, 4H), 3.58 (td, J = 7.7, 4.1 Hz, 2H), 3.53 (t, J = 5.4 Hz, 2H), 3.27 (q, J =5.5 Hz, 2H), 3.20 (s, 6H), 2.90 (ddd, J = 14.7, 9.4, 5.2 Hz, 1H), 2.77 (t, J =12.2 Hz, 1H), 2.51 (dt, J = 15.0, 4.4 Hz, 1H), 2.07 (dd, J = 34.4, 12.5 Hz, 2H),1.73 (qd, J = 11.8, 4.0 Hz, 1H), 1.47 (s, 1H), 1.44 (s, 9H).

[0085] 2) Synthesis of Compound 8 The obtained compound 7 (100 mg, 141.68 μmol) was dissolved in 300 μL of ethyl acetate, and then ethyl hydrochloride solution (2 M HCl) was added. . The reaction was carried out at room temperature for 2 hours with 0.5 mL of ethyl acetate (EA) until complete. The solid was filtered and washed three times each with ethyl acetate and dichloromethane. The filter cake was collected and dried at 45 °C to give crude compound 8 (60 mg, 99.04 μmol). 1 H NMR (400MHz, DMSO- d 6) δ 9.81 (s, 1H), 8.85 (d, J = 7.8 Hz, 1H), 8.05 (s, 2H), 7.76 –7.68 (m, 1H), 6.94 – 6.82 (m, 2H), 4.21 – 4.10 (m, 2H), 3.89 (d, J = 14.1 Hz,1H), 3.67 – 3.62 (m, 4H), 3.58 (s, 4H), 3.54 (d, J = 4.4 Hz, 10H), 3.24 (t, J =12.0 Hz, 1H), 2.98 (d, J = 5.5 Hz, 2H), 2.96 (s, 2H), 2.94 (s, 6H), 2.63 (td,J =6.6, 3.4 Hz, 2H), 1.96 (d, J = 17.6 Hz, 2H), 1.59 – 1.34 (m, 2H).

[0086] 3) Synthesis of compound PP4D Compound 8 (4 mg, 6.60 μmol) was dissolved in 400 μL of dimethyl sulfoxide (DMSO), and DIPEA (4.27 mg, 33.02 μmol) and DOTA-activated ester (starting material 3, 6.62 mg, 13.21 μmol) were added sequentially. The reaction was carried out at room temperature for 12 hours. The crude product was separated by high performance liquid chromatography (HPLC) (column type: C18 reversed-phase column, 250 × 10 mm). 2 Thermo Scientific; Mobile phase: A: ultrapure water (containing 0.1% trifluoroacetic acid), D: acetonitrile (containing 0.1% trifluoroacetic acid); Mobile phase conditions: 5-95% D, 20 min, 3 mL / min). The separated product was lyophilized to obtain PP4D white solid powder (2.5 mg, 2.52 μmol). MS (ESI) m / z: calcd for C 45 H 70 N9O 16 + [M + H] + , 992.4935; found, 992.4875; calcd for[M + 2H] 2+ , 496.7504; found, 496.7476, see Figure 9 and Figure 10 .

[0087] Preparation Example 6 Synthesis of PP4SD: Specifically, the following steps are included: Compound 8 (4 mg, 6.60 μmol) was dissolved in dimethyl sulfoxide (DMSO, 400 μL), and then SCN-DOTA activated ester (starting material 6, 8.22 mg, 13.21 μmol) and N,N-diisopropylethylamine (DIPEA, 4.27 mg, 33.02 μmol) were added sequentially. The mixture was stirred at room temperature for 12 hours. The crude product was separated by high performance liquid chromatography (HPLC) (column type: C18 reversed-phase column, 250 × 10 mm). 2Thermo Scientific; Mobile phase: A: ultrapure water (containing 0.1% trifluoroacetic acid), D: acetonitrile (containing 0.1% trifluoroacetic acid); Mobile phase conditions: 5-95% D, 20 min, 3 mL / min). The separated product was lyophilized to obtain PP4SD white solid powder (2 mg, 1.63 μmol). MS (ESI) m / z: calcd for C 56 H 82 N 11 O 18 S + [M + H] + , 1228.5555; found,1228.5483; calcd for [M + 2H] 2+ , 614.7814; found, 614.7778, see Figure 11 and Figure 12 .

[0088] Preparation Example 7 Synthesis of PP4SN: Specifically, the following steps are included: Compound 8 (3 mg, 4.95 μmol) was dissolved in dimethyl sulfoxide (DMSO, 400 μL), and then SCN-NOTA activated ester (starting material 7, 4.46 mg, 9.91 μmol) and N,N-diisopropylethylamine (DIPEA, 3.20 mg, 24.77 μmol) were added sequentially. The mixture was stirred at room temperature for 12 hours. The crude product was separated by high performance liquid chromatography (HPLC) (column type: C18 reversed-phase column, 250 × 10 mm). 2 Thermo Scientific; Mobile phase: A: ultrapure water (containing 0.1% trifluoroacetic acid), D: acetonitrile (containing 0.1% trifluoroacetic acid); Mobile phase conditions: 5-95% D, 20 min, 3 mL / min). The separated product was lyophilized to obtain PP4SN white solid powder (2 mg, 1.63 μmol). MS (ESI) m / z: calcd for C 49 H 70 N9O 15 S + [M + H] + , 1056.4704; found, 1056.4907, see Figure 13 and Figure 14 .

[0089] Preparation Example 8 Synthesis of Amyl-PP4D: Specifically, the following steps are included: 1) Synthesis of compound 13 Diethyl squaric acid (raw material 1, 440 mg, 2.59 mmol) was dissolved in ethanol (7 mL), and tert-butyl(2-(3-amino-2-hydroxy-)) was slowly added under ice bath conditions. N A solution of methylbenzamide ethyl carbamate (compound 12, 400 mg, 1.29 mmol) (previously dissolved in 7 mL of ethanol) was reacted at room temperature for 12 h until compound 12 was completely reacted. The ethanol was removed by rotary evaporation, followed by extraction three times with ethyl acetate and water. The organic phase was collected, washed once with saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The concentrated crude product was subjected to column chromatography (dichloromethane:methanol = 1:0-50:1) to give compound 13 (0.5 g, 1.15 mmol).

[0090] 2) Synthesis of compound 14 The obtained compound 13 (0.5 g, 1.15 mmol) was dissolved in 2 mL of ethyl acetate, and then ethyl acetate hydrochloride solution (4 M HCl) was added. . The mixture was reacted with ethyl acetate (2 mL, 8 mmol) at room temperature for 2 hours until complete. The solid was filtered and washed three times each with ethyl acetate and dichloromethane. The filter cake was collected and dried at 45 °C to give crude compound 14 (380 mg, 1.14 mmol).

[0091] 3) Synthesis of Compound 15 Starting material 7 (625 mg, 1.71 mmol), HATU (867 mg, 2.28 mmol), and DIPEA (295 mg, 2.28 mmol) were dissolved in 5 mL of DMF. After stirring at room temperature for 0.5 hours, compound 14 (380 mg, 1.14 mmol) was added, and the reaction was continued for 6 hours. After the reaction was completed, water was added to quench the reaction. The DMF was evaporated using a rotary evaporator. The mixture was extracted three times with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was purified by rotary evaporation under reduced pressure and column chromatography (DCM:MeOH = 1:0-20:1) to give compound 15 (700 mg, 1.03 mmol).

[0092] 4) Synthesis of Compound 16 Compound 15 (700 mg, 1.03 mmol) and starting material 10 (pentane-3-amine, 108 mg, 1.23 mmol) were dissolved in ethanol, and DIPEA (27 mg, 0.21 mmol) was added. The mixture was reacted overnight at room temperature. The ethanol was removed by rotary evaporation. The mixture was extracted three times with water and ethyl acetate, and the organic phase was collected, washed with saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The concentrated crude product was subjected to column chromatography (dichloromethane:methanol = 1:0-50:1) to obtain compound 16 (500 mg, 693 μmol).

[0093] 5) Synthesis of Compound 17 The obtained compound 16 (0.5 g, 693 μmol) was dissolved in 2 mL of ethyl acetate, and then ethyl hydrochloride solution (4 M HCl) was added. . The mixture was reacted with ethyl acetate (1.5 mL, 6 mmol) at room temperature for 2 hours until complete. The solid was filtered and washed three times each with ethyl acetate and dichloromethane. The filter cake was collected and dried at 45 °C to give crude compound 17 (400 mg, 643 μmol).

[0094] 6) Synthesis of Amyl-PP4D Compound 17 (5 mg, 8.04 μmol) was dissolved in 400 μL of dimethyl sulfoxide (DMSO), and DIPEA (5.20 mg, 40.21 μmol) and DOTA-activated ester (starting material 3, 8.07 mg, 16.08 μmol) were added sequentially. The reaction was carried out at room temperature for 12 hours. The crude product was separated by high-performance liquid chromatography (HPLC) (column type: C18 reversed-phase column, 250 × 10 mm). 2 Thermo Scientific; Mobile phase: A: ultrapure water (containing 0.1% trifluoroacetic acid), D: acetonitrile (containing 0.1% trifluoroacetic acid); Mobile phase conditions: 5-95% D, 20 min, 3 mL / min). The separated product was lyophilized to obtain Amyl-PP4D white solid powder (3.5 mg, 3.47 μmol). MS (ESI) m / z: calcd for C 46 H 74 N9O 16 + [M + H] + , 1008.5248; found, 1008.5400, see Figure 15 and Figure 16 .

[0095] Preparation Example 9 Py-PP4D synthesis: Py-PP4D Synthesis Compound 18 (5 mg, 8.24 μmol) was dissolved in 400 μL of dimethyl sulfoxide (DMSO), followed by the sequential addition of DIPEA (5.33 mg, 41.21 μmol) and DOTA-activated ester (starting material 3, 8.27 mg, 16.48 μmol). The reaction was carried out at room temperature for 12 hours. The crude product was separated by high-performance liquid chromatography (HPLC) (C18 reversed-phase column, 250 × 10 mm). 2 Thermo Scientific; Mobile phase: A: ultrapure water (containing 0.1% trifluoroacetic acid), D: acetonitrile (containing 0.1% trifluoroacetic acid); Mobile phase conditions: 5-95% D, 20 min, 3 mL / min). The separated product was lyophilized to obtain Py-PP4D white solid powder (3.5 mg, 3.47 μmol). MS (ESI) m / z: calcd for C 44 H 69 N 10 O 16 + [M + H] + , 993.4888.; found, 993.4975, see Figure 17 and Figure 18 .

[0096] Preparation Example 10: 68 The Ga nuclide labeling process is as follows: by[ 68 Taking Ga-PP4D as an example: Wet method: to 68 Ga hydrochloric acid solution (740 MBq, rinsed from the germanium-gallium generator) was added to a centrifuge tube containing 2 mL of acetate-sodium acetate buffer solution of compound PP4D (50 µg) from Preparation Example 5. The mixture was reacted at 95 °C for 15 minutes and then cooled to room temperature. After dilution with physiological saline or ultrapure water, the mixture was sterilely filtered to obtain the labeled compound injection solution.

[0097] Freeze-drying method: 68 A Ga hydrochloric acid solution (740 MBq, rinsed from the germanium-gallium generator) and a certain amount of acetate-sodium acetate buffer solution were mixed evenly and added to a lyophilized kit containing compound PP4D (50 µg). The mixture was reacted at 95°C for 15 minutes, diluted with physiological saline or ultrapure water, and then sterile filtered to obtain the labeled compound injection solution.

[0098] For the labeled compound [ 68 Ga]Ga-PP4D samples were taken for HPLC analysis and identification. The HPLC system was as follows: Column type: C18 reversed-phase column, 250×4.6mm 2Thermo Scientific; Mobile phase: A: ultrapure water (containing 0.1% trifluoroacetic acid), D: acetonitrile (containing 0.1% trifluoroacetic acid); Mobile phase conditions: 5-95% D, 20 min, 1 mL / min). The retention time of the radiolabeled compound was approximately 10.72 minutes, and based on this, the radiochemical purity was calculated to be greater than 95%. Figure 19 .

[0099] Preparation Examples 11-16: According to Preparation Example 10 [ 68 The method for preparing Ga]Ga-PP4D involves replacing PP4D with: PD prepared in Preparation Example 1; PP2D prepared in Preparation Example 2; PP2SD prepared in Preparation Example 4; PP4SD prepared in Preparation Example 6; Amyl-PP4D prepared in Preparation Example 8; Py-PP4D prepared in Preparation Example 9; They were obtained respectively 68 Ga nuclide-labeled compounds [ 68 Ga]Ga-PD (Preparation Example 11), [ 68 Ga]Ga-PP2D (Preparation Example 12), [ 68 Ga]Ga-PP2SD (Preparation Example 13), [ 68 Ga]Ga-PP4SD (Preparation Example 14), [ 68 Ga]Ga-Amyl-PP4D (Preparation Example 15) and [ 68 Ga]Ga-Py-PP4D (Preparation Example 16). The radio-HPLC results of the radionuclide-labeled complexes prepared in Preparation Examples 11-16 are shown below. Figures 19-20 It can be seen that the synthesized by this invention 68 The radiochemical purity of Ga-labeled compound probes is greater than 95%.

[0100] Preparation Example 17: 18 The F nuclide labeling process is as follows: by[ 18 Taking F]AlF-PP4SN as an example 740MBq 18 F -The solution, consisting of 10 μL of 2 mM AlCl3 sodium acetate solution (0.1 M, pH=4.0), compound PP4SN (50 µg) from Preparation Example 7, and 200 µL of acetonitrile, was added to a vial and shaken at 100 °C for 20 minutes. A Sep-Pak C18 separation column was activated by rinsing with 10 mL of anhydrous ethanol and 10 mL of ultrapure water, respectively. The reaction solution was diluted with approximately 10 mL of ultrapure water before being loaded onto the C18 separation column. The column was washed with 20 mL of ultrapure water to remove unreacted components. 18 F ions were then used to rinse the labeled compound into a vial with 1.5 mL of ethanol. The organic solvent was removed by nitrogen evaporation at 60°C, and the solution was diluted with physiological saline or ultrapure water and then aseptically filtered to obtain the labeled compound injection solution.

[0101] For the labeled compound [ 18 F]AlF-PP4SN samples were taken for HPLC analysis and identification. The HPLC system is as follows: Column type: C18 reversed-phase column, 250×4.6mm 2 Thermo Scientific; Mobile phase: A: ultrapure water (containing 0.1% trifluoroacetic acid), D: acetonitrile (containing 0.1% trifluoroacetic acid); Mobile phase conditions: 5-95% D, 20 min, 1 mL / min). The retention time of the radiolabeled compound was approximately 11.52 minutes, and based on this, the radiochemical purity was calculated to be greater than 95%. Figure 20 .

[0102] Preparation Example 18: [ 18 Preparation of F]AlF-PP2N According to Preparation Example 17 [ 18 The method for preparing F]AlF-PP4SN was used, in which compound PP4SN was replaced with PP2N prepared in Preparation Example 3, to obtain 18 F-labeled compounds [ 18 F]AlF-PP2N. Radio-HPLC results are shown in […]. Figure 20 It can be seen that the [synthetic product] synthesized in this invention 18 The radiochemical purity of F]AlF-PP2N is greater than 95%.

[0103] Test Example 1 1. Stability test: Stability in physiological saline: The labeled compounds were incubated in physiological saline at room temperature, and samples were taken at the measured time points (0.5 h, 1 h, and 2 h) for HPLC analysis and identification. The radiochemical purity of each probe was >95%, indicating that the probes were stable in physiological saline and not easily decomposed. 68 Ga]Ga-PP4D (Preparation Example 10), [ 68The HPLC results for the in vitro stability of Ga]Ga-PP4SD (Preparation Example 14) in physiological saline are as follows: Figure 21 As shown in A and 21B, it maintains high stability (>95%) in physiological saline system up to 2 hours.

[0104] Serum stability: The labeled compounds were incubated with serum at room temperature for different times (0.5 h, 1 h, and 2 h). Acetonitrile was added to denature the proteins, and the supernatant was collected by centrifugation and analyzed by HPLC. At the tested time points, the radiochemical purity of each probe remained >95%, indicating that they were stable and not easily decomposed in the specified solution. 68 Ga]Ga-PP4D and [ 68 The in vitro serum stability of Ga]Ga-PP4SD was determined by HPLC as follows: Figure 21 As shown in C and 21D, it maintains high stability (>95%) in the serum system for 2 hours.

[0105] 2. Lipid-water distribution coefficient (Log) P pH=7.4 ) Measurement A portion of the synthesized radiolabeled compound was diluted to 100 µL with phosphate-buffered saline (PBS, pH 7.4), and then added to a centrifuge tube containing 0.9 mL of PBS and 1 mL of n-octanol mixture. The mixture was vortexed for 3 minutes, centrifuged at 12000 rpm for 5 minutes, and 100 µL each from the aqueous and n-octanol phases were taken for radioactivity counting using a gamma counter. The experiment was repeated three times, and the average value was taken. The formula for calculating Log P is: P=I 有机相 / I 水相 Where I 有机相 Represents the radioactivity count measured in the organic phase, I 水相 The radioactivity counts measured in the aqueous phase are represented. The lipid-water distribution coefficients of each radiolabeled target probe were calculated. The results are shown in Table 1 below; the labeled compounds tested exhibit water solubility.

[0106] Table 1

[0107] The experimental results show that 68 Ga-labeled probes are hydrophilic. By linking radioactive metal chelating groups to the parent structure using connectives of varying lengths and polarities, the logarithm of the probe can be appropriately adjusted. P This allows probes with different structures to possess different pharmacokinetic properties, making them suitable for different application scenarios.

[0108] Test Example 2 Micro-PET imaging of normal mice: Prepared according to the preparation example 68 A Ga-labeled compound solution, 0.2 mL (approximately 7.4 MBq), was injected via the tail vein of normal female mice. Micro-PET imaging was performed at 0.5 hours and 1 hour, and regions of interest (ROIs) were delineated on the obtained whole-body decay-corrected images to calculate the probe's in vivo metabolism. Figure 22 As shown, the present invention 68 Ga-labeled probes [ 68 Ga]Ga-PP4D and [ 68 In normal mice, Ga-PP4SD is primarily metabolized via the kidneys. The probe is essentially metabolized in normal mice within one hour.

[0109] Test Example 3 PET imaging of mice bearing melanoma B16F10 model: Validation in a murine melanoma B16F10 model [ 68 Ga]Ga-PP2D, [ 68 Ga]Ga-PP2SD, [ 68 Ga]Ga-PP4D and [ 68 The targeting ability of Ga]Ga-PP4SD to tumors. 0.2 mL of freshly prepared radiocomplex (approximately 7.4 MBq) was injected via the tail vein into male black mice bearing B16F10 tumors. Micro-PET imaging was performed at 0.5 hours, 1 hour, and 2 hours, and regions of interest (ROIs) were delineated on the obtained whole-body decay-corrected images. Figure 23 As shown, [ 68 Ga]Ga-PP2D and [ 68 Ga-PP2SD reached peak uptake at the tumor site half an hour after tail vein injection (circled area indicates tumor location), with high radioactive signals also observed at the bladder and kidney sites. 68 Ga]Ga-PP4D and [ 68 Ga]Ga-PP4SD showed significantly increased uptake at the tumor site compared to [ 68 Ga]Ga-PP2D and [ 68 Ga]Ga-PP2SD, modified with polyethylene glycol chains, alters the probe's metabolic pathway, primarily through renal metabolism. The probe is rapidly cleared from normal organs such as muscle, heart, liver, and kidneys, exhibiting a high target-to-non-target ratio. Figure 24 and Figure 25 Showing [ 68 Ga]Ga-PP2D,[ 68 Ga]Ga-PP2SD, [68 Ga]Ga-PP4D, [ 68 The uptake values ​​and target / non-target ratios of Ga]Ga-PP4SD in vital organs of mice were measured. These data indicate that changes in the length and properties of the linker strand alter the metabolic pathways of the probe in a B16F10-bearing tumor model, and also result in altered uptake at the tumor site.

[0110] Test Example 4 PET imaging of a mouse model of pancreatic cancer PANC-02: Validation in a murine pancreatic cancer PANC-02 model [ 68 The targeting ability of Ga]Ga-PP4SD to tumors. Studies have shown that CXCR2 is highly expressed in PANC-02 cells. High expression of CXCR2 is associated with tumor progression and poor prognosis in pancreatic cancer (including the PANC-02 cell line). 0.2 mL of freshly prepared radiocomplex (approximately 7.4 MBq) was injected via the tail vein into male C57BL / 6 mice of the PANC-02 model. Micro-PET imaging was performed at 0.5 h, 1 h, and 1.5 h, and regions of interest (ROIs) were delineated on the obtained whole-body decay-corrected images. Figure 26 As shown, [ 68 Ga]Ga-PP4SD shows significant uptake at tumor sites and rapid clearance in normal tissues such as muscle, heart, and kidneys, with a high target-to-non-target ratio. Figure 27 show,[ 68 The target-to-muscle ratio of Ga-PP4SD reached approximately 6, and non-target sites were almost completely cleared one hour after probe injection. A high radioactive signal was observed in the bladder, indicating that the probe was metabolized by the kidneys and excreted in urine.

[0111] At the same time, we will further [ 18 F]AlF-PP4SN was used in PET imaging studies of the PANC-02 tumor-bearing mouse model. Figure 28 and Figure 29 show,[ 18 [F]AlF-PP4SN also showed high uptake at the tumor site and good tumor imaging performance. Compared to [ 68 Ga]Ga-PP4SD, [ 18 F]AlF-PP4SN exhibits a more persistent retention capacity at the tumor site, maintaining high tumor uptake and excellent target-to-non-target contrast even at delayed time points, suggesting superior in vivo pharmacokinetic characteristics and potential clinical application value.

[0112] Test Example 5 PET imaging of a mouse model of human lung cancer A549: To further evaluate the in vivo imaging capability of the CXCR2 targeting probe, an in vivo imaging study was conducted in a human lung cancer A549 tumor-bearing nude mouse model. 68 Ga]Ga-Py-PP4D Micro-PET Imaging Study. Previous studies have shown that CXCR2 is abnormally expressed in various tumors and is closely related to tumor invasion, metastasis, and poor prognosis.

[0113] 0.2 mL of freshly prepared radioactive complex (approximately 7.4 MBq) was injected via the tail vein into A549 tumor-bearing nude mice. Micro-PET imaging was performed at 0.5 h, 1 h, and 1.5 h post-injection, and the Region of Interest (ROI) was delineated on the decay-corrected whole-body images for quantitative analysis. The results showed that... Figure 30 As shown, [ 68 Ga]Ga-Py-PP4D exhibits significant uptake at tumor sites, while clearing is relatively rapid in normal tissues such as muscle, heart, and kidneys, demonstrating a high target-to-non-target ratio. Figure 31 Further evidence indicates that the probe achieves a high tumor / muscle ratio within 1 hour post-injection, and the radioactive signal in non-target tissues is significantly reduced, suggesting that the probe possesses favorable in vivo pharmacokinetic characteristics and high tumor-targeting ability. Additionally, a high radioactive signal was observed in the bladder region, indicating that the probe is primarily metabolized by the kidneys and excreted in urine.

[0114] Test Example 6 PET imaging of MC38 mouse model with colorectal cancer: To further evaluate the in vivo imaging capability of the CXCR2 targeting probe, an in vivo imaging study was conducted in an MC38 colon cancer-bearing mouse model. 68 Ga]Ga-Amyl-PP4D Micro-PET Imaging Study. Previous studies have shown that CXCR2 and its related chemokine axes are widely involved in neutrophil recruitment, inflammation regulation, and tumor progression in the MC38 tumor microenvironment, and therefore can serve as important targets for molecular imaging of the tumor microenvironment.

[0115] 0.2 mL of freshly prepared radioactive complex (approximately 7.4 MBq) was injected via the tail vein into MC38 tumor-bearing mice. Micro-PET imaging was performed at 0.5 h, 1 h, and 2 h post-injection, and ROIs were delineated on decay-corrected whole-body images for quantitative analysis. Results showed that... Figure 32 As shown, [ 68Ga-Amyl-PP4D rapidly accumulates at the tumor site, reaching peak uptake 0.5 hours post-injection. While the radioactive signal within the tumor gradually decreases over time, it maintains a clear and discernible imaging effect. Simultaneously, the probe is rapidly cleared from normal tissues such as muscle, heart, and kidneys, resulting in a sustained decrease in background radioactivity levels and demonstrating high contrast between tumor and non-target tissues.

[0116] Figure 33 This further demonstrates that, 68 Ga-Amyl-PP4D achieved a high tumor / muscle ratio early on, suggesting that the probe rapidly targets tumors and is quickly cleared from non-target tissues, exhibiting good in vivo pharmacokinetic properties and high tumor-targeting ability. Furthermore, high radioactive uptake was observed in the gallbladder region during later imaging, suggesting that in addition to renal excretion, the probe may also be metabolized in the hepatobiliary pathway. This result indicates that... 68 Ga]Ga-Amyl-PP4D possesses both excellent tumor imaging capabilities and high imaging contrast, providing a basis for its further research and application as a CXCR2-targeting molecular imaging probe.

[0117] Test Example 7 PET images of a mouse model of myocarditis: Validated in a mouse model of myocarditis induced by doxorubicin (DOX) 68 The targeting of inflammatory factors by Ga]Ga-PP4SD. 0.2 mL of freshly prepared radiocomplex (approximately 7.4 MBq) was injected via the tail vein into DOX-induced myocarditis-inducing male Balb / c mice and normal male Balb / c mice. Micro-PET imaging was performed at 0.5 hours, and regions of interest were delineated on the obtained whole-body decay-corrected images. Figure 34 and Figure 35 As shown, compared to normal Balb / c male rats, [ 68 In a mouse model of myocarditis, Ga-PP4SD was uptaken in the heart at a rate of approximately 2 compared to normal mice. It was also rapidly cleared from the heart and kidneys. The ratio of uptake in the heart to muscle reached approximately 3.

[0118] Test Example 8 PET images of a mouse model of gouty arthritis: Validated in a mouse model of gouty arthritis [ 68 Ga]Ga-PP2D,[ 68 Ga]Ga-PP2SD, [ 68 Ga]Ga-PP4D and [ 68The targeting of inflammatory factors by Ga]Ga-PP4SD. 0.2 mL of freshly prepared radiocomplex (approximately 7.4 MBq) was injected via the tail vein into male Balb / c rats with a gouty arthritis model. Micro-PET imaging was performed at 0.5 hours, and regions of interest were delineated on the resulting whole-body decay-corrected images. Figure 36 As shown, the four radioactive probes showed significant uptake at the right ankle joint after injection of sodium urate (MSU), and were rapidly cleared from normal tissues such as muscles, liver, and kidneys, with a high target-to-non-target ratio. Figure 37 show,[ 68 Ga]Ga-PP2SD and [ 68 The ratio of the right ankle joint to the left ankle joint of Ga]Ga-PP4SD reached about 2.7, and the non-target area was almost completely cleared 1 hour after probe injection.

[0119] Test Example 9 PET imaging of a mouse model of ischemia-reperfusion-acute kidney injury: Validated in a mouse model of acute kidney injury due to ischemia-reperfusion. 68 Ga]Ga-PP4SD and [ 68 The targeting of inflammatory factors by Ga]Ga-PP4D. 0.2 mL of freshly prepared radiocomplex (approximately 7.4 MBq) was injected via the tail vein into C57BL / 6 male mice with an acute kidney injury model. Micro-PET imaging was performed at 0.5 hours and 1 hour, and regions of interest were delineated on the obtained whole-body decay-corrected images. Figure 38 and Figure 39 As shown, compared to the normal left kidney, the probe showed higher uptake in the right kidney undergoing ischemia-reperfusion, with an uptake ratio of approximately 1.5–3 between the two kidneys. Clearance was rapid in sites such as muscle, heart, liver, and kidneys. It exhibited a good target-to-non-target ratio. [Among them...] 68 Ga]Ga-PP4SD showed high uptake in the kidneys of mice with acute kidney injury, approximately [ 68 Ga]Ga-PP4D is 3 times more potent than other drugs. Furthermore, the uptake of Ga-PP4D can be effectively blocked using the CXCR2 small molecule inhibitor Navarixin, with the left-right kidney ratio after blocking being approximately 1.1~1.2.

[0120] Test Case 10 PET images of a cisplatin-induced acute kidney injury model mouse: Validated in mice with acute kidney injury induced by different doses of cisplatin [ 68The targeting of Ga]Ga-PP4SD to CXCR2. Acute kidney injury models were established in male C57BL / 6 mice by treating them with cisplatin (5 mg / kg, 10 mg / kg, 20 mg / kg) and saline, respectively. At 12, 24, 48, and 72 hours post-modeling, 0.2 mL of the newly prepared radioactive complex was injected via the tail vein. 68 Ga]Ga-PP4SD (approximately 7.4 MBq). Micro-PET imaging was performed 0.5 h post-injection, and regions of interest were delineated on the whole-body decay-corrected images for semi-quantitative analysis. Figure 40 and Figure 41 It was observed that, compared with the saline control group 12 hours after modeling, the uptake of radioactive probes at the renal lesion site in cisplatin-induced acute kidney injury mice was significantly increased, and the degree of probe enrichment was positively correlated with the severity of kidney injury. Even at the lowest modeling dose of 5 mg / kg, the renal probe uptake at 12 hours after modeling was significantly higher than that in the control group, approximately twice that of the control group, indicating that […]. 68 Ga]Ga-PP4SD can identify cisplatin-induced inflammatory kidney injury at an early stage. With increasing cisplatin dosage, probe uptake in mouse kidney lesions gradually increased, suggesting that the severity of acute kidney injury is significantly dose-dependent.

[0121] In the 20 mg / kg group, the renal lesion uptake of CXCR2 peaked at 48 h after modeling. At 72 h, renal probe accumulation significantly decreased, while cardiac tissue uptake significantly increased, suggesting that the late-stage severe injury may be accompanied by systemic inflammatory response and upregulation of CXCR2 expression in multiple organs. In addition to the kidneys, CXCR2 probe uptake in the heart and liver of cisplatin-treated mice was also significantly higher than in the saline control group. Particularly in the 20 mg / kg group at 72 h, probe accumulation in the heart and liver was much higher than in the control group. This result is consistent with previous reports on the involvement of CXCR2 in cisplatin-related inflammatory damage.

[0122] Further, a specific blockade experiment was conducted using the CXCR2 small molecule inhibitor Navarixin in a 20 mg / kg cisplatin-induced model. PET imaging and quantitative analysis results showed that […] in the renal lesion site of the blockade group. 68 The Ga]Ga-PP4SD uptake was significantly lower in the Ga-PP4SD group than in the unblocked group, demonstrating that this probe has good targeting specificity for CXCR2 in vivo. Figure 42 and Figure 43 .

[0123] Test Example 11 Autoradiography experiment: The cisplatin-induced acute kidney injury model mice were constructed as described above. Immediately after Micro-PET imaging was completed, the animals were sacrificed, and the heart, liver, and kidney tissues were rapidly dissected and separated. Each organ tissue was wrapped in plastic wrap and exposed to a high-resolution storage phosphorus screen for 60 minutes. Autoradiography signals were then acquired using the accompanying imaging system.

[0124] The autoradiography results were consistent with the trends observed in PET imaging: the 20 mg / kg group showed the highest accumulation of probes in the heart and liver, while the 10 mg / kg group exhibited the most significant renal probe uptake, suggesting that local renal inflammation was most pronounced during the moderate injury stage. Figure 44 and Figure 45 The CXCR2 / CXCL1 signaling axis has been shown to mediate inflammatory cell recruitment and renal tissue damage during cisplatin-induced acute kidney injury, therefore [ 68 Ga]Ga-PP4SD shows promise as a molecular imaging probe for evaluating the degree and dynamic changes of cisplatin-related inflammatory kidney injury.

[0125] Test Example 12 Biological distribution experiment: In vitro biodistribution experiments were conducted 72 hours after administration of physiological saline or cisplatin (5 mg / kg, 10 mg / kg, 20 mg / kg). Mice were injected via tail vein with approximately 7.4 MBq of […]. 68 Following Ga-PP4SD, the animals were euthanized after 0.5 hours. To minimize interference from residual blood in tissue radioactivity assays, the systemic circulation was flushed with physiological saline perfusion via the heart after euthanasia to remove residual radioactivity from blood vessels as much as possible. Subsequently, blood and tissues from major organs, including the heart, liver, lungs, kidneys, spleen, stomach, bones, muscles, small intestine, and brain, were collected. Surface liquid was blotted dry with filter paper before weighing. The radioactivity of each tissue sample was measured using a gamma counter and corrected for radioactive decay. The probe uptake level was expressed as a percentage of the injected dose per unit tissue mass (%ID / g).

[0126] Figure 46 The results showed that, consistent with Micro-PET imaging, the 10 mg / kg cisplatin group exhibited the highest renal uptake, significantly higher than the 20 mg / kg group. Conversely, the 20 mg / kg group showed significantly higher probe uptake in the heart and liver than the 10 mg / kg group, and both were significantly higher than the saline control group. These results suggest that under conditions of severe cisplatin-induced injury, [ 68 The uptake of Ga]Ga-PP4SD may gradually change from simple local inflammation of the kidney to a state accompanied by systemic inflammatory response and upregulation of CXCR2 expression in multiple organs.

[0127] The above results indicate that the labeled compounds of the present invention are based on the structural modification of CXCR2 inhibitors: on the one hand, they exhibit significant aggregation in tumor and inflammatory regions where CXCR2 protein is highly expressed; on the other hand, by altering the length and properties of the linker chain, the pharmacokinetic properties of the probe can be changed.

[0128] In summary, the CXCR2-targeting radiolabeled complexes provided by this invention exhibit specificity for binding to the CXCR2 protein in vivo. Currently, there are no reports of CXCR2-related small molecule radioactive probes in tumor and inflammation models. PET imaging of tumor model mice showed that CXCR2 had high uptake in murine melanoma B16F10, murine pancreatic cancer PANC-02, and murine MC38.

[0129] In various mouse tumor models, including but not limited to melanoma and pancreatic cancer, and in various inflammatory mouse models, including but not limited to myocarditis, arthritis, and kidney injury, the radioligands were enriched in tumors and inflammatory regions with high CXCR2 expression. Furthermore, some compounds exhibited low background uptake and good PET imaging performance, indicating that the designed compounds have the potential to become novel diagnostic and therapeutic agents based on the CXCR2 target.

[0130] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A CXCR2-targeting compound, characterized in that, Including compounds with the structure described in Formula I or the structure shown in Formula II: Formula I; Formula II; Among them, X1, X2, and X3 independently include CH or N; R1 includes any of the following structures: ; R2 and R5 are either present or absent independently; when R2 or R5 exists, it independently includes any of the following structures: a and b are integers from 0 to 10; R3 includes any of the following structures: -H, -CH3, -CH2CH3, -CH(CH3)2, -CH(CH2CH3)2, -CH(CH2CH2CH3)2. , , , ; R4 includes any of the following structures: Where G is -H or -CH3; x and y are integers from 0 to 10; t and f are integers from 0 to 5; Z is the group to be labeled, including any of the following chelate structures: X and Y independently include -OH or -NH2.

2. The method for preparing the CXCR2-targeting compound according to claim 1, characterized in that, The preparation method of the compound with the structure shown in Formula I includes the following steps: Step 1: Reaction of aminophenol compounds with diethyl squaric acid yields monosubstituted intermediate 1 of squaric acid; Step 2: React the monosubstituted squaric acid intermediate 1 with an R1 linker compound containing an amino group to obtain squaric amide intermediate 2 with a linker introduced. Step 3: Deprotect the squaramide intermediate 2 to obtain the free amine intermediate 3; When R2 is present, step 4: the free amine intermediate 3 is coupled with an elongation reagent containing R2 and the protecting group is removed to obtain squaric acid intermediate 4; the squaric acid intermediate 4 is substituted with the Z active compound to obtain the compound with the structure shown in Formula I. When R2 is absent, step 4: the free amine intermediate 3 is subjected to a substitution reaction with the Z active compound to obtain the compound with the structure shown in Formula I; The structural formula of the aminophenolic compound is: ; The structural formula of the compound containing the amino group and its R1 linker arm is as follows: ; The structural formula of the R2-containing elongation reagent is: ; The preparation method of the compound with the structure shown in Formula II includes the following steps: Step a: Reaction of Boc-terminated aminophenolic compounds with diethyl squaric acid yields monosubstituted squaric acid intermediate 5; When R5 is not present, step b: react the monosubstituted squaric acid intermediate 5 with an R3 linker compound containing an amino group, and remove the protecting group to obtain intermediate 6. When R5 is present, step b1: remove the protecting group from the monosubstituted squaric acid intermediate 5 and react it with the R5 linker with the protecting group to obtain intermediate 7; step b2: react the intermediate 7 with the R3 linker compound containing an amino group and remove the protecting group to obtain intermediate 8. Step c: The intermediate 6 or the intermediate 8 is subjected to a substitution reaction with the Z active compound to obtain the compound with the structure shown in Formula II; The structural formula of the aminophenolic compound protected by the terminal Boc group is: ; The structural formula of the compound containing the amino group and its R3 linker arm is as follows: ; The structural formula of the R5 binder with a protective group is: ; R1, R2, R3, R4, R5, X1, X2, X3 in each raw material and intermediate are the same as R1, R2, R3, R4, R5, X1, X2, X3 in the compound with the structure shown in Formula I and Formula II.

3. A radionuclide-labeled probe, characterized in that, Including compounds with the structure shown in Formula III or compounds with the structure shown in Formula IV; Formula III; Formula IV; Wherein, R1, R2, R3, R4, R5, X1, X2, X3 are the same as R1, R2, R3, R4, R5, X1, X2, X3 in the structural compound of Formula I and the structural compound of Formula II; Q is a radionuclide labeling group, obtained by coordinating the Z labeling group in the CXCR2 targeting compound of claim 1 or the CXCR2 targeting compound prepared by the preparation method of claim 2 with a radionuclide.

4. The radionuclide-labeled probe according to claim 3, characterized in that, The radionuclides include 18 F, 43 Sc、 44 Sc、 47 Sc、 51 Cr 55 Co、 57 Co、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 72 As、 72 Se、 89 Zr、 86 Y、 89 Sr、 90 Y、 97 Ru、 99m Tc, 105 Rh、 101m Rh、 103 Pd, 109 Pd, 111 In、 119 Sb, 128 Ba、 139 La、 140 La、 142 Pr、 149 Pm, 149 Tb, 151 Tb, 151 Eu、 153 Eu、 169 Eu、 153 Sm、 152 Gd, 153 Gd, 157 Gd, 159 Gd, 161 Tb, 165 Dy、 166 Ho、 169 Er、 175 Yb、 177 Lu、 186 Re、 188 Re、 197 Hg, 198 Au、 201 Tl、 203 Pb, 211 At、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 227 Th and 225 At least one of Ac.

5. The method for preparing the radionuclide-labeled probe according to claim 3 or 4, characterized in that, Includes the following steps: The CXCR2 targeting compound of claim 1 or the CXCR2 targeting compound prepared by the preparation method of claim 2, and the radionuclide are prepared by wet labeling or freeze-drying labeling.

6. A pharmaceutically acceptable form of existence, characterized in that, The pharmaceutically acceptable forms include the CXCR2 targeting compound of claim 1 and / or the radionuclide-labeled probe of claim 3 or 4, wherein the pharmaceutically acceptable forms include one or more of salts, tautomers, stereoisomers, racemates, hydrates and solvates.

7. A pharmaceutical composition, characterized in that, It includes an active ingredient and pharmaceutically acceptable excipients; the active ingredient includes one or more of the CXCR2 targeting compound of claim 1, the radionuclide-labeled probe of claim 3 or 4, and the pharmaceutically acceptable form of claim 6.

8. A lyophilized reagent kit, characterized in that, It includes a precursor and a pharmaceutically acceptable excipient, said precursor comprising one or more of the pharmaceutically acceptable forms of the CXCR2-targeting compound of claim 1 and the CXCR2-targeting compound of claim 6.

9. The use of the CXCR2 targeting compound of claim 1, the radionuclide-labeled probe of claim 3 or 4, the pharmaceutically acceptable form of claim 6, the pharmaceutical composition of claim 7, or the lyophilized kit of claim 8 in the preparation of therapeutic agents, therapeutic drugs, diagnostic agents, or diagnostic drugs for CXCR2 protein-mediated diseases.

10. The application according to claim 9, characterized in that, The diseases mediated by the CXCR2 protein include tumors or immune-related diseases; The tumors include breast cancer, lung cancer, colorectal cancer, prostate cancer, bone and connective tissue sarcoma, renal cell carcinoma, gastric cancer, pancreatic cancer, nasopharyngeal carcinoma, head and neck cancer, neuroendocrine tumors, or skin melanoma. The immune-related diseases include lupus, atherosclerosis, rheumatoid arthritis, kidney damage, diabetes, hepatitis, autoimmune encephalomyelitis, scleroderma, psoriasis, Sjögren's syndrome, polyarteritis nodosa, or asthma.

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