A compound targeting PD-L1, a radiotracer and a preparation method and application thereof

Abstract

The application is suitable for the fields of biological medicine and nuclear medicine technology, and provides a compound targeting PD-L1, a radioactive tracer and a preparation method and application thereof. The radioactive tracer provided by the application has strong binding force with PD-L1, can accurately position PD-L1 in the body, has excellent in-vivo targeting performance, and can realize the tumor molecular imaging purpose through nuclear medical imaging. Meanwhile, the radioactive tracer realizes non-invasive visualization of PD-L1 expression, can effectively complete non-invasive diagnosis of tumors through small animal PET / CT verification, and has wide clinical application prospect. In addition, the compound targeting PD-L1 and the radioactive tracer derived therefrom also have advantages of simple preparation process, low cost, high specificity, good in-vivo and in-vitro stability, long imaging cycle and easy clinical transformation, and have outstanding comprehensive performance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine and nuclear medicine technology, and particularly relates to a compound targeting PD-L1, a radioactive tracer and a preparation method and application thereof. BACKGROUND

[0002] Programmed Death-Ligand 1 (PD-L1) is a key molecule mediating tumor immune escape. Its abnormally high expression inhibits T cell function by binding to PD-1 on the surface of T cells, thereby promoting the occurrence and development of tumors. At present, PD-1 / PD-L1 inhibitors have been widely used in clinical practice, but some patients develop drug resistance due to tumor microenvironment regulation or alternative pathway activation, affecting the treatment effect.

[0003] The existing PD-L1 detection methods have obvious limitations: image technology examination (such as X-ray, CT, MRI, and ultrasound) evaluates the efficacy by morphological changes of the tumor, and significant changes in tumor volume after chemotherapy usually lag for more than two weeks, making it difficult to reflect the treatment response in a timely manner; tumor marker detection is interfered by many factors such as inflammation, infection, and drugs, and the expression level fluctuates greatly and individual differences are significant, limiting the accuracy; molecular biology detection (including fluorescence in situ hybridization, reverse transcription polymerase chain reaction, high-throughput sequencing, and immunohistochemistry) requires invasive sampling of patients, and PD-L1 expression in different parts of the tumor tissue is heterogeneous, so local specimens cannot fully reflect the overall condition of the disease, and the detection standards are not uniform, limiting its application as a predictive biomarker.

[0004] With the development of medical imaging technology, nuclear medicine PET / CT imaging technology based on molecular recognition and tracing provides a new direction for tumor treatment efficacy evaluation. This technology does not require invasive procedures and can achieve in vivo visualization and tracking of target points in a non-invasive manner, can accurately diagnose lesions to overcome tumor heterogeneity and sampling bias, can reveal early pathological changes after treatment, and can provide guidance for individualized treatment plans, and can evaluate the dynamic changes of pathophysiology in vivo without the need for ex vivo detection. Based on these advantages, the use of nuclear medicine PET / CT imaging technology to achieve non-invasive monitoring of PD-L1 activity has important value for evaluating the efficacy of PD-L1 targeted therapy and accurately determining the effect of anti-tumor drugs in a timely manner. SUMMARY

[0005] Therefore, the present application provides a compound targeting PD-L1 and a radioactive tracer derived therefrom, aiming to solve the problems of existing PD-L1 detection methods such as lag, invasiveness, difficulty in reflecting dynamic changes, and incomplete evaluation.

[0006] The first aspect of the embodiments of the present application provides a compound targeting PD-L1, the compound targeting PD-L1 has a structure shown in the following formula (I) or formula (II) or a stereoisomer, a tautomer, a pharmaceutically acceptable salt, a hydrate thereof:

[0007] The formula (I) is:

[0008] ;

[0009] The formula (II) is:

[0010] .

[0011] The second aspect of the embodiments of the present application provides an intermediate of the compound targeting PD-L1 in the first aspect, the intermediate of the compound shown in the formula (II) is shown in the following formula (IV);

[0012] The formula (IV) is:

[0013] ;

[0014] In the formula (IV), the structure of -NHBoc is

[0015] The third aspect of the embodiments of the present application provides a preparation method of the compound targeting PD-L1 in the first aspect, comprising:

[0016] After removing the Boc protection of the compound shown in the following formula (III), tetraazacyclododecane tetraacetic acid-succinimidyl ester and N,N-diisopropylethylamine are added to carry out a condensation reaction to obtain the compound targeting PD-L1 shown in the formula (I);

[0017] After removing the Boc protection of the compound shown in the following formula (IV), tetraazacyclododecane tetraacetic acid-succinimidyl ester and N,N-diisopropylethylamine are added to carry out a condensation reaction to obtain the compound targeting PD-L1 shown in the formula (II);

[0018] The formula (III) is:

[0019] ;

[0020] The formula (IV) is:

[0021] .

[0022] The fourth aspect of the embodiments of the present application provides a radioactive tracer, comprising the compound targeting PD-L1 in the first aspect or the compound targeting PD-L1 prepared by the preparation method in the third aspect and a radionuclide.

[0023] The fifth aspect of the embodiments of the present application provides a preparation method of the radioactive tracer described in the fourth aspect, comprising:

[0024] Mixing and reacting the radionuclide and the compound targeting PD-L1 to obtain the radioactive tracer.

[0025] The sixth aspect of the embodiments of the present application provides application of the radioactive tracer described in the fourth aspect or the radioactive tracer prepared by the preparation method described in the fifth aspect in preparation of a preparation for detecting expression level of PD-L1 in a tumor or in preparation of a preparation for diagnosing a tumor with high expression of PD-L1.

[0026] Compared with the prior art, the embodiments of the present application have the beneficial effects that:

[0027] The radioactive tracer provided by the embodiments of the present application has strong binding force with PD-L1, can accurately locate PD-L1 in vivo, has excellent in-vivo targeting performance, and can realize the purpose of tumor molecular imaging through nuclear medical imaging; at the same time, it realizes non-invasive visualization of PD-L1 expression, can effectively complete non-invasive diagnosis of tumors verified by small animal PET / CT, and has broad clinical application prospects; in addition, the compound targeting PD-L1 and the radioactive tracer derived therefrom also have advantages of simple preparation process, low cost, high specificity, good in-vivo and in-vitro stability, long imaging cycle and easy clinical transformation, and outstanding comprehensive performance. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 A synthesis route of the compound CB01 targeting PD-L1 provided for the embodiment 1 of the present application;

[0029] Figure 2 A nuclear magnetic hydrogen spectrum of the compound 1 provided for the embodiment 1 of the present application;

[0030] Figure 3 A high-resolution mass spectrum of the compound 1 provided for the embodiment 1 of the present application;

[0031] Figure 4 A high-resolution mass spectrum of the compound CB01 targeting PD-L1 provided for the embodiment 1 of the present application;

[0032] Figure 5 A synthesis route of the compound CB02 targeting PD-L1 provided for the embodiment 2 of the present application;

[0033] Figure 6 A nuclear magnetic hydrogen spectrum of the intermediate 4 provided for the embodiment 2 of the present application;

[0034] Figure 7 A high-resolution mass spectrum of the intermediate 4 provided for the embodiment 2 of the present application;

[0035] Figure 8 NMR spectrum of intermediate 5 provided for Example 2 of the present application;

[0036] Figure 9 High resolution mass spectrum of intermediate 5 provided for Example 2 of the present application;

[0037] Figure 10 High resolution mass spectrum of compound CB02 targeting PD-L1 provided for Example 2 of the present application;

[0038] Figure 11 Radio-HPLC analysis results of radiotracer [ 68 Ga]Ga-CB01 provided for Example 3 of the present application;

[0039] Figure 12 Radio-HPLC analysis results of radiotracer [ 68 Ga]Ga-CB02 provided for Example 3 of the present application;

[0040] Figure 13 In vitro stability study results of radiotracer [ 68 Ga]Ga-CB01 provided for Example 4 of the present application;

[0041] Figure 14 In vitro stability study results of radiotracer [ 68 Ga]Ga-CB02 provided for Example 4 of the present application;

[0042] Figure 15 Saturation curves of radiotracer [ 68 Ga]Ga-CB01 and radiotracer [ 68 Ga]Ga-CB02 provided for Example 6 of the present application;

[0043] Figure 16 Uptake curves of radiotracer [ 68 Ga]Ga-CB01 and radiotracer [ 68 Ga]Ga-CB02 provided for Example 6 of the present application;

[0044] Figure 17 Blood clearance curves of radiotracer [ 68 Ga]Ga-CB01 and radiotracer [ 68 Ga]Ga-CB02 provided for Example 7 of the present application;

[0045] Figure 18 Comparison of small animal PET / CT imaging provided for Example 8 of the present application;

[0046] Figure 19 Radioactive tracer provided for Example 9 of the present application 68 Ga]Ga-CB01 and radioactive tracer 68 Ga]Ga-CB02 in the radioactive distribution column chart of each tissue in HCC78 tumor-bearing mice;

[0047] Figure 20 Target / non-target ratio column chart provided for Example 9 of the present application;

[0048] Figure 21 Phosphor screen autoradiogram of the corresponding tissue after injection of radioactive tracer 68 Ga]Ga-CB01 provided for Example 10 of the present application;

[0049] Figure 22 Phosphor screen autoradiogram of the corresponding tissue after injection of radioactive tracer 68 Ga]Ga-CB02 provided for Example 10 of the present application;

[0050] Figure 23 Tumor tissue section immunofluorescence staining results provided for Example 11 of the present application. DETAILED DESCRIPTION

[0051] It should be noted that the following detailed description is merely exemplary in nature and is intended to provide further description of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0052] It is to be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments consistent with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, devices, components and / or combinations thereof, but do not preclude the presence or addition of one or more other features, steps, operations, devices, components and / or combinations thereof.

[0053] In order to make the purposes, technical solutions and advantages of the present application more clear, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0054] In order to solve the problems of existing PD-L1 detection methods, such as hysteresis, invasiveness, difficulty in reflecting dynamic changes, and incomplete evaluation, the present application provides a PD-L1 targeting compound, which has the structure shown in the following formula (I) or formula (II), or a stereoisomer, tautomer, pharmaceutically acceptable salt, hydrate thereof:

[0055] Formula (I):

[0056] ;

[0057] Formula (II):

[0058] .

[0059] The pharmaceutically acceptable salt refers to the organic salt and inorganic salt of the compound of the present application. The pharmaceutically acceptable salt is well known to the person skilled in the art. The pharmaceutically acceptable salt includes but is not limited to inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, etc., and organic acid salts such as acetate, oxalate, maleate, tartrate, citrate, succinate, malonate, etc., or salts obtained by other methods such as ion exchange method as recorded in the literature.

[0060] Similarly, in addition to the pharmaceutically acceptable salt of the compound, the stereoisomer (such as enantiomer, diastereomer) of the compound, the tautomer (such as keto-enol tautomer) of the compound, the hydrate (crystalline water form) of the compound, etc. usually have similar biological activity, and can be naturally formed during preparation or storage, and also belong to the technical solutions under the same concept of the first aspect of the present application.

[0061] The present application further provides the intermediates of the above-mentioned PD-L1 targeting compound, the intermediate of the compound represented by Formula (I) is represented by Formula (III), and the intermediate of the compound represented by Formula (II) is represented by Formula (IV).

[0062] Formula (III):

[0063] ;

[0064] Formula (IV):

[0065] .

[0066] In Formula (III) and Formula (IV), the structure of -NHBoc is (the structure of -NHBoc in the specific embodiment 1-2). The intermediate represented by Formula (III) can be prepared by the person skilled in the art based on the prior art, such as by the method of the following specific embodiment 1.

[0067] The present application further provides the preparation method of the above-mentioned PD-L1 targeting compound, which comprises:

[0068] After removing the Boc protection of the compound shown in the following formula (III), tetraazacyclododecane tetraacetic acid-succinimidyl ester and N,N-diisopropylethylamine are added to carry out condensation reaction to obtain the compound shown in formula (I) targeting PD-L1.

[0069] After removing the Boc protection of the compound shown in the following formula (IV), tetraazacyclododecane tetraacetic acid-succinimidyl ester and N,N-diisopropylethylamine are added to carry out condensation reaction to obtain the compound shown in formula (II) targeting PD-L1.

[0070] Formula (III):

[0071] ;

[0072] Formula (IV):

[0073] .

[0074] In one or more embodiments, the method for removing the Boc protecting group of the compound shown in formula (III) or formula (IV) can be: dissolving the compound shown in formula (III) or formula (IV) in dichloromethane, adding trifluoroacetic acid to react to obtain the first intermediate after removing the Boc protection.

[0075] In one or more embodiments, the molar ratio of the compound shown in formula (III) or formula (IV) to tetraazacyclododecane tetraacetic acid-succinimidyl ester (DOTA-NHS) and N,N-diisopropylethylamine (DIPEA) is (23-27):(35-40):(135-160), preferably 25:38:150.

[0076] In one or more embodiments, the preparation method of the compound shown in formula (IV) comprises:

[0077] After removing the Boc protection of the compound shown in formula (III), tert-butyloxycarbonyl 6-aminohexanoic acid, 2-(7-azobenzo triazole)-N,N,N',N'-tetramethyl urea hexafluorophosphate and N,N-diisopropylethylamine are added to carry out condensation reaction to obtain a first condensation intermediate;

[0078] After removing the Boc protection of the first condensation intermediate, tert-butyloxycarbonyl 6-aminohexanoic acid, 2-(7-azobenzo triazole)-N,N,N',N'-tetramethyl urea hexafluorophosphate and N,N-diisopropylethylamine are added to carry out condensation reaction to obtain the compound shown in formula (IV).

[0079] The application further provides a radioactive tracer, comprising the compound targeting PD-L1 or the compound targeting PD-L1 prepared by the preparation method described above and a radionuclide.

[0080] In one or more embodiments, the radionuclide is selected from one of 18 F, 94 Tc, 99m Tc, 90 In, 111 In, 67 Ga, 68 Ga, 86 Y, 90 Y, 177 Lu, 151 Tb, 186 Re, 188 Re, 64 Cu, 67 Cu, 55 Co, 57 Co, 43 Sc, 44 Sc, 47 Sc, 225 Ac, 213 Bi, 212 Bi, 212 Pb 、227 Th, 153 Sm, 166 Ho, 152 Gd, 153 Gd, 157 Gd and 166 Dy; preferably 68 Ga.

[0081] Preferably, the structure of the radioactive tracer is shown in formula (V) and formula (VI):

[0082] Formula (V):

[0083] ;

[0084] Formula (VI):

[0085] .

[0086] The application further provides a preparation method of the radioactive tracer, comprising:

[0087] mixing and reacting the radionuclide with the PD-L1 targeting compound to obtain the radioactive tracer.

[0088] In one or more embodiments, the radionuclide is 68 Ga, for example, 3.5-10 nmol of the PD-L1 targeting compound is mixed with 200 µL 68The GaCl3 eluate (74 MBq~100 MBq) is mixed with 200 μL of acetic acid-sodium acetate buffer solution (0.25 M), the pH of the reaction system is adjusted to 4~4.6, and then the mixture is uniformly mixed at 95°C for 10-15 min. After the reaction is completed, the mixture is cooled to room temperature to obtain the radioactive tracer.

[0089] The application further provides use of the radioactive tracer or the radioactive tracer prepared by the preparation method in preparation of a preparation for detecting expression levels of PD-L1 in tumors or in preparation of a preparation for diagnosing tumors with high expression of PD-L1.

[0090] The tumors include any one of lymphoma, multiple myeloma and solid tumors, for example, lung cancer, liver cancer, pancreatic cancer, gastric cancer, colon cancer, thyroid cancer and head and neck tumors, but are not limited thereto.

[0091] The following specifically describes the compound targeting PD-L1 and the radioactive tracer derived therefrom by means of specific examples. Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the materials, reagents and the like used, unless otherwise specified, can be obtained from commercial channels.

[0092] Cell culture:

[0093] HCC78 cells are cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-gentamicin mixture (P-S-G); and cultured in a 37 °C, 5% CO2 humidified incubator.

[0094] Establishment of HCC78 tumor-bearing mouse model:

[0095] HCC78 cells are subcutaneously injected into the right shoulder of NSG mice, 2×10 6 cells are injected into each mouse, and the growth of the tumor is observed every day within two weeks. When the tumor grows to a limited size, imaging and in vivo distribution experiments are carried out.

[0096] Example 1

[0097] Figure 1 The synthesis route of the compound targeting PD-L1 shown in formula (I) is described with reference to Figure 1 , and the compound CB01 targeting PD-L1 is synthesized.

[0098] Compound 1 (100 mg, 0.25 mmol) was dissolved in DCM (11.8 mL), N-tert- butyloxycarbonyl ethylenediamine (78.98 mg, 0.49 mmol), acetic acid (10 mg, 0.17 mmol) were added, after stirring at room temperature for 4 hours, sodium triacetoxyborohydride (156.98 mg, 0.74 mmol) was added, and stirring was continued at room temperature for 12 hours, and the reaction was monitored by TLC. After the reaction was completed, water was added for quenching, and DCM was used for extraction, and the combined organic phase was dried over anhydrous Na2SO4, filtered, and the solvent was evaporated by a rotary evaporator. The reaction crude product was separated by silica gel column chromatography to obtain intermediate 2 (110 mg, yield 83%).

[0099] The nuclear magnetic hydrogen spectrum of compound 1 is shown in Figure 2 The high-resolution mass spectrum of compound 1 is shown in Figure 3

[0100] Intermediate 2 (20 mg, 0.04 mmol) was dissolved in DCM (0.3 ml), TFA (25.31 mg, 0.22 mmol) was added, and the reaction was carried out at room temperature for 4 hours, and the reaction progress was monitored by TLC. After the reaction was completed, saturated K2CO3 aqueous solution was added for quenching, and DCM was used for extraction, and the combined organic phase was dried over anhydrous Na2SO4, filtered, and the solvent was evaporated by a rotary evaporator. Then, DMF (0.3 mL), DOTA-NHS (17.21 mg, 0.03 mmol), and DIPEA (14.75 mg, 0.11 mmol) were added to the reaction system, and stirring was continued at room temperature for 8 hours, and the reaction was monitored by HPLC. After the reaction was completed, the reaction system was purified by HPLC (see Table 1), and compound 3 (CB01) (4.7 mg, yield 25%) was obtained after freeze-drying.

[0101] The high-resolution mass spectrum of compound 3 (CB01) is shown in Figure 4

[0102] Table 1 HPLC purification method (flow rate: 3 mL / min)

[0103]

[0104] Example 2

[0105] Figure 5 The synthesis route of the compound targeting PD-L1 shown in formula (II) was used to synthesize compound CB02 targeting PD-L1.

[0106] ​​Intermediate 2 (300 mg, 0.56 mmol) was dissolved in DCM (6 ml), TFA (383.04 mg, 3.36 mmol) was added and the reaction was allowed to proceed at room temperature for 4 h, monitoring the reaction progress by TLC. After completion of the reaction, saturated aqueous K2CO3solution was added to quench the reaction and extracted with DCM. The organic layers were combined, dried over anhydrous Na2SO4, filtered and the solvent was evaporated using a rotary evaporator. The residue was then dissolved in DMF (6 ml) and N-(tert-butoxycarbonyl)-L-proline (180.92 mg, 0.84 mmol), HATU (319.77 mg, 0.84 mmol) and DIPEA (434.21 mg, 3.37 mmol) were added and stirred at room temperature for 12 h, monitoring the reaction by TLC. After completion of the reaction, water was added to quench the reaction and extracted with ethyl acetate. The combined extracts were dried over Na2SO4, filtered and the solvent was evaporated using a rotary evaporator. Column chromatography afforded intermediate 4 (156 mg, 44% yield).

[0107] The1H NMR of intermediate 4 is shown in Figure 6 The high resolution mass spectrum is shown in Figure 7

[0108] Intermediate 4 (100 mg, 0.159 mmol) was dissolved in DCM (2 mL), TFA (108.57 mg, 0.95 mmol) was added and the reaction was allowed to proceed at room temperature for 4 h, monitoring the reaction progress by TLC. After completion of the reaction, saturated aqueous K2CO3solution was added to quench the reaction and extracted with DCM. The organic layers were combined, dried over anhydrous Na2SO4, filtered and the solvent was evaporated using a rotary evaporator. The residue was then dissolved in DMF (6 ml) and tert-butoxycarbonyl 6-aminohexanoic acid (51.9 mg, 0.24 mmol), HATU (120.61 mg, 0.32 mmol) and DIPEA (81.89 mg, 0.63 mmol) were added and stirred at room temperature for 2 h, monitoring the reaction by TLC. After completion of the reaction, water was added to quench the reaction and extracted with ethyl acetate. The combined extracts were dried over Na2SO4, filtered and the solvent was evaporated using a rotary evaporator. Column chromatography afforded intermediate 5 (59 mg, 29.3% yield).

[0109] The1H NMR of intermediate 5 is shown in Figure 8 The high resolution mass spectrum is shown in Figure 9

[0110] ​​Intermediate 5 (15 mg, 0.02 mmol) was dissolved in DCM (0.3 mL), and TFA (13.99 mg, 0.12 mmol) was added. The reaction was carried out at room temperature for 4 h, and the reaction progress was monitored by TLC. After the reaction was completed, saturated K2CO3 aqueous solution was added to quench the reaction, and the mixture was extracted with DCM. The organic phases of the extracts were combined, dried over anhydrous Na2SO4, filtered, and the solvent was evaporated by rotary evaporator. Then, DMF (0.3 mL), DOTA-NHS (15.04 mg, 0.03 mmol), and DIPEA (3.87 mg, 0.03 mmol) were added to the reaction system, and the mixture was stirred at room temperature for 4 h. The reaction was monitored by HPLC. After the reaction was completed, the reaction system was purified by HPLC and lyophilized to give compound 6 (CB02) (2.3 mg, yield 11.1%).

[0111] The high-resolution mass spectrum of compound 6 (CB02) is shown below. Figure 10 As shown.

[0112] Example 3

[0113] Use 4 mL of high-purity hydrochloric acid (0.1 M) from 68 Ge- 68 Ga obtained by rinsing in Ga generator 68 GaCl3 eluent;

[0114] 10 nmol of compound CB01 was mixed with 200 µL 68 After mixing GaCl3 eluent (74 MBq~100 MBq), 200 µL of acetate-sodium acetate buffer (0.25 M) was added to adjust the pH of the reaction system to 4.6. The mixture was then kept at 95 °C and mixed for 15 min. After the reaction was complete, the mixture was cooled to room temperature to obtain the radioactive tracer. 68 Ga]Ga-CB01.

[0115] 3.5 nmol of compound CB02 was mixed with 200 µL 68 GaCl3 eluent (74 MBq~100 MBq) was mixed with 200 µL of acetate-sodium acetate buffer (0.25 M) to adjust the pH of the reaction system to 4.6, and then the mixture was kept at 95 °C for 15 min. After the reaction was completed, the mixture was cooled to room temperature to obtain the radioactive tracer. 68 Ga]Ga-CB02.

[0116] Detection of radioactive tracers using radioactive HPLC [ 68 Ga]Ga-CB01 and [ 68 The radiochemical purity (RCP) of Ga]Ga-CB02 and the analytical method of radioactive HPLC are shown in Table 2.

[0117] Table 2 Radio-HPLC analysis method (flow rate: 1 mL / min)

[0118]

[0119] [ 68 Ga]Ga-CB01 and 68 Ga]Ga-CB02 were analyzed by radio-HPLC. As shown in Figure 11 and Figure 12 , it can be seen from Figure 11 that the retention time of 68 Ga]Ga-CB01 was 14.67 min, RCP > 99%, and the specific activity was 9 ± 0.1 GBq / µmol; it can be seen from Figure 12 that the retention time of 68 Ga]Ga-CB02 was 14.82 min, RCP > 99%, and the specific activity was 28 ± 0.21 GBq / µmol.

[0120] The formula for calculating the specific activity is:

[0121] Specific activity (GBq / µmol) = radioactivity (GBq) / amount of substance (µmol) of the precursor compound.

[0122] Example 4

[0123] The in vitro stability of the radiotracers 68 Ga]Ga-CB01 and 68 Ga]Ga-CB02 was studied.

[0124] PBS stability study of the radiotracers: 100 µL of the radiotracer was mixed with 100 µL of PBS and incubated at 37 °C for 2 h; after incubation, 100 µL of the mixed system was taken to analyze the RCP of the radiotracer by radio-HPLC.

[0125] In vitro serum stability study of the radiotracers: 100 µL of the radiotracer was mixed with 100 µL of human serum and incubated at 37 °C for 2 h; after incubation, 200 µL of absolute ethanol was first added to the incubation system, and then the incubation system was centrifuged at 12000 rpm for 5 min; the supernatant was filtered and the RCP of the radiotracer was analyzed by radio-HPLC.

[0126] The results of the in vitro stability study of the radiotracer 68 Ga]Ga-CB01 are shown in Figure 13 , where Figure 13 (a) is the PBS stability study, Figure 13(b) is the in vitro serum stability study. It can be seen from Figure 13 The in vitro PBS stability of the radiotracer [ 68 Ga]Ga-CB01 and the in vitro human serum stability are both good, and the RCPs at 1 h are both greater than 99%.

[0127] The in vitro stability study results of the radiotracer [ 68 Ga]Ga-CB02 are shown in Figure 14 Figure 14 (a) is the PBS stability study, Figure 14 (b) is the in vitro serum stability study. It can be seen from Figure 14 The in vitro PBS stability of the radiotracer [ 68 Ga]Ga-CB02 and the in vitro human serum stability are both good, and the RCPs at 1 h are both greater than 99%.

[0128] Example 5

[0129] Hydrophilic-lipophilic study:

[0130] 5 μL of the radiotracer was added to a mixture of 495 μL of HEPES buffer (pH = 7.4) and 500 μL of n-octanol, mixed vigorously, and centrifuged at 5000 rpm for 5 min; after centrifugation, 400 μL of the upper liquid (n-octanol) and 400 μL of the lower liquid (HEPES buffer solution) were taken from the centrifuge tube and centrifuged at 12000 rpm for 5 min; finally, 100 μL of the upper liquid (n-octanol) and 100 μL of the lower liquid (HEPES buffer solution) were taken from the re-centrifuged system, and the radioactivity counts were measured with a γ counter to calculate the oil-water distribution coefficient D (log D ). The calculation formula is: log D =lg ((γ counts in n-octanol) / (γ counts in HEPES buffer)).

[0131] The oil-water distribution coefficients D (log D ) of the radiotracer [ 68 Ga]Ga-CB01 and the radiotracer [ 68 Ga]Ga-CB02 were calculated to be -1.80 ± 0.08 and -1.66 ± 0.06, respectively; both of them have excellent hydrophilicity.

[0132] Example 6

[0133] Cell receptor affinity study of the radiotracer:

[0134] (1) The equilibrium dissociation constant (Kd) was determined by saturation experiment K ​d To evaluate the binding affinity of the radiotracer to PD-L1, a saturation binding assay was performed on HCC78 cells. HCC78 cells (4 × 10⁻⁶) were used. 5 Cells were incubated with different concentrations of radioactive tracer (0.625–60 nM) for 1 h to determine total binding. To determine nonspecific binding, cells were pretreated with an excess of PD-L1 antibody (20 μg) before the experiment. Cells in both the total binding and nonspecific binding groups were washed three times with cold PBS, followed by lysis with 200 μL / well of NaOH solution (1 M). The radioactivity count of cell lysis products was measured using a gamma counter. All experiments were performed in triplicate.

[0135] (2) Cell uptake experiment: HCC78 cells (4×10⁻⁶) were added to the cell uptake test. 5 Cells were seeded in 24-well plates (number per well) and incubated with a radiotracer for 15, 30, 60, 90, and 120 min to assess uptake over time. For blocking experiments, cells were pre-incubated with PD-L1 antibody (20 μg) for 1 hour before adding the radiotracer. After incubation, the culture medium was removed, cells were washed with PBS, and then lysed with 200 μL of 1 M NaOH. Radioactivity was then measured using a gamma counter. All experiments were performed in triplicate.

[0136] Radioactive tracers [ 68 Ga]Ga-CB01 and radioactive tracers[ 68 The saturation curve of Ga-CBO2 is as follows: Figure 15 As shown, the radioactive tracer [ 68 Ga]Ga-CB01 K d The concentration is 98.96 nM, and the radioactive tracer [ 68 Ga]Ga-CB02 K d It is 51.67 nM; from Figure 15 as well as K d The values ​​show that the radioactive tracer [ 68 Ga]Ga-CB01 and radioactive tracers[ 68 Ga]Ga-CB02 has a good affinity for HCC78 cells. K d The values ​​are all in the nM range.

[0137] Radioactive tracers [ 68 Ga]Ga-CB01 and radioactive tracers[ 68 The uptake curve of Ga-CBO2 is as follows: Figure 16 As shown, from Figure 16As can be seen from 68 Ga]Ga-CB01 and radiotracer 68 Ga]Ga-CB02 both showed rapid and specific uptake within 15 min, and gradually increased over time until reaching a plateau at 90 min; the uptake inhibition of PD-L1 antibody confirmed that the binding of radiotracer 68 Ga]Ga-CB01 and radiotracer 68 Ga]Ga-CB02 to PD-L1 was specific.

[0138] Example 7

[0139] Normal BALB / c female mice (n=3) were injected with radiotracer 68 Ga]Ga-CB01, 68 Ga]Ga-CB02 (7.4 MBq each) via tail vein. Subsequently, the mice blood was taken at 1, 3, 5, 10, 15, 30, 45, 60, 90, 120 min, respectively, weighed, and the radioactivity count was measured using a gamma counter. The blood clearance half-life of each radiotracer was obtained by fitting the blood clearance curve using Drug And Statistics (DAS) software. The calculation formula of %ID / g was: %ID / g= ( (measured radioactivity count) / (total radioactivity count injected) ) / the mass of the tissue (including blood).

[0140] The blood clearance curves of radiotracer 68 Ga]Ga-CB01 and radiotracer 68 Ga]Ga-CB02 are shown in Figure 17 As can be seen from Figure 17 The pharmacokinetic curves of each radiotracer in normal mice accorded with a two-compartment model, the distribution phase half-life (t 68 ) of radiotracer 1 / 2α Ga]Ga-CB01 was 0.55 min, the elimination phase half-life (t 1 / 2β ) was 8.878 min, and the blood metabolism time was moderate. The distribution phase half-life (t 1 / 2α ) of radiotracer 68 Ga]Ga-CB02 was 0.646 min, the elimination phase half-life (t 1 / 2β ) was 11.273 min, and the blood metabolism time was moderate.

[0141] Example 8

[0142] Small animal PET / CT imaging:

[0143] HCC78 tumor-bearing mice were randomly divided into experimental group (n=3) and blocking group (n=3), and then injected with radioactive tracer 68 Ga]Ga-CB01 or radioactive tracer 68 Ga]Ga-CB02 for small animal PET / CT imaging. In the experimental group, each tumor-bearing mouse was injected with 200 μL of radioactive tracer (3.7 MBq) through the tail vein under 2% isoflurane oxygen. In the blocking group, the tumor-bearing mouse was first injected with PD-L1 antibody (50 μg) through the tail vein, and then injected with radioactive tracer through the tail vein 24 h later, and PET / CT imaging was performed. After imaging, the PET / CT images were reconstructed using Nucline NanScan 3.00 software, and image analysis was performed using InterView FUSION 3.0 software.

[0144] As shown in Figure 18 , the experimental group had obvious 68 Ga]Ga-CB01 and 68 Ga]Ga-CB02 radioactive uptake at the tumor site, and the radioactive accumulation of the inhibition group was significantly reduced; indicating that the radioactive tracer specifically targets PD-L1 protein and can be used for imaging, efficacy monitoring, etc. of PD-L1 protein expressing tumors.

[0145] Example 9

[0146] Biodistribution:

[0147] Under gaseous anesthesia (2% isoflurane in oxygen), HCC78 tumor-bearing mice (n=3) were injected with 100 μL of radioactive tracer 68 Ga]Ga-CB01 or radioactive tracer 68 Ga]Ga-CB02 (3.7 MBq) through the tail vein. After 1 h of injection, the HCC78 tumor-bearing mice were subjected to ocular blood sampling, and after sacrifice, important tissues / organs including tumor, muscle, bone, kidney, spleen, liver, intestine, heart, lung and stomach were collected. The collected blood and tissues were weighed, and then the radioactivity counts were measured by a gamma counter. By comparing the tissue counts with the calibrated counts of the injected radioactive tracer, the %ID / g and target / non-target (T / NT) ratios were obtained.

[0148] As shown in Figure 19 , Figure 20 , Table 3 and Table 4, in the HCC78 tumor-bearing mouse model, the injection of radioactive tracer 68 Ga]Ga-CB01 and 68 Ga]Ga-CB02 showed high tumor uptake and low non-target uptake, and the blocking group showed significantly reduced radioactive accumulation in the tumor, indicating that the radioactive tracer specifically targets PD-L1 protein and can be used for imaging, efficacy monitoring, etc. of PD-L1 protein expressing tumors.Sixty minutes after Ga]Ga-CB02 administration, tumor uptake was 0.38±0.03%ID / g and 0.47±0.29%ID / g, respectively, indicating that the radiotracer has tumor targeting properties in PD-L1 positive tumors.

[0149] Table 3 Radioactive tracers [ 68 Ga]Ga-CB01、[ 68 Biological distribution study of Ga-CB02

[0150]

[0151] Table 4 Radioactive tracers [ 68 Ga]Ga-CB01、[ 68 Ga]Ga-CBO2 TNT ratio

[0152]

[0153] Example 10

[0154] Phosphorus screen autoradiography: Under gas anesthesia (oxygen containing 2% isoflurane), 100 μL of radioactive tracer was injected via the tail vein into HCC78 tumor-bearing mice (n=3). 68 Ga]Ga-CB01 or radioactive tracer[ 68 Ga]Ga-CB02 (3.7 MBq). One h after injection, blood was collected from the eyeballs of HCC78 tumor-bearing mice. After sacrifice, the tumors, muscles, and kidneys were collected. The tumors, muscles, and kidneys were placed on a phosphorescent screen and exposed to darkness for 10 min. Images were then obtained by scanning with a phosphorescent screen imaging system.

[0155] The results are as follows Figure 21 and 22 As shown, the phosphorus screen analysis results are highly consistent with PET / CT imaging and biodistribution results, further confirming the effectiveness of the radioactive tracer. 68 Ga]Ga-CB01 and radioactive tracers [ 68 The specific distribution pattern of Ga]Ga-CB02 in vivo.

[0156] Example 11

[0157] Immunohistochemical staining to verify PD-L1 expression in the HCC78 tumor-bearing mouse model:

[0158] The tumor tissue of tumor-bearing mice was fixed in 4% paraformaldehyde for 24 h, then embedded to make paraffin sections; the paraffin sections were placed in a 60°C oven for 30 min, then sequentially immersed in xylene I and xylene II for 10 min each; the sections were sequentially placed in ethanol from high to low for 2 min each (ethanol gradient: 100%, 95%, 90%, 80%, 70%); then the sections were placed in citric acid antigen retrieval solution, heated to boiling in a microwave oven for 15 min, then naturally cooled to room temperature;

[0159] The endogenous peroxidase blocker was added dropwise to the tissue, and incubated at room temperature for 15 min, and washed with double distilled water; normal sheep serum was added dropwise for blocking, and incubated at room temperature for 20 min; the section was added dropwise with PD-L1 antibody or IgG antibody (rabbit anti-mouse, 1:200), and incubated at 4°C overnight, then removed and incubated at room temperature for 30 min, washed with PBS for 5 min x 3 times, to complete the incubation of the first antibody; the secondary antibody (goat anti-rabbit, 1:200) was added dropwise, and incubated at 37°C for 30 min, washed with PBS for 5 min x 3 times, to complete the incubation of the secondary antibody; DAB color developing solution working solution was added dropwise, and incubated for 10 s, then washed with water; Mayor's hematoxylin was added dropwise, and incubated at room temperature for 2 min; hydrochloric acid alcohol differentiation, alkaline solution returned to blue, and washed with water; the sections were sequentially placed in ethanol from high to low for a few seconds (ethanol gradient: 80%, 90%, 95%), anhydrous ethanol for 10 min, then immersed in xylene for 10 min, 1 drop of neutral resin was added for mounting, and the results were observed under a microscope and photographed.

[0160] The results of immunofluorescence staining of tumor tissue sections are shown in Figure 23 The results show that the tumor tissue in the constructed HCC78 tumor-bearing mouse model highly expresses PD-L1, and is negatively expressed in IgG tumor staining.

[0161] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A compound targeting PD-L1, characterized in that, The compound targeting PD-L1 has a structure as shown in formula (I) or formula (II) or a pharmaceutically acceptable salt thereof: Equation (I): ； Formula (II): 。 2. An intermediate for the compound targeting PD-L1 according to claim 1, characterized in that, The intermediate of the compound shown in formula (II) is shown in formula (IV); Formula (Ⅳ): ； In the formula (IV), the structure of -NHBoc is .

3. The method for preparing the PD-L1-targeting compound according to claim 1, characterized in that, include: After removing Boc protection from the compound shown in formula (III), tetraazacyclododecanetetraacetic acid-succinimide ester and N,N-diisopropylethylamine were added to carry out a condensation reaction to obtain the compound shown in formula (I) that targets PD-L1. After removing Boc protection from the compound shown in formula (Ⅳ), tetraazacyclododecanetetraacetic acid-succinimide ester and N,N-diisopropylethylamine were added to carry out a condensation reaction to obtain the compound showing formula (Ⅱ) that targets PD-L1. Formula (III): ； Formula (Ⅳ): 。 4. The method for preparing the PD-L1-targeting compound according to claim 3, characterized in that, The molar ratio of the compound shown in formula (III) or formula (IV), tetraazacyclododecanetetraacetic acid-succinimide ester and N,N-diisopropylethylamine is (23~27):(35~40):(135~160).

5. The method for preparing the PD-L1-targeting compound according to claim 3, characterized in that, The preparation method of the compound shown in formula (Ⅳ) includes: After removing the Boc protection from the compound shown in formula (III), tert-butoxycarbonyl 6-aminohexanoic acid, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine were added to carry out a condensation reaction to obtain a first-order condensation intermediate. After removing the Boc protection from the first-order condensation intermediate, tert-butoxycarbonyl 6-aminohexanoic acid, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine were added to carry out a condensation reaction to obtain the compound shown in formula (Ⅳ).

6. A radioactive tracer, characterized in that, This includes the PD-L1-targeting compound of claim 1 or the PD-L1-targeting compound prepared by the preparation method of any one of claims 3-5, as well as radionuclides.

7. The radioactive tracer according to claim 6, characterized in that, The structural formula of the radioactive tracer is shown in formula (V) or formula (VI) below: Formula (V): ； Formula (VI): 。 8. The radioactive tracer according to claim 6, characterized in that, The radionuclide is selected from the group consisting of 18 F, 94 Tc, 99m Tc, 90 In, 111 In, 67 Ga, 68 Ga, 86 Y, 90 Y, 177 Lu, 151 Tb, 186 Re, 188 Re, 64 Cu, 67 Cu, 55 Co, 57 Co, 43 Sc, 44 Sc, 47 Sc, 225 Ac, 213 Bi, 212 Bi, 212 Pb 、227 Th, 153 Sm, 166 Ho, 152 Gd, 153 Gd, 157 Gd and 166 Dy.

9. The method for preparing the radioactive tracer according to any one of claims 6-8, characterized in that, include: The radionuclide is mixed with the compound that targets PD-L1 to obtain a radioactive tracer.

10. The use of the radiotracer according to any one of claims 6-8 or the radiotracer prepared by the preparation method according to claim 9 in the preparation of formulations for detecting PD-L1 expression levels in tumors or in the preparation of formulations for diagnosing tumors with high PD-L1 expression, characterized in that, The tumor is selected from any one of lung cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, thyroid cancer, and head and neck tumors.

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