A compound targeting CDK4 / 6 and a radioactive tracer derived therefrom

Abstract

The present invention relates to the fields of medicinal chemistry, radiopharmaceutical chemistry, and clinical nuclear medicine, and in particular to a compound targeting CDK4 / 6 and a radioactive tracer derived therefrom. The CDK4 / 6-targeting compound has the structures shown in the following formulas (I) and (II). The radioactive tracer provided by the present invention has strong binding affinity with CDK4 / 6, can accurately locate CDK4 / 6 in vivo, exhibits excellent in vivo targeting performance, and achieves tumor molecular imaging through nuclear medicine imaging. Formula (I); Formula (II).

Description

Technical Field

[0001] The present invention relates to the technical fields of medicinal chemistry, radiopharmaceutical chemistry and clinical nuclear medicine, and in particular to a compound targeting CDK4 / 6 and a radioactive tracer derived therefrom. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Cyclin-dependent kinases 4 / 6 (CDK4 / 6) are essential regulators of the cell cycle. Dysfunction of CDK4 / 6 leads to uncontrolled cell proliferation, thereby promoting tumor development and progression. CDK4 / 6 inhibitors are small molecule drugs that directly target the cyclin-dependent kinases CDK4 and CDK6, arresting the cell cycle in the G1 phase, thereby halting cell proliferation and inhibiting tumor growth. However, some tumor cells can activate resistance-associated signaling pathways, ultimately leading to reduced sensitivity to CDK4 / 6 inhibitors. Therefore, dynamic and specific monitoring of CDK4 / 6 expression levels, timely assessment of therapeutic efficacy, and guidance of personalized treatment strategies are crucial for prolonging patient survival and improving prognosis.

[0004] Common methods for monitoring CDK4 / 6 in tumors include imaging techniques (X-rays, CT, MRI, and ultrasound), tumor marker testing, and molecular biological testing (fluorescence in situ hybridization, reverse transcription polymerase chain reaction, high-throughput sequencing, and immunohistochemistry). However, these methods have the following problems: Imaging techniques typically evaluate the efficacy of anti-tumor drugs by measuring morphological changes in tumor lesions, but tumor volume changes only become noticeable two weeks after chemotherapy, resulting in a certain lag; tumor marker expression levels are affected by multiple factors such as inflammation, infection, and medications, and are subject to certain fluctuations, with significant individual differences; molecular biological testing requires invasive sampling of patients, and CDK4 / 6 expression is heterogeneous in different parts of tumor tissue, making local tissue specimens unable to fully assess the overall disease status.

[0005] With the rapid development and in-depth exploration of medical imaging research, nuclear medicine PET / CT imaging, which utilizes the interaction of radioactive tracers with specific gene products, has provided new insights into the efficacy of cancer treatment. Building on molecular recognition and molecular tracing technologies, it enables the visual tracking of targets in vivo without the need for invasive procedures. It can comprehensively and accurately diagnose lesions, overcoming tumor heterogeneity and sampling bias. It can reveal early pathological changes after treatment, providing guidance for personalized treatment plans. Furthermore, it eliminates the need for ex vivo testing, allowing for in vivo visualization and assessment of dynamic pathophysiological changes through molecular imaging. Based on these advantages, nuclear medicine PET / CT imaging can noninvasively monitor CDK4 / 6 kinase activity to assess the efficacy of CDK4 / 6-targeted therapies, providing the potential for timely and accurate assessment of the efficacy of anti-cancer drugs. Summary of the Invention

[0006] In order to overcome the above problems, the present invention provides a compound targeting CDK4 / 6 and a radioactive tracer derived therefrom.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a compound targeting CDK4 / 6, wherein the compound targeting CDK4 / 6 has the structure shown in the following formula (I) and formula (II):

[0009]

[0010] Formula (I);

[0011]

[0012] Formula (II).

[0013] The compounds represented by formula (I) and formula (II), in addition to the small molecule entities having the above-mentioned structures, also include physiologically acceptable salts of the compounds that belong to the technical solutions under the same concept as the first aspect of the present invention and belong to the technical content of the present invention.

[0014] Physiologically acceptable salts refer to organic and inorganic salts of the compounds of the present invention. Physiologically acceptable salts are well known to those skilled in the art. Physiologically acceptable salts include, but are not limited to, inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, perchlorates, and the like, and organic acid salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates, and the like, or salts obtained by other methods described in the literature, such as ion exchange methods.

[0015] The second aspect of the present invention provides an intermediate of the compound targeting CDK4 / 6 described in the first aspect, 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);

[0016]

[0017] Formula (III);

[0018]

[0019] Formula (IV);

[0020] The structure of -NHCbz in formula (III) and formula (IV) is .

[0021] The third aspect of the present invention provides a method for preparing the compound targeting CDK4 / 6 according to the first aspect, comprising the following steps:

[0022] The Cbz protection of the compound represented by formula (III) is removed, and then a chelating agent tetraazacyclododecane tetraacetic acid-succinimidyl ester (DOTA-NHS) and N,N-diisopropylethylamine (DIPEA) are added for condensation reaction to obtain the compound represented by formula (I) targeting CDK4 / 6;

[0023] The Cbz protection of the compound represented by formula (IV) is removed, and then a chelating agent tetraazacyclododecane tetraacetic acid-succinimidyl ester (DOTA-NHS) and N,N-diisopropylethylamine (DIPEA) are added for condensation reaction to obtain a compound represented by formula (II) targeting CDK4 / 6;

[0024] Wherein, the structural formula of formula (III) is as follows:

[0025] ;

[0026] The structural formula of formula (IV) is shown below:

[0027] .

[0028] In one or more embodiments, the method for removing Cbz protection from the compound represented by formula (III) comprises:

[0029] The compound represented by formula (III) is dissolved in methanol and reacted with palladium carbon under a hydrogen atmosphere to obtain the first intermediate without Cbz protection.

[0030] In one or more embodiments, the molar ratio of the compound represented by formula (III) to tetraazacyclododecane tetraacetic acid-succinimidyl ester (DOTA-NHS) and N,N-diisopropylethylamine (DIPEA) is: (23-27): (35-40): (135-160), preferably 25:38:150.

[0031] In one or more embodiments, the method for preparing the compound represented by formula (III) comprises:

[0032] Compound 1 is dissolved in a first organic solvent, and N-carbobenzoxy-6-aminohexanoic acid, N,N-diisopropylethylamine (DIPEA), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) are added to react to obtain a compound represented by formula (III);

[0033] The structural formula of compound 1 is shown below:

[0034] .

[0035] Preferably, the first organic solvent is N,N-dimethylformamide (DMF).

[0036] Preferably, the molar ratio of compound 1, N-benzyloxycarbonyl-6-aminohexanoic acid, N,N-diisopropylethylamine (DIPEA) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) is (110-120):(165-180):(220-240):(165-180), preferably 115:173:230:173.

[0037] Preferably, the concentration of compound 1 in the first organic solvent is 0.367-0.4 mol / L, preferably 0.38 mol / L.

[0038] In one or more embodiments, the method for removing Cbz protection from the compound represented by formula (IV) comprises:

[0039] The compound represented by formula (IV) is dissolved in methanol and reacted with palladium carbon under a hydrogen atmosphere to obtain a second intermediate without Cbz protection.

[0040] In one or more embodiments, the molar ratio of the compound represented by formula (IV) to tetraazacyclododecane tetraacetic acid-succinimide ester (DOTA-NHS) and N,N-diisopropylethylamine (DIPEA) is: (23-27): (35-40): (135-160), preferably 25:38:150.

[0041] In one or more embodiments, the method for preparing the compound represented by formula (IV) comprises:

[0042] Compound 2 was deprotected by Boc, and then N-carbobenzoxy-6-aminohexanoic acid, N,N-diisopropylethylamine (DIPEA), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) were added to react to obtain the compound represented by formula (IV);

[0043] The structural formula of compound 2 is shown below:

[0044] .

[0045] Preferably, the molar ratio of compound 2, N-benzyloxycarbonyl-6-aminohexanoic acid, N,N-diisopropylethylamine (DIPEA) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) is (6.5-7.5):(7.5-9):(14-16):(75-90), preferably 7:8.3:15.4:83.

[0046] The fourth aspect of the present invention provides a radioactive tracer comprising the CDK4 / 6 targeting compound described in the first aspect or the CDK4 / 6 targeting compound prepared by the preparation method described in the third aspect and a radionuclide.

[0047] In one or more embodiments, the radionuclide is selected from 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, 67Cu, 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 One of Dy; preferably 68 Ga.

[0048] Preferably, the structural formula of the radioactive tracer is as shown in formula (V) and formula (VI):

[0049]

[0050] Formula (V);

[0051]

[0052] Formula (VI).

[0053] A fifth aspect of the present invention provides a method for preparing the radioactive tracer described in the fourth aspect, comprising the following steps:

[0054] The radioactive nuclide is mixed with the compound targeting CDK4 / 6 to react and obtain a radioactive tracer.

[0055] In a sixth aspect, the present invention provides use of the radioactive tracer described in the fourth aspect or the radioactive tracer prepared by the preparation method described in the fifth aspect in preparing a preparation for detecting the expression level of CDK4 / 6 in tumors.

[0056] The seventh aspect of the present invention provides use of the radioactive tracer described in the fourth aspect or the radioactive tracer prepared by the preparation method described in the fifth aspect in preparing a preparation for diagnosing CDK4 / 6-overexpressing tumors.

[0057] The above-mentioned tumors include any one of lymphoma, multiple myeloma and solid tumors, such as lung cancer, liver cancer, pancreatic cancer, gastric cancer, colon cancer, thyroid cancer and head and neck tumors, but are not limited thereto.

[0058] The beneficial effects of the present invention are:

[0059] (1) The radioactive tracer provided by the present invention has a strong binding affinity with CDK4 / 6 and can accurately locate CDK4 / 6 in vivo. It has excellent in vivo targeting performance and can achieve the purpose of tumor molecular imaging through nuclear medicine imaging.

[0060] (2) The present invention realizes non-invasive visualization of CDK4 / 6 expression, investigates the tumor imaging effect of radioactive tracers through small animal PET / CT, and realizes non-invasive diagnosis of tumors, thus having good clinical application prospects.

[0061] (3) The CDK4 / 6 targeting compounds and derived radiotracers provided by the present invention have the advantages of simple preparation process, low cost, high specificity, high stability in vivo and in vitro, long imaging cycle, and easy clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0063] Figure 1 The synthetic route of the compound targeting CDK4 / 6 represented by formula (I);

[0064] Figure 2 is the H NMR spectrum of the compound represented by formula (III);

[0065] Figure 3 is the C NMR spectrum of the compound represented by formula (III);

[0066] Figure 4 is a high-resolution mass spectrum of the compound represented by formula (III);

[0067] Figure 5 is a high-resolution mass spectrum of the compound represented by formula (I);

[0068] Figure 6 A synthetic route for a compound targeting CDK4 / 6 as shown in formula (II);

[0069] Figure 7 is the H NMR spectrum of the compound represented by formula (Ⅳ);

[0070] Figure 8 is the NMR carbon spectrum of the compound represented by formula (Ⅳ);

[0071] Figure 9 is a high-resolution mass spectrum of the compound represented by formula (IV);

[0072] Figure 10 is a high-resolution mass spectrum of the compound represented by formula (II);

[0073] Figure 11for[ 68 Ga]Ga-PYO2 radio-HPLC analysis results;

[0074] Figure 12 for[ 68 Ga]Ga-PYO4 radio-HPLC analysis results;

[0075] Figure 13 For radioactive tracers [ 68 In vitro stability study results of Ga]Ga-PY02, where a is the in vitro PBS stability and b is the in vitro human serum stability;

[0076] Figure 14 For radioactive tracers [ 68 In vivo stability study results of Ga]Ga-PY02, where a represents serum stability, b represents liver stability, and c represents kidney stability;

[0077] Figure 15 For radioactive tracers [ 68 In vitro stability study results of Ga]Ga-PY04, where a is the in vitro PBS stability and b is the in vitro human serum stability;

[0078] Figure 16 For radioactive tracers [ 68 In vivo stability study results of Ga]Ga-PY04, where a represents serum stability, b represents liver stability, and c represents kidney stability;

[0079] Figure 17 For radioactive tracers [ 68 Ga]Ga-PY02 and radioactive tracers [ 68 Ga]Ga-PY04 saturation curve, where a is the radioactive tracer [ 68 Ga]Ga-PY02, b is a radioactive tracer [ 68 Ga]Ga-PY04;

[0080] Figure 18 For radioactive tracers [ 68 Ga]Ga-PY02 and radioactive tracers [ 68 Ga]Ga-PY04 uptake curve, where a is the radioactive tracer [ 68 Ga]Ga-PY02, b is a radioactive tracer [ 68 Ga]Ga-PY04;

[0081] Figure 19 For radioactive tracers [ 68 Ga]Ga-PY02 and radioactive tracers [ 68Blood clearance curve of Ga]Ga-PY04; where a is the radioactive tracer [ 68 Ga]Ga-PY02, b is a radioactive tracer [ 68 Ga]Ga-PY04;

[0082] Figure 20 Small animal PET / CT imaging, where a is the experimental group and b is the inhibition group;

[0083] Figure 21 is the biodistribution result;

[0084] Figure 22 is phosphor screen radioautography, where a is a radioactive tracer [ 68 Ga]Ga-PY02, b is a radioactive tracer [ 68 Ga]Ga-PY04;

[0085] Figure 23 The expression of CDK4 and CDK6 in HCC78 cells was determined by Western blot. A is the HCC78 CDK4 / 6 Western blot; B is the grayscale value ratio data of the target protein / reference protein in the protein blot. DETAILED DESCRIPTION

[0086] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0087] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0088] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0089] Cell culture:

[0090] HCC78 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-gentamicin (PSG) in a humidified incubator at 37°C and 5% CO2.

[0091] Establishment of HCC78 tumor-bearing mouse model:

[0092] HCC78 cells were injected subcutaneously into the right shoulder of NSG mice at a rate of 2 × 10 6 To construct a lung metastasis model, HCC78 cells (4×10 5 A lung metastasis model was established by injecting 100 μL of tumor cells (100 μL of tumor cells suspended in 50 μL of PBS) into the tail vein. Imaging and in vivo distribution experiments were performed after tumors grew to a defined size.

[0093] Example 1

[0094] Figure 1 The synthetic route of the compound targeting CDK4 / 6 shown in formula (I) is as follows: Figure 1 , synthesized the CDK4 / 6 targeting compound PY02.

[0095] Compound 1 (50 mg, 0.115 mmol) was dissolved in DMF (0.3 mL), and N-carbobenzoxy-6-aminohexanoic acid (45 mg, 0.173 mmol), DIPEA (29 mg, 0.23 mmol), and HATU (65 mg, 0.173 mmol) were added. The mixture was stirred at room temperature for 8 h, and the reaction was monitored by thin-layer chromatography (TLC). After completion of the reaction, the mixture was quenched with water and extracted with ethyl acetate. The combined extracts were dried over Na₂SO₄, filtered, and the solvent was evaporated on a rotary evaporator. Column chromatography afforded the compound represented by formula (III) (59 mg, 75% yield).

[0096] The H NMR spectrum of the compound represented by formula (III) is as follows Figure 2 As shown, the NMR carbon spectrum is Figure 3 As shown in the high-resolution mass spectrum Figure 4 shown.

[0097] The compound represented by formula (III) (17 mg, 0.025 mmol) was dissolved in methanol (1 mL), and Pd / C (Pd loading of 10%, 2 mg) was added. The reaction was allowed to proceed under a hydrogen atmosphere for 8 h, monitored by TLC. After completion of the reaction, the reaction solution was filtered through celite and rinsed with a 5:1 (volume ratio) DCM:CH3OH mixture. The solvent was then evaporated to dryness to obtain the first intermediate without Cbz protection. The first intermediate without Cbz protection was then dissolved in DMF (0.3 mL), and DOTA-NHS (19 mg, 0.038 mmol) and DIPEA (19 mg, 0.15 mmol) were added. The mixture was stirred at room temperature for 5 h. After completion of the reaction, the reaction system was purified by HPLC. The HPLC purification method is shown in Table 1. After lyophilization, the CDK4 / 6-targeting compound PY02, represented by formula (I), was obtained (7 mg, 30% yield).

[0098] Table 1 HPLC purification method (flow rate: 3 mL / min)

[0099]

[0100] The high-resolution mass spectrum of the compound represented by formula (I) is as follows Figure 5 shown.

[0101] Example 2

[0102] Figure 6 The synthetic route of the compound targeting CDK4 / 6 shown in formula (II) is as follows: Figure 6 , synthesized the CDK4 / 6 targeting compound PY04.

[0103] Compound 2 (44 mg, 0.07 mmol) was dissolved in DCM (0.7 mL), and trifluoroacetic acid (TFA) (159 mg, 1.4 mmol) was added. The mixture was stirred at room temperature for 0.5 h and monitored by TLC. After the reaction, the solvent was evaporated and DMF (0.7 mL) was added. N-carbobenzoxy-6-aminohexanoic acid (22 mg, 0.083 mmol), DIPEA (19 mg, 0.154 mmol), and HATU (58 mg, 0.83 mmol) were then added. The mixture was allowed to react at room temperature for 8 h and monitored by TLC. After the reaction was completed, the mixture was quenched with water and extracted with ethyl acetate. The combined extracts were dried over Na2SO4, filtered, and the solvent was evaporated on a rotary evaporator. Column chromatography afforded the compound represented by formula (IV) (41 mg, 75% yield).

[0104] The H NMR spectrum of the compound represented by formula (Ⅳ) is as follows Figure 7 As shown, the NMR carbon spectrum is Figure 8 As shown in the high-resolution mass spectrum Figure 9 shown.

[0105] The compound represented by formula (IV) (20 mg, 0.025 mmol) was dissolved in methanol (1 mL), and Pd / C (Pd loading of 10%, 2 mg) was added. The reaction was allowed to proceed under a hydrogen atmosphere for 8 h, monitored by TLC. After completion of the reaction, the reaction solution was filtered through celite and rinsed with a 5:1 (volume ratio) DCM:CH3OH mixture. The solvent was then evaporated to obtain the Cbz-deprotected second intermediate. The Cbz-deprotected second intermediate was then dissolved in DMF (0.3 mL), and DOTA-NHS (19 mg, 0.038 mmol) and DIPEA (19 mg, 0.15 mmol) were added. The mixture was stirred at room temperature for 5 h. After completion of the reaction, the reaction mixture was purified by HPLC and lyophilized to obtain the CDK4 / 6-targeting compound PY04 (represented by formula (II)) (11 mg, 42% yield).

[0106] The high-resolution mass spectrum of the compound represented by formula (II) is shown in FIG. Figure 10 shown.

[0107] Example 3

[0108] 4 mL of high-purity hydrochloric acid (0.1 M) was used to 68 Ge- 68 Ga generator eluted 68 GaCl3 eluent; 3.7 nmol of compound PY02 or PY04 was mixed with 200 µL 68 The GaCl3 eluate (74 MBq~100 MBq) was mixed, 200 μL of acetic acid-sodium acetate buffer solution (0.25 M) was added, the pH of the reaction system was adjusted to 4~4.6, 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-PY02 or [ 68 Ga]Ga-PY04.

[0109] Radioactive tracer detection by radio-HPLC [ 68 Ga]Ga-PY02 and [ 68 The radiochemical purity (RCP) of Ga]Ga-PYO4 and the analysis method of radio-HPLC are shown in Table 2.

[0110] Table 2 Radioactive HPLC analysis method (flow rate: 1 mL / min)

[0111]

[0112] [68 Ga]Ga-PY02 and [ 68 The radioactive HPLC analysis results of Ga]Ga-PYO4 are as follows Figure 11 and Figure 12 As shown, from Figure 11 It can be seen that [ 68 The retention time of Ga]Ga-PY02 was 10.90 min, RCP>98%, and specific activity was 39±0.2 GBq / µmol; Figure 12 It can be seen that [ 68 The retention time of Ga]Ga-PY04 was 11.67 min, the RCP was > 98%, and the specific activity was 31 ± 0.21 GBq / µmol.

[0113] The calculation formula for specific activity is:

[0114] Specific activity (GBq / µmol) = radioactivity (GBq) / amount of substance of the precursor compound (µmol).

[0115] Example 4

[0116] Radioactive tracers 68 Ga]Ga-PY02 and [ 68 Study on the stability of Ga]Ga-PY04 in vitro and in vivo:

[0117] (1) In vitro stability study:

[0118] PBS stability study of radiotracer: 100 μL of radiotracer was mixed with 100 μL of PBS and incubated at 37 °C for 1 h. After the incubation, 100 μL of the mixture was taken and the RCP of the radiotracer was analyzed by radio-HPLC.

[0119] In vitro serum stability study of the radiotracer: 100 μL of radiotracer was mixed with 100 μL of human serum and incubated at 37°C for 1 h. After the incubation, 200 μL of anhydrous ethanol was added to the incubation system, and then the incubation system was centrifuged at 12,000 rpm for 5 min. The supernatant was collected, filtered, and the RCP of the radiotracer was analyzed by radio-HPLC.

[0120] (2) In vivo stability study:

[0121] In vivo serum stability study of the radiotracer: Normal BALB / c mice were injected with 37 MBq of radiotracer via the tail vein, and the mice were sacrificed 1 hour later, and serum samples were collected. Subsequently, two volumes of anhydrous ethanol were added to the serum samples, and the samples were centrifuged at 12,000 rpm for 5 minutes. The supernatant was collected, filtered, and the RCP of the radiotracer was analyzed by radio-HPLC.

[0122] In vivo renal and liver stability studies of the radiotracer: Normal BALB / c mice were injected with 37 MBq of the radiotracer via the tail vein and sacrificed 1 hour later. Kidney and liver samples were collected. The kidney and liver samples were ground to obtain renal and liver tissue fluids, and then appropriate amounts of anhydrous ethanol were added to the two tissue fluids. The mixtures were centrifuged at 12,000 rpm for 5 minutes. The supernatant was collected, filtered, and the RCP of the radiotracer was analyzed by radio-HPLC.

[0123] Radioactive tracers 68 The results of in vitro stability study of Ga]Ga-PY02 are as follows Figure 13 As shown, from Figure 13 As can be seen in the figure, the radiotracer [ 68 The in vitro stability of Ga]Ga-PY02 in PBS and human serum was good, and the RCP at 1 h was greater than 90%.

[0124] Radioactive tracers 68 The results of the in vivo stability study of Ga]Ga-PY02 are as follows Figure 14 As shown, from Figure 14 As can be seen in the figure, the radiotracer [ 68 Ga]Ga-PY02 has good stability in serum, liver and kidney in vivo.

[0125] Radioactive tracers 68 The results of the in vitro stability study of Ga]Ga-PY04 are as follows Figure 15 As shown, from Figure 15 As can be seen in the figure, the radiotracer [ 68 The in vitro stability of Ga]Ga-PY04 in PBS and human serum was good, and the RCP at 1 h was greater than 90%.

[0126] Radioactive tracers 68 The results of the in vivo stability study of Ga]Ga-PY04 are as follows Figure 16 As shown, from Figure 16 As can be seen in the figure, the radiotracer [ 68 Ga]Ga-PY04 has good stability in serum, liver and kidney in vivo.

[0127] Example 5

[0128] Hydrophilicity and lipophilicity study:

[0129] 5 μL of radioactive tracer 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 layer (n-octanol) and 400 μL of the lower layer (HEPES buffer solution) were collected in centrifuge tubes and centrifuged at 12000 rpm for 5 min. Finally, 100 μL of the upper layer (n-octanol) and 100 μL of the lower layer (HEPES buffer solution) were collected from the re-centrifuged system, and the radioactivity count was measured using 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)).

[0130] Calculation of radioactive tracer [ 68 Ga]Ga-PY02 and radioactive tracers [ 68 The oil-water distribution coefficient D (log D ) were -2.24±0.10 and -2.49±0.04, respectively; both have excellent hydrophilicity.

[0131] Example 6

[0132] Radiotracer Cell Receptor Affinity Studies:

[0133] (1) Saturation experiment to determine the equilibrium dissociation constant ( K d To evaluate the binding affinity of radiotracer to CDK4 / 6, saturation binding experiments were performed on HCC78 cells. 5 Total binding was determined by incubating cells with different concentrations of radiotracer (0.625-40 nM) for 1 h. To determine nonspecific binding, cells were pretreated with a 2000-fold molar excess of compound 1 before the experiment. Both the total and nonspecific binding groups were washed three times with cold PBS and then lysed with 200 μL / well of NaOH solution (1 M). The radioactivity counts of the cell lysates were measured using a gamma counter. All experiments were repeated three times.

[0134] (2) Cell uptake and internalization experiment: HCC78 cells (4×10 5Cells were seeded in 24-well plates (100 μL / well) and incubated with the radiotracer for 15, 30, 60, 90, and 120 minutes to assess its uptake over time. For blocking experiments, cells were pre-incubated with compound 1 (400 nM, 2000-fold molar excess) for 1 hour before the addition of the radiotracer. After incubation, the medium was removed, and the cells were washed with PBS and lysed with 200 μL of 1 M NaOH. Radioactivity was then measured using a gamma counter. All experiments were performed in triplicate.

[0135] Radioactive tracers 68 Ga]Ga-PY02 and radioactive tracers [ 68 The saturation curve of Ga]Ga-PYO4 is as follows Figure 17 As shown, the radiotracer [ 68 Ga]Ga-PY02 K d was 49.73±0.62 nM, and the radiotracer [ 68 Ga]Ga-PY04 K d was 52.51±0.43 nM; Figure 17 as well as K d The values ​​show that the radiotracer [ 68 Ga]Ga-PY02 and radioactive tracers [ 68 Ga]Ga-PY04 has a good affinity with HCC78 cells. K d The values ​​are all in nM level.

[0136] Radioactive tracers 68 Ga]Ga-PY02 and radioactive tracers [ 68 The uptake curve of Ga]Ga-PYO4 is as follows Figure 18 As shown, from Figure 18 As can be seen in the figure, the radiotracer [ 68 Ga]Ga-PY02 and radioactive tracers [ 68 Ga]Ga-PY04 showed rapid and specific uptake within 15 min, which gradually increased over time until reaching a plateau at 90 min; the uptake inhibition of compound 1 confirmed the radiotracer [ 68 Ga]Ga-PY02 and radioactive tracers [ 68 Ga]Ga-PY04 binds specifically to CDK4 / 6.

[0137] Example 7

[0138] Normal BALB / c male mice (n=3) were injected with radioactive tracer via the tail vein.68 Ga]Ga-PY02 or [ 68 Ga]Ga-PY04 (7.4 MBq each). Blood was then collected from the mice at 1, 2, 5, 10, 15, 30, 45, 60, 90, and 120 min, weighed, and radioactivity counted using a gamma counter. %ID / g was calculated, and the blood elimination half-life of each radiotracer was obtained by fitting the blood clearance curve using Drug And Statistics (DAS) software. %ID / g was calculated as follows: %ID / g = ((measured radioactivity count) / (total injected radioactivity count)) / tissue mass (including blood).

[0139] Radioactive tracers 68 Ga]Ga-PY02 and radioactive tracers [ 68 The blood clearance curve of Ga]Ga-PY04 is as follows Figure 19 As shown, from Figure 19 It can be seen that the pharmacokinetic curves of each radiotracer in normal mice conform to the two-compartment model. 68 The half-life of the distribution phase of Ga]Ga-PYO2 (t 1 / 2α ) is 1.684 minutes, and the elimination phase half-life (t 1 / 2β ) is 51.933 minutes, and the blood metabolism time is moderate. Radioactive tracer [ 68 The half-life of the distribution phase of Ga]Ga-PY04 (t 1 / 2α ) is 1.854 minutes, and the elimination phase half-life (t 1 / 2β ) is 61.461 minutes, and the blood metabolism time is moderate.

[0140] Example 8

[0141] Small Animal PET / CT Imaging:

[0142] HCC78 tumor-bearing mice were randomly divided into experimental group (n=3) and blocking group (n=3), and then injected with radiotracer [ 68 Ga]Ga-PY02 or radioactive tracer [ 68 Small animal PET / CT imaging was performed using Ga]Ga-PY04. In the experimental group, each tumor-bearing mouse received a 200 μL injection of the radiotracer (3.7 MBq) via the tail vein under 2% isoflurane in oxygen. In the blockade group, tumor-bearing mice received a tail vein injection of the radiotracer and unlabeled compound 1 (20 mg / kg), followed by 1 hour of PET / CT imaging. Following imaging, PET / CT images were reconstructed using Nucline NanScan 3.00 software, and image analysis was performed using InterView FUSION 3.0 software.

[0143] The results are as follows Figure 20 As shown, from Figure 20 It can be seen that the tumor sites in the experimental group had obvious [ 68 Ga]Ga-PY02 and [ 68 The radioactive uptake of Ga]Ga-PY04 was significantly inhibited, and the radioactive accumulation of tumors in the inhibition group was significantly reduced, indicating that the radioactive tracer specifically targets CDK4 / 6 protein and can be used for imaging and efficacy monitoring of CDK4 / 6 protein-expressing tumors.

[0144] Example 9

[0145] Biological distribution:

[0146] Under gas anesthesia (2% isoflurane in oxygen), HCC78 tumor-bearing mice (n=3) were injected with 100 μL of radiotracer via the tail vein. 68 Ga]Ga-PY02 or radioactive tracer [ 68 Ga]Ga-PY04 (3.7 MBq). 1.5 hours after injection, HCC78 tumor-bearing mice were bled by eye. Following sacrifice, vital tissues and organs, including tumor, muscle, bone, kidney, spleen, liver, intestine, heart, lung, and stomach, were collected. The collected blood and tissues were weighed, and radioactivity counts were measured using a gamma counter. Tissue counts were compared with calibration counts of the injected radiotracer to obtain %ID / g and target / non-target (T / NT) ratios.

[0147] The results are as follows Figure 21 As shown in Table 3, in the HCC78 tumor-bearing mouse model, the injection of radiotracer [ 68 Ga]Ga-PY02 and [ 68 60 min after administration of Ga]Ga-PY04, tumor uptake was 1.66±0.47%ID / g and 2.77±0.29%ID / g, respectively, indicating tumor targeting of the radiotracer in CDK4 / 6-positive tumors.

[0148] Table 3 Radioactive tracers [ 68 Ga]Ga-PY02, [ 68 Study on the Biodistribution of Ga]Ga-PY04

[0149]

[0150] Example 10

[0151] Phosphorus autoradiography: Under gas anesthesia (2% isoflurane in oxygen), 100 μL of radiotracer was injected into the tail vein of HCC78 tumor-bearing mice (n=3). 68 Ga]Ga-PY02 or radioactive tracer [ 68Ga]Ga-PY04 (3.7 MBq). One and a half hours after injection, HCC78 tumor-bearing mice were bled from their eyeballs. Following sacrifice, tumors, muscles, and kidneys were collected. The tumors, muscles, and kidneys were placed on a phosphor screen and exposed to light for 10 minutes in the dark. Images were then acquired using a phosphor screen imaging system.

[0152] The results are as follows Figure 22 As shown, from Figure 22 As can be seen in the results of phosphor screen analysis, the results were highly consistent with the PET / CT imaging and biodistribution results, further confirming the radiotracer[ 68 Ga]Ga-PY02 and radioactive tracers [ 68 Specific distribution pattern of Ga]Ga-PY04 in vivo.

[0153] Example 11

[0154] Western blot analysis of CDK4 and CDK6 expression in HCC78 cells:

[0155] Western blot analysis was performed to assess the expression of CDK4 and CDK6 in HCC78 cells. Total protein was extracted from HCC78 cells using RIPA lysis buffer on ice. After protein quantification and heat denaturation, equal amounts of protein (5 µL per sample) were loaded onto a 10% SDS-PAGE gel, electrophoresed at 80 V for the stacking gel and 120 V for the separating gel. Proteins were transferred to a 0.45 µm PVDF membrane pre-activated with methanol (200 mA, 90 min), blocked with 5% nonfat dry milk for 30 min, and incubated with a CDK4 antibody (Bioworld Technology, MB10716) at room temperature for 2 h. The membrane was washed three times with TBST for 10 min each.

[0156] To detect CDK6, residual ECL and antibodies were washed away after development, the membrane was blocked again, and incubated with CDK6 antibody (BosterBiological Technology, MB1616) and HRP-conjugated goat anti-rabbit secondary antibody (Servicebio™, GB23303), and the washing and development steps were repeated.

[0157] Subsequently, to detect the internal control β-actin, the elution, blocking, and antibody incubation steps were repeated using a β-actin antibody (Servicebio™, GB15003) and the same secondary antibody. Finally, bands were captured using a TANON 4200 imaging system and quantitatively analyzed using ImageJ software.

[0158] The results are as follows Figure 23 As shown, from Figure 23As can be seen in Figure 3, CDK4 and CDK6 are specifically expressed in HCC78 cells.

[0159] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A radioactive tracer, characterized in that The structural formulas of the radioactive tracer are shown in formula (V) and formula (VI): Formula (V); Formula (VI).

2. The method for preparing the radioactive tracer according to claim 1, characterized in that: The steps include: Radionuclides 68 Ga is mixed with the compound targeting CDK4 / 6 to react and obtain a radioactive tracer; Wherein, the compound targeting CDK4 / 6 has the structure shown in the following formula (I) and formula (II): Formula (I); Formula (II).

3. The method for preparing the radioactive tracer according to claim 2, characterized in that: The method for preparing the compound targeting CDK4 / 6 comprises the following steps: The Cbz protection of the compound represented by formula (III) is removed, and then a chelating agent tetraazacyclododecane tetraacetic acid-succinimide ester and N,N-diisopropylethylamine are added to carry out a condensation reaction to obtain a compound represented by formula (I) targeting CDK4 / 6; The Cbz protection of the compound represented by formula (IV) is removed, and then a chelating agent tetraazacyclododecane tetraacetic acid-succinimide ester and N,N-diisopropylethylamine are added to carry out a condensation reaction to obtain a compound represented by formula (II) targeting CDK4 / 6; Wherein, the structural formula of formula (III) is as follows: ； The structural formula of formula (IV) is shown below: 。 4. The method for preparing a radioactive tracer according to claim 3, wherein: The preparation method of the compound represented by formula (III) comprises: Dissolving compound 1 in a first organic solvent, adding N-benzyloxycarbonyl-6-aminocaproic acid, N,N-diisopropylethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and reacting to obtain a compound represented by formula (III); The structural formula of compound 1 is shown below: ； The preparation method of the compound represented by formula (IV) comprises: Compound 2 was deprotected by Boc, and then N-benzyloxycarbonyl-6-aminohexanoic acid, N,N-diisopropylethylamine, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate were added to react to obtain the compound represented by formula (IV); The structural formula of compound 2 is shown below: 。 5. The method for preparing a radioactive tracer according to claim 4, wherein: The molar ratio of the compound 1, N-benzyloxycarbonyl-6-aminohexanoic acid, DIPEA and HATU is (110-120): (165-180): (220-240): (165-180); the molar ratio of the compound 2, N-benzyloxycarbonyl-6-aminohexanoic acid, DIPEA and HATU is (6.5-7.5): (7.5-9): (14-16): (75-90).

6. Use of the radioactive tracer according to claim 1 in preparing a preparation for detecting the expression level of CDK4 / 6 in tumors.

7. Use of the radioactive tracer according to claim 1 in the preparation of a preparation for diagnosing CDK4 / 6 overexpressing tumors.

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