A molecular imaging probe targeting cdk4 / 6, a labeling precursor, a probe and a preparation method and application thereof

Abstract

The present application relates to the fields of medicinal chemistry, radiopharmaceutical chemistry and clinical nuclear medicine technology, in particular to a kind of molecular imaging probe targeting CDK4 / 6 labeled precursor, probe and preparation method and application thereof.The molecular imaging probe provided by the present application [68 Ga]Ga-PY03 is based on Ribociclib structure, by introducing PEG1 chain and proline with envelope conformation, not only can fill the hydrophobic region in protein binding pocket, enhance van der waals force, so that the molecular imaging probe [68 Ga]Ga-PY03 can be stably combined with CDK4 / 6, thereby ensuring the targeting of the molecular imaging probe to CDK4 / 6;It can also regulate the hydrophilicity of the molecular imaging probe, reduce kidney uptake;And excellent in tumor / liver and tumor / muscle ratio, can effectively improve tumor uptake.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of pharmaceutical chemistry, radiopharmaceutical chemistry and clinical nuclear medicine technology, in particular to a kind of molecular imaging probe labeled precursor targeting CDK4 / 6, probe and its preparation method and application. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission or a recognition that any of this information constitutes prior art merely by virtue of its inclusion in this section.

[0003] The occurrence and development of tumors are related to cell cycle, and abnormal activation of cell cycle leads to uncontrolled proliferation of cancer cells. Cyclin-dependent kinase 4 / 6 (CDK4 / 6) is a key kinase that regulates the transition of cells from the G1 phase to the S phase of the cell cycle. After binding with cyclin D, CDK4 / 6 phosphorylates RB1, releases E2F transcription factor, and thus increases the transcription of E2F target genes CCNE1 and CCNE2, which jointly promote the complete cell cycle process. Studies have shown that the continuous activation of CDK4 / 6 promotes the occurrence and development of various malignant tumors. Therefore, CDK4 / 6 has been regarded as an important target for anti-tumor drug therapy in recent years, and CDK4 / 6 inhibitors are also popular in drug research. The clinical application of CDK4 / 6 inhibitors has achieved breakthroughs in the treatment of breast cancer first. In addition to breast cancer, clinical trials of CDK4 / 6 inhibitors for the treatment of non-small cell lung cancer, liver cancer, melanoma, ovarian cancer, glioblastoma, mantle cell lymphoma, etc. are in progress. CDK4 / 6 inhibitors are new targeted drugs with great clinical application prospects, so how to accurately determine the activity of CDK4 / 6 and the efficacy of anti-tumor drugs at an early stage is an important problem in clinical practice.

[0004] Common diagnostic methods for monitoring CDK4 / 6 in tumors include imaging technology examination (X-ray, CT, MRI and ultrasound), tumor marker detection, molecular biology detection (fluorescence in situ hybridization, reverse transcription polymerase chain reaction, high-throughput sequencing technology and immunohistochemistry), etc. These methods have the following problems: imaging technology examination usually measures morphological changes of tumor lesions to evaluate the efficacy of anti-tumor drugs, but the tumor volume will not change significantly until two weeks after chemotherapy treatment, which has a certain lag; the expression level of tumor markers is affected by many factors such as inflammation, infection and drugs, and there is a certain fluctuation, and the individual level difference is large; molecular biology detection requires invasive sampling of patients, and CDK4 / 6 expression is heterogeneous in different parts of tumor tissue, so local tissue specimens cannot fully evaluate the overall condition of the disease.

[0005] With the rapid development and in-depth exploration of medical imaging research, nuclear medicine PET / CT imaging, which utilizes the interaction of molecular imaging probes with specific gene products, has provided new insights into evaluating the efficacy of tumor 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 can visually assess dynamic pathophysiological changes in vivo through molecular imaging, eliminating the need for ex vivo testing. 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.

[0006] However, the currently available molecular imaging probes targeting CDK4 / 6 have problems such as poor targeting, unclear distinction between tumor and non-tumor areas, and poor hydrophilicity. Summary of the Invention

[0007] In order to overcome the above problems, the present invention provides a molecular imaging probe labeling precursor targeting CDK4 / 6, a probe, and a preparation method and application thereof.

[0008] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0009] In the first aspect of the present invention, a molecular imaging probe labeling precursor targeting CDK4 / 6 is provided, the structural formula of which is shown in formula (I):

[0010]

[0011] Formula (I).

[0012] The second aspect of the present invention provides an intermediate of the molecular imaging probe labeling precursor targeting CDK4 / 6 described in the first aspect, the structural formula of which is shown in formula (II);

[0013]

[0014] Formula (II).

[0015] The third aspect of the present invention provides a method for preparing a molecular imaging probe labeled precursor targeting CDK4 / 6 as described in the first aspect, comprising the following steps:

[0016] The compound represented by formula (II) is subjected to Boc deprotection to obtain a Boc deprotected intermediate, which is then subjected to a condensation reaction with a chelating agent tetraazacyclododecane tetraacetic acid-succinimidyl ester (DOTA-NHS) and N,N-diisopropylethylamine (DIPEA) to obtain the molecular imaging probe labeling precursor targeting CDK4 / 6;

[0017] Wherein, the structural formula of formula (II) is as follows:

[0018]

[0019] Formula (II).

[0020] In one or more embodiments, the method for removing Boc protection from the compound represented by formula (II) to obtain a Boc-protected intermediate includes:

[0021] The compound represented by formula (II) is dissolved in dichloromethane, and trifluoroacetic acid (TFA) is added to react to obtain a Boc-protected intermediate.

[0022] In one or more embodiments, the molar ratio of the compound represented by formula (II), the chelating agent tetraazacyclododecane tetraacetic acid-succinimidyl ester (DOTA-NHS) and N,N-diisopropylethylamine (DIPEA) is (22-27): (35-40): (140-160), preferably 25:38:150.

[0023] The fourth aspect of the present invention provides a molecular imaging probe targeting CDK4 / 6, comprising the molecular imaging probe labeled precursor targeting CDK4 / 6 described in the first aspect or the molecular imaging probe labeled precursor targeting CDK4 / 6 prepared by the preparation method described in the third aspect and a radioactive nuclide.

[0024] In one or more embodiments, the radionuclide is selected from 18 F. 94 Tc, 99 mTc, 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 One of Dy; preferably 68 Ga.

[0025] Preferably, the molecular imaging probe targeting CDK4 / 6 has a structural formula as shown in formula (III),

[0026]

[0027] Formula (III).

[0028] The fifth aspect of the present invention provides a method for preparing the molecular imaging probe targeting CDK4 / 6 according to the fourth aspect, comprising the following steps:

[0029] The radioactive nuclide is mixed with the molecular imaging probe labeling precursor targeting CDK4 / 6 to obtain the molecular imaging probe targeting CDK4 / 6.

[0030] The sixth aspect of the present invention provides the use of the molecular imaging probe targeting CDK4 / 6 described in the fourth aspect or the molecular imaging probe targeting CDK4 / 6 prepared by the preparation method described in the fifth aspect in the preparation of a preparation for detecting the expression level of CDK4 / 6 in tumors.

[0031] The seventh aspect of the present invention provides the use of the molecular imaging probe targeting CDK4 / 6 described in the fourth aspect or the molecular imaging probe targeting CDK4 / 6 prepared by the preparation method described in the fifth aspect in the preparation of a preparation for diagnosing CDK4 / 6-overexpressing tumors.

[0032] 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.

[0033] The beneficial effects of the present invention are:

[0034] (1) The molecular imaging probe provided by the present invention [ 68 Ga]Ga-PY03, based on the Ribociclib structure, introduces PEG1 chains and proline with an envelope conformation, which can not only fill the hydrophobic area in the protein binding pocket and enhance the van der Waals force, but also make the molecular imaging probe [ 68Ga]Ga-PY03 can stably bind to CDK4 / 6, thereby ensuring the targeting of the molecular imaging probe to CDK4 / 6; it can also regulate the hydrophilicity of the molecular imaging probe to reduce renal uptake; and it performs excellently in tumor / liver and tumor / muscle ratios, which can effectively enhance tumor uptake.

[0035] (2) In the present invention, U87MG, ACHN, OVCAR8, PC3, HT29, A549, 4T1, MCF-7 and A375 tumor-bearing mice were tested. 68 Ga]Ga-PY03 PET / CT imaging achieved good imaging results, further proving that the molecular imaging probe [ 68 Ga]Ga-PY03 has high targeting property, thus proving that it can be used as a molecular imaging probe [ 68 Ga]Ga-PY03 can be used for tumor diagnosis and treatment evaluation, and has great clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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.

[0037] Figure 1 A synthetic route for a molecular imaging probe labeled precursor targeting CDK4 / 6 as shown in formula (I);

[0038] Figure 2 is the H NMR spectrum of compound 1;

[0039] Figure 3 is the C NMR spectrum of compound 1;

[0040] Figure 4 is the high-resolution mass spectrum of compound 1;

[0041] Figure 5 This is the H NMR spectrum of the compound represented by formula (II);

[0042] Figure 6 is the C NMR spectrum of the compound represented by formula (II);

[0043] Figure 7 is a high-resolution mass spectrum of the compound represented by formula (II);

[0044] Figure 8 This is the high-resolution mass spectrum of PY03;

[0045] Figure 9 Molecular imaging probes[ 68 Radio-HPLC analysis results of Ga]Ga-PYO3;

[0046] Figure 10 Molecular imaging probes[ 68 In vitro stability study results of Ga]Ga-PY03, where a is the in vitro PBS stability and b is the in vitro human serum stability;

[0047] Figure 11 Molecular imaging probes[ 68 In vivo stability study results of Ga]Ga-PY03, where a represents serum stability, b represents liver stability, and c represents kidney stability;

[0048] Figure 12 Molecular imaging probes[ 68 Ga]Ga-PY01, [ 68 Ga]Ga-PY02, [ 68 Ga]Ga-PY04, [ 68 Ga]Ga-PY05, [ 68 Ga]Ga-PY06,[ 68 Ga]Ga-PY07 and [ 68 The structural formula of Ga]Ga-PY08; where a~g are [ 68 Ga]Ga-PY01, [ 68 Ga]Ga-PY02, [ 68 Ga]Ga-PY04, [ 68 Ga]Ga-PY05, [ 68 Ga]Ga-PY06,[ 68 Ga]Ga-PY07 and [ 68 Ga]Ga-PY08;

[0049] Figure 13 Molecular imaging probes[ 68 Saturation curve of Ga]Ga-PYO3;

[0050] Figure 14 Molecular imaging probes[ 68 Ga]Ga-PY03 uptake curve;

[0051] Figure 15 Molecular imaging probes[ 68 Blood clearance curve of Ga]Ga-PY03;

[0052] Figure 16 for[ 68 Ga]Ga-PY03 and CDK4 binding mode diagram; where (a) is [ 68 Ga]Ga-PY03 position in the three-dimensional structure of the protein; (b) is [ 68 Three-dimensional binding pattern of Ga]Ga-PY03 and protein;

[0053] Figure 17 for[ 68 The binding mode diagram of Ga]Ga-PY03 and CDK6; (a) is [ 68 Ga]Ga-PY03 position in the three-dimensional structure of the protein; (b) is [ 68 Three-dimensional binding pattern of Ga]Ga-PY03 and protein;

[0054] Figure 18 For small animal PET / CT imaging;

[0055] Figure 19 Molecular imaging probes[ 68 Ga]Ga-PY01~[ 68 Radioactive uptake of Ga]Ga-PY08 in different parts;

[0056] Figure 20 Molecular imaging probes[ 68 Ga]Ga-PY01~[ 68 In vivo biodistribution study of Ga]Ga-PY08;

[0057] Figure 21 Molecular imaging probes[ 68 Ga]Ga-PY03 phosphor screen radioautography;

[0058] Figure 22 Western blot analysis of CDK4 and CDK6 expression in HCC78 cells. A is the HCC78 CDK4 / 6 Western blot; B is the target protein / reference protein grayscale ratio data in the Western blot.

[0059] Figure 23 Molecular imaging probes[ 68 Small animal PET / CT imaging of various tumor models using Ga]Ga-PY03;

[0060] Figure 24 PET / CT images of mice with lung orthotopic tumors and healthy mice, where a is lung orthotopic tumors and b is healthy mouse controls;

[0061] Figure 25 Injection of molecular imaging probes into normal lung and in situ tumor tissues[ 68 Ga]Ga-PY03 tissue uptake at 30 min;

[0062] Figure 26 For the injection of molecular imaging probes[ 68PET imaging study of HCC78 tumor-bearing mice in the control group and ribociclib-treated mice 1 h after Ga]Ga-PY03, where a is the ribociclib-treated group and b is the control group;

[0063] Figure 27 is the response of tumor volume to different treatments;

[0064] Figure 28 For PET imaging-based [ 68 Quantitative analysis of tumor uptake of Ga]Ga-PY03;

[0065] Figure 29 The expression status of CDK4 / 6 in A549, MCF-7, HT29, U87MG, HCC78, PC3, ACHN, OVCAR8 and 4T1 tumor tissues. DETAILED DESCRIPTION

[0066] 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.

[0067] 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.

[0068] 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.

[0069] Cell culture:

[0070] U87MG and ACHN cells were cultured in MEM medium containing 1% non-essential amino acid mixture (NEAA; non-essential amino acids include glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-gentamicin mixture (PSG) at 37°C in a humidified incubator with 5% CO2. MCF-7 cells were cultured in MEM medium supplemented with 1% NEAA, 10% FBS, 1% GlutaMAX™, 1% sodium pyruvate, and 1% PSG. A549 cells were cultured in Ham's F-12 medium supplemented with 10% FBS and 1% PSG. HT29 cells were cultured in McCoy's 5A medium supplemented with 10% FBS and 1% PSG. HCC78 cells were cultured in DMEM supplemented with 10% FBS and 1% PSG. PC3, 4T1, and OVCAR8 cells were cultured in RPMI-1640 supplemented with 10% FBS and 1% PSG. % represents the volume fraction.

[0071] Establishment of tumor-bearing mouse model and orthotopic tumor model:

[0072] Tumor cells (U87MG, A549, MCF-7, HT29, U87MG, HCC78, PC3, ACHN, OVCAR8, A375, and 4T1) were subcutaneously injected 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.

[0073] The structural formula of Ribociclib is as follows:

[0074] .

[0075] Example 1

[0076] Figure 1 The synthetic route of the molecular imaging probe labeled precursor targeting CDK4 / 6 as shown in formula (I) is as follows: Figure 1 , synthesized a molecular imaging probe targeting CDK4 / 6 and labeled the precursor PY03.

[0077] Ribociclib (50 mg, 0.115 mmol) was dissolved in DMF (0.3 mL), and tert-butyloxycarbonyl-L-proline (50 mg, 0.23 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 monitored by TLC. After completion, the reaction 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. Compound 1 (54 mg, 74% yield) was obtained by column chromatography.

[0078] The H NMR spectrum of compound 1 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.

[0079] Compound 1 (44 mg, 0.07 mmol) was dissolved in dichloromethane (DCM) (0.7 mL), and 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 removed by rotary evaporation, and DMF (0.7 mL) was added. Boc-NH-PEG1-CH2COOH (18 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 quenched with water, the mixture was extracted with ethyl acetate, and 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 (II) (37 mg, 71% yield).

[0080] The H NMR spectrum of the compound represented by formula (II) is as follows Figure 5 As shown, the NMR carbon spectrum is Figure 6 As shown in the high-resolution mass spectrum Figure 7 shown.

[0081] The compound represented by formula (II) (18 mg, 0.025 mmol) was dissolved in DCM (0.15 mL), and TFA (57 mg, 0.5 mmol) was added. The reaction was allowed to react at room temperature for 0.5 h, monitored by TLC. After completion of the reaction, the solvent was evaporated to dryness. DMF (0.3 mL), DOTA-NHS (19 mg, 0.038 mmol), and DIPEA (19 mg, 0.15 mmol) were then added to the reaction system, and 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 precursor of the molecular imaging probe targeting CDK4 / 6, PY03, was obtained, represented by formula (I) (12 mg, 47% yield).

[0082] Table 1 HPLC purification method (flow rate: 3 mL / min)

[0083]

[0084] The high-resolution mass spectrum of PY03 is as follows Figure 8 shown.

[0085] Example 2

[0086] 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 precursor compound PY03 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, and the pH of the reaction system was adjusted to 4~4.6. The mixture was then kept at 95 °C for 15 minutes. After the reaction was completed, it was cooled to room temperature to obtain a molecular imaging probe targeting CDK4 / 6. 68 Ga]Ga-PY03.

[0087] Detection of molecular imaging probes targeting CDK4 / 6 by radio-HPLC[ 68 The radiochemical purity (RCP) of Ga]Ga-PYO3 and the analysis method of radio-HPLC are shown in Table 2.

[0088] Table 2 Radioactive HPLC analysis method (flow rate: 1 mL / min)

[0089]

[0090] Molecular imaging probes[ 68 The radioactive HPLC analysis results of Ga]Ga-PYO3 are as follows Figure 9 As shown, [68 The retention time of Ga]Ga-PY03 was 10.90 min, the RCP was > 98%, and the specific activity was 39±0.2 GBq / µmol.

[0091] The calculation formula for specific activity is:

[0092] Specific activity (GBq / µmol) = radioactivity (GBq) / amount of substance of the precursor compound (µmol).

[0093] Example 3

[0094] Molecular imaging probes[ 68 Study on the stability of Ga]Ga-PY03 in vitro and in vivo:

[0095] (1) In vitro stability study:

[0096] PBS stability study: 100 µL of the molecular imaging probe 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 molecular imaging probe was analyzed by radio-HPLC.

[0097] In vitro serum stability study: 100 µL of the molecular imaging probe 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 molecular imaging probe was analyzed by radioactive HPLC.

[0098] (2) In vivo stability study:

[0099] In vivo serum stability study of the molecular imaging probe: 37 MBq of the molecular imaging probe was injected into normal BALB / c mice via the tail vein, and the mice were killed 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 molecular imaging probe was analyzed by radioactive HPLC.

[0100] In vivo renal and liver stability studies of the molecular imaging probe: 37 MBq of the molecular imaging probe was injected into normal BALB / c mice via the tail vein, and the mice were killed 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 mixture was centrifuged at 12,000 rpm for 5 minutes. The supernatant was collected, filtered, and the RCP of the molecular imaging probe was analyzed by radioactive HPLC.

[0101] Molecular imaging probes[68 The results of the in vitro stability study of Ga]Ga-PY03 are as follows Figure 10 As shown, from Figure 10 As can be seen in the molecular imaging probe [ 68 The in vitro stability of Ga]Ga-PY03 in PBS and human serum was good, and the RCP at 1 h was greater than 90%.

[0102] Molecular imaging probes[ 68 The results of the in vivo stability study of Ga]Ga-PY03 are as follows Figure 11 As shown, from Figure 11 As can be seen in the molecular imaging probe [ 68 Ga]Ga-PY03 has good stability in serum, liver and kidney in vivo.

[0103] Example 4

[0104] Hydrophilicity and lipophilicity study:

[0105] 5 μL of molecular imaging probe 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 a centrifuge tube 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)).

[0106] Computationally obtained molecular imaging probes[ 68 The oil-water distribution coefficient D (log D ) is -3.00±0.06; it has excellent hydrophilicity.

[0107] In PET / CT imaging applications, the advantages of hydrophilic molecules are particularly prominent because they can effectively reduce the nonspecific uptake of molecular imaging probes in non-target tissues such as the abdomen, thereby improving the imaging signal-to-noise ratio.

[0108] For comparison, the oil-water distribution coefficient D (log D ), as shown in the following table, it can be seen from Table 3 that molecular imaging probes [ 68 Ga]Ga-PY03 exhibits strong hydrophilicity.

[0109] Table 3 Oil-water distribution coefficient D (log D )

[0110]

[0111] Molecular imaging probes[ 68 Ga]Ga-PY01, [ 68 Ga]Ga-PY02, [ 68 Ga]Ga-PY04, [ 68 Ga]Ga-PY05, [ 68 Ga]Ga-PY06,[ 68 Ga]Ga-PY07, [ 68 The structural formula of Ga]Ga-PY08 Figure 12 shown.

[0112] Example 5

[0113] Study on cell receptor affinity of molecular imaging probes:

[0114] (1) Saturation experiment to determine the equilibrium dissociation constant ( K d To evaluate the binding affinity of molecular imaging probes to CDK4 / 6, saturation binding experiments were performed on HCC78 cells. 5 Cells were incubated with different concentrations of the molecular imaging probe (0.625-40 nM) for 1 h to determine total binding. To determine nonspecific binding, cells were pretreated with a 2000-fold molar excess of ribociclib before the experiment. Cells in 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 performed in triplicate.

[0115] (2) Cell uptake and internalization experiment: HCC78 cells (4×10 5 Cells were seeded in 24-well plates (100 μL / well) and incubated with the molecular imaging probe for 15, 30, 60, 90, and 120 minutes to assess its uptake over time. For blocking experiments, cells were pre-incubated with ribociclib (400 nM, 2000-fold molar excess) for 1 hour before adding the molecular imaging probe. 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.

[0116] Molecular imaging probes[ 68The saturation curve of Ga]Ga-PYO3 is as follows Figure 13 As shown, molecular imaging probes [ 68 Ga]Ga-PY01~[ 68 The equilibrium dissociation constant of Ga]Ga-PY08 ( K d ) As shown in Table 4, it can be seen from Table 4 that [ 68 Ga]Ga-PY03 has the best affinity for HCC78 cells.

[0117] Table 4 Equilibrium dissociation constants of molecular imaging probes ( K d )

[0118]

[0119] Molecular imaging probes[ 68 Ga]Ga-PY03 uptake curve is as follows Figure 14 As shown, from Figure 14 As can be seen in the molecular imaging probe [ 68 Ga]Ga-PY03 showed rapid and specific uptake within 15 min, which gradually increased over time until reaching a plateau at 90 min; the uptake inhibition of Ribociclib confirmed the molecular imaging probe [ 68 Ga]Ga-PY03 binds specifically to CDK4 / 6.

[0120] Example 6

[0121] Normal BALB / c male mice (n=3) were injected with molecular imaging probes via the tail vein. 68 Ga]Ga-PY03 (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. The %ID / g was calculated, and the blood clearance curve was fitted using Drug And Statistics (DAS) software to obtain the blood elimination half-life of the molecular imaging probe. The %ID / g formula is: %ID / g = ((measured radioactivity count) / (total injected radioactivity count)) / tissue mass (including blood).

[0122] The blood clearance curve of molecular imaging probes is as follows Figure 15 As shown, from Figure 15 It can be seen that the pharmacokinetic curve of the molecular imaging probe in normal mice conforms to the two-compartment model. 68 The half-life of the distribution phase of Ga]Ga-PY03 (t 1 / 2α ) is 0.636 minutes, and the elimination phase half-life (t1 / 2β ) is 39.604 minutes, and the blood metabolism time is moderate.

[0123] Example 7

[0124] Computer molecular docking simulation analysis:

[0125] Molecular imaging probes[ 68 Docking simulation of Ga]Ga-PY03 and CDK4 / 6 protein. Figure 16 As shown, [ nat In the binding model of Ga]Ga-PY03 and CDK4, [ nat The interaction pattern diagram between Ga]Ga-PY03 and various amino acids around the pocket shows that [ nat There are 20 amino acids involved in the formation of hydrophobic interactions between Ga]Ga-PY03 and proteins; at the same time, the two N atoms in the aminopyrimidine structure form two groups of hydrogen bonds with Ile12 and Arg101, further enhancing the [ nat The affinity between Ga]Ga-PY03 and protein. nat Ga]Ga-PY03 in its binding mode with CDK6 ( Figure 17 ), [ nat There are 17 amino acids involved in the formation of hydrophobic interactions between Ga]Ga-PY03 and the protein. At the same time, the carbonyl group of the amide, the N atom in the aminopyrimidine structure, and the hydroxyl group of the DOTA group form four groups of hydrogen bonds with Asp163, Val101, and Asp104, respectively. According to the prediction of Autodock software, the results show that [ nat The binding energy between Ga]Ga-PY03 and CDK4 protein is -8.969 kcal / mol, and the binding energy between Ga]Ga-PY03 and CDK6 protein is -8.924 kcal / mol. The strong binding energy results indicate that the small molecule can stably bind to the protein.

[0126] Example 8

[0127] Small Animal PET / CT Imaging:

[0128] HCC78 tumor-bearing mice were randomly divided into experimental group (n=3) and blocking group (n=3), and then injected with molecular imaging probes[ 68 Ga]Ga-PY01~[ 68Small animal PET / CT imaging was performed using Ga]Ga-PY08. In the experimental group, each tumor-bearing mouse received a tail vein injection of 200 μL of the molecular imaging probe (3.7 MBq) under 2% isoflurane in oxygen. In the blocking group, tumor-bearing mice received a tail vein injection of the molecular imaging probe and unlabeled ribociclib (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.

[0129] Small animal PET / CT imaging Figure 18 As shown, molecular imaging probes [ 68 Ga]Ga-PY01~[ 68 The radioactive uptake of Ga]Ga-PY08 in different parts of the body is as follows Figure 19 As shown, from Figure 18 and Figure 19 It can be seen that molecular imaging probes all show high radioactive accumulation in the tumor site, however, molecular imaging probes[ 68 The radioactivity uptake of Ga]Ga-PY03 in the tumor site was highest 1 hour after injection.

[0130] Example 9

[0131] Biological distribution:

[0132] Under gas anesthesia (2% isoflurane in oxygen), HCC78 tumor-bearing mice (n=3) were injected via the tail vein with 100 μL of the molecular imaging probe [ 68 Ga]Ga-PY01~[ 68 Ga]Ga-PY08 (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 molecular imaging probe to obtain %ID / g and target / non-target (T / NT) ratios.

[0133] The results are as follows Figure 20 As shown, from Figure 20 As can be seen in the molecular imaging probe [ 68 Ga]Ga-PY03 significantly enhanced tumor uptake (5.12±0.68% ID / g) and exhibited excellent tumor / liver and tumor / muscle ratios.

[0134] Based on the structure of Ribociclib, a flexible group module (Linker) was first introduced for structural modification, and molecular imaging probes containing only PEG1 or Ahx hydrophilic chains were constructed. 68 Ga]Ga-PY01 and [ 68 Ga]Ga-PY02. Thanks to the optimization of the Linker structure, these two molecular imaging probes showed significant differences in liver uptake. 68 Ga]Ga-PY02 showed a high radioactive accumulation in the liver (7.43±0.50%ID / g), while [ 68 Ga]Ga-PY01 significantly reduced liver uptake (2.84±0.27%ID / g) and exhibited a superior tumor-to-liver ratio (4.19±0.43). Notably, there was no significant difference in tumor tissue uptake between the two (2.14±0.15%ID / g and 2.33±0.20%ID / g, respectively).

[0135] On this basis, amino acid and flexible group modules were further introduced to design and synthesize three molecular imaging probes based on PEG1. The biodistribution results showed that their distribution characteristics in vivo were significantly different: 68 Ga]Ga-PY03 significantly improved tumor uptake (5.12±0.68% ID / g) and showed excellent tumor / liver and tumor / muscle ratios; [ 68 Although the tumor uptake of Ga]Ga-PY05 was slightly increased (2.92±0.72%ID / g), it was accompanied by a significant increase in liver uptake (7.97±0.87%ID / g), resulting in a significant decrease in the tumor / liver ratio (0.30±0.02); 68 Ga]Ga-PY07 is similar to [ 68 The accumulation levels of Ga]Ga-PY05 in the liver were similar (11.65±1.06%ID / g, 12.21±0.34%ID / g), but the radioactive accumulation in the kidney was significantly enhanced (25.93±3.30%ID / g).

[0136] Similarly, among the three new molecular imaging probes designed based on Ahx, [ 68 Ga]Ga-PY04 exhibited a favorable tumor / non-target tissue (T / NT) ratio, which was mainly attributed to its rapid radioactive clearance rate in non-target tissues. 68 Ga]Ga-PY06 and [ 68 Ga]Ga-PY08 and its PEG1 counterpart [ 68 Ga]Ga-PY05 and [ 68Ga]Ga-PY07 showed a similar distribution trend, indicating that the structural features of amino acids (structural modifications) have certain similarities in the mechanism of action on different flexible chain platforms. Blocking experiments showed that when ribociclib was co-administered, tumor uptake was significantly reduced, confirming target specificity.

[0137] Example 10

[0138] Phosphorus screen autoradiography: Under gas anesthesia (2% isoflurane in oxygen), 100 μL of molecular imaging probe was injected into the tail vein of HCC78 tumor-bearing mice (n=3). 68 Ga]Ga-PY03 (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.

[0139] The results are as follows Figure 21 As shown, from Figure 21 As can be seen from the results of phosphor screen radioautography analysis, the results were highly consistent with PET / CT imaging and biodistribution, further confirming the molecular imaging probe[ 68 Specific distribution pattern of Ga]Ga-PY03 in vivo.

[0140] Example 11

[0141] Western blot analysis of CDK4 and CDK6 expression in HCC78 cells:

[0142] 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 minutes), blocked with 5% nonfat dry milk for 30 minutes, and incubated with a CDK4 antibody (Bioworld Technology, MB10716) at room temperature for 2 hours. The membrane was washed three times with TBST for 10 minutes each.

[0143] 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.

[0144] 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.

[0145] The results are as follows Figure 22 As shown, from Figure 22 As can be seen in Figure 3, CDK4 and CDK6 are specifically expressed in HCC78 cells.

[0146] Example 12

[0147] Based on molecular imaging probes[ 68 Ga]Ga-PY03 Micro-PET / CT Imaging Application Research (Various Tumors):

[0148] To investigate whether the difference in relative expression of CDK4 / 6 can be measured in vivo, this example utilized molecular imaging probes [ 68 PET / CT imaging of mice bearing tumors of U87MG, ACHN, OVCAR8, PC3, HT29, A549, 4T1, MCF-7, and A375 was performed using Ga]Ga-PY03. Quantitative PET analysis was performed 1 hour after injection ( Figure 23 ) showed that the tumor uptake rates of U87MG, ACHN, OVCAR8, PC3, HT29, A549, 4T1, MCF-7 and A375 were 1.00±0.09 %ID / g, 0.75±0.06 %ID / g, 0.58±0.06 %ID / g, 0.72±0.09 %ID / g, 0.79±0.06 %ID / g, 0.66±0.04 %ID / g, 0.78±0.05 %ID / g, 0.62±0.03 %ID / g and 0.92±0.04 %ID / g, respectively.

[0149] Example 13

[0150] Based on molecular imaging probes[ 68 Application of Ga]Ga-PY03 in Micro-PET / CT Imaging (Tumor in Situ):

[0151] To investigate the expression of CDK4 / 6 in in situ tumor lesions, molecular imaging probes [ 68 PET imaging of Ga]Ga-PY03 in the HCC78 lung metastasis model. Figure 24 and 25 As shown, 30 min after tail vein injection of HCC78 lung metastasis model, molecular imaging probe [ 68The lung uptake of Ga]Ga-PY03 was significantly higher in the model group than in normal mice, with absolute uptake values ​​of 0.62±0.05%ID / g and 0.38±0.02%ID / g, respectively. Statistical analysis showed a significant difference in uptake between the two groups (***p<0.001). After PET imaging, lung tissue was isolated and subjected to autoradiography. The radioactivity in the model group was approximately 1.52 times that of the control group (***p<0.001).

[0152] Example 14

[0153] Micro-PET / CT monitoring of changes in CDK4 / 6 in mice during Ribociclib treatment:

[0154] The study evaluated molecular imaging probes[ 68 Ga]Ga-PY03 was shown to be able to monitor changes in CDK4 / 6 expression after ribociclib blockade. The HCC78 tumor-bearing mouse model, considered a typical CDK4 / 6 "hot tumor," was used in the experiment. Ribociclib was administered intraperitoneally every other day, and molecular imaging probes were performed on days 1 and 21. 68 Ga]Ga-PY03 PET imaging. Figures 26-28 As shown, compared with the control group (tumor volume increased from 339 ± 69 mm 3 Increased to 953 ± 119mm 3 ) compared to the ribociclib-treated group (from 350 ± 13 mm 3 Increased to 580 ± 68 mm 3 ) was significantly inhibited, indicating that it has good anti-tumor activity. On day 1, the molecular imaging probe [ 68 Tumor uptake of Ga]Ga-PY03 was similar in both groups (0.67 ± 0.005 and 0.52 ± 0.05, respectively). However, by day 21, uptake in the control group increased to 0.76 ± 0.05, while uptake in the treatment group decreased significantly to 0.32 ± 0.06.

[0155] Example 15

[0156] Immunofluorescence staining to verify CDK4 / 6 expression in various tumor-bearing mouse models:

[0157] To assess CDK4 and CDK6 expression levels, tumor tissues were immunofluorescently stained. Paraffin sections were deparaffinized and rehydrated, followed by microwave retrieval of antigens using EDTA (pH 8.0). After blocking with 5% BSA, sections were incubated with primary antibodies overnight at 4°C, followed by incubation with fluorescently labeled secondary antibodies for 50 minutes at room temperature in the dark. Cell nuclei were stained with DAPI. Fluorescence detection parameters were as follows: DAPI (excitation wavelength: 330-380 nm / emission wavelength: 420 nm), FITC (excitation wavelength: 465-495 nm / emission wavelength: 515-555 nm), Cy3 (excitation wavelength: 510-560 nm / emission wavelength: 590 nm), and Cy5 (excitation wavelength: 608-648 nm / emission wavelength: 672-712 nm).

[0158] Immunofluorescence staining results of tumor tissue sections Figure 29 The results showed that the tumor tissues of U87MG, ACHN, OVCAR8, PC3, HT29, A549, 4T1, MCF-7 and A375 tumor-bearing mice highly expressed CDK4 / 6.

[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 molecular imaging probe targeting CDK4 / 6, characterized in that: The structural formula is shown in formula (III), Formula (III).

2. The method for preparing a molecular imaging probe targeting CDK4 / 6 according to claim 1, characterized in that: The steps include: Radionuclides 68 Ga is mixed with a precursor labeled with a molecular imaging probe targeting CDK4 / 6 to obtain a molecular imaging probe targeting CDK4 / 6; Wherein, the molecular imaging probe targeting CDK4 / 6 is labeled with a precursor, and its structural formula is shown in formula (I). Formula (I).

3. The method for preparing a molecular imaging probe targeting CDK4 / 6 according to claim 2, characterized in that: The method for preparing the molecular imaging probe labeling precursor targeting CDK4 / 6 comprises the following steps: The compound represented by formula (II) is subjected to Boc-deprotection to obtain a Boc-deprotected intermediate, which is then subjected to a condensation reaction with a chelating agent tetraazacyclododecane tetraacetic acid-succinimide ester and N,N-diisopropylethylamine to obtain the molecular imaging probe labeling precursor targeting CDK4 / 6; The structural formula of formula (II) is as follows: Formula (II).

4. The method for preparing a molecular imaging probe targeting CDK4 / 6 according to claim 3, characterized in that: The molar ratio of the compound represented by formula (II), the chelating agent tetraazacyclododecane tetraacetic acid-succinimidyl ester (DOTA-NHS) and N,N-diisopropylethylamine (DIPEA) is (22-27): (35-40): (140-160).

5. Use of the molecular imaging probe targeting CDK4 / 6 according to claim 1 in the preparation of a preparation for detecting the expression level of CDK4 / 6 in tumors.

6. Use of the molecular imaging probe targeting CDK4 / 6 according to claim 1 in the preparation of a preparation for diagnosing tumors with high CDK4 / 6 expression.

Images