Preparation method and application of a radionuclide-labeled reagent targeting CDK19

Abstract

The application discloses a radionuclide diagnostic and therapeutic agent for targeting CDK19, and belongs to the technical field of biological medicines. Bone metastatic prostate cancer has a poor prognosis in men and is significantly associated with mortality. CDK19 is specifically highly expressed in prostate cancer cells, and a complex formed by CDK19 is involved in the occurrence and development of bone metastasis of prostate cancer, and is a new specific target for treatment and diagnosis of bone metastatic prostate cancer. The radionuclide conjugated drug for targeting CDK19 has a good application prospect in the fields of preparation of diagnostic, staging and therapeutic agents for prostate cancer and medicines.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a radionuclide diagnostic and therapeutic reagent and drug targeting CDK19, a method for preparing the compound, and the application of the compound as a radionuclide conjugate drug targeting CDK19 in the preparation of diagnostic, staging and therapeutic reagents and drugs for prostate cancer. Background Technology

[0002] The following background information related to this invention is provided to aid in understanding the invention, but should not be considered as prior art. All cited sources are referenced in full.

[0003] Prostate cancer (PCa) is the most common malignant tumor in men, and its leading cause of death is metastasis, with 80% of metastatic prostate cancers closely associated with bone metastasis. For androgen-sensitive advanced prostate cancer, approximately 50% of men will develop bone metastasis within two years. 56% of prostate cancer patients without bone metastasis are still alive at 5 years, compared to 3% of those with bone metastasis. Therefore, bone metastasis in prostate cancer has a poor prognosis in men and is significantly associated with mortality. Current treatment strategies for prostate cancer focus on early diagnosis and treatment. Radiotherapy is a radical treatment for early and locally advanced prostate cancer, and inhibiting bone metastasis is key to improving patient survival rates.

[0004] CDK19 and its analog CDK8 are members of the cyclin-dependent kinase (CDK) family, playing crucial roles in regulating fundamental cellular processes such as cell proliferation, survival, differentiation, transcription, and metabolism. CDK8 / 19 primarily regulates RNA polymerase II activity and is associated with the transcription factor complex, co-regulating various cancer-related transcription factors. Inhibition of CDK8 / 19 kinase activity specifically inhibits phosphorylation of the C-terminal domain of RNA polymerase II, which is required for transcriptional elongation. Studies have shown that CDK19 is specifically highly expressed in prostate cancer and can serve as a targeted biomarker for prostate cancer. The complex CYCLINL1 / CDK19NEAT 1-1 in prostate cancer cells is involved in the occurrence and development of bone metastases in prostate cancer, potentially representing a novel specific target for the treatment and diagnosis of bone-metastatic prostate cancer.

[0005] PET-CT imaging, using radiopharmaceuticals based on prostate-specific membrane antigen (PSMA), is an effective method for predicting cancer progression risk and response to specific treatments. However, its limitation lies in the inability to accurately obtain diagnostic information and provide treatment options for PSMA-negative / low-expressing prostate cancer. There is a need to develop new specific targets for effective diagnosis and staging of prostate cancer. Small molecule drugs that specifically bind to CDK19 can increase the targeting of prostate cancer. Inhibiting CDK19 activity is expected to interfere with the function of the CYCLINL1 / CDK19 / NEAT1-1 complex, thereby inhibiting the occurrence of bone metastasis in prostate cancer. Successful conjugation of radionuclides can enable specific imaging at the target site of prostate cancer, providing new possibilities for the diagnosis and treatment of prostate cancer. Therefore, the development of radionuclide conjugates targeting CDK19 has promising applications in the diagnosis, staging, and treatment of prostate cancer. Summary of the Invention

[0006] The present invention aims to address the deficiencies of existing prostate diagnosis and treatment technologies by providing a novel method for preparing and applying a CDK19-targeting radionuclide-coupled drug, thereby solving the technical problem of the lack of a similar compound in the prior art.

[0007] Another technical problem that this invention aims to solve is that there are no reports of radionuclide conjugates targeting CDK19;

[0008] Another technical problem to be solved by the present invention is to provide the application of the compound in the fields of diagnosis, staging and treatment of prostate tumors.

[0009] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0010] A compound represented by formula I:

[0011]

[0012] in:

[0013] a) X and Y are selected from C atoms and N atoms, and at least one of X and Y is a nitrogen atom.

[0014] b) R1 and R2 are independently selected from hydrogen atoms, cyano groups, C1-C5 alkyl groups, C1-C5 alkoxy groups, C1-C5 ester groups, C1-C5 amide groups, C1-C5 haloalkanes, C2-C5 alkenyl groups, C3-C6 heterocyclic alkyl groups, and C3-C6 cycloalkyl groups.

[0015] c) R3 is a linker composed of alkyl chains, heteroalkyl chains, cycloalkyl chains, heterocycloalkyl chains, aryl chains, heteroaryl chains, and chains of random combinations of the above-mentioned substituents with a carbon chain length of 1-10 atoms.

[0016] d) M is selected from radioactive or non-radioactive isotopes of metallic elements.

[0017] As a preferred option, X and Y are selected from C atoms and N atoms, and at least one of X and Y is an N atom;

[0018] As a preferred option, R3 is independently selected with the following chemical structure:

[0019]

[0020] e) Preferably, M is selected from radioactive or non-radioactive isotopes of metallic elements; including but not limited to gallium, copper, lutetium, actinium, yttrium, and gadolinium.

[0021] Preferably, the compound or a pharmaceutically usable salt thereof is selected from:

[0022]

[0023]

[0024] The present invention provides a pharmaceutical composition comprising the above-described compounds and / or their pharmaceutically usable salts.

[0025] This invention provides a pharmaceutical composition comprising the above-described compound and / or a pharmaceutically usable salt thereof, and / or a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is, for example, a diluent.

[0026] Meanwhile, the present invention provides the use of the above-mentioned compounds or their pharmaceutically usable salts, and the above-mentioned pharmaceutical compositions, in the preparation of radionuclide diagnostic and therapeutic reagents targeting CDK19.

[0027] Meanwhile, the present invention provides the application of the above-mentioned compounds or their pharmaceutically usable salts, and the above-mentioned pharmaceutical compositions, in the fields of prostate cancer diagnosis, staging, and treatment.

[0028] Meanwhile, this invention provides a prodrug of the above-mentioned compound, or a labeled prodrug:

[0029]

[0030]

[0031] Meanwhile, the present invention provides a method for preparing the above-mentioned compound, comprising the following steps:

[0032] 1) Take haloquinoline, isoquinoline or its derivative (A), and react with (4-bromophenyl)boronic acid (B) in dioxane under alkaline conditions with palladium catalysis to obtain intermediate compound C;

[0033] 2) Compound C obtained in step 1) was subjected to a palladium-catalyzed Suzuki coupling reaction with 4-(4,4,5,5-tetramethyl-1,3,2-dioxane-2-yl)-1H-pyrazole (D) in dioxane under alkaline conditions to give intermediate compound E:

[0034] 3) 2-Chloroacetyl chloride (F) and linker compound (G) were coupled in high yield with triethylamine as a base under DCM and at room temperature to give intermediate compound H:

[0035] 4) In the presence of potassium carbonate and acetonitrile, compound H and compound E are coupled at room temperature, and then the BOC protection is removed under acidic conditions to obtain compound I;

[0036] 5) Compound I obtained in step 4) undergoes an acid-ammonia condensation reaction with tri-tert-butyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid under HATU and DIPEA conditions to obtain compound J:

[0037] 6) The compound J obtained in step 5) was hydrolyzed under acidic conditions to obtain the labeled precursor compound IRM-X-DOTA:

[0038] 7) The labeled precursor compound IRM-X-DOTA obtained in step 6) can be complexed with the corresponding metal salt in water to obtain a non-radioactive cold-labeled compound.

[0039] The structures of each compound are shown below:

[0040]

[0041]

[0042] Preferably, the palladium catalyst for the reactions described in steps 1) and 2) includes, but is not limited to, the following: palladium acetate, palladium chloride, tetra(triphenylphosphine)palladium, bis(acetonitrile)palladium dichloride, bis(triphenylphosphine)palladium dichloride, 1,1'-[bis(diphenylphosphine)ferrocene]palladium dichloride, bis(benzonitrile)palladium dichloride, 1,1'-[bis(di-tert-butylphosphine)ferrocene]palladium dichloride, bis(tricyclohexylphosphine)palladium dichloride, and bis(o-toluenephosphine)palladium dichloride. More preferably, the catalyst is 1,1'-[bis(diphenylphosphine)ferrocene]palladium dichloride.

[0043] Preferably, the reactions described in steps 1) and 2) are carried out under alkaline conditions, and the alkaline is including, but is not limited to, sodium carbonate, potassium carbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, sodium fluoride, and potassium fluoride. More preferably, sodium carbonate is used.

[0044] Preferably, the reactions described in steps 3), 5), and 6) are carried out at room temperature.

[0045] Preferably, the reactions described in steps 4) and 6) are carried out under acidic conditions to deprotect the product to trifluoroacetic acid.

[0046] Preferably, the solvent for the reaction in step 7) is water.

[0047] As a scrambler, the reaction described in step 7) is carried out under heating conditions, preferably in the temperature range of 80-100°C.

[0048] In addition to the preparation methods provided above, the compounds of the present invention can also be prepared according to the following process. It should be clearly stated that the following methods are only used to illustrate the preparation process, and the claims of the present invention are not limited thereto. The typical synthetic method of general formula (I) can be summarized into several basic steps: (1) preparing a key intermediate (E) from haloquinoline, isoquinoline or its derivative (A) as starting material through a two-step coupling reaction; (2) preparing a linker of the target compound from chloroacetyl chloride and various nitrogen-containing basic compounds, and coupling it with the above intermediate E to obtain the key intermediate (I); (3) coupling intermediate (I) with tert-butyl-protected DOTA and then hydrolyzing it to obtain a labeled precursor compound; (4) chelating it with the corresponding metal salt in aqueous solution to obtain a non-radioactive cold-labeled compound.

[0049]

[0050] Halogenated quinolines, isoquinolines, or their derivatives (A) undergo two coupling reactions with (4-bromophenyl)boronic acid (B) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxane-2-yl)-1H-pyrazole (D) to prepare intermediate compound E. 2-Chloroacetyl chloride (F) is coupled sequentially with linker compound (G) and compound E, followed by deprotection to obtain compound I. Compound I undergoes an ammonium condensation reaction with tri-tert-butyl-protected DOTA, followed by hydrolysis to obtain a labeled precursor compound. The labeled precursor compound is then complexed with the corresponding metal salt in water to yield a non-radioactive cold-labeled compound. The reaction flow of this preparation method is shown in the above reaction equations.

[0051] Unless otherwise specified, the technical terms related to the above technical solutions shall follow the definitions below.

[0052] The term "alkyl" refers to a straight-chain or branched hydrocarbon group having a specified number of carbon atoms; therefore, for example, the terms "C1-C4 alkyl" and "C1-C4 alkyl" are used herein. 10"Alkyl" refers to an alkyl group having at least one and at most four or ten carbon atoms. Examples of such branched or straight-chain alkyl groups used in this invention include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, isobutyl, n-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl, and branched analogs of the following five n-alkanes.

[0053] When the term "alkenyl" (or "alkenylene") is used, it refers to a straight-chain or branched hydrocarbon chain containing a specified number of carbon atoms and at least one to five carbon-carbon double bonds. Examples include vinyl (or vinylene) and propenyl (or propenylene).

[0054] When the term "alkynyl" (or "ethynylene") is used, it refers to a straight-chain or branched hydrocarbon chain containing a specified number of carbon atoms and at least one to five carbon-carbon triple bonds. Examples include ethynyl (or ethynylene) and propynyl (or propynylene).

[0055] When the term "cycloalkyl" is used, it refers to a non-aromatic, saturated, cyclic hydrocarbon ring containing a specified number of carbon atoms. Thus, for example, the term "C3-C8 cycloalkyl" refers to a non-aromatic cyclic hydrocarbon ring having 3-8 carbon atoms. Exemplary "C3-C8 cycloalkyl" groups used in this invention include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0056] When the term "C5-C8 cycloalkenyl" is used, it refers to a non-aromatic monocyclic carbon ring having a specified carbon atom and at most three carbon-carbon double bonds. Exemplary "cycloalkenyl" includes cyclopentenyl and cyclohexenyl.

[0057] When the term "C3-C8 heterocyclic alkyl" is used, it refers to a non-aromatic heterocycle containing a specified number of ring atoms, which is saturated or has one or more degrees of unsaturation, and contains one or more heteroatoms selected from O, S, or N. Such a ring may optionally be fused to one or more other "heterocyclic" or cycloalkyl groups. Examples of "heterocyclic" groups include, but are not limited to, azirrocyclopropane, thiocyclopropane, oxacyclopropane, azirrocyclobutane, thiocyclobutane, oxacyclobutane, tetrahydrofuran, pyran, 1,4-dioxane, 1,4-dithiaane, 1,3-dioxane, 1,3-dioxane, piperidine, piperazine, 2,4-piperazinedione, pyrrolidine, 2-imidazoline, imidazoline, pyrazolidine, pyrazolidine, pyrazoline, morpholine, thiomorpholine, tetrahydrofuran, tetrahydrothiophene, etc.

[0058] The term "aryl" refers to an aromatic group containing 5-14 ring atoms, with at least one ring possessing a conjugated π-electron system. This includes aromatic rings with all carbon atoms, aromatic heterocycles, and fused or biaromatic rings, and may contain substituents. Aryl groups can contain 1-6 substituents. A heteroaromatic ring or aromatic heterocycle refers to a group containing 5-14 ring atoms, of which 1-4 heteroatoms are aromatic ring atoms, and the remaining ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, nitrogen, and selenium atoms. Suitable aromatic heterocycles include furan, thiophene, pyridine, pyrrolidine, pyrrolidines with low-carbon alkyl substituents on the nitrogen atom, pyridine nitrides, pyrimidines, pyrazines, imidazoles, and other similar heterocycles, all of which may contain substituents.

[0059] The terms "arbitrary substitution" or "substitution" refer to groups having 1-4 different substituents, which can be: low-carbon alkyl, low-carbon aryl, low-carbon aralkyl, low-carbon cyclic alkyl, low-carbon heterocyclic alkyl, hydroxyl, low-carbon alkoxy, low-carbon aryloxy, polyhaloalkoxy, arylalkoxy, low-carbon heteroaryl, low-carbon heteroarylepoxy, low-carbon heteroarylalkyl, low-carbon heteroarylalkoxy, azide, nitrogen, halogen, low-carbon alkylthiol, oxy, low-carbon acylalkyl. Low carbon number carboxylic acid ester group, carboxylic acid, amide group, nitro group, low carbon number acyloxy group, low carbon number amine alkyl group, low carbon number alkylamine aryl group, low carbon number alkylaryl group, low carbon number alkylamine alkyl group, low carbon number alkoxyaryl group, low carbon number arylamine group, low carbon number arylalkylamine group, sulfonyl group, low carbon number amide alkylaryl group, low carbon number amide aryl group, low carbon number hydroxyalkyl group, low carbon number haloalkyl group, low carbon number alkylamine alkyl acid group, low carbon number urea alkyl group, cyano group, low carbon number alkoxyalkyl group, low carbon number polyhaloalkyl group, low carbon number arylalkoxyalkyl group.

[0060] "Substituted aryl" and "substituted heteroaryl" refer to aromatic rings or heteroaryl groups with 1 to 6 substituents. These substituents can be low-carbon alkyl groups, low-carbon alkoxy groups, low-carbon polyhaloalkyl groups, halogens, hydroxyl groups, and amino groups.

[0061] Intermediates are chemical raw materials or chemical products used in the process of drug synthesis.

[0062] Prodrugs, also known as precursors, are compounds obtained by chemically modifying drugs. These compounds are inactive or have low activity in vitro, but release their active pharmaceutical ingredient in vivo through enzymatic or non-enzymatic conversion to exert their therapeutic effect.

[0063] Labeled prodrugs, also known as labeled precursors, are intermediates prepared for the synthesis of the target compound of this invention or its pharmaceutically usable salt. They generally contain chemical structures that can chelate metal nuclides, and have a certain degree of stability and can be stored for a long time. Attached Figure Description

[0064] Figure 1Radiochemical purity of radiolabeled compounds:

[0065] Figure 2 Schematic diagram of IRM-015-DOTA molecular docking results and enzyme activity assay results.

[0066] Figure 3 Mass cytometry results of Ga-IRM-015-DOTA cellular uptake assay:

[0067] Figure 4 : 68 Results of PET / CT experiments on Ga-IRM-015-DOTA in tumor-bearing mice:

[0068] Figure 5 Gallium-68 labeled compounds 68 Distribution of Ga-IRM-015-DOTA in tumor-bearing mice:

[0069] Figure 6 Imaging study of Gallium-68 labeled compound 36-39 in healthy mice; where A is PET imaging 1h, 2h and 3h after administration of compound 36-39; B is the statistical analysis of SUVmean values ​​of major organs and tumors.

[0070] Figure 7 Imaging study of prostate cancer xenograft model of gallium-68 labeled compound 36-38: AC represents PET images after 1h, 2h, and 3h of compound 36 administration; DG represents the SUVmean values ​​of the three compounds after 1h, 2h, and 3h of administration to the tumor, heart, liver, and kidney, respectively.

[0071] Figure 8 Investigation into the optimal in vivo imaging time for Gallium-68 labeled compound 37: A shows PET scan images at different time points after administration of compound 37; B shows the statistical values ​​of SUVmean of compound 37 in major organs and tumors; C shows the change in the ratio of radioactive signal in tumors to liver and kidneys over time.

[0072] Figure 9 In vivo blocking imaging and tissue distribution study of Gallium-68 labeled compound 37: A shows the comparison of PET images between the drug-treated group and the blocking group; B shows the results of the tissue distribution experiment, showing the radioactivity counts of each tissue at 1 hour and 2 hours after dissection, and calculating %ID / g. Detailed Implementation

[0073] The compounds and their preparation in this invention can be better illustrated by the following examples. These examples should not be construed as limiting the invention, and variations of these compounds, whether now known or developed in the future, should also be considered to fall within the scope of this invention and are protected thereon.

[0074] The specific embodiments of the present invention will be described in detail below. To avoid excessive and unnecessary details, well-known structures or functions will not be described in detail in the following embodiments.

[0075] The approximate language used in the following embodiments is for quantitative expression, indicating that a certain degree of variation in quantity is permissible without changing the basic function. Therefore, the values ​​corrected using terms such as "approximately," "around," etc., are not limited to the exact value itself. In some embodiments, "approximately" indicates that the value being corrected is allowed to vary within a range of plus or minus ten percent (±10%); for example, "approximately 100" represents any value between 90 and 110. Furthermore, in the expression "approximately from the first value to the second value," both the first and second values ​​are corrected simultaneously. In some cases, the approximate language may be related to the accuracy of the measuring instrument.

[0076] Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0077] Example 1 4-(4-bromophenyl)isoquinoline (3)

[0078]

[0079] Compound 1 (10 mmol, 1 eq), compound 2 (6 mmol, 0.6 eq), Na₂CO₃ (20 mmol, 2 eq), and 20 mL of dioxane were placed in a 50 mL three-necked flask. The reaction system was evacuated and backfilled three times with argon. Pd(dppf)Cl₂ (0.5 mmol, 0.05 eq) was added to the reaction flask under an argon flow. The resulting solution was heated to 90 °C and stirred at this temperature for 2 hours. The reaction was monitored by TLC. The reaction solution was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, concentrated to obtain the crude product, and purified by column chromatography to obtain compound 3. H NMR (300MHz, DMSO-d6) δ: 937 (s, 1H), 8.45 (s, 1H), 8.24 (d, J=7.7Hz, 1H), 7.8 8-7.71 (m, 5H), 7.57-7.48 (m, 2H); HRMS (ESI): m / z=284.0072, 286.0053[M+H] + .

[0080] Example 2 4-(4-(1H-pyrazol-4-yl)phenyl)isoquinoline (5)

[0081]

[0082] Compound 3 (5 mmol, 1 eq), compound 4 (5.25 mmol, 1.05 eq), Na₂CO₃ (10 mmol, 2 eq), and 10 mL of dioxane were placed in a 50 mL three-necked flask. The reaction system was evacuated and backfilled three times with argon gas. Pd(dppf)Cl₂ (0.25 mmol, 0.05 eq) was added to the reaction flask under an argon gas flow. Subsequent steps were similar to the preparation of compound 3, and the crude product was purified by column chromatography to obtain compound 5. 1 H NMR (300MHz, DMSO-d6) δ: 13.03 (s, 1H), 9.35 (s, 1H), 8.48 (s, 1H), 8.32 (s, 1H), 8.23 ​​(d, J=7.7Hz, 1H), 8.0 4 (s, 1H), 7.93 (d, J=8.1Hz, 1H), 7.87-7.70 (m, 4H), 7.54 (d, J=8.1Hz, 2H): HRMS (ESI): m / z=272.1184[M+H] + .

[0083] Example 3: 4-(2-chloroacetyl)piperazine-1-carboxylic acid tert-butyl ester (8)

[0084]

[0085] Compound 7 (5 mmol, 1 eq), TEA (5.25 mmol, 1.05 eq), and DCM (15 mL) were added sequentially to a 50 mL dry reaction flask. The mixture was cooled to 0 °C, and a DCM solution of compound 6 (5 mmol, 1 eq) in 10 mL was slowly added dropwise. After the addition was complete, the reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, the mixture was diluted with water and extracted with DCM. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated to obtain the crude product, which was then purified by column chromatography to obtain compound 8. 1 H NMR (300MHz, DMSO-d6) δ: 4.40 (s, 2H), 3.48-3.41 (m, 4H), 3.40-3.32 (m, 4H), 1.42 (s, 9H): HRMS (ESI): m / z=285.0980[M+Na] + .

[0086] Example 4 2-(4-(4-(isoquinoline-4-yl)phenyl)-1H-pyrazol-1-yl)-1-(piperazin-1-yl)ethane-1-one (9)

[0087]

[0088] K₂CO₃ (8 mmol, 2 eq) was added to a CAN solution of compound 5 (4 mmol, 1.0 eq) and compound 8 (5.2 mmol, 1.3 eq). The mixture was stirred at 85 °C for 4 hours and filtered. The filtrate was evaporated to dryness, dissolved in DCM, and 2 mL of TFA was added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the solvent was evaporated off, and the crude product was concentrated by washing with aqueous Na₂CO₃ solution and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered, concentrated, and then purified by column chromatography to obtain the target compound. 1 H NMR (300MHz, DMSO-d6) δ: 935 (s, 1H), 8.48 (s, 1H), 8.27-8.18 (m, 2H), 8.00 (s, 1H), 7.93 (d, J=8.4Hz , 1H), 7.85-7.71 (m, 4H), 7.55 (d, J=8.2Hz, 2H), 5.18 (s, 2H), 3.50-3.36 (m, 4H), 2.77-2.63 (m, 4H); 13 C NMR (101MHz, DMSO-d6) δ165.15, 151.86, 142.41, 136.36, 133.86, 133.20, 132.44, 132.28, 131.09, 130.43, 12 8.92, 128.13, 128.08, 127.52, 125.24, 124.11, 121.48, 53.00, 45, 34, 42.71: HRMS (ESI): m / z=398.1978[M+H] + 420.1796 [M+Na] + .

[0089] Example 5 2,2′,2″-(10-(4-(2-(4-(4-(isoquinoline-4-yl)phenyl)-1H-pyrazol-1-yl)acetyl)piperazin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tritert-butyl ester (10)

[0090]

[0091] DOTA ester (3 mmol, 1 eq), compound 9 (3 mmol, 1 eq), and DIPEA (6 mmol, 2 eq) were dissolved in CAN (20 mL), and HATU (3 mmol, 1 eq) was added to the solution. The reaction system was stirred at room temperature for 5 hours. After the reaction was completed, the mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine and dried over anhydrous Na2SO4, concentrated to give the crude product, and purified by column chromatography to give compound 10. 1H NMR (300MHz, DMSO-d6) δ: 9.36 (s, 1H), 8.48 (s, 1H), 8.26-8.20 (m, 2H), 8.02 (s, 1H), 7.93 (d, J=8.2H7, 1H), 7 .85-7.72 (m, 4H), 7.56 (d, J=8.2Hz, 2H), 5.23 (s, 2H), 3.68-3.05 (m, 8H), 1.44 (s, 27H), 1.28-1.23 (m, 24H).

[0092] Example 6 2,2′,2″-(10-(4-(2-(4-(4-(isoquinoline-4-yl)phenyl)-1H-pyrazol-1-yl)acetyl)piperazin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (IRM-015-DOTA)

[0093]

[0094] TFA (2 mL) was added to a mixed solution of compound 10 (2 mmol, 1 eq) and DCM (4 mL), and the mixture was stirred at room temperature for 6 hours. The reactants were concentrated under reduced pressure, dissolved in hot methanol, and ethyl acetate was added to produce a light, pale yellow precipitate. 1 H NMR (300MHz, D2O) δ: 9.38 (s, 1H), 8.31 (d, J-8.1Hz, 1H), 8.23 ​​(s, 1H), 8.03-7.94 (m, 2H), 7.94-7.84 (m, 2H), 7.81 (s, 1H) , 7.51 (d, J=7.9Hz, 2H), 7.39 (d, J=7.9Hz, 2H), 5.17 (d, J=7.5Hz, 2H), 3.82-2.96 (m, 32H); HRMS (ESI): m / z=782.3634[MH] - .

[0095] Example 7 Ga-IRM-015-DOTA

[0096]

[0097] Ga-IRM-015-DOTA was obtained by reacting a stoichiometric amount of gallium chloride with the precursor ligand IRM-015-DOTA in an acidic (pH 5) acetate buffer to prevent the formation of Ga(OH)3 precipitate. Generally, 50 mg of the precursor compound was weighed, reacted at 90 °C for 20 min, and the target compound was separated by gel column chromatography after monitoring the reaction using thin-layer chromatography. 1H NMR (300MHz, D2O) δ: 9.43 (s, 1H), 8.40-8.27 (m, 2H), 8.04 (s, 2H), 7.98-7.85 (m, 3H), 7.60 (d, J=7. 5Hz, 2H), 7.45 (d, J=8.1Hz, 2H), 5.20 (s, 2H), 4.09-3.01 (m, 32H); HRMS (ESI): m / z=850.2785[M+H] + 872.2607 [M+Na] + .

[0098] Example 8 (2-(2-chloroacetamide)ethyl)carbamate tert-butyl ester (11)

[0099]

[0100] The intermediate compound in this example was obtained by referring to the preparation and purification method of compound 8 in Example 3. HRMS (ESI): m / z = 259.0824 [M + Na] + .

[0101] Example 9 (2-(2-chloro-N-methylacetamido)ethyl)carbamate tert-butyl ester (12)

[0102]

[0103] Following the preparation and purification method of compound 8 in Example 3, the intermediate compound in this example was obtained. HRMS (ESI): m / z = 273.0979 [M + Na] + .

[0104] Example 10 (5-(2-chloroacetamido)pentyl)carbamate tert-butyl ester (13)

[0105]

[0106] The intermediate compound in this example was obtained by referring to the preparation and purification method of compound 8 in Example 3. HRMS (ESI): m / z = 301.1294 [M + Na] + .

[0107] Example 11 4-(2-(2-chloroacetamido)ethyl)piperazine-1-carboxylic acid tert-butyl ester (14)

[0108]

[0109] The intermediate compound in this example was obtained by referring to the preparation and purification method of compound 8 in Example 3. HRMS (ESI): m / z = 306.1581 [M + Na]+ .

[0110] Example 12 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (15)

[0111]

[0112] The intermediate compound in this example was obtained by referring to the preparation and purification method of compound 8 in Example 3. HRMS (ESI): m / z = 335.1136 [M + Na] + .

[0113] Example 13 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (16)

[0114]

[0115] Following the preparation and purification method of compound 9 in Example 4, the intermediate compound in this example was obtained. HRMS (ESI): m / z = 372.1828 [M+H] + .

[0116] Example 14 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (17)

[0117]

[0118] Following the preparation and purification method of compound 9 in Example 4, the intermediate compound in this example was obtained. HRMS (ESI): m / z = 386.1986 [M+H] + .

[0119] Example 15 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (18)

[0120]

[0121] Following the preparation and purification method of compound 9 in Example 4, the intermediate compound in this example was obtained. HRMS (ESI): m / z = 414.2299 [M+H] + 436.2199 [M+Na] + .

[0122] Example 16 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (19)

[0123]

[0124] The intermediate compound in this example was obtained by referring to the preparation and purification method of compound 9 in Example 4. HRMS (ESI): m / z = 441.2410 [M+H] + .

[0125] Example 17 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (20)

[0126]

[0127] The intermediate compound in this example was obtained by referring to the preparation and purification method of compound 9 in Example 4. HRMS (ESI): m / z = 448.2142 [M+H] + .

[0128] Example 18 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (21)

[0129]

[0130] The intermediate compound in this example was obtained by referring to the preparation and purification method of compound 10 in Example 5. HRMS (ESI): m / z -926.5512 [M+H] + 948.5333[M+Na] + .

[0131] Example 19 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (22)

[0132]

[0133] The intermediate compound in this example was obtained by referring to the preparation and purification method of compound 10 in Example 5. HRMS (ESI): m / z = 940.5667 [M+H] + 962.5494 [M+Na] + .

[0134] Example 20 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (23)

[0135]

[0136] The intermediate compound in this example was obtained by referring to the preparation and purification method of compound 10 in Example 5. HRMS (ESI): m / z = 968.5975 [M+H] + 990.5803 [M+Na] + .

[0137] Example 21 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (24)

[0138]

[0139] Following the preparation and purification method of compound 10 in Example 5, the intermediate compound in this example can be obtained. HRMS (ESI): m / z = 995.6077 [M+H] + .

[0140] Example 22 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (25)

[0141]

[0142] The intermediate compound in this example was obtained by referring to the preparation and purification method of compound 10 in Example 5. HRMS (ESI): m / z = 1002.5827 [M+H] + 1024.5648 [M+Na] + .

[0143] Example 23 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (26)

[0144]

[0145] Following the preparation and purification methods of the compound in Example 6, the labeled precursor compound containing the DOTA structure of this example can be obtained. HRMS (ESI): m / z = 756.3453 [MH] + .

[0146] Example 24 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (27)

[0147]

[0148] Following the preparation and purification methods of the compound in Example 6, the labeled precursor compound containing the DOTA structure of this example can be obtained. HRMS (ESI): m / z = 770.3622 [MH] + .

[0149] Example 25 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (28)

[0150]

[0151] Following the preparation and purification methods of the compound in Example 6, the labeled precursor compound containing the DOTA structure of this example can be obtained. HRMS (ESI): m / z = 798.3933 [MH] + .

[0152] Example 26 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (29)

[0153]

[0154] Following the preparation and purification methods of the compound in Example 6, the labeled precursor compound containing the DOTA structure of this example can be obtained. HRMS (ESI): m / z = 825.4175 [M + Na] + .

[0155] Example 27 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (30)

[0156]

[0157] Following the preparation and purification methods of the compound in Example 6, the labeled precursor compound containing the DOTA structure of this example can be obtained. HRMS (ESI): m / z = 832.3781 [MH] + .

[0158] Example 28 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (31)

[0159]

[0160] Following the preparation and purification methods of the compound in Example 7, the non-radioactive cold-labeled compound of this example can be obtained. HRMS (ESI): m / z = 824.2636 [M+H] + .

[0161] Example 29 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (32)

[0162]

[0163] Following the preparation and purification methods of the compound in Example 7, the non-radioactive cold-labeled compound of this example can be obtained. HRMS (ESI): m / z = 838.2787 [M+H] + 860.2604 [M+Na] + .

[0164] Example 30 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (33)

[0165]

[0166] Following the preparation and purification methods of the compound in Example 7, the non-radioactive cold-labeled compound of this example can be obtained. HRMS (ESI): m / z = 866.3095 [M+H] + .888.2912[M+Na] + .

[0167] Example 31 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (34)

[0168]

[0169] Following the preparation and purification methods of the compound in Example 7, the non-radioactive cold-labeled compound of this example can be obtained. HRMS (ESI): m / z = 893.3213 [M+H] + .

[0170] Example 32 (4-(2-chloroacetamido)phenethyl)carbamate tert-butyl ester (35)

[0171]

[0172] Following the preparation and purification methods of the compound in Example 7, the non-radioactive cold-labeled compound of this example can be obtained. HRMS (ESI): m / z = 900.2945 [M+Na] + .

[0173] Example 33 Radiolabeling

[0174] 68 Ga-IRM-015-DOTA and 68 Ga-PSMA-11 was adjusted with sodium acetate (1.25M). 68 The pH of the GaCl3 solution was adjusted to 4.5 in 0.05 M hydrochloric acid. 10 μg of the labeled precursor was added to 1 mL of [amount missing]. 68 The labeled product was obtained by heating at 100°C for 10 min in GaCl3 solution (pH 4.5). The radiochemical purity of the product was determined by radiometric thin-layer chromatography, and the purity was above 97% (see...). Figure 1 No further purification is required.

[0175] Example 34 Molecular docking and enzyme inhibition experiments

[0176] Molecular docking studies were conducted on the labeled precursor IRM-015-DOTA. Its binding conformation largely overlapped with that of the control drug BI-1347, and it bound via a similar mode of action. The introduction of the DOTA structure did not affect the binding of the drug to the target protein. Figure 2 A); The cold-labeled compound Ga-IRM-015-DOTA exhibited nanomolar inhibitory activity against both CDK19 and CDK8, with CDK19 showing superior activity compared to CDK8. Figure 2 B).

[0177] Example 35 Cell Uptake Experiment

[0178] We complexed gadolinium with the precursor compound IRM-015-DOTA to obtain Gd-IRM-015-DOTA, and then used imaging mass cytometry to locate Gd-IRM-015-DOTA and compare the drug uptake by different cells. We selected previously obtained CDK19 high-expression (C4-2) and low-expression (PC-3) cells for cell uptake experiments. The cells used in the experiment were cultured on sterile glass slides in culture dishes for 24 hours and wrapped with PAP pens. Then, Gd-IRM015-DOTA (10 μM) was added and incubated with the cells for 12 hours. The samples were washed three times with DPBS, fixed with 4% paraformaldehyde at room temperature for 20 min, and then washed three times with DPBS. Then, they were washed with 0.3% Triton-X100 in DPBS for 15 min, and washed three times with DPBS. The samples were stained with Ir-intercalation agent (1:400) in DPBS at RT for 30 min. Rinse in ddH2O for 5 minutes. Air dry the slides at RT for at least 20 minutes and store at 4°C. Finally, scan and image the slides using imaging mass cytometry on the Hyperion system (Fluidigm), and perform data analysis using MCD Viewer 1.0. Six regions were selected for testing for each sample, with a sampling area of ​​150µm x 150µm.

[0179] See results Figure 3 IRM-015-DOTA exhibits high specific uptake in C4-2 cells with high CDK19 expression.

[0180] Example 36: In vivo PET / CT experiment in tumor-bearing mice

[0181] A tumor-bearing mouse model was established in NOD-SCID mice using C4-2 (high expression of CDK19, high expression of PSMA) and PC-3 (low expression of CDK19, low expression of PSMA) cells. After the tumors grew to a suitable size for 3 weeks, they were... 68 Ga-IRM-015-DOTA and 68 Ga-PSMA-11 was administered via tail vein injection at a dose of 100 μCi. PET / CT scans were performed at 30 min, 1 h, 2 h, and 4 h after administration. The scan results were recorded, and the in vivo imaging effects were compared. The distribution of the drug in various organs and tumors in mice was also recorded. See Figure 4and Figure 5 . 68 Ga-IRM-015-DOTA was successfully visualized in a CDK19-expressing tumor model, and the signal intensity of the lesions was correlated with the CDK19 expression level. 68 Ga-PSMA-11 was only visualized in the C4-2 model where PSMA expression was high, demonstrating that... 68 The Ga-IRM-015-DOTA may enable a wider range of imaging capabilities. Furthermore, 68 The optimal display time for Ga-IRM-015-DOTA is 2 hours, while 68 The optimal imaging time for Ga-PSMA-11 is 1 hour. Therefore, drug structure optimization was also performed in subsequent embodiments.

[0182] Example 37: IC50 of the compound against the target protein CDK19 50 Value determination

[0183] Compounds 16-20, 26-30, and 31-35 from the above embodiments were subjected to IC50 analysis. 50 The results of the measurements are shown in Table 1.

[0184] Table 1. IC50 of the compounds against the target protein CDK19 50 Value determination

[0185]

[0186] Under the same detection conditions, the IC50 of most compounds 50 The value was below 1 μM, while the IC50 value of the control compound BI-1347 was lower. 50 The values ​​were 0.8 μM, indicating that the activities of the precursor compounds and cold-labeled compounds were comparable to those of the control compound BI-1347. Furthermore, compounds 29 (0.43 μM), 30 (0.72 μM), 32 (0.66 μM), and 33 (0.76 μM) showed higher activities than BI-1347. The IC50 values ​​for non-radioactively labeled compounds 32-35 were also significant. 50 Below 1 μM, given that gallium chelate products are mainly used for CDK19 binding in tumors, the corresponding precursor compounds 27-30 were selected for gallium-68 labeling and mouse imaging.

[0187] Radiolabeling of precursor compounds 27-30 in Example 38

[0188] Referring to the radionuclide labeling method in Example 33, precursor compounds 27-30 were labeled to obtain gallium-68 labeled products 36-39. The radiochemical purity of the products was determined by radio-thin-layer scanning, and the purity was over 97%, which can be used for subsequent imaging studies without further purification.

[0189] Example 39: Imaging study of gallium-68 labeled compounds 36-39 in healthy mice.

[0190] The tissue distribution of 36-39 was rapidly studied in healthy BALB / c nude mice. Results Figure 6 As shown, compounds 36, 37, and 38 were less distributed in normal tissues, and their average SUV values ​​were less than 0.5. One hour after injection, most of the radiopharmaceutical was rapidly metabolized to the bladder, with greater distribution in the kidneys, liver, and heart. Conversely, compound 39 showed greater uptake in normal tissues than in other tissues, particularly in the digestive system organs. One hour after injection, except for brain tissue, the average SUV values ​​in all other tissues were above 0.5, with the liver showing the highest average SUV value (~2.5), which remained above 1.0 for up to 3 hours. These data suggest that aromatic linkers are not conducive to improving tissue specificity, and compounds 36-38 were selected for PET / CT imaging in tumor-bearing mice.

[0191] Example 40: Imaging study of a prostate cancer xenograft model using gallium-68 labeled compounds 36-38

[0192] A NOD / SCID mouse model of prostate cancer xenograft was established by injecting 100 μCi of labeled product 36-38 via the tail vein. Images were acquired at 0.5, 1, 2, 3, and 4 hours post-injection.

[0193] See results Figure 7 As attached Figure 7 As shown in A / B / C, the lesions are clearly visible after the three compounds are absorbed into the tumor. The optimal imaging time for compounds 36 and 38 is 2 hours after injection. 68 Similar to Ga-IRM-015-DOTA. However, the average value of SUVs 36 and 38 is lower than the latter (see attached image). Figure 7 (D). However, compound 37 maintained a relatively stable tumor signal over 1–4 hours, providing a longer detection period. Myocardial uptake of compound 36 gradually increased, reaching a maximum at 2 hours, and then decreased, while the uptake of compounds 37 and 38 gradually decreased after 1 hour (see appendix). Figure 7 E). Hepatic uptake of compounds 36 and 37 peaked 2 hours after injection, while uptake of compound 38 gradually increased over 3 hours (see appendix). Figure 7 F). The kidneys are one of the main metabolic organs for the three compounds, and the absorption duration of 38 in the kidneys is shorter than that of 36 and 37 (see appendix). Figure 7 G).

[0194] Example 41: Investigation of the optimal in vivo imaging time for Gallium-68 labeled compound 37

[0195] Compound 37 exhibited a strong and prolonged absorption signal in tumors; therefore, comprehensive imaging studies were conducted from 5 minutes to 3 hours post-injection, with results as follows: Figure 8 A / B / C.

[0196] The results showed that compound 37 was rapidly absorbed by the tumor within 5 minutes of intravenous injection and slowly eliminated from the tumor lesion within 3 hours, providing physicians with greater flexibility in choosing scan timing. In non-tumor areas, the uptake levels of 37 in the heart, liver, and kidneys were higher than in tumors, with cardiac signals being rapidly cleared within 30 minutes of injection. Uptake in the liver and kidneys remained consistently high, indicating that these two organs are major metabolic sites.

[0197] Example 42: In vivo blocking imaging and tissue distribution study of gallium-68 labeled compound 37

[0198] Blockade experiments were conducted using a 100-fold dose of the CDK19 inhibitor BI.1347. Figure 9 A) The disappearance of tumor signal further confirms the CDK19 targeting of radionuclide 37. To further evaluate the metabolic characteristics of 37 in vivo, tissue distribution studies were conducted. Mice were sacrificed 1 hour and 2 hours after administration, tumors and major organs were dissected, weighed, and radioactivity was measured using a gamma counter to calculate %ID / g. Results are shown below. Figure 9 B, the results were consistent with the SUV values ​​obtained from in vivo PET / CT imaging, showing drug distribution in the heart, liver, kidneys, and blood, in addition to the tumor. 37 is slowly cleared from the tumor, providing stable tumor imaging, but its signal in the heart, lungs, and blood decreases rapidly within 1 to 2 hours post-injection. Furthermore, 37 is simultaneously metabolized in the liver and kidneys, which promises to alleviate metabolic stress and improve tissue safety compared to single-organ administration.

Claims

1. A nuclide of Formula I and its pharmaceutically usable salt: Ⅰ in: a) X and Y are selected from C atoms and N atoms, and at least one of X and Y is an N atom; b) R1 and R2 are independently selected from hydrogen atoms; c) R3 is independently selected from: ； d) M is selected from Ga and Gd.

2. The compound according to claim 1 and its pharmaceutically usable salt, characterized in that... R3 is selected independently from: 。 3. The compound according to claim 1, or a pharmaceutically usable salt thereof, selected from: 。 4. A method for preparing the compound according to any one of claims 1-3, characterized in that... Includes the following steps: 1) Take A and react it with B in dioxane under alkaline conditions via palladium catalysis to give intermediate compound C; 2) Compound C obtained in step 1) undergoes a Suzuki coupling reaction with D in dioxane under alkaline conditions with palladium catalysis to obtain intermediate compound E; 3) F was coupled with linker compound G in high yield at DCM and room temperature using triethylamine as a base to obtain intermediate compound H; 4) In the presence of potassium carbonate and acetonitrile, compound H and compound E are coupled at room temperature, and then the BOC protection is removed under acidic conditions to obtain compound I; 5) Compound I obtained in step 4) was reacted with tri-tert-butyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid under HATU and DIPEA conditions to undergo an acid-ammonia condensation reaction to obtain compound J; 6) The compound J obtained in step 5) was hydrolyzed under acidic conditions to obtain the labeled precursor compound IRM-X-DOTA; 7) The labeled precursor compound IRM-X-DOTA obtained in step 6) is complexed with the corresponding metal salt in water to obtain a non-radioactive cold-labeled compound I; The structures of the compounds are as follows: 。 5. A prodrug of the compound according to any one of claims 1-3 or a pharmaceutically usable salt thereof: 。 6. A composition, characterized in that, It includes the compound of any one of claims 1-3 and / or its pharmaceutically usable salt.

7. A composition, characterized in that, It comprises the compound of any one of claims 1-3 and / or a pharmaceutically usable salt thereof, and / or a pharmaceutically acceptable carrier.

8. The use of the compound of any one of claims 1-3 and its pharmaceutically usable salt in the preparation of radionuclide diagnostic and therapeutic reagents targeting CDK19.

9. The use of the compound of claim 8 and its pharmaceutically usable salt in the preparation of reagents for the diagnosis, staging and treatment of prostate cancer.

10. The use of the compound of any one of claims 1-3 and its pharmaceutically usable salt in the preparation of radionuclide diagnostic and therapeutic drugs targeting CDK19.

11. Use of the compound of claim 10 and its pharmaceutically usable salts in the preparation of medicaments for the diagnosis, staging and treatment of prostate cancer.

Images