CD38-targeted small-molecule radioactive probe as well as preparation method and application of CD38-targeted small-molecule radioactive probe

Abstract

The invention relates to a CD38-targeted micromolecule radioactive probe as well as a preparation method and application thereof, and belongs to the technical field of radiopharmaceutical chemistry. The invention aims to solve the problems that in the prior art, a targeted CD38 imaging agent lacks small molecule adaptability, and high specificity and tissue penetrability cannot be considered at the same time; unreasonable coupling and marking strategies lead to insufficient retention of target activity or pharmacokinetic disorder; the preparation process is complicated, and the large-area clinical popularization feasibility is low. The invention provides a small molecule radioactive probe targeting CD38, direct visualization of a CD38 target spot is realized, the image resolution and the specificity are obviously superior to those of a traditional antibody tracer, the tracer is good in stability, and the safety and the reliability meet clinical application requirements.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of radiopharmaceutical chemistry, and particularly relates to a small-molecule radioactive probe targeting CD38 and a preparation method and application thereof. BACKGROUND

[0002] CD38 belongs to a type II transmembrane glycoprotein with a molecular weight of 45 kDa, has enzymatic activity, can regulate intracellular calcium ion signal transmission, and is involved in mediating receptor-dependent lymphocyte adhesion and signal transduction process. The molecule is significantly highly expressed on the membrane surface of plasma cells, especially in multiple myeloma cells (MM), and the expression level is low in most resting natural killer (NK) cells, monocytes and other hematopoietic cell lines, indicating that CD38 can be used as an ideal targeting biomarker for plasma cell-related tumors. It is worth noting that CD38 also presents a high expression characteristic in various hematological malignancies such as acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia and non-Hodgkin's lymphoma, and studies have confirmed that the expression intensity is related to the prognosis of patients with chronic lymphocytic leukemia. Therefore, detecting the expression intensity of CD38 in tumor tissues, especially for malignant lesions with high expression of CD38, has important clinical value for early identification of diseases, tracking of treatment effect and prognosis.

[0003] At present, CD38-targeted PET imaging agents are divided into the following categories: (1) antibody / antibody fragment type: the molecular weight of monoclonal antibody is too large (150 kDa), the tissue penetration is poor, and it cannot reach the micro-lesions (such as bone lesions of myeloma with a diameter of <5 mm); and it cannot be filtered through the glomerulus, so the circulation and retention time in the body is long, and it needs to be labeled with a long half-life nuclide, but the above metal nuclides need to be prepared by solid target, and the preparation is generally expensive; and the imaging cycle of immune PET imaging is long, the radiation dose is large, there is a potential immunogenic risk, and long-term or repeated use may cause immune response of the body. (2) Polypeptide type: although the molecular weight is smaller than that of antibody (2-3 kDa), the chelating agent is coupled to the amino acid side chain of the polypeptide backbone, the structural design flexibility is low, and the polypeptide is easily degraded by peptidase in the body, resulting in a narrow imaging window; and the scale production and quality control are difficult, the production cost is higher than that of small-molecule probes, and it is relatively difficult to promote to large-area clinical application; there is a potential immunogenic risk, and long-term or repeated use may cause immune response of the body.

[0004] And, the antibody class CD38 targeting PET imaging agent coupling and labeling strategy is unreasonable, leading to insufficient target point activity reservation or pharmacokinetic disorder. On the one hand, the amino random coupling is adopted, the coupling site cannot be consistent each time, the key structural domain of CD38 binding can be occupied, the affinity can be influenced, a single probe structure cannot be obtained, the labeling rate is low, the product mass clinical application is influenced, and the cost is high. On the other hand, because the molecular weight is large, the clearance is slow, and the polypeptide class scheme is prone to degradation, the half-life is short, and the balance of ''fast imaging + low background noise'' cannot be realized.

[0005] Therefore, the person skilled in the art is eager to develop a small molecule radioactive probe targeting CD38, which has high specificity and tissue penetration, reasonable coupling and labeling strategy, simple preparation process, low cost, and can be widely clinically promoted. SUMMARY

[0006] The present application provides a small molecule radioactive probe targeting CD38, a preparation method and application, to solve the problems in the prior art that the imaging agent targeting CD38 lacks small molecule adaptability, cannot balance high specificity and tissue penetration, the coupling and labeling strategy is unreasonable, leading to insufficient target point activity reservation or pharmacokinetic disorder, the preparation process is complex, and the large-area clinical promotion feasibility is low.

[0007] One of the purposes of the present application is to provide a small molecule radioactive probe precursor targeting CD38, and the structure of the small molecule radioactive probe precursor is shown in formula I: Formula I; Or a pharmaceutically acceptable salt thereof, wherein R in formula I is any one of formula II or formula III: Formula II, Formula III; in formula III, n is an integer from 1 to 5.

[0008] The second purpose of the present application is to provide a small molecule radioactive probe targeting CD38, which is obtained by radionuclide labeling of the above-mentioned small molecule radioactive probe precursor targeting CD38.

[0009] In a preferred embodiment of the present application, the radionuclide is located in the NOTA chelating group in formula I, and the radionuclide is any one of 68 Ga, 67 Ga, 111 In, 64 Cu or 18 F.

[0010] The third object of the present application is to provide a preparation method of the CD38-targeting small molecule radioactive probe, which is prepared by a wet method or a freeze-drying method. The wet preparation step is as follows: the CD38-targeting small molecule radioactive probe precursor is dissolved in a sodium acetate buffer, a nuclide solution is added, and the reaction is oscillated at 40-100 DEG C for 15-30 min to obtain a reaction mixture; the reaction mixture is purified by an activated Sep-Pak C18 solid-phase extraction column to obtain the CD38-targeting small molecule radioactive probe. The freeze-drying preparation step is as follows: the CD38-targeting small molecule radioactive probe precursor is dissolved in a solvent, is divided into freeze-drying containers, is sealed into a freeze-dried medicine box after freeze-drying, is dissolved in a sodium acetate buffer, a nuclide solution is added, and the reaction is oscillated at 40-100 DEG C for 15-30 min to obtain a reaction mixture; the reaction mixture is purified by an activated Sep-Pak C18 solid-phase extraction column to obtain the CD38-targeting small molecule radioactive probe.

[0011] The fourth object of the present application is to provide a pharmaceutical composition comprising any one of the CD38-targeting small molecule radioactive probe precursor or the CD38-targeting small molecule radioactive probe and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0012] The fifth object of the present application is to provide a kit comprising any one or more of the CD38-targeting small molecule radioactive probe precursor or the CD38-targeting small molecule radioactive probe.

[0013] The sixth object of the present application is to provide the use of the CD38-targeting small molecule radioactive probe precursor or the CD38-targeting small molecule radioactive probe in the preparation of an imaging agent or imaging detection.

[0014] In a preferred embodiment of the present application, the imaging agent is an agent for imaging detection of cancer, tumor or metastasis; the cancer is any one or several of lymphoma, multiple myeloma, plasmacytoma, leukemia.

[0015] In a preferred embodiment of the present application, the imaging detection is performed by positron emission tomography (PET) or single photon emission computed tomography (SPECT).

[0016] In a preferred embodiment of the present application, the imaging detection object is a cell or tissue in vivo or in vitro.

[0017] Compared with the prior art, the present application has the beneficial effects that: the present application provides a small molecule radioactive probe targeting CD38, the non-active modification site of the small molecule radioactive probe is selected as the coupling site, that is, the 2-methoxyethoxy side chain at the end of the MK-0159 molecule, the site-specific coupling is carried out through the piperazine ring and the NOTA chelator (1,4,7-triazacyclononane-1,4,7-triacetic acid), to form a small molecule radioactive probe precursor, and the molecular structure of the small molecule radioactive probe precursor meets the following characteristics: the connection of the NOTA chelator and the MK-0159 does not change the binding conformation of the MK-0159 and the CD38, the inhibition activity IC 50 of the small molecule radioactive probe precursor on the human CD38 is maintained at 70-120 nM; the molecular lipid-water partition coefficient Log D 7.4 P is -1.30 to -1.60, which ensures that the small molecule radioactive probe precursor has certain water solubility (to avoid excessive accumulation in the liver or kidney) and tissue penetration (to reach the tumor microlesions) in the body.

[0018] Compared with the conventional CD38-targeted PET imaging agent, the present application has the following advantages: 1. Filling the blank of CD38 small molecule PET tracer: for the first time, the CD38 small molecule inhibitor is converted into a PET tracer to realize the direct visualization of the CD38 target, the site that does not affect the target binding is precisely modified, and the high affinity (target IC 50 is maintained at the nanomolar level) of the MK-0159 to the CD38 is retained, which provides a special tool for the diagnosis, target quantification and treatment monitoring of multiple myeloma and other CD38-positive diseases, solves the limitation of the conventional "indirect inference of target activity in vitro experiment", and can quantitatively evaluate the distribution heterogeneity of the CD38 in the body to provide a basis for precise treatment.

[0019] 2. Balancing high target activity and excellent pharmacokinetics: through site-specific coupling design (designing a "linker + site-specific coupling" strategy), the NOTA chelator is connected through the piperazine ring at the non-active site (such as the end alkyl chain) of the MK-0159, the interference of the space steric hindrance on the CD38 binding is avoided, the high affinity (IC 50 =72.58 nM) of the MK-0159 to the CD38 is retained, and the molecular lipid-water partition coefficient is optimized to ensure that the tracer is rapidly cleared in the body (the terminal elimination half-life t 1 / 2z =30-60 minutes), the tumor / muscle ratio (T / M) is high (T / M=3.95±0.51 at 60 minutes of imaging), there is no obvious liver / kidney accumulation (the uptake is all <10 %ID / g), and the image resolution and specificity are significantly better than those of the conventional antibody tracer.

[0020] 3. High clinical transformation feasibility: the molecular weight of the small molecule radioactive probe is less than 1 kDa, which is suitable for the synthesis of radiopharmaceuticals, and the synthesis process is simple and the cost is low. 68The Ga chelation reaction conditions are simple and rapid (total time ≤30 min), achieving a labeling rate of >90% within 15 minutes. The product can be purified by simple solid-phase extraction (no complex HPLC required), with a purity >99% after purification. The tracer also exhibits good stability (after 2 hours of incubation in physiological saline / serum, the radiochemical purity remains ≥98%; in vivo, the proportion of intact structures in blood / urine within 2 hours is ≥95%). Its safety and reliability meet the requirements for clinical applications, solving the labeling efficiency and application problems of short-half-life radionuclides.

[0021] The method for preparing small molecule radioactive probes targeting CD38 provided by this invention has the following advantages: (1) The reaction at 40 degrees Celsius avoids high temperature damage to the MK-0159 structure; (2) Sodium acetate buffer maintains a neutral to slightly acidic environment, ensuring both... 68 The efficient chelation of Ga and Nota (labeling rate ≥93%) avoids molecular degradation caused by strong acids and bases; (3) the solid phase extraction purification steps are simple and fast (total labeling + purification time ≤30 minutes), which is suitable for 68 Ga's short half-life characteristic can meet the needs of clinical on-demand preparation. Attached Figure Description

[0022] Figure 1 A schematic diagram of the synthesis steps for a small molecule radioactive probe precursor targeting CD38; Figure 2 Mass spectrometry results of the small molecule radioactive probe precursor NOTA-MK targeting CD38; Figure 3 The small molecule radioactive probe precursor NOTA-MK targeting CD38 1 Image of H NMR detection results; Figure 4 The small molecule radioactive probe precursor NOTA-MK targeting CD38 13 Image of C NMR detection results; Figure 5 Mass spectrometry results of the small molecule radioactive probe precursor NOTA-P3MK targeting CD38; Figure 6 The small molecule radioactive probe precursor NOA-P3MK targeting CD38 1 Image of H NMR detection results; Figure 7 The HPLC results of a small molecule radioactive probe targeting CD38 are shown in Figure A; A represents... 68 The detection results of the Ga-NOTA-MK small molecule radioactive probe are shown in Figure B, where B represents... 68 Figure showing the detection results of the Ga-NOTA-P3MK small molecule radioactive probe. Figure 8IC of small molecule radioprobes targeting CD38 NOTA-MK 50 Detection results figure; A is mCD38 detection results figure, B is hCD38 detection results figure; Figure 9 In vitro stability detection results figure of small molecule radioprobes targeting CD38; A and B are respectively 68 Ga-NOTA-MK small molecule radioprobes detection results figure in saline and serum, C and D are respectively 68 Ga-NOTA-P3MK small molecule radioprobes detection results figure in saline and serum; Figure 10 In vivo stability detection results figure of small molecule radioprobes targeting CD38; A and B are respectively 68 Ga-NOTA-MK small molecule radioprobes detection results figure in blood and urine, C and D are respectively 68 Ga-NOTA-P3MK small molecule radioprobes detection results figure in blood and urine; Figure 11 In vivo pharmacokinetics detection results figure of small molecule radioprobes targeting CD38; A is 68 Ga-NOTA-MK small molecule radioprobes detection results figure, B is 68 Ga-NOTA-P3MK small molecule radioprobes detection results figure; Figure 12 Small molecule radioprobes targeting CD38 68 Ga-NOTA-MK animal PET imaging results figure. DETAILED DESCRIPTION

[0023] Those skilled in the art can improve the process parameters according to the content herein, and make appropriate modifications. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content and scope of the present application, to realize and apply the present application technology.

[0024] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below in combination with specific embodiments. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by commercial channels by those skilled in the art.

[0025] Example 1: Preparation of a small molecule radioactive probe precursor targeting CD38 1. The structure of the small molecule radioactive probe precursor targeting CD38 described in this embodiment is shown in Formula I. When R in Formula I is Formula II, the preparation steps are as follows: S1: To a solution of methyl 1H-pyrrolo[2,3-b]pyridine-4-carboxylic acid (5.0 g, 28.4 mmol, 1.0 equivalence) dissolved in DME (1,2-dimethoxyethane, 50 mL), m-chloroperoxybenzoic acid (14.4 g, 71.0 mmol, 85% purity, 2.5 equivalence) was added. The mixture was stirred at 20 °C for 12 h, poured into water (300 mL), and extracted with ethyl acetate (twice, 300 mL each time). The organic phase was filtered and concentrated under vacuum to obtain intermediate 1, which was 4-(methoxycarbonyl)-1H-pyrrolo[2,3-b]pyridine 7-oxide (3.5 g, yield 55.19%, purity 86%). S2: Intermediate 1 (3.5 g, 18.2 mmol, 1.0 equivalence) and hexamethyldisilazane (2.9 g, 18.2 mmol, 3.8 mL, 1.0 equivalence) were dissolved in THF (tetrahydrofuran, 50 mL), and then ethyl chloroformate (8.0 g, 73.2 mmol, 7 mL, 4.0 equivalence) was added. The mixture was stirred at 50 °C for 12 h and concentrated under vacuum to obtain residue 1. Residue 1 was dissolved in DCM (dichloromethane, 200 mL), washed with saturated sodium bicarbonate aqueous solution, and the organic phase was dried and concentrated under vacuum to obtain residue 2. Residue 2 was purified by silica gel column chromatography (ISCO®; 80 g SepaFlash® silica gel rapid column, using petroleum ether and 0%-30% ethyl acetate as mixed eluent, with a volume ratio of petroleum ether to ethyl acetate of 3:1, and a flow rate of 120. (mL / min) Residue 2, to obtain intermediate product 2, which is a mixture of methyl 6-chloro-1H-pyrrolo[2,3-b]pyridine-4-carboxylate and methyl 6-chloro-1H-pyrrolo[2,3-b]pyridine-1,4-dicarboxylic acid 1-ethyl ester 4-methyl ester, with a mixing ratio of 1:2 (2.5 g, crude product); S3: Intermediate 2 (2.5 g, 8.8 mmol, 1.0 eq), thiazole-5-boronic acid pinacol ester (2.8 g, 13.3 mmol, 1.5 eq), cuprous iodide (1.0 g, 5.3 mmol, 0.6 eq), potassium carbonate (3.7 g, 26.5 mmol, 3.0 eq) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II), dichloromethane complex (361 mg, 442 μmol, 0.05 eq) were dissolved in ethanol aqueous solution (ethanol 36 mL, water 4 mL), degassed and replaced by nitrogen for 3 times, then stirred at 100 °C for 12 h under nitrogen atmosphere, poured into water (200 mL), extracted with ethyl acetate (100 mL each time), the aqueous phase was acidified with 3N HC1 to pH = 3, concentrated under vacuum to obtain intermediate 3, which was 6-(thiazol-5-yl)-1H-pyrrolo[2,3-b]pyridine-4-carboxylic acid (2 g, 80.22% yield, 87.19% purity); S4: To a solution of trans-4-aminocyclohexanol (25 g, 217.06 mmol, 1 eq) in acetonitrile (350 mL) was added potassium carbonate (90.00 g, 651.19 mmol, 3 eq) and benzyl bromide (74.25 g, 434.13 mmol, 51.56 mL, 2 eq), stirred at 75 °C for 5 h, diluted with water (1000 mL), extracted with ethyl acetate (3 times, 400 mL each time), the organic phases were combined and washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, slurried with petroleum ether (200 mL) at 20 °C for 30 min to obtain intermediate 4, which was trans-4-benzylaminocyclohexanol (44.4 g, 145.79 mmol, 67.16% yield, 97% purity); S5: To a solution of intermediate 4 (10 g, 33.85 mmol, 1 eq) in N,N- dimethylformamide (150 mL) was added sodium hydride (5.42 g, 135.4 mmol, 60% purity, 4 eq) at 0 °C with stirring for 0.5 h; then 2-(3-bromopropyl)tetrahydro-2H-pyran (15.10 g, 67.70 mmol, 2 eq) was added at 0 °C with stirring for 11.5 h at 90 °C. After dilution with water (1 L), extraction was performed with ethyl acetate (2 times, 500 mL each time), the organic phases were combined and washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to give residue 3. Purification of residue 3 by silica gel column chromatography (330 g SepaFlash® silica gel flash column, eluent: petroleum ether-ethyl acetate mixture, gradient elution with 0-10% ethyl acetate, flow rate 150 mL / min) gave intermediate 5, which was (1R,4S)-4-[(3-(tetrahydro-2H-pyran-2- yloxy)propyl)oxy]cyclohexyldibenzylamine (11.8 g, 25.35 mmol, 24.96% yield, 94% purity); S6: Intermediate 5 (11.8 g, 26.96 mmol, 1 eq) was added to a solution of hydrogen chloride 1,4-dioxane (4 mol / L, 50 mL, 7.42 eq) at 20 °C with stirring for 1 h. Extraction was performed with ethyl acetate (50 mL each time), the organic phase was adjusted to pH 8 with sodium hydroxide, poured into water (80 mL), and extracted with ethyl acetate (2 times, 100 mL each time). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to give residue 4. Purification of residue 4 by preparative high performance liquid chromatography (chromatography column Phenomenex luna C18 100*40 mm*5 μm; mobile phase: water containing 0.2% formic acid-acetonitrile; gradient: 1%-40% B, 8.0 min) gave intermediate 6, which was (1R,4S)-4-(3-hydroxypropoxy)cyclohexyldibenzylamine (8 g, 22.40 mmol, 83.09% yield, 99% purity); S7: To a solution of intermediate 6 (8 g, 22.63 mmol, 1 eq) in dichloromethane (120 mL) was added triethylamine (6.87 g, 67.89 mmol, 9.45 mL, 3 eq) and 4-methylbenzenesulfonyl chloride (6.47 g, 33.95 mmol, 1.5 eq) sequentially at 0 °C. The reaction mixture was stirred at 20 °C for 12 h, diluted with water (100 mL) and extracted with dichloromethane (3 times, 100 mL each time). The combined organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give a residue 5. The residue 5 was purified by column chromatography on silica gel (4 g SepaFlash® silica gel flash column, eluent: petroleum ether-ethyl acetate mixture, gradient elution with 0-25% ethyl acetate at a flow rate of 40 mL / min) to give intermediate 7, (1R,4S)-4-(3-p-tolylsulfonyloxypropoxy)cyclohexyl dibenzylamine (9.45 g, 16.38 mmol, 72.38% yield, 88% purity); S8: To a solution of intermediate 7 (8 g, 15.76 mmol, 1 eq) in acetonitrile (160 mL) was added potassium carbonate (4.36 g, 31.52 mmol, 2 eq) and 1-(tert-butoxycarbonyl)piperazine (3.52 g, 18.91 mmol, 1.2 eq) sequentially. The reaction mixture was stirred at 90 °C for 12 h, diluted with water (100 mL) and extracted with ethyl acetate (3 times, 100 mL each time). The combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give a residue 6. The residue 6 was purified by column chromatography on silica gel (4 g SepaFlash® silica gel flash column, eluent: petroleum ether-ethyl acetate mixture, gradient elution with 0-100% ethyl acetate at a flow rate of 40 mL / min) to give intermediate 8, (1R,4S)-4-[3-(4-tert-butoxycarbonylpiperazin-1-yl)propoxy]cyclohexyl dibenzylamine (7.5 g, 87% purity); S9: Intermediate product 8 (7 g, 13.42 mmol, 1 eq) was dissolved in tetrahydrofuran (140 mL) and stirred at 20 °C until a clear solution was formed; the solution was pumped into a fixed bed reactor (the fixed bed reactor was: a fixed bed reactor with 13.42 mmol, 5% purity of palladium hydroxide on alumina catalyst, 1 / 4 inch stainless steel, 20 mL in volume, heated to 70 °C, hydrogen back pressure regulator set to 2 MPa) at a flow rate of 1 mL / min, the hydrogen flow rate was 80 mL / min, the system was operated for 3.3 min before the reaction solution was collected, and the collection was continued for 140 min before stopping, and the reaction solution was concentrated under reduced pressure to obtain intermediate product 9, which was (1R,4S)-4-[3-(4-tert-butoxycarbonyl piperazin-1-yl)propoxy]cyclohexylamine (3.9 g, crude); S10: To a solution of intermediate product 3 (590 mg, 2.41 mmol, 1 eq) in N,N- dimethylformamide (15 mL) were added 1-hydroxybenzotriazole (487.59 mg, 3.61 mmol, 1.5 eq), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (691.75 mg, 3.61 mmol, 1.5 eq), N,N-diisopropylethylamine (932.73 mg, 7.22 mmol, 1.26 mL, 3 eq) and intermediate product 9 (821.50 mg, 2.41 mmol, 1 eq) successively, and stirred at 20 °C for 12 h, diluted with water (50 mL) and extracted with ethyl acetate (3 times, 50 mL each time), the organic phases were combined and washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue, which was purified by silica gel column chromatography (4 g SepaFlash® silica gel flash separation column, eluent was a mixture of ethyl acetate-methanol, eluted with a gradient of 0-20% methanol, flow rate was 40 mL / min) to obtain intermediate product 10, which was (1R,4S)-4-[3-(4-tert-butoxycarbonyl piperazin-1-yl)propoxy]cyclohexyl-(4- thiazolyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (400 mg, 82% purity); S11: Intermediate product 10 (350 mg, 615.41 μmol, 1 eq) was dissolved in hydrogen chloride ethyl acetate solution (4 mol / L, 14.00 mL, 91.00 eq), stirred at 20 °C for 1 h, filtered, and the filter cake was concentrated under reduced pressure to obtain intermediate product 11, which was (1R,4S)-4-[3-(piperazin-1-yl)propoxy]cyclohexyl-(4-thiazolyl)-1H-pyrrolo[2,3-b]pyridine-5- carboxamide (370 mg, 90% purity, hydrochloride salt); S12: To a solution of 1,4,7-triazacyclononane-1,4-diacetic acid di-tert-butyl ester (150 mg, 360.99 μmol, 1 eq) in N,N-dimethylformamide (2 mL) was added 1-hydroxybenzotriazole (73.17 mg, 541.49 μmol, 1.5 eq), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (103.80 mg, 541.49 μmol, 1.5 eq), N,N-diisopropylethylamine (139.97 mg, 1.08 mmol, 188.63 μL, 3 eq) and intermediate 11 (182.33 mg, 360.99 μmol, 1 eq, hydrochloride salt), stirred at 20 °C for 12 h, filtered, the filtrate was purified by preparative high performance liquid chromatography (column WePure Biotech XP tC18 150*40*7 μm; mobile phase: water containing 10 mmol / L ammonium bicarbonate-acetonitrile; gradient: 30%-70% B, 8.0 min) to obtain intermediate 12, which was (1R,4S)-4-[3-(4-{[1,4,7-tris(tert-butoxycarbonylmethyl)-1,4,7-triazacyclononan-1-yl]carbonyl}piperazin-1-yl)propoxy]cyclohexyl-(4-thiazolyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (170 mg, 174.69 μmol, 48.39% yield, 89% purity); S13: To a solution of intermediate 12 (170 mg, 196.28 μmol, 1 eq) in dichloromethane (4 mL) was added trifluoroacetic acid (6.14 g, 53.85 mmol, 4 mL, 274.35 eq), stirred at 20 °C for 12 h, concentrated under reduced pressure to obtain residue 8, which was purified by preparative high performance liquid chromatography (column Phenomenex luna C18 100*40 mm*5 μm; mobile phase: water containing 0.2% formic acid-acetonitrile; gradient: 3%-20% B, 10.0 min) to obtain the small-molecule radioprobes precursor targeting CD38 1 (33.5 mg, 44.22 μmol, 22.53% yield, 99.51% purity), abbreviated as NOTA-MK Figure 1 ).

[0026] 2, The structure of a small-molecule radioprobes precursor targeting CD38 according to the embodiment is shown in formula I, wherein when R in formula I is formula III and n = 3, the preparation steps are as follows: S1 : To a solution of methyl 1H-pyrrolo[2,3-b]pyridine-4-carboxylate (5.0 g, 28.4 mmol, 1.0 eq) in DME (1,2-dimethoxyethane, 50 mL) was added m-chloroperoxybenzoic acid (14.4 g, 71.0 mmol, 85% purity, 2.5 eq) at 20 °C and stirred for 12 h. After pouring into water (300 mL), it was extracted with ethyl acetate (2 times, 300 mL each time), the organic phase was filtered, and concentrated in vacuo to give intermediate 1, which was 4-(methoxycarbonyl)-1H-pyrrolo[2,3-b]pyridine 7-oxide (3.5 g, yield 55.19%, purity 86%); S2: Intermediate 1 (3.5 g, 18.2 mmol, 1.0 eq) and hexamethyldisilazane (2.9 g, 18.2 mmol, 3.8 mL, 1.0 eq) were dissolved in THF (tetrahydrofuran, 50 mL), and then ethyl chloroformate (8.0 g, 73.2 mmol, 7 mL, 4.0 eq) was added at 50 °C and stirred for 12 h. The residue 1 was obtained by concentration in vacuo. The residue 1 was dissolved in DCM (dichloromethane, 200 mL), washed with saturated aqueous sodium bicarbonate solution, and then concentrated in vacuo after drying to give residue 2. Residue 2 was purified by silica gel column chromatography (ISCO®; 80 g SepaFlash® silica gel flash column, using petroleum ether and 0-30% ethyl acetate as mixed eluent, the volume ratio of petroleum ether and ethyl acetate was 3:1, and the flow rate was 120 mL / min) to give intermediate 2, which was a mixture of 6-chloro-1H-pyrrolo[2,3-b]pyridine-4-carboxylic acid methyl ester and 6-chloro-1H-pyrrolo[2,3-b]pyridine-1,4-dicarboxylic acid 1-ethyl ester 4-methyl ester with a mixing ratio of 1:2 (2.5 g, crude product); S3: Intermediate 2 (2.5 g, 8.8 mmol, 1.0 eq), thiazole-5-boronic acid pinacol ester (2.8 g, 13.3 mmol, 1.5 eq), cuprous iodide (1.0 g, 5.3 mmol, 0.6 eq), potassium carbonate (3.7 g, 26.5 mmol, 3.0 eq), and [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride dichloromethane complex (361 mg, 442 μmol, 0.05 eq) were dissolved in an ethanol aqueous solution (ethanol 36 mL, water 4 mL), degassed and replaced with nitrogen for 3 times, and then stirred at 100 °C for 12 h under a nitrogen atmosphere. After pouring into water (200 mL), it was extracted with ethyl acetate (100 mL each time), the aqueous phase was acidified with 3N HCl to pH=3, and concentrated in vacuo to give intermediate 3, which was 6-(thiazol-5-yl)-1H-pyrrolo[2,3-b]pyridine-4-carboxylic acid (2 g, yield 80.22%, purity 87.19%); S4: To (1R,4R)-4-[(2-(2-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethoxy)ethoxy]cyclohexylamine (70 mg, 179 μmol, 1.1 eq) and intermediate product 3 (40 mg, 163 μmol, 1.0 eq), 1-hydroxybenzotriazole (33 mg, 244 μmol, 1.5 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (47 mg, 244 μmol, 1.5 eq) and diisopropylethylamine (63 mg, 489 μmol, 0.1 mL, 3.1 eq) were added, stirred at 20 °C for 12 h; diluted with water, extracted with ethyl acetate, combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered to obtain residue 9, residue 9 was purified by silica gel column chromatography to obtain intermediate product 13 (100 mg, yield 85.09%, purity 85.66%); S5: To the solution of intermediate product 13 (100 mg, 162 μmol, 1.0 eq) in ethyl acetate (2 mL), hydrogen chloride ethyl acetate solution (4 M, 4 mL) was added, stirred at 20 °C for 1 h, concentrated under reduced pressure to obtain intermediate product 14 (90 mg, crude, purity 91.72%, HC1); S6: To the solution of 1,4,7-triazacyclononane-1,4-di-tert-butyl ester (67 mg, 162 μmol, 1.0 eq) in N,N-dimethylformamide (1 mL), 1-hydroxybenzotriazole (33 mg, 244 μmol, 1.5 eq), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (62 mg, 325 μmol, 2.0 eq), N,N-diisopropylethylamine (105 mg, 812 μmol, 0.1 mL, 5.0 eq) and intermediate product 14 (90 mg, 162 μmol, 1.0 eq, HC1) were added, stirred at 20 °C for 12 h, filtered, the filtrate was purified by preparative high performance liquid chromatography to obtain intermediate product 15 (40 mg, yield 23.34%, purity 86.73%); S7: To the solution of intermediate 15 (40 mg, 44 μmol, 1.0 eq) in dichloromethane (1 mL), trifluoroacetic acid (1.54 g, 13 mmol, 1 mL) was added, stirred at 20 °C for 12 h, concentrated under reduced pressure to obtain residue 10, purified by preparative high performance liquid chromatography (chromatography column WePure Biotech XP tC18 150*40*7 μm; mobile phase: water containing 10 mmol / L ammonium bicarbonate-acetonitrile; gradient: 30%-70% B, 8.0 min) to obtain the small-molecule radioactive probe precursor 2 targeting CD38 (6.5 mg, yield 17.76%, purity 95.89%), referred to as NOTA-P3MK Figure 1 ).

[0027] Example 2: Preparation of a small-molecule radioactive probe targeting CD38 The preparation method is prepared by wet method or freeze-drying method; The wet preparation step is: 50 μg of the small-molecule radioactive probe precursor (NOTA-MK or NOTA-P3MK) prepared in Example 1 is dissolved in 500 μL of 0.25 M sodium acetate buffer (pH=4.0-4.5), and then 1 mL of GaCl3 solution (radioactivity 0.74-1.11 GBq, dissolved in 0.1M HCl) is added, and the reaction is oscillated at 40 °C for 15 min to obtain a reaction mixture; the reaction mixture is purified by using an activated Sep-Pak C18 solid phase extraction column), to obtain the small-molecule radioactive probe (Ga-NOTA-MK or Ga-NOTA-P3MK) targeting CD38; 68 68 Ga-NOTA-MK or Ga-NOTA-P3MK); 68 Ga-NOTA-P3MK); The freeze-drying preparation step is: 50 μg of the small-molecule radioactive probe precursor (NOTA-MK or NOTA-P3MK) prepared in Example 1 is dissolved in a solvent, and then is divided into freeze-drying containers, and is sealed into a freeze-dried medicine box after freeze-drying; the freeze-dried medicine box is dissolved in 500 μL of 0.25 M sodium acetate buffer (pH=4.0-4.5), and then 1 mL of GaCl3 solution (radioactivity 0.74-1.11 GBq, dissolved in 0.1M HCl) is added, and the reaction is oscillated at 40 °C for 15 min to obtain a reaction mixture; the reaction mixture is purified by using an activated Sep-Pak C18 solid phase extraction column (first washed with 10 mL of ultrapure water to remove unreacted Ga 68 68 Ga 3+ ​​, eluted with 1 mL 85% ethanol, and then diluted with normal saline after the ethanol was blown dry, to obtain the small-molecule radioactive probe targeting CD38 68 Ga-NOTA-MK or 68 Ga-NOTA-P3MK).

[0028] Effect experiment: 1. The small-molecule radioactive probe precursor targeting CD38 prepared in Example 1 (NOTA-MK and NOTA-P3MK) was detected by mass spectrometry and 1 H NMR, respectively. The results are shown in Table 1. Figures 2-6 The structure of the synthesized small-molecule radioactive probe precursor targeting CD38 was correct.

[0029] 2. The purity of the small-molecule radioactive probe targeting CD38 prepared in Example 2 (Ga-NOTA-MK and Ga-NOTA-P3MK) was detected by HPLC. The results are shown in Table 2. 68 Ga-NOTA-MK and 68 Ga-NOTA-P3MK) were labeled with Ga, and the purity of the obtained small-molecule radioactive probe targeting CD38 was >98%. Figure 7 68 Ga-NOTA-MK and 68 Ga-NOTA-P3MK) were labeled with Ga, and the purity of the obtained small-molecule radioactive probe targeting CD38 was >98%.

[0030] 3. Binding energy calculation: The extracellular domain crystal structure of human CD38 wild type (hCD38, WT; PDB ID: 2EF1) and E226Q mutant (PDB ID: 4OGW) was used to dock the NOTA-MK small-molecule radioactive probe precursor prepared in Example 1 by AutoDock Vina (version 1.1.2); the protein structure was treated by protonation state assignment (pH=7.4) and conformational minimization; the small-molecule structure was preprocessed to generate stereoisomers, assign tautomer forms, and define the protonation state under the condition of pH=7.4; the protein backbone was kept as a rigid structure during the docking process.

[0031] The results are shown in Table 1. The scoring value of the NOTA-MK small-molecule radioactive probe precursor prepared in Example 1 for human CD38 was lower than that of the CD38 small-molecule inhibitor MK-0159, indicating better binding effect and higher conformational matching degree.

[0032] Table 1

[0033] 4. IC 50 detection: IC 50The values ​​were determined using a fluorescent CD38 inhibitor screening kit (hydrolase activity; BPS Bioscience, CA, USA), strictly following the manufacturer's instructions. Fluorescence signals were detected using a multi-well plate reader. Data were analyzed using GraphPad Prism 10 software (GraphPad Software, CA, USA) to generate concentration curves for the small molecule radioactive probe precursor NOA-MK prepared in Example 1 and to calculate the IC50 values ​​of mCD38 and hCD38. 50 value.

[0034] The results are as follows Figure 8 As shown, the small molecule radioactive probe precursor NOTA-MK prepared in Example 1 has IC50 activity against hCD38 and mCD38. 50 The values ​​are 72.58 nM and 7.55 nM, respectively.

[0035] 5. In vitro stability: 100 μL of the solution prepared in Example 2 was tested. 68 Ga-NOTA-MK and 68 Ga-NOTA-P3MK samples were placed in 900 μL of physiological saline or fetal bovine serum (FBS) and incubated at 37°C for 30, 60, or 120 minutes, respectively, before detection. For physiological saline samples, the samples were directly analyzed by radio-HPLC. For FBS samples, an equal volume of precipitant (acetonitrile:methanol, 1:1 volume ratio) was added to completely precipitate the proteins. After centrifugation at 13,000 rpm for 5 minutes, the supernatant was collected and analyzed by radio-HPLC.

[0036] The results are as follows Figure 9 As shown, the in vitro stability results indicated that after incubation in saline and serum for 2 hours, the product prepared in Example 2... 68 Ga-NOTA-MK and 68 Ga-NOTA-P3MK still maintains a radiochemical purity of >97%.

[0037] 6. In vivo stability: In vivo metabolic stability studies were conducted in normal BALB / c nude mice (purchased from Jicui Pharmaceutical Co., Ltd.), with intravenous injection of 37 MBq radioactive tracer (prepared in Example 2). 68 Ga-NOTA-MK or 68 Following the administration of the Ga-NOTA-P3MK small molecule radioactive probe, blood and urine samples were collected at 30, 60, and 120 minutes. Urine samples were diluted for radio-HPLC analysis. Blood samples were centrifuged, and the supernatant was mixed with an equal volume of precipitant (acetonitrile:methanol = 1:1 volume ratio) to precipitate proteins. This mixture was centrifuged at 13,000 rpm for 5 minutes, and the supernatant was collected for subsequent radio-HPLC analysis.

[0038] The results are as follows Figure 10 As shown, in vivo stability results indicated that after injection of 2, the small molecule radioactive probe was present in the blood and urine of mice. 68 Ga-NOTA-MK and 68 Ga-NOTA-P3MK still maintains a radiochemical purity of >95%.

[0039] 7. Pharmacokinetics: Normal mice (n=3, purchased from Jicui Pharmaceutical Co., Ltd.) were injected via tail vein with the preparation of Example 2. 68 Ga-NOTA-MK and 68 Ga-NOTA-P3MK (~7.4 MBq). Tail vein blood samples were collected at different time points (3, 5, 10, 15, 30, 60, and 120 minutes) after injection. The weight of the collected blood was measured, and the radioactivity was determined using a gamma counter. After attenuation correction to the injection time, the probe content in the blood was expressed as %ID / g. Time-activity (%ID / g) data were plotted in Origin software (OriginLab, Massachusetts, USA) and fitted using a double exponential decay model. Pharmacokinetic parameters were derived using DAS 2.0 software (Chinese Society of Mathematical Pharmacology, Shanghai) based on a single-dose two-compartment model.

[0040] The results are as follows Figure 11 As shown, the in vivo pharmacokinetic results indicate... 68 Ga-NOTA-MK and 68 The Ga-NOTA-P3MK has an in vivo half-life of about 30-60 minutes, making it suitable for diagnostic imaging applications.

[0041] 8. Animal Models: All experimental animals were purchased from Jicui Pharmaceutical Co., Ltd. For the subcutaneous xenograft model, H929 cell suspension (5×10⁻⁶) was used. 6 MM.1S or U266 cell suspensions (5 × 10⁶ cells, containing 50% PBS and 50% Matrigel) were injected subcutaneously into the right hind limb of female BALB / c nude mice; 6 One cell (containing 50% PBS and 50% matrix gel) was injected subcutaneously into the right hind limb of NCG mice.

[0042] 9. Small animal PET imaging: When the tumor volume reaches 300-500 cm³ 3 Micro PET / CT was performed (n=3 per group). Each tumor-bearing mouse model was injected via tail vein with 5.55-7.4 MBq of radiotracer (molecular radioactive probe prepared in Example 2). 68Ga-NOTA-MK (200 μL dissolved in PBS). PET or PET / CT scans were performed at 30, 60, and 120 minutes after injection, with anesthesia maintained continuously by inhalation of isoflurane. Subcutaneous tumor tissue was dissected after imaging for HE staining and human CD38 immunohistochemistry.

[0043] The results are as follows Figure 12 As shown, micro PET / CT imaging of tumor-bearing mouse models after injection of small-molecule radioactive probes targeting CD38 showed that the uptake of small-molecule radioactive probes targeting CD38 at the tumor site was proportional to CD38 expression; the subcutaneous tumor area is marked with a red dashed line.

[0044] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. These embodiments are selected and specifically described in this specification to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A small molecule radioactive probe precursor targeting CD38, characterized in that, The structure of the small molecule radioactive probe precursor is shown in Formula I: Equation I; Or a pharmaceutically acceptable salt thereof, wherein R in Formula I is any one of the structures of Formula II or Formula III: Formula II, Equation III; in Equation III, n is an integer from 1 to 5.

2. A small molecule radioactive probe targeting CD38, characterized in that, The small molecule radioactive probe targeting CD38 is obtained by radiolabeling the small molecule radioactive probe precursor targeting CD38 as described in claim 1.

3. The small molecule radioactive probe according to claim 2, characterized in that, The radiolabeled nuclide is located in the NOA chelate group of Formula I, and the nuclide is... 68 Ga、 67 Ga、 111 In、 64 Cu or 18 Any one of F.

4. The method for preparing the small molecule radioactive probe targeting CD38 according to any one of claims 2 to 3, characterized in that, The preparation method employs either a wet method or a freeze-drying method; The wet preparation step is as follows: the small molecule radioactive probe precursor targeting CD38 described in claim 1 is dissolved in sodium acetate buffer, a radionuclide solution is added, and the reaction is shaken at 40℃-100℃ for 15-30 min to obtain a reaction mixture; the reaction mixture is purified using an activated Sep-Pak C18 solid-phase extraction column to obtain the small molecule radioactive probe targeting CD38. The freeze-drying preparation steps are as follows: the small molecule radioactive probe precursor targeting CD38 described in claim 1 is dissolved in a solvent, dispensed into a freeze-drying container, and sealed into a freeze-dried kit after freeze-drying; the freeze-dried kit is dissolved in sodium acetate buffer, a radionuclide solution is added, and the reaction is shaken at 40℃-100℃ for 15-30 min to obtain a reaction mixture; the reaction mixture is purified using an activated Sep-Pak C18 solid-phase extraction column to obtain the small molecule radioactive probe targeting CD38.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises any one of the small molecule radioactive probe precursor targeting CD38 as described in claim 1 or any one of the small molecule radioactive probes targeting CD38 as described in any one of claims 2 to 3, as well as a pharmaceutically acceptable carrier, diluent, excipient or excipient.

6. A reagent kit, characterized in that, The kit contains one or more of the small molecule radioactive probe precursor targeting CD38 as described in claim 1 or the small molecule radioactive probe targeting CD38 as described in any one of claims 2 to 3.

7. The use of the CD38-targeting small molecule radioactive probe precursor of claim 1 or the CD38-targeting small molecule radioactive probe of any one of claims 2 to 3 in the preparation of imaging agents or imaging detection.

8. The application according to claim 7, characterized in that, The imaging agent is a reagent used for imaging detection of cancer, tumors, or metastatic lesions; the cancer is any one or more of lymphoma, multiple myeloma, plasmacytoma, and leukemia.

9. The application according to claim 7, characterized in that, The imaging detection is performed using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

10. The application according to claim 7, characterized in that, The imaging detection targets cells or tissues, whether inside or outside the body.

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