DOTA-HX2-DZ compound and use thereof

Abstract

The present invention relates to the field of biopharmaceutical technology, and in particular, to a DOTA-HX2-DZ compound and use thereof. In the present invention, by optimizing the molecular structure of DZ-HX2-DOTA, special linkers are designed and used, thereby solving the problem in the prior art of limited anti-tumor efficacy caused by poor stability, lipid solubility, retention time in tumors, and other properties of radiolabeled drugs. The compound of the present invention can significantly reduce the delabeling risk of radioactive metal ions in an in vivo environment and ensure a stable effect at the target site. The compound of the present invention has excellent lipid solubility, and can penetrate cell membranes and vascular barriers more easily, thereby improving the bioavailability of drugs, prolonging the in vivo circulation time, prolonging the retention time in tumors, and further improving the treatment efficiency. The DZ-HX2-DOTA of the present invention has a broad-spectrum tumor targeting property, and can be used for diagnosing and treating various types of malignant tumors.

Description

Compound DOTA-HX2-DZ and its applications Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the compound DOTA-HX2-DZ and its applications. Background Technology

[0002] With the rising incidence of cancer year by year, the development of diagnostic and treatment methods for malignant tumors has become an important topic in the medical field. Currently, radionuclide drugs play a crucial role in cancer diagnosis and treatment. Radionuclide drugs utilize the energy of radioactive isotopes to release radiation at the tumor site, killing cancer cells while minimizing damage to surrounding normal tissues. The advantage of this treatment method lies in its ability to target tumor cells, providing more precise and effective treatment. Radionuclide drugs have achieved good results in the diagnosis and treatment of various cancers, especially some difficult-to-treat malignant tumors, such as prostate cancer and neuroendocrine tumors.

[0003] In the development of radionuclide drugs, the stability and specificity of the compounds are two key factors. To effectively target tumor cells, radionuclide drugs typically need to be bound to specific target molecules, such as antibodies, peptides, or small molecule compounds, which can recognize and bind to specific receptors or antigens on tumor cells. However, these conjugates usually face two main challenges: first, the problem of radioisotope detargeting, where the radioisotope detaches from the target molecule, causing the radiopharmaceutical to circulate in the bloodstream and increasing radiation damage to normal tissues; and second, the rate of drug metabolism and targeting in the body, i.e., how to ensure that the drug rapidly reaches the tumor site after entering the body and remains within the tumor for a prolonged period to exert its therapeutic effect.

[0004] Chinese patent CN112870389A discloses a DZ-1-Lys-DOTA conjugate combined with a radioactive metal, which contains a heptamethylcarbocyanine dye group coupled to the DOTA group through a lysine crosslinking agent. However, it has shortcomings in terms of stability of radiolabeling, lipophilicity and tumor retention time, which limits its anti-tumor therapeutic effect. Summary of the Invention

[0005] The purpose of this invention is to provide the compound DOTA-HX2-DZ and its applications. By improving the connection method and structural design, it significantly enhances lipid solubility, prolongs in vivo circulation and tumor retention time, improves the accumulation efficiency and anti-tumor effect of radiotherapy, and reduces non-specific radiation damage.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a compound comprising a DOTA group and a heptamethylcarbocyanine dye group in its structure;

[0008] The DOTA group and the heptamethylcarbocyanine dye group are coupled via an aliphatic chain;

[0009] The DOTA group can complex radioactive or non-radioactive metal nuclides. Complexing radioactive metal nuclides is used to prepare diagnostic or therapeutic drugs, while complexing non-radioactive metal nuclides is used to bind other radioactive nuclides.

[0010] Preferably, the compound is a compound having the structure shown in Formula A:

[0011]

[0012] Formula A

[0013] This invention also provides a method for preparing the compound, characterized by comprising the following steps:

[0014] 4-(2-((E)-2-((E)-3-(2-((E)-1-(5-carboxypentyl)-3,3-dimethylindol-2-ylidene)vinyl)-2-chlorocyclohexyl-1-enyl)vinyl)-3,3-dimethyl-3H-indol-1-onium-1-yl)butane-1-sulfonate, HOBt, and EDCI were dissolved in dichloromethane and reacted for 20–40 min. Then, tri-tert-butyl 2,2',2”-(10-(2-((4-aminobutyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate was added, and the reaction was continued for 0.5–1.5 h. After the reaction was completed, the solvent was removed and the product was purified to obtain intermediate 1, as shown in Formula 1 below:

[0015]

[0016] Formula 1

[0017] The intermediate product 1 was dissolved in a mixed solution of dichloromethane and trifluoroacetic acid and reacted for 4–8 hours. After the reaction was completed, the solvent was removed and the product was purified to obtain the compound.

[0018] The present invention also provides a radionuclide drug comprising a compound having the structure shown in Formula B:

[0019]

[0020] Formula B;

[0021] In the formula, M is a radioactive metal.

[0022] Preferably, the radioactive metal is selected from one or more of terbium-161, lutetium-177, and copper-64.

[0023] This invention also provides a method for preparing a radionuclide drug, comprising the following steps:

[0024] The compound was dissolved to obtain solution 1;

[0025] The compound is a compound having the structure shown in Formula A:

[0026]

[0027] Formula A

[0028] The radioactive metal, solution 1, and buffer solution are mixed and heated for 10–30 min to obtain a radionuclide drug.

[0029] The temperature of the heating reaction is 80–100°C.

[0030] Preferably, the radioactive metal is selected from one or more of terbium-161, lutetium-177, and copper-64.

[0031] The present invention also provides the application of the above-mentioned radionuclide drug, or the radionuclide drug prepared by the above-mentioned preparation method, in the preparation of antitumor drugs.

[0032] The present invention also provides the application of the above-mentioned radionuclide drug, or the radionuclide drug prepared by the above preparation method, in the preparation of tumor diagnostic reagents or tumor tracers.

[0033] Preferably, the tumor is cervical cancer, pancreatic cancer, glioma, liver cancer, lung cancer, and / or breast cancer.

[0034] The beneficial effects of this invention are:

[0035] 1. Significantly Improved Stability of Radiolabeling: This invention enhances the chelating ability of the chelating agent DOTA to radionuclides by carefully selecting and optimizing the linker between the chelating agent DOTA and the target molecule DZ-HX2. The optimized linker design improves the binding strength between the radionuclide and the chelating agent, significantly reducing the risk of delabeling of radioactive metal ions in the in vivo environment, ensuring the stable function of the radionuclide at the target site. The improved labeling method enables the labeling rate of the radionuclide and DZ-HX2-DOTA to exceed 95%, and exhibits high radiochemical stability in both in vivo and in vitro environments, ensuring the efficacy and safety of the drug.

[0036] 2. Prolongs the time of internal circulation and the time of tumor retention.

[0037] Optimization of the DZ-HX2-DOTA molecular structure increased its lipid solubility, making it easier to penetrate cell membranes and vascular barriers, thus improving drug bioavailability. Increased lipid solubility also allows the drug to remain in the bloodstream for a longer period, increasing the chance of reaching the tumor site and enhancing its accumulation in tumor tissue. The prolonged residence time of the drug within tumor tissue ensures the radionuclide continues to exert its therapeutic effect at the tumor site, enhancing its killing effect on tumor cells. The extended circulation and tumor residence time increase the cumulative dose of the drug at the tumor site, improving the efficiency of radiotherapy and reducing the number of treatments and dosage requirements.

[0038] 3. Broad-spectrum tumor targeting characteristics with minimal toxic side effects.

[0039] The DZ-HX2-DOTA of this invention possesses broad-spectrum tumor targeting, enabling the diagnosis and treatment of various types of malignant tumors, thus expanding the scope of drug applications. Due to its optimized molecular structure, the drug is less distributed in normal tissues and is rapidly cleared via the liver and kidneys, reducing radiation exposure to non-target tissues. The low distribution and rapid clearance of the drug in normal tissues reduce the risk of radiation damage, with negligible toxic side effects, improving patient tolerability and safety. In various tumor models, DZ-HX2-DOTA has demonstrated significant anti-tumor activity, effectively inhibiting tumor growth and improving patient survival rates and quality of life, showing broad application prospects. Attached Figure Description

[0040] Figure 1 is 177 The labeling and stability test results of Lu-DOTA-HX2-DZ are shown in the figure. A: HPLC detection. 177 Radiochemical purity of Lu-DOTA-HX2-DZ; B: 177 Stability of Lu-DOTA-HX2-DZ in PBS and FBS;

[0041] Figure 2 is 177 The results of in vitro cell-killing ability and blood clearance rate of Lu-DOTA-HX2-DZ are shown in the figure. Among them, A: 177 Lu-DOTA-HX2-DZ's in vitro cell-killing ability against lung cancer, cervical cancer, and pancreatic cancer; B: 177 Blood clearance rate curve of Lu-DOTA-HX2-DZ;

[0042] Figure 3 is 177 The in vivo distribution results of Lu-DOTA-HX2-DZ are shown in the figure, where A & B: 177Attenuation-corrected in vivo distribution data and tumor / normal tissue ratio of Lu-DOTA-HX2-DZ in the H975 lung cancer tumor-bearing animal model; C&D: 177 In vivo distribution data of Lu-DOTA-HX2-DZ after attenuation correction and tumor / normal tissue ratio in the Hela cervical cancer animal model;

[0043] Figure 4 shows the in vivo distribution results of DZ-Glucose-DOTA.

[0044] Figure 5 is 177 The therapeutic effects of Lu-DOTA-HX2-DZ on H1975 lung cancer and HeLa cervical cancer-bearing mice were evaluated. AC: tumor volume growth curves, relative body weight changes, and representative images of tumor tissue in H1975 xenograft tumor mice during the 16-day treatment period (n=5 per group); DG: tumor volume growth curves, relative body weight changes, representative images of tumors, and tumor weight in HeLa xenograft tumor mice during the 16-day treatment period (n=5 per group).

[0045] Figure 6 shows the stability test results of DZ-lys-DOTA (Comparative Example 2), where A: Schematic diagram of the structure of DZ-lys-DOTA; B: Screening of labeling conditions for DZ-lys-DOTA; C: Detection by HPLC. 68 The marking rate of Ga-DOTA-DZ-1; D: 68 Stability of Ga-DOTA-DZ-1 in in vitro FBS; E: TLC assay 177 Markup rate of Lu-DOTA-DZ-1; F: 177 Stability of Lu-DOTA-DZ-1 in PBS and FBS in vitro. Detailed Implementation

[0046] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0047] Example: Preparation of DZ-HX2-DOTA

[0048] Synthesis of Intermediate Product 1: 4-[2-[(E)-2-[(E)-2-chloro-3-[2-[(E)-3,3-dimethyl-1-(6-oxo-6-{[4-[2-[4,7,10-tris[2-(tert-butoxy)-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1-yl]acetamido]butyl]amino}hexyl)indoline-2-yl]vinyl]cyclohex-1-en-1-yl]vinyl]-3,3-dimethyl-3H-indo-1-on-1-yl]butane-1-sulfonate, as shown in Formula 1:

[0049]

[0050] Formula 1

[0051] DZ-1 (24 mg, 0.034 mmol), HOBt (7 mg, 0.051 mmol), and EDCI (9.8 mg, 0.051 mmol) were dissolved in dichloromethane (3 mL) and stirred at room temperature for 30 minutes. Then, tri-tert-butyl 2,2',2”-(10-(2-((4-aminobutyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (22 mg, 0.034 mmol) was added, and stirring was continued at room temperature for 1 hour.

[0052] The solvent was removed at 30°C, and the product was purified by preparative high-performance liquid chromatography (Prep-HPLC) [using water containing 10 μM trifluoroacetic acid (TFA) as the mobile phase, with an acetonitrile gradient increasing from 0% to 36%] to give intermediate 1 (32 mg, yield 71.1%), which was a green solid.

[0053] LCMS (Liquid Chromatography-Mass Spectrometry): m / z 665.3 [1 / 2M+H] + .

[0054] Synthesis of the final product DZ-HX2-DOTA: 4-[2-[(E)-2-[(E)-2-chloro-3-[2-[(E)-3,3-dimethyl-1-(6-oxo-6-[(4-[2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetamido]butyl)amino]hexyl)indoline-2-ylidene]vinyl]cyclohex-1-en-1-yl]vinyl]-3,3-dimethyl-3H-indo-1-on-1-yl]butane-1-sulfonate—2,2,2-trifluoroacetic acid (1 / 1), as shown in Formula 2:

[0055]

[0056] Formula 2

[0057] Intermediate product 1 was dissolved in a mixed solution of dichloromethane (DCM, 8 mL) and trifluoroacetic acid (TFA, 16 mL) and stirred at room temperature for 6 hours. After the reaction was complete, the solvent was removed, and the residue was purified by preparative high-performance liquid chromatography (Prep-HPLC, using TFA as the mobile phase) to obtain the final product DZ-HX2-DOTA (11.8 mg, yield 42.2%), which was a green solid.

[0058] LCMS (Liquid Chromatography-Mass Spectrometry): m / z 1161.6 [M+H] + And 581.3 [1 / 2M+H] +

[0059] Preparation of DZ-Glucose-DOTA (Comparative Example)

[0060] Preparation method of DZ-Glucose-DOTA:

[0061] Synthesis of Intermediate Product 1: Tri-tert-butyl 2,2',2”-(10-((S)-5-((((2R,3R,4R,5S,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl)oxy)methyl)-1-(9H-fluorenyl)-3,6,13-trioxo-2-oxa-4,7,12-triazatetradecyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate, as shown in Formula 3 below:

[0062]

[0063] Formula 3.

[0064] N-(((9H-fluorene-9-yl)methoxy)carbonyl)-O-((2R,3R,4R,5S,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl)-L-serine (200 mg, 0.30 mmol) and tri-tert-butyl-2,2',2”-(10-(2-((4-aminobutyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (193 mg, 0.30 mmol) were dissolved in 8 mL of DMF. DIEA (38.7 mg, 0.30 mmol) and PyBOP (312 mg, 0.6 mmol) were added, and the mixture was stirred at 20 °C for 1 hour. After the reaction, the reaction solvent was filtered, and the mixture was purified by preparative HPLC (column: Welch Xtimate). 21.2*250mm C18, 10μm, mobile phase: A: water (0.2% formic acid) B: acetonitrile; B%: 30%-70% over 15.0 min), yielded tri-tert-butyl 2,2',2”-(10-((S)-5-((((2R,3R,4R,5S,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl)oxy)methyl)-1-(9H-fluorenyl)-3,6,13-trioxo-2-oxa-4,7,12-triazatetradecyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (200 mg, yield 52%), as a white solid. LCMS: m / z 641.4(M+H)+

[0065] Synthesis of Intermediate Product 2: Tri-tert-butyl 2,2',2”-(10-(2-((4-((S)-3-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl)oxy)-2-aminopropionylamino)butyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate, as shown in Formula 4 below:

[0066]

[0067] Formula 4.

[0068] To a solution of tri-tert-butyl 2,2',2”-(10-((S)-5-((((2R,3R,4R,5S,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl)oxy)methyl)-1-(9H-fluorene-9-yl)-3,6,13-trioxo-2-oxa-4,7,12-triazatetradecane-14-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (150 mg, 0.117 mmol) (8 mL methanol and 8 mL water), lithium hydroxide monohydrate (39.3 mg, 0.936 mmol) was added, and the reaction was stirred at room temperature for 1 hour. After completion, the solvent was removed, and the residue was suspended in water (20 mL). The mixture was extracted with a dichloromethane / methanol solution (volume ratio = 20:1, 200 mL x 2). The organic layers were combined and concentrated, and the residue was dried under reduced pressure to give tri-tert-butyl 2,2',2”-(10-(2-((4-((S)-3-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl)oxy)-2-aminopropamido)butyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (98 mg, 89.9% yield), a brown solid. LCMS: m / z 467.3 [1 / 2 M + H]+.

[0069] Synthesis of intermediate product 3

[0070] 4-(2-((E)-2-((E)-3-(2-((E)-1-(6-(((S)-3-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-1-oxo-1-((4-(2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetamido)butyl)amino)propyl)amino)-6-oxohexyl)-3,3-dimethylindol-2-yl)vinyl)-2-chlorocyclohex-1-enyl)vinyl)-3,3-dimethyl-3H-indol-1-onthium-1-yl)butane-1-sulfonate, as shown in Formula 5 below:

[0071]

[0072] Formula 5.

[0073] 4-(2-((E)-2-((E)-3-(2-((E)-1-(5-carboxypentyl)-3,3-dimethylindol-2-yl)vinyl)-2-chlorocyclohex-1-enyl)vinyl)-3,3-dimethyl-3H-indol-1-onthium-1-yl)butane-1-sulfonate (DZ-1) (76 mg, 0.082 mmol), HOBT (16.5 mg, 0.122 mmol) and EDCI (23.4 mg, 0.122 mmol) were dissolved in dichloromethane (DCM, 20 mL) and stirred at room temperature for 30 minutes. Then, tri-tert-butyl 2,2',2”-(10-(2-((4-((S)-3-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-2-aminopropionylamino)butyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (57 mg, 0.082 mmol) was added, and stirring was continued at room temperature for 1 hour. The solvent was evaporated at 30 °C, and the residue was purified by preparative high-performance liquid chromatography (Prep-HPLC) [acetonitrile in water (containing 0.1% TFA) from 0% to 34%] to give 4-(2-((E)-2- ((E)-3-(2-((E)-1-(6-(((S)-3-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-1-oxo-1-((4-(2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetamido)butyl)amino)propyl)amino)-6-oxohexyl)-3,3-dimethylindol-2-ethylene)vinyl)-2-chlorocyclohex-1-enyl)vinyl)-3,3-dimethyl-3H-indol-1-onthium-1-yl)butane-1-sulfonate (65 mg, 49.2% yield) is a green solid.

[0074] The synthesis of the final product DOTA-Glucose-DZ is shown in Equation 6 below:

[0075]

[0076] Formula 6.

[0077] 4-(2-((E)-2-((E)-3-(2-((E)-1-(6-(((S)-3-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-1-oxo-1-((4-(2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecyl) Alkyl)acetamido)butyl)amino)propyl)amino)-6-oxohexyl)-3,3-dimethylindol-2-ethylene)vinyl)-2-chlorocyclohex-1-enyl)vinyl)-3,3-dimethyl-3H-indol-1-on-1-yl)butane-1-sulfonate (55 mg, 0.034 mmol) was dissolved in dichloromethane (DCM, 18 mL) and trifluoroacetic acid (TFA, 6 mL) and stirred at room temperature for 6 hours. The solvent was then removed, and the residue was purified by preparative high-performance liquid chromatography (Prep-HPLC, TFA) to obtain 4-(2-((E)-2-((E)-3-(2-((E)-1-(6-(((S)-3-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-1-oxo-1-((4-(2-(4,7,1) 0-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetamido)butyl)amino)propyl)amino)-6-oxohexyl)-3,3-dimethylindol-2-ethylene)vinyl)-2-chlorocyclohex-1-enyl)vinyl)-3,3-dimethyl-3H-indol-1-onthium-1-yl)butane-1-sulfonate-2,2,2-trifluoroacetic acid (1 / 1) (20.7 mg, 35.6% yield), is a green solid.

[0078] Liquid chromatography-mass spectrometry (LCMS): m / z 1451.7 [M+H]+ and 726.3 [1 / 2M+H]+.

[0079] Comparative Example 2

[0080] Direct coupling of DZ-1 68 Ga / 177 Lu, designed and synthesized 68 Ga / 177 Lu-DOTA-DZ-1. A system was established by filtering and marking criteria. 68 Ga / 177 The marking process of Lu-DOTA-DZ-1.

[0081] The results of the study (Figure 6) show 68 Ga / 177 Lu-DOTA-DZ-1 is unstable in vitro.68 The labeling efficiency of the Ga-DOTA-lys-DZ-1 compound in FBS decreased from 94.9% to 90.04% after 4 hours, while... 177 The Lu-DOTA-lys-DZ-1 compound is extremely unstable; its labeling efficiency in FBS decreased from 88.85% to 22.83% after 24 hours. These results indicate that direct coupling with DZ-1 as the core... 177 The radiopharmaceutical constructed by Lu exhibited severe off-labeling.

[0082] Investigation into the therapeutic effects of experimental case DZ-HX2-DOTA

[0083] 1. 177 The markup of Lu-DOTA-HX2-DZ

[0084] 200μL (approximately 37MBq) 177 LuCl3 was added to 50 μL of LOTA-HX2-DZ aqueous solution and 50 μL of sodium acetate buffer (1 mmol / L). The mixture was heated in a 90°C oil bath for 15–20 minutes, and the corresponding labeled product was obtained after natural cooling. Radioactivity was detected by HPLC using a Raytest Gabistar radioactivity detector, an Agilent-35900E digital-to-analog converter, and a Kinetex C18 column (4.6 × 100 mm, 3.5 μm). 177 Radiochemical Purity (RCP) of the standard drug was determined. The HPLC mobile phase A was deionized water (containing 0.1% HCOOH), and mobile phase B was acetonitrile, with a flow rate of 1 mL / min. The HPLC gradient used was as follows: 90% A and 10% B for 0–5 minutes, 40% A and 60% B for 15 minutes, and 90% A and 10% B for 25 minutes.

[0085] 2. 177 In vitro stability of Lu-DOTA-HX2-DZ

[0086] 400 μCi was concentrated using a C18 column. 177 Lu-DOTA-HX2-DZ was placed in a suitable EP tube, and then 500 μL of PBS and 500 μL of fetal bovine serum (FBS) were added respectively. After mixing thoroughly, the mixture was incubated at 37°C. Samples were taken at 0 h, 4 h, 24 h, 48 h, 120 h, and 168 h of incubation. Using iTLC-SG as the stationary phase and 0.1 mmol / L citric acid as the mobile phase, the product was subjected to radiochemical purity determination, and its stability at the time points was measured.

[0087] 3. 177Lipid-water partition coefficient of Lu-DOTA-HX2-DZ

[0088] 177 The lipid-water partition coefficient (Log P value) of Lu-DOTA-HX2-DZ was determined by its radioactive distribution in n-octanol and phosphate buffer. 2 μCi was taken. 177 Lu-DOTA-HX2-DZ was added to a two-phase system of 0.5 mL n-octanol and 0.5 mL PBS buffer, vortexed for 2 min, and centrifuged at 6000 g for 20 min to ensure complete separation of the liquids. 100 μL of each layer was taken, and the radioactivity count (CPM) was measured using a Wizard radiometric counter. The lipid-water partition coefficient was calculated using the formula Log P = Log(CO / CW) (where CO is the radioactivity count of n-octanol and CW is the radioactivity count of the PBS buffer).

[0089] 4. 177 In vitro cell-killing ability of Lu-DOTA-HX2-DZ

[0090] Detection using CCK-8 method 177 The cytotoxicity of Lu-DOTA-HX2-DZ was assessed. H1975, HeLa, and MIA-paca2 cells were subjected to 10... 4 Cells per well were seeded in 96-well plates and incubated overnight at 37°C with 5% CO2. After cell attachment, the original culture medium was discarded. Different activities of [unspecified substance] were added to the experimental groups. 177 Lu-DOTA-HX2-DZ (0MBq, 0.4625MBq, 0.925MBq, 1.85MBq, 2.775MBq, and 3.7MBq) and different activities 177 Incubate with LuCl3 (0 MBq, 0.4625 MBq, 0.925 MBq, 1.85 MBq, 2.775 MBq, and 3.7 MBq) for 48 h, with 5 replicates per cell activity level. Negative control: HeLa cells with blank culture medium; Blank control: Blank culture medium. After 24 h, remove the 96-well plate, discard the culture medium, and wash three times with PBS. Then add 100 μL of fresh culture medium and 10 μL of CCK-8 solution to each well, and incubate in the dark for 4 h. Measure the absorbance (optical density, OD) of each well at 450 nm using a microplate reader. Cell viability = (OD experimental group - OD blank control group) / (OD negative control group - OD blank group) × 100%

[0091] 5. 177 Blood purification experiment of Lu-DOTA-HX2-DZ

[0092] Six ICR mice, each weighing approximately 20g, were injected via the tail vein with 20μCi.177 Lu-DOTA-HX2-DZ. Blood samples of 10 μL were collected from the tail of mice at 2 min, 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h post-injection using a microcapillary tube, and the wet weight of the blood was recorded. The CPM value was determined using a Wizard radioactive gamma counter, and the percentage of injected dose per gram (%ID / g) was calculated. The %ID / g values ​​at different time points were analyzed using nonlinear regression analysis with GraphPad Prism 10 software. The rapid and slow half-lives were calculated using a two-compartment model, and the circulating drug volume was calculated using the area under the curve (AUC).

[0093] 6. 177 In vivo distribution experiment of Lu-DOTA-HX2-DZ

[0094] Twenty-four H1975 and HeLa tumor-bearing animal models were randomly divided into six groups of four each. Each tumor-bearing model was injected via the tail vein with 1.11 MBq of [a specific drug / method / etc.]. 177 Mice were sacrificed at 4h, 24h, 48h, 72h, 96h, and 168h using Lu-DOTA-HX2-DZ. Blood, heart, liver, spleen, lungs, kidneys, stomach, intestines, muscles, bones, brain, and tumor tissues and organs were collected, weighed, and their radioactivity counts were measured using a gamma counter. The results were then calculated. 177 Distribution of Lu-DOTA-HX2-DZ in various tissues and organs of Hela and H1975 tumor-bearing animal models.

[0095] 7. 177 In vivo distribution experiment of Lu-DOTA-Glucose-DZ

[0096] Twenty-four HeLa tumor-bearing animal models were randomly divided into 6 groups of 4 each. Each tumor-bearing model was injected with 1.11 MBq via the tail vein. 177 Mice were sacrificed at 1h, 4h, 24h, 48h, 72h, and 96h using Lu-DOTA-Glucose-DZ. Blood, heart, liver, spleen, lungs, kidneys, stomach, intestines, muscles, bones, brain, and tumor tissues and organs were collected, weighed, and their radioactivity counts were measured using a gamma counter. The results were then calculated. 177 Distribution of Lu-DOTA-Glucose-DZ in various tissues and organs of the Hela tumor-bearing animal model.

[0097] 8. 177 Treatment research on Lu-DOTA-HX2-DZ

[0098] Four- to six-week-old Balb / c Nude mice were randomly divided into four groups (n=5 per group). After anesthetizing with 2.5% isoflurane, they were subcutaneously injected with 5 × 10⁻⁶ ppm of isoflurane. 5 H1975 and HeLa cells. After 10 days, the tumor volume was approximately 50-70 mm. 3 At that time, each group of tumor-bearing animal models was injected via the tail vein with radiolabeled DZ-1 solutions of different specific activities:

[0099] (1) 177 Lu-DOTA-HX2-DZ high-activity treatment group: 37MBq, 18.5MBq;

[0100] (2) 177 Lu-DOTA-HX2-DZ low activity treatment group: 11.1 MBq, 7.4 MBq;

[0101] (3) PBS group.

[0102] After drug injection, the mental state and appetite of the mice in each group were observed daily. The body weight and tumor volume of the H1975 tumor-bearing animal model were measured every two days. Tumor size was measured using calipers. The tumor volume was calculated using the formula: Volume = Length × Width. 2 ×1 / 2. Tumor-bearing animal models were sacrificed on day 16 after administration. Hematoxylin-eosin (HE) staining was performed on the major organs (heart, liver, spleen, lung, and kidney) of each tumor-bearing model to evaluate the toxicity of radionuclide therapy to normal organs. Finally, tumor tissue from each tumor-bearing animal model was collected and subjected to HE and TUNEL staining to assess tumor necrosis and tumor development.

[0103] Experimental results:

[0104] 1. 177 Markings and stability of Lu-DOTA-HX2-DZ

[0105] HPLC analysis showed that... 177 The labeling rate of Lu-DOTA-HX2-DZ was 96%; after 7 days in PBS and FBS, the labeling rates were 98.66±0.36% and 94.88±0.37%, respectively, as shown in Figure 1.

[0106] 2. 177 Lipid-water partition coefficient of Lu-DOTA-HX2-DZ

[0107] 177 The LogD of Lu-DOTA-HX2-DZ is 0.44±0.03, indicating that the probe is lipophilic.

[0108] 3. 177In vitro cell-killing ability and blood clearance rate of Lu-DOTA-HX2-DZ

[0109] 177 48 hours after treatment with Lu-DOTA-HX2-DZ, the viability of H1975 cells in the experimental group was 20.4%, the viability of HeLa cells in the experimental group was 17.2%, and the viability of MIA-Paca2 cells in the experimental group was 12.59%. Free... 177 The cell viability of LuCl3 was 68.5%, indicating that the probe has a strong killing ability against lung cancer, cervical cancer and pancreatic cancer cells in vitro.

[0110] The results of the blood clearance experiment (Figure 2) show 177 Lu-DOTA-HX2-DZ is a lipophilic probe that is slowly metabolized in the blood, exhibiting a biphasic metabolic pattern. Its fast half-life in the blood is 29.76 minutes, and its slow half-life in the blood is 401.2 minutes.

[0111] 4. 177 In vivo distribution experiment of Lu-DOTA-HX2-DZ

[0112] 177 The in vivo distribution results of Lu-DOTA-HX2-DZ (Figure 3) show that compared with compound DZ-Glucose-DOTA (Comparative Example 1, whose in vivo distribution results are shown in Figure 4), the drug is cleared more slowly in vivo and is mainly metabolized by the kidneys. At the same time, the peak tumor uptake is higher (27.78±4.61% ID / g vs. 20.49±4.89% ID / g).

[0113] For the lung cancer model: 72 hours post-injection, the uptake values ​​in the tumor, spleen, and liver were 11.91±2.87%ID / g, 5.87±0.77%ID / g, and 8.06±0.85%ID / g, respectively. Tumor uptake was highest at 48 hours, at 14.56±2.16%ID / g. Within 96 hours post-injection, the tumor / muscle ratios were 5.38±0.64, 4.87±0.47, 6.49±1.88, 8.72±2.00, 6.45±0.06, and 6.14±1.60, respectively.

[0114] For the cervical cancer model: Compared with the lung cancer model, the cervical cancer model showed higher drug uptake. The tumor uptake value peaked at 72 hours, at 27.78±4.61%ID / g, and at 168 hours, it was 13.54±2.84%ID / g.

[0115] 5. 177 Treatment trials of Lu-DOTA-HX2-DZ

[0116] The results of the treatment trial (Figure 5) show that,177 Lu-DOTA-HX2-DZ effectively inhibits the growth of lung and cervical cancer tumors without toxic side effects. Relative tumor inhibition rate for lung cancer: TGI = 91.6%, initial treatment volume 38.6 mm². 3 The high-dose group had a 16% rate of weight loss. Relative tumor inhibition rate for cervical cancer: TGI = 94.0%, initial treatment volume 97.3 mm. 3 The high-dose group experienced a 4% weight loss rate. This was compared to the drug in Comparative Example 1. 177 Lu-DOTA-Glucose-DZ, which has a higher relative tumor inhibition rate (94.0% vs. 91.4%). 177 Lu-DOTA-HX2-DZ showed better therapeutic effects against cervical cancer, possibly because cervical cancer expresses more OATP and HIF-1, which are related to DZ-1 uptake. HE staining showed no significant abnormalities in the heart, liver, spleen, lungs, and kidneys of both the treatment and control groups.

[0117] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound, characterized in that, The compound comprises a DOTA group and a heptamethylcarbocyanine dye group; The DOTA group and the heptamethylcarbocyanine dye group are coupled via an aliphatic chain; The DOTA group can complex radioactive or non-radioactive metal nuclides. Complexing radioactive metal nuclides is used to prepare diagnostic or therapeutic drugs, and complexing non-radioactive metal nuclides is used to bind other radioactive nuclides. The compound is a compound having the structure shown in Formula A:

2. The method for preparing the compound according to claim 1, characterized in that, Includes the following steps: 4-(2-((E)-2-((E)-3-(2-((E)-1-(5-carboxypentyl)-3,3-dimethylindol-2-ylidene)vinyl)-2-chlorocyclohexyl-1-enyl)vinyl)-3,3-dimethyl-3H-indol-1-onium-1-yl)butane-1-sulfonate, HOBt, and EDCI were dissolved in dichloromethane and reacted for 20–40 min. Then, tri-tert-butyl 2,2',2”-(10-(2-((4-aminobutyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate was added, and the reaction was continued for 0.5–1.5 h. After the reaction was completed, the solvent was removed and the product was purified to obtain intermediate 1, as shown in Formula 1 below: The intermediate product 1 was dissolved in a mixed solution of dichloromethane and trifluoroacetic acid and reacted for 4–8 hours. After the reaction was completed, the solvent was removed and the product was purified to obtain the compound.

3. A radiopharmaceutical, characterized in that, Compounds having the structure shown in Formula B: In the formula, M is a radioactive metal; The radioactive metal is selected from terbium-161 or lutetium-177.

4. A method for preparing a radionuclide drug, characterized in that, Includes the following steps: Dissolve the compound according to claim 1 to obtain solution 1; The radioactive metal, solution 1, and buffer solution are mixed and heated for 10–30 min to obtain a radionuclide drug. The temperature of the heating reaction is 80–100°C; The radioactive metal is selected from terbium-161 or lutetium-177.

5. The use of the radionuclide drug according to claim 3, or the radionuclide drug prepared by the preparation method according to claim 4, in the preparation of antitumor drugs.

6. The use of the radionuclide drug of claim 3, or the radionuclide drug prepared by the preparation method of claim 4, in the preparation of tumor diagnostic reagents or tumor tracers.

7. The application according to claim 5, characterized in that, The tumors are cervical cancer, pancreatic cancer, glioma, liver cancer, lung cancer, and / or breast cancer.

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