Glutamate urea compound, preparation method therefor and use thereof, nuclide targeting probe, preparation method therefor and use thereof, and pharmaceutical composition

Abstract

The present application relates to the technical field of biomedicine, and in particular to a glutamate urea compound, a preparation method and a use thereof, a nuclide targeting probe, a preparation method and a use thereof, and a pharmaceutical composition. The glutamate urea compound and the nuclide targeting probe provided by the present application have excellent in vivo biological properties, high specific uptake in lesions having high PSMA protein expression, high target-to-non-target ratio, low non-specific background activity, and significantly enhanced tumor uptake and retention time, are suitable for use as nuclide treatment and imaging of tumors, can also reduce unnecessary radioactive damage to normal tissues and organs, can overcome the defects of relatively low target organ uptake and excessively short retention time of small molecules acting on PSMA, exhibit improved targeted PSMA nuclide treatment and imaging effects, and have the potential for clinical promotion and application.

Description

Glutamic acid urea compounds, preparation method and application thereof, nuclide targeting probe, preparation method and application thereof, and pharmaceutical composition

[0001] The present application claims priority to the Chinese patent application No. CN202411035514.0, filed on July 31, 2024, and entitled "Glutamic acid urea compounds, preparation method and application thereof, nuclide targeting probe, preparation method and application thereof, and pharmaceutical composition", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the technical field of biological medicine, and specifically relates to glutamic acid urea compounds, preparation method and application thereof, pharmaceutically acceptable salts of glutamic acid urea compounds, nuclide targeting probe, preparation method and application thereof, pharmaceutically acceptable salts of nuclide targeting probe, and pharmaceutical composition. BACKGROUND

[0003] Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein that is overexpressed in about 90% of prostate cancers. Therefore, PSMA-targeted radioligand therapy (RLT) has become a potentially valuable treatment strategy for metastatic castration-resistant prostate cancer (mCRPC). In addition, PSMA expression has also been found in other solid tumors, so PSMA can be used as a therapeutic target for a variety of tumors. In recent years, various PSMA-targeted radioligands have been developed, which have shown good prospects in early clinical evaluation, and the leading position in this field is [ 177 Lu]Lu-PSMA617, and current studies have shown that, 177 Lu]Lu-PSMA617 RLT has good safety and effectiveness in a large number of mCRPC patients. However, despite this, due to the rapid metabolism of the drug in the body, the single dose is often large, and many patients do not respond adequately to radioligand therapy, and disease progression occurs during or after treatment.

[0004] One of the strategies to enhance the therapeutic effect is to improve the delivery of radioligands. The commonly used method at present is to combine an albumin-binding group with a PSMA-targeted radioligand to prolong the blood residence time, thereby increasing the tumor uptake dose. For example, an albumin-binding group such as iodo-phenyl butyric acid, Evans blue or ibuprofen can be modified into a PSMA probe to achieve higher tumor uptake. However, this strategy often also increases the irradiation dose of healthy organs and tissues, including the kidneys and bone marrow. Therefore, the pharmacokinetic properties of the probe in the body must be carefully regulated. In summary, it is of great significance to develop a PSMA-targeted probe with a short blood circulation period, high absolute tumor uptake, long lesion retention, and low background in non-target organs.

[0005] Deferasirox (DFX) is an oral iron chelator, which is often used to reduce the iron content in patients with transfusion-dependent anemia and non-transfusion-dependent thalassemia. Studies have shown that deferasirox has certain tumor targeting properties, and after enrichment in the tumor site, it has an anti-tumor cell proliferation effect, and it can also be used as a chemotherapeutic drug, and as an antifungal drug and antibacterial drug. However, how to modify deferasirox to have better tumor targeting properties, higher lesion enrichment rate, and better disease treatment effect is a difficult problem facing researchers. In addition, the value of the structure of deferasirox in the design of radionuclide targeting drugs has not been developed, and it is not yet known how much the combination of deferasirox structure and other receptor targeting groups affects the pharmacokinetic properties of the probe. Its role in the field of radionuclide targeted diagnosis and treatment also needs to be explored. SUMMARY

[0006] Therefore, the purpose of the present application is to provide glutamic acid urea compounds and preparation methods and applications thereof, pharmaceutically acceptable salts of glutamic acid urea compounds, radionuclide targeting probes and preparation methods and applications thereof, pharmaceutically acceptable salts of radionuclide targeting probes, and pharmaceutical compositions. The glutamic acid urea compounds and pharmaceutically acceptable salts thereof, radionuclide targeting probes and pharmaceutically acceptable salts thereof provided by the present application all have PSMA targeting properties, long target organ retention time, high tumor uptake dose, and low background.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions.

[0008] The present application provides a glutamic acid urea compound, which has the structure shown in formula I-1 and formula I-2:

[0009] wherein, either R1 or R2 is the other is a to-be-labeled group, which includes any one of the following structures:

[0010] X is present or absent, when X is present, it includes a linker group 1 or a linker group 2, and the -NH- end of the linker group 1 and the linker group 2 is connected to R1;

[0011] The linker group 1 includes any one of the following structures:

[0012] The linker group 2 includes any one of the following structures:

[0013] wherein, n, m, y, z, p and q are independently integers between 0 and 10.

[0014] The present application provides a preparation method of the glutamic acid urea compound as described in the above technical solution,

[0015] (i) When X is absent or a linking group 1, the preparation method comprises the following steps:

[0016] carrying out a first substitution reaction of the polypeptide compound 1 and an R1 active compound to obtain an intermediate 1;

[0017] carrying out a second substitution reaction of the intermediate 1 after a de-R3 protection group reaction and an R2 active compound to obtain the glutamic acid urea compound;

[0018] Any one of the R1 active compound and the R2 active compound is When the R1 active compound is

[0019] When the polypeptide compound 1 is the intermediate 1 is

[0020] When the polypeptide compound 1 is the intermediate 1 is

[0021] X and R1 in the polypeptide compound 1 and the intermediate 1 are the same as X and R1 in the formula I-1 and the formula I-2; R3 includes a Boc protection group, a DDE protection group or a Fmoc protection group;

[0022] (ii) When X is a linking group 2, the preparation method comprises the following steps:

[0023] carrying out a third substitution reaction of the polypeptide compound 2 and R1-L to obtain an intermediate 2;

[0024] carrying out a fourth substitution reaction of the intermediate 2 and an R2 active compound to obtain the glutamic acid urea compound;

[0025] L in the R1-L includes any one of the following structures:

[0026] p and q in the L are the same as in the linking group 2;

[0027] When R1 in the R1-L is the R2 active compound is any one of the following structures:

[0028] When R1 in the R1-L is the to-be-labeled group, the R2 active compound is

[0029] when the polypeptide compound 2 is , the intermediate 2 is

[0030] when the polypeptide compound 2 is , the intermediate 2 is

[0031] X and R1 in the polypeptide compound 2 and the intermediate 2 are the same as X and R1 in the formula I-1 and the formula I-2.

[0032] The application provides a pharmaceutically acceptable salt of a glutamic acid urea compound, which is obtained by reacting a glutamic acid urea compound with an acid or a base; the glutamic acid urea compound is the glutamic acid urea compound in the technical solution or the glutamic acid urea compound prepared by the preparation method.

[0033] The application provides a nuclide targeting probe, which is obtained by coordination reaction of a labeling nuclide with a labeling group in the glutamic acid urea compound or the pharmaceutically acceptable salt of the glutamic acid urea compound.

[0034] Preferably, the labeling nuclide comprises at least one of 18 F, 47 Sc, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 89 Zr, 86 Y, 89 Sr, 90 Y, 99m Tc, 105 Rh, 109 Pd, 111 In, 119 Sb, 149 Tb, 153 Sm, 157 Gd, 161 Tb, 166 Ho, 177 Lu, 186 Re, 188 Re, 201 Tl, 203 Pb, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 227 Th and 225 Ac.

[0035] The application provides a preparation method of the radionuclide targeting probe, comprising the following steps: performing a coordination reaction on a glutamic acid urea compound or a pharmaceutically acceptable salt thereof and a labeling radionuclide to obtain the radionuclide targeting probe or a pharmaceutically acceptable salt of the radionuclide targeting probe; the glutamic acid urea compound or the pharmaceutically acceptable salt thereof is the glutamic acid urea compound in the technical solution or the glutamic acid urea compound prepared by the preparation method in the technical solution or the pharmaceutically acceptable salt of the glutamic acid urea compound in the technical solution.

[0036] The application provides a pharmaceutically acceptable salt of a radionuclide targeting probe, which is obtained by performing a salt formation reaction on a radionuclide targeting probe or a glutamic acid urea compound; the radionuclide targeting probe is the radionuclide targeting probe in the technical solution or the radionuclide targeting probe prepared by the preparation method in the technical solution; and the glutamic acid urea compound is the glutamic acid urea compound in the technical solution or the glutamic acid urea compound prepared by the preparation method in the technical solution.

[0037] The application provides a pharmaceutical composition, which comprises an active component and a pharmaceutically acceptable adjuvant; the active component comprises one or more of a glutamic acid urea compound, a radionuclide targeting probe, a pharmaceutically acceptable salt of the glutamic acid urea compound and a pharmaceutically acceptable salt of the radionuclide targeting probe in the technical solution; the glutamic acid urea compound is the glutamic acid urea compound in the technical solution or the glutamic acid urea compound prepared by the preparation method in the technical solution; and the radionuclide targeting probe is the radionuclide targeting probe in the technical solution or the radionuclide targeting probe prepared by the preparation method in the technical solution.

[0038] The application provides an application of the glutamic acid urea compound in the technical solution, the glutamic acid urea compound prepared by the preparation method in the technical solution, the pharmaceutically acceptable salt of the glutamic acid urea compound in the technical solution, the radionuclide targeting probe in the technical solution, the radionuclide targeting probe prepared by the preparation method in the technical solution, the pharmaceutically acceptable salt of the radionuclide targeting probe in the technical solution or the pharmaceutical composition in the technical solution in the preparation of a therapeutic drug or a diagnostic drug for a PSMA protein-mediated disease.

[0039] Preferably, the PSMA protein-mediated disease comprises a tumor.

[0040] The glutamate urea compounds provided in this application are deferasirox-modified compounds. Defererasirox has a certain targeting ability for tumor lesions and has been approved for clinical use as an iron chelating agent with good safety. This application modifies the deferasirox structure into a PSMA-targeting probe, significantly increasing the uptake of the probe at the target site and prolonging the probe's residence time at the target site. The glutamate urea compounds provided in this application can be used for labeling various diagnostic and therapeutic radionuclides and can also be used to construct imaging therapy platforms based on diagnostic and therapeutic radionuclide pairs, showing great promise in the preparation of therapeutic or diagnostic drugs for PSMA protein-mediated diseases.

[0041] The glutamate-urea compounds provided in this application can chelate with radionuclides through the labeled groups to form PSMA-targeting radionuclide probes with high affinity and high specificity. These probes exhibit strong labeling ability, short labeling time, and high labeling yield, which is beneficial for the commercial application and clinical promotion of radionuclide-targeting probes. Compared with existing PSMA-targeting probes, the radionuclide-targeting probes provided in this application have suitable metabolic kinetic properties (different pharmacokinetic properties) and high lesion uptake and retention time, demonstrating excellent diagnostic and therapeutic effects on PSMA protein-mediated diseases. They are highly promising radionuclide-targeted diagnostic and therapeutic drugs. As shown in the test results of the examples, the probes provided in this application… 177 The absolute uptake of Lu-labeled radionuclide targeting probes in tumors is [ 177 Lu-PSMA617, which is 4-5 times more potent than the current gold standard, is a highly promising radionuclide-targeted therapy drug. It overcomes the shortcomings of existing small-molecule PSMAs, such as rapid metabolism and short target organ retention time, thus improving the efficacy of PSMA radionuclide-targeted therapy and possessing the potential for widespread clinical application. In addition to treatment, the radionuclide-targeting probe provided in this application distributes within the body, forming a concentration gradient. If the emitted radiation or changes in relaxation rate are detected by external instruments, the reconstructed image can provide diagnostic information for the disease, achieving an integrated diagnostic and therapeutic effect. Furthermore, by adjusting the appropriate specific activity or drug combination, a better target / non-target ratio can be obtained, enhancing the uptake of the radionuclide-targeting probe in tumors. Attached Figure Description

[0042] Figure 1 shows the mass spectrometry spectrum of compound PKND01.

[0043] Figure 2 shows the mass spectrometry spectrum of compound PKND02.

[0044] Figure 3 shows the mass spectrometry spectrum of compound PKSD01.

[0045] Figure 4 shows the mass spectrometry spectrum of compound PKSD02.

[0046] Figure 5 is a mass spectrometry identification profile of compound PKSP2D01;

[0047] Figure 6 is a mass spectrometry identification profile of compound Gd-PKND01;

[0048] Figure 7 is an HPLC identification profile of compounds PKND01 (a), PKND02 (b), PKSD01 (c), PKSD02 (d) and PKSP2D01 (e);

[0049] Figure 8 is an HPLC identification profile of probe Gd-PKND01;

[0050] Figure 9 is an HPLC identification profile of probe 68 Ga]Ga-PKND01 (a), [ 68 Ga]Ga-PKND02 (b), [ 68 Ga]Ga-PKSD01 (c) and [ 68 Ga]Ga-PKSP2D01 (d);

[0051] Figure 10 is an HPLC identification profile of probe 177 Lu]Lu-PKND01 (a), [ 177 Lu]Lu-PKND02 (b) and [ 177 Lu]Lu-PKSD01 (c);

[0052] Figure 11 is an HPLC identification profile of stability of probe 68 Ga]Ga-PKND01 (a), [ 68 Ga]Ga-PKND02 (b) and [ 68 Ga]Ga-PKSD01 (c);

[0053] Figure 12 is an HPLC identification profile of stability of probe 177 Lu]Lu-PKND01 (a), [ 177 Lu]Lu-PKND02 (b) and [ 177 Lu]Lu-PKSD01 (c);

[0054] Figure 13 is a cellular uptake and inhibition result of probe 177 Lu]Lu-PKND01 (a) and [ 177 Lu]Lu-PKND02 (b);

[0055] Figure 14 is a cellular uptake and inhibition result of probe 177 Lu]Lu-PKSD01 (a) and [ 177 Lu]Lu-PKSD02 (b);

[0056] Figure 15 shows [ 68 PET imaging results of Ga-PKND01 (a) and uptake quantification of the tissue of interest (b);

[0057] Figure 16 is [ 68 PET imaging results of Ga-PKND02 (a) and uptake quantification of the tissue of interest (b);

[0058] Figure 17 is [ 68 PET imaging results of Ga]Ga-PKSD01 (a) and uptake quantification of the tissue of interest (b);

[0059] Figure 18 is [ 68 PET imaging results of Ga-PKSP2D01 (a) and uptake quantification of the tissue of interest (b);

[0060] Figure 19 is [ 177 SPECT imaging results of Lu-PKND01 (a) and target / non-target ratio (b);

[0061] Figure 20 is [ 177 SPECT imaging results of Lu-PKND02 (a) and target / non-target ratio (b);

[0062] Figure 21 is [ 177 SPECT imaging results of Lu-PKSD01 (a) and target / non-target ratio (b);

[0063] Figure 22 is [ 177 SPECT imaging results of Lu-PSMA617 (a), [ 177 Lu]Lu-PKND01 and [ 177 Lu]Lu-PKSD01 uptake count at tumor sites and [ 177 The ratio of Lu]Lu-PSMA617 (b);

[0064] Figure 23 shows the specific activities at different […]. 177 SPECT imaging results of Lu-PKND01 tumor uptake (a) and tumor / kidney ratio (b);

[0065] Figure 24 shows the magnetic resonance imaging results of tumor-bearing mice at different time points before and after tail vein injection of Gd-PKND01;

[0066] Figure 25 is [ 177 Biodistribution of Lu-PKND01 in tumor-bearing mice (a) and tumor / kidney ratio at different time points (b);

[0067] Figure 26 is [177 Lu]Biodistribution results of Lu-PSMA617 in tumor-bearing mice (a) and 177 Lu]Uptake values of Lu-PSMA617 in tumors and 177 Lu]Comparison results of Lu-PKND01 (b);

[0068] Figure 27 is a 177 Lu]Lu-PKND01, 177 Lu]Lu-PKSD01 and 177 Lu]Lu-PSMA617 in tumor-bearing mice;

[0069] Figure 28 is a mass spectrum identification of compound PK2ND01;

[0070] Figure 29 is a mass spectrum identification of compound PK2NGD01;

[0071] Figure 30 is a mass spectrum identification of compound PKED01;

[0072] Figure 31 is a mass spectrum identification of compound PKE3D01;

[0073] Figure 32 is a mass spectrum identification of compound PKP 22 D01;

[0074] Figure 33 is a radiochemical purity HPLC identification of probes 68 Ga]Ga-PK2ND01 and 68 Ga]Ga-PK2NGD01;

[0075] Figure 34 is a radiochemical purity HPLC identification of probes 68 Ga]Ga-PKED01, 68 Ga]Ga-PKE3D01 and 68 Ga]Ga-PKP 22 D01;

[0076] Figure 35 is a cell uptake and inhibition results of probe 177 Lu]Lu-PKE3D01 (a), intracellular internalization and membrane uptake rate (b);

[0077] Figure 36 is a PET imaging results of 68 Ga]Ga-PK2ND01 (a) and uptake quantification values of tissues of interest (b);

[0078] Figure 37 is a PET imaging results of 68 Ga]Ga-PK2NGD01 (a) and uptake quantification values of tissues of interest (b);

[0079] Figure 38 is the PET imaging result (a) and uptake quantification value (b) of the tissue of interest of [68Ga]Ga-PKED01; 68 Ga]Ga-PKE3D01;

[0080] Figure 39 is the PET imaging result (a) and uptake quantification value (b) of the tissue of interest of [68Ga]Ga-PKE3D01;

[0081] Figure 40 is the PET imaging result (a) and uptake quantification value (b) of the tissue of interest of [68Ga]Ga-PKP 68 Ga]Ga-PKE3D01;

[0081] Figure 40 is the PET imaging result (a) and uptake quantification value (b) of the tissue of interest of [68Ga]Ga-PKP 68 Ga]Ga-PKE3D01; 22 Ga]Ga-PKE3D01. DETAILED DESCRIPTION

[0082] The present application provides a glutamic acid urea compound having the structure shown in Formula I-1 and Formula I-2:

[0083] wherein either R1 or R2 is and the other is a labeling group, which includes any one of the following structures:

[0084] X is present or absent, when X is present, includes Linker 1 or Linker 2, and the -NH- end of Linker 1 and Linker 2 is connected to R1;

[0085] Linker 1 includes any one of the following structures:

[0086] Linker 2 includes any one of the following structures:

[0087] wherein n, m, y, z, p and q are independently an integer between 0 and 10.

[0088] In the present application, when X is absent, either R1 or R2 is and the other is The glutamic acid urea compound includes PKND01, PKND02, PK2ND01 or PK2NGD01:

[0089] In the present application, when X is either R1 or R2 is and the other is The glutamic acid urea compound includes PKSD01, PKSD02, PKSP2D01 or PKSP2D02:

[0090] In the present application, when X is either of said R1 and R2 is the other is said glutamic acid urea compound includes PKP 23 D01, PKP 23 D02, PKP 22 D01, PKP 22 D02, PKED01, PKED02, PKDD01, PKDD02, PKD2D01, PKD3D01, PKE2D01or PKE3D01:

[0091] The present application provides a preparation method of the glutamic acid urea compound described in the above technical solution, when X is absent or is a linking group 1, the preparation method comprises the following steps:

[0092] carrying out a first substitution reaction on the polypeptide compound 1 with an R1 active compound to obtain an intermediate 1;

[0093] carrying out a second substitution reaction on the intermediate 1 after a de-R3 protection group reaction with an R2 active compound to obtain the glutamic acid urea compound;

[0094] either of said R1 active compound and R2 active compound is the other is any one of the following structures:

[0095] when said polypeptide compound 1 is , said intermediate 1 is

[0096] when said polypeptide compound 1 is , said intermediate 1 is

[0097] X and R1 in said polypeptide compound 1 and intermediate 1 are the same as X and R1 in said formula I-1 and formula I-2; R3 includes a Boc protection group, a DDE protection group or a Fmoc protection group.

[0098] Unless otherwise specified, the materials and equipment used in the present application are commercially available in the art.

[0099] The present application carries out a first substitution reaction on the polypeptide compound 1 with an R1 active compound to obtain an intermediate 1.

[0100] In the present application, the molar ratio of the polypeptide compound 1 to the R1 active compound is preferably 1:1-5, more preferably 1:2-3. In the present application, the first substitution reaction is preferably carried out in the presence of a high-boiling-point solvent and a basic reagent; the high-boiling-point solvent preferably includes one or more of N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF); the present application does not have a special limitation on the amount of the high-boiling-point solvent, which can only be required to dissolve the polypeptide compound 1 and ensure the smooth progress of the first substitution reaction. In the present application, the basic reagent is preferably an organic base, which preferably includes triethylamine (TEA) and / or N,N-diisopropylethylamine (DIPEA); the molar ratio of the polypeptide compound 1 to the basic reagent is preferably 1:1-10, more preferably 1:3-5. In the present application, the temperature of the first substitution reaction is preferably 25-60°C, more preferably 25-40°C, and the time of the first substitution reaction is preferably 1-24 h, more preferably 5-12 h.

[0101] After the first substitution reaction, the present application preferably further includes post-treatment, which preferably includes: freezing-drying the obtained first substitution system after reverse-phase high-performance liquid chromatography column purification to obtain intermediate 1. In the present application, the conditions of the reverse-phase high-performance liquid chromatography column purification include: the chromatography column is a reverse-phase C18 semi-preparative column; the mobile phase A is preferably water + 0.1% trifluoroacetic acid (TFA), and the mobile phase B is preferably acetonitrile + 0.1% TFA; the elution mode is preferably gradient elution, and the conditions of the gradient elution are preferably 0-30 min: the volume fraction of the mobile phase B is increased from 10% to 90%, and the flow rate of the mobile phase is preferably 3 mL / min. The present application does not have a special limitation on the temperature and time of the freezing-drying, which can only be required to freeze-dry to a constant weight (i.e., lyophilization).

[0102] After obtaining the intermediate 1, the present application carries out a second substitution reaction of the intermediate 1 with an R2 active compound after a de-R3 protecting group reaction to obtain the glutamic acid urea compound.

[0103] In the present application, the de-R3 protecting group reaction is preferably carried out in the presence of a hydrazine hydrate solution or trifluoroacetic acid; the mass fraction of hydrazine hydrate in the hydrazine hydrate solution is preferably 1-10%, more preferably 3-5%; the present application does not have a special limitation on the amount of the hydrazine hydrate solution and trifluoroacetic acid, which can only be required to remove the protecting group (DDE, Boc, or Fmoc). In the present application, the temperature of the de-R3 protecting group reaction is preferably 0-37°C, more preferably 25°C, and the time of the de-R3 protecting group reaction is preferably 1-12 h, more preferably 2-5 h.

[0104] In the present application, the conditions of the second substitution reaction and the post-treatment after the second substitution reaction are preferably the same as the conditions of the first substitution reaction and the post-treatment, which are not repeated here.

[0105] In the present application, when X is absent or is Linker 1, the preparation route of the glutamic acid urea compound is as follows:

[0106] The preparation route of the glutamic acid urea compound is as follows:

[0107] The preparation route of the glutamic acid urea compound is as follows.

[0108] The present application provides a preparation method of the glutamic acid urea compound in the above technical solution, when X is Linker 2, the preparation method comprises the following steps:

[0109] carrying out a third substitution reaction on the polypeptide compound 2 and R1-L to obtain an intermediate 2;

[0110] carrying out a fourth substitution reaction on the intermediate 2 and R2-active compound to obtain the glutamic acid urea compound;

[0111] L in the R1-L comprises any one of the following structures:

[0112] p and q in the L are the same as in the Linker 2;

[0113] when R1 in the R1-L is , the R2-active compound is any one of the following structures:

[0114] when R1 in the R1-L is a labeling group, the R2-active compound is

[0115] when the polypeptide compound 2 is , the intermediate 2 is

[0116] when the polypeptide compound 2 is , the intermediate 2 is

[0117] X and R1 in the polypeptide compound 2 and the intermediate 2 are the same as X and R1 in the formula I-1 and formula I-2.

[0118] In the present application, when X is Linker 2, the preparation route of the glutamic acid urea compound is as follows:

[0119] Preparation route of the compound 2;

[0120] Preparation route of the compound 2.

[0121] In the present application, the polypeptide compound 2 is subjected to a third substitution reaction with R1-L to obtain an intermediate 2.

[0122] In the present application, the R1-L preferably includes DFX-MAL, DOTA-MAL or DFX-P2-MAL:

[0123] In the present application, the molar ratio of the polypeptide compound 2 to R1-L is preferably 1:1-5, and more preferably 1:1.5-2. In the present application, the third substitution reaction is preferably carried out in the presence of a solvent, which preferably includes a high-boiling-point solvent and / or PBS (phosphate buffer solution), and more preferably a mixed solvent of a high-boiling-point solvent and PBS; the high-boiling-point solvent preferably includes one or more of N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF); the PBS has a pH value of preferably 7-8, and more preferably 7.4-7.6; the present application does not have a special limitation on the amount of the solvent, which can only be used to dissolve the polypeptide compound 2 and ensure the smooth progress of the third substitution reaction. In the present application, the temperature and time of the third substitution reaction and the post-treatment after the third substitution reaction are preferably the same as those of the first substitution reaction, which will not be described here one by one.

[0124] After obtaining the intermediate 2, the present application carries out a fourth substitution reaction of the intermediate 2 with an R2 active compound to obtain the glutamic acid urea compound.

[0125] In the present application, the R2 active compound preferably includes a DOTA or DFX active compound with an active reaction group -NHS or -SCN. In the present application, the molar ratio of the intermediate 2 to the R2 active compound is preferably 1:1-5, and more preferably 1:2-2.5. In the present application, the conditions of the fourth substitution reaction and the post-treatment after the fourth substitution reaction are preferably the same as those of the first substitution reaction, which will not be described here one by one.

[0126] The present application provides a pharmaceutically acceptable salt of a glutamic acid urea compound, which is obtained by reacting a glutamic acid urea compound with an acid or a base; the glutamic acid urea compound is the glutamic acid urea compound described in the above technical solution or obtained by the preparation method described in the above technical solution. In the present application, the pharmaceutically acceptable salt preferably includes a trifluoroacetate, a phosphate, a formate, an acetate, a potassium salt or a sodium salt.​

[0127] The present application provides a nuclide targeting probe, which is obtained by coordination reaction of a labeling nuclide with a labeling group in the glutamic acid urea compound or pharmaceutically acceptable salt thereof in the technical solution described above. In the present application, the labeling nuclide includes at least one of 18 F、 47 Sc、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 89 Zr、 86 Y、 89 Sr, 90 Y、 99m Tc、 105 Rh、 109 Pd、 111 In、 119 Sb、 149 Tb、 153 Sm、 157 Gd、 161 Tb、 166 Ho、 177 Lu、 186 Re、 188 Re、 201 Tl、 203 Pb、 212 Pb、 212 Bi、 213 Bi、 223 Ra、 227 Th and 225 Ac, more preferably 177 Lu、 68 Ga、 64 Cu、 18 F、 90 Y or 225 Ac.

[0128] The present application provides a preparation method of the nuclide targeting probe in the technical solution described above, which comprises the following steps:

[0129] Coordination reaction of the glutamic acid urea compound and the labeling nuclide to obtain the nuclide targeting probe; the glutamic acid urea compound is the glutamic acid urea compound in the technical solution described above or the glutamic acid urea compound prepared by the preparation method in the technical solution described above. In the present application, the nuclide targeting probe is preferably prepared by wet labeling method or freeze-drying labeling method.

[0130] In the present application, the preparation of the radionuclide targeting probe by wet labeling method preferably comprises the following steps: mixing a solution of glutamic acid urea compound with a solution of labeling radionuclide, diluting after coordination reaction to obtain an injection of radionuclide targeting probe.

[0131] In the present application, the solvent in the solution of glutamic acid urea compound preferably comprises one or more of buffer solution, water and organic solvent; the buffer solution preferably comprises acetic acid-acetate solution or aluminum chloride-acetate solution, the pH value of the buffer solution is preferably 3-7, more preferably 4-6.5; the concentration of aluminum chloride in the aluminum chloride-acetate solution is preferably 0.2-1 g / L, more preferably 0.4 g / L; the acetate in the acetic acid-acetate solution and the aluminum chloride-acetate solution independently comprises one or more of sodium acetate, potassium acetate and ammonium acetate; the concentration of the solution of glutamic acid urea compound is preferably 0.001-1000 mg / mL, more preferably 0.01-1 mg / mL. In the present application, the ratio of the mass of the glutamic acid urea compound to the radioactivity of the labeling radionuclide in the solution of labeling radionuclide is preferably 20-400 μg: 1 kBq-1000 GBq, more preferably 20-400 μg: 0.037-74000 MBq, further preferably more preferably 20-200 μg: 0.037-7400 MBq. In the present application, the present application does not have special limitations on the solution of labeling radionuclide, and the solution of labeling radionuclide known to those skilled in the art can be used, such as gadolinium chloride hexahydrate (GdCl3·6H2O) solution, 68 GaCl3 hydrochloric acid solution or 177 LuCl3 solution; the 68 The GaCl3 hydrochloric acid solution is preferably obtained by elution from a germanium-gallium generator.

[0132] In the present application, the temperature of the coordination reaction is preferably 25-100℃, more preferably 80-100℃, and the time of the coordination reaction is preferably 10-60 min, more preferably 20-30 min. When the temperature of the coordination reaction is higher than room temperature, the present application preferably further comprises cooling the obtained coordination reaction system to room temperature, and the present application does not have special limitations on the cooling, and the cooling method known to those skilled in the art can be used, such as natural cooling. After the dilution, the present application preferably further comprises filtering the obtained dilution system through a sterile membrane to obtain an injection of radionuclide targeting probe. In the present application, the dilution preferably utilizes normal saline or water for injection. In the present application, the radioconcentration of the injection of radionuclide targeting probe is preferably 0.037-3700 MBq / mL.

[0133] In the present application, when the solution of the labeling nuclide is the solution of gadolinium chloride hexahydrate, the pH value of the mixed solution of the glutamic acid urea compound and the solution of gadolinium chloride hexahydrate is preferably adjusted to 5.0-6.5, more preferably 5.5-6.0, by using an alkali, preferably KOH solution, preferably having a concentration of 1-4 mol / L, more preferably 2-3 mol / L.

[0134] In the present application, the preparation of the nuclide-targeting probe by the freeze-drying method preferably comprises the following steps: freezing and drying the solution of the glutamic acid urea compound to obtain a freeze-dried kit; adding a solvent to the freeze-dried kit to dissolve, then adding the solution of the labeling nuclide to coordinate and dilute to obtain an injection of the nuclide-targeting probe. In the present application, the freezing and drying is preferably performed by dispensing the solution of the glutamic acid urea compound into a freeze-drying container and then freezing and drying; the conditions of the freezing and drying are not particularly limited in the present application, and the freezing and drying conditions well known to those skilled in the art can be used. The freeze-dried kit preferably contains an auxiliary material, which is preferably at least one of an excipient, an antioxidant and an acid-base regulator, and the excipient, antioxidant and acid-base regulator are not particularly limited in the present application, and those well known to those skilled in the art can be used. In the present application, the other preparation conditions of the nuclide-targeting probe are preferably the same as those of the wet labeling method described above, and will not be described herein.

[0135] In the present application, when the radiochemical purity of the injection of the nuclide-targeting probe prepared by the wet labeling method or the freeze-drying method is less than 95%, the injection is preferably purified, and the purification is preferably performed by using a Sep-Pak C18 separation column, which is preferably activated and eluted with anhydrous ethanol and water in sequence before use. In the present application, the eluents used in the purification are preferably water and anhydrous ethanol in sequence, the eluate of the anhydrous ethanol is collected and the solvent is removed, and then diluted to obtain an injection of the high-purity nuclide-targeting probe. In the present application, the dilution is preferably performed by using normal saline or water for injection. In the present application, the radioactivity concentration of the injection of the high-purity nuclide-targeting probe is preferably 0.037-3700 MBq / mL.

[0136] The preparation method provided by the present application has the advantages of simple labeling, good stability of the obtained nuclide-targeting probe, high tumor uptake, etc., and is suitable for industrial production and clinical promotion.

[0137] In the present application, a pharmaceutically acceptable salt of a nuclide targeting probe is provided, which is obtained by a salt formation reaction of a nuclide targeting probe or a glutamic acid urea compound; the nuclide targeting probe is the nuclide targeting probe described in the above technical solution or the nuclide targeting probe prepared by the preparation method described in the above technical solution; and the glutamic acid urea compound is the glutamic acid urea compound described in the above technical solution or the glutamic acid urea compound prepared by the preparation method described in the above technical solution. In the present application, the pharmaceutically acceptable salt preferably includes trifluoroacetate, phosphate, formate, acetate, potassium salt or sodium salt. The present application does not have special limitations on the preparation method of the pharmaceutically acceptable salt of the nuclide targeting probe, and the preparation method of the pharmaceutically acceptable salt known to those skilled in the art can be used.

[0138] The present application provides a pharmaceutical composition, which comprises an active ingredient and a pharmaceutically acceptable excipient; the active ingredient comprises one or more of a glutamic acid urea compound, a nuclide targeting probe, a pharmaceutically acceptable salt of the glutamic acid urea compound described in the above technical solution, and a pharmaceutically acceptable salt of the nuclide targeting probe described in the above technical solution; the glutamic acid urea compound is the glutamic acid urea compound described in the above technical solution or the glutamic acid urea compound prepared by the preparation method described in the above technical solution; and the nuclide targeting probe is the nuclide targeting probe described in the above technical solution or the nuclide targeting probe prepared by the preparation method described in the above technical solution. The present application does not have special limitations on the pharmaceutically acceptable excipient, and the pharmaceutically acceptable excipient known to those skilled in the art can be used. In the present application, when the active ingredient comprises a nuclide targeting probe and / or a pharmaceutically acceptable salt of a nuclide targeting probe, the dosage form of the pharmaceutical composition is preferably an injection, which is preferably administered by intravenous injection; when the active ingredient does not contain a nuclide targeting probe and a pharmaceutically acceptable salt thereof, the present application does not have special limitations on the dosage form and administration method of the pharmaceutical composition, and the dosage form and administration method known to those skilled in the art can be used.

[0139] The application also provides the use of the glutamic acid urea compound, the glutamic acid urea compound prepared by the preparation method, the pharmaceutically acceptable salt of the glutamic acid urea compound, the radionuclide targeting probe, the radionuclide targeting probe prepared by the preparation method, the pharmaceutically acceptable salt of the radionuclide targeting probe or the pharmaceutical composition in the preparation of a therapeutic drug or a diagnostic drug for a PSMA protein-mediated disease. In the application, the PSMA protein-mediated disease preferably includes a tumor; the tumor preferably includes one or more of prostate cancer, breast cancer, ovarian cancer, liver cancer, lung cancer, colorectal cancer, skeletal tissue sarcoma, connective tissue sarcoma, renal cell carcinoma, gastric cancer, pancreatic cancer, nasopharyngeal carcinoma, head and neck cancer, neuroendocrine tumor and skin melanoma. In the application, the diagnostic method preferably includes single photon emission computed tomography (SPECT), positron emission computed tomography (PET) and magnetic resonance imaging (MRI); and the treatment method preferably includes radionuclide targeted therapy and / or chemotherapy.

[0140] The application also provides a treatment method for a PSMA protein-mediated disease, including the following steps: a patient ingests the glutamic acid urea compound, the glutamic acid urea compound prepared by the preparation method, the pharmaceutically acceptable salt of the glutamic acid urea compound, the radionuclide targeting probe, the radionuclide targeting probe prepared by the preparation method, the pharmaceutically acceptable salt of the radionuclide targeting probe or the pharmaceutical composition, and the patient is treated by a treatment method including radionuclide targeted therapy and / or chemotherapy.

[0141] The application also provides a diagnosis method for a PSMA protein-mediated disease, including the following steps: the glutamic acid urea compound, the glutamic acid urea compound prepared by the preparation method, the pharmaceutically acceptable salt of the glutamic acid urea compound, the radionuclide targeting probe, the radionuclide targeting probe prepared by the preparation method, the pharmaceutically acceptable salt of the radionuclide targeting probe or the pharmaceutical composition are distributed in a patient's body, the emitted rays or magnetic resonance relaxation rates are detected by an instrument, an image is reconstructed and obtained, and the image provides diagnostic information for a disease; and the instrument is one or more of an instrument capable of single photon emission computed tomography, an instrument capable of positron emission computed tomography and an instrument capable of magnetic resonance imaging. In the application, the active ingredient in the pharmaceutical composition is one or more of the radionuclide targeting probe and the pharmaceutically acceptable salt of the radionuclide targeting probe.

[0142] In order to further illustrate the present application, the glutamic acid urea compound, the preparation method and application thereof, the nuclide targeting probe, the preparation method and application thereof, and the pharmaceutical composition are described in detail below in combination with the drawings and examples, but they cannot be understood as limiting the protection scope of the present application.

[0143] Example 1

[0144] Synthesis of PKND01

[0145] (1) Synthesis of compound 2: Compound 1 (3.16 μmol) was weighed in a 1.5 mL centrifuge tube, dissolved in DMSO (0.5 mL), and then raw material DFX-NHS (9.49 μmol) and N, N-diisopropylethylamine (DIPEA, 15.82 μmol) were added. The reaction was carried out at 25°C for 12 h, and then HPLC purification and freeze-drying (freeze-drying temperature -65°C) were performed to obtain white solid compound 2 (4 mg, yield 97%, purity 98%). HPLC purification conditions: reversed-phase C18 semi-preparative column (10 mm x 250 mm); mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA; gradient elution conditions: 0-30 min: mobile phase B volume fraction increased from 10% to 90%, mobile phase flow rate 3 mL / min. Compound 2: ESIMS [M+H] + for C 70 H 83 N 10 O 15 , calcd 1303.60, found 1303.25.

[0146] (2) Synthesis of PKND01: Compound 2 (3.06 μmol) was weighed in a 1.5 mL centrifuge tube, and then hydrazine hydrate (0.5 mL, mass fraction 3%) was added. After the reaction was carried out at 25°C for 2 h, DMSO (0.5 mL) was added, and then DOTA-NHS (5.48 μmol) and DIPEA (10.98 μmol) were added. The reaction was carried out at 25°C for 12 h, and then HPLC purification and freeze-drying (freeze-drying temperature -65°C) were performed to obtain compound PKND01 (1.6 mg, yield 47.7%), which was identified to have a purity greater than 95%. HPLC purification conditions: reversed-phase C18 semi-preparative column (10 mm x 250 mm); mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA; gradient elution conditions: 0-30 min: mobile phase B volume fraction increased from 10% to 90%, mobile phase flow rate 3 mL / min. PKND01: ESIMS [M+H] +for C 76 H 97 N 14 O 20 , calcd 1525.69, found 1525.88, as shown in Figure 1; HPLC purity analysis is shown in Figure 7a, with purity greater than 95%.

[0147] Prepared according to the preparation method of PKND01, replacing the DDE protected lysine fragment in compound 1 with a 2,7-diaminoheptanoic acid fragment (compound 1-K2), to obtain the PK2ND01 product.

[0148] Prepared according to the preparation method of PKND01, replacing the DDE protected lysine fragment in compound 1 with a 2,7-diaminoheptanoic acid fragment (compound 1-K2), and replacing DOTA-NHS with DOTA-GA, to obtain the PK2NGD01 product.

[0149] Prepared according to the preparation method of PKND01, replacing the DDE protected lysine fragment in compound 1 with compound 1-KE, to obtain the PKED01 product.

[0150] Prepared according to the preparation method of PKND01, replacing the DDE protected lysine fragment in compound 1 with compound 1-KE3, to obtain the PKE3D01 product.

[0151] Prepared according to the preparation method of PKND01, replacing the DDE protected lysine fragment in compound 1 with compound 1-KP22, to obtain the PKP 22 D01 product.

[0152] Mass spectrometric identification of compounds PK2ND01, PK2NGD01, PKED01, PKE3D01, PKP 22 D01 are shown in Figures 28, 29, 30, 31, and 32, respectively.

[0153] Example 2

[0154] Synthesis of compound PKND02

[0155] (1) Synthesis of compound 3: Compound 1 (3.16 μmol) was weighed into a 1.5 mL centrifuge tube, dissolved with DMSO (0.5 mL), raw material DOTA-NHS (7.91 μmol) and N, N-diisopropyl ethylamine (DIPEA, 15.82 μmol) were added, and the reaction was carried out at 25°C for 12 h, HPLC purification and freeze-drying (freeze-drying temperature -65°C) to obtain white solid compound 3 (3.7 mg, yield 88%, purity 98%). HPLC purification conditions: reversed phase C18 semi-preparative column (10 mm x 250 mm); mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA; gradient elution conditions: 0-30 min: mobile phase B volume fraction increased from 10% to 90%, mobile phase flow rate was 3 mL / min. Compound 3: ESIMS [M+H] + for C 65 H 96 N 11 O 19 , calcd 1335.53, found 1335.24.

[0156] (2) Synthesis of PKND02: Compound 3 (3.06 μmol) was weighed into a 1.5 mL centrifuge tube, hydrazine hydrate (0.5 mL, mass fraction 3%) was added, and the reaction was carried out at 25°C for 2 h, then DMSO (0.5 mL) was added, DOTA-NHS (5.34 μmol) and DIPEA (6.41 μmol) were added, and the reaction was carried out at 25°C for 12 h, HPLC purification and freeze-drying (freeze-drying temperature -65°C) to obtain compound PKND02 (2 mg, yield 61.53%), and the purity was identified to be greater than 95%. HPLC purification conditions: reversed phase C18 semi-preparative column (10 mm x 250 mm); mobile phase B: acetonitrile + 0.1% TFA; gradient elution conditions: 0-30 min: mobile phase B volume fraction increased from 10% to 90%, mobile phase flow rate was 3 mL / min. PKND02: ESIMS [M+H] + for C 76 H 97 N 14 O 20 , calcd 1525.69, found 1525.39, as shown in Figure 2; HPLC purity analysis is shown in Figure 7b, and the purity is greater than 95%.

[0157] Example 3

[0158] Synthesis of PKSD01 compound

[0159] (1) Synthesis of compound 5: Compound 4 (3.03 pmol) was weighed in a 1.5 mL centrifuge tube, dissolved in DMSO (0.5 mL), raw material DFX-MAL (6.05 pmol) was added, then PBS solution (0.3 mL) was added, and the reaction was carried out at 25 °C for 12 h. HPLC purification and freeze-drying (freeze-drying temperature -65 °C) were performed to obtain white solid compound 5 (2 mg, yield 48.78%, purity 98%). HPLC purification conditions: reversed-phase C18 semi-preparative column (10 mm x 250 mm); mobile phase B: acetonitrile + 0.1% TFA; gradient elution conditions: 0-30 min: mobile phase B volume fraction increased from 10% to 90%, mobile phase flow rate was 3 mL / min. ESI MS [M+H] + for C 68 H 81 N 12 O 16 S, calcd 1354.52, found 1354.34.

[0160] (2) Synthesis of compound PKSD01: Compound 5 (2 mg, 1 eq., 1.48 pmol) was weighed in a 1.5 mL centrifuge tube, DMSO (0.5 mL) was added, DOTA-NHS (1.85 mg, 2.5 eq., 3.69 pmol) and DIPEA (0.7 mg, 4 eq., 5.91 pmol) were added, and the reaction was carried out at 25 °C for 12 h. HPLC purification and freeze-drying (freeze-drying temperature -65 °C) were performed to obtain compound PKSD01 (1 mg, yield 38.91%), and the purity was identified to be greater than 95%. HPLC purification conditions: reversed-phase C18 semi-preparative column (10 mm x 250 mm); mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA; gradient elution conditions: 0-30 min: mobile phase B volume fraction increased from 10% to 90%, mobile phase flow rate was 3 mL / min. PKSD01: ESI MS [M+H] + for C 76 H 97 N 14 O 20 , calcd 1739.73, found 1739.70, as shown in Figure 3; HPLC purity analysis is shown in Figure 7c, purity is greater than 95%.

[0161] Example 4

[0162] Synthesis of compound PKSD02

[0163] (1) Synthesis of compound 6: Compound 4 (2.33 μmol) was weighed in a 1.5 mL centrifuge tube, dissolved in DMSO (0.5 mL), raw material DOTA-MAL (4.66 μmol) was added, then PBS solution (0.3 mL) was added, and the reaction was carried out at 25 °C for 12 h. HPLC purification and freeze-drying (freeze-drying temperature -65 °C) were performed to obtain white solid compound 6 (2.1 mg, yield 65.01%, purity 98%). HPLC purification conditions: reversed-phase C18 semi-preparative column (10 mm x 250 mm); mobile phase B: acetonitrile + 0.1% TFA; gradient elution conditions: 0-30 min: mobile phase B volume fraction increased from 10% to 90%, mobile phase flow rate was 3 mL / min. Compound 6: ESIMS [M+H] + for C 63 H 94 N 13 O 20 S, calcd 1385.57, found 1385.48.

[0164] (2) Synthesis of compound PKSD02: Compound 6 (1.52 μmol) was weighed in a 1.5 mL centrifuge tube, DMSO (0.5 mL) was added, DFX-NHS (3.79 μmol) and DIPEA (4.55 μmol) were added, and the reaction was carried out at 25 °C for 12 h. HPLC purification and freeze-drying (freeze-drying temperature -65 °C) were performed to obtain compound PKSD02 (1 mg, yield 37.87%), and the purity was identified to be greater than 95%. HPLC purification conditions: reversed-phase C18 semi-preparative column (10 mm x 250 mm); mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA; gradient elution conditions: 0-30 min: mobile phase B volume fraction increased from 10% to 90%, mobile phase flow rate was 3 mL / min. PKSD02: ESIMS [M+H] + for C 84 H 107 N 16 O 23 S, calcd 1740.92, found 1740.63, as shown in Figure 4; HPLC purity analysis is shown in Figure 7d, purity is greater than 95%.

[0165] Example 5

[0166] Synthesis of compound PKSP2D01

[0167] (1) Synthesis of compound 7: Compound 4 (3.5 pmol) and DFX-P2-MAL (5.24 pmol) were weighed in a 1.5 mL centrifuge tube, DMSO (0.3 mL) and PBS (pH = 7.4) were added for dissolution, and the reaction was carried out at 25 °C for 12 h. HPLC purification and freeze-drying (freeze-drying temperature -65 °C) were performed to obtain white solid compound 7 (1.3 mg, yield 24.6%, purity 95%). HPLC purification conditions: reversed-phase C18 semi-preparative column (10 mm x 250 mm); mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA; gradient elution conditions: 0-30 min: mobile phase B volume fraction increased from 10% to 90%, mobile phase flow rate 3 mL / min. Compound 7: ESIMS [M+H] + for C 75 H 94 N 13 O 19 S, calcd 1513.70, found 1513.53.

[0168] (2) Synthesis of PKSP2D01: Compound 7 (0.86 pmol) was weighed in a 1.5 mL centrifuge tube, and DOTA-NHS (1.72 pmol) and DIPEA (4.3 pmol) were added thereto. The reaction was carried out at 25 °C for 12 h with stirring. HPLC purification and freeze-drying (freeze-drying temperature -65 °C) were performed to obtain compound PKSP2D01 (1.0 mg, yield 59.9%), which was identified to have a purity greater than 95%. HPLC purification conditions: reversed-phase C18 semi-preparative column (10 mm x 250 mm); mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA; gradient elution conditions: 0-30 min: mobile phase B volume fraction increased from 10% to 90%, mobile phase flow rate 3 mL / min. PKSP2D01: ESIMS [M+H] + for C 91 H 120 N 17 O 26 S, calcd 1900.11, found 1899.83, as shown in Figure 5; HPLC purity analysis is shown in Figure 7e, with a purity greater than 95%.

[0169] Example 6

[0170] Gd radionuclide labeling

[0171] Compound PKND01 and gadolinium chloride hexahydrate (GdCl3·6H2O) were dissolved in a mixed solvent (DMSO:H2O volume ratio = 1:1) at a molar ratio of 1:2. The pH was then adjusted to 6.0 with KOH solution, and the mixture was heated to 60℃ and shaken for 12 h. After the reaction was completed, the mixture was purified by HPLC to obtain the radionuclide targeting probe Gd-PKND01. The volume ratio of compound PKND01 to the mixed solvent was 1 mg: 1 mL.

[0172] HPLC analysis conditions: reversed-phase C18 column (4.6 mm × 250 mm); mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA; gradient elution conditions: 0–30 min: mobile phase B volume fraction increased from 10% to 90%, mobile phase flow rate 1 mL / min. Mass spectrometry identification of Gd-PKND01 and HPLC identification of chemical purity are shown in Figures 6 and 8, respectively. ESI MS [M+H] + for C 76 H 93 GdN 14 O 20 ,calcd 1679.91,found 1680.55.

[0173] Example 7

[0174] 68 Ga nuclide labeling

[0175] Wet method: Add 370MBq 68 GaCl3 hydrochloric acid solution (rinsed from the germanium-gallium generator) was added to an acetic acid-acetate solution containing 0.5 mL of the glutamate urea compounds (20 μg) prepared in Examples 1-5. The mixture was reacted at 90 °C for 20 min, then cooled to room temperature, diluted with physiological saline or water for injection, and sterile filtered to obtain the radionuclide-targeted probe injection solution (the concentration of the radionuclide-targeted probe was 110 MBq / mL).

[0176] Lyophilization method: A certain amount of buffer solution (sodium acetate buffer, pH 5.5, 0.4 mL) and 370 MBq were added. 68 GaCl3 eluent (eluted from a germanium-gallium generator) was added to a lyophilized kit containing 20 μg of glutamate-urea compounds. The mixture was reacted at 90°C for 20 min, cooled to room temperature, diluted with physiological saline or water for injection, and sterile filtered to obtain the radionuclide-targeted probe injection solution (the concentration of the radionuclide-targeted probe was 110 MBq / mL).

[0177] If the radiochemical purity is below 95%, it can be purified using a C18 separation column to remove unreacted substances. 68 Ga 3+C18 separation column purification step: Sep-Pak C18 separation column was taken, and activated and washed with 10 mL of anhydrous ethanol and 10 mL of water in sequence. The labeled solution was diluted with 10 mL of water and then loaded onto the separation column. The separation column was washed with water to remove unreacted 68 Ga ions, and then eluted with an ethanol solution to obtain 68 Ga-labeled nuclide targeting probes.

[0178] [ 68 Ga]Ga-PKND01, 68 Ga]Ga-PKND02, 68 Ga]Ga-PKSD01, and 68 Ga]Ga-PKSP2D01. The radiochemical purity HPLC identification results of the four nuclide targeting probes are shown in Figures 9a, 9b, 9c, and 9d, respectively. The radiochemical purity of each nuclide targeting probe was greater than 95%.

[0179] [ 68 Ga]Ga-PK2ND01, 68 Ga]Ga-PK2NGD01. The radiochemical purity HPLC identification results of the two nuclide targeting probes are shown in Figure 33. 68 Ga]Ga-PKED01, 68 Ga]Ga-PKE3D01, and 68 Ga]Ga-PKP 22 D01. The radiochemical purity HPLC identification results of the four nuclide targeting probes are shown in Figure 34.

[0180] Example 8

[0181] 177 Lu nuclide labeling

[0182] Wet method: 370 MBq of a 177 LuCl3solution was added to 0.2 mL of the acetic acid-acetate solution containing the compound prepared in Examples 1-3 (20 μg), and the mixture was reacted at 90°C for 20 min and then cooled to room temperature. The solution was diluted with normal saline or water for injection, and then sterilized to obtain a nuclide targeting probe injection solution (the concentration of the nuclide targeting probe was 110 MBq / mL).

[0183] Freeze-drying method: 0.2 mL of a buffer (sodium acetate buffer, pH = 5.5) and 370 MBq of a 177 LuCl3solution were added to a freeze-dried kit containing the glutamic acid urea compound (20 μg), and the mixture was dissolved and reacted at 90°C for 20 min and then cooled to room temperature. The solution was diluted with normal saline or water for injection, and then sterilized to obtain a nuclide targeting probe injection solution (the concentration of the nuclide targeting probe was 110 MBq / mL).

[0184] If the radiochemical purity is below 95%, it can be purified using a C18 separation column to remove unreacted substances. 177 Lu 3+ The purification steps for the C18 separation column are as follows: Take a Sep-Pak C18 separation column and activate and rinse it successively with 10 mL of anhydrous ethanol and 10 mL of water. Dilute the labeled solution with 10 mL of water and load the sample onto the separation column. Rinse the separation column with water to remove unreacted components. 177 Lu ions were obtained by rinsing with ethanol solution. 177 Lu-labeled nuclide targeting probe.

[0185] [ 177 Lu]Lu-PKND01、[ 177 Lu]Lu-PKND02、[ 177 Lu]Lu-PKSD01 and [ 177 The HPLC identification results of the radiochemical purity of Lu-PKE3D01 are shown in Figure 10 (a, b, c, and d), and the radiochemical purity of each probe is greater than 95%.

[0186] Test Example 1

[0187] Evaluation of stability and lipid-water distribution properties

[0188] 1. Stability test of the injection solution: The radionuclide-targeting probe diluted with physiological saline was placed at room temperature for different times, and samples were analyzed by HPLC. HPLC analysis conditions: reversed-phase C18 column (4.6 mm × 250 mm); mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA; gradient elution conditions: 0–30 min: the volume fraction of mobile phase B increased from 10% to 90%, and the flow rate of the mobile phase was 1 mL / min.

[0189] [ 68 Ga]Ga-PKND01、[ 68 Ga]Ga-PKND02 and [ 68 The HPLC results of the stability identification of Ga-PKSD01 are shown in Figures 11a, b, and c, respectively; 177 Lu]Lu-PKND01、[ 177 Lu]Lu-PKND02 and [ 177 The HPLC results of the stability identification of Lu-Lu-PKSD01 are shown in Figures 12a, b, and c, respectively. As can be seen from Figures 11-12, at the tested time points, the radiochemical purity of each nuclide targeting probe was greater than 95%, indicating that it was stable in the specified solution.

[0190] 2. Determination of the lipid-water distribution coefficient (logP):

[0191] The 100 μL diluted radionuclide-labeled targeting probe solution was added into a centrifuge tube containing 2.9 mL PBS and 3 mL n-octanol mixture, vortexed for 3 min, and then centrifuged at 10,000 rpm for 3 min. 100 μL liquid was taken from the water phase and the n-octanol phase, respectively, and the radioactivity count was measured by a γ-counter. The experiment was repeated three times and the average value was taken. The calculation formula of logP is: P = I 有机相 / I 水相 ;

[0192] wherein I 有机相 represents the radioactivity count measured in the organic phase, I 水相 represents the radioactivity count measured in the water phase. Through calculation, the lipid-water distribution coefficient of each radionuclide-labeled targeting probe was finally determined, and the results are shown in Table 1. The radionuclide-labeled targeting probes measured were water-soluble

[0193] Table 1 Lipid-water distribution coefficient of radionuclide-labeled targeting probes

[0194] As can be seen from Table 1, the above five radionuclide-labeled targeting probes are water-soluble, indicating that the radionuclide-labeled targeting probes prepared in the present application can be metabolized by the kidneys, avoiding non-specific uptake of radioactivity by normal tissues. 117 The radionuclide-labeled targeting probes labeled with Lu are water-soluble, indicating that the radionuclide-labeled targeting probes labeled with Lu prepared in the present application can be metabolized by the kidneys, avoiding non-specific uptake of radioactivity by normal tissues. 117

[0195] Test Example 2

[0196] Cell uptake and inhibition experiment

[0197] PC3 PIP cells with high expression of PSMA and PC3 flu cells with negative expression were plated in a 24-well plate containing culture medium (containing fetal bovine serum and double antibodies) (the number of cells was counted by a cell counting plate, about 200,000 cells / well) and cultured for 24 h. At the beginning of the uptake experiment, the original culture medium was removed, washed twice with PBS (500 μL), and the PBS was removed. An equal amount of the tested probe diluted with culture medium was added to each well, and incubated at 37°C for 0.5, 1, 2, 4, and 8 h. After the incubation at each time point was completed, the culture medium was removed, and sodium hydroxide (NaOH) solution (500 μL, 1M) was added to each well to lyse the cells. After 5 min, the lysed cells were placed in a disposable centrifuge tube to measure the radioactivity count. The percentage of cell uptake was obtained by dividing the count by the total amount of radioactivity added.

[0198] ​To investigate the targeting specificity of the probe PSMA, an inhibition group was set up, i.e. an appropriate amount of inhibitor PSMA617 was added to each well of cells before the addition of the nuclide-labeled probe, and incubated at 37°C for 2h and 4h; after the incubation was completed, the radioactive medium was aspirated, and sodium hydroxide solution (500μL, 1M) was added to each well to lyse the cells, and after 5min, the lysed cells were placed in a disposable centrifuge tube to measure the radioactivity count; the percentage of cell uptake was obtained by dividing the total amount of radioactivity added.

[0199] [ 177 Lu]Lu-PKND01, 177 Lu]Lu-PKND02, 177 Lu]Lu-PKSD01 and 177 Lu]Lu-PKSD02. The cell uptake and inhibition results of the probes are shown in Figures 13-14. Taking the results at 4h as an example, each nuclide-targeting probe was significantly taken up in PSMA-positive expressing cells, and the uptake was significantly reduced in negative cells. The uptake of each nuclide-targeting probe in PSMA-positive expressing cells could be inhibited by PSMA617, indicating that the targeting of the nuclide-targeting probes provided by the present application to PSMA protein has specificity. 177 The cell uptake and inhibition results of Lu]Lu-PKE3D01 are shown in Figure 35. The uptake of the nuclide-targeting probe at each time point in PSMA-positive expressing cells was significantly higher than that in PSMA-negative expressing cells and the inhibition group. In addition, 177 The internalization rate of Lu]Lu-PKE3D01 in PSMA-positive expressing cells was significantly higher than the membrane binding rate, proving that the probe can enter the cells to function.

[0200] Test Example 3

[0201] PET imaging experiment

[0202] The radiolabeled nuclide-targeting probes prepared in the examples have a radiochemical purity of greater than 95% 68 After the Ga-labeled nuclide-targeting probe was diluted with physiological saline, 0.2mL (1MBq) of the injection solution was injected through the tail vein of a PC3 PIP model mouse, MicroPET imaging was performed at different time points, and the region of interest (ROI) was outlined on the image to obtain the probe distribution value. 68 Ga]Ga-PKND01, 68 Ga]Ga-PKND02, 68 Ga]Ga-PKSD01, 68 Ga]Ga-PKSP2D01, 68 Ga]Ga-PK2ND01, 68 Ga]Ga-PK2NGD01, 68Ga]Ga-PKED01,[ 68 Ga]Ga-PKE3D01 and [ 68 Ga]Ga-PKP 22 The PET imaging results of D01 are shown in Figures 15, 16, 17, 18, 36, 37, 38, 39, and 40, respectively. It can be seen that the radionuclide-targeted probe exhibits high uptake at the tumor site. High radioactive signals are observed in the bladder and kidney locations, indicating that the radionuclide-targeted probe is excreted in urine. Over time, the absolute uptake by the tumor and the target / non-target ratio significantly increase.

[0203] Test Example 4

[0204] SPECT imaging experiment

[0205] Tumor-bearing model mice were injected with 14 MBq via the tail vein. 177 Lu-labeled radionuclide targeting probes were used for static SPECT imaging at different time points after injection, with CT scans assisting in localization. After imaging, the images were reconstructed, and regions of interest (ROIs) in the mouse images were delineated to obtain radioactivity counts. The target / non-target ratio of the probe distribution was then calculated. 177 Lu]Lu-PKND01、[ 177 Lu]Lu-PKND02 and [ 177 The SPECT imaging results of Lu-PKSD01 are shown in Figures 19, 20, and 21, respectively. [Control group] 177 The SPECT imaging results of Lu-PSMA617 are shown in Figure 22. Within the monitoring time range, [ 177 Lu]Lu-PKND01、[ 177 Lu]Lu-PKND02 and [ 177 The enrichment of the Lu-PKSD01 radionuclide targeting probe at the tumor site was significantly higher than that in the control group. 177 The high contrast and clear lesion outline of Lu-PSMA617 demonstrate that the radionuclide targeting probe provided in this application has good tumor uptake effect. Over time, the background radiation in the blood pool and other normal organs is gradually cleared, and the target / non-target ratio continuously increases. The above data indicate that the radionuclide targeting probe provided in this application, compared to […], has a high imaging contrast and clear lesion outline, proving that the radionuclide targeting probe provided in this application has good tumor uptake effect. 177 Lu-PSMA617 has superior potential for radionuclide-targeted therapy.

[0206] Figure 23 shows the specific activities at different […]. 177 SPECT imaging results of Lu-PKND01 tumor uptake (a) and tumor / kidney ratio (b), comparing different specific activities using SPECT imaging experiments. 177Lu-PKND01 (distributed in tumor-bearing mice by adding different masses of PKND01 to the labeled solution). Figure 23 shows that when the specific activity is 14 MBq / nmol, [ 177 Lu]Lu-PKND01 combines good tumor uptake with a high tumor / kidney ratio.

[0207] Test Example 5

[0208] MRI Imaging Experiment

[0209] The Gd-PKND01 compound was dissolved in PBS buffer and injected into tumor-bearing mice via the tail vein at a dose of 11.9 μmol / kg. MRI imaging was performed at 1, 3, 6, 12, 24, and 48 hours post-injection. As shown in Figure 24, the area indicated by the arrow is the tumor location. Compared with before injection, the PC3-pip tumor showed a gradual increase in signal after injection, indicating that the radionuclide-targeting probe provided in this application has a good enrichment effect at the tumor site.

[0210] Test Example 6

[0211] Biodistribution Experiment

[0212] Tumor-bearing mice were injected with 1.5 MBq via the tail vein. 177 Lu]Lu-PKND01 or [ 177 Lu-PSMA617. Mice were sacrificed at different time points after injection, and tumor and other organ tissue samples were obtained by dissection, weighed, and radioactivity counted using a gamma counter. Results are expressed as percentage uptake dose per gram of tissue or organ (%ID / g). 177 Lu]Lu-PKND01 and [ 177 The biodistribution results of Lu-PSMA617 are shown in Figures 25 and 26, respectively. 4 hours after injection, [ 177 Lu-PKND01 showed tumor uptake exceeding 80% ID / g. 24 hours post-injection, tumor uptake reached 150% ID / g and remained at 60% ID / g for 96 hours. This demonstrates that compared to […] 177 Lu]Lu-PSMA617, the radionuclide targeting probe provided in this application [ 177 Lu]Lu-PKND01 significantly enhances tumor uptake and prolongs retention time.

[0213] Test Example 7

[0214] Radionuclide targeted therapy experiment

[0215] Tumor-bearing mice were divided into experimental group, [ 177Lu]Lu-PSMA617 control group and saline group, 6-8 in each group. The experimental group each mouse through the tail vein injection of different doses of [ 177 Lu]Lu-PKND01 or [ 177 Lu]Lu-PKSD01; 177 Lu]Lu-PSMA617 control group each mouse tail vein injection of 37 MBq [ 177 Lu]Lu-PSMA617; saline group each mouse tail vein injection of the same volume of saline, daily monitoring of tumor size and body weight changes. The results of treatment as shown in Figure 27, compared with the saline group, the experimental group tumor volume decreased significantly over time, each dose of [ 177 Lu]Lu-PKND01 has a significant tumor treatment effect. And low dose of [ 177 Lu]Lu-PKND01 or [ 177 Lu]Lu-PKSD01 (9.5 MBq) and high dose [ 177 Lu]Lu-PSMA617 (37 MBq) compared with the efficacy of the same, the radionuclide targeting probes provided by the application shows good potential for application.

[0216] Although the above examples make a detailed description of the present application, it is only a part of the embodiments of the present application, rather than all the embodiments, people can also be obtained according to the embodiments of the present application without creative labor under the premise of other embodiments, which belong to the scope of protection of the present application.

Claims

A glutamic acid urea compound characterized by having the structure of Formula I-1 or Formula I-2: wherein either R1or R2is Another is a group to be labeled, said group to be labeled comprising any one of the following structures: X is present or not, when X is present, X comprises Linker 1 or Linker 2, and the -NH- end of Linker 1 and Linker 2 is connected to R1; The linking group 1 includes any one of the following structures: The linking group 2 comprises any one of the following structures: wherein n, m, y, z, p and q are independently an integer between 0 and 10. The glutamic acid urea-based compound according to claim 1, characterized in that, when X is not present, either R1or R2is Another is When said X is at the time, either R1or R2is Another is When X is at the time, either of said R1 and R2 is Another is The preparation method of the glutamic acid urea compound according to claim 1 or 2, characterized in that, (i) when X is not present or is Linker 1, the preparation method comprises the following steps: carrying out a first substitution reaction of polypeptide compound 1 and R1 active compound to obtain intermediate 1; carrying out a deprotection reaction of R3 of the intermediate 1 and a second substitution reaction with R2 active compound to obtain the glutamic acid urea compound; either of the R1 active compound and the R2 active compound is at a time, and the other is any one of the following structures: when the polypeptide compound 1 is at the time, the intermediate 1 is when the polypeptide compound 1 is At this time, intermediate 1 is X and R1 in the polypeptide compound 1 and intermediate 1 are the same as X and R1 in the formula I-1 and formula I-2; R3 comprises a Boc protecting group, a DDE protecting group or a Fmoc protecting group; (ii) when X is Linker 2, the preparation method comprises the following steps: carrying out a third substitution reaction of polypeptide compound 2 and R1-L to obtain intermediate 2; carrying out a fourth substitution reaction of the intermediate 2 and R2 active compound to obtain the glutamic acid urea compound; The L in R1-L includes any one of the following structures: p and q in the L are the same as in the Linker 2; when R1in said R1-L is At this time, the R2 active compound is any one of the following structures: When R1in said R1-L is a label to be attached, said R2active compound is when the polypeptide compound 2 is At this time, the intermediate 2 is when the polypeptide compound 2 is At this time, the intermediate 2 is X and R1 in the polypeptide compound 2 and intermediate 2 are the same as X and R1 in the formula I-1 and formula I-2. The production method according to claim 3, characterized in that The molar ratio of the polypeptide compound 1 and R1 active compound is 1:1-5; The first substitution reaction is carried out in the presence of a high-boiling solvent and a basic reagent; the high-boiling solvent comprises one or more of N-methyl pyrrolidone, dimethyl sulfoxide and N,N-dimethylformamide; the basic reagent is an organic base; the molar ratio of the polypeptide compound 1 and the basic reagent is 1:1-10; the temperature of the first substitution reaction is 25-60°C, and the time is 1-24h. The production method according to claim 3, characterized in that The deprotection reaction of R3 is carried out in the presence of a hydrazine hydrate solution or trifluoroacetic acid; the mass percentage of hydrazine hydrate in the hydrazine hydrate solution is 1-10%; The temperature of the deprotection reaction of R3 is 0-37°C, and the time is 1-12h; The second substitution reaction is carried out in the presence of a high-boiling solvent and a basic reagent; the high-boiling solvent comprises one or more of N-methyl pyrrolidone, dimethyl sulfoxide and N,N-dimethylformamide; the basic reagent is an organic base; the molar ratio of the intermediate 1 and the basic reagent is 1:1-10; the temperature of the second substitution reaction is 25-60°C, and the time is 1-24h. The production method according to claim 3, characterized in that The molar ratio of the polypeptide compound 2 and R1-L is 1:1-5; The third substitution reaction is carried out in the presence of a solvent, which comprises a high-boiling organic solvent and / or a phosphate buffer solution; the high-boiling organic solvent comprises one or more of N-methyl pyrrolidone, dimethyl sulfoxide and N,N-dimethylformamide; the pH value of the phosphate buffer solution is 7-8; the temperature of the third substitution reaction is 25-60°C, and the time is 1-24h. The production method according to claim 3, characterized in that The molar ratio of the intermediate 2 and R2 active compound is 1:1-5; The fourth substitution reaction is carried out in the presence of a high-boiling solvent and a basic reagent; the high-boiling solvent includes one or more of N-methyl pyrrolidone, dimethyl sulfoxide and N,N-dimethylformamide; the basic reagent is an organic base; the molar ratio of the intermediate 2 to the basic reagent is 1:1-10; the temperature of the fourth substitution reaction is 25-60℃, and the time is 1-24h. A pharmaceutically acceptable salt of a glutamic acid urea compound, characterized in that, The glutamic acid urea compound is prepared by the method of any one of claims 3-7. The pharmaceutically acceptable salt of glutamic acid urea compound according to claim 8, characterized in that, The pharmaceutically acceptable salt includes a trifluoroacetate, a phosphate, a formate, an acetate, a potassium salt or a sodium salt. A nuclear species targeting probe, characterized in that The glutamic acid urea compound is prepared by the method of any one of claims 3-7. The nuclide targeting probe according to claim 10, wherein The marker nuclide includes 18 F, 47 Sc, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 89 Zr, 86 Y, 89 Sr, 90 Y, 99m Tc, 105 Rh, 109 Pd, 111 In, 119 Sb, 149 Tb, 153 Sm, 157 Gd, 161 Tb, 166 Ho, 177 Lu, 186 Re, 188 Re, 201 Tl, 203 Pb, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 227 Th, and 225 Ac. The method of claim 10 or 11, wherein the method comprises the steps of: The method comprises the following steps: The glutamic acid urea compound is prepared by the method of any one of claims 3-7. A pharmaceutically acceptable salt of a nuclide-targeting probe, characterized in that, The method comprises the following steps: The pharmaceutically acceptable salt of the nuclide targeting probe according to claim 13, characterized in that, The glutamic acid urea compound is prepared by the method of any one of claims 3-7. A pharmaceutical composition, characterized in that, The PSMA protein-mediated disease includes a tumor. ​ The use according to claim 16, characterized in that ​ The use according to claim 17, characterized in that The tumor includes one or several of prostate cancer, breast cancer, ovarian cancer, liver cancer, lung cancer, colorectal cancer, skeletal tissue sarcoma, connective tissue sarcoma, renal cell carcinoma, gastric cancer, pancreatic cancer, nasopharyngeal carcinoma, head and neck cancer, neuroendocrine tumor and skin melanoma. The use according to claim 16, characterized in that The treatment includes nuclide targeted therapy and / or chemotherapy. The use according to claim 16, characterized in that The diagnosis includes one or several of single photon emission computed tomography, positron emission tomography and nuclear magnetic resonance imaging. A method of treatment of a PSMA protein-mediated disease, characterized in that, The method comprises the following steps: The patient is treated by taking the glutamic acid urea compound of claim 1 or 2, the glutamic acid urea compound prepared by the method of any one of claims 3-7, the pharmaceutically acceptable salt of the glutamic acid urea compound of claim 8 or 9, the nuclide targeted probe of claim 10 or 11, the nuclide targeted probe prepared by the method of claim 12, the pharmaceutically acceptable salt of the nuclide targeted probe of claim 13 or 14 or the pharmaceutical composition of claim 15, and the treatment includes nuclide targeted therapy and / or chemotherapy. A method of diagnosing a PSMA protein-mediated disease, characterized in that, The method comprises the following steps: the nuclide targeted probe of claim 10 or 11, the nuclide targeted probe prepared by the method of claim 12, the pharmaceutically acceptable salt of the nuclide targeted probe of claim 13 or 14 or the pharmaceutical composition of claim 15 is distributed in the patient's body, the emitted rays or relaxation rate is detected by an instrument, the image is reconstructed, the image provides diagnostic information for the disease, the instrument is one or several of an instrument capable of single photon emission computed tomography, an instrument capable of positron emission tomography and an instrument capable of nuclear magnetic resonance imaging; the active ingredient in the pharmaceutical composition is one or several of a nuclide targeted probe and a pharmaceutically acceptable salt of a nuclide targeted probe.

Images