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

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to glutamic acid urea compounds and a preparation method and application thereof, a nuclide targeting probe and a preparation method and application thereof, and a pharmaceutical composition. The glutamic acid urea compounds and the nuclide targeting probe provided by the application have excellent in-vivo biological performance, have high specific uptake in a lesion with high expression of PSMA protein, have a high target-to-non-target ratio, have low non-specific background activity, have significantly enhanced tumor uptake and retention time, are suitable for being used as nuclide treatment and imaging of tumors, can reduce unnecessary radioactive damage to normal tissues and organs, can overcome the defects of low uptake of small-molecule PSMA target organs and short retention time, can improve the effect of targeted PSMA nuclide treatment and imaging, and have potential for clinical popularization and application.

Description

[0001] This application claims priority to Chinese Patent Application No. CN2023110358835, filed on August 17, 2023, entitled "Glutamic Acid Urea Compounds and Their Preparation Methods and Applications, Radionuclide Targeting Probes and Their Preparation Methods and Applications, Pharmaceutical Compositions", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biomedical technology, specifically relating to glutamate urea compounds and their preparation methods and applications, pharmaceutically acceptable salts of glutamate urea compounds, radionuclide targeting probes and their preparation methods and applications, pharmaceutically acceptable salts of radionuclide targeting probes, and pharmaceutical compositions. Background Technology

[0003] Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein that is overexpressed in approximately 90% of prostate cancers. Therefore, PSMA-targeted radioligand therapy (RLT) has emerged as a potentially valuable therapeutic strategy for metastatic castration-resistant prostate cancer (mCRPC). Furthermore, PSMA expression has been found in other solid tumors, thus making it a potential therapeutic target for various cancers. In recent years, various PSMA-targeting radioligands have been developed, showing promising results in early clinical evaluations. [The leading company in this field is...] 177 Lu]Lu-PSMA617, current studies have shown that, 177 Lu-PSMA617 RLT has demonstrated good safety and efficacy 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, resulting in disease progression during or after treatment.

[0004] One strategy to enhance therapeutic efficacy is to improve the delivery of radioligands. A common approach is to prolong the bloodstream residence time of PSMA-targeting radioligands by binding albumin-binding groups, thereby increasing the uptake dose by the tumor. For example, albumin-binding groups such as p-iodophenylbutyrate, Evans blue, or ibuprofen can be modified into PSMA probes to achieve higher tumor uptake. However, this strategy often also increases the radiation dose to healthy organs and tissues, including the kidneys and bone marrow. Therefore, the pharmacokinetic properties of the probe in vivo must be carefully controlled. In conclusion, developing a PSMA-targeting probe with a short blood circulation period, high absolute tumor uptake, long lesion retention, and low background radiation to non-target organs is of great significance.

[0005] Deferrasirox (DFX) is an oral iron chelator commonly used to reduce iron levels in patients with transfusion-dependent anemia and non-transfusion-dependent thalassemia. Studies have shown that deferasirox possesses certain tumor-targeting properties; after accumulation at tumor sites, it exhibits anti-tumor cell proliferation effects and can be used as a chemotherapy drug. Its role as an antifungal and antibacterial agent has also been reported. However, modifying deferasirox to achieve better tumor targeting, higher lesion accumulation rates, and improved disease treatment outcomes remains a challenge for researchers. Furthermore, the value of the deferasirox structure in the design of radionuclide-targeted drugs has not yet been fully explored, the extent to which its hybridization with other receptor-targeting groups affects the pharmacokinetic properties of probes is unknown, and its role in radionuclide-targeted diagnosis and therapy requires further investigation. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide glutamate urea compounds, their preparation methods and applications, pharmaceutically acceptable salts of glutamate urea compounds, radionuclide-targeting probes, their preparation methods and applications, pharmaceutically acceptable salts of radionuclide-targeting probes, and pharmaceutical compositions. The glutamate urea compounds and their pharmaceutically acceptable salts, as well as the radionuclide-targeting probes and their pharmaceutically acceptable salts provided by this invention, all possess PSMA targeting properties, exhibiting long target organ retention time, high tumor uptake dose, and low background.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0008] This invention provides a glutamate urea compound having the structures shown in Formula I-1 and Formula I-2:

[0009] Formula I-1

[0010] Formula I-2;

[0011] Either R1 or R2 is or The other is the labeling group, which includes any one of the following structures:

[0012] ;

[0013] X may or may not exist, and when X exists, it includes linking group 1 or linking group 2;

[0014] The linking group 1 includes any one of the following structures:

[0015] ;

[0016] The linking group 2 includes any one of the following structures:

[0017] ;

[0018] Where n, m, y, z, p, and q are independent integers between 0 and 10.

[0019] This invention provides a method for preparing the glutamate urea compounds described in the above technical solution.

[0020] (i) When X is absent or is a linking group 1, the preparation method includes the following steps:

[0021] The polypeptide compound 1 was subjected to a first substitution reaction with the active compound R1 to obtain intermediate 1;

[0022] After the intermediate 1 undergoes a deprotection reaction of the R3 protecting group, it is subjected to a second substitution reaction with the R2 active compound to obtain the glutamic acid urea compound.

[0023] Either the R1 active compound or the R2 active compound is or When, the other is any of the following structures:

[0024] ;

[0025] When the polypeptide compound 1 is At that time, the intermediate 1 is

[0026] ;

[0027] When the polypeptide compound 1 is At that time, the intermediate 1 is

[0028] ;

[0029] In the polypeptide compound 1 and intermediate 1, X and R1 are the same as X and R1 in formula I-1 and formula I-2; R3 includes a Boc protecting group, a DDE protecting group or an Fmoc protecting group.

[0030] (ii) When X is a linking group 2, the preparation method includes the following steps:

[0031] The polypeptide compound 2 was subjected to a third substitution reaction with R1-L to obtain intermediate 2;

[0032] The intermediate 2 was subjected to a fourth substitution reaction with the active compound R2 to obtain the glutamate urea compound.

[0033] In the R1-L, L includes any one of the following structures:

[0034] ;

[0035] In L, p and q are the same as in the linking group 2;

[0036] When R1 in R1-L is or When the R2 active compound is any one of the following structures:

[0037] ;

[0038] When R1 in R1-L is the group to be labeled, the active compound R2 is... or ;

[0039] When the polypeptide compound 2 is At that time, the intermediate 2 is ;

[0040] When the polypeptide compound 2 is At that time, the intermediate 2 is ;

[0041] In the polypeptide compound 2 and intermediate 2, X and R1 are the same as X and R1 in formula I-1 and formula I-2.

[0042] This invention provides a pharmaceutically acceptable salt of a glutamate urea compound, obtained by reacting the glutamate urea compound with an acid or base; wherein the glutamate urea compound is the glutamate urea compound described in the above technical solution or the glutamate urea compound prepared by the preparation method described in the above technical solution.

[0043] This invention provides a radionuclide targeting probe, which is obtained by coordinating the labeling group in a glutamate urea compound or a pharmaceutically acceptable salt of the glutamate urea compound described in the above technical solution with a labeled radionuclide.

[0044] Preferably, the labeled 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、 149Tb, 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 At least one of Ac.

[0045] This invention provides a method for preparing the radionuclide-targeting probe described in the above-mentioned technical solution, comprising the following steps: coordinating a glutamate urea compound or its pharmaceutically acceptable salt with a labeled radionuclide to obtain a radionuclide-targeting probe or a pharmaceutically acceptable salt of a radionuclide-targeting probe; wherein the glutamate urea compound or its pharmaceutically acceptable salt is the glutamate urea compound described in the above-mentioned technical solution or the glutamate urea compound prepared by the above-mentioned technical solution or a pharmaceutically acceptable salt of the glutamate urea compound described in the above-mentioned technical solution.

[0046] This invention provides a pharmaceutically acceptable salt of a radionuclide-targeting probe, obtained by a salt-forming reaction of a radionuclide-targeting probe or a glutamate-urea compound; wherein the radionuclide-targeting probe is the radionuclide-targeting probe described in the above technical solution or the radionuclide-targeting probe prepared by the preparation method described in the above technical solution; and the glutamate-urea compound is the glutamate-urea compound described in the above technical solution or the glutamate-urea compound prepared by the preparation method described in the above technical solution.

[0047] This invention provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients; the active ingredient comprises one or more of a glutamate urea compound, a radionuclide-targeting probe, a pharmaceutically acceptable salt of a glutamate urea compound, and a pharmaceutically acceptable salt of a radionuclide-targeting probe as described in the above-described technical solutions; the glutamate urea compound is a glutamate urea compound as described in the above-described technical solutions or a glutamate urea compound prepared by the preparation method described in the above-described technical solutions; the radionuclide-targeting probe is a radionuclide-targeting probe as described in the above-described technical solutions or a radionuclide-targeting probe prepared by the preparation method described in the above-described technical solutions.

[0048] This invention provides the use of the glutamate urea compounds described in the above-described technical solutions, the glutamate urea compounds prepared by the above-described technical solutions, pharmaceutically acceptable salts of the glutamate urea compounds described in the above-described technical solutions, the radionuclide targeting probes described in the above-described technical solutions, the radionuclide targeting probes prepared by the above-described technical solutions, the pharmaceutically acceptable salts of the radionuclide targeting probes described in the above-described technical solutions, or the pharmaceutical compositions described in the above-described technical solutions in the preparation of therapeutic or diagnostic drugs for PSMA protein-mediated diseases.

[0049] Preferably, the diseases mediated by the PSMA protein include tumors.

[0050] The glutamate urea compounds provided by this invention 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 invention 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 by this invention are applicable to the labeling of various diagnostic and therapeutic radionuclides and can also be used to construct imaging therapy platforms based on diagnostic and therapeutic radionuclide pairs. They have excellent application prospects in the preparation of therapeutic or diagnostic drugs for PSMA protein-mediated diseases.

[0051] The glutamate-urea compounds provided by this invention can chelate with radionuclides through the labeled group 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 by this invention 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 by this invention… 177 The absolute uptake of Lu-labeled radionuclide targeting probes in tumors is [ 177Lu-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-based radionuclide-targeted therapy and possessing the potential for widespread clinical application. In addition to treatment, the radionuclide-targeting probes provided by this invention distribute within the body, forming a concentration gradient. If the emitted radiation or changes in magnetic inertia 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 appropriate specific activity or drug combinations, a better target / non-target ratio can be obtained, enhancing the uptake of the radionuclide-targeting probes in tumors. Attached Figure Description

[0052] Figure 1 The mass spectrometry spectrum for identifying compound PKND01;

[0053] Figure 2 The mass spectrometry spectrum for identifying compound PKND02;

[0054] Figure 3 The mass spectrometry spectrum for identifying compound PKSD01;

[0055] Figure 4 The mass spectrometry spectrum for identifying compound PKSD02;

[0056] Figure 5 The mass spectrometry spectrum for identifying compound PKSP2D01;

[0057] Figure 6 The mass spectrometry spectrum for identifying compound Gd-PKND01;

[0058] Figure 7 HPLC identification chromatograms of compounds PKND01(a), PKND02(b), PKSD01(c), PKSD02(d) and PKSP2D01(e);

[0059] Figure 8 The HPLC identification chromatogram for probe Gd-PKND01 is shown.

[0060] Figure 9 For probe [ 68 Ga]Ga-PKND01(a), [ 68 Ga]Ga-PKND02(b), [ 68 Ga]Ga-PKSD01(c) and [ 68 HPLC identification chromatogram of radiochemical purity of Ga]Ga-PKSP2D01(d);

[0061] Figure 10 For probe [177 Lu]Lu-PKND01(a), [ 177 Lu]Lu-PKND02(b), [ 177 Lu]Lu-PKSD01(c) and [ 177 HPLC chromatogram of radiochemical purity identification of Lu]Lu-PKE3D01(d);

[0062] Figure 11 For probe [ 68 Ga]Ga-PKND01(a), [ 68 Ga]Ga-PKND02(b) and [ 68 HPLC results of the stability of Ga]Ga-PKSD01(c);

[0063] Figure 12 For probe [ 177 Lu]Lu-PKND01(a), [ 177 Lu]Lu-PKND02(b) and [ 177 HPLC results of stability identification of Lu]Lu-PKSD01(c);

[0064] Figure 13 For probe [ 177 Lu]Lu-PKND01(a) and [ 177 Cellular uptake and inhibition results of Lu]Lu-PKND02(b);

[0065] Figure 14 For probe [ 177 Lu]Lu-PKSD01(a) and [ 177 Cellular uptake and inhibition results of Lu]Lu-PKSD02(b);

[0066] Figure 15 for[ 68 PET imaging results of Ga-PKND01 (a) and uptake quantification of the tissue of interest (b);

[0067] Figure 16 for[ 68 PET imaging results of Ga-PKND02 (a) and uptake quantification of the tissue of interest (b);

[0068] Figure 17 for[ 68 PET imaging results of Ga]Ga-PKSD01 (a) and uptake quantification of the tissue of interest (b);

[0069] Figure 18 for[ 68PET imaging results of Ga-PKSP2D01 (a) and uptake quantification of the tissue of interest (b);

[0070] Figure 19 for[ 177 SPECT imaging results of Lu-PKND01 (a) and target / non-target ratio (b);

[0071] Figure 20 for[ 177 SPECT imaging results of Lu-PKND02 (a) and target / non-target ratio (b);

[0072] Figure 21 for[ 177 SPECT imaging results of Lu-Lu-PKSD01 (a) and target / non-target ratio (b);

[0073] Figure 22 for[ 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);

[0074] Figure 23 For different specific activities [ 177 SPECT imaging results of Lu-PKND01 tumor uptake (a) and tumor / kidney ratio (b);

[0075] Figure 24 Magnetic resonance imaging results at different time points before and after tail vein injection of Gd-PKND01 in tumor-bearing mice;

[0076] Figure 25 for[ 177 Biodistribution of Lu-PKND01 in tumor-bearing mice (a) and tumor / kidney ratio at different time points (b);

[0077] Figure 26 for[ 177 Biodistribution of Lu-PSMA617 in tumor-bearing mice (a) and [ 177 Lu]Lu-PSMA617 uptake value in tumors and [ 177 Comparison results of Lu]Lu-PKND01 (b);

[0078] Figure 27 for[ 177 Lu]Lu-PKND01、[ 177Lu]Lu-PKSD01 and [ 177 Treatment results of Lu-PSMA617 in tumor-bearing mice;

[0079] Figure 28 This is the mass spectrometry identification chromatogram of compound PK2ND01;

[0080] Figure 29 The mass spectrometry chromatogram for the identification of compound PK2NGD01;

[0081] Figure 30 This is the mass spectrometry chromatogram for the compound PKED01.

[0082] Figure 31 This is the mass spectrometry chromatogram for the compound PKE3D01.

[0083] Figure 32 The compound PKP 22 Mass spectrometry identification chromatogram of D01;

[0084] Figure 33 For probe [ 68 Ga]Ga-PK2ND01 and [ 68 HPLC identification chromatogram of radiochemical purity of Ga]Ga-PK2NGD01;

[0085] Figure 34 For probe [ 68 Ga]Ga-PKED01,[ 68 Ga]Ga-PKE3D01 and [ 68 Ga]Ga-PKP 22 HPLC chromatogram of radiochemical purity identification of D01;

[0086] Figure 35 For probe [ 177 Cellular uptake and inhibition results of Lu-Lu-PKE3D01 (a), cell internalization and membrane uptake rate (b);

[0087] Figure 36 for[ 68 PET imaging results of Ga-PK2ND01 (a) and uptake quantification of the tissue of interest (b);

[0088] Figure 37 for[ 68 PET imaging results of Ga]Ga-PK2NGD01 (a) and uptake quantification of the tissue of interest (b);

[0089] Figure 38 for[ 68 PET imaging results of Ga-PKED01 (a) and uptake quantification of the tissue of interest (b);

[0090] Figure 39 for[ 68 PET imaging results of Ga-PKE3D01 (a) and uptake quantification of the tissue of interest (b);

[0091] Figure 40 for[ 68 Ga]Ga-PKP 22 PET imaging results of D01 (a) and uptake quantification of tissue of interest (b). Detailed Implementation

[0092] This invention provides a glutamate urea compound having the structures shown in Formula I-1 and Formula I-2:

[0093] Formula I-1

[0094] Formula I-2;

[0095] Either R1 or R2 is or The other is the labeling group, which includes any one of the following structures:

[0096] ;

[0097] X may or may not exist, and when X exists, it includes linking group 1 or linking group 2;

[0098] The linking group 1 includes any one of the following structures:

[0099] ;

[0100] The linking group 2 includes any one of the following structures:

[0101] ;

[0102] Where n, m, y, z, p, and q are independent integers between 0 and 10.

[0103] In this invention, when X is absent, either R1 or R2 is... or The other is The glutamate urea compounds include PKND01, PKND02, PK2ND01, or PK2NGD01:

[0104] PKND01;

[0105] PKND02;

[0106] PK2ND01;

[0107] PK2NGD01.

[0108] In this invention, when X is or When either R1 or R2 is The other is The glutamate urea compounds include PKSD01, PKSD02, PKSP2D01, or PKSP2D02:

[0109] PKSD01;

[0110] PKSD02;

[0111] PKSP2D01;

[0112] PKSP2D02.

[0113] In this invention, when X is , , , , , , or When either R1 or R2 is The other is The glutamate urea compounds include PKP. 23 D01、PKP 23 D02, PKP 22 D01、PKP 22 D02, PKED01, PKED02, PKDD01, PKDD02, PKD2D01, PKD3D01, PKE2D01 or PKE3D01:

[0114] PKP 23 D01;

[0115] PKP 23 D02;

[0116] PKP 22 D01;

[0117] PKP 22 D02;

[0118] PKDD01;

[0119] PKDD02;

[0120] PKED01;

[0121] PKED02;

[0122] PKD2D01;

[0123] PKD3D01;

[0124] PKE2D01;

[0125] PKE3D01.

[0126] This invention provides a method for preparing the glutamate urea compounds described in the above technical solution. When X is absent or is a linking group 1, the preparation method includes the following steps:

[0127] The polypeptide compound 1 was subjected to a first substitution reaction with the active compound R1 to obtain intermediate 1;

[0128] After the intermediate 1 undergoes a deprotection reaction of the R3 protecting group, it is subjected to a second substitution reaction with the R2 active compound to obtain the glutamic acid urea compound.

[0129] Either the R1 active compound or the R2 active compound is or When, the other is any of the following structures:

[0130] ;

[0131] When the polypeptide compound 1 is At that time, the intermediate 1 is ;

[0132] When the polypeptide compound 1 is At that time, the intermediate 1 is ;

[0133] In the polypeptide compound 1 and intermediate 1, X and R1 are the same as X and R1 in formula I-1 and formula I-2; R3 includes a Boc protecting group, a DDE protecting group or an Fmoc protecting group.

[0134] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0135] In this invention, polypeptide compound 1 is subjected to a first substitution reaction with active compound R1 to obtain intermediate 1.

[0136] In this invention, the molar ratio of polypeptide compound 1 to active compound R1 is preferably 1:1 to 5, more preferably 1:2 to 3. In this invention, 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 amount of the high-boiling-point solvent is not particularly limited in this invention, as long as it is sufficient to dissolve polypeptide compound 1 and ensure the smooth progress of the first substitution reaction. In this invention, the basic reagent is preferably an organic base, preferably including triethylamine (TEA) and / or N,N-diisopropylethylamine (DIPEA); the molar ratio of polypeptide compound 1 to the basic reagent is preferably 1:1 to 10, more preferably 1:3 to 5. In this invention, the temperature of the first substitution reaction is preferably 25 to 60°C, more preferably 25 to 40°C, and the time of the first substitution reaction is preferably 1 to 24 hours, more preferably 5 to 12 hours.

[0137] Following the first substitution reaction, the present invention preferably further includes post-treatment, which preferably includes: purifying the obtained first substitution system by reversed-phase high-performance chromatography (RP-HPLC) followed by freeze-drying to obtain intermediate 1. In the present invention, the conditions for the RPI purification include: the column is a reversed-phase C18 semi-preparative column; mobile phase A is preferably water + 0.1% trifluoroacetic acid (TFA), and mobile phase B is preferably acetonitrile + 0.1% TFA; the elution method is preferably gradient elution, and the gradient elution conditions are preferably 0~30 min: the volume fraction of mobile phase B increases from 10% to 90%, and the flow rate of the mobile phase is preferably 3 mL / min. The present invention does not have specific limitations on the temperature and time of the freeze-drying; freeze-drying to constant weight (i.e., lyophilization) is sufficient.

[0138] After obtaining intermediate 1, the present invention performs a deprotection reaction of intermediate 1 with R3 protecting group and then a second substitution reaction with R2 active compound to obtain the glutamic acid urea compound.

[0139] In this invention, the R3 deprotection reaction is preferably carried out in the presence of 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%; this invention does not have a particular limitation on the amount of hydrazine hydrate solution and trifluoroacetic acid used, as long as it is sufficient to remove the protecting group (DDE, Boc, or Fmoc). In this invention, the temperature of the R3 deprotection reaction is preferably 0-37°C, more preferably 25°C, and the time of the R3 deprotection reaction is preferably 1-12 hours, more preferably 2-5 hours.

[0140] In this invention, the conditions for the second substitution reaction and the post-treatment after the second substitution reaction are preferably the same as those for the first substitution reaction, and will not be described in detail here.

[0141] In this invention, when X is absent or is a linking group 1, the preparation route of the glutamate urea compound is as follows:

[0142] Preparation route of Formula I-1;

[0143] The preparation route of Formula I-2.

[0144] This invention provides a method for preparing the glutamate urea compounds described above. When X is a linking group 2, the preparation method includes the following steps:

[0145] The polypeptide compound 2 was subjected to a third substitution reaction with R1-L to obtain intermediate 2;

[0146] The intermediate 2 was subjected to a fourth substitution reaction with the active compound R2 to obtain the glutamate urea compound.

[0147] In the R1-L, L includes any one of the following structures:

[0148] ;

[0149] In L, p and q are the same as in the linking group 2;

[0150] When R1 in R1-L is or When the R2 active compound is any one of the following structures:

[0151] ;

[0152] When R1 in R1-L is the group to be labeled, the active compound R2 is... or ;

[0153] When the polypeptide compound 2 is At that time, the intermediate 2 is ;

[0154] When the polypeptide compound 2 is At that time, the intermediate 2 is ;

[0155] In the polypeptide compound 2 and intermediate 2, X and R1 are the same as X and R1 in formula I-1 and formula I-2.

[0156] In this invention, when X is a linking group 2, the preparation route of the glutamate urea compound is as follows:

[0157] Preparation route of Formula I-1;

[0158] The preparation route of Formula I-2.

[0159] In this invention, polypeptide compound 2 is subjected to a third substitution reaction with R1-L to obtain intermediate 2.

[0160] In this invention, R1-L preferably includes DFX-MAL, DOTA-MAL, or DFX-P2-MAL:

[0161] .

[0162] In this invention, the molar ratio of the polypeptide compound 2 to the third active compound is preferably 1:1 to 5, more preferably 1:1.5 to 2. In this invention, the third substitution reaction is preferably carried out in the presence of a solvent, preferably including a high-boiling-point solvent and / or PBS (phosphate buffered saline), more preferably a mixture 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 pH of the PBS is preferably 7 to 8, more preferably 7.4 to 7.6; this invention does not have a particular limitation on the amount of solvent used, as long as it is sufficient to dissolve the polypeptide compound 2 and ensure the smooth progress of the third substitution reaction. In this invention, the temperature and time of the third substitution reaction, as well as the post-treatment after the third substitution reaction, are preferably the same as those of the first substitution reaction, and will not be described in detail here.

[0163] After obtaining intermediate 2, the present invention performs a fourth substitution reaction between intermediate 2 and active compound R2 to obtain the glutamic acid urea compound.

[0164] In this invention, the R2 active compound preferably comprises a DOTA or DFX active compound with an active reactive group -NHS or -SCN. In this invention, the molar ratio of intermediate 2 to the R2 active compound is preferably 1:1 to 5, more preferably 1:2 to 2.5. In this invention, the conditions for the fourth substitution reaction and the post-treatment following the fourth substitution reaction are preferably the same as the conditions and post-treatment for the first substitution reaction, and will not be described in detail here.

[0165] This invention provides a pharmaceutically acceptable salt of a glutamate urea compound, obtained by reacting the glutamate urea compound with an acid or base; wherein the glutamate urea compound is the glutamate urea compound described in the above-described technical solution or a glutamate urea compound prepared by the preparation method described in the above-described technical solution. In this invention, the pharmaceutically acceptable salt preferably includes trifluoroacetate, phosphate, formate, acetate, potassium salt, or sodium salt. In this invention, the acid preferably includes trifluoroacetic acid, hydrochloric acid, formic acid, or acetic acid; the base preferably includes potassium hydroxide or sodium hydroxide. This invention does not specifically limit the reaction conditions; conditions for salt-forming reactions well known to those skilled in the art can be used.

[0166] This invention provides a radionuclide-targeting probe, obtained by coordinating the labeling group in a glutamate urea compound or its pharmaceutically acceptable salt as described in the above-described technical solution with a labeled radionuclide. In this invention, the labeled radionuclide 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 and225 At least one of Ac, more preferably 177 Lu、 68 Ga、 64 Cu、 18 F, 90 Y or 225 Ac.

[0167] This invention provides a method for preparing the radionuclide targeting probe described in the above technical solution, comprising the following steps:

[0168] A glutamate-urea compound is coordinated with a labeled radionuclide to obtain a radionuclide-targeted probe; the glutamate-urea compound is the glutamate-urea compound described in the above technical solution or a glutamate-urea compound prepared by the preparation method described in the above technical solution. In this invention, the radionuclide-targeted probe is preferably prepared by a wet labeling method or a lyophilization labeling method.

[0169] In this invention, the wet labeling method for preparing radionuclide-targeting probes preferably includes the following steps: mixing a solution of glutamate-urea compounds with a labeled radionuclide solution, performing a coordination reaction, and then diluting to obtain an injection solution of the radionuclide-targeting probe.

[0170] In this invention, the solvent in the solution of the glutamate urea compound preferably includes one or more of a buffer solution, water, and an organic solvent; the buffer solution preferably includes an acetic acid-acetate solution or an aluminum chloride-acetate solution, and 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 includes one or more of sodium acetate, potassium acetate, and ammonium acetate; the concentration of the solution of the glutamate urea compound is preferably 0.001-1000 mg / mL, more preferably 0.01-1 mg / mL. In this invention, the ratio of the mass of the glutamate urea compound to the radioactivity of the labeled radionuclide in the labeled radionuclide solution is preferably 20-400 µg: 1 kBq-1000 GBq, more preferably 20-400 µg: 0.037-74000 MBq, and even more preferably 20-200 µg: 0.037-7400 MBq. In this invention, there is no particular limitation on the labeled radionuclide solution; any labeled radionuclide solution well 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 rinsing from a germanium-gallium generator.

[0171] In this invention, the temperature of the coordination reaction is preferably 25-100°C, more preferably 80-100°C, 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 invention preferably further includes cooling the obtained coordination reaction system to room temperature after the coordination reaction. The invention does not have a specific limitation on the cooling method; any cooling method well known to those skilled in the art can be used, such as natural cooling. After dilution, the invention preferably further includes sterile membrane filtration of the obtained diluted system to obtain an injection solution of the radionuclide-targeting probe. In this invention, the dilution preferably uses physiological saline or water for injection. In this invention, the radioactivity concentration of the injection solution of the radionuclide-targeting probe is preferably 0.037-3700 MBq / mL.

[0172] In this invention, when the labeled radionuclide solution is a gadolinium chloride hexahydrate solution, it is preferable to first adjust the pH of the mixed solution of glutamate urea compound and gadolinium chloride hexahydrate solution to 5.0-6.5, more preferably 5.5-6.0, using an alkali. The alkali preferably includes KOH solution, and the concentration of the KOH solution is preferably 1-4 mol / L, more preferably 2-3 mol / L.

[0173] In this invention, the preferred method for preparing radionuclide-targeted probes using lyophilization labeling includes the following steps: lyophilizing a solution of a glutamate-urea compound and sealing it to obtain a lyophilized kit; dissolving the compound in a solvent, then adding a labeled radionuclide solution for coordination reaction and dilution to obtain an injection solution of the radionuclide-targeted probe. In this invention, the lyophilization is preferably performed by dispensing the glutamate-urea compound solution into a lyophilization container before lyophilization. This invention does not specifically limit the lyophilization conditions; lyophilization conditions well-known to those skilled in the art can be used. Preferably, excipients are added to the lyophilized kit as needed. These excipients preferably include at least one of excipients, antioxidants, and acid-base regulators. This invention does not specifically limit the excipients, antioxidants, and acid-base regulators; excipients, antioxidants, and acid-base regulators well-known to those skilled in the art can be used. Other preparation conditions for the radionuclide-targeted probes in this invention are preferably the same as those described in the wet labeling method, and will not be repeated here.

[0174] In this invention, when the radiochemical purity of the injection solution of the radionuclide targeting probe prepared by the wet labeling method and the lyophilization labeling method is less than 95%, it is preferable to further purify the injection solution of the radionuclide targeting probe. The purification is preferably performed using a Sep-Pak C18 separation column, which is preferably activated and rinsed sequentially with anhydrous ethanol and water before use. In this invention, the eluent used for purification is preferably water and anhydrous ethanol sequentially. The eluent of anhydrous ethanol is collected and the solvent is removed, followed by dilution to obtain a high-purity injection solution of the radionuclide targeting probe. In this invention, the dilution is preferably performed using physiological saline or water for injection. In this invention, the radioactivity concentration of the high-purity injection solution of the radionuclide targeting probe is preferably 0.037~3700 MBq / mL.

[0175] The preparation method provided by this invention has the advantages of simple and readily available labeling, good stability of the obtained radionuclide targeting probe, and high tumor uptake, making it suitable for industrial production and clinical application.

[0176] In this invention, a pharmaceutically acceptable salt of a radionuclide-targeting probe is provided, obtained by a salt-forming reaction of the radionuclide-targeting probe or a glutamate-urea compound. The radionuclide-targeting probe is either the radionuclide-targeting probe described in the above-described technical solution or a radionuclide-targeting probe prepared by the above-described technical solution. The glutamate-urea compound is either the glutamate-urea compound described in the above-described technical solution or a glutamate-urea compound prepared by the above-described technical solution. In this invention, the pharmaceutically acceptable salt preferably includes trifluoroacetate, phosphate, formate, acetate, potassium salt, or sodium salt. This invention does not specifically limit the preparation method of the pharmaceutically acceptable salt of the radionuclide-targeting probe; any pharmaceutically acceptable salt preparation method well known to those skilled in the art can be used.

[0177] This invention provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients. The active ingredient comprises one or more of the following: a glutamate urea compound, a radionuclide-targeting probe, a pharmaceutically acceptable salt of the glutamate urea compound described in the above-described technical solution, and a pharmaceutically acceptable salt of the radionuclide-targeting probe described in the above-described technical solution. The glutamate urea compound is either the glutamate urea compound described in the above-described technical solution or a glutamate urea compound prepared by the preparation method described in the above-described technical solution. The radionuclide-targeting probe is either the radionuclide-targeting probe described in the above-described technical solution or a radionuclide-targeting probe prepared by the preparation method described in the above-described technical solution. This invention does not specifically limit the pharmaceutically acceptable excipients; any pharmaceutically acceptable excipient well-known to those skilled in the art can be used. In this invention, when the active ingredient includes a radionuclide-targeting probe and / or a pharmaceutically acceptable salt of a radionuclide-targeting probe, the dosage form of the pharmaceutical composition is preferably an injection, preferably administered intravenously. When the active ingredient does not contain a radionuclide-targeting probe or its pharmaceutically acceptable salt, this invention does not specifically limit the dosage form and administration method of the pharmaceutical composition; any dosage form and administration method well-known to those skilled in the art can be used.

[0178] This invention also provides the use of the glutamate urea compounds described in the above-mentioned technical solutions, the glutamate urea compounds prepared by the preparation methods described in the above-mentioned technical solutions, pharmaceutically acceptable salts of the glutamate urea compounds described in the above-mentioned technical solutions, the radionuclide targeting probes described in the above-mentioned technical solutions, the radionuclide targeting probes prepared by the preparation methods described in the above-mentioned technical solutions, the pharmaceutically acceptable salts of the radionuclide targeting probes described in the above-mentioned technical solutions, or the pharmaceutical compositions described in the above-mentioned technical solutions in the preparation of therapeutic or diagnostic drugs for PSMA protein-mediated diseases. In this invention, the PSMA protein-mediated diseases preferably include tumors; the tumors preferably include one or more of prostate cancer, breast cancer, ovarian cancer, liver cancer, lung cancer, colorectal cancer, bone sarcoma, connective tissue sarcoma, renal cell carcinoma, gastric cancer, pancreatic cancer, nasopharyngeal carcinoma, head and neck cancer, neuroendocrine tumors, and cutaneous melanoma. In this invention, the diagnostic methods preferably include single-photon emission computed tomography (SPECT), positron emission tomography (PET), and magnetic resonance imaging (MRI); the treatment methods preferably include radionuclide targeted therapy and / or chemotherapy.

[0179] To further illustrate the present invention, the following detailed descriptions, in conjunction with the accompanying drawings and embodiments, describe the glutamate urea compounds and their preparation methods and applications, the radionuclide targeting probes and their preparation methods and applications, and the pharmaceutical compositions of the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0180] Example 1

[0181] Synthesis of PKND01

[0182]

[0183] (1) Synthesis of Compound 2: Compound 1 (3.16 μmol) was weighed into a 1.5 mL centrifuge tube, dissolved in 0.5 mL DMSO, and then added with raw material DFX-NHS (9.49 μmol) and N,N-diisopropylethylamine (DIPEA, 15.82 μmol). The reaction was carried out at 25 °C for 12 h, purified by HPLC and lyophilized (lyophilization temperature -65 °C) to obtain white solid compound 2 (4 mg, yield 97%, purity 98%). HPLC purification conditions: reversed-phase C18 semi-preparative column (10 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 3 mL / min. Compound 2: ESI MS [M+H] + for C 70 H 83 N 10 O 15 , calcd 1303.60, found1303.25.

[0184] (2) Synthesis of PKND01: Compound 2 (3.06 μmol) was weighed into a 1.5 mL centrifuge tube, and hydrazine hydrate (0.5 mL, mass fraction 3%) was added. After reacting at 25°C for 2 h, DMSO (0.5 mL), DOTA-NHS (5.48 μmol) and DIPEA (10.98 μmol) were added. The mixture was stirred at 25°C for 12 h, purified by HPLC and lyophilized (lyophilization temperature -65°C) to obtain compound PKND01 (1.6 mg, yield 47.7%), and the purity was identified as greater than 95%. HPLC purification conditions: reversed-phase C18 semi-preparative column (10 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 3 mL / min. PKND01: ESIMS [M+H] + for C 76 H 97 N 14 O 20 , calcd 1525.69, found 1525.88, such as Figure 1 As shown; HPLC purity analysis is as follows. Figure 7 As shown in Figure a, the purity is greater than 95%.

[0185]

[0186] The product PK2ND01 was prepared by replacing the DDE-protected lysine fragment in compound 1 with a 2,7-diaminoheptanoic acid fragment (compound 1-K2).

[0187] The product PK2NGD01 was prepared by 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.

[0188] The product PKND01 was prepared by replacing the DDE-protected lysine fragment in compound 1 with compound 1-KE according to the preparation method of PKND01.

[0189] The product PKE3D01 was prepared by replacing the DDE-protected lysine fragment in compound 1 with compound 1-KE3 according to the preparation method of PKND01.

[0190] Prepared according to the method for PKND01, the DDE-protected lysine fragment in compound 1 was replaced with compound 1-KP22 to obtain PKP. 22 Product D01

[0191] Compounds PK2ND01, PK2NGD01, PKED01, PKE3D01, PKP 22 The mass spectrometry identification chromatograms of D01 are as follows: Figure 28 , Figure 29 , Figure 30 , Figure 31 , Figure 32 As shown.

[0192] Example 2

[0193] Synthesis of PKND02 compounds

[0194]

[0195] (1) Synthesis of Compound 3: Compound 1 (3.16 μmol) was weighed into a 1.5 mL centrifuge tube, dissolved in 0.5 mL DMSO, and then DOTA-NHS (7.91 μmol) and N,N-diisopropylethylamine (DIPEA, 15.82 μmol) were added. The mixture was reacted at 25 °C for 12 h, purified by HPLC, and lyophilized (lyophilization temperature -65 °C) to obtain a white solid compound 3 (3.7 mg, yield 88%, purity 98%). HPLC purification conditions: reversed-phase C18 semi-preparative column (10 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 3 mL / min. Compound 3: ESI MS [M+H] + for C 65 H 96 N 11 O 19 ,calcd 1335.53,found 1335.24.

[0196] (2) Synthesis of PKND02: Compound 3 (3.06 μmol) was weighed into a 1.5 mL centrifuge tube, and hydrazine hydrate (0.5 mL, 3% by mass) was added. After reacting at 25 °C for 2 h, DMSO (0.5 mL), DOTA-NHS (5.34 μmol), and DIPEA (6.41 μmol) were added. The mixture was stirred at 25 °C for 12 h, purified by HPLC, and lyophilized (lyophilization temperature -65 °C) to obtain compound PKND02 (2 mg, yield 61.53%), with a purity greater than 95%. HPLC purification conditions: reversed-phase C18 semi-preparative column (10 mm × 250 mm); 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 3 mL / min. PKND02: ESI MS [M+H] + for C 76 H 97 N 14 O 20 , calcd1525.69, found 1525.39, such as Figure 2 As shown; HPLC purity analysis is as follows. Figure 7 The purity shown in b is greater than 95%.

[0197] Example 3

[0198] Synthesis of PKSD01 compound

[0199]

[0200] (1) Synthesis of compound 5: Compound 4 (3.03 μmol) was weighed into a 1.5 mL centrifuge tube, dissolved in 0.5 mL DMSO, followed by 6.05 μmol of raw material DFX-MAL, and then 0.3 mL of PBS solution. The mixture was reacted at 25 °C for 12 h, purified by HPLC, and lyophilized (lyophilization temperature -65 °C) to obtain a white solid compound 5 (2 mg, yield 48.78%, purity 98%). HPLC purification conditions: reversed-phase C18 semi-preparative column (10 mm × 250 mm); 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 was 3 mL / min. ESI MS [M+H] + forC 68 H 81 N 12 O 16 S, cald 1354.52, found 1354.34.

[0201] (2) Synthesis of compound PKSD01: Compound 5 (2 mg, 1 eq., 1.48 μmol) was weighed into a 1.5 mL centrifuge tube, DMSO (0.5 mL) was added, followed by DOTA-NHS (1.85 mg, 2.5 eq., 3.69 μmol) and DIPEA (0.7 mg, 4 eq., 5.91 μmol). The mixture was stirred at 25 °C for 12 h, purified by HPLC and lyophilized (lyophilization temperature -65 °C) to obtain compound PKSD01 (1 mg, yield 38.91%), with a purity greater than 95%. HPLC purification conditions: reversed-phase C18 semi-preparative column (10 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 3 mL / min. PKSD01: ESI MS [M+H] + for C 76 H 97 N 14 O 20 , calcd 1739.73, found 1739.70, such as Figure 3 As shown; HPLC purity analysis is as follows. Figure 7 The purity shown in c is greater than 95%.

[0202] Example 4

[0203] Synthesis of PKSD02 compounds

[0204]

[0205] (1) Synthesis of Compound 6: Compound 4 (2.33 μmol) was weighed into a 1.5 mL centrifuge tube, dissolved in 0.5 mL DMSO, followed by 4.66 μmol of DOTA-MAL and 0.3 mL of PBS solution. The mixture was reacted at 25 °C for 12 h, purified by HPLC, and lyophilized (lyophilization temperature -65 °C) to obtain a white solid, Compound 6 (2.1 mg, yield 65.01%, purity 98%). HPLC purification conditions: reversed-phase C18 semi-preparative column (10 mm × 250 mm); 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 was 3 mL / min. Compound 6: ESI MS [M+H] + for C 63 H 94 N 13 O 20 S, cald 1385.57, found 1385.48.

[0206] (2) Synthesis of compound PKSD02: Compound 6 (1.52 μmol) was weighed into a 1.5 mL centrifuge tube, DMSO (0.5 mL) was added, followed by DFX-NHS (3.79 μmol) and DIPEA (4.55 μmol). The mixture was stirred at 25 °C for 12 h, purified by HPLC, and lyophilized (lyophilization temperature -65 °C) to obtain compound PKSD02 (1 mg, yield 37.87%), with a purity greater than 95%. HPLC purification conditions: reversed-phase C18 semi-preparative column (10 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 was 3 mL / min. PKSD02: ESI MS [M+H] + for C 84 H 107 N 16 O 23 S, calcd 1740.92, found 1740.63, such as Figure 4 As shown; HPLC purity analysis is as follows. Figure 7 The purity shown in d is greater than 95%.

[0207] Example 5

[0208] Synthesis of PKSP2D01 compound

[0209]

[0210] (1) Synthesis of Compound 7: Compound 4 (3.5 μmol) and DFX-P2-MAL (5.24 μmol) were weighed into a 1.5 mL centrifuge tube, dissolved in DMSO (0.3 mL) and PBS (pH=7.4), and reacted at 25 °C for 12 h. The mixture was purified by HPLC and lyophilized (lyophilization temperature -65 °C) to obtain a white solid compound 7 (1.3 mg, yield 24.6%, purity 95%). HPLC purification conditions: reversed-phase C18 semi-preparative column (10 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 was 3 mL / min. Compound 7: ESI MS [M+H] + for C 75 H 94 N 13 O 19 S, cald 1513.70, found 1513.53.

[0211] (2) Synthesis of PKSP2D01: Compound 7 (0.86 μmol) was weighed into a 1.5 mL centrifuge tube, and DOTA-NHS (1.72 μmol) and DIPEA (4.3 μmol) were added. The mixture was stirred at 25 °C for 12 h, purified by HPLC, and lyophilized (lyophilization temperature -65 °C) to obtain compound PKSP2D01 (1.0 mg, yield 59.9%), with a purity greater than 95%. HPLC purification conditions: reversed-phase C18 semi-preparative column (10 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 3 mL / min. PKSP2D01: ESI MS [M+H] + for C 91 H 120 N 17 O 26 S, calcd 1900.11, found 1899.83, such as Figure 5 As shown; HPLC purity analysis is as follows. Figure 7 The purity shown in equation e is greater than 95%.

[0212] Example 6

[0213] Gd nuclide labeling

[0214] 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.

[0215] 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 and HPLC identification of chemical purity of Gd-PKND01 are shown below. Figure 6 and Figure 8 ESI MS [M+H] + for C 76 H 93 GdN 14 O 20 , calcd 1679.91, found 1680.55.

[0216] Example 7

[0217] 68 Ga nuclide labeling

[0218] Wet method: Add 370 MBq 68 GaCl3 hydrochloric acid solution (rinsed from the germanium-gallium generator) was added to an acetic acid-acetate solution containing 0.5 mL of glutamic acid 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).

[0219] Lyophilization method: A certain amount of buffer solution (sodium acetate buffer, pH 5.5, 0.4 mL) and 370 MBq were added. 68 GaCl3 elution buffer (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).

[0220] 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 procedure: 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. 68 Ga ions were obtained by rinsing with ethanol solution. 68 Ga-labeled nuclide targeting probe.

[0221] [ 68 Ga]Ga-PKND01、[ 68 Ga]Ga-PKND02、[ 68 Ga]Ga-PKSD01 and [ 68 The HPLC results for the radiochemical purity of Ga]Ga-PKSP2D01 are shown below. Figure 9 In samples a, b, c, and d, the radiochemical purity of each nuclide targeting probe is greater than 95%.

[0222] [ 68 Ga]Ga-PK2ND01、[ 68 The HPLC results for the radiochemical purity of Ga]Ga-PK2NGD01 are shown below. Figure 33 ;[ 68 Ga]Ga-PKED01,[ 68 Ga]Ga-PKE3D01 and [ 68 Ga]Ga-PKP 22 The HPLC results for the radiochemical purity of D01 are shown below. Figure 34 .

[0223] Example 8

[0224] 177 Lu nuclide labeling

[0225] Wet method: Add 370MBq 177 LuCl3 solution was added to an acetic acid-acetate solution containing 0.2 mL of the compound prepared in Examples 1-3 (20 µg). The mixture was reacted at 90 °C for 20 min and then cooled to room temperature. The solution was diluted with physiological saline or water for injection and then filtered aseptically to obtain the radionuclide-targeting probe injection solution (the concentration of the radionuclide-targeting probe was 110 MBq / mL).

[0226] Lyophilization method: Add 0.2 mL of buffer (sodium acetate buffer, pH=5.5) and 370 MBq of... 177LuCl3 solution was added to a lyophilized kit containing 20µg of glutamate urea compounds. After mixing and dissolving, the mixture was reacted at 90°C for 20 min and then cooled to room temperature. The solution was diluted with physiological saline or water for injection and then filtered aseptically to obtain the radionuclide-targeted probe injection solution (the concentration of the radionuclide-targeted probe was 110MBq / mL).

[0227] 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.

[0228] [ 177 Lu]Lu-PKND01、[ 177 Lu]Lu-PKND02、[ 177 Lu]Lu-PKSD01 and [ 177 The HPLC results for the radiochemical purity of Lu-PKE3D01 are shown below. Figure 10 In samples a, b, c, and d, the radiochemical purity of each probe is greater than 95%.

[0229] Test Example 1

[0230] Evaluation of stability and lipid-water distribution properties

[0231] 1. Stability test of the injection solution: The radionuclide-targeted 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.

[0232] [ 68 Ga]Ga-PKND01、[ 68 Ga]Ga-PKND02 and [ 68 The stability HPLC identification results of Ga]Ga-PKSD01 are shown in the following figures. Figure 11 a, b, and c; 177 Lu]Lu-PKND01、[ 177 Lu]Lu-PKND02 and [ 177The stability HPLC results of Lu-PKSD01 are shown in the following figures. Figure 12 a, b, and c. From Figure 11~12 It can be seen that 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.

[0233] 2. Lipid-water distribution coefficient (log P Measurement:

[0234] 100 μL of the diluted solution containing the radionuclide targeting probe was added to a centrifuge tube containing a mixture of 2.9 mL PBS and 3 mL n-octanol. The mixture was vortexed for 3 min, then centrifuged at 10000 rpm for 3 min. 100 μL of liquid was taken from both the aqueous and n-octanol phases, and the radioactivity count was determined using a γ-counter. The experiment was repeated three times, and the average value was taken. P The calculation formula is:

[0235] P = I 有机相 / I 水相 ;

[0236] Where I 有机相 Represents the radioactivity count measured in the organic phase, I 水相 This represents the radioactivity count measured in the aqueous phase. Through calculation, the lipid-water distribution coefficient of each radiolabeled target probe was finally determined, and the results are shown in Table 1. The measured radionuclide target probes exhibit water solubility.

[0237] Table 1. Lipid-water distribution coefficient of radionuclide targeting probes

[0238]

[0239] As shown in Table 1, the above five 117 The Lu-labeled radionuclide targeting probe exhibits water solubility, indicating that the probe prepared in this invention... 117 Lu-labeled radionuclide targeting probes can be metabolized by the kidneys, avoiding non-specific uptake of radioactivity by normal tissues.

[0240] Test Example 2

[0241] Cell uptake and inhibition experiments

[0242] PC3 PIP cells with high PSMA expression and PC3 flu cells with negative expression were seeded in 24-well plates containing culture medium (fetal bovine serum and antibiotics) and cultured for 24 h (cell count using a cell counting chamber, approximately 200,000 cells / well). At the start of the uptake experiment, the original culture medium was aspirated, and the cells were washed twice with PBS (500 µL), and the PBS was aspirated. An equal volume of the test probe diluted with culture medium was added to each well, and the cells were incubated at 37 °C for 0.5, 1, 2, 4, and 8 h. After incubation at each time point, the culture medium was aspirated, and sodium hydroxide (NaOH) solution (500 µL, 1 M) was added to each well to lyse the cells. After 5 min, the lysed cells were transferred to disposable centrifuge tubes to measure the radioactivity count. The percentage of cell uptake was obtained by dividing the count by the total amount of radioactivity added.

[0243] To investigate the targeting specificity of the probe PSMA, this study also included an inhibition group. Before adding the radionuclide-labeled probe, an appropriate amount of the inhibitor PSMA617 was added to each well of cells, and the cells were incubated at 37°C for 2 h and 4 h. After incubation, the radioactive medium was aspirated, and sodium hydroxide solution (500 µL, 1 M) was added to each well to lyse the cells. After 5 min, the lysed cells were placed in disposable centrifuge tubes to measure the radioactivity count. The cell uptake percentage was obtained by dividing the count by the total amount of radioactivity added.

[0244] [ 177 Lu]Lu-PKND01、[ 177 Lu]Lu-PKND02、[ 177 Lu]Lu-PKSD01 and [ 177 The results of cellular uptake and inhibition of Lu-PKSD02 are as follows: Figure 13~14 As shown. Taking the results after 4 hours as an example, each radionuclide targeting probe showed significant uptake in PSMA-positive cells, while uptake was significantly reduced in PSMA-negative cells. The uptake of each radionuclide targeting probe in PSMA-positive cells was inhibited by PSMA617, indicating that the radionuclide targeting probes provided by this invention have specific targeting for PSMA protein. 177 The results of cellular uptake and inhibition of Lu-PKE3D01 are as follows: Figure 35 As shown. The uptake of the radionuclide-targeting probe in PSMA-positive cells at each time point was significantly higher than that in PSMA-negative cells and the inhibition group. Furthermore, [ 177 The internalization rate of Lu]Lu-PKE3D01 in PSMA-positive cells was significantly higher than that of membrane binding, demonstrating that the probe can enter the cells and exert its function.

[0245] Test Example 3

[0246] PET imaging experiment

[0247] The radiochemical purity prepared in the examples is greater than 95%. 68 Ga-labeled radionuclide targeting probes were diluted with physiological saline, and 0.2 mL (1 MBq) of the injection solution was injected via the tail vein of PC3 PIP model mice. MicroPET imaging was performed at different time points, and regions of interest (ROIs) were delineated on the images. The probe distribution values ​​were then calculated. 68 Ga]Ga-PKND01、[ 68 Ga]Ga-PKND02、[ 68 Ga]Ga-PKSD01, [ 68 Ga]Ga-PKSP2D01、[ 68 Ga]Ga-PK2ND01、[ 68 Ga]Ga-PK2NGD01、[ 68 Ga]Ga-PKED01,[ 68 Ga]Ga-PKE3D01 and [ 68 Ga]Ga-PKP 22 The PET imaging results of D01 are as follows: Figure 15 , Figure 16 , Figure 17 , Figure 18 , Figure 36 , Figure 37 , Figure 38 , Figure 39 and Figure 40 As shown, the radionuclide-targeted probe exhibits high uptake at the tumor site. High radioactivity signals are observed in the bladder and kidneys, 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.

[0248] Test Example 4

[0249] SPECT imaging experiment

[0250] 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-Lu-PKSD01 are as follows: Figure 19 , Figure 20 and Figure 21 As shown. Control group [ 177SPECT imaging results of Lu-PSMA617 are as follows: Figure 22 As shown. 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-targeted probe provided by this invention 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-targeted probe provided by this invention, compared to […], exhibits good tumor uptake effect. 177 Lu-PSMA617 has superior potential for radionuclide-targeted therapy.

[0251] Figure 23 For different specific activities [ 177 SPECT imaging results of Lu-PKND01 tumor uptake (a) and tumor / kidney ratio (b), comparing different specific activities using SPECT imaging experiments. 177 Lu-PKND01 (distributed in tumor-bearing mice by adding different masses of PKND01 to a labeled solution) was analyzed. Figure 23 It can be seen that when the specific activity is 14 MBq / nmol, [ 177 Lu]Lu-PKND01 combines good tumor uptake with a high tumor / kidney ratio.

[0252] Test Example 5

[0253] MRI Imaging Experiment

[0254] Gd-PKND01 compound was dissolved in PBS buffer and injected intravenously into tumor-bearing mice at a dose of 11.9 μmol / kg. MRI imaging was performed at 1, 3, 6, 12, 24, and 48 hours post-injection. Figure 24 As shown, the area indicated by the arrow is the location of the tumor. Compared with before injection, the PC3-pip tumor showed a gradual increase in signal after injection, indicating that the radionuclide targeting probe provided by the present invention has a good enrichment effect at the tumor site.

[0255] Test Example 6

[0256] Biodistribution experiment

[0257] Tumor-bearing mice were injected with 1.5 MBq via the tail vein. 177 Lu]Lu-PKND01 or [ 177Lu-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]Lu-PSMA617 are as follows: Figure 25 and Figure 26 As shown. 4 hours after injection, [ 177 Lu-PKND01 uptake by tumors exceeded 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 by this invention [ 177 Lu]Lu-PKND01 significantly enhances tumor uptake and prolongs retention time.

[0258] Test Example 7

[0259] Radionuclide targeted therapy experiment

[0260] Tumor-bearing mice were divided into experimental group, [ 177 The experimental group consisted of a Lu-PSMA617 control group and a saline group, with 6-8 mice in each group. Each mouse in the experimental group was injected with a different dose of [Lu-PSMA617] via the tail vein. 177 Lu]Lu-PKND01 or [ 177 Lu]Lu-PKSD01;[ 177 In the Lu-PSMA617 control group, each mouse received a tail vein injection of 37 MBq. 177 Lu]Lu-PSMA617; In the saline group, each mouse received an equal volume of saline via the tail vein, and tumor size and body weight changes were monitored daily. Treatment results were as follows: Figure 27 As shown, compared with the saline group, the tumor volume in the experimental group decreased significantly over time, and the […] of each dose […] 177 Lu-PKND01 and Lu-PKND01 both have significant therapeutic effects on tumors. Furthermore, low doses of […]. 177 Lu]Lu-PKND01 or [ 177 Lu]Lu-PKSD01 (9.5 MBq) and high dose [ 177 The therapeutic effect is comparable to that of Lu-PSMA617 (37MBq), indicating that the radionuclide targeting probe provided by this invention shows good application potential.

[0261] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A glutamate urea-like compound, characterized in that, It has the structure shown in Equation I-1 or Equation I-2: Equation I-1; Equation I-2; Where R1 and R2 are The other is the labeling group, which is selected from any of the following structures: ； X may or may not exist, and when X exists, it is selected from linking group 1 or linking group 2; The linking group 1 is selected from any of the following structures: ； The linking group 2 is selected from any of the following structures: ； Where n, y, z and p are independent integers between 0 and 10.

2. The method for preparing the glutamate urea compound according to claim 1, characterized in that, (i) When X is absent or is a linking group 1, the preparation method includes the following steps: The polypeptide compound 1 was subjected to a first substitution reaction with the active compound R1 to obtain intermediate 1; After the intermediate 1 undergoes a deprotection reaction of the R3 protecting group, it is subjected to a second substitution reaction with the R2 active compound to obtain the glutamic acid urea compound. Either the R1 active compound or the R2 active compound is When, the other is any of the following structures: ； When the polypeptide compound 1 is At that time, the intermediate 1 is ; When the polypeptide compound 1 is At that time, intermediate 1 is ; X in polypeptide compound 1 and X and R1 in intermediate 1 are the same as X and R1 in formula I-1 and formula I-2; R3 is selected from Boc protecting group, DDE protecting group or Fmoc protecting group; (ii) When X is a linking group 2, the preparation method includes the following steps: The polypeptide compound 2 was subjected to a third substitution reaction with R1-L to obtain intermediate 2; The intermediate 2 was subjected to a fourth substitution reaction with the active compound R2 to obtain the glutamate urea compound. In R1-L, L is selected from any of the following structures: ； In L, p is the same as in the linking group 2; When R1 in R1-L is When the R2 active compound is any one of the following structures: ； When R1 in R1-L is the group to be labeled, the active compound R2 is... ; When the polypeptide compound 2 is At that time, the intermediate 2 is ; When the polypeptide compound 2 is At that time, the intermediate 2 is ; In intermediate 2, X and R1 are the same as X and R1 in formulas I-1 and I-2.

3. A pharmaceutically acceptable salt of a glutamate urea compound, characterized in that, It is obtained by reacting a glutamate urea compound with an acid or a base; the glutamate urea compound is the glutamate urea compound of claim 1 or the glutamate urea compound prepared by the preparation method of claim 2.

4. A radionuclide targeting probe, characterized in that, It is obtained by coordinating the labeling group in a pharmaceutically acceptable salt of the glutamate urea compound of claim 1 with a labeling nuclide; The labeled nuclide is selected from 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 At least one of Ac.

5. The method for preparing the radionuclide targeting probe according to claim 4, characterized in that, Includes the following steps: A glutamate urea compound or its pharmaceutically acceptable salt is subjected to a coordination reaction with a labeled nuclide to obtain a nuclide-targeting probe or a pharmaceutically acceptable salt of a nuclide-targeting probe; wherein the glutamate urea compound or its pharmaceutically acceptable salt is the glutamate urea compound of claim 1 or the glutamate urea compound prepared by the preparation method of claim 2 or the pharmaceutically acceptable salt of the glutamate urea compound of claim 3. The labeled nuclide is selected from 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 At least one of Ac.

6. A pharmaceutically acceptable salt for a radionuclide targeting probe, characterized in that, It is obtained by a salt formation reaction of a radionuclide-targeting probe or a glutamate-urea compound; the radionuclide-targeting probe is the radionuclide-targeting probe of claim 4 or the radionuclide-targeting probe prepared by the preparation method of claim 5; the glutamate-urea compound is the glutamate-urea compound of claim 1 or the glutamate-urea compound prepared by the preparation method of claim 2.

7. A pharmaceutical composition, characterized in that, It includes an active ingredient and pharmaceutically acceptable excipients; the active ingredient is selected from one or more of glutamate urea compounds, radionuclide targeting probes, pharmaceutically acceptable salts of glutamate urea compounds according to claim 3, and pharmaceutically acceptable salts of radionuclide targeting probes according to claim 6; the glutamate urea compound is the glutamate urea compound according to claim 1 or the glutamate urea compound prepared by the preparation method according to claim 2; the radionuclide targeting probe is the radionuclide targeting probe according to claim 4 or the radionuclide targeting probe prepared by the preparation method according to claim 5.

8. The use of the glutamate urea compound of claim 1, the glutamate urea compound prepared by the method of claim 2, a pharmaceutically acceptable salt of the glutamate urea compound of claim 3, the radionuclide targeting probe of claim 4, the radionuclide targeting probe prepared by the method of claim 5, a pharmaceutically acceptable salt of the radionuclide targeting probe of claim 6, or the pharmaceutical composition of claim 7 in the preparation of therapeutic or diagnostic drugs for PSMA protein-mediated diseases; The diseases mediated by the PSMA protein are selected from tumors; the tumors are selected from one or more of the following: prostate cancer, breast cancer, ovarian cancer, liver cancer, lung cancer, colorectal cancer, bone tissue sarcoma, connective tissue sarcoma, renal cell carcinoma, gastric cancer, pancreatic cancer, head and neck cancer, neuroendocrine tumors, and skin melanoma.

9. The application according to claim 8, characterized in that, The head and neck cancer mentioned is nasopharyngeal carcinoma.

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