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

Abstract

The present invention relates to the technical field of biomedicine, and particularly relates to a glutamic acid urea compound and a preparation method therefor and a use thereof, a nuclide targeting probe and a preparation method therefor and a use thereof, and a pharmaceutical composition. The glutamic acid urea compound and the nuclide targeting probe provided by the present invention have excellent in-vivo biological performance, high specific uptake in lesions with high expression of PSMA protein, a high target-nontarget ratio, low non-specific background activity, and remarkably enhanced tumor uptake and retention time, are suitable for nuclide therapy and imaging of tumors, can reduce unnecessary radioactive damage to normal tissues and organs, can overcome the defects of low uptake and short retention time in small-molecule PSMA targeting organs, improve the PSMA targeting nuclide treatment and imaging effects, and have the potential of clinical popularization and application.

Description

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

[0001] This application claims priority to the Chinese patent application filed with the State Intellectual Property Office on August 17, 2023, with application number CN202311035883.5 and invention name “Glutamic acid urea compounds, preparation methods and applications thereof, radionuclide targeted probes, preparation methods and applications thereof, and pharmaceutical compositions”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention belongs to the field of biomedicine technology, and specifically relates to glutamate urea compounds and preparation methods and applications thereof, pharmaceutically acceptable salts of glutamate urea compounds, radionuclide targeting probes and preparation methods and applications thereof, pharmaceutically acceptable salts of radionuclide targeting probes, and pharmaceutical compositions. Background Art

[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 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. The leading position in this field is [ 177 Lu]Lu-PSMA617, multiple studies have shown that [ 177 Lu]Lu-PSMA617RLT has shown good safety and efficacy in a large number of mCRPC patients. However, due to the rapid metabolism of the drug in the body, single-dose administration is often large, and many patients have an insufficient response to radioligand therapy, resulting in disease progression during or after treatment.

[0004] One of the strategies to enhance therapeutic efficacy is to improve the delivery of radioligands. The commonly used method is to increase the tumor uptake dose by conjugating albumin-binding groups to PSMA-targeted radioligands to prolong the blood residence time. For example, albumin-binding groups such as p-iodophenylbutyric acid, Evans blue or ibuprofen can be modified into PSMA probes to achieve higher tumor uptake. However, this strategy often also increases the irradiation dose to healthy organs and tissues (including kidneys and bone marrow). Therefore, the pharmacokinetic properties of the probe in vivo must be carefully regulated. In summary, it is of great significance to develop a PSMA-targeted probe with a short blood circulation cycle, high absolute tumor uptake, long lesion retention, and low background in non-target organs.

[0005] Deferasirox (DFX) is an oral iron chelator that 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. After it is enriched in the tumor site, it has an anti-tumor cell proliferation effect. It can be used as a chemotherapy drug. It has also been reported as an antifungal and antibacterial drug. However, how to modify deferasirox so that it can have better tumor targeting, a higher lesion enrichment rate, and better disease treatment effects is a difficult problem facing researchers. In addition, the value of the deferasirox structure in the design of radionuclide-targeted drugs has not yet been developed. It is still unknown how much influence it will have on the pharmacokinetic properties of the probe after hybridization with other receptor-targeting groups. Its role in the field of radionuclide-targeted diagnosis and treatment is even more to be explored.

[0006] Summary of the Invention

[0007] In view of this, the present invention aims to provide glutamate urea compounds, preparation methods and uses thereof, pharmaceutically acceptable salts thereof, radionuclide targeting probes, preparation methods and uses thereof, pharmaceutically acceptable salts thereof, and pharmaceutical compositions. The glutamate urea compounds and pharmaceutically acceptable salts thereof, radionuclide targeting probes and pharmaceutically acceptable salts thereof provided by the present invention all exhibit PSMA targeting properties, have long target organ retention times, high tumor uptake doses, and low background.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0009] The present invention provides a glutamic acid urea compound having a structure shown in Formula I:

[0010] In the formula I, either R1 or R2 is The other is a group to be labeled, which includes any one of the following structures:

[0011] X is present or absent, and when present, includes Linker 1 or Linker 2;

[0012] The linking group 1 includes any one of the following structures:

[0013] The linking group 2 includes any one of the following structures:

[0014] wherein n, n′, m, y, z, p, and q are independently integers between 0 and 10.

[0015] The present invention provides a method for preparing the glutamic acid urea compound described in the above technical solution.

[0016] (i) When X does not exist or is a linking group 1, the preparation method comprises the following steps:

[0017] The polypeptide compound 1 is subjected to a first substitution reaction with the R1 active compound to obtain an intermediate 1;

[0018] The intermediate 1 is subjected to a R3 protecting group removal reaction and then subjected to a second substitution reaction with an R2 active compound to obtain the glutamic acid urea compound;

[0019] Any one of the R1 active compound and the R2 active compound is When , the other one is any of the following structures:

[0020] X and R1 in the polypeptide compound 1 and intermediate 1 are the same as X and R1 in the formula I; the R3 protecting group includes a Boc protecting group, a DDE protecting group or an Fmoc protecting group;

[0021] (ii) When X is a linking group 2, the preparation method comprises the following steps:

[0022] The polypeptide compound 2 is subjected to a third substitution reaction with R1-L to obtain intermediate 2;

[0023] The intermediate 2 is subjected to a fourth substitution reaction with the R2 active compound to obtain the glutamic acid urea compound;

[0024] In the R1-L, L includes any one of the following structures:

[0025] p and q in said L are the same as those in said linker 2;

[0026] When R1 in the R1-L is When the R2 active compound is any one of the following structures:

[0027] When R1 in the R1-L is the group to be labeled, the R2 active compound is

[0028] X and R1 in the polypeptide compound 2 and intermediate 2 are the same as X and R1 in formula I.

[0029] The present invention provides a pharmaceutically acceptable salt of a glutamic acid urea compound, which is obtained by reacting the 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 the glutamic acid urea compound prepared by the preparation method described in the above technical solution.

[0030] The present invention provides a nuclide targeting probe, which is obtained by a coordination reaction between a to-be-labeled group in the glutamic acid urea compound or a pharmaceutically acceptable salt of the glutamic acid urea compound described in the above technical solution and a labeled nuclide.

[0031] Preferably, the labeled nuclides include 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.

[0032] The present invention provides a method for preparing the nuclide targeting probe described in the above technical solution, comprising the following steps: subjecting a glutamate urea compound or a pharmaceutically acceptable salt thereof to a coordination reaction with a labeled nuclide to obtain a nuclide targeting probe or a pharmaceutically acceptable salt of the nuclide targeting probe; the glutamate urea compound or a pharmaceutically acceptable salt thereof is the glutamate urea compound described in the above technical solution or the glutamate urea compound obtained by the preparation method described in the above technical solution or the pharmaceutically acceptable salt of the glutamate urea compound described in the above technical solution.

[0033] The present invention provides a pharmaceutically acceptable salt of a nuclide targeting probe, which is obtained by a salt-forming 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; 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.

[0034] The present invention provides a pharmaceutical composition comprising an active component and a pharmaceutically acceptable excipient; the active component 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 the radionuclide targeting probe described in the above technical solution; 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; 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.

[0035] The present invention provides the use of the glutamate urea compound described in the above technical solution, the glutamate urea compound prepared by the preparation method described in the above technical solution, the pharmaceutically acceptable salt of the glutamate urea compound described in the above technical solution, the nuclide targeting probe described in the above technical solution, the nuclide targeting probe prepared by the preparation method described in the above technical solution, the pharmaceutically acceptable salt of the nuclide targeting probe described in the above technical solution, or the pharmaceutical composition described in the above technical solution in the preparation of therapeutic or diagnostic drugs for PSMA protein-mediated diseases.

[0036] Preferably, the PSMA protein-mediated disease includes tumors.

[0037] The glutamic acid urea compound provided by the present invention is a modified compound of deferasirox. Deferasirox has a certain targeting ability for tumor lesions and has been approved as an iron chelator for clinical use with good safety. The present invention modifies the structure of deferasirox into a PSMA-targeting probe, significantly improving the probe's uptake at the target site and prolonging its retention time at the target site. The glutamic acid urea compound provided by the present invention can be used to label a variety of diagnostic and therapeutic nuclides and can also be used to construct imaging and therapeutic platforms based on diagnostic and therapeutic nuclide pairs. It has great application prospects in the preparation of therapeutic or diagnostic drugs for PSMA protein-mediated diseases.

[0038] The glutamic acid urea compound having the structure shown in Formula I provided by the present invention can chelate with the nuclide through the group to be labeled to form a PSMA-targeted nuclide probe with high affinity and high specificity, which has the characteristics of strong labeling ability, short labeling time, high labeling yield, etc., and is conducive to the commercial application and clinical promotion of nuclide-targeted probes. Compared with the existing PSMA-targeted probes, the nuclide-targeted probes provided by the present invention have suitable metabolic kinetic properties (different pharmacokinetic properties) and high lesion uptake and retention time, and have excellent diagnostic and therapeutic effects on diseases mediated by PSMA protein. It is a nuclide-targeted diagnostic and therapeutic drug with great application prospects. As shown in the test results of the embodiment, the nuclide-targeted probes provided by the present invention have suitable metabolic kinetic properties (different pharmacokinetic properties) and high lesion uptake and retention time. It has excellent diagnostic and therapeutic effects on diseases mediated by PSMA protein, and is a nuclide-targeted diagnostic and therapeutic drug with great application prospects. As shown in the test results of the embodiment, the nuclide-targeted probes provided by the present invention have good metabolic kinetic properties (different pharmacokinetic properties) and high lesion uptake and retention time. It has excellent diagnostic and therapeutic effects on diseases mediated by PSMA protein, and is a nuclide-targeted diagnostic and therapeutic drug with great application prospects. 177 The absolute value of uptake of Lu-labeled radionuclide targeting probe in tumor is [ 177 Lu]Lu-PSMA617 (one of the current gold standards) is 4 to 5 times that of Lu-PSMA617 (one of the current gold standards). It is a radionuclide targeted therapy drug with great application prospects. It overcomes the defects of the existing small molecule PSMA, such as rapid metabolism and short retention time in target organs, improves the effect of PSMA radionuclide targeted therapy, and has the potential for clinical promotion and application. In addition to treatment, the radionuclide targeted probe provided by the present invention is distributed in the body to form a concentration difference. If the emitted radiation or magnetic flux change is detected by an external instrument, an image is formed after reconstruction, which can provide diagnostic information for the disease and achieve the effect of integrated diagnosis and treatment. In addition, by adjusting the appropriate specific activity or drug combination, it is helpful to obtain a better target / non-target ratio and enhance the uptake of radionuclide targeted probes in tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG1 is a mass spectrometric identification diagram of compound PKND01;

[0040] FIG2 is a mass spectrometric identification diagram of compound PKND02;

[0041] FIG3 is a mass spectrometric identification pattern of compound PKSD01;

[0042] FIG4 is a mass spectrometric identification pattern of compound PKSD02;

[0043] FIG5 is a mass spectrometric identification pattern of compound PKSP2D01;

[0044] FIG6 is a mass spectrometric identification diagram of compound Gd-PKND01;

[0045] FIG7 is an HPLC identification pattern of compounds PKND01 (a), PKND02 (b), PKSD01 (c), PKSD02 (d) and PKSP2D01 (e);

[0046] FIG8 is a HPLC identification diagram of probe Gd-PKND01;

[0047] Figure 9 shows the probe [68 Ga]Ga-PKND01(a), [ 68 Ga]Ga-PKND02(b), [ 68 Ga]Ga-PKSD01(c) and [ 68 HPLC identification chart of the radiochemical purity of Ga]Ga-PKSP2D01(d);

[0048] Figure 10 shows the probe [ 177 Lu]Lu-PKND01(a),[ 177 Lu]Lu-PKND02(b) and [ 177 Lu] Lu-PKSD01 (c) radiochemical purity HPLC identification chart;

[0049] Figure 11 shows the probe [ 68 Ga]Ga-PKND01(a), [ 68 Ga]Ga-PKND02(b) and [ 68 HPLC identification results of the stability of Ga]Ga-PKSD01(c);

[0050] Figure 12 shows the probe [ 177 Lu]Lu-PKND01(a),[ 177 Lu]Lu-PKND02(b) and [ 177 HPLC identification results of the stability of Lu]Lu-PKSD01(c);

[0051] Figure 13 shows the probe [ 177 Lu]Lu-PKND01(a) and [ 177 Lu]Lu-PKND02(b) cellular uptake and inhibition results;

[0052] Figure 14 shows the probe [ 177 Lu]Lu-PKSD01(a) and [ 177 Lu]Lu-PKSD02(b) cellular uptake and inhibition results;

[0053] Figure 15 is [ 68 PET imaging results of Ga]Ga-PKND01 (a) and quantitative uptake values ​​of tissues of interest (b);

[0054] Figure 16 is [ 68 PET imaging results of Ga]Ga-PKND02 (a) and quantitative uptake values ​​of tissues of interest (b);

[0055] Figure 17 is [ 68 PET imaging results of Ga]Ga-PKSD01 (a) and quantitative uptake values ​​of tissues of interest (b);

[0056] Figure 18 is [ 68 PET imaging results of Ga]Ga-PKSP2D01 (a) and quantitative uptake values ​​of tissues of interest (b);

[0057] Figure 19 is [ 177 SPECT imaging results (a) and target / non-target ratio (b) of Lu]Lu-PKND01;

[0058] Figure 20 is [ 177 SPECT imaging results (a) and target / non-target ratio (b) of Lu]Lu-PKND02;

[0059] Figure 21 is [ 177 SPECT imaging results (a) and target / non-target ratio (b) of Lu]Lu-PKSD01;

[0060] Figure 22 is [ 177 SPECT imaging results of Lu]Lu-PSMA617 (a), [ 177 Lu]Lu-PKND01 and [ 177 The uptake counts of Lu]Lu-PKSD01 in tumor sites were similar to those of [ 177 Lu]Lu-PSMA617 ratio (b);

[0061] Figure 23 shows different specific activities [ 177 SPECT imaging of tumor uptake results (a) and tumor / kidney ratio (b) of Lu]Lu-PKND01;

[0062] FIG24 shows the MRI results of tumor-bearing mice at different time points before and after tail vein injection of Gd-PKND01;

[0063] Figure 25 is [ 177 Biodistribution results of Lu]Lu-PKND01 in tumor-bearing mice (a) and tumor / kidney ratios at different time points (b);

[0064] Figure 26 is [ 177 Lu]Lu-PSMA617 biodistribution results in tumor-bearing mice (a) and [ 177 The uptake values ​​of Lu]Lu-PSMA617 in tumors were similar to those of [ 177 Lu]Lu-PKND01 comparison results (b);

[0065] Figure 27 is [ 177 Lu]Lu-PKND01, [ 177 Lu]Lu-PKSD01 and [ 177 [Lu]Lu-PSMA617 therapeutic outcomes in tumor-bearing mice. DETAILED DESCRIPTION

[0066] The present invention provides a glutamic acid urea compound having a structure shown in Formula I:

[0067] In the formula I, either R1 or R2 is The other is a group to be labeled, which includes any one of the following structures:

[0068] X is present or absent, and when present, includes Linker 1 or Linker 2;

[0069] The linking group 1 includes any one of the following structures:

[0070] The linking group 2 includes any one of the following structures:

[0071] wherein n, n′, m, y, z, p, and q are independently integers between 0 and 10.

[0072] In the present invention, when X does not exist, either R1 or R2 is Another one is The glutamate urea compound includes PKND01 or PKND02:

[0073] In the present invention, when X is When either R1 or R2 is Another one is Among them, the Preferably The glutamate urea compound includes PKSD01, PKSD02, PKSP2D01 or PKSP2D02:

[0074] In the present invention, when X is When any one of R1 and R2 is Another one is Among them, the Preferably The glutamate urea compounds include PKP 23 D01, PKP 23 D02, PKP 22 D01, PKP 22 D02, PKED01 or PKED02:

[0075] The present invention provides a method for preparing the glutamic acid urea compound described in the above technical solution. When X does not exist or is a linker group 1, the preparation method comprises the following steps:

[0076] The polypeptide compound 1 is subjected to a first substitution reaction with the R1 active compound to obtain an intermediate 1;

[0077] The intermediate 1 is subjected to a R3 protecting group removal reaction and then subjected to a second substitution reaction with an R2 active compound to obtain the glutamic acid urea compound;

[0078] Any one of the R1 active compound and the R2 active compound is When , the other one is any of the following structures:

[0079] X and R1 in the polypeptide compound 1 and intermediate 1 are the same as X and R1 in the formula I; the R3 protecting group includes a Boc protecting group, a DDE protecting group or an Fmoc protecting group.

[0080] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0081] In the present invention, the polypeptide compound 1 is subjected to a first substitution reaction with the R1 active compound to obtain an intermediate 1.

[0082] In the present invention, 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 invention, the first substitution reaction is preferably carried out in the presence of a high-boiling-point solvent and an alkaline 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 used is not particularly limited, as long as it can dissolve the polypeptide compound 1 and ensure the smooth progress of the first substitution reaction. In the present invention, the alkaline reagent is preferably an organic base, preferably triethylamine (TEA) and / or N,N-diisopropylethylamine (DIPEA); the molar ratio of the polypeptide compound 1 to the alkaline reagent is preferably 1:1-10, more preferably 1:3-5. In the present invention, 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 hours, more preferably 5-12 hours.

[0083] After the first substitution reaction, the present invention preferably further comprises a post-treatment, and the post-treatment preferably comprises: subjecting the obtained first substitution system to a reverse-phase high-performance chromatography column purification and then freeze-drying to obtain intermediate 1. In the present invention, the conditions for the reverse-phase high-performance chromatography column purification include: the chromatographic 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 method is preferably gradient elution, and the conditions for the gradient elution are preferably 0 to 30 minutes: the volume fraction of the mobile phase B increases from 10% to 90%, and the flow rate of the mobile phase is preferably 3 mL / min. The present invention has no special restrictions on the temperature and time of the freeze-drying, and freeze-drying to constant weight (i.e., freeze-drying) is sufficient.

[0084] After obtaining the intermediate 1, the present invention performs a deprotection reaction on the intermediate 1 at R3 and then performs a second substitution reaction with an R2 active compound to obtain the glutamic acid urea compound.

[0085] In the present invention, the R3 protecting group removal 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 to 10%, more preferably 3 to 5%; the present invention does not specifically limit the amount of the hydrazine hydrate solution and trifluoroacetic acid used, as long as the protecting group (DDE, Boc or Fmoc) can be removed. In the present invention, the temperature of the R3 protecting group removal reaction is preferably 0 to 37°C, more preferably 25°C, and the time of the R3 protecting group removal reaction is preferably 1 to 12 hours, more preferably 2 to 5 hours.

[0086] In the present invention, the conditions of the second substitution reaction and the post-treatment after the second substitution reaction are preferably the same as the conditions and post-treatment of the first substitution reaction, and are not described in detail here.

[0087] In the present invention, when X does not exist or is a linker group 1, the preparation route of the glutamic acid urea compound is as follows:

[0088] The present invention provides a method for preparing the glutamic acid urea compound described in the above technical solution. When X is a linker group 2, the preparation method comprises the following steps:

[0089] The polypeptide compound 2 is subjected to a third substitution reaction with R1-L to obtain intermediate 2;

[0090] The intermediate 2 is subjected to a fourth substitution reaction with the R2 active compound to obtain the glutamic acid urea compound;

[0091] In the R1-L, L includes any one of the following structures:

[0092] p and q in said L are the same as those in said linker 2;

[0093] When R1 in the R1-L is When the R2 active compound is any one of the following structures:

[0094] When R1 in the R1-L is the group to be labeled, the R2 active compound is

[0095] X and R1 in the polypeptide compound 2 and intermediate 2 are the same as X and R1 in formula I.

[0096] In the present invention, when X is a linker group 2, the preparation route of the glutamic acid urea compound is as follows:

[0097] In the present invention, the polypeptide compound 2 is subjected to a third substitution reaction with R1-L to obtain an intermediate 2.

[0098] In the present invention, the R1-L preferably includes DFX-MAL, DOTA-MAL or DFX-P2-MAL:

[0099] In the present invention, the molar ratio of the polypeptide compound 2 to R1-L is preferably 1:1 to 5, more preferably 1:1.5 to 2. In the present invention, the third substitution reaction is preferably carried out in the presence of a solvent, and the solvent preferably includes a high-boiling point organic solvent and / or PBS (phosphate buffer solution), more preferably a mixed solvent of a high-boiling point organic 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 value of the PBS is preferably 7 to 8, more preferably 7.4 to 7.6; the present invention has no special limitation on the amount of the solvent, as long as it can dissolve the polypeptide compound 2 and ensure that the third substitution reaction proceeds smoothly. In the present invention, the temperature and time of the third substitution reaction and the post-treatment after the third substitution reaction are preferably the same as the temperature, time and post-treatment of the first substitution reaction, and will not be repeated here.

[0100] After obtaining the intermediate 2, the present invention conducts a fourth substitution reaction between the intermediate 2 and the R2 active compound to obtain the glutamic acid urea compound.

[0101] In the present invention, the R2 reactive compound preferably comprises a DOTA or DFX reactive compound bearing an active reactive group -NHS or -SCN. In the present invention, the molar ratio of the intermediate 2 to the R2 reactive compound is preferably 1:1-5, more preferably 1:2-2.5. In the present invention, the conditions for the fourth substitution reaction and the post-treatment after the fourth substitution reaction are preferably the same as those for the first substitution reaction and are not further described here.

[0102] The present invention provides a pharmaceutically acceptable salt of a glutamic acid urea compound, 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 the glutamic acid urea compound prepared by the preparation method described in the above technical solution. In the present invention, the pharmaceutically acceptable salt preferably includes trifluoroacetate, phosphate, formate, acetate, potassium salt, or sodium salt. In the present invention, the acid preferably includes trifluoroacetic acid, hydrochloric acid, formic acid, or acetic acid; the base preferably includes potassium hydroxide or sodium hydroxide. The present invention does not particularly limit the reaction conditions; the conditions for salt-forming reactions familiar to those skilled in the art can be used.

[0103] The present invention provides a radionuclide targeting probe, which is obtained by a coordination reaction between a labeled group in the glutamic acid urea compound or a pharmaceutically acceptable salt thereof and a labeled radionuclide. In the present 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.

[0104] The present invention provides a method for preparing the nuclide targeting probe described in the above technical solution, comprising the following steps:

[0105] A glutamic acid urea compound is subjected to a coordination reaction with a labeled nuclide to obtain a nuclide-targeted probe; 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 invention, the nuclide-targeted probe is preferably prepared by a wet labeling method or a freeze-drying labeling method.

[0106] In the present invention, the wet labeling method for preparing the nuclide targeting probe preferably includes the following steps: mixing a solution of glutamic acid urea compound with a labeled nuclide solution, performing a coordination reaction, and then diluting to obtain an injection solution of the nuclide targeting probe.

[0107] In the present 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 to 7, more preferably 4 to 6.5; the concentration of aluminum chloride in the aluminum chloride-acetate solution is preferably 0.2 to 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 to 1000 mg / mL, more preferably 0.01 to 1 mg / mL. In the present invention, the ratio of the mass of the glutamic acid urea compound to the radioactivity of the labeled nuclide in the labeled nuclide solution is preferably 20-400 μg: 1 kBq-1000 GBq, more preferably 20-400 μg: 0.037-74000 MBq, and further preferably 20-200 μg: 0.037-7400 MBq. In the present invention, there is no particular limitation on the labeled nuclide solution, and any labeled nuclide 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; 68 The GaCl3 hydrochloric acid solution is preferably obtained by elution from a gallium germanium generator.

[0108] In the present invention, the temperature of the coordination reaction is preferably 25 to 100°C, more preferably 80 to 100°C, and the time of the coordination reaction is preferably 10 to 60 minutes, more preferably 20 to 30 minutes. When the temperature of the coordination reaction is higher than room temperature, the present invention preferably further comprises cooling the obtained coordination reaction system to room temperature after the coordination reaction. The present invention has no special limitation on the cooling, and a cooling method well known to those skilled in the art may be adopted, such as natural cooling. After the dilution, the present invention preferably further comprises aseptically filtering the obtained dilution system to obtain an injection of a radionuclide targeted probe. In the present invention, the dilution preferably utilizes physiological saline or water for injection. In the present invention, the radioactivity concentration of the injection of the radionuclide targeted probe is preferably 0.037 to 3700 MBq / mL.

[0109] In the present invention, when the labeled nuclide solution is a gadolinium chloride hexahydrate solution, the present invention preferably first uses a base to adjust the pH value of the mixed solution of the glutamic acid urea compound and the gadolinium chloride hexahydrate solution to 5.0 to 6.5, more preferably 5.5 to 6.0. The base preferably includes a KOH solution, and the concentration of the KOH solution is preferably 1 to 4 mol / L, more preferably 2 to 3 mol / L.

[0110] In the present invention, the preparation of a radionuclide-targeted probe using a freeze-drying labeling method preferably includes the following steps: freeze-drying a solution of a glutamic acid urea compound and then sealing it to obtain a freeze-dried kit; adding a solvent to the freeze-dried kit to dissolve it, then adding a labeled radionuclide solution for coordination reaction and diluting it to obtain an injection of the radionuclide-targeted probe. In the present invention, the freeze-drying method preferably involves dispensing the solution of the glutamic acid urea compound into freeze-drying containers and then freeze-drying it. The present invention does not specifically limit the freeze-drying conditions; freeze-drying conditions familiar to those skilled in the art can be used. In the present invention, excipients are preferably added to the freeze-dried kit as needed. The excipients preferably include at least one of an excipient, an antioxidant, and an acid-base modifier. The present invention does not specifically limit the excipients, antioxidants, and acid-base modifiers; excipients, antioxidants, and acid-base modifiers familiar to those skilled in the art can be used. In the present invention, the other preparation conditions for the radionuclide-targeted probe are preferably the same as those for the aforementioned wet labeling method and will not be detailed here.

[0111] In the present invention, when the radiochemical purity of the injection of the radionuclide targeted probe prepared by the wet labeling method and the freeze-drying labeling method is lower than 95%, it is preferred to further include purifying the injection of the radionuclide targeted probe, and the purification is preferably Sep-Pak C18 separation column purification, and the Sep-Pak C18 separation column is preferably activated and eluted with anhydrous ethanol and water in sequence before use. In the present invention, the eluents used for the purification are preferably water and anhydrous ethanol in sequence, and the eluate of anhydrous ethanol is collected and the solvent is removed, and then diluted to obtain an injection of a high-purity radionuclide targeted probe. In the present invention, the dilution is preferably carried out using physiological saline or water for injection. In the present invention, the radioactivity concentration of the injection of the high-purity radionuclide targeted probe is preferably 0.037 to 3700 MBq / mL.

[0112] The preparation method provided by the present invention has the advantages of simple and easy labeling, good stability of the obtained radionuclide targeting probe, high tumor uptake, etc., and is suitable for industrial production and clinical promotion.

[0113] In the present invention, a pharmaceutically acceptable salt of a nuclide targeting probe is provided, which is obtained by a salt-forming 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; 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 invention, the pharmaceutically acceptable salt preferably includes trifluoroacetate, phosphate, formate, acetate, potassium salt or sodium salt. The present invention does not specifically limit the preparation method of the pharmaceutically acceptable salt of the nuclide targeting probe, and the preparation method of the pharmaceutically acceptable salt well known to those skilled in the art can be used.

[0114] The present invention provides a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable excipient; the active ingredient comprises one or more of a glutamic acid urea compound, a radionuclide 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 radionuclide 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 a glutamic acid urea compound produced by the preparation method described in the above technical solution; and the radionuclide targeting probe is the radionuclide targeting probe described in the above technical solution or a radionuclide targeting probe produced by the preparation method described in the above technical solution. The present invention does not specifically limit the pharmaceutically acceptable excipient; pharmaceutically acceptable excipients well known to those skilled in the art may be used. In the present invention, when the active ingredient comprises 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 by intravenous injection; when the active ingredient does not contain a radionuclide targeting probe or a pharmaceutically acceptable salt thereof, the present invention does not specifically limit the dosage form and administration method of the pharmaceutical composition; dosage forms and administration methods well known to those skilled in the art may be used.

[0115] The present invention also provides the use of the glutamic acid urea compound described in the above technical solution, the glutamic acid urea compound prepared by the preparation method described in the above technical solution, the pharmaceutically acceptable salt of the glutamic acid urea compound described in the above technical solution, the radionuclide targeting probe described in the above technical solution, the radionuclide targeting probe prepared by the preparation method described in the above technical solution, the pharmaceutically acceptable salt of the radionuclide targeting probe described in the above technical solution, or the pharmaceutical composition described in the above technical solution in the preparation of therapeutic or diagnostic drugs for PSMA protein-mediated diseases. In the present invention, the PSMA protein-mediated disease preferably includes tumors; 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 tumors, and skin melanoma. In the present invention, the diagnostic method preferably includes one or more of single photon emission computed tomography (SPECT), positron emission tomography (PET) and magnetic resonance imaging (MRI); the treatment method preferably includes radionuclide targeted therapy and / or chemotherapy.

[0116] To further illustrate the present invention, the glutamate urea compounds of the present invention, their preparation methods and applications, the radionuclide targeting probes, their preparation methods and applications, and the pharmaceutical compositions are described in detail below with reference to the accompanying drawings and examples. However, these should not be construed as limiting the scope of protection of the present invention.

[0117] Example 1

[0118] Synthesis of PKND01

[0119] (1) Synthesis of Compound 2: Compound 1 (3.16 μmol) was weighed into a 1.5 mL centrifuge tube and dissolved in DMSO (0.5 mL). DFX-NHS (9.49 μmol) and N,N-diisopropylethylamine (DIPEA, 15.82 μmol) were added. The mixture was 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 2 (4 mg, yield 97%, purity 98%). HPLC purification conditions: reverse 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 mobile phase flow rate was 3 mL / min. Compound 2: ESI MS [M+H] + for C 70 H 82 N 10 O 15 ,calcd 1302.60,found 1303.25.

[0120] (2) Synthesis of PKND01: Compound 2 (3.06 μmol) was weighed into a 1.5 mL centrifuge tube, and hydrazine hydrate (0.5 mL, 3% by mass) was added. The mixture was reacted at 25°C for 2 h, and then DMSO (0.5 mL) was added. DOTA-NHS (5.48 μmol) and DIPEA (10.98 μmol) were added. The mixture was stirred at 25°C for 12 h. The mixture was purified by HPLC and lyophilized (lyophilization temperature -65°C) to obtain compound PKND01 (1.6 mg, yield 47.7%), which was identified as having a purity greater than 95%. HPLC purification conditions: reverse 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 mobile phase flow rate was 3 mL / min. PKND01:ESI MS[M+H]+for C 76 H 96 N 14 O 20 ,calcd 1524.69,found 1525.88, as shown in Figure 1; HPLC purity analysis is shown in Figure 7a, the purity is greater than 95%.

[0121] Example 2

[0122] Synthesis of PKND02 compound

[0123] (1) Synthesis of compound 3: Compound 1 (3.16 μmol) was weighed into a 1.5 mL centrifuge tube, dissolved in DMSO (0.5 mL), and then added with DOTA-NHS (7.91 μmol) and N,N-diisopropylethylamine (DIPEA, 15.82 μmol). The reaction was carried out at 25°C for 12 h. The mixture was 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: reverse 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 mobile phase flow rate was 3 mL / min. Compound 3: ESI MS [M+H] + for C 65 H 95 N 11 O 19 ,calcd 1334.53,found 1335.24.

[0124] (2) Synthesis of PKND02: Compound 3 (3.06 μmol) was weighed and placed in a 1.5 mL centrifuge tube. Hydrazine hydrate (0.5 mL, 3% by mass) was added thereto. The mixture was reacted at 25°C for 2 h. DMSO (0.5 mL) was then added, followed by DOTA-NHS (5.34 μmol) and DIPEA (6.41 μmol). The mixture was stirred at 25°C for 12 h. The mixture was purified by HPLC and lyophilized (lyophilization temperature -65°C) to obtain compound PKND02 (2 mg, yield 61.53%). The purity was determined to be greater than 95%. HPLC purification conditions: reverse 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 mobile phase flow rate was 3 mL / min. PKND02: ESI MS [M+H]+ for C 76 H 96 N 14 O 20 , calcd 1524.69, found 1525.39, as shown in FIG2 ; HPLC purity analysis shows that the purity is greater than 95% as shown in FIG7 b.

[0125] Example 3

[0126] Synthesis of PKSD01 compounds

[0127] (1) Synthesis of Compound 5: Compound 4 (3.03 μmol) was weighed into a 1.5 mL centrifuge tube, dissolved in DMSO (0.5 mL), and then added with DFX-MAL (6.05 μmol) and PBS solution (0.3 mL). 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: reverse 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 mobile phase flow rate was 3 mL / min. ESI MS [M+H] + for C 68 H 80 N 12 O 16 S,calcd 1353.52,found 1354.34.

[0128] (2) Synthesis of compound PKSD01: Compound 5 (2 mg, 1 eq., 1.48 μmol) was weighed and placed in a 1.5 mL centrifuge tube. DMSO (0.5 mL) was added, along with 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%), which was identified as having a purity greater than 95%. HPLC purification conditions: reverse-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 mobile phase flow rate was 3 mL / min. PKSD01:ESI MS[M+H]+for C 76 H 96 N 14 O 20 , calcd 1738.73, found 1739.70, as shown in FIG3 ; HPLC purity analysis shows that the purity is greater than 95% as shown in FIG7 c.

[0129] Example 4

[0130] Synthesis of PKSD02 compound

[0131] (1) Synthesis of compound 6: Compound 4 (2.33 μmol) was weighed into a 1.5 mL centrifuge tube, dissolved in DMSO (0.5 mL), and then added with raw material DOTA-MAL (4.66 μmol). PBS solution (0.3 mL) was then added. The mixture was reacted at 25°C for 12 h, purified by HPLC, and lyophilized (lyophilization temperature -65°C) to obtain white solid compound 6 (2.1 mg, yield 65.01%, purity 98%). HPLC purification conditions: reverse 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 mobile phase flow rate was 3 mL / min. Compound 6: ESI MS [M+H] + for C 63 H 93 N 13 O 20 S,calcd 1384.57,found 1385.48.

[0132] (2) Synthesis of compound PKSD02: Compound 6 (1.52 μmol) was weighed and placed in 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%), which was identified as having a purity greater than 95%. HPLC purification conditions: reverse 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 mobile phase flow rate was 3 mL / min. PKSD02: ESI MS [M+H]+ for C 84 H 106 N 16 O 23 S, calcd 1739.92, found 1740.63, as shown in FIG4 ; HPLC purity analysis shows that the purity is greater than 95% as shown in FIG7 d.

[0133] Example 5

[0134] Synthesis of PKSP2D01 compounds

[0135] (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, and DMSO (0.3 mL) and PBS (pH = 7.4) were added to dissolve the mixture. 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 7 (1.3 mg, yield 24.6%, purity 95%). HPLC purification conditions: reverse 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 mobile phase flow rate was 3 mL / min. Compound 7: ESI MS [M+H]+ for C 75 H 93 N 13 O 19 S,calcd 1512.70,found 1513.53.

[0136] (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 thereto. 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%), which was identified as having a purity greater than 95%. HPLC purification conditions: reverse 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 mobile phase flow rate was 3 mL / min. PKSP2D01: ESI MS [M+H]+ for C 91 H 119 N 17 O 26 S, calcd 1899.11, found 1899.83, as shown in FIG5 ; HPLC purity analysis shows that the purity is greater than 95% as shown in FIG7 e.

[0137] Example 6

[0138] Gd nuclide labeling

[0139] The 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 a KOH solution. The mixture was heated to 60°C and shaken for 12 hours. After completion of the reaction, it was purified by HPLC to obtain the radionuclide targeted probe Gd-PKND01. The amount ratio of the compound PKND01 to the mixed solvent was 1 mg:1 mL.

[0140] HPLC analysis conditions: Reversed-phase C18 analytical column (4.6 mm × 250 mm); Mobile phase A: Water + 0.1% trifluoroacetic acid (TFA); Mobile phase B: Acetonitrile + 0.1% TFA; Gradient elution: 0-30 min: Mobile phase B volume fraction increased from 10% to 90%, mobile phase flow rate 1 mL / min. Mass spectrometric identification and HPLC chemical purity of Gd-PKND01 are shown in Figures 6 and 8, respectively. ESI MS [M+H]+ for C 76 H 93 GDJ 14 O 20 ,calcd 1679.91,found 1680.55.

[0141] Example 7

[0142] 68 Ga nuclide labeling

[0143] Wet method: 370MBq 68 GaCl3 hydrochloric acid solution (eluted from a germanium-gallium generator) was added to 0.5 mL of acetic acid-acetate solution containing glutamic acid urea compounds (20 μg) prepared in Examples 1 to 5, respectively. The mixture was reacted at 90°C for 20 minutes and then cooled to room temperature. The mixture was diluted with physiological saline or water for injection and sterile filtered to obtain a radionuclide targeted probe injection (the concentration of the radionuclide targeted probe was 110 MBq / mL).

[0144] Lyophilization method: a certain amount of buffer (sodium acetate buffer, pH 5.5, 0.4 mL) and 370 MBq of 68 GaCl3 eluent (eluted from a gallium germanium generator) was added to a freeze-dried drug box containing 20 μg of glutamic acid urea compound, reacted at 90°C for 20 minutes, cooled to room temperature, diluted with normal saline or water for injection, and sterile filtered to obtain a radionuclide targeted probe injection solution (the concentration of the radionuclide targeted probe was 110 MBq / mL).

[0145] If the radiochemical purity is less than 95%, it can be purified by C18 separation column to remove unreacted 68 Ga 3+Purification steps of C18 separation column: Take Sep-Pak C18 separation column and activate it by eluting it with 10 mL of anhydrous ethanol and 10 mL of water. Dilute the labeled solution with 10 mL of water and load it onto the separation column. Rinse the separation column with water to remove unreacted 68 Ga ions, and then washed with ethanol solution to obtain 68 Ga-labeled radionuclide targeting probe.

[0146] [ 68 Ga]Ga-PKND01, [ 68 Ga]Ga-PKND02, [ 68 Ga]Ga-PKSD01 and [ 68 The HPLC identification results of the radiochemical purity of Ga]Ga-PKSP2D01 are shown in Figures 9a, b, c, and d, respectively. The radiochemical purity of each radionuclide targeting probe is greater than 95%.

[0147] Example 8

[0148] 177 Lu nuclide labeling

[0149] Wet method: 370MBq 177 LuCl3 solution was added to 0.2 mL of acetic acid-acetate solution containing the compound prepared in Examples 1 to 3 (20 μg), respectively, and 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 a radionuclide targeting probe injection (the concentration of the radionuclide targeting probe was 110 MBq / mL).

[0150] Lyophilization method: 0.2 mL of buffer (sodium acetate buffer, pH = 5.5) and 370 MBq of 177 LuCl3 solution was added to a freeze-dried drug box containing glutamic acid urea compound (20 μg), mixed and dissolved, and then placed at 90°C for 20 minutes, cooled to room temperature, diluted with normal saline or water for injection, and sterile filtered to obtain the radionuclide targeted probe injection (the concentration of the radionuclide targeted probe was 110 MBq / mL).

[0151] If the radiochemical purity is less than 95%, it can be purified by C18 separation column to remove unreacted 177 Lu 3+ The purification steps of C18 separation column are as follows: take Sep-PakC18 separation column, activate and elute it with 10mL of anhydrous ethanol and 10mL of water. Dilute the labeled solution with 10mL of water and load it onto the separation column. Rinse the separation column with water to remove unreacted 177 Lu ions, and then washed with ethanol solution to obtain 177 Lu-labeled radionuclide targeting probe.

[0152] [ 177 Lu]Lu-PKND01, [ 177 Lu]Lu-PKND02 and [ 177 The radiochemical purity HPLC identification results of Lu]Lu-PKSD01 are shown in Figure 10 a, b and c, respectively. The radiochemical purity of each probe is greater than 95%.

[0153] Test Example 1

[0154] Evaluation of stability and lipid-water distribution properties

[0155] 1. Injection Stability Test: The radionuclide-targeted probe diluted with saline was left at room temperature for various periods of time, and samples were then analyzed by HPLC. HPLC analysis conditions: reversed-phase C18 analytical column (4.6 mm × 250 mm); mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA; gradient elution: 0-30 min: mobile phase B volume fraction increased from 10% to 90%, mobile phase flow rate 1 mL / min.

[0156] [ 68 Ga]Ga-PKND01, [ 68 Ga]Ga-PKND02 and [ 68 The HPLC identification results of the stability of Ga]Ga-PKSD01 are shown in Figure 11a, b and c respectively; [ 177 Lu]Lu-PKND01, [ 177 Lu]Lu-PKND02 and [ 177 The HPLC stability evaluation results of Lu]Lu-PKSD01 are shown in Figures 12a, b, and c. As shown in Figures 11 and 12, the radiochemical purity of each radionuclide-targeted probe was greater than 95% at the tested time points, indicating its stability in the specified solution.

[0157] 2. Determination of lipid-water distribution coefficient (logP):

[0158] Add 100 μL of the diluted solution containing the radionuclide targeting probe to a centrifuge tube containing a mixture of 2.9 mL of PBS and 3 mL of n-octanol. Vortex for 3 minutes and centrifuge at 10,000 rpm for 3 minutes. Take 100 μL of each of the aqueous and n-octanol phases and measure the radioactivity count using a γ-counter. Repeat the experiment three times and take the average value. The logP calculation formula is: P = I 有机相 / I 水相 ;

[0159] Among them I 有机相 represents the radioactive count measured in the organic phase, I 水相Represents the radioactive count measured in the aqueous phase. By calculation, the lipid-water distribution coefficient of each radiolabeled targeting probe was finally determined. The results are shown in Table 1. The measured radionuclide targeting probes are water-soluble.

[0160] Table 1 Lipid-water distribution coefficient of radionuclide targeting probe

[0161] As can be seen from Table 1, the above four 117 The Lu-labeled nuclide targeting probe is water-soluble, indicating that the 117 Lu-labeled radionuclide targeting probes can be metabolized by the kidneys, avoiding nonspecific uptake of radioactivity by normal tissues.

[0162] Test Example 2

[0163] Cellular uptake and inhibition experiments

[0164] PSMA-overexpressing PC3PIP cells and negatively expressing PC3flu cells were plated in 24-well plates containing culture medium (including fetal bovine serum and dual antibody) (cell counts were performed using a cell counter, approximately 200,000 cells / well) and cultured for 24 hours. At the start of the uptake experiment, the original culture medium was aspirated, and the cells were washed twice with PBS (500 μL). The PBS was then aspirated. An equal amount of the probe to be tested, diluted in culture medium, was added to each well and incubated at 37°C for 0.5, 1, 2, 4, and 8 hours. 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 minutes, the lysed cells were placed in a disposable centrifuge tube and the radioactivity count was measured. This count was divided by the total amount of added radioactivity to obtain the percentage of cellular uptake.

[0165] To investigate the PSMA targeting specificity of the probe, an inhibition group was also set up in this study. That is, an appropriate amount of the inhibitor PSMA617 was added to each well of cells before the addition of the radionuclide-labeled probe, and the cells were incubated at 37°C for 2 hours and 4 hours. After the incubation was completed, the radioactive culture medium was aspirated, and sodium hydroxide solution (500 μL, 1 M) was added to each well to lyse the cells. After 5 minutes, the lysed cells were placed in a disposable centrifuge tube to measure the radioactivity count; the count was divided by the total amount of added radioactivity to obtain the percentage of cellular uptake.

[0166] [ 177 Lu]Lu-PKND01, [ 177 Lu]Lu-PKND02, [ 177 Lu]Lu-PKSD01 and [ 177The cellular uptake and inhibition results of Lu]Lu-PKSD02 are shown in Figures 13-14. Taking the 4-hour results as an example, each radionuclide-targeted probe showed significant uptake in PSMA-positive cells, while uptake was significantly reduced in PSMA-negative cells. The uptake of each radionuclide-targeted probe in PSMA-positive cells was inhibited by PSMA617, demonstrating that the radionuclide-targeted probes provided by the present invention are specific for PSMA protein.

[0167] Test Example 3

[0168] PET imaging experiment

[0169] The radiochemical purity of the prepared embodiment is greater than 95% 68 After the Ga-labeled radionuclide targeted probe was diluted with physiological saline, 0.2 mL (1 MBq) of the injection solution was injected through the tail vein of PC3PIP model mice. MicroPET imaging was performed at different time points, and the region of interest (ROI) was outlined on the image. The probe distribution value was obtained by calculation. 68 Ga]Ga-PKND01, [ 68 Ga]Ga-PKND02, [ 68 Ga]Ga-PKSD01 and [ 68 The PET imaging results of Ga]Ga-PKSP2D01 are shown in Figures 15, 16, 17, and 18, respectively. Figures 15-18 demonstrate high uptake of the radionuclide-targeted probe at the tumor site. High radioactivity signals were observed in the bladder and kidneys, indicating that the radionuclide-targeted probe was excreted in the urine. Both the absolute tumor uptake and the target / non-target ratio increased significantly over time.

[0170] Test Example 4

[0171] SPECT imaging experiment

[0172] Tumor-bearing mice were injected with 14 MBq via tail vein 177 Lu-labeled radionuclide targeting probes were subjected to static SPECT scanning at different time points after injection, with CT scanning used for auxiliary positioning. After imaging, the images were reconstructed and the regions of interest (ROI) in the mouse images were delineated to obtain radioactivity counts. The target / non-target ratio of the probe distribution was calculated. 177 Lu]Lu-PKND01, [ 177 Lu]Lu-PKND02 and [ 177 The SPECT imaging results of Lu]Lu-PKSD01 are shown in Figures 19, 20 and 21 respectively. 177 The SPECT imaging results of Lu]Lu-PSMA617 are shown in Figure 22. Within the monitoring time range, [ 177Lu]Lu-PKND01, [ 177 Lu]Lu-PKND02 and [ 177 The enrichment of Lu-PKSD01 nuclide targeted probe in tumor site was significantly higher than that in control group. 177 Lu]Lu-PSMA617, and the imaging contrast is high and the lesion outline is clear, which proves that the radionuclide targeted probe provided by the present invention has a good tumor uptake effect. Over time, the radioactive background in the blood pool and other normal organs is gradually cleared, and the target / non-target ratio continues to increase. The above data show that the radionuclide targeted probe provided by the present invention is better than [ 177 Lu]Lu-PSMA617 has better radionuclide targeted therapy potential.

[0173] Figure 23 shows different specific activities [ 177 SPECT imaging results of tumor uptake (a) and tumor / kidney ratio (b) of Lu]Lu-PKND01. SPECT imaging experiments were used to compare different specific activities [ 177 The distribution of Lu]Lu-PKND01 (by adding different masses of PKND01 into the labeling solution) in tumor-bearing mice. As shown in Figure 23, when the specific activity is 14MBq / nmol, [ 177 Lu]Lu-PKND01 has both good tumor uptake and a high tumor / kidney ratio.

[0174] Test Example 5

[0175] MRI imaging experiment

[0176] The Gd-PKND01 compound was dissolved in PBS 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 after injection. As shown in Figure 24, the arrows indicate the tumor location. PC3-pip tumors showed a gradual increase in signal after injection compared to pre-injection levels, demonstrating that the radionuclide-targeted probe provided by the present invention has a good tumor enrichment effect.

[0177] Test Example 6

[0178] Biodistribution experiments

[0179] Tumor-bearing mice were injected with 1.5 MBq [ 177 Lu]Lu-PKND01 or [ 177 Lu]Lu-PSMA617. Mice were sacrificed at different time points after injection, and tumor and other organ tissue samples were obtained by dissection. The samples were weighed and radioactivity counts were measured using a gamma counter. Results are expressed as percent uptake dose per gram of tissue or organ (%ID / g). 177Lu]Lu-PKND01 and [ 177 The biodistribution results of Lu]Lu-PSMA617 are shown in Figures 25 and 26, respectively. 4 hours after injection, [ 177 Lu] Lu-PKND01 tumor uptake was greater than 80% ID / g. 24 hours after injection, the tumor uptake value reached 150% ID / g and remained at 60% ID / g at 96 hours. 177 Lu]Lu-PSMA617, the nuclide targeting probe provided by the present invention [ 177 Lu]Lu-PKND01 has significantly enhanced tumor uptake and prolonged retention time.

[0180] Test Example 7

[0181] Radionuclide targeted therapy trial

[0182] Tumor-bearing mice were divided into experimental group, 177 There were 6 to 8 mice in each group of the control group and the normal saline group. Each mouse in the experimental group was injected with different doses of [ 177 Lu]Lu-PKND01 or [ 177 Lu]Lu-PKSD01;[ 177 Each mouse in the Lu]Lu-PSMA617 control group was injected with 37MBq[ 177 Lu]Lu-PSMA617; Each mouse in the saline group was injected with the same volume of saline via the tail vein, and the tumor size and body weight were monitored daily. The treatment results are shown in Figure 27. Compared with the saline group, the tumor volume in the experimental group decreased significantly over time. 177 Lu]Lu-PKND01 has obvious tumor therapeutic effect. And low dose [ 177 Lu]Lu-PKND01 or [ 177 Lu]Lu-PKSD01 (9.5 MBq) and high dose [ 177 The therapeutic effect of the probe was comparable to that of Lu]Lu-PSMA617 (37 MBq), indicating that the radionuclide targeting probe provided by the present invention showed good application potential.

[0183] Although the above embodiments provide a detailed description of the present invention, they are only part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on the embodiments of the present invention without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A glutamic acid urea compound, characterized in that: It has the structure shown in Formula I: In the formula I, either R1 or R2 is The other is a group to be labeled, which includes any one of the following structures: X is present or absent, and when X is present, it includes Linker 1 or Linker 2; The linking group 1 includes any one of the following structures: The linking group 2 includes any one of the following structures: wherein n, n′, m, y, z, p and q are independently integers between 0 and 10.

2. The glutamate urea compound according to claim 1, characterized in that: In the formula I, when X is absent, either R1 or R2 is Another one is When X is When either R1 or R2 is Another one is When X is When any one of R1 and R2 is Another one is 3. The method for preparing glutamic acid urea compounds according to claim 1 or 2, characterized in that: (i) When X does not exist or is a linking group 1, the preparation method comprises the following steps: The polypeptide compound 1 is subjected to a first substitution reaction with the R1 active compound to obtain an intermediate 1; The intermediate 1 is subjected to a R3 protecting group removal reaction and then subjected to a second substitution reaction with an R2 active compound to obtain the glutamic acid urea compound; Any one of the R1 active compound and the R2 active compound is When , the other is any of the following structures: X and R1 in the polypeptide compound 1 and intermediate 1 are the same as X and R1 in the formula I; the R3 protecting group includes a Boc protecting group, a DDE protecting group or a Fmoc protecting group; (ii) When X is a linking group 2, the preparation method comprises the following steps: The polypeptide compound 2 is subjected to a third substitution reaction with R1-L to obtain an intermediate 2; The intermediate 2 is subjected to a fourth substitution reaction with the R2 active compound to obtain the glutamic acid urea compound; In the R1-L, L includes any one of the following structures: p and q in said L are the same as those in said 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 R2 active compound is X and R1 in the polypeptide compound 2 and intermediate 2 are the same as X and R1 in formula I.

4. The preparation method according to claim 3, characterized in that: The molar ratio of the polypeptide compound 1 to the R1 active compound is 1:1-5; The first substitution reaction is carried out in the presence of a high boiling point solvent and an alkaline reagent; the high boiling point solvent includes one or more of N-methylpyrrolidone, dimethyl sulfoxide and N,N-dimethylformamide; the alkaline reagent is an organic base; the molar ratio of the polypeptide compound 1 to the alkaline reagent is 1:1-10; the temperature of the first substitution reaction is 25-60°C, and the time is 1-24h.

5. The preparation method according to claim 3, characterized in that: The R3 protecting group removal reaction 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 to 10; The temperature of the R3 protection group removal reaction is 0 to 37°C and the time is 1 to 12 hours; The second substitution reaction is carried out in the presence of a high boiling point solvent and an alkaline reagent; the high boiling point solvent includes one or more of N-methylpyrrolidone, dimethyl sulfoxide and N,N-dimethylformamide; the alkaline reagent is an organic base; the molar ratio of the intermediate 1 to the alkaline reagent is 1:1 to 10; the temperature of the second substitution reaction is 25 to 60°C, and the time is 1 to 24 hours.

6. The preparation method according to claim 3, characterized in that: The molar ratio of the polypeptide compound 2 to R1-L is 1:1-5; The third substitution reaction is carried out in the presence of a solvent, wherein the solvent includes a high boiling point organic solvent and / or PBS; the high boiling point organic solvent includes one or more of N-methylpyrrolidone, dimethyl sulfoxide and N,N-dimethylformamide; the pH value of the PBS is 7 to 8; the temperature of the third substitution reaction is 25 to 60° C., and the time is 1 to 24 hours.

7. The preparation method according to claim 3, characterized in that: The molar ratio of the intermediate 2 to the R2 active compound is 1:1-5; The fourth substitution reaction is carried out in the presence of a high boiling point solvent and an alkaline reagent; the high boiling point solvent includes one or more of N-methylpyrrolidone, dimethyl sulfoxide and N,N-dimethylformamide; the alkaline reagent is an organic base; the molar ratio of the intermediate 2 to the alkaline reagent is 1:1 to 10; the temperature of the fourth substitution reaction is 25 to 60°C, and the time is 1 to 24 hours.

8. A pharmaceutically acceptable salt of a glutamic acid urea compound, characterized in that: The glutamate urea compound is obtained by reacting a glutamate urea compound with an acid or a base; the glutamate urea compound is the glutamate urea compound according to claim 1 or 2 or the glutamate urea compound obtained by the preparation method according to any one of claims 3 to 7.

9. The pharmaceutically acceptable salt of glutamate urea compound according to claim 8, characterized in that: The pharmaceutically acceptable salts include trifluoroacetate, phosphate, formate, acetate, potassium salt or sodium salt.

10. A nuclide targeting probe, characterized in that: The labeled nuclide is obtained by a coordination reaction between the labeled group in the glutamate urea compound according to claim 1 or 2 or the pharmaceutically acceptable salt of the glutamate urea compound according to claim 8 or 9.

11. The nuclide targeting probe according to claim 10, characterized in that: The labeled nuclides include 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.

12. The method for preparing the nuclide targeting probe according to claim 10 or 11, characterized in that: The following steps are involved: A glutamate urea compound or a pharmaceutically acceptable salt thereof is subjected to a coordination reaction with a labeled nuclide to obtain a nuclide targeting probe or a pharmaceutically acceptable salt of the nuclide targeting probe; the glutamate urea compound or a pharmaceutically acceptable salt thereof is the glutamate urea compound described in claim 1 or 2, or the glutamate urea compound prepared by the preparation method described in any one of claims 3 to 7, or a pharmaceutically acceptable salt of the glutamate urea compound described in claim 8 or 9.

13. A pharmaceutically acceptable salt of a nuclide targeting probe, characterized in that: It is obtained by salt-forming reaction of a nuclide targeting probe or a glutamate urea compound; the nuclide targeting probe is the nuclide targeting probe described in any one of claims 10 to 11 or the nuclide targeting probe obtained by the preparation method described in claim 12; the glutamate urea compound is the glutamate urea compound described in claim 1 or 2 or the glutamate urea compound obtained by the preparation method described in any one of claims 3 to 7.

14. A pharmaceutical composition, characterized in that It comprises active components and pharmaceutically acceptable excipients; the active components comprise one or more of glutamate urea compounds, radionuclide targeting probes, pharmaceutically acceptable salts of glutamate urea compounds according to claim 8 or 9, and pharmaceutically acceptable salts of radionuclide targeting probes according to claim 13; the glutamate urea compounds are glutamate urea compounds according to claim 1 or 2, or glutamate urea compounds obtained by the preparation method described in any one of claims 3 to 7; the radionuclide targeting probes are radionuclide targeting probes according to any one of claims 10 to 11, or radionuclide targeting probes obtained by the preparation method described in claim 12.

15. Use of the glutamate urea compound according to claim 1 or 2, the glutamate urea compound obtained by the preparation method according to any one of claims 3 to 7, the pharmaceutically acceptable salt of the glutamate urea compound according to claim 8 or 9, the nuclide targeting probe according to any one of claims 10 to 11, the nuclide targeting probe obtained by the preparation method according to claim 12, the pharmaceutically acceptable salt of the nuclide targeting probe according to claim 13, or the pharmaceutical composition according to claim 14 in the preparation of therapeutic drugs or diagnostic drugs for diseases mediated by PSMA protein.

16. The use according to claim 15, characterized in that The PSMA protein-mediated diseases include tumors.

17. The use according to claim 16, characterized in that The tumor includes one or more of 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, nasopharyngeal carcinoma, head and neck cancer, neuroendocrine tumors and skin melanoma.

18. The use according to claim 15, characterized in that: The treatment methods include radionuclide targeted therapy and / or chemotherapy.

19. The use according to claim 15, characterized in that The diagnostic method includes one or more of single photon emission computed tomography, positron emission tomography and magnetic resonance imaging.

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