WO2025035608A1 - 谷氨酸尿素类化合物及其制备方法和应用、核素靶向探针及其制备方法和应用、药物组合物 - Google Patents

谷氨酸尿素类化合物及其制备方法和应用、核素靶向探针及其制备方法和应用、药物组合物 Download PDF

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WO2025035608A1
WO2025035608A1 PCT/CN2023/128478 CN2023128478W WO2025035608A1 WO 2025035608 A1 WO2025035608 A1 WO 2025035608A1 CN 2023128478 W CN2023128478 W CN 2023128478W WO 2025035608 A1 WO2025035608 A1 WO 2025035608A1
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urea compound
compound
nuclide
preparation
targeting probe
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French (fr)
Inventor
郭志德
程星星
张现忠
刘宏武
王雪琪
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Xiamen University
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Xiamen University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
    • A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/021—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)n-C(=0)-, n being 5 or 6; for n > 6, classification in C07K5/06 - C07K5/10, according to the moiety having normal peptide bonds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link

Definitions

  • 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.
  • PSMA Prostate-specific membrane antigen
  • RLT radioligand therapy
  • mCRPC metastatic castration-resistant prostate cancer
  • PSMA expression has also been found in other solid tumors, so PSMA can be used as a therapeutic target for a variety of tumors.
  • various PSMA-targeted radioligands have been developed, which have shown good prospects in early clinical evaluations.
  • the leader in this field is [ 177Lu ]Lu-PSMA617.
  • One of the strategies to enhance the therapeutic effect 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.
  • albumin binding groups such as iodophenylbutyric acid, Evans blue or ibuprofen can be modified into PSMA probes to achieve higher tumor uptake.
  • this strategy often also increases the irradiation dose of 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.
  • Deferasirox is an oral iron chelator, which is often used to reduce the iron content in patients with transfusion-dependent anemia and non-transfusion-dependent thalassemia.
  • DFX Deferasirox
  • how to modify deferasirox so that it can have better tumor targeting, higher lesion enrichment rate, and better disease treatment effect is a difficult problem facing researchers.
  • 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 has on the pharmacokinetic properties of the probe after hybridization with other receptor targeting groups, and its role in the field of radionuclide targeted diagnosis and treatment needs to be explored.
  • the object of the present invention is to provide 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.
  • the glutamate urea compounds and pharmaceutically acceptable salts thereof, radionuclide targeting probes and pharmaceutically acceptable salts thereof provided by the present invention all have PSMA targeting performance, have long target organ retention time, high tumor uptake dose and low background.
  • the present invention provides the following technical solutions.
  • the present invention provides a glutamic acid urea compound having a structure shown in Formula I:
  • 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:
  • n, n′, m, y, z, p and q are independently integers between 0 and 10.
  • the present invention provides a method for preparing the glutamic acid urea compound described in the above technical scheme.
  • the polypeptide compound 1 is subjected to a first substitution reaction with the R 1 active compound to obtain an intermediate 1;
  • the intermediate 1 is subjected to a deprotection reaction of the R 3 protecting group and then subjected to a second substitution reaction with an R 2 active compound to obtain the glutamic acid urea compound;
  • R1 active 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 R 1 in the polypeptide compound 1 and intermediate 1 are the same as X and R 1 in the formula I;
  • the R 3 protecting group includes a Boc protecting group, a DDE protecting group or a Fmoc protecting group;
  • the polypeptide compound 2 is subjected to a third substitution reaction with R 1 -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;
  • L includes any one of the following structures:
  • R 1 in the R 1 -L is When R 1 in the R 1 -L is When the R2 active compound is any one of the following structures:
  • R 1 in the R 1 -L is the group to be labeled
  • the R 2 active compound is
  • X and R 1 in the polypeptide compound 2 and intermediate 2 are the same as X and R 1 in formula I.
  • the present invention provides a pharmaceutically acceptable salt of a glutamate urea compound, which is obtained by reacting a glutamate urea compound with an acid or a base; the glutamate urea compound is the glutamate urea compound described in the above technical scheme or the glutamate urea compound prepared by the preparation method described in the above technical scheme.
  • the present invention provides a nuclide targeting probe, which is obtained by a coordination reaction between a group to be labeled in the glutamate urea compound described in the above technical solution or a pharmaceutically acceptable salt of the glutamate urea compound described in the above technical solution and a labeled nuclide.
  • the labeled nuclide includes at least one of18F , 47Sc, 64Cu , 67Cu , 67Ga , 68Ga , 89Zr , 86Y , 89Sr , 90Y , 99mTc , 105Rh , 109Pd , 111In , 119Sb, 149Tb , 153Sm , 157Gd , 161Tb , 166Ho , 177Lu , 186Re , 188Re , 201Tl , 203Pb , 212Pb , 212Bi , 213Bi , 223Ra , 227Th and 225Ac .
  • the present invention provides a method for preparing the nuclide targeting probe described in the above technical scheme, 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 scheme or the glutamate urea compound obtained by the preparation method described in the above technical scheme or the pharmaceutically acceptable salt of the glutamate urea compound described in the above technical scheme.
  • 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 glutamate urea compound;
  • the nuclide targeting probe is the nuclide targeting probe described in the above technical scheme or the nuclide targeting probe prepared by the preparation method described in the above technical scheme;
  • the glutamate urea compound is the glutamate urea compound described in the above technical scheme or the glutamate urea compound prepared by the preparation method described in the above technical scheme.
  • 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 scheme;
  • the glutamate urea compound is the glutamate urea compound described in the above technical scheme or the glutamate urea compound obtained by the preparation method described in the above technical scheme;
  • the radionuclide targeting probe is the radionuclide targeting probe described in the above technical scheme or the radionuclide targeting probe obtained by the preparation method described in the above technical scheme.
  • the present invention provides use of the glutamate urea compounds described in the above technical scheme, the glutamate urea compounds prepared by the preparation method described in the above technical scheme, the pharmaceutically acceptable salts of the glutamate urea compounds described in the above technical scheme, the nuclide targeting probe described in the above technical scheme, the nuclide targeting probe prepared by the preparation method described in the above technical scheme, the pharmaceutically acceptable salts of the nuclide targeting probe described in the above technical scheme, or the pharmaceutical composition described in the above technical scheme in the preparation of therapeutic drugs or diagnostic drugs for PSMA protein-mediated diseases.
  • the PSMA protein-mediated disease includes tumors.
  • the glutamic acid urea compound provided by the present invention is a compound modified by deferasirox, which has a It has certain targeting ability, and has been approved as an iron chelator for clinical use, and has good safety.
  • the present invention modifies the structure of deferasirox into the PSMA targeting probe, significantly improving the uptake value of the probe at the target site and prolonging the retention time of the probe at the target site.
  • the glutamate urea compound provided by the present invention can be applied to the labeling of a variety of diagnostic and therapeutic nuclides, and can also be used to construct an imaging treatment platform based on diagnostic and therapeutic nuclides, and has a good application prospect in the preparation of therapeutic drugs or diagnostic drugs for PSMA protein-mediated diseases.
  • the glutamic acid urea compound having the structure shown in Formula I provided by the present invention can be chelated with the nuclide through the labeled group to form a PSMA-targeted nuclide probe with high affinity and high specificity, and has the characteristics of strong labeling ability, short labeling time, high labeling yield, etc., which is conducive to the commercial application and clinical promotion of the nuclide targeted probe.
  • the nuclide targeted probe provided by the present invention has suitable metabolic kinetic properties (different pharmacokinetic properties) and high lesion uptake and retention time, and has excellent diagnostic and therapeutic effects on PSMA protein-mediated diseases, and is a nuclide targeted diagnostic and therapeutic drug with great application prospects.
  • the absolute value of the uptake of the 177 Lu-labeled nuclide targeted probe provided by the present invention in the tumor is 4 to 5 times that of [ 177 Lu] Lu-PSMA617 (one of the current gold standards), which is a nuclide targeted therapeutic drug with great application prospects, overcoming the defects of the existing small molecule PSMA being too fast in metabolism and short in target organ retention time, improving the effect of PSMA nuclide targeted therapy, and having the potential for clinical promotion and application.
  • the radionuclide targeted probe provided by the present invention is distributed in the body to form a concentration difference.
  • an image is formed after reconstruction, which can provide diagnostic information for the disease and achieve the effect of integrated diagnosis and treatment.
  • an image is formed after reconstruction, which can provide diagnostic information for the disease and achieve the effect of integrated diagnosis and treatment.
  • it is helpful to obtain a better target/non-target ratio and enhance the uptake of radionuclide targeted probes in tumors.
  • FIG1 is a mass spectrometry identification spectrum of compound PKND01
  • FIG2 is a mass spectrometry identification spectrum of compound PKND02
  • FIG3 is a mass spectrometry identification spectrum of compound PKSD01
  • FIG4 is a mass spectrometry identification spectrum of compound PKSD02
  • FIG5 is a mass spectrometry identification spectrum of compound PKSP 2 D01
  • FIG6 is a mass spectrometry identification spectrum of compound Gd-PKND01
  • FIG7 is the HPLC identification spectra of compounds PKND01 (a), PKND02 (b), PKSD01 (c), PKSD02 (d) and PKSP 2 D01 (e);
  • FIG8 is a HPLC identification diagram of probe Gd-PKND01
  • FIG9 is a graph showing the radiochemical purity HPLC identification of probes [ 68 Ga]Ga-PKND01 (a), [ 68 Ga]Ga-PKND02 (b), [ 68 Ga]Ga-PKSD01 (c) and [ 68 Ga]Ga-PKSP 2 D01 (d);
  • FIG10 is a graph showing the radiochemical purity HPLC identification of probes [ 177 Lu]Lu-PKND01 (a), [ 177 Lu]Lu-PKND02 (b) and [ 177 Lu]Lu-PKSD01 (c);
  • FIG11 shows the stability HPLC identification results of probes [ 68 Ga]Ga-PKND01 (a), [ 68 Ga]Ga-PKND02 (b) and [ 68 Ga]Ga-PKSD01 (c);
  • FIG12 shows the stability HPLC identification results of probes [ 177 Lu]Lu-PKND01 (a), [ 177 Lu]Lu-PKND02 (b) and [ 177 Lu]Lu-PKSD01 (c);
  • FIG13 shows the cell uptake and inhibition results of probes [ 177 Lu]Lu-PKND01 (a) and [ 177 Lu]Lu-PKND02 (b);
  • FIG14 shows the cell uptake and inhibition results of probes [ 177 Lu]Lu-PKSD01 (a) and [ 177 Lu]Lu-PKSD02 (b);
  • FIG15 shows the PET imaging results of [ 68 Ga]Ga-PKND01 (a) and the quantitative uptake values of the tissue of interest (b);
  • FIG16 shows the PET imaging results of [ 68 Ga]Ga-PKND02 (a) and the quantitative uptake values of the tissue of interest (b);
  • FIG17 shows the PET imaging results of [ 68 Ga]Ga-PKSD01 (a) and the quantitative uptake values of the tissue of interest (b);
  • FIG18 shows the PET imaging results of [ 68 Ga]Ga-PKSP 2 D01 (a) and the quantitative uptake value of the tissue of interest (b);
  • FIG19 shows the SPECT imaging results (a) and target/non-target ratio (b) of [ 177 Lu]Lu-PKND01;
  • FIG20 shows the SPECT imaging results (a) and target/non-target ratio (b) of [ 177 Lu]Lu-PKND02;
  • FIG21 shows the SPECT imaging results (a) and target/non-target ratio (b) of [ 177 Lu]Lu-PKSD01;
  • FIG22 shows the SPECT imaging results of [ 177 Lu]Lu-PSMA617 (a), and the ratio of the uptake counts of [ 177 Lu]Lu-PKND01 and [ 177 Lu]Lu-PKSD01 to [ 177 Lu]Lu-PSMA617 at the tumor site (b);
  • FIG23 shows the SPECT imaging tumor uptake results (a) and tumor/kidney ratio (b) of [ 177 Lu]Lu-PKND01 with different specific activities;
  • FIG24 shows the results of MRI at different time points before and after tail vein injection of Gd-PKND01 in tumor-bearing mice
  • FIG25 shows the biodistribution results of [ 177 Lu]Lu-PKND01 in tumor-bearing mice (a) and the tumor/kidney ratio at different time points (b);
  • FIG26 shows the biodistribution results of [ 177 Lu]Lu-PSMA617 in tumor-bearing mice (a) and the comparison results of the uptake values of [ 177 Lu]Lu-PSMA617 in tumors and [ 177 Lu]Lu-PKND01 (b);
  • FIG. 27 shows the treatment results of [ 177 Lu]Lu-PKND01, [ 177 Lu]Lu-PKSD01 and [ 177 Lu]Lu-PSMA617 in tumor-bearing mice.
  • the present invention provides a glutamic acid urea compound having a structure shown in Formula I:
  • 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:
  • n, n′, m, y, z, p and q are independently integers between 0 and 10.
  • the glutamate urea compound includes PKND01 or PKND02:
  • the glutamate urea compounds include PKSD01, PKSD02, PKSP 2 D01 or PKSP 2 D02:
  • the glutamate urea compounds include PKP 23 D01, PKP 23 D02, PKP 22 D01, PKP 22 D02, PKED01 or PKED02:
  • the present invention provides a method for preparing the glutamic acid urea compound described in the above technical solution.
  • the preparation method comprises the following steps:
  • the polypeptide compound 1 is subjected to a first substitution reaction with the R 1 active compound to obtain an intermediate 1;
  • the intermediate 1 is subjected to a deprotection reaction of the R 3 protecting group and then subjected to a second substitution reaction with an R 2 active compound to obtain the glutamic acid urea compound;
  • R1 active 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 R 1 in the polypeptide compound 1 and intermediate 1 are the same as X and R 1 in the formula I;
  • the R 3 protecting group includes a Boc protecting group, a DDE protecting group or a Fmoc protecting group.
  • the polypeptide compound 1 is subjected to a first substitution reaction with an R 1 active compound to obtain an intermediate 1.
  • the molar ratio of the polypeptide compound 1 to the R 1 active compound is preferably 1:1-5, more preferably 1:2-3.
  • the first substitution reaction is preferably carried out in the presence of a high boiling point solvent and an alkaline agent; the high boiling point solvent preferably includes one or more of N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF); the present invention has no special limitation on the amount of the high boiling point solvent, as long as the polypeptide compound 1 can be dissolved and the first substitution reaction can be smoothly carried out.
  • NMP N-methylpyrrolidone
  • DMSO dimethyl sulfoxide
  • DMF N,N-dimethylformamide
  • the alkaline agent is preferably an organic base, and the organic base preferably includes triethylamine (TEA) and/or N,N-diisopropylethylamine (DIPEA); the molar ratio of the polypeptide compound 1 to the alkaline agent is preferably 1:1-10, more preferably 1:3-5.
  • 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 1-24h, more preferably 5-12h.
  • 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.
  • 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 min: 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.
  • the present invention After obtaining the intermediate 1, the present invention performs a deprotection reaction of the R 3 protecting group on the intermediate 1 and then performs a second substitution reaction with an R 2 active compound to obtain the glutamic acid urea compound.
  • the R 3 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 has no special restrictions on the amount of the hydrazine hydrate solution and trifluoroacetic acid, as long as the protecting group (DDE, Boc or Fmoc) can be removed.
  • the temperature of the R 3 protecting group removal reaction is preferably 0 to 37°C, more preferably 25°C, and the time of the R 3 protecting group removal reaction is preferably 1 to 12h, more preferably 2 to 5h.
  • 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 will not be described in detail here.
  • the preparation route of the glutamic acid urea compound is as follows:
  • the present invention provides a method for preparing the glutamic acid urea compound described in the above technical solution.
  • the preparation method comprises the following steps:
  • the polypeptide compound 2 is subjected to a third substitution reaction with R 1 -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;
  • L includes any one of the following structures:
  • R 1 in the R 1 -L is When R 1 in the R 1 -L is When the R2 active compound is any one of the following structures:
  • R 1 in the R 1 -L is the group to be labeled
  • the R 2 active compound is
  • X and R 1 in the polypeptide compound 2 and intermediate 2 are the same as X and R 1 in formula I.
  • the polypeptide compound 2 is subjected to a third substitution reaction with R 1 -L to obtain an intermediate 2.
  • the R 1 -L preferably includes DFX-MAL, DOTA-MAL or DFX-P2-MAL:
  • the molar ratio of the polypeptide compound 2 to R 1 -L is preferably 1:1-5, more preferably 1:1.5-2.
  • the third substitution reaction is preferably carried out in the presence of a solvent, 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 7-8, more preferably 7.4-7.6; the present invention has a solvent
  • NMP N-methylpyrrolidone
  • DMSO dimethyl sulfoxide
  • DMF N,N-dimethylformamide
  • the pH value of the PBS is preferably 7-8, more preferably 7.4-7.6
  • the present invention has a solvent
  • the amount is not particularly limited, as long as it can dissolve
  • the present invention conducts a fourth substitution reaction between the intermediate 2 and the R 2 active compound to obtain the glutamic acid urea compound.
  • the R2 active compound preferably includes DOTA or DFX active compounds with active reactive groups -NHS or -SCN.
  • the molar ratio of the intermediate 2 to the R2 active compound is preferably 1:1 to 5, more preferably 1:2 to 2.5.
  • the conditions of the fourth substitution reaction and the post-treatment after the fourth substitution reaction are preferably the same as the conditions and post-treatment of the first substitution reaction, and will not be repeated here.
  • the present invention provides a pharmaceutically acceptable salt of a glutamic acid urea compound, which is obtained by reacting a glutamic acid urea compound with an acid or a base; the glutamic acid urea compound is the glutamic acid urea compound described in the above technical scheme or the glutamic acid urea compound prepared by the preparation method described in the above technical scheme.
  • the pharmaceutically acceptable salt preferably includes trifluoroacetate, phosphate, formates, acetates, potassium salts or sodium salts.
  • 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 specifically limit the conditions of the reaction, and the conditions of the salt-forming reaction well known to those skilled in the art can be used.
  • the present invention provides a nuclide targeting probe, which is obtained by a coordination reaction between a group to be labeled in the glutamic acid urea compound or a pharmaceutically acceptable salt thereof described in the above technical solution and a labeled nuclide.
  • the labeling nuclide includes at least one of18F , 47Sc, 64Cu , 67Cu , 67Ga , 68Ga , 89Zr , 86Y , 89Sr , 90Y , 99mTc , 105Rh , 109Pd , 111In , 119Sb , 149Tb , 153Sm , 157Gd , 161Tb , 166Ho , 177Lu , 186Re , 188Re , 201Tl , 203Pb , 212Pb , 212Bi , 213Bi , 223Ra , 227Th and 225Ac , more preferably177Lu
  • the present invention provides a method for preparing the nuclide targeting probe described in the above technical solution, comprising the following steps:
  • the glutamate urea compound is subjected to a coordination reaction with a labeled nuclide to obtain a nuclide targeting probe;
  • 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 nuclide targeting probe is preferably prepared by a wet labeling method or a freeze-drying labeling method.
  • the wet labeling method for preparing the nuclide targeting probe preferably includes the following steps: mixing a solution of glutamate urea compound with a labeled nuclide solution, performing coordination reaction and then diluting to obtain an injection solution of the nuclide targeting probe.
  • the solvent in the solution of glutamate urea compounds 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 glutamate urea compounds is preferably 0.001-1000 mg/mL, more preferably 0.01-1 mg/mL.
  • 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 ⁇ g: 1 kBq-1000 GBq, more preferably 20-400 ⁇ g: 0.037-74000 MBq, and further preferably 20-200 ⁇ g: 0.037-7400 MBq.
  • the labeled nuclide solution there is no special limitation on the labeled nuclide solution, and any labeled nuclide solution known to those skilled in the art can be used, such as gadolinium chloride hexahydrate (GdCl 3 ⁇ 6H 2 O) solution, 68 GaCl 3 hydrochloric acid solution or 177 LuCl 3 solution; the 68 GaCl 3 hydrochloric acid solution is preferably obtained by eluting from a germanium gallium generator.
  • Gadolinium chloride hexahydrate GdCl 3 ⁇ 6H 2 O
  • 68 GaCl 3 hydrochloric acid solution or 177 LuCl 3 solution
  • the 68 GaCl 3 hydrochloric acid solution is preferably obtained by eluting from a germanium gallium generator.
  • 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.
  • 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 familiar to those skilled in the art may be adopted, such as natural cooling.
  • the present invention preferably further comprises aseptically filtering the obtained dilution system to obtain an injection of a radionuclide targeted probe.
  • the dilution preferably utilizes physiological saline or water for injection.
  • the radioactivity concentration of the injection of the radionuclide targeted probe is preferably Select 0.037 ⁇ 3700MBq/mL.
  • 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 5.0-6.5, more preferably 5.5-6.0, the base preferably includes a KOH solution, and the concentration of the KOH solution is preferably 1-4 mol/L, more preferably 2-3 mol/L.
  • the preparation of the nuclide targeting probe by freeze-drying labeling method preferably includes the following steps: freeze-drying the solution of glutamic acid urea compound and sealing it to obtain a freeze-dried medicine box; adding a solvent to the freeze-dried medicine box to dissolve it, and then adding the labeled nuclide solution to carry out coordination reaction and dilution to obtain the injection of the nuclide targeting probe.
  • the freeze drying is preferably to pack the solution of glutamic acid urea compound into a freeze-drying container and then freeze-dry it; the present invention has no special restrictions on the freeze-drying conditions, and the freeze-drying conditions familiar to those skilled in the art can be used.
  • the present invention preferably adds auxiliary materials to the freeze-dried medicine box as needed, and the auxiliary materials preferably include at least one of excipients, antioxidants and acid-base regulators.
  • the present invention has no special restrictions on the excipients, antioxidants and acid-base regulators, and the excipients, antioxidants and acid-base regulators familiar to those skilled in the art can be used.
  • the other preparation conditions of the nuclide targeting probe are preferably the same as those of the aforementioned wet labeling method, and will not be repeated here.
  • the radiochemical purity of the injection of the nuclide 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 nuclide 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.
  • the eluents used for the purification are preferably water and anhydrous ethanol in sequence, and the eluent of anhydrous ethanol is collected and the solvent is removed, and then diluted to obtain an injection of a high-purity nuclide targeted probe.
  • the dilution is preferably performed using physiological saline or water for injection.
  • the radioactivity concentration of the injection of the high-purity nuclide targeted probe is preferably 0.037 to 3700 MBq/mL.
  • 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.
  • a pharmaceutically acceptable salt of a nuclide targeting probe which is obtained by a salt-forming reaction of a nuclide targeting probe or a glutamate 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 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 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 adopted.
  • 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 nuclide targeting probe, a pharmaceutically acceptable salt of the glutamate urea compound described in the above technical solution, and a pharmaceutically acceptable salt of the nuclide targeting probe described in the above technical solution;
  • the 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 nuclide targeting probe is the nuclide targeting probe described in the above technical solution or the nuclide targeting probe prepared by the preparation method described in the above technical solution.
  • the present invention has no special restrictions on the pharmaceutically acceptable excipients, and pharmaceutically acceptable excipients well known to those skilled in the art can be used.
  • the dosage form of the pharmaceutical composition is preferably an injection, preferably administered by intravenous injection; when the active component does not contain a nuclide targeting probe and a pharmaceutically acceptable salt thereof, the present invention has no special restrictions on the dosage form and administration method of the pharmaceutical composition, and dosage forms and administration methods well known to those skilled in the art can be used.
  • the present invention also provides 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.
  • 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, bone tissue sarcoma, connective tissue sarcoma, renal cell carcinoma, gastric cancer, pancreatic cancer, nasopharyngeal carcinoma, head and neck cancer, neuroendocrine tumors and skin melanoma.
  • 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.
  • SPECT single photon emission computed tomography
  • PET positron emission tomography
  • MRI magnetic resonance imaging
  • 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 increases from 10% to 90%, and the mobile phase flow rate is 3 mL/min.
  • Compound 2 ESI MS [M+H] + for C 70 H 82 N 10 O 15 , calcd 1302.60, found 1303.25.
  • 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 flow rate of the mobile phase 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 7 a, the purity is 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 increases from 10% to 90%, and the mobile phase flow rate is 3 mL/min.
  • Compound 3 ESI MS [M+H] + for C 65 H 95 N 11 O 19 , calcd 1334.53, found 1335.24.
  • 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 Figure 2; HPLC purity analysis As shown in FIG. 7 b, the purity is 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.
  • 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, as shown in FIG7 c , showed a purity 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.
  • Compound 6 ESI MS [M+H] + for C 63 H 93 N 13 O 20 S, calcd 1384.57, found 1385.48.
  • 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, as shown in FIG7 d , showed 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.
  • Compound 7 ESI MS [M+H]+ for C 75 H 93 N 13 O 19 S, calcd 1512.70, found 1513.53.
  • 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.
  • PKSP 2 D01 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, as shown in FIG7 e, showed a purity greater than 95%.
  • HPLC analysis conditions reverse 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 conditions: 0-30 min: the volume fraction of mobile phase B increases from 10% to 90%, and the mobile phase flow rate is 1 mL/min.
  • the mass spectrometry identification and HPLC identification of chemical purity of Gd-PKND01 are shown in Figures 6 and 8, respectively.
  • Lyophilization method Add a certain amount of buffer (sodium acetate buffer, pH 5.5, 0.4 mL) and 370 MBq of 68 GaCl 3 eluent (eluted from a germanium gallium generator) to a lyophilized drug box containing 20 ⁇ g of glutamic acid urea compounds, react at 90°C for 20 minutes, cool to room temperature, dilute with physiological saline or water for injection, and obtain the radionuclide targeted probe injection solution (the concentration of the radionuclide targeted probe is 110 MBq/mL) by sterile filtration.
  • buffer sodium acetate buffer, pH 5.5, 0.4 mL
  • 370 MBq of 68 GaCl 3 eluent eluted from a germanium gallium generator
  • C18 separation column purification steps Take Sep-PakC18 separation column, activate and elute it with 10mL of anhydrous ethanol and 10mL of water. Dilute the labeling solution with 10mL of water and load it onto the separation column. Rinse the separation column with water to remove unreacted 68 Ga ions, and then elute it with ethanol solution to obtain 68 Ga labeled nuclide targeting probe.
  • the radiochemical purity HPLC identification results of [ 68 Ga]Ga-PKND01, [ 68 Ga]Ga-PKND02, [ 68 Ga]Ga-PKSD01 and [ 68 Ga]Ga-PKSP 2 D01 are shown in FIG9 a, b, c and d, respectively.
  • the radiochemical purity of each radionuclide targeted probe is greater than 95%.
  • 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 anhydrous ethanol and 10mL water. Dilute the labeling solution with 10mL water and load it onto the separation column. Rinse the separation column with water to remove unreacted 177 Lu ions, and then elute it with ethanol solution to obtain 177 Lu labeled nuclide targeting probe.
  • the radiochemical purity HPLC identification results of [ 177 Lu]Lu-PKND01, [ 177 Lu]Lu-PKND02 and [ 177 Lu]Lu-PKSD01 are shown in FIG10 a, b and c, respectively.
  • the radiochemical purity of each probe is greater than 95%.
  • the stability HPLC identification results of [ 68 Ga]Ga-PKND01, [ 68 Ga]Ga-PKND02 and [ 68 Ga]Ga-PKSD01 are shown in Figure 11 a, b and c, respectively; the stability HPLC identification results of [ 177 Lu]Lu-PKND01, [ 177 Lu]Lu-PKND02 and [ 177 Lu]Lu-PKSD01 are shown in Figure 12 a, b and c, respectively.
  • the radiochemical purity of each radionuclide targeted probe was greater than 95%, indicating that it was stable in the specified solution.
  • I organic phase represents the radioactive count measured in the organic phase
  • I aqueous phase represents the radioactive count measured in the aqueous phase.
  • the lipid-water distribution coefficient of each radiolabeled targeting probe was finally determined by calculation. The results are shown in Table 1.
  • the measured radionuclide targeting probes are water-soluble.
  • the above four 117 Lu-labeled nuclide targeting probes are water-soluble, indicating that the 117 Lu-labeled nuclide targeting probes prepared by the present invention can be metabolized by the kidneys and avoid nonspecific uptake of radioactivity by normal tissues.
  • PC3PIP cells with high PSMA expression and PC3flu cells with negative expression were plated in a 24-well plate containing culture medium (containing fetal bovine serum and double antibody) (cell counting plate counted the number of cells, about 200,000/well) and cultured for 24 hours.
  • culture medium containing fetal bovine serum and double antibody
  • the original culture medium was aspirated, washed twice with PBS (500 ⁇ L), and the PBS was aspirated; an equal amount of the probe to be tested diluted with culture medium was added to each well, and incubated at 37°C for 0.5, 1, 2, 4 and 8 hours; after the incubation at each time point was completed, the culture medium was aspirated, and sodium hydroxide (NaOH) solution (500 ⁇ L, 1M) 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 cell uptake percentage.
  • NaOH sodium hydroxide
  • an inhibition group was also set up in this study, that is, an appropriate amount of inhibitor PSMA617 was added to each well of cells before adding the radionuclide-labeled probe, and the cells were incubated at 37°C for 2h and 4h; after the incubation, the radioactive culture medium was aspirated, and sodium hydroxide solution (500 ⁇ L, 1M) 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 cell uptake percentage.
  • each radionuclide targeting probe was significantly taken up in PSMA-positive cells, while the uptake in negative cells was significantly reduced.
  • the uptake of each radionuclide targeting probe in PSMA-positive cells can be inhibited by PSMA617, indicating that the radionuclide targeting probe provided by the present invention has specificity for targeting PSMA protein.
  • the radionuclide targeting probe labeled with 68 Ga and having a radiochemical purity greater than 95% prepared in the embodiment was diluted with physiological saline, and 0.2 mL (1 MBq) of the injection solution was injected through the tail vein of the PC3PIP model mouse.
  • MicroPET imaging was performed at different time points, and the region of interest (ROI) was outlined on the image, and the probe distribution value was obtained by calculation.
  • the PET imaging results of [ 68 Ga]Ga-PKND01, [ 68 Ga]Ga-PKND02, [ 68 Ga]Ga-PKSD01 and [ 68 Ga]Ga-PKSP 2 D01 are shown in Figures 15, 16, 17 and 18, respectively.
  • the radionuclide targeting probe has a higher uptake in the tumor site.
  • the bladder and kidney sites have higher radioactive signals, which means that the radionuclide targeting probe is excreted through urine.
  • the absolute uptake of the tumor and the target/non-target ratio increase significantly.
  • Tumor-bearing mice were injected with 14MBq 177Lu -labeled radionuclide targeting probes through the tail vein.
  • Static SPECT scanning imaging was performed at different time points after injection, and CT scanning was used to assist positioning. After imaging, the images were reconstructed and the mouse images were analyzed. The region of interest (ROI) in the image was delineated to obtain the radioactivity count value, and the target/non-target ratio of the probe distribution was calculated.
  • the SPECT imaging results of [ 177 Lu]Lu-PKND01, [ 177 Lu]Lu-PKND02 and [ 177 Lu]Lu-PKSD01 are shown in Figures 19, 20 and 21, respectively.
  • the SPECT imaging results of the control group [ 177 Lu]Lu-PSMA617 are shown in Figure 22.
  • the enrichment of the radionuclide targeted probes [ 177 Lu]Lu-PKND01, [ 177 Lu]Lu-PKND02 and [ 177 Lu]Lu-PKSD01 in the tumor site was significantly higher than that of the control group [ 177 Lu]Lu-PSMA617, and the imaging contrast was high and the lesion outline was clear, which proved that the radionuclide targeted probe provided by the present invention has a good tumor uptake effect.
  • 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 indicate that the radionuclide targeted probe provided by the present invention has better radionuclide targeted therapeutic potential than [ 177 Lu]Lu-PSMA617.
  • FIG23 shows the SPECT imaging tumor uptake results (a) and tumor/kidney ratio (b) of [ 177Lu ]Lu-PKND01 with different specific activities.
  • the SPECT imaging experiment compares the in vivo distribution of [ 177Lu ]Lu-PKND01 with different specific activities (by adding different masses of PKND01 into the labeling solution) in tumor-bearing mice.
  • the specific activity is 14MBq/nmol
  • [ 177Lu ]Lu-PKND01 has both good tumor uptake and a high tumor/kidney ratio.
  • the Gd-PKND01 compound was dissolved in PBS buffer and injected into tumor-bearing mice via the tail vein at a dose of 11.9 ⁇ mol/kg.
  • MRI imaging was performed at 1, 3, 6, 12, 24, and 48 h after injection.
  • the area indicated by the arrow is the tumor location.
  • the PC3-pip tumor showed a gradually enhanced signal after injection, indicating that the radionuclide targeting probe provided by the present invention has a good enrichment effect at the tumor site.
  • Tumor-bearing mice were injected with 1.5MBq [ 177Lu ]Lu-PKND01 or [ 177Lu ]Lu-PSMA617 via the tail vein.
  • the mice were killed at different time points after injection, and tumor and other organ tissue samples were obtained by dissection.
  • the samples were weighed and the radioactivity counts were measured using a gamma counter. The results were expressed as the percentage of uptake dose per gram of tissue or organ (%ID/g).
  • the biodistribution results of [ 177Lu ]Lu-PKND01 and [ 177Lu ]Lu-PSMA617 are shown in Figures 25 and 26, respectively.
  • the tumor uptake of [ 177Lu ]Lu-PKND01 was greater than 80%ID/g.
  • the tumor uptake value reached 150%ID/g and remained at 60%ID/g at 96h. It can be seen that compared with [ 177 Lu]Lu-PSMA617, the radionuclide targeting probe [ 177 Lu]Lu-PKND01 provided by the present invention has significantly enhanced tumor uptake and prolonged retention time.
  • Tumor-bearing mice were divided into an experimental group, a [ 177 Lu]Lu-PSMA617 control group, and a saline group, with 6 to 8 mice in each group.
  • Each mouse in the experimental group was injected with different doses of [ 177 Lu]Lu-PKND01 or [ 177 Lu]Lu-PKSD01 through the tail vein;
  • each mouse in the [ 177 Lu]Lu-PSMA617 control group was injected with 37MBq [ 177 Lu]Lu-PSMA617 through the tail vein;
  • each mouse in the saline group was injected with the same volume of saline through the tail vein, and the tumor size and body weight changes were monitored every day.
  • the treatment results are shown in Figure 27.
  • the tumor volume of the experimental group decreased significantly over time, and each dose of [ 177 Lu]Lu-PKND01 had a significant tumor treatment effect.
  • the therapeutic effects of low-dose [ 177 Lu]Lu-PKND01 or [ 177 Lu]Lu-PKSD01 (9.5 MBq) and high-dose [ 177 Lu]Lu-PSMA617 (37 MBq) were comparable, indicating that the radionuclide targeted probe provided by the present invention showed good application potential.

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Abstract

本发明属于生物医药技术领域，具体涉及谷氨酸尿素类化合物及其制备方法和应用、核素靶向探针及其制备方法和应用、药物组合物。本发明提供的谷氨酸尿素类化合物以及核素靶向探针具有优良的体内生物学性能，在PSMA蛋白高表达的病灶中具有高的特异性摄取，靶与非靶比值高，非特异性背景活性低，具有显著增强的肿瘤摄取和保留时间，适合用作肿瘤的核素治疗和显像，还可以减少正常组织与器官不必要的放射性损伤，能够克服小分子PSMA靶器官摄取较低、保留时间过短的缺陷，改善靶向PSMA核素治疗和显像效果，具有在临床上推广应用的潜力。

Description

谷氨酸尿素类化合物及其制备方法和应用、核素靶向探针及其制备方法和应用、药物组合物
本申请要求于2023年8月17日提交国家知识产权局、申请号为CN202311035883.5、发明名称为“谷氨酸尿素类化合物及其制备方法和应用、核素靶向探针及其制备方法和应用、药物组合物”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物医药技术领域，具体涉及谷氨酸尿素类化合物及其制备方法和应用、谷氨酸尿素类化合物的药学上可接受的盐、核素靶向探针及其制备方法和应用、核素靶向探针的药学上可接受的盐、药物组合物。
背景技术
前列腺特异性膜抗原(PSMA)是一种跨膜糖蛋白，在大约90％的前列腺癌中过度表达。因此靶向PSMA放射性配体疗法(RLT)已成为转移性去势抵抗性前列腺癌(mCRPC)的一种潜在有价值的治疗策略。此外，在其他的实体瘤中也发现了PSMA的表达，因此，PSMA可作为多种肿瘤的治疗靶点。近年来开发了各种PSMA靶向放射性配体，其在早期临床评估中表现出良好的前景，在该领域处于领先地位的是[177Lu]Lu-PSMA617，目前的多项研究表明，[177Lu]Lu-PSMA617RLT在大量mCRPC患者中具有良好的安全性和有效性。但尽管如此，由于药物在体内代谢较快，单次给药剂量往往较大，且许多患者对放射性配体治疗的反应不足，在治疗期间或治疗后会出现疾病进展。
增强治疗效果的策略之一是改善放射性配体的递送。目前常用的方法是通过将白蛋白结合基团与PSMA靶向放射性配体结合以延长血液停留时间，从而增加肿瘤的摄取剂量。例如，可以将对碘苯基丁酸、伊文思蓝或布洛芬等白蛋白结合基团修饰到PSMA探针中，以实现肿瘤的较高摄取。然而，该策略往往也增加了健康器官和组织(包括肾脏和骨髓)的辐照剂量。因此，必须谨慎调控探针在体内的药代动力学性质。综上所述，开发一种血液循环周期短、肿瘤绝对摄取高、病灶滞留久、非靶器官本底低的PSMA靶向探针具有重要的意义。
地拉罗司(DFX)是一种口服铁螯合剂，常被用于降低输血依赖型贫血和非输血依赖型地中海贫血患者体内铁含量。研究地拉罗司具有一定的肿瘤靶向性，其富集到肿瘤部位后具有抗肿瘤细胞增殖作用，可作为化疗药物，作为抗真菌药物和抗菌药物等的角色亦有报道。但如何对地拉罗司进行改造，使之能够具有更好的肿瘤靶向性、更高的病灶富集率，更好的疾病治疗效果，是摆在科研人员面前的难题。此外，地拉罗司结构在核素靶向药物设计中的价值还未见被开发，其与其他受体靶向基团组合杂交之后对探针药代动力学性质的影响有多大还未可知，其在核素靶向诊疗领域的作用更是有待探索。
发明内容
有鉴于此，本发明的目的在于提供谷氨酸尿素类化合物及其制备方法和应用、谷氨酸尿素类化合物的药学上可接受的盐、核素靶向探针及其制备方法和应用、核素靶向探针的药学上可接受的盐、药物组合物。本发明提供的谷氨酸尿素类化合物及其药学上可接受的盐、核素靶向探针及其药学上可接受的盐均具有PSMA靶向性能，其靶器官保留时间长，肿瘤摄取剂量高且背景低。
为了实现上述发明目的，本发明提供以下技术方案。
本发明提供了一种谷氨酸尿素类化合物，具有式I所示的结构：
所述式I中，R1和R2中任一个为另一个为待标记基团，所述待标记基团包括以下结构中的任意一种：
X存在或不存在，当所述X存在时包括连接基团1或连接基团2；
所述连接基团1包括以下结构中的任意一种：
所述连接基团2包括以下结构中的任意一种：
其中，n、n'、m、y、z、p和q独立地为0～10之间的整数。
本发明提供了上述技术方案所述谷氨酸尿素类化合物的制备方法，
(i)当X不存在或为连接基团1时，所述制备方法包括以下步骤：
将多肽化合物1与R1活性化合物进行第一取代反应，得到中间体1；
将所述中间体1进行脱R3保护基反应后与R2活性化合物进行第二取代反应，得到所述谷氨酸尿素类化合物；
所述R1活性化合物和R2活性化合物中任意一个为 时，另一个为以下结构中的任意一种：
所述多肽化合物1和中间体1中X和R1与所述式I中X和R1相同；R3保护基包括Boc保护基、DDE保护基或Fmoc保护基；
(ii)当X为连接基团2时，所述制备方法包括以下步骤：
将多肽化合物2与R1-L进行第三取代反应，得到中间体2；
将所述中间体2与R2活性化合物进行第四取代反应，得到所述谷氨酸尿素类化合物；
所述R1-L中L包括以下结构中的任意一种：
所述L中p和q与所述连接基团2中相同；
当所述R1-L中的R1为时，所述R2活性化合物为以下结构中的任意一种：
当所述R1-L中的R1为所述待标记基团时，所述R2活性化合物为
所述多肽化合物2和中间体2中X和R1与所述式I中X和R1相同。
本发明提供了一种谷氨酸尿素类化合物的药学上可接受的盐，由谷氨酸尿素类化合物与酸或碱进行反应得到；所述谷氨酸尿素类化合物为上述技术方案所述的谷氨酸尿素类化合物或上述技术方案所述制备方法制得的谷氨酸尿素类化合物。
本发明提供了一种核素靶向探针，由上述技术方案所述的谷氨酸尿素类化合物或上述技术方案所述谷氨酸尿素类化合物的药学上可接受的盐中待标记基团与标记核素进行配位反应得到。
优选的，所述标记核素包括18F、47Sc、64Cu、67Cu、67Ga、68Ga、89Zr、86Y、89Sr，90Y、99mTc、105Rh、109Pd、111In、119Sb、149Tb、153Sm、157Gd、161Tb、166Ho、177Lu、186Re、188Re、201Tl、203Pb、212Pb、212Bi、213Bi、223Ra、227Th和225Ac中的至少一种。
本发明提供了上述技术方案所述核素靶向探针的制备方法，包括以下步骤：将谷氨酸尿素类化合物或其药学上可接受的盐与标记核素进行配位反应，得到核素靶向探针或核素靶向探针的药学上可接受的盐；所述谷氨酸尿素类化合物或其药学上可接受的盐为上述技术方案所述的谷氨酸尿素类化合物或上述技术方案所述制备方法制得的谷氨酸尿素类化合物或上述技术方案所述谷氨酸尿素类化合物的药学上可接受的盐。
本发明提供了一种核素靶向探针的药学上可接受的盐，由核素靶向探针或谷氨酸尿素类化合物进行成盐反应得到；所述核素靶向探针为上述技术方案所述的核素靶向探针或上述技术方案所述制备方法制得的核素靶向探针；所述谷氨酸尿素类化合物为上述技术方案所述的谷氨酸尿素类化合物或上述技术方案所述制备方法制得的谷氨酸尿素类化合物。
本发明提供了一种药物组合物，包括活性组分和药学上可接受的辅料；所述活性组分包括谷氨酸尿素类化合物、核素靶向探针、谷氨酸尿素类化合物药学上可接受的盐和上述技术方案所述的核素靶向探针的药学上可接受的盐中的一种或几种；所述谷氨酸尿素类化合物为上述技术方案所述的谷氨酸尿素类化合物或上述技术方案所述制备方法制得的谷氨酸尿素类化合物；所述核素靶向探针为上述技术方案所述的核素靶向探针或上述技术方案所述制备方法制得的核素靶向探针。
本发明提供了上述技术方案所述的谷氨酸尿素类化合物、上述技术方案所述制备方法制得的谷氨酸尿素类化合物、上述技术方案所述的谷氨酸尿素类化合物的药学上可接受的盐、上述技术方案所述的核素靶向探针、上述技术方案所述制备方法制得的核素靶向探针、上述技术方案所述的核素靶向探针的药学上可接受的盐或上述技术方案所述的药物组合物在制备PSMA蛋白介导的疾病的治疗药物或诊断药物中的应用。
优选的，所述PSMA蛋白介导的疾病包括肿瘤。
本发明提供的谷氨酸尿素类化合物为地拉罗司修饰的化合物，地拉罗司对于肿瘤病灶具 有一定的靶向能力，且其已被批准为一种铁螯合剂应用于临床，具有良好的安全性，本发明将地拉罗司结构修饰到PSMA靶向探针中，显著提高了靶部位对探针的摄取值、延长了探针在靶部位的滞留时间。本发明提供的谷氨酸尿素类化合物可适用于多种诊断及治疗核素的标记，还可以用于构建基于诊疗核素对的显像治疗平台，在制备PSMA蛋白介导的疾病的治疗药物或诊断药物中具有很好的应用前景。
本发明提供的具有式I所示结构的谷氨酸尿素类化合物通过待标记基团能够与核素螯合形成具有高亲和力、高特异性的PSMA靶向核素探针，具有标记能力强、标记时间短、标记产率高等特点，有利于核素靶向探针的商业化应用与临床推广。与现有的PSMA靶向探针相比，本发明提供的核素靶向探针，具有适宜的代谢动力学性质(不同的药代动力学性质)及高的病灶摄取和滞留时间，对PSMA蛋白介导的疾病具有优异的诊断及治疗效果，是非常具有应用前景的核素靶向诊疗药物。如实施例测试结果所示，本发明提供的177Lu标记的核素靶向探针在肿瘤中的摄取绝对值是[177Lu]Lu-PSMA617(现行金标准之一)的4～5倍，是非常具有应用前景的核素靶向治疗药物，克服了现有的小分子PSMA代谢过快以及靶器官保留时间短的缺陷，改善了PSMA核素靶向治疗的效果，具有在临床上推广应用的潜力。除了治疗之外，本发明提供的核素靶向探针在生物体内分布，形成浓度差，如果发出的射线或者磁豫率变化被外面仪器检测到，重建之后形成图像，可为疾病提供诊断信息，达到诊疗一体化的效果。此外，通过调整合适的比活度或药物组合，有助于获得更好的靶/非靶比值，增强核素靶向探针在肿瘤中的摄取。
附图说明
图1为化合物PKND01的质谱鉴定图谱；
图2为化合物PKND02的质谱鉴定图谱；
图3为化合物PKSD01的质谱鉴定图谱；
图4为化合物PKSD02的质谱鉴定图谱；
图5为化合物PKSP2D01的质谱鉴定图谱；
图6为化合物Gd-PKND01的质谱鉴定图谱；
图7为化合物PKND01(a)、PKND02(b)、PKSD01(c)、PKSD02(d)及PKSP2D01(e)的HPLC鉴定图谱；
图8为探针Gd-PKND01的HPLC鉴定图；
图9为探针[68Ga]Ga-PKND01(a)、[68Ga]Ga-PKND02(b)、[68Ga]Ga-PKSD01(c)及[68Ga]Ga-PKSP2D01(d)的放射化学纯度HPLC鉴定图；
图10为探针[177Lu]Lu-PKND01(a)、[177Lu]Lu-PKND02(b)及[177Lu]Lu-PKSD01(c)的放射化学纯度HPLC鉴定图；
图11为探针[68Ga]Ga-PKND01(a)、[68Ga]Ga-PKND02(b)及[68Ga]Ga-PKSD01(c)的稳定性HPLC鉴定结果；
图12为探针[177Lu]Lu-PKND01(a)、[177Lu]Lu-PKND02(b)及[177Lu]Lu-PKSD01(c)的稳定性HPLC鉴定结果；
图13为探针[177Lu]Lu-PKND01(a)及[177Lu]Lu-PKND02(b)的细胞摄取及抑制结果；
图14为探针[177Lu]Lu-PKSD01(a)及[177Lu]Lu-PKSD02(b)的细胞摄取及抑制结果；
图15为[68Ga]Ga-PKND01的PET成像结果(a)及感兴趣组织的摄取定量值(b)；
图16为[68Ga]Ga-PKND02的PET成像结果(a)及感兴趣组织的摄取定量值(b)；
图17为[68Ga]Ga-PKSD01的PET成像结果(a)及感兴趣组织的摄取定量值(b)；
图18为[68Ga]Ga-PKSP2D01的PET成像结果(a)及感兴趣组织的摄取定量值(b)；
图19为[177Lu]Lu-PKND01的SPECT成像结果(a)及靶/非靶比值(b)；
图20为[177Lu]Lu-PKND02的SPECT成像结果(a)及靶/非靶比值(b)；
图21为[177Lu]Lu-PKSD01的SPECT成像结果(a)及靶/非靶比值(b)；
图22为[177Lu]Lu-PSMA617的SPECT成像结果(a)，[177Lu]Lu-PKND01及[177Lu]Lu-PKSD01在肿瘤部位的摄取计数与[177Lu]Lu-PSMA617的比值(b)；
图23为不同比活度[177Lu]Lu-PKND01的SPECT成像肿瘤摄取结果(a)及瘤/肾比值(b)；
图24为荷瘤小鼠尾静脉注射Gd-PKND01前后不同时间点的核磁共振成像结果；
图25为[177Lu]Lu-PKND01在荷瘤小鼠中的生物分布结果(a)及不同时间点瘤/肾的比值(b)；
图26为[177Lu]Lu-PSMA617在荷瘤小鼠中的生物分布结果(a)以及[177Lu]Lu-PSMA617在肿瘤中的摄取值与[177Lu]Lu-PKND01对比结果(b)；
图27为[177Lu]Lu-PKND01、[177Lu]Lu-PKSD01及[177Lu]Lu-PSMA617在荷瘤小鼠中的治疗结果。
具体实施方式
本发明提供了一种谷氨酸尿素类化合物，具有式I所示的结构：
所述式I中，R1和R2中任一个为另一个为待标记基团，所述待标记基团包括以下结构中的任意一种：
X存在或不存在，当所述X存在时包括连接基团1或连接基团2；
所述连接基团1包括以下结构中的任意一种：
所述连接基团2包括以下结构中的任意一种：
其中，n、n'、m、y、z、p和q独立地为0～10之间的整数。
在本发明中，当X不存在时，R1和R2中任一个为另一个为所述谷氨酸尿素类化合物包括PKND01或PKND02：
在本发明中，当所述X为时，R1和R2中任一个为另一个为其中，所述优选为所述谷氨酸尿素类化合物包括PKSD01、PKSD02、PKSP2D01或PKSP2D02：

在本发明中，当X为时，所述R1和R2中任一个为另一个为其中，所述优选为所述谷氨酸尿素类化合物包括PKP23D01、PKP23D02、PKP22D01、PKP22D02、PKED01或PKED02：

本发明提供了上述技术方案所述谷氨酸尿素类化合物的制备方法，当X不存在或为连接基团1时，所述制备方法包括以下步骤：
将多肽化合物1与R1活性化合物进行第一取代反应，得到中间体1；
将所述中间体1进行脱R3保护基反应后与R2活性化合物进行第二取代反应，得到所述谷氨酸尿素类化合物；
所述R1活性化合物和R2活性化合物中任意一个为 时，另一个为以下结构中的任意一种：
所述多肽化合物1和中间体1中X和R1与所述式I中X和R1相同；R3保护基包括Boc保护基、DDE保护基或Fmoc保护基。
若无特殊说明，本发明使用的材料和设备均为本领域市售商品。
本发明将多肽化合物1与R1活性化合物进行第一取代反应，得到中间体1。
在本发明中，所述多肽化合物1与R1活性化合物的摩尔比优选为1：1～5，更优选为1：2～3。在本发明中，所述第一取代反应优选在高沸点溶剂和碱性试剂存在条件下进行；所述高沸点溶剂优选包括N-甲基吡咯烷酮(NMP)、二甲基亚砜(DMSO)和N,N-二甲基甲酰胺(DMF)中的一种或几种；本发明对于所述高沸点溶剂的用量没有特殊限定，能够将多肽化合物1溶解且保证第一取代反应顺利进行即可。在本发明中，所述碱性试剂优选为有机碱，所述有机碱优选包括三乙胺(TEA)和/或N,N-二异丙基乙胺(DIPEA)；所述多肽化合物1与碱性试剂的摩尔比优选为1：1～10，更优选为1：3～5。在本发明中，所述第一取代反应的温度优选为25～60℃，更优选为25～40℃，所述第一取代反应的时间优选为1～24h，更优选为5～12h。
所述第一取代反应后，本发明优选还包括后处理，所述后处理优选包括：将所得第一取代体系进行反相高效色谱柱纯化后冷冻干燥，得到中间体1。在本发明中，所述反相高效色谱柱纯化的条件包括：色谱柱为反相C18半制备柱；流动相A优选为水+0.1％三氟乙酸(TFA)，流动相B优选为乙腈+0.1％TFA；洗脱方式优选为梯度洗脱，所述梯度洗脱的条件优选为0～30min：流动相B体积分数由10％增加至90％，所述流动相的流速优选为3mL/min。本发明对于所述冷冻干燥的温度及时间没有特殊限定，冷冻干燥至恒重(即冻干)即可。
得到中间体1后，本发明将所述中间体1进行脱R3保护基反应后与R2活性化合物进行第二取代反应，得到所述谷氨酸尿素类化合物。
在本发明中，所述脱R3保护基反应优选在水合肼溶液或三氟乙酸存在条件下进行；所述水合肼溶液中水合肼的质量分数优选为1～10％，更优选为3～5％；本发明对于所述水合肼溶液和三氟乙酸的用量没有特殊限定，能够将保护基(DDE、Boc或Fmoc)脱去即可。在本发明中，所述脱R3保护基反应的温度优选为0～37℃，更优选为25℃，所述脱R3保护基反应的时间优选为1～12h，更优选为2～5h。
在本发明中，所述第二取代反应的条件以及第二取代反应之后的后处理均优选与所述第一取代反应的条件以及后处理相同，在此不再一一赘述。
在本发明中，当X不存在或为连接基团1时，所述谷氨酸尿素类化合物的制备路线如下：
本发明提供了上述技术方案所述谷氨酸尿素类化合物的制备方法，当X为连接基团2时，所述制备方法包括以下步骤：
将多肽化合物2与R1-L进行第三取代反应，得到中间体2；
将所述中间体2与R2活性化合物进行第四取代反应，得到所述谷氨酸尿素类化合物；
所述R1-L中L包括以下结构中的任意一种：
所述L中p和q与所述连接基团2中相同；
当所述R1-L中的R1为时，所述R2活性化合物为以下结构中的任意一种：
当所述R1-L中的R1为所述待标记基团时，所述R2活性化合物为
所述多肽化合物2和中间体2中X和R1与所述式I中X和R1相同。
在本发明中，当X为连接基团2时，所述谷氨酸尿素类化合物的制备路线如下：
本发明将多肽化合物2与R1-L进行第三取代反应，得到中间体2。
在本发明中，所述R1-L优选包括DFX-MAL、DOTA-MAL或DFX-P2-MAL：
在本发明中，所述多肽化合物2与R1-L的摩尔比优选为1：1～5，更优选为1：1.5～2。在本发明中，所述第三取代反应优选在溶剂存在条件下进行，所述溶剂优选包括高沸点有机溶剂和/或PBS(磷酸盐缓冲溶液)，更优选为高沸点有机溶剂和PBS的混合溶剂；所述高沸点溶剂优选包括N-甲基吡咯烷酮(NMP)、二甲基亚砜(DMSO)和N,N-二甲基甲酰胺(DMF)中的一种或几种；所述PBS的pH值优选为7～8，更优选为7.4～7.6；本发明对于所述溶剂的 用量没有特殊限定，能够将多肽化合物2溶解且保证第三取代反应顺利进行即可。在本发明中，所述第三取代反应的温度和时间以及第三取代反应之后的后处理优选与所述第一取代反应的温度、时间以及后处理相同，在此不再一一赘述。
得到中间体2后，本发明将所述中间体2与R2活性化合物进行第四取代反应，得到所述谷氨酸尿素类化合物。
在本发明中，所述R2活性化合物优选包括带有活性反应基团-NHS或-SCN的DOTA或DFX活性化合物。在本发明中，所述中间体2与R2活性化合物的摩尔比优选为1：1～5，更优选为1：2～2.5。在本发明中，所述第四取代反应的条件以及第四取代反应之后的后处理均优选与所述第一取代反应的条件和后处理相同，在此不再一一赘述。
本发明提供了一种谷氨酸尿素类化合物的药学上可接受的盐，由谷氨酸尿素类化合物与酸或碱进行反应得到；所述谷氨酸尿素类化合物为上述技术方案所述的谷氨酸尿素类化合物或上述技术方案所述制备方法制得的谷氨酸尿素类化合物。在本发明中，所述药学上可接受的盐优选包括三氟乙酸盐、磷酸盐、甲酸盐、醋酸盐、钾盐或钠盐。在本发明中，所述酸优选包括三氟乙酸、盐酸、甲酸或醋酸；所述碱优选包括氢氧化钾或氢氧化钠。本发明对于所述反应的条件没有特殊限定，采用本领域技术人员熟知的成盐反应的条件即可。
本发明提供了一种核素靶向探针，由上述技术方案所述的谷氨酸尿素类化合物或其药学上可接受的盐中的待标记基团与标记核素进行配位反应得到。在本发明中，所述标记核素包括18F、47Sc、64Cu、67Cu、67Ga、68Ga、89Zr、86Y、89Sr，90Y、99mTc、105Rh、109Pd、111In、119Sb、149Tb、153Sm、157Gd、161Tb、166Ho、177Lu、186Re、188Re、201Tl、203Pb、212Pb、212Bi、213Bi、223Ra、227Th和225Ac中的至少一种，更优选为177Lu、68Ga、64Cu、18F、90Y或225Ac。
本发明提供了上述技术方案所述核素靶向探针的制备方法，包括以下步骤：
将谷氨酸尿素类化合物与标记核素进行配位反应，得到核素靶向探针；所述谷氨酸尿素类化合物为上述技术方案所述的谷氨酸尿素类化合物或上述技术方案所述制备方法制得的谷氨酸尿素类化合物。在本发明中，所述核素靶向探针优选采用湿法标记法或冻干法标记法制备。
在本发明中，采用湿法标记法制备核素靶向探针优选包括以下步骤：将谷氨酸尿素类化合物的溶液与标记核素溶液混合，进行配位反应后稀释，得到核素靶向探针的注射液。
在本发明中，所述谷氨酸尿素类化合物的溶液中的溶剂优选包括缓冲溶液、水和有机溶剂中的一种或几种；所述缓冲溶液优选包括醋酸-醋酸盐溶液或氯化铝-醋酸盐溶液，所述缓冲溶液的pH值优选为3～7，更优选为4～6.5；所述氯化铝-醋酸盐溶液中氯化铝的浓度优选为0.2～1g/L，更优选为0.4g/L；所述醋酸-醋酸盐溶液和氯化铝-醋酸盐溶液中的醋酸盐独立地包括醋酸钠、醋酸钾和醋酸铵中的一种或几种；所述谷氨酸尿素类化合物的溶液的浓度优选为0.001～1000mg/mL，更优选为0.01～1mg/mL。在本发明中，所述谷氨酸尿素类化合物的质量与标记核素溶液中标记核素的放射性活度之比优选为20～400μg：1kBq～1000GBq，更优选为20～400μg：0.037～74000MBq，进一步优选为20～200μg：0.037～7400MBq。在本发明中，本发明对于所述标记核素溶液没有特殊限定，采用本领域技术人员熟知的标记核素溶液即可，具体如六水合氯化钆(GdCl3·6H2O)溶液、68GaCl3盐酸溶液或177LuCl3溶液；所述68GaCl3盐酸溶液优选由淋洗自锗镓发生器得到。
在本发明中，所述配位反应的温度优选为25～100℃，更优选为80～100℃，所述配位反应的时间优选为10～60min，更优选为20～30min。当所述配位反应的温度高于室温时，所述配位反应后本发明优选还包括将所得配位反应体系冷却至室温，本发明对于所述冷却没有特殊限定，采用本领域技术人员熟知的冷却方式即可，具体如自然冷却。所述稀释后，本发明优选还包括将所得稀释体系进行无菌膜过滤，得到核素靶向探针的注射液。在本发明中，所述稀释优选利用生理盐水或注射用水。在本发明中，所述核素靶向探针的注射液的放射浓度优 选为0.037～3700MBq/mL。
在本发明中，当标记核素溶液为六水合氯化钆溶液时，本发明优选先利用碱调节谷氨酸尿素类化合物与六水合氯化钆溶液的混合溶液的pH值至5.0～6.5，更优选为5.5～6.0，所述碱优选包括KOH溶液，所述KOH溶液的浓度优选为1～4mol/L，更优选为2～3mol/L。
在本发明中，采用冻干法标记法制备核素靶向探针优选包括以下步骤：将谷氨酸尿素类化合物的溶液进行冷冻干燥后密封，得到冻干药盒；在所述冻干药盒中加入溶剂进行溶解，然后加入标记核素溶液进行配位反应后稀释，得到核素靶向探针的注射液。在本发明中，所述冷冻干燥优选为将谷氨酸尿素类化合物的溶液分装于冻干容器中后再冷冻干燥；本发明对于所述冷冻干燥的条件没有特殊限定，采用本领域技术人员熟知的冷冻干燥条件即可。本发明优选根据需要在所述冻干药盒中加入辅料，所述辅料优选包括赋形剂、抗氧化剂和酸碱调节剂中的至少一种，本发明对于所述赋形剂、抗氧化剂和酸碱调节剂没有特殊限定，采用本领域技术人员熟知的赋形剂、抗氧化剂和酸碱调节剂即可。在本发明中，所述核素靶向探针的其他制备条件优选与前述湿法标记法相同，在此不再一一赘述。
在本发明中，当所述湿法标记法以及冻干法标记法制备的核素靶向探针的注射液的放射化学纯度低于95％时，优选还包括将所述核素靶向探针的注射液进行纯化，所述纯化优选为Sep-Pak C18分离小柱纯化，所述Sep-Pak C18分离小柱在使用前优选依次利用无水乙醇和水进行活化淋洗。在本发明中，所述纯化采用的洗脱剂优选依次为水和无水乙醇，收集无水乙醇的洗脱液并除去溶剂，然后进行稀释，得到高纯核素靶向探针的注射液。在本发明中，所述稀释优选利用生理盐水或注射用水进行。在本发明中，所述高纯核素靶向探针的注射液的放射性浓度优选为0.037～3700MBq/mL。
本发明提供的制备方法具有标记简单易得、所得核素靶向探针的稳定性好、肿瘤摄取高等优点，适于工业化生产和临床推广。
在本发明中，本发明提供了一种核素靶向探针的药学上可接受的盐，由核素靶向探针或谷氨酸尿素类化合物进行成盐反应得到；所述核素靶向探针为上述技术方案所述的核素靶向探针或上述技术方案所述制备方法制得的核素靶向探针；所述谷氨酸尿素类化合物为上述技术方案所述的谷氨酸尿素类化合物或上述技术方案所述制备方法制得的谷氨酸尿素类化合物。在本发明中，所述药学上可接受的盐优选包括三氟乙酸盐、磷酸盐、甲酸盐、醋酸盐、钾盐或钠盐。本发明对于所述核素靶向探针的药学上可接受的盐的制备方法没有特殊限定，采用本领域技术人员熟知的药学可接受的盐的制备方法即可。
本发明提了一种药物组合物，包括活性组分和药学上可接受的辅料；所述活性组分包括谷氨酸尿素类化合物、核素靶向探针、上述技术方案所述的谷氨酸尿素类化合物药学上可接受的盐和上述技术方案核素靶向探针的药学上可接受的盐中的一种或几种；所述谷氨酸尿素类化合物为上述技术方案所述的谷氨酸尿素类化合物或上述技术方案所述制备方法制得的谷氨酸尿素类化合物；所述核素靶向探针为上述技术方案所述的核素靶向探针或上述技术方案所述制备方法制得的核素靶向探针。本发明对于所述药学上可接受的辅料没有特殊限定，采用本领域技术人员熟知的药学上可接受的辅料即可。在本发明中，当所述活性组分包括核素靶向探针和/或核素靶向探针的药学上可接受的盐时，所述药物组合物的剂型优选为注射剂，优选通过静脉注射给药；当所述活性组分不含核素靶向探针及其药学上可接受的盐时，本发明对于所述药物组合物的剂型和给药方式没有特殊限定，采用本领域技术人员熟知的剂型以及给药方式即可。
本发明还提供提了上述技术方案所述的谷氨酸尿素类化合物、上述技术方案所述制备方法制得的谷氨酸尿素类化合物、上述技术方案所述的谷氨酸尿素类化合物的药学上可接受的盐、上述技术方案所述的核素靶向探针、上述技术方案所述制备方法制得的核素靶向探针、上述技术方案所述的核素靶向探针的药学上可接受的盐或上述技术方案所述的药物组合物在 制备PSMA蛋白介导的疾病的治疗药物或诊断药物中的应用。在本发明中，所述PSMA蛋白介导的疾病优选包括肿瘤；所述肿瘤优选包括前列腺癌、乳腺癌、卵巢癌、肝癌、肺癌、结直肠癌、骨骼组织肉瘤、结缔组织肉瘤、肾细胞癌、胃癌、胰腺癌、鼻咽癌、头颈癌、神经内分泌瘤和皮肤黑色素瘤中的一种或几种。在本发明中，所述诊断的方式优选包括单光子发射计算机断层成像(SPECT)、正电子发射断层成像(PET)和核磁共振成像(MRI)中的一种或几种；所述治疗的方式优选包括核素靶向治疗和/或化学治疗。
为了进一步说明本发明，下面结合附图和实施例对本发明的谷氨酸尿素类化合物及其制备方法和应用、核素靶向探针及其制备方法和应用、药物组合物进行详细地描述，但不能将它们理解为对本发明保护范围的限定。
实施例1
PKND01的合成
(1)化合物2的合成：称取化合物1(3.16μmol)于1.5mL离心管中，加入DMSO(0.5mL)溶解，加入原料DFX-NHS(9.49μmol)，和N,N-二异丙基乙胺(DIPEA，15.82μmol)，在25℃条件下，反应12h，HPLC纯化并冻干(冻干温度-65℃)，得到白色固体化合物2(4mg，收率97％，纯度98％)。HPLC纯化条件：反相C18半制备柱(10mm×250mm)；流动相A：水+0.1％三氟乙酸(TFA)；流动相B：乙腈+0.1％TFA；梯度淋洗条件：0～30min：流动相B体积分数由10％增加至90％，流动相流速为3mL/min。化合物2：ESI MS[M+H]+for C70H82N10O15,calcd 1302.60,found 1303.25。
(2)PKND01的合成：称取化合物2(3.06μmol)于1.5mL离心管中，向其中加入水合肼(0.5mL，质量分数为3％)，在25℃条件下反应2h后，加入DMSO(0.5mL)，加入DOTA-NHS (5.48μmol)和DIPEA(10.98μmol)，在25℃条件下，搅拌12h，HPLC纯化并冻干(冻干温度-65℃)，得到化合物PKND01(1.6mg，收率为47.7％)，并经鉴定纯度大于95％。HPLC纯化条件：反相C18半制备柱(10mm×250mm)；流动相A：水+0.1％三氟乙酸(TFA)；流动相B：乙腈+0.1％TFA；梯度淋洗条件：0～30min：流动相B体积分数由10％增加至90％，流动相流速为3mL/min。PKND01：ESI MS[M+H]+for C76H96N14O20,calcd 1524.69,found 1525.88，如图1所示；HPLC纯度分析如图7中a所示，纯度大于95％。
实施例2
PKND02化合物的合成
(1)化合物3的合成：称取化合物1(3.16μmol)于1.5mL离心管中，加入DMSO(0.5mL)溶解，加入原料DOTA-NHS(7.91μmol)，和N,N-二异丙基乙胺(DIPEA，15.82μmol)的，在25℃条件下，反应12h，HPLC纯化并冻干(冻干温度-65℃)，得到白色固体化合物3(3.7mg，收率88％，纯度98％)。HPLC纯化条件：反相C18半制备柱(10mm×250mm)；流动相A：水+0.1％三氟乙酸(TFA)；流动相B：乙腈+0.1％TFA；梯度淋洗条件：0～30min：流动相B体积分数由10％增加至90％，流动相流速为3mL/min。化合物3：ESI MS[M+H]+for C65H95N11O19,calcd 1334.53,found 1335.24。
(2)PKND02的合成：称取的化合物3(3.06μmol)于1.5mL离心管中，向其中加入水合肼(0.5mL，质量分数为3％)，在25℃条件下反应2h后，加入DMSO(0.5mL)，加入DOTA-NHS(5.34μmol)和DIPEA(6.41μmol)，在25℃条件下，搅拌12h，HPLC纯化并冻干(冻干温度-65℃)，得到化合物PKND02(2mg，收率为61.53％)，并经鉴定纯度大于95％。HPLC纯化条件：反相C18半制备柱(10mm×250mm)；流动相B：乙腈+0.1％TFA；梯度淋洗条件：0～30min：流动相B体积分数由10％增加至90％，流动相流速为3mL/min。PKND02：ESI MS[M+H]+for C76H96N14O20,calcd 1524.69,found 1525.39，如图2所示；HPLC纯度分析 如图7中b所示纯度大于95％。
实施例3
PKSD01化合物的合成
(1)化合物5的合成：称取化合物4(3.03μmol)于1.5mL离心管中，加入DMSO(0.5mL)溶解，加入原料DFX-MAL(6.05μmol)，再加入PBS溶液(0.3mL)，在25℃条件下，反应12h，HPLC纯化并冻干(冻干温度-65℃)，得到白色固体化合物5(2mg，收率48.78％，纯度98％)。HPLC纯化条件：反相C18半制备柱(10mm×250mm)；流动相B：乙腈+0.1％TFA；梯度淋洗条件：0～30min：流动相B体积分数由10％增加至90％，流动相流速为3mL/min。ESI MS[M+H]+for C68H80N12O16S,calcd 1353.52,found 1354.34。
(2)化合物PKSD01的合成：称取的化合物5(2mg,1eq.,1.48μmol)于1.5mL离心管中，加入DMSO(0.5mL)，加入DOTA-NHS(1.85mg,2.5eq.,3.69μmol)和DIPEA(0.7mg,4eq.,5.91μmol)，在25℃条件下，搅拌12h，HPLC纯化并冻干(冻干温度-65℃)，得到化合物PKSD01(1mg，收率为38.91％)，并经鉴定纯度大于95％。HPLC纯化条件：反相C18半制备柱(10mm×250mm)；流动相A：水+0.1％三氟乙酸(TFA)；流动相B：乙腈+0.1％TFA；梯度淋洗条件：0～30min：流动相B体积分数由10％增加至90％，流动相流速为3mL/min。PKSD01：ESI MS[M+H]+for C76H96N14O20,calcd 1738.73,found 1739.70，如图3所示；HPLC纯度分析如图7中c所示纯度大于95％。
实施例4
PKSD02化合物的合成
(1)化合物6的合成：称取化合物4(2.33μmol)于1.5mL离心管中，加入DMSO(0.5mL)溶解，加入原料DOTA-MAL(4.66μmol)，再加入PBS溶液(0.3mL)，在25℃条件下，反应12h，HPLC纯化并冻干(冻干温度-65℃)，得到白色固体化合物6(2.1mg，收率65.01％，纯度98％)。HPLC纯化条件：反相C18半制备柱(10mm×250mm)；流动相B：乙腈+0.1％TFA；梯度淋洗条件：0～30min：流动相B体积分数由10％增加至90％，流动相流速为3mL/min。化合物6：ESI MS[M+H]+for C63H93N13O20S,calcd 1384.57,found 1385.48。
(2)化合物PKSD02的合成：称取的化合物6(1.52μmol)于1.5mL离心管中，加入DMSO(0.5mL)，加入DFX-NHS(3.79μmol)和DIPEA(4.55μmol)，在25℃条件下，搅拌12h，HPLC纯化并冻干(冻干温度-65℃)，得到化合物PKSD02(1mg，收率为37.87％)，并经鉴定纯度大于95％。HPLC纯化条件：反相C18半制备柱(10mm×250mm)；流动相A：水+0.1％三氟乙酸(TFA)；流动相B：乙腈+0.1％TFA；梯度淋洗条件：0～30min：流动相B体积分数由10％增加至90％，流动相流速为3mL/min。PKSD02：ESI MS[M+H]+for C84H106N16O23S,calcd 1739.92,found 1740.63，如图4所示；HPLC纯度分析如图7中d所示纯度大于95％。
实施例5
PKSP2D01化合物的合成
(1)化合物7的合成：称取化合物4(3.5μmol)和DFX-P2-MAL(5.24μmol)于1.5mL离心管中，加入DMSO(0.3mL)和PBS(pH＝7.4)溶解，在25℃条件下，反应12h，HPLC纯化并冻干(冻干温度-65℃)，得到白色固体化合物7(1.3mg，收率24.6％，纯度95％)。HPLC纯化条件：反相C18半制备柱(10mm×250mm)；流动相A：水+0.1％三氟乙酸(TFA)；流动相B：乙腈+0.1％TFA；梯度淋洗条件：0～30min：流动相B体积分数由10％增加至90％，流动相流速为3mL/min。化合物7：ESI MS[M+H]+for C75H93N13O19S,calcd 1512.70,found 1513.53。
(2)PKSP2D01的合成：称取化合物7(0.86μmol)于1.5mL离心管中，向其中加入DOTA-NHS(1.72μmol)和DIPEA(4.3μmol)，在25℃条件下，搅拌12h，HPLC纯化并冻干(冻干温度-65℃)，得到化合物PKSP2D01(1.0mg，收率为59.9％)，并经鉴定纯度大于95％。HPLC纯化条件：反相C18半制备柱(10mm×250mm)；流动相A：水+0.1％三氟乙酸(TFA)；流动相B：乙腈+0.1％TFA；梯度淋洗条件：0～30min：流动相B体积分数由10％增加至90％，流动相流速为3mL/min。PKSP2D01：ESI MS[M+H]+for C91H119N17O26S,calcd 1899.11,found 1899.83，如图5所示；HPLC纯度分析如图7中e所示纯度大于95％。
实施例6
Gd核素标记
将化合物PKND01与六水合氯化钆(GdCl3·6H2O)以1:2的摩尔比例溶于混合溶剂(DMSO:H2O体积比＝1:1)中，后用KOH溶液调节pH至6.0，加热至60℃震荡反应12h，反应完成后通过HPLC进行纯化，得到核素靶向探针Gd-PKND01，其中，化合物PKND01与混合溶剂的用量比为1mg:1mL。
HPLC分析条件：反相C18分析柱(4.6mm×250mm)；流动相A：水+0.1％三氟乙酸(TFA)；流动相B：乙腈+0.1％TFA；梯度淋洗条件：0～30min：流动相B体积分数由10％增加至90％，流动相流速为1mL/min。Gd-PKND01质谱鉴定及化学纯度的HPLC鉴定分别见图6和图8。 ESI MS[M+H]+for C76H93GdN14O20,calcd 1679.91,found 1680.55。
实施例7
68Ga核素标记
湿法：将370MBq 68GaCl3盐酸溶液(淋洗自锗-镓发生器)分别加入到含0.5mL实施例1～5所制备谷氨酸尿素类化合物(20μg)的醋酸-醋酸盐溶液中，置于90℃下反应20min后冷却至室温，用生理盐水或注射用水稀释，并经无菌过滤即得核素靶向探针注射液(核素靶向探针的浓度为110MBq/mL)。
冻干法：将一定量的缓冲液(醋酸钠缓冲液，pH 5.5，0.4mL)及370MBq的68GaCl3淋洗液(淋洗自锗镓发生器)加入到含20μg谷氨酸尿素类化合物的冻干药盒中，90℃下反应20min，冷却至室温，用生理盐水或注射用水稀释，经无菌过滤即得核素靶向探针注射液(核素靶向探针的浓度为110MBq/mL)。
如放射化学纯度低于95％，则可通过C18分离小柱进行纯化以除去未反应的68Ga3+。C18分离小柱纯纯化步骤：取Sep-PakC18分离小柱，先后通过10mL无水乙醇及10mL水进行活化淋洗。用10mL水将标记液稀释后，上样到分离柱上。用水冲洗分离柱除去未反应的68Ga离子，再用乙醇溶液淋洗得到68Ga标记的核素靶向探针。
[68Ga]Ga-PKND01、[68Ga]Ga-PKND02、[68Ga]Ga-PKSD01及[68Ga]Ga-PKSP2D01的放射化学纯度HPLC鉴定结果分别见图9中a、b、c和d，各核素靶向探针的放射化学纯度均大于95％。
实施例8
177Lu核素标记
湿法：将370MBq 177LuCl3溶液分别加入到含0.2mL实施例1～3所制备化合物(20μg)的醋酸-醋酸盐溶液中，90℃下反应20min后冷却至室温，用生理盐水或注射用水稀释，并经无菌过滤即得核素靶向探针注射液(核素靶向探针的浓度为110MBq/mL)。
冻干法：将0.2mL缓冲液(醋酸钠缓冲液，pH＝5.5)及370MBq的177LuCl3溶液加入到含谷氨酸尿素类化合物(20μg)的冻干药盒中，混匀溶解后置于90℃下反应20min后冷却至室温，用生理盐水或注射用水稀释，并经无菌过滤即得核素靶向探针注射液(核素靶向探针的浓度为110MBq/mL)。
如放射化学纯度低于95％，则可通过C18分离小柱进行纯化以除去未反应的177Lu3+。C18分离小柱纯化步骤为：取Sep-PakC18分离小柱，先后通过10mL无水乙醇及10mL水进行活化淋洗。用10mL水将标记液稀释后，上样到分离柱上。用水冲洗分离柱除去未反应的177Lu离子，再用乙醇溶液淋洗得到177Lu标记的核素靶向探针。
[177Lu]Lu-PKND01、[177Lu]Lu-PKND02及[177Lu]Lu-PKSD01的放射化学纯度HPLC鉴定结果分别见图10中a、b和c，各探针的放射化学纯度均大于95％。
测试例1
稳定性及脂水分布性质评价
1、注射液稳定性实验：生理盐水稀释的核素靶向探针在室温下放置不同时间，取样通过HPLC进行分析。HPLC分析条件：反相C18分析柱(4.6mm×250mm)；流动相A：水+0.1％三氟乙酸(TFA)；流动相B：乙腈+0.1％TFA；梯度淋洗条件：0～30min：流动相B体积分数由10％增加至90％，流动相流速为1mL/min。
[68Ga]Ga-PKND01、[68Ga]Ga-PKND02及[68Ga]Ga-PKSD01的稳定性HPLC鉴定结果分别见图11中a、b和c；[177Lu]Lu-PKND01、[177Lu]Lu-PKND02及[177Lu]Lu-PKSD01的稳定性HPLC鉴定结果分别见图12中a、b和c。由图11～12可知，在所测试时间点，各核素靶向探针的放射化学纯度均大于95％，表明其在指定溶液中性质稳定。
2、脂水分布系数(logP)测定：
将100μL稀释后的含放射性核素靶向探针的溶液加入到含有2.9mLPBS和3mL正辛醇混合液的离心管中，涡旋震荡3min之后，10000rpm离心3min，从水相及正辛醇相中各取100μL液体并通过γ-counter测定放射性计数。实验重复三次取平均值。logP的计算公式为：
P＝I有机相/I水相；
其中I有机相代表有机相中测定的放射性计数、I水相代表水相中测定的放射性计数。通过计算，最终测定各放射性标记的靶向探针的脂水分布系数，结果如表1所示，所测核素靶向探针呈现水溶性
表1核素靶向探针的脂水分布系数
由表1可知，上述4个117Lu标记的核素靶向探针呈现水溶性，表明本发明制备的117Lu标记的核素靶向探针能够通过肾脏代谢，避免正常组织放射性的非特异性摄取。
测试例2
细胞摄取及抑制实验
将PSMA高表达的PC3PIP细胞和阴性表达的PC3flu细胞铺在含培养基(含胎牛血清和双抗)的二十四孔板中(细胞计数板计数细胞数，约20万个/孔)培养24h。在摄取实验开始时，吸走原有培养基，用PBS(500μL)洗两次，吸去PBS；每孔加入等量的已用培养基稀释好的待测探针，在37℃条件下，孵育0.5、1、2、4和8h；在各时间点孵育完成后将培养基吸走，向每孔中加入氢氧化钠(NaOH)溶液(500μL，1M)裂解细胞，5min后将裂解的细胞放入一次性离心管中测量放射性计数；将该计数除以加入放射性的总量即得细胞摄取百分比。
为考察探针PSMA靶向特异性，本研究同时设置抑制组，即在加入核素标记探针之前向每孔细胞加入适量的抑制剂PSMA617，在37℃条件下共孵育2h及4h；孵育完成后将放射性培养基吸走，向每孔中加入氢氧化钠溶液(500μL，1M)裂解细胞，5min后将裂解的细胞放入一次性离心管中测量放射性计数；将该计数除以加入放射性的总量即得细胞摄取百分比。
[177Lu]Lu-PKND01、[177Lu]Lu-PKND02、[177Lu]Lu-PKSD01及[177Lu]Lu-PKSD02的细胞摄取及抑制结果如图13～14所示。以4h的结果为例，各核素靶向探针在PSMA阳性表达的细胞中均有显著摄取，而在阴性细胞中摄取明显降低。各核素靶向探针在PSMA阳性表达细胞中的摄取均能被PSMA617抑制，表明本发明提供的核素靶向探针对PSMA蛋白的靶向具有特异性。
测试例3
PET显像实验
实施例制备的放射化学纯度大于95％的68Ga标记的核素靶向探针经生理盐水稀释后，取0.2mL(1MBq)注射液通过PC3PIP模型小鼠尾静脉进行注射，在不同时间点进行MicroPET成像，在图像上勾画感兴趣区(ROI)，经过计算获得探针分布值。[68Ga]Ga-PKND01、[68Ga]Ga-PKND02、[68Ga]Ga-PKSD01及[68Ga]Ga-PKSP2D01的PET成像结果分别如图15、图16、图17及图18所示。由图15～18可知，核素靶向探针在肿瘤部位具有较高的摄取。膀胱及肾脏位置有较高的放射性信号，意味着核素靶向探针经尿液排出。随时间点推移，肿瘤的绝对摄取及靶/非靶比值显著升高。
测试例4
SPECT显像实验
荷瘤模型鼠通过尾静脉注射14MBq 177Lu标记的核素靶向探针，于注射后不同时间点进行静态SPECT扫描成像，并以CT扫描辅助定位。成像结束后对图像进行重建并对小鼠图像 中的感兴趣区(ROI)进行勾画获得放射性计数值，经计算获得探针分布的靶/非靶比值。[177Lu]Lu-PKND01、[177Lu]Lu-PKND02及[177Lu]Lu-PKSD01的SPECT成像结果分别如图19、图20及图21所示。对照组[177Lu]Lu-PSMA617的SPECT成像结果如图22所示。在监测时间范围内，[177Lu]Lu-PKND01、[177Lu]Lu-PKND02及[177Lu]Lu-PKSD01核素靶向探针在肿瘤部位的富集明显高于对照组[177Lu]Lu-PSMA617，且成像对比度高，病灶轮廓清晰，证明本发明提供的核素靶向探针具有良好的肿瘤摄取效果。随时间推移，血池及其他正常脏器中的放射性本底逐渐清除，靶/非靶比值不断提升。以上数据表明，本发明提供的核素靶向探针相对于[177Lu]Lu-PSMA617具有更优的核素靶向治疗潜力。
图23为不同比活度[177Lu]Lu-PKND01的SPECT成像肿瘤摄取结果(a)及瘤/肾比值(b)，通过SPECT成像实验比较不同比活度[177Lu]Lu-PKND01(通过在标记溶液中加入不同质量的PKND01)在荷瘤小鼠的体内分布。由图23可以看出，当比活度为14MBq/nmol时，[177Lu]Lu-PKND01兼具有良好的肿瘤摄取及较高的瘤/肾比值。
测试例5
MRI显像实验
取Gd-PKND01化合物溶于PBS缓冲液中，按11.9μmol/kg的剂量通过尾静脉注射至荷瘤小鼠体内。于注射后1、3、6、12、24、48h时间点进行MRI成像。如图24所示，箭头所指区域为肿瘤位置处，与注射前相比，PC3-pip肿瘤在注射后显示信号逐渐增强，表明了本发明提供的核素靶向探针在肿瘤部位具有较好的富集效果。
测试例6
生物分布实验
荷瘤小鼠通过尾静脉分别注射1.5MBq[177Lu]Lu-PKND01或[177Lu]Lu-PSMA617。在注射后不同时间点将小鼠处死，解剖获得肿瘤及其他脏器组织样本，称重并用γ计数器测量放射性计数。结果以每克组织或器官的百分摄取剂量表示(％ID/g)。[177Lu]Lu-PKND01及[177Lu]Lu-PSMA617的生物分布结果分别如图25及图26所示。注射后4h，[177Lu]Lu-PKND01肿瘤摄取即大于80％ID/g。注射后24h，肿瘤摄取值达到150％ID/g，并在96h依然维持在60％ID/g。可见，相比于[177Lu]Lu-PSMA617，本发明提供的核素靶向探针[177Lu]Lu-PKND01具有显著增强的肿瘤的摄取与延长的滞留时间。
测试例7
核素靶向治疗实验
将荷瘤小鼠分为实验组、[177Lu]Lu-PSMA617对照组和生理盐水组，每组6～8只。实验组每只小鼠通过尾静脉注射不同剂量的[177Lu]Lu-PKND01或[177Lu]Lu-PKSD01；[177Lu]Lu-PSMA617对照组每只小鼠尾静脉注射37MBq[177Lu]Lu-PSMA617；生理盐水组每只小鼠尾静脉注射相同体积的生理盐水，每天监测肿瘤大小和体重变化。治疗结果如图27所示，与生理盐水组相比，实验组肿瘤体积随着时间推移明显减小，各剂量的[177Lu]Lu-PKND01均具有明显的肿瘤治疗效果。且低剂量的[177Lu]Lu-PKND01或[177Lu]Lu-PKSD01(9.5MBq)和高剂量[177Lu]Lu-PSMA617(37MBq)相比疗效相当，说明本发明提供的核素靶向探针显示出良好的应用潜力。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本发明实施例在不经创造性劳动前提下获得其他实施例，这些实施例都属于本发明保护范围。

Claims (19)

  1. 一种谷氨酸尿素类化合物，其特征在于，具有式I所示的结构：
    所述式I中，R1和R2中任一个为另一个为待标记基团，所述待标记基团包括以下结构中的任意一种：
    X存在或不存在，当所述X存在时包括连接基团1或连接基团2；
    所述连接基团1包括以下结构中的任意一种：
    所述连接基团2包括以下结构中的任意一种：
    其中，n、n'、m、y、z、p和q独立地为0～10之间的整数。
  2. 根据权利要求1所述的谷氨酸尿素类化合物，其特征在于，所述式I中，当X不存在时， R1和R2中任一个为另一个为
    当X为时，R1和R2中任一个为另一个为
    当X为时，所述R1和R2中任一个为另一个为
  3. 权利要求1或2所述谷氨酸尿素类化合物的制备方法，其特征在于，
    (i)当X不存在或为连接基团1时，所述制备方法包括以下步骤：
    将多肽化合物1与R1活性化合物进行第一取代反应，得到中间体1；
    将所述中间体1进行脱R3保护基反应后与R2活性化合物进行第二取代反应，得到所述谷氨酸尿素类化合物；
    所述R1活性化合物和R2活性化合物中任意一个为时，另一个为以下结构中的任意一种：
    所述多肽化合物1和中间体1中X和R1与所述式I中X和R1相同；R3保护基包括Boc保护基、DDE保护基或Fmoc保护基；
    (ii)当X为连接基团2时，所述制备方法包括以下步骤：
    将多肽化合物2与R1-L进行第三取代反应，得到中间体2；
    将所述中间体2与R2活性化合物进行第四取代反应，得到所述谷氨酸尿素类化合物；
    所述R1-L中L包括以下结构中的任意一种：
    所述L中p和q与所述连接基团2中相同；
    当所述R1-L中的R1为时，所述R2活性化合物为以下结构中的任意一种：
    当所述R1-L中的R1为所述待标记基团时，所述R2活性化合物为
    所述多肽化合物2和中间体2中X和R1与所述式I中X和R1相同。
  4. 根据权利要求3所述的制备方法，其特征在于，所述多肽化合物1与R1活性化合物的摩尔比为1：1～5；
    所述第一取代反应在高沸点溶剂和碱性试剂存在条件下进行；所述高沸点溶剂包括N-甲基吡咯烷酮、二甲基亚砜和N,N-二甲基甲酰胺中的一种或几种；所述碱性试剂为有机碱；所述多肽化合物1与碱性试剂的摩尔比为1：1～10；所述第一取代反应的温度为25～60℃，时间为1～24h。
  5. 根据权利要求3所述的制备方法，其特征在于，所述脱R3保护基反应在水合肼溶液或三氟乙酸存在条件下进行；所述水合肼溶液中水合肼的质量百分含量为1～10；
    所述脱R3保护基反应的温度为0～37℃，时间为1～12h；
    所述第二取代反应在高沸点溶剂和碱性试剂存在条件下进行；所述高沸点溶剂包括N-甲基吡咯烷酮、二甲基亚砜和N,N-二甲基甲酰胺中的一种或几种；所述碱性试剂为有机碱；所述中间体1与碱性试剂的摩尔比为1：1～10；所述第二取代反应的温度为25～60℃，时间为1～24h。
  6. 根据权利要求3所述的制备方法，其特征在于，所述多肽化合物2与R1-L的摩尔比为1：1～5；
    所述第三取代反应在溶剂存在条件下进行，所述溶剂包括高沸点有机溶剂和/或PBS；所述高沸点有机溶剂包括N-甲基吡咯烷酮、二甲基亚砜和N,N-二甲基甲酰胺中的一种或几种；所述PBS的pH值为7～8；所述第三取代反应的温度为25～60℃，时间为1～24h。
  7. 根据权利要求3所述的制备方法，其特征在于，所述中间体2与R2活性化合物的摩尔比为1：1～5；
    所述第四取代反应在高沸点溶剂和碱性试剂存在条件下进行；所述高沸点溶剂包括N-甲基吡咯烷酮、二甲基亚砜和N,N-二甲基甲酰胺中的一种或几种；所述碱性试剂为有机碱；所述中间体2与碱性试剂的摩尔比为1：1～10；所述第四取代反应的温度为25～60℃，时间为1～24h。
  8. 一种谷氨酸尿素类化合物的药学上可接受的盐，其特征在于，由谷氨酸尿素类化合物与酸或碱进行反应得到；所述谷氨酸尿素类化合物为权利要求1或2所述的谷氨酸尿素类化合物或权利要求3～7任一项所述制备方法制得的谷氨酸尿素类化合物。
  9. 根据权利要求8所述的谷氨酸尿素类化合物的药学上可接受的盐，其特征在于，所述药学上可接受的盐包括三氟乙酸盐、磷酸盐、甲酸盐、醋酸盐、钾盐或钠盐。
  10. 一种核素靶向探针，其特征在于，由权利要求1或2所述的谷氨酸尿素类化合物或权利要求8或9所述谷氨酸尿素类化合物的药学上可接受的盐中待标记基团与标记核素进行配位反应得到。
  11. 根据权利要求10所述的核素靶向探针，其特征在于，所述标记核素包括18F、47Sc、64Cu、67Cu、67Ga、68Ga、89Zr、86Y、89Sr，90Y、99mTc、105Rh、109Pd、111In、119Sb、149Tb、153Sm、157Gd、161Tb、166Ho、177Lu、186Re、188Re、201Tl、203Pb、212Pb、212Bi、213Bi、223Ra、227Th和225Ac中的至少一种。
  12. 权利要求10或11所述核素靶向探针的制备方法，其特征在于，包括以下步骤：
    将谷氨酸尿素类化合物或其药学上可接受的盐与标记核素进行配位反应，得到核素靶向探针或核素靶向探针的药学上可接受的盐；所述谷氨酸尿素类化合物或其药学上可接受的盐为权利要求1或2所述的谷氨酸尿素类化合物或权利要求3～7任一项所述制备方法制得的谷氨酸尿素类化合物或权利要求8或9所述谷氨酸尿素类化合物的药学上可接受的盐。
  13. 一种核素靶向探针的药学上可接受的盐，其特征在于，由核素靶向探针或谷氨酸尿素类化合物进行成盐反应得到；所述核素靶向探针为权利要求10～11任一项所述的核素靶向探针或权利要求12所述制备方法制得的核素靶向探针；所述谷氨酸尿素类化合物为权利要求1或2所述的谷氨酸尿素类化合物或权利要求3～7任一项所述制备方法制得的谷氨酸尿素类化合物。
  14. 一种药物组合物，其特征在于，包括活性组分和药学上可接受的辅料；所述活性组分包括谷氨酸尿素类化合物、核素靶向探针、权利要求8或9所述的谷氨酸尿素类化合物药学上可接受的盐和权利要求13核素靶向探针的药学上可接受的盐中的一种或几种；所述谷氨酸尿素类化合物为权利要求1或2所述的谷氨酸尿素类化合物或权利要求3～7任一项所述制备方法制得的谷氨酸尿素类化合物；所述核素靶向探针为权利要求10～11任一项所述的核素靶向探针或权利要求12所述制备方法制得的核素靶向探针。
  15. 权利要求1或2所述的谷氨酸尿素类化合物、权利要求3～7任一项所述制备方法制得 的谷氨酸尿素类化合物、权利要求8或9所述的谷氨酸尿素类化合物的药学上可接受的盐、权利要求10～11任一项所述的核素靶向探针、权利要求12所述制备方法制得的核素靶向探针、权利要求13所述的核素靶向探针的药学上可接受的盐或权利要求14所述的药物组合物在制备PSMA蛋白介导的疾病的治疗药物或诊断药物中的应用。
  16. 根据权利要求15所述的应用，其特征在于，所述PSMA蛋白介导的疾病包括肿瘤。
  17. 根据权利要求16所述的应用，其特征在于，所述肿瘤包括前列腺癌、乳腺癌、卵巢癌、肝癌、肺癌、结直肠癌、骨骼组织肉瘤、结缔组织肉瘤、肾细胞癌、胃癌、胰腺癌、鼻咽癌、头颈癌、神经内分泌瘤和皮肤黑色素瘤中的一种或几种。
  18. 根据权利要求15所述的应用，其特征在于，所述治疗的方式包括核素靶向治疗和/或化学治疗。
  19. 根据权利要求15所述的应用，其特征在于，所述诊断的方式包括单光子发射计算机断层成像、正电子发射断层成像和核磁共振成像中的一种或几种。
PCT/CN2023/128478 2023-08-17 2023-10-31 谷氨酸尿素类化合物及其制备方法和应用、核素靶向探针及其制备方法和应用、药物组合物 Pending WO2025035608A1 (zh)

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CN118812544A (zh) * 2024-06-19 2024-10-22 厦门大学 一种腺苷a2ar靶向小分子化合物、核素标记探针及其制备方法、应用和药物组合物
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