Compound containing linking group, chelating agent and ligand binding entity as well as preparation method and application thereof

Abstract

The invention relates to a compound comprising a linking group, a chelating agent and a ligand binding entity, a preparation method and application thereof. In particular to a ligand compound of a targeted PSMA antigen and a chelate thereof, and application of the compound and the chelate thereof in diagnosis and treatment of prostatic cancer.

Description

Technical Field

[0001] The present invention relates to a compound comprising a linker, a chelating agent, a ligand binding entity, a preparation method and use thereof, and in particular to a compound binding to prostate specific membrane antigen, which can be used for preparing therapeutic radiopharmaceuticals, and belongs to the field of pharmaceutical chemistry. Background Art

[0002] Prostate cancer is one of the most common malignant tumors affecting men's health, with approximately 1.3 million men newly diagnosed with prostate cancer each year worldwide. In February 2021, the 2020 edition of the Global Cancer Statistics Report published by the WHO International Agency for Research and Development on Cancer showed that there were 1,414,259 new cases of prostate cancer worldwide in 2020, accounting for 7.3% of all malignant tumors in the body, and the incidence rate ranked third after breast cancer and lung cancer. There were 375,304 deaths from prostate cancer, and the mortality rate ranked eighth (Sung H, Ferlay J, Siegel RL, et al. CA Cancer J Clin. 2021; 10.3322 / caac.21660.). Epidemiological data show that the occurrence of prostate cancer has racial and familial genetic tendencies, especially in the United States, the Caribbean, and people of African descent with the highest incidence rate (Rebbeck TR, Devesa SS, Chang BL, et al. Prostate Cancer. 2013; 2013: 560857). In addition, advanced age, obesity, and fat intake are also correlated with the occurrence of prostate cancer.

[0003] The etiology and pathogenesis of prostate cancer are very complex. Early prostate cancer often has no typical clinical manifestations. Patients in the middle and late stages often experience clinical symptoms such as frequent urination, urgency, urinary retention, interrupted urinary flow, and labored urination due to local tumor invasion or obstruction. Therefore, early diagnosis is crucial for the treatment of prostate cancer. Current diagnosis of prostate cancer includes computed tomography (CT), digital rectal examination (DRE), prostate-specific antigen (PSA) levels, prostate magnetic resonance imaging (MRI), prostate biopsy, etc. However, these tests are complex to operate and cannot effectively monitor and manage the disease, and overdiagnosis often occurs. Therefore, there is an urgent need to develop effective tools for early diagnosis and treatment of prostate cancer.

[0004] The main treatments for prostate cancer currently include active monitoring, radical surgery, endocrine therapy, radiotherapy, and chemotherapy. However, almost all patients with advanced prostate cancer will eventually progress to castration-resistant prostate cancer (CRPC) after endocrine therapy, and metastatic castration-resistant prostate cancer (mCRPC) is the main cause of death in patients. Although docetaxel and cabazitaxel have successfully improved the survival rate of CRPC patients as first-line chemotherapy drugs, and androgen receptor blockers (AR) such as abiraterone and enzalutamide have been approved and widely used in clinical practice for the treatment of CRPC, the 5-year survival rate of CRPC patients is still very low, and new treatment methods are urgently needed (Barata PC, Sartor AO. Cancer. 2019 Jun 1; 125(11): 1777-1788.).

[0005] Recent studies have found that prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GDPII) or folate hydrolase I (FOLH1), is located on stain 11 and is a transmembrane 750-amino acid type II glycoprotein (Wang F, Li Z, Feng X, Yang D, Lin M. Prostate Cancer Prostatic Dis. 2022 Mar; 25 (1): 11-26.). PSMA is expressed at low levels in certain non-prostate tissues (such as kidneys, small intestines, and salivary glands), and is abnormally highly expressed in prostate cancer cells. More than 80% of prostate cancer patients highly express PSMA, and its high expression is associated with poor prognosis. The extracellular domain of PSMA is large, making it easy to design targeting molecules. These characteristics make PSMA an ideal target for immune-related therapies (eitner TM, Babich JW, Kelly JM. Transl Oncol. 2022 Aug; 22: 101450.). Currently, anti-tumor therapy using PSMA mainly focuses on PSMA-targeted radioligands, antibody-drug conjugates, antibodies, or α / β radioisotopes, such as 111 In-PSMA is approved by the FDA as an anti-PSMA antibody targeting a cell epitope, calomab pendetide, on the linking of beta-emitting particles 177 Lu-PSMA-617 has been widely used in clinical practice (Garje R, Rumble RB, Parikh RA. J Clin Oncol. 2022 Nov 1; 40(31): 3664-3666.), and has been approved by the FDA for recurrent / metastatic / castration-resistant prostate cancer in recent years. However, due to the macromolecular structure of the antibody and the limited intracellular domain of PSMA, 177Lu-PSMA-617 has a short metabolic half-life in the blood, a short retention time in the tumor, limited uptake by target organs, and frequent administration increases the possibility of adverse reactions. Although existing technologies attempt to increase the half-life of drugs by increasing albumin binding in order to increase the radioactive exposure of tumors, simply extending the half-life of drugs will also greatly increase the radioactive exposure of normal tissues, and there is a risk of serious toxic and side effects; at the same time, drugs that are tightly bound to albumin will also cause the drug to be unable to be effectively enriched in the target tumor targeting area, making it difficult to achieve the expected tumor inhibitory effect. It will continue to circulate in the human body with the blood, and will further increase the risk of radioactive exposure of normal tissues. Therefore, there is still a need for a drug with a longer half-life, better effects, and lower risk of toxic and side effects. Summary of the invention

[0006] The present invention provides a compound represented by formula (I), a pharmaceutically acceptable salt thereof, or an ester thereof, or a solvate thereof (such as a hydrate), or a radiolabeled complex thereof,

[0007]

[0008] n is selected from any integer from 1 to 5,

[0009] m is selected from 8, 9, 10, 11, 12, 13, 14, 15, 16,

[0010] p is selected from any integer from 0 to 6 (e.g., 0, 1, 2, 3, 4, 5, 6), each R1 is independently selected from C1 to C6 alkyl, halogen, C1 to C6 alkoxy,

[0011] R is a chelating group or a chelating group residue.

[0012] In certain embodiments of the present invention, the n is preferably any integer selected from 1 to 3 (eg, 1, 2, 3); in certain specific embodiments of the present invention, the n is 2.

[0013] In certain embodiments of the present invention, m is preferably selected from 11, 12, 13, 14, 15, 16, more preferably 11, 12, 13, 14. In certain embodiments of the present invention, m is selected from 8, 11, 14, 16, preferably 11, 14.

[0014] In certain embodiments of the present invention, the chelating group is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), N,N"-bis[2-hydroxy-5-(carboxyethyl)-benzyl]ethylenediamine-N,N"-diacetic acid (HBED-CC), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7-bis(carboxymethyl)-1,4,7-triazolidine-1-yl) glutaric acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)-glutaric acid (DOTAGA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (N ... 4,7-Triazacyclononanephosphinic acid (TRAP), 1,4,7-triazacyclononane-1-[methyl(2-carboxyethyl)-phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,9,15-tetraazabicyclo[9,3,1]pentadecan-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), N'-{5-[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO), diethyltriaminepentaacetic acid (DTPA). In certain specific embodiments of the present invention, the chelating group is selected from DOTA, NOTA, DOTAGA, and NODAGA. In certain specific embodiments of the present invention, the chelating group is DOTA.

[0015] In certain embodiments of the present invention, the chelating group residue is selected from (DOTA residue), (NOTA residue), (NODAGA residue 1), (NODAGA residue 2), (NODAGA residue 3), (DOTAGA residue 1), (DOTAGA residue 2), (DOTAGA residue 3), (DOTAGA residue 4), (TETA residue), (TE2A residues), (CB-TE2A residues), (PCTA residue 1), (PCTA residue 2), (PAPA residue), (HEHA residue). In certain specific embodiments of the present invention, the chelating group residue is selected from DOTA residue, NOTA residue, DOTAGA residue 1, DOTAGA residue 2, NODAGA residue 1, NODAGA residue 2. In certain specific embodiments of the present invention, the chelating group residue is a DOTA residue.

[0016] The present invention also provides a compound represented by formula (II), a pharmaceutically acceptable salt thereof, or an ester thereof, or a solvate thereof (such as a hydrate), or a radiolabeled complex thereof:

[0017]

[0018] m is selected from 8, 9, 10, 11, 12, 13, 14, 15, 16,

[0019] p is selected from any integer between 0 and 4 (e.g., 0, 1, 2, 3, 4), each R 1 Each is independently selected from C1-C6 alkyl, halogen, C1-C6 alkoxy,

[0020] R is a chelating group residue.

[0021] In certain embodiments of the present invention, m is preferably selected from 11, 12, 13, 14, 15, 16, more preferably 11, 12, 13, 14. In certain embodiments of the present invention, m is selected from 8, 11, 14, 16, preferably 11, 14.

[0022] In certain embodiments of the present invention, the chelating group residue is selected from the group consisting of DOTA residue, NOTA residue, NODAGA residue 1, NODAGA residue 2, NODAGA residue 3, DOTAGA residue 1, DOTAGA residue 2, DOTAGA residue 3, DOTAGA residue 4, TETA residue, TE2A residue, CB-TE2A residue, PCTA residue 1, PCTA residue 2, PAPA residue, HEHA residue. In certain specific embodiments of the present invention, the chelating group residue is selected from the group consisting of DOTA residue, NOTA residue, DOTAGA residue 1, DOTAGA residue 2, NODAGA residue 1, NODAGA residue 2. In certain specific embodiments of the present invention, the chelating group residue is a DOTA residue.

[0023] The present invention also provides a compound represented by formula (III), a pharmaceutically acceptable salt thereof, or an ester thereof, or a solvate thereof (such as a hydrate), or a radiolabeled complex thereof:

[0024]

[0025] n is selected from any integer from 1 to 3,

[0026] m is selected from 8, 9, 10, 11, 12, 13, 14, 15, 16,

[0027] p is selected from any integer between 0 and 4 (e.g., 0, 1, 2, 3, 4), each R 1 Each is independently selected from C1-C6 alkyl, halogen, and C1-C6 alkoxy.

[0028] In certain specific embodiments of the present invention, n is 2.

[0029] In certain embodiments of the present invention, the m is preferably selected from 11, 12, 13, 14, 15, 16, more preferably from 11, 12, 13, 14; in certain specific embodiments of the present invention, the m is selected from 8, 11, 14, 16.

[0030] In certain embodiments of the present invention, the compound represented by any one of the aforementioned formulas (I), (II), (III), its pharmaceutically acceptable salt, or ester, or solvate (e.g., hydrate), or radiolabeled complex thereof, wherein p is 0. In certain embodiments of the present invention, the compound represented by any one of the aforementioned formulas (I), (II), (III), its pharmaceutically acceptable salt, or ester, or solvate (e.g., hydrate), or radiolabeled complex thereof, wherein R 1 Each is independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, fluorine, chlorine, bromine, and iodine.

[0031] In some embodiments of the present invention, the present invention provides compounds as shown in (i) to (ix) below, or pharmaceutically acceptable salts thereof, or esters thereof, or solvates thereof (e.g., hydrates), or radiolabeled complexes thereof:

[0032]

[0033]

[0034]

[0035] In some specific embodiments of the present invention, the present invention provides the following compounds (x) to (xiii), or pharmaceutically acceptable salts thereof, or solvates thereof (such as hydrates), or radiolabeled complexes thereof:

[0036]

[0037]

[0038] After extensive research, the inventors found that in the embodiment of the present invention, the linking group The number of has an important influence on the plasma protein binding, tumor uptake enrichment, and tissue biodistribution spectrum (e.g., tumor and kidney, tumor and liver, tumor and blood) of the compounds of the present invention. The inventors have found that the linking group When the number is too large (for example, when the number is greater than or equal to 6), the affinity of the compound targeting PSMA may be weakened, thereby failing to effectively target and bind to the PSMA ligand. It is also easy to over-enhance the albumin binding ability of the long-chain fatty diacid in the embodiment of the present invention, resulting in the compound being too tightly bound to albumin, increasing the risk of radioactive exposure to the blood circulation system and normal tissues; and when the linking group When the amount is too small (for example, when the amount is 0), the compound will lose its shielding protection for the PSMA-targeted binding part, which may cause the compound to quickly strip albumin and quickly bind to the PSMA ligand, which may not only destroy the original intention of extending the half-life, but also reduce tumor uptake and increase normal tissue uptake. In the embodiment of the present invention, the linking group When the number is 1 to 5, the compound of the present invention can obtain balanced PSMA and albumin affinity, achieve the excellent half-life performance, PSMA binding performance, higher tumor / normal tissue distribution ratio and lower toxic side effects required by the inventor; especially in certain embodiments of the present invention, the linking group When the amount is 2, the compound of the present invention can have more excellent aforementioned efficacy.

[0039] In certain embodiments of the present invention, when the linking group When the number of terminal fatty diacid alkyl groups is 2 and the number of terminal fatty diacid alkyl groups is 13 to 18 (for example, the number of terminal fatty diacid alkyl groups is 13, 14, 15, 16, 17, 18), the compound exhibits excellent solubility and system stability, and has a better half-life and plasma albumin binding ability; in certain embodiments of the present invention, the compound of the present invention also exhibits better plasma albumin binding stability (for example, the linking group The number of the terminal fatty diacid alkyl group is 2 and the number of the terminal fatty diacid alkyl group is 13, 14, 15, 16) of the present invention); in certain embodiments of the present invention, the tumor / normal tissue ratio of the present invention compound is higher and the toxic side effect is smaller; when the connecting group When the number is 2 and the number of terminal fatty diacid alkyl groups is 13 to 16 (for example, the number of terminal fatty diacid alkyl groups is 13, 14, 15, or 16), it has the ability to inhibit tumors more quickly, causing tumors to disappear more quickly and maintaining tumor growth.

[0040] In certain embodiments of the present invention, the compound represented by any one of the above formulae (I), (II), (III), (i), (ii), (iii), (iv), (v), (vi), (vii), (viii), (ix), or a pharmaceutically acceptable salt thereof, or a solvate thereof (e.g., a hydrate), or a radiolabeled complex thereof, wherein the It can be in the trans configuration (e.g. ), It can be an S configuration (e.g. ), the lysine residue and glutamic acid residue may be in S configuration.

[0041] The present invention also provides a radiolabeled complex, wherein the complex is a chelate formed by chelating a compound represented by any one of the above formulas (I), (II), (III), (i), (ii), (iii), (iv), (v), (vi), (vii), (viii), (ix), (x), (xi), (xii), (xiii), a pharmaceutically acceptable salt thereof, or a solvate thereof (such as a hydrate) with a metal radionuclide (also known as a radioactive metal). In the embodiment of the present invention, the metal radionuclide can be selected from 51 Cr, 94 Tc, 94m Tc, 99m Tc, 110m In, 111 In, 113m In, 114m In, 117m Sn, 121 Sn, 67 Ga, 68 Ga, 86 Y. 87 Y. 90 Y. 177 Lu, 186 Re, 187 Re, 188 Re, 62 Cu, 64 Cu, 67 Cu, 55 Co. 57 Co. 58 Co. 60 Co. 43 Sc, 44 Sc, 47 Sc, 48 Sc, 153 Sm, 225 Ac, 213 Bi, 212 Bi, 227 Th,153 Sm, 166 Ho, 152 Gd, 153 Gd, 155 Gd, 157 Gd, 166 Dy, 223 Ra, 224 Ra, 82 Rb, 89 Zr, 59 Fe, 90 In certain embodiments of the present invention, the metal radionuclide is selected from 186 Re, 187 Re, 177 Lu, 68 Ga, 111 In, 90 Y. 153 In certain specific embodiments of the present invention, the metal radionuclide is 177 Lu.

[0042] The present invention also provides a non-radioactive labeled complex, wherein the complex is a chelate formed by chelating a compound represented by any one of the above formulas (I), (II), (III), (i), (ii), (iii), (iv), (v), (vi), (vii), (viii), (ix), (x), (xi), (xii), (xiii), a pharmaceutically acceptable salt thereof, or a solvate thereof (such as a hydrate) with a non-radioactive metal nuclide (also known as a cold metal nuclide). In certain embodiments of the present invention, the non-radioactive metal nuclide is selected from 175 Lu, 113 In, 115 In, 89 Y. 144 Sm, 147 Sm, 148 Sm, 149 Sm, 150 Sm, 152 Sm, 154 Sm, 185 Re, 69 Ga, 71 Ga, 63 Cu, 65 Cu.

[0043] The present invention also covers solutions obtained by any combination, deletion or replacement of the above-mentioned embodiments.

[0044] Another aspect of the present invention is to provide a pharmaceutical composition comprising at least one of the aforementioned compounds, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or at least one of the aforementioned radiolabeled complexes, and at least one pharmaceutically acceptable excipient.

[0045] Another aspect of the present invention is to provide a use of the aforementioned compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a radiolabeled complex thereof, or a pharmaceutical composition thereof for preparing a therapeutic agent or a diagnostic agent. Alternatively, the present invention provides a aforementioned compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a radiolabeled complex thereof, or a pharmaceutical composition thereof, for use as a therapeutic agent or a diagnostic agent. Alternatively, the present invention provides a method of treatment or diagnosis, comprising administering the aforementioned compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a radiolabeled complex thereof, or a pharmaceutical composition thereof to a subject in need thereof, as a method of using it as a therapeutic agent or a diagnostic agent.

[0046] In certain embodiments of the present invention, the therapeutic agent is used to treat cancer, and the diagnostic agent is used to diagnose cancer. In certain specific embodiments of the present invention, the cancer is a solid tumor or a hematological tumor. In certain specific embodiments of the present invention, the cancer includes prostate cancer, pancreatic cancer, kidney cancer or bladder cancer, liver cancer, neuroblastoma, non-Hodgkin's lymphoma. In certain specific embodiments of the present invention, the cancer is prostate cancer.

[0047] definition

[0048] Unless stated otherwise, the following terms used in the specification and claims have the following meanings.

[0049] "Coordination bond" refers to an interaction in which a multi-electron pair donor is bound to a central atom or ion (e.g., a metal nuclide), that is, the multi-electron pair donor supplies electron pairs alone, and the central atom or ion (e.g., a metal nuclide) provides empty orbitals, forming a special bonding form in which the electron pair donor and a central atom or ion (e.g., a metal nuclide) share two or more unshared electron pairs. In the present invention, the metal nuclide can be a radioactive metal nuclide (i.e., a radioactive metal nuclide, a radioactive metal) or a non-radioactive metal nuclide (i.e., a non-radioactive metal nuclide, a cold metal nuclide).

[0050] The term "coordinating atom" refers to a donor atom that can independently donate an electron pair to form a coordination bond.

[0051] "Chelate group" refers to a group with two or more coordinating atoms that can chelate with the same central atom or ion (such as a metal nuclide) through a coordination bond to obtain a ring-shaped substance. The group is connected to the parent compound by a covalent single bond after removing a hydrogen atom from any of its carbon atoms. Common chelate groups include but are not limited to Ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), N,N"-bis[2-hydroxybenzyl]ethylenediamine-N,N"-diacetic acid (HBED), N,N"-bis[2-hydroxy-5-(carboxyethyl)-benzyl]ethylenediamine-N,N"-diacetic acid (HBED-CC), N-2-Hydroxy-5-(tetrafluorophenylethyl)-benzyl-N"-2'-hydroxy-5'-(carboxyethyl)-benzylethylenediamine-N,N"-diacetic acid (HBED-CC-TFP),

[0052] N,N"-bis[3-carboxyl)pyridylmethyl]ethylenediamine (DEDPA), 1,4,7-Triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7-bis(carboxymethyl)-1,4,7-triazolidine-1-yl)glutaric acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)-pentanedioic acid (DOTAGA), 1,4,8,11-Tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA),

[0053] 1,4,8,11-Tetraazacyclotetradecane-1,8-diacetic acid (TE2A), 4,11-Bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6,6,2]hexadecane (CB-TE2A), 1,4,7-Triazacyclononane-1,4,7-trimethylphosphinic acid (TRAP), 1,4,7-Triazacyclononane-1,4,7-tri[methyl(2-hydroxymethyl)phosphinic acid](TRAP-OH), 1,4,7-Triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid](TRAP-Pr), 1,4,7-Triazacyclononane-1-[methyl(2-carboxyethyl)phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid](NOPO), 3,6,9,15-Tetraazabicyclo[9,3,1]pentadecan-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), Diethyltriaminepentaacetic acid (DTPA),

[0054] 1,4,7,10,13-pentaazacyclopentadecane-1,4,7,10,13-pentaacetic acid (PAPA),

[0055] 1,4,7,10,13,16-hexaazacyclooctadecane-1,4,7,10,13,16-hexaacetic acid (HEHA),

[0056] 10-phosphonomethyl-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3AP),

[0057] 10-(2-carboxyethyl)phosphinate methyl-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3APPrA), 6-bis(carboxymethyl)amino-6-methyl-1,4-diazepane-1,4-diacetic acid (AAZTA), etc. The chelating group in the present invention also includes their derivatives (such as carboxylate derivatives).

[0058] "Chelate group residue" refers to a group that is covalently connected to the parent compound by forming an amide, phosphonamide, phosphinyl, etc. after removing any "-OH" group of the carboxyl, phosphonic acid or phosphinic acid group from the chelate group. The "chelate group residue" in the present invention includes but is not limited to (EDTA residue), (DOTA residue),

[0059] (HBED residues), (HBED-CC residue 1), (HBED-CC residue 2), (HBED-CC-TFP residue 1), (HBED-CC-TFP residue 2),

[0060] (HBED-CC-TFP residue 3), (DEDPA residue), (NOTA residue), (NODAGA residue 1), (NODAGA residue 2),

[0061] (NODAGA residue 3), (DOTAGA residue 1),

[0062] (DOTAGA residue 2), (DOTAGA residue 3),

[0063] (DOTAGA residue 4), (TETA residue), (TE2A residues), (CB-TE2A residues), (TRAP residues), (TRAP-OH residue), (TRAP-Pr residue 1), (TRAP-Pr residue 2),

[0064] (PCTA residue 1), (PCTA residue 2), (DTPA residue 1), (DTPA residue 2), (PAPA residue),

[0065] (HEHA residue), etc. The chelating group residue of the present invention also includes their derivatives (eg, carboxylate derivatives).

[0066] "Derivative" refers to a compound or group (such as a chelating group) whose hydrogen atoms or atomic groups (such as carboxyl, amine, hydroxyl, aldehyde, etc.) are replaced by other atoms or atomic groups, which still retains some of its inherent properties. For example, the carboxylate derivative of the chelating group is a derivative in which the carboxyl group in the chelating group is replaced by an ester group.

[0067] In the present invention, the carboxylate derivative may be a fully esterified derivative (i.e., all carboxylic acid groups of the chelating group form carboxylate), or a partially esterified derivative (i.e., at least one carboxyl group in the chelating group is retained without forming an ester, and the remaining carboxylic acid groups form carboxylate). In certain embodiments of the present invention, the carboxylate derivative is a partially esterified derivative.

[0068] "Chelate" (or "complex") refers to a substance with a ring structure obtained by chelating a chelate group or a chelate group residue with the same central atom or ion (such as a metal nuclide) in the form of a coordination bond.

[0069] "Metal complex" (or "metal chelate") refers to a substance with a ring structure obtained by chelating a compound with a metal nuclide (for example, a metal radionuclide or a non-radioactive metal nuclide) through a chelating group or a chelating group residue in the form of a coordination bond.

[0070] "Radionuclide" (or "radioisotope"), also known as unstable nuclide, means an isotope of natural or artificial origin having an unstable neutron to proton ratio, which decomposes with the emission of particles (i.e. protons (α-radiation) or electrons (β-radiation) or electromagnetic radiation (γ-radiation), i.e. the radionuclide undergoes radioactive decay. The radionuclide may be a non-metallic radionuclide (also known as radioactive non-metallic nuclide, e.g. 18 F. 14 C. 13 N. 15 O. 125 I. 211 At, etc.), or a radioactive nuclide of a metal (also called a radioactive metal nuclide, a radioactive metal, such as 99m Tc, 67 Ga, 68 Ga, 225 Ac, 111 In, 177 Lu, etc.). Radioactive non-metallic nuclides can be covalently linked to organic molecules, and radioactive metal nuclides are usually linked to chelate groups in the form of metal ions through coordination bonds to obtain chelates. Chelate groups can be linked to any known radioactive metal nuclides through coordination bonds to obtain chelates.

[0071] The radioactive metal nuclides can be preferably used for cancer / tumor imaging or treatment. Common radioactive metal nuclides include but are not limited to 51 Cr, 94 Tc, 94m Tc, 99m Tc, 110m In, 111 In, 113m In, 114m In, 117m Sn, 121 Sn, 67 Ga, 68 Ga, 86 Y. 89 Y. 90 Y.177 Lu, 186 Re, 188 Re, 64 Cu, 67 Cu, 55 Co. 57 Co. 58 Co. 60 Co. 43 Sc, 44 Sc, 47 Sc, 48 Sc, 225 Ac, 213 Bi, 212 Bi, 227 Th, 153 Sm, 166 Ho, 152 Gd, 153 Gd, 155 Gd, 157 Gd, 166 Dy, 223 Ra, 224 Ra, 82 Rb, 89 Zr, 59 Fe, 90 Sr, etc. The selection of a suitable radiometal nuclide may depend, inter alia, on the chemical structure and chelating capacity of the chelator group, and the intended application (eg, diagnosis and therapy) of the resulting chelate.

[0072] "Radioactive labeled complex" refers to a chelate containing a radioactive nuclide in its structure. The "radioactive labeled complex" in the present invention may be a chelate formed by chelation between a radioactive metal nuclide and the chelating group of the compound of the present invention; it may also be a radioactive complex formed by covalently bonding a radioactive non-metal nuclide to a chelate formed by chelation between the chelating group of the compound of the present invention and a non-radioactive metal nuclide; it may also be a radioactive chelate containing both a radioactive metal nuclide and a radioactive non-metal nuclide to form a chelate formed by a radioactive metal nuclide and the compound of the present invention, and then covalently bonding a radioactive non-metal nuclide.

[0073] The "non-radioactive labeled complex" refers to a substance having a ring structure obtained by chelating a compound with a non-radioactive metal nuclide via a chelating group or a chelating group residue in the form of a coordination bond.

[0074] "Alkyl" refers to a saturated aliphatic chain hydrocarbon group, including straight-chain alkyl and branched-chain alkyl. For example, the C1-C6 alkyl used in the present invention refers to a straight-chain alkyl or branched-chain alkyl consisting of 1 to 6 carbon atoms. Typical alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl, n-hexyl, etc.

[0075] "Alkoxy" refers to -O-alkyl; the C1-C6 alkoxy used in the present invention refers to a straight chain alkoxy or branched chain alkoxy consisting of 1 to 6 carbon atoms. Typical alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentoxy, isopentyl, tert-pentyl, n-hexyl, and the like.

[0076] "Ring" refers to any cyclic covalently closed structure, including, for example, carbocycle (e.g., aromatic ring or alicyclic ring), heterocycle (e.g., aromatic heterocycle or alicyclic heterocycle). Carbocycle refers to a ring consisting of only carbon atoms, and heterocycle refers to a closed structure formed by covalent bonding of carbon atoms and heteroatoms. Depending on the number of rings, a "ring" can be a monocyclic, bicyclic, tricyclic or polycyclic ring. When the ring is bicyclic, tricyclic or polycyclic, the relationship between the rings may include fused rings, spirocyclic rings, and bridged rings.

[0077] "Heteroatom" refers to any atom other than a carbon atom that can be covalently bonded to a carbon atom. Common heteroatoms include, but are not limited to, O, S, N, P, Si, etc.

[0078] "Member" refers to the number of atoms constituting the ring. Typical 5-membered rings include, but are not limited to, cyclopentane, pyrrole, imidazole, thiazole, furan, and thiophene; typical 6-membered rings include, but are not limited to, cyclohexane, pyridine, pyran, pyrazine, thiopyran, pyridazine, pyrimidine, benzene, and the like.

[0079] "Aromatic ring" or "aryl" refers to a completely unsaturated carbon ring whose planar ring has a delocalized π electron system and contains 4n+2 π electrons, where n is an integer. An aromatic ring can be composed of six, eight, ten or more carbon atoms, and can be a monocyclic ring, a bicyclic ring, a tricyclic ring or a polycyclic ring. Common aromatic rings include, but are not limited to, benzene rings, naphthalene rings, phenanthrene rings, anthracene rings, tetraphenyl rings, pyrene rings, pentphenyl rings, etc.

[0080] "Halogen" or "halo" refers to fluorine, chlorine, bromine or iodine.

[0081] "Haloalkyl" means that at least one hydrogen in the alkyl group is replaced by a halogen atom. The C1-C6 haloalkyl used in the present invention refers to a straight-chain alkyl or branched alkyl composed of 1 to 6 carbon atoms, and at least one hydrogen on the alkyl group is arbitrarily replaced by a halogen atom.

[0082] "Haloalkoxy" refers to an alkoxy group in which at least one hydrogen is replaced by a halogen atom. The C1-C6 haloalkoxy group used in the present invention refers to a straight-chain alkyl group or a branched-chain alkyl group consisting of 1 to 6 carbon atoms, and at least one hydrogen on the alkyl group is arbitrarily replaced by a halogen atom.

[0083] "Amine" or "amine" refers to a group having -NR S R T The chemical structure of S , R T Each is independently selected from hydrogen, deuterium, tritium, alkyl, cycloalkyl, common "amino groups" include but are not limited to -NH2, -NHCH3, -N(CH3)2, -NHCH2CH3, -N(CH2CH3)2.

[0084] "Iminyl" or "imine" refers to a group having =NR W The chemical structure of W Selected from hydrogen, deuterium, tritium, C1-C6 alkyl or 3-12 membered cycloalkyl, common imino groups include but are not limited to =NH, =NCH3, =CH2CH3, =NCH(CH3)2.

[0085] "Carbonyl" refers to a group having -COR Z The chemical structure of Z Selected from alkyl, cycloalkyl, heterocycloalkyl, common carbonyl groups include but are not limited to -COCH3, -COCH2CH3, -CO(CH2)2CH3, -COCH(CH3)2.

[0086] "Amide" or "amido" refers to a group having -C(O)NR U R V or -NR U C(O)R V The chemical structure of U , R V Each is independently selected from hydrogen, deuterium, tritium, alkyl, cycloalkyl, heterocycloalkyl, common amide groups include but are not limited to -CONH2, -CONHCH3, -CON(CH3)2, -NHCOH, -NHCOCH3, -N(CH3)COCH3.

[0087] "Ester", "ester group" or "carboxylate" refers to a ester having the formula -C(O)OR a or -OC(O)R b The chemical structure of a , R b Selected from alkyl, cycloalkyl, heterocycloalkyl, common ester groups include but are not limited to -C(O)OCH3, -C(O)OCH2CH3,

[0088] -C(O)O(CH2)2CH3, -C(O)OCH(CH3)2, -OC(O)CH3, -OC(O)CH2CH3, -OC(O)(CH2)2CH3, -OC(O)CH(CH3)2. "Ester" in the present invention refers to an ester derivative formed by condensation of the compound of the present invention with an alcohol, which may be, for example, one carboxyl group to form an ester, two carboxyl groups to form an ester, three carboxyl groups to form an ester, four carboxyl groups to form an ester, five carboxyl groups to form an ester, six carboxyl groups to form an ester, seven carboxyl groups to form an ester, or eight carboxyl groups to form an ester; the ester may be, for example, a methyl ester, an ethyl ester, a n-propyl ester, an isopropyl ester, a n-butyl ester, an isobutyl ester, a tert-butyl ester, etc. The ester of the compound of the present invention may be used as a prodrug of the compound of the present invention (also known as a prodrug, a drug precursor, a prodrug, etc., which refers to a compound obtained by chemically modifying the drug structure, which is inactive or less active in vitro, and releases an active drug through enzymatic or non-enzymatic conversion in vivo to exert its pharmacological effect).

[0089] "Terminal fatty diacid alkyl" refers to a saturated fatty chain hydrocarbon group (i.e., alkyl) and two carboxyl groups, and one of the carboxyl groups removes the "-OH" group and is covalently connected to the parent compound through an amide, ester, etc., and the other carboxyl group is retained as the end of the entire compound structure. For example In the present invention, the "number of terminal fatty diacid alkyl groups" refers to the number of all carbon atoms constituting the terminal fatty diacid alkyl group, that is, the number of alkyl carbons plus the number of carboxyl carbons, specifically the number of alkyl carbons plus two, for example The number of terminal fatty diacid alkyl groups is 10. The number of terminal fatty diacid alkyl groups is 13. The number of terminal fatty diacid alkyl groups is 16.

[0090] "Replacement" refers to that one or more hydrogen atoms in a group are replaced by a corresponding number of substituents independently of one another. It goes without saying that substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible replacements without making too much effort. For example, amino or hydroxyl with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (such as olefinic) bond. Each and independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, hydroxyl, alkoxy, alkylthio, aryloxy, nitro, acyl, halogen, haloalkyl, amino, etc.

[0091] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0092] "Pharmaceutical composition" means a mixture containing one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0093] "Pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0094] As the pharmaceutically acceptable salt, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids and the like can be mentioned.

[0095] "Stereoisomers" refer to isomers caused by the same order of connection between atoms or atomic groups in a molecule, but different arrangements of atoms in space. It can be divided into two categories: conformational isomers, cis-trans isomers, and chiral isomers. Chiral isomers can be divided into two categories: enantiomers and diastereomers. The spatial arrangement of atoms in the structure of a compound in "stereoisomers" is usually represented by a wedge-shaped covalent bond (bold wedge-shaped bond) Dashed wedge key ), where a bold wedge-shaped bond indicates that it faces out of the paper, and a dotted wedge-shaped bond indicates that it faces inward. In the present invention, a straight covalent bond "—" in a compound structure may represent being in the same plane as the paper. In the present invention, when stereoisomers exist in atoms connected by a straight covalent bond, the straight covalent bond indicates that the arrangement of the atoms connected thereto may include being in the same plane as the paper, facing out of the paper, facing inward, or a mixture of various arrangements. For example, in the present invention include Trans configuration (trans), cis configuration, or their mixed configurations.

[0096] Obviously, according to the above contents of the present invention, in accordance with the common technical knowledge and means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0097] Unless otherwise specified, the terms "comprise, comprises and comprising" or their equivalents (contain, contains, containing, include, includes, including) used herein are open-ended expressions and mean that in addition to the listed elements, components and steps, other unspecified elements, components and steps may also be included.

[0098] Unless otherwise indicated, all numbers used herein expressing the amounts of ingredients, measurements or reaction conditions are to be understood as being modified in all cases by the term "about". When connected with a percentage, the term "about" can mean, for example, ±1%, preferably ±0.5%, more preferably ±0.1%.

[0099] Unless the context clearly indicates otherwise, singular terms herein include plural referents and vice versa. Similarly, the word "or" herein is intended to include "and" unless the context clearly indicates otherwise.

[0100] Obviously, based on the above content of this application, according to the common technical knowledge and means in this field, without departing from the above basic technical ideas of this application, other various forms of modifications, replacements or changes can be made.

[0101] The abbreviations in the present invention have the following meanings:

[0102] BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 is the NMR image of Example 3, 1 H NMR (600 MHz, DMSO-d6).

[0104] Figure 2 The labeled compound obtained in 3.3 of Experimental Example 3 177 Lu-Example 1, 177 Lu-Example 2, 177 Lu-Example 3, 177 Radiochemical purity TLC scanning spectrum of Lu-Example 4.

[0105] Figure 3a for 177 Tissue distribution diagram of Lu-Example 1 injection LNCaP clone FGC tumor-bearing mice.

[0106] Figure 3b for 177 Tissue distribution diagram of Lu-Example 2 injection LNCaP clone FGC tumor-bearing mice.

[0107] Figure 3c for 177 Tissue distribution diagram of Lu-Example 3 injection LNCaP clone FGC tumor-bearing mice.

[0108] Figure 3d for 177 Tissue distribution diagram of Lu-Example 4 injection LNCaP clone FGC tumor-bearing mice.

[0109] Figure 3e for 177 Tissue distribution of Lu-PSMA-617 injection in LNCaP clone FGC tumor-bearing mice.

[0110] Figure 4 This is a graph showing the changes in tumor volume in the tumor-bearing mouse model over 40 days. DETAILED DESCRIPTION

[0111] Synthesis method

[0112] The present invention also provides a method for synthesizing the above-mentioned compound. The method for synthesizing the present invention is mainly based on the preparation method reported in chemical literature or uses commercially available chemical reagents as starting materials for related synthesis.

[0113]

[0114]

[0115]

[0116] According to the above synthesis method, N-[fluorenylmethoxycarbonyl]-N 2 -[(2-propenyloxy)carbonyl]-L-lysine reacts with solid phase resin intermediate 9 to obtain intermediate P1B, and then uses piperidine to remove the protecting group "Fmoc-" of intermediate P1B, and then reacts with intermediate L to obtain the intermediate shown in formula (PD), and then removes the protecting group "Alloc-" of the intermediate shown in formula (PD), and then reacts with intermediate (

[0117] uO) o -R'-OH" condensation reaction to obtain the intermediate shown in formula (PF), and then use a solvent to elute the intermediate from the solid phase resin to obtain the intermediate shown in formula (PG), and finally remove the group "tBu" (tert-butyl) to obtain the compound shown in formula (I),

[0118] Wherein, n can be 1 to 5, m can be 8 to 16; R is a chelating group residue; the intermediate "(tBu-O) o-R'-OH" is a "-OH" group that retains any one carboxyl, phosphonic acid or phosphinic acid group, and the remaining "o" carboxyl, phosphonic acid or phosphinic acid groups react with tert-butyl alcohol through an esterification condensation reaction to form a chelating group of a protecting group tert-butyloxy "tBu-O", wherein the "o" corresponds to the number of carboxyl, phosphonic acid or phosphinic acid groups in the chelating group minus 1; the "" (tBu-O) o -R'-" is a chelating group residue formed by the esterification reaction between the carboxyl group and o tert-butyl alcohols to form a protective group tert-butyloxy "tBu-O".

[0119] The compounds of the present invention can be purified by a suitable method, such as reverse phase preparative liquid purification. The compounds of the present invention can be characterized by analytical HPLC and ESI-MS. The analytical HPLC method can be characterized by, for example, Agilent 1260 high performance liquid chromatograph, Dacao UG80 chromatographic column (250×4.6mm, 5μm), flow rate 1.2mL / min, column temperature 30°C, ultraviolet wavelength 254 / 214nm for analytical HPLC characterization. The specific method can be, for example, 1) HPLC method A, 10mmol ammonium bicarbonate aqueous solution as mobile phase A, acetonitrile as mobile phase B, mobile phase elution gradient: from 0min to 12.5min, linearly change from 90% mobile phase A + 10% mobile phase B to 100% mobile phase B, from 12.5min to 14.5min, maintain 100% mobile phase B, from 14.5min to 16.01min, linearly change from 100% mobile phase B to 90% mobile phase A + 10% mobile phase B, from 16.01min to 19min, maintain 90% mobile phase A + 10% mobile phase B; 2) HP LC method B, 0.1% trifluoroacetic acid aqueous solution as mobile phase A, 0.1% trifluoroacetic acid containing acetonitrile solution as mobile phase B, mobile phase elution gradient: 0min-12.5min linearly changes from 90% mobile phase A+10% mobile phase B to 10% mobile phase A+90% mobile phase B, 12.5-16min linearly changes to 100% mobile phase B, 16min-16.01min linearly changes from 100% mobile phase B to 90% mobile phase A+10% mobile phase B, 16.01min-19min maintains 90% mobile phase A+10% mobile phase B. The compounds of the present invention are characterized by HPLC method, wherein R f represents the retention time of the compound on analytical HPLC.

[0120] The structure of the ligand compound PSMA-617 in the present invention is The PSMA-617 in the present invention is prepared by referring to the prior art method or purchased from commercial sources.

[0121] The following examples illustrate the synthesis methods of the compounds and intermediates of the present invention. The following examples are only examples of the present invention and should not be used to limit the scope of the present invention. Unless otherwise specified, the raw materials and reagents involved in the present invention can be obtained through commercial channels, and the specific channel sources do not affect the implementation of the technical solution of the present invention. Using the corresponding commercial reagents, products prepared by existing literature methods, etc. as raw materials, and referring to the above-mentioned synthesis methods, the following intermediates and compounds are prepared. Preparation Example L1: Preparation of (S)-22-(tert-butoxycarbonyl)-35,35-dimethyl-10,19,24,33-tetraoxo-3,6,12,15,34-pentaoxa-9,18,23-triazatriacontriacontanoic acid

[0122]

[0123] Step 1: Preparation of intermediate L1B

[0124] The intermediate L1B was prepared by solid phase resin loading of compound L1A. The specific operation was as follows: 2-chloro-trityl chloride resin (1.182mmol / g, 1.61g) was weighed into a solid phase synthesizer, swollen with dichloromethane (50mL) for 1 hour, and then the dichloromethane was removed by suction filtration, and then washed with N,N-dimethylformamide, filtered, and set aside. Subsequently, [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (2.36g) was weighed and dissolved in N,N-dimethylformamide (50mL), and added to the resin. Subsequently, N,N-diisopropylethylamine (2.37g) was added to the reaction system, and the reaction was shaken at room temperature for 3 hours. After the reaction was completed, the resin was filtered, and washed with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in turn. The washed resin is the solid phase resin loaded intermediate L1B.

[0125] Step 2: Preparation of intermediate L1C

[0126] Add N,N-dimethylformamide (40 mL) and hexahydropyridine (9.3 g) to the resin obtained in the previous step (intermediate L1B, 2.45 g). After the addition, place the system at room temperature for 1 hour for shaking reaction. After the reaction is completed, filter and wash the resin with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in sequence. The washed resin is intermediate L1C.

[0127] Step 3: Preparation of intermediate L1D

[0128] Weigh [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (2.77 g) and dissolve it in N,N-dimethylformamide (50 mL). Add O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (3.01 g) under ice bath conditions; after addition, stir and activate for 15 minutes, then add the reaction solution to the resin (intermediate L1C) obtained in the previous step, add N,N-diisopropylethylamine (2.79 g) to adjust the pH to 8-9, and then shake and react at room temperature for 1 hour. After the reaction is completed, filter and wash the resin with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in turn. The washed resin is intermediate L1D.

[0129] MS (ESI) m / z (M+H) + =531.2.

[0130] Step 4: Preparation of intermediate L1E

[0131] At room temperature, add piperidine in N,N-dimethylformamide solution (V:V=1:4, 100 mL) to the resin (intermediate L1D) obtained in the previous step. After the addition, react on a shaker at room temperature for 1.5 hours. After the reaction is completed, filter and wash the resin with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in sequence. The washed resin is intermediate L1E.

[0132] Step 5: Preparation of intermediate L1F

[0133] Weigh 1-tert-butyl fluorenylmethoxycarbonyl-L-glutamic acid ester (3.09 g) and dissolve it in N,N-dimethylformamide (40 mL). Add O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (3.00 g) under ice bath conditions; after addition, stir and activate for 15 minutes, then add the reaction solution to the resin (intermediate L1E) obtained in the previous step, add N,N-diisopropylethylamine (2.35 g), adjust the pH to 8-9, and then shake and react at room temperature for 1 hour. After the reaction is completed, filter and wash the solid phase resin with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in turn. The washed resin is intermediate L1F.

[0134] MS (ESI) m / z (M+H) + =716.2.

[0135] Step 6: Preparation of intermediate L1G

[0136] At room temperature, add 20% piperidine in N,N-dimethylformamide solution (V:V=1:4, 100 mL) to the resin obtained in the previous step (intermediate L1F), and react on a shaker at room temperature for 1.5 hours. After the reaction is completed, filter and wash the resin with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in sequence. The washed resin is intermediate L1G.

[0137] Step 7: Preparation of intermediate L1H

[0138] Weigh 10-(tert-butoxy)-10-oxodecanoic acid (1.86 g) and dissolve it in N,N-dimethylformamide (40 mL). Add O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (2.97 g) under ice bath conditions; after addition, stir and activate for 15 minutes, then add the reaction solution to the resin obtained in the previous step (intermediate L1G), add N,N-diisopropylethylamine (2.33 g) to adjust the pH to 8-9, and then shake the reaction at room temperature for 1 hour. After the reaction is completed, filter and wash the resin with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in turn. The washed resin is intermediate L1H. MS (ESI) m / z (M+H) + =734.5.

[0139] Step 8: Preparation of Preparation Example L1

[0140] Hexafluoroisopropanol (20 mL) and dichloromethane (30 mL) were added to the resin (L1H) obtained in the previous step. After the addition, the system was placed at room temperature for 0.5 hours of shaking reaction, and the reaction was repeated twice. After the reaction was completed, the filtrate was filtered and concentrated, and the crude product was dissolved in N, N-dimethylformamide, and the target product (S)-22-(tert-butoxycarbonyl)-35,35-dimethyl-10,19,24,33-tetraoxo-3,6,12,15,34-pentaoxa-9,18,23-triazatriacontahedral acid (Preparation Example L1, 2.9 g) was obtained.

[0141] MS (ESI) m / z (M+H) + =734.5.

[0142] Preparation Example L2: Preparation of (S)-22-(tert-butoxycarbonyl)-38,38-dimethyl-10,19,24,36-tetraoxo-3,6,12,15,37-pentaoxa-9,18,23-triazanonatriacontadecanoic acid

[0143]

[0144] According to the preparation method of Preparation Example L1, a resin intermediate L1G was prepared. Then, referring to steps 7 and 8 of the preparation method of Preparation Example L1, 13-(tert-butoxy)-13-oxotridecanoic acid (2.67 g) was dissolved in N,N-dimethylformamide (40 mL). Under ice bath conditions, O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.97 g) was added, and the mixture was stirred and activated for 15 minutes. The reaction solution was added to the resin intermediate L1G, and N,N-diisopropylethylamine (2.33 g) was added to adjust the pH to 8-9. The mixture was shaken and reacted at room temperature for 1 hour. The mixture was filtered and the resin was washed with N,N-dimethylformamide, dichloromethane and N,N-dimethylformamide in sequence. The washed resin was the intermediate L2H. MS (ESI) m / z (M+H) + =776.2.

[0145] Then add hexafluoroisopropanol (20 mL) and dichloromethane (30 mL) to the resin intermediate L2H. After the addition, place the system at room temperature for 0.5 hours of shaking reaction, repeat twice. After the reaction is completed, filter and concentrate the filtrate, dissolve the crude product with N,N-dimethylformamide, and prepare liquid phase purification to obtain the target product (S)-22-(tert-butoxycarbonyl)-38,38-dimethyl-10,19,24,36-tetraoxo-3,6,12,15,37-pentaoxa-9,18,23-triazatriacontadecanoic acid (Preparation Example L2, 2.3 g). MS (ESI) m / z (M+H) + =776.2.

[0146] Preparation Example L3: Preparation of (S)-22-(tert-butyloxycarbonyl)-41,41-dimethyl-10,19,24,39-tetraoxo-3,6,12,15,40-pentaoxa-9,18,23-triazatetradecanoic acid

[0147]

[0148] According to the preparation method of Preparation Example L1, a resin intermediate L1G was prepared. Then, referring to steps 7 and 8 of the preparation method of Preparation Example L1, 16-(tert-butoxy)-16-oxohexadecanoic acid (2.61 g) was dissolved in N,N-dimethylformamide (40 mL). Under ice bath conditions, O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.97 g) was added, and the mixture was stirred and activated for 15 minutes. The reaction solution was added to the resin intermediate L1G, and N,N-diisopropylethylamine (2.33 g) was added to adjust the pH to 8-9. The mixture was shaken and reacted at room temperature for 1 hour, filtered, and the resin was washed with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in sequence. The washed resin was the intermediate L3H. MS (ESI) m / z (M+H)+ =818.2;

[0149] Then add hexafluoroisopropanol (20 mL) and dichloromethane (30 mL) to the resin intermediate L3H. After the addition, place the system at room temperature for 0.5 hours of shaking reaction, repeat twice. After the reaction is completed, filter and concentrate the filtrate, dissolve the crude product with N,N-dimethylformamide, and prepare liquid phase purification to obtain the target product (S)-22-(tert-butyloxycarbonyl)-41,41-dimethyl-10,19,24,39-tetrahydro-3,6,12,15,40-pentaoxa-9,18,23-triazatetradecanoic acid (2.32 g). MS (ESI) m / z (M+H) + =818.2.

[0150] Preparation Example L4: Preparation of (S)-22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42-pentaoxa-9,18,23-triazatetradecanoic acid

[0151]

[0152] According to the preparation method of Preparation Example L1, the resin intermediate L1G was prepared, and then referring to steps 7 and 8 of the preparation method of Preparation Example L1, 18-(tert-butoxy)-18-oxooctadecanoic acid (2.67 g) was dissolved in N,N-dimethylformamide (40 mL), and O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.97 g) was added under ice bath conditions, and the mixture was stirred and activated for 15 minutes. The reaction solution was added to the resin intermediate L1G, and N,N-diisopropylethylamine (2.33 g) was added to adjust the pH to 8-9, and the reaction was shaken at room temperature for 1 hour, filtered, and the resin was washed with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in turn. The washed resin is the intermediate L4H. MS (ESI) m / z (M+H) + =846.2.

[0153] Then add hexafluoroisopropanol (20 mL) and dichloromethane (30 mL) to the resin intermediate L4H. After the addition, place the system at room temperature for 0.5 hours of shaking reaction, repeat twice. After the reaction is completed, filter and concentrate the filtrate, dissolve the crude product with N,N-dimethylformamide, and prepare liquid phase purification to obtain the target product (S)-22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42-pentaoxa-9,18,23-triazatetradecanoic acid (2.12 g). MS (ESI) m / z (M+H) + =846.2.

[0154] Preparation of solid phase resin intermediate 9:

[0155]

[0156] Step 1: Preparation of intermediate 2

[0157] Intermediate 2 was prepared by solid phase resin loading of intermediate 1. The specific operation was as follows: 2-chlorotrityl chloride resin (1.182mmol / g, 4.55g) was weighed into a solid phase synthesizer, swollen with dichloromethane (50mL) for 1 hour, and then the dichloromethane was removed by suction filtration, and then washed with N,N-dimethylformamide, filtered, and set aside. Then N-[(9H-fluorene-9-methoxy)carbonyl]-N'-[(2-propyleneoxy)carbonyl]-L-lysine (2.31g) was weighed and dissolved in N,N-dimethylformamide (50mL), and then added to the resin, followed by N,N-diisopropylethylamine (1.98g), and the system was shaken and reacted at room temperature overnight. After the reaction was completed, the resin was filtered, and the resin was washed with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in sequence. The washed resin was intermediate 2.

[0158] Step 2: Preparation of intermediate 3

[0159] To the treated resin (Intermediate 2, 2.38 g), N,N-dimethylformamide (40 mL) and piperidine (10 mL) were added in sequence. After the addition, the system was shaken and reacted at room temperature for 2 hours. After the reaction was completed, the resin was filtered and washed with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in sequence. The washed resin was Intermediate 3.

[0160] Step 3: Preparation of intermediate 4

[0161] Under ice bath, di-tert-butyl L-glutamic acid hydrochloride (4.48g) was dissolved in ultra-dry dichloromethane (50mL), and N,N-diisopropylethylamine (7.84g) was added; then triphosgene (1.50g) was weighed and dissolved in dichloromethane (20mL), and the solution was slowly added dropwise to the reaction system, and the reaction was maintained under ice bath conditions overnight. The prepared isocyanate was added to the resin in the previous step, and then N,N-diisopropylethylamine was added to make the pH of the system 8-9. After the reaction system was shaken at room temperature for 2 hours, it was filtered and the resin was washed with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in turn. The washed resin is intermediate 4.

[0162] MS (ESI) m / z (M+H) + =516.2.

[0163] Step 4: Preparation of intermediate 5

[0164] At room temperature, tetrakistriphenylphosphine palladium (1.18 g), morpholine (10.10 g), and dichloromethane (90 mL) were added to the resin obtained in the previous step (intermediate 4), and then placed on a shaker to react at room temperature for 3 hours. After the reaction was completed, the resin was filtered and washed with N, N-dimethylformamide, dichloromethane, and N, N-dimethylformamide in sequence. The washed resin is intermediate 5.

[0165] MS (ESI) m / z (M+H) + =432.2.

[0166] Step 5: Preparation of intermediate 6

[0167] Weigh (2S)-2-({[(9H-fluorene-9-yl)methoxy]carbonyl}amino)-3-(naphthalene-2-yl)propionic acid (3.29 g) and dissolve it in N,N-dimethylformamide (100 mL). Add benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.86 g) at room temperature and stir for 10 minutes. Then, add the reaction solution to the resin (intermediate 5) obtained in the previous step, add N,N-diisopropylethylamine (1.94 g), adjust the pH of the reaction system to 8-9, and react at room temperature for 2 hours. After the reaction is completed, filter and wash the resin with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in turn. The washed resin is intermediate 6.

[0168] MS (ESI) m / z (M+H) + =851.2.

[0169] Step 6: Preparation of intermediate 7

[0170] At room temperature, add piperidine in N,N-dimethylformamide solution (V:V=1:4, 100 mL) to the resin obtained in the previous step (Intermediate 6), and react on a shaker at room temperature for 1.5 hours. After the reaction is completed, filter and wash the resin with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in sequence. The washed resin is Intermediate 7.

[0171] MS (ESI) m / z (M+H) + =629.2.

[0172] Step 7: Preparation of intermediate 8

[0173] Under room temperature, weigh trans-4-[[[[(9H-fluorene-9-yl)methoxy]carbonyl]amino]methyl]cyclohexanecarboxylic acid (1.89 g), dissolve it in N,N-dimethylformamide (80 mL), add benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.86 g), and stir for 10 minutes. Then add the reaction solution to the resin obtained in the previous step, add N,N-diisopropylethylamine (1.94 g), adjust the pH of the reaction system to 8-9, and react at room temperature for 2 hours. After the reaction is completed, filter and wash the resin with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in turn. The washed resin is intermediate 8.

[0174] MS (ESI) m / z (M+H) + =990.2.

[0175] Step 8: Preparation of solid phase resin intermediate 9

[0176] At room temperature, add piperidine in N,N-dimethylformamide solution (V:V=1:4, 100 mL) to the resin (intermediate 8) obtained in the previous step, and react on a shaker at room temperature for 1.5 hours. After the reaction is completed, filter and wash the resin with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in sequence. The washed resin is the solid phase resin intermediate 9. Example 1: Preparation of (((S)-1-carboxy-5-((S)-2-((1,4-trans)-4-((4S,31S)-31,41-dicarboxy-3,10,19,28,33-pentaoxo-4-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetamide)-12,15,21,24-tetraoxa-2,9,18,27,32-pentaazatetradecanoyl)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propionamido)pentyl)carbamoyl)-L-glutamic acid

[0177]

[0178]

[0179] Step 1: Preparation of intermediate P1B

[0180] Weigh N-[Fluorenylmethoxycarbonyl]-N 2-[(2-propenyloxy)carbonyl]-L-lysine (P1A, 4.34g), add N,N-dimethylformamide (50mL) to dissolve. Under ice bath conditions, add O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (3.95g). After the addition, stir and activate for 15 minutes. Then add the reaction solution to the solid phase resin intermediate 9, add N,N-diisopropylethylamine (3.10g), adjust the pH to 8-9, and then shake the resin at room temperature for 1 hour. After the reaction is completed, filter and wash the solid phase resin with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in turn. The washed resin is the intermediate P1B. MS (ESI) m / z (M+H) + =1202.6.

[0181] Step 2: Preparation of intermediate P1C

[0182] Add N,N-dimethylformamide (40 mL) and piperidine (10 mL) to the resin obtained in the previous step (intermediate P1B). After the addition, shake the system at room temperature for 1 hour. After the reaction is completed, filter and wash the resin with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in sequence. The washed resin is intermediate P1C. MS (ESI) m / z (M+H) + =980.2.

[0183] Step 3: Preparation of intermediate P1D

[0184] Weigh (S)-22-(tert-butoxycarbonyl)-35,35-dimethyl-10,19,24,33-tetraoxo-3,6,12,15,34-pentaoxa-9,18,23-triazatriacontriacontanoic acid (Preparation Example L1, 1.76 g), add N,N-dimethylformamide (20 mL) to dissolve. Add O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (0.99 g) under ice bath conditions; after addition, stir and activate for 15 minutes, then add the reaction solution to the resin (intermediate P1C) obtained in the previous step. Add N,N-diisopropylethylamine (0.78 g) to adjust the pH to 8-9, then shake and react at room temperature for 1 hour. After the reaction is completed, the mixture is filtered and the resin is washed with N,N-dimethylformamide, dichloromethane and N,N-dimethylformamide in sequence. The washed resin is the intermediate P1D.

[0185] MS (ESI) m / z (M+H) + =1696.2; MS (ESI) m / z (M+H) + / 2=848.1.

[0186] Step 4: Preparation of intermediate P1E

[0187] Add morpholine (10 mL), dichloromethane (90 mL), and then tetrakistriphenylphosphine palladium (0.46 g) to the resin obtained in the previous step (intermediate P1D). After the addition, shake the system at room temperature for 1.5 hours. After the reaction is completed, filter and wash the resin with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in sequence. The washed resin is intermediate P1E.

[0188] MS (ESI) m / z (M+H) + =1612.2; MS (ESI) m / z (M+H) + / 2=806.2.

[0189] Step 5: Preparation of intermediate P1F

[0190] Weigh tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (1.37 g) and add N,N-dimethylformamide (30 mL) to dissolve. Add O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (0.99 g) under ice bath conditions. After the addition, stir and activate for 15 minutes. Then add the reaction solution to the resin (P1E) obtained in the previous step, add N,N-diisopropylethylamine (0.78 g), adjust the pH to 8-9, and then shake the resin at room temperature for 1 hour. After the reaction is completed, filter and wash the resin with N,N-dimethylformamide, dichloromethane, and N,N-dimethylformamide in turn. The washed resin is P1F.

[0191] MS (ESI) m / z (M+H) + =2166.3; MS (ESI) m / z (M+H) + / 2=1084.1.

[0192] Step 6: Preparation of intermediate P1G

[0193] Hexafluoroisopropanol (20 mL) and dichloromethane (30 mL) were added to the resin (P1F) obtained in the previous step. After the addition, the system was shaken and reacted at room temperature for 1 hour, and repeated twice. After the reaction was completed, the resin was filtered and washed with dichloromethane, and the filtrate was collected and concentrated to obtain a crude product, which was purified by medium pressure Pre-HPLC (TFA system) to obtain the target product N 6-((2S)-2-((1,4-trans)-4-((4S,31S)-31-(tert-butoxycarbonyl)-44,44-dimethyl-3,10,19,28,33,42-hexaoxo-4-(2-(4,7,10-tris(tert-butyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododec-1-yl)acetamido)-12,15,21,24,43-pentaoxa-2,9,18,27,32-pentaazatetrapentadecanyl)-cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propanoyl)-N-((2S)-2-((1,4-trans)-4-((4S,31S)-31-(tert-butoxycarbonyl)-44,44-dimethyl-3,10,19,28,33,42-hexaoxo-4-(2-(4,7,10-tris(tert-butyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododec-1-yl)acetamido)-12,15,21,24,43-pentaoxa-2,9,18,27,32-pentaazatetrapentadecanyl)-cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propanoyl)- 2 -(((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)-L-lysine (Intermediate P1G, 3.0 g).

[0194] MS (ESI) m / z (M+H) + =2166.3; MS (ESI) m / z (M+H) + / 2=1084.1.

[0195] Step 7: Preparation of (((S)-1-carboxy-5-((S)-2-((1,4-trans)-4-((4S,31S)-31,41-dicarboxy-3,10,19,28,33-pentaoxo-4-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetamide)-12,15,21,24-tetraoxa-2,9,18,27,32-pentaazatetradecanoyl)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propionamido)pentyl)carbamoyl)-L-glutamic acid (Example 1)

[0196] The intermediate P1G (1.1 g) was dissolved in a 1,4-dioxane solution of hydrochloric acid (14 mL). After the addition, the reaction was allowed to proceed at room temperature for 2 hours. The reaction of the raw material was complete as monitored by LC-MS. The reaction solution was concentrated, and the crude product was dissolved in N,N-dimethylformamide. The title compound (Example 1) was obtained by preparative purification in an amount of 300 mg.

[0197] MS (ESI) m / z (M+H) + =1773.9; MS (ESI) m / z (M+H) + / 2=887.6;HPLC method A:R f =4.32

[0198] min, purity 98.4%.

[0199] Example 2: Preparation of (((S)-1-carboxy-5-((S)-2-((1,4-trans)-4-((4S,31S)-31,44-dicarboxy-3,10,19,28,33pentaoxo-4-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetamido)-12,15,21,24-tetraoxa-2,9,18,27,32-pentaazatetradecanoyl)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propionamido)pentyl)carbamoyl)-L-glutamic acid

[0200]

[0201]

[0202]

[0203] The intermediate P1C was prepared according to the same preparation method as in Example 1, and then the intermediate L1 in the preparation method of Example 1 was replaced by the intermediate L2. Referring to the preparation method of steps 3 to 7 of Example 1, 30 mg of Example 2 was synthesized.

[0204] MS (ESI) m / z (M+H) + =1815.9; MS (ESI) m / z (M+H) + / 2=908.5;HPLC method B:R f =8.35min,

[0205] Purity 99.2%.

[0206] Example 3: Preparation of (((S)-1-carboxy-5-((S)-2-((1,4-trans)-4-((4S,31S)-31,47-dicarboxy-3,10,19,28,33-pentaoxo-4-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetamide)-12,15,21,24-tetraoxa-2,9,18,27,32-pentaazatetraheptadecyl)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propionamido)pentyl)carbamoyl)-L-glutamic acid

[0207]

[0208]

[0209]

[0210] The intermediate P1C was prepared according to the same preparation method as in Example 1, and then the intermediate L1 in the preparation method of Example 1 was replaced by the intermediate L3. Referring to the preparation method of steps 3 to 7 of Example 1, 165 mg of Example 3 was synthesized.

[0211] MS (ESI) m / z (M+H) + =1858.2; MS (ESI) m / z (M+H) + / 2=929.6;HPLC method B:R f =9.20min,

[0212] Purity 99.6%.

[0213] Example 4: Preparation of (((S)-1-carboxy-5-((S)-2-((1,4-trans)-4-((4S,31S)-31,49-dicarboxy-3,10,19,28,33-pentaoxo-4-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetamido)-12,15,21,24-tetraoxa-2,9,18,27,32-pentaazatetranonadecanyl)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propionamido)pentyl)carbamoyl)-L-glutamic acid

[0214]

[0215]

[0216]

[0217] The intermediate P1C was prepared according to the same preparation method as in Example 1, and then the intermediate L1 in the preparation method of Example 1 was replaced by the intermediate L4. Referring to the preparation method of steps 3 to 7 of Example 1, 150 mg of Example 3 was synthesized.

[0218] MS (ESI) m / z (M+H) + =1886.2; (M+H) + / 2=943.6;HPLC method B:R f =9.99min, purity 99.2%.

[0219] Biological tests

[0220] Test Example 1 Solubility Test

[0221] 1.1 Solution preparation

[0222] The compounds of Examples 1 to 4 were dissolved in DMSO to a final concentration of 10 mM to prepare stock solutions.

[0223] 1.2 Solubility test

[0224] (1) Take 15 μL of the stock solution (10 mM) and add it to the corresponding 96-well plate as the sample plate, and then add 485 μL of pH 7.4 or pH 2.0 phosphate buffer to the corresponding vial of the sample plate. Then put the sample plate into a constant temperature mixer and shake it at 1100 rpm and 25°C for 2 hours. After 2 hours, filter the solution in the sample plate. Transfer 5 μL of the filtrate and 5 μL of blank DMSO to a new 96-well plate, and then add 490 μL of internal standard water (internal standard water is a mixed solvent of water: methanol = 1:1). Depending on the peak shape, a certain proportion of internal standard water may be used to dilute the sample diluent to obtain a better peak shape. The dilution factor may be adjusted depending on the solubility of the analyte or the strength of its LC-MS response signal.

[0225] (2) Transfer 6 μL of the stock solution of Examples 1 to 4 to a blank 96-well plate, and then add 194 μL of DMSO to prepare a 300 μM standard solution. Transfer 5 μL of the standard solution to another blank 96-well plate, and then add 5 μL of phosphate buffer (i.e., PBS, pH 7.4 or pH 2.0) and 490 μL of internal standard water to a final concentration of 3 μM. Depending on the peak shape, a certain proportion of internal standard water may be used to dilute the standard solution to obtain a better peak shape. The concentration of the standard solution may be adjusted depending on the strength of its LC-MS response signal.

[0226] (3) Identify and quantify the peak of the standard solution of known concentration by LC-MS. Calculate the solubility as follows: Sample solubility = [sample peak area × dilution factor × concentration of standard solution] / peak area of ​​standard solution.

[0227] The results are shown in Table 1.

[0228] Table 1 Solubility results of compounds at pH 2.0 and pH 7.4

[0229] Compound No. Solubility at pH 2.0 (μM) Solubility at pH 7.4 (μM) Example 4 35.50 275.61 Example 1 261.54 275.92 Example 2 253.34 295.66 Example 3 150.22 302.40

[0230] It can be seen that the compounds of the present invention have good solubility. For example, under the experimental conditions, in a pH 2.0 solution, Examples 1 to 3 have high solubility, and Example 4 has good solubility; in a pH 7.4 solution, Examples 1 to 4 all have high solubility.

[0231] Test Example 2: Plasma protein binding test

[0232] In vitro test examples 1 to 4 were tested for plasma protein binding rates in human plasma (source: People's Hospital of Weifang High-tech Industrial Development Zone, Shandong Province, batch number: P-KB (173-222) -20210728), monkey plasma (source: Beijing Rixin Technology Co., Ltd., batch number: MK20220620) and mouse plasma (source: Beijing Huizhiheyuan Biotechnology Co., Ltd., batch number: M220093).

[0233] 2.1 Test methods

[0234] (1) Preparation of working stock solutions of compounds in various examples

[0235] The compounds of Examples 1 to 4 were weighed respectively, dissolved in DMSO, and diluted to prepare 200 μM working stock solutions of each Example.

[0236] (2) Preparation of incubation system

[0237] 3 μL of the working stock solution of each example was fully mixed with human, monkey and mouse plasma (597 μL each) to obtain an incubation system, so that the final organic solvent content of the incubation system was 0.5%, and then 50 μL of the incubation system was transferred to a blank 96-well plate as a sample at the T0 time point.

[0238] (3) Dialysis treatment

[0239] Using the equilibrium dialysis method, 120 μL of the incubation system was added to each well on one side of the dialysis plate as a plasma sample, and an equal volume of PBS (phosphate buffer) with a pH of 7.4 was placed on the other side as a buffer sample. All samples were prepared in parallel, the dialysis plate was sealed, and placed in an incubator at 37°C and 5% CO2, and incubated at 100 rpm for 6 hours. After the equilibrium incubation, 50 μL of plasma samples and buffer samples were taken for processing.

[0240] (4) Sample preparation at time point T6 (protein binding stability sample)

[0241] The remaining incubation system in step (2) was placed in an incubator at 37°C and 5% CO2 and incubated at 100 rpm for 6 hours. After the incubation, 50 μL of the incubation system of the second incubation was transferred, an equal volume of PBS (phosphate buffer) at pH 7.4 was added, mixed evenly, and 400 μL of stop solution (the stop solution was a mixture of phosphate buffer and methanol in a ratio of 1:1) was added as the sample at time point T6 for detecting the albumin binding stability of the compound of the present invention.

[0242] (5) Treatment of plasma samples and buffer samples

[0243] An equal volume of blank plasma of the corresponding species was added to the buffer sample taken out in step (3), and an equal volume of PBS (phosphate buffer) at pH 7.4 was added to the plasma sample taken out in step 3), and the mixture was mixed evenly, and then 400 μL of stop solution (the stop solution was a mixture of phosphate buffer and methanol in a ratio of 1:1) was added to each sample.

[0244] (6) Sample analysis

[0245] The plasma sample and buffer sample obtained in step (5) were vortexed together with the sample at time point T0 and the sample at time point T6 for 10 minutes, and then centrifuged at 3220g for 30 minutes for protein precipitation. 120 μL of the supernatant of each sample was transferred and added to an equal volume of purified water for mixing, and then used for LC-MS analysis, with tolbutamide as the internal standard.

[0246] All data calculations were performed using Microsoft Excel software. The concentration of the drug in the buffer sample and plasma sample was determined. The calculation formula is as follows:

[0247] Free rate (%) = [peak area ratio of buffer sample / peak area ratio of plasma sample] × 100%;

[0248] Binding rate (%) = 100% - free percentage;

[0249] 6-hour residual binding rate (%) = [sample peak area ratio at T6 time point / sample peak area ratio at T0 time point] × 100%;

[0250] The aforementioned peak area ratio is the ratio of the sample peak area to the internal standard peak area.

[0251] 2.3 Test results

[0252] Examples 1 to 4 were highly bound to human, monkey and mouse plasma proteins. After 6 hours, the plasma protein binding of Example 1 was comparable to that of PSMA-617, but Examples 2 to 4 still had high plasma protein binding rates, as shown in Table 2.

[0253] Table 2 Plasma protein binding rates of Examples 1 to 4 in humans, monkeys and mice

[0254]

[0255]

[0256] The albumin binding rate of the compounds of the present invention is increased relative to PSMA-617, among which the increase rate of mouse albumin is the highest; when the number of terminal fatty diacid alkyl groups of the compounds of the present invention is 13-16, such as 13 and 16 in Examples 2 and 3, the albumin binding rate increases while the 6-hour residual binding rate is basically unchanged, that is, the albumin binding stability is better.

[0257] Test Example 3: 177 Lu-labeled compounds and their radiochemical purity analysis

[0258] Under sterile conditions, PSMA-617 and Examples 1 to 4 were dissolved in DMSO to prepare 2000 μM stock solutions. 3.1 Stability Test 177 Lu-labeled compounds

[0259] Take 2 μL (4 nmol) of the stock solutions of compound PSMA-617, Example 1, and Example 4, respectively, add 40 μL of 0.5 M sodium acetate buffer solution at pH 4.5, shake thoroughly, and then add 1 mcI of 177 LuCl3 (source: Isotope Technologies Muncich SE, batch number: Lu-23-277-01) injection solution was mixed (specific activity 0.25 mci / nmol), heated upright in a metal bath at 95°C for 15 min under sealed conditions, then taken out and cooled to room temperature. A small amount of solution was taken to detect the radioactivity concentration (i.e., radiochemical purity analysis). 177 Lu-PSMA-617, 177 Lu-Example 1, 177 Lu-Example 4 were placed in lead pots and stored at room temperature.

[0260] 3.2 Cellular uptake and internalization assay 177 Lu-labeled compounds

[0261] The labeled product obtained in 3.1 above 177 Lu-Example 1 and 177 Lu-Example 4 0.7mci was taken and added to a phosphate buffer solution (pH 8.0) containing 7mg / mL sodium vitamin C and 0.25% bovine serum albumin, and mixed evenly as a mother solution, with a specific activity of about 1.4mci / mL (0.25mci / nmol). Binding buffer (a mixed solution of RPMI-1640 culture medium and 0.25% bovine serum albumin) was added to dilute the mother solution to a 60nM solution for use.

[0262] 3.3 PSMA ligand tissue distribution assay in tumor-bearing mice 177 Lu-labeled compounds

[0263] Take 6 μL (12 nmol) of the compound PSMA-617 and the stock solutions of Examples 1 to 4, add 60 μL of 0.5 M sodium acetate buffer solution (pH 4.5), shake thoroughly, and then add 3 mcI 177LuCl3 (source: Isotope Technologies Muncich SE, batch number: Lu-23-277-01) injection solution was mixed (specific activity 0.25 mci / nmol), heated upright in a metal bath at 95°C for 15 min under sealed conditions, then taken out and cooled to room temperature. A small amount of solution was taken to detect the radioactivity concentration (i.e., radiochemical purity analysis), and then the labeled compound was obtained. 177 Lu-PSMA-617, 177 Lu-Example 1, 177 Lu-Example 2, 177 Lu-Example 3, 177 Lu-Example 4 was diluted to 1.5 μCi / μL by adding physiological salt, and placed in a lead bottle and stored at room temperature for later use.

[0264] 3.4 Drug efficacy test in tumor-bearing mice 177 Lu-labeled compounds

[0265] Take 4 μL (8 nmol) of PSMA-617 and the stock solution of Example 2, add 80 μL of 0.5 M sodium acetate buffer solution at pH 4.5, shake thoroughly, and then add 13 mcg of 177 Mix the LuCl3 (source: Isotope Technologies Muncich SE, batch number: Lu-23-585-01) injection solution (specific activity 1.625mci / nmol, 60MBq / nmol), heat it upright in a metal bath at 95°C for 15min under sealed conditions, take it out, cool it to room temperature, take a small amount of solution to detect the radioactivity concentration (radiochemical purity analysis), then dilute it with saline to 16.2mCi / mL, put it in a lead bottle and store it at room temperature for later use.

[0266] Take 5 μL (10 nmol) of the stock solutions of compounds in Example 3 and Example 4, add 100 μL of 0.5 M sodium acetate buffer solution at pH 4.5, shake thoroughly, and then add 16.2 mcg of 177 The LuCl3 injection solution was mixed (specific activity 1.62mci / nmol, 60MBq / nmol), heated upright in a metal bath at 95°C for 15min under sealed conditions, taken out, cooled to room temperature, and a small amount of the solution was taken to detect the radioactivity concentration (i.e., radiochemical purity analysis), and then diluted with normal saline to 16.2mCi / mL as the high-dose (60MBq) group test sample. Another part of the high-dose (60MBq) group test sample was diluted with normal saline to 5.5mCi / mL as the low-dose (20MBq) group test sample, which was placed in a lead can and stored at room temperature for later use.

[0267] 3.5 Radiochemical purity quality control analysis

[0268] Use the above 177 A small amount of Lu-labeled solution was drawn with a capillary and spotted on the bottom of a polyamide thin-layer paper chromatographic plate at about 1 cm. After drying, the chromatographic plate was placed in a developing cylinder. After the developing agent (1M ammonium acetate solution: anhydrous methanol = 1:4) was developed to about 10 cm, it was taken out and blown dry, and the chromatographic peak was measured with a thin-layer scanner (source: BIOSCAN, USA, model: MINIscan). The radiochemical purity was calculated using the area normalization method.

[0269] 177 Lu-labeled compounds 177 Lu-Example 1, 177 Lu-Example 2, 177 Lu-Example 3, 177 The radiochemical purity of Lu-Example 4 can reach more than 95%. 177 The Lu-labeled compound is obtained as in 3.3 of Experimental Example 3. 177 Lu-Example 1, 177 Lu-Example 2, 177 Lu-Example 3, 177 The radiochemical purity of Lu-Example 4 reached 100%. The radiochemical purity TLC scanning spectrum is shown in the attached Figure 2 (where A is 177 Lu-Example 1, retention time 1.502min; B is 177 Lu-Example 2, retention time 1.481min; C is 177 Lu-Example 3, retention time 1.497min; D is 177 Lu-Example 4, retention time 1.426 min).

[0270] Test Example 4: Stability Test

[0271] 4.1 Test steps

[0272] The labeled compound obtained in 3.1 of Experimental Example 3 177 Lu-PSMA, 177 Lu-Example 1, 177 Lu-Example 3 and 177 Lu-Example 4 was diluted with physiological saline of pH 7.4 to 3.0 mCi / 0.6 mL, and then 1 mL of the solution was respectively aspirated into a low-adsorption centrifuge tube and incubated at room temperature, and 25 μL of the incubation solution was respectively aspirated at 0 h, 3 h, 24 h, 48 h, 72 h, 96 h, 120 h and 144 h, and immediately detected by radio-HPLC (Company: Shimadzu Corporation, Japan; Model: LC-20AT).

[0273] 4.2 Radio-HPLC detection method

[0274] Table 3 Radio-HPLC detection method

[0275]

[0276]

[0277] Data processing and analysis:

[0278] Remaining percentage of the labeled compound (%) = (peak area of ​​the main peak of Radio-HPLC at a specific time / total peak area of ​​Radio-HPLC at a specific time) × 100%

[0279] The incubation time was used as the horizontal axis and the remaining percentage of the labeled compound was used as the vertical axis. GraphPad Prism 8.0.1 was used for fitting. According to the fitting curve, T 1 / 2 (ie the time required for 50% of the labeled compound to be degraded).

[0280] 4.3 Test results

[0281] Labeled compounds 177 Lu-PSMA-617, 177 Lu-Example 1 and 177 The remaining percentage of Lu-Example 4 and the degradation time of 50% of the labeled compound (T 1 / 2 ) The results are shown in Table 4 and Table 5.

[0282] Table 4 Remaining percentage of labeled compounds (%)

[0283] Theoretical time (h) 0 3 24 48 72 96 120 144 <![CDATA[ 177 Lu-Example 4]]> 100 100 100 100 100 100 100 100 <![CDATA[ 177 Lu-Example 1]]> 100 100 100 100 100 100 100 100 <![CDATA[ 177 Lu-Example 3]]> 100 100 100 100 100 100 100 100 <![CDATA[ 177 Lu-PSMA-617]]> 100 100 100 100 89.63 92.66 86.22 84.98

[0284] Table 5 50% degradation time of labeled compounds T 1 / 2 (Non-linear fitting)

[0285] Compound <![CDATA[T 1 / 2 (h)]]> <![CDATA[ 177 Lu-Example 4]]> ∞ <![CDATA[ 177 Lu-Example 1]]> ∞ <![CDATA[ 177 Lu-Example 3]]> ∞ <![CDATA[ 177 Lu-PSMA-617]]> 415.5

[0286] The stability of the labeled compound of the present invention in a pH 7.4 solution is good, for example 177 Lu-Example 1, 177 Lu-Example 3, 177 Lu-Example 4 did not degrade within 144 hours; according to the nonlinear fitting results, the labeled compound of the present invention will not degrade to 50% in a pH 7.4 solution. 177 Lu-PSMA-617 has poor stability and begins to degrade to a certain extent after 48 hours. According to the nonlinear fitting results, 177Lu-PSMA-617 degraded by 50% after 415 h in pH 7.4 solution.

[0287] Test Example 5 Cellular Uptake and Internalization Test

[0288] 5.1 Test steps

[0289] (1) LNCaP cells (source: ATCC, USA; supplier: Nanjing Kebai Biotechnology Co., Ltd.) and PC-3 cells (source: ATCC, USA) were cultured at 2×10 5 The cells were inoculated at a density of 100 / ml in two 24-well plates coated with poly-lysine (one plate for each time point, with 3 replicates), 1 ml of RPMI-1640 medium (Roswell Park Memorial Institute-1640 cell culture medium, supplier: Gibco, USA, catalog number: 8122158) / well, and cultured overnight at 37°C (PC-3 was the negative control group);

[0290] (2) The RPMI-1640 medium was removed, and the cells were washed with 500 μL of phosphate buffered saline (pH 7.4), and 200 μL of binding buffer (a mixed solution of RPMI-1640 and 0.25% bovine serum albumin) was added to each well, and the mixture was equilibrated at 37° C. for more than 15 min; the solution was removed, and 175 μL of binding buffer and 25 μL of the labeled compound (1.5 pmol) of 3.2 of Experimental Example 3 were added to each well, so that the final concentration of the labeled compound reached 7.5 nM;

[0291] (3) Incubate at 37°C, then place two 24-well plates on ice after incubation for 2 h and 4 h, respectively, to terminate the reaction.

[0292] (4) Free ligand: Collect the solutions after 2 h and 4 h of incubation, wash the cells with 250 μL of phosphate buffer solution (pH 7.4), mix the washing solution with the solution after incubation, and use it as the activity of free ligand (solution 1);

[0293] (5) Cell surface bound ligand: add 250 μL of acid buffer (0.05 M glycine buffer dissolved in 100 mM NaCl solution, pH 2.8) and incubate for 10 min, collect the supernatant, wash the cells with 250 μL of ice-cold phosphate buffer solution (pH 7.4), and mix the washing solution and supernatant as the radioactivity of the cell surface bound ligand (solution 2);

[0294] (6) Cell endocytosis of ligand: Recover cells with 250 μL 1 M NaOH solution, wash each well with 250 μL 1 M NaOH solution, transfer the solution twice to a 0.6 mL centrifuge tube and mix (Solution 3);

[0295] (7) The activities of solutions 1, 2, and 3 were measured using γ-counting (source: PerkinElmer, USA, model: 2480WIZARD2).

[0296] 5.2 Data processing and analysis:

[0297] Uptake internalization rate = solution 3 / (solution 2+solution 3)*100%

[0298] 5.3 Test results:

[0299] The labeled compounds of the present invention all have good specific affinity and uptake and internalization ability for LNCaP cells, for example, 177 Lu-PSMA-617, 177 The uptake and internalization rates of Lu-Example 4 in LNCaP cells were 29.5% and 36.4% at 2 hours, and 29.6% and 39.0% at 4 hours. 177 Lu-Example 4 has a stronger uptake and internalization rate in LNCaP cells with high PSMA expression than 177 Lu-PSMA-617.

[0300] Experimental Example 6 Biological tissue distribution test in tumor-bearing mice

[0301] 6.1 Test methods

[0302] (1) Tumor-bearing mouse model

[0303] B-NDG mice were obtained from Biocytogen Jiangsu Gene Biotechnology Co., Ltd., China, male, 6-8 weeks old; human prostate cancer LNCaP clone FGC cells (ATCC, USA, batch number: CRL-1740). The aforementioned cells were cultured in an incubator at 37°C and 5% CO2, and the culture medium was Dulbecco's Modified Eagle's medium (source: Corning, USA, catalog number 10-013-CV) with 10% inactivated fetal bovine serum. When the cell confluence reached about 90%, the cells were passaged 2-3 times a week.

[0304] LNCaP clone FGC cells resuspended in phosphate buffer solution (pH 7.4) were plated at 5×10 6 100 μg / 0.1 mL (phosphate buffer solution and matrix gel (source: Corning, USA, catalog number: Corning-354248) 1:1 mixture) / mouse, inoculated subcutaneously on the right side of the back of B-NDG mice. Two weeks later, the tumor reached a volume of about 200-300 mm, which is suitable for biodistribution and imaging studies. 3 .

[0305] (2) Biodistribution studies

[0306] 100-200 μL of the labeled compound diluted with physiological saline in 3.3 of the above Experimental Example 3 was injected into the tail vein of LNCaP clone FGC tumor-bearing mice, and then 177 The LNCaP clone FGC tumor-bearing mice of Lu-Example 1 were euthanized at 0.25h, 1h, 4h and 24h respectively; 177 Lu-LNCaP clone FGC tumor-bearing mice of Example 2 were euthanized at 0.5h, 2h, 4h and 24h respectively; 177 The mice bearing the Lu-LNCaP clone FGC tumors of Examples 3 and 4 were euthanized at 4h, 24h, 48h and 96h, respectively.

[0307] Tissues and organs at each time point were then collected, weighed and γ-counted (source: PerkinElmer, USA, model: 2480WIZARD2). The results were decay-corrected and listed as the percentage of injected activity per gram of tissue mass (%ID / g).

[0308] The results of the biological tissue distribution of the labeled compounds of the present invention are shown in Tables 6 to 10 below.

[0309] Table 6 177 Lu-Example 4 Injection LNCaP clone FGC tumor-bearing mouse tissue distribution %ID / g

[0310]

[0311] Table 7 177 Lu-Example 1 Injection LNCaP clone FGC tumor-bearing mouse tissue distribution %ID / g

[0312]

[0313] Table 8 177 Lu-Example 2 Injection LNCaP clone FGC tumor-bearing mouse tissue distribution %ID / g

[0314]

[0315] Table 9 177 Lu-Example 3 Injection LNCaP clone FGC tumor-bearing mouse tissue distribution %ID / g

[0316]

[0317]

[0318] Table 10 177 Tissue distribution of Lu-PSMA-617 injection in LNCaP clone FGC tumor-bearing mice %ID / g

[0319]

[0320] The distribution ratios of the labeled compounds of the present invention in tumor-bearing mouse tissues at 4h, 24h, 48h, and 96h are shown in Tables 11 to 14 below.

[0321] Table 11 Distribution ratios of tumor and main tissues in tumor-bearing mice at 4 h

[0322]

[0323]

[0324] Table 12 Distribution ratios of tumor and main tissues in tumor-bearing mice at 24 hours

[0325] Labeled compounds <![CDATA[ 177 Lu-PSMA-617]]> <![CDATA[ 177 Lu-Example 4]]> <![CDATA[ 177 Lu-Example 1]]> <![CDATA[ 77 Lu-Example 2]]> <![CDATA[ 177 Lu-Example 3]]> Tumor / Kidney 1.82±0.7 7.36±2.38 0.75±0.19 4.54±2.00 2.10±0.13 Tumor / liver 1.73±0.10 22.55±6.41 0.98±0.37 51.20±4.11 145.51±94.29 Tumor / blood 128±14 5.09±0.11 97.25±34.25 492.63±115.88 47.13±6.15

[0326] Table 13 Distribution ratios of tumor and main tissues in tumor-bearing mice at 48h

[0327] Labeled compounds <![CDATA[ 177 Lu-Example 4]]> <![CDATA[ 177 Lu-Example 3]]> Tumor / Kidney 42.45±35.44 2.03±0.85 Tumor / liver 28.47±2.82 45.61±5.97 Tumor / blood 13.35±1.33 175.76±42.23

[0328] Table 14 Distribution ratios of tumor and main tissues in tumor-bearing mice at 96h

[0329] Labeled compounds <![CDATA[ 177 Lu-Example 4]]> <![CDATA[ 177 Lu-Example 3]]> Tumor / Kidney 16.80±7.38 3.59±0.99 Tumor / liver 26.37±2.80 37.40±10.56 Tumor / blood 30.19±8.45 560.67±238.66

[0330] The distribution diagram of the labeled compound of the present invention in LNCaP clone FGC tumor-bearing mice is shown in the attached Figure 3a to 3eThe results of the test on the distribution of the labeled compounds of the present invention in biological tissues show that the compounds of the present invention, such as Examples 1, 2, 3, and 4, have a longer half-life than PSMA-617 and tend to be more enriched in tumors. For example, at 48h and 96h, the distribution of Examples 3 and 4 in tumors is still high and can maintain the therapeutic effect, while there is little or even no enrichment in normal tissues such as blood, liver, and kidneys. According to the tissue distribution test, after administration, the compounds of the present invention (such as Examples 2, 3, and 4) have a significantly higher tumor distribution ratio than the blood distribution ratio, and from the fastest 2 hours (such as Example 2), the blood distribution suddenly decreases and is continuously enriched in the tumor in large quantities, which can last up to 96 hours, greatly reducing the risk of systemic blood exposure and effectively prolonging the therapeutic effect; after administration, the compounds of the present invention (such as Examples 2, 3, and 4) have a significantly higher tumor distribution ratio than the liver, and have a lower toxic effect on the liver; the compounds of the present invention and PSMA-617 are mainly excreted through the kidneys after administration, so there is a certain distribution in the kidneys in the initial stage (such as 4 hours after administration), and as time goes on, after 24 hours, the kidney distribution of the compounds of the present invention (such as Examples 2, 3, and 4) is significantly reduced, and the compounds of the present invention have lower persistent kidney toxicity.

[0331] Test Example 7: Drug efficacy test in tumor-bearing mouse model

[0332] 7.1 Materials and methods

[0333] 7.1.1 Establishment of tumor mouse model

[0334] M-NSG mice (6-8 weeks, male) were subcutaneously inoculated with LNCaP clone FGC cells (5*10 6 / pc, source: American ATCC, catalog number: CRL-1740), wait until the tumor volume grows to 200-300mm 3 The tumor-bearing mice had an average weight of 25 g and were randomly divided into 6 groups, with 6 mice in each group.

[0335] 7.1.2 Treatment in mouse models

[0336] The labeled compound obtained in 3.4 of the above Experimental Example 3 and saline were respectively injected into the tail vein of tumor-bearing mice, wherein 100 μL saline was given to the G1 group and 100 μL saline was given to the G2 group. 177 Lu-PSMA-617 (60 MBq), 100 μL was given to the G3 group 177 Lu-Example 2 (60 MBq), 100 μL given to G4 group 177 Lu-Example 4 (60 MBq), 100 μL given to G5 group 177 Lu-Example 4 (20 MBq), 100 μL given to G7 group177 Lu-Example 3 (20 MBq). The tumor volume was measured every Monday and Thursday for 40 days. After the experiment, the mice were euthanized on the 42nd day. The tumor size was determined by measuring the long axis (a) and the vertical axis (b) of the tumor with a vernier caliper. According to the formula [V = 0.5*(a*b 2 )] Calculate the tumor volume (V).

[0337] 7.2 Test results

[0338] As time went on, mice in group G1 (normal saline control group) showed continuous tumor growth. On the 3rd day after administration, all groups of the labeled compound of the present invention effectively inhibited tumor growth. On the 6th day, all mice in group G4 died due to excessive dosage; on the 19th day after administration, mice in group G7 ( 177 Lu-Example 3, 20MBq dose) that is, the tumors of 4 mice completely disappeared, accounting for 67.7%. On the 36th day, the tumors of all mice completely disappeared, and the disappeared tumors did not grow again until the end of the experiment; at this time, the G2 group ( 177 Lu-PSMA-617) only 2 mice had tumor disappearance, which shows that the labeled compound of the present invention is superior to high dose at relatively low dose. 177 The effect of Lu-PSMA-617. The statistical data of the efficacy test on the tumor-bearing mouse model are shown in Table 14, and the changes in tumor volume on the 40th day of the tumor-bearing mouse model are shown in Appendix Figure 4 .

[0339] Table 15 Statistics of drug efficacy test data in tumor-bearing mouse model

[0340]

[0341]

[0342] “——” means not applicable.

[0343] Experimental Example 8: Competitive Binding Affinity Assay

[0344] By using 177 Lu-PSMA-617 (0.4 μCi / mL) was used as a competitive cell binding assay for the radioligand to determine the binding affinity of the test compounds.

[0345] Select LNCaP cells (source: ATCC, USA, supplier: Nanjing Kebai Biotechnology Co., Ltd.) with good growth in logarithmic phase, add binding buffer (RPMI-1640 medium mixed with 0.25% bovine serum albumin) to prepare cell suspension (cell concentration is about 1-3×10 6 / mL).

[0346] Use DMSO to prepare respective 2000 μM stock solutions of Examples 1 to 4, respectively, and aspirate 5 μL of the stock solution, add 245 μL of binding buffer to prepare working solution stock solutions with a final concentration of 40 μM, and then dilute the working solution stock solutions of each example with binding buffer to prepare working solutions with concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM, and 10000 nM.

[0347] Add 100 μL of cell suspension and 50 μL of radiolabeled compound to the 96-well filter plate. 177 Lu-PSMA-617 (0.4 μCi / mL) and 50 μL of one of the working solutions of Example 1 (or Example 2, or Example 3, or Example 4), 200 μL per well. Incubate in a 96-well filter plate system at 37°C incubator for 1 hour, remove the culture solution by suction, add 200 μL of binding buffer to each well, and repeat the washing and suction filtration 3 times. Remove the plate holder and dry it naturally, place the filter membrane at the bottom of a centrifuge tube (0.5 mL), and detect the count by γ-counting (source: PerkinElmer, USA, model: 2480WIZARD2).

[0348] According to the test results, the compound of the present invention and the labeled compound have good competitive binding ability.

[0349] Test Example 9: Low-dose efficacy test in tumor-bearing mouse model

[0350] 9.1 Materials and methods

[0351] 9.1.1 Tumor Mouse Model

[0352] M-NSG mice (6-8 weeks, male) were subcutaneously inoculated with LNCaP clone FGC cells (5*10 6 / pc, source: American ATCC, catalog number: CRL-1740), wait until the tumor volume grows to 200-300mm 3 The tumor-bearing mice had an average weight of 25 g and were randomly divided into 4 groups, with 6 mice in each group.

[0353] 9.1.2 Treatment in mouse models

[0354] The labeled compound obtained in 3.4 of the above Experimental Example 3 was diluted with saline to a certain concentration, and then injected into the tail vein of each group of tumor-bearing mice with saline, so that 100 μL of saline was given to group C1 and 100 μL of saline was given to group C2. 177 Lu-PSMA-617 (20 MBq), 100 μL was given to group C3 177 Lu-Example 3 (3 MBq), 100 μL given to C4 group177 Lu-Example 3 (10 MBq). The experiment was terminated after 30 days of observation or when the vehicle control group died. During this period, the tumor volume was measured twice a week and the number of surviving tumor-bearing mice was recorded. The tumor size was determined by measuring the long axis (a) and its vertical axis (b) with a vernier caliper. According to the formula [V = 0.5*(a*b 2 )] Calculate the tumor volume (V).

[0355] According to the test results, the labeled compound of the present invention can still effectively inhibit tumor growth at a low dose.

[0356] Over time, mice in group C1 (saline control group) showed sustained tumor growth, while mice in group C2 ( 177 Lu-PSMA-617, 20MBq dose) mice died on the 13th day after administration, and 50% of the mice died on the 23rd day. Only 2 mice survived on the 30th day. Meanwhile, although the C2 group showed a weak tumor inhibition effect on the 20th day, the tumor size rebounded on the 27th day. The labeled compounds of the present invention significantly prolonged the survival rate of mice. On the 20th day, the C3 group ( 177 Lu-Example 3, 3MBq dose), C4 group ( 177 No mice died in the Lu-Example 3, 10MBq dose) group. Although the tumor inhibition effect of the C3 group was delayed and the tumor size rebounded on the 30th day, the survival rate and tumor inhibition ratio of the mice in the C3 group were significantly better than those in the high-dose C2 group; in addition, the C4 group showed continuous and effective tumor inhibition, and no tumor size rebound was observed until the end of the experiment (30 days), and the mice maintained a higher survival rate. It can be seen that the labeled compound of the present invention is better than the high-dose at a relatively low dose. 177 The superior effect of Lu-PSMA-617. The statistical data of the efficacy test on the tumor-bearing mouse model are shown in Table 16.

[0357] Table 16 Statistics of low-dose efficacy test data in tumor-bearing mouse model

[0358]

[0359]

[0360] “——” means not applicable.

Claims

1. A compound represented by formula (I), a pharmaceutically acceptable salt thereof, an ester thereof, a solvate thereof, or a radiolabeled complex thereof, n is selected from any integer from 1 to 5, m is selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, p is selected from any integer between 0 and 6, and each R 1 Each is independently selected from C1-C6 alkyl, halogen, C1-C6 alkoxy, R is a chelating group or a chelating group residue.

2. The compound according to claim 1, its pharmaceutically acceptable salt, or its ester, or its solvate, or its radiolabeled complex, characterized in that: The n is selected from any integer from 1 to 3.

3. The compound according to claim 1, its pharmaceutically acceptable salt, or its ester, or its solvate, or its radiolabeled complex, characterized in that: The m is selected from 11, 12, 13, 14, 15, and 16.

4. The compound according to claim 1, its pharmaceutically acceptable salt, or its ester, or its solvate, or its radiolabeled complex, characterized in that: The m is selected from 8, 11, 14, and 16.

5. The compound according to claim 1, its pharmaceutically acceptable salt, or its ester, or its solvate, or its radiolabeled complex, characterized in that: The compound is a compound represented by formula (II), m is selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, p is selected from any integer between 0 and 4, and each R 1 Each is independently selected from C1-C6 alkyl, halogen, C1-C6 alkoxy, R is a chelating group residue.

6. The compound according to claim 5, its pharmaceutically acceptable salt, or its ester, or its solvate, or its radiolabeled complex, characterized in that: The m is selected from 11, 12, 13, 14, 15, and 16.

7. The compound according to claim 5, its pharmaceutically acceptable salt, or its ester, or its solvate, or its radiolabeled complex, characterized in that: The m is selected from 8, 11, 14, and 16.

8. The compound according to any one of claims 1 to 4, its pharmaceutically acceptable salt, or its ester, or its solvate, or its radiolabeled complex, characterized in that: The chelate group is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, N,N"-bis[2-hydroxy-5-(carboxyethyl)-benzyl]ethylenediamine-N,N"-diacetic acid, 1,4,7-triazacyclononane-1,4,7-triacetic acid, 2-(4,7-bis(carboxymethyl)-1,4,7-triazol N-1-yl)pentanedioic acid, 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)-pentanedioic acid, 1,4,7-triazacyclononane Phosphinic acid, 1,4,7-triazacyclononane-1-[methyl(2-carboxyethyl)-phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid], 3,6,9,15-tetraazabicyclo[9,3,1]pentadecan-1(15),11,13-triene-3,6,9-triacetic acid, N'-{5-[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide, diethyltriaminepentaacetic acid.

9. The compound according to any one of claims 1 to 7, its pharmaceutically acceptable salt, or its ester, or its solvate, or its radiolabeled complex, characterized in that: The chelating group residue is selected from 10. The compound according to claim 1, its pharmaceutically acceptable salt, or its ester, or its solvate, or its radiolabeled complex, characterized in that: The compound is a compound represented by formula (III), n is selected from any integer from 1 to 3, m is selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, p is selected from any integer between 0 and 4, and each R 1 Each is independently selected from C1-C6 alkyl, halogen, and C1-C6 alkoxy.

11. The compound according to claim 10, its pharmaceutically acceptable salt, or its ester, or its solvate, or its radiolabeled complex, characterized in that: The n is selected from 2.

12. The compound according to claim 10, its pharmaceutically acceptable salt, or its ester, or its solvate, or its radiolabeled complex, characterized in that: The m is selected from 11, 12, 13, 14, 15, and 16.

13. The compound according to claim 10, its pharmaceutically acceptable salt, or its ester, or its solvate, or its radiolabeled complex, characterized in that: The m is selected from 8, 11, 14, and 16.

14. The compound according to any one of claims 1 to 13, its pharmaceutically acceptable salt, or its ester, or its solvate, or its radiolabeled complex, characterized in that: The p is 0.

15. The compound according to any one of claims 1 to 13, its pharmaceutically acceptable salt, or its ester, or its solvate, or its radiolabeled complex, characterized in that: The R 1 Each is independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, fluorine, chlorine, bromine, and iodine.

16. The compound according to any one of claims 1 to 15, its pharmaceutically acceptable salt, or its ester, or its solvate, or its radiolabeled complex, characterized in that: Selected from the following compounds, 17. A radiolabeled complex, characterized in that The complex is a chelate formed by the compound according to any one of claims 1 to 16, its pharmaceutically acceptable salt, ester or solvate thereof and a metal radionuclide. Preferably, the metal radionuclide is selected from 51 Cr, 94 Tc, 94m Tc, 99m Tc, 110m In, 111 In, 113m In, 114m In, 117m Sn, 121 Sn, 67 Ga, 68 Ga, 86 Y. 87 Y. 90 Y. 177 Lu, 186 Re, 187 Re, 188 Re, 62 Cu, 64 Cu, 67 Cu, 55 Co. 57 Co. 58 Co. 60 Co. 43 Sc, 44 Sc, 47 Sc, 48 Sc, 153 Sm, 225 Ac, 213 Bi, 212 Bi, 227 Th, 153 Sm, 166 Ho, 152 Gd, 153 Gd, 155 Gd, 157 Gd, 166 Dy, 223 Ra, 224 Ra, 82 Rb, 89 Zr, 59 Fe, 90 Sr.

18. A non-radioactive labeled complex, characterized in that The complex is a chelate formed by the compound according to any one of claims 1 to 16, its pharmaceutically acceptable salt, ester or solvate thereof and a non-radioactive metal nuclide. Preferably, the non-radioactive metal nuclide is selected from 175 Lu, 113 In, 115 In, 89 Y. 144 Sm, 147 Sm, 148 Sm, 149 Sm, 150 Sm, 152 Sm, 154 Sm, 185 Re, 69 Ga, 71 Ga, 63 Cu, 65 Cu.

19. A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 16, a pharmaceutically acceptable salt thereof, or an ester thereof, or a solvate thereof, or at least one complex according to any one of claims 17 to 20, and at least one pharmaceutically acceptable excipient.

20. Use of a compound according to any one of claims 1 to 16, a pharmaceutically acceptable salt thereof, an ester thereof, or a solvate thereof for preparing a therapeutic or diagnostic agent, or use of a complex according to any one of claims 17 to 20 or a pharmaceutical composition thereof for preparing a therapeutic or diagnostic agent, or use of a pharmaceutical composition according to claim 21 for preparing a therapeutic or diagnostic agent. Preferably, the therapeutic agent is used to treat cancer, and the diagnostic agent is used to diagnose cancer. More preferably, the cancer is a solid tumor or a hematological tumor. More preferably, the cancer comprises prostate cancer, pancreatic cancer, kidney cancer or bladder cancer, liver cancer, neuroblastoma, non-Hodgkin's lymphoma.

Images