Macrocyclic compounds and their radiolabeled complexes as ligands for targeted radiotherapy applications

Abstract

The present invention relates to compounds of formula (I) that have the potential to be used as metal complexes in radiotherapy. TIFF2024538619000043.tif85132 (wherein A is CO2R 1 , and PO3R 1 and B is selected from the group consisting of COR 2 , and PO3R 2 R 1 , R 2 and R 3 are each independently H and C1 to C 12 alkyl, and each R a is independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN; b are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN, and L is a linker having 1 to 20 atoms in the normal chain), or a pharma- ceutically acceptable salt thereof.

Description

[Technical Field]

[0001]

[0001] The present invention relates to macroring-containing compounds that can be used as ligands in targeted radiotherapy applications. The present invention also relates to certain metal complexes of these compounds and methods of using them. [Background technology]

[0002]

[0002] Over the past 200 years, remarkable progress has been made in medical research, resulting in the development of effective medical treatments for a significant number of diseases / conditions, which has dramatically increased life expectancy. Unfortunately, this success in providing improved treatments for certain conditions has meant that humans now live long enough to develop and suffer from other conditions that were previously relatively rare, typically being very slow-developing or “old age” diseases.

[0003]

[0003] For example, cancer is the largest contributor to the disease burden in Australia (16%) and is estimated to be the second leading cause of death globally. According to the World Health Organization, an estimated 10 million people died from cancer in 2020, although this number is likely significantly lower than the actual total due to potential underreporting in developing countries. Nevertheless, the magnitude of the problem is highlighted by the estimated 1.8 million deaths from lung cancer, 935,000 from colorectal cancer, 830,000 from liver cancer, 769,000 from stomach cancer, and 685,000 from breast cancer in 2020. Thus, the economic impact of cancer is significant and increasing. The global annual economic cost associated with cancer is estimated to exceed US$1.2 trillion, although the exact cost is difficult to quantify. This figure is expected to increase as life expectancy increases and as lifestyle, diet, and / or environmental factors change over time.

[0004]

[0004] Cancer is a somewhat general term used to describe a large group of diseases that can affect any part (port) of the body. A common feature of almost all cancers is the rapid production by the body of abnormal cells that can grow beyond normal boundaries and then invade other parts of the body. Thus, cancer can be described as the uncontrolled proliferation of cells that can invade and spread to other parts of the body. The causes of cancer are generally thought to be environmental or genetic factors. More than 100 different types of cancer are known, and more new types are characterized each year.

[0005]

[0005] Cancer cells can exist in several different forms. For example, they can exist as a solid tumor, where cancer cells are clustered together, or as dispersed cells, as in leukemia. Cancer cells are often called "malignant" because they divide uncontrollably and eventually push aside nearby cells and spread to other parts of the body. The tendency of cancer cells to spread from one organ to another or from one part of the body to another distinguishes cancer cells from benign tumor cells that overgrow but do not spread to other organs or other parts of the body. Malignant cancer cells can eventually metastasize through the bloodstream or lymphatic system, spreading to other parts of the body, where they proliferate and form new tumors. This type of tumor progression makes cancer a fatal disease.

[0006]

[0006] Although the diagnosis and treatment of cancer have improved significantly, many people still die from cancer every year. Therefore, several treatments have been developed that have been used to treat cancer patients, and ongoing efforts are being made to develop further improved cancer treatments.

[0007]

[0007] One area of ​​growing interest is the use of radiotherapy in the treatment of cancer. Radiotherapy is based on the observation that high doses of radiation kill cancer cells or slow their growth as a result of radiation damaging the DNA of cancer cells. Cancer cells, like other cells, stop dividing and die when their DNA is damaged beyond repair. Following cell death, the cells are destroyed and removed from the body.

[0008]

[0008] Radiotherapy is typically divided into two main types, namely (1) external beam radiotherapy and (2) internal radiotherapy, and in either case, the type of radiotherapy used depends on several factors specific to the patient.

[0009]

[0009] External beam radiotherapy involves the use of a machine that irradiates cancer in a patient. This type of radiotherapy is intended to treat a specific part of the body, and as a result, only the area where the cancer is located is exposed to radiation, rather than the whole body. This type of therapy can be very effective for tumors located deep within the body, but because it targets cancer cells within the body, it is inevitable that some healthy cells will be exposed to radiation.

[0010]

[0010] Internal radiotherapy involves either seeding a radioactive material at the site of a tumor or using a chemotherapy agent that "targets" the site of cancer. In these targeted methods, the ligand of a radionuclide is typically conjugated with an antibody that targets the specific cancer to be treated. For example, targeted alpha particle therapy has been developed for the treatment of soft tissue metastases. In these therapies, a radionuclide that emits lethal alpha particles is conjugated with a tumor-targeting vector using a chelator. This therapy allows the delivery of cytotoxic levels of alpha radiation to be selectively delivered to cancer cells (cancer calls).

[0011]

[0011] As a result, there has been considerable research interest in developing conjugates that can be used to bind to radionuclides that emit alpha particles, and which can then be further processed to include a portion that targets cancer. Several potential conjugates have been developed.

[0012]

[0012] One of the best conjugates currently in use is H4DOTA(1).

[0013] [ka]

[0014]

[0013] This conjugate is relatively easy to obtain, but it has been plagued by the observation that the thermodynamic stability of the H4DOTA complex decreases as the number of ionic metal ions increases, resulting in Ac, the largest +3 (trivalent) ion in the periodic table. 3+ It is not ideally suited to all metal complexes, such as those mentioned above. In addition, the radiolabeling reaction rate of this ligand is not suitable for some radionuclides (e.g., Ac 3 The process is relatively slow at +, which means that if a short labeling time is required, labeling at high temperatures is necessary.

[0015]

[0014] As a result, several studies have been conducted on the development of alternative conjugates. One alternative conjugate developed is N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6(2), which is colloquially known as H2macropa.

[0016] [ka]

[0017]

[0015] Another conjugate developed is an analog of H2macropa. That is, H2macropa-NCS(3):

[0018] [ka]

[0019] That is the case.

[0016] These conjugates could be more radiolabeled without the need for high temperatures, but there were problems, as aggregation of the conjugates was observed.

[0020]

[0017] Thus, there is great interest in the development of additional compounds that can be used as radionuclide conjugates in targeted therapy.

Summary of the Invention

Problems to be Solved by the Invention

[0021]

[0018] The present applicants have conducted research aimed at developing compounds that can be used as potential radionuclide conjugates and can thus be utilized in targeted therapy.

Means for Solving the Problems

[0022]

[0019] As a result of these studies, the present applicants have identified compounds that show great potential as conjugates for radionuclides.

[0020] Thus, in one embodiment, the present invention provides a compound of formula (I):

[0023]

Chemical formula

[0024]

[0021] (wherein,

[0022] A is selected from the group consisting of CO2R 1 , and PO3R 1 ,

[0023] B is selected from the group consisting of CO2R 2 , and PO3R 2 ,

[0024] R 1 , R 2 and R 3 are each independently selected from the group consisting of H and C1-C 12 alkyl,

[0025] each R a is independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NHCI2, and CN,

[0026] Each R b These are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN.

[0027] L is a linker that has 1 to 20 atoms in its normal chain.

[0028] or provide a pharmaceutically acceptable salt thereof.

[0025]

[0029] The compounds of the present invention have been found to have the ability to form metal complexes with radionuclides, and therefore can be used in targeted therapy.

[0030] In yet another embodiment, the present invention relates to a compound of formula (Ia):

[0026] [ka]

[0027]

[0031] (In the formula,

[0032] A is CO2R 1 , and PO3R 1 Selected from the group consisting of,

[0033] B is CO2R 2 , and PO3R 2 Selected from the group consisting of,

[0034] R 1 , R 2 and R 3 These are H and C1-C, each independently. 12 Selected from the group consisting of alkyl groups,

[0035] Each R a These are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN.

[0036] Each R bThese are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN.

[0037] L is a linker having 1 to 20 atoms in its normal chain.

[0038] M is a radioactive nuclide.

[0039] or provide a pharmaceutically acceptable salt thereof.

[0028]

[0040] The compounds of formula (I) or (Ia) described herein have the ability to react with antibodies to form targeting conjugates. Therefore, in yet further embodiments, the present invention relates to the compounds of formula (Ib):

[0029] [ka]

[0030]

[0041] (In the formula,

[0042] A is CO2R 1 , and PO3R 1 Selected from the group consisting of,

[0043] B is CO2R 2 , and PO3R 2 Selected from the group consisting of,

[0044] R 1 , and R 2 These are H and C1-C, each independently. 12 Selected from the group consisting of alkyl groups,

[0045] R 4 It is a monoclonal antibody,

[0046] Each R a These are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN.

[0047] Each R bThese are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN.

[0048] L is a linker having 1 to 20 atoms in its normal chain.

[0049] M is a radioactive nuclide.

[0050] or provide a pharmaceutically acceptable salt thereof.

[0031]

[0051] In yet another embodiment, the present invention relates to a method for synthesizing the compound of formula (I) described above,

[0052] (a) Compound of formula (10):

[0032] [ka]

[0033]

[0053] (A is CO2R 1 , and PO3R 1 Selected from the group consisting of,

[0054] B is CO2R 2 , and PO3R 2 Selected from the group consisting of,

[0055] R 1 , and R 2 These are H and C1-C, each independently. 12 Selected from the group consisting of alkyl groups,

[0056] Each R a These are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN.

[0057] Each R b This involves independently preparing (selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN),

[0058] (b) The compound of formula (10) is the compound of formula (11)

[0034] [ka]

[0035]

[0059] (In the formula, R 3 H and C1~C 12 Selected from the group consisting of alkyl groups,

[0060] L is a linker that has 1 to 20 atoms in its normal chain,

[0061] A step in which the reaction is carried out in the presence of a copper(I) catalyst. This provides a method that includes [something].

[0036]

[0062] In yet another embodiment, the present invention provides a pharmaceutical composition comprising a compound according to formula (I), formula (Ia), or formula (Ib) and a pharmaceutically acceptable diluent, excipient, or carrier.

[0037]

[0063] In yet another embodiment, the present invention provides a method for treating a subject, comprising the step of administering a therapeutically effective amount of a compound of formula (Ib) to the subject. In a particular embodiment, the subject is suffering from cancer. [Brief explanation of the drawing]

[0038] [Figure 1]

[0064] This figure shows the TLC analysis of free Ac-225 using 0.4M sodium citrate pH 4 + 10% methanol (origin at 15 mm, solvent tip at 80 mm; free Ac-225 is located at the solvent tip: Rf = 0.8~1.0). [Figure 2]

[0065] This figure shows the iTLC analysis of free Ac-225 using 50 mM citrate pH 5 (origin at 15 mm, solvent tip at 80 mm; free Ac-225 is located at the solvent tip: Rf = 0.8~1.0). [Figure 3]

[0066] This figure shows the TLC analysis of crude [225Ac]Ac-macropa at a chelator concentration of 10⁻³M using 0.4M sodium citrate pH 4 + 10% methanol (origin at 10 mm, solvent tip at 80 mm; [225Ac]Ac-macropa remains at the origin: Rf=0; free Ac-225 is at the solvent tip: Rf=0.8~1.0). [Figure 4]

[0067] Unless otherwise noted, this figure outlines the radiochemical yields of various Ac-225-labeled chelators at different chelator concentrations after incubation at ambient temperature for 2 hours. Radiochemical yields were determined by TLC analysis of the crude reaction mixture using 0.4 M sodium citrate, pH 4, and 10% methanol. [Figure 5]

[0068] This figure outlines the radiochemical yields of [225Ac]Ac-macropa-tzPEG3SqOEt at different chelator concentrations over time, incubated at ambient temperature. Radiochemical yields were determined by TLC analysis of the crude reaction mixture using 0.4 M sodium citrate, pH 4, and 10% methanol. [Figure 6]

[0069] This figure outlines the radiochemical yields of [225Ac]Ac-DOTA-methyltetrazine at different chelator concentrations over time, incubated at 90°C. Radiochemical yields were determined by TLC analysis of the crude reaction mixture using 0.4 M sodium citrate, pH 4, and 10% methanol. [Figure 7A]

[0070] This figure shows iTLC analysis of (a) [225Ac]Ac-macropa-tzPEG3Sq-EGFRVIII IgG1 and (b) [225Ac]Ac-macropa-tzPEG3Sq-EphA3 IgG1 at an antibody concentration of 10⁻⁶ M after incubation at ambient temperature for 15 minutes (50 mM citrate pH 5; origin at 10 mm, solvent tip at 80 mm; [225Ac]Ac-macropa-tzPEG3Sq-conjugate monoclonal antibody remains at origin: Rf=0; free Ac-225 is at solvent tip: Rf=0.8~1.0). [Figure 7B] This figure shows iTLC analysis of (a) [225Ac]Ac-macropa-tzPEG3Sq-EGFRVIII IgG1 and (b) [225Ac]Ac-macropa-tzPEG3Sq-EphA3 IgG1 at an antibody concentration of 10⁻⁶ M after incubation at ambient temperature for 15 minutes (50 mM citrate pH 5; origin at 10 mm, solvent tip at 80 mm; [225Ac]Ac-macropa-tzPEG3Sq-conjugate monoclonal antibody remains at origin: Rf=0; free Ac-225 is at solvent tip: Rf=0.8~1.0). [Figure 8]

[0071] This figure shows the SE-HPLC chromatogram (UV280) of H2macropa-tzPEG3Sq-immunoconjugate. [Figure 9]

[0072] This figure shows the SE-HPLC chromatogram (UV280 and radiation) of [225Ac]Ac-macropa-tzPEG3Sq-immunoconjugate. [Figure 10]

[0073] Figure 10A shows the serum stability studies of [225Ac]Ac-macropa-tzPEG3Sq- and [225Ac]Ac-DOTA-dhPzPEG4-conjugate monoclonal antibodies at different chelator-antibody ratios, with Figure 10A showing the radiochemical purity determined using iTLC (50mM EDTA pH5), and Figure 10B showing the serum stability studies of [225Ac]Ac-macropa-tzPEG3Sq- and [225Ac]Ac-DOTA-dhPzPEG4-conjugate monoclonal antibodies at different chelator-antibody ratios, with the immunoreactivity determined for EGFRVIII IgG1 and EphA3 IgG1 using U87MG.de2-7 and U251 cells, respectively. [Figure 11A]

[0074] This figure shows (a) tumor growth curves and (b) Kaplan-Meier survival curves for U251 mouse xenografts (n=5) treated with varying amounts of [225Ac]Ac-macropa-tzPEG3Sq-EphA3 IgG1 (0.25~1.0 μg / 9.25~37.0 kBq) or vehicle. [Figure 11B] This figure shows (a) tumor growth curves and (b) Kaplan-Meier survival curves for U251 mouse xenografts (n=5) treated with varying amounts of [225Ac]Ac-macropa-tzPEG3Sq-EphA3 IgG1 (0.25~1.0 μg / 9.25~37.0 kBq) or vehicle. [Figure 12A]

[0075] This figure shows (a) tumor growth curves and (b) Kaplan-Meier survival curves for U87MG.de2-7 mouse xenografts (n=5) treated with varying amounts of [225Ac]Ac-macropa-tzPEG3Sq-EGFRVIII IgG1 (0.25~1.0 μg / 9.25~37.0 kBq) or vehicle. [Figure 12B] This figure shows (a) tumor growth curves and (b) Kaplan-Meier survival curves for U87MG.de2-7 mouse xenografts (n=5) treated with varying amounts of [225Ac]Ac-macropa-tzPEG3Sq-EGFRVIII IgG1 (0.25~1.0 μg / 9.25~37.0 kBq) or vehicle. [Modes for carrying out the invention]

[0039]

[0076] This specification uses several terms that are well known to those skilled in the art. Nevertheless, for clarity, some terms are defined.

[0077] In some of the definitions of substituents below, it is stated that "the group may be a terminal group or a bridging group." This is intended to indicate that the use of this term is intended to encompass situations in which the group is a linker between two other parts of a molecule and situations in which it is a terminal part. Using the term alkyl as an example, some publications use the term alkylene for bridging groups, and therefore, in these other publications, there is a distinction between the terms alkyl (terminal group) and alkylene (bridging group). In this application, no such distinction is made, and most groups may be either bridging groups or terminal groups.

[0040]

[0078] Unless otherwise noted, "alkyl" as a group or part of a group refers to a linear or branched aliphatic hydrocarbon group, preferably C1-C1. 12 Alkyl, more preferably C1-C 10 Alkyl, most preferably C1-C6, is the term. Examples of suitable linear and branched C1-C6 alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t-butyl, and hexyl. The group may be a terminal group or a crosslinking group.

[0041]

[0079] The term "normal chain" refers to a straight chain connecting the two ends of a linkage.

[0080] The term “pharmaceutically acceptable salt” refers to salts, including acid and base addition salts, that retain the desired biological activity of the compounds identified above and are pharmaceutically acceptable. Suitable pharmaceutically acceptable acid addition salts of the compounds of formula (I) can be prepared from inorganic or organic acids. Examples of such inorganic acids are hydrochloric acid, sulfuric acid, and phosphoric acid. Suitable organic acids may be selected from the aliphatic, alicyclic, aromatic, and heterocyclic carboxylic acid and sulfonic acid classes, examples of which are formic acid, acetic acid, propanoic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, fumaric acid, maleic acid, alkylsulfonic acid, and arylsulfonic acid. Similarly, base addition salts can be prepared using organic or inorganic bases in ways well known in the art. Examples of suitable organic bases include simple amines, such as methylamine, ethylamine, and triethylamine. Examples of suitable inorganic bases include NaOH and KOH. Additional information regarding pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 19th edition, Mack Publishing Co., Easton, PA 1995. For solid pharmaceuticals, it will be understood by those skilled in the art that the compounds, pharmaceuticals, and salts of the present invention may exist in different crystalline or polymorphic forms, all of which are intended to fall within the scope of the present invention and the defined formulas.

[0042]

[0081] The term "therapeutic dose" or "effective dose" refers to an amount sufficient to produce a beneficial or desired clinical outcome. An effective dose may be administered in one or more doses. Typically, an effective dose is sufficient to alleviate, improve, stabilize, reverse, slow, or delay the progression of a disease.

[0043]

[0082] As described above, the compounds of the present invention have formulas (I), (Ia), or (Ib). As with any group of structurally related compounds that possess specific utility, certain embodiments of the compounds of formula (I), (Ia), or (Ib) with variable elements are particularly useful in their end use.

[0044]

[0083] In the compound of the present invention, A is CO2R 1 , and PO3R 1 Selected from the group consisting of the following. In some embodiments, A is CO2R 1 In some embodiments, A is PO3R 1 That is the case.

[0045]

[0084] In the compound of the present invention, B is CO2R 2 , and PO3R 2 Selected from the group consisting of the following. In some embodiments, B is CO2R 2 In some embodiments, B is PO3R 2 That is the case.

[0046]

[0085] In the compound of the present invention, R 1 , R 2 and R 3 These are H and C1-C, each independently. 12 Selected from the group consisting of alkyl groups.

[0086] In some embodiments, R 1 is H. In some embodiments, R 1 C1~C 12 It is alkyl. In some embodiments, R 1 The compound is selected from the group consisting of methyl, ethyl, isopropyl, propyl, 2-ethyl-propyl, 3,3-dimethyl-propyl, butyl, isobutyl, 3,3-dimethyl-butyl, 2-ethyl-butyl, pentyl, 2-methyl, pentyl, and hexyl.

[0047]

[0087] In some embodiments, R 2 H is H. In some embodiments, R 2 C1~C 12 It is alkyl. In some embodiments, R 2 The compound is selected from the group consisting of methyl, ethyl, isopropyl, propyl, 2-ethyl-propyl, 3,3-dimethyl-propyl, butyl, isobutyl, 3,3-dimethyl-butyl, 2-ethyl-butyl, pentyl, 2-methyl, pentyl, and hexyl.

[0048]

[0088] In some embodiments of the compound of the present invention, A is CO2R 1 Therefore, B is CO2R 2 And R 1 H is R 2 is H. This gives compounds of formulas (II), (IIa), and (IIb), respectively:

[0049] [ka]

[0050]

[0089] (In the formula, R a , R b , L and R 3 (This is as defined above).

[0051] [ka]

[0052]

[0090] (In the formula, R a , R b , LR 3 (and M is as defined above).

[0053] [ka]

[0054]

[0091] (In the formula, R a , R b , LR 4 (and M is as defined above).

[0092] In the compound of the present invention, each R a Each R is independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN. In some embodiments, each R ais independently selected from H, F, Cl, Br, I, CH3, CH2CH3, OH, OCH3, OCH2CH3, and CN. In some embodiments, each R a is independently selected from H, CH3, CH2CH3, and OH. In some embodiments, each R a is H.

[0055]

[0093] In the compounds of the present invention, each R b is independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN. In some embodiments, each R b is independently selected from H, F, Cl, Br, I, CH3, CH2CH3, OH, OCH3, OCH2CH3, and CN. In some embodiments, each R b is independently selected from H, CH3, CH2CH3, and OH. In some embodiments, each R b is H.

[0056]

[0094] In some embodiments of the compounds of the present invention, A is CO2R 1 where B is CO2R 2 where R 1 is H, R 2 is H, each R a is H, and each R b is H. This gives the compounds of formulas (III), (IIIa), and (IIIb), respectively.

[0057]

Chemical formula

[0058]

[0095] (where L and R 3 are as defined above).

[0059]

Chemical formula

[0060]

[0096] (where L, R 3 and M are as defined above).

[0061]

Chemical Formula

[0062]

[0097] (where L, R 4 and M are as defined above).

[0098] In some embodiments, R 3 is H. In some embodiments, R 3 is C1-C 12 alkyl. In some embodiments, R 3 is selected from the group consisting of methyl, ethyl, isopropyl, propyl, 2-ethyl-propyl, 3,3-dimethyl-propyl, butyl, isobutyl, 3,3-dimethyl-butyl, 2-ethyl-butyl, pentyl, 2-methyl, pentyl, and hexyl. In some embodiments, R 3 is methyl. In some embodiments, R 3 is ethyl.

[0063]

[0099] R 4 In the compounds of the present invention containing the moiety, R 4 is an antibody. In certain embodiments, R 4 is a monoclonal antibody. In certain embodiments, R 4These include penplimab, cintilimab, tripalimab, omblutamab, tisotumab, retifanlimab, ubrituximab, aniflorumab, lonkastuximab, valstilimab, dostallimab, oporutuzumab, marjetuximab, naxitamab, verantamab, tafacitamab, sacituzumab, isatuximab, trastuzumab (Herceptin), gilentuximab, ifabotuzumab, depatuxizumab, enfortumab, polatuzumab, emaparmab, semiplimab, moxetumomab, mogamulizumab, durvalumab, and ave The following drugs are selected from the group consisting of rumab, atezolizumab, oraratumab, daratumumab, elotuzumab, necitumumab, dinutuximab, nivolumab, blinatumomab, blinatumomab, ramucirumab, obinutuzumab, ad-trastuzumab, pertuzumab, brentuximab, ipilimumab, ofatumumab, katumakisomab, panitumumab, bevacizumab, cetuximab, tositumomab-I131, ibritumomab, alemtuzumab, gemtuzumab, rituximab, edrecolomab, nimotuzumab, prorugolimab, and cetuximab.

[0064]

[0100] In one embodiment, the antibody is Herceptin. In one embodiment, the antibody is EphA3 IgG1 monoclonal antibody. In one embodiment, the antibody is EGFRVIII IgG1 monoclonal antibody. In one embodiment, the antibody is isotype control IgG1.

[0065]

[0101] In the compounds of the present invention, L is a linker having 1 to 20 atoms in the normal chain. In some embodiments, L is a linker having 2 to 19 atoms in the normal chain. In some embodiments, L is a linker having 3 to 18 atoms in the normal chain. In some embodiments, L is a linker having 4 to 17 atoms in the normal chain. In some embodiments, L is a linker having 5 to 16 atoms in the normal chain. In some embodiments, L is a linker having 6 to 15 atoms in the normal chain. In some embodiments, L is a linker having 7 to 14 atoms in the normal chain. In some embodiments, L is a linker having 8 to 13 atoms in the normal chain. In some embodiments, L is a linker having 9 to 12 atoms in the normal chain.

[0066]

[0102] In some embodiments, L is a linker having 1 atom in the normal chain. In some embodiments, L is a linker having 2 atoms in the normal chain. In some embodiments, L is a linker having 3 atoms in the normal chain. In some embodiments, L is a linker having 4 atoms in the normal chain. In some embodiments, L is a linker having 5 atoms in the normal chain. In some embodiments, L is a linker having 6 atoms in the normal chain. In some embodiments, L is a linker having 7 atoms in the normal chain. In some embodiments, L is a linker having 8 atoms in the normal chain. In some embodiments, L is a linker having 9 atoms in the normal chain. In some embodiments, L is a linker having 10 atoms in the normal chain. In some embodiments, L is a linker having 11 atoms in the normal chain. In some embodiments, L is a linker having 12 atoms in the normal chain. In some embodiments, L is a linker having 13 atoms in the normal chain. In some embodiments, L is a linker having 14 atoms in its normal chain. In some embodiments, L is a linker having 15 atoms in its normal chain. In some embodiments, L is a linker having 16 atoms in its normal chain. In some embodiments, L is a linker having 17 atoms in its normal chain. In some embodiments, L is a linker having 18 atoms in its normal chain. In some embodiments, L is a linker having 19 atoms in its normal chain. In some embodiments, L is a linker having 20 atoms in its normal chain.

[0067]

[0103] In some embodiments, L is expressed by the formula: -(CH2) m - A linker having m is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0068]

[0104] In some embodiments, L is -(CH2)7-, -(CH2)8-, -(CH2)9-, -(CH2) 10 -,-(CH2) 11 -,-(CH2) 12 -,-(CH2) 13 -,-(CH2) 14 -, and -(CH2) 15 - Selected from the group consisting of these.

[0069]

[0105] In some embodiments, L is expressed by the formula: -(CH2CH2O) n -CH2CH2-

[0106] The linker is such that n is an integer from the group consisting of 0, 1, 2, 3, 4, and 5.

[0070]

[0107] In some embodiments, L is selected from the group consisting of -(CH2CH2O)-CH2CH2-, -(CH2CH2O)2-CH2CH2-, -(CH2CH2O)3-CH2CH2-, -(CH2CH2O)4-CH2CH2-, and -(CH2CH2O)5-CH2CH2-.

[0071]

[0108] In some embodiments, L is -(CH2CH2O)-CH2CH 2- In some embodiments, L is -(CH2CH2O)2-CH2CH 2- In some embodiments, L is -(CH2CH2O)3-CH2CH 2- In some embodiments, L is -(CH2CH2O)4-CH2CH 2- In some embodiments, L is -(CH2CH2O)5-CH2CH-2.

[0072]

[0109] In a particularly preferred embodiment, L is -(CH2CH2O)3-CH2CH 2- That is the case.

[0110] The macro-ring of the present invention can, in principle, bond to several metals. For applications, the compound is used in radiotherapy, and therefore, M is preferably a radionuclide. In certain embodiments, M is selected from the group consisting of actinium-225, lutetium-177, zirconium-89, terbium-149, terbium-152, terbium-155, terbium-161, radium-223, bismuth-212, indium-111, yttrium-86, yttrium-89, yttrium-90, and lead-212.

[0073]

[0111] In some embodiments, M is actinium-225. In some embodiments, M is lutetium-177. In some embodiments, M is zirconium-89. In some embodiments, M is terbium-149. In some embodiments, M is terbium-152. In some embodiments, M is terbium-155. In some embodiments, M is terbium-161. In some embodiments, M is radium-223. In some embodiments, M is bismuth-212. In some embodiments, M is indium-111. In some embodiments, M is yttrium-86. In some embodiments, M is yttrium-89. In some embodiments, M is yttrium-90. In some embodiments, M is lead-212.

[0074]

[0112] In a particularly preferred embodiment, M is actinium-225.

[0113] The compounds of the present invention, containing radionuclide M, are typically formed by incubation of a metal with a conjugate of a suitable formula. Incubation can be carried out at any temperature, but the bonding of the radionuclide with the compounds of the present invention is very rapid, and as a result, bonding can occur at room temperature, and is still found to be extremely rapid. Once bonded, the resulting complex is found to be very stable.

[0075]

[0114] In principle, metals can complex with antibody-containing compounds, but it is typically found to be more efficient to complex the radionuclide with the compound before adding the antibody. While we do not wish to dwell on theory, this is likely because the antibody site may compete with the macroring for binding to the metal, thus ensuring more efficient complexation. Therefore, it is common practice to react the complex with the radionuclide before adding the antibody.

[0076]

[0115] Antibody-containing compounds are typically formed by reacting compounds of formulas (I), (Ia), (II), (IIa), (III), and (IIIa) with the pendant amine group on the antibody to form the antibody-containing compound. As will be recognized by those skilled in the art, many antibodies contain multiple amine residues available for reactivity with the squaramid moiety on the compounds of the present invention. Therefore, in some embodiments, each antibody is conjugated with two or more compounds of the present invention. In some embodiments, each antibody is conjugated with two compounds of the present invention. In some embodiments, each antibody is conjugated with three compounds of the present invention. In some embodiments, each antibody is conjugated with four compounds of the present invention. In some embodiments, each antibody is conjugated with five compounds of the present invention. Control of reaction stoichiometry and general reaction conditions may be used to control the formation of the final antibody conjugate.

[0077]

[0116] Radionuclides (M) and targeting antibodies (R) 4 The compounds of the present invention, which contain ), have the ability to be used in targeted radiotherapy for cancer. As a result of the presence of antibodies on the compound, the compound targets (i.e., selectively binds to) cancer cells. As a result of the compound selectively binding to cancer cells, the radiation produced by the radionuclide is emitted in close proximity to the cancer cells, thereby causing greater damage to the cancer cells than to other cells in the body. This has been found to be particularly effective when the radionuclide emits alpha particles.

[0078]

[0117] Therefore, the compounds of the present invention are expected to have useful therapeutic properties in the treatment of cancer. Examples of cancers include prostate cancer, breast cancer, pancreatic cancer, colon cancer, non-small cell lung cancer, hepatocellular carcinoma, intrahepatic cholangiocarcinoma, renal cell carcinoma, endometrial cancer, esophageal cancer, esophageal / esophagogastric junction cancer, osteosarcoma, Wilms' tumor, mesothelioma, squamous cell carcinoma, glioblastoma multiforme, melanoma, and ovarian cancer.

[0079]

[0118] The compounds of the present invention may be administered to humans by any of the accepted parenteral administration methods, such as subcutaneous, intramuscular, intravenous, and intradermal routes. Injections may be by bolus or by continuous or intermittent infusion. The active compound is typically contained in a pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver a therapeutically effective dose to the patient.

[0080]

[0119] The compounds of the present invention may be administered in any form or manner that makes them available for binding to desired target cells. Those skilled in the art of pharmaceutical preparation can readily select an appropriate form and manner of administration depending on the specific properties of the selected compound, the condition to be treated, the stage of the condition to be treated, and other relevant circumstances. For further information, see Remingtons Pharmaceutical Sciences, 19th edition, Mack Publishing Co. (1995).

[0081]

[0120] The compounds of the present invention may be administered alone or in the form of pharmaceutical compositions in combination with pharmaceutically acceptable carriers, diluents, or excipients. Although the compounds of the present invention are effective on their own, they are typically formulated and administered in the form of their pharmaceutically acceptable salts, for these forms are typically more stable, more readily crystallized, and have increased solubility.

[0082]

[0121] However, the compounds are typically used in the form of pharmaceutical compositions formulated according to a desired dosage regimen. Therefore, in some embodiments, the present invention provides pharmaceutical compositions comprising the compounds of the present invention and pharmaceutically acceptable carriers, diluents, or excipients. The compositions are prepared in ways well known in the art.

[0083]

[0122] The pharmaceutical compositions of the present invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injection solutions or dispersions immediately before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injection organic esters, such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0084]

[0123] These compositions may also contain adjuvants, such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial action can be ensured by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. The inclusion of isotonic agents, such as sugars and sodium chloride, may also be desirable. Long-term absorption of the injectable drug form can be achieved by including substances that slow absorption, such as aluminum monostearate and gelatin.

[0085]

[0124] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use.

[0086]

[0125] The amount of compound administered is preferably such that it treats, alleviates, or mitigates the condition. The therapeutically effective dose can be readily determined by the attending physician by observing the results obtained using conventional techniques and under similar circumstances. When determining the therapeutically effective dose, several factors should be considered, including but not limited to the animal species, its size, age, and overall health status; the specific condition involved; the severity of the condition; the patient's response to treatment; the specific compound administered; the method of administration; the bioavailability of the administered preparation; the chosen dosing regimen; the use of other drug therapies; and other relevant circumstances.

[0087]

[0126] The preferred dosage is in the range of approximately 0.01 to 300 mg per kilogram of body weight per day. A more preferred dosage is in the range of 0.1 to 100 mg per kilogram of body weight per day, more preferably 0.2 to 80 mg per kilogram of body weight per day, and even more preferably 0.2 to 50 mg per kilogram of body weight per day. The preferred dose may be administered in multiple partial doses per day. Synthesis of the Compound of the Present Invention

[0127] The compounds of the present invention may be prepared using known organic synthesis techniques and can be synthesized using readily available starting materials and techniques available in the art, following any of a number of possible synthesis routes, including the reaction routes and synthesis schemes described below. The preparation of the compounds of the embodiments is described in detail in the following examples, but those skilled in the art will find that the described chemical reactions can be readily adapted to prepare other agents of various embodiments.

[0088]

[0128] The reaction for preparing the compounds of the present invention may be carried out in a suitable solvent that can be readily selected by those skilled in the art of organic synthesis. The suitable solvent may be substantially inactive with the starting materials (reactants), intermediates, or products at a temperature in which the reaction takes place, for example, a temperature ranging from the freezing temperature to the boiling temperature of the solvent. A given reaction may be carried out in one solvent or a mixture of two or more solvents. Depending on the reaction step, a solvent suitable for a particular reaction step may be selected by those skilled in the art.

[0089]

[0129] The preparation of the compounds of the present invention may involve the protection and deprotection of various chemical groups. The need for protection and deprotection, as well as the selection of appropriate protecting groups, can be readily determined by those skilled in the art. The chemistry of protecting groups can be found, for example, in TW Greene and PGMWuts, Protective Groups in Organic Synthesis, 3rd edition, Wiley & Sons, Inc., New York (1999), which is incorporated herein by reference in its entirety.

[0090]

[0130] The reaction can be monitored according to any suitable method known in the art. For example, the formation of the product can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) It can be monitored by infrared spectroscopy, spectrophotometrics (e.g., ultraviolet-visible), or mass spectrometry, or by chromatography, such as high-performance liquid chromatography (HPLC) or thin-layer chromatography.

[0091]

[0131] As used herein, the terms “ambient temperature,” “room temperature,” and “rt” refer to a temperature that is close to the temperature of the room in which the reaction takes place, for example, the reaction temperature, for example, a temperature between approximately 20°C and approximately 30°C, as understood in the art.

[0092]

[0132] In yet another embodiment, the present invention relates to a compound of formula (I):

[0093] [ka]

[0094]

[0133] (In the formula,

[0134] A is CO2R 1 , and PO3R 1 Selected from the group consisting of,

[0135] B is CO2R 2 , and PO3R 2 Selected from the group consisting of,

[0136] R 1 , R 2 and R 3 These are H and C1-C, each independently. 12 Selected from the group consisting of alkyl groups,

[0137] Each R a These are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN.

[0138] Each R b These are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN.

[0139] L is a linker that has 1 to 20 atoms in its normal chain.

[0140] or a method for synthesizing a pharmaceutically acceptable salt thereof,

[0141]

[0142] (a) Compound of formula (II):

[0095] [ka]

[0096]

[0143] (In the formula, A is CO2R 1 , and PO3R 1 Selected from the group consisting of,

[0144] B is selected from the group consisting of CO2R 2 , and PO 3 R 2 and is selected from the group consisting of

[0145] R 1 , and R 2 are each independently selected from the group consisting of H and C1-C 12 alkyl

[0146] Each R a is independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN

[0147] Each R b is independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN), a step of preparing

[0148] (b) A compound of formula (II) is reacted with a compound of formula (III):

[0097]

Chemical formula

[0098]

[0149] (wherein R 3 is selected from the group consisting of H and C1-C 12 alkyl

[0150] L is a linker having 1 to 20 atoms in the normal chain) and

[0151] reacting in the presence of a copper(I) catalyst A method is provided that includes

[0099]

[0152] The reaction can be carried out in any suitable solvent. Examples of suitable solvents include polar organic solvents and combinations thereof. Examples of polar solvents that can be used include dimethylformamide (DMF), alcohols (e.g., methanol, ethanol, propanol, and t-butanol), dimethyl sulfoxide, tetrahydrofuran, and acetonitrile. In some embodiments, the solvent is a combination of the polar organic solvents discussed above and water. In some embodiments, the ratio of polar organic solvent to water is 10:1 to 1:1. In one embodiment, the ratio of polar organic solvent to water is 8:1 to 1:1. In one embodiment, the ratio of polar organic solvent to water is 6:1 to 1:1. In one embodiment, the ratio of polar organic solvent to water is 5:1 to 3:1. In one embodiment, the ratio of polar organic solvent to water is about 4:1. In a preferred embodiment, the solvent is DMF:water in a volume ratio of 4:1.

[0100]

[0153] The reaction can take place over a wide range of temperatures. In some embodiments, the reaction takes place at temperatures from 5°C to 50°C. In one embodiment, the reaction takes place at temperatures from 10°C to 30°C. In one embodiment, the reaction takes place at temperatures from 15°C to 25°C. In one embodiment, the reaction takes place at temperatures from 20°C to 25°C.

[0101]

[0154] The reaction is typically carried out for a period of time sufficient to achieve a complete reaction of the starting materials. In some embodiments, the reaction is carried out for 1 to 24 hours. In some embodiments, the reaction is carried out for 4 to 20 hours. In some embodiments, the reaction is carried out for 8 to 16 hours. In some embodiments, the reaction is carried out for 10 to 14 hours.

[0102]

[0155] As described above, the reaction is carried out in the presence of a copper(I) catalyst. Examples of suitable copper catalysts include copper(I) salts (iodide, bromide, chloride, acetate), copper(I) complexes: e.g., [Cu(CH3CN)4]PF6 and [Cu(CH3CN)4]BF4 or triflate counterions, copper(II) sulfate pentahydrate, and copper(II) acetate. In one embodiment, the copper catalyst is generated in situ by reduction of copper sulfate.

[0103]

[0156] In certain embodiments, the reaction is carried out in the presence of a copper(I) ligand that helps stabilize copper(I) in solution, in which case the copper(I) ligand is selected from the group consisting of TBTA, TEOTA, THPTA, BTTES, BTTAA, BTTP, BTTPS, (BimH)3, (Bth)3, BPS, and 4,4'-dimethyl;-2,2'-bipyrimidine.

[0104]

[0157] Following the completion of the reaction, the reaction medium is post-treated in a manner known in the art, and the resulting reaction product is purified to obtain the desired final product.

[0158] The present invention will be illustrated by examples, but these examples should not be construed as limiting the invention to them. Additional compounds other than those listed below can be prepared using the methods and synthesis protocols described herein or their appropriate variations or modifications. [Examples]

[0105] experiment Materials and Reagents

[0159] All solvents and reagents were purchased from standard commercial suppliers and used as received. device

[0160] 1 H, 13 All C, COSY, HSQC, and HMBC were recorded using a Varian FT-NMR 400 spectrometer (Varian, California, USA). 1 The 1H NMR spectrum was acquired at 400 MHz or 500 MHz. 13 C spectra were acquired at 101 MHz or 126 MHz. All reported peaks were referenced to the solvent peak on the order of parts per million at 25°C.

[0106]

[0161] ESI-QTOF MS data was collected using an Exactive Plus Orbitrap Infusion mass spectrometer (Exactive series, 2.8 Build 268801, ThermoFisher Scientific). Analysis was performed using Xcalibur 4.0.27.10 (ThermoFisher Scientific).

[0107]

[0162] Protein samples were analyzed using an Agilent 6220 ESI-TOF LC / MS mass spectrometer connected to an Agilent 1200 LC system (Agilent, Palo Alto, CA). All data were acquired using a dual-spray electrospray ionization (ESI) source and corrected for reference mass. Acquisition was performed using Agilent Mass Hunter Acquisition software version B.02.01 (B2116.30). Ionization mode: Electrospray ionization; Dry gas flow rate: 7 L / min; Nebulizer: 241317 Pa (35 psi); Dry gas temperature: 325 °C; Capillary voltage (Vcap): 4000 V; Fragmenter: 300 V; Skimmer: 65 V; OCT RFV: 250 V; Acquisition scan range: 300~3200 m / z; Internal reference ions: Cation mode = m / z = 121.050873 and 922.009798. Protein desalting and chromatographic separation were performed using an Agilent Poroshell C18 2.1 × 75 mm, 5 μm column with 5% (v / v) acetonitrile, which leads to waste (0~5 min). After desalting the sample, the flow was reconnected to the ESI source for subsequent gradient elution using (5% (v / v) to 100% (v / v)) acetonitrile / 0.1% formic acid at 0.25 mL / min for 8 minutes. Analysis was performed using Mass Hunter version B.06.00 with BioConfirm software using the maximal entropy protein deconvolution algorithm; mass step 1Da; baseline coefficient 3.00; and indeterminately set peak width.

[0108]

[0163] Non-radioactive HPLC analysis was performed on an Agilent 1200 series HPLC system equipped with an Alltech Hypersil BDS-C18 (4.6 × 150 nm, 5 μm, column A) or a Phenomonex Luna C18(2) column (4.6 mm × 150 mm, 5 μm, column B) and a Phenomonex SecurityGuard™ C18 guard cartridge (4 mm × 30 mm), at a flow rate of 1 mL / min; System A: gradient elution of 0.1% TFA in buffer A = H2O and 0.1% TFA in buffer B = acetonitrile (0 to 100% B in A over 25 minutes), with UV detection at λ220, 254, 280 nm and 350 nm.

[0109]

[0164] Multiple chromatography systems were used in the purification process. Semi-preparative RP-HPLC at a flow rate of 8 mL / min (Agilent 1200 series HPLC system with Lunar C18 column, 100 Å 21.2 × 250 mm, 5 μm). System B: Gradient elution of 0.1% TFA in buffer A = H2O and 0.1% TFA in buffer B = acetonitrile (0 to 40% B in A at 30 min, 40 to 100% B in A at 35 min, and 100% B at 40 min), and detection at 214 and 254 nm. System C: System B with the following gradient (0 to 100% B in A at 40 min, 100% B at 44 min, and 100 to 0% B in A at 45 min).

[0110]

[0165] Radioactivity was measured using either a Capintec CRC-55tPET dose calibrator set to cal#108 or a PerkinElmer Wizard 2-2470 automated gamma counter set to an energy window of 320-500 keV (Bi-213).

[0111]

[0166] Protein concentrations were determined using a Thermo Scientific NanoDrop Lite spectrophotometer, and blank readings for each vehicle buffer prior to measurement were subtracted.

[0112]

[0167] Thin-layer chromatography (TLC) uses silica gel with an aluminum backing, 60°F. 254 The procedure was performed using strips (Merck, Darmstadt, Germany) and 0.4 M sodium citrate pH 4 + 10% methanol as the mobile phase. The developed TLC strips were measured using an Elysia-Raytest Gina Star TLC reader at permanent equilibrium (minimum 8 hours after development). The chromatogram of the control (i.e., without chelator) is shown in Figure 1. This shows that, as expected, the solution containing actinium reacted with the solvent tip (R) in the absence of H2macropa or H2macropa-tzPEG3SqOEt. f This indicates that it moves together with (=1).

[0113]

[0168] Instant TLC (iTLC) was performed using glass microfiber iTLC-SG chromatography paper strips (Agilent, CA, USA) and either 50 mM EDTA pH 5 or 50 mM citrate pH 5 as the mobile phase. The developed iTLC strips were measured using an Elysia-Raytest Gina Star TLC reader at permanent equilibrium (minimum 8 hours after development). The chromatogram of the control (i.e., without chelator) is shown in Figure 2. This was expected, as the actinium-containing solution showed a solvent tip (R) in the absence of the H2macropa-tzPEG3Sq-conjugate monoclonal antibody. f This indicates that it moves together with (=1).

[0114]

[0169] Size exclusion HPLC (SE-HPLC) was performed at a flow rate of 1 mL / min using a Phenomenex BioSep-SEC-S3000 5 μm 300 × 7.8 mm column and a mobile phase consisting of 50 mM phosphate buffer pH 7.2, 0.2 M NaCl, 5% isopropanol, and 0.02% NaN3, on an Agilent 1200 Series system equipped with a fraction collector and diode array detector.

[0115]

[0170] The immunoreactive fraction was determined by incubating the radioimmunoconjugate (20 ng) in the relevant cell line (5 x 10 6 cells) at ambient temperature for 45 minutes, followed by spinning the cell suspension (2000 rcf for 2 minutes) and washing the resulting cell pellet with medium (1 mL). The washing was repeated a further two times and the radioactivity in the final cell pellet was measured using an automated gamma counter at permanent equilibrium (at least 8 hours after washing). The immunoreactivity or immunoreactive fraction (IRF) was calculated as the ratio of the radioactivity in the cell pellet compared to a standard using the radioimmunoconjugate (20 ng, 500 μL, mean of triplicates). Nonspecific binding (NSB) was determined by incubating the radioimmunoconjugate (20 ng) with each unconjugated monoclonal antibody (60 μg) according to the above procedure.

[0116]

[0171] Ac-225 generated from thorium-229 was purchased as nitrate from Oak Ridge National Laboratory (USA). 225 The Ac]Ac-nitrate (approximately 25 - 30 MBq) was reconstituted in hydrochloric acid (100 μL, 0.2 M) prepared by diluting 30% Suprapur HCl (Sigma) with Ultrapur water (Sigma). A stock solution of reconstituted 225 Ac]Ac-nitrate with a similar radioactivity concentration was prepared by diluting in 0.15 M sodium acetate pH 5.5 (1:9 - 1:1 depending on the decay of Ac-225).

[0117]

[0172] Purified mouse-human IgG1 chimeric monoclonal antibodies EGFRVIII IgG1, EphA_{3} IgG_{1}, and chimeric IgG1 isotype control, and humanized IgG1 isotype control antibodies were provided by the Olivia Newton-John Cancer Research Institute.

[0118]

[0173] The U251 glioblastoma cell line was obtained from the American Type Culture Collection (ATCC) and cultured in RPMI medium containing 10% fetal bovine serum (FCS). The U87MG.de2-7 glioblastoma cell line was provided by the Ludwig Institute for Cancer Research and has been previously described (Nishikawa R, Ji XD, Harmon RC, CS Lazar CS, Gill GN, Cavenee WK, Huang HJ. A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc Natl Acad Sci USA 1994;91:7727~31). Cells were cultured in DMEM medium containing 10% FCS and 0.4 mg / mL Geneticin. The SK-RC-52 renal cell carcinoma cell line was provided by the Catholic University of Nijmegen (Netherlands) and cultured in RPMI medium containing 10% FCS, 2 mM GlutaMAX (Gibco), and 100 units / mL penicillin and 100 μg / mL streptomycin. All cultures were incubated at 37°C with 5% CO2.

[0174] Most of the materials were commercially purchased as reagent grade from readily available suppliers.

[0119] Example 1 Dimethylpyridine-2,6-dicarboxylate (100)

[0120] [ka]

[0121]

[0175] Compound 100 was prepared according to a modified version of the procedure described in the literature (Tetrahedron 2015, 71(33), 5321-5336). Pyridine-2,6-dicarboxylic acid (4.23 g, 25.3 mmol) and sulfuric acid (1 mL) were heated under reflux for 6 hours in methanol (30 mL). The solid was filtered as a colorless crystalline solid. A second yield was obtained by removing the solvent under reduced pressure, dissolved in dichloromethane (100 mL), and washed with saturated NaHCO3 solution (2 × 50 mL) and water (50 mL). The combined organic layers were dried over MgSO4, and the solvent was removed under reduced pressure to obtain a colorless solid (3.94 g, 80%). ESI-MS{M+H +}:196.0599 (C9H 10 NO4) + The calculated value is 196.0605. 1 H NMR (400 MHz, CDCl3) δ 8.30 (d, J = 7.8 Hz, 2H), 8.01 (t, J = 7.8 Hz, 1H), 4.01 (s, 6H). 13 ¹³C NMR (10¹ MHz, CDCl₃) δ 165.0, 148.2, 138.4, 128.0, 53.2. Rt = 9.70 mins (Method A, Column B).

[0122] Example 2 Methyl 6-(hydroxymethyl)picolinate (101)

[0123] [ka]

[0124]

[0176] Methyl 6-(hydroxymethyl)picolinate was synthesized using a protocol adapted from the literature. Inorg. Chem. 2008, 47(17), 7840-51. Dimethyl 2,6-pyridine-2,6-dicarboxylate (6.0 g, 30.6 mmol) was added to methanol (200 mL) at 0°C with sodium borohydride (2.32 g, 62 mmol) over 1 hour. The reaction mixture was stirred at room temperature for 5 hours. The solution was quenched with saturated NH4Cl (100 mL) at 0°C, and methanol was removed under reduced pressure. The aqueous layer was extracted with dichloromethane (3 × 100 mL). The combined organic layers were dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The colorless solid was purified by column chromatography (0-2% MeOH in DCM) to obtain compound 101 (3.37 g, 57%). ESI-MS{M+H +}:168.0652 (C8H 10 NO3) + The calculated value is 168.0655. 1 H NMR (400 MHz, CDCl3): δ 8.01 (d, J = 7.7 Hz, 1H), 7.83 (t, J = 7.7 Hz, 1H), 7.53 (d, J = 7.8 Hz, 1H), 4.85 (s, 2H), 3.97 (d, J = 0.7 Hz, 3H). 13 ¹³C NMR (125.7 MHz, CDCl₃): δ 165.0, 148.2, 138.4, 128.0, 53.2. Rt = 6.67 mins (Method A, Column A).

[0125] Example 3 Methyl-6-chloromethyl-pyridine-2-carboxylate (102)

[0126] [ka]

[0127]

[0177] Methyl-6-chloromethyl-pyridine-2-carboxylate was synthesized using a protocol from the literature that was adapted. 2Thionyl chloride (6 mL) was slowly added to methyl-6-hydroxymethyl-2-pyridinecarboxylate (2.5 g, 15 mmol) at 0°C under an N2 atmosphere, and the mixture was stirred for 1 hour. After 1 hour, the thionyl chloride was removed under vacuum. The residue was dissolved in toluene (50 mL) and washed with saturated NaHCO3 (50 mL). The organic fraction was dried over MgSO4, filtered, and the solvent was removed under reduced pressure to obtain an oily substance, which precipitated to form an off-white solid (2.42 g, 90%). ESI-MS{M+H +}:186.0319 (C8H9ClNO2) + The calculated value is 186.0244. 1 H NMR (500 MHz, CDCl3) δ 8.09 (d, J = 7.7 Hz, 1H), 7.91 (t, J = 7.8 Hz, 1H), 7.74 (d, J = 7.8 Hz, 1H), 4.79 (s, 2H), 4.02 (s, 3H). 13 ¹³C NMR (10¹ MHz, CDCl₃): δ 165.3, 157.2, 147.5, 138.1, 126.2, 124.5, 53.1, 46.3. Rt = 7.65 mins (Method A, Column A).

[0128] Example 4 Diethyl-4-hydroxypyridine-2,6-carboxylate (103)

[0129] [ka]

[0130]

[0178] Diethyl-4-hydroxypyridine-2,6-carboxylate was synthesized using a protocol adapted from the literature. 3 Chelidamic acid (4.0 g, 21.8 mmol) was dissolved in ethanol (150 mL), sulfuric acid (6 drops) was added, and the mixture was refluxed for 16 hours. The ethanol was removed under reduced pressure, and the solution was used in the next reaction without further purification (3.98 g, 76%). ESI-MS{M+H +}:240.0866 (C 11 H 14 NO5)+ Calculated value: 240.0867. 124℃. 1 H NMR (400 MHz, CDCl3): δ 9.40 (br, 1H), 7.45 (s, 2H), 4.45 (q, 4H), 1.41(t, 6H). Rt= 7.85 min (Method A, Column A).

[0131] Example 5 Diethyl 4-(propa-2-in-1-yloxy)pyridine-6-dicarboxylate (104)

[0132] [ka]

[0133]

[0179] Diethyl 4-(propa-2-in-1-yloxy)pyridine-2,6-dicarboxylate was synthesized using a protocol from the literature that was adapted. 4 A suspension of diethyl chelidamate (4) (3.15 g, 13.2 mmol) and K2CO3 (3.6 g, 26.4 mmol) in DMF (30 ml) was mixed with propargyl bromide solution (80 wt% - 4.69 mL, 52.3 mmol in toluene). To aid solubility, the mixture was heated at 80°C for 3 hours, then stirred overnight at room temperature. The mixture was then filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0-2% MeOH in DCM) to obtain compound 104 as a pale yellow or brown solid (2.47 g, 68%). ESI-MS{M+H +}:278.1021 (C 14 H 16 NO5) + Calculated value: 278.1023 1 H NMR (400 MHz, DMSO-d6) δ 7.80 (s, 2H), 5.11 (s, 2H), 4.38 (q, J = 7.1 Hz, 4H), 3.75 (s, 1H), 1.34 (t, J = 7.1 Hz, 6H). 13¹³C NMR (10¹ MHz, DMSO-d6): δ 165.5, 164.4, 150.1, 114.9, 80.3, 78.1, 62.1, 57.0, 39.7, 14.5. Rt = 8.75 mins (Method A, Column A).

[0134] Example 6 2-Hydroxymethyl 4-(propa-2-in-1-yloxy)pyridine-6-ethylcarboxylate (105)

[0135] [ka]

[0136]

[0180] 2-Hydroxymethyl-4-(propa-2-in-1-yloxy)pyridine-6-ethylcarboxylate was synthesized using a protocol adapted from the literature. 2 Diethyl 4-(propa-2-in-1-yloxy)pyridine-2,6-dicarboxylate (1.81 g, 6.49 mmol) was added to ethanol (100 mL) at 0°C with sodium borohydride (0.319 g, 8.44 mmol) over 0.5 hours. The reaction mixture was stirred at room temperature for 3 hours and monitored by MS until no starting material remained. The solution was quenched with saturated NH4Cl (50 mL) at 0°C, and the ethanol was removed under reduced pressure. The aqueous layer was extracted with ethyl acetate (3 × 70 mL). The combined organic layers were dried over MgSO4, filtered, and the solvent was removed under reduced pressure to obtain compound 6, an off-white color, without further purification (1.2 g, 75%). ESI-MS{M+H +}:236.0919 (C 12 H 14 NO4) + The calculated value is 236.0918. 1 ¹H NMR (500 MHz, chloroform-d): δ 7.62 (s, 1H), 7.09 (s, 1H), 4.81 (s, 4H), 4.45 (q, J = 7.1 Hz, 2H), 2.60 (t, J = 2.3 Hz, 1H), 1.42 (t, J = 7.1 Hz, 3H). 13¹³C NMR (126 MHz, chloroform-d): δ 165.2, 164.7, 162.1, 148.8, 111.4, 109.7, 76.7, 64.5, 62.1, 56.0, 14.3. Rt = 11.32 mins (Method A, Column A).

[0137] Example 7 2-Chloromethyl 4-(propa-2-in-1-yloxy)pyridine-6-ethylcarboxylate (106)

[0138] [ka]

[0139]

[0181] 6 mL of thionyl chloride was slowly added to 2-hydroxymethyl 4-(propa-2-in-1-yloxy)pyridine-6-ethyl carboxylate (0.4 g, 1.7 mmol) under an N2 atmosphere at 0°C, and the mixture was stirred for 3 hours. After 3 hours, the thionyl chloride was removed under vacuum to obtain a pale yellow residue, which was dissolved in ethyl acetate (20 mL) and washed with saturated NaHCO3 (30 mL) and water (30 mL). The organic fraction was dried over MgSO4, filtered, and the solvent was removed under reduced pressure to obtain a pale yellow powder (0.33 g, 77%). ESI-MS{M+H +}:254.0578 (C 12 H 14 NO4) + The calculated value is 254.0578. 1 ¹H NMR (500 MHz, chloroform-d): δ 7.62 (s, 1H), 7.09 (s, 1H), 4.81 (s, 4H), 4.45 (q, J = 7.1 Hz, 2H), 2.60 (t, J = 2.3 Hz, 1H), 1.42 (t, J = 7.1 Hz, 3H). 13 ¹³C NMR (126 MHz, chloroform-d): δ 165.2, 164.7, 162.1, 148.8, 111.4, 109.7, 76.7, 64.5, 62.1, 56.0, 14.3. Rt = 14.942 mins (Method A, Column B).

[0140] Example 8 Methyl 6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinate (107)

[0141] [ka]

[0142]

[0182] Methyl 6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinate was synthesized using a protocol adapted from the literature. 5 To a solution of 1,7,10,16-tetraoxa-4,13-diazacyclooctadecane (0.77 g, 3.02 mmol) and DIPEA (0.34 g, 3.93 mmol) in 350 mL of dry ACN at 75°C, a solution of 102 (0.37 g, 2.03 mmol) in 80 mL of dry ACN was added dropwise over 4 hours under a nitrogen atmosphere, and the mixture was heated under reflux for 40 hours. The solution was concentrated under reduced pressure at 60°C to obtain a pale yellow oil. The crude oil was then purified by semi-preparative HPLC (Method C), the appropriate fractions were combined, and freeze-dried to obtain a pale oil (626 mg, 76%). ESI-MS{M+2H +}:206.6257 (C 20 H 33 N3O6) 2+ The calculated value is 206.6257. 1 H NMR (500 MHz, chloroform-d) δ 9.57 (s, 1H), 8.07 (d, J = 7.7 Hz, 1H), 7.91 (t, J = 7.7 Hz, 1H), 7.85 (d, J = 7.7 Hz, 1H), 4.97 (s, 2H), 3.97 (s, 3H), 3.89 (s, 4H), 3.82 (s, 4H), 3.75 (s, 3H), 3.61 (s, 9H), 3.33 (s, 4H). 13¹³C NMR (126 MHz, cdcl3) δ values: 165.0, 161.6-160.7 (TFA, q), 152.0, 146.9, 138.5, 128.5, 124.6, 119.6-112.6 (TFA, q), 70.1, 69.4, 65.8, 65.5, 54.5, 52.9, 52.5, 48.6. Rt = 7.26 mins (Method A, Column A).

[0143] Example 9 Ethyl 4-(propa-2-in-1-yloxy)-6-((16-((6-(methoxycarbonyl)pyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinate (108)

[0144] [ka]

[0145]

[0183] Cesium carbonate (0.65 g, 2.0 mmol) was added to a solution of methyl 6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl) picolinate (0.149 g, 0.4 mmol) in anhydrous DMF (10 mL) under a nitrogen atmosphere, and the mixture was stirred at room temperature for 30 minutes. To the stirred reaction mixture, a solution of 2-chloromethyl 4-(propa-2-in-1-yloxy)pyridine-6-ethyl carboxylate (0.138 g, 0.54 mmol) in anhydrous DMF (2 mL) was added under nitrogen, and the reaction mixture was heated at 50°C for 40 hours. The resulting solution was filtered and dried under reduced pressure to obtain an orange oily substance, which was purified by semi-preparative HPLC, the appropriate fractions were combined, and freeze-dried to obtain a yellow oily substance. ESI-MS{M+2H +}:315.1627 (C 32 H 46 N4O9) 2+ The calculated value is 315.1627. 1H NMR (500 MHz, CDCl3) δ 8.11 (d, J = 7.2 Hz, 1H), 7.94 (t, J = 7.6 Hz, 1H), 7.80 (d, J = 6.7 Hz, 1H), 7.69 (s, 1H), 7.44 (s, 1H), 4.84 (s, 2H), 4.75 (s, 2H), 4.69 (s, 2H), 4.47 - 4.39 (m, 2H), 3.98 (d, J = 2.7 Hz, 3H), 3.95 (s, 8H), 3.65 (d, J = 3.0 Hz, 16H), 2.61 (s, 1H), 1.40 (t, J = 8.3 Hz, 3H). Rt = 10.23 minutes (Method A, Column A).

[0146] Example 10 4-(propa-2-in-1-yloxy)-6-((16-((6-carboxypyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinic acid (109)

[0147] [ka]

[0148]

[0184] 4-(propa-2-in-1-yloxy)-6-((16-((6-carboxypyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinic acid was synthesized using a protocol from the literature that was adapted. 6 To a solution of 10⁸ (18.5 mg, 0.03 mmol) in DCM (450 μL), a sonicated suspension of NaOH in methanol (50 μL, 3 M) was added to achieve a final NaOH concentration of 0.3 M and a final DCM:MeOH ratio of 9:1. The reaction mixture was stirred at room temperature for 1 hour, then the solvent was removed under reduced pressure, and the mixture was used in the next step without further purification. ESI-MS{M+2H +}:294.1393 (C 29 H 40 N4O9) 2+Calculated value: 294.1393. Rt = 8.53 minutes (Method A, Column A).

[0149] Example 11 N3-PEG3-Squalate (110)

[0150] [ka]

[0151]

[0185] Freshly prepared diethyl squalate (0.23 g, 1.32 mmol) was dissolved in ethanol (4 mL), and then 1-amino-11-azido-3,6,9-trioxaundecane (0.28 g, 1.29 mmol) and DIPEA (1 equivalent) were added under nitrogen. The mixture was stirred at room temperature for 8 hours, and then the colorless solution was allowed to dry under reduced pressure and purified by semi-preparative HPLC (Method C). The fractions were combined and freeze-dried to obtain a colorless oil (209 mg, 49%). ESI-MS{M+H +}:343.1613 (C 14 H 23 N4O6) + Calculated value: 343.1612 1 13-C NMR (126 MHz; cdcl3): δ 188.8, 183.3, 177.4, 177.2, 172.7, 70.7, 70.3, 70.0, 69.73, 69.61, 50.7, 44.4, 44.1, 15.8. RT= 7.23 minutes (Method A, Column A).

[0152] Example 12 CuAAC between N3-PEG3-squalate and 4-(propa-2-in-1-yloxy)-6-((16-((6-carboxypyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinic acid (111)

[0153] [ka]

[0154]

[0186] CuSO4 (0.22 mg, 0.0014 mmol, 8% mmol) and sodium ascorbate (0.64 mg, 0.0032 mmol, 18% mmol) were combined and shaken for 2 minutes to ensure complete reduction to copper(I) species. Then, TBTA (2.8 mg, 0.0057 mmol, 32% mmol in DMF) was added along with N3-PEG3-squalate (6.42 mg, 0.018 mmol) as a stabilizing ligand, and the mixture was reacted for 10 minutes. 109 (11 mg, 0.018 mmol) was added, and the reaction mixture was purged with a nitrogen stream for 15 minutes to reduce the oxygen content in the reaction mixture. The reaction was monitored by HPLC (280 nm), and additional 2% mmol CuSO4 and 18% mmol sodium ascorbate were added after 12 hours to ensure that the reaction proceeded to completion. The yellow solution was dried under reduced pressure and then dried in a vacuum. The obtained precipitate was purified by semi-preparative HPLC, the fractions were combined, and freeze-dried to obtain an off-white powder (5 mg, 40%). ESI-MS{M+2H +}:465.2161 (C 43 H 62 N8O 15 ) 2+ The calculated value is 465.2162. 1H NMR (500 MHz, chloroform-d) δ 8.11 (d, J = 7.2 Hz, 1H), 7.94 (t, J = 7.6 Hz, 1H), 7.80 (d, J = 6.7 Hz, 1H), 7.69 (s, 1H), 7.44 (s, 1H), 4.84 (d, J = 2.8 Hz, 2H), 4.75 (s, 2H), 4.69 (s, 2H), 4.47 - 4.39 (m, 2H), 3.98 (d, J = 2.7 Hz, 3H), 3.95 (s, 8H), 3.72 - 3.58 (m, 16H), 2.61 (s, 1H), 1.40 (t, J = 8.3 Hz, 3H). Rt = 10.04 mins (Method A, Column A).

[0155] Example 13 Protein conjugation - Herceptin

[0187] Lyophilized Herceptin (trastuzumab-500 mg) was reconstituted to a final concentration of 10 mg / mL in borate buffer (0.2 M, pH 9.0). Macropa-PEG3-SqOEt (compound 111, 5 mg / mL storage solution in DMSO, 6 μL, 10 equivalents) was added, and the reaction mixture was incubated in the dark at room temperature for 6 hours before removing excess reagent. The buffer was then changed by spin filtration (MW cutoff at 50 kDa) (HEPES, 0.1 M, pH 7.4).

[0156] Example 14 Conjugation of EGFRVIII IgG1 and EphA3 IgG1 antibodies

[0188] EGFRVIII IgG1 (9.4 mg / mL) and EphA3 IgG1 antibody (3.1 mg / mL; 3 mg, 2 × 10) in PBS -5The mmol of EGFRVIII IgG1 was exchanged for borate buffer (0.2 M, pH 9.0), divided into two equal aliquots, and the final concentration was 5 mg / mL (300 μL for EGFRVIII IgG1 and 320 μL for EphA3 IgG1). H2macropa-tzPEG3Sq OEt (111, 10 mg / mL storage solution in DMSO - final DMSO concentration less than 4%) was added to the reaction mixture, and the mixture was shaken overnight at 4°C, then shaken again overnight at room temperature. Excess reagent was removed, and the mixture was exchanged for buffer by spin filtration (MW cutoff at 50 kDa) (sodium acetate, 0.1 M, pH 5.5).

[0157] [Table 1]

[0158] Example 15 Ac-225 label concentration study

[0189] Ac-225 storage solution (approximately 74 kBq, 2.3 μL) was added to a solution of H2 macropa, DOTA (2,2',2'',2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid), DTPA (diethylenetriamine pentaacetate), EDTA (ethylenediaminetetraacetic acid), and a buffer control, and the chelator concentration was 10 -3 M~10 -10 A total volume of 100 μL of mixture between M was obtained. Unless otherwise noted, the reaction mixture was incubated at ambient temperature for 2 hours and analyzed by TLC (0.4M sodium citrate pH 4 + 10% MeOH, product R f A sample for (=0) was taken at the end of the reaction. 225 An exemplary chromatogram of Ac]Ac-macropa is shown in Figure 3. This shows that actinium was retained at the baseline (i.e., the "origin") by being complexed with macropa. The radiochemical yield is shown in Figure 4.

[0159]

[0190] [ 225The radiochemical yield of Ac]Ac-macropa is significantly higher at ambient temperature compared to other chelators investigated. H2macropa yields 10 -6 M can be quantitatively radiolabeled down to the chelator concentration. Other chelators, such as DOTA, DTPA, and EDTA, can be [ 225 Ac]Ac complexation was not observed, similar to the control (without chelator). Radiolabeling of DOTA showed quantitative radiolabeling yield when heated to 90°C, but was slightly inferior to H2macropa at lower chelator concentrations. These findings are consistent with published observations: NAThiele, V. Brown, JMKelly, A. Amor-Coarasa, U. Jermilova, S. MacMillan, A. Nikolopoulou, S. Ponnala, C. FRamogida, A. Akhrobertson, C. Rodriguez-Rodriguez, P. Schaffer, C. Williams, J. W. Babich, V. Radchenko, J. J. Wilson, Angew. Chem. Int. Ed. 2017, 56, 14712.

[0160] Example 16 Comparative study of the complexation rates between compound 111 and Ac-225.

[0191] Ac-225 storage solution (approximately 74 kBq, 2.3 μL) was added to a solution of H2macropa-tzPEG3SqOEt or DOTA-methyltetrazine, and the chelator concentration was reduced to 10 in each case. -5 M, 10 -6 M, 10 -7 M and 10 -4 M, 10 -5 M, 10 -6 A total volume of 100 μL of mixture M was obtained. The reaction mixtures of H2macropa-tzPEG3SqOEt and DOTA-methyltetrazine were incubated at ambient temperature and 90°C, respectively, and analyzed by TLC (0.4M sodium citrate pH 4 + 10% MeOH, product R fSamples for (=0) were taken at 5, 15, 30, and 60 minutes. Radiochemical yields are shown in Figures 5 and 6.

[0161]

[0192] Radiolabeling of H2macropa-tzPEG3SqOEt proceeded in quantitative yield at all concentrations after a 15-minute incubation. 10 -7 At chelator concentrations of M, the radiochemical yield began to decline after 5 minutes of incubation. Therefore, 10 -7 and 10 -6 For radiolabeling studies using H2macropa-tzPEG3Sq-conjugate monoclonal antibodies, a 15-minute incubation at the chelator concentration of M and ambient temperature was selected.

[0162]

[0193] The radioactive labeling of DOTA-methyltetrazine is 10 -4 At chelator concentration M, the process proceeded with quantitative yield at all incubation times. 10 -5 At chelator concentration M, the radiochemical yield was 88.5% after 15 minutes of incubation. -6 In M, the maximum radiochemical yield was 65.3% after a 60-minute incubation. Therefore, 10 -5 and 10 -4 The chelator concentration of M was selected for radiolabeling studies using a TCOPEG4-conjugate monoclonal antibody (where TCOPEG- is an abbreviation for "transcyclooctene polyethylene glycol").

[0163] Example 17 [ 225 Ac]Ac-macropa-tzPEG3Sq-EGFRVIII IgG1 and [ 225 Synthesis of Ac]Ac-macropa-tzPEG3Sq-EphA3 IgG1

[0194] Ac-225 preservation solution (555 kBq, 30 μL) was added to a solution of H2macropa-tzPEG3Sq-conjugated monoclonal antibody, and the conjugate concentration was 10 -7M and 10 -6 A total volume of 100 μL of mixture M was obtained. The reaction mixture was incubated at ambient temperature for 15 minutes to obtain radiochemical purity (RP; iTLC: 50 mM citrate pH 5, product R). f Samples were taken to determine the chelator-antibody ratio (=0) and immunoreactivity (IR). Specific activity (SA) was calculated by dividing the initial activity of Ac-225 by the amount of conjugate and multiplying by the radiochemical purity. The results are summarized in Table 1. Numerical notations (2×, 5×) refer to the mean chelator-antibody ratio for each conjugate. 225 Ac]Ac-macropa-tzPEG3Sq-EGFRVIII IgG1 and [ 225 The chromatograms of Ac]Ac-macropa-tzPEG3Sq-EphA3 IgG1 are shown in Figures 7A and 7B, respectively. This indicates that actinium was retained at the baseline (i.e., the "origin") by being complexed with macropa-tzPEG3Sq-EGFRVIII IgG1 or macropa-tzPEG3Sq-EphA3 IgG1.

[0164]

[0195] Radiolabeling of H2macropa-tzPEG3Sq-conjugated monoclonal antibodies with Ac-225 is 10 -6 The study proceeded with a radiochemical yield of >99.5% at antibody concentration M. At this antibody concentration, the specific activity was close to the theoretical maximum of 37 MBq / mg. Radioimmunoconjugates with an average of two chelators per antibody showed higher immunoreactivity compared to radioimmunoconjugates with an average of five chelators per antibody. Therefore, the former was selected for further study.

[0165]

[0196]

[0166] [Table 2]

[0167] Example 18 Size exclusion HPLC of various H2macropa-tzPEG3Sq-conjugate monoclonal antibodies

[0197] H2macropa-tzPEG3Sq-conjugate monoclonal antibody (20 μg) was analyzed using size exclusion HPLC (SE-HPLC). Retention times are summarized in Table 2. SE-HPLC chromatogram (UV 280nm ) is shown in Figure 8.

[0168]

[0198] SE-HPLC demonstrated excellent antibody integrity for all immunoconjugates, with aggregation being negligible.

[0199]

[0169] [Table 3]

[0170] Example 19 Size exclusion HPLC of various Ac225-labeled monoclonal antibodies

[0200] [ 225 Ac]Ac-macropa-tzPEG3Sq-conjugate monoclonal antibody (37 kBq, approximately 1 μg) was analyzed using SE-HPLC. HPLC fractions were collected continuously in Eppendorf tubes every 30 seconds (0.5 mL). Radioactivity in the tubes was determined using an automated gamma counter at permanent equilibrium (minimum 8 hours after collection). UV 280 nm and radiation retention times are summarized in Table 3. SE-HPLC chromatogram (UV 280nm Figure 9 shows the RAD (and other related data).

[0171]

[0201] SE-HPLC demonstrated excellent antibody integrity for all radioimmunoconjugates, with negligible levels of aggregation. No free Ac-225 was detected by SE-HPLC, which is consistent with iTLC analysis.

[0172]

[0202]

[0173] [Table 4]

[0174] Example 20 [ 225 Ac]Ac-macropa-tzPEG3Sq-EGFRVIII IgG1 and La 3+ and competitive studies with EDTA

[0203] [ 225 Ac]Ac-macropa-tzPEG3Sq-EGFRVIII IgG1 (37kBq, 1μg, 5μL) is mixed with various gentisic acid salts (0 / 25mg / mL), La 3+ The mixture was added to a PBS containing (5 / 50 / 500 × molar excess relative to the antibody) and EDTA (50 × molar excess relative to the antibody) to obtain a solution with a total volume of 20 μL. The mixture was incubated at ambient temperature and prepared using iTLC (50 mM citrate, pH 5, product R). f =0) Samples for analysis were taken at 1, 24, 48, and 168 hours. Radiochemical purity is shown in Table 4.

[0175]

[0204] [ 225 Ac]Ac-macropa-tzPEG3Sq-EGFRVIII IgG1 is added to PBS with and without sodium gentisate up to a 50-fold molar excess. 3+ It competed with and showed superior stability. 500-fold molar excess of La 3+ In addition, with EDTA in a 50-fold molar excess, the radiochemical purity dropped to less than 95% over a period of 24-48 hours.

[0176]

[0205]

[0177] [Table 5]

[0178] Example 21 Serum stability analysis and [ 225 Comparison with Ac]Ac-DOTA-dhPzPEG4-conjugate monoclonal antibody

[0206] TCOPEG4-conjugate monoclonal antibodies were prepared in accordance with the procedures published by O. Keinanen, K. Fung, J. Pourat, V. Jallinoja, D. Vivier, NK Pillarsetty, AJAiraksinen, JS Lewis, BM Zeglis, M. Sarparanta, EJ NMMI Res 2017, 7, 95 and Z. Zhou, N. Devoogdt, MR Zalutsky, G. Vaidyanathan, Bioconjug Chem. 2018, 29, 4090-4103. In short, monoclonal antibodies in sodium bicarbonate (0.1 M, pH 8.5) were incubated with TCO-PEG4-NHS (4 × / 10 × / 40 × molar equivalents) at 37°C for 1 hour, and then purified by centrifugation filtration (50 kDa MWCO) using a formulation buffer consisting of sodium acetate (50 mM, pH 5.6), sorbitol (5 w / v%), and Tween 20 (0.02 w / v%). TCOPEG4-EGFRVIII IgG1 (150 μg) and TCOPEG4-EphA3 IgG1 (150 μg) were purified in sodium acetate (0.15 M, pH 5.5) [ 225 [Ac]Ac-DOTA-methyltetrazine (555kBq) was incubated with it at ambient temperature for 15 minutes, and then purified by spin filtration (MWCO of 10kDa). 225 Ac]Ac-DOTA-dhPzPEG4-EGFRVIII IgG1 and [ 225 Ac]Ac-DOTA-dhPzPEG4-EphA3 IgG1 (where -dhPzPEG- is an abbreviation for "methyldihydropyridazine polyethylene glycol") was synthesized. 225 Ac]Ac-macropa-tzPEG3Sq-and[ 225 Ac]Ac-DOTA-dhPzPEG4-conjugate monoclonal antibody was incubated in healthy donor human serum (100 μL) at 37°C. Samples were collected at EOS and after incubation at 2, 7, and 14 days. Samples were then tested in iTLC (50 mM EDTA pH 5, product R). fThe immunoreactivity was analyzed using the formula (=0), and the immunoreactivity rate was determined for EGFRVIII IgG1 and EphA3 IgG1 radioimmunoconjugates using U87MG.de2-7 and U251 cells, respectively. The results for radiochemical purity are shown in Figure 10A, and for immunoreactivity in Figure 10B. The numerical values ​​(2×, 5×, 1×, 2×, 9×) refer to the mean chelator-antibody ratio for each conjugate.

[0179]

[0207] In human serum [ 225 Ac]Ac-macropa-tzPEG3Sq-EGFRVIII IgG1 and [ 225 The radiochemical purity of Ac]Ac-macropa-tzPEG3Sq-EphA3 IgG1 was >95% at the end of the experiment (14 days). 225 Ac]Ac-DOTA-dhPzPEG4-conjugate monoclonal antibodies collectively showed inferior stability over the time frame investigated. The mean chelator-antibody ratio was 9. 225 Except for the Ac]Ac-DOTA-dhPzPEG4 conjugate showing inferior properties, other radioimmunoconjugates could not be distinguished based on immunoreactivity. The mean chelator-antibody ratio was 2. 225 The Ac]Ac-macropa-tzPEG3Sq-conjugate was used in the in vivo studies described in Examples 23 and 24.

[0180] Example 22 Serum stability analysis

[0208] The mean chelator-antibody ratio is 2. 225 Ac]Ac-macropa-tzPEG3Sq-conjugate monoclonal antibody was incubated in healthy donor human serum (100 μL) at 37°C. Samples were collected at EOS and after incubation at 2, 7, and 14 days. Samples were then subjected to iTLC (50 mM EDTA pH 5, product R) fThe immune response rate was analyzed using U87MG.de2-7 (EGFRVIII IgG1), U251 (EphA3 IgG1), or SK-RC-52 (chimeric isotype control IgG1) cells. The results are shown in Table 5.

[0181]

[0209] Radiochemical yields and specific activity were consistently high for all three radioimmunoconjugates, and radiochemical purity was excellent over the time frame investigated (>99% after 14 days). Immunoreceptor rates gradually decreased at each time point for all radioimmunoconjugates. Nonspecific binding was low in all cases.

[0182]

[0210]

[0183] [Table 6]

[0184] Example 23 In mice with U251 xenografts, [ 225 Dose escalation study of Ac]Ac-macropa-tzPEG3Sq-EphA3 IgG1

[0211] Female 4-6 week old BALB / c nu / nu mice (Animal Research Centre, Western Australia, Perth, Australia) were inoculated with U251 cells (5.5 × 10^6 cells) in PBS to induce subcutaneous tumors in the left flank. At 11 or 14 days post-inoculation, the tumors were approximately 100 mm in size. 3 Mice with tumor xenografts (n=5) were given [ 225 Ac]Ac-macropa-tzPEG3Sq-EphA3 IgG1 (0.25~1.0 μg / 9.25~37.0 kBq) or vehicle PBS was administered intravenously. mm 3 Measure the unit tumor volume (TV) at least twice a week (biweekly) and use the following formula: TV = (L × W 2The calculation was performed using ) / 2. Data were expressed as mean tumor volume ± SD. Statistical analysis was performed using an unpaired t-test at a given time point. Mice were subjected to an ethical endpoint (TV > 1000 mm). 3 Alternatively, animals were humanely euthanized if they experienced weight loss >10%. Tumor growth curve results and survival rate data are presented in Figures 11A and 11B, respectively. This research project was approved by the Austin Health Animal Ethics Committee.

[0185]

[0212] [ 225 A significant dose-response effect on tumor growth inhibition was observed within a few days of treatment following a single dose of Ac]Ac-macropa-tzPEG3Sq-EphA3 IgG1. All treatment groups showed a significant difference from the control group at 34 days post-inoculation, when the vehicle control group reached the ethical endpoint of tumor volume (P ≤ 0.0002). The duration of antitumor efficacy was also dose-dependent. The effects of radiotoxicity were demonstrated by weight loss. No radiotoxicity was observed at doses up to 18.5 kBq. At the highest dose of 37 kBq, radiotoxicity in the form of acute weight loss was observed in 60% of the cohort within a few days post-treatment. At 27.8 kBq, radiotoxicity was evident in 20% of the cohort by 14 days post-treatment. 225 The maximum tolerated dose of Ac]Ac-macropa-tzPEG3Sq-EphA3 IgG1 was determined.

[0186] Example 24 In mice with U87MG.de2-7 xenografts, [ 225 Dose escalation study of Ac]Ac-macropa-tzPEG3Sq-EGFRVIII IgG1

[0213] Female 4-6 week old BALB / c nu / nu mice (Animal Research Centre, Western Australia, Perth, Australia) were inoculated with U87MG.de2-7 cells (2.2 × 10^6 cells) to induce subcutaneous tumors in the left flank. At 7 or 11 days post-inoculation, the tumors were approximately 100 mm in size. 3Mice with tumor xenografts (n=5 / group) were given [ 225 Ac-macropa-tzPEG3Sq-EGFRVIII IgG1 (0.25~1.0 μg / 9.25~37.0 kBq) or vehicle PBS was administered intravenously. mm 3 The unit tumor volume (TV) is measured twice a week, and the following formula is used: TV = (L × W 2 Calculations were performed using ) / 2. Data were expressed as mean tumor volume ± SD. Mice were subjected to the ethical endpoint (TV > 1000 mm). 3 Alternatively, animals were humanely euthanized if they experienced weight loss >10%. Tumor growth curve results and survival rate data are presented in Figures 12A and 12B, respectively. This research project was approved by the Austin Health Animal Ethics Committee.

[0187]

[0214] In rapidly growing U87MG.de2-7 glioblastoma xenografts, no dose-response antitumor efficacy was observed. Only the highest dose of 37 kBq demonstrated long-term delay of tumor growth over the duration of the study in 40% of the cohort, and radiotoxicity was again observed in 60% of the cohort within two weeks of single-dose treatment. [18.5 kBq / 0.5 μg] 225 The maximum tolerated dose of Ac]Ac-macropa-tzPEG3Sq-EGFRVIII IgG1 was determined.

[0188]

[0215] Throughout this specification, unless otherwise required by context, the word “comprise,” or variations such as “comprises” or “comprising,” is understood to mean that it includes the element or integer or group of elements or integers described, but not to mean that it excludes any other element or integer or group of elements or integers.

[0189]

[0216] When a range of values ​​is expressed, it should be clearly understood that this range includes the upper and lower limits of the range, as well as all numbers or subranges between these limits, just as if each number and subrange were explicitly enumerated. The statement "approximately X% to Y%" is equivalent to "approximately X% to approximately Y%" unless otherwise indicated.

[0190]

[0217] As used herein, the term “about” means approximately or near, and in the context of any number or range specified herein, it is intended to include variations of no more than + / -10%, + / -5%, + / -1%, or + / -0.1% from the enumerated or claimed number or range.

[0191]

[0218] It should also be noted that, as used herein, the singular forms “a,” “an,” and “the” include multiple aspects unless otherwise indicated by the context.

[0192]

[0219] The headings used herein are included solely for the convenience of the reader and should not be used to limit the subject matter found throughout this disclosure or the claims. The headings should not be used in interpreting the claims or any limitations of the claims.

[0193]

[0220] The descriptions provided herein relate to multiple embodiments that may share common properties and features. It should be understood that one or more features of one embodiment may be combined with one or more features of other embodiments. In addition, a single feature or combination of features of an embodiment may constitute an additional embodiment.

[0194]

[0221] All methods described herein may be carried out in any preferred order unless otherwise indicated herein or unless it is clearly inconsistent with the context. Any examples or illustrative language provided herein (e.g., “e.g., “etc.”) are intended solely to better illustrate examples of embodiments and, unless otherwise claimed, do not impose any limitation on the scope of the claimed invention. Nothing in this specification should be construed as indicating that any unclaimed element is essential.

[0195]

[0222] Although the present invention has been described in some detail for clarity and understanding, it will be apparent to those skilled in the art that various modifications and changes can be made to the embodiments and methods described herein without departing from the scope of the inventive concept disclosed herein.

[0196]

[0223] Those skilled in the art will recognize that variations and modifications other than those specifically described are possible with respect to the inventions described herein. It should be understood that the present invention includes all such variations and modifications. The present invention also includes, individually or collectively, all of the steps, features, compositions and compounds mentioned or indicated herein, as well as any combination of any two or more of the steps or features.

[0197]

[0224] Future patent applications may be filed in Australia or abroad based on this application, for example, by claiming priority of this application, by claiming divisional status, and / or by claiming continuation status. The following provisional claims are provided merely as examples. It should be understood that this disclosure is not intended to limit the scope of what may be claimed in any future application. Furthermore, the claims should not be considered to limit (or exclude) any understanding of the invention as inherent in this disclosure. Features may be added to or excluded from the provisional claims at a later date to further define the invention. The following is a description of the claims as they were at the time of filing the application. [Claim 1] Compound of formula (I) [ka] (In the formula, A is CO2R 1 , and PO3R 1 Selected from the group consisting of, B is CO2R 2 , and PO3R 2 Selected from the group consisting of, R 1 , R 2 and R 3 These are H and C1-C, each independently. 12 Selected from the group consisting of alkyl groups, Each R a These are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN. Each R b These are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN. L is a linker that has 1 to 20 atoms in its normal chain. or a pharmaceutically acceptable salt thereof. [Claim 2] A is CO2R 1 The compound according to claim 1. [Claim 3] B is CO2R 2 The compound according to claim 1 or 2. [Claim 4] R 1 The compound according to any one of claims 1 to 3, wherein H is present. [Claim 5] R 2 The compound according to any one of claims 1 to 4, wherein H is present. [Claim 6] Each R aThe compound according to any one of claims 1 to 5, wherein H is present. [Claim 7] Each R b The compound according to any one of claims 1 to 6, wherein H is present. [Claim 8] R 3 However, C1~C 12 A compound according to any one of claims 1 to 7, wherein it is alkyl. [Claim 9] R 3 The compound according to any one of claims 1 to 8, wherein the compound is selected from the group consisting of methyl, ethyl, propyl, and butyl. [Claim 10] L is the formula: -(CH2CH2O) n -CH2CH2- The compound according to any one of claims 1 to 9, wherein n is a linker, where n is an integer from the group consisting of 0, 1, 2, 3, 4, and 5. [Claim 11] The compound according to claim 10, wherein n is 3. [Claim 12] Compound of formula (Ia): [ka] (In the formula, A is CO2R 1 , and PO3R 1 Selected from the group consisting of, B is CO2R 2 , and PO3R 2 Selected from the group consisting of, R 1 , R 2 and R 3 These are H and C1-C, each independently. 12 Selected from the group consisting of alkyl groups, Each R a These are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN. Each Rb These are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN. L is a linker having 1 to 20 atoms in its normal chain. M is a radioactive nuclide. or a pharmaceutically acceptable salt thereof. [Claim 13] A is CO2R 1 The compound according to claim 12. [Claim 14] B is CO2R 2 The compound according to claim 12 or 13. [Claim 15] R 1 The compound according to any one of claims 12 to 14, wherein H is present. [Claim 16] R 2 The compound according to any one of claims 12 to 15, wherein H is present. [Claim 17] Each R a The compound according to any one of claims 12 to 16, wherein H is present. [Claim 18] Each R b The compound according to any one of claims 12 to 17, wherein H is present. [Claim 19] R 3 However, C1~C 12 The compound according to any one of claims 12 to 18, wherein it is alkyl. [Claim 20] R 3 The compound according to any one of claims 12 to 19, wherein the compound is selected from the group consisting of methyl, ethyl, propyl, and butyl. [Claim 21] L is the formula: -(CH2CH2O) n -CH2CH2- The compound according to any one of claims 12 to 20, wherein n is a linker, where n is an integer from the group consisting of 0, 1, 2, 3, 4, and 5. [Claim 22] The compound according to claim 21, wherein n is 3. [Claim 23] A compound according to any one of claims 12 to 22, wherein the radionuclide is selected from the group consisting of actinium-225, lutetium-177, zirconium-89, terbium-149, terbium-152, terbium-155, terbium-161, radium-223, bismuth-212, indium-111, yttrium-86, yttrium-89, yttrium-90, and lead-212. [Claim 24] The compound according to any one of claims 12 to 23, wherein M is actinium-225. [Claim 25] Compound of formula (Ib): [ka] (In the formula, A is CO2R 1 , and PO3R 1 Selected from the group consisting of, B is CO2R 2 , and PO3R 2 Selected from the group consisting of, R 1 , and R 2 These are H and C1-C, each independently. 12 Selected from the group consisting of alkyl groups, R 4 It is a monoclonal antibody, Each R a These are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN. Each R bThese are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN. L is a linker having 1 to 20 atoms in its normal chain. M is a radioactive nuclide. or a pharmaceutically acceptable salt thereof. [Claim 26] A is CO2R 1 The compound according to claim 25. [Claim 27] B is CO2R 2 The compound according to claim 25 or 26. [Claim 28] R 1 The compound according to any one of claims 25 to 27, wherein H is present. [Claim 29] R 2 The compound according to any one of claims 25 to 28, wherein H is present. [Claim 30] Each R a The compound according to any one of claims 25 to 29, wherein H is present. [Claim 31] Each R b The compound according to any one of claims 25 to 30, wherein H is present. [Claim 32] L is the formula: -(CH2CH2O) n -CH2CH2- The compound according to any one of claims 25 to 31, wherein the linker is an integer from the group consisting of 0, 1, 2, 3, 4, and 5. [Claim 33] The compound according to claim 32, wherein n is 3. [Claim 34] A compound according to any one of claims 25 to 33, wherein the radionuclide is selected from the group consisting of actinium-225, lutetium-177, zirconium-89, terbium-149, terbium-152, terbium-155, terbium-161, radium-223, bismuth-212, indium-111, yttrium-86, yttrium-89, yttrium-90, and lead-212. [Claim 35] The compound according to any one of claims 25 to 34, wherein M is actinium-225. [Claim 36] A compound according to any one of claims 25 to 35, wherein the monoclonal antibody is penprimab, cintilimab, tripalimab, omblutamab, tisotumab, retifanlimab, ubrituximab, aniflorumab, roncasutuximab, valstilimab, dostallimab, oportuzumab, marjetuximab, naxitamab, verantamab, tafacitamab, sacituzumab, isatsuki Simab, trastuzumab (Herceptin), gilentuximab, ifabotuzumab, depatuxidimab, enfortumab, polatuzumab, emaparmab, semiprimab, moxetumomab, mogamulizumab, durvalumab, avelumab, atezolizumab, oraratumumab, daratumumab, elotuzumab, necitumumab, dinutuximab, nivolumab, blinatumomab, blinatumomab, ramucirumab, o A compound selected from the group consisting of binutuzumab, ad-trastuzumab, pertuzumab, brentuximab, ipilimumab, ofatumumab, catumakisomab, panitumumab, bevacizumab, cetuximab, ibritumomab, alemtuzumab, gemtuzumab, rituximab, edrecolomab, nimotuzumab, prorugolimab, and cetuximab. [Claim 37] A pharmaceutical composition comprising a compound according to any one of claims 25 to 36 and a pharmaceutically acceptable carrier. [Claim 38] A method for treating a subject, comprising the step of administering an effective amount of a compound according to any one of claims 25 to 36 to the subject. [Claim 39] The method according to claim 38, wherein the subject has cancer. [Claim 40] Compound of formula (I): (In the formula, [ka] A is CO2R 1 , and PO3R 1 Selected from the group consisting of, B is CO2R 2 , and PO3R 2 Selected from the group consisting of, R 1 , R 2 and R 3 These are H and C1-C, each independently. 12 Selected from the group consisting of alkyl groups, Each R a These are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN. Each R b These are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN. L is a linker that has 1 to 20 atoms in its normal chain. or a method for synthesizing a pharmaceutically acceptable salt thereof, (a) Compound of formula (10): [ka] (A is CO2R 1 , and PO3R 1 Selected from the group consisting of, B is CO2R 2 , and PO3R 2 Selected from the group consisting of, R 1 and R 2These are H and C1-C, each independently. 12 Selected from the group consisting of alkyl groups, Each R a These are independently selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN. Each R b The process involves independently providing a substance selected from the group consisting of H, F, Cl, Br, I, CH3, CH2CH3, CH(CH3)2, OH, OCH3, OCH2CH3, CF3, OCF3, NO2, NH2, and CN. (b) Compound of formula (10) to compound of formula (11): [ka] (In the formula, R 3 H and C1~C 12 Selected from the group consisting of alkyl groups, L is a linker that has 1 to 20 atoms in its normal chain, A step in which the reaction is carried out in the presence of a copper(I) catalyst. Methods that include... [Claim 41] A is CO2R 1 The method according to claim 40. [Claim 42] B is CO2R 2 The method according to claim 40 or 41. [Claim 43] R 1 The method according to any one of claims 40 to 42, wherein H is the case. [Claim 44] R 2 The method according to any one of claims 40 to 43, wherein H is the case. [Claim 45] Each R a The method according to any one of claims 40 to 44, wherein H is the case. [Claim 46] Each R b The method according to any one of claims 40 to 45, wherein H is the case. [Claim 47] R 3 However, C1~C 12 The method according to any one of claims 40 to 46, wherein the alkyl is... [Claim 48] R 3 The method according to any one of claims 40 to 47, wherein the element is selected from the group consisting of methyl, ethyl, propyl, and butyl. [Claim 49] L is the formula: -(CH2CH2O) n -CH2CH2- The method according to any one of claims 40 to 48, wherein the linker is an integer from the group consisting of 0, 1, 2, 3, 4, and 5. [Claim 50] The method according to claim 49, wherein n is 3. [Claim 51] The method according to any one of claims 40 to 50, wherein step (B) is carried out in the presence of a copper(I) ligand. [Claim 52] The method according to claim 51, wherein the copper(I) ligand is selected from the group consisting of TBTA, TEOTA, THPTA, BTTES, BTTAA, BTTP, BTTPS, (BimH)3, (Bth)3, BPS, and 4,4'-dimethyl-2,2'-bipyrimidine.

Claims

1. Compound of formula (I) 【Chemistry 1】 (In the formula, A is CO ２ R １ , and PO ３ R １ Selected from the group consisting of, B is CO ２ R ２ , and PO ３ R ２ Selected from the group consisting of, R １ , R ２ and R ３ are each independently selected from the group consisting of H and C １ ~C １２ alkyl, Each R ａ These are independently H, F, Cl, Br, I, and CH ３ ,CH ２ CH ３ , CH (CH ３ ) ２ OH, OCH ３ , OCH ２ CH ３ CF ３ OCF ３ NO ２ NH ２ Selected from the group consisting of , and CN, Each R ｂ These are independently H, F, Cl, Br, I, and CH ３ ,CH ２ CH ３ , CH (CH ３ ) ２ OH, OCH ３ , OCH ２ CH ３ CF ３ OCF ３ NO ２ NH ２ Selected from the group consisting of , and CN, L is a linker having 1 to 20 atoms in its normal chain. or a pharmaceutically acceptable salt thereof.

2. A is CO ２ R １ And B is CO ２ R ２ The compound according to claim 1.

3. R １ However, it is H; R ２ However, H is; each R ａ H is, and each R ｂ The compound according to claim 1, wherein H is present.

4. R ３ However, C １ ~C １２ The compound according to claim 1, wherein it is alkyl.

5. L is the formula: -(CH) ２ CH ２ O) ｎ -CH ２ CH ２ - The compound according to claim 1, wherein n is a linker, where n is an integer from the group consisting of 0, 1, 2, 3, 4, and 5.

6. Compound of formula (Ia): 【Chemistry 2】 (In the formula, A is CO ２ R １ , and PO ３ R １ Selected from the group consisting of, B is CO ２ R ２ , and PO ３ R ２ Selected from the group consisting of, R １ , R ２ and R ３ H and C are independent of each other. １ ~C １２ Selected from the group consisting of alkyl groups, Each R ａ These are independently H, F, Cl, Br, I, and CH ３ ,CH ２ CH ３ , CH (CH ３ ) ２ OH, OCH ３ , OCH ２ CH ３ CF ３ OCF ３ NO ２ NH ２ Selected from the group consisting of , and CN, Each R ｂ is independently selected from the group consisting of H, F, Cl, Br, I, CH ３ , CH ２ CH ３ , CH(CH ３ ), ２ OH, OCH ３ , OCH ２ CH ３ , CF ３ , OCF ３ , NO ２ , NH ２ , and CN L is a linker having 1 to 20 atoms in its normal chain. M is a radioactive nuclide.) or a pharmaceutically acceptable salt thereof.

7. A is CO ２ R １ and B is CO ２ R ２ The compound according to claim 6, wherein

8. R １ However, it is H; R ２ However, H is; each R ａ H is; and each R ｂ The compound according to claim 6, wherein H is present.

9. R ３ However, C １ ~C １２ The compound according to claim 6, wherein it is alkyl.

10. L is the formula: -(CH) ２ CH ２ O) ｎ -CH ２ CH ２ - The compound according to claim 6, wherein n is a linker, where n is an integer from the group consisting of 0, 1, 2, 3, 4, and 5.

11. A compound according to claim 6, wherein the radioactive nuclide is selected from the group consisting of actinium-225, lutetium-177, zirconium-89, terbium-149, terbium-152, terbium-155, terbium-161, radium-223, bismuth-212, indium-111, yttrium-86, yttrium-89, yttrium-90, and lead-212.

12. Compound of formula (Ib): 【Transformation 3】 (In the formula, A is CO ２ R １ , and PO ３ R １ Selected from the group consisting of, B is CO ２ R ２ , and PO ３ R ２ Selected from the group consisting of, R １ , and R ２ H and C are independent of each other. １ ~C １２ Selected from the group consisting of alkyl groups, R ４ It is a monoclonal antibody, Each R ａ These are independently H, F, Cl, Br, I, and CH ３ ,CH ２ CH ３ , CH (CH ３ ) ２ OH, OCH ３ , OCH ２ CH ３ CF ３ OCF ３ NO ２ NH ２ Selected from the group consisting of , and CN, Each R ｂ These are independently H, F, Cl, Br, I, and CH ３ ,CH ２ CH ３ , CH (CH ３ ) ２ OH, OCH ３ , OCH ２ CH ３ CF ３ OCF ３ NO ２ NH ２ Selected from the group consisting of , and CN, L is a linker having 1 to 20 atoms in its normal chain. M is a radioactive nuclide.) or a pharmaceutically acceptable salt thereof.

13. A is CO ２ R １ And B is CO ２ R ２ The compound according to claim 12.

14. R １ However, it is H; R ２ However, H is; each R ａ H is, and each R ｂ The compound according to claim 12, wherein H is present.

15. L is the formula: -(CH) ２ CH ２ O) ｎ -CH ２ CH ２ - The compound according to claim 12, wherein the linker is an integer from the group consisting of 0, 1, 2, 3, 4, and 5.

16. A compound according to claim 12, wherein the radioactive nuclide is selected from the group consisting of actinium-225, lutetium-177, zirconium-89, terbium-149, terbium-152, terbium-155, terbium-161, radium-223, bismuth-212, indium-111, yttrium-86, yttrium-89, yttrium-90, and lead-212.

17. The compound according to claim 12, wherein the monoclonal antibody is penprimab, cintilimab, tripalimab, omblutamab, tisotumab, retifanlimab, ubrituximab, aniflorumab, roncustuximab, valstilimab, dostallimab, oportuzumab, marjetuximab, naxitamab, verantamab, tafacitamab, sacituzumab, isatuximab, trastuzumab (Herceptin), gylenetuximab, ifabotuzumab, depatuxidimab, enfortumab, polatuzumab, emapalmab, semiprimab, moxetumomab, A compound selected from the group consisting of mogamulizumab, durvalumab, avelumab, atezolizumab, olaratumab, daratumumab, elotuzumab, necitumumab, dinutuximab, nivolumab, blinatumomab, blinatumomab, ramucirumab, obinutuzumab, ad-trastuzumab, pertuzumab, brentuximab, ipilimumab, ofatumumab, catumakisomab, panitumumab, bevacizumab, cetuximab, ibritumomab, alemtuzumab, gemtuzumab, rituximab, edrecolomab, nimotuzumab, prorugolimab, and cetuximab.

18. A pharmaceutical composition comprising the compound described in claim 12 and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition according to claim 18 for use in the treatment of cancer.

20. Compound of formula (I): (In the formula, 【Chemistry 4】 A is CO ２ R １ , and PO ３ R １ Selected from the group consisting of, B is CO ２ R ２ , and PO ３ R ２ Selected from the group consisting of, R １ , R ２ and R ３ H and C are independent of each other. １ ~C １２ Selected from the group consisting of alkyl groups, Each R ａ These are independently H, F, Cl, Br, I, and CH ３ ,CH ２ CH ３ , CH (CH ３ ) ２ OH, OCH ３ , OCH ２ CH ３ CF ３ OCF ３ NO ２ NH ２ Selected from the group consisting of , and CN, Each R ｂ These are independently H, F, Cl, Br, I, and CH ３ ,CH ２ CH ３ , CH (CH ３ ) ２ OH, OCH ３ , OCH ２ CH ３ CF ３ OCF ３ NO ２ NH ２ Selected from the group consisting of , and CN, L is a linker having 1 to 20 atoms in its normal chain. or a method for synthesizing a pharmaceutically acceptable salt thereof, (a) Compound of formula (10): 【Transformation 5】 (A is CO) ２ R １ , and PO ３ R １ Selected from the group consisting of, B is CO ２ R ２ , and PO ３ R ２ Selected from the group consisting of, R １ and R ２ H and C are independent of each other. １ ~C １２ Selected from the group consisting of alkyl groups, Each R ａ These are independently H, F, Cl, Br, I, and CH ３ ,CH ２ CH ３ , CH (CH ３ ) ２ OH, OCH ３ , OCH ２ CH ３ CF ３ OCF ３ NO ２ NH ２ Selected from the group consisting of , and CN, Each R ｂ These are independently H, F, Cl, Br, I, and CH ３ ,CH ２ CH ３ , CH (CH ３ ) ２ OH, OCH ３ , OCH ２ CH ３ CF ３ OCF ３ NO ２ NH ２ A process of providing (selected from the group consisting of , and CN), (b) Compound of formula (10) to compound of formula (11): 【Transformation 6】 (In the formula, R ３ H and C １ ~C １２ Selected from the group consisting of alkyl groups, L is a linker having 1 to 20 atoms in its normal chain. A step in which the reaction is carried out in the presence of a copper(I) catalyst. Methods that include...

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