Macrocycle-containing compounds and their radiolabeled complexes as ligands in 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] This invention relates to macrocycle-containing compounds that can be used as ligands in targeted radiotherapy applications. The invention also relates to certain metal complexes of these compounds and methods of their use. [Background technology]

[0002]

[0002] During the past 200 years, significant advances have been made in medical research, resulting in the development of effective medical treatments for a significant number of diseases / conditions, and thus a dramatic increase in 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 because they develop very slowly or are "older adult" diseases.

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

[0004] Cancer is a somewhat general term used to describe a large group of diseases that can affect any port of the body. A common feature of nearly 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 cause of cancer is generally believed to be environmental or genetic factors. Over 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 solid tumors where the cancer cells are clumped together, or dispersed as in leukemia. Cancer cells are often called "malignant" because they divide endlessly, eventually crowding out nearby cells and spreading 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 them from benign tumor cells, which overgrow but do not spread to other organs or other parts of the body. Malignant cancer cells eventually metastasize via the bloodstream or lymphatic system and spread to other parts of the body where they can grow and form new tumors. This type of tumor progression makes cancer a deadly disease.

[0006]

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

[0007]

[0007] One area of ​​growing interest is the use of radiation therapy in the treatment of cancer. Radiation therapy is based on the observation that, at high doses, radiation does kill cancer cells or slow their growth as a result of it damaging their DNA. 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] Radiation therapy is typically divided into two major types: (1) external beam radiation therapy and (2) internal radiation therapy; in either case, the type of radiation therapy used depends on several factors specific to the patient.

[0009]

[0009] External beam radiation therapy involves the use of a machine that delivers radiation to the cancer in a patient. This type of radiation therapy is aimed at treating specific parts of the body, so that only the area with the cancer is exposed to radiation, rather than the entire body. This type of therapy can be very successful for tumors deep within the body, but since it targets cancer cells within the body, some healthy cells will inevitably be exposed to radiation.

[0010]

[0010] Internal radiation therapy involves either seeding the tumor site with radioactive material or using chemotherapy agents that are "targeted" to the cancer site. In these targeting approaches, radionuclide ligands are typically conjugated to antibodies that target 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, radionuclides that emit lethal alpha particles are conjugated with tumor-targeting vectors using chelators. This therapy allows the delivery of cytotoxic levels of alpha radiation selectively to cancer calls.

[0011] As a result, there has been great research interest in developing conjugates that can be used to bind alpha particle-emitting radionuclides and which can then be further engineered to contain cancer-targeting moieties. Several potential conjugates have been developed.

[0012]

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

[0013] [ka]

[0014]

[0013] Although this conjugate is relatively easy to obtain, it suffers from the observation that the thermodynamic stability of the complexes of H4DOTA decreases with increasing ionic metal ion, resulting in the formation of complexes of H4DOTA with the most highly ionized cation, Ac, the most highly ionized cation in the periodic table. 3+ In addition, the radiolabeling kinetics of this ligand may be slower than that of some radionuclides, e.g., Ac 3 +), which necessitates labeling at elevated temperatures if short labeling times are required.

[0015] As a result, some research has been directed to the development of alternative conjugates. One alternative conjugate that has been developed is N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6 (2), colloquially known as H2macropa.

[0016] [ka]

[0017] Another conjugate that has been developed is an analogue of H2macropa, namely, H2macropa-NCS (3):

[0018] [ka]

[0019] It is.

[0016] Although these conjugates could be better radiolabeled without the need for high temperatures, they were not without problems as aggregation of the conjugates was observed to occur.

[0020]

[0017] There is therefore great interest in the development of additional compounds that can be used as radionuclide conjugates in targeted therapy. Summary of the Invention [Problem to be solved by the invention]

[0021]

[0018] The applicants have carried out studies aimed at the development of compounds which can be used as potential radionuclide conjugates and thus be utilized in targeted therapy. [Means for solving the problem]

[0022] As a result of these studies, applicants have identified compounds which exhibit great potential as conjugates for radionuclides. Thus, in one embodiment, the present invention provides a compound of formula (I):

[0023] [ka]

[0024]

[0021] (In the formula,

[0022] A is CO2R 1 , and PO3R 1 is selected from the group consisting of B is COR 2 , and PO3R 2 is selected from the group consisting of

[0024] R 1 , R 2 and R 3 are each independently H and C1 to C 12 is selected from the group consisting of 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, NH2, and CN;

[0026] 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;

[0027] L is a linker having 1 to 20 atoms in the normal chain;

[0028] or a pharma- ceutically acceptable salt thereof.

[0025]

[0029] It has been found that compounds of the present invention have the ability to form metal complexes with radionuclides and thus may be used in targeted therapy.

[0030] In a still yet further aspect, the present invention provides a compound of formula (Ia):

[0026] [ka]

[0027]

[0031] (In the formula,

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

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

[0034] R 1 , R 2 and R 3 are each independently H and C1 to C 12 is selected from the group consisting of alkyl,

[0035] 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;

[0036] Each R bis 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 the normal chain,

[0038] M is a radionuclide;

[0039] or a pharma- ceutically 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. Thus, in yet a further aspect, the present invention provides a compound of formula (Ib):

[0029] [ka]

[0030]

[0041] (In the formula,

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

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

[0044] R 1 , and R 2 are each independently H and C1 to C 12 is selected from the group consisting of alkyl,

[0045] R 4 is a monoclonal antibody,

[0046] 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;

[0047] Each R bis 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 the normal chain,

[0049] M is a radionuclide;

[0050] or a pharma- ceutically acceptable salt thereof.

[0031]

[0051] In a still yet further aspect, the present invention provides a method for the synthesis of a compound of formula (I) above, comprising the steps of:

[0052] (a) A compound of formula (10):

[0032] [ka]

[0033]

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

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

[0055] R 1 , and R 2 are each independently H and C1 to C 12 is selected from the group consisting of alkyl,

[0056] 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;

[0057] Each R 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;

[0058] (b) reacting a compound of formula (10) with a compound of formula (11)

[0034] [ka]

[0035]

[0059] (In the formula, R 3 H and C1-C 12 is selected from the group consisting of alkyl,

[0060] L is a linker having 1 to 20 atoms in the normal chain;

[0061] reacting in the presence of a copper(I) catalyst The present invention provides a method comprising:

[0036]

[0062] In a still yet further aspect, the present invention provides a pharmaceutical composition comprising a compound according to Formula (I), Formula (Ia) or Formula (Ib) and a pharma- ceutically acceptable diluent, excipient or carrier.

[0037]

[0063] In yet a further aspect, the present invention provides a method of treating a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula (Ib). In certain embodiments, the subject is suffering from cancer. [Brief description of the drawings]

[0038] [Figure 1]

[0064] Figure 13. TLC analysis of free Ac-225 using 0.4 M sodium citrate pH 4 + 10% methanol (origin is at 15 mm, solvent front is at 80 mm; free Ac-225 is at solvent front: Rf = 0.8-1.0). [Diagram 2]

[0065] Figure 13. iTLC analysis of free Ac-225 using 50 mM citric acid pH 5 (origin is at 15 mm, solvent front is at 80 mm; free Ac-225 is at solvent front: Rf = 0.8-1.0). [Diagram 3]

[0066] Figure 1 shows TLC analysis of crude [225Ac]Ac-macropa at chelator concentration of 10-3M using 0.4M sodium citrate pH 4 + 10% methanol (origin is at 10mm, solvent front is at 80mm; [225Ac]Ac-macropa remains at origin: Rf=0; free Ac-225 is at solvent front: Rf=0.8-1.0). [Figure 4]

[0067] Figure 1 shows a summary of the radiochemical yields of various Ac-225 labeled chelators at different chelator concentrations incubated at ambient temperature for 2 hours unless otherwise noted. Radiochemical yields were determined by TLC analysis of crude reaction mixtures using 0.4M sodium citrate pH 4 + 10% methanol. [Diagram 5]

[0068] Figure 13 shows a summary of the radiochemical yield of [225Ac]Ac-macropa-tzPEG3SqOEt at different chelator concentrations over time upon incubation at ambient temperature. The radiochemical yield was determined by TLC analysis of the crude reaction mixture using 0.4 M sodium citrate pH 4 + 10% methanol. [Figure 6]

[0069] Figure 13 shows a summary of the radiochemical yield of [225Ac]Ac-DOTA-methyltetrazine at different chelator concentrations over time upon incubation at 90° C. Radiochemical yields were determined by TLC analysis of the crude reaction mixture using 0.4 M sodium citrate pH 4 + 10% methanol. [Figure 7A]

[0070] 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, incubated for 15 min at ambient temperature (50 mM citric acid pH 5; origin at 10 mm, solvent front at 80 mm; [225Ac]Ac-macropa-tzPEG3Sq-conjugated monoclonal antibody remains at origin: Rf=0; free Ac-225 is at solvent front: Rf=0.8-1.0). [Figure 7B] 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, incubated for 15 min at ambient temperature (50 mM citric acid pH 5; origin at 10 mm, solvent front at 80 mm; [225Ac]Ac-macropa-tzPEG3Sq-conjugated monoclonal antibody remains at origin: Rf=0; free Ac-225 is at solvent front: Rf=0.8-1.0). [Figure 8]

[0071] FIG. 1 shows the SE-HPLC chromatogram (UV280) of H2macropa-tzPEG3Sq-immunoconjugate. [Figure 9]

[0072] FIG. 1 shows the SE-HPLC chromatogram (UV280 and radiation) of [225Ac]Ac-macropa-tzPEG3Sq-immunoconjugate. [Figure 10]

[0073] FIG. 10A shows the radiochemical purity determined using iTLC (50 mM EDTA pH 5), and FIG. 10B shows the serum stability study of [225Ac]Ac-macropa-tzPEG3Sq- and [225Ac]Ac-DOTA-dhPzPEG4-conjugated monoclonal antibodies at different chelator-antibody ratios, with immunoreactivity determined using U87MG.de2-7 and U251 cells for EGFRVIII IgG1 and EphA3 IgG1, respectively. [Figure 11A]

[0074] Figure 1 shows (a) tumor growth curves and (b) Kaplan-Meier survival curves of 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] Figure 1 shows (a) tumor growth curves and (b) Kaplan-Meier survival curves of 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] Figure 1 shows (a) tumor growth curves and (b) Kaplan-Meier survival curves of 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] Figure 1 shows (a) tumor growth curves and (b) Kaplan-Meier survival curves of 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039]

[0076] In this specification, certain terms are used that are well known to those of ordinary skill in the art. Nevertheless, for the sake of clarity, certain terms are defined.

[0077] In the definitions of some of the 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 the situation where the group is a linker between two other parts of the molecule as well as the situation where it is a terminal moiety. Using the term alkyl as an example, some publications use the term "alkylene" for a bridging group, and therefore in these other publications there is a distinction between the term "alkyl" (terminal group) and the term "alkylene" (bridging group). In this application, no such distinction is made and most groups may be either a bridging group or a terminal group.

[0040]

[0078] "Alkyl" as a group or part of a group, unless otherwise noted, means a straight or branched chain aliphatic hydrocarbon group, preferably C1-C 12 Alkyl, more preferably C1-C 10 It refers to alkyl, most preferably C1-C6. Examples of suitable straight and branched C1-C6 alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t-butyl, hexyl, etc. The group may be a terminal group or a bridging group.

[0041]

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

[0080] The term "pharmaceutically acceptable salt" refers to salts that retain the desired biological activity of the compounds identified above, including pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts of the compounds of formula (I) can be prepared from inorganic acids or from organic acids. Examples of such inorganic acids are hydrochloric acid, sulfuric acid, and phosphoric acid. Suitable organic acids may be selected from aliphatic, alicyclic, aromatic, heterocyclic carboxylic and sulfonic classes of organic acids, 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, arylsulfonic acid. Similarly, base addition salts can be prepared in a manner well known in the art using organic or inorganic bases. Examples of suitable organic bases include simple amines, such as methylamine, ethylamine, triethylamine, and the like. Examples of suitable inorganic bases include NaOH, KOH, and the like. Additional information regarding pharma- ceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co., Easton, PA 1995. For drugs that are solid, it will be understood by those of skill in the art that the compounds, drugs and salts of the invention may exist in different crystalline or polymorphic forms, all of which are intended to be within the scope of the invention and the defined formulas.

[0042]

[0081] The term "therapeutically effective amount" or "effective amount" is an amount sufficient to effect a beneficial or desired clinical result. An effective amount may be administered in one or more administrations. An effective amount is typically sufficient to palliate, ameliorate, stabilize, reverse, slow or retard the progression of a disease condition.

[0043]

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

[0044]

[0083] In the compounds of the present invention, A is COR 1 , and PO3R 1 In some embodiments, A is selected from the group consisting of: 1 In some embodiments, A is PO3R 1 It is.

[0045]

[0084] In the compounds of the present invention, B is COR 2 , and PO3R 2 In some embodiments, B is selected from the group consisting of: 2 In some embodiments, B is PO3R 2 It is.

[0046]

[0085] In the compounds of the present invention, R 1 , R 2 and R 3 are each independently H and C1 to C 12 alkyl.

[0086] In some embodiments, R 1 is H. In some embodiments, R 1 is C1~C 12 In some embodiments, R 1 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 is H. In some embodiments, R 2 is C1~C 12 In some embodiments, R 2 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 compounds of the invention, A is COR 1 and B is CO2R 2 and R 1 is H and R 2 is H. This gives compounds of formula (II), (IIa) and (IIb), respectively:

[0049] [ka]

[0050]

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

[0051] [ka]

[0052]

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

[0053] [ka]

[0054]

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

[0092] In the compounds of the present invention, each R a is independently selected from the group consisting of H, F, Cl, Br, I, CH, CHCH, CH(CH), OH, OCH, OCHCH, CF, OCF, NO, NH, and CN. ais independently selected from H, F, Cl, Br, I, CH, CHCH, OH, OCH, OCHCH, and CN. a is independently selected from H, CH, CHCH, and OH. 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, CH, CHCH, CH(CH), OH, OCH, OCHCH, CF, OCF, NO, NH, and CN. b is independently selected from H, F, Cl, Br, I, CH, CHCH, OH, OCH, OCHCH, and CN. b is independently selected from H, CH, CHCH, and OH. b is H.

[0056]

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

[0057] [ka]

[0058]

[0095] (Wherein, L and R 3 is as defined above).

[0059] [ka]

[0060]

[0096] (In the formula, LR 3 and M is as defined above).

[0061] [ka]

[0062]

[0097] (In the formula, L, R 4 and M is as defined above).

[0098] In some embodiments, R 3 is H. In some embodiments, R 3 is C1~C 12 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. 3 is methyl. In some embodiments, R 3 is ethyl.

[0063]

[0099] R 4 In compounds of the invention containing the moiety, R 4 is an antibody. In certain embodiments, R 4 is a monoclonal antibody. In certain embodiments, R 4are penprimab, sintilimab, toripalimab, ombultamab, tisotumab, retifanlimab, ublituximab, anifrolumab, loncastuximab, valstilimab, dostallimab, oportuzumab, marjetuximab, naxitamab, belantamab, tafasitamab, sacituzumab, isatuximab, trastuzumab (Herceptin), girentuximab, ifavotuzumab, depatuxizumab, enfortumab, polatuzumab, emapalumab, cemiplimab, moxetumomab, mogamulizumab, durvalumab, abetuximab, ramucirumab, atezolizumab, olaratumumab, daratumumab, elotuzumab, necitumumab, dinutuximab, nivolumab, blinatumomab, blinatumomab, ramucirumab, obinutuzumab, ado-trastuzumab, pertuzumab, brentuximab, ipilimumab, ofatumumab, catumaxomab, 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 an EphA3 IgG1 monoclonal antibody. In one embodiment, the antibody is an EGFRVIII IgG1 monoclonal antibody. In one embodiment, the antibody is an 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 the normal chain. In some embodiments, L is a linker having 15 atoms in the normal chain. In some embodiments, L is a linker having 16 atoms in the normal chain. In some embodiments, L is a linker having 17 atoms in the normal chain. In some embodiments, L is a linker having 18 atoms in the normal chain. In some embodiments, L is a linker having 19 atoms in the normal chain. In some embodiments, L is a linker having 20 atoms in the normal chain.

[0067]

[0103] In some embodiments, L is of the formula: -(CH2) m - wherein 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 - is selected from the group consisting of

[0069]

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

[0106] where 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- It is.

[0110] The macrocycles of the present invention can in principle bind to several metals. In application, the compounds are 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 invention containing a radionuclide M are typically formed by incubating a metal with a conjugate of the appropriate formula. The incubation can be carried out at any temperature, but it is found that the binding of the radionuclide to the compounds of the invention is very rapid, so that the binding can be carried out at room temperature and still be extremely rapid. Once bound, the complexes so formed are found to be very stable.

[0075]

[0114] In principle, the metal can also be complexed with the compound containing the antibody, but it is typically found to be more efficient to complex the radionuclide with the compound before the addition of the antibody. Without wishing to be bound by theory, this is thought to ensure more efficient complexation, since sites on the antibody may compete with the macrocycle for binding to the metal. Therefore, it is common to react the complex with the radionuclide before the addition of the antibody.

[0076]

[0115] Antibody-containing compounds are typically formed by reacting a compound of formula (I), (Ia), (II), (IIa), (III) and (IIIa) with a pendant amine group on an antibody to form an antibody-containing compound. As will be appreciated by those skilled in the art, many antibodies contain multiple amine residues available for reaction with the squaramide moiety on the compound of the present invention. Thus, in some embodiments, each antibody is conjugated to two or more compounds of the present invention. In some embodiments, each antibody is conjugated to two compounds of the present invention. In some embodiments, each antibody is conjugated to three compounds of the present invention. In some embodiments, each antibody is conjugated to four compounds of the present invention. In some embodiments, each antibody is conjugated to five compounds of the present invention. Control of reaction stoichiometry and general reaction conditions can 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 containing the antibody have the potential to be used in targeted radiotherapy of cancer. As a result of the presence of the antibody on the compound, the compound targets (i.e., selectively binds to) the cancer cells. As a result of the compound selectively binding to the cancer cells, the radiation produced by the radionuclide is emitted in close proximity to the cancer cells, thereby causing more damage to the cancer cells than to other cells of the body. This is 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 cancer 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] Administration of the compounds of the present invention to humans can be by any of the recognized modes for parenteral administration, such as subcutaneous, intramuscular, intravenous and intradermal routes.Injection can be by bolus or continuous or intermittent infusion.The active compound is typically included in a pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver a therapeutically effective dose to the patient.

[0080]

[0119] The compound of the present invention can be administered in any form or manner that makes the compound available for binding to desired target cells.Those skilled in the art of preparation of formulations can easily select suitable form and manner of administration according to the specific nature of selected compound, the condition to be treated, the stage of the condition to be treated and other relevant circumstances.For further information, please refer to Remingtons Pharmaceutical Sciences, 19th edition, Mack Publishing Co. (1995).

[0081]

[0120] The compound of the present invention can be administered alone or in the form of pharmaceutical composition in combination with pharmaceutically acceptable carrier, diluent or excipient.Although the compound of the present invention is effective in itself, it is typically formulated and administered in the form of its pharmaceutically acceptable salt, because these forms are typically more stable, more easily crystallized and have increased solubility.

[0082]

[0121] However, the compound is typically used in the form of a pharmaceutical composition that is formulated according to the desired mode of administration.Therefore, in some embodiments, the present invention provides a pharmaceutical composition that includes the compound of the present invention and a pharma-ceutically acceptable carrier, diluent or excipient.The composition is prepared in a manner well known in the art.

[0083]

[0122] The pharmaceutical composition of the present invention for parenteral injection comprises sterilized aqueous or non-aqueous solution, dispersion, suspension or emulsion, and sterile powder for reconstitution into sterile injectable solution or dispersion immediately before use.Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyol (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oil (e.g., olive oil), and injectable organic esters such as ethyl oleate.Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants.

[0084]

[0123] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action may be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc. Prolonged absorption of injectable pharmaceutical forms may be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0085]

[0124] Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which 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 to treat and relieve or alleviate the condition.The therapeutically effective amount can be easily determined by the attending diagnostician using conventional techniques and observing the results obtained under similar circumstances.In determining the therapeutically effective amount, several factors should be considered, including but not limited to the species of animal, its size, age and general health condition, the specific condition involved, the severity of the condition, the patient's response to treatment, the specific compound administered, the mode of administration, the bioavailability of the administered preparation, the selected dosing schedule, the use of other drug therapies and other relevant circumstances.

[0087]

[0126] A preferred dosage is in the range of about 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. A suitable dose may be administered in multiple subdoses per day. Synthesis of Compounds of the Invention

[0127] The compounds of the present invention may be prepared using known organic synthesis techniques, and may be synthesized using readily available starting materials and techniques available in the art according to any of a number of possible synthetic routes, including the reaction routes and synthetic 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 appreciate that the chemical reactions described can be easily adapted to prepare other agents of the various embodiments.

[0088]

[0128] The reaction for preparing the compound of the present invention can be carried out in a suitable solvent that can be easily selected by those skilled in the art of organic synthesis.A suitable solvent can be substantially non-reactive with the starting material (reactant), intermediate, or product at the temperature at which the reaction is carried out, which can range, for example, from the freezing temperature of the solvent to the boiling temperature of the solvent.A given reaction can be carried out in one solvent or a mixture of two or more solvents.Depending on the reaction step, a suitable solvent for a particular reaction step can 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, and the selection of suitable protecting groups, can be easily determined by those skilled in the art. The chemistry of protecting groups can be found, for example, in TW Greene and PG M Huts, 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), infrared spectroscopy, spectrophotometry (eg, UV-visible), or mass spectrometry, or by chromatography, such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0091]

[0131] As used herein, the expressions "ambient temperature," "room temperature," and "rt" are understood in the art and generally refer to a temperature that is about the temperature of the room in which the reaction is carried out, e.g., the reaction temperature, e.g., a temperature of about 20° C. to about 30° C.

[0092]

[0132] In a still yet further aspect, the present invention provides a compound of formula (I):

[0093] [ka]

[0094]

[0133] (In the formula,

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

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

[0136] R 1 , R 2 and R 3 are each independently H and C1 to C 12 is selected from the group consisting of alkyl,

[0137] 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;

[0138] Each R b is 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 having 1 to 20 atoms in the normal chain;

[0140] or a pharma- ceutically acceptable salt thereof, comprising the steps of:

[0141]

[0142] (a) A compound of formula (II):

[0095] [ka]

[0096]

[0143] (Wherein, A is COR 1 , and PO3R 1 is selected from the group consisting of

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

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

[0148] (b) reacting a compound of formula (II) with a compound of formula (III):

[0097] [ka]

[0098]

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

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

[0151] reacting in the presence of a copper(I) catalyst The present invention provides a method comprising:

[0099]

[0152] The reaction may be carried out in any suitable solvent. Examples of suitable solvents include polar organic solvents and combinations thereof. Examples of polar solvents that may 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 solvent discussed above and water. In some embodiments, the ratio of the polar organic solvent to water is 10:1 to 1:1. In one embodiment, the ratio of the polar organic solvent to water is 8:1 to 1:1. In one embodiment, the ratio of the polar organic solvent to water is 6:1 to 1:1. In one embodiment, the ratio of the polar organic solvent to water is 5:1 to 3:1. In one embodiment, the ratio of the 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 be carried out over a wide range of temperatures. In some embodiments, the reaction is carried out at a temperature of 5° C. to 50° C. In one embodiment, the reaction is carried out at a temperature of 10° C. to 30° C. In one embodiment, the reaction is carried out at a temperature of 15° C. to 25° C. In one embodiment, the reaction is carried out at a temperature of 20° C. to 25° C.

[0101]

[0154] The reaction is typically carried out for a period of time sufficient to achieve 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 mentioned 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 such as [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 serves to stabilize copper(I) in solution, where 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 completion of the reaction, the reaction medium is worked up 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 now be illustrated by examples which should not be construed as limitations thereon. Using the methods and synthetic protocols described herein, or suitable variations or modifications thereof, additional compounds other than those described below may be prepared. 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 NMR500 FT-NMR400 spectrometer (Varian, California, USA). 1 H NMR spectra were acquired at 400 MHz or 500 MHz. 13 C spectra were acquired at 101 MHz or 126 MHz. All reported peaks are referenced to the solvent peak in parts per million at 25 °C.

[0106]

[0161] ESI-QTOF MS were collected on an Exactive Plus Orbitrap Infusion mass spectrometer (Exactive Series, 2.8 Build 268801, ThermoFisher Scientific). Analyses were performed using Xcalibur 4.0.27.10 (ThermoFisher Scientific).

[0107]

[0162] Protein samples were analyzed on an Agilent 6220 ESI-TOF LC / MS mass spectrometer coupled to an Agilent 1200 LC system (Agilent, Palo Alto, CA). All data were acquired with a dual-spray electrospray ionization (ESI) source and were reference mass corrected. Acquisition was performed using Agilent Mass Hunter Acquisition software version B.02.01 (B2116.30). Ionization mode: electrospray ionization; drying gas flow rate: 7 L / min; nebulizer: 35 psi; drying gas temperature: 325 °C; capillary voltage (Vcap): 4000 V; fragmentor: 300 V; skimmer: 65 V; OCT RFV: 250 V; acquisition scan range: 300-3200 m / z. Internal reference ions: positive ion mode = m / z = 121.050873 and 922.009798. Desalting and chromatographic separation of proteins was performed using an Agilent Poroshell C18 2.1 × 75 mm, 5 μm column using 5% (v / v) acetonitrile, diverted to waste (0-5 min). After desalting the samples, the flow was reconnected to the ESI source for a subsequent gradient elution with acetonitrile / 0.1% formic acid (5% (v / v) to 100% (v / v)) over 8 min at 0.25 mL / min. Analysis was performed using Mass Hunter version B.06.00 with BioConfirm software using a maximum entropy protein deconvolution algorithm; mass step 1 Da; baseline factor 3.00; peak widths set to uncertain.

[0108]

[0163] Non-radioactive analytical HPLC 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 Phenomenex Luna C18(2) column (4.6 mm×150 mm, 5 μm, column B) and a Phenomenex SecurityGuard™ C18 guard cartridge (4 mm×30 mm) at a flow rate of 1 mL / min; System A: Buffer A=0.1% TFA in HO and Buffer B=0.1% TFA in acetonitrile gradient elution (0 to 100% B in A over 25 min) and UV detection at λ 220, 254, 280 nm and 350 nm.

[0109]

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

[0110]

[0165] Radioactivity was measured using either a Capintec CRC-55t PET 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 the respective vehicle buffers were subtracted prior to measurement.

[0112]

[0167] Thin-layer chromatography (TLC) was performed using aluminum-backed silica gel 60 F 254 The experiments were carried out 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 equilibration (minimum 8 hours after development). The chromatogram of the control (i.e., no chelator) is shown in Figure 1. This shows that, as expected, the actinium-containing solution did not move at the solvent front (R) in the absence of H2macropa or H2macropa-tzPEG3SqOEt. f =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 citric acid 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., no chelator) is shown in Figure 2. This shows that, as expected, the actinium-containing solution did not move beyond the solvent front (R) in the absence of H2macropa-tzPEG3Sq-conjugated monoclonal antibody. f =1).

[0114]

[0169] Size-exclusion HPLC (SE-HPLC) was performed on a 1200 series Agilent system equipped with a fraction collector and a diode array detector 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 at a flow rate of 1 mL / min.

[0115]

[0170] The immunoreactive fraction was determined by administering the radioimmunoconjugate (20 ng) to the relevant cell line (5 x 10 6 The radioactivity was determined by incubation in 1000 rpm (1000 rpm, 1000 cells) at ambient temperature for 45 min, followed by spinning the cell suspension (2000 rcf for 2 min) and washing the resulting cell pellet with medium (1 mL). The wash was repeated two more times and the radioactivity in the final cell pellet was measured using an automatic gamma counter at permanent equilibration (minimum 8 h after washing). The immunoreactivity or immunoreactivity ratio (IRF) was calculated as the ratio of radioactivity in the cell pellet compared to the standard using radioimmunoconjugates (20 ng, 500 μL, average of triplicates). Nonspecific binding (NSB) was determined by incubating the radioimmunoconjugates (20 ng) with the respective unconjugated monoclonal antibodies (60 μg) following the procedure described above.

[0116]

[0171] Ac-225, derived from thorium-229, was purchased as the nitrate salt from Oak Ridge National Laboratory (USA). 225 [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). Reconstituted [Ac]Ac-nitrate with similar radioactivity concentrations was 225 A stock solution of Ac]Ac-nitrate was prepared by dilution in 0.15 M sodium acetate pH 5.5 (1:9 to 1:1 depending on the decay of Ac-225).

[0117]

[0172] Purified mouse-human IgG1 chimeric monoclonal antibodies EGFRVIII IgG1, EphA3 IgG1, and chimeric IgG1 isotype control, and humanized IgG1 isotype control antibodies were provided by 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 calf 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 (The 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 purchased commercially as reagent grade from readily available sources.

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

[0120] [ka]

[0121]

[0175] 100 was prepared following an adaptation of a literature procedure (Tetrahedron 2015, 71(33), 5321-5336). Pyridine-2,6-dicarboxylic acid (4.23 g, 25.3 mmol) and sulfuric acid (1 mL) in methanol (30 mL) were heated at reflux for 6 h. The solid was filtered as a colorless crystalline solid. A second crop was obtained by removing the solvent under reduced pressure, dissolved in dichloromethane (100 mL), and washed with saturated NaHCO3 solution (2 x 50 mL) and water (50 mL). The combined organic layers were dried over MgSO4 and the solvent was removed under reduced pressure to give a colorless solid (3.94 g, 80%). ESI-MS {M+H +}:196.0599 (C9H 10 No. 4) + Calculated value: 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 C NMR (101 MHz, CDCl3) δ 165.0, 148.2, 138.4, 128.0, 53.2. Rt= 9.70 min (Method A, Column B).

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

[0123] [ka]

[0124]

[0176] Methyl 6-(hydroxymethyl)picolinate was synthesized using an adapted literature protocol. Inorg.Chem.2008, 47(17), 7840-51. To dimethyl 2,6-pyridine-2,6-dicarboxylate (6.0 g, 30.6 mmol) in methanol (200 mL) at 0 °C was added sodium borohydride (2.32 g, 62 mmol) over 1 h. The reaction mixture was stirred at room temperature for 5 h. The solution was quenched using 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 give compound 101 (3.37 g, 57%). ESI-MS {M+H +}:168.0652 (C8H 10 No. 3) + Calculated value: 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 C NMR (125.7 MHz, CDCl3): δ 165.0, 148.2, 138.4, 128.0, 53.2. Rt = 6.67 min (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 an adapted literature protocol. 2Thionyl chloride (6 mL) was added slowly to methyl-6-hydroxymethyl-2-pyridinecarboxylate (2.5 g, 15 mmol) at 0° C. under N2 atmosphere and stirred for 1 h. After 1 h, thionyl chloride was removed in vacuo. 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 give an oil that precipitated to give an off-white solid (2.42 g, 90%). ESI-MS {M+H +}:186.0319 (C8H9ClNO2) + Calculated value: 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 C NMR (101 MHz, CDCl3) δ 165.3, 157.2, 147.5, 138.1, 126.2, 124.5, 53.1, 46.3. Rt = 7.65 min (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 an adapted literature protocol. 3 Chelidamic acid (4.0 g, 21.8 mmol) was dissolved in ethanol (150 mL), sulfuric acid (6 drops) was added, and refluxed for 16 h. The ethanol was removed under reduced pressure and used in the next reaction without further purification (3.98 g, 76%). ESI-MS {M+H +}:240.0866 (C 11 H 14 No. 5)+ Calculated value: 240.0867. 124°C. 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-(prop-2-yn-1-yloxy)pyridine-6-dicarboxylate (104)

[0132] [ka]

[0133]

[0179] Diethyl 4-(prop-2-yn-1-yloxy)pyridine-2,6-dicarboxylate was synthesized using an adapted literature protocol. 4 To 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 added a propargyl bromide solution (80 wt% in toluene - 4.69 mL, 52.3 mmol). The mixture was heated at 80 °C for 3 h to aid solubility and then stirred at room temperature overnight. The mixture was then filtered and the filtrate concentrated under reduced pressure. The residue was purified by column chromatography (0-2% MeOH in DCM) to give compound 104 as either a pale yellow or brown solid (2.47 g, 68%). ESI-MS {M+H +}:278.1021 (C 14 H 16 No. 5) + 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). 13C 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 min (Method A, Column A).

[0134] Example 6 2-Hydroxymethyl 4-(prop-2-yn-1-yloxy)pyridine-6-ethyl carboxylate (105)

[0135] [ka]

[0136]

[0180] 2-Hydroxymethyl 4-(prop-2-yn-1-yloxy)pyridine-6-ethyl carboxylate was synthesized using an adapted literature protocol. 2 To diethyl 4-(prop-2-yn-1-yloxy)pyridine-2,6-dicarboxylate (1.81 g, 6.49 mmol) in ethanol (100 mL) at 0° C. was added sodium borohydride (0.319 g, 8.44 mmol) over 0.5 h. The reaction mixture was stirred at room temperature for 3 h and monitored by MS until no starting material remained. The solution was quenched using 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 give the off-white compound 6 (1.2 g, 75%) without further purification. ESI-MS {M+H +}:236.0919 (C 12 H 14 No. 4) + Calculated value: 236.0918. 1 H 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). 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 = 11.32 min (Method A, Column A).

[0137] Example 7 2-Chloromethyl 4-(prop-2-yn-1-yloxy)pyridine-6-ethyl carboxylate (106)

[0138] [ka]

[0139]

[0181] Thionyl chloride (6 mL) was added slowly to 2-hydroxymethyl 4-(prop-2-yn-1-yloxy)pyridine-6-ethylcarboxylate (0.4 g, 1.7 mmol) at 0° C. under N2 atmosphere and stirred for 3 h. After 3 h, thionyl chloride was removed in vacuo to give 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 give a pale yellow powder (0.33 g, 77%). ESI-MS {M+H +}:254.0578 (C 12 H 14 No. 4) + Calculated value: 254.0578. 1 H 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 C 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 min (Method A, Column B).

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

[0141] [ka]

[0142]

[0182] Methyl 6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate was synthesized using an adapted literature protocol. 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 dry ACN (350 mL) at 75 °C was added a solution of 102 (0.37 g, 2.03 mmol) in dry ACN (80 mL) dropwise over 4 h under nitrogen atmosphere and heated at reflux for 40 h. The solution was concentrated under reduced pressure at 60 °C to a pale yellow oil. The crude oil was then purified by semi-preparative HPLC (Method C) and the appropriate fractions were combined and lyophilized to give a pale oil (626 mg, 76%). ESI-MS {M+2H +}:206.6257 (C 20 H 33 N3O6) 2+ Calculated value: 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). 13C NMR (126 MHz, cdcl3) δ 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 min (Method A, Column A).

[0143] Example 9 Ethyl 4-(prop-2-yn-1-yloxy)-6-((16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-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-diazacyclooctadecan-7-yl)methyl)picolinate (0.149 g, 0.4 mmol) in anhydrous DMF (10 mL) under nitrogen atmosphere and stirred at room temperature for 30 min. To the stirred reaction mixture was added a solution of 2-chloromethyl 4-(prop-2-yn-1-yloxy)pyridine-6-ethylcarboxylate (0.138 g, 0.54 mmol) in anhydrous DMF (2 mL) under nitrogen and the reaction was heated at 50° C. for 40 h. The resulting solution was filtered and taken to dryness under reduced pressure to give an orange oil which was purified by semi-preparative HPLC and the appropriate fractions were combined and lyophilized to give a yellow oil. ESI-MS{M+H +}:315.1627 (C 32 H 46 N4O9) 2+ Calculated value: 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 min (Method A, Column A).

[0146] Example 10 4-(prop-2-yn-1-yloxy)-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid (109)

[0147] [ka]

[0148]

[0184] 4-(prop-2-yn-1-yloxy)-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid was synthesized using an adapted literature protocol. 6 To a solution of 108 (18.5 mg, 0.03 mmol) in DCM (450 μL) was added a sonicated suspension of NaOH in methanol (50 μL, 3 M) to 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 h, then the solvent was removed under reduced pressure and used in the next step without further purification. ESI-MS {M+H +}:294.1393 (C 29 H 40 N4O9) 2+Calculated for: 294.1393. Rt=8.53 min (Method A, Column A).

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

[0150] [ka]

[0151]

[0185] Freshly prepared diethyl squarate (0.23 g, 1.32 mmol) was dissolved in ethanol (4 mL) followed by the addition of 1-amino-11-azido-3,6,9-trioxaundecane (0.28 g, 1.29 mmol) and DIPEA (1 eq.) under nitrogen. The mixture was stirred at room temperature for 8 h, then the colorless solution was taken to dryness under reduced pressure and purified by semi-preparative HPLC (Method C). The fractions were combined and lyophilized to give 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-squarate and 4-(prop-2-yn-1-yloxy)-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-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 min to ensure complete reduction to copper(I) species. TBTA (2.8 mg, 0.0057 mmol, 32% mmol in DMF) was then added along with N3-PEG3-squarate (6.42 mg, 0.018 mmol) as a stabilizing ligand and allowed to react for 10 min. 109 (11 mg, 0.018 mmol) was added and the reaction mixture was purged with a stream of nitrogen for 15 min 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 h to ensure the reaction had gone to completion. The yellow solution was taken to dryness under reduced pressure and dried in vacuum. The resulting precipitate was purified by semi-preparative HPLC and the fractions were combined and lyophilized to give an off-white powder (5 mg, 40%). ESI-MS {M+2H +}:465.2161 (C 43 H 62 N8O 15 ) 2+ Calculated value: 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 min (Method A, Column A).

[0155] Example 13 Protein conjugation – Herceptin

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

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

[0188] EGFRVIII IgG1 (9.4 mg / mL) and EphA3 IgG1 antibodies (3.1 mg / mL; 3 mg, 2 × 10 -5IgG1 (10 mmol) was buffer exchanged into borate buffer (0.2 M, pH 9.0) and split into two equal aliquots to a final concentration of 5 mg / mL (total volume of 300 μL for EGFRVIII IgG1 and 320 μL for EphA3 IgG1. To the reaction mixture was added H2macropa-tzPEG3SqOEt (111, 10 mg / mL stock solution in DMSO - final DMSO concentration <4%) and shaken overnight at 4°C, then another overnight at room temperature. Excess reagent was removed and buffer exchanged (sodium acetate, 0.1 M, pH 5.5) by spin filtration (MW cutoff of 50 KDa).

[0157] [Table 1]

[0158] Example 15 Ac-225 label concentration study

[0189] Ac-225 stock solution (approximately 74 kBq, 2.3 μL) was added to solutions of H2macropa, DOTA (2,2',2'',2''''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid), DTPA (diethylenetriaminepentaacetate), EDTA (ethylenediaminetetraacetic acid), and buffer control to obtain a chelator concentration of 10 -3 M~10 -10 A total volume of 100 μL of the mixture was obtained, with the product being between 1 M and 1 M. The reaction mixture was incubated at ambient temperature for 2 h, unless otherwise noted, and analyzed by TLC (0.4 M sodium citrate pH 4 + 10% MeOH, product R f A sample for [0.05%] was taken at the end of the reaction. 225 An exemplary chromatogram of [Ac]Ac-macropa is shown in Figure 3, which shows that actinium was retained at the baseline (i.e., "origin") by being complexed to macropa. The radiochemical yield is presented in Figure 4.

[0159]

[0190] [ 225The radiochemical yield of Ac]Ac-macropa is significantly higher than that of the other chelators investigated at ambient temperature. -6 Other chelators, such as DOTA, DTPA, and EDTA, can be quantitatively radiolabeled at lower chelator concentrations. 225 [Ac]Ac complexation is not observed, similar to the control (no chelator). Radiolabeling of DOTA shows quantitative radiolabeling yields when heated to 90 °C, but slightly inferior compared to H2macropa at lower chelator concentrations. These findings are in line with published observations: NAThiele, V. Brown, JM Kelly, A. Amor-Coarasa, U. Jermilova, SN MacMillan, A. Nikolopoulou, S. Ponnala, CFR Amogida, AKH Rodertson, C. Rodriguez-Rodriguez, P. Schaffer, C. Williams, JW Babich, V. Radchenko, JJ Wilson, Angew. Chem. Int. Ed. 2017, 56, 14712.

[0160] Example 16 Comparative study of the complexation kinetics of compound 111 with Ac-225.

[0191] Ac-225 stock solution (approximately 74 kBq, 2.3 μL) was added to the solution of H2macropa-tzPEG3SqOEt or DOTA-methyltetrazine to obtain a chelator concentration of 10 -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 was obtained with a concentration of 100 μL of H2macropa-tzPEG3SqOEt and DOTA-methyltetrazine. The reaction mixtures of H2macropa-tzPEG3SqOEt and DOTA-methyltetrazine were incubated at ambient temperature and 90 °C, respectively, and analyzed by TLC (0.4 M sodium citrate pH 4 + 10% MeOH, product R fSamples for NMR (NMR = 0) were taken at 5, 15, 30, and 60 min. The radiochemical yields are presented in Figures 5 and 6.

[0161]

[0192] Radiolabeling of H2macropa-tzPEG3SqOEt proceeded in quantitative yields after 15 min of incubation at all concentrations. -7 At chelator concentrations of 10 M, the radiochemical yield began to decline after 5 min of incubation. -7 and 10 -6 A chelator concentration of M as well as a 15 min incubation at ambient temperature was chosen for radiolabeling studies with H2macropa-tzPEG3Sq-conjugated monoclonal antibodies.

[0162]

[0193] Radiolabeling of DOTA-methyltetrazine was performed at 10 -4 At a chelator concentration of 10 M, the reaction proceeded in quantitative yield for all incubation times. -5 At a chelator concentration of 1 M, the radiochemical yield was 88.5% after 15 min of incubation; -6 At M, the maximum radiochemical yield was 65.3% after 60 min of incubation. -5 and 10 -4 A chelator concentration of M was chosen for radiolabeling studies with TCOPEG4-conjugated monoclonal antibodies (where TCOPEG- = an abbreviation for "transcyclooctene polyethylene glycol").

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

[0194] Ac-225 stock solution (555 kBq, 30 μL) was added to the solution of H2macropa-tzPEG3Sq-conjugated monoclonal antibody to obtain a conjugate concentration of 10 -7M and 10 -6 A total volume of 100 μL of the mixture was obtained, with the product being 100 μL. The reaction mixture was incubated at ambient temperature for 15 min and the radiochemical purity (RP; iTLC: 50 mM citric acid pH 5, product R f Samples were taken for determination of antibody titer (=0) and immunoreactivity (IR). Specific activity (SA) was calculated by dividing the starting radioactivity of Ac-225 by the amount of conjugate and multiplying by the radiochemical purity. The results are summarized in Table 1. The numerical designation (2x, 5x) refers to the average chelator-antibody ratio for each conjugate. 225 Ac]Ac-macropa-tzPEG3Sq-EGFRVIII IgG1 and [ 225 Chromatograms of [Ac]Ac-macropa-tzPEG3Sq-EphA3 IgG1 are shown in Figures 7A and 7B, respectively, which show that actinium was retained at baseline (i.e., "origin") by being complexed to macropa-tzPEG3Sq-EGFRVIII IgG1 or macropa-tzPEG3Sq-EphA3 IgG1.

[0164]

[0195] Radiolabeling of H2macropa-tzPEG3Sq-conjugated monoclonal antibodies with Ac-225 was performed for 10 min. -6 Radiochemical yields of >99.5% were achieved at an antibody concentration of 1000 M. At this antibody concentration, the specific radioactivity was close to the theoretical maximum of 37 MBq / mg. Radioimmunoconjugates with an average of two chelators per antibody resulted in higher immunoreactivity compared to radioimmunoconjugates with an average of five chelators per antibody. Thus, the former were selected for further studies.

[0165]

[0196]

[0166] [Table 2]

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

[0197] H2macropa-tzPEG3Sq-conjugated 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 negligible levels of aggregation.

[0199]

[0169] [Table 3]

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

[0200] [ 225 The Ac]Ac-macropa-tzPEG3Sq-conjugated monoclonal antibody (37 kBq, approximately 1 μg) was analyzed using SE-HPLC. HPLC fractions were collected consecutively every 30 seconds (0.5 mL) into Eppendorf tubes. Radioactivity in the tubes was determined using an automatic gamma counter at permanent equilibrium (minimum 8 hours after collection). UV280nm and radiation retention time are summarized in Table 3. SE-HPLC chromatograms (UV 280nm and RAD) are shown in Figure 9.

[0171]

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

[0172]

[0202]

[0173] [Table 4]

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

[0203] [ 225 Ac]Ac-macropa-tzPEG3Sq-EGFRVIII IgG1 (37 kBq, 1 μg, 5 μL) was treated with various gentisate salts (0 / 25 mg / mL), La 3+ (5 / 50 / 500x molar excess relative to antibody), and EDTA (50x molar excess relative to antibody) in PBS to give a solution with a total volume of 20 μL. The mixture was incubated at ambient temperature and analyzed by iTLC (50 mM citric acid pH 5, product R f = 0) Samples for analysis were taken at 1, 24, 48, and 168 hours. The radiochemical purity is presented in Table 4.

[0175]

[0204] [ 225 Ac]Ac-macropa-tzPEG3Sq-EGFRVIII IgG1 was incubated with up to a 50-fold molar excess of La in PBS with and without sodium gentisate. 3+ It showed excellent stability in competition with La. 3+ and at a 50-fold molar excess of EDTA, the radiochemical purity dropped to less than 95% between 24 and 48 hours.

[0176]

[0205]

[0177] [Table 5]

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

[0206] TCOPEG4-conjugated monoclonal antibodies were prepared similarly to published procedures by O. Keinanen, K. Fung, J. Pourat, V. Jallinoja, D. Vivier, NK Pillarsetty, AJ Airaksinen, JS Lewis, BM Zeglis, M. Sarparanta, EJNMMI Res 2017, 7, 95 and Z. Zhou, N. Devoogdt, MR Zalutsky, G. Vaidyanathan, Bioconjug Chem. 2018, 29, 4090-4103. Briefly, monoclonal antibodies in sodium bicarbonate (0.1 M, pH 8.5) were incubated with TCO-PEG4-NHS (4× / 10× / 40× molar equivalents) for 1 h at 37°C, followed by purification by centrifugal filtration (MWCO of 50 kDa) 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 diluted in sodium acetate (0.15 M, pH 5.5) [ 225 Ac]Ac-DOTA-methyltetrazine (555 kBq) for 15 min at ambient temperature, followed by purification by spin filtration (10 kDa MWCO) 225 Ac]Ac-DOTA-dhPzPEG4-EGFRVIII IgG1 and [ 225 Ac]Ac-DOTA-dhPzPEG4-EphA3 IgG1 (wherein -dhPzPEG- is the abbreviation for "methyldihydropyridazinepolyethylene glycol") was synthesized. 225 Ac]Ac-macropa-tzPEG3Sq- and [ 225 Ac]Ac-DOTA-dhPzPEG4-conjugated monoclonal antibodies were incubated in human serum (100 μL) from healthy donors at 37°C. Samples were taken at EOS and after 2, 7, and 14 days of incubation. Samples were analyzed by iTLC (50 mM EDTA pH 5, product R f=0) and the immune response rates were determined using U87MG.de2-7 and U251 cells for EGFRVIII IgG1 and EphA3 IgG1 radioimmunoconjugates, respectively. The results are presented in Figure 10A for radiochemical purity and in Figure 10B for immune reactivity. The numerical designations (2x, 5x, 1x, 2x, 9x) refer to the average 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 (day 14). 225 The Ac]Ac-DOTA-dhPzPEG4-conjugated monoclonal antibodies collectively exhibited poorer stability over the time frame examined. 225 No distinction could be made on the basis of immunoreactivity between the other radioimmunoconjugates, except that the Ac]Ac-DOTA-dhPzPEG4-conjugate showed inferior properties. 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 average chelator-antibody ratio is 2[ 225 Ac]Ac-macropa-tzPEG3Sq-conjugated monoclonal antibodies were incubated in human serum (100 μL) from healthy donors at 37°C. Samples were taken at EOS and after 2, 7, and 14 days of incubation. Samples were analyzed by iTLC (50 mM EDTA pH 5, product R f= 0) and immune response rates were determined using U87MG.de2-7 (EGFRVIII IgG1), U251 (EphA3 IgG1), or SK-RC-52 (chimeric isotype control IgG1) cells. The results are presented in Table 5.

[0181]

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

[0182]

[0210]

[0183] [Table 6]

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

[0211] Female 4- to 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 generate subcutaneous tumors in the left flank. At 11 or 14 days postinoculation, tumors of approximately 100 mm 3 Mice (n=5) bearing tumor xenografts of 225 [Ac]Ac-macropa-tzPEG3Sq-EphA3 IgG1 (0.25–1.0 μg / 9.25–37.0 kBq) or vehicle PBS was injected intravenously. 3 The tumor volume (TV) in units was measured at least twice a week (biweekly) and calculated according to the following formula: TV = (L × W 2) / 2. Data are expressed as mean tumor volume ± SD. Statistical analysis was performed using unpaired t-tests at given time points. Mice were cultured to reach the ethical endpoint (TV>1000mm). 3 Mice were humanely euthanized at baseline or weight loss >10%). Tumor growth curve results and survival data are presented in Figure 11A and Figure 11B, respectively. This research project was approved by Austin Health's Animal Ethics Committee.

[0185]

[0212] [ 225 Following a single dose of Ac]Ac-macropa-tzPEG3Sq-EphA3 IgG1, a striking dose-response effect was observed for tumor growth inhibition within days of treatment. All treatment groups were significantly different from controls 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. Radiotoxic effects were demonstrated by weight loss. No radiotoxicity was observed at doses up to 18.5 kBq. At the highest dose, 37 kBq, radiotoxicity in the form of acute weight loss was observed in 60% of the cohort within days of treatment. At 27.8 kBq, radiotoxicity was evident in 20% of the cohort by 14 days post-treatment. At 18.5 kBq / 0.5 μg [ 225 The dose of Ac]Ac-macropa-tzPEG3Sq-EphA3 IgG1 was determined as the maximum tolerated dose.

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

[0213] Female 4- to 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 generate subcutaneous tumors in the left flank. At 7 or 11 days postinoculation, tumors of approximately 100 mm 3Mice (n=5 / group) bearing tumor xenografts of 225 Ac]Ac-macropa-tzPEG3Sq-EGFRVIII IgG1 (0.25–1.0 μg / 9.25–37.0 kBq) or vehicle PBS was injected intravenously. 3 The tumor volume (TV) in units was measured twice weekly and calculated according to the following formula: TV = (L × W 2 ) / 2. Data are expressed as mean tumor volume ± SD. Mice were randomly assigned to the ethical endpoint (TV>1000mm). 3 Mice were humanely euthanized at baseline or weight loss >10%). Tumor growth curve results and survival data are presented in Figure 12A and Figure 12B, respectively. This research project was approved by Austin Health's Animal Ethics Committee.

[0187]

[0214] In the rapidly growing U87MG.de2-7 glioblastoma xenografts, no dose response of antitumor efficacy was observed, with only the highest dose, 37 kBq, demonstrating long-term delay in tumor growth over the duration of the study in 40% of the cohort, and radiotoxicity was observed again in 60% of the cohort within 2 weeks of single-dose treatment. 225 The dose of Ac]Ac-macropa-tzPEG3Sq-EGFRVIII IgG1 was determined as the maximum tolerated dose.

[0188]

[0215] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" are understood to imply the inclusion of a stated element or integer or group of elements or integers, but not the exclusion of any other element or integer or group of elements or integers.

[0189]

[0216] It should be noted that when a range of values ​​is expressed, the range is expressly understood to include the upper and lower limits of the range, and every numerical value or subrange between those limits, as if each numerical value and subrange were explicitly recited. The statement "about X% to Y%" has the same meaning as "about X% to about Y%," unless otherwise indicated.

[0190]

[0217] The term "about" as used herein means approximately or near, and in the context of numerical values ​​or ranges set forth herein, is intended to encompass variations of no more than + / - 10%, no more than + / - 5%, no more than + / - 1%, or no more than + / - 0.1% from the recited or claimed numerical value or range.

[0191]

[0218] It should also be noted that as used herein, the singular forms "a," "an," and "the" include plural aspects unless the context already dictates otherwise.

[0192]

[0219] The headings used herein are included merely for ease of reference to 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 claim limitations.

[0193]

[0220] The description provided herein relates to multiple embodiments that may share common properties and characteristics. It should be understood that one or more features of one embodiment may be combinable 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 performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to more fully clarify exemplary embodiments and do not impose limitations on the scope of the claimed invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential.

[0195]

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

[0196]

[0223] Those skilled in the art will recognize that the invention described herein can be modified and altered in other ways than those specifically described.The present invention should be understood to include all such modifications and alterations.The present invention also includes all of the steps, features, compositions and compounds mentioned or shown herein, individually or collectively, and any combination of two or more of any 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 to this application, by claiming divisional status, and / or by claiming continuation status. It should be understood that the provisional claims below are provided merely as examples and are not intended to limit the scope of what may be claimed in any such future application. Moreover, the claims should not be considered to limit (or exclude) the understanding of the invention inherent in this disclosure. Features may be added to or excluded from the provisional claims at a later date so as to further define the invention.

Claims

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

2. A is CO ２ R １ and B is CO ２ R ２ 2. The compound of claim 1, wherein:

3. R １ is H; R ２ is H; each R ａ is H, and each R ｂ The compound of claim 1 , wherein is H.

4. R ３ But C １ ~C １２ The compound of claim 1 , wherein the aryl group is alkyl.

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

6. Compounds of formula (Ia): 【Chemistry 2】 (In the formula, A is CO ２ R １ , and P.O. ３ R １ is selected from the group consisting of B is CO ２ R ２ , and P.O. ３ R ２ is selected from the group consisting of R １ , R ２ and R ３ are each independently H and C １ ~C １２ is selected from the group consisting of alkyl, Each R ａ are independently H, F, Cl, Br, I, CH ３ , C.H. ２ CH ３ , CH(CH ３ ) ２ , OH, OCH ３ , OCH ２ CH ３ , C.F. ３ , OCF ３ , NO ２ , N.H. ２ and CN; Each R ｂ are independently H, F, Cl, Br, I, CH ３ , C.H. ２ CH ３ , CH(CH ３ ) ２ , OH, OCH ３ , OCH ２ CH ３ , C.F. ３ , OCF ３ , NO ２ , N.H. ２ and CN; L is a linker having 1 to 20 atoms in the normal chain; M is a radionuclide), or a pharmaceutically acceptable salt thereof.

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

8. R １ is H; R ２ is H; each R ａ is H; and each R ｂ The compound of claim 6 , wherein is H.

9. R ３ But C １ ~C １２ The compound of claim 6, wherein the aryl group is alkyl.

10. L is of the formula: -(CH ２ CH ２ O) ｎ -CH ２ CH ２ - 7. The compound of claim 6, wherein n is an integer from the group consisting of 0, 1, 2, 3, 4, and 5.

11. 7. The compound of claim 6, 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.

12. Compound of formula (Ib): 【Chemistry 3】 (In the formula, A is CO ２ R １ , and P.O. ３ R １ is selected from the group consisting of B is CO ２ R ２ , and P.O. ３ R ２ is selected from the group consisting of R １ , and R ２ are each independently H and C １ ~C １２ is selected from the group consisting of alkyl, R ４ is a monoclonal antibody, Each R ａ are independently H, F, Cl, Br, I, CH ３ , C.H. ２ CH ３ , CH(CH ３ ) ２ , OH, OCH ３ , OCH ２ CH ３ , C.F. ３ , OCF ３ , NO ２ , N.H. ２ and CN; Each R ｂ are independently H, F, Cl, Br, I, CH ３ , C.H. ２ CH ３ , CH(CH ３ ) ２ , OH, OCH ３ , OCH ２ CH ３ , C.F. ３ , OCF ３ , NO ２ , N.H. ２ and CN; L is a linker having 1 to 20 atoms in the normal chain; M is a radionuclide), or a pharmaceutically acceptable salt thereof.

13. A is CO ２ R １ and B is CO ２ R ２ 13. The compound of claim 12, wherein:

14. R １ is H; R ２ is H; each R ａ is H, and each R ｂ The compound of claim 12, wherein is H.

15. L is of the formula: -(CH ２ CH ２ O) ｎ -CH ２ CH ２ - 13. The compound of claim 12, wherein n is an integer from the group consisting of 0, 1, 2, 3, 4, and 5.

16. 13. The compound of claim 12, 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.

17. 13. The compound of claim 12, wherein the monoclonal antibody is selected from the group consisting of penprimab, sintilimab, toripalimab, omburtamab, tisotumab, retifanlimab, ublituximab, anifrolumab, loncastuximab, balstilimab, dostallimab, oportuzumab, marjetuximab, naxitamab, belantamab, tafasitamab, sacituzumab, isatuximab, trastuzumab (Herceptin), girentuximab, ifavotuzumab, depatuximab, enfortumab, polatuzumab, emapalumab, cemiplimab, moxetumomab, A compound selected from the group consisting of mogamulizumab, durvalumab, avelumab, atezolizumab, olaratumumab, daratumumab, elotuzumab, necitumumab, dinutuximab, nivolumab, blinatumomab, blinatumomab, ramucirumab, obinutuzumab, ado-trastuzumab, pertuzumab, brentuximab, ipilimumab, ofatumumab, catumaxomab, panitumumab, bevacizumab, cetuximab, ibritumomab, alemtuzumab, gemtuzumab, rituximab, edrecolomab, nimotuzumab, prorugolimab, and cetuximab.

18. 13. A pharmaceutical composition comprising a compound of claim 12 and a pharmaceutically acceptable carrier.

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

20. Compounds of formula (I): (In the formula, 【Chemistry 4】 A is CO ２ R １ , and P.O. ３ R １ is selected from the group consisting of B is CO ２ R ２ , and P.O. ３ R ２ is selected from the group consisting of R １ , R ２ and R ３ are each independently H and C １ ~C １２ is selected from the group consisting of alkyl, Each R ａ are independently H, F, Cl, Br, I, CH ３ , C.H. ２ CH ３ , CH(CH ３ ) ２ , OH, OCH ３ , OCH ２ CH ３ , C.F. ３ , OCF ３ , NO ２ , N.H. ２ and CN; Each R ｂ are independently H, F, Cl, Br, I, CH ３ , C.H. ２ CH ３ , CH(CH ３ ) ２ , OH, OCH ３ , OCH ２ CH ３ , C.F. ３ , OCF ３ , NO ２ , N.H. ２ and CN; L is a linker having 1 to 20 atoms in the normal chain; or a pharmaceutically acceptable salt thereof, comprising: (a) A compound of formula (10): 【Chemistry 5】 (A is CO ２ R １ , and P.O. ３ R １ is selected from the group consisting of B is CO ２ R ２ , and P.O. ３ R ２ is selected from the group consisting of R １ and R ２ are each independently H and C １ ~C １２ is selected from the group consisting of alkyl, Each R ａ are independently H, F, Cl, Br, I, CH ３ , C.H. ２ CH ３ , CH(CH ３ ) ２ , OH, OCH ３ , OCH ２ CH ３ , C.F. ３ , OCF ３ , NO ２ , N.H. ２ and CN; Each R ｂ are independently H, F, Cl, Br, I, CH ３ , C.H. ２ CH ３ , CH(CH ３ ) ２ , OH, OCH ３ , OCH ２ CH ３ , C.F. ３ , OCF ３ , NO ２ , N.H. ２ and CN; (b) reacting a compound of formula (10) with a compound of formula (11): 【Chemistry 6】 (In the formula, R ３ is H and C １ ~C １２ is selected from the group consisting of alkyl, L is a linker having 1 to 20 atoms in the normal chain; reacting in the presence of a copper (I) catalyst A method comprising:

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