Therapeutic conjugate

Abstract

Provided herein is a dendrimer-targeting agent conjugate, or a salt thereof, further comprising: (a) a dendrimer comprising: i) a core unit (C); and ii) a building block unit (BU), wherein the dendrimer has generations 2 to 6 of the building blocks, the core unit being covalently attached to at least two of the building blocks; and b) a targeting agent covalently linked to the dendrimer by a spacer group; c) one or more first terminal groups attached to the outermost building block unit of the dendrimer, the first terminal group comprising a complexing group for complexing a radionuclide; and d) one or more second terminal groups attached to the outermost building block unit of the dendrimer, the second terminal group comprising a pharmacokinetic-modifying moiety. Also provided are compositions comprising the dendrimer-targeting agent conjugates and methods of using the dendrimer-targeting agent conjugates and compositions comprising them in therapeutic and imaging applications.

Description

[Technical Field]

[0001] This disclosure relates to targeted agent-dendrimer conjugates for therapeutic and imaging applications. The conjugates are used in therapeutic applications, such as the treatment of tumors. This disclosure also relates to pharmaceutical compositions comprising the conjugates and therapeutic methods using the conjugates. [Background technology]

[0002] According to the WHO, cancer is the second leading cause of death worldwide, accounting for an estimated 9.6 million deaths in 2018. The most common types of cancer include those affecting the lungs, breasts, colorectal cancer, prostate, skin, and stomach.

[0003] Significant efforts are being made in the research and development of novel and effective oncology therapies. However, so far, modern cancer therapies have proven only partially successful in treating and extending the lifespan of patients with many common types of cancer. This limited success is largely attributable to the lack of relative specificity found in many primary class anticancer drugs and cytotoxic technologies. In fact, most oncology therapies available today operate on the premise that they simply destroy any cells that exhibit uncontrolled growth. Focusing on such nonspecific cell division results in non-selective treatment, which can inadvertently damage rapidly dividing non-tumor cells (e.g., cells present in the gut). As a result, the administration of oncology therapies often causes irreversible damage to the patient's healthy cells, leading to a multitude of side effects that are detrimental to the patient's quality of life.

[0004] One such nonspecific tumor therapy is radionuclide therapy. Radionuclide therapy is a systemic treatment that uses molecules labeled with radionuclides to deliver high levels of radiation to tumor cells to treat certain cancers. This therapy uses ionizing radiation to kill cancer cells and shrink tumors by damaging their DNA, thereby preventing the continued growth and division of these cells. Existing methods for delivering radiotherapy to desired sites include mimetic therapies such as Xifigo (Ra223, Bayer) and radioactive beads (SIR-Spheres (Y-90 Sirtex), etc.), and targeted therapies (Lutathera (AAA / Novartis), etc.).

[0005] While radionuclides may be effective in reducing the growth and spread of cancer cells, they can also inadvertently damage the patient's healthy tissues. Significant challenges remain in providing safe, long-term radionuclide therapy that achieves and maintains therapeutically appropriate levels at the target site (e.g., tumor cells) for the radionuclides to be effective. This challenge of safe, long-lasting radionuclide therapy is compounded by the properties of the radionuclides themselves, which inadvertently damage healthy cells (e.g., blood cells and other cells of the immune system) that are exposed to them for extended periods. Such undesirable exposure to healthy cells often manifests as unbearable side effects in cancer patients, further limiting the effectiveness of the therapy. Essentially, for at least some radionuclide therapies, the pharmacokinetic / pharmacodynamic properties and / or side effect profiles of the radionuclide therapy are suboptimal.

[0006] Therefore, there remains a need to develop safe and effective radionuclide therapies that, based on pharmacokinetic / pharmacodynamic properties, deliver radionuclides potently, selectively, for extended periods, and with overall controlled delivery, while minimizing patient side effects throughout. [Overview of the project]

[0007] In the first embodiment, a dendrimer-targeting agent conjugate, a) A dendrimer, i) Core unit (C), and ii) comprising a constituent unit (BU), where each constituent unit is a lysine residue or an analog thereof, A dendrimer having 2 to 6 generations of constituent units, with a core unit covalently attached to at least two constituent units, b) A targeting agent covalently linked to the dendrimer by a spacer group, c) One or more first terminal groups attached to the outermost constituent unit of the dendrimer, wherein the first terminal group includes a complexing group for complexing a radionuclide, d) One or more second terminal groups attached to the outermost constituent unit of the dendrimer, wherein the second terminal group includes a pharmacokinetic modification moiety, and the second terminal group includes a conjugate Or the salt is provided.

[0008] The dendrimer-targeting agent conjugate of the first embodiment may have a radionuclide that complexes with a complexing group to form a dendrimer-targeting agent therapeutic conjugate.

[0009] In the first embodiment, a dendrimer-targeting agent conjugate, a) A dendrimer, i) Core unit (C), and ii) comprising a constituent unit (BU), where each constituent unit is a lysine residue or an analog thereof, A dendrimer having 2 to 6 generations of constituent units, with a core unit covalently attached to at least two constituent units, b) A targeting agent covalently linked to the dendrimer by a spacer group, c) One or more first terminal groups attached to the outermost constituent unit of the dendrimer, wherein the first terminal group includes a complexing group that has complexed with a radionuclide, d) One or more second terminal groups attached to the outermost constituent unit of the dendrimer, wherein the second terminal group includes a pharmacokinetic modification moiety, and the second terminal group includes a conjugate Or the salt is provided.

[0010] In some embodiments, the targeting agent is a peptide moiety containing an antigen-binding site, having a molecular weight of up to approximately 150 kDa, or up to approximately 110 kDa, or up to approximately 80 kDa, or up to approximately 55 kDa, or up to approximately 20 kDa, or up to approximately 16 kDa.

[0011] In some embodiments, the targeting agent is a peptide moiety having a molecular weight of up to approximately 80 kDa and containing an antigen-binding site.

[0012] In some embodiments, the targeting agent is selected from antibodies, heavy chain antibodies, ScFV-Fc, Fab, Fab2, Fv, scFv, or single-domain antibodies. In some embodiments, the targeting agent is a heavy chain variable (V H ) contains or consists of a domain. In some embodiments, the targeting agent is light chain variable (V L ) Includes or consists of a domain.

[0013] In some embodiments, the targeting agent has a molecular weight of about 5 kDa to about 30 kDa. In some embodiments, the targeting agent has a molecular weight of about 5 kDa to about 20 kDa.

[0014] In some embodiments, the targeting agent contains fewer than 120 amino acid residues.

[0015] In some embodiments, the targeting agent is a HER2 targeting agent or an EGFR targeting agent.

[0016] In some embodiments, the targeting agent comprises or consists of any of the targeting agent amino acid sequences defined herein.

[0017] In some embodiments, the targeting agent is a small molecule.

[0018] In some embodiments, the targeting agent is a small molecule that binds to PSMA.

[0019] In some embodiments, the targeting agent is a DUPA analog.

[0020] In some embodiments, the targeting agent is one that binds to FAP.

[0021] In some embodiments, the covalent linkage between the targeting agent and the spacer group is formed by a reaction between complementary reactive functional groups present on the intermediate containing the targeting agent and the intermediate containing the dendrimer.

[0022] In some embodiments, the intermediate containing the targeting agent includes a non-natural amino acid residue, the non-natural amino acid residue having a side chain containing a reactive functional group. In some embodiments, the non-natural amino acid residue is a 4-azidophenylalanine residue.

[0023] In some embodiments, the spacer group includes a PEG group.

[0024] In some embodiments, the targeting agent is covalently linked to a spacer group at or near the C-terminus of the peptide moiety.

[0025] In some embodiments, the dendrimer-containing intermediate contains a reactive functional group which is an alkyne group. In some embodiments, the alkyne group is a dibenzocyclooctin-amine group.

[0026] In some embodiments, as described above, the first terminal group includes a complexing group that is complexed with a radionuclide. This can be considered to form a radionuclide complexed moiety. In some embodiments, the complexing group is DOTA, benzyl-DOTA, NOTA, DTPA, macropa, sarcofazine, DFO, EDTA, or PEPA group.

[0027] In some embodiments, the radionuclide in the radionuclide complex is lutetium, gadolinium, gallium, zirconium, actinium, bismuth, astatine, technetium, lead, yttrium, or copper radionuclides. In some embodiments, the radionuclide is gadolinium, gallium, zirconium, lead, or lutetium radionuclide. In some embodiments, the radionuclide is an α-emitting material. In some embodiments, the radionuclide is a β-emitting material.

[0028] In some embodiments, the pharmacokinetic modification moiety is a polyethylene glycol (PEG) group, a polyethyl oxazoline (PEOX) group, a poly-(2)methyl-(2)-oxazoleamine (POZ), a poly(2-hydroxypropyl) methacrylamide (pHPMA) group, or polysarcosine. In some embodiments, the pharmacokinetic modification moiety is a polyethylene glycol (PEG) group. In some embodiments, the pharmacokinetic modification moiety is a PEG group having an average molecular weight in the range of 400 to 2400 daltons, 400 to 2200 daltons, or 400 to 1400 daltons.

[0029] In some embodiments, the dendrimer has four generations of constituent units. In some embodiments, the generations of constituent units are complete generations.

[0030] In some embodiments, the core unit is as follows: [ka]

[0031] In some embodiments, the core unit includes the following structure: [ka]

[0032] In some embodiments, the core unit is as follows: [ka]

[0033] In some embodiments, the constituent units are lysine residues or analogs thereof. In some embodiments, the constituent units are as follows: [ka]

[0034] In some embodiments, 1 to 3 nitrogen atoms present in the surface constituent unit are attached to the first end group. In some embodiments, at least one-third of the nitrogen atoms present in the surface constituent unit are attached to the second end group. In some embodiments, at least one-third of the nitrogen atoms present in the surface constituent unit are attached to the third end group.

[0035] In some embodiments, the dendrimer includes a surface constituent unit containing nitrogen atoms capped with acetyl groups.

[0036] In some embodiments, the dendrimer is one of the exemplary conjugates.

[0037] In a further embodiment, a composition comprising a plurality of conjugates as defined herein is provided.

[0038] In a further embodiment, a pharmaceutical composition, i) Conjugates described herein, ii) A pharmaceutical composition is provided comprising a pharmaceutically acceptable excipient.

[0039] In some embodiments, the conjugate or pharmaceutical composition is for use in therapy. In some embodiments, the conjugate or composition is for use in the treatment of cancer. In such therapeutic embodiments, the dendrimer-targeting agent conjugate may be complexed with a radionuclide to become a dendrimer-targeting agent therapeutic conjugate that is distributed to a target requiring such treatment. References to “conjugate” or “dendrimer conjugate” herein may include both dendrimer-targeting agent conjugates and dendrimer-targeting agent therapeutic conjugates.

[0040] In a further embodiment, the use of the conjugate or pharmaceutical composition described herein in the manufacture of a pharmaceutical product for the treatment of cancer is provided.

[0041] In further embodiments, a method is provided for treating a target cancer, comprising administering a therapeutically effective amount of a conjugate or pharmaceutical composition as defined herein to the target. In some embodiments, the cancer is prostate cancer, pancreatic cancer, breast cancer, or brain tumor In some embodiments, the cancer is a glioblastoma, meningioma, pituitary gland, nerve sheath, astrocytoma, oligodendroglioma, ependymoma, medulloblastoma, or craniopharyngioma. brain tumor That is the case.

[0042] In a further embodiment, a method, use, or conjugate or composition for use is provided herein, in which the conjugate is administered in combination with a further activator.

[0043] In a further embodiment, a kit for generating a therapeutic conjugate as defined herein, a) A conjugate of the first embodiment as defined herein, b) A kit containing radionuclides is provided.

[0044] In a further embodiment, a process is provided for generating a therapeutic conjugate as described herein, comprising contacting a conjugate of the first embodiment as defined herein with a radionuclide to generate a therapeutic conjugate. [Brief explanation of the drawing]

[0045] [Figure 1a] The image shows dendrimer-nanobody conjugation reactions (Layn reaction mixtures) that produce mixtures of product dendrimers linked to one, two, three, four, or more nanobodies, as visualized by SDSPAGE and fluorescence imaging. The SDSPAGE size marker (blue) represents the approximate kilodalton mass. [Figure 1b] The image shows dendrimer-nanobody conjugation reactions (Layn reaction mixtures) that produce mixtures of product dendrimers linked to one, two, three, four, or more nanobodies, as visualized by SDSPAGE and fluorescence imaging. The SDSPAGE size marker (blue) represents the approximate kilodalton mass. [Figure 1c] The image shows dendrimer-nanobody conjugation reactions (Layn reaction mixtures) that produce mixtures of product dendrimers linked to one, two, three, four, or more nanobodies, as visualized by SDSPAGE and fluorescence imaging. The SDSPAGE size marker (blue) represents the approximate kilodalton mass. [Figure 1d] The image shows dendrimer-nanobody conjugation reactions (lane M) that produce mixtures of product dendrimers linked to one, two, three, four, or more nanobodies, as visualized by SDSPAGE and fluorescence imaging. The SDSPAGE size marker (blue) represents the approximate kilodalton mass. [Figure 1e]The image shows dendrimer-nanobody conjugation reactions (lane M) that produce mixtures of product dendrimers linked to one, two, three, four, or more nanobodies, as visualized by SDSPAGE and fluorescence imaging. The SDSPAGE size marker (blue) represents the approximate kilodalton mass. [Figure 1f] The image shows dendrimer-nanobody conjugation reactions (lane M) that produce mixtures of product dendrimers linked to one, two, three, four, or more nanobodies, as visualized by SDSPAGE and fluorescence imaging. The SDSPAGE size marker (blue) represents the approximate kilodalton mass. [Figure 1g] The image shows dendrimer-nanobody conjugation reactions (lane M) that produce mixtures of product dendrimers linked to one, two, three, four, or more nanobodies, as visualized by SDSPAGE and fluorescence imaging. The SDSPAGE size marker (blue) represents the approximate kilodalton mass. [Figure 1h] The SDS-PAGE of the fraction obtained from size exclusion is shown. The SDS-PAGE marker (blue) represents the approximate kilodalton mass, and the red represents the Cy5 fluorescence emitted from the dendrimer. The predicted molecular weight of the nanobody-dendrimer is approximately 25 kDa. [Figure 1i] The SDS-PAGE of the fraction obtained from size exclusion is shown. The SDS-PAGE marker (blue) represents the approximate kilodalton mass, and the red represents the Cy5 fluorescence emitted from the dendrimer. The predicted molecular weight of the nanobody-dendrimer is approximately 25 kDa. [Figure 2] The mean fluorescence intensity values ​​over 24 hours for compound 71 (control) and compound 123 (target) in MDA-MB-231, MDA-MB-231 / HER2, and SKOV-3 cells are shown. At least 10,000 cells were counted for each measurement. Values ​​are mean ± standard deviation (SD; n=3). [Figure 3]Flow cytometry analysis of dendrimers incubated with HER2-positive cells (MDA-MB-231 / HER2) and HER2-negative cells (MDA-MB-231) at 3.33 nM and 37°C for 24 hours is shown. At least 10,000 cells were counted for each measurement. Values ​​are mean ± standard deviation (SD; n=3). [Figure 4] a) Confocal microscope images of MDA-MB-231 cells treated with compound 71 (control) or b) compound 123 (target) at a concentration of 3.33 nM for 24 hours are shown. Green, blue, and red fluorescence represent the cell membrane stained with AF-488-WGA, the nucleus stained with DAPI, and the dendrimer labeled with Cy5, respectively. Scale bar = 50 μm. [Figure 5] a) Confocal microscopy images of MDA-MB-231 / HER2 cells treated with compound 71 (control) or b) compound 123 (target) at a concentration of 3.33 nM for 24 hours are shown. Green, blue, and red fluorescence represent the cell membrane stained with AF-488-WGA, the nucleus stained with DAPI, and the dendrimer labeled with Cy5, respectively. Scale bar = 50 μm. [Figure 6] a) Confocal microscopy images of SKOV-3 cells treated with compound 71 (control) or b) compound 123 (target) at a concentration of 3.33 nM for 24 hours are shown. Green, blue, and red fluorescence represent the cell membrane stained with AF-488-WGA, the nucleus stained with DAPI, and the dendrimer labeled with Cy5, respectively. Scale bar = 50 μm. [Figure 7] The tumor and blood distribution data of 3H-labeled dendrimers of compound 72 and compound 127 after slaughter 48 hours later are shown. All data represent mean ± SEM (n=5); (NS = not significant; * = p value < 0.05). [Figure 8] The typical ex vivo tumor distribution of compounds 71 ​​and 123 after euthanasia 48 hours later is shown. The data represent typical fields of view for (a) non-targeted dendrimer (compound 71) and (b) targeted dendrimer (compound 123). [Figure 9]The image shows that the target dendrimer (compound 123) was taken up in the core and surrounding regions of the tumor, while the control compound 71 was not taken up. [Figure 10] This plot shows the mean tumor volume over time in mice inoculated with SKOV3 cells and subsequently treated with a vehicle, control compound 71, target compound 123, Kadcyla®, or Herceptin®. [Figure 11] This shows the time course survival rates of mice inoculated with SKOV3 cells and subsequently treated with a vehicle, control compound 71, target compound 123, Kadcyla®, or Herceptin®. [Figure 12] This shows the mean percentage body weight change over time in mice inoculated with SKOV3 cells and subsequently treated with a vehicle, control compound 71, target compound 123, Kadcyla®, or Herceptin®. [Figure 13] This paper demonstrates the internalization rates of fourth-generation dendrimers and single (compound 91, single MFI) or multiple (compound 92, multiple MFI) conjugate anti-HER2 nanobodies. [Figure 14] The images show confocal microscopy images of SKOV-3 cells incubated with compound 92 (multiple 2D3-dendrimer conjugates) at 37°C for a) 1 hour, b) 3 hours, c) 6 hours, or d) 24 hours. Compound 92 was labeled with Cy5 (magenta), the cell membrane was stained with AF488-WGA (green), and the nucleus was labeled with Hoechst33342 (blue). Scale bar = 30 μM. [Figure 15] The images show confocal microscopy images of SKOV-3 cells incubated with compound 91 (a single 2D3-dendrimer conjugate) at 37°C for a) 1 hour, b) 3 hours, c) 6 hours, or d) 24 hours. Compound 91 was labeled with Cy5 (magenta), the cell membrane was stained with AF488-WGA (green), and the nucleus was labeled with Hoechst33342 (blue). Scale bar = 30 μM. [Figure 16] This is a radioactive TLC image of compound 73, showing that 89Zr is bound to the dendrimer. [Figure 17A] The graphs show the percentage zirconium injection dose per gram for compounds 89, 91, and 93 in (a) the kidney, (b) the liver, and (c) tumors over a 9-day period. [Figure 17B] The graphs show the percentage zirconium injection dose per gram for compounds 89, 91, and 93 in (a) the kidney, (b) the liver, and (c) tumors over a 9-day period. [Figure 17C] The graphs show the percentage zirconium injection dose per gram for compounds 89, 91, and 93 in (a) the kidney, (b) the liver, and (c) tumors over a 9-day period. [Figure 18] This shows representative maximum intensity projections of PET images of animals with radioisotope conjugates from 4 hours to 9 days after imaging. Data are expressed in becquerels / voxels (cm³), and thresholds have been set to highlight tumor uptake. [Figure 19] This plot shows the percentage change in tumor volume over time in balb / c nude mice inoculated with BT474 cells and subsequently treated with a vehicle and test articles delivering 15 MBq177 Lu (trastuzumab KY-3-310, HER2 nanobody-targeted SRS-2-304, and non-targeted RH-3-160). [Figure 20] This plots the percentage change in tumor volume over time in balb / c nude mice inoculated with BT474 cells and subsequently treated with a vehicle, trastuzumab KY-3-310, or HER2 nanobody-targeted SRS-2-304, varying doses of 177 Lu. [Figure 21] The SDS page gels of reduced and unreduced KY-2a are shown. [Figure 22] The SDS page gels for dendrimer conjugates SRS-15, SRS-16, and SRS-17 (and their corresponding starting materials) are shown. [Figure 23] The SDS page gels for dendrimer conjugates SRS-20, SRS-21, and SRS-22 (and their corresponding starting materials) are shown. [Figure 24](Gels for compounds 85-97, 123-127, SRS-20, and SRS-22 are shown.)

[0046] Legend for sequence listings The single-domain antibody of Sequence ID No. 1 has a 2D3 amino acid sequence. Sequence ID 2: Example of a single-domain antibody amino acid sequence. Sequence ID 3: Example of a single-domain antibody amino acid sequence. Sequence ID 4: Example of a single-domain antibody amino acid sequence. Sequence ID 5: Example of a single-domain antibody amino acid sequence. Sequence ID 6: Example of a single-domain antibody amino acid sequence. Sequence ID 7: Example of a single-domain antibody amino acid sequence. Sequence ID 8: Example of a single-domain antibody amino acid sequence. Sequence ID 9: Example of a single-domain antibody amino acid sequence. Sequence ID No. 10: Example of a single-domain antibody amino acid sequence.

[0047] explanation General definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art (e.g., chemistry, biochemistry, medicinal chemistry, polymer chemistry, etc.).

[0048] When used herein, the term "and / or," for example "X and / or Y," is understood to mean either "X and Y" or "X or Y," and is deemed to explicitly support both meanings or either meaning.

[0049] When used herein, unless otherwise stated, the term “about” means ±20%, more preferably ±10%, of the specified value.

[0050] As used herein, the terms "a," "an," and "the" include both singular and plural aspects unless the context clearly indicates otherwise.

[0051] Unless otherwise indicated, terms such as “first,” “second,” etc., are used herein solely as labels and are not intended to impose any sequential, positional, or hierarchical requirements on the items they refer to. Furthermore, references to “second” items do not require or exclude the presence of items with lower numbers (e.g., “first” items) and / or items with higher numbers (e.g., “third” items).

[0052] As used herein, the phrase “at least one of ~” when used with a list of items means that various combinations of one or more of the enumerated items may be used, and only one of the items in the list may be required. An item can be a specific object, thing, or category. In other words, “at least one of ~” means that any combination of items or any number of items may be used from the list, but not all items in the list may be required. For example, “at least one of item A, item B, and item C” could mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C. In some cases, “at least one of item A, item B, and item C” could mean, for example, two of item A, one of item B, and ten of item C; four of item B, and seven of item C; or several other suitable combinations.

[0053] As used herein, the term “subject” refers to any organism susceptible to disease or condition. For example, a subject may be an animal, mammal, primate, domestic animal (e.g., sheep, cattle, horse, pig), companion animal (e.g., dog, cat), or laboratory animal (e.g., mouse, rabbit, rat, guinea pig, hamster). In one example, the subject is a mammal. In one embodiment, the subject is a human. In one embodiment, the subject is a non-human animal.

[0054] In this specification, the term “treating” includes alleviating symptoms associated with a particular disorder or condition. For example, as used herein, the term “treating cancer” includes alleviating symptoms associated with cancer. In one embodiment, the term “treating cancer” refers to reducing the size of a cancerous tumor. In one embodiment, the term “treating cancer” refers to extending progression-free survival. In this specification, the term “progression-free survival” refers to the length of time during and after treatment for cancer in which a patient has the disease, i.e., cancer, but does not experience a recurrence of the disease or an increase in the symptoms of the disease.

[0055] In this specification, the term “prevention” includes preventing a particular disorder or condition. For example, in this specification, the term “prevent cancer” means preventing the onset or persistence of symptoms associated with cancer. In one embodiment, the term “prevent cancer” means slowing or stopping the progression of cancer. In one embodiment, the term “prevent cancer” means slowing or preventing metastasis.

[0056] As used herein, “therapeutic effective dose” means an amount of dendrimer administered that is sufficient to alleviate or prevent, to some extent, one or more symptoms of the disorder or condition being treated. The result may be a reduction and / or mitigation of signs, symptoms or causes of the disease or condition, or any other desirable change in the biological system. In one embodiment, the term “therapeutic effective dose” means an amount of dendrimer administered that is sufficient to result in a reduction in the size of a cancerous tumor. In one embodiment, the term “therapeutic effective dose” means an amount of dendrimer administered that is sufficient to result in an extension of progression-free survival. As used herein, “effective dose” means an amount of dendrimer that is effective to achieve the desired pharmacological effect or therapeutic improvement without excessive side effects. “Therapeutic effective dose” includes, for example, a prophylactic effective dose. In one embodiment, a prophylactic effective dose is an amount sufficient to prevent metastasis. It is understood that “effective dose” or “therapeutic effective dose” may vary from subject to subject due to variations in the compound’s metabolism, age, weight, the subject’s overall health, the condition being treated, the severity of the condition being treated, and the prescribing physician’s judgment.

[0057] The term “diagnosis,” as used herein, may include a process of administering the conjugates of the Disclosure to a subject having or suspected having a condition, disease, or disorder, and then using techniques such as single-photon emission, positron emission tomography, and / or positron emission tomography-magnetic resonance imaging to provide information about the levels of radioactivity in various parts of the body, for example, by imaging some or more parts of the subject’s body so that a determination can be made regarding the presence of a disease, disorder, or condition (e.g., cancer), and / or regarding the status, stage, and / or extent of the disease, disorder, or condition. In some embodiments, the term “diagnosis” may include the act of identifying and / or classifying the status, stage, or extent of a disease, disorder, or condition from signs or symptoms. For example, as used herein, the term “diagnosing cancer” may include identifying and / or classifying the status, stage, or extent of cancer in a subject.

[0058] Suitable salts of dendrimers include those formed with organic or inorganic acids or bases. In this specification, the expression "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt. Examples of acid addition salts include, but are not limited to, salts of sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acidic phosphates, isonicotinates, lactates, salicylates, acidic citrates, tartrates, oleates, tannates, pantothenates, vitalates, ascorbicates, succinates, maleates, gentisinates, fumarates, glucons, glucurons, saccharates, formates, benzoates, glutamates, methanesulfons, ethanesulfons, benzenesulfons, p-toluenesulfons, and pamoates (i.e., salts of 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). Examples of base addition salts, but not limited to these, include ammonium salts, alkali metal salts (e.g., potassium and sodium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), salts with organic bases (e.g., dicyclohexylamine, N-methyl-D-glucomine, morpholine, thiomorpholine, piperidine, pyrrolidine), mono, di, and tri lower alkylamines (e.g., ethyl, tert-butyl, diethyl, diisopropyl-, triethyl-, tributyl-, or dimethyl-propylamine), or mono-, di-, and tri-hydroxy lower alkylamines (e.g., mono-, di-, and triethanolamine). A pharmaceutically acceptable salt may contain other molecules, such as acetate ions, succinate ions, or other counterions. The counterion can be any organic or inorganic part that stabilizes the charge of the parent compound. Furthermore, a pharmaceutically acceptable salt may have two or more charged atoms in its structure. If multiple charged atoms are part of the pharmaceutically acceptable salt, it may have multiple counterions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.Furthermore, non-pharmaceutically acceptable salts may be useful as intermediates in the preparation of pharmaceutically acceptable salts, or as useful during storage or transport, and will therefore be understood to be within the scope of this disclosure.

[0059] Those skilled in organic chemistry and medicinal chemistry will understand that many organic compounds can form complexes with solvents that react with, precipitate, or crystallize them. These complexes are known as “solvates.” For example, a complex with water is known as a “hydrate.” In this specification, the terms “pharmaceutically acceptable solvate” or “solvate” refer to the association of one or more solvent molecules with a compound of the disclosure. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0060] As used herein, the term “5- to 10-membered monocyclic or bicyclic heterocyclic group” refers to a monocyclic or bicyclic aromatic or unaromatic cyclic group that is similar to a carbocyclyl group but in which one or more carbon atoms are replaced by one or more heteroatoms independently selected from nitrogen, oxygen, or sulfur. Polycyclic heterocyclyls may, for example, contain fused rings. In bicyclic heterocyclyl groups, one or more heteroatoms may be present in each ring, or a heteroatom may be present in only one of the rings. The heteroatoms may be N, O, or S. A suitable nitrogen-containing heterocyclyl group contains the corresponding N-oxide. In one example, the heterocyclic group has 5 to 10 atoms (i.e., a 5- to 10-membered heterocycle). Examples of monocyclic non-aromatic heterocyclic groups include azilidinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, tetrahydrofuranyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, and azepanyl. Examples of bicyclic heterocyclic groups in which one of the rings is non-aromatic include dihydrobenzofuranyl, indanyl, indlinyl, isoindlinyl, tetrahydroisoquinolinyl, tetrahydroquinolyl, and benzoazepanyl. Examples of monocyclic aromatic heterocyclic groups (also called monocyclic heteroaryl groups) include furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl. Examples of bicyclic aromatic heterocyclic groups (also called bicyclic heteroaryl groups) include quinoxalinyl, quinazolinyl, pyridopyradinyl, benzoxazolyl, benzothiophenyl, benzimidazolyl, naphthilidinyl, quinolinyl, benzofuranyl, indolyl, indazolyl, benzothiazolyl, oxazolyl[4,5-b]pyridyl, pyridopyrimidinyl, isoquinolinyl, and benzohydroxazole.

[0061] As used herein, the term “saturated” refers to a group in which all available valence bonds of a main chain atom are attached to other atoms. Typical examples of saturated groups include, but are not limited to, butyl, cyclohexyl, and piperidine.

[0062] As used herein, the term “unsaturated” refers to a group in which at least one valence bond between two adjacent main chain atoms is not attached to the other atom. Typical examples, but not limited to, include alkenes (e.g., -CH2-CH2CH=CH), phenyl, and pyrrole.

[0063] As used herein, the term “substituted” refers to a group having one or more hydrogens or other atoms that have been removed from a carbon or preferred heteroatom and substituted with a further group (i.e., substituent).

[0064] In this specification, the term "dendrimer" refers to a molecule comprising a core and dendrons attached to that core. Each dendron consists of generations of branched constituent units, with the number of branches increasing with each generation of constituent units. Dendrimers may include pharmaceutically acceptable salts or solvates as defined above.

[0065] As used herein, the term “constituent unit” refers to a branched molecule containing functional groups. One functional group is for attachment to the core or a previous generation constituent unit, and at least two functional groups are for attachment to a next generation constituent unit or for forming the surface of a dendrimer molecule.

[0066] As used herein, the term “attached” refers to a connection between chemical components by covalent bonds. The term “covalent bond” is used interchangeably with the term “covalent attachment.”

[0067] Conjugate In the first embodiment, a dendrimer-targeting agent conjugate, a) i) Core unit (C), and ii) Including constituent units (BUs), A dendrimer having 2 to 6 generations of constituent units, with a core unit covalently attached to at least two constituent units, b) A targeting agent covalently linked to the dendrimer by a spacer group, c) One or more first terminal groups attached to the outermost constituent unit of the dendrimer, wherein the first terminal group includes a complexing group for complexing a radionuclide, d) One or more second terminal groups attached to the outermost constituent unit of the dendrimer, wherein the second terminal group includes a pharmacokinetic modification moiety, and the second terminal group includes a conjugate Or the salt is provided.

[0068] The dendrimer-targeting agent conjugate of the first embodiment may have a radionuclide that complexes with a complexing group to form a dendrimer-targeting agent therapeutic conjugate. The dendrimer-targeting agent therapeutic conjugate may be used for therapeutic or imaging / diagnostic purposes.

[0069] In the first embodiment, a dendrimer-targeting agent conjugate, a) A dendrimer, i) Core unit (C), and ii) comprising a constituent unit (BU), where each constituent unit is a lysine residue or an analog thereof, A dendrimer having 2 to 6 generations of constituent units, with a core unit covalently attached to at least two constituent units, b) A targeting agent covalently linked to the dendrimer by a spacer group, c) One or more first terminal groups attached to the outermost constituent unit of the dendrimer, wherein the first terminal group includes a complexing group that has complexed with a radionuclide, d) One or more second terminal groups attached to the outermost constituent unit of the dendrimer, wherein the second terminal group includes a pharmacokinetic modification moiety, and the second terminal group includes a conjugate Or the salt is provided.

[0070] The conjugates of this disclosure, comprising a dendrimer skeleton incorporating a targeting agent and a first terminal group containing a complexing group for complexing a radionuclide, are used as pharmaceutical agents with pharmaceutically useful applications, for example, as therapeutic agents useful in the treatment of cancer. Certain combinations of dendrimer skeletons having pharmacokinetic modifying groups such as PEG or PEOX groups conjugated to the targeting agent are thought to provide delivery of the radionuclide to the site of action in a manner that can provide a sustained therapeutic effect. The design of the conjugates also enables the synthesis of the targeting agent, the complexing group for complexing the radionuclide, and, in particular, the radionuclide in a manner that allows for the subsequent introduction of the radionuclide.

[0071] Dendrimer Core unit The dendrimer core unit (C) provides attachment points for the dendrons formed by the constituent units. Any suitable core unit containing functional groups that can form covalent bonds with functional groups present on the constituent units may be used.

[0072] In some embodiments, the core unit is covalently attached to at least two constituent units via amide bonds. In some embodiments, each amide bond is formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in the constituent unit. In other embodiments, each amide bond is formed between a carbon atom of an acyl group present in the core unit and a nitrogen atom present in the constituent unit.

[0073] In some embodiments, the core unit is covalently attached to two, three, or four constituent units. In one particular embodiment, the core unit is covalently attached to two constituent units. The core unit may be formed from a core unit precursor containing, for example, an amino group. As another example, the core unit may be formed from a core unit precursor containing a carboxylic acid group. In the case of a core unit attached to two constituent units, the dendrimer core unit may be formed from, for example, a core unit precursor containing two amino groups.

[0074] In some embodiments, the core unit can be derived from a precursor having three reactive nitrogen atoms, two of which may be used to attach the constituent units and one of which may be used to attach the spacer group. In some embodiments, the core unit is [ka] Therefore, by using a suitable protecting group strategy, the terminal nitrogen can be functionalized with a different group from the central nitrogen; for example, the constituent unit can be attached to the terminal nitrogen, and the central nitrogen can be functionalized with a spacer group.

[0075] In some embodiments, the core unit can be derived from a precursor having two reactive nitrogen atoms, which can be used to attach the constituent units. For example, in some embodiments, the core unit may be derived from ethylenediamine, 1,4-diaminobutane, or 1,6-diaminohexane.

[0076] In some embodiments, the core unit is [ka] Thus, the core unit contains a lysine residue (BHA-Lys) in which the acid portion is capped with benzhydrylamine to form the corresponding amide, for example, a core unit precursor having two reactive (amino)nitrogens: [ka] It can be formed from.

[0077] When using core unit precursors that have only two reactive nitrogen atoms, such as BHA-Lys, the two amino groups are typically functionalized in the constituent unit, and the spacer group is typically attached to the surface constituent unit.

[0078] The dendrimer of the present invention allows for the presentation of multiple terminal groups on the surface of the dendrimer in a controlled manner. In particular, when a lysine constituent unit is used, the arrangement of the constituent unit on the alpha or epsilon nitrogen atom can be predetermined as described below. In some preferred embodiments, all residues of the complexing group (radionuclide-containing portion), pharmacokinetic group, targeting agent, and, if present, pharmaceutically active agent are provided to the surface of the dendrimer via attachment through the constituent unit. In other words, in those embodiments, the core unit does not provide attachment sites for terminal groups other than via the constituent unit. In such embodiments, it will be understood that any functional groups present in the core unit that are not used for covalent attachment to the constituent unit are either unreacted (i.e., unreacted under the conditions to which the conjugate is exposed) or capped with a suitable capping group to prevent further reaction. An example of such a core unit is the BHA-Lys group described above.

[0079] Maleimide core unit In some embodiments, the core is [ka] And, Dotted lines adjacent to bonds from lysine nitrogen atoms indicate the attachment of constituent units, and dotted lines adjacent to bonds from maleimide nitrogen may indicate, for example, attachment points of functional parts discussed herein.

[0080] The advantage of using such a core is that the properties of each arm extending from the thioether bond attached to the maleimide may be the same or different. This increases flexibility in dendrimer synthesis, allowing for adjustments, for example, of the dendrimer generation, the type of constituent units, and the terminal groups.

[0081] This can be achieved in several ways, but in one embodiment, it may be desirable to start with a simple cystamine core in which the dendrimer is first constructed via a conjugation of lysine residues, as shown in Scheme 1. The dendrimer can be incorporated into any desired number of generations using the cystamine core, but 2, 3, 4, or 5 generations may be most appropriate. As shown in Scheme 2, the cystamine dendrimer can be cleaved into two distinct dendrons by reduction of the disulfide bond. This provides the dendrons with a thiol moiety for subsequent coupling to a maleimide unit.

[0082] This coupling step can also be achieved in several ways, but the embodiment shown in Scheme 2 has been found useful when a dibromomaleimide reagent is provided. The nitrogen of the maleimide ring provides an opportunity for functionalization with, for example, functional moieties, spacer groups, or their precursors or components, which are discussed below. In Scheme 2, this is shown as a PEG moiety activated with a tetrazine functional group, but it will be understood that a wide range of other spacer or linker groups may be used. By reacting the thiol group of the dendron with the dibromomaleimide unit, a new dendrimer core is effectively formed. Scheme 2 then shows that further iterations can be added to the dendrimer via additional constituent units up to the desired generation. The spacer group on the maleimide nitrogen can be further functionalized at the appropriate time or otherwise developed to conjugate a targeting agent, therapeutic agent, or other desired moiety.

[0083] When dendrons are exposed to dibromomaleimide units, it is possible to add mixtures of different dendrons, and thus it will be understood that different dendrimer arms will be present on the core that is subsequently formed. Separate batches of different dendrons can be synthesized from a cystamine core, removed via reduction, and then a selected relative amount of dendrons can be mixed with maleimide units to obtain the desired dendrimer having a maleimide core.

[0084] In one embodiment, the core unit may be a methylmaleimide unit or may contain one, and therefore one carbon of the ring may have methyl attached to it, and thus the double bond may be located between the ring carbons, which may have the advantage of stability.

[0085] In one embodiment, the nitrogen of the maleimide ring may simply offer hydrogen. In one embodiment, the nitrogen of the maleimide ring may be attached to a functional moiety. When the nitrogen is attached to a functional moiety, the maleimide core essentially has a further "synthetic handle" to which additional functionality can be introduced into the core. For example, therapeutic agents, targeting agents, or pharmacokinetic modifiers may be conjugated to the core as a functional moiety.

[0086] The functional moiety may be directly conjugated to the maleimide core. Alternatively, the functional moiety may be conjugated to the maleimide core via a spacer group, as defined herein. The advantage of conjugating the functional moiety to the maleimide core via a spacer group is that the functional moiety is distally attached to the dendrimer, reducing any steric hindrance from the dendrimer that might otherwise impair the functional moiety's ability to perform its function (i.e., the ability of the therapeutic or targeting agent to interact with its target receptor or molecule).

[0087] In one embodiment, the nitrogen of the maleimide ring is attached to a functional moiety containing a targeting agent. The targeting agent may be conjugated directly to the maleimide core, as described herein, or otherwise via any suitable spacer group. In one example, R 3 This is a HER2 targeting agent conjugated to a maleimide core. For example, R 3 This is a HER2 targeting agent conjugated to a maleimide core via a spacer group as described herein. For example, R 3 This is an FAP binding group conjugated to a maleimide core. For example, R 3 This is an FAP binding group conjugated to a maleimide core via a spacer group as described herein. [ka] [ka]

[0088] Scheme 2: [[(ε-NHCO-PEG 1100 )8(α-NHDOTA) 4.75 (α-NHCy5) 0.5 (α-NH2) 2.75 [Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2MAL-PEG3-NHCO-PEG 24 -CONH-PEG4-(PhTzMe) / TCO-NHCO-PEG 24 -(MAL)Me / Cys-nanobody SRS-15 (where G2(NH2) = Lys[Lys]2[Lys]4[(α-NH2.TFA)4(ε-NH2.TFA)4)], G3(NHBoc) = Lys[Lys]2[Lys]4[Lys]8(α-NHBoc)8(ε-NHPEG 1100 )8)], G3(NH2) = Lys[Lys]2[Lys]4[Lys]8(α-NH2)8(ε-NHPEG 1100 )8)], G3(DOTA) = Lys[Lys]2[Lys]4[Lys]8(α-NHCy5) 0.5 (α-NHDOTA) 4.75 (α-NH2) 2.75 (ε-NHPEG 1100 )8)]).

[0089] Structural unit Any suitable structural unit (BU) contains a first functional group capable of forming a linkage with a functional group present on another structural unit or core unit, and at least two additional functional groups capable of forming a linkage with a functional group present on another structural unit (e.g., deprotection as follows), and may be used to generate a dendrimer as long as it contains such functional groups.

[0090] In some preferred embodiments, different generations of constituent units are covalently bonded to one another via amide bonds formed between nitrogen atoms present in one constituent unit and carbon atoms of an acyl group present in another constituent unit. For example, in some embodiments, the constituent units are lysine residues or analogs thereof and may be formed from suitable constituent unit precursors, e.g., lysine or lysine analogs containing suitable protecting groups. The lysine analog has two amino nitrogen atoms for bonding to a later generation of constituent units and an acyl group for bonding to a previous generation of constituent units or core. Examples of suitable constituent units include: [ka] These include, Each acyl group of a component unit provides a covalent attachment site for attachment to the core or a previous generation component unit, and each nitrogen atom provides a covalent attachment site for attachment to a subsequent generation component unit or terminal group.

[0091] In some preferred embodiments, the constituent units are, respectively; [ka] Each acyl group of a component unit provides a covalent attachment site for attachment to the core or a previous generation component unit, and each nitrogen atom provides a covalent attachment site for attachment to a subsequent generation component unit or terminal group.

[0092] In some preferred embodiments, the constituent units are as follows: [ka]

[0093] In other embodiments, the constituent units are aspartic acid residues, glutamic acid residues, or analogs thereof, i.e., formed from a suitable precursor containing a suitable protecting group, such as aspartic acid, glutamic acid, or analogs thereof. In such embodiments, the core unit may be formed from a core unit precursor containing a carboxylic acid group (i.e., capable of reacting with an amino group present in the aspartic acid / glutamic acid / analog).

[0094] The outermost layer of generational constituent units (BU) outer The outermost layer of constituent units (BU) may be formed by constituent units used in other generations of constituent units (BU) as described above, for example, lysine or lysine analog constituent units. outer ) are the outermost generational constituent units from the dendrimer core, i.e., the outermost generational constituent units (BU) outer Subsequent generation component units will not be attached to it.

[0095] It will be understood that the dendrons of the dendrimer can be synthesized to the required number of generations by, for example, attaching constituent units (BUs) as appropriate. In some embodiments, the constituent units (BUs) of each generation may be formed from the same constituent units, for example, all generations of the constituent units may be lysine constituent units. In some other embodiments, the constituent units of one or more generations may be formed from different constituent units than the constituent units of the other generations.

[0096] Dendrimers have 2 to 6 generations of constituent units, i.e., 2, 3, 4, 5, or 6 generations of constituent units.

[0097] In some embodiments, the dendrimer has three generations of constituent units. A three-generation constituent unit dendrimer is a dendrimer having a structure that includes three constituent units covalently linked to one another, for example, when the constituent units are lysine, and this may include the following substructures. [ka]

[0098] In some embodiments, the dendrimer has five generations of constituent units. A five-generation constituent unit dendrimer is a dendrimer having a structure that includes five constituent units covalently linked to one another, for example, when the constituent units are lysine, and this may include the following substructures. [ka]

[0099] In some embodiments, the dendrimer has four generations of constituent units. A four-generation constituent unit dendrimer is a dendrimer having a structure that includes four constituent units covalently linked to one another, for example, when the constituent units are lysine, and this may include the following substructures. [ka]

[0100] In some embodiments, the generation of constituent units is a complete generation. For example, if a dendrimer has three generations of constituent units, in some embodiments the dendrimer has three complete generations of constituent units. In the case of a core having two reactive amine groups, such a dendrimer would contain 14 constituent units (i.e., core unit + 2BU + 4BU + 8BU).

[0101] Similarly, for example, if the dendrimer has four generations of constituent units, in some embodiments the dendrimer has four complete generations of constituent units. In the case of a core having two reactive amine groups, such a dendrimer would contain 30 constituent units (i.e., core unit + 2BU + 4BU + 8BU + 16BU).

[0102] Similarly, for example, if a dendrimer has five generations of constituent units, in some embodiments the dendrimer has five complete generations of constituent units. In the case of a core having two reactive amine groups, such a dendrimer would contain 62 constituent units (i.e., core unit + 2BU + 4BU + 8BU + 16BU + 32BU).

[0103] However, given the nature of the synthetic process for generating dendrimers, it is understandable that one or more reactions performed to produce a dendrimer may not be fully completed. Therefore, in some embodiments, the dendrimer may contain constituent units of an incomplete generation. For example, it is possible to obtain a population of dendrimers having a distribution of the number of constituent units per dendrimer.

[0104] In some embodiments, when a dendrimer has three generations of constituent units, a group of dendrimers is obtained in which each dendrimer has an average number of constituent units of at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13. In some embodiments, a group of dendrimers is obtained in which at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers have 10 or more constituent units. In some embodiments, a group of dendrimers is obtained in which at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers have 12 or more constituent units.

[0105] In some embodiments, if a dendrimer has four generations of constituent units, a group of dendrimers is obtained in which each dendrimer has an average number of constituent units of at least 25, or at least 26, or at least 27, or at least 28, or at least 29. In some embodiments, a group of dendrimers is obtained in which at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers have 25 or more constituent units. In some embodiments, a group of dendrimers is obtained in which at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers have 29 or more constituent units.

[0106] In some embodiments, if a dendrimer has five generations of constituent units, a group of dendrimers is obtained in which each dendrimer has an average number of constituent units of at least 55, or at least 56, or at least 57, or at least 58, or at least 59, or at least 60. In some embodiments, a group of dendrimers is obtained in which at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers have 55 or more constituent units. In some embodiments, a group of dendrimers is obtained in which at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers have 60 or more constituent units.

[0107] In some embodiments, each reactive (amino) group of the core unit precursor represents a conjugation site of a dendron containing the constituent units.

[0108] In some embodiments, each generation of constituent units in each dendron (X) is given by Equation [BU]2 (b-1)It may also be expressed as (where b is the number of generations). A dendron (X) with 3 complete generations of constituent units is represented as [BU]1-[BU]2-[BU]4. A dendron (X) with 4 complete generations of constituent units is represented as [BU]1-[BU]2-[BU]4-[BU]8. A dendron (X) with 5 complete generations of constituent units is represented as [BU]1-[BU]2-[BU]4-[BU]8-[BU] 16 It is expressed as follows.

[0109] In some embodiments, the dendrimer includes two or more dendrons. In some embodiments, the dendrons are the same. In some embodiments, the dendrons are different. In some embodiments, the dendrons are the same or different at the level of constituent units, surface groups, generation size, first terminal groups, or second terminal groups.

[0110] First terminal group The first terminal group (T1) contains a complexing group for complexing a radionuclide. After exposure to a suitable radionuclide, the complexing group for complexing the radionuclide includes the radionuclide and the complexing group, and may be referred to as the radionuclide-containing portion.

[0111] In the embodiment, the radionuclide-containing portion may include radionuclides chelated with a complexing group.

[0112] In this embodiment, the radionuclide-containing portion may contain the radionuclide in the coordination complex with the complexing group.

[0113] In the embodiment, the radionuclide-containing portion may include radionuclides chelated to at least two different atoms of the complexing group.

[0114] In the embodiment, the radionuclide-containing portion may include a radionuclide that is datively bonded to the complexing group.

[0115] Radioactive nuclides Any suitable radionuclides may be used in the dendrimer of the present invention. Radionuclides, also known as radioisotopes, are unstable forms of chemical elements that undergo radioactive decay to emit nuclear radiation.

[0116] Radionuclides have applications in the treatment of diseases such as cancer. In such cases, when a substance containing a radionuclide is administered to a patient, the radionuclide is delivered to the tumor, and after radioactive decay and radiation exposure, the tumor cells die.

[0117] Preferably, the radionuclide is a metallic or metalloid (for example, astatine is considered a metalloid for the purposes of this invention) radionuclide, such as a metal ion or metalloid ion. In some embodiments, the radionuclide is an alpha emitter (α emitter). In some embodiments, the radionuclide is a beta emitter (β emitter). In some embodiments, the radionuclide is a beta and gamma emitter (γ emitter).

[0118] In this embodiment, the radioactive nuclide is not an isotope of hydrogen, including deuterium and tritium.

[0119] In some embodiments, the radioactive nuclide is actinium (e.g., Ac 225 ), astatine (for example, As 211 ), bismuth (for example, Bi 212 , Bi 213 ), lead (e.g., Pb 212 ), technetium (for example, Tc 99m ), Thorium (for example, Th 227 ), radium (for example, Ra 223 ), lutetium (for example, Lu 177 ), Yttrium (for example, Y 90 ), indium (for example, In 111 In 114 ), gadolinium (for example, Gd 153 ), gallium (for example, Ga 68 ), zirconium (for example, Zr 89 ), rhenium (for example, Re 186), iodine (for example, I 131 ), or copper (for example, Cu 60 Cu 61 Cu 62 Cu 64 Cu 67 ) is a radioactive nuclide. In some embodiments, the radioactive nuclide is lutetium (e.g., Lu 177 ), gallium (for example, Ga 68 ), zirconium (for example, Zr 89 ), actinium (for example, Ac 225 ), bismuth (for example, Bi 212 , Bi 213 ), astatine (for example, As 211 ), technetium (for example, Tc 99m ), or copper (for example, Cu 60 Cu 61 Cu 62 Cu 64 Cu 67 ) is a radioactive nuclide. In some embodiments, the radioactive nuclide is lutetium (e.g., Lu 177 ), gallium (for example, Ga 68 ), zirconium (for example, Zr 89 ), or copper (for example, Cu 60 Cu 61 Cu 62 Cu 64 Cu 67 ) is a radioactive nuclide. In some embodiments, the radioactive nuclide is gallium (e.g., Ga 68 ), zirconium (for example, Zr 89 ), or lutetium (for example, Lu 177 It is a radioactive nuclide.

[0120] In some embodiments, radionuclides are used for the treatment of conditions (e.g., cancer). Examples of such radionuclides include actinium (e.g., Ac 225 ), astatine (for example, As 211 ), bismuth (for example, Bi 212 , Bi 213 ), Thorium (for example, Th 227 ), radium (for example, Ra 223) Ruthenium (e.g., Lu 177 ) Yttrium (e.g., Y 90 ) Gadolinium (e.g., Gd 153 ) Lead (e.g., Pb 212 ) and copper (e.g., Cu 60 , Cu 61 , Cu 62 , Cu 64 ) are included.

[0121] In some embodiments, the radionuclide is an alpha emitter selected from actinium (e.g., Ac 225 ), astatine (e.g., At 211 ), bismuth (e.g., Bi 212 , Bi 213 ), and lead (e.g., Pb 212 ).

[0122] In some embodiments, the radionuclide is a beta emitter selected from ruthenium (e.g., Lu 177 ), yttrium (e.g., Y 90 ), iodine (e.g., I 131 ), copper (e.g., Cu 67 ), and rhenium (e.g., Re 186 ).

[0123] Ideally, the radiation characteristics of the therapeutic radionuclide should have a suitable half-life for concentrating the energy within the tumor considering the lesion size and for the extended delivery of the dendrimer. In some embodiments, the radionuclide is an alpha emitter having a half-life of less than 20 days or less than 12 days. In some embodiments, the radionuclide is a beta emitter having a half-life of 2 - 20 days or 5 - 10 days. 177 Lu is a medium energy beta emitter (490 keV) with a maximum energy of 0.5 MeV and a maximum tissue penetration of <2 mm. 177Lu also emits low-energy gamma rays at 208 and 113 keV, enabling ex vivo imaging and, as a result, collection of information regarding tumor localization and dosimetry. In some embodiments, the injected dose of the therapeutic radionuclide is 1 - 50 GBq per injection. In other embodiments, the injected dose is 2 - 20 GBq per single injection / infusion. In other embodiments, the injected dose is 2 - 10 GBq per single injection. The dose calculation for an individual patient can be determined from a combination of disease burden, patient weight, and renal function. Image-based dosimetry in each treatment cycle is recommended, for example, using SPECT-CT.

[0124] Radionuclides are also utilized in the field of medical diagnosis. Using techniques such as single photon emission, positron emission tomography (PET) imaging, and positron emission tomography - magnetic resonance imaging (PET-MRI), radionuclides within a subject administered with a suitable radionuclide-containing substance can be detected, and images can be generated to indicate the presence and / or progression of diseases such as tumors.

[0125] In some embodiments, the radionuclide is for the diagnosis or imaging of a condition (e.g., cancer). Examples of such radionuclides include gallium (e.g., Ga 68 ), indium (e.g., In 111 ), zirconium (e.g., Zr 89 ), iodine (e.g., 123 I, 131 I), technetium (e.g., Tc 99m ), yttrium (e.g., Y 86 ), fluorine (e.g., F 18 ), and copper (e.g., Cu 60 , Cu 61 , Cu 62 , Cu 64 ). In some embodiments, the radionuclide is gallium (e.g., Ga 68 ), technetium (e.g., Tc 99m ), zirconium (e.g., Zr 89 ), and copper (e.g., Cu 60Cu 61 Cu 62 Cu 64 The imaging agent is selected from ). In some embodiments, the radionuclide is gallium (e.g., Ga 68 ), zirconium (for example, Zr 89 ), and copper (e.g., Cu 64 ) will be selected from.

[0126] In some embodiments, the radioactive nuclide is not gadolinium.

[0127] In some embodiments, the radioactive nuclide is not a paramagnetic agent.

[0128] The type of radionuclide used may be tailored to the dendrimer structure to optimize the level of radioactive exposure received by the subject. For example, the body is typically considered to have greater exposure to dendrimers with a larger number of generations of constituent units. Therefore, by pairing such dendrimers with radionuclides having appropriate half-lives, optimal therapeutic and / or diagnostic activity can be achieved while avoiding or reducing side effects associated with the body's exposure to radioactivity.

[0129] In some embodiments, the dendrimer is a 4th or 5th generation dendrimer, and the radionuclide has a half-life of 10 days or less, preferably less than 5 days. In some embodiments, the dendrimer is a 4th generation dendrimer, and the radionuclide has a half-life of 10 days or less, preferably less than 5 days. In some embodiments, the dendrimer is a 5th generation dendrimer, and the radionuclide has a half-life of less than 10 days, preferably less than 5 days. In some embodiments, the dendrimer is a 4th generation dendrimer, and the radionuclide is Y 90 , Tc 99m Th 201 , Rb 82 , Lt 177 , Ga 67 , Ga 68 , and In 111Selected from the group consisting of . In some embodiments, the dendrimer is a third-generation dendrimer, and the radionuclide has a half-life of 10 days or less. In some embodiments, the dendrimer is a third-generation dendrimer, and the radionuclide is Y 90 , Tc 99m Th 201 , Rb 82 , Lt 177 , Ga 67 , Ga 68 Ac 225 , and In 111 It is selected from the group consisting of the following.

[0130] Radioactive nuclide complex group Any suitable complexing group may be used, as long as it is appropriate for forming a complex with the desired radionuclide. The complexing group for complexing a radionuclide provides a functional moiety that can complex the radionuclide. Examples of such functional moieties include carboxylic acids, amines, amides, hydroxyl groups, thiol groups, urea, thiourea, -N-OH groups, phosphates, and phosphinate groups that form complexes with radionuclides.

[0131] In some embodiments, the complexing group is directly complexed with the radionuclide.

[0132] In some embodiments, the complexing group directly complexes with the radionuclide to form a coordination complex. That is, the complexing group forms a complex by coordinating with the radionuclide.

[0133] In some embodiments, the complexing group forms only donor covalent bonds to the radionuclide. For example, the complexing group may form at least two distinct donor covalent bonds with the radionuclide.

[0134] In some embodiments, complexing groups that form chelates with radionuclides are used. As used herein, the phrase "complexing groups that form chelates with radionuclides" means that the complexing group forms at least two distinct bonds (i.e., bonds from at least two different atoms of the complexing group) with the radionuclides.

[0135] Examples of suitable complexing groups are shown in the table below: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0136] In some embodiments, the complexing group is DOTA, NOTA, DTPA, sarcofazine, or DFO.

[0137] In some embodiments, the complexing group is a macropa group. A macropa group is, for example, Ac 225 It is particularly suitable for use with radionuclides. In some embodiments, the complexing group is a macropa group, and the radionuclide is Ac 225 That is the case.

[0138] In some embodiments, the complexing group is EDTA or PEPA.

[0139] The first terminal group is attached to the outermost constituent unit, for example, a constituent unit that is a lysine residue or an analog thereof, via the nitrogen atom of the outermost constituent unit. In some embodiments, the complexing group may react directly with the constituent unit if the complexing group contains a group suitable for direct reaction with the outermost constituent unit. In other embodiments, the loading group may be used to load the complexing group into a dendrimer, i.e., a group whose first terminal is covalently attached to the complexing group and whose second terminal has a functional group suitable for reaction with a functional group present on the outermost constituent unit (for example, when the first terminal group is attached via the nitrogen atom of the outermost constituent unit). For example, the loading group may have a functional group suitable for reaction with an amino group.

[0140] To form an adhesion between the outermost constituent unit and the first terminal group, a reaction may be carried out between a suitable complexing precursor group and a dendrimer intermediate having a functional group available for the reaction (e.g., an amine group). In some embodiments, the complexing precursor is a DOTA-containing group, a NOTA-containing group, a DTPA-containing group, a sarcofazine-containing group, or a DFO-containing group.

[0141] In some embodiments, the complexing group is structure [ka] It is a DOTA-containing group, and the DOTA-containing group is attached to the conjugate.

[0142] In some embodiments, the complexing group is structure [ka] It is a NOTA-containing group, and the NOTA-containing group is attached to the conjugate.

[0143] In some embodiments, the complexing group is structure [ka] It is a DTPA-containing group, and the DTPA-containing group is attached to the conjugate.

[0144] In some embodiments, the complexing group is structure [ka] It is a DFO-containing group, and the DFO-containing group is attached to the conjugate.

[0145] In some embodiments, the complexing group is structure [ka] It is a sarcofadin-containing group, and the sarcofadin-containing group is attached to the conjugate.

[0146] In some embodiments, the complexing group is structure [ka] It is a sarcofadin-containing group, and the sarcofadin-containing group is attached to the conjugate.

[0147] In some embodiments, the complexing group is structure [ka] It is a macropa-containing group having the macropa-containing group attached to the conjugate.

[0148] Specific examples of suitable complexing precursor groups include the following: [ka] [ka]

[0149] Such groups can react with amine groups present on the outermost constituent unit to form a first terminal group that links to thiourea.

[0150] Second terminal group This conjugate contains multiple second terminal groups (T2), each terminal group containing a pharmacokinetic modification moiety, i.e., a moiety that can modify or modulate the pharmacokinetic profile of this conjugate. The pharmacokinetic modification moiety can modulate the absorption, distribution, metabolism, excretion, and / or toxicity of the dendrimer.

[0151] The pharmacokinetic modification moiety (T2) can alter the solubility profile of the dendrimer, increasing or decreasing its solubility in pharmaceutically acceptable carriers. For example, the pharmacokinetic modification moiety (T2) can reduce the clearance of the dendrimer.

[0152] If a dendrimer contains a third terminal group containing a pharmaceutically active agent, the pharmacokinetic modification moiety (T2) may affect the release rate of the pharmaceutically active agent by slowing or increasing the rate at which the agent is released from the dendrimer, either through a chemical (e.g., hydrolysis) or enzymatic degradation pathway. The pharmacokinetic modification moiety (T2) may help the dendrimer deliver the pharmaceutically active agent to a specific tissue (e.g., a tumor).

[0153] The pharmacokinetic modification portion may be, for example, a biocompatible, water-soluble oligomeric group or polymer group. In some embodiments, the pharmacokinetic modification portion is a water-soluble oligomer or polymer having a molecular weight in the range of 300 to 5000 daltons.

[0154] In some preferred embodiments, the pharmacokinetic modification moiety is a polyethylene glycol (PEG) group, or a polyethyl oxazoline (PEOX) group, or poly-(2)methyl-(2)-oxazolamine (POZ), or polysarcosine (poly(n-methylated glycine)), or poly(2-hydroxypropyl)methacrylamide (pHPMA) group.

[0155] In some embodiments, the second terminal group comprises a PEG group. The PEG group is a polyethylene glycol group, i.e., a group comprising a repeating unit of the formula -CH2CH2O-. The PEG material used to produce the dendrimers of this disclosure typically comprises a mixture of PEGs having some variability in molecular weight (i.e., ±10%), and therefore, when a molecular weight is specified, it is typically an approximation of the average molecular weight of the PEG composition. For example, "PEG ~2100 The term "PEG" refers to an average molecular weight of approximately 2100 daltons, i.e., ±10% (PEG). 1890 ~PEG 2310 This refers to polyethylene glycol containing PEG. ~2300 "This refers to an average molecular weight of approximately 2300 daltons, or ±10% (PEG). 2070 ~PEG 2530 This refers to polyethylene glycol having polyethylene glycol of ) . Three commonly used methods for calculating the MW average are the number average, weight average, and Z average molecular weight. As used herein, the term “molecular weight” is intended to refer to the weight average molecular weight that can be measured using techniques well known in the art, such as NMR, mass spectrometry, matrix-assisted laser desorption / ionization time of flight (MALDI-TOF), gel permeation chromatography, or other liquid chromatography techniques, light scattering techniques, ultracentrifugation, and viscosity measurement, but is not limited to these.

[0156] In some embodiments, the second terminal group comprises a PEG group having an average molecular weight of about 200 to 5000 daltons, or 200 to 4000 daltons, or 300 to 3000 daltons, or 300 to 2000 daltons, or 400 to 1500 daltons, or 400 to 1200 daltons, or 400 to 1000 daltons, or 400 to 800 daltons, or 400 to 600 daltons. In some embodiments, the second terminal group comprises a PEG group having an average molecular weight of about 400, about 450, about 500, about 550, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, or about 1500 daltons. In some embodiments, the second terminal group comprises a PEG group having an average molecular weight of about 470 daltons. In some embodiments, the second terminal group includes a PEG group having an average molecular weight in the range of 500 to 3000 daltons, or 1500 to 2500 daltons. In some embodiments, the second terminal group includes a PEG group having an average molecular weight in the range of 220 to 2500 daltons, or 570 to 2500 daltons, or 220 to 1100 daltons, or 570 to 1100 daltons, or 1000 to 5500 daltons, or 1000 to 2500 daltons, or 1000 to 2300 daltons. In some embodiments, the second terminal group includes a PEG group having an average molecular weight in the range of 1900 to 2300 daltons. In some embodiments, the second terminal group includes a PEG group having an average molecular weight in the range of 2100 to 2500 daltons. In some embodiments, the second terminal group includes a PEG group having an average molecular weight in the range of 2400 to 2800 daltons. In some embodiments, the second terminal group comprises a PEG group having an average molecular weight of about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, or about 2800 daltons.

[0157] In some embodiments, the PEG group has a polydispersity index (PDI) of about 1.00 to about 1.50, about 1.00 to about 1.25, or about 1.00 to about 1.10. In some embodiments, the PEG group has a polydispersity index (PDI) of about 1.05. The term "polydispersity index" is an indicator of the distribution of molecular weight in a polymer sample. The polydispersity index (PDI) is the weight-average molecular weight (M w ) number average molecular weight (M n This is calculated by dividing by ( ) and shows the distribution of individual molecular weights within a batch of polymers. The polydispersity index (PDI) has a value of 1 or greater, but as the polymer approaches a uniform change in length and average molecular weight, the polydispersity index (PDI) approaches 1.

[0158] If the second terminal group includes a PEG group, the PEG group may be linear or branched. An end-capped PEG group may be used if desired. In some embodiments, the PEG group is a methoxy-terminated PEG.

[0159] In some embodiments, the second terminal group includes a polysarcosine group, i.e., a group containing a repeating unit of the following formula. [ka]

[0160] In some embodiments, the second terminal group comprises a polysarcosine group having an average molecular weight of at least 750 daltons, at least 1000 daltons, or at least 1500 daltons. In some embodiments, the second terminal group comprises a polysarcosine group having an average molecular weight in the range of 750 to 2500 daltons, or 1000 to 2500 daltons.

[0161] In some embodiments, the second terminal group includes a PEOX group, which is a polyethyloxazoline group, i.e., a group containing repeating units of the following formula. [ka]

[0162] The PEOX group is so named because it can be produced by polymerization of ethyl oxazoline. The PEOX material used to produce the dendrimers of this disclosure typically comprises a mixture of PEOX having some variability in molecular weight (i.e., ±10%), and therefore, where a molecular weight is specified, it is typically an approximation of the average molecular weight of the PEOX composition. In some embodiments, the second terminal group comprises a PEOX group having an average molecular weight of at least 750 daltons, at least 1000 daltons, or at least 1500 daltons. In some embodiments, the second terminal group comprises a PEOX group having an average molecular weight in the range of 750 daltons to 2500 daltons, or 1000 daltons to 2000 daltons. End-capped PEOX groups may be used if desired. In some embodiments, the PEOX group is methoxy-terminated PEOX.

[0163] In some embodiments, the second terminal group includes a poly-(2)methyl-(2)-oxazoleamine (POZ) group.

[0164] In some embodiments, the second terminal group includes a poly(2-hydroxypropyl)methacrylamide (pHPMA) group.

[0165] The second terminal group may be attached to the outermost component unit by any preferred means. In some embodiments, where the second terminal group includes a PEG group, a PEOX group, a POZ group, or a pHPMA group, a linking group is used to attach the PEG group, PEOX group, POZ group, or pHPMA group to the outer component unit.

[0166] The second terminal group is typically attached by using a second terminal group precursor containing a reactive group that is reactive with an amine group, such as a reactive acyl group (which can form an amide bond) or an aldehyde (which can form an amine group under reductive amination conditions).

[0167] In some embodiments, each second terminal group includes a PEG group, the PEG group is covalently attached to the PEG linking group (L1) via an ether linkage formed between the carbon atom present in the PEG group and the oxygen atom present in the PEG linking group, and each second terminal group is covalently attached to the constituent unit via an amide linkage formed between the nitrogen atom present in the constituent unit and the carbon atom of the acyl group present in the PEG linking group. In some embodiments, the second terminal groups are, [ka] The PEG group is a methoxy-terminated PEG having an average molecular weight in the range of approximately 500-3000 daltons or 2000-2700 daltons.

[0168] In some embodiments, each second terminal group includes a PEOX group, the PEOX group is covalently attached to the PEOX linking group (L1') via a bond formed between the nitrogen atom present in the PEOX group and the carbon atom present in the PEOX linking group, and each second terminal group is covalently attached to the constituent unit via an amide bond formed between the nitrogen atom present in the constituent unit and the carbon atom of the acyl group present in the PEOX linking group. In some embodiments, the second terminal groups are, [ka] That is the case.

[0169] In some embodiments, the second terminal group is, for example, a polysarcosine group of the following formula: [ka] It is attached to the constituent unit via an amide bond formed between the nitrogen atom present in the constituent unit and the carbon atom of the acyl group present in the polysarcosine group.

[0170] Targeting agent The dendrimer-targeting agent conjugates described herein comprise at least one targeting agent for localization and enrichment of the conjugate at a site or target of interest in the body. Examples of targeting agents include antibodies, antibody fragments, peptide sequences, and other motifs that can selectively bind to a target of interest.

[0171] This interaction can arise from any kind of bond or association, such as covalent bonds, ionic bonds, hydrogen bonds, and van der Waals forces.

[0172] As used herein, "peptide" refers to a molecule containing two or more amino acids linked by peptide bonds.

[0173] The targeting agents described herein are useful for targeting the disclosed dendrimer-targeting agent conjugate to targets such as tumors, cancer cells, and / or the tumor microenvironment.

[0174] The targeting agent may include, for example, an antigen-binding site or antigen-binding domain that specifically binds to and / or has affinity for a target molecule (also referred to herein as "target" or "antigen").

[0175] In one embodiment, the target is selected from one or more of the following: human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), vascular epidermal growth factor (VEGF) receptor, G protein-coupled receptor 161 (GPR161), fibroblast growth factor receptor (FGFR2, etc.), hepatocyte growth factor (HGF), hepatocyte growth factor receptor (HGFR), tyrosine protein kinase met (C-met), atypical chemokine receptor 3 (CXCR7), CXC-motif chemokine receptor 4 (CXCR4), carcinoembryonic antigen, mucin 1 (MUC-1), mucin 16 (MUC16), epidermal cell adhesion molecule (EpCAM), trophoblast glycoprotein (5T4), interleukin-2 (IL-2), glycoprotein (gpNMB), syndecane (S yndecan)1 (CD138), prostate-specific membrane antigen (PSMA), carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), solute carrier family 44 member 4 (CSLC44A4), granulocyte colony-stimulating factor receptor (G-CSFR), ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), mesothelin, nectin cell adhesion molecule 4 (nectin-4), fibroblast-activating protein (FAP), folate receptor, hyaluronic acid receptor, integrin receptor (αvβ3), lectin-binding glycoprotein, TfR, VEGFR-1 and VEGFR-2, cytokines, CD56, CD19, CD16, CD74, CD37, CD70, CD52, CD19, CD22, CD20, CD30, CD3, and CD79b.

[0176] In one embodiment, the target is HER2, also known as ERBB2, gene ID number 2064 (NCBI).

[0177] In one embodiment, the target is the epidermal growth factor receptor (EGFR), also known as ERBB1 or HER1, gene ID number 1956 (NCBI).

[0178] In one embodiment, the target is prostate-specific membrane antigen (PSMA), gene ID number 2346 (NCBI).

[0179] In one embodiment, the target is fibroblast-activating protein (FAP). Overexpression of serine protease fibroblast-activating protein in cancer facilitates selective targeting of tumors (Loktev et al, J Nucl Med, 2019, 60(10), p1421-1429).

[0180] In one embodiment, the targeting agent contains a molecular weight of up to about 200 kDa, or up to about 150 kDa, or up to about 110 kDa, or up to about 80 kDa, or up to about 55 kDa, or up to about 16 kDa. In one embodiment, the targeting agent contains a molecular weight of up to about 200 kDa. In one embodiment, the targeting agent contains a molecular weight of up to about 150 kDa. In one embodiment, the targeting agent contains a molecular weight of up to about 110 kDa. In one embodiment, the targeting agent contains a molecular weight of up to about 80 kDa. In one embodiment, the targeting agent contains a molecular weight of up to about 55 kDa. In some embodiments, the targeting agent contains a molecular weight of up to about 16 kDa. In one embodiment, the targeting agent has a molecular weight of about 3 kDa to about 80 kDa. In one embodiment, the targeting agent has a molecular weight of about 3 kDa to about 60 kDa. In one embodiment, the targeting agent has a molecular weight of approximately 3 kDa to approximately 50 kDa. In one embodiment, the targeting agent has a molecular weight of approximately 3 kDa to approximately 40 kDa. In one embodiment, the targeting agent has a molecular weight of approximately 3 kDa to approximately 30 kDa. In one embodiment, the targeting agent has a molecular weight of approximately 3 kDa to approximately 20 kDa. In one embodiment, the targeting agent has a molecular weight of approximately 3 kDa to approximately 15 kDa. In one embodiment, the targeting agent has a molecular weight of approximately 3 kDa to approximately 13 kDa. In one embodiment, the targeting agent has a molecular weight of approximately 5 kDa to approximately 15 kDa. In one embodiment, the targeting agent has a molecular weight of approximately 5 kDa to approximately 12 kDa. In one embodiment, the targeting agent has a molecular weight of approximately 5 kDa to approximately 10 kDa.

[0181] As used herein, "kDA" or "kilodalton" refers to a unit of molecular weight consisting of 1,000 daltons.

[0182] In one embodiment, the targeting agent is selected from an antibody, a heavy chain antibody, ScFV-Fc, Fab, Fab2, Fv, scFv, or a single-domain antibody. In another embodiment, the targeting agent is selected from an antibody or an antibody fragment.

[0183] In one embodiment, the targeting agent is an antibody. For the purposes of this disclosure, the term “antibody” includes a four-chain protein, such as two light chains and two heavy chains, including recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, primate-like antibodies, deimmunized antibodies, and half-antibodies, bispecific antibodies) that can specifically bind to one or more closely related antigens by Fv. Antibodies generally include a constant domain, which can be arranged into a constant region and a constant fragment, a crystallizable fragment (Fc). Exemplary forms of antibodies include a four-chain structure as the basic unit. Full-length antibodies include two heavy chains (about 50-70 kDa) and two light chains (about 23 kDa each) linked by covalent bonds. Light chains generally include a variable region and a constant domain, which in mammals is either a κ light chain or a λ light chain. Heavy chains generally include a variable region and one or two constant domains, which are linked to additional constant domains by a hinge region. Mammalian heavy chains consist of one of the following types: α, δ, ε, γ, or μ. Each light chain is covalently linked to one of the heavy chains. For example, two heavy chains, and a heavy chain and a light chain, are held together by interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds can vary depending on the type of antibody. Each chain has an N-terminal variable region (V H or V LThe light chain has a constant domain (CL: approximately 110 amino acids in length) and one or more constant domains at its C-terminus. The constant domain of the light chain (CL: approximately 110 amino acids in length) is aligned with the first constant domain of the heavy chain (CH: approximately 330-440 amino acids in length) and is disulfide-bonded to it. The variable region of the light chain is aligned with the variable region of the heavy chain. The antibody heavy chain may contain two or more additional CH domains (e.g., CH2, CH3, etc.) and may contain a hinge region between the CH1 constant domain and the CH2 constant domain. The antibody may be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a subclass. In one example, the antibody may be a human antibody, its immunized form, germline form, or its affinity mature form. The terms "full-length antibody" or "whole antibody" are used synonymously to refer to an antibody in its substantially intact form, as opposed to an antigen-binding fragment. Specifically, whole antibodies include antibodies that have heavy and light chains, such as a constant region. The constant region may be the wild-type sequence constant region (e.g., the human wild-type sequence constant region) or an amino acid sequence variant thereof.

[0184] In one embodiment, the targeting agent is a fusion protein. As used herein, “fusion protein” is a protein produced by the conjugation of two or more nucleic acid sequences that originally encode separate proteins or parts thereof (for example, the fusion of a part of a protein receptor and a part of an antibody (etanercept)).

[0185] In some embodiments, the targeting agent is an antibody fragment. As used herein, the term “antibody fragment” means a part or fragment of an antibody that can specifically bind to an antigen, for example, F as defined herein. V , V H , V Lor a variable region is included. This term will be understood to encompass fragments directly derived from antibodies, as well as proteins produced using recombinant means. In one embodiment, the antibody fragment is selected from Fab, Fab2, Fv, scFv, heavy chain antibody, domain antibody, heavy chain antibody, diabody, or triabody.

[0186] As used herein, the term "Fv" means V whether it consists of multiple polypeptides or a single polypeptide (scFV). L and V H This refers to any protein that associates to form a complex having an antigen-binding domain, i.e., any protein that can specifically bind to an antigen. H and V L This may be a single polypeptide chain or different polypeptide chains. In one embodiment, Fv of the Disclosure (as well as any protein of the Disclosure) may have multiple antigen-binding sites that may or may not bind the same antigen. This term will be understood to encompass fragments directly derived from antibodies, as well as proteins produced using recombinant means. In some examples, V H It is not linked to the heavy chain constant domain CH1, and / or V L The light chain constant domain (CL), for example, is not linked to the domain antibody. Exemplary Fv-containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, diabodies, triabodies, etc. Fab fragments consist of monovalent antigen-binding fragments of immunoglobulin and can be produced by digesting the whole antibody with the enzyme papain to produce a fragment consisting of the intact light chain and part of the heavy chain, or by using recombinant means. Fab fragments are generally V H and C H 1 and V L and C L It contains or is composed of these. The "Fab' fragment" of the antibody is obtained by reducing the intact light chain and V after treating the entire antibody with pepsin. HThis can be obtained by generating a molecule consisting of a portion of the heavy chain containing a single constant domain. Two Fab' fragments are obtained from antibodies processed in this manner. Fab' fragments can also be generated by recombinant means. A "single-chain Fv" or "scFv" is a recombinant molecule containing a variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable and flexible polypeptide linker.

[0187] In some embodiments, the antibody fragment is selected from heavy chain antibodies, Fab, Fab2, Fv, scFv, or single-domain antibodies.

[0188] As used herein, a "single-domain antibody (sdAb)," also called a "domain antibody (dAb)" or "nanobody," is a single variable region heavy chain V H or light chain V L It consists of. In one embodiment, the variable region is derived from the camelid family. In one embodiment, the variable region is derived from the shark. In one embodiment, V H V, which originates from the camelid family. H That is the case.

[0189] In some embodiments, the targeting agent is a single-domain antibody. In some embodiments, the targeting agent is a VH single-domain antibody. In some embodiments, the targeting agent is a VL single-domain antibody.

[0190] In one embodiment, the single-domain antibody comprises a single-domain amino acid sequence described, for example, EP2215125(A1), US2011 / 0028695, Hussack et al. (2018), Arezumand et al. (2017), and Chanier et al. (2019). In one embodiment, the single-domain antibody comprises a single-domain amino acid sequence described in US2011 / 0028695.

[0191] In one embodiment, the single-domain antibody comprises the single-domain amino acid sequence disclosed in Example 7. In one embodiment, the single-domain antibody is 2D3 comprising the amino acid sequence shown in SEQ ID NO: 1986 of US2011 / 0028695.

[0192] In one embodiment, the single-domain antibody comprises the amino acid sequence EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS (SEQ ID NO: 1). In one embodiment, the sequence comprises additional cysteine ​​residues for conjugation.

[0193] In one embodiment, a single-domain antibody has the following amino acid sequence: The amino acid sequence is EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS#ENLYFQGHHHHHH, where # represents a non-natural amino acid, preferably a 4-azidophenylalanine residue (SEQ ID NO: 2).

[0194] In one embodiment, a single-domain antibody has the following amino acid sequence: The amino acid sequence is EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS#, where # represents a non-natural amino acid, preferably a 4-azidophenylalanine residue (SEQ ID NO: 3).

[0195] In one embodiment, the single-domain antibody contains the amino acid sequence:GGSHHHHHHGMASMTGGQQMGRDLYENLYFQGEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS (SEQ ID NO: 4).

[0196] In one embodiment, the single-domain antibody comprises the amino acid sequence:GGSHHHHHHGMASMTGGQQMGRDLYENLYFQGEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS#, where # represents a non-natural amino acid, preferably a 4-azidophenylalanine residue (SEQ ID NO: 5).

[0197] In one embodiment, the single-domain antibody has the amino acid sequence:EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS[X] n The molecule contains an amino acid sequence in which C is any amino acid and n = 0 to 20. In one embodiment, n = 0, 1, 2, 3, 4, or 5. In one embodiment, n = 4 (SEQ ID NO: 6).

[0198] In one embodiment, a single-domain antibody has the following amino acid sequence: C[X] nEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS, which comprises an amino acid sequence where X is any amino acid and n = 0 to 20. In one embodiment, n = 0, 1, 2, 3, 4, or 5 (SEQ ID NO: 7).

[0199] In one embodiment, the single-domain antibody has the amino acid sequence:EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS[X] n C[X] m The amino acid sequence includes an amino acid sequence in which X is any amino acid, m = 0 to 20, and n = 0 to 20. In one embodiment, n = 0, 1, 2, 3, 4, or 5 (SEQ ID NO: 8).

[0200] In one embodiment, the single-domain antibody has the amino acid sequence: [X] n C[X] m EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS, comprising an amino acid sequence where X is any amino acid, m=0-20, and n=0-20. In one embodiment, n=0, 1, 2, 3, 4, or 5 (SEQ ID NO: 9). In one embodiment, a single-domain antibody comprises the amino acid sequence: Includes EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSLGTLCTPSRENLYFQGHHHHHH (sequence number 10).

[0201] In some embodiments, the targeting agent is a single-domain antibody having a molecular weight of approximately 4 kDa to approximately 80 kDa, or approximately 5 kDa to approximately 80 kDa, or approximately 5 kDa to approximately 60 kDa, or approximately 5 kDa to approximately 50 kDa, or approximately 5 kDa to approximately 40 kDa, or approximately 5 kDa to approximately 30 kDa, or approximately 5 kDa to approximately 20 kDa, or approximately 5 kDa to approximately 16 kDa, or approximately 5 kDa to approximately 15 kDa, or approximately 5 kDa to approximately 12 kDa, or approximately 10 kDa to approximately 16 kDa, or approximately 15 kDa to approximately 20 kDa.

[0202] In some embodiments, the targeting agent contains less than 500, less than 400, less than 300, less than 200, less than 150, less than 140, less than 130, less than 120, less than 110, or less than 100 amino acid residues. In some embodiments, the targeting agent contains more than 50, more than 75, more than 100, or more than 120 amino acid residues. In some embodiments, the targeting agent contains less than 120 amino acid residues. In some embodiments, the targeting agent contains about 100 to about 120 amino acid residues.

[0203] In some embodiments, the targeting agent is an antibody mimetic. In this specification, the terms “mimetic” or “mimetics” refer to compounds that can bind an antigen but are not structurally related to an antibody, such as an antibody or antibody fragment. This term is understood to be exclusive to the antibodies or antibody fragments described herein. This term is understood to be exclusive to synthetic (in vitro) mimetics and mimetics produced using recombinant means. This term is understood to be exclusive to protein mimetics.

[0204] In one embodiment, the mimetic is selected from afibody, aptamer, affin, afimer, afitin, antikalin, avimer, alphabody, monobody, DARPin, aptamer, Fyomer, fibronectin type III-derived protein scaffold, phytocystatin-derived protein scaffold, and paratope mimetic peptide. Similar to antibodies, the mimetic can also be used as a targeting site.

[0205] In one embodiment, the mimetic is derived from one of the following protein scaffolds: the z domain of protein A, γ-B crystallin, ubiquitin, cystatin, sac7d, triple helix, coiled coil, lipocalin, cyclotide, the A domain of membrane receptors, ankyrin repeat motif, the sh3 domain of Fym, the Kunits domian of protease inhibitors, the type III domain of fibronectin, the IgG-like form of Clostridium perfringens, or one of the thermostable carbohydrate-binding module family 32 (CBM32).

[0206] In one embodiment, the mimetic range is approximately 3kDa to approximately 20kDa, or approximately 4kDa to approximately 18kDa, or approximately 6kDa to approximately 16kDa, or approximately 6kDa to approximately 14kDa, or approximately 6kDa to approximately 12kDa, or approximately 6kDa to approximately 10kDa, or approximately 6kDa to approximately 8kDa. In one embodiment, the mimetic range is approximately 3kDa to approximately 20kDa. In one embodiment, the mimetic range is approximately 3kDa to approximately 20kDa. In one embodiment, the mimetic range is approximately 4kDa to approximately 18kDa. In one embodiment, the mimetic range is approximately 6kDa to approximately 16kDa. In one embodiment, the mimetic range is approximately 6kDa to approximately 14kDa. In one embodiment, the mimetic range is approximately 6kDa to approximately 12kDa. In one embodiment, the mimetic value is approximately 6 kDa to approximately 10 kDa. In another embodiment, the mimetic value is approximately 6 kDa to approximately 8 kDa.

[0207] In some embodiments, the targeting agent is an affibody. Hereinafter, the term “affibody” refers to any of the classes of very small (approximately 6 kDa) polypeptide antibody mimetics based on a domain of three α-helix bundles, approximately 58 amino acids in length, known as the “Z domain.” Typically, the scaffold of an affibody is based on a modified B domain of protein A. Affibodies are characterized by extremely high stability (tolerant to high temperatures of 90°C and possessing target affinity ranging from nanomolar to picomolar). See, for example, Nord et al. (1995), Protein Eng., 8:601-608. Examples of known affibodies include, for example, an affibody against HER2 (e.g., Antibody-HER2 Antibody®, AFFIBODY AB, Bromma, Sweden; U.S. Patent No. 7,993,650).

[0208] In some embodiments, the targeting agent is an affibody having a molecular weight in the range of about 3 kDa to about 10 kDa, or about 3 kDa to about 8 kDa, or about 4 kDa to about 8 kDa, or about 4 kDa to about 7 kDa, or about 5 kDa to about 7 kDa, or about 6 kDa.

[0209] In some embodiments, the targeting agent is an affibody having fewer than 80 amino acid residues, or fewer than 70 amino acid residues, or fewer than 65 amino acid residues, or fewer than 60 amino acid residues. In some embodiments, the targeting agent consists of 40 to 80 amino acid residues, or 50 to 70 amino acid residues, or 55 to 65 amino acid residues, or 56 to 60 amino acid residues, or about 58 amino acid residues.

[0210] For the purposes of this disclosure, the term “antibody” includes four-chain proteins, such as two light chains and two heavy chains, including recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, primate-like antibodies, deimmunized antibodies, and half-antibodies, bispecific antibodies) that can specifically bind to one or more closely related antigens by Fv. Antibodies generally include a constant domain, which can be arranged into a constant region and a constant fragment, a crystallizable fragment (Fc). Exemplary forms of antibodies include a tetrachain structure as a basic unit. Full-length antibodies include two covalently linked heavy chains (about 50-70 kDa) and two light chains (about 23 kDa each). Light chains generally include a variable region and a constant domain, which in mammals are either κ-light chains or λ-light chains. Heavy chains generally include a variable region and one or two constant domains, which are linked to additional constant domains by a hinge region. Mammalian heavy chains are one of the following types: α, δ, ε, γ, or μ. Each light chain is covalently linked to one of the heavy chains. For example, two heavy chains, and heavy and light chains, are held together by interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds may vary depending on the type of antibody. Each chain has an N-terminal variable region (VH or VL, approximately 110 amino acids long each) and one or more constant domains at the C-terminus. The constant domain of the light chain (CL: approximately 110 amino acids long) is aligned with and disulfide-bonded to the first constant domain of the heavy chain (CH: approximately 330-440 amino acids long). The variable region of the light chain is aligned with the variable region of the heavy chain. Antibody heavy chains can contain two or more additional CH domains (e.g., CH2, CH3, etc.) and may include a hinge region between the CH1 and CH2 constant domains. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. For example, an antibody may be a human antibody, its immunized form, germline form, or affinity mature form.

[0211] The terms "full-length antibody" or "whole antibody" are used synonymously to refer to an antibody in its substantially intact form, as opposed to an antigen-binding fragment. Specifically, whole antibodies include antibodies that have heavy and light chains, such as a constant region. The constant region may be the wild-type sequence constant region (e.g., the human wild-type sequence constant region) or an amino acid sequence variant thereof.

[0212] As used herein, the term “variable region” refers to a portion of the light chain and / or heavy chain of an antibody as defined herein, or a portion of an antibody consisting only of the heavy chain that can specifically bind to an antigen (e.g., an antibody from a camelid family or an immunoglobulin neoantigen receptor (IgNAR) from a cartilaginous fish), and includes the amino acid sequences of the complementary determining region “CDR”; i.e., CDR1, CDR2, and CDR3, and the framework region “FR”. FR is a variable region residue other than the CDR residues. For example, the variable region includes three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) along with the three CDRs. H This refers to the variable region of the heavy chain. L This refers to the variable region of the light chain.

[0213] As used herein, the term “complementarity-determining region” (synonym CDR, i.e., CDR1, CDR2, and CDR3) refers to amino acid residues in the variable region of an antibody, whose presence is a major contributor to specific antigen binding. Each variable region typically has three CDR regions, which are identified as CDR1, CDR2, and CDR3. Each complementarity-determining region may contain amino acid residues of a “complementarity-determining region” as defined by Kabat et al., (1987 and / or 1991). For example, in the heavy chain variable region, CDRH1 is located at residues 31–35, CDRH2 at residues 50–65, and CDRH3 at residues 95–102. In the light chain, CDRL1 is located at residues 24–34, CDRL2 at residues 50–56, and CDRL3 at residues 89–97. These CDRs may also include numerous inserts, as described, for example, in Kabat (1987 and / or 1991). This disclosure covers FRs and CDRs defined by all numbering systems, including, but not limited to, the Kabat numbering system, canonical numbering systems, or the numbering systems of Chothia and Lesk (1987), Chothia et al. (1989), and / or Al-Lazikani et al., (1997), Honnegher and Plukthun (2001), the IMGT system discussed in Giudicelli et al., (1997), or the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html). In one example, CDRs and / or FRs are defined according to the Kabat numbering system, as shown in bold in Figures 9A–9D. Optionally, the heavy chain CDR2, as assigned by the Kabat numbering system, may not contain any of the five C-terminal amino acids described herein, or one or more of those amino acids may be substituted with other naturally occurring amino acids. Additionally or alternatively, the light chain CDR1 may not contain any of the four N-terminal amino acids described herein, or one or more of those amino acids may be substituted with other naturally occurring amino acids.In this regard, Padlan et al. established in 1995 that the five C-terminal amino acids of the heavy chain CDR2 and / or the four N-terminal amino acids of the light chain CDR1 are generally not involved in antigen binding. For example, CDRs and / or FRs are defined according to Chothia's numbering system, as depicted in the underlined text in Figures 9A-9D, for example.

[0214] As used herein, the term “Kabat numbering system” refers to the scheme described by Kabat et al. (1987 and / or 1991) for numbering the variable regions of antibodies and identifying the CDR (hypervariable region).

[0215] As used herein, the term “Chothia numbering system” refers to the scheme described in Chothia and Lesk (1987) or Al-Lazikani et al. (1997) for numbering the variable region of an antibody and identifying the CDR (structural loop).

[0216] As used herein, the term “antigen-binding domain” means a region of a targeting agent that can specifically bind to an antigen (e.g., HER2).

[0217] As used herein, the terms “bind” or “binding” in relation to the interaction between a protein or its antigen-binding domain and an antigen mean that the interaction depends on the presence of a specific structure on the antigen (e.g., an antigenic determinant or epitope). For example, an antibody recognizes and binds to a specific protein structure, rather than a general protein. If an antibody is bound to epitope “A”, the presence of a molecule containing epitope “A” (or free, unlabeled “A”) and the antibody in a reaction involving labeled “A” reduces the amount of labeled “A” that binds to the antibody.

[0218] When used herein, “specifically bind,” “bind specifically,” or similar phrases mean that the proteins of this disclosure react to or associate with a particular antigen (such as HER2) or a cell expressing it more frequently, more rapidly, more persistently, and / or with higher affinity than alternative antigens or cells. For example, a protein that specifically binds to one antigen will bind to that antigen with higher affinity (e.g., 20, 40, 60, 80, or 100, or 150, or 200 times or more affinity), avidity, ease, and / or longer persistence than a protein that binds to another antigen. Also, by reading this definition, it will be understood that, for example, a protein that specifically binds to a first antigen may or may not specifically bind to a second antigen. Since such “specific binding” does not necessarily require exclusive or undetectable binding to another antigen, this is meant by the term “selective binding.”

[0219] In some embodiments, the targeting agent comprises or consists of an amino acid sequence corresponding to the targeting agent amino acid sequence defined herein.

[0220] In some embodiments, the targeting agent is, for example, an oligomeric peptide sequence with a length of up to 20 amino acids, or includes such a sequence. In some embodiments, the targeting agent is a peptide sequence of 5 to 20 amino acids, 7 to 18 amino acids, or 9 to 15 amino acids. In some embodiments, the targeting agent is a peptide sequence of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids.

[0221] In some embodiments, the targeting agent has a molecular weight of less than 2000 Da, less than 1000 Da, or less than 500 Da. In some embodiments, the targeting agent is a small molecule that may be considered to have a molecular weight of less than 1000 Da, less than 750 Da, or less than 500 Da.

[0222] In one example, the targeting agent is a small molecule that binds to PSMA. In one embodiment, the small molecule that binds to PSMA may be a peptide. Such binding peptides are known in the art. In another example, the targeting agent is a small molecule that binds to fibroblast-activating protein (FAP).

[0223] In some embodiments, the targeting agent is a targeting agent that specifically binds to prostate-specific membrane antigen (PSMA). For example, the targeting agent may be a DUPA group, an analogue thereof, or may contain one. DUPA has the following structure: [ka]

[0224] In some embodiments, the targeting agent is a DUPA group conjugated via a carboxyl group, for example, the following, or contains the following: [ka]

[0225] In some embodiments, the targeting agent is a targeting agent that specifically binds to fibroblast-activating protein (FAP). In some embodiments, the targeting agent may be or contain a group that inhibits fibroblast-activating protein (FAP). For example, the targeting gent may be or contain an FAP-binding group having the following structure: [ka] In the formula, R is a substituent of formula I, [ka] Equation I During the ceremony, X is selected from O, NH, N(CH3), S, and CH2. Y is selected from N and C. n is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6. A is a monocyclic or bicyclic heterocyclic group with 5 to 10 members. R 1 This is selected from the group consisting of C1-C6 alkyl groups substituted with one or more 5-10 membered cyclic groups by arbitrary selection. [ka] This represents the conjugation point to the dendrimer.

[0226] In some embodiments, R is a substituent of formula I, where X is O. In some embodiments, R is a substituent of formula I, where X is NH. In some embodiments, R is a substituent of formula I, where X is N(CH3). In some embodiments, R is a substituent of formula I, where X is S. In some embodiments, R is a substituent of formula I, where X is CH2.

[0227] In some embodiments, R is a substituent of formula I, where Y is N. In some embodiments, R is a substituent of formula I, where Y is C. In these embodiments where Y is C, it will be understood that A may be an aromatic or saturated 5- to 10-membered monocyclic or bicyclic heterocyclic group. In some embodiments, R is a substituent of formula I, where Y is CH. In these embodiments where Y is C, it will be understood that A may be a partially or fully saturated 5- to 10-membered monocyclic or bicyclic heterocyclic group.

[0228] In some embodiments, R is a substituent of formula I, where n is 0. In this embodiment, when n is 0, it will be understood that X is directly bonded to Y. In some embodiments, R is a substituent of formula I, where n is 1. In some embodiments, R is a substituent of formula I, where n is 2. In some embodiments, R is a substituent of formula I, where n is 3. In some embodiments, R is a substituent of formula I, where n is 4. In some embodiments, R is a substituent of formula I, where n is 5. In some embodiments, R is a substituent of formula I, where n is 6.

[0229] In some embodiments, R is a substituent of formula I, where A is a 5- to 10-membered monocyclic or bicyclic heterocyclic group. In one example, A is a 5-membered monocyclic heterocyclic group. In another example, A is a 6-membered monocyclic heterocyclic group. In another example, A is a 7-membered monocyclic heterocyclic group. In another example, A is a 7-membered bicyclic heterocyclic group. In another example, A is an 8-membered bicyclic heterocyclic group. In another example, A is a 9-membered bicyclic heterocyclic group. In another example, A is a 10-membered bicyclic heterocyclic group.

[0230] An example of a group A is [ka] These include, but are not limited to, the following:

[0231] In some embodiments, R is a substituent of formula I, where R 1 It is a C1-C6 alkyl group. For example, R 1 is a C1-alkyl group (i.e., CH3). In some embodiments, R is a substituent of formula I, where R 1 is a C1-C6 alkyl group optionally substituted with one or more 5-10 membered cyclic groups. In some embodiments, R 1 R is a C1-alkyl group substituted with a 6-membered cyclic group. For example, R 1 It is CH2-phenyl.

[0232] In one example, R is a substituent in the following formula I. [ka]

[0233] In one example, R is a substituent in the following formula I. [ka]

[0234] Further examples of R substituents include those published by Loktev et al. (Loktev, A. et al., The Journal of Nuclear Medicine, 60(10), 2019, pp. 1421-1429).

[0235] Therefore, in one embodiment, the targeting agent is an FAP binding group having the following structure. [ka] And, In the formula, X, Y, n, A, and R 1 This is as described in this specification.

[0236] In one example, the targeting agent is an FAP binding group having the following structure. [ka]

[0237] In one example, the targeting agent is an FAP binding group having the following structure. [ka]

[0238] In some embodiments, the targeting agent is selective for one or more of HER2, EGFR, PSMA, or FAP. In one embodiment, the targeting agent is selective for HER2. In one embodiment, the targeting agent is selective for EGFR. In one embodiment, the targeting agent is selective for PSMA. In one embodiment, the targeting agent is selective for FAP.

[0239] In some embodiments, the targeting agent is competitive with other targeting agents, such as commercially available ones. In one example, the targeting agent is competitive with commercially available antibody therapies. In another example, the targeting agent is selective for HER2 and competitive with HER2 antibodies, such as trastuzumab, pertuzumab, and margetuximab. In yet another example, the targeting agent is selective for EGFR and competitive with EGFR antibodies, such as cetuximab, panitumumab, nimotuzumab, and nesitumumab. In yet another example, the targeting agent is selective for FAP and competitive with FAP antibodies, such as cibrotuzumab.

[0240] In some embodiments, the conjugate comprises a single targeting agent. In other embodiments, the conjugate comprises multiple targeting agents, for example, two, three, four, or five targeting agents. In some embodiments, the conjugate comprises 1 to 32 targeting agents. In some embodiments, the conjugate comprises at least five targeting agents. In some embodiments, the conjugate comprises 5 to 30 targeting agents.

[0241] The targeting agent is attached to the remainder of the conjugate via a spacer. In some embodiments, the covalent attachment or linkage between the targeting agent and the spacer group is formed by a reaction between complementary reactive functional groups present on the intermediate containing the targeting agent and the intermediate containing the dendrimer.

[0242] In some embodiments, the targeting agent is covalently bonded to a spacer group at its C-terminus.

[0243] In one embodiment, the FAP binding group is conjugated to the dendrimer via a spacer group as described herein. Thus, in one embodiment, the targeting agent is an FAP binding group conjugated to the dendrimer via a spacer group containing polyethylene glycol (PEG). In one embodiment, the targeting agent is an FAP binding group conjugated to the dendrimer via a spacer group containing polyethylene glycol (PEG) having the following structure: [ka] , In the formula, the spacer group is conjugated to the dendrimer via the terminal carboxylic acid group of the spacer.

[0244] Covalent attachment sites for attaching the targeting agent to the spacer and therefore to the dendrimer may be, for example, cysteine, lysine, N-terminal amine, tyrosine, carbohydrate, unnatural amino acid or transaminase, or a recognition sequence. Binding sites for covalent attachment to proteins are known in the art (e.g., Milla P., et al, Current Drug Metabolism (2012) V13, 1:105-119). In some embodiments, the intermediate containing the targeting agent includes an unnatural amino acid residue for attachment to the spacer. The unnatural amino acid residue may be located on the spacer group or on the intermediate containing the dendrimer to which the spacer group is attached, and may have a side chain having a reactive functional group complementary to the reactive functional group. In some embodiments, the unnatural amino acid residue contains an azide group, for example, a 4-azidophenylalanine residue, for example, [ka] This may also be the case. The azide group can undergo cycloaddition reactions with alkyne groups that may be present in the spacer precursor group. In some embodiments, the non-natural amino acid is a diene-containing amino acid, such as a spirocyclopentadiene-containing amino acid, including the following: [ka] The diene group can undergo cycloaddition reactions with alkene groups, such as those found in maleimides (e.g., Diels-Alder reactions). Further examples of unnatural amino acids that can be used to attach to spacers include those containing carbonyl groups, such as ketones, and those containing methylcyclopropylene groups. Additional examples of unnatural amino acids include: [ka] These are some examples.

[0245] In some embodiments, the targeting agent comprises or consists of any of the amino acid sequences defined herein.

[0246] Spacer base As used herein, the term “spacer group” refers to a chemical entity that plays a role in attaching a targeting agent to a dendrimer; that is, the spacer group conjugates the targeting agent to the dendrimer. In some embodiments, the spacer may simply be an atom or small chemical linking group to which the targeting agent binds to the dendrimer. In other embodiments, the spacer group may be broader. In some embodiments, the spacer group is intended to position the targeting agent so that it can bind, for example, to a HER2 receptor without being excessively adversely affected by other components of the dendrimer.

[0247] In some embodiments, the targeting agent is attached to the dendrimer via the dendrimer's core. That is, the targeting agent, such as an antibody fragment, is covalently attached to the dendrimer's core by a spacer group. Attaching the targeting agent to the dendrimer via a spacer attached to the dendrimer's core can be beneficial when the dendrimers are sterically densely packed, and the spacer group can be long enough to protrude beyond the dendrimer's surface, thereby enabling the targeting agent to bind to its receptor in vivo. For example, if the core is a maleimide-containing core, the targeting agent may attach to the maleimide ring nitrogen, in which case the spacer group would be beneficial.

[0248] In some other embodiments, the targeting agent is attached to the dendrimer via the surface constituent units of the dendrimer. For example, the targeting agent may be linked to the surface nitrogen of the lysine residue via a spacer group, such as by an amide bond formed between the amine group of the lysine residue and the carboxylic acid group present on the intermediate containing the targeting agent and the spacer group.

[0249] Any suitable chemical group may be used that plays a role in moving the targeting agent away from the dendrimer at an appropriate distance so that the dendrimer can bind to its target. Exemplary spacer groups include polyethylene glycol (PEG), polypropylene glycol, polyaryl, amide linkages, peptides, amino acids, alkyloxy, alkylamino, alkyl and alkenyl chains, and sugars (mono, oligo, and poly), or residues thereof. In some embodiments, the spacer group comprises one or more PEG groups, such as 2 to 60 ethyleneoxy repeating units, e.g., 2 to 20 or 20 to 48 repeating units. In one embodiment, the PEG is 8 to 36 repeating units. In further embodiments, the PEG is 12, 16, 20, 24, or 36 repeating units.

[0250] In some embodiments, the spacer group comprises multiple PEG groups sandwiched between other functional groups. For example, the spacer group may comprise PEG groups linked via, for example, an amide group or other functional groups useful for connecting portions of the spacer group.

[0251] Any suitable means may be used to attach the intermediate containing the spacer group to the intermediate containing the targeting agent. For example, sites for covalent attachment include, but are not limited to, cysteine ​​residues, lysine residues, C-terminal amino acid residues, N-terminal amines, tyrosine residues, carbohydrates, suitable non-natural amino acid residues, or transaminases, or recognition sequences. Binding sites for covalent attachment of targeting agents to proteins are known in the art (e.g., Milla P., et al., 2012). For example, the intermediate containing the spacer group may react with the intermediate containing the targeting agent at the C-terminus of the targeting agent such that the spacer group is attached to the targeting agent via its C-terminus. In some embodiments, the targeting agent is attached to the spacer group via its C-terminus. In one embodiment, the targeting agent is covalently attached to or linked to the spacer group via its C-terminus.

[0252] Therefore, in order to attach the spacer group to the targeting agent and / or dendrimer, the precursor containing the spacer group may contain one or more reactive functional groups.

[0253] In certain embodiments, the reactive functional group may be a complementary reactive group selected from the group consisting of active esters such as hydroxy, carboxy, NHS, or pentafluorophenol esters; alkyne-containing groups such as amino, azide, maleimide (sulfomaleimide, etc.), diene (cyclopentadiene, e.g., spiro[2.4]hepta-4,6-diene group); alkyne-containing groups such as tetrazine, citracomide, BCN (bisicle[6.1.0]non-4-in-9-yl), DBCO (dibenzocyclooctin-amine); carbonyl groups such as thiol, aldehyde, and ketone; alkoxyamines, haloacetates, biotin, tetrazine, TCO (trans-cyclooctene); methyl-cyclopropylene groups; and PTAD or other tyrosine-reactive groups.

[0254] For example, in some embodiments, the spacer group intermediate may contain two reactive groups (e.g., one at each orthogonal end). That is, at least one of the reactive groups can react with a complementary group present on either the intermediate containing the targeting agent or the intermediate containing the dendrimer, under conditions where the other reactive group is stable and substantially unreactive, to attach the spacer group to the components of the conjugate. This allows the spacer to be covalently attached to either the dendrimer or the targeting agent, and then the other reactive groups can react with complementary groups present on the remaining components to link the targeting agent to the dendrimer.

[0255] In some embodiments, the spacer group is directly or indirectly attached to the targeting agent by the reaction of precursors containing alkyne and azide groups, respectively (for example, the intermediate containing the targeting group may contain an azide group, and the intermediate containing the spacer group may contain an alkyne group). Such reactions result in the formation of triazole-containing groups, for example, the following: [ka] It can be formed by the reaction of a precursor having the following structure. [ka]

[0256] As another example, a spacer group may be attached via the formation of a triazole-containing group, for example, as follows: [ka] It can be formed by the reaction of a precursor having the following structure. [ka]

[0257] In some embodiments, the spacer group is attached to the targeting agent by the reaction of a precursor containing an alkene (e.g., a strained alkene such as trans-cyclooctene) and a tetrazine group, respectively. Such a reaction results in the formation of a pyridazine-containing group having, for example, the following nitrogen protrusions, [ka] It can be formed by the reaction of a precursor having the following structure. [ka]

[0258] In some embodiments, the spacer group is attached to the dendrimer by the reaction of a precursor containing a carboxylic acid group and an amine group, for example, the spacer group intermediate may contain a carboxylic acid group that is present as part of the core unit or can react with an amine group extending from the core unit to form, for example, an amide linkage.

[0259] In some embodiments, spacer groups are attached to the dendrimer by the reaction of precursors containing carboxylic acid groups and amine groups. For example, the spacer group intermediate may contain a carboxylic acid group that exists as part of the core unit or reacts with an amine group extending from the core unit to form, for example, an amide linkage, and is attached to the targeting agent by the reaction of precursors containing alkyne groups and azide groups, respectively (for example, the intermediate containing the target group may contain an azide group, and the intermediate containing the spacer group may contain an alkyne group).

[0260] As described above, the precursor containing the targeting agent may contain a non-natural amino acid residue. This non-natural amino acid residue may be, for example, any non-natural amino acid capable of exhibiting a reactive side chain, the reactive side chain holding a functional group complementary to a functional group present on the spacer group intermediate. In this way, the complementary functional groups react, resulting in the attachment of the targeting moiety to the spacer group. In some embodiments, the non-natural amino acid residue is a 4-azidophenylalanine residue. In some embodiments, the spacer group-containing intermediate contains an alkyne group for conjugation to an intermediate containing a targeting moiety containing an azido group-containing non-natural amino acid residue. In some embodiments, the spacer group-containing intermediate contains an alkyne group, which is a dibenzylcyclooctin-amine (DBCO) group, for conjugation to an intermediate containing a targeting moiety containing a 4-azidophenylalanine residue.

[0261] In some embodiments, one end of the spacer group forms part of the DBCO group and the targeted portion (for example, below: [ka] By cycloaddition with the azide moiety on a 4-phenylalanine residue (which forms a triazole-containing group like the one shown below), or by a BCN((bisicle 6.1.0]non-4-in-9-yl) group which forms part of the targeting moiety (for example, the following: [ka] It attaches to the target site by reacting with the azide moiety on the 4-phenylalanine residue (which forms a triazole-containing group like the one shown).

[0262] In some embodiments, one end of the spacer group is attached to the dendrimer by amidation reactions (e.g., by the reaction of an activated ester) between amino groups present on the core or on the surface constituent unit, and between carboxyl groups present on the spacer group. In some embodiments, the intermediate containing the spacer group contains a tetrazine group. In some embodiments, the intermediate containing the spacer group contains a maleimide group for conjugation to a diene (cyclopentadiene, e.g., spiro[2,4]hepta-4,6-diene group).

[0263] In some embodiments, the spacer group intermediate comprises a PEG group and a carboxyl group for reacting with an amine that forms part of or extends from the core of the dendrimer, and an alkyne group for reacting with an azide group present in the intermediate containing the targeting moiety. In some embodiments, the spacer group intermediate comprises a PEG chain having a reactive carboxyl group for bonding to an amine in the core of the dendrimer, and an azide group for conjugating to a targeting agent intermediate containing a reactive alkyne moiety. In some embodiments, the spacer group intermediate comprises a PEG chain having a reactive amine group for bonding to a carboxyl group in the core of the dendrimer, and an azide group for conjugating to a targeting agent intermediate containing a reactive alkyne moiety. In some embodiments, the spacer group intermediate comprises a PEG chain having a reactive carboxyl group for bonding to an amine in the core of the dendrimer, and a maleimide group for conjugating to a targeting agent intermediate containing a reactive thiol moiety. In some embodiments, the spacer group intermediate comprises a PEG chain having a reactive amine group for bonding to a carboxyl group at the core of the dendrimer, and a thiol or masked thiol group for conjugating to a targeting agent intermediate containing a reactive maleimide moiety. In some embodiments, the spacer group intermediate contains a PEG chain having a reactive carboxyl group for bonding to an amine at the core of the dendrimer, and a tetrazine group for conjugating to a targeting agent intermediate containing a reactive alkene moiety. In some embodiments, the spacer group intermediate comprises a PEG chain having a reactive carboxyl group for bonding to an amine at the core of the dendrimer, and a maleimide group for conjugating to a targeting agent intermediate containing a reactive diene (cyclopentadiene, e.g., spiro[2,4]hepta-4,6-diene group).

[0264] In some embodiments, linking of a targeting agent to a dendrimer can be achieved by attaching a first spacer group to a targeting agent intermediate and a second spacer group to the dendrimer (e.g., to the core of the dendrimer), and then reacting complementary functional groups present on the first and second spacer groups together to link the targeting agent and the dendrimer. Such an approach may be provided to facilitate the connection between the dendrimer and the targeting agent. For example, the first spacer group intermediate may include a first reactive group at one end that is complementary to a reactive group on the targeting agent (e.g., an alkyne group complementary to an azide group, which can react together to form a triazole group) and a second reactive group complementary to a reactive group on the second spacer group (e.g., a tetrazine-containing group complementary to the reaction with a trans-cyclooctene-containing group). The second spacer group intermediate may, for example, contain a third reactive group at one end that is complementary to the reaction with the reactive group on the dendrimer (e.g., a carboxylic acid group complementary to the reaction with an amine group) and a fourth reactive group at the other end that is complementary to the reaction with the reactive group on the first spacer group intermediate (e.g., a trans-cyclooctene-containing group complementary to the reaction with a tetrazine group). For example, the first group and the spacer group may be attached via a group produced by reacting a trans-cyclooctene group with a tetrazine group, and may include the following structure. [ka]

[0265] In some embodiments, the targeted portion may be linked to the dendrimer via a spacer group formed by the reaction of an azide portion present on the targeting agent with an alkyne-containing group (e.g., DBCO, BCN) at one end of a spacer group, and the reaction of a tetrazine portion attached to the dendrimer with a strained alkene group at the other end of a spacer group (e.g., transcyclooctene).

[0266] Third terminal group In some embodiments, the dendrimer comprises one or more third terminal groups (T3) attached to the outermost constituent unit, the third terminal group comprising a pharmaceutically active agent residue that is not a radionuclide-containing portion. If the constituent unit is a lysine residue or an analogue thereof, the third terminal group may be attached, for example, to the nitrogen atom of the outermost constituent unit. Incorporating a pharmaceutically active agent into the dendrimer improves the therapeutic properties and allows the same dendrimer agent to be used for both diagnostic / therapeutic imaging and disease therapy. For example, in a subject suspected of having cancer or diagnosed with cancer, the dendrimer of the Disclosure may be administered first, and imaging of the relevant part of the subject's body may be performed to diagnose the patient's condition by imaging and / or, if cancer is present, to determine the potential susceptibility of the cancer to a course of dendrimer therapy. If the tumor is likely to be susceptible to dendrimer therapy, further courses of the same dendrimer or another dendrimer of the Disclosure (e.g., containing a different radionuclide) may then be administered, for example, to the subject.

[0267] Pharmaceutical activators Any suitable pharmaceutically active agent may be conjugated to the dendrimer as a third terminal group, for example, via a linking group. In some embodiments, the pharmaceutically active agent is an anticancer agent. Examples of anticancer agents, but not limited to, include hypercytotoxic agents, taxanes, and topoisomerase inhibitors. In some embodiments, the anticancer agent is a hypercytotoxic agent. In some embodiments, the anticancer agent is auristatin. In some embodiments, the anticancer agent is a meitansinoid. In some embodiments, the anticancer agent is a taxane. In some embodiments, the anticancer agent is a topoisomerase inhibitor.

[0268] As used herein, "supercytotoxic agent" refers to an agent that exhibits very potent chemotherapeutic properties but is so highly toxic that it cannot be administered alone as an anticancer agent. In other words, supercytotoxic agents exhibit chemotherapeutic properties, but the harmful and toxic side effects outweigh the benefits of chemotherapy, and therefore they generally cannot be safely administered to the target. In some embodiments, supercytotoxic agents are used in in vitro IC against cancer cell lines (e.g., SKBR3 cells and / or HEK293 cells and / or MCF7 cells). 50 However, the concentration is less than 100 nM, or less than 10 nM, or less than 5 nM, or less than 3 nM, or less than 2 nM, or less than 1 nM, or less than 0.5 nM. Examples of hypercytotoxic agents include, in particular, dorastatins (e.g., dorastatin-10, dorastatin-15), auristatins (e.g., monomethyl auristatin-E, monomethyl auristatin-F), meitansinoids (e.g., meitansin, meltansin / emtansin (DM1, labtansin (DM4))), calichiamicins (e.g., calichiamicin γ1), esperamycins (e.g., esperamycin A1), and pyrrolobenzodiazepines (PDB).

[0269] In some embodiments, the pharmaceutically active agent is auristatin. In some embodiments, the pharmaceutically active agent is monomethyl auristatin. In one embodiment, the pharmaceutically active agent is monomethyl auristatin E (MMAE). In one embodiment, the pharmaceutically active agent is monomethyl auristatin F (MMAF). Both MMAE and MMAF are understood to inhibit cell division by blocking the polymerization of tubulin.

[0270] In some embodiments, the hypercytotoxic agent is a meitansinoid. In one embodiment, the hypercytotoxic agent is meitansin. In one embodiment, the hypercytotoxic agent is ansamitosin. In one embodiment, the hypercytotoxic agent is emtansine / meltansine (DM1). In one embodiment, the hypercytotoxic agent is labtansine (DM4). It is understood that meitansinoids inhibit microtubule assembly by binding to tubulin.

[0271] Examples of taxanes include paclitaxel, cabazitaxel, and docetaxel. In some embodiments, the pharmaceutically active agent is paclitaxel. In some embodiments, the pharmaceutically active agent is cabazitaxel. In some embodiments, the pharmaceutically active agent is docetaxel.

[0272] Examples of topoisomerase inhibitors include, but are not limited to, camptothecin activators. In some embodiments, the pharmaceutically active agent is a camptothecin activator. Examples of camptothecin activators include, but are not limited to, SN-38, irinotecan (CPT-11), topotecan, siratecan, cocitecan, exatecan, lulutotecan, gimatecan, berotecan, and rubitecan. In some embodiments, the pharmaceutically active agent is SN-38. In some embodiments, the pharmaceutically active agent is irinotecan.

[0273] In some embodiments, the pharmaceutically active agent is an anticancer agent selected from the group consisting of cabazitaxel, docetaxel, SN-38, monomethyl auristatin A, and monomethyl auristatin F.

[0274] Linker In some embodiments, the dendrimer includes a third terminal group (T3) containing a residue of a pharmaceutically active agent, and the residue of the pharmaceutically active agent is attached to the outermost constituent unit via a linker. In one embodiment, the linker is a cleavable linker. In another embodiment, the linker is a non-cleavable linker. The linker group can be used to provide a suitable group for attaching the pharmaceutically active agent to the dendrimer, for example, when the available functionality of the pharmaceutically active agent is not suitable for direct attachment to the constituent unit. Alternatively, the linker group can also be used to facilitate the controlled release of the pharmaceutically active agent from the dendrimer scaffold, thereby providing a therapeutically effective concentration and a desirable pharmacokinetic profile of the pharmaceutically active agent for a suitable period (e.g., a long period).

[0275] Those skilled in the art will understand that any one of several suitable linkers may be used. The linker should provide sufficient stability during systemic circulation while, for example, enabling the rapid and efficient release of the active form of a cytotoxic drug at its site of action once internalized in cancer cells.

[0276] In some embodiments, the linker is a cleavable linker comprising one or more ester groups, hydrazone groups, oxime groups, imine groups, or disulfide groups, either by itself or in conjunction with its linkage to a pharmaceutically active agent. In some embodiments, the linker is cleavable in a tumor environment and is unstable to acid, unstable in a reducing environment, unstable to hydrolysis, or unstable to proteases.

[0277] Chemically unstable linkers include, but are not limited to, acid-unstable linkers (i.e., hydrazones) and disulfide linkers. Enzymatically cleavable linkers include, but are not limited to, peptide linkers (e.g., dipeptide linkers such as those containing Val-Cit or Phe-Lys groups) and β-glucuronide linkers. Because lysosomal proteolytic enzymes have very low activity in the blood, peptide linkers and their peptide bonds are expected to have favorable serum stability. Both Val-Cit and Phe-Lys linkers are rapidly hydrolyzed by cathepsin B. In some embodiments, the linker is an enzymatically cleavable linker. For example, in some embodiments, the linker contains amino acid residues that can be recognized and cleaved enzymatically.

[0278] In some embodiments, the linker includes a peptide group. In some embodiments, the linker includes a dipeptide group. In some embodiments, the linker includes, for example, a valine-citrulline-para-aminobenzyl alcohol-containing group (Val-Cit-PAB) having the following structure. [ka]

[0279] For example, the PAB group may be covalently bonded to the amine group present in the therapeutic portion via a carbonyl group to form a carbamate linkage, or it may be bonded to the amine group present in the outer constituent unit via a diacyl linker that forms an amide bond with the valine amino group and the amine group present in the outer constituent unit.

[0280] In some embodiments, the linker comprises, for example, a glutaric acid-valine-citrulline-para-aminobenzyl alcohol group having the following structure: [ka]

[0281] In some embodiments, the pharmaceutically active agent comprises a hydroxyl group, and the residue of the pharmaceutically active agent is attached to the linker via the oxygen atom of the hydroxyl group. In this approach, it is possible to attach to the linker via an ester group, and such an ester group has been found to be cleavable in vivo, releasing the pharmaceutically active agent at a desired rate.

[0282] In some embodiments, the core unit is formed from a core unit precursor containing an amino group, the constituent units are lysine residues or analogs thereof, the pharmaceutically active agent contains a hydroxyl group, the residue of the pharmaceutically active agent is attached via the oxygen atom of the hydroxyl group, and the cleavable linker is a diacyl linker, thereby resulting in an ester linkage between the residue of the pharmaceutically active agent and the linker, and an amide linkage between the linker and the nitrogen atom present on the outermost constituent unit. In some embodiments, the pharmaceutically active agent contains a hydroxyl group, the residue of the pharmaceutically active agent is attached via the oxygen atom of the hydroxyl group, and the cleavable linker is a diacyl linker group of the following formula: [ka] In the formula, A is C2~C interrupted by O, S, SS, NH, or N(Me). 10 A is either an alkylene group, or A is a heterocycle selected from the group consisting of tetrahydrofuran, tetrahydrothiophene, pyrrolidine, and N-methylpyrrolidine.

[0283] As used herein, the term “alkyl” refers to a monovalent linear or branched saturated hydrocarbon group. For example, an alkyl group may have 1 to 10 carbon atoms (i.e., C 1~10 It contains alkyl groups. For example, an alkyl group has 1 to 6 carbon atoms (i.e., C 1~6It includes alkyl groups. Examples of alkyl groups include methyl, ethyl, propyl (e.g., n-propyl, iso-propyl), butyl (e.g., n-butyl, sec-butyl, tert-butyl), pentyl, and hexyl groups.

[0284] As used herein, the term "alkylene" refers to a divalent linear (i.e., linear) or branched saturated hydrocarbon group. For example, an alkylene group has 2 to 10 carbon atoms (i.e., C 2~10 It contains alkylene. For example, an alkylene group has 2 to 6 carbon atoms (i.e., C 2~6 It contains alkylenes. Examples of alkylene groups include -CH2CH2-, -CH2CH2CH2-, -CH2CH(CH3)-, -CH2CH2CH2CH2-, and -CH2CH(CH3)CH2-.

[0285] In some embodiments, the pharmaceutically active agent comprises a hydroxyl group, the residues of the pharmaceutically active agent are attached via the oxygen atom of the hydroxyl group, and the cleavable linker is a diacyl linker group of the following formula: [ka] A is C2~C interrupted by O, S, NH, or N(Me). 10 It is an alkylene group.

[0286] In some embodiments, the pharmaceutically active agent has a hydroxyl group, and the residue of the pharmaceutically active agent is attached via the oxygen atom of the hydroxyl group, and the diacyl linker is [ka] That is the case.

[0287] Certain types of cleavable linkers include a disulfide moiety. Such linkers are susceptible to cleavage by glutathione. For example, this type of linker may contain two acyl groups linked via an alkyl chain interrupted by the disulfide moiety.

[0288] In some embodiments, the linker comprises an alkyl chain interrupted by a disulfide moiety, where one or both carbon atoms adjacent to the disulfide group are substituted with one or more methyl groups. For example, one of the carbon atoms adjacent to the disulfide moiety may be substituted with a gem-dimethyl group, and the linker may contain that group, for example. [ka]

[0289] A non-cleavable linker is a linking group that is inert to cleavage, or substantially inert to cleavage, even when exposed to in vivo conditions for the required period. Non-cleavable linkers are not cleaved under biological conditions.

[0290] For example, alkylene groups or cycloalkylene groups (e.g., C 1~10 Alkylene group or C 3-10 Examples include diacyl linkers crosslinked by cycloalkylene groups. Further examples of non-cleavable linkers include thioether linkers. A specific example of a non-cleavable linker is one formed using SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). SMCC can be used to form thioether links by reacting the maleimide functionality with thiol groups present in or attached to the therapeutic portion. The carboxylic acid functionality can be used to react with amino groups present on the outer constituent units.

[0291] composition In some embodiments, the conjugate is presented as a composition, preferably a pharmaceutical composition. Thus, compositions comprising multiple conjugates as defined herein are also provided.

[0292] The composition or pharmaceutical composition may comprise a plurality of dendrimer-targeting agent conjugates and / or dendrimer-targeting agent therapeutic conjugates. If the composition or pharmaceutical composition comprises only dendrimer-targeting agent conjugates, the composition may be exposed to a suitable radionuclide before administration for radiotherapy, imaging, or similar purposes to form dendrimer-targeting agent therapeutic conjugates.

[0293] It will be understood that, as a result of the nature of the synthetic process for generating the conjugates, there may be some variation in the molecular composition of the conjugates present in a given composition. For example, as described above, one or more synthetic steps used to generate the conjugates may not proceed completely to completion, and as a result, there may be conjugates that do not all contain the same number of targeting agents, first terminal groups, second terminal groups, or third terminal groups, or that contain incomplete generations of constituent units in the dendrimer components of the conjugates.

[0294] In some embodiments of a composition comprising a conjugate containing three generations of constituent units, the average number of targeting agents per conjugate is about 1. In some embodiments of a composition comprising a conjugate containing three generations of constituent units, the average number of first terminal groups per conjugate in the composition is in the range of 1 to 4. In some embodiments of a composition comprising a conjugate containing three generations of constituent units, the average number of second terminal groups per conjugate in the composition is in the range of 4 to 7. In some embodiments of a composition comprising a conjugate containing three generations of constituent units as defined herein, the average number of targeting agents per conjugate is about 1, the average number of first terminal groups per conjugate in the composition is in the range of 1 to 4, and the average number of second terminal groups per conjugate in the composition is in the range of 4 to 7.

[0295] In some embodiments of a composition comprising a conjugate containing four generations of constituent units, the average number of targeting agents per conjugate is about 1. In some embodiments of a composition comprising a conjugate containing four generations of constituent units, the average number of first terminal groups per conjugate in the composition is in the range of 1 to 4. In some embodiments of a composition comprising a conjugate containing four generations of constituent units, the average number of second terminal groups per conjugate in the composition is in the range of 4 to 7. In some embodiments of a composition comprising a conjugate containing four generations of constituent units as defined herein, the average number of targeting agents per conjugate is about 1, the average number of first terminal groups per conjugate in the composition is in the range of 1 to 4, and the average number of second terminal groups per conjugate in the composition is in the range of 4 to 7.

[0296] In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the conjugate in the composition contains a targeting agent.

[0297] In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the conjugate in the composition contains the first terminal group.

[0298] In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the conjugate in the composition contains a second terminal group.

[0299] In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the conjugate in the composition contains a third terminal group.

[0300] This disclosure also provides pharmaceutical formulations or compositions for both veterinary and human medical applications, which comprise the conjugate of this disclosure or a pharmaceutically acceptable salt thereof together with one or more pharmaceutically acceptable carriers and optionally any other therapeutic components, stabilizers, etc. Thus, in some embodiments, the composition is a pharmaceutical composition comprising the conjugate as defined herein and a pharmaceutically acceptable excipient.

[0301] Excipients and / or carriers must be pharmaceutically acceptable in the sense that they are compatible with the other components of the formulation and are not excessively harmful to its recipient. The compositions of this disclosure may also include polymeric excipients / additives or carriers, e.g., polyvinylpyrrolidone, derivatized cellulose, e.g., hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficol (polymeric sugars), hydroxyethyl starch (HES), dextrates (e.g., cyclodextrins (e.g., 2-hydroxypropyl-β-cyclodextrin and sulfobutyl ether-β-cyclodextrin, etc.)), polyethylene glycol, and pectin. The compositions may further include diluents, buffers, citrates, trehaloses Examples of excipients and additives suitable for use in compositions according to this disclosure include: cereals, binders, disintegrants, thickeners, lubricants, preservatives (such as antioxidants), inorganic salts (e.g., sodium chloride), antimicrobial agents (e.g., benzalkonium chloride), sweeteners, antistatic agents, sorbitan esters, lipids (phospholipids such as lecithin, and other phosphatidylcholine, phosphatidylethanolamine, fatty acids, and fatty acid esters, steroids (e.g., cholesterol)), and chelating agents (e.g., EDTA, zinc, and other suitable cations). Other pharmaceutical excipients and / or additives suitable for use in compositions according to this disclosure are described in “Remington: The They are listed in "Science & Practice of Pharmacy," 19th sup.th ed., Williams & Williams, (1995), "Physician's Desk Reference," 52nd sup.nd ed., Medical Economics, Montvale, NJ (1998), and "Handbook of Pharmaceutical Excipients," Third Ed., Ed. AH Kibbe, Pharmaceutical Press, 2000.

[0302] The conjugates of this disclosure can be formulated into compositions suitable for administration by any preferred route, such as parenteral administration (including intraperitoneal, intravenous, subcutaneous, or intramuscular injection).

[0303] The compositions may be provided in convenient unit dosage forms or prepared by any method well known in the field of pharmaceuticals. All methods involve the step of associating a dendrimer with a carrier constituting one or more accessory components. Generally, compositions are prepared by associating a dendrimer with a liquid carrier to form a solution or suspension, or by associating a dendrimer with a solid, optionally particulate product suitable for forming a particulate product, and, where appropriate, shaping the product into a desired delivery form. When the solid dosage forms of the present disclosure are micronized, they typically contain particles in the size range of about 1 nanometer to about 500 microns. Generally, for solid dosage forms intended for intravenous administration, the particles are in the size range of about 1 nm to about 10 microns in diameter. The compositions may contain the dendrimers of the present disclosure which are nanoparticles having particle sizes of 1000 nm or less, for example, 5 to 1000 nm, particularly 5 to 500 nm, more particularly 5 to 400 nm, for example 5 to 50 nm, particularly 5 to 20 nm. In one example, the composition contains dendrimers with an average size of 5 to 20 nm. In some embodiments, the dendrimer is polydisperse in the composition, and the PDI is 1.01–1.8, particularly 1.01–1.5, and more particularly 1.01–1.2. In one example, the dendrimer is monodisperse in the composition.

[0304] In some preferred embodiments, the composition is formulated for parenteral delivery. For example, in one embodiment, the formulation may be a sterile lyophilized composition that is suitable for reconstitution with an aqueous vehicle before injection.

[0305] In one embodiment, a formulation suitable for parenteral administration conveniently comprises a sterile aqueous preparation of a dendrimer, which may be formulated, for example, to be isotonic with the recipient's blood.

[0306] In some embodiments, the composition is formulated for intertumoral delivery. Other suitable delivery means may be used. For example, in some embodiments, delivery may be by lavage solution or aerosol. In one embodiment, the composition is formulated for intraperitoneal delivery and is for the treatment of intraperitoneal cancers such as malignant epithelial tumors (e.g., ovarian cancer) and cancerous peritonitis (e.g., gastrointestinal cancers, particularly colorectal cancer, stomach cancer, gynecological cancers, and primary peritoneal neoplasms).

[0307] Furthermore, pharmaceutical formulations suitable for administration by inhalation as an aerosol are also provided. These formulations comprise a solution or suspension of the desired conjugate or its salt. The desired formulation may be placed in a small chamber and nebulized. Nebulization can be performed using compressed air or ultrasonic energy to form a plurality of droplets or solid particles containing the dendrimer or its salt.

[0308] As described below, the conjugates of this disclosure can be administered, for example, in combination with one or more additional pharmaceutically active agents. In some embodiments, the conjugates are provided in combination with further activators. In some embodiments, a composition is provided comprising a conjugate as defined herein or a pharmaceutically acceptable salt thereof, one or more pharmaceutically acceptable carriers, and one or more additional pharmaceutically active agents (e.g., additional anti-cancer / tumor agents such as small molecule cytotoxic agents, checkpoint inhibitors, or antibody therapies). The conjugates of this disclosure can be administered not only with other chemotherapeutic agents, but also in combination with other pharmaceuticals such as corticosteroids, antihistamines, analgesics, and drugs that aid in recovery or protect against hematological toxicity, such as cytokines.

[0309] In some embodiments, the composition is formulated for parenteral infusion as part of a chemotherapy regimen.

[0310] Therapeutic use of conjugates The conjugates and compositions described herein can be used in a variety of applications in the medical field. For example, the conjugates can be used to treat various conditions, such as cancer.

[0311] Accordingly, conjugates or pharmaceutical compositions as defined herein are provided for use in therapy, and more specifically for use in the therapy of cancer. In some embodiments, the conjugates are used in methods of treating or preventing cancer, for example, by inhibiting tumor growth. In some embodiments, the conjugates are for use in the treatment of cancer. Also provided are methods of treating cancer, comprising administering a therapeutically effective amount of a conjugate or pharmaceutical composition as defined herein to a subject in need of cancer treatment. Also provided are the use of a conjugate or composition as defined herein in the manufacture of a pharmaceutical for the treatment of cancer.

[0312] In some embodiments, cancer is a solid tumor. Cancer can be a primary or metastatic tumor. In some embodiments, cancer is a primary tumor. In some embodiments, cancer is a metastatic tumor.

[0313] In some embodiments, cancer is characterized by abnormal or overexpression of HER2 (also known as ERBB2). Such abnormal or overexpression of HER2 is known to occur in, for example, breast cancer, testicular cancer, ovarian cancer, stomach cancer, lung adenocarcinoma, gastric cancer, pancreatic cancer, salivary duct cancer, esophageal cancer, and uterine cancer (e.g., severe endometrial cancer).

[0314] In some embodiments, the cancer is characterized by abnormal or overexpression of EGFR. In some embodiments, the cancer is characterized by abnormal or overexpression of PSMA. In some embodiments, the cancer is characterized by abnormal or overexpression of FAP.

[0315] In some embodiments, cancer is prostate cancer. brain tumor The cancer is selected from the group consisting of breast cancer, testicular cancer, ovarian cancer, stomach cancer, lung adenocarcinoma, gastric cancer, pancreatic cancer, salivary duct cancer, esophageal cancer, and uterine cancer (e.g., severe endometrial cancer).

[0316] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, gastric cancer, pancreatic cancer, prostate cancer, and breast cancer.

[0317] In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is testicular cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is stomach cancer. In some embodiments, the cancer is lung adenocarcinoma. In some embodiments, the cancer is stomach cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is salivary duct cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is uterine cancer.

[0318] In some embodiments, cancer is brain tumor That is the case. brain tumor Examples include, but are not limited to, glioblastoma, meningioma, pituitary gland, nerve sheath, astrocytoma, oligodendroglioma, epithelioma, medulloblastoma, or craniopharyngioma. In some embodiments, the cancer is selected from the group consisting of glioblastoma, meningioma, pituitary gland, nerve sheath, astrocytoma, oligodendroglioma, epithelioma, medulloblastoma, and craniopharyngioma. brain tumor In some embodiments, brain tumor It is a glioblastoma. In some embodiments, brain tumor This is a meningioma. In some embodiments, brain tumor This is the pituitary gland. In some embodiments, brain tumor This is a nerve sheath. In some embodiments, brain tumor This is an astrocytoma. In some embodiments, brain tumor This is an oligodendroglioma. In some embodiments, brain tumorIt is an epithelial tumor. In some embodiments, brain tumor It is a medulloblastoma. In some embodiments, brain tumor This is a craniopharyngioma.

[0319] A therapeutically effective dose of the conjugate or composition is used in the treatment method and for use. The term “therapeutically effective dose” is understood to mean a conjugate or composition containing such conjugate administered in an amount sufficient to alleviate or prevent to some extent one or more symptoms of the disorder or condition being treated.

[0320] The dendrimer-targeted therapeutic conjugate may be administered by any preferred route, including, for example, intravenous administration. In some embodiments, the dendrimer-targeted therapeutic conjugate is delivered as an IV bolus. In some embodiments, the dendrimer-targeted therapeutic conjugate is administered IV over a period of 0.5 to 60 minutes, or 0.5 to 30 minutes, or 0.5 to 15 minutes, or 0.5 to 5 minutes. In another example, the dendrimer-targeted therapeutic conjugate may be administered intraperitoneally. The route of administration may target, for example, a disease or disorder in the subject. For example, in some embodiments, the disease or disorder may be an intraperitoneal malignancy such as gynecological cancer or gastrointestinal cancer, and the conjugate may be administered intraperitoneally. In some embodiments, the conjugate may be for the treatment of cancers of the peritoneal cavity, such as malignant epithelial tumors (e.g., ovarian cancer) or peritoneal carcinomatosis (e.g., gastrointestinal cancer, especially colorectal cancer, stomach cancer, gynecological cancers, and primary peritoneal neoplasms), and the conjugate is administered intraperitoneally.

[0321] When used for therapeutic purposes, the conjugate is administered in an amount sufficient to deliver a therapeutically effective dose of radioactivity to the target (e.g., a tumor) while avoiding unacceptable radiation exposure to other parts of the body (e.g., other organs). The precise dosage may depend on the properties of the radionuclide (e.g., alpha or beta emitters) and the condition being treated.

[0322] In some embodiments, the dose of the conjugate contains an amount of radionuclide having radioactivity of up to about 10 GBq, or up to about 7.5 GBq, or up to about 5 GBq, or up to about 2.5 GBq, or up to about 1 GBq, or up to about 500 MBq, or up to about 250 MBq, or up to about 10 MBq, or up to about 100 MBq, or up to about 50 MBq, or up to about 25 MBq, or up to about 10 MBq, or up to about 5 MBq. In some embodiments, the dosage of the conjugate or each dosage is 0.1 MBq to 10 GBq, 0.1 MBq to 7.5 GBq, 0.1 MBq to 5 GBq, 0.1 MBq to 2.5 GBq, 0.1 MBq to 1 GBq, 0.1 MBq to 500 MBq, 0.1 MBq to 250 MBq, 0.1 MBq to 100 MBq, 0.1 MBq to 50 MBq, 0.1 MB The conjugate contains an amount of radionuclide having radioactivity in the range of q~25MBq, 0.1MBq~10MBq, 0.1MBq~5MBq, 0.1MBq~2MBq, 0.1MBq~1MBq, 0.5MBq~10MBq, 1~10MBq, 1~5MBq, 5~10MBq, or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10MBq. In some embodiments, the dose of the conjugate or each dose contains an amount of radionuclide having radioactivity in the range of 0.1MBq~10GBq, 1MBq~10GBq, 10MBq~10GBq, 100MBq~10GBq, 500MBq~10GBq, 1GBq~10GBq, or 5GBq~10GBq.

[0323] For example, if the radioactive nuclide is Lu 177 In some embodiments, the dose of the conjugate or each dose contains an amount of radionuclide having radioactivity in the range of 1 GBq to 25 GBq, more preferably in the range of 4 to 25 GBq, or in the range of 4 to 10 GBq. In some embodiments, Lu 177 The conjugate containing [the substance] is administered to provide a radioactivity level ranging from 5 to 20 MBq per kg of body weight of the subject.

[0324] As another example, if the radioactive nuclide is Ga 68In some embodiments, the dose of the conjugate or each dose contains an amount of radionuclide having radioactivity in the range of 50 MBq to 1 GBq. In some embodiments, Ga 68 The conjugate containing is administered in an amount ranging from 1 to 5 MBq per kg of body weight of the subject, more preferably in the range of 1 to 3 MBq per kg, or approximately 2 MBq per kg.

[0325] As a further example, if the radionuclide is Y 90 In some embodiments, the dose of the conjugate or each dose contains an amount of radionuclide having radioactivity in the range of 500 MBq to 20 GBq, or 10 to 20 GBq. In some embodiments, Y 90 The conjugate containing is administered in an amount ranging from 5 to 50 MBq per kg of body weight, more preferably in the range of 10 to 15 MBq per kg.

[0326] Radioactive nuclide Ac 225 In this case, the dosage of the conjugate or each dosage may contain, for example, an amount of radionuclide having radioactivity in the range of 1 MBq to 20 MBq. In some embodiments, Ac 225 Conjugates containing this substance are administered in doses ranging from 15 to 200 KBq per kg of body weight.

[0327] Radioactive nuclides are At 211 In this case, the dosage of the conjugate or each dosage may contain, for example, an amount of radionuclide having radioactivity in the range of 50 MBq to 400 MBq.

[0328] As described above, the dose of the conjugate administered is sufficient to deliver a therapeutically effective dose of radioactivity to the target (e.g., a tumor) while avoiding unacceptable exposure to other parts of the body (e.g., other organs).

[0329] In some embodiments, the amount of conjugate administered in a single dose is such that the average radiation absorbed per organ of the group of organs consisting of the lungs, spleen, bladder, kidneys, heart, bone marrow, liver, and gastrointestinal tract is less than 5 mGy, or less than 5 mBq, or less than 2 mGy, or less than 2 mBq, or less than 1 mGy, or less than 1 mBq, or less than 0.5 mGy.

[0330] If the conjugate contains a third terminal group which is a further pharmaceutically active agent, in some embodiments the amount of conjugate administered is 2 to 100 mg / m². 2 The activator, 2-50 mg / m² 2 The activator, 2-40 mg / m² 2 The activator, 2-30 mg / m² 2 The activator, 2-25 mg / m² 2 Activating agent, 2-20 mg / m² 2 The activator, 5-50 mg / m² 2 The activator, 10-40 mg / m² 2 The activator, 15-35 mg / m² 2 The activator, 10-20 mg / m² 2 , 20-30 mg / m² 2 , or 25-35 mg / m² 2 This is sufficient to deliver the activator. A mouse dose of 10 mg / kg of the activator is equivalent to a human dose of 30 mg / m². 2 This should roughly correspond to the dose (FDA guidance 2005). (The human mg / kg dose is mg / m 2 To convert it, you can multiply by 37 (FDA Guidance 2005).

[0331] In some embodiments, a therapeutically effective dose of the conjugate is administered to the subject in need at a predetermined frequency. In some embodiments, the conjugate is administered to the subject in need according to a dosing regimen administered once every 1 to 4 weeks. In some embodiments, the conjugate is administered to the subject in need according to a dosing regimen administered once every 3 to 4 weeks. In some embodiments, a dosing regimen is used that involves a total of 2, 3, 4, 5, 6, 7, 8, 8, 9, or 10 doses administered once every 3 to 4 weeks.

[0332] combination Drugs are often administered in combination with other drugs, particularly during chemotherapy. Therefore, in some embodiments, the conjugate is administered in combination with one or more additional pharmaceutically active agents, such as one or more additional anticancer agents / drugs. The dendrimer-targeting agent conjugate and one or more additional pharmaceutically active agents can be administered simultaneously, sequentially, or separately. For example, they may be administered as part of the same composition or as separate compositions.

[0333] One or more additional pharmaceutically active agents, for example, prostate cancer, brain tumor It may also be an anticancer agent for the treatment of breast cancer, testicular cancer, ovarian cancer, gastric cancer, lung adenocarcinoma, pancreatic cancer, salivary duct cancer, esophageal cancer, or uterine cancer (e.g., severe endometrial cancer of the uterine body).

[0334] One or more additional pharmaceutically active agents may be, for example, anticancer agents for the treatment of colorectal cancer, gastric cancer, pancreatic cancer, prostate cancer, or breast cancer.

[0335] Further examples of pharmaceutically active agents include chemotherapeutic agents, cytotoxic agents, small molecule cytotoxic agents, tyrosine kinase inhibitors, checkpoint inhibitors, EGFR inhibitors, antibody therapies, taxanes (e.g., paclitaxel, docetaxel, cabazitaxel, nab-paclitaxel), topoisomerase inhibitors (e.g., SN-38, irinotecan (CPT-11), topotecan, siratecan, cocitecan, exatecan, lulutotecan, gimatecan, berotecan, or rubitecan), and aromatase inhibitors.

[0336] Diagnosis / Imaging The conjugates and compositions described herein can also be used as diagnostic agents, such as imaging agents. Examples of diagnostic applications include imaging, theranostics, companion diagnostic therapy, monitoring disease progression, evaluating the effectiveness of therapy, determining patient outcomes, and developing treatment regimens for specific patients or patient groups.

[0337] Therefore, a method for determining whether a subject has cancer, To administer to a subject a conjugate as defined herein or a pharmaceutical composition containing such conjugate, The process of creating images of the subject's body or a part thereof, A method is provided which includes determining whether a subject has cancer based on the imaging results.

[0338] A method for imaging the target cancer, To administer to a subject a conjugate as defined herein or a pharmaceutical composition containing such conjugate, A method is also provided which includes imaging the body or a part thereof of the subject.

[0339] A method for determining the progression of cancer in a subject, Administering a first amount of a conjugate as defined herein or a pharmaceutical composition containing such conjugate to a subject, Performing a first imaging step on the subject's body or a part thereof, Next, a second amount of the conjugate as defined herein or a pharmaceutical composition containing the conjugate is administered to the subject, The second imaging step is performed on the subject's body or a part thereof, A method is also provided which includes determining whether cancer has progressed based on the first and second imaging results.

[0340] A method for determining appropriate therapy for a patient with cancer, To administer to a subject a conjugate as defined herein or a pharmaceutical composition containing such conjugate, The process of creating images of the subject's body or a part thereof, A method is also provided which includes administering a therapy to a target when imaging results indicate that the cancer is sensitive to the therapy.

[0341] A method for determining the effectiveness of cancer therapy administered to a subject with cancer, Administering a first amount of a conjugate or pharmaceutical composition as defined herein, Performing a first imaging step on the subject's body or a part thereof, Administering cancer therapy to the target, Next, a second amount of the conjugate or pharmaceutical composition as defined herein is administered to the target, The second imaging step is performed on the subject's body or a part thereof, A method is also provided which includes determining the effectiveness of cancer therapy based on first and second imaging results.

[0342] Also provided are conjugates as defined herein, or pharmaceutical compositions containing such conjugates, for use in the diagnosis of cancer in a subject, for use in determining appropriate therapy for a subject with cancer, for use in determining the effectiveness of cancer therapy administered to a subject, for use in determining the progression of cancer in a subject, or for use in the treatment of cancer.

[0343] Furthermore, the invention also provides conjugates as defined herein, or pharmaceutical compositions containing such conjugates, for use in the manufacture of pharmaceuticals for the diagnosis of cancer, for determining appropriate therapy for a subject with cancer, for determining the effectiveness of cancer therapy administered to a subject, for determining the progression of cancer in a subject, or for the treatment of cancer.

[0344] The cancer may be any of the cancers described above, for example, in relation to the therapeutic use of conjugates. For example, in some embodiments, the cancer is a solid tumor. The cancer may be a primary or metastatic tumor. In some embodiments, the cancer is a primary tumor. In some embodiments, the cancer is a metastatic tumor.

[0345] In some embodiments, cancer is characterized by abnormal or overexpression of HER2 (also known as ERBB2). Such abnormal or overexpression of HER2 is known to occur in, for example, breast cancer, testicular cancer, ovarian cancer, stomach cancer, lung adenocarcinoma, gastric cancer, pancreatic cancer, salivary duct cancer, esophageal cancer, and uterine cancer (e.g., severe endometrial cancer).

[0346] In some embodiments, cancer is prostate cancer. brain tumor The cancer is selected from the group consisting of breast cancer, testicular cancer, ovarian cancer, stomach cancer, lung adenocarcinoma, gastric cancer, pancreatic cancer, salivary duct cancer, esophageal cancer, and uterine cancer (e.g., severe endometrial cancer).

[0347] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, gastric cancer, pancreatic cancer, prostate cancer, and breast cancer.

[0348] In the above methods and uses, any suitable means may be used to administer a sufficient amount of the conjugate or composition for diagnostic use. For example, the conjugate or composition may be administered intravenously to the subject.

[0349] Suitable techniques for imaging samples containing radionuclides, or subjects to which radionuclides have been administered, and for analyzing the results, are known to those skilled in the art and can be used in the methods and uses described above.

[0350] Radionuclide-based imaging methods, particularly PET (positron emission tomography), remain an active area for both diagnostic and therapeutic applications due to their high sensitivity (picomole level) and unrestricted tissue penetration. In some embodiments, PET imaging is used. In some embodiments, PET-MRI, SPECT, SPECT-CT, CT, scintography, or PET-CT imaging is used.

[0351] Typically, when used for imaging and diagnostic purposes, the conjugate is administered, and then the subject, or a relevant portion of the subject, is imaged after a suitable period of time. The time between the administration step and the imaging step may depend on the embodiment, including the properties of the targeting agent. For example, in some cases where small molecule targeting agents are used, it may be beneficial to image the subject within 2 hours, 1 hour, or 30 minutes after administration. As a further example, when antibody targeting agents are used, or when the dendimer is large, for example, G4 or G5, it may be preferable to allow an additional time, e.g., 1, 2, 3, 4, 5, 6, or 7 days, after administration before imaging. In some embodiments, the conjugate is administered, and imaging is performed about 24 hours later, or about 48 hours later.

[0352] In some embodiments, the conjugate used for diagnosis and imaging is a conjugate having two or three generations of constituent units.

[0353] The conjugates and compositions containing them exhibit good selectivity for the target of interest (e.g., tumor tissue). To further improve selectivity and reduce the levels of conjugates present in other tissues or organs, such as the kidneys or liver, the diagnostic and therapeutic methods may include additional steps as part of the administration regimen.

[0354] For example, a drug that reduces the potential for nephrotoxicity associated with renal exposure to a radioactive agent may be administered beforehand or concurrently. In some embodiments of the therapeutic and diagnostic methods provided in the present invention, the conjugate or the pharmaceutical composition providing the conjugate is administered in combination with a drug that reduces the potential for nephrotoxicity.

[0355] Examples of such agents include amino acids, such as basic amino acids like lysine and / or arginine. For example, an aqueous solution containing 18-24 g of L-lysine and 18-24 g of L-arginine per 1.5-2.2 L of solution is used. Such a solution may have an osmolality of, for example, less than 1200 mOsmol, less than 1100 mOsmol, or less than 1060 mOsmol. Further examples of suitable agents include succinylated gelatin (a 4% w / v solution is sold by Hausmann Laboratories Ltd under the trade name Gelofusine). Further examples of such agents include furosemide (sold under the brand name Lasix) and spironolactone (sold under the brand name Aldactone).

[0356] In some embodiments, pre-administration or co-administration of an amino acid such as lysine or arginine may be utilized. Accordingly, in some embodiments of the therapeutic and diagnostic methods provided herein, the conjugate or the pharmaceutical composition providing the conjugate is administered in combination with an amino acid, such as lysine or arginine. In some embodiments, the amino acid (e.g., lysine, arginine) is administered before administration of the conjugate or the composition containing the conjugate. In some embodiments, the amino acid (e.g., lysine, arginine) is administered simultaneously with the conjugate or the composition containing the conjugate.

[0357] In some embodiments, succinylated gelatin is administered in combination with a conjugate or a pharmaceutical composition providing the conjugate. In some embodiments, succinylated gelatin is administered before administration of the conjugate or a composition containing the conjugate.

[0358] In some embodiments, the combination of succinyl gelatin and an amino acid (e.g., lysine, arginine) is administered before or simultaneously with the administration of the conjugate.

[0359] In some embodiments, furosemide is administered before or at the same time as the administration of the conjugate.

[0360] In some embodiments, spironolactone is administered before or simultaneously with the administration of the conjugate.

[0361] Drugs (e.g., amino acids such as lysine and arginine) are typically administered in the form of pharmaceutical compositions, such as aqueous compositions. Drugs may also be administered intravenously, for example, by injection or infusion.

[0362] Conjugates suitable for diagnostic, therapeutic, imaging, and other purposes requiring the presence of radionuclides will be understood to be therapeutic conjugates as described herein, even though their end use may be diagnostic or otherwise used for imaging, rather than being essentially therapeutic.

[0363] Preparation of therapeutic conjugates Radioactive materials are hazardous substances, and handling steps involving such materials should ideally be minimized. Introducing radioactive nuclide components into a conjugate should be limited to a later stage, ideally immediately before the conjugate is used.

[0364] Therefore, a process for generating a therapeutic conjugate as defined herein, The process involves contacting a suitable dendrimer-targeting agent conjugate as defined above with a radionuclide to generate a therapeutic conjugate, wherein the dendrimer-targeting agent complex or a salt thereof is a) A dendrimer, i) Core unit (C), and ii) Including constituent units (BUs), A dendrimer having 2 to 6 generations of constituent units, with a core unit covalently attached to at least two constituent units, b) A targeting agent covalently linked to the dendrimer by a spacer group, c) One or more first terminal groups attached to the outermost constituent unit of the dendrimer, wherein the first terminal group includes a complexing group for complexing a radionuclide, d) One or more second terminal groups attached to the outermost constituent unit of the dendrimer, wherein the second terminal group includes a pharmacokinetic modification moiety, The process is also provided.

[0365] The present invention also provides a kit for generating the therapeutic conjugate defined above, comprising: a) the dendrimer-targeting agent conjugate defined above; and b) a radionuclide.

[0366] It will be understood that any one or more different embodiments or examples described herein relating to the conjugate, for example, the core unit (C), constituent units (BU), end groups, targeting agents, or dendrimers, may also be provided for intermediates. Similarly, any of the radionuclides described above in relation to the conjugate may be used in the process for generating the conjugate.

[0367] Any suitable means for generating a therapeutic conjugate from a dendrimer-targeting agent conjugate and a radionuclide may be used. For example, the dendrimer-targeting agent conjugate and the radionuclide (e.g., in the form of a metal salt) may be mixed in a suitable solvent, preferably a solvent suitable for administration to a patient. For example, in some embodiments, an aqueous solvent may be used.

[0368] In some embodiments, radionuclides (e.g., Zr) in aqueous solution 89 A preferred salt form of the oxalate may be mixed with a solution of the dendrimer-targeting agent conjugate or intermediate in a preferred buffer (e.g., HEPES). Any preferred molar ratio of the intermediate to the radionuclide salt, for example, at least 25:1, at least 50:1, or about 100:1, may be used. If necessary, purification may be performed to separate the unbound radionuclide. If desired, the solution may be changed before administration, for example, by changing the buffer to phosphate-buffered saline.

[0369] If other metal ion species are present (for example, if the intermediate contains significant levels of chelate metal), these may be removed as needed before labeling with radionuclides. For example, iron contaminants may be removed by treatment with EDTA (ethylenediaminetetraacetic acid) before labeling with radionuclides.

[0370] Labeling of dendrimer-targeting agent conjugates or intermediates may be carried out, for example, according to the procedure described in Verel et al, J. Nucl. Med., 2003, 44(8), pp. 1271-1281.

[0371] The kits, intermediates, and processes described above can be used to provide effective preparation of pharmaceutical compositions in a clinical setting by enabling radiolabeling of the intermediates and the generation of conjugates in the clinical setting immediately before administration.

[0372] Those skilled in the art will understand that numerous variations and / or modifications can be made to the embodiments described above without departing from the broad general scope of this disclosure. Accordingly, these embodiments should be considered in all respects as illustrative and not limiting. [Examples]

[0373] In this specification, the following nomenclature is used in relation to the synthesis of dendrimer conjugates. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 3-1] [Table 3-2]

[0374] Preparative HPLC Preparative HPLC was performed on a Gilson HPLC system using a Waters XBridge® BEH300 Prep C 185μm OBD® 30×150mm column, using a two-component solvent system consisting of solvent A (water, water containing formic acid, or water containing TFA) and solvent B (acetonitrile, or acetonitrile containing formic acid or TFA). Peaks were detected at wavelengths of 214 nm, 243 nm, or 254 nm using a UV detector.

[0375] Preparative HPLC method Preparative HPLC method: 5-60% TFA: Solvent A, 0.05% TFA (v / v) in water; Solvent B, 0.05% TFA (v / v) in MeCN; Flow rate: 8.0 mL / min; Gradient: 0-5 min, 5% B; 5-35 min, 5-60%, 35-47 min, 60% B; 47-50 min, 60-5% B; 50-60 min, 5%. Peaks were detected at wavelengths of 214 and 254 nm using a UV detector.

[0376] Preparative HPLC method: 30-50% TFA: Solvent A, 0.05% TFA (v / v) in water; Solvent B, 0.05% TFA (v / v) in MeCN; Flow rate: 8.0 mL / min; Gradient: 0-5 min, 30% B; 5-35 min, 30-50%, 35-47 min, 50% B; 47-50 min, 50-30% B, 50-60 min, 30%. Peaks were detected at wavelengths of 214 and 254 nm using a UV detector.

[0377] Preparative HPLC method: 40-70% TFA: Solvent A, 0.05% TFA (v / v) in water; Solvent B, 0.05% TFA (v / v) in MeCN; Flow rate: 8.0 mL / min; Gradient: 0-5 min, 40% B; 5-35 min, 40-70%, 35-47 min, 70% B; 47-50 min, 70-40% B; 50-60 min, 40%. Peaks were detected at wavelengths of 214 and 254 nm using a UV detector.

[0378] Preparative HPLC was performed at 20-60°C, with TFA: 0.05% TFA (v / v) in solvent A (water); 0.05% TFA (v / v) in solvent B (MeCN); flow rate: 8.0 mL / min; gradient: 0-5 min, 20% B; 5-35 min, 20-60%; 35-40 min, 60-100% B; 40-47 min, 100% B; 47-50 min, 100-10% B; 50-60 min, 10%. Peaks were detected at wavelengths of 214 and 254 nm using a UV detector.

[0379] Preparative HPLC method 20-60: Solvent A, water; Solvent B, MeCN; Flow rate: 8.0 mL / min; Gradient: 0-7 min, 20% B; 7-37 min, 20-60%, 37-47 min, 60% B; 47-54 min, 60-20% B; 54-60 min, 20%. λ = 214 nm and 254 nm.

[0380] Preparative HPLC method 20-60(2): Solvent A, water; Solvent B, MeCN; Flow rate: 8.0 mL / min; Gradient: 0-5 min, 20% B; 5-55 min, 20-60%, 55-60 min, 60-20% B. Detection at λ=214 nm and 254 nm.

[0381] Preparative HPLC was performed at 20-90°C, with TFA: 0.05% TFA (v / v) in solvent A (water); 0.05% TFA (v / v) in solvent B (MeCN); flow rate: 8.0 mL / min; gradient: 0-6 min, 20% B; 6-40 min, 20-90%; 40-47 min, 90% B; 47-51 min, 90-20% B; 51-60 min, 20% B. Peaks were detected at wavelengths of 214 and 254 nm using a UV detector.

[0382] Preparative HPLC was performed at 30-90°C, with TFA: 0.05% TFA (v / v) in solvent A (water); 0.05% TFA (v / v) in solvent B (MeCN); flow rate: 8.0 mL / min; gradient: 0-5 min, 30% B; 5-40 min, 30-90%; 40-45 min, 90% B; 45-50 min, 90-30% B; 50-60 min, 30% B. Peaks were detected at wavelengths 214 and 254 nmd using a UV detector.

[0383] Preparative HPLC method 5-60: Solvent A, water; Solvent B, MeCN; Flow rate: 8.0 mL / min; Gradient: 0-5 min, 5% B; 5-42 min, 5-60%, 42-49.5 min, 60-80% B; 49.5-55 min, 80%, 55-57 min, 80-5% B; 57-60 min, 5%. Peaks were detected at wavelengths of 214 and 254 nm using a UV detector.

[0384] Preparative HPLC method 5-60(2): Solvent A, water; Solvent B, MeCN; Flow rate: 8.0 mL / min; Gradient: 0-9 min, 5% B; 9-38 min, 5-60%, 38-48 min, 60% B; 48-54 min, 60-5% B; 54-60 min, 5% B. Peaks were detected at wavelengths of 214 and 254 nm using a UV detector.

[0385] Preparative HPLC method 40-60: Solvent A, water; Solvent B, MeCN; Flow rate: 8.0 mL / min; Gradient: 0-9 min, 40% B; 9-37 min, 40-60%, 37-47.5 min, 60% B; 47.5-54 min, 60-40% B; 54-60 min, 40% B. Peaks were detected at wavelengths of 214 and 254 nm using a UV detector.

[0386] Preparative HPLC method 20-40: Solvent A, water; Solvent B, MeCN; Flow rate: 8.0 mL / min; Gradient: 0-10 min, 20% B; 10-38 min, 20-40%, 38-47.5 min, 40% B; 47.5-54 min, 40-20% B; 54-60 min, 20%. Detection at λ=214 nm and 254 nm.

[0387] Automated flash chromatography: Automated flash purification was performed on a Biotage® Selekt5 automated flash chromatography system using a standard phase or reversed phase silica cartridge.

[0388] Automated flash chromatography method Auto-flush method 1: Cartridge = Biotage Sfar C18 D (Duo100 A30μm) 12g cartridge; Solvent A, water; Solvent B, methanol; Flow rate: 12 mL / min; Gradient: 0~3 CV, 10% B; 3~5 CV, 10~60% B; 5~9.8 CV, 60~74% B; 9.8~11.4 CV, 74% B; 11.4~13.6 CV, 74~80% B; 13.6~15.6 CV, 80~100% B; 15.6~20.6 CV, 100% B; 20.6~21.6 CV, 100~10% B, and 21.6~24.6 CV, 10% B. λ = 214 nm and 254 nm and λ all Detection in the 198-810nm range.

[0389] Auto-flush method 2: Cartridge = Biotage Sfar C18 D (Duo100 A30μm) 12g cartridge; Solvent A: water; Solvent B: methanol; Flow rate: 12 mL / min; Gradient: 0-3 CV, 5% B; 3-4 CV, 5-30% B; 4-14 CV, 30-100% B, and 14-19 CV, 100% B. λ = 214 nm and 254 nm and λ all Detection in the 198-810nm range.

[0390] analyticalLCMS LC-MS was recorded using a Waters 2795 HPLC equipped with a Waters 2996 diode array detector using a Waters XBridge® 3.5 μm 3 × 100 mm C8 column or a Phenomenex Kinetex® 2.6 μm 2.1 × 75 mm C18 column. The flow rate was typically 0.4 mL / min, and the injection volume was typically 5–10 μL. Peaks were detected at λ = 214 nm, 243 nm, or 254 nm using a UV detector (unless otherwise specified).

[0391] MS-Waters ZQ4000 (with ESI probe), inlet flow was branched to approximately 50 μL / min to the MS. Mass spectrometry data were obtained in positive or negative electrospray ionization mode as shown. Raw data were deconvoluted using the Maximum Entropy algorithm (MaxEnt) implemented in MassLynx software v4.0 provided by Waters Corporation. The data reported in the experimental details correspond to the observed values ​​after deconvolution to the theoretical zero-charge state.

[0392] LCMS method LCMS method (hydrophilic TFA / formate buffer): The gradients were as follows: 0-1 min, 5%B; 1-10 min, 5-60%B; 10-11 min, 60%B; 11-13 min, 60-5%B; 13-15 min, 5%B.

[0393] LC-MS method (hydrophobic TFA / formate buffer): The gradients were as follows: 0-1 min, 40%B; 1-7 min, 40-90%B; 7-9 min, 90%B; 9-11 min, 90-40%B; 11-15 min, 40%B.

[0394] LCMS method 40-65, TFA buffer: The gradient was 40%B at 0-1 min; 40-65%B at 1-10 min; 65%B at 10-11 min; 45%B at 11-12 min; 45%B at 12-15 min.

[0395] LC-MS method (20-90, 15 min): The gradients were as follows: 0-1 min, 20%B; 1-9 min, 20-90%B; 9-11 min, 90%B; 11-13 min, 90-20%B; 13-15 min, 20%B (including 0.1% HCOOH acid).

[0396] LCMS method, 5-60, 8 min, TFA: The gradients were 0-1 min, 5%B; 1-5 min, 5-60%B; 5-6 min, 60%B; 6-6.1 min, 60-5%B; 6.1-8 min, 5%B.

[0397] LCMS method, 20-90, 8 min, TFA: The gradients were 0-0.5 min, 5%B; 0.5-1 min, 5-20%B; 1-5 min, 20-90%B; 5-6 min, 90%B; 6-6.1 min, 90-5%B; 6.1-8 min, 5%B.

[0398] LCMS method, 40-90, 8 min, TFA: The gradients were 0-0.5 min, 5%B; 0.5-1 min, 5-40%B; 1-5 min, 40-90%B; 5-6 min, 90%B; 6-6.1 min, 90-5%B; 6.1-8 min, 5%B.

[0399] LCMS method, 20-60, 8 min, TFA: The gradients were 0-0.5 min, 5%B; 0.5-1 min, 5-20%B; 1-5 min, 20-60%B; 5-6 min, 60%B; 6-6.1 min, 60-5%B; 6.1-8 min, 5%B.

[0400] LCMS method, 60-90, 8 min, TFA: The gradients were 0-0.5 min, 5%B; 0.5-1 min, 5-60%B; 1-5 min, 60-90%B; 5-6 min, 90%B; 6-6.1 min, 90-5%B; 6.1-8 min, 5%B.

[0401] LCMS method, 40-60, 8 min, TFA: The gradients were 0-0.5 min, 5%B; 0.5-1 min, 5-40%B; 1-5 min, 40-60%B; 5-6 min, 60%B; 6-6.1 min, 60-5%B; 6.1-8 min, 5%B.

[0402] LCMS method, 5-80, 8 min, TFA: The gradients were 0-1 min, 5%B; 1-5 min, 5-80%B; 5-6 min, 80%B; 6-6.1 min, 80-5%B; 6.1-8 min, 5%B.

[0403] LCMS method, 5-80, 15 minutes, TFA: The gradients were 5%B at 0-1 minute; 5-80%B at 1-10 minutes; 80%B at 10-11 minutes; 80%B at 11-13 minutes; 5%B at 13-15 minutes; 0.1%TFA.

[0404] analyticalHPLC HPLC data were recorded using a Waters 2695 separation module equipped with a 2996 PDA detector and either a Waters XBridge® C8 3.5 μm 3 × 100 mm column or a Phenomenex Kinetex® 2.6 μm 2.1 × 75 mm C18 column. The instrument control software was Waters Empower3. The three mobile phases used were a) 1% v / v TFA buffer or 1% v / v formate buffer or 100 mM ammonium formate, b) water, and c) acetonitrile. The flow rate was typically 0.4 mL / min, and the injection volume was typically 5–10 μL. Peaks were detected at λ = 214 nm, 243 nm, or 254 nm using a UV detector (unless otherwise specified).

[0405] HPLC method HPLC analysis: For formate / TFA gradient at 5–80°C, 15 minutes: The gradient at a flow rate of 0.40 mL / min was 5% B at 0–1 min, 5–80% B at 1–7 min, 80% B at 7–12 min, 80% B at 12–13 min, 80% B at 13–15 min, and 5% B at 13–15 min. Peaks were detected at wavelengths of 214, 243, and 254 nm using a UV detector.

[0406] HPLC method, 5–80%, 8 min, TFA: The gradient at a flow rate of 0.40 mL / min was 0–0.5 min, 5%B; 0.5–3.5 min, 5–80%B; 3.5–6 min, 80%B; 6–6.5 min, 80–5%B; 6.5–8 min, 5%B. Peaks were detected at wavelengths of 214, 243, and 254 nm using a UV detector.

[0407] HPLC-hydrophilic method, formate / TFA buffer, 15 minutes: The gradients were 0-1 min, 5% B; 1-10 min, 5-60% B; 10-11 min, 60% B; 11-13 min, 60-5% B; 13-15 min, 5% B.

[0408] Analytical ULC-ToF (Ultra-High Pressure Liquid Chromatography - Time of Flight) UPLC-ToF data were recorded using a Waters Aquity UPLC binary separation module equipped with a Waters Aquity PDA detector and a Waters LCT Premiere (ToF) mass spectrometer. The column used was a Phenomenex Kinetex EVO C18 2.6 μm 2.1 × 100 mm column. The instrument control software was Waters Masslynx Version 4.1. The two mobile phases used were a) 0.01% v / v TFA in water and b) 0.01% v / v TFA in acetonitrile. The flow rate was typically 0.2 mL / min or 0.4 mL / min, and the injection volume was typically 2–5 μL. Peaks were detected within 200 nm–400 nm (unless otherwise specified).

[0409] UPLC-ToF method Method 1: The gradient consisted of 15-35% MeCN / H2O (1-9 mins), 35% MeCN / H2O (9-11 mins), 35-15% MeCN / H2O (11-12 mins), 15% MeCN / H2O (12-15 mins), and 0.01% TFA buffer, with UV detection at 254 nm. Method 2: The gradient was 20-80% MeCN / H2O (1-10 min), 80% MeCN / H2O (10-11 min), 80-20% MeCN / H2O (11-13 min), 20% MeCN / H2O (13-15 min), 0.01% TFA buffer), and UV detection was performed at 254 nm.

[0410] Size exclusion chromatography (SEC) Size exclusion chromatography was performed on a Sephadex® LH-20 column under gravity at a flow rate of approximately 50–60 drops / min using methanol or acetonitrile as an eluent. Each fraction size consisted of 400–600 drops. Fractions containing PEGylated compounds were detected by TLC [TLC plates were developed in 5% (w / v) BaCl2 aqueous solution followed by I2 solution in ethanol] or analyzed by HPLC.

[0411] Tangent flow filtration Tangent flow filtration uses water as the elution medium in a 50 cm³ solution. 2 Pellicon® XL Cassette Ultracel® Regenerated Cellulose Membrane, or a 0.11m² Ultracel® Regenerated Cellulose Membrane equipped with water or acetonitrile as an elution medium. 2 The test was performed using either Pellicon(registered trademark)3 Cassette.

[0412] Centrifugal ultrafiltration Centrifugation was performed using an Amicon® Ultra centrifugation filter equipped with a specified molecular weight cutoff (MWCO) Ultracel® regenerated cellulose membrane, at 4000 rpm on an Eppendorf 5810R centrifuge or 14000 rpm on a 5415R centrifuge.

[0413] NMR NMR spectra were recorded using CD3OD, CDCl3, D2O, CD3CN, or otherwise described using a Bruker (Bruker Daltonics Inc., NSW, Australia) 300 UltraShield® 300MHz NMR instrument.

[0414] IR The IR spectrum was recorded using a Cary 630 FTIR Agilent Technologies diamond ATR accessory with 16 scans.

[0415] General procedure: The preparation of carboxy-reactive dendrimer skeletons has been previously described; see, in particular, WO2008 / 017125. Those skilled in the art can adapt these methods to prepare the various dendrimers outlined herein. In the following examples, [Lys] in the formulas refers to the lysine constituent units in the surface layer of the dendrimer.

[0416] General Procedure A. Boc Deprotection To a Boc compound (1.0 equivalent) / aqueous suspension, which had been cooled on ice and stirred, TFA (40-200 equivalents / Boc group) was added. After 5 minutes, the ice bath was removed, and the reaction mixture was stirred at room temperature overnight. Volatile substances were removed by vacuum, and the remaining aqueous solution was further diluted with water, freeze-dried, and the deprotected product was obtained in quantitative yield.

[0417] General procedure B. Addition of a lysine layer to the dendrimer surface. Under a nitrogen atmosphere, TEA (6.0 equivalents / NH2) was added to a TFA dendrimer (1.0 equivalent) / DMF stirred solution, followed by DBL-ONp (2.0 equivalents / NH2). The resulting reaction mixture was then stirred at room temperature overnight. Volatile substances were removed by vacuum, and the resulting crude product was purified using standard methods.

[0418] General procedure C.HO-Lys[(α-NHBoc)(ε-NH-COPEG 1100 PEGylation of dendrimer surfaces using a wedge. Under a nitrogen atmosphere, PyBOP (2.0 equivalents / NH2) and DIPEA (8.0 equivalents / NH2) were added to a TFA dendrimer (1.0 equivalent) / DMF stirred solution. After 10 minutes, HO-Lys[(α-NHBoc)(ε-NH-COPEG 1100 (1.35 equivalents / NH2) / DMF solution was added, and the resulting reaction mixture was stirred overnight at room temperature. Volatile substances were removed by vacuum, and the resulting crude product was purified using standard methods.

[0419] The general procedure involves capping the dendrimer surface with a D.HO-Lys[(α-NHBoc)(ε-NHFmoc)] wedge, followed by Fmoc deprotection. Step 1: PyBOP (1.4 equivalents / NH2) was added to a stirred solution of HO-Lys[(α-NHBoc)(ε-NHFmoc)] (1.5 equivalents / NH2) and NMM (2.5 equivalents / NH2) in DMF. The resulting reaction mixture was then stirred at room temperature for 15 minutes, and TFA-dendrimer (1.0 equivalent) and NMM (2.5 equivalents / NH2) / DMF solution were added. The resulting reaction mixture was then stirred at room temperature for 1 hour and allowed to stand, after which it was slowly added to ice-cold MeCN and stirred for 15 minutes. The resulting solid was collected by filtration, washed with MeCN (3 times), and then freeze-dried.

[0420] Step 2: Piperidine (21 equivalents / Fmoc) was added to a DMF solution containing Fmoc / Boc dendrimer (1.0 equivalent). This solution was stirred at room temperature for 90 minutes, then slowly added to ice-cold Et2O. After 15 minutes, the precipitated solid was collected by filtration, washed with Et2O, dissolved in H2O, and frozen for storage.

[0421] General procedure E. Glu-vc-PAB-MMAE or DGA-MMAF(OMe) for dendrimer surface capping Azido-PEG 24 CO-[N(PN)2[Lys]2[Lys]4[Lys]8[(α-NH2.TFA)(ε-NH-COPEG 570 / 1100 / 2000 Compound 10, Compound 14, or Compound 16 (1.0 equivalent) was dissolved in a mixture of DMF and NMM (5.0 equivalents / NH2) at room temperature. This solution was added to HO-Glu-vc-PAB-MMAE (Levena Biopharma) or DGA-MMAF (OMe) (Concortis Biosystems) (1.2 equivalents / NH2) and PyBOP (2.0 equivalents / NH2), and left at room temperature. The resulting crude material was purified by SEC.

[0422] General Procedure F. Conjugation of Affibody to MMAE / MMAF Dendrimers Step 1: A solution of afibody protein (HER2, afibody AB, 1.0 mg / mL PBS) was treated with TCEP (50 mM, 39.0 equivalents), and the reaction mixture was shaken at 650 rpm for 2 hours at room temperature. The resulting solution was purified by SEC.

[0423] Step 2: The collected permeate was treated with a solution of ((1R,8S,9s)-bicyclo[6.1.0]non-4-in-9-yl)methyl(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propanamide)ethyl)carbamate (Mal-BCN) (compound 117) (20.0 equivalents) in DMSO. The resulting reaction mixture was then shaken at 650 rpm for 2 hours at room temperature. The resulting solution was purified by SEC.

[0424] Step 3: Affibody-BCN solution, Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NHGlu-vc-PAB-MMAE)(ε-NH-COPEG 570 / 1100 / 2000 )]8 Compound 64, Compound 65, Compound 66 (240 μm in PBS) or Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NHDGA-MMAF(OMe))(ε-NH-COPEG 1100 Compound 67 (365 μm in PBS) was treated with a solution of (1.3 equivalents of afibody-BCN / dendrimer). The resulting mixture was then shaken at 650 rpm overnight at room temperature, and then treated with a 9.38 mM (30% EtOH / water) solution of DBCO agarose (5.0 equivalents / dendrimer). The resulting suspension was then shaken at 1200 rpm overnight at room temperature. This suspension was purified using SEC.

[0425] General procedure for conjugation of nanobodies into G.MMAE dendrimers Step 1: Dissolve the linker (1 mg) in 20:80 (DMSO / 10 mM PBS, 1 mL) and dissolve the linker (BCN-PEG2NH-Glu-NHPEG 24CO-NHPEG3-TCO compound 50 or DBCO-Glu-NHPEG 24 A solution of compound 51 (CO-NHPEG3-TCO) was prepared.

[0426] Step 2: One equivalent of TCO-linker solution was added to a solution of tetrazine-functionalized dendrimer (1.0 equivalent, 8 mg / mL) in PBS(1). The reaction mixture was left at room temperature for 30 minutes. Completion of the reaction was indicated by the disappearance of the pink tetrazine color. This reaction was monitored by HPLC.

[0427] Step 3: Once the reaction was complete, the contents were diluted with PBS (to a final volume of 0.5 mL). A portion of the BCN / DBCO-MMAE-dendrimer (1.0 equivalent) was added to a solution of nanobody-N3 (1.0 equivalent, 9.2 mg / ml) in Tris buffer (20 mM, 1 mL). The resulting solution was left at room temperature for 7 hours, followed by overnight at 4°C. The nanobody-dendrimer construct was purified by anion exchange chromatography and subsequent SEC.

[0428] General Procedure H.N3-PEG 570 / 1100 - Capping of the dendrimer surface with NHS ester, followed by removal of Boc groups. Step 1: BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [(α-NHBoc) 32 (ε-NH2) 32 ](Reference 1, WO2007 / 082331A1, J.Controlled Release 2011, 152, 241-248) and N3-PEG in a stirred DMF solution of DIPEA (2.0 equivalents / NH2) 570 / 1100 -NHS ester (1.5 equivalents / NH2), or N3-PEG 570 / 1100- A DMF solution of acid (1.3 equivalents / NH2) and PyBOP (1.3 equivalents / NH2) was added. The resulting reaction mixture was then stirred at room temperature for 15 hours. Deionized water was added to the reaction mixture, and the resulting solution was filtered (0.45 μm Acrodisc syringe filter). The filtrate was ultrafiltered through a 10 kDa regenerated cellulose Pellicone membrane using water as the circulation medium until 20 dialysate filtration volumes (DV) were collected as permeate. The residue was collected, pooled with line washing, and freeze-dried.

[0429] Step 2: Trifluoroacetic acid (321 equivalents / NHBoc) (TFA / DCM 1:1v / v) was added to a DCM solution of azidodendrimer (1.0 equivalent). The solution was stirred at room temperature for 15 hours, and volatile components were removed by vacuum.

[0430] General Procedure I. Conjugation of cyanine 5 to dendrimers, followed by acetylation. Step 1: BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [(α-NH2.TFA) 32 (ε-NH-COPEG 570 / 1100 N3) 32 To a stirred DMF solution of compounds 32 and 33 and DIPEA (4.0 equivalents / NH2), cyanine 5-NHS ester (2.0 equivalents) was added. The reaction mixture was stirred at ambient temperature for 3 hours. Volatile components were removed by vacuum, and the residue was used without further purification in step 2.

[0431] Step 2: To the stirred pyridine (1 mL) solution of the dried residue obtained in Step 1, acetic anhydride (2 mL) was added. The reaction mixture was stirred at ambient temperature for 15 hours. Volatile components were removed by vacuum, and the residue was purified using SEC (Sephadex LH-20) with methanol as the eluent.

[0432] General Procedure: Click reaction between J.DUPA-BCN and azidodendrimer BHALys[Lys]2[Lys]4[Lys]8[Lys] in acetonitrile and water (1:1)16 [Lys] 32 [(α-NHCy5)1(α-NHAc) 31 (ε-NH-COPEG 570 / 1100 N3) 32 DUPA-BCN(19) was added to a stirred solution of compounds 34 and 35. The reaction mixture was stirred at ambient temperature for 15 hours. The reaction mixture was freeze-dried, and the residue obtained after freeze-drying was purified by SEC(LH-20) using methanol as the eluent, or by ultrafiltration using water on a 10 kDa MWCO Pellicone regenerated cellulose TFF membrane.

[0433] General Procedure: Conjugation of K.DOTA Dendrimer and Nanobody-Cys SRS-13 Step 1: Reduction of nanobody dimer: To a solution of nanobody-Cys dimer (3.33 mg / mL, 10 mM PBS, pH 7.4; 1 equivalent), 10 equivalents of 0.5 M TCEP aqueous solution were added. The reaction mixture was incubated at 37°C for 2 hours. Excess TCEP was removed from the reaction mixture using an Amicon® Ultra centrifugal filter with a 10 kDa MWCO Ultracel® regenerated cellulose membrane, and the mixture was concentrated by centrifugation at 4000 rpm for 10 minutes. The residue was washed by centrifugation at 4000 rpm with pH 7.4 PBS and buffer (×5), and the resulting residue was replaced with pH 7.2 PBS buffer (PBS 10 mM; EDTA 5 mM; degassed with nitrogen).

[0434] Step 2: Nanobody-Cys / Me(MAL)-PEG 24 Synthesis of -CONH-PEG3-TCO SRS-14. Reduced nanobodies (Step 1, 3.16 mg / mL) are dissolved in aqueous PBS pH 7.2 buffer (10 mM PBS, 5 mM EDTA) and Me(MAL)-PEG3. 24A deionized aqueous solution of -CONH-PEG3-TCO SRS-12 (2 equivalents; 10 mg / mL) was added. The resulting solution was left to stand at 4°C, and the reaction was monitored by UPLC analysis (UPLC method 2). After 18 hours, excess linker SRS-12 was removed from the reaction mixture using an Amicon® Ultra centrifugal filter equipped with a 10 kDa MWCO Ultracel® regenerated cellulose membrane, and the mixture was concentrated by centrifugation at 4000 rpm for 10 minutes. The residue was washed with aqueous PBS buffer (pH 7.2) (10 mM PBS, 5 mM EDTA) by centrifugation at 4000 rpm (×5).

[0435] Step 3: Conjugation reaction: Deionized aqueous solution of tetrazine-substituted dendrimer (10 mg / mL; 1 equivalent) is converted to nanobody-Cys / Me(MAL)-PEG 24 -CONH-PEG3-TCO SRS-14 (1.2 equivalents) was added to a solution at room temperature. After 18 hours, the nanobody-dendrimer constructs were purified by nickel affinity column chromatography and subsequent SEC.

[0436] General Procedure for Quantification of L.Cy5-labeled Dendrimer-Nanobody Conjugates The purified conjugates were quantified by interpolating the 650 nm absorbance of the measured samples against a standard curve prepared using the corresponding non-conjugate dendrimer. Absorbance measurements were performed on a Nanodrop ND-1000 spectrophotometer (Thermo Fisher). For each dendrimer-nanobody conjugate, the amount of dry non-conjugate dendrimer, weighed using a digital microbalance (Mettler-Toledo), was dissolved in an appropriate volume of 10 mM HEPES pH8 buffer. Standard solutions of known concentrations (10 mg / ml to 0.05 mg / ml) were prepared by diluting them in 10 mM HEPES pH8 buffer, and their absorbance was measured at 650 nm. The standard curves and interpolations generated by linear regression were performed using Prism9 software (Graphpad). [Table 4-1] Table 4-2 Table 4-3 Table 4-4 Table 4-5 Table 4-6 Table 4-7 Table 4-8 Table 4-9 Table 5-1 Table 5-2 Table 5-3 Table 5-4 Table 5-5 Table 5-6 [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8] [Table 6-9]

[0437] Example 1: Synthesis of intermediates and controls 1a. Synthesis of bifunctional linkers and lysine wedges 1a.1 BCN-PEG2-Glu-CO-NHPEG 24 CO2H, compound 49 NH2-PEG in a water / THF (1:1, 4 mL) mixture 24Sodium bicarbonate (15.2 mg, 0.181 mmol) was added to a solution of -COOH (93.7 mg, 0.082 mmol). The mixture was stirred at room temperature for 5 minutes, and then a solution of BCN-PEG2-Glu-NHS ester (50 mg, 0.093 mmol) in THF (2 mL) was added. The resulting reaction mixture was stirred at room temperature for 15 hours. After that, volatile components were removed under reduced pressure, and MeCN (2.5 mL) was added to the resulting aqueous suspension. This solution was subjected to preparative HPLC (preparative HPLC method 5-60% TFA) R t After purification for 32.2 to 34.3 minutes and freeze-drying, compound 49 was obtained as a white solid (42 mg, 33%). 1 H NMR(300MHz,D2O)δ(ppm):0.85-0.92(m,2H);1.25-1.35(m,1H);1.43-1.57(m,2H);1.73-1.83(m,2H);2 .09-2.25(m,9H);2.39(t,J=9.0Hz,2H);3.21-3.31(m,6H);3.35-3.85(m,106H);4.10(d,J=9.0Hz,2H). LCMS (hydrophilic method, formic acid buffer) R t =10.28 min, ESI MS(+ve)m / z 1566.7[M] + .

[0438] 1a.2 BCN-PEG2-Glu-CO-NHPEG 24 CO-NHPEG3-TCO, compound 50 BCN-PEG2-Glu-CO-NHPEG in DMF (3.0 mL) 24 -COOH, compound 49 (10.0 mg, 0.006 mmol) was stirred, to which PyBOP (3.64 mg, 0.007 mmol), NMM (1.31 μL, 0.012 mmol), and then TCO-PEG3-NH2 (Click Chemistry Tools, 2.41 mg, 0.007 mmol) were added. The resulting reaction mixture was then stirred overnight at room temperature. After that, the solvent was removed under reduced pressure, and the resulting residue was dissolved in MeCN (2 mL) and filtered through a 0.45 μm filter. The collected filtrate was subjected to preparative HPLC (preparative HPLC method 30-50% TFA) R tThe product was purified for 40-42 minutes, and the product-containing fraction was concentrated under reduced pressure to remove MeCN. The remaining aqueous solution was freeze-dried overnight to obtain compound 50 as a white solid (4.7 mg, 39%). 1 H NMR(300MHz,CD3OD)δ(ppm): 0.89-1.05(m,2H),1.26-1.48(m,2H),1.52-1.79(m,5H),1.83-2.06(m,6H),2.11-2.39(m,12H),2.48(t,2H,J=6.0Hz),3.35-3. 44(m,6H),3.47-3.79(m,102H),3.83-3.92(m,1H),4.16(d,2H,J=6.0Hz),4.26-4.41(m,1H),4.58(bs,10H),5.39-5.74(m,3H). LCMS (hydrophilic method, formic acid buffer) R t =11.0 minutes. ESI MS(+ve)1894.0[M] + ;C 90 H 165 N5O 36 [M] + m / z calculation value for: 1894.2

[0439] 1a.3 DBCO-Glu-NHPEG 24 CO-NHPEG3-TCO, compound 51 DBCO-Glu-NHPEG in DMF (3 mL) 24 To a stirred solution of COOTFP (50.0 mg, 0.031 mmol), TCO-PEG3-NH2 (Click Chemistry Tools, 10.7 mg, 0.031 mmol) was added, followed by NMM (4.08 μL, 0.037 mmol). The resulting reaction mixture was then stirred at room temperature for 3 hours and then concentrated under reduced pressure. The resulting residue was dissolved in MeCN (2 mL), filtered through a 0.45 μm filter, and the filtrate was purified by preparative HPLC (preparative HPLC method, 40-70% TFA) with a Rt of 27-29 minutes. The product-containing fraction was concentrated under reduced pressure to remove MeCN, and the remaining aqueous solution was freeze-dried overnight to obtain compound 51 as a viscous, colorless liquid (25.0 mg, 42%). 1H NMR(300MHz,CD3OD)δ(ppm): 1.31-1.62(m,4H),1.60-1.83(m,6H),1.92-2.16(m,4H),2.25(t,2H,J=6.0Hz),2.91-3.03(m,4H),3.12-3. 58(m,78H),3.59-3.69(m,1H),4.01-4.18(m,1H),4.92(d,2H,J=15H),5.15-5.49(m,3H),6.95-7.59(m,8H). LCMS (hydrophilic method, formic acid buffer) R t =7.0 minutes. ESI MS(+ve)1775.0.0[M] + ;C 88 H 148 N4O 32 [M] + m / z calculated value for this: 1775.12

[0440] 1a.4 N3-PEG7-NHCO-Lys[(α-NHBoc)(ε-NHFmoc)], compound SRS-3 To a stirred solution of N3-PEG7-NH2 (QuantaBiodesign, 514 mg, 1.30 mmol), HO-Lys[(α-NHBoc)(ε-NHFmoc)] (488 mg, 1.04 mmol), and NMM (286 μL, 2.60 mmol) in DMF (5 mL), PyBOP (811 mg, 1.56 mmol) was added, and the reaction mixture was stirred at room temperature for 18 hours. Volatile substances were removed under reduced pressure, and the residue was purified by column chromatography on silica gel eluted with dichloromethane:MeOH (100:0~90:10 v / v) to obtain SRS-3 as a viscous colorless oil (410 mg, 47%). LC-MS (LC-MS method 20~90, 15 min):R t =9.35 minutes, ESI MS(+ve)845[M+H] + ;C 42 H 64 N6O 12 [M+H] + The calculated m / z value for this is 845; 1H NMR(300MHz,MeOD)δ(ppm): 1.17-1.94(m,16H),3.12(t,2H,J=6.0Hz),3.33-3.44(m,4H),3.45-3.74(m,28H),3.89-4.65(m,1H),4.12(t,1H,J=9. 0Hz),4.38(d,2H,J=6.0Hz),7.33(t,2H,J=9.0Hz),7.41(t,2H,J=9.0Hz),7.66(d,2H,J=6.0Hz),7.81(d,2H,J=9.0Hz).

[0441] 1a.5N3-PEG7-NHCO-Lys[(α-NH2.HCl)(ε-NHFmoc)], compound SRS-3a Compound SRS-3, N3-PEG7-NHCO-Lys[(α-NHBoc)(ε-NHFmoc)] (200 mg, 0.236 mmol), was dissolved in 1.5 M HCl methanol (4 mL), and the reaction mixture was stirred at room temperature for 3 hours. Volatile components were removed under reduced pressure to obtain N3-PEG7-NHCO-Lys[(α-NH2.HCl)(ε-NHFmoc)]SRS-3a as a white solid (180 mg, 98%). LC-MS (LC-MS method 20-90°C, 15 min): R t =6.13 minutes, ESI MS(+ve)745[M+H] + ;C 37 H 56 N6O 10 [M+H] + The calculated m / z value for this is 745.

[0442] 1a.6 N3-PEG7-NHCO-Lys[(α-NHCy5)(ε-NHFmoc)], compound SRS-4 To a stirred solution of N3-PEG7-NHCO-Lys[(α-NH2.HCl)(ε-NHFmoc)]SRS-3a (58 mg, 0.074 mmol) and NMM (16 μL, 0.148 mmol) in DMF (5 mL), Cy5 NHS ester (50 mg, 0.074 mmol) was added. The reaction mixture was stirred at room temperature for 20 hours, and volatile matter was removed under reduced pressure. The residue was dissolved in MQ water:MeCN (2 mL, 1:1 v / v), filtered (through a 0.45 μm Acrodisc filter), and purified by preparative HPLC to obtain compound SRS-4 as a blue solid (30 mg, 33%). HPLC: C18 column with gradients of 30% MeCN / H2O (1-10 min), 30-90% MeCN / H2O (10-35 min), 90% MeCN / H2O (35-48 min), 90-30% MeCN / H2O (48-55 min), and 0.05% formate buffer), and UV detection at 254 nm. t =35~40 minutes. LCMS (LCMS method 20~90, 15 minutes) R t =8.20 minutes, ESI MS(+ve)1210[M+H] + ;C 69 H 93 N8O 11 [M+H] + The calculated m / z value for this is 1210.

[0443] 1a.7 N3-PEG7-NHCO-Lys[(α-NHCy5)(ε-NH2)], compound SRS-4a N3-PEG7-NHCO-Lys[(α-NHCy5)(ε-NHFmoc)] compound SRS-4 (30 mg, 0.024 mmol) was dissolved in DMF:piperzine (3.0 mL, 4:1 v / v), and the reaction mixture was stirred at room temperature. After 18 hours, volatile matter was removed under reduced pressure, and the residue was purified by preparative HPLC to obtain N3-PEG7-NHCO-Lys[(α-NHCy5)(ε-NH2)]SRS-4a as a blue solid (18.0 mg, 75%). HPLC: C18 column with gradients of 20% MeCN / H2O (1-10 min), 20-60% MeCN / H2O (10-35 min), 60% MeCN / H2O (35-48 min), 60-20% MeCN / H2O (48-55 min), and 0.05% formate buffer), and UV detection at 214 and 254 nm.t =24~28 minutes. LCMS (LCMS method 20~90, 15 minutes) R t =4.91 min, ESI MS(+ve)988[M+H] + ;C 54 H 83 N8O9[M+H] + The calculated m / z value for this is 988.

[0444] 1a.8 N3-PEG7-NHCO-Lys[(α-NHCy5)(ε-NHDOTA)] Compound SRS-5 To a stirred solution of amine SRS-4a (18.0 mg, 0.018 mmol) in DMF (4 mL), solutions of NMM (4 μL, 0.036 mmol) and p-SCN-Bn-DOTA (13 mg, 0.018 mmol) in DMSO (500 μL) were added. After stirring for 18 hours, volatile matter was removed under reduced pressure, and the residue was purified by preparative HPLC to obtain SRS-5 as a blue solid (9.0 mg, 32%). HPLC: C18 column with gradients of 40% MeCN / H2O (1-10 min), 40-80% MeCN / H2O (10-35 min), 80% MeCN / H2O (35-48 min), 80-40% MeCN / H2O (48-55 min), and 0.05% formate buffer), and UV detection at 214 and 254 nm. t =26~28 minutes. LCMS (LCMS method 20~90, 15 minutes) R t =3.95 minutes, ESI MS(+ve)1538[M+H] + ;C 78 H 116 N 13 O 17 S[M+H] + The calculated m / z value for this is 1538.

[0445] 1a.9 DFO-PEG4-sulfo-DBCO, compound 55 A suspension of deferoxamine-SCN (22.3 mg, 29.7 mmol) and sulfo-PEG3-DBCO (20.0 mg, 29.7 mmol) in DMF (2 mL) was mixed with NMM (6.5 μL, 59.1 μmol). Sonication did not yield a homogeneous solution. Addition of NMM (10 μL, 91.0 μmol) and DIPEA (10 μL, 57.4 μmol) did not further solubilize the reaction mixture. DMSO (2 mL) was added, and the contents were sonicated for 1 minute to obtain a clear solution. The resulting reaction mixture was then stirred overnight at room temperature. The contents were concentrated under reduced pressure, and MeCN (12 mL) was added to the residual solution, after which the contents were filtered (0.45 μm acrodisc). The filtrate was subjected to preparative HPLC; injection volume 1000 μL, 5-80% MeCN, 60 minutes, 0.1% formic acid, R t The product was purified for 33.5 to 34.5 minutes. The product fractions were combined, MeCN was removed under reduced pressure, and the remaining aqueous solution was freeze-dried to obtain the title product as a white solid of 12.7 mg (30%). LC-MS (hydrophilic method, formate buffer) R t =4.70 min, ESI MS(+ve) 1428[M] + ;C 65 H 94 N 12 O 18 S3[M] + The calculated m / z value for this is 1427.7. 1 H NMR(300MHz,D2O)δ(ppm): 7.71-6.98(m,12H),5.11-4.95(m,1H),3.90-3.01(m,36H),2.87-2.30(m,11H),2.21-1.90(m,4H),1.80-1.16(m,17H).

[0446] 1a.10 Synthesis of DFO-DBCO compound 56 To a stirred solution of deferoxamine mesylate (Macrocyclics, 200.0 mg, 0.304 mmol) in DMSO (1 mL), NMM (100.0 μL, 0.912 mmol) and DBCO-NHS ester (130.7 mg, 304 mmol) were added. The reaction mixture was stirred overnight at room temperature, and then concentrated by blowing a nitrogen gas stream over the solution for 2 hours. The residue was dissolved in a mixture of MQ water:acetonitrile (3 mL, 1:1 v / v) and then filtered (0.45 μm Acrodisc syringe filter). The filtrate was purified by preparative HPLC. MQ water 30-40% MeCN + 0.1% formic acid (60 min, R t The product was obtained as a colorless viscous liquid (195.0 mg, 73%) by 42-44 minutes. LC-MS (hydrophilic method, TFA buffer) R t = 5.64 min, ESI MS(+ve) 876[M] + ;C 46 H 65 N7O 10 [M] + The calculated m / z value for [M+Fe] is 876. + =929; 1 H NMR(300MHz,DMSO-d6)δ(ppm): 9.96-9.46(bs,2H),7.92-7.24(m,9H),5.09(d,1H,J=15.0Hz),3.66(d,1H,J=12.0Hz),3.56-3.43(m,10H),3.13-2 .87(m,4H),2.69-2.58(m,4H),2.40-2.27(m,3H),2.13(s,6H),2.02(s,2H),1.94-1.75(m,2H),1.68-1.09(m,17H).

[0447] 1a.11 Synthesis of TCO-PEG8-dibromomaleimide compound 57 To a stirred solution of 3,4-dibromo-2,5-dioxo-2H-pyrrole-1(5H)-carboxylate ethyl (25.0 mg, 0.077 mmol) in THF (2.0 mL), TCO-PEG8-NH2 (Broadpharm; 44.0 mg, 0.077 mmol) was added, and the reaction mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure, and the residue was purified by column chromatography on silica gel eluted with dichloromethane:MeOH (gradient elution of 100:0 to 97:3 v / v). Subsequently, compound 57 was obtained as a colorless oil by preparative HPLC eluting with 50-90% MeCN in MQ water over 60 minutes (8.0 mg, 13%). 1 H NMR(300MHz,MeOD)5.79-5.42(m,2H),4.77-4.62(m,1H),3.80(t,2H,J=6.0Hz),3.70-3.57( m,29H),3.53(t,2H,J=6.0Hz),3.27(t,2H,J=6.0Hz),2.48-2.30(m,1H),2.26-1.46(m,9H).

[0448] 1a.12 Synthesis of HO-Lys[(α-NHCy5)(ε-NHDFO)] wedge and compound 58 To a stirred solution of HO-Lys[(α-NH2.TFA)(ε-NHFmoc)] (57.0 mg, 0.118 mmol) in DMF (5 mL), NMM (52 μL, 0.472 mmol) and Cy5-NHS ester (Lumiprobes, 40.0 mg, 0.059 mmol) were added. The reaction mixture was stirred overnight at room temperature, and volatile components were removed by vacuum. The residue was dissolved in MeCN:MQ water (3 mL 1:1 v / v), and the solution was filtered (0.45 μm Acrodisc syringe filter). The filtrate was subjected to preparative HPLC; 20-90% MeCN in MQ water + 0.1% formic acid (60 min, R) t The compound 58 was purified by 39.0-42.0 minutes to obtain 33 mg (67%) of compound 58 as a blue solid. LC-MS (hydrophilic method, TFA buffer) R t =10.54 min, ESI MS(+ve)833[M] + ;C 53 H 61 N4O5[M] + The calculated m / z value for this is 833.46.1 H NMR(300MHz,D2O)δ(ppm): 8.21(t,2H,J=15.0Hz),7.79(d,2H,J=6.0Hz),7.63(d,2H,J=6.0Hz),7.49-7.24( m,10H),6.61(t,1H,J=12.0Hz),6.29-6.22(m,2H),4.49-4.26(m,2H),.18(t,1H, J=6.0Hz),4.07(t,2H,J=6.0Hz),3.68-3.60(m,2H),3.60(s,3H),3.12(t,2H,J=6 .0Hz),2.28(t,2H,J=6.0Hz),1.94-1.61(m,6H),1.71(s,12H),1.62-1.19(m,6H).

[0449] 1a.13 HO-Lys[(α-NHCy5)(ε-NH2)], compound 59 To a stirred solution of compound 58 (36 mg, 0.043 mmol) in DMF (4 mL), piperidine (1.5 mL) was added, and the reaction mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the residue was separated and HPLC analyzed; MQ water + 0.1% formic acid in 20-90% MeCN (60 min, R) t The compound 59 was purified by 32.0-33.0 minutes to obtain 12 mg (44%) of the product, compound 59, as a blue solid. LC-MS (hydrophilic method, TFA buffer) R t =7.65 min, ESI MS(+ve)611[M] + ;C 38 H 51 N4O3[M] + The calculated m / z value is 611.

[0450] 1a.14 HO-Lys[(α-NHCy5)(ε-NHDFO)], compound 60 To a stirred solution of compound 59 (20.0 mg, 0.032 mmol) in DMSO (5 mL), DIPEA (32 μL, 0.224 mmol) was added, followed by p-SCN-deferoxamine (Macrocyclics, 24.0 mg, 0.032 mmol). The reaction mixture was stirred at room temperature for 4 hours, and then concentrated by blowing a nitrogen gas stream over the solution for several hours. The resulting residue was then purified by preparative HPLC. (30-60% MeCN in MQ water + 0.1% TFA for 60 minutes, R) t 39-42 minutes) 15 mg (34%) of the product, compound 60, was obtained as a blue solid. LC-MS (hydrophilic method, TFA buffer) R t = 5.93 min, ESI MS(+ve) 1364[M] + ;C 71 H 103 N 12 O 11 S2[M] + The calculated m / z value for this is 1364. 1 H NMR(300MHz,D2O)δ(ppm): 8.25(t,2H,J=12.0Hz),7.56-7.18(m,9H),6.65(t,1H,J=12.0Hz),6.37-6.15(m,2H),4.43-4.32(m,1H),4.11(t,2H,J=6.0Hz),3. 70-3.46(m,8H),3.22-3.10(m,3H),2.86-2.69(m,3H),2.56-2.38(m,3H),2.31(t,2H,J=6.0Hz),2.11(s,2H),1.96-1.18(m,32H). 1a.15 Synthesis of Compound 61, DUPA-BCN linker (17S,21S)-1-((1R,8S,9S)-bicyclo[6.1.0]non4-in-9-yl)-3,14,19-trioxo-2,7,10-trioxa-4,13,18,20-tetraazathricosan-17,21,23-tricarboxylic acid

[0451] To a stirred solution of 3.3(13S,17S)-1-amino-10,15-dioxo-3,6-dioxa-9,14,16-triazanonadecane-13,17,19-tricarboxylic acid compound 48 (149 mg, 0.26 mmol) and triethylamine (184 μL, 1.32 mmol) in a mixture of tetrahydrofuran and water (1:1, 4 mL), ((1R,8S,9s)-bicyclo[6.1.0]non4-in-9-yl)methyl(2,5-dioxopyrrolidine-1-yl)carbonate dichloromethane (BCN-NHS ester) (84 mg, 0.29 mmol) was gradually added. The reaction mixture was stirred at ambient temperature for 2.5 hours, after which volatile components were removed by vacuum. The residue was purified by preparative HPLC (Gilson HPLC system; column: X-Bridge BEH300 Prep C18 5um OBD 30×150 mm; solvent: A = deionized water containing 0.05% formic acid; B = MeCN containing 0.05% formic acid; flow rate: 8 mL / min), and after lyophilization, the title product, compound 61 (120 mg, 73%), was obtained as a white solid. 1 H NMR(300MHz,D2O)δ(ppm): 0.77-0.90(m,2H);1.13-1.36(m,1H);1.45-1.49(m,2H);1.79-1.93(m,2H);2.01-2.19(m,9H);2.24-2.3 1(m,2H);2.39-2.44(m,2H);3.20-3.30(m,4H);3.48-3.52(m,4H);3.56-3.62(m,4H);4.06-4.19(m,4H). LCMS (hydrophilic method, formic acid buffer) R t =8.92 minutes. ESI MS(+ve)627[M+1] + ;C 28 H 42 N4O 12 [M] + The calculated m / z value for this is 626.28.

[0452] 1 a.16 BocHN-PEG3-NHCO-PEG 24 -CONH-PEG4-(PhTzMe) SRS-7 HO2C-PEG in DMF (12.0 mL) 24A stirred solution of -CONH-PEG4-(PhTzMe), SRS-5a (500 mg, 0.32 mmol), BocNH-PEG3-NH2, SRS-6 (1-2.5 mg, 0.32 mmol), and NMM (53 μL, 0.48 mmol) was mixed with PyBOP (166 mg, 0.32 mmol) at room temperature. The reaction mixture was stirred for 16 hours, after which volatile components were removed by vacuum. The residue was dissolved in MeCN (3.0 mL), filtered (on a 0.45 μm filter disc), and subjected to preparative HPLC (preparative HPLC method 20-60°C, R2). t Purified by (34-47 minutes), BocHN-PEG3-NHCO-PEG 24 CONH-PEG4-(PhTzMe)SRS-7 was obtained as a red solid (550 mg, 92%). 1 H NMR(300MHz,CD3OD):δ(ppm)1.78(q,J=6.7Hz,2H),1.95(q,J=6.2Hz,2H),2.46(t,J=6.2Hz,4H),3.02(s,3H),3.13(t,J=6.8Hz,2H),3.26- 3.43(m,4H),3.50-3.58(m,4H),3.58-3.78(m,128H),3.89-3.95(m,2H),4.25-4.32(m,2H),7.20(d,J=9.0Hz,2H) and 8.52(d,J=9.0Hz,2H). LCMS (LCMS method 20~60, 8 minutes, TFA)R t =6.12 minutes. ESI MS(+ve)1885 [M+H2O] + ;C 86 H 159 N7O 36 [M+H2O] + The calculated m / z value for this is 1885.

[0453] 1 a.17 HCl.H2N-PEG3-NHCO-PEG 24 -CONH-PEG4-(PhTzMe)SRS-8 BocHN-PEG3-NHCO-PEG 24-CONH-PEG4-(PhTzMe)SRS-7 (450 mg, 0.241 mmol) was mixed with 2.0 M HCl in methanol (4.0 mL) at room temperature. The resulting solution was stirred for 18 hours, and volatile components were removed by vacuum. The residue was dissolved in water (4.0 mL), freeze-dried, and then HCl.H2N-PEG3-NHCO-PEG. 24 -CONH-PEG4-(PhTzMe)SRS-8 was obtained as a pink solid of 420 mg (97%). LC-MS (LC-MS method 5-60°C, 8 mins, TFA):R t =5.72 minutes, ESI MS(+ve)1769[MH] + ;C 81 H 151 N7O 34 [MH] + The calculated m / z value for this is 1769.

[0454] 1a.18 Me(MAL)-PEG 24 -CO2H, SRS-10 Amino-PEG in glacial acetic acid (15 mL) 24 - To a stirred solution of acid RL-11 (510 mg, 0.44 mmol), citraconic anhydride (50 μL, 0.53 mmol) was added. The reaction mixture was then heated at 120°C for 2 hours with stirring, and after cooling to room temperature, an additional aliquot of citraconic anhydride (50 μL, 0.53 mmol) was added. The reaction mixture was heated at 120°C for 2 hours and then cooled to room temperature. After 16 hours, the reaction mixture was concentrated under vacuum, and the residual brown oil was purified by preparative HPLC (preparative HPLC method 5-60, Rt=35-37.5 min) to obtain Me(MAL)-PEG. 24 -CO2H SRS-10 was obtained as a light brown solid (236 mg, 43%). 1 H NMR (300MHz, CD3OD): δ (ppm) 2.07 (d, J = 1.8 Hz, 3 H), 2.56 (t, J = 6.3 Hz, 2 H), 3.48-3.72 (m, 98 H), 3.75 (t, J = 6.3 Hz, 2 H) and 6.46 (q, J = 1.8 Hz, 1 H). LCMS (LCMS method 60~90, 8 minutes, TFA):R t =5.54 minutes. ESI MS(+ve)1258[M+H2O] + ;C 56H 107 NO 29 The calculated m / z value for [M+H2O] is 1258.

[0455] 1a.19 Me(MAL)-PEG 24 -CONH-PEG3-TCO, SRS-12 Me(MAL)-PEG in DMF (2.0 mL) 24 To a stirred solution of -CO2H SRS-10 (130 mg, 0.105 mmol), NH2-PEG3-TCO SRS-11 (47 mg, 0.126 mmol), PyBOP (65 mg, 0.126 mmol), and NMM (23 μL, 0.126 mmol) were added at room temperature. After stirring for 3.5 hours, volatile components were removed by vacuum, and the residue was dissolved in MeCN:water (1:2 v / v, 3 mL) and subjected to preparative HPLC (Preparative HPLC method 5~60(2), R t Purified by (41-43 minutes), then Me(MAL)-PEG 24 -CONH-PEG3-TCO SRS-12 was obtained as a colorless oil (77 mg, 46%). 1 H NMR(300MHz,CD3OD):δ(ppm)1.53-2.40(m,15H),2.07(d,J=1.85Hz,3H),2.45(t,J=6.2Hz,2H), 3.13-3.21 (m, 2H), 3.28 (t, J=6.8Hz, 2H), 3.47-3.81 (m, 118H), 5.42-5.84 (m, 2H) and 6.46 (m, 1H). LCMS (LCMS method 5~60, 8 minutes, TFA)R t =6.51 minutes. ESI MS(+ve)1612 [M+H2O] + ;C 75 H 141 N3O 33 [M+H2O] + The calculated m / z value for this is 1612.

[0456] 1a.20 Br2(MAL)-PEG3-NHCO-PEG 24 -CONH-PEG4-(PhTzMe), RL-15 HCl, H2N-PEG3-NHCO-PEG in THF (2.0 mL) 24To a solution of -CONH-PEG4-(PhTzMe)SRS-8 (100 mg, 0.057 mmol), TEA (15 μL, 0.068 mmol) and 1H-pyrrole-1-carboxylic acid,3,4-dibromo-2,5-dihydro-2,5-dioxo-ethyl ester RL-12 (22 mg, 0.068 mmol) were added at room temperature. The reaction mixture was stirred for 18 hours and then concentrated under vacuum. The residue was dissolved in MeCN:water (2.0 ml, 1:1 v / v) and preparative HPLC (40-60°C, R) was used. t Purify by (25-27 minutes) and Br2(MAL)-PEG3-NHCO-PEG 24 -CONH-PEG4-(PhTzMe)RL-15 (42 mg, 37%) was obtained.

[0457] Alternatively, in a solution of Br2(MAL)-PEG3-NH2.TFA RL-17 (202 mg, 0.35 mmol) in DMF (3.0 mL), add HO2C-PEG 24 CONH-PEG4-(PhTzMe) SRS-5a (461 mg, 0.30 mmol), PyBOP (184 mg, 0.35 mmol), and NMM (85 μL, 0.78 mmol) were added at room temperature. The reaction mixture was stirred for 18 hours and then concentrated under vacuum. The residue was dissolved in MeCN:water (1:1 v / v, 4.0 mL) and purified by preparative HPLC [Preparative HPLC method 20~60(2)] to obtain Br2(MAL)-PEG3-NHCO-PEG 24 CONH-PEG4-(PhTzMe)RL-15 was obtained as a pink solid (37 mg, 6%). 1 H NMR(300MHz,CD3OD):δ(ppm)1.70-1.82(m,2H),1.82-1.93(m,2H),2.45(m,4H),3.02(s,3H),3.20-3.4 3(m,4H), 3.47-3.89(m,132H), 3.91(m,2H), 4.28(m,2H), 7.20(d,J=9.0Hz,2H) and 8.50(d,J=9.0Hz,2H). LCMS (LCMS method 40~60, 8 minutes, TFA)R t =5.12 minutes. ESI MS(+ve)2024 [M+H3O] + ;C 85 H 149Br2N7O 36 [M+H3O] + The m / z calculation value for this is 2024.

[0458] 1a.21 Br2(MAL)-PEG3-NHBoc, RL-16 A solution of 1H-pyrrole-1-carboxylic acid,3,4-dibromo-2,5-dihydro-2,5-dioxo-ethyl ester RL-12 (212 mg, 0.66 mmol) in dichloromethane (2.0 mL) was mixed with a solution of BocHN-PEG3-NH2SRS-6 at room temperature. The reaction mixture was stirred for 2 days, after which volatile components were removed by vacuum. The residue was purified by column chromatography on silica gel that elutes methanol [gradient elution; % methanol (v / v)]: ​​0%~3.3%~6.7% in dichloromethane to obtain Br2(MAL)-PEG3-NHBoc RL-16 as an off-white solid (220 mg, 59%). 1 H NMR (300MHz, CD3CN): δ (ppm) 1.41 (s, 9H), 1.67 (m, 2H), 1.82 (m, 2H), 3.10 (q, J = 6.7Hz, 2H), 3.42-4.58 (m, 12H) and 3.64 (t, J = 7.1Hz, 2H). LCMS (LCMS method 60~90, 8 minutes, TFA)R t =3.99 minutes. ESI MS(+ve)459 [MH-Boc] + ;C 14 H 23 Br2N2O5[MH-Boc] + The calculated m / z value for this is 459.

[0459] 1a.22 Br2(MAL)-PEG3-NH2.TFA, RL-17 To a solution of Br2(MAL)-PEG3-NHBoc RL-16 (220 mg, 0.40 mmol) in dichloromethane (6.0 mL), TFA (609 μL, 7.79 mmol) was added at room temperature. The reaction mixture was stirred for 2 hours and then vacuum concentrated. The residue was dissolved in deionized water (approximately 2 mL), and the resulting solution was freeze-dried to obtain Br2(MAL)-PEG3-NH2.TFA RL-17 as an off-white solid (202 mg, 88%). 1H NMR (300MHz, CD3OD): δ (ppm) 1.84-1.98 (m, 4H), 3.12 (t, J = 6.4Hz, 2H) and 3.49-3.74 (m, 14H). LCMS (LCMS method 60~90, 8 minutes, TFA)R t =3.19 minutes. ESI MS(+ve)459 [MH] + ;C 14 H 23 Br2N2O5[MH] + The calculated m / z value for this is 459.

[0460] 1a.23 Me(MAL)-PEG 24 -CONH-Bn-Tz(Me), RL-18 Me(MAL)-PEG in DMF (2.0 mL) 24 To a solution of -CO2H SRS-10 (50 mg, 0.04 mmol) and 4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzenemethaneamine RL-13 (11 mg, 0.06 mmol), PyBOP (24 mg, 0.06 mmol) and NMM (6 μL, 0.06 mmol) were added at room temperature. The reaction mixture was stirred for 16 hours, after which 4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzenemethaneamine RL-13 (5 mg, 0.02 mmol) was added to DMF (2 mL), followed by PyBOP (12 mg, 0.02 mmol) and NMM (6 μL, 0.06 mmol). After 2 hours, the reaction mixture was concentrated under vacuum, and the residue was subjected to preparative HPLC (preparative HPLC method 20-40, R). t Purified by (45-48 minutes), then Me(MAL)-PEG 24 -CONH-Bn-Tz(Me)RL-18 was obtained as a pink solid (24 mg, 42%). 1 H NMR(300MHz,CD3OD):δ(ppm)2.06(d,J=1.9Hz,3H),2.56(t,J=6.0Hz,2H),3.05(s,3H),3. 58-3.84(m,96H),4.55(s,2H),6.46(m,1H),7.59(d,J=8.6Hz,2H) and 8.53(d,J=8.6Hz,2H). LCMS (LCMS method 60~90, 8 minutes, TFA)R t= 6.07 minutes. ESI MS(+ve)1424[M] + ;C 66 H 114 N6O 27 [M] + The calculated m / z value for this is 1424.

[0461] 1b. Synthesis of dendrimer intermediates 1b.1 Azide-PEG 24 CO-[N(PNBoc)2], compound 1 Azide-PEG in DMF (20 mL) under N2 atmosphere 24 - To a stirred solution of acid (Quanta Biodesign, 2.00 g, 1.71 mmol) and PyBOP (1.33 g, 2.56 mmol), NMM (563 μL, 5.12 mmol) was added. After 10 minutes, a DMF (5 mL) solution containing N(PNBoc)2 (622 mg, 1.88 mmol) was added, and the resulting reaction mixture was stirred overnight at room temperature. After removing volatile components by vacuum, the resulting oil was dissolved in MeCN and preparative HPLC (27-50-70% MeCN, R t The mixture was purified by 47-50 minutes to obtain a pale yellow oily solid (1.37 g, 54%). 1 H NMR(300MHz,CD3OD)δ(ppm): 1.44(m,18H);1.65-1.84(m,4H);2.63(t,J=6.3Hz,2H);3.05(dt,J=6.9 and 14.7Hz,4H);3.36-3.41(m,6H);3.60-3.78(m,98H). LCMS (hydrophilic method, formic acid buffer) R t =9.32 minutes. ESI MS(+ve)1486.3[M] + ;C 67 H 132 N6O 29 [M] + The calculated m / z value for this is 1486.8.

[0462] 1b.2 Azide-PEG 24 CO-[N(PNH2.TFA)2], compound 2 Azido-PEG 24CO-[N(PNBoc)2], compound 1 (1.37 g, 922 μmol) was used to prepare the compound according to general procedure A. The lyophilized product, compound 2, was obtained as a pale yellow oil (1.67 g, 119%). 1 H NMR(300MHz,D2O)δ(ppm): 1.88-2.06(m,4H);2.74(t,J=6.0Hz,2H);2.96(t,J=7.2Hz,2H);3.04(apparent t,J=7.5Hz,2H);3.42-3.52(m,6H);3.67-3.95(m,93H). LCMS (hydrophilic method, TFA buffer) R t =8.47 min, ESI MS(+ve) 1286.0[M] + ;C 57 H 116 N6O 25 [M] + The calculated m / z value is 1286.6.

[0463] 1b.3 Azide-PEG 24 CO-[N(PN)2][Lys]2[NHBoc]4, G1, Compound 3 Azido-PEG 24 The material was prepared using CO-[N(PNH2.TFA)2] and compound 2 (186 mg, 145 μmol) according to general procedure B. The crude material was dissolved in MeCN and preparative HPLC (30-80% MeCN, R) was performed. t The compound was purified for 33.5 to 36 minutes to obtain compound 3 as a pale yellow oil (224 mg, 80%). 1 H NMR(300MHz,CD3OD)δ(ppm): 1.22-1.87(m,56H);2.64(t,J=6.0Hz,2H);3.03(t,J=6.6Hz,4H);3.13-3.23(m,4H),3.36-3. 45(m,6H);3.60-3.69(m,100H);3.77(t,J=6.0Hz,2H);3.85-3.88(m,1H);3.92-4.02(m,2H). LCMS (hydrophobic method, formic acid buffer) R t =6.74 minutes, ESI MS(+ve)1942.4[M] + ;C 89 H 172 N 10 O 35 + [M+H]+ The calculated m / z value for this is 1942.4.

[0464] 1b.4 Azide-PEG 24 CO-[N(PN)2][Lys]2[NH2.TFA]4, G1, compound 4 Azido-PEG 24 Compound 3 (220 mg, 113 μmol) was prepared using CO-[N(PN)2[Lys]2[NHBoc]4] according to general procedure A. The lyophilized product, compound 4, was obtained as a pale yellow oil (251 mg, 111%). LC-MS (hydrophilic method, formate buffer) R t =6.49 min, ESI MS(+ve) 1542.1[M] + ;C 69 H 140 N 10 O 27 [M] + The calculated m / z value is 1541.90.

[0465] 1b.5 Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[(α-NHBoc)(ε-NH-COPEG 1100 )]4, G2, compound 5 Azido-PEG 24 The material was prepared using CO-[N(PN)2[Lys]2[NH2.TFA]4, compound 4 (120 mg, 60.1 μmol) according to general procedure C. The crude material was dissolved in MeCN / H2O (1:1) and preparative HPLC (20-70% MeCN, R) was performed. t The compound was purified over 31-32.5 minutes to obtain compound 5 as a pale yellow oil (244 mg, 59%). 1 H NMR(300MHz,CD3OD)δ(ppm): 1.24-1.91(m,74H);2.42-2.47(m,8H);2.62-2.66(m,2H);3.13-3.25(m,12H);3.36(s ,12H);3.52-3.78(m,490H);3.85-3.88(m,4H);3.94-4.11(m,4H);4.25-4.31(m,2H). LCMS (hydrophilic method, formic acid buffer) R t =8.70 minutes, ESI MS(+ve)1714.0[M+4H]4+ / 4,1371.7[M+5H] 5+ / 5;1143.0[M+6H] 6+ / 6,980.0[M+7H] 7+ Convert to / 7.6852.

[0466] 1b.6 Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[(α-NH2.TFA)(ε-NH-COPEG 1100 )]4, G2, compound 6 Azido-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[(α-NHBoc)(ε-NH-COPEG 1100 )]4. Compound 5 (244 mg, 35.6 μmol) was used to prepare according to general procedure A. The lyophilized crude material was redissolved in water and preparative HPLC (22-70% MeCN, 0.01% TFA, R t The compound 6 was purified by (27 minutes) to obtain the product as a pale yellow viscous solid (173 mg, 67%). 1 H NMR(300MHz,D2O)δ(ppm): 1.31-1.98(m,40H);2.51-2.56(m,8H);2.72(broad t,J=6.0Hz,2H);3.16-3.30(m,16H);3.40(s,12H);3.46-3.53(m,4H);3.62-3.97(m,490H);4.03(t,J=6.6Hz,2H);4.24-4.29(m,2H). LCMS (hydrophilic method, TFA buffer) R t =9.85 minutes, ESI MS(+ve)1614.1[M+4H] 4+ / 4,1291.6[M+5H] 5+ / 5;1076.5[M+6H] 6+ Convert / 6 to 6452.

[0467] 1b.7 Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[NHBoc]8, G2, compound 7 Azido-PEG 24CO-[N(PN)2][Lys]2[NH2.TFA]4, compound 6 (117 mg, 58.6 μmol) was used to prepare the compound according to general procedure B. The crude compound 7 was obtained as a pale yellow oil (167 mg, 100%). LC-MS (hydrophobic method, formate buffer) R t =8.35 minutes, ESI MS(+ve)1328.6[M+2H] 2+ / 2-Boc;C 133 H 252 N 18 O 47 [M] + The calculated m / z value for this is 2855.6.

[0468] 1b.8 Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[NH2.TFA]8, G2, compound 8 Azide, PEG 24 CO-[N(PN)2][Lys]2[Lys]4[NHBoc]8, compound 7 (167 mg, 58.6 μmol) was prepared according to general procedure A. The crude aqueous solution was preparatively HPLC (10-60% MeCN, 0.1% TFA buffer; R) t The compound 8 was purified by (27-29 minutes) to obtain the product as a pale yellow viscous solid (124 mg, 71%) in 2 steps. 1 H NMR(300MHz,D2O)δ(ppm): 1.30-1.96(m,42H);2.71(t,J=6.0Hz,2H);2.98-3.04(m,8H);3.13-3.30(m,8H);3.36-3.53(m ,7H);3.68-3.84(m,100H);3.93(t,J=6.6Hz,2H);4.04(t,J=6.6Hz,2H);4.25(t,J=7.2Hz,2H). LCMS (hydrophilic method, TFA buffer) R t =7.76 min, ESI MS(+ve)1028.3 [M+2H] 2+ / 2, 685.9 [M+3H] 3+ / 3;C 93 H 190 N 18 O 31 2+ [M+2H] 2+ m / z calculation value for / 2: 1028.3, C93 H 191 N 18 O 31 3+ [M+3H] 3+ m / z calculation value for / 3: 685.9.

[0469] 1b.9 Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NHBoc)(ε-NH-COPEG1100)]8, G3, compound 9 Azido-PEG 24 Compound 9, the crude material, was obtained as brown oil by preparing compound 8 (123 mg, 41.5 μmol) using CO-[N(PN)2][Lys]2[Lys]4[NH2.TFA]8 according to general procedure C. LCMS (hydrophilic method, formate buffer) R t =11.52 minutes, ESI MS(+ve)2113[M+6H] 6+ / 6,1812[M+7H] 7+ / 7,1585[M+8H] 8+ / 8,1409[M+9H] 9+ / 9,1268[M+10H] 10+ / 10,1153[M+11H] 11+ / 11,1057[M+12H] 12+ Convert to 12,673 ( / 12).

[0470] 1b.10 Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NH2.TFA)(ε-NH-COPEG 1100 )]8, G3, compound 10 Azido-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NHBoc)(ε-NH-COPEG 1100 )]8, compound 9 (526 mg, 41.5 μmol) was prepared according to general procedure A. The crude aqueous solution was separated and HPLC (3-60% MeCN, 0.1% TFA buffer; R tThe compound 10 was purified by LCMS (hydrophilic method, TFA buffer) for 38-39 minutes to obtain the product as a pale yellow viscous solid (359 mg, 68% in 2 steps). t = 10.27 minutes, converted to 11,880.

[0471] 1b.11 Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NHBoc)(ε-NH-Fmoc)]8, G3, compound 11 Azido-PEG 24 Compound 8 (105 mg, 35.4 μmol) was prepared using CO-[N(PN)2][Lys]2[Lys]4[NH2.TFA]8 according to general procedure D. The product, compound 11, was obtained as a white solid (166 mg, 83%). 1 H NMR(300MHz,(CD3)2S=O)δ(ppm): 1.23-1.49(m,160H);2.73-2.95(m,36H);3.44-3.60(m,94H);3.83(m,8H);4.05-4.28(m,29H);6.33-6.90(m,8H,NH);7.24-7.87(m,84H).

[0472] 1b.12 Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NHBoc)(ε -NH2)]8, G3, compound 12 Azido-PEG 24 Using CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NHBoc)(ε-NHFmoc)]8 and compound 11 (169 mg, 29.9 μmol), compound 12 was prepared according to general procedure D to obtain compound 12 as a cottony solid (95 mg, 82%). 1 H NMR(300MHz,CD3OD)δ(ppm): 1.46-1.49(m,160H);2.69(br s,14H);3.09-3.19(m,18H);3.38-3.41(m,8H);3.55-3.78(m,96H),3.89-4.33(m,14H).

[0473] 1b.13 Azide-PEG24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NHBoc)(ε-NH-COPEG 570 )8, G3, Compound 13 and Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NH2.TFA)(ε-NH-COPEG 570 )]8, G3, compound 14 mPEG in DMF (1.5 mL) 570 A solution of -CO2H (205 mg, 348 μmol), NMM (60 μL, 546 μmol), and PyBOP (171 mg, 329 μmol) is mixed with azido-PEG in DMF (0.5 mL). 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NHBoc)(ε-NH2)]8 and compound 12 were added. The resulting reaction mixture was then stirred overnight at room temperature, and subsequently concentrated under vacuum to obtain crude compound 13. This was dissolved in water, treated with TFA, and stirred overnight at room temperature. The mixture was concentrated and incorporated into water, then purified using a Millipore Centrifugation filtration unit (3K MWCO regenerated cellulose) to obtain compound 14, a lyophilized product, as an off-white, cottony material (68% in 2 steps). 1 H NMR(300MHz,D2O)δ(ppm): 1.33-1.90(m,88H);2.51-2.55(m,16H);2.67-2.75(m,4H),3.16-3.23 (m,32H);3.40-3.53(m,32H),3.62-4.03(m,470H);4.21-4.39(m,7H). LCMS (hydrophilic method, formic acid buffer) R t =7.50 minutes.

[0474] 1b.14 Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NHBoc)(ε-NH-COPEG 2000 )]8, G3, compound 15 Azide-PEG in DMF (4 mL) 24CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NHBoc)(ε-NH2)]8, compound 12 (95.0 mg, 24.5 μmol) is stirred, then DIPEA (85 μL, 488 μmol) is added, followed by mPEG. 2000 -NHS (720 mg, 313 μmol) was added. The resulting reaction mixture was then stirred overnight at room temperature. The crude residue was dissolved in water and purified by ultrafiltration (5K, Pall PES membrane). The residue was collected and freeze-dried to obtain compound 15 as an off-white, cottony material (76%). 1 H NMR (300MHz,D2O)δ(ppm): 1.33-1.63(m,160H);3.05-3.15(m,35H);3.29(s,24H);3.35-3.96(m,1370H);4.13-4.19(m,6H). LCMS (hydrophilic method, formic acid buffer) R t =11.24 minutes.

[0475] 1b.15 Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NH2.TFA)(ε-NH-COPEG 2000 )]8, G3, compound 16 Azido-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NHBoc)(ε-NH-COPEG 2000 )]8. Compound 15 (40.0 mg, 1.87 μmol) was used and prepared according to general procedure A to obtain the product as an off-white, cottony material (35 mg, 88%). 1 H NMR(300MHz,D2O)δ(ppm): 1.28-1.79(m,88H);2.51-2.58(m,4H);3.04-3.18(m,35H);3.28(s,24H);3.35-3.97(m,1348H),4.14-4.25(m,6H). LCMS (hydrophilic method, formic acid buffer) R t =9.12 minutes.

[0476] 1b.16 BHALys[Lys]2[Lys]4[((α-NH-COPEG 24NH-COPEG4(PhTzMe))1(α-NH2)3)(ε-NH-COPEG 1000 )4], G2, compound 17 Except for wrapping the reaction vessel in foil to remove light, BHALys[Lys]2[Lys]4[(α-NH2.TFA)4(ε-NH-COPEG 1000 )4](Reference 1)(50.0 mg, 0.007 mmol) and HOOCPEG 24 Using NH-COPEG4(PhTzMe) (Click Chemistry Tools, 13.39 mg, 0.010 mmol), preparation was carried out according to general procedure C. The residue was then purified using methanol as the eluent with SEC (Sephadex® LH-20) to obtain compound 17 (44.00 mg, 77%), which is the product. 1 H NMR(300MHz,D2O)δ(ppm):8.42-8.26(m,2H),7.44-7.11(m,12H),6.04(bs,1H),4.44-4.07(m,8H),4. 07-3.37(m,556H),3.33(s,12H),3.25-2.90(m,17H),2.62-2.43(m,2H),1.96-0.97(m,48H);HPLC(C8 XBridge, 3×100mm) Gradient (formate buffer): 5% MeCN / H2O (0-1 min), 5-80% MeCN (1-7 min), 80% MeCN (7-12 min), 80-5% MeCN (12-13 min), 5% MeCN (13-15 min), 214nm, 0.4mL / min, R t =8.08~9.01 minutes.

[0477] 1b.17 BHALys[Lys]2[Lys]4[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhTzMe))1(α-NH2)7)(ε-NH-COPEG 412 )8], G3, compound 18 Except for wrapping the reaction vessel in foil to block out light, BHALys[Lys]2[Lys]4[Lys]8[(α-NH 2. TFA)8(ε-NH-COPEG 412 )8](Reference 1)(50.0 mg, 0.008 mmol) and HOOC-PEG24 NH-COPEG4(PhTzMe) (Click Chemistry Tools, 11.2 mg, 0.008 mmol) was used to prepare the compound according to general procedure C, and the residue was purified using SEC (Sephadex® LH-20) with methanol as the eluent to obtain the product compound 18 (29 mg, 55%). 1 H NMR(300MHz,D2O)δ(ppm):8.43-8.20(m,2H),7.50-7.09(m,10H),6.03(bs,1H),4.41-4.05(m,10H), 4.01-3.41(m,258H),3.31(s,16H),3.24-2.83(m,28H),2.41(bs,13H),2.00-0.98(m,75H);HPLC(C8 XBridge, 3×100mm) Gradient (formate buffer): 5% MeCN / H2O (0-1 min), 5-80% MeCN (1-7 min), 80% MeCN (7-12 min), 80-5% MeCN (12-13 min), 5% MeCN (13-15 min), 214nm, 0.4mL / min, R t =8.16~8.93 minutes.

[0478] 1b.18 BHALys[Lys]2[Lys]4[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhTzMe))1(α-NH2)7)(ε-NH-COPEG 1000 )8], G3, compound 19 Except for wrapping the reaction vessel in foil to remove light, BHALys[Lys]4[Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 1000 )8] (Reference 1) (100.0 mg, 0.007 mmol) and HOOCPEG 24 NH-COPEG4(PhTzMe) (Click Chemistry Tools, 15.97 mg, 0.010 mmol) was used to prepare the compound according to general procedure C, and the residue was purified using methanol as the eluent with SEC (Sephadex® LH-20) to obtain the product compound 19 (69 mg, 66%). 1H NMR(300MHz,D2O)δ(ppm):8.45-8.25(m,2H),7.47-7.07(m,12H),6.07(bs,1H),4.45-4.05(m,12H),4 .04-3.37(m,936H),3.32(s,25H),3.23-2.88(m,32H),2.59-2.32(m,6H),1.90-0.98(m,90H);HPLC(C8 XBridge, 3 x 100 mm) gradient (formate buffer): 5% MeCN / H2O (0-1 min), 5-80% MeCN (1-7 min), 80% MeCN (7-12 min), 80-5% MeCN (12-13 min), 5% MeCN (13-15 min), 214 nm, 0.4 mL / min, R t =8.21~9.15 minutes.

[0479] 1b.19 BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [((α-NH-COPEG 24 NH-COPEG4(PhTzMe)1(α-NH2) 15 )(ε-NH-COPEG 1000 ) 16 ], G4, compound 20 Except for wrapping the reaction vessel in foil to block out light, BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [(α-NH2.TFA) 16 (ε-NH-COPEG 1000 ) 16 ](Reference 1) (100.0 mg, 0.004 mmol) and HOOCPEG 24 NH-COPEG4(PhTzMe) (Click Chemistry Tools, 6.74 mg, 0.005 mmol) was used to prepare the compound according to general procedure C, and the residue was purified using SEC (Sephadex® LH-20) with methanol as the eluent to obtain the product, compound 20 (63 mg, 65%). 1H NMR(300MHz,D2O)δ(ppm):8.45-8.28(m,2H),7.47-7.07(m,12H),6.03(bs,1H),4.40-4.08(m,23H),4. 06-3.38(m,1906H),3.33(s,56H),3.29-2.91(m,78H),2.63-2.45(m,3H),1.98-0.98(m,200H);HPLC(C8 XBridge, 3×100mm) Gradient (formate buffer): 5% MeCN / H2O (0-1 min), 5-80% MeCN (1-7 min), 80% MeCN (7-12 min), 80-5% MeCN (12-13 min), 5% MeCN (13-15 min), 243 nm, 0.4 mL / min, R t =8.51~9.02 minutes.

[0480] 1b.20 BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [((α-NH-COPEG 24 NH-COPEG4(PhTzMe)1(α-NH2) 31 )(ε-NH-COPEG 1000 ) 32 ], G5, compound 21 Except for wrapping the reaction vessel in foil to block out light, BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [(α-NH2.TFA) 32 (ε-NH-COPEG 1000 ) 32 ](Reference 1) (100.0 mg, 0.002 mmol) and HOOCPEG 24 NH-COPEG4(PhTzMe) (Click Chemistry Tools, 3.38 mg, 0.005 mmol) was used to prepare the compound according to general procedure C, and the residue was purified using SEC (Sephadex® LH-20) with methanol as the eluent to obtain the product, compound 21 (68 mg, 72%). 1H NMR(300MHz,D2O)δ(ppm):8.44-8.29(m,2H),7.44-7.09(m,12H),6.02(bs,1H),4.37-4.11(m,31H),4.0 8-3.37(m,2937H),3.32(s,83H),3.27-2.88(m,116H),2.59-2.42(m,3H),2.18-0.92(m,313H);HPLC(C8 XBridge, 3×100mm) Gradient (formate buffer): 5% MeCN / H2O (0-1 min), 5-80% MeCN (1-7 min), 80% MeCN (7-12 min), 80-5% MeCN (12-13 min), 5% MeCN (13-15 min), 214nm, 0.4mL / min, R t =8.77 minutes.

[0481] 1b.21 BHALys[Lys]2[Lys]4[(α-NHBoc)4(ε-NH-COPEG 24 NH-COPEG4(PhTzMe)4], G2, compound 22 A stirred solution of BHALys[Lys]2[Lys]4[(α-NHBoc)4(ε-NH2)4] (Reference 1) (46.0 mg, 0.031 mmol) in DMF is mixed with NMM (68.0 μL, 0.620 mmol) and HOOCPEG. 24 NH-COPEG4 (PhTzMe) (Click Chemistry Tools; 43.0 mg, 0.155 mmol) and PyBOP (81.0 mg, 0.155 mmol) were added at room temperature. After 16 hours, the reaction mixture was dissolved in MeCN:MQ water (3 mL, 1:1 v / v), and the solution was filtered (0.45 μm Acrodisc syringe filter). The filtrate was separated and subjected to HPLC; 30-80% MeCN, 60 minutes, mobile phase: MQ water and acetonitrile, R t The compound 22 was purified for 33.0-36.0 minutes and obtained as a pink solid of 52 mg (22%). LC-MS (hydrophilic method, TFA buffer) R t =5.66 minutes, 1H NMR(300MHz,D2O)δ(ppm): 8.52(d,8H,J=9.0Hz),7.40-7.26(m,10H),7.20(d,8H,J=9.0Hz),6.23-6.17(m,1H),4.36-4.21(m,10H),3.99-3.83(m,1 3H),3.82-3.46(m,487H),3.46-3.32(m,22H),3.27-3.02(m,15H),3.02(s,12H),2.54-2.38(m,17H),1.92-1.14(m,89H).

[0482] 1b.22 BHALys[Lys]2[Lys]4[(α-NH2.HCl)4(ε-NH-COPEG 24 NH-COPEG4(PhTzMe)4], G2, compound 23 BHALys[Lys]2[Lys]4[(α-NHBoc)4(ε-NH-COPEG] in methanol (2 mL) 24 To a stirred solution of NH-COPEG4(PhTzMe)4), G2, and compound 22 (48.0 mg), a 3M HCl methanol (2 mL) solution was added, and the reaction mixture was stirred at room temperature for 20 hours. Volatile components were removed under reduced pressure, and BHALys[Lys]2[Lys]4[(α-NH2.HCl)4(ε-NH-COPEG 24 NH-COPEG4(PhTzMe)4], G2, compound 23 was obtained as a pink solid of 41.0 mg (91%). LC-MS (LC-MS method, 20-90°C, 8 mins, TFA) t =5.27 minutes.

[0483] 1b.23 BHALys[Lys]2[Lys]4[((α-Lys(α-NHCy5)( α-NHDFO))1(α-NH2)3)(ε-NH-COPEG 24 NH-COPEG4(PhTzMe)4], G2, compound 24 BHALys[Lys]2[Lys]4[(α-NH2.HCl)4(ε-NH-COPEG) in DMF (3 mL) 24To a stirred solution of NH-COPEG4(PhTzMe)4], G2, and compound 23 (11.0 mg, 0.0015 mmol), NMM (3.32 μL, 0.039 mmol), HO-Lys[(α-NHCy5)(ε-NHDFO)] compound 60 (2.06 mg, 0.0015 mmol), and PyBOP (1.18 mg, 0.0022 mmol) were added at room temperature. After 18 hours, volatile matter was removed under reduced pressure, the blue solid residue was dissolved in MQ water, and the solution was filtered (0.45 μm Acrodisc syringe filter). The filtrate was concentrated using a spin column (Amicon Ultra, 0.5 mL, 3 kDa MW cutoff), and the retained solution was repeatedly washed with MQ water (10 x 450 μL) to obtain compound 24 (concentration in MQ water 10 mg / mL; 1.3 mL). LCMS (LCMS method 20~90, 8 minutes, TFA)R t =5.64 minutes; 1 H NMR(300MHz,D2O)δ(ppm): 8.52(d,8H,J=9.0Hz),8.32(s,1H),8.29-8.17(m,2H),7.78-7.70(m,2H),7.69-7.62(m,2H),7.57-7.47(m,2H) ,7.48-7.25(m,16H),7.21(d,8H,J=9.9Hz),7.15-7.08(m,1H),7.06-7.00(m,1H),6.70-6.60(m,2H),6.21-6.12 (m,2H),4.37-4.20(m,14H),4.18-4.06(m,8H),3.98-3.50(m,480H),3.48-3.34(m,15H),3.27-3.06(m,18H),3 .06-2.97(m,19H),2.88(s,8H),2.54-2.40(m,19H),2.08-1.98(m,28H),1.93-1.12(m,99H),1.00-0.82(m,8H).

[0484] 1b.24 BHALys[Lys]2[Lys]4[((α-NH-COPEG 24 NH-COPEG4(PhTzMe))1(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2)2)(ε-NH-COPEG 1000 )4], G2, compound 25 BHALys[Lys]2[Lys]4[(α-NH-COPEG 24 NH-COPEG4(PhTzMe))1(α-NH2)3)(ε-NH-COPEG 1000 Using compound 17 (3.0 mg, 0.414 μmol), and compound 60 (0.56 mg, 0.414 μmol) of HO-Lys[(α-NHCy5)(ε-NHDFO)], preparations were made according to general procedure C and purified by spin column (10 kDa MW cutoff, washed with 10 × 450 μL MQ water) to obtain the desired product, compound 25 (final concentration of 3.56 mg in 300 μL MQ water).

[0485] 1b.25 BHALys[Lys]2[Lys]4[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhTzMe))1(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2)6)(ε-NH-COPEG 412 )8], G3, compound 26 BHALys[Lys]2[Lys]4[Lys]8[((α-NHCOPEG 24 NH-COPEG4(PhTzMe))1(α-NH2)7))(ε-NH-COPEG 412 Using compound 18 (2.97 mg, 0.452 μmol) and compound 60 (0.61 mg, 0.452 μmol) of HO-Lys[(α-NHCy5)(ε-NHDFO)], preparations were made according to general procedure C and purified by spin column (10 kDa MW cutoff, washed with 450 μL MQ water) to obtain the product compound 26 (final concentration of 3.60 mg in 300 μL MQ water).

[0486] 1b.26 BHALys[Lys]2[Lys]4[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhTzMe))1(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2)6)(ε-NH-COPEG 1000 )8], G3, compound 27 BHALys[Lys]2[Lys]4[Lys]8[((α-NHCOPEG24 NH-COPEG4(PhTzMe))1(α-NH2)7)(ε-NHCOPEG 1000 Using )8], G3, compound 19 (3.14 mg, 0.234 μmol), and HO-Lys(α-NHCy5)(ε-NHDFO) compound 60 (0.32 mg, 0.234 μmol), preparations were made according to general procedure C, and the compounds were purified by spin column (10 kDa MW cutoff, washed with 10 × 450 μL MQ water) to obtain the product compound 27 (final concentration of 3.45 mg in 300 μL MQ water).

[0487] 1b.27 BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [((α-NH-COPEG 24 NH-COPEG4(PhTzMe))1(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2) 14 )(ε-NH-COPEG 1000 ) 16 ], G4, compound 28 BHALys[Lys]2[Lys]4[Lys]8-[Lys] 16 [((α-NH-COPEG 24 NH-COPEG4(PhTzMe)1(α-NH2) 15 )(ε-NH-COPEG 1000 ) 16 Compound 28 was prepared according to general procedure C using G4, compound 20 (11.8 mg, 0.454 μmol), and HO-Lys(α-NHCy5)(ε-NHDFO) compound 60 (0.62 mg, 0.454 μmol), and purified using a spin column (10 kDa MW cutoff, washed with 10 x 450 μL MQ water) to obtain 9.3 mg of compound 28 as a blue solid (after lyophilization).

[0488] 1b.28 BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [((α-NH-COPEG 24NH-COPEG4(PhTzMe))1(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2) 30 )(ε-NH-COPEG 1000 ) 32 ], G5, compound 29 BHALys[Lys]2[Lys]4[Lys]8-[Lys] 16 [((α-NH-COPEG 24 NH-COPEG4(PhTzMe)1(α-NH2) 30 )(ε-NH-COPEG 1000 ) 32 Compound 29 was prepared according to general procedure C using G4, compound 21 (12.9 mg, 0.271 μmol), and HO-Lys(α-NHCy5)(ε-NHDFO) compound 60 (0.37 mg, 0.271 μmol), and purified using a spin column (10 kDa MW cutoff, washed with 10 x 450 μL MQ water) to obtain 8.4 mg of compound 29 as a blue solid (after lyophilization).

[0489] 1b.29 BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [(α-NHBoc) 32 (ε-NH-COPEG 570 N3) 32 ], compound 30 BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [(α-NHBoc) 32 (ε-NH2) 32 (Reference 1)) was used to prepare the compound according to general procedure H, step 1. Compound 30, the lyophilized product, was obtained as an off-white sticky solid (40 mg, 19%). 1 HNMR(300MHz,D2O)δ(ppm): 0.39-2.11(m,666H);2.25-2.53(m,58H);2.53-2.70(m,12H);2.70-4.42(m,1540H);6.89-7.48(m,12H). HPLC (HPLC method 5~80, 15 minutes, formate)R t =10.21 minutes.

[0490] 1b.30 BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [(α-NHBoc) 32 (ε-NH-COPEG 1100 N3) 32 ], compound 31 BHALys-[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [(α-NHBoc) 32 (ε-NH2) 32 ](Reference 1) (0.50 mg, 4.4 μmol) and N3-PEG 1100 Using -COOH, the compound was prepared according to general procedure H, step 1. The lyophilized product, compound 31, was obtained as an off-white sticky solid (159 mg, 75%). 1 H NMR(300MHz,CD3OD)δ(ppm): 0.91-2.08(m,666H);2.27-2.58(m,64H);3.01-3.27(m,113H);3.33-3.92(m,3018H) );3.95-4.18(m,33H);4.20-4.50(m,31H);7.10-7.55(m,12H),7.62-8.15(m,24H). HPLC (HPLC method 5~80, 15 minutes, formate)R t =9.62 minutes.

[0491] 1b.31 BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [(α-NH2.TFA) 32 (ε-NH-COPEG 570 N3) 32 ], compound 32 General procedure H, step 2, BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [(α-NHBoc) 32 (ε-NH-COPEG 570 N3) 32Compound 30 (40 mg, 1.3 μmol) was used to prepare the solution. The product, compound 32, was obtained as a pale yellow oil (41 mg, quantitative). 1 H NMR(300MHz,D2O)δ(ppm): 0.87-1.93(m,378H);2.24-2.51(m,67H);2.52-2.69(m,20H);2.77-3.20(m,122 H);3.20-4.05(m,1312H);4.05-4.32(m,34H);5.97(s,1H);6.98-7.34(m,10H).

[0492] 1b.32 BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [(α-NH2.TFA) 32 (ε-NH-COPEG 1100 N3) 32 ], compound 33 BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [(α-NHBoc) 32 (ε-NH-COPEG 1100 N3) 32 Compound 31 (145 mg, 3.0 μmol) was used to prepare the compound according to general procedure H, step 2. The product, compound 33, was obtained as a pale yellow, viscous solid (146 mg, quantitative). 1 H NMR(300MHz,CD3OD)δ(ppm): 1.20-2.03(m,378H);2.37-2.59(m,63H);3.00-3.28(m,121H);3.35-3.96(m,30 36H);3.96-4.13(m,22H);4.18-4.56(m,39H);6.19(s,1H);7.20-7.42(m,10H). HPLC HPLC method 5~80, 15 minutes, formate)R t =9.41 minutes.

[0493] 1b.33 BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [(α-NHCy5)1(α-NHAc) 31 (ε-NH-COPEG570 N3) 32 ], compound 34 BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [(α-NH2.TFA) 32 (ε-NH-COPEG 570 N3) 32 The compound was prepared using (46.1 mg, 1.6 μmol) according to general procedure I, steps 1 and 2. The product, compound 34, was obtained as a blue solid (31 mg, 72%). 1 H NMR(300MHz,D2O)δ(ppm): 0.93-1.85(m,400H);1.89-2.02(m,89H);2.04-2.09(m,23H);2.30-2.52(m,59H);2.53-2.73(m,13H); 2.91-3.21(m,121H);3.24-3.94(m,1313H);3.97-4.37(m,67H);5.87-6.31(m,6H);6.83-7.60(m,25H). IR(cm -1 ): 2100(-N3).

[0494] 1b.34 BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [(α-NHCy5)1(α-NHAc) 31 (ε-NH-COPEG 1100 N3) 32 ], compound 35 BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [(α-NH2.TFA) 32 (ε-NH-COPEG 1100 N3) 32 Compound 35 was prepared using compound 33 (112.5 mg, 2.3 μmol) according to general procedure I, steps 1 and 2. The product, compound 35, was obtained as a blue solid (98 mg, 85%). 11H NMR (300 MHz, CD3OD) δ (ppm): 1.04 - 2.18 (m, 507H); 2.38 - 2.55 (m, 64H); 2.99 - 3.27 (m, 122H); 3.37 - 3.96 (m, 3026H); 4.15 - 4.51 (m, 67H); 6.10 - 6.48 (m, 5H); 7.15 - 7.58 (m, 24H). HPLC method: 5 - 80, 15 minutes, formate) R t = 9.52 minutes.

[0495] 1b.35 BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [((α-NH-COPEG 24 NH-COPEG4(PhTzMe))1(α-NHCy5)1(α-NHDOTA) 10 (α-NH2)4(ε-NH-COPEG 1000 ) 16 , G4, compound SRS-1 BHALys[Lys]2[Lys]4[Lys]8[Lys] in DMF (4 mL) 16 [((α-NH-COPEG 24 NH-COPEG4(PhTzMe))1(α-NH2) 15 )(ε-NH-COPEG 1000 ) 16To a stirred solution of ], G4, compound 20 (148.0 mg, 0.0054 mmol), and NMM (38.0 μL, 0.474 mmol), Cy5-NHS ester (Lumiprobes, 3.66 mg, 0.0054 mmol) was added, and the reaction mixture was stirred at room temperature, while the consumption of Cy5 NHS ester was monitored by LC-MS. After 18 hours, a solution of p-SCN-Bn-DOTA (Macrocyclics, 75.0 mg, 0.109 mmol) in DMSO (1 mL) was added, and stirring was continued for 24 hours. Volatile components were removed by vacuum, the residue was dissolved in MQ water (15.0 mL), and the solution was filtered (0.45 μm Acrodisc filter). The filtrate was purified using a spin column (Amicon Ultra-15, 10 kDa MWCO), and the residue was repeatedly washed with MQ water (5 × 15 mL). The residue was dried by freeze-drying to obtain SRS-1 as a blue solid (173.0 mg, 96%). HPLC: XBridge C8 column with gradients of 5-80% MeCN / H2O (1-7 min), 80% MeCN / H2O (7-12 min), 80-5% MeCN / H2O (12-13 min), 5% MeCN / H2O (13-15 min), and 10 mM HCOONH4), and UV detection at 214 nm. t =5.07 minutes. 1 H NMR (300MHz, D2O) δ (ppm): 0.33-2.08 (m, 177H), 2.26-4.46 (m, 1979H), 6.87-7.66 (bs, 59H), 8.34 (bs, 2H).

[0496] 1b.36 BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [(α-NH2) 30 (α-NHDOTA)2(ε-NHCOPEG 2000 ) 32 ], G5, RH-2 BHALys[Lys]2[Lys]4[Lys]8[Lys] in DMF (6.0mL) 16 [Lys] 32 [(α-NH2.TFA) 32 (α-(ε-NHCOPEG 2000 ) 32To a stirred solution of RH-1 (301 mg, 3.97 μmol) (as described in Example 1 of WO2020 / 0014750), p-SCN-Bn-DOTA (8.13 mg, 11.8 μmol, 2.98 equivalents) was added, followed by NMM (114 μL, 1.03 mmol). The resulting reaction mixture was stirred at ambient temperature for 4.5 hours, after which a portion of the reaction mixture (2.0 mL) was removed and placed in another reaction flask equipped with a stirring bar, where stirring was continued. After 24 hours, a smaller aliquot of the reaction mixture was concentrated from vacuum to dry, dissolved in MeOH (1.0 mL), and SEC (stationary phase = Sepahdex LH-20™, mobile phase = acetonitrile, elution rate = approximately 1 drop per second) was used. -1 The product was purified by (fraction size = 400 drops). The product-containing fractions were combined and vacuum concentrated, and the resulting residue was dissolved in MQ water, filtered (0.45 μm Acrodisc filter), and freeze-dried to obtain compound RH-2 as a white solid (82.5 mg). 1 ¹H NMR (300MHz, CD3OD-d4) δ (ppm): 1.17-2.29 (m, 401H), 3.36 (s, 96H), 3.39-3.43 (m, 43H), 3.50-4.08 (m, 5564H), 4.21-4.67 (m, 84H), 6.17 (broad, 1H), 7.18-7.64 (m, 18H), 8.09 (s, 1H). 1 1H NMR analysis suggests approximately 2.1 DOTA / dendrimer; DOTA %(w / w) = 2.0%.

[0497] 1b.37 [(ε-NHBoc)(α-NHBoc)][Lys]-CONH-CH2-CH2-S-]2, RL-3 To a stirred solution of cystamine hydrochloride RL-1 (1.26 g, 5.6 mmol) and Boc-Lys(Boc)-ONp RL-2 (5.68 g, 12.2 mmol) in DMF (100 mL), TEA (5.44 mL, 39.0 mmol) was added. The reaction mixture was stirred at room temperature for 3 days, and a solution of glycine (1.82 g, 24.4 mmol) in deionized water (10 mL) was added, causing a white solid to precipitate from the solution. The suspension was stirred for 2 hours, and volatile components were removed by vacuum. The residue was suspended in ethyl acetate (50 mL), and the organic matter was sequentially washed with saturated sodium carbonate aqueous solution (5 × 20 mL), 0.1 M HCl aqueous solution (20 mL), and brine (20 mL), dried, and (MgSO4), and volatile components were removed by vacuum. The residue was dissolved in dichloromethane (50 mL), washed with 0.2 M NaOH aqueous solution (4 × 50 mL) and brine (50 mL), dried (MgSO4), and volatile components were removed by vacuum to obtain [(ε-NHBoc)(α-NHBoc)][Lys]-NHCO-CH2-CH2-S-]2RL-3 as an off-white solid (3.66 g, 81%). 1 H NMR (300MHz, CD3OD): δ(ppm)1.13-1.82(m,48H),2.83(t,J=6.6Hz,4H),3.02(t,J=6.6Hz,4H),3.52(m,4H),3.98(m,2H). LCMS (LCMS method 40~90, 8 minutes, TFA):R t = 5.36 min, ESI MS(+ve) 809[M] + ;C 36 H 68 N6O 10 S2[M] + The calculated m / z value for this is 809.

[0498] 1b.38 [(ε-NH2.TFA)(α-NH2.TFA)][Lys]-CONH-CH2-CH2-S-]2, RL-4 To a 30 mL solution of [(ε-NHBoc)(α-NHBoc)][Lys]-NHCO-CH2-CH2-S-]2RL-3 (3.66 g, 4.5 mmol) in dichloromethane, TFA (27.7 mL, 362.0 mmol) was added dropwise for 5 minutes at room temperature. The reaction mixture was stirred at room temperature for 18 hours and then vacuum concentrated. The residue was dissolved in 50 mL of deionized water and freeze-dried to obtain [(ε-NH2.TFA)(α-NH2.TFA)][Lys]-CONH-CH2-CH2-S-]2RL-4 as a light brown solid (3.91 g, quantitative). 1 H NMR(300MHz,CD3OD):δ(ppm)1.28-1.42(m,4H),1.54-1.68(m,4H),1.76-1.88(m,4H) ,2.70-2.86(m,4H),2.90(t,J=7.7Hz,2H),3.39-3.62(m,4H),3.88(t,J=6.6Hz,2H). LCMS (LCMS method 5~60, 8 minutes, TFA)R t = 0.80 minutes. ESI MS(+ve)409[M] + ;C 16 H 36 N6O2S2[M] + The calculated m / z value for this is 409.

[0499] 1b.39 [[(ε-NHBoc)2(α-NHBoc)2][Lys]2[Lys]-CONH-CH2-CH2-S-]2, RL-5 To a solution of [(ε-NH2.TFA)(α-NH2.TFA)][Lys]-CONH-CH2-CH2-S-]2RL-4 (3.9 g, 4.52 mmol) in DMF (18 mL), Boc-Lys(Boc)-ONp RL-2 (10.13 g, 21.7 mmol) was added. This mixture was heated at 40°C until a clear solution was obtained, then cooled to room temperature, and TEA (7.55 mL, 54.2 mmol) was added. After stirring at room temperature for 18 hours, an aqueous solution of glycine (509 mg, 4.46 mmol) (1.28 mL) was added, and the reaction mixture was heated at 40°C while stirring. After 2 hours, the reaction mixture was cooled to room temperature, and then added dropwise to water over 20 minutes (while stirring). The resulting precipitate was collected by filtration, washed with deionized water (5 × 30 mL), and dried in an air stream for 30 minutes. The solid was dissolved in DMF (18 mL), and the solution was added dropwise to water (180 mL) while stirring. The resulting white solid was collected by filtration, washed with water (3 × 30 mL), and dried in a vacuum oven at 40°C for 20 hours to obtain [[(ε-NHBoc)2(α-NHBoc)2][Lys]2[Lys]-CONH-CH2-CH2-S-]2RL-5 as a white solid (6.49 g, 83.3%). 1 H NMR (300MHz, CD3OD): δ (ppm) 1.26-1.93 (m, 108H), 2.84 (m, 4H), 3.04 (m, 8H), 3.20 (m, 4H), 3.51 (m, 4H), 4.00 (m, 4H), 4.33 (m, 2H). LCMS (LCMS method 40~90, 8 minutes, TFA)R t = 6.34 minutes. ESI MS(+ve)1722[M] + ;C 80 H 148 N 14 O 22 S2[M] + The calculated m / z value for this is 1722.

[0500] 1b.40 [[(ε-NH2.TFA)2(α-NH2.TFA)2][Lys]2[Lys]-CONH-CH2-CH2-S-]2, RL-6 To a stirred suspension of [[(ε-NHBoc)2(α-NHBoc)2][Lys]2[Lys]-CONH-CH2-CH2-S-]2RL-5 (6.49 g, 3.8 mmol) in dichloromethane (65 mL), TFA (34.6 mL, 451.2 mmol) was added dropwise over 10 minutes at 0°C. The resulting solution was warmed to room temperature, stirred for 4 hours, and then volatile components were removed by vacuum. The residue was dissolved in the minimum amount of deionized water and freeze-dried twice (Note: During the second freeze-drying process, the turbid aqueous solution was filtered through a 0.45 μm filter disc, and the filtrate was freeze-dried) to obtain [[(ε-NH2.TFA)2(α-NH2.TFA)2][Lys]2[Lys]-CONH-CH2-CH2-S-]2RL-6 as a light brown foam (6.20 g, 96.9%). 1 H NMR(300MHz,CD3OD):δ(ppm)1.13-1.87(m,36H),2.72(m,4H),2.87(m,8H),3.09(t,J=7.0Hz ,4H),3.30-3.53(m,4H),3.79(t,J=6.6Hz,2H),3.90(t,J=6.5Hz,2H),4.13(t,J=7.1Hz,2H).

[0501] 1b.41 [[(ε-NHBoc)4(α-NHBoc)4][Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2RL-7 To a solution of [[(ε-NH2.TFA)2(α-NH2.TFA)2][Lys]2[Lys]-CONH-CH2-CH2-S-]2RL-6 (4.02 g, 2.20 mmol) in DMF (16 mL), Boc-Lys(Boc)-ONp RL-2 (9.02 g, 19.3 mmol) was added. This mixture was heated at 40°C until a clear solution was obtained, then cooled to room temperature, and TEA (7.35 mL, 52.8 mmol) was added. After stirring at room temperature for 18 hours, DMF (25 mL) was added to dissolve any precipitated solids, and the resulting solution was heated at 55°C, and a solution of glycine (250 mg, 3.3 mmol) in water (1.20 mL) was added. After stirring for 2 hours, the reaction mixture was cooled to room temperature, and then added dropwise to water (200 mL) while stirring for 10 minutes. The obtained precipitate was collected by filtration and dried in an airflow for 30 minutes. The solid was dissolved in DMF (40 mL), and the resulting solution was added dropwise to deionized water (200 mL) while stirring. The resulting white solid was collected by filtration, dried in an airflow for 30 minutes, suspended in MeCN, and volatile components were removed by vacuum to obtain [[(ε-NHBoc)4-(α-NHBoc)4][Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2RL-7 as a white solid (4.80 g, 37.0%). 1 H NMR (300MHz, CD3OD): δ(ppm)1.17-1.95(m,228H),2.86(m,4H),3.05(m,16H),3.20(m,12H),3.53(m,4H),4.03 & 4.32(14H). LCMS (LCMS method 40~90, 8 minutes, TFA)R t = 7.69 minutes. ESI MS(+ve)1773[M] + ;C 84 H 154 N 15 O 23 S1[M / 2] + The calculated m / z value for this is 1773.

[0502] 1b.42 [[(ε-NH2.TFA)4(α-NH2.TFA)4][Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2, RL-8 To a stirred suspension of [[(ε-NHBoc)4-(α-NHBoc)4][Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2RL-7 (4.80 g, 1.4 mmol) in dichloromethane (50 mL), TFA (50.0 mL, 648.0 mmol) was added dropwise at 0°C over 15 minutes. The resulting solution was warmed to room temperature, stirred for 4 hours, and then volatile components were removed by vacuum. The residue was dissolved in the minimum amount of deionized water and freeze-dried (twice) to obtain a pale brown foam (5.38 g), which was then dissolved in methanol (approximately 15 mL). The resulting solution was added dropwise to diethyl ether (300 mL) while stirring. The white precipitate formed in this manner was recovered by filtration, dissolved in methanol, and volatile matter was removed by vacuum to obtain [[(ε-NH2.TFA)4(α-NH2.TFA)4]4[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2RL-8 as an off-white hygroscopic foam (3.50 g, 74.1%). 1 H NMR(300MHz,CD3OD):δ(ppm)1.27-2.01(m,84H),2.82(m,4H),2.90-3.04(m,16H),3.06-3 .29(m,12H),3.52(m,4H),3.87(m,4H),3.99(t,J=6.32Hz,4H),4.30(m,4H),4.38(m,2H). LCMS (LCMS method 5~60, 8 minutes, TFA)R t =0.65 minutes. ESI MS(+ve)975(M / 2)+H] + ;C 44 H 91 N 15 O7S1[(M / 2)+H] + The calculated m / z value for this is 975.

[0503] 1b.43 [[(ε-NHBoc)8(α-NHBoc)8][Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2, RL-9 and [ [(ε-NH2.TFA)8(α-NH2.TFA)8][Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2, RL-10 To a solution of [(ε-NH2.TFA)4(α-NH2.TFA)4]4[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2RL-8 (1.5 g, 0.398 mmol) in DMF (17 mL), Boc-Lys(Boc)-ONp RL-2 (3.27 g, 6.99 mmol) and NMM (2.10 mL, 19.1 mmol) were added at room temperature. After stirring the reaction mixture for 2 days, a solution of glycine (66 mg) in water (1.0 mL) was added. After 4 hours, the reaction mixture was added dropwise to water (200 mL) while stirring. The resulting yellow precipitate was collected by filtration, washed with water (5 × 50 mL), and dried in a stream of air. The solid was dissolved in DMF (20 mL), and the resulting solution was added to water (200 mL) to obtain an off-white precipitate, which was collected and dried as described above. The solid was dissolved in DMF (4.0 mL) and TEA (2.1 mL), heated with stirring at 55°C, and a solution of glycine (66 mg) in water (1 mL) was added. After 4 hours, the reaction mixture was cooled to room temperature, stirred for 2 days, and then added dropwise to water (250 mL) while stirring. The resulting precipitate was collected by filtration, washed with water (3 × 50 mL), and vacuum-dried to obtain an off-white solid [[(ε-NHBoc)8(α-NHBoc)8][Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2RL-9 (approximately 0.8 g), which was used without further purification.

[0504] To a solution of [[(ε-NHBoc)8(α-NHBoc)8][Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2RL-9 (approximately 0.8 g) in dichloromethane, TFA (16 mL) was added at 0°C (16 mL). The reaction mixture was warmed to room temperature and stirred for 18 hours, after which volatile matter was removed by vacuum. The residue was dissolved in water (20 mL) and concentrated by centrifugation at 4000 rpm for 20 minutes using a 15 mL Amicon® Ultra centrifugal filter equipped with a 3 kDa MWCO Ultracel® regenerated cellulose membrane. The residue was diluted with water, the centrifugation / concentration process was repeated (10 times), and the final residue was freeze-dried to obtain [[(ε-NH2.TFA)8(α-NH2.TFA)8][Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2RL-10 as an off-white solid (606 mg, 20%, 2 steps). 1 H NMR(300MHz,CD3OD):δ(ppm)1.24-2.00(m,180H),2.81(m,4H),2.90-3.04(m,32H) ,3.12-3.33(m,28H),3.52(m,4H),3.83(m,8H),3.94(m,8H) and 4.23-4.42(m,14H). LCMS (LCMS method 5~60, 8 minutes, TFA)R t =3.76 minutes. ESI MS(+ve)1000[M / 4+H] + ;[C 184 H 373 N 62 O 30 S2 / 4[M / 4+H] + The calculated m / z value for this is 1000.

[0505] 1b.44 [(ε-NH2.TFA)8(α-NH2.TFA)8][Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-Me(MAL)-PEG 24 -CONH-Bn-Tz(Me)RP-1 To a stirred solution of [[Lys(ε-NH2.TFA)(α-NH2.TFA)]8-[Lys]4-[Lys]2-[Lys]-CONH-CH2-CH2-S-]2RL-10 (128 mg, 0.017 mmol) in water (6.0 mL), an aqueous solution of TCEP 0.5 M (335 μL, 0.170 mmol) was added at room temperature. After stirring the reaction mixture for 1 hour, the pH of the reaction mixture was measured (pH 5.2), and adjusted to pH 6.5 by adding 0.1 M NaOH aqueous solution dropwise. Then, Me(MAL)-PEG was added. 24 -CONH-Bn-Tz(Me)RL-18 (28.6 mg, 0.020 mmol) was added in a single portion, and stirring was continued. After 2 hours, the reaction mixture was transferred to an Amicon® Ultra centrifugal filter equipped with a 3 kDa MWCO Ultracel® regenerated cellulose membrane and concentrated by centrifugation at 4000 rpm for 20 minutes. The residue was washed with water (×5) by centrifugation at 4000 rpm and lyophilized to obtain a yellow solid (109 mg). The solid was dissolved in water (6 mL), and air was passed through the solution and the mixture was stirred for 18 hours to obtain a pink solution. The solution was freeze-dried to obtain a pink solid (111 mg), which was further purified by preparative HPLC (HPLC method 5-80°C, 8 min, TFA) to obtain [(ε-NH2.TFA)8(α-NH2.TFA)8][Lys]8[Lys]4[Lys]2[Lys]CONH-CH2-CH2-S-(Me)MAL-Me(MAL)-PEG 24 -CONH-Bn-Tz(Me)RP-1 was obtained as a pink solid (32.5 mg, 32.5%). 1 H NMR(300MHz,CD3OD):δ(ppm)1.21-2.05(m,87H),2.90-3.04(m,14H),3.06(s,3H),3.06-3.26( m, 11H), 3.48-4.07 (m, 89H), 4.17-4.53 (m, 11H), 7.59 (d, J = 7.6Hz, 2H) and 8.53 (d, J = 8.0Hz, 2H). LCMS (LCMS method 40~90, 8 minutes, TFA)R t =3.98 minutes. ESI MS(+ve)1141 [(M / 3)+H] + ;C 158 H 301 N 37 O42 S[(M / 3)+H] + The calculated m / z value for this is 1141.

[0506] 1b.45 [(ε-NHBoc) 16 (α-NHCOPEG 25 ) 16 [Lys] 16 [Lys]8[Lys]4[Lys]2[Lys]CONH-CH2-CH2-S-Me(MAL)-PEG 24 -CONH-Bn-Tz(Me), RP-3 [(ε-NH2.TFA)8(α-NH2.TFA)8][Lys]8[Lys]4[Lys]2[Lys]CONH-CH2-CH2-S-(Me)MAL-PEG in anhydrous DMF (3.0 mL) 24 -CONH-Bn-Tz(Me)RP-1 (31.4 mg, 6 μmol) is stirred into a solution of NMM (105 μL, 955 μmol), followed by PyBOP (59.8 mg, 115 μmol) and HO-Lys(Boc)(PEG) in anhydrous DMF (2.0 mL). 1100 )RP-2 solution was added. The reaction mixture was stirred for 18 hours and then purified by TFF (Pellicon 10kDa MWCO membrane) using water as the circulation medium until 10 dialysie filtration volumes were collected as permeate. The residue was collected, pooled in line washing, and freeze-dried to obtain [(ε-NHBoc) 16 (α-NHCOPEG 25 ) 16 [Lys] 16 [Lys]8[Lys]4[Lys]2[Lys]CONH-CH2-CH2-S-(Me)MAL-PEG 24 -CONH-Bn-Tz(Me)RP-3 was obtained as a pink solid (104 mg, 65%). 1H NMR(300MHz,CD3OD):δ(ppm)1.19-1.97(m,383H),2.38-2.54(m,36H),3.06( s,3H),3.09-3.29(m,64H),3.38(s,56H),3.39-3.48(m,8H),3.51-3.59(m,3 6H),3.59-3.70(m,1889H),3.70-3.80(m,32H),3.85-3.92(m,6H),3.94-4.1 5(m,7H), 4.21-4.44(m,10H), 7.60(d,J=8.1Hz,2H) and 8.53(d,J=8.1Hz,2H). HPLC (HPLC method 5-80°C, 8 min, TFA) t= 5.10 minutes.

[0507] 1b.46 [(ε-NH2.HCl) 16 (α-NHCOPEG 25 ) 16 [Lys] 16 [Lys]8[Lys]4[Lys]2[Lys]CONH-CH2-CH2-S-Me(MAL)-PEG 24 -CONH-Bn-Tz(Me), RP-4 [(ε-NHBoc) in a cooled vial 16 (α-NHCOPEG 25 ) 16 [Lys] 16 [Lys]8[Lys]4[Lys]2[Lys]CONH-CH2-CH2-S-(Me)MAL-PEG 24 -CONH-Bn-Tz(Me)RP-3 (83 mg, 3.3 μmol) was mixed with 3.0 M HCl in methanol (1.75 mL). The resulting solution was stirred for 18 hours, warmed to room temperature, and then volatile components were removed by vacuum. The residue was dissolved in water and freeze-dried to obtain [(ε-NH2.HCl) 16 (α-NHCOPEG 25 ) 16 [Lys] 16 [Lys]8[Lys]4[Lys]2[Lys]CONH-CH2-CH2-S-Me(MAL)-PEG 24 -CONH-Bn-Tz(Me)RP-4 was obtained as a pink solid (82.6 mg, quantitative). 11H NMR (300 MHz, CD3OD): δ (ppm) 1.27 - 2.05 (m, 198H), 2.39 - 2.57 (m, 36H), 3.15 - 3.29 (m, 44H), 3.38 (s, 39H), 3.39 - 3.59 (m, 9H), 3.59 - 3.73 (m, 32H), 3.59 - 3.70 (m, 1217H), 3.73 - 3.83 (m, 33H), and 3.85 - 4.15 (m, 31H), and 4.21 - 4.55 (m, 26H). HPLC (HPLC method 5 - 80, 8 minutes, TFA) R t = 4.78 minutes.

[0508] 1b.47 [(ε-NHCO-PEG 1100 ) 16 (α-NHCy5)1(α-NHDOTA)8(α-NH2)7]][Lys] 16 [Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-(Me)MAL-PEG 24 -CONH-Bn-Tz(Me), RP-5 [(ε-NHCO-PEG 1100 ) 16 (α-NH2.HCl) 16 [Lys] 16 [Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-(Me)MAL-PEG 24To a stirred solution of -CONH-Bn-Tz(Me)RP-4 (82.6 mg, 3.2 μmol) and NMM (24 μL, 0.21 mmol), cyanine 5 NHS ester (2.3 mg, 3.2 μmol) in DMF (1.0 mL) was added at room temperature. The reaction mixture was monitored by LC-MS (LC-MS method 20-90°C, 8 min, TFA) for the consumption of cyanine 5 NHS ester. After 18 hours, a solution of p-SCN-Bn-DOTA (44.7 mg, 68 μmol) in DMSO solution (1.0 mL) was added to the reaction mixture, and stirring was continued. After 2 days, water (50 mL) was added, and the resulting solution was filtered through a 0.45 μm filter disc and purified by TFF (Pellicon 10 kDa MWCO membrane) using water as the circulation medium until 11 diafiltration volumes were collected as permeate. The residue is collected, pooled in line washing, freeze-dried, and then [(ε-NHCO-PEG 1100 ) 16 (α-NHCy5)1(α-NHDOTA)8(α-NH2)7][Lys] 16 [Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-(Me)MAL-PEG 24 -CONH-Bn-Tz(Me)RP-5 was obtained as a blue solid (74.5 mg). 1 H NMR(300MHz,CD3OD):δ(ppm)0.74-2.23(m,230H),2.36-2.65(m,53H),2.91-3.28(m,102H),3. 38(s,63H),3.40-3.46(m,19H),3.52-3.94(m,1959H),6.17-6.86(m,3H),6.96-7.74(m,46H). The integral of the aromatic region (δ6.96~7.74ppm) is calculated using the internal standard aromatic proton (δ8.62ppm, 2H ISThe DOTA loading was determined by qNMR using 3,4,5-trichloropyridine as an internal standard, by comparison with the above. Thus, the number of DOTA groups per molecule was found to be 8.6. Since complete consumption of cyanine 5 NHS ester was observed, the number of cyanine 5 groups per dendrimer was set to 1. The molecular weight of the dendrimer was calculated to be 26,665 Da. HPLC (HPLC method 5-80°C, 8 min, TFA) t =4.76 minutes.

[0509] 1b.48 [[(ε-NH2)4(α-NH2)4][Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2MAL-PEG3-NHCO-PEG 24 -CONH-PEG4-(PhTzMe)SRS-1-Mal To a solution of [[(ε-NH2.TFA)4(α-NH2.TFA)4][Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2RL-8 (65 mg, 17.2 μmol) in water (1.0 mL), an aqueous solution of 0.5 M TCEP (17.0 μL, 8.5 μmol, pH 7.0) was added at room temperature. The reaction reaction was monitored by LC-MS (LC-MS method 2), and the 0.5 M TCEP solution was gradually added until complete reduction of the disulfide bond was observed (a total of 17.0 μL, 8.5 μmol of TCEP was added over approximately 1 hour). The pH of the reaction mixture was adjusted to 6.2 using 0.1 N NaOH (aqueous solution), and then Br2(MAL)-PEG3-NHCO-PEG in water (0.5 mL) was added. 24A solution of -CONH-PEG4-(PhTzMe)RL-15 (32 mg, 16.4 μmol) was added. The reaction mixture was stirred for 1 hour, diluted with water (30 mL), filtered (0.45 μm syringe filter disc), and the filtrate was transferred to a 15 mL Amicon® Ultra centrifuge filter equipped with a 3 kDa MWCO Ultracel® regenerated cellulose membrane and concentrated by centrifugation at 4000 rpm for 20 minutes. The residue was washed with water (6 × 15 mL) by centrifugation at 4000 rpm, lyophilized, and then [[(ε-NH2)4(α-NH2)4][Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2MAL-PEG3-NHCO-PEG 24 -CONH-PEG4-Bn-Tz(Me)SRS-1-Mal was obtained as an orange solid (65 mg, 99%). 1 H NMR(300MHz,CD3OD):δ(ppm)1.31-1.66(m,37H),1.66-2.02(m,45H),2.05-2.26(m,7H),2.38-2.59(11H),2.91-3.08(m,18H),3.12-3.26(m,8H) ,3.27-3.41(m,8H),3.40-3.62(m,20H),3.68-3.80(m,20H),3.80-4.05( m,9H), 4.23-4.47(m,7H), 7.21(d,J=9.0Hz,2H) and 8.52(d,J=9.0Hz,2H). LCMS (LCMS method 5~60, 8 minutes, TFA)R t =5.04. ESI MS(+ve)1265[(M / 3)+H] + ;C 171 H 327 N 39 O 50 S2[(M / 3)+H] + The calculated m / z value for this is 1265.

[0510] 1b.49 [[(ε-NHCO-PEG 1100 )8(α-NH2)8][Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2MAL-PEG3-NHCO-PEG 24 -CONH-PEG4-(PhTzMe), SRS-2 [[(ε-NH2)4(α-NH2)4][Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2MAL-PEG3-NHCO-PEG in DMF (4.0 mL) 24 -CONH-PEG4-Bn-Tz(Me)SRS-1-Mal(65mg, 17.1μmol), HO-Lys(Boc)(PEG 1100 PyBOP (108 mg, 208 μmol) was added to a solution of RP-2 (289 mg, 208 μmol) and NMM (119 μL, 1.08 mmol) at room temperature. After 18 hours, the reaction mixture was diluted with water (50 mL), filtered (using a 0.45 μm syringe filter disc), and purified by TFF (Pellicon 10 kDa MWCO membrane) using water as the circulating medium until 10 dialysate filtration volumes were collected as permeate. The residue was collected, pooled in line washing, and freeze-dried to obtain [[(ε-NHCO-PEG 1100 )8(α-NH2)8][Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2MAL-PEG3-NHCO-PEG 24 -CONH-PEG4-(PhTzMe)SRS-2 was obtained as a dark orange solid (158 mg, 36%). 1 H NMR(300MHz,CD3OD):δ(ppm): 1.19-2.01(m,307H),2.40-2.55(m,34H),3.03(s,3H)3.10-3.30(m,52H),3.38(s,45H),3.39-3.44(m,9H)3.53-3.59(m,35H),3.59-3. 70(m,1522H),3.70-3.80(m,37H),3.84-3.94(m,7H),3.95-4.14(m,8H),4.26-4.44(m,11H),7.22(d,J=9Hz,2H) and 8.53(d,J=9Hz,2H). HPLC (HPLC method 5-80°C, 8 min, TFA) t =5.09 minutes.

[0511] 1b.50 [[(ε-NHCO-PEG 1100)8(α-NH2.HCl)8][Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2MAL-PEG3-NHCO-PEG 24 -CONH-PEG4-(PhTzMe), SRS-3-Mal [[(ε-NHCO-PEG 1100 )8(α-NH2)8][Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2MAL-PEG3-NHCO-PEG 24 -CONH-PEG4-(PhTzMe)SRS-2 (156 mg, 6.1 μmol) was dissolved in 3.0 M HCl in methanol (3.23 mL) at room temperature. The resulting solution was stirred for 18 hours, and then volatile components were removed by vacuum. The residue was dissolved in water (approximately 10 mL), and the solution was freeze-dried to obtain [[(ε-NHCO-PEG 1100 )8(α-NH2.HCl)8][Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2MAL-PEG3-NHCO-PEG 24 CONH-PEG4-(PhTzMe)SRS-3-Mal was obtained as a dark orange solid (142 mg, 95%). HPLC (HPLC method 5-80°C, 8 min, TFA) t =4.77 minutes.

[0512] 1b.51 [[(ε-NHCO-PEG 1100 )8(α-NHDOTA) 4.75 (α-NHCy5) 0.5 (α-NH2) 2.75 ][Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2MAL-PEG3-NHCO-PEG 24 -CONH-PEG4-(PhTzMe), SRS-4-Mal [[(ε-NHCO-PEG in DMF(mL) 1100 )8(α-NH2.HCl)8][Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2MAL-PEG3-NHCO-PEG 24To a solution of -CONH-PEG4-(PhTzMe)SRS-3-Mal (130 mg, 5.3 μmol) and NMM (37 μL, 337 μmol), cyanine 5 NHS ester (3.5 mg, 5.2 μmol) was added. The reaction mixture was stirred at room temperature for 18 hours, then a solution of p-SCN-Bn-DOTA (72 mg, 105 μmol) in DMSO (500 μL) was added, and the mixture was stirred for a further 24 hours. After removing volatile components by vacuum, the residue was dissolved in water (50 mL), and the solution was filtered (using a 0.45 μm syringe filter disc). The mixture was purified by TFF (Pellicon 10 kDa MWCO membrane) using water as the circulating medium until 10 dialysate filtration volumes were collected as permeate. The residue was collected, pooled by line washing, and freeze-dried to obtain [[(ε-NHCO-PEG 1100 )8(α-NHDOTA) 4.75 (α-NHCy5) 0.5 (α-NH2) 2.75 ][Lys]8[Lys]4[Lys]2[Lys]-CONH-CH2-CH2-S-]2MAL-PEG3-NHCO-PEG 24 CONH-PEG4-(PhTzMe)SRS-4-Mal was obtained as a blue solid (120 mg). 1 H NMR(300MHz,CD3OD):δ(ppm): 1.02-2.14(m,110H),2.36-2.61(m,26H),3.03(s,3H),3.07-3.30(m,39H),3.38(s,32H),3.39-3 .45(m,11H),3.53-3.59(m,33H),3.69-3.97(m,61H),6.87-7.79(m,22H) and 8.52(d,J=9.0Hz,2H). The integral of the aromatic region (δ6.80~7.92ppm) is calculated using the internal standard aromatic proton (δ8.62ppm, 2H ISThe DOTA loading was determined by qNMR using 3,4,5-trichloropyridine as an internal standard, by comparison with the above. Thus, the number of DOTA groups per molecule was found to be 9.5. Since complete consumption of cyanine 5 NHS ester was observed, the number of cyanine 5 groups per dendrimer was set to 1. The molecular weight of the dendrimer was calculated to be 29,817 Da. LC-MS (LC-MS method 20-90°C, 8 mins, TFA, no MS): R t =5.03 minutes.

[0513] 1b.52 [(ε-NHCO-PEG 1100 ) 16 (α-DOTA)7(α-NHCy5)1(α-NH2)8][Lys] 16 [Lys]8[Lys]4[Lys]2[Lys]-(PN)NCO-PEG 24 -CONH-PEG4-(PhTzMe), HH-2 [(ε-NHCO-PEG) in DMF (4.0 mL) 1100 ) 16 (α-NH2.HCl) 16 [Lys] 16 [Lys]8[Lys]4[Lys]2[Lys]-(PN)NCO-PEG 24 To a stirred solution of -CONH-PEG4-(PhTzMe)HH-1 (129 mg, 5.6 μmol), NMM (39 μL, 357 μmol), followed by cyanine 5 NHS ester (3.7 mg, 5.5 μmol), was added. After stirring for 18 hours, analysis of the reaction mixture (LCMS method 3) showed complete consumption of cyanine 5 NHS ester. A DMSO solution (1.0 mL) of p-SCN-Bn-DOTA (77 mg, 112 μmol) was added, and stirring was continued for a further 24 hours. The reaction mixture was then diluted, and the resulting solution was transferred to an Amicon® Ultra centrifugal filter equipped with a 10 kDa MWCO Ultracel® regenerated cellulose membrane and concentrated by centrifugation at 4000 rpm for 10 minutes. The residue was washed with water (×5) by centrifugation at 4000 rpm, and the residue was freeze-dried to obtain blue gum (117 mg). 11H NMR (300 MHz, CD3OD): δ (ppm): 1.05 - 2.16 (m, 172H), 2.37 - 2.58 (m, 32H), 2.98 - 3.29 (m, 61H), 3.38 (s, 48H), 3.40 - 3.44 (m, 9H), 3.58 - 3.70 (m, 1351H), 3.70 - 3.83 (m, 43H), 3.83 - 3.97 (m, 15H) and 6.99 - 7.70 (m, 25H). The integration of the aromatic region (δ 6.99 - 7.70 ppm) was compared with that of the aromatic protons of the internal standard (δ 8.62 ppm, 2H IS ) and the DOTA loading was determined by qNMR using 3,4,5-trichloropyridine as the internal standard. In this way, the number of DOTA groups per molecule was found to be 7. Since complete consumption of the cyanine 5 NHS ester was observed, the number of cyanine 5 groups per dendrimer was set to 1. The molecular weight of the dendrimer was calculated to be 27,420 Da. LCMS (LCMS method 20 - 90, 8 min, TFA, MS not available): R t = 5.11 min.

[0514] 1b.53 (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[((α-NHCy5)1(α-NHAc)7)(ε-NH-COPEG 1100 )8], G3, Compound 52 A solution of Cy5-NHS ester (1.0 mL of a 1 mg / mL DMF solution; 1.0 mg, 1.59 μmol) was added to (MeTzPh)PEG4CO-NHPEG in DMF (0.5 mL) 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[((α-NH2)8)(ε-NH-COPEG 1100)8] was added to a vial containing compound 113 (20 mg, 1.59 μmol). NMM (10 μL, 91.0 μmol) was added to this solution, and the resulting reaction mixture was protected from light and stirred at room temperature. After 3.5 h, acetic anhydride (20 μL, 212 μmol) was added, and the reaction mixture was stirred overnight and allowed to stand. After concentration of the reaction mixture under reduced pressure, it was taken up in MQ water (5 mL), divided into two portions, and purified by two (pre-equilibrated) PD10 columns. When the sample entered the column bed, it was eluted with 3.5 mL of MQ water, and the filtrate was collected. The filtrates were combined and lyophilized overnight. Lyophilization gave 19.3 mg (93%) of the title product as a bright blue powder. HPLC (C8 XBridge, 3 × 100 mm) gradient: 5% MeCN / H2O (0 - 1 min), 5 - 80% MeCN (1 - 7 min), 80% MeCN (7 - 12 min), 80 - 5% MeCN (12 - 13 min), 5% MeCN (13 - 15 min), 214 nm, 0.4 mL / min, R t = 8.30 min.

[0515] 1b.54 (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[Lys] 16 [((α-NHCy5)1(α-NHAc) 15 )(ε-NH-COPEG 1100 ) 16 , G4, compound 53 A solution of Cy5-NHS ester (520 μL of a 1 mg / mL DMF solution; 0.52 mg, 844 nmol) was added to (MeTzPh)PEG4CO-NHPEG in DMF (1.0 mL) 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[Lys] 16 [((α-NH2.HCl) 16 )(ε-NH-COPEG 1100 ) 16 ​The compound was added to a vial containing compound 120 (20 mg, 844 nmol). NMM (10 μL, 94.5 μmol) was added to this solution, and the resulting reaction mixture was then protected from light and stirred at room temperature. After 3.5 hours, acetic anhydride (20 μL, 230 μmol) was added, and the reaction mixture was stirred and left to stand overnight. The reaction mixture was concentrated under reduced pressure, then taken up with MQ water (5 mL) and divided into two parts, which were purified using two (pre-equilibriumized) PD10 columns. Upon entering the column bed, the sample was eluted with 3.5 mL of MQ water, and the filtrate was collected. The filtrates were combined and freeze-dried overnight. Freeze-drying yielded 19.9 mg (98%) of the title product as a bright blue powder. HPLC (C8 t =8.40 minutes.

[0516] 1b.55 (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [((α-NHCy5)1(α-NHAc) 31 )(ε-NH-COPEG 1100 ) 32 ], G5, compound 54 A solution of Cy5-NHS ester (300 μL of 1 mg / mL DMF solution; 0.30 mg, 487 nmol) is mixed with (MeTzPh)PEG4CO-NHPEG in DMF (1.2 mL). 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [(α-NH2.TFA) 32 (ε-NH-COPEG 1100 ) 32The compound 122 (20 mg, 435 nmol) was added to a vial. NMM (11 μL, 97.5 μmol) was added to this solution, and the resulting reaction mixture was protected from light and stirred at room temperature. After 3.5 hours, acetic anhydride (22 μL, 237 μmol) was added, and the reaction mixture was stirred and left to stand overnight. The reaction mixture was concentrated under reduced pressure, then taken up with MQ water (5 mL) and divided into two parts, which were purified using two (pre-equilibriumized) PD10 columns. When the sample entered the column bed, it was eluted with 3.5 mL of MQ water, and the filtrate was collected. The filtrates were combined and freeze-dried overnight. Freeze-drying yielded 18.7 mg (91%) of the title product as a bright blue powder. HPLC (C8 t =8.50 minutes.

[0517] 1b.56 Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NHGlu-vc-PAB-MMAE)8(ε-NH-COPEG 570 )8], G3, compound 64 Azido-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 570 Compound 64 was obtained as a product of 14.6 mg / 3.5 mL (240 μM) by preparing compound 14 (7.2 mg, 842 nmol) and HO-Glu-vc-PAB-MMAE (10.0 mg, 8.08 μmol) according to general procedure E.

[0518] 1b.57 Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NHGlu-vc-PAB-MMAE)8(ε-NH-COPEG 1100 )8], G3, compound 65 Azido-PEG 24CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 1100 Compound 65 was obtained at a concentration of 18 mg / 3.5 mL (240 μM) using compound 10 (10.8 mg, 842 nmol) and HO-Glu-vc-PAB-MMAE (10.0 mg, 8.08 μmol) according to general procedure E.

[0519] 1b.58 Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NHGlu-vc-PAB-MMAE)8(ε-NH-COPEG 2000 )8], G3, compound 66 Azido-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 2000 Compound 66 was obtained at a concentration of 25.5 mg / 3.5 mL (240 μM) using compound 16 (18.1 mg, 842 nmol) and HO-Glu-vc-PAB-MMAE (10.0 mg, 8.08 μmol) according to general procedure E.

[0520] 1b.59 Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NHDGA-MMAF(OMe))8(ε-NH-COPEG 1100 )8], G3, compound 67 Azido-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 1100 Compound 67 was obtained at a concentration of 23.8 mg / 3.5 mL (365 μM) using compound 10 (16.3 mg, 1.28 μmol) and DGA-MMAF(OMe) (10.6 mg, 12.3 μmol) according to general procedure E.

[0521] 1b.60 BHA[Lys]2[Lys]4[Lys]8[((α-NH-COPEG24 NH-COPEG4(PhMeTz))1(α-NH2)7)( ε-NH-COPEG 1000 )8], G3, compound 69 BHA[Lys]2[Lys]4[Lys]8[(α-NH2.TFA)8(ε-NH-COPEG) in DMF (300 μL) 1000 A stirred solution of (MeTzPh)PEG4CO-NHPEG (100 mg, 0.00786 mmol, 1.0 equivalent) was prepared at room temperature. 24 CO2H (Click Chemistry Tools; 16 mg, 0.01 mmol, 1.3 equivalents), PyBOP (8 mg, 0.013 mmol, 1.6 equivalents), and DMF (200 μL) were added. After stirring the reaction mixture for 3 minutes, NMM (40 mg, 50 μL, 0.38 mmol, 48 equivalents) was added. The contents were protected from light and stirred overnight at room temperature. The reaction mixture was diluted with MQ water and frozen overnight. The lyophilized material was taken up in MeOH (1 mL) and purified by SEC (400 drops / tube, MeOH sephadex LH20, 35 drops / min). The product-containing fraction was identified by HPLC and collected in two different fractions. Each fraction was concentrated under reduced pressure, and the resulting residue was taken up in MQ water, filtered (through a 0.45 μm Acrodisc filter), and lyophilized to obtain compound 69 as a pink solid (69 mg, 66%). HPLC (C8 t = 8.4 minutes (broad peak). 1 H NMR(300MHz,D2O)δ(ppm): 1.00-2.00(m,90H),2.51(t,3H),2.60(br s,3H),3.00-3.12(m,6H),3.12-3.35(br s,27H),3.35-3.45(m,26H),3.45-4.15(m,937H),4.15-4.45(m,12H),6.12(s,1H),7.15-7.50(m,12H),8.40-8.50(m,2H).

[0522] 1b.61 BHA[Lys]2[Lys]4[Lys]8[((α-NH-COPEG 24 NH-COPEG2-BCN)1(α-NH2)7)(ε-NH-COPEG 1000 )8], G3, compound 70 BCN-PEG2CO-NHPEG in DMF (200 μL) 24 A stirred solution of -CO2H and compound 19 (9.6 mg, 0.006 mmol, 1.3 equivalents) was prepared at room temperature. To this, PyBOP (4 mg, 0.008 mmol, 1.6 equivalents) and NMM (23 mg, 25 μL, 0.226 mmol, 48 equivalents) were added, and after 5 minutes, BHA[Lys]2[Lys]4[Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 1000 )8](Reference 1) (60 mg, 0.006 mmol, 1.0 equivalent), followed by DMF (200 μL). The contents were protected from light and stirred overnight at room temperature. The reaction mixture was diluted with MeCN (10 mL) and then purified by SEC (400 drops / tube, MeCN sephadex LH20, 35 drops / min). The product-containing fraction was identified by HPLC, recovered, filtered (0.45 μm Acrodisc filter), concentrated under reduced pressure, and lyophilized overnight to obtain compound 70 as a pale yellow solid (58 mg, 92% yield). HPLC (C8 t = 8.43 minutes (broad peak). 1 H NMR(300MHz,CD3OD)δ(ppm): 0.75-1.12(m,15H), 1.12-2.15(m,95H),2.15-2.35(m,7H), 2.55(br s,3H),3.00-3.35(m,90H),3.35-3.38(s,17H),3.38-4.09(m,592H),4.13(d,2H),4.18-4.63(br s,7H),6.18(s,0.9H),7.12-7.48(m,7H).

[0523] 1b.62 (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[((α-NHCy5)1(α-NHGlu-VC-PAB-MMAE)7)( ε-NH-COPEG 1100 )8], G3, compound 71 A solution of Cy5-NHS (4.2 mg / mL solution in DMF, 214 μL, 1.43 μmol, 1.0 equivalent) is prepared by adding neat (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NH2.HCl)8(ε-NH-COPEG 1100 )8] was added to compound 113 (18 mg, 1.43 μmol). After complete dissolution, NMM (10 μL, 91.0 μmol) was added, and the resulting reaction mixture was stirred and protected from light. After 2 hours, NMM (10 μL, 91.0 μmol) and PyBOP (7.3 mg, 14.0 μmol) were added to the dendrimer solution, and then to the Glu-VC-PAB-MMAE solution in DMF (290 μL of 60 mg / mL solution, 14.0 μmol, 9.8 equivalents). The resulting reaction mixture was then protected from light and stirred overnight at room temperature. The reaction mixture was diluted with PBS (2.0 mL) to a final volume of 2.5 mL. The diluted solution was then passed through a PD10 desalting column (pre-equilibrated with PBS). After the entire solution entered the column bed, PBS (3.5 mL) was added to elute the product (which appeared as a blue band). The final theoretical concentration of compound 71 = 8.7 mg / mL in PBS. The materials were frozen and stored at -80°C. HPLC (C8 XBridge, 3×100mm) gradient: 5% MeCN / H2O (0-1 min), 5-80% MeCN (1-7 min), 80% MeCN (7-12 min), 80-5% MeCN (12-13 min), 5% MeCN (13-15 min), 214 nm, 0.4 mL / min, R t =95-10 minutes (broad peak); 83% conjugate-related peak including 17% MMAE / MMAE linker

[0524] 1b.63 (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]2[3 H-Lys]4[Lys]8[(α-NHGlu-VC-PAB-MMAE)8(ε-NH-COPEG 1100 )8], G3, Compound 72 (MeTzPh)PEG4CO-NHPEG in NMM / DMF (7.8 μL / 0.5 mL) 24 CO-[N(PN)2][Lys]2 3 H-Lys]4[Lys]8[(α-NH2.HCl)8(ε-NH-COPEG 1100 )8](Synthesized according to the procedure used for the synthesis of Compound 114, but using tritiated DBL-OPNP (Reference 1) for the synthesis of the G2 layer) (36.8 mg, 2.93 μmol) solution, solid PyBOP (24.7 mg, 47.5 μmol) was added. After complete dissolution, this dendrimer solution was added to a solution of HO-Glu-VC-PAB-MMAE (40 mg, 32.3 μmol) in NMM / DMF (7.8 μL / 0.5 mL). Subsequently, the resulting reaction mixture was protected from light and stirred at room temperature overnight. The reaction mixture was diluted with PBS (4.0 mL) to a final volume of 5 mL. Then, the diluted solution was passed through two PD10 desalting columns (pre-equilibrated with PBS, 2.5 mL each column). When all the solution had entered the column bed, PBS (3.5 mL) was added to each column to elute the product. The resulting crude mixture was further purified using a regenerated cellulose Amicon Ultra-0.5 mL centrifugal unit (10K MWCO). Final theoretical concentration of Compound 72 = 29 - 30 mg / mL in PBS. The material was stored frozen at -20 °C. HPLC (C8 XBridge, 3 × 100 mm) gradient: 5% MeCN / H2O (0 - 1 min), 5 - 80% MeCN (1 - 7 min), 80% MeCN (7 - 12 min), 80 - 5% MeCN (12 - 13 min), 5% MeCN (13 - 15 min), 214 nm, 0.4 mL / min, R t = 9.5 - 12 min (broad peak); 91.4% conjugate-related peak including 8.6% MMAE / MMAE linker-related peak.

[0525] 1b.64 (MeTzPh)PEG4CO-NHPEG 24CO-[N(PN)2][Lys]2[Lys]4[Lys]8[((α-NHDFO)2(α-NHGlu-VC-PAB-MMAE)6)(ε-NH-COPEG 1100 )8], compound 73 A stirred solution of p-SCN-deferoxamine (2.1 mg, 2.79 μmol) in DMSO (100 μL) was prepared at room temperature. To this, (MeTzPh)PEG4CO-NHPEG in DMF (200 μL) was added. 24 CO[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NH2.HCl)8(ε-NH-COPEG 1100 Compound 113 (17.0 mg, 1.35 μmol) was added. The resulting reaction mixture was then stirred for 3 minutes, after which NMM (10 μL, 91.0 μmol) was added. The resulting solution was protected from light and stirred at room temperature for 4 hours. PyBOP (7.0 mg, 13.5 μmol) was added, and after 5 minutes, the reaction mixture was added to neat HO-Glu-VC-PAB-MMAE (9.76 mg, 7.89 μmol). The resulting reaction mixture was then left to stand overnight. The reaction mixture was diluted with PBS buffer (4.5 mL), divided into four Amicon Ultra centrifugation filters (10K MWCO), and the filters were centrifuged (14K rcf, 15 min). The residue was dialyzed against PBS (400 μL, 14K rcf, 15 min x 10 times). The retaining solution was mixed to obtain a pink solution. The concentration of compound 73 is approximately 16 mg in 2 mL. HPLC (C8 t = 8.7-9.8 minutes (broad peak).

[0526] 1b.65 BHA[Lys]2[Lys]4[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhMeTz))1(α-NHDFO)2(α-NHGlu-VC-PAB-MMAE)5)(ε-NH-COPEG 1100 )8], compound 74 A stirred solution of p-SCN-deferoxamine (2.0 mg, 2.66 μmol) in DMSO (100 μL) was prepared at room temperature. To this, BHALys[Lys]2[Lys]4[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhMeTz))1(α-NH2)7)( ε-NHPEG 1100 Compound 69 (17.0 mg, 1.27 μmol) was added to [8]. The resulting reaction mixture was then stirred for 3 minutes, after which NMM (10 μL, 91.0 μmol) was added. The resulting solution was protected from light and stirred at room temperature for 4 hours. PyBOP (7.0 mg, 13.5 μmol) was added, and after 5 minutes, the reaction mixture was added to neat HO-Glu-VC-PAB-MMAE (9.17 mg, 7.41 μmol). The resulting reaction mixture was then left to stand overnight. The reaction mixture was diluted with PBS buffer (4.5 mL), divided into four Amicon Ultra centrifugation filters (10K MWCO), and the filters were centrifuged (14K rcf, 15 min). The residue was dialyzed against PBS (400 μL, 14K rcf, 15 min x 10 times). The retaining solution was mixed to obtain a pink solution. The concentration of compound 74 is approximately 16 mg in 2 mL. HPLC (C8 t = 9.3-9.7 minutes (broad peak).

[0527] 1b.66 (MeTzPh)-PEG 24 -CO[N(PNBoc)2], compound 106 (MeTzPh)-PEG in DMF (3 mL) 24-CO2H (0.402 g, 0.305 mmol), PyBOP (0.205 g, 0.394 mmol), and NMM (130 μL, 1.18 mmol) were stirred together, and NH(PNBoc)2 (0.147 g, 0.444 mmol) was added under an N2 atmosphere. The resulting reaction mixture was then stirred at room temperature and left to stand overnight. Volatile components were removed by vacuum, and the resulting oil was purified by silica chromatography (5%~10% MeOH / DCM) to obtain the desired product, compound 106, as a red residue (0.474 g, 95%). 1 H NMR(300MHz,CD3OD)δ(ppm): 1.43-1.44(m,18H); 1.64-1.80(m,4H); 2.63(t,J 6.0Hz,2H); 3.00(s,3H); 3.02-3.09(m,4H); 3.34-3.40(m,4H),3.58-3.77(m,99H); 3.88-3.91(m,2H); 4.25-4.28(m,2H); 7.15-7.20(m,2H); 8.46-8.51(m,2H). LCMS (hydrophobic method, formic acid buffer) R t =6.20 minutes. ESI MS(+ve)1631.0[M+H]+;C 76 H 140 N7O 30 [M+H] + The calculated m / z value for this is 1631.0.

[0528] 1b.67 (MeTzPh)-PEG 24 -CO[N(PNH2.HCl)2], compound 107 (MeTzPh)-PEG in ice / water bath 24 -CO[N(PNBoc)2] Compound 106 (0.420 g, 0.258 mmol) was slowly mixed with 1.25 M HCl / MeOH solution (8 mL, 10.0 mmol). After 5 minutes, the ice bath was removed, and the resulting reaction mixture was stirred overnight at room temperature. Volatile components were removed by vacuum, and the product, Compound 107, was obtained as a red residue (0.388 g, 100%). 1H NMR(300MHz,CD3OD)δ(ppm): 1.91-2.07(m,4H);2.68(t,J6.0Hz,2H);2.94-3.09(m,7H);3.53-3.80(m,108H );3.88-3.91(m,2H);4.25-4.28(m,2H);7.16-7.20(m,2H);8.47-8.51(m,2H). LCMS (hydrophilic method, formic acid buffer) R t =8.12 minutes. ESI MS(+ve)1430.9[M+H] + ;C 66 H 124 N7O 26 [M+H] + The calculated m / z value for this is 1430.8.

[0529] 1b.68 (MeTzPh)-PEG4-PEG 24 -CO[N(PNH2.HCl)2] Compound 108 H2N-PEG in DMF (2.0 mL) 24 To a stirred solution of -CO[N(PNBoc)2](0.418g, 0.286 mmol), (MeTzPh)-PEG4-CO2H(0.15g, 0.344 mmol), PyBOP(0.178g, 0.342 mmol), and NMM(80 μL, 0.727 mmol) were added. The resulting reaction mixture was then stirred at room temperature overnight. After removing volatile components under vacuum and cooling the resulting oil in an ice / water bath, a 1.25 M HCl / MeOH solution (10.0 mL, 12.5 mmol) was slowly added. After 5 minutes, the ice bath was removed, and the resulting reaction mixture was then stirred at room temperature overnight. The volatile components were removed by vacuum, and the resulting oil was dissolved in MeCN / H2O (8 mL, 1:1). It was then purified by preparative HPLC (10-50% MeCN, 0.1% formate buffer, RT 35 min) to obtain the red solid compound 108 (1.37 g, 54%). 1H NMR(300MHz,CD3OD)δ(ppm): 1.91-2.04(m,4H);2.43(t,J6.0Hz,2H);2.68(t,J6.0Hz,2H);2.94-3.07(m,4H);3.00(s,3H);3.35(t,J6. 0Hz,2H);3.51-3.80(m,119H);3.88-3.91(m,2H);4.25-4.28(m,2H);7.16-7.20(m,2H);8.46-8.51(m,2H). LCMS (hydrophilic method, formic acid buffer) R t =8.15 minutes. ESI MS(+ve)1677.9[M+H] +...

Claims

1. A dendrimer-targeting agent conjugate, The aforementioned dendrimer-targeting agent conjugate is a) Dendrimers and, b) Targeting agent, c) One or more first terminal groups, d) One or more second terminal groups and Includes, a) The dendrimer is, i) Core unit (C), and ii) Including the component unit (BU), The aforementioned core unit is 【Chemistry 1】 And, Each of the aforementioned component units is: 【Chemistry 2】 And, The aforementioned dendrimer has 2 to 5 generations of constituent units, The core unit is attached to at least two constituent units by covalent bonds. b) The targeting agent is A targeting agent which is a single-domain antibody having a molecular weight of 5 kDa to 30 kDa, and is covalently linked to the dendrimer by a spacer group containing a polyethylene glycol (PEG) group. c) The one or more first terminal groups are One or more first terminal groups attached to the outermost constituent unit of the dendrimer, wherein the first terminal group includes a complexing group that is complexed with a radionuclide, and the complexing group is a DOTA, benzyl-DOTA, NOTA, DTPA, sarcofazine, macropa, DFO, PEPA, or EDTA group. d) The one or more second terminal groups are One or more second terminal groups attached to the outermost constituent unit of the dendrimer, the pharmacokinetic modification moiety comprising a polyethylene glycol (PEG) group, The dendrimer-targeting agent conjugate, or a salt thereof.

2. The conjugate according to claim 1, wherein the targeting agent is covalently bonded to the spacer group at or near its C-terminus.

3. The conjugate according to claim 1 or 2, wherein the complexing group is a DOTA or DFO group.

4. The conjugate according to any one of claims 1 to 3, wherein the radioactive nuclide in the radioactive nuclide-containing portion is lutetium, gallium, zirconium, actinium, bismuth, astatine, technetium, lead, yttrium, or copper radioactive nuclide.

5. The conjugate according to any one of claims 1 to 4, wherein the radioactive nuclide is an α-emitting or β-emitting material.

6. The conjugate according to any one of claims 1 to 5, wherein the pharmacokinetic modification portion is a polyethylene glycol (PEG) group having an average molecular weight of 1,000 to 2,300 Da.

7. The aforementioned component unit, 【Transformation 3】 The conjugate according to any one of claims 1 to 6.

8. A pharmaceutical composition, i) A conjugate according to any one of claims 1 to 7, ii) A pharmaceutical composition comprising a pharmaceutically acceptable excipient.

9. A conjugate according to any one of claims 1 to 7, for use in therapy or imaging.

10. A pharmaceutical composition according to claim 8 for use in the treatment of cancer.

11. The pharmaceutical composition according to claim 10, wherein the cancer is prostate cancer, pancreatic cancer, gastrointestinal cancer, lung cancer, breast cancer, or brain tumor.

12. Use of the conjugate according to any one of claims 1 to 7 in the manufacture of a pharmaceutical product for the treatment of cancer.

13. Use of the pharmaceutical composition according to claim 8 in the manufacture of a pharmaceutical for the treatment of cancer.

14. The use according to claim 12 or 13, wherein the cancer is prostate cancer, pancreatic cancer, gastrointestinal cancer, lung cancer, breast cancer, or brain tumor.

15. A kit for generating therapeutic conjugates, The aforementioned kit is a) Dendrimer-targeting agent conjugate or a salt thereof, b) Radionuclides and Includes, The aforementioned dendrimer-targeting agent conjugate is a1) Dendrimer and b1) Targeting agent, c1) One or more first terminal groups, d1) One or more second terminal groups and Includes, a1) The dendrimer is, i) Core unit (C), and ii) Including the component unit (BU), The aforementioned core unit is 【Chemistry 4】 And, Each of the aforementioned component units is: 【Transformation 5】 And, The aforementioned dendrimer has 2 to 5 generations of constituent units, The core unit is attached to at least two constituent units by covalent bonds. b1) The targeting agent is A targeting agent which is a single-domain antibody having a molecular weight of 3 kDa to 30 kDa, and is covalently linked to the dendrimer by a spacer group containing a polyethylene glycol (PEG) group. c1) The one or more first terminal groups are One or more first terminal groups attached to the outermost constituent unit of the dendrimer, wherein the first terminal groups include a complexing group for complexing a radionuclide, and the complexing group is a DOTA, benzyl-DOTA, NOTA, DTPA, sarcofazine, macropa, DFO, PEPA, or EDTA group. d1) The one or more second terminal groups are One or more second terminal groups attached to the outermost constituent unit of the dendrimer, the pharmacokinetic modification moiety comprising a polyethylene glycol (PEG) group, The aforementioned kit.

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