Macrocyclic chelators and methods of use thereof

Abstract

Macrocyclic chelators (I) are provided for the chelation of alpha-emitting radioactive metal ions, such as actinium-225. Radiometal complexes comprising alpha-emitting radioactive metal ions bound to the macrocyclic chelators by coordinate bonds, and radioimmunoconjugates comprising the radiometal complexes covalently bound to targeting ligands, such as antibodies or antigen-binding fragments thereof, are also provided. The radioimmunoconjugates can be produced by click chemistry reactions. Methods of using the radiocomplexes and radioimmunoconjugates for selectively targeting neoplastic cells for radiotherapy and for treating neoplastic diseases and disorders are also described.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 846,044, filed May 10, 2019, which is incorporated herein by reference in its entirety for all purposes.

[0002] (Reference to electronically submitted sequence listing) This application contains a Sequence Listing that has been submitted electronically via EFS-Web as an ASCII sequence listing with the filename "JBI6072WOPCT1_SeqListing.txt", created on May 1, 2020, and having a size of 26 kb. The Sequence Listing submitted via EFS-Web is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] Alpha-particle-emitting radionuclides hold great promise for cancer therapy due to their combination of high energy and short-range action, offering the potential for potent killing, largely confined to tumor cells (Kim, Y. Sand and M.W. Brechbiel, An overview of targeted alpha therapy. Tumor Biol, 2012. 33(3): pp. 573-90). Targeted delivery of alpha-emitters using antibodies, scaffold proteins, small molecule ligands, aptamers, or other binding moieties specific for cancer antigens offers a method for selective delivery of radionuclides to tumors, enhancing their efficacy and reducing off-target effects. In common practice, the binding moiety is attached to a chelator that binds to an alpha-emitting radiometal to generate a radioconjugate. Many such examples use monoclonal antibodies (mAbs) as targeting ligands, generating what are known as radioimmunoconjugates.

[0004] Actinium-225( 225Actinium-225 (Ac) is an alpha-emitting radioisotope of particular interest for medical applications (Miederer et al., Realizing the potential of the Actinium-225 radionuclide generator in targeted alpha particle therapy applications. Adv Drug Deliv Rev, 2008. 60(12):71-82). 225 The 10-day half-life of Ac is long enough to facilitate the generation of radioconjugates, yet short enough to match the circulating pharmacokinetics of delivery vehicles such as antibodies. 225 Radioimmunoconjugates of Ac are of particular interest. 225 Ac is a stable isotope 209 Its potency increases as it decays in a series of steps, ultimately emitting four alpha particles before reaching Bi. Another radioisotope of interest for medical applications is lutetium-177 ( 177 Lu). 177 Lu-labeled peptides showed reduced damage to normal tissues, 177 Lu labeling has been shown to allow the use of a single radiolabeled agent for both therapy and imaging (Kwekkeboom DJ, et al. 177 Lu-DOTA 0 ,Tyr 3 ]octreotate:comparison with[ 111 In-DTPA 0 ]octreotide in patients. Eur J Nucl Med. 2001;28:pp.1319-1325). Other radioisotopes used in therapeutic applications include, for example, beta-emitters or alpha-emitters, e.g., 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111Ag, 131 I, 149 Tb, 152 Tb, 155 Tb, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 255 Fm and 227 Other radioisotopes used in imaging applications include, for example, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr and 111 Examples include gamma-ray emitting radioisotopes such as In.

[0005] Currently, the most widely used chelator for actinium-225 and lanthanides is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; tetraxathen). Previous clinical and preclinical programs have primarily used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for actinium chelation. However, DOTA chelation of actinium is known to be challenging (Deal, K.A., et al., Improved in vivo stability of actinium-225 macrocyclic complexes. J. Med. Chem., 1999, 42(15):2988-92). For example, when conjugated to targeting ligands such as proteins or antibodies, DOTA only allows a maximum DOTA:actinium-225 chelation ratio of >500:1, often requiring either harsh conditions or high levels of DOTA per antibody. Other macrocyclic chelators for lanthanides and actinium-225 are described, for example, in WO 2018 / 183906, Thiele et al., "An Eighteen-Membered Macrocyclic Ligand for Actinium-225 Targeted Alpha Therapy," Angew. Chem. Int. Ed. (2017) 56, 14712-14717, and Roca-Sabio et al., "Macrocyclic Receptor Exhibiting Unprecedented Selectivity for Light Lanthanides," J. Am. Chem. Soc. (2009) 131, 3331-3341.

[0006] Site specificity has become a major area of ​​focus in the antibody-drug conjugate (ADC) field, as it has been demonstrated that site-specific approaches can enhance both the efficacy and safety of ADCs compared to random conjugation (Agarwal, P. and C.R. Bertozzi, Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development, Bioconjug Chem, 2015. 26(2): pp. 176-92). It is believed that similar safety and efficacy benefits may be achieved with radioimmunoconjugates. Summary of the Invention [Means for solving the problem]

[0007] Therefore, radioactive metals, preferably actinium-225 ( 225 There is a need in the art for novel chelators that can be used to bind alpha-emitting radiometals, such as α-Ac, and generate stable radioimmunoconjugates with high specific activity and high yields. The present invention provides a method for binding alpha-emitting radiometals, particularly α-Ac, regardless of their specific activity or the most common metal impurities. 225 This need is met by providing macrocyclic chelators capable of binding radiometals such as Ac. The chelators of the invention can be conjugated to targeting ligands, such as antibodies, proteins, aptamers, and small molecules, preferably in a site-specific manner using "click chemistry," to generate radioimmunoconjugates with high in vitro and in vivo stability. Radioimmunoconjugates generated by conjugating the chelators of the invention to targeting ligands can be used for targeted therapy, such as targeted radiotherapy of neoplastic cells and / or targeted therapy of neoplastic diseases or disorders, including cancer.

[0008] In one general aspect, the present invention relates to a chelator of formula (I):

[0009] [ka] During the ceremony, Each of ring A and ring B is independently a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl, and each of ring A and ring B is independently a halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN, -OC(O)N(R 13 Optionally substituted with one or more substituents selected from the group consisting of X and Each of Z1 and Z2 independently represents -(C(R 12 )2) m -or-(CH2) n -C(R 12 )(X)-(CH2) n - and Each X is independently -L1-R 11 and each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5; each p is independently 0 or 1; L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12 are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; Each R 13 are independently hydrogen or alkyl; R 14 , R 15 , R 16 and R 17 each is independently hydrogen, alkyl or X; Alternatively, R 14 and R 15 and / or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring optionally substituted with X; provided that the chelator contains at least one X, and when X is present on ring A or ring B, L1 is a linker or R 12 and R 14 ~R 17 At least one of them is not hydrogen.

[0010] In an alternative embodiment, it is contemplated that each of Ring A and Ring B is an optionally substituted heterocyclyl ring, for example, oxazoline.

[0011] In one embodiment, the chelator of the present invention is a chelator of formula (II):

[0012] [ka] During the ceremony, A1 is N or CR1 or absent; A2 is N or CR2; A3 is N or CR3; A4 is N or CR4; A5 is N or CR5; A6 is N or CR6 or absent; A7 is N or CR7; A8 is N or CR8; A9 is N or CR9, A 10 is N or CR 10 and However, three or less of A1, A2, A3, A4 and A5 are N, and A6, A7, A8, A9 and A 10 Not more than three of R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 each independently represents hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN, -OC(O)N(R 13 )2 and -X; Alternatively, any two immediately adjacent R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 together with the atoms to which they are attached form a 5- or 6-membered substituted or unsubstituted carbocyclic or nitrogen-containing ring; Each of Z1 and Z2 independently represents -(C(R 12 )2) m -or-(CH2) n -C(R 12 )(X)-(CH2) n - and Each X is independently -L1-R 11 and each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5; each p is independently 0 or 1; L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12 are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; Each R 13are independently hydrogen or alkyl; R 14 , R 15 , R 16 and R 17 each is independently hydrogen, alkyl or X; Alternatively, R 14 and R 15 and / or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring optionally substituted with X; provided that the chelator contains at least one X and R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 When any one of is X, L1 is a linker.

[0013] In one embodiment, the chelator of the present invention is a chelator of formula (III):

[0014] [ka] During the ceremony, Each A 11 are independently O, S, NMe, or NH; Each of Z1 and Z2 independently represents -(C(R 12 )2) m -or-(CH2) n -C(R 12 )(X)-(CH2) n - and Each X is independently -L1-R 11 and each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5; each p is independently 0 or 1; L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; Each R 13 are independently hydrogen or alkyl; R 14 , R 15 , R 16 and R 17 each is independently hydrogen, alkyl or X; Alternatively, R 14 and R 15 and / or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring optionally substituted with X; Each R 18 are independently hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN, -OC(O)N(R 13 )2 and -X; provided that the chelator contains at least one X and R 18 is X, L1 is a linker, or R 12 and R 14 ~R 17 At least one of them is not hydrogen.

[0015] In certain embodiments, the chelator is

[0016] [ka] wherein: L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11comprises a targeting ligand, Each R 12 are independently hydrogen, -CH3, or -CH2CH3, provided that at least one R 12 is -CH3 or CH2CH3.

[0017] In some embodiments, R 11 -NH2, -NCS, -NCO, -N3, alkynyl, cycloalkynyl, C(O)R 13 , -COOR 13 , -CON(R 13 ) 2, maleimide, acyl halide, tetrazine or trans-cyclooctene.

[0018] In certain embodiments, R 11 is a cyclooctynyl or cyclooctynyl derivative selected from the group consisting of bicyclononynyl (BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, biarylazacyclooctynonyl (BARAC), dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO), and tetramethoxy dibenzocyclooctynyl (TMDIBO).

[0019] In certain embodiments, R 11 is DBCO or BCN.

[0020] In some embodiments, R 11comprises a targeting ligand, which comprises an antibody or antigen-binding fragment thereof, a scaffold protein, a small molecule, or an aptamer.

[0021] In certain embodiments, the targeting ligand is an antibody or an antigen-binding fragment thereof.

[0022] In another aspect, the present invention relates to radiometal complexes comprising a chelator of the present invention, which comprise a radiometal ion bound to the chelator by a coordinate bond.

[0023] In one embodiment, a radiometal complex of the present invention has the structure of formula (Im):

[0024] [ka] During the ceremony, M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion; Each of ring A and ring B is independently a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl, and each of ring A and ring B is independently a halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 Optionally substituted with one or more substituents selected from the group consisting of X and Each of Z1 and Z2 independently represents -(C(R 12 )2) m -or-(CH2) n -C(R 12 )(X)-(CH2) n - and Each X is independently -L1-R 11 and each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5; each p is independently 0 or 1; L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12 are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; Each R 13 are independently hydrogen or alkyl; R 14 , R 15 , R 16 and R 17 each is independently hydrogen, alkyl or X; Alternatively, R 14 and R 15 and / or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring optionally substituted with X; provided that when the radiometal complex contains at least one X and X is in ring A or ring B, L1 is a linker or R 12 and R 14 ~R 17 At least one of them is not hydrogen.

[0025] In an alternative embodiment, it is contemplated that each of Ring A and Ring B is an optionally substituted heterocyclyl ring, for example, oxazoline.

[0026] In one embodiment, the radiometal complex of the present invention is a radiometal complex of formula (II-m):

[0027] [ka] During the ceremony, M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion; A1 is N or CR1 or absent; A2 is N or CR2; A3 is N or CR3; A4 is N or CR4; A5 is N or CR5; A6 is N or CR6 or absent; A7 is N or CR7; A8 is N or CR8; A9 is N or CR9, A 10 is N or CR 10 and However, three or less of A1, A2, A3, A4 and A5 are N, and A6, A7, A8, A9 and A 10 Not more than three of R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 each independently represents hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 )2 and -X; Alternatively, any two immediately adjacent R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 together with the atoms to which they are attached form a 5- or 6-membered substituted or unsubstituted carbocyclic or nitrogen-containing ring; Each of Z1 and Z2 independently represents -(C(R 12 )2) m -or-(CH2) n -C(R 12 )(X)-(CH2) n - and Each X is independently -L1-R 11 and each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5; each p is independently 0 or 1; L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12 are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; Each R 13 are independently hydrogen or alkyl; R 14 , R 15 , R 16 and R 17 each is independently hydrogen, alkyl or X; Alternatively, R 14 and R 15 and / or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring optionally substituted with X; provided that the radiometal complex contains at least one X and R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 When any one of is X, L is a linker or at least one or R 12 and R 14 ~R 17 is not hydrogen.

[0028] In one embodiment, the radiometal complex of the present invention is a radiometal complex of formula (III-m):

[0029] [ka] During the ceremony, M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion; Each A 11 are independently O, S, NMe, or NH; Each of Z1 and Z2 independently represents -(C(R 12 )2) m -or-(CH2) n -C(R 12 )(X)-(CH2) n - and Each X is independently -L1-R 11 and each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5; each p is independently 0 or 1; L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12 are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; Each R 13 are independently hydrogen or alkyl; R 14 , R 15 , R 16 and R 17 each is independently hydrogen, alkyl or X; Alternatively, R 14 and R 15 and / or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring optionally substituted with X; Each R 18 are independently hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13, -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 )2 and -X; provided that the radioactive metal complex contains at least one X and R 18 is X, L1 is a linker, or R 12 and R 14 ~R 17 At least one of them is not hydrogen.

[0030] In certain embodiments, the alpha-emitting radioactive metal ion is actinium-225 ( 225 Ac).

[0031] In certain embodiments, the radiometal complexes of the present invention are

[0032] [ka] and

[0033] [ka] is selected from the group consisting of During the ceremony, M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion, more preferably actinium-225 ( 225 Ac), and L1 is absent or a linker; R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12 are independently hydrogen, -CH3, or -CH2CH3, provided that at least one R 12 is -CH3 or CH2CH3.

[0034] In another general aspect, the invention provides a compound comprising: 11The present invention also relates to immunoconjugates comprising a chelator of the present invention covalently attached via a chelator to a targeting ligand, preferably an antibody or antigen-binding fragment thereof.

[0035] In yet another general aspect, the invention provides a compound comprising: 11 The present invention relates to a radioimmunoconjugate comprising a radiometal complex of the present invention covalently bound to a targeting ligand, preferably an antibody or antigen-binding fragment thereof, via

[0036] In one embodiment, the radioimmunoconjugate comprises a radiometal complex of the invention covalently attached via a triazole moiety to a targeting ligand, particularly an antibody or antigen-binding fragment thereof.

[0037] In a specific embodiment, the radioimmunoconjugate of the present invention comprises:

[0038] [ka] is selected from the group consisting of wherein L1 is a linker; mAb is an antibody or an antigen-binding fragment thereof; preferably, the mAb is an antibody or antigen-binding fragment that specifically binds to a tumor cell, more preferably to a tumor antigen selected from the group consisting of prostate-specific membrane antigen (PSMA), BCMA, Her2, EGFR, KLK2, CD19, CD22, CD30, CD33, Cd79b, and Nectin-4; and each R 12 are independently hydrogen, -CH3, or -CH2CH3, provided that at least one R 12 is -CH3 or -CH2CH3.

[0039] In another general aspect, the present invention provides a method for covalently linking a chelator or radiometal complex of the invention to a targeting ligand, preferably the R 11The present invention relates to a method for preparing an immunoconjugate or radioimmunoconjugate of the present invention, comprising covalently linking the immunoconjugate or radioimmunoconjugate to an antibody or antigen-binding fragment thereof via

[0040] In a specific embodiment, the method for preparing a radioimmunoconjugate of the present invention comprises: (i) providing a modified polypeptide comprising a polypeptide (e.g., an antibody or antigen-binding fragment thereof) covalently attached to a first click reaction partner (e.g., an azide group); (ii) providing a chelator complex comprising a chelator of the invention covalently bonded to a second Click reaction partner (e.g., an alkynyl or cycloalkynyl group); (iii) contacting the modified polypeptide with a chelator complex under conditions that allow the first Click reaction partner (e.g., an azide group) to react with a second Click reaction partner (e.g., an alkynyl or cycloalkynyl group) to form a polypeptide-chelator complex (i.e., an immunoconjugate); (iv) contacting the polypeptide-chelator complex with a radioactive metal ion, thereby preparing a radioimmunoconjugate (the radioimmunoconjugate comprises a radioactive metal ion-labeled polypeptide, e.g., a modified antibody or antigen-binding fragment thereof, labeled with an alpha-emitting radioactive metal ion, bound to the chelator by a coordinate bond).

[0041] According to certain embodiments, step (iv) is carried out under metal-free conditions. Preferably, the method is carried out in a site-specific manner as described herein.

[0042] In an alternative embodiment, the method for preparing a radioimmunoconjugate of the present invention comprises: (i) providing a modified antibody or antigen-binding fragment thereof, comprising an antibody or antigen-binding fragment thereof covalently bound to an azido group; (ii) providing a radioactive complex of the present invention comprising an α-emitting radioactive metal ion bound by a coordinate bond to a chelator, wherein the chelator is covalently bound to an alkynyl or cycloalkynyl group; (iii) contacting the modified antibody or antigen-binding fragment thereof with a radioactive complex under conditions that allow the azido group to react with the alkynyl or cycloalkynyl group, thereby preparing a radioimmunoconjugate (e.g., as shown in Figure 2D).

[0043] In some embodiments, the cycloalkynyl group is cyclooctynyl or a cyclooctynyl derivative selected from the group consisting of bicyclononynyl (BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, biarylazacyclooctynyl (BARAC), dibenzoazacyclooctynyl (DIBAC), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO), and tetramethoxydibenzocyclooctynyl (TMDIBO).

[0044] In another general aspect, the present invention relates to a pharmaceutical composition comprising a radioimmunoconjugate of the invention and a pharmaceutically acceptable carrier. The pharmaceutical composition may also include one or more pharmaceutically acceptable carriers.

[0045] In yet another general aspect, the present invention relates to methods of using the radioimmunoconjugates and pharmaceutical compositions of the invention for targeted radiotherapy.

[0046] In one embodiment, there is provided a method for selectively targeting neoplastic cells for radiation therapy in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition of the present invention.

[0047] In one embodiment, the present invention provides a method of treating a neoplastic disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the present invention.

[0048] The foregoing Summary of the Invention and the following Detailed Description of the Invention will be better understood when read in conjunction with the accompanying drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.

[0049] The drawings are as follows: [Brief explanation of the drawings]

[0050] [Figure 1A] HPLC chromatograms of the La chelation study described in Example 1 are shown. HPLC chromatograms of H2bp18c6-benzyl-phenyl before mixing (top) and after mixing with La are shown; the shift in retention time from 14.137 min to 12.047 min after mixing with La indicates rapid chelation of La by H2bp18c6-benzyl-phenyl. [Figure 1B] HPLC chromatograms of the chelation study with La described in Example 1 are shown. HPLC chromatograms of H2bp18c6-benzyl-isopentyl before mixing (top) and after mixing with La are shown; the shift in retention time from 17.181 min to 15.751 min after mixing with La indicates rapid chelation of La by H2bp18c6-benzyl-isopentyl. [Figure 2]1A-1D show schematic diagrams of radiolabeling of antibodies to generate radioimmunoconjugates according to embodiments of the present invention by random conjugation methods (e.g., labeling of lysine residues, cysteine ​​residues, etc.) or site-specific conjugation methods (e.g., glycan-specific methods, conjugation tag methods, or engineered cysteine ​​methods). A shows random conjugation by one-step direct radiolabeling. B shows random conjugation by click radiolabeling. C shows site-specific conjugation by one-step direct radiolabeling. D shows site-specific conjugation by click radiolabeling. DETAILED DESCRIPTION OF THE INVENTION

[0051] Various publications, articles, and patents are cited or described in the Background and throughout this specification. Each of these references is incorporated herein by reference in its entirety. Any discussion of documents, operations, materials, devices, articles and the like which has been included in the specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these items constitute part of the prior art to any invention disclosed or claimed.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise defined, specific terms cited herein have the meanings set forth herein. All patents, published patent applications, and publications cited herein are incorporated by reference as if set forth in their entireties.

[0053] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0054] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of the specified integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the word "comprising" can be replaced with the words "containing" or "including," or, as sometimes used herein, can also be replaced with the word "having."

[0055] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. When used herein in connection with aspects or embodiments of the present invention, any of the above terms "comprising," "containing," "including," and "having" can be substituted with the terms "consisting of" or "essentially consisting of" to vary the scope of the disclosure.

[0056] As used herein, the connective term "and / or" between multiple listed elements is understood to encompass both individual and combined alternatives. For example, when two elements are connected by "and / or," the first alternative refers to the applicability of the first element without the second element. The second alternative refers to the applicability of the second element without the first element. The third alternative refers to the applicability of the first and second elements together. Any one of these alternatives is understood to be included within the meaning and, therefore, meets the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more of the alternatives is also understood to be included within the meaning and, therefore, meets the requirements of the term "and / or."

[0057] To assist the reader of this application, the description of the specification is divided into various paragraphs or sections or directed to various embodiments of the application. These separations should not be considered as separating the substance of a paragraph, section, or embodiment from the substance of another paragraph, section, or embodiment. To the contrary, those skilled in the art will understand that the description herein has broad applicability and encompasses all combinations of the various paragraphs, paragraphs, and sentences that may be envisioned. The discussion of any embodiment is meant to be merely illustrative and is not intended to suggest that the scope of the present disclosure, including the claims, is limited to these examples.

[0058] Unless otherwise specified, any numerical values, such as concentrations or concentration ranges described herein, should be understood in all cases to be modified by the word "about." Thus, numerical values ​​typically include ±10% of the described value. For example, a description of "10 times" includes 9 times and 11 times. As used herein, the use of numerical ranges expressly includes all possible subranges, including integers and fractions of values ​​within the range, and all individual numerical values ​​within the range, unless the context clearly indicates otherwise.

[0059] As used herein, "subject" refers to any animal, preferably a mammal, most preferably a human, to which a radioimmunoconjugate of the present invention is or has been administered. As used herein, the term "mammal" encompasses all mammals. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, and the like, more preferably humans.

[0060] As used herein, the term "alkyl" refers to a saturated, monovalent unbranched or branched hydrocarbon chain. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like.

[0061] The term "cycloalkyl" refers to a monocyclic or polycyclic alkyl group having 3 to 12, more preferably 3 to 8, carbon atoms in the ring. Monocyclic cycloalkyl rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0062] As used herein, the term "alkoxy" refers to an -O-alkyl or -OR group, where R is alkyl, where alkyl is as defined above. An alkoxy group is attached to the parent molecule via an oxygen atom. Examples of alkoxy include methoxy, ethoxy, propoxy (e.g., n-propoxy, isopropoxy), butoxy (e.g., n-butoxy, isobutoxy, tert-butoxy), pentyloxy (e.g., n-pentyloxy, isopentyloxy, neopentyloxy), and the like. An alkoxy group can be unsubstituted or substituted with one or more suitable substituents. Similarly, "alkylthio" or "thioalkoxy" refers to an -SR group, where R is alkyl, such as -S-methyl, -S-ethyl, and the like, attached to the parent molecule via a sulfur bridge. Representative examples of alkylthio include, but are not limited to, -SCH, -SCHCH, and the like.

[0063] As used herein, the term "halogen" means fluorine, chlorine, bromine, or iodine. Correspondingly, the term "halo" means fluoro, chloro, bromo, or iodo.

[0064] The terms "hydroxy" and "hydroxyl" can be used interchangeably and refer to --OH.

[0065] The term "carboxy" refers to --COOH.

[0066] The term "cyano" refers to -CN.

[0067] The term "nitro" refers to -NO2.

[0068] The term "isothiocyanate" refers to -N=C=S.

[0069] The term "isocyanate" refers to -N=C=O.

[0070] The term "azido" refers to -N3.

[0071] The term "alkenyl" refers to a straight or branched hydrocarbon chain having at least two carbon atoms, such as 2 to 10 carbon atoms, and containing at least one double bond between the two carbon atoms. An alkenyl can have one carbon-carbon double bond or multiple carbon-carbon double bonds, such as 2, 3, 4, or more carbon-carbon double bonds. Examples of alkenyl groups include, but are not limited to, methenyl, ethenyl, propenyl, butenyl, and the like.

[0072] The term "cycloalkenyl" refers to a monocyclic or polycyclic alkyl group having 3 to 12, more preferably 3 to 8, carbon atoms in the ring and containing at least one double bond between two carbon atoms. A cycloalkenyl can have one carbon-carbon double bond or multiple carbon-carbon double bonds, such as 2, 3, 4, or more carbon-carbon double bonds. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cycloheptenyl, cyclohexenyl, and the like.

[0073] As used herein, the term "alkynyl," "alkyne group," or "alkyne moiety" refers to a straight or branched hydrocarbon chain having at least two carbon atoms, such as 2 to 10 carbon atoms, and containing at least one triple bond between the two carbon atoms. An alkynyl group can be a terminal alkynyl group or a cyclic alkynyl group. A terminal alkyne has at least one hydrogen atom attached to the triple-bonded carbon atom. A "cyclic alkyne" or "cycloalkynyl" is a cycloalkyl ring containing at least one triple bond between two carbon atoms. Examples of cyclic alkyne or cycloalkynyl groups include, but are not limited to, cyclooctyne and cyclooctyne derivatives such as bicyclononyne (BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, biarylazacyclooctynonyl (BARAC), dibenzoazacyclooctyne (DIBAC), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctyne (DIFBO), monobenzocyclooctynyl (MOBO), and tetramethoxyDIBO (TMDIBO).

[0074] The term "amino" refers to NH. The term "alkylamino" refers to an amino group in which one or both of the hydrogen atoms bonded to the nitrogen are replaced with an alkyl group. An alkylamine group can be represented as -NR, where each R is independently hydrogen or an alkyl group. For example, alkylamines include methylamine (-NHCH), dimethylamine (-N(CH), -NHCH-CH, and the like. As used herein, the term "aminoalkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups substituted with one or more amino groups. Representative examples of aminoalkyl groups include, but are not limited to, -CHNH, -CHCHNH, and -CHCH(NH)CH.

[0075] As used herein, "amide" refers to -C(O)N(R)2, where each R is independently an alkyl group or hydrogen. Examples of amides include, but are not limited to, -C(O)NH2, -C(O)NHCH3, and -C(O)N(CH3)2.

[0076] The terms "hydroxylalkyl" and "hydroxyalkyl" are used interchangeably and refer to an alkyl group substituted with one or more hydroxyl groups. The alkyl can be a branched or straight chain aliphatic hydrocarbon. Examples of hydroxylalkyl include, but are not limited to, hydroxylmethyl (-CHOH), hydroxylethyl (-CHCHOH), and the like.

[0077] As used herein, the term "aryl" refers to a group containing any carbon-based aromatic group, including, but not limited to, phenyl, naphthyl, anthracenyl, phenanthranyl, etc. Aryl moieties are well known and are described, for example, in Lewis, RJ, ed., Hawley's Condensed Chemical Dictionary, 13th Edition, John Wiley & Sons, Inc., New York (1997). Aryl groups can be single ring structures (i.e., monocyclic) or can contain multiple ring structures (i.e., polycyclic) that are fused ring structures. Preferably, the aryl group is a monocyclic aryl group.

[0078] As used herein, the term "heterocyclyl" includes stable monocyclic and polycyclic hydrocarbons containing at least one heteroatom ring member, such as sulfur, oxygen, or nitrogen. As used herein, the term "heteroaryl" includes stable monocyclic and polycyclic aromatic hydrocarbons containing at least one heteroatom ring member, such as sulfur, oxygen, or nitrogen. Heteroaryls can be monocyclic or polycyclic, e.g., bicyclic or tricyclic. Each ring of a heteroatom-containing heterocyclyl or heteroaryl group can contain one or two oxygen or sulfur atoms and / or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less, and each ring has at least one carbon atom. Heteroaryl groups that are polycyclic, e.g., bicyclic or tricyclic, must contain at least one fully aromatic ring, but other fused rings or rings can be aromatic or non-aromatic. The heterocyclyl or heteroaryl group can be attached to any available nitrogen or carbon atom of any ring of the heterocyclyl or heteroaryl group. Preferably, the term "heteroaryl" refers to 5- or 6-membered monocyclic and 9- or 10-membered bicyclic groups having at least one heteroatom (O, S, or N) in at least one of the rings, with the heteroatom-containing ring preferably having 1, 2, or 3 heteroatoms, more preferably 1 or 2 heteroatoms selected from O, S, and / or N. The nitrogen heteroatom of a heteroaryl can be substituted or unsubstituted. Additionally, the nitrogen and sulfur heteroatoms of a heteroaryl can be optionally oxidized (i.e., N→O and S(O)). r where r is 0, 1 or 2).

[0079] The term "ester" refers to -C(O)R, where R is alkyl.

[0080] The term "carbamate" refers to -OC(O)NR2, where each R is independently alkyl or hydrogen.

[0081] The term "aldehyde" refers to -C(O)H.

[0082] The term "carbonate" refers to -OC(O)OR, where R is alkyl.

[0083] The term "maleimide" refers to a group having the chemical formula H2C2(CO)2NH. The term "maleimide" refers to a maleimide group covalently bonded to another group or molecule. Preferably, the maleimide group is, for example, N-linked.

[0084] [ka]

[0085] The term "acyl halide" refers to -C(O)X, where X is halo (e.g., Br, Cl). Exemplary acyl halides include acyl chloride (-C(O)Cl) and acyl bromide (-C(O)Br).

[0086] As referred to herein, the term "substituted" means that at least one hydrogen atom has been replaced with a non-hydrogen group, provided that all normal valences are maintained and the substitution results in a stable compound. When a particular group is "substituted," the group can have one or more substituents independently selected from a list of substituents, preferably 1 to 5 substituents, more preferably 1 to 3 substituents, and most preferably 1 to 2 substituents. The term "independently," when used with reference to a substituent, means that when two or more such substituents are possible, such substituents can be the same or different from one another. Any of the substituents described herein (e.g., alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heterocyclyl, heteroaryl, etc.) can be unsubstituted or substituted with one or more suitable substituents. Examples of suitable substituents include, but are not limited to, alkyl, halogen, hydroxy, alkoxy, amido, alkylthio, amino, alkylamino, aminoalkyl, hydroxyalkyl, hydroxyl, carboxyl, etc.

[0087] According to the convention used in the art,

[0088] [ka] is used in structural formulas herein to indicate the bond that is the point of attachment of a moiety, functional group, or substituent to a core, parent, or backbone structure, such as a chelator or targeting ligand.

[0089] When any variable occurs more than one time in any constituent or formula for a compound, its definition on each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0 to 3 R groups, that group may be optionally substituted with up to 3 R groups, and each occurrence of R is independently selected from the definitions of R.

[0090] When a bond to a substituent is shown to cross a bond connecting two atoms in a ring, then such substituent may be bonded to any atom on the ring.

[0091] As used herein, the term "radiometal ion" or "radioactive metal ion" refers to one or more isotopes of an element that emit particles and / or photons. Any chelator known to those of skill in the art in light of the present disclosure can be used in the present invention. Examples of radiometals suitable for use in the present invention include: 32 P, 47 Sc, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 77 As, 86 Y, 89 Zr, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 111 In, 117 Sn, 131 I, 149 Tb, 152 Tb,155 Tb, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 177 Lu, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227 Th and 255 Preferably, the radioactive metal ion is a "therapeutic emitter," which refers to a radioactive metal ion useful in therapeutic applications. Examples of therapeutic emitters include, but are not limited to, beta or alpha emitters, e.g., 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I, 149 Tb, 152 Tb, 155 Tb, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 177 Lu, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 255 Fm and 227Preferably, the radioactive metal ion used in the present invention is an α-ray emitting radioactive metal ion, such as actinium-225 ( 225 Ac).

[0092] As used herein, the term "chelate" or "chelating agent" refers to a chemical compound to which a metal, preferably a radioactive metal, can be chelated by a coordinate bond. In a typical embodiment, the chelator is a macrocycle containing one or more heteroatoms, e.g., oxygen and / or nitrogen, as ring atoms. Preferably, the chelator is a derivative of 4,13-diaza-18-crown-6.

[0093] As used herein, a "radiometal complex" refers to a complex comprising a radioactive metal ion associated with a chelator. Typically, the radioactive metal ion is bound or coordinated to the chelator by a coordinate bond. A heteroatom of the macrocycle may participate in the coordinate bonding of the radioactive metal ion to the chelator. The chelator may be substituted with one or more substituents, and one or more substituents may also participate in the coordinate bonding of the radioactive metal ion to the chelator in addition to or instead of the heteroatom of the macrocycle.

[0094] As used herein, the term "click chemistry" refers to a chemical principle introduced by Sharpless, which describes chemistry tailored to rapidly and reliably generate covalent bonds by linking together small units containing reactive groups (see Kolb, et al., Angewandte Chemie International Edition (2001) 40:2004-2021). Click chemistry does not refer to a specific reaction, but rather to a concept that includes, but is not limited to, reactions that mimic reactions found in nature. In some embodiments, click chemistry reactions are modular, have a wide range, produce high chemical yields, produce inactive by-products, are stereospecific, exhibit large thermodynamic driving forces that favor reactions with a single reaction product, and / or can be performed under physiological conditions. In some embodiments, click chemistry reactions can be performed under simple reaction conditions, use readily available starting materials and reagents, do not use toxic solvents or use innocuous or easily removed solvents such as water, and / or provide for simple product isolation by non-chromatographic methods such as crystallization or distillation.

[0095] Click chemistry reactions utilize reactive groups that are rarely found in naturally occurring biomolecules and are chemically inert toward biomolecules. However, when click chemistry partners are reacted together, the reaction can occur efficiently under biologically relevant conditions, such as cell culture conditions, without excessive heat and / or harsh reagents. Generally, click chemistry reactions require at least two molecules, including click reaction partners, that can react with each other. Such mutually reactive click reaction partners are sometimes referred to herein as click chemistry handle pairs or click chemistry pairs. In some embodiments, the click reaction partners are azides and strained alkynes, such as cyclooctyne or cyclooctyne derivatives, or any other alkynes. In other embodiments, the click reaction partners are reactive dienes and suitable tetrazine dienophiles. For example, trans-cyclooctene, norbornene, or biscyclononene can be paired with suitable tetrazine dienophiles as click reaction pairs. In yet another embodiment, tetrazole can be paired with an unactivated alkene in the presence of ultraviolet light to create a click reaction pair, referred to as a "photoclick" reaction pair. In another embodiment, the click reaction partners are cysteine ​​and maleimide. For example, a cysteine ​​(e.g., GGGC) derived from a peptide can be reacted with a maleimide conjugated to a chelator (e.g., NOTA). Other suitable click chemistry handles are known to those skilled in the art (e.g., Spicer et al., Selective chemical protein modification. Nature Communications. 2014;5:4740). In another embodiment, the click reaction partners are Staudinger ligation components, such as a phosphine and an azide. In another embodiment, the click reaction partners are Diels-Alder reaction components, such as a diene (e.g., tetrazine) and an alkene (e.g., trans-cyclooctene (TCO) or norbornene).Exemplary click reaction partners are described in U.S. Patent Application Publication No. 20130266512 and WO 2015073746, the relevant descriptions of both click reaction partners being incorporated herein by reference.

[0096] According to a preferred embodiment, the click chemistry reaction utilizes an azide group and an alkyne group, more preferably a strained alkyne group, e.g., a cycloalkyne such as a cyclooctyne or cyclooctyne derivative, as a click chemistry pair or reaction partner. In certain embodiments, the click chemistry reaction is a Huisgen cycloaddition or 1,3-dipolar cycloaddition between an azide (-N3) and an alkyne or alkyne moiety to form a 1,2,3-triazole linker. Click chemistry reactions between alkynes and azides typically require the addition of a copper catalyst to promote the 1,3-cycloaddition reaction, known as the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. However, click chemistry reactions between cyclooctyne or cyclooctyne derivatives and azides typically do not require the addition of a copper catalyst and instead proceed via strain-promoted azide-alkyne cycloaddition (SPAAC) (Debets, M.F., et al., Bioconjugation with strained alkenes and alkynes. Acc Chem Res, 2011. 44(9): pp. 805-15).

[0097] As used herein, the term "targeting ligand" refers to any molecule that enhances affinity for a selected target, e.g., an antigen, cell, cell type, tissue, organ, body region, or compartment (e.g., a cell, tissue, or organ compartment). Targeting ligands include, but are not limited to, antibodies or antigen-binding fragments thereof, small molecules, aptamers, polypeptides, and scaffold proteins. Preferably, the targeting ligand is a polypeptide, more preferably an antibody or antigen-binding fragment thereof, an engineered domain, or a scaffold protein.

[0098] As used herein, the term "polypeptide" refers to a polymer composed of naturally occurring structural variants and their synthetic, non-naturally occurring analogs linked via peptide bonds. The term "polypeptide" refers to a polypeptide of any size, structure, or function. Typically, a polypeptide is at least three amino acids in length. A polypeptide can be naturally occurring, recombinant, or synthetic, or any combination thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. According to a preferred embodiment, the polypeptide is an antibody, preferably a monoclonal antibody, or a fragment thereof, an antigen-binding fragment thereof, or the like. According to a preferred embodiment, the antibody or fragment thereof is specific for a cancer antigen. According to another embodiment, the polypeptide is a genetically engineered domain or scaffold protein.

[0099] As used herein, the term "antibody" or "immunoglobulin" is used broadly and includes immunoglobulin or antibody molecules, including polyclonal antibodies, monoclonal antibodies, including murine, human, human-adapted, humanized, and chimeric monoclonal antibodies, and antigen-binding fragments thereof.

[0100] Generally, an antibody is a protein or peptide chain that exhibits binding specificity to a specific antigen, referred to herein as a "target." The structure of an antibody is known. Immunoglobulins can be assigned to five major classes, namely, IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes, IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Thus, antibodies used in the present invention can be of any of the five major classes or corresponding subclasses. Antibody light chains of any vertebrate species can be assigned to one of two distinct types, namely, kappa and lambda, based on the amino acid sequence of their constant domains. According to certain embodiments, antibodies used in the present invention include heavy and / or light chain constant regions of murine or human antibodies. Each of the four IgG subclasses has a distinct biological function known as an effector function. These effector functions are generally mediated by interaction with Fc receptors (FcγR) or by binding of C1q and fixation of complement. Binding to FcγR can result in antibody-dependent cell-mediated cytolysis, while binding to complement factors can result in complement-mediated cytolysis. Antibodies useful in the present invention may have no or minimal effector functions but retain their ability to bind to FcRn.

[0101] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, such as, for example, a diabody, Fab, Fab', F(ab'), Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (dsdiabody), single-chain antibody molecule (scFv), single-domain antibody (sdab), scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody containing one or more CDRs, camelized single-domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain the entire antibody structure. An antigen-binding fragment can bind to the same antigen as the parent antibody or parent antibody fragment. As used herein, the term "single-chain antibody" refers to a conventional single-chain antibody in the art, which contains a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids. As used herein, the term "single domain antibody" refers to a conventional single domain antibody in the art, which comprises a heavy chain variable region and a heavy chain constant region, or which comprises only a heavy chain variable region.

[0102] As used herein, the term "scaffold" or "scaffold protein" refers to any protein that has a target-binding domain and can bind to a target. A scaffold includes a "framework," which is mostly structural, and a "binding domain," which contacts the target and provides specific binding. The binding domain of a scaffold need not be defined by a single continuous sequence of the scaffold. In certain cases, a scaffold may be part of a larger binding protein, which itself may be part of a multimeric binding protein comprising multiple scaffolds. Certain binding proteins may be bispecific or multispecific, in that they can bind to two or more different epitopes. A scaffold may be derived from a single-chain antibody, or the scaffold may not be derived from an antibody.

[0103] As used herein, the term "aptamer" refers to a single-stranded oligonucleotide (a single-stranded DNA or RNA molecule) that can specifically bind to its target with high affinity. Aptamers can be used as targeting molecules for a variety of organic and inorganic substances.

[0104] As used herein, the term "small molecule ligand" refers to a low molecular weight organic compound. As used herein, a small molecule ligand can refer to a compound having a size of less than about 1000 daltons, and can refer to a compound that can be synthesized in a laboratory or found in nature.

[0105] Chelater: In one general aspect, the present invention relates to a chelator, preferably a chelator in which a radiometal is chelated by a coordinate bond. According to an embodiment of the invention, the chelator has the structure of formula (I):

[0106] [ka] During the ceremony, Each of ring A and ring B is independently a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl, and each of ring A and ring B is independently a halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 Optionally substituted with one or more substituents selected from the group consisting of X and Each of Z1 and Z2 independently represents -(C(R 12 )2) m -or-(CH2) n -C(R 12 )(X)-(CH2) n - and Each X is independently -L1-R 11 and each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5; each p is independently 0 or 1; L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12 are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; Each R 13 are independently hydrogen or alkyl; R 14 , R 15 , R 16 and R 17 each is independently hydrogen, alkyl or X; Alternatively, R 14 and R 15 and / or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring optionally substituted with X; provided that the chelator contains at least one X, and when X is present on ring A or ring B, L1 is a linker or R 12 and R 14 ~R 17 At least one of them is not hydrogen.

[0107] According to an embodiment of the present invention, the chelator comprises at least one X group, where X is -L1-R 11 wherein L1 is absent or a linker, and R 11 is an electrophilic or nucleophilic moiety, or R 11 R contains a targeting ligand. 11When is a nucleophilic or electrophilic moiety, such a moiety can be used to attach the chelator to a targeting ligand directly or indirectly via a linker.

[0108] In certain embodiments, the chelator comprises a single X group, and preferably L1 of the X group is a linker.

[0109] The chelators of the present invention can be substituted with X at any one of the carbon atoms of the macrocycle, at the Z1 or Z2 position, or on ring A or ring B, provided that when ring or ring B contains an X group, L1 is a linker or is R 12 and R 14 ~R 17 is not hydrogen (i.e., at least one of the carbon atoms of Z, Z and / or the carbon of the macrocycle is substituted with an alkyl group such as, for example, methyl or ethyl). Preferably, substitution at such positions is to provide a radioactive metal ion, especially 225 The substitution does not affect the chelating efficiency of the chelator Ac, and in some embodiments, can increase the chelation efficiency.

[0110] In some embodiments, L is absent. When L is absent, R 11 is directly attached (eg, via a covalent bond) to the chelator.

[0111] In some embodiments, L1 is a linker. As used herein, the term "linker" refers to a chemical moiety that connects a chelator to a nucleophilic moiety, an electrophilic moiety, or a targeting ligand. Any suitable linker known to those of skill in the art in light of the present disclosure can be used in the present invention. The linker can include, for example, a substituted or unsubstituted alkyl, a substituted or unsubstituted heteroalkyl moiety, a substituted or unsubstituted aryl or heteroaryl, a polyethylene glycol (PEG) linker, a peptide linker, a sugar-based linker, or a cleavable linker, such as a disulfide bond or a protease cleavage site, e.g., valine-citrulline-p-aminobenzyl (PAB). Exemplary linker structures suitable for use in the present invention include:

[0112] [ka] In the formula, N is an integer of 0 to 10, preferably an integer of 1 to 4; m is an integer of 0 to 12, preferably an integer of 0 to 6;

[0113] In some embodiments, R 11 is a nucleophilic moiety or an electrophilic moiety. A "nucleophilic moiety" or "nucleophilic group" refers to a functional group that donates an electron pair to form a covalent bond in a chemical reaction. An "electrophilic moiety" or "electrophilic group" refers to a functional group that accepts an electron pair to form a covalent bond in a chemical reaction. A nucleophilic group reacts with an electrophilic group to form a new covalent bond in a chemical reaction, and vice versa. Reaction of the nucleophilic or electrophilic group of a chelator of the invention with a targeting ligand or other chemical moiety (e.g., a linker), including a corresponding reaction partner, allows the targeting ligand or chemical moiety to be covalently attached to the chelator of the invention.

[0114] Examples of nucleophilic groups include, but are not limited to, azides, amines, and thiols. Examples of electrophilic groups include, but are not limited to, amine-reactive groups, thiol-reactive groups, alkynyls, and cycloalkynyls. Amine-reactive groups preferably react with primary amines, including those present at the N-terminus of each polypeptide chain and in the side chains of lysine residues. Examples of amine-reactive groups suitable for use in the present invention include, but are not limited to, N-hydroxy succinimide (NHS), substituted NHS (such as sulfo-NHS), isothiocyanate (-NCS), isocyanate (-NCO), esters, carboxylic acids, acyl halides, amides, alkylamides, and tetrafluorophenyl esters and perfluorophenyl esters. Thiol-reactive groups react with thiols or sulfhydryls, preferably with thiols present in the side chains of cysteine ​​residues in polypeptides. Examples of thiol-reactive groups suitable for use in the present invention include, but are not limited to, Michael acceptors (e.g., maleimides), haloacetyls, acyl halides, activated disulfides, and phenyloxadiazole sulfones.

[0115] In certain embodiments, R 11 is -NH2, -NCS (isothiocyanate), -NCO (isocyanate), -N3 (azide), alkynyl, cycloalkynyl, carboxylic acid, ester, amide, alkylamide, maleimide, acyl halide, tetrazine or trans-cyclooctene, more specifically, -NCS, -NCO, -N3, alkynyl, cycloalkynyl, -C(O)R 13 , -COOR 13 , -CON(R 13 )2, maleimide, acyl halide (e.g., —C(O)Cl, —C(O)Br), tetrazine, or transcyclooctene, wherein each R 13 is independently hydrogen or alkyl.

[0116] In some embodiments, R 11is an alkynyl group, a cycloalkynyl group, or an azide group, which allows the chelator to be attached to a targeting ligand or other chemical moiety (e.g., a linker) using a click chemistry reaction. In such embodiments, a click chemistry reaction that can be performed is a Huisgen cycloaddition or a 1,3-dipolar cycloaddition between an azide (-N3) and an alkynyl or cycloalkynyl group to form a 1,2,4-triazole linker or moiety. In one embodiment, the chelator comprises an alkynyl or cycloalkynyl group, and the targeting ligand or other chemical moiety comprises an azide group. In another embodiment, the chelator comprises an azide group, and the targeting ligand or other chemical moiety comprises an alkynyl or cycloalkynyl group.

[0117] In certain embodiments, R 11 is an alkynyl group, more preferably a terminal alkynyl group or a cycloalkynyl group that is particularly reactive with an azide group via strain-promoted azide-alkyne cyclocycloaddition (SPAAC). Examples of cycloalkynyl groups that can react with an azide group via SPAAC include, but are not limited to, cyclooctynyl or a cyclooctynyl derivative selected from the group consisting of bicyclononynyl (BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, biarylazacyclooctynyl (BARAC), dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO), and tetramethoxydibenzocyclooctynyl (TMDIBO).

[0118] In certain embodiments, R 11 is dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO) having the following structure:

[0119] [ka]

[0120] R 11 In such embodiments where is a DBCO, the DBCO can be covalently attached to the chelator directly or indirectly via a linker, and is preferably attached to the chelator indirectly via a linker.

[0121] In some embodiments, R 11 comprises a targeting ligand. The targeting ligand can be covalently bound directly to the chelator or indirectly via a linker. The targeting ligand can be a polypeptide, such as an antibody or antigen-binding fragment thereof, a small molecule, an aptamer, or a scaffold protein. In a preferred embodiment, the targeting ligand is an antibody or antigen-binding fragment thereof, e.g., a monoclonal antibody (mAb) or antigen-binding fragment thereof that specifically binds to an antigen associated with a neoplastic disease or disorder, such as a cancer antigen, which can be prostate-specific membrane antigen (PSMA), BCMA, Her2, EGFR, KLK2, CD19, CD22, CD30, CD33, CD79b, or Nectin-4.

[0122] According to an embodiment of the present invention, each of rings A and B is independently a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl. In an alternative embodiment, each of rings A and B is contemplated to be an optionally substituted heterocyclyl ring, such as oxazoline. Each of rings A and B is optionally and independently selected from halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13)2 and X. Examples of 6- to 10-membered aryl groups suitable for this purpose include, but are not limited to, phenyl and naphthyl. Examples of 5- to 10-membered heteroaryl groups suitable for this purpose include, but are not limited to, pyridinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, and imidazolyl. Examples of suitable substituents for 5- to 10-membered heteroaryl and 6- to 10-membered aryl groups include, but are not limited to, -COOH, tetrazolyl, and -CHCOOH. In a preferred embodiment, the substituent is -COOH or tetrazolyl, which is an isostere of -COOH.

[0123] In certain embodiments, each of Ring A and Ring B is independently and optionally substituted with one or more carboxyl groups, including, but not limited to, -COOH and -CH2COOH.

[0124] In certain embodiments, each of ring A and ring B is independently and optionally substituted with tetrazolyl.

[0125] In one embodiment, ring A and ring B are the same, e.g., both ring A and ring B are pyridinyl. In another embodiment, ring A and ring B are different, e.g., ring A and one of ring A and ring B is pyridinyl and the other is phenyl.

[0126] In certain embodiments, both ring A and ring B are pyridinyl substituted with -COOH.

[0127] In certain embodiments, both Ring A and Ring B are pyridinyl substituted with tetrazolyl.

[0128] In another particular embodiment, both Ring A and Ring B are picolinic acid groups having the following structure:

[0129] [ka]

[0130] According to an embodiment of the present invention, each of Z1 and Z2 is independently -(C(R 12 )2) m -or-(CH2) n -C(R 12 )(X)-(CH2) n and each X is independently -L1-R 11 and each R 12 is independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; each n is independently 0, 1, 2, 3, 4, or 5; and each m is independently 1, 2, 3, 4, or 5.

[0131] In some embodiments, each R 12 are independently hydrogen or alkyl, more preferably hydrogen, —CH 3 or —CH 2 CH 3 .

[0132] In some embodiments, each R 12 is hydrogen.

[0133] In some embodiments, both Z and Z are -(CH) m -, where each m is preferably 1. In such embodiments, a carbon atom of the macrocycle, ring A or ring B is substituted with an X group.

[0134] In some embodiments, one of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and the other is -(CH2) m -It is.

[0135] In some embodiments, one of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and the other is -(CH2) m -, where each n is 0, m is 1, and X is -L1-R 11 and L1 is a linker.

[0136] In some embodiments, both Z and Z are -(CH) m -, each m is independently 0, 1, 2, 3, 4, or 5, preferably each m is 1, and R 14 , R 15 , R 16 and R 17 One of them is X and the other is R 14 , R 15 , R 16 and R 17 The remainder of each is hydrogen.

[0137] In some embodiments, R 14 and R 15 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring (i.e., cyclopentyl or cyclohexyl). Such 5- or 6-membered cycloalkyl rings can be substituted with an X group.

[0138] In some embodiments, R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring (i.e., cyclopentyl or cyclohexyl). Such 5- or 6-membered cycloalkyl rings can be substituted with an X group.

[0139] In certain embodiments, the chelator has the structure of formula (II):

[0140] [ka] During the ceremony, A1 is N or CR1 or absent; A2 is N or CR2; A3 is N or CR3; A4 is N or CR4; A5 is N or CR5; A6 is N or CR6 or absent; A7 is N or CR7; A8 is N or CR8; A9 is N or CR9, A 10 is N or CR 10 and However, three or less of A1, A2, A3, A4 and A5 are N, and A6, A7, A8, A9 and A 10 Not more than three of R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 each independently represents hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN, -OC(O)N(R 13 )2 and -X; Alternatively, any two immediately adjacent R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 together with the atoms to which they are attached form a 5- or 6-membered substituted or unsubstituted carbocyclic or nitrogen-containing ring; Z1, Z2, X, n, m, p, L1 and R 11 ~R 17 is as described above for formula (I), provided that the chelator contains at least one X and R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 When any one of is X, L is a linker, or R 12 and R 14 ~R 17 At least one of them is not hydrogen.

[0141] In some embodiments, any two immediately adjacent R, R, R, R, R, R, R, R, R, R, R, and R 10may, together with the atoms to which they are attached, form a 5- or 6-membered substituted or unsubstituted carbocyclic or nitrogen-containing ring. Examples of such carbocyclic rings that may be formed include, but are not limited to, naphthyl. Examples of such nitrogen-containing rings that may be formed include, but are not limited to, quinolinyl. The carbocyclic or nitrogen-containing ring may be unsubstituted or substituted with one or more suitable substituents, such as -COOH, -CHCOOH, tetrazolyl, etc.

[0142] In some embodiments, L is absent. When L is absent, R 11 is directly attached (eg, via a covalent bond) to the chelator.

[0143] In some embodiments, L1 is a linker. Any suitable linker known to those of skill in the art in light of the present disclosure, such as those described above, can be used in the present invention.

[0144] In some embodiments, one of A1, A2, A3, A4, and A5 is nitrogen, one of A1, A2, A3, A4, and A5 is a carbon substituted with -COOH, and the remainder are CH, i.e., forming a pyridinyl ring substituted with a carboxylic acid.

[0145] In some embodiments, A6, A7, A8, A9 and A 10 One of them is nitrogen, and A6, A7, A8, A9 and A 10 One of the carbons is substituted with -COOH and the rest are CH, i.e., forming a pyridinyl ring substituted with a carboxylic acid.

[0146] In one embodiment, at least one of R1, R2, R3, R4, and R5 is -COOH. In one embodiment, at least one of R6, R7, R8, R9, and R 10 In another embodiment, at least one of R1, R2, R3, R4, and R5 is -COOH, and at least one of R6, R7, R8, R9, and R10 At least one of the is -COOH.

[0147] In some embodiments, A and A 10 each is nitrogen, A2 is CR2 and R2 is -COOH; A9 is CR9, R9 is -COOH, each of A3 to A8 is CR2, CR3, CR4, CR5, CR6, CR7 and CR8, respectively, and each of R3 to R8 is hydrogen.

[0148] In some embodiments, one of A1, A2, A3, A4, and A5 is nitrogen, one of A1, A2, A3, A4, and A5 is carbon substituted with tetrazolyl, and the remainder are CH.

[0149] In some embodiments, A6, A7, A8, A9 and A 10 One of them is nitrogen, and A6, A7, A8, A9 and A 10 One of the carbons is substituted with tetrazolyl, and the rest are CH.

[0150] In one embodiment, at least one of R1, R2, R3, R4, and R5 is tetrazolyl. In one embodiment, at least one of R6, R7, R8, R9, and R 10 In another embodiment, at least one of R1, R2, R3, R4, and R5 is tetrazolyl, and at least one of R6, R7, R8, R9, and R 10 At least one of is tetrazolyl.

[0151] In some embodiments, each R 12 is hydrogen.

[0152] In some embodiments, R 11 is an alkynyl or cycloalkynyl group, preferably cyclooctynyl or a cyclooctynyl derivative, such as DBCO.

[0153] In certain embodiments of the chelator of formula (II), A1 and A 10 each is nitrogen; A2 is CR2 and R2 is -COOH; A9 is CR9 and R9 is -COOH; A3 to A8 are CR2, CR3, CR4, CR5, CR6, CR7, and CR8, respectively; Each of R3 to R8 is hydrogen; One of Z1 and Z2 is -(CH2) m -, and the other of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and R 12 is hydrogen, m is 1, Each n is 0, X is -L1-R 11 wherein L1 is a linker and -R 11 is an electrophilic group, for example, cyclooctynyl or a cyclooctynyl derivative such as DBCO; R 14 ~R 17 Each of is hydrogen or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring.

[0154] In certain embodiments, the compound has the structure of formula (III):

[0155] [ka] During the ceremony, Each A 11 are independently O, S, NMe, or NH; Each R 18 are independently hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR13 , -COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 )2 and -X; Z1, Z2, X, n, m, L1, R 11 -R 17 is as described above for formula (I), provided that the chelator contains at least one X and R 18 is X, L1 is a linker, or R 12 and R 14 ~R 17 At least one of them is not hydrogen.

[0156] In some embodiments, each A 11 is the same, and each A 11 is O, S, NMe, or NH. For example, each A 11 can be S. In other embodiments, each A 11 are different and each is independently selected from O, S, NMe, and NH.

[0157] In some embodiments, each R 18 are independently -(CH2) p -COOR 13 or tetrazolyl, wherein R 13 is hydrogen, and each p is independently 0 or 1.

[0158] In some embodiments, each R 18 is -COOH.

[0159] In some embodiments, each R 18 is -CH2COOH.

[0160] In some embodiments, each R 18 is tetrazolyl.

[0161] In certain embodiments of the chelator of formula (III), Each R 18 is COOH, One of Z1 and Z2 is -(CH2) m -, and the other of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and R 12 is hydrogen, m is 1 and each n is 0; X is -L1-R 11 wherein L1 is a linker and -R 11 is an electrophilic group, for example, cyclooctynyl or a cyclooctynyl derivative such as DBCO or BCN; R 14 ~R 17 Each of is hydrogen or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring.

[0162] Particular embodiments of the present invention are chelators selected from the group consisting of:

[0163] [ka] During the ceremony, L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12 are independently hydrogen, -CH3, or -CH2CH3, provided that at least one R 12 is -CH3 or -CH2CH3.

[0164] In some embodiments, R 11 -NH2, -NCS, -NCO, -N3, alkynyl, cycloalkynyl, -C(O)R13 , -COOR 13 , -CON(R 13 ) 2, maleimide, acyl halide, tetrazine or trans-cyclooctene.

[0165] In certain embodiments, R 11 is cyclooctynyl or a cyclooctynyl derivative selected from the group consisting of bicyclononynyl (BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, biarylazacyclooctynyl (BARAC), dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO), and tetramethoxydibenzocyclooctynyl (TMDIBO).

[0166] Preferably, R 11 is an alkynyl group or a cycloalkynyl group, more preferably a cycloalkynyl group, such as DBCO or BCN.

[0167] Exemplary chelators of the present invention include:

[0168] [ka]

[0169] [ka] These include, but are not limited to:

[0170] Such chelators can be covalently attached to a targeting ligand (e.g., an antibody or antigen-binding fragment thereof) to form an immunoconjugate or radioimmunoconjugate by reacting the chelator with the azide-labeled targeting ligand to form a 1,2,3-triazole linker via a click chemistry reaction, described in more detail below.

[0171] The chelators of the present invention can be prepared by any method known in the art in light of the present disclosure, for example, the pendant aromatic / heteroaromatic group can be attached to the macrocycle moiety by methods known in the art, such as those exemplified and described below.

[0172] Radioactive metal complexes In another general aspect, the present invention relates to a radiometal complex comprising a radiometal ion coordinated to a chelator of the invention by a coordinate bond. Any of the chelators of the invention described herein can comprise a radiometal ion. Preferably, the radiometal ion is an alpha-emitting radiometal ion, more preferably an alpha-emitting radiometal ion. 225 The chelators of the present invention can chelate radioactive metal ions, especially radioactive metal ions, at any specific activity, regardless of metal impurities. 225 It is able to tightly chelate Ac, thus forming radiometal complexes with high chelate stability in vivo and in vitro and that are stable to challenge agents such as diethylene triamine pentaacetic acid (DTPA).

[0173] According to an embodiment of the present invention, the radiometal complex has the structure of formula (Im):

[0174] [ka] wherein the variables are as defined above in the chelators of the invention, e.g., the chelators of formula (I), and M is a radioactive metal ion. The radioactive metal ion, M, is bound to the chelator by a coordinate bond to form a radioactive metal complex. The heteroatoms of the macrocycle of the chelator and any functional groups of the pendant arms (i.e., -Z1-ring A and / or -Z2-ring B) can participate in the coordinate bonding of the radioactive metal ion.

[0175] Any of the chelators of formula (I) above can be used to form radiometal complexes of formula (Im).

[0176] In certain embodiments, the radioactive metal ion M is an alpha-emitting radioactive metal ion. Preferably, the alpha-emitting radioactive metal ion is 225 It is Ac.

[0177] According to an embodiment of the present invention, the radiometal complex comprises at least one X group, wherein X is -L-R 11 wherein L1 is absent or a linker, and R 11 is an electrophilic or nucleophilic moiety, or R 11 R contains a targeting ligand. 11 When is a nucleophilic or electrophilic moiety, such a moiety can be used to attach the radiometal complex to the targeting ligand directly or indirectly via a linker.

[0178] In certain embodiments, the radiometal comprises a single X group, and preferably L1 of the X group is a linker.

[0179] In certain embodiments, R 11 is -NH2, -NCS (isothiocyanate), -NCO (isocyanate), -N3 (azide), alkynyl, cycloalkynyl, carboxylic acid, ester, amide, alkylamide, maleimide, acyl halide, tetrazine or trans-cyclooctene, more specifically, -NCS, -NCO, -N3, alkynyl, cycloalkynyl, -C(O)R 13 , -COOR 13 , -CON(R 13 )2, maleimide or acyl halide (e.g., —C(O)Cl or —C(O)Br), where each R 13 is independently hydrogen or alkyl.

[0180] In some embodiments, R 11is an alkynyl group, a cycloalkynyl group, or an azido group, which allows the chelator to be attached to a targeting ligand or other chemical moiety (e.g., a linker) using a click chemistry reaction.

[0181] In certain embodiments, R 11 is an alkynyl group, more preferably a terminal alkynyl group or a cycloalkynyl group that is reactive with azide groups, particularly via strain-promoted azide-alkyne cyclocycloaddition (SPAAC). Examples of cycloalkynyl groups that can react with azide groups via SPAAC include, but are not limited to, cyclooctynyl or a cyclooctynyl derivative selected from the group consisting of bicyclononynyl (BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, biarylazacyclooctynyl (BARAC), dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO), and tetramethoxydibenzocyclooctynyl (TMDIBO).

[0182] In certain embodiments, R 11 is dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO) having the following structure:

[0183] [ka]

[0184] R 11 In such embodiments where is a DBCO, the DBCO can be covalently attached to the chelator directly or indirectly via a linker, and is preferably attached to the chelator indirectly via a linker.

[0185] In another particular embodiment, R 11 is bicrononyl (BCN).

[0186] According to an embodiment of the present invention, each of rings A and B is independently a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl. In an alternative embodiment, each of rings A and B is contemplated to be an optionally substituted heterocyclyl ring, such as oxazoline. Each of rings A and B is optionally and independently selected from halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 )2 and X. Examples of 6- to 10-membered aryl groups suitable for this purpose include, but are not limited to, phenyl and naphthyl. Examples of 5- to 10-membered heteroaryl groups suitable for this purpose include, but are not limited to, pyridinyl, isothiazolyl, isoxazolyl, and imidazolyl. Examples of suitable substituents for 5- to 10-membered heteroaryl and 6- to 10-membered aryl groups include, but are not limited to, -COOH, tetrazolyl, and -CHCOOH.

[0187] In certain embodiments, each of Ring A and Ring B is independently and optionally substituted with one or more carboxyl groups, including, but not limited to, -COOH and -CH2COOH.

[0188] In one embodiment, ring A and ring B are the same, e.g., both ring A and ring B are pyridinyl. In another embodiment, ring A and ring B are different, e.g., ring A and one of ring A and ring B is pyridinyl and the other is phenyl.

[0189] In certain embodiments, both ring A and ring B are pyridinyl substituted with -COOH.

[0190] In certain embodiments, both Ring A and Ring B are pyridinyl substituted with tetrazolyl.

[0191] In another particular embodiment, both Ring A and Ring B are picolinic acid groups having the following structure:

[0192] [ka]

[0193] According to an embodiment of the present invention, each of Z1 and Z2 is independently -(C(R 12 )2) m -or-(CH2) n -C(R 12 )(X)-(CH2) n and each X is independently -L1-R 11 wherein each n is independently 0, 1, 2, 3, 4, or 5; and each m is independently 1, 2, 3, 4, or 5.

[0194] In some embodiments, both Z and Z are -(CH) m -, where each m is preferably 1. In such embodiments, a carbon atom of the macrocycle, ring A or ring B is substituted with an X group.

[0195] In some embodiments, one of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and the other is -(CH2) m -It is.

[0196] In some embodiments, one of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and the other is -(CH2) m -, where each n is 0, m is 1, and X is -L1-R 11 and L1 is a linker.

[0197] In some embodiments, both Z and Z are -(CH) m -, each m is independently 0, 1, 2, 3, 4, or 5, preferably each m is 1, and R 14 , R 15 , R 16 and R 17 One of them is X and the other is R 14 , R 15 , R 16 and R 17 The remainder of each is hydrogen.

[0198] In some embodiments, R 14 and R 15 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring (e.g., cyclopentyl or cyclohexyl). Such 5- or 6-membered cycloalkyl rings can be substituted with an X group.

[0199] In some embodiments, R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring (e.g., cyclopentyl or cyclohexyl). Such 5- or 6-membered cycloalkyl rings can be substituted with an X group.

[0200] In certain embodiments, the radiometal complex has the structure of Formula (II-m):

[0201] [ka] wherein the variables are as defined above in the chelators of the invention, e.g., the chelators of formula (II), and M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion, more preferably 225 It is Ac.

[0202] Any of the chelators of formula (II) above can be used to form radiometal complexes of formula (II-m).

[0203] In some embodiments, one of A1, A2, A3, A4, and A5 is nitrogen, one of A1, A2, A3, A4, and A5 is a carbon substituted with -COOH, and the remainder are CH, i.e., forming a pyridinyl ring substituted with a carboxylic acid.

[0204] In some embodiments, A6, A7, A8, A9 and A 10 One of them is nitrogen, and A6, A7, A8, A9 and A 10 One of the carbons is substituted with -COOH and the rest are CH, i.e., forming a pyridinyl ring substituted with a carboxylic acid.

[0205] In one embodiment, at least one of R1, R2, R3, R4, and R5 is -COOH. In one embodiment, at least one of R6, R7, R8, R9, and R 10 In another embodiment, at least one of R1, R2, R3, R4, and R5 is -COOH, and at least one of R6, R7, R8, R9, and R 10 At least one of the is -COOH.

[0206] In some embodiments, A and A 10 each is nitrogen, A2 is CR2 and R2 is -COOH; A9 is CR9, R9 is -COOH, each of A3 to A8 is CR2, CR3, CR4, CR5, CR6, CR7 and CR8, respectively, and each of R3 to R8 is hydrogen.

[0207] In one embodiment, at least one of R1, R2, R3, R4, and R5 is tetrazolyl. In one embodiment, at least one of R6, R7, R8, R9, and R 10 In another embodiment, at least one of R1, R2, R3, R4, and R5 is tetrazolyl, and at least one of R6, R7, R8, R9, and R 10 At least one of is tetrazolyl.

[0208] In some embodiments, each R 12 is hydrogen.

[0209] In some embodiments, R 11 is an alkynyl or cycloalkynyl group, preferably cyclooctynyl or a cyclooctynyl derivative, such as DBCO.

[0210] In certain embodiments of the radiometal complex of formula (II-m), M is 225 Ac, A1 and A 10 each is nitrogen; A2 is CR2 and R2 is -COOH; A9 is CR9 and R9 is -COOH; A3 to A8 are CR2, CR3, CR4, CR5, CR6, CR7, and CR8, respectively; Each of R3 to R8 is hydrogen; One of Z1 and Z2 is -(CH2) m -, and the other of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and R 12 is hydrogen, m is 1, Each n is 0, X is -L1-R 11 wherein L1 is a linker and -R 11 is an electrophilic group, for example, cyclooctynyl or a cyclooctynyl derivative such as DBCO; R 14 ~R 17 Each of is hydrogen or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring.

[0211] In certain embodiments, the radiometal complex has the structure of formula (III-m):

[0212] [ka] wherein the variables are as defined above in the chelators of the invention, e.g., the chelators of formula (III), and M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion, more preferably 225 It is Ac.

[0213] Any of the chelators of formula (III) above can be used to form radiometal complexes of formula (III-m).

[0214] In some embodiments, each A 11 is the same, and each A 11 is O, S, NMe, or NH. For example, each A 11 can be S. In other embodiments, each A 11 are different and each is independently selected from O, S, NMe, and NH.

[0215] In some embodiments, each R 18 are independently -(CH2) p -COOR 13 where R 13 is hydrogen, and each p is independently 0 or 1.

[0216] In some embodiments, each R 18 is -COOH.

[0217] In some embodiments, each R 18 is -CH2COOH.

[0218] In some embodiments, each R 18 is tetrazolyl.

[0219] In certain embodiments of the radiometal complex of formula (III-m), Each R 18is COOH, One of Z1 and Z2 is -(CH2) m -, and the other of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and R 12 is hydrogen, m is 1 and each n is 0; X is -L1-R 11 wherein L1 is a linker and -R 11 is an electrophilic group, for example, cyclooctynyl or a cyclooctynyl derivative such as DBCO; R 14 ~R 17 Each of is hydrogen or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring.

[0220] In certain embodiments of the invention, the radiometal complex has one of the following structures:

[0221] [ka] During the ceremony, M is actinium-225( 225 Ac), and L1 is absent or a linker; R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12 are independently hydrogen, -CH3, or -CH2CH3, provided that at least one R 12 is -CH3 or -CH2CH3.

[0222] The radiometal complexes can be prepared by any method known in the art in view of the present disclosure. For example, a chelator of the present invention can be mixed with a radiometal ion and the mixture can be incubated to form the radiometal complex. In an exemplary embodiment, the chelator can be 225 It was mixed with a solution of Ac(NO3)3 and bound to the chelator by coordination bonds. 225 As noted above, the chelators of the present invention form radioactive complexes with radiometals, especially Ac. 225 Thus, in certain embodiments, the chelators of the present invention are used in combination with chelators at a ratio of 1:1000, 1:500, 1:400, 1:300, 1:200, 1:100, 1:50, 1:10, or 1:5, preferably 1:5 to 1:200, more preferably 1:5 to 1:100. 225 The concentration ratio with Ac ions is 225 Thus, in some embodiments, the chelators of the present invention can be used to form radiometal complexes. 225 The ratio of Ac to other known 225 This is much lower than the ratio achievable with Ac chelators such as DOTA. The radioactive complexes can be characterized by instant thin layer chromatography (e.g., iTLC-SG), HPLC, LC-MS, etc. Exemplary methods are described herein, for example, in the Examples below.

[0223] Immunoconjugates and radioimmunoconjugates In another general aspect, the present invention relates to immunoconjugates and radioimmunoconjugates. The chelators and radioactive metal complexes of the present invention can be conjugated (i.e., covalently attached) to targeting ligands, such as immunological agents, to produce immunoconjugates and / or radioimmunoconjugates suitable for medical applications in subjects, such as targeted radiotherapy. The chelators and radioactive metal complexes of the present invention can be used to specifically label targeting ligands, particularly antibodies or antigen-binding fragments thereof, capable of specifically binding to a target of interest (such as cancer cells), with radioactive metal ions to produce radioimmunoconjugates. Specifically, the chelators and / or radioactive metal complexes of the present invention can be used to specifically label radioactive metal ions, particularly antibodies or antigen-binding fragments thereof, capable of specifically binding to a target of interest (such as cancer cells). 225 Radioimmunoconjugates can be produced with high yield chelation of Ac and desirable chelator-to-antibody ratios (CARs). According to certain embodiments, the methods of the invention provide an average CAR of less than 10, less than 8, less than 6, or less than 4, or about 2 to about 8, or about 2 to about 6, or about 2 to about 4, or about 2 to about 3, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8.

[0224] As used herein, an "immunoconjugate" is an antibody or antigen-binding fragment thereof conjugated (e.g., covalently attached) to a second molecule, such as a toxin, a drug, a radioactive metal ion, a chelator, a radioactive metal complex, etc. A "radioimmunoconjugate" is, in particular, an immunoconjugate in which the antibody or antigen-binding fragment thereof is labeled with a radioactive metal or conjugated to a radioactive metal complex.

[0225] According to embodiments of the invention, an immunoconjugate comprises a chelator of the invention, e.g., a chelator of Formula (I), Formula (II), or Formula (III) described herein, covalently attached, preferably via a linker, to an antibody or antigen-binding fragment thereof. Many linkage configurations with different linkages between the chelator and the antibody or antigen-binding fragment thereof are possible depending on the reactive functional groups (i.e., nucleophilic and electrophilic) on the chelator and the antibody or antigen-binding fragment thereof.

[0226] According to an embodiment of the present invention, a radioimmunoconjugate comprises a radiometal complex of the present invention, e.g., a radiometal complex of Formula (Im), Formula (II-m), or Formula (III-m) described herein, covalently attached, preferably via a linker, to an antibody or antigen-binding fragment thereof.

[0227] Any of the chelators or radiometal complexes of the invention, such as those described herein, can be used to produce immunoconjugates or radioimmunoconjugates of the invention.

[0228] In some embodiments, the radiometal complexes of the radioimmunoconjugates of the present invention comprise an α-emitting radiometal ion coordinated to the chelating moiety of the radiocomplex. Preferably, the α-emitting radiometal ion is 225 It is Ac.

[0229] In certain embodiments, the antibody or antigen-binding fragment thereof is conjugated to the radioconjugate via a triazole moiety to form a radioimmunoconjugate of the invention.

[0230] In certain embodiments, the antibody or antigen-binding fragment in the immunoconjugate or radioimmunoconjugate of the present application can specifically bind to a tumor antigen. Preferably, the antibody or antigen-binding fragment specifically binds to a cancer antigen. Examples of cancer antigens include, but are not limited to, prostate-specific membrane antigen (PSMA), BCMA, Her2, EGFR, KLK2, CD19, CD22, CD30, CD33, CD79b, and Nectin-4.

[0231] In one embodiment, the antibody specifically binds to PSMA. Preferably, the antibody is PSMB127. A human IgG4 antibody that binds to human prostate-specific membrane antigen (PSMA), referred to herein as "anti-PSMA mAb" and designated "PSMB127," has a heavy chain (HC) CDR1 sequence of SEQ ID NO: 3, a HC CDR2 sequence of SEQ ID NO: 4, a HC CDR3 sequence of SEQ ID NO: 5, a light chain (LC) CDR1 sequence of SEQ ID NO: 6, a LC CDR2 sequence of SEQ ID NO: 7, and a LC CDR3 sequence of SEQ ID NO: 8, and has a HC sequence of SEQ ID NO: 9 and a LC sequence of SEQ ID NO: 10. The anti-PSMA mAb was expressed and purified using standard chromatographic methods. The antibody PSMB127, its biological activity, uses, and other related information are described, for example, in U.S. Patent Application Publication No. 20200024360(A1), the contents of which are incorporated herein by reference in their entirety.

[0232] In another embodiment, the antibody specifically binds to human kallikrein-2 (KLK2). Preferably, the antibody is H11B6. The H11B6 antibody, its biological activity, uses, and other related information are described in U.S. Patent No. 10,100,125, the contents of which are incorporated herein by reference in their entirety. As described herein, the H11B6 antibody polypeptide comprises a heavy chain (HC) variable region comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 13, and a light chain (LC) variable region comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 16. The anti-H11B6 antibody may further comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18, or a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 20, or a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 21 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 22.

[0233] Commercially available antibodies, trastuzumab (Herceptin), cetuximab (Erbitux), pertuzumab (Perjeta), and panitumumab (Vectibix), were purchased from Roche, Lilly, Roche, and Amgen, respectively. Trastuzumab and pertuzumab bind to human Her2. Cetuximab and panitumumab bind to human EGFR.

[0234] The immunoconjugates and radioimmunoconjugates of the present invention can be prepared by any method known in the art in light of the present disclosure for conjugating a ligand, e.g., an antibody, to a chelator, including chemical and / or enzymatic methods. For example, immunoconjugates and radioimmunoconjugates can be prepared by coupling reactions, including the formation of esters, thioesters, or amides from activated acids or acyl halides; nucleophilic substitution reactions (e.g., nucleophilic substitution of a ring halide or ring opening of a ring system with strain); azide-alkyne Huisgen cycloaddition (e.g., 1,3 dipolar cycloaddition between an azide and an alkyne to form a 1,2,3-triazole linker); thiophosphorus addition reactions; imine formation; Diels-Alder reaction between tetrazine and trans-cycloctene (TCO); and Michael additions (e.g., maleimide additions). Depending on the reactive functional group used, many other attachment modes with different linkages are possible. Ligand attachment can be performed to a chelator coordinated to a radiometal ion or to a chelator not coordinated to a radiometal ion.

[0235] According to one embodiment, radiometal conjugates of the present invention can be produced by covalently attaching a radiometal complex of the present invention to an antibody or antigen-binding fragment thereof, e.g., by a click chemistry reaction (see, e.g., Figures 2B and 2D, referred to as "click radiolabeling"). Alternatively, radioimmunoconjugates can be produced by first preparing an immunoconjugate of the present invention by covalently attaching a chelator of the present invention to an antibody or antigen-binding fragment thereof, e.g., by a click chemistry reaction, and subsequently labeling the immunoconjugate with a radiometal ion to produce the radioimmunoconjugate (see, e.g., Figures 2A and 2C, referred to as "one-step direct radiolabeling"). Both residue-specific (e.g., Figures 2A and 2B) and site-specific (e.g., Figures 2C and 2D) methods of conjugation can be used to produce the immunoconjugates and radioimmunoconjugates of the present invention.

[0236] Residue-specific methods for conjugation to proteins are well established and most commonly involve either lysine side chains using activated esters or isothiocyanates, or cysteine ​​side chains with maleimides, haloacetyl derivatives, or activated disulfides (Brinkley Bioconjugate Chem 1992:2). Because most proteins contain multiple lysine and cysteine ​​residues, heterogeneous mixtures of products with different numbers of conjugate molecules at various amino acid positions are typically obtained using such methods. Additional methods have been established, including tyrosine-specific conjugation (Ban et al. Bioconjugate Chemistry 2013:520), methionine-specific methods (Lin et al. Science 2017(355)597), and approaches focusing on additional cysteines (Toda et al. Angew Chemie 2013:12592).

[0237] More recently, site-selective and site-specific conjugation methods have been established for monoclonal antibodies and other proteins (Agarwal, P. and C.R. Bertozzi, Bioconjug Chem, 2015. 26(2): p. 176-92; Rabuka et al. Curr Opin Chem Biol 2010: 790). These include the incorporation of natural amino acids; fusion of the protein of interest to a "self-labeling tag" such as SNAP or DHFR or a tag that is specifically recognized and modified by another enzyme such as sortase A, lipoic acid ligase, and formylglycine generating enzyme; enzymatic modification of glycans to allow conjugation of a desired payload (Hu et al. Chem Soc Rev 2016:1691); the use of microbial transglutaminase to selectively recognize defined locations on antibodies; and additional methods that use molecular recognition and / or chemical approaches to affect selective conjugation (Yamada et al. 2019:5592; Park et al. Bioconjugate Chem 2018:3240; Pham et al. Chembiochem 2018:799).

[0238] In some embodiments, immunoconjugates or radioimmunoconjugates of the invention are produced using residue-specific methods for conjugating a chelator of the invention to an antibody or antigen-binding fragment thereof. Such residue-specific methods typically result in immunoconjugates or radioimmunoconjugates covalently attached to the chelator or radiometal complex at various positions on the antibody. Any residue-specific method for forming protein or antibody conjugates known to those of skill in the art in light of the present disclosure can be used. Examples of residue-specific methods for conjugation that can be used include, but are not limited to, conjugating a chelator or radioactive metal complex to a lysine residue of an antibody, e.g., using a chelator or radioactive metal complex containing an activated ester or isothiocyanate group; conjugating a chelator or radioactive metal complex to a cysteine ​​residue of an antibody, e.g., using a chelator or radioactive metal complex containing a maleimide, haloacetyl derivative, acyl halide, activated disulfide group, or methylsulfonylphenyloxadiazole group; conjugating a tyrosine residue of an antibody, e.g., using a chelator or radioactive metal complex containing 4-phenyl-3H-1,2,4-triazoline-3,5-(4H)-dione (PTAD); or conjugating a methionine residue of an antibody, e.g., using a chelator or radioactive metal complex containing an oxaziridine derivative. Antibodies can also be labeled with biorthogonally reactive functional groups at specific residues using one or more of the above methods prior to conjugation to a chelator or radioactive metal complex of the invention. For example, tyrosine residues can be site-specifically labeled at biorthogonal reactive functional groups using oxaziridine derivatives attached to biorthogonal reactive functional groups, such as azide, alkynyl, or cycloalkynyl, and then antibodies containing labeled tyrosine residues can be conjugated to the chelators or radiometal complexes of the invention using chelators or radiometal complexes with compatible reactive functional groups.

[0239] In some embodiments, immunoconjugates or radioimmunoconjugates of the invention are generated using site-specific or site-selective methods for conjugating a chelator of the invention to an antibody or antigen-binding fragment thereof. In contrast to residue-specific methods, "site-specific" or "site-selective" methods typically result in immunoconjugates or radioimmunoconjugates covalently linked to a chelator or radiometal complex at a specific position on the antibody. Any site-specific method for forming protein or antibody conjugates known to those of skill in the art in light of the present disclosure can be used. For example, unnatural amino acids (e.g., azidoamino acids or alkynyl amino acids) can be specifically incorporated into antibodies using mutant aminoacyl-tRNA synthetases that can selectively aminoacrylate tRNAs bearing the unnatural amino acid of interest. Mutant acylated tRNAs can then be used in conjunction with amber suppressor tRNAs to site-specifically incorporate the unnatural amino acid into proteins in response to amber nonsense codons. Antibodies specifically labeled by one or more of the above methods can subsequently be conjugated to a chelator or radiometal complex of the invention bearing a compatible reactive functional group.

[0240] According to an embodiment of the present invention, a method for producing a radioimmunoconjugate comprises the steps of: 11 is a nucleophilic or electrophilic moiety with an antibody or antigen-binding fragment thereof, or a modified antibody or antigen-binding fragment thereof containing a nucleophilic or electrophilic moiety.

[0241] In one embodiment, a method comprises reacting a chelator of the invention with an antibody or antigen-binding fragment thereof, or a modified antibody or antigen-binding fragment thereof comprising a nucleophilic or electrophilic functional group, to form an immunoconjugate having a covalent bond between the chelator and the antibody or antigen-binding fragment thereof, or the modified antibody or antigen-binding fragment, and reacting the immunoconjugate with a radioactive metal ion, such that the radioactive metal ion is bound to the chelator of the immunoconjugate by a coordinate bond, thereby forming a radioimmunoconjugate. This embodiment may be referred to as a "one-step direct radiolabeling" method (e.g., as shown schematically in Figure 2C) because there is only one chemical reaction step involving the radioactive metal.

[0242] In another embodiment, the method comprises reacting a radioconjugate of the present invention with an antibody or antigen-binding fragment thereof, or a modified antibody or antigen-binding fragment thereof comprising a nucleophilic or electrophilic functional group, thereby forming a radioimmunoconjugate. This embodiment may be referred to as a "click radiolabeling" method (e.g., as shown schematically in Figure 2D). The modified antibody or antigen-binding fragment thereof can be produced by any method known in the art in view of this disclosure, for example, by labeling an antibody with two orthogonally reactive functional groups at specific residues using one or more of the methods described above, or by specifically incorporating an unnatural amino acid (e.g., an azido amino acid or an alkynyl amino acid) into an antibody using one or more of the methods described above. The degree of labeling (DOL), sometimes referred to as the degree of substitution (DOS), is a particularly useful parameter for characterizing and optimizing bioconjugates, such as antibodies modified with unnatural amino acids. It can be expressed as the average number of unnatural amino acids attached to a protein molecule (e.g., an antibody) or as a molar ratio of label / protein. The DOL can be determined from the absorbance spectrum of the labeled antibody by any method known in the art.

[0243] In certain embodiments, as described herein, immunoconjugates and radioimmunoconjugates of the present invention are prepared using click chemistry reactions. For example, radioimmunoconjugates of the present invention can be prepared using a click chemistry reaction referred to as "click radiolabeling" (see, e.g., Figures 2B and 2D). Click radiolabeling uses click chemistry reaction partners, preferably azides and alkynes (e.g., cyclooctyne or cyclooctyne derivatives), to form a triazole covalent bond between a radiocomplex (a radioactive metal ion bound to a chelator) and an antibody or antigen-binding fragment thereof. Methods for click radiolabeling of antibodies are described, for example, in International Patent Application No. PCT / US18 / 65913, entitled "Radiolabeling of Polypeptides," the relevant disclosure of which is incorporated herein by reference. In another embodiment, referred to as "one-step direct radiolabeling," an immunoconjugate is prepared using a click chemistry reaction between an antibody or antigen-binding fragment thereof and a chelator, and the immunoconjugate is then contacted with a radioactive metal ion to form a radioimmunoconjugate (see, e.g., Figures 2A and 2C).

[0244] According to one embodiment, a method for preparing a radioimmunoconjugate comprises binding (eg, by coordination) a radiometal ion to a chelator of the invention.

[0245] One embodiment of the "one-step direct radiolabeling" method may be described as a method of preparing a radioimmunoconjugate, comprising contacting an immunoconjugate (i.e., a polypeptide-chelator complex) with a radioactive metal ion, thereby forming a radioimmunoconjugate, wherein the immunoconjugate comprises a chelator of the present invention. According to certain embodiments, the immunoconjugate is formed by a click chemistry reaction between a chelator of the present invention and a polypeptide. According to certain embodiments, the radioimmunoconjugate is formed without metal-free conditions (e.g., without any step of removing or actively eliminating common metal impurities from the reaction mixture). This is in contrast to certain conventional methods that require radiolabeling antibodies under strict metal-free conditions to avoid competitive (non-productive) chelation of common metals such as iron, zinc, and copper, which poses significant challenges to the manufacturing process.

[0246] In a specific embodiment, the method for preparing a radioimmunoconjugate of the present invention comprises: (i) providing a polypeptide (e.g., an antibody or antigen-binding fragment thereof) covalently attached to a first click reaction partner (e.g., an azide group); (ii) providing a chelator complex comprising a chelator of the invention covalently bonded to a second Click reaction partner (e.g., an alkynyl or cycloalkynyl group); (iii) contacting the modified polypeptide with a chelator complex under conditions that allow the first Click reaction partner (e.g., an azide group) to react with a second Click reaction partner (e.g., an alkynyl or cycloalkynyl group) to form a polypeptide-chelator complex (i.e., an immunoconjugate); (iv) contacting the polypeptide-chelator complex with a radioactive metal ion, thereby preparing a radioimmunoconjugate (the radioimmunoconjugate comprises a radioactive metal ion-labeled polypeptide, e.g., a modified antibody or antigen-binding fragment thereof, labeled with an alpha-emitting radioactive metal ion that is coordinatively bound to the chelator).

[0247] According to a particular embodiment, step (iv) is carried out under metal-free conditions.

[0248] In another embodiment, the method for preparing a radioimmunoconjugate comprises: (i) providing a modified antibody or antigen-binding fragment thereof, comprising an antibody or antigen-binding fragment thereof covalently bound to an azido group; (ii) providing a radioactive complex comprising an alpha-emitting radioactive metal ion bound by a coordinate bond to a chelator, wherein the chelator is covalently bound to an alkynyl or cycloalkynyl group; (iii) contacting the modified antibody or antigen-binding fragment thereof with a radioactive complex under conditions that allow the azido group to react with the alkynyl or cycloalkynyl group, thereby preparing a radioimmunoconjugate (e.g., as shown in Figure 2D).

[0249] Conditions for carrying out click chemistry reactions are known in the art, and any conditions for carrying out click chemistry reactions known to one of skill in the art in light of the present disclosure can be used in the present invention. Exemplary conditions include, but are not limited to, incubating the modified polypeptide and radioactive complex in a ratio of 1:1 to 1000:1 at a pH of 4 to 10 and a temperature of 20°C to 70°C.

[0250] The click radiolabeling method described above allows for radiometal ion chelation under low or high pH and / or high temperature conditions to maximize yield, and this can be achieved without the risk of inactivating the alkyne reaction partner. Efficient chelation between azide-labeled antibodies or antigen-binding fragments thereof and radioactive complexes and efficient SPAAC reactions allow for the production of radioimmunoconjugates in high radiochemical yields even at low azide:antibody ratios. The only step that requires the exclusion of trace metals is the radiometal ion chelation to the chelating moiety; antibody production, purification, and conjugation steps do not need to be performed under metal-free conditions.

[0251] The chelators and radiometal complexes of the present invention can also be used to generate site-specific radiolabeled polypeptides (e.g., antibodies). The click radiolabeling method described herein utilizes established methods for site-specifically introducing azide groups onto antibodies, facilitating the site-specific generation of radioimmunoconjugates (Li, X., et al. Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions. Angew Chem Int Ed Engl, 2014. 53(28): pp. 7179-82; Xiao, H., et al., Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells. Angew Chem Int Ed Engl, 2013. 52(52): pp. 14080-3). Methods for attaching molecules to proteins or antibodies in a site-specific manner are known in the art, and any method for site-specifically labeling antibodies known to one of skill in the art can be used in the present invention in light of the present disclosure. Examples of methods for site-specifically modifying antibodies suitable for use in the present invention include, but are not limited to, the incorporation of modified cysteine ​​residues (e.g., THIOMAB™), unnatural amino acids or glycans (e.g., selenocysteine, p-AcPhe, formylglycine-generating enzyme (FGE, SMARTag™), etc.), and the use of enzymatic methods (e.g., glycotransferases, endoglycosidases, microbial or bacterial transglutaminases (MTG or BTG), sortase A, etc.).

[0252] According to some embodiments, modified antibodies or antigen-binding fragments thereof for use in producing immunoconjugates or radioimmunoconjugates of the invention can be obtained by trimming the antibody or antigen-binding fragment thereof with a bacterial endoglycosidase specific for β-1,4 linkages between core GlcNAc residues in the Fc-glycosylation site of the antibody, such as GlycINATOR (Genovis), which leaves the innermost GlcNAc on the Fc intact and allows site-specific incorporation of an azido sugar at that site. The truncated antibody or antigen-binding fragment thereof is then reacted with an azido-labeled sugar, such as UDP-N-azidoacetylgalactosamine (UDP-GalNaz) or UDP-6-azido6-deoxyGalNac, in the presence of a glycosyltransferase, such as GalT galactosyltransferase or GalNAc transferase, thereby obtaining the modified antibody or antigen-binding fragment thereof.

[0253] In other embodiments, modified antibodies or antigen-binding fragments thereof for use in producing immunoconjugates or radioimmunoconjugates of the present invention can be obtained by deglycosylating the antibody or antigen-binding fragment thereof with an amidase. The resulting deglycosylated antibody or antigen-binding fragment thereof can then be reacted with an azidoamine, preferably 3-azidopropylamine, 6-azidohexylamine, or any azido linker amine or any azidoalkyl / heteroalkylamine, such as an azido-polyethylene glycol (PEG)-amine, for example, O-(2-aminoethyl)-O'-(2-azidoethyl)tetraethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl)pentaethylene glycol, or O-(2-aminoethyl)-O'-(2-azidoethyl)triethylene glycol, or in the presence of a microbial transglutaminase, to obtain the modified antibody or antigen-binding fragment thereof.

[0254] Any of the radiometal complexes described herein can be used to generate the radioimmunoconjugates of the invention. In certain embodiments, the radiometal complex has a structure of Formula (Im), Formula (II-m), or Formula (III-m). In preferred embodiments, the radiometal complex has a structure selected from the group consisting of:

[0255] [ka] In the formula, M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion, more preferably actinium-225 ( 225 Ac) and R 11 is cyclooctynyl or a cyclooctynyl derivative selected from the group consisting of bicyclononynyl (BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, biarylazacyclooctynyl (BARAC), dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO) and tetramethoxydibenzocyclooctynyl (TMDIBO).

[0256] In some embodiments, the antibody or antigen-binding fragment thereof is covalently attached to the azide group using any method for chemical or enzymatic modification of antibodies and polypeptides known to those of skill in the art in light of the present disclosure. The azide-labeled antibody or antigen-binding fragment thereof is reacted with a chelator or radiometal complex of the invention comprising an alkynyl or cycloalkynyl group, preferably a cyclooctynyl group, more preferably DBCO, under conditions sufficient to subject the azide and alkynyl or cycloalkynyl group to a click chemistry reaction to form a 1,2,3-triazole moiety.

[0257] In certain embodiments, the radioimmunoconjugates of the present application include:

[0258] [ka] These include, but are not limited to: wherein mAb is an antibody or antigen-binding fragment thereof, L is absent or a linker, preferably a linker, and each R 12 are independently hydrogen, CH3, or CH2CH3, and at least one R 12 is —CH 3 or —CH 2 CH 3 , and M is an alpha-emitting radionuclide, preferably 225 It is Ac.

[0259] Examples of radioimmunoconjugates of the present application include:

[0260] [ka] These include, but are not limited to: Preferably, the mAb is selected from PSMB127, pertuzumab, cetuximab, panitumumab, herceptin, or H11B6.

[0261] Radioimmunoconjugates produced by the methods described herein can be analyzed using methods known to those of skill in the art in light of the present disclosure. For example, LC / MS analysis can be used to determine the ratio of chelator to labeled polypeptide, e.g., antibody or antigen-binding fragment thereof; analytical size-exclusion chromatography can be used to determine the oligomeric state of polypeptides and polypeptide conjugates, e.g., antibodies and antibody conjugates; radiochemical yield can be determined by instant thin-layer chromatography (e.g., iTLC-SG); and radiochemical purity can be determined by size-exclusion HPLC. Exemplary methods are described herein, e.g., in the Examples below.

[0262] Pharmaceutical Compositions and Methods of Use In another general aspect, the invention relates to a pharmaceutical composition comprising a chelator, radiometal complex, immunoconjugate, or radioimmunoconjugate of the invention and a pharmaceutically acceptable carrier. The pharmaceutical composition may also include one or more pharmaceutically acceptable carriers.

[0263] In one embodiment, the pharmaceutical composition comprises a radiometal complex of the present invention and a pharmaceutically acceptable carrier.

[0264] In another embodiment, a pharmaceutical composition comprises a radioimmunoconjugate of the present invention and a pharmaceutically acceptable carrier.

[0265] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulate, or other material known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the efficacy or biological activity of the compositions of the present invention. According to certain embodiments, any pharmaceutically acceptable carrier suitable for use in antibody-based or radioconjugate-based pharmaceutical compositions in light of the present disclosure can be used in the present invention.

[0266] According to certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein can be formulated to be suitable for parenteral administration, e.g., intravenous, subcutaneous, intramuscular, or intratumoral administration.

[0267] In another general aspect, the present invention relates to radiation therapy and methods of selectively targeting neoplastic cells to treat neoplastic diseases or disorders. Any of the radiocomplexes or radioimmunoconjugates described herein and pharmaceutical compositions thereof can be used in the methods of the invention.

[0268] A "neoplasm" is an abnormal mass of tissue that forms when cells divide more than necessary or do not die when they should. Tumors can be benign (not cancerous) or malignant (cancer). Neoplasm is also called a tumor. A neoplastic disease or disorder is a disease or disorder associated with a tumor, such as cancer. Examples of neoplastic diseases or disorders include, but are not limited to, disseminated cancer and solid tumor cancer.

[0269] According to one embodiment, a method of treating prostate cancer (e.g., metastatic prostate cancer or metastatic castration-resistant prostate cancer) in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a radioimmunoconjugate described herein, wherein the radioimmunoconjugate comprises a radiometal complex described herein conjugated to H11B6.

[0270] Other examples of diseases that may be targeted for treatment or radiation therapy by the methods described herein include, but are not limited to, hypertrophy, coronary artery disease, or vascular occlusive disease, diseases or disorders associated with infected cells, microorganisms, or viruses, or diseases or disorders associated with inflammatory cells, such as rheumatoid arthritis (RA).

[0271] In one embodiment of the present invention, a method for selectively targeting neoplastic cells for radiation therapy comprises administering to a subject a radioimmunoconjugate or pharmaceutical composition of the present invention.

[0272] In one embodiment of the present invention, a method for treating a neoplastic disease or disorder comprises administering to a subject in need thereof a radioimmunoconjugate or pharmaceutical composition of the present invention to the subject.

[0273] In one embodiment of the present invention, a method of treating cancer in a subject in need thereof comprises administering to the subject a radioimmunoconjugate or pharmaceutical composition of the present invention.

[0274] The radioimmunoconjugate delivers radiation directly to, for example, cells targeted by a targeting ligand. Preferably, the radioimmunoconjugate 225 When targeted, the alpha-emitting radioactive metal ions, e.g., Ac, are carried. 225 Alpha particles from Ac and its daughters are delivered to targeted cells, causing a cytotoxic effect thereon, thereby selectively targeting neoplastic cells for radiation therapy and / or treating neoplastic diseases or disorders.

[0275] A pre-targeting approach for selectively targeting neoplastic cells for radiation therapy and to treat neoplastic diseases or disorders is also contemplated by the present invention. According to the pre-targeting approach, an azide-labeled antibody or antigen-binding fragment thereof is administered, binds to cells bearing the antibody's target antigen, and is removed from the circulation over time or with a removal agent. Subsequently, a radioactive complex of the present invention, preferably a radioactive complex containing a cyclooctyne or cyclooctyne derivative (e.g., DBCO), is administered and undergoes a SPAAC reaction with the azide-labeled antibody bound to the target site, and the remaining unbound radioactive complex is rapidly removed from the circulation. This pre-targeting technique provides a method for enhancing radioactive metal ion localization at the target site in a subject.

[0276] In other embodiments, the modified polypeptide, e.g., an azido-labeled antibody or antigen-binding fragment thereof, and the radioconjugate of the invention are administered in the same composition or in different compositions to a subject in need of targeted radiation therapy or treatment of a neoplastic disease or disorder.

[0277] As used herein, the term "therapeutically effective amount" refers to that amount of an active ingredient or component that elicits a desired biological or pharmacological response in a subject. A therapeutically effective amount can be determined empirically and routinely for the stated purpose. For example, in vitro assays can optionally be used to help identify optimal dosage ranges. Selection of a specific effective dose can be determined (e.g., by clinical trials) by one of ordinary skill in the art based on consideration of several factors, including the disease to be treated or prevented, accompanying symptoms, the patient's weight, the patient's immune status, and other factors known to those of skill in the art. The precise dose to be employed in the formulation will also depend on the route of administration and the severity of the disease, and should be determined according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0278] As used herein, the terms "treat," "treating," and "treatment" are all intended to refer to the improvement or amelioration of at least one measurable physical parameter associated with a disease, disorder, or condition that may be discernible in a subject, but is not necessarily discernible in the subject, and that may benefit from the administration of a radioactive metal ion, such as a neoplastic disease or disorder. The terms "treat," "treating," and "treatment" may also refer to causing regression of, preventing the progression of, or at least slowing the progression of, a disease, disorder, or condition. In certain embodiments, "treat," "treating," and "treatment" refer to the amelioration of, preventing the development or onset of, or shortening the duration of, one or more symptoms associated with a disease, disorder, or condition that may benefit from the administration of a radioactive metal ion, such as a neoplastic disease or disorder. In certain embodiments, "treat," "treating," and "treatment" refer to preventing the recurrence of a disease, disorder, or condition. In certain embodiments, "treat," "treating," and "treatment" refer to improving the survival rate of a subject with a disease, disorder, or condition. In certain embodiments, "treat," "treating," and "treatment" refer to eliminating the disease, disorder, or condition in a subject.

[0279] In some embodiments, a therapeutically effective amount of a radioimmunoconjugate or pharmaceutical composition of the present invention is administered to a subject to treat a neoplastic disease or disorder in the subject, such as cancer.

[0280] In other embodiments of the present invention, the radioimmunoconjugates and pharmaceutical compositions of the present invention may be used in combination with other agents effective in the treatment of neoplastic diseases or disorders.

[0281] Also provided are the radioimmunoconjugates and pharmaceutical compositions described herein for use in selectively targeting neoplastic cells for radiation therapy and / or treatment of a neoplastic disease or disorder, and the use of a radioimmunoconjugate or pharmaceutical composition described herein in the manufacture of a medicament for selectively targeting neoplastic cells for radiation therapy and / or treatment of a neoplastic disease or disorder. [Example]

[0282] The following examples of the present invention are presented to further illustrate the principles of the present invention, but it should be understood that the following examples do not limit the present invention, the scope of which is defined by the appended claims.

[0283] Example 1: Synthesis and chelation efficiency of macrocyclic chelates with linker substitutions at different positions Actinium-225( 225 To investigate the effect of linker position on the chelating efficiency of Ac), two chelators based on the macrocyclic chelator N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6 (H2bp18c6) were synthesized. Specifically, H2bp18c6-benzyl-isopentyl and H2bp18c6-benzyl-phenyl were synthesized.

[0284] [ka]

[0285] Synthesis and characterization of H2bp18c6-benzyl-isopentyl H2bp18c6-benzyl-isopentyl was synthesized according to Scheme 1.

[0286] [ka]

[0287] To a solution of ZnCl (1.9 M in 2-methyltetrahydrofuran, 3.50 mL, 6.65 mmol) and THF (25 mL) at −78 °C was added isopentylmagnesium chloride (2 M in EtO, 3.33 mL, 6.66 mmol) dropwise. The cloudy mixture was stirred at room temperature for 1 h and then cooled to 0 °C. A solution of methyl 6-formylpicolinate (1.00 g, 6.1 mmol) in THF (10 mL) was added. The mixture was heated at 50 °C for 3 h. The cooled reaction mixture was poured into saturated aqueous NH Cl and extracted three times with EtOAc. The combined extracts were dried over Na SO . Filtration and concentration of the filtrate afforded the crude product as a brown oil. Silica gel chromatography (heptane to 50% EtOAc in heptane) afforded 693 mg (48% yield) of the product as a yellow oil.

[0288] To a solution of methyl 6-(1-hydroxy-4-methylpentyl)picolinate (91 mg, 0.38 mmol) in CHCl (3 mL) at room temperature was added PPh (120 mg, 0.46 mmol) and NBS (56 mg, 0.42 mmol). The reaction solution was stirred at room temperature for 1 h and then concentrated. Chromatography on silica gel (heptane to 30% EtOAc in heptane) afforded 85 mg (87% yield) of the product as a colorless oil.

[0289] To a stirred mixture of 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (315 mg, 1.2 mmol) and KCO (691 mg, 5 mmol) in ACN (30 mL) at 60 °C, a solution of methyl 6-(chloromethyl)picolinate in ACN (5 mL) was slowly added over 1 h using a syringe pump. After the addition, the reaction mixture was stirred at 60 °C for 6 h and then filtered. The filtrate was concentrated, and the residue was purified by chromatography on silica gel (CHCl to 10% MeOH in CHCl) to give 206 mg (50% yield) of the product as a yellowish foamy solid.

[0290] To a mixture of methyl 6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (31 mg, 0.075 mmol), NaCO (40 mg, 0.38 mmol), and NaI (1.5 mg) in DMF (0.5 mL) was added 6-(1-chloro-4-methylpentyl)picolinate (29 mg, 0.11 mmol). The reaction mixture was heated at 100 °C for 20 h. Chromatography on silica gel (EtOAc to 10% MeOH in CHCl) afforded 18.9 mg (40% yield) of the product as a yellow film.

[0291] A solution of methyl 6-((16-(1-(6-(methoxycarbonyl)pyridin-2-yl)-4-methylpentyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (3 mg, 0.005 mmol) in THF / MeOH / HO (4:1:1 v / v / v, 0.6 mL) was treated with LiOH (1 N, 0.1 mL). The reaction mixture was stirred at room temperature for 1 h, and then concentrated to dryness. The residue was dissolved in 0.95 mL of metal-free water and neutralized with 0.05 mL of 2 N HCl to give a solution of H2bp18c6-benzyl-isopentyl in water (approximately 3 mg / mL = approximately 5 mM, pH approximately 6).

[0292] H2bp18c6-benzyl-isopentyl (MW=602 Da) was characterized by high performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS). HPLC analysis showed a major peak at 17.181 min elution time, corresponding to the free chelator. LC-MS analysis showed a peak at 603 [M+H + ], 625[M+Na + ] and 302[M+2H + ] showed a mass ion peak (ES, m / z) at 1000 kJ / min, confirming the synthesis of H2bp18c6-benzyl-isopentyl.

[0293] HPLC method: XBridge C18 3.5 μm 150 × 4.6 mm, 100 Å column; Mobile phase A: 0.1% TFA in HO, B: 0.1% TFA in ACN; gradient 10% to 30% B from 0 to 20 min, gradient 30% to 100% B from 20 to 20.1 min, isocratic at 100% B from 20.1 to 25 min, gradient 100% to 10% B from 25 to 25.1 min, isocratic at 10% B from 25.1 to 30 min; 3 min after run; flow rate 1 mL / min, column temperature: 30 °C; injection volume: 5 μL.

[0294] Synthesis and characterization of H2bp18c6-benzyl-phenyl H2bp18c6-benzyl-phenyl was synthesized according to Scheme 2.

[0295] [ka]

[0296] Methyl 6-formylpicolinate (165 mg, 1.0 mmol), phenylboronic acid (244 mg, 2.0 mmol), Cs2CO3 (326 mg, 1.0 mmol), Pd2(dba)3·CHCl3 (52 mg, 0.05 mmol), and PPh 3( A mixture of 26 mg (0.1 mmol) of HCl (H 2 Cl 2 HCl) was placed in a sealed vial under N 2 . Toluene (3 mL) was added via syringe, and the mixture was heated in a microwave at 100 °C for 4 h. The cooled reaction mixture was filtered through Celite, and the filtrate was concentrated. Chromatography on silica gel (heptane to 50% EtOAc in heptane) afforded 146 mg (60% yield) of the product as a yellow oil.

[0297] A solution of methyl 6-(hydroxy(phenyl)methyl)picolinate (141 mg, 0.58 mmol), PPh3 (183 mg, 0.70 mmol), and NBS (113 mg, 0.64 mmol) in CHCl2 (8 mL) was stirred at room temperature for 1 h. Additional PPh3 (133 mg, 0.70 mmol) and NBS (113 mg, 0.64 mmol) were added, and the solution was stirred at room temperature for an additional 1 h. The reaction mixture was concentrated and purified by chromatography on silica gel (heptane to 30% EtOAc in heptane) to give 73 mg (41% yield) of the product as a colorless oil.

[0298] A mixture of methyl 6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (33 mg, 0.080 mmol), methyl 6-(bromo(phenyl)methyl)picolinate (37 mg, 0.12 mmol), and Na2CO3 (42 mg, 0.40 mmol) in ACN (1.0 mL) was heated at 80 °C for 3 h. The reaction mixture was filtered, and the filtrate was concentrated. Chromatography on silica gel (EtOAc to 10% MeOH in CHCl2) afforded 51 mg (64% yield) of the product as a white solid.

[0299] A solution of methyl 6-((16-((6-(methoxycarbonyl)pyridin-2-yl)(phenyl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (16 mg, 0.025 mmol) in THF / MeOH / HO (4:1:1 v / v / v, 1.2 mL) was treated with LiOH (1 N, 0.2 mL). The reaction mixture was stirred at room temperature for 1 h, and then concentrated to dryness. The residue was dissolved in 5.2 mL of metal-free water and neutralized with 0.10 mL of 2 N HCl to give a solution of H2bp18c6-benzyl-phenyl in water (approximately 3 mg / mL = 5 mM, approximately pH 6).

[0300] H2bp18c6-benzyl-phenyl (MW=608 Da) was characterized by high-performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS). HPLC analysis was performed as described above for isopentane-H2bp18c6. HPLC analysis showed a major peak at 14:137 min elution time, corresponding to the free chelator. LC-MS analysis showed a peak at 609 [M+H + ], 631 [M+Na + ] and 305 [M+2H + ] showed a mass ion peak (ES, m / z) at 1000 kJ / min, confirming the synthesis of H2bp18c6-benzyl-phenyl.

[0301] Chelation test with lanthanum(III) An aqueous solution of H2bp18c6-benzyl-isopentyl (approximately 3 mg / mL = approximately 5 mM, 20 μL, 0.1 μmol) was treated with La(NO3)3 (10 mM, 50 μL, 0.5 μmol in metal-free water). An aqueous solution of H2bp18c6-benzyl-phenyl in water (approximately 3 mg / mL = approximately 5 mM, 20 μL, 0.1 μmol) was treated with La(NO3)3 (10 mM, 50 μL, 0.5 μmol in metal-free water). After thorough mixing, each solution was analyzed by LCMS and HPLC to determine the chelator and La 3+ It was determined whether a complex with

[0302] Both isopentane-H2bp18c6 and H2bp18c6-benzyl-phenyl chelates exhibited La activity at room temperature as indicated by significant shifts in HPLC peak retention times analyzed according to the method described above for the synthesis of H2bp18c6. 3+ The complex formation was also confirmed by LCMS. LCMS analysis of isopentane-H2bp18c6 after mixing with La(NO3)3 showed a mass ion peak (ES, m / z) at 739 (H2bp18c6-benzyl-isopentyl + La +3 -2H +LC-MS analysis of H2bp18c6-benzyl-phenyl after mixing with LA(NO3)3 showed a mass ion peak (ES, m / z) at 745 (H2bp18c6-benzyl-phenyl + La +3 -2H + ), confirming complex formation with both chelators.

[0303] 225 Chelation of H2bp18c6-benzyl-isopentyl with Ac(III) (i) Low 225 Ac / chelate ratio 225 Chelation with Ac(III) In a plastic vial, tetramethylammonium acetate (1 M in water, 10 μL), H2bp18c6-benzyl-isopentyl (1.66 mM in water, 2 μL, approximately 3.32 nmol) and 225 Ac(NO3)3 (10 mCi / mL in 0.1 N HCl, 3 μL, 30 μCi) was added sequentially. After mixing, the pH was approximately 6.5 as determined by pH paper. The reaction solution was left at room temperature for 1.5 hours.

[0304] iTLC-SG analysis: 0.5 μL of the reaction solution was spotted onto iTLC-SG, which was developed with 10 mM EDTA. The dried iTLC-SG was left overnight at room temperature and then scanned with a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, bound Ac-225 remained above the baseline of iTLC-SG, while free Ac-225 migrated to the solvent front with the solvent. Radioactivity was not observed at the solvent front of iTLC-SG, which suggests that the chelator 225 This indicates that Ac ions were successfully chelated.

[0305] HPLC analysis: 5 μL of the reaction mixture was diluted with 95 μL of PBS buffer. The diluted mixture was analyzed by HPLC. After HPLC, fractions were collected at 1-minute intervals. The collected fractions were left at room temperature overnight and then counted in a gamma counter. HPLC radio-traces were constructed from the activity of the fractions.

[0306] HPLC analysis revealed a retention time shift similar to that shown in the HPLC chromatogram in Figure 1A. 225 It was confirmed that an Ac complex was formed.

[0307] DTPA loading: 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA solution, and the mixture was incubated for 30 minutes. 10 μL of the mixture was spotted onto iTLC-SG, which was developed with 10 mM EDTA. The dried iTLC-SG was left overnight at room temperature and then scanned with a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, stably chelated Ac-225 remained on the baseline of iTLC-SG, while free Ac-225 migrated to the solvent front with the solvent. After DTPA loading, no radioactivity was observed at the solvent front of iTLC-SG, which may be due to the following: 225 This indicates that a stable complex with Ac ions was formed.

[0308] (ii) High 225 Ac / chelate ratio 225 Chelation with Ac(III) In a plastic vial, tetramethylammonium acetate (1 M in water, 10 μL), H2bp18c6-benzyl-isopentyl (0.33 mM in water, 2 μL, approximately 0.66 nmol) and 225 Ac(NO3)3 (10 mCi / mL in 0.1 N HCl, 5 μL, 50 μCi) was added sequentially. After mixing, the pH was approximately 6.5 by pH paper. The reaction solution was left at room temperature for 1.5 hours. The reaction was then analyzed by iTLC-SG and DTPA loading as described above. The released HCl was detected before or after DTPA loading. 225 Ac was not detected, which is 225 This indicates that a stable complex with Ac ions was formed.

[0309] 225 Chelation of H2bp18c6-benzyl-phenyl with Ac(III) (i) Low 225 Ac / chelate ratio 225Chelation with Ac(III) In a plastic vial, tetramethylammonium acetate (1 M solution in water, 10 μL), H2bp18c6-benzyl-phenyl (1.64 mM in water, 2 μL, approximately 3.28 nmol) and 225 Ac(NO3)3 (10 mCi / mL in 0.1 N HCl, 3 μL, 30 μCi) was added sequentially. After mixing, the pH was approximately 6.5 according to pH test paper. The reaction solution was left at room temperature for 2 hours. 225 No Ac ions were detected, which indicates that the chelator was successful. 225 This indicates that Ac ions were chelated.

[0310] iTLC-SG analysis: 0.5 μL of the reaction solution was spotted onto iTLC-SG, which was developed with 10 mM EDTA. The dried iTLC-SG was left overnight at room temperature and then scanned with a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, bound Ac-225 remained above the baseline of iTLC-SG, while free Ac-225 migrated with the solvent to the solvent front. No X-ray activity was observed at the solvent front of iTLC-SG, indicating good chelation of Ac-225.

[0311] HPLC analysis: 5 μL of the reaction mixture was diluted with 95 μL of PBS buffer. The diluted mixture was analyzed by HPLC. After HPLC, fractions were collected at 1-minute intervals. The collected fractions were left at room temperature overnight and then counted in a gamma counter. HPLC radioactivity traces were constructed from the activity of the fractions. HPLC analysis revealed a shift in retention time similar to that shown in the HPLC chromatogram in Figure 1B. 225 It was confirmed that an Ac complex was formed.

[0312] DTPA loading: 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA solution, and the mixture was incubated for 30 minutes. 10 μL of the mixture was spotted onto iTLC-SG, which was developed with 10 mM EDTA. The dried iTLC-SG was left overnight at room temperature and then scanned with a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, stably chelated Ac-225 remained on the baseline of iTLC-SG, while free Ac-225 migrated to the solvent front with the solvent. After DTPA loading, free Ac-225 225 Ac was not detected, which is 225 No X-ray activity was observed at the solvent front of iTLC-SG before or after DTPA loading, indicating that a stable complex with Ac ions was formed. 225 This indicates that a stable complex with Ac ions was formed.

[0313] (ii) High 225 Ac / chelate ratio 225 Chelation with Ac(III) In a plastic vial, tetramethylammonium acetate (1 M solution in water, 10 μL), H2bp18c6-benzyl-phenyl (0.16 mM in water, 2 μL, approximately 0.33 nmol) and 225 Ac(NO3)3 (10 mCi / mL in 0.1 N HCl, 5 μL, 50 μCi) was added sequentially. After mixing, pH was approximately 6.5 by pH paper. The reaction solution was left at room temperature for 2 hours. The reaction was analyzed by iTLC-SG and DTPA loading as described above. The released HCl was analyzed before or after DTPA loading. 225 Ac was not detected, which is 225 This indicates that a stable complex with Ac ions was formed.

[0314] summary Taken together, the above results demonstrate that H2bp18c6 derivatized at the "benzyl" carbon exhibits rapid chelation kinetics at room temperature. 225 The results also demonstrate high specific activity (i.e., higher specific activity with lower chelators). 225It shows that the ratio of α- and β-Ac to β-Ac can be achieved by binding through the "benzyl" position.

[0315] Example 2: Synthesis and chelation efficiency of H2bp18c6 derivatives with DBCO "click" linkers The following H2bp18c6 derivatives can then be synthesized with a DBCO linker for conjugation to a targeting ligand via a click chemistry reaction:

[0316] [ka]

[0317] For example, H2bp18c6-acetate-DBCO can be synthesized according to Scheme 3.

[0318] [ka]

[0319] Methyl 6-bromopicolinate can be reacted with 2-tert-butoxy-2-oxoethylzinc bromide in the presence of a palladium catalyst to give methyl 6-(2-(tert-butoxy)-2-oxoethyl)picolinate. Subsequent bromination with NBS and AIBN can give methyl 6-(1-bromo-2-(tert-butoxy)-2-oxoethyl)picolinate. Substitution reaction of methyl 6-(1-bromo-2-(tert-butoxy)-2-oxoethyl)picolinate with methyl 6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate under basic reaction conditions can produce methyl 6-(2-(tert-butoxy)-1-(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)-2-oxoethyl)picolinate. In the presence of TFA, the tert-butyl ester can be hydrolyzed to the carboxylic acid. Amide bond formation with dibenzocyclooctyne-amine followed by hydrolysis of the methyl ester with lithium hydroxide can afford H2bp18c6-benzyl-acetate-DBCO.

[0320] H2bp18c6-benzyl-phenyl-DBCO is synthesized according to Scheme 4.

[0321] [ka]

[0322] THF (5 mL) was added to a mixture of methyl 6-formylpicolinate (165 mg, 1.0 mmol), (4-(tert-butoxycarbonyl)phenyl)boronic acid (444 mg, 2.0 mmol), PdCl (8.9 mg, 0.05 mmol), tri(naphthalen-1-yl)phosphane (20.6 mg, 0.05 mmol), and KCO (415 mg, 3.0 mmol) at −78 °C under N. The mixture was purged with N and stirred at room temperature for 0.5 h, then heated at 65 °C for 24 h. The cooled reaction mixture was filtered through Celite, and the filtrate was concentrated. Silica gel chromatography (heptane to 50% EtOAc in heptane) afforded 116 mg (34% yield) of the product as a yellow oil.

[0323] A solution of methyl 6-((4-(tert-butoxycarbonyl)phenyl)(hydroxy)methyl)picolinate (138 mg, 0.40 mmol), PPh3 (126 mg, 0.48 mmol), and NBS (79 mg, 0.44 mmol) in CHCl2 (5 mL) was stirred at room temperature for 1 h. Additional PPh3 (63 mg, 0.24 mmol) and NBS (39 mg, 0.22 mmol) were added, and this was stirred for another 1 h. The reaction solution was loaded onto a silica gel column. Chromatography (heptane to 30% EtOAc in heptane) afforded 62 mg (38% yield) of the product as a yellowish film attached to the flask wall.

[0324] A mixture of methyl 6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (56 mg, 0.14 mmol), methyl 6-(bromo(4-(tert-butoxycarbonyl)phenyl)methyl)picolinate (60 mg, 0.15 mmol), and NaCO (72 mg, 0.68 mmol) in ACN (1 mL) was heated at 80 °C for 13 h. The cooled reaction mixture was filtered, and the filtrate was concentrated. Chromatography on silica gel (CHCl to 10% MeOH in CHCl) afforded 37 mg (37% yield) of the product as a white solid.

[0325] To a solution of methyl 6-((4-(tert-butoxycarbonyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (22 mg, 0.03 mmol) in CHCl (1.5 mL) was added TFA (0.5 mL). The solution was stirred for 1 hour. The reaction solution was concentrated to give the crude product as a yellowish residue, which was used in the next step reaction without further purification.

[0326] To a solution of the above crude product in CHCl (0.5 mL) was added EtN (42 μL, 0.3 mmol) followed by HBTU (15 mg, 0.04 mmol) at 0 °C. After the solution was stirred at 0 °C for 5 min, dibenzocyclooctyne-amine in CHCl (0.5 mL) was added. The cold bath was removed, and it was stirred at room temperature for 18 h. Water was added to the reaction mixture, which was extracted three times with CHCl. ​​The combined extracts were washed with saturated aqueous NaHCO, then brine, dried (NaSO), and filtered. The filtrate was concentrated to give the crude product. Chromatography on silica gel (CHCl to 10% MeOH in CHCl) afforded 13.2 mg (47% yield) of the product as a colorless film attached to the flask wall.

[0327] To a solution of H2bp18c6-benzyl-phenyl-DBCO (4.8 mg, 0.005 mmol) in THF / MeOH / HO (4:1:1 v / v / v, 0.6 mL) was added NaOH (1 N, 0.1 mL). After stirring the reaction mixture at room temperature for 1 h, it was neutralized with HCl (1 N) to pH = 6.5. The reaction mixture was concentrated on a rotary evaporator at room temperature to remove volatile solvents. The residue was dissolved in HO (4 mL) and ACN (1 mL). After lyophilization, the crude product was obtained as a white solid.

[0328] Chelation of H2bp18c6-benzyl-phenyl-DBCO with La(III) A solution of H2bp18c6-benzyl-phenyl-DBCO (about 1.55 mg / mL = about 1.7 mM in 4:1 v / v H2O / ACN, about pH 6.5 by pH paper) was prepared from the crude product above.

[0329] A solution of H2bp18c6-benzyl-phenyl (approximately 1.55 mg / mL = approximately 1.7 mM, 50 μL, 0.085 μmol) was treated with La(NO3)3 (10 mM in metal-free water, 50 μL, 0.5 μmol). After thorough mixing, the solution was analyzed by LCMS and HPLC. MS (ES, m / z) 1047 (H2bp18c6-benzyl-phenyl-DBCO + La +3 -2H + ).

[0330] HPLC method: XBridge C18 3.5 μm 150 × 4.6 mm, 100 Å column; mobile phase A: 0.1% TFA in HO, B: 0.1% TFA in ACN; gradient 10% to 50% B from 0 to 20 min, gradient 50% to 100% B from 20 to 20.1 min, isocratic at 100% B from 20.1 to 25 min, gradient 100% to 10% B from 25 to 25.1 min, isocratic at 10% B from 25.1 to 30 min; flow rate 1 mL / min, column temperature: 30 °C; injection volume: 5 μL.

[0331] 225 Chelation of H2bp18c6-benzyl-phenyl with Ac(III) (i) Low 225 Ac / chelate ratio 225 Chelation with Ac(III) In a plastic vial, tetramethylammonium acetate (1 M solution in water, 10 μL), H2bp18c6-benzyl-phenyl-DBCO (1.7 mM in HO / ACN, 2 μL, approximately 3.4 nmol) and 225 Ac(NO3)3 (10 mCi / mL in 0.1 N HCl, 3 μL, 30 μCi) was added sequentially. After mixing, the pH was approximately 6.5 as determined by pH test paper. The reaction solution was left at room temperature for 1 hour.

[0332] iTLC-SG analysis: 0.5 μL of the reaction solution was spotted onto iTLC-SG and developed with 10 mM EDTA. The dried iTLC-SG was left overnight at room temperature and then scanned with a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates to the solvent front along with the solvent. No radioactivity was observed at the solvent front of iTLC-SG, indicating that no free Ac-225 was present in the reaction solution after 1 h.

[0333] HPLC analysis: 5 μL of the reaction mixture was diluted with 95 μL of PBS buffer. The diluted mixture was analyzed by HPLC. After HPLC, fractions were collected at 1-minute intervals. The collected fractions were left at room temperature overnight and then counted in a gamma counter. HPLC radioactivity traces were constructed from the activity of the fractions.

[0334] DTPA loading: 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA solution, and the mixture was incubated for 30 minutes. 10 μL of the mixture was spotted onto iTLC-SG, which was developed with 10 mM EDTA. The dried iTLC-SG was left at room temperature overnight and then scanned with a Bioscan AR-2000 radio-TLC scanner. No radioactivity was detected at the solvent front of the iTLC-SG.

[0335] (ii) High 225 Ac / chelate ratio 225 Chelation with Ac(III) In a plastic vial, tetramethylammonium acetate (1 M solution in water, 10 μL), H2bp18c6-benzyl-phenyl-DBCO (0.17 mM in water, 2 μL, approximately 0.34 nmol) and 225 Ac(NO3)3 (10 mCi / mL in 0.1 N HCl, 5 μL, 50 μCi) was added sequentially. After mixing, the pH was approximately 6.5 by pH paper. The reaction solution was left at room temperature for 2 hours. The reaction was analyzed by iTLC-SG and DTPA loading as described above.

[0336] Example 3: Preparation of H2bp18c6-benzyl-phenyl-DBCO-IgG4 and 225 Ac(III) labeling General method for preparing radioimmunoconjugates: Radioimmunoconjugates comprising a radiometal complex of the invention covalently bound to an antibody are prepared by click radiolabeling. See Figures 2A-2D for a schematic diagram of an exemplary method of click radiolabeling to generate a radioimmunoconjugate of the invention.

[0337] Random conjugation of azide handles to antibodies A stock solution of antibody (1–10 mg / mL) in 10 mM sodium acetate (pH 5.2), phosphate-buffered saline (pH 7), or other compatible buffer was mixed with 20% (v / v) of 1 M sodium carbonate buffer (pH 9) to a final pH of approximately 9. NHS-PEG4-azide (Thermo catalog no. 26130) was dissolved in DMSO to a final concentration of 100 mM, and a 0.2% (v / v) stock solution was added to generate an approximately 3–10 molar excess over antibody (Ab). The reaction was incubated at 22 °C for 10 min and then quenched by adding 1 M Tris (pH 7.5) to a final concentration of 50 mM Tris.

[0338] The azide-mAb conjugate was purified and exchanged into a compatible buffer (PBS; 20 mM Hepes, 150 mM NaCl, pH 7.5; or 10 mM sodium acetate, pH 5.2) using methods such as a Zeba desalting column (Thermo) with a 7K MW cutoff, dialysis, standard Protein A affinity chromatography, or another suitable method. After purification, the conjugate was concentrated to 10-20 mg / mL using an Amicon concentrator (Millipore) with a 50K MW cutoff. Conjugation efficiency was determined by LC-MS.

[0339] Site-specific incorporation of azido sugars into antibody glycans Antibody glycans were trimmed with GlycINATOR (Genovis), a bacterial endoglycosidase specific for β-1,4 linkages between core GlcNAc residues within the Fc glycosylation site(s), leaving the innermost GlcNAc intact on the Fc, which could later be used for site-specific incorporation of azido sugars. More specifically, GlycINATOR immobilized on agarose beads packed in a column (Genovis) was equilibrated in Tris-buffered saline, pH 7.4 (TBS). 1 mL of 5–10 mg / mL mAb was added to the resin, incubated on a rocker at room temperature for 1 h, and eluted by spinning at 100 x g for 1 min. The column was eluted three times with 0.5 mL of TBS. The eluates containing the trimmed mAb were pooled and loaded with the supplied buffer additive (Genovis) along with the UDP-GALNaz azido sugar substrate and GalT galactosyltransferase enzyme. The reaction mixture was stirred overnight at 30° C. The final azido mAb was purified using a mAb Select column (GE) on an AKTA Avant instrument. Azide modification was confirmed by LC-MS.

[0340] Conjugation of chelators to azido-Ab Chelators of the invention containing DBCO groups, e.g.

[0341] [ka] as described in Example 1 225The azide-modified antibody is coordinated to a radioactive metal ion, such as Ac, to form a radioactive complex. A randomly or site-specifically azide-modified antibody in PBS or another compatible buffer (10-20 mg / mL) is added to the radioactive complex solution. The reaction solution is gently stirred and left at room temperature for 3 hours, after which it is purified, for example, on a PD-10 column (GE Healthcare) preconditioned with 15 mL of NaOAc buffer (10 mM, pH 6-6.5) or another compatible buffer. Purity is assessed by iTLC-SG. The product solution is analyzed by HPLC for chemical and radiochemical purity. The antibody concentration in the product solution is determined by UV absorption using a calibration curve. The activity of the product solution is quantified using a Capintec CRC-55TW dose calibrator.

[0342] Analytical characterization of click-labeled radioimmunoconjugates The radiochemical conversion (% RA conversion) is determined by iTLC-SG (Instant Thin Layer Chromatography (iTLC)). The radiochemical purity (% RA purity) of the Ac-225 chelate is determined by SE-HPLC (Size Exclusion HPLC).

[0343] Direct chelation of 225Ac(III) to H2bp18c6-phenyl-IgG4 The following method for preparing radioimmunoconjugates can be referred to as a one-step direct radiolabeling method (e.g., as shown schematically in Figure 2C). Radiolabeling was successfully performed under non-metal-free conditions, further illustrating the ability of the chelator to tolerate metal contaminants.

[0344] The IgG4 used in this example is an isotype control that binds to respiratory syncytial virus (RSV) antigens. The amino acid sequences of the heavy chain (HC) and light chain (LC) of IgG4 are provided below as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0345] SEQ ID NO: 1 mAb HC QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0346] Sequence number 2 mAb LC DIVMTQSPDSLAVSLGERNATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0347]

Chem.

[0348] Azide modification and click reaction of mAb: The above mAb IgG4 was site-selectively modified with a 100-fold molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37°C. The addition of two azides onto the heavy chain of the mAb was monitored by intact mass ESI-TOF LC-MS on an Agilent G224 instrument. Excess 3-azidopropylamine and MTG were purified and removed using a 1 mL GE Healthcare MabSelect column. The azido-mAb was eluted from the resin using 100 mM sodium citrate (pH 3.0) and then exchanged into 20 mM Hepes, 100 mM NaCl (pH 7.5) using a 7K Zeba desalting column. A 10-fold molar excess of H2bp18c6-benzyl-phenyl-DBCO was reacted with site-specific azide-IgG4 (DOL=2) for 1 hour at 37°C without shaking. The completion of the DBCO-azide click reaction was monitored by intact mass spectrometry. Excess free chelator was removed by desalting the conjugate into 20 mM Hepes, 100 mM NaCl (pH 7.5) on a Zeba7K desalting column, followed by concentration into 20 mM Hepes, 100 mM NaCl (pH 7.5) using three 15-fold serial dilutions and a 30K MWCO Amicon concentrator device by spinning at 3800 x g. This yielded the final site-specific H2bp18c6-benzyl-phenyl-DBCO-IgG4 conjugate with a CAR=2. The final conjugate was confirmed to be monomeric by analytical size exclusion chromatography using a Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 u column, eluting the column with DPBS buffer (x1, calcium and magnesium free), flow rate: 0.7 mL / min, 18 min run, injection volume: 18 μL, at room temperature.

[0349] Labeling: To a solution of NaOAc (3 M in H2O, 20 μL) in a plastic vial, 225Ac(NO3)3 (approximately 5 mCi / mL in 0.1 N HCl, 20 μL, 0.098 mCi) and H2bp18c6-benzyl-phenyl-DBCO-IgG4 (site-specific, CAR=2, 36 μL of 1.7 mg / mL HCl in 20 mM Hepes, 100 mM NaCl (pH 7.5), and 61.2 μg of HCl were added sequentially. After mixing, the pH was approximately 6.5 as determined by pH paper. The reaction solution was left at 37°C for 2 hours. 0.5 μL of the reaction mixture was then loaded onto iTLC-SG, which was developed with 10 mM EDTA. The dried iTLC-SG was left at room temperature overnight and then scanned with a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates to the solvent front with the solvent. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that all Ac-225 was completely chelated in the reaction solution after 2 hours.

[0350] Purification: The reaction mixture was purified on a PD-10 column. The PD-10 resin was conditioned with NaOAc buffer solution by passing 5 mL of 10 mM NaOAc buffer (pH 6-6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluate was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL of 10 mM NaOAc buffer (pH 6-6.5) solution, and the wash solution was pipetted into the PD-10 column reservoir, and the eluate was collected. Each tube contained approximately 1 mL of eluate. Continuous application of NaOAc buffer (10 mM, pH 6-6.5) to the PD-10 column reservoir was performed until a total elution volume of 10 mL was reached.

[0351] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto iTLC-SG, which was developed with 10 mM EDTA and allowed to dry overnight. This was scanned using a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that no free Ac-225 was present in fraction #3.

[0352] HPLC Analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC Method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column, column temperature: room temperature. The column was eluted with DPBS buffer (x1, calcium and magnesium free); flow rate: 0.7 mL / min, 20 min run, injection volume: 30 μL. After HPLC, fractions were collected at 30 s or 1 min time intervals. The collected HPLC fractions were left overnight at room temperature. The radioactivity of each collected fraction was counted in a gamma counter. HPLC radioactivity traces were constructed from the radioactivity in each HPLC fraction. The HPLC radioactivity trace showed a radioactivity peak corresponding to the H2bp18c6-benzyl-phenyl-DBCO-IgG4 peak on the HPLC UV trace.

[0353] Example 4: Preparation of H2bp18c6-benzyl-phenyl-DBCO-PSMB127 and 225 Ac(III) labeling

[0354] [ka] Azide modification and click reaction of mAb: PSMB127 was site-selectively modified with a 100-fold molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37 °C. The addition of two azides onto the heavy chain of the mAb was monitored by intact mass ESI-TOF LC-MS on an Agilent G224 instrument. Excess 3-azidopropylamine and MTG were purified and removed using a 1 mL GE Healthcare MabSelect column. The azido-mAb was eluted from the resin using 100 mM sodium citrate (pH 3.0) and then exchanged into 20 mM Hepes, 100 mM NaCl (pH 7.5) using a 7K Zeba desalting column. A 10-fold molar excess of H2bp18c6-benzyl-phenyl-DBCO was reacted with site-specific azide-PSMB127 (DOL=2) for 1 hour at 37°C without shaking. The completion of the DBCO-azide click reaction was monitored by intact mass spectrometry. Excess free chelator was removed by desalting the conjugate into 20 mM Hepes, 100 mM NaCl (pH 7.5) on a Zeba7K desalting column, followed by concentration into 20 mM Hepes, 100 mM NaCl (pH 7.5) using three 15-fold serial dilutions and a 30K MWCO Amicon concentrator device by spinning at 3800 x g. This yielded the final site-specific H2bp18c6-benzyl-phenyl-DBCO-PSMB127 conjugate with a CAR=2. The final conjugate was confirmed to be monomeric by analytical size exclusion chromatography using a Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 u column, eluting the column with DPBS buffer (x1, calcium and magnesium free), flow rate: 0.7 mL / min, 18 min run, injection volume: 18 μL, at room temperature.

[0355] Labeling: To a solution of NaOAc (3 M in H2O, 20 μL) in a plastic vial, 225Ac(NO3)3 (approximately 5 mCi / mL in 0.1 N HCl, 20 μL, 0.098 mCi) and H2bp18c6-benzyl-phenyl-DBCO-PSMB127 (site-specific, CAR = 2, 2.8 mg / mL in 20 mM Hepes, 100 mM NaCl, pH 7.5, 22 μL, 61.6 μg) were added sequentially. After mixing, the pH was approximately 6.5 as determined by pH paper. The reaction solution was left at 37 °C for 2 h. Then, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC-SG column was left at room temperature overnight and then scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that all Ac-225 was completely chelated in the reaction solution after 2 h.

[0356] Purification: The reaction mixture was purified on a PD-10 column. The PD-10 resin was conditioned with NaOAc buffer solution by passing 5 mL of 10 mM NaOAc buffer (pH 6-6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluate was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL of 10 mM NaOAc buffer (pH 6-6.5) solution, and the wash solution was pipetted into the PD-10 column reservoir, and the eluate was collected. Each tube contained approximately 1 mL of eluate. Continuous application of NaOAc buffer (10 mM, pH 6-6.5) to the PD-10 column reservoir was performed until a total elution volume of 10 mL was reached.

[0357] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto iTLC-SG, which was developed with 10 mM EDTA and allowed to dry overnight. This was scanned using a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that no free Ac-225 was present in fraction #3.

[0358] HPLC Analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC Method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column, column temperature: room temperature. The column was eluted with DPBS buffer (x1, calcium and magnesium free), flow rate: 0.7 mL / min, 20 min run, injection volume: 30 μL. After HPLC, fractions were collected at 30 s or 1 min time intervals. The collected HPLC fractions were left overnight at room temperature. The radioactivity of each collected fraction was counted in a gamma counter. HPLC radioactivity traces were constructed from the radioactivity in each HPLC fraction. The HPLC radioactivity traces showed a radioactive peak corresponding to the H2bp18c6-benzyl-phenyl-DBCO-PSMB127 peak on the HPLC UV trace.

[0359] Example 5: Preparation of H2bp18c6-benzyl-phenyl-DBCO-pertuzumab and 225 Ac(III) labeling

[0360] [ka] Azide modification and click reaction of mAb: Pertuzumab was site-selectively modified with a 100-fold molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37°C. The addition of two azides onto the heavy chain of the mAb was monitored by intact mass ESI-TOF LC-MS on an Agilent G224 instrument. Excess 3-azidopropylamine and MTG were purified and removed using a 1 mL GE Healthcare MabSelect column. The azido-mAb was eluted from the resin using 100 mM sodium citrate (pH 3.0) and then exchanged into 1x dPBS using a 7K Zeba desalting column. A 10-fold molar excess of H2bp18c6-benzyl-phenyl-DBCO was reacted with site-selective azido-pertuzumab (DOL=2) in 1x dPBS at 37°C for 1 hour. The completion of the DBCO-azide click reaction was monitored by intact mass spectrometry. Excess free chelator was removed by desalting the conjugate into 1x dPBS on a Zeba7K desalting column, followed by concentration into PBS by three 15-fold serial dilutions and spinning at 3800xg using a 30K MWCO Amicon concentrator device. This yielded the final site-specific H2bp18c6-benzyl-phenyl-DBCO-pertuzumab conjugate with a CAR of 2. Analytical size-exclusion chromatography was performed on a Tosoh TSKgel G3000SWxl 7.8mm x 30cm, 5u column. The final conjugate was confirmed to be monomeric by eluting the column with 1x DPBS buffer (calcium- and magnesium-free) at room temperature, a flow rate of 0.7mL / min, an 18-minute run, and an injection volume of 18µL.

[0361] Labeling: To a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial, 225Ac(NO3)3 (approximately 5 mCi / mL in 0.1 N HCl, 10 μL, 0.042 mCi) and H2bp18c6-benzyl-phenyl-DBCO-pertuzumab (site-specific, CAR=2, 2.4 mg / mL in PBS buffer, 12.5 μL, 30 μg) were added sequentially. After mixing, the pH was approximately 6.5 as determined by pH paper. The reaction solution was left at 37 °C for 2 hours. Then, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC-SG column was left at room temperature overnight and then scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that all Ac-225 was completely chelated in the reaction solution after 2 h.

[0362] Purification: The reaction mixture was purified on a PD-10 column. The PD-10 resin was conditioned with NaOAc buffer solution by passing 5 mL of 10 mM NaOAc buffer (pH 6-6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluate was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL of 10 mM NaOAc buffer (pH 6-6.5) solution, and the wash solution was pipetted into the PD-10 column reservoir, and the eluate was collected. Each tube contained approximately 1 mL of eluate. Continuous application of NaOAc buffer (10 mM, pH 6-6.5) to the PD-10 column reservoir was performed until a total elution volume of 10 mL was reached.

[0363] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto iTLC-SG, which was developed with 10 mM EDTA and allowed to dry overnight. This was scanned using a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that no free Ac-225 was present in fraction #3.

[0364] HPLC Analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC Method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column, column temperature: room temperature. The column was eluted with DPBS buffer (x1, calcium and magnesium free), flow rate: 0.7 mL / min, 20 min run, injection volume: 30 μL. After HPLC, fractions were collected at 30 s or 1 min time intervals. The collected HPLC fractions were left overnight at room temperature. The radioactivity of each collected fraction was counted in a gamma counter. HPLC radioactivity traces were constructed from the radioactivity in each HPLC fraction. The HPLC radioactivity traces showed radioactive peaks corresponding to the H2bp18c6-benzyl-phenyl-DBCO-pertuzumab peak on the HPLC UV trace.

[0365] Example 6: Preparation of H2bp18c6-benzyl-phenyl-DBCO-cetuximab and 225 Ac(III) labeling

[0366] [ka] Azide modification and click reaction of mAb: Cetuximab was site-selectively modified with a 100-fold molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37°C. The addition of two azides onto the heavy chain of the mAb was monitored by intact mass ESI-TOF LC-MS on an Agilent G224 instrument. Excess 3-azidopropylamine and MTG were purified and removed using a 1 mL GE Healthcare MabSelect column. The azido-mAb was eluted from the resin using 100 mM sodium citrate (pH 3.0) and then exchanged into 1x dPBS using a 7K Zeba desalting column. A 10-fold molar excess of H2bp18c6-benzyl-phenyl-DBCO was reacted with site-selective azido-cetuximab (DOL=2) in 1x dPBS at 37°C for 1 hour. The completion of the DBCO-azide click reaction was monitored by intact mass spectrometry. Excess free chelator was removed by desalting the conjugate into 1x dPBS on a Zeba7K desalting column, followed by concentration into PBS by three 15-fold serial dilutions and spinning at 3800xg using a 30K MWCO Amicon concentrator device. This afforded the final site-specific H2bp18c6-benzyl-phenyl-DBCO-cetuximab conjugate with a CAR of 2. The final conjugate was confirmed to be monomeric by analytical size-exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8mm x 30cm, 5u column, eluting with DPBS buffer (1x, calcium- and magnesium-free), flow rate: 0.7mL / min, 18min run, injection volume: 18µL, at room temperature.

[0367] Labeling: To a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial, 225Ac(NO3)3 (approximately 5 mCi / mL in 0.1 N HCl, 10 μL, 0.044 mCi) and H2bp18c6-benzyl-phenyl-DBCO-cetuximab (site-specific, CAR=2, 1.8 mg / mL in PBS buffer, 16.7 μL, 30 μg) were added sequentially. After mixing, the pH was approximately 6.5 as determined by pH paper. The reaction solution was left at 37 °C for 2 hours. 0.5 μL of the reaction mixture was then loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC-SG column was left at room temperature overnight and then scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that all Ac-225 was completely chelated in the reaction solution after 2 h.

[0368] Purification: The reaction mixture was purified on a PD-10 column. The PD-10 resin was conditioned with NaOAc buffer solution by passing 5 mL of 10 mM NaOAc buffer (pH 6-6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluate was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL of 10 mM NaOAc buffer (pH 6-6.5) solution, and the wash solution was pipetted into the PD-10 column reservoir, and the eluate was collected. Each tube contained approximately 1 mL of eluate. Continuous application of NaOAc buffer (10 mM, pH 6-6.5) to the PD-10 column reservoir was performed until a total elution volume of 10 mL was reached.

[0369] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto iTLC-SG, which was developed with 10 mM EDTA and allowed to dry overnight. This was scanned using a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that no free Ac-225 was present in fraction #3.

[0370] HPLC Analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC Method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column, column temperature: room temperature. The column was eluted with DPBS buffer (x1, calcium and magnesium free), flow rate: 0.7 mL / min, 20 min run, injection volume: 30 μL. After HPLC, fractions were collected at 30 s or 1 min time intervals. The collected HPLC fractions were left overnight at room temperature. The radioactivity of each collected fraction was counted in a gamma counter. HPLC radioactivity traces were constructed from the radioactivity in each HPLC fraction. The HPLC radioactivity traces showed radioactive peaks corresponding to the H2bp18c6-benzyl-phenyl-DBCO-cetuximab peak on the HPLC UV trace.

[0371] Example 7: Preparation of H2bp18c6-benzyl-phenyl-DBCO-panitumumab and 225 Ac(III) labeling

[0372] [ka] Azide modification and click reaction of mAb: Panitumumab was site-selectively modified with a 100-fold molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37°C. The addition of two azides onto the heavy chain of the mAb was monitored by intact mass ESI-TOF LC-MS on an Agilent G224 instrument. Excess 3-azidopropylamine and MTG were purified and removed using a 1 mL GE Healthcare MabSelect column. The azido-mAb was eluted from the resin using 100 mM sodium citrate (pH 3.0) and then exchanged into 1x dPBS using a 7K Zeba desalting column. A 10-fold molar excess of H2bp18c6-benzyl-phenyl-DBCO was reacted with site-selective azido-panitumumab (DOL=2) in 1x dPBS at 37°C for 1 hour. The completion of the DBCO-azide click reaction was monitored by intact mass spectrometry. Excess free chelator was removed by desalting the conjugate into 1x dPBS on a Zeba7K desalting column, followed by concentration into PBS by three 15-fold serial dilutions and spinning at 3800xg using a 30K MWCO Amicon concentrator device. This yielded the final site-specific H2bp18c6-benzyl-phenyl-DBCO-panitumumab conjugate with a CAR of 2. The column was then loaded with DPBS buffer (1x, calcium and magnesium free) on a Tosoh TSKgel G3000SWxl 7.8mm x 30cm, 5u column at room temperature, with a flow rate of 0.7mL / min, 18min run, and an injection volume of 18µL. Analytical size exclusion chromatography eluting at 1000 s confirmed that the final conjugate was monomeric.

[0373] Labeling: To a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial, 225Ac(NO3)3 (approximately 5 mCi / mL in 0.1 N HCl, 10 μL, 0.043 mCi) and H2bp18c6-benzyl-phenyl-DBCO-panitumumab (site-specific, CAR=2, 2.6 mg / mL in PBS buffer, 11.5 μL, 30 μg) were added sequentially. After mixing, the pH was approximately 6.5 as determined by pH paper. The reaction solution was left at 37°C for 2 hours. Then, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC-SG column was left at room temperature overnight and then scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that all Ac-225 was completely chelated in the reaction solution after 2 h.

[0374] Purification: The reaction mixture was purified on a PD-10 column. The PD-10 resin was conditioned with NaOAc buffer solution by passing 5 mL of 3x NaOAc buffer (10 mM, pH 6-6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluate was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL of 3x NaOAc buffer (10 mM, pH 6-6.5), and the wash solution was pipetted into the PD-10 column reservoir, and the eluate was collected. Each tube contained approximately 1 mL of eluate. Continuous application of NaOAc buffer (10 mM, pH 6-6.5) to the PD-10 column reservoir was performed until a total elution volume of 10 mL was reached.

[0375] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto iTLC-SG, which was developed with 10 mM EDTA and allowed to dry overnight. This was scanned using a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that no free Ac-225 was present in fraction #3.

[0376] HPLC Analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC Method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column, column temperature: room temperature. The column was eluted with DPBS buffer (x1, calcium and magnesium free) at a flow rate of 0.7 mL / min for 20 minutes with an injection volume of 30 μL. After HPLC, fractions were collected at 30 second or 1 minute time intervals. The collected HPLC fractions were left overnight at room temperature. The radioactivity of each collected fraction was counted in a gamma counter. HPLC radioactivity traces were constructed from the radioactivity in each HPLC fraction. The HPLC radioactivity traces showed radioactive peaks corresponding to the H2bp18c6-benzyl-phenyl-DBCO-panitumumab peak on the HPLC UV trace.

[0377] Example 8: Preparation of H2bp18c6-benzyl-phenyl-DBCO-Herceptin and 225 Ac(III) labeling

[0378] [ka] Azide modification and click reaction of mAb: Herceptin was site-selectively modified with a 100-fold molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37°C. The addition of two azides onto the heavy chain of the mAb was monitored by intact mass ESI-TOF LC-MS on an Agilent G224 instrument. Excess 3-azidopropylamine and MTG were purified and removed using a 1 ml GE Healthcare MabSelect column. The azido-mAb was eluted from the resin using 100 mM sodium citrate (pH 3.0) and then exchanged into 1x dPBS using a 7K Zeba desalting column. A 10-fold molar excess of H2bp18c6-benzyl-phenyl-DBCO was reacted with site-selective azido-Herceptin (DOL=2) in 1x dPBS at 37°C for 1 hour. The completion of the DBCO-azide click reaction was monitored by intact mass spectrometry. Excess free chelator was removed by desalting the conjugate into 1x dPBS on a Zeba7K desalting column, followed by concentration into PBS by three 15-fold serial dilutions and spinning at 3800xg using a 30K MWCO Amicon concentrator device. This yielded the final site-specific H2bp18c6-benzyl-phenyl-DBCO-Herceptin conjugate with a CAR of 2. The final conjugate was confirmed to be monomeric by analytical size-exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8mm x 30cm, 5u column, eluting with DPBS buffer (1x, calcium- and magnesium-free), flow rate: 0.7mL / min, 18min run, injection volume: 18µL, at room temperature.

[0379] Labeling: To a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial, 225Ac(NO3)3 (approximately 5 mCi / mL in 0.1 N HCl, 10 μL, 0.041 mCi) and H2bp18c6-benzyl-phenyl-DBCO-Herceptin (site-specific, CAR=2, 1.7 mg / mL in PBS buffer, 17.6 μL, 30 μg) were added sequentially. After mixing, the pH was approximately 6.5 as determined by pH paper. The reaction solution was left at 37 °C for 2 hours. Then, 0.5 μL of the reaction mixture was loaded onto iTLC-SG, which was developed with 10 mM EDTA. The dried iTLC-SG was left at room temperature overnight and then scanned with a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrated to the solvent front along with the solvent. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that all Ac-225 was completely chelated in the reaction solution after 2 hours.

[0380] Purification: The reaction mixture was purified on a PD-10 column. The PD-10 resin was conditioned with NaOAc buffer solution by passing 5 mL of 10 mM NaOAc buffer (pH 6-6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluate was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL of 10 mM NaOAc buffer (pH 6-6.5) solution, and the wash solution was pipetted into the PD-10 column reservoir, and the eluate was collected. Each tube contained approximately 1 mL of eluate. Continuous application of NaOAc buffer (10 mM, pH 6-6.5) to the PD-10 column reservoir was performed until a total elution volume of 10 mL was reached.

[0381] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto iTLC-SG, which was developed with 10 mM EDTA and allowed to dry overnight. This was scanned using a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that no free Ac-225 was present in fraction #3.

[0382] HPLC Analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC Method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column, column temperature: room temperature. The column was eluted with DPBS buffer (x1, calcium and magnesium free), flow rate: 0.7 mL / min, 20 min run, injection volume: 30 μL. After HPLC, fractions were collected at 30 s or 1 min time intervals. The collected HPLC fractions were left overnight at room temperature. The radioactivity of each collected fraction was counted in a gamma counter. HPLC radioactivity traces were constructed from the radioactivity in each HPLC fraction. The HPLC radioactivity traces showed radioactive peaks corresponding to the H2bp18c6-benzyl-phenyl-DBCO-Herceptin peak on the HPLC UV trace.

[0383] Example 9: Preparation of H2bp18c6-benzyl-phenyl-DBCO-H11B6 and 225 Ac(III) labeling

[0384] [ka] Azide modification and click reaction of mAb: H11B6 was site-selectively modified with a 100-fold molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37°C. The addition of two azides onto the heavy chain of the mAb was monitored by intact mass ESI-TOF LC-MS on an Agilent G224 instrument. Excess 3-azidopropylamine and MTG were purified and removed using a 1 ml GE Healthcare MabSelect column. The azido-mAb was eluted from the resin using 100 mM sodium citrate (pH 3.0) and then exchanged into 1x dPBS using a 7K Zeba desalting column. A 10-fold molar excess of H2bp18c6-benzyl-phenyl-DBCO was reacted with the site-selective azido-H11B6 (DOL = 1.82) in 1x dPBS at 37°C for 1 hour. The completion of the DBCO-azide click reaction was monitored by intact mass spectrometry. Excess free chelator was removed by desalting the conjugate into 1x dPBS on a Zeba7K desalting column, followed by concentration into PBS by three 15-fold serial dilutions and spinning at 3800xg using a 30K MWCO Amicon concentrator device. This yielded the final site-specific H2bp18c6-benzyl-phenyl-DBCO-H11B6 conjugate with a CAR of 1.82. The final conjugate was confirmed to be monomeric by analytical size-exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8mm x 30cm, 5u column eluted with DPBS buffer (1x, calcium- and magnesium-free), flow rate: 0.7mL / min, 18min run, injection volume: 18µL, at room temperature.

[0385] Labeling: To a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial, 225Ac(NO3)3 (approximately 5 mCi / mL in 0.1 N HCl, 10 μL, 0.043 mCi) and H2bp18c6-phenyl-DBCO-H11B6 (site-specific, CAR = 1.82, 1.2 mg / mL in PBS buffer, 25.0 μL, 30 μg) were added. After mixing, the pH was approximately 6.5 as determined by pH paper. The reaction solution was left at 37 °C for 2 h. Then, 0.5 μL of the reaction mixture was loaded onto iTLC-SG, which was developed with 10 mM EDTA. The dried iTLC-SG was left at room temperature overnight and then scanned with a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrated to the solvent front along with the solvent. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that all Ac-225 was completely chelated in the reaction solution after 2 h.

[0386] Purification: The reaction mixture was purified on a PD-10 column. The PD-10 resin was conditioned with NaOAc buffer solution by passing 5 mL of 10 mM NaOAc buffer (pH 6-6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluate was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL of 10 mM NaOAc buffer (pH 6-6.5) solution, and the wash solution was pipetted into the PD-10 column reservoir, and the eluate was collected. Each tube contained approximately 1 mL of eluate. Continuous application of NaOAc buffer (10 mM, pH 6-6.5) to the PD-10 column reservoir was performed until a total elution volume of 10 mL was reached.

[0387] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto iTLC-SG, which was developed with 10 mM EDTA and allowed to dry overnight. This was scanned using a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that no free Ac-225 was present in fraction #3.

[0388] HPLC Analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC Method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column, column temperature: room temperature. The column was eluted with DPBS buffer (x1, calcium and magnesium free), flow rate: 0.7 mL / min, 20 min run, injection volume: 30 μL. After HPLC, fractions were collected at 30 s or 1 min time intervals. The collected HPLC fractions were left overnight at room temperature. The radioactivity of each collected fraction was counted in a gamma counter. HPLC radioactivity traces were constructed from the radioactivity in each HPLC fraction. The HPLC radioactivity trace showed a radioactive peak corresponding to the H2bp18c6-benzyl-phenyl-DBCO-H11B6 peak on the HPLC UV trace.

[0389] Example 10: Synthesis of H2bp18c6-phenyl-BCN

[0390] [ka] To a solution of 4-((6-(methoxycarbonyl)pyridin-2-yl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)benzoic acid (50 mg, 0.073 mmol) and EtN (0.1 mL, 0.73 mmol) in CHCl (2.5 mL) was added HATU (37 mg, 0.10 mmol) at 0 °C. The solution was stirred at 0 °C for 5 min, and then N-[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl]-1,8-diamino-3,6-dioxaoctane in CHCl (1 mL) was added. The cooling bath was removed and the mixture was stirred at room temperature for 18 h. The mixture was concentrated, and the residue was purified by chromatography on amine-functionalized silica gel (CH2Cl2 to 10% MeOH in CH2Cl2) to give 19.8 mg (27% yield) of product as a colorless film attached to the flask wall. LC-MS analysis showed 987.6 [M+H + ] indicates the mass / ion peaks.

[0391] To a solution of 6-((4-((1-((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecan-12-yl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (14.5 mg, 0.015 mmol) in THF / MeOH / HO (4:1:1 v / v / v, 1.8 mL) at room temperature was added LiOH (1 N, 0.3 mL). The reaction mixture was stirred at room temperature for 1 hour, after which it was neutralized to pH=6.5 with HCl (1 N). The reaction mixture was concentrated in vacuo. The residue was purified by chromatography on amine-functionalized silica gel (CHCl to approximately 10% MeOH in CHCl) to give 9.5 mg (48% yield) of Hbp18c6-benzyl-phenyl-BCN as a colorless film, which was dissolved in HO (4 mL) and ACN (1 mL). After lyophilization, this gave the product as a white solid. H NMR (CDOD, 400 MHz) δ 7.91(d,J=8Hz,2H), 7.84(t,J=8Hz,2H), 7.81(d,J=8Hz,2H), 7.60(d,J=8Hz,2H), 7.53( d,J=8Hz,1H), 7.46(d,J=8Hz,1H), 5.24(s,1H), 4.10(d,J=8Hz,2H), 3.91(brs,2H), 3.7 5-3.58(m,22H), 3.56(t,J=4Hz,2H), 3.51(t,J=4Hz,2H), 3.23(t,J=4Hz,2H), 2.97(dt, J=8,4Hz,2H), 2.92-2.82(m,4H), 2.78(dt,J=8,4Hz,2H), 2.28-2.08(m,6H), 1.65-1.50 9m,2H), 1.39-1.27(m,1H), 0.96-0.84(m,2H). MS(ESI)981.4[M+Na + ].

[0392] Example 11: Preparation of H2bp18c6-benzyl-phenyl-BCN-PSMB127 and 225 Ac(III) labeling

[0393] [ka] Azide modification and click reaction of mAb: PSMB127 was site-selectively modified with a 100-fold molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37°C. The addition of two azides onto the heavy chain of the mAb was monitored by intact mass ESI-TOF LC-MS on an Agilent G224 instrument. Excess 3-azidopropylamine and MTG were purified and removed using a 1 mL GE Healthcare MabSelect column. The azido-mAb was eluted from the resin using 100 mM sodium citrate (pH 3.0) and then exchanged into 1x dPBS using a 7K Zeba desalting column. A 10-fold molar excess of H2bp18c6-benzyl-phenyl-BCN was reacted with the site-selective azido-PSMB127 (DOL=2) in 1x dPBS at 37°C for 1 hour without shaking. The completion of the BCN-azide click reaction was monitored by intact mass spectrometry. Excess free chelator was removed by desalting the conjugate into 1x dPBS on a Zeba7K desalting column, followed by concentration into PBS by three 15-fold serial dilutions and spinning at 3800xg using a 30K MWCO Amicon concentrator device. This yielded the final site-specific H2bp18c6-benzyl-phenyl-BCN-PSMB127 conjugate with a CAR of 2. The final conjugate was confirmed to be monomeric by analytical size-exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8mm x 30cm, 5u column, eluting with 1x DPBS buffer (calcium- and magnesium-free) at room temperature, a flow rate of 0.7mL / min, an 18-minute run, and an injection volume of 18µL.

[0394] Labeling: To a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial, 225Ac(NO3)3 (approximately 5 mCi / mL in 0.1 N HCl, 10 μL, 0.046 mCi) and H2bp18c6-benzyl-phenyl-BCN-PSMB127 (site-specific, CAR = 2, 2.3 mg / mL in PBS buffer, 13.0 μL, 30 μg) were added sequentially. After mixing, the pH was approximately 6.5 as determined by pH paper. The reaction solution was left at 37 °C for 2 h. Then, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC-SG column was left at room temperature overnight and then scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that all Ac-225 was completely chelated in the reaction solution after 2 h.

[0395] Purification: The reaction mixture was purified on a PD-10 column. The PD-10 resin was conditioned with NaOAc buffer solution by passing 5 mL of 10 mM NaOAc buffer (pH 6-6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluate was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL of 10 mM NaOAc buffer (pH 6-6.5) solution, and the wash solution was pipetted into the PD-10 column reservoir, and the eluate was collected. Each tube contained approximately 1 mL of eluate. Continuous application of NaOAc buffer (10 mM, pH 6-6.5) to the PD-10 column reservoir was performed until a total elution volume of 10 mL was reached.

[0396] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto iTLC-SG, which was developed with 10 mM EDTA and allowed to dry overnight. This was scanned using a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that no free Ac-225 was present in fraction #3.

[0397] HPLC Analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC Method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column, column temperature: room temperature. The column was eluted with DPBS buffer (x1, calcium and magnesium free), flow rate: 0.7 mL / min, 20 min run, injection volume: 40 μL. After HPLC, fractions were collected at 30 s or 1 min time intervals. The collected HPLC fractions were left overnight at room temperature. The radioactivity of each collected fraction was counted in a gamma counter. HPLC radioactivity traces were constructed from the radioactivity in each HPLC fraction. The HPLC radioactivity traces showed a radioactive peak corresponding to the H2bp18c6-benzyl-phenyl-BCN-PSMB127 peak on the HPLC UV trace.

[0398] Example 12: Synthesis of H2bp18c6-off macrocycle-ethyl sulfide-DBCO H2bp18c6 derivatives substituted at the carbon atoms of the macrocycle bearing linkers for conjugation to targeting ligands are generally synthesized according to Scheme 14a and Scheme 14b below:

[0399] [ka]

[0400] The synthesis of -CH2OR1 substituted diaza-18-crown-6 is performed using the procedure described in Org. Lett. 2005, 7(6), 1105-1108, followed by reaction with a derivative of 6-(halomethyl)picolinic acid as described above with respect to Schemes 3 and 4. The functional group OR1 can be converted to another functional group WR2, including but not limited to NH2, N3, aldehyde, carboxylate, alkyne, etc., with or without a linker W, which may be a heteroatom, alkyl, alkyl with a heteroatom, substituted aryl, etc., for ligation or conjugation to a linker, targeting ligand, etc. In Scheme 6a, X is a leaving group such as halo, mesylate, tosylate, etc.; R is allyl, benzyl, alcohol, etc.; W is absent or a linker such as a heteroatom, alkyl, heteroalkyl, substituted aryl, etc.; and R is NH, N aldehyde, carboxylate, alkynyl, etc.

[0401] [ka]

[0402] Diaza-18-crown-6 substituted with -CH2OR1 can be synthesized using the procedures described in Journal of Organic Chemistry, 1988, 53(14), 3190-5 and Journal of Heterocyclic Chemistry, 1986, 23(2), 609-13, followed by reaction with a derivative of 6-(halomethyl)picolinic acid as described above with respect to Schemes 3 and 4. The functional group OR1 can be converted to another functional group R2, including but not limited to NH2, N3, aldehyde, carboxylate, alkyne, etc., with or without a linker W, which can be a heteroatom, alkyl, alkyl with a heteroatom, substituted aryl, etc., for ligation or conjugation to a linker, targeting ligand, etc.

[0403] Next, the synthesis of H2bp18c6-off macrocycle-ethyl sulfide-DBCO is described.

[0404] [ka]

[0405] Sodium hydride (18.16 g, 60% in mineral oil, 454 mmol) was added to a solution of (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol (50 g, 378 mmol) in anhydrous DMF (500 mL) at 0 °C. After stirring at this temperature for 10 min, benzyl bromide (77.63 g, 454 mmol) was added slowly to the suspension. The reaction was allowed to reach room temperature after 30 min and stirred for an additional 8 h. The reaction mixture was quenched with saturated ammonium chloride solution and extracted with dichloromethane (2 × 500 mL). The combined organic layers were washed with brine and dried over sodium sulfate. After removal of the solvent, the residue was purified by flash column chromatography (silica gel, 230-400 mesh) using petroleum ether and ethyl acetate to give (S)-4-((benzyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (72 g, 86%) as a colorless liquid.

[0406] To a solution of (S)-4-((benzyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (72 g, 324 mmol) in THF (100 mL) was added 1.5 N HCl (100 mL), followed by stirring at room temperature for 16 h. The reaction mixture was diluted with ethyl acetate (500 mL) and neutralized with 10% sodium bicarbonate solution. The reaction mixture was extracted with ethyl acetate (2 × 500 mL), washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 230-400 mesh) using petroleum ether and ethyl acetate to give (R)-3-(benzyloxy)propane-1,2-diol (51 g, 85%) as a colorless oil.

[0407] To a suspension of sodium hydride (26.29 g, 60% in mineral oil, 686 mmol) in DMF (20 mL) was added (R)-3-(benzyloxy)propane-1,2-diol (25.0 g, 137 mmol) in DMF (100 mL) dropwise at 0° C. The reaction mixture was stirred at room temperature for 2 hours. It was cooled again to 0° C., and 2-(2-bromoethoxy)tetrahydro-2H-pyran (85.89 g, 411 mmol) in DMF (100 mL) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred for 16 hours. The reaction was quenched with saturated ammonium chloride and extracted with ethyl acetate (3×300 mL). The combined organic layers were washed with water, dried over sodium sulfate, filtered, and concentrated. The crude oil was purified by silica gel (230-400 mesh) flash chromatography using ethyl acetate (0-40%) in petroleum ether to give 2,2'-(((((R)-3-(benzyloxy)propane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(tetrahydro-2H-pyran) (32 g, 53%) as a colorless liquid.

[0408] To a solution of 2,2'-(((((R)-3-(benzyloxy)propane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(tetrahydro-2H-pyran) (32 g, 73.05 mmol) in 500 mL of methanol was added 5 mL of HCl in dioxane. The reaction was stirred at reflux for 1 h, cooled, and evaporated. The crude product (R)-2,2'-((3-(benzyloxy)propane-1,2-diyl)bis(oxy))bis(ethan-1-ol) (20 g) was used in the next step without purification.

[0409] To a solution of (R)-2,2'-((3-(benzyloxy)propane-1,2-diyl)bis(oxy))bis(ethan-1-ol) (20 g, 74.07 mmol) in dichloromethane (250 mL) and triethylamine (53 mL, 370 mmol) at 10°C was added solid p-toluenesulfonyl chloride (42.2 g, 222 mmol) in portions. The mixture was stirred at ambient temperature for 16 h. After completion of the reaction, the suspension was diluted with 1000 mL of dichloromethane and washed with cold 1 M HCl (3 x 100 mL), followed by 2 x 500 mL of ice-cold water, dried over sodium sulfate, and evaporated to a solid gum. The crude material was purified by flash chromatography (silica gel, 230-400 mesh) using ethyl acetate in petroleum ether (0-40%) to give (R)-((3-(benzyloxy)propane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (30 g, 70%) as a colorless liquid.

[0410] A mixture of (R)-((3-(benzyloxy)propane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (30 g, 51.9 mmol) and cesium carbonate (50.76 g, 155.7 mmol) in 200 mL of dry DMF was stirred at ambient temperature for 1.5 hours. To the suspension was added N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(4-methylbenzenesulfonamide) (23.56 g, 51.9 mmol) in 200 mL of DMF dropwise over 2 hours. The mixture was stirred at ambient temperature for an additional 20 hours. The solvent was removed under reduced pressure to give a solid paste, which was suspended in 1000 mL of dichloromethane and stirred for 30 minutes. The precipitated solid was filtered off, and the filtrate was evaporated under high vacuum. The crude material was purified by flash chromatography (silica gel, 230-400 mesh) using ethyl acetate in petroleum ether (0-40%) to give (R)-2-((benzyloxy)methyl)-7,16-ditosyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (24 g, 67%) as a colorless liquid.

[0411] To a solution of (R)-2-((benzyloxy)methyl)-7,16-ditosyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (24 g, 34.78 mmol) in acetic acid (50%, 100 mL) was added phenol (16.35 g, 174 mmol) at room temperature. The reaction was heated at 60 °C for 6 h. After completion of the reaction, it was cooled to room temperature and the acetic acid was removed under high vacuum. The crude product was purified on a reverse-phase column using 0-100% acetonitrile in water (0.1% TFA) to give (S)-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2-yl)methyl acetate (8.0 g, 69%) as a colorless liquid.

[0412] A suspension of (S)-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2-yl)methyl acetate (8.0 g, 23.95 mmol), methyl 6-(chloromethyl)picolinate (11.07 g, 59.88 mmol), and sodium carbonate (12.69 g, 119.75 mmol) in dry acetonitrile (100 mL) was heated at 90° C. for 16 h. After completion of the reaction, the reaction mixture was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (silica gel, 230-400 mesh) using methanol (0-10%) in dichloromethane to give dimethyl 6,6'-((2-(acetoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (5.0 g, 33%) as a brown liquid.

[0413] To a solution of 6,6'-((2-(acetoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene)) (S)-dipicolinate (5.0 g, 7.91 mmol) in methanol (50 mL), potassium carbonate (0.11 g, 0.79 mmol) was added at room temperature and stirred for 10 minutes. After completion of the reaction, it was concentrated under reduced pressure. The residue was purified by flash column chromatography (230-400 mesh) eluting with a gradient of 0-10% methanol in dichloromethane to give 6,6'-((2-(hydroxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene)) (R)-dipicolinate (3.5 g, 75%) as a brown liquid.

[0414] To a solution of dimethyl 6,6'-((2-(hydroxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(R)-dipicolinate (1.0 g, 1.7 mmol) in dichloromethane (20 mL) was added triethylamine (0.51 g, 0.70 mL, 5.1 mmol). Mesyl chloride (0.39 g, 0.26 mL, 3.4 mmol) was added dropwise to this solution at 0°C. The reaction was stirred at room temperature for 30 minutes. The progress of the reaction was monitored by TLC. After completion of the reaction, it was concentrated and the crude product was purified by column chromatography (alumina-neutral) using methanol (1-2%) in dichloromethane as the eluent to give dimethyl 6,6'-((2-(((methylsulfonyl)oxy)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (0.7 g, 62%) as a brown liquid.

[0415] To a solution of tert-butyl (2-mercaptoethyl)carbamate (53 mg, 0.3 mmol) in DMF (2 mL) was added sodium hydride (12 mg, 60% in mineral oil, 0.3 mmol) at 0° C. The reaction mixture was stirred at room temperature for 10 minutes. To the reaction mixture was added dimethyl 6,6′-((2-(((methylsulfonyl)oxy)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (100 mg, 0.15 mmol) in DMF (1 mL) at 0° C. The reaction was stirred at room temperature for 2 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was quenched with saturated ammonium chloride solution and extracted with ethyl acetate (3×5 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and filtered. The crude product was purified by preparative HPLC using acetonitrile in water (0.1% TFA) to give dimethyl 6,6'-((2-(((2-((tert-butoxycarbonyl)amino)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (20 mg, 18%) as a brown liquid.

[0416] A cold solution of dimethyl 6,6'-((2-(((2-((tert-butoxycarbonyl)amino)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (100 mg, 0.15 mmol) and HCl in methanol (2 mL, 4 N) was added and the solution was stirred for 2 h. The reaction mixture was concentrated under reduced pressure to give dimethyl 6,6'-((2-(((2-aminoethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate as a yellow liquid (55 mg, 64%).

[0417] To a solution of dimethyl 6,6'-((2-(((2-aminoethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (60 mg, 0.09 mmol) in dichloromethane (0.5 mL) at 0 °C, triethylamine (0.04 mL, 0.27 mmol) was added, followed by HATU (51 mg, 0.13 mmol). The solution was stirred at 0 °C for 5 min, followed by DBCO-acid (27 mg, 0.09 mmol) in dichloromethane (0.5 mL). The cooling bath was removed and the mixture was stirred at room temperature for 18 h. Water was added to the reaction mixture, which was then extracted with dichloromethane (2 mL × 3). The combined extracts were washed with saturated aqueous NaHCO3, brine, dried over sodium sulfate, and filtered. The solution was concentrated to give the crude product, which was purified by chromatography on silica gel with dichloromethane and methanol to give the dimethyl ester of H2bp18c6-off macrocycle-ethylsulfide-DBCO (20 mg, 23%) as a colorless liquid.

[0418] To a solution of H2bp18c6-off macrocycle-ethyl sulfide-DBCO (20 mg, 0.02 mmol) in methanol (0.5 mL) at room temperature was added LiOH (0 . To the resulting solution was added 64 mL of 0.1N acetic acid (0.06 mmol). After stirring at room temperature for 16 hours, it was neutralized with acetic acid to pH 6.5. The reaction mixture was concentrated on an evaporator at room temperature to remove volatile solvents. The residue was purified by preparative HPLC to give H2bp18c6-off macrocycle-ethyl sulfide-DBCO (6 mg, 31%) as an off-white solid. LC-MS APCI: calculated for C48H56N6O10S 909.07; observed m / z [M+H] + 909.4. Purity by LC-MS: 92.92% RT: 1.86. Purity by HPLC: 91.56% RT: 3.87. 1H NMR(400MHz,D2O):δ 7.83-7.76(m,4H), 7.54-7.17(m,10H), 4.97-4.90(m,1H), 4.80(s,4H), 4.23(s,4H), 3.78- 3.42(m,18H), 3.04-3.00(m,2H), 2.51-2.39(m,3H), 2.31-2.29(m,2H), 2.10-2.04(m,3H).

[0419] Example 13: Modulation and synthesis of H2bp18c6-Efda Ring-Etacron-DBCO-PSMB127 225 Ac(III) labeling

[0420]

change

[0421] Labeling: To a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial, 225Ac(NO3)3 (approximately 5 mCi / mL in 0.1 N HCl, 10 μL, 0.048 mCi) and H2bp18c6-off macrocycle-ethyl sulfide-DBCO-PSMB127 (site-specific, CAR = 2, 2.4 mg / mL in PBS buffer, 10 μL, 24 μg) were added sequentially. After mixing, the pH was approximately 6.5 as determined by pH paper. The reaction solution was left at 37 °C for 2 h. Then, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC-SG column was left at room temperature overnight and then scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that all Ac-225 was completely chelated in the reaction solution after 2 h.

[0422] Purification: The reaction mixture was purified on a PD-10 column. The PD-10 resin was conditioned with NaOAc buffer solution by passing 5 mL of 10 mM NaOAc buffer (pH 6-6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluate was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL of 10 mM NaOAc buffer (pH 6-6.5) solution, and the wash solution was pipetted into the PD-10 column reservoir. The eluate was collected. Each tube contained approximately 1 mL of eluate. Continuous application of NaOAc buffer (10 mM, pH 6-6.5) to the PD-10 column reservoir was performed until a total elution volume of 10 mL was reached.

[0423] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto iTLC-SG, which was developed with 10 mM EDTA and allowed to dry overnight. This was scanned using a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that no free Ac-225 was present in fraction #3.

[0424] HPLC Analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC Method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column, column temperature: room temperature. The column was eluted with DPBS buffer (x1, calcium and magnesium free), flow rate: 0.7 mL / min, 20 min run, injection volume: 40 μL. After HPLC, fractions were collected at 30 s or 1 min time intervals. The collected HPLC fractions were left overnight at room temperature. The radioactivity of each collected fraction was counted in a gamma counter. HPLC radioactivity traces were constructed from the radioactivity in each HPLC fraction. The HPLC radioactivity traces showed a radioactive peak corresponding to the H2bp18c6-off macrocycle-ethyl sulfide-DBCO-PSMB127 peak on the HPLC UV trace.

[0425] Example 14: Synthesis of H2bp18c6-off macrocycle-pentyl sulfide-DBCO

[0426] [ka] To a solution of tert-butyl (5-mercaptopentyl)carbamate (65 mg, 0.3 mmol) in DMF (2 mL) was added sodium hydride (12 mg, 60% in mineral oil, 0.3 mmol) at 0° C. The resulting reaction mixture was stirred at room temperature for 10 minutes. To the reaction mixture was added dimethyl 6,6′-((2-(((methylsulfonyl)oxy)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (100 mg, 0.15 mmol) in DMF (1 mL) at 0° C. The reaction was stirred at room temperature for 2 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, it was concentrated and the residue was purified by preparative HPLC using acetonitrile and 0.1% TFA to give dimethyl 6,6'-((2-(((5-((tert-butoxycarbonyl)amino)pentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (15 mg, 13%) as a brown liquid.

[0427] Dimethyl 6,6'-((2-(((5-((tert-butoxycarbonyl)amino)pentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (120 mg, 0.15 mmol) was added to a cold solution of HCl in methanol (2 mL, 4N) and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure to give dimethyl 6,6'-((2-(((5-aminopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (70 mg, 66%) as a yellow liquid.

[0428] To a solution of dimethyl 6,6'-((2-(((5-aminopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (50 mg, 0.07 mmol) in dichloromethane (0.5 mL) at 0 °C, triethylamine (0.03 mL, 0.21 mmol) was added, followed by HATU (38 mg, 0.10 mmol). The solution was stirred at 0 °C for 5 min, and then DBCO-acid (21 mg, 0.07 mmol) in dichloromethane (0.5 mL) was added. The cooling bath was removed, and the mixture was stirred at room temperature for 18 h. Water was added to the reaction mixture, which was then extracted with dichloromethane (2 mL × 3). The combined extracts were washed with saturated aqueous NaHCO3, brine, dried over sodium sulfate, and filtered. The solution was concentrated to give the crude product, which was purified by chromatography on silica gel with dichloromethane and methanol to give the dimethyl ester of H2bp18c6-off macrocycle-pentylsulfide-DBCO (16 mg, 23%) as a colorless liquid.

[0429] A solution of H2bp18c6-off macrocycle-pentyl sulfide-DBCO (16 mg, 0.01 mmol) in methanol (0.5 mL) was added to a solution of LiOH (0 . To the resulting solution was added 49 mL of 0.1N, 0.05 mmol). After stirring at room temperature for 16 hours, it was neutralized with acetic acid to pH=6.5. The reaction mixture was concentrated on an evaporator at room temperature to remove volatile solvents. The residue was purified by preparative HPLC to give H2bp18c6-off macrocycle-pentylsulfide-DBCO (5 mg, 33%) as an off-white solid. LC-MS APCI: calculated for C51H62N6O10S; 951.15; observed m / z [M+H] + 951.4. Purity by LC-MS: 94.51% RT: 1.98. Purity by HPLC: 98.41% RT: 4.13. 1H NMR(400MHz,D2O):δ 7.81-7.75(m,4H), 7.52-7.17(m,10H), 4.97-4.90(m,1H), 4.80(s,4H), 4.14(s,3H), 3.77-3.46(m,16H), 3.10(s,7H) ), 2.83-2.80(m,2H), 2.55-2.53(m,2H), 2.42-2.38(m,3H), 2.11-2.08(m,3H), 1.39-1.35(m,2H), 1.20-1.10(m,4H).

[0430] Example 15: Modulation and preparation of H2bp18c6-Efda member ring-Hypercone-DBCO-PSMB127 225 Ac(III) labeling

[0431]

change

[0432] Labeling: To a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial, 225Ac(NO3)3 (approximately 5 mCi / mL in 0.1 N HCl, 10 μL, 0.047 mCi) and H2bp18c6-off macrocycle-pentyl sulfide-DBCO-PSMB127 (site-specific, CAR = 2, 2.8 mg / mL in PBS buffer, 8 μL, 22 μg) were added sequentially. After mixing, the pH was approximately 6.5 as determined by pH paper. The reaction solution was left at 37 °C for 2 h. Then, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC-SG column was left at room temperature overnight and then scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that all Ac-225 was completely chelated in the reaction solution after 2 h.

[0433] Purification: The reaction mixture was purified on a PD-10 column. The PD-10 resin was conditioned with NaOAc buffer solution by passing 5 mL of 10 mM NaOAc buffer (pH 6-6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluate was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL of 10 mM NaOAc buffer (pH 6-6.5) solution, and the wash solution was pipetted into the PD-10 column reservoir, and the eluate was collected. Each tube contained approximately 1 mL of eluate. Continuous application of NaOAc buffer (10 mM, pH 6-6.5) to the PD-10 column reservoir was performed until a total elution volume of 10 mL was reached.

[0434] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto iTLC-SG, which was developed with 10 mM EDTA and allowed to dry overnight. This was scanned using a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that no free Ac-225 was present in fraction #3.

[0435] HPLC Analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC Method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column, column temperature: room temperature. The column was eluted with DPBS buffer (x1, calcium and magnesium free), flow rate: 0.7 mL / min, 20 min run, injection volume: 40 μL. After HPLC, fractions were collected at 30 s or 1 min time intervals. The collected HPLC fractions were left overnight at room temperature. The radioactivity of each collected fraction was counted in a gamma counter. HPLC radioactivity traces were constructed from the radioactivity in each HPLC fraction. The HPLC radioactivity trace showed a radioactive peak corresponding to the H2bp18c6-off macrocycle-pentylsulfide-DBCO-PSMB127 peak on the HPLC UV trace.

[0436] Example 16: Preparation of DOTA-DBCO-H11B6 (site-specific) and 225 Ac(III) labeling

[0437] [ka] Azide modification and click reaction of mAb: H11B6 was site-selectively modified with a 200-fold molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37°C. The addition of two azides onto the heavy chain of the mAb was monitored by intact mass ESI-TOF LC-MS on an Agilent G224 instrument. Excess 3-azidopropylamine and MTG were purified and removed using a 1 ml GE Healthcare MabSelect column. The azido-mAb was eluted from the resin using 100 mM sodium citrate (pH 3.0) and then exchanged into 1x dPBS using a 7K Zeba desalting column. A 10-fold molar excess of DOTA-DBCO was reacted with the site-selective azide H11B6 (DOL = 1.94) in 1x dPBS at 37°C for 1 hour without shaking. The completion of the DBCO-azide click reaction was monitored by intact mass spectrometry. Excess free chelator was removed by desalting the conjugate into 1x dPBS on a Zeba7K desalting column, followed by concentration into PBS by three 15-fold serial dilutions and spinning at 3800xg using a 30K MWCO Amicon concentrator device. This yielded the final site-specific DOTA-DBCO-H11B6 conjugate with a CAR of 1.94. The final conjugate was confirmed to be monomeric by analytical size-exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8mm x 30cm, 5u column, eluting with 1x DPBS buffer (calcium- and magnesium-free) at room temperature, a flow rate of 0.7mL / min, an 18-minute run, and an injection volume of 18µL.

[0438] Labeling at a ratio of 50:1: H11B6:Ac-225: To a solution of NaOAc (3 M in HO, 10 μL) in a plastic vial, 225Ac(NO3)3 (approximately 10 mCi / mL in 0.1 N HCl, 5 μL) and DOTA-H11B6 (site-specific, CAR = 1.94, 2.4 mg / mL in PBS buffer, 12.5 μL, 30 μg) were added sequentially. The H11B6:Ac-225 ratio was 50:1. After mixing, the pH was approximately 6.5 as determined by pH paper. The reaction solution was left at 37 °C for 2 h. Then, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC-SG column was left at room temperature overnight and then scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. Approximately 21% of the radioactive signal was observed at the solvent front of iTLC-SG, indicating that approximately 79% of Ac-225 was chelated in the reaction solution after 2 hours.

[0439] Example 17: Targeting various SAs 225 Labeling of DOTA-DBCO-H11B6 (random conjugation) with Ac(III)

[0440] [ka] Labeling at a ratio of 880:1 H11B6:Ac-225: To a solution of NaOAc (3 M in HO, 20 μL) in a plastic vial, 225Ac(NO3)3 (approximately 10 mCi / mL in 0.1 N HCl, 5 μL), DOTA-H11B6 (random conjugate, CAR approximately 2.5, 10 mg / mL in 25 mM acetate buffer, pH 5.5, 50 μL, 500 μg), and NaOH solution (0.1 M, 2 μL) were added sequentially. The H11B6:Ac-225 ratio was 880:1. After mixing, the pH was approximately 6.5 as determined by pH paper. The reaction solution was left at 37 °C for 2 hours. Then, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC-SG column was left at room temperature overnight and then scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that all Ac-225 was completely chelated in the reaction solution after 2 h.

[0441] Labeling at a ratio of 440:1 H11B6:Ac-225: To a solution of NaOAc (3 M in HO, 20 μL) in a plastic vial, 225 Ac(NO3)3 (approximately 10 mCi / mL in 0.1 N HCl, 5 μL), DOTA-H11B6 (random conjugate, CAR approximately 2.5, 10 mg / mL in 25 mM acetate buffer, pH 5.5, 25 μL, 250 μg), and NaOH solution (0.1 M, 1 μL) were added sequentially. The H11B6:Ac-225 ratio was 440:1. After mixing, the pH was approximately 6.5 as determined by pH paper. The reaction solution was left at 37 °C for 2 hours. 0.5 μL of the reaction mixture was then loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC-SG was left overnight at room temperature and then scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, indicating that all Ac-225 was completely chelated in the reaction solution after 2 h.

[0442] Labeling at a ratio of 220:1 H11B6:Ac-225: To a solution of NaOAc (3 M in HO, 20 μL) in a plastic vial, 225 Ac(NO3)3 (approximately 10 mCi / mL in 0.1 N HCl, 5 μL), DOTA-H11B6 (random conjugate, CAR approximately 2.5, 10 mg / mL in 25 mM acetate buffer, pH 5.5, 12.5 μL, 125 μg), and NaOH solution (0.1 M, 1 μL) were added sequentially. The H11B6:Ac-225 ratio was 220:1. After mixing, the pH was approximately 6.5 as determined by pH paper. The reaction solution was left at 37 °C for 2 hours. 0.5 μL of the reaction mixture was then loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC-SG was left overnight at room temperature and then scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. A 5% radioactive signal was observed at the solvent front of iTLC-SG, indicating that 95% of Ac-225 was chelated in the reaction solution after 2 h.

[0443] Labeling at a ratio of 110:1 H11B6:Ac-225: To a solution of NaOAc (3 M in HO, 10 μL) in a plastic vial, 225Ac(NO3)3 (approximately 10 mCi / mL in 0.1 N HCl, 5 μL), DOTA-H11B6 (random conjugate, CAR approximately 2.5, 10 mg / mL in 25 mM acetate buffer, pH 5.5, 6.25 μL, 62.5 μg), and NaOH solution (0.1 M, 1 μL) were added sequentially. The H11B6:Ac-225 ratio was 110:1. After mixing, the pH was approximately 6.5 as determined by pH paper. The reaction solution was left at 37 °C for 2 hours. 0.5 μL of the reaction mixture was then loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC-SG was left overnight at room temperature and then scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. A 71% radioactive signal was observed at the solvent front of iTLC-SG, indicating that 29% of Ac-225 was chelated in the reaction solution after 2 h.

[0444] Labeling at a ratio of 55:1 H11B6:Ac-225: To a solution of NaOAc (3 M in HO, 10 μL) in a plastic vial, 225 Ac(NO3)3 (approximately 10 mCi / mL in 0.1 N HCl, 5 μL), DOTA-H11B6 (random conjugate, CAR approximately 2.5, 10 mg / mL in 25 mM acetate buffer, pH 5.5, 3.13 μL, 31.3 μg), and NaOH solution (0.1 M, 1 μL) were added sequentially. The H11B6:Ac-225 ratio was 55:1. After mixing, the pH was approximately 6.5 as determined by pH paper. The reaction solution was left at 37 °C for 2 hours. 0.5 μL of the reaction mixture was then loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC-SG was left overnight at room temperature and then scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. A 64% radioactive signal was observed at the solvent front of iTLC-SG, indicating that 36% of Ac-225 was chelated in the reaction solution after 2 h.

[0445] Conclusion: Examples 9, 16 and 17 demonstrated that DOTA containing a chelator has lower actinium chelation potency compared to H2bp18c6 containing a chelator.

[0446] Example 18: H2bp18c6-cis-cyclopentyl-fused macrocycle and 225 Synthesis of Ac(III) chelates

[0447] [ka] The above H2bp18c6 derivatives having ring bonds on the macrocycle are generally synthesized according to Scheme 21 below.

[0448] [ka]

[0449] A linker for conjugation to a targeting ligand can be introduced at the "benzyl" position by reacting intermediate 7 with methyl 6-(bromo(4-(tert-butoxycarbonyl)phenyl)methyl)picolinate, for example, as described above with respect to Schemes 3 and 4. Cyclopentane 1,2-diol (compound 1) can be replaced with cyclohexane 1,2-diol to provide H2bp18c6 derivatives with cyclohexyl ring fusions.

[0450] Next, the synthesis of the H2bp18c6-cis-cyclopentyl-fused macrocycle is described.

[0451] [ka]

[0452] To a suspension of sodium hydride (5.63 g, 60% in mineral oil, 147.05 mmol) in DMF (30 mL) was added (1R,2S)-cyclopentane-1,2-diol (3.0 g, 29.41 mmol) in DMF (30 mL) dropwise at 0° C. The reaction mixture was stirred at room temperature for 2 hours. It was cooled again to 0° C., and 2-(2-bromoethoxy)tetrahydro-2H-pyran (18.44 g, 88.23 mmol) in DMF (30 mL) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred for 16 hours. The reaction was quenched with saturated ammonium chloride and extracted with ethyl acetate (3×500 mL). The combined organic layers were washed with water, dried over sodium sulfate, filtered, and concentrated. The crude oil was purified by silica gel (230–400 mesh) flash chromatography using ethyl acetate (0–40%) in petroleum ether to give (1R,2S)-1,2-bis(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)cyclopentane (3.5 g, 33%) as a colorless liquid.

[0453] To a solution of ((1R,2S)-1,2-bis(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)cyclopentane (3.5 g, 9.77 mmol) in 100 mL of methanol was added 1 mL of a solution of HCl in dioxane, stirred at reflux for 1 h, cooled, and evaporated to give 2,2′-(((1R,2S)-cyclopentane-1,2-diyl)bis(oxy))bis(ethan-1-ol) (2.0 g), which was used in the next step without purification.

[0454] To a solution of 2,2'-(((1R,2S)-cyclopentane-1,2-diyl)bis(oxy))bis(ethan-1-ol)2 (2.0 g, 10.52 mmol) in dichloromethane (50 mL) and triethylamine (7.60 mL, 52.63 mmol) at 10 °C, p-toluenesulfonyl chloride (6.0 g, 31.56 mmol) was added portionwise. The mixture was stirred at ambient temperature for 16 h. After completion of the reaction, the suspension was diluted with 200 mL of dichloromethane and washed with cold 1 M HCl (3 × 100 mL), followed by ice-cold water (2 × 100 mL), dried over sodium sulfate, and evaporated to give a solid gum. The crude material was purified by flash chromatography (silica gel, 230-400 mesh) using ethyl acetate in petroleum ether (0-40%) to give (((1R,2S)-cyclopentane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (2.2 g, 42%).

[0455] A mixture of (((1R,2S)-cyclopentane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (2.2 g, 4.42 mmol) and cesium carbonate (4.32 g, 13.25 mmol) in 50 mL of dry DMF was stirred at ambient temperature for 1.5 hours. To the suspension was added 1,2-bis(2-(tosyl-λ))-2-methyl-2-propanol in 50 mL of DMF. 2 (16aR,19aS)-4,13-ditosyltetradecahydro-2H,11H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.8 g, 67%) was added dropwise over 2 h. The mixture was stirred at ambient temperature for an additional 20 h. The solvent was removed under reduced pressure to give a solid paste, which was suspended in 200 mL of dichloromethane and stirred for 30 min. The precipitated solid was filtered off, and the filtrate was evaporated under high vacuum. The crude material was purified by flash chromatography (silica gel, 230-400 mesh) using ethyl acetate in petroleum ether (0-40%) to give (16aR,19aS)-4,13-ditosyltetradecahydro-2H,11H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.8 g, 67%) as a colorless liquid.

[0456] To a solution of (16aR,19aS)-4,13-ditosyltetradecahydro-2H,11H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.8 g, 2.95 mmol) in acetic acid and hydrobromic acid (50%, 10 mL) was added phenol (1.38 g, 14.75 mmol) at room temperature. The reaction was heated at 60 °C for 6 hours. After completion of the reaction, the reaction was cooled to room temperature and the acetic acid was removed under high vacuum. The residue was purified by reverse-phase column purification using 0–100% acetonitrile in water (0.1% TFA) to give (16aR,19aS)-tetradecahydro-2H,11H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (0.7 g, 78%) as a colorless liquid.

[0457] A suspension of (16aR,19aS)-tetradecahydro-2H,11H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (0.7 g, 2.32 mmol), methyl 6-(chloromethyl)picolinate (1.73 g, 5.80 mmol), and sodium carbonate (0.74 g, 6.96 mmol) in dry acetonitrile (10 mL) was heated at 90° C. for 16 h. After completion of the reaction, the reaction mixture was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (silica gel, 230-400 mesh) using methanol (0-10%) in dichloromethane to give dimethyl 6,6'-(((16aR,19aS)-tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (0.55 g, 39%) as a brown liquid.

[0458] A solution of dimethyl 6,6'-(((16aR,19aS)-tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (0.55 g, 0.92 mmol) in 6 N hydrochloric acid (5 mL) was heated at 80 °C for 5 h. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC to give H2bp18c6-cis-cyclopentyl-fused macrocycle (0.3 g, 58%) as a colorless sticky solid. LC-MS APCI: calculated for C29H40N4O8 572.66; observed m / z [M+H] + 572.9. Purity by LC-MS: 99.27% ​​RT: 1.17. Purity by HPLC: 99.05% RT: 2.12. 1 H NMR(400MHz,DMSO-d6):δ 7.86-7.84(m,4H), 7.59-7.56(m,2H), 3.80(s,4H), 3.60-3.49(m,10H), 3.44-3.41(m,4H), 2.75-2.68(m,8H), 1.57-1.47(m,6H).

[0459] [ka]

[0460] 225 Chelation with Ac(III): tetramethylammonium acetate (1 M, 10 μL), H2bp18c6-cis-cyclopentyl-fused macrocycle (2 mg / mL in water, 3 μL) in 0.1 N HCl (10 mCi / mL, 3 μL, 30 μCi); 225 Ac(NO3)3 was added continuously to the plastic vial. pH was approximately 6.5 by pH paper. The vial was heated at 37°C for 2 hours.

[0461] 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dried iTLC was scanned after 20 hours on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC showed 99% chelated Ac-225.

[0462] 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG, which was developed with 10 mM EDTA. The dried iTLC was scanned after 20 hours on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC showed 99% chelated Ac-225.

[0463] Example 19: H2bp18c6-trans-cyclopentyl-fused macrocycle and 225 Synthesis of Ac(III) chelates

[0464] [ka] To a suspension of sodium hydride (5.63 g, 60% in mineral oil, 147.05 mmol) in DMF (30 mL) was added (1R,2R)-cyclopentane-1,2-diol (3.0 g, 29.41 mmol) in DMF (30 mL) dropwise at 0° C. The reaction mixture was stirred at room temperature for 2 hours. It was again cooled to 0° C., and 2-(2-bromoethoxy)tetrahydro-2H-pyran (18.44 g, 88.23 mmol) in DMF (30 mL) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred for 16 hours. The reaction was quenched with saturated ammonium chloride and extracted with ethyl acetate (3×500 mL). The combined organic layers were washed with water, dried over sodium sulfate, filtered, and concentrated. The crude oil was purified by silica gel (230-400 mesh) flash chromatography using ethyl acetate (0-40%) in petroleum ether to give (1R,2R)-1,2-bis(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)cyclopentane (4.8 g, 46%) as a colorless liquid.

[0465] To a solution of (1R,2R)-1,2-bis(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)cyclopentane (4.8 g, 13.40 mmol) in 100 mL of methanol was added 1 mL of HCl in dioxane, stirred at reflux for 1 h, cooled, and evaporated to give 2'-(((1R,2R)-cyclopentane-1,2-diyl)bis(oxy))bis(ethan-1-ol) (2.9 g), which was used in the next step without purification.

[0466] To 2'-(((1R,2R)-cyclopentane-1,2-diyl)bis(oxy))bis(ethan-1-ol) (2.9 g, 10.52 mmol) in dichloromethane (50 mL) and triethylamine (11.03 mL, 76.31 mmol) at 10 °C was added p-toluenesulfonyl chloride (8.70 g, 45.78 mmol) in portions. The mixture was stirred at ambient temperature for 16 h. After completion of the reaction, the suspension was diluted with 200 mL of dichloromethane and washed with cold 1 M HCl (3 × 100 mL), followed by ice-cold water (2 × 100), dried over sodium sulfate, and evaporated to give a solid gum. The crude material was purified by flash chromatography (silica gel, 230-400 mesh) using ethyl acetate in petroleum ether (0-40%) to give (((1R,2R)-cyclopentane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (3.5 g, 46%) as a colorless liquid.

[0467] A mixture of (((1R,2R)-cyclopentane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (3.5 g, 7.03 mmol) and cesium carbonate (6.87 g, 21.08 mmol) in 50 mL of dry DMF was stirred at ambient temperature for 1.5 hours. To the suspension was added 1,2-bis(2-(tosyl-λ))-2-methyl-2-propanol in 50 mL of DMF. 2 (16aR,19aR)-4,13-ditosyltetradecahydro-2H,11H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (3.5 g, 82%) was added dropwise over 2 h. The mixture was stirred at ambient temperature for an additional 20 h. The solvent was removed under reduced pressure to give a solid paste, which was suspended in 200 mL of dichloromethane and stirred for 30 min. The precipitated solid was filtered off, and the filtrate was evaporated under high vacuum. The crude material was purified by flash chromatography (silica gel, 230-400 mesh) using ethyl acetate in petroleum ether (0-40%) to give (16aR,19aR)-4,13-ditosyltetradecahydro-2H,11H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (3.5 g, 82%) as a colorless liquid.

[0468] To a solution of (16aR,19aR)-4,13-ditosyltetradecahydro-2H,11H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (3.5 g, 5.73 mmol) in hydrobromic acid (50%, 15 mL) in acetic acid was added phenol (2.70 g, 28.68 mmol) at room temperature. The reaction was heated at 60 °C for 6 hours. After completion of the reaction, the reaction was cooled to room temperature and the acetic acid was removed under high vacuum. The crude material was purified by reverse-phase column purification using 0–100% acetonitrile in water (0.1% TFA) to give (16aR,19aR)-tetradecahydro-2H,11H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.3 g, 75%) as a colorless liquid.

[0469] A suspension of (16aR,19aS)-tetradecahydro-2H,11H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.3 g, 4.30 mmol), methyl 6-(chloromethyl)picolinate (1.99 g, 10.76 mmol), and sodium carbonate (1.37 g, 12.90 mmol) in dry acetonitrile (20 mL) was heated at 90° C. for 16 h. After completion of the reaction, the reaction mixture was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (silica gel, 230-400 mesh) using methanol (0-10%) in dichloromethane to give dimethyl 6,6'-(((16aR,19aR)-tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (1.0 g, 39%) as a brown liquid.

[0470] A solution of dimethyl 6,6'-(((16aR,19aR)-tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (1.0 g, 1.66 mmol) in 6 N hydrochloric acid (10 mL) was heated at 80 °C for 5 h. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC to give H2bp18c6-trans-cyclopentyl-fused macrocycle (0.8 g, 84%) as a colorless sticky solid. LC-MS APCI: calculated for C29H40N4O8 572.66; observed m / z [M+H] + 573.0. Purity by LC-MS: 96.10% RT: 1.18. Purity by HPLC: 97.77% RT: 2.29. 1 H NMR (400MHz, DMSO-d6): δ13.42(s,1H), 9.70(s,1H), 8.15-8.09(m,4H), 7.80-7.78(m,2H), 4.69(s,4H), 3.93-3.55(m,22H), 1.90-1.85(m,2H), 1.57-1.52(m,2H), 1.46-1.39(m,2H).

[0471] [ka]

[0472] 225 Chelation with Ac(III): tetramethylammonium acetate (1 M, 10 μL), H2bp18c6-trans-cyclopentyl-fused macrocycle (2 mg / mL in water, 3 μL), 225 Ac(NO3)3 was added to 0.1 N HCl (10 mCi / mL, 3 μL, 30 μCi). pH was approximately 6.5 by pH paper. The vial was heated at 37°C for 2 hours.

[0473] 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dried iTLC was scanned after 20 hours on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC showed 99% chelated Ac-225.

[0474] 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG, which was developed with 10 mM EDTA. The dried iTLC was scanned after 20 hours on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrated with the solvent to the solvent front, and the iTLC showed 99% chelated Ac-225.

[0475] Example 20: H2bp18c6-cis-cyclohexyl-fused macrocycle and 225 Synthesis of Ac(III) chelates

[0476] [ka] To a suspension of sodium hydride (3.3 g, 60% in mineral oil, 86.20 mmol) in DMF (20 mL) was added (1R,2S)-1,2-cyclohexane-diol (2.0 g, 17.24 mmol) in DMF (20 mL) dropwise at 0 °C. The reaction mixture was stirred at room temperature for 2 hours. It was cooled again to 0 °C, and 2-(2-bromoethoxy)tetrahydro-2H-pyran (10.81 g, 51.72 mmol) in DMF (20 mL) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred for 16 hours. The reaction was quenched with saturated ammonium chloride and extracted with ethyl acetate (3 × 500 mL). The combined organic phase was washed with brine, dried over sodium sulfate, filtered, and concentrated. The crude oil was purified by silica gel (230–400 mesh) flash chromatography using ethyl acetate (0–40%) in petroleum ether to give (1R,2S)-1,2-bis(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)cyclohexane (2.5 g, 39%) as a colorless liquid.

[0477] To a solution of 1R,2S)-1,2-bis(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)cyclohexane (2.5 g, 6.7 mmol) in 100 mL of methanol, 1 mL of HCl in dioxane was added, stirred at reflux for 1 h, cooled, and evaporated. The crude 2,2'-(((1R,2S)-cyclohexane-1,2-diyl)bis(oxy))bis(ethan-1-ol) (1.5 g) was used in the next step without purification.

[0478] To a solution of 2,2'-(((1R,2S)-cyclohexane-1,2-diyl)bis(oxy))bis(ethan-1-ol) (1.5 g, 7.35 mmol) in dichloromethane (25 mL) and triethylamine (5.31 mL, 36.76 mmol) at 10°C, p-toluenesulfonyl chloride (4.20 g, 22.05 mmol) was added portionwise. The mixture was stirred at ambient temperature for 16 h. After completion of the reaction, the suspension was diluted with 200 mL of dichloromethane and washed with cold 1 M HCl (3 x 100 mL), followed by ice-cold water (2 x 100), dried over sodium sulfate, and evaporated to give a solid gum. The crude material was purified by flash chromatography (silica gel, 230-400 mesh) using ethyl acetate in petroleum ether (0-40%) to give (((1R,2S)-cyclohexane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (1.9 g, 50%) as a colorless liquid.

[0479] A mixture of (((1R,2S)-cyclohexane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (1.9 g, 3.71 mmol) and cesium carbonate (3.63 g, 11.13 mmol) in 50 mL of dry DMF was stirred at ambient temperature for 1.5 hours. To the suspension was added 1,2-bis(2-(tosyl-λ))-2-methyl-2-propanol in 50 mL of DMF. 2 (16aR,20aS)-4,13-ditosylhexadecahydro-2H,11H-benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.2 g, 52%) was added dropwise over 2 hours. The mixture was stirred at ambient temperature for an additional 20 hours. The solvent was removed under reduced pressure to give a solid paste, which was suspended in 200 mL of dichloromethane and stirred for 30 minutes. The precipitated solid was filtered off, and the filtrate was evaporated under high vacuum. The crude material was purified by flash chromatography (silica gel, 230-400 mesh) using ethyl acetate in petroleum ether (0-40%) to give (16aR,20aS)-4,13-ditosylhexadecahydro-2H,11H-benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.2 g, 52%) as a colorless liquid.

[0480] To a solution of (16aR,20aS)-4,13-ditosylhexadecahydro-2H,11H-benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.2 g, 1.92 mmol) in hydrobromic acid (50%, 5 mL) in acetic acid was added phenol (0.9 g, 9.61 mmol) at room temperature. The reaction was heated at 60 °C for 6 hours. After completion of the reaction, the reaction was cooled to room temperature and the acetic acid was removed under high vacuum. The residue was purified by reverse-phase column purification using 0–100% acetonitrile in water (0.1% TFA) to give (16aR,20aS)-hexadecahydro-2H,11H-benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (0.45 g, 74%) as a colorless liquid.

[0481] A suspension of (16aR,20aS)-hexadecahydro-2H,11H-benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (0.45 g, 1.42 mmol), methyl 6-(chloromethyl)picolinate (0.65 g, 3.56 mmol), and sodium carbonate (0.45 g, 4.26 mmol) in dry acetonitrile (10 mL) was heated at 90° C. for 16 h. The reaction mixture was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (silica gel, 230-400 mesh) using methanol (0-10%) in dichloromethane to give dimethyl 6,6'-(((16aR,20aS)-hexadecahydro-4H,13H-benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (0.26 g, 30%) as a brown liquid.

[0482] A solution of dimethyl 6,6'-(((16aR,20aS)-hexadecahydro-4H,13H-benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (0.26 g, 0.42 mmol) in 6 N hydrochloric acid (5 mL) was heated at 80°C for 5 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC to give the product H2bp18c6-cis-cyclohexyl-fused macrocycle (0.11 g, 44%) as an off-white solid. LC-MS APCI: calculated for C30H42N4O8 586.69; observed m / z [M+H] + 587.0. Purity by LC-MS: 98.15% RT: 1.31. Purity by HPLC: 97.78% RT: 2.32. 1 H NMR (400MHz, DMSO-d6): δ13.35(s,1H), 9.84(s,1H), 8.15-8.09(m,4H), 7.80-7.78(m,2H), 4.70(s,4H), 3.97-3.54(m,22H), 1.70-1.23(m,8H).

[0483] [ka]

[0484] 225 Chelation with Ac(III): Tetramethylammonium acetate (1 M, 10 μL), H2bp18c6-cis-cyclohexyl-fused macrocycle (2 mg / mL in water, 3 μL), in 0.1 N HCl 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi) was added sequentially to the plastic vial. pH was approximately 6.5 by pH paper. The vial was heated at 37°C for 2 hours.

[0485] 0.5 μL of the reaction mixture was loaded onto an iTLC-SG column and developed with 10 mM EDTA. The dried iTLC was scanned after 20 hours on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC showed 99% chelated Ac-225.

[0486] 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC was scanned after 20 hours on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC showed 99% chelated Ac-225.

[0487] Example 21: H2bp18c6-trans-cyclohexyl-fused macrocycle and 225 Synthesis of Ac(III) chelates

[0488] [ka] To a suspension of sodium hydride (3.3 g, 60% in mineral oil, 86.20 mmol) in DMF (20 mL) was added (1R,2R)-cyclohexane-1,2-diol (2.0 g, 17.24 mmol) in DMF (20 mL) dropwise at 0 °C. The reaction mixture was stirred at room temperature for 2 hours. It was cooled again to 0 °C, and 2-(2-bromoethoxy)tetrahydro-2H-pyran (10.81 g, 51.72 mmol) in DMF (20 mL) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred for 16 hours. The reaction was quenched with saturated ammonium chloride and extracted with ethyl acetate (3 × 500 mL). The combined organic phase was washed with brine, dried over sodium sulfate, filtered, and concentrated. The crude oil was purified by silica gel (230-400 mesh) flash chromatography using ethyl acetate (0-40%) in petroleum ether to give (1R,2R)-1,2-bis(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)cyclohexane (2.45 g, 38%) as a colorless liquid.

[0489] To a solution of (1R,2R)-1,2-bis(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)cyclohexane (2.45 g, 6.5 mmol) in 100 mL of methanol, 1 mL of HCl in dioxane was added, stirred at reflux for 1 h, cooled, and evaporated. The crude 2,2'-(((1R,2R)-cyclohexane-1,2-diyl)bis(oxy))bis(ethan-1-ol) (1.5 g) was used in the next step without purification.

[0490] To a solution of 2,2'-((((1R,2R-cyclohexane-1,2-diyl)bis(oxy))bis(ethan-1-ol) (1.5 g, 7.35 mmol) in dichloromethane (25 mL) and triethylamine (5.31 mL, 36.76 mmol) at 10°C was added p-toluenesulfonyl chloride (4.20 g, 22.05 mmol) in portions. The mixture was stirred at ambient temperature for 16 h. The suspension was further diluted with 200 mL of dichloromethane and added to cold 1 M After washing with HCl (3 × 100 mL) followed by ice-cold water (2 × 100 mL), drying over sodium sulfate, and evaporation gave a solid gum. The crude material was purified by flash chromatography (silica gel, 230–400 mesh) using ethyl acetate in petroleum ether (0–40%) to give (((1R,2R)-cyclohexane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (2.1 g, 56%) as a colorless liquid.

[0491] A mixture of (((1R,2R)-cyclohexane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (2.1 g, 4.1 mmol) and cesium carbonate (4.01 g, 12.30 mmol) in 30 mL of dry DMF was stirred at ambient temperature for 1.5 hours. To the suspension was added 1,2-bis(2-(tosyl-λ))-2-methyl-2-propanol in 30 mL of DMF. 2 (16aR,20aR)-4,13-ditosylhexadecahydro-2H,11H-benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.35 g, 54%) was added dropwise over 2 h. The mixture was stirred at ambient temperature for an additional 20 h. The solvent was removed under reduced pressure to give a solid paste, which was suspended in 200 mL of dichloromethane and stirred for 30 min. The precipitated solid was filtered off, and the filtrate was evaporated under high vacuum. The residue was purified by flash chromatography (silica gel, 230-400 mesh) using ethyl acetate in petroleum ether (0-40%) to give (16aR,20aR)-4,13-ditosylhexadecahydro-2H,11H-benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.35 g, 54%) as a colorless liquid.

[0492] To a solution of (16aR,20aR)-4,13-ditosylhexadecahydro-2H,11H-benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.35 g, 2.16 mmol) in hydrobromic acid (50%, 5 mL) in acetic acid was added phenol (1.01 g, 1.81 mmol) at room temperature. The reaction was heated at 60 °C for 6 h. After cooling to room temperature, the acetic acid was removed under high vacuum. The residue was purified by reverse-phase column chromatography using 0–100% acetonitrile in water (0.1% TFA) to give (16aR,20aR)-hexadecahydro-2H,11H-benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (0.5 g, 72%) as a colorless liquid.

[0493] A suspension of (16aR,20aR)-hexadecahydro-2H,11H-benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (0.5 g, 1.58 mmol), methyl 6-(chloromethyl)picolinate (0.73 g, 3.95 mmol), and sodium carbonate (0.5 g, 4.74 mmol) in dry acetonitrile (10 mL) was heated at 90° C. for 16 h. The reaction mixture was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 230–400 mesh) using methanol (0–10%) in dichloromethane to give dimethyl 6,6′-(((16aR,20aR)-hexadecahydro-4H,13H-benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (0.3 g, 31%) as a brown liquid.

[0494] A solution of dimethyl 6,6'-(((16aR,20aR)-hexadecahydro-4H,13H-benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (0.3 g, 0.49 mmol) in 6 N hydrochloric acid (5 mL) was heated at 80°C for 5 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC to give H2bp18c6-trans-cyclohexyl-fused macrocycle (0.16 g, 56%) as an off-white solid. LC-MS APCI: calculated for C30H42N4O8 586.69; observed m / z [M+H] + 587.0. Purity by LC-MS: 98.97% RT: 1.31. Purity by HPLC: 97.08% RT: 2.27. 1 H NMR (400MHz, DMSO-d6): δ13.35(s,1H), 9.84(s,1H), 8.16-8.10(m,4H), 7.80-7.78(m,2H), 4.71(s,4H), 4.00- 3.82(m,8H), 3.73-3.53(m,12H), 3.20-3.18(m,2H), 2.03-2.00(m,2H), 1.61-1.60(m,2H), 1.15-1.02(m,4H).

[0495] [ka]

[0496] 225 Chelation with Ac(III): Tetramethylammonium acetate (1 M, 10 μL), H2bp18c6-trans-cyclohexyl-fused macrocycle (2 mg / mL in water, 3 μL), in 0.1 N HCl 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi) was added sequentially to the plastic vial. pH was approximately 6.5 by pH paper. The vial was heated at 37°C for 2 hours.

[0497] 0.5 μL of the reaction mixture was loaded onto an iTLC-SG column and developed with 10 mM EDTA. The dried iTLC was scanned after 20 hours on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC showed 99% chelated Ac-225.

[0498] 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC was scanned after 20 hours on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC showed 99% chelated Ac-225.

[0499] Example 22: H2bp18c6-off macrocycle-hydroxymethyl isomer I and 225 Synthesis of Ac(III) chelates

[0500] [ka] To a suspension of sodium hydride (3.33 g, 60% in mineral oil, 83.33 mmol) in DMF (20 mL) was added 3-ethoxypropane-1,2-diol (2.0 g, 16.66 mmol) in DMF (20 mL) dropwise at 0 °C. The reaction mixture was stirred at room temperature for 2 hours. It was cooled again to 0 °C, and 2-(2-bromoethoxy)tetrahydro-2H-pyran (10.44 g, 49.98 mmol) in DMF (20 mL) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred for 16 hours. The reaction was quenched with saturated ammonium chloride and extracted with ethyl acetate (3 × 500 mL). The combined organic phase was washed with brine, dried over sodium sulfate, filtered, and concentrated. The residual oil was purified by silica gel (230-400 mesh) flash chromatography using ethyl acetate (0-40%) in petroleum ether to give 2,2'-((((3-ethoxypropane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(tetrahydro-2H-pyran) (2.2 g, 35%) as a colorless liquid.

[0501] To a solution of 2,2'-((((3-ethoxypropane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(tetrahydro-2H-pyran) (2.2 g, 5.85 mmol) in 50 mL of methanol was added 1 mL of HCl in dioxane. The reaction was stirred at reflux for 1 h, cooled, and evaporated. The crude 2,2'-((3-ethoxypropane-1,2-diyl)bis(oxy))bis(ethan-1-ol) (1.3 g) was used in the next step without purification.

[0502] To a solution of 2,2'-((3-ethoxypropane-1,2-diyl)bis(oxy))bis(ethan-1-ol) (1.3 g, 6.25 mmol) in dichloromethane (30 mL) and triethylamine (4.51 mL, 31.25 mmol) at 10°C was added p-toluenesulfonyl chloride (3.56 g, 18.75 mmol) in portions. The mixture was stirred at ambient temperature for 16 h. The suspension was diluted with 200 mL of dichloromethane and washed with cold 1 M HCl (3 x 100 mL) followed by ice-cold water (1 x 100 mL), dried over sodium sulfate, and evaporated to give a solid gum. The crude material was purified by flash chromatography (silica gel, 230-400 mesh) using ethyl acetate in petroleum ether (0-40%) to give ((3-ethoxypropane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (2.0 g, 62%) as a colorless liquid.

[0503] A mixture of ((3-ethoxypropane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (2.0 g, 3.87 mmol) and cesium carbonate (3.79 g, 11.62 mmol) in 25 mL of dry DMF was stirred at ambient temperature for 1.5 hours. To the suspension was added 1,2-bis(2-(tosyl-λ))-2-methyl-2-propanediol in 25 mL of DMF. 2(1.75 g, 3.87 mmol) was added dropwise over 2 h. The mixture was stirred at ambient temperature for an additional 20 h. The solvent was removed under reduced pressure to give a solid paste, which was suspended in 200 mL of dichloromethane and stirred for 30 min. The precipitated solid was filtered off, and the filtrate was evaporated under high vacuum. The residue was purified by flash chromatography (silica gel, 230-400 mesh) using ethyl acetate in petroleum ether (0-40%) to give 2-(ethoxymethyl)-7,16-ditosyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (1.1 g, 45%) as a colorless liquid. The product was further purified by SFC to resolve the two enantiomeric isomers, Isomer-I (0.3 g, 12%) and Isomer-II (0.26 g, 11%). The stereochemistry of isomers I and II was arbitrarily assigned.

[0504] To a solution of (S)-2-(ethoxymethyl)-7,16-ditosyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (isomer I, 0.3 g, 0.48 mmol) in acetic acid (50%, 1 mL) was added phenol (0.22 g, 2.39 mmol) at room temperature. The reaction was heated at 60 °C for 6 h. After cooling to room temperature, the acetic acid was removed under high vacuum. The residue was purified by reverse-phase column chromatography using 0–100% acetonitrile in water (0.1% TFA) to give (R)-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2-yl)methyl acetate (arbitrarily assigned stereochemistry) (0.1 g, 63%) as a colorless liquid.

[0505] (R)-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2-yl)methyl acetate (arbitrarily assigned stereochemistry) (100 mg, 0.30 mmol), methyl 6-(chloromethyl)picolinate (138 mg, 0.75 mmol), and sodium carbonate (159 mg, 1.5 mmol) in dry acetonitrile (3 mL) were heated at 90° C. for 16 h. The reaction mixture was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 230-400 mesh) using methanol (0-10%) in dichloromethane to give dimethyl 6,6'-((2-(acetoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene)-(R)-dipicolinate (arbitrarily assigned stereochemistry) (50 mg, 26%) as a brown liquid.

[0506] To a solution of 6,6'-((2-(acetoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene)-(R)-dipicolinate (arbitrarily assigned stereochemistry) (50 mg, 0.08 mmol) in methanol (1 mL) was added potassium carbonate (1 mg, 0.008 mmol) at room temperature and stirred for 10 min. The mixture was concentrated under reduced pressure. The residue was treated with dichloromethane (HCl). Purification by flash chromatography (silica gel, 230-400 mesh) using methanol (0-10%) in dichloromethane gave 6,6'-((2-(hydroxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))-(S)-dipicolinate (arbitrarily assigned stereochemistry) (22 mg, 48%) as a brown liquid.

[0507] A solution of 6,6'-((2-(hydroxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))-(S)-dipicolinate (arbitrarily assigned stereochemistry) (22 mg, 0.04 mmol) in 6 N hydrochloric acid (0.5 mL) was heated at 80 °C for 5 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC to give H2bp18c6-off macrocycle-hydroxymethyl isomer I (12 mg, 57%) as a sticky solid. LC-MS APCI: calculated for C27H38N4O9 562.62; observed m / z [M+H] + 562.8. Purity by LC-MS: 96.89% RT: 1.61. Purity by HPLC: 96.47% RT: 1.57. 1 H NMR (400MHz, DMSO-d6): δ 3.39(s,1H), 9.81(s,1H), 8.14-8.09(m,4H), 7.80-7.78(m,2H), 4.70(s,4H), 3.98-3.85(m,10H), 3.60-3.42(m,15H).

[0508] [ka]

[0509] 225 Chelation with Ac(III): Tetramethylammonium acetate (1 M, 10 μL), H2bp18c6-off macrocycle-hydroxylmethyl isomer I (2 mg / mL in water, 3 μL), in 0.1 N HCl 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi) was added sequentially to the plastic vial. pH was approximately 6.5 by pH paper. The vial was heated at 37°C for 2 hours.

[0510] 0.5 μL of the reaction mixture was loaded onto an iTLC-SG column and developed with 10 mM EDTA. The dried iTLC was scanned after 20 hours on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC showed 99% chelated Ac-225.

[0511] 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC was scanned after 20 hours on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC showed 99% chelated Ac-225.

[0512] Example 23: H2bp18c6-off macrocycle-hydroxymethyl isomer II and 225 Synthesis of Ac(III) chelates

[0513] [ka] To a solution of (R)-2-(ethoxymethyl)-7,16-ditosyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (arbitrarily assigned stereochemistry) (isomer II, 0.26 g, 0.42 mmol) in acetic acid (50%, 1 mL) was added phenol (197 mg, 2.07 mmol) at room temperature. The reaction was heated at 60 °C for 6 h. After cooling to room temperature, the acetic acid was removed under high vacuum. The residue was purified by reverse-phase column chromatography using 0–100% acetonitrile in water (0.1% TFA) to give (S)-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2-yl)methyl acetate (arbitrarily assigned stereochemistry) (0.06 g, 43%) as a colorless liquid.

[0514] (S)-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2-yl)methyl acetate (arbitrarily assigned stereochemistry) (0.06 g, 0.18 mmol), methyl 6-(chloromethyl)picolinate (0.083 g, 0.45 mmol) in dry acetonitrile (3 mL), and sodium carbonate (0.095 g, 0.9 mmol) were heated at 90° C. for 16 h. The reaction mixture was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 230-400 mesh) using methanol (0-10%) in dichloromethane to give dimethyl 6,6'-((2-(acetoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))-(S)-dipicolinate (arbitrarily assigned stereochemistry) (0.025 g, 22%) as a brown liquid.

[0515] To a solution of dimethyl 6,6'-((2-(acetoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))-(S)-dipicolinate (arbitrarily assigned stereochemistry) (0.025 g, 0.04 mmol) in methanol (0.5 mL) was added potassium carbonate (0.5 mg, 0.004 mmol) at room temperature. The mixture was stirred for 10 minutes. It was concentrated under reduced pressure. The crude product was purified by flash chromatography on silica (230-400 mesh) eluting with a gradient of 0-10% methanol in dichloromethane to give dimethyl 6,6'-((2-(hydroxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))-(R)-dipicolinate (0.01 g, 43%) as a brown liquid.

[0516] A solution of dimethyl 6,6'-((2-(hydroxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))-dipicolinate (0.01 g, 0.02 mmol) in 6 N hydrochloric acid (0.5 mL) was heated at 80 °C for 3 h. The reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC to give H2bp18c6-off macrocycle-hydroxymethyl isomer II (4 mg, 44%) as a viscous liquid. LC-MS APCI: calculated for C27H38N4O9 562.62; observed m / z [M+H] + 562.8. Purity by LC-MS: 96.89% RT: 6.31. Purity by HPLC: 93.97% RT: 6.67. 1 H NMR (400MHz, DMSO-d6): δ13.34(s,1H), 9.86(s,1H), 8.13-8.11(m,4H), 7.80-7.78(m,2H), 4.70(s,4H), 3.95-3.84(m,10H), 3.58-3.42(m,15H).

[0517] [ka]

[0518] 225 Chelation with Ac(III): Tetramethylammonium acetate (1 M, 10 μL), H2bp18c6-off macrocycle-hydroxylmethyl isomer II (2 mg / mL in water, 3 μL), in 0.1 N HCl 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi) was added sequentially to the plastic vial. pH was approximately 6.5 by pH paper. The vial was heated at 37°C for 2 hours.

[0519] 0.5 μL of the reaction mixture was loaded onto an iTLC-SG column and developed with 10 mM EDTA. The dried iTLC was scanned after 20 hours on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC showed 99% chelated Ac-225.

[0520] 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC was scanned after 20 hours on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC showed 99% chelated Ac-225.

[0521] Example 24: H2bp18c6-off macrocycle-ethoxymethyl isomer I and 225 Synthesis of Ac(III) chelates

[0522] [ka] To a stirred solution of dimethyl 6,6'-((2-(hydroxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (100 mg, 0.17 mmol) (arbitrarily assigned stereochemistry) in DMF (2 mL) at 0 °C, sodium hydride (10 mg, 60% dispersion in mineral oil, 0.25 mmol) was added slowly. The reaction mixture was cooled to room temperature and then stirred for 10 hours. The reaction mixture was again cooled to 0 °C, and a solution of ethyl iodide (39 mg, 0.25 mmol, 20 μL) in DMF (1 ml) was added dropwise. The reaction mixture was allowed to reach room temperature and then stirred at this temperature for 2 hours. After completion of the reaction (monitored by LCMS), the reaction mixture was quenched with saturated NH4Cl solution and extracted with ethyl acetate. The combined organic extracts were then dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel (230-400 mesh) flash column chromatography using 5% methanol in dichloromethane as the eluent to give the product dimethyl 6,6'-((2-(ethoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (arbitrarily assigned stereochemistry) (45 mg, 42%) as a brown liquid.

[0523] To a stirred solution of dimethyl 6,6'-((2-(ethoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (45 mg, 0.07 mmol) (arbitrarily assigned stereochemistry) in THF (0.25 mL), water (0.5 mL), and methanol (0.25 mL) was added lithium hydroxide monohydrate (9 mg, 0.21 mmol). The reaction mixture was stirred at room temperature for 4 h. After completion of the reaction (monitored by UPLC-MS), the pH of the reaction was adjusted to 3-4 using 1.5 M aqueous HCl and concentrated. The residue was purified by preparative HPLC to afford H2bp18c6-off macrocycle-ethoxymethyl isomer I (28 mg, 66%) as a viscous liquid. LC-MS APCI: calculated for C29H42N4O9 590.30; observed m / z [M+H] + 591.2. Purity by LC-MS: 99.59% RT: 1.19. Purity by HPLC: 96.12% RT: 2.01. 1 H NMR (400MHz, DMSO-d6): δ13.40(s,1H), 9.75(s,1H), 8.16-8.09(m,4H), 7.80-7.78(m,2H), 4.69(s,4H), 3.98-3.34(m,28H), 1.06(t,J=6.80Hz,3H).

[0524] [ka]

[0525] 225 Chelation with Ac(III): Tetramethylammonium acetate (1 M, 10 μL), H2bp18c6-off macrocycle-hydroxylmethyl isomer I (1 mg / mL in water, 1 μL), in 0.1 N HCl 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi) was added sequentially to the plastic vial. pH was approximately 6.5 by pH paper. The vial was heated at 37°C for 2 hours.

[0526] 0.5 μL of the reaction mixture was loaded onto an iTLC-SG column and developed with 10 mM EDTA. The dried iTLC was scanned after 20 hours on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC showed 99% chelated Ac-225.

[0527] 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC was scanned after 20 hours on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC showed 99% chelated Ac-225.

[0528] Example 25: H2bp18c6-off macrocycle-ethoxymethyl isomer II and 225 Synthesis of Ac(III) chelates

[0529] [ka] To a stirred solution of 6,6'-((2-(hydroxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(R)-dipicolinate dimethyl (arbitrarily assigned stereochemistry) (60 mg, 0.1 mmol) in DMF (1.5 mL) at 0 °C, sodium hydride (6 mg, 60% dispersion in mineral oil, 0.15 mmol) was added slowly. The reaction mixture was cooled to room temperature and then stirred for 10 hours. The reaction mixture was again cooled to 0 °C, and a solution of ethyl iodide (23 mg, 0.15 mmol, 12 uL) in DMF (0.5 ml) was added dropwise. The reaction mixture was allowed to reach room temperature and then stirred at this temperature for 2 hours. After completion of the reaction (monitored by LCMS), the reaction mixture was quenched with saturated NH4Cl solution and extracted with ethyl acetate. The combined organic extracts were then dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel (230-400 mesh) flash column chromatography using 5% methanol in dichloromethane as the eluent to give 6,6'-((2-(ethoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(R)-dipicolinate dimethyl (arbitrarily assigned stereochemistry) (20 mg, 32%) as a brown liquid.

[0530] To a solution of 6,6'-((2-(ethoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))(R)-dipicolinate dimethyl (arbitrarily assigned stereochemistry) (20 mg, 0.03 mmol) in THF (0.1 mL), water (0.25 mL), and methanol (0.1 mL) was added lithium hydroxide monohydrate (4 mg, 0.09 mmol). The reaction mixture was stirred at room temperature for 4 h. After completion of the reaction (monitored by UPLC-MS), the pH of the reaction was adjusted to 3-4 using 1.5 M aqueous HCl and concentrated. The residue was purified by preparative HPLC to afford H2bp18c6-off macrocycle-ethoxymethyl isomer II (5 mg, 26%) as a viscous liquid. LC-MS APCI: calculated for C29H42N4O9 590.30; observed m / z [M+H]+ 591.3. Purity by LC-MS: 99.28%, RT: 1.199. Purity by HPLC: 97.60% RT: 2.01. 1H NMR 1H-NMR (400 MHz, DMSO-d6): δ 13.40 (s, 1H), 9.72 (s, 1H), 8.16-8.00 (m, 4H), 7.80-7.77 (m, 2H), 4.69 (s, 4H), 4.34-3.38 (m, 28H), 1.06 (t, J = 6.80 Hz, 3H).

[0531] [ka]

[0532] 225 Chelation with Ac(III): Tetramethylammonium acetate (1 M, 10 μL), H2bp18c6-off macrocycle-hydroxylmethyl isomer II (1 mg / mL in water, 1 μL), in 0.1 N HCl 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi) was added sequentially to the plastic vial. pH was approximately 6.5 by pH paper. The vial was heated at 37°C for 2 hours.

[0533] 0.5 μL of the reaction mixture was loaded onto an iTLC-SG column and developed with 10 mM EDTA. The dried iTLC was scanned after 20 hours on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC showed 99% chelated Ac-225.

[0534] 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC was scanned after 20 hours on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC showed 99% chelated Ac-225.

[0535] Example 26: Synthesis of H2bp18c6-off macrocycle-NCS

[0536] [ka] Step 1: Sodium hydride (18.16 g, 60% in mineral oil, 454 mmol) was added to a solution of (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol (50 g, 378 mmol) in anhydrous DMF (500 mL) at 0 °C. After stirring at this temperature for 10 min, benzyl bromide (77.63 g, 454 mmol) was slowly added to the suspension. The reaction was allowed to reach room temperature after 30 min and stirred for an additional 8 h. The reaction mixture was quenched with saturated ammonium chloride solution and extracted with dichloromethane (2 × 500 mL). The combined organic layers were washed with brine and dried over sodium sulfate. After removal of the solvent, the residue was purified by flash column chromatography (silica gel, 230–400 mesh) using petroleum ether and ethyl acetate to give compound 1 (72 g, 86%) as a colorless liquid.

[0537] Step 2: Compound 1 (72 g, 324 mmol) was dissolved in THF (100 mL), and aqueous HCl (1.5 N, 100 mL) was added and stirred at room temperature for 16 h. The reaction mixture was diluted with ethyl acetate (500 mL) and neutralized with 10% sodium bicarbonate solution. The solution was extracted with ethyl acetate (2 × 500 mL), and the combined organic layers were washed with water, brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 230-400 mesh) using petroleum ether and ethyl acetate to give compound 2 (51 g, 85%) as a colorless oil.

[0538] Step 3: To a suspension of sodium hydride (26.29 g, 60% in mineral oil, 686 mmol) in DMF (20 mL) was added compound 2 (25.0 g, 137 mmol) in DMF (100 mL) dropwise at 0 °C. The reaction mixture was stirred at room temperature for 2 h. It was cooled again to 0 °C, and 2-(2-bromoethoxy)tetrahydro-2H-pyran (85.89 g, 411 mmol) in DMF (100 mL) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred for 16 h. The reaction was quenched using saturated ammonium chloride solution and extracted with ethyl acetate (3 × 300 mL). The combined organic layers were washed with water, dried over sodium sulfate, filtered, and concentrated. The crude oil was purified by flash chromatography on silica gel (230–400 mesh) using ethyl acetate (0–40%) in petroleum ether. Product 3 (32 g, 53%) was obtained as a colorless liquid.

[0539] Step 4: To a solution of compound 3 (32 g, 73.05 mmol) in 500 mL of methanol, 5 mL of HCl in dioxane was added, stirred at reflux for 1 h, cooled, and evaporated. The crude material 4 (20 g) was used in the next step without purification.

[0540] Step 5: Compound 4 (20 g, 74.07 mmol) was dissolved in dichloromethane (250 mL) and triethylamine (53 mL, 370 mmol). The solution was cooled to 10 °C, and solid p-toluenesulfonyl chloride (42.20 g, 222 mmol) was added portionwise. The mixture was stirred at ambient temperature for 16 h. After completion of the reaction, the suspension was further diluted with 1000 mL of dichloromethane and washed with cold aqueous HCl (1 M, 3 × 200 mL), ice-cold water (2 × 200 mL), dried over sodium sulfate, and evaporated to a solid gum. The crude material was purified by flash chromatography (silica gel, 230-400 mesh) using ethyl acetate (0-40%) in petroleum ether. Product 5 (30 g, 70%) was obtained as a colorless liquid.

[0541] Step 6: A mixture of compound 5 (30 g, 51.9 mmol) and cesium carbonate (50.76 g, 155.7 mmol) in 200 mL of dry DMF was stirred at ambient temperature for 1.5 h. To the suspension, compound 6 (23.56 g, 51.9 mmol) in 200 mL of DMF was added dropwise over 2 h. The mixture was stirred at ambient temperature for an additional 20 h. The solvent was removed under reduced pressure to give a solid paste. This was suspended in 1000 mL of dichloromethane and stirred for 30 min. The precipitated solid was filtered off, and the filtrate was evaporated under high vacuum. The crude material was purified by flash chromatography (silica gel, 230-400 mesh) using ethyl acetate in petroleum ether (0-40%). Product 7 (24 g, 67%) was obtained as a colorless liquid.

[0542] Step 7: To a solution of compound 7 (24 g, 34.78 mmol) in hydrobromic acid (50% acetic acid, 100 mL), phenol (16.35 g, 174 mmol) was added at room temperature. The reaction was heated at 60 °C for 6 hours. After completion of the reaction, it was cooled to room temperature and the acetic acid was removed under high vacuum. The crude product was purified by reverse-phase column chromatography using acetonitrile and 0.1% TFA in water to give compound 8 (8.0 g, 69%) as a colorless liquid.

[0543] Step 8: A suspension of compound 8 (8.0 g, 23.95 mmol), compound 9 (11.07 g, 59.88 mmol), and sodium carbonate (12.69 g, 119.75 mmol) in dry acetonitrile (100 mL) was heated at 90 °C for 16 h. After completion of the reaction, the reaction mixture was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. The crude material was purified by flash chromatography (silica gel, 230-400 mesh) using methanol (0-10%) in dichloromethane. Product 10 (5.0 g, 33%) was obtained as a brown liquid.

[0544] Step 9: To a solution of compound 10 (5.0 g, 7.91 mmol) in methanol (50 mL), potassium carbonate (0.11 g, 0.79 mmol) was added at room temperature and stirred for 10 minutes. After completion of the reaction, it was concentrated under reduced pressure. The residue was purified by flash chromatography on silica (230-400 mesh) eluting with a gradient of 0-10% methanol in dichloromethane. Product 11 (3.5 g, 75%) was obtained as a brown liquid.

[0545] Step 10: To a solution of compound 11 (1.0 g, 1.7 mmol) in dichloromethane (20 mL) was added triethylamine (0.51 g, 0.70 mL, 5.1 mmol). Mesyl chloride (0.39 g, 0.26 mL, 3.4 mmol) was added dropwise to this solution at 0 °C. The reaction was stirred at room temperature for 30 minutes. The progress of the reaction was monitored by TLC. After completion of the reaction, it was concentrated, and the crude product was purified by column chromatography (alumina-neutral) using methanol (1-2%) in dichloromethane as the eluent to give compound 12 (0.7 g, 62%) as a brown liquid.

[0546] Step 11: To a solution of compound 13 (53 mg, 0.3 mmol) in DMF (2 mL) was added sodium hydride (12 mg, 60% in mineral oil, 0.3 mmol) at 0° C. The reaction mixture was stirred at room temperature for 10 minutes. To the reaction mixture was added compound 12 (100 mg, 0.15 mmol) in DMF (1 mL) at 0° C. The reaction was stirred at room temperature for 2 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was quenched with saturated ammonium chloride solution and extracted with ethyl acetate (3×5 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and filtered. The crude product was purified by preparative HPLC using acetonitrile and 0.1% TFA to give compound 14 (20 mg, 18%) as a brown liquid.

[0547] Step 12: A solution of compound 14 (20 mg, 0.02 mmol) in 6N hydrochloric acid (0.5 mL) was stirred overnight at room temperature. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC to give the product H2bp18c6-off macrocycle-ethylsulfideamine (6 mg, 36%) as a sticky solid. LC-MS APCI: calculated for C29H43N5O8S 621.75; observed m / z [M+H] + 622.2. Purity by LC-MS: 97.94% RT: 1.38. Purity by HPLC: 94.11% RT: 2.94. 1 H NMR (400MHz, DMSO-d6): δ11.02-11.00(m,2H), 8.21(s,2H), 8.16-8.09(m,4H), 7.93-7.90(m,2H), 4.78-4. 75(m,4H), 4.15-3.93(m,9H), 3.56-3.50(m,14H), 2.97-2.96(m,2H), 2.81-2.79(m,2H), 2.70-2.67(m,2H).

[0548] [ka]

[0549] Step 1: Compound 14 (100 mg, 0.15 mmol) was added to a cold solution of HCl in methanol (2 mL, 4N) and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure to give compound 2 (55 mg, 64%) as a yellow liquid.

[0550] Step 2: To a solution of compound 15 (50 mg, 0.08 mmol) and triethylamine (24 mg, 0.24 mmol) in dry dichloromethane (2 mL) was added carbon disulfide (12 mg, 0.16 mmol). The vial was subjected to MW irradiation (150 W power) at 90 °C for 30 min. The reaction mixture was then diluted with dichloromethane (10 mL) and washed successively with water (5 mL), 1 M HCl (5 mL), and water (5 mL), and dried over anhydrous sodium sulfate. After concentration, the crude product was purified by flash chromatography on silica gel (230-400 mesh) using 0-10% methanol in dichloromethane as the eluent to give compound 1616 (20 mg, 38%) as a yellow solid.

[0551] Step 3: A solution of compound 16 (20 mg, 0.03 mmol) in hydrochloric acid (6N, 0.5 mL) was stirred at room temperature overnight. After completion of the reaction, it was concentrated under reduced pressure. The residue was purified by preparative HPLC to give H2bp18c6-off macrocycle-ethyl sulfide NCS (6 mg, 31%) as a white solid. LC-MS APCI: calculated for C30H41N5O8S2: 663.81; observed m / z [M+H] + 664.2. Purity by LC-MS: 99.94% RT: 1.41. Purity by HPLC: 98.77% RT: 2.76. 1 H NMR(400MHz,DMSO-d6):δ 9.78(s,1H), 8.10(s,4H), 7.78(d,J=6.00Hz,2H), 4.69(s,4H), 3.96-3.52(m,23H), 2.85(t,J=6.40Hz,2H), 2.70(t,J=8.00Hz,2H).

[0552] [ka]

[0553] Step 1: To a solution of compound 17 (1.0 g, 1.7 mmol) in dichloromethane (20 mL) was added triethylamine (0.51 g, 0.70 mL, 5.1 mmol). Mesyl chloride (0.39 g, 0.26 mL, 3.4 mmol) was added dropwise to this solution at 0 °C. The reaction was stirred at room temperature for 30 minutes. The progress of the reaction was monitored by TLC. After completion of the reaction, it was concentrated, and the residue was purified by column chromatography (alumina-neutral) using methanol in dichloromethane (1-2%) as the eluent to give compound 18 (0.7 g, 62%) as a brown liquid.

[0554] Step 2: To a solution of compound 19 (65 mg, 0.3 mmol) in DMF (2 mL) was added sodium hydride (12 mg, 60% in mineral oil, 0.3 mmol) at 0° C. The resulting reaction mixture was stirred at room temperature for 10 minutes. To the reaction mixture was added compound 18 (100 mg, 0.15 mmol) in DMF (1 mL) at 0° C. The reaction was stirred at room temperature for 2 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, it was concentrated, and the residue was purified by preparative HPLC using acetonitrile and 0.1% TFA to give compound 20 (15 mg, 13%) as a brown liquid.

[0555] Step 3: A solution of compound 20 (15 mg, 0.02 mmol) in hydrochloric acid (6N, 0.5 mL) was stirred overnight at room temperature. After completion of the reaction, it was concentrated under reduced pressure. The residue was purified by preparative HPLC to give the product H2bp18c6-off macrocycle-pentylsulfidamine (4 mg, 33%) as a viscous liquid. LC-MS APCI: calculated for C32H49N5O8S 663.83; observed m / z [M+H] + 664.2. Purity by LC-MS: 90.31% RT: 1.43. Purity by HPLC: 90.69% RT: 3.11. 1H NMR(400MHz,DMSO-d6):δ 7.85-7.83(m,2H), 7.79-7.75(m,2H), 7.30(dd,J=6.80,24.00Hz,2H), 4.0 1-3.38(m,29H), 2.89-2.85(m,4H), 1.80-1.60(m,2H), 1.44-1.39(m,4H).

[0556] [ka]

[0557] Step 1: Compound 20 (120 mg, 0.15 mmol) was added with a cold solution of HCl in methanol (2 mL, 4N) and stirred for 2 h. The reaction mixture was concentrated under reduced pressure to give compound 21 (70 mg, 66%) as a yellow liquid.

[0558] Step 2: To a solution of compound 21 (70 mg, 0.1 mmol) and triethylamine (20 mg, 0.2 mmol) in dry dichloromethane (2 mL) was added carbon disulfide (15 mg, 0.2 mmol). The vial was subjected to MW irradiation (150 W power) at 90 °C for 30 min. The reaction mixture was then diluted with dichloromethane (10 mL) and washed successively with water (5 mL), 1 M HCl (5 mL), and water (5 mL), and dried over anhydrous sodium sulfate. After concentration, the crude product was purified by flash chromatography on silica gel (230-400 mesh) using 0-10% methanol in dichloromethane as the eluent to give compound 22 (30 mg, 40%) as a yellow solid.

[0559] Step 3: A solution of compound 22 (30 mg, 0.04 mmol) in hydrochloric acid (6N, 0.5 mL) was stirred at room temperature overnight. After completion of the reaction, it was concentrated under reduced pressure. The residue was purified by preparative HPLC to give H2bp18c6-off macrocycle-pentyl sulfide NCS (12 mg, 41%) as a white solid. LC-MS APCI: calculated for C33H47N5O8S2: 705.89; observed m / z [M+H] +706.2. Purity by LC-MS: 99.33% RT: 1.58. Purity by HPLC: 98.92% RT: 2.76. 1 H NMR1H-NMR(400MHz,DMSO-d6):δ13.40(s,1H), 9.90(s,1H), 8.17-8.09(m,4H), 7.78(d,J=6.80Hz,2H), 4.7 0(s,4H), 3.93-3.17(m,27H), 2.68-2.67(m,2H), 1.64-1.60(m,2H), 1.53-1.49(m,2H), 1.40-1.38(m,2H).

[0560] Example 26: Characterization of radioimmunoconjugates Table 1 summarizes the chelation efficiencies of the radioimmunoconjugates prepared in the Examples.

[0561] [Table 1]

[0562] four 225 The human serum stability of the Ac-chelator-mAb conjugate is measured according to the following method. 225 Ac-H2bp18c6-benzyl-phenyl-DBCO-PSMB127 225 Ac-H2bp18c6-off macrocycle-ethyl sulfide-DBCO-PSMB127 225 Ac-H2bp18c6-off macrocycle-pentyl sulfide-DBCO-PSMB127 225 Ac-H2bp18c6-benzyl-phenyl-BCN-PSMB127

[0563] To each solution of 900 μL of pooled human serum (BioIVT), 100 μL of one of the four conjugates (approximately 300 μCi / mg) was added. The individual conjugate solutions were mixed, and 10 μL aliquots were withdrawn at various time points over a 7-day period. Each 10 μL sample was transferred to a separate plastic vial containing 30 μL of 50 mM DTPA and allowed to stand at room temperature for 5 minutes. 10 μL of the mixture was then withdrawn and loaded onto an iTLC-SG column, which was developed with 10 mM EDTA. The dried iTLC-SG was left at room temperature overnight before scanning on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 migrated with the solvent to the solvent front. Stability results are listed in Table 2.

[0564] [Table 2]

[0565] Example 27: Radioimmunoconjugate cell binding assay The cell binding of azide-modified antibodies and azide-modified antibodies conjugated to chelators or radiometal complexes of the invention is compared to the parent antibody. Cells expressing the antigen target of the antibody are incubated with the parent antibody, azide-modified antibody, or conjugated antibody diluted in buffer, and then analyzed for cell binding by flow cytometry.

[0566] The examples and embodiments described herein are for illustrative purposes only and are not intended to be limiting of the scope of the invention as described above. Changes to the embodiments may be made without departing from the broad inventive concept thereof. It is understood, therefore, that the invention is not limited to the particular embodiments disclosed, but ... and the like, including modifications within the spirit and scope of the invention as defined by the appended claims. is understood to be intended to Various embodiments of the present invention are described below. 1. A chelator of formula (I): [ka] During the ceremony, Each of ring A and ring B is independently a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl, and each of ring A and ring B is independently a halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 Optionally substituted with one or more substituents selected from the group consisting of X and Each of Z1 and Z2 independently represents -(C(R 12 )2) m -or-(CH2) n -C(R 12 )(X)-(CH2) n - and Each X is independently -L1-R 11 and each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5; each p is independently 0 or 1; L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12 are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; Each R 13 are independently hydrogen or alkyl; R 14 , R 15 , R 16 and R 17each is independently hydrogen, alkyl or X; Alternatively, R 14 and R 15 and / or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring optionally substituted with X; provided that the chelator comprises at least one X, and when X is present on ring A or ring B, L1 is a linker or R 12 and R 14 ~R 17 at least one of which is not hydrogen. 2. A chelator of formula (II): [ka] During the ceremony, A1 is N or CR1 or absent; A2 is N or CR2; A3 is N or CR3; A4 is N or CR4; A5 is N or CR5; A6 is N or CR6 or absent; A7 is N or CR7; A8 is N or CR8; A9 is N or CR9, A 10 is N or CR 10 and However, three or less of A1, A2, A3, A4 and A5 are N, and A6, A7, A8, A9 and A 10 Not more than three of R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 each independently represents hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN, -OC(O)N(R 13 )2 and -X; or Alternatively, any two immediately adjacent R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 together with the atoms to which they are attached form a 5- or 6-membered substituted or unsubstituted carbocyclic or nitrogen-containing ring; Each of Z1 and Z2 independently represents -(C(R 12 )2) m -or-(CH2) n -C(R 12 )(X)-(CH2) n - and Each X is independently -L1-R 11 and each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5; each p is independently 0 or 1; L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12 are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; Each R 13 are independently hydrogen or alkyl; R 14 , R 15 , R 16 and R 17 each is independently hydrogen, alkyl or X; Alternatively, R 14 and R 15 and / or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring optionally substituted with X; provided that the chelator comprises at least one X and R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 When any one of the groups is X, L1 is a linker or R 12 and R 14 ~R 17 10. The chelator of claim 1, wherein at least one of is not hydrogen. 3. A chelator of formula (III): [ka] During the ceremony, Each A 11 are independently O, S, NMe, or NH; Each of Z1 and Z2 independently represents -(C(R 12 )2) m -or-(CH2) n -C(R 12 )(X)-(CH2) n - and Each X is independently -L1-R 11 and each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5; each p is independently 0 or 1; L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12 are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; Each R 13 are independently hydrogen or alkyl; R 14 , R 15 , R 16 and R 17 each is independently hydrogen, alkyl or X; Alternatively, R 14 and R15 and / or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring optionally substituted with X; Each R 18 are independently hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 )2 and -X; provided that the chelator contains at least one X and R 18 is X, L1 is a linker, or R 12 and R 14 ~R 17 10. The chelator of claim 1, wherein at least one of is not hydrogen. 4. The chelator is [ka] is selected from the group consisting of During the ceremony, L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12 are independently hydrogen, -CH3, or -CH2CH3, provided that at least one R 12 3. The chelator according to claim 1, wherein is -CH3 or -CH2CH3. 5.R 11 -NH2, -NCS, -NCO, -N3, alkynyl, cycloalkynyl, -C(O)R 13 , -COOR 13 , -CON(R 132) The chelator according to any one of the above 1 to 4, which is maleimide, acyl halide, tetrazine or trans-cyclooctene. 6.R 11 6. The chelator according to claim 5, wherein is cyclooctynyl or a cyclooctynyl derivative selected from the group consisting of bicyclononynyl (BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, biarylazacyclooctynyl (BARAC), dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO) and tetramethoxydibenzocyclooctynyl (TMDIBO). 7.R 11 7. The chelator according to claim 6, wherein is DBCO or BCN. 8.R 11 5. The chelator according to any one of 1 to 4 above, wherein the chelator comprises a targeting ligand, and the targeting ligand comprises an antibody or an antigen-binding fragment thereof, a scaffold protein, a small molecule, or an aptamer. 9.L1 is, [ka] is selected from the group consisting of 9. The chelator according to any one of 1 to 8 above, wherein in the formula, n is an integer of 0 to 10, preferably an integer of 1 to 4, and m is an integer of 0 to 12, preferably an integer of 0 to 6. 10. [ka] A chelator selected from the group consisting of: 11. The chelator according to any one of claims 1 to 10, wherein the chelator comprises a radioactive metal ion bound to the chelator by a coordinate bond, thereby forming a radioactive metal complex. 12. A radiometal complex comprising a chelator bound by a coordinate bond to an alpha-emitting radiometal ion, said radiometal complex having the structure of formula (Im): [ka] During the ceremony, M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion, more preferably actinium-225 ( 225 Ac), Each of ring A and ring B is independently a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl, and each of ring A and ring B is independently a halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 Optionally substituted with one or more substituents selected from the group consisting of X and Each of Z1 and Z2 independently represents -(C(R 12 )2) m -or-(CH2) n -C(R 12 )(X)-(CH2) n - and Each X is independently -L1-R 11 and each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5; each p is independently 0 or 1; L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; Each R 13 are independently hydrogen or alkyl; R 14 , R 15 , R 16 and R 17 each is independently hydrogen, alkyl or X; Alternatively, R 14 and R 15 and / or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring optionally substituted with X; provided that the radiometal complex contains at least one X, and when X is present on ring A or ring B, L1 is a linker or R 12 and R 14 ~R 17 A radioactive metal complex, wherein at least one of the 13. A radioactive metal complex of formula (II-m): [ka] During the ceremony, M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion, more preferably actinium-225 ( 225 Ac), A1 is N or CR1 or absent; A2 is N or CR2; A3 is N or CR3; A4 is N or CR4; A5 is N or CR5; A6 is N or CR6 or absent; A7 is N or CR7; A8 is N or CR8; A9 is N or CR9, A 10 is N or CR 10 and However, three or less of A1, A2, A3, A4 and A5 are N, and A6, A7, A8, A9 and A 10 Not more than three of R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 each independently represents hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 )2 and -X; or Alternatively, any two immediately adjacent R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 together with the atoms to which they are attached form a 5- or 6-membered substituted or unsubstituted carbocyclic or nitrogen-containing ring; Each of Z1 and Z2 independently represents -(C(R 12 )2) m -or-(CH2) n -C(R 12 )(X)-(CH2) n - and Each X is independently -L1-R 11 and each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5; each p is independently 0 or 1; L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12 are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; Each R 13are independently hydrogen or alkyl; R 14 , R 15 , R 16 and R 17 each is independently hydrogen, alkyl or X; Alternatively, R 14 and R 15 and / or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring optionally substituted with X; provided that the radiometal complex contains at least one X, and R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 When any one of the groups is X, L1 is a linker or R 12 and R 14 ~R 17 13. The radiometal complex of claim 12, wherein at least one of 14. A radioactive metal complex of formula (III-m): [ka] During the ceremony, M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion, more preferably actinium-225 ( 225 Ac), Each A 11 are independently O, S, NMe, or NH; Each of Z1 and Z2 independently represents -(C(R 12 )2) m -or-(CH2) n -C(R 12 )(X)-(CH2) n - and Each X is independently -L1-R 11 and each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5; each p is independently 0 or 1; L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12 are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; Each R 13 are independently hydrogen or alkyl; R 14 , R 15 , R 16 and R 17 each is independently hydrogen, alkyl or X; Alternatively, R 14 and R 15 and / or R 16 and R 17 together with the carbon atoms to which they are attached form a 5- or 6-membered cycloalkyl ring optionally substituted with X; Each R 18 are independently hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 , -(CH2) p COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN-OC(O)N(R 13 )2 and -X; provided that the radiometal complex contains at least one X and R 18 is X, L1 is a linker, or R 12 and R 14 ~R 17 13. The radiometal complex of claim 12, wherein at least one of 15. The radioactive metal complex is [ka] is selected from the group consisting of During the ceremony, M is actinium-225( 225 Ac), and L1 is absent or a linker; R 11 is a nucleophilic or electrophilic moiety, or R 11 comprises a targeting ligand, Each R 12 are independently hydrogen, -CH3, or -CH2CH3, provided that at least one R 12 15. The radiometal complex according to any one of the above 12 to 14, wherein is —CH3 or —CH2CH3. 16. A radioimmunoconjugate comprising a radiometal complex according to any one of 12 to 15 above conjugated to an antibody or an antigen-binding fragment thereof. 17. The antibody or antigen-binding fragment thereof is linked to the R of the radioconjugate via a triazole moiety. 11 17. The radioimmunoconjugate according to claim 16, wherein the radioimmunoconjugate is bound to 18. [ka] A radioimmunoconjugate having a structure selected from the group consisting of: During the ceremony, M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion, more preferably actinium-225 ( 225 Ac), L1 is a linker, mAb is an...

Claims

【Request 1】 【Chemical 1】 A chelator selected from the group consisting of: During the ceremony, L １ is not present or L １ is a substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl or heteroaryl, a polyethylene glycol (PEG) linker, a peptide linker, a sugar-based linker, or a cleavable linker which is a disulfide bond or valine-citrulline-p-aminobenzyl (PAB); or L 1 is: 【Chemistry 2】 is selected from the group consisting of In the formula, n is an integer from 0 to 10, and m is an integer from 0 to 12. R １１ But -NH ２ , -NCS, -NCO, -N ３ , alkynyl, cycloalkynyl, cyclooctynyl derivatives, C(O)R １３ , -COOR １３ , -CON(R １３ ) ２ , maleimide, acyl halide, tetrazine, or trans-cyclooctene, wherein the cyclooctynyl derivative is selected from the group consisting of bicyclononynyl (BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, biarylazacyclooctynyl (BARAC), dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO), and tetramethoxydibenzocyclooctynyl (TMDIBO), and each R １３ is independently hydrogen or alkyl.

2. R １１ 2. The chelator of claim 1, wherein is -NCS, cyclooctynyl, or a derivative thereof.

3. R １１ The chelator of claim 2, wherein is DBCO or BCN.

4. R １１ is further conjugated to a targeting ligand, wherein the targeting ligand is an antibody or antigen-binding fragment thereof, a scaffold protein, a small molecule, or an aptamer.

5. L １ but, 【Chemistry 3】 is selected from the group consisting of 5. A chelator according to any one of claims 1 to 4, wherein n is an integer from 0 to 10, preferably from 1 to 4, and m is an integer from 0 to 12, preferably from 0 to 6. 【Request 6】 【Chemical 4】 2. The chelator of claim 1 selected from the group consisting of:

7. 7. The chelator of any one of claims 1 to 6, wherein the chelator further comprises a radiometal ion bound to the chelator by a coordinate bond, thereby forming a radiometal complex. 【Request 8】 【Chemical 5】 A radioactive metal complex selected from the group consisting of: During the ceremony, M is actinium-225 ( ２２５ Ac), L １ is not present or L １ is a substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl or heteroaryl, a polyethylene glycol (PEG) linker, a peptide linker, a sugar-based linker, or a cleavable linker which is a disulfide bond or valine-citrulline-p-aminobenzyl (PAB); or L 1 is: 【Chemistry 6】 is selected from the group consisting of In the formula, n is an integer from 0 to 10, and m is an integer from 0 to 12. R １１ is -NH ２ , -NCS, -NCO, -N ３ , alkynyl, cycloalkynyl, cyclooctynyl derivatives, C(O)R １３ , -COOR １３ , -CON(R １３ ) ２ , maleimide, acyl halide, tetrazine or trans-cyclooctene, and the cyclooctynyl derivative is selected from the group consisting of bicyclononynyl (BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, biarylazacyclooctynyl (BARAC), dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO) and tetramethoxydibenzocyclooctynyl (TMDIBO); Each R １３ is independently hydrogen or alkyl.

9. A radioimmunoconjugate comprising the radiometal complex of claim 8 further conjugated to an antibody or antigen-binding fragment thereof.

10. The antibody or antigen-binding fragment thereof is linked to the R of the radioconjugate via a triazole moiety. １１ 10. The radioimmunoconjugate of claim 9, wherein the radioimmunoconjugate is bound to

11. 【Chemical 7】 【change】 10. The radioimmunoconjugate of claim 9 having a structure selected from the group consisting of: During the ceremony, M is a radioactive metal ion, preferably an α-emitting radioactive metal ion, more preferably actinium-225 ( ２２５ Ac), L １ is the linker, mAb is an antibody or antigen-binding fragment thereof; Each R １２ is hydrogen, radioimmunoconjugate.

12. the radioimmunoconjugate comprising: 【Chemistry 8】 is selected from the group consisting of 10. The radioimmunoconjugate of claim 9, wherein the mAb is an antibody or an antigen-binding fragment thereof, preferably an antibody or an antigen-binding fragment thereof that specifically binds to a tumor antigen, more preferably the mAb is selected from PSMB127, pertuzumab, cetuximab, panitumumab, herceptin and H11B6.

13. 10. A method for preparing a radioimmunoconjugate, comprising contacting a chelator according to claim 4 with a radioactive metal ion, thereby forming a radioactive metal complex bound to a targeting ligand.

14. 14. The method of claim 13, wherein the targeting ligand is an antibody or an antigen-binding fragment thereof.

15. A pharmaceutical composition comprising the radioimmunoconjugate of any one of claims 9 to 12 and a pharmaceutically acceptable carrier.

16. 16. The pharmaceutical composition of claim 15 for use in a method for selectively targeting neoplastic cells for radiation therapy in a subject in need thereof.

17. 16. The pharmaceutical composition of claim 15 for use in a method for treating a neoplastic disease or disorder in a subject in need thereof.

18. 16. The pharmaceutical composition of claim 15 for use in a method for treating cancer in a subject in need thereof.

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