Macrocyclic chelators and methods of use thereof

Abstract

To provide novel chelators that can bind radiometals, preferably α-emitting radiometals such as actinium-225(225Ac), and can be used to generate stable radioimmunoconjugates having high specific activity and high yield.SOLUTION: Provided are chelators of the following formula (I). Also provided are radiometal complexes containing an α-emitting radiometal ion bound to a macrocyclic chelator via coordinate bonding, and radioimmunoconjugates containing the radiometal complexes covalently linked to a targeting ligand such as an antibody. In the formula, each of a ring A and a ring B independently is a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl which may have a substituent, and each of Z1 and Z2 independently is -(C(R12)2)m- or -(CH2)n-C(R12)(X)-(CH2)n-.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is a continuation of U.S. Provisional Patent Application No. 62 / 846,044, filed May 10, 2019. No. 6,399,499, filed on Oct. 1, 2002, which claims the benefit of priority to U.S. Provisional Patent Application No. 6,399,499, filed on Oct. 1, 2002, which application is hereby incorporated by reference in its entirety for all purposes. and is hereby incorporated by reference for the purposes of this application.

[0002] (Reference to electronically submitted sequence listing) This application is filed under the name "JBI6072WOPCT1_SeqListing.tx t", created on May 1, 2020, with a size of 26kb, in ASCII format. Sequence listings submitted electronically via EFS-Web as formula sequence listings are included. The sequence listing submitted by JP 2004-2023634 is incorporated herein by reference in its entirety. To be incorporated. [Background technology]

[0003] Alpha particle-emitting radionuclides are promising candidates for cancer therapy due to their combination of high energy and short-range action. Very promising, offering the potential for potent killing that is largely localized to tumor cells (Kim,YSand MWBrechbiel,An overview o f targeted alpha therapy.Tumour Biol,201 2.33(3):p.573-90). Antibodies, scaffold proteins, small molecule ligands Targeted delivery of alpha emitters using alpha emitters, aptamers, or other binding moieties specific for cancer antigens We provide a method for selective delivery of radionuclides to tumors, enhancing their efficacy and reducing the In general practice, the binding moiety is an alpha-emitting radiation. Many such examples are , using a monoclonal antibody (mAb) as a targeting ligand. , producing what are known as radioimmunoconjugates.

[0004] Actinium-225( 225 Ac) are alpha emitters that are of particular interest for medical applications. It is a radioisotope (Miederer et al., Realizing the potential of the Actinium-225 radionucli de 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 gate generation but not to disrupt the circulating pharmacokinetics of a delivery vehicle such as an antibody. It is short enough to match the voice. 225 Radioimmunoconjugates of Ac Of particular interest are: 225 Ac is a stable isotope 209 Before reaching Bi Its potency increases as it decays through a series of processes, ultimately emitting four alpha particles. Other radioisotopes of interest are suitable for gamma irradiation and radiotherapy. Lutetium-177 ( 177 Lu). 177 Lu-labeled peptides showed reduced damage to normal tissues, 177 Lu labeling is a treatment and It has been shown that a single radiolabeled agent can be used for both imaging and imaging (Kwek keboom DJ,et al.[ 177 Lu-DOTA 0 ,Tyr 3 ]octreo tate:comparison with[ 111 In-DTPA 0 ]octreot ide in patients.Eur J Nucl Med.2001;28:p 1319-1325). Other radioisotopes used in therapeutic applications include, for example, β ray emitters 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, 15 2 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 P b. 212 Bi, 213 Bi, 223 Ra, 255 Fm and 227 Th etc. Other radioisotopes used in imaging applications include, for example: 62 Cu, 64 Cu, 6 7 Ga, 68 Ga, 86 Y,89 Zr and 111 Examples of gamma-ray emitting radioisotopes include In can be done.

[0005] Currently the most widely used chelators for actinium-225 and lanthanides are , DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) ; tetraxathen), and previous clinical and preclinical programs have included actinium chelates For the synthesis, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ( However, the chelation of actinium with DOTA has It is known that there may be challenges (Deal, KA, et al., Improv ed in vivo stability of actinium-225 mac rocyclic complexes.J Med Chem,1999.42(15 For example, DOTA can be used as a targeting ligand, such as a protein or antibody. When bound to a chelation bond, DOTA:actinium-225 chelation ratios of up to >500:1 only possible, requiring either harsh conditions or high levels of DOTA per antibody. Other macrocyclic chelators for lanthanides and actinium-225 are e.g. For example, International Patent Application Publication No. 2018 / 183906, Thiele et al. "A n Eighteen-Membered Macrocyclic Ligand f or Actinium-225 Targeted Alpha Therapy”A ngew.Chem.Int.Ed.(2017) 56,14712-14717, R oca-Sabio et al. “Macrocyclic Receptor Ex hibiting Unprecedented Selectivity for L ight Lanthanides” J.Am.Chem.Soc.(2009)131 , 3331-3341.

[0006] Site specificity is achieved by site-specific methods compared to random conjugation. It has been demonstrated that antibody-drug conjugates can enhance both the efficacy and safety of C. This has become a major focus area in the field of antibody-drug conjugates (ADCs). (Agarwal, P. and CR Bertozzi, Site-speci fic antibody-drug conjugates:the nexus o f bioorthogonal chemistry, protein engine ering,and drug development,Bioconjug Che m,2015.26(2):p.176-92). Similar safety and efficacy benefits are seen in It is believed that this can be achieved with respect to radioactive immunoconjugates. Summary of the Invention [Means for solving the problem]

[0007] Therefore, radioactive metals, preferably actinium-225 ( 225 Ac) and other alpha rays A stable compound that binds released radioactive metals and has high specific activity and high yield. Novel chelators are now described that can be used to generate radioimmunoconjugates. There is a need in the art. The present invention provides a method for the preparation of soluble cellulose, regardless of specific activity or the most common metal impurities. Alpha-emitting radioactive metals, especially 225A macromolecule capable of binding to radioactive metals such as Ac This need is met by providing a ring chelator. The chelator of the present invention can be used with antibodies, Targeting ligands such as proteins, aptamers, and small molecules are preferably synthesized using "click chemistry" By conjugating to a targeting ligand in a site-specific manner using To generate radioimmunoconjugates with high in vitro and in vivo stability The chelators of the present invention can be conjugated to targeting ligands. and radioimmunoconjugates produced by the method are useful for targeted radiotherapy of neoplastic cells and and / or for use in targeted therapy, such as targeted therapy of neoplastic diseases or disorders, including cancer. can.

[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. ring A and ring B are each independently selected from halo, alkyl, alkenyl, cycloalkenyl, alkyl, 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 the group consisting of X and optionally substituted with one or more substituents selected from 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 is the targeting ligand Including, 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 independently is hydrogen, alkyl, or X mosquito, Alternatively, R 14 and R 15 and / or R 16 and R 17 are combined together with the carbon atoms form a 5- or 6-membered cycloalkyl ring optionally substituted with X. Formation However, the chelator contains at least one X, and when X is present on ring A or ring B, L 1 is a linker or R 12 and R 14 ~R 17 At least one of them is hydrogen There is no.

[0010] In an alternative embodiment, each of ring A and ring B is optionally substituted heterocyclyl. It is contemplated that the ring may be, for example, an 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, and A 8, A9 and A 10 Not more than three of R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 Each of these is independent. and 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, -C N, -OC(O)N(R13 )2 and -X; Alternatively, any two directly adjacent R1, R2, R3, R4, R5, R6, R7, R 8, R9 and R 10 together with the atoms to which they are attached form a 5- or 6-membered substituent or forms an 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 is the targeting ligand Including, 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 independently is hydrogen, alkyl, or X mosquito, Alternatively, R 14 and R 15 and / or R 16 and R 17 are combined together with the carbon atoms form a 5- or 6-membered cycloalkyl ring optionally substituted with X. Formation provided that the chelator contains at least one X and R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 If any one of L1 is X, then L1 is a linker. do.

[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 is the targeting ligand Including, 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 independently is hydrogen, alkyl, or X mosquito, Alternatively, R 14 and R 15 and / or R 16 and R 17 are combined together with the carbon atoms form a 5- or 6-membered cycloalkyl ring optionally substituted with X. Formation Each R 18 are independently hydrogen, halo, alkyl, alkenyl, cycloalkyl, cyclo Alkenyl, 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] where: L1 is absent or a linker, R 11 is a nucleophilic or electrophilic moiety, or R 11 is the targeting ligand Including, Each R 12are 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, alkyl Nyl, cycloalkynyl, C(O)R 13 , -COOR 13 , -CON(R 13 )2, Ma The compound is selected from the group consisting of tetrazine, trans-cyclooctene, tetraimide, acyl halide, tetrazine, and trans-cyclooctene.

[0018] In certain embodiments, R 11 is cyclooctynyl or bicyclononynyl onynyl, BCN), difluorinated cyclooctynyl (DIF O), dibenzocyclooctynyl (DIBO), keto-DIBO , biarylazacyclooctynonyl (BARAC), dibenzyl Dibenzoazacyclooctynyl (DIBAC, DBCO, ADIB O), dimethoxyazacyclooctynyl (DIMAC), di Fluorobenzocyclooctynyl (difluorobenzocyclooctynyl, DIFBO), monobenzyl Monobenzocyclooctynyl (MOBO) and tetramethoxydibenzo From the group consisting of tetramethoxy dibenzocyclooctynyl (TMDIBO) A cyclooctynyl derivative selected from the group consisting of:

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

[0020] In some embodiments, R 11 comprises a targeting ligand, the targeting ligand being antibodies or antigen-binding fragments, scaffold proteins, small molecules, or aptamers Includes.

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

[0022] In another aspect, the present invention comprises a radioactive metal ion bound to a chelator by a coordinate bond. , relates to radiometal complexes containing the chelators of the present invention.

[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. ring A and ring B are each independently selected from halo, alkyl, alkenyl, cycloalkenyl, alkyl, 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 optionally substituted with one or more substituents selected from 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 is the targeting ligand Including, 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 independently is hydrogen, alkyl, or X mosquito, Alternatively, R 14 and R 15 and / or R 16 and R 17 are combined together with the carbon atoms form a 5- or 6-membered cycloalkyl ring optionally substituted with X. Formation However, when the radioactive metal complex contains at least one X, and X is present in ring A or ring B, , L1 is a linker, or R 12 and R 14 ~R 17 At least one of them is water Not original.

[0025] In an alternative embodiment, each of ring A and ring B is optionally substituted heterocyclyl. It is contemplated that the ring may be, for example, an 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, and A 8, A9 and A 10 Not more than three of R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 Each of these is independent. and 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, -C N-OC(O)N(R 13 )2 and -X; Alternatively, any two directly adjacent R1, R2, R3, R4, R5, R6, R7, R 8, R9 and R 10 together with the atoms to which they are attached form a 5- or 6-membered substituent or forms an 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 is the targeting ligand Including, 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 independently is hydrogen, alkyl, or X mosquito, Alternatively, R 14 and R 15and / or R 16 and R 17 But they are combined together with the carbon atoms form a 5- or 6-membered cycloalkyl ring optionally substituted with X. Formation However, the radioactive metal complex contains at least one X, and R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 If any one of the is X, L1 is the 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): the law of nature,

[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 11is the targeting ligand Including, 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 independently is hydrogen, alkyl, or X mosquito, Alternatively, R 14 and R 15 and / or R 16 and R 17 But they are combined together with the carbon atoms form a 5- or 6-membered cycloalkyl ring optionally substituted with X. Formation Each R 18 are independently hydrogen, halo, alkyl, alkenyl, cycloalkyl, cyclo Alkenyl, 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 If X, L1 is the 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 A c).

[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 an alpha-emitting radioactive metal ion. Cu-225( 225 Ac), and L1 is absent or a linker; R 11 is a nucleophilic or electrophilic moiety, or R 11 is the targeting ligand Including, 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: 11 via a targeting ligand, preferably an antibody or an immunoconjugate comprising a chelator of the invention covalently attached to an antigen-binding fragment thereof; Regarding.

[0035] In yet another general aspect, the invention provides a compound comprising: 11 via a targeting ligand, preferably an antibody or an antigen-binding fragment thereof. Concerning epidemic conjugates.

[0036] In one embodiment, the radioimmunoconjugate is linked to a targeting ligation via a triazole moiety. The radiometal complexes of the present invention are covalently bound to a radioactive metal bond, particularly an antibody or antigen-binding fragment thereof. include.

[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 and mAb is an antibody or an antigen-binding fragment thereof. Preferably, the mAb binds to tumor cells, more preferably to prostate-specific membrane antigen (prostate-specific membrane antigen). pecific membrane antigen, PSMA), BCMA, Her2, EGFR, KLK2, C D19, CD22, CD30, CD33, Cd79b and nectin-4 An antibody or antigen-binding fragment that specifically binds to a tumor antigen of interest, 12 teeth , 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 combining a chelator or radiometal complex of the invention with a targeting ligand. Covalently bonded to the R of the chelator or radiometal complex, preferably 11 via anti The immunoconjugates of the present invention include covalently linking the antibody or antigen-binding fragment thereof to a The present invention relates to a method for preparing a radioimmunoconjugate or radioimmunoconjugate.

[0040] In a specific embodiment, the method for preparing a radioimmunoconjugate of the present invention comprises: (i) a polypeptide covalently linked to a first click reaction partner (e.g., an azide group); and providing a modified polypeptide comprising the polypeptide (e.g., an antibody or antigen-binding fragment thereof). And, (ii) a second Click reaction partner (e.g., an alkynyl group or a cycloalkynyl providing a chelator complex comprising a chelator of the present invention covalently bonded to a chelate group; (iii) converting a first click reaction partner (e.g., an azide group) into a second click reaction partner; It is possible to react with a partner (e.g., an alkynyl group or a cycloalkynyl group). and contacting the modified polypeptide with a chelator complex under conditions that result in the formation of a polypeptide- forming a chelator complex (i.e., an immunoconjugate); (iv) contacting the polypeptide-chelator complex with a radioactive metal ion, thereby releasing Preparing a radioimmunoconjugate (a radioimmunoconjugate is a compound containing a radioactive metal ion) A polypeptide labeled with an alpha-emitting radioactive isomer, e.g., an alpha-emitting radioactive isomer, which is coordinatively bound to a chelator. (including modified antibodies or antigen-binding fragments thereof labeled with radioactive metal ions) , a "one-step direct radiolabeling" method (e.g., as shown in Figure 2C), which includes:

[0041] According to a particular embodiment, step (iv) is carried out under metal-free conditions. In some embodiments, 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) a modified antibody comprising an antibody or antigen-binding fragment thereof covalently bound to an azido group; providing an antibody or antigen-binding fragment thereof; (ii) a compound of the present invention comprising an α-emitting radioactive metal ion bound to a chelator by a coordinate bond; providing a radioactive complex, wherein the chelator is an alkynyl or cycloalkynyl group; covalently bonded, providing (iii) allowing the azide group to react with an alkynyl or cycloalkynyl group; The modified antibody or antigen-binding fragment thereof is contacted with a radioactive complex under conditions and preparing a radioimmunoconjugate by "click radiolabeling." " method (e.g., as shown in Figure 2D).

[0043] In some embodiments, the cycloalkynyl group is cyclooctynyl, or bicyclono Difluorinated cyclooctynyl (BCN), dibenzocyclooctynyl (DIFO), DIBO, keto-DIBO, biarylazacyclooctynonyl (BARAC), Dibenzoazacyclooctynyl (DIBAC), dimethoxyazacyclooctynyl (DI MAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl tetramethoxydibenzocyclooctynyl (MOBO) and tetramethoxydibenzocyclooctynyl (TMDIBO) The cyclooctynyl derivative is selected from the group consisting of:

[0044] In another general aspect, the invention provides the radioimmunoconjugates and pharmaceutical compositions of the invention. The present invention relates to a pharmaceutical composition comprising a physiologically acceptable carrier. The pharmaceutical composition also includes one or more The composition may comprise a pharmaceutically acceptable carrier.

[0045] In yet another general aspect, the present invention provides a method for the preparation of a radioimmunoassay of the present invention for targeted radiotherapy. The present invention relates to methods of using the immunoconjugates and pharmaceutical compositions.

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

[0047] In one embodiment, the present invention provides a method for treating a neoplastic disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a tumor suppressant. A method for treating a sexual disease or disorder, comprising administering to a subject a pharmaceutical composition of the present invention. Well, we provide a method.

[0048] The above Summary of the Invention and the following Detailed Description of the Invention should be read in conjunction with the accompanying drawings. The invention will be better understood by reading the accompanying drawings, in which: It should be understood that the present invention is not limited to the above.

[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 referenced 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 or the like which is included in the specification is indicative of the context of this invention. Such discussion is intended to provide a basis for understanding whether any or all of these matters are disclosed or implied. is admitted to be part of the prior art to any claimed invention. isn't it.

[0052] Unless otherwise defined, all technical and scientific terms used herein are defined by the It has the same meaning as commonly understood by a person skilled in the art to which the invention pertains. Unless otherwise specified, specific terms referred to herein shall have the meanings ascribed to them herein. All patents, published patent applications and publications cited herein are hereby incorporated by reference. No. 6,027,797, filed Dec. 1, 2004, which is incorporated herein in its entirety as if set forth herein.

[0053] As used in this specification and the appended claims, the singular forms "a," "an," and " It should be noted that "the" includes multiple referents unless the context makes clear otherwise. There is.

[0054] Throughout this specification and the claims that follow, unless the context otherwise requires, the term "comprises" "comprises" and variations such as "comprises" and "comprising" are used to refer to Contains the specified integer or step or group of integers or steps, but does not include any other integer or step. It will be understood that the term "integer" means that no step or integer or group of steps is excluded. As used herein, the term "comprising" is used in conjunction with the term "containing." "including" or "including" may be substituted or are sometimes used herein. When used, it can be substituted with the term "having."

[0055] As used herein, "consisting of" means that a claim element As used herein, the term "excludes" any element, step, or ingredient not specified in the preceding paragraph. When "consisting essentially of" is used, The present invention does not exclude materials or steps that do not substantially affect the novel characteristics. or when used herein in connection with an embodiment, to vary the scope of the disclosure, " "comprising," "containing," "including," and " Any of the above terms "comprising" or "comprising" may be replaced with the terms "consisting of" or "consisting essentially of." It can be replaced.

[0056] As used herein, the conjunctive term "and / or" between multiple listed elements means It is understood to encompass both individual and combined options. For example, When elements are connected by "and / or," the first option is the first option without the second option. The second option refers to the applicability of the first element. The second option refers to the applicability of the second element without the first element. The third option indicates that the first and second elements are applicable together. Any one of these alternatives is within the meaning and therefore is used herein. When two of the options are used, it is understood that the requirement of the term "and / or" is met. The above simultaneous applicability is also included in the meaning and therefore meets the requirements of the term "and / or." It is understood that

[0057] To assist the reader of this application, the description is divided into various paragraphs or sections. These separations are not intended to be limiting unless they are followed by a paragraph or a section. Separating a section or embodiment entity from another paragraph or section or embodiment entity On the contrary, those skilled in the art will appreciate that the description herein has broad application. It includes all possible combinations of paragraphs, paragraphs, and sentences. It will be understood that the discussion of any embodiment is meant to be exemplary only. It is to be understood that the scope of the present disclosure, including the claims, is limited to these examples. It is not intended to be suggestive.

[0058] Unless otherwise specified, all numerical values, such as concentrations or concentration ranges, described herein are the total In all cases, the terms "about" and "about" should be understood as being modified by the term "about." Therefore, numerical values ​​typically include ±10% of the stated value. For example, "10 The reference to "times" includes 9 times and 11 times. As used herein, the use of numerical ranges is Unless the context clearly indicates otherwise, all values ​​within the range, including integers and fractions of values, are Any possible subranges of any of the above, expressly including all individual numerical values ​​within that range.

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

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

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

[0062] As used herein, the term "alkoxy" refers to an -O-alkyl group or an -OR group. wherein R is alkyl, alkyl being as defined above. is attached to the parent molecule through an oxygen atom. Examples of alkoxy include methoxy, ethoxy, and propoxy (e.g., n-propoxy, isopropoxy), butoxy (e.g., n- butoxy, isobutoxy, tert-butoxy), pentyloxy (e.g., n-pentyl Alkoxy groups include alkoxy, isopentyloxy, neopentyloxy, etc. can be unsubstituted or substituted with one or more suitable substituents. Similarly, "alkylthio" or "thioalkoxy" refers to the group -SR, where R is Alkyl attached to the parent molecule through a sulfur bridge, e.g., -S-methyl, -S-ethyl, etc. Representative examples of alkylthio include -SCH3 and -SCH2CH3. Examples include, but are not limited to:

[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. Point.

[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 an alkyl group having at least two carbon atoms, such as from 2 to 10 carbon atoms. and a straight or branched chain hydrocarbon containing at least one double bond between two carbon atoms. Alkenyl refers to a chain having one carbon-carbon double bond or two, three, four or more carbon atoms. Alkenyl groups may have multiple carbon-carbon double bonds, such as a carbon-carbon double bond. Examples of the aryl group include methenyl, ethenyl, propenyl, butenyl, etc. Not limited to.

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

[0073] As used herein, the terms "alkynyl," "alkyne group," or "alkyne moiety" has at least two carbon atoms, such as 2 to 10 carbon atoms, and has a bond between the two carbon atoms An alkynyl group refers to a straight or branched hydrocarbon chain containing at least one triple bond. The alkynyl group may be a terminal alkynyl group or a cyclic alkynyl group. A terminal alkyne is a group having a triple-bonded carbon atom. "Cyclic alkyne" or "cycloalkynyl" has at least one hydrogen atom bonded to it. A "cycloalkyl" is a cycloalkyl ring containing at least one triple bond between two carbon atoms. Examples of cyclic alkyne or cycloalkynyl groups include cyclooctyne and cyclooctyne. Derivatives such as bicyclononyne (BCN), difluorinated cyclooctyl difluorinated cyclooctynyl (DIFO), dibenzocyclooctyne octynyl, DIBO), keto-DIBO, biarylazacyclooctynone (biarylazacy clooctynonyl, BARAC), dibenzoazacyclooctyne, DIBAC), dimethoxyazacyclooctynyl (DIMAC) ), difluorobenzocyclooctyne (DIFBO), mono Monobenzocyclooctynyl (MOBO) and tetramethoxy DIB O(TMDIBO).

[0074] The term "amino" refers to NH2. The term "alkylamino" refers to a hydrogen atom attached to a nitrogen. An alkylamine group refers to an amino group in which one or both of the radicals are substituted with an alkyl group. It can be represented as —NR 2 , where each R is independently hydrogen or an alkyl group. For example, alkylamines include methylamine (-NHCH3), dimethylamine (-N(C As used herein, the term " "Aminoalkyl" refers to branched and straight chain saturated fatty acids substituted with one or more amino groups. Representative examples of aminoalkyl groups include: Examples include -CH2NH2, -CH2CH2NH2 and -CH2CH(NH2)CH3. , but not limited to these.

[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 -C(O)NH2, -C These include, but are not limited to, -(O)NHCH3 and -C(O)N(CH3)2. .

[0076] The terms "hydroxylalkyl" and "hydroxyalkyl" are used interchangeably and are intended to mean a group consisting of hydroxylalkyl, hydroxy ... Alkyl refers to an alkyl group substituted with one or more hydroxyl groups. The hydroxyl alkyl may be a straight chain aliphatic hydrocarbon. Examples of hydroxyl alkyl include hydroxymethyl. Examples include hydroxyethyl (-CH2OH), hydroxyethyl (-CH2CH2OH), etc. Not limited to these.

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

[0078] As used herein, the term "heterocyclyl" refers to an alkyl group containing at least one of the following: sulfur, oxygen, or nitrogen. The term "hydrocarbon" includes stable monocyclic and polycyclic hydrocarbons containing at least one heteroatom ring member. As used herein, the term "heteroaryl" refers to a heteroaryl group containing at least one heteroatom, such as sulfur, oxygen, or nitrogen. Heteroaryl includes stable monocyclic and polycyclic aromatic hydrocarbons containing heteroatom ring members. It may be monocyclic or polycyclic, for example bicyclic or tricyclic. Each ring of the alkyl or heteroaryl group may contain one or two oxygen or sulfur atoms and / or It may contain 1 to 4 nitrogen atoms, provided that the total number of heteroatoms in each ring is 4 or less. and each ring has at least one carbon atom. A heteroaryl group must contain at least one fully aromatic ring, but may contain other fused or unfused rings. The ring may be aromatic or non-aromatic. A heterocyclyl or heteroaryl group is a heterocyclyl. attached to any available nitrogen or carbon atom of any ring of the aryl or heteroaryl group Preferably, the term "heteroaryl" refers to a heteroaryl group having at least one ring. 5- or 6-membered monocyclic groups containing at least one heteroatom (O, S, or N) in each ring, and refers to a 9- or 10-membered bicyclic group, the heteroatom-containing ring preferably containing 1, 2 or 3 It has heteroatoms, more preferably 1 or 2 heteroatoms selected from O, S and / or N. The nitrogen heteroatom of a heteroaryl may be substituted or unsubstituted. Additionally, the nitrogen and sulfur heteroatoms of a heteroaryl can be optionally oxidized (also referred to as aryl). That is, 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 an alkyl group. Kill 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. "Imido" refers to a maleimide group covalently bonded to another group or molecule. The alkyl group may, for example, be N-linked.

[0084] [ka]

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

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

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

[0088] [ka] However, the addition of moieties, functional groups or substituents to the core, parent or backbone structure, such as chelators or targeting ligands, It is used in structural formulas herein to indicate the bond that is the point of attachment of a substituent.

[0089] When any variable occurs more than once in any structure or formula of a compound, each occurrence Its definition in one occurrence is independent of its definition in all other occurrences. For example, if a group is shown to be substituted with 0 to 3 R groups, the group may be substituted with up to 3 R groups. R is optionally substituted, and in each instance, 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, A substituent such as may be attached to any atom on the ring.

[0091] As used herein, the term "radioactive metal ion" or "radioactive metal ion" refers to a metal ion that is refers to one or more isotopes of an element that emit particles and / or photons. Any chelator known to those skilled in the art may be used in the present invention. Examples of suitable radioactive metals 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 R h, 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, 1 77 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 25 5 Preferably, the radioactive metal ion is a therapeutic "Therapeutic radiators" refer to radioactive metal ions useful in therapeutic applications. Examples of radiation include, but are not limited to, beta or alpha emissions, e.g., 32 P, 47 Sc, 67 C u, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 1 31 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 A u, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 22 5 Ac, 255 Fm and227 Preferably, the radiation used in the present invention is The radioactive metal ions are alpha-emitting radioactive metal ions, such as actinium-225 ( 225 A c).

[0092] As used herein, the term "chelator" or "chelating agent" refers to a compound that binds a metal, preferably a radioactive material. In an exemplary embodiment, the term "radiative metal" refers to a chemical compound that can be chelated by a coordinate bond. The chelator may include one or more heteroatoms, e.g., oxygen and / or nitrogen, as ring atoms. Preferably, the chelator is a 4,13-diaza-18-crown. It is a derivative of -6.

[0093] As used herein, a "radiometal complex" refers to a radiometal ion associated with a chelator. Typically, the radioactive metal ion is bound to the chelator by a coordinate bond or The heteroatoms of the macrocycle are involved in the coordination of the radioactive metal ion to the chelator. The chelator may be substituted with one or more substituents, and may include one or more The substituents may also provide a radiometal bond to the chelator in addition to or instead of the heteroatom of the macrocycle. It can participate in coordination bonding of the on.

[0094] As used herein, the term "click chemistry" refers to the synthesis of small units containing reactive groups. Chemistry tailored to rapidly and reliably create covalent bonds by joining This refers to the chemical principle introduced by Sharpless that explains al., Angewandte Chemie International Edit ion (2001) 40:2004-2021). Click chemistry does not refer to a specific reaction, but includes reactions that mimic reactions found in nature. In some embodiments, the click chemistry reaction is It is modular, has a wide range, provides high chemical yields, and produces no inert by-products. It is stereospecific and exhibits a large thermodynamic driving force that favors reactions with a single reaction product. In some embodiments, the method may be performed under physiological conditions. The reaction can be carried out under simple reaction conditions and is readily available. Uses decomposing materials and reagents, without using toxic solvents or non-toxic or easily removed materials such as water. using solvents that are suitable for this purpose and / or by non-chromatographic methods such as crystallization or distillation It provides for simple product isolation.

[0095] Click chemistry reactions are rarely seen in naturally occurring biomolecules and have not been shown to be effective in biomolecules. Utilizing reactive groups that are chemically inert to the molecule but not to the click chemistry partner When reacted together, the reaction occurs under biologically relevant conditions, e.g., excessive heating and / or This can occur efficiently under certain cell culture conditions, such as in the absence of excessively strong reagents. The click chemistry reaction involves click reaction partners that can react with each other. At least two molecules are required to be reactive with each other. The handles are 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 In another embodiment, the click reaction partner is a reactive diene. and suitable tetrazine dienophiles. Cyclonene or biscyclononene is a suitable tetrazine dienophile as a click reaction partner. In yet another embodiment, the tetrazole is activated in the presence of ultraviolet light. When paired with an unmodified alkene, the click reaction pair is called a "photo-click" reaction pair. In another embodiment, the click reaction partners are cysteine ​​and malic acid. For example, a cysteine ​​(e.g., GGGC) derived from a peptide can be converted to a chelate. It can be reacted with maleimides associated with other amines (e.g., NOTA). Suitable click chemistry handles are known to those skilled in the art (e.g., Spicer et al.,Selective chemical protein modification cation.Nature Communications.2014;5:p.47 40) In other embodiments, the click reaction partners are silyl groups such as phosphines and azides. In another embodiment, the Click reaction partner is a Taudinger ligation component. Dienes (e.g., tetrazines) and alkenes (e.g., trans-cyclooctene Diels-Alder reaction components such as trans-cyclooctene (TCO) or norbornene Exemplary click reaction partners are described in U.S. Patent Application Publication No. 2013026651 2 and WO 2015073746, and both click reaction parts The relevant discussion of toners is incorporated herein by reference.

[0096] According to a preferred embodiment, the click chemistry reaction is a click chemistry pair or As reaction partners, an azide group and an alkyne group are preferred, and more preferably, a strained alkyne group. Utilizing a quinone group, for example, a cycloalkyne such as cyclooctyne or a cyclooctyne derivative In certain embodiments, the click chemistry reaction is carried out using a 1,2,3-triazole derivative. Hydration between azide (-N3) and alkyne or alkyne moiety to form a linker The click chemistry between alkynes and azides is a cycloaddition or a 1,3-dipolar cycloaddition. The Street reaction typically involves the addition of a copper catalyst to promote the 1,3-cycloaddition reaction. This reaction requires a procedure known as copper-catalyzed azide-alkyne cycloaddition (CuAAC). However, click chemistry between cyclooctyne or a cyclooctyne derivative and azide The Storey reaction typically does not require the addition of a copper catalyst, but instead is strain-promoted. -promoted azide-alkyne cycloaddition ion, SPAAC) (Debets, MF, et al., Bio conjugation with strained alkenes and al kynes.Acc Chem Res, 2011.44(9):p.805-15).

[0097] As used herein, the term "targeting ligand" refers to a ligand that targets a selected target, e.g., an antigen. , cell, cell type, tissue, organ, body region or compartment (e.g., cell, tissue or refers to any molecule that enhances affinity for a targeting ligand (organ compartment). Examples include antibodies or antigen-binding fragments thereof, small molecules, aptamers, polypeptides, and serotypes. Preferably, the targeting linker is a scaffold protein. The antibody may be a polypeptide, more preferably an antibody or antigen-binding fragment thereof, an engineered The domain or scaffold protein is a protein derived from a protein.

[0098] As used herein, the term "polypeptide" refers to a group of polypeptides linked together through peptide bonds. Polypeptides consisting of naturally occurring structural variants and their synthetic non-naturally occurring analogues. The term "polypeptide" refers to a polypeptide of any size, structure, or function. Typically, a polypeptide is at least three amino acids long. 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, etc. In other embodiments, the antibody or fragment thereof is specific for a cancer antigen. A polypeptide is a genetically engineered domain or scaffold protein.

[0099] As used herein, the terms "antibody" or "immunoglobulin" are used broadly. Immunoglobulin or antibody molecules, including polyclonal antibodies, murine, human, human-adapted, human Monoclonal antibodies, including homogenized and chimeric monoclonal antibodies and antigen-binding fragments thereof Includes clonal antibodies.

[0100] In general, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. The structure of an antibody is known. Immunoglobulins are composed of multiple There are five major classes depending on the amino acid sequence of the chain constant domain: IgA, IgD, IgA and IgG can be assigned to the isotypes IgE, IgG, and IgM. Further subdivided as IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4 Therefore, the antibodies used in the present invention are classified into five major classes or corresponding subclasses. The antibody light chains of any vertebrate species can be of any of the following constant domains: Based on the amino acid sequence of the polypeptide, it is one of two distinct types: kappa and lambda. According to a particular embodiment, the antibodies used in the present invention can be assigned to The heavy and / or light chain constant regions of the four IgG subunits are those of mouse or human antibodies. Each subclass has a different biological function known as an effector function. These effector functions are generally mediated by interactions with Fc receptors (FcγR) or Binding to FcγRs mediates antibody-dependent cell death. Binding to complement factors can result in complement-mediated cytolysis. Antibodies useful in the present invention may have no or minimal effector functions. but retains its ability to bind to FcRn.

[0101] As used herein, the term "antigen-binding fragment" refers to, for example, a diabody, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv flag ment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), Disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), single Single domain antibody (sdab), scFv dimer (bivalent diabody), one or two Multispecific antibodies formed from portions of antibodies containing the above CDRs, camelized single domains Antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or antigen-binding but intact antibodies Antibody fragments refer to antibody fragments such as any other antibody fragment that does not contain the antigen-binding structure. The 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 short antibody of about 15 to about 20 amino acids. The present invention relates to a method for producing a peptide comprising a heavy chain variable region and a light chain variable region connected by a peptide. As used herein, the term "single domain antibody" refers to a heavy chain variable domain antibody. The conventional method in the field is to use a heavy chain constant region and a heavy chain constant region, or a heavy chain variable region only. Refers to a single domain antibody.

[0102] As used herein, the term "scaffold" or "scaffold protein" refers to any protein that has a target binding domain and is capable of binding to a target. The scaffold consists of a "framework" that is mostly structural and a target-specific binding domain. The binding domain of the scaffold is the binding domain of the scaffold. A scaffold does not have to be defined by a single contiguous sequence of scaffolds. In certain cases, a scaffold , may be part of a larger binding protein, which itself comprises multiple scaffolds A particular binding protein may be part of a multimeric binding protein comprising two or Bispecific or multispecific antibodies are capable of binding to more than one different epitope. The scaffold may be derived from a single chain antibody or may be a scaffold The gene may not be derived from an antibody.

[0103] As used herein, the term "aptamer" refers to an aptamer that specifically binds to its target with high affinity. It refers to a single-stranded oligonucleotide (a single-stranded DNA or RNA molecule) that can be used to Ptamers 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 is a compound having a size of less than about 1000 daltons. It can refer to compounds that can be synthesized in a laboratory or found in nature. This can be done.

[0105] Chelater: In one general aspect, the invention provides a method for the preparation of a radioactive metal complex comprising: According to an embodiment of the present invention, the chelator is of formula (I): having the structure

[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. ring A and ring B are each independently selected from halo, alkyl, alkenyl, cycloalkenyl, alkyl, 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 optionally substituted with one or more substituents selected from 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 is the targeting ligand Including, 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 independently is hydrogen, alkyl, or X mosquito, Alternatively, R 14 and R 15 and / or R 16 and R 17 are combined together with the carbon atoms form a 5- or 6-membered cycloalkyl ring optionally substituted with X. Formation However, the chelator contains at least one X, and when X is present on ring A or ring B, L 1 is a linker or R 12 and R 14 ~R 17 At least one of them is hydrogen There is no.

[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 electrophilic a nucleophilic moiety or a R 11 R contains a targeting ligand. 11 is nucleophilic When the moiety is an electrophilic or electrophilic moiety, such moiety may be attached directly or indirectly via a linker. can be used to attach a chelator to a targeting ligand.

[0108] In certain embodiments, the chelator comprises a single X group, preferably wherein L1 of the X group is phosphorus. It's a car.

[0109] The chelators of the present invention may be located at any one of the carbon atoms of the macrocycle, at the Z1 or Z2 position, or It can be substituted on ring A or ring B with X, provided that when ring A or ring B contains an X group, In this case, L1 is a linker or R 12 and R 14 ~R 17 At least one of is not hydrogen (i.e., at least one of the carbon atoms of Z1, Z2 and / or the carbon of the macrocycle) At least one of the groups is substituted with an alkyl group, such as, for example, methyl or ethyl. Substitution at such positions is beneficial for radioactive metal ions, especially 225 Ac chelator chelator In some embodiments, the substitutions do not affect chelation efficiency, and in some embodiments, the substitutions can increase chelation efficiency. This can be done.

[0110] In some embodiments, L is absent. When L is absent, R 11 Is, Ki The catalyst is directly bonded (e.g., via a covalent bond) to the catalyst.

[0111] In some embodiments, L is a linker. A "linker" is a chemical compound 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 may be used in the present invention. The linker can be, for example, a substituted or unsubstituted alkyl, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted alkyl group. substituted heteroalkyl moieties, substituted or unsubstituted aryl or heteroaryl, polyethylene Polyethylene glycol (PEG) linkers, peptide linkers, sugar-based linkers, or cleavable linkers, e.g., disulfide bonds or protease-cleavable linkers. Cleavage sites, 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 1 to 4. and m is an integer of 0 to 12, preferably an integer of 0 to 6.

[0113] In some embodiments, R 11 is a nucleophilic or electrophilic moiety. A "nucleophile" or "nucleophilic group" is a 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 forms a covalent bond in a chemical reaction. Nucleophiles are functional groups that accept electron pairs to form new covalent bonds in chemical reactions. and vice versa. or other chemical moieties (e.g., targeting ligands or corresponding reaction partners) that target electrophilic groups. , linker) to attach targeting ligands or chemical moieties to the chelators of the present invention. It is possible to covalently bond to

[0114] Examples of nucleophilic groups include, but are not limited to, azides, amines, and thiols. Examples of electrophilic groups include amine-reactive groups, thiol-reactive groups, alkynyl and silyl groups. The amine reactive groups are preferably selected from the group consisting of, but not limited to, chloroalkynyl. , a primary amine present at the N-terminus of each polypeptide chain and in the side chain of a lysine residue, Examples of amine-reactive groups suitable for use in the present invention include N-hydroxybenzoates, ... N-hydroxy succinimide (NHS), substituted NHS (sulfo-NHS etc.), isothiocyanate (-NCS), isocyanate (-NCO), ester, Carboxylic acids, acyl halides, amides, alkylamides, and tetrafluorophenyl Examples of phenyl esters include, but are not limited to, esters and perfluorophenyl esters. The ol-reactive group reacts with thiols or sulfhydryls, preferably of the polypeptide. Reacts with thiols present in the side chains of cysteine ​​residues. Thiols suitable for use in the present invention Examples of reactive groups include Michael acceptors (e.g., maleimides), haloacetyls, and halogenated groups. Examples include activated acyl sulfones, activated disulfides, and phenyloxadiazole sulfones. , but not limited to these.

[0115] In certain embodiments, R 11are -NH2, -NCS (isothiocyanate), -NC O(isocyanate), -N3(azide), alkynyl, cycloalkynyl, carboxylic acid , ester, amide, alkylamide, maleimide, acyl halide, tetrazine or trans-cyclooctene, more specifically -NCS, -NCO, -N3, alkenyl Alkynyl, cycloalkynyl, -C(O)R 13 , -COOR 13 , -CON(R 13 )2 , maleimide, acyl halides (e.g., -C(O)Cl, -C(O)Br), tetramethyl cyclooctene, or trans cyclooctene, 13 are independently hydrogen or alkane It's a kill.

[0116] In some embodiments, R 11 is an alkynyl group, a cycloalkynyl group, or an azido group which allows the chelator to be linked to a targeting ligand or can be attached to other chemical moieties (e.g., linkers). In some embodiments, click chemistry reactions that can be performed include 1,2,4-triazoles. Azide (-N3) and alkynyl or cycloalkynyl groups to form a linker or moiety In one embodiment, the cycloaddition is a Huisgen cycloaddition or a 1,3-dipolar cycloaddition between a cyclopentyl group and a cyclopentyl group. The chelator contains an alkynyl or cycloalkynyl group and is capable of binding to a targeting ligand or other chemical moiety. In another embodiment, the chelator comprises an azide group and is a targeting ligand or Other chemical moieties include alkynyl or cycloalkynyl groups.

[0117] In certain embodiments, R 11 is an alkynyl group, more preferably an alkynyl group, particularly a strain-promoting group. Terminus reactive with azide groups via sequential azide-alkyne cyclocycloaddition (SPAAC) It is an alkynyl or cycloalkynyl group. It can react with an azide group by SPAAC. Examples of cycloalkynyl groups that can be used include cyclooctynyl and bicyclononynyl ( BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DI BO), keto-DIBO, biarylazacyclooctynonyl (BARAC), dibenzo Azacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl Difluorobenzocyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzo Dibenzocyclooctynyl (MOBO) and tetramethoxydibenzocyclooctynyl (TMD) cyclooctynyl derivatives selected from the group consisting of, but not limited to, Not determined.

[0118] In certain embodiments, R 11 is a dibenzoazacyclooctynyl ( dibenzoazacyclooctynyl, DIBAC, DBCO, ADIBO).

[0119] [ka]

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

[0121] In some embodiments, R 11 The targeting ligand comprises a covalently The target can be attached to the chelator directly by a bond or indirectly through a linker. The targeting ligand may be a polypeptide, e.g., an antibody or an antigen-binding fragment thereof, a small molecule, or a polypeptide. In a preferred embodiment, the antibody may be a molecule, an aptamer, or a scaffold protein. The targeting ligand may be an antibody or antigen-binding fragment thereof, such as a prostate-specific membrane antigen ( PSMA), BCMA, Her2, EGFR, KLK2, CD19, CD22, CD30 In tumor diseases or disorders, such as cancer antigens, which may be CD33, CD79b, or Nectin-4. Monoclonal antibodies (mAbs) that specifically bind to the relevant antigen or their antigen-binding flags It is a ment.

[0122] According to an embodiment of the present invention, each of rings A and B is independently a 6- to 10-membered aryl or 5-10 membered heteroaryl. In an alternative embodiment, each of ring A and ring B is , an optionally substituted heterocyclyl ring, e.g., oxazoline. Each of rings A and B is optionally and independently selected from halo, alkyl, alkenyl, cyclohex ... 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 The substituted aryl group may be substituted with one or more substituents selected from the group consisting of 6 to 1. Examples of 0-membered aryl groups include, but are not limited to, phenyl and naphthyl. Examples of 5- to 10-membered heteroaryl groups suitable for this purpose include pyridinyl, thiazole, and the like. Examples include azolyl, isothiazolyl, oxazolyl, isoxazolyl and imidazolyl. Examples include, but are not limited to, 5- to 10-membered heteroaryl and 6- to 10-membered aryl. Examples of suitable substituents for the group include -COOH, tetrazolyl, and -CH2COOH. In a preferred embodiment, the substituent is -COOH, or It is a tetrazolyl, an isostere of -COOH.

[0123] In certain embodiments, each of ring A and ring B is independently and optionally selected from -COOH and substituted with one or more carboxyl groups, including but not limited to -CH2COOH It will be exchanged.

[0124] In certain embodiments, each of ring A and ring B is independently and optionally tetrazolyl. It has been replaced.

[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 rings is pyridinyl and the other is phenyl.

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

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

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

[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 Te,-L1-R 11 and each R 12 are independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl, and 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 It is hydrogen, -CH3 or -CH2CH3.

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

[0133] In some embodiments, both Z and Z are -(CH) m - in which each m is preferably 1. In such embodiments, the carbon atom of the macrocycle, ring A or ring B is It 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 and 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 - and each m is independently 0, 1, 2, 3, 4, or 5, preferably each m is 1; 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 and a 5- or 6-membered cycloalkyl ring (i.e., cyclopentyl or cyclohexyl). Such 5- or 6-membered cycloalkyl rings may be substituted with X groups. can be done.

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

[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, and A 8, A9 and A 10 Not more than three of R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 Each of these is independent. and 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, -C N, -OC(O)N(R 13 )2 and -X; Alternatively, any two directly adjacent R1, R2, R3, R4, R5, R6, R7, R 8, R9 and R 10together with the atoms to which they are attached form a 5- or 6-membered substituent or forms an 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). That is correct, provided that the chelator contains at least one X and R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 If any one of L1 is X, then L1 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 R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 together with the atoms to which they are attached form a five-membered or form a 6-membered substituted or unsubstituted carbocyclic or nitrogen-containing ring. Examples of such carbocycles include, but are not limited to, naphthyl. Examples of such nitrogen-containing rings that can be substituted include, but are not limited to, quinolinyl. The carbocyclic or nitrogen-containing ring may be unsubstituted or may have one or more Suitable substituents such as -COOH, -CH2COOH, tetrazolyl, etc. That's fine.

[0142] In some embodiments, L is absent. When L is absent, R 11 Is, Ki The catalyst is directly bonded (e.g., via a covalent bond) to the catalyst.

[0143] In some embodiments, L is a linker. In view of the present disclosure, such as that described above, Any suitable linker known to those of skill in the art 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. , the remainder are C—H, 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. A6, A7, A8, A9 and A 10 One of the carbon atoms is substituted with -COOH. and the remainder 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 -COO H. In one embodiment, R6, R7, R8, R9 and R 10 At least one of In another embodiment, at least one of R1, R2, R3, R4, and R5 is —COOH. At least one of R6, R7, R8, R9 and R 10 At least one of One is -COOH.

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

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

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

[0150] In one embodiment, at least one of R1, R2, R3, R4, and R5 is a tetrazolium salt. In one embodiment, R, R, R, R and R are 10 At least one of In another embodiment, one of R1, R2, R3, R4 and R5 is tetrazolyl. At least one is tetrazolyl, and R6, R7, R8, R9 and R 10 A small number of At least one is tetrazolyl.

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

[0152] In some embodiments, R 11 is an alkynyl group or a cycloalkynyl group, preferably is 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, 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 are combined together with the surrounding carbon atoms to 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, cyclo Alkenyl, aryl, heterocyclyl, heteroaryl, -OR 13 , -SR 13 ,- COOR 13 , -OC(O)R 13 , -N(R 13)2, -CON(R 13 )2, -NO 2. -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). and 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 N H. For example, each A 11 can be S. In other embodiments, each A 11 is different each independently selected from O, S, NMe, and NH.

[0157] In some embodiments, each R 18 are independently -(CH2) p -COOR 13 or tetrazolyl, where 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. and R 14 ~R 17 Each of is hydrogen or R 16 and R 17 are combined together with the surrounding carbon atoms to 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 is the targeting ligand Including, 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, alkyl Nyl, cycloalkynyl, -C(O)R 13 , -COOR 13 , -CON(R 13 )2, It is maleimide, acyl halide, tetrazine or trans-cyclooctene.

[0165] In certain embodiments, R 11 is cyclooctynyl or bicyclononynyl (BCN) , difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DIBO), Keto-DIBO, biarylazacyclooctynonyl (BARAC), dibenzoazacyclo Dimethoxyazacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclo octynyl (MOBO), and tetramethoxydibenzocyclooctynyl (TMDIBO ) is a cyclooctynyl derivative selected from the group consisting of

[0166] Preferably, R 11 is an alkynyl group or a cycloalkynyl group, more preferably A cycloalkynyl group, for example DBCO or BCN.

[0167] Exemplary chelators of the present invention include:

[0168] [ka]

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

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

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

[0172] Radioactive metal complexes In another general aspect, the present invention provides a radioactive isomer of a chelator of the present invention coordinated by a coordinate bond. The present invention relates to a radioactive metal complex containing a radioactive metal ion. Both may contain radioactive metal ions. Preferably, the radioactive metal ions are α ray-emitting radioactive metal ions, more preferably 225 The chelator of the present invention is a metal Radioactive metal ions, especially radioactive metal ions, at any specific activity, regardless of impurities 225 Ac firmly clean and therefore has high chelating stability in vivo and in vitro. and a challenge agent, such as diethylenetriaminepentaacetic acid (DTA). e. Forming radioactive metal complexes that are stable to triamine pentaacetic acid (DTPA) do.

[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 The heteroatom of the macrocycle of the chelator and the and any functional groups in the pendant arms (i.e., -Z1-ring A and / or -Z2-ring B). can participate in the coordination of radioactive metal ions.

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

[0176] In certain embodiments, the radioactive metal ion M is an alpha-emitting radioactive metal ion. Or, alpha-emitting radioactive metal ions are 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 -L1-R 11 wherein L1 is absent or a linker, and R 11 is seeking electricity a nucleophilic or nucleophilic moiety, or R 11 R contains a targeting ligand. 11 is wanted In the case of a nuclear or electrophilic moiety, such moiety may be directly or indirectly linked via a linker. can be used to effectively bind radiometal complexes to targeting ligands.

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

[0179] In certain embodiments, R 11 are -NH2, -NCS (isothiocyanate), -NC O(isocyanate), -N3(azide), alkynyl, cycloalkynyl, carboxylic acid , ester, amide, alkylamide, maleimide, acyl halide, tetrazine or trans-cyclooctene, more specifically -NCS, -NCO, -N3, alkenyl 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). In the formula, each R 13 is independently hydrogen or alkyl.

[0180] In some embodiments, R 11 is an alkynyl group, a cycloalkynyl group, or an azido group which allows the chelator to be linked to a targeting ligand or can be attached to other chemical moieties (e.g., linkers).

[0181] In certain embodiments, R 11 is an alkynyl group, more preferably a strain-promoting terminal, reactive with azide groups via SPAAC-type azide-alkyne cyclocycloaddition It is an alkynyl or cycloalkynyl group. It can react with an azide group by SPAAC. Examples of cycloalkynyl groups that can be used include cyclooctynyl and bicyclononynyl ( BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclooctynyl (DI BO), keto-DIBO, biarylazacyclooctynonyl (BARAC), dibenzo Azacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl Difluorobenzocyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzo Dibenzocyclooctynyl (MOBO) and tetramethoxydibenzocyclooctynyl (TMD) cyclooctynyl derivatives selected from the group consisting of, but not limited to, Not determined.

[0182] In certain embodiments, R 11 is a dibenzoazacyclooctynyl ( DIBAC, DBCO, ADIBO).

[0183] [ka]

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

[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 5-10 membered heteroaryl. In an alternative embodiment, each of ring A and ring B is , an optionally substituted heterocyclyl ring, e.g., oxazoline. Each of rings A and B is optionally and independently selected from halo, alkyl, alkenyl, cyclohex ... 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 The substituted aryl group may be substituted with one or more substituents selected from the group consisting of 6 to 1. Examples of 0-membered aryl groups include, but are not limited to, phenyl and naphthyl. Examples of 5- to 10-membered heteroaryl groups suitable for this purpose include pyridinyl, iridyl, and methyl. Examples include, but are not limited to, isothiazolyl, isoxazolyl, and imidazolyl. Suitable examples of the substituents for the 5- to 10-membered heteroaryl and 6- to 10-membered aryl groups include: Examples include, but are not limited to, -COOH, tetrazolyl, and -CH2COOH. do not have.

[0187] In certain embodiments, each of ring A and ring B is independently and optionally selected from -COOH and substituted with one or more carboxyl groups, including but not limited to -CH2COOH It will be exchanged.

[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 rings is pyridinyl and the other is phenyl.

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

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

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

[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 Te,-L1-R 11 and 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 - in which each m is preferably 1. In such embodiments, the carbon atom of the macrocycle, ring A or ring B is It 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 and 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 - and each m is independently 0, 1, 2, 3, 4, or 5, preferably each m is 1; 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 and a 5- or 6-membered cycloalkyl ring (e.g., cyclopentyl or cyclohexyl). Such 5- or 6-membered cycloalkyl rings may be substituted with X groups. can.

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

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

[0201] [ka] wherein the variable groups are as defined above in the chelators of the invention, e.g., the chelators of formula (II). M is a radioactive metal ion, preferably an α-emitting radioactive metal ion; More preferably 225 It is Ac.

[0202] Using any of the chelators of formula (II) above, a radioactive metal complex of formula (II-m) can be formed.

[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. , the remainder are C—H, 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. A6, A7, A8, A9 and A 10 One of the carbon atoms is substituted with -COOH. and the remainder 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 -COO H. In one embodiment, R6, R7, R8, R9 and R 10 At least one of In another embodiment, at least one of R1, R2, R3, R4, and R5 is —COOH. At least one of R6, R7, R8, R9 and R 10 At least one of One is -COOH.

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

[0207] In one embodiment, at least one of R1, R2, R3, R4, and R5 is a tetrazolium salt. In one embodiment, R, R, R, R and R are 10 At least one of In another embodiment, one of R1, R2, R3, R4 and R5 is tetrazolyl. At least one is tetrazolyl, and R6, R7, R8, R9 and R 10 A small number of At least one is tetrazolyl.

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

[0209] In some embodiments, R 11 is an alkynyl group or a cycloalkynyl group, preferably is 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, 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 are combined together with the surrounding carbon atoms to 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 variable groups are as defined above in the chelators of the invention, e.g., the chelators of formula (III). is as defined, 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 may be used to chelate a radiometal of formula (III-m) Complexes can be formed.

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

[0215] In some embodiments, each R 18 are independently -(CH2) p -COOR 13 Yes In the formula, 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 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, R 14 ~R 17 Each of is hydrogen or R 16 and R17 are combined together with the surrounding carbon atoms to 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 - and R 11 is a nucleophilic or electrophilic moiety, or R 11 is the targeting ligand Including, Each R 12 are independently hydrogen, -CH3, or -CH2CH3, provided that at least One R 12 is -CH3 or -CH2CH3.

[0222] The radiometal complexes may be prepared by any method known in the art in light of the present disclosure. For example, the chelators of the present invention can be mixed with radioactive metal ions. The mixture can be incubated to form the radioactive metal complex. In terms of form, chelators 225 When mixed with a solution of Ac(NO3)3, the chelate bound to the 225 As described above, the chelators of the present invention form radioactive complexes containing Ac. radioactive metals, especially 225 Therefore, in certain embodiments, In some embodiments, the chelators of the present invention may be used in concentrations of 1:1000, 1:500, 1:400, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:10 ...200, 1:300 :200, 1:100, 1:50, 1:10, or 1:5, preferably 1:5 to 1:20 0, more preferably 1:5 to 1:100 with a chelator 225 The concentration ratio with Ac ions is 2 25 Ac ions are mixed with a solution of the radioactive metal. The chelators of the present invention that can be used to form complexes 225 The ratio of Ac to other known 2 25 This is much lower than the ratio achievable with Ac chelators, e.g., DOTA. , instant thin layer chromatography (e.g., iTLC-SG), HPLC, LC- Exemplary methods are described herein, for example, below. This is described in the examples.

[0223] Immunoconjugates and radioimmunoconjugates In another general aspect, the invention provides immunoconjugates and radioimmunoconjugates. The chelators and radioactive metal complexes of the present invention are coupled to targeting ligands such as immunological agents. Conjugated (i.e., covalently attached) to target molecules for use in targeted radiation therapy, etc. and generating immunoconjugates and / or radioimmunoconjugates suitable for medical use. The chelators and radiometal complexes of the present invention can be used to target targeting ligands, especially an antibody or its antigen-binding fragment that can specifically bind to a target of interest (such as a cancer cell); Specific labeling of the fragment with a radioactive metal ion to generate a radioimmunoconjugate Specifically, the chelator and / or radioactive metal complex of the present invention can be used to Radioactive metal ions, especially 225 High yield chelation of Ac and desirable chelator-antibody ratios (CA R) can be produced. For example, the methods of the invention can achieve an average CAR of less than 10, less than 8, less than 6, or less than 4, or about a CAR of 2 to about 8, or about 2 to about 6, or about 2 to about 4, or about 2 to about 3, or is about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or It offers approximately 8 cars.

[0224] As used herein, an "immunoconjugate" refers to a compound that is capable of binding to a toxin, drug, radioactive metal ion, or conjugated (e.g., covalently bonded) to a second molecule such as a chelator, radioactive metal complex, or the like. "Radioimmunoconjugates" are antibodies or antigen-binding fragments thereof (conjugated by a radioimmunoconjugate). The "gate" is particularly useful when the antibody or antigen-binding fragment thereof is labeled with a radioactive metal or It is an immunoconjugate conjugated to a radioactive metal complex.

[0225] According to an embodiment of the invention, the immunoconjugate comprises an antibody or an antigen-binding fragment thereof. a chelator of the invention, e.g., a chelator as described herein, covalently attached, preferably via a linker, to a The chelator and an antibody or a chelator of formula (I), formula (II), or formula (III) described above may be used. Many binding forms with different linkages between the chelator and its antigen-binding fragment are available. and reactive functional groups (i.e., nucleophilic and electrophilic) on the antibody or antigen-binding fragment thereof. ) is possible depending on the

[0226] According to an embodiment of the present invention, the radioimmunoconjugate comprises a radiometal complex of the present invention, For example, a radioactive compound of formula (Im), formula (II-m) or formula (III-m) described herein and a radioactive metal complex, preferably linked to an antibody or antigen-binding fragment thereof via a linker. is covalently bonded to

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

[0228] In some embodiments, the radiometal complexes of the radioimmunoconjugates of the present invention are The radioactive metal ion comprises an α-emitting radioactive metal ion coordinated to the chelating portion of the radioactive complex. Radiation-emitting radioactive metal ions 225 It is Ac.

[0229] In certain embodiments, the antibody or antigen-binding fragment thereof is linked to the hydroxyl group via a triazole moiety. The radioactive complex is bound to the antibody to form the radioimmunoconjugate of the present invention.

[0230] In certain embodiments, in the immunoconjugates or radioimmunoconjugates of the present application The antibody or antigen-binding fragment is capable of specifically binding to a tumor antigen. The antibody or antigen-binding fragment specifically binds to a cancer antigen. Examples of cancer antigens include , prostate-specific membrane antigen (PSMA), BCMA, Her2, EGFR, KLK2, CD1 9, CD22, CD30, CD33, CD79b, and nectin-4. Not limited to:

[0231] In one embodiment, the antibody specifically binds to PSMA. Preferably, the antibody specifically binds to PSMB. 127, herein referred to as "anti-PSMA mAb" and designated "PSMB127." The human IgG4 antibody that binds to human prostate-specific membrane antigen (PSMA) is SEQ ID NO: 3 heavy chain (HC) CDR1 sequence of SEQ ID NO: 4, HC CDR2 sequence of SEQ ID NO: HC CDR3 sequence of SEQ ID NO: 5, light chain (LC) CDR1 sequence of SEQ ID NO: 6, LC CDR2 sequence of sequence number 7 and LC CDR3 sequence of sequence number 8, and It has the HC sequence of SEQ ID NO: 9 and the LC sequence of SEQ ID NO: 10. The antibody PSMB127 and its biological activity were purified using standard chromatographic methods. , its use or other related information can be found, 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. H11B6 antibody, biological activity, uses or other Relevant information is found 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 The polypeptide is a complex comprising the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13. Chain (HC) variable region and amino acids of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 The anti-H11B6 antibody further comprises a light chain (LC) variable region comprising the 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 the amino acid sequence of SEQ ID NO: 20 a light chain constant region comprising the amino acid sequence of SEQ ID NO: 21, or a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 22. The antibody can have a light chain constant region comprising the amino acid sequence of SEQ ID NO:22.

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

[0234] The immunoconjugates and radioimmunoconjugates of the present invention may be chemically and / or enzymatically The present disclosure includes methods for conjugating a ligand, e.g., an antibody, to a chelator. The antibody can be prepared by any method known in the art, taking into account the above-mentioned factors. For example, by immunoassay. Conjugates and radioimmunoconjugates are prepared from activated acids or acyl halides. coupling reactions, including the formation of esters, thioesters, or amides; nucleophilic substitution reactions (e.g., nucleophilic substitution of halogenated rings or ring opening of strained ring systems; azide-alkyne huisgen Cycloaddition (e.g., azides and alkyl groups to form 1,2,3-triazole linkers) 1,3 dipolar cycloaddition between tetrazines; thiophosphorus addition reaction; imine formation; tetrazines and tetrazines Diels-Alder reaction between cycloctene (TCO) and cyclohexene (CCO); and Michael addition (e.g. Depending on the reactive functional group used, Many other attachment modes with different linkages are possible. Chelators coordinated to metal ions or chelators not coordinated to radioactive metal ions This can be done for.

[0235] According to one embodiment, the radiometal conjugates of the present invention are prepared by, for example, click chemistry. The Lee reaction (see, e.g., Figures 2B and 2D, also referred to as "click radiolabeling") The radiometal complexes of the present invention are covalently attached to the antibody or antigen-binding fragment thereof by Alternatively, the radioimmunoconjugate can be produced by, for example, For example, the chelators of the present invention can be attached to antibodies or their antigen-binding fragments by a click chemistry reaction. The immunoconjugates of the present invention are first prepared by covalently linking the To generate a radioimmunoconjugate, the immunoconjugate is then fused to a radioactive metal ion. On-labeling (see, e.g., Figures 2A and 2C, "one-step direct radiolabeling") Residue-specific methods of conjugation (e.g., Figures 2A and 2B) and site-specific methods (e.g., Figures 2C and 2D). , used to produce the immunoconjugates and radioimmunoconjugates of the present invention. It is possible.

[0236] Residue-specific methods for conjugation to proteins are well established and include: Most commonly, lysine side chains are modified using activated esters or isothiocyanates, or cysteine ​​side chains bearing either acetylimides, haloacetyl derivatives or activated disulfides (Brinkley Bioconjugate Chem 1992:2). Most proteins contain multiple lysine and cysteine ​​residues, so various amino acid positions A heterogeneous mixture of products with different numbers of conjugate molecules in different positions is typically can be obtained using such methods. et al.Bioconjugate Chemistry 2013:520), Thionine-specific method (Lin et al. Science 2017(355)597 ), an approach focusing on additional cysteines (Toda et al. Angew Additional methods have been established, including (Chemie 2013:12592).

[0237] More recently, site-selective and site-specific antibody targeting for monoclonal antibodies and other proteins has been reported. Specific conjugation methods have been established (Agarwal, P. and CR .Bertozzi,Bioconjug Chem,2015.26(2):p.17 6-92;Rabuka et al.Curr Opin Chem Biol 20 10:790). These include the incorporation of natural amino acids; such as SNAP or DHFR. "Self-labeling tag" or sortase A, lipoic acid ligase and formylglycine generating enzyme fusion of the protein of interest to a tag that is specifically recognized and modified by another enzyme, such as; Enzymatic modification of glycans to allow conjugation of desired payloads (Hu et al. al.Chem Soc Rev 2016:1691); defined positions on the antibody Use of microbial transglutaminase for selective recognition; and selective conjugation Additional methods using molecular recognition and / or chemical approaches to affect the Yamada et al.2019:5592;Park et al.Biocon jugate Chem 2018:3240;Pham et al.Chembio chem 2018:799).

[0238] In some embodiments, the immunoconjugates or radioimmunoconjugates of the invention for conjugating the chelators of the present invention to antibodies or antigen-binding fragments thereof. Such residue-specific methods typically produce Immunoconjugates covalently linked to chelators or radioactive metal complexes at various positions on the antibody Protein conjugates or radioimmunoconjugates known to those of skill in the art in light of this disclosure are obtained. Any residue-specific method for forming protein or antibody conjugates can be used. Examples of residue-specific methods for conjugation that can be used include, for example, For example, chelators or radioactive metal complexes containing activated ester or isothiocyanate groups are used. conjugating a chelator or a radioactive metal complex to a lysine residue of the antibody, e.g. , maleimides, haloacetyl derivatives, acyl halides, activated disulfide groups or methyl using a chelator or radioactive metal complex containing a sulfonylphenyloxadiazole group, Conjugation to cysteine ​​residues of antibodies, e.g., 4-phenyl-3H-1,2, Chelators containing 4-triazoline-3,5-(4H)-dione (PTAD) or radioactive gold conjugation to tyrosine residues of antibodies using oxaziridines, Conjugates can be attached to methionine residues of antibodies using chelators or radioactive metal complexes containing methionine derivatives. The chelators or radioactive Prior to conjugation to a metal complex, the compound may be prepared using one or more of the methods described above. Antibodies can also be labeled with biorthogonal reactive functional groups at specific residues. For example, biorthogonal reactive functional groups such as azide, alkynyl, or cycloalkane are possible. Tyrosine residues were linked to biorthogonal reactive groups using quinyl-linked oxaziridine derivatives. can be site-specifically labeled at functional groups and then transferred to a substrate bearing a compatible reactive functional group. The antibody containing the labeled tyrosine residue is then reacted with the antibody of the present invention using a chelator or radioactive metal complex. The radioactive metal complexes can be conjugated to a variety of chelators or radioactive metal complexes.

[0239] In some embodiments, the immunoconjugates or radioimmunoconjugates of the invention for conjugating the chelators of the present invention to antibodies or antigen-binding fragments thereof. In contrast to residue-specific methods, Typically, the enzyme is introduced into a specific location of the antibody by a "site-specific" or "site-selective" method. Immunoconjugates or radioimmunoconjugates covalently bound to a radioactive metal complex or Protein or antibody conjugates known to those of skill in the art in light of this disclosure are obtained. Any site-specific method for forming a non-natural amino acid sequence of interest can be used. A mutant aminoacid that can selectively aminoacrylate tRNA containing amino acids Using a tRNA synthetase, unnatural amino acids (e.g., azidoamino acids or azidoamino acids) can be synthesized. Then, amber suppressors can be specifically incorporated into the antibody. Responds to amber nonsense codons using mutant acylated tRNAs along with tRNAs By using the above method, unnatural amino acids can be site-specifically incorporated into proteins. The antibody, specifically labeled with one or more of these, is then coupled to a suitable reactive group. can be conjugated to a chelator or radioactive metal complex of the present invention having a functional group .

[0240] According to an embodiment of the present invention, a method for producing a radioimmunoconjugate comprises the steps of: 11 is nucleophilic or a chelator or radioactive complex of the present invention, which is an electrophilic moiety, is Binding fragments or modified antibodies containing nucleophilic or electrophilic moieties or The method comprises reacting the antibody with an antigen-binding fragment of the antibody.

[0241] In one embodiment, the method comprises administering a chelator of the invention to an antibody or antigen-binding fragment thereof. or modified antibodies or their antigen binding moieties containing nucleophilic or electrophilic functional groups The chelator is reacted with the antibody or antigen-binding fragment thereof or modified fragment. Immunoconjugates having a covalent bond between the antibody or antigen-binding fragment thereof The radioactive metal ion is then attached to the chelator of the immunoconjugate by a coordinate bond. The immunoconjugate is reacted with a radioactive metal ion so that it binds, thereby releasing and forming a radioactive immunoconjugate. This embodiment includes a method for treating a subject with a radioactive metal. Since there is only one chemical reaction step, it is possible to use a "one-step direct radiolabeling" method (e.g., Figure 2C (schematically shown).

[0242] In another embodiment, the method comprises administering a radioconjugate of the present invention to an antibody or its antigen-binding fragment. or modified antibodies or their antigen-binding domains containing nucleophilic or electrophilic functional groups. The method comprises reacting the fragments to form a radioimmunoconjugate. This embodiment is similar to the "click radiolabeling" method (e.g., as shown schematically in Figure 2D). The modified antibodies or antigen-binding fragments thereof may be referred to as By any method known in the art, for example, by one or more of the methods described above. Labeling an antibody with two orthogonally reactive functional groups at specific residues using the above or by using one or more of the methods described above. by specifically incorporating amino acids (e.g., azido or alkynyl amino acids) into antibodies. The degree of labeling (DOL) is sometimes called the degree of substitution (DOS). In some cases, bioconjugates such as antibodies modified with unnatural amino acids have been characterized. This is a particularly useful parameter to determine and optimize the activity of protein molecules (antibodies) or as the average number of unnatural amino acids bound to the The DOL is expressed as a ratio of the absorbance of the labeled antibody by any method known in the art. It can be determined from the absorption spectrum.

[0243] In certain embodiments, the immunoconjugates and Radioimmunoconjugates are prepared using click chemistry reactions. For example, The radioimmunoconjugates of the present invention are synthesized using click chemistry, which is referred to as "click radiolabeling." They can be prepared using a silane reaction (see, for example, Figures 2B and 2D). Click radiolabeling is performed using click chemistry reaction partners, preferably azides and alkanes. A quinone (e.g., cyclooctyne or a cyclooctyne derivative) is used to produce a radioactive complex (quinone). A radioactive metal ion bound to a radiotransferase (radioactive metal ion) is transferred to an antibody or its antigen-binding fragment. The method for click radiolabeling of antibodies is described, for example, in "Radi International Patent Application No. PCT / PCT10 / 01004 entitled "Labeling of Polypeptides" / US18 / 65913, the relevant disclosure of which is incorporated herein by reference. In another embodiment, referred to as "one-step direct radiolabeling," the immunoconjugate The antibody or antigen-binding fragment thereof is a chelator. reaction, and then contacting the immunoconjugate with a radioactive metal ion to A radioimmunoconjugate is formed (see, for example, Figures 2A and 2C).

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

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

[0246] In a specific embodiment, the method for preparing a radioimmunoconjugate of the present invention comprises: (i) a polypeptide covalently linked to a first click reaction partner (e.g., an azide group); providing a target (e.g., an antibody or antigen-binding fragment thereof); (ii) a second Click reaction partner (e.g., an alkynyl group or a cycloalkynyl providing a chelator complex comprising a chelator of the present invention covalently bonded to a chelate group; (iii) converting a first click reaction partner (e.g., an azide group) into a second click reaction partner; It is possible to react with a partner (e.g., an alkynyl group or a cycloalkynyl group). The modified polypeptide is contacted with a chelator complex under conditions that favor the formation of a polypeptide therefrom. forming a tide-chelator complex (i.e., an immunoconjugate); (iv) contacting the polypeptide-chelator complex with a radioactive metal ion, thereby releasing Preparing a radioimmunoconjugate (a radioimmunoconjugate is a compound containing a radioactive metal ion) A polypeptide labeled with an alpha-emitting radioactive isomer, e.g., an alpha-emitting radioactive isomer, which is coordinatively bound to a chelator. (including modified antibodies or antigen-binding fragments thereof labeled with radioactive metal ions) The present invention includes a "one-step direct radiolabeling" method, which includes:

[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) a modified antibody comprising an antibody or antigen-binding fragment thereof covalently bound to an azido group; providing an antibody or antigen-binding fragment thereof; (ii) a radioactive complex containing an α-emitting radioactive metal ion bound to a chelator by a coordinate bond. a chelator covalently bonded to an alkynyl or cycloalkynyl group; It is in line with, and provides, (iii) A compound that allows the azido group to react with an alkynyl or cycloalkynyl group. The modified antibody or antigen-binding fragment thereof is contacted with the radioactive complex under conditions thereby preparing a radioimmunoconjugate, This includes "conversion" methods (e.g., as shown in Figure 2D).

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

[0250] The above click radiolabeling method can be used under conditions of low or high pH and / or high temperature. This maximizes the yield by allowing chelation of reactive metal ions, which is essential for the alkyne reaction partner. This can be achieved without the risk of inactivating the azide-labeled antibody or its antigen-binding Efficient chelation between the fragment and the radioactive complex and efficient SPAAC reaction This allows the generation of radioactive immunoconjugates with high radiochemical yields even at low azide:antibody ratios. The only step that must be taken to remove trace metals is the addition of chelating moieties. Radioactive metal ion chelation to α- and β-glucan-1-phosphate dehydrogenase (GD)-1, ... The process does not need to be carried out under metal-free conditions.

[0251] The chelators and radiometal complexes of the present invention also can be used to site-specifically radiolabeled polypeptides ( For example, the click radiolabeling described herein can be used to generate antibodies. The method utilizes established methods for site-specifically introducing azide groups onto antibodies. facilitates the site-specific generation of radioimmunoconjugates (Li, X., et al. al.Preparation of well-defined antibodies -drug conjugates through glycan remodeling ng and strain-promoted azide-alkyne cycl oadditions.Angew Chem Int Ed Engl,2014.5 3(28):p.7179-82, Xiao, H., et al., Genetic i ncorporation of multiple unnatural amino acids into proteins in mammalian cells. Angew Chem Int Ed Engl,2013.52(52):p.140 80-3). Methods for attaching molecules to proteins or antibodies in a site-specific manner are well known in the art. Any method for site-specific labeling of antibodies known in the art and known to those skilled in the art may be used in the present invention. Antibodies suitable for use in the present invention may be site-specific. Examples of methods for modifying the cysteine ​​residue include, but are not limited to, modified cysteine ​​residues (e.g., THIO MAB™), incorporation of unnatural amino acids or glycans (e.g., selenocysteine , p-AcPhe, formylglycine generating enzyme (FGE, SMARTag™), etc. ), and enzymatic methods (e.g., glycotransferases, endoglycosidases, microbial or Examples of the use of bacterial transglutaminase (MTG or BTG), sortase A, etc. can be done.

[0252] According to some embodiments, the immunoconjugates or radioimmunoconjugates of the invention The modified antibodies or antigen-binding fragments thereof for use in generating the antibody or The antigen-binding fragment is isolated from the antibody by isolating the core GlcNAc at the Fc-glycosylation site of the antibody. Bacterial endoglycosidases specific for β-1,4 interresidue bonds, e.g., GlycINA TOR (Genovis) (which retains the innermost GlcNAc on Fc intact) and shortening (trim) the nucleotide sequence by a nucleotide sequence that allows for site-specific incorporation of an azido sugar at that site. The truncated antibody or antigen-binding fragment thereof is then isolated by the Ga Transglycosylation enzymes such as galactosyltransferase or GalNAc transferase In the presence of the enzyme UDP-N-azidoacetylgalactosamine (UDP-GalNaz) or by reacting with an azide-labeled sugar such as UDP-6-azido 6-deoxyGalNac, This allows for the production of modified antibodies or antigen-binding fragments thereof.

[0253] In another embodiment, production of an immunoconjugate or radioimmunoconjugate of the invention The modified antibody or antigen-binding fragment thereof for use in The resulting fragment is then deglycosylated with amidase. The resulting deglycosylated antibody or antigen-binding fragment thereof is treated with azidoamine, preferably 3 -Azidopropylamine, 6-azidohexylamine or any azido linker amine or or any azidoalkyl / heteroalkylamine, e.g., azido-polyethylene glycol Lithium (PEG)-amines, e.g., O-(2-aminoethyl)-O'-(2-azide Ethyl)tetraethylene glycol, O-(2-aminoethyl)-O'-(2-azido O-(2-aminoethyl)-O'-(2-azidoethyl)pentaethylene glycol (I) react with triethylene glycol or in the presence of microbial transglutaminase The modified antibody or antigen-binding fragment thereof can be obtained by reacting the modified antibody or antigen-binding fragment thereof under the conditions below.

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

[0255] [ka] In the formula, M is a radioactive metal ion, preferably an α-ray emitting radioactive metal ion, more preferably is actinium-225( 225 Ac) and R 11 is cyclooctynyl or biphenyl Cyclononyl (BCN), difluorinated cyclooctynyl (DIFO), dibenzocyclo Octynyl (DIBO), keto-DIBO, biarylazacyclooctynonyl (BAR AC), dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO), dimethicone Dibenzocyclooctynyl (DIMAC), Difluorobenzocyclooctynyl (DIF BO), monobenzocyclooctynyl (MOBO) and tetramethoxydibenzocyclooctynyl The cyclooctynyl derivative is selected from the group consisting of octynyl (TMDIBO).

[0256] In some embodiments, the antibody or antigen-binding fragment thereof is Any method for chemical or enzymatic modification of antibodies and polypeptides known to those skilled in the art may be used. The azide and alkynyl or cycloalkynyl groups are then covalently bonded to the azide group. under conditions sufficient to form a 1,2,3-triazole moiety by subjecting the compound to a chemistrochemical reaction. the azido-labeled antibody or antigen-binding fragment thereof is reacted with an alkynyl or cycloalkynyl group. The chelates of the present invention include aryl groups, preferably cyclooctynyl groups, more preferably DBCO. reacting it with a metal complex or a radioactive metal complex.

[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, and L1 is absent or or a linker, preferably a linker, and each R 12 are independently hydrogen, CH3 or CH2CH3, and at least one R 12 is -CH3 or -CH2CH3 , 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 PSMB127, pertuzumab, cetuximab, panitumumab , Herceptin, or H11B6.

[0261] Radioimmunoconjugates produced by the methods described herein are useful in treating cancer in the context of this disclosure. and analyzed using methods known to those skilled in the art. For example, LC / MS analysis can be used. The chelator and the labeled polypeptide, e.g., an antibody or antigen-binding fragment thereof, are then coupled to each other. The ratio of the PO to the PO can be determined using analytical size exclusion chromatography. polypeptide and polypeptide conjugates, e.g., antibodies and antibody conjugates; The oligomeric state can be determined and the radiochemical yield can be measured using instant thin layer chromatography. The radiochemical purity can be determined by PCR (e.g., iTLC-SG). Exemplary methods are described herein, e.g., by size exclusion HPLC. As described in the examples below.

[0262] Pharmaceutical Compositions and Methods of Use In another general aspect, the invention provides the chelators, radiometal complexes, immunoconjugates, and the like of the invention. and a pharmaceutical composition comprising the radioimmunoconjugate 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. Includes.

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

[0265] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, Liquids, stabilizers, solubilizers, oils, lipids, lipid-containing vesicles, microspheres, liposome encapsulates, or other materials well known in the art for use in pharmaceutical formulations. It will be appreciated that the characteristics of the diluent will depend on the route of administration for a particular application. When used, the term "pharmaceutically acceptable carrier" refers to a compound or a pharmaceutical composition according to the present invention. According to certain embodiments, the present invention refers to a non-toxic material that does not interfere with the biological activity of the composition. In view of the disclosure, any suitable antibody-based or radiocomplex-based pharmaceutical composition may be used. Any pharmaceutically acceptable carrier can be used in the present invention.

[0266] According to certain embodiments, the compositions described herein are administered in a manner consistent with the intended route of administration to a subject. For example, the compositions described herein can be administered parenterally, For example, it can be formulated to be suitable for intravenous, subcutaneous, intramuscular, or intratumoral administration. do.

[0267] In another general aspect, the present invention provides a method for treating radiation therapy and neoplastic diseases or disorders. The present invention also relates to a method for selectively targeting neoplastic cells to a radioconjugate or radioactive compound as described herein. and the use of any of the immunoconjugates and pharmaceutical compositions thereof in the methods of the present invention. can be done.

[0268] A "neoplasm" is formed when cells divide more than necessary or when cells need to die. Tumors are abnormal masses of tissue that form when cells do not die in the proper way. It can be malignant (cancer). Neoplasm is also called tumor. Neoplasm or The disorder is a tumor-related disease or disorder, such as cancer. Examples of tumor-related diseases or disorders include: Cancers include, but are not limited to, disseminated cancers and solid tumor cancers.

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

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

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

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

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

[0274] The radioimmunoconjugate can be administered to, for example, cells targeted by a targeting ligand. Preferably, the radioimmunoconjugate delivers direct radiation. 225 α such as Ac When targeted, they carry alpha-emitting radioactive metal ions, e.g. , 225 Alpha particles from Ac and its daughters are delivered to target cells, causing cytotoxic effects. and thereby causing side effects in radiation therapy and / or to treat neoplastic diseases or disorders. To selectively target neoplastic cells.

[0275] Selectively targeting neoplastic cells for radiation therapy and to treat neoplastic diseases or disorders A pre-targeting approach to targeting is also contemplated by the present invention. According to the targeting approach, an azide-labeled antibody or antigen-binding fragment thereof is administered. The antibody binds to cells bearing the antibody's target antigen and is cleared or removed from the circulation over time. Subsequently, the radioactive complex of the present invention, preferably cyclooctyne or cyclooctyne, is removed with an agent. A radioactive complex containing a cutin derivative (e.g., DBCO) is administered and binds to the target site. undergoes a SPAAC reaction with the azide-labeled antibody, and the remaining unbound radioactive complex is rapidly released from the circulation. This pre-targeting technique allows the radioactive metal ions to be delivered to the target site in the subject. Provides a way to strengthen presence.

[0276] In other embodiments, modified polypeptides, such as azide-labeled antibodies or antigens thereof, The binding fragments and radioactive complexes of the present invention can be used in targeted radiotherapy or to treat tumor diseases or They may be administered to a subject in need of treatment for a disorder in the same composition or in different compositions.

[0277] As used herein, the term "therapeutically effective amount" refers to an amount that induces a desired biological or pharmacological response in a subject. A therapeutically effective amount refers to the amount of active ingredient or component that elicits a therapeutic effect for the stated purpose. It can be determined empirically and routinely, for example, by in vitro assays. can be used to help identify optimal dose ranges. The selection will depend on the disease to be treated or prevented, the accompanying symptoms, the patient's weight, the patient's immune status, and the availability of a suitable antibody to the compound of formula (I). It may be determined by one of skill in the art (e.g., clinical practice) based on consideration of several factors, including other factors known in the art. The exact dose to be used in the formulation can be determined by the route of administration and It depends on the severity of the disease and should be decided at the discretion of the doctor and according to the circumstances of each patient. Effective doses can be estimated from dose-response curves derived from in vitro or animal model test systems. It is possible.

[0278] As used herein, the terms "treat," "treating," and Both "treatment" and "treatment" refer to a condition that is not necessarily discernible in the subject, but is a symptom of the condition. and the administration of radioactive metal ions to treat tumors or disorders such as neoplastic diseases or disorders that may be recognizable in at least one measurable physical parameter associated with a disease, disorder, or condition that may lead to The term "treat" or "treat" is intended to refer to the improvement or recovery of a disease. "Treatment" and "treatment" also refer to the treatment of a disease, disorder, or condition that causes regression or remission of the disease, disorder, or condition. It can also refer to preventing or at least slowing the progression of a disease. In embodiments, the terms "treat," "treating," and "treatment" are used interchangeably. ) refers to a disease or disorder that may benefit from the administration of radioactive metal ions, such as a neoplastic disease or disorder. or ameliorating, preventing the development or onset of one or more symptoms associated with the condition; or In certain embodiments, "treat" or "treatment" refers to a reduction in the duration of the disease. "Treating" and "treatment" refer to the prevention of recurrence of a disease, disorder or condition. In certain embodiments, the terms "treat," "treating," and "treatment" are used interchangeably. "Treatment" refers to improving the survival rate of a subject with a disease, disorder, or condition. In embodiments, the terms "treat," "treating," and "treatment" are used interchangeably. "nt") refers to the elimination of a 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. The composition is administered to a subject to treat a neoplastic disease or disorder in the subject, such as cancer.

[0280] In another embodiment of the present invention, the radioimmunoconjugates and pharmaceutical compositions of the present invention are administered to tumors. It may be used in combination with other agents effective in the treatment of neoplastic diseases or disorders.

[0281] Selective targeting of neoplastic cells for radiation therapy and / or treatment of neoplastic diseases or disorders and the radioimmunoconjugates and medicaments described herein for use in targeting Compositions and methods for the treatment of neoplastic cells for radiation therapy and / or the treatment of neoplastic diseases or disorders and the use of a radioimmunoconjugate as described herein in the manufacture of a medicament for selectively targeting a Use of the adjugate or pharmaceutical composition is also provided. [Example]

[0282] The following examples of the present invention are intended to further illustrate the principles of the present invention. The examples do not limit the invention, the scope of which is defined by the appended claims. I would like you to understand this.

[0283] Example 1: Synthesis and chelating efficiency of macrocyclic chelates with linker substitutions at different positions rate Actinium-225( 225 The effect of linker position on the chelation efficiency of Ac To study this, we used the macrocyclic chelator N,N'-bis[(6-carboxy-2-pyridyl)methyl]methyl. Two chiral compounds based on [H2Bp18c6]-4,13-diaza-18-crown-6 (H2Bp18c6) Specifically, H2bp18c6-benzyl-isopentyl and H2b p18c6-benzyl-phenyl was synthesized.

[0284] [ka]

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

[0286] [ka]

[0287] ZnCl2 (1.9 M in 2-methyltetrahydrofuran, 3.50 mL, 6.65 mm ol) and THF (25 mL) at -78 °C. (2M in Et2O, 3.33 mL, 6.66 mmol) was added dropwise. The mixture was stirred at room temperature for 1 hour and then cooled to 0° C. Methyl 6-formyl in THF (10 mL) A solution of picolinate (1.00 g, 6.1 mmol) was added, and the mixture was heated at 50° C. for 3 hours. The cooled reaction mixture was poured into saturated aqueous NH4Cl and extracted three times with EtOAc. The combined extracts were dried over Na2SO4. Filtration and concentration of the filtrate gave the crude product. The product was obtained as a brown oil. 0% EtOAc) to give 693 mg (48% yield) of the product as a yellow oil.

[0288] Methyl 6-(1-hydroxy-4-methylpentyl) ) picolinate (91 mg, 0.38 mmol) in a solution of PPh3 (120 mg, 0. The reaction solution was cooled to room temperature. The mixture was stirred at rt for 1 h and then concentrated. Chromatography on silica gel (heptane to heptane) 30% EtOAc in ethanol to give 85 mg (87% yield) of product as a colorless oil Got it.

[0289] 1,4,10,13-Tetraoxa-7,16-diazacyclo in ACN (30 mL) Octadecane (315 mg, 1.2 mmol) and K2CO3 (691 mg, 5 mmol) ) at 60 °C using a syringe pump to add methyl 6 in ACN (5 mL). The solution of 2-(chloromethyl)picolinate was added slowly over 1 hour. The reaction mixture was stirred at 60°C for 6 hours and then filtered. The filtrate was concentrated and the residue was purified by silica gel chromatography. Purification was performed by chromatography (CH2Cl2 to 10% MeOH in CH2Cl2) 206 mg (50% yield) of product was obtained as a yellowish foamy solid.

[0290] Methyl 6-((1,4,10,13-tetraoxa-7,1 6-diazacyclooctadecan-7-yl)methyl)picolinate (31 mg, 0.07 5 mmol), Na2CO3 (40 mg, 0.38 mmol) and NaI (1.5 mg) To the mixture, 6-(1-chloro-4-methylpentyl)picolinate (29 mg, 0.1 1 mmol) was added. The reaction mixture was heated at 100°C for 20 hours. Chromatography (EtOAc to 10% MeOH in CH2Cl2) gave 18.9 m g (40% yield) of the product was obtained as a yellow film.

[0291] Methyl 6-(( 16-(1-(6-(methoxycarbonyl)pyridin-2-yl)-4-methylpentyl )-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl A solution of (methyl)picolinate (3 mg, 0.005 mmol) in LiOH (1 N, The reaction mixture was stirred at room temperature for 1 hour, after which it was concentrated to dryness. The residue was dissolved in 0.95 mL of metal-free water and neutralized with 0.05 mL of 2N HCl. A solution of H2bp18c6-benzyl-isopentyl in water (approximately 3 mg / mL = approximately 5 mM, approximately pH 6).

[0292] H2bp18c6-benzyl-isopentyl (MW=602 Da) was purified by high performance liquid chromatography. The results were characterized by liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). HPLC analysis revealed a major chelator with an elution time of 17.181 min, corresponding to the free chelator. LC-MS analysis showed a peak at 603 [M+H + ], 625[M+Na + ] and 3 02[M+2H + ] shows a mass ion peak (ES, m / z), and H2bp18c The synthesis of 6-benzyl-isopentyl was confirmed.

[0293] HPLC method: XBridge C18 3.5 μm 150 × 4.6 mm, 100 Å column ram, mobile phase A: 0.1% TFA in HO, B: 0.1% TFA in ACN; 0–20 min The gradient is 10% to 30% B, and the gradient is 30% to 100% B in 20 to 20.1 minutes. Isocratic at 100% B for 5 min, gradient 100% to 10% B for 25–25.1 min %B, 25. Isocratic at 10%B from 1 to 30 min; 3 min after run; 1 mL flow rate / min, column temperature: 30°C; injection volume: 5uL.

[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 (244mg, 2.0mmol), Cs2CO3(326mg, 1.0mmol), Pd 2(dba)3·CHCl3 (52 mg, 0.05 mmol) and PPh 3( 26mg, A mixture of 1000 mg of toluene (3 mL) was placed in a sealed vial under N2. was added via syringe and the mixture was heated in a microwave at 100°C for 4 hours. The reaction mixture was filtered through Celite, and the filtrate was concentrated. Feeding (heptane to 50% EtOAc in heptane) gave 146 mg (60% yield) The product was obtained as a yellow oil.

[0297] Methyl 6-(hydroxy(phenyl)methyl)picolinate in CH2Cl2 (8 mL) (141 mg, 0.58 mmol), PPh3 (183 mg, 0.70 mmol) and A solution of NBS (113 mg, 0.64 mmol) was stirred at room temperature for 1 hour. h3 (133 mg, 0.70 mmol) and NBS (113 mg, 0.64 mmol) The reaction mixture was concentrated and subjected to chromatography on silica gel. Purification by HPLC (heptane to 30% EtOAc in heptane) gave 73 mg ( The product was obtained in a 41% yield as a colorless oil.

[0298] Methyl 6-((1,4,10,13-tetraoxa-7,1 6-diazacyclooctadecan-7-yl)methyl)picolinate (33 mg, 0.08 0 mmol), methyl 6-(bromo(phenyl)methyl)picolinate (37 mg, 0. A mixture of Na2CO3 (42 mg, 0.40 mmol) and Na2CO3 (42 mg, 0.40 mmol) was heated at 80 °C for 3 The reaction mixture was filtered and the filtrate was concentrated. Chromatography on silica gel (EtOAc to 10% MeOH in CH2Cl2) to give 51 mg (64% yield) of The product was obtained as a white solid.

[0299] Methyl 6-(( 16-((6-(methoxycarbonyl)pyridin-2-yl)(phenyl)methyl)-1 ,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl A solution of (16 mg, 0.025 mmol) picolinate was dissolved in LiOH (1 N, 0.2 The reaction mixture was stirred at room temperature for 1 hour, and then concentrated to dryness. Dissolve in 5.2 mL of metal-free water, neutralize with 0.10 mL of 2N HCl, and A solution of H2bp18c6-benzyl-phenyl (approximately 3 mg / mL = 5 mM, approximately pH 6 ) was obtained.

[0300] H2bp18c6-benzyl-phenyl (MW=608 Da) was analyzed by high performance liquid chromatography. Characterization was performed by liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). HPLC analysis was performed as described above for isopentane-H2bp18c6. HPLC analysis revealed a major peak at an elution time of 14.137 min, corresponding to the free chelator. LC-MS analysis showed 609 [M+H + ], 631 [M+Na + ] and 305[M +2H + ] showed a mass ion peak (ES, m / z) in The synthesis of di-phenyl was confirmed.

[0301] Chelation test with lanthanum(III) Aqueous solution of H2bp18c6-benzyl-isopentyl (approximately 3 mg / mL = approximately 5 mM, 2 0 μL, 0.1 μmol) in La(NO3)3 (10 mM in metal-free water, 50 μL , 0.5 μmol). Aqueous solution of H2bp18c6-benzyl-phenyl in water (approximately 3 mg / mL = approximately 5 mM, 20 μL, 0.1 μmol) La(NO3)3 (metal After thorough mixing, each The solution was analyzed by LCMS and HPLC to identify the chelator and La 3+ A complex was formed with It was decided whether

[0302] Isopentane-H2bp18c6 and H2bp18c6-benzyl-phenyl chelate Both were analyzed by HPLC following the method described above for the synthesis of H2bp18c6. At room temperature, La 3+ Faster and more efficient Stoichiometric chelation was demonstrated (Figures 1A and 1B). Complex formation was also confirmed by LCMS. LCMS analysis of isopentane-H2bp18c6 after mixing with La(NO3)3 showed 7 A mass ion peak (ES, m / z) was observed at 39 (H2bp18c6-benzyl -Isopentyl + La +3 -2H + ). H2bp18c after mixing with LA(NO3)3 LC-MS analysis of 6-benzyl-phenyl showed a mass ion peak at 745 (ES, m / z) indicates (H2bp18c6-benzyl-phenyl + La +3 -2H + ), both Complex formation by the chelator was confirmed.

[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, add 10 μL of tetramethylammonium acetate (1 M in water). ), H2bp18c6-benzyl-isopentyl (1.66 mM in water, 2 μL, ca. 3.3 2 nmol) and 225 Ac(NO3)3 (10 mCi / mL in 0.1N HCl, 3μ After mixing, the pH was approximately 6.5 according to the pH test 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 on iTLC-SG. The dried iTLC-SG was left at room temperature overnight and then developed with 0 mM EDTA. Scanned with an AR-2000 radio-TLC scanner. Conditions described herein Below, the bound Ac-225 remained on the baseline of iTLC-SG but was released The isolated Ac-225 moves to the solvent front along with the solvent. This was not observed at the edge, but this is because the chelator 225 Successfully chelate Ac ions This indicates that

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

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

[0307] DTPA loading: Mix 0.5 μL of the reaction mixture with 15 μL of 10 mM DTPA solution. The mixture was incubated for 30 minutes. 10 μL of the mixture was spotted onto iTLC-SG. The dried iTLC-SG was left at room temperature overnight. Afterwards, the samples were scanned on a Bioscan AR-2000 radio-TLC scanner. Under the conditions described above, stably chelated Ac-225 was found to be the baseline of iTLC-SG. The free Ac-225 remained on the solvent front, while the free Ac-225 migrated with the solvent to the solvent front. After loading, no radioactivity was observed at the solvent front of iTLC-SG, which is 225 A This indicates that a stable complex with the c ion was formed.

[0308] (ii) High 225 Ac / chelate ratio 225 Chelation with Ac(III) In a plastic vial, add 10 μL of tetramethylammonium acetate (1 M in water). ), H2bp18c6-benzyl-isopentyl (0.33 mM in water, 2 μL, approximately 0.6 6 nmol) and 225 Ac(NO3)3 (10 mCi / mL in 0.1N HCl, 5μ After mixing, the pH was approximately 6.5 according to the pH test paper. The reaction solution was left at room temperature for 1.5 hours. Then, the same iTLC-SG and DTPA-negative Reactions were analyzed according to the loading. 225Ac is not detected However, this 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 225 Chelation with Ac(III) In a plastic vial, add tetramethylammonium acetate (1 M solution in water, 10 μL), H2bp18c6-benzyl-phenyl (1.64 mM in water, 2 μL, approximately 3.2 8 nmol) and 225 Ac(NO3)3 (10 mCi / mL in 0.1N HCl, 3μ After mixing, the pH was approximately 6.5 according to the pH test paper. The reaction solution was left at room temperature for 2 hours. 225 Although Ac ions were not detected, this 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 on iTLC-SG. The dried iTLC-SG was left at room temperature overnight and then developed with 0 mM EDTA. Scanning was performed with an ioscan AR-2000 radio-TLC scanner. Under these conditions, the bound Ac-225 remained at the baseline of iTLC-SG. At the solvent front of iTLC-SG, free Ac-225 migrates with the solvent. No X-ray activity was observed, indicating good chelation of Ac-225. do.

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

[0312] DTPA loading: Mix 0.5 μL of the reaction mixture with 15 μL of 10 mM DTPA solution. The mixture was incubated for 30 minutes. 10 μL of the mixture was spotted onto iTLC-SG. The dried iTLC-SG was left at room temperature overnight. Afterwards, the samples were scanned on a Bioscan AR-2000 radio-TLC scanner. Under the conditions described above, stably chelated Ac-225 was found to be the baseline of iTLC-SG. The free Ac-225 remained on the solvent front, while the free Ac-225 migrated with the solvent to the solvent front. Released after loading 225 Ac was not detected, which is 225 Stable complex with Ac ion The solvent front of iTLC-SG showed no formation of DTPA complexes before or after DTPA loading. No X-ray activity was observed, which is 225 A stable complex with Ac ions is formed This indicates that

[0313] (ii) High 225 Ac / chelate ratio 225 Chelation with Ac(III) In a plastic vial, add tetramethylammonium acetate (1 M solution in water, 10 μL), H2bp18c6-benzyl-phenyl (0.16 mM in water, 2 μL, approximately 0.3 3 nmol) and225 Ac(NO3)3 (10 mCi / mL in 0.1N HCl, 5μ After mixing, the pH was approximately 6.5 according to the pH test paper. The reaction solution was left at room temperature for 2 hours. The reaction mixture was analyzed before and after DTPA loading. 225 No Ac was detected, but this teeth, 225 This indicates that a stable complex with Ac ions was formed.

[0314] summary Taken together, the above results indicate that H2bp18c6 derivatized at the "benzyl" carbon However, with rapid chelation kinetics, 225 Efficiently chelates and stabilizes Ac The results also show that the complex formed by the hydroxylase exhibited a high specific activity (i.e., a lower kinetic energy). Data and 225 Ac) can be achieved by bonding through the "benzyl" position. This indicates that

[0315] Example 2: Synthesis of H2bp18c6 derivatives with DBCO "click" linkers and Chelation Efficiency for subsequent conjugation to targeting ligands via click chemistry reactions. The following H2bp18c6 derivatives can be synthesized with a DBCO linker of:

[0316] [ka]

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

[0318] [ka]

[0319] In the presence of a palladium catalyst, methyl 6-bromopicolinate was converted to 2-tert-butoxy -2-oxoethyl zinc bromide to give methyl 6-(2-(tert-butoxy)- 2-oxoethylpicolinate is obtained, which is then brominated with NBS and AIBN. and methyl 6-(1-bromo-2-(tert-butoxy)-2-oxoethyl) Methyl 6-(1-bromo-2-picolinate) can be obtained under basic reaction conditions. (tert-Butoxy)-2-oxoethyl)picolinate and methyl 6-((1,4,1 0,13-Tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)pi Substitution reaction with cholinate yields 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 can be produced. In the presence of TFA, the tert-butyl ester can be hydrolyzed to the carboxylic acid. Dibenzocyclooctyne-amine amide Bond formation and subsequent hydrolysis of the methyl ester with lithium hydroxide results in H2bp 18c6-Benzyl-acetate-DBCO can be obtained.

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

[0321] [ka]

[0322] THF (5 mL) was dissolved in methyl 6-formylpicolinate (165 mg, 1.0 mmol) ), (4-(tert-butoxycarbonyl)phenyl)boronic acid (444 mg, 2.0 mmol), PdCl2 (8.9 mg, 0.05 mmol), tri(naphthalene-1-yl) 1)phosphane (20.6 mg, 0.05 mmol) and K2CO3 (415 mg, 3. The mixture was purged with N2 and heated at room temperature. The reaction mixture was stirred for 0.5 h and then heated at 65° C. for 24 h. The cooled reaction mixture was filtered through Celite. The mixture was filtered through a filtration medium and the filtrate was concentrated. in 50% EtOAc) to give 116 mg (34% yield) of the product as a yellow oil. Ta.

[0323] Methyl 6-((4-(tert-butoxycarbonyl)fluor)fluoride in CH2Cl2 (5 mL) (phenyl)(hydroxy)methyl)picolinate (138 mg, 0.40 mmol), PP H3 (126 mg, 0.48 mmol) and NBS (79 mg, 0.44 mmol) The mixture was stirred at room temperature for 1 hour. Additional PPh3 (63 mg, 0.24 mmol) and NBS were added. (39 mg, 0.22 mmol) was added and the mixture was stirred for another hour. The column was packed with PEG and then chromatographed (heptane to 30% EtOAc in heptane). c) yielded 62 mg (38% yield) of product from the yellowish film adhering to the flask wall. I got it as a room.

[0324] Methyl 6-((1,4,10,13-tetraoxa-7,16- Diazacyclooctadecan-7-yl)methyl)picolinate (56 mg, 0.14 mm ol), methyl 6-(bromo(4-(tert-butoxycarbonyl)phenyl)methyl ) picolinate (60 mg, 0.15 mmol) and Na2CO3 (72 mg, 0.68 The mixture was heated at 80° C. for 13 hours. The cooled reaction mixture was filtered and the filtrate was Concentrated. Chromatography on silica gel (CH2Cl2 to 10% in CH2Cl2) MeOH) to give 37 mg (37% yield) of the product as a white solid.

[0325] Methyl 6-((4-(tert-butoxycarbonyl) )phenyl)(16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)- 1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl To a solution of methyl)picolinate (22 mg, 0.03 mmol), 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. The product was obtained and used in the next step reaction without further purification.

[0326] To a solution of the above crude product in CH2Cl2 (0.5 mL) was added Et3N (42 μL) at 0 °C. , 0.3 mmol), followed by HBTU (15 mg, 0.04 mmol). was stirred at 0 °C for 5 min, and then dibenzocyclooctyne- The amine was added. The cold bath was removed and it was stirred at room temperature for 18 hours. Water was added to the reaction mixture. This was extracted three times with CH2Cl2. The combined extracts were washed with saturated aqueous NaHCO3. The mixture was washed with water, then with brine, dried (Na2SO4), and filtered. The filtrate was concentrated to give The crude product was obtained by chromatography on silica gel (CH2Cl2 to CH2Cl2 13.2 mg (47% yield) of product was deposited on the flask wall by filtration (10% MeOH in 10% MeOH). The resulting film was a colorless film.

[0327] H2bp18c in THF / MeOH / H2O (4:1:1 v / v / v, 0.6 mL) A solution of 6-benzyl-phenyl-DBCO (4.8 mg, 0.005 mmol) was added to Na OH (1N, 0.1 mL) was added. The reaction mixture was stirred at room temperature for 1 hour, after which it was diluted with H The reaction mixture was neutralized with Cl (1N) to pH = 6.5. The mixture was then evaporated on a rotary evaporator at room temperature. The mixture was concentrated at rt to remove volatile solvents. The residue was dissolved in H2O (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) H2bp18c6-benzyl-phenyl-DBCO (approximately 1.55 mg / mL = 4:1v 1.7 mM in 1000 ppm H2O / ACN (approximately pH 6.5, measured by pH paper) It was prepared from the crude product of

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

[0330] HPLC method: XBridge C18 3.5 μm 150 × 4.6 mm, 100 Å column ram, mobile phase A: 0.1% TFA in HO, B: 0.1% TFA in ACN; 0–20 min The gradient is 10% to 50% B, and the gradient is 50% to 100% B in 20 to 20.1 minutes. Isocratic at 100% B for 5 min, gradient 100% to 10% B for 25–25.1 min %B, 25.1-30 min isocratic at 10%B; flow rate 1 mL / min, column Temperature: 30℃; Injection volume: 5μL.

[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, add tetramethylammonium acetate (1 M solution in water, 10 μL), H2bp18c6-benzyl-phenyl-DBCO (1.7 m in HO / ACN), M, 2 μL, approximately 3.4 nmol) and 225 Ac(NO3)3(10 in 0.1N HCl After mixing, the pH was measured using pH test paper. The pH was about 6.5. The reaction solution was left at room temperature for 1 hour.

[0332] iTLC-SG analysis: 0.5 μL of the reaction solution was spotted on iTLC-SG. The dried iTLC-SG was left at room temperature overnight and then developed with 0 mM EDTA. Scanning was performed using an ioscan AR-2000 radio-TLC scanner. Conditions described herein Below, free Ac-225 migrates with the solvent to the solvent front. At the end, no radioactivity was observed, which is because there was no free Ac-22 in the reaction solution after 1 hour. Indicates that 5 was not present.

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

[0334] DTPA loading: Mix 0.5 μL of the reaction mixture with 15 μL of 10 mM DTPA solution. The mixture was incubated for 30 minutes. 10 μL of the mixture was spotted onto iTLC-SG. The dried iTLC-SG was left at room temperature overnight. Afterwards, the samples were scanned with a Bioscan AR-2000 radio-TLC scanner. At the solvent front of G, no radioactivity was detected.

[0335] (ii) High 225 Ac / chelate ratio 225 Chelation with Ac(III) In a plastic vial, add 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 / in 0.1N HCl mL, 5 μL, and 50 μCi were added sequentially. After mixing, the pH was approximately 6. The reaction solution was left at room temperature for 2 hours. The reaction was analyzed by

[0336] Example 3: Preparation of H2bp18c6-benzyl-phenyl-DBCO-IgG4 and 2 25 Ac(III) labeling General method for preparing radioimmunoconjugates: A radioimmunoconjugate comprising a radiometal complex of the present invention covalently bound to an antibody is prepared. The radioactive labeling of the antibody used to produce the radioimmunoconjugates of the present invention is carried out. See Figures 2A-2D for a schematic of an exemplary method for radiolabeling.

[0337] Random conjugation of azide handles to antibodies 10 mM sodium acetate (pH 5.2), phosphate buffered saline (pH 7) or other suitable Stock solutions of antibodies (1–10 mg / mL) in synthesis buffer were diluted to 1 M with 20% (v / v) It was mixed with sodium carbonate buffer (pH 9) to a final pH of approximately 9. Dissolve azide (Thermo Cat. No. 26130) in DMSO to a final concentration of 100 Make the antibody (Ab) approximately 3 to 10 mM and add a 0.2% (v / v) stock solution. A molar excess of 10 was generated. The reaction was incubated at 22°C for 10 minutes, after which 1M T Quench the reaction by adding Tris (pH 7.5) to a final concentration of 50 mM Tris. Ta.

[0338] Purify the azide-mAb conjugate using a Zeba desalting column with a 7K MW cutoff. Thermo, dialysis, standard protein A affinity chromatography, or another suitable method, in a suitable buffer (PBS; 20 mM Hep es 150 mM, NaCl (pH 7.5); or 10 mM sodium acetate pH 5.2) After purification, the eluate was concentrated in an Amicon concentrator (Millipore) with a 50K MW cutoff. The conjugate was concentrated to 10-20 mg / mL using a centrifuge tube (pore). The conversion efficiency was determined by LC-MS.

[0339] Site-specific incorporation of azido sugars into antibody glycans Antibody glycans are bound to core GlcNac residues within the Fc glycosylation site(s). GlycINATOR, a bacterial endoglycosidase specific for β-1,4 intergroup linkages (Genovis) and later used for site-specific incorporation of azido sugars. The innermost GlcNac was left intact on the Fc. GlycINATOR immobilized on agarose beads packed in (Genovis) Equilibrated in Tris-buffered saline pH 7.4 (TBS). 5-10 mg / mL Add 1 mL of mAb to the resin and incubate on a rocker at room temperature for 1 hour. The column was eluted by spinning at 100×g for 1 minute. The eluates containing the trimmed mAb were pooled and diluted with the supplied buffer. A liquid additive (Genovis) was added to the UDP-GALNaz azido-sugar substrate and GalT galactose. The reaction mixture was stirred at 30° C. overnight. The final azido mAb was purified using a mAb selective column (GE) on an AKTA Avant instrument. The 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 1 225 It coordinates with radioactive metal ions such as Ac to form radioactive complexes. Generate random or partial solutions in PBS or other compatible buffer (10-20 mg / mL). The site-specific azide-modified antibody is added to the solution of the radioactive complex. The reaction solution is gently stirred. After leaving it at room temperature for 3 hours, add, for example, 15 mL of NaOAc buffer (10 mM, pH 6.6). 5) or a PD-10 column (GE He) preconditioned with another compatible buffer. The product was purified by iTLC-SG. The solution was analyzed for chemical and radiochemical purity by HPLC. The antibody concentration in the product solution was determined by linear absorption. The activity of the product solutions was quantified using W dose calibrators.

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

[0343] Direct chelation of 225Ac(III) to H2bp18c6-phenyl-IgG4 The following method for preparing radioimmunoconjugates is a one-step direct radiolabeling method (e.g. For example, the radiolabeling may be referred to as a "metal-free" condition. This has been successfully demonstrated by demonstrating the ability of the chelator to tolerate metal contaminants. Further examples are given.

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

[0345] SEQ ID NO: 1 mAb HC QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWI RQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQ VVLTMTNMPDPVDTATYYCARLYGFTYGFAYWGQGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK

[0346] SEQ ID NO:2 mAb LC DIVMTQSPDSLAVSLGERNATINCRASQSVDYNGISYMH WYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLT ISSLQAEDVAVYYCQQIIEDPWTFGQGTKVEIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC

[0347] [ka]

[0348] Azide modification of mAb and click reaction: The above mAb IgG4 was added to a 100-fold molar excess of 3-Azidopropylamine and microbial transglutaminase (MTG; Activa The intact DNA was then subjected to site-selective modification at 37°C using an Agilent G224 instrument. Mass ESI-TOF LC-MS monitors the addition of two azides on the heavy chain of a mAb A 1 mL GE Healthcare MabSelect column was used. The excess 3-azidopropylamine and MTG were purified and removed. The azido-mAb was eluted from the resin using sodium chloride (pH 3.0), and then 7 Using a K Zeba desalting column, dehydrate the solution in 20 mM Hepes, 100 mM NaCl (p H7.5) and a 10-fold molar excess of H2bp18c6-benzyl-phenyl-DB CO was reacted with site-specific azido-IgG4 (DOL=2) at 37°C for 1 hour without shaking. The completion of the DBCO-azide click reaction was monitored by intact mass spectrometry. The excess free chelator was removed by desalting the conjugate to 20 mM on a Zeba7K desalting column. Desalted into Hepes, 100 mM NaCl (pH 7.5), followed by three 15x steps Stepwise dilution steps and 3800x using a 30K MWCO Amicon concentrator device By spinning at 47°C, the solution was diluted with 20 mM Hepes, 100 mM NaCl (pH 7.5 ) to remove the final site-specific H2bp fragment with CAR=2. 18c6-benzyl-phenyl-DBCO-IgG4 conjugate was obtained. TSKgel G3000SWxl 7.8mm x 30cm, 5u column, column temperature : At room temperature, the column is filled with DPBS buffer (x1, calcium and magnesium free). ), flow rate: 0.7 mL / min, 18 min run, injection volume: 18 μL Chromatography confirmed that the final conjugate was monomeric.

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

[0350] Purification: The reaction mixture was purified on a PD-10 column: 5 mL x 3 NaOAc buffer (1 PD-1 was purified by passing a 100 mM HCl solution (pH 6-6.5) through the column and discarding the wash solution. The resin was conditioned with NaOAc buffer solution. The reaction mixture was applied to a reservoir and the eluate was collected in pre-numbered plastic tubes. Wash the al with 0.2 mL of NaOAc buffer (10 mM, pH 6-6.5) solution. The washing solution was then pipetted into the reservoir of the PD-10 column, and the eluate was collected. The tube contained approximately 1 mL of eluate. Continued application of 10 mM HCl (pH 6-6.5) was performed until a total elution volume of 10 mL was reached. Ta.

[0351] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was added to 15 μL of 10 The mixture was mixed with 10 μL of DTPA solution (pH 6.5) and incubated for 30 minutes. 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, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of G, which is likely due to the presence of free A in fraction #3. Indicates that c-225 was not present.

[0352] HPLC analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. PLC method: Tosoh TSKgel G3000SWxl 7.8mm×30cm, 5 μm column, column temperature: room temperature, this column was and magnesium free); Flow rate: 0.7 mL / min, 20 min run, Injection volume: 30 μL After HPLC, fractions were collected at 30 second or 1 minute time intervals. Fraction C was left at room temperature overnight. The radioactivity of each collected fraction was counted in a gamma counter. LC radioactivity traces were constructed from the radioactivity in each HPLC fraction. The race corresponds to the H2bp18c6-benzyl-phenyl-DBCO-IgG4 peak. The radioactive peaks were shown on the HPLC UV trace.

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

[0354] [ka] Azide modification of mAb and click reaction: PSMB127 was added to a 100-fold molar excess of 3-azide. Didopropylamine and microbial transglutaminase (MTG; Activa TI) The intact mass E was measured on an Agilent G224 instrument at 37°C. SI-TOF LC-MS monitoring the addition of two azides on the heavy chain of a mAb A 1 mL GE Healthcare MabSelect column was used to The remaining 3-azidopropylamine and MTG were removed by purification. The azido-mAb was eluted from the resin using sodium (pH 3.0) followed by 7K Ze Using a desalting column, the lysate was purified by centrifugation in 20 mM Hepes, 100 mM NaCl (pH 7.5 ) and a 10-fold molar excess of H2bp18c6-benzyl-phenyl-DBCO was React with site-specific azido-PSMB127 (DOL=2) at 37°C for 1 hour without shaking The completion of the DBCO-azide click reaction was monitored by intact mass spectrometry. The excess free chelator was removed by desalting the conjugate to 20 mM on a Zeba7K desalting column. Desalted into Hepes, 100 mM NaCl (pH 7.5), followed by three 15-fold steps Dilution step and 30K MWCO Amicon concentrator device were used to achieve 3800Xg 20 mM Hepes, 100 mM NaCl (pH 7.5) by rotation This resulted in the final site-specific H2bp1 8c6-Benzyl-phenyl-DBCO-PSMB127 conjugate was obtained. oh TSKgel G3000SWxl 7.8mm x 30cm, 5u column, column Temperature: At room temperature, the column is filled with DPBS buffer (x1, containing calcium and magnesium) (not included), flow rate: 0.7 mL / min, 18 min run, injection volume: 18 μL analysis size eluted Exclusion chromatography confirmed that the final conjugate was monomeric. .

[0355] Labeling: To a solution of NaOAc (3 M in H2O, 20 μL) in a plastic vial, 2 25 Ac(NO3)3 (approximately 5 mCi / mL in 0.1N HCl, 20 μL, 0.098 m Ci) and H2bp18c6-benzyl-phenyl-DBCO-PSMB127 (site specific Heterogeneous, CAR=2, 2 in 20mM Hepes, 100mM NaCl (pH 7.5). After mixing, the pH was measured using pH test paper. The pH was approximately 6.5. The reaction solution was left at 37°C for 2 hours. Then, 0.5 μL of the reaction mixture was added. The reaction mixture was loaded onto iTLC-SG and developed with 10 mM EDTA. The TLC-SG was left at room temperature overnight and then analyzed by Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 was removed by the solvent. At the solvent front of iTLC-SG, no radioactive signal is observed. This is because all Ac-225 was completely chelated in the reaction solution after 2 hours. Indicates that it has been

[0356] Purification: The reaction mixture was purified on a PD-10 column: 5 mL x 3 NaOAc buffer (1 PD-1 was purified by passing a 100 mM HCl solution (pH 6-6.5) through the column and discarding the wash solution. The resin was conditioned with NaOAc buffer solution. The reaction mixture was applied to a reservoir and the eluate was collected in pre-numbered plastic tubes. Wash the al with 0.2 mL of NaOAc buffer (10 mM, pH 6-6.5) solution. The washing solution was then pipetted into the reservoir of the PD-10 column, and the eluate was collected. The tube contained approximately 1 mL of eluate. Continued application of 10 mM HCl (pH 6-6.5) was performed until a total elution volume of 10 mL was reached. Ta.

[0357] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was added to 15 μL of 10 The mixture was mixed with 10 μL of DTPA solution (pH 6.5) and incubated for 30 minutes. 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, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of G, which is likely due to the presence of free A in fraction #3. Indicates that c-225 was not present.

[0358] HPLC analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. PLC method: Tosoh TSKgel G3000SWxl 7.8mm×30cm, 5 μm column, column temperature: room temperature, the column was filled with DPBS buffer (×1, calcium and (without magnesium), flow rate: 0.7 mL / min, 20 min run, injection volume: 30 μL After HPLC, fractions were collected at 30-second or 1-minute time intervals. The fractions were left at room temperature overnight. The radioactivity of each collected fraction was counted in a gamma counter. C radioactivity traces were constructed from the radioactivity in each HPLC fraction. HPLC radioactivity tray The peak corresponds to the H2bp18c6-benzyl-phenyl-DBCO-PSMB127 peak. The corresponding radioactive peaks were shown on the HPLC UV trace.

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

[0360] [ka] Azide modification of mAb and click reaction: Pertuzumab was added to a 100-fold molar excess of 3-azide. with isopropylamine and microbial transglutaminase (MTG; Activa TI) Site-selective modification was performed at 37°C. Intact mass ES on an Agilent G224 instrument The addition of two azides onto the heavy chain of the mAb was monitored by I-TOF LC-MS. A 1 mL GE Healthcare MabSelect column was used to separate excess The 3-azidopropylamine and MTG were purified and removed. The azido-mAb was eluted from the resin using HCl (pH 3.0) and then eluted with 7K Zeb a Use a desalting column to exchange into 1x dPBS. Benzyl-phenyl-DBCO was incubated in 1x dPBS at 37°C for 1 hour with site-specific azides. -Pertuzumab (DOL=2). Intact mass spectrometry revealed that DBCO- The completion of the di-click reaction was monitored. Excess free chelator was desalted using Zeba7K. The conjugate was desalted on a column into 1x dPBS, followed by three 15-fold serial dilution steps and and a 30K MWCO Amicon concentrator device, spun at 3800Xg. This resulted in a final concentration of CAR = 2. Site-specific H2bp18c6-benzyl-phenyl-DBCO-pertuzumab conjugates Tosoh TSKgel G3000SWxl 7.8mm x 30cm, 5u column. Column temperature: At room temperature, the column was filled with DPBS buffer (1x, calcium and (without magnesium), flow rate: 0.7 mL / min, 18 min run, injection volume: 18 μL Analytical size exclusion chromatography eluting at 1000 s determined that the final conjugate was a monomer. It has been confirmed that there is.

[0361] Labeling: To a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial, 225 Ac(NO3)3 (approximately 5 mCi / mL in 0.1N HCl, 10 μL, 0.042 mCi) and H2bp18c6-benzyl-phenyl-DBCO-pertuzumab (site-specific (specifically, CAR=2, 2.4 mg / mL in PBS buffer solution, 12.5 μL, 30 μg) After mixing, the pH was approximately 6.5 as determined by pH test paper. The reaction solution was then heated at 37°C. The reaction mixture was then left for 2 hours. Then, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG. The dried iTLC-SG was left at room temperature overnight and then developed with 0 mM EDTA. Scan AR-2000 radio-TLC scanner. Elution conditions as described herein Below, any free Ac-225 migrates with the solvent to the solvent front. iTLC-SG No radioactive signal was observed at the solvent front of , indicating complete chelation in the reaction solution after 2 hours.

[0362] Purification: The reaction mixture was purified on a PD-10 column: 5 mL x 3 NaOAc buffer (1 PD-1 was purified by passing a 100 mM HCl solution (pH 6-6.5) through the column and discarding the wash solution. The resin was conditioned with NaOAc buffer solution. The reaction mixture was applied to a reservoir and the eluate was collected in pre-numbered plastic tubes. Wash the al with 0.2 mL of NaOAc buffer (10 mM, pH 6-6.5) solution. The washing solution was then pipetted into the reservoir of the PD-10 column, and the eluate was collected. The tube contained approximately 1 mL of eluate. Continued application of 10 mM HCl (pH 6-6.5) was performed until a total elution volume of 10 mL was reached. Ta.

[0363] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was added to 15 μL of 10 The mixture was mixed with 10 μL of DTPA solution (pH 6.5) and incubated for 30 minutes. 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, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of G, which is likely due to the presence of free A in fraction #3. Indicates that c-225 was not present.

[0364] HPLC analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. PLC method: Tosoh TSKgel G3000SWxl 7.8mm×30cm, 5 μm column, column temperature: room temperature, the column was filled with DPBS buffer (×1, calcium and (without magnesium), flow rate: 0.7 mL / min, 20 min run, injection volume: 30 μL After HPLC, fractions were collected at 30-second or 1-minute time intervals. The fractions were left at room temperature overnight. The radioactivity of each collected fraction was counted in a gamma counter. C radioactivity traces were constructed from the radioactivity in each HPLC fraction. HPLC radioactivity tray The peak corresponds to the H2bp18c6-benzyl-phenyl-DBCO-pertuzumab peak. The radioactive peaks were shown on the HPLC UV trace.

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

[0366] [ka] Azide modification of mAb and click reaction: Cetuximab was added to a 100-fold molar excess of 3-azide. with isopropylamine and microbial transglutaminase (MTG; Activa TI) Site-selective modification was performed at 37°C. Intact mass ES on an Agilent G224 instrument The addition of two azides onto the heavy chain of the mAb was monitored by I-TOF LC-MS. A 1 mL GE Healthcare MabSelect column was used to separate excess The 3-azidopropylamine and MTG were purified and removed. The azido-mAb was eluted from the resin using HCl (pH 3.0) and then eluted with 7K Zeb a Use a desalting column to exchange into 1x dPBS. Benzyl-phenyl-DBCO was incubated in 1x dPBS at 37°C for 1 hour with site-specific azides. The DBCO-α was reacted with cetuximab (DOL=2). The completion of the di-click reaction was monitored. Excess free chelator was desalted using Zeba7K. The conjugate was desalted on a column into 1x dPBS, followed by three 15-fold serial dilution steps and and a 30K MWCO Amicon concentrator device, spun at 3800Xg. This resulted in a final concentration of CAR = 2. Site-specific H2bp18c6-benzyl-phenyl-DBCO-cetuximab conjugates Tosoh TSKgel G3000SWxl 7.8mm x 30cm, 5u column, column temperature: room temperature, this column was filled with DPBS buffer (x1, calcium and (without magnesium), flow rate: 0.7 mL / min, 18 min run, injection volume: 18 μL Analytical size exclusion chromatography of the eluted nucleotides confirmed that the final conjugate was a monomer. It was confirmed that this is the case.

[0367] Labeling: To a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial, 225 Ac(NO3)3 (approximately 5 mCi / mL in 0.1N HCl, 10 μL, 0.044 mCi) and H2bp18c6-benzyl-phenyl-DBCO-cetuximab (site-specific (specifically, CAR=2, 1.8 mg / mL in PBS buffer solution, 16.7 μL, 30 μg) After mixing, the pH was approximately 6.5 as determined by pH test paper. The reaction solution was then heated at 37°C. The reaction mixture was then left for 2 hours. Then, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG. The dried iTLC-SG was left at room temperature overnight and then developed with 0 mM EDTA. Scan AR-2000 radio-TLC scanner. Elution conditions as described herein Below, any free Ac-225 migrates with the solvent to the solvent front. iTLC-SG No radioactive signal was observed at the solvent front of , indicating complete chelation in the reaction solution after 2 hours.

[0368] Purification: The reaction mixture was purified on a PD-10 column: 5 mL x 3 NaOAc buffer (1 PD-1 was purified by passing a 100 mM HCl solution (pH 6-6.5) through the column and discarding the wash solution. The resin was conditioned with NaOAc buffer solution. The reaction mixture was applied to a reservoir and the eluate was collected in pre-numbered plastic tubes. Wash the al with 0.2 mL of NaOAc buffer (10 mM, pH 6-6.5) solution. The washing solution was then pipetted into the reservoir of the PD-10 column, and the eluate was collected. The tube contained approximately 1 mL of eluate. Continued application of 10 mM HCl (pH 6-6.5) was performed until a total elution volume of 10 mL was reached. Ta.

[0369] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was added to 15 μL of 10 The mixture was mixed with 10 μL of DTPA solution (pH 6.5) and incubated for 30 minutes. 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, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of G, which is likely due to the presence of free A in fraction #3. Indicates that c-225 was not present.

[0370] HPLC analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. PLC method: Tosoh TSKgel G3000SWxl 7.8mm×30cm, 5 μm column, column temperature: room temperature, the column was filled with DPBS buffer (×1, calcium and (without magnesium), flow rate: 0.7 mL / min, 20 min run, injection volume: 30 μL After HPLC, fractions were collected at 30-second or 1-minute time intervals. The fractions were left at room temperature overnight. The radioactivity of each collected fraction was counted in a gamma counter. C radioactivity traces were constructed from the radioactivity in each HPLC fraction. HPLC radioactivity tray The peak corresponds to the H2bp18c6-benzyl-phenyl-DBCO-cetuximab peak. The corresponding radioactive peak was shown on the HPLC UV trace.

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

[0372] [ka] Azide modification of mAb and click reaction: Panitumumab was added to a 100-fold molar excess of 3-azide. with isopropylamine and microbial transglutaminase (MTG; Activa TI) Site-selective modification was performed at 37°C. Intact mass ES on an Agilent G224 instrument The addition of two azides onto the heavy chain of the mAb was monitored by I-TOF LC-MS. A 1 mL GE Healthcare MabSelect column was used to separate excess The 3-azidopropylamine and MTG were purified and removed. The azido-mAb was eluted from the resin using HCl (pH 3.0) and then eluted with 7K Zeb a Use a desalting column to exchange into 1x dPBS. Benzyl-phenyl-DBCO was incubated in 1x dPBS at 37°C for 1 hour with site-specific azides. - panitumumab (DOL=2). Intact mass spectrometry revealed DBCO- The completion of the di-click reaction was monitored. Excess free chelator was desalted using Zeba7K. The conjugate was desalted on a column into 1x dPBS, followed by three 15-fold serial dilution steps and and a 30K MWCO Amicon concentrator device, spun at 3800Xg. This resulted in a final concentration of CAR = 2. Site-specific H2bp18c6-benzyl-phenyl-DBCO-panitumumab conjugate Tosoh TSKgel G3000SWxl 7.8mm x 30cm, 5u column, column temperature: room temperature, this column was filled with DPBS buffer (x1, calcium and (Magnesium not included), Flow rate: 0.7 mL / min, 18 min run, Injection volume: 18 μL The final conjugate was isolated from the monomer by analytical size exclusion chromatography eluting at 1000kJ / min. It was confirmed that this is the case.

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

[0374] Purification: The reaction mixture was purified on a PD-10 column: 5 mL x 3 NaOAc buffer (1 PD-1 was purified by passing a 100 mM HCl solution (pH 6-6.5) through the column and discarding the wash solution. The resin was conditioned with NaOAc buffer solution. The reaction mixture was applied to a reservoir and the eluate was collected in pre-numbered plastic tubes. Wash the al with 0.2 mL of 3x NaOAc buffer (10 mM, pH 6-6.5) solution. The washing solution was pipetted into the reservoir of the PD-10 column, and the eluate was collected. The NaOAc buffer solution ( Successive applications of 10 mM HCl, pH 6–6.5) were 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 added to 15 μL of 10 The mixture was mixed with 10 μL of DTPA solution (pH 6.5) and incubated for 30 minutes. 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, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of G, which is likely due to the presence of free A in fraction #3. Indicates that c-225 was not present.

[0376] HPLC analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. PLC method: Tosoh TSKgel G3000SWxl 7.8mm×30cm, 5 μm column, column temperature: room temperature, the column was filled with DPBS buffer (×1, calcium and (Magnesium not included) Flow rate: 0.7 mL / min, 20 min run, Injection volume: 30 μL After HPLC, fractions were collected at time intervals of 30 seconds or 1 minute. The fractions were left at room temperature overnight. The radioactivity of each of the collected fractions was counted in a gamma counter. Radioactive traces were constructed from the radioactivity in each HPLC fraction. corresponds to the H2bp18c6-benzyl-phenyl-DBCO-panitumumab peak The radioactive peak was shown on the HPLC UV trace.

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

[0378] [ka] Azide modification of mAb and click reaction: Herceptin was added to a 100-fold molar excess of 3-azide. with isopropylamine and microbial transglutaminase (MTG; Activa TI) Site-selective modification was performed at 37°C. Intact mass ES on an Agilent G224 instrument The addition of two azides onto the heavy chain of the mAb was monitored by I-TOF LC-MS. A 1 ml GE Healthcare MabSelect column was used to separate excess The 3-azidopropylamine and MTG were purified and removed. The azido-mAb was eluted from the resin using HCl (pH 3.0) and then eluted with 7K Zeb a Use a desalting column to exchange into 1x dPBS. Benzyl-phenyl-DBCO was incubated in 1x dPBS at 37°C for 1 hour with site-specific azides. -Herceptin (DOL=2). Intact mass spectrometry revealed that DBCO- The completion of the di-click reaction was monitored. Excess free chelator was desalted using Zeba7K. The conjugate was desalted on a column into 1x dPBS, followed by three 15-fold serial dilution steps and and a 30K MWCO Amicon concentrator device, spun at 3800Xg. This resulted in a final concentration of CAR = 2. Site-specific H2bp18c6-benzyl-phenyl-DBCO-Herceptin conjugates Tosoh TSKgel G3000SWxl 7.8mm x 30cm, 5u column, column temperature: room temperature, this column was filled with DPBS buffer (x1, calcium and (without magnesium), flow rate: 0.7 mL / min, 18 min run, injection volume: 18 μL Analytical size exclusion chromatography of the eluted nucleotides confirmed that the final conjugate was a monomer. It was confirmed that this is the case.

[0379] Labeling: To a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial, 225 Ac(NO3)3 (approximately 5 mCi / mL in 0.1N HCl, 10 μL, 0.041 mCi) and H2bp18c6-benzyl-phenyl-DBCO-Herceptin (site-specific (specifically, CAR=2, 1.7 mg / mL in PBS buffer solution, 17.6 μL, 30 μg) After mixing, the pH was approximately 6.5 as determined by pH test paper. The reaction solution was then heated at 37°C. The reaction mixture was then left for 2 hours. Then, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG. The dried iTLC-SG was left at room temperature overnight and then developed with 0 mM EDTA. Scan AR-2000 radio-TLC scanner. Elution conditions as described herein Below, any free Ac-225 migrates with the solvent to the solvent front. iTLC-SG No radioactive signal was observed at the solvent front of , indicating complete chelation in the reaction solution after 2 hours.

[0380] Purification: The reaction mixture was purified on a PD-10 column: 5 mL x 3 NaOAc buffer (1 PD-1 was purified by passing a 100 mM HCl solution (pH 6-6.5) through the column and discarding the wash solution. The resin was conditioned with NaOAc buffer solution. The reaction mixture was applied to a reservoir and the eluate was collected in pre-numbered plastic tubes. Wash the al with 0.2 mL of NaOAc buffer (10 mM, pH 6-6.5) solution. The washing solution was then pipetted into the reservoir of the PD-10 column, and the eluate was collected. The tube contained approximately 1 mL of eluate. Continued application of 10 mM HCl (pH 6-6.5) was performed until a total elution volume of 10 mL was reached. Ta.

[0381] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was added to 15 μL of 10 The mixture was mixed with 10 μL of DTPA solution (pH 6.5) and incubated for 30 minutes. 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, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of G, which is likely due to the presence of free A in fraction #3. Indicates that c-225 was not present.

[0382] HPLC analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. PLC method: Tosoh TSKgel G3000SWxl 7.8mm×30cm, 5 μm column, column temperature: room temperature, the column was filled with DPBS buffer (×1, calcium and (without magnesium), flow rate: 0.7 mL / min, 20 min run, injection volume: 30 μL After HPLC, fractions were collected at 30-second or 1-minute time intervals. The fractions were left at room temperature overnight. The radioactivity of each collected fraction was counted in a gamma counter. C radioactivity traces were constructed from the radioactivity in each HPLC fraction. HPLC radioactivity tray The peak corresponds to the H2bp18c6-benzyl-phenyl-DBCO-Herceptin peak. The radioactive peaks were shown on the HPLC UV trace.

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

[0384] [ka] Azide modification of mAb and click reaction: H11B6 was added to a 100-fold molar excess of 3-azide 3 with propylamine and microbial transglutaminase (MTG; Activa TI) Site-selective modification was performed at 7°C. Intact mass ESI on an Agilent G224 instrument. The addition of two azides onto the heavy chain of the mAb was monitored by -TOF LC-MS. A 1 ml GE Healthcare MabSelect column was used to remove excess 3-Azidopropylamine and MTG were removed by purification. The azido-mAb was eluted from the resin using HCl (pH 3.0) and then eluted with 7K Zeba Exchange into 1x dPBS using a desalting column. Add 10-fold molar excess of H2bp18c6-beta phenyl-DBCO was reacted with the site-specific azido- The reaction was carried out with H11B6 (DOL=1.82). The completion of the azide click reaction was monitored. Excess free chelator was removed by Zeba7K desorption cleavage. Desalt the conjugate into 1x dPBS with a salt column, followed by three 15-fold serial dilution steps. and spun at 3800Xg using a 30K MWCO Amicon concentrator device. This resulted in a CAR of 1.8. 2. Final Site-Specific H2bp18c6-Benzyl-phenyl-DBCO-H11B6 Conjugate Tosoh TSKgel G3000SWxl 7.8mm x 30 cm, 5u column, column temperature: room temperature, this column was filled with DPBS buffer (x1, calcium (does not contain methyl or magnesium), flow rate: 0.7 mL / min, 18 min run, injection volume: 18 The final conjugate was purified by analytical size exclusion chromatography eluting in μL. It was confirmed that this was the case.

[0385] Labeling: To a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial, 225 Ac(NO3)3 (approximately 5 mCi / mL in 0.1N HCl, 10 μL, 0.043 mCi) and H2bp18c6-phenyl-DBCO-H11B6 (site-specific, CAR = 1.82, 1.2 mg / mL in PBS buffer solution, 25.0 μL, 30 μg) was added. After mixing, the pH was approximately 6.5 as measured by pH test 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 then diluted with 10 mM E The dried iTLC-SG was left at room temperature overnight and then analyzed by Bioscan The elution conditions described herein were analyzed using an AR-2000 radio-TLC scanner. Any free Ac-225 migrates to the solvent front along with the solvent. No radioactive signal was observed in the 225-Ac solution, which indicates that all of the Ac-225 was released after 2 hours. indicates that the compound was completely chelated in the reaction solution.

[0386] Purification: The reaction mixture was purified on a PD-10 column: 5 mL x 3 NaOAc buffer (1 PD-1 was purified by passing a 100 mM HCl solution (pH 6-6.5) through the column and discarding the wash solution. The resin was conditioned with NaOAc buffer solution. The reaction mixture was applied to a reservoir and the eluate was collected in pre-numbered plastic tubes. Wash the al with 0.2 mL of NaOAc buffer (10 mM, pH 6-6.5) solution. The washing solution was then pipetted into the reservoir of the PD-10 column, and the eluate was collected. The tube contained approximately 1 mL of eluate. Continued application of 10 mM HCl (pH 6-6.5) was performed until a total elution volume of 10 mL was reached. Ta.

[0387] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was added to 15 μL of 10 The mixture was mixed with 10 μL of DTPA solution (pH 6.5) and incubated for 30 minutes. 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, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of G, which is likely due to the presence of free A in fraction #3. Indicates that c-225 was not present.

[0388] HPLC analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. PLC method: Tosoh TSKgel G3000SWxl 7.8mm×30cm, 5 μm column, column temperature: room temperature, the column was filled with DPBS buffer (×1, calcium and (without magnesium), flow rate: 0.7 mL / min, 20 min run, injection volume: 30 μL After HPLC, fractions were collected at 30-second or 1-minute time intervals. The fractions were left at room temperature overnight. The radioactivity of each collected fraction was counted in a gamma counter. C radioactivity traces were constructed from the radioactivity in each HPLC fraction. HPLC radioactivity tray The peak corresponds to the H2bp18c6-benzyl-phenyl-DBCO-H11B6 peak. The radioactive peak was shown on the HPLC UV trace.

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

[0390] [ka] 4-((6-(methoxycarbonyl)pyridine-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) To a solution of 100 mg (0.10 mmol) was added HATU (37 mg, 0.10 mmol) at 0° C. The solution was After stirring at 5 °C for 5 min, N-[(1R,8S,9s)-bis(2-methyl-2-propanol)-2-yl]-2-propanol (1 mL) was added to CH2Cl2 (1 mL). Chlo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl]-1,8-diazo Add 3,6-diamino-3,6-dioxaoctane, remove the cold bath, and let the mixture stand at room temperature for 18 hours. The mixture was concentrated and the residue was purified by amine-functionalized silica gel (CH2Cl2 to CH2Cl2). Purification by chromatography with 10% MeOH in 120 ml gave 19.8 mg (yield 2 7%) of the product was obtained as a colorless film adhering to the flask wall. LC-MS analysis showed 987.6[M+H + ] indicates the mass / ion peaks.

[0391] 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 A solution of methyl)picolinate (14.5 mg, 0.015 mmol) was added to LiOH at room temperature. (1N, 0.3 mL) was added. The reaction mixture was stirred at room temperature for 1 hour, after which it was diluted with HCl The reaction mixture was concentrated in vacuo and the residue was purified by amine functionalization. Chromatography on fluorinated silica gel (CH2Cl2 to approximately 10% MeO in CH2Cl2) H) and purified to obtain 9.5 mg (48% yield) of H2bp18c6-benzyl-phenyl -BCN was obtained 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. ,400MHz)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,1 H), 4.10(d,J=8Hz,2H), 3.91(brs,2H), 3.75-3. 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), 2.2 8-2.08(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 of mAb and click reaction: PSMB127 was added to a 100-fold molar excess of 3-azide. Didopropylamine and microbial transglutaminase (MTG; Activa TI) The intact mass E was measured on an Agilent G224 instrument at 37°C. SI-TOF LC-MS monitoring the addition of two azides on the heavy chain of a mAb A 1 mL GE Healthcare MabSelect column was used to The remaining 3-azidopropylamine and MTG were removed by purification. The azido-mAb was eluted from the resin using sodium (pH 3.0) followed by 7K Ze Exchange into 1x dPBS using a desalting column. -benzyl-phenyl-BCN in 1x dPBS at 37°C for 1 hour without shaking. The specific azido-PSMB127 (DOL=2) was reacted with the azide. The completion of the BCN-azide click reaction was monitored by HCl. The excess free chelator was then converted to Z. Desalt the conjugate into 1x dPBS with an eba7K desalting column, followed by three 15x desalting steps. Using a serial dilution process and a 30K MWCO Amicon concentrator device, The cells were removed by concentrating them in PBS by spinning at 375°C. Final site-specific H2bp18c6-benzyl-phenyl-BCN-PSMB1 with AR=2 27 conjugates were obtained. Tosoh TSKgel G3000SWxl 7.8m m × 30 cm, 5u column, column temperature: room temperature, this column was filled with DPBS buffer (× 1, (Calcium and magnesium free), Flow rate: 0.7 mL / min, 18 min run, Infusion Volume: 18 μL elution by analytical size exclusion chromatography of the final conjugate was confirmed to be a monomer.

[0394] Labeling: To a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial, 225 Ac(NO3)3 (approximately 5 mCi / mL in 0.1N HCl, 10 μL, 0.046 mCi) and H2bp18c6-benzyl-phenyl-BCN-PSMB127 (site-specific (specifically, CAR=2, 2.3 mg / mL in PBS buffer solution, 13.0 μL, 30 μg) After mixing, the pH was approximately 6.5 as determined by pH test paper. The reaction solution was then heated at 37°C. The reaction mixture was then left for 2 hours. Then, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG. The dried iTLC-SG was left at room temperature overnight and then developed with 0 mM EDTA. Scan AR-2000 radio-TLC scanner. Elution conditions as described herein Below, any free Ac-225 migrates with the solvent to the solvent front. iTLC-SG No radioactive signal was observed at the solvent front of , indicating complete chelation in the reaction solution after 2 hours.

[0395] Purification: The reaction mixture was purified on a PD-10 column: 5 mL x 3 NaOAc buffer (1 PD-1 was purified by passing a 100 mM HCl solution (pH 6-6.5) through the column and discarding the wash solution. The resin was conditioned with NaOAc buffer solution. The reaction mixture was applied to a reservoir and the eluate was collected in pre-numbered plastic tubes. Wash the al with 0.2 mL of NaOAc buffer (10 mM, pH 6-6.5) solution. The washing solution was then pipetted into the reservoir of the PD-10 column, and the eluate was collected. The tube contained approximately 1 mL of eluate. Continued application of 10 mM HCl (pH 6-6.5) was performed until a total elution volume of 10 mL was reached. Ta.

[0396] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was added to 15 μL of 10 The mixture was mixed with 10 μL of DTPA solution (pH 6.5) and incubated for 30 minutes. 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, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of G, which is likely due to the presence of free A in fraction #3. Indicates that c-225 was not present.

[0397] HPLC analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. PLC method: Tosoh TSKgel G3000SWxl 7.8mm×30cm, 5 μm column, column temperature: room temperature, the column was filled with DPBS buffer (×1, calcium and (without magnesium), flow rate: 0.7 mL / min, 20 min run, injection volume: 40 μL After HPLC, fractions were collected at 30-second or 1-minute time intervals. The fractions were left at room temperature overnight. The radioactivity of each collected fraction was counted in a gamma counter. C radioactivity traces were constructed from the radioactivity in each HPLC fraction. HPLC radioactivity tray The peak corresponds to the H2bp18c6-benzyl-phenyl-BCN-PSMB127 peak. The corresponding radioactive peak was shown on the HPLC UV trace.

[0398] Example 12: H2bp18c6-off macrocycle-ethyl sulfide-DBCO Synthesis of At the carbon atom of the macrocycle there is a linker for conjugation to a targeting ligand. The substituted H2bp18c6 derivatives can generally be prepared according to the following schemes 14a and 14b: Synthesize according to Step 14b:

[0399] [ka]

[0400] The synthesis of -CH2OR1-substituted diaza-18-crown-6 was reported in Org. Lett. 2005,7(6),1105-1108, followed by skimming. This was reacted with a derivative of 6-(halomethyl)picolinic acid as described above for 3 and 4. The functional group OR1 is a heteroatom, an alkyl, an alkyl having a heteroatom, a substituted The linker, the targeting ligand, with or without a linker W, which may be, for example, aryl, NH2, N3, aldehydes for ligation or conjugation to amides, etc. Convert WR2 to another functional group, including but not limited to, carboxylates, alkynes, etc. In Scheme 6a, X is a leaving group, e.g., halo, mesylate, tosylate, etc., R1 is allyl, benzyl, alcohol, etc., and W is not present. or a linker, e.g., a heteroatom, alkyl, heteroalkyl, substituted aryl and the like, and R2 is NH2, N3 aldehyde, carboxylate, alkynyl, etc. do.

[0401] [ka]

[0402] -CH2OR1-substituted diaza-18-crown-6 was synthesized in the Journal of Organic Chemistry,1988,53(14),3190-5 and J Ournal of Heterocyclic Chemistry,1986,23 (2), 609-13, followed by the synthesis of Scheme 3 and This is reacted with a derivative of 6-(halomethyl)picolinic acid as described above for 4 and 5. The functional group OR1 is a heteroatom, an alkyl, an alkyl having a heteroatom, a substituted aryl, or a substituted aryl. a linker, targeting ligand, etc., with or without a linker W, such as a linker For ligation or conjugation to NH2, N3, aldehyde, carboxyl Converting to another functional group R2, including but not limited to carboxylates, alkynes, etc. can be done.

[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 dissolved in anhydrous DMF ( (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methacrylamide gel (500 mL) The mixture was added to a solution of ethanol (50 g, 378 mmol) at 0°C. The mixture was stirred at this temperature for 10 min. After this, benzyl bromide (77.63 g, 454 mmol) was slowly added to the suspension. The reaction mixture was allowed to reach room temperature after 30 minutes and stirred for an additional 8 hours. The mixture was quenched with a ammonium chloride solution and extracted with dichloromethane (2 x 500 mL). The organic layer was washed with brine and dried over sodium sulfate. After removing the solvent, the residue was extracted with petroleum Flash column chromatography (silica gel, 2 30-400 mesh) to obtain (S)-4-((benzyloxy)methyl)- 2,2-Dimethyl-1,3-dioxolane (72 g, 86%) was obtained as a colorless liquid.

[0406] (S)-4-((benzyloxy)methyl)-2,2-dimethylbenzyloxy ... A solution of ethyl-1,3-dioxolane (72 g, 324 mmol) was added to 1.5 N HCl ( 100 mL) was added, followed by stirring at room temperature for 16 hours. The reaction mixture was diluted with ethyl acetate (50 The reaction mixture was diluted with 100 mL of ethyl acetate ( 2 x 500 mL), washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography using petroleum ether and ethyl acetate. The compound was purified by chromatography (silica gel, 230-400 mesh) to give (R)-3-(benzyl)- (Benzyloxy)propane-1,2-diol (51 g, 85%) was obtained as a colorless oil. .

[0407] Sodium hydride (26.29 g, 60% in mineral oil, 686 mmHg) in DMF (20 mL) To a suspension of (R)-3-(benzyloxy)propane (100 mL) in DMF (100 mL) 1,2-Diol (25.0 g, 137 mmol) was added dropwise at 0° C. The reaction mixture was stirred at room temperature. The mixture was stirred at room temperature for 2 hours. This was cooled to 0°C again and 2-(2-bromo-2-methyl-2-methylpropional) in DMF (100 mL) was added. (bromoethoxy)tetrahydro-2H-pyran (85.89 g, 411 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. The reaction was quenched with saturated ammonium chloride. The combined organic layers were washed with water and extracted with sodium sulfate. The crude oil was dissolved in ethyl acetate (0-10%) in petroleum ether and concentrated. Silica gel (230-400 mesh) flash chromatography using 40% The product was purified by 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%) was obtained as a colorless liquid.

[0408] 2,2'-(((((R)-3-(benzyloxy)propyl)propanol in 500 mL of methanol bis(ethane-1,2-diyl)bis(oxy)bis(ethane-2,1-diyl)bis(oxy) To a solution of bis(tetrahydro-2H-pyran) (32 g, 73.05 mmol), 5 mL of HCl in dioxane was added. The reaction was stirred at reflux for 1 hour, cooled, evaporated and 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 further purification. did.

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

[0410] (R)-((3-(benzyloxy)propane-1,2- Diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenes sulfonate) (30 g, 51.9 mmol) and cesium carbonate (50.76 g, 155. 7 mmol) was stirred at ambient temperature for 1.5 hours. The suspension was added with 200 mL of DMSO. N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1- Diyl))bis(4-methylbenzenesulfonamide) (23.56g, 51.9mmol l) was added dropwise over 2 hours. The mixture was stirred at ambient temperature for a further 20 hours. The solvent was reduced The solid was removed under pressure to give a solid paste, which was suspended in 1000 mL of dichloromethane. The mixture was stirred for 30 minutes. The precipitated solid was filtered off and the filtrate was evaporated under high vacuum. , flash chromatography using ethyl acetate in petroleum ether (0–40%) (silica gel, 230-400 mesh) to purify the (R)-2-((benzyloxy) (Ci)methyl)-7,16-ditosyl-1,4,10,13-tetraoxa-7,16-di Azacyclooctadecane (24 g, 67%) was obtained as a colorless liquid.

[0411] (R)-2-((benzyloxy)methyl)-7,1 in acetic acid (50%, 100 mL) 6-Ditosyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadeca A solution of phenol (16.35 g, 174 mmol) was added to a solution of ethanol (24 g, 34.78 mmol). l) was added at room temperature. The reaction was heated at 60°C for 6 hours. After the reaction was complete, it was cooled to room temperature and The acetic acid was removed under high vacuum. The crude product was purified by 0-100% acetone in water (0.1% TFA). Purification was carried out on a reversed-phase column using nitrile to give (S)-(1,4,10,13-tetraoxo-1,4-dioxane). sa-7,16-diazacyclooctadecan-2-yl)methyl acetate (8.0 g, 6 9%) as a colorless liquid.

[0412] (S)-(1,4,10,13-tetraoxa-2,4-dioxazolidinyl) -7,16-diazacyclooctadecan-2-yl)methyl acetate (8.0 g, 23 0.95mmol), 6-(chloromethyl)picolinate methyl (11.07g, 59.8 A suspension of sodium carbonate (12.69 g, 119.75 mmol) was added to 9 The mixture was heated at 0° C. for 16 hours. After the reaction was complete, the reaction mixture was cooled to room temperature and filtered through Celite. The crude material was purified by filtration and concentrated under reduced pressure using methanol (0-10%) in dichloromethane. By using flash chromatography (silica gel, 230-400 mesh) Purified and purified dimethyl 6,6'-((2-(acetoxymethyl)-1,4,10,13-tetramethyl-2-methyl ... (laoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene) )(S)-dipicolinate (5.0 g, 33%) was obtained as a brown liquid.

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

[0414] Dimethyl 6,6'-((2-(hydroxymethyl)-) in dichloromethane (20 mL) 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16- (R)-dipicolinate (1.0 g, 1.7 mmol) To the solution 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 reaction progress was monitored by TLC. After completion, the mixture was concentrated, and the crude product was extracted with methanol (1-2%) in dichloromethane as an eluent. ) and purified by column chromatography (alumina-neutral) to obtain dimethyl 6 ,6'-((2-(((methylsulfonyl)oxy)methyl)-1,4,10,13-tetramethyl Dioxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene) )) (S)-dipicolinate (0.7 g, 62%) was obtained as a brown liquid.

[0415] Tert-butyl (2-mercaptoethyl)carbamate (53) in DMF (2 mL) mg, 0.3 mmol) was added to a solution of sodium hydride (12 mg, 60% in mineral oil, 0.3 (mmol) was added at 0°C. The reaction mixture was stirred at room temperature for 10 minutes. Dimethyl 6,6'-((2-(((methylsulfonyl)oxy)methyl)methyl)methyl in MF (1 mL) 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7, 16-diyl)bis(methylene)(S)-dipicolinate (100 mg, 0.15 mm ol) was added at 0°C. The reaction was stirred at room temperature for 2 hours. The progress of the reaction was monitored by TLC. After the reaction was completed, the reaction mixture was quenched with saturated ammonium chloride solution and The combined organic layers were washed with brine and extracted with anhydrous sodium sulfate. The crude product was purified by elution with acetonitrile in water (0.1% TFA). The dimethyl 6,6'-((2-(((2-((ter t-Butoxycarbonyl)amino)ethyl)thio)methyl)-1,4,10,13-tet (laoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene) )(S)-dipicolinate (20 mg, 18%) was obtained as a brown liquid.

[0416] Dimethyl 6,6'-((2-(((2-((tert-butoxycarbonyl)amino) Ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclo Octadecane-7,16-diyl)bis(methylene)(S)-dipicolinate(100 mg, 0.15 mmol), a cold solution of HCl in methanol (2 mL, 4 N) was added, The solution was stirred for 2 hours. 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)-dipicolin The ester was obtained as a yellow liquid (55 mg, 64%).

[0417] Dimethyl 6,6'-((2-(((2-aminoethyl) (I)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacycloocta Tadecane-7,16-diyl)bis(methylene))(S)-dipicolinate (60 mg, To a solution of 0.09 mmol of triethylamine (0.04 mL, 0.27 mmol) at 0 °C l) was added, followed by HATU (51 mg, 0.13 mmol). The solution was heated to 0°C. After stirring at rt for 5 min, DBCO-acid (27 mg, 0.5 mL) in dichloromethane (0.5 mL) was added. 09 mmol) was added. The cold bath was removed and the mixture was stirred at room temperature for 18 hours. Water was added to the reaction mixture. The mixture was extracted with dichloromethane (2 mL x 3). The combined extracts were washed with saturated NaH The solution was washed with aqueous CO3, brine, dried over sodium sulfate, and filtered. The crude product was obtained by chromatography on silica gel with dichloromethane and methanol. Fee, H2bp18c6-off macrocycle-ethyl sulfide-DBCO (20 mg , 23%) of the dimethyl ester was obtained as a colorless liquid.

[0418] H2bp18c6-off macrocycle-ethyl sulfide in methanol (0.5 mL) at room temperature A solution of do-DBCO (20 mg, 0.02 mmol) was added to LiOH (0 . 64 mL, 0.1N, 0.06 mmol) was added. After stirring at room temperature for 16 hours, this was diluted with acetic acid. The reaction mixture was neutralized with acid to pH 6.5. The reaction mixture was concentrated at room temperature using an evaporator to obtain a volatile The solvent was removed, and the residue was purified by preparative HPLC to give H2bp18c6-off macrocycle-ene. Thiosulfide-DBCO (6 mg, 31%) was obtained as an off-white solid. MS APCI: Calculated for C48H56N6O10S 909.07; Observed m / z[M+H] + 909.4. Purity by LC-MS: 92.92% RT: 1.86. H Purity by PLC: 91.56% RT: 3.87. 1 H NMR (400 MHz, D2 O):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: H2bp18c6-off macrocycle-ethyl sulfide-DBCO-PSMB Preparation of 127 and 225 Ac(III) labeling

[0420] [ka] Azide modification of mAb and click reaction: PSMB127 was added to a 100-fold molar excess of 3-azide. Didopropylamine and microbial transglutaminase (MTG; Activa TI) The intact mass E was measured on an Agilent G224 instrument at 37°C. SI-TOF LC-MS monitoring the addition of two azides on the heavy chain of a mAb A 1 ml GE Healthcare MabSelect column was used to The excess 3-azidopropylamine and MTG were removed by purification. The azido-mAb was eluted from the resin using sodium (pH 3.0) followed by 7K Ze Exchange into 1x dPBS using a desalting column. -off macrocycle-ethyl sulfide-DBCO in 1x dPBS at 37 °C for 1 h. The reaction was carried out with specific azido-PSMB127 (DOL=2). The completion of the DBCO-azide click reaction was monitored. The excess free chelator was converted to Z. Desalt the conjugate into 1x dPBS with an eba7K desalting column, followed by three 15x desalting steps. Using a serial dilution process and a 30K MWCO Amicon concentrator device, The cells were removed by concentrating them in PBS by spinning at 375°C. Final site-specific H2bp18c6-off macrocycle-ethyl sulfide-DBCO with AR=2 The -PSMB127 conjugate was obtained using Tosoh TSKgel G3000SWx. 7.8mm x 30cm, 5u column. Column temperature: Room temperature. Refrigerate the column in DPBS. Buffer solution (x1, calcium and magnesium free), flow rate: 0.7 mL / min, 18 min The final concentration was determined by analytical size exclusion chromatography using an injection volume of 18 μL. The conjugate was confirmed to be a monomer.

[0421] Labeling: To a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial, 225 Ac(NO3)3 (approximately 5 mCi / mL in 0.1N HCl, 10 μL, 0.048 mCi) and H2bp18c6-off macrocycle-ethyl sulfide-DBCO-PSMB1 27 (site-specific, CAR=2, 2.4 mg / mL in PBS buffer, 10 μL, 24 μ g) were added successively. After mixing, the pH was about 6.5 as measured by pH test paper. The mixture was left at 37°C for 2 hours. Then, 0.5 μL of the reaction mixture was loaded onto iTLC-SG. This was developed with 10 mM EDTA. After leaving the dried iTLC-SG at room temperature overnight, , and scanned on a Bioscan AR-2000 radio-TLC scanner. Under elution conditions, any free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of C-SG, which is likely due to the fact that all Ac- This shows that 225 was completely chelated in the reaction solution after 2 hours.

[0422] Purification: The reaction mixture was purified on a PD-10 column: 5 mL x 3 NaOAc buffer (1 PD-1 was purified by passing a 100 mM HCl solution (pH 6-6.5) through the column and discarding the wash solution. The resin was conditioned with NaOAc buffer solution. The reaction mixture was applied to a reservoir and the eluate was collected in pre-numbered plastic tubes. Wash the vial with 0.2 mL of NaOAc buffer (10 mM, pH 6-6.5) solution. The washing solution was then pipetted into the reservoir of the PD-10 column, and the eluate was collected. The probe contained approximately 1 mL of eluate. Continue applying elution solution (10 mM, pH 6–6.5) until a total elution volume of 10 mL is reached. It was.

[0423] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was added to 15 μL of 10 The mixture was mixed with 10 μL of DTPA solution (pH 6.5) and incubated for 30 minutes. 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, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of G, which is likely due to the presence of free A in fraction #3. Indicates that c-225 was not present.

[0424] HPLC analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. PLC method: Tosoh TSKgel G3000SWxl 7.8mm×30cm, 5 μm column, column temperature: room temperature, the column was filled with DPBS buffer (×1, calcium and (without magnesium), flow rate: 0.7 mL / min, 20 min run, injection volume: 40 μL After HPLC, fractions were collected at 30-second or 1-minute time intervals. The fractions were left at room temperature overnight. The radioactivity of each collected fraction was counted in a gamma counter. C radioactivity traces were constructed from the radioactivity in each HPLC fraction. HPLC radioactivity tray The enzyme is H2bp18c6-off macrocycle-ethyl sulfide-DBCO-PSMB127 The radioactive peak corresponding to the peak was shown on the HPLC UV trace.

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

[0426] [ka] Tert-butyl (5-mercaptopentyl)carbamate (6 mL) in DMF (2 mL) A solution of sodium hydride (12 mg, 60% in mineral oil, 0.5 mg, 0.3 mmol) was added to the solution. 3 mmol) was added at 0° C. The resulting reaction mixture was stirred at room temperature for 10 minutes. The mixture was treated with dimethyl 6,6'-((2-(((methylsulfonyl)o)methyl)propanol in DMF (1 mL). (xy)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctade Can-7,16-diyl)bis(methylene))(S)-dipicolinate (100 mg, 0 0.15 mmol) was added at 0° C. The reaction was stirred at room temperature for 2 hours. The progress of the reaction was monitored by TL After the reaction was completed, it was concentrated and the residue was dissolved in acetonitrile and 0. Purification by preparative HPLC using 1% TFA gave 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%) was obtained as a brown liquid. Ta.

[0427] Dimethyl 6,6'-((2-(((5-((tert-butoxycarbonyl)amino) Pentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacycl (S)-dipicolinate (12 To the HCl solution (0.0 mg, 0.15 mmol) was added a cold solution of HCl in methanol (2 mL, 4 N). 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)-dipicolin The ester (70 mg, 66%) was obtained as a yellow liquid.

[0428] Dimethyl 6,6'-((2-(((5-aminopentane) in dichloromethane (0.5 mL) (ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooxo (Cutadecane-7,16-diyl)bis(methylene)(S)-dipicolinate (50mg To a solution of triethylamine (0.03 mL, 0.21 mmol) at 0 °C, ol) was added, followed by HATU (38 mg, 0.10 mmol). After stirring at 4°C for 5 min, DBCO-acid (21 mg, 0 The cold bath was removed and the mixture was stirred at room temperature for 18 hours. Water was added to the reaction mixture. This was extracted with dichloromethane (2 mL x 3). The combined extracts were washed with saturated Na The solution was washed with aqueous HCO3, brine, dried over sodium sulfate, and filtered. The crude product was obtained by chromatography on silica gel with dichloromethane and methanol. Raffi et al., synthesized H2bp18c6-off macrocycle-pentyl sulfide-DBCO(16 mg, 23%) of the dimethyl ester was obtained as a colorless liquid.

[0429] H2bp18c6-off macrocycle-pentyl sulfide- in methanol (0.5 mL) A solution of DBCO (16 mg, 0.01 mmol) was added to LiOH (0 . 49mL After stirring at room temperature for 16 hours, this was diluted with acetic acid. The reaction mixture was neutralized to pH 6.5. The reaction mixture was concentrated at room temperature using an evaporator to remove volatile solvents. The residue was purified by preparative HPLC to obtain H2bp18c6-off macrocycle-plier Disulfide-DBCO (5 mg, 33%) was obtained as an off-white solid. S APCI: Calculated for C51H62N6O10S; 951.15; Observed m / z[M+H] + 951.4. Purity by LC-MS: 94.51% RT: 1.98. H Purity by PLC: 98.41% RT: 4.13. 1 H NMR (400 MHz, D2 O):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), 2.11-2.0 8(m,3H), 1.39-1.35(m,2H), 1.20-1.10(m,4H).

[0430] Example 15: H2bp18c6-off macrocycle-pentylsulfide-DBCO-PSM Preparation of B127 and 225 Ac(III) labeling

[0431] [ka] Azide modification of mAb and click reaction: PSMB127 was added to a 100-fold molar excess of 3-azide. Didopropylamine and microbial transglutaminase (MTG; Activa TI) The intact mass E was measured on an Agilent G224 instrument at 37°C. SI-TOF LC-MS monitoring the addition of two azides on the heavy chain of a mAb A 1 mL GE Healthcare MabSelect column was used to The remaining 3-azidopropylamine and MTG were removed by purification. The azido-mAb was eluted from the resin using sodium (pH 3.0) followed by 7K Ze Exchange into 1x dPBS using a desalting column. -off macrocycle-pentyl sulfide-DBCO, in 1x dPBS, without shaking, at 37 °C The intact DNA was reacted with site-specific azido-PSMB127 (DOL=2) at RT for 1 hour. The completion of the DBCO-azide click reaction was monitored by mass spectrometry. The chelator was prepared by desalting the conjugate into 1x dPBS on a Zeba7K desalting column, followed by , using three 15-fold serial dilution steps and a 30K MWCO Amicon concentrator device The precipitate was removed by a step of concentrating it in PBS by spinning at 3800×g. This resulted in the final site-specific H2bp18c6-off macrocycle-pentylsulfate synthesis of CAR=2. The fid-DBCO-PSMB127 conjugate was obtained. G3000SWxl 7.8mm x 30cm, 5u column, column temperature: room temperature, The rum was resuspended in DPBS buffer (x1, calcium and magnesium free), flow rate: 0.7 Analytical size exclusion chromatography eluting at mL / min, 18 min run, 18 μL injection volume This confirmed that the final conjugate was monomeric.

[0432] Labeling: To a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial, 225 Ac(NO3)3 (approximately 5 mCi / mL in 0.1N HCl, 10 μL, 0.047 mCi) and H2bp18c6-off macrocycle-pentyl sulfide-DBCO-PSMB 127 (site specific, CAR=2, 2.8mg / mL in PBS buffer, 8μL, 22μ g) were added successively. After mixing, the pH was about 6.5 as measured by pH test paper. The mixture was left at 37°C for 2 hours. Then, 0.5 μL of the reaction mixture was loaded onto iTLC-SG. This was developed with 10 mM EDTA. After leaving the dried iTLC-SG at room temperature overnight, , and scanned on a Bioscan AR-2000 radio-TLC scanner. Under elution conditions, any free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of C-SG, which is likely due to the fact that all Ac- This shows that 225 was completely chelated in the reaction solution after 2 hours.

[0433] Purification: The reaction mixture was purified on a PD-10 column: 5 mL x 3 NaOAc buffer (1 PD-1 was purified by passing a 100 mM HCl solution (pH 6-6.5) through the column and discarding the wash solution. The resin was conditioned with NaOAc buffer solution. The reaction mixture was applied to a reservoir and the eluate was collected in pre-numbered plastic tubes. Wash the al with 0.2 mL of NaOAc buffer (10 mM, pH 6-6.5) solution. The washing solution was then pipetted into the reservoir of the PD-10 column, and the eluate was collected. The tube contained approximately 1 mL of eluate. Continued application of 10 mM HCl (pH 6-6.5) was performed until a total elution volume of 10 mL was reached. Ta.

[0434] DTPA loading: 10 μL of fraction #3 collected after the PD-10 column was added to 15 μL of 10 The mixture was mixed with 10 μL of DTPA solution (pH 6.5) and incubated for 30 minutes. 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, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of G, which is likely due to the presence of free A in fraction #3. Indicates that c-225 was not present.

[0435] HPLC analysis: Fraction #3 collected after the PD-10 column was analyzed by HPLC. PLC method: Tosoh TSKgel G3000SWxl 7.8mm×30cm, 5 μm column, column temperature: room temperature, the column was filled with DPBS buffer (×1, calcium and (without magnesium), flow rate: 0.7 mL / min, 20 min run, injection volume: 40 μL After HPLC, fractions were collected at 30-second or 1-minute time intervals. The fractions were left at room temperature overnight. The radioactivity of each collected fraction was counted in a gamma counter. C radioactivity traces were constructed from the radioactivity in each HPLC fraction. HPLC radioactivity tray The enzyme is H2bp18c6-off macrocycle-pentyl sulfide-DBCO-PSMB127 The radioactive peaks corresponding to the peaks were indicated on the HPLC UV trace.

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

[0437] [ka] Azide modification of mAb and click reaction: H11B6 was added to a 200-fold molar excess of 3-azide 3 with propylamine and microbial transglutaminase (MTG; Activa TI) Site-selective modification was performed at 7°C. Intact mass ESI on an Agilent G224 instrument. The addition of two azides onto the heavy chain of the mAb was monitored by -TOF LC-MS. A 1 ml GE Healthcare MabSelect column was used to remove excess 3-Azidopropylamine and MTG were removed by purification. The azido-mAb was eluted from the resin using HCl (pH 3.0) and then eluted with 7K Zeba Use a desalting column to exchange the solution into 1x dPBS. Add a 10-fold molar excess of DOTA-DBCO. , without shaking, in 1x dPBS at 37°C for 1 hour, followed by the site-specific azide H11B6 (DOL =1.94). Intact mass analysis confirmed the DBCO-azide click reaction. The completion of the conjugate reaction was monitored. Excess free chelator was removed by desalting on a Zeba7K desalting column. The gates were desalted into 1x dPBS, followed by three 15-fold serial dilution steps and 30K MWC o Using an Amicon concentrator device, purify the P by spinning at 3800Xg. This resulted in a final site-specific CAR of 1.94. DOTA-DBCO-H11B6 conjugate was obtained. Tosoh TSKgel G 3000SWxl 7.8mm x 30cm, 5u column, column temperature: room temperature, this column The tube was filled with DPBS buffer (x1, calcium and magnesium free), flow rate: 0.7 ml Analytical size exclusion chromatography eluting at 1 L / min, 18 min run, 18 μL injection volume confirmed that the final conjugate was monomeric.

[0438] Labeling at a ratio of 50:1:H11B6:Ac-225:N in plastic vials To a solution of aOAc (3 M in H2O, 10 μL), 225 Ac(NO3)3(0.1NH approximately 10 mCi / mL in Cl, 5 μL) and DOTA-H11B6 (site-specific, CAR= 1.94, 2.4 mg / mL in PBS buffer solution, 12.5 μL, 30 μg) were added sequentially. The ratio of H11B6 to Ac-225 was 50:1. After mixing, pH was measured using pH test paper. The H value was approximately 6.5. The reaction solution was left at 37°C for 2 hours. Then, 0.5 μL of the reaction mixture was added. The mixture was loaded onto iTLC-SG, which was developed with 10 mM EDTA. The LC-SG was left at room temperature overnight and then analyzed using a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 was eluted with the solvent. Approximately 21% of the radioactive signal was observed at the solvent front of iTLC-SG. It was observed that about 79% of Ac-225 was chelated in the reaction solution after 2 hours. This indicates that

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

[0440] [ka] Labeling at a ratio of 880:1 H11B6:Ac-225:N in plastic vials To a solution of aOAc (3 M in H2O, 20 μL), 225 Ac(NO3)3(0.1NH approximately 10 mCi / mL in Cl, 5 μL), DOTA-H11B6 (random conjugate CAR approximately 2.5, 10 mg / mL in 25 mM acetate buffer, pH 5.5, 50 μL, 5 00 μg) and NaOH solution (0.1 M, 2 μL) were added sequentially. The ratio of 225 is 880:1. After mixing, the pH was approximately 6.5 according to pH paper. The reaction solution was left at 37°C for 2 hours. Then, 0.5 μL of the reaction mixture was analyzed by iTLC-SG The dried iTLC-SG was loaded onto a column and developed with 10 mM EDTA. After standing, the samples were scanned using a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described in the paper, any free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, which is due to the This indicates that all Ac-225 was completely chelated in the reaction solution after 2 hours.

[0441] Labeling at a ratio of 440:1 H11B6:Ac-225:N in plastic vials To a solution of aOAc (3 M in H2O, 20 μL), 225 Ac(NO3)3(0.1NH approximately 10 mCi / mL in Cl, 5 μL), DOTA-H11B6 (random conjugate , CAR ~2.5, 10 mg / mL in 25 mM acetate buffer, pH 5.5, 25 μL, 2 50 μg) and NaOH solution (0.1 M, 1 μL) were added sequentially. The ratio of 225 is 440:1. After mixing, the pH was approximately 6.5 according to pH paper. The reaction solution was left at 37°C for 2 hours. Then, 0.5 μL of the reaction mixture was analyzed by iTLC-SG The dried iTLC-SG was loaded onto a column and developed with 10 mM EDTA. After standing, the samples were scanned using a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described in the paper, any free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of iTLC-SG, which is due to the This indicates that all Ac-225 was completely chelated in the reaction solution after 2 hours.

[0442] Labeling at a ratio of 220:1 H11B6:Ac-225:N in plastic vials To a solution of aOAc (3 M in H2O, 20 μL), 225 Ac(NO3)3(0.1NH approximately 10 mCi / mL in Cl, 5 μL), DOTA-H11B6 (random conjugate , CAR ~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 ratio of c-225 to HCl was 220:1. After mixing, the pH was approximately 6.5 using pH test paper. The reaction solution was left at 37°C for 2 hours. Then, 0.5 μL of the reaction mixture was analyzed by iTLC- The dried iTLC-SG was loaded onto the column and developed with 10 mM EDTA. After standing overnight, the samples were scanned using 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, which is It shows that 5% of Ac-225 was chelated in the reaction solution after 2 hours.

[0443] Labeling at a ratio of 110:1 H11B6:Ac-225:N in plastic vials To a solution of aOAc (3 M in H2O, 10 μL), 225 Ac(NO3)3(0.1NH approximately 10 mCi / mL in Cl, 5 μL), DOTA-H11B6 (random conjugate , CAR ~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 ratio of Ac-225 to Ac-225 is 110:1. After mixing, the pH was approximately 6.5 using pH test paper. The reaction solution was left at 37°C for 2 hours. Then, 0.5 μL of the reaction mixture was analyzed by iTLC. The dried iTLC-SG was loaded onto the column and developed with 10 mM EDTA. After standing overnight at RT, the samples were scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 will migrate to the solvent front along with the solvent. 71% of the radioactive signal was observed at the solvent front of iTLC-SG, which is , indicating that 29% of Ac-225 was chelated in the reaction solution after 2 hours.

[0444] Labeling at a ratio of 55:1 H11B6:Ac-225:Na in plastic vials To a solution of OAc (3 M in H2O, 10 μL), 225 Ac(NO3)3(0.1N HC Approximately 10 mCi / mL in 1, 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 ratio of c-225 to HCl was 55:1. After mixing, the pH was approximately 6.5 according to pH test paper. The reaction solution was left at 37°C for 2 hours. Then, 0.5 μL of the reaction mixture was analyzed by iTLC-S. The dried iTLC-SG was loaded onto a column of iTLC-SG and developed with 10 mM EDTA. After leaving overnight, the samples were scanned using a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described in the specification, any free Ac-225 migrates with the solvent to the solvent front. 64% of the radioactive signal was observed at the solvent front of iTLC-SG, which is It shows that 6% of Ac-225 was chelated in the reaction solution after 2 hours.

[0445] Conclusion: Examples 9, 16 and 17 demonstrate that chelator-containing DOTA inhibits chelator-containing H2b It was demonstrated to have a lower actinium chelating efficacy compared to p18c6.

[0446] Example 18: H2bp18c6-cis-cyclopentyl-fused macrocycle and 225 Ac( III) Chelate synthesis

[0447] [ka] The above H2bp18c6 derivatives having ring bonds on the macrocycle can generally be prepared according to the following scheme: Synthesize according to formula 21.

[0448] [ka]

[0449] Linkers for conjugation to targeting ligands can be prepared, for example, as shown in Scheme 3 and Intermediate 7 was converted to methyl 6-(bromo)(4-(tert-butoxy)methyl)-4-(tert-butoxy)methyl ... By reacting with (carbonyl)phenyl)methyl)picolinate, the "benzyl" position Cyclopentane 1,2-diol (compound 1) can be introduced into the cyclohexane position. H2bp18c6 derivatives with cyclohexyl ring fusion were used instead of 1,2-diol. A conductor can be obtained.

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

[0451] [ka]

[0452] Sodium hydride (5.63 g, 60% in mineral oil, 147.05 mL) in DMF (30 mL) To a suspension of (1R,2S)-cyclopentane-1, 2-Diol (3.0 g, 29.41 mmol) was added dropwise at 0° C. The reaction mixture was stirred at room temperature. This was stirred for 2 hours, cooled to 0°C again, and 2-(2-bromoethylene)-2-methylpropional in DMF (30 mL) was added. (2H-pyran) (18.44 g, 88.23 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. The reaction was quenched with saturated ammonium chloride. The combined organic layers were washed with water and added sodium sulfate. The crude oil was extracted with ethyl acetate (0-4%) in petroleum ether, dried over silica gel, filtered, and concentrated. Silica gel (230-400 mesh) flash chromatography using 0% and purified to give (1R,2S)-1,2-bis(2-((tetrahydro-2H-pyran -2-yl)oxy)ethoxy)cyclopentane (3.5 g, 33%) as a colorless liquid Got it.

[0453] ((1R,2S)-1,2-bis(2-((tetrahydrofuran))) in 100 mL of methanol -2H-pyran-2-yl)oxy)ethoxy)cyclopentane (3.5 g, 9.77 m To a solution of 100 mol of HCl in dioxane, add 1 mL of the solution, stir under reflux for 1 hour, and then cool. Cool and evaporate to give 2,2'-(((1R,2S)-cyclopentane-1,2-diyl) Bis(oxy)bis(ethan-1-ol) (2.0 g) was obtained, which was carried on to the next step without further purification. It was used in the process.

[0454] Dichloromethane (50 mL) and triethylamine (7.60 mL, 52.63 mmol) 2,2'-(((1R,2S)-cyclopentane-1,2-diyl)bis(oxo) A solution of bis(ethan-1-ol)2 (2.0 g, 10.52 mmol) was heated at 10°C. Then, p-toluenesulfonyl chloride (6.0 g, 31.56 mmol) was added in small portions. The mixture was stirred at ambient temperature for 16 hours. After the reaction was complete, the suspension was poured into 200 mL of dichloromethane. Dilute with hexane and wash with cold 1 M HCl (3 × 100 mL) followed by ice-cold water (2 × 1 00 mL), dried over sodium sulfate and evaporated to give a solid gum. The material was purified by flash chromatography using ethyl acetate (0-40%) in petroleum ether. The product was purified by silica gel (230-400 mesh) to give (((1R,2S)-silica gel). (Clopentane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis (4-methylbenzenesulfonate) (2.2 g, 42%) was obtained.

[0455] ((1R,2S)-cyclopentane-1,2-diyl)bis(2-methyl-2-pyridin-1-yl)-2-methylpropional in 50 mL of dry DMF (oxy)bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) ) (2.2 g, 4.42 mmol) and cesium carbonate (4.32 g, 13.25 mmol) The mixture of 1,2) was stirred at ambient temperature for 1.5 hours. -bis(2-(tosyl-λ 2 -Azanil)ethoxy)ethane (2.06g, 4.42m mol) was added dropwise over 2 hours. The mixture was stirred at ambient temperature for a further 20 hours. The HCl was removed under reduced pressure to give a solid paste, which was suspended in 200 mL of dichloromethane. The mixture was stirred for 30 minutes. The precipitated solid was filtered off and the filtrate was evaporated under high vacuum. was purified by flash chromatography using ethyl acetate (0-40%) in petroleum ether. (silica gel, 230-400 mesh) to obtain (16aR,19aS)-4 ,13-Ditosyltetradecahydro-2H,11H,17H-cyclopenta[b][1, 4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.8 g, 67 %) was obtained as a colorless liquid.

[0456] (16aR,19aS)-4,13-dithiocarbamate in acetic acid and hydrobromic acid (50%, 10 mL) Siltetradecahydro-2H,11H,17H-cyclopenta[b][1,4,10,1 3]Tetraoxa[7,16]diazacyclooctadecine (1.8 g, 2.95 mmol) To a solution of 1.38 g of phenol (14.75 mmol) was added at room temperature. The mixture was heated at 60° C. for 6 hours. After completion of the reaction, the mixture was cooled to room temperature and the acetic acid was removed under high vacuum. The residue was purified by reversed-phase column chromatography using 0-100% acetonitrile in water (0.1% TFA). Purification by ram purification gave (16aR,19aS)-tetradecahydro-2H,11H, 17H-Cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazamine Clooctadecine (0.7 g, 78%) was obtained as a colorless liquid.

[0457] (16aR,19aS)-tetradecahydro-2 in dry acetonitrile (10 mL) H,11H,17H-Cyclopenta[b][1,4,10,13]tetraoxa[7,1 6] Diazacyclooctadecine (0.7 g, 2.32 mmol), methyl 6-(chloromethyl) ethyl) picolinate (1.73 g, 5.80 mmol) and sodium carbonate (0.74 g A suspension of 1,2-dimethyl-3-(2-methyl-2-propanol, 6.96 mmol) was heated at 90° C. for 16 hours. After the reaction was complete, the reaction mixture was Cool to room temperature, filter through Celite and concentrate under reduced pressure. Flash chromatography (silica gel) using methanol (0–10%) in ethanol , 230-400 mesh) and purified by dimethyl 6,6'-(((16aR, 19a S)-Tetradecahydro-4H,13H,17H-cyclopenta[b][1,4,10, 13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis( methylene)) dipicolinate (0.55 g, 39%) was obtained as a brown liquid.

[0458] Dimethyl 6,6'-(((16aR,19aS)-tetradecamethyl)propanol in 6N hydrochloric acid (5 mL) Hydro-4H,13H,17H-cyclopenta[b][1,4,10,13]tetraoxo sa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene)dipyridine A solution of cholinate (0.55 g, 0.92 mmol) was heated at 80° C. for 5 hours. After completion, the reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC to give H2b p18c6-cis-cyclopentyl-fused macrocycle (0.3 g, 58%) was added to a colorless, sticky Obtained as a solid. LC-MS APCI: calculated for C29H40N4O8 572 .66; Observed m / z [M+H] + 572.9. Purity by LC-MS: 99.27%R T: 1.17. Purity by HPLC: 99.05% RT: 2.12. 1 H NMR(4 00MHz,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), 3.4 4-3.41(m,4H), 2.75-2.68(m,8H), 1.57-1.47(m ,6H).

[0459] [ka]

[0460] 225 Chelation with Ac(III): 0.1 N HCl (10 mCi / mL, 3 μL , 30 μCi) in tetramethylammonium acetate (1 M, 10 μL), H2bp 18c6-cis-cyclopentyl-fused macrocycle (2 mg / mL in water, 3 μL), 225 A c(NO3)3 was added continuously to a plastic vial. The pH was measured by pH test paper. The pH was approximately 6.5. The vials were heated at 37°C for 2 hours.

[0461] 0.5 μL of the reaction mixture was loaded onto iTLC-SG and developed with 10 mM EDTA. The dried iTLC was scanned for 2 min using a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 is released from the solvent. The solvent migrated to the front surface with iTLC, which 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 3 min. The mixture was then incubated for 10 min. 10 μL of the mixture was loaded onto an iTLC-SG and The dried iTLC was developed with 100 μL of EDTA. The TLC scanner was scanned after 20 hours. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC shows that 99% of the chelated Ac It showed -225.

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

[0464] [ka] Sodium hydride (5.63 g, 60% in mineral oil, 147.05 mL) in DMF (30 mL) To a suspension of (1R,2R)-cyclopentane-1, 2-Diol (3.0 g, 29.41 mmol) was added dropwise at 0° C. The reaction mixture was stirred at room temperature. This was stirred for 2 hours, cooled to 0°C again, and 2-(2-bromoethylene)-2-methylpropional in DMF (30 mL) was added. (2H-pyran) (18.44 g, 88.23 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. The reaction was quenched with saturated ammonium chloride. The combined organic layers were washed with water and added sodium sulfate. The crude oil was extracted with ethyl acetate (0-4%) in petroleum ether, dried over silica gel, filtered, and concentrated. Silica gel (230-400 mesh) flash chromatography using 0% and purified to give (1R,2R)-1,2-bis(2-((tetrahydro-2H-pyran -2-yl)oxy)ethoxy)cyclopentane (4.8 g, 46%) as a colorless liquid Got it.

[0465] (1R,2R)-1,2-bis(2-((tetrahydro- 2H-pyran-2-yl)oxy)ethoxy)cyclopentane (4.8 g, 13.40 m To a solution of 100 mol of HCl in dioxane was added 1 mL of HCl, the mixture was refluxed for 1 hour, cooled, and Evaporation gave 2'-(((1R,2R)-cyclopentane-1,2-diyl)bis(oxomethylidynamido) bis(ethan-1-ol) (2.9 g) was obtained, which was used in the next step without purification. Used.

[0466] Dichloromethane (50 mL) and triethylamine (11.03 mL, 76.31 mm 2'-(((1R,2R)-cyclopentane-1,2-diyl)bis(oxy) )) Bis(ethan-1-ol) (2.9 g, 10.52 mmol) was added to p-toluidine at 10 °C. To the mixture was added benzenesulfonyl chloride (8.70 g, 45.78 mmol) in small portions. The mixture was stirred at ambient temperature for 16 hours. After the reaction was complete, 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 The crude material was washed, dried over sodium sulfate and evaporated to give a solid gum. Flash chromatography (silica) using ethyl acetate in ether (0-40%) The resulting product was purified by filtration (gel, 230-400 mesh) to give (((1R,2R)-cyclopentane -1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methyl Benzene sulfonate) (3.5 g, 46%) was obtained as a colorless liquid.

[0467] ((1R,2R)-cyclopentane-1,2-diyl)bis(2-methyl-2-pyridin-1-yl)-2-methylpropional in 50 mL of dry DMF (oxy)bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) ) (3.5 g, 7.03 mmol) and cesium carbonate (6.87 g, 21.08 mmol) The mixture was stirred at ambient temperature for 1.5 hours. Bis(2-(tosyl-λ) 2 -azanyl)ethoxy)ethane ((3.19 g, 7.03 mm ol) 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. The mixture was stirred for 30 minutes. The precipitated solid was filtered off and the filtrate was evaporated under high vacuum. , flash chromatography using ethyl acetate (0–40%) in petroleum ether ( The resulting mixture was purified by silica gel (230-400 mesh) to give (16aR,19aR)-4, 13-Ditosyltetradecahydro-2H,11H,17H-cyclopenta[b][1,4 ,10,13]tetraoxa[7,16]diazacyclooctadecine (3.5g, 82% ) was obtained as a colorless liquid.

[0468] (16aR,19aR)-4,13-dithiocarbamate in hydrobromic acid (50%, 15 mL) in acetic acid Siltetradecahydro-2H,11H,17H-cyclopenta[b][1,4,10,1 3]Tetraoxa[7,16]diazacyclooctadecine (3.5 g, 5.73 mmol) To a solution of 2.70 g of phenol (28.68 mmol) was added at room temperature. The mixture was heated at 60° C. for 6 hours. After completion of the reaction, the mixture was cooled to room temperature and the acetic acid was removed under high vacuum. The crude material was purified by reverse chromatography using 0-100% acetonitrile in water (0.1% TFA). The (16aR,19aR)-tetradecahydro-2H,11 was purified by phase column purification to give H,17H-Cyclopenta[b][1,4,10,13]tetraoxa[7,16]dia Zacyclooctadecine (1.3 g, 75%) was obtained as a colorless liquid.

[0469] (16aR,19aS)-tetradecahydro-2 in dry acetonitrile (20 mL) H,11H,17H-Cyclopenta[b][1,4,10,13]tetraoxa[7,1 6] Diazacyclooctadecine (1.3 g, 4.30 mmol), methyl 6-(chloromethyl) ethyl) picolinate (1.99 g, 10.76 mmol) and sodium carbonate (1.37 A suspension of 100 mg of HCl (12.90 mmol) was heated at 90°C for 16 hours. The mixture was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. Flash chromatography (silica) using methanol (0-10%) in fluoromethane The product was purified by gel chromatography (230-400 mesh) to obtain 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%) was obtained as a brown liquid.

[0470] Dimethyl 6,6'-(((16aR,19aR)-tetradecane) in 6N hydrochloric acid (10 mL) 4H,13H,17H-cyclopenta[b][1,4,10,13]tetrahydro- (7,16)diazacyclooctadecine-4,13-diyl)bis(methylene)di A solution of picolinate (1.0 g, 1.66 mmol) was heated at 80° C. for 5 hours. After completion, the reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC to give H2bp 18c6-trans-cyclopentyl-fused macrocycle (0.8 g, 84%) was obtained as a colorless sticky LC-MS APCI: calculated for C29H40N4O8 57 2.66; 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,4 H), 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 (1M , 10 μL), H2bp18c6-trans-cyclopentyl-fused macrocycle (2 mg in water / mL, 3μL) 225 Ac(NO3)3 in 0.1N HCl (10mCi / mL, 3 The pH was approximately 6.5 according to pH test paper. was heated at 37°C for 2 hours.

[0473] 0.5 μL of the reaction mixture was loaded onto iTLC-SG and developed with 10 mM EDTA. The dried iTLC was scanned for 2 min using a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 is released from the solvent. The solvent migrated to the front surface with iTLC, which 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 3 min. The mixture was then incubated for 10 min. 10 μL of the mixture was loaded onto an iTLC-SG and The dried iTLC was developed with 100 μL of EDTA. The TLC scanner was used to scan the sample after 20 hours. Ac-225 migrates with the solvent to the solvent front, and iTLC shows that 99% of Ac-22 is chelated. 5 was shown.

[0475] Example 20: H2bp18c6-cis-cyclohexyl-fused macrocycle and 225 Ac(I II) Chelate synthesis

[0476] [ka] Sodium hydride (3.3 g, 60% in mineral oil, 86.20 mmHg) in DMF (20 mL) To a suspension of (1R,2S)-1,2-cyclohexane- The diol (2.0 g, 17.24 mmol) was added dropwise at 0° C. The reaction mixture was stirred at room temperature for 2 hours. This was cooled to 0°C again and 2-(2-bromoethoxy)-2-methyl-2-propanol was added to the reaction mixture in DMF (20 mL). (c) Tetrahydro-2H-pyran (10.81 g, 51.72 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. The reaction was quenched with saturated ammonium chloride and The combined organic phase was washed with brine and extracted with sodium sulfate. The crude oil was extracted with ethyl acetate (0-4%) in petroleum ether, dried over silica gel, filtered, and concentrated. Silica gel (230-400 mesh) flash chromatography using 0% and purified to give (1R,2S)-1,2-bis(2-((tetrahydro-2H-pyran (2.5 g, 39%) as a colorless liquid Got it.

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

[0478] Dichloromethane (25 mL) and triethylamine (5.31 mL, 36.76 mmol) 2,2'-(((1R,2S)-cyclohexane-1,2-diyl)bis(oxadiene) To a solution of bis(ethan-1-ol) (1.5 g, 7.35 mmol) at 10°C, p-Toluenesulfonyl chloride (4.20 g, 22.05 mmol) was added in small portions. The mixture was stirred at ambient temperature for 16 hours. After the reaction was complete, the suspension was diluted with 200 mL of dichloromethane. Dilute with methane and wash with cold 1 M HCl (3 × 100 mL), followed by ice-cold water (2 × 10 0), dried over sodium sulfate and evaporated to give a solid gum. , flash chromatography using ethyl acetate (0–40%) in petroleum ether ( The resulting mixture was purified using silica gel (230-400 mesh) to give (((1R,2S)-cyclohexyl) bis(ethane-1,2-diyl)bis(oxy)bis(ethane-2,1-diyl)bis(4- Methyl benzenesulfonate) (1.9 g, 50%) was obtained as a colorless liquid.

[0479] (((1R,2S)-cyclohexane-1,2-diyl)bis(2-methyl-2-methyl-1 ... (oxy)bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) ) (1.9 g, 3.71 mmol) and cesium carbonate (3.63 g, 11.13 mmol) The mixture was stirred at ambient temperature for 1.5 hours. Bis(2-(tosyl-λ) 2 -Azanil)ethoxy)ethane (1.68g, 3.71mm ol) 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. The mixture was stirred for 30 minutes. The precipitated solid was filtered off and the filtrate was evaporated under high vacuum. , flash chromatography using ethyl acetate in petroleum ether (0–40%) (silica gel, 230-400 mesh) to purify (16aR, 20aS)-4. 13-Ditosylhexadecahydro-2H,11H-benzo[b][1,4,10,13] Tetraoxa[7,16]diazacyclooctadecine (1.2 g, 52%) was treated as a colorless liquid. And got it.

[0480] (16aR,20aS)-4,13-dithiophene in hydrobromic acid (50%, 5 mL) in acetic acid Hexadecahydro-2H,11H-benzo[b][1,4,10,13]tetraoxa A solution of [7,16]diazacyclooctadecine (1.2 g, 1.92 mmol) was added to the solution of phenanthroline. The reaction mixture 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 dissolved in water (0 Purification was performed by reversed-phase column purification using 0-100% acetonitrile in 0.1% TFA. (16aR,20aS)-hexadecahydro-2H,11H-benzo[b][1,4 ,10,13]tetraoxa[7,16]diazacyclooctadecine (0.45g, 74 %) was obtained as a colorless liquid.

[0481] (16aR,20aS)-Hexadecahydro-2 in dry acetonitrile (10 mL) H,11H-benzo[b][1,4,10,13]tetraoxa[7,16]diazacycl Ioctadecine (0.45 g, 1.42 mmol), methyl 6-(chloromethyl)picolinate nate (0.65 g, 3.56 mmol) and sodium carbonate (0.45 g, 4.26 mmol) A suspension of 1,2-dimethyl-3,4-trimethyl-2,4-trimethyl-1 ... The crude material was extracted with methanol (0-1%) in dichloromethane. 0%) using flash chromatography (silica gel, 230-400 mesh) ) and purified by dimethyl 6,6'-(((16aR,20aS)-hexadecahydrochloride -4H,13H-benzo[b][1,4,10,13]tetraoxa[7,16]diaza Cyclooctadecine-4,13-diyl)bis(methylene)dipicolinate (0.26 g, 30%) as a brown liquid.

[0482] Dimethyl 6,6'-(((16aR,20aS)-hexadecamethylpropional in 6N hydrochloric acid (5 mL) Hydro-4H,13H-benzo[b][1,4,10,13]tetraoxa[7,16] Diazacyclooctadecine-4,13-diyl)bis(methylene)dipicolinate(0 A solution of 0.26 g (0.42 mmol) was heated at 80° C. for 5 hours. The reaction mixture was cooled under reduced pressure. The residue was purified by preparative HPLC to give the product H2bp18c6-cis-cyclohexyl The xyl-fused macrocycle (0.11 g, 44%) was obtained as an off-white solid. S APCI: Calculated for C30H42N4O8 586.69; Observed m / z [M +H] + 587.0. Purity by LC-MS: 98.15% RT: 1.31. Purity: 97.78% RT: 2.32. 1 H NMR (400 MHz, DMSO-d 6):δ13.35(s,1H), 9.84(s,1H), 8.15-8.09(m,4 H), 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] 225Chelation with Ac(III): Tetramethylammonium acetate (1M , 10 μL), H2bp18c6-cis-cyclohexyl-fused macrocycle (2 mg / m in water L, 3 μL) in 0.1 N HCl 225 Ac(NO3)3 (10 mCi / mL, 3 μ The pH was measured by pH test paper. The pH was 6.5. The vials were heated at 37°C for 2 hours.

[0485] 0.5 μL of the reaction mixture was loaded onto iTLC-SG and developed with 10 mM EDTA. The dried iTLC was scanned for 2 min using a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 is released from the solvent. The solvent front migrated with iTLC, which 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 3 min. The mixture was then incubated for 10 min. 10 μL of the mixture was loaded onto an iTLC-SG and The dried iTLC was developed with 100 μL of EDTA. The TLC scanner was scanned after 20 hours. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC shows that 99% of the chelated Ac It showed -225.

[0487] Example 21: H2bp18c6-trans-cyclohexyl-fused macrocycle and 225 Ac (III) Chelate synthesis

[0488] [ka] Sodium hydride (3.3 g, 60% in mineral oil, 86.20 mmHg) in DMF (20 mL) To a suspension of (1R,2R)-cyclohexane-1,2-ol in DMF (20 mL) The diol (2.0 g, 17.24 mmol) was added dropwise at 0° C. The reaction mixture was stirred at room temperature for 2 hours. This was cooled to 0°C again and 2-(2-bromoethoxy)-2-methyl-2-propanol was added to the reaction mixture in DMF (20 mL). (c) Tetrahydro-2H-pyran (10.81 g, 51.72 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. The reaction was quenched with saturated ammonium chloride. Extraction was performed with ethyl acetate (3 x 500 mL). The combined organic phase was washed with brine and The crude oil was dissolved in ethyl acetate (0-10%) in petroleum ether and concentrated. Silica gel (230-400 mesh) flash chromatography using 40% The product was purified by (2.45 g, 38%) as a colorless liquid. And got it.

[0489] (1R,2R)-1,2-bis(2-((tetrahydro- 2H-pyran-2-yl)oxy)ethoxy)cyclohexane (2.45 g, 6.5 mm To a solution of 1 mL of HCl in dioxane was added, the mixture was refluxed for 1 hour, 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] Dichloromethane (25 mL) and triethylamine (5.31 mL, 36.76 mmol) 2,2'-((((1R,2R-cyclohexane-1,2-diyl)bis(oxadiene) To a solution of bis(ethan-1-ol) (1.5 g, 7.35 mmol) at 10°C, p-Toluenesulfonyl chloride (4.20 g, 22.05 mmol) was added in small portions. The mixture was stirred at ambient temperature for 16 hours. The suspension was further diluted with 200 mL of dichloromethane. Dilute and wash with cold 1 M HCl (3 × 100 mL), followed by ice-cold water (2 × 100 mL). The crude material was washed with hexane, dried over sodium sulfate and evaporated to give a solid gum. Flash chromatography (silica) using ethyl acetate (0-40%) in petroleum ether The product was purified by gel chromatography (230-400 mesh) to give ((1R,2R)-cyclohexyl ethane-1,2-diyl)bis(oxy)bis(ethane-2,1-diyl)bis(4-methyl To obtain 2.1 g (56%) of 1,2-dimethylphenylsulfonate (2.1 g, 56%) as a colorless liquid.

[0491] (((1R,2R)-cyclohexane-1,2-diyl)bis(2-methyl-2-methyl-1 ... (oxy)bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) ) (2.1 g, 4.1 mmol) and cesium carbonate (4.01 g, 12.30 mmol) The mixture was stirred at ambient temperature for 1.5 hours. The suspension was added with 1,2-bis(2,4-dimethylamino)propanol in 30 mL of DMF. s(2-(tosyl-λ 2 -(azanyl)ethoxy)ethane (1.86 g, 4.1 mmol) was added dropwise over 2 hours. The mixture was stirred at ambient temperature for a further 20 hours. The solvent was removed under reduced pressure. The solution was removed with 500 ml of water to give a solid paste, which was suspended in 200 mL of dichloromethane and The mixture was stirred for 1 minute. The precipitated solid was filtered off and the filtrate was evaporated under high vacuum. The residue was dissolved in petroleum ether. Flash chromatography (silica gel) using ethyl acetate (0-40%) in ether (16aR,20aR)-4,13-dithiocarbamate was purified by filtration (230-400 mesh). hexadecahydro-2H,11H-benzo[b][1,4,10,13]tetraoxo sa[7,16]diazacyclooctadecine (1.35 g, 54%) was obtained as a colorless liquid. .

[0492] (16aR,20aR)-4,13-dithiocarbamate in hydrobromic acid (50%, 5 mL) in acetic acid Hexadecahydro-2H,11H-benzo[b][1,4,10,13]tetraoxa A solution of [7,16]diazacyclooctadecine (1.35 g, 2.16 mmol) was added to The reaction mixture was heated at 60°C for 6 hours. After cooling to room temperature, the acetic acid was removed under high vacuum. The residue was dissolved in water (0.1% TFA) The product was purified by reversed-phase column purification using 0 to 100% acetonitrile (16aR ,20aR)-Hexadecahydro-2H,11H-benzo[b][1,4,10,13] Tetraoxa[7,16]diazacyclooctadecine (0.5 g, 72%) was treated as a colorless liquid. And got it.

[0493] (16aR,20aR)-Hexadecahydro-2 in dry acetonitrile (10 mL) H,11H-benzo[b][1,4,10,13]tetraoxa[7,16]diazacycl Octadecin (0.5 g, 1.58 mmol), methyl 6-(chloromethyl)picolinate sodium carbonate (0.5 g, 4.74 mmol) The suspension of l) was heated at 90°C for 16 hours. The reaction mixture was cooled to room temperature and passed through Celite. The residue was extracted with methanol (0-10%) in dichloromethane. By using flash chromatography (silica gel, 230-400 mesh) Purification yielded dimethyl 6,6'-(((16aR,20aR)-hexadecahydro-4H, 13H-benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclo[2,3-dimethyl-1,4 ... Cutadecin-4,13-diyl)bis(methylene))dipicolinate (0.3g, 31% ) was obtained as a brown liquid.

[0494] Dimethyl 6,6'-(((16aR,20aR)-hexadecamethyl)propanol in 6N hydrochloric acid (5 mL) Hydro-4H,13H-benzo[b][1,4,10,13]tetraoxa[7,16] Diazacyclooctadecine-4,13-diyl)bis(methylene)dipicolinate(0 A solution of 1.3 g (0.49 mmol) was heated at 80° C. for 5 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC to give H2bp18c6-trans-cyclohexanediol. The sil-fused macrocycle (0.16 g, 56%) was obtained 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: 97.08% RT: 2.27. 1 H NMR (400 MHz, DMSO-d ):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 (1M , 10 μL), H2bp18c6-trans-cyclohexyl-fused macrocycle (2 mg in water / mL, 3 μL) in 0.1 N HCl 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi) were added to plastic vials sequentially. The RI was approximately 6.5. The vials were heated at 37°C for 2 hours.

[0497] 0.5 μL of the reaction mixture was loaded onto iTLC-SG and developed with 10 mM EDTA. The dried iTLC was scanned for 2 min using a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 is released from the solvent. The solvent front migrated with iTLC, which 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 3 min. The mixture was then incubated for 10 min. 10 μL of the mixture was loaded onto an iTLC-SG and The dried iTLC was developed with 100 μL of EDTA. The TLC scanner was scanned after 20 hours. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC shows that 99% of the chelated Ac It showed -225.

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

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

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

[0502] Dichloromethane (30 mL) and triethylamine (4.51 mL, 31.25 mmol) l) in 2,2'-((3-ethoxypropane-1,2-diyl)bis(oxy))bis A solution of (ethan-1-ol) (1.3 g, 6.25 mmol) was added to p-toluene at 10 °C. Sulfonyl chloride (3.56 g, 18.75 mmol) was added in portions. The mixture was stirred at ambient temperature for 16 hours. The suspension was diluted with 200 mL of dichloromethane and Wash with HCl (3 x 100 mL), followed by ice-cold water (1 x 100 mL), and add sodium sulfate. The crude material was extracted with ethyl acetate in petroleum ether and dried over ice and evaporated to give a solid gum. Flash chromatography (silica gel, 230-400 Purification by filtration (fiber mesh) gave ((3-ethoxypropane-1,2-diyl)bis(oxy) ) bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (2.0g , 62%) as a colorless liquid.

[0503] ((3-ethoxypropane-1,2-diyl)bis(oxy)methylpropane) in 25 mL of dry DMF ))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate)(2.0 g, 3.87 mmol) and cesium carbonate (3.79 g, 11.62 mmol) The mixture was stirred at ambient temperature for 1.5 hours. The suspension was added with 25 mL of 1,2-bis(2- (Tosyl-λ 2(-azanyl)ethoxy)ethane (1.75 g, 3.87 mmol) for 2 h The mixture was stirred at ambient temperature for an additional 20 hours, and the solvent was removed under reduced pressure. This gave 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 residue was dissolved in petroleum ether Flash chromatography (silica gel, 23 0-400 mesh) to obtain 2-(ethoxymethyl)-7,16-ditosyl- 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (1.1 g, The product was further purified by SFC to separate the two enantiomers. Decomposition of isomer-I (0.3 g, 12%) and isomer-II (0.26 g, 11%) The stereochemistry of isomers I and II was arbitrarily assigned.

[0504] (S)-2-(ethoxymethyl)-7,16-ditosyl- in acetic acid (50%, 1 mL) 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (isomer I, phenol (0.22 g, 2.39 mmol) was added at room temperature. The reaction was heated at 60° C. for 6 hours. After cooling to room temperature, acetic acid was removed under high vacuum. The residue was purified by 0-100% acetonitrile in water (0.1% TFA). Purification by reverse phase column gave (R)-(1,4,10,13-tetraoxa-7,16- Diazacyclooctadecan-2-yl)methyl acetate (arbitrarily assigned stereo Chemical) (0.1 g, 63%) was obtained as a colorless liquid.

[0505] (R)-(1,4,10,13-tetraoxa-7 ,16-diazacyclooctadecan-2-yl)methyl acetate (arbitrarily assigned (selected stereochemistry) (100 mg, 0.30 mmol), methyl 6-(chloromethyl)picolinate nate (138 mg, 0.75 mmol) and sodium carbonate (159 mg, 1.5 mmol) The mixture was heated at 90° C. for 16 hours. The reaction mixture was cooled to room temperature and filtered through Celite. The residue was purified by distillation with methanol (0-10%) in dichloromethane. Purification by flash chromatography (silica gel, 230-400 mesh) Dimethyl 6,6'-((2-(acetoxymethyl)-1,4,10,13-tetramethyl-2-methyl ... (hexa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene)-( R)-dipicolinate (arbitrarily assigned stereochemistry) (50 mg, 26%) was added to brown Obtained as a liquid.

[0506] 6,6'-((2-(acetoxymethyl)-1,4,10, 13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis( methylene)-(R)-dipicolinate (arbitrarily assigned stereochemistry) (50 mg, To a solution of 0.08 mmol of potassium carbonate (1 mg, 0.008 mmol) was added at room temperature. The mixture was added and stirred for 10 minutes. It was concentrated under reduced pressure. The residue was dissolved in methanol in dichloromethane. Flash chromatography (silica gel, 230-40%) with ethanol (0-10%) 00 mesh) to give 6,6'-((2-(hydroxymethyl)-1,4,1 0,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis (S)-(methylene)-(S)-dipicolinate (arbitrarily assigned stereochemistry) (22 mg, 48%) as a brown liquid.

[0507] 6,6'-((2-(hydroxymethyl)-1,4,10) in 6N hydrochloric acid (0.5 mL) ,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis (Methylene)-(S)-dipicolinate (arbitrarily assigned stereochemistry) (22m g, 0.04 mmol) was heated at 80°C for 5 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC to obtain H2bp18c6-off macrocycle-hydroxymethyl The isomer I (12 mg, 57%) was obtained as a sticky solid. LC-MS APCI:C Calculated for 27H38N4O9 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 (400 MHz, DMSO-d): δ 3.3 9(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 (1M , 10 μL), H2bp18c6-off macrocycle-hydroxylmethyl isomer I (2 ml in water g / mL, 3 μL) in 0.1 N HCl225 Ac(NO3)3 (10 mCi / mL , 3 μL, 30 μCi) were added to a plastic vial in sequence. H was approximately 6.5. The vial was heated at 37° C. for 2 hours.

[0510] 0.5 μL of the reaction mixture was loaded onto iTLC-SG and developed with 10 mM EDTA. The dried iTLC was scanned for 2 min using a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 is released from the solvent. The solvent front migrated with iTLC, which 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 3 min. The mixture was then incubated for 10 min. 10 μL of the mixture was loaded onto an iTLC-SG and The dried iTLC was developed with 100 μL of EDTA. The TLC scanner was scanned after 20 hours. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC shows that 99% of the chelated Ac It showed -225.

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

[0513] [ka] (R)-2-(ethoxymethyl)-7,16-ditosyl- in acetic acid (50%, 1 mL) 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (arbitrarily divided) To a solution of isomer II (assigned stereochemistry) (isomer II, 0.26 g, 0.42 mmol) The reaction mixture was heated at 60°C for 6 hours. After cooling to room temperature, the acetic acid was removed under high vacuum. The residue was dissolved in water (0.1% TFA) Purification was performed by reverse-phase column purification using 0–100% acetonitrile to obtain (S)-(1, 4,10,13-tetraoxa-7,16-diazacyclooctadecan-2-yl)methyl Acetate (arbitrarily assigned stereochemistry) (0.06 g, 43%) as a colorless liquid Got it.

[0514] (S)-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecane) -2-yl)methyl acetate (arbitrarily assigned stereochemistry) (0.06 g, 0. 18 mmol), methyl 6-(chloromethyl)picolinate (0.083 g, dry acetone nitrile (3 mL), 0.45 mmol) and sodium carbonate (0.095 g, 0.9 The reaction mixture was cooled to room temperature and filtered through Celite. The residue was purified by filtration and concentrated under reduced pressure using methanol (0-10%) in dichloromethane. The mixture was purified by flash chromatography (silica gel, 230-400 mesh) using Dimethyl 6,6'-((2-(acetoxymethyl)-1,4,10,13-tetramethyl-2-methyl ... Oxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene) -(S)-dipicolinate (arbitrarily assigned stereochemistry) (0.025 g, 22% ) was obtained as a brown liquid.

[0515] Dimethyl 6,6'-((2-(acetoxymethyl)-1)-methyl)-1 in methanol (0.5 mL) ,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-di (yl)bis(methylene)-(S)-dipicolinate (arbitrarily assigned stereochemistry ) (0.025 g, 0.04 mmol) was added to a solution of potassium carbonate (0.5 mg, 0.00 4 mmol) was added at room temperature. The mixture was stirred for 10 minutes and then concentrated under reduced pressure. The crude product was purified by column chromatography on silica (230 Purification by flash chromatography on a 400 mesh (~400 mesh) afforded dimethyl 6,6 '-((2-(hydroxymethyl)-1,4,10,13-tetraoxa-7,16-di (Azacyclooctadecane-7,16-diyl)bis(methylene)-(R)-dipicolinone The compound (0.01 g, 43%) was obtained as a brown liquid.

[0516] Dimethyl 6,6'-((2-(hydroxymethyl)-1, 4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl A solution of 0.01 g (0.02 mmol) of bis(methylene)-dipicolinate was added to 8 The mixture was heated at 0° C. for 3 hours. The reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC. Purification yielded H2bp18c6-off macrocycle-hydroxymethyl isomer II (4 mg, 44 %) was obtained as a viscous liquid. LC-MS APCI: C27H38N4O9 Calculated value 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,15 H).

[0517] [ka]

[0518] 225 Chelation with Ac(III): Tetramethylammonium acetate (1M , 10 μL), H2bp18c6-off macrocycle-hydroxylmethyl isomer II (in water mg / mL, 3 μL) in 0.1 N HCl 225 Ac(NO3)3(10mCi / m The pH values ​​were measured by pH test paper. The pH was approximately 6.5. The vials were heated at 37° C. for 2 hours.

[0519] 0.5 μL of the reaction mixture was loaded onto iTLC-SG and developed with 10 mM EDTA. The dried iTLC was scanned for 2 min using a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 is released from the solvent. The solvent front migrated with iTLC, which 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 3 min. The mixture was then incubated for 10 min. 10 μL of the mixture was loaded onto an iTLC-SG and The dried iTLC was developed with 100 μL of EDTA. The TLC scanner was scanned after 20 hours. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC shows that 99% of the chelated Ac It showed -225.

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

[0522] [ka] Dimethyl 6,6'-((2-(hydroxymethyl)-1,4,1 0,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis (methylene)(S)-dipicolinate (100 mg, 0.17 mmol) (arbitrarily To a stirred solution of 10 mg of 1,000 sucrose (6% sucrose, 10 ... The reaction mixture was cooled to room temperature and then The reaction mixture was cooled to 0°C again and stirred at rt for 10 h. A solution of ethyl acetate (39 mg, 0.25 mmol, 20 uL) was added dropwise. The reaction mixture was allowed to cool to room temperature. After the reaction was completed (monitored by LCMS), the reaction The reaction mixture was quenched with saturated NH4Cl solution and extracted with ethyl acetate. The organic extract was dried over anhydrous sodium sulfate and concentrated. The residue was extracted with dichloromethane as eluent. Silica gel (230-400 mesh) flash column with 5% methanol in ethanol. The product dimethyl 6,6'-((2-(ethoxymethyl)methyl)methyl) (ethyl)-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] Dimethyl in THF (0.25 mL), water (0.5 mL) and methanol (0.25 mL) 6,6'-((2-(ethoxymethyl)-1,4,10,13-tetraoxa-7, 16-Diazacyclooctadecane-7,16-diyl)bis(methylene)(S)-dipyridine Stirring of cholinate (45 mg, 0.07 mmol) (arbitrarily assigned stereochemistry) To the solution was added lithium hydroxide monohydrate (9 mg, 0.21 mmol). The mixture was stirred at room temperature for 4 hours. After the reaction was completed (monitored by UPLC-MS), the p The concentration of H was adjusted to 3-4 with 1.5M aqueous HCl and concentrated. The residue was subjected to preparative HPLC. Purified from H2bp18c6-off macrocycle-ethoxymethyl isomer I (28 mg, 6 6%) was obtained as a viscous liquid. LC-MS APCI: C29H42N4O9 Calculated value 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), 4.6 9(s,4H), 3.98-3.34(m,28H), 1.06(t,J=6.80Hz ,3H).

[0524] [ka]

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

[0526] 0.5 μL of the reaction mixture was loaded onto iTLC-SG and developed with 10 mM EDTA. The dried iTLC was scanned for 2 min using a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 is released from the solvent. The solvent front migrated with iTLC, which 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 3 min. The mixture was then incubated for 10 min. 10 μL of the mixture was loaded onto an iTLC-SG and The dried iTLC was developed with 100 μL of EDTA. The TLC scanner was scanned after 20 hours. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC shows that 99% of the chelated Ac It showed -225.

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

[0529] [ka] 6,6'-((2-(hydroxymethyl)-1,4,10, 13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis( (R)-Dipicolinate Dimethyl (Arbitrarily Assigned Stereochemistry) (6 To a stirred solution of 100 mg of sodium hydride (6 mg, 60 in mineral oil) was added at 0°C. % dispersion, 0.15 mmol) was slowly added. The reaction mixture was cooled to room temperature and then The reaction mixture was cooled to 0°C again and stirred for 10 hours. A solution of ethyl acetate (23 mg, 0.15 mmol, 12 uL) was added dropwise. The reaction mixture was allowed to stand at room temperature. The reaction was 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 organic extract was dried over anhydrous sodium sulfate and concentrated. The residue was extracted with dichloromethane as eluent. Silica gel (230-400 mesh) flushing with 5% methanol in methyl ether The compound was purified by column chromatography to give 6,6'-((2-(ethoxymethyl)- 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16- (R)-dipicolinate dimethyl (arbitrarily assigned stereochemistry) (20 mg, 32%) as a brown liquid.

[0530] 6,6' in THF (0.1 mL), water (0.25 mL), and methanol (0.1 mL) -((2-(ethoxymethyl)-1,4,10,13-tetraoxa-7,16-diaza Cyclooctadecane-7,16-diyl)bis(methylene)(R)-dipicolinate To a solution of methyl (arbitrarily assigned stereochemistry) (20 mg, 0.03 mmol), Lithium hydroxide monohydrate (4 mg, 0.09 mmol) was added and the reaction mixture was stirred at room temperature. After the reaction was completed (monitored by UPLC-MS), the pH of the reaction was adjusted to 1. The solution was adjusted to 3-4 with 0.5M aqueous HCl and concentrated. The residue was purified by preparative HPLC. Prepared by H2bp18c6-off macrocycle-ethoxymethyl isomer II (5 mg, 26%) was obtained as a viscous liquid. LC-MS APCI: calculated for C29H42N4O9 Value 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 NMR1H-NMR(400MHz,DMSO-d6):δ13.40(s,1H), 9.72(s,1H), 8.16-8.00(m,4H), 7.80-7.77(m,2 H), 4.69(s,4H), 4.34-3.38(m,28H), 1.06(t,J= 6.80Hz,3H).

[0531] [ka]

[0532] 225 Chelation with Ac(III): Tetramethylammonium acetate (1M , 10 μL), H2bp18c6-off macrocycle-hydroxylmethyl isomer II (in water mg / mL, 1 μL) in 0.1 N HCl 225 Ac(NO3)3(10mCi / m The pH values ​​were measured by pH test paper. The pH was approximately 6.5. The vials were heated at 37° C. for 2 hours.

[0533] 0.5 μL of the reaction mixture was loaded onto iTLC-SG and developed with 10 mM EDTA. The dried iTLC was scanned for 2 min using a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 is released from the solvent. The solvent front migrated with iTLC, which 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 3 min. The mixture was then incubated for 10 min. 10 μL of the mixture was loaded onto an iTLC-SG and The dried iTLC was developed with 100 μL of EDTA. The TLC scanner was scanned after 20 hours. Under the elution conditions described herein, any free Ac-225 migrates with the solvent to the solvent front. iTLC shows that 99% of the chelated Ac It showed -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 dissolved in anhydrous D (S)-(+)-2,2-Dimethyl-1,3-dioxolane-4 in MF (500 mL) - was added to a solution of methanol (50 g, 378 mmol) at 0 °C and kept at this temperature for 10 min. After stirring, benzyl bromide (77.63 g, 454 mmol) was slowly added to the suspension. The reaction mixture was allowed to reach room temperature after 30 minutes and stirred for an additional 8 hours. The reaction mixture was quenched with ammonium hydroxide solution and extracted with dichloromethane (2 x 500 mL). The combined organic layers were washed with brine and dried over sodium sulfate. After removing the solvent, the residue The eluate was analyzed by flash column chromatography (silica gel) using petroleum ether and ethyl acetate. Compound 1 (72 g, 86%) was purified by filtration using a filtration filter (230-400 mesh). was obtained as.

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

[0538] Step 3: Sodium hydride (26.29 g, 60% in mineral oil, 6%) in DMF (20 mL) To a suspension of compound 2 (25.0 g, 137 mmol) in DMF (100 mL) mol) was added dropwise at 0°C. The reaction mixture was stirred at room temperature for 2 hours. It was then cooled to 0°C again. and 2-(2-bromoethoxy)tetrahydro-2H-pyran in DMF (100 mL). (85.89 g, 411 mmol) 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 diluted with ethyl acetate (3×30 The combined organic layers were washed with water, dried over sodium sulfate, and filtered. The crude oil was purified by silica gel chromatography using ethyl acetate in petroleum ether (0-40%). The product was purified by gel (230-400 mesh) flash chromatography. 3 (32 g, 53%) was obtained as a colorless liquid.

[0539] Step 4: Solution of compound 3 (32 g, 73.05 mmol) in 500 mL of methanol To this was added 5 mL of HCl in dioxane, stirred at reflux for 1 hour, cooled and evaporated. 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 The solution was cooled to 10° C. and dissolved in triethylamine (53 mL, 370 mmol). Add solid p-toluenesulfonyl chloride (42.20 g, 222 mmol) in small portions The mixture was stirred at ambient temperature for 16 hours. After the reaction was complete, the suspension was poured into a 1000 mL flask. The solution was further diluted with dichloromethane, cooled aqueous HCl (1 M, 3 × 200 mL), ice-cold water (2 × 20 0 mL), dried over sodium sulfate and evaporated to a solid gum. The product was purified by flash chromatography using ethyl acetate in petroleum ether (0-40%). The product was purified by silica gel (230-400 mesh). ) was obtained as a colorless liquid.

[0541] Step 6: Compound 5 (30 g, 51.9 mmol) and carbonate in 200 mL of dry DMF A mixture of cesium (50.76 g, 155.7 mmol) was stirred at ambient temperature for 1.5 hours. To the suspension, compound 6 (23.56 g, 51.9 mmol) in 200 mL of DMF was added. The mixture was stirred at ambient temperature for an additional 20 hours. The solvent was removed under reduced pressure. The solution was removed to give a solid paste, which was suspended in 1000 mL of dichloromethane and The mixture was stirred for 1 minute. The precipitated solid was filtered off and the filtrate was evaporated under high vacuum. The crude material was extracted with petroleum Flash chromatography (silica) using ethyl acetate in ether (0-40%) The product 7 (24 g, 67%) was purified by filtration (gel, 230-400 mesh). Got it as a body.

[0542] Step 7: Compound 7 (24 g, 34.78 m) in hydrobromic acid (acetic acid 50%, 100 mL) To a solution of 16.35 g (174 mmol) of phenol was added at room temperature. The reaction was heated at 60° C. for 6 hours. After the reaction was complete, 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. The product was purified by conventional methods to obtain compound 8 (8.0 g, 69%) as a colorless liquid.

[0543] Step 8: Compound 8 (8.0 g, 23.95 mmHg) in dry acetonitrile (100 mL) ol), compound 9 (11.07 g, 59.88 mmol) and sodium carbonate (12.6 A suspension of 9 g (119.75 mmol) was heated at 90°C for 16 hours. The mixture was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. Flash chromatography (Schiff) using methanol (0–10%) in dichloromethane The product was purified by silica gel (230-400 mesh). Product 10 (5.0 g, 33%) was obtained as a brown liquid.

[0544] Step 9: Dissolution of compound 10 (5.0 g, 7.91 mmol) in methanol (50 mL) Potassium carbonate (0.11 g, 0.79 mmol) was added to the solution at room temperature and stirred for 10 minutes. After the reaction was completed, it was concentrated under reduced pressure. The residue was dissolved in 0-10% methanol in dichloromethane. Flash chromatography on silica (230-400 mesh) eluted with a gradient of ethanol. The product 11 (3.5 g, 75%) was obtained as a brown liquid.

[0545] Step 10: Compound 11 (1.0 g, 1.7 mmol) in dichloromethane (20 mL) To the solution 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 the reaction was completed, it was concentrated and the crude product was extracted with methanol (1- The compound was purified by column chromatography (alumina-neutral) using 2% HCl. 12 (0.7 g, 62%) was obtained as a brown liquid.

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

[0547] Step 12: Compound 14 (20 mg, 0.02 mmol) in 6N hydrochloric acid (0.5 mL) The solution was stirred at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. Purify the product H2bp18c6-off macrocycle-ethyl sulfide by preparative HPLC. Mine (6 mg, 36%) was obtained as a sticky solid. LC-MS APCI: C29H4 Calculated for 3N5O8S: 621.75; observed m / z [M+H] + 622.2. L Purity by C-MS: 97.94% RT: 1.38. Purity by HPLC: 94.11% RT:2.94. 1 H NMR(400MHz,DMSO-d6):δ11.02-1 1.00(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 dissolved in methanol (2 mL, 4N A cold solution of HCl in HCl was added and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure to give the compound Product 2 was obtained as a yellow liquid (55 mg, 64%).

[0550] Step 2: Compound 15 (50 mg, 0.08 mmol) in dry dichloromethane (2 mL) ) and triethylamine (24 mg, 0.24 mmol), g, 0.16 mmol) was added to the vial. The vial was irradiated with MW radiation (150 W power) at 90 °C. The reaction mixture was then diluted with dichloromethane (10 mL) and water (5 ml). The mixture was washed successively with 1M HCl (5 mL), water (5 mL), and anhydrous sodium sulfate. After concentration, the crude product was purified by elution with 0-10% methanol in dichloromethane. Flash chromatography on silica gel (230-400 mesh) using Further purification gave compound 1616 (20 mg, 38%) as a yellow solid.

[0551] Step 3: Compound 16 (20 mg, 0.03 mmol) in hydrochloric acid (6 N, 0.5 mL) The mixture was stirred at room temperature overnight. After the reaction was completed, it was concentrated under reduced pressure. The residue was purified by preparative HPLC. and purified to obtain H2bp18c6-off macrocycle-ethyl sulfide NCS (6 mg, 31%). ) was obtained as a white solid. LC-MS APCI: calculated for C30H41N5O8S2 Calculated: 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), 3.9 6-3.52(m,23H), 2.85(t,J=6.40Hz,2H), 2.70(t ,J=8.00Hz,2H).

[0552] [ka]

[0553] Step 1: Compound 17 (1.0 g, 1.7 mmol) in dichloromethane (20 mL) To the solution 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, it was concentrated and the residue was purified by elution with methanol in dichloromethane (1-2%). Compound 18 (0 0.7g, 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 water Sodium chloride (12 mg, 60% in mineral oil, 0.3 mmol) was added at 0°C. The reaction mixture was stirred at room temperature for 10 minutes. Compound 1 in DMF (1 mL) was added to the reaction mixture. 8 (100 mg, 0.15 mmol) was added at 0° C. The reaction was stirred at room temperature for 2 h. The progress of the reaction was monitored by TLC. After completion of the reaction, it was concentrated and the residue was Compound 20 was purified by preparative HPLC using 2-methylpropyltrimonial and 0.1% TFA to give compound 20( 15 mg, 13%) was obtained as a brown liquid.

[0555] Step 3: Compound 20 (15 mg, 0.02 mmol) in hydrochloric acid (6 N, 0.5 mL) The solution was stirred at room temperature overnight. After the reaction was completed, it was concentrated under reduced pressure. The residue was purified by preparative HPLC. The product H2bp18c6-off macrocycle-pentyl sulfide amine (4m g, 33%) as a viscous liquid. LC-MS APCI: C32H49N5O8 Calculated for S 663.83; observed m / z [M+H] + 664.2. LC-MS Pur...

Claims

1. A chelator of formula (I): 【Chemistry 1】 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. ring A and ring B are each independently halo, alkyl, alkenyl, cycloalkenyl, alkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR １３ , -SR １３ 、-(CH ２ ) ｐ COOR １３ 、-OC(O)R １３ 、-N(R １３ ) ２ 、- CON (R １３ ) ２ , -NO ２ , -CN-OC(O)N(R １３ ) ２ and the group consisting of X optionally substituted with one or more substituents selected from Z １ and Z ２ Each of the groups independently represents -(C(R １２ ) ２ ) ｍ - or - (CH ２ ) ｎ -C (R １２ ) (X)-(CH ２ ) ｎ - and Each X is independently -L １ -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; L １ is absent or is a linker, R １１ is a nucleophilic or electrophilic moiety, or R １１ is the targeting ligand Including, 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 of is independently hydrogen, alkyl, or X mosquito, Or, R １４ and R １５ and / or R １６ and R １７ But they are combined together with the carbon atoms form a 5- or 6-membered cycloalkyl ring optionally substituted by X. Formation provided that the chelator contains at least one X, and when X is present on ring A or ring B, , L １ is a linker, or R １２ and R １４ ~R １７ At least one of them is water Not an element, a chelator.

2. is a chelator of formula (II), 【Chemistry 2】 During the ceremony, A １ is N or CR １ or not present, A ２ is N or CR ２ and A ３ is N or CR ３ and A ４ is N or CR ４ and A ５ is N or CR ５ and A ６ is N or CR ６ or not present, A ７ is N or CR ７ and A ８ is N or CR ８ and A ９ is N or CR ９ and A １０ is N or CR １０ and However, A １ , A ２ , A ３ , A ４ and A ５ Three or fewer of the ６ , A ７ , A ８ , A ９ and A １０ 3 or less of the R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ and R １０ Each of these is independent. and hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl , heterocyclyl, heteroaryl, -OR １３ , -SR １３ , -(CH ２ ) ｐ COOR １３ 、-OC(O)R １３ 、-N(R １３ ) ２ 、-CON(R １３ ) ２ 、-NO ２ 、-C N, -OC(O)N(R １３ ) ２ and -X; Alternatively, any two directly adjacent R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ and R １０ together with the atoms to which they are attached form a 5- or 6-membered substituent or forms an unsubstituted carbocyclic or nitrogen-containing ring, Z １ and Z ２ Each of the groups independently represents -(C(R １２ ) ２ ) ｍ - or - (CH ２ ) ｎ -C (R １２ ) (X)-(CH ２ ) ｎ - and Each X is independently -L １ -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; L １ is absent or is a linker, R １１ is a nucleophilic or electrophilic moiety, or R １１ contains a targeting ligand fruit, 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 of is independently hydrogen, alkyl, or X mosquito, Or, R １４ and R １５ and / or R １６ and R １７ But they are combined together with the carbon atoms form a 5- or 6-membered cycloalkyl ring optionally substituted by X. Formation provided that the chelator contains at least one X and R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ and R １０ If any one of １ is the linker or R １２ and R １４ ~R １７ at least one of which is not hydrogen. Item 1. A chelator according to Item 1.

3. is a chelator of formula (III), 【Transformation 3】 During the ceremony, Each A １１ are independently O, S, NMe, or NH; Z １ and Z ２ Each of the groups independently represents -(C(R １２ ) ２ ) ｍ - or - (CH ２ ) ｎ -C (R １２ ) (X)-(CH ２ ) ｎ - and Each X is independently -L １ -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; L １ is absent or is a linker, R １１ is a nucleophilic or electrophilic moiety, or R １１ is the targeting ligand Including, 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 of is independently hydrogen, alkyl, or X mosquito, Or, R １４ and R １５ and / or R １６ and R １７ But they are combined together with the carbon atoms form a 5- or 6-membered cycloalkyl ring optionally substituted by X. Formation Each R １８ are independently hydrogen, halo, alkyl, alkenyl, cycloalkyl, cyclo Alkenyl, aryl, heterocyclyl, heteroaryl, -OR １３ , -SR １３ ,- (CH ２ ) ｐ COOR １３ 、-OC(O)R １３ 、-N(R １３ ) ２ 、-CON(R １３ ) ２ , -NO ２ , -CN-OC(O)N(R １３ ) ２ and -X; provided that the chelator contains at least one X and R １８ If is X, then L １ Is Lin Car or R １２ and R １４ ~R １７ at least one of which is not hydrogen; The chelator of claim 1.

4. The chelator is 【Chemistry 4】 is selected from the group consisting of During the ceremony, L １ is absent or is a linker, R １１ is a nucleophilic or electrophilic moiety, or R １１ is the targeting ligand Including, Each R １２ are independently hydrogen, —CH ３ or -CH ２ CH ３ However, at least One R １２ But -CH ３ or -CH ２ CH ３ 2. The chelator of claim 1, wherein:

5. R １１ But -NH ２ , -NCS, -NCO, -N ３ , alkynyl, cycloalkynyl, -C(O)R １３ , -COOR １３ , -CON(R １３ ) ２ , maleimide, halogenated 5. The compound according to claim 1, wherein the compound is cyclohexyl, tetrazine, or trans-cyclooctene. The chelator described.

6. R １１ cyclooctynyl, bicyclononynyl (BCN), difluorinated cyclooctynyl Octynyl (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, bia Arylazacyclooctynonyl (BARAC), dibenzoazacyclooctynyl (DIB AC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), dif Difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO) ) and tetramethoxydibenzocyclooctynyl (TMDIBO), 6. The chelator of claim 5, which is a cyclooctynyl derivative of

7. R １１ The chelator of claim 6, wherein is DBCO or BCN.

8. R １１ comprises a targeting ligand, wherein the targeting ligand is an antibody or an antigen binding fragments, scaffold proteins, small molecules or aptamers according to claims 1 to 4 The chelator according to any one of the preceding claims.

9. L1 is, 【Transformation 5】 is selected from the group consisting of 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.

9. A chelator according to any one of claims 1 to 8, wherein R is an integer between 0 and 6, preferably between 0 and 6. 【Request Item 10】 【Chemistry 6】 A chelator selected from the group consisting of:

11. the chelator comprises a radioactive metal ion bound to the chelator by a coordinate bond, The chelator according to any one of claims 1 to 10, which thereby forms a radioactive metal complex.

12. A radiometal complex containing a chelator bound by a coordinate bond to an alpha-emitting radiometal ion. wherein the radioactive metal complex has a structure of formula (I-m): 【Transformation 7】 During the ceremony, M is a radioactive metal ion, preferably an α-emitting radioactive metal ion, more preferably an α-emitting radioactive metal ion. Cu-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. ring A and ring B are each independently halo, alkyl, alkenyl, cycloalkenyl, alkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR １３ , -SR １３ 、-(CH ２ ) ｐ COOR １３ 、-OC(O)R １３ 、-N(R １３ ) ２ 、- CON (R １３ ) ２ , -NO ２ , -CN-OC(O)N(R １３ ) ２ and the group consisting of X optionally substituted with one or more substituents selected from Z １ and Z ２ Each of the groups independently represents -(C(R １２ ) ２ ) ｍ - or - (CH ２ ) ｎ -C (R １２ ) (X)-(CH ２ ) ｎ - and Each X is independently -L １ -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; L １ is absent or is a linker, R １１ is a nucleophilic or electrophilic moiety, or R １１ contains a targeting ligand fruit, 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 of is independently hydrogen, alkyl, or X mosquito, Or, R １４ and R １５ and / or R １６ and R １７ But they are combined together with the carbon atoms form a 5- or 6-membered cycloalkyl ring optionally substituted by X. Formation However, the radiometal complex contains at least one X, and X is present on ring A or ring B. In this case, L １ is a linker, or R １２ and R １４ ~R １７ At least one of A radioactive metal complex in which one is not hydrogen.

13. A radiometal complex of formula (II-m): 【Transformation 8】 During the ceremony, M is a radioactive metal ion, preferably an α-emitting radioactive metal ion, more preferably an α-emitting radioactive metal ion. Cu-225 ( ２２５ Ac), A １ is N or CR １ or not present, A ２ is N or CR ２ and A ３ is N or CR ３ and A ４ is N or CR ４ and A ５ is N or CR ５ and A ６ is N or CR ６ or not present, A ７ is N or CR ７ and A ８ is N or CR ８ and A ９ is N or CR ９ and A １０ is N or CR １０ and However, A １ , A ２ , A ３ , A ４ and A ５ Three or fewer of the ６ , A ７ , A ８ , A ９ and A １０ 3 or less of the R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ and R １０ Each of these is independent. and hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl , heterocyclyl, heteroaryl, -OR １３ , -SR １３ , -(CH ２ ) ｐ COOR １３ 、-OC(O)R １３ 、-N(R １３ ) ２ 、-CON(R １３ ) ２ 、-NO ２ 、-C N-OC(O)N(R １３ ) ２ and -X; Alternatively, any two directly adjacent R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ and R １０ together with the atoms to which they are attached form a 5- or 6-membered substituent or forms an unsubstituted carbocyclic or nitrogen-containing ring, Z １ and Z ２ Each of the groups independently represents -(C(R １２ ) ２ ) ｍ - or - (CH ２ ) ｎ -C (R １２ ) (X)-(CH ２ ) ｎ - and Each X is independently -L １ -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; L １ is absent or is a linker, R １１ is a nucleophilic or electrophilic moiety, or R １１ is the targeting ligand Including, 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 of is independently hydrogen, alkyl, or X mosquito, Or, R １４ and R １５ and / or R １６ and R １７ But they are combined together with the carbon atoms form a 5- or 6-membered cycloalkyl ring optionally substituted by X. Formation provided that the radiometal complex contains at least one X and R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ and R １０ If any one of １ Is Is a runner or １２ and R １４ ~R １７ At least one of the The radioactive metal complex of claim 12.

14. A radiometal complex of formula (III-m): 【Chemistry 9】 During the ceremony, M is a radioactive metal ion, preferably an α-emitting radioactive metal ion, more preferably an α-emitting radioactive metal ion. Cu-225 ( ２２５ Ac), Each A １１ are independently O, S, NMe, or NH; Z １ and Z ２ Each of the groups independently represents -(C(R １２ ) ２ ) ｍ - or - (CH ２ ) ｎ -C (R １２ ) (X)-(CH ２ ) ｎ - and Each X is independently -L １ -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; L １ is absent or is a linker, R １１ is a nucleophilic or electrophilic moiety, or R １１ is the targeting ligand Including, 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 of is independently hydrogen, alkyl, or X mosquito, Or, R １４ and R １５ and / or R １６ and R １７ But they are combined together with the carbon atoms form a 5- or 6-membered cycloalkyl ring optionally substituted by X. Formation Each R １８ are independently hydrogen, halo, alkyl, alkenyl, cycloalkyl, cyclo Alkenyl, aryl, heterocyclyl, heteroaryl, -OR １３ , -SR １３ ,- (CH ２ ) ｐ COOR １３ 、-OC(O)R １３ 、-N(R １３ ) ２ 、-CON(R １３ ) ２ , -NO ２ , -CN-OC(O)N(R １３ ) ２ and -X; provided that the radioactive metal complex contains at least one X and R １８ If X, then L １ Is Is a runner or １２ and R １４ ~R １７ At least one of the The radioactive metal complex of claim 12.

15. The radioactive metal complex is 【Chemistry 10】 is selected from the group consisting of During the ceremony, M is actinium-225 ( ２２５ Ac), and L １ is not present or phosphorus It is a car, R １１ is a nucleophilic or electrophilic moiety, or R １１ is the targeting ligand Including, Each R １２ are independently hydrogen, —CH ３ or -CH ２ CH ３ However, at least One R １２ is -CH ３ or -CH ２ CH ３ Any one of claims 12 to 14, The radioactive metal complex according to claim 1.

16. Any of claims 12 to 15 conjugated to an antibody or antigen-binding fragment thereof A radioimmunoconjugate comprising the radiometal complex of any one of claims 1 to 4.

17. The antibody or antigen-binding fragment thereof is attached to the radioactive complex via a triazole moiety. R １１ 17. The radioimmunoconjugate of claim 16, wherein the radioimmunoconjugate is bound to 【Request Item 18】 【Chemistry 11】 A radioimmunoconjugate 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 an α-emitting radioactive metal ion. Cu-225 ( ２２５ Ac), L １ is the linker, mAb is an antibody or antigen-binding fragment thereof; Each R １２ are independently hydrogen, —CH ３ or -CH ２ CH ３ However, at least One R １２ is -CH ３ or -CH ２ CH ３ A radioimmunoconjugate.

19. the radioimmunoconjugate comprising: 【Chemistry 12】 is selected from the group consisting of where mAb is an antibody or antigen-binding fragment thereof, preferably binding to a tumor antigen. and more preferably, the antibody or antigen-binding fragment thereof specifically binds to the mAbs include PSMB127, pertuzumab, cetuximab, panitumumab, and Herceptin. and H11B6.

20. A method for preparing a radioimmunoconjugate comprising: irradiating a chelator according to claim 8 with and contacting the targeting ligand with a radioactive metal ion, thereby forming a radioactive metal complex bound to the targeting ligand. The method includes:

21. 21. The method of claim 20, wherein the targeting ligand is an antibody or an antigen-binding fragment thereof. How to do it.

22. 1. A method for preparing a radioimmunoconjugate, said method comprising: (i) a modified polypeptide comprising an antibody or antigen-binding fragment thereof covalently bound to an azido group; Providing chids and (ii) a chelator comprising a chelator covalently bonded to an alkynyl or cycloalkynyl group. providing a complex; (iii) reacting the azide group with the alkynyl group or cycloalkynyl group contacting the modified polypeptide with the chelator complex under conditions that allow forming an immunoconjugate therewith; (iv) contacting the immunoconjugate with an α-emitting radioactive metal ion, thereby and forming said radioimmunoconjugate by said radioimmunoconjugate. The compound contains a radioactive complex comprising the α-emitting radioactive metal ion bound to the chelator. the radioactive complex has the structure of formula (I-m): 【Chemistry 13】 During the ceremony, M is a radioactive metal ion, preferably an α-emitting radioactive metal ion, more preferably an α-emitting radioactive metal ion. Cu-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. ring A and ring B are each independently halo, alkyl, alkenyl, cycloalkenyl, alkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR １３ , -SR １３ 、-(CH ２ ) ｐ COOR １３ 、-OC(O)R １３ 、-N(R １３ ) ２ 、- CON (R １３ ) ２ , -NO ２ , -CN-OC(O)N(R １３ ) ２ and the group consisting of X optionally substituted with one or more substituents selected from Z １ and Z ２ Each of the groups independently represents -(C(R １２ ) ２ ) ｍ - or - (CH ２ ) ｎ -C (R １２ ) (X)-(CH ２ ) ｎ - and Each X is independently -L １ -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; L １ is absent or is a linker, R １１ is an alkynyl group or a cycloalkynyl group, 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 of is independently hydrogen, alkyl, or X mosquito, Or, R １４ and R １５ and / or R １６ and R １７ But they are combined together with the carbon atoms form a 5- or 6-membered cycloalkyl ring optionally substituted by X. Formation However, the radioactive metal complex contains at least one X, and X is present in ring A or ring B. In case, L １ is a linker, or R １２ and R １４ ~R １７ At least one of But hydrogen is not, how.

23. 1. A method for preparing a radioimmunoconjugate, said method comprising: (i) a modified antibody comprising an antibody or antigen-binding fragment thereof covalently bound to an azide group; providing an antibody or antigen-binding fragment thereof; (ii) a radioactive complex comprising an α-emitting radioactive metal ion bound to a chelator by a coordinate bond. the chelator is covalently linked to an alkynyl or cycloalkynyl group. providing a coupled (iii) reacting the azide group with the alkynyl group or cycloalkynyl group The modified antibody or antigen-binding fragment thereof is reacted with the radioactive complex under conditions that allow and contacting the radioimmunoconjugate with the body, thereby preparing the radioimmunoconjugate; the radioactive complex has the structure of formula (I-m): 【Chemistry 14】 During the ceremony, M is an α-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. ring A and ring B are each independently halo, alkyl, alkenyl, cycloalkenyl, alkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, -OR １３ , -SR １３ 、-(CH ２ ) ｐ COOR １３ 、-OC(O)R １３ 、-N(R １３ ) ２ 、- CON (R １３ ) ２ , -NO ２ , -CN-OC(O)N(R １３ ) ２ and the group consisting of X optionally substituted with one or more substituents selected from Z １ and Z ２ Each of the groups independently represents -(C(R １２ ) ２ ) ｍ - or - (CH ２ ) ｎ -C (R １２ ) (X)-(CH ２ ) ｎ - and Each X is independently -L １ -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; L １ is absent or is a linker, R １１ is an alkynyl group or a cycloalkynyl group, 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 of is independently hydrogen, alkyl, or X mosquito, Or, R １４ and R １５ and / or R １６ and R １７ But they are combined together with the carbon atoms form a 5- or 6-membered cycloalkyl ring optionally substituted by X. Formation However, the radioactive metal complex contains at least one X, and X is present in ring A or ring B. In case, L １ is a linker, or R １２ and R １４ ~R １７ At least one of But hydrogen is not, how.

24. R １１ cyclooctynyl, bicyclononynyl (BCN), difluorinated cyclooctynyl Octynyl (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, bia Arylazacyclooctynonyl (BARAC), dibenzoazacyclooctynyl (DIB AC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), dif Difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO) ) and tetramethoxydibenzocyclooctynyl (TMDIBO), 24. The method of claim 22 or 23, wherein the cyclooctynyl derivative is

25. A radioimmunoconjugate according to any one of claims 16 to 19 and a pharmaceutically acceptable A pharmaceutical composition comprising a carrier.

26. Selectively targeting neoplastic cells for radiation therapy in a subject in need thereof - Patent Application 20070122999 26. A method for treating a patient suffering from atopic dermatitis, comprising administering to said subject a therapeutically effective amount of the pharmaceutical composition of claim 25. and

27. Methods for treating a neoplastic disease or disorder in a subject in need thereof 26. A method for treating a patient, comprising administering to said subject a therapeutically effective amount of the pharmaceutical composition of claim 25. Hmm, a method.

28. 10. A method for treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of claim 2 6. A method comprising administering to the subject the pharmaceutical composition described in 5.

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