Macrocyclic Chelate and Method of Using the Same

Abstract

A macrocyclic chelator (I) for chelating an alpha-emitting radioactive metal ion, e.g., actinium-225, is provided. A radiometal complex containing an alpha-emitting radioactive metal ion coupled to the macrocyclic chelator via coordination bonding, and a radioimmunoconjugate containing the radiometal complex covalently linked to a targeting ligand, e.g., an antibody or its antigen-binding fragment, are also provided. The radioimmunoconjugate may be generated by a click chemical reaction. Methods of using the radiocomplex and radioimmunoconjugate for selectively targeting neoplastic cells for radiotherapy and for treating neoplastic diseases and disorders are also described. [Chemical Formula (I)]

Description

Technology Field

[0001] Cross-reference with related applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 846,044 filed May 10, 2019, which is incorporated herein by reference in whole and for all purposes.

[0003] Reference to the electronically submitted sequence list

[0004] The present application includes a sequence list submitted electronically via EFS-Web as an ASCII format sequence list with the filename "JBI6072WOPCT1_SeqListing.txt", a creation date of May 1, 2020, and a size of 26 kb. The sequence list submitted via EFS-Web is part of this specification and is incorporated herein by reference in its entirety. Background Technology

[0005] Alpha particle-emitting radionuclides are very promising in cancer therapy due to the combination of their high energy and short-range action, offering the potential for potent killing primarily localized to tumor cells (Kim, YS and MW Brechbiel, An overview of targeted alpha therapy. Tumour Biol[ , 2012. 33(3): p. 573-90]). Targeted delivery of alpha-emitting molecules using antibodies, scaffold proteins, small molecule ligands, aptamers, or other binding moieties specific to cancer antigens provides a method for selectively delivering radionuclides to tumors to enhance their efficacy and mitigate off-target effects. In common practice, radiocomplexes are prepared by attaching a binding moiety to a chelator that binds to an alpha-emitting radiometal. Many such examples use a monoclonal antibody (mAb) as a targeting ligand to prepare what is known as a radioimmunoconjugate.

[0006] Actinium-225 ( 225 Actinium-225 (Ac) is an alpha-emitting radioisotope of particular interest for medical applications (Miederer et al., Realizing the potential of the Actinium-225 radionuclide generator in targeted alpha particle therapy applications). Adv Drug Deliv Rev , 2008. 60(12):71-82]). 225 The 10-day half-life of Ac is long enough to facilitate the preparation of radioconjugates, but short enough to be consistent with the circulating pharmacokinetics of delivery vehicles such as antibodies. As such, 225 Ac radioimmunoconjugates are particularly interesting. Additionally, 225 Ac is a stable isotope 209 It increases efficacy by decaying into a series of stages that ultimately emit four alpha particles before reaching Bi. Another interesting radioisotope for medical applications is Lutetium-177, which emits both gamma irradiation suitable for imaging and intermediate-energy beta irradiation suitable for radiotherapy. 177 Lu) is. 177Lu-labeled peptides show reduced normal tissue damage, and 177 Lu-labeling has been shown to enable the use of a single radiolabeled agent for both therapy and imaging (Literature [Kwekkeboom DJ, et al. [ 177 Lu-DOTA 0 ,Tyr 3 ]octreotate: comparison with [ 111 In-DTPA 0 ]octreotide in patients. Eur J Nucl Med [ . 2001;28: p. 1319-1325]). Other radioisotopes used in therapeutic applications include, for example, beta or alpha emitters, e.g. 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I, 149 Tb, 152 Tb, 155 Tb, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 255 Fm, and 227 Th is included. Other radioisotopes used in imaging applications include gamma-emitting radioisotopes, such as 62 Cu, 64 Cu,67 Ga, 68 Ga, 86 Y, 89 Zr, and 111 In is included.

[0007] Currently, the most widely used chelator for actinium-225 and lanthanides is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; tetraxaten), and previous clinical and preclinical programs have primarily used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for actinium chelation. However, DOTA chelation of actinium is known to be a challenge (Literature [Deal, KA, et al., Improved in vivo stability of actinium-225 macrocyclic complexes. J Med Chem [, 1999. 42(15): p. 2988-92]). For example, DOTA enables chelation ratios of DOTA:actinium-225 exceeding 500:1 when attached to targeting ligands such as proteins or antibodies, often requiring harsh conditions or high levels of DOTA per antibody. Other macrocyclic chelators for lanthanides and actinium-225 are, for example, International Patent Application Publication No. WO 2018 / 183906; literature [Thiele et al. "An Eighteen-Membered Macrocyclic Ligand for Actinium-225 Targeted Alpha Therapy" Angew. Chem . Int . Ed . (2017) 56, 14712-14717]; Roca-Sabio et al. “Macrocyclic Receptor Exhibiting Unprecedented Selectivity for Light Lanthanides” J. Am. Chem. SocIt is described in

[2009] 131, 3331-3341.

[0008] Since it has been demonstrated that site-specific methods can increase both the efficacy and safety of antibody-drug conjugates (ADCs) compared to random conjugation, site-specificity has become a key focus area in the field of antibody-drug conjugates (literature [Agarwal, P. and CR Bertozzi, Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development, Bioconjug Chem [ , 2015. 26(2): p. 176-92]). It is thought that similar safety and efficacy benefits can be achieved for radioimmunoconjugates.

[0009] Therefore, radioactive metal, preferably an alpha-emitting radioactive metal, such as actinium-225 ( 225 There is a need in the art for novel chelators that can bind to Ac and be used to produce stable radioimmunoconjugates with high specific activity and high yield. The present invention relates to alpha-emitting radioactive metals, particularly, regardless of specific activity or the most common metal impurities. 225This need is met by providing a macrocyclic chelator capable of binding to a radioactive metal such as Ac. The chelator of the present invention can be used to generate radioimmunoconjugates having high stability in vitro and in vivo by conjugating to targeting ligands, such as antibodies, proteins, aptamers, small molecules, etc., preferably in a site-specific manner using "click chemistry." The radioimmunoconjugate generated by the conjugation of the chelator of the present invention to a targeting ligand can be used for targeted radiotherapy, e.g., targeted radiotherapy of neoplastic cells and / or targeted treatment of neoplastic diseases or disorders including cancer.

[0010] In general, the present invention relates to a chelator of formula (I):

[0011] [Chemical Formula (I)]

[0012]

[0013] In the above formula,

[0014] Rings A and B are each independently a 6 to 10-membered aryl or a 5 to 10-membered heteroaryl, wherein rings A and B are each a halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclil, 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 arbitrarily substituted with one or more substituents independently selected from the group consisting of X;

[0015] Z1 and Z2 each independently -(C(R 12 )2) m - or -(CH2) n-C(R 12 )(X)-(CH2) n -is;

[0016] Each X is independently -L1-R 11 And;

[0017] Each n is independently 0, 1, 2, 3, 4, or 5;

[0018] Each m is independently 1, 2, 3, 4, or 5;

[0019] Each p is independently 0 or 1;

[0020] L1 is absent or a linker;

[0021] R 11 is a nucleophilic moiety or an electrophilic moiety, or R 11 It includes a targeting ligand;

[0022] Each R 12 is independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclil, or heteroaryl;

[0023] Each R 13 is independently hydrogen or alkyl;

[0024] R 14 , R 15 , R 16 , and R 17 Each is independently hydrogen, alkyl, or X, or

[0025] Alternatively, R 14 and R 15 and / or R 16 and R 17 They form a 5- or 6-membered cycloalkyl ring arbitrarily substituted with X together with the carbon atom to which they are attached;

[0026] However, the chelator includes one or more Xs, and if X exists on ring A or ring B, L1 is a linker or R 12 and R 14 to R 17 One or more of them are not hydrogen.

[0027] In an alternative embodiment, ring A and ring B are each optionally substituted heterocyclil rings, such as oxazoline.

[0028] In one embodiment, the chelator of the present invention is a chelator of formula (II):

[0029] [Chemical Formula (II)]

[0030]

[0031] In the above formula,

[0032] A1 is N or CR1 or is absent;

[0033] A2 is N or CR2;

[0034] A3 is N or CR3;

[0035] A4 is N or CR4;

[0036] A5 is N or CR5;

[0037] A6 is N or CR6 or is absent;

[0038] A7 is N or CR7;

[0039] A8 is N or CR8;

[0040] A9 is N or CR9;

[0041] A 10 is N or CR 10 This is it;

[0042] However, three or fewer of A1, A2, A3, A4, and A5 are N, and A6, A7, A8, A9, and A 10 Three or fewer of them are N;

[0043] R1, R2, R3, R4, R5, R6, R7, R8, R9, and R 10 Each is hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclil, heteroaryl, -OR 13 , -SR 13 , -(CH2) pCOOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN, -OC(O)N(R 13 Independently selected from the group consisting of )2, and -X, or

[0044] Alternatively, any two directly adjacent R1, R2, R3, R4, R5, R6, R7, R8, R9, and R 10 They form substituted or unsubstituted 5- or 6-membered carbocyclic or nitrogen-containing rings together with the atoms to which they are attached;

[0045] Z1 and Z2 each independently -(C(R 12 )2) m - or -(CH2) n -C(R 12 )(X)-(CH2) n -is;

[0046] Each X is independently -L1-R 11 And;

[0047] Each n is independently 0, 1, 2, 3, 4, or 5;

[0048] Each m is independently 1, 2, 3, 4, or 5;

[0049] Each p is independently 0 or 1;

[0050] L1 is absent or a linker;

[0051] R 11 is a nucleophilic moiety or an electrophilic moiety, or R 11 It includes a targeting ligand;

[0052] Each R 12 is independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclil, or heteroaryl;

[0053] Each R 13 is independently hydrogen or alkyl;

[0054] R 14 , R 15 , R 16 , and R 17 Each is independently hydrogen, alkyl, or X, or

[0055] Alternatively, R 14 and R 15 and / or R 16 and R 17 They form a 5- or 6-membered cycloalkyl ring arbitrarily substituted with X together with the carbon atom to which they are attached;

[0056] However, the chelator includes one or more X, R1, R2, R3, R4, R5, R6, R7, R8, R9, and R 10 If any one of them is X, L1 is a linker.

[0057] In one embodiment, the chelator of the present invention is a chelator of formula (III):

[0058] [Chemical Formula (III)]

[0059]

[0060] In the above formula,

[0061] Each A 11 is independently O, S, NMe, or NH;

[0062] Z1 and Z2 each independently -(C(R 12 )2) m - or -(CH2) n -C(R 12 )(X)-(CH2) n -is;

[0063] Each X is independently -L1-R 11 And;

[0064] Each n is independently 0, 1, 2, 3, 4, or 5;

[0065] Each m is independently 1, 2, 3, 4, or 5;

[0066] Each p is independently 0 or 1;

[0067] L1 is absent or a linker;

[0068] R 11 is a nucleophilic moiety or an electrophilic moiety, or R 11 It includes a targeting ligand;

[0069] Each R 12 is independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclil, or heteroaryl;

[0070] Each R 13 is independently hydrogen or alkyl;

[0071] R 14 , R 15 , R 16 , and R 17 Each is independently hydrogen, alkyl, or X, or

[0072] Alternatively, R 14 and R 15 and / or R 16 and R 17 They form a 5- or 6-membered cycloalkyl ring arbitrarily substituted with X together with the carbon atom to which they are attached;

[0073] Each R 18 -OR hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclil, 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 Independently selected from the group consisting of )2, and -X,

[0074] However, the chelator includes one or more X, and R 18 If this is X, L1 is a linker or R 12 and R14 to R 17 One or more of them are not hydrogen.

[0075] In a specific embodiment, the chelator is

[0076]

[0077]

[0078]

[0079]

[0080] is,

[0081] In the above formula,

[0082] L1 is absent or a linker;

[0083] R 11 is a nucleophilic moiety or an electrophilic moiety, or R 11 It includes a targeting ligand;

[0084] Each R 12 is independently hydrogen, -CH3, or -CH2CH3, provided that one or more R 12 is -CH3 or CH2CH3.

[0085] In some embodiments, R 11 -NH2, -NCS, -NCO, -N3, alkynyl, cycloalkynyl, -C(O)R 13 , -COOR 13 , -CON(R 13 )2, maleimido, acyl halide, tetrazine, or trans-cyclooctene.

[0086] In a given embodiment, R 11is a cyclooctinyl or cyclooctinyl derivative selected from the group consisting of bicyclononinyl (BCN), difluorinated cyclooctinyl (DIFO), dibenzocyclooctinyl (DIBO), keto-DIBO, biaryl-azacyclooctinyl (BARAC), dibenzo-azacyclooctinyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctinyl (DIMAC), difluorobenzocyclooctinyl (DIFBO), monobenzocyclooctinyl (MOBO), and tetramethoxydibenzocyclooctinyl (TMDIBO).

[0087] In a specific embodiment, R 11 It is DBCO or BCN.

[0088] In some embodiments, R 11 It includes a targeting ligand, wherein the targeting ligand includes an antibody or its antigen-binding fragment, a scaffold protein, a small molecule, or an aptamer.

[0089] In a specific embodiment, the targeting ligand is an antibody or its antigen-binding fragment.

[0090] In another aspect, the present invention relates to a radiometal complex comprising a chelator of the present invention comprising a radioactive metal ion coupled to the chelator through a coordination bond.

[0091] In one embodiment, the radioactive metal complex of the present invention has the structure of the chemical formula (Im):

[0092] [Chemical Formula (Im)]

[0093]

[0094] In the above formula,

[0095] M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion;

[0096] Rings A and B are each independently a 6 to 10-membered aryl or a 5 to 10-membered heteroaryl, wherein rings A and B are each a halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclil, 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 arbitrarily substituted with one or more substituents independently selected from the group consisting of X;

[0097] Z1 and Z2 each independently -(C(R 12 )2) m - or -(CH2) n -C(R 12 )(X)-(CH2) n -is;

[0098] Each X is independently -L1-R 11 And;

[0099] Each n is independently 0, 1, 2, 3, 4, or 5;

[0100] Each m is independently 1, 2, 3, 4, or 5;

[0101] Each p is independently 0 or 1;

[0102] L1 is absent or a linker;

[0103] R 11 is a nucleophilic moiety or an electrophilic moiety, or R 11 It includes a targeting ligand;

[0104] Each R 12 is independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclil, or heteroaryl;

[0105] Each R 13is independently hydrogen or alkyl;

[0106] R 14 , R 15 , R 16 , and R 17 Each is independently hydrogen, alkyl, or X, or

[0107] Alternatively, R 14 and R 15 and / or R 16 and R 17 They form a 5- or 6-membered cycloalkyl ring arbitrarily substituted with X together with the carbon atom to which they are attached;

[0108] However, the radioactive metal complex contains one or more Xs, and if X exists on ring A or ring B, L1 is a linker or R 12 and R 14 to R 17 One or more of them are not hydrogen.

[0109] In an alternative embodiment, ring A and ring B are each optionally substituted heterocyclil rings, such as oxazoline.

[0110] In one embodiment, the radioactive metal complex of the present invention is a radioactive metal complex of the formula (II-m):

[0111] [Chemical Formula (II-m)]

[0112]

[0113] In the above formula,

[0114] M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion;

[0115] A1 is N or CR1 or is absent;

[0116] A2 is N or CR2;

[0117] A3 is N or CR3;

[0118] A4 is N or CR4;

[0119] A5 is N or CR5;

[0120] A6 is N or CR6 or is absent;

[0121] A7 is N or CR7;

[0122] A8 is N or CR8;

[0123] A9 is N or CR9;

[0124] A 10 is N or CR 10 This is it;

[0125] However, three or fewer of A1, A2, A3, A4, and A5 are N, and A6, A7, A8, A9, and A 10 Three or fewer of them are N;

[0126] R1, R2, R3, R4, R5, R6, R7, R8, R9, and R 10 Each is hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclil, 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 Independently selected from the group consisting of )2, and -X, or

[0127] Alternatively, any two directly adjacent R1, R2, R3, R4, R5, R6, R7, R8, R9, and R 10 They form substituted or unsubstituted 5- or 6-membered carbocyclic or nitrogen-containing rings together with the atoms to which they are attached;

[0128] Z1 and Z2 each independently -(C(R 12 )2) m - or -(CH2) n -C(R 12 )(X)-(CH2) n-is;

[0129] Each X is independently -L1-R 11 And;

[0130] Each n is independently 0, 1, 2, 3, 4, or 5;

[0131] Each m is independently 1, 2, 3, 4, or 5;

[0132] Each p is independently 0 or 1;

[0133] L1 is absent or a linker;

[0134] R 11 is a nucleophilic moiety or an electrophilic moiety, or R 11 It includes a targeting ligand;

[0135] Each R 12 is independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclil, or heteroaryl;

[0136] Each R 13 is independently hydrogen or alkyl;

[0137] R 14 , R 15 , R 16 , and R 17 Each is independently hydrogen, alkyl, or X, or

[0138] Alternatively, R 14 and R 15 and / or R 16 and R 17 They form a 5- or 6-membered cycloalkyl ring arbitrarily substituted with X together with the carbon atom to which they are attached;

[0139] However, the radioactive metal complex contains one or more Xs, and R1, R2, R3, R4, R5, R6, R7, R8, R9, and R 10 If either of them is X, L1 is a linker or R 12 and R 14 to R 17 One or more of them are not hydrogen.

[0140] In one embodiment, the radioactive metal complex of the present invention is a radioactive metal complex of the formula (III-m):

[0141] [Chemical Formula (III-m)]

[0142]

[0143] In the above formula,

[0144] M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion;

[0145] Each A 11 is independently O, S, NMe, or NH;

[0146] Z1 and Z2 each independently -(C(R 12 )2) m - or -(CH2) n -C(R 12 )(X)-(CH2) n -is;

[0147] Each X is independently -L1-R 11 And;

[0148] Each n is independently 0, 1, 2, 3, 4, or 5;

[0149] Each m is independently 1, 2, 3, 4, or 5;

[0150] Each p is independently 0 or 1;

[0151] L1 is absent or a linker;

[0152] R 11 is a nucleophilic moiety or an electrophilic moiety, or R 11 It includes a targeting ligand;

[0153] Each R 12 is independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclil, or heteroaryl;

[0154] Each R 13 is independently hydrogen or alkyl;

[0155] R 14 , R 15 , R 16 , and R 17 Each is independently hydrogen, alkyl, or X, or

[0156] Alternatively, R 14 and R 15 and / or R 16 and R 17 They form a 5- or 6-membered cycloalkyl ring arbitrarily substituted with X together with the carbon atom to which they are attached;

[0157] Each R 18 -OR hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclil, 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 Independently selected from the group consisting of )2, and -X,

[0158] However, the radioactive metal complex contains one or more Xs, and R 18 If this is X, L1 is a linker or R 12 and R 14 to R 17 One or more of them are not hydrogen.

[0159] In a given embodiment, the alpha-emitting radioactive metal ion is actinium-225 ( 225 It is Ac).

[0160] In certain embodiments, the radioactive metal complex of the present invention

[0161]

[0162]

[0163]

[0164] and

[0165] It is selected from a group consisting of,

[0166] In the above formula,

[0167] M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion, more preferably actinium-225 ( 225 Ac) and L1 is absent or a linker;

[0168] R 11 is a nucleophilic moiety or an electrophilic moiety, or R 11 It includes a targeting ligand;

[0169] Each R 12 is independently hydrogen, -CH3, or -CH2CH3, provided that one or more R 12 is -CH3 or CH2CH3.

[0170] In other general suns, the present invention R 11 The present invention relates to an immunoconjugate comprising a chelator of the present invention that is covalently linked to a targeting ligand, preferably an antibody or an antigen-binding fragment thereof.

[0171] In another general aspect, the present invention R 11 The present invention relates to a radioimmunoconjugate comprising a radioactive metal complex of the present invention that is covalently linked to a targeting ligand, preferably an antibody or an antigen-binding fragment thereof.

[0172] In one embodiment, the radioimmunoconjugate comprises a radioactive metal complex of the present invention covalently linked to a targeting ligand, in particular an antibody or its antigen-binding fragment, via a triazole moiety.

[0173] In a specific embodiment, the radioimmunoconjugate of the present invention

[0174] , ,

[0175] , ,

[0176] , ,

[0177] , ,

[0178] , and It is selected from a group consisting of,

[0179] In the above formula,

[0180] L1 is a linker; mAb is an antibody or an antigen-binding fragment thereof, preferably mAb is an antibody or antigen-binding fragment that specifically binds to a tumor antigen selected from the group consisting of tumor cells, more preferably prostate-specific membrane antigen (PSMA), BCMA, Her2, EGFR, KLK2, CD19, CD22, CD30, CD33, CD79b, and Nectin-4; and each R 12 is independently hydrogen, -CH3, or -CH2CH3, provided that one or more R 12 is -CH3 or -CH2CH3.

[0181] In another general aspect, the present invention comprises the step of covalently linking a chelator or radioactive metal complex of the present invention with a targeting ligand, preferably R of the chelator or radioactive metal complex. 11 The present invention relates to a method for manufacturing an immunoconjugate or a radioimmunoconjugate, comprising the step of covalently linking to an antibody or an antigen-binding fragment thereof through [a specific method].

[0182] In a specific embodiment, the method for preparing a radioimmunoconjugate of the present invention comprises a "one-step direct radiolabeling" method comprising the following steps (e.g., as illustrated in FIG. 2c):

[0183] (i) providing a modified polypeptide comprising a polypeptide (e.g., an antibody or an antigen-binding fragment thereof) covalently linked to a first click reaction partner (e.g., an azido group);

[0184] (ii) providing a chelator complex comprising a chelator of the present invention covalently connected to a second click reaction partner (e.g., an alkynyl group or a cycloalkynyl group);

[0185] (iii) a step of contacting the modified polypeptide with the chelator complex under conditions that enable the first click reaction partner (e.g., an azido group) to react with the second click reaction partner (e.g., an alkynyl group or a cycloalkynyl group) to form a polypeptide-chelator complex (i.e., an immunoconjugate); and

[0186] (iv) a step of preparing a radioimmunoconjugate by contacting a polypeptide-chelator complex with a radioactive metal ion (wherein the radioimmunoconjugate comprises a polypeptide labeled with a radioactive metal ion, e.g., a modified antibody labeled with an alpha-emitting radioactive metal ion that is bound to a chelator via coordination bonding or an antigen-binding fragment thereof).

[0187] According to a specific embodiment, step (iv) is performed without metal-free conditions. Preferably, the method is carried out in a site-specific manner as described herein.

[0188] In an alternative embodiment, the method for preparing a radioimmunoconjugate of the present invention comprises a "click radiolabeling" method (e.g., as illustrated in FIG. 2d):

[0189] (i) providing a modified antibody or an antigen-binding fragment thereof comprising an antibody covalently linked to an azido group or an antigen-binding fragment thereof;

[0190] (ii) providing a radioactive complex of the present invention comprising an alpha-emitting radioactive metal ion coupled to a chelator through a coordination bond, wherein the chelator is covalently connected to an alkynyl group or a cycloalkynyl group; and

[0191] (iii) a step of preparing a radioimmunoconjugate by contacting a modified antibody or its antigen-binding fragment with a radioactive complex under conditions that enable the azido group to react with an alkynyl group or a cycloalkynyl group.

[0192] In some embodiments, the cycloalkinyl group is a cyclooctinyl or cyclooctinyl derivative selected from the group consisting of bicyclononinyl (BCN), difluorinated cyclooctinyl (DIFO), dibenzocyclooctinyl (DIBO), keto-DIBO, biaryl-azacyclooctinyl (BARAC), dibenzo-azacyclooctinyl (DIBAC), dimethoxyazacyclooctinyl (DIMAC), difluorobenzocyclooctinyl (DIFBO), monobenzocyclooctinyl (MOBO), and tetramethoxydibenzocyclooctinyl (TMDIBO).

[0193] In another general aspect, the present invention relates to a pharmaceutical composition comprising the radioimmunoconjugate of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition may comprise one or more pharmaceutically acceptable excipients.

[0194] In another general aspect, the present invention relates to a method of using the radioimmunoconjugate and pharmaceutical composition of the present invention for targeted radiotherapy.

[0195] In one embodiment, a method for selectively targeting neoplastic cells for radiotherapy is provided, comprising the step of administering a pharmaceutical composition of the present invention to a subject who requires selective targeting of neoplastic cells for radiotherapy.

[0196] In one embodiment, a method for treating a neoplastic disease or disorder in a subject requiring treatment of the neoplastic disease or disorder is provided, comprising the step of administering a pharmaceutical composition of the present invention to the subject. Brief explanation of the drawing

[0197] In addition to the foregoing summary, the following detailed description of the invention will be better understood when read together with the accompanying drawings. It should be understood that the invention is not limited to the exact embodiments shown in the drawings. In the drawing: FIGS. 1a and FIGS. 1b is La described in Example 1 3+ Showing the HPLC chromatogram from the chelation test with; Fig. 1a is before mixing (top) and La 3+ Shows the HPLC chromatogram of H2bp18c6-benzyl-phenyl after mixing with; La 3+ The shift in residence time from 14.137 minutes to 12.047 minutes after mixing with La due to H2bp18c6-benzyl-phenyl 3+ Indicates rapid chelation of; Fig. 1b is before mixing (top) and La 3+ Shows the HPLC chromatogram of H2bp18c6-benzyl-isopentyl after mixing with; La 3+ The shift in residence time from 17.181 minutes to 15.751 minutes after mixing with La caused by H2bp18c6-benzyl-isopentyl 3+ Indicates rapid chelation of; Fig. 2 ...represents a schematic representation of the radiolabeling of an antibody for generating a radioimmunoconjugate according to an embodiment of the present invention by a random conjugation method (e.g., a labeling method for lysine residues, cysteine ​​residues, etc.) or a site-specific conjugation method (e.g., a glycan-specific method, a conjugation tag method, or a engineered cysteine ​​method); Fig. 2a ... schematically illustrates random splicing through one-step direct radiolabeling; Fig. 2b... schematically illustrates random splicing through click radiolabeling; Fig. 2c ... schematically illustrates site-specific splicing through one-step direct radiolabeling; Fig. 2d This schematically illustrates site-specific pairing through click radiolabeling. Specific details for implementing the invention

[0198] Various publications, papers, and patents are cited or described in the background art and throughout this specification; each of these references is incorporated herein by reference in its entirety. Discussions of literature, actions, materials, devices, articles, etc., included in this specification are intended to provide context for the invention. Such discussions are not to acknowledge that any or all of these objects constitute part of the prior art with respect to any of the disclosed or claimed inventions.

[0199] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art. Otherwise, certain terms cited herein have the meaning as set forth herein. All patents, published patent applications, and publications cited herein are incorporated by reference as fully described herein.

[0200] It should be noted that, as used in this specification and the appended claims, the singular form (indefinite and definite articles) includes plural objects unless the context clearly indicates otherwise.

[0201] Throughout this specification and the claims set forth below, unless otherwise required by the context, the word “comprising” and its variations, such as “comprising” and “comprising,” will be understood to imply that it includes the mentioned integer or step or group of integers or steps, but does not exclude any other integer or step or group of integers or steps. As used herein, the term “comprising” may be substituted with the terms “containing” or “comprising,” or, in some cases, with the term “having” as used herein.

[0202] As used herein, “consisting of” excludes any element, step, or component not specified in the claim elements. As used herein, “essentially consisting of” does not exclude materials or steps that do not substantially affect the basic and novel features of the claim. In all instances where used herein in relation to a shape or embodiment of the invention, any of the foregoing terms “comprising,” “containing,” “having,” and “having” may be replaced with the terms “consisting of” or “essentially consisting of” to vary the scope of the invention.

[0203] As used herein, the connecting term “and / or” between a number of mentioned elements is understood to include both individual options and combined options. For example, where two elements are combined by “and / or,” the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first element and the second element together. Any one of these options is understood to fall within this meaning and thus satisfy the requirements of the term “and / or” as used herein. The simultaneous applicability of more than one of these options is also understood to fall within this meaning and thus satisfy the requirements of the term “and / or”.

[0204] In an attempt to assist the reader of this application, the detailed description of the invention has been separated into various paragraphs or sections or relates to various embodiments of this application. These separations should not be construed as separating the essence of a paragraph or section or embodiment from the essence of another paragraph or section or embodiment. On the contrary, those skilled in the art will understand that the detailed description of the invention has a wide range of applications and includes all combinations of various sections, paragraphs, and sentences that may be considered. Discussion of any embodiments is intended merely to be illustrative and is not intended to imply that the scope of the disclosure, including the claims, is limited to these embodiments.

[0205] Unless otherwise noted, any numerical value, such as a concentration or concentration range described herein, should be understood in all cases to be modified by the term “about.” Accordingly, numerical values ​​typically include ±10% of the stated value. For example, a reference to “10-fold” includes 9-fold and 11-fold. As used herein, unless the context clearly indicates otherwise, the use of a numerical range explicitly includes all possible sub-ranges, all individual numerical values ​​within such range, e.g., integers and fractions of values ​​within such range.

[0206] As used herein, “subject” means any animal to be administered or has been administered the radioimmunoconjugate of the present invention, preferably a mammal, most preferably a human. As used herein, the term “mammal” includes all mammals. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs), such as monkeys or apes, humans, etc., more preferably humans.

[0207] As used herein, the term “alkyl” means a saturated monovalent unbranched or branched hydrocarbon chain. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl).

[0208] The term "cycloalkyl" refers to a mono- or polycyclic alkyl group having 3 to 12, more preferably 3 to 8, carbon atoms within the ring(s). Examples of monocyclic cycloalkyl rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.

[0209] As used herein, the term “alkoxy” refers to an -O-alkyl group or an -OR group where R is alkyl, wherein the alkyl is defined as above. The alkoxy group is attached to the parent molecule via an oxygen atom. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy, isopropoxy), butoxy (e.g., n-butoxy, isobutoxy, tert-butoxy), and pentyloxy (e.g., n-pentyloxy, isopentyloxy, neopentyloxy). The alkoxy group may be unsubstituted or substituted with one or more suitable substituents. Similarly, “alkylthio” or “thioalkoxy” refers to an -SR group, where R is an alkyl attached to the parent molecule via a sulfur crosslink, e.g., -S-methyl, -S-ethyl, etc. Representative examples of alkylthio include -SCH3, -SCH2CH3, etc. Includes, but is not limited to.

[0210] As used herein, the term “halogen” means fluorine, chlorine, bromine, or iodine. Correspondingly, the term “halo” means fluoro, chloro, bromo, or iodine.

[0211] The terms "hydroxy" and "hydroxyl" may be used interchangeably and refer to -OH.

[0212] The term "carboxy" refers to -COOH.

[0213] The term "cyano" refers to -CN.

[0214] The term "nitro" refers to -NO2.

[0215] The term "isothiocyanate" refers to -N=C=S.

[0216] The term "isocyanate" refers to -N=C=O.

[0217] The term "azido" refers to -N3.

[0218] The term “alkenyl” refers to a linear or branched hydrocarbon chain having two or more carbon atoms, e.g., 2 to 10 carbon atoms, and containing one or more double bonds between the two carbon atoms. An alkenyl may have one carbon-carbon double bond or multiple carbon-carbon double bonds, e.g., two, three, four or more carbon-carbon double bonds. Examples of alkenyl groups include, but are not limited to, methanel, ethenyl, propenyl, butenyl, etc.

[0219] The term "cycloalkenyl" refers to a mono- or polycyclic alkyl group having 3 to 12, more preferably 3 to 8, carbon atoms within the ring(s) and comprising one or more double bonds between two carbon atoms. The cycloalkenyl may have one carbon-carbon double bond or multiple carbon-carbon double bonds, e.g., two, three, four or more carbon-carbon double bonds. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cycloheptenyl, cyclohexenyl, etc.

[0220] As used herein, the terms “alkynyl,” “alkyne group,” or “alkyne moiety” refer to a linear or branched hydrocarbon chain having two or more carbon atoms, e.g., 2 to 10 carbon atoms, and containing one or more triple bonds between two carbon atoms. The alkynyl group may be a terminal alkynyl group or a cyclic alkynyl group. The terminal alkyne has one or more hydrogen atoms bonded to the triple-bonded carbon atom. A “cyclic alkyne” or “cycloalkynyl” is a cycloalkyl ring containing one or more triple bonds between two carbon atoms. Examples of cyclic alkynes or cycloalkynyl groups include, but are not limited to, cyclooctine and cyclooctine derivatives, such as bicyclononine (BCN), difluorinated cyclooctine (DIFO), dibenzocyclooctine (DIBO), keto-DIBO, biaryl-azacyclooctine (BARAC), dibenzo-azacyclooctine (DIBAC), dimethoxyazacyclooctine (DIMAC), difluorobenzocyclooctine (DIFBO), monobenzocyclooctine (MOBO), and tetramethoxy DIBO (TMDIBO).

[0221] The term "amino" refers to -NH2. The term "alkylamino" refers to an amino group in which one or both of the hydrogen atoms attached to the nitrogen are substituted with an alkyl group. An alkylamine group is -NR2. It may be represented as, wherein each R is independently a hydrogen or alkyl group. For example, alkylamines include methylamine (-NHCH3), dimethylamine (-N(CH3)2), -NHCH-2CH3, etc. As used herein, the term "aminoalkyl" is intended to include both branched-chain and straight-chain saturated aliphatic hydrocarbon groups substituted with one or more amino groups. Representative examples of aminoalkyl groups include, but are not limited to, -CH2NH2, -CH2CH2NH2, and -CH2CH(NH2)CH3.

[0222] As used herein, "amide" refers to -C(O)N(R)2, where each R is independently an alkyl group or hydrogen. Examples of amides include, but are not limited to, -C(O)NH2, -C(O)NHCH3, and -C(O)N(CH3)2.

[0223] The terms "hydroxyalkyl" and "hydroxyalkyl" are used interchangeably and refer to an alkyl group substituted with one or more hydroxyl groups. The alkyl may be a branched-chain or straight-chain aliphatic hydrocarbon. Examples of hydroxyalkyl include hydroxylmethyl (-CH2OH), Hydroxylethyl (-CH2CH2OH), etc. are included but not limited thereto.

[0224] As used herein, the term "aryl" is a group containing any carbon-based aromatic group, including but not limited to phenyl, naphthyl, anthracenyl, phenanthranil, etc. Aryl moiety is well known, for example, in the literature [Lewis, RJ, ed., Hawley's Condensed Chemical Dictionary , 13 th It is described in Edition, John Wiley & Sons, Inc., New York (1997). The aryl group may include a single ring structure (i.e., monocyclic) or a multi-ring structure that is a fused ring structure (i.e., polycyclic). Preferably, the aryl group is a monocyclic aryl group.

[0225] As used herein, the term “heterocyclile” comprises stable monocyclic and polycyclic hydrocarbons containing one or more heteroatom ring members such as sulfur, oxygen, or nitrogen. As used herein, the term “heteroaryl” comprises stable monocyclic and polycyclic aromatic hydrocarbons containing one or more heteroatom ring members such as sulfur, oxygen, or nitrogen. Heteroaryls may be monocyclic or polycyclic, for example, bicyclic or tricyclic. Each ring of a heterocyclile or heteroaryl group containing heteroatoms may contain one or two oxygen or sulfur atoms and / or one to four nitrogen atoms, provided that the total number of heteroatoms within each ring is four or fewer and each ring has one or more carbon atoms. A polycyclic, e.g. bicyclic or tricyclic heteroaryl group must contain one or more fully aromatic rings, but other fused rings or rings may be aromatic or non-aromatic. A heterocyclile or heteroaryl group may be attached to any available nitrogen or carbon atom of any ring of the heterocyclile or heteroaryl group. Preferably, the term “heteroaryl” refers to a 5- or 6-membered monocyclic group and a 9- or 10-membered bicyclic group having one or more heteroatoms (O, S, or N) in one or more of the rings, wherein the heteroatom-containing ring has 1, 2, or 3 heteroatoms selected from O, S, and / or N, preferably 1 or 2 heteroatoms, more preferably 1 or 2 heteroatoms. The nitrogen heteroatom(s) of the heteroaryl may be substituted or unsubstituted. Additionally, the nitrogen and sulfur heteroatom(s) of the heteroaryl can be oxidized at will (i.e., N→O and S(O) r (Here, r is 0, 1, or 2)).

[0226] The term "ester" refers to -C(O)2R, where R is an alkyl.

[0227] The term "carbamate" refers to -OC(O)NR2, where each R is independently an alkyl or hydrogen.

[0228] The term "aldehyde" refers to the group -C(O)H.

[0229] The term "carbonate" refers to -OC(O)OR, where R is an alkyl.

[0230] The term "maleimide" refers to a group having the chemical formula H2C2(CO)2NH. The term "maleimido" refers to a maleimide group covalently linked to another group or molecule. Preferably, the maleimido group is N-linked, for example, as follows:

[0231]

[0232] The term "acyl halide" refers to -C(O)X, where X is a halo (e.g., Br, Cl). Exemplary acyl halides include acyl chloride (-C(O)Cl) and acyl bromide (-C(O)Br).

[0233] As stated herein, the term “substituted” means that one or more hydrogen atoms are replaced by non-hydrogen groups, provided that all normal valencies are maintained and the substitution produces a stable compound. Where a specific group is “substituted,” that group may have one or more substituents independently selected from the list of substituents, preferably 1 to 5 substituents, more preferably 1 to 3 substituents, and most preferably 1 to 2 substituents. When used in relation to substituents, the term “independently” means that, where more than one of such substituents is possible, such substituents may be identical or different from one another. Any of the substituents described herein (e.g., alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heterocyclil, heteroaryl, etc.) may be unsubstituted or substituted with one or more suitable substituents. Examples of suitable substituents include, but are not limited to, alkyl, halogen, hydroxy, alkoxy, amide, alkylthio, amino, alkylamino, aminoalkyl, hydroxyalkyl, hydroxyl, carboxyl, etc.

[0234] According to industry practice:

[0235]

[0236] It is used in the structural formulas of this specification to illustrate a bond that is a core structure, parent structure, or skeletal structure, such as a moiety, functional group, or substituent attachment site to a chelator or targeting ligand.

[0237] In any configuration or formula of a compound where any variable appears more than once, its limitation in each case is independent of its limitation in all other cases. Thus, for example, if a group is shown to be substituted with 0 to 3 R groups, said group may be arbitrarily substituted with up to 3 R groups, and in each case R is selected independently of the limitation of R.

[0238] If the bond to a substituent appears to cross a bond connecting two atoms within the ring, such a substituent can be bonded to any atom on the ring.

[0239] As used herein, the terms “radioactive metal ion” or “radioactive metal ion” refer to one or more isotopes of an element that emit particles and / or photons. Any radioactive metal ion known to those skilled in the art in the context of this disclosure may be used in the present invention. Examples of radioactive metal ions suitable for use in the present invention include 32 P, 47 Sc, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 77 As, 86 Y, 89 Zr, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 111 In, 117 Sn, 131 I, 149 Tb, 152 Tb, 155 Tb, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 177 Lu, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227 Th, and 255Fm is included but not limited thereto. Preferably, the radioactive metal ion is a "therapeutic emitter," meaning a radioactive metal ion useful for therapeutic applications. Examples of therapeutic emitters include beta or alpha emitters, such as 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I, 149 Tb, 152 Tb, 155 Tb, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 177 Lu, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 255 Fm, and 227 Th is included but not limited thereto. Preferably, The radioactive metal ions used in the present invention are alpha-emitting radioactive metal ions, such as actinium-225 ( 225 It is Ac).

[0240] As used herein, the terms “chelator” or “kilant” refer to a chemical compound in which a metal, preferably a radioactive metal, can be chelated through coordination bonding. In a typical embodiment, the chelator is a macrocyclic ring containing one or more heteroatoms, e.g., oxygen and / or nitrogen, as ring atoms. Preferably, the chelator is a derivative of 4,13-diaza-18-crown-6.

[0241] As used herein, "radiometallic complex" refers to a complex comprising a radioactive metal ion associated with a chelator. Typically, the radioactive metal ion is bound to or coordinated to the chelator through a coordinate bond. The heteroatom of the macrocyclic ring may participate in the coordinate bonding of the radioactive metal ion to the chelator. The chelator may be substituted with one or more substituents, and one or more substituents may also participate in the coordinate bonding of the radioactive metal ion to the chelator, either by adding to the heteroatom of the macrocyclic ring or alternatively.

[0242] As used herein, the term “click chemistry” refers to a chemical philosophy introduced by Sharpless that describes chemistry tuned to rapidly and reliably generate covalent bonds by joining together small units containing reactive groups (Reference [Kolb, et al., Angewandte Chemie International Edition[See (2001) 40: 2004-2021]). Click chemistry refers to a concept that includes, but is not limited to, reactions that mimic reactions found in nature, rather than specific reactions. In some embodiments, click chemical reactions may be modular, / or broad in scope, provide high chemical yields, / or produce inert by-products, / or stereospecific, / or exhibit a large thermodynamic driving force that promotes reactions with a single reaction product, or be carried out under physiological conditions. In some embodiments, click chemical reactions may / are carried out under simple reaction conditions, use readily available materials and / or reagents, use non-toxic solvents or harmless or easily removable solvents such as water, / or provide simple product isolation by non-chromatographic methods, such as crystallization or distillation.

[0243] Click chemistry reactions utilize reactive groups that are rarely found in naturally occurring biomolecules and are chemically inert to the biomolecule; however, when click chemistry partners are reacted together, the reaction can occur efficiently under biologically relevant conditions, such as cell culture conditions, for example, in the absence of excess heat and / or harsh reagents. Generally, a click chemistry reaction requires two or more molecules comprising click reaction partners capable of reacting with each other. Such mutually reactive click reaction partners are sometimes referred to herein as click chemistry handle pairs or click chemistry pairs. In some embodiments, the click reaction partners are an azide and a strained alkyne, for example, a cycloalkyne, such as cyclooctine or a cyclooctine derivative, or any other alkyne. In other embodiments, the click reaction partners are a reactive diene and a suitable tetrazine dieneophile. For example, trans-cyclooctene, norbornene, or biscyclononene can be paired with a tetrazine dieneine suitable as a click reaction pair. In another embodiment, tetrazole can act as a potential source of nitrile imine capable of pairing with an alkene inactivated in the presence of ultraviolet light to produce a click reaction pair referred to as a "photo-click" reaction pair. In another embodiment, the click reaction partners are cysteine ​​and maleimide. For example, cysteine ​​from a peptide (e.g., GGGC) can react with maleimide associated with a chelating agent (e.g., NOTA). Other suitable click chemical handles are known to those skilled in the art (e.g., in the literature [Spicer et al., Selective chemical protein modification. Nature Communications[. 2014; 5: p. 4740]). In another embodiment, the click reaction partner is a Staudinger ligation component, e.g., phosphine and azide. In another embodiment, the click reaction partner is a Diels-Alder reaction component, e.g., a diene (e.g., tetrazine) and an alkene (e.g., trans-cyclooctene (TCO) or norbornene). Exemplary click reaction partners are described in US20130266512 and WO2015073746, and related descriptions of the click reaction partners in both are incorporated herein by reference.

[0244] According to a preferred embodiment, the click chemical reaction utilizes an azide group and an alkyne group, more preferably a shunt alkyne group, e.g., a cycloalkyne, e.g., cyclooctine or a cyclooctine derivative, as the click chemical pair or reaction partner. In such an embodiment, the click chemical reaction is a Huisgen cycloaddition or 1,3-dipolar cycloaddition between an azide (-N3) and an alkyne moiety to form a 1,2,3-triazole linker. The click chemical reaction between an alkyne and an azide typically requires the addition of a copper catalyst to promote the 1,3-cycloaddition reaction and is known as a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. However, the click chemical reaction between cyclooctine or cyclooctine derivatives and azide typically does not require the addition of a copper catalyst, but instead proceeds via strain-promoted azide-alkyne cycloaddition (SPAAC) (Debets, MF, et al., Bioconjugation with strained alkenes and alkynes. Acc Chem Res , 2011. 44(9): p. 805-15]).

[0245] As used herein, the term “targeting ligand” refers to any molecule that provides enhanced affinity for a selected target, e.g., an antigen, a cell, a cell type, a tissue, an organ, a region of the body, or a compartment (e.g., a cell, tissue, or organ compartment). Targeting ligands include, but are not limited to, antibodies or their antigen-binding fragments, small molecules, aptamers, polypeptides, and scaffold proteins. Preferably, the targeting ligand is a polypeptide, more preferably an antibody or its antigen-binding fragment, an engineered domain, or a scaffold protein.

[0246] As used herein, the term “polypeptide” refers to a polymer composed of amino acid residues linked by peptide bonds, associated naturally occurring structural variants, and synthetic non-natural analogues thereof. The term refers to a polypeptide of any size, structure, or function. Typically, a polypeptide is three or more amino acids long. A polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. Synthetic polypeptides may 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, such as its antigen-binding fragment. According to a preferred embodiment, the antibody or its fragment is specific to a cancer antigen. According to another embodiment, the polypeptide is an engineered domain or scaffold protein.

[0247] As used herein, the terms “antibody” or “immunoglobulin” are used in a broad sense and include polyclonal antibodies, monoclonal antibodies, e.g., rat, human, human-adapted, humanized, and chimeric monoclonal antibodies, and immunoglobulin or antibody molecules comprising antigen-binding fragments thereof.

[0248] Generally, an antibody is a protein or peptide chain that exhibits binding specificity to a specific antigen referred to herein as a "target." The structure of antibodies is well known. Immunoglobulins can be classified into five major classes, namely IgA, IgD, IgE, IgG, and IgM, based on the amino acid sequence of their heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibodies used in the present invention may be any of the five major classes or corresponding subclasses. The light chains of antibodies of any vertebrate species may be classified into one of two distinct types, kappa and lambda, based on the amino acid sequence of their constant domains. According to certain embodiments, the antibodies used in the present invention comprise heavy chain and / or light chain constant regions from mouse antibodies or human antibodies. Each of the four IgG subclasses has a different biological function known as an effector function. These effector functions are generally mediated through interaction with Fc receptors (FcγR) or by C1q binding and complement binding. Binding to FcγR can lead to antibody-dependent cell-mediated lysis, while binding to complement factors can lead to complement-mediated lysis. Antibodies useful in the present invention may not have effector functions or may have minimal functions, but may possess the ability to bind to FcRn.

[0249] As used herein, the term “antigen-binding fragment” refers to an antibody fragment, such as a diabody, Fab, Fab’, F(ab’)2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv’), disulfide-stabilized diabody (ds diabody), single-stranded antibody molecule (scFv), single-domain antibody (sdab), scFv dimer (divalent diabody), multispecific antibody formed from a portion of an antibody containing one or more CDRs, camelized single-domain antibody, nanobody, domain antibody, divalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. An antigen-binding fragment may bind to the same antigen to which the parent antibody or parent antibody fragment binds. As used herein, the term “single-chain antibody” refers to a single-chain antibody conventional in the art, comprising a heavy-chain variable region and a light-chain variable region connected by a short peptide of about 15 to about 20 amino acids. As used herein, the term “single-domain antibody” refers to a single-domain antibody conventional in the art, comprising a heavy-chain variable region and a heavy-chain constant region, or comprising only a heavy-chain variable region.

[0250] As used herein, the term “scaffold” or “scaffold protein” refers to any protein having a target binding domain and capable of binding to a target. The scaffold comprises a primarily structural “framework” and a “binding domain” that contacts the target and provides specific binding. The binding domain of the scaffold does not need to be defined by a single adjacent sequence of the scaffold. In certain cases, the scaffold may be part of a larger binding protein, which may itself be part of a multimeric binding protein containing multiple scaffolds. The certain binding protein may be bispecific or multispecific in that it can bind to two or more different epitopes. The scaffold may be derived from a single-strand antibody, or the scaffold may not be antibody-derived.

[0251] As used herein, the term “aptamer” refers to a single-stranded oligonucleotide (single-stranded DNA or RNA molecule) capable of specifically binding to its target with high affinity. Aptamers can be used as molecules to target various organic and inorganic materials.

[0252] As used herein, the term “small molecule ligand” refers to a low molecular weight organic compound. As used herein, a small molecule ligand may refer to a compound with a size of less than about 1,000 daltons and may be synthesized in a laboratory or found in nature.

[0253] Chelate

[0254] In a general aspect, the present invention relates to a chelator, preferably a chelator capable of chelating a radioactive metal through a coordination bond. According to an embodiment of the present invention, the chelator has the structure of formula (I):

[0255] [Chemical Formula (I)]

[0256]

[0257] In the above formula,

[0258] Rings A and B are each independently a 6 to 10-membered aryl or a 5 to 10-membered heteroaryl, wherein rings A and B are each a halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclil, 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 arbitrarily substituted with one or more substituents independently selected from the group consisting of X;

[0259] Z1 and Z2 each independently -(C(R 12 )2) m - or -(CH2) n -C(R 12 )(X)-(CH2) n -is;

[0260] Each X is independently -L1-R 11 And;

[0261] Each n is independently 0, 1, 2, 3, 4, or 5;

[0262] Each m is independently 1, 2, 3, 4, or 5;

[0263] Each p is independently 0 or 1;

[0264] L1 is absent or a linker;

[0265] R 11 is a nucleophilic moiety or an electrophilic moiety, or R 11 It includes a targeting ligand;

[0266] Each R 12 is independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclil, or heteroaryl;

[0267] Each R 13 is independently hydrogen or alkyl;

[0268] R 14 , R 15 , R 16 , and R 17 Each is independently hydrogen, alkyl, or X, or

[0269] Alternatively, R 14 and R 15 and / or R 16 and R 17 They form a 5- or 6-membered cycloalkyl ring arbitrarily substituted with X together with the carbon atom to which they are attached;

[0270] However, the chelator includes one or more Xs, and if X exists on ring A or ring B, L1 is a linker or R 12 and R 14 to R 17 One or more of them are not hydrogen.

[0271] According to an embodiment of the present invention, the chelator comprises one or more X groups, wherein X is L1-R 11 and, where L1 is absent or a linker, and R 11 is an electrophilic moiety or a nucleophilic moiety, or R 11 It includes a targeting ligand. R 11 In the case of this nucleophilic or electrophilic moiety, such a moiety can be used to attach a chelator to a targeting ligand directly or indirectly through a linker.

[0272] In a given embodiment, the chelator comprises a single X group, preferably X L1 of the device is a linker.

[0273] The chelator of the present invention may be substituted with X at any one of the carbon atoms of a macrocyclic ring, at the Z1 or Z2 position, or on ring A or ring B, provided that, if ring or ring B contains an X group, L1 is a linker or R 12 and R 14 to R 17 One or more of them are not hydrogen (i.e., carbon atoms of Z1, Z2, and / or one or more carbons of the macrocyclic ring are substituted, for example, with alkyl groups, e.g., methyl or ethyl). Preferably, substitution at such positions is a radioactive metal ion, particularly 225 It does not affect the chelation efficiency of the chelator for Ac, and in some embodiments, substitution can improve the chelation efficiency.

[0274] In some embodiments, L1 is absent. If L1 is absent, R 11 It is directly coupled to the chelator (e.g., via a shared connection).

[0275] In some embodiments, L1 is a linker. As used herein, the term “linker” refers to a nucleophilic moiety, an electrophilic moiety, or a chemical moiety that binds a chelator to a targeting ligand. Any suitable linker known to those skilled in the art in the context of this disclosure may be used in the present invention. The linker may contain, for example, a substituted or unsubstituted alkyl, a substituted or unsubstituted heteroalkyl moiety, a substituted or unsubstituted aryl or heteroaryl, a polyethylene glycol (PEG) linker, a peptide linker, a sugar-based linker, or a cleavable linker, such as a disulfide linkage or a protease cleavage site, such as valine-citrulline-p-aminobenzyl (PAB). Exemplary linker structures suitable for use in the present invention include, but are not limited to, the following:

[0276] , , ,

[0277] , ,

[0278] , , and ,

[0279] In the above formula,

[0280] n is an integer from 0 to 10, preferably an integer from 1 to 4; and m is an integer from 0 to 12, preferably an integer from 0 to 6.

[0281] In some embodiments, R 11 is a nucleophilic moiety or an electrophilic moiety. "Nucleophilic moiety" or "nucleophilic group" refers to a functional group that donates an electron pair to form a covalent bond in a chemical reaction. "Electrophilic moiety" or "electrophilic group" refers to a functional group that accepts an electron pair to form a covalent bond in a chemical reaction. In a chemical reaction, a nucleophilic group reacts with an electrophilic group to form a new covalent bond, and vice versa. The reaction of the nucleophilic group or electrophilic group of the chelator of the present invention with a targeting ligand or another chemical moiety (e.g., a linker) containing a corresponding reaction partner enables the covalent linkage of the targeting ligand or chemical moiety to the chelator of the present invention.

[0282] Exemplary examples of nucleophilic groups include, but are not limited to, azides, amines, and thiols. Exemplary examples of electrophilic groups include, but are not limited to, amine-reactive groups, thiol-reactive groups, alkynoyls, and cycloalkynoyls. The amine-reactive group preferably reacts with a primary amine, which includes a primary amine present at the N-terminus of each polypeptide chain and on the side chain of a lysine residue. Examples of amine-reactive groups suitable for use in the present invention include, but are not limited to, N-hydroxysuccinimide (NHS), substituted NHS (e.g., sulfo-NHS), isothiocyanate (-NCS), isocyanate (-NCO), esters, carboxylic acids, acyl halides, amides, alkylamides, and tetra- and per-fluorophenyl esters. The thiol-reactive group reacts with a thiol or a sulfhydryl, preferably a thiol present in the side chain of a cysteine ​​residue of the polypeptide. Examples of thiol-reactive groups suitable for use in the present invention include, but are not limited to, Michael acceptors (e.g., maleimide), haloacetyls, acyl halides, activated disulfides, and phenyloxadiazole sulfones.

[0283] In a specific embodiment, R 11 -NH2, -NCS(isothiocyanate), -NCO(isocyanate), -N3(azido), alkynyl, cycloalkynyl, carboxylic acid, ester, amido, alkylamide, maleimido, acyl halide, tetrazine, or trans-cyclooctene, more particularly -NCS, -NCO, -N3, alkynyl, cycloalkynyl, -C(O)R 13 , -COOR 13 , -CON(R 13 )2, maleimido, acyl halide (e.g., -C(O)Cl, -C(O)Br), tetrazine, or trans-cyclooctene, where each R 13 is independently hydrogen or alkyl.

[0284] In some embodiments, R11 The group is an alkynyl, cycloalkynyl, or azido group, and thus enables the attachment of the chelator to a targeting ligand or other chemical moiety (e.g., a linker) using a click chemical reaction. In such embodiments, the click chemical reaction that may be performed is a huigen cyclization addition or a 1,3-bipolar cyclization addition between the azido (-N3) and the alkynyl or cycloalkynyl group to form a 1,2,4-triazole linker or moiety. In one embodiment, the chelator comprises an alkynyl or cycloalkynyl group, and the targeting ligand or other chemical moiety comprises an azido group. In another embodiment, the chelator comprises an azido group, and the targeting ligand or other chemical moiety comprises an alkynyl or cycloalkynyl group.

[0285] In a given embodiment, R 11 The alkynyl group is a terminal alkynyl group or a cycloalkynyl group that is reactive with an azide group, more preferably, particularly through strain-promoted azide-alkyne cyclization addition (SPAAC). Examples of cycloalkynyl groups capable of reacting with an azide group through SPAAC include, but are not limited to, cyclooctinyl or bicyclononinyl (BCN), difluorinated cyclooctinyl (DIFO), dibenzocyclooctinyl (DIBO), keto-DIBO, biaryl-azaccyclooctinyl (BARAC), dibenzo-azaccyclooctinyl (DIBAC, DBCO, ADIBO), dimethoxyazaccyclooctinyl (DIMAC), difluorobenzocyclooctinyl (DIFBO), monobenzocyclooctinyl (MOBO), and tetramethoxydibenzocyclooctinyl (TMDIBO).

[0286] In a specific embodiment, R 11 It is dibenzoazacyclooctinyl (DIBAC, DBCO, ADIBO), which has the following structure:

[0287] . R 11In such an embodiment of this DBCO, the DBCO may be covalently connected to the chelator directly or indirectly through a linker, and preferably attached to the chelator indirectly through a linker.

[0288] In some embodiments, R 11 The targeting ligand comprises a targeting ligand. The targeting ligand may be connected to the chelator directly via a covalent linkage or indirectly via a linker. The targeting ligand may be a polypeptide, e.g., an antibody or its antigen-binding fragment, a small molecule, an aptamer, or a scaffold protein. In a preferred embodiment, the targeting ligand is an antibody or its antigen-binding fragment, e.g., a monoclonal antibody (mAb) or its antigen-binding fragment, which specifically binds to an antigen associated with a neoplastic disease or disorder, such as a cancer antigen, which may be prostate-specific membrane antigen (PSMA), BCMA, Her2, EGFR, KLK2, CD19, CD22, CD30, CD33, CD79b, or Nectin-4.

[0289] According to an embodiment of the present invention, ring A and ring B are each independently a 6 to 10-membered aryl or a 5 to 10-membered heteroaryl. In an alternative embodiment, ring A and ring B are each optionally substituted heterocyclil rings, e.g., oxazolin. Ring A and ring B are each halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclil, 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 13It may be optionally and independently substituted with one or more substituents independently selected from the group consisting of )2, and X. Examples of 6 to 10-membered aryl groups suitable for this purpose include, but are not limited to, phenyl and naphthyl. Examples of 5 to 10-membered heteroaryl groups suitable for this purpose include, but are not limited to, pyridinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, and imidazolyl. Examples of suitable substituents for 5 to 10-membered heteroaryl and 6 to 10-membered aryl groups include -COOH, tetrazolyl, and -CH2COOH Includes, but is not limited to. In a preferred embodiment, the substituent is -COOH or tetrazolyl, which is a homologue of -COOH.

[0290] In a given embodiment, ring A and ring B each have -COOH and -CH2COOH It is independently and arbitrarily substituted with one or more carboxyl groups, including but not limited to.

[0291] In a given embodiment, ring A and ring B are each independently and optionally substituted with tetrazolyl.

[0292] In one embodiment, ring A and ring B are identical, for example, both ring A and ring B are pyridinyl. In another embodiment, ring A and ring B are different, for example, one of ring A and ring B is pyridinyl and the other is phenyl.

[0293] In a specific embodiment, both ring A and ring B are pyridinyls substituted with -COOH.

[0294] In a specific embodiment, both ring A and ring B are pyridinyl substituted with tetrazolyl.

[0295] In another specific embodiment, both ring A and ring B are picolinic acid groups having the following structure:

[0296] .

[0297] According to an embodiment of the present invention, Z1 and Z2 each independently -(C(R 12 )2) m - or -(CH2) n -C(R 12 )(X)-(CH2) n - and; each X is independently -L1-R 11 and; each R 12 is independently hydrogen, alkyl, cycloalkyl, aryl, heterocyclil, or heteroaryl; each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 1, 2, 3, 4, or 5.

[0298] In some embodiments, each R 12 is independently hydrogen or alkyl, more preferably hydrogen, -CH3, or -CH2CH3.

[0299] In some embodiments, each R 12 is hydrogen.

[0300] In some embodiments, both Z1 and Z2 are -(CH2) m - and, where each m is preferably 1. In such an embodiment, the carbon atom of the macrocyclic ring, ring A, or ring B is substituted with an X group.

[0301] In some embodiments, one of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and the other one is -(CH2) m -am.

[0302] In some embodiments, one of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and the other one is -(CH2) m-and; each n is 0 and; m is 1 and; X is -L1-R 11 And; L1 is the linker.

[0303] In some embodiments, both Z1 and Z2 are -(CH2) 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 R 14 , R 15 , R 16 , and R 17 The rest are hydrogen.

[0304] In some embodiments, R 14 and R 15 They form a 5- or 6-membered cycloalkyl ring (i.e., cyclopentyl or cyclohexyl) together with the carbon atom to which they are attached. Such a 5- or 6-membered cycloalkyl ring can be substituted with an X group.

[0305] In some embodiments, R 16 and R 17 They form a 5- or 6-membered cycloalkyl ring (i.e., cyclopentyl or cyclohexyl) together with the carbon atom to which they are attached. Such a 5- or 6-membered cycloalkyl ring can be substituted with an X group.

[0306] In a given embodiment, the chelator has the structure of chemical formula (II):

[0307] [Chemical Formula (II)]

[0308]

[0309] In the above formula,

[0310] A1 is N or CR1 or is absent;

[0311] A2 is N or CR2;

[0312] A3 is N or CR3;

[0313] A4 is N or CR4;

[0314] A5 is N or CR5;

[0315] A6 is N or CR6 or is absent;

[0316] A7 is N or CR7;

[0317] A8 is N or CR8;

[0318] A9 is N or CR9;

[0319] A 10 is N or CR 10 This is it;

[0320] However, three or fewer of A1, A2, A3, A4, and A5 are N, and A6, A7, A8, A9, and A 10 Three or fewer of them are N;

[0321] R1, R2, R3, R4, R5, R6, R7, R8, R9, and R 10 Each is hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclil, 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 Independently selected from the group consisting of )2, and -X, or

[0322] Alternatively, any two directly adjacent R1, R2, R3, R4, R5, R6, R7, R8, R9, and R 10 They form substituted or unsubstituted 5- or 6-membered carbocyclic or nitrogen-containing rings together with the atoms to which they are attached;

[0323] Z1, Z2, X, n, m, p, L1, and R 11 to R17 The formula (I) is as described above, but,

[0324] However, the chelator includes one or more X, and R1, R2, R3, R4, R5, R6, R7, R8, R9, and R 10 If either of them is X, L1 is a linker or R 12 and R 14 to R 17 One or more of them are not hydrogen.

[0325] In some embodiments, any two directly adjacent R1, R2, R3, R4, R5, R6, R7, R8, R9, and R 10 They form a substituted or unsubstituted 5- or 6-membered carbocyclic or nitrogen-containing ring together with the atoms to which they are attached. Examples of such carbocyclic rings that may be formed include, but are not limited to, naphthyl. Examples of such nitrogen-containing rings that may be formed include, but are not limited to, quinolinyl. The carbocyclic or nitrogen-containing ring may be unsubstituted or substituted with one or more suitable substituents, e.g., -COOH, -CH2COOH, tetrazolyl, etc.

[0326] In some embodiments, L1 is absent. If L1 is absent, R 11 It is directly coupled to the chelator (e.g., via a shared connection).

[0327] In some embodiments, L1 is a linker. Any suitable linker known to those skilled in the art in the context of the present disclosure, such as those described above, may be used in the present invention.

[0328] In some embodiments, one of A1, A2, A3, A4, and A5 is nitrogen, one of A1, A2, A3, A4, and A5 is a carbon substituted with -COOH, and the remainder is CH, i.e., forms a pyridinyl ring substituted with a carboxylic acid.

[0329] In some embodiments, A6, A7, A8, A9, and A 10 One of them is nitrogen, and A6, A7, A8, A9, and A 10 One is a carbon substituted with an -COOH group, and the other is a CH group, that is, forming a pyridinyl ring substituted with a carboxylic acid.

[0330] In one embodiment, one or more of R1, R2, R3, R4, and R5 are -COOH. In one embodiment, R6, R7, R8, R9, and R 10 One or more of them are -COOH. In another embodiment, one or more of R1, R2, R3, R4, and R5 are -COOH; R 6, R7, R8, R9, and R 10 One or more of them are -COOH.

[0331] In some embodiments, A1 and A 10 Each is nitrogen; A2 is CR2 and R2 is -COOH;

[0332] A9 is CR9 and R9 is -COOH; A3 to A8 are each CR2, CR3, CR4, CR5, CR6, CR7, and CR8, respectively; and R3 to R8 are each hydrogen.

[0333] 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 tetrazolyl, and the rest are CH.

[0334] In some embodiments, A6, A7, A8, A9, and A 10 One of them is nitrogen, and A6, A7, A8, A9, and A 10 One of them is a carbon substituted with tetrazolyl, and the rest are CH.

[0335] In one embodiment, one or more of R1, R2, R3, R4, and R5 are tetrazolyl. In one embodiment, R 6, R7, R8, R9, and R 10One or more of them are tetrazolyl. In another embodiment, one or more of R1, R2, R3, R4, and R5 are tetrazolyl; R 6, R7, R8, R9, and R 10 One or more of them are tetrazolyl.

[0336] In some embodiments, each R 12 is hydrogen.

[0337] In some embodiments, R 11 It is an alkynyl group or a cycloalkynyl group, preferably cyclooctinyl or a cyclooctinyl derivative, e.g., DBCO.

[0338] In a specific embodiment of the chelator of formula (II),

[0339] A1 and A 10 Each is nitrogen;

[0340] A2 is CR2 and R2 is -COOH;

[0341] A9 is CR9 and R9 is -COOH;

[0342] A3 through A8 are each CR2, CR3, CR4, CR5, CR6, CR7, and CR8;

[0343] R3 through R8 are each hydrogen;

[0344] One of Z1 and Z2 is -(CH2) m - and the other one of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n -is;

[0345] R 12 is hydrogen;

[0346] m is 1 and;

[0347] Each n is 0 and;

[0348] X is -L1-R 11 and, where L1 is the linker and -R 11is an electrophilic group, for example, cyclooctinyl or a cyclooctinyl derivative, e.g., DBCO;

[0349] R 14 to R 17 Each is hydrogen, or alternatively R 16 and R 17 They form a 5- or 6-membered cycloalkyl ring with the carbon atom to which they are attached.

[0350] In a given embodiment, the chelator has the structure of chemical formula (III):

[0351] [Chemical Formula (III)]

[0352]

[0353] In the above formula,

[0354] Each A 11 is independently O, S, NMe, or NH;

[0355] Each R 18 -OR hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclil, heteroaryl, -OR 13 , -SR 13 , -COOR 13 , -OC(O)R 13 , -N(R 13 )2, -CON(R 13 )2, -NO2, -CN -OC(O)N(R 13 Independently selected from the group consisting of )2, and -X, and

[0356] Z1, Z2, X, n, m, L1, R 11 to R 17 The formula (I) is as described above, but,

[0357] However, the chelator includes one or more X, and R 18 If this is X, L1 is a linker or R 12 and R 14 to R 17 One or more of them are not hydrogen.

[0358] In some embodiments, each A 11 is identical, and each A 11 is O, S, NMe, or NH. For example, each A 11 can be S. In other embodiments, each A 11 The values ​​are different and each is independently selected from O, S, NMe, and NH.

[0359] In some embodiments, each R 18 -(CH2) independently p -COOR 13 or tetrazolyl, where R 13 is hydrogen, and each p is independently 0 or 1.

[0360] In some embodiments, each R 18 It is -COOH.

[0361] In some embodiments, each R 18 It is -CH2COOH.

[0362] In some embodiments, each R 18 It is tetrazolyl.

[0363] In a specific embodiment of the chelator of formula (III),

[0364] Each R 18 It is COOH;

[0365] One of Z1 and Z2 is -(CH2) m - and the other one of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n -is;

[0366] R 12 is hydrogen;

[0367] m is 1; and each n is 0;

[0368] X is -L1-R 11 and, where L1 is the linker and -R 11is an electrophilic group, for example, cyclooctinyl or a cyclooctinyl derivative, e.g., DBCO, or BCN;

[0369] R 14 to R 17 Each is hydrogen, or alternatively R 16 and R 17 They form a 5- or 6-membered cycloalkyl ring with the carbon atom to which they are attached.

[0370] In a specific embodiment of the present invention, the chelator is selected from the group consisting of the following:

[0371]

[0372]

[0373]

[0374]

[0375]

[0376] In the above formula,

[0377] L1 is absent or a linker;

[0378] R 11 is a nucleophilic moiety or an electrophilic moiety, or R 11 It includes a targeting ligand;

[0379] Each R 12 is independently hydrogen, -CH3, or -CH2CH3, provided that one or more R 12 is -CH3 or -CH2CH3.

[0380] In some embodiments, R 11 -NH2, -NCS, -NCO, -N3, alkynyl, cycloalkynyl, -C(O)R 13 , -COOR 13 , -CON(R 13)2, maleimido, acyl halide, tetrazine, or trans-cyclooctene.

[0381] In a given embodiment, R 11 is a cyclooctinyl or cyclooctinyl derivative selected from the group consisting of bicyclononinyl (BCN), difluorinated cyclooctinyl (DIFO), dibenzocyclooctinyl (DIBO), keto-DIBO, biaryl-azacyclooctinyl (BARAC), dibenzo-azacyclooctinyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctinyl (DIMAC), difluorobenzocyclooctinyl (DIFBO), monobenzocyclooctinyl (MOBO), and tetramethoxydibenzocyclooctinyl (TMDIBO).

[0382] Preferably, R 11 It is an alkynyl group or a cycloalkynyl group, more preferably a cycloalkynyl group, for example, DBCO or BCN.

[0383] An exemplary chelator of the present invention

[0384] , , , , , , , , , , , , , , , , and This is included but not limited to.

[0385] As described in more detail below, by reacting a chelator with an azide-labeled targeting ligand via a click chemical reaction to form a 1,2,3-triazole linker, such a chelator can covalently attach to a targeting ligand (e.g., an antibody or its antigen-binding fragment) to form an immunoconjugate or a radioimmunoconjugate.

[0386] The chelator of the present invention may be produced by any method known in the art in the context of the present disclosure. For example, the pendant aromatic / heteroaromatic group may be attached to the macrocyclic ring portion by methods known in the art, such as those exemplified and described below.

[0387] radioactive metal complex

[0388] In another general aspect, the present invention relates to a radioactive metal complex comprising a radioactive metal ion coordinated to a chelator of the present invention via a coordination bond. Any of the chelators of the present invention described herein may comprise a radioactive metal ion. Preferably, the radioactive metal ion is an alpha-emitting radioactive metal ion, more preferably 225 It is Ac. The chelator of the present invention chelates radioactive metal ions, particularly at any specific activity regardless of metal impurities. 225 By firmly chelating Ac, it is possible to form a radioactive metal complex that has high chelation stability in vivo and in vitro and is stable against a test agent, e.g., diethylenetriamine pentaacetic acid (DTPA).

[0389] According to an embodiment of the present invention, the radioactive metal complex has the structure of the chemical formula (Im):

[0390]

[0391] In the above formula,

[0392] The variable group is as defined above in the chelator of the present invention, for example, the chelator of formula (I); M is a radioactive metal ion. The radioactive metal ion M is bonded to the chelator through a coordinate bond to form a radioactive metal complex. Any functional group of the pendant arm (i.e., -Z1-ring A and / or -Z2-ring B), as well as the heteroatom of the macrocyclic ring of the chelator, may participate in the coordinate bonding of the radioactive metal ion.

[0393] A radioactive metal complex of formula (Im) can be formed using any of the chelators of formula (I) described above.

[0394] In a given embodiment, the radioactive metal ion M is an alpha-emitting radioactive metal ion. Preferably, the alpha-emitting radioactive metal ion is 225 It is Ac.

[0395] According to an embodiment of the present invention, the radioactive metal complex comprises one or more X groups, wherein X is -L1-R 11 and, where L1 is absent or a linker, and R 11 is an electrophilic moiety or a nucleophilic moiety, or R 11 It includes a targeting ligand. R 11 In the case of this nucleophilic or electrophilic moiety, such a moiety can be used to attach a radioactive metal complex to a targeting ligand directly or indirectly through a linker.

[0396] In a given embodiment, the radioactive metal comprises a single X group, preferably X L1 of the device is a linker.

[0397] In a specific embodiment, R 11-NH2, -NCS(isothiocyanate), -NCO(isocyanate), -N3(azido), alkynyl, cycloalkynyl, carboxylic acid, ester, amido, alkylamide, maleimido, acyl halide, tetrazine, or trans-cyclooctene, more particularly -NCS, -NCO, -N3, alkynyl, cycloalkynyl, -C(O)R 13 , -COOR 13 , -CON(R 13 )2, maleimido, or acyl halide (e.g., -C(O)Cl or -C(O)Br), where each R 13 is independently hydrogen or alkyl.

[0398] In some embodiments, R 11 It is an alkynyl, cycloalkynyl, or azido group, and thus enables the attachment of the chelator to a targeting ligand or other chemical moiety (e.g., linker) using a click chemical reaction.

[0399] In a given embodiment, R 11 The alkynyl group is a terminal alkynyl group or a cycloalkynyl group that is reactive with the azide group, more preferably, particularly through strain-promoted azide-alkyne cyclization addition (SPAAC). Examples of cycloalkynyl groups capable of reacting with the azide group through SPAAC include, but are not limited to, cyclooctinyl or cyclooctinyl derivatives, such as bicyclononinyl (BCN), difluorinated cyclooctinyl (DIFO), dibenzocyclooctinyl (DIBO), keto-DIBO, biaryl-azaccyclooctinyl (BARAC), dibenzo-azaccyclooctinyl (DIBAC, DBCO, ADIBO), dimethoxyazaccyclooctinyl (DIMAC), difluorobenzocyclooctinyl (DIFBO), monobenzocyclooctinyl (MOBO), and tetramethoxydibenzocyclooctinyl (TMDIBO).

[0400] In a specific embodiment, R 11It is dibenzoazacyclooctinyl (DIBAC, DBCO, ADIBO), which has the following structure:

[0401]

[0402] R 11 In such an embodiment of this DBCO, the DBCO can be covalently linked to the radioactive metal complex directly or indirectly through a linker, and preferably attached indirectly to the radioactive metal complex through a linker.

[0403] In other specific embodiments, R 11 It is bicyclononinil (BCN).

[0404] According to an embodiment of the present invention, ring A and ring B are each independently a 6 to 10-membered aryl or a 5 to 10-membered heteroaryl. In an alternative embodiment, ring A and ring B are each optionally substituted heterocyclil rings, e.g., oxazolin. Ring A and ring B are each halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclil, 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 13It may be optionally and independently substituted with one or more substituents independently selected from the group consisting of )2, and X. Examples of 6 to 10-membered aryl groups suitable for this purpose include, but are not limited to, phenyl and naphthyl. Examples of 5 to 10-membered heteroaryl groups suitable for this purpose include, but are not limited to, pyridinyl, isothiazolyl, isoxazolyl, and imidazolyl. Examples of suitable substituents for 5 to 10-membered heteroaryl and 6 to 10-membered aryl groups include -COOH, tetrazolyl, and -CH2COOH Includes, but is not limited to.

[0405] In a given embodiment, ring A and ring B each have -COOH and -CH2COOH It is independently and arbitrarily substituted with one or more carboxyl groups, including but not limited to.

[0406] In one embodiment, ring A and ring B are identical, for example, both ring A and ring B are pyridinyl. In another embodiment, ring A and ring B are different, for example, one of ring A and ring B is pyridinyl and the other is phenyl.

[0407] In a specific embodiment, both ring A and ring B are pyridinyls substituted with -COOH.

[0408] In a specific embodiment, both ring A and ring B are pyridinyl substituted with tetrazolyl.

[0409] In another specific embodiment, both ring A and ring B are picolinic acid groups having the following structure:

[0410] .

[0411] According to an embodiment of the present invention, Z1 and Z2 each independently -(C(R 12 )2) m - or -(CH2) n-C(R 12 )(X)-(CH2) n - and; each X is independently -L1-R 11 and; each n is independently 0, 1, 2, 3, 4, or 5; and each m is independently 1, 2, 3, 4, or 5.

[0412] In some embodiments, both Z1 and Z2 are -(CH2) m - and, where each m is preferably 1. In such an embodiment, the carbon atom of the macrocyclic ring, ring A, or ring B is substituted with an X group.

[0413] In some embodiments, one of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and the other one is -(CH2) m -am.

[0414] In some embodiments, one of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n - and the other one is -(CH2) m -and; each n is 0 and; m is 1 and; X is -L1-R 11 And; L1 is the linker.

[0415] In some embodiments, both Z1 and Z2 are -(CH2) 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 R 14 , R 15 , R 16 , and R 17 The rest are hydrogen.

[0416] In some embodiments, R 14 and R 15They form a 5- or 6-membered cycloalkyl ring (e.g., cyclopentyl or cyclohexyl) together with the carbon atom to which they are attached. Such a 5- or 6-membered cycloalkyl ring can be substituted with an X group.

[0417] In some embodiments, R 16 and R 17 They form a 5- or 6-membered cycloalkyl ring (e.g., cyclopentyl or cyclohexyl) together with the carbon atom to which they are attached. Such a 5- or 6-membered cycloalkyl ring can be substituted with an X group.

[0418] In a given embodiment, the radioactive metal complex has the structure of the chemical formula (II-m):

[0419] [Chemical Formula (II-m)]

[0420]

[0421] In the above formula,

[0422] The variable group is as defined above in the chelator of the present invention, for example, the chelator of formula (II); M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion, more preferably 225 It is Ac.

[0423] A radioactive metal complex of formula (II-m) can be formed using any of the chelators of formula (II) described above.

[0424] In some embodiments, one of A1, A2, A3, A4, and A5 is nitrogen, one of A1, A2, A3, A4, and A5 is a carbon substituted with -COOH, and the remainder is CH, i.e., forms a pyridinyl ring substituted with a carboxylic acid.

[0425] In some embodiments, A6, A7, A8, A9, and A 10 One of them is nitrogen, and A6, A7, A8, A9, and A 10One is a carbon substituted with an -COOH group, and the other is a CH group, that is, forming a pyridinyl ring substituted with a carboxylic acid.

[0426] In one embodiment, one or more of R1, R2, R3, R4, and R5 are -COOH. In one embodiment, R6, R7, R8, R9, and R 10 One or more of them are -COOH. In another embodiment, one or more of R1, R2, R3, R4, and R5 are -COOH; R 6, R7, R8, R9, and R 10 One or more of them are -COOH.

[0427] In some embodiments, A1 and A 10 Each is nitrogen; A2 is CR2 and R2 is -COOH;

[0428] A9 is CR9 and R9 is -COOH; A3 to A8 are each CR2, CR3, CR4, CR5, CR6, CR7, and CR8, respectively; and R3 to R8 are each hydrogen.

[0429] In one embodiment, one or more of R1, R2, R3, R4, and R5 are tetrazolyl. In one embodiment, R 6, R7, R8, R9, and R 10 One or more of them are tetrazolyl. In another embodiment, one or more of R1, R2, R3, R4, and R5 are tetrazolyl; R 6, R7, R8, R9, and R 10 One or more of them are tetrazolyl.

[0430] In some embodiments, each R 12 is hydrogen.

[0431] In some embodiments, R 11 It is an alkynyl group or a cycloalkynyl group, preferably cyclooctinyl or a cyclooctinyl derivative, e.g., DBCO.

[0432] In a specific embodiment of a radioactive metal complex of the chemical formula (II-m),

[0433] M is 225 Ac and;

[0434] A1 and A 10 Each is nitrogen;

[0435] A2 is CR2 and R2 is -COOH;

[0436] A9 is CR9 and R9 is -COOH;

[0437] A3 through A8 are each CR2, CR3, CR4, CR5, CR6, CR7, and CR8;

[0438] R3 through R8 are each hydrogen;

[0439] One of Z1 and Z2 is -(CH2) m - and the other one of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n -is;

[0440] R 12 is hydrogen;

[0441] m is 1 and;

[0442] Each n is 0 and;

[0443] X is -L1-R 11 and, where L1 is the linker and -R 11 is an electrophilic group, for example, cyclooctinyl or a cyclooctinyl derivative, e.g., DBCO;

[0444] R 14 to R 17 Each is hydrogen, or alternatively R 16 and R 17 They form a 5- or 6-membered cycloalkyl ring with the carbon atom to which they are attached.

[0445] In a given embodiment, the radioactive metal complex has the structure of the chemical formula (III-m):

[0446] [Chemical Formula (III-m)]

[0447]

[0448] In the above formula,

[0449] The variable group is as defined above in the chelator of the present invention, for example, the chelator of formula (III); M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion, more preferably 225 It is Ac.

[0450] A radioactive metal complex of formula (III-m) can be formed using any of the chelators of formula (III) described above.

[0451] In some embodiments, each A 11 is identical, and each A 11 is O, S, NMe, or NH. For example, each A 11 can be S. In other embodiments, each A 11 The values ​​are different and each is independently selected from O, S, NMe, and NH.

[0452] In some embodiments, each R 18 -(CH2) independently p -COOR 13 and, here R 13 is hydrogen, and each p is independently 0 or 1.

[0453] In some embodiments, each R 18 It is -COOH.

[0454] In some embodiments, each R 18 It is -CH2COOH.

[0455] In some embodiments, each R 18 It is tetrazolyl.

[0456] In a specific embodiment of a radioactive metal complex of the chemical formula (III-m),

[0457] Each R 18 It is COOH;

[0458] One of Z1 and Z2 is -(CH2) m - and the other one of Z1 and Z2 is -(CH2) n -C(R 12 )(X)-(CH2) n -is;

[0459] R 12 is hydrogen;

[0460] m is 1; each n is 0;

[0461] X is -L1-R 11 and, where L1 is the linker and -R 11 is an electrophilic group, for example, cyclooctinyl or a cyclooctinyl derivative, e.g., DBCO;

[0462] R 14 to R 17 Each is hydrogen, or alternatively R 16 and R 17 They form a 5- or 6-membered cycloalkyl ring with the carbon atom to which they are attached.

[0463] In a specific embodiment of the present invention, the radioactive metal complex has one of the following structures:

[0464]

[0465]

[0466]

[0467]

[0468]

[0469] In the above formula,

[0470] M is Actinium-225 ( 225 Ac) and, L1 is absent or a linker;

[0471] R 11 is a nucleophilic moiety or an electrophilic moiety, or R 11 It includes a targeting ligand;

[0472] Each R 12 is independently hydrogen, -CH3, or -CH2CH3, provided that one or more R 12 is -CH3 or -CH2CH3.

[0473] Radioactive metal complexes can be produced by any method known in the art in the context of the present disclosure. For example, the chelator of the present invention can be mixed with a radioactive metal ion and the mixture can be incubated to enable the formation of a radioactive metal complex. In an exemplary embodiment, the chelator is 225 Mixed with a solution of Ac(NO3)3, it is coupled to a chelator through coordination bonding. 225 It forms a radioactive complex containing Ac. As described above, the chelator of the present invention is a radioactive metal, particularly 225 Ac is efficiently chelated. Accordingly, in certain embodiments, a chelator of 1:1000, 1:500, 1:400, 1:300, 1:200, 1:100, 1:50, 1:10, or 1:5, preferably 1:5 to 1:200, more preferably 1:5 to 1:100. 225 The chelator of the present invention according to the concentration ratio of Ac ions 225 It is mixed with a solution of Ac ions. Thus, in some embodiments, the chelator of the present invention, which can be used to form a radioactive metal complex 225 The ratio of Ac is other known 225 It is much lower than what can be achieved with an Ac chelator, e.g., DOTA. Radioactive complexes can be characterized by transient thin-layer chromatography (e.g., iTLC-SG), HPLC, LC-MS, etc. Exemplary methods are described herein, for example, in the following examples.

[0474] Immunoconjugates and radioimmunoconjugates

[0475] In another general aspect, the present invention relates to immunoconjugates and radioimmunoconjugates. The chelator and radiometal complex of the present invention can be conjugated (i.e. covalently linked) to a targeting ligand, such as an immune substance, to produce an immunoconjugate and / or radioimmunoconjugate suitable for medical applications, for example, in a target, for example, in humans, such as targeted radiotherapy. Using the chelator and radiometal complex of the present invention, a radioimmunoconjugate can be produced by site-specifically labeling a targeting ligand, in particular an antibody or its antigen-binding fragment, capable of specifically binding to a target of interest (e.g., cancer cells), with a radiometal ion. In particular, using the chelator and / or radiometal complex of the present invention, a radiometal ion, in particular 225 High-yield chelation of Ac and a radioimmunoconjugate having a desired chelator-antibody ratio (CAR) can be produced. According to a specific embodiment, the method of the present invention provides an average CAR of less than 10, less than 8, less than 6, or less than 4; or a CAR of about 2 to about 8, or about 2 to about 6, or about 2 to about 4, or about 2 to about 3; or a CAR of about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8.

[0476] As used herein, "immunoconjugate" is an antibody or its antigen-binding fragment conjugated (e.g., via covalent bonding) to a second molecule such as a toxin, drug, radioactive metal ion, chelator, radioactive metal complex, etc. In particular, "radioimmunoconjugate" is an immunoconjugate in which an antibody or its antigen-binding fragment is labeled with a radioactive metal or conjugated to a radioactive metal complex.

[0477] According to an embodiment of the present invention, the immunoconjugate comprises, preferably, a chelator of the present invention covalently linked to an antibody or its antigen-binding fragment via a linker, for example, a chelator of Formula (I), Formula (II), or Formula (III) as described herein. Depending on the reactive functional groups (i.e., nucleophiles and electrophiles) on the chelator and the antibody or its antigen-binding fragment, a plurality of attachment modes having different connections between the chelator and the antibody or its antigen-binding fragment are possible.

[0478] According to an embodiment of the present invention, the radioimmunoconjugate comprises, preferably, a radioactive metal complex of the present invention covalently linked to an antibody or its antigen-binding fragment through a linker, for example, a radioactive metal complex of formula (Im), formula (II-m), or formula (III-m) as described herein.

[0479] The immunoconjugate or radioimmunoconjugate of the present invention can be produced using any of the chelators or radioactive metal complexes of the present invention, such as those described in this specification.

[0480] In some embodiments, the radiometal complex of the radioimmunoconjugate of the present invention comprises an alpha-emitting radiometal ion coordinated to a chelator moiety of the radiocomplex. Preferably, the alpha-emitting radiometal ion is 225 It is Ac.

[0481] In a specific embodiment, an antibody or an antigen-binding fragment thereof is linked to a radioactive complex through a triazole moiety to form the radioimmunoconjugate of the present invention.

[0482] In certain embodiments, the antibody or antigen-binding fragment within the immunoconjugate or radioimmunoconjugate of the present application may specifically bind to a tumor antigen. Preferably, the antibody or antigen-binding fragment specifically binds to a cancer antigen. Examples of cancer antigens include, but are not limited to, prostate-specific membrane antigen (PSMA), BCMA, Her2, EGFR, KLK2, CD19, CD22, CD30, CD33, CD79b, and Nectin-4.

[0483] In one embodiment, the antibody specifically binds to PSMA. Preferably, the antibody is PSMB127. The human IgG4 antibody binding to human prostate-specific membrane antigen (PSMA), referred to herein as "anti-PSMA mAb" and denoted as "PSMB127", has the heavy chain (HC) CDR1 sequence of SEQ ID NO. 3, the HC CDR2 sequence of SEQ ID NO. 4, the HC CDR3 sequence of SEQ ID NO. 5, the light chain (LC) CDR1 sequence of SEQ ID NO. 6, the LC CDR2 sequence of SEQ ID NO. 7, and the LC CDR3 sequence of SEQ ID NO. 8, and has the HC sequence of SEQ ID NO. 9 and the LC sequence of SEQ ID NO. 10. The anti-PSMA mAb was expressed and purified using a standard chromatography method. Antibody PSMB127, its biological activity, uses thereof, or other related information is described, for example, in U.S. Patent Application Publication No. US 20200024360A1, the contents of which are incorporated herein by reference in their entirety.

[0484] In another embodiment, the antibody specifically binds to human kallikrein-2 (KLK2). Preferably, the antibody is H11B6. The H11B6 antibody, its biological activity, uses, or other related information is described in U.S. Patent No. 10,100,125, the contents of which are incorporated herein by reference in whole. As described herein, the H11B6 antibody polypeptide comprises a heavy chain (HC) variable region comprising the amino acid sequences of SEQ ID NO. 11, SEQ ID NO. 12, and SEQ ID NO. 13, and a light chain (LC) variable region comprising the amino acid sequences of SEQ ID NO. 14, SEQ ID NO. 15, and SEQ ID NO. 16. The H11B6 antibody may additionally have a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 17 and a light chain variable region containing the amino acid sequence of SEQ ID NO. 18, or have a heavy chain constant region containing the amino acid sequence of SEQ ID NO. 19 and a light chain constant region containing the amino acid sequence of SEQ ID NO. 20, or have a heavy chain containing the amino acid sequence of SEQ ID NO. 21 and a light chain containing the amino acid sequence of SEQ ID NO. 22.

[0485] The commercial antibodies trastuzumab (Herceptin), cetuximab (Erbitux), pertuzumab (Perjeta), and panitumumab (Vectibix) were purchased from Roche, Lilly, Roche, and Amgen, respectively. Trastuzumab and pertuzumab bind to human Her2. Cetuximab and panitumumab bind to human EGFR.

[0486] Immunoconjugates and radioimmunoconjugates of the present invention may be prepared by any method known in the art in the context of the present disclosure for conjugating a ligand, e.g., an antibody, to a chelator, comprising chemical and / or enzymatic methods. For example, immunoconjugates and radioimmunoconjugates may be prepared by coupling reactions, including but not limited to the formation of esters, thioesters, or amides from activated acids or acyl halides; nucleophilic substitution reactions (e.g., nucleophilic substitution of a halide ring or ring opening of a tension ring system); azide-alkyne huigen cyclization addition (e.g., 1,3-bipolar cyclization addition between an azide and an alkyne to form a 1,2,3-triazole linker); thiolin addition; imine formation; Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO); and Michael addition (e.g., maleimide addition). A number of different attachment modes having different linkages are possible depending on the reactive functional group used. The attachment of the ligand can be performed on a chelator coordinated to a radioactive metal ion, or on a chelator not coordinated to a radioactive metal ion.

[0487] According to one embodiment, a radioimmunoconjugate may be produced by a step of covalently linking a radioactive metal complex of the present invention to an antibody or its antigen-binding fragment by a click chemical reaction (see, for example, FIG. 2b and 2d, referred to as "click radiolabeling"). Alternatively, a radioimmunoconjugate may be produced by a step of first producing an immunoconjugate of the present invention by covalently linking a chelator of the present invention to an antibody or its antigen-binding fragment by a click chemical reaction; the immunoconjugate may subsequently be labeled with a radioactive metal ion to produce a radioimmunoconjugate (see, for example, FIG. 2a and 2c, referred to as "1-step direct radiolabeling"). Both residue-specific (e.g., FIG. 2a and 2b) and site-specific (e.g., FIG. 2c and 2d) conjugation methods may be used to produce the immunoconjugate and radioimmunoconjugate of the present invention.

[0488] Residue-specific methods for protein conjugation are well established and most commonly include lysine side chains using activated esters or isothiocyanates, or cysteine ​​side chains using maleimides, haloacetyl derivatives, or activated disulfides (Brinkley Bioconjugate Chem [1992:2]). Since most proteins have multiple lysine and cysteine ​​residues, heterogeneous mixtures of products having different numbers of conjugated molecules at various amino acid positions are typically obtained using such methods. Tyrosine-specific conjugation (Reference [Ban et al. Bioconjugate Chemistry 2013:520]), methionine-specific methods (Lin et al. Science 2017 (355) 597]), additional cysteine-focused approaches (Toda et al. Angew Chemie Additional methods including [2013:12592]) etc. have been established.

[0489] More recently, site-selective and site-specific conjugation methods have been established for monoclonal antibodies and other proteins (reference [Agarwal, P. and CR Bertozzi, Bioconjugation Chemistry , 2015. 26(2): p. 176-92]; literature[Rabuka et al. Curr Opin Chem Biol These include the incorporation of non-natural amino acids; the fusion of the protein of interest to a tag specifically recognized and modified by other enzymes such as sorbase A, lipoic acid ligase, and formylglycine-synthesizing enzyme, or to a 'self-labeling tag' such as DHFR or SNAP; and the enzymatic modification of glycans to enable the conjugation of the payload of interest (reference [Hu et al. Chem Soc Rev 2016:1691]; use of microbial transglutaminases to selectively recognize limited sites on antibodies; and additional methods influencing selective conjugation using molecular recognition and / or chemical approaches (Literature [Yamada et al. 2019:5592]; Literature [Park et al. Bioconjugate Chem 2018:3240]; Literature[Pham et al. Chembiochem Includes 2018:799]).

[0490] In some embodiments, the immunoconjugate or radioimmunoconjugate of the present invention is produced using a residue-specific method for conjugating a chelator of the present invention to an antibody or its antigen-binding fragment. Such residue-specific methods typically produce an immunoconjugate or radioimmunoconjugate covalently linked to a chelator or radiometal complex at various sites of the antibody. Any residue-specific method for forming a protein or antibody conjugate known to those skilled in the art in the context of this disclosure may be used. Examples of residue-specific conjugation methods that may be used include, for example, conjugation of a chelator or radiometal complex to a lysine residue of an antibody using a chelator or radiometal complex comprising an activated ester or isothiocyanate group; for example, conjugation to a cysteine ​​residue of an antibody using a chelator or radiometal complex comprising a maleimide, a haloacetyl derivative, an acyl halide, an activated disulfide group, or a methylsulfonylphenyloxadiazole group; For example, conjugation to a tyrosine residue of an antibody using a chelator or radioactive metal complex comprising 4-phenyl-3 H-1,2,4-triazolin-3,5(4 H)-dione (PTAD); and, for example, conjugation to a methionine residue of an antibody using a chelator or radioactive metal complex comprising an oxaziridine derivative, are included but not limited thereto. It is also possible to label the antibody with a biorthogonal reactive functional group at a specific residue using one or more of the methods described above before conjugation to the chelator or radioactive metal complex of the present invention.For example, a tyrosine residue can be site-specifically labeled with a hexazyridine derivative linked to a hexazyridine derivative, such as an azido, alkynyl, or cycloalkynyl, and subsequently, an antibody containing the labeled tyrosine residue can be conjugated to the chelator or radiometal complex of the present invention using a chelator or radiometal complex having a compatible reactive functional group.

[0491] In some embodiments, the immunoconjugate or radioimmunoconjugate of the present invention is produced using a site-specific or site-selective method for conjugating the chelator of the present invention to an antibody or its antigen-binding fragment. In contrast to residue-specific methods, "site-specific" or "site-selective" methods typically produce an immunoconjugate or radioimmunoconjugate covalently linked to the chelator or radiometal complex at a specified site on the antibody. Any site-specific method for forming a protein or antibody conjugate known to those skilled in the art in the context of this disclosure may be used. For example, a non-natural amino acid (e.g., azido- or alkynyl-amino acid) may be site-specifically incorporated into the antibody using a mutant aminoacyl t-RNA synthetase capable of selectively aminoacylating its tRNA to the non-natural amino acid of interest. Subsequently, a mutant acylated tRNA can be used in conjunction with an amber repressor tRNA to react with an amber nonsense codon and site-specifically incorporate non-natural amino acids into the protein. An antibody site-specifically labeled by one or more of the methods described above can subsequently be conjugated to the chelator or radioactive metal complex of the present invention having a compatible reactive functional group.

[0492] According to an embodiment of the present invention, a method for generating a radioimmunoconjugate is R 11The method includes the step of reacting the chelator or radioactive complex of the present invention, which is the nucleophilic or electrophilic moiety, with an antibody or its antigen-binding fragment, or a modified antibody or its antigen-binding fragment comprising the nucleophilic or electrophilic moiety.

[0493] In one embodiment, the method comprises the steps of: reacting the chelator of the present invention with an antibody or its antigen-binding fragment, or a modified antibody or its antigen-binding fragment comprising a nucleophilic or electrophilic functional group to form an immunoconjugate having a covalent link between the chelator and the antibody or its antigen-binding fragment, or the modified antibody or its antigen-binding fragment; and reacting the immunoconjugate with a radioactive metal ion to form a radioimmunoconjugate such that the radioactive metal ion binds to the chelator of the immunoconjugate through a coordination bond. This embodiment may be referred to as a "one-step direct radiolabeling" method (e.g., as schematically illustrated in FIG. 2c) because there is only one chemical reaction step involving a radioactive metal.

[0494] In another embodiment, the method comprises the step of forming a radioimmunoconjugate by reacting the radiocomplex of the present invention with an antibody or its antigen-binding fragment, or with a modified antibody or its antigen-binding fragment comprising a nucleophilic or electrophilic functional group. Such embodiments may be referred to as "click radiolabeling" methods (e.g., as schematically illustrated in FIG. 2d). The modified antibody or its antigen-binding fragment may be produced by any method known in the art in the context of the present disclosure, for example, by labeling the antibody with a biorthogonal reactive functional group at a specific residue using one or more of the methods described above, or by site-specifically incorporating a non-natural amino acid (e.g., azido- or alkynyl-amino acid) into the antibody using one or more of the methods described above. The degree of labeling (DOL), sometimes referred to as the degree of substitution (DOS), is a particularly useful parameter for characterizing and optimizing bioconjugates, such as antibodies modified by non-natural amino acids. It is expressed as the average number of non-natural amino acids coupled to a protein molecule (e.g., antibody), or as a molar ratio of label / protein form. DOL can be determined from the absorption spectrum of a labeled antibody by any known method in the art.

[0495] In a given embodiment, as described herein, the immunoconjugate and radioimmunoconjugate of the present invention are prepared using a click chemical reaction. For example, the radioimmunoconjugate of the present invention may be prepared using a click chemical reaction referred to as "click radiolabeling" (see, for example, FIG. 2b and FIG. 2d). Click radiolabeling forms a covalent triazole link between a radioactive complex (a radioactive metal ion bound to a chelator) and an antibody or its antigen-binding fragment using a click chemical reaction partner, preferably an azide and an alkyne (e.g., cyclooctine or a cyclooctine derivative). A method for click radiolabeling of an antibody is described, for example, in International Patent Application No. PCT / US18 / 65913, titled "Radiolabeling of Polypeptides," in which the relevant description is incorporated herein by reference. In another embodiment referred to as "one-step direct radiolabeling," the immunoconjugate is prepared using a click chemical reaction between an antibody or its antigen-binding fragment and a chelator; Next, the immunoconjugate is brought into contact with a radioactive metal ion to form a radioimmunoconjugate (see, for example, FIG. 2a and FIG. 2c).

[0496] According to one embodiment, a method for manufacturing a radioimmunoconjugate comprises the step of binding a radioactive metal ion to the chelator of the present invention (e.g., through coordination bonding).

[0497] One embodiment of the "one-step direct radiolabeling" method forms a radioimmunoconjugate by contacting an immunoconjugate (i.e., a polypeptide-chelator complex) with a radioactive metal ion, wherein the immunoconjugate may be described as a method for producing a radioimmunoconjugate comprising the step of including the chelator of the present invention. According to a specific embodiment, the immunoconjugate is formed through a click chemical reaction between the chelator of the present invention and the polypeptide. According to a specific embodiment, the radioimmunoconjugate is formed without metal-free conditions (e.g., without any step(s) of removing or actively excluding common metal impurities from the reaction mixture). This is in contrast to a conventional method that requires radiolabeling antibodies under strict metal-free conditions to avoid competitive (non-productive) chelation of common metals such as iron, zinc, and copper (which introduces significant difficulties into the production process).

[0498] In a specific embodiment, the method for preparing a radioimmunoconjugate of the present invention comprises a "one-step direct radiolabeling" method comprising the following steps:

[0499] (i) providing a modified polypeptide comprising a polypeptide (e.g., an antibody or an antigen-binding fragment thereof) covalently linked to a first click reaction partner (e.g., an azido group);

[0500] (ii) providing a chelator complex comprising a chelator of the present invention covalently connected to a second click reaction partner (e.g., an alkynyl group or a cycloalkynyl group);

[0501] (iii) a step of contacting the modified polypeptide with the chelator complex under conditions that enable the first click reaction partner (e.g., an azido group) to react with the second click reaction partner (e.g., an alkynyl group or a cycloalkynyl group) to form a polypeptide-chelator complex (i.e., an immunoconjugate); and

[0502] (iv) a step of preparing a radioimmunoconjugate by contacting a polypeptide-chelator complex with a radioactive metal ion (wherein the radioimmunoconjugate comprises a polypeptide labeled with a radioactive metal ion, e.g., a modified antibody labeled with an alpha-emitting radioactive metal ion that is bound to a chelator via coordination bonding or an antigen-binding fragment thereof).

[0503] According to a specific embodiment, step (iv) is performed without metal-free conditions.

[0504] In an alternative embodiment, a method for manufacturing a radioimmunoconjugate comprises a "click radiolabeling" method (e.g., as illustrated in FIG. 2d) comprising the following steps:

[0505] (i) providing a modified antibody or an antigen-binding fragment thereof comprising an antibody covalently linked to an azido group or an antigen-binding fragment thereof;

[0506] (ii) providing a radioactive complex comprising an alpha-emitting radioactive metal ion coupled to a chelator via a coordination bond, wherein the chelator is covalently connected to an alkynyl group or a cycloalkynyl group; and

[0507] (iii) a step of preparing a radioimmunoconjugate by contacting a modified antibody or its antigen-binding fragment with a radioactive complex under conditions that enable the azido group to react with an alkynyl group or a cycloalkynyl group.

[0508] Conditions for carrying out click chemistry reactions are known in the art, and any conditions for carrying out click chemistry reactions known to those skilled in the art in the context of this disclosure may be used in the present invention. Examples of conditions include, but are not limited to, the step of incubating the modified polypeptide and the radioactive complex at a pH of 4 to 10 and a temperature of 20°C to 70°C in a ratio of 1:1 to 1000:1.

[0509] The click radiolabeling method described above maximizes efficiency by enabling the chelation of radioactive metal ions under low or high pH and / or high temperature conditions, which can be performed without the risk of inactivating alkyne reaction partners. Efficient chelation between an azide-labeled antibody or its antigen-binding fragment and a radioactive complex, along with an efficient SPAAC reaction, enables the generation of radioimmunoconjugates in high radiochemical yields even at low azide:antibody ratios. The only step where trace metals must be excluded is the chelation of radioactive metal ions to the chelation moiety; the antibody preparation, purification, and conjugation steps do not need to be performed under metal-free conditions.

[0510] The chelator and radiometal complex of the present invention may also be used to produce site-specific radiolabeled polypeptides, e.g., antibodies. The click radiolabeling method described herein facilitates the site-specific production of radioimmunoconjugates by utilizing established methods for site-specific installation of azide groups on antibodies (Li, X., et al. Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions. Angew Chem Int Ed Engl , 2014. 53(28): p. 7179-82]; Xiao, H., et al., Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells. Angew Chem Int Ed Engl[ , 2013. 52(52): p. 14080-3]). Methods of attaching molecules to proteins or antibodies in a site-specific manner are known in the art, and any method known to those skilled in the art of site-specific labeling of antibodies may be used in the present invention in the context of the present disclosure. Examples of methods for site-specifically modifying antibodies suitable for use in the present invention include, but are not limited to, the incorporation of engineered cysteine ​​residues (e.g., THIOMAB™), the use of non-natural amino acids or glycans (e.g., selenocysteine, p-AcPhe, formylglycine synthetase (FGE, SMARTag™), etc.), and enzymatic methods (e.g., the use of glycotransferase, endoglycosidase, microbial or bacterial transglutaminase (MTG or BTG), sorbase A, etc.).

[0511] In some embodiments, a modified antibody or its antigen-binding fragment for use in generating an immunoconjugate or radioimmunoconjugate of the present invention is obtained by trimming the antibody or its antigen-binding fragment with a bacterial endoglycosidase specific to the β-1,4 linkage between core GlcNac residues at the Fc-glycosylation site of the antibody, such as GlycINATOR (Genovis), which leaves the innermost GlcNAc intact on the Fc, thereby enabling site-specific incorporation of an azido sugar into that site. Subsequently, the modified antibody or its antigen-binding fragment can be obtained by reacting the trimmed antibody or its antigen-binding fragment with an azide-labeled sugar, such as UDP-N-azidoacetylgalactosamine (UDP-GalNAz) or UDP-6-azido 6-deoxy GalNAc, in the presence of a sugar transferase, such as GalT-galactosyltransferase or GalNAc transferase.

[0512] In another embodiment, a modified antibody or its antigen-binding fragment for use in generating an immunoconjugate or radioimmunoconjugate of the present invention is obtained by deglycosylating the antibody or its antigen-binding fragment with an imidase. Subsequently, the resulting deglycosylated antibody or its antigen-binding fragment may be reacted with an azidoamine, preferably 3-azidopropylamine, 6-azidohexylamine, or any azido-linker-amine or any azido-alkyl / heteroalkyl-amine, such as azido-polyethylene glycol (PEG)-amine, for example, O-(2-aminoethyl)-O′-(2-azidoethyl)tetraethylene glycol, O-(2-aminoethyl)-O′-(2-azidoethyl)pentaethylene glycol, O-(2-aminoethyl)-O′-(2-azidoethyl)triethylene glycol, etc., or in the presence of a microbial transglutaminase, to obtain the modified antibody or its antigen-binding fragment.

[0513] The radioimmunoconjugate of the present invention may be produced using any radioactive metal complex described herein. In certain embodiments, the radioactive metal complex has a structure of formula (Im), formula (II-m), or formula (III-m). In preferred embodiments, the radioactive metal complex has a structure selected from the group consisting of the following:

[0514]

[0515]

[0516]

[0517]

[0518]

[0519] In the above formula,

[0520] M is a radioactive metal ion, preferably an alpha-emitting radioactive metal ion, more preferably actinium-225 ( 225 Ac) and, R 11 is cyclooctinyl or cyclooctinyl derivatives, such as bicyclononinil (BCN), difluorinated cyclooctinyl (DIFO), dibenzocyclooctinyl (DIBO), keto-DIBO, biaryl-azacyclooctinyl (BARAC), dibenzo-azacyclooctinyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctinyl (DIMAC), difluorobenzocyclooctinyl (DIFBO), monobenzocyclooctinyl (MOBO), and tetramethoxydibenzocyclooctinyl (TMDIBO).

[0521] In some embodiments, an antibody or its antigen-binding fragment is covalently linked to an azido group using any method for chemical or enzymatic modification of antibodies and polypeptides known to those skilled in the art in the context of the present disclosure. The azido-labeled antibody or its antigen-binding fragment is reacted with an alkynyl or cycloalkynyl group, preferably a cyclooctinyl group, more preferably a DBCO, comprising the chelator or radioactive metal complex of the present invention, under conditions sufficient for the azido and the alkynyl or cycloalkynyl group to undergo a click chemical reaction to form a 1,2,3-triazole moiety.

[0522] In a specific embodiment, the radioimmunoconjugate of the present application is,

[0523] , ,

[0524] , ,

[0525] , ,

[0526] , ,

[0527] , and Includes, but not limited to,

[0528] In the above formula,

[0529] mAb is an antibody or its antigen-binding fragment; L1 is absent or a linker, preferably a linker; and each R 12 is independently hydrogen, CH3, or CH2CH3, provided that one or more R 12 is -CH3 or -CH2CH3; M is an alpha-emitting radionuclide, preferably 225 It is Ac.

[0530] Examples of the radioimmunoconjugates of the present application include

[0531] ,

[0532] , , and

[0533] This is included but not limited to,

[0534] Preferably, the mAb is selected from PSMB127, pertuzumab, cetuximab, panitumumab, Herceptin, or H11B6.

[0535] Radioimmunoconjugates produced by the methods described herein may be analyzed using methods known to those skilled in the art in the context of the present disclosure. For example, LC / MS analysis may be used to determine the ratio of chelators to labeled polypeptides, e.g., antibodies or their antigen-binding fragments; analytical size-exclusion chromatography may be used to determine the oligomeric state of polypeptides and polypeptide conjugates, e.g., antibodies and antibody conjugates; radiochemical yields may be determined by instantaneous thin-layer chromatography (e.g., iTLC-SG); and radiochemical purity may be determined by size-exclusion HPLC. Exemplary methods are described herein, for example, in the following examples.

[0536] Pharmaceutical composition and method of use

[0537] In another general aspect, the present invention relates to a pharmaceutical composition comprising the chelator of the present invention, a radioactive metal complex, an immunoconjugate, or a radioimmunoconjugate, and a pharmaceutically acceptable carrier. The pharmaceutical composition may comprise one or more pharmaceutically acceptable excipients.

[0538] In one embodiment, the pharmaceutical composition comprises the radioactive metal complex of the present invention and a pharmaceutically acceptable carrier.

[0539] In another embodiment, the pharmaceutical composition comprises the radioimmunoconjugate of the present invention and a pharmaceutically acceptable carrier.

[0540] As used herein, the term “carrier” refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application. As used herein, the term “pharmaceutically acceptable carrier” refers to a non-toxic material that does not interfere with the efficacy or biological activity of the composition according to the present invention. According to specific embodiments, any pharmaceutically acceptable carrier suitable for use in antibody-based or radiocomplex-based pharmaceutical compositions may be used in the present invention.

[0541] According to specific embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to the subject. For example, the compositions described herein may be formulated to be suitable for parenteral administration, e.g., intravenously, subcutaneously, intramuscularly, or intratumorally.

[0542] In another general aspect, the present invention relates to a method for selectively targeting neoplastic cells for radiotherapy and a method for treating neoplastic diseases or disorders. Any of the radioactive complexes or radioimmunoconjugates described herein and pharmaceutical compositions thereof may be used in the method of the present invention.

[0543] A "neoplasm" is an abnormal mass of tissue caused when cells divide more than they should or fail to die when they should. Neoplasms can be benign (not cancerous) or malignant (cancer). Neoplasms are also referred to as tumors. Neoplastic diseases or disorders are diseases or disorders associated with neoplasms, such as cancer. Examples of neoplastic diseases or disorders include, but are not limited to, disseminated cancer and solid tumor cancers.

[0544] According to one embodiment, a method for treating prostate cancer (e.g., metastatic prostate cancer, or metastatic castration-resistant prostate cancer) in a subject requiring treatment for prostate cancer comprises the step of administering a therapeutically effective amount of a radioimmunoconjugate as described herein to the subject, wherein the radioimmunoconjugate comprises a radioactive metal complex as described herein conjugated to H11B6.

[0545] Other examples of diseases to be treated by the methods described herein or targeted for radiotherapy include, but are not limited to, hypertrophy, coronary artery disease, or vascular occlusion disease, diseases or disorders associated with infected cells, microorganisms, or viruses, or diseases or disorders associated with inflammatory cells, such as rheumatoid arthritis (RA).

[0546] In one embodiment of the present invention, a method for selectively targeting neoplastic cells for radiotherapy comprises the step of administering the radioimmunoconjugate or pharmaceutical composition of the present invention to a subject who requires it.

[0547] In one embodiment of the present invention, a method for treating a neoplastic disease or disorder comprises the step of administering the radioimmunoconjugate or pharmaceutical composition of the present invention to a subject in need thereof.

[0548] In one embodiment of the present invention, a method for treating cancer in a subject requiring treatment for cancer comprises the step of administering a radioimmunoconjugate or a pharmaceutical composition of the present invention to the subject.

[0549] A radioimmunoconjugate directly delivers radiation to, for example, cells, etc., targeted by a targeting ligand. Preferably, the radioimmunoconjugate 225 It supports alpha-emitting radioactive metal ions such as Ac. Upon targeting, alpha-emitting radioactive metal ions, for example 225 Alpha particles from Ac and its daughter nuclides are delivered to targeted cells and cause cytotoxic effects thereon, thereby selectively targeting neoplastic cells for radiotherapy and / or treating neoplastic diseases or disorders.

[0550] A pre-targeting approach for selectively targeting neoplastic cells for radiotherapy and treating neoplastic diseases or disorders is also considered by the present invention. According to the pre-targeting approach, an azide-labeled antibody or an antigen-binding fragment thereof is administered, binds to a cell possessing the target antigen of the antibody, and is removed from circulation over time or by a removal agent. Subsequently, a radiocomplex of the present invention, preferably comprising cyclooctine or a cyclooctine derivative, e.g., DBCO, is administered and undergoes a SPAAC reaction with the azide-labeled antibody bound to the target site, while the remaining unbound radiocomplex is rapidly removed from circulation. The pre-targeting technique provides a method for enhancing the localization of radiometal ions at a target site within a target.

[0551] In another embodiment, a modified polypeptide, e.g., an azide-labeled antibody or an antigen-binding fragment thereof, and a radiocomplex of the present invention are administered to a subject requiring targeted radiotherapy or treatment of a neoplastic disease or disorder in the same composition or in a different composition.

[0552] As used herein, the term "therapeutic effective dose" refers to the amount of an active ingredient or component that elicits a desired biological or medical response in a subject. The therapeutic effective dose may be determined empirically and routinely with respect to the stated purpose. For example, to help identify the optimal dosage range, in vitro ( in vitro ) The assay may be used optionally. The selection of a specific effective dose may be determined by a person skilled in the art (e.g., through clinical trials) based on the consideration of several factors, including the disease to be treated or prevented, concomitant symptoms, the patient's body weight, the patient's immune status, and other factors known to those skilled in the art. The exact dose used in the formulation will also depend on the route of administration and the severity of the disease, and must be determined according to the judgment of a specialist and the situation of each individual patient. The effective dose may be estimated from dose-response curves derived from in vitro or animal model test systems.

[0553] As used herein, the terms “treat,” “treating,” and “treatment” are all intended to refer to the improvement or reversal of one or more measurable physical parameters associated with a disease, disorder, or pathological condition, such as a neoplastic disease or disorder, for which the administration of radioactive metal ions is beneficial, which may not necessarily be identifiable in the subject, but may be identifiable in the subject. The terms “treat,” “treating,” and “treatment” may also refer to causing regression, preventing progression, or at least slowing the progression of a disease, disorder, or condition. In certain embodiments, “treat,” “treating,” and “treatment” refer to the alleviation, prevention of onset or onset, or reduction in duration of one or more symptoms associated with a disease, disorder, or pathological condition, such as a neoplastic disease or disorder, for which the administration of radioactive metal ions is beneficial. In certain embodiments, “treat,” “treating,” and “treatment” refer to the prevention of recurrence of a neoplastic disease, disorder, or pathological condition. In certain embodiments, "treat," "treating," and "treatment" refer to an increase in the survival rate of a subject having a neoplastic disease, disorder, or pathological condition. In certain embodiments, "treat," "treating," and "treatment" refer to the removal of a neoplastic disease, disorder, or pathological condition from a subject.

[0554] In some embodiments, a therapeutically effective amount of the radioimmunoconjugate or pharmaceutical composition of the present invention is administered to a subject to treat a neoplastic disease or disorder, e.g., cancer, in the subject.

[0555] In another embodiment of the present invention, the radioimmunoconjugate and pharmaceutical composition of the present invention may be used in combination with other agents effective in treating neoplastic diseases or disorders.

[0556] Radioimmunoconjugates and pharmaceutical compositions as described herein for use in selectively targeting neoplastic cells for radiotherapy and / or treating neoplastic diseases or disorders; and uses of radioimmunoconjugates or pharmaceutical compositions as described herein in the manufacture of medicines for selectively targeting neoplastic cells for radiotherapy and / or treating neoplastic diseases or disorders are also provided.

[0557] Examples

[0558] The following examples of the present invention are intended to further illustrate the nature of the invention. It should be understood that the following examples do not limit the invention, and that the scope of the invention is determined by the appended claims.

[0559] Example 1: Synthesis of macrocyclic chelators having different linker substitution positions and chelation efficiency

[0560] Actinium-225 ( 225 To investigate the effect of linker position on the chelation efficiency of Ac), two chelators based on the macrocyclic chelator N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6 (H2bp18c6) were synthesized. Specifically, H2bp18c6-benzyl-isopentyl and H2bp18c6-benzyl-phenyl were synthesized:

[0561]

[0562] Synthesis and Characterization of H2bp18c6-Benzyl-Isopentyl

[0563] H2bp18c6-benzyl-isopentyl was synthesized according to Reaction Scheme 1.

[0564] Reaction Scheme 1: Synthesis of H2bp18c6-benzyl-isopentyl

[0565]

[0566] Isopentyl magnesium chloride (2 M in Et2O, 3.33 mL, 6.66 mmol) was added dropwise at -78°C to a solution of ZnCl2 (1.9 M in 2-methyltetrahydrofuran, 3.50 mL, 6.65 mmol) and THF (25 mL). The milky white mixture was stirred at room temperature for 1 hour and then cooled to 0°C. A solution of methyl 6-formylpicolinate (1.00 g, 6.1 mmol) in THF (10 mL) was added. The mixture was heated at 50°C for 3 hours. The cooled reaction mixture was poured into a saturated aqueous solution of NH4Cl and extracted three times with EtOAc. The combined extracts were dried over Na2SO4. Filtration and concentration of the filtrate yielded an unrefined product as a brown oil. Chromatography on silica gel (heptane to 50% EtOAc in heptane) yielded 693 mg (48% yield) of the product as yellow oil.

[0567] PPh3 (120 mg, 0.46 mmol) and NBS (56 mg, 0.42 mmol) were added at room temperature to a solution of methyl 6-(1-hydroxy-4-methylpentyl)picolinate (91 mg, 0.38 mmol) in CH2Cl2 (3 mL). The reaction solution was stirred at room temperature for 1 hour and then concentrated. Chromatography on silica gel (heptane to 30% EtOAc in heptane) yielded 85 mg (87% yield) of the product as a colorless oil.

[0568] A solution of methyl 6-(chloromethyl)picolinate in ACN (5 mL) was slowly added at 60°C using a syringe pump within 1 hour to a stirred mixture of 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (315 mg, 1.2 mmol) and K2CO3 (691 mg, 5 mmol) in ACN (30 mL). After addition, the reaction mixture was stirred at 60°C for 6 hours and then filtered. The filtrate was concentrated, and the residue was purified by chromatography on silica gel (CH2Cl2 to 10% MeOH in CH2Cl2) to yield 206 mg (50% yield) of the product as a yellowish foamy solid.

[0569] Methyl 6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinate (31 mg, 0.075 mmol), Na2CO3 (40 mg, 0.38 mmol), and NaI (1.5 mg) in DMF (0.5 mL) were mixed with methyl 6-(1-chloro-4-methylpentyl)picolinate (29 mg, 0.11 mmol). The reaction mixture was heated at 100°C for 20 hours. Chromatography on silica gel (10% MeOH in EtOAc to CH2Cl2) yielded 18.9 mg (40% yield) of the product as a yellow film adhering to the flask wall.

[0570] A solution of methyl 6-((16-(1-(6-(methoxycarbonyl)pyridine-2-yl)-4-methylpentyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinate (3 mg, 0.005 mmol) in THF / MeOH / H2O (4:1:1 v / v / v, 0.6 mL) was treated with LiOH (1 N, 0.1 mL). The reaction mixture was stirred at room temperature for 1 hour, after which it was concentrated and dried. The residue was dissolved in 0.95 mL of metal-free water and neutralized with 0.05 mL of 2 N HCl to provide a solution of H2bp18c6-benzyl-isopentyl (approx. 3 mg / mL = approx. 5 mM, pH approx. 6) in water.

[0571] H2bp18c6-benzyl-isopentyl (MW = 602 Da) was characterized by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). HPLC analysis showed a major peak at an elution time of 17.181 min, which corresponds to a glass chelator. LC-MS analysis showed 603 [M+H + ], 625 [M+Na + ], and 302 [M+2H + Mass ion peak (ES, ) at ] m / z It showed ) and confirmed the synthesis of H2bp18c6-benzyl-isopentyl.

[0572] HPLC Method: XBridge C18 3.5 μm 150 x 4.6 mm, 100 Å column; mobile phase A: 0.1% TFA in H2O, B: 0.1% TFA in ACN; isosolvent with gradient 10% to 30% B from 0 to 20 min, gradient 30% to 100% B from 20 to 20.1 min, isosolvent with 100% B from 20.1 to 25 min, gradient 100% to 10% B from 25 to 25.1 min, isosolvent with 10% B from 25.1 to 30 min; 3 min after run; flow rate 1 mL / min; column temperature 30°C; injection volume 5 μL.

[0573] Synthesis and Characterization of H2bp18c6-Benzyl-Phenyl

[0574] H2bp18c6-benzyl-phenyl was synthesized according to reaction scheme 2.

[0575] Reaction Scheme 2: Synthesis of H2bp18c6-Benzyl-Phenyl

[0576]

[0577] Methyl 6-formylpicolinate (165 mg, 1.0 mmol), phenylboronic acid (244 mg, 2.0 mmol), Cs2CO3 (326 mg, 1.0 mmol), Pd2(dba) 3- A mixture of CHCl3 (52 mg, 0.05 mmol) and PPh3 (26 mg, 0.1 mmol) was placed in a sealed vial under N2. Toluene (3 mL) was added via a syringe, and the mixture was heated in a microwave at 100°C for 4 hours. The cooled reaction mixture was filtered through Celite, and the filtrate was concentrated. Chromatography on silica gel (heptane to 50% EtOAc in heptane) yielded 146 mg (60% yield) of the product as a yellow oil.

[0578] A solution of methyl 6-(hydroxy(phenyl)methyl)picolinate (141 mg, 0.58 mmol), PPh3 (183 mg, 0.70 mmol), and NBS (113 mg, 0.64 mmol) in CH2Cl2 (8 mL) was stirred at room temperature for 1 hour. Additional PPh3 (133 mg, 0.70 mmol) and NBS (113 mg, 0.64 mmol) were added, and the solution was further stirred at room temperature for 1 hour. The reaction mixture was concentrated and purified by chromatography on silica gel (30% EtOAc in heptane to 30% EtOAc in heptane) to yield 73 mg (41% yield) of the product as a colorless oil.

[0579] A mixture of methyl 6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinate (33 mg, 0.080 mmol), methyl 6-(bromo(phenyl)methyl)picolinate (37 mg, 0.12 mmol), and Na2CO3 (42 mg, 0.40 mmol) in ACN (1.0 mL) was heated at 80°C for 3 hours. The reaction mixture was filtered, and the filtrate was concentrated. Chromatography on silica gel (10% MeOH in EtOAc to CH2Cl2) yielded 51 mg (64% yield) of the product as a white solid.

[0580] A solution of methyl 6-((16-((6-(methoxycarbonyl)pyridine-2-yl)(phenyl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinate (16 mg, 0.025 mmol) in THF / MeOH / H2O (4:1:1 v / v / v, 1.2 mL) was treated with LiOH (1 N, 0.2 mL). The reaction mixture was stirred at room temperature for 1 hour, after which it was concentrated and dried. The residue was dissolved in 5.2 mL of metal-free water and neutralized with 0.10 mL of 2 N HCl to obtain a solution of H2bp18c6-benzyl-phenyl (approx. 3 mg / mL = approx. 5 mM, pH approx. 6) in water.

[0581] H2bp18c6-benzyl-phenyl (MW= 608 Da) was characterized by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). HPLC analysis was performed on isopentane-H2bp18c6 as described above. The HPLC analysis showed a major peak at an elution time of 14.137 minutes, which corresponds to a glass chelator. The LC-MS analysis showed 609 [M+H + ], 631 [M+Na + ], and 305 [M+2H + Mass ion peak (ES, ) at ] m / zIt showed ) and confirmed the synthesis of H2bp18c6-benzyl-phenyl.

[0582] Chelation test using lanthanum (III)

[0583] An aqueous solution of H2bp18c6-benzyl-isopentyl in water (approx. 3 mg / mL = approx. 5 mM, 20 μL, 0.1 μmol) was treated with La(NO3)3 (10 mM, 50 μL, 0.5 μmol in metal-free water). An aqueous solution of H2bp18c6-benzyl-phenyl in water (approx. 3 mg / mL = approx. 5 mM, 20 μL, 0.1 μmol) was treated with La(NO3)3 (10 mM, 50 μL, 0.5 μmol in metal-free water). After thorough mixing, each solution was analyzed by LCMS and HPLC to determine the chelator and La 3+ It was determined whether a complex of was formed.

[0584] As evidenced by the significant shift in HPLC peak retention times analyzed according to the method described above for the synthesis of H2bp18c6, both isopentane-H2bp18c6 and H2bp18c6-benzyl-phenyl chelator are La at room temperature 3+ It exhibited rapid and stoichiometric chelation with ( FIGS. 1a and FIGS. 1b ). Complex formation was also confirmed by LCMS. LCMS analysis of isopentane-H2bp18c6 after mixing with La(NO3)3 was 739(H2bp18c6-benzyl-isopentyl + La +3 - 2H + Mass ion peak (ES, ) m / z ) showed; LC-MS analysis of H2bp18c6-benzyl-phenyl after mixing with La(NO3)3 was 745(H2bp18c6-benzyl-phenyl + La +3 - 2H + Mass ion peak (ES, ) m / z It showed ) and confirmed the formation of complexes with both chelators.

[0585] H2bp18c6-benzyl-isopentyl and 225 Chelation of Ac(III)

[0586] (i) low 225 In the Ac / Chelator ratio 225 Chelation with Ac(III)

[0587] Tetramethylammonium acetate (1 M in water, 10 μL), H2bp18c6-benzyl-isopentyl (1.66 mM in water, 2 μL, approx. 3.32 nmol), and in a plastic vial 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi in 0.1 N HCl) was added sequentially. After mixing, the pH was approximately 6.5 according to pH paper. The reaction solution was left to stand at room temperature for 1.5 hours.

[0588] iTLC-SG analysis : 0.5 μL of the reaction solution was spotted onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, bound Ac-225 remains at the baseline of the iTLC-SG, while free Ac-225 migrates with the solvent to the solvent front. No radioactivity was observed at the solvent front of the iTLC-SG, indicating that the chelator 225 This indicates that Ac ions were successfully chelated.

[0589] HPLC analysis 5 μL of the reaction mixture was diluted with 95 μL of PBS buffer. The diluted mixture was analyzed by HPLC. After HPLC, fractions were collected at 1-minute intervals. The collected fractions were left overnight at room temperature and then counted using a gamma counter. HPLC radio-trace was constructed from the activity of the fractions.

[0590] Based on the shift in retention time similar to that shown in the HPLC chromatogram in Fig. 1a, the HPLC analysis225 It was confirmed that an Ac complex was formed.

[0591] DTPA test ( DTPA challenge ): 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA solution, and the mixture was incubated for 30 minutes. 10 μL of the mixture was spotted onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, stably chelated Ac-225 remains at the baseline of the iTLC-SG, while free Ac-225 migrates with the solvent to the solvent front. No radioactivity was observed at the solvent front of the iTLC-SG after the DTPA test, which is 225 This indicates that a stable complex with Ac ions has been formed.

[0592] (ii) high 225 In the Ac / Chelator ratio 225 Chelation with Ac(III)

[0593] In a plastic vial, tetramethylammonium acetate (1 M in water, 10 μL), H2bp18c6-benzyl-isopentyl (0.33 mM in water, 2 μL, approx. 0.66 nmol), and 225 Ac(NO3)3 (10 mCi / mL, 5 μL, 50 μCi in 0.1 N HCl) was added sequentially. After mixing, the pH was approximately 6.5 according to pH paper. The reaction solution was left to stand at room temperature for 1.5 hours. Subsequently, the reaction was analyzed by iTLC-SG and DTPA tests as described above. Glass before or after the DTPA test 225 Ac was not detected, and this 225 This indicates that a stable complex with Ac ions has been formed.

[0594] H2bp18c6-benzyl-phenyl and 225 Chelation of Ac(III)

[0595] (i) low 225 In the Ac / Chelator ratio 225 Chelation with Ac(III)

[0596] Tetramethylammonium acetate (1 M solution in water, 10 μL), H2bp18c6-benzyl-phenyl (1.64 mM in water, 2 μL, approx. 3.28 nmol), and in a plastic vial 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi in 0.1 N HCl) was added sequentially. After mixing, the pH was approximately 6.5 according to pH paper. The reaction solution was left to stand at room temperature for 2 hours. Glass 225 Ac ions were not detected, which means the chelator 225 This indicates that Ac ions were successfully chelated.

[0597] iTLC-SG analysis : 0.5 μL of the reaction solution was spotted onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, bound Ac-225 remains at the baseline of the iTLC-SG, while free Ac-225 migrates with the solvent to the solvent front. No radioactivity was observed at the solvent front of the iTLC-SG, indicating successful chelation of Ac-225.

[0598] HPLC analysis: 5 μL of the reaction mixture was diluted with 95 μL of PBS buffer. The diluted mixture was analyzed by HPLC. After HPLC, fractions were collected at 1-minute intervals. The collected fractions were left overnight at room temperature and then counted using a gamma counter. HPLC radioactive traces were constructed from the activities of the fractions. Based on the shift in residence time similar to that shown in the HPLC chromatogram in Figure 1b, the HPLC analysis 225 It was confirmed that an Ac complex was formed.

[0599] DTPA test 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA solution, and the mixture was incubated for 30 minutes. 10 μL of the mixture was spotted onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, stably chelated Ac-225 remains at the baseline of the iTLC-SG, whereas free Ac-225 migrates with the solvent to the solvent front. Free after the DTPA test 225 Ac was not detected, and this 225 This indicates that a stable complex with Ac ions was formed. No radioactivity was observed in the solvent front of iTLC-SG before or after the DTPA test, which is 225 This indicates that a stable complex with Ac ions has been formed.

[0600] (ii) high 225 In the Ac / Chelator ratio 225 Chelation with Ac(III)

[0601] In a plastic vial, tetramethylammonium acetate (1 M solution in water, 10 μL), H2bp18c6-benzyl-phenyl (0.16 mM in water, 2 μL, approx. 0.33 nmol), and225 Ac(NO3)3 (10 mCi / mL, 5 μL, 50 μCi in 0.1 N HCl) was added sequentially. After mixing, the pH was approximately 6.5 according to pH paper. The reaction solution was left to stand at room temperature for 2 hours. Subsequently, the reaction was analyzed by iTLC-SG and DTPA tests as described above. Glass before or after the DTPA test 225 Ac was not detected, and this 225 This indicates that a stable complex with Ac ions has been formed.

[0602] summation

[0603] In summary, the above results show that H2bp18c6 derivatized from the "benzyl" carbon exhibits rapid chelation kinetics at room temperature. 225 It indicates that Ac is efficiently chelated to form a stable complex. The results also show high specific activity (i.e., chelator versus) through linkage via the "benzyl" position. 225 It indicates that a lower ratio of Ac can be achieved.

[0604] Example 2: H having a DBCO "click" linker 2 Synthesis and Chelation Efficiency of bp18c6 Derivatives

[0605] The following H2bp18c6 derivatives having a DBCO linker for subsequent conjugation to a targeting ligand via a click chemical reaction can be synthesized:

[0606]

[0607] H2bp18c6-Benzyl-Acetate-DBCO H2bp18c6-Benzyl-Phenyl-DBCO

[0608] For example, H2bp18c6-acetate-DBCO can be synthesized according to reaction scheme 3.

[0609] Reaction Equation 3: H 2 bp18c6 - benzyl -acetate- DBCO's synthesis

[0610]

[0611] Methyl 6-bromopicolinate 2- in the presence of a palladium catalyst tert Methyl 6-(2-(tert-butoxy)-2-oxoethyl)picolinate can be provided by reacting with butoxy-2-oxoethyl zinc bromide. Methyl 6-(1-bromo-2-(tert-butoxy)-2-oxoethyl)picolinate can be obtained by subsequent bromination using NBS and AIBN. Under basic reaction conditions, the substitution reaction of methyl 6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinate with methyl 6-(1-bromo-2-(tert-butoxy)-2-oxoethyl)picolinate can produce methyl 6-(2-(tert-butoxy)-1-(16-((6-(methoxycarbonyl)pyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)-2-oxoethyl)picolinate. In the presence of TFA, tert -Butyl esters can be hydrolyzed into carboxylic acids. The formation of an amide bond using dibenzocyclooctine-amine and subsequent hydrolysis of the methyl ester by lithium hydroxide can yield H2bp18c6-benzyl-acetate-DBCO.

[0612] H2bp18c6-benzyl-phenyl-DBCO is synthesized according to reaction scheme 4.

[0613] Reaction Equation 4: H 2 Synthesis of bp18c6-Besyl-phenyl-DBCO

[0614]

[0615] Methyl 6-formylpicolinate (165 mg, 1.0 mmol), (4-( tertTHF (5 mL) was added to a mixture of -butoxycarbonyl)phenyl)boronic acid (444 mg, 2.0 mmol), PdCl2 (8.9 mg, 0.05 mmol), tri(naphthalene-1-yl)phosphane (20.6 mg, 0.05 mmol), and K2CO3 (415 mg, 3.0 mmol) at -78°C under N2. The mixture was purged with N2 and stirred at room temperature for 0.5 hours, then heated at 65°C for 24 hours. The cooled reaction mixture was filtered through Celite, and the filtrate was concentrated. Chromatography on silica gel (heptane to 50% EtOAc in heptane) yielded 116 mg (34% yield) of the product as a yellow oil.

[0616] Methyl 6-((4-( in CH2Cl2(5 mL) tert A solution of -butoxycarbonyl)phenyl)(hydroxy)methyl)picolinate (138 mg, 0.40 mmol), PPh3 (126 mg, 0.48 mmol), and NBS (79 mg, 0.44 mmol) was stirred at room temperature for 1 hour. Additional PPh3 (63 mg, 0.24 mmol) and NBS (39 mg, 0.22 mmol) were added and stirred for an additional 1 hour. The reaction solution was loaded onto a silica gel column. Chromatography (heptane to 30% EtOAc in heptane) yielded 62 mg (38% yield) of the product as a yellowish film adhering to the flask wall.

[0617] Methyl 6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinate (56 mg, 0.14 mmol), methyl 6-(bromo(4-( tertA mixture of -butoxycarbonyl)phenyl)methyl)picolinate (60 mg, 0.15 mmol) and Na2CO3 (72 mg, 0.68 mmol) 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% MeOH in CH2Cl2) yielded 37 mg (37% yield) of the product as a white solid.

[0618] Methyl 6-((4-( in CH2Cl2 (1.5 mL) tert TFA (0.5 mL) was added at room temperature to a solution of -butoxycarbonyl)phenyl)(16-((6-(methoxycarbonyl)pyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinate (22 mg, 0.03 mmol). The solution was stirred for 1 hour. The reaction solution was concentrated to provide an unrefined product as a yellow residue, which was used in the next step of the reaction without further purification.

[0619] Et3N (42 μL, 0.3 mmol) and then HBTU (15 mg, 0.04 mmol) were added to the solution of the above-mentioned unrefined product in CH2Cl2 (0.5 mL) at 0°C. After stirring the solution at 0°C for 5 minutes, dibenzocyclooctine-amine in CH2Cl2 (0.5 mL) was added. The low-temperature bath was removed, and the mixture was stirred at room temperature for 18 hours. Water was added to the reaction mixture, and it was extracted three times with CH2Cl2. The combined extracts were washed with a saturated aqueous solution of NaHCO3 followed by saline, dried (Na2SO4), and filtered. The filtrate was concentrated to yield the unrefined product. Chromatography on silica gel (CH2Cl2 to 10% MeOH in CH2Cl2) yielded 13.2 mg (47% yield) of the product as a colorless film adhering to the flask wall.

[0620] NaOH (1 N, 0.1 mL) was added at room temperature to a solution of dimethyl ester of H2bp18c6-benzyl-phenyl-DBCO (4.8 mg, 0.005 mmol) in THF / MeOH / H2O (4:1:1 v / v / v, 0.6 mL). The reaction mixture was stirred at room temperature for 1 hour, after which it was neutralized with HCl (1 N) to pH = 6.5. The reaction mixture was concentrated at room temperature over a rotary evaporator to remove volatile solvents. The residue was dissolved in H2O (4 mL) and ACN (1 mL). After freeze-drying, it yielded an unrefined product as a white solid.

[0621] H 2 Chelation of bp18c6-benzyl-phenyl-DBCO and La(III)

[0622] A solution of H2bp18c6-benzyl-phenyl-DBCO (about 1.55 mg / mL = about 1.7 mM in 4:1 v / v H2O / ACN, pH about 6.5 according to pH paper) was prepared from the above unrefined product.

[0623] The above solution of H2bp18c6-benzyl-phenyl (approx. 1.55 mg / mL = approx. 1.7 mM, 50 μL, 0.085 μmol) was treated with La(NO3)3 (10 mM, 50 μL, 0.5 μmol in metal-free water). After thorough mixing, the solution was analyzed by LCMS and HPLC. MS (ES, m / z ) 1047 (H2bp18c6-benzyl-phenyl-DBCO + La +3 - 2H + ).

[0624] HPLC method: XBridge C18 3.5 μm 150X4.6 mm, 100 Å column; mobile phase A: 0.1% TFA in H2O, B: 0.1% TFA in ACN; isosolvent with a gradient of 10% to 50% B from 0 to 20 min, a gradient of 50% to 100% B from 20 to 20.1 min, and 100% B from 20.1 to 25 min; isosolvent with a gradient of 100% to 10% B from 25 to 25.1 min, and 10% B from 25.1 to 30 min; flow rate 1 mL / min; column temperature 30℃; injection volume 5 μL.

[0625] H 2 bp18c6-benzyl-phenyl-DBCO and 225 Chelation of Ac(III)

[0626] (i) low 225 In the Ac / Chelator ratio 225 Chelation with Ac(III)

[0627] In a plastic vial, tetramethylammonium acetate (1 M solution in water, 10 μL), H2bp18c6-benzyl-phenyl-DBCO (1.7 mM in H2O / ACN, 2 μL, approx. 3.4 nmol), and 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi in 0.1 N HCl) was added sequentially. After mixing, the pH was approximately 6.5 according to pH paper. The reaction solution was left to stand at room temperature for 1 hour.

[0628] iTLC-SG analysis: 0.5 μL of the reaction solution was spotted onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates along with the solvent to the solvent front. No radioactive activity was observed at the solvent front of the iTLC-SG, indicating that there was no free Ac-225 in the reaction solution after 1 hour.

[0629] HPLC analysis 5 μL of the reaction mixture was diluted with 95 μL of PBS buffer. The diluted mixture was analyzed by HPLC. After HPLC, fractions were collected at 1-minute intervals. The collected fractions were left overnight at room temperature and then counted using a gamma counter. HPLC radiotraces were constructed from the activity of the fractions.

[0630] DTPA test 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA solution, and the mixture was incubated for 30 minutes. 10 μL of the mixture was spotted onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG at room temperature overnight, it was scanned on a Bioscan AR-2000 radio-TLC scanner. No radioactivity was detected in the solvent front of the iTLC-SG.

[0631] (ii) high 225 In the Ac / Chelator ratio 225 Chelation with Ac(III)

[0632] In a plastic vial, tetramethylammonium acetate (1 M solution in water, 10 μL), H2bp18c6-benzyl-phenyl-DBCO (0.17 mM in water, 2 μL, approx. 0.34 nmol), and 225Ac(NO3)3 (10 mCi / mL, 5 μL, 50 μCi in 0.1 N HCl) was added sequentially. After mixing, the pH was approximately 6.5 according to pH paper. The reaction solution was left to stand at room temperature for 2 hours. Subsequently, the reaction was analyzed by iTLC-SG and DTPA tests as described above.

[0633] Example 3: Preparation of H2bp18c6-benzyl-phenyl-DBCO-IgG4 and 225 Ac(III) Signage

[0634] General method for manufacturing radioimmunoconjugates:

[0635] A radioimmunoconjugate containing the radioactive metal complex of the present invention covalently linked to an antibody is produced by click radiolabeling. Refer to FIGS. 2a through 2d for a schematic representation of an exemplary method of click radiolabeling for producing the radioimmunoconjugate of the present invention.

[0636] Random conjugation of azide-handles to antibodies

[0637] A stock solution of the antibody (1 to 10 mg / mL) in 10 mM sodium acetate pH 5.2, phosphate-buffered saline pH 7, or other commercial buffers was mixed with 20% (v / v) of 1 M sodium carbonate buffer pH 9 to achieve a final pH of about 9. NHS-PEG4-azide (Thermo Catalog No. 26130) was dissolved in DMSO to a final concentration of 100 mM, and 0.2% (v / v) of stock was added to produce a molar excess of about 3 to 10 for the antibody (Ab). After incubating the reaction mixture at 22°C for 10 minutes, quenching was performed by adding 1 M Tris pH 7.5 to a final concentration of 50 mM Tris.

[0638] The azide-mAb conjugate was purified using a Zeba desalting column (Thermo) with a 7K MW cutoff; dialysis; standard protein A affinity chromatography; or other compatible methods, and exchanged with compatible buffer (PBS; 20 mM HEPES 150 mM, NaCl pH 7.5; or 10 mM sodium acetate pH 5.2). After purification, the conjugate was concentrated to 10 to 20 mg / mL using an Amicon concentrator (Millipore) with a 50 K MW cutoff. Conjugation efficiency was determined by LC-MS.

[0639] Site-specific incorporation of azido sugars into antibody glycans

[0640] The antibody glycan was trimmed with GlycINATOR (Genovis), a bacterial endoglycosidase specific to the β-1,4 bond between core GlcNac residues at the Fc-glycosylation site(s), leaving the innermost GlcNAc intact on the Fc so that it could subsequently be used for site-specific incorporation of azido sugars. More specifically, GlycINATOR immobilized on agarose beads packed into the column (Genovis) was equilibrated in Tris-buffered saline pH 7.4 (TBS). 1 mL of 5 to 10 mg / mL mAb was added to the resin and incubated on a shaker at RT for 1 hour. The mAb was eluted by spinning at 100 xg for 1 minute. The column was eluted three more times with 0.5 mL of TBS. The eluent containing the trimmed mAb was mixed, and the supplied buffer additive (Genovis) was added along with the UDP-GalNaz azido sugar substrate and the GalT galactosyltransferase enzyme. The reaction mixture was incubated overnight at 30°C. The final azido mAb was purified using an mAb Select column (GE) on an AKTA Avant instrument. Azide modification was confirmed by LC-MS.

[0641] Conjugation of chelators for azido-Ab

[0642] The chelator of the present invention comprising a DBCO group, e.g.:

[0643] and radioactive metal ions, as described in Example 1, for example 225 A radioactive complex is formed by coordination to Ac. Random or site-specific azide-modified antibodies in PBS or other commercial buffer (10 to 20 mg / mL) are added to the solution of the radioactive complex. The reaction solution is gently stirred and allowed to stand at room temperature for 3 hours, after which it is purified using a PD-10 column (GE Healthcare) pre-conditioned with, for example, 15 mL of NaOAc buffer, 10 mM, pH 6 to 6.5, or other commercial buffer. Purity is evaluated by iTLC-SG. The product solution is analyzed by HPLC for chemical and radiochemical purity. The antibody concentration in the product solution is determined by UV absorption using a standard curve. The activity of the product solution is quantified using a Capintec CRC-55TW dosimeter.

[0644] Analytical Characterization of Click-Labeled Radioimmunoconjugates

[0645] Radiochemical conversion (%RA conversion rate) is determined by iTLC-SG (instantaneous thin-layer chromatography (iTLC)). The radiochemical purity (% RA purity) of Ac-225 chelate is determined by SE-HPLC (size exclusion HPLC).

[0646] Direct chelation of 225Ac(III) on H2bp18c6-phenyl-IgG4

[0647] The following method for preparing a radioimmunoconjugate may be referred to as a "one-step direct radiolabeling" method (e.g., as schematically illustrated in FIG. 2c). Radiolabeling was successfully performed under non-metallic conditions, which further exemplifies the ability of the chelator to be resistant to metal contaminants.

[0648] The IgG4 used in these examples is a homologous control that binds to respiratory syncytial virus (RSV) antigens. The amino acid sequences of the heavy chain (HC) and light chain (LC) of IgG4 are provided below as Sequence No. 1 and Sequence No. 2, respectively:

[0649] Sequence No. 1 mAb HC

[0650] QITLKESGPTLVKPTQTLTLTCTFSGFSLTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0651] Sequence No. 2 mAb LC

[0652] DIVMTQSPDSLAVSLGERNATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0653] Scheme 5: Direct chelation of 225Ac(III) to H2bp18c6-phenyl-IgG4 (formed by the click reaction of H2bp18c6-benzyl-phenyl-DBCO to site-specific azide-IgG4)

[0654]

[0655] Azide modification of mAb and click response The above mAb IgG4 was site-selectively modified at 37°C with 100x molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI). The addition of two azides to the heavy chain of the mAb was monitored by intact mass ESI-TOF LC-MS on an Agilent G224 instrument. The excess 3-azidopropylamine and MTG were purified using a 1 mL GE Healthcare MabSelect column. After eluting the azido-mAb from the resin using 100 mM sodium citrate pH 3.0, the solution was exchanged with 20 mM Hepes, 100 mM NaCl pH 7.5 using a 7K Zeba desalting column. A 10x molar excess of H2bp18c6-benzyl-phenyl-DBCO was reacted with site-specific azide-IgG4 (DOL = 2) without shaking for 1 hour at 37°C. The completion of the DBCO-azide click reaction was monitored by circular mass spectrometry. Excess free chelators were removed by a desalting step of the conjugate in 20 mM Hepes, 100 mM NaCl, pH 7.5 on a Zeba 7K desalting column, followed by a concentration step in 20 mM Hepes, 100 mM NaCl, pH 7.5 using a 30K MWCO Amicon concentrator apparatus by three sequential 15x dilutions and spinning at 3800 xg. This yielded a final site-specific H2bp18c6-benzyl-phenyl-DBCO-IgG4 conjugate with a CAR of 2. The final conjugate was confirmed to be monomeric by analytical size exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μ column; the column temperature was room temperature; the column was eluted with DPBS buffer (X1, without calcium and magnesium); the flow rate was 0.7 mL / min and the column was run for 18 minutes; and the injection volume was 18 μL.

[0656] Cover art : In a solution of NaOAc (3 M in H2O, 20 μL) in a plastic vial 225 Ac(NO3)3 (approx. 5 mCi / mL in 0.1 N HCl, 20 μL, 0.098 mCi) and H2bp18c6-benzyl-phenyl-DBCO-IgG4 (site-specific, CAR = 2, 1.7 mg / mL in 20 mM Hepes, 100 mM NaCl pH 7.5, 36 μL, 61.2 μg) were added sequentially. After mixing, the pH was approximately 6.5 as measured by pH paper. The reaction solution was incubated at 37°C for 2 hours. Subsequently, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. No radioactive signal was observed at the solvent front of the iTLC-SG, indicating that all Ac-225 in the reaction solution was completely chelated after 2 hours.

[0657] tabletThe reaction mixture was purified on a PD-10 column: The PD-10 resin was conditioned in the NaOAc buffer solution by passing 5 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluent was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) solution, the wash solution was pipetteed into the PD-10 column reservoir, and the eluent was collected. Each tube contained approximately 1 mL of eluent. Continuous application of NaOAc buffer (10 mM, pH 6 to 6.5) into the PD-10 column reservoir occurred until a total elution volume of 10 mL was reached.

[0658] DTPA test 10 μL of Fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG, developed with 10 mM EDTA, and dried overnight. It was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of the iTLC-SG, indicating that there was no free Ac-225 in Fraction #3.

[0659] HPLC analysisFraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column; column temperature: room temperature; column eluted with DPBS buffer (X1, without calcium and magnesium); flow rate: 0.7 mL / min; 20-minute run; injection volume: 30 μL. After HPLC, fractions were collected at time intervals of 30 seconds or 1 minute. The collected HPLC fractions were left overnight at room temperature. The radioactivity in each collected fraction was counted using a gamma counter. HPLC radiation traces were constructed from the radioactivity in each HPLC fraction. The HPLC radiation traces exhibited radioactive peaks corresponding to the H2bp18c6-benzyl-phenyl-DBCO-IgG4 peak on the HPLC UV trace.

[0660] Example 4: Preparation of H2bp18c6-benzyl-phenyl-DBCO-PSMB127 and 225 Ac(III) Signage

[0661] Reaction Scheme 6. For H2bp18c6-benzyl-phenyl-DBCO-PSMB127 225 Direct chelation of Ac(III) (formed by the click reaction of H2bp18c6-benzyl-phenyl-DBCO to site-specific azide-PSMB127)

[0662]

[0663] Azide modification of mAb and click responsePSMB127 was site-selectively modified with a 100x molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37°C. The addition of two azides to the heavy chain of the mAb was monitored by circular mass ESI-TOF LC-MS on an Agilent G224 instrument. The excess 3-azidopropylamine and MTG were purified using a 1 mL GE Healthcare MabSelect column. After eluting the azido-mAb from the resin using 100 mM sodium citrate pH 3.0, the solution was exchanged with 20 mM Hepes, 100 mM NaCl pH 7.5 using a 7K Zeba desalting column. A 10x molar excess of H2bp18c6-benzyl-phenyl-DBCO was reacted with site-specific azide-PSMB127 (DOL = 2) for 1 hour at 37°C without shaking. The completion of the DBCO-azide click reaction was monitored by circular mass spectrometry. Excess free chelators were removed by a concentration step in 20 mM Hepes, 100 mM NaCl, pH 7.5 using a 30K MWCO Amicon concentrator apparatus, which involved desalting the conjugate on a Zeba 7K desalting column with 20 mM Hepes, 100 mM NaCl, pH 7.5, followed by three sequential 15x dilutions and spinning at 3800 xg. This yielded a final site-specific H2bp18c6-benzyl-phenyl-DBCO-PSMB127 conjugate with CAR 2. The final conjugate was confirmed to be monomeric by analytical size exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 u column; the column temperature was room temperature; the column was eluted with DPBS buffer (X1, without calcium and magnesium); The flow rate was 0.7 mL / min; it was run for 18 minutes; and the injection volume was 18 μL.

[0664] Cover art: In a solution of NaOAc (3 M in H2O, 20 μL) in a plastic vial 225 Ac(NO3)3 (approx. 5 mCi / mL in 0.1 N HCl, 20 μL, 0.098 mCi) and H2bp18c6-benzyl-phenyl-DBCO-PSMB127 (site-specific, CAR = 2, 2.8 mg / mL in 20 mM Hepes, 100 mM NaCl pH 7.5, 22 μL, 61.6 μg) were added sequentially. After mixing, the pH was approximately 6.5 as measured by pH paper. The reaction solution was incubated at 37°C for 2 hours. Subsequently, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. No radioactive signal was observed at the solvent front of the iTLC-SG, indicating that all Ac-225 in the reaction solution was completely chelated after 2 hours.

[0665] tabletThe reaction mixture was purified on a PD-10 column: The PD-10 resin was conditioned in the NaOAc buffer solution by passing 5 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluent was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) solution, the wash solution was pipetteed into the PD-10 column reservoir, and the eluent was collected. Each tube contained approximately 1 mL of eluent. Continuous application of NaOAc buffer (10 mM, pH 6 to 6.5) into the PD-10 column reservoir occurred until a total elution volume of 10 mL was reached.

[0666] DTPA test 10 μL of Fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG, developed with 10 mM EDTA, and dried overnight. It was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of the iTLC-SG, indicating that there was no free Ac-225 in Fraction #3.

[0667] HPLC analysisFraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column; column temperature: room temperature; column eluted with DPBS buffer (X1, without calcium and magnesium); flow rate: 0.7 mL / min; 20-minute run; injection volume: 30 μL. After HPLC, fractions were collected at time intervals of 30 seconds or 1 minute. The collected HPLC fractions were left overnight at room temperature. The radioactivity in each collected fraction was counted using a gamma counter. HPLC radiation traces were constructed from the radioactivity in each HPLC fraction. The HPLC radiation traces exhibited radioactive peaks corresponding to the H2bp18c6-benzyl-phenyl-DBCO-PSMB127 peak on the HPLC UV trace.

[0668] Example 5: Preparation of H2bp18c6-benzyl-phenyl-DBCO-pertuzumab and 225 Ac(III) Signage

[0669] Reaction Scheme 7. For H2bp18c6-benzyl-phenyl-DBCO-pertuzumab 225 Direct chelation of Ac(III) (formed by the click reaction of H2bp18c6-benzyl-phenyl-DBCO to site-specific azid-pertuzumab)

[0670]

[0671] Azide modification of mAb and click responsePertuzumab was site-selectively modified with a 100x molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37°C. The addition of two azides to the heavy chain of the mAb was monitored by round-mass ESI-TOF LC-MS on an Agilent G224 instrument. The excess 3-azidopropylamine and MTG were purified using a 1 mL GE Healthcare MabSelect column. After eluting the azido-mAb from the resin using 100 mM sodium citrate pH 3.0, the solution was exchanged with 1x dPBS using a 7K Zeba desalting column. A 10x molar excess of H2bp18c6-benzyl-phenyl-DBCO was reacted with site-specific azide-pertuzumab (DOL = 2) in 1x dPBS for 1 hour at 37°C without shaking. The completion of the DBCO-azide click reaction was monitored by circular mass spectrometry. Excess free chelators were removed by a concentration step in PBS using a 30K MWCO Amicon concentrator device, consisting of three sequential 15x dilutions and rotation at 3800 xg, following a step of desalting the conjugate with 1x dPBS on a Zeba 7K desalting column. This yielded a final site-specific H2bp18c6-benzyl-phenyl-DBCO-pertuzumab conjugate with CAR 2. The final conjugate was confirmed to be monomeric by analytical size exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μ column; the column temperature was room temperature; the column was eluted with DPBS buffer (X1, without calcium and magnesium); the flow rate was 0.7 mL / min; the run was 18 minutes; and the injection volume was 18 μL.

[0672] Cover art : In a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial 225Ac(NO3)3 (about 5 mCi / mL in 0.1 N HCl, 10 μL, 0.042 mCi) and H2bp18c6-benzyl-phenyl-DBCO-pertuzumab (site-specific, CAR = 2, 2.4 mg / mL in PBS buffer, 12.5 μL, 30 μg) were added sequentially. After mixing, the pH was about 6.5 as measured by pH paper. The reaction solution was incubated at 37°C for 2 hours. Subsequently, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. No radioactive signal was observed in the solvent front of iTLC-SG, which indicates that all Ac-225 in the reaction solution was completely chelated after 2 hours.

[0673] tablet The reaction mixture was purified on a PD-10 column: The PD-10 resin was conditioned in the NaOAc buffer solution by passing 5 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluent was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) solution, the wash solution was pipetteed into the PD-10 column reservoir, and the eluent was collected. Each tube contained approximately 1 mL of eluent. Continuous application of NaOAc buffer (10 mM, pH 6 to 6.5) into the PD-10 column reservoir occurred until a total elution volume of 10 mL was reached.

[0674] DTPA test10 μL of Fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG, developed with 10 mM EDTA, and dried overnight. It was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of the iTLC-SG, indicating that there was no free Ac-225 in Fraction #3.

[0675] HPLC analysis Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column; column temperature: room temperature; column eluted with DPBS buffer (X1, without calcium and magnesium); flow rate: 0.7 mL / min; 20-minute run; injection volume: 30 μL. After HPLC, fractions were collected at time intervals of 30 seconds or 1 minute. The collected HPLC fractions were left overnight at room temperature. The radioactivity in each collected fraction was counted using a gamma counter. HPLC radiation traces were constructed from the radioactivity in each HPLC fraction. The HPLC radiation traces exhibited radioactive peaks corresponding to the H2bp18c6-benzyl-phenyl-DBCO-pertuzumab peak on the HPLC UV trace.

[0676] Example 6: Preparation of H2bp18c6-benzyl-phenyl-DBCO-cetuximab and 225 Ac(III) Signage

[0677] Reaction Equation 8. For H2bp18c6-benzyl-phenyl-DBCO-cetuximab 225 Direct chelation of Ac(III) (formed by the click reaction of H2bp18c6-benzyl-phenyl-DBCO to site-specific azid-cetuximab)

[0678]

[0679] Azide modification of mAb and click responseCetuximab was site-selectively modified with a 100x molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37°C. The addition of two azides to the heavy chain of the mAb was monitored by round-mass ESI-TOF LC-MS on an Agilent G224 instrument. The excess 3-azidopropylamine and MTG were purified using a 1 mL GE Healthcare MabSelect column. After eluting the azido-mAb from the resin using 100 mM sodium citrate pH 3.0, the solution was exchanged with 1x dPBS using a 7K Zeba desalting column. A 10x molar excess of H2bp18c6-benzyl-phenyl-DBCO was reacted with site-specific azide-cetuximab (DOL = 2) in 1x dPBS for 1 hour at 37°C without shaking. The completion of the DBCO-azide click reaction was monitored by circular mass spectrometry. Excess free chelators were removed by a concentration step in PBS using a 30K MWCO Amicon concentrator device, consisting of three sequential 15x dilutions and spinning at 3800 xg, following a step of desalting the conjugate with 1x dPBS on a Zeba 7K desalting column. This yielded a final site-specific H2bp18c6-benzyl-phenyl-DBCO-cetuximab conjugate with CAR 2. The final conjugate was confirmed to be monomeric by analytical size exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μ column; the column temperature was room temperature; the column was eluted with DPBS buffer (X1, without calcium and magnesium); the flow rate was 0.7 mL / min; the column was run for 18 minutes; and the injection volume was 18 μL.

[0680] Cover art : In a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial 225Ac(NO3)3 (approx. 5 mCi / mL in 0.1 N HCl, 10 μL, 0.044 mCi) and H2bp18c6-benzyl-phenyl-DBCO-cetuximab (site-specific, CAR = 2, 1.8 mg / mL in PBS buffer, 16.7 μL, 30 μg) were added sequentially. After mixing, the pH was approximately 6.5 as measured by pH paper. The reaction solution was incubated at 37°C for 2 hours. Subsequently, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. No radioactive signal was observed in the solvent front of iTLC-SG, which indicates that all Ac-225 in the reaction solution was completely chelated after 2 hours.

[0681] tablet The reaction mixture was purified on a PD-10 column: The PD-10 resin was conditioned in the NaOAc buffer solution by passing 5 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluent was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) solution, the wash solution was pipetteed into the PD-10 column reservoir, and the eluent was collected. Each tube contained approximately 1 mL of eluent. Continuous application of NaOAc buffer (10 mM, pH 6 to 6.5) into the PD-10 column reservoir occurred until a total elution volume of 10 mL was reached.

[0682] DTPA test10 μL of Fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG, developed with 10 mM EDTA, and dried overnight. It was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of the iTLC-SG, indicating that there was no free Ac-225 in Fraction #3.

[0683] HPLC analysis Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column; column temperature: room temperature; column eluted with DPBS buffer (X1, without calcium and magnesium); flow rate: 0.7 mL / min; 20-minute run; injection volume: 30 μL. After HPLC, fractions were collected at time intervals of 30 seconds or 1 minute. The collected HPLC fractions were left overnight at room temperature. The radioactivity in each collected fraction was counted using a gamma counter. HPLC radiation traces were constructed from the radioactivity in each HPLC fraction. The HPLC radiation traces exhibited radioactive peaks corresponding to the H2bp18c6-benzyl-phenyl-DBCO-cetuximab peak on the HPLC UV trace.

[0684] Example 7: Preparation of H2bp18c6-benzyl-phenyl-DBCO-panitumumab and 225 Ac(III) Signage

[0685] Reaction Scheme 9. For H2bp18c6-benzyl-phenyl-DBCO-panitumumab 225 Direct chelation of Ac(III) (formed by the click reaction of H2bp18c6-benzyl-phenyl-DBCO to site-specific azide-panitumumab)

[0686]

[0687] Azide modification of mAb and click responsePanitumumab was site-selectively modified with a 100x molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37°C. The addition of two azides to the heavy chain of the mAb was monitored by round-mass ESI-TOF LC-MS on an Agilent G224 instrument. The excess 3-azidopropylamine and MTG were purified using a 1 mL GE Healthcare MabSelect column. After eluting the azido-mAb from the resin using 100 mM sodium citrate pH 3.0, the solution was exchanged with 1x dPBS using a 7K Zeba desalting column. A 10x molar excess of H2bp18c6-benzyl-phenyl-DBCO was reacted with site-specific azide-panitumumab (DOL = 2) in 1x dPBS for 1 hour at 37°C without shaking. The completion of the DBCO-azide click reaction was monitored by circular mass spectrometry. Excess free chelators were removed by a concentration step in PBS using a 30K MWCO Amicon concentrator device, consisting of three sequential 15x dilutions and rotation at 3800 xg, following a step of desalting the conjugate with 1x dPBS on a Zeba 7K desalting column. This yielded a final site-specific H2bp18c6-benzyl-phenyl-DBCO-panitumumab conjugate with CAR 2. The final conjugate was confirmed to be monomeric by analytical size exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μ column; the column temperature was room temperature; the column was eluted with DPBS buffer (X1, without calcium and magnesium); the flow rate was 0.7 mL / min; the run was 18 minutes; and the injection volume was 18 μL.

[0688] Cover art : In a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial 225Ac(NO3)3 (approx. 5 mCi / mL in 0.1 N HCl, 10 μL, 0.043 mCi) and H2bp18c6-benzyl-phenyl-DBCO-panitumumab (site-specific, CAR = 2, 2.6 mg / mL in PBS buffer, 11.5 μL, 30 μg) were added sequentially. After mixing, the pH was approximately 6.5 as measured by pH paper. The reaction solution was incubated at 37°C for 2 hours. Subsequently, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. No radioactive signal was observed in the solvent front of iTLC-SG, which indicates that all Ac-225 in the reaction solution was completely chelated after 2 hours.

[0689] tablet The reaction mixture was purified on a PD-10 column: The PD-10 resin was conditioned in the NaOAc buffer solution by passing 5 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluent was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) solution, the wash solution was pipetteed into the PD-10 column reservoir, and the eluent was collected. Each tube contained approximately 1 mL of eluent. Continuous application of NaOAc buffer (10 mM, pH 6 to 6.5) into the PD-10 column reservoir occurred until a total elution volume of 10 mL was reached.

[0690] DTPA test10 μL of Fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG, developed with 10 mM EDTA, and dried overnight. It was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of the iTLC-SG, indicating that there was no free Ac-225 in Fraction #3.

[0691] HPLC analysis Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column; column temperature: room temperature; column eluted with DPBS buffer (X1, without calcium and magnesium); flow rate: 0.7 mL / min; 20-minute run; injection volume: 30 μL. After HPLC, fractions were collected at time intervals of 30 seconds or 1 minute. The collected HPLC fractions were left overnight at room temperature. The radioactivity in each collected fraction was counted using a gamma counter. HPLC radiation traces were constructed from the radioactivity in each HPLC fraction. The HPLC radiation traces exhibited radioactive peaks corresponding to the H2bp18c6-benzyl-phenyl-DBCO-panitumumab peak on the HPLC UV trace.

[0692] Example 8: Preparation of H2bp18c6-benzyl-phenyl-DBCO-Herceptin and 225 Ac(III) Signage

[0693] Reaction Scheme 10. For H2bp18c6-benzyl-phenyl-DBCO-Herceptin 225 Direct chelation of Ac(III) (formed by the click reaction of H2bp18c6-benzyl-phenyl-DBCO to site-specific azide-Herceptin)

[0694]

[0695] Azide modification of mAb and click responseHerceptin was site-selectively modified with a 100x molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37°C. The addition of two azides to the heavy chain of the mAb was monitored by circular mass ESI-TOF LC-MS on an Agilent G224 instrument. The excess 3-azidopropylamine and MTG were purified using a 1 mL GE Healthcare MabSelect column. After eluting the azido-mAb from the resin using 100 mM sodium citrate pH 3.0, the solution was exchanged with 1x dPBS using a 7K Zeba desalting column. A 10x molar excess of H2bp18c6-benzyl-phenyl-DBCO was reacted with site-specific azide-Herceptin (DOL = 2) in 1x dPBS for 1 hour at 37°C without shaking. The completion of the DBCO-azide click reaction was monitored by circular mass spectrometry. Excess free chelators were removed by a concentration step in PBS using a 30K MWCO Amicon concentrator device, consisting of three sequential 15x dilutions and rotation at 3800 xg, following a step of desalting the conjugate with 1x dPBS on a Zeba 7K desalting column. This yielded a final site-specific H2bp18c6-benzyl-phenyl-DBCO-Herceptin conjugate with CAR 2. The final conjugate was confirmed to be monomeric by analytical size exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μ column; the column temperature was room temperature; the column was eluted with DPBS buffer (X1, without calcium and magnesium); the flow rate was 0.7 mL / min; the column was run for 18 minutes; and the injection volume was 18 μL.

[0696] Cover art : In a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial 225Ac(NO3)3 (about 5 mCi / mL in 0.1 N HCl, 10 μL, 0.041 mCi) and H2bp18c6-benzyl-phenyl-DBCO-Herceptin (site-specific, CAR = 2, 1.7 mg / mL in PBS buffer, 17.6 μL, 30 μg) were added sequentially. After mixing, the pH was about 6.5 as measured by pH paper. The reaction solution was incubated at 37°C for 2 hours. Subsequently, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. No radioactive signal was observed in the solvent front of iTLC-SG, which indicates that all Ac-225 in the reaction solution was completely chelated after 2 hours.

[0697] tablet The reaction mixture was purified on a PD-10 column: The PD-10 resin was conditioned in the NaOAc buffer solution by passing 5 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluent was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) solution, the wash solution was pipetteed into the PD-10 column reservoir, and the eluent was collected. Each tube contained approximately 1 mL of eluent. Continuous application of NaOAc buffer (10 mM, pH 6 to 6.5) into the PD-10 column reservoir occurred until a total elution volume of 10 mL was reached.

[0698] DTPA test10 μL of Fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG, developed with 10 mM EDTA, and dried overnight. It was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of the iTLC-SG, indicating that there was no free Ac-225 in Fraction #3.

[0699] HPLC analysis Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column; column temperature: room temperature; column eluted with DPBS buffer (X1, without calcium and magnesium); flow rate: 0.7 mL / min; 20-minute run; injection volume: 30 μL. After HPLC, fractions were collected at time intervals of 30 seconds or 1 minute. The collected HPLC fractions were left overnight at room temperature. The radioactivity in each collected fraction was counted using a gamma counter. HPLC radiation traces were constructed from the radioactivity in each HPLC fraction. The HPLC radiation traces exhibited radioactive peaks corresponding to the H2bp18c6-benzyl-phenyl-DBCO-Herceptin peak on the HPLC UV trace.

[0700] Example 9: Preparation of H2bp18c6-Benzyl-Phenyl-DBCO-H11B6 and 225 Ac(III) Signage

[0701] Reaction Scheme 11. For H2bp18c6-benzyl-phenyl-DBCO-H11B6 225 Direct chelation of Ac(III) (formed by the click reaction of H2bp18c6-benzyl-phenyl-DBCO to site-specific azide-H11B6)

[0702]

[0703] Azide modification of mAb and click responseH11B6 was site-selectively modified with a 100x molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37°C. The addition of two azides to the heavy chain of the mAb was monitored by circular mass ESI-TOF LC-MS on an Agilent G224 instrument. The excess 3-azidopropylamine and MTG were purified using a 1 mL GE Healthcare MabSelect column. After eluting the azido-mAb from the resin using 100 mM sodium citrate pH 3.0, the solution was exchanged with 1x dPBS using a 7K Zeba desalting column. A 10x molar excess of H2bp18c6-benzyl-phenyl-DBCO was reacted with site-specific azide-H11B6 (DOL = 1.82) in 1x dPBS for 1 hour at 37°C without shaking. The completion of the DBCO-azide click reaction was monitored by circular mass spectrometry. Excess free chelators were removed by a concentration step in PBS using a 30K MWCO Amicon concentrator, consisting of three sequential 15x dilutions and rotation at 3800 xg, following a step of desalting the conjugate with 1x dPBS on a Zeba 7K desalting column. This yielded a final site-specific H2bp18c6-benzyl-phenyl-DBCO-H11B6 conjugate with a CAR of 1.82. The final conjugate was confirmed to be monomeric by analytical size exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 u column; the column temperature was room temperature; the column was eluted with DPBS buffer (X1, without calcium and magnesium); the flow rate was 0.7 mL / min; and the column was run for 18 minutes; The injection volume was 18 μL.

[0704] Cover art : In a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial 225Ac(NO3)3 (approx. 5 mCi / mL in 0.1 N HCl, 10 μL, 0.043 mCi) and H2bp18c6-phenyl-DBCO-H11B6 (site-specific, CAR = 1.82, 1.2 mg / mL in PBS buffer, 25.0 μL, 30 μg) were added sequentially. After mixing, the pH was approximately 6.5 as measured by pH paper. The reaction solution was incubated at 37°C for 2 hours. Subsequently, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. No radioactive signal was observed in the solvent front of iTLC-SG, which indicates that all Ac-225 in the reaction solution was completely chelated after 2 hours.

[0705] tablet The reaction mixture was purified on a PD-10 column: The PD-10 resin was conditioned in the NaOAc buffer solution by passing 5 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluent was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) solution, the wash solution was pipetteed into the PD-10 column reservoir, and the eluent was collected. Each tube contained approximately 1 mL of eluent. Continuous application of NaOAc buffer (10 mM, pH 6 to 6.5) into the PD-10 column reservoir occurred until a total elution volume of 10 mL was reached.

[0706] DTPA test10 μL of Fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG, developed with 10 mM EDTA, and dried overnight. It was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of the iTLC-SG, indicating that there was no free Ac-225 in Fraction #3.

[0707] HPLC analysis Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column; column temperature: room temperature; column eluted with DPBS buffer (X1, without calcium and magnesium); flow rate: 0.7 mL / min; 20-minute run; injection volume: 30 μL. After HPLC, fractions were collected at time intervals of 30 seconds or 1 minute. The collected HPLC fractions were left overnight at room temperature. The radioactivity in each collected fraction was counted using a gamma counter. HPLC radiation traces were constructed from the radioactivity in each HPLC fraction. The HPLC radiation traces exhibited radioactive peaks corresponding to the H2bp18c6-benzyl-phenyl-DBCO-H11B6 peak on the HPLC UV trace.

[0708] Example 10: Synthesis of H2bp18c6-phenyl-BCN

[0709] Reaction Equation 12.

[0710]

[0711] HATU (37 mg, 0.10 mmol) was added at 0°C to a solution of 4-((6-(methoxycarbonyl)pyridine-2-yl)(16-((6-(methoxycarbonyl)pyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)benzoic acid (50 mg, 0.073 mmol) and Et3N (0.1 mL, 0.73 mmol) in CH2Cl2 (2.5 mL). After stirring the solution at 0°C for 5 minutes, HATU in CH2Cl2 (1 mL) N -[(1 R ,8 S ,9 s )-bicyclo[6.1.0]non-4-phosphor-9-ylmethyloxycarbonyl]-1,8-diamino-3,6-dioxaoctane was added. The low-temperature bath was removed, and the mixture was stirred at room temperature for 18 hours. The mixture was concentrated, and the residue was purified by chromatography on amine-functionalized silica gel (CH2Cl2 to 10% MeOH in CH2Cl2) to yield 19.8 mg (27% yield) of the product as a colorless film adhering to the flask wall. LC-MS analysis was 987.6 [M+H + Mass / ion peaks were shown in ]

[0712] Methyl 6-((4-((1-((1 in THF / MeOH / H2O (4:1:1 v / v / v, 1.8 mL) R ,8 SLiOH (1 N, 0.3 mL) was added at room temperature to a solution of ,9s)-bicyclo[6.1.0]non-4-in-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecane-12-yl)carbamoyl)phenyl)(16-((6-(methoxycarbonyl)pyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinate (14.5 mg, 0.015 mmol). The reaction mixture was stirred at room temperature for 1 hour, after which it was neutralized to pH = 6.5 with HCl (1 N). The reaction mixture was concentrated under vacuum. The residue was purified by chromatography on amine-functionalized silica gel (CH2Cl2 to 10% MeOH in CH2Cl2) to yield 9.5 mg (48% yield) of H2bp18c6-benzyl-phenyl-BCN as a colorless film, which was dissolved in H2O (4 mL) and ACN (1 mL). After freeze-drying, the product yielded a white solid. H NMR (CD3OD, 400 MHz) δ 7.91 (d, J = 8 Hz, 2H), 7.84 (t, J = 8 Hz, 2H), 7.81 (d, J = 8 Hz, 2H), 7.60 (d, J = 8 Hz, 2H), 7.53 (d, J = 8 Hz, 1H), 7.46 (d, J = 8 Hz, 1H), 5.24 (s, 1H), 4.10 (d, J = 8 Hz, 2H), 3.91 (brs, 2H), 3.75-3.58 (m, 22H), 3.56 (t, J = 4 Hz, 2H), 3.51 (t, J = 4 Hz, 2H), 3.23 (t, J = 4 Hz, 2H), 2.97 (dt, J = 8, 4 Hz, 2H), 2.92-2.82 (m, 4H), 2.78 (dt, J= 8, 4 m², 2H), 2.28-2.08 (m, 6H), 1.65-1.50 (m, 2H), 1.39-1.27 (m, 1H), 0.96-0.84 (m, 2H). MS (ESI) 981.4 [M+Na + ].

[0713] Example 11: Preparation of H2bp18c6-Benzyl-Phenyl-BCN-PSMB127 and 225 Ac(III) Signage

[0714] Reaction Scheme 13. For H2bp18c6-benzyl-phenyl-BCN-PSMB127 225 Direct chelation of Ac(III) (formed by the click reaction of H2bp18c6-benzyl-phenyl-BCN to site-specific azide-PSMB127)

[0715]

[0716] Azide modification of mAb and click responsePSMB127 was site-selectively modified with a 100x molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37°C. The addition of two azides to the heavy chain of the mAb was monitored by circular mass ESI-TOF LC-MS on an Agilent G224 instrument. The excess 3-azidopropylamine and MTG were purified using a 1 mL GE Healthcare MabSelect column. After eluting the azido-mAb from the resin using 100 mM sodium citrate pH 3.0, the solution was exchanged with 1x dPBS using a 7K Zeba desalting column. A 10x molar excess of H2bp18c6-benzyl-phenyl-BCN was reacted with site-specific azide-PSMB127 (DOL = 2) in 1x dPBS for 1 hour at 37°C without shaking. The completion of the BCN-azide click reaction was monitored by circular mass spectrometry. Excess free chelators were removed by a concentration step in PBS using a 30K MWCO Amicon concentrator device, consisting of three sequential 15x dilutions and rotation at 3800 xg, following a step of desalting the conjugate with 1x dPBS on a Zeba 7K desalting column. This yielded a final site-specific H2bp18c6-benzyl-phenyl-BCN-PSMB127 conjugate with CAR 2. The final conjugate was confirmed to be monomeric by analytical size exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μ column; the column temperature was room temperature; the column was eluted with DPBS buffer (X1, without calcium and magnesium); the flow rate was 0.7 mL / min; the column was run for 18 minutes; and the injection volume was 18 μL.

[0717] Cover art : In a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial 225Ac(NO3)3 (about 5 mCi / mL in 0.1 N HCl, 10 μL, 0.046 mCi) and H2bp18c6-benzyl-phenyl-BCN-PSMB127 (site-specific, CAR = 2, 2.3 mg / mL in PBS buffer, 13.0 μL, 30 μg) were added sequentially. After mixing, the pH was about 6.5 as measured by pH paper. The reaction solution was incubated at 37°C for 2 hours. Subsequently, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. No radioactive signal was observed in the solvent front of iTLC-SG, which indicates that all Ac-225 in the reaction solution was completely chelated after 2 hours.

[0718] tablet The reaction mixture was purified on a PD-10 column: The PD-10 resin was conditioned in the NaOAc buffer solution by passing 5 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluent was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) solution, the wash solution was pipetteed into the PD-10 column reservoir, and the eluent was collected. Each tube contained approximately 1 mL of eluent. Continuous application of NaOAc buffer (10 mM, pH 6 to 6.5) into the PD-10 column reservoir occurred until a total elution volume of 10 mL was reached.

[0719] DTPA test10 μL of Fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG, developed with 10 mM EDTA, and dried overnight. It was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of the iTLC-SG, indicating that there was no free Ac-225 in Fraction #3.

[0720] HPLC analysis Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column; column temperature: room temperature; column eluted with DPBS buffer (X1, without calcium and magnesium); flow rate: 0.7 mL / min; 20-minute run; injection volume: 40 μL. After HPLC, fractions were collected at time intervals of 30 seconds or 1 minute. The collected HPLC fractions were left overnight at room temperature. The radioactivity in each collected fraction was counted using a gamma counter. HPLC radiation traces were constructed from the radioactivity in each HPLC fraction. The HPLC radiation traces exhibited radioactive peaks corresponding to the H2bp18c6-benzyl-phenyl-BCN-PSMB127 peak on the HPLC UV trace.

[0721] Example 12: Synthesis of H2bp18c6-off-macrocycle-ethyl sulfide-DBCO

[0722] H2bp18c6 derivatives substituted at carbon atoms of a macrocyclic ring having a linker for conjugation to a targeting ligand are generally synthesized according to the following Reaction Schemes 14a and 14b:

[0723] Scheme 14a: H substituted at the carbon atom of a macrocyclic ring having a linker for conjugation 2 Synthesis of bp18c6 ring derivatives

[0724]

[0725] The synthesis of diazah-18-crown-6 substituted with -CH2OR1 is [in the literature Org. Lett . 2005 , 7 It is synthesized using the procedure described in [6] 1105-1108, and subsequently reacted with a derivative of 6-(halomethyl)picolinic acid as described above for Reaction Schemes 3 and 4. The functional group OR1 may be converted to another functional group WR2 for ligation or conjugation to a linker, targeting ligand, etc., including but not limited to NH2, N3, aldehyde, carboxylate, alkyne, etc., in the presence or absence of a linker W, which is a heteroatom, alkyl, alkyl having heteroatom(s), substituted aryl, etc. In Reaction Scheme 6a, X is a leaving group, e.g., halo, mesylate, tosylate, etc.; R1 is an allyl, benzyl, alcohol, etc.; W is absent or a linker, e.g., heteroatom, alkyl, heteroalkyl, substituted aryl, etc.; R2 is NH2, N3 aldehyde, carboxylate, alkynyl, etc.

[0726] Scheme 14b: H substituted at the carbon atom of a macrocyclic ring having a linker for conjugation 2 Synthesis of bp18c6 ring derivatives

[0727]

[0728] Diaza-18-crown-6 substituted with -CH2OR1 is also [ Journal of Organic Chemistry , 1988, 53 (14), 3190-5] and literature[ Journal of Heterocyclic Chemistry , 1986, 23It can be synthesized using the procedure described in [2], 609-13, and subsequently reacted with a derivative of 6-(halomethyl)picolinic acid as described above for Reaction Schemes 3 and 4. The functional group OR1 can be converted to another functional group R2 for ligation or conjugation to a linker, targeting ligand, etc., including but not limited to NH2, N3, aldehyde, carboxylate, alkyne, etc., in the presence or absence of a linker W, which is a heteroatom, alkyl, alkyl having heteroatom(s), substituted aryl, etc.

[0729] Next, the synthesis of H2bp18c6-off-macrocycle-ethyl sulfide-DBCO is described.

[0730] Reaction Equation 15.

[0731]

[0732] (in anhydrous DMF (500 mL) S Sodium hydride (18.16 g, 60% of the mineral oil, 454 mmol) was added to a solution of )-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol (50 g, 378 mmol) at 0°C. After stirring for 10 minutes at this temperature, benzyl bromide (77.63 g, 454 mmol) was slowly added to the suspension. After 30 minutes, the reaction mixture was allowed to reach room temperature and stirred for an additional 8 hours. The reaction mixture was quenched with a saturated ammonium chloride solution and extracted with dichloromethane (2 x 500 mL). The combined organic layer was washed with brine and dried over sodium sulfate. After removing the solvent, the residue is purified by flash column chromatography using petroleum ether and ethyl acetate (silica gel, 230 to 400 mesh) ( S )-4-((benzyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (72 g, 86%) was obtained as a colorless liquid.

[0733] (in THF (100 mL) S After adding 1.5 N HCl (100 mL) to a solution of )-4-((benzyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (72 g, 324 mmol), the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with ethyl acetate (500 mL) and neutralized with a 10% sodium bicarbonate solution. The reaction mixture was extracted with ethyl acetate (2 x 500 mL), washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 230 to 400 mesh) using petroleum ether and ethyl acetate ( R )-3-(benzyloxy)propane-1,2-diol (51 g, 85%) was obtained as a colorless oil.

[0734] In a suspension of sodium hydride (26.29 g, 60% in mineral oil, 686 mmol) in DMF (20 mL), (in DMF (100 mL) R )-3-(benzyloxy)propane-1,2-diol (25.0 g, 137 mmol) was added dropwise at 0°C. The reaction mixture was stirred at room temperature for 2 hours. It was then cooled back to 0°C, and 2-(2-bromoethoxy)tetrahydro-2 in DMF (100 mL) H Pyran (85.89 g, 411 mmol) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred for 16 hours. The reaction was quenched with saturated ammonium chloride and extracted with ethyl acetate (3 x 300 mL). The combined organic layer was washed with water, dried over sodium sulfate, filtered, and concentrated. The unrefined oil was purified by silica gel (230 to 400 mesh) flash chromatography using ethyl acetate (0 to 40%) in petroleum ether to obtain 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 provided as a colorless liquid.

[0735] 2,2'-((((( in 500 mL of methanol R 5 mL of HCl in dioxane was added to a solution of )-3-(benzyloxy)propane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(tetrahydro-2H-pyran) (32 g, 73.05 mmol). The reaction mixture was stirred under reflux for 1 hour, cooled, and evaporated. Unpurified product ( R )-2,2'-((3-(benzyloxy)propane-1,2-diyl)bis(oxy))bis(ethanol-1-ol)(20 g) was used in the next step without purification.

[0736] (in dichloromethane (250 mL) and triethylamine (53 mL, 370 mmol) R Solid in a solution of )-2,2'-((3-(benzyloxy)propane-1,2-diyl)bis(oxy))bis(ethanol-1-ol) (20 g, 74.07 mmol) p -Toluenesulfonyl chloride (42.2 g, 222 mmol) was added in portions at 10°C. The mixture was stirred at ambient temperature for 16 hours. After the reaction was complete, the suspension was diluted with 1000 mL of dichloromethane, washed with 3 x 100 mL of cold 1 M HCl, followed by 2 x 500 mL of ice-cold water, dried over sodium sulfate, and evaporated to yield a solid gum. The unrefined material was purified by flash chromatography (silica gel, 230 to 400 mesh) using ethyl acetate (0 to 40%) in petroleum ether ( R )-((3-(benzyloxy)propane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (30 g, 70%) was provided as a colorless liquid.

[0737] (in 200 mL of dry DMF) R A mixture of )-((3-(benzyloxy)propane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (30 g, 51.9 mmol) and cesium carbonate (50.76 g, 155.7 mmol) was stirred at ambient temperature for 1.5 hours. In 200 mL of DMF N , N' -((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(4-methylbenzenesulfonamide) (23.56 g, 51.9 mmol) was added dropwise to the suspension over a period of 2 hours. The mixture was stirred at ambient temperature for an additional 20 hours. The solvent was removed under reduced pressure to obtain a solid paste. This was suspended in 1000 mL of dichloromethane and stirred for 30 minutes. The precipitated solid was filtered, and the filtrate was evaporated under high vacuum. The unrefined material was purified by flash chromatography (silica gel, 230 to 400 mesh) using ethyl acetate (0 to 40%) in petroleum ether ( R )-2-((benzyloxy)methyl)-7,16-diatosyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (24 g, 67%) was provided as a colorless liquid.

[0738] (in hydrobromide (50%, 100 mL) in acetic acid R Phenol (16.35 g, 174 mmol) was added at room temperature to a solution of )-2-((benzyloxy)methyl)-7,16-ditosyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (24 g, 34.78 mmol). The reaction mixture was heated at 60°C for 6 hours. After the reaction was complete, it was cooled to room temperature, and acetic acid was removed under high vacuum. The unrefined product was purified on a reverse-phase column using 0 to 100% acetonitrile (0.1% TFA) in water ( S)-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-2-yl)methyl acetate (8.0 g, 69%) was provided as a colorless liquid.

[0739] (in dry acetonitrile (100 mL) S A suspension of )-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-2-yl)methyl acetate (8.0 g, 23.95 mmol), methyl 6-(chloromethyl)picolinate (11.07 g, 59.88 mmol), and sodium carbonate (12.69 g, 119.75 mmol) was heated at 90°C for 16 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. Unpurified material is purified by flash chromatography (silica gel, 230 to 400 mesh) using methanol (0 to 10%) in dichloromethane to obtain dimethyl 6,6'-((2-(acetoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))( S )-dipicolinate (5.0 g, 33%) was provided as a brown liquid.

[0740] Dimethyl 6,6'-((2-(acetoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))( in methanol (50 mL) S Potassium carbonate (0.11 g, 0.79 mmol) was added to a solution of )-dipicolinate (5.0 g, 7.91 mmol) at room temperature and stirred for 10 minutes. After the reaction was completed, it was concentrated under reduced pressure. The residue was purified by flash chromatography on silica (230-400 mesh) eluted with a 0 to 10% methanol gradient in dichloromethane to obtain dimethyl 6,6'-((2-(hydroxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))( R)-dipicolinate (3.5 g, 75%) was provided as a brown liquid.

[0741] Dimethyl 6,6'-((2-(hydroxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))( in dichloromethane (20 mL) R Triethylamine (0.51 g, 0.70 mL, 5.1 mmol) was added to a solution of )-dipicolinate (1.0 g, 1.7 mmol). Methyl chloride (0.39 g, 0.26 mL, 3.4 mmol) was added dropwise to this solution at 0°C. The reaction mixture was stirred at room temperature for 30 minutes. The progress of the reaction was monitored by TLC. After the completion of the reaction, the product was concentrated, and the unrefined material was purified by column chromatography (alumina-neutral) using methanol (1 to 2%) in dichloromethane as the eluent to obtain dimethyl 6,6'-((2-(((methylsulfonyl)oxy)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))( S )-dipicolinate (0.7 g, 62%) was provided as a brown liquid.

[0742] in DMF (2 mL) tert Sodium hydride (12 mg, 60% of mineral oil, 0.3 mmol) was added to a solution of butyl (2-mercaptoethyl)carbamate (53 mg, 0.3 mmol) at 0°C. The reaction mixture was stirred at room temperature for 10 minutes. To the reaction mixture, dimethyl 6,6'-((2-(((methylsulfonyl)oxy)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))( in DMF (1 mL) S)-dipicolinate (100 mg, 0.15 mmol) was added at 0°C. The reaction mixture was stirred at room temperature for 2 hours. The progress of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was quenched with a saturated ammonium chloride solution and extracted with ethyl acetate (3 x 5 mL). The combined organic layer was washed with water and brine, dried over anhydrous sodium sulfate, and concentrated. The unrefined product was purified by preparative HPLC using acetonitrile (0.1% TFA) in water to obtain dimethyl 6,6'-((2-(((2-(( tert -Butoxycarbonyl)amino)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazcyclooctadecane-7,16-diyl)bis(methylene))( S )-dipicolinate (20 mg, 18%) was provided as a brown liquid.

[0743] Dimethyl 6,6'-((2-(((2-(( tert -Butoxycarbonyl)amino)ethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazcyclooctadecane-7,16-diyl)bis(methylene))( S A low-temperature solution of HCl in methanol (2 mL, 4 N) was added to )-dipicolinate (100 mg, 0.15 mmol), and the solution was stirred for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain dimethyl 6,6'-((2-(((2-aminoethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))( S )-dipicolinate was obtained as a yellow liquid (55 mg, 64%).

[0744] Dimethyl 6,6'-((2-(((2-aminoethyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene)) in dichloromethane (0.5 mL) STo a solution of )-dipicolinate (60 mg, 0.09 mmol), HATU (51 mg, 0.13 mmol) was added following triethylamine (0.04 mL, 0.27 mmol) at 0°C. After stirring the solution at 0°C for 5 minutes, DBCO-acid (27 mg, 0.09 mmol) in dichloromethane (0.5 mL) was added. The low-temperature bath was removed, and the mixture was stirred at room temperature for 18 hours. Water was added to the reaction mixture, and it was extracted with dichloromethane (2 mL x 3). The combined extracts were washed with a saturated aqueous NaHCO3 solution and brine, dried over sodium sulfate, and filtered. The filtrate was concentrated to yield an unrefined product. Dimethyl ester of H2bp18c6-off-macrocycle-ethyl sulfide-DBCO (20 mg, 23%) was obtained as a colorless liquid by chromatography on silica gel using dichloromethane and methanol.

[0745] LiOH (0.64 mL, 0.1 N, 0.06 mmol) in H2O was added at room temperature to a solution of dimethyl ester of H2bp18c6-off-macrocyclic-ethyl sulfide-DBCO (20 mg, 0.02 mmol) in methanol (0.5 mL). After stirring at room temperature for 16 hours, it was neutralized with acetic acid to pH = 6.5. The reaction mixture was concentrated at room temperature over a rotary evaporator to remove volatile solvents. The residue was purified by preparative HPLC to obtain H2bp18c6-off-macrocyclic-ethyl sulfide-DBCO (6 mg, 31%) It was provided as an off-white solid. LC-MS APCI: Calculated value for C48H56N6O10S 909.07; observed m / z [M+H] + 909.4. Purity by LC-MS: 92.92% RT: 1.86. Purity by HPLC: 91.56% RT: 3.87. 1H NMR (400 ㎒, D2O): δ 7.83-7.76 (m, 4H), 7.54-7.17 (m, 10H), 4.97-4.90 (m, 1H), 4.80 (s, 4H), 4.23 (s, 4H), 3.78-3.42 (m, 18H), 3.04-3.00 (m, 2H), 2.51-2.39 (m, 3H), 2.31-2.29 (m, 2H), 2.10-2.04 (m, 3H)

[0746] Example 13: Preparation of H2bp18c6-off-macrocycle-ethyl sulfide-DBCO-PSMB127 and 225 Ac(III) Signage

[0747] Reaction Scheme 16. For H2bp18c6-off-macrocycle-ethyl sulfide-DBCO-PSMB127 225 Direct chelation of Ac(III) (formed by the click reaction of H2bp18c6-off-macrocycle-ethyl sulfide-DBCO to site-specific azide-PSMB127)

[0748]

[0749] Azide modification of mAb and click responsePSMB127 was site-selectively modified with a 100x molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37°C. The addition of two azides to the heavy chain of the mAb was monitored by circular mass ESI-TOF LC-MS on an Agilent G224 instrument. The excess 3-azidopropylamine and MTG were purified using a 1 mL GE Healthcare MabSelect column. After eluting the azido-mAb from the resin using 100 mM sodium citrate pH 3.0, the solution was exchanged with 1x dPBS using a 7K Zeba desalting column. A 10x molar excess of H2bp18c6-off-macrocycled-ethyl sulfide-DBCO was reacted with site-specific azide-PSMB127 (DOL = 2) in 1x dPBS for 1 hour at 37°C without shaking. The completion of the DBCO-azide click reaction was monitored by circular mass spectrometry. Excess free chelators were removed by a concentration step in PBS using a 30K MWCO Amicon concentrator, consisting of three sequential 15x dilutions and rotation at 3800 xg, following a step of desalting the conjugate with 1x dPBS on a Zeba 7K desalting column. This yielded a final site-specific H2bp18c6-off-macrocyclic-ethyl sulfide-DBCO-PSMB127 conjugate with a CAR of 2. The final conjugate was confirmed to be monomeric by analytical size exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 u column; the column temperature was room temperature; the column was eluted with DPBS buffer (X1, without calcium and magnesium); the flow rate was 0.7 mL / min; and the column was run for 18 minutes; The injection volume was 18 μL.

[0750] Cover art : In a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial 225Ac(NO3)3 (about 5 mCi / mL in 0.1 N HCl, 10 μL, 0.048 mCi) and H2bp18c6-off-macrocycle-ethyl sulfide-DBCO-PSMB127 (site-specific, CAR = 2, 2.4 mg / mL in PBS buffer, 10 μL, 24 μg) were added sequentially. After mixing, the pH was about 6.5 as measured by pH paper. The reaction solution was incubated at 37°C for 2 hours. Subsequently, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. No radioactive signal was observed in the solvent front of iTLC-SG, which indicates that all Ac-225 in the reaction solution was completely chelated after 2 hours.

[0751] tablet The reaction mixture was purified on a PD-10 column: The PD-10 resin was conditioned in the NaOAc buffer solution by passing 5 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluent was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) solution, the wash solution was pipetteed into the PD-10 column reservoir, and the eluent was collected. Each tube contained approximately 1 mL of eluent. Continuous application of NaOAc buffer (10 mM, pH 6 to 6.5) into the PD-10 column reservoir occurred until a total elution volume of 10 mL was reached.

[0752] DTPA test10 μL of Fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG, developed with 10 mM EDTA, and dried overnight. It was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of the iTLC-SG, indicating that there was no free Ac-225 in Fraction #3.

[0753] HPLC analysis Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column; column temperature: room temperature; column eluted with DPBS buffer (X1, without calcium and magnesium); flow rate: 0.7 mL / min; 20-minute run; injection volume: 40 μL. After HPLC, fractions were collected at time intervals of 30 seconds or 1 minute. The collected HPLC fractions were left overnight at room temperature. The radioactivity in each collected fraction was counted using a gamma counter. HPLC radiation traces were constructed from the radioactivity in each HPLC fraction. The HPLC radiation traces exhibited radioactive peaks corresponding to the H2bp18c6-off-macrocycle-ethyl sulfide-DBCO-PSMB127 peak on the HPLC UV trace.

[0754] Example 14: Synthesis of H2bp18c6-off-macrocycle-pentyl sulfide-DBCO

[0755] Reaction Equation 17.

[0756]

[0757] in DMF (2 mL) tertSodium hydride (12 mg, 60% of mineral oil, 0.3 mmol) was added to a solution of butyl (5-mercaptopentyl)carbamate (65 mg, 0.3 mmol) at 0°C. The reaction mixture was stirred at room temperature for 10 minutes. To the reaction mixture, dimethyl 6,6'-((2-(((methylsulfonyl)oxy)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))( in DMF (1 mL) S )-dipicolinate (100 mg, 0.15 mmol) was added at 0°C. The reaction mixture was stirred at room temperature for 2 hours. The progress of the reaction was monitored by TLC. After the completion of the reaction, it was concentrated, and the residue was purified by preparative HPLC using acetonitrile in water and 0.1% TFA to obtain dimethyl 6,6'-((2-(((5-(( tert -Butoxycarbonyl)amino)pentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazcyclooctadecane-7,16-diyl)bis(methylene))( S )-dipicolinate (15 mg, 13%) was provided as a brown liquid.

[0758] Dimethyl 6,6'-((2-(((5-(( tert -Butoxycarbonyl)amino)pentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazcyclooctadecane-7,16-diyl)bis(methylene))( S To )-dipicolinate (120 mg, 0.15 mmol), a low-temperature solution of HCl in methanol (2 mL, 4 N) was added and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain dimethyl 6,6'-((2-(((5-aminopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))( S )-dipicolinate (70 mg, 66%) was obtained as a yellow liquid.

[0759] Dimethyl 6,6'-((2-(((5-aminopentyl)thio)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene)) in dichloromethane (0.5 mL) S To a solution of )-dipicolinate (50 mg, 0.07 mmol), HATU (38 mg, 0.10 mmol) was added following triethylamine (0.03 mL, 0.21 mmol) at 0°C. After stirring the solution at 0°C for 5 minutes, DBCO-acid (21 mg, 0.07 mmol) in dichloromethane (0.5 mL) was added. The low-temperature bath was removed, and the mixture was stirred at room temperature for 18 hours. Water was added to the reaction mixture, and it was extracted with dichloromethane (2 mL x 3). The combined extracts were washed with a saturated aqueous NaHCO3 solution and brine, dried over sodium sulfate, and filtered. The filtrate was concentrated to yield an unrefined product. Dimethyl ester of H2bp18c6-off-macrocycle-pentyl sulfide-DBCO (16 mg, 23%) was obtained as a colorless liquid by chromatography on silica gel using dichloromethane and methanol.

[0760] LiOH (0.49 mL, 0.1 N, 0.05 mmol) in H2O was added at room temperature to a solution of dimethyl ester of H2bp18c6-off-macrocycle-pentyl sulfide-DBCO (16 mg, 0.01 mmol) in methanol (0.5 mL). After stirring at room temperature for 16 hours, it was neutralized with acetic acid to pH = 6.5. The reaction mixture was concentrated at room temperature over a rotary evaporator to remove volatile solvents. The residue was purified by preparative HPLC to obtain H2bp18c6-off-macrocycle-pentyl sulfide-DBCO (5 mg, 33%) It was provided as an off-white solid. LC-MS APCI: Calculated for C51H62N6O10S; 951.15; Observed m / z [M+H] +951.4. Purity by LC-MS: 94.51% RT: 1.98. Purity by HPLC: 98.41% RT: 4.13. 1 H NMR (400 MHz, DO): δ 7.81-7.75 (m, 4H), 7.52-7.17 (m, 10H), 4.97-4.90 (m, 1H), 4.80 (s, 4H), 4.14 (s, 3H), 3.77-3.46 (m, 16H), 3.10 (s, 7H), 2.83-2.80 (m, 2H), 2.55-2.53 (m, 2H), 2.42-2.38 (m, 3H), 2.11-2.08 (m, 3H), 1.39-1.35 (m, 2H), 1.20-1.10 (m, 4H).

[0761] Example 15: Preparation of H2bp18c6-off-macrocycle-pentyl sulfide-DBCO-PSMB127 and 225 Ac(III) Signage

[0762] Reaction Equation 18. For H2bp18c6-off-macrocycle-pentyl sulfide-DBCO-PSMB127 225 Direct chelation of Ac(III) (formed by the click reaction of H2bp18c6-off-macrocycle-pentyl sulfide-DBCO to site-specific azide-PSMB127)

[0763]

[0764] Azide modification of mAb and click responsePSMB127 was site-selectively modified at 37°C with 100x molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI). The addition of two azides to the heavy chain of the mAb was monitored by round-mass ESI-TOF LC-MS on an Agilent G224 instrument. The excess 3-azidopropylamine and MTG were purified using a 1 mL GE Healthcare MabSelect column. After eluting the azido-mAb from the resin using 100 mM sodium citrate pH 3.0, the solution was exchanged with 1x dPBS using a 7K Zeba desalting column. A 10x molar excess of H2bp18c6-off-macrocycle-pentyl sulfide-DBCO was reacted with site-specific azide-PSMB127 (DOL = 2) in 1x dPBS without shaking for 1 hour at 37°C. The completion of the DBCO-azide click reaction was monitored by circular mass spectrometry. Excess free chelators were removed by a concentration step in PBS using a 30K MWCO Amicon concentrator apparatus, followed by desalting the conjugate with 1x dPBS on a Zeba 7K desalting column, three sequential 15x dilutions, and spinning at 3800 xg. This yielded a final site-specific H2bp18c6-off-macrocycle-pentyl sulfide-DBCO-PSMB127 conjugate with a CAR of 2. The final conjugate was confirmed to be monomeric by analytical size exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μ column; the column temperature was room temperature; the column was eluted with DPBS buffer (X1, without calcium and magnesium); the flow rate was 0.7 mL / min; the column was run for 18 minutes; and the injection volume was 18 μL.

[0765] Cover art : In a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial 225Ac(NO3)3 (approx. 5 mCi / mL in 0.1 N HCl, 10 μL, 0.047 mCi) and H2bp18c6-off-macrocycle-pentyl sulfide-DBCO-PSMB127 (site-specific, CAR = 2, 2.8 mg / mL in PBS buffer, 8 μL, 22 μg) were added sequentially. After mixing, the pH was approximately 6.5 as measured by pH paper. The reaction solution was incubated at 37°C for 2 hours. Subsequently, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. No radioactive signal was observed in the solvent front of iTLC-SG, which indicates that all Ac-225 in the reaction solution was completely chelated after 2 hours.

[0766] tablet The reaction mixture was purified on a PD-10 column: The PD-10 resin was conditioned in the NaOAc buffer solution by passing 5 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) through the column and discarding the wash solution. The entire reaction mixture was applied to the column reservoir, and the eluent was collected in pre-numbered plastic tubes. The reaction vial was washed with 0.2 mL x 3 of NaOAc buffer (10 mM, pH 6 to 6.5) solution, the wash solution was pipetteed into the PD-10 column reservoir, and the eluent was collected. Each tube contained approximately 1 mL of eluent. Continuous application of NaOAc buffer (10 mM, pH 6 to 6.5) into the PD-10 column reservoir occurred until a total elution volume of 10 mL was reached.

[0767] DTPA test10 μL of Fraction #3 collected after the PD-10 column was mixed with 15 μL of 10 mM DTPA solution (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG, developed with 10 mM EDTA, and dried overnight. It was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the conditions described herein, free Ac-225 migrates with the solvent to the solvent front. No radioactive signal was observed at the solvent front of the iTLC-SG, indicating that there was no free Ac-225 in Fraction #3.

[0768] HPLC analysis Fraction #3 collected after the PD-10 column was analyzed by HPLC. HPLC method: Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μm column; column temperature: room temperature; column eluted with DPBS buffer (X1, without calcium and magnesium); flow rate: 0.7 mL / min; 20-minute run; injection volume: 40 μL. After HPLC, fractions were collected at time intervals of 30 seconds or 1 minute. The collected HPLC fractions were left overnight at room temperature. The radioactivity in each collected fraction was counted using a gamma counter. HPLC radiation traces were constructed from the radioactivity in each HPLC fraction. The HPLC radiation traces exhibited radioactive peaks corresponding to the H2bp18c6-off-macrocycle-pentyl sulfide-DBCO-PSMB127 peak on the HPLC UV trace.

[0769] Example 16: Preparation of DOTA-DBCO-H11B6 (site-specific) and 225 Ac(III) Signage

[0770] Reaction Scheme 19. For DOTA-H11B6 225 Direct chelation of Ac(III) (site-specific, formed by the click reaction of DOTA-DBCO to site-specific azide-H11B6)

[0771]

[0772] Azide modification of mAb and click responseH11B6 was site-selectively modified with a 200x molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; Activa TI) at 37°C. The addition of two azides to the heavy chain of the mAb was monitored by circular mass ESI-TOF LC-MS on an Agilent G224 instrument. The excess 3-azidopropylamine and MTG were purified using a 1 mL GE Healthcare MabSelect column. After eluting the azido-mAb from the resin using 100 mM sodium citrate pH 3.0, the solution was exchanged with 1x dPBS using a 7K Zeba desalting column. A 10x molar excess of DOTA-DBCO was reacted with site-specific azide-H11B6 (DOL = 1.94) in 1x dPBS without shaking for 1 hour at 37°C. The completion of the DBCO-azide click reaction was monitored by circular mass spectrometry. Excess free chelators were removed by a concentration step in PBS using a 30K MWCO Amicon concentrator device, consisting of three sequential 15x dilutions and rotation at 3800 xg, following a step of desalting the conjugate with 1x dPBS on a Zeba 7K desalting column. This yielded a final site-specific DOTA-DBCO-H11B6 conjugate with a CAR of 1.94. The final conjugate was confirmed to be monomeric by analytical size exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8 mm x 30 cm, 5 μ column; the column temperature was room temperature; the column was eluted with DPBS buffer (X1, without calcium and magnesium); the flow rate was 0.7 mL / min; the column was run for 18 minutes; and the injection volume was 18 μL.

[0773] 50:1 of H11B6 : having the ratio of Ac-225 Cover art : In a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial 225Ac(NO3)3 (about 10 mCi / mL in 0.1 N HCl, 5 μL) and DOTA-H11B6 (site-specific, CAR = 1.94, 2.4 mg / mL in PBS buffer, 12.5 μL, 30 μg) were added sequentially. The ratio of H11B6 to Ac-225 was 50:1. After mixing, the pH was about 6.5 as measured by pH paper. The reaction solution was incubated at 37°C for 2 hours. Subsequently, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. About 21% of the radioactivity signal was observed in the solvent front of iTLC-SG, which indicates that about 79% of Ac-225 in the reaction solution was chelated after 2 hours.

[0774] Example 17: Targeting various SAs 225 Labeling of DOTA-DBCO-H11B6 (random junction) using Ac(III)

[0775] Reaction Scheme 20. For DOTA-H11B6 (random site) 225 Direct chelation of Ac(III)

[0776]

[0777] 880:1 of Labeling with a ratio of H11B6:Ac-225: to a solution of NaOAc (3 M in H2O, 20 μL) in a plastic vial 225Ac(NO3)3 (approx. 10 mCi / mL in 0.1 N HCl, 5 μL), DOTA-H11B6 (random conjugate, CAR approx. 2.5, 10 mg / mL in 25 mM acetate buffer, pH 5.5, 50 μL, 500 μg), and NaOH solution (0.1 M, 2 μL) were added sequentially. The ratio of H11B6 to Ac-225 was 880:1. After mixing, the pH was approximately 6.5 as measured by pH paper. The reaction solution was incubated at 37°C for 2 hours. Subsequently, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. No radioactive signal was observed at the solvent front of the iTLC-SG, indicating that all Ac-225 in the reaction solution was completely chelated after 2 hours.

[0778] 440:1 of Labeling with a ratio of H11B6:Ac-225: to a solution of NaOAc (3 M in H2O, 20 μL) in a plastic vial 225Ac(NO3)3 (approx. 10 mCi / mL in 0.1 N HCl, 5 μL), DOTA-H11B6 (random conjugate, CAR approx. 2.5, 10 mg / mL in 25 mM acetate buffer, pH 5.5, 25 μL, 250 μg), and NaOH solution (0.1 M, 1 μL) were added sequentially. The ratio of H11B6 to Ac-225 was 440:1. After mixing, the pH was approximately 6.5 as measured by pH paper. The reaction solution was incubated at 37°C for 2 hours. Subsequently, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. No radioactive signal was observed at the solvent front of the iTLC-SG, indicating that all Ac-225 in the reaction solution was completely chelated after 2 hours.

[0779] Marker with a ratio of H11B6:Ac-225 of 220:1 : In a solution of NaOAc (3 M in H2O, 20 μL) in a plastic vial 225Ac(NO3)3 (approx. 10 mCi / mL in 0.1 N HCl, 5 μL), DOTA-H11B6 (random conjugate, CAR approx. 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 H11B6 to Ac-225 was 220:1. After mixing, the pH was approximately 6.5 as measured by pH paper. The reaction solution was incubated at 37°C for 2 hours. Subsequently, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on 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. A 5% radioactivity signal was observed at the solvent front of the iTLC-SG, indicating that 95% of the Ac-225 in the reaction solution was chelated after 2 hours.

[0780] 110:1 of Labeling with a ratio of H11B6:Ac-225: to a solution of NaOAc (3 M in H2O, 10 μL) in a plastic vial 225Ac(NO3)3 (approx. 10 mCi / mL in 0.1 N HCl, 5 μL), DOTA-H11B6 (random conjugate, CAR approx. 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 H11B6 to Ac-225 was 110:1. After mixing, the pH was approximately 6.5 as measured by pH paper. The reaction solution was incubated at 37°C for 2 hours. Subsequently, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on a Bioscan AR-2000 radio-TLC scanner. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. A radioactivity signal of 71% was observed at the solvent front of the iTLC-SG, indicating that 29% of the Ac-225 in the reaction solution was chelated after 2 hours.

[0781] 55:1 of H11B6: Labeling with a ratio of Ac-225: to a solution of NaOAc (3 M, μL in H2O) in a plastic vial 225Ac(NO3)3 (approx. 10 mCi / mL in 0.1 N HCl, 5 μL), DOTA-H11B6 (random conjugate, CAR approx. 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 H11B6 to Ac-225 was 55:1. After mixing, the pH was approximately 6.5 as measured by pH paper. The reaction solution was incubated at 37°C for 2 hours. Subsequently, 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. After leaving the dried iTLC-SG overnight at room temperature, it was scanned on 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. A 64% radioactivity signal was observed at the solvent front of the iTLC-SG, indicating that 36% of the Ac-225 in the reaction solution was chelated after 2 hours.

[0782] Conclusion: Examples 9, 16, and 17 demonstrated that DOTA containing a chelator has poorer actinium chelation efficacy compared to H2bp18c6 containing a chelator.

[0783] Example 18: H2bp18c6- Sis Synthesis of -cyclopentyl-fused macrocycles and 225 Ac(III) chelation

[0784]

[0785] The above H2bp18c6 derivative having ring fusion on a macrocyclic ring is generally synthesized according to the following reaction scheme 21:

[0786] Reaction Equation 21: H 2 Synthesis of bp18c6 fusion ring derivatives

[0787]

[0788] A linker for conjugation to a targeting ligand can be introduced at the "benzyl" position by reacting intermediate 7 with methyl 6-(bromo(4-(tert-butoxycarbonyl)phenyl)methyl)picolinate as described above in relation to Reaction Schemes 3 and 4. To obtain an H2bp18c6 derivative having a cyclohexyl ring fusion, cyclopentane 1,2-diol (compound 1) can be replaced with cyclohexane 1,2-diol.

[0789] Next, the synthesis of an H2bp18c6-cis-cyclopentyl-fused macrocycle is described.

[0790] Reaction Scheme 22. Synthesis of H2bp18c6-Cis-Cyclopentyl-Fused Macrocycle

[0791]

[0792] In a suspension of sodium hydride (5.63 g, 60% in mineral oil, 147.05 mmol) in DMF (30 mL), (in DMF (30 mL) 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 for 2 hours. It was then cooled back to 0°C, and 2-(2-bromoethoxy)tetrahydro-2 in DMF (30 mL) H Pyran (18.44 g, 88.23 mmol) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred for 16 hours. The reaction was quenched with saturated ammonium chloride and extracted with ethyl acetate (3 x 500 mL). The combined organic layer was washed with water, dried over sodium sulfate, filtered, and concentrated. The unrefined oil was purified by silica gel (230 to 400 mesh) flash chromatography using ethyl acetate (0 to 40%) in petroleum ether ( 1R,2S )-1,2-bis(2-((tetrahydro-2 H Pyran-2-yl oxy ethoxy cyclopentane (3.5 g, 33%) was provided as a colorless liquid.

[0793] (in 100 mL of methanol 1R,2S )-1,2-bis(2-((tetrahydro-2 H 1 mL of HCl in dioxane was added to a solution of -pyran-2-yl)oxy)ethoxy)cyclopentane (3.5 g, 9.77 mmol), stirred under reflux for 1 hour, cooled, and evaporated to 2,2'-((( 1R,2S )-cyclopentane-1,2-diyl)bis(oxy))bis(ethanol-1-ol) (2.0 g) was provided and used in the next step without purification.

[0794] 2,2'-((( in dichloromethane (50 mL) and triethylamine (7.60 mL, 52.63 mmol) 1R,2S )-cyclopentane-1,2-diyl)bis(oxy))bis(ethanol-1-ol) 2 In a solution of (2.0 g, 10.52 mmol) p -Toluenesulfonyl chloride (6.0 g, 31.56 mmol) was added in portions at 10°C. The mixture was stirred at ambient temperature for 16 hours. After the reaction was complete, the suspension was diluted with 200 mL of dichloromethane, washed with low-temperature 1 M HCl (3 x 100 mL) followed by ice-cold water (2 x 100 mL), dried over sodium sulfate, and evaporated to yield a solid gum. The unrefined material was purified by flash chromatography (silica gel, 230 to 400 mesh) using ethyl acetate (0 to 40%) in petroleum ether ((( 1R,2S )-cyclopentane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (2.2 g, 42%) was provided as a colorless liquid.

[0795] ((( in 50 mL of dry DMF 1R,2SA mixture of )-cyclopentane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (2.2 g, 4.42 mmol) and cesium carbonate (4.32 g, 13.25 mmol) was stirred at ambient temperature for 1.5 hours. 1,2-bis(2-(tosyl-λ) in 50 mL of DMF 2 -azanyl)ethoxy)ethane (2.06 g, 4.42 mmol) was added dropwise to the suspension over a period of 2 hours. The mixture was stirred at ambient temperature for an additional 20 hours. The solvent was removed under reduced pressure to obtain a solid paste. This was suspended in 200 mL of dichloromethane and stirred for 30 minutes. The precipitated solid was filtered, and the filtrate was evaporated under high vacuum. The unrefined material was purified by flash chromatography (silica gel, 230 to 400 mesh) using ethyl acetate (0 to 40%) in petroleum ether (16a R ,19a S )-4,13-ditosyltetradecahydro-2 H ,11 H ,17 H -Cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.8 g, 67%) was provided as a colorless liquid.

[0796] (16a in hydrobromide (50%, 10 mL) in acetic acid R ,19a S )-4,13-ditosyltetradecahydro-2 H ,11 H ,17 HPhenol (1.38 g, 14.75 mmol) was added at room temperature to a solution of cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.8 g, 2.95 mmol). The reaction mixture was heated at 60°C for 6 hours. After the reaction was complete, it was cooled to room temperature and acetic acid was removed under high vacuum. The residue was purified by reverse-phase column purification using 0 to 100% acetonitrile (0.1% TFA) in water (16a R ,19a S )-tetradecahydro-2 H ,11 H ,17 H -Cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (0.7 g, 78%) was obtained as a colorless liquid.

[0797] (16a in dry acetonitrile (10 mL) R ,19a S )-tetradecahydro-2 H ,11 H ,17 H A suspension of cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (0.7 g, 2.32 mmol), methyl 6-(chloromethyl)picolinate (1.73 g, 5.80 mmol), and sodium carbonate (0.74 g, 6.96 mmol) was heated at 90°C for 16 hours. After the reaction was complete, the reaction mixture was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. The unrefined material was purified by flash chromatography (silica gel, 230 to 400 mesh) using methanol (0 to 10%) in dichloromethane to obtain dimethyl 6,6'-(((16a R ,19a S )-tetradecahydro-4 H ,13 H ,17 H-Cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (0.55 g, 39%) was provided as a brown liquid.

[0798] Dimethyl 6,6'-(((16a in 6 N hydrochloric acid (5 mL) R ,19a S )-tetradecahydro-4 H ,13 H ,17 H A solution of -cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (0.55 g, 0.92 mmol) was heated at 80°C for 5 hours. After the reaction was complete, the reaction mixture was concentrated under reduced pressure. The unrefined material was purified by preparative HPLC to H2bp18c6- Sis- Cyclopentyl-fused macrocycles (0.3 g, 58%) were obtained as a colorless gummy solid. LC-MS APCI: Calculated value for C29H40N4O8 572.66; observed m / z [M+H] + 572.9. Purity by LC-MS: 99.27% ​​RT: 1.17. Purity by HPLC: 99.05% RT: 2.12. 1 ¹H NMR (400 MHz, DMSO- d 6 ): δ 7.86-7.84 (m, 4H), 7.59-7.56 (m, 2H), 3.80 (s, 4H), 3.60-3.49 (m, 10H), 3.44-3.41 (m, 4H), 2.75-2.68 (m, 8H), 1.57-1.47 (m, 6H).

[0799] Reaction Scheme 23. H2bp18c6-cis-cyclopentyl-fused macrocycle and 225 Chelation of Ac(III)

[0800]

[0801] 225 Ac(III) with Chelation: Tetramethylammonium acetate (1 M, 10 μL), H2bp18c6- in a plastic vial Sis- Cyclopentyl-fused macrocycle (2 mg / mL in water, 3 μL), in 0.1 N HCl 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi) was added sequentially. The pH was approximately 6.5 according to pH paper. The vial was heated at 37°C for 2 hours.

[0802] 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dry iTLC was scanned on a Bioscan AR-2000 radio-TLC scanner after 20 hours. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. The iTLC showed 99% chelated Ac-225.

[0803] 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dry iTLC was scanned on a Bioscan AR-2000 radio-TLC scanner after 20 hours. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. The iTLC showed 99% chelated Ac-225.

[0804] Example 19: H2bp18c6- Trans Synthesis of -cyclopentyl-fused macrocycles and 225 Ac(III) chelation

[0805] Reaction Scheme 24. Synthesis of H2bp18c6-trans-cyclopentyl-fused macrocycle

[0806]

[0807] In a suspension of sodium hydride (5.63 g, 60% in mineral oil, 147.05 mmol) in DMF (30 mL), (1 in DMF (30 mL) R ,2 R )-cyclopentane-1,2-diol (3.0 g, 29.41 mmol) was added dropwise at 0°C. The reaction mixture was stirred at room temperature for 2 hours. It was then cooled back to 0°C, and 2-(2-bromoethoxy)tetrahydro-2 in DMF (30 mL) H Pyran (18.44 g, 88.23 mmol) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred for 16 hours. The reaction was quenched with saturated ammonium chloride and extracted with ethyl acetate (3 x 500 mL). The combined organic layer was washed with water, dried over sodium sulfate, filtered, and concentrated. The unrefined oil was purified by silica gel (230 to 400 mesh) flash chromatography using ethyl acetate (0 to 40%) in petroleum ether ( 1R, 2R )-1,2-bis(2-((tetrahydro-2 H Pyran-2-yl oxy ethoxy cyclopentane (4.8 g, 46%) was obtained as a colorless liquid.

[0808] (in 100 mL of methanol 1R, 2R )-1,2-bis(2-((tetrahydro-2 H 1 mL of HCl in dioxane was added to a solution of -pyran-2-yl)oxy)ethoxy)cyclopentane (4.8 g, 13.40 mmol), stirred under reflux for 1 hour, cooled, and evaporated to 2'-((( 1R, 2R )-cyclopentane-1,2-diyl)bis(oxy))bis(ethanol-1-ol) (2.9 g) was provided and used in the next step without purification.

[0809] 2'-((( in dichloromethane (50 mL) and triethylamine (11.03 mL, 76.31 mmol) 1R, 2RIn )-cyclopentane-1,2-diyl)bis(oxy))bis(ethanol-1-ol) (2.9 g, 10.52 mmol) p -Toluenesulfonyl chloride (8.70 g, 45.78 mmol) was added in portions at 10°C. The mixture was stirred at ambient temperature for 16 hours. After the reaction was complete, the suspension was diluted with 200 mL of dichloromethane, washed with low-temperature 1 M HCl (3 x 100 mL) followed by ice-cold water (2 x 100 mL), dried over sodium sulfate, and evaporated to yield a solid gum. The unrefined material was purified by flash chromatography (silica gel, 230 to 400 mesh) using ethyl acetate (0 to 40%) in petroleum ether ((( 1R, 2R )-cyclopentane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (3.5 g, 46%) was obtained as a colorless liquid.

[0810] ((( in 50 mL of dry DMF 1R, 2R A mixture of )-cyclopentane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (3.5 g, 7.03 mmol) and cesium carbonate (6.87 g, 21.08 mmol) was stirred at ambient temperature for 1.5 hours. 1,2-bis(2-(tosyl-λ) in 50 mL of DMF 2 -azanyl)ethoxy)ethane (3.19 g, 7.03 mmol) was added dropwise to the suspension over a period of 2 hours. The mixture was stirred at ambient temperature for an additional 20 hours. The solvent was removed under reduced pressure to obtain a solid paste. This was suspended in 200 mL of dichloromethane and stirred for 30 minutes. The precipitated solid was filtered, and the filtrate was evaporated under high vacuum. The unrefined material was purified by flash chromatography (silica gel, 230 to 400 mesh) using ethyl acetate (0 to 40%) in petroleum ether (16a R ,19aR )-4,13-ditosyltetradecahydro-2 H ,11 H ,17 H -Cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (3.5 g, 82%) was obtained as a colorless liquid.

[0811] (16a in hydrobromide (50%, 15 mL) in acetic acid R ,19a R )-4,13-ditosyltetradecahydro-2 H ,11 H ,17 H Phenol (2.70 g, 28.68 mmol) was added at room temperature to a solution of -cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (3.5 g, 5.73 mmol). The reaction mixture was heated at 60°C for 6 hours. After the reaction was completed, it was cooled to room temperature and acetic acid was removed under high vacuum. The unrefined material was purified by reverse-phase column purification using 0 to 100% acetonitrile (0.1% TFA) in water (16a R ,19a R )-tetradecahydro-2 H ,11 H ,17 H -Cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.3 g, 75%) was obtained as a colorless liquid.

[0812] (16a in dry acetonitrile (20 mL) R ,19a S )-tetradecahydro-2 H ,11 H ,17 HA suspension of cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.3 g, 4.30 mmol), methyl 6-(chloromethyl)picolinate (1.99 g, 10.76 mmol), and sodium carbonate (1.37 g, 12.90 mmol) was heated at 90°C for 16 hours. After the reaction was complete, the reaction mixture was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. The unrefined material was purified by flash chromatography (silica gel, 230 to 400 mesh) using methanol (0 to 10%) in dichloromethane to obtain dimethyl 6,6'-(((16a R ,19a R )-tetradecahydro-4 H ,13 H ,17 H -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.

[0813] Dimethyl 6,6'-(((16a in 6 N hydrochloric acid (10 mL) R ,19a R )-tetradecahydro-4 H ,13 H ,17 H A solution of -cyclopenta[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (1.0 g, 1.66 mmol) was heated at 80°C for 5 hours. After the reaction was complete, the reaction mixture was concentrated under reduced pressure. The unrefined material was purified by preparative HPLC to yield H2bp18c6-trans-cyclopentyl-fused macrocycle (0.8 g, 84%) as a colorless gummy solid. LC-MS APCI: 572.66 for C29H40N4O8; 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 ¹H NMR (400 MHz, DMSO- d 6 ): δ 13.42 (s, 1H), 9.70 (s, 1H), 8.15-8.09 (m, 4H), 7.80-7.78 (m, 2H), 4.69 (s, 4H), 3.93-3.55 (m, 22H), 1.90-1.85 (m, 2H), 1.57-1.52 (m, 2H), 1.46-1.39 (m, 2H).

[0814] Reaction Scheme 25. H2bp18c6-trans-cyclopentyl-fused macrocycle and 225 Chelation of Ac(III)

[0815]

[0816] 225 Chelation with Ac(III): Tetramethylammonium acetate (1 M, 10 μL), H2bp18c6-trans-cyclopentyl-fused macrocyclone (2 mg / mL in water, 3 μL), and 0.1 N HCl in a plastic vial 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi) was added sequentially. The pH was approximately 6.5 according to pH paper. The vial was heated at 37°C for 2 hours.

[0817] 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dry iTLC was scanned on a Bioscan AR-2000 radio-TLC scanner after 20 hours. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. The iTLC showed 99% chelated Ac-225.

[0818] 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dry iTLC was scanned on a Bioscan AR-2000 radio-TLC scanner after 20 hours. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. The iTLC showed 99% chelated Ac-225.

[0819] Example 20: H2bp18c6- Sis Synthesis of -cyclohexyl-fused macrocycles and 225 Ac(III) chelation

[0820] Reaction Scheme 26. Synthesis of H2bp18c6-cis-cyclohexyl-fused macrocycle

[0821]

[0822] In a suspension of sodium hydride (3.3 g, 60% in mineral oil, 86.20 mmol) in DMF (20 mL), (1 in DMF (20 mL) R ,2 S )-cyclohexane-1,2-diol (2.0 g, 17.24 mmol) was added dropwise at 0°C. The reaction mixture was stirred at room temperature for 2 hours. It was then cooled back to 0°C, and 2-(2-bromoethoxy)tetrahydro-2H-pyran (10.81 g, 51.72 mmol) in DMF (20 mL) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred for 16 hours. The reaction was quenched with saturated ammonium chloride and extracted with ethyl acetate (3 x 500 mL). The combined organic layer was washed with water, dried over sodium sulfate, filtered, and concentrated. The unrefined oil was purified by silica gel (230 to 400 mesh) flash chromatography using ethyl acetate (0 to 40%) in petroleum ether (1 R ,2 S)-1,2-bis(2-((tetrahydro-2 H Pyran-2-yl)oxy)ethoxy)cyclohexane (2.5 g, 39%) was provided as a colorless liquid.

[0823] 1 in 100 mL of methanol R ,2 S )-1,2-bis(2-((tetrahydro-2 H 1 mL of HCl in dioxane was added to a solution of -pyran-2-yl)oxy)ethoxy)cyclohexane (2.5 g, 6.7 mmol), stirred under reflux for 1 hour, cooled, and evaporated. Unpurified 2,2'-(((1 R ,2 S )-cyclohexane-1,2-diyl)bis(oxy))bis(ethanol-1-ol) (1.5 g) was used in the next step without purification.

[0824] 2,2'-(((1 in dichloromethane (25 mL) and triethylamine (5.31 mL, 36.76 mmol) R ,2 S In a solution of )-cyclohexane-1,2-diyl)bis(oxy))bis(ethanol-1-ol) (1.5 g, 7.35 mmol) p -Toluenesulfonyl chloride (4.20 g, 22.05 mmol) was added in portions at 10°C. The mixture was stirred at ambient temperature for 16 hours. After the reaction was complete, the suspension was diluted with 200 mL of dichloromethane, washed with low-temperature 1 M HCl (3 x 100 mL) followed by ice-cold water (2 x 100 mL), dried over sodium sulfate, and evaporated to yield a solid gum. The unrefined material was purified by flash chromatography (silica gel, 230 to 400 mesh) using ethyl acetate (0 to 40%) in petroleum ether (((1 R ,2 S )-cyclohexane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (1.9 g, 50%) was obtained as a colorless liquid.

[0825] (((1 in 50 mL of dry DMF R ,2 S A mixture of )-cyclohexane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (1.9 g, 3.71 mmol) and cesium carbonate (3.63 g, 11.13 mmol) was stirred at ambient temperature for 1.5 hours. 1,2-bis(2-(tosyl-λ) in 50 mL of DMF 2 -azanyl)ethoxy)ethane (1.68 g, 3.71 mmol) was added dropwise to the suspension over a period of 2 hours. The mixture was stirred at ambient temperature for an additional 20 hours. The solvent was removed under reduced pressure to obtain a solid paste. This was suspended in 200 mL of dichloromethane and stirred for 30 minutes. The precipitated solid was filtered, and the filtrate was evaporated under high vacuum. The unrefined material was purified by flash chromatography (silica gel, 230 to 400 mesh) using ethyl acetate (0 to 40%) in petroleum ether (16a R ,20a S )-4,13-ditosylhexadecahydro-2 H ,11 H Benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.2 g, 52%) was obtained as a colorless liquid.

[0826] (16a in hydrobromide (50%, 5 mL) in acetic acid R ,20a S )-4,13-ditosylhexadecahydro-2 H ,11 HPhenol (0.9 g, 9.61 mmol) was added at room temperature to a solution of benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.2 g, 1.92 mmol). The reaction mixture was heated at 60°C for 6 hours. After the reaction was complete, it was cooled to room temperature and acetic acid was removed under high vacuum. The residue was purified by reverse-phase column purification using 0 to 100% acetonitrile (0.1% TFA) in water (16a R ,20a S )-hexadecahydro-2 H ,11 H -Benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (0.45 g, 74%) was obtained as a colorless liquid.

[0827] (16a in dry acetonitrile (10 mL) R ,20a S )-hexadecahydro-2 H ,11 H A suspension of benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (0.45 g, 1.42 mmol), methyl 6-(chloromethyl)picolinate (0.65 g, 3.56 mmol), and sodium carbonate (0.45 g, 4.26 mmol) was heated at 90°C for 16 hours. The reaction mixture was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. The unrefined material was purified by flash chromatography (silica gel, 230 to 400 mesh) using methanol (0 to 10%) in dichloromethane to obtain dimethyl 6,6'-(((16a R ,20a S )-hexadecahydro-4 H ,13 H -Benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (0.26 g, 30%) was obtained as a brown liquid.

[0828] Dimethyl 6,6'-(((16a in 6 N hydrochloric acid (5 mL) R ,20a S )-hexadecahydro-4 H ,13 H A solution of benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (0.26 g, 0.42 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 the product H2bp18c6-cis-cyclohexyl-fused macrocycle (0.11 g, 44%) as an off-white solid. LC-MS APCI: Calculated value for C30H42N4O8 586.69; observed m / z [M+H] + 587.0. Purity by LC-MS: 98.15% RT: 1.31. Purity by HPLC: 97.78% RT: 2.32. 1 H NMR (400 MHz, DMSO-d6): δ 13.35 (s, 1H), 9.84 (s, 1H), 8.15-8.09 (m, 4H), 7.80-7.78 (m, 2H), 4.70 (s, 4H), 3.97-3.54 (m, 22H), 1.70-1.23 (m, 8H).

[0829] Reaction Scheme 27. H2bp18c6-cis-cyclohexyl-fused macrocycle and 225 Chelation of Ac(III)

[0830]

[0831] 225 Chelation with Ac(III): Tetramethylammonium acetate (1 M, 10 μL), H2bp18c6-cis-cyclohexyl-fused macrocycle (2 mg / mL in water, 3 μL), and 0.1 N HCl in a plastic vial 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi) was added sequentially. The pH was approximately 6.5 according to pH paper. The vial was heated at 37°C for 2 hours.

[0832] 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dry iTLC was scanned on a Bioscan AR-2000 radio-TLC scanner after 20 hours. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. The iTLC showed 99% chelated Ac-225.

[0833] 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dry iTLC was scanned on a Bioscan AR-2000 radio-TLC scanner after 20 hours. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. The iTLC showed 99% chelated Ac-225.

[0834] Example 21: H2bp18c6- Trans Synthesis of -cyclohexyl-fused macrocycles and 225 Ac(III) chelation

[0835] Reaction Scheme 28. Synthesis of H2bp18c6-trans-cyclohexyl-fused macrocycles

[0836]

[0837] In a suspension of sodium hydride (3.3 g, 60% in mineral oil, 86.20 mmol) in DMF (20 mL), (1 in DMF (20 mL) R ,2 R )-cyclohexane-1,2-diol (2.0 g, 17.24 mmol) was added dropwise at 0°C. The reaction mixture was stirred at room temperature for 2 hours. It was then cooled back to 0°C, and 2-(2-bromoethoxy)tetrahydro-2 in DMF (20 mL) HPyran (10.81 g, 51.72 mmol) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred for 16 hours. The reaction was quenched with saturated ammonium chloride and extracted with ethyl acetate (3 x 500 mL). The combined organic layer was washed with water, dried over sodium sulfate, filtered, and concentrated. The unrefined oil was purified by silica gel (230 to 400 mesh) flash chromatography using ethyl acetate (0 to 40%) in petroleum ether (1 R ,2 R )-1,2-bis(2-((tetrahydro-2 H Pyran-2-yl)oxy)ethoxy)cyclohexane (2.45 g, 38%) was obtained as a colorless liquid.

[0838] (1 in 100 mL of methanol R ,2 R )-1,2-bis(2-((tetrahydro-2 H 1 mL of HCl in dioxane was added to a solution of -pyran-2-yl)oxy)ethoxy)cyclohexane (2.45 g, 6.5 mmol), stirred under reflux for 1 hour, cooled, and evaporated. Unpurified 2,2'-(((1 R ,2 R )-cyclohexane-1,2-diyl)bis(oxy))bis(ethanol-1-ol) (1.5 g) was used in the next step without purification.

[0839] 2,2'-(((1 in dichloromethane (25 mL) and triethylamine (5.31 mL, 36.76 mmol) R ,2 R In a solution of )-cyclohexane-1,2-diyl)bis(oxy))bis(ethanol-1-ol) (1.5 g, 7.35 mmol) p-Toluenesulfonyl chloride (4.20 g, 22.05 mmol) was added in portions at 10°C. The mixture was stirred at ambient temperature for 16 hours. The suspension was diluted with an additional 200 mL of dichloromethane, washed with cold 1 M HCl (3 x 100 mL) followed by ice-cold water (2 x 100 mL), dried over sodium sulfate, and evaporated to provide a solid gum. The unrefined material was purified by flash chromatography (silica gel, 230 to 400 mesh) using ethyl acetate (0 to 40%) in petroleum ether (((1 R ,2 R )-cyclohexane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (2.1 g, 56%) was obtained as a colorless liquid.

[0840] (((1 in 30 mL of dry DMF R ,2 R A mixture of )-cyclohexane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (2.1 g, 4.1 mmol) and cesium carbonate (4.01 g, 12.30 mmol) was stirred at ambient temperature for 1.5 hours. 1,2-bis(2-(tosyl-λ) in 30 mL of DMF 2 -azanyl)ethoxy)ethane (1.86 g, 4.1 mmol) was added dropwise to the suspension over a period of 2 hours. The mixture was stirred at ambient temperature for an additional 20 hours. The solvent was removed under reduced pressure to obtain a solid paste. This was suspended in 200 mL of dichloromethane and stirred for 30 minutes. The precipitated solid was filtered, and the filtrate was evaporated under high vacuum. The residue was purified by flash chromatography (silica gel, 230 to 400 mesh) using ethyl acetate (0 to 40%) in petroleum ether (16a R ,20a R )-4,13-ditosylhexadecahydro-2 H,11 H Benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.35 g, 54%) was obtained as a colorless liquid.

[0841] (16a in hydrobromide (50%, 5 mL) in acetic acid R ,20a R )-4,13-ditosylhexadecahydro-2 H ,11 H Phenol (1.01 g, 1.81 mmol) was added to a solution of benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (1.35 g, 2.16 mmol) at room temperature. The reaction mixture was heated at 60°C for 6 hours. It was cooled to room temperature and acetic acid was removed under high vacuum. The residue was purified by reverse-phase column purification using 0 to 100% acetonitrile (0.1% TFA) in water (16a R ,20a R )-hexadecahydro-2 H ,11 H -Benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (0.5 g, 72%) was obtained as a colorless liquid.

[0842] (16a in dry acetonitrile (10 mL) R ,20a R )-hexadecahydro-2 H ,11 HA suspension of benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine (0.5 g, 1.58 mmol), methyl 6-(chloromethyl)picolinate (0.73 g, 3.95 mmol), and sodium carbonate (0.5 g, 4.74 mmol) was heated at 90°C for 16 hours. The reaction mixture was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 230 to 400 mesh) using methanol (0 to 10%) in dichloromethane to obtain dimethyl 6,6'-(((16a R ,20a R )-hexadecahydro-4 H ,13 H -Benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (0.3 g, 31%) was obtained as a brown liquid.

[0843] Dimethyl 6,6'-(((16a in 6 N hydrochloric acid (5 mL) R ,20a R )-hexadecahydro-4 H ,13 H A solution of benzo[b][1,4,10,13]tetraoxa[7,16]diazacyclooctadecine-4,13-diyl)bis(methylene))dipicolinate (0.3 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 obtain H2bp18c6-trans-cyclohexyl-fused macrocycle (0.16 g, 56%) as an off-white solid. LC-MS APCI: 586.69 for C30H42N4O8; observed m / z [M+H] + 587.0. Purity by LC-MS: 98.97% RT: 1.31. Purity by HPLC: 97.08% RT: 2.27. 1 ¹H NMR (400 MHz, DMSO- d6 ): δ 13.35 (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).

[0844] Reaction Scheme 29. H2bp18c6-trans-cyclohexyl-fused macrocycle and 225 Chelation of Ac(III)

[0845]

[0846] 225 Chelation with Ac(III): Tetramethylammonium acetate (1 M, 10 μL), H2bp18c6-trans-cyclohexyl-fused macrocycle (2 mg / mL in water, 3 μL), in 0.1 N HCl in a plastic vial 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi) was added sequentially. The pH was approximately 6.5 according to pH paper. The vial was heated at 37°C for 2 hours.

[0847] 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dry iTLC was scanned on a Bioscan AR-2000 radio-TLC scanner after 20 hours. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. The iTLC showed 99% chelated Ac-225.

[0848] 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dry iTLC was scanned on a Bioscan AR-2000 radio-TLC scanner after 20 hours. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. The iTLC showed 99% chelated Ac-225.

[0849] Example 22: Synthesis of H2bp18c6-off-macrocycle-hydroxylmethyl isomer I and 225 Ac(III) chelation

[0850] Reaction Scheme 30. Synthesis of H2bp18c6-off-macrocycle-hydroxylmethyl isomer I

[0851]

[0852] 3-ethoxypropane-1,2-diol (2.0 g, 16.66 mmol) in DMF (20 mL) was added dropwise at 0°C to a suspension of sodium hydride (3.33 g, 60% of mineral oil, 83.33 mmol) in DMF (20 mL). The reaction mixture was stirred at room temperature for 2 hours. It was then cooled back to 0°C and 2-(2-bromoethoxy)tetrahydro-2 in DMF (20 mL) H Pyran (10.44 g, 49.98 mmol) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred for 16 hours. The reaction was quenched with saturated ammonium chloride and extracted with ethyl acetate (3 x 500 mL). The combined organic layer was washed with water, dried over sodium sulfate, filtered, and concentrated. The residue oil was purified by silica gel (230 to 400 mesh) flash chromatography using ethyl acetate (0 to 40%) in petroleum ether to obtain 2,2'-((((3-ethoxypropane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(tetrahydro-2 H-Pyran)(2.2 g, 35%) was obtained as a colorless liquid.

[0853] 2,2'-((((3-ethoxypropane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(tetrahydro-2) in 50 mL of methanol H 1 mL of HCl in dioxane was added to a solution of 2,2'-pyran) (2.2 g, 5.85 mmol). The reaction mixture was stirred under reflux for 1 hour, cooled, and evaporated. Unpurified 2,2'-((3-ethoxypropane-1,2-diyl)bis(oxy))bis(ethanol-1-ol) (1.3 g) was used in the next step without purification.

[0854] In a solution of 2,2'-((3-ethoxypropane-1,2-diyl)bis(oxy))bis(ethanol-1-ol) (1.3 g, 6.25 mmol) in dichloromethane (30 mL) and triethylamine (4.51 mL, 31.25 mmol) p -Toluenesulfonyl chloride (3.56 g, 18.75 mmol) was added in portions at 10°C. The mixture was stirred at ambient temperature for 16 hours. The suspension was diluted with 200 mL of dichloromethane, washed with low-temperature 1 M HCl (3 x 100 mL) and ice-cold water (1 x 100 mL), dried over sodium sulfate, and evaporated to yield a solid gum. The unrefined material was purified by flash chromatography (silica gel, 230 to 400 mesh) using ethyl acetate (0 to 40%) in petroleum ether to obtain ((3-ethoxypropane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (2.0 g, 62%) as a colorless liquid.

[0855] A mixture of ((3-ethoxypropane-1,2-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (2.0 g, 3.87 mmol) and cesium carbonate (3.79 g, 11.62 mmol) in 25 mL of dry DMF was stirred at ambient temperature for 1.5 hours. 1,2-bis(2-(tosyl-λ) in 25 mL of DMF 2 -Azanyl)ethoxy)ethane (1.75 g, 3.87 mmol) was added dropwise to the suspension over a period of 2 hours. The mixture was stirred at ambient temperature for an additional 20 hours. The solvent was removed under reduced pressure to obtain a solid paste. This was suspended in 200 mL of dichloromethane and stirred for 30 minutes. The precipitated solid was filtered, and the filtrate was evaporated under high vacuum. The residue was purified by flash chromatography (silica gel, 230 to 400 mesh) using ethyl acetate (0 to 40%) in petroleum ether to obtain 2-(ethoxymethyl)-7,16-ditosyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (1.1 g, 45%) as a colorless liquid. The product was further purified by SFC to isolate two enantiomers, isomer-I (0.3 g, 12%) and isomer-II (0.26 g, 11%). Isomers I and isomers II The stereochemistry of was assigned arbitrarily.

[0856] (in hydrobromide (50%, 1 mL) in acetic acid S )-2-(ethoxymethyl)-7,16-ditosyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane(isomer) IPhenol (0.22 g, 2.39 mmol) was added at room temperature to a solution of (0.3 g, 0.48 mmol). The reaction mixture was heated at 60°C for 6 hours. It was cooled to room temperature and acetic acid was removed under high vacuum. The residue was purified by a reverse-phase column using 0 to 100% acetonitrile (0.1% TFA) in water ( R )-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-2-yl)methyl acetate (stereochemistry assigned arbitrarily) (0.1 g, 63%) was obtained as a colorless liquid.

[0857] (in dry acetonitrile (3 mL) R A suspension of )-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-2-yl)methyl acetate (stereochemistry assigned arbitrarily) (100 mg, 0.30 mmol), methyl 6-(chloromethyl)picolinate (138 mg, 0.75 mmol), and sodium carbonate (159 mg, 1.5 mmol) was heated at 90°C for 16 hours. The reaction mixture was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 230 to 400 mesh) using methanol (0 to 10%) in dichloromethane to obtain dimethyl 6,6'-((2-(acetoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene)-( R )-dipicolinate (stereochemistry assigned arbitrarily) (50 mg, 26%) was obtained as a brown liquid.

[0858] Dimethyl 6,6'-((2-(acetoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene)-( RPotassium carbonate (1 mg, 0.008 mmol) was added at room temperature to a solution of )-dipicolinate (stereochemistry assigned arbitrarily) (50 mg, 0.08 mmol) and stirred for 10 minutes. The mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 230 to 400 mesh) using methanol (0 to 10%) in dichloromethane to obtain dimethyl 6,6'-((2-(hydroxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))-( S )-dipicolinate (stereochemistry assigned arbitrarily) (22 mg, 48%) was obtained as a brown liquid.

[0859] Dimethyl 6,6'-((2-(hydroxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))-( in 6 N hydrochloric acid (0.5 mL) S A solution of )-dipicolinate (stereochemistry arbitrarily assigned) (22 mg, 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 H2bp18c6-off-macrocyclic-hydroxylmethyl isomer I (12 mg, 57%) as a gum-like solid. LC-MS APCI: Calculated value for C27H38N4O9 562.62; observed m / z [M+H] + 562.8. Purity by LC-MS: 96.89% RT: 1.61. Purity by HPLC: 96.47% RT: 1.57. 1 ¹H NMR (400 MHz, DMSO- d 6 ): δ 3.39 (s, 1H), 9.81 (s, 1H), 8.14-8.09 (m, 4H), 7.80-7.78 (m, 2H), 4.70 (s, 4H), 3.98-3.85 (m, 10H), 3.60-3.42 (m, 15H).

[0860] Reaction Scheme 31. H2bp18c6-off-macrocycle-hydroxylmethyl isomer I and 225 Chelation of Ac(III)

[0861]

[0862] 225 Chelation with Ac(III) : In a plastic vial, tetramethylammonium acetate (1 M, 10 μL), H2bp18c6-off-macrocycl-hydroxymethyl isomer I (2 mg / mL in water, 3 μL), and in 0.1 N HCl 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi) was added sequentially. The pH was approximately 6.5 according to pH paper. The vial was heated at 37°C for 2 hours.

[0863] 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dry iTLC was scanned on a Bioscan AR-2000 radio-TLC scanner after 20 hours. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. The iTLC showed 99% chelated Ac-225.

[0864] 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dry iTLC was scanned on a Bioscan AR-2000 radio-TLC scanner after 20 hours. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. The iTLC showed 99% chelated Ac-225.

[0865] Example 23: Synthesis of H2bp18c6-off-macrocycle-hydroxylmethyl isomer II and 225 Ac(III) chelation

[0866] Reaction Scheme 32. Synthesis of H2bp18c6-off-macrocycle-hydroxylmethyl isomer II

[0867]

[0868] (in hydrobromide (50%, 1 mL) in acetic acid R Phenol (197 mg, 2.07 mmol) was added at room temperature to a solution of )-2-(ethoxymethyl)-7,16-ditosyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (stereochemistry assigned arbitrarily) (isomer II, 0.26 g, 0.42 mmol). The reaction mixture was heated at 60°C for 6 hours. It was cooled to room temperature and acetic acid was removed under high vacuum. The residue was purified by reverse-phase column purification using 0 to 100% acetonitrile (0.1% TFA) in water ( S )-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-2-yl)methyl acetate (stereochemistry assigned arbitrarily) (0.06 g, 43%) was obtained as a colorless liquid.

[0869] (in dry acetonitrile (3 mL) S A suspension of )-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-2-yl)methyl acetate (stereochemistry assigned arbitrarily) (0.06 g, 0.18 mmol), methyl 6-(chloromethyl)picolinate (0.083 g, 0.45 mmol), and sodium carbonate (0.095 g, 0.9 mmol) was heated at 90°C for 16 hours. The reaction mixture was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 230 to 400 mesh) using methanol (0 to 10%) in dichloromethane to obtain dimethyl 6,6'-((2-(acetoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))-( S )-dipicolinate (stereochemistry assigned arbitrarily) (0.025 g, 22%) was provided as a brown liquid.

[0870] Dimethyl 6,6'-((2-(acetoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))-( S Potassium carbonate (0.5 mg, 0.004 mmol) was added at room temperature to a solution of )-dipicolinate (stereochemistry assigned arbitrarily) (0.025 g, 0.04 mmol). The mixture was stirred for 10 minutes. It was concentrated under reduced pressure. The unrefined material was purified by flash chromatography on silica (230–400 mesh) eluting with a 0–10% methanol gradient in dichloromethane to obtain dimethyl 6,6'-((2-(hydroxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))( R )-dipicolinate (0.01 g, 43%) was obtained as a brown liquid.

[0871] A solution of dimethyl 6,6'-((2-(hydroxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))-dipicolinate (0.01 g, 0.02 mmol) in 6 N hydrochloric acid (0.5 mL) was heated at 80°C for 3 hours. The reaction mixture was concentrated under reduced pressure. The unrefined material was purified by preparative HPLC to obtain H2bp18c6-off-macrocyclo-hydroxymethyl isomer II (4 mg, 44%) as a gum-like liquid. LC-MS APCI: Calculated value for C27H38N4O9 562.62; observed m / z [M+H] + 562.8. Purity by LC-MS: 96.89% RT: 6.31. Purity by HPLC: 93.97% RT: 6.67. 1 ¹H NMR (400 MHz, DMSO- d 6): δ 13.34 (s, 1H), 9.86 (s, 1H), 8.13-8.11 (m, 4H), 7.80-7.78 (m, 2H), 4.70 (s, 4H), 3.95-3.84 (m, 10H), 3.58-3.42 (m, 15H).

[0872] Reaction Scheme 33. H2bp18c6-off-macrocycle-hydroxylmethyl isomer II and 225 Chelation of Ac(III)

[0873]

[0874] 225 Ac(III) Chelation with: Tetramethylammonium acetate (1 M, 10 μL), H2bp18c6-off-macrocyclo-hydroxymethyl isomer II (2 mg / mL in water, 3 μL), in 0.1 N HCl in a plastic vial 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi) was added sequentially. The pH was approximately 6.5 according to pH paper. The vial was heated at 37°C for 2 hours.

[0875] 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dry iTLC was scanned on a Bioscan AR-2000 radio-TLC scanner after 20 hours. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. The iTLC showed 99% chelated Ac-225.

[0876] 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dry iTLC was scanned on a Bioscan AR-2000 radio-TLC scanner after 20 hours. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. The iTLC showed 99% chelated Ac-225.

[0877] Example 24: Synthesis of H2bp18c6-off-macrocycle-ethoxylmethyl isomer I and 225 Ac(III) chelation

[0878] Reaction Scheme 34. Synthesis of H2bp18c6-off-macrocycle-ethoxylmethyl isomer I

[0879]

[0880] Dimethyl 6,6'-((2-(hydroxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))( in DMF (2 mL) SSodium hydride (10 mg, 60% dispersion in mineral oil, 0.25 mmol) was slowly added to a stirred solution of )-dipicolinate (stereochemistry assigned arbitrarily) (100 mg, 0.17 mmol) under nitrogen at 0°C. The reaction mixture was stirred for 10 minutes after reaching room temperature. The reaction mixture was cooled back to 0°C, and a solution of ethyl iodide (39 mg, 0.25 mmol, 20 μL) in DMF (1 ml) was added dropwise. The reaction mixture was stirred for 2 hours after reaching room temperature. After the reaction was completed (monitored by LCMS), the reaction mixture was quenched with a saturated NH4Cl solution and extracted with ethyl acetate. Subsequently, the combined organic extract was dried and concentrated over anhydrous sodium sulfate. The residue was purified by silica gel (230 to 400 mesh) flash column chromatography using 5% methanol in dichloromethane as the eluent to obtain the product dimethyl 6,6'-((2-(ethoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))( S )-dipicolinate (stereochemistry assigned arbitrarily) (45 mg, 42%) was obtained as a brown liquid.

[0881] Dimethyl 6,6'-((2-(ethoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))( in THF (0.25 mL), water (0.5 mL), and methanol (0.25 mL) SLithium hydroxide monohydrate (9 mg, 0.21 mmol) was added to a stirred solution of )-dipicolinate (stereochemistry assigned arbitrarily) (45 mg, 0.07 mmol). The reaction mixture was stirred at room temperature for 4 hours. After the completion of the reaction (monitored by UPLC-MS), the pH of the reaction mixture was adjusted to 3 to 4 using 1.5 M aqueous HCl solution and concentrated. The residue was purified by preparative HPLC to provide H2bp18c6-off-macrocycle-ethoxylmethyl isomer I (28 mg, 66%) as a gum-like liquid. LC-MS APCI: Calculated value for C29H42N4O9 590.30; observed m / z [M+H] + 591.2. Purity by LC-MS: 99.59% RT: 1.19. Purity by HPLC: 96.12% RT: 2.01. 1 ¹H NMR (400 MHz, DMSO- d 6 ): δ 13.40 (s, 1H), 9.75 (s, 1H), 8.16-8.09 (m, 4H), 7.80-7.78 (m, 2H), 4.69 (s, 4H), 3.98-3.34 (m, 28H), 1.06 (t, J = 6.80 ㎐, 3H).

[0882] Reaction Scheme 35. H2bp18c6-off-macrocycle-ethoxylmethyl isomer I and 225 Chelation of Ac(III)

[0883]

[0884] 225 Chelation with Ac(III) : In a plastic vial, tetramethylammonium acetate (1 M, 10 μL), H2bp18c6-off-macrocyclo-ethoxymethyl isomer I (1 mg / mL in water, 1 μL), and in 0.1 N HCl 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi) was added sequentially. The pH was approximately 6.5 according to pH paper. The vial was heated at 37°C for 2 hours.

[0885] 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dry iTLC was scanned on a Bioscan AR-2000 radio-TLC scanner after 20 hours. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. The iTLC showed 99% chelated Ac-225.

[0886] 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dry iTLC was scanned on a Bioscan AR-2000 radio-TLC scanner after 20 hours. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. The iTLC showed 99% chelated Ac-225.

[0887] Example 25: Synthesis of H2bp18c6-off-macrocycle-ethoxylmethyl isomer II and 225 Ac(III) chelation

[0888] Reaction Scheme 36. Synthesis of H2bp18c6-off-macrocycle-ethoxylmethyl isomer II

[0889]

[0890] Dimethyl 6,6'-((2-(hydroxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))( in DMF (1.5 mL) RSodium hydride (6 mg, 60% dispersion in mineral oil, 0.15 mmol) was slowly added to a stirred solution of )-dipicolinate (stereochemistry assigned arbitrarily) (60 mg, 0.1 mmol) under nitrogen at 0°C. The reaction mixture was stirred for 10 minutes after reaching room temperature. The reaction mixture was cooled back to 0°C, and a solution of ethyl iodide (23 mg, 0.15 mmol, 12 μL) in DMF (0.5 ml) was added dropwise. The reaction mixture was stirred for 2 hours after reaching room temperature. After the reaction was completed (monitored by LCMS), the reaction mixture was quenched with a saturated NH4Cl solution and extracted with ethyl acetate. Subsequently, the combined organic extract was dried and concentrated over anhydrous sodium sulfate. The residue was purified by silica gel (230 to 400 mesh) flash column chromatography using 5% methanol in dichloromethane as the eluent to obtain dimethyl 6,6'-((2-(ethoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))( R )-dipicolinate (stereochemistry assigned arbitrarily) (20 mg, 32%) was obtained as a brown liquid.

[0891] Dimethyl 6,6'-((2-(ethoxymethyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))( in THF (0.1 mL), water (0.25 mL), and methanol (0.1 mL) RLithium hydroxide monohydrate (4 mg, 0.09 mmol) was added to a stirred solution of )-dipicolinate (stereochemistry assigned arbitrarily) (20 mg, 0.03 mmol). The reaction mixture was stirred at room temperature for 4 hours. After the completion of the reaction (monitored by UPLC-MS), the pH of the reaction mixture was adjusted to 3 to 4 using 1.5 M aqueous HCl solution and concentrated. The residue was purified by preparative HPLC to provide H2bp18c6-off-macrocycle-ethoxylmethyl isomer II (5 mg, 26%) as a gum-like liquid. LC-MS APCI: Calculated value for C29H42N4O9 590.30; observed m / z [M+H]+ 591.3. Purity by LC-MS: 99.28%, RT: 1.199. Purity by HPLC: 97.60% RT: 2.01. 1H NMR 1H-NMR (400 MHz, DMSO- d 6 ): δ 13.40 (s, 1H), 9.72 (s, 1H), 8.16-8.00 (m, 4H), 7.80-7.77 (m, 2H), 4.69 (s, 4H), 4.34-3.38 (m, 28H), 1.06 (t, J = 6.80 ㎐, 3H).

[0892] Reaction Scheme 37. H2bp18c6-off-macrocycle-ethoxylmethyl isomer II and 225 Chelation of Ac(III)

[0893]

[0894] 225 Chelation with Ac(III) : In a plastic vial, tetramethylammonium acetate (1 M, 10 μL), H2bp18c6-off-macrocyclo-ethoxymethyl isomer II (1 mg / mL in water, 1 μL), and in 0.1 N HCl 225 Ac(NO3)3 (10 mCi / mL, 3 μL, 30 μCi) was added sequentially. The pH was approximately 6.5 according to pH paper. The vial was heated at 37°C for 2 hours.

[0895] 0.5 μL of the reaction mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dry iTLC was scanned on a Bioscan AR-2000 radio-TLC scanner after 20 hours. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. The iTLC showed 99% chelated Ac-225.

[0896] 0.5 μL of the reaction mixture was mixed with 15 μL of 10 mM DTPA (pH 6.5) and incubated for 30 minutes. 10 μL of the mixture was loaded onto an iTLC-SG and developed with 10 mM EDTA. The dry iTLC was scanned on a Bioscan AR-2000 radio-TLC scanner after 20 hours. Under the elution conditions described herein, any free Ac-225 will migrate with the solvent to the solvent front. The iTLC showed 99% chelated Ac-225.

[0897] Example 26: Synthesis of H2bp18c6-off-macrocycle-NCS

[0898] Reaction Scheme 38. Synthesis of H2bp18c6-Off-Macrocycle-Ethyl Sulfide Amine

[0899]

[0900] Step 1:Sodium hydride (18.16 g, 60% of mineral oil, 454 mmol) was added at 0°C to a solution of (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol (50 g, 378 mmol) in anhydrous DMF (500 mL). After stirring for 10 minutes at this temperature, benzyl bromide (77.63 g, 454 mmol) was slowly added to the suspension. After 30 minutes, the reaction mixture was allowed to reach room temperature and stirred for an additional 8 hours. The reaction mixture was quenched with a saturated ammonium chloride solution and extracted with dichloromethane (2 x 500 mL). The combined organic layer was washed with brine and dried over sodium sulfate. After removing the solvent, the residue is purified by flash column chromatography using petroleum ether and ethyl acetate (silica gel, 230 to 400 mesh) to obtain the compound 1 (72 g, 86%) was obtained as a colorless liquid.

[0901] Step 2: compound 1 (72 g, 324 mmol) was dissolved in THF (100 mL), aq. HCl (1.5 N, 100 mL) was added, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with ethyl acetate (500 mL) and neutralized with a 10% sodium bicarbonate solution. The solution was extracted with ethyl acetate (2 x 500 mL), and the combined organic layer was washed with water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 230 to 400 mesh) using petroleum ether and ethyl acetate to obtain the compound 2 (51 g, 85%) was obtained as a colorless oil.

[0902] Step 3: Compound in DMF (100 mL) in a suspension of sodium hydride (26.29 g, 60% in mineral oil, 686 mmol) in DMF (20 mL) 2(25.0 g, 137 mmol) was added dropwise at 0°C. The reaction mixture was stirred at room temperature for 2 hours. It was then cooled back to 0°C, and 2-(2-bromoethoxy)tetrahydro-2H-pyran (85.89 g, 411 mmol) in DMF (100 mL) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred for 16 hours. The reaction was quenched with a saturated ammonium chloride solution and extracted with ethyl acetate (3 x 300 mL). The combined organic layer was washed with water, dried over sodium sulfate, filtered, and concentrated. The unrefined oil was purified by silica gel (230 to 400 mesh) flash chromatography using ethyl acetate (0 to 40%) in petroleum ether. The product 3 (32 g, 53%) was obtained as a colorless liquid.

[0903] Step 4: Compound in 500 mL of methanol 3 5 mL of HCl in dioxane was added to a solution of (32 g, 73.05 mmol), stirred under reflux for 1 hour, cooled, and evaporated. Unpurified material 4 (20 g) was used in the next step without refining.

[0904] Step 5: compound 4(20 g, 74.07 mmol) was dissolved in dichloromethane (250 mL) and triethylamine (53 mL, 370 mmol). The solution was cooled to 10°C, and solid p-toluenesulfonyl chloride (42.20 g, 222 mmol) was added in portions. The mixture was stirred at ambient temperature for 16 hours. After the reaction was complete, the suspension was further diluted with 1000 mL of dichloromethane, washed with low-temperature aq. HCl (1 M, 3 x 200 mL) and ice-cold water (2 x 200 mL), dried over sodium sulfate, and evaporated into a solid gum. The unrefined material was purified by flash chromatography (silica gel, 230 to 400 mesh) using ethyl acetate (0 to 40%) in petroleum ether. The product 5 (30 g, 70%) was obtained as a colorless liquid.

[0905] Step 6: Compounds in 200 mL of dry DMF 5 A mixture of (30 g, 51.9 mmol) and cesium carbonate (50.76 g, 155.7 mmol) was stirred at ambient temperature for 1.5 hours. Compounds in 200 mL of DMF 6 (23.56 g, 51.9 mmol) was added dropwise to the suspension over a period of 2 hours. The mixture was stirred at ambient temperature for an additional 20 hours. The solvent was removed under reduced pressure to obtain a solid paste. This was suspended in 1000 mL of dichloromethane and stirred for 30 minutes. The precipitated solid was filtered, and the filtrate was evaporated under high vacuum. The unrefined material was purified by flash chromatography (silica gel, 230 to 400 mesh) using ethyl acetate (0 to 40%) in petroleum ether. Product 7 (24 g, 67%) was obtained as a colorless liquid.

[0906] Step 7:Compounds in hydrobromide (50% of acetic acid, 100 mL) 7 Phenol (16.35 g, 174 mmol) was added to a solution of (24 g, 34.78 mmol) at room temperature. The reaction mixture was heated at 60°C for 6 hours. After the reaction was complete, it was cooled to room temperature, and acetic acid was removed under high vacuum. The unrefined material was purified by reverse-phase column purification using acetonitrile in water and 0.1% TFA to obtain the compound 8 (8.0 g, 69%) was obtained as a colorless liquid.

[0907] Step 8: Compound in dry acetonitrile (100 mL) 8 (8.0 g, 23.95 mmol), compound 9 A suspension of (11.07 g, 59.88 mmol), and sodium carbonate (12.69 g, 119.75 mmol) was heated at 90°C for 16 hours. After the reaction was complete, the reaction mixture was cooled to room temperature, filtered through celite, and concentrated under reduced pressure. The unrefined material was purified by flash chromatography (silica gel, 230 to 400 mesh) using methanol (0 to 10%) in dichloromethane. The product 10 (5.0 g, 33%) was obtained as a brown liquid.

[0908] Step 9: Compound in methanol (50 mL) 10 Potassium carbonate (0.11 g, 0.79 mmol) was added to a solution of (5.0 g, 7.91 mmol) at room temperature and stirred for 10 minutes. After the reaction was complete, it was concentrated under reduced pressure. The residue was purified by flash chromatography on silica (230 to 400 mesh) eluted by a 0 to 10% methanol gradient in dichloromethane. The product 11 (3.5 g, 75%) was obtained as a brown liquid.

[0909] Step 10:Compound in dichloromethane (20 mL) 11 Triethylamine (0.51 g, 0.70 mL, 5.1 mmol) was added to a solution of (1.0 g, 1.7 mmol). Methyl chloride (0.39 g, 0.26 mL, 3.4 mmol) was added dropwise to this solution at 0°C. The reaction mixture was stirred at room temperature for 30 minutes. The progress of the reaction was monitored by TLC. After the completion of the reaction, the product was concentrated, and the unrefined material was purified by column chromatography (alumina-neutral) using methanol (1 to 2%) in dichloromethane as the eluent to obtain the compound 12 (0.7 g, 62%) was obtained as a brown liquid.

[0910] Step 11: Compound in DMF (2 mL) 13 Sodium hydride (12 mg, 60% of mineral oil, 0.3 mmol) was added to a solution of (53 mg, 0.3 mmol) at 0°C. The reaction mixture was stirred at room temperature for 10 minutes. To the reaction mixture, the compound in DMF (1 mL) 12 (100 mg, 0.15 mmol) was added at 0°C. The reaction mixture was stirred at room temperature for 2 hours. The progress of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was quenched with a saturated ammonium chloride solution and extracted with ethyl acetate (3 x 5 mL). The combined organic layer was washed with water and brine, dried over anhydrous sodium sulfate, and concentrated. The unrefined product was purified by preparative HPLC using acetonitrile in water and 0.1% TFA to obtain the compound 14 (20 mg, 18%) was obtained as a brown liquid.

[0911] Step 12: Compound in 6 N hydrochloric acid (0.5 mL) 14A solution of (20 mg, 0.02 mmol) was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was concentrated under reduced pressure. The unrefined product was purified by preparative HPLC to obtain the product H2bp18c6-off-macrocyclo-ethylsulfide amine (6 mg, 36%) as a gum-like solid. LC-MS APCI: Calculated value for C29H43N5O8S 621.75; observed m / z [M+H] + 622.2. Purity by LC-MS: 97.94% RT: 1.38. Purity by HPLC: 94.11% RT: 2.94. 1 H NMR (400 MHz, DMSO-d6): δ 11.02-11.00 (m, 2H), 8.21 (s, 2H), 8.16-8.09 (m, 4H), 7.93-7.90 (m, 2H), 4.78-4.75 (m, 4H), 4.15-3.93 (m, 9H), 3.56-3.50 (m, 14H), 2.97-2.96 (m, 2H), 2.81-2.79 (m, 2H), 2.70-2.67 (m, 2H).

[0912] Reaction Scheme 39. Synthesis of H2bp18c6-off-macrocycle-ethylsulfide NCS

[0913]

[0914] Step 1 : Compound 14 A low-temperature solution of HCl (2 mL, 4 N) in methanol was added to (100 mg, 0.15 mmol) and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain compound 2 as a yellow liquid (55 mg, 64%).

[0915] Step 2 : Compound in dry dichloromethane (2 mL) in a pressure vial 15Carbon disulfide (12 mg, 0.16 mmol) was added to a solution of (50 mg, 0.08 mmol) and triethylamine (24 mg, 0.24 mmol). MW-irradiation (150 W power) was applied to the vial at 90°C for 30 minutes. Afterward, the reaction mixture was diluted with dichloromethane (10 mL), washed successively with water (5 mL), 1 M HCl (5 mL), and water (5 mL), and dried on anhydrous sodium sulfate. After concentration, the unrefined product was purified by flash chromatography on silica gel (230 to 400 mesh) using 0 to 10% methanol in dichloromethane as the eluent to obtain the compound 16 (20 mg, 38%) was obtained as a yellow solid.

[0916] Step 3: Compounds in hydrochloric acid (6 N, 0.5 mL) 16 A solution of (20 mg, 0.03 mmol) was stirred overnight at room temperature. After the reaction was complete, it was concentrated under reduced pressure. The residue was purified by preparative HPLC to obtain H2bp18c6-off-macrocyclic-ethylsulfide NCS (6 mg, 31%). It was obtained as a white solid. LC-MS APCI: Calculated value for C30H41N5O8S2: 663.81; Observed m / z [M+H] + 664.2. Purity by LC-MS: 99.94% RT: 1.41. Purity by HPLC: 98.77% RT: 2.76. 1 H NMR (400 MHz, DMSO-d6): δ 9.78 (s, 1H), 8.10 (s, 4H), 7.78 (d, J = 6.00 Hz, 2H), 4.69 (s, 4H), 3.96-3.52 (m, 23H), 2.85 (t, J = 6.40) ㎐, 2H), 2.70 (t, J = 8.00 ㎐, 2H).

[0917] Reaction Scheme 40. Synthesis of H2bp18c6-Off-Macrocycle-Pentylsulfide Amine

[0918]

[0919] Step 1: Compound in dichloromethane (20 mL) 17 Triethylamine (0.51 g, 0.70 mL, 5.1 mmol) was added to a solution of (1.0 g, 1.7 mmol). Methyl chloride (0.39 g, 0.26 mL, 3.4 mmol) was added dropwise to this solution at 0°C. The reaction mixture was stirred at room temperature for 30 minutes. The progress of the reaction was monitored by TLC. After the completion of the reaction, it was concentrated, and the residue was purified by column chromatography (alumina-neutral) using methanol (1 to 2%) in dichloromethane as the eluent to obtain the compound 18 (0.7 g, 62%) was obtained as a brown liquid.

[0920] Step 2: Compound in DMF (2 mL) 19 Sodium hydride (12 mg, 60% of mineral oil, 0.3 mmol) was added to a solution of (65 mg, 0.3 mmol) at 0°C. The reaction mixture was stirred at room temperature for 10 minutes. To the reaction mixture, the compound in DMF (1 mL) 18 (100 mg, 0.15 mmol) was added at 0°C. The reaction mixture was stirred at room temperature for 2 hours. The progress of the reaction was monitored by TLC. After the completion of the reaction, it was concentrated, and the residue was purified by preparative HPLC using acetonitrile in water and 0.1% TFA to obtain the compound 20 (15 mg, 13%) was obtained as a brown liquid.

[0921] Step 3: Compounds in hydrochloric acid (6 N, 0.5 mL) 20 A solution of (15 mg, 0.02 mmol) was stirred overnight at room temperature. After the reaction was complete, it was concentrated under reduced pressure. The residue was purified by preparative HPLC to obtain the product H2bp18c6-off-macrocycle-pentylsulfide amine (4 mg, 33%) as a gum-like liquid. LC-MS APCI: Calculated value for C32H49N5O8S 663.83; observed m / z [M+H] + 664.2. Purity by LC-MS: 90.31% RT: 1.43. Purity by HPLC: 90.69% RT: 3.11. 1 H NMR (400 MHz, DMSO-d6): δ 7.85-7.83 (m, 2H), 7.79-7.75 (m, 2H), 7.30 (dd, J = 6.80, 24.00 Hz, 2H), 4.01-3.38 (m, 29H), 2.89-2.85 (m, 4H), 1.80-1.60 (m, 2H), 1.44-1.39 (m, 4H).

[0922] Reaction Scheme 41. Synthesis of H2bp18c6-Off-Macrocycle-Pentylsulfide NCS

[0923]

[0924] Step 1 : Compound 20 A cold solution of HCl (2 mL, 4 N) in methanol was added to (120 mg, 0.15 mmol) and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain the compound 21 It was obtained as a yellow liquid (70 mg, 66%).

[0925] Step 2 : Compound in dry dichloromethane (2 mL) in a pressure vial 21Carbon disulfide (15 mg, 0.2 mmol) was added to a solution of (70 mg, 0.1 mmol) and triethylamine (20 mg, 0.2 mmol). MW-irradiation (150 W power) was applied to the vial at 90°C for 30 minutes. Afterward, the reaction mixture was diluted with dichloromethane (10 mL), washed successively with water (5 mL), 1 M HCl (5 mL), and water (5 mL), and dried on anhydrous sodium sulfate. After concentration, the unrefined product was purified by flash chromatography on silica gel (230 to 400 mesh) using 0 to 10% methanol in dichloromethane as the eluent to obtain the compound 22 (30 mg, 40%) was obtained as a yellow solid.

[0926] Step 3: Compounds in hydrochloric acid (6 N, 0.5 mL) 22 A solution of (30 mg, 0.04 mmol) was stirred overnight at room temperature. After the reaction was complete, it was concentrated under reduced pressure. The residue was purified by preparative HPLC to obtain H2bp18c6-off-macrocycle-pentylsulfide NCS (12 mg, 41%) as a white solid. LC-MS APCI: Calculated value for C33H47N5O8S2: 705.89; Observed m / z [M+H] + 706.2. Purity by LC-MS: 99.33% RT: 1.58. Purity by HPLC: 98.92% RT: 2.76. 1 H NMR 1H-NMR (400 MHz, DMSO-d6): δ 13.40 (s, 1H), 9.90 (s, 1H), 8.17-8.09 (m, 4H), 7.78 (d, J = 6.80 Hz, 2H), 4.70 (s, 4H), 3.93-3.17 (m, 27H), 2.68-2.67 (m, 2H), 1.64-1.60 (m, 2H), 1.53-1.49 (m, 2H), 1.40-1.38 (m, 2H).

[0927] Example 26: Characteristics of a radioimmunoconjugate

[0928] Table 1 summarizes the chelation efficiency of the radioimmunoconjugates prepared in the examples.

[0929] [Table 1]

[0930]

[0931] 4 225 Human serum stability of the Ac-chelator-mAb conjugate is measured according to the following method:

[0932] 225 Ac-H2bp18c6-benzyl-phenyl-DBCO-PSMB127

[0933] 225 Ac-H2bp18c6-off-macrocycle-ethyl sulfide-DBCO-PSMB127

[0934] 225 Ac-H2bp18c6-off-macrocycle-pentyl sulfide-DBCO-PSMB127

[0935] 225 Ac-H2bp18...

Claims

Claim 1 Chelating agent selected from the army composed of: , , , , , , and In the above formula, L1 is absent, or L1 is a substituted or unsubstituted alkyl, a substituted or unsubstituted heteroalkyl moiety, a substituted or unsubstituted aryl or heteroaryl, a polyethylene glycol (PEG) linker, a peptide linker, a sugar-based linker, or a cleavable linker that is a disulfide linkage or valine-citrulline-p-aminobenzyl (PAB) (wherein the “substituted or unsubstituted” group means that the corresponding group is substituted or unsubstituted with an alkyl, hydroxy, alkoxy, amide, alkylthio, amino, alkylamino, aminoalkyl, hydroxyalkyl, or carboxyl group), or L1 is selected from the group consisting of: , , , , , , , and (where n is an integer from 0 to 10 and m is an integer from 0 to 12);R 11 -NH2, -NCS, -NCO, -N3, alkynyl, cycloalkynyl, cyclooctinyl derivatives, -C(O)R 13 , -COOR 13 , -CON(R 13 )2, maleimido, acyl halide, tetrazine, or trans-cyclooctene, or R 11 ... comprises an antibody or its antigen-binding fragment, scaffold protein, small molecule, or aptamer, wherein the cyclooctinyl derivative is selected from the group consisting of bicyclononinil (BCN), difluorinated cyclooctinyl (DIFO), dibenzocyclooctinyl (DIBO), keto-DIBO, biaryl-azacyclooctinyl (BARAC), dibenzo-azacyclooctinyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctinyl (DIMAC), difluorobenzocyclooctinyl (DIFBO), monobenzocyclooctinyl (MOBO), and tetramethoxydibenzocyclooctinyl (TMDIBO); and each R 13 is independently hydrogen or alkyl. Claim 2 In paragraph 1, R 11 This -NH2, -NCS, -NCO, -N3, alkynyl, cycloalkynyl, -C(O)R 13 , -COOR 13 , -CON(R 13 )2, maleimido, acyl halide, tetrazine, or trans-cyclooctene, chelator. Claim 3 In paragraph 1, R 11 This -NCS, cyclooctinyl, or a chelator that is a cyclooctinyl derivative. Claim 4 In paragraph 3, R 11 This is a chelator that is -NCS, DBCO, or BCN. Claim 5 In paragraph 1, R 11 A chelator additionally conjugated to this antibody or its antigen-binding fragment, scaffold protein, small molecule, or aptamer. Claim 6 delete Claim 7 In paragraph 1, a chelator selected from the group consisting of the following: , , , and . Claim 8 A chelator that forms a radiometal complex by additionally including a radioactive metal ion coupled to the chelator through coordination bonding in any one of claims 1 to 5 and 7. Claim 9 Radioactive metal complexes selected from the group consisting of: , , , , , , and , in the above formula, M is a radioactive metal ion; L1 is absent, or L1 is a cleavable linker that is a substituted or unsubstituted alkyl, a substituted or unsubstituted heteroalkyl moiety, a substituted or unsubstituted aryl or heteroaryl, a polyethylene glycol (PEG) linker, a peptide linker, a sugar-based linker, or a disulfide linkage or valine-citrulline-p-aminobenzyl (PAB) (wherein the “substituted or unsubstituted” group means that the corresponding group is substituted or unsubstituted with an alkyl, hydroxy, alkoxy, amide, alkylthio, amino, alkylamino, aminoalkyl, hydroxyalkyl, or carboxyl group), or L1 is selected from the group consisting of: , , , , , , , and (where n is an integer from 0 to 10 and m is an integer from 0 to 12);R 11 -NH2, -NCS, -NCO, -N3, alkynyl, cycloalkynyl, cyclooctinyl derivatives, -C(O)R 13 , -COOR 13 , -CON(R 13 )2, maleimido, acyl halide, tetrazine, or trans-cyclooctene, or R 11 ... comprises an antibody or its antigen-binding fragment, scaffold protein, small molecule, or aptamer, wherein the cyclooctinyl derivative is selected from the group consisting of bicyclononinil (BCN), difluorinated cyclooctinyl (DIFO), dibenzocyclooctinyl (DIBO), keto-DIBO, biaryl-azacyclooctinyl (BARAC), dibenzo-azacyclooctinyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctinyl (DIMAC), difluorobenzocyclooctinyl (DIFBO), monobenzocyclooctinyl (MOBO), and tetramethoxydibenzocyclooctinyl (TMDIBO); and each R 13 is independently hydrogen or alkyl. Claim 10 In paragraph 9, the radioactive metal ion is actinium-225 ( 225 Ac)phosphorus, radioactive metal complex. Claim 11 A radioimmunoconjugate comprising the radioactive metal complex of claim 9 additionally conjugated to an antibody or its antigen-binding fragment. Claim 12 In claim 11, the antibody or its antigen-binding fragment is R of the radioactive metal complex through a triazole moiety. 11 A radioimmunoconjugate connected to. Claim 13 In paragraph 11, a radioimmunoconjugate having a structure selected from the group consisting of: , , , , , , , and , in the above formula, M is a radioactive metal ion; L1 is absent, or L1 is a cleavable linker that is a substituted or unsubstituted alkyl, a substituted or unsubstituted heteroalkyl moiety, a substituted or unsubstituted aryl or heteroaryl, a polyethylene glycol (PEG) linker, a peptide linker, a sugar-based linker, or a disulfide linkage or valine-citrulline-p-aminobenzyl (PAB) (wherein the “substituted or unsubstituted” group means that the corresponding group is substituted or unsubstituted with an alkyl, hydroxy, alkoxy, amide, alkylthio, amino, alkylamino, aminoalkyl, hydroxyalkyl, or carboxyl group), or L1 is selected from the group consisting of: , , , , , , , and (where n is an integer from 0 to 10 and m is an integer from 0 to 12); mAb is an antibody or an antigen-binding fragment thereof; and each R 12 is independently hydrogen, -CH3, or -CH2CH3, provided that one or more R 12 is -CH3 or -CH2CH3. Claim 14 In paragraph 11, a radioimmunoconjugate selected from the group consisting of the following: , , and In the above formula, mAb is an antibody or its antigen-binding fragment. Claim 15 In paragraph 14, the antibody or its antigen-binding fragment is a radioimmunoconjugate that specifically binds to a tumor antigen. Claim 16 In paragraph 14, the mAb is a radioimmunoconjugate selected from PSMB127, pertuzumab, cetuximab, panitumumab, Herceptin, and H11B6. Claim 17 A method for manufacturing a radioimmunoconjugate, comprising the step of forming a radiometal complex bound to an antibody or its antigen-binding fragment, a scaffold protein, a small molecule, or an aptamer by contacting the chelator of claim 5 with a radioactive metal ion. Claim 18 A method according to claim 17, wherein a radioactive metal complex is bound to an antibody or its antigen-binding fragment. Claim 19 A pharmaceutical composition for selectively targeting neoplastic cells for radiotherapy in subjects requiring selective targeting of neoplastic cells for radiotherapy, comprising a radioimmunoconjugate of any one of claims 11 to 16 and a pharmaceutically acceptable carrier. Claim 20 A pharmaceutical composition for treating a neoplastic disease or disorder in a subject requiring treatment of a neoplastic disease or disorder, comprising a radioimmunoconjugate of any one of claims 11 to 16 and a pharmaceutically acceptable carrier. Claim 21 A pharmaceutical composition for treating cancer in a subject requiring treatment for cancer, comprising a radioimmunoconjugate of any one of claims 11 to 16 and a pharmaceutically acceptable carrier. Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete

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