CN118201925A

CN118201925A - Macrocyclic compounds and methods of making the same

Info

Publication number: CN118201925A
Application number: CN202280074126.6A
Authority: CN
Inventors: E·克利特; W·M·玛顿; R·萨尔特
Original assignee: Janssen Biotech Inc
Current assignee: Janssen Biotech Inc
Priority date: 2021-11-09
Filing date: 2022-11-08
Publication date: 2024-06-14

Abstract

The present invention relates to the preparation of key intermediates and the synthesis of compounds (macrocyclic compounds) and pharmaceutically acceptable salts thereof, immunoconjugates, radioimmunoconjugates, pharmaceutical compositions containing the compounds and immunoconjugates, radioimmunoconjugates.

Description

Macrocyclic compounds and methods of making the same

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application No. 63/277,278 filed on day 11 and 9 of 2021 and U.S. provisional application No. 63/338,949 filed on day 5 and 6 of 2022, both of which are incorporated herein by reference in their entireties for all purposes.

Electronically submitted reference sequence listing

The present application comprises a computer readable sequence listing submitted with the present application in XML file format, the entire contents of which are incorporated herein by reference in their entirety. The XML file name of the sequence table submitted with the application is JBI6638WOPCT1_SL.xml, which is created on month 11 and 2 of 2022 and has the size of 27422 bytes.

Technical Field

The present invention relates to the preparation of key intermediates and synthesis of macrocyclic compounds and pharmaceutically acceptable salts thereof, and immunoconjugates and radioimmunoconjugates comprising the same.

Background

Radionuclides emitting alpha particles show good promise for cancer treatment due to their combination of highly linear energy transfer and short-range action, providing the possibility of potent killing that is localized primarily to tumor cells (Kim, y.s. And m.w. brechbiel, an overview of TARGETED ALPHA therapy. Tumourbiol, 2012.33 (3): p.573-90). Targeting delivery of alpha emitters using antibodies, scaffold proteins, small molecule ligands, nucleic acid aptamers, or other binding moieties specific for cancer antigens provides a method of selectively delivering radionuclides to tumors to enhance their efficacy and mitigate off-target effects. Conventionally, the binding moiety is linked to a chelator that binds to the alpha-emitting radiometal to produce a radioactive complex. Many such examples use monoclonal antibodies (mabs) as targeting vectors to produce compounds known as radioimmunoconjugates.

Actinium-225 (²²⁵ Ac) is a radioisotope of particular interest for medical applications that emits alpha (Miederer et al ,Realizing the potential of the Actinium-225radionuclide generator in targeted alpha particle therapy applications.Adv Drug Deliv Rev,2008.60(12):71-82).²²⁵Ac has a10 day half-life long enough to facilitate radioconjugate production, but also short enough to match the circulating pharmacokinetics of delivery vehicles such as antibodies, and thus ²²⁵ Ac radioimmunoconjugates are of particular interest, in addition, ²²⁵ Ac decays In a series of steps that collectively emit 4 alpha particles for each ²²⁵ Ac decay before reaching stable isotope ²⁰⁹ Bi, thereby increasing potency.

Currently, the most widely used chelator for actinides-225 and lanthanides is DOTA (1, 4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid; tetraxaten), and previous clinical and preclinical procedures have used mainly 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA) for actinide chelation. However, it is known that DOTA chelation of actinides can be challenging (Deal, K.A. et al Improved in vivo stability of actinium-225macrocyclic complexes.JMed Chem,1999.42 (15): p.2988-92). For example, DOTA allows for a chelation ratio of up to >500:1DOTA to actinium-225 when attached to a targeting ligand (such as a protein or antibody), and often requires harsh conditions or high levels of DOTA per antibody. Other macrocyclic chelators of lanthanoids and actinides-225 have been described, for example, in international patent application publication WO 2018/183906; as described in Thiele et al "An Eighteen-Membered Macrocyclic Ligand for Actinium-225TargetedAlphaTherapy"Angew.Chem.Int.Ed.(2017)56,14712-14717.;Roca-Sabio et al "Macrocyclic Receptor Exhibiting Unprecedented Selectivity for Light Lanthanides"J.Am.Chem.Soc.(2009)131,3331-3341.

Site-specificity has become a critical focal area in the field of Antibody Drug Conjugates (ADCs) (Agarwal, p. And C.R.Bertozzi,Site-specific antibody-drug conjugates:the nexus ofbioorthogonalchemistry,proteinengineering,anddrugdevelopment,Bioconjug Chem,2015.26(2): pages 176-92) because it has been demonstrated that site-specific approaches can increase the efficacy and safety of ADCs compared to random conjugation. It is believed that the radioimmunoconjugates may achieve similar safety and efficacy benefits.

Disclosure of Invention

Described herein are methods of making key intermediates for use in preparing compounds, immunoconjugates, radioimmunoconjugates of the invention.

The novel compounds of the present invention bind to radiometals, preferably alpha-emitting radiometals such as actinium-225 (²²⁵ Ac), and are useful in the production of stable radioimmunoconjugates with high specific activity and high yields. The present invention provides macrocyclic compounds capable of binding radiometals such as alpha emitting radiometals, e.g., ²²⁵ Ac, regardless of specific activity or most common metal impurities, and having the ability to sequester radiometals, e.g., ¹³⁴ Ce. The compounds of the invention can be used to generate radioimmunoconjugates with high stability in vitro and in vivo by conjugation to targeting ligands (such as antibodies, proteins, nucleic acid aptamers, etc.), preferably in a site-specific manner using "click chemistry". The radioimmunoconjugates produced by conjugating the compounds of the present invention to targeting ligands can be used for targeted radiotherapy, such as targeted radiotherapy of neoplastic cells and/or targeted treatment of neoplastic diseases or disorders, including cancers.

One embodiment of the present invention provides a method for preparing compound 14:

One embodiment of the present invention provides an intermediate in a step for preparing compounds that are capable of chelating a radioactive metal.

In one embodiment of the invention, compound 14:

or a pharmaceutically acceptable salt or solvate thereof, comprising the steps of:

Reacting 7, 16-dibenzyl-1,4,10,13-tetraoxa-7, 16-diazacyclooctadecane (1) with a reducing agent in an organic solvent or mixture thereof at a temperature in the range of from ambient to-78 ℃ to obtain compound 2;

Reacting methyl 6- (hydroxymethyl) pyridine carboxylate with thionyl chloride in an organic solvent or mixture thereof at a temperature in the range of about ambient temperature to about-78 ℃ to provide compound 3;

reacting 3 with 2 in an organic solvent or mixture thereof at a temperature ranging from about ambient temperature to about-78 ℃ to provide compound 4 (6- ((1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridinecarboxylate);

Reacting methyl 6-formylpicolinate 5 with (4- ((tert-butoxycarbonyl) amino) phenyl) boronic acid 6 in an organic solvent or mixture thereof under reducing conditions at a temperature ranging from about ambient temperature to about-78 ℃ to give compound 7;

Reacting methyl 6- ((4- ((tert-butoxycarbonyl) amino) phenyl) (hydroxy) methyl) picolinate 7 with methanesulfonyl chloride in an organic solvent or mixture thereof at a temperature ranging from about ambient temperature to about-78 ℃ to give methyl 6- ((4- ((tert-butoxycarbonyl) amino) phenyl) - ((methanesulfonyl) oxy) methyl) picolinate;

Reacting methyl 6- ((4- ((tert-butoxycarbonyl) amino) phenyl) - ((methylsulfonyl) oxy) methyl) pyridinecarboxylate 8 with methyl 6- ((1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridinecarboxylate 4 with sodium carbonate in an organic solvent or a mixture thereof at a temperature in the range of about ambient temperature to about-78 ℃ to give compound 9;

reacting compound 9 with N, O-bis (trimethylsilyl) acetamide (BSA) in an organic solvent or mixture thereof at a temperature ranging from about ambient temperature to about-78 ℃, adding thereto a solution of trimethylsilicone triflate (TMSOTf) in an organic solvent to give compound 10;

compound 10 is reacted in an organic solvent or mixture thereof under basic conditions at a temperature in the range of about ambient temperature to about-78 ℃ to provide 13, and is reacted with thiocarbonyldiimidazole in an organic solvent or mixture thereof at a temperature in the range of about ambient temperature to about-78 ℃ to provide compound 14.

In another embodiment of compound 14, a method for preparing compound 10

A method of making a pharmaceutical composition comprising:

Reacting compound 9 with N, O-bis (trimethylsilyl) acetamide (BSA) in an organic solvent or mixture thereof at a temperature ranging from about ambient temperature to about-78 ℃ for 5 minutes to 60 minutes; with trimethylsilyl triflate (TMSOTf) in an organic solvent or mixture thereof at a temperature in the range of about ambient temperature to about-78 ℃ to give compound 10.

Removal of the protecting group t-butoxycarbonyl has proven challenging and many conditions have been tried but none have been successful. Attempts were made under acidic and basic conditions (HCl, TFA, MSA, phosphoric acid, KOAc/AcOH, tsCl-DMAP, BF ₃.OEt₂, TMSCl and CsCO ₃). All of this resulted in rapid decomposition of compound 9.

The conditions for successful deprotection using BSA and TMSOTf reagents are unexpected and novel.

One aspect of the present invention is an intermediate of formula (14) as a free base compound:

or a pharmaceutically acceptable salt or solvate thereof.

Another embodiment of the invention is a compound of formula (11)

Or a pharmaceutically acceptable salt or solvate thereof.

Another aspect of the invention is intermediate compound 12 (TOPA- [ C7] -phenylisothiocyanate sodium salt):

or a pharmaceutically acceptable salt or solvate thereof.

One embodiment of the present invention encompasses the preparation of compound 12 (TOPA- [ C7] -phenyl isothiocyanate sodium salt)

Reacting compound 10 with sodium hydroxide in an organic solvent or mixture thereof at a temperature ranging from about ambient temperature to about-78 ℃ to provide compound 11;

Reacting compound 11 with thiocarbonyldiimidazole in an organic solvent or mixture thereof at a temperature in the range of about ambient temperature to about-78 ℃ to provide compound 12.

Furthermore, the present invention encompasses compounds, radiometal complexes and radioimmunoconjugates capable of forming complexes with radiometals, as described below.

In one embodiment of the invention are compounds of formula (I):

Or a pharmaceutically acceptable salt thereof, wherein:

R ₁ is hydrogen and R ₂ is-L ₁-R₄;

Alternatively, R ₁ is-L ₁-R₄ and R ₂ is hydrogen;

R ₃ is hydrogen;

Alternatively, R ₂ and R ₃ together with the carbon atom to which they are attached form a 5-or 6-membered cycloalkyl, wherein the 5-or 6-membered cycloalkyl is optionally substituted by-L ₁-R₄;

L ₁ is absent or a linker; and

R ₄ is a nucleophilic moiety, electrophilic moiety, or targeting ligand.

In one embodiment, the present invention relates to one or more compounds independently selected from the group consisting of:

Wherein the method comprises the steps of

L ₁ is absent or a linker; and

R ₄ is a nucleophilic moiety, electrophilic moiety, or targeting ligand.

In certain embodiments, R ₄ is-NH ₂、-NCS、-NCO、-N₃, alkynyl, cycloalkynyl, -C (O) R ₁₃、-COOR₁₃、-CON(R₁₃)₂, maleimide, acyl halide, tetrazine, or trans-cyclooctene.

In certain embodiments, R ₄ is cyclooctynyl or a cyclooctynyl derivative selected from the group consisting of: dicyclononynyl (BCN), cyclooctynyl Difluoride (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, diarylazedoxynyl (BARAC), dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO) and Tetramethoxydibenzocyclooctynyl (TMDIBO).

In certain embodiments, R ₄ is DBCO or BCN.

In certain embodiments, R ₄ comprises a targeting ligand, wherein the targeting ligand is selected from the group consisting of: antibodies, antibody fragments (e.g., antigen binding fragments), binding peptides, binding polypeptides (such as selectively targeted oligopeptides containing up to 50 amino acids), binding proteins, enzymes, nucleobase-containing moieties (such as oligonucleotides, DNA or RNA vectors, or nucleic acid aptamers), and lectins.

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

In another embodiment, the invention is a radiometal complex comprising a radiometal ion complexed with a compound of formula I.

In another embodiment, the invention relates to a radiometal complex of formula (I-M ⁺):

Or a pharmaceutically acceptable salt thereof, wherein:

M ⁺ is a radioactive metal ion selected from the group consisting of: actinium-225 (²²⁵ Ac), radium-223 (²³³ Ra), bismuth-213 (²¹³ Bi), lead-212 (²¹² Pb (II) and/or ²¹² Pb (IV)), terbium-149 (¹⁴⁹ Tb), terbium-152 (¹⁵² Tb), terbium-155 (¹⁵⁵ Tb), thorium-255 (²⁵⁵ Fm), thorium-227 (²²⁷ Th), thorium-226 (²²⁶Th⁴⁺), astatin-211 (²¹¹ At), cerium-134 (¹³⁴ Ce), neodymium-144 (¹⁴⁴ Nd), lanthanum-132 (¹³² La), lanthanum-135 (¹³⁵ La) and uranium-230 (²³⁰ U);

R ₁ is hydrogen and R ₂ is-L ₁-R₄;

Alternatively, R ₁ is-L ₁-R₄ and R ₂ is hydrogen;

R ₃ is hydrogen;

L ₁ is absent or a linker;

r ₄ is a nucleophilic moiety, electrophilic moiety, or targeting ligand.

In some embodiments, R ₄ is-NH ₂、-NCS、-NCO、-N₃, alkynyl, cycloalkynyl, -C (O) R ₁₃、-COOR₁₃、-CON(R₁₃)₂, maleimide, acyl halide, tetrazine, or trans-cyclooctene.

In certain embodiments, R ₄ is DBCO or BCN.

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

In another embodiment, the invention relates to a radioimmunoconjugate of formula (I-M ⁺), or a pharmaceutically acceptable salt thereof, wherein

M ⁺ is a radioactive metal ion, wherein M ⁺ is selected from the group consisting of: actinium-225 (²²⁵ Ac), radium-223 (²³³ Ra), bismuth-213 (²¹³ Bi), lead-212 (²¹² Pb (II) and/or ²¹² Pb (IV)), terbium-149 (¹⁴⁹ Tb), terbium-152 (¹⁵² Tb), terbium-155 (¹⁵⁵ Tb), 255 (²⁵⁵ Fm), thorium-227 (²²⁷ Th), thorium-226 (²²⁶Th⁴⁺), astatine-211 (²¹¹ At), cerium-134 (¹³⁴ Ce), neodymium-144 (¹⁴⁴ Nd), lanthanum-132 (¹³² La), lanthanum-135 (¹³⁵ La), and uranium-230 (²³⁰ U);

R ₁ is hydrogen and R ₂ is-L ₁-R₄;

Alternatively, R ₁ is-L ₁-R₄ and R ₂ is hydrogen;

R ₃ is hydrogen;

L ₁ is absent or a linker; and

R ₄ is a targeting ligand; wherein the targeting ligand is selected from the group consisting of: antibodies, antibody fragments (e.g., antigen binding fragments), binding moieties, binding peptides, binding polypeptides (such as selectively targeted oligopeptides containing up to 50 amino acids), binding proteins, enzymes, nucleobase containing moieties (such as oligonucleotides, DNA or RNA vectors or nucleic acid aptamers), and lectins.

In another embodiment, the invention relates to an immunoconjugate comprising a compound of the invention covalently attached to a targeting ligand (preferably an antibody or antigen binding fragment thereof) via R ₄.

In another embodiment, the radioimmunoconjugate comprises a radiometal complex of the present invention covalently linked to an antibody or antigen binding fragment thereof via a triazole moiety.

In another embodiment, the invention relates to a method of preparing an immunoconjugate or radioimmunoconjugate of the invention, comprising covalently linking a compound or radiometal complex of the invention to a targeting ligand, preferably via R ₄ of the compound or radiometal complex, to an antibody or antigen-binding fragment thereof.

In another embodiment, the invention relates to a pharmaceutical composition comprising a compound, immunoconjugate or radioimmunoconjugate of the invention and a pharmaceutically acceptable carrier. The pharmaceutical composition may comprise one or more pharmaceutically acceptable excipients.

In another embodiment, the invention also provides compositions (e.g., pharmaceutical compositions) and medicaments comprising one of the compounds (or a pharmaceutically acceptable salt thereof) as described herein and any one of a pharmaceutically acceptable carrier or one or more excipients or fillers. In similar embodiments, the invention also provides compositions (e.g., pharmaceutical compositions) and medicaments comprising any of the embodiments of the modified antibodies, modified antibody fragments, or modified binding peptides of the technology disclosed herein and a pharmaceutically acceptable carrier or one or more excipients or fillers.

In another embodiment, the invention relates to methods of targeted radiation therapy using the radioimmunoconjugates and pharmaceutical compositions of the invention.

In one embodiment, the invention relates to a method of selectively targeting neoplastic cells for radiation therapy in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of the invention.

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

Drawings

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.

In the drawings:

FIGS. 1A-1B show HPLC chromatograms from a chelation test using La ³⁺; FIG. 1A shows HPLC chromatograms of 6- ((16- ((6-carboxypyridin-2-yl) (phenyl) methyl) -1,4,10,13-tetraoxa-7, 16-diazadioctadec-7-yl) methyl) pyridinecarboxylic acid (TOPA- [ C7] -phenyl) before (top) and after (middle) mixing with La ³⁺; the retention time after mixing with La ³⁺ and Ac-225 shifted from 14.137 minutes to 12.047 minutes, indicating rapid chelation of La ³⁺; FIG. 1B shows HPLC chromatograms of TOPA- [ C7] -isopentyl before (top) and after (bottom) mixing with La ³⁺; the retention time after mixing with La ³⁺ was shifted from 17.181 minutes to 15.751 minutes, indicating that La ³⁺ and Ac-225 were rapidly sequestered by TOPA- [ C7] -isopentyl;

FIG. 2 shows a schematic representation of radiolabeling an antibody by a random conjugation method (e.g., a method for labeling lysine residues, cysteine residues, etc.) or a site-specific conjugation method (e.g., a glycan-specific method, a conjugation tag method, or an engineered cysteine method) to produce a radioimmunoconjugate according to an embodiment of the present invention; figure 2A schematically shows random conjugation via one-step direct radiolabeling; fig. 2B schematically shows random conjugation via click radiolabeling; figure 2C schematically shows site-specific conjugation via one-step direct radiolabeling; and

Fig. 2D schematically shows site-specific conjugation via click radiolabeling.

Figures 3A-3B show HPLC chromatograms from chelation assays using Ac-225. FIG. 3A shows an HPLC chromatogram (RA (radioactivity) trace of 6- ((16- ((6-carboxypyridin-2-yl) (phenyl) methyl) -1,4,10,13-tetraoxa-7, 16-diazadioctadec-7-yl) methyl) pyridine carboxylic acid (TOPA- [ C7] -phenyl) chelated with Ac-225, drawn by cleavage-counting-reconstruction. Fig. 3B shows an HPLC chromatogram (RA (radioactivity) trace of 6- ((16- (1- (6-carboxypyridin-2-yl) -4-methylpentyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridine carboxylic acid (TOPA- [ C7] -isopentyl) chelated with Ac-225, drawn by cleavage-counting-reconstruction.

Figures 4A-4B show HPLC chromatograms from chelation assays using Ac-225. FIG. 4A shows an HPLC chromatogram (UV) of TOPA- [ C7] -phenylthiourea-H11B 6 chelated with Ac-225. FIG. 4B shows an HPLC chromatogram of TOPA- [ C7] -phenylthiourea-H11B 6 chelated with Ac-225, with RA (radioactivity) traces drawn by cleavage-counting-reconstruction.

FIG. 5 shows a scan of transient thin layer chromatography (iTLC) indicating the percentage of Ac-225 bound to TOPA- [ C7] -phenylthiourea-h 11b6 in the presence of metallic impurities, as described in example 12.

FIG. 6 shows a scan of iTLC indicating the percentage of Ac-225 bound to TOPA- [ C7] -phenylthiourea-h 11b6 in the presence of metallic impurities, as described in example 12.

FIG. 7 shows a scan of iTLC indicating the percentage of Ac-225 bound to TOPA- [ C7] -phenylthiourea-h 11b6 in the presence of metallic impurities, as described in example 12.

FIG. 8 shows a scan of iTLC indicating the percentage of Ac-225 bound to TOPA- [ C7] -phenylthiourea-h 11b6 in the presence of metallic impurities, as described in example 12.

FIG. 9 shows a scan of iTLC indicating the percentage of Ac-225 sequestered with DOTA-h11b6 in the presence of metallic impurities, as described in example 12.

FIG. 10 shows a scan of iTLC indicating the percentage of Ac-225 bound to DOTA-h11b6 in the presence of metallic impurities, as described in example 12.

FIG. 11 shows a scan of iTLC indicating the percentage of Ac-225 sequestered with DOTA-h11b6 in the presence of metallic impurities, as described in example 12.

FIG. 12 shows a scan of iTLC indicating the percentage of Ac-225 bound to DOTA-h11b6 in the presence of metallic impurities, as described in example 12.

Detailed Description

Various publications, articles and patents are cited or described throughout the specification; each of these references is incorporated by reference herein in its entirety. The discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is intended to provide a context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art base with respect to any of the inventions disclosed or claimed.

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

As defined below, the following terms are used throughout the specification.

As used herein and in the appended claims, the singular forms "a," "an," and "the" and similar referents in the context of describing elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Unless otherwise indicated, any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the claims. No language in the specification should be construed as indicating any non-claimed element as essential.

In general, reference to an element such as hydrogen or H is intended to include all isotopes of that element. For example, if an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Compounds comprising radioisotopes such as tritium, C ¹⁴、P³² and S ³⁵ are therefore within the scope of the present technology. Methods of inserting such markers into compounds of the present technology will be apparent to those skilled in the art based on the disclosure herein.

The term "substituted" means that at least one hydrogen atom is replaced with a non-hydrogen group, provided that all normal valences are maintained and that the substitution results in a stable compound. When a particular group is "substituted," the group may have one or more substituents, preferably one to five substituents, more preferably one to three substituents, most preferably one to two substituents, independently selected from the list of substituents. For example, "substituted" refers to an organic group (e.g., an alkyl group) as defined below in which one or more bonds to a hydrogen atom contained therein are replaced with a bond to a non-hydrogen atom or a non-carbon atom. Substituted groups also include those wherein one or more bonds to a carbon atom or a hydrogen atom are replaced with one or more bonds to a heteroatom, including double or triple bonds. Thus, unless otherwise indicated, a substituted group is substituted with one or more substituents. In some embodiments, a substituted group is substituted with 1,2,3,4, 5, or 6 substituents. Examples of substituent groups include: halogen (i.e., F, cl, br and I); a hydroxyl group; alkoxy, alkenyloxy, aryloxy, aralkoxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy and heterocyclyloxy groups; carbonyl (oxo); a carboxylate salt; an ester; a carbamate; an oxime; a hydroxylamine; an alkoxyamine; aralkoxy amines; a mercaptan; a sulfide; sulfoxide; sulfone; a sulfonyl group; pentafluorosulfanyl (i.e., SF), sulfonamide; an amine; an N-oxide; hydrazine; a hydrazide; hydrazone; an azide; an amide; urea; an amidine; guanidine; enamines; an imide; an isocyanate; isothiocyanate; cyanate ester; thiocyanate esters; an imine; a nitro group; nitrile (i.e., CN); etc. When used with reference to substituents, the term "independently" means that when there may be more than one such substituent, such substituents may be the same or different from each other.

Substituted ring groups such as substituted cycloalkyl, aryl, heterocyclyl, and heteroaryl groups also include rings and ring systems in which the bond to a hydrogen atom is replaced by a bond to a carbon atom. Thus, substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups may also be substituted with substituted or unsubstituted alkyl, alkenyl and alkynyl groups as defined below.

As used herein, cm-Cn, such as C ₁-C₁₁、C₁-C₈ or C ₁-C₆, when used before a group, refers to a group containing m to n carbon atoms.

Alkyl groups include straight and branched alkyl groups having from 1 to 12 carbon atoms, and typically from 1 to 10 carbons, or in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Examples of the straight-chain alkyl group include groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2, 2-dimethylpropyl groups. The alkyl group may be substituted or unsubstituted. Representative substituted alkyl groups can be substituted one or more times with substituents such as those listed above, and include, but are not limited to, haloalkyl (e.g., trifluoromethyl), hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, carboxyalkyl, and the like.

Cycloalkyl groups include monocyclic, bicyclic, or tricyclic alkyl groups having 3 to 12 carbon atoms in the ring, or in some embodiments, 3 to 10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms. Exemplary monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, cycloalkyl groups have 3 to 8 ring members, while in other embodiments the number of ring carbon atoms ranges from 3 to 5, 3 to 6, or 3 to 7. Bicyclic and tricyclic ring systems include bridged cycloalkyl groups and fused rings such as, but not limited to, bicyclo [2.1.1] hexane, adamantyl, decalinyl, and the like. Cycloalkyl groups may be substituted or unsubstituted. The substituted cycloalkyl groups may be substituted one or more times with non-hydrogen groups and non-carbon groups as defined above. However, substituted cycloalkyl groups also include rings substituted with straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups may be monosubstituted or substituted more than once, such as, but not limited to, a2, 2-disubstituted, 2, 3-disubstituted, 2, 4-disubstituted, 2, 5-disubstituted or 2, 6-disubstituted cyclohexyl group that may be substituted with substituents such as those listed above.

A cycloalkylalkyl group is an alkyl group as defined above, wherein the hydrogen bond or carbon bond of the alkyl group is replaced by a bond to a cycloalkyl group as defined above. In some embodiments, the cycloalkylalkyl group has 4 to 16 carbon atoms, 4 to 12 carbon atoms, and typically 4 to 10 carbon atoms. The cycloalkylalkyl group may be substituted or unsubstituted. The substituted cycloalkylalkyl group may be substituted at the alkyl portion, the cycloalkyl portion, or both the alkyl portion and the cycloalkyl portion of the group. Representative substituted cycloalkylalkyl groups may be monosubstituted or substituted more than once, such as but not limited to monosubstituted, disubstituted or trisubstituted with substituents such as those listed above.

Alkenyl groups include straight and branched alkyl groups as defined above, except that there is at least one double bond between two carbon atoms. The alkenyl group has 2 to 12 carbon atoms, and typically 2 to 10 carbons, or in some embodiments, 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, an alkenyl group may have one carbon-carbon double bond or multiple carbon-carbon double bonds, such as 2, 3, 4, or more carbon-carbon double bonds. Examples of alkenyl groups include, but are not limited to, methine, ethenyl, propenyl, butenyl, and the like. The alkenyl group may be substituted or unsubstituted. Representative substituted alkenyl groups may be monosubstituted or substituted more than once, such as but not limited to monosubstituted, disubstituted or trisubstituted with substituents such as those listed above.

Cycloalkenyl groups include cycloalkyl groups as defined above having at least one double bond between two carbon atoms. Cycloalkenyl groups may be mono-or multicyclic alkyl groups having 3 to 12, more preferably 3 to 8, carbon atoms in the ring and containing at least one double bond between the two carbon atoms. Cycloalkenyl groups may be substituted or unsubstituted. In some embodiments, cycloalkenyl groups may have one, two, or three double bonds or multiple carbon-carbon double bonds, such as 2, 3, 4, or more carbon-carbon double bonds, but do not include aromatic compounds. Cycloalkenyl groups have 3 to 14 carbon atoms, or in some embodiments, 5 to 14 carbon atoms, 5 to 10 carbon atoms, or even 5,6, 7, or 8 carbon atoms. Examples of cycloalkenyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, cyclobutenyl and cyclopentadienyl.

Cycloalkenyl alkyl groups are alkyl groups as defined above wherein the hydrogen bond or carbon bond of the alkyl group is replaced by a bond to the cycloalkenyl group as defined above. Cycloalkenyl alkyl groups may be substituted or unsubstituted. The substituted cycloalkenylalkyl group may be substituted at the alkyl portion, the cycloalkenyl portion, or both the alkyl portion and the cycloalkenyl portion of the group. Representative substituted cycloalkenylalkyl groups may be substituted one or more times with substituents such as those listed above.

Alkynyl groups include straight and branched alkyl groups as defined above except that there is at least one triple bond between two carbon atoms. Alkynyl groups have 2 to 12 carbon atoms, and typically 2 to 10 carbons, or in some embodiments, 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, the alkynyl group has one, two, or three carbon-carbon triple bonds. Examples include, but are not limited to, -c=ch, -c=cch ₃、-CH₂C＝CCH₃、-C＝CCH₂CH(CH₂CH₃)₂, and the like. Alkynyl groups may be substituted or unsubstituted. The terminal alkyne has at least one hydrogen atom bonded to a triple bond carbon atom. Representative substituted alkynyl groups may be monosubstituted or substituted more than once, such as but not limited to monosubstituted, disubstituted or trisubstituted with substituents such as those listed above. A "cyclic alkyne" or "cycloalkynyl" is a cycloalkyl ring that contains at least one triple bond between two carbon atoms. Examples of cyclic alkyne or cycloalkynyl groups include, but are not limited to, cyclooctyne, bicyclonyne (BCN), cyclooctyne Difluoride (DIFO), dibenzocyclooctyne (DIBO), keto-DIBO, diarylazedox (BARAC), dibenzoazacyclooctyne (DIBAC), dimethoxyazacyclooctyne (DIMAC), difluorobenzocyclooctyne (DIFBO), monobenzocyclooctyne (MOBO), and Tetramethoxydibo (TMDIBO).

Aryl groups are cyclic aromatic hydrocarbons that contain no heteroatoms. Aryl groups herein include monocyclic, bicyclic and tricyclic ring systems. Thus, aryl groups include, but are not limited to, phenyl, azulenyl (azulenyl), cycloheptenyl (heptalenyl), biphenyl, fluorenyl, phenanthryl, anthracyl, indenyl, indanyl, pentalenyl (pentalenyl), and naphthyl groups. In some embodiments, aryl groups contain 6-14 carbons in the ring portion of the group, and in other embodiments 6 to 12 or even 6-10 carbon atoms. In some embodiments, the aryl group is phenyl or naphthyl. The aryl group may be substituted or unsubstituted. The phrase "aryl group" includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, etc.). Representative substituted aryl groups may be monosubstituted or substituted more than once. For example, monosubstituted aryl groups include, but are not limited to, 2-substituted, 3-substituted, 4-substituted, 5-substituted or 6-substituted phenyl or naphthyl groups that may be substituted with substituents such as those listed above. Aryl moieties are well known and are described, for example, in Lewis, R.J. editions, hawley's Condensed Chemical Dictionary, 13 th edition, john Wiley & Sons, inc., new York (1997). The aryl group may be a monocyclic structure (i.e., monocyclic) or comprise a polycyclic structure (i.e., polycyclic) which is a fused ring structure. Preferably, the aryl group is a monocyclic aryl group.

An alkoxy group is a hydroxyl group (-OH) in which the bond to a hydrogen atom is replaced by a bond to a carbon atom of a substituted or unsubstituted alkyl group as defined above. Examples of straight chain alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, and the like. Examples of branched alkoxy groups include, but are not limited to, isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cycloalkyloxy groups include, but are not limited to, cyclopropyloxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, and the like. The alkoxy group may be substituted or unsubstituted. Representative substituted alkoxy groups may be substituted one or more times with substituents such as those listed above.

Similarly, alkylthio or thioalkoxy refers to an-SR group, wherein R is an alkyl group attached to the parent molecule through a sulfur bridge, such as-S-methyl, -S-ethyl, and the like. Representative examples of alkylthio groups include, but are not limited to, -SCH ₃、-SCH₂CH₃ and the like.

The term "halogen" as used herein refers to bromine, chlorine, fluorine or iodine. Accordingly, the term "halo" refers to fluoro, chloro, bromo or iodo. In some embodiments, the halogen is fluorine. In other embodiments, the halogen is chlorine or bromine.

The terms "hydroxy" and "hydroxyl" are used interchangeably and refer to-OH.

The term "carboxy" refers to-COOH.

The term "cyano" refers to-CN.

The term "nitro" refers to-NO ₂.

The term "isothiocyanate" refers to-n=c=s.

The term "isocyanate" refers to-n=c=o.

The term "azido" refers to-N ₃.

The term "amino" refers to-NH ₂. The term "alkylamino" refers to an amino group in which one or both of the hydrogen atoms attached to the nitrogen is replaced with an alkyl group. The alkylamine group may be represented as-NR ₂, wherein each R is independently hydrogen or an alkyl group. For example, alkylamines include methylamine (-NHCH ₃), dimethylamine (-N (CH ₃)₂)、-NHCH-₂CH₃, etc. As used herein, the term "aminoalkyl" is intended to include both branched saturated aliphatic hydrocarbon groups and straight saturated aliphatic hydrocarbon groups substituted with one or more amino groups.

As used herein, "amide" refers to-C (O) N (R) ₂, wherein each R is independently an alkyl group or hydrogen. Examples of amides include, but are not limited to, -C (O) NH ₂、-C(O)NHCH₃ and-C (O) N (CH ₃)₂).

The terms "hydroxyalkyl" and "hydroxyalkyl" are used interchangeably and refer to an alkyl group substituted with one or more hydroxyl groups. Alkyl groups may be branched or straight chain aliphatic hydrocarbons. Examples of hydroxyalkyl groups include, but are not limited to, hydroxymethyl (-CH ₂ OH), hydroxyethyl (-CH ₂CH₂ OH), and the like.

As used herein, the term "heterocyclyl" includes stable mono-and polycyclic hydrocarbons containing at least one heteroatom ring member, such as sulfur, oxygen or nitrogen. As used herein, the term "heteroaryl" includes stable monocyclic and polycyclic aromatic hydrocarbons containing at least one heteroatom ring member, such as sulfur, oxygen, or nitrogen. Heteroaryl groups may be monocyclic or polycyclic, for example bicyclic or tricyclic. Each ring of a heterocyclyl or heteroaryl group containing a heteroatom may contain one or two oxygen or sulfur atoms and/or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less and that each ring has at least one carbon atom. A polycyclic (e.g., bicyclic or tricyclic) heteroaryl group must include at least one fully aromatic ring, but another one or more fused rings may be aromatic or non-aromatic. The heterocyclyl or heteroaryl group may be attached at any available nitrogen or carbon atom of any ring of the heterocyclyl 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 at least one heteroatom (O, S or N) in at least one ring, wherein the heteroatom-containing ring preferably has 1,2 or 3 heteroatoms, more preferably 1 or 2 heteroatoms, selected from O, S and/or N. The nitrogen heteroatom of the heteroaryl group may be substituted or unsubstituted. In addition, the nitrogen and sulfur heteroatoms of the heteroaryl groups can optionally be oxidized (i.e., N→O and S (O) _r, where r is 0,1, or 2).

The term "ester" refers to-C (O) ₂ R, wherein R is alkyl.

The term "carbamate" refers to the group-OC (O) NR ₂, where each R is independently an alkyl group or hydrogen.

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

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

The term "maleimide" refers to a group of the formula H ₂C₂(CO)₂ NH. The term "maleimide group" refers to a maleimide group covalently linked to another group or molecule. Preferably, the maleimide group is N-linked, for example:

The term "acyl halide" refers to-C (O) X, wherein X is halo (e.g., br, cl). Exemplary acid halides include acid chloride (-C (O) Cl) and acid bromide (-C (O) Br).

According to the convention used in the art:

As used in the formulae herein to describe bonds as attachment points of moieties, functional groups or substituents to core, parent or backbone structures such as the compounds or targeting ligands of the invention.

When any variable occurs more than once in any component or formula of a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-3R groups, then the group may optionally be substituted with up to three R groups, and at each occurrence R is independently selected from the definition of R.

When the bond to a substituent is shown as intersecting a bond connecting two atoms in a ring, then such substituent may be bound to any atom on the ring.

As used herein, the term "radioactive metal ion (radiometal ion)" or "radioactive metal ion (radioactive metal ion)" refers to one or more isotopes of an element that emits particles and/or photons. Any radioactive metal ion known to those skilled in the art may be used in the present invention in light of the present disclosure. Examples of radioactive metal ions suitable for use in the present invention include, but are not limited to ：⁴⁷Sc、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁷Ga、⁶⁸Ga、⁸⁶Y、⁸⁹Zr、⁸⁹Sr、⁹⁰Y、⁹⁹Tc、¹⁰⁵Rh、¹⁰⁹Pd、¹¹¹Ag、¹¹¹In、¹¹⁷Sn、¹⁴⁹Tb、¹⁵²Tb、¹⁵⁵Tb、¹⁵³Sm、¹⁵⁹Gd、¹⁶⁵Dy、¹⁶⁶Ho、¹⁶⁹Er、¹⁷⁷Lu、¹⁸⁶Re、¹⁸⁸Re、¹⁹⁴Ir、¹⁹⁸Au、¹⁹⁹Au、²¹¹At、²¹²Pb、²¹²Bi、²¹³Bi、²²³Ra、²²⁵Ac、²²⁷Th and ²⁵⁵ Fm. Preferably, the radioactive metal ion is a "therapeutic emitter", meaning a radioactive metal ion that can be used in therapeutic applications. Examples of therapeutic emitters include, but are not limited to, beta emitters or alpha emitters, such as ¹³²La、¹³⁵La、¹³⁴Ce、¹⁴⁴Nd、¹⁴⁹Tb、¹⁵²Tb、¹⁵⁵Tb、¹⁵³Sm、¹⁵⁹Gd、¹⁶⁵Dy、¹⁶⁶Ho、¹⁶⁹Er、¹⁷⁷Lu、¹⁸⁶Re、¹⁸⁸Re、¹⁹⁴Ir、¹⁹⁸Au、¹⁹⁹Au、²¹¹At、²¹²Pb、²¹²Bi、²¹³Bi、²²³Ra、²²⁵Ac、²⁵⁵Fm and ²²⁷Th、²²⁶Th、²³⁰ U. Preferably, the radioactive metal ions used in the present invention are alpha emitting radioactive metal ions such as actinium-225 (²²⁵ Ac).

The compounds of the present invention are macrocyclic compounds with which metals, preferably radiometals, can complex. In certain embodiments, the compounds are macrocycles (macrocycle/macrocyclic ring) containing one or more heteroatoms (e.g., oxygen and/or nitrogen) as ring atoms. Preferably, the compound is a macrocyclic ring of a derivative of 4, 13-diaza-18-crown-6.

As used herein, "radiometal complex" refers to a complex comprising a radiometal ion associated with a macrocyclic compound. The radioactive metal ion is bound or coordinated to the macrocycle via a coordinate bond. The heteroatoms of the macrocycle may be involved in coordinate bonding of the radioactive metal ion to the macrocyclic compound. The macrocyclic compound may be substituted with one or more substituent groups, and the one or more substituent groups may also participate in coordination bonding of the radiometal ion to the macrocyclic compound, in addition to or alternatively to the heteroatom of the macrocyclic compound.

As used herein, the term "TOPA" refers to a macrocyclic ring known in the art as H ₂ bp18c6, and may alternatively be referred to as N, N' -bis [ (6-carboxy-2-pyridinyl) methyl ] -4, 13-diaza-18-crown-6. See, for example, roca-Sabio et al ,"Macrocyclic Receptor Exhibiting Unprecedented SelectivityforLightLanthanides,"J.Am.Chem.Soc.(2009)131,3331-3341,, which is incorporated herein by reference.

As used herein, the term "click chemistry" refers to the chemical concept introduced by Sharpless, which describes a chemistry that is modulated to rapidly and reliably generate covalent bonds by linking together small units that contain reactive groups (see Kolb et al ANGEWANDTE CHEMIE International Edition (2001) 40:2004-2021). Click chemistry does not refer to a specific reaction, but rather refers to a concept that includes, but is not limited to, a reaction that mimics a reaction that exists in nature. In some embodiments, click chemistry reactions are modular, broad in scope, give high chemical yields, produce inert byproducts, are stereospecific, exhibit a large thermodynamic driving force to facilitate reaction with a single reaction product, and/or can be performed under physiological conditions. In some embodiments, click chemistry reactions can be performed under simple reaction conditions, using readily available starting materials and reagents, using non-toxic solvents or using benign or readily removable solvents such as water, and/or providing simple product isolation by non-chromatographic methods such as crystallization or distillation.

Click chemistry reactions utilize reactive groups that are rarely present in naturally occurring biomolecules and are chemically inert to biomolecules, but when click chemistry partners are reacted together, the reaction can occur effectively under biologically relevant conditions, e.g., under cell culture conditions, such as in the absence of excessive heat and/or harsh reagents. Generally, click chemistry reactions require at least two molecules comprising click reaction partners that can react with each other. Such click reaction partners that react with each other are sometimes referred to herein as click chemistry handle pairs or click chemistry pairs. In some embodiments, the click reaction partner is an azide or strained alkyne, e.g., a cycloalkyne such as cyclooctyne or cyclooctyne derivative, or any other alkyne. In other embodiments, the click reaction partner is a reactive diene and a suitable tetrazine dienophile. For example, trans-cyclooctene, norbornene, or bicyclononene can be paired with a suitable tetrazine dienophile as a click reaction pair. In other embodiments, tetrazoles may serve as a potential source of nitrilimine, which may pair with unactivated olefins in the presence of ultraviolet light to produce click reaction pairs, referred to as "light click" reaction pairs. In other embodiments, the click reaction partners are cysteine and maleimide. For example, a cysteine from a peptide (e.g., GGGC (SEQ ID NO: 23)) can be reacted with a maleimide that is associated with a chelator (e.g., NOTA). Other suitable click chemistry handles are known to those skilled in the art (see, e.g., spicer et al, SELECTIVE CHEMICAL protein modification. Nature communications.2014;5: p.4740). In other embodiments, the click reaction partner is a staudinger ligation (Staudinger ligation) component, such as phosphine and azide. In other embodiments, the click reaction partner is a diels-alder reaction component, such as 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, the relevant descriptions of which are both incorporated herein by reference.

According to a preferred embodiment, the click chemistry reaction utilizes an azide group and an alkyne group, more preferably a strained alkyne group, for example a cycloalkyne such as cyclooctyne or cyclooctyne derivative, as a click chemistry pair or reaction partner. In such embodiments, the click chemistry reaction is a Huisgen cycloaddition or a1, 3-dipolar cycloaddition between an azide (-N ₃) and an alkyne moiety to form a1, 2, 3-triazole linker. Click chemistry reactions between alkynes and azides typically require the addition of copper catalysts to promote the 1, 3-cycloaddition reaction, and are known as copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions. However, click chemistry between cyclooctyne or cyclooctyne derivatives and azides does not typically require the addition of copper catalysts, but rather proceeds via strain-promoted azide-alkyne cycloaddition (sparc) (Debets, m.f. et al, bioconjugationwith strainedalkenes analkynes. Acc ChemRes,2011.44 (9): p.805-15).

The term "targeting ligand" as used herein refers to any molecule that provides enhanced affinity for a selected target (e.g., antigen, cell type, tissue, organ, body region or compartment (e.g., cell, tissue or organ compartment).

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

Generally, an antibody is a protein or peptide chain that exhibits binding specificity for a particular antigen (referred to herein as a "target"). Antibody structures are well known. Immunoglobulins can be assigned to five major classes, igA, igD, igE, igG and IgM, based on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified into isotypes IgA1, igA2, igG1, igG2, igG3 and IgG4. Antibodies used in the present invention may be of any of five major classes or corresponding subclasses. Based on the amino acid sequence of its constant domain, the antibody light chain of any spinal species can be assigned to one of two completely different types, namely kappa and lambda. According to particular embodiments, antibodies for use in the present invention include heavy and/or light chain constant regions from a mouse or human antibody. Each of the four IgG subclasses has a different biological function, which is called effector function. These effector functions are typically mediated through interactions with Fc receptors (fcγr) or by binding C1q and fixing complement. Binding to fcγr can result in antibody dependent cell-mediated lysis, while binding to complement factors can result in complement-mediated cell lysis. Antibodies useful in the invention may have no or minimal effector function, but retain their ability to bind FcRn.

As used herein, the term "antigen binding fragment" refers to an antibody fragment, such as, for example, a diabody, fab ', F (ab ') 2, fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv) 2, bispecific dsFv (dsFv-dsFv '), disulfide stabilized diabody (ds diabody), single chain antibody molecule (scFv), single domain antibody (sdab), scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody comprising one or more CDRs, camelized single domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise an intact antibody structure. The antigen binding fragment is capable of binding to the same antigen as the parent antibody or parent antibody fragment binds to. As used herein, the term "single chain antibody" refers to a conventional single chain antibody in the art comprising a heavy chain variable region and a light chain variable region linked by a short peptide of about 15 to about 20 amino acids. As used herein, the term "single domain antibody" refers to conventional single domain antibodies in the art that comprise a heavy chain variable region and a heavy chain constant region or comprise only a heavy chain variable region.

As used herein, the term "scaffold" or "scaffold protein" refers to any protein that has a target binding domain and that can bind to a target. The scaffold contains a "framework" that is largely structured, and a "binding domain" that contacts the target and provides specific binding. The binding domain of the scaffold need not be defined by one continuous sequence of the scaffold. In some cases, the scaffold may be part of a larger binding protein, which itself may be part of a multimeric binding protein comprising multiple scaffolds. Some binding proteins may be bispecific or multispecific in that they may bind to two or more different epitopes. The scaffold may be derived from a single chain antibody, or the scaffold may not be derived from an antibody.

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

Pharmaceutically acceptable salts of the compounds described herein are within the scope of the present technology and include acid addition salts or base addition salts that retain the desired pharmacological activity and are not biologically undesirable (e.g., the salts are not overly toxic, allergic or irritating, and are bioavailable). When the compounds of the present technology have basic groups such as, for example, amino groups, pharmaceutically acceptable salts can be formed with inorganic acids (such as hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid), organic acids (such as alginate, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and p-toluenesulfonic acid), or acidic amino acids (such as aspartic acid and glutamic acid). When a compound of the present technology has an acidic group such as, for example, a carboxylic acid group, it may form salts with metals such as alkali metals and alkaline earth metals (e.g., na ⁺、Li⁺、K⁺、Ca²⁺、Mg²⁺、Zn²⁺), ammonia or organic amines (e.g., dicyclohexylamine, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine), or basic amino acids (e.g., arginine, lysine, and ornithine). Such salts may be prepared in situ during isolation and purification of the compounds, or by reacting the purified compounds in the form of the free base or free acid, respectively, with a suitable acid or base, respectively, and isolating the salt thus formed.

Those skilled in the art will appreciate that the compounds of the present technology may exhibit tautomerism, conformational isomerism, geometric isomerism and/or stereoisomerism. Since the chemical formulas within the specification and claims may represent only one of the possible tautomeric, conformational, stereoisomeric or geometric forms, it is to be understood that the present technology encompasses any tautomeric, conformational, stereoisomeric and/or geometric form of a compound having one or more of the utility as described herein, as well as mixtures of these different forms.

Stereoisomers (also known as optical isomers) of a compound include all chiral, diastereomeric, and racemic forms of the structure unless a specific stereochemical structure is specifically indicated. Thus, compounds used in the present technology include optical isomers that are enriched or resolved at any or all asymmetric atoms, as will be apparent from the description. Both racemic and diastereomeric mixtures, as well as individual optical isomers, can be separated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these stereoisomers are all within the scope of the technology.

The present technology provides novel macrocyclic complexes that are significantly more stable than those of conventional technology. Thus, these novel complexes can advantageously target cancer cells more effectively with significantly less toxicity to non-targeted tissues than the complexes of the art. Furthermore, the new complexes may advantageously be produced at room temperature compared to DOTA-type complexes which typically require elevated temperatures (e.g. at least 80 ℃) for complexation with radionuclides. The present technology also specifically uses alpha emitting radionuclides instead of beta radionuclides. The alpha emitting radionuclide has a much higher energy than the beta emitting radionuclide and is therefore significantly more efficient.

While certain embodiments have been illustrated and described, modifications, substitutions of equivalents, and other types of modifications may be made to the compounds of the present technology or salts, pharmaceutical compositions, derivatives, prodrugs, metabolites, tautomers, or racemic mixtures thereof as described herein by one of ordinary skill in the art after reading the foregoing description. Each of the aspects and embodiments described above may also include or incorporate such variations or aspects disclosed in relation to any or all of the other aspects and embodiments.

The present technology is also not limited to the specific implementations described herein, which are intended as single illustrations of various aspects of the technology. Many modifications and variations of this technique may be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. It is to be understood that the present technology is not limited to particular methods, reagents, compounds, compositions, labeling compounds, or biological systems, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It is intended, therefore, that the specification be considered as exemplary only, with a breadth, scope and spirit of the present technology being indicated only by the appended claims, their definitions and any equivalents.

The embodiments illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like are to be construed broadly and without limitation. In addition, the terms and expressions which have been employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the technology claimed. In addition, the phrase "consisting essentially of … …" will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase "consisting of … …" excludes any elements not specified.

All publications, patent applications, issued patents, and other documents (e.g., journals, articles, and/or textbooks) mentioned in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent or other document was specifically and individually indicated to be incorporated by reference in its entirety. The definitions contained in the text incorporated by reference are excluded to the extent that they contradict the definitions in this disclosure.

In one embodiment of the invention, intermediate compound 14 is prepared:

Reacting compound 9 with N, O-bis (trimethylsilyl) acetamide (BSA) in an organic solvent or mixture thereof at a temperature ranging from about ambient temperature to about-78 ℃, adding a solution of trimethylsilicone triflate (TMSOTf) in an organic solvent to the reaction to give compound 10;

In another embodiment of the invention, compound 14 is prepared:

a preferred synthetic procedure in the process of the pharmaceutically acceptable salt or solvate thereof is: the method comprises the following steps:

In another embodiment, compound 10

Or a pharmaceutically acceptable salt or solvate thereof, by a process comprising the steps of:

Removal of the protecting group t-butoxycarbonyl has proven challenging and many conditions have been tried but none have been successful. Attempts were made under acidic and basic conditions (HCl, TFA, MSA, phosphoric acid, KOAc/AcOH, tsCl-DMAP, BF ₃.OEt₂, TMSCl and CsCO ₃). All of this results in rapid decomposition.

To overcome this synthetic challenge, it was determined that the conditions and reagents used in the deprotection step of the t-butoxycarbonyl were successfully completed using BSA and TMSOTf reagents. The mild conditions using BSA and TMSOTf reagents were unexpected, giving compound 10.

One embodiment of the present invention is a compound of formula (14):

or a pharmaceutically acceptable salt or solvate thereof.

Another embodiment of the invention is a compound of formula (11)

Or a pharmaceutically acceptable salt or solvate thereof.

or a pharmaceutically acceptable salt or solvate thereof.

Compounds of the invention (macrocyclic compounds)

In one embodiment, the present invention relates to compounds of formula (I)

Or a pharmaceutically acceptable salt thereof, wherein:

R ₁ is hydrogen and R ₂ is-L ₁-R₄;

Alternatively, R ₁ is-L ₁-R₄ and R ₂ is hydrogen;

R ₃ is hydrogen;

L ₁ is absent or a linker; and

R ₄ is a nucleophilic moiety, electrophilic moiety, or targeting ligand.

In some embodiments, L ₁ is absent. When L ₁ is absent, R ₄ is directly bound (e.g., via covalent bonding) to the compound.

In some embodiments, L ₁ is a linker. As used herein, the term "linker" refers to a chemical moiety that binds a compound of the invention to a nucleophilic moiety, electrophilic moiety, or targeting ligand. Any suitable linker known to those skilled in the art may be used in the present invention in light of the present disclosure. The linker may have, for example, a substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl moiety, substituted or unsubstituted aryl or heteroaryl, a polyethylene glycol (PEG) linker, a peptide linker, a sugar-based linker, or a cleavable linker, such as a disulfide bond or a protease cleavage site, such as valine-citrulline-p-aminobenzyl (PAB). Exemplary linker structures suitable for use in the present invention include, but are not limited to:

Wherein m is an integer from 0 to 12.

In some embodiments, R ₄ is a nucleophilic moiety or electrophilic moiety. "nucleophilic moiety" or "nucleophilic group" refers to a functional group that provides an electron pair in a chemical reaction to form a covalent bond. "electrophilic moiety" or "electrophilic group" refers to a functional group that accepts an electron pair in a chemical reaction to form a covalent bond. The nucleophilic group reacts with the electrophilic group and vice versa to form a new covalent bond in the chemical reaction. The reaction of the nucleophilic or electrophilic group of the compounds of the invention with a targeting ligand or other chemical moiety (e.g., linker) comprising a corresponding reaction partner allows covalent bonding of the targeting ligand or chemical moiety to the compounds of the invention.

Illustrative examples of nucleophilic groups include, but are not limited to, azides, amines, and thiols. Illustrative examples of electrophilic groups include, but are not limited to, amine-reactive groups, thiol-reactive groups, alkynyl groups, and cycloalkynyl groups. The amine reactive groups preferably react with primary amines, including primary amines present in the N-terminus of each polypeptide chain and in the side chains of lysine residues. Examples of amine-reactive groups suitable for use in the present invention include, but are not limited to, N-hydroxysuccinimide (NHS), substituted NHS (such as sulfo-NHS), isothiocyanate (-NCS), isocyanate (-NCO), ester, carboxylic acid, acid halide, amide, alkylamide, and tetrafluorophenyl and perfluorophenyl esters. The thiol reactive group reacts with a thiol or thiol group (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), haloacetyl, acyl halides, activated disulfides, and phenyloxadiazole sulfones.

In certain embodiments, R ₄ is-NH ₂, -NCS (isothiocyanate), -NCO (isocyanate), -N ₃ (azido), alkynyl, cycloalkynyl, carboxylic acid, ester, amido, alkylamide, maleimide, acyl halide, tetrazine, or trans-cyclooctene, more specifically, -NCS, -NCO, -N ₃, alkynyl, cycloalkynyl, -C (O) R ₁₃、-COOR₁₃、-CON(R₁₃)₂, maleimide, acyl halide (e.g., -C (O) Cl, -C (O) Br), tetrazine, or trans-cyclooctene, wherein each R ₁₃ is independently hydrogen or alkyl.

In some embodiments, R ₄ is an alkynyl, cycloalkynyl, or azido group, allowing the compounds of the invention to be attached to a targeting ligand or other chemical moiety (e.g., linker) using a click chemistry reaction. In such embodiments, the click chemistry that can be performed is Huisgen cycloaddition or 1, 3-dipolar cycloaddition between an azide (-N ₃) and an alkynyl or cycloalkynyl group to form a1, 2, 4-triazole linker or moiety. In one embodiment, the compounds of the invention comprise an alkynyl or cycloalkynyl group, and the targeting ligand or other chemical moiety comprises an azido group. In another embodiment, the compounds of the invention comprise an azido group and the targeting ligand or other chemical moiety comprises an alkynyl or cycloalkynyl group.

In certain embodiments, R ₄ is an alkynyl group that reacts with an azide group, more preferably a terminal alkynyl group or a cycloalkynyl group, specifically via strain-promoted azide-alkyne cycloaddition (sparc). Examples of cycloalkynyl groups that can be reacted with azide groups via sparc include, but are not limited to, cyclooctynyl or Bicyclononyl (BCN), cyclooctynyl Difluoride (DIFO), dibenzocyclooctynyl (DIBO), keto-DIBO, diarylazedoxynyl (BARAC), dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO), dimethoxyazacyclooctynyl (DIMAC), difluorobenzocyclooctynyl (DIFBO), monobenzocyclooctynyl (MOBO), and Tetramethoxydibenzocyclooctynyl (TMDIBO).

In certain embodiments, R ₄ is dibenzoazacyclooctynyl (DIBAC, DBCO, ADIBO), which has the structure:

In embodiments wherein R ₄ is DBCO, DBCO may be covalently linked to the compound directly or indirectly via a linker, and preferably is linked to the compound indirectly via a linker.

In certain embodiments, R ₄ is a targeting ligand. The targeting ligand may be attached to the compound directly via covalent bonding, or indirectly via a linker. The targeting ligand may be a polypeptide, such as an antibody or antigen binding fragment thereof, a nucleic acid aptamer, or a scaffold protein, or the like. In preferred embodiments, the targeting ligand is an antibody or antigen binding fragment thereof, such as an antibody or antigen binding fragment thereof, e.g., a monoclonal antibody (mAb) or antigen binding fragment thereof, that specifically binds 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.

According to particular embodiments, the targeting ligand specifically binds to a prostate specific antigen (e.g., PSMA or KLK 2).

In another embodiment, the invention relates to a compound of formula (II):

Or a pharmaceutically acceptable salt thereof, wherein:

L ₁ is absent or a linker; and

R ₄ is a nucleophilic moiety, electrophilic moiety, or targeting ligand.

In another embodiment the invention relates to compounds of formula (III):

Or a pharmaceutically acceptable salt thereof, wherein:

L ₁ is absent or a linker; and

R ₄ is a nucleophilic moiety, electrophilic moiety, or targeting ligand.

In another embodiment, the invention relates to a compound wherein: r ₁ is-L ₁-R₄;R₂ and R ₃ taken together with the carbon atom to which they are attached to form a 5-or 6-membered cycloalkyl; l ₁ is absent or a linker; and R ₄ is a nucleophilic moiety, electrophilic moiety, or targeting ligand; or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention relates to a compound, wherein R ₁ is H; r ₂ and R ₃ together with the carbon atom to which they are attached form a 5-or 6-membered cycloalkyl substituted by-L ₁-R₄; l ₁ is absent or a linker; and R ₄ is a nucleophilic moiety, electrophilic moiety, or targeting ligand; or a pharmaceutically acceptable salt thereof.

Additional embodiments include those wherein R ₄ is a targeting ligand, wherein the targeting ligand is selected from the group consisting of: antibodies, antigen binding fragments of antibodies, scaffold proteins, and nucleic acid aptamers.

In one embodiment, the compounds of the present invention are any one or more compounds independently selected from the group consisting of:

/>

wherein n is 1 to 10.

The compound can be covalently linked to a targeting ligand (e.g., an antibody or antigen binding fragment thereof) to form an immunoconjugate or radioimmunoconjugate (when complexed with a metal) by reacting the compound with an azide-labeled targeting ligand to form a 1,2, 3-triazole linkage via a click chemistry reaction, as described in more detail below.

In accordance with the present disclosure, the compounds of the present invention may be produced by any method known in the art. For example, pendant aromatic/heteroaromatic groups may be attached to the macrocyclic moiety by methods known in the art, such as those illustrated and described below.

Radioactive metal complex

In certain embodiments, the present invention relates to a radiometal complex comprising a radiometal ion complexed with a compound of the present invention via a coordinate bond. Any of the compounds of the invention described herein may comprise a radioactive metal ion. Preferably, the radioactive metal ion is an alpha emitting radioactive metal ion, more preferably ²²⁵ Ac. The compounds of the invention can complex with radioactive metal ions, particularly ²²⁵ Ac, at any specific activity, irrespective of metal impurities, to form radioactive metal complexes that have high chelation stability in vivo and in vitro and are stable to test reagents such as diethylenetriamine pentaacetic acid (DTPA).

In certain embodiments, the invention relates to a radiometal complex structure of formula (I-M ⁺):

Or a pharmaceutically acceptable salt thereof, wherein:

R ₁ is hydrogen and R ₂ is-L ₁-R₄;

Alternatively, R ₁ is-L ₁-R₄ and R ₂ is hydrogen;

R ₃ is hydrogen;

L ₁ is absent or a linker;

r ₄ is a nucleophilic moiety, electrophilic moiety, or targeting ligand.

In another embodiment, the invention relates to a radiometal complex of formula (II-M ⁺):

Or a pharmaceutically acceptable salt thereof, wherein:

L ₁ is absent or a linker;

r ₄ is a nucleophilic moiety, electrophilic moiety, or targeting ligand.

In another embodiment, the invention relates to a radiometal complex of formula (III-M ⁺):

Or a pharmaceutically acceptable salt thereof, wherein:

M ⁺ is a radioactive metal ion, wherein M ⁺ is selected from the group consisting of: actinium-225 (²²⁵ Ac), radium-223 (²³³ Ra), bismuth-213 (²¹³ Bi), lead-212 (²¹² Pb (II) and/or ²¹² Pb (IV)), terbium-149 (¹⁴⁹ Tb), terbium-152 (¹⁵² Tb), terbium-155 (¹⁵⁵ Tb), 255 (²⁵⁵ Fm), thorium-227 (²²⁷ Th), thorium-226 (²²⁶Th⁴⁺), astatine-211 (²¹¹ At), cerium-134 (¹³⁴ Ce), neodymium-144 (¹⁴⁴ Nd), lanthanum-132 (¹³² La), lanthanum-135 (¹³⁵ La) and uranium-230 (²³⁰U);L₁ is absent or a linker; and

R ₄ is a nucleophilic moiety, electrophilic moiety, or targeting ligand.

In another embodiment, the invention relates to a radiometal complex, wherein:

R ₁ is-L ₁-R₄;

R ₂ and R ₃ together with the carbon atom to which they are attached form a 5-or 6-membered cycloalkyl;

L ₁ is absent or a linker; and

R ₄ is a nucleophilic moiety, electrophilic moiety, or targeting ligand;

Or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention relates to a radioactive metal complex wherein

R ₁ is H;

R ₂ and R ₃ together with the carbon atom to which they are attached form a 5-or 6-membered cycloalkyl substituted by-L ₁-R₄;

L ₁ is absent or a linker; and

R ₄ is a nucleophilic moiety, electrophilic moiety, or targeting ligand;

Or a pharmaceutically acceptable salt thereof.

In certain embodiments, the invention relates to any one or more radioactive metal complexes selected from the group consisting of:

/>

Wherein n is 1 to 10 and M ⁺ is a radioactive metal ion, wherein M ⁺ is selected from the group consisting of: actinium-225 (²²⁵ Ac), radium-223 (²³³ Ra), bismuth-213 (²¹³ Bi), lead-212 (²¹² Pb (II) and/or ²¹² Pb (IV)), terbium-149 (¹⁴⁹ Tb), terbium-152 (¹⁵² Tb), terbium-155 (¹⁵⁵ Tb), lead-212 (²¹² Pb (II) and/or ²¹² Pb (IV), Is selected from the group consisting of (A) thorium-255 (²⁵⁵ Fm), thorium-227 (²²⁷ Th), thorium-226 (²²⁶Th⁴⁺), astatine-211 (²¹¹ At), cerium-134 (¹³⁴ Ce), neodymium-144 (¹⁴⁴ Nd), lanthanum-132 (¹³² La), lanthanum-135 (¹³⁵ La) and uranium-230 (²³⁰ U).

According to the present disclosure, the radioactive metal complex may be produced by any method known in the art. For example, the macrocyclic compounds of the invention can be mixed with a radioactive metal ion and the mixture incubated to allow the formation of a radioactive metal complex. In one exemplary embodiment, a compound is mixed with a solution of ²²⁵Ac(NO₃)₃ to form a radioactive complex comprising ²²⁵ Ac bound to the compound via a coordinate bond. As mentioned above, the compounds of the present invention are effective in sequestering radiometals, particularly ²²⁵ Ac. Thus, in particular embodiments, a solution of a compound of the invention and ²²⁵ Ac ions is mixed at a concentration ratio of the compound of the invention to ²²⁵ Ac ions 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. Thus, in some embodiments, the ratio of the compounds of the present invention to ²²⁵ Ac useful in forming the radiometal complex is much lower than that achievable with other known ²²⁵ Ac chelators (e.g., DOTA). The radioactive complex can be characterized by transient thin layer chromatography (e.g., iTLC-SG), HPLC, LC-MS, and the like. Exemplary methods are described herein, for example, in the following examples.

Immunoconjugates and radioimmunoconjugates

In another embodiment, the invention relates to immunoconjugates and radioimmunoconjugates. The compounds of the invention and the radiometal complexes of the invention can be conjugated (i.e., covalently linked) to a targeting ligand, such as an immune substance, to produce immunoconjugates and/or radioimmunoconjugates suitable for medical applications, such as targeted radiation therapy, e.g., in a subject (e.g., a human). Using the macrocyclic compounds, radiometal complexes and radioimmunoconjugates of the present invention, particularly antibodies or antigen-binding fragments thereof that specifically bind to a target of interest, such as a cancer cell, site-specific labeling with radiometal ions can be performed to produce the radioimmunoconjugates. In particular, using the compounds of the invention and/or the radiometal complexes of the invention, a radioimmunoconjugate having high yields of complexation to radiometal ions, particularly ²²⁵ Ac, and a desired compound-to-antibody ratio (CAR) can be produced.

According to particular embodiments, the methods of the invention provide an average CAR of less than 10, less than 8, less than 6, or less than 4; or between about 2 and about 8, or about 2 to about 6, or about 2 to about 4, or about 2 to about 3; or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8.

As used herein, an "immunoconjugate" is an antibody or antigen-binding fragment thereof conjugated (e.g., bound via a covalent bond) to a second molecule such as a toxin, drug, radiometal ion, radiometal complex, or the like. In particular, a "radioimmunoconjugate" (which may also be referred to as a radio conjugate) is an immunoconjugate in which the antibody or antigen-binding fragment thereof is labeled with or conjugated to a radiometal complex.

In certain embodiments of the invention, the immunoconjugate comprises a compound of the invention, e.g., a compound of formula (I) as described herein, covalently attached to an antibody or antigen binding fragment thereof, preferably via a linker. Many modes of attachment are possible according to the reactive functional groups (i.e., nucleophiles and electrophiles) on the compounds of formula (I) and antibodies or antigen binding fragments thereof, with different linkages between the compounds of the invention and the antibodies or antigen binding fragments thereof.

In certain embodiments of the invention, the radioimmunoconjugate comprises a radiometal complex of the invention, such as a radiometal complex as described herein, covalently attached, preferably via a linker, to an antibody or antigen binding fragment thereof.

Any of the compounds or radiometal complexes of the invention described herein may be used to produce immunoconjugates or radioimmunoconjugates of the invention.

In certain embodiments, the radiometal complexes or radioimmunoconjugates of the present invention comprise an alpha-emitting radiometal ion coordinated to a compound moiety of the radiocomplex. Preferably, the alpha emitting radioactive metal ion is ²²⁵ Ac.

In certain embodiments, the antibody or antigen binding fragment in an immunoconjugate or radioimmunoconjugate of the application can specifically bind to a tumor antigen. Preferably, the antibody or antigen binding fragment specifically binds to a cancer antigen. Examples of cancer antigens include, but are not limited to, prostate Specific Membrane Antigen (PSMA), BCMA, her2, EGFR, KLK2, CD19, CD22, CD30, CD33, CD79b, and Nectin-4.

In one embodiment, the antibody specifically binds PSMA. Preferably, the antibody is PSMB127. A human IgG4 antibody that binds to human Prostate Specific Membrane Antigen (PSMA) is referred to herein as an "anti-PSMA mAb," designated "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. anti-PSMA mabs were expressed and purified using standard chromatographic methods. Antibody PSMB127, its biological activity, use, or other relevant information is described, for example, in U.S. patent application publication US20200024360A1, the contents of which are hereby incorporated by reference in their entirety.

In another embodiment, the antibody specifically binds human kallikrein-2 (KLK 2). KLK2 may also be referred to as hK2. Preferably, the antibody is H11B6 (also known as H11B 6). The H11B6 antibody, its biological activity, use, or other relevant information is described in U.S. patent 10,100,125, the contents of which are hereby incorporated by reference in their entirety. As described therein, the H11B6 antibody polypeptide comprises a Heavy Chain (HC) variable region comprising the amino acid sequences of SEQ ID NO. 11 and 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 and SEQ ID NO. 15 and SEQ ID NO. 16.

Thus, according to a particular embodiment, the radioactive conjugate of the present invention comprises an h11B6 antibody comprising (a) a heavy chain variable region (VH) comprising VH CDR1 having the amino acid sequence of SEQ ID No. 11 (SDYAWN), VHCDR2 having the amino acid sequence of SEQ ID No. 12 (YISYSGSTTYNPSLKS) and VH CDR3 having the amino acid sequence of SEQ ID No. 13 (GYYYGSGF); and (b) a light chain variable region (VL) comprising a VL CDR1 having the amino acid sequence of SEQ ID NO. 14 (KASESVEYFGTSLMH), a VL CDR2 having the amino acid sequence of SEQ ID NO. 15 (AASNRES) and a VL CDR3 having the amino acid sequence of SEQ ID NO. 16 (QQTRKVPYT).

The H11B6 antibody may also have a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 18, or a heavy chain constant region comprising the amino acid sequence of SEQ ID NO. 19 and a light chain constant region comprising the amino acid sequence of SEQ ID NO. 20, or a heavy chain comprising the amino acid sequence of SEQ ID NO. 21 and a light chain comprising the amino acid sequence of SEQ ID NO. 22.

The Kabat numbering scheme (Kabat et al, 1991) is incorporated by reference throughout this specification ("Sequences of Immunological Interest", 5 th edition, NIH, bethesda, md).

According to particular embodiments, the antibodies of the invention comprise a heavy chain variable region (VH) having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to the amino acid sequence of SEQ ID No. 17 and/or a light chain variable region (VL) having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to the amino acid sequence of SEQ ID No. 18.

According to particular embodiments, the antibodies of the invention have a heavy chain constant region having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to the amino acid sequence of SEQ ID NO. 19 and/or a light chain constant region having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to the amino acid sequence of SEQ ID NO. 20.

According to particular embodiments, the antibodies of the invention comprise a heavy chain having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to the amino acid sequence of SEQ ID NO. 21 and/or a light chain having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to the amino acid sequence of SEQ ID NO. 22.

According to a particular embodiment, the antibodies of the invention (e.g. h11B 6) comprise or consist of intact (i.e. complete) antibodies, such as IgA, igD, igE, igG or IgM molecules.

According to a particular embodiment, the antibody of the invention (e.g. h11B 6) comprises or consists of an intact IgG molecule or variant thereof. The IgG molecules may be of any known subtype, such as IgG1, igG2, igG3 or IgG4.

According to a particular embodiment, the antibodies of the invention include h11B6 antibodies, which are IgG1 antibodies. According to a particular embodiment, the antibodies of the invention comprise h11B6 antibodies, which are IgG1 kappa isotypes. According to a particular embodiment, the antibodies of the invention comprise h11B6 antibodies, which are IgG1 antibodies or variants thereof, such as Fc variants.

In any of the embodiments disclosed herein (hereinafter described as "in any of the embodiments disclosed herein," etc., for simplicity), the antibody may include, but is not limited to, belimumab, mo Geli bead mab, bonafuzumab, temozolomab, oxuzumab, rituximab, oxganigzumab, mositumomab, rituximab, daritumumab, ceritumumab, cetuximab, rituximab, cermetuzumab, panitumumab, rituximab, pertuzumab, siltuzumab, cimitumumab, cimitumomab Li Shan, nal Wu Shankang, panitumumab, oxuzumab, at Zhu Shan, abauzumab, duluzumab, ceruzumab, etotuzumab, etomizumab, deluximab, alemtuzumab, bevacizumab, oxuzumab, cetuximab, alemtuzumab, aleuxb 25, gemtuzumab, or other than 25. In any of the embodiments disclosed herein, it is possible that the antibody fragment comprises an antigen binding fragment of: belimumab, mo Geli bead mab, bolamiumab, timox, oxuzumab, oxfamuzumab, rituximab, oxetaxeb, moxitimox, bentuximab, darimab, moprimumab, cetuximab, toxitimab, panitumumab, pertuzumab, trastuzumab, enmtuzumab, stetuximab, cimapr Li Shan, nat Wu Shankang, pamuzumab, olantimab, att Zhu Shan, abauzumab, duluzumab, caruzumab, etomizumab, disomizumab, temozolomab, alonguzumab, bezelmab, berrimuzumab, tolizumab, gemtuzumab, zolizumab, cetuximab, ji Tuo, oxuzumab, caruximab, or Ada beads. In any of the embodiments disclosed herein, the binding peptide can include, but is not limited to, a prostate specific membrane antigen ("PSMA") binding peptide, a somatostatin receptor agonist, a bombesin receptor agonist, a seprase binding compound, or binding fragments thereof.

The immunoconjugates and radioimmunoconjugates of the invention may be prepared according to any method known in the art for conjugating ligands (e.g., antibodies) to the compounds of the invention, including chemical and/or enzymatic methods, in accordance with the present disclosure. 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 acid halides; nucleophilic displacement reactions (e.g., nucleophilic displacement such as halide rings or ring opening of strained ring systems); azido-alkyne Huisgen cycloaddition (e.g., 1, 3-dipolar cycloaddition between azide and alkyne to form a1, 2, 3-triazole linkage); addition of mercaptoalkyne; imine formation; diels-alder reaction (Diels-Alderreaction) between tetrazine and trans-cyclooctene (TCO); and Michael addition (e.g., maleimide addition). Many other modes of attachment with different linkages are also possible, depending on the reactive functional groups used. The attachment of the ligand may be performed on compounds that coordinate to the radioactive metal ion or on compounds that do not coordinate to the radioactive metal ion.

In one embodiment, the radioimmunoconjugates may be produced by covalently linking the radiometal complexes of the invention to antibodies or antigen binding fragments thereof, for example, by click chemistry reactions (see, e.g., fig. 2B and 2D, referred to as "click radiolabels"). Alternatively, the radioimmunoconjugate may be produced by: the immunoconjugates of the invention are first prepared by covalently attaching a compound of the invention to an antibody or antigen-binding fragment thereof, e.g., by click chemistry; the immunoconjugate can then be labeled with a radiometal ion to produce a radioimmunoconjugate (see, e.g., fig. 2A and 2C, referred to as "one-step direct radiolabeling"). Both the residue-specific method of conjugation (e.g., fig. 2A and 2B) and the site-specific method (e.g., fig. 2C and 2D) can be used to produce immunoconjugates and radioimmunoconjugates of the invention.

Residue-specific methods for conjugation to proteins are well established and most commonly involve the use of lysine side chains of activated esters or isothiocyanates, or cysteine side chains with maleimides, haloacetyl derivatives, or activated disulfides (BrinkleyBioconjugate Chem 1992: 2). Since most proteins have multiple lysine and cysteine residues, such methods are typically used to obtain heterogeneous mixtures of products with different numbers of conjugated molecules at multiple amino acid positions. Additional methods have been established, including tyrosine-specific conjugation (Ban et al, bioconjugate Chemistry 2013:520), methionine-specific methods (Lin et al, science 2017 (355) 597), additional cysteine-focused methods (Toda et al, ANGEW CHEMIE 2013:12592), and the like.

Recently, site-selective and site-specific conjugation methods for monoclonal antibodies and other proteins have been established (Agarwal, P. And C.R.Bertozzi, bioconjugChem,2015.26 (2): p.176-92; rabuka et al Curr Opin ChemBiol 2010:790). These methods include incorporating unnatural amino acids; fusing the protein of interest to a "self-labeling tag" such as SNAP or DHFR, or a tag specifically recognized and modified by another enzyme such as sortase a, lipoic acid ligase, and formylglycine generating enzyme; enzymatic modification of glycans to allow for conjugated payloads of interest (Hu et al Chem Soc Rev 2016:1691); selectively recognizing a defined position on the antibody using microbial transglutaminase; and additional methods that use molecular recognition and/or chemical methods to affect selective conjugation (Yamada et al, 2019:5592; park et al, bioconjugate Chem 2018:3240; pham et al, chembiochem, 2018:799).

In certain embodiments, the immunoconjugates or radioimmunoconjugates of the invention are produced using residue-specific methods for conjugating the compounds of the invention to antibodies or antigen binding fragments thereof. Such residue-specific methods typically result in the covalent attachment of an immunoconjugate or radioimmunoconjugate to a compound or radiometal complex of the invention at multiple positions of the antibody. Any residue-specific method known to those of skill in the art for forming protein or antibody conjugates can be used in accordance with the present disclosure. Examples of residue-specific methods for conjugation that may be used include, but are not limited to, conjugation of a compound or radiometal complex of the invention to a lysine residue of an antibody using a compound or radiometal complex of the invention comprising, for example, an activating ester or isothiocyanate group; conjugation to cysteine residues of an antibody using a compound or radiometal complex of the invention comprising, for example, maleimide, haloacetyl derivatives, acyl halides, activated disulfide groups, or methylsulfonylphenyl oxadiazole groups; conjugation to tyrosine residues of antibodies using a compound or radiometal complex of the invention comprising, for example, 4-phenyl-3H-1, 2, 4-triazolin-3, 5 (4H) -dione (PTAD); and methionine residues conjugated to antibodies using a compound of the invention comprising, for example, an oxaziridine derivative or a radiometal complex. Antibodies can also be labeled with a biorthogonal reactive functional group at a specific residue using one or more of the methods described above prior to conjugation to the compounds of the invention or the radiometal complexes of the invention. For example, tyrosine residues may be site-specifically labeled with a biorthogonal reactive functional group using an oxaziridine derivative attached to the biorthogonal reactive functional group (e.g., azido, alkynyl or cycloalkynyl), and then an antibody containing the labeled tyrosine residues may be conjugated to a compound of the present invention or a radioactive metal complex of the present invention using a compound of the present invention or a radioactive metal complex bearing a compatible reactive functional group.

In certain embodiments, the immunoconjugates or radioimmunoconjugates of the invention may be produced using a site-specific or site-selective method for conjugating the compounds of the invention to an antibody or antigen binding fragment thereof. In contrast to residue-specific methods, "site-specific" or "site-selective" methods generally result in an immunoconjugate or radioimmunoconjugate being covalently attached to a compound or radiometal complex of the invention at a designated position of the antibody. Any site-specific method known to those of skill in the art for forming protein or antibody conjugates can be used in accordance with the present disclosure. For example, unnatural amino acids (e.g., azido-or alkynyl-amino acids) can be site-specifically incorporated into antibodies using mutant aminoacyl t-RNA synthetases that can selectively aminoacylate their tRNA's with the unnatural amino acid of interest. The mutant acylated tRNA together with the amber suppressor tRNA can then be used to site-specifically incorporate an unnatural amino acid into a protein in response to an amber nonsense codon. Antibodies site-specifically labeled by one or more of the above methods may then be conjugated to a compound of the invention or a radioactive metal complex of the invention bearing compatible reactive functionalities.

In certain embodiments, the invention relates to a method of producing a radioimmunoconjugate, comprising reacting a compound of the invention or a radioactive complex of the invention, wherein R ₄ is a nucleophilic or electrophilic moiety, with an antibody or antigen-binding fragment thereof, or a modified antibody or antigen-binding fragment thereof, comprising a nucleophilic or electrophilic moiety.

In one embodiment, the invention relates to a method comprising reacting a compound of the invention with an antibody or antigen-binding fragment thereof, or a modified antibody or antigen-binding fragment thereof, comprising a nucleophilic or electrophilic functional group to form an immunoconjugate having a covalent bond between the compound of the invention and the antibody or antigen-binding fragment thereof, or the modified antibody or antigen-binding fragment thereof, and then reacting the immunoconjugate with a radiometal ion such that the radiometal ion binds to the compound of the invention of the immunoconjugate via a coordinate bond, thereby forming a radioimmunoconjugate. This embodiment may be referred to as a "one-step direct radiolabelling" method (e.g., as schematically shown in fig. 2C) because there is only one chemical reaction step involving the radioactive metal.

In another embodiment, the invention relates to a method comprising reacting a radioactive complex of the invention with an antibody or antigen-binding fragment thereof, or a modified antibody or antigen-binding fragment thereof, comprising a nucleophilic or electrophilic functional group, thereby forming a radioimmunoconjugate. This embodiment may be referred to as a "click radiolabel" method (e.g., as schematically shown in fig. 2D). According to the present disclosure, modified antibodies or antigen-binding fragments thereof can be produced by any method known in the art, for example, by labeling the antibody with a biorthogonal reactive functional group at a particular residue using one or more of the methods described above, or by site-specifically incorporating an unnatural amino acid (e.g., an azido-or alkynyl-amino acid) into the antibody using one or more of the methods described above. Degree of labelling (DOL), sometimes referred to as degree of substitution (DOS), is a particularly useful parameter for characterizing and optimizing bioconjugates, such as antibodies modified by unnatural amino acids. It is expressed as the average number of unnatural amino acids coupled to a protein molecule (e.g., an antibody), or as the molar ratio of label/protein form. DOL can be determined from the absorption spectrum of the labeled antibody by any method known in the art.

In certain embodiments of the invention, the immunoconjugates and radioimmunoconjugates of the invention are prepared using click chemistry reactions. For example, the radioimmunoconjugates of the present invention may be prepared using a click chemistry reaction known as "click radiolabeling" (see, e.g., fig. 2B and 2D). Click radiolabelling uses click chemistry reaction partners, preferably azides and alkynes (e.g., cyclooctyne or cyclooctyne derivatives) to form covalent triazole linkages between a radioactive complex (a radioactive metal ion bound to a compound of the invention) and an antibody or antigen-binding fragment thereof. The click radiolabeling method of antibodies is described, for example, in International patent application PCT/US18/65913, entitled "Radiolabeling of Polypeptides", the relevant description of which is incorporated herein by reference. In other embodiments, known as "one-step direct radiolabeling", an immunoconjugate is prepared using a click chemistry reaction between an antibody or antigen binding fragment thereof and a compound of the invention; the immunoconjugate is then contacted with a radiometal ion to form the radioimmunoconjugate (see, e.g., fig. 2A and 2C).

In one embodiment, the invention relates to a method of preparing a radioimmunoconjugate comprising associating (e.g., via coordinate bonding) a radiometal ion with a compound of the invention.

In one embodiment, a "one-step direct radiolabeling" method of preparing a radioimmunoconjugate, the method comprising contacting an immunoconjugate (i.e., a polypeptide-compound complex of the invention) with a radiometal ion to form the radioimmunoconjugate, wherein the immunoconjugate comprises a compound of the invention. According to a particular embodiment, the immunoconjugate is formed by a click chemistry reaction between a compound of the invention and a polypeptide. According to particular embodiments, the radioimmunoconjugate is formed in the presence of a metal (e.g., without any step of removing or actively excluding common metal impurities from the reaction mixture). This is in contrast to some conventional methods in which the antibody must be radiolabeled under stringent metal-free conditions to avoid competitive (non-productive) chelation of common metals such as iron, zinc and copper, which presents a significant challenge to the production process.

In one embodiment, the invention relates to a method of preparing a radioimmunoconjugate (including a "one-step direct radiolabeling" method) comprising:

(i) Reacting a modified polypeptide with a compound of the invention to produce an immunoconjugate, wherein the modified polypeptide is an antibody or antigen-binding fragment thereof consisting of an azido group; and

(Ii) The immunoconjugate is reacted with a radiometal ion to produce the radioimmunoconjugate.

In another embodiment, the invention relates to a method of preparing a radioimmunoconjugate (including a "one-step direct radiolabeling" method) comprising:

(i) Reacting a modified antibody consisting of an azido group or antigen-binding fragment thereof with a compound of formula I to produce an immunoconjugate; and

In certain embodiments, the invention relates to a method of preparing a radioimmunoconjugate (including a "click radiolabel" method, e.g., as shown in fig. 2D), comprising:

(i) Reacting the modified antibody or antigen binding fragment thereof with a radioactive complex under conditions wherein the azido group reacts with an alkynyl group or a cycloalkynyl group to produce a radioimmunoconjugate.

The conditions for performing the click chemistry reaction are known in the art, and any conditions for performing the click chemistry reaction known to those skilled in the art can be used in the present invention in light of the present disclosure. Examples of conditions include, but are not limited to, incubating the modified polypeptide and the radioactive complex at a ratio of 1:1 to 1000:1 at a pH of 4 to 10 and a temperature of 20 ℃ to 70 ℃.

The click radiolabelling method described above allows complexing of the radioactive metal ions under low or high pH and/or high temperature conditions to maximize efficiency, which can be achieved without the risk of inactivating the alkyne reaction partner. The efficient complexation and efficient sparc reaction between the azide-labeled antibody or antigen-binding fragment thereof and the radioactive complex allows the production of radioimmunoconjugates in high radiochemical yields even though the azide to antibody ratio is low. The only step that must be excluded from trace metals is the complexation of the radioactive metal ion with the macrocyclic compound moiety; the antibody production, purification and conjugation steps need not be performed under metal-free conditions.

The compounds of the invention and the radiometal complexes of the invention can also be used to produce site-specific radiolabeled polypeptides, such as antibodies. Click radiolabelling methods described herein facilitate site-specific production of radioimmunoconjugates by site-specifically installing azide groups on antibodies using established methods (methods of Li, x. Et al ,Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions.Angew ChemInt 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). attaching molecules to proteins or antibodies in a site-specific manner are known in the art and any method of site-specifically labeling antibodies known to those of skill in the art may be used in the present invention in accordance with the present disclosure examples of methods suitable for site-specific modification of antibodies in the present invention include, but are not limited to, incorporation of engineered cysteine residues (e.g., THIOMAB ^TM), use of unnatural amino acids or glycans (e.g., selenocysteine, p-AcPhe, formylglycine generating enzymes (FGE, smatag ^TM), etc.), and enzymatic methods (e.g., use of glycosyltransferases, endoglycosidases, microbial or bacterial transglutaminases (MTG or BTG), transpeptidase a, etc.).

In certain embodiments, the modified antibodies or antigen binding fragments thereof used in the production of the immunoconjugates or radioimmunoconjugates of the invention are obtained by: trimming the antibody or antigen binding fragment thereof with a bacterial endoglycosidase (such as GlycINATOR (Genovis)) specific for the β -1,4 bond between core GlcNac residues in the Fc glycosylation site of the antibody, which leaves the innermost GlcNac intact on the Fc, allowing site-specific incorporation of the azido sugar at that site. The trimmed antibody or antigen binding fragment thereof may then be reacted with an azide-labeled sugar, such as UDP-N-azidoacetylgalactosamine (UDP-GalNAz) or UDP-6-azido 6-deoxygalnac, in the presence of a glycosyltransferase, such as GalT galactosyltransferase or GalNAc transferase, to obtain a modified antibody or antigen binding fragment thereof.

In other embodiments, the modified antibodies or antigen-binding fragments thereof for use in producing the immunoconjugates or radioimmunoconjugates of the invention are obtained by deglycosylating the antibodies or antigen-binding fragments thereof with amidase. The resulting deglycosylated antibody or antigen binding fragment thereof may then be reacted with an azidoamine (preferably 3-azidopropylamine, 6-azidohexamine, or any azido-linker-amine or any azido-alkyl/heteroalkyl-amine, such as azido-polyethylene glycol (PEG) -amine, e.g., 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 microbial transglutaminase, to obtain a modified antibody or antigen binding fragment thereof.

Any of the radiometal complexes described herein may be used to produce the radioimmunoconjugates of the present invention. In a particular embodiment, the radiometal complex has the structure of formula (I-M ⁺).

In certain embodiments, the radioimmunoconjugate is any one or more structures independently selected from the group consisting of:

Wherein:

L ₁ is absent or a linker; and

The mAb is an antibody or antigen-binding fragment thereof.

In another embodiment, the radioimmunoconjugate is any one or more selected from the group consisting of:

wherein the mAb is an antibody or antigen-binding fragment thereof.

It should be noted that in the structures of the radioimmunoconjugates described herein that comprise "mabs," these structures do not show residues of the mAb (e.g., lysine residues of the mAb) attached to the radiometal complex.

One embodiment of the present invention provides a radioimmunoconjugate having the structure:

(also referred to herein as TOPA- [ C7] -phenylthiourea-h 11B6 antibody conjugate), wherein M ⁺ is actinium-225 (²²⁵ Ac), and

Wherein the mAb has binding specificity for hK 2; for example, the number of the cells to be processed,

(I) Wherein the mAb is an h11B6 antibody polypeptide comprising a Heavy Chain (HC) variable region comprising the amino acid sequences of SEQ ID NO. 11 and 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 and SEQ ID NO. 15 and SEQ ID NO. 16; and/or

(Ii) Wherein the mAb comprises a heavy chain variable region (VH) having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:17 and/or a light chain variable region (VL) having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18.

In accordance with the present disclosure, radioimmunoconjugates produced by the methods described herein can be analyzed using methods known to those of skill in the art. For example, LC/MS analysis can be used to determine the ratio of compounds to labeled polypeptides (e.g., antibodies or antigen binding fragments thereof); analytical size exclusion chromatography can be used to determine the oligomerization status of polypeptides and polypeptide conjugates (e.g., antibodies and antibody conjugates); radiochemical yields can be determined by transient thin layer chromatography (e.g., iTLC-SG) and radiochemical purity can be determined by size exclusion HPLC. Exemplary methods are described herein, for example, in the following examples.

Pharmaceutical compositions and methods of use

In another embodiment, the present invention relates to a pharmaceutical composition comprising: the compounds of the invention, the radiometal complexes, immunoconjugates or radioimmunoconjugates of the invention, and a pharmaceutically acceptable carrier. The pharmaceutical composition may comprise one or more pharmaceutically acceptable excipients.

In one embodiment, the pharmaceutical composition comprises a compound of the invention and a pharmaceutically acceptable carrier.

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

In another embodiment, the pharmaceutical composition comprises an immunoconjugate of the invention and a pharmaceutically acceptable carrier.

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

As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid-containing vesicle, microsphere, liposome encapsulation, or other material known in the art for use in pharmaceutical formulations. It will be appreciated that the characteristics of the carrier, excipient or diluent will depend upon 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 effect of the composition according to the invention or the biological activity of the composition according to the invention. According to the present disclosure, any pharmaceutically acceptable carrier suitable for use in an antibody-based or radioactive complex-based pharmaceutical composition may be used in the present invention, according to particular embodiments.

According to particular embodiments, the compositions described herein are formulated for the intended route of administration to a subject. For example, the compositions described herein may be formulated for parenteral administration, e.g., intravenous, subcutaneous, intramuscular, or intratumoral administration.

In certain embodiments, the invention relates to methods of selectively targeting neoplastic cells for radiation therapy and treatment of neoplastic diseases or disorders. Any of the radioactive complexes or radioimmunoconjugates described herein and pharmaceutical compositions thereof may be used in the methods of the present invention.

"Tumor" is an abnormal mass of tissue that is produced when cells divide beyond the extent to which they should divide or when they should die without dying. Tumors may be benign (not cancer) or malignant (cancer). Tumors are also known as tumors. A neoplastic disease or disorder is a disease or disorder associated with a tumor, such as cancer. Examples of neoplastic diseases or disorders include, but are not limited to, disseminated cancer and solid tumor cancer.

In certain embodiments, the invention relates to a method of treating prostate cancer (e.g., metastatic prostate cancer or metastatic castration-resistant prostate cancer) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an immunoconjugate or radioimmunoconjugate as described herein, wherein the immunoconjugate or radioimmunoconjugate comprises a radiometal complex as described herein conjugated to H11B 6.

Embodiments of the invention are particularly useful in treating patients who have been diagnosed with prostate cancer; for example, patients with advanced prostate cancer. According to one embodiment, the cancer is non-restricted prostate cancer. According to another embodiment, the cancer is metastatic prostate cancer. According to another embodiment, the cancer is Castration Resistant Prostate Cancer (CRPC). According to another embodiment, the cancer is metastatic castration-resistant prostate cancer (mCRPC). According to another embodiment, the cancer is mCRPC with an adenocarcinoma.

Other examples of diseases to be treated or targeted by radiation therapy by the methods of the invention described herein include, but are not limited to, hypertrophy, coronary artery disease or vascular occlusive disease, diseases or disorders associated with infected cells, microorganisms or viruses, or diseases or disorders associated with inflammatory cells, such as Rheumatoid Arthritis (RA).

In one embodiment, the invention relates to a method of selectively targeting neoplastic cells for radiation therapy, the method comprising administering to a subject in need thereof a radioimmunoconjugate or pharmaceutical composition of the invention.

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

In one embodiment, the present invention relates to a method of treating cancer in a subject in need thereof, the method comprising administering to the subject in need thereof a radioimmunoconjugate or pharmaceutical composition of the present invention.

The radioimmunoconjugate carries the radiation directly to, for example, cells targeted by the targeting ligand, etc. Preferably, the radioimmunoconjugate carries an alpha-emitting radioactive metal ion, such as ²²⁵ Ac. Upon targeting, alpha particles from alpha emitting radioactive metal ions (e.g., ²²⁵ Ac and its daughter) are delivered to and exert a cytotoxic effect on the targeted cells, thereby selectively targeting neoplastic cells for radiation therapy and/or treatment of neoplastic diseases or disorders.

The invention also includes pre-targeting methods for selectively targeting neoplastic cells for radiation therapy and for treating neoplastic diseases or disorders. According to the pretargeting method, azide-labeled antibodies or antigen binding fragments thereof are administered, which bind to the cells carrying the target antigen of the antibody and allow for clearance from the circulation over time or removal with a detergent. Subsequently, the radiometal complex of the invention, preferably comprising cyclooctyne or a cyclooctyne derivative (e.g., DBCO), is administered and allowed to react with the azide-labeled antibody bound at the target site, sparc, while the remaining unbound radiometal complex is rapidly cleared from the circulation. The pretargeting technique provides a method of enhancing the localization of radioactive metal ions at a target site in a subject.

In other embodiments, the modified polypeptide (e.g., azide-labeled antibody or antigen-binding fragment thereof) and the radiometal complex of the invention are administered to a subject in need of targeted radiation therapy or treatment of a neoplastic disease or disorder in the form of the same composition or different compositions.

As used herein, the term "therapeutically effective amount" refers to the amount of an active ingredient or component that elicits the desired biological or medicinal response in a subject. The therapeutically effective amount can be determined empirically and in a conventional manner with respect to the intended purpose. For example, in vitro assays may optionally be employed to help determine optimal dosage ranges. The selection of a particular effective dose can be determined by one of skill in the art (e.g., via a clinical trial) based on a number of factors including the disease to be treated or prevented, the symptoms involved, the weight of the patient, the immune status of the patient, and other factors known to the skilled artisan. The precise dosage to be employed in the formulation will also depend on the route of administration and the severity of the disease and should be determined according to the judgment of the practitioner and each patient's circumstances. The effective dose can be deduced from dose response curves derived from in vitro or animal model test systems.

As used herein, the terms "treatment" and "treatment" are both intended to refer to an improvement or reversal of at least one measurable physical parameter associated with a disease, disorder or condition in which administration of a radioactive metal ion would be beneficial, such as a neoplastic disease or disorder, which is not necessarily identifiable in a subject, but may be identifiable in a subject. The terms "treatment" and "treatment" may also refer to causing regression, preventing progression, or at least slowing the progression of a disease, disorder, or condition. In particular embodiments, "treating" and "treatment" refer to alleviating one or more symptoms associated with a disease, disorder, or condition in which administration of a radioactive metal ion would be beneficial, such as a neoplastic disease or disorder, preventing the development or onset of the one or more symptoms, or shortening the duration of the one or more symptoms. In particular embodiments, "treating" and "treatment" refer to preventing recurrence of a neoplastic disease, disorder, or condition. In particular embodiments, "treating" and "treatment" refer to an increase in survival of a subject suffering from a neoplastic disease, disorder or condition. In particular embodiments, "treating" and "treatment" refer to the elimination of a neoplastic disease, disorder or condition in a subject.

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

In other embodiments of the invention, the radioimmunoconjugates and pharmaceutical compositions of the present invention may be administered in combination with other agents effective to treat neoplastic diseases or disorders.

In further embodiments, the present invention relates to radioimmunoconjugates and pharmaceutical compositions as described herein for use in selectively targeting neoplastic cells for radiation therapy and/or treating neoplastic diseases or disorders; and the use of a radioimmunoconjugate or pharmaceutical composition as described herein in the manufacture of a medicament for selectively targeting neoplastic cells for radiation therapy and/or for the treatment of neoplastic diseases or disorders.

Detailed description of the illustrated embodiments

Exemplary numbered embodiments of the invention are provided below.

1. Preparation of Compound 14 (6- ((16- ((6-carboxypyridin-2-yl) (4-isothiocyanatophenyl) methyl) -1,4,10,13-tetraoxa-7, 16-diazacyclooctadec-7-yl) methyl) pyridinecarboxylic acid)

A method of making a pharmaceutical composition comprising:

reacting compound 9 with N, O-bis (trimethylsilyl) acetamide (BSA) in an organic solvent or mixture thereof; adding trimethyl silicone triflate (TMSOTF) into the organic solvent

A solution in the reagent to obtain a compound 10;

Reacting compound 10 in an organic solvent or mixture thereof under basic conditions to provide compound 13; compound 13 is reacted with thiocarbonyldiimidazole in an organic solvent or mixture thereof to afford compound 14.

2. Preparation of Compound 14 (6- ((16- ((6-carboxypyridin-2-yl) (4-isothiocyanatophenyl) methyl) -1,4,10,13-tetraoxa-7, 16-diazacyclooctadec-7-yl) methyl) pyridine carboxylic acid) according to embodiment 1

A method of making a pharmaceutical composition comprising:

Reacting compound 9 with N, O-bis (trimethylsilyl) acetamide (BSA) in an organic solvent or mixture thereof at a temperature in the range of about ambient temperature to about-78deg.C, adding a solution of trimethyltrisulfonate (TMSOTF) in the organic solvent to give the compound

An object 10;

Compound 10 is reacted in an organic solvent or mixture thereof under basic conditions at a temperature in the range of about ambient temperature to about-78 ℃ to provide compound 13. Reacting compound 13 with thiocarbonyldiimidazole in an organic solvent or mixture thereof at a temperature in the range of about ambient temperature to about-78 ℃ to provide compound 14.

3. Preparation of Compound 14 (6- ((16- ((6-carboxypyridin-2-yl) (4-isothiocyanatophenyl) methyl) -1,4,10,13-tetraoxa-7, 16-diazadioctadec-7-yl) methyl) pyridinecarboxylic acid according to embodiment 1 or 2

A method of making a pharmaceutical composition comprising:

reacting 7, 16-dibenzyl-1,4,10,13-tetraoxa-7, 16-diazacyclooctadecane (1) with a reducing agent in an organic solvent or mixture thereof to give compound 2; /(I)

Reacting methyl 6- (hydroxymethyl) pyridine carboxylate with thionyl chloride in an organic solvent or mixture thereof to give compound 3;

Reacting 3 with 2 in an organic solvent or mixture thereof to provide compound 4 (6- ((1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridine carboxylate);

Reacting methyl 6-formylpicolinate 5 with (4- ((tert-butoxycarbonyl) amino) phenyl) boronic acid 6 in an organic solvent or a mixture thereof under reducing conditions to give compound 7;

reacting methyl 6- ((4- ((tert-butoxycarbonyl) amino) phenyl) (hydroxy) methyl) pyridine carboxylate 7 with methanesulfonyl chloride in an organic solvent or a mixture thereof to give methyl compound 8 (6- ((4- ((tert-butoxycarbonyl) amino) phenyl) - ((methanesulfonyl) oxy) methyl) pyridine carboxylate;

Reacting methyl 6- ((4- ((tert-butoxycarbonyl) amino) phenyl) - ((methylsulfonyl) oxy) methyl) pyridinecarboxylate 8 with methyl 6- ((1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridinecarboxylate 4 with sodium carbonate in an organic solvent or a mixture thereof to give compound 9;

Reacting compound 9 with N, O-bis (trimethylsilyl) acetamide (BSA) in an organic solvent or a mixture thereof, and adding a solution of trimethylsilyl triflate (TMSOTf) in the organic solvent to the reaction to give compound 10;

4. The method of any one of embodiments 1-3 for preparing compound 14 (6- ((16- ((6-carboxypyridin-2-yl) (4-isothiocyanatophenyl) methyl) -1,4,10,13-tetraoxa-7, 16-diazacyclooctadec-7-yl) methyl) pyridinecarboxylic acid

A method of making a pharmaceutical composition comprising:

Reacting compound 9 with N, O-bis (trimethylsilyl) acetamide (BSA) in an organic solvent or mixture thereof at a temperature in the range of about ambient temperature to about-78 ℃, adding to said reaction a solution of trimethylsilicone triflate (TMSOTf) in an organic solvent

Obtaining a compound 10 from the solution;

Compound 10 is reacted in an organic solvent or mixture thereof under basic conditions at a temperature in the range of about ambient temperature to about-78 ℃ to provide compound 13, which is reacted with thiocarbonyldiimidazole in an organic solvent or mixture thereof at a temperature in the range of about ambient temperature to about-78 ℃ to provide compound 14.

5. The method for preparing compound 14 according to any one of embodiments 1-4

/>

A method of making a pharmaceutical composition comprising:

Reacting methyl 6- ((4- ((tert-butoxycarbonyl) amino) phenyl) (hydroxy) methyl) pyridinecarboxylate 7 with methanesulfonyl chloride in an organic solvent or mixture thereof at a temperature ranging from about ambient temperature to about-78 ℃ to give compound 8 (6- ((4- ((tert-butoxycarbonyl) amino) phenyl) propanoic acid

((Methylsulfonyl) oxy) methyl) pyridine carboxylic acid methyl ester;

Methyl 6- ((4- ((tert-butoxycarbonyl) amino) phenyl) - ((methylsulfonyl) oxy) methyl) pyridinecarboxylate 8 and 6- ((1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridinecarboxylate 4 with sodium carbonate at a temperature in the range of about ambient temperature to about-78 DEG C

Reacting in an organic solvent or a mixture thereof to obtain a compound 9;

Obtaining a compound 10 from the solution;

6. For preparing compound 10

A method of making a pharmaceutical composition comprising:

Reacting compound 9 with N, O-bis (trimethylsilyl) acetamide (BSA) in an organic solvent or mixture thereof; a solution of trimethylsilicone triflate (TMSOTf) in an organic solvent was added to give compound 10.

7. Use in the preparation of Compound 10 according to embodiment 6

A method of making a pharmaceutical composition comprising:

Compound 9 is reacted with N, O-bis (trimethylsilyl) acetamide (BSA) in an organic solvent or mixture thereof at a temperature ranging from about ambient temperature to about-78 ℃ to give compound 10 by the addition of a solution of trimethylsilicone triflate (TMSOTf) in an organic solvent.

8. The method of embodiment 6 or embodiment 7 for preparing Compound 10

A method of making a pharmaceutical composition comprising:

9. Method for preparing compound 12 (TOPA- [ C7] -phenylisothiocyanate sodium salt)

The method comprises the following steps:

Reacting compound 10 (having a structure as described herein) with sodium hydroxide in an organic solvent or mixture thereof to yield compound 11 (having a structure as described herein); compound 11 is reacted with thiocarbonyldiimidazole in an organic solvent or mixture thereof to give compound 12.

10. The process for preparing compound 12 (TOPA- [ C7] -phenylisothiocyanate sodium salt) according to embodiment 9

The method comprises the following steps:

Contacting compound 10 with sodium hydroxide at a temperature in the range of about ambient temperature to about-78 DEG C

Reacting in an organic solvent or a mixture thereof to obtain a compound 11;

11. The method of embodiment 10 for preparing TOPA- [ C7] -phenylthiourea-h 11B6 antibody

Method of conjugate:

The method comprises the following steps:

Reacting an 8-to 12-fold excess of compound 12 with h11b6mAb (e.g., reacting a 10-fold excess of compound 12 therewith); and removing the excess free chelator to give TOPA- [ C7] -phenylthiourea-h 11B6 antibody conjugate.

12. The method of embodiment 10 or embodiment 11 for preparing TOPA- [ C7] -phenylthiourea-h 11B6 antibody conjugate:

The method comprises the following steps:

Reacting an 8-fold to 12-fold excess of compound 12 (e.g., 10-fold excess of compound 12) with h11b6mAb in a buffer adjusted to about pH 8-10 (e.g., about pH 9), and incubating at room temperature; and removing excess free chelator by desalting the reaction to give TOPA- [ C7] -phenylthiourea-h 11B6 antibody conjugate.

13. The method for preparing TOPA- [ C7] -phenylthiourea according to any one of embodiments 10-12

-Method of h11B6 antibody conjugate:

The method comprises the following steps:

Reacting an about 10-fold excess of compound 12 with h11b6mAb in 10mM sodium acetate buffer at pH of about 5.2, adjusted to pH of about 9 with sodium bicarbonate buffer, and incubated at room temperature without shaking for about 1 hour; quenching by adding 1M Tris at pH about 8.5 to a final concentration of about 100 mM; removing excess free chelator by desalting the reaction to 10mM sodium acetate at a pH of about 5.2; and removing the excess chelator to give TOPA- [ C7] -phenylthiourea-h 11B6 antibody conjugate.

14. The method for preparing TOPA- [ C7] -phenylthiourea according to any one of embodiments 10-13

-Method of h11B6 antibody conjugate:

The method comprises the following steps:

A 10-fold excess of compound 12 was reacted with h11b6mAb in 10mM sodium acetate pH5.2 buffer, immediately adjusted to pH 9 with sodium bicarbonate buffer, and incubated at room temperature without shaking for about 1 hour. Then quenched by adding 1M Tris at pH about 8.5 to a final concentration of 100 mM. Excess free chelator was removed by desalting the reaction to 10mM sodium acetate pH5.2 using a 7K Zeba desalting column. The sample was diluted to 15ml by 3x rounds and then concentrated to 1ml using a 50000MWCO Amicon concentration device to remove excess chelator, after which the sample was adjusted to its final concentration to produce

TOPA- [ C7] -phenylthiourea-h 11B6 antibody conjugate.

15. A compound of formula (12) (TOPA- [ C7] -phenylisothiocyanate sodium salt)

16. A compound of formula (14)

Or a pharmaceutically acceptable salt or solvate thereof.

17. A compound of formula (11)

Generalized synthetic scheme:

Methods for preparing compound 2 have been described in a number of publications, for example US5247078;Justus Liebigs Annalen der Chemie(1977),(8),1344-6;Zhurnal Organicheskoi Khimii(1988),24(8),1731-42;Journal of the Chemical Society,Perkin Transactions 2:Physical Organic Chemistry(1972-1999)(1994),(3),513-20;Organic Syntheses(1990),68,227-33;Journal ofOrganic Chemistry(1987),52(23),5172-6;Russian Journal of General Chemistry(2010),80(5),1007-1010;Chinese Chemical Letters(1992),3(12),963-4;Journal of Organic Chemistry(1986),51(26),5373-84; and Journal of THE CHEMICAL Society, chemical Communications (1991), (14), 956-7.

Synthetic schemes for preparing 4 (A) compounds

Wherein the method comprises the steps of

X is selected from the group consisting of: cl, br, I, OSO ₂ R and P (O) (OR ") ₂;

R is selected from the group consisting of: primary linear or branched (C ₁-C₈) alkyl, alkenyl, alkynyl, aryl or heteroaryl alcohols; zhong Zhilian or branched (C ₃-C₈) alkyl, alkenyl, alkynyl, aryl or heteroaryl alcohols; tertiary linear or branched (C ₄-C₈) alkyl, alkenyl, alkynyl, aryl or heteroaryl alcohols;

R' is selected from the group consisting of: a halogenated linear or branched group of formula (CY 2) nCY3, wherein y=f or Cl, and n=0 to 8

The process for preparing the above 4 (a) compound is prepared as described above for compound 4, with the reaction conditions modified as necessary as will be appreciated by those skilled in the art in view of this disclosure. These compounds are prepared by using a suitable solvent in the presence or absence of a base and optionally in the presence of additives. Solvents such as ACN, DMF, NMP, THF, meTHF, dioxane, DMAc, DMSO, meOH, etOH, IPA, tert-BuOH, tert-AMOH, DCM, etOAc, IPAc or toluene. The base may be an inorganic base (e.g., Na₂CO₃、NaHCO₃、K₂CO₃、KHCO₃、CsCO₃、KOH、NaOH、LiOH、K₃PO₄、K₂HPO₄ or KH ₂PO₄) or an organic base (e.g., DBU, lutidine, PMP, DMAP, DCMA, DIPEA, or TEA). The additive may be NaCl, naBr, naI, KCl, KI, KBr or CsCl.

Synthetic schemes for preparing 9 (A) compounds

Wherein the method comprises the steps of

The process for preparing the above 9 (a) compound is prepared as described above for compound 9, with the reaction conditions modified as necessary as will be appreciated by those skilled in the art in view of this disclosure. These compounds are prepared by using a suitable solvent in the presence of a base. Solvents such as ACN, DMF, NMP, THF, meTHF, dioxane, DMAc, DMSO, meOH, etOH, IPA, tert-BuOH, tert-AMOH, DCM, etOAc, IPAc or toluene. The base may be an inorganic base (e.g., Na₂CO₃、NaHCO₃、K₂CO₃、KHCO₃、CsCO₃、KOH、NaOH、LiOH、K₃PO₄、K₂HPO₄ or KH ₂PO₄) or an organic base (e.g., DBU, lutidine, PMP, DMAP, DCMA, DIPEA, or TEA). The additive may be NaCl, naBr, naI, KCl, KI, KBr or CsCl.

Synthetic schemes for preparing 10 (A) compounds

Wherein:

The process for preparing the above 10 (a) compound is prepared as described above for compound 10, with the reaction conditions modified as necessary as will be appreciated by those skilled in the art in view of this disclosure. These compounds are prepared by using a suitable solvent in the presence of a base. Solvents such as ACN, DMF, NMP, THF, meTHF, dioxane, DMAc, DMSO, meOH, etOH, IPA, tert-BuOH, tert-AMOH, DCM, etOAc, IPAc or toluene. The base may be an inorganic base (e.g., Na₂CO₃、NaHCO₃、K₂CO₃、KHCO₃、CsCO₃、KOH、NaOH、LiOH、K₃PO₄、K₂HPO₄ or KH ₂PO₄) or an organic base (e.g., DBU, lutidine, PMP, DMAP, DCMA, DIPEA, piperidine, or TEA). The acid may be HCl, TFA, MSA, phosphoric acid, KOAc/AcOH, tsCl-DMAP, BF ₃·OEt₂, TMSI, TMSCl or TMSOTF/BSA. Other reactants may be used, such as Pd/H ₂、Pt/H₂、Pd(OH₂)/H₂ or CAN.

Synthetic schemes for preparing 13 (A) compounds

Wherein:

The process for preparing the above 13 (a) compound is prepared as described above for compound 13, with the reaction conditions modified as necessary as will be appreciated by those skilled in the art in view of this disclosure. These compounds are prepared by using a suitable solvent in the presence of a base and a suitable reagent. Solvents such as ACN, DMF, NMP, THF, meTHF, dioxane, DMAc, DMSO, meOH, etOH, IPA, tert-BuOH, tert-AMOH, DCM, etOAc, IPAc or toluene. The base may be an inorganic base (e.g., Na₂CO₃、NaHCO₃、K₂CO₃、KHCO₃、CsCO₃、KOH、NaOH、LiOH、K₃PO₄、K₂HPO₄ or KH ₂PO₄) or an organic base (e.g., DBU, lutidine, PMP, DMAP, DCMA, DIPEA, piperidine, or TEA). These reagents may be Cl ₂CS、CS₂, imidazole ₂CS、NH₂C(S)NH₂EtOC(S)SK、ClC(S)NMe₂、Me₂N(S)CSSC(S)NMe₂、ClC(S)OR'、R'OC(S), where R' =substituted and unsubstituted phenyl or heteroaryl groups.

Synthetic schemes for preparing 14 (A) compounds

Wherein:

Ra is selected from the group consisting of: H. na, K, cs, li and an amine salt, wherein the amine salt is selected from the group consisting of: pyridine, H ₂NR'、HNR'₂ and NR' ₃;

R' is selected from the group consisting of: linear, branched, cyclic (substituted or unsubstituted) alkyl, alkenyl, alkynyl, aryl, and heteroaryl groups (C ₁-C₈);

In one embodiment, R' is selected from the group consisting of: containing one or more bases with or without a cyclic structure of heteroatoms (examples are described below).

Wherein,

W is selected from the group consisting of: o, NR, S, S (O), SO ₂, S (O) NH, S (O) NR ' and SN (R ') N (R ' ₂). R' is the same as described above.

The process for preparing the above 14 (a) compound is prepared as described above for compound 14, with the reaction conditions modified as necessary as will be appreciated by those skilled in the art in view of this disclosure. These compounds are prepared by using a suitable solvent in the presence of a base and a suitable reagent. Solvents such as ACN, DMF, NMP, THF, meTHF, dioxane, DMAc, DMSO, meOH, etOH, IPA, tert-BuOH, tert-AMOH, DCM, etOAc, IPAc or toluene. The base may be an inorganic base (e.g., Na₂CO₃、NaHCO₃、K₂CO₃、KHCO₃、CsCO₃、KOH、NaOH、LiOH、K₃PO₄、K₂HPO₄ or KH ₂PO₄).

Examples

The following examples are presented to aid in the understanding of the invention and are not intended to, and should not be construed to, limit in any way the invention as set forth in the claims following the examples.

In the examples below, some synthetic products are listed which have been isolated as residues. Those of ordinary skill in the art will appreciate that the term "residue" does not limit the physical state of the product as it is isolated and may include, for example, solids, oils, foams, gels, slurries, and the like.

Abbreviations used in the specification, particularly schemes and examples, are listed in table a below:

Table a: abbreviations (abbreviations)

/>

As used herein, ambient temperature refers to room temperature, which is typically in the range of about 20 ℃ to about 25 ℃ (about 68°f to about 77°f), or about 25 ℃.

As used herein, unless otherwise indicated, the term "isolated form" shall mean that the compound exists in any solid mixture with another compound, separate from the solvent system, or separate from the biological environment. In one embodiment of the invention, any of the compounds as described herein are present in isolated form.

As used herein, unless otherwise indicated, the term "substantially pure form" shall mean that the mole percent of impurities in the isolated compound is less than about 5 mole percent, preferably less than about 2 mole percent, more preferably less than about 0.5 mole percent, and most preferably less than about 0.1 mole percent. In one embodiment of the invention, the compound of formula (I) is present in substantially pure form.

As used herein, unless otherwise indicated, the term "substantially free of the corresponding salt form" when used to describe a compound of formula (I) shall mean that the mole percent of the corresponding salt form in the isolated base of formula (I) is less than about 5 mole percent, preferably less than about 2 mole percent, more preferably less than about 0.5 mole percent, and most preferably less than about 0.1 mole percent. In one embodiment of the invention, the compound of formula (I) is present in a form substantially free of the corresponding salt form.

Example 1

4- ((6- (Methoxycarbonyl) pyridin-2-yl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diazadioctadec-7-yl) methyl) benzoic acid

(TOPA- [ C-7] -phenyl-carboxylic acid)

Scheme 1

Step 1: tetrahydrofuran (100 mL) was added in one portion to a mixture of methyl 6-formylpicolinate (4.00 g,24.2 mmol), (4- (tert-butoxycarbonyl) phenyl) boronic acid (10.7 g,48.5 mmol), pdCl ₂ (0.21 g,1.2 mmol), tris (naphthalen-1-yl) phosphine (0.50 g,1.2 mmol) and potassium carbonate (10.0 g,72.7 mmol) in a 500mL three-necked round bottom flask under nitrogen. The mixture was purged with nitrogen and stirred at room temperature for 30 minutes and then heated at 65 ℃ for 24 hours. The reaction mixture was cooled to room temperature, filtered through a pad of Celite, and the filtrate was concentrated to dryness. The crude product was purified by silica gel chromatography (0% -50% etoac/petroleum ether) to give methyl 6- ((4- (tert-butoxycarbonyl) phenyl) (hydroxy) methyl) pyridinecarboxylate as a yellow oil (2.5 g,30% yield).

Step 2: a stirrer, methyl 6- ((4- (tert-butoxycarbonyl) phenyl) (hydroxy) methyl) pyridine carboxylate (2.50 g,7.30 mmol), PPh ₃ (3.43 g,13.1 mmol), N-bromosuccinimide (2.13 g,12.0 mmol) and methylene chloride (30 mL) were added to a 250mL three-necked round bottom flask under nitrogen at room temperature and stirred for 1 hour. The reaction solution was loaded onto a silica gel column and chromatographed (0% -30% etoac/petroleum ether) to give compound 6- (bromo (4- (tert-butoxycarbonyl) phenyl) methyl) picolinate (1.65 g,56% yield) as a yellow oil.

Step 3: a stirrer, methyl 6- (bromo (4- (tert-butoxycarbonyl) phenyl) methyl) pyridinecarboxylate (1.52 g,3.69 mmol), methyl 6- ((1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridinecarboxylate (1.50 g,3.69 mmol), na ₂CO₃ (1.17 g,11.1 mmol) and acetonitrile (30 mL) were added to a 250mL three-necked round bottom flask under a nitrogen atmosphere, and the resulting heterogeneous mixture was heated at 90℃for 16 hours. The reaction mixture was then cooled to room temperature, filtered through celite pad, and concentrated to dryness in vacuo to give the crude product. The crude product was purified by silica gel chromatography (0% -10% meoh in dichloromethane) to give methyl 6- ((4- (tert-butoxycarbonyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridinecarboxylate as a brown oil (1.2 g,44% yield).

Step 4: stir bar, methyl 6- ((4- (tert-butoxycarbonyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-n-7-yl) methyl) pyridinecarboxylate (1.2 g,1.6 mmol), TFA (0.62 mL,8.1 mmol) and DCM (20 mL) were added to a 100mL three-necked round bottom flask at room temperature and stirred for 1 hour. The reaction mixture was concentrated to dryness and the resulting crude product was subjected to preparative HPLC (column: XBRIDGE C18 (19 mm. Times.150 mm) 5.0 μm; mobile phase: 0.1% aqueous TFA/ACN; flow rate: 15.0 mL/min) to give TOPA- [ C-7] -phenyl-carboxylic acid (0.8 g, 72%) as a brown oil. LC-MS APCI: calculated for C ₃₅H₄₄N₄O₁₀: 680.31; actual measurement value: m/z [ M+H ] ⁺ 681.5. Purity as measured by LC-MS: 99.87%. Purity by HPLC: 97.14% (97.01% at 210nm, 97.20% at 254nm and 97.21% at 280 nm), column ATLANTIS DC (250 mm. Times.4.6 mm), 5 μm, mobile phase A0.1% TFA in water, mobile phase B acetonitrile, flow rate 1.0 mL/min .％.¹H NMR(400MHz,DMSO-d6):δ8.12-8.07(m,4H),8.00-7.98(m,2H),7.75-7.73(m,4H),6.10(s,1H),4.67(s,2H),3.96(s,3H),3.91(s,3H),3.82(s,8H),3.56(s,8H),3.52(s,8H).

Example 2

6- ((16- ((6-Carboxypyridin-2-yl) (4- ((2- (2- (2-isothiocyanato ethoxy) ethyl) carbamoyl) phenyl) methyl) -1,4,10,13-tetraoxa-7, 16-diazacyclooctadec-7-yl) methyl) pyridinecarboxylic acid

Scheme 2

Step 1: to a 25mL three-necked round bottom flask was added, under a nitrogen atmosphere, tert-butyl 4- ((6- (methoxycarbonyl) pyridin-2-yl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) benzoate (0.40 g,0.60 mmol), (2- (2- (2-aminoethoxy) ethoxy) ethyl) carbamate (0.15 g,0.60 mmol), triethylamine (0.18 g,0.76 mmol), HATU (0.33 g,0.90 mmol) and DCM (4.0 mL) at 0deg.C. The mixture was stirred at room temperature overnight. The reaction was treated with water (10 mL) and extracted with dichloromethane (10 mL. Times.3). The combined extracts were washed with 10% aqueous nahco ₃ (10 mL), brine (10 mL), dried over anhydrous Na ₂SO₄, filtered and concentrated to dryness to give a concentrate which was purified by silica gel chromatography (0% -10% meoh/DCM) to give methyl 6- ((4- ((2, 2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-n-7-yl) methyl) pyridinecarboxylate (0.18 g).

Step 2: a solution of stirrer, 6- ((4- ((2, 2-dimethyl-4-oxo-3,8,11-azatridecan-13-yl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadecan-7-yl) methyl) pyridine carboxylic acid methyl ester (0.18 g,0.20 mmol), meOH (1.8 mL) and HCl in methanol (4M, 1.0mL,4.0 mmol) was added to a 10mL single neck round bottom flask at 0deg.C, then warmed to room temperature and stirred for 2 hours. The volatiles were removed in vacuo to give methyl 6- ((4- ((2- (2- (2-aminoethoxy) ethoxy) ethyl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridinecarboxylate (0.15 g), which was used without purification.

Step 3: methyl 6- ((4- ((2- (2- (2-aminoethoxy) ethoxy) ethyl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridinecarboxylate (0.10 g,0.12 mmol), triethylamine (37 mg,0.37 mmol), anhydrous DCM (2 mL) and carbon disulphide (14 mg,0.18 mmol) were added to a pressure vial at room temperature under a nitrogen atmosphere. The vials were subjected to microwave irradiation (150W power) at 90 ℃ for 30 minutes. The vial was then cooled to room temperature, the reaction mixture was diluted with dichloromethane (10 mL), then washed sequentially with water (5 mL), 1M HCl (5 mL) and water (5 mL), dried over anhydrous Na ₂SO₄, filtered and concentrated to dryness to give methyl 6- ((4- ((2- (2- (2-isothiocyanato ethoxy) ethyl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diazacyclooctadec-7-yl) methyl) pyridinecarboxylate (100 mg) which was used directly without purification.

Step 4: a stirrer, methyl 6- ((4- ((2- (2- (2-isothiocyanato ethoxy) ethyl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridinecarboxylate (0.10 g,0.12 mmol) and aqueous HCl (6N, 0.4mL,2.34 mmol) were added to a 10mL single neck round bottom flask and stirred at 50℃for 3 hours. The reaction mixture was cooled to room temperature and concentrated to dryness in vacuo to give an oil which was purified by preparative HPLC (column: XBRIDGE C1819 mm. Times.150 mm,5.0 μm; mobile phase: 0.1% TFA in water/acetonitrile; flow rate: 15.0 mL/min) to give 6- ((6-carboxypyridin-2-yl) (4- ((2- (2- (2-isothiocyanato ethoxy) ethyl) carbamoyl) phenyl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-n-7-yl) methyl) pyridine carboxylic acid (5.0 mg). LC-MS APCI: calculated value of C ₄₀H₅₂N₆O₁₁ S: 824.34; actual measurement value ：m/z[M+H]⁺824.8.¹HNMR(400MHz,CD₃OD):δ8.22–8.20(m,2H),8.14-8.05(m,2H),7.94(d,J＝8.00Hz,2H),7.79(d,J＝8.00Hz,2H),7.73-7.67(m,2H),6.16(s,1H),4.77(s,2H),3.93-4.00(m,8H),3.59-3.70(m,27H),3.47–3.44(m,2H).

Example 3

6- ((4- ((6-Aminohexyl) carbamoyl) phenyl) (16- ((6-carboxypyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diazadioctadec-7-yl) methyl) pyridinecarboxylic acid

And

Example 4

6- ((16- ((6-Carboxypyridin-2-yl) (4- ((6-isothiocyanatohexyl) carbamoyl) phenyl) methyl) -1,4,10,13-tetraoxa-7, 16-diazacyclooctadec-7-yl) methyl) pyridine carboxylic acid

Scheme 3

Step 1: to a 25mL three-necked round bottom flask was added, under a nitrogen atmosphere, stirring, 4- ((6- (methoxycarbonyl) pyridin-2-yl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) benzoic acid (0.12 g,0.18 mmol), (6-aminohexyl) carbamic acid tert-butyl ester (38 mg,0.18 mmol), triethylamine (54 mg,0.54 mmol), HATU (0.10 g,0.27 mmol) and DCM (4.0 mL) at 0deg.C. The reaction mixture was then warmed to room temperature and stirred overnight. The reaction was then treated with water (10 mL) and extracted with dichloromethane (10 mL. Times.3). The combined extracts were washed with 10% aqueous nahco ₃ (10 mL) and brine (10 mL), dried over anhydrous Na ₂SO₄, filtered and concentrated to dryness to give an oil. The oil was purified by silica gel chromatography (0% -10% meoh/DCM) to give methyl 6- ((4- ((6- ((tert-butoxycarbonyl) amino) hexyl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-n-7-yl) methyl) pyridinecarboxylate (70 mg) as a viscous oil.

Step 2: methyl ester (70 mg,0.080 mmol) of 6- ((4- ((6- ((tert-butoxycarbonyl) amino) hexyl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridine carboxylate (70 mg,0.080 mmol), meOH (1.5 mL) and a solution of HCl in methanol (4M, 0.4mL,1.6 mmol) were added to a 25mL round bottom flask which was then allowed to warm to room temperature and stirred for 2 hours. The volatiles were removed in vacuo to give methyl 6- ((4- ((6-aminohexyl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridinecarboxylate (30 mg) which was used directly without purification.

Step 3: stir bar, 6- ((4- ((6-aminohexyl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diazadioctadec-7-yl) methyl) pyridine carboxylic acid methyl ester (30 mg,0.038 mmol), aqueous LiOH (1.1 mL,0.1n,0.11 mmol) and MeOH (1.0 mL) were added to an 8mL reaction vial at room temperature and stirred overnight. The reaction mixture was then treated with acetic acid until pH 6.5, then concentrated to dryness in vacuo at room temperature. The resulting product was subjected to preparative HPLC (column: XBRIDGE C1819 mM. Times.150 mM,5.0 μm; mobile phase: 10mM ammonium acetate in water/ACN; flow rate: 15.0 mL/min) to give example 3:6- ((4- ((6-aminohexyl) carbamoyl) phenyl) (16- ((6-carboxypyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridinecarboxylic acid (10 mg). LC-MS APCI: calculated for C ₃₉H₅₄N₆O₉: 750.40; actual measurement value ：m/z[M+H]⁺751.3.¹H NMR(400MHz,CD₃OD):δ8.22(d,J＝1.60Hz,2H),8.21-8.06(m,2H),7.92(d,J＝8.40Hz,2H),7.80(d,J＝8.40Hz,2H),7.75-7.69(m,2H),6.20(s,1H),4.70(s,2H),4.02-3.92(m,8H),3.76-3.62(m,14H),3.51-3.32(m,4H),2.93(t,J＝8.00Hz,2H),1.67-1.64(m,4H),1.46-1.45(m,4H).

Step 4: methyl 6- ((4- ((6-aminohexyl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-n-7-yl) methyl) pyridinecarboxylate (0.10 g,0.13 mmol), triethylamine (39 mg,0.38 mmol), anhydrous DCM (2 mL) and carbon disulphide (15 mg,0.19 mmol) were added to a pressure vial at room temperature under a nitrogen atmosphere. The vials were subjected to microwave irradiation (150W power) at 90 ℃ for 30 minutes. The vial was then cooled to room temperature and the reaction mixture was diluted with dichloromethane (10 mL), washed with water (5 mL), 1M HCl (5 mL) and water (5 mL), dried over anhydrous Na ₂SO₄, filtered and concentrated to dryness to give methyl 6- ((4- ((6-isothiocyanatohexyl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diazacyclooctadec-7-yl) methyl) pyridinecarboxylate (0.1 g) which was used directly without purification.

Step 5: a stirrer, methyl 6- ((4- ((6-isothiocyanatohexyl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diazadioctadec-7-yl) methyl) pyridinecarboxylate (0.10 g,0.12 mmol) and aqueous HCl (6N, 0.4mL,2.4 mmol) were added to a 10mL round bottom flask and then stirred at 50℃for 3 hours. The reaction mixture was then cooled to room temperature and concentrated to dryness in vacuo to give a residue which was purified by preparative HPLC (column: XBRIDGE C1819mm x 150mm,3.5 μm; mobile phase: 0.1% tfa in water/acetonitrile; flow rate: 2.0 mL/min) to give example 4:6- ((16- ((6-carboxypyridin-2-yl) (4- ((6-isothiocyanatohexyl) carbamoyl) phenyl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridinecarboxylic acid (15 mg). LC-MS APCI: calculated value of C ₄₀H₅₂N₆O₉ S: 792.35; actual measurement value ：m/z[M+H]⁺792.8.¹H NMR(400MHz,CD₃OD):δ8.23-8.20(m,2H),8.15-8.06(m,2H),7.92(d,J＝8.40Hz,2H),7.79(d,J＝8.40Hz,2H),7.74–7.68(m,2H),6.17(s,1H),4.77(s,2H),4.01-3.93(m,8H),3.75-3.56(m,16H),3.42-3.33(m,5H),1.74-1.64(m,4H),1.50-1.44(m,4H).

Example 5

6- ((16- ((6-Carboxypyridin-2-yl) (4- ((4-isothiocyanatophenethyl) carbamoyl) phenyl) methyl) -1,4,10,13-tetraoxa-7, 16-diazacyclooctadec-7-yl) methyl) pyridinecarboxylic acid

Scheme 4

Step 1: a stirrer, 4- ((6- (methoxycarbonyl) pyridin-2-yl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) benzoic acid (0.25 g,0.37 mmol), 4- (2-aminoethyl) aniline (60 mg,0.37 mmol), TEA (0.11 g,0.15mL,1.1 mmol), HATU (0.21 g,0.55 mmol) and DCM (5 mL) were added to a 25mL three-necked round bottom flask under a nitrogen atmosphere. The reaction mixture was stirred at room temperature overnight, then treated with water (10 mL) and extracted with dichloromethane (10 mL. Times.3). The combined extracts were washed with 10% aqueous nahco ₃ (10 mL) and brine (10 mL), dried over anhydrous Na ₂SO₄, filtered and concentrated to dryness to give the product, which was purified by silica gel chromatography (0-10% meoh/DCM) to give methyl 6- ((4- ((4-aminophenylethyl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-l-7-yl) methyl) pyridinecarboxylate (0.12 g).

Step 2: methyl 6- ((4- ((4-aminophenylethyl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridinecarboxylate (0.12 g,0.15 mmol), TEA (45 mg, 65. Mu.L, 0.45 mmol), DCM (3 mL) and CS ₂ (17 mg,0.23 mmol) were added to a 10mL microwave pressure vial at room temperature under a nitrogen atmosphere. The reaction mixture was subjected to microwave irradiation (150W power) at 90 ℃ for 30 minutes. The reaction mixture was then cooled to room temperature, diluted with dichloromethane (10 mL), washed sequentially with water (5 mL), 1M HCl (5 mL) and water (5 mL), dried over anhydrous Na ₂SO₄ and concentrated to dryness to give methyl 6- ((4- ((4-isothiocyanatoethyl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diazacyclooctadec-7-yl) methyl) pyridinecarboxylate (0.12 g), which was used directly without purification.

Step 3: a stirrer, methyl 6- ((4- ((4-isothiocyanatophenethyl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diazadioctadec-7-yl) methyl) pyridinecarboxylate (0.12 g,0.14 mmol) and aqueous HCl (0.50 mL,6N,2.8 mmol) were added to a 10mL single neck round bottom flask and stirred at 50℃for 3 hours. The reaction mixture was cooled to room temperature, concentrated to dryness in vacuo, and the crude product was subjected to preparative HPLC (column: XBRIDGE C1819 ×150mm,5.0 μm; mobile phase: 0.1% TFA in water/acetonitrile; flow rate: 15.0 mL/min) to give 6- ((16- ((6-carboxypyridin-2-yl) (4- ((4-isothiocyanatophenyl ethyl) carbamoyl) phenyl) methyl) -1,4,10,13-tetraoxa-7, 16-diazacyclooctadec-7-yl) methyl) pyridine carboxylic acid (30 mg). LC-MS APCI: calculated value of C ₄₂H₄₈N₅O₁₀ S: 812.32; actual measurement value ：m/z[M+H]⁺812.9.¹H NMR(400MHz,CD₃OD):δ8.22(d,J＝0.80Hz,2H),8.06-8.21(m,2H),7.85(d,J＝8.40Hz,2H),7.68-7.78(m,4H),7.31(d,J＝8.40Hz,2H),7.21(d,J＝2.00Hz,2H),6.18(s,1H),4.77(s,2H),3.70-4.00(m,7H),3.60-3.67(m,16H),3.44-3.49(m,2H),2.90-3.10(m,3H).

Example 6

6- ((4- ((2- (2- (2-Aminoethoxy) ethoxy) ethyl) carbamoyl) phenyl) (16- ((6-carboxypyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diazadioctadec-7-yl) methyl) pyridinecarboxylic acid

Scheme 9

Step 1: to a 25mL three-necked round bottom flask was added, under a nitrogen atmosphere, tert-butyl 4- ((6- (methoxycarbonyl) pyridin-2-yl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) benzoate (0.40 g,0.60 mmol), (2- (2- (2-aminoethoxy) ethoxy) ethyl) carbamate (0.15 g,0.60 mmol), triethylamine (0.18 g,0.76 mmol), HATU (0.33 g,0.90 mmol) and DCM (4.0 mL) at 0deg.C. The mixture was stirred at room temperature overnight, diluted with water (10 mL) and extracted with dichloromethane (10 ml×3). The combined extracts were washed with 10% aqueous nahco ₃ (10 mL) and brine (10 mL), dried over anhydrous Na ₂SO₄, filtered and concentrated to dryness to give a concentrate which was purified by silica gel chromatography (0% -10% meoh/DCM) to give methyl 6- ((4- ((2, 2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-n-7-yl) methyl) pyridinecarboxylate (0.18 g).

Step 3: methyl 6- ((4- ((2- (2- (2-aminoethoxy) ethoxy) ethyl) carbamoyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridinecarboxylate (0.1 g,0.1 mmol), aqueous LiOH (3 mL,0.1n,0.3 mmol) and MeOH (1.0 mL) were added to an 8mL reaction vial at room temperature and stirred overnight. The reaction mixture was adjusted to pH 6.5 with acetic acid and then concentrated to dryness in vacuo at room temperature to give a concentrate which was purified by preparative HPLC (column: XBRIDGE C1819 mm. Times.150 mm,5.0 μm; mobile phase: 0.1% TFA aqueous solution/ACN; flow rate: 15.0 mL/min) to give 6- ((4- ((2- (2-aminoethoxy) ethyl) carbamoyl) phenyl) (16- ((6-carboxypyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diazacyclooctadec-7-yl) methyl) pyridine carboxylic acid (40 mg). LC-MS APCI: calculated for C ₃₉H₅₄N₆O₁₁: 782.39; actual measurement value: m/z [ M+H ] +783.0.

Example 7

N-acyl-DBCO labeled 6- ((4- ((6-aminoethyl) carbamoyl) phenyl) 16- ((6-carboxypyridin-2-yl) -1,4,10,13-tetraoxa-7, 16-diazacyclooctadec-7-yl) methyl) pyridinecarboxylic acid (TOPA- [ C7] -phenylimino-DBCO)

Scheme 15

Step 1: tetrahydrofuran (100 mL) was added in one portion to a mixture of methyl 6-formylpicolinate (4.00 g,24.2 mmol), (4- (tert-butoxycarbonyl) phenyl) boronic acid (10.7 g,48.5 mmol), pdCl ₂ (0.21 g,1.2 mmol), tris (naphthalen-1-yl) phosphine (0.50 g,1.2 mmol) and potassium carbonate (10.0 g,72.7 mmol) in a 500mL three-necked round bottom flask under nitrogen. The mixture was purged with nitrogen and stirred at room temperature for 30 minutes and then heated at 65 ℃ for 24 hours. The reaction mixture was cooled to room temperature byThe pad was filtered and the filtrate was concentrated to dryness. The crude product was chromatographed on silica gel (0% -50% EtOAc/petroleum ether) to give methyl 6- ((4- (tert-butoxycarbonyl) phenyl) (hydroxy) methyl) pyridinecarboxylate as a yellow oil (2.5 g, 30%).

Step 2: a stirrer, methyl 6- ((4- (tert-butoxycarbonyl) phenyl) (hydroxy) methyl) pyridinecarboxylate (2.50 g,7.30 mmol), PPh ₃ (3.43 g,13.1 mmol), N-bromosuccinimide (2.13 g,12.0 mmol) and DCM (30 mL) were placed in a 250mL three-necked round bottom flask and stirred for 1 hour under a nitrogen atmosphere at room temperature. The reaction solution was applied to a silica gel column and purified using 0% -30% ethyl acetate/petroleum ether to give methyl 6- (bromo (4- (tert-butoxycarbonyl) phenyl) methyl) pyridinecarboxylate (1.65 g, 56%) as a yellow oil.

Step 3: a stirrer, methyl 6- ((1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridine carboxylate (1.52 g,3.69 mmol), methyl 6- (bromo (4- (tert-butoxycarbonyl) phenyl) pyridine carboxylate (1.50 g,3.69 mmol), na ₂CO₃ (1.17 g,11.1 mmol) and acetonitrile (30 mL) were added to a 250mL three-necked round bottom flask under nitrogen atmosphere and the resulting heterogeneous mixture was heated at 90℃for 16 hours under nitrogen. The reaction mass was then cooled to room temperature byFiltration over a pad and concentration to dryness in vacuo afforded the crude product. The crude product was purified by silica gel chromatography (0% -10% meoh/DCM) to give methyl 6- ((4- (tert-butoxycarbonyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridinecarboxylate as a brown oil (1.2 g,44% yield).

Step 4: stir bar, methyl 6- ((4- (tert-butoxycarbonyl) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-n-7-yl) methyl) pyridinecarboxylate (1.2 g,1.6 mmol), TFA (0.62 mL,8.1 mmol) and DCM (20 mL) were added to a 100mL three-necked round bottom flask at room temperature and stirred for 1 hour. The reaction mixture was concentrated to dryness and the resulting crude product was subjected to preparative HPLC (column: XBRIDGE C (19 mm. Times.150 mm) 5.0 μm; mobile phase: 0.1% TFA in water/ACN; flow rate: 15.0 mL/min) to give 4- ((6- (methoxycarbonyl) pyridin-2-yl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-n-7-yl) methyl) benzoic acid (0.8 g, 72%) as a brown oil. LC-MS APCI: calculated for C ₃₅H₄₄N₄O₁₀: 680.31; actual measurement value: m/z [ M+H ] ⁺ 681.5. Purity as measured by LC-MS: 99.87%. Purity by HPLC: 97.14% (97.01% at 210nm, 97.20% at 254nm and 97.21% at 280 nm), column ATLANTIS DC (250 mm. Times.4.6 mm), 5 μm, mobile phase A0.1% TFA in water, mobile phase B acetonitrile, flow rate 1.0 mL/min .％.¹H NMR(400MHz,DMSO-d6):δ8.12-8.07(m,4H),8.00-7.98(m,2H),7.75-7.73(m,4H),6.10(s,1H),4.67(s,2H),3.96(s,3H),3.91(s,3H),3.82(s,8H),3.56(s,8H),3.52(s,8H).

Step 5: a stirrer, 4- ((6- (methoxycarbonyl) pyridin-2-yl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-n-7-yl) methyl) benzoic acid (0.25 g,0.37 mmol), DBCO (0.10 g,0.37 mmol), triethylamine (0.16 mL,1.1 mmol), HBTU (0.21 g,0.55 mmol) and DCM (10 mL) were added to a 25mL three-necked round bottom flask under a nitrogen atmosphere and stirred at room temperature for 16 h. The reaction was quenched with water (20 mL) and extracted with DCM (3X 20 mL). The combined extracts were washed with 10% aqueous nahco ₃ (20 mL), brine (20 mL), dried over anhydrous Na ₂SO₄, filtered and concentrated to dryness to give the crude product as an oil. The crude product was chromatographed on silica gel (0% -10% MeOH/DCM) to give TOPA dimethyl- [ C7] -phenyl-DBCO (0.12 g, 35%) as a colorless viscous oil.

Step 6: a stirrer, TOPA dimethyl- [ C7] -phenyl-DBCO (0.1 g,0.1 mmol), liOH.H ₂ O in water (3 mL,0.1N,0.3 mmol) and THF/MeOH/H ₂ O (4:1:1 v/v/v,2 mL) were added to an 8mL reaction vial at room temperature and stirred for 2 hours. The reaction mixture was neutralized to pH 6.56.5 with aqueous HCl (1N). The reaction mixture was concentrated to dryness in vacuo at room temperature and the resulting crude product was subjected to preparative HPLC (column: XBRIDGE C18 (19 mM. Times.150 mM) 5.0 μm; mobile phase: 10mM aqueous ammonium acetate/ACN; flow rate: 15.0 mL/min) to give TOPA- [ C7] -phenyl-DBCO (20 mg, 21%) as an off-white solid. LC-MS APCI: calculated for C ₅₁H₅₄N₆O₁₀: 910.39; actual measurement value: m/z [ M-H ] ⁺ 909.3. Purity as measured by LC-MS: 92.47%. Purity by HPLC: 90.68% (88.04% at 210nm, 90.43% at 254nm and 93.56% at 280 nm), column XBRIDGE C (50X 4.6 mM), 3.5 μm, mobile phase A10 mM ammonium bicarbonate aqueous solution, mobile phase B acetonitrile, flow rate 1.0 mL/min .¹HNMR(400MHz,DMSO-d6):δ7.84-7.82(m,4H),7.60-7.29(m,12H),7.13-7.10(m,2H),5.12-5.02(m,2H),3.97(s,2H),3.59-3.44(m,20H),2.85(s,4H),2.73-2.68(m,6H).

Example 8

TOPA- [ C7] -phenylimino-DBCO-triazole-PSMB-127 antibody conjugates

Azide modification and click reaction of mab: PSMB127 was site-selectively modified with a 100 molar excess of 3-azidopropylamine and microbial transglutaminase (MTG; active TI) at 37 ℃. The addition of two azides on the heavy chain of the mAb was monitored by full mass ESI-TOF LC-MS on an Agilent G224 instrument. Excess 3-azidopropylamine and MTG were removed and azide-modified mAb (azido-mAb) was purified using a 1mL GE Healthcare MabSelect column. azido-mAb was eluted from the resin using 100mM sodium citrate (pH 3.0) followed by 7KThe desalting column was exchanged into 20mM Hepes, 100mM NaCl (pH 7.5). A 10x molar excess of TOPA- [ C7] -phenyl-DBCO was reacted with site-specific azide-PSMB 127 (dol=2) at 37 ℃ for 1 hour without shaking. Completion of the DBCO-azide click reaction was monitored by complete mass spectrometry. By the method disclosed in Zeba/>The conjugate was desalted onto a desalting column into 20mM Hepes, 100mM NaCl (pH 7.5) and then the excess free chelator was removed using a 30KMWCO Amicon concentration device by three consecutive 15 Xdilution and concentration steps at 3800 Xg spin in 20mM Hepes, 100mM NaCl (pH 7.5). This provides the final site-specific TOPA- [ C7] -phenyl-DBCO-PSMB 127 conjugate, where car=2. The final conjugate was confirmed as monomer by analytical size exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8.8 mm x 30cm,5u column using the following conditions; column temperature: room temperature; eluting the column with DPBS buffer (1 x, free of calcium and magnesium); flow rate: 0.7mL/min;18Min operation; sample injection volume: 18. Mu.L.

Step 2, chelation: a stock solution of the following metal salts was prepared in pure water:

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the metal solution was added to a 5x molar excess of TOPA- [ C7] -phenyl-DBCO-PSMB 127 (6.8. Mu.M antibody, 34. Mu.M metal ion) in 10mM sodium acetate buffer (pH 5.2) and incubated for 2 hours at 37 ℃. By using Column (/ >)) Desalination followed by 50K MWCO Amicon concentrator (EMD/>)) Two 10x dilution and concentration cycles were performed to remove excess metal. Chelation was assessed by LC-MS of intact and simplified mass.

Step 3, stability measurement: to determine the stability of the chelate, a DTPA test was performed. mu.L of sample (6.3. Mu.M antibody) was mixed with 50. Mu.L of 10mM DTPPH 6.5 and incubated overnight at 37 ℃. Chelation was assessed by LC-MS of intact and simplified mass. LC-MS was performed on an Agilent 1260HPLC system connected to an Agilent G6224 MS-TOF mass spectrometer. LC was run on AGILENTRP-mAb C4 column (2.1 mm x 50mm,3.5 microns) at a flow rate of 1 mL/min with mobile phase of 0.1% formic acid in water (a) and 0.1% formic acid in acetonitrile (Sigma-Aldrich, cat No. 34688) (B) and gradient of 20% B (0 to 2 min), 20% -60% B (2 to 3 min), 60% -80% B (3 to 5.5 min). The instrument was operated in positive electrospray ionization mode and scanned from m/z 600 to 6000. The mass-to-charge ratio spectrum is deconvolved using a maximum entropy algorithm, and the relative amounts of the related substances are estimated by deconvolving the peak heights of the masses. The instrument arrangement comprises: capillary voltage 3500V; a fragmentation voltage 175V; skimming tool voltage 65V; a gas temperature 325C; the flow rate of the drying gas is 5.0L/min; sprayer pressure 30psig; acquisition mode range 100-7000, scan rate 0.42.

MW changes relative to TOPA- [ C7] -phenyl-DBCO-PSMB 127 were observed for cerium and neodymium samples. The complete mass of the conjugates incubated with cerium showed either a MW increase of 139 (20%, peak area) or 276 (77%). Da corresponds to the addition of 1 or 2 cerium ions. After DTPA testing, the masses remained similar, with similar abundances (30% and 67% for +138 species and +274 species).

Example 9

6- ((16- ((6-Carboxypyridin-2-yl) (4-isothiocyanatophenyl) methyl) -1,4,10,13-tetraoxa-7, 16-diazadioctadec-7-yl) methyl) pyridine carboxylic acid (H2 bp18C 6-benzyl-phenyl) (TOPA- [ C7] -phenyl isothiocyanate and sodium salt forms

Compound 2 was prepared in a similar manner to the prior literature methods, see j.org.chem;1987, 52, 5172.

Compound 3 was prepared in a similar manner to the prior literature methods, see Chemistry-aEuropean Journal;2015, 21, 10179.

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Preparation of Compound 4:

1,4,10,13-tetraoxa-7, 16-diazaoctadecane (494 g,1.88mol,2.5 eq), naCl (44.1 g,0.75mol,1.0 eq), H ₂ O (140 mL,1 vol. With respect to Compound 3) and acetonitrile (2.1L, 15 vol.) are charged to a 10L reactor at 15℃to 20℃under an atmosphere of N ₂. To the resulting mixture was added dropwise a solution of compound 3 (140 g,0.75 mol) in acetonitrile (280 ml,2 volumes) at 65℃over 1 hour. The solution was aged at 65℃for 0.5 hours. LCMS analysis of the mixture showed the reaction was complete. The mixture was cooled to room temperature and concentrated in vacuo. Acetone (700 ml,5 volumes) was added to the mixture and the suspension was stirred for an additional 1 hour. The mixture was filtered (the filtered solid was unreacted compound 2). The filtrate was concentrated in vacuo and then dissolved in DCM (1.4 l,10 vol). The organic phase was washed with water (3×750 mL) and dried over Na ₂SO₄, then concentrated in vacuo to give compound 4, 212g (63% yield, assay) ：85％w/w).LCMS:(ES,m/z):412.15[M+H]⁺¹H-NMR(300MHz,DMSO-d₆,ppm):δ7.98–7.87(m,2H),7.81(dd,J＝6.4,2.6Hz,1H),3.87(s,3H),3.81(s,2H),3.61–3.38(m,16H),2.77(dt,J＝19.0,5.2Hz,8H).

Preparation of compound 7:

Methyl 6-formylpyridinium carboxylate 5 (250 g,1.0 eq), (4- ((tert-butoxycarbonyl) amino) phenyl) boronic acid 6 (538 g,1.5 eq) and degassed THF (6.5L, 26 volumes relative to 5) were charged to a 10L reactor at 15 ℃ to 20 ℃ under an atmosphere of N ₂. PdCl ₂ (14.0 g,0.05 eq), tris (naphthalen-1-yl) -phosphane (31 g,0.05 eq) and K ₂CO₃ (650 g,3.1 eq) were then added. The resulting solution was stirred at 20℃for 0.5 h. The mixture was then heated to 65 ℃ and aged for 17 hours. Analysis by LCMS showed the reaction was complete. The resulting solution was cooled at room temperature and diluted with ice water (2.5 l,10 volumes) and ethyl acetate (5 l,20 volumes). The mixture was stirred and then filtered through a pad of Celite. The solution was separated and the aqueous layer was discarded. The organic phase was washed with water (2X 1.5L,12 vol). The layers were separated and the organic layer was dried over Na ₂SO₄ and concentrated in vacuo. The resulting residue was treated with heptane (1.25L, 5 volumes) and the resulting suspension was stirred for 0.5 hours. The mixture was filtered and the filter cake was washed with n-heptane (500 ml,2 volumes) to give 530g (98% yield, LCAP purity: 90%) of the desired product 7 as a yellow solid which was used in the next step without further purification .LCMS:(ES,m/z):381.10[M+Na]⁺¹H-NMR(300MHz,DMSO-d₆,ppm):δ9.27(s,1H),8.03–7.85(m,2H),7.79(dd,J＝7.7,1.4Hz,1H),7.39(d,J＝8.4Hz,2H),7.26(d,J＝8.4Hz,2H),6.13(d,J＝4.0Hz,1H),5.72(d,J＝3.9Hz,1H),3.87(s,3H),1.46(s,9H).

Preparation of compound 8;

6- ((4- ((tert-butoxycarbonyl) amino) phenyl) (hydroxy) methyl) pyridine carboxylic acid methyl ester 7 (310 g,1.0 eq), triethylamine (219 g,2.5 eq) and DCM (6.2L, 20 vol. Relative to 7) were charged to a 10L reactor at 15℃to 20℃under a nitrogen atmosphere and the solution was cooled to 0 ℃. Methanesulfonyl chloride (99.2 g,1.0 eq.) was added dropwise over 30 minutes, maintaining the temperature at 0 ℃. The cooling bath was removed and the temperature was brought to ambient temperature and then aged at that temperature for 1 hour. The solution was concentrated in vacuo at 10 ℃ to 15 ℃ and the residue was then dissolved in acetonitrile (438 ml,2 volumes). The resulting solution was concentrated in vacuo to give 518g (crude) of the desired product 8. The crude product was used directly in the next step without further purification.

Preparation of Compound 9:

Methyl 6- ((4- ((tert-butoxycarbonyl) amino) phenyl) - ((methylsulfonyl) oxy) methyl) pyridinecarboxylate 8 (212 g,1.0 eq, purity 85% as determined by Q-NMR), na ₂CO₃ (137.6 g,3.0 eq) and acetonitrile (3.56L, 20 volumes relative to 8) were charged to a 10L reactor at room temperature under nitrogen atmosphere, and the mixture was then heated to 65 ℃ and aged for 1 hour. A solution of methyl 6- ((1,4,10,13-tetraoxa-7, 16-diaza-octadeca-7-yl) methyl) pyridine carboxylate 4 (377.8 g,2.0 eq.) in acetonitrile (3L, 10 vol.) was added dropwise over 0.5 hours at 65 ℃. The mixture was aged at this temperature until HPLC analysis showed the reaction was complete. The resulting solution was cooled at room temperature and then filtered, and the filter cake was washed with MeOH (2 x 1 volumes). The filtrate was concentrated in vacuo and the resulting residue was dissolved in EA (700 mL) followed by addition of silica gel (800 g, model number ZCX-2, 100 mesh-200 mesh, 2.11 w/w). The mixture was concentrated in vacuo while maintaining the temperature below 35 ℃. Silica gel (9.6 kg, model ZCX-2, 100 mesh-200 mesh, 26.3 w/w) was charged into the column followed by the prepared dry silica gel containing adsorbed crude product 9. The column was eluted with ethyl acetate: petroleum ether: dichloromethane (3:3:1)/methanol: dichloromethane (1:1) (gradient 100:0 to 90:10, samples collected every 4 L.+ -. 0.5L). Fractions were analyzed by TLC (ethyl acetate: petroleum ether: dichloromethane: methanol=4:4:1:1). The product-containing fractions were combined and concentrated to give 260g of compound 9 as a yellow solid (HPLC: 94%, QNMR: 92%). An additional 70g of Compound 9 were obtained as a yellow oil (HPLC; 75%, QNMR: 60%). LCMS (ES, m/z): 752.30[ M+H ] ⁺ found m/z ¹H-NMR(400MHz,CDCl₃,ppm):δ7.53–7.32(m,3H),7.28–7.18(m,3H),6.86(d,J＝8.4Hz,2H),6.76(d,J＝8.4Hz,2H),6.09(s,1H),4.63(s,1H),3.48(s,3H),3.44(bs,5H),3.17–2.92(m,16H),2.38(dq,J＝25.0,7.2,6.8Hz,8H),0.97(s,9H).

Preparation of compound 10:

Compound 9 (260 g, qnmr:92%,1.0 eq), N, O-bis (trimethylsilyl) acetamide (BSA, 6.0 eq) and acetonitrile (4L, 15 volumes) were charged to a 10L reaction vessel under nitrogen atmosphere at 15-20 ℃. The mixture was stirred at 20℃for 40 min. A solution of TMSOTF (212.9 g,3.0 eq.) in acetonitrile (1.3L, 5 volumes) was added dropwise over 0.5 hours, the internal temperature being maintained between 15℃and 20 ℃. The solution was aged at 15℃to 20℃for 1 hour. When process analysis (sample preparation 0.1mL of system+0.9 mL acn+one drop of diisopropylethylamine) showed complete conversion of the starting material, the mixture was quenched with diisopropylethylamine (611 g,15.0 eq.) and the temperature was maintained between 5 ℃ and 10 ℃. The mixture was stirred at 5-10 ℃ for 20 min, then saturated aqueous NH ₄ Cl (2.6 l,10 volumes) was added and the temperature was maintained between 5-10 ℃. The mixture was aged at this temperature for an additional 30 minutes. The aqueous phase (containing solids) was collected and extracted with 2-MeTHF (520 ml,2 volumes). The organic phases were combined and checked for water content with KF (KF: 9.18%) and then dried over anhydrous Na ₂SO₄ (500 g,10.0 eq). The solids were removed by filtration and the filter cake was washed with acetonitrile (2X 520ml,2 volumes). The filtrate was then dried over anhydrous Na ₂SO₄ (500 g,10.0 eq). After filtration, the filter cake was washed with acetonitrile (2X 520ml,2 vol) and checked for water content with KF (KF: 8.15%). The 10 acetonitrile/2-MeTHF stream was used directly in the next step. (the product is unstable under LCMS conditions)

Preparation of Compound 14 (free acid)

6- ((4- ((Tert-Butoxycarbonyl) amino) phenyl) (16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10,13-tetraoxa-7, 16-diaza-octadeca-n-7-yl) methyl) pyridinecarboxylic acid methyl ester (6.0 g,1.0 eq), BSA (9.7 g,6.0 eq) and MeCN (120 mL,20 vol. Relative to 9) were added to a 500mL reactor at room temperature under a nitrogen atmosphere. A solution of TMSOTF (5.4 g,2.3 eq.) in MeCN (120 mL,20 volumes) was added dropwise over 30 minutes at room temperature. The mixture was aged at room temperature overnight. Analysis of the mixture (sample preparation 0.1mL of system +0.9mL ACN + one drop of diisopropylethylamine) showed that the reaction was complete. The mixture was quenched with diisopropylethylamine (15.4 g,15.0 eq.) and the temperature was maintained between 0 ℃ and 5 ℃. The mixture was stirred at 0 ℃ to 5 ℃ for 5 minutes, then saturated aqueous NH ₄ Cl (60 ml,10 volumes) was added dropwise, and the temperature was maintained between 0 ℃ to 5 ℃. The aqueous phase was removed by extraction and the organic phase was collected and used directly in the next step. The organic phase was charged to a 500mL three-necked round bottom flask and a solution of LiOH (1.15 g,6.0 eq.) in water (60 mL, 10V) was added to the solution at room temperature. The solution was stirred at this temperature for 1 hour. Analysis of the mixture (sample preparation, 0.1mL system +0.9mL acetonitrile) showed no complete conversion. Another portion of LiOH (576 mg,3.0 eq.) was added and the solution was stirred at room temperature for an additional 1 hour. Analysis of the mixture (sample preparation, 0.1mL system +0.9mL acetonitrile) showed that the reaction was complete. TCDI (5.6 g,3.9 eq) was then added and the solution stirred at room temperature for 1 hour. Analysis of the mixture (sample preparation, 0.1mL system +0.9mL acetonitrile) showed no complete conversion. Another portion of TCDI (2.8 g,2.0 eq.) was added and the solution stirred at room temperature for an additional 1 hour. Analysis of the mixture (sample preparation, 0.1mL system +0.9mL acetonitrile) showed that the reaction was complete. The reaction solution was separated by reverse phase Combi-Flash. The method comprises the following steps: column C18, solution a H ₂ O (containing 0.01% formic acid), solution B ACN. 5% to 35% in 40 min, flow rate (100 mL/min), product in 20 min-25 min. The solution was collected. The solution was concentrated to remove ACN and again isolated by reverse phase Combi-Flash. The method comprises the following steps: column C18, solution a, H ₂ O, solution B, ACN. Flow rate: 5%10 min, 5% to 35%5 min, 95%10 min, flow rate (100 mL/min), product 13 min-25 min. The solution was collected. The solution was concentrated in vacuo at <20 ℃ and dried by freeze-drying. This gave 2.5g (47% yield over 3 steps) of compound 14 as a yellow solid. Compound 14 (6- ((16- ((6-carboxypyridin-2-yl) (4-isothiocyanatophenyl) methyl) -1,4,10,13-tetraoxa-7, 16-diazacyclooctadec-7-yl) methyl) pyridine carboxylic acid) needs to be stored in -80℃.LCMS:(ES,m/z):666.3[M+H]⁺ ¹H-NMR:(400MHz,D₂O,ppm):7.94-7.84(m,4H),7.56-7.40(m,4H),7.16-7.14(m,2H),5.83(s,1H),4.56(s,2H),3.80-3.75(m,8H),3.60-3.49(m,14H),3.36-3.33(m,2H).

Preparation of compound 11 (sodium salt):

A solution of the prepared compound 10 in ACN and 2-MeTHF was charged to a 10L four-necked reactor and the solution was cooled to 5-10 ℃. Powdered NaOH (56.9 g,4.5 eq.) was added and the temperature was maintained between 5℃and 10 ℃. The solution was stirred at 15℃to 20℃for 0.5 h. Analysis of the mixture (sample preparation, 0.1mL system +0.9mL acetonitrile) showed no conversion. Additional powdered NaOH (25.3 g,2.0 eq.) was added at 5℃to 10 ℃. The solution was further aged at 15℃to 20℃for 0.5 hours. The second IPC was analyzed and showed 50% conversion. The final amount of powdered NaOH (25.3 g,2.0 eq.) was added at 5℃to 10 ℃. The mixture was stirred for an additional 0.5 hours at 15℃to 20 ℃. Analysis showed complete conversion of starting material 10. The mixture was filtered and the filter cake was washed with acetonitrile (2 x 520ml,2 volumes). The final solution (about 7.5L,28.8 volumes) was concentrated to 1-2 volumes and the temperature was maintained between 15℃and 20 ℃. The residue was then treated with acetonitrile (2L, 7.7 vol) and checked for water content with KF (KF: 5.7%). The mixture was filtered and the filter cake was washed with ACN (2 x 520ml,2 volumes). The solution was then concentrated to 1V-2V in vacuo at 15-20 ℃. The water content was checked again with KF (KF: 5.5%). The solution was diluted with acetonitrile (390 ml,1.5 volumes) and added dropwise to MTBE (2.6 l,10 volumes) over 0.5 hours, maintaining the temperature between 15 ℃ and 20 ℃. The solvent was decanted off leaving a viscous oil which was redissolved in acetonitrile (520 ml,2 volumes) and added to MTBE (2.6 l,10 volumes). This process was repeated four more times. A viscous oil was obtained which was finally dissolved in acetonitrile (520 ml,2 volumes) and dried, then concentrated under reduced pressure at 15℃to 20 ℃. The residual solvent was then removed by evaporation with an oil pump at 15-20 ℃. After drying, 335g of compound 11 were obtained as a pale yellow solid (QNMR: 70%, overall yield of the two steps 87%). LCMS (ES, m/z): 624.3[ M-TfONa-2Na+3H ] ⁺ ¹ H-NMR (300 MHz, methanol) -d₄,ppm):δ7.97(dd,J＝7.8,2.1Hz,2H),7.84(t,J＝7.7Hz,1H),7.75(t,J＝7.8Hz,1H),7.36(dd,J＝7.8,1.1Hz,1H),7.23(d,J＝7.7Hz,1H),7.11(d,J＝8.5Hz,2H),6.72(d,J＝8.5Hz,2H),3.96(s,1H),3.83-3.36(m,18H),3.03–2.62(m,6H),2.55(d,J＝14.3Hz,2H).

Preparation of Compound 12 (TOPA- [ C7] -phenylisothiocyanate sodium salt):

TCDI (68.7 g,1.4 eq) and acetonitrile (2.6L, 8 volumes) were charged to a 10L reactor at 15 ℃ -20 ℃ under nitrogen atmosphere. A solution of Compound 11 (330 g, na ⁺ salt, QNMR:70%,1.0 eq.) in acetonitrile (660 mL,2 volumes) was added dropwise over 30 minutes, the temperature being maintained between 15℃and 20 ℃. The mixture was aged at 15℃to 20℃for 0.5 h. Analysis of the mixture (sample preparation: 30. Mu.L of system +300. Mu. LACN +one drop of water) showed that the reaction was complete. The water content was checked with KF (KF: 0.19%). The system was dried and concentrated at 15 ℃ to 20 ℃ under reduced pressure. The resulting residue was dissolved in acetonitrile (945 ml,2.9 vol) and the water content was measured with KF (KF: 0.34%). Isopropyl acetate (660 ml,2 volumes) was added to the solution over 40 minutes at 15 ℃ -20 ℃. No nucleation was observed and additional isopropyl acetate (6.6 l,18 volumes) was slowly added dropwise over 40 minutes at 15 ℃ -20 ℃ resulting in precipitation of product 12, which was collected by filtration as a pale yellow solid. The solid was dissolved in acetonitrile (330 ml,1 vol) and IPAc (6.6 l,20 vol) was slowly added dropwise over 40 minutes at 15 ℃ -20 ℃. The mixture was filtered to give 230g of product as a pale yellow solid (LCAP: 80.99%, QNMR:59%,10% IPAc). The wet cake was dried in vacuo at 15℃to 20℃for 2 hours to give 224g of crude product 12 as a pale yellow solid (LCAP: 80.9%, QNMR:60.4%, ca. 6% IPAc). The crude product 12 was redissolved in acetonitrile (330 ml,1 vol) and isopropyl acetate (412 ml,1.25 vol) was slowly added dropwise over 40 minutes at 15 ℃ to 20 ℃. The resulting mixture was filtered and 12 (30.5 g, hplc=60.9%, assay: 25.5%) was collected. The mother liquor was diluted with isopropyl acetate (6.6L, 20 volumes) added over 40 minutes at 15℃to 20 ℃. The mixture was filtered and the filter cake was dried to give 173.5g of crude product 12 as a pale yellow solid (LCAP: 85.4%, QNMR:66%,3.9% IPAc, RRT 1.19=3.9%). 190g of crude product 12 were dissolved in 760mL of acetonitrile-isopropyl acetate (2:1) and the mixture was passed through a silica gel column (380 g, 2X). The silica was rinsed with acetonitrile-isopropyl acetate (2:1, 5.7L) followed by 12L acetonitrile (very little product). The product-containing fraction was concentrated to give 118g of product 12 as a pale yellow solid (LCAP: 95%). The silica pad was then rinsed with MeCN/H ₂ O (12L, 10:1). The solvent was removed in vacuo to give an additional 60g of crude product 12 as a pale yellow solid, which was dissolved in acetonitrile (1.5L), stirred for 30 min, and then filtered. The mother liquor was then concentrated to give 24g of crude product 12 as a pale yellow solid (lcap=92%). The crude product 12 (118 g) and the crude product 12 (24 g) prepared above were dissolved in acetonitrile (330 ml,1 volume), and isopropyl acetate (6.6 l,20 volume) was added dropwise at 15 ℃ to 20 ℃ over 40 minutes. The mixture was then filtered to give 133g of product 12 as a pale yellow solid of appropriate purity (LCAP: 95%, QNMR:60.8%,7.8% IPAc). Note that compound 12 needs to be stored at-20 ℃. LCMS (ES, m/z): 666.61[ M-TfONa-2Na+3H ] ⁺ ¹ H-NMR (400 MHz, methanol) -d₄,ppm):δ8.00(ddd,J＝13.8,7.7,1.0Hz,2H),7.84(dt,J＝20.4,7.7Hz,2H),7.58–7.49(m,2H),7.40(dd,J＝7.6,1.0Hz,1H),7.36–7.28(m,2H),7.28–7.20(m,1H),4.96(hept,J＝6.3Hz,1H),3.96–3.88(m,1H),3.83(d,J＝15.1Hz,1H),3.70–3.52(m,11H),3.55–3.39(m,4H),3.07–2.73(m,6H),2.62(dt,J＝15.1,3.6Hz,2H).

Example 10

TOPA- [ C7] -phenylthiourea-h 11B6 antibody conjugates

(In the TOPA- [ C7] -phenylthiourea-h 11B6 antibody conjugate described above, the structure does not show the lysine residue of h11B6 attached to the phenylthiourea moiety.)

TOPA- [ C7] -phenylthiourea modification of mAb:

H11b6mAb (10.2 mg/ml) was diluted to 1mg/ml in 10mM sodium acetate (pH 5.2) buffer. Immediately prior to conjugation, the pH was adjusted to pH 9 with sodium bicarbonate buffer (VWR 144-55-8). The pH was confirmed with pH paper. Then, 10x molar excess of disodium salt TOPA- [ C7] -phenylisothiocyanate sodium salt (50 mM stock solution in water) was added to the h11b6mAb, and the mixture of antibody and TOPA- [ C7] -phenylisothiocyanate sodium salt was incubated at room temperature for about 1 hour without shaking. At the position of The addition of TOPA- [ C7] -phenylisothiocyanate sodium salt was monitored on a G224 instrument by complete mass ESI-TOF LC-MS until the CAR value was 1.5-2.0. The mixture was then immediately quenched by addition of 1M Tris pH 8.5 (Teknova T1085) to a final concentration of 100 mM. By using 7K/>The reaction was desalted into 10mM sodium acetate (pH 5.2) to remove excess free chelator. To confirm the absence of excess chelator, 3 rounds of sample dilution to 15ml were performed using a 50,000mwco amicon concentrator device, followed by concentration to 1ml. The sample is then concentrated to its final concentration for radiolabeling. The final conjugate was confirmed as monomer by analytical size exclusion chromatography on a Tosoh TSKgel G3000SWxl 7.8.8 mm x 30cm,5u column using the following conditions; column temperature: room temperature; the column was eluted with 0.2M sodium phosphate pH 6.8; flow rate: 0.8mL/min;18Min operation; injection volume: 18. Mu.L.

Example 11

Ac-225-labeled TOPA- [ C7] -phenylthiourea-h 11B6 antibody conjugates

(In the Ac-225-labeled TOPA- [ C7] -phenylthiourea-h 11b6 antibody conjugate described above, the structure does not show the lysine residue of h11b6 attached to the phenylthiourea moiety.)

(I) Labeling TOPA- [ C7] -phenylthiourea in 3M NaOAc buffer

h11B6：

To a NaOAc solution (3M H ₂ O solution, 60. Mu.L) in a plastic vial were added Ac-225 (10 mCi/mL of 0.1N HCl solution, 15. Mu.L) and TOPA- [ C7] -phenylthiourea-h 11B6 (1.13 mg/mL of 10mM NaOAc solution, pH=5.5, 441. Mu.L, 0.5 mg) in sequence. After mixing, the pH was measured by pH paper to about 6.5. The vials were allowed to stand at 37℃for 2 hours.

ITLC labeling the reaction mixture:

mu.L of the labeled reaction mixture was loaded onto iTLC-SG and developed with 10mM EDTA (pH 5-6). The dried iTLC-SG was left overnight at room temperature and then scanned on a BioscanAR-2000 radioactive TLC scanner. Under the elution conditions described herein TOPA- [ C7] -phenylthiourea-h 11B6 bound Ac-225 remained at the origin and any free Ac-225 would migrate with the solvent to the solvent front. iTLC scans showed 99.9% of TOPA- [ C7] -phenylthiourea-h 11B6 bound Ac-225.

DTPA test of the labeling reaction mixture:

Also, 0.5 μl of the labeled reaction mixture was mixed with 10mM DTPA (pH=6.5, 15 μl) at 37deg.C. After 30 minutes, 10. Mu.L of the mixture was spotted on iTLC-SG and developed with 10mM EDTA. The dried iTLC-SG was left overnight at room temperature and then scanned on a BioscanAR-2000 radioactive TLC scanner. Under the elution conditions described herein, TOPA- [ C7] -phenylthiourea-h 11B6 chelated Ac-225 remained at the origin, and any free Ac-225 would migrate with the solvent to the solvent front. iTLC scans showed 99.7% of TOPA- [ C7] -phenylthiourea-h 11B6 sequestered Ac-225.

Purification on PD10 column:

PD-10 resin was adjusted in NaOAc buffer by passing 5mL×3NaOAc buffer (25 mM NaOAc,0.04% PS-20, pH 5.5) through the column and discarding the wash. The entire labeled reaction mixture was applied to the reservoir of the column and the eluate was collected in pre-numbered plastic tubes. The reaction vials were washed with 0.2mL×3NaOAc buffer (25mM NaOAc,0.04%PS-20, pH 5.5) and the washes were removed to the reservoir of the PD-10 column and the eluate collected. Each tube contains about 1mL of eluent. The NaOAc buffer (25mM NaOAc,0.04%PS-20, pH 5.5) was continued to be applied to the reservoir of the PD-10 column until a total elution volume of 10mL was reached. The radiochemical purity of the collected fractions was checked by iTLC: mu.L of each fraction collected was spotted on iTLC-SG and developed with 10mM EDTA. The dried iTLC-SG was left overnight at room temperature and then scanned on a BioscanAR-2000 radioactive TLC scanner. The pure fraction should have no radioactive signal at the solvent front of iTLC-SG.

DTPA test of purified ²²⁵ Ac-TOPA- [ C7] -phenylthiourea-h 11B 6:

10. Mu.L of fraction #3 collected after PD-10 column was mixed with 15. Mu.L of 10mMDTPA solution (pH 6.5) and incubated for 30 minutes. mu.L of the mixture was loaded onto iTLC-SG, developed with 10mM EDTA and dried overnight. It was scanned on a Bioscan AR-2000 radioactive TLC scanner. No radioactivity signal was observed at the solvent front of iTLC-SG, indicating the absence of free Ac-225 in fraction # 3.

HPLC analysis of purified ²²⁵ Ac-TOPA- [ C7] -phenylthiourea-h 11B 6:

Fraction #3 collected after PD-10 column was analyzed by HPLC. HPLC method: tosoh TSKgel G3000SWxl 7.8.8mm.times.30cm, 5 μm column; column temperature: room temperature; eluting the column with DPBS buffer (X1, free of calcium and magnesium); flow rate: 0.7mL/min; running for 20min; sample injection volume: 40. Mu.L. After HPLC, fractions were collected at 30 second or 1 minute intervals. The collected HPLC fractions were left overnight at room temperature. Radioactivity in each fraction collected was counted in a gamma counter. HPLC radial traces were constructed from radioactivity in each HPLC fraction. The HPLC radiation trace shows a radioactive peak corresponding to the TOPA- [ C7] -phenylthiourea-h 11B6 peak on the HPLCUV trace.

(Ii) Higher concentrations of TOPA- [ C7] -phenylthiourea-h 11B6 were labeled with Ac-225 in 1.5M NaOAc buffer:

To a NaOAc solution (1.5M H ₂ O solution containing 0.04% PS-20, 63. Mu.L) in a plastic vial was added Ac-225 (10 mCi/mL of 0.1N HCl solution, 10. Mu.L) and TOPA- [ C7] -phenylthiourea-h 11B6 (9.36 mg/mL of 10mM NaOAc solution, pH=5.2, 0.04% PS-20, 36. Mu.L, 337. Mu.g) in sequence. After mixing, the pH was measured by pH paper to about 6.5. The vials were allowed to stand at 37℃for 2 hours.

ITLC labeling the reaction mixture:

Then 0.5. Mu.L of the labeled reaction mixture was loaded onto iTLC-SG, which was developed with 10mM EDTA. The dried iTLC-SG was left overnight at room temperature and then scanned on a Bioscan AR-2000 radioTLC scanner. Under the elution conditions described herein, TOPA- [ C7] -phenylthiourea-h 11B6 chelated Ac-225 will remain at the origin, and any free Ac-225 will migrate with the solvent to the solvent front. iTLC scans showed 99.9% of TOPA- [ C7] -phenylthiourea-h 11B6 sequestered Ac-225.

DTPA test of the labeling reaction mixture:

Also, 0.5 μl of the labeled reaction mixture was mixed with 10mM DTPA (pH=6.5, 15 μl) at 37deg.C. After 30 minutes, 10. Mu.L of the mixture was spotted on iTLC-SG and developed with 10mM EDTA. The dried iTLC-SG was left overnight at room temperature and then scanned on a BioscanAR-2000 radioactive TLC scanner. Under the elution conditions described herein, TOPA- [ C7] -phenylthiourea-h 11B6 chelated Ac-225 will remain at the origin, and any free Ac-225 will migrate with the solvent to the solvent front. iTLC scans showed 99.9% of TOPA- [ C7] -phenylthiourea-h 11B6 sequestered Ac-225.

(Iii) Higher concentrations of TOPA- [ C7] -phenylthiourea-h 11B6 were labelled with Ac-225 in 1M NaOAc buffer:

To a NaOAc solution (1.0M H ₂ O solution containing 0.04% PS-20, 63. Mu.L) in a plastic vial was added Ac-225 (10 mCi/mL of 0.1N HCl solution, 10. Mu.L) and TOPA- [ C7] -phenylthiourea-h 11B6 (9.36 mg/mL of 10mM NaOAc solution, pH=5.2, 0.04% PS-20, 36. Mu.L, 337. Mu.g) in sequence. After mixing, the pH was measured by pH paper to about 6.5. The vials were allowed to stand at 37℃for 2 hours.

ITLC labeling the reaction mixture:

DTPA test of the labeling reaction mixture:

Higher concentrations of TOPA- [ C7] -phenylthiourea-h 11B6 were labeled with Ac-225 in 25mM NaOAc (pH 5.5) containing 0.4% tween-20:

To a solution of NaOAc (25 mM H ₂ O solution containing 0.04% PS-20, pH 5.5, 10. Mu.L) in a plastic vial were added Ac-225 (10 mCi/mL of 0.1N HCl solution, 5. Mu.L), TOPA- [ C7] -phenylthiourea-H11B 6 (10.4 mg/mL of 10mM NaOAc solution, pH=5.2, 16. Mu.L, 166. Mu.g) and NaOH (0.1M, 5. Mu.L) in this order. After mixing, the pH was measured to be about 6.0 by pH paper. The vials were allowed to stand at 37℃for 2 hours.

ITLC labeling the reaction mixture:

DTPA test of the labeling reaction mixture:

Also, 0.5 μl of the labeled reaction mixture was mixed with 10mM DTPA (pH=6.5, 15 μl) at 37deg.C. After 30 minutes, 10. Mu.L of the mixture was spotted on iTLC-SG and developed with 10mM EDTA. The dried iTLC-SG was left overnight at room temperature and then scanned on a BioscanAR-2000 radioactive TLC scanner. Under the elution conditions described herein, TOPA- [ C7] -phenylthiourea-h 11B6 chelated Ac-225 will remain at the origin, and any free Ac-225 will migrate with the solvent to the solvent front. iTLC scans showed 99.8% of TOPA- [ C7] -phenylthiourea-h 11B6 sequestered Ac-225.

Reaction conditions for labeling TOPA- [ C7] -phenylthiourea-h 11B6 with Ac-225

Example 12

The following set of experiments was performed to examine the effect of the presence of non-radioactive metallic contaminants accompanying actinium sources on TOPA and DOTA chelating macrocycles. Four of the most common contaminants found in the ORNL source of ²²⁵Ac(NO₃)₃ by ICP-MS; al ³⁺、Ca²⁺、Zn²⁺、Mg²⁺ was used as an admixture standard in the chelation reaction of DOTA and TOPA conjugated to h11b 6. The chelation results were monitored with iTLC and then tested with DTPA.

TOPA- [ C7] -phenylthiourea-h 11b6 was chelated with Ac-225 in the presence of metallic impurities (lower levels of impurities).

Ac-225 was dissolved in 0.1M HCl and mixed with AlCl ₃、CaCl₂、ZnCl₂ and MgCl ₂ to form a 5mCi/mL solution. The concentrations of aluminum, calcium, zinc and magnesium were 9.76. Mu.g/mCi, 3.83. Mu.g/mCi, 0.61. Mu.g/mCi and 0.27. Mu.g/mCi, respectively. To a NaOAc solution (3 MH ₂ O solution, 20. Mu.L) in a plastic vial were added Ac-225 (5 mCi/mL,0.1N HCl, 10. Mu.L containing the added metal impurities) and TOPA- [ C7] -phenylthiourea-h 11b6 (1.17 mg/mL of 10mM NaOAc solution, pH=5.5, 143. Mu.L, 0.167 mg) in sequence. After mixing, the pH was measured by pH paper to about 6.5. The vials were kept at 37℃for 2 hours.

ITLC labeling the reaction mixture:

Then 0.5. Mu.L of the labeled reaction mixture was loaded onto iTLC-SG and developed with 10mM EDTA solution. iTLC-SG was dried overnight at room temperature and then scanned on a Bioscan AR-2000 radioTLC scanner. Under the elution conditions described herein, ac-225 binds to TOPA- [ C7] -phenylthiourea-h 11b6, ac-225 will remain at the origin of TLC (baseline), and any free Ac-225 will migrate with the solvent to the solvent front. iTLC scans showed 99.5% of Ac-225 bound to TOPA- [ C7] -phenylthiourea-h 11b6 (scan 1, shown in FIG. 5).

DTPA test of the labeling reaction mixture:

Also, 0.5 μl of the labeled reaction mixture was mixed with 10mM DTPA (pH=6.5, 15 μl) at 37deg.C. After 30 minutes, 10. Mu.L of the mixture was spotted on iTLC-SG and developed in 10mM EDTA eluate. iTLC-SG was air dried and left overnight at room temperature before scanning on a Bioscan AR-2000 radioTLC scanner. Under the elution conditions described herein, TOPA- [ C7] -phenylthiourea-h 11b6 chelated Ac-225 will remain at the origin, and any free Ac-225 will migrate with the solvent to the solvent front. iTLC scan showed 99.4% of Ac-225 bound to TOPA- [ C7] -phenylthiourea-h 11b6 (scan 2, shown in FIG. 6).

Metal doping experiments: TOPA- [ C7] -phenylthiourea-h 11b6 was labeled with Ac-225 in the presence of metallic impurities (higher levels of impurities, 5-fold increase in concentration compared to lower levels of impurities).

Ac-225 was dissolved in 0.1M HCl and mixed with AlCl ₃、CaCl₂、ZnCl₂ and MgCl ₂ to form a 5mCi/mL solution. The concentrations of aluminum, calcium, zinc and magnesium were 45.0 μg/mCi, 17.3 μg/mCi, 3.01 μg/mCi and 1.15 μg/mCi, respectively. To a NaOAc solution (3M H ₂ O solution, 20. Mu.L) in a plastic vial were added Ac-225 (5 mCi/mL,0.1N HCl, 10. Mu.L containing added metallic impurities) and TOPA- [ C7] -phenylthiourea-h 11b6 (1.17 mg/mL of 10mM NaOAc solution, pH=5.5, 143. Mu.L, 0.167 mg) in sequence. After mixing, the pH was measured by pH paper to about 6.5. The vials were kept at 37℃for 2 hours.

ITLC labeling the reaction mixture:

then 0.5. Mu.L of the labeled reaction mixture was loaded onto iTLC-SG and developed with 10mM EDTA solution. iTLC-SG was dried overnight at room temperature and then scanned on a Bioscan AR-2000 radioTLC scanner. Under the elution conditions described herein, ac-225 binds to TOPA- [ C7] -phenylthiourea-h 11b6, ac-225 will remain at the origin of TLC (baseline), and any free Ac-225 will migrate with the solvent to the solvent front. iTLC scan showed 98.9% of Ac-225 bound to TOPA- [ C7] -phenylthiourea-h 11b6 (scan 3, shown in FIG. 7).

DTPA test of the labeling reaction mixture:

Also, 0.5 μl of the labeled reaction mixture was mixed with 10mM DTPA (pH=6.5, 15 μl) at 37deg.C. After 30 minutes, 10. Mu.L of the mixture was spotted on iTLC-SG and developed in 10mM EDTA eluate. iTLC-SG was air dried and left overnight at room temperature before scanning on a Bioscan AR-2000 radioTLC scanner. Under the elution conditions described herein, TOPA- [ C7] -phenylthiourea-h 11b6 chelated Ac-225 will remain at the origin, and any free Ac-225 will migrate with the solvent to the solvent front. iTLC scan showed 99.8% of Ac-225 bound to TOPA- [ C7] -phenylthiourea-h 11b6 (scan 4, shown in FIG. 8).

DOTA-h11b6 was labeled with Ac-225 in the presence of metallic impurities (lower levels of impurities).

Ac-225 was dissolved in 0.1M HCl and mixed with AlCl ₃、CaCl₂、ZnCl₂ and MgCl ₂ to form a 5mCi/mL solution. The concentrations of aluminum, calcium, zinc and magnesium were 9.76. Mu.g/mCi, 3.83. Mu.g/mCi, 0.61. Mu.g/mCi and 0.27. Mu.g/mCi, respectively. Ac-225 (5 mCi/mL,0.1N HCl, 10. Mu.L containing added metallic impurities) and DOTA-h11b6 (10 mg/mL of 25mM NaOAc solution, pH=5.5, 16.7. Mu.L, 0.167 mg) were added sequentially to NaOAc solution (3M H ₂ O solution, 20. Mu.L) in a plastic vial. After mixing, the pH was measured by pH paper to about 6.5. The vials were kept at 37℃for 2 hours.

ITLC labeling the reaction mixture:

Then 0.5. Mu.L of the labeled reaction mixture was loaded onto iTLC-SG and developed with 10mM EDTA solution. iTLC-SG was dried overnight at room temperature and then scanned on a Bioscan AR-2000 radioTLC scanner. Under the elution conditions described herein, ac-225 binds to DOTA-h11b6, ac-225 will remain at the origin of TLC (baseline), and any free Ac-225 will migrate with the solvent to the solvent front. iTLC scan showed 43.6% of Ac-225 chelated with DOTA-h11b6 (scan 5, shown in FIG. 9).

DTPA test of the labeling reaction mixture:

also, 0.5 μl of the labeled reaction mixture was mixed with 10mM DTPA (pH=6.5, 15 μl) at 37deg.C. After 30 minutes, 10. Mu.L of the mixture was spotted on iTLC-SG and developed in 10mM EDTA eluate. iTLC-SG was air dried and left overnight at room temperature before scanning on a Bioscan AR-2000 radioTLC scanner. Under the elution conditions described herein, DOTA-h11b6 chelated Ac-225 will remain at the origin, and any free Ac-225 will migrate with the solvent to the solvent front. iTLC scan showed 18.1% of Ac-225 chelated with DOTA-h11b6 (scan 6, shown in FIG. 10).

Metal doping experiments: DOTA-h11b6 was labeled with Ac-225 in the presence of metallic impurities (higher levels of impurities, 5-fold increase in concentration compared to lower levels of impurities).

Ac-225 was dissolved in 0.1M HCl and mixed with AlCl ₃、CaCl₂、ZnCl₂ and MgCl ₂ to form a 5mCi/mL solution. The concentrations of aluminum, calcium, zinc and magnesium were 45.0 μg/mCi, 17.3 μg/mCi, 3.01 μg/mCi and 1.15 μg/mCi, respectively. Ac-225 (5 mCi/mL,0.1N HCl, 10. Mu.L containing added metallic impurities) and DOTA-h11b6 (10 mg/mL of 25mM NaOAc solution, pH=5.5, 16.7. Mu.L, 0.167 mg) were added sequentially to NaOAc solution (3M H ₂ O solution, 20. Mu.L) in a plastic vial. After mixing, the pH was measured by pH paper to about 6.5. The vials were kept at 37℃for 2 hours.

ITLC labeling the reaction mixture:

Then 0.5. Mu.L of the labeled reaction mixture was loaded onto iTLC-SG and developed with 10mM EDTA solution. iTLC-SG was dried overnight at room temperature and then scanned on a Bioscan AR-2000 radioTLC scanner. Under the elution conditions described herein, ac-225 binds to DOTA-h11b6, ac-225 will remain at the origin of TLC (baseline), and any free Ac-225 will migrate with the solvent to the solvent front. iTLC scan showed 52.7% of Ac-225 chelated with DOTA-h11b6 (scan 7, shown in FIG. 11).

DTPA test of the labeling reaction mixture:

also, 0.5 μl of the labeled reaction mixture was mixed with 10mM DTPA (pH=6.5, 15 μl) at 37deg.C. After 30 minutes, 10. Mu.L of the mixture was spotted on iTLC-SG and developed in 10mM EDTA eluate. iTLC-SG was air dried and left overnight at room temperature before scanning on a Bioscan AR-2000 radioTLC scanner. Under the elution conditions described herein, DOTA-h11b6 chelated Ac-225 will remain at the origin, and any free Ac-225 will migrate with the solvent to the solvent front. iTLC scan showed 14.0% of Ac-225 chelated with DOTA-h11b6 (scan 8, shown in FIG. 12).

While the foregoing description is directed to the principles of the present invention, by way of example provided herein, it is to be understood that the practice of the invention encompasses all of the generic variations, alterations and/or modifications which come within the scope of the following claims and the equivalents thereto.

Throughout this disclosure, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this application pertains.

Claims

2. For preparing compound 14

Methyl 6- ((4- ((tert-butoxycarbonyl) amino) phenyl) (hydroxy) methyl) pyridinecarboxylate 7 is reacted with methanesulfonyl chloride in an organic solvent or mixture thereof at a temperature in the range of about ambient temperature to about-78 DEG C

Reacting to obtain methyl picolinate of compound 8 (6- ((4- ((tert-butoxycarbonyl) amino) phenyl) - ((methylsulfonyl) oxy) methyl);

3. For preparing compound 10

4. A process for preparing compound 12 (TOPA- [ C7] -phenylisothiocyanate sodium salt)

5. A method for preparing TOPA- [ C7] -phenylthiourea-h 11B6 antibody conjugate:

The method comprises the following steps:

Reacting an about 10-fold excess of compound 12 with h11b6mAb in 10mM sodium acetate buffer at pH of about 5.2, adjusted to pH of about 9 with sodium bicarbonate buffer, and incubated at room temperature without shaking for about 1 hour; quenching by adding 1M Tris at a pH of about 8.5 to a final concentration of about 100 mM; removing excess free chelator by desalting the reaction to 10mM sodium acetate at a pH of about 5.2; and removing the excess chelator to give TOPA- [ C7] -phenylthiourea-h 11B6 antibody conjugate.

6. A compound of formula (12) (TOPA- [ C7] -phenylisothiocyanate sodium salt)

7. A compound of formula (14)

Or a pharmaceutically acceptable salt or solvate thereof.

8. A compound of formula (11)