Theragnostics folate conjugates

Abstract

The present disclosure relates to novel folic acid conjugates comprising 5-methyltetrahydrofolic acid, a radiometal chelator that optionally coordinates a radiometal M, and the albumin binder 5-(p-iodophenyl)pentanoate, and further provides uses of such conjugates and / or pharmaceutical compositions thereof in diagnostic imaging, radionuclide therapy, or theragnostic applications.

Description

[Technical field]

[0001] The present disclosure relates to novel folic acid conjugates comprising 5-methyltetrahydrofolic acid, a radiometal chelator that optionally coordinates a radiometal M, and the albumin binder 5-(p-iodophenyl)pentanoate, and further provides uses of such conjugates and / or pharmaceutical compositions thereof in diagnostic imaging, radionuclide therapy, or theragnostic applications. [Background technology]

[0002] Folate receptor alpha (herein referred to as FR) is a membrane-bound glycoprotein that is overexpressed in various types of tumors, among them ovarian, lung, breast, renal, and colon cancers (Parker, N. et al., Anal Bioch, 2005, 338, (2), 284-93; Low, PS et al., Curr Opin Chem Biol, 2009, 13, (3), 256-62). In healthy tissues, FR is expressed at only a few sites, most importantly in the proximal tubule cells of the kidney (Holm, J. et al., Kidney Int, 1992, 41, (1), 50-5; Birn, H. et al., J Am Soc Nephrol: JASN 2005, 16, (3), 608-15). It is therefore a promising tumor-associated protein for nuclear imaging and targeted radionuclide therapy (Low, PS et al., Acc Chem Res, 2008, 41, (1), 120-9; Muller, C. Curr Pharm Design, 2012, 18, (8), 1058-83). Folic acid (the oxidized form of vitamin folate) and 5-methyltetrahydrofolate (5-MTHF; a reduced form of folic acid that can be prepared as one of four stereoisomers [6R- / 6S- and L-Glu / D-Glu]) bind with high affinity to the FR (K in the nanomolar range). D), FR is internalized via endocytosis (Kamen, BA et al., Adv Drug Deliv Rev, 2004, 56, (8), 1085-97). Therefore, folic acid has been used as a targeting agent to deliver accompanying diagnostic and therapeutic payloads for imaging and treatment of FR-expressing cancers (Low, PS et al., Acc Chem Res 2008, 41, (1)). Only a few folate radioconjugates have been developed for clinical use, including [ 111 In]In-DTPA-folate and [ 99m Tc]Tc-EC20 (Etarfolatide (trademark), Endocyte Inc.) (Siegel, BA et al., J Nucl Med, 2003, 44, (5), 700-7; Fisher, RE et al., J Nucl Med, 2008, 49, (6), 899-906), and 18 F-AzaFol(3'-aza-2'-[ 18 F]fluorofolate) (Gnesin, S. et al., EJNMMI Res, 2020, 10, (1), 32).

[0003] Based on preclinical studies in mice, the main concern for therapeutic use of folate radioconjugates is the potential risk of kidney damage (radionephrotoxicity) as a result of high accumulation of folate radioconjugates in the kidney (Muller, C. et al., Nucl Med Biol, 2011, 38, (5), 715-23). ​​4-(p-iodophenyl)butanoic acid (Dumelin, CE et al., Angew Chem Int Ed Engl, 2008, 47, (17), 3196-201) as an albumin binder was used to treat folate radioconjugates ([ 177Lu]Lu-cm09(Muller, C. et al., J Nucl Med, 2013, 54, (1), 124-31) and [ 177 Lu]Lu-cm10 (Muller et al., J Nucl Med, 2014, 55, (10), 1658-64, herein [ 177 Incorporation of 5-methyltetrahydrofolate (5-MTHF) into the structure of the folate-based compound [Lu]Lu-OxFol-1 had a positive effect on their pharmacokinetic properties. Albumin-binding properties promoted the blood circulation of the folate radioconjugates, resulting in increased accumulation in the tumor and decreased retention in the kidney, thus significantly improving the tumor-to-kidney ratio (Muller, C. et al., J Nucl Med, 2013, 54, (1), 124-31; Siwowska, K. et al., Mol Pharm, 2017, 14, (2), 523-532). The use of albumin-binding radioconjugates with 5-methyltetrahydrofolate (5-MTHF) as a targeting agent was shown to be more efficient than the respective folate-based compound [ 177 [Lu]Lu-OxFol-1, which showed a higher tumor-to-kidney ratio and resulted in superior therapeutic efficacy (Guzik, P. et al., Eur J Nucl Med Mol Imaging, 2021, 48, 972-983). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Parker, N. et al., Anal. Bioch., 2005, 338, (2), 284-93 [Non-Patent Document 2] Low, PS et al., Curr. Opin. Chem. Biol., 2009, 13, (3), 256-62 [Non-Patent Document 3] Holm, J. et al., Kidney Int., 1992, 41, (1), 50-5 [Non-Patent Document 4] Birn, H. et al., J. Am. Soc. Nephrol.: JASN 2005, 16, (3), 608-15

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[0005] In a first aspect, the present disclosure is directed to novel folate conjugates comprising 5-methyltetrahydrofolic acid, a radiometal chelator that optionally coordinates a radiometal M, and 5-(p-iodophenyl)pentanoate as an albumin binder.

[0006] In one specific embodiment, the novel folic acid conjugate is a compound of formula I, or a stereoisomer (or combination of stereoisomers) thereof, or a pharma- ceutically acceptable salt thereof.

[0007] [ka]

[0008] Here, Y is a radiometal chelator that optionally coordinates a radiometal M, n is 1-8, and m is 1-8.

[0009] In some embodiments, the radiometal chelator is a linear or macrocyclic polyaminocarboxylate, such as DTPA, DOTA (and its derivatives, such as p-SCN-DOTA, maleimide-DOTA, DOTA-NHS-ester), DFO, *, DO3A, AAZTA, HP-DO3A, EDTA, TETA, EHPG, HBED, NOTA (and derivatives such as p-SCN-NOTA), DOTAGA, DOTMA, TETMA, PDTA, TTHA, LICAM, MECAM, AAZTA, preferably macrocyclic polyaminocarboxylates, e.g., NOTA, DOTA, DTPA, DO3A, HP-DO3A, EDTA, TETA, DOTMA, AAZTA. The radiometal chelator, e.g., macrocyclic polyaminocarboxylate, is covalently attached to the compound of the disclosure via amide coupling to one of its carboxylate groups.

[0010] The radiometal chelator may or may not coordinate the radiometal M. In some embodiments, the optionally coordinated radiometal M is 51 Cr, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 47 Sc, 167 Tm, 141 Ce, 111 In, 168 Yb, 175 Yb, 140 La, 89 Zr, 90 Y, 88 Y, 153 Sm, 166 Ho, 52 Mn, 165 Dy, 166 Dy, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 97 Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 211 Bi, 212 Bi, 213 Bi, 214 Bi, 105 Rh, 109 Pd, 212 Pb, 117m Sn, 149 Pm,161 Tb, 149 Tb, 152 Tb, 155 Tb, 99m Tc, 165 Er, 169 Er, 172 Yb, 165 Tm, 177 Lu, 225 Ac, 198 Au, 199 Au, and 227 Th.

[0011] In some embodiments, the optionally coordinated radiometal M is 67 Ga, 68 Ga, 64 Cu, 67 Cu, 43 Sc, 44 Sc, 99m Tc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 90 Y, 177 Lu, and 225 Ac.

[0012] In some embodiments, the optionally coordinated radiometal M for use in diagnostic imaging is 61 Cu, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 52 Mn, 89 Zr, 99m Tc, 111 In, 152 Tb, and 155 Tb.

[0013] In some embodiments, the optionally coordinated radiometal M for use in diagnostic imaging is 67 Ga, 68 Ga, 64 Cu, 43 Sc,44 Sc, 99m Tc, 152 Tb, and 155 Tb.

[0014] In some embodiments, the optionally coordinated radiometal M for use in radionuclide therapy is 67 Cu, 90 Y, 105 Rh, 117m Sn, 149 Pm, 153 Sm, 161 Tb, 149 Tb, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 186 Re, 188 Re, 225 Ac, and 213 Bi.

[0015] In some embodiments, the optionally coordinated radiometal M for use in radionuclide therapy is 67 Cu, 90 Y, 161 Tb, 149 Tb, 177 Lu, and 225 Ac.

[0016] In some embodiments, n is 2, 3, 4, 5, or 6, preferably 4. In some embodiments, m is 2, 3, 4, 5, or 6, preferably 4. In some embodiments, n is 4 and m is 4.

[0017] In a further aspect, the present disclosure provides the use of the compound and / or pharmaceutical composition of the present disclosure in diagnostic imaging, radionuclide therapy or theragnostics applications.In some embodiments, the subject of the method of the present disclosure is a mammal, such as an animal or a human.In some embodiments, the subject of the method of the present disclosure is a human.

[0018] In a further aspect, the present disclosure provides single or multi-vial kits containing all the components necessary to prepare the compounds of the present disclosure.

[0019] Other features and advantages of the disclosure will become apparent from the following detailed description, and from the claims.

[0020] It should be understood that the specification and embodiments are intended to provide an overview or framework for understanding the nature and character of the present disclosure without limiting the scope thereof. [Brief description of the drawings]

[0021] [Figure 1] Synthesis scheme of RedFol-24: a) Fmoc-Lys(Alloc)-OH, DIPEA in DMF; o / n; b) 50% piperidine in DMF; 2 × 5 min; c) Dde-Lys(Fmoc)-OH, HBTU, DIPEA in DMF; 1 h; d) DOTA-tris(tBu) ester, HBTU, DIPEA in DMF; 3 h; e) Pd(PPh3)4, morpholine in DCM; 1 h; f) 5-(p-iodo h) Fmoc-Glu-OtBu, HBTU, DIPEA in DMF; 1.5 h; i) 10-formyl-5-methyltetrahydropteroic acid, HBTU; DIPEA in DMF; 2 h; j) TFA, TIPS, HO (95:2.5:2.5); 2 h; k) NaOH in water (1M), 5 h. [Diagram 2] Uptake and internalization of folate radioconjugate in KB tumor cells after 2 and 4 hours of incubation at 37° C. (A) Uptake of folate radioconjugate in KB tumor cells. (B) Internalized fraction of folate radioconjugate in KB tumor cells. [Diagram 3] Decay-corrected uptake of folate radioconjugates in KB tumors and various other tissues of tumor-bearing mice 1 h, 4 h, and 24 h post-injection. [Figure 4] Tumor-to-blood ratios determined based on biodistribution data obtained at 1, 4, and 24 hours post-injection of each radioconjugate. [Diagram 5] Tumor-to-kidney ratios determined based on biodistribution data obtained at 1, 4, and 24 hours post-injection of each radioconjugate. [Figure 6] Tumor-to-liver ratios determined based on biodistribution data obtained at 1, 4, and 24 hours post-injection of each radioconjugate. [Figure 7] SPECT / CT images of KB tumor-bearing mice 1, 4, and 24 hours after injection of 177Lu-folate radioconjugate (25MBq; 0.5nmol per mouse) are shown as maximum intensity projections (MIPs). (A) SPECT / CT scan of [177Lu]Lu-OxFol radioconjugate; (B) SPECT / CT scan of 6R-5-MTHF-based radioconjugate; (C) SPECT / CT scan of 6S-5-MTHF-based radioconjugate; Tu=KB tumor; Ki=kidney; H=heart. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description of the Disclosure In a first aspect, the present disclosure is directed to a novel folate conjugate (hereinafter also referred to as the compound or conjugate of the present disclosure) comprising 5-methyltetrahydrofolate (5-MTHF), a radiometal chelator that optionally coordinates a radiometal M, and the albumin binder 5-(p-iodophenyl)pentanoate.

[0023] The term "radiometal chelator" (or (metal) chelator) may refer to any metal chelator known in the art for complexing (and useful for the intended application of) a radiometal or radionuclide. The binding of the chelator to the radiometal can be determined by measuring the dissociation constant between the chelator and the radiometal. For purposes of this disclosure, the dissociation constant K between the chelator and the radiometal is D is about 10 -3 ~about 10 -15 M -1 Preferably, the dissociation constant K between the chelator and the radioactive metal is D is about 10 -6 ~about 10 -15 M -1 In some embodiments, the radiometal for use in the present disclosure is one that can be externally detected in a non-invasive manner after administration in vivo. In some embodiments, the radiometal is particularly suitable for imaging using SPECT or PET.

[0024] Examples of chelators are well known in the art and include bidentate, tridentate, and tetradentate ligands in linear, tripodal, and macrocyclic forms. Typical examples include bipyridyl (bipy); terpyridyl (terpy); crown ethers; azacrown ethers; succinic acid; citric acid; salicylic acid; histidine; imidazole; ethylene glycol-bis-(β-aminoethyl ether) N,N'-tetraacetic acid (EGTA); nitrilotriacetic acid (nitroloacetic acid). acid);acetylacetonate (acac);sulfuric acid;dithiocarbamate;carboxylate;alkyldiamine;ethylenediamine(en);diethylenetriamine(dien);nitrate;nitro;nitroso;(C6H5)2PCH2CH2P(C6H5)2(diphos);glyme;diglyme;bis(acetylacetonate)ethylenediamine(acacen);ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA);N-[2-[bis(carboxymethyl)amino]-3-(4-ethoxyphenyl)propyl]-N-[2-[bis(carboxymethyl)-amino]ethyl]-L-glycine (EOB-DTPA);N,N-bis[2-[bis(carboxymethyl)amino]-ethyl]-L-glutamic acid (DT PA-Glu;N,N-bis[2-[bis(carboxymethyl)amino]-ethyl]-L-lysine (DTPA-Lys);N,N-bis[2-[carboxymethyl[(methylcarbamoyl)methyl]amino]-ethyl]glycine (DTPA-BMA), and other mono- or bis-amide derivatives of DTPA;N'-[5-(acetyl-hydroxy-amino)pentyl]-N-{5-[3-(5-aminopentyl-hydroxy-carbamoyl)propanoylamino]pentyl}-N-hydroxy-butanediamide (DFO);N1-(27-amino-11,22-dihydroxy-7,10,18,21-tetraoxo-6,11,17,22-tetraazaheptacosyl)-N1-hydroxy-N4-(5-(N-hydroxyacetamido)pentyl)succinamide (DFO) *);4-Carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecane(triazamidecan)-13-oic acid (BOPTA);2,2'-(6-(bis(carboxymethyl)amino)-6-(4-carboxybutyl)-1,4-diazepane-1,4-diyl)diacetic acid (AAZTA);1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A);1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (DO3A) acetic acid (DOTA); 1,4,7,10-tetraazacyclododecane, 1-(glutaric acid)-4,7,10-triacetic acid (DOTAGA); 10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (HPDO3A); 2-methyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (MCTA); tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA); 3,6,9,15-tetraazabicyclo[9.3.1]Pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA);PCTA12;Cyclo-PCTA12;N,N'-bis(2-aminoethyl)-1,2-ethanediamine (TETA);1,4,7,10-tetraazacyclotridecane-N,N',N'',N'''-tetraacetic acid (TRITA);1,12-dicarbonyl, 15-(4-isothiocyanatobenzyl) 1,4,7,10,13-pentaazacylic acid Hexadecane-N,N',N''-triacetic acid (HETA); 1,4,7,10-tetraazacyclododecane-N,N',N'',N''-tetraacetic acid mono(N-hydroxysuccinimidyl) ester (DOTA-NHS); N,N'-bis(2-aminoethyl)-1,2-ethanediamine-N-hydroxysuccinimide ester (TETA-NHS); [(2S,5S,8S,11S)-4,7,10-tris-carboxymethyl 2,5,8,11-Tetramethyl-1,4,7,10-tetraazacyclododecan-1-yl]ethyl-2,5,8,11-tetramethyl-1,4,7,10-tetraazacyclododecan-1-yl]acetic acid (M4DOTA);[(2S,5S,8S,11S)-4,7-Biscarboxymethyl-2,5,8,11-tetramethyl-1,4,7,10-tetraazacyclododecan-1-yl]acetic acid;(M4DO3A);(R)-2-[(2S,5S,8S,11S)-4,7,10-Tris-((R)-1-carboxyethyl)- 2,5,8,11-tetramethyl-1,4,7,10-tetraazacyclododecan-1-yl]propionic acid (M4DOTMA); 10-phosphonomethyl-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (MPDO3A); hydroxybenzyl-ethylenediamine-diacetic acid (HBED), and N,N'-ethylenebis-[2-(o-hydroxyphenol)glycine] (EHPG).

[0025] Metal chelators suitable for use in the compounds of the present disclosure include bidentate, tridentate, and tetradentate ligands in linear, tripodal, and macrocyclic forms, as specified herein. In some embodiments, the metal chelators used for the present disclosure include linear or macrocyclic polyaminocarboxylates, such as DTPA, DOTA (and derivatives thereof, such as p-SCN-DOTA, maleimide-DOTA, DOTA-NHS-ester), DFO, DFO, and the like. * , DO3A, HP-DO3A, AAZTA, EDTA, TETA, EHPG, HBED, NOTA (and derivatives such as p-SCN-NOTA), DOTAGA, DOTMA, TETMA, PDTA, TTHA, LICAM, MECAM, etc. In some embodiments, the metal chelator used for the present disclosure comprises a macrocyclic polyaminocarboxylate such as NOTA, DOTA, DTPA, DO3A, HP-DO3A, EDTA, TETA, DOTMA, AAZTA, etc. The radiometal chelator as defined herein is linked to the compound of the present disclosure via one of its carboxylate groups, for example via coupling with an amino group to give an amide bond.

[0026] The radiometal chelator may or may not coordinate the radiometal M. As used herein, the term "radiometal" (also referred to as a radionuclide) refers to an atom capable of undergoing radioactive decay and may be used as a diagnostic imaging or therapeutic agent, as described below. In some embodiments, a radiometal for nuclear imaging or radionuclide therapy is 51 Cr, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 47 Sc, 167 Tm, 141 Ce, 111 In, 168 Yb, 175 Yb, 140 La, 89 Zr, 90 Y, 88 Y,153 Sm, 166 Ho, 52 Mn, 165 Dy, 166 Dy, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 97 Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 211 Bi, 212 Bi, 213 Bi, 214 Bi, 105 Rh, 109 Pd, 212 Pb, 117m Sn, 149 Pm, 161 Tb, 149 Tb, 152 Tb, 155 Tb, 99m Tc, 165 Er, 169 Er, 172 Yb, 165 Tm, 177 Lu, 225 Ac, 198 Au, 199 Au, and 227 Contains Th.

[0027] In some embodiments, the optionally coordinated radiometal M is 67 Ga, 68 Ga, 64 Cu, 67 Cu, 43 Sc, 44 Sc, 99m Tc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 90 Y, 177 Lu, and 225 Ac.

[0028] In some embodiments, the optionally coordinated radiometal M is 67 Ga, 68 Ga,64 Cu, 43 Sc, 44 Sc, 99m Tc, 152 Tb, 155 Tb, 67 Cu, 90 Y, 161 Tb, 149 Tb, 177 Lu, and 225 Ac.

[0029] The choice of metal is determined based on the intended therapeutic or diagnostic application, and one of skill in the art would know which radiometal to choose for the intended application.

[0030] In some embodiments, the optionally coordinated radiometal M for use in diagnostic imaging is 61 Cu, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 52 Mn, 89 Zr, 99m Tc, 111 In, 152 Tb, and 155 Tb.

[0031] In some embodiments, the optionally coordinated radiometal M for use in diagnostic imaging is 67 Ga, 68 Ga, 64 Cu, 43 Sc, 44 Sc, 99m Tc, 152 Tb, and 155 Tb.

[0032] In some embodiments, the optionally coordinated radiometal M for use in radionuclide therapy is 67 Cu, 90 Y, 105 Rh, 117m Sn, 149 Pm, 153Sm, 161 Tb, 149 Tb, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 186 Re, 188 Re, 225 Ac, and 213 Bi.

[0033] In some embodiments, the optionally coordinated radiometal M for use in radionuclide therapy is 67 Cu, 90 Y, 161 Tb, 149 Tb, 177 Lu, and 225 Ac.

[0034] The term "compounds of the disclosure" includes compounds of Formulas (I)-(III) and any stereoisomers thereof (e.g., explicitly (6R)- or (6S)-isomers), as well as any pharma- ceutically acceptable salts thereof.

[0035] The present disclosure also encompasses compounds of the present disclosure in which one or more atoms have been replaced with a specific isotope of the corresponding atom, for example, compounds of the present disclosure in which one or more or all hydrogen atoms have been replaced with a deuterium atom, D, to form deuterium-enriched compounds of the present disclosure.

[0036] In some embodiments, the compound of the disclosure is a compound of Formula I, or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0037] [ka]

[0038] Where: Y is a radiometal chelator that optionally coordinates the radiometal M; n is an integer from 1 to 8, and m is an integer from 1 to 8.

[0039] In some embodiments, the radiometal chelator is as defined herein above. In some embodiments, the radiometal chelator is a linear or macrocyclic polyaminocarboxylate, such as DTPA, DOTA (and its derivatives, such as p-SCN-DOTA, maleimide-DOTA, DOTA-NHS-ester), DFO, DFO * , DO3A, HP-DO3A, AAZTA, EDTA, TETA, EHPG, HBED, NOTA (and derivatives such as p-SCN-NOTA), DOTAGA, DOTMA, TETMA, PDTA, TTHA, LICAM, MECAM.

[0040] In some embodiments, metal chelators used for the present disclosure include macrocyclic polyaminocarboxylates such as NOTA, DOTA, DTPA, DO3A, HP-DO3A, AAZTA, EDTA, TETA, DOTMA, and the like.

[0041] In some embodiments, the optionally coordinated radiometal M is 51 Cr, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 47 Sc, 167 Tm, 141 Ce, 111 In, 168 Yb, 175 Yb, 140 La, 89 Zr, 90 Y, 88 Y, 153 Sm, 166 Ho, 52 Mn, 165 Dy, 166 Dy, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 97 Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 211 Bi, 212Bi, 213 Bi, 214 Bi, 105 Rh, 109 Pd, 212 Pb, 117m Sn, 149 Pm, 161 Tb, 149 Tb, 152 Tb, 155 Tb, 99m Tc, 165 Er, 169 Er, 172 Yb, 165 Tm, 177 Lu, 225 Ac, 198 Au, 199 Au, and 227 Th.

[0042] In some embodiments, the optionally coordinated radiometal M is 67 Ga, 68 Ga, 64 Cu, 67 Cu, 43 Sc, 44 Sc, 99m Tc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 90 Y, 177 Lu, and 225 Ac.

[0043] In some embodiments, the optionally coordinated radiometal M is 67 Ga, 68 Ga, 64 Cu, 43 Sc, 44 Sc, 99m Tc, 152 Tb, 155 Tb, 67 Cu, 90 Y, 161 Tb, 149 Tb, 177 Lu, and 225 Ac.

[0044] In some embodiments, the optionally coordinated radiometal M for use in diagnostic imaging is 61 Cu, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 52 Mn, 89 Zr, 99m Tc, 111 In, 152 Tb, and 155 Tb.

[0045] In some embodiments, the optionally coordinated radiometal M for use in diagnostic imaging is 67 Ga, 68 Ga, 64 Cu, 43 Sc, 44 Sc, 99m Tc, 152 Tb, and 155 Tb.

[0046] In some embodiments, the optionally coordinated radiometal M for use in radionuclide therapy is 67 Cu, 90 Y, 105 Rh, 117m Sn, 149 Pm, 153 Sm, 161 Tb, 149 Tb, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 186 Re, 188 Re, 225 Ac, and 213 Bi.

[0047] In some embodiments, the optionally coordinated radiometal M for use in radionuclide therapy is 67 Cu, 90 Y, 161 Tb, 149 Tb, 177 Lu, and 225Ac.

[0048] In some embodiments, n is 2, 3, 4, 5, or 6, preferably 4. In some embodiments, m is 2, 3, 4, 5, or 6, preferably 4. In some embodiments, n is 4 and m is 4.

[0049] In some embodiments, the compound of formula I has formula Ia or Ib.

[0050] [ka]

[0051] [ka]

[0052] Where: Y is a radiometal chelator that optionally coordinates the radiometal M; n is an integer from 1 to 8, and m is an integer from 1 to 8.

[0053] In some embodiments, the radiometal chelator is as defined herein above. In some embodiments, the radiometal chelator is a linear or macrocyclic polyaminocarboxylate, such as DTPA, DOTA (and its derivatives, such as p-SCN-DOTA, maleimide-DOTA, DOTA-NHS-ester), DFO, DFO * , DO3A, HP-DO3A, AAZTA, EDTA, TETA, EHPG, HBED, NOTA (and derivatives such as p-SCN-NOTA), DOTAGA, DOTMA, TETMA, PDTA, TTHA, LICAM, MECAM.

[0054] In some embodiments, metal chelators used for the present disclosure include macrocyclic polyaminocarboxylates such as NOTA, DOTA, DTPA, DO3A, HP-DO3A, AAZTA, EDTA, TETA, DOTMA, and the like.

[0055] In some embodiments, the optionally coordinated radiometal M is 51 Cr, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 47 Sc, 167 Tm, 141 Ce, 111 In, 168 Yb, 175 Yb, 140 La, 89 Zr, 90 Y, 88 Y, 153 Sm, 166 Ho, 52 Mn, 165 Dy, 166 Dy, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 97 Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 211 Bi, 212 Bi, 213 Bi, 214 Bi, 105 Rh, 109 Pd, 212 Pb, 117m Sn, 149 Pm, 161 Tb, 149 Tb, 152 Tb, 155 Tb, 99m Tc, 165 Er, 169 Er, 172 Yb, 165 Tm, 177 Lu, 225 Ac, 198 Au, 199 Au, and227 Th.

[0056] In some embodiments, the optionally coordinated radiometal M is 67 Ga, 68 Ga, 64 Cu, 67 Cu, 43 Sc, 44 Sc, 99m Tc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 90 Y, 177 Lu, and 225 Ac.

[0057] In some embodiments, the optionally coordinated radiometal M is 67 Ga, 68 Ga, 64 Cu, 43 Sc, 44 Sc, 99m Tc, 152 Tb, 155 Tb, 67 Cu, 90 Y, 161 Tb, 149 Tb, 177 Lu, and 225 Ac.

[0058] In some embodiments, the optionally coordinated radiometal M for use in diagnostic imaging is 61 Cu, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 52 Mn, 89 Zr, 99m Tc, 111 In, 152 Tb, and 155 Tb.

[0059] In some embodiments, the optionally coordinated radiometal M for use in diagnostic imaging is67 Ga, 68 Ga, 64 Cu, 43 Sc, 44 Sc, 99m Tc, 152 Tb, and 155 Tb.

[0060] In some embodiments, the optionally coordinated radiometal M for use in radionuclide therapy is 67 Cu, 90 Y, 105 Rh, 117m Sn, 149 Pm, 153 Sm, 161 Tb, 149 Tb, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 186 Re, 188 Re, 225 Ac, and 213 Bi.

[0061] In some embodiments, the optionally coordinated radiometal M for use in radionuclide therapy is 67 Cu, 90 Y, 161 Tb, 149 Tb, 177 Lu, and 225 Ac.

[0062] In some embodiments, n is 2, 3, 4, 5, or 6, preferably 4. In some embodiments, m is 2, 3, 4, 5, or 6, preferably 4. In some embodiments, n is 4 and m is 4.

[0063] In some embodiments, the compound of formula I has formula II.

[0064] [ka]

[0065] where Y is a radiometal chelator that optionally coordinates the radiometal M.

[0066] In some embodiments, the radiometal chelator is as defined herein above. In some embodiments, the radiometal chelator is a linear or macrocyclic polyaminocarboxylate, such as DTPA, DOTA (and its derivatives, such as p-SCN-DOTA, maleimide-DOTA, DOTA-NHS-ester), DFO, DFO * , DO3A, HP-DO3A, AAZTA, EDTA, TETA, EHPG, HBED, NOTA (and derivatives such as p-SCN-NOTA), DOTAGA, DOTMA, TETMA, PDTA, TTHA, LICAM, MECAM.

[0067] In some embodiments, metal chelators used for the present disclosure include macrocyclic polyaminocarboxylates such as NOTA, DOTA, DTPA, DO3A, HP-DO3A, AAZTA, EDTA, TETA, DOTMA, and the like.

[0068] In some embodiments, the optionally coordinated radiometal M is 51 Cr, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 47 Sc, 167 Tm, 141 Ce, 111 In, 168 Yb, 175 Yb, 140 La, 89 Zr, 90 Y, 88 Y, 153 Sm, 166 Ho, 52 Mn, 165 Dy, 166 Dy, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 97Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 211 Bi, 212 Bi, 213 Bi, 214 Bi, 105 Rh, 109 Pd, 212 Pb, 117m Sn, 149 Pm, 161 Tb, 149 Tb, 152 Tb, 155 Tb, 99m Tc, 165 Er, 169 Er, 172 Yb, 165 Tm, 177 Lu, 225 Ac, 198 Au, 199 Au, and 227 Th.

[0069] In some embodiments, the optionally coordinated radiometal M is 67 Ga, 68 Ga, 64 Cu, 67 Cu, 43 Sc, 44 Sc, 99m Tc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 90 Y, 177 Lu, and 225 Ac.

[0070] In some embodiments, the optionally coordinated radiometal M is 67 Ga, 68 Ga, 64 Cu, 43 Sc, 44 Sc, 99m Tc, 152 Tb, 155 Tb, 67 Cu, 90 Y, 161 Tb, 149Tb, 177 Lu, and 225 Ac.

[0071] In some embodiments, the optionally coordinated radiometal M for use in diagnostic imaging is 61 Cu, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 52 Mn, 89 Zr, 99m Tc, 111 In, 152 Tb, and 155 Tb.

[0072] In some embodiments, the optionally coordinated radiometal M for use in diagnostic imaging is 67 Ga, 68 Ga, 64 Cu, 43 Sc, 44 Sc, 99m Tc, 152 Tb, and 155 Tb.

[0073] In some embodiments, the optionally coordinated radiometal M for use in radionuclide therapy is 67 Cu, 90 Y, 105 Rh, 117m Sn, 149 Pm, 153 Sm, 161 Tb, 149 Tb, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 186 Re, 188 Re, 225 Ac, and 213 Bi.

[0074] In some embodiments, the optionally coordinated radiometal M for use in radionuclide therapy is67 Cu, 90 Y, 161 Tb, 149 Tb, 177 Lu, and 225 Ac.

[0075] In some embodiments, the compound of formula I has formula IIa or IIb.

[0076] [ka]

[0077] [ka]

[0078] where Y is a radiometal chelator that optionally coordinates the radiometal M.

[0079] In some embodiments, the radiometal chelator is as defined herein above. In some embodiments, the radiometal chelator is a linear or macrocyclic polyaminocarboxylate, such as DTPA, DOTA (and its derivatives, such as p-SCN-DOTA, maleimide-DOTA, DOTA-NHS-ester), DFO, DFO * , DO3A, HP-DO3A, AAZTA, EDTA, TETA, EHPG, HBED, NOTA (and derivatives such as p-SCN-NOTA), DOTAGA, DOTMA, TETMA, PDTA, TTHA, LICAM, MECAM.

[0080] In some embodiments, metal chelators used for the present disclosure include macrocyclic polyaminocarboxylates such as NOTA, DOTA, DTPA, DO3A, HP-DO3A, AAZTA, EDTA, TETA, DOTMA, and the like.

[0081] In some embodiments, the optionally coordinated radiometal M is 51Cr, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 47 Sc, 167 Tm, 141 Ce, 111 In, 168 Yb, 175 Yb, 140 La, 89 Zr, 90 Y, 88 Y, 153 Sm, 166 Ho, 52 Mn, 165 Dy, 166 Dy, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 97 Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 211 Bi, 212 Bi, 213 Bi, 214 Bi, 105 Rh, 109 Pd, 212 Pb, 117m Sn, 149 Pm, 161 Tb, 149 Tb, 152 Tb, 155 Tb, 99m Tc, 165 Er, 169 Er, 172 Yb, 165 Tm, 177 Lu, 225 Ac, 198 Au, 199 Au, and 227 Th.

[0082] In some embodiments, the optionally coordinated radiometal M is 67 Ga, 68 Ga, 64 Cu, 67 Cu, 43 Sc, 44 Sc,99m Tc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 90 Y, 177 Lu, and 225 Ac.

[0083] In some embodiments, the optionally coordinated radiometal M is 67 Ga, 68 Ga, 64 Cu, 43 Sc, 44 Sc, 99m Tc, 152 Tb, 155 Tb, 67 Cu, 90 Y, 161 Tb, 149 Tb, 177 Lu, and 225 Ac.

[0084] In some embodiments, the optionally coordinated radiometal M for use in diagnostic imaging is 61 Cu, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 52 Mn, 89 Zr, 99m Tc, 111 In, 152 Tb, and 155 Tb.

[0085] In some embodiments, the optionally coordinated radiometal M for use in diagnostic imaging is 67 Ga, 68 Ga, 64 Cu, 43 Sc, 44 Sc, 99m Tc, 152 Tb, and 155 Tb.

[0086] In some embodiments, the optionally coordinated radiometal M for use in radionuclide therapy is 67 Cu, 90 Y, 105 Rh, 117m Sn, 149 Pm, 153 Sm, 161 Tb, 149 Tb, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 186 Re, 188 Re, 225 Ac, and 213 Bi.

[0087] In some embodiments, the optionally coordinated radiometal M for use in radionuclide therapy is 67 Cu, 90 Y, 161 Tb, 149 Tb, 177 Lu, and 225 Ac.

[0088] In some embodiments, the compound of formula I has formula III and is optionally coordinated with a radiometal M.

[0089] [ka]

[0090] In some embodiments, the optionally coordinated radiometal M is 51 Cr, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 47 Sc, 167 Tm, 141 Ce, 111 In, 168 Yb, 175 Yb, 140 La, 89 Zr, 90 Y, 88 Y, 153 Sm,166 Ho, 52 Mn, 165 Dy, 166 Dy, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 97 Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 211 Bi, 212 Bi, 213 Bi, 214 Bi, 105 Rh, 109 Pd, 212 Pb, 117m Sn, 149 Pm, 161 Tb, 149 Tb, 152 Tb, 155 Tb, 99m Tc, 165 Er, 169 Er, 172 Yb, 165 Tm, 177 Lu, 225 Ac, 198 Au, 199 Au, and 227 Th.

[0091] In some embodiments, the optionally coordinated radiometal M is 67 Ga, 68 Ga, 64 Cu, 67 Cu, 43 Sc, 44 Sc, 99m Tc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 90 Y, 177 Lu, and 225 Ac.

[0092] In some embodiments, the optionally coordinated radiometal M is 67 Ga, 68 Ga, 64 Cu,43 Sc, 44 Sc, 99m Tc, 152 Tb, 155 Tb, 67 Cu, 90 Y, 161 Tb, 149 Tb, 177 Lu, and 225 Ac.

[0093] In some embodiments, the optionally coordinated radiometal M is for use in diagnostic imaging, 61 Cu, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 52 Mn, 89 Zr, 99m Tc, 111 In, 152 Tb, and 155 Tb.

[0094] In some embodiments, the optionally coordinated radiometal M is for use in diagnostic imaging, 67 Ga, 68 Ga, 64 Cu, 43 Sc, 44 Sc, 99m Tc, 152 Tb, and 155 Tb.

[0095] In some embodiments, the optionally coordinated radiometal M is for use in radionuclide therapy, 67 Cu, 90 Y, 105 Rh, 117m Sn, 149 Pm, 153 Sm, 161 Tb, 149 Tb, 166 Dy, 166 Ho, 175 Yb, 177 Lu,186 Re, 188 Re, 225 Ac, and 213 Bi.

[0096] In some embodiments, the optionally coordinated radiometal M is for use in radionuclide therapy, 67 Cu, 90 Y, 161 Tb, 149 Tb, 177 Lu, and 225 Ac.

[0097] In some embodiments, the compound of formula I has formula IIIa or IIIb and is optionally coordinated with a radiometal M.

[0098] [ka]

[0099] [ka]

[0100] In some embodiments, the optionally coordinated radiometal M is 51 Cr, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 47 Sc, 167 Tm, 141 Ce, 111 In, 168 Yb, 175 Yb, 140 La, 89 Zr, 90 Y, 88 Y, 153 Sm, 166 Ho, 52 Mn, 165 Dy, 166 Dy, 61 Cu, 62 Cu, 64 Cu, 67 Cu,97 Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 211 Bi, 212 Bi, 213 Bi, 214 Bi, 105 Rh, 109 Pd, 212 Pb, 117m Sn, 149 Pm, 161 Tb, 149 Tb, 152 Tb, 155 Tb, 99m Tc, 165 Er, 169 Er, 172 Yb, 165 Tm, 177 Lu, 225 Ac, 198 Au, 199 Au, and 227 Th.

[0101] In some embodiments, the optionally coordinated radiometal M is 67 Ga, 68 Ga, 64 Cu, 67 Cu, 43 Sc, 44 Sc, 99m Tc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 90 Y, 177 Lu, and 225 Ac.

[0102] In some embodiments, the optionally coordinated radiometal M is 67 Ga, 68 Ga, 64 Cu, 43 Sc, 44 Sc, 99m Tc, 152 Tb, 155 Tb, 67 Cu, 90 Y, 161 Tb,149 Tb, 177 Lu, and 225 Ac.

[0103] In some embodiments, the optionally coordinated radiometal M is for use in diagnostic imaging, 61 Cu, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 52 Mn, 89 Zr, 99m Tc, 111 In, 152 Tb, and 155 Tb.

[0104] In some embodiments, the optionally coordinated radiometal M is for use in diagnostic imaging, 67 Ga, 68 Ga, 64 Cu, 43 Sc, 44 Sc, 99m Tc, 152 Tb, and 155 Tb.

[0105] In some embodiments, the optionally coordinated radiometal M is for use in radionuclide therapy, 67 Cu, 90 Y, 105 Rh, 117m Sn, 149 Pm, 153 Sm, 161 Tb, 149 Tb, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 186 Re, 188 Re, 225 Ac, and 213 Bi.

[0106] In some embodiments, the optionally coordinated radiometal M is for use in radionuclide therapy, 67 Cu, 90 Y, 161 Tb, 149 Tb, 177 Lu, and 225 Ac.

[0107] The compounds of the present disclosure can be prepared by methods known in the art of synthetic chemistry and by methods described in the examples of the present disclosure.

[0108] In a further aspect, the present disclosure provides a pharmaceutical composition comprising a diagnostically or therapeutically effective amount of at least one compound of the present disclosure and a pharma- ceutically acceptable carrier therefor.As used herein, the pharma-ceutically acceptable carrier present in an appropriate dosage includes physiologically acceptable solvents, dispersion media, antibacterial and antifungal agents, isotonicity agents, etc.The use of such media and agents is well known in the art.

[0109] In further aspects, the present disclosure provides compounds and / or pharmaceutical compositions of the present disclosure (i) for use as diagnostic imaging agents, (ii) for use in radionuclide therapy, or (iii) for use as theragnostic agents, e.g., for use in targeting, visualization, and treatment of tumors, particularly for use in monitoring and assessing different stages, progression, and migration of tumors and determining appropriate radionuclide therapy regimens (frequency, dose, etc.).

[0110] In some embodiments, the subject of the disclosed methods is a mammal, such as an animal or a human. In some embodiments, the subject is a human.

[0111] In some embodiments, the compounds and / or pharmaceutical compositions of the present disclosure may be used for use in diagnostic imaging, i.e., diagnostic imaging of cells or cell populations expressing folate receptors in vitro or in vivo, i.e., for convenient and effective administration to a subject in need of diagnostic imaging.

[0112] In some embodiments, the compounds and / or pharmaceutical compositions of the present disclosure may be used for diagnostic imaging of a cell or cell population expressing a folate receptor, comprising administering a diagnostically effective amount of at least one compound and / or pharmaceutical composition of the present disclosure and obtaining a diagnostic image of the cell or cell population expressing the folate receptor.

[0113] In some embodiments, the compounds and / or pharmaceutical compositions of the present disclosure may be used as diagnostic imaging agents for monitoring the effectiveness of therapeutic regimens and / or ongoing therapeutic treatment.

[0114] In some embodiments, the present disclosure provides a method for diagnostic imaging of a cell or cell population expressing a FR, the method comprising administering a diagnostically effective amount of at least one compound or composition of the present disclosure and obtaining a diagnostic image of the cell or cell population.

[0115] In some embodiments, the present disclosure provides a method for detecting cells, e.g., tumor cells, expressing folate receptors in a tissue sample, e.g., a tissue biopsy taken from a subject, in vitro, the method comprising contacting the tissue sample with a diagnostically effective amount of a compound or composition of the present disclosure for a time and under conditions sufficient for binding to occur, and detecting such binding by an imaging technique, such as PET imaging.

[0116] In some embodiments, the present disclosure provides a method for imaging or monitoring a subject (e.g., cancer treatment), comprising: (i) administering to the subject a diagnostically effective amount of at least one compound and / or pharmaceutical composition of the present disclosure; and (ii) performing imaging using PET by detecting a signal from said at least one compound and / or pharmaceutical composition of the present disclosure (to track the course of cancer treatment and / or to determine a therapeutically effective amount of at least one compound of an additional composition of the present disclosure to be administered for treatment).

[0117] In some embodiments, the compounds and / or pharmaceutical compositions of the present disclosure may be used for radionuclide therapy, i.e., for convenient and effective administration to a subject in need of radionuclide therapy.

[0118] In some embodiments, the compounds and / or pharmaceutical compositions of the present disclosure may be used for radionuclide therapy, comprising administering a therapeutically effective amount of at least one compound and / or pharmaceutical composition of the present disclosure to a subject in need thereof, localizing said at least one compound and / or pharmaceutical composition to the tissue to be treated, and irradiating the tissue to achieve the desired therapeutic effect.

[0119] In some embodiments, the present disclosure provides a method for radionuclide therapy comprising administering a therapeutically effective amount of at least one compound or pharmaceutical composition of the present disclosure to a subject in need thereof, and after localization of said at least one compound or pharmaceutical composition in a desired tissue, irradiating the tissue to achieve a desired therapeutic effect.

[0120] In some embodiments, the compounds and / or pharmaceutical compositions of the present disclosure may be used as theragnostic agents for theragnostic applications.

[0121] The term "theragnostic" is derived from therapy and diagnostics, and refers to a strategy of using the same radiolabeled agent, which ultimately contains different radionuclides, for diagnosis and treatment, in terms of use and agent. This allows the compound of the present disclosure coordinated with a radionuclide effective for diagnostic and treatment planning purposes to be used to image the disease. Thus, the disease can be treated by converting it into a radionuclide effective for tumor treatment. This is the so-called "treat what you see" principle. More specifically, the compound of the present disclosure can be used as a theragnostic agent, firstly, as a diagnostically effective amount of an imaging agent with a radionuclide effective for tumor localization, evaluation, monitoring, or treatment planning ("diagnostic compound of the present disclosure"), and secondly, as a therapeutic agent with a therapeutically effective amount of a radionuclide effective for tumor treatment ("therapeutic compound of the present disclosure"). By combining both functions, for example, one can optimize selectivity (i.e., biodistribution) and efficacy (i.e., effective dose) by first localizing and monitoring tumors or cancerous tissues using the diagnostic compounds of the present disclosure, and then tailoring an appropriate administration regimen (i.e., frequency, dose, etc.) of the therapeutic compounds of the present disclosure according to the needs of the individual subject.

[0122] Thus, in some embodiments, the compounds of the present disclosure may be used in treatment planning and / or treatment and / or monitoring of a tumor (or cancerous tissue) by administering to a subject in need thereof (i) a diagnostically effective amount (i.e., an amount effective for obtaining diagnostic images and / or for treatment planning, i.e., for establishing a treatment regimen) of at least one compound or composition of the present disclosure, and (ii) a therapeutically effective amount of at least one additional compound or composition of the present disclosure for tumor treatment (by irradiating the tumor (or cancerous tissue)) to achieve the desired therapeutic effect.

[0123] A diagnostically effective amount is an amount effective for diagnostic imaging, i.e., an amount effective to obtain a diagnostic image for treatment planning, i.e., an amount effective to obtain a diagnostic image based on which a treatment regimen may be designed (i.e., a therapeutically effective amount of at least one further compound or composition of the present disclosure to be administered may be determined or calculated) or an ongoing treatment regimen may be altered.

[0124] In some embodiments, a diagnostically effective amount of at least one compound or composition of the present disclosure is a diagnostic compound that coordinates a radiometal M for use in diagnostic imaging as defined herein.

[0125] In some embodiments, a therapeutically effective amount of at least one additional compound or composition of the present disclosure is a therapeutic compound that coordinates a radiometal M for use in radionuclide therapy as defined herein.

[0126] A "diagnostically effective amount" of a compound or composition of the present disclosure to be administered is an amount sufficient to produce a diagnostic image of a tumor, cancerous tissue, organ, or other site of a subject, and / or to determine a therapeutically effective amount for treatment. A diagnostically effective amount of a compound or composition of the present disclosure is administered to monitor tumor growth or size before, during, and after radionuclide therapy, allowing for planning, adjusting, and regulating treatment during the course of treatment. In theragnostics applications, the results obtained by administering a diagnostically effective amount of a compound or composition of the present disclosure are used to calculate a therapeutically effective amount.

[0127] A "therapeutically effective amount" of a compound or composition of the present disclosure to be administered is an amount sufficient to produce a desired radiotherapeutic effect. More specifically, a therapeutically effective amount is an amount of at least one compound of the present disclosure sufficient to substantially ameliorate, i.e., alleviate, reduce, or inhibit, at least one symptom associated with a disease or condition, and / or delay, inhibit, or prevent the onset of a disease or condition.

[0128] In some embodiments, the diagnostic compound of the present disclosure is a compound of the present disclosure, in which the radiometal M is a diagnostic radionuclide that allows for the diagnosis of a tumor. In some embodiments, the diagnostic radionuclide is 61 Cu, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 52 Mn, 89 Zr, 99m Tc, 111 In, 152 Tb, and 155 In some embodiments, the diagnostic radionuclide is selected from 67 Ga, 68 Ga, 64 Cu, 43 Sc, 44 Sc, 99m Tc, 152 Tb, and 155 Tb.

[0129] In some embodiments, the therapeutic compound of the present disclosure is a compound of the present disclosure, in which the radiometal M is a therapeutic radionuclide that allows for the treatment of tumors. In some embodiments, the therapeutic radionuclide is 67 Cu, 90 Y, 105 Rh, 117m Sn, 149 Pm, 153 Sm, 161 Tb, 149 Tb, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 186 Re, 188 Re, 225 Ac, and 213 In some embodiments, the therapeutic radionuclide is selected from 67 Cu, 90 Y, 161 Tb, 149 Tb, 177 Lu, and 225Ac.

[0130] It is understood that the diagnostic compounds of the disclosure and the therapeutic compounds of the disclosure are administered sequentially to the subject.

[0131] In some embodiments, the present disclosure provides a method for treatment planning in a subject, comprising the steps of (i) administering to a subject in need thereof a diagnostically effective amount of at least one compound or composition of the present disclosure to obtain a diagnostic image, and (ii) determining a therapeutically effective amount of at least one additional compound or composition of the present disclosure to be administered for treatment.

[0132] In some embodiments, the present disclosure provides a method for theragnostic applications in a subject, comprising: (i) administering to a subject in need thereof a diagnostically effective amount of at least one compound or composition of the present disclosure for obtaining diagnostic images; and (ii) administering a therapeutically effective amount of at least one additional compound or composition of the present disclosure for treatment (i.e., by irradiating the tissue to achieve a desired therapeutic effect).

[0133] In some embodiments, the diagnostic images obtained of at least one compound or composition in the tissue to be treated are used to determine (or calculate) a therapeutically effective amount of a compound or composition of the invention to be used for treatment.

[0134] In some embodiments, the present disclosure provides a method for theragnostic applications, i.e., encompassing both diagnostics and radionuclide therapy, comprising the steps of: (i) administering to a subject in need thereof a diagnostically effective amount of at least one compound or composition of the present disclosure; (ii) obtaining a diagnostic image of said at least one compound or composition in the tissue to be treated; (iii) administering a therapeutically effective amount of at least one additional compound or composition of the present disclosure; and (iv) irradiating the tissue to achieve the desired therapeutic effect.

[0135] In some embodiments, a diagnostically effective amount of at least one compound or composition of the present disclosure is a diagnostic compound that coordinates a radiometal M for use in diagnostic imaging as defined herein.

[0136] In some embodiments, the diagnostically effective amount of at least one additional compound or composition of the present disclosure is a therapeutic compound that coordinates a radiometal M for use in radionuclide therapy, as defined herein.

[0137] In some embodiments, the diagnostic images of at least one compound or composition in the tissue to be treated are used to determine (e.g., calculate) a therapeutically effective amount of at least one additional compound or composition to be administered to obtain a therapeutic effect.

[0138] The image of the cell or tissue expressing FR, i.e., tumor cell or tissue, labeled with one or more compounds or compositions of the present disclosure can be detected using radiation detector, for example, γ-radiation detector.One such method is scintigraphy.Tomographic imaging such as SPECT can also be used to improve visualization.The selection and use of such radiation detector is within the skill of the person skilled in the art.

[0139] In determining the dose for diagnostic imaging or radionuclide therapy, the selected radioimaging metal ion, e.g. 51 Cr, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 47 Sc, 167 Tm, 141 Ce, 111 In, 168 Yb, 175 Yb, 140 La, 89 Zr, 90 Y, 88 Y, 153 Sm, 166 Ho, 52 Mn, 165 Dy, 166 Dy, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 97 Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 211 Bi, 212 Bi, 213 Bi, 214 Bi, 105 Rh, 109 Pd, 212 Pb, 117m Sn, 149 Pm, 161 Tb, 149 Tb, 152 Tb, 155 Tb, 99m Tc, 165 Er, 169 Er, 172 Yb, 165 Tm, 177 Lu, 225 Ac, 198 Au, 199 Au, and 227 It is understood that the specific activity of Th is taken into consideration.

[0140] In some embodiments, the unit dose administered is from about 0.1 MBq to about 10 4 In some embodiments, the unit dose administered for diagnostic imaging has a radioactivity of about 1 MBq to about 1,000 MBq, for example, about 100 MBq to about 600 MBq. In some embodiments, the unit dose administered for radionuclide therapy has a radioactivity of about 1,000 MBq to about 10 4 The radioactivity is about 5,000 MBq to about 8,000 MBq. In the case of an injected solution, the preferred unit dose is about 0.01 mL to about 10 mL. For example, after intravenous administration, imaging of an organ or tumor can be performed in vivo for a period of minutes to hours or longer, as desired, after the radiolabeled reagent is administered to a subject.

[0141] The compound and / or composition of the present disclosure can be administered by suitable route, such as parenterally (e.g., intravenously), intramuscularly, or intraperitoneally, or by any other suitable method.For example, the compound and / or composition of the present disclosure can be administered to a subject by bolus injection or slow infusion intravenous injection.The form suitable for injection includes sterile aqueous solution or dispersion and sterile powder of the above compound and / or composition of the present disclosure.

[0142] The compounds or pharmaceutical compositions are typically sterile, which may be accomplished by any technique recognized by those of skill in the art, including, but not limited to, sterile filtration, the addition of antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.

[0143] Samples may be obtained by procedures known to those skilled in the art, such as tissue biopsy, collection of body fluids, aspiration of tracheal or lung samples, and the like.

[0144] The tissue samples to be examined include tumor cells, epithelial cells, renal, gastrointestinal, or hepatobiliary tissues, or any other tissue suspected of containing cells expressing FRs. To facilitate microscopic examination and observation of the bound complex, the sample can be sectioned, for example, by a microtome. The sample can also be fixed with a suitable fixative either before or after incubation with one of the compounds or compositions of the present disclosure, to improve the histological quality of the sample tissue.

[0145] The time and conditions sufficient for the complex of the present disclosure to bind to FR on cells include standard tissue culture conditions.That is, the sample can be cultured in vitro and cultured with one of the compounds or compositions of the present disclosure in physiological medium.Such conditions are well known to those skilled in the art.Alternatively, the sample can be fixed and then cultured with the complex or composition of the present disclosure in isotonic buffer or physiological buffer.

[0146] A typical amount of the conjugate of the present disclosure for in vitro detection of tumor cells can range from about 1 ng / L to about 1,000 μg / L. In some embodiments, the amount is from about 1 μg / L to about 100 μg / L.

[0147] In some embodiments, the compounds of the present disclosure for use in in vitro diagnosis of tumor cells and in in vivo applications include compounds comprising a radioactive metal chelator: 51 Cr, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 47 Sc, 167 Tm, 141 Ce, 111 In, 168 Yb, 175 Yb, 140 La, 89 Zr, 90 Y, 88 Y, 153 Sm, 166 Ho, 52 Mn, 165 Dy, 166 Dy,61 Cu, 62 Cu, 64 Cu, 67 Cu, 97 Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 211 Bi, 212 Bi, 213 Bi, 214 Bi, 105 Rh, 109 Pd, 212 Pb, 117m Sn, 149 Pm, 161 Tb, 149 Tb, 152 Tb, 155 Tb, 99m Tc, 165 Er, 169 Er, 172 Yb, 165 Tm, 177 Lu, 225 Ac, 198 Au, 199 Au, and 227 The compounds of the present disclosure coordinate a radiometal M site selected from Th.

[0148] In some embodiments, the optionally coordinated radiometal M is 67 Ga, 68 Ga, 64 Cu, 67 Cu, 43 Sc, 44 Sc, 99m Tc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 90 Y, 177 Lu, and 225 Ac.

[0149] In some embodiments, the optionally coordinated radiometal M is 67 Ga, 68 Ga, 64 Cu, 43 Sc, 44 Sc,99m Tc, 152 Tb, 155 Tb, 67 Cu, 90 Y, 161 Tb, 149 Tb, 177 Lu, and 225 Ac.

[0150] In some embodiments, the optionally coordinated radiometal M for use in diagnostic imaging is 61 Cu, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 43 Sc, 44 Sc, 52 Mn, 89 Zr, 99m Tc, 111 In, 152 Tb, and 155 Tb.

[0151] In some embodiments, the optionally coordinated radiometal M for use in diagnostic imaging is 67 Ga, 68 Ga, 64 Cu, 43 Sc, 44 Sc, 99m Tc, 152 Tb, and 155 Tb.

[0152] In some embodiments, the optionally coordinated radiometal M for use in radionuclide therapy is 67 Cu, 90 Y, 105 Rh, 117m Sn, 149 Pm, 153 Sm, 161 Tb, 149 Tb, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 186 Re, 188 Re, 225 Ac, and213 Bi.

[0153] In some embodiments, the optionally coordinated radiometal M for use in radionuclide therapy is 67 Cu, 90 Y, 161 Tb, 149 Tb, 177 Lu, and 225 Ac.

[0154] To detect cell binding of one of the compounds of the present disclosure, samples can be incubated in the presence of the compound, washed, and counted in a standard scintillation counter. Alternative methods are applicable and known to those skilled in the art.

[0155] It is understood that the methods of the present disclosure may be practiced in combination with any other method of cancer diagnosis or treatment, including methods using other diagnostic and / or therapeutic agents already developed, as well as methods utilizing X-ray computed tomography (CT), magnetic resonance imaging (MRI), functional magnetic resonance imaging (fMRI), SPECT, optical imaging, and ultrasound.

[0156] In diagnostic imaging, radionuclide therapy, or theragnostics applications, it may be convenient to prepare the compounds of the present disclosure at or near the site where they are to be used. Thus, in a further aspect, the present disclosure provides single or multi-vial kits that contain all the components necessary to prepare the compounds or compositions of the present disclosure, except the radioactive metal ions themselves. Thus, a preferred single kit of the present disclosure includes the compounds of the present disclosure that are not coordinated with a radioactive metal chelator, and a source of a pharma-ceutically acceptable reducing agent, such as a tin(II) salt. In addition, the kit may optionally include additional additives, for example, the kit may be buffered with a pharma-ceutically acceptable acid or base to adjust the pH to a desired value for complex formation. Such single vial kits may optionally include an exchange ligand, such as glucoheptonic acid, gluconic acid, mannitol, maleic acid, citric acid, or tartaric acid, and may also include a reaction modifier, such as diethylenetriaminepentaacetic acid or ethylenediaminetetraacetic acid. Additional additives such as solubilizers (e.g., cyclodextrin), antioxidants (e.g., ascorbic acid), and / or bulking agents (e.g., NaCl) can be used to improve the radiochemical purity and stability of the final product or to aid in the manufacture of the kit. The radiometal is usually added separately in the form of a solution.

[0157] Similarly, a preferred multi-vial kit of the present disclosure contains in one vial components other than the radiometal itself, i.e., an exchange ligand and a pharma- ceutically acceptable reducing agent such as a tin(II) salt. In the compositions of the present disclosure, the radiometal chelator is contained in a second vial, as well as any additives, such as a suitable buffer to adjust the pH to an optimal value. In some cases, the radiometal is provided in the form of a solution to be added.

[0158] All components of the kit may be in liquid, frozen or dried form, hi some embodiments, the components of the kit are provided in lyophilized form.

[0159] All compounds, compositions, and / or methods disclosed and claimed herein can be made and carried out without undue experimentation in light of this disclosure. It will be apparent to those skilled in the art that modifications can be applied to this disclosure without departing from the scope of this disclosure. The examples provided herein are intended to be illustrative and are not exhaustive. Therefore, the illustrated examples should not be considered as limiting this disclosure in any way. EXAMPLES

[0160] Methods: General: All commercial solvents and chemicals were purchased from abcr, Bachem, Fluka, CheMatech, Iris Biotech, Merck KGaA, or Sigma Aldrich and used without further purification. Folate and pteroate precursors were obtained from Merck&Cie, Schaffhausen, Switzerland.

[0161] Cleavage from the resin and deprotection. Cleavage from the resin was performed with 2% trifluoroacetic acid (TFA) (v / v) in dichloromethane (DCM), followed by removal of the tri-tert-butyl and formyl protecting groups in phosphoric acid (60%, aq., 24 h). Sodium hydroxide (NaOH) solution (32%, w / v, aq.) was added until the pH was about 4. Then, hydrochloric acid solution (1 M, aq.) was added to pH 4, precipitating the final product. The mixture was centrifuged at 14000 rpm for 5 min, after which the supernatant was discarded and the crude folate conjugate was redissolved and purified using RP-HPLC.

[0162] Purification of folate conjugates. Purification of the final product was performed using a Merck-Hitachi LaChrom HPLC system equipped with a D-7000 interface, an L-7200 autosampler, an L-7400UV detector, an L-7100 pump, and a semi-preparative reversed-phase C18 column (5 μm, 10 × 150 mm, Sunfire™, Waters, Milford, US-MA). The product was eluted using variable gradient conditions with Milli-Q water / acetonitrile (MeCN) / TFA or Milli-Q water / MeCN / NH4HCO3 systems at a flow rate of 4 mL / min. Fractions containing pure product were collected in a round-bottom flask, frozen in liquid nitrogen, and lyophilized for more than 24 h.

[0163] Characterization of the folate conjugates. The final products were characterized using high-resolution MALDI-TOF-MS (Bruker UltraFlex II; Billarica, US-MA).

[0164] Radiolabeling: The folate conjugate was dissolved in Milli-Q water containing 4-6% sodium L-ascorbate (0.5 M) to a final folate concentration of 1 mM and frozen at -20 °C as a stock solution. The conjugate was labeled with lutetium-177 (carrier-free in 0.04 M HCl; ITM Medical Isotopes GmbH, Munich, Germany) to a molar activity of 50 MBq / nmol. Radiolabeling was performed after thawing the stock solution in a mixture of sodium acetate (0.5 M), hydrochloric acid (0.05 M), and ascorbic acid (0.5 M) at a pH of about 4.5. The reaction mixture was incubated at 95 °C for 10-15 min.

[0165] Quality control: An aliquot of the radiolabeled conjugate was diluted in Milli-Q water containing pentasodium diethylenetriaminepentaacetic acid (Na5-DTPA; 50 μM) and assessed using a Merck Hitachi LaChrom HPLC system consisting of an L-7100 pump, a D7000 interface, an L-7200 autosampler, a radioactivity detector (LB506B, Berthold Technologies GmbH), and a reversed-phase C18 column (5 μm, 4.6 × 150 mm, Xterra (trademark), Waters, USA). A linear gradient of Milli-Q water containing 0.1% TFA (95–20% in 15 min) in MeCN (5–80% in 15 min) was used at a flow rate of 1.0 mL / min.

[0166] Radiolytic stability: After quality control of the radioconjugate (50MBq / nmol) using HPLC (t0=0h), the radioconjugate was diluted with PBS pH 7.4 to obtain an activity (radioactivity) concentration of 100MBq / 500μL. The radioconjugate dilutions were incubated at room temperature. The integrity of the radioconjugate was evaluated using HPLC after 4 and 24 hours, respectively. The HPLC chromatograms were analyzed by determining the peak areas of the radiolabeled product, the released lutetium-177, and a degradation product of unknown structure. The amount of intact product was expressed as a percentage of the sum of the integrated peak areas of the entire chromatogram and was set in relation to the original value determined at t0 (radiochemical purity ≧95% was set as 100%).

[0167] [Example 1: Solid-phase synthesis of 6R-RedFol-24 and 6S-RedFol-24]

[0168] [ka]

[0169] 2-Chlorotrityl chloride (2-CTC) resin (0.1 mmol) was weighed into a filtered 5 mL syringe and swollen in anhydrous DCM for 45 min. Nα-fluorenylmethyloxycarbonyl-Nε-(4-allyloxycarbonyl)-L-lysine (Fmoc-Lys(Alloc)-OH, 0.12 mmol, 1.2 equiv.) was dissolved in dry DCM in the presence of diisopropylethylamine (DIPEA, 0.8 mmol, 8.0 equiv.) and added to the resin and stirred overnight (o / n). After each reaction step, residual reactants were removed by washing the resin three times with dimethylformamide (DMF) or DCM depending on the solvent used. Potential unreacted carbocations of the 2-CTC resin were capped with a solution of DCM, methanol, and DIPEA (17:2:1, v / v / v). After conditioning in DMF, the Fmoc protecting group was removed by shaking twice for 5 min in a mixture of DMF and piperidine in a ratio of 1:1 (v / v) to give compound 1. Next, Nα-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-Nε-Fmoc-L-lysine (Dde-Lys(Fmoc)-OH, 0.4 mmol, 4.0 equiv.) was activated with O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium-hexafluorophosphate (HBTU, 0.396 mmol, 3.96 equiv.) in the presence of DIPEA (0.8 mmol, 8.0 equiv.) in dry DMF for 1 min, and then added to the resin-immobilized compound 1 and reacted for 1 h. After removal of the Fmoc protecting group to give compound 2, activated tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-tris(tert-butyl)ester, 0.3 mmol, 3.0 equiv.) was coupled to the resin-immobilized compound 2 for 3 h. Cleavage of the Alloc protecting groups of the lysine residues was carried out with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4, 0.045 mmol, 0.45 equiv.) in the presence of morpholine (1.0 mmol, 10 equiv.) in dry DCM in the dark for 1 h.To remove residual palladium, the resin-immobilized compound was washed with 1% DIPEA (v / v) in DMF and a solution of sodium diethyldithiocarbamate (35 mg / mL) in DMF to give resin-immobilized compound 3.

[0170] Activated 5-(p-iodophenyl)pentanoic acid (0.4 mmol, 4.0 equiv.) was added to compound 3 and stirred for 1 h, followed by stirring in hydrazine solution (2% in DMF, v / v) for 1.5 h to remove the Dde protecting group to give compound 4. Activated Fmoc-L-glutamic acid 5-tert-butyl ester (Fmoc-Glu-O(tert-butyl), 0.4 mmol, 4.0 equiv.) was coupled for 1.5 h to remove the Fmoc protecting group to give compound 5. 10-formyl-5-methyl-(6S)-tetrahydropteroic acid (6S-5-MTHP) was activated and added to resin-bound compound 5 and stirred for 2 h. The resulting resin-bound compound 6 was washed with DMF, DCM, and diethyl ether (Et2O) and dried under reduced pressure. Cleavage from the resin and removal of the protecting groups were performed as described below. The precipitate was dissolved in a mixture of MeCN and aqueous ammonium hydrogen solution (NH4HCO3, 50 mM; 1:1, v / v) and purified by reversed-phase high-performance liquid chromatography (RP-HPLC).

[0171] The synthesis of the reduced folate conjugate 6S-RedFol-24 was carried out according to the procedure described for 6R-RedFol-24, except that 10-formyl-5-methyl-(6S)-tetrahydropteroic acid (6R-5-MTHP) instead of 10-formyl-5-methyl-(6R)-tetrahydropteroic acid (6S-5-MTHP) was conjugated to the resin-immobilized conjugate precursor under a nitrogen atmosphere and protected from light.

[0172] Example 2: Characterization of folic acid conjugates of the present disclosure Table 1: Chemical characteristics of folate conjugates ( * Measured by MALDI-MS, [M+H]+ Detected as; ** Measured by ESI-MS, [M+2H] 2+ nd = not detected).

[0173] [Table 1]

[0174] [Example 3: Stability of folic acid radioactive conjugates] Table 2: Product peaks are expressed as a percentage of the sum of integrated peak areas over the entire chromatogram (taken as 100%) relative to the value obtained immediately after labeling (average of experiments performed individually with n = 2–3).

[0175] [Table 2]

[0176] [Example 4: PBS / n-octanol partition coefficient (LogD value)] To assess hydrophilicity / hydrophobicity, the n-octanol / PBS partition coefficient (logD value) was measured for each folic acid radioconjugate of the present disclosure. The logD value of the folic acid radioconjugate of the present disclosure (50MBq / nmol) was determined as previously reported after dilution in PBS pH 7.4 to an active concentration of 10MBq / 500μL. A sample of each radioactive folic acid (approximately 0.5MBq, 25μL, 0.01nmol) was added to a mixture of 1475μL PBS pH 7.4 and 1500μL n-octanol. The vials were vortexed vigorously for 1 min and then centrifuged at 2500rpm for 6 min to separate the phases. Aliquots were taken from each phase and measured in a gamma counter (Perkin Elmer, Wallac Wizard 1480). The partition coefficient was calculated as the logarithm of the ratio of counts per minute (cpm) measured in the n-octanol phase to that measured in the PBS phase. Results are presented as the mean ± SD of data from three independent experiments, each performed in five replicates.

[0177] The logD values ​​of the folate radioconjugates of the present disclosure indicate that they are generally hydrophilic (Table 3). However, the additional methyl group in the 5-MTHF-based targeting molecule and the additional methylene group in the 5-(p-iodophenyl)pentanoate unit increase the hydrophobicity of the radioconjugates of the present disclosure.

[0178] Table 3: LogD values ​​of folic acid radioconjugates of the present disclosure.

[0179] [Table 3]

[0180] [Example 5: Albumin binding properties (filter assay)] Albumin binding properties were measured in mouse and human plasma. Albumin binding properties of the disclosed folic acid radioconjugates in mouse plasma (Rockland Immunochemicals, Inc., USA) and human plasma (Stiftung Blutspende SRK Aargau-Solothurn, Switzerland) were measured using ultrafiltration (Deberle, LM et al., Molecules, 2020, 25, (11)). The serum albumin amounts in mouse (MSA) and human (HSA) plasma were defined as 550 μM and 800 μM, respectively, based on measurements using a dry chemistry analyzer (DRI-CHEM 4000i, Fujifilm, Japan). The disclosed folic acid radioconjugate (50MBq / nmol, approximately 300kBq, 0.006nmol in 15μL) was added to mouse and human plasma samples (150μL), after which the samples were incubated at 37℃ for 30 minutes. The samples were loaded onto Amicon centrifugal filters (cutoff 10kDa; Merck Millipore) and then centrifuged (14,000rcf, 30min, 4℃) to separate the plasma-bound and plasma-unbound (free) fractions of each sample. The inserts of the filter devices were inverted and centrifuged at 200rcf for 3min to collect the protein-bound fraction. The radioactivity in the filtrate and filter units, as well as the radioactivity in the protein-bound fraction, were measured separately in a gamma counter (Perkin Elmer, Wallac Wizard 1480). The percentage of radioconjugate bound to mouse and human albumin, respectively, was established in relation to the total radioactivity measured (Table 4).

[0181] Table 4: Albumin-bound fraction of folate radioconjugates of the present disclosure in mouse and human plasma. Data presented as the average of albumin-bound fraction from triplicate experiments.

[0182] [Table 4]

[0183] [Example 6: Cellular uptake and internalization] The experiment was performed as previously reported (Deberle, LM et al., Bioconj. Chem., 2021, 32, p. 1617). KB tumor cells (human cervical cancer cell line, ACC-136) were seeded in 12-well plates (0.5 × 10 cells in 2 mL per well) using folate-free RPMI (FFRPMI) medium supplemented with 10% fetal bovine serum, L-glutamine, and antibiotics. 6 Tumor cells were cultured overnight to allow attachment and were grown overnight at 37°C, 5% CO2. After removing the supernatant, the KB cells were washed with PBS and supplement-free FFRPMI medium was added (975 μL / well). 25 μL (0.75 pmol, 38 kBq) of the disclosed folate radioconjugate (50 MBq / nmol) was added to each well. In some wells, KB tumor cells were co-cultured with excess folate (100 μM) to block FRs on the cell surface. After culturing the well plates at 37°C, 5% CO2 for 2 or 4 hours, the KB tumor cells were washed three times with ice-cold PBS and the total uptake of the disclosed folate radioconjugate was measured. To assess the internalized fraction, a stripping buffer (a solution of 0.1 M acetic acid and 0.15 M NaCl, in water, pH 3) was applied to release the disclosed FR-bound folate radioconjugate from the cell surface. Cell samples were lysed by adding NaOH solution (1M, aqueous, 1 mL) to each well. Radioactivity was counted in the cell lysates using a gamma counter (Perkin Elmer, Wallac Wizard 1480). After homogenizing the cell suspension by vortexing, the protein concentration of each sample was measured using a Micro BCA Protein Assay Kit (Pierce, Thermo Scientific) to normalize the measured radioactivity to the average protein content in a single well. The uptake and internalization fractions were expressed as a percentage of the total radioactivity added and shown as the mean ± SD of n = 2–6 independent experiments (Figure 2A / B).

[0184] [Example 7: FR binding affinity (K D value)] K DDetermination of the value was performed as previously reported (Deberle, LM et al., Bioconj. Chem., 2021, 32, p. 1617). FR-positive IGROV-1 cells (human ovarian cancer cell line, kindly provided by Dr. Gerrit Janssen, Vrije Universiteit Medical Center Amsterdam, The Netherlands) were seeded (2.5 × 10 5 Cells were cultured overnight at 37°C and 5% CO2 to allow cell adhesion. Experiments were performed on ice using ice-cold medium and buffer. After removing the supernatant, cells were washed once with PBS and supplement-free FFRPMI medium was added (450 μL / well). Each folate radioconjugate of the present disclosure (20 MBq / nmol) was added to each well at various concentrations (ranging from 0.1 to 500 nM, 50 μL per well). To measure non-specific binding of the folate radioconjugate of the present disclosure, half of the cell sample was co-cultured with excess folic acid (100 μM) to block FR on the cell surface. IGROV-1 tumor cells were cultured on a shaker at 4°C for 1 hour, after which the supernatant was removed and the cells were washed twice with PBS. After lysing the cells with NaOH solution (1M, aqueous, 500 μL), the radioactivity of the samples was counted in a γ-counter (Wallac Wizard 1480, Perkin Elmer). The specific binding counts per minute (cpm, determined by subtracting the non-specific binding cpm from the total binding cpm) were plotted against the molar concentration of the folic acid radioconjugate of the present disclosure added. D Nonlinear regression analysis to determine K values ​​was performed using GraphPad Prism software (version 8). Results are presented for three experiments performed in triplicate. D All folate radioconjugates showed activity in the low nanomolar range (K D = 1.4 to 5.6 nM) (Table 6).

[0185] Table 6: K determined for each folate radioconjugate of the present disclosure. D value.

[0186] [Table 5]

[0187] [Example 8: Biodistribution test] Five-week-old female athymic nude mice (CD-1 Foxn1 nu ) were purchased from Charles River Laboratories (Sulzfeld, Germany) and fed folate-deficient rodent chow (ssniff Spezialdiaten GmbH, Germany) ad libitum. After an acclimation period of at least 7 days, KB tumor cells (5 × 10 in 100 μL PBS) were inoculated into the shoulders of the mice. 6 Cells) were inoculated subcutaneously. Approximately 2 weeks later, biodistribution studies were performed using n=3-4 mice per time point after intravenous administration of the disclosed folate radioconjugate (3MBq, 0.5nmol, 100μL PBS containing 0.05% BSA). Mice were sacrificed at 1, 4, and 24 hours post-injection (pi), and selected tissues and organs were harvested, weighed, and counted for radioactivity using a gamma counter (PerkinElmer Wallac Wizard 1480). Results are reported as percentage of injected radioactivity per gram of tissue mass (%IA / g), with counts of a defined amount of the original injection solution measured simultaneously to obtain decay-corrected data.

[0188] Biodistribution data, particularly uptake in KB tumor and various other tissues, as well as blood retention, were obtained at various time points following injection of the folate radioconjugate of the present disclosure (FIG. 3).

[0189] The tumor-to-blood, tumor-to-kidney, and tumor-to-liver ratios observed for the conjugates of the present disclosure are shown in Figures 4, 5, and 6, respectively.

[0190] [Example 9: SPECT / CT imaging] SPECT / CT imaging was performed to examine the whole-body distribution of the disclosed folate radioconjugate in KB tumor-bearing mice ( FIG. 7 ). SPECT / CT experiments showed that approximately 2 weeks after tumor cell inoculation, tumors had grown to approximately 300 mm 3 The mice were injected with the disclosed folate radioconjugate (25MBq, 0.5nmol, 100μL, diluted in PBS with 0.05% BSA) and scanned at 1, 4, 24, and 48 hours after injection. Imaging was performed using a four-head, multiplexing, multi-pinhole small animal SPECT camera (NanoSPECT / CT(trademark), Mediso Medical Imaging Systems, Budapest, Hungary) as previously reported. Each head contained nine 1.4mm diameter pinholes fitted with 10mm thick tungsten-based apertures. A CT scan of approximately 7.5 minutes was followed by a SPECT scan of approximately 40 minutes. Images were acquired using Nucline software (version 1.02, Mediso Ltd., Budapest, Hungary). Cone-beam filtered backprojection was used for real-time CT reconstruction. Reconstruction of SPECT data was performed by HiSPECT software (version 1.4.3049, Scivis GmbH, Göttingen, Germany) using lutetium-177 gamma energies of 56.1 keV (±10%), 112.9 keV (±10%), and 208.4 keV (±10%). Images were generated using VivoQuant post-processing software (version 3.5, invicro Imaging Services and Software, Boston, USA). A Gaussian post-reconstruction filter (FWHM=1.0 mm) was applied and the radioactivity scale was set as shown in the images (min=2 Bq / voxel to max=30 Bq / voxel).

[0191] Figure 7 shows 177SPECT / CT images of KB tumor-bearing mice 1, 4, and 24 hours after injection of Lu-folate radioconjugate (25 MBq; 0.5 nmol per mouse) are shown as maximum intensity projections (MIPs). (A) SPECT / CT scan of 6R-5-MTHF-based radioconjugate; (B) SPECT / CT scan of 6S-5-MTHF-based radioconjugate; Tu=KB tumor; Ki=kidney; H=heart.

Claims

1. A compound of formula I, Ia, or Ib, or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 I 【Chemistry 2】 Ia 【Transformation 3】 Ib where: Y is a radiometal chelator that optionally coordinates the radiometal M; n is 1 to 8; m is 1 to 8.

2. The radiometal chelator is a linear or macrocyclic polyaminocarboxylate, such as DTPA, DOTA (and its derivatives, such as p-SCN-DOTA, maleimide-DOTA, DOTA-NHS-ester), DFO, DFO ＊ , DO3A, HP-DO3A, AAZTA, EDTA, TETA, EHPG, HBED, NOTA (and derivatives such as p-SCN-NOTA), DOTAGA, DOTMA, TETMA, PDTA, TTHA, LICAM, and MECAM.

3. The optionally coordinated radioactive metal M is ５１ Cr, ６７ Ga, ６８ Ga, ４３ Sc, ４４ Sc, ４７ Sc, １６７ Tm, １４１ Ce, １１１ In, １６８ Yb, １７５ Yb, １４０ La, ８９ Zr, ９０ Y. ８８ Y. １５３ Sm, １６６ Ho, ５２ Mn, １６５ Dy, １６６ Dy, ６１ Cu, ６２ Cu, ６４ Cu, ６７ Cu, ９７ Ru, １０３ Ru, １８６ Re, １８８ Re, ２０３ Pb, ２１１ Bi, ２１２ Bi, ２１３ Bi, ２１４ Bi, １０５ Rh, １０９ Pd, ２１２ Pb, １１７ｍ Sn, １４９ Pm, １６１ Tb, １４９ Tb, １５２ Tb, １５５ Tb, ９９ｍ Tc, １６５ Er, １６９ Er, １７２ Yb, １６５ Tm, １７７ Lu, ２２５ Ac, １９８ Au, １９９ Au, and ２２７ 2. The compound of claim 1, wherein the compound is selected from the group consisting of:

4. 2. The compound of claim 1, wherein n is 2, 3, 4, 5, or 6.

5. 2. The compound of claim 1, wherein m is 2, 3, 4, 5, or 6.

6. 2. The compound of claim 1, wherein n is 4 and m is 4.

7. The compound of claim 1 having formula II, IIa, or IIb: 【Chemistry 4】 II 【Transformation 5】 IIa 【Transformation 6】 IIb Here, Y is a radiometal chelator that optionally coordinates the radiometal M.

8. The radiometal chelator is a linear or macrocyclic polyaminocarboxylate, such as DTPA, DOTA (and its derivatives, such as p-SCN-DOTA, maleimide-DOTA, DOTA-NHS-ester), DFO, DFO ＊ 8. The compound of claim 7, selected from: DO3A, HP-DO3A, AAZTA, EDTA, TETA, EHPG, HBED, NOTA (and derivatives such as p-SCN-NOTA), DOTAGA, DOTMA, TETMA, PDTA, TTHA, LICAM, and MECAM.

9. The optionally coordinated radioactive metal M is ５１ Cr, ６７ Ga, ６８ Ga, ４３ Sc, ４４ Sc, ４７ Sc, １６７ Tm, １４１ Ce, １１１ In, １６８ Yb, １７５ Yb, １４０ La, ８９ Zr, ９０ Y. ８８ Y. １５３ Sm, １６６ Ho, ５２ Mn, １６５ Dy, １６６ Dy, ６１ Cu, ６２ Cu, ６４ Cu, ６７ Cu, ９７ Ru, １０３ Ru, １８６ Re, １８８ Re, ２０３ Pb, ２１１ Bi, ２１２ Bi, ２１３ Bi, ２１４ Bi, １０５ Rh, １０９ Pd, ２１２ Pb, １１７ｍ Sn, １４９ Pm, １６１ Tb, １４９ Tb, １５２ Tb, １５５ Tb, ９９ｍ Tc, １６５ Er, １６９ Er, １７２ Yb, １６５ Tm, １７７ Lu, ２２５ Ac, １９８ Au, １９９ Au, and ２２７ 8. The compound of claim 7, wherein the compound is selected from the group consisting of:

10. 10. The compound of claim 1 having formula III, IIIa, or IIIb, optionally coordinated with a radiometal M: 【Transformation 7】 III 【Transformation 8】 IIIa 【Chemistry 9】 IIIb.

11. The optionally coordinated radioactive metal M is ５１ Cr, ６７ Ga, ６８ Ga, ４３ Sc, ４４ Sc, ４７ Sc, １６７ Tm, １４１ Ce, １１１ In, １６８ Yb, １７５ Yb, １４０ La, ８９ Zr, ９０ Y. ８８ Y. １５３ Sm, １６６ Ho, ５２ Mn, １６５ Dy, １６６ Dy, ６１ Cu, ６２ Cu, ６４ Cu, ６７ Cu, ９７ Ru, １０３ Ru, １８６ Re, １８８ Re, ２０３ Pb, ２１１ Bi, ２１２ Bi, ２１３ Bi, ２１４ Bi, １０５ Rh, １０９ Pd, ２１２ Pb, １１７ｍ Sn, １４９ Pm, １６１ Tb, １４９ Tb, １５２ Tb, １５５ Tb, ９９ｍ Tc, １６５ Er, １６９ Er, １７２ Yb, １６５ Tm, １７７ Lu, ２２５ Ac, １９８ Au, １９９ Au, and ２２７ The compound of claim 10, wherein the compound is selected from the group consisting of:

12. A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 11.

13. A compound according to any one of claims 1 to 11 for use as a diagnostic imaging agent, or as a radiotherapeutic agent, or as a theragnostic agent.

14. A compound according to any one of claims 1 to 11 for use in the diagnostic imaging of cells or cell populations expressing folate receptors in vitro or in vivo.

15. 12. A compound according to any one of claims 1 to 11 for use in diagnostic imaging of a cell or cell population expressing a folate receptor, comprising the steps of administering a diagnostically effective amount of at least one compound according to any one of claims 1 to 11 and obtaining a diagnostic image of the cell or cell population expressing the folate receptor.

16. A compound according to any one of claims 1 to 11 for use as a diagnostic imaging agent for monitoring the effect of a treatment regimen and / or ongoing therapeutic treatment.

17. A compound according to any one of claims 1 to 11 for use in radionuclide therapy in a subject in need thereof.

18. 12. A compound according to any one of claims 1 to 11 for use in radionuclide therapy, comprising the steps of administering to a subject in need thereof a therapeutically effective amount of at least one compound according to any one of claims 1 to 11, allowing said at least one compound to localize in the tissue to be treated, and irradiating the tissue to achieve the desired therapeutic effect.

19. 12. A compound according to any one of claims 1 to 11 for use in the treatment planning and / or monitoring and / or treatment of a tumor comprising the steps of: (i) administering a diagnostically effective amount of at least one compound according to any one of claims 1 to 11 for obtaining diagnostic images; and (ii) administering a therapeutically effective amount of at least one further compound according to any one of claims 1 to 11 for tumor treatment.

20. 20. The compound of claim 19, wherein the at least one compound in step (i) is a diagnostic compound and the at least one further compound in step (ii) is a radiotherapeutic compound.

21. 20. The compound of claim 19, wherein the diagnostic image obtained from the diagnostically effective amount of the at least one compound administered in step (i) is used to determine a therapeutically effective amount of the at least one further compound administered for treatment in step (ii).

22. 12. A method for diagnostic imaging of a cell or cell population expressing a folate receptor, comprising administering a diagnostically effective amount of at least one compound according to any one of claims 1 to 11, and obtaining a diagnostic image of said cell or cell population.

23. 23. The method of claim 22, wherein the imaging is performed on a cell or cell population that expresses a folate receptor in vitro or in vivo.

24. 12. A method for the in vitro detection of cells expressing folate receptors in a tissue sample, comprising contacting said tissue sample with a diagnostically effective amount of a compound of any one of claims 1 to 11 for a time and under conditions sufficient for binding to occur, and detecting such binding by PET imaging.

25. 12. A method for imaging or monitoring a subject, comprising the steps of: (i) administering a diagnostically effective amount of at least one compound according to any one of claims 1 to 11; and (ii) performing imaging using PET by detecting a signal from said at least one compound.

26. 12. A method for treatment planning in a subject, comprising the steps of: (i) administering to a subject in need thereof a diagnostically effective amount of at least one compound according to any one of claims 1 to 11 in order to obtain a diagnostic image; and (ii) determining a therapeutically effective amount of at least one further compound according to any one of claims 1 to 11 to be administered for treatment.

27. 12. A method for theragnostic applications, comprising the steps of: (i) administering to a subject in need thereof a diagnostically effective amount of at least one compound according to any one of claims 1 to 11; (ii) obtaining a diagnostic image of said at least one compound according to any one of claims 1 to 11 in the tissue to be treated; (iii) administering a therapeutically effective amount of at least one further compound according to any one of claims 1 to 11; and (iv) irradiating said tissue to achieve a desired therapeutic effect.

28. 28. The method of claim 27, wherein the at least one compound in step (i) is a diagnostic imaging compound and the at least one additional compound in step (iii) is a radiotherapeutic compound.

29. 28. The method of claim 27, wherein the diagnostic image obtained in step (ii) is used to determine a therapeutically effective amount of the at least one additional compound administered in step (iii) for treatment.

30. 23. The method of claim 22 used in combination with any other method of cancer diagnosis or treatment.

Images