Pharmacokinetic Enhancement of Bifunctional Chelates and Their Use

Abstract

The present invention relates to conjugates comprising a metal complex chelating moiety and a therapeutic or targeting moiety, methods for their preparation, and uses thereof.

Description

Technical Field

[0001] Related Applications This application claims priority and the benefit thereof to U.S. Provisional Patent Application No. 62 / 502,260, filed on May 5, 2017, entitled "Pharmacokinetic Enhancement of Bifunctional Chelates and Their Use", the entire content of which is incorporated herein by reference for all purposes.

Background Art

[0002] Radiolabeled targeting moieties, or radioconjugates, are typically prepared by using bifunctional chelating agents to add a radiolabel to a biomolecule while maintaining target affinity. Bifunctional chelates may structurally contain a chelate, a linker, and a crosslinking group or targeting moiety. By modifying the linker region of the bifunctional chelate, pharmacokinetic advantages can be obtained that increase the excretion of radioactivity.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

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Non-Patent Document 19

Non-Patent Document 20

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention relates to a linker that enhances the excretion of a chelating moiety or its metal complex when conjugated to a therapeutic moiety, a targeting moiety, or a crosslinking group.

Means for Solving the Problems

[0006] Accordingly, in a first aspect, the present invention has the structure: A-L 1 -(L 2 ) n -B Formula I [wherein A is a chelating moiety or its metal complex; L 1is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl or heteroaryl; B is a therapeutic moiety, a targeting moiety, or a crosslinking group, or a pharmaceutically acceptable salt thereof; n is from 1 to 5; L 2 each independently has the structure: (-X 1 -L 3 -Z 1 -) Formula II (wherein X 1 is C=O(NR 1 ), C=S(NR 1 ), OC=O(NR 1 ), NR 1 C=O(O), NR 1 C=O(NR 1 ), -CH2PhC=O(NR 1 ), -CH2Ph(NH)C=S(NR 1 ), O, NR 1 ; R 1 is H or optionally substituted C1-C6 alkyl or optionally substituted C1-C6 heteroalkyl, optionally substituted aryl or heteroaryl; L 3 is optionally substituted C1-C 50 alkyl or optionally substituted C1-C 50 heteroalkyl or C5-C 20 polyethylene glycol; Z 1 is CH2, C=O, C=S, OC=O, NR 1 C=O, NR 1 ; R 1 is hydrogen or optionally substituted C1-C6 alkyl, pyrrolidine-2,5-dione) having] characterized by a compound having.

[0007] In some embodiments, the chelating moiety is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA ((1R,4R,7R,10R)-α,α',α'',α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid), DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetic acid), DOTA-GA anhydride (2,2',2''-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid), DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid)), DOTMP (1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylenephosphonic acid), DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonic acid)), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2) hexadecan-4,11-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP (1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid)), TETPA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), HEHA (1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid), PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N''''-pentaacetic acid), H4Octapa (N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid), H2Dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane), H6phospa (N,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane), TTHA (triethylenetetramine-N,N,N',N'',N''',N'''-hexaacetic acid), DO2P (tetraazacyclododecanedimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecane triacetic acid), EDTA (ethylenediaminetetraacetic acid), deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), HOPO (octadentate hydroxypyridinone) or porphyrin.

[0008] One skilled in the art can understand that the use of the chelating moiety in the practice of the present invention is not limited to the specific constructs disclosed herein, but rather may include other known chelating moieties.

[0009] In some embodiments, the chelating moiety has the structure:

[0010]

Chemical formula

[0011] (wherein Y 1 is -CH2OCH2(L 2 ) n -B, C=O(L 2 ) n -B, or C=S(L 2 ) n -B, and Y 2 is -CH2CO2H; wherein Y 1 is H, and Y 2 is L 1 -(L 2 ) n -B) has.

[0012] In some embodiments, L 1 has the structure:

[0013]

Chemical formula

[0014] (wherein R 2 is optionally substituted hydrogen or -CO2H) has.

[0015] In some embodiments, the metal can be selected from Bi, Pb, Y, Mn, Cr, Fe, Co, Zn, Ni, Tc, In, Ga, Cu, Re, Sm, lanthanides, or actinides for use as an imaging agent or a therapeutic agent. Specific examples of radionuclides suitable for complexation with the compound of formula (I) include 47 Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga, 68 Ga, 82 Rb, 86 Y, 87 Y, 90 Y, 97 Ru, 105 Rh,109 Pd, 111 In, 117m Sn, 149 Pm, 149 Tb, 153 Sm, 177 Lu, 186 Re, 188 Re, 199 Au, 201 Tl, 203 Pb, 212 Pb, 212 Bi, 213 Bi, 225 Ac, and 227 Th are included.

[0016] In some embodiments, B is a therapeutic moiety or a targeting moiety.

[0017] In some embodiments, the therapeutic moiety or the targeting moiety is an antibody, an antigen-binding fragment thereof or a nanobody, an affibody, and other targeting proteins such as consensus sequences derived from type III fibronectin domains.

[0018] In some embodiments, the antibody, or an antigen-binding fragment thereof, specifically binds to the insulin-like growth factor-1 receptor (IGF-1R).

[0019] In some embodiments, the crosslinking group is an amino-reactive crosslinking group, a methionine-reactive crosslinking group, a thiol-reactive crosslinking group or a sortase-mediated coupling sequence.

[0020] In some embodiments, the amino-reactive, methionine-reactive or thiol-reactive crosslinking group comprises an activated ester such as a hydroxysuccinimide ester, N-hydroxysulfosuccinimide, 2,3,5,6-tetrafluorophenol ester, 4-nitrophenol ester or imidate, anhydride, thiol, disulfide, maleimide, azide, alkyne, strained alkyne, strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide, diazirine, phosphine, tetrazine, isothiocyanate, or oxaziridine.

[0021] In some embodiments, the sortase recognition sequence may include a terminal glycine-glycine-glycine (GGG) and / or an LPTXG amino acid sequence, where X is any amino acid.

[0022] One skilled in the art can understand that the use of crosslinking groups in the practice of the present invention is not limited to the specific constructs disclosed herein, but rather may include other known crosslinking groups.

[0023] In some embodiments, the crosslinking group is

[0024]

Chemical formula

[0025] selected from the group consisting of.

[0026] In some embodiments, Y 1 is H.

[0027] In some embodiments, X 1 is C=O(NR 1 ) and R 1 is H.

[0028] In some embodiments, Z 1 is -CH2.

[0029] In some embodiments, L 2 has an n value of 1.

[0030] In some embodiments, the compound is

[0031]

Chemical formula

[0032] selected from the group consisting of.

[0033] In some embodiments, the metal is a radionuclide.

[0034] In some embodiments, the radionuclide is 111 In.

[0035] In some embodiments, the radionuclide is 68 Ga.

[0036] In some embodiments, the radionuclide is 86 Y.

[0037] In some embodiments, the metal is a beta-emitting radionuclide.

[0038] In some embodiments, the radionuclide is 67 Cu, 177 Lu', or 90 Y.

[0039] In some embodiments, the metal is an alpha-emitting radionuclide.

[0040] In some embodiments, the radionuclide is 225 Ac, 212 Pb, 227 Th or progeny (daughter isotope) thereof.

[0041] In another aspect, the present invention features a pharmaceutical composition comprising any of the above compounds and a pharmaceutically acceptable excipient.

[0042] In another aspect, the present invention features a method of radiation treatment planning and / or radiation treatment, comprising administering to a subject in need thereof any of the above compounds or pharmaceutical compositions.

[0043] In another aspect, the present invention features a method of detecting and / or treating cancer, comprising administering to a subject in need thereof a first dose of any of the above compounds or pharmaceutical compositions effective for a radiation treatment plan, and then administering a subsequent dose of any of the above compounds or pharmaceutical compositions in a therapeutically effective amount.

[0044] In some embodiments, the compound or composition administered at the first dose is the same as the compound or composition administered at the second dose.

[0045] In some embodiments, the compound or composition administered at the first dose is different from the compound or composition administered at the second dose.

[0046] In some embodiments, the cancer is a solid tumor or a hematological (liquid) cancer.

[0047] In some embodiments, the solid tumor cancer is breast cancer, non-small cell lung cancer, small cell lung cancer, pancreatic cancer, head and neck cancer, prostate cancer, colorectal cancer, sarcoma, adrenocortical carcinoma, neuroendocrine cancer, Ewing sarcoma, multiple myeloma, or acute myeloid leukemia.

[0048] In some embodiments, the method further comprises administering an anti-proliferative agent, a radiosensitizer, or an immunomodulatory or immunoregulatory agent.

[0049] In some embodiments, either the compound or its composition and the anti-proliferative agent or radiosensitizer are administered within 28 days (e.g., within 14, 7, 6, 5, 4, 3, 2, or 1 day) of each other.

[0050] In some embodiments, either the compound or its composition and the immunomodulatory or immunoregulatory agent are administered within 90 days (e.g., within 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 day) of each other.

[0051] In another aspect, the present invention features a method of making a radioactive conjugate (e.g., any of the radioactive conjugates described herein). The method includes (a) conjugating a bifunctional chelate to a biomolecule, (b) purifying the conjugate produced in step (a), and (c) chelating one or more radionuclides (e.g., one or more Ac-225 radionuclides) using the purified conjugate of step (b) at a temperature below 35°C (e.g., 20 - 25°C) to produce a radioactive conjugate (e.g., an actinium radioactive conjugate).

[0052] In some embodiments, the radioactive conjugate is a radioimmunoconjugate (e.g., any of the radioimmunoconjugates described herein).

[0053] In some embodiments, the pH of the reaction mixture in conjugation step (a) is less than 6.4 (e.g., 6.3, 6.2, 6.1, 6.0, 5.9, or 5.8 or less).

[0054] In some embodiments, the pH of the reaction mixture in conjugation step (c) is less than 5.5 (e.g., 5.4, 5.3, 5.2, 5.1, or 5.0 or less) or greater than 7.0 (e.g., 7.1, 7.2, 7.3, 7.4, 7.5 or greater).

[0055] In some embodiments, the temperature of the reaction mixture in conjugation step (c) is 20 - 34°C (e.g., 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, or 34°C).

[0056] Chemical terms: As used herein, the term "acyl" refers to a hydrogen or an alkyl group (e.g., a haloalkyl group) as defined herein, bonded to a parent molecular group via a carbonyl group as defined herein, and is exemplified by formyl (i.e., carboxaldehyde group), acetyl, trifluoroacetyl, propionyl, butanoyl, etc. Exemplary unsubstituted acyl groups contain from 1 to 7, from 1 to 11, or from 1 to 21 carbons. In some embodiments, the alkyl group is further substituted with 1, 2, 3, or 4 substituents as described herein.

[0057] As used herein, the term "alkyl" includes both straight-chain and branched-chain saturated groups of 1 to 20 carbons (e.g., 1 to 10 or 1 to 6), unless otherwise specified. Alkyl groups are exemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso- and tert-butyl, neopentyl, etc., and (1) C 1~6 alkoxy; (2) C 1~6 alkylsulfinyl; (3) amino as defined herein (e.g., unsubstituted amino (i.e., -NH2) or substituted amino (i.e., -N(R N1 ))2, where R N1 is as defined for amino); (4) C 6~10 aryl-C 1~6 alkoxy; (5) azide; (6) halo; (7) (C 2~9 heterocyclyl)oxy; (8) hydroxy optionally substituted with an O-protecting group; (9) nitro; (10) oxo (e.g., carboxaldehyde or acyl); (11) C 1~7 spirocyclic; (12) thioalkoxy; (13) thiol; (14) -CO2R A' optionally substituted with an O-protecting group, where R A' is (a) C 1~20 alkyl (e.g., C 1~6 alkyl), (b) C 2~20 alkenyl (e.g., C 2~6 alkenyl), (c) aryl, (d) hydrogen, (e) C 6~10 alk-C 1~6 aryl, (f) amino-C 6~10 ​1~20 alkyl, (g) (CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), s2 and s3 are each independently integers from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 alkyl), polyethylene glycol, and (h) -NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), s2 and s3 are each independently integers from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R N1 is each independently hydrogen or optionally substituted C 1~6 alkyl); (15) -C(O)NR B' R C' (wherein R B' and R C' are each independently (a) hydrogen, (b) C 1~6 alkyl, (c) C 6~10 aryl, and (d) C 1~6 alk-C 6~10 aryl); (16) -SO2R D' (wherein R D' is (a) C 1~6 alkyl, (b) C 6~10 aryl, (c) C 1~6 alk-C 6~10 aryl, and (d) hydroxy); (17) -SO2NR E' R F' (wherein R E' and R F' are each independently (a) hydrogen, (b) C 1~6 alkyl, (c) C 6~10 aryl and (d) C 1~6 alk-C 6~10selected from the group consisting of aryl); (18)-C(O)R G' (wherein R G' is (a) C 1~20 alkyl (e.g., C 1~6 alkyl), (b) C 2~20 alkenyl (e.g., C 2~6 alkenyl), (c) C 6~10 aryl, (d) hydrogen, (e) C 1~6 alk-C 6~10 aryl, (f) amino-C 1~20 alkyl, (g)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer from 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 alkyl), polyethylene glycol, and (h)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer from 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R N1 are each independently hydrogen or optionally substituted C 1~6 alkyl); (19)-NR H' C(O)R I' (wherein R H' is selected from the group consisting of (a1) hydrogen and (b1) C 1~6 alkyl, and R I' is (a2) C 1~20 alkyl (e.g., C 1~6 alkyl), (b2) C 2~20 alkenyl (e.g., C 2~6 alkenyl), (c2) C 6~10 aryl, (d2) hydrogen, (e2) C 1~6 alk-C 6~10Aryl, (f2)-amino-C 1~20 Alkyl, (g2)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), s2 and s3 are each independently an integer from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 alkyl), polyethylene glycol, and (h2)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), s2 and s3 are each independently an integer from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R N1 is each independently hydrogen or optionally substituted C 1~6 alkyl); (20)-NR J' C(O)OR K' (wherein R J' is selected from the group consisting of (a1) hydrogen and (b1)C 1~6 alkyl, R K' is (a2)C 1~20 alkyl (for example, C 1~6 alkyl), (b2)C 2~20 alkenyl (for example, C 2~6 alkenyl), (c2)C 6~10 aryl, (d2) hydrogen, (e2)C 1~6 alk-C 6~10 aryl, (f2)-amino-C 1~20 alkyl, (g2)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), s2 and s3 are each independently an integer from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20a polyethylene glycol which is alkyl), and (h2)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), s2 and s3 are each independently an integer from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R N1 is each independently hydrogen or an optionally substituted C 1~6 alkyl); and (21) independently selected from the group consisting of an amidine, 1, 2, 3 substituents, or in the case of an alkyl group of 2 or more carbons, optionally substituted with 4 substituents. In some embodiments, these groups can each be further substituted as described herein. For example, an alkylene group of C1-aryl can be further substituted with an oxo group to obtain the corresponding aryloyl substituent.

[0058] As used herein, the terms "alkylene" and the prefix "alk-" represent a saturated divalent hydrocarbon group derived from a straight-chain or branched-chain saturated hydrocarbon by removal of two hydrogen atoms, exemplified by methylene, ethylene, isopropylene, etc. The term "C x~y alkylene" and the prefix "C x~y alk-" represent an alkylene group having x to y carbons. Exemplary values of x are 1, 2, 3, 4, 5 and 6, and exemplary values of y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20 (for example, C 1~6 , C 1~10 , C 2~20 , C 2~6 , C 2~10 , or C 2~20 alkylene). In some embodiments, alkylene can be further substituted with 1, 2, 3, or 4 substituents as defined herein to form an alkyl group.

[0059] As used herein, the term "alkenyl", unless otherwise specified, refers to a monovalent straight-chain or branched-chain group of 2 to 20 carbons (e.g., 2 to 6 or 2 to 10 carbons) containing one or more carbon-carbon double bonds, exemplified by ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, etc. Alkenyl includes both cis and trans isomers. The alkenyl group may be substituted with one, two, three, or four substituents independently selected from amino, aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl) as defined herein, or any of the exemplary alkyl substituents described herein.

[0060] As used herein, the term "alkynyl" refers to a monovalent straight-chain or branched-chain group of 2 to 20 carbon atoms (e.g., 2 to 4, 2 to 6, or 2 to 10 carbons) containing a carbon-carbon triple bond, exemplified by ethynyl, 1-propynyl, etc. The alkynyl group may be substituted with one, two, three, or four substituents independently selected from aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl) as defined herein, or any of the exemplary alkyl substituents described herein.

[0061] As used herein, the term "amino" refers to -N(R N1 )2, where each R N1 is independently H, OH, NO2, N(R N2 )2, SO2OR N2 , SO2R N2 , SOR N2, N - protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, aralkyl, cycloalkyl, alkcycloalkyl, carboxyalkyl (e.g., optionally substituted arylalkoxycarbonyl group or optionally substituted with an O - protecting group such as any of those described herein), sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), alkoxycarbonylalkyl (e.g., optionally substituted arylalkoxycarbonyl group or optionally substituted with an O - protecting group such as any of those described herein), heterocyclyl (e.g., heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), where these described R N1 groups may each be optionally substituted as defined herein for each group; or two R N1 are combined to form a heterocyclyl or N - protecting group, and R N2 each independently represents H, alkyl, or aryl). The amino group of the present invention may be an unsubstituted amino (i.e., -NH2) or a substituted amino (i.e., -N(R N1 )2). In a preferred embodiment, the amino is -NH2 or NHR N1 (wherein R N1 independently is OH, NO2, NH2, NR N2 2, SO2OR N2 , SO2R N2 , SOR N2 , alkyl, carboxyalkyl, sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), alkoxycarbonylalkyl (e.g., t - butoxycarbonylalkyl) or aryl, and R N2 each is independently H, C 1~20 alkyl (e.g., C 1~6 alkyl), or C 6~10 aryl and may be).

[0062] As used herein, the term "amino acid" refers to a molecule having a side chain, an amino group, and an acid group (e.g., a carboxy group of -CO2H or a sulfo group of -SO3H), where the amino acid is attached to a parent molecule by the side chain, amino group, or acid group (e.g., the side chain). In some embodiments, the amino acid is attached to a parent molecular group by a carbonyl group, where the side chain or amino group is attached to the carbonyl group. Exemplary side chains include optionally substituted alkyl, aryl, heterocyclyl, alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl. Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolidine, selenocysteine, serine, taurine, threonine, tryptophan, tyrosine, and valine. An amino acid group is (1) C 1~6 alkoxy; (2) C 1~6 alkylsulfinyl; (3) amino as defined herein (e.g., unsubstituted amino (i.e., -NH2) or substituted amino (i.e., -N(R N1 2 (wherein R N1 is as defined for amino)); (4) C 6~10 aryl-C 1~6 alkoxy; (5) azide; (6) halo; (7) (C 2~9 heterocyclyl)oxy; (8) hydroxy; (9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C 1~7 spirocyclic; (12) thioalkoxy; (13) thiol; (14) -CO2R A' (wherein R A' is (a) C 1~20 alkyl (e.g., C 1~6 alkyl), (b) C 2~20 alkenyl (e.g., C 2~6 alkenyl), (c) C 6~10 aryl, (d) hydrogen, (e) C 1~6 alk-C 6~10Aryl, (f)amino-C 1~20 Alkyl, (g)(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), s2 and s3 are each independently integers from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 alkyl), polyethylene glycol, and (h)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), s2 and s3 are each independently integers from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R N1 are each independently hydrogen or optionally substituted C 1~6 alkyl); (15)-C(O)NR B' R C' (wherein R B' and R C' are each independently (a) hydrogen, (b)C 1~6 alkyl, (c)C 6~10 aryl, and (d)C 1~6 alk-C 6~10 aryl); (16)-SO2R D' (wherein R D' is (a)C 1~6 alkyl, (b)C 6~10 aryl, (c)C 1~6 alk-C 6~10 aryl, and (d)hydroxy); (17)-SO2NR E' R F' (wherein R E' and R F' are each independently (a) hydrogen, (b)C 1~6 alkyl, (c)C 6~10 aryl and (d)C 1~6 alk-C 6~10selected from the group consisting of aryl); (18)-C(O)R G' (wherein R G' is (a) C 1~20 alkyl (e.g., C 1~6 alkyl), (b) C 2~20 alkenyl (e.g., C 2~6 alkenyl), (c) C 6~10 aryl, (d) hydrogen, (e) C 1~6 alk-C 6~10 aryl, (f) amino-C 1~20 alkyl, (g)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently integers from 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 alkyl), polyethylene glycol, and (h)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently integers from 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R N1 are each independently hydrogen or optionally substituted C 1~6 alkyl); (19)-NR H' C(O)R I' (wherein R H' is selected from the group consisting of (a1) hydrogen and (b1) C 1~6 alkyl, and R I' is (a2) C 1~20 alkyl (e.g., C 1~6 alkyl), (b2) C 2~20 alkenyl (e.g., C 2~6 alkenyl), (c2) C 6~10 aryl, (d2) hydrogen, (e2) C 1~6 alk-C 6~10Aryl, (f2)-amino-C 1~20 Alkyl, (g2)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently integers from 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 alkyl); polyethylene glycol, and (h2)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently integers from 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R N1 is each independently hydrogen or optionally substituted C 1~6 alkyl); selected from the group consisting of amino-polyethylene glycol; (20)-NR J' C(O)OR K' (wherein R J' is selected from the group consisting of (a1) hydrogen and (b1) C 1~6 alkyl, R K' is selected from the group consisting of (a2) C 1~20 alkyl (e.g., C 1~6 alkyl), (b2) C 2~20 alkenyl (e.g., C 2~6 alkenyl), (c2) C 6~10 aryl, (d2) hydrogen, (e2) C 1~6 alk-C 6~10 aryl, (f2) amino-C 1~20 alkyl, (g2)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently integers from 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20a polyethylene glycol which is alkyl), and (h2)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), and s2 and s3 are each independently an integer from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R N1 is each independently hydrogen or optionally substituted C 1~6 alkyl); and selected from the group consisting of amino - polyethylene glycols); and (21) in the case of amino acid groups of 1, 2, 3, or 2 or more carbons independently selected from the group consisting of amidines, may be substituted with 4 substituents. In some embodiments, these groups can each be further substituted as described herein.

[0063] As used herein, the term "aryl" represents a monocyclic, bicyclic, or polycyclic carbocyclic system having one or two aromatic rings, exemplified by phenyl, naphthyl, 1,2 - dihydronaphthyl, 1,2,3,4 - tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl, indanyl, indenyl, etc., and (1) C 1~7 acyl (for example, carboxyaldehyde); (2) C 1~20 alkyl (for example, C 1~6 alkyl, C 1~6 alkoxy - C 1~6 alkyl, C 1~6 alkylsulfinyl - C 1~6 alkyl, amino - C 1~6 alkyl, azido - C 1~6 alkyl, (carboxyaldehyde) - C 1~6 alkyl, halo - C 1~6 alkyl (for example, perfluoroalkyl), hydroxy - C 1~6 alkyl, nitro - C 1~6 alkyl, or C 1~6 thioalkoxy - C 1~6 alkyl); (3) C 1~20Alkoxy (e.g., C such as perfluoroalkoxy, etc.) 1~6 Alkoxy); (4) C 1~6 Alkylsulfinyl; (5) C 6~10 Aryl; (6) Amino; (7) C 1~6 Alk-C 6~10 Aryl; (8) Azido; (9) C 3~8 Cycloalkyl; (10) C 1~6 Alk-C 3~8 Cycloalkyl; (11) Halo; (12) C 1~12 Heterocyclyl (e.g., C 1~12 Heteroaryl); (13) (C 1~12 Heterocyclyl)oxy; (14) Hydroxy; (15) Nitro; (16) C 1~20 Thioalkoxy (e.g., C 1~6 Thioalkoxy); (17) -(CH2) q CO2R A' (wherein q is an integer from 0 to 4, and R A' is selected from the group consisting of (a) C 1~6 alkyl, (b) C 6~10 aryl, (c) hydrogen, and (d) C 1~6 Alk-C 6~10 aryl); (18) -(CH2) q CONR B' R C' (wherein q is an integer from 0 to 4, and R B' and R C' are independently selected from the group consisting of (a) hydrogen, (b) C 1~6 alkyl, (c) C 6~10 aryl, and (d) C 1~6 Alk-C 6~10 aryl); (19) -(CH2) q SO2R D' (wherein q is an integer from 0 to 4, and R D' is selected from the group consisting of (a) alkyl, (b) C 6~10 aryl, and (c) Alk-C 6~10 aryl); (20) -(CH2) q SO2NR E' R F' (wherein q is an integer from 0 to 4, and R E' and RF' are each independently selected from the group consisting of (a) hydrogen, (b) C 1~6 alkyl, (c) C 6~10 aryl, and (d) C 1~6 alk-C 6~10 aryl); (21) thiol; (22) C 6~10 aryloxy; (23) C 3~8 cycloalkoxy; (24) C 6~10 aryl-C 1~6 alkoxy; (25) C 1~6 alk-C 1~12 heterocyclyl (e.g., C 1~6 alk-C 1~12 heteroaryl); (26) C 2~20 alkenyl; and (27) C 2~20 alkynyl, and may be substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group. In some embodiments, these groups may each be further substituted as described herein. For example, an alkylene group of C1-aryl or C1-alk-heterocyclyl can be further substituted with an oxo group to obtain the corresponding aryl oil and (heterocyclyl) oil substituents.

[0064] As used herein, the term "arylalkyl" represents an aryl group as defined herein attached to the parent molecular group via an alkylene group as defined herein. Exemplary unsubstituted arylalkyl groups have 7 to 30 carbons (e.g., 7 to 16 or 7 to 20 carbons, e.g., C 1~6 alk-C 6~10 aryl, C 1~10 alk-C 6~10 aryl, or C 1~20 alk-C 6~10 aryl). In some embodiments, alkylene and aryl may each be further substituted with 1, 2, 3, or 4 substituents as defined herein for the corresponding groups. Other groups after the prefix "alk-" are defined in the same manner, and "alk" refers to C 1~6 alkylene, unless otherwise noted, and the chemical structure of the bond is as defined herein.

[0065] As used herein, the term "carbonyl" refers to a C(O) group, which can also be represented as C=O.

[0066] As used herein, the term "carboxy" means -CO2H.

[0067] As used herein, the term "cyano" refers to a -CN group.

[0068] As used herein, the term "cycloalkyl" refers to a monovalent saturated or unsaturated non-aromatic cyclic hydrocarbon group having 3 to 8 carbons, exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicycloheptyl, etc. When a cycloalkyl group contains one carbon-carbon double bond or one carbon-carbon triple bond, the cycloalkyl group can be referred to as a "cycloalkenyl" or "cycloalkynyl" group, respectively. Exemplary cycloalkenyl and cycloalkynyl groups include cyclopentenyl, cyclohexenyl, cyclohexynyl, etc. The cycloalkyl group of the present invention is (1) C 1~7 acyl (e.g., carboxyaldehyde); (2) C 1~20 alkyl (e.g., C 1~6 alkyl, C 1~6 alkoxy-C 1~6 alkyl, C 1~6 alkylsulfinyl-C 1~6 alkyl, amino-C 1~6 alkyl, azido-C 1~6 alkyl, (carboxyaldehyde)-C 1~6 alkyl, halo-C 1~6 alkyl (e.g., perfluoroalkyl), hydroxy-C 1~6 alkyl, nitro-C 1~6 alkyl, or C 1~6 thioalkoxy-C 1~6 alkyl); (3) C 1~20 alkoxy (e.g., C 1~6 alkoxy such as perfluoroalkoxy); (4) C 1~6 alkylsulfinyl; (5) C6~10 Aryl; (6) Amino; (7) C 1~6 Alk-C 6~10 Aryl; (8) Azido; (9) C 3~8 Cycloalkyl; (10) C 1~6 Alk-C 3~8 Cycloalkyl; (11) Halo; (12) C 1~12 Heterocyclyl (e.g., C 1~12 Heteroaryl); (13) (C 1~12 Heterocyclyl) oxy; (14) Hydroxy; (15) Nitro; (16) C 1~20 Thioalkoxy (e.g., C 1~6 Thioalkoxy); (17)-(CH2) q CO2R A' (wherein q is an integer from 0 to 4, and R A' is (a) C 1~6 Alkyl, (b) C 6~10 Aryl, (c) Hydrogen, and (d) C 1~6 Alk-C 6~10 Aryl selected from the group consisting of); (18)-(CH2) q CONR B' R C' (wherein q is an integer from 0 to 4, and R B' and R C' are independently selected from the group consisting of (a) Hydrogen, (b) C 1~6 Alkyl, (c) C 6~10 Aryl, and (d) C 1~6 Alk-C 6~10 Aryl); (19)-(CH2) q SO2R D' (wherein q is an integer from 0 to 4, and R D' is (a) C 6~10 Alkyl, (b) C 6~10 Aryl, and (c) C 1~6 Alk-C 6~10 Aryl selected from the group consisting of); (20)-(CH2) q SO2NR E' R F' (wherein q is an integer from 0 to 4, and R E' and R F' are each independently (a) Hydrogen, (b) C 6~10 Alkyl, (c) C6~10 Aryl, and (d)C 1~6 Alk-C 6~10 Selected from the group consisting of aryl); (21) Thiol; (22)C 6~10 Aryloxy; (23)C 3~8 Cycloalkoxy; (24)C 6~10 Aryl-C 1~6 Alkoxy; (25)C 1~6 Alk-C 1~12 Heterocyclyl (e.g., C 1~6 Alk-C 1~12 Heteroaryl); (26) Oxo; (27)C 2~20 Alkenyl; and (28)C 2~20 May be substituted with alkynyl. In some embodiments, these groups may each be further substituted as described herein. For example, an alkylene group of C1-aryl or C1-alkheterocyclyl can be further substituted with an oxo group to obtain the corresponding aryl oil and (heterocyclyl) oil substituents.

[0069] As used herein, the term "diastereomer" means stereoisomers that are not mirror images of each other and cannot be superimposed on each other.

[0070] As used herein, the term "enantiomer" means each individual optically active form of the compounds of the present invention having an optical purity or enantiomeric excess of at least 80% (i.e., at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least 90% and more preferably at least 98% (determined by standard methods in the art).

[0071] As used herein, the term "halogen" represents a halogen selected from bromine, chlorine, iodine, or fluorine.

[0072] As used herein, the term "heteroalkyl" refers to an alkyl group as defined herein in which one or two of the constituent carbon atoms are each replaced by nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkyl group can be further substituted with 1, 2, 3, or 4 substituents as described herein for alkyl groups. As used herein, the terms "heteroalkenyl" and "heteroalkynyl" refer to alkenyl and alkynyl groups as defined herein in which one or two of the constituent carbon atoms are each replaced by nitrogen, oxygen, or sulfur, respectively. In some embodiments, the heteroalkenyl and heteroalkynyl groups can be further substituted with 1, 2, 3, or 4 substituents as described herein for alkyl groups.

[0073] As used herein, the term "heteroaryl" represents a subset of heterocyclyl as defined herein that is aromatic, i.e., they contain 4n+2 pi electrons within a monocyclic or polycyclic system. Exemplary unsubstituted heteroaryl groups are those having 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. In some embodiments, heteroaryl is substituted with 1, 2, 3, or 4 substituents as defined for heterocyclyl groups.

[0074] The term "heteroarylalkyl" refers to a heteroaryl group as defined herein attached to a parent molecular group via an alkylene group as defined herein. Exemplary unsubstituted heteroarylalkyl groups have 2 to 32 (e.g., 2 to 22, 2 to 18, 2 to 17, 2 to 16, 3 to 15, 2 to 14, 2 to 13, or 2 to 12) carbons, e.g., C 1~6 alk-C 1~12 heteroaryl, C 1~10 alk-C 1~12 heteroaryl, or C 1~20 alk-C 1~12Those of (heteroaryl). In some embodiments, alkylene and heteroaryl can each be further substituted with 1, 2, 3, or 4 substituents as defined herein for the corresponding groups. A heteroarylalkyl group is a subset of a heterocyclylalkyl group.

[0075] As used herein, the term "heterocyclyl" refers to a 5-, 6- or 7-membered ring containing 1, 2, 3 or 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur, unless otherwise specified. The 5-membered ring has 0 to 2 double bonds, and the 6- and 7-membered rings have 0 to 3 double bonds. Exemplary unsubstituted heterocyclyl groups have 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. The term "heterocyclyl" also refers to a heterocyclic compound having a bridged polycyclic structure in which one or more carbons and / or heteroatoms bridge two non-adjacent members of a monocyclic ring, such as a quinuclidinyl group. The term "heterocyclyl" includes bicyclic, tricyclic and tetracyclic groups in which any of the above heterocycles is fused to 1, 2 or 3 carbocyclic rings, such as aryl rings, cyclohexane rings, cyclohexene rings, cyclopentane rings, cyclopentene rings, or another monocyclic heterocycle, such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl, etc. Examples of fused heterocyclyls include tropane and 1,2,3,5,8,8a-hexahydroindolizine. Heterocycles include dihydro and tetrahydro types in which one or more double bonds are reduced and replaced by hydrogen, including pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl, quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl, triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl), purinyl, thiadiazolyl (e.g., 1,2,3-thiadiazolyl), tetrahydrofuranyl, dihydrofuranyl,Tetrahydrothienyl, dihydrothienyl, dihydroindolyl, dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl, isobenzofuranyl, benzothienyl, and the like. Further exemplary heterocyclyls include 2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1H-imidazolyl; 2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g., 2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl); 2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g., 2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl); 2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g., 2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl); 4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino-5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g., 1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl); 2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl); 1,6-dihydro-6-oxopyriminyl; 1,6-dihydro-4-oxopyriminyl (e.g., 2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl); 1,2,3,4-tetrahydro-2,4-dioxopyriminyl (e.g., 1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl); 1,6-dihydro-6-oxo-pyridazinyl (e.g., 1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl (e.g., 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl);2,3-dihydro-2-oxo-1H-indolyl (e.g., 3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and 2,3-dihydro-2-oxo-3,3'-spiropropane-1H-indol-1-yl); 1,3-dihydro-1-oxo-2H-iso-indolyl; 1,3-dihydro-1,3-dioxo-2H-iso-indolyl; 1H-benzopyrazolyl (e.g., 1-(ethoxycarbonyl)-1H-benzopyrazolyl); 2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g., 3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl); 2,3-dihydro-2-oxo-benzoxazolyl (e.g., 5-chloro-2,3-dihydro-2-oxo-benzoxazolyl); 2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl; 1,4-benzodioxanyl; 1,3-benzodioxanyl; 2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl; 3,4-dihydro-4-oxo-3H-quinazolinyl (e.g., 2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl); 1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g., 1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl); 1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g., 1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl); 1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g., 1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl); 2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphtha[1,8-c,d]isothiazolyl;and 1,8-naphthalenedicarboxamide. Further heterocycles include 3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl and 2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanyl), tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl, thiepanyl, azocanyl, oxecanyl, and thiacanyl. The heterocyclic group also has the formula;

[0076] [Chemical formula]

[0077] (wherein E' is selected from the group consisting of -N- and -CH-; F' is selected from the group consisting of -N=CH-, -NH-CH2-, -NH-C(O)-, -NH-, -CH=N-, -CH2-NH-, -C(O)-NH-, -CH=CH-, -CH2-, -CH2CH2-, -CH2O-, -OCH2-, -O-, and -S-; G' is selected from the group consisting of -CH- and -N-) and contains a group of. Any of the heterosilyl groups described herein can be (1) C 1~7 acyl (e.g., carboxaldehyde); (2) C 1~20 alkyl (e.g., C 1~6 alkyl, C 1~6 alkoxy-C 1~6 alkyl, C 1~6 alkylsulfinyl-C 1~6 alkyl, amino-C 1~6 alkyl, azido-C 1~6 alkyl, (carboxaldehyde)-C 1~6 alkyl, halo-C 1~6 alkyl (e.g., perfluoroalkyl), hydroxy-C 1~6 alkyl, nitro-C 1~6 alkyl, or C 1~6 thioalkoxy-C 1~6 alkyl); (3) C 1~20 alkoxy (e.g., C 1~6 alkoxy such as perfluoroalkoxy); (4) C1~6 Alkylsulfinyl; (5) C 6~10 Aryl; (6) Amino; (7) C 1~6 Alk-C 6~10 Aryl; (8) Azido; (9) C 3~8 Cycloalkyl; (10) C 1~6 Alk-C 3~8 Cycloalkyl; (11) Halo; (12) C 1~12 Heterocyclyl (e.g., C 2~12 Heteroaryl); (13) (C 1~12 Heterocyclyl)oxy; (14) Hydroxy; (15) Nitro; (16) C 1~20 Thioalkoxy (e.g., C 1~6 Thioalkoxy); (17)-(CH2) q CO2R A' (wherein q is an integer from 0 to 4, and R A' is selected from the group consisting of (a) C 1~6 alkyl, (b) C 6~10 aryl, (c) hydrogen, and (d) C 1~6 Alk-C 6~10 aryl); (18)-(CH2) q CONR B' R C' (wherein q is an integer from 0 to 4, and R B' and R C' are independently selected from the group consisting of (a) hydrogen, (b) C 1~6 alkyl, (c) C 6~10 aryl, and (d) C 1~6 Alk-C 6~10 aryl); (19)-(CH2) q SO2R D' (wherein q is an integer from 0 to 4, and R D' is selected from the group consisting of (a) C 1~6 alkyl, (b) C 6~10 aryl, and (c) C 1~6 Alk-C 6~10 aryl); (20)-(CH2) q SO2NR E' R F' (wherein q is an integer from 0 to 4, and R E' and R F'are each independently, (a) hydrogen, (b) C 1~6 alkyl, (c) C 6~10 aryl, and (d) C 1~6 alk-C 6~10 aryl selected from the group consisting of); (21) thiol; (22) C 6~10 aryloxy; (23) C 3~8 cycloalkoxy; (24) arylalkoxy; (25) C 1~6 alk-C 1~12 heterocyclyl (e.g., C 1~6 alk-C 1~12 heteroaryl); (26) oxo; (27) (C 1~12 heterocyclyl)imino; (28) C 2~20 alkenyl; and (29) C 2~20 alkynyl, and may be substituted with 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of. In some embodiments, these groups may each be further substituted as described herein. For example, an alkylene group of C1-aryl or C1-alkheterocyclyl can be further substituted with an oxo group to obtain the corresponding aryl oil and (heterocyclyl) oil substituents.

[0078] As used herein, the term "hydrocarbon" represents a group consisting of only carbon and hydrogen atoms.

[0079] As used herein, the term "hydroxyl" represents an -OH group. In some embodiments, the hydroxyl group can be substituted with 1, 2, 3 or 4 substituents (e.g., O-protecting groups) defined herein for alkyl.

[0080] As used herein, the term "isomer" means any tautomer, stereoisomer, enantiomer, or diastereomer of any of the compounds of the present invention. The compounds of the present invention have one or more chiral centers and / or double bonds and thus may exist as stereoisomers such as double bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). According to the present invention, the chemical structures described herein, and thus the compounds of the present invention, include all corresponding stereoisomers, i.e., in stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure), as well as mixtures of enantiomers and stereoisomers, such as racemates. Mixtures of enantiomers and stereoisomers of the compounds of the present invention can typically be resolved into their component enantiomers or stereoisomers by known methods such as chiral phase gas chromatography, chiral phase high performance liquid chromatography, crystallization of the compound as a chiral salt complex, or crystallization of the compound in a chiral solvent. Enantiomers and stereoisomers can also be obtained from stereoisomerically or enantiomerically pure intermediates, reagents, and catalysts by known asymmetric synthesis methods.

[0081] As used herein, the term "N-protected amino" refers to an amino group as defined herein bonded to one or two N-protecting groups as defined herein.

[0082] As used herein, the term "N-protecting group" refers to a group intended to protect an amino group against unwanted reactions during synthetic procedures. Commonly used N-protecting groups are disclosed in Greene, "Protective Groups in Organic Synthesis", 3rd Edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference.Examples of N-protecting groups include acyl, aryloyl, or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliary groups such as protected or unprotected D, L, or D,L-amino acids such as alanine, leucine, phenylalanine; sulfonyl-containing groups such as benzenesulfonyl, p-toluenesulfonyl; carbamate-forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl; aralkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl; and silyl groups such as trimethylsilyl. Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanine, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

[0083] As used herein, the term "O-protecting group" refers to a group intended to protect an oxygen-containing (e.g., phenol, hydroxyl, or carbonyl) group against unwanted reactions during a synthetic procedure. Commonly used O-protecting groups are disclosed in Greene, "Protective Groups in Organic Synthesis", 3rd Edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference. Exemplary O-protecting groups include acyl, aryloyl, or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, t-butyldimethylsilyl, tri-iso-propylsilyloxymethyl, 4,4'-dimethoxytrityl, isobutyryl, phenoxyacetyl, 4-isopropylphenoxyacetyl, dimethylformamidine, and 4-nitrobenzoyl; alkylcarbonyl groups such as acyl, acetyl, propionyl, pivaloyl; optionally substituted arylcarbonyl groups such as benzoyl; silyl groups such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), triisopropylsilyl (TIPS); groups that form an ether with hydroxyl such as methyl, methoxymethyl, tetrahydropyranyl, benzyl, p-methoxybenzyl, trityl; alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-isopropoxycarbonyl, n-butyloxycarbonyl, isobutyloxycarbonyl, sec-butyloxycarbonyl, t-butyloxycarbonyl, 2-ethylhexyloxycarbonyl, cyclohexyloxycarbonyl, methyloxycarbonyl;Alkoxyalkoxycarbonyl groups such as methoxymethoxycarbonyl, ethoxymethoxycarbonyl, 2-methoxyethoxycarbonyl, 2-ethoxyethoxycarbonyl, 2-butoxyethoxycarbonyl, 2-methoxyethoxymethoxycarbonyl, allyloxycarbonyl, propargyloxycarbonyl, 2-butenoxycarbonyl, 3-methyl-2-butenoxycarbonyl; Haloalkoxycarbonyl such as 2-chloroethoxycarbonyl, 2-chloroethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl; Optionally substituted arylalkoxycarbonyl groups such as benzyloxycarbonyl, p-methylbenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2,4-dinitrobenzyloxycarbonyl, 3,5-dimethylbenzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, fluorenylmethyloxycarbonyl; And optionally substituted aryloxycarbonyl groups such as phenoxycarbonyl, p-nitrophenoxycarbonyl, o-nitrophenoxycarbonyl, 2,4-dinitrophenoxycarbonyl, p-methyl-phenoxycarbonyl, m-methylphenoxycarbonyl, o-bromophenoxycarbonyl, 3,5-dimethylphenoxycarbonyl, p-chlorophenoxycarbonyl, 2-chloro-4-nitrophenoxy-carbonyl; Substituted alkyl, aryl, and aralkyl ethers (e.g., trityl; methylthiomethyl; methoxymethyl; benzyloxymethyl; siloxymethyl; 2,2,2-trichloroethoxymethyl; tetrahydropyranyl; tetrahydrofuranyl; ethoxyethyl; 1-[2-(trimethylsilyl)ethoxy]ethyl; 2-trimethylsilylethyl; t-butyl ether; p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl, and nitrobenzyl); Silyl ethers (e.g., trimethylsilyl; triethylsilyl; triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl; t-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; and diphenylmethylsilyl);Carbonates (e.g., methyl, methoxymethyl, 9-fluorenylmethyl; ethyl; 2,2,2-trichloroethyl; 2-(trimethylsilyl)ethyl; vinyl, allyl, nitrophenyl; benzyl; methoxybenzyl; 3,4-dimethoxybenzyl; and nitrobenzyl); carbonyl protecting groups (e.g., acetal and ketal groups such as dimethyl acetal, 1,3-dioxolane; acylal groups; and dithiane groups such as 1,3-dithiane, 1,3-dithiolane); carboxylic acid protecting groups (e.g., ester groups such as methyl ester, benzyl ester, t-butyl ester, ortho ester); and oxazoline groups.

[0084] As used herein, the term "oxo" represents =O.

[0085] As used herein, the term "polyethylene glycol" represents an alkoxy chain containing one or more monomer units, each monomer unit consisting of -OCH2CH2-. Polyethylene glycol (PEG) may also be referred to as polyethylene oxide (PEO) or polyoxyethylene (POE), and these terms may be considered interchangeable for the purposes of the present invention. For example, polyethylene glycol has the structural formula -(CH2) s2 (OCH2CH2) s1 (CH2) s3 O- (wherein s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), and s2 and s3 are each independently integers from 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10)). Polyethylene glycol may also be -NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), and s2 and s3 are each independently integers from 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R N1 each independently is hydrogen or optionally substituted C 1~6It may be considered to contain amino - polyethylene glycol which is alkyl).

[0086] As used herein, the term "stereoisomer" refers to all possible different isomers and conformational forms that a compound (e.g., a compound of any formula described herein) can have, in particular, all possible stereochemical and conformational isomeric forms of the basic molecular structure, all diastereomers, enantiomers and / or conformers. Some compounds of the present invention may exist in different tautomeric forms, and all of the latter are included within the scope of the present invention.

[0087] As used herein, the term "sulfonyl" represents a -S(O2)- group.

[0088] As used herein, the term "thiol" represents a -SH group.

[0089] Definitions As used herein, the term "administered in combination" or "combination administration" means that two or more agents are administered to a subject simultaneously or within an interval such that there may be an overlap in the effects of each agent on the patient. In some embodiments, they are within 90 days of each other (e.g., within 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 day), within 28 days (e.g., within 14, 7, 6, 5, 4, 3, 2, or 1 day), within 24 hours (e.g., within 12, 6, 5, 4, 3, 2, or 1 hour), or within about 60, 30, 15, 10, 5, or 1 minute. In some embodiments, the administration of the agents is done at intervals close enough so that a combined (e.g., synergistic) effect is achieved.

[0090] As used herein, "antibody" refers to a polypeptide whose amino acid sequence, including immunoglobulins and fragments thereof, specifically binds to a designated antigen or a fragment thereof. Antibodies according to the present invention may be of any class (e.g., IgA, IgD, IgE, IgG, or IgM) or subtype (e.g., IgA1, IgA2, IgG1, IgG2, IgG3, or IgG4). One of ordinary skill in the art will understand that characteristic sequences or portions of an antibody may include amino acids found in one or more regions of the antibody (e.g., variable regions, hypervariable regions, constant regions, heavy chains, light chains, and combinations thereof). Further, one of ordinary skill in the art will understand that characteristic sequences or portions of an antibody may include one or more polypeptide chains and may include sequence elements found in the same polypeptide chain or different polypeptide chains.

[0091] As used herein, "antigen-binding fragment" refers to a portion of an antibody that retains the binding characteristics of the parent antibody.

[0092] The terms "bifunctional chelate" or "bifunctional conjugate" as used interchangeably herein refer to a compound containing a chelate group or its metal complex, a linker group, and a therapeutic moiety, targeting moiety, or crosslinking group.

[0093] The term "cancer" refers to any cancer caused by the proliferation of malignant neoplastic cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias, and lymphomas. "Solid tumor cancer" is a cancer that includes an abnormal mass of tissue, e.g., sarcoma, carcinoma, and lymphoma. "Blood cancer" or "liquid cancer," used interchangeably herein, is a cancer that exists in body fluids, e.g., lymphoma and leukemia.

[0094] The term "chelate" as used herein refers to an organic compound or a portion thereof that can bind to a central metal or radioactive metal atom at two or more points.

[0095] As used herein, the term "conjugate" refers to a molecule containing a chelating group or its metal complex, a linker group, and optionally a therapeutic moiety, a targeting moiety, or a crosslinking group.

[0096] As used herein, the term "compound" is meant to include all stereoisomers, geometric isomers, and tautomers of the described structures.

[0097] The compounds described herein may be asymmetric (e.g., having one or more stereocenters). Unless otherwise indicated, all stereoisomers such as enantiomers and diastereomers are intended. The compounds of the disclosure containing asymmetrically substituted carbon atoms can be isolated in optically active form or in racemic form. Methods for preparing optically active forms from optically active starting materials, such as by resolution of racemic mixtures or stereoselective synthesis, are known in the art. Many geometric isomers, such as olefins, C=N double bonds, etc., may also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. The cis and trans geometric isomers of the compounds of the disclosure are described and can be isolated as mixtures of isomers or in separated isomeric forms.

[0098] The compounds of the disclosure also include tautomeric forms. Tautomeric forms result from the exchange of a single bond with an adjacent double bond and the simultaneous movement of a proton. Tautomeric forms include proton tautomers that are isomeric protonation states having the same empirical formula and total charge. Examples of proton tautomers include keto-enol pairs, amide-imino acid pairs, lactam-lactim pairs, amide-imino acid pairs, enamine-imine pairs, and cyclic forms in which a proton can occupy two or more positions of a heterocyclic system, such as 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms may be in equilibrium or can be stereochemically fixed in one form by appropriate substitution.

[0099] At various places in this specification, substituents of the compounds of the present disclosure are disclosed in groups or ranges. The present disclosure is specifically intended to include each and every individual sub-combination of the members of such groups and ranges. For example, the term "C 1~6 alkyl" is specifically intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl. The phrase form "optionally substituted X" as used herein (e.g., optionally substituted alkyl) is intended to be equivalent to "X (wherein X is optionally substituted)" (e.g., "alkyl (wherein the alkyl is optionally substituted)"). It is not intended to mean that the feature "X" (e.g., alkyl) itself is optional.

[0100] As used herein, "detecting agent" refers to a molecule or atom useful in diagnosing a disease by searching for cells containing an antigen. Various methods of labeling polypeptides with detecting agents are known in the art. Examples of detecting agents include, but are not limited to, radioisotopes and radionuclides, dyes (such as those including biotin-streptavidin complexes), contrast agents, luminescent agents (e.g., FITC, rhodamine, lanthanide phosphors, cyanines, and near-IR dyes), and magnetic agents such as gadolinium chelates.

[0101] As used herein, the term "radionuclide" refers to an atom that can undergo radioactive decay (e.g., 3 H, 14 C, 15 N, 18 F, 35 S, 47 Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 75 Br, 76 Br, 77 Br, 89 Zr, 86 Y, 87 Y, 90 Y,97 Ru, 99 Tc, 99m Tc, 105 Rh, 109 Pd, 111 In, 123 I, 124 I, 125 I, 131 I, 149 Pm, 149 Tb, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 198 Au, 199 Au, 203 Pb, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227 Th, 229 Th, 66 Ga, 67 Ga, 68 Ga, 82 Rb, 117m Sn, 201 Tl). The term radioactive nuclide, radioisotope, or radioactive isotope can also be used to describe a radionuclide. A radionuclide can be used as a detecting agent as described above. In some embodiments, the radionuclide may be an alpha-emitting radionuclide.

[0102] As used herein, the term "effective amount" of a drug (e.g., any of the conjugates) is an amount sufficient to obtain a beneficial or desired result such as a clinical outcome, and thus the "effective amount" depends on the context in which it is applied.

[0103] As used herein, the term "immunoconjugate" refers to a conjugate comprising a targeting moiety, such as an antibody, nanobody, affibody, or consensus sequence derived from a type III fibronectin domain. In some embodiments, the immunoconjugate comprises, on average, at least 0.10 conjugates per targeting moiety (e.g., on average, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5 conjugates per targeting moiety).

[0104] As used herein, the term "radioactive conjugate" refers to any conjugate comprising a radioisotope or radionuclide, such as any of the radioisotopes or radionuclides described herein. In some embodiments, the radioisotope or radionuclide is a metal chelate.

[0105] As used herein, the term "radioactive immunoconjugate" refers to any immunoconjugate comprising a radioisotope or radionuclide, such as any of the radioisotopes or radionuclides described herein. In some embodiments, the radioisotope or radionuclide is a metal chelate.

[0106] As used herein, the term "radioimmunotherapy" refers to a method of effecting a therapeutic effect using a radioactive immunoconjugate. In some embodiments, radioimmunotherapy may include administration of a radioactive immunoconjugate to a subject in need thereof, wherein administration of the radioactive immunoconjugate effects a therapeutic effect in the subject. In some embodiments, radioimmunotherapy may include administration of a radioactive immunoconjugate to cells, wherein administration of the radioactive immunoconjugate kills the cells. When radioimmunotherapy includes selective killing of cells, in some embodiments, the cells are cancer cells in a subject having cancer.

[0107] As used herein, the term "pharmaceutical composition" refers to a composition containing a compound described herein formulated with a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is manufactured or sold under the approval of a government regulatory authority as part of a treatment regimen for the treatment of diseases in mammals. The pharmaceutical composition can be formulated, for example, for oral administration in unit dosage form (e.g., tablets, capsules, caplets, gelcaps, or syrups); for topical administration (e.g., as creams, gels, lotions, or ointments); for intravenous administration (e.g., as a sterile solution free of particulate embolisms and in a solvent system suitable for intravenous use); or for any other formulation described herein.

[0108] As used herein, "pharmaceutically acceptable excipient" refers to any component other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving the active compound) that has the property of being non-toxic and non-inflammatory in a patient. Excipients may include, for example, anti-adhesives, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavoring agents, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, radiation protectants, adsorbents, suspending or dispersing agents, sweeteners, or water of hydration. Exemplary excipients include, but are not limited to, ascorbic acid, histidine, phosphate buffer, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propyl paraben, retinol palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0109] As used herein, the term "pharmaceutically acceptable salt" refers to salts of the compounds described herein that are within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, or allergic response, and are known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and Pharmaceutical Salts: Properties, Selection, and Use (P.H. Stahl and C.G. Wermuth (eds.)), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein, or separately by reacting the free base with a suitable organic acid.

[0110] The compounds of the present invention may have ionizable groups so that they can be prepared as pharmaceutically acceptable salts. These salts may be acid addition salts containing inorganic or organic acids, or the salts can be prepared from inorganic or organic bases in the case of the compounds of the present invention in acidic form. Frequently, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases such as hydrochloric acid, sulfuric acid, hydrobromic acid, acetic acid, lactic acid, citric acid, or tartaric acid for forming acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, etc. for forming basic salts are known in the art. Methods for the preparation of appropriate salts are established in the art.

[0111] Representative acid addition salts include, in particular, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as non-toxic ammonium, quaternary ammonium, and, without limitation, amine cations such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0112] As used herein, the term "therapeutic moiety" refers to any molecule or any part of a molecule that provides a therapeutic benefit. In some embodiments, the therapeutic moiety is a protein or polypeptide, such as an antibody, an antigen-binding fragment thereof. In some embodiments, the therapeutic moiety is a small molecule.

[0113] As used herein, the term "targeting moiety" refers to any molecule or any part of a molecule that binds to a given target. In some embodiments, the targeting moiety is a protein or polypeptide, such as an antibody or an antigen-binding fragment thereof, a nanobody, an affibody, or a consensus sequence derived from a type III fibronectin domain.

[0114] As used herein, the term "crosslinking group" refers to any reactive group capable of linking two or more molecules by a covalent bond. In some embodiments, the crosslinking group is an amino-reactive or thiol-reactive crosslinking group. In some embodiments, the amino-reactive or thiol-reactive crosslinking group includes activated esters or imidates such as hydroxysuccinimide ester, 2,3,5,6-tetrafluorophenol ester, 4-nitrophenol ester, anhydrides, thiols, disulfides, maleimides, azides, alkynes, strained alkynes, strained alkenes, halogens, sulfonates, haloacetyls, amines, hydrazides, diazirines, phosphines, tetrazines, isothiocyanates. In some embodiments, the crosslinking group may be glycine-glycine-glycine and / or leucine-proline-(any amino acid)-threonine-glycine, which is a recognition sequence for coupling a targeting agent to a linker using a sortase-mediated coupling reaction. Those skilled in the art will understand that the use of crosslinking groups in the practice of the present invention is not limited to the specific constructs disclosed herein, but rather may include other known crosslinking groups.

[0115] As used herein, the term "polypeptide" refers to a filamentous chain of at least two amino acids joined to each other by peptide bonds. In some embodiments, the polypeptide may each contain at least 3-5 amino acids that are each joined to other amino acids by at least one peptide bond. Those skilled in the art will understand that a polypeptide may include one or more "non-natural" amino acids or other entities that can be incorporated into the polypeptide chain nonetheless. In some embodiments, the polypeptide may be glycosylated, for example, the polypeptide may contain one or more covalently linked sugar moieties. In some embodiments, a single "polypeptide" (e.g., an antibody polypeptide) may include two or more individual polypeptide chains, and in some cases, may be linked to each other by, for example, one or more disulfide bonds or other means.

[0116] "Subject" means a human or non-human animal (e.g., a mammal).

[0117] "Substantial identity" or "substantially identical" each mean a polypeptide sequence that has the same polypeptide sequence as a reference sequence or that has a particular percentage of amino acid residues that are the same at corresponding positions within the reference sequence when the two sequences are optimally aligned. For example, an amino acid sequence that is "substantially identical" to a reference sequence has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the reference amino acid sequence. For polypeptides, the length of the comparison sequence is generally at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 100, 150, 200, 250, 300, or 350 contiguous amino acids (e.g., the full-length sequence). Sequence identity can be measured using sequence analysis software with default settings (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Such software can align similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.

[0118] As used herein, and as is well understood in the art, "treating" a condition or "treatment" of a condition (e.g., a condition described herein such as cancer) is a method for obtaining a beneficial or desired result, such as a clinical outcome. Beneficial or desired results include, but are not limited to, reduction or amelioration of one or more symptoms or conditions, whether detectable or undetectable; diminution of the extent of a disease, disorder, or condition; stabilization (i.e., non-worsening) of a disease, disorder, or condition; prevention of the spread of a disease, disorder, or condition; delay or slowing of the progression of a disease, disorder, or condition; improvement or alleviation of a disease, disorder, or condition; and remission (partial or complete). "Alleviation" of a disease, disorder, or condition means that the extent and / or undesirable clinical findings of the disease, disorder, or condition are reduced, and / or the time course of progression is slowed or lengthened, as compared to the absence of treatment.

Brief Description of the Drawings

[0119]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0120] Radiolabeled targeting moieties (also known as radioimmunoconjugates) are designed to target proteins or receptors that are upregulated in disease states to deliver a radioactive payload to kill the target cells (radioimmunotherapy). The method of delivering such a payload by radioactive decay can result in the emission of alpha, beta, or gamma particles or Auger electrons that can cause direct effects on DNA (such as single-stranded or double-stranded DNA breaks) or indirect effects such as the bystander effect or the crossfire effect.

[0121] Radioimmunoconjugates typically contain a biological targeting moiety, a radioisotope, and a conjugate that links the two. The conjugate is formed when a bifunctional chelate is added to the biological targeting molecule such that structural changes to the compound are minimized while maintaining target affinity. Once radiolabeled, the final radioimmunoconjugate is formed.

[0122] The bifunctional chelate structurally contains a chelate, a linker, and a crosslinking group (Figure 1). When developing a new bifunctional chelate, much effort focuses on the chelating portion of the molecule. Some examples of bifunctional chelates with various cyclic and acyclic structures when conjugated to a targeting moiety are described [Bioconjugate Chem. 2000, 11, pp. 510 - 519, Bioconjugate Chem. 2012, 23, pp. 1029 - 1039, Mol Imaging Biol (2011) 13:215 - 221, Bioconjugate Chem. 2002, 13, pp. 110 - 115].

[0123] One important factor in developing a safe and effective radioimmunoconjugate is to maximize efficacy while minimizing off - target toxicity in normal tissues. This view is one of the core tenets in developing new drugs, but its application to radioimmunotherapy presents new challenges. Radioimmunoconjugates do not need to block receptors as required for therapeutic antibodies or release cytotoxic payloads intracellularly as required for antibody - drug conjugates to have therapeutic efficacy. However, the release of toxic particles occurs as a result of primary (radioactive) decay and can happen randomly anywhere in the body after administration. Once the particle is released, damage to surrounding cells within the range of the radiation occurs, leading to the potential for off - target toxicity. Therefore, limiting the exposure of normal tissues to these emissions is the key to developing new drugs [Bioconjugate Chem. 2006, 17, pp. 1551 - 1560, Bioconjugate Chem. 2003, 14, pp. 927 - 933].

[0124] One potential way to reduce off-target exposure is to more efficiently remove radioactivity from the body. The most obvious mechanism is to increase the rate of clearance of the biological targeting agent. This approach also likely requires identifying ways to shorten the half-life of the biological targeting agent, a topic that has been less well described for biological targeting agents. Regardless of the mechanism, increasing drug clearance also negatively impacts pharmacokinetics / efficacy in that more rapid removal of the drug from the body decreases the effective concentration at the site of action, which in turn requires a higher total dose and does not achieve the desirable result of decreasing the total radioactivity dose to normal tissue.

[0125] Other efforts have focused on accelerating the metabolism of portions of the molecule containing the radioactive moiety. For this purpose, some efforts have been made to increase the rate of cleavage of radioactivity from the biological targeting agent using what has been referred to as a "cleavable linker." However, cleavable linkers are taken in different meanings in that they relate to radioactive immunoconjugates. Cornelissen et al. described a cleavable linker as a bifunctional conjugate that binds to the biological targeting agent via a reducing cysteine, while others have described the use of an enzyme cleavage system that requires co-administration of the radioactive immunoconjugate and a cleaving agent / enzyme for release [Mol Cancer Ther; 12(11) November 2013, Methods in Molecular Biology, 2009, 539, 191-211, Bioconjugate chemistry, Volume 14, Issue 5, 927-33 (2003)]. These methods are not practical from a drug development perspective (enzyme cleavage system) as in the case of cysteine conjugation, they alter the nature of the biological targeting moiety or in the case of the cited references, they require the administration of two agents.

[0126] The focus of the embodiments described herein is centered around more efficiently removing radioactivity from the body after the catabolism and / or metabolism of radioimmunoconjugates by modifying the linker region of the bifunctional chelate.

[0127] In particular, since there is little information available describing the in vivo effects of the linker, as it applies to radioimmunoconjugates, this is a novel approach. One possible reason is that after the catabolism / metabolism of radioimmunoconjugates, one of ordinary skill in the art would expect the radiolabeled conjugate to undergo rapid whole body clearance. When the bifunctional chelate was administered alone, speculation was advanced experimentally; it cleared the bloodstream faster than radioimmunoconjugates with the same bifunctional chelate. Based on these data, one of ordinary skill in the art would expect metabolites containing the bifunctional chelate to also be rapidly removed after the catabolism / metabolism of radioimmunoconjugates.

[0128] However, rapid clearance of metabolites containing the radiolabeled conjugate does not necessarily occur in vivo. Based on the results described below, the linker region of the bifunctional chelate does not affect the overall in vitro properties or in vivo pharmacokinetics and pharmacodynamics of radioimmunoconjugates, but can directly affect the removal of radioactivity from the body after the catabolism of the radiolabeled conjugate. Data are presented below demonstrating that certain commercially available bifunctional chelates result in slower rates and lower extents of removal of total radioactivity from the body when compared to the embodiments described herein.

[0129] The excretion profiles of the embodiments described in the examples show unexpected findings. As previously reported by Quadri and Vriesendorp [Q. J. Nucl. Med. 1998, 42, 250-261], simple modifications to the linker region of bifunctional chelates did not affect the urinary excretion of radioactivity, despite their hydrophobicity. The results provided below clearly show that both hydrophobic and hydrophilic linkers can affect the excretion pattern. Furthermore, the following examples show that hepatobiliary clearance also plays a role in excretion.

[0130] Thus, according to the embodiments described herein, when a bifunctional chelate is bound to a biological targeting moiety, it has been identified that it can achieve a reduction in whole-body radioactivity by increasing the degree of excretion of catabolic / metabolic products while maintaining the pharmacokinetics of intact molecules compared to similar bifunctional chelates known in the art. This reduction in whole-body radioactivity was determined to be due to the clearance of catabolic / metabolic by-products and did not affect other in vitro and in vivo properties such as specificity (binding in vitro), cell retention, and tumor uptake in vivo. Taken as a whole, these embodiments achieve the desired properties of radioactive immunoconjugates by reducing the in-body burden of radioactivity while maintaining on-target activity.

[0131] Therapeutic moiety and targeting moiety The therapeutic moiety includes any molecule or any part of a molecule that provides a therapeutic benefit. In some embodiments, the therapeutic moiety is a protein or polypeptide, such as an antibody, an antigen-binding fragment thereof. In some embodiments, the therapeutic moiety is a small molecule. The targeting moiety includes any molecule or any part of a molecule that binds to a given target. In some embodiments, the targeting moiety is a protein or polypeptide such as an antibody or an antigen-binding fragment thereof, a nanobody, an affibody, and a consensus sequence derived from a type III fibronectin domain (e.g., Centyrin or Adnectin).

[0132] Polypeptide The polypeptide includes, for example, any of various blood agents (including, for example, erythropoietin, blood coagulation factors, etc.), interferon, colony stimulating factor, antibody, enzyme, and hormone. The identity of a particular polypeptide is not intended to limit the present disclosure, and any polypeptide of interest may be the polypeptide in the method of the present invention.

[0133] The reference polypeptides described herein may include a target binding domain that binds to a target of interest (e.g., binds to an antigen). For example, a polypeptide such as an antibody can bind to a transmembrane polypeptide (e.g., a receptor) or a ligand (e.g., a growth factor). Exemplary molecular targets (e.g., antigens) for the polypeptides (e.g., antibodies) described herein include CD proteins such as CD2, CD3, CD4, CD8, CD11, CD19, CD20, CD22, CD25, CD33, CD34, CD40, CD52; members of the ErbB receptor such as the EGF receptor (EGFR, HER1, ErbB1), HER2 (ErbB2), HER3 (ErbB3) or HER4 (ErbB4) receptor; macrophage receptors such as CRIg; tumor necrosis factors such as TNFα or TRAIL / Apo-2; cell adhesion molecules such as LFA-1, Mac1, p150,95, VLA-4, ICAM-1, VCAM and ανβ3 integrin (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibody) including either the α or β subunit; growth factors and receptors such as EGF, EGFR (e.g., FGFR3) and VEGF; IgE; cytokines such as IL1; cytokine receptors such as the IL2 receptor; blood group antigens; the flk2 / flt3 receptor; the obesity (OB) receptor; the mpl receptor; CTLA-4; protein C; neutrophilin; ephrin and receptor; netrin and receptor; slit and receptor; chemokines and chemokine receptors such as CCL5, CCR4, CCR5; amyloid beta; complement factors such as complement factor D; lipoproteins such as oxidized LDL (oxLDL); lymphotoxins such as lymphotoxin alpha (LTa). Other molecular targets include Tweak, B7RP-1, proprotein convertase subtilisin / kexin type 9 (PCSK9), sclerostin, c-kit, Tie-2, c-fms, and anti-M1.

[0134] antibody An IgG antibody consists of two identical light chain polypeptides and two identical heavy chain polypeptides linked together by disulfide bonds. The first domain located at the amino terminus of each chain is variable in amino acid sequence and provides the antibody binding specificity found in each individual antibody. These are known as the variable heavy chain (VH) and variable light chain (VL) regions. The other domains of each chain are relatively invariant in amino acid sequence and are known as the constant heavy chain (CH) and constant light chain (CL) regions. For IgG antibodies, the light chain contains one variable region (VL) and one constant region (CL). The IgG heavy chain contains a variable region (VH), a first constant region (CH1), a hinge region, a second constant region (CH2), and a third constant region (CH3). In IgE and IgM antibodies, the heavy chain contains an additional constant region (CH4).

[0135] The antibodies described herein may include, for example, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, single-chain Fv (scFv), disulfide-linked Fv (sdFv), and anti-idiotype (anti-Id) antibodies, as well as antigen-binding fragments of any of the foregoing. The antibodies may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0136] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, scFv fragments, dAb fragments (Ward et al. (1989) Nature 341:544-546), and isolated complementarity determining regions (CDRs). These antibody fragments can be obtained using conventional techniques known to those of skill in the art and the fragments can be screened for use in the same manner as intact antibodies.

[0137] The antibodies or fragments described herein can be produced by any method known in the art for the synthesis of antibodies (e.g., Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Brinkman et al., 1995, J. Immunol. Methods 182:41-50; WO92 / 22324; WO98 / 46645). Chimeric antibodies can be produced using, for example, the method described in Morrison, 1985, Science 229:1202, and humanized antibodies can be produced by the method described in U.S. Patent No. 6,180,370.

[0138] Further antibodies described herein are, for example, bispecific and multispecific antibodies such as those described in Segal et al., J. Immunol. Methods 248:1-6 (2001); and Tutt et al., J. Immunol. 147: 60 (1991).

[0139] Insulin-like growth factor 1 (IGF-1R) antibody Insulin-like growth factor 1 receptor is a transmembrane protein found on the surface of human cells that is activated by insulin-like growth factors 1 (IGF-1) and 2 (IGF-2). IGF-1R is involved in several cancers including breast cancer, non-small cell lung cancer, prostate cancer, colon cancer, sarcoma, and adrenocortical carcinoma, and high levels of IGF-1R are expressed on the surface of tumor cells of these cancers.

[0140] In some embodiments, the antibody, or antigen-binding fragment thereof, specifically binds to insulin-like growth factor-1 receptor (IGF-1R).

[0141] Nanobody A nanobody is an antibody fragment consisting of a single monomeric variable antibody domain. A nanobody may also be referred to as a single-domain antibody. Like an antibody, a nanobody selectively binds to a specific antigen. A nanobody may be a heavy-chain variable domain or a light-chain domain. A nanobody may occur naturally or may be a product of biological manipulation. A nanobody can be biologically manipulated by site-directed mutagenesis or mutagenicity screening (e.g., phage display, yeast display, bacterial display, mRNA display, ribosome display).

[0142] Affibody An Affibody is a polypeptide or protein engineered to bind to a specific antigen. Thus, an Affibody may be considered to mimic a particular function of an antibody. An Affibody may be an engineered variant of the B domain in the immunoglobulin-binding region of staphylococcal protein A. An Affibody may be an engineered variant of the Z domain, which is a B domain with lower affinity for the Fab region. An Affibody can be biologically manipulated by site-directed mutagenesis or mutagenicity screening (e.g., phage display, yeast display, bacterial display, mRNA display, ribosome display).

[0143] Affibody molecules that exhibit specific binding to various different proteins (e.g., insulin, fibrinogen, transferrin, tumor necrosis factor-α, IL-8, gp120, CD28, human serum albumin, IgA, IgE, IgM, HER2, and EGFR) have been generated and show affinities (K d ) in the μM to pM range.

[0144] Type III fibronectin domain Type III fibronectin domains are evolutionarily conserved protein domains found in various extracellular proteins. Type III fibronectin domains have been used as a molecular scaffold for producing molecules that can selectively bind to specific antigens. Variants of the Type III fibronectin domain (FN3) engineered for selective binding can also be referred to as monobodies. The FN3 domain can be biologically engineered by site-directed mutagenesis or mutagenicity screening (e.g., CIS display, phage display, yeast display, bacterial display, mRNA display, ribosome display).

[0145] Modified polypeptide The polypeptide of the present invention may have a modified amino acid sequence. The modified polypeptide may be substantially identical to the corresponding reference polypeptide (e.g., the amino acid sequence of the modified polypeptide may have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of the reference polypeptide). In certain embodiments, the modification does not significantly disrupt the desired biological activity. The modification may decrease the biological activity of the original polypeptide (e.g., by at least 5%, 10%, 20%, 25%, 35%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%), have no effect on it, or increase it (e.g., by at least 5%, 10%, 25%, 50%, 100%, 200%, 500%, or 1000%). The modified polypeptide may have or optimize characteristics of the polypeptide such as in vivo stability, bioavailability, toxicity, immunological activity, immunological identity, and conjugation properties.

[0146] Modifications include those resulting from natural processes such as post-translational processing or chemical modification techniques known in the art. Modifications may occur anywhere on the polypeptide, including the polypeptide backbone, amino acid side chains, and amino or carboxy termini. The same type of modification may be present in the same or different degrees at several sites in a given polypeptide, and a polypeptide may contain more than one type of modification. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branches. Cyclic, branched, and branched-cyclic polypeptides may result from natural post-translational processes or may be produced synthetically. Other modifications include pegylation, acetylation, acylation, addition of an acetomidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, carboxyethylation, esterification, covalent attachment to flavin, covalent attachment to a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a drug, covalent attachment of a marker (e.g., a fluorescent or radioactive marker), covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of a covalent cross-link, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, and tRNA-mediated addition of amino acids to proteins such as ubiquitination.

[0147] The modified polypeptide may also contain insertions, deletions, or substitutions of amino acids that are conservative or non-conservative (e.g., D-amino acids, desamino acids) in the polypeptide sequence (e.g., when such changes do not substantially alter the biological activity of the polypeptide). In particular, the addition of one or more cysteine residues to either the amino or carboxy terminus of any of the polypeptides of the present invention can facilitate the conjugation of these polypeptides, for example, by disulfide bonds. For example, the polypeptide can be modified to contain a single cysteine residue at the amino terminus or a single cysteine residue at the carboxy terminus. Amino acid substitutions can be conservative (i.e., when the residue is replaced by another of the same general type or group) or non-conservative (i.e., when the residue is replaced by an amino acid of another type). Furthermore, naturally occurring amino acids can be replaced with non-naturally occurring amino acids (i.e., non-naturally occurring conservative amino acid substitutions or non-naturally occurring non-conservative amino acid substitutions).

[0148] Synthetically produced polypeptides may contain substitutions of amino acids not naturally encoded by DNA (e.g., non-naturally occurring or unnatural amino acids). Examples of non-naturally occurring amino acids include D-amino acids, N-protected amino acids, amino acids in which an acetylaminomethyl group is attached to the sulfur atom of cysteine, pegylated amino acids, omega amino acids of the formula NH2(CH2) n COOH (where n is from 2 to 6), sarcosine, t-butylalanine, t-butylglycine, N-methylisoleucine, and neutral non-polar amino acids such as norleucine. Phenylglycine may be substituted for Trp, Tyr, or Phe; citrulline and methionine sulfoxide are neutral non-polar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline, which retains the conformation that provides the properties.

[0149] Analogs can be generated by substitution mutagenesis, which retains the biological activity of the original polypeptide. Examples of substitutions identified as "conservative substitutions" are shown in Table 1. If such substitutions result in undesirable changes, other types of substitutions named "exemplary substitutions" in Table 1 or further described herein with reference to amino acid classes are introduced and the products are screened.

[0150] [Table 1]

[0151] Substantial modifications of function or immunological identity are achieved by selecting substitutions that are significantly different in (a) the structure of the polypeptide backbone in the region of substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) its effect on the maintenance of the bulk of the side chain.

[0152] Crosslinking group A crosslinking group is a reactive group capable of linking two or more molecules by a covalent bond. Using the crosslinking group, a linker and a chelating moiety can be attached to a therapeutic moiety or a targeting moiety. Also, using the crosslinking group, a linker and a chelating moiety can be attached to a target in vivo. In some embodiments, the crosslinking group is an amino-reactive or thiol-reactive crosslinking group, or sortase-mediated coupling. In some embodiments, the amino-reactive, methionine-reactive or thiol-reactive crosslinking group includes an activated ester such as a hydroxysuccinimide ester, 2,3,5,6-tetrafluorophenol ester, 4-nitrophenol ester or an imidate, an anhydride, a thiol, a disulfide, a maleimide, an azide, an alkyne, a strained alkyne, a strained alkene, a halogen, a sulfonate, a haloacetyl, an amine, a hydrazide, a diazirine, a phosphine, a tetrazine, an isothiocyanate, or an oxaziridine. In some embodiments, the sortase recognition sequence may include a terminal glycine-glycine-glycine (GGG) and / or LPTXG amino acid sequence (wherein X is any amino acid). One skilled in the art can understand that the use of the crosslinking group in the practice of the present invention is not limited to the specific constructs disclosed herein, but rather may include other known crosslinking groups.

[0153] Detection agent The detection agent is a molecule or atom that is conjugated to a polypeptide, such as an antibody or an antigen-binding fragment thereof, and administered to search for cells containing an antigen, and is useful in the diagnosis of diseases, radiation treatment planning, or treatment of diseases. Useful detection agents include, but are not limited to, radioisotopes, dyes (such as those containing a biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules, luminescent agents, and enhancers for magnetic resonance imaging (MRI) (e.g., paramagnetic ions). To load the detection agent on the polypeptide component, it may be necessary to react it with a reagent having a linker that binds to the detection agent or a plurality of detection agents.

[0154] Radioisotopes and radionuclides Radioactive isotopes and radionuclides known in the art for their use as detection agents include, but are not limited to, 3 H, 14 C, 15 N, 18 F, 35 S, 47 Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 75 Br, 76 Br, 77 Br, 89 Zr, 86 Y, 87 Y, 90 Y, 97 Ru, 99 Tc, 99m Tc, 105 Rh, 109 Pd, 111 In, 123 I, 124 I, 125 I, 131 I, 149 Pm, 149 Tb, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 198 Au, 199 Au, 203 Pb, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227 Th, 229 Th, 66 Ga, 67 Ga, 68 Ga, 82 Rb, 117m Sn, 201 Tl.

[0155] Chelating moiety Chelating moieties known in the art for its use as a detecting agent include, but are not limited to, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA ((1R,4R,7R,10R)-α,α',α'',α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid), DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetic acid), DOTA-GA anhydride (2,2',2''-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid)), DOTMP (1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylenephosphonic acid), DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonic acid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2) hexadecan-4,11-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP (1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid)), TETPA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), HEHA (1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid), PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N''''-pentaacetic acid), H4Octapa (N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid), H2Dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane), H6phospa (N,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane), TTHA (triethylenetetramine-N,N,N',N'',N''',N'''-hexaacetic acid), DO2P (tetraazacyclododecanedimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecane triacetic acid), EDTA (ethylenediaminetetraacetic acid), deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), HOPO (octadentate hydroxypyridinone), or porphyrin. The chelating group is a metal such as manganese, iron, and gadolinium, and... 47 Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga, 68 Ga, 82 Rb, 86 Y, 87 Y, 90 Y, 97 Ru, 99m Tc, 105 Rh,109 Pd, 111 In, 117m Sn, 149 Tb, 149 Pm, 153 Sm, 177 Lu, 186 Re, 188 Re, 199 Au, 201 Tl, 203 Pb, 212 Pb, 212 Bi, 213 Bi, 225 Ac, and 227 can be used in metal chelate combinations with isotopes such as Th (e.g., isotopes in the general energy range of 60 - 4,000 keV).

[0156] Linker The linker of the present invention has the formula I: A - L 1 -(L 2 ) n -B Formula I [Wherein, A is a chelating moiety or its metal complex; L 1 is optionally substituted C1 - C6 alkyl, optionally substituted C1 - C6 heteroalkyl, optionally substituted aryl or heteroaryl; B is a therapeutic moiety, a targeting moiety, or a cross - linking group, or a pharmaceutically acceptable salt thereof; n is 1 - 5; L 2 each independently has the structure: (-X 1 -L 3 -Z 1 ) Formula II (Wherein, X 1 is C = O(NR 1 ), C = S(NR 1 ), OC = O(NR 1 ), NR 1 C = O(O), NR 1 C = O(NR 1 ), -CH2PhC = O(NR 1), -CH2Ph(NH)C=S(NR 1 ), O, NR 1 wherein R 1 is H or optionally substituted C1-C6 alkyl or optionally substituted C1-C6 heteroalkyl, optionally substituted aryl or heteroaryl; L 3 is optionally substituted C1-C 50 alkyl or optionally substituted C1-C 50 heteroalkyl or C5-C 20 polyethylene glycol; Z 1 is CH2, C=O, C=S, OC=O, NR 1 C=O, NR 1 wherein R 1 is hydrogen or optionally substituted C1-C6 alkyl, pyrrolidine-2,5-dione) having] may have the structure of.

[0157] The conjugate of the present invention comprises three different modules that together result in an increase in its effectiveness compared to those known in the art.

[0158] 1. Chelating moiety or its metal complex: Module A is included for the incorporation of a detecting agent (e.g., a chelating moiety or its metal complex). The metal complex may contain an imaging radionuclide.

[0159] 2. Linker: The linker of the present invention has the formula I: A-L 1 -(L 2 ) n -B Formula I [wherein A is a chelating moiety or its metal complex; L 1 is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl or heteroaryl; B is a therapeutic moiety, a targeting moiety, or a linking group, or a pharmaceutically acceptable salt thereof; n is from 1 to 5; L 2 each independently has the structure: (-X 1 -L 3 -Z 1 -) Formula II (wherein X 1 is C=O(NR 1 ), C=S(NR 1 ), OC=O(NR 1 ), NR 1 C=O(O), NR 1 C=O(NR 1 ), -CH2PhC=O(NR 1 ), -CH2Ph(NH)C=S(NR 1 ), O, NR 1 ; R 1 is H or optionally substituted C1-C6 alkyl or optionally substituted C1-C6 heteroalkyl, optionally substituted aryl or heteroaryl; L 3 is optionally substituted C1-C 50 alkyl or optionally substituted C1-C 50 heteroalkyl or C5-C 20 polyethylene glycol; Z 1 is CH2, C=O, C=S, OC=O, NR 1 C=O, NR 1 ; R 1 is hydrogen or optionally substituted C1-C6 alkyl, pyrrolidine-2,5-dione) having] may have the structure of.

[0160] 3. Therapeutic moiety, targeting moiety, or linking group: Module B is a therapeutic moiety (e.g., an antibody, antigen-binding fragment), a targeting moiety (e.g., a nanobody, an affibody, a consensus sequence derived from a type III fibronectin domain), or a crosslinking group (e.g., an amino-reactive, thiol-reactive crosslinking group, or sortase-mediated coupling).

[0161] Administration and Dosage The present invention also features a pharmaceutical composition containing a therapeutically effective amount of a compound of the present invention. The composition can be formulated for use in various drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for appropriate formulations. Suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th edition, 1985. For a brief overview of methods for drug delivery, see, for example, Langer (Science 249:1527-1533, 1990).

[0162] The pharmaceutical composition is intended for parenteral, intranasal, topical, oral, or local administration by transdermal means or the like for prophylactic and / or therapeutic treatment. The pharmaceutical composition can be administered parenterally (e.g., by intravenous, intramuscular, or subcutaneous injection), or by oral ingestion, or by topical application or intra-articular injection in the region affected by a vascular or cancerous condition. Further routes of administration include intravascular, intra-arterial, intratumoral, intraperitoneal, intraventricular, intradural, as well as intranasal, ophthalmic, intrascleral, intraorbital, rectal, topical, or aerosol inhalation administration. Sustained release administration by means of depot injection or erodible implants or components, etc., is also particularly included in the present invention. Thus, the present invention provides a composition for parenteral administration comprising the above drug dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, such as water, buffered water, saline, or PBS. The composition may particularly contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as a pH adjuster and buffer, an isotonicity adjuster, a wetting agent, or a surfactant. The present invention also provides a composition for oral delivery which may contain inert components such as a binder or filler for formulation into unit dosage forms such as tablets or capsules. Further, the present invention provides a composition for local administration which may contain inert components such as a solvent or emulsifier for formulation into creams, ointments, gels, pastes, or ophthalmic solutions.

[0163] These compositions can be sterilized by conventional sterilization techniques or by sterile filtration. The resulting aqueous solution can be packaged for use as is or lyophilized, and the lyophilized preparation can be mixed with a sterile aqueous carrier prior to administration. The pH of the preparation is typically from 3 to 11, more preferably from 5 to 9 or 6 to 8, most preferably from 6 to 7, for example, from 6 to 6.5. The resulting composition in solid form can be packaged into a plurality of single dosage units each containing a fixed amount of the above drug or drugs, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible amount, such as in a squeeze tube designed for a topically applicable cream or ointment.

[0164] A composition containing an effective amount can be administered for a radiation treatment plan, diagnosis, or therapeutic treatment. When administered for a radiation treatment plan or diagnostic purpose, the conjugate is administered to the subject in an amount effective to determine a diagnostic effective dose and / or a therapeutic effective dose. In a therapeutic application, the composition is administered to a subject (e.g., a human) already suffering from a condition (e.g., cancer) in an amount sufficient to cure or at least partially arrest the symptoms of the disorder and its complications. The amount sufficient to achieve this purpose is defined as a "therapeutically effective amount," which is the amount of the compound sufficient to substantially improve at least one symptom associated with the disease or medical condition. For example, in the treatment of cancer, an agent or compound that reduces, prevents, delays, suppresses, or arrests any symptom of the disease or condition is therapeutically effective. The therapeutically effective amount of an agent or compound need not cure the disease or condition, but provides treatment for the disease or condition such that the onset of the disease or condition is delayed, impeded, or prevented, or the symptoms of the disease or condition are improved, or the duration of the disease or condition is altered, or, for example, becomes less severe, or recovery in the individual is accelerated. The conjugate of the present invention can be used for the treatment of cancer by administering to the subject a first dose of either the conjugate or the composition in an amount effective for a radiation treatment plan, and then administering a second dose of either the conjugate or the composition in a therapeutically effective amount.

[0165] The effective amount for these uses can depend on the severity of the disease or condition as well as the weight and general condition of the subject. A person skilled in the art can determine the therapeutically effective amount of the compositions of the present invention and of the methods of the present invention used in mammals (e.g., humans) taking into account the individual differences in the age, weight, and condition of the mammal. Certain conjugates of the present invention exhibit an enhanced ability to target and remain in cancer cells, so the dosage of the compounds of the present invention may be less than the equivalent dosage required for the therapeutic effect of the unconjugated agent (e.g., about 90%, 75%, 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, less than 0.5%, or 0.1% or equivalent thereto). The agents of the present invention are administered to a subject (e.g., a mammal such as a human) in an effective amount, which is an amount that produces a desired result in the subject being treated.

[0166] Single or multiple administrations of the compositions of the present invention containing an effective amount can be carried out at dosage levels and patterns selected by the treating physician. The dosage and administration schedule can be determined and adjusted based on the severity of the disease or condition in the subject and monitored throughout the course of treatment according to methods generally practiced by a clinician or those described herein.

[0167] The conjugates of the present invention can be used in combination with conventional treatment methods or therapies or separately from conventional treatment methods or therapies.

[0168] When the compounds of the present invention are administered in combination therapy with other agents, they can be administered to the individual sequentially or simultaneously. Alternatively, the pharmaceutical compositions according to the present invention may comprise a combination of a compound of the present invention bound to a pharmaceutically acceptable excipient as described herein and another therapeutic or prophylactic agent known in the art.

[0169] As used interchangeably herein, "anti-proliferative" or "anti-proliferative agent" means any anti-cancer agent, including those listed in Table 2, all of which can be used in combination with the conjugates of the present invention for treating the medical conditions described herein. Anti-proliferative agents also include organoplatinum derivatives, naphthoquinone and benzoquinone derivatives, chrysophanic acid and its anthraquinone derivatives.

[0170] As used interchangeably herein, "immunomodulatory agent" or "immunomodulator" means any immunomodulator, including those listed in Table 2, all of which can be used in combination with the conjugates of the present invention for treating the medical conditions described herein.

[0171] As used herein, "radiosensitizer" includes any agent that increases the sensitivity of cancer cells to radiotherapy. Radiosensitizers include, but are not limited to, 5-fluorouracil, platinum analogs (e.g., cisplatin, carboplatin, oxaliplatin), gemcitabine, EGFR antagonists (e.g., cetuximab, gefitinib), farnesyltransferase inhibitors, COX-2 inhibitors, bFGF antagonists, and VEGF antagonists.

[0172] [Table 2A]

[0173] [Table 2B]

[0174] [Table 2C]

[0175] [Table 2D]

[0176]

Table 2E

[0177]

Table 2F

[0178]

Table 2G

[0179] The following examples are intended to illustrate the synthesis of a representative number of conjugates and the use of these conjugates for the treatment of cancer. Accordingly, the examples are intended to illustrate, not limit, the invention. Additional compounds not specifically illustrated can be synthesized using conventional methods in combination with the methods described herein.

Examples

[0180] (Example 1) General Materials and Methods The antibody used was HuMIgG (Aldrich, I4506). The lutetium-177 was received from Perkin Elmer as lutetium chloride in 0.05 N hydrochloric acid solution.

[0181] Analytical HPLC-MS was performed using a Waters Acquity HPLC-MS system comprising a Waters Acquity Binary Solvent Manager, a Waters Acquity Sample Manager (cooling the sample to 10 °C), a Waters Acquity Column Manager (column temperature 30 °C), a Waters Acquity Photodiode Array Detector (monitoring at 254 nm and 214 nm), a Waters Acquity TQD using electrospray ionization, and a Waters Acquity BEH C18, 2.1×50 (1.7 μm) column. Preparative HPLC was performed using a Waters HPLC system comprising a Waters 1525 Binary HPLC pump, a Waters 2489 UV / Visible Detector (monitoring at 254 nm and 214 nm), and a Waters XBridge Prep phenyl or C18 19×100 mm (5 μm) column.

[0182] HPLC elution method 1: Waters Acquity BEH C18 2.1×50 mm (1.7 μm) column; mobile phase A: H2O (0.1% v / v TFA); mobile phase B: acetonitrile (0.1% v / v TFA); flow rate = 0.3 mL / min; initial = 90% A, 3 - 3.5 min = 0% A, 4 min = 90% A, 5 min = 90% A.

[0183] HPLC elution method 2: Waters XBridge Prep phenyl 19×100 mm (5 μm) column; mobile phase A: H2O (0.1% v / v TFA); mobile phase B: acetonitrile (0.1% v / v TFA); flow rate = 10 mL / min; initial = 80% A, 13 min = 0% A.

[0184] HPLC Dissolution Method 3: Waters Acquity BEH C18 2.1×50mm (1.7μm) column; Mobile phase A: H20 (0.1% v / v TFA); Mobile phase B: Acetonitrile (0.1% v / v TFA); Flow rate = 0.3 mL / min; Initial = 90% A, 8 min = 0% A, 10 min = 0% A, 11 min = 90% A, 12 min = 90% A.

[0185] HPLC Dissolution Method 4: Waters XBridge Prep C18 OBD 19×100mm (5μm) column; Mobile phase A: H20 (0.1% v / v TFA); Mobile phase B: Acetonitrile (0.1% v / v TFA); Flow rate: 10 mL / min; Initial = 80% A, 3 min = 80% A, 13 min = 20% A, 18 min = 0% A.

[0186] HPLC Dissolution Method 5: Waters XBridge Prep C18 OBD 19×100mm (5μm) column; Mobile phase A: H20 (0.1% v / v TFA); Mobile phase B: Acetonitrile (0.1% v / v TFA); Flow rate: 10 mL / min; Initial = 90% A, 3 min = 90% A, 13 min = 0% A, 20 min = 0% A.

[0187] HPLC Dissolution Method 6: Waters XBridge Prep C18 OBD 19×100mm (5μm) column; Mobile phase A: H20 (0.1% v / v TFA); Mobile phase B: Acetonitrile (0.1% v / v TFA); Flow rate: 10 mL / min; Initial = 75% A, 13 min = 0% A, 15 min = 0% A.

[0188] HPLC Dissolution Method 7: Waters XBridge Prep C18 OBD 19×100mm (5μm) column; Mobile phase A: H20 (0.1% v / v TFA); Mobile phase B: Acetonitrile (0.1% v / v TFA); Flow rate: 10 mL / min; Initial = 80% A, 12 min = 0% A, 15 min = 0% A.

[0189] HPLC Dissolution Method 8: Waters XBridge Prep C18 OBD 19×100 mm (5 μm) column; Mobile Phase A: H20 (0.1% v / v TFA); Mobile Phase B: Acetonitrile (0.1% v / v TFA); Flow rate: 10 mL / min; Initial = 90% A, 12 min = 0% A, 15 min = 0% A.

[0190] Analytical size exclusion chromatography (SEC) was performed using a Waters system including a Waters 1525 Binary HPLC pump, a Waters 2489 UV / Visible Detector (monitored at 280 nm), a Bioscan Flow Count radiation detector (FC-3300), and a TOSOH TSKgel G3000SWxl, 7.8×300 mm column. The isocratic SEC method has a flow rate = 1 mL / min and uses a mobile phase of 0.1 M phosphoric acid, 0.6 M NaCl, 0.025% sodium azide, pH = 7.

[0191] MALDI-MS (positive ion) was performed using a MALDI Bruker Ultraflextreme Spectrometer.

[0192] Radioactive thin layer chromatography (radioactive TLC) was performed using a Bioscan AR-2000 Imaging Scanner and run on an iTLC-SG glass microfiber chromatography paper (Agilent Technologies, SGI0001) plate using a citrate buffer (0.1 M, pH 5.5).

[0193] (Example 2) Synthesis of 4-{[11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound B) The bifunctional chelate, 4-{[11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound B) was synthesized according to the scheme provided in Figure 2. To a solution of 5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanoic acid (DOTA-GA-(tBu)4, 50 mg, 0.07 mmol) in ACN (2.0 mL) was added DSC (50 mg, 0.21 mmol), followed by pyridine (0.20 mL, 2.48 mmol). The reaction was stirred at room temperature for 1 hour. To the reaction mixture was added 11-aminoundecanoic acid (70 mg, 0.36 mmol), followed by PBS solution (1.0 mL) at room temperature. The reaction was stirred at room temperature for 72 hours. The reaction mixture was filtered using a syringe filter and directly purified by preparative HPLC using Method 6 to give Intermediate 2-A (71 mg, 74.8%).

[0194] To a solution of Intermediate 2-A (40 mg, 0.03 mmol), TFP (90 mg, 0.54 mmol) and EDC (40 mg, 0.27 mmol) in ACN (1.0 mL) was added pyridine (0.05 mL, 50 mg, 0.62 mmol) at room temperature. The solution was stirred at room temperature for 24 hours. The reaction was directly purified by preparative HPLC using Method 7 and concentrated using a Biotage V10 Rapid Evaporator to give Intermediate 2-B (33 mg, 82.5%) as a wax.

[0195] Intermediate 2-B (33 mg, 0.022 mmol) was dissolved in DCM / TFA (1.0 mL / 2.0 mL) and stirred at room temperature for 24 h. The reaction mixture was concentrated by a stream of air and directly purified by preparative HPLC using Method 8 to give Compound B (14 mg, 50.0%) as a clear wax after concentration. An aliquot was analyzed by HPLC-MS elution method 3; retention time: 4.15 min; MS (positive ESI); found m / z 808.1 [M+H] + ; C 36 H 54 F4N5O 11 (calcd 808.8).

[0196]

Number

[0197] (Example 3) Synthesis of 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound C) The bifunctional chelate, 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound C), was synthesized according to the scheme provided in Figure 3. To a solution of 5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanoic acid (DOTA-GA(tBu)4, 100 mg, 0.143 mmol) in ACN (8.0 mL) was added DSC (73 mg, 0.285 mmol) and pyridine (0.80 mL, 9.89 mmol). The reaction mixture was stirred at ambient temperature for 90 minutes. This solution was added to a half-solution of amino-PEG3-acid (63 mg, 0.285 mmol in 1.2 mL of DMF) in a 100 mL round-bottom flask. After 4 hours at ambient temperature, the reaction was finished by concentrating under a stream of air until dry. The crude material was purified by HPLC elution method 2 (the crude material was dissolved in 6 mL of 20% ACN / H2O). The fractions containing the product were pooled, concentrated under reduced pressure, and then co-evaporated with ACN (3 x 2 mL). Intermediate 1-A was obtained in 82% yield.

[0198] To a vial containing Intermediate 1-A (82 mg, 60 μmol) was added ACN (2 mL), NEt3 (50 μL, 360 μmol, 6 eq), HBTU (23 mg, 60 μmol, 1 eq), and a TFP solution (50 mg, 300 μmol, 5 eq, dissolved in 250 μL of ACN). The resulting clear solution was stirred at ambient temperature for 3 hours. The reaction was finished by concentrating under a stream of air until dry, then diluted with ACN / H2O (1:1, 3 mL total) and purified on preparative HPLC using elution method 4. The fractions containing the product were pooled, concentrated under reduced pressure, and then co-evaporated with ACN (3 x 2 mL). Intermediate 1-B was obtained as a clear residue (67 mg, 74% yield).

[0199] To a vial containing Intermediate 1-B (67 mg, 64 μmol), DCM (2 mL) and TFA (2 mL) were added, and the resulting solution was stirred at ambient temperature for 16 h. Additional TFA (2 mL) was added and the reaction was stirred at ambient temperature for 6 h. The reaction was concentrated under a stream of air until dry, and the crude product was finally dissolved in ACN / H2O (1 mL of 10% ACN / H2O). The crude reaction solution was then purified by preparative HPLC using elution method 5. The fractions containing the product were pooled, frozen, and lyophilized. Compound C was obtained as a white solid (36 mg, 63% yield). An aliquot was analyzed by HPLC-MS elution method 3: retention time: 3.11 min; MS (positive ESI): found m / z 828.4 [M+H] + ; C 34 H 50 F4N5O 14 (calculated 828.3).

[0200]

Number

[0201] (Example 4) 177 Synthesis of [[Lu]-Compound A-Human IgG Compound A (1.34 μmol) was dissolved in sodium acetate buffer (20 μL, pH 6.5) and added to a solution containing antibody human IgG antibody (6.7 nmol) in bicarbonate buffer (pH 8.5). After 45 min at ambient temperature, the antibody conjugate product was purified by HPLC SEC column (1 mL / min, eluting with acetate buffer (pH 6.5, 1 mM ascorbic acid)). MALDI-TOF-MS (positive ion): Compound A-Human IgG: found m / z 150360 [M+H] + ; Human IgG: found m / z 148339 [M+H] + .

[0202] ​As a typical reaction, Lu-177 (1.1 mCi, 5 μL) was added to a solution of compound A-human IgG (90 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5))). The radiolabeled reactant was incubated at 37 °C for 90 minutes. The crude product, 177 The [Lu]-compound A-human IgG was purified by a Sephadex G-50 resin-packed column and eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). Radiochemical purity by radioactive TLC: 98%; radiochemical yield: 45%; specific activity: 15.1 mCi / mg.

[0203] (Example 5) 177 Synthesis of [Lu]-compound B-human IgG Compound B (1.17 μmol) was dissolved in sodium acetate buffer (0.117 mL, pH 6.5). An aliquot (2 μL, 10 nmol) of the compound B solution was added to a solution containing human IgG antibody (6.7 nmol) in bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the antibody conjugate product was purified by a Sephadex G-50 resin-packed column. The antibody conjugate compound A-human IgG was eluted from the column using acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Observed m / z 149949 [M + H] for compound B-human IgG + ; Observed m / z 148540 [M + H] for human IgG + .

[0204] As a typical reaction, Lu-177 (1.1 mCi, 5 μL) was added to a solution of compound B-human IgG (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5))). The radiolabeled reactant was incubated at 37 °C for 30 minutes. The crude product, 177 ​The Lu-compound B-human IgG was purified by an HPLC SEC column (eluted at 1 mL / min with an acetate buffer (pH 6.5, 1 mM ascorbic acid)) and concentrated by ultrafiltration (Vivaspin, 10 kDa). Radiochemical purity by radioactive TLC: 98%; radiochemical yield: 51%; inactivity: 9.68 mCi / mg.

[0205] (Example 6) 177 Synthesis of Compound C (0.96 μmol) was dissolved in a sodium acetate buffer (95 μL, pH 6.5). An aliquot (2 μL, 20 nmol) of the compound C solution was added to a solution containing a human IgG antibody (6.7 nmol) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the antibody conjugate product was purified by a Sephadex G-50 resin-packed column. The antibody conjugate compound C-human IgG was eluted from the column using an acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Observed m / z 150095 [M+H] for compound C-human IgG + ; Observed m / z 148540 [M+H] for human IgG + 。

[0206] As a typical reaction, Lu-177 (1.1 mCi, 5 μL) was added to a solution of compound C-human IgG (100 μg in an acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in an acetate buffer (pH 6.5))). The radiolabeled reactant was incubated at 37 °C for 30 minutes. The crude product, 177 the

[0207] (Example 7) Pharmacokinetic and metabolic studies of compounds based on human IgG ​Groups of 4 or 5 mice (normal CD-1 mice or athymic CD-1 nude mice) were injected intravenously with approximately 15 microcuries of a radiolabeled test compound. Test compounds with various linkers were synthesized and radiolabeled with lutetium-177. For pharmacokinetic studies, animals were sacrificed at specific time points and blood and tumors (if applicable) were analyzed for total radioactivity. For metabolism studies, animals were placed in metabolic cages (4 - 5 per cage) for urine and feces collection every 24 hours up to 7 days maximum. The radioactive content of urine and feces samples was quantified and converted to total urine or feces output based on body weight. Excretion profiles for urine, feces, or total excretion (urine + feces) were generated by plotting the cumulative injected dose % (%ID) versus time.

[0208] Unlabeled human IgG antibody was used for metabolism excretion studies to demonstrate that changes in the radiolabeled excretion profile directed by conjugation with linker compounds B and C are a common process. The human IgG preparations used consisted of a purified mixture of all IgG isotypes (IgG1 - 4).

[0209] Unlabeled human IgG antibody conjugate 177 Lu]-Compound B - HuMIgG, and 177 The metabolism excretion profiles of Lu]-Compound C - HuMIgG were 177 compared to Lu]-Compound A - HuMIgG. 177 Lu]-Compound A - HuMIgG was excreted slowly, with only 13% of the injected dose (ID) removed over 7 days by low - level urinary excretion. Compounds B and C targeted distinctly different excretion pathways and increased the total excretion of radioactivity over 7 days when compared to Compound A - HuMIgG. 177 Lu]-Compound B - HuMIgG was removed via feces, 177The removal of [Lu]-Compound C-HuMIgG was roughly equally divided between urine and feces. Thus, although the linker type affects the route, rate, and extent of compound excretion (Figure 4), it was found that it does not change the overall pharmacokinetics of the total radioactivity associated with the radiolabeled immunoconjugate in the blood. Also, when conjugated to an antibody, an improved excretion profile of Compound B or Compound C was observed to be a common and reproducible effect.

[0210] (Example 8) 225 Synthesis of [Ac]-Compound A-Human IgG Compound A (1.34 μmol) was dissolved in sodium acetate buffer (20 μL, pH 6.5) and added to a solution containing human IgG antibody (6.7 nmol) in bicarbonate buffer (pH 8.5). After 45 minutes at ambient temperature, the antibody conjugate product was purified by an HPLC SEC column (1 mL / min, eluting with acetate buffer (pH 6.5, 1 mM ascorbic acid)). MALDI-TOF-MS (positive ion): [Ac]-Compound A-Human IgG measured m / z 150360 [M+H] + ; Human IgG measured m / z 148339 [M+H] + .

[0211] As a typical reaction, [Ac]-225 (1.1 mCi, 5 μL) is added to a solution of [Ac]-Compound A-Human IgG (90 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5))). The radiolabeled reactant is incubated at ambient temperature (e.g., 20 - 25 °C) for 90 minutes. The crude product, 225 [Ac]-Compound A-Human IgG was purified by a Sephadex G-50 resin-packed column and eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid).

[0212] (Example 9) 225 Synthesis of [Ac]-Compound B-Human IgG ​​Compound B (1.17 μmol) was dissolved in sodium acetate buffer (0.117 mL, pH 6.5). An aliquot (2 μL, 10 nmol) of the compound B solution was added to a solution containing human IgG antibody (6.7 nmol) in bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the antibody conjugate product was purified by a Sephadex G-50 resin-packed column. The antibody conjugate, compound A-human IgG, was eluted from the column using acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Measured value of compound B-human IgG m / z 149949 [M+H] + ; Measured value of human IgG m / z 148540 [M+H] + .

[0213] As a typical reaction, Ac-225 (1.1 mCi, 5 μL) is added to a solution of compound B-human IgG (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5))). The radiolabeled reactant is incubated for 30 minutes at ambient temperature (e.g., 20 - 25 °C). The crude product, 225 [[Ac]]-compound B-human IgG was purified by an HPLC SEC column (eluting at 1 mL / min with acetate buffer (pH 6.5, 1 mM ascorbic acid)) and concentrated by ultrafiltration (Vivaspin, 10 kDa).

[0214] (Example 10) 225 Synthesis of [[[Ac]]-compound C-human IgG Compound C (0.96 μmol) was dissolved in sodium acetate buffer (95 μL, pH 6.5). An aliquot (2 μL, 20 nmol) of the compound C solution was added to a solution containing human IgG antibody (6.7 nmol) in bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the antibody conjugate product was purified by a Sephadex G-50 resin-packed column. The antibody conjugate, compound C-human IgG, was eluted from the column using acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Measured value of compound C-human IgG m / z 150095 [M+H] + ​; Measured value of human IgG, m / z 148540 [M+H] + .

[0215] As a typical reaction, Ac-225 (1.1 mCi, 5 μL) is added to a solution of compound C-human IgG (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5))). The radiolabeled reactant is incubated at ambient temperature (e.g., 20-25 °C) for 30 minutes. The crude product, 225 [Ac]-compound C-human IgG was purified by HPLC SEC column (eluted at 1 mL / min with acetate buffer (pH 6.5, 1 mM ascorbic acid)) and concentrated by ultrafiltration (Vivaspin, 10 kDa).

[0216] Other embodiments Although the present invention has been described in connection with its specific embodiments, it can be further modified, and this application is generally intended to cover any modifications, uses, or adaptations of the present invention that follow the principles of the present invention, and it is understood that the present disclosure includes such departures from the present disclosure that are within the scope of known or customary practice in the art to which the present invention pertains and can be applied to the essential features described above.

Claims

1. Structure of Formula I: A-L 1 -(L 2 ) n -B Formula I [wherein, A is a moiety derived from a chelating agent selected from DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA ((1R,4R,7R,10R)-α,α',α'',α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid), DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetic acid), DOTA-GA anhydride (2,2',2''-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid), DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenephosphonic acid)), DOTMP (1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylenephosphonic acid), DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonic acid)), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP (1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid)), TETPA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), HEHA (1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid), PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N''''-pentaacetic acid), DO2P (tetraazacyclododecanedimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecane triacetic acid), or porphyrin, or a metal complex thereof; L 1 is an optionally substituted C 1 - C 6 alkylene; B is a human or humanized IgG antibody, or an antigen-binding fragment thereof; n is 1; L 2 are each independently of the structure: (-X 1 - L 3 - Z 1 - ) of formula II (wherein X 1 is C=O(NR 1 ) or NR 1 , R 1 is H or an optionally substituted C 1 - C 6 alkyl or an optionally substituted aryl or heteroaryl; L 3 is an optionally substituted C 1 - C 50 alkylene or C4 - C 20 polyethylene oxide - ethylene; Z 1 is CH 2 , C=O, C=S, OC=O, NR1'C=O, or NR1', where R1' is hydrogen or an optionally substituted C 1 - C 6 alkyl, or pyrrolidine - 2,5 - dione) having, wherein Z 1 and B are directly bonded by a covalent bond, and L 1 is bonded to one atom constituting the ring in the chelating agent of A) having a compound, or a pharmaceutically acceptable salt thereof (provided that the human or humanized IgG antibody is an IGF - 1R antibody selected from the group consisting of figitumumab, cixutumumab, ganitumab, AVE1642, BIIB002, robatumumab, and teprotumumab).

2. The moiety derived from the chelating agent has the structure: 【Chemical Formula 1】 (wherein, Y 1 is -CH 2 OCH 2 (L 2 ) n -B, or C=O(L 2 ) n -B, and Y 2 is -CH 2 CO 2 H; Or, Y 1 is H, and Y 2 is L 1 -(L 2 ) n -B) The compound according to claim 1, having

3. L 1 has the structure: 【Chemical Formula 2】 (wherein, R 2 is hydrogen or -CO 2 H, and the wavy line on the upper left indicates the bond to A) The compound according to claim 1 or 2, having

4. The metal complex contains a metal selected from the group consisting of Bi, Pb, Y, Mn, Cr, Fe, Co, Zn, Ni, Tc, In, Ga, Cu, Re, Sm, lanthanides, and actinides, or the metal complex is 47 Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga, 68 Ga, 82 Rb, 86 Y, 87 Y, 90 Y, 97 Ru, 99m Tc, 105 Rh, 109 Pd, 111 In, 117m Sn, 149 Pm, 149 Tb, 153 Sm, 177 Lu, 186 Re, 188 Re, 199 Au, 201 Tl, 203 Pb, 212 Pb, 212 Bi, 213 Bi, 225 Ac, and 227 Th, and a compound according to any one of claims 1 to 3, comprising a radionuclide selected from the group consisting of.

5. A compound according to any one of claims 1 to 4, wherein B is an antibody that specifically binds to the insulin-like growth factor-1 receptor (IGF-1R), or an antigen-binding fragment thereof.

6. X 1 is C=O(NR 1 ), R 1 is H, L 3 is C 4 -C 50 alkylene or C4-C 20 polyethylene oxide-ethylene, and a compound according to any one of claims 1 to 5.

7. Z 1 is -CH 2 and a compound according to any one of claims 1 to 6.

8. The following: 【Chemical Formula 3】 and a compound according to any one of claims 1 to 7, selected from the group consisting of.

9. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 and a pharmaceutically acceptable excipient.

10. Use in a method of radiotherapy planning and / or radiotherapy for the composition according to claim 9.

11. The composition according to claim 9 for use in a method of treating cancer, wherein the method comprises administering to a subject in need thereof a first dose of the composition in an amount effective for a radiotherapy treatment plan, and then administering subsequent doses of the composition in a therapeutically effective amount.

12. A composition for use in a method of treating cancer according to claim 9, wherein the composition administered in the first dose is different from the composition administered in the second dose.

13. The composition for use in a method of treating cancer according to claim 11 or 12, wherein the method further comprises administering an anti-proliferative agent, a radiosensitizer, or an immunomodulatory or immunoregulatory agent.

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