EA045232B1 - STRENGTHENING THE PHARMACOKINETICS OF BIFUNCTIONAL CHELATES AND THEIR APPLICATION - Google Patents

Description

Related applications

The invention claims priority and benefit from U.S. Provisional Patent Application No. 62/502,260, entitled Pharmacokinetic enhancements of bifunctional chelates and uses thereof, filed May 5, 2017

g., the entire contents of which are incorporated herein by reference for all purposes.

State of the art

Radiolabeled targeting moieties, or radioconjugates, are typically prepared using a bifunctional chelating agent to attach a radiolabel to a biological molecule while maintaining target affinity. Bifunctional chelates may contain in their structure a chelate, a linker and a cross-linking group or targeting moiety. By modifying the linker region of the bifunctional chelate, it is possible to provide pharmacokinetic advantages that can enhance the removal of radioactive elements.

Brief description of the invention

The present invention relates to linkers that enhance the clearance of a chelating moiety, or its metal complex, when conjugated to a therapeutic moiety, targeting moiety, or cross-linking group.

Accordingly, in a first aspect, the present invention relates to a compound having the structure:

AL ¹ -(L ² )nB,

Formula I where A represents a chelating moiety or its complex with a metal;

wherein said chelating moiety is selected from the group consisting of DOTA (1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R,4R,7R,10R)-α,α',α, α'-tetramethyl1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA ( 1,4,7,10-tetraazacyclododecane1,4,7,10-tetrapropionic acid), DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)1,4,7, 10-tetraazacyclododecan-1-yl)acetic acid), DOTA-GA anhydride (2,2',2-(10-(2,6dioxotetrahydro-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)), DOTA-4AMP (1,4,7,10 -tetraazacyclododecane-1,4,7,10-tetrakis(acetamidomethylenephosphonic acid), NOTA (1,4,7triazacyclononane-1,4,7-triacetic acid), and HP-DO3A (hydroxypropyltetraazacyclododecanetriacetic acid), where the metal of the specified complex with the metal is a radionuclide selected from the group consisting of 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, ⁸⁹ Zr, ⁹⁰ Y, ⁹⁷ Ru, ⁹⁹ Tc, ^99m Tc, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹ⁿ In, ^117m Sn, ¹⁴⁹ Pm, ¹⁴⁹ Tb, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au, ¹⁹⁹ Au, ²⁰¹ Tl, ²⁰³ Pb, ²¹¹ At, ²¹² Pb, ²¹² Bi, ²¹³ Bi, ²²³ Ra, ²²⁵ Ac, ²²⁷ Th, and ²²⁹ Th,

L ¹ represents C ₁ -C ₆ -alkyl or C ₁ -C ₆ -heteroalkyl;

B is a human IgG antibody or a humanized IgG antibody or an antigen binding fragment thereof, or a pharmaceutically acceptable salt thereof;

n is 1;

each L ² independently has the structure:

(-X ¹ -L ³ - _Z 1-),

Formula II where X ¹ represents C=O(NR1), in which R1 represents H or C1-C6 alkyl or C1C6 heteroalkyl; L ³ represents C1-C ₅₀ -alkyl or C ₁ -C ₅₀ -heteroalkyl; Z ¹ represents C=O, C=S, OC=O or NR1C=O in which R ¹ represents hydrogen or C ₁ -C ₆ -alkyl or C1-C6 heteroalkyl;

where in each case

C ₁ -C ₆ -alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of C ₁ . ₆ alkoxy, amino, halogen, hydroxy, nitro, C ₂ . ₆ αlkenyl, C ₆ . ₁₀ aryl, C ₅ -C ₂₀ polyethylene glycol and -CO ₂ R ^A , where R ^A is selected from the group consisting of hydrogen, C ₁ . ₆ alkyl, C ₂ . ₆ alkenyl and C ₅ -C ₂₀ -polyethylene glycol,

C ₁ -C ₆ -heteroalkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of C ₁ . ₆ αlkoxy, amino, halogen, hydroxy, nitro, C ₁ . ₆ alkyl, C ₂ . ₆ alkenyl, C ₆ _ ₁₀ aryl, C ₅ -C ₂₀ -polyethylene glycol and -CO ₂ ^RA , where ^{RA is} selected from the group consisting of hydrogen, C1. ₆ alkyl, C ₂ . ₆ α-alkenyl and C ₅ -C ₂₀ -polyethylene glycol,

C ₆ -C ₁₀ -aryl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of C ₁ . ₆ alkoxy, amino, halogen, hydroxy, nitro, C ₁ . ₆ alkyl, C ₂ . ₆ αlkenyl, C ₆ . ₁₀ aryl, C ₅ -C ₂₀ -polyethylene glycol and -CO ₂ R ^A , where R ^A is selected from the group consisting of hydrogen, C ₁ . ₆ alkyl, C2- 1 045232 ₆ alkenyl and C ₅ -C ₂₀ polyethylene glycol, and

C ₁ -C ₁₂ -heteroaryl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of C ₁ - ₆ alkoxy, amino, halogen, hydroxy, nitro, C ₁ . ₆ alkyl, C ₂ . ₆ alkenyl, C _6-10 aryl, C ₅ -C ₂₀ -polyethylene glycol and -CO2RA, where R ^A is selected from _the group consisting of hydrogen, Cp ₆ alkyl, C ₂ . ₆ alkenyl and C ₅ -C ₂₀ -polyethylene glycol, where in each case heteroalkyl represents an alkyl group in which one or more of its constituent carbon atoms is each replaced by nitrogen, oxygen or sulfur, heteroaryl represents 5-, 6- or 7- ring member containing one, two, three or four heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur

One of ordinary skill in the art will appreciate that the use of chelating moieties in the practice of the present invention is not limited to the specific constructs disclosed herein, but rather may include the use of any known chelating moieties.

In some embodiments, the chelating moiety has the structure:

where Y ¹ represents -CH2OCH2(L ² )nB, and Y ² represents -CH2CO2H; or where Y ¹ represents H, Y ² represents L ¹ -(L2) _n -B.

In some implementations, L ¹ has the structure:

R'

Formula III where R ² represents hydrogen or -CO ₂ H.

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

In some embodiments, Y ¹ is H.

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

In some embodiments, L ² has a value of n equal to 1.

In some embodiments, the connection is selected from the group consisting of:

In some embodiments, the radionuclide is In some embodiments, the radionuclide is In some embodiments, the radionuclide is

111In.

⁶⁸ Ga. 86γ

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

In some embodiments, the radionuclide is ⁶⁷ Cu, ¹⁷⁷ Lu, or ⁹⁰ Y.

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

In some embodiments, the radionuclide is ²²⁵ Ac, ²¹² Pb, ²²⁷ Th.

In another aspect of the present invention, there is provided a pharmaceutical composition containing any of the above compounds and a pharmaceutically acceptable excipient.

- 2 045232

In another aspect of the present invention, there is provided a method for planning radiation treatment and/or a method for radiation treatment itself, wherein said method comprises administering to a subject in need thereof any of the foregoing compounds or pharmaceutical compositions.

In another aspect of the present invention, there is provided a method of detecting and/or treating cancer, comprising administering to a subject in need thereof a first dose of any of the foregoing compounds or pharmaceutical compositions in an amount effective for planning radiation treatment, and then administering subsequent doses of any of the foregoing compounds or pharmaceutical compositions. pharmaceutical compositions in a therapeutically effective amount.

In some embodiments, the compound or composition administered in the first dose and the compound or composition administered in the second dose are the same.

In some embodiments, the compound or composition administered in the first dose and the compound or composition administered in the second dose are different.

In some embodiments, said cancer is a solid tumor or hematologic (liquid) cancer.

In some embodiments, the solid tumor 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's sarcoma, multiple myeloma, or acute myeloid leukemia.

In some embodiments, the above methods further comprise administering an antiproliferative agent, a radiosensitizer, or an immunoregulatory or immunomodulatory agent.

In some embodiments, any of the above compounds or compositions thereof and the antiproliferative agent or radiosensitizer are administered within 28 days (eg, within 14, 7, 6, 5, 4, 3, 2, or 1 day) of each other.

In some embodiments, any of the above-described compounds or compositions thereof and the immunoregulatory or immunomodulatory agent are administered for 90 days (e.g., for 80, 70, 60, 50, 4o, 30, 20, 10, 5, 4, 3, 2, or 1 day) after the previous one.

In another aspect of the present invention, a method for producing a radioconjugate (eg, any of the radioconjugates described herein) is provided. The method includes the steps of (a) conjugating a bifunctional chelate with a biological molecule, (b) purifying the conjugate obtained in step (a), and (c) chelating one or more radionuclides (for example, one or more Ac-225 radionuclides) with the purified conjugate with step (b) at a temperature less than 35°C (eg, 20-25°C) to obtain a radioconjugate (eg, an actinium radioconjugate).

In some embodiments, the radioconjugate is a radioimmunoconjugate (eg, any of the radioimmunoconjugates described herein).

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

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 more).

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

Chemical terms.

The term acyl as used herein means hydrogen or an alkyl group (eg, a haloalkyl group) as defined herein, which is attached to a group of the parent molecule via a carbonyl group as defined herein. Examples are formyl (ie, carboxyaldehyde group), acetyl, trifluoroacetyl, propionyl, butanoyl and the like. Examples of unsubstituted acyl groups include 1 to 7, 1 to 11, or 1 to 21 carbon atoms. In some embodiments, the alkyl group is further substituted with 1,2,3, or 4 substituents as described herein.

The term alkyl as used herein includes saturated groups, both straight and branched chain, containing from 1 to 20 carbon atoms (eg, 1 to 10 or 1 to 6), unless otherwise noted. Examples of alkyl groups are methyl, ethyl, n- and isopropyl, n-, sec-, iso- and tert-butyl, neopentyl and the like, and may optionally involve one, two, three or, in the case of alkyl groups with two carbon atoms or more, four substituents independently selected from the group consisting of: (1) C ₁ . ₆ αlkoxy; (2) C ₁ . ₆ alkylsulfinyl; (3) amino, as defined herein (e.g., unsubstituted amino (i.e., -NH ₂ ) or substituted amino (i.e., -N(R ⁿ¹ )2, where the meanings of R ^N1 are given in the definition of the term amino); (4) C610aryl-C1.6αalkoxy; (5) _{azido; (6) halogen; (7) (C2.9 heterocyclyl ) oxy} ; (8) hydroxy, optionally substituted with an O-protecting group; (9 ) nitro; (10) oxo (for example, carboxyaldehyde or acyl); (11) C1 ₇ spirocyclyl; (12) thioalkoxy; (13) thiol; (14) -CO2RA, optionally substituted with an O-protecting group, and R ^a selected from the group _consisting of (a) _C1.20 alkyl (e.g. _C1.6 alkyl), ( _b ) _C2-

- 3 045232 ₂₀ alkenyl (for example, C _2- balkenyl), (c) Sb-1oaryl, (d) hydrogen, (e) Cl. _b αlk-C _b .l _O αrylα, (f) amino-C1— ₂₀ alkyl, (g) polyethylene glycol of the formula -(CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR', where s1 is an integer from 1 to 10 (for example, from 1 to 6 or from 1 to 4), each of s2 and s3 independently represents an integer from 0 to 10 (for example, from 0 to 4, for example, from 0 to 6, from 1 to 4, 1 to 6 or 1 to 10), and R' is H or C _1-20 alkyl and (h) aminopolyethylene glycol of the formula NR ^N1 (CH2)s2(CH2CH2O)s1(CH2)s3NR ^N1 , where s1 represents an integer from 1 to 10 (for example, from 1 to 6 or from 1 to 4), each of s2 and s3 independently represents an integer from 0 to 10 (for example, from 0 to 4, from 0 to 6, from 1 to 4, 1 to 6, or 1 to 10), and each R ^N1 independently represents hydrogen or represents an optionally substituted C1-6alkyl; (15) -C(O)NR ^B R ^C wherein each of R ^B and R ^C is independently selected from the group consisting of (a) hydrogen, (b) C1-6αalkyl, (c) C6-10aryl, and (d) C1.6alc-C6. 10arila; (16) -SO2RD, where RD is selected from the group consisting of (a) C1-6alkyl, (b) C6-10aryl, (c) C1.6alk-C6. 10aryl and (d) hydroxy; (17) -SO2NR ^E R ^F wherein each of R ^E and R ^F is independently selected from the group consisting of (a) hydrogen, (b) C1-6αalkyl, (c) _C6-10 aryl, and (d) _{C1 -6} alk-C _6-10 aryl; (18) -C(O)R ^G wherein R ^G is selected from the group consisting of (a) C1-20αalkyl (e.g. C1-6alkyl), (b) C2-20alkenyl (e.g. C2-6alkenyl), (c ) C6-10aryl, (d) hydrogen, (e) C1-6alk-C6-10aryl, (f) amino-C1-20alkyl, (g) polyethylene glycol of the formula -(CH2)s2(OCH2CH2)s1(CH2)s3OR', where s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), each of s2 and s3 is independently an integer from 0 to 10 (for example, 0 to 4, 0 to 6 , from 1 to 4, from 1 to 6, or from 1 to 10), and R' represents H or C1-20alkyl and (h) aminopolyethylene glycol of the formula -NR ^N1 (CH2) _s2 (CH ₂ CH ₂ O) _s1 ( CH ₂ ) _s3 NR ^N1 where s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), each of s2 and s3 independently represents 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 each R ^N1 independently represents hydrogen or optionally substituted C1-6alkyl; (19) -NR ^H C(O)R ^I , where R ^H is selected from the group consisting of (a1) hydrogen and (b1) C1-6αalkyl, and RI is selected from the group consisting of (a2) _C1-20αalkyl (for example, C _1-6 alkyl), (b2) C _2-20 αlkenyl (for example, C _2-6 alkenyl), (c2) C _6-10 aryl, (d2) hydrogen, (e2) C _1-6 αlk -C _6-10 aryl, (f2) amino-C _1-20 alkyl, (g2) polyethylene glycol (CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR', where s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), each of s2 and s3 independently represents an integer from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6 or from 1 to 10), and R' is H or C _1-20 αlkyl and (h2) aminopolyethylene glycol of the formula -NR ^N1 (CH2)s2(CH2CH2O)s1(CH2)s3NR ^N1 where s1 is an integer 1 to 10 (for example, 1 to 6 or 1 to 4), each of s2 and s3 independently represents an integer from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, from 1 to 6 or from 1 to 10), and each R ^N1 independently represents hydrogen or optionally substituted C1-6alkyl; (20) -NR ^J C(O)OR ^K , where R ^J is selected from the group consisting of (a1) hydrogen and (b1) C1-6 alkyl, and R ^K is selected from the group consisting of (a2) C120 alkyl (for example, C _1-6 αlkyl), (b2) C _2-20 alkenyl (for example, C _2-6 αlkenyl), (c2) C _6-10 aryl, (d2) hydrogen, (e2) C ₁ . ₆ alk-C ₆ . ₁₀ aryl, (f2) amino-C ₁ . ₂₀ alkyl, (g2) polyethylene glycol of the formula (CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR', where s1 is an integer from 1 to 10 (for example, from 1 to 6 or from 1 to 4 ), each of s2 and s3 independently represents 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' represents is H or C ₁ . ₂₀ alkyl and (h2) aminopolyethylene glycol of the formula -NR ^N1 (CH2)s2(CH2CH2O)s1(CH2)s3NR ^N1 , where s1 is an integer from 1 to 10 (for example, from 1 to 6 or from 1 to 4), each of s2 and s3 is 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 each R ^N1 is independently hydrogen or optionally substituted C1. ₆ αlkyl; and (21) amidine. In some embodiments, each of these groups may be further substituted as described herein. For example, the alkylene group of a C ₁ -alkaryl group can be further substituted with an oxo group to produce the corresponding aryloyl substituent.

The term alkylene and the prefix alk- as used herein is a saturated divalent hydrocarbon group derived from a straight or branched chain saturated hydrocarbon by the removal of two hydrogen atoms, examples of which are methylene, ethylene, isopropylene and the like. The term C _x - _y alkyl and the prefix C _x - _y alk- means an alkylene group containing x to y carbons. Exemplary x values are 1, 2, 3, 4, 5, and 6, and exemplary y values are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 (for example , C _1-6 , C ₂ - ₁₂ , C ₂ - ₂₀ , C ₂ - ₆ , C ₂ - ₁₀ or C ₂ - ₂₀ alkylene). In some embodiments, the alkylene may be further substituted with 1,2,3, or 4 substituent groups as defined herein for an alkyl group.

The term alkenyl as used herein is a straight or branched chain monovalent group containing, unless otherwise indicated, 2 to 20 carbon atoms (e.g., 2 to 6 or 2 to 10 carbon atoms), and having one or more carbon-carbon double bonds. Examples are ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl and the like. Alkenyls include cis and trans isomers. Alkenyl groups may be optionally substituted with 1,2,3, or 4 substituents that are independently selected from amino, aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl) as defined herein, or any of the exemplary alkyl substituents described herein document

- 4 045232 mente.

The term alkynyl, as used herein, is a straight- or branched-chain monovalent group containing 2 to 20 carbon atoms (e.g., 2 to 4, 2 to 6, or 2 to 10 carbon atoms) and having a carbon triple bond -carbon. Examples are ethynyl, 1-propynyl and the like. Alkynyl groups may be optionally substituted with 1, 2, 3, or 4 substituent groups that are independently selected from aryl, cycloalkyl, or heterocyclyl (eg, heteroaryl) as defined herein, or any of the exemplary alkyl substituent groups described herein.

The term amino as used herein represents -N( ^Rn1 )2, wherein each ^RN1 is independently H, OH, NO2, N( ^RN2 )2, SO2ORN2, SO2RN2, SORN2, N-protecting group, alkyl, alkenyl , alkynyl, alkoxy, aryl, alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl (e.g., optionally substituted with an O-protecting group such as optionally substituted arylalkoxycarbonyl groups or any groups described herein), sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or other groups described herein), alkoxycarbonylalkyl (for example, optionally substituted with an O-protecting group, such as optionally substituted arylalkoxycarbonyl groups or any described herein), heterocyclyl (for example, heteroaryl) or alkheterocyclyl (for example, alkheteroaryl), where each of these groups, R ^N1 may be optionally substituted, as defined herein for each group; or two R ^N1 are combined to form a heterocyclyl or N-protecting group, and wherein each R N2 is independently H, alkyl or aryl. The amino groups of the present invention may be unsubstituted amino (ie -NH2) or substituted amino (ie -N(R ^N1 )2). In a preferred embodiment, amino is -NH2 or -NHRN ¹ wherein ^RN1 is independently OH, NO2, NH2, NR ^N2 2, SO2ORN2, SO2RN2, SORN2, alkyl, carboxyalkyl, sulfoalkyl, acyl (e.g. acetyl, trifluoroacetyl or other substituents described herein), alkoxycarbonylalkyl (eg, t-butoxycarbonylalkyl) or aryl, and each RN2 may be H, C1 ₂₀ alkyl (eg, C1_ ₆ alkyl) or C ₆ . ₁₀ aryl.

The term amino acid as used herein refers to a molecule containing a side chain, an amino group, and an acid group (e.g., a -CO2H carboxy group or a SO3H sulfo group), wherein said amino acid is attached to the parent molecular group via a side chain, an amino group, or an acid group (e.g., side chain). In some embodiments, the amino acid is attached to the parent molecular group through a carbonyl group, wherein 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, pyrrolysine, selenocysteine, serine, taurine, threonine , tryptophan, tyrosine and valine. The amino acid groups may be optionally substituted with one, two, three or, in the case of amino acid groups with two carbon atoms or more, four substituents independently selected from the group consisting of: (1) C1_ ₆ alkoxy; (2) C1_ 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) C6. ₁₀ aryl-C1. ₆ alkoxy; (5) azido; (6) halogen; (7) (C ₂ _ ₉ heterocyclyl)oxy; (8) hydroxy; (9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) _{C1.7 spirocyclyl} ; (12) thioalkoxy; (13) thiol; (14) -CO2RA', wherein R ^A is selected from the group consisting of (a) C1.20αalkyl (for example, C16alkyl), (b) C2.20αalkenyl (for example, C2.6alkenyl), (c) C6.10aryl, (d) hydrogen, (e) C1.6αalk-C6.10aryl, (f) amino-C1. 20 alkyl, (g) polyethylene glycol of the formula (CH2)s2(OCH2CH2)s1(CH2)s3OR', where s1 is an integer from 1 to 10 (for example, from 1 to 6 or from 1 to 4), each of s2 and s3 is 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 C1.20αalkyl, and (h) an aminopolyethylene glycol of the formula NR ^N1 (CH2) _s2 (CH ₂ CH ₂ O) _s1 (CH ₂ ) _s3 NR ^N1 , where s1 is an integer from 1 to 10 (for example, from 1 to 6 or from 1 to 4 ), each of s2 and s3 is 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 each R ^{N1 is} independently represents hydrogen or optionally substituted C1.6alkyl; (15) -C(O)NR ^b R ^c , where each of R ^B and R ^C is independently selected from _the group consisting of (a) hydrogen, (b) C1_6alkyl, (c) _{C6_10aryl} , and (d) C ₁ _ ₆ alk-C ₆ _ ₁₀ aryl; (16) -SO2RD, where RD is selected from the group consisting of (a) C1. ₆ alkyl, (b) C ₆ - ₁₀ aryl, (c) C ₁ - ₆ alk-C ₆ - ₁₀ aryl and (d) hydroxy; (17) -SO ₂ NR ^e R ^f , where each of R ^E and R ^F is independently selected from the group consisting of (a) hydrogen, (b) C1.6alkyl, (c) C6-10aryl, and (d) C1_6alk- C6_10aryla; (18) -C(O)R ^G , where R ^G is selected from the group consisting of (a) C1_20alkyl ₍ for example, _C1.6 alkyl ₎ , (b) _C2-20 alkenyl (for example, _C2-6 alkenyl) , (c) C ₆ - ₁₀ aryl, (d) hydrogen, (e) C ₁ _ ₆ alk-C ₆ _ ₁₀ aryl, (f) amino-C1. ₂₀ alkyl, (g) polyethylene glycol of the formula (CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR' where s1 is an integer from 1 to 10 (for example, from 1 to 6 or from 1 to 4) , each of s2 and s3 independently represents 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' represents H or _{C1_20} alkyl, and (h) aminopoly

- 5 045232 ethylene glycol of the formula -NR ^N1 (CH2)s2(CH2CH2O)s1(CH2)s3NR ^N1 , where si is an integer from 1 to 10 (for example, from 1 to 6 or from 1 to 4), each of s2 and s3 is 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 each R ^N1 is independently hydrogen or optionally substituted C1. ₆ alkyl; (19) -NR ^H C(O)RI, where ^RH is selected from the group consisting of (a1) hydrogen and (b1) C1.6 alkyl, and RI is selected from the group consisting of (a2) C120 alkyl (for example, C1. 6alkyl), (b2)C2.20alkenyl (for example, C2.6alkenyl), (c2)C6.10aryl, (d2) hydrogen, (e2)C1.6alk-C6.10aryl, (f2) amino-C1.20alkyl, ( g2) polyethylene glycol of the formula (CH2)s2(OCH2CH2)s1(CH2)s3OR', where s1 is an integer from 1 to 10 (for example, from 1 to 6 or from 1 to 4), each of s2 and s3 independently represents an integer from 0 to 10 (for example, from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R' represents H or C1-20alkyl and (n2) aminopolyethylene glycol formula -NR ^N1 (CH2) _s2 (CH ₂ CH ₂ O) _s1 (CH ₂ ) _s3 NR ^N1 , where s1 is an integer from 1 to 10 (for example, from 1 to 6 or from 1 to 4), each of s2 and s3 independently represent 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 each R ^N1 independently represents hydrogen or optionally substituted C1-6alkyl; (20) -NR ^J C(O)OR ^K , where R ^J is selected from the group consisting of (a1) hydrogen and (b1) C1-6 alkyl, and R ^K is selected from the group consisting of (a2) C120 alkyl (for example, C1-6alkyl), (b2)C2-20alkenyl (e.g. C2-6alkenyl), (c2)C6-10aryl, (d2) hydrogen, (e2) C1.6alk-C6.10aryl, (f2) amino-C1.20alkyl , (g2) polyethylene glycol of the formula (CH2)s2(OCH2CH2)s1(CH2)s3OR', where s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), each of s2 and s3 independently is 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 C1.20alkyl, and ( h2) aminopolyethylene glycol of the formula -NR ^N1 (CH2) _s2 (CH ₂ CH ₂ O) _s1 (CH2) _s3 NR ^N1 , where s1 is an integer from 1 to 10 (for example, from 1 to 6 or from 1 to 4), each of s2 and s3 is 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 each R ^N1 is independently hydrogen or optionally substituted C ₁ . ₆ alkyl; and (21) amidine. In some embodiments, each of these groups may be further substituted as described herein.

The term aryl as used herein is a mono-, bicyclic or multicyclic carbocyclic ring system containing one or two aromatic rings. Examples are phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl, indanyl, indenyl and the like. Said system may be optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of: (1) C ₁ . ₇ acyl (eg carboxyaldehyde); (2) C ₁ . ₂₀ alkyl (e.g. _C1.6 alkyl, _C1.6 alkoxy _{- C1.6} alkyl _{, C1.6 alkylsulfinyl} - _C1.6 alkyl _, amino _{- C1.6} alkyl, azido _{- C1.6 alkyl} , ₍ carboxylaldehyde) _-C1.6alkyl , halo- _C1.6alkyl ( _e.g. perfluoroalkyl), _{hydroxy - C1.6alkyl , nitro - C1.6alkyl} or _{C1.6thioalkoxyC1.6alkyl} ) ; (3) C ₁ . ₂₀ alkoxy (for example, C ₁ . ₆ alkoxy, such as perfluoroalkoxy); (4) C1 ₆ alkylsulfinyl; (5) C ₆ . ₁₀ aryla; (6) amino; (7) C ₁ . ₆ alk-C ₆ . ₁₀ aryla; (8) azido; (9) C ₃ . ₈ cycloalkyl; (10) C1 ₆ alk-C ₃ . ₈ cycloalkyl; (11) halogen; (12) C ₁ . ₁₂ heterocyclyl (for example, C ₁ . ₁₂ heteroaryl); (13) (C1 ₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C ₁ . _{20 thioalkoxy (for example, C 1.6 thioalkoxy} ); (17) (CH ₂ ) _q CO ₂ R ^A where q is an integer from zero to four 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 aryla; (18) -(CH ₂ ) _q CONR R where q is an integer from zero to four, and where RB and R ^C are independently selected from the group consisting of (a) hydrogen, (b) C1.6alkyl, (c ) C6.10aryl and (d) C1.6alk-C6.10aryl; (19) -(CH2)qSO2R ^D where q is an integer from zero to four, and where R ^D is selected from the group consisting of (a) alkyl, (b) C6. ₁₀ aryl and (c) alk-C ₆ . ₁₀ aryla; (20) -(CH ₂ ) _q SO ₂ NR ^E R ^F where q is an integer from zero to four, and where each of R ^E and R ^F is independently selected from the group consisting of (a) hydrogen, (b ) C1 ₆ alkyl, (c) C ₆ . ₁₀ aryl and (d) C ₁ . ₆ alk-C ₆ . ₁₀ aryla; (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 (for example, C ₁ . ₆ alk-C ₁ . ₁₂ heteroaryl); (26) C2 ₂₀ alkenyl and (27) C ₂ . ₂₀ alkynyl. In some embodiments, each of these groups may be further substituted as described herein. For example, the alkylene group of a C 1 alkaryl or C ₁ -alkheterocyclyl group may be further substituted with an oxo group to give the corresponding aryloyl and (heterocyclyl)oyl substituent groups.

The term arylalkyl as used herein is an aryl group, as defined herein, attached to the parent molecular group via an alkylene group, as defined herein. Individual unsubstituted arylalkyl groups contain from 7 to 30 carbon atoms (e.g. 7 to 16 _or 7 _{to 20 carbon} atoms, examples are _C1.6alk - _C6.10aryl , _C2.12alk - _C6.10 aryl or _C1.20 alk- _{C6.10 aryl} ) _. In some embodiments, alkylene and aryl may each be further substituted with 1, 2, 3, or 4 substituent groups, as defined herein for the respective groups. Other groups preceded by the prefix alk- are defined in the same way, where alk refers to _C1 . ₆ alkylene, unless otherwise noted, and the attached chemical structure is as defined herein.

The term carbonyl as used herein represents a C(O) group, which may also be represented as C=O.

- 6 045232

The term carboxy as used herein means -CO2H.

The term cyano as used herein represents a -CN group.

The term cycloalkyl as used herein is a monovalent saturated or unsaturated non-aromatic cyclic hydrocarbon group containing from three to eight carbon atoms, unless otherwise indicated. Examples are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicycloheptyl and the like. When the cycloalkyl group includes one carbon-carbon double bond or one carbon-carbon triple bond, the cycloalkyl group may be referred to as a cycloalkenyl group or a cycloalkynyl group, respectively. Examples of cycloalkenyl and cycloalkynyl groups include cyclopentenyl, cyclohexenyl, cyclohexynyl and the like. The cycloalkyl groups of the present invention may be optionally substituted by: (1) C ₁ . ₇ acyl (for example, carboxyaldehyde); (2) C ₁ . ₂₀ alkyl (e.g. Lbalkyl, 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, halogen-C _{1 .6} alkyl (e.g. perfluoroalkyl), hydroxy- _Cl .alkyl, nitro-C _{1 .6} alkyl or C _{1 .6} thioalkoxy-C _{1 .6} alkyl); (3) C ₁ . ₂₀ alkoxy (for example, Cl. _b alkoxy, such as perfluoroalkoxy); (4) C ₁ . ₆ alkylsulfinyl; (5) C _b . ₁₀ aryl; (6) amino; (7) C1. _b αlk-C _b . ₁₀ aryl; (8) azido; (9) C ₃ . ₈ cycloalkyl; (10) C ₁ . ₆ αlk-C ₃ . ₈ cycloalkyl; (11) halogen; (12) C1. ₁₂ heterocyclyl (for example, C ₁ . ₁₂ heteroaryl); (13) (C ₁ . ₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C ₁ . _{20 thioalkoxy (for example, C 1.6 thioalkoxy} ); (17) -(CH ₂ ) _q CO ₂ R ^A where q is an integer from zero to four and R is selected from the group consisting of (a) C ₁ . ₆ alkyl, (b) C ₆ . ₁₀ aryl, (c) hydrogen and (d) C ₁ . ₆ αlk-C ₆ . ₁₀ aryla; (18) -(CH ₂ ) _q CONR ^B R ^C where q is an integer from zero to four, and B'T-»C' where R and R are independently selected from the group consisting of (a) hydrogen, ( b) C ₆ . ₁₀ alkyl, (c) C ₆ . ₁₀ aryl and (d) C ₁ . ₆ αlk-C ₆ . ₁₀ aryla; (19) -(CH ₂ )qSO ₂ R ^D where q is an integer from zero to four, and where R ^d is selected from the group consisting of (a) C6.10alkyl, (b) C6.10aryl and (c ) C1.6alk-C6.10aryl; (20) (CH2)qSO2NR ^E R ^F where q is an integer from zero to four, and where each of R ^E and R ^F is independently selected from the group consisting of (a) hydrogen, (b) C6. ₁₀ αlkyl, (c) C ₆ . ₁₀ aryl and (d) C ₁ . ₆ alk-C ₆ . ₁₀ aryla; (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 (for example, C ₁ . ₆ alk-C ₁ . ₁₂ heteroaryl); (26) oxo; (27) C ₂ . ₂₀ alkenyl and (28) C ₂ _ ₂₀ alkynyl. In some embodiments, each of these groups may be further substituted as described herein. For example, the alkylene group of a C ₁ -alkaryl or C 1 -alkheterocyclyl group may be further substituted with an oxo group to produce the corresponding aryloyl and (heterocyclyl)oyl substituent group.

The term diastereomer as used herein means stereoisomers that are not mirror images of each other and do not overlap each other.

The term enantiomer as used herein means each individual optically active form of a compound of the present invention having an optical purity or enantiomeric excess (determined by methods standard in the art) 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%.

The term halogen as used herein is a halogen selected from bromine, chlorine, iodine or fluorine.

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

The term heteroaryl as used herein means that subgroup of heterocyclyls as defined herein which are aromatic: that is, they contain 4n+2 pieelectrons in a mono- or multicyclic ring system. Examples of unsubstituted heteroaryl groups are those with 1-12 (eg 1-11, 1-10, 1-9, 2-12, 2-11, 2-10 or 2-9) carbon atoms. In some embodiments, the heteroaryl is substituted with 1, 2, 3, or 4 substituent groups as defined for a heterocyclyl group.

The term heteroarylalkyl refers to a heteroaryl group, as defined herein, attached to the parent molecular group via an alkylene group, as defined herein. Examples of unsubstituted heteroarylalkyl groups contain from 2 to 32 carbon atoms (for example, from 2 to 22, from 2 to 18, from 2 to 17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or 2 to 12 carbon _{atoms ,} examples are _C1.6αlk - _C1.12 heteroaryl, _C2.12alk - _C1.12 heteroaryl or _{C1.20alk - C1.12 heteroaryl )} . In some embodiments, each of alkylene and heteroaryl may

- 7 045232 be additionally substituted with 1, 2, 3 or 4 substituent groups defined in the present description for the corresponding group. Heteroarylalkyl groups are a subgroup of heterocyclylalkyl groups.

The term heterocyclyl as used herein is a 5-, 6-, or 7-membered ring containing, unless otherwise indicated, one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. A 5-membered ring contains zero to two double bonds, while 6- and 7-membered rings contain zero to three double bonds. Examples of unsubstituted heterocyclyl groups contain 1 to 12 (eg, 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbon atoms. The term heterocyclyl also represents a heterocyclic compound containing a bridged multicyclic structure in which one or more carbon atoms and/or heteroatoms links two non-adjacent monocyclic ring members, such as a quinuclidinyl group. The term heterocyclyl includes bicyclic, tricyclic and tetracyclic groups in which any of the above heterocyclic rings is fused to one, two or three carbocyclic rings, for example, an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring or another monocyclic heterocyclic ring, such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl and the like. Examples of fused heterocyclyls include tropanes and 1,2,3,5,8,8a-hexahydrondolysine. Heterocyclyls include pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morph olinyl, 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, including their dihydro- and tetrahydro forms, where one or more double bonds are reduced and replaced by hydrogens. Other 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 (for example, 2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl); 2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (for example, 2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5phenyl-1H-imidazolyl); 2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (for example, 2,3-dihydro-2-thioxo-5phenyl-1,3,4-oxadiazolyl); 4,5-dihydro-5-oxo-1H-triazolyl (for example, 4,5-dihydro-3-methyl-4-amino5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (for example, 1,2,3,4-tetrahydro-2,4dioxo-3,3-diethylpyridinyl); 2,6-dioxopiperidinyl (eg 2,6-dioxo-3-ethyl-3phenylpiperidinyl); 1,6-dihydro-6-oxopyrimidinyl; 1,6-dihydro-4-oxopyrimidinyl (for example, 2(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl); 1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (for example, 1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl); 1,6-dihydro-6-oxo-pyridazinyl (for example, 1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl (for example, 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl); 2,3-dihydro-2-oxo-III-indolyl (for example, 3,3-dimethyl2,3-dihydro-2-oxo-1H-indolyl and 2,3-dihydro-2-oxo-3,3'-spiropropane -1H-indol-1-yl); 1,3-dihydro-1oxo-2H-isoindolyl; 1,3-dihydro-1,3-dioxo-2H-isoindolyl; 1H-benzopyrazolyl (eg 1(ethoxycarbonyl)-1H-benzopyrazolyl); 2,3-dihydro-2-oxo-1H-benzimidazolyl (for example, 3-ethyl-2,3dihydro-2-oxo-1H-benzimidazolyl); 2,3-dihydro-2-oxobenzoxazolyl (eg 5-chloro-2,3-dihydro-2oxobenzoxazolyl); 2,3-dihydro-2-oxobenzoxazolyl; 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 (for example, 2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl); 1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (for example, 1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl); 1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (for example, 1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl); 1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (for example, 1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl); 2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naph[1,8-c,d]isothiazolyl and 1,8-naphthylenedicarboxamido. Additional heterocyclyls include 3,3a,4,5,6,6a-hexahydropyrrolo[3,4-b]pyrrol-(2H)-yl and 2,5-diazabicyclo[2.2.1]heptan-2yl, homopiperazinyl (or diazepanil) , tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanil, tiepanil, azocanil, oxecanil and thiocanil. Heterocyclic groups also include groups of the formula where

E' is selected from the group consisting of -N- and -CH-; F' selected from the group consisting of -N=CH-, -NH- 8 045232

CH ₂ -, -NH-C(O)-, -NH-, -CH=N-, -CH2-NH-, -C(O)-NH-, -CH=CH-, -CH ₂ -, - CH2CH2-, -CH2O, -OCH2-, -O- and S-; and G' is selected from the group consisting of -CH- and -N-. Any of the heterocyclyl groups specified herein may be optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of: (1) C _1-7 acyl (eg, carboxyaldehyde); (2) C _1-20 alkyl (for example, C _1-6 alkyl, C _1-6 α-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 (for example, C _1-6 alkoxy, such as perfluoroalkoxy); (4) C _1-6 alkylsulfinyl; (5) C _6-10 aryl; (6) amino; (7) C _1- 6αlk-C6 _-1 oaryl; (8) azido; (9) C _3-8 cycloalkyl; (10) C _1-6 alk-C _3-8 cycloalkyl; (11) halogen; (12) C1 ₁₂ heterocyclyl (eg C _2-12 heteroaryl); (13) (C _1-12 heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C _1-20 thioalkoxy (for example, C _1-6 thioalkoxy); (17) -(CH ₂ ) _q CO ₂ R ^A where q is an integer from zero to four and R is selected from the group consisting of (a) C _1-6 alkyl, (b) C _{6- 10} aryl, (c) hydrogen and (d) C _1-6 αlk-C _6-10 aryl; (18) -(CH2) _q CONR ^B R ^C where q is an integer from zero to four, and B'C' where R and R 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) -(CH ₂ ) _q SO ₂ R ^D where q is an integer from zero to four, and where D'

R 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) (CH ₂ ) _q SO ₂ NR ^E R ^F where q is an integer from zero to four, and where each of R ^E and R ^F is independently selected from the group consisting of (a) hydrogen, (b) C _1-6 αlkyl, (c) C _6-10 aryl and (d) C _1-6 alk-C ₆₁₀ aryl; (21) thiol; (22) C _6-10 aryloxy; (23) C _3-8 cycloalkoxy; (24) arylalkoxy; (25) C _1-6 αlk-C _1-12 heterocyclyl (for example, C _1-6 alk-C _1-12 heteroaryl); (26) oxo; (27) (C _1-12 heterocyclyl)imino; (28) C ₂₂₀ alkenyl and (29) C _2-20 α alkenyl. In some embodiments, each of these groups may be further substituted as described herein. For example, the alkylene group of a C 1 alkaryl or C ₁ -alkheterocyclyl group may be further substituted with an oxo group to provide the corresponding aryl and (heterocyclyl)oyl substituent group.

The term hydrocarbon as used herein is a group consisting only of carbon and hydrogen atoms.

The term hydroxyl as used herein represents an -OH group. In some embodiments, a hydroxyl group may be substituted with 1, 2, 3, or 4 substituent groups (eg, O-protecting groups) as defined herein for alkyl.

The term isomer as used herein means any tautomer, stereoisomer, enantiomer or diastereomer of any compound of the present invention. It is understood that the compounds of the present invention may have one or more chiral centers and/or double bonds and thus 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). In accordance with the present invention, the chemical structures depicted herein, and therefore the compounds of the present invention, include all corresponding stereoisomers, which are either stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and mixtures of enantiomers and stereoisomers, for example racemates. Enantiomeric and stereoisomeric mixtures of the compounds of the present invention can generally be separated into their enantiomer or stereoisomer components by well-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 a compound in a chiral solvent. Enantiomers and stereoisomers can be prepared from stereomerically or enantiomerically pure intermediates, reagents and catalysts by well known asymmetric synthetic methods.

The term N-protected amino as used herein refers to an amino group as defined herein to which one or two N-protecting groups as defined herein are attached.

The term N-protecting group as used herein represents groups designed to protect the amino group from unwanted reactions during synthesis. Conventionally used N-protecting groups are disclosed in Greene, Protective groups in Organic Synthesis, ^3rd Edition (John Wiley & Sons, New York, 1999), the contents of which are incorporated herein by reference. 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 auxiliaries such as protected or unprotected D, L or D, L-amino acids such as alanine, leucine, phenylalanine and the like; sulfonyl-containing groups such as benzenesulfonyl, p-toluenesulfonyl and the like; carbamate-forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, pnitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxy benzyloxycarbonyl, 2-nitro-4 ,5-dimethoxybenzyloxycarbonyl, 3,4,5- 9 045232 trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, a,a-dimethyl-3,5dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl silt, methoxycarbonyl , allyloxycarbonyl, 2,2,2,trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like and silyl groups , such as trimethylsilyl and the like. Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).

The term O-protecting group as used herein refers to groups designed to protect an oxygen-containing (eg phenol, hydroxyl or carbonyl) group from unwanted reactions during synthesis. Traditionally used 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 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, αchlorobutyryl, benzoyl, 4 -chlorobenzoyl, 4-bromobenzoyl, t-butyldimethylsilyl, triisopropylsilyloxymethyl, 4,4'-dimethoxytrityl, isobutyryl, phenoxyacetyl, 4isopropylpegenoxyacetyl, dimethylformamidino and 4-nitrobenzoyl; alkylcarbonyl groups such as acyl, acetyl, propionyl, pivaloyl and the like; optionally substituted arylcarbonyl groups such as benzoyl; silyl groups such as trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS), tri-isopropylsilyloxymethyl (TOM), triisopropylsilyl (TIPS), and the like; hydroxyl ester groups such as methyl, methoxymethyl, tetrahydropyranyl, benzyl, p-methoxybenzyl, trityl and the like; alkoxycarbonyls such as methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-isopropoxycarbonyl, n-butyloxycarbonyl, isobutyloxycarbonyl, sec-butyloxycarbonyl, t-butyloxycarbonyl, 2-ethylhexyloxycarbonyl, cyclohexyloxycarbonyl, methyloxycarbonyl and the like; alkoxyalkoxycarbonyl groups such as methoxymethoxycarbonyl, ethoxymethoxycarbonyl, 2-methoxyethoxycarbonyl, 2-ethoxyethoxycarbonyl, 2-butoxyethoxycarbonyl, 2-methoxyethoxymethoxycarbonyl, allyloxycarbonyl, propargyloxycarbonyl, 2-butenoxycarbonyl, 3-methyl-2-butenoxycarbonyl and the like; haloalkoxycarbonyls such as 2-chloroethoxycarbonyl, 2-chloroethoxycarbonyl, 2,2,2trichloroethoxycarbonyl and the like; optionally substituted arylalkoxycarbonyl groups such as benzyloxycarbonyl, p-methylbenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, pnitrobenzyloxycarbonyl, 2,4-dinitrobenzyloxycarbonyl, 3,5-dimethylbenzyloxycarbonyl, pchlorobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, fluorenylmethyloxycarbonyl and the like; and optionally substituted aryloxycarbonyl groups such as phenoxycarbonyl, pnitrophenoxycarbonyl, o-nitrophenoxycarbonyl, 2,4-dinitrophenoxycarbonyl, pmethylphenoxycarbonyl, m-methylphenoxycarbonyl, o-bromophenoxycarbonyl, 3,5dimethylphenoxycarbonyl, p-chlorophenoxycarbonyl, 2-chloro-4-nitrophenoxycarbonyl silt and the like); substituted alkyl, aryl and alkaryl 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 (eg, trimethylsilyl; triethylsilyl; triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl; t-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; and diphenylmethylsilyl); carbonates (eg 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 (eg, acetal and ketal groups such as dimethyl acetal, 1,3-dioxolane and the like; acyl groups; and dithian groups such as 1,3-dithiane, 1,3-dithiolane and the like); carboxylic acid protecting groups (eg, ester groups such as methyl ester, benzyl ester, t-butyl ester, orthoesters and the like) and oxazoline groups.

The term oxo as used herein represents =O.

The term polyethylene glycol as used herein is an alkoxy alkoxy chain containing one or more monomer units, and each monomer unit consists of OCH ₂ CH ₂ -. Polyethylene glycol (PEG) is also sometimes referred to as polyethylene oxide (PEO) or polyoxyethylene (POE), and these terms may be considered interchangeable for purposes of the present invention. For example, polyethylene glycol may have the structure -(CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 O-, where s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4) , and each of s2 and s3 is independently an integer from 0 to 10 (eg, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10). Polyethylene glycol may also be considered to include aminopolyethylene glycol of the formula

- 10 045232

NR ^N1 (CH ₂ ) _s2 (CH ₂ CH ₂ O) _s1 (CH ₂ ) _s3 NR ^N1 -, where s1 is an integer from 1 to 10 (for example, from 1 to 6 or from 1 to 4), each of s2 and s3 independently represent 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 each R ^N1 independently represents hydrogen or optionally substituted C _1-6 alkyl.

The term stereoisomer as used herein refers to any possible different isomeric as well as conformational forms in which a compound (e.g., a compound of any of the formulas described herein) may occur, in particular all stereochemical and conformational isomeric forms, all diastereomers, enantiomers and/or conformers of the basic molecular structure. Some compounds of the present invention may exist in various tautomeric forms, and all of the above are included within the scope of the present invention.

The term sulfonyl as used herein represents the group -S(O) ₂ -.

The term thiol as used herein represents a -SH group.

Definitions.

As used herein, the term administered in combination means that two or more agents are administered to a subject at the same time or interval such that the effects of each agent on the patient may be superimposed upon each other. In some embodiments, the agents are administered over 90 days (e.g., over 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 day(s)), over 28 days (for example, within 14, 7, 6, 5, 4, 3, 2 or 1 day(s), within 24 hours (for example, 12,6,5,4,3,2 or 1 hour, or in for about 60, 30, 15, 10, 5 or 1 minute after each other.In some embodiments, the agents are administered sufficiently close in time to each other that a combinational (eg, synergistic) effect is achieved.

As used herein, an antibody refers to a polypeptide whose amino acid sequence includes immunoglobulins and fragments thereof that specifically bind to said antigen, or a fragment thereof. Antibodies in accordance with the present invention can be of any type (eg, IgA, IgD, IgE, IgG or IgM) or subtype (eg, IgA1, IgA2, IgG1, IgG2, IgG3 or IgG4). Those of ordinary skill in the art will appreciate that the characteristic sequence or portion of an antibody may include amino acids found in one or more regions of the antibody (eg, variable region, hypervariable region, constant region, heavy chain, light chain, and combinations thereof). Moreover, those of ordinary skill in the art will appreciate that the characteristic sequence or portion of an antibody may include one or more polypeptide chains, and may include sequence elements found on the same polypeptide chain or on different polypeptide chains.

As used herein, an antigen binding fragment refers to a portion of an antibody that retains the binding characteristics of the original antibody.

The terms bifunctional chelate or bifunctional conjugate are used interchangeably herein and refer to a compound that contains a chelating moiety or a metal complex thereof, a linker moiety, and a therapeutic moiety, targeting moiety, or crosslinking moiety.

The term cancer refers to any type of cancer caused by the proliferation of malignant neoplastic cells such as tumors, neoplasms, carcinomas, sarcomas, leukemias and lymphomas. Solid tumor refers to cancer that includes an abnormal mass of tissue, such as sarcomas, carcinomas, and lymphomas. The terms hematologic cancer or fluid cancer, used interchangeably herein, mean cancers occurring in any body fluid, such as lymphomas and leukemias.

The term chelate as used herein refers to an organic compound or portion thereof that may be bonded to a central metal atom or a radioactive metal atom at two or more locations.

The term conjugate as used herein refers to a molecule that contains a chelating group or a metal complex thereof, a linker group, and which optionally contains a therapeutic moiety, a targeting moiety, or a cross-linking group.

As used herein, the term compound is intended to include all stereoisomers, geometric isomers, and tautomers of the structures depicted.

The compounds described herein may be asymmetric (eg, having one or more stereocenters). Unless otherwise stated, all stereoisomers, such as enantiomers and diastereomers, are meant. Compounds according to the present description, which contain asymmetrically substituted carbon atoms, can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically active starting materials are known in the art, examples being the resolution of racemic mixtures or stereoselective synthesis. For the compounds described herein, a variety of geometric olefin isomers, C=N double bond isomers may occur, and all such stable isomers are included herein. Geometric cis- and trans-isomers of the compounds according to the present description are described and can be isolated as a mixture of isomers or as individual isomeric forms.

The compounds herein also include tautomeric forms. Tautomeric forms result from the rearrangement of a single bond and an adjacent double bond and the corresponding migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonated states having the same empirical formula and net charge. Examples of prototropic tautomers include keto-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-imine pairs, and cyclic forms where the proton can occupy two or more positions of the 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 may be sterically capped by the corresponding substituent as a single form.

At various places herein, substituents of the compounds herein are disclosed in groups or ranges. The specification is hereby specifically intended to include each and every distinct subcombination of members of such groups and ranges. For example, it is assumed that the term C ₁ . ₆ alkyl specifically includes separately methyl, ethyl, ^alkyl, C ₄ alkyl, C ₅ alkyl and C ₆ alkyl. As used herein, a phrase of the form X is optionally substituted (eg, is an optionally substituted alkyl) is intended to be equivalent to the phrase X where X is optionally substituted (eg, alkyl, wherein said alkyl is optionally substituted). But this does not imply that feature X (eg, alkyl) is itself optional.

As used herein, a detection agent refers to a molecule or atom that is suitable for use in diagnosing a disease by determining the location of cells containing an antigen. Various methods for labeling polypeptides with detection agents are known in the art. Examples of detection agents include, but are not limited to, radioactive isotopes and radionuclides, dyes (such as biotinstreptavidin complex), contrast agents, luminescent agents (for example, fluorescein isothiocyanate (FITC), rhodamine, lanthanide-based phosphors, cyanine and dyes operating in near-infrared spectrum) and magnetic agents such as gadolinium chelates.

As used herein, the term radionuclide refers to an atom capable of undergoing 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, ¹ⁿ 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 terms radioactive nuclide, radioisotope or radioactive isotope can also be used to describe a radionuclide. Radionuclides can be used as detection agents as described above. In some embodiments, the radionuclide may be an alpha-emitting radionuclide.

The term effective amount of an agent (eg, any of the above conjugates) as used herein is an amount sufficient to achieve preferred or desired results, such as clinical results, and as such, the effective amount depends on the context in which the term is used.

The term immunoconjugate as used herein refers to a conjugate that includes a targeting moiety, such as an antibody, nanobody, affibody, or fibronectin type III domain consensus sequence. In some embodiments, the immunoconjugate contains an average of at least 0.10 conjugates per targeting moiety (e.g., an average of 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).

The term radioconjugate as used herein refers to any conjugate that includes a radioactive isotope or radionuclide, such as any of the radioactive isotopes or radionuclides described herein. In some embodiments, the radioactive isotope or radionuclide is a metal chelate.

The term radioimmunoconjugate as used herein refers to any immunoconjugate that includes a radioactive isotope or radionuclide, such as any of the radioactive isotopes or radionuclides described herein. In some embodiments, the radioactive isotope or radionuclide is a metal chelate.

The term radioimmunotherapy as used herein means a method in which a radioimmunoconjugate is used to achieve a therapeutic effect. In some embodiments, radioimmunotherapy may include administering a radioimmunoconjugate to a subject in need thereof, wherein administration of the radioimmunoconjugate achieves a therapeutic effect in the subject. In some embodiments, radioimmunotherapy may include introducing a radioimmunoconjugate into a cell, wherein administration of said radioimmunoconjugate kills said cell. In this case, radioimmunotherapy involves the selective destruction of a cell, in some embodiments, said cell is a cancer cell of a subject having cancer.

- 12 045232

The term pharmaceutical composition as used herein is a composition containing a compound described herein formulated with a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is prepared or sold with regulatory approval as part of a therapeutic regimen for treating a disease in a mammal. Pharmaceutical compositions can be prepared, for example, for oral administration in unit dosage form (eg, tablet, capsule, gelatin capsule, or syrups); for topical use (eg, as a cream, gel, lotion, or ointment); for intravenous administration (eg, as a sterile solution containing no particulate emboli and as a solvent system suitable for intravenous use); or in any other composition described herein.

The term pharmaceutically acceptable excipient as used herein means any ingredient other than the compounds described herein (eg, a carrier capable of suspending or solubilizing the active compound) that is not toxic and does not cause inflammation in a patient. Excipients may include, for example: release agents, antioxidants, binders, coatings, pressing aids, disintegrants, dyes, emollients, emulsifiers, fillers, film formers or coatings, flavors, fragrances, glidants. , lubricants, preservatives, printing inks, radioprotectors, sorbents, suspending or dispersing agents, sweeteners or water of hydration. Examples of 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, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, qi sodium waste, sodium starch glycolate , sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C and xylitol.

The term "pharmaceutically acceptable salt" as used herein means those salts of the compounds described herein which, in the judgment of medical professionals, are suitable for use in contact with tissues of humans and animals without undesirable toxicity, irritation or allergic reactions. Pharmaceutically acceptable salts are well 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, (Eds. P.H. Stahl and C.G. Wermuth), Wiley- VCH, 2008. These salts can be prepared in situ during the final isolation and purification of the compounds described herein, or separately by reaction of the free base group with a suitable organic acid.

The compounds of the present invention may have ionizable groups to provide pharmaceutically acceptable salts. These salts may be acid addition salts, including inorganic or organic acids, or, in the case of acidic forms of the compounds, the salts may be derived from inorganic or organic bases. Often the compounds are prepared or used in the form of pharmaceutically acceptable salts obtained as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well known in the art, examples are hydrochloric, sulfuric, hydrobromic, acetic, lactic, citric or tartaric acids for the preparation of acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines for obtaining basic salts. Methods for preparing suitable salts are well known in the art.

Exemplary acid addition salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glu coheptonate, glycerophosphate, hemisulfate , heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate and valerate salts. Exemplary alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium and magnesium salts, as well as non-toxic ammonium, quaternary ammonium and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, three

- 13 045232 ethylamine and ethylamine.

The term therapeutic moiety as used herein 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, or an antigen-binding fragment thereof. In some embodiments, the therapeutic moiety is a small molecule.

The term targeting moiety as used herein 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 antigen binding fragment thereof, nanobody, affibody, or fibronectin type III domain consensus sequences.

The term cross-linking group as used herein refers to any reactive group that is capable of joining two or more molecules through a covalent bond. In some embodiments, the cross-linking group is an amino-reactive or thiol-reactive cross-linking group. In some embodiments, the amino-reactive or thiol-reactive cross-linking group includes an activated ester, such as a hydroxysuccinimide ester, a 2,3,5,6-tetrafluorophenol ester, a 4-nitrophenol ester or imidate, an anhydride, a thiol, a disulfide, a maleimide, an azide, an alkyne, strained alkyne, strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide, diazirine, phosphine, tetrazine, isothiocyanate. In some embodiments, the crosslinking group may be glycine-glycine-glycine and/or leucine-proline-(any amino acid)-threonine-glycine, which sequences are recognition sequences for attaching targeting agents via a linker using a sortase-mediated coupling reaction. One of ordinary skill in the art will appreciate that the use of crosslinking groups in the practice of the present invention is not limited to the specific designs disclosed herein, but rather may include the use of other known crosslinking groups.

The term polypeptide as used herein refers to a chain of at least two amino acids linked to each other by a peptide bond. In some embodiments, the polypeptide may include at least 3-5 amino acids, each of which is attached to the others as at least one peptide bond. Those of ordinary skill in the art will appreciate that polypeptides may include one or more unnatural amino acids or other compounds that are capable of being integrated into a polypeptide chain. 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 (eg, an antibody polypeptide) may contain two or more distinct polypeptide chains, which for some classes may be linked to each other, for example, through one or more disulfide bonds or other means.

Subject refers to a human or non-human animal (such as a mammal).

By substantially identical or essentially identical is meant a polypeptide sequence that has a polypeptide sequence that is the same as that of the reference sequence or has a certain percentage of amino acid residues that are the same at the same location as the reference sequence when the two sequences are optimally superimposed . 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 sequences compared will generally be 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 (example is full length sequence). Sequence identities can be determined using sequence analysis software with standard settings (eg, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Such software can find similar sequences and determine the degree of homology of various substitutions, deletions and other modifications.

As used herein, and as is apparent in the art, the term treat a condition or treat a condition (eg, the conditions described herein, such as cancer) means an approach to obtain preferred or desired results, such as clinical results. Preferred or desired results may include, but are not limited to, alleviation or improvement of one or more symptoms or conditions, reduction in the severity of a disease, disorder or condition; stabilization (i.e. no deterioration) of the condition

- 14 045232 diseases, disorders or conditions; preventing the spread of a disease, disorder or condition; delaying or slowing the progression of a disease, disorder or condition; improvement or mitigation of a disease, disorder or condition and remission (partial or complete), detectable or undetectable. Alleviation of a disease, disorder or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder or condition are reduced and/or the progression of the disease is attenuated or prolonged compared to the extent or duration of the disease in the absence of treatment.

Brief description of drawings

In fig. 1 schematically depicts the general structure of a conjugate containing a chelate, a linker and a cross-linking group (top) and a conjugate containing a chelate, a linker and a targeting moiety (bottom).

In fig. Figure 2 schematically shows the synthesis of the bifunctional chelate, 4-{[11-oxo-11-(2,3,5,6tetrafluorophenoxy)undecyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7 ,10-tetraazacyclododecan-1yl]butanoic acid (compound B). The synthesis of compound B is described in example 3.

In fig. Figure 3 schematically depicts the synthesis of the bifunctional chelate, 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4 ,7,10-tris(carboxymethyl)-1,4,7,10tetraazacyclododecan-1-yl]butanoic acid (compound C). The synthesis of compound C is described in example 4.

In fig. 4 is a series of graphs depicting the metabolic clearance profile of untargeted human IgG antibody conjugates [ ¹⁷⁷ Lu]-Compound B-HuMIgG and [ ¹⁷⁷ Lu]-Compound CHuMIgG compared to [ ¹⁷⁷ Lu]-Compound A-HuMIgG, the procedures and results are described in detail in example 7.

Detailed description

Radiolabeled targeting moieties (also known as radioimmunoconjugates) are designed to target a protein or receptor that is activated by a disease to deliver a radioactive load to kill target cells (radioimmunotherapy). The process of delivering such a load, through radioactive decay, is accompanied by the emission of alpha, beta or gamma particles or Auger electrons, which can cause direct effects on DNA (such as single- or double-strand breaks) or indirect effects such as side effects. observer or crossfire effects.

Radioimmunoconjugates typically contain a biological targeting moiety, a radioisotope, and a conjugate that binds them. Conjugates are formed when a bifunctional chelate is attached to a biological targeting molecule in such a way that structural changes to the compound remain minimal while maintaining affinity for the target. After the introduction of a radioactive label, the final radioimmunoconjugate is formed.

The structures of bifunctional chelates contain a chelate, a linker, and a cross-linking group (Figure 1). When developing new bifunctional chelates, the greatest effort is concentrated on the chelating region of the molecule. Several examples of bifunctional chelates with various cyclic and acyclic structures have been described and conjugated to a targeting moiety [Bioconjugate Chem. 2000, 11, 510-519, Bioconjugate Chem. 2012, 23, 1029-1039, Mol Imaging Biol (2011) 13:215-221, Bioconjugate Chem. 2002, 13, 110-115].

One of the key factors in developing safe and effective radioimmunoconjugates is to maximize efficacy while minimizing off-target toxicity in normal tissue. Considering that this statement is one of the fundamental principles in the development of new drugs, its application to radioimmunotherapy agents poses new challenges. To be therapeutically effective, radioimmunoconjugates must not block the receptor, which is required in the case of a therapeutic antibody, or release a cytotoxic load intracellularly, which is required in the case of an antibody-drug conjugate. However, the release of a toxic particle is an event that occurs as a result of first-order (radioactive) decay and can occur randomly anywhere in the body after administration. Once the particle is released, damage to surrounding cells within the emission range may occur, leading to potential off-target toxicity. Accordingly, limiting the exposure of normal tissue to this emission is a key factor in the development of new drugs [Bioconjugate Chem. 2006, 17, 1551-1560, Bioconjugate Chem. 2003, 14, 927-933].

One possible way to reduce off-target exposure is to remove radioactivity from the body more efficiently. The most obvious mechanism is an increase in the rate of clearance of the biological targeting agent. This approach also likely requires finding ways to reduce the half-life of the biological agent, an issue that has not been well documented for biological targeting agents. Regardless of the mechanism, increasing drug clearance will also negatively impact pharmacodynamics/efficacy in that faster clearance of the drug from the body will result in lower effective concentrations at the site of action, which in turn will require more administration. high total dose and will not achieve the desired results in reducing the total radioactive dose to normal tissues.

Other studies have been aimed at speeding up the metabolism of the region of the molecule containing the radioactive fragment. To this end, some attempts have been made to increase the rate of cleavage of the radioactive fragment from biological targeting agents by using so-called cleavable linkers. However, in relation to radioimmunoconjugates, the term cleavable linker has acquired different meanings. Cornelissen, et al. described cleavable linkers as those by which a bifunctional conjugate is attached to a biological targeting agent via a reduced cysteine residue, while other authors have described the use of enzymatically cleavable systems, which involves co-administration of a radioimmunoconjugate with a cleaving agent/enzyme to achieve release [Mol Cancer Ther; 12(11) November 2013, Methods in Molecular Biology, 2009, 539, 191-211, Bioconjugate chemistry, Volume 14, Issue 5, p. 92733 (2003)]. These methods either lead to a change in the nature of the biological targeting moiety, in the case of a cysteine bond, or are not promising for drug development (enzymatically degradable systems) because, as described in the above sources, these methods require the administration of 2 agents.

The main idea of the embodiments described herein is to more efficiently remove radioactivity from the body after catabolism and/or metabolism of the radioimmunoconjugate by making modifications to the linker region of the bifunctional chelate.

This approach is novel because there appears to be little information describing the effects of the linker in vivo, particularly with respect to radioimmunoconjugates. One possible reason for this finding is the expectation that, following catabolism/metabolism of the radioimmunoconjugate, the radiolabeled conjugate will undergo rapid systemic clearance. This assumption was developed experimentally when the bifunctional chelate was administered separately; it was cleared from the bloodstream more quickly than a radioimmunoconjugate with the same bifunctional chelate. Based on these data, it can be expected that after catabolism/metabolism of the radioimmunoconjugate, the metabolite containing the bifunctional chelate will also undergo rapid clearance.

However, rapid clearance of metabolites containing the radiolabeled conjugate does not necessarily occur in vivo. Based on the results described below, it was found that the linker region of bifunctional chelates can directly influence the removal of radioactivity from the body after catabolism of the radioconjugate, but does not affect the general properties of the radioimmunoconjugate in vitro or its pharmacokinetic and pharmacodynamic parameters in vivo. The following data indicate that certain commercially available bifunctional chelates exhibit slower rates and lower rates of removal of total radioactivity from the body compared to the compounds of the embodiments described herein.

The release profiles of the compounds according to the embodiments described in the examples present unexpected results. As previously reported, Quadri and Vriesendorp [Q. J. Nucl. Med. 1998, 42, 250-261], simple modifications of the linker region of the bifunctional chelate, regardless of their hydrophobicity, did not affect the excretion of radioactivity in the urine. The results presented below clearly indicate that both hydrophobic and hydrophilic linkers can influence clearance profiles. Additionally, the examples below demonstrate that hepatobiliary clearance also plays a role in the elimination process.

Thus, in the embodiments described herein, bifunctional chelates have been identified that, when attached to biological targeting moieties, provide a reduction in total radioactivity in the body by increasing the rate of elimination of catabolic/metabolic products while maintaining pharmacokinetic parameters for the intact molecule when compared with similar bifunctional chelates available in the public domain. It was found that this decrease in total radioactivity in the body is due to the clearance of catabolic/metabolic by-products and does not affect other in vitro and in vivo properties, such as the degree of specificity (in vitro binding), cellular retention and in vivo tumor uptake. In general, these embodiments provide the desired properties of radioimmunoconjugates by reducing the radioactive load on the body while maintaining activity on the target.

Therapeutic moieties and targeting moieties.

Therapeutic moieties include any molecule or any fragment 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. Targeting moieties include any molecule or any fragment of a molecule that binds to a given target. In some embodiments, the targeting fragment is

- 16 045232 protein or polypeptide, such as antibodies or antigen-binding fragments thereof, nanobodies, affibodies and consensus sequences from fibronectin type III domains (for example, centirins or adnectins).

Polypeptides.

Polypeptides include, for example, any of a variety of hematological agents (including, for example, erythropoietin, coagulation factors, etc.), interferons, colony-stimulating factors, antibodies, enzymes and hormones. The identity of a particular polypeptide is not limited by the scope of the present invention, and any target polypeptide can be used in the methods of the present invention.

A reference polypeptide as described herein may comprise a target binding domain that binds to a desired target (eg, binds an antigen). For example, a polypeptide, such as an antibody, can bind to a transmembrane polypeptide (eg, receptor) or ligand (eg, growth factor). Exemplary molecular targets (eg, antigens) for the polypeptides described herein (eg, antibodies) include CD proteins such as CD2, CD3, CD4, CD8, CD11, CD19, CD20, CD22, CD25, CD33, CD34, CD40 , CD52; members of the ErbB receptor family, such as epidermal growth factor receptor (EGF) (EGFR, HER1, ErbB1), HER receptor2 (ErbB2), HER3 (ErbB3) or HER4 (ErbB4); 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 αγβ3 integrin, including either their α or β subunits (e.g., anti-CD11a, anti-CD18, or anti-CD11b antibodies) ; growth factors and receptors such as EGF, fibroblast growth factor receptor (FGF) (eg, FGFR3) and vascular endothelial growth factor (VEGF); IgE; cytokines such as IL1; cytokine receptors such as IL2 receptor; blood group antigens; flk2/flt3 receptor; obesity receptor (OB); mpl receptor; CTLA-4; protein C; neuropilins; ephrins and their receptors; netrins and their receptors; fusion proteins and their receptors; 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 9 (PCSK9), sclerostin, c-kit, Tie-2, c-fms, and anti-M1.

Antibodies.

An IgG antibody consists of two identical light polypeptide chains and two identical heavy polypeptide chains linked through disulfide bonds. The first domain, located at the N-terminus of each chain, is variable in amino acid sequence, which provides the antibody binding specificity found for each individual antibody. Variable domains are known as variable heavy (VH) and variable light (VL) regions. The other domains of each chain are relatively invariant in amino acid sequence and are known as constant heavy (CH) and constant light (CL) regions. In the case of an IgG antibody, the light chain contains one variable region (VL) and one constant region (CL). An 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).

Antibodies according to the present description 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-idiotypic (anti-Id) antibodies and antigen-binding fragments of any of the above antibodies. Antibodies can be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

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

The antibodies or fragments described herein can be prepared by any antibody synthesis method known in the art (see, for example, 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; WO 92/22324; WO 98/46645). Chimeric antibodies can be prepared using methods described in, for example, Morrison, 1985, Science 229:1202, and humanized antibodies using methods described, for example, in US Patent No. 6,180,370.

Other antibodies described herein are bispecific antibodies and multivalent antibodies described in, for example, Segal et al., J. Immunol. Methods 248:1-6 (2001); And

Tutt et al., J. Immunol. 147:60 (1991).

Antibodies to insulin-like growth factor receptor 1 (IGF-1R)

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

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

Nanobodies.

Nanobodies are antibody fragments consisting of a single monomeric antibody variable domain. Nanobodies can also be called single-domain antibodies. Like antibodies, nanobodies selectively bind to a specific antigen. Nanobodies may be heavy chain variable domains or light chain domains. Nanobodies can occur naturally or be the product of biological engineering. Nanobodies can be biologically engineered through site-directed mutagenesis or mutagenic screening (eg, phage display, yeast display, bacterial display, mRNA display, ribosomal display).

Affitel.

Affibodies are polypeptides or proteins designed to bind to a specific antigen. Thus, affibodies can be considered to mimic some of the functions of antibodies. Affibodies may be engineered variants of the B domain in the immunoglobulin binding region of Staphylococcus protein A. Affibodies may be engineered variants of the Z-domain, B-domain, which has lower affinity for the Fab region. Affibodies can be biologically engineered through site-directed mutagenesis or mutagenic screening (eg, phage display, yeast display, bacterial display, mRNA display, ribosomal display).

Affibody molecules have been produced that exhibit specific binding to a variety of different proteins (e.g., insulin, fibrinogen, transferrin, tumor necrosis factor-α, IL-8, gp120, CD28, human serum albumin, IgA, IgE, IgM, HER2, and EGFR) and characterized by the degree of affinity (K _d ) in the range from µM to pM.

Fibronectin type III domains

The fibronectin type III domain is an evolutionarily conserved protein domain found in a variety of extracellular proteins. The fibronectin type III domain has been used as a molecular scaffold to produce molecules capable of selectively binding a specific antigen. Variants of fibronectin type III (FN3) domains that have been engineered to bind selectively may also be referred to as monobodies. FN3 domains can be biologically engineered through site-directed mutagenesis or mutagenic screening (eg, CIS display, phage display, yeast display, bacterial display, mRNA display, ribosomal display).

Modified polypeptides.

The polypeptides of the present invention may contain a modified amino acid sequence. The modified polypeptides may be substantially identical to the corresponding reference polypeptide (e.g., the amino acid sequence of the modified polypeptide may be at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical amino acid sequence of the reference polypeptide). In some embodiments, the modification does not significantly interfere with the desired biological activity. The modification may reduce (e.g., by at least 5, 10, 20, 25, 35, 50, 60, 70, 75, 80, 90, or 95%), may have no effect, or may increase (e.g., by at least 5 , 10, 25, 50, 100, 200, 500 or 1000%) biological activity of the original polypeptide. The modified polypeptide may have the same or optimized characteristics of the parent polypeptide, such as in vivo stability, bioavailability, toxicity, immunological activity, immunological identity and conjugation ability.

Modifications include modifications due to natural processes such as post-translational processing, or modifications by chemical methods known in the art. Modifications may be present at any portion of the polypeptide, including the polypeptide backbone, amino acid side chains, and the N- or C-terminal amino acid. Modifications of the same type may be present to the same or varying degrees at multiple sites on a given polypeptide, and a polypeptide may contain modifications of more than one type. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with

- 18 045232 with or without branching. Cyclic, branched and branched cyclic polypeptides can be formed by natural post-translational processes or can be produced synthetically. Other modifications include pegylation, acetylation, acylation, addition of an acetamidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, carboxyethylation, esterification, covalent attachment to a 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 (eg, fluorescent or radioactive), covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-linking, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI-anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, tRNA-mediated addition of amino acids to proteins such as arginylation and ubiquitination.

The modified polypeptide may also contain an amino acid insertion, deletion or substitution, either conservative or non-conservative (eg, D-amino acids, deamino acids) in the polypeptide sequence (eg, in cases where such changes do not substantially affect the biological activity of the polypeptide). In particular, the addition of one or more cysteine residues to the N- or C-terminus of any of the polypeptides of the present invention may facilitate conjugation of these polypeptides, through, for example, the formation of a disulfide bond. For example, the polypeptide may be modified to contain a single cysteine residue at the N-terminus or a single cysteine residue at the C-terminus. Amino acid substitutions can be conservative (ie, where the residue is replaced by another residue belonging to the same general type or group) or non-conservative (ie, where the residue is replaced by an amino acid of a different type). In addition, a naturally occurring amino acid may be replaced by a non-natural amino acid (ie, a conservative non-natural amino acid substitution or a non-conservative non-natural amino acid substitution).

Synthetically produced polypeptides may contain substitutions for amino acids not naturally encoded by DNA (eg, non-naturally occurring or unnatural amino acids). Examples of non-naturally occurring amino acids include D-amino acids, N-protected amino acids, an amino acid containing an acetylaminomethyl group attached to a cysteine sulfur atom, a pegylated amino acid, omega amino acids of the formula NH2(CH2) _n COOH, where n is 2-6, neutral non-polar amino acids such as sarcosine, tert-butylalanine, tert-butylglycine, N-methylisoleucine and norleucine. Phenylglycine can be a substitute for Tip, Tyr or Phe; citrulline and methionine sulfoxide are neutral non-polar, cysteic acid is acidic, and ornithine is basic. Proline can be replaced by hydroxyproline while maintaining the ability to provide conformation.

Analogs can be obtained using replacement mutagenesis and retain the biological activity of the original polypeptide. Examples of substitutions designated as conservative substitutions are presented in Table. 1. If such substitutions lead to an undesirable change, then substitutions of a different type are introduced, indicated in the table. 1 as exemplary substitutions further described herein with reference to amino acid classes, and screen products.

- 19 045232

Table 1

Amino acid substitutions

Original balance Examples of substitutions Conservative substitution Ala (A) Val, Leu, Ile Val Arg(R) Lys, Gln, Asn Lys Asn(Ν) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys(C) Ser Ser Gln (Q) Asn Asn Glu(E) Asp Asp Gly (G) Pro Pro N represents (Η) Asn, Gln, Lys, Arg Arg lle(l) Leu, Val, Met, Ala, Phe, pogleucine Leu Leu (L) Mogleucine, Ile, Val, Met, Ala, Phe lie Lys (K) Arg, Gln, Asn Arg Met(M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala Leu Pro (P) Gly Gly Ser(S) Thr Thr Thr (T) Ser Ser Trp(W) Tyr Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val(V) Ile, Leu, Met, Phe, Ala, norleucine Leu

Significant modifications of function or immunological properties are achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the backbone structure of the polypeptide at the site of the substitution, such as a sheet or helical conformation, (b) a change or hydrophobicity of the molecule at the target site, or (c) side chain volume.

Stitching groups.

A cross-linking group is a reactive group that is capable of joining two or more molecules through a covalent bond. Crosslinkers can be used to attach a linker and chelating moiety to a therapeutic or targeting moiety. Cross-linking groups can also be used to attach a linker and chelating moiety to a target in vivo. In some embodiments, the cross-linking group is an amino- or thiol-reactive cross-linking group or sequence for sortase-mediated addition. In some embodiments, the amino-reactive, methionine-reactive, or thiol-reactive cross-linking group comprises an activated ester, such as a hydroxysuccinimide ester, a 2,3,5,6-tetrafluorophenol ester, a 4-nitrophenol ester or an imidate, an anhydride, a thiol, a disulfide, a maleimide, azide, alkyne, strained alkyne, strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide, diazirine, phosphine, tetrazine, isothiocyanate or oxaziridine. In some embodiments, the sortase recognition sequence may comprise a terminal glycine-glycine-glycine (GGG) sequence and/or the amino acid sequence LPTXG, where X is any amino acid. One of ordinary skill in the art will appreciate that the use of cross-linking groups in the practice of the present invention is not limited to the specific designs disclosed herein, but rather may include the use of other known cross-linking groups.

Detection agents.

A detection agent is a molecule or atom that is administered conjugated to a polypeptide, such as an antibody or an antigen-binding fragment thereof, and that is useful in diagnosing a disease by locating cells containing the antigen, planning radiation treatment, or treating a disease. Suitable for

- 20 045232 applications detection agents include, but are not limited to, radioactive isotopes, dyes (such as in the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules, luminescent agents and enhancing agents (such as paramagnetic ions) for magnetic resonance imaging imaging (MRI). To connect a polypeptide moiety to a detection agent, it may be necessary to react it with a reagent containing a linker to which the detection agent or multiple detection agents are attached.

Radioactive isotopes 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.

Chelating moieties.

Chelating moieties known in the art for their usefulness as detection agents include, but are not limited to, DOTA (1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R, 4R,7R,10R)-a, α', α, α'tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10tetrakis(carbamoylmethyl )-1,4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane1,4,7,10-tetrapropionic acid), DO3AM-acetic acid (2-(4,7,10-tris (2-amino-2-oxoethyl)1,4,7,10-tetraazacyclododecan-1-yl)acetic acid), DOTA-GA anhydride (2,2',2-(10-(2,6dioxotetrahydro-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 (1,4,6,10tetraazacyclododecane-1,4,7,10-tetramethylenephosphonic acid, DOTA-4AMP (1,4,7,10tetraazacyclododecane-1,4,7,10-tetrakis(acetamidomethylenephosphonic acid) , CB-TE2A (1,4,8,11 tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7triacetic 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, 11tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), HEHA (1,4,7,10,13,16hexaazacyclohexadecane-1,4,7,10,13,16-hexacetic acid), PEPA (1,4, 7,10,13pentaazacyclopentadecane-N,N',N,N', N-pentaacetic acid), H ₄ octapa (N,N'-bis(6-carboxy-2pyridylmethyl)-ethylenediamine-N,N'-diacetic acid) , H ₂ dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]methylamino]ethane), H ₆ hospa (N,N'-(methylenephosphonate)-N,N'-[6-( methoxycarbonyl)pyridin-2-yl]-methyl1,2-diaminoethane), TTHA (triethylenetetramine-N,N,N',N,N', N'''-hexacetic acid), DO2P (tetraazacyclododecane dimethanephosphonic acid), HP- DO3A (hydroxypropyltetraazacyclododecanetriacetic acid), EDTA (ethylenediaminetetraacetic acid), deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), HOPO (hydroxypyridinone octadentate) or porphyrins. Chelating groups can be used as metal-chelate combinations with metals such as manganese, iron and gadolinium, and isotopes (e.g. isotopes with a general energy range of 60 to 4000 keV) such as 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, ⁿ¹ 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 Th.

Linkers.

Linkers according to the present invention may have the structure of formula I:

IA-L ¹ -(L ² )nB,

Formula I where A represents a chelating moiety or its complex with a metal;

L ¹ represents optionally substituted C ₁ -C ₆ -alkyl, substituted C ₁ -C ₆ -heteroalkyl, substituted aryl or heteroaryl;

B is a therapeutic moiety targeting moiety or cross-linking group, or a pharmaceutically acceptable salt thereof;

n is 1-5;

each L ² independently has the structure:

(-X ¹ -L ³ -Z ¹ -),

Formula 0 where X ¹ represents 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(NR1), -CH2Ph(NH)C=S(NR1), O, NR ¹ , and R ¹ is H or optionally substituted C1-C ₆ alkyl or optionally substituted C ₁ -C ₆ heteroalkyl, substituted aryl or heteroaryl;

L ³ represents optionally substituted C ₁ -C ₅₀ -alkyl or optionally substituted C1C ₅₀ -heteroalkyl or C ₅ -C ₂₀ -polyethylene glycol;

Z ¹ represents CH ₂ , C=O, C=S, OC=O, NR1C=O, NR ¹ , and R ¹ represents hydrogen or

- 21 045232 is an optionally substituted C ₁ -C ₆ -alkyl, pyrrolidine-2,5-dione.

The conjugates of the present invention contain three separate modules, which together provide increased efficiency compared to solutions known in the art.

1. Chelating fragment or its complex with a metal:

Module A is intended to be included in a detection agent (eg, a chelating moiety or a metal complex thereof). The metal complex may include an imaging radionuclide.

2. Linkers:

The linkers according to the present invention have the structure of formula I:

IA-L ¹ -(L ² ) _n -B,

Formula I where A represents a chelating moiety or its complex with a metal;

L ¹ represents optionally substituted C ₁ -C ₆ -alkyl, substituted C ₁ -C ₆ -heteroalkyl, substituted aryl or heteroaryl;

B is a therapeutic moiety targeting moiety or cross-linking group, or a pharmaceutically acceptable salt thereof;

n is 1-5;

each L ² independently has the structure:

(-X ¹ -L ³ -Z ¹ -),

Formula 0 where X ¹ represents 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), -CH2Ph(NH)C=S(NR ¹ ), O, NR ¹ , and R ¹ is H or optionally substituted C1-C ₆ alkyl or optionally substituted C ₁ -C ₆ heteroalkyl , substituted aryl or heteroaryl;

L ³ represents optionally substituted C ₁ -C ₅₀ -alkyl or optionally substituted C _{1 -C 50} -heteroalkyl or C ₅ -C ₂₀ -polyethylene glycol;

Z ¹ is CH2, C=O, C=S, OC=O, NR ¹ C=O, NR ¹ , and R ¹ is hydrogen or optionally substituted C1-C ₆ alkyl, pyrrolidine-2,5-dione .

3. Therapeutic fragment, targeting fragment or cross-linking group:

module B is a therapeutic moiety (eg, antibodies, antigen binding fragments), a targeting moiety (eg, nanobodies, affibodies, fibronectin type III domain consensus sequences), or a cross-linker (eg, an amino-reactive, thiol-reactive cross-linker, or sortase-mediated coupling sequence) .

Administration and doses.

The present invention also provides pharmaceutical compositions that contain a therapeutically effective amount of a compound of the present invention. The compositions can be prepared for use in various drug delivery systems. To obtain suitable formulations, one or more physiologically acceptable excipients or carriers may also be included in the composition. Suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed., 1985. A summary of drug delivery methods can be found, for example, in Langer (Science 249:1527- 1533, 1990).

The pharmaceutical compositions are intended for parenteral, intranasal, topical, oral or topical administration, such as through the skin, for prophylactic and/or therapeutic treatment. The pharmaceutical compositions may be administered parenterally (eg, by intravenous, intramuscular, or subcutaneous injection), or by oral administration, or by topical application, or intra-articular injection at sites affected by a vascular or cancerous condition. Additional routes of administration include intravascular, intraarterial, intratumoral, intraperitoneal, intraventricular, intraepidural, as well as nasal, ophthalmic, intrascleral, intraorbital, rectal, topical, or aerosol inhalation. Sustained release administration is also specifically included in the present invention through means such as depot injections or disintegrable implants or components. Thus, the present invention provides compositions for parenteral administration that contain the above agents dissolved or suspended in a suitable vehicle, preferably an aqueous vehicle, such as, but not limited to, water, buffered water, saline or phosphate buffered saline (PBS). . The compositions may contain pharmaceutically acceptable auxiliary substances necessary to adjust conditions to physiological values, such as, but not limited to, pH adjusting agents, buffering agents, tonicity adjusting agents, wetting agents or detergents. The present invention also provides compositions for oral delivery, which may contain inert ingredients, such as binders or excipients, to form a unit dosage form, such as a tablet or capsule. In addition, the present invention provides compositions for topical administration, which may contain inert ingredients such as solvents or emulsifiers to form a cream, ointment, gel, paste or eye drops.

Such compositions may be sterilized using conventional sterilization techniques or may be subjected to sterile filtration. The resulting aqueous solution may be packaged for use as is or lyophilized, and the lyophilized preparation is combined with a sterile aqueous vehicle prior to administration. The pH of the preparations will generally be from 3 to 11, more preferably from 5 to 9 or from 6 to 8, and most preferably from 6 to 7, for example from 6 to 6.5. The resulting compositions in solid form may be packaged in multiple unit dosages, each containing a fixed amount of the above agent or agents, such as in sealed tablets or capsules. The composition in solid form may also be packaged in a variable quantity container, such as a compressible tube designed for a topically applied cream or ointment.

Compositions containing an effective amount may be administered for radiation treatment planning, diagnostic or therapeutic purposes. When administered for radiation treatment planning or diagnostic purposes, the conjugate is administered to the subject at a diagnostically effective dose and/or in an amount effective to determine a therapeutically effective dose. For therapeutic purposes, the compositions are administered to a subject (eg, a human) already suffering from a condition (eg, cancer) in an amount sufficient to treat or at least partially arrest the symptoms of the disorder and its complications. An amount adequate to achieve the stated purpose is referred to as a therapeutically effective amount, and is an amount of a compound sufficient to significantly improve at least one symptom associated with a 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 a disease or condition will be therapeutically effective. A therapeutically effective amount of an agent or compound is not that amount required to cure a disease or condition, but is an amount that will treat the disease or condition to the extent that the disease or condition is delayed, delayed, or prevented, or the symptoms of the disease or condition are reduced or condition, or the duration of the disease or condition changes, or, for example, it becomes less severe, or the individual's recovery is accelerated. The conjugates of the present invention can be used to treat cancer by administering to a subject a first dose of any of the foregoing conjugates or compositions in an amount effective for planning radiation treatment, followed by a second dose of any of the above conjugates or compositions in a therapeutically effective amount.

Amounts effective for these purposes may depend on the severity of the disease or condition, as well as the weight and general condition of the subject. The therapeutically effective amount of the compositions of the present invention used in the methods of the present invention in mammals (eg, humans) can be determined by one of ordinary skill in the art, taking into account individual differences in the age, weight and condition of the mammal. Because some of the conjugates of the present invention have an enhanced ability to target and release cancer cells, the dosage of the compounds of the present invention may be lower than (e.g., be or be less than about 90, 75, 50, 40, 30, 20, 15, 12 , 10, 8, 7, 6, 5%, 4, 3, 2, 1, 0.5 or 0.1%) equivalent dose of the unconjugated agent required to achieve a therapeutic effect. The agents of the present invention are administered to a subject (eg, a mammal, such as a human) in an effective amount, which is an amount that produces the desired result in the subject being treated. Therapeutically effective amounts can also be determined empirically by those skilled in the art.

One or more administrations of the compositions of the present invention, including an effective amount, can be administered at dosage levels and schedules selected by the attending physician. The dosage and schedule of administration can be determined and adjusted depending on the severity of the disease or condition in the subject, which can be monitored during treatment in accordance with methods commonly used by physicians or described herein.

The conjugates of the present invention may be used in combination with any conventional treatment or therapy, or may be used separately from conventional treatment or therapy.

When the compounds of the present invention are administered as combination therapy with other agents, they may be administered to an individual sequentially or in parallel. Alternatively, the pharmaceutical compositions of the present invention may contain a combination of a compound of the present invention together with a pharmaceutically acceptable excipient as described herein and another therapeutic or

- 23 045232 prophylactic agent known in the art.

The terms antiproliferative or antiproliferative agent, used interchangeably herein, mean any anticancer agent, including the antiproliferative agents listed in Table. 2, any of which can be used in combination with a conjugate of the present invention for the treatment of medical conditions listed herein. Antiproliferative agents also include organoplatinum derivatives, naphthoquinone and benzoquinone derivatives, chrysophanic acid and anthroquinone derivatives.

The terms immunoregulatory agent or immunomodulatory agent, used interchangeably herein, mean any immunomodulator, including the immunomodulators listed in table. 2, any of which can be used in combination with a conjugate of the present invention for the treatment of medical conditions described herein.

As used herein, the term radiosensitizer includes any agent that increases the sensitivity of cancer cells to radiation therapy. Radiosensitizers may include, but are not limited to, 5'-fluorouracil, platinum analogs (eg, cisplatin, carboplatin, oxaliplatin), gemcitabine, epidermal growth factor receptor (EGFR) antagonists (eg, cetuximab, gefitinib), farnesyltransferase inhibitors, cyclooxygenase inhibitors, 2 (COX2), basic fibroblast growth factor (bFGF) antagonists and vascular endothelial growth factor (VEGF) antagonists.

table 2

Alkylating agents Busulfan dacarbazine ifosfamide hexamethylmelamine thiotepa dacarbazine lomustine cyclophosphamide Chlorambucil procarbazine altretamine estramustine phosphate mechlorethamine streptozocin temozolamide semustine Platinum based agents spiroplatin tetraplatin ormaplatin iproplatin picoplatin oxaliplatin carboplatin lobaplatin (Aeterna) satraplatin (Johnson Matthey) BBR-3464 (Hoffmann-La Roche) SM-11355 (Sumitomo) AP-5280 (Access) cisplatin Antimetabolites azacitidine floxuridine 2-chlorodeoxyadenosine 6-mercaptopurine 6-thioguanine cytarabine 2-fluorodeoxycytidine methotrexate tomudex fludarabine raltitrexed trimetrexate deoxycoformycin lentostatin hydroxyurea decitabine (SuperGen) clofarabine (Bioenvision) irofulven (MGI Pharma) DMDC (Hoffman-La Roche) ethynylcytidine (Taiho) gemcitabine capecitabine

-24045232

Topoisomerase inhibitors amsacrine epirubicin etoposide teniposide or mitoxantrone 7-ETHYL-10- hydroxy camptothecin dexrazoxanet (TopoTarget) pixantrone (Novuspharma) rebeccamycin analogue (Exelixis) BBR-3576 (Novuspharma) Rubitecan (SuperGen) irinotecan (CPT-11) topotecan exatecan mesylate (Daiichi) quinamed (ChemGenex) gimatecan (Sigma-Tau) diphlomothecan (Beaufour-lpsen) TAS-103 (Taiho) elsamitrucin (Spectrum) J-107088 (Merck & Co) BNP-1350 (BioNumerik) CKD-602 (Chong Kun Dang) KW-2170 (Kyowa Hakko) hydroxycamptothecin (SN-38) Antitumor antibiotics valrubicin terarubicin idarucibin rubidazone plicamycin porphyromycin mitoxantrone (novantrone) amonafide azonafide anthrapyrazole oxantrazole losoxantrone MEN-10755 (Menarini) GPX-100 (Gem Pharmaceuticals) Epirubicin mitoxantrone doxorubicin Antimitotic agents colchicine vinblastine vindesine dolastatin 10 (NCI) rhizoxin (Fujisawa) mivobulin (Warner-Lambert) cematodine (BASF) RPR 109881A (Aventis) TXD 258 (Aventis) epothilone B (Novartis) T 900607 (Tularik) T 138067 (Tularik) cryptophycin 52 (Eli Lil ly ) vinflunine (Fabre) auristatin PE (Teikoku Hormone) BMS 247550 (BMS) BMS 184476 (BMS) BMS 188797 (BMS) taxoprexin (Protarga) SB 408075 (GlaxoSmithKline) Vinorelbine Trichostatin A E7010 (Abbott) PG-TXL (Cell Therapeutics) IDN 5109 (Bayer) A 105972 (Abbott) A 204197 (Abbott) LU 223651 (BASF) D 24851 (ASTAMedica) ER-86526 (Eisai) combretastatin A4 (BMS) isohomohalichondrion-B (PharmaMar) ZD 6126 (AstraZeneca) AZ10992 (Asahi) IDN-5109 (Indena) AVLB (Prescient NeuroPharma) azaepothilone B (BMS) BNP-7787 (BioNumerik) CA-4 prodrug (OXiGENE) dolastatin-10 (NIH) CA-4 (OXiGENE) docetaxel vincristine paclitaxel Aromatase inhibitors aminoglutethimide atamestane (BioMedicines) letrozole anastrazole YM-511 (Yamanouchi) formestane exemestane

-25045232

Thymid ntase inhibitors pemetrexed (Eli Lilly) ZD-9331 (BTG) nolatrexed (Eximias) CoFactor™ (BioKeys) DNA antagonists Trabectedin (PharmaMar) Glufosfamide (Baxter International) Albumin + 32P (Isotope Solutions) Timektacin (NewBiotics) edotreotide (Novartis) mafosfamide (Baxter International) apasichone (Spectrum Pharmaceuticals) O benzylguanine (Paligent) Farnesyltransferase inhibitors arglabin (NuOncology Labs) lonafarnib (Schering-Plough) BAY-43-9006 (Bayer) tipifarnib (Johnson & Johnson) perillyl alcohol (DOR BioPharma) Pump inhibitors CBT-1 (CBA Pharma) tarihidar (Xenova) MS-209 (Schering AG) Zosuquidar trihydrochloride (Eli Lilly) biricodar dicitrate (Vertex) Histone acetyltransferase inhibitors tacedinaline (Pfizer) SAHA (Aton Pharma) MS-275 (Schering AG) pivaloyloxymethylbutyrate (Titan) depsipeptide (Fujisawa) Metalloproteinase inhibitors inhibitors Geovastat (Aeterna Laboratories) marimastat (British Biotech) CMT-3 (CollaGenex) BMS-275291 (Celltech) Ribonucleoside reductase inhibitors gallium maltolate (Titan) triapine (Vion) tezacitabine (Aventis) didox (Molecules for Health) TNF-alpha agonists/antagonists virulysin (Lorus Therapeutics) CDC-394 (Celgene) revimid (Celgene) Endothelin A receptor antagonist atrasentan (Abbott) ZD-4054 (AstraZeneca) YM-598 (Yamanouchi) Retinoic acid receptor agonists fenretinide (Johnson & Johnson) LGD-1550 (Ligand) alitretinoin (Ligand)

- 26 045232

Immunomodulators interferon oncophage (Antigenics) GMK (Progenies) adenocarcinoma vaccine (Biomira) STP-37 (AVI BioPharma) IRX-2 (Immuno-Rx) PEP-005 (PERNp Biotech) synchrovax vaccines (CTL Immuno) melanoma vaccine (CTL Immuno) p21 RAS vaccine (GemVax) MAGE-A3 (GSK) nivolumab (BMS) abatacept (BMS) pembrolizumab (Merck) dexosomal therapy (Anosys) pentrix (Australian Cancer technology) ISF-154 (Tragen) cancer vaccine (Intercell) norelin (Biostar) BLP-25 (Biomira) MGV (Progenies) β-alethine (Dovetail) CLL therapy (Vasogen) Ipilimumab (BMS), CM-10 (cCam Biotherapeutics) atezolizumab (Genentech) Hormonal and antihormonal agents estrogens conjugated estrogens ethinyl estradiol chlorotrianisene idenestrol hydroxyprogesterone caproate medroxyprogesterone testosterone testosterone propionate fluoxymesterone methyltestosterone diethylstilbestrol megestrol bicalutamide flutamide nilutamide dexamethasone prednisone methylprednisolone prednisolone aminoglutethimide leuporide octreotide mitotane P-04 (Novogen) 2-methoxyestradiol (EntreMed) arzoxifene (Eli Lilly) tamoxifen toremophine goserelin Leuprorelin bicalutamide Photodynamic agents talaporphine (Light Sciences) Theralux (Theratechnologies) Motexafin gadolinium (Pharmacyclics) Pd-bacteriopheophorbide (Yeda) lutetium texaphyrin (Pharmacyclics) hypericin Kinase inhibitors imatinib (Novartis) leflunomide (Sugen/Pharmacia) ZD1839 (AstraZeneca) erlotinib (Oncogene Science) canertinib (Pfizer) squalamine (Genaera) SU5416 (Pharmacia) SU6668 (Pharmacia) ZD4190 (AstraZeneca) ZD6474 (AstraZeneca) vatalanib ( Novartis) RKP 66 (Novartis) GW2016 (GlaxoSmithKline) EKB-509 (Wyeth) Trastuzumab (Genentech) OSI-774 (Tarceva™) CI-1033 (Pfizer) SU11248 (Pharmacia) RH3 (York Medical) Genistein Radicinol Met-MAb (Roche) EKB-569 (Wyeth) kagalide F (PharmaMar) CEP-701 (Cephalon) CEP-751 (Cephalon) MLN518 (Millenium) PKC412 (Novartis) Phenoxodiol (Novogen) C225 (ImClone) rhu-Mab (Genentech) MDX-H210 (Medarex) 2C4 (Genentech) MDX-447 (Medarex) ABX-EGF (Abgenix) IMC-1C11 (ImClone) Tyrphostins Gefitinib (Iressa) PTK787 (Novartis) EMD 72000 ( Merck) Emodin Radicinol Vemurafenib (B-Raf enzyme inhibitor, Daiichi Sankyo)

- 27 045232

SR-27897 (cholecystokinin A (CCA) inhibitor, Sanofi-Synthelabo) tocladesine (cyclic AMP agonist, Ribapharm) alvocidib (cyclin-dependent kinase (CDK) inhibitor, Aventis)

CV-247 (COX-2 inhibitor, Ivy Medical) P54 (COX-2 inhibitor, Phytopharm) CapCell™ (CYP450 stimulator, Bavarian Nordic)

GCS-100 (gal3 antagonist, GlycoGenesys) immunogen G17DT (gastrin inhibitor, Aphton) efaproxiral (oxidizing agent, Alios Therapeutics) PI-88 (heparanase inhibitor, Progen) tesmilifen (histamine antagonist, YM BioSciences) histamine (histamine H2 receptor agonist, Maxim) tiazofurin (IMPDH inhibitor, Ribapharm) cilengitide (integrin antagonist, Merck KGaA) SR-31747 (IL-1 antagonist, Sanofi-Synthelabo)

CCI-779 (mTOR kinase inhibitor, Wyeth) exisulind (PDE V inhibitor, Cell Pathways) CP-461 (PDE V inhibitor, Cell Pathways) AG-2037 (GART inhibitor, Pfizer) WX-UK1 (plasminogen activator inhibitor, Wilex)

PBI-1402 (cnbvekznjh PMN, ProMetic LifeSciences) bortezomib (proteasome inhibitor, Millennium) SRL-172 (T cell stimulator, SR Pharma) TLK-286 (glutathione S-transferase inhibitor, Telik)

PT-100 (growth factor agonist, Point Therapeutics) midostaurin (PKC inhibitor, Novartis) bryostatin-1 (PKC stimulator, GPC Biotech) CDA-II (apoptosis promoter, Everlife) SDX-101 (apoptosis promoter, Salmedix) rituximab (antibody CD20, Genentech carmustine Mitoxantrone Bleomycin Absinthine

Chrysophanic acid Cesium oxides BRAF inhibitors PD-L1 inhibitors MEK inhibitors bevacizumab Angiogenesis inhibitors dabrafenib ceflatonin (apoptosis promoter, ChemGenex)

BCX-1777 (PNP inhibitor, BioCryst) ranpirnase (ribonuclease stimulator, Alfacell) galarubicin (RNA synthesis inhibitor, Dong-A) tirapazamine (reducing agent, SRI International) N-acetylcysteine (reducing agent, Zambon) R-flurbiprofen (NFkappaB inhibitor, Encore)

ZCPA (NF-kappaB inhibitor, Active Biotech) seocalcitol (vitamin D receptor agonist, Leo) 131-I-TM-601 (DNA agonist, TransMolecular) eflornithine (ODC inhibitor, ILEX Oncology) minodronic acid (osteoclast inhibitor, Yamanouchi) indizulam ( p53 stimulator, Eisai) aplidine (PPT inhibitor, PharmaMar) gemtuzumab (anti-CD33 antibody, Wyeth Ayerst)

PG2 (hematopoiesis enhancer, Pharmagenesis) Immunol™ (triclosan, mouth rinse, Endo) triacetyluridine (uridine prodrug, Wellstat)

SN-4071 (anti-sarcoma agent, Signature BioScience) TransMID-107™ (immunotoxin, KS Biomed ix)

RCK-3145 (apoptosis promoter, Procyon) doranidazole (apoptosis promoter, Pola) CHS-828 (cytotoxic agent, Leo) trans-retinoic acid (differentiator, NIH) MX6 (apoptosis promoter, MAXIA) apomin (apoptosis promoter, ILEX Oncology) urocidin (apoptosis promoter, Bioniche) Ro-31-7453 (apoptosis promoter, La Roche) brostallicin (apoptosis promoter, Pharmacia) β-lapachone gelonin cafestol kahweol caffeic acid Tyrphostin AG PD-1 inhibitors CTLA-4 inhibitors sorafenib

The following examples are intended to illustrate the synthesis of representative amounts of conjugates and the use of said conjugates for the treatment of cancer. Accordingly, these examples are intended to be illustrative and not limiting of the present invention. Additional compounds not specifically presented may be synthesized using conventional methods in combination with the methods described herein.

Examples

Example 1: General materials and methods.

HuMIgG antibody (Aldrich, I4506) was used. Lutetium-177 was obtained from Perkin Elmer as lutetium chloride in a 0.05 N solution. of hydrochloric acid.

Analytical HPLC-MS was performed using a Waters Acquity HPLC-MS system containing Waters Acquity Binary Solvent Manager, Waters Acquity Sample Manager (samples cooled to 10°C), Waters Acquity Column Manager (column temperature 30°C), Waters Acquity detector Photodiode Array Detector (monitoring at 254 nm and 214 nm), Waters Acquity TQD with electrospray ionization and Waters Acquity BEH C18, 2.1x50 (1.7 µm) column. Preparative HPLC was performed using a Waters system containing a Waters 1525 Binary HPLC pump, a Waters 2489 UV/Vis detector (for monitoring at 254 nm and 214 nm) and a Waters XBri column for preparative chromatography with phenyl or C18 19x100 mm (5 µm).

HPLC elution method 1: Waters Acquity BEH C18 2.1x50 mm (1.7 µm) column; mobile phase A: H ₂ O (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.

HPLC elution method 2: Waters XBridge Prep Phenyl column 19x100 mm (5 µm); 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.

HPLC elution method 3: Waters Acquity BEH C18 2.1x50 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, 8 min = 0% A, 10 min = 0% A, 11 min = 90% A, 12 min = 90% A.

HPLC elution method 4: Waters XBridge Prep C18 OBD 19x100 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, 3 min = 80% A, 13 min = 20% A, 18 min = 0% A.

HPLC elution method 5: Waters XBridge Prep C18 OBD column 19x100 mm (5 µm); mobile phase A: H2O (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.

HPLC elution method 6: Waters XBridge Prep C18 OBD column 19x100 mm (5 µm); mobile phase A: H2O (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.

HPLC elution method 7: Waters XBridge Prep C18 OBD column 19x100 mm (5 µm); 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, 12 min = 0% A, 15 min = 0% A.

HPLC elution method 8: Waters XBridge Prep C18 OBD column 19x100 mm (5 µm); mobile phase A: H2O (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.

Analytical size exclusion chromatography (SEC) was performed using a Waters system containing a Waters 1525 Binary HPLC pump, a Waters 2489 UV/Vis detector (for monitoring at 280 nm), a Bioscan Flow Count (FC- 3300) and column TOSOH TSKgel G3000SWxI, 7.8x300 mm. When performing SEC in isocratic mode, flow rate = 1 ml/min, mobile phase 0.1 M phosphate, 0.6 M NaCl, 0.25% sodium azide, pH = 7 was used.

MALDI-MS (positive ion) was performed using a Bruker Ultraflextreme MALDI spectrometer.

Radio-thin layer chromatography (radio-TLC) was performed using a Bioscan AR-2000 scanner on iTLC-SG glass microfiber chromatography paper plates (Agilent Technologies, SGI0001) using citrate buffer (0.1 M, pH 5.5).

Example 2. Synthesis of 4-{[11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2-[4,7,10tris(carboxymethyl)-1,4,7,10- tetraazacyclododecan-1-yl]butanoic acid (compound B).

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 shown in FIG. 2. To a solution of 5-(tert-butoxy)-5-oxo4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-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 mixture was stirred at room temperature for 1 hour. 11-Aminoundecanoic acid (70 mg, 0.36 mmol) was added to the reaction mixture, followed by phosphate-buffered saline (PBS) solution (1.0 mL) at room temperature. The reaction mixture was stirred for 72 hours at room temperature. The reaction mixture was filtered through a syringe filter and purified directly using Preparative HPLC Method 6 to give Intermediate 2-A (71 mg, 74.8%).

Pyridine (0 .05 ml, 50 mg, 0.62 mmol) at room temperature. The solution was stirred at room temperature for 24 hours. The reaction mixture was purified directly using Preparative HPLC Method 7, yielding Intermediate 2-B (33 mg, 82.5%) as a wax after concentration using a Biotage V10 Rapid Evaporator.

Intermediate 2-B (33 mg, 0.022 mmol) was dissolved in DCM/TFA (1.0 mL/2.0 mL) and left stirring at room temperature for 24 hours. The reaction mixture was concentrated under a stream of air and purified directly by preparative HPLC using method 8, obtaining compound B (14 mg, 50.0%) as a clear wax after concentration. An aliquot was analyzed by HPLC-MS using elution method 3; retention time: 4.15 min;

- 29 045232

MS (positive ESI), result: m/z 808.1 [M+H]+; C ₃₆ H ₅₄ F ₄ N ₅ O11 (calc. 808.8).

1H NMR (600 MHz, DMSO-d ₆ ) δ 7.99 - 7.88 (m, 1H), 7.82 (t, J=5.5 Hz, 1H), 3.78 (broad s, 4H ), 3.43 (broad s, 12H), 3.08 (broad s, 4H), 3.00 (m, 3H), 2.93 (broad s, 3H), 2.77 (t, J=7.2 Hz, 2h), 2.30 (broad s, 2H), 1.88 (broad s, 2H), 1.66 (p, J=7.3 Hz, 2H), 1, 36 (m, 4H), 1.32 - 1.20 (m, 9H).

Example 3. Synthesis of 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7, 10-Tris(carboxymethyl)-1,4,7,10tetraazacyclododecan-1-yl]butanoic acid (compound C).

Bifunctional chelate, 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10 -Tris(carboxymethyl)-1,4,7,10tetraazacyclododecan-1-yl]butanoic acid (compound C) was synthesized according to the scheme shown in FIG. 3. To a solution of 5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2oxoethyl)-1,4,7,10-tetraazacyclododecane-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 for 90 min at ambient temperature. This solution was added to a half solution of amino-PEG3 acid (63 mg, 0.285 mmol in 1.2 mL DMF) in a 100 mL round bottom flask. After 4 hours at ambient temperature, the reaction mixture was concentrated to dryness under a stream of air. The crude material was purified by HPLC using method 2 elution (the crude material was dissolved in 6 ml of 20% ACN/H ₂ O). Fractions containing product were combined and concentrated in vacuo and then co-evaporated with ACN (3x2 ml). Intermediate 1-A was obtained in 82% yield.

To the vial containing Intermediate 1-A (82 mg, 60 µmol), ACN (2 ml), NEt ₃ (50 µl, 360 µmol, 6 equiv.), HBTU (23 mg, 60 µmol, 1 equiv.) was added. and TFP solution (50 mg, 300 μmol, 5 equiv., dissolved in 250 μl ACN). The resulting clear solution was stirred at ambient temperature for 3 hours. The reaction mixture was concentrated to dryness under a stream of air and then diluted with ACN/H2O (1:1, total amount 3 ml) and purified by preparative HPLC eluting with method 4. Fractions containing the product were combined and concentrated in vacuo and then co-evaporated with ACN (3x2 ml). Intermediate 1-B was obtained as a clear residue (67 mg, 74% yield).

DCM (2 mL) and TFA (2 mL) were added to the vial containing intermediate 1-B (67 mg, 64 μmol) and the resulting solution was stirred at ambient temperature for 16 hours. Additional TFA (2 mL) was added ), and the resulting reaction mixture was stirred at ambient temperature for 6 hours. The reaction mixture was concentrated to dryness under a stream of air, and the resulting crude product was ultimately dissolved in ACN/H ₂ O (1 ml 10% ACN/H ₂ O ). The crude reaction solution was then purified by preparative HPLC using elution method 5. Product containing fractions were pooled, frozen and lyophilized. Compound C was obtained as a white solid (36 mg, 63% yield). An aliquot was analyzed by HPLC-MS using elution method 3; retention time: 3.11 min; MS (positive ESI), m/z result. 828.4 [M+H]+; C ₃₄ H ₅₀ F ₄ N ₅ O ₁₄ (calc. 828.3).

¹ H NMR (DMSO-d ₆ , 600 MHz) δ 7.97-7.91 (m, 2H), 3.77 (t, 2H, J=6.0 Hz), 3.58-3.55 ( m, 2H), 3.53-3.48 (m, 8H), 3.44-3.38 (m, 10H), 3.23-3.08 (m, 11H), 3.02 (t, 2H, J=6.0 Hz), 2.93 (broad s, 4H), 2.30 (broad s, 2H), 1.87 (broad s, 2H).

Example 4. Synthesis of [ ¹⁷⁷ 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 human IgG antibody (6.7 nmol) in bicarbonate buffer (pH 8.5). After 45 min at ambient temperature, the antibody conjugate product was purified using a size exclusion HPLC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). MALDI-TOF-MS (positive ion): Compound A-human IgG, result m/z: 150360 [M+H]+; Human IgG, result m/z: 148339 [M+H] ⁺ .

For a typical reaction, Lu-177 (1.1 mCi, 5 μl) was added to a solution of human Compound A-IgG conjugate (90 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μl, 0.1 M in acetate buffer (pH 6.5). The reaction mixture of the radiolabeling reaction was incubated at 37°C for 90 minutes. The crude product, [ ¹⁷⁷ Lu]-human A-IgG compound, was purified on a Sephadex G-50 resin column, eluting with acetate buffer (pH 6.5, 1 mM ascorbic acid. Radiochemical purity according to radio-TLC: 98%; radiochemical yield: 45%; specific activity: 15.1 mCi/mg.

Example 5. Synthesis of [ ¹⁷⁷ Lu]-human B-IgG conjugate.

Compound B (1.17 μmol) was dissolved in sodium acetate buffer (0.117 ml, pH 6.5). An aliquot of compound B solution (2 μl, 10 nmol) 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 on a Sephadex G-50 resin column. The antibody conjugate compound A-human IgG was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): human B-IgG compound, result m/z: 149949

[M+H]+; Human IgG, result m/z: 148540 [M+H] ⁺ .

For a typical Lu-177 reaction (1.1 mCi, 5 μl), human B-IgG compound (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μl, 0.1 M in acetate buffer) was added to the solution (pH 6.5). The radiolabeling reaction mixture was incubated at 37°C for 30 minutes. The crude product, ¹⁷⁷ Lu compound human B-IgG, was purified using a size exclusion HPLC column (1 ml/min, eluted acetate buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa) Radiochemical purity by radioTLC: 98%, radiochemical yield: 51%, specific activity: 9.68 mCi/mg.

Example 6. Synthesis of [ ¹⁷⁷ Lu]-human C-IgG conjugate.

Compound C (0.96 µmol) was dissolved in sodium acetate buffer (95 µl, pH 6.5). An aliquot of compound C solution (2 μl, 20 nmol) 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 on a Sephadex G-50 resin column. The human C-IgG antibody conjugate was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Human C-IgG compound, m/z result: 150095 [M+H]+; Human IgG, m/z result: 148540 [M+H] ⁺ .

For a typical Lu-177 reaction (1.1 mCi, 5 μl), human C-IgG (100 μg in acetate buffer (pH 6.5)) and ascorbic acid (1 μl, 0.1 M in acetate buffer) were added to the solution (pH 6.5). The radiolabeling reaction mixture was incubated at 37°C for 30 minutes. The crude product, [ ¹⁷⁷ Lu]-human C-IgG compound, was purified using a size exclusion HPLC column (1 ml/min , eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa). Radio-TLC radiochemical purity: 98%; radiochemical yield: 37%; specific activity: 9.99 mCi/ mg.

Example 7. Pharmacokinetics and metabolism studies of human IgG-based compounds.

Groups of 4 or 5 mice (normal CD-1 or athymic CD-1 nude) were intravenously injected with a radiolabeled test compound with a radioactivity of approximately 15 microcuries. Test compounds with various linkers were synthesized and radiolabeled with lutetium-177. For pharmacokinetic studies, animals were sacrificed at specified times and blood and tumors (where applicable) were analyzed for total radioactivity. To study metabolism, animals were placed in metabolic cages (4-5 per cage) and urine and feces were collected every 24 hours for 7 days. The radioactivity of urine and feces samples was determined and the total results in urine and feces were calculated on a mass basis. Elimination profiles for urine, feces, and total excretion (urine + feces) were obtained by plotting the cumulative % of administered dose (%ID) as a function of time.

Untargeted human IgG antibodies were used for metabolic clearance studies to demonstrate changes in radioactivity clearance profiles provided by conjugation to linker compound B and compound C in the overall process. The human IgG preparation used consisted of a purified mixture of all IgG isotypes (IgG1-4).

The metabolic clearance profile of the untargeted human IgG antibody conjugates [ ¹⁷⁷ Lu] compound B-HuMIgG and [ ¹⁷⁷ Lu] compound C-HuMIgG were compared with the [ ¹⁷⁷ Lu] compound AHuMIgG conjugate. The [ ¹⁷⁷ Lu]-compound A-HuMIgG conjugate was excreted slowly, with only 13% of the administered dose (ID) recovered over 7 days, with low urinary excretion rates. Compounds B and C exited via completely different clearance pathways and increased the total amount of radioactivity excreted over a 7-day period compared to the compound A-HuMIgG conjugate. [ ¹⁷⁷ Lu]-BHuMIgG conjugate was excreted through feces, and excretion of [ ¹⁷⁷ Lu]-C-HuMIgG conjugate was roughly equally distributed between urine and feces. Thus, it was found that although the type of linker influenced the route, rate and extent of clearance of the compound (Fig. 4), it did not change the overall pharmacokinetics in the blood for all radioactivity associated with the radioimmunoconjugate. It was also observed that the improved clearance profile of Compound B or Compound C when conjugated to an antibody was a general and reproducible effect.

Example 8. Synthesis of [ ²²⁵ Ac]-Compound A-human IgG conjugate.

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 min at ambient temperature, the antibody conjugate product was purified using a size exclusion HPLC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). MALDI-TOF-MS (positive ion): Compound A-human IgG, result m/z: 150360 [M+H]+; Human IgG, result m/z: 148339 [M+H] ⁺ .

For a typical reaction, Ac-225 (1.1 mCi, 5 μl) was added to a solution of Human Compound A-IgG conjugate (90 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μl, 0.1 M in acetate buffer (pH 6.5)). The reaction mixture of the reaction for obtaining a radioactive label was incubated at ambient temperature (for example, 20-25 ° C) for 90 minutes. The crude product, conjugated

Claims (25)

gat[ ²²⁵ Ac]-human IgG compound was purified using a Sephadex G-50 resin column and eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). Example 9. Synthesis of [ ²²⁵ Ac]-human B-IgG conjugate. Compound B (1.17 µmol) was dissolved in sodium acetate buffer (0.117 ml, pH 6.5). An aliquot of compound B solution (2 μl, 10 nmol) 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 on a Sephadex G-50 resin column. The antibody conjugate compound A-human IgG was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Human B-IgG compound, m/z result: 149949 [M+H]+; Human IgG, result m/z: 148540 [M+H] ⁺ . To perform a typical Ac-225 reaction (1.1 mCi, 5 μl), human B-IgG compound (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μl, 0.1 M in acetate buffer) was added to the conjugate solution buffer (pH 6.5). The radiolabeling reaction mixture was incubated at ambient temperature (eg, 20-25°C) for 30 minutes. The crude product, [ ²²⁵ Ac]Human B-IgG Compound, was purified using size exclusion HPLC columns (1 ml/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa). Example 10. Synthesis of [ ²²⁵ Ac]-human C-IgG conjugate. Compound C (0.96 µmol) was dissolved in sodium acetate buffer (95 µl, pH 6.5). An aliquot of compound C solution (2 μl, 20 nmol) 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 on a Sephadex G-50 resin column. The human C-IgG antibody conjugate was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): human C-IgG compound: result m/z 150095 [M+H]+; Human IgG: result m/z 148540 [M+H] ⁺ . To perform a typical Ac-225 reaction (1.1 mCi, 5 μl), human C-IgG compound (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μl, 0.1 M in acetate buffer) were added to the conjugate solution (pH 6.5). The radiolabeling reaction mixture was incubated at ambient temperature (eg, 20-25°C) for 30 minutes. The crude product, [ ²²⁵ Ac]Human C-IgG Compound, was purified by column for size exclusion HPLC (1 ml/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa). Other implementation options. While the present invention has been described in the context of its specific embodiments, it is to be understood that further modifications are possible thereto, and the present invention is intended to cover all variations, uses and adaptations of the present invention generally consistent with the principles of the present invention and includes such deviations from the present invention, which may be made due to known or customary experience available in the field of technology to which the present invention relates, and may be applied to the previously stated essential features. CLAIM 1. A compound having the structure or a pharmaceutically acceptable salt thereof: IA-L ¹ -(L ² ) _n -B, Formula I wherein A is a chelating moiety or a metal complex thereof, wherein said chelating moiety is selected from the group consisting of DOTA (1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R, 4R,7R,10R)-α,α',α,α'-tetramethyl1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10tetrakis(carbamoylmethyl) -1,4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane1,4,7,10-tetrapropionic acid), DO3AM-acetic acid (2-(4,7,10-tris( 2-amino-2-oxoethyl)1,4,7,10-tetraazacyclododecan-1-yl)acetic acid), DOTA-GA anhydride (2,2',2-(10-(2,6dioxotetrahydro-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 )), DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamidomethylenephosphonic acid), NOTA (1,4,7triazacyclononane-1,4,7-triacetic acid), and HP-DO3A (hydroxypropyltetraazacyclododecanetriacetic acid), wherein the metal of said metal complex is a radionuclide selected from the group consisting of 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, ⁸⁹ Zr, ⁹⁰ Y, ⁹⁷ Ru, ⁹⁹ Tc, ^99m Tc, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹ⁿ In, ^117m Sn, ¹⁴⁹ Pm, ¹⁴⁹ Tb, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au, ¹⁹⁹ Au, ²⁰¹ Tl, ²⁰³ Pb, ²¹¹ At, ²¹² Pb, ²¹² Bi, ²¹³ Bi, ²²³ Ra, ²²⁵ Ac, ²²⁷ Th and ²²⁹ Th, - 32 045232 L ¹ represents C ₁ -C ₆ -alkyl or C ₁ -C ₆ -heteroalkyl; B is a human IgG antibody or a humanized IgG antibody or an antigen-binding fragment thereof; n is 1; each L ² independently has the structure: (-X ¹ -L ³ -Z ¹ -), Formula II where X ¹ represents C=O(NR ¹ ), in which R ¹ represents H or C ₁ -C ₆ -alkyl or CiC ₆ -heteroalkyl; L ³ represents C ₁ -C ₅₀ -alkyl or C ₁ -C ₅₀ -heteroalkyl; Z ¹ represents C=O, C=S, OC=O or NR1C=O, in which R ¹ represents hydrogen or C ₁ -C ₆ -alkyl or C ₁ -C ₆ -heteroalkyl; where in each case C ₁ -C ₆ -alkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of C _1-6 alkoxy, amino, halogen, hydroxy, nitro, C _1-6 alkenyl, C _6-10 aryl, C5-C20 polyethylene glycol and -CO2RA, where R ^A is selected from the group consisting of hydrogen, C _1-6 alkyl, C _2-6 alkenyl and C ₅ -C ₂₀ polyethylene glycol, C ₁ -C ₆ -heteroalkyl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of C _1-6 alkoxy, amino, halogen, hydroxy, nitro, C _1-6 alkyl, C _2-6 alkenyl, C _6-10 aryl, C ₅ -C ₂₀ -polyethylene glycol and -CO2RA, where R ^A is selected from the group consisting of hydrogen, C _1-6 alkyl, C _2-6 alkenyl and C ₅ -C ₂₀ -polyethylene glycol, C ₆ -C ₁₀ -aryl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of C _1-6 alkoxy, amino, halogen, hydroxy, nitro, C _1-6 alkyl, C _2-6 alkenyl, C _6-10 aryl, C ₅ -C ₂₀ -polyethylene glycol and -CO2RA, where R ^A is selected from the group consisting of hydrogen, C _1-6 alkyl, C _2-6 alkenyl and C ₅ -C ₂₀ -polyethylene glycol, and C ₁ -C ₁₂ -heteroaryl is unsubstituted or substituted with one, two or three substituents selected from the group consisting of C _1-6 alkoxy, amino, halogen, hydroxy, nitro, C _1-6 alkyl, C _2-6 alkenyl, C _6-10 aryl, C ₅ -C ₂₀ -polyethylene glycol and -CO2RA, where R ^A is selected from the group consisting of hydrogen, C _1-6 alkyl, C _2-6 alkenyl and C ₅ -C ₂₀ -polyethylene glycol, where in In each case, heteroalkyl is an alkyl group in which one or more of its constituent carbon atoms are each replaced by nitrogen, oxygen or sulfur; heteroaryl is a 5-, 6-, or 7-membered ring containing one, two, three, or four heteroatoms, independently selected from the group consisting of nitrogen, oxygen and sulfur. 2. A compound according to claim 1, wherein said chelating moiety is DOTA (1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid). 3. A compound according to claim 2, wherein the compound of formula (I) has the structure: where Y ¹ represents -CH2OCH2(L ² )nB, and Y ² represents -CH2CO2H; or where Y ¹ represents H and Y ² represents L ¹ -(L2) _n -B. 4. A compound according to any one of claims 1-3, where L ¹ has the structure: R' Formula III where R ² represents hydrogen or -CO2H. 5. A compound according to any one of claims 1 to 4, wherein said metal complex contains a radionuclide selected from the group consisting of ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁹⁰ Y, 1111^, ¹⁷⁷ Lu, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac and ²²⁷ Th. 6. The compound of claim 1, wherein said antibody or antigen-binding fragment thereof specifically binds to the insulin-like growth factor-1 receptor (IGF-1R). 7. A compound according to any one of claims 3 to 6, where Y ¹ represents H. 8. A compound according to any one of claims 1 to 7, where X ¹ represents C=O(NR1) and R ¹ represents H. 9. A compound according to any one of claims 1 to 8, wherein said compound is one of the following - 33 045232 blowing or its complex with metal: 10. The compound according to any one of claims 1 to 9, where the specified metal in the specified complex with the metal is a radionuclide. 11. The compound according to claim 10, wherein said radionuclide is In ¹¹¹ . 12. The compound according to claim 10, wherein said radionuclide is Ga ⁶⁸ . 13. A compound according to any one of claims 1 to 9, wherein said metal in said complex with the metal is an alpha-emitting radionuclide. 14. The compound of claim 13, wherein said alpha-emitting radionuclide is ^Ac225 . 15. A pharmaceutical composition containing a compound according to claims 1 to 14 and a pharmaceutically acceptable excipient. 16. A method of treating cancer, comprising administering to a subject in need thereof a compound according to any one of claims 1 to 14 or a composition according to claim 15. 17. A method of treating cancer, comprising administering to a subject in need thereof a first dose of a compound according to any one of claims 1 to 14 or a composition according to claim 15 in an amount effective for planning radiation treatment, and then administering subsequent doses of a compound according to any one of claims 1-14 or compositions according to claim 15 in a therapeutically effective amount. 18. The method of claim 17, wherein the compound or composition administered in the first dose and the compound or composition administered in the second dose are the same. 19. The method of claim 17, wherein the compound or composition administered in the first dose and the compound or composition administered in the second dose are different. 20. The method according to any one of claims 17-19, wherein said cancer is a solid tumor or hematological (liquid) cancer. 21. The method of claim 20, wherein said solid tumor 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, Ewing's sarcoma, multiple myeloma or acute myeloid leukemia. 22. The method according to any one of claims 17-21, further comprising the administration of an antiproliferative agent, a radiosensitizer or an immunoregulatory or immunomodulatory agent. 23. The method according to claim 22, wherein the compound according to any one of claims 1 to 14 or the composition according to claim 15 and the specified antiproliferative agent or radiosensitizer are administered within 28 days of each other. 24. The method according to claim 23, where the compound according to any one of claims 1 to 14 or the composition according to claim 15 and the immunoregulatory or immunomodulatory agent are administered within 90 days of each other. 25. The compound according to claim 1, where L ³ represents C ₁ -C ₅₀ -heteroalkyl and C ₁ -C ₅₀ -heteroalkyl represents C ₅ -C ₂₀ -polyethylene glycol. -