Reactive conjugates

Abstract

The present invention relates to compounds (reactive conjugates) for the chemical modification of therapeutic antibodies or proteins. These compounds are capable of selectively attaching a payload region to an antibody or antibody fragment in a single step, thereby generating a modified antibody or modified antibody fragment that can be used for the diagnosis, monitoring, imaging or treatment of a disease.

Description

Technical Field

[0001] This invention relates to compounds for the chemical modification of therapeutic antibodies (hereinafter sometimes referred to as "reactive conjugates"). These compounds are capable of selectively attaching an effective load region to an antibody or antibody fragment in a single step, thereby producing a modified antibody or modified antibody fragment that can be used for the diagnosis, monitoring, imaging, or treatment of diseases. Background Technology

[0002] Traditional cancer treatments (e.g., chemotherapy) are not only extremely arduous (due to their toxicity causing severe side effects), but can also be highly unpredictable, with the treatment working for one patient but completely ineffective for another. Therefore, there is a constant need to develop new, less toxic, and / or more effective treatments, as well as the ability to monitor treatment efficacy, such as distinguishing between "responders" and "non-responders."

[0003] To address these needs, novel therapeutic agents called antibody-drug conjugates (ADCs) have emerged. ADCs utilize the targeting capabilities of antibodies (e.g., monoclonal antibodies, mAbs) to deliver a payload (e.g., a cytotoxic agent or marker) directly to cancer cells. This targeting of cancer cells maximizes the therapeutic effect of the payload while minimizing toxicity to healthy cells. Depending on the payload, ADCs can achieve various functions, such as diagnosis, monitoring, and / or treatment.

[0004] ADCs can be prepared using a variety of methods. However, most of these methods result in heterogeneous mixtures of ADCs with different chemical properties, varying payload (drug) antibody ratios (DARs) and conjugation sites. This heterogeneity complicates manufacturing, leading to high batch-to-batch variability and sometimes unpredictable safety and efficacy. Therefore, methods that can produce homogeneous mixtures (e.g., regioselective or site-specific conjugation methods) are gaining increasing attention. Such methods can significantly improve the predictability of DARs and payload (drug) binding sites and can be used to streamline the development and manufacture of more defined ADC products with more predictable safety or efficacy.

[0005] Several methods have been developed for region- and site-specific conjugation of payloads with antibodies. However, commonly known methods require antibody modification / engineering, such as by incorporating non-natural amino acids or by modifying carbohydrate moieties. This modification can negatively impact the therapeutic efficacy / safety of the corresponding ADC, for example, due to undesirable effects related to antibody activity, targeting, metabolism and / or excretion, and the immune response to the antibody. Other methods involve multiple steps, such as those listed in WO2018 / 199337. Such multi-step approaches can be costly and / or laborious, making them less attractive or even unsuitable for applications that expect a rapid and simple antibody modification process (e.g., for point-of-care diagnostic applications).

[0006] Therefore, there remains a need to find alternative methods for the region- or site-specific conjugation of payloads with antibodies or antibody fragments, particularly methods that do not require prior engineering of the antibodies or antibody fragments. Furthermore, there is a need to find methods for preparing antibody-drug conjugates in as few steps as possible (preferably in a single step).

[0007] In view of the above, one object of the present invention is to provide compounds (reactive conjugates) that enable the selective conjugation of a payload region to an antibody or antibody fragment in a single step, without prior engineering and / or modification of the antibody or antibody fragment. Another object is to provide a kit comprising such a compound.

[0008] Another object of the present invention is to provide a method for preparing modified antibodies or modified antibody fragments (e.g., ADCs) that can be used in methods for diagnosing, monitoring, imaging or treating diseases. Summary of the Invention

[0009] This invention provides a compound capable of selectively attaching a payload region to an antibody (e.g., a therapeutic antibody) or optionally incorporating an antibody fragment into an Fc-fusion protein. This region-selective attachment can be performed in a single step. The resulting modified antibody or modified antibody fragment (e.g., an ADC or antibody-radioactive conjugate) can be used in methods for diagnosing, monitoring, imaging, or treating diseases, particularly cancer.

[0010] The compounds (reactive conjugates) of the present invention can be represented by the following formula (1):

[0011] PYSV (1)

[0012] in,

[0013] P represents the payload;

[0014] Y is a reactive moiety capable of reacting with the side chain of an amino acid (e.g., lysine or cysteine), preferably a moiety capable of reacting with the side chain of lysine.

[0015] V is a carrier that can interact with the crystallizable (Fc) region of an antibody or its fragment, which is optionally incorporated into the Fc-fusion protein;

[0016] S is a spacer of length Z, where Z is the length such that when the carrier V interacts with the Fc region of the antibody or its fragment, the reactive portion Y can react with the side chain of amino acid residues on the antibody or antibody fragment.

[0017] The present invention also relates to a kit for regioselectively modifying an antibody or antibody fragment, the antibody fragment optionally incorporated into an Fc-fusion protein, wherein the kit comprises a compound and a buffer as described above, the compound optionally immobilized on a solid matrix (e.g., beads).

[0018] Furthermore, the present invention relates to a method for regioselectively modifying an antibody or antibody fragment, the antibody fragment optionally incorporated into an Fc-fusion protein, wherein the method uses the compounds described above.

[0019] Furthermore, the present invention relates to a modified antibody or modified antibody fragment (e.g., obtainable or acquired by the methods described above), the antibody fragment optionally incorporated into an Fc-fusion protein, the modified antibody or modified antibody fragment for use in methods for diagnosing, monitoring, imaging and / or treating diseases (especially cancer).

[0020] The present invention specifically includes the following embodiments (“projects”):

[0021] 1. A compound represented by the following formula (1):

[0022] PYSV (1)

[0023] in,

[0024] P represents the payload;

[0025] Y is a reactive moiety capable of reacting with the side chain of an amino acid, preferably a moiety capable of reacting with the side chain of lysine.

[0026] V is a carrier that can interact with the crystallizable fragment (Fc) region of an antibody or a fragment thereof, the antibody fragment optionally being incorporated into the Fc-fusion protein;

[0027] S is a spacer of length Z, where Z is the length such that when the carrier V interacts with the Fc region of the antibody or its fragment, the reactive portion Y can react with the side chain of amino acid residues on the antibody or antibody fragment.

[0028] 2. The compound according to Project 1, wherein the payload comprises a fraction selected from the group consisting of a radioisotope of the fraction and / or a pharmaceutically acceptable salt of the fraction:

[0029] (i) Selected from the following:

[0030] The labeling portion may include a radionuclide, preferably a chelating agent, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriamine pentaaceticacid (DTPA), cyclohexyl diethylenetriamine pentaaceticacid (CH-X-DTPA), 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (3,6,9,15-Tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid). The chelating agent may be PCTA or deferrioxamine (DFO), wherein the chelating agent optionally chelates a radionuclide;

[0031] Hair color clusters;

[0032] Fluorescein, such as fluorescein or rhodamine; and

[0033] Contains such as 125 I, 123 I, 131 I, 18 F, 11 C 15 O、 18 The labeled portion of the radionuclide of F, for example, derived from those containing, such as 125 I, 123 I or 131 The 4-hydroxyphenylpropionate fraction of radionuclide I;

[0034] (ii) A portion selected from the portion containing a bonding group, the portion containing a bonding group including optionally substituted conjugated dienes, optionally substituted tetrazines, optionally substituted alkynes or azides, optionally substituted dibenzocyclooctyne (DBCO), optionally substituted trans-cyclooctene (TCO), optionally substituted bicyclo[6.1.0]nonyne (BCN), optionally substituted aldehydes, optionally substituted ketones, and optionally substituted hydrazines;

[0035] (iii) Derivatives from the following drugs

[0036] Antitumor agents, such as DNA-alkylating agents, for example, duocarmycin;

[0037] Topoisomerase inhibitors, such as doxorubicin;

[0038] RNA polymerase II inhibitors, such as α-amanitin;

[0039] DNA cleavage agents, such as calicheamicin;

[0040] Antimitotic agents or microtubule disruptors, such as taxane, auristatin, or maytansinoid;

[0041] Antimetabolites;

[0042] Kinase inhibitors, such as partasertib;

[0043] Immunomodulators;

[0044] Anti-infective agents.

[0045] 3. The compound according to item 1 or 2, wherein the active loading is optionally a chelating agent for chelating radionuclides, the chelating agent preferably being derived from the following moieties: DTPA, CH-X-DTPA, DFO, 1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-tetraacetic acid (NODAGA), 1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid. 2,2'-(1,4,7-triazacyclononane-1,4-diyl)diacetate (NO2A), DOTA, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), ethylenediaminetetraacetic acid (EDTA), ethylenediamine diacetic acid, triethylenetetraminehexaacetic acid acid, TTHA), 1,4,8,11-tetraazacyclotetradecane (CYCLAM), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diaceticacid (CB-TE2A), 2,2',2''-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetamide (2,2',2''-(1,4,7,10- tetraazacyclododecane-1,4,7-triyl)triacetamide (DO3AM), 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A), 1,5,9-triazacyclododecane (TACD), (3a1s,5a1s)-decahydro-3a,5a,8a,10a-tetraazapyrene (cis-glyoxal-cyclam), 1,4,7-triazacyclononane (TACN), 1,4,7,10-tetraazacyclododecane (cyclen), tri(hydroxypyridinone) (tri(hydroxypyridinone), THP), 3-(((4,7-bis((hydroxy(hydroxymethyl)phosphoryl)methyl)-1,4,7-triazonan-1-yl)methyl)(hydroxy)phosphoryl)propanoic acid ((((4,7-bis((hydroxy(hydroxymethyl) phosphoryl)methyl)-1,4,7-triazonan-1-yl)methyl)(hydroxy)phosphoryl)propanoic acid, NOPO), PCTA, 2,2',2'',2'''-(1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetraacetic acid (2,2',2'',2'''-(1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetraacetic acid (TRITA), 2,2',2'',2'''-(1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetraacetamide (TRITAM), 2,2' ... TRITRAM (2,2',2''-(1,4,7,10-tetraazacyclotridecane-1,4,7-triyl)triacetamide), trans-N-dimethylcyclopamide, 2,2',2''-(1,4,7-triazacyclononane-1,4,7-triyl)triacetamide (2,2',2''-(1,4,7-triazacyclononane-1,4,7-triyl)triacetamide)7-triyl)triacetamide (NOTAM), oxocyclam, dioxocyclam, 1,7-dioxa-4,10-diazacyclododecane, cross-bridged-cyclam (CB-cyclam), triazacyclononane phosphinate (TRAP), dipyridoxyl diphosphate (DPDP), meso-tetra-(4-sulfanotophenyl)porphine (TPPS4), ethylenebishydroxyphenylglycine (EHPG), hexamethylenediaminetetraacetic acid, dimethylphosphinomethane (DMPE), methylene diphosphate, dimercaptosuccinic acid (DMPA), or derivatives thereof; more preferably, the fraction derived from DTPA, DOTA, DFO, NODAGA, PCTA, CH-X-DTPA, NODAGA, or DOTAGA.

[0046] 4. The compound according to item 2 or 3, wherein the radionuclide is selected from... 124 I, 131 I, 86 Y、 90 Y、 177 Lu、 111 In、 188 Re、 55 Co、 64 Cu、 67 Cu、 68 Ga、 89 Zr、 203 Pb, 212 Pb, 212 Bi、 213 Bi、 72 As、 211 At、 225 Ac、 223 Ra、 97 Ru、 149 Tb, 152 Tb, 161 Tb, 99m Tc, 226 Th、 227 Th、 201 Tl、 89 Sr、 44 / 43 Sc、47 Sc、 153 Sm、 133 Xe and Al 18 F, preferably selected from 89 Zr、 111 In、 64 Cu、 177 Lu、 68 Ga、 99m Tc, 203 Pb, 72 As、 55 Co、 97 Ru、 201 Tl、 152 Tb, 133 Xe, 86 Y and Al 18 F, more preferably selected from 89 Zr、 111 In、 64 Cu、 177 Lu、 68 Ga and 99m Tc, especially 111 In.

[0047] 5. Compounds according to item 2 or 3, wherein the active loading is derived from, or a radioisotope of, said fraction and / or a pharmaceutically acceptable salt of said fraction: eczema, PNU-159682, muscarinic acid, docamycin, auristatin, maytansine, tubulolysin, calcitrazine, SN-38, paclitaxel, daunomycin, vincristine, doxorubicin, methotrexate, pyrrolobenzodiazepine, pyrrole-based kinesin spindle protein (KSP) inhibitors, indolino-benzodiazepine dimers.

[0048] 6. A compound from any one of items 1 to 5, wherein P is represented by the following formula (2):

[0049] P1-L-- (2)

[0050] in,

[0051] P1 is the payload defined in any of items 2 to 5;

[0052] L is a connector, preferably a connector containing one or more atoms selected from carbon, nitrogen, oxygen and sulfur, which is optionally pyrolyzable;

[0053] 'Refers to covalent attachment to the reactive portion (Y).

[0054] 7. The compound of item 6, wherein the connector is selected from:

[0055] (a1) An alkylene group having 1 to 12 carbon atoms, preferably an alkylene group having 2 to 6 carbon atoms, such as a propylene group;

[0056] (b1) A polyalkyleneoxide group having 2 or 3 carbon atoms and 1 to 36 repeating units; preferably of the formula -NH-(CH2CH2O). n1 -CH2CH2–, where n1 is an integer from 0 to 35, for example, an integer from 1 to 20;

[0057] (c1) A peptide group having 2 to 12 amino acids.

[0058] 8. A compound according to any one of items 1 to 7, wherein the reactive part is represented by the following formula (3a):

[0059] --(F1-RC-F2)-- (3a)

[0060] in,

[0061] RC is the reaction center, preferably an electrophilic reaction center, and more preferably a group selected from C=O and C=S;

[0062] F1 is a single covalent bond, atom, or atomic group; preferably an atom selected from O and S, or an atomic group containing one or more atoms selected from C, N, O, and S; more preferably an atom selected from O and S.

[0063] F2 represents an atom or an atomic group; preferably an atom selected from O and S, or an atomic group containing one or more atoms selected from C, N, O and S; more preferably an atom selected from O and S;

[0064] Refers to covalent attachment to the spacer (S); and

[0065] Refers to covalent attachment to the payload (P).

[0066] 9. The compound according to item 8, wherein the reactive portion is represented by one of the following formulas (4a) to (4m):

[0067] (4a) (4b) (4c)

[0068] (4d) (4e) (4f)

[0069] (4g) (4h)

[0070] (4i) (4j) (4k)

[0071] (4l) (4m);

[0072] in Refers to covalent attachment to the spacer (S). Refers to covalent attachment to the payload (P).

[0073] 10. A compound according to any one of items 1 to 7, wherein the reactive part is represented by the following formula (3b):

[0074] --(F1-RC-F2)-(M)-- (3b)

[0075] in,

[0076] RC is the reaction center, preferably an electrophilic reaction center, and more preferably a group selected from C=O and C=S;

[0077] F1 is a single covalent bond, atom, or atomic group; preferably an atom selected from O and S, or an atomic group containing one or more atoms selected from C, N, O, and S; more preferably an atom selected from O and S.

[0078] F2 represents an atom or an atomic group; preferably an atom selected from O and S, or an atomic group containing one or more atoms selected from C, N, O and S; more preferably an atom selected from O and S;

[0079] M is a group that can adjust the electron density and stability of F2, preferably a group that can withdraw electrons;

[0080] Refers to covalent attachment to the spacer (S); and

[0081] Refers to covalent attachment to the payload (P).

[0082] 11. According to the compound of Project 10, the group capable of regulating the electron density and stability of F2 is represented by the following formula (3c):

[0083] --M'—B—C-- (3c)

[0084] in,

[0085] M' is an aryl group having a 6-membered, 10-membered, or 14-membered ring and one, two, or three fused rings, or a heteroaryl group having a 5- to 20-membered ring, one, two, or three fused rings, and one to four heteroatoms independently selected from N, O, and S. M' may be substituted with one or more substituents; preferably, it is a phenyl, naphthyl, pyridyl, quinolinyl, isoquinolinyl, or benzotriazolyl substituent that may be substituted with one or more substituents, with each substituent preferably selected from -F, -Br, -Cl, -I, -NO2, -CN, and -C. 1-6 -alkyl, -C 1-6 -alkoxy group, -C 1-6 -amino groups (such as -C(=O)NH2), and combinations thereof (such as -CCl3, -CF3 or -CH2NO2);

[0086] B represents a single covalent bond, O, S, NR', where R' represents a hydrogen atom, OH, alkyl or cycloalkyl, and C. 2-6 - Idemenyl, C 2-6 -Imyynyl, a group having the following general formula or any combination thereof:

[0087] –(CH2) n1 -(H 1 ) x1 -(CH2) n2 -(H 2 ) x2 -(CH2) n3 -(H 3 ) x3 -(CH2) n4 -(3c')

[0088] in,

[0089] Each of n1, n2, n3, and n4 represents an integer independently selected from 0 to 10, such that n1 + n2 + n3 + n4 is 10 or less.

[0090] Each of x1, x2, and x3 is independently selected from 0 and 1, and

[0091] H 1H 2 and H 3 Each of them is an atom independently selected from N, O, and S.

[0092] The conditions are: if x1+x2=2, then n2≥1; if x2+x3=2, then n3≥0; if x1+x3=2, then n2≥1 or n3≥1; if x1+x2+x3 is 3, then n2≥1 and n3≥1.

[0093] B is preferably a single covalent bond, NH, or C. 1-10 -alkylene; more preferably a single covalent bond;

[0094] C can be C=O, C=S, or C(=NR''), where R'' represents a hydrogen atom, OH, alkyl or cycloalkyl, S=O or S(=O)2; preferably C=O;

[0095] Refers to covalent attachment to the spacer (S); and

[0096] It refers to covalent attachment to F2.

[0097] 12. According to the compounds of item 10 or 11, wherein a portion (F1-RC-F2) is represented by one of the following formulas (4a') to (4m'), and / or M is independently represented by one of the following formulas (5a) to (5j'):

[0098] (4a') (4b') (4c')

[0099] (4d') (4e')

[0100] (4f') (4g')

[0101] (4h') (4i') (4j')

[0102] (4k') (4l'), (4m')

[0103] (5a) (5b) (5c)

[0104] (5d) (5e)

[0105] (5f) (5g)

[0106] (5h) (5i) (5j) (5k) (5l) (5m) (5n) (5o) (5p) (5q) (5r) (5s) (5t) (5u) (5V) (5w) (5x) (5y) (5z) (5a') (5b') (5c') (5d') (5e') (5f') (5g') (5h') (5i') (5j');

[0107] Wherein refers to covalent attachment to the spacer (S), Refers to covalent attachment to the payload (P). Refers to covalent attachment to M. It refers to covalent attachment to F2.

[0108] 13. A compound according to any one of items 10 to 12, wherein the reactive portion is represented by one of the following formulas (6a) to (6l'):

[0109] (6a) (6b) (6c) (6d) (6e) (6f) (6g) (6h) (6i) (6j) (6k) (6l) (6m) (6n) (6o) (6p) (6q) (6r) (6s) (6t) (6u) (6v) (6w) (6x) (6y) (6z) (6a') (6b') (6c') (6d') (6e') (6f') (6g') (6h')

[0110] (6i') (6j')

[0111] (6k') (6l');

[0112] in Refers to covalent attachment to the spacer (S). Refers to covalent attachment to the payload (P).

[0113] 14. A compound according to any one of items 1 to 13, wherein the spacer has a length of 10 to 35 Å; and preferably a group having 12 to 120 atoms (e.g., 16 to 80 atoms) in the main chain, the atoms being selected from carbon, nitrogen, oxygen, and sulfur; more preferably selected from groups including:

[0114] (a2) A polyepoxyalkyl group having 6 to 36 repeating units, for example 8 to 24 repeating units; preferably a group represented by the following formula (7):

[0115] –X 1–(CH2CH2O) n2 –CH2CH2–X 2 –(7)

[0116] in,

[0117] X 1 It can be NH, O, or S; NH is preferred;

[0118] X 2 For NH or C=O, if X 2 If X covalently binds to the carrier, then X 2 Preferably, C=O; and

[0119] n2 is an integer from 4 to 28, preferably an integer from 6 to 20, such as 10;

[0120] (b2) A peptide group having 6 to 25 amino acids in the main chain, for example, having 9 amino acids in the main chain, wherein the amino acids are preferably selected from Pro, Gly, Ala, Asn, Asp, Thr, Glu, Gln and Ser; more preferably Pro, Gly or Ser.

[0121] 15. The compound according to any one of items 1 to 13, wherein the spacer comprises a polyethylene oxide having 4 to 36 repeating units, preferably 6 to 28 repeating units, more preferably 7 to 24 repeating units.

[0122] 16. A compound according to any one of items 1 to 15, wherein the carrier is a peptide comprising a sequence of 11 to 17 amino acids, for example 13 to 17 amino acids, preferably a peptide represented by one of the following formulas (8a) and (8b):

[0123]

[0124]

[0125] in,

[0126] Each of Bxx, Cxx, Dxx, Exx, and Fxx independently represents an amino acid;

[0127] Axx represents an amino acid, a dicarboxylic acid, or a peptide moiety represented by the following formula (9a):

[0128] ---Axx1–Axx2–Axx3--- (9a)

[0129] In equation (9a),

[0130] Axx1 represents a single covalent bond or amino acid, such as Arg;

[0131] Axx2 represents amino acids, such as Gly or Cys; and

[0132] Axx3 represents amino acids, such as Asp or Asn;

[0133] Gxx represents an amino acid, or a peptide moiety represented by the following formula (9b):

[0134] ---Gxx1–Gxx2–Gxx3---(9b)

[0135] In equation (9b),

[0136] Gxx1 represents amino acids, such as Thr;

[0137] Gxx2 represents amino acids, such as Tyr or Cys; and

[0138] Gxx3 represents a single covalent bond or amino acid, such as His; and

[0139] The side chains of Axx2 can covalently bind to the side chains of Gxx2 to form a ring;

[0140] If Axx2 is Cys and Gxx2 is Cys, then it is preferable to connect the side chains of Axx2 and Gxx2 together to form the formula -(SX). 4 -S)- groups, where X 4 It indicates a single covalent bond or a divalent group containing one or more atoms selected from carbon, nitrogen, and oxygen, such as a divalent maleimide group, a divalent acetone group, or a divalent aryl group, preferably a single covalent bond;

[0141] Hxx represents a single covalent bond, or a trifunctional amino acid, such as diaminocarboxylic acid;

[0142] Z1 indicates

[0143] If Hxx is a single covalent bond, then Z1 represents a group covalently bonded to the C-terminus of Gxx, which is selected from: -N(H)(R), where R represents a hydrogen atom, alkyl or cycloalkyl, and a portion derived from a compound containing a bonding group, which is selected from biotin, DBCO, TCO, BCN, alkynes, azides, bromoacetamide, maleimide and thiols.

[0144] If Hxx is a trifunctional amino acid and Y' is bound to the side chain of Hxx, then Z1 represents a group covalently bound to the C-terminus of Hxx, preferably N(H)(R), wherein if Z1 is covalently bound to the C-terminus of Hxx, then R represents a hydrogen atom, an alkyl group, or a cycloalkyl group; or

[0145] If Hxx is a trifunctional amino acid and Y' is bound to the C-terminus of Hxx, then Z1 represents a hydrogen atom bound to the side chain of Hxx.

[0146] Z2 indicates

[0147] If Hxx is a single covalent bond, then Z2 represents a group covalently bonded to the N-terminus of Axx, which is selected from hydrogen atoms, carbonyl groups such as acetyl groups, and groups containing coupling moieties such as biotin.

[0148] If Hxx is a trifunctional amino acid and Y' is bound to the side chain of Hxx, then Z2 represents a group covalently bound to the N-terminus of Hxx, selected from hydrogen atoms and carbonyl groups such as acetyl groups; or

[0149] If Hxx is a trifunctional amino acid and Y' is bound to the N-terminus of Hxx, then Z2 represents a hydrogen atom bound to the side chain of Hxx.

[0150] If Hxx is a trifunctional amino acid, then only Y' exists; and

[0151] If Z1 binds to the C-terminus of Hxx, or if Z2 binds to the N-terminus of Hxx, then Y' represents the portion covalently bound to the side chain of Hxx.

[0152] If Z1 is bound to the side chain of Hxx, then Y' represents the portion covalently bound to the C-terminus of Hxx, or

[0153] If Z2 is bound to the side chain of Hxx, then Y' represents the portion covalently bound to the N-terminus of Hxx;

[0154] Y' is derived from compounds containing a binding group, which is preferably selected from biotin, DBCO, TCO, BCN, alkynes, azides, bromoacetamide, maleimide and thiols;

[0155] X 3 This refers to a single covalent bond or a divalent group containing one or more atoms selected from carbon, nitrogen, and oxygen, such as a divalent maleimide group, a divalent acetone group, or a divalent aryl group, preferably X. 3 It is a single covalent bond;

[0156] Refers to covalent attachment to a spacer (S).

[0157] 17. According to the compounds in Item 16, at least one of Axx, Bxx, Cxx, Dxx, Exx, Fxx, Gxx, and Hxx is defined as follows:

[0158] Axx represents an amino acid selected from Ala, 2,3-diamino-propionic acid (Dap), Asp, Glu, 2-aminooctanoic acid, α-aminobutyric acid, Asn, and Gln; a dicarboxylic acid selected from succinic acid, glutaric acid, and adicarboxylic acid; or a peptide moiety of formula (9a); Axx is preferably Ala, Asp, or Asn, more preferably Asp; wherein Axx1 is a single covalent bond, Axx2 is Cys, and Axx3 is Asp;

[0159] Bxx represents an amino acid selected from Trp, Phe, Tyr, phenylglycine (Phg), 3-benzothiophene-2-yl-L-alanine, 3-naphthyl-2-yl-L-alanine, 3-biphenyl-4-yl-L-alanine, and 3-naphthyl-1-yl-L-alanine; preferably Trp;

[0160] Cxx represents an amino acid selected from His, Ala, 3-pyridin-2-yl-L-alanine, meta-tyrosine (mTyr), and Phe; preferably His, Ala, or mTyr; more preferably His;

[0161] Dxx represents an amino acid selected from Ala, Abu, Gly, Leu, Ile, Val, Met, cyclohexylalanine (Cha), Phe, Thr, Cys, Tyr, and norleucine (Nle); preferably Ala, Nle, or Leu; more preferably Leu;

[0162] Exx represents an amino acid selected from Ala, Gly, Asn, Ser, Abu, and Asp; preferably Ala or Gly; more preferably Gly;

[0163] Fxx represents an amino acid selected from Ala, Glu, Asp, Gln, His, Arg, Ser, and Asn; preferably Asp or Glu; more preferably Glu;

[0164] Gxx represents an amino acid selected from Thr, Ser, Ala, Asn, Val, 2-amino-butyric acid (Abu), Ile, Met, Leu, Pro, Gln, and Cys, or a peptide moiety of formula (9b); Gxx is preferably Thr or Ser, more preferably Thr; wherein Gxx1 is Thr, Gxx2 is Cys, and Gxx3 is a single covalent bond; and

[0165] Hxx represents amino acids selected from Dap, Dab, Lys, Orn and homo-lysine (homo-lysine, homo-Lys), preferably selected from Dap, Dab, Lys, Orn and homo-Lys.

[0166] 18. A compound according to any one of items 1 to 17, wherein the carrier is a peptide represented by one of the following formulas (8a') to (8d'):

[0167]

[0168] in,

[0169] Z1, Z2, X 3 X 4 and As defined in Item 16;

[0170] Preferably, it is a peptide represented by formula (8a') or (8b').

[0171] 19. Based on any one of items 1 to 18, the compound is selected from...

[0172] ,

[0173] ,

[0174] ,

[0175] ,

[0176] ,

[0177] ,

[0178] ,

[0179] as well as,

[0180] ;

[0181] Wherein, P is defined as in any of items 1 to 5, and Y' is defined as in item 16.

[0182] 20. The compound selected from any one of items 1 to 19.

[0183] ,

[0184] ,

[0185] ,

[0186] ,

[0187] ,

[0188] ,

[0189] ,

[0190] ,

[0191] ,

[0192] ,

[0193] ,

[0194] ,

[0195] ,

[0196] ,

[0197] ,

[0198] ,

[0199] ,

[0200] ,

[0201] ,

[0202] ,

[0203] ,

[0204] ,

[0205] ,

[0206] ,

[0207] ,

[0208] ,

[0209] ,

[0210] ,

[0211] ,

[0212] ,

[0213] ,

[0214] ,

[0215] ,

[0216] ,

[0217] ,

[0218] ,

[0219] ,and

[0220] .

[0221] 21. A kit for site-specific modification of an antibody or a fragment thereof, the antibody fragment optionally incorporated into an Fc-fusion protein, the kit comprising a compound of any one of items 1 to 20 and a buffer; wherein the pH of the buffer is preferably 5.5 to 11, more preferably 7.5 to 9.5.

[0222] 22. A kit for regioselectively modifying antibodies or fragments thereof according to item 21, wherein the compound is immobilized on a solid matrix, such as beads.

[0223] 23. A method for regioselectively modifying an antibody or a fragment thereof, the method comprising reacting the antibody or a fragment thereof with a compound according to any one of items 1 to 20, the antibody fragment optionally being incorporated into an Fc-fusion protein.

[0224] 24. According to the method in Project 23, where,

[0225] This antibody is a monoclonal antibody, preferably selected from the group consisting of: adalimumab, aducanumab, alemtuzumab, attumomab pentetate, atezolizumab, anetumab, avelumab, bapineuzumab, basiliximab, bectumomab, bermekimab, besilesomab, bevacizumab, bezlotoxumab, and brentuximab. brentuximab vedotin), Brodalumab, Blinatumomab, Catumaxomab, Cemipril, Cetuximab, Cinpanemab, Clivatuzumab, Clivatuzumab tetraxetan, Crenezumab tetraxetan, Daclizumab, Daratumumab, Denosumab, Dinutuximab, Durvalumab, Eedrecolomab, Elotuzumab, Elapalumab, Enfortumab, Vitin-Enfortumab vedotin), epratuzumab, epratuzumab-SN-38, etaracizumab, gemtuzumab, gemtuzumab-oczomicin, girentuximab, gosuranemab, ibritumomab, inebilizumab, infliximab, inotuzumab, inotuzumab ozogamicin, ipilimumab, isatuximab, ixekizumab, J591PSMA-antibody, labetuzumab, lecanemab, mogamulizumab, necitumumab, nimotuzumab, natalizumab, nivolumab, ocrelizumab, ofatumumab, olaratumab, oregovomab, panitumumab, pembrolizumab, pertuzumab, polotuzumab, vebolutuzumab vedotin, prasinezumab, racotumomab, ramucirumab, rituximab, siltuximab, sacituzumab, sacituzumab govitecan, semorinemab, siltuximab, solanezumab, tacatuzumab, teprotumumab, tilavonemab, tocilizumab, tositumomab, trastuzumab, trastuzumab deruxtecan, trastuzumab emtansine emtansine), TS23, ustekinumab, vedolizumab, votumumab, zagotenemab, zalutumumab, fragments and derivatives thereof;

[0226] The antibody fragment is incorporated into the Fc-fusion protein, which is preferably selected from belatacept, aflibercept, ziv-aflibercept, dulaglutide, rilonacept, romiplostim, abatacept, and alefacept.

[0227] 25. A modified antibody or modified antibody fragment obtained by reacting an antibody or antibody fragment with a compound according to any one of items 1 to 20, the antibody fragment optionally being incorporated into an Fc-fusion protein, wherein the antibody or antibody fragment preferably has the same definition as in item 24.

[0228] 26. A method for diagnosing, monitoring, imaging, or treating a disease, as defined in Item 25, comprising administering the modified antibody or modified antibody fragment to a subject.

[0229] 27. A method for diagnosing, monitoring, imaging, or treating a disease, the method comprising administering a modified antibody or modified antibody fragment according to item 25 to a subject in need.

[0230] 28. The modified antibody or modified antibody fragment for use according to Item 26, or the method according to Item 27, wherein the disease is a neurological disease, a cardiovascular disease, an autoimmune disease, or cancer.

[0231] 29. A modified antibody or modified antibody fragment for use according to item 26 or 28, or the method according to item 27 or 28, wherein the disease or treatment thereof is selected from the group consisting of: Alzheimer's disease, amyotrophic lateral sclerosis, cerebral arteriosclerosis, encephalopathy, Huntington's disease, multiple sclerosis, Parkinson's disease, progressive multifocal leukoencephalopathy, systemic lupus erythematosus, systemic sclerosis, angina pectoris including unstable angina, aortic aneurysm, atherosclerosis, heart transplantation, diagnosis of cardiotoxicity, coronary artery bypass grafting, including heart Atrial fibrillation can terminate systolic heart failure, hypercholesterolemia, local ischemia, myocardial infarction, thromboembolism, thrombosis, ankylosing spondylitis, autoimmune cytopenia, autoimmune myocarditis, Crohn's disease, graft-versus-host disease, granulomatous polyangiitis, idiopathic thrombocytopenic purpura, juvenile arthritis, juvenile diabetes (type 1 diabetes), lupus, microscopic polyangiitis, multiple sclerosis, plaque psoriasis, psoriasis, psoriatic arthritis, rheumatoid arthritis, ulcerative colitis (UC), uveitis, and vasculitis.

[0232] 30. A modified antibody or modified antibody fragment for use according to item 26 or 28, or a method according to item 27 or 28, wherein the disease involves cells selected from lymphoma cells, myeloma cells, renal cell carcinoma cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cell carcinoma cells, small cell lung cancer cells, testicular cancer cells, pancreatic cancer cells, liver cancer cells, melanoma, head and neck cancer cells; and any cells that cause cancer by growing and dividing at an unregulated and accelerated rate; preferably selected from breast cancer cells, small cell lung cancer cells, lymphoma cells, colorectal cancer cells, and head and neck cancer cells. Attached Figure Description

[0233] Figure 1 – A schematic diagram of an antibody conjugation method using the compounds of this invention. A carrier capable of interacting with the Fc region of the antibody binds to the Fc region, thereby bringing the reactive portion close to the side chain of a lysine residue exposed on the antibody surface. The reaction between the side chain of the lysine residue and the reactive portion causes the payload (via a linker) to covalently attach to the antibody, and causes the carrier to be released concurrently.

[0234] Figure 2 – The synthesis of compound 29, which includes a labeled moiety (DOTA) and PEG as the active ingredient. 10The spacer compounds are: a) 1. HATU, DMF, DIEA (pre-activated for 3-5 minutes), 2. Compound 1, 3. 20% piperidine in DMF (yield after 2 steps: 42%); b) 1. HATU, DMF, DIEA (pre-activated for 3-5 minutes), 2. Compound 7, 3. TFA (+HPLC purification).

[0235] Figure 3 – Fluorescence polarization (FP) binding assay. Binding isotherms at 5 nM for the fluorescein derivative of ligand Fc-III (Fc-III-FAM) with the therapeutic monoclonal antibodies trastuzumab, alemtuzumab, bevacizumab, and rituximab. These lines are obtained by fitting the data using the Hill equation to obtain the half-maximal effect concentration (EC50). 50 This study confirmed that the Fc-binding ligand Fc-III-FAM binds to each antibody (trastuzumab: 14 nM, alemtuzumab: 13 nM, bevacizumab: 7 nM, rituximab: 11 nM) with high affinity.

[0236] Figure 4 – Competitive FP binding assay. The propensity of the Fc-binding vectors of Example 1 (compounds 1 (Fc III), 2, 9-11, 15, and 16) to bind to the Fc region of trastuzumab against Fc-III-FAM was evaluated. These lines are data fitted using the Hill equation to obtain the half-maximal inhibitory concentration (IC50). 50 The results are also given in Table 3.

[0237] Figure 5 – Synthesis of compounds 17 and 19, namely fluorescein- and DOTA-carbonate (ester) derivatives: a) CH3CN of Et3N at 40 °C, b) CH2Cl2 of DMAP at 25 °C, c) TFA / CH2Cl2 (1 / 3, v / v), d) CH3CN of DIPEA at 25 °C, e) CH3CN / DMF of DIPEA at 25 °C (1 / 1, v / v).

[0238] Figure 6 – High-resolution mass spectrometry (HRMS) of trastuzumab and trastuzumab modified with compound 31, i.e., trastuzumab-DOTA conjugates. Peaks D0 to D3 correspond to trastuzumab fragments with different degrees of conjugation. The sample was deglycosylated prior to HRMS measurement.

[0239] Figure 7– Trastuzumab DOTA conjugate with Gingis Khan ® Enzymatic digestion yielded HRMS with Fab and Fc regions. Peaks D0 to D2 corresponded to trastuzumab with different conjugation levels.

[0240] Figure 8 – Affinity of trastuzumab-DOTA conjugate and trastuzumab to SK-BR-3 (HER2+) and MD-MB-231 (HER2-) cells. For SK-BR-3 cells, the concentration range of antibody or antibody-drug conjugate was 0.003 to 30 µg / mL (after 1 / 10 dilution). For MD-MB-231 cells, only 3 µg / mL and 30 µg / mL were used. Trastuzumab and trastuzumab-DOTA conjugate were stained with a secondary rat anti-human IgG Fc antibody conjugated to Alexa 488. Dead cells were excluded using DRAQ7. Error bar: SD (n=2).

[0241] Figure 9 – Synthesis of compound 38, which includes PEG 20 Reactive conjugates for spacers and labeled moieties (fluorescein (FL)) as active loads: a) 1. HATU, DMF, DIEA (pre-activated for 3-5 min), 2. Compound 1, 3. 3. 20% piperidine in DMF (yield after 2 steps: 50%), b) 1. HATU, DMF, DIEA (pre-activated for 3-5 min), 2. Compound 10, 3. TFA (+HPLC purification; yield: 19%).

[0242] Figure 10 – Non-reducing SDS-PAGE analysis of trastuzumab-FL conjugates prepared by the reaction of compounds 35-41 with trastuzumab (IgGT). Conjugates after reduction (A) or IdeS protease digestion (B) were analyzed using Coomassie blue staining and fluorescence analysis.

[0243] Figure 11 – BT-474 cells were incubated with 10 µg / ml FITC-trastuzumab (conjugate 12, random conjugate, dashed line) and FL-trastuzumab (conjugate 11, solid color), as well as increased concentrations of unlabeled trastuzumab. The plotted data represent the mean MFI scores from two independent experiments. The maximum MFI for each antibody was normalized to 1.

[0244] Figure 12 – Through compounds 38 and 40 with trastuzumab (IgGT), the commercial trastuzumab (Herceptin) ®Non-reducing SDS-PAGE analysis was performed on trastuzumab-FL conjugates prepared by reacting with IgGH, alemtuzumab (IgGA), bevacizumab (IgGB), and rituximab (IgGR). The conjugates after IdeS protease digestion were analyzed using fluorescence and Coomassie blue staining.

[0245] Figure 13 – A schematic diagram of a reactive coupling material fixed on a solid support.

[0246] Figure 14 – A schematic diagram of an antibody conjugation method using a peptide conjugate containing a DBCO group (compound 43) and any payload containing an azide group. Detailed Implementation

[0247] 1. Definition

[0248] As used herein, the term "payload" refers to a substance (e.g., naturally occurring or synthetic) that, when attached (coupled) to an antibody or antibody fragment, can confer new functions. In some embodiments, the term "payload" as used herein should be understood as a labeled portion (e.g., a chromophore, fluorophore, radiolabeled portion) capable of and / or facilitating the detection and / or visualization of its attached complementary portion (e.g., an antibody). For example, the labeled portion can be detected and / or visualized using functionalized (physiological) imaging techniques known in the art, such as computed tomography (CT), positron emission tomography (PET), etc. In some embodiments, the term "payload" as used herein should be understood as a pharmacologically active substance capable of inhibiting or preventing cellular function and / or killing cells. In some embodiments, the term "payload" should be understood as synonymous with other terms commonly used in the art, such as "cytotoxic agent," "toxin," or "drug" as used in the field of cancer treatment. Alternatively, a payload is a portion selected from those containing a conjugating group. The effective load may include a group derived from a functional group that allows the effective load to be covalently attached to the remainder of the compound (e.g., to the reactive portion Y in formula (1)), such as a carboxylic acid, primary amine, secondary amine, hydroxyl group, thiol group, etc.

[0249] As used herein, the term "peptide" can be understood as a compound comprising a continuous sequence of at least three amino acids linked together by peptide bonds. In this respect, the term "peptide bond" refers to both the (skeletal) amide bond and the modifying bond, which can be obtained if a non-natural amino acid is introduced into the peptide sequence. In this case, the modifying bond replaces the (skeletal) amide bond, which is formed by the reaction of the amino and carboxyl groups of two amino acid residues (NH2-CR). 1 -COOH+NH2-CR 2 -COOH NH2-CR 1 -(C=O)-NH-CR 2 The modified bond is in the continuous peptide sequence of (-COOH). For example, the modified bond can be an ester (NH2-CR). 1 -(C=O)-O-CR 2 -COOH), thioesters (NH2-CR) 1 -(C=O)-S-CR 2 -COOH or NH2-CR 1 -(C=S)-O-CR 2 -COOH), urea (NH2-CR) 1 -NH-(C=O)-NH-CR 2 -COOH), thiourea (NH2-CR) 1 -NH-(C=S)-NH-CR 2 -COOH) or triazole bond (e.g., NH2-CR) 1 -C≡CH + N3-CR 2 -COOH NH2-CR 1 -X-CR 2 -COOH, where X represents a 1,4-disubstituted-1,2,3-triazole moiety. Preferably, the amino acids forming the continuous peptide sequence are linked to each other by skeletal amide bonds. The peptide can be linear or branched. On the one hand, the peptide can be a ring formed, for example, from a linear chain of amino acids modified to form a ring (e.g., "head-to-tail" cyclization), or a ring formed from a linear chain of amino acids having side chains covalently attached to each other (e.g., formed by disulfide bonds or any other modification). In this document, amino acids include naturally occurring amino acids as well as non-natural (synthetic) amino acids, as described below.

[0250] As used herein, the term "labeled portion" (or the synonym "label" or "labeling group") refers to a portion containing a group that enables and / or facilitates the detection and / or visualization of its attached complementary portion (e.g., antibody) by visual or instrumental means. Examples of labeled portions include radiolabels (e.g., radionuclides), contrast agents for magnetic resonance imaging (MRI), and light-absorbing or light-emitting chemicals (e.g., chromophores and fluorophores).

[0251] As used herein, the term "drug-derived portion" refers to the portion corresponding to a natural drug, other than having structural modifications for incorporating a natural drug into the reactive group or linker contained in the compounds of the present invention. Depending on the functional groups available in the natural drug, one of the functional groups already present in the natural drug can be used to influence the incorporation, or the natural drug can be modified by incorporating a new functional group to influence the incorporation. Therefore, the drug can be used for incorporation in its unmodified form, or the drug can be chemically modified to incorporate a functional group that allows covalent attachment to the reactive portion or linker contained in the compounds of the present invention. As used herein, the term "drug-derived portion" encompasses both meanings.

[0252] In a similar manner, the term "derivative" is used in conjunction with other parts to characterize the presence of covalent bonds required for bonding to adjacent or other parts that are chemically modified to incorporate a functional group that allows covalent attachment to the adjacent parts. In other words, the term "derivative" can characterize parts that bond to adjacent parts, distinguishing them from the molecules from which they are derived only by the structural elements responsible for bonding to the adjacent parts. This could include, for example, covalent bonds formed by existing functional groups after removing a hydrogen atom to provide the free valence required for bonding, or newly introduced covalent bonds and adjacent functional groups for this purpose.

[0253] The term "natural medicine" refers to a compound whose therapeutic efficacy has been demonstrated through in vitro and / or in vivo studies. In a preferred embodiment, a natural medicine is a compound whose therapeutic efficacy has been demonstrated through clinical trials. Most preferably, a natural medicine is a commercially available drug. The type of therapeutic efficacy to be demonstrated and the appropriate trials to be applied, of course, depend on the type of medical indication to be treated.

[0254] When referring to specific classes of drug molecules, such as antitumor agents, topoisomerase inhibitors, RNA polymerase II inhibitors, DNA cleavage agents, antimitotic agents or microtubule disruptors, antimetabolites, kinase inhibitors, immunomodulators, or anti-infective agents, these terms are intended to have a generally accepted meaning in the medical field, for example, by […]. Mosby's Medical Dictionary Mosby, Elsevier 10 th ed. (2016), or Oxford Textbook of Oncology David J. Kerr, OUP Oxford 3 rd As reflected in ed. (2016).

[0255] As used herein, a chelating agent is a molecule containing two or more electron-donating atoms that can form coordinate bonds with a single central metal ion (e.g., a radionuclide). Typically, chelating agents coordinate the metal ion via oxygen or nitrogen donor atoms, or both. After the formation of the first coordinate bond, the bonded successive donor atoms create a ring containing the metal ion. Chelating agents can be bidentate, tripentate, tetradentate, etc., depending on whether they contain 2, 3, 4, or more donor atoms capable of binding to the metal ion. However, the chelation mechanism is not fully understood and depends on the chelating agent and / or the radionuclide. For example, it is believed that DOTA can coordinate a radionuclide via a carboxylate and an amino group (donor group), thereby forming a highly stable complex (Dai et al. Nature Com. 2018, 9, 857). The term "chelating agent" should be understood to include both the chelating agent and its salts. For example, chelating agents having carboxyl groups (e.g., DOTA, TRITA, HETA, HEXA, EDTA, DTPA, etc.) can be derivatized to convert one or more carboxyl groups into an amino group for attachment to the compound (attached to the reactive moiety or linker). Alternatively, for example, the compound can be derivatized to be able to attach to the compound via one of the CH2 groups in the chelating ring.

[0256] As used herein, the term "radionoid" refers to an atom having an unstable nucleus, characterized by its ability to impart excess energy to newly generated radioactive particles or atomic electrons within the nucleus. Radionoids may occur naturally or be produced artificially. In some embodiments, the radionoids used in this invention are medically useful radionoids, including, for example, radioactive metals with positively charged ions, such as Y, In, Cu, Lu, Tc, Re, Co, and Fe. Preferably, the radionoids are selected from... 89 Zr、 111 In、 64 Cu、 177 Lu、 68 Ga、 99m Tc, 203 Pb, 72 As、 55 Co、 97 Ru、 201 Tl、152 Tb, 133 Xe, 86 Y and Al 18 F, more preferably selected from 89 Zr、 111 In、 64 Cu、 177 Lu、 68 Ga and 99m Tc, especially 111 In.

[0257] As used herein, the term "chromophore" refers to an organic or organometallic compound that can absorb electromagnetic radiation in the range of 350 nm to 1100 nm or its sub-ranges, such as 350-500 nm, 500-850 nm, or 350-850 nm.

[0258] As used herein, the term "fluorophore" refers to a compound that emits light of different (higher) wavelengths when excited by exposure to light of a specific wavelength. Fluorophores are typically described by their emission distribution or "color." For example, green fluorophores such as Cy3 or FITC typically emit wavelengths in the 515–540 nm range, while red fluorophores such as Cy5 or tetramethylrhodamine typically emit wavelengths in the 590–690 nm range. As used herein, the term "fluorophore" should be understood to specifically encompass organic fluorescent dyes such as fluorescein, rhodamine, or AMCA, as well as biofluorescent dyes.

[0259] As used herein, "pharmaceutically acceptable salt" refers to a derivative of the disclosed compound (including reactive conjugates) wherein the parent compound is modified by preparing its acid salt or base salt. Pharmaceutically acceptable salts include, for example, non-toxic salts or quaternary ammonium salts of parent compounds formed from non-toxic inorganic acids or bases, or non-toxic organic acids or bases. A list of suitable salts can be found below: Remington's Pharmaceutical Sciences, 17 thed., Mack Publishing Company, Easton, PA, 1985, page 1418; SMBerge, LMBighley, and D.C. Monkhouse, “Pharmaceutical Salts,” J. Pharm.Sci.66 (1), 1–19 (1977); PH Stahl and CG Wermuth, editors, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zürich, Wiley-VCH, 2008 and in A.K. Bansal et al., Pharmaceutical Technology, 3(32), 2008. Pharmaceutical salts can be synthesized by conventional chemical methods from parent compounds containing basic or acidic moieties. For reactive conjugates, this can be done before or after incorporating the pharmaceutical moiety into the compounds of this application. Unless the context otherwise requires, all references to compounds of the present invention (conjugates, modified antibodies, etc.) should also be understood as references to pharmaceutically acceptable salts of the corresponding compounds.

[0260] As used herein, the term "reactive moiety" refers to a portion that readily reacts with a binding partner on another molecule (e.g., a nucleophile). This contrasts with portions that require the addition of a catalyst or very impractical reaction conditions (i.e., "non-reactive" or "inert" portions). Specifically, the term "reactive moiety" refers to a portion of a reactive conjugate that is coupled with an antibody (preferably trastuzumab, which can be purchased from Roche's Herceptin). ® The side chain of Lys was reacted in 50 mM NaHCO3 at pH 9.0 with a molar ratio of conjugate to trastuzumab of 2:1 at 1000 rpm at room temperature for 2 hours, such that at least 25% of the conjugate reacted, preferably at least 50%, and more preferably at least 70% (e.g., attachment of the active load to trastuzumab). The attachment of the active load to trastuzumab can be determined by high-resolution mass spectrometry according to the method described in Section 9.3.5 below.

[0261] As used herein, "side chain of an amino acid" refers to the portion attached to the α-carbon of the amino acid. For example, the side chain of Ala is methyl, the side chain of Phe is benzyl, the side chain of Cys is thiomethyl, and the side chain of Tyr is 4-hydroxybenzyl, etc. Both naturally occurring and non-naturally occurring side chains are included in this definition. In the case of non-natural amino acids, the side chain can also exist in different positions, such as attached to the skeletal nitrogen in a peptide-like structure or attached to the β-carbon in some forms of β-amino acids.

[0262] As used herein, the term "amino acid" refers to a compound containing or derived from at least one amino group and at least one acidic group (preferably a carboxyl group). There is no particular limitation on the distance between the amino group and the acidic group; α-amino acids, β-amino acids, and γ-amino acids are suitable, but α-amino acids, especially α-aminocarboxylic acids, are particularly preferred. The term encompasses naturally occurring amino acids as well as synthetic amino acids not found in nature. In the following text, amino acids may be referred to by three-letter amino acid codes (Arg, Phe, Ala, Cys, Gly, Gln, etc.) or by one-letter amino acid codes (R, F, A, C, G, Q, etc.). In the following text, the amino acid sequence is written from the N-terminus to the C-terminus (from left to right).

[0263] As used herein, the term "trifunctional group" refers to a compound or part having three functional groups that can form or have formed three covalent bonds with adjacent parts. Therefore, the term "trifunctional amino acid" refers to a compound that contains or is derived from a compound that contains at least an amino group, an acid group (e.g., a carboxyl group), and another functional group (such as an amino or carboxyl group).

[0264] As used herein, the term "C-terminus" refers to the C-terminus of an amino acid (peptide) chain. Binding to the "C-terminus" means the formation of a covalent bond between the acid group in the backbone (backbone) of the amino acid residue and the binding partner. For example, the group "X" binds to the C-terminus of amino acid residue Axx to produce an ester or amide-type structural element –C(O)–X, where the carbonyl group is derived from the acid group of Axx.

[0265] As used herein, the term "N-terminus" refers to the N-terminus of an amino acid (peptide) chain. "N-terminus" binding refers to the formation of a covalent bond between an amino group in the backbone (backbone) of an amino acid residue and a binding partner (which replaces a hydrogen atom). For example, the group "X" binds to the N-terminus of amino acid residue Axx to create the structural element X-NH-, where the amino group is derived from Axx.

[0266] As used herein, the expression “capable of interacting with the crystallizable fragment (Fc) region of an antibody or a fragment thereof” indicates that the carrier can bind to the Fc region of an antibody or antibody fragment as defined above. This interaction / binding can produce a targeting effect, i.e., a localized increase in the concentration of the reactive portion near the amino acid (e.g., lysine residue) side chain of the antibody or antibody fragment. The interaction (binding) of the carrier with the Fc region of the antibody or antibody fragment can be evaluated using fluorescence polarization techniques known in the art and further described below. In some aspects, the expression “compound capable of interacting with the Fc region of an antibody or a fragment thereof” refers to a compound that retains at least 20%, preferably at least 50%, more preferably at least 80% of the binding affinity of the ligand “Fc-III” for the Fc-region of IgG, such binding affinity as that described by DeLano et al. ( Science Described in 2000, 287, 1279-1283, and measured by fluorescence polarization. This compound is capable of interacting with the Fc region of antibodies or fragments thereof, and therefore, compared with Fc-III, this compound can have a superior binding affinity to the Fc region.

[0267] As used herein, the term "antibody" (also synonymously "immunoglobulin" (Ig)) encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), eneered antibodies, and small immunoproteins, provided that it contains at least one crystallizable fragment (Fc) region. Antibodies are proteins produced by the immune system that recognize and bind to specific antigens. Target antigens typically have numerous binding sites, also known as epitopes, recognized by complementary-determining regions on multiple antibodies. Antibodies that specifically bind to different epitopes have different structures. Therefore, an antigen can have more than one corresponding antibody. Antibodies comprise full-length immunoglobulin molecules or the immunoactive portion of full-length immunoglobulin molecules, i.e., molecules containing antigen-binding sites that immune-specifically bind to the target antigen or a portion of the target antigen. Antibodies can be IgG, such as IgG1, IgG2, IgG3, and IgG4. Preferably, the antibody is an IgG protein, more preferably IgG1, IgG2, or IgG4 protein. Most preferably, the antibody is an IgG1 protein. The antibody can be human or derived from other species. Preferably, the antibody is a human antibody.

[0268] As used in this article, “monoclonal antibody” refers to the same antibody because they are produced by one type of immune cell and are clones of a single parent cell.

[0269] As used herein, “antibody fragment” refers to a molecule containing at least one polypeptide chain derived from an antibody that is not full-length and has at least one crystallizable fragment region capable of interacting with a ligand.

[0270] As used herein, "commercially formulated antibody" refers to a commercially available formulation comprising a therapeutic antibody and one or more excipients. Preferably, a commercially formulated antibody is a formulation commercially available in the European Union. Examples of commercially formulated antibodies include Humira. ® Lemtrada ® Camppath ® Tecentriq ® Bavencio ® Simulect ® LymphoScan ® Xilonix ® Scintimun ® Avastin ® Zinplava ® Blincyto ® Libtayo ® Erbitux ® hPAM4-Cide ® Zenapax ® Darzalex ® Prolia ® Unituxin ® Imfinzi ® Panorex ® Empliciti ® Gamifant ® Rencarex ® Remicade ® Besponsa ® Yervoy ® CEA-Cide ® Poteligeo ® Tysabri ® Portrazza ® Theracim ® Opdivo ® Arzerra ® Lartruvo ®Omnitarg ® Vaxira ® Cyramza ® MabThera ® Rituxan ® Sylvant ® , Bexxar ® Herceptin ® Kadcyla ® Stelara ® HuMax-EGFr ® HuMax-CD4 ® and their biosimilars. Information on commercially formulated antibodies can be found, for example, in... Allgemeine and Spezielle Pharmakologie und Toxicologie , Thomas Karow and Ruth Lang-Roth, Karow, 27 th Found in ed. (2018).

[0271] Preferably, the commercially formulated antibody is Herceptin® (a trastuzumab-containing formulation) approved for sale in the European Union by the European Medicines Agency (EMA) with authorization numbers EU / 1 / 00 / 145 / 001 and EU / 1 / 00 / 145 / 002 (available from Roche), or Rituximab® (a rituximab-containing formulation) approved for sale in the European Union by the EMA with authorization numbers EU / 1 / 98 / 067 / 001, EU / 1 / 98 / 067 / 002, EU / 1 / 98 / 067 / 003 and EU / 1 / 98 / 067 / 004.

[0272] As used herein, "Fc-fusion protein" refers to a protein comprising at least an Fc-containing antibody fragment—i.e., an immunoglobulin-derived portion containing at least one Fc region—and a portion derived from a second non-immunoglobulin. The Fc-containing antibody fragment forms part of the Fc-fusion protein, thereby being incorporated into the Fc-fusion protein. The Fc-containing antibody fragment may be derived from the antibodies described above, particularly from IgG, such as IgG1, IgG2, IgG3, and IgG4. Preferably, the Fc-containing portion is derived from IgG1 protein, more preferably from human IgG1 protein. Non-Ig proteins can be therapeutic proteins, such as those derived from: erythropoietin (EPO), thrombopoietin (THPO) such as THPO-binding peptide, growth hormone, interferon (IFN) such as IFNα, IFNβ, or IFNγ, platelet-derived growth factor (PDGF), interleukin (IL) such as IL1α or IL1β, transforming growth factor (TGF) such as TGFα or TGFβ, or tumor necrosis factor (TNF) such as TNFα or TNFβ, or therapeutic proteins derived from receptors, particularly those derived from ligand-binding fragments of the receptor's extracellular domain, such as those derived from cluster of differentiation 2. 2, CD2), CD4, CD8, CD11, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD58 (LFA3), CD80, CD86, CD147, CD164, IL2 receptor, IL4 receptor, IL6 receptor, IL12 receptor, epidermal growth factor (EGF) receptor, vascular endothelial growth factor (VEGF) receptor, epithelial cell adhesion molecule (EpCAM), or cytotoxic T-lymphocyte-associated protein 4 (CTLA4). Examples of Fc-fusion proteins include beraccept (Nulojix). ® Eyla ®), Linasip (Arcalyst ® ), Romistrin (NPlate) ® ), Abtacept (Orencia) ® Amevine ® ) and Enbrel ® ).

[0273] As used herein, the term "cancer" refers to a physiological condition in mammals characterized by unregulated cell growth. A tumor includes one or more cancer cells. Examples of cancer include carcinoma, lymphoma, germ cell tumor, sarcoma, and leukemia or lymphoid malignancies. Other examples of cancer include: squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma; peritoneal cancer; hepatocellular carcinoma; gastric antrum cancer or gastric cancer, including gastrointestinal cancer and gastrointestinal stromal tumors; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; hepatocellular carcinoma; breast cancer; colon cancer; rectal cancer; colorectal cancer; endometrial cancer or uterine cancer; salivary gland cancer; kidney cancer or renal cell carcinoma; prostate cancer; thyroid cancer; and liver cancer.

[0274] As used herein, the term "solid matrix" (or synonyms "solid support," "solid phase," or "solid material") characterizes a material that is insoluble or can become insoluble through a subsequent reaction. Representative examples of solid materials include polymers or glass beads, microparticles, tubes, sheets, plates, glass slides, pores, and tapes.

[0275] As used herein, the term "alkyl" refers to a straight-chain or branched hydrocarbon group having 1 to 20 carbon atoms, preferably methyl or ethyl; or a cycloalkyl group having 3 to 20 carbon atoms, preferably 5 to 8 carbon atoms. A cycloalkyl group may consist of a single ring, but may also be formed from two or more fused rings.

[0276] As used herein, the term "aryl" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system having 6-14 ring carbon atoms (e.g., having 6, 10, or 14 π electrons shared in the cyclic array) and providing zero heteroatoms in the aromatic ring system. In some embodiments, the aryl group has 6 ring carbon atoms (e.g., phenyl). In some embodiments, the aryl group has 10 ring carbon atoms (e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms (e.g., anthracene). As used herein, the term "aryl" is intended to cover a ring system in which the aromatic ring is fused with one or more carbocyclic or heterocyclic groups, wherein the group or attachment site is located on the aromatic ring (in which case the number of carbon atoms represents the number of carbon atoms in the aromatic ring system). Unless otherwise stated, the aryl group can be unsubstituted ("unsubstituted aryl") or substituted by one or more (e.g., 1 to 5) substituents ("substituted aryl"). Non-limiting examples of aryl groups include groups derived from benzene, naphthalene, anthracene, biphenyl, etc.

[0277] As used herein, the term "heteroaryl" refers to a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having a ring carbon atom and 1-4 heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryls containing one or more nitrogen atoms, the attachment site can be a carbon atom or a nitrogen atom, if the valence allows. Heteroaryl polynuclear ring systems may contain one or more heteroatoms in one or two rings. As used herein, the term "heteroaryl" is intended to encompass ring systems in which a heteroaryl ring is fused with one or more carbocyclic or heterocyclic groups, wherein the attachment site is on the heteroaryl ring (in which case the number of ring members indicates the number of ring members in the heteroaryl ring system). The term “heteroaryl” is also intended to include ring systems in which a heteroaryl ring is fused with one or more aryl groups, wherein the attachment point is on the aryl or heteroaryl ring (in which case the number of ring members represents the number of ring members in a fused multinucleate (aryl / heteroaryl) ring system).

[0278] As used herein, "substituted aryl" refers to an aryl group in which one or more hydrogen atoms are independently replaced by a substituent. Non-limiting examples of substituents include -Z, -R, -OR, -SR, -NR2, -NR3, -CZ3, -CN, -OCN, -SCN, -NO2, -C(O)R, -C(O)NR2, -SO3, -S(O)2R, -C(S)R, -C(O)OR, and -C(O)SR, where each Z is independently a halogen (i.e., -F, -Cl, -Br, or -I), and each R is independently -H, -C, or -C. 1-20 Alkyl or alkoxy, -C 6-20Aryl or -C 5-14 Heteroaryl groups. The heteroaryl groups described above can be similarly substituted.

[0279] The term "divalent aryl" refers to a divalent moiety derived from an optionally substituted aryl or heteroaryl group as defined above, wherein two hydrogen atoms are covalently substituted, allowing attachment to adjacent moieties. Divalent aryl disulfide bridges (e.g., of formula -SX) 3 -S- / -SX 4 -S- divalent groups, where X 3 / X 4 (Representing a divalent aryl group) can be based on techniques known in the art (see Stefanucci et al.). ACS Med.Chem.Lett. 2017, 8, 449-454, and Beard et al. Bioorg. & Med. Chem. (2018, 26, 3039-3045) was obtained via side-chain to side-chain cyclization.

[0280] As used herein, "divalent xylene group" refers to the divalent portion of one of the three isomers derived from xylene (i.e., ortho-xylene, meta-xylene, and para-xylene), wherein a hydrogen atom of each methyl group is covalently substituted, thereby allowing attachment to adjacent portions. Preferably, the divalent xylene group is a divalent meta-xylene group. Divalent xylene-type disulfide bridges (e.g., of formula -SX) 3 -S- / -SX 4 -S- divalent groups, where X 3 / X 4 (Representing a divalent xylene group) can be obtained, for example, in the presence of dibromoxylene, via side-chain-to-side-chain cyclization, such as in... Stefanucci Waiting for someone ACS Med.Chem.Lett. As described in 2017, 8, 449-454.

[0281] As used herein, "divalent maleimide group" refers to the divalent portion derived from maleimide, in which the free covalent bonds of the hydrogen atoms at positions 2 and 3 are substituted, thereby allowing attachment to adjacent portions. Divalent maleimide-type disulfide bridges (e.g., of formula -SX) 3 -S- / -SX 4 -S- divalent groups, where X 3 / X 4 The divalent maleimide group can be obtained, for example, in the presence of 2,3-dibromomaleimide or another suitable reagent, via a side-chain-to-side-chain cyclization, as described by Kuan et al. Chem.Eur.J. As described in 2016, 22, 17112-17129.

[0282] As used herein, "divalent acetone group" refers to the divalent moiety derived from acetone (ACE), in which one hydrogen atom of each methyl group is covalently substituted, allowing attachment to adjacent moieties. Divalent ACE-type disulfide bridges (e.g., formula -SX) 3 -S- / -SX 4 -S- divalent groups, where X 3 / X 4 (Representing a divalent ACE group) can be obtained, for example, in the presence of dibromoacetone or dichloroacetone, by side-chain-to-side-chain cyclization (see, for example, Assem et al.). Angew.Chem.Int. Ed.Engl. 2015, 54(30), 8665-8668).

[0283] As used herein, "a group capable of modulating the electron density and stability of X" refers to a group that can modulate (increase or decrease) the properties (electron density / stability) of an adjacent group (X), such as part (F2) in formula (3b). The modulating group (M) can, for example, absorb or supply electrons to the adjacent group through inductive and / or mediating effects (see International Union of Pure and Applied Chemistry, Compendium of Chemical Technology, Gold Book 2012, 477-480). Preferably, the inductive and mediating effects can cause a shift in the electron density distribution toward the modulating group, thereby modulating the electron density and stability of the adjacent group (e.g., F2). The modulation of electron density can be achieved through… 13 CNMR spectroscopy determination, for example, by measuring the shifts of carbon atoms in the carbonate (ester) group and comparing them with the shifts of a reference compound (e.g., compound 31), is performed. A higher NMR shift (compared to the shift of the reference compound) in the carbonate (ester) signal indicates a decrease in electron density, and therefore decreased stability. A lower NMR shift (compared to the shift of the reference compound) in the carbonate (ester) signal indicates an increase in electron density and increased stability. Such adjustment of electron density can be used to optimize the reactivity and stability of the conjugates of this invention.

[0284] According to embodiments of the invention, groups capable of adjusting the electron density and stability of X are selected such that the conjugate is stable against degradation (e.g., hydrolysis) in the absence of other reagents. This means that when the conjugate is mixed with water / DMSO (95 / 5, v / v) at a concentration of 1 mg / mL and pH 9 and stirred at 500 rpm for 1 hour at 25°C, the conjugate exhibits less than 50% degradation as determined by HPLC, preferably less than 25% degradation, more preferably less than 10% degradation, and particularly less than 5% degradation.

[0285] As used herein, "electron-withdrawing group" refers to a group or substituent that can absorb electrons from the part to which it is bonded; that is, the electron-withdrawing group reduces the electron density of that part compared to the same part carrying hydrogen atoms rather than the electron-withdrawing group. Electron-withdrawing groups typically include, but are not limited to, cyano, nitro, haloalkyl, carboxyl, aryl, sulfonyl, etc. Electron-withdrawing groups can exert their electron-withdrawing effect through inductive and / or mediating effects (as shown above). As used herein, the term "electron-withdrawing" is intended to encompass both meanings. Electron-withdrawing groups / substituents are known in the art and, for example, by Carey and Sundberg in [the field of electron-withdrawing research]. Advanced Organic Chemistry , Part A: Structure and Mechanisms,4 th As described in Edition.

[0286] As used herein, "leaving group" refers to an atom or group (which may be charged or uncharged) that detaches from the remaining or main part of a molecule that is considered to participate in a particular reaction, such as a nucleophilic substitution reaction. Pure Appl.Chem. (1994, 66, 1134). Examples of leaving groups include thiophenes (esters), phenolates (esters), carboxylates (esters), and sulfonates (esters).

[0287] Where “preferred” embodiments / features are mentioned in this description, such combinations of “preferred” embodiments / features shall also be considered disclosed, provided that such combinations are technically meaningful.

[0288] In the following description and claims, the terms “comprising” and “including” should be understood to mean that other unmentioned elements may be present in addition to the elements mentioned. However, as a more limited embodiment, these terms should also be understood to disclose the term “composed of”, thus making it impossible for other unmentioned elements to be present, provided that this is technically meaningful.

[0289] Unless otherwise stated or the context otherwise requires, references to “substituted” or “optionally substituted” groups shall be construed as references to the presence (or optional presence, as the case may be) of at least one substituent selected from the following: F, Cl, Br, I, CN, NO2, NH2, NH-C 1-6 -alkyl, N(C) 1-6 -alkyl)2、-XC 1-6 -alkyl, -XC 2-6 -Alkenyl, -XC 2-6 -alkynyl group, -XC 6-14 -aryl, -X- (a 5-14 membered heteroalkyl group having 1-3 heteroatoms selected from N, O, S), where X represents a single bond, -(CH2)-, -O-, -S-, -S(O)-, -S(O)2-, -NH-, -CO-, or any combination thereof, including, for example, -C(O)-NH-, -NH-C(O)-. The number of substituents is not particularly limited and can range from 1 to the maximum valence that can be saturated by the substituent. The number of substituents is typically 1, 2, or 3, usually 1 or 2, and most typically 1.

[0290] Unless otherwise stated, all valences of individual atoms in the compounds or portions described herein are saturated. In particular, they are saturated with the binding pairs shown. If a binding pair is not shown or the number of binding pairs is too small, the remaining valences of the individual atoms are saturated with the corresponding number of hydrogen atoms.

[0291] Unless otherwise stated, chiral compounds and portions may be present as pure stereoisomers or as mixtures of stereoisomers, including 50:50 racemates. In the context of this invention, references to specific stereoisomers should be understood as references to compounds or portions, wherein the specified stereoisomer is present in an enantiomeric excess of at least 90%, more preferably at least 95%, and most preferably 100%, where %ee is defined as (RS) / (R+S)*100%, where R and S represent the molar amounts of their respective enantiomers.

[0292] Unless otherwise stated or the context otherwise requires, all connections between adjacent amino acid groups are formed by peptide (amide) bonds.

[0293] Unless the context otherwise requires, and / or an alternative meaning is expressly provided herein, all terms shall have the meanings generally accepted in the art, such as those in the IUPAC Gold Book (status as of November 1, 2019) or the Dictionary of Chemistry, Oxford, 6 th As shown in Ed.

[0294] 2. Overview

[0295] This invention is based on the surprising discovery that the region-selective attachment of a payload to an antibody or antibody fragment can be accomplished using the compounds of this invention. More specifically, the region-selective attachment can be accomplished in a single step, for example without further chemical reactions to cleave the covalent bonds between the carrier and the antibody or antibody fragment.

[0296] 3. Compounds of formula (1)

[0297] This invention relates to compounds represented by general formula (1):

[0298] PYSV (1)

[0299] The compound of formula (1) comprises a carrier V, a spacer S, a reactive moiety Y, and a payload P, wherein the carrier V is capable of interacting with the Fc-region of an antibody or a fragment thereof (having binding affinity for the Fc-region), the antibody fragment optionally being incorporated into an Fc-fusion protein, and the spacer S having a length Z.

[0300] 3.1 Payload (P)

[0301] There are no particular restrictions on the payload to be used, and any molecule that is labeled and / or pharmaceutically active can be used, as long as it can be attached to the reactive part.

[0302] According to one embodiment, the payload comprises a portion selected from the following, along with a radioisotope of said portion and / or a pharmaceutically acceptable salt of said portion:

[0303] (i) Selected from the following:

[0304] The labeling portion includes a radionuclide, preferably a chelating agent such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), cyclohexyldiethylenetriaminepentaacetic acid (CH-X-DTPA), 3,6,9,15-tetraazabicyclo[9.3.1]pentadecane-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) or deferoxamine (DFO), wherein the chelating agent optionally chelates the radionuclide;

[0305] Hair color clusters;

[0306] Fluoresceins, such as fluorescein or rhodamine; and

[0307] Contains radioactive nuclides (such as 125 I, 123 I, 131 I,18 F, 11 C 15 O、 18 The marked portion of F), for example, is derived from a radioactive nuclide (such as, 125 I, 123 I or 131 I) is the 4-hydroxyphenylpropionate ester (also known as the Bolton-Hunter reagent);

[0308] (ii) A portion selected from the part containing the bonding group, thereby allowing for the later attachment of a payload as specified in items (i) and (iii) herein. This may be a portion selected from the group consisting of: optionally substituted conjugated dienes, optionally substituted tetraazines, optionally substituted alkynes or azides, optionally substituted dibenzocyclooctyne (DBCO), optionally substituted trans-cyclooctene (TCO), optionally substituted bicyclic [6.1.0]nonyne (BCN), optionally substituted aldehydes, optionally substituted ketones, and optionally substituted hydrazides;

[0309] (iii) Derivatives from the following drugs

[0310] Antitumor agents, such as DNA-alkylating agents, for example, duocarmycin;

[0311] Topoisomerase inhibitors, such as doxorubicin;

[0312] RNA polymerase II inhibitors, such as α-amanitin;

[0313] DNA cleavage agents, such as calicheamicin;

[0314] Antimitotic agents or microtubule disruptors, such as taxane, auristatin, or maytansinoid;

[0315] Antimetabolites;

[0316] Kinase inhibitors, such as partasertib;

[0317] Immunomodulators;

[0318] Anti-infective agents;

[0319] According to one embodiment, the payload is optionally a chelating agent for chelating radionuclides, which is preferably derived from the following: diethylenetriaminepentaacetic acid (DTPA), 3,6,9,15-tetraazabicyclo[9.3.1]pentadecane-1(15),11,13-trien-3,6,9-triacetic acid (PCTA), cyclohexyldiethylenetriaminepentaacetic acid (CH-X-DTPA), deferoxamine (DFO), 1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-tetraacetic acid (NODAGA), 1 4,7,10-Tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid (DOTAGA), 2,2'-(1,4,7-triazacyclononane-1,4-diyl)diacetic acid salt (ester) (NO2A), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), ethylenediaminetetraacetic acid (EDTA), ethylenediaminediacetic acid, triethylenetetraminehexaacetic acid (TTHA), 1,4,8,11- Tetraazacyclotetradecane (CYCLAM), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A), 2,2',2''-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetamide (DO3AM), 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A), 1,5,9-triazacyclododecane (TAC) D), (3a1s,5a1s)-decahydro-3a,5a,8a,10a-tetraazapyrene (cis-glyoxal-cyclopyramine), 1,4,7-triazacyclononane (TACN), 1,4,7,10-tetraazacyclododecane (cyclen), tri(hydroxypyridinone) (THP), 3-(((4,7-bis((hydroxy(hydroxymethyl)phosphoryl)methyl)-1,4,7-triazolidine-1-yl)methyl)(hydroxy)phosphoryl)propionic acid (NOPO), 3,6,9,15-tetraazabicyclo[9.3].[1] Pentadecane-1(15),11,13-trien-3,6,9-triacetic acid (PCTA), 2,2',2'',2'''-(1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetraacetic acid (TRITA), 2,2',2'',2'''-(1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetraacetamide (TRITAM), 2,2',2''-(1,4,7,10-tetraazacyclotridecane-1,4,7-triyl)triacetamide (TRITRAM), trans-N-dimethylcyclopamide, 2,2', 2''-(1,4,7-triazacyclononane-1,4,7-triyl)triacetamide (NOTAM), oxocyclopentamine, dioxocyclopentamine, 1,7-dioxa-4,10-diazacyclododecane, cross-linked bridged cyclopentamine (CB-cyclopentamine), triazacyclononane phosphonite (TRAP), bispyridoxine diphosphate (DPDP), meso-tetra(4-sulfonylphenyl)porphyrin (TPPS4), ethylidene dihydroxyphenylglycine (EHPG), hexamethylenediaminetetraacetic acid, dimethylphosphomethane (DMPE), methylene diphosphate, dimercaptosuccinic acid (DMPA), or derivatives thereof.

[0320] According to a preferred embodiment, the payload is a chelating agent optionally used to chelate a radionuclide. This chelating agent is derived from a portion of DTPA, DOTA, DFO, NOOTA, PCTA, CH-X-DTPA, NODAGA, or DOTAGA, preferably from a portion of DTPA, DOTA, DFO, NOOTA, PCTA, CH-X-DTPA, or NODAGA, and more preferably from a portion of DTPA, DOTA, DFO, or PCTA. Most preferably, the chelating agent is DTPA.

[0321] According to one implementation scheme, the chelating agent chelates a radionuclide, which is selected from... 124 I, 131 I, 86 Y、 90 Y、 177 Lu、 111 In、 188 Re、 55 Co、 64 Cu、 67 Cu、 68 Ga、 89 Zr、 203 Pb, 212 Pb, 212 Bi、 213 Bi、 72 As、 211 At、 225 Ac、223 Ra、 97 Ru、 149 Tb, 152 Tb, 161 Tb, 99m Tc, 226 Th、 227 Th、 201 Tl、 89 Sr、 44 / 43 Sc、 47 Sc、 153 Sm、 133 Xe and Al 18 F, preferably selected from 89 Zr、 111 In、 64 Cu、 177 Lu、 68 Ga、 99m Tc, 203 Pb, 72 As、 55 Co、 97 Ru、 201 Tl、 152 Tb, 133 Xe, 86 Y and Al 18 F, more preferably 89 Zr、 111 In、 64 Cu、 177 Lu、 68 Ga and 99m Tc, especially 111 In.

[0322] In a preferred embodiment, the payload is a chelate. 111 In DTPA.

[0323] In another preferred embodiment, the payload is selected from:

[0324] Derived from chelation 111 The portion (i) of DOTA, PCTA, DTPA, or CH-X-DTPA in In is most preferably chelated. 111 In CH-X-DTPA;

[0325] Derived from chelation 64 The portion (i) of Cu containing Nota, Nodaga, or PCTA is most preferably chelated. 64 Cu's Nota;

[0326] Derived from chelation 89The portion (i) of Zr consisting of DOTA, DFO, DFO', or DFO-cyclo' is most preferably chelated. 89 Zr DFO.

[0327] According to one embodiment, the payload is a portion selected from the portion containing the binding group, thereby allowing for the later attachment of payloads as specified in items (i) and (iii) herein. This could be a portion containing a binding group suitable for a “click chemistry” that rapidly and reliably generates covalent bonds by reacting with another portion containing a coupling group of the “click chemistry” (i.e., the payload containing the coupling coupling group), for example via strain-promoted cycloaddition, [2+3] dipolar cycloaddition, or Diels-Alder cycloaddition.

[0328] In one embodiment, the portion is a portion comprising a binding group selected from the group consisting of: optionally substituted conjugated dienes, optionally substituted tetraazines, optionally substituted alkynes or azides, optionally substituted dibenzocyclooctyne (DBCO), optionally substituted trans-cyclooctene (TCO), optionally substituted bicyclic [6.1.0]nonyne (BCN), optionally substituted aldehydes, optionally substituted ketones, and optionally substituted hydrazines.

[0329] In one embodiment, the portion is a portion comprising a bonding group that can react to form a covalent bond in the absence of a metal catalyst (“metal-free”), as described by Becer et al. in “Click Chemistry beyond Metal-Catalysed Cycloaddition” Angewandte Chemie Int. Ed. 2009, 48(27), 4900-4908. Examples of bonding groups that can react in the absence of a metal catalyst include electron-deficient alkynes, strained alkynes such as cyclooctyne, tetrazine, and azides. Preferably, the portion is a portion comprising a bonding group selected from the group consisting of azides (N3), TZ, TCO, BCN, and DBCO, more preferably BCN or DBCO, and most preferably DBCO.

[0330] According to one embodiment, the payload is a portion derived from a drug substance. The following are exemplary drugs that can be used as a payload in the compounds of this invention:

[0331] (A) Such as DNA-alkylating agents antitumor agentsExamples include docalamycin (including synthetic analogs: adolaxine, carzecillin, pyrazin, KW-2189, and CBI-TMI), and nitrogen mustard analogs (e.g., cyclophosphamide-chlorambucil, melphalan, dichloroethyl methylamine, ifosfamide, troprophosphatide, prednimustine, bendamustine, naphthylmustine, estradiol, dichloromethyldiethylamine, and mechlorethamine oxide hydrochloride). Hydrochloride, mannomustine, dibromocerinol, neo-embezzin, benzethonol, uramustine; alkyl sulfonates (e.g., busulfan, succinyl sulfonate, mannosulfan, indomethacin, and piperosulfan); ethyleneimine (e.g., thiotepa, triaminequinone, carbaquinone); nitrosoureas (e.g., carmustine, lomustine, semustine, streptozotocin, chlorhexidine, formustine, nimustine, ramustine); epoxides (e.g., etogluconol); other alkylating agents (e.g., dibromomannitol, piperobromane, temozolomide, dacarbazine);

[0332] (B) topoisomerase inhibitors For example, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin, etoposide, etoposide phosphate, irinotecan and its metabolites (such as SN-38, teniposide, topotecan, resveratrol, epipodophyllin) (e.g., 9-aminocamptothecin, camptothecin, cristatol, donomycin, mitoxantrone, novate, retinoic acid (retinol), 9-nitrocamptothecin (RFS 2000)).

[0333] (C) RNA polymerase II inhibitors For example, α-amaminine, other amanitaines;

[0334] (D) DNA lysis agent For example, calicizidoxime;

[0335] (F) Antimitotic agents or microtubule disruptorsExamples include vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine, navelbin, vinflunide, vintafolide); taxanes (e.g., paclitaxel, docetaxel, polyglutamic acid paclitaxel). Polyglumex, cabazitaxel and their analogues; maytansine alkaloids (e.g., DM1, DM2, DM3, DM4, maytansine and anserine) and their analogues; cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); epothilone, eleutherobin, disccodermolide, bryostatin, dolostatin, aurestatin (e.g., monomethylaurestatin E, monomethylaurestatin F), tubulolysin, cephalostatin; sarcodictyin, spongistatin, dimethicone, mitomycin;

[0336] (F) Antimetabolites Examples of such inhibitors include DHFR inhibitors (e.g., methotrexate, trimethoprim, folate, pteroxetine, aminopterin (4-aminopteric acid) or other folic acid analogs such as raltitrexed, pemetrexed, pralatrexed); IMP dehydrogenase inhibitors (e.g., mycophenolic acid, thiazofuran, ribavirin, EICAR); ribonucleotide reductase inhibitors (e.g., hydroxyurea, deferoxamine); and pyrimidine analogs (e.g., cytarabine, fluorouracil, 5-fluorouracil and its metabolites, nitrofurantoin, carmoflurane). Gemcitabine, capecitabine, azacitidine, decitabine, fluorouracil compositions, uridine compositions, trifluorouridine compositions, cytosine arabinoside, ancitabine, fluorouridine, deoxyfluorouridine), uracil analogs (e.g., 6-azauridine, deoxyuridine); cytosine analogs (e.g., enoxabin); purine analogs (e.g., imidazothiopurine, fludarabine, mercaptopurine, thioimidazopurine, thioguanine, cladribine, clofarabine, nerabine); folic acid supplements, such as folic acid;

[0337] (G) kinase inhibitorsExamples include ipatasertib, BIBW 2992 (anti-EGFR / Erb2), imatinib, gefitinib, pilgatanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, afatinib, vemurafenib, crizotinib, regorafenib, masitinib, dabrafenib, trametinib, ibrutinib, ceritinib. Lenvatinib, Nintedanib, Sildenafil, Palbociclib, Osimertinib, Alectinib, Alectinib, Rociletinib, Cobitinib, Midotutolin, Ometinib, E7080 (anti-VEGFR2), Mubritinib, Panatinib (AP24534), Baflutinib (INNO-406), Bosutinib (SKI-606), Cabozantinib, Vismodegib, Iniparib, Ruxotinib, CYT387, Tivazanib, Ispinesib, Temsirolimus, Everolimus, Ridaforolimus;

[0338] (H) Immunomodulators These include immunostimulants, immunosuppressants, cyclosporine, cyclosporine A, aminocaproic acid, imidazoline, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g., ancinonide, betamethasone, budesonide, hydrocortisone, flunisolone, fluticasone propionate, fluocortolonedanazol, dexamethasone, prednisone, triamcinolone, beclomethasone propionate), DHEA, hydroxychloroquine, meloxicam, methotrexate, mofetil, mycophenylate, sirolimus, tacrolimus, everolimus, fingolimod, and ibrutinib;

[0339] (I) Anti-infectious disease drugs This includes antibacterial drugs, anti-mycobacterial drugs, and antiviral drugs. Non-limiting examples of antibiotics used in antibiotic-antibody drug conjugates include rifalogue derivatives and rafamycin derivatives.

[0340] According to one embodiment, the payload is derived from, or from, a radioisotope of, said portion and / or a pharmaceutically acceptable salt of said portion: eczema, PNU-159682, (α-)amycin, docamycin, auristatin, maytansine, tubulolysin, calcitrazine, SN-38, paclitaxel, doxorubicin, vincristine, doxorubicin, methotrexate, pyrrolobenzodiazepines, pyrrolokinase spindle protein (KSP) inhibitors, and indoline-benzodiazepine dimers.

[0341] In some aspects of the invention, it is preferred to use a payload with a certain degree of hydrophilicity, for example, in the case of a chelating agent that chelates radionuclides, to avoid and / or prevent possible aggregation. For example, high aggregation can be overcome by adding / increasing / increasing the number of PEG units present as a linker between the antibody and the payload.

[0342] This attachment of the effective load to the reactive group can be achieved via a linking group (or “connector”). In the context of this disclosure, the linking group can be considered as part of the effective load. Thus, in one embodiment, the effective load is represented by the following formula (2):

[0343] P 1 -L-- (2)

[0344] in,

[0345] P 1 This refers to the payload as described above—for example, a chelating agent that optionally chelates radionuclides (such as...). 177 Lu-DOTA, or a component derived from the drug.

[0346] L indicates a connector.

[0347] Refers to covalent attachment to the reactive portion.

[0348] The connector is a divalent group, preferably containing one or more atoms selected from carbon, nitrogen, oxygen and sulfur.

[0349] In one implementation, the connector can be selected from...

[0350] (a1) An alkylene group having 1 to 12 carbon atoms, preferably an alkylene group having 2 to 6 carbon atoms, such as ethylene or propylene;

[0351] (b1) A polyepoxyalkyl group having 2 or 3 carbon atoms and having 1 to 36 repeating units; preferably of the formula -NH-(CH2CH2O). n1–CH2CH2– represents a group, where n1 is an integer from 0 to 35, for example, an integer from 1 to 20; and

[0352] (c1) A peptide group having 2 to 12 amino acids.

[0353] In a more specific implementation, the connector is selected from...

[0354] (a1) Alkylene groups having 2 to 6 carbon atoms (-(CH2)) 2-6 -);

[0355] (b1) Formula -NH-(CH2CH2O) n1 -CH2CH2- polyalkylene groups, where n1 is an integer from 0 to 35; and

[0356] (c1) A peptide linker comprising 2 to 12 amino acids, wherein the amino acids are optionally cleavable, preferably cleavable peptide linkers comprising Val-Cit units, Val-Ala units, Val-Cit-PABC or Val-Cit-PABC-DMEA units.

[0357] The connector can be either pyrolytic or non-pyrolytic. In one embodiment, the connector is non-pyrolytic. In another embodiment, the connector is pyrolytic.

[0358] The cleavable linker can be a linker capable of specifically releasing the payload upon internalization in target cells. It can leverage the inherent properties of target cells (e.g., cancer cells) to selectively release the payload from modified antibodies or modified antibody fragments, namely (1) protease sensitivity (enzyme-triggered release linker system), (2) pH sensitivity, (3) glutathione sensitivity, or (4) glucuronidase sensitivity. In a specific embodiment, the linker is a cleavable linker comprising a valine-citrulline (Val-Cit) or valine-alanine (Val-Ala) dipeptide, which can serve as a substrate for intracellular cleavage of cathepsin B (Cat B).

[0359] In another specific embodiment, the linker is a cleavable linker comprising a self-immolative moiety capable of releasing the payload via elimination-type or cyclization-type mechanisms. An example of a cleavable linker containing a self-immolative moiety is the para-aminobenzyloxycarbonyl (PABC) linker, such as the benzyloxycarbonyl conjugate acestril. ® (bremtuximab-vedotin conjugate Adcetris ® The text appears to be a mix of Chinese characters and symbols, possibly related to a document or document. A direct translation wouldn't be meaningful without further context or clarification. Engl. J. Med. 2010, 363, 1812-1821; Jain et al. Pharm.Res. 2015, 32(11), 3526–3540). The PABC-containing linker comprises a protease-sensitive Val-Cit-PABC dipeptide linker unit that can be recognized and cleaved by Cat B. This linker unit can be attached to the reactive moiety via the maleimide propionyl moiety (and after antibody modification to the antibody). This linker helps avoid steric conflicts in substrate recognition caused by Cat B. Following enzymatic cleavage of the citrulline-PABC amide bond, the resulting PABC-substituted payload spontaneously undergoes 1,6-elimination, which releases the free payload as a product into the target cells. Therefore, the group according to formula (2) can represent benzoxicam, i.e., a group consisting of a payload moiety derived from monomethylaurestatin E that is attached to the reactive moiety via a linker containing a Val-Cit-PABC unit.

[0360] In another specific embodiment, the linker is a cleavable linker comprising a C-terminal dipeptide unit, which can be used as a highly specific substrate for the exo-Cat B peptide chain degradative activity (exo-Cat B). Examples of exo-Cat B cleavable linker systems are described in WO 2019 / 096867 A1. In particular, the linker L may comprise a C-terminal dipeptide unit (“Axx-Ayy” or “Ayy-Axx”) as defined in WO 2019 / 096867 A1.

[0361] 3.2 Reactivity Component (Y)

[0362] The compounds of the present invention comprise a reactive moiety (Y) capable of reacting with amino acid side chains exposed on the surface of an antibody or antibody fragment (e.g., via nucleophilic substitution). Preferably, the reactive moiety is capable of reacting with a lysine side chain. This reaction causes a payload (P) to covalently attach to the antibody or antibody fragment, simultaneously releasing a spacer (S) and a carrier (V). When the reactive moiety (Y) reacts with the side chains of amino acids exposed on the surface of the antibody or antibody fragment to form covalent bonds, the covalent bonds within Y, or between Y and S, spontaneously cleave to release the peptide (without further chemical reactions such as hydrolysis or reduction).

[0363] The reactive moiety includes a reactive center (RC) that can react with the side chain of an amino acid, preferably with the side chain of a lysine residue, for example, through a nucleophilic substitution reaction. Preferably, the reactive center is electrophilic. Non-limiting examples of electrophilic reactive centers capable of reacting with the side chain of an amino acid include C=O and C=S. Preferred reactive centers are carbonyl (C=O) or thiocarbonyl (C=S), with carbonyl (C=O) being particularly preferred.

[0364] Part (F1) is covalently attached to one side of the reaction center, through which the reaction center (RC) is attached to the effective load (P). Part (F2) is covalently attached to the other side of the reaction center, through which the reaction center (RC) is attached to the support (V) via the spacer (S). Therefore, the reactive part (Y) can be represented by the following equation (3a):

[0365] --F1-RC-F2-- (3a)

[0366] in,

[0367] RC is a reaction center, preferably an electrophilic reaction center, more preferably a group selected from C=O and C=S, and most preferably C=O;

[0368] F1 represents a single covalent bond, atom, or atomic group; preferably an atom selected from O and S, or an atomic group containing one or more atoms selected from C, N, O, and S; more preferably an atom selected from O and S;

[0369] F2 represents an atom or an atomic group; preferably an atom selected from O and S, or an atomic group containing one or more atoms selected from C, N, O and S; more preferably an atom selected from O and S.

[0370] Refers to the attachment to the payload (P), and

[0371] Refers to attachment to a spacer (S).

[0372] F1 and F2 can be the same atom or atomic group. However, preferably, the atom or atomic group constituting F2 is a leaving group that is better / preferred than F1 in nucleophilic substitution reactions. This ensures that F2 is the preferred leaving group when the reaction center reacts with the side chain of an amino acid residue on the antibody or antibody fragment (e.g., with the side chain of a lysine residue) via a nucleophilic substitution reaction; thus enabling the payload to attach to the antibody or antibody fragment, rather than the carrier / spacer construct.

[0373] According to one implementation, the reactive part of equation (3a) is represented by one of the following equations (4a) to (4m).

[0374] (4a) (4b) (4c)

[0375] (4d) (4e) (4f)

[0376] (4g) (4h)

[0377] (4i) (4j) (4k)

[0378] (4l) (4m);

[0379] in Refers to the attachment to the payload (P). Refers to attachment to a spacer (S).

[0380] To ensure that F2 is a better or more preferred leaving group than F1 in nucleophilic substitution reactions, especially if F1 and F2 are the same atom or atomic group, F2 can be linked to a modifying group (M), where M is, for example, a group that absorbs or donates electrons and can modulate the electronegativity and / or stability of the adjacent F2 moiety.

[0381] Therefore, in the implementation scheme, the reactive component (Y) is represented by the following formula (3b):

[0382] --(F1-RC-F2)-(M)-- (3b)

[0383] Among them, RC, F1, F2, , As defined in formula (3a) above, M represents a group that can adjust the electron density and stability of F2, preferably a group that can absorb electrons.

[0384] In the implementation scheme, M is represented by the following formula (3c):

[0385] --M'—B—C-- (3c)

[0386] in,

[0387] M' is an aryl group having a 6-membered, 10-membered, or 14-membered ring and one, two, or three fused rings, or a heteroaryl group having a 5- to 20-membered ring, one, two, or three fused rings, and one to four heteroatoms independently selected from N, O, and S. M' may be substituted with one or more substituents; preferably phenyl, naphthyl, pyridyl, quinolinyl, isoquinolinyl, or benzotriazolyl substituents that may be substituted with one or more substituents, with each substituent preferably selected from -F, -Br, -Cl, -I, -NO2, -CN, and -C. 1-6 -alkyl, -C 1-6 -alkoxy groups, such as -C(O)NH2, -C 1-6 -amino groups, and combinations thereof such as -CCl3, -CF3, or -CH2NO2;

[0388] B represents a single covalent bond, O, S, NR', where R' represents a hydrogen atom, OH, alkyl or cycloalkyl, and C. 2-6 - Idemenyl, C 2-6 - Hydrinyl, groups having the following general formulas, or any combination thereof:

[0389] –(CH2) n1 -(H 1 ) x1 -(CH2) n2 -(H 2 ) x2 -(CH2) n3 -(H 3 ) x3 -(CH2) n4 -(3c')

[0390] in,

[0391] Each of n1, n2, n3, and n4 represents an integer independently selected from 0 to 10, such that n1 + n2 + n3 + n4 is 10 or less.

[0392] Each of x1, x2, and x3 is independently selected from 0 and 1, and

[0393] H 1 H 2 and H 3 Each of them is an atom independently selected from N, O, and S.

[0394] The conditions are: if x1+x2=2, then n2≥1; if x2+x3=2, then n3≥0; if x1+x3=2, then n2≥1 or n3≥1; if x1+x2+x3 is 3, then n2≥1 and n3≥1.

[0395] B is preferably a single covalent bond, NH, or C.1-10 -alkylene; more preferably a single covalent bond;

[0396] C is C=O, C=S, or C(NR''), where R'' represents a hydrogen atom, OH, alkyl or cycloalkyl, S(=O) or S(=O)2; preferably C=O;

[0397] Refers to covalent attachment to the spacer (S); and

[0398] It refers to covalent attachment to F2.

[0399] According to one implementation, in equation (3b), part (F1-RC-F2) is represented by one of equations (4a') to (4m'), and / or M is independently represented by one of equations (5a) to (5j'):

[0400] (4a') (4b') (4c')

[0401] (4d') (4e')

[0402] (4f') (4g')

[0403] (4h') (4i') (4j')

[0404] (4k') (4l'), (4m')

[0405] (5a) (5b) (5c)

[0406] (5d) (5e)

[0407] (5f) (5g)

[0408] (5h) (5i) (5j) (5k) (5l) (5m) (5n) (5o) (5p) (5q) (5r) (5s) (5t) (5u) (5V) (5w) (5x) (5y) (5z) (5a') (5b') (5c') (5d') (5e') (5f') (5g') (5h') (5i') (5j');

[0409] in Refers to covalent attachment to the spacer (S). Refers to covalent attachment to the payload (P). Refers to covalent attachment to the modifying group (M). 'Refers to covalent attachment to F2.'

[0410] In a preferred embodiment, the reactive component is represented by one of the following formulas (6a) to (6l'):

[0411] (6a) (6b) (6c) (6d) (6e) (6f) (6g) (6h) (6i) (6j) (6k) (6l) (6m) (6n) (6o) (6p) (6q) (6r) (6s) (6t) (6u) (6v) (6w) (6x) (6y) (6z) (6a') (6b') (6c') (6d') (6e') (6f') (6g') (6h') (6i') (6j') (6k') (6l')

[0412] in Refers to covalent attachment to the spacer (S). Refers to covalent attachment to the payload (P).

[0413] Most preferably, the reactive portion is represented by one of formulas (6a), (6b) and (6m), particularly by formula (6a).

[0414] 3.3 Spacer (S)

[0415] The compounds of the present invention comprise a spacer (S) having a length Z, wherein the length Z is such that when the carrier interacts with the Fc region of an antibody or a fragment thereof, the reactive portion is capable of reacting with the side chains of amino acid residues exposed on the surface of the antibody or antibody fragment, thereby selectively attaching the payload (and optionally the linker) region to the antibody or antibody fragment. The spacer is attached to the carrier (V) via functional groups (e.g., amino, carboxyl) of the carrier's chemical structure. If the carrier is a peptide, the spacer is attached to the N-terminus or C-terminus of the peptide (as further described below). For example, the spacer may be attached to the amino or carboxyl functional groups at the N-terminus or C-terminus of the peptide backbone, or to the N-terminal or C-terminal amino acid side chains. Particularly in the case of non-peptide carrier molecules, it is preferable to recognize the attachment sites of the spacer (S) such that the binding affinity (denoted as Kd) is not significantly reduced (<20%) compared to a carrier without an attached spacer.

[0416] Length Z refers to the length of the spacer in its natural conformation (not its maximum tensile length). The natural conformation can be used when the spacer is linked to a carrier and a reactive portion that is part of the construct of the reactive coupling of the present invention.

[0417] The appropriate length Z can be determined using computer modeling (Molecular Operating Environment (MOE) from the Chemical Computing Group) or X-ray crystallography to calculate the approximate distance in angstroms (Å) between the binding site of the carrier on the Fc domain of the antibody or its fragment and the target amino acid, such as a lysine or cysteine ​​residue, with lysine being the most preferred. In the case of polymers, the length Z can be determined by applying a worm-like-chain (WLC) model, which will be further described below. The three-dimensional structure of the Fc-III / Fc-region complex at a resolution of 2.7 Å can be obtained under the PDB identifier 1DN2 (DeLano et al. Science 2000, vol. 287, no. 5456, 1279-1283).

[0418] In one embodiment, the length Z is 13 to 30 Å, preferably 14 to 25 Å, more preferably 16 to 18 Å.

[0419] The inventors believe that a length Z in the range of 13 to 30 Å (as described above) may lead to the targeting of amino acids on the Fc region of the antibody or antibody fragment (a highly conserved region in the antibody, particularly IgG antibodies), such as one or more lysine residues found at positions 317, 326, 338, 340, and 439 (especially at positions 317 and / or 326), and that a length Z in the range of 13 to 30 Å may lead to the reaction of the reactive portion and the attachment of a payload with high regioselectivity. In embodiments, high regioselectivity is achieved if the payload loading ratio Fc / F(ab)2 is greater than 1.0, greater than 1.5, greater than 2.0, and particularly greater than 2.5. The degree of regioselectivity can be determined by measuring the payload loading ratio (selective Fc / F(ab)2) between the Fc and F(ab)2 regions, as will be further described below.

[0420] The spacer can be any group having the aforementioned length Z that can link the carrier and the reactive portion. Preferably, the spacer is chemically inert.

[0421] In one embodiment, the spacer is preferably selected from...

[0422] (a2) A polyepoxyalkyl group having 6 to 36 repeating units, for example having 8 to 24 repeating units; preferably a group represented by the following formula (7):

[0423] –X 1 –(CH2CH2O) n2 –CH2CH2–X 2 –(7)

[0424] in,

[0425] X 1 It can be NH, O, or S; NH is preferred;

[0426] X 2 For NH or C=O, if X 2 If X covalently binds to the carrier, then X 2 Preferably, C=O; and

[0427] n2 is an integer from 4 to 28, preferably an integer from 6 to 20, more preferably an integer from 8 to 12, and especially 10;

[0428] (b2) A peptide group having 6 to 25 amino acids in the main chain, for example having 9 amino acids in the main chain, wherein the amino acids are preferably selected from Pro, Gly, Ala, Asn, Asp, Thr, Glu, Gln and Ser; more preferably Pro, Gly or Ser.

[0429] In equation (7), in some implementations, X 2 Covalently bound to the carrier, X 1 Covalently bonded to the reactive portion; in some other embodiments, X 1 Covalently bound to the carrier, X 2 Covalently bonded to the reactive portion. In some specific embodiments, the spacer's attachment point to the support, and X... 1 and X 2 Each component is independently selected to form an amide bond with the support. For example, if the support is attached to the spacer via its N-terminus (i.e., via the amino group of the N-terminal amino acid), then X... 2 It can be chosen as C=O, and if the carrier is attached to the spacer via the C-terminus (i.e., via the carboxyl group of the C-terminal amino acid), then X 1 (or X) 2 ) can be selected as NH.

[0430] According to one embodiment, the spacer comprises a polyoxyethylene group having 4 to 36 repeating units, preferably 6 to 28 repeating units, more preferably 7 to 24 repeating units, such as 10 or 20 repeating units. Most preferably, the spacer comprises a polyethylene oxide having 10 repeating units.

[0431] 3.4 Carrier (V)

[0432] The compounds of the present invention comprise a carrier (V) (or "ligand") capable of interacting (binding) with a crystallizable fragment (Fc) region of an antibody or a fragment thereof, the antibody fragment optionally incorporated into an Fc-fusion protein. The interaction between the carrier and the Fc region increases the concentration of reactive portions near the amino acid side chains exposed on the surface of the antibody or antibody fragment, thereby enabling the payload to covalently attach to the side chains. In some aspects, the interaction between the carrier and the Fc region induces targeting, as the reactive portions react with the side chains of specific amino acids exposed on the surface of the antibody or antibody fragment (e.g., amino acid residue at position 317), allowing the payload region to selectively attach to the antibody or antibody fragment.

[0433] Vectors capable of interacting with the Fc region of antibodies or fragments thereof are known in the art and are described, for example, in Choe et al., Materials 2016, 9, 994. Suitable vectors are also disclosed in WO 2018 / 199337A1. Non-limiting examples of vectors capable of interacting with the Fc region of antibodies or fragments thereof include proteins Z and Fc-III. In particular, the cyclic peptide Fc-III has been described as a peptide carrier / ligand with high affinity for the Fc region of IgG proteins, with a reported dissociation constant Kd of about 16 nm (DeLano et al. Science 2000, 287, 1279-1283).

[0434] In one embodiment, the carrier used in the compounds of the present invention is a peptide containing a sequence of 11 to 17 amino acids, preferably 13 to 17 amino acids. In some specific embodiments, the spacer is attached to the carrier via its N-terminus or C-terminus (i.e., attached to the aforementioned peptide sequence). In some more specific embodiments, the carrier is not further modified to attach the spacer to the N-terminus or C-terminus.

[0435] According to a preferred embodiment, the carrier is a peptide represented by one of the following formulas (8a) and (8b):

[0436]

[0437] in,

[0438] Each of Bxx, Cxx, Dxx, Exx, and Fxx independently represents an amino acid;

[0439] Axx represents an amino acid, a dicarboxylic acid, or a peptide moiety represented by the following formula (9a):

[0440] ---Axx1–Axx2–Axx3--- (9a)

[0441] in,

[0442] Axx1 represents a single covalent bond or amino acid, such as Arg;

[0443] Axx2 represents an amino acid, such as Gly or Cys; and

[0444] Axx3 represents amino acids, such as Asp or Asn;

[0445] Gxx represents an amino acid or a peptide moiety represented by the following formula (9b):

[0446] ---Gxx1–Gxx2–Gxx3---(9b)

[0447] in,

[0448] Gxx1 represents amino acids, such as Thr;

[0449] Gxx2 represents an amino acid, such as Tyr or Cys; and

[0450] Gxx3 represents a single covalent bond or amino acid, such as His;

[0451] Furthermore, the side chain of Axx2 in equation (9a) can be covalently bonded to the side chain of Gxx2 in equation (9b) to form a ring; if Axx2 is Cys and Gxx2 is Cys, then it is preferable to link the side chains of Axx2 and Gxx2 together to form equation –(S–X 4 –S)– groups, where X 4 This represents a single covalent bond or a divalent group containing one or more atoms selected from carbon, nitrogen, and oxygen, such as a divalent maleimide group, a divalent acetone group, or a divalent aryl group (e.g., a divalent xylyl group). Preferably, X 4 This represents a single covalent bond.

[0452] Hxx represents a single covalent bond or a trifunctional amino acid, such as diaminocarboxylic acid.

[0453] Z1 means:

[0454] If Hxx is a single covalent bond, then Z1 represents a group covalently bonded to the C-terminus of Gxx, which is selected from -N(H)(R), where R represents a hydrogen atom, alkyl or cycloalkyl, and a portion derived from a compound containing a bonding group, which is selected from biotin, DBCO, TCO, BCN, alkynes, azides, bromoacetamide, maleimide and thiols.

[0455] If Hxx is a trifunctional amino acid and Y' is bound to the side chain of Hxx, then Z1 represents a group covalently bound to the C-terminus of Hxx, preferably N(H)(R), wherein if Z1 is covalently bound to the C-terminus of Hxx, then R represents a hydrogen atom, an alkyl group, or a cycloalkyl group; or

[0456] If Hxx is a trifunctional amino acid and Y' is bound to the C-terminus of Hxx, then Z1 represents a hydrogen atom bound to the side chain of Hxx.

[0457] Z2 means:

[0458] If Hxx is a single covalent bond, then Z2 represents a group covalently bonded to the N-terminus of Axx, which is selected from hydrogen atoms, carbonyl groups such as acetyl groups, and groups containing coupling moieties such as biotin.

[0459] If Hxx is a trifunctional amino acid and Y' is bound to the side chain of Hxx, then Z2 represents a group covalently bound to the N-terminus of Hxx, selected from hydrogen atoms and carbonyl groups such as acetyl groups; or

[0460] If Hxx is a trifunctional amino acid and Y' is bound to the N-terminus of Hxx, then Z2 represents a hydrogen atom bound to the side chain of Hxx.

[0461] If Hxx is a trifunctional amino acid, then only Y' exists; and

[0462] If Z 1 If Z2 is attached to the C-terminus of Hxx in Equation (8a), or if Z2 is attached to the N-terminus of Hxx in Equation (8b), then Y' represents the portion covalently attached to the Hxx side chain;

[0463] If Z1 is attached to the side chain of Hxx in equation (8a), then Y' represents the portion covalently attached to the C-terminus of Hxx;

[0464] If Z2 is attached to the side chain of Hxx in equation (8b), then Y' represents the portion covalently attached to the N-terminus of Hxx;

[0465] Y' is derived from compounds containing a binding group, preferably selected from biotin, DBCO, TCO, BCN, alkynes, azides, bromoacetamide, maleimide, and thiols.

[0466] X 3 This refers to a single covalent bond or a divalent group containing one or more atoms selected from carbon, nitrogen, and oxygen, such as a divalent maleimide group, a divalent acetone group, or a divalent aryl group (e.g., a divalent xylyl group). Preferably, X 3 This represents a single covalent bond.

[0467] **** refers to covalent attachment to the spacer (S).

[0468] In the implementation scheme, part of Y' is represented by the following formula (9c):

[0469] Y 1 -L 1 -- (9c)

[0470] in,

[0471] Y 1 It is a part derived from the bonding group, which is selected from biotin, DBCO, TCO, BCN, alkynes, azides, bromoacetamide, maleimide and thiols;

[0472] L 1 It is a divalent group, preferably containing one or more atoms selected from C, N, O, and S, more preferably containing a polyoxyethylene group having 1-12 repeating units (e.g., 4 repeating units); and

[0473] Refers to covalent attachment to Hxx.

[0474] The connector is a divalent group, preferably containing one or more atoms selected from carbon, nitrogen, oxygen and sulfur.

[0475] In the embodiment, connector L 1 Can be selected

[0476] (a1) An alkylene group having 1 to 12 carbon atoms, preferably an alkylene group having 2 to 6 carbon atoms, such as ethylene or propylene;

[0477] (b1) A polyepoxyalkylene compound having 2 or 3 carbon atoms and having 1 to 36 repeating units; preferably of the formula –NH–(CH2CH2O). n1 –CH2CH2– represents a group, where n1 is an integer from 0 to 35, for example, 1 to 20; and

[0478] (c1) A peptide group having 2 to 12 amino acids.

[0479] According to a preferred embodiment, at least one of Axx, Bxx, Cxx, Dxx, Exx, Fxx, Gxx and Hxx in equations (8a) and (8b) is defined as follows:

[0480] Axx represents an amino acid selected from Ala, 2,3-diamino-propionic acid (Dap), Asp, Glu, 2-aminooctanoic acid, α-aminobutyric acid, Asn, and Gln; a dicarboxylic acid selected from succinic acid, glutaric acid, and adipic acid; or a peptide moiety of formula (9a); Axx is preferably Ala, Asp, or Asn, more preferably Asp; wherein Axx1 is a single covalent bond, Axx2 is Cys, and Axx3 is Asp;

[0481] Bxx represents an amino acid selected from Trp, Phe, Tyr, phenylglycine (Phg), 3-benzothiophene-2-yl-L-alanine, 3-naphthyl-2-yl-L-alanine, 3-biphenyl-4-yl-L-alanine, and 3-naphthyl-1-yl-L-alanine; preferably Trp;

[0482] Cxx represents an amino acid selected from His, Ala, 3-pyridin-2-yl-L-alanine, meta-tyrosine (mTyr), and Phe; preferably His, Ala, or mTyr; more preferably His;

[0483] Dxx represents an amino acid selected from Ala, Abu, Gly, Leu, Ile, Val, Met, cyclohexylalanine (Cha), Phe, Thr, Cys, Tyr, and norleucine (Nle); preferably Ala, Nle, or Leu; more preferably Leu;

[0484] Exx represents an amino acid selected from Ala, Gly, Asn, Ser, Abu, and Asp; preferably Ala or Gly; more preferably Gly;

[0485] Fxx represents an amino acid selected from Ala, Glu, Asp, Gln, His, Arg, Ser, and Asn; preferably Asp or Glu; more preferably Glu;

[0486] Gxx represents an amino acid selected from Thr, Ser, Ala, Asn, Val, 2-amino-butyric acid (Abu), Ile, Met, Leu, Pro, Gln, and Cys, or a peptide moiety of formula (9b); Gxx is preferably Thr or Ser; more preferably Thr; wherein Gxx1 is Thr, Gxx2 is Cys, and Gxx3 is a single covalent bond;

[0487] Hxx represents amino acids selected from Dap, Dab, Lys, Orn and homo-Lys, preferably selected from Dap, Dab, Lys, Orn and homo-Lys.

[0488] According to one embodiment, the ligand V capable of interacting with the Fc region of an antibody or its fragment is a peptide represented by one of the following formulas (8a') to (8d'):

[0489]

[0490] In the above equations (8a'), (8b'), (8c'), and (8d'), Z1, Z2, and X 3 X 4 and As described above with respect to equations (8a) and (8b).

[0491] In the implementation scheme, the disulfide bridge between cysteine ​​residues in the above formula (i.e., formula -(SX)) 3 -S)- or -(SX) 4 The disulfide bridges of the -S)- can each be independently replaced by divalent groups suitable for the cyclization of the side chains (sometimes referred to as "cysteine ​​rebridging"; see, for example, Stefanucci et al., Scientific Reports 2019, 9:5771). Examples of suitable divalent groups include divalent xylene groups, divalent maleimide groups, divalent triazole groups, divalent carbonyl groups (e.g., divalent acetone groups), divalent succinimide groups (which can be obtained by reacting the cysteine ​​side chain with, for example, an aryloxymaleimide reagent; see Marculescu et al., Chem. Commun. 2014, 50, 7139), divalent thioether groups (which can be obtained by reacting the cysteine ​​side chain with, for example, a disulfone or allyl sulfone reagent; see Brocchini et al., Nat. Protoc. 2006, 1, 2241-2252), and divalent piperazine-dion groups (which can be obtained by reacting the cysteine ​​side chain with, for example, a dibromopiperazine-dion reagent; see Chudamasa et al., Chem. Commun. 2011, 47, 8781-8783). Specifically, the disulfide bridges can each be independently substituted with a divalent triazole-containing group obtainable through "click" chemistry. In this case, the cysteine ​​residue (which forms the bridge in the above formula) can be substituted with an amino acid having a side chain containing a functional group suitable for click chemistry, namely, an alkynyl or azide group, which can react to generate a divalent triazole moiety (e.g., a 1,4-disubstituted-1,2,3-triazole moiety).

[0492] Preferably, the carrier is a peptide represented by formula (8a') or (8b').

[0493] According to one embodiment, the compounds of the present invention are compounds selected from the following formulas: VS 1 -(O-(C=O)-O)-P、VS 1 -(O-(C=O))-P、VS 1 -(S-(C=O))-P、VS 1 -(S-(C=O)-O)-P、VS 1 -(O-(C=S)-O)-P、VS 1 -(O-(C=O)-S)-P、VS 1 -(S-(C=O)-S)-P、VS 1 -(S-(C=S)-O)-P、VS 1 -(O-(C=S)-S)-P、VS 1 -(S-(C=S))-P、VS 1 -(O-(C=O)-NH)-P、VS 1 -(S-(C=S)-S)-P、VS 1 -(MO-(C=O)-O)-P、VS 1 -(MO-(C=O))-P、VS 1 -(MS-(C=O))-P、VS 1 -(MS-(C=O)-O)-P、VS 1 -(MO-(C=S)-O)-P、VS 1 -(MO-(C=O)-S)-P、VS 1 -(MS-(C=O)-S)-P、VS 1 -(MS-(C=S)-O)-P、VS 1 -(MO-(C=S)-S)-P、VS 1 -(MS-(C=S))-P、VS 1 -(MO-(C=O)-NH)-P、VS 1 -(MS-(C=S)-S)-P、VS 1 -(O-(C=O)-O)-LP 1 VS 1 -(O-(C=O))-LP 1 VS 1 -(S-(C=O))-LP 1 VS 1-(S-(C=O)-O)-L-P 1 、V-S 1 -(O-(C=S)-O)-L-P 1 、V-S 1 -(O-(C=O)-S)-L-P 1 、V-S 1 -(S-(C=O)-S)-L-P 1 、V-S 1 -(S-(C=S)-O)-L-P 1 、V-S 1 -(O-(C=S)-S)-L-P 1 、V-S 1 -(S-(C=S))-L-P 1 、V-S 1 -(O-(C=O)-NH)-L-P 1 、V-S 1 -(S-(C=S)-S)-L-P 1 、V-S 1 -(M-O-(C=O)-O)-L-P 1 、V-S 1 -(M-O-(C=O))-L-P 1 、V-S 1 -(M-S-(C=O))-L-P 1 、V-S 1 -(M-S-(C=O)-O)-L-P 1 、V-S 1 -(M-O-(C=S)-O)-L-P 1 、V-S 1 -(M-O-(C=O)-S)-L-P 1 、V-S 1 -(M-S-(C=O)-S)-L-P 1 、V-S 1 -(M-S-(C=S)-O)-L-P 1 、V-S 1 -(M-O-(C=S)-S)-L-P 1 、V-S 1 -(M-S-(C=S))-L-P 1 、V-S 1 -(M-O-(C=O)-NH)-L-P 1 、and V-S 1 -(M-S-(C=S)-S)-L-P 1 ,where V, P, P 1 and L are as defined above, S1 The spacer S is as defined above, and preferably V, S l P, P l At least one of L and M (e.g., two, three, four or more) is defined as follows:

[0494] (α) V is a peptide of formula (8a) or (8b), preferably a peptide of formula (8a') or (8b');

[0495] (β) S 1 The groups are selected from the following:

[0496] (a2) A polyepoxyalkyl group having 6 to 36 repeating units; preferably a group represented by formula (7):

[0497] –X 1 –(CH2CH2O) n2 –CH2CH2–X 2 –(7)

[0498] in,

[0499] X 1 It can be NH, O, or S; NH is preferred;

[0500] X 2 For NH or C=O, if X 2 If covalently bonded to the carrier, then X is preferred. 2 For C=O; and

[0501] n2 is an integer from 4 to 28, preferably an integer from 6 to 20, more preferably 10; and

[0502] (b2) A peptide group having 6 to 25 amino acids on the main chain, wherein the amino acids are preferably selected from Pro, Gly, Ala, Asn, Asp, Thr, Glu, Gln and Ser; more preferably Pro, Gly or Ser;

[0503] (γ) P or P 1 It is derived from the following parts:

[0504] (γ1) NOTA, DOTA, NODAGA, DTPA, each of which may optionally chelate radionuclides selected from the following: 89 Zr、 111 In、 64 Cu、 177 Lu、 68 Ga and 99m Tc, preferably selected from 89 Zr、 111 In、 64 Cu,

[0505] (γ2) N3, TZ, TCO, DBCO, BCN,

[0506] (γ3) Auristatin (e.g., MMAE) or PNU-159582;

[0507] (δ) L is a connector selected from the following:

[0508] (a1) Alkylene groups having 2 to 6 carbon atoms (-(CH2)) 2-6 -),

[0509] (b1) Formula -NH-(CH2CH2O) n1 -CH2CH2- polyalkylene groups, n1 being an integer from 0 to 35, and

[0510] (c1) A peptide linker comprising 2 to 12 amino acids, wherein the peptide linker is optionally cleavable, preferably a cleavable peptide linker comprising a Val-Cit unit, a Val-Ala unit, a Val-Cit-PABC unit, or a Val-Cit-PABC-DMEA unit; and

[0511] (ε) M is a group of formula (5a) or (5e), ​​preferably a group of formula (5a).

[0512] According to the preferred implementation scheme, in the above formula, V and S 1 M is defined as follows:

[0513] (α) V is a peptide of formula (8a') or (8b');

[0514] (β) S 1 For groups represented by the following formula (7):

[0515] –X 1 –(CH2CH2O) n2 –CH2CH2–X 2 –(7)

[0516] in,

[0517] X 1 It can be NH, O, or S; NH is preferred;

[0518] X 2 For NH or C=O, if X 2 If X covalently binds to the carrier, then X 2 Preferably, C=O; and

[0519] n2 is an integer from 6 to 20, preferably 10; and

[0520] (ε) M is a group of formula (5a).

[0521] If (γ)P 1 For a derivative of auristatin (e.g., MMAE), (δ)L preferably represents a cleavable linker comprising a Val-Cit unit, a Val-Ala unit, or a Val-Cit-PABC unit, more preferably a cleavable linker comprising a Val-Cit-PABC unit. If (γ)P1 is a derivative of PNU-159582, then (δ)L preferably represents a cleavable linker comprising a Val-Cit-PABC-DMEA unit.

[0522] According to one embodiment, the compound of the present invention is a compound selected from compounds represented by the following formula: V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MO-(C=O)-O)-P、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MO-(C=O))-P、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MS-(C=O))-P、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MS-(C=O)-O)-P、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MO-(C=S)-O)-P、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MO-(C=O)-S)-P、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MS-(C=O)-S)-P、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MS-(C=S)-O)-P、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MO-(C=S)-S)-P、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MS-(C=S))-P、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(MO-(C=O)-NH)-P,V 1-NH-(CH2CH2O) n2 -CH2CH2-NH-(M-S-(C=S)-S)-P、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(O-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(O-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(S-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(S-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(O-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(O-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(S-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(S-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(O-(C=S)-S)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(S-(C=S))-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(O-(C=O)-NH)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(S-(C=S)-S)-L-P1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-O-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-O-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-S-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-S-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-O-(C=S)-O)-L-P 1 、V-NH-(CH2CH2O) n2 -CH2CH2-NH-(M-O-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-S-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-S-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-O-(C=S)-S)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-S-(C=S))-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-O-(C=O)-NH)-L-P 1 、V 1 -NH-(CH2CH2O) n2 -CH2CH2-NH-(M-S-(C=S)-S)-L-P 1、P-(O-(C=O)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-((C=O)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-((C=O)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-(O-(C=O)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-(O-(C=S)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-(S-(C=O)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-(S-(C=O)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-(O-(C=S)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-(S-(C=S)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-((C=S)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-(NH-(C=O)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P-(S-(C=S)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=O)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-((C=O)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2、P 1 -L-((C=O)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=O)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=S)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(S-(C=O)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(S-(C=O)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=S)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(S-(C=S)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-((C=S)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(NH-(C=O)-O-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、P 1 -L-(S-(C=S)-S-M)-NH-(CH2CH2O) n2 -CH2CH2-(C=O)-V 2 、V-AA 6-25 -(M-O-(C=O)-O)-P、V-AA 6-25 -(M-O-(C=O))-P、V-AA 6-25 -(M-S-(C=O))-P、V-AA 6-25 -(M-S-(C=O)-O)-P、V-AA 6-25-(MO-(C=S)-O)-P、V-AA 6-25 -(MO-(C=O)-S)-P、V-AA 6-25 -(MS-(C=O)-S)-P、V-AA 6-25 -(MS-(C=S)-O)-P、V-AA 6-25 -(MO-(C=S)-S)-P、V-AA 6-25 -(MS-(C=S))-P、V-AA 6-25 -(MO-(C=O)-NH)-P、V-AA 6-25 -(MS-(C=S)-S)-P、V-AA 6-25 -(O-(C=O)-O)-LP 1 V-AA 6-25 -(O-(C=O))-LP 1 V-AA 6-25 -(S-(C=O))-LP 1 V-AA 6-25 -(S-(C=O)-O)-LP 1 V-AA 6-25 -(O-(C=S)-O)-LP 1 V-AA 6-25 -(O-(C=O)-S)-LP 1 V-AA 6-25 -(S-(C=O)-S)-LP 1 V-AA 6-25 -(S-(C=S)-O)-LP 1 V-AA 6-25 -(O-(C=S)-S)-LP 1 V-AA 6-25 -(S-(C=S))-LP 1 V-AA 6-25 -(O-(C=O)-NH)-LP 1 V-AA 6-25 -(S-(C=S)-S)-LP 1 V-AA 6-25 -(MO-(C=O)-O)-LP 1 V-AA 6-25 -(MO-(C=O))-LP 1 V-AA 6-25 -(MS-(C=O))-LP 1 V-AA 6-25 -(MS-(C=O)-O)-LP1 V-AA 6-25 -(MO-(C=S)-O)-LP 1 V-AA 6-25 -(MO-(C=O)-S)-LP 1 V-AA 6-25 -(MS-(C=O)-S)-LP 1 V-AA 6-25 -(MS-(C=S)-O)-LP 1 V-AA 6-25 -(MO-(C=S)-S)-LP 1 V-AA 6-25 -(MS-(C=S))-LP 1 V-AA 6-25 -(MO-(C=O)-NH)-LP 1 and V-AA 6-25 -(MS-(C=S)-S)-LP 1 Among them, V, P, P 1 As defined above, and L, V 1 For the peptide of formula (8b), V 2 The peptide of formula (8a), wherein preferably V, V 1 V 2 n2, AA, P, P 1 At least one of L and M (e.g., two, three, four or more) is defined as follows:

[0523] (α) V is a peptide of formula (8a) or (8b), preferably a peptide of formula (8a') or (8b'); V 1 For a peptide of formula (8b'), V 2 The peptide of formula (8a');

[0524] (β) n2 is an integer from 6 to 20, preferably 10; or

[0525] Each amino acid (AA) is independently selected from Pro, Gly, Ala, Asn, Asp, Thr, Glu, Gln, and Ser, preferably from Pro, Gly, and Ser;

[0526] (γ)P or P 1 It is derived from the following parts:

[0527] (γ1) NOTA, DOTA, NODAGA, DTPA, each of which may optionally chelate radionuclides selected from the following: 89 Zr、 111 In、 64Cu、 177 Lu、 68 Ga and 99m Tc, preferably selected from 89 Zr、 111 In、 64 Cu,

[0528] (γ2) N3, TZ, TCO, DBCO, BCN,

[0529] (γ3) Auristatin (e.g., MMAE) or PNU-159582;

[0530] (δ)L is a connector selected from the following:

[0531] (a1) Alkylene groups having 2 to 6 carbon atoms (-(CH2)) 2-6 -),

[0532] (b1) Formula -NH-(CH2CH2O) n1 -CH2CH2- polyalkylene groups, n1 being an integer from 0 to 35, and

[0533] (c1) A peptide linker comprising 2 to 12 amino acids, wherein the peptide linker is optionally cleavable, preferably a cleavable peptide linker comprising a Val-Cit unit, a Val-Ala unit, a Val-Cit-PABC unit, or a Val-Cit-PABC-DMEA unit; and

[0534] (ε) M is a group of formula (5a) or (5e), ​​preferably a group of formula (5a).

[0535] According to the preferred implementation scheme, in the above formula, V and V 1 V 2 n2, AA, and M are defined as follows:

[0536] (α) V is a peptide of formula (8a') or (8b'); V 1 For a peptide of formula (8b'), V 2 The peptide of formula (8a');

[0537] (β) n2 is an integer from 6 to 20, preferably 10; or

[0538] Each amino acid (AA) is independently selected from Pro, Gly, Ala, Asn, Asp, Thr, Glu, Gln, and Ser, preferably from Pro, Gly, and Ser; and

[0539] (ε) M is a group of formula (5a).

[0540] If (γ)P 1For a derivative derived from auristatin (e.g., MMAE), (δ)L preferably represents a cleavable linker comprising a Val-Cit unit, a Val-Ala unit, or a Val-Cit-PABC unit, more preferably a cleavable linker comprising a Val-Cit-PABC unit. If (γ)P 1 If it is a part derived from PNU-159582, then (δ)L preferably represents a pyrolytic connector containing a Val-Cit-PABC-DMEA unit.

[0541] According to one embodiment, the compounds of the present invention are compounds represented by the following formula: V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MO-(C=O)-O)-P、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MO-(C=O))-P、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MS-(C=O))-P、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MS-(C=O)-O)-P、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MO-(C=S)-O)-P、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MO-(C=O)-S)-P、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MS-(C=O)-S)-P、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MS-(C=S)-O)-P、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MO-(C=S)-S)-P、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MS-(C=S))-P、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(MO-(C=O)-NH)-P,V 1 -NH-(CH2CH2O)6-20 -CH2CH2-NH-(M-S-(C=S)-S)-P、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(O-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(O-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(S-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(S-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(O-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(O-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(S-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(S-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(O-(C=S)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(S-(C=S))-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(O-(C=O)-NH)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(S-(C=S)-S)-L-P 1 、V1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-O-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-O-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-S-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-S-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-O-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-O-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-S-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-S-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-O-(C=S)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-S-(C=S))-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-O-(C=O)-NH)-L-P 1 、V 1 -NH-(CH2CH2O) 6-20 -CH2CH2-NH-(M-S-(C=S)-S)-L-P 1、P-(O-(C=O)-O-M)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-((C=O)-O-M)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-((C=O)-S-M)-NH- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-(O-(C=O)-S-M)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-(O-(C=S)-O-M)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-(S-(C=O)-O-M)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-(S-(C=O)-S-M)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-(O-(C=S)-S-M)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-(S-(C=S)-O-M)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-((C=S)-S-M)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-(NH-(C=O)-O-M)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P-(S-(C=S)-S-M)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=O)-O-M)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 、P 1 -L-((C=O)-O-M)- NH-(CH2CH2O) 6-20-CH2CH2-(C=O)-V 2 P 1 -L-((C=O)-SM)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 P 1 -L-(O-(C=O)-SM)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 P 1 -L-(O-(C=S)-OM)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 P 1 -L-(S-(C=O)-OM)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 P 1 -L-(S-(C=O)-SM)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 P 1 -L-(O-(C=S)-SM)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 P 1 -L-(S-(C=S)-OM)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 P 1 -L-((C=S)-SM)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 P 1 -L-(NH-(C=O)-OM)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 and P 1 -L-(S-(C=S)-SM)- NH-(CH2CH2O) 6-20 -CH2CH2-(C=O)-V 2 Among them, V, P, P 1 As defined above, and L, V 1 For the peptide of formula (8b), V 2 The peptide is of formula (8a), and preferably, V 1 V 2P, P 1 At least one of L and M (e.g., two, three, four or more) is defined as follows:

[0542] (α) V 1 For a peptide of formula (8b'), V 2 The peptide of formula (8a');

[0543] (γ) P or P 1 It is derived from the following parts:

[0544] (γ1)NOTA, DOTA, NODAGA, DTPA, each of which may optionally chelate radionuclides selected from the following: 89 Zr、 111 In、 64 Cu、 177 Lu、 68 Ga and 99m Tc, preferably selected from 89 Zr、 111 In、 64 Cu,

[0545] (γ2) N3, TZ, TCO, DBCO, BCN,

[0546] (γ3) Auristatin (e.g., MMAE) or PNU-159582;

[0547] (δ) L is a connector selected from the following:

[0548] (a1) Alkylene groups having 2 to 6 carbon atoms (-(CH2)) 2-6 -),

[0549] (b1) Formula -NH-(CH2CH2O) n1 -CH2CH2- polyalkylene groups, n1 being an integer from 0 to 35, and

[0550] (c1) A peptide linker comprising 2 to 12 amino acids, wherein the peptide linker is optionally cleavable, preferably a cleavable peptide linker comprising a Val-Cit unit, a Val-Ala unit, a Val-Cit-PABC unit, or a Val-Cit-PABC-DMEA unit; and

[0551] (ε) M is a group of formula (5a) or (5e), ​​preferably a group of formula (5a).

[0552] According to the preferred implementation scheme, in the above formula, V 1 V 2 M is defined as follows:

[0553] (a) V1 For a peptide of formula (8b'), V 2 A peptide of formula (8a'); and

[0554] (ε) M is a group of formula (5a).

[0555] If (γ)P 1 For a derivative derived from auristatin (e.g., MMAE), (δ)L preferably represents a cleavable linker comprising a Val-Cit unit, a Val-Ala unit, or a Val-Cit-PABC unit, more preferably a cleavable linker comprising a Val-Cit-PABC unit. If (γ)P 1 If it is a part derived from PNU-159582, then (δ)L preferably represents a pyrolytic connector containing a Val-Cit-PABC-DMEA unit.

[0556] According to one embodiment, the compounds of the present invention are compounds represented by the following formula: V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(MO-(C=O)-O)-P、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(MO-(C=O))-P、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(MS-(C=O))-P、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(MS-(C=O)-O)-P、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(MO-(C=S)-O)-P、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(MO-(C=O)-S)-P、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(MS-(C=O)-S)-P、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(MS-(C=S)-O)-P、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(MO-(C=S)-S)-P、V 1 -NH-(CH2CH2O)10 -CH2CH2-NH-(M-S-(C=S))-P、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-O-(C=O)-NH)-P、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-S-(C=S)-S)-P、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(O-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(O-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(S-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(S-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(O-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(O-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(S-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(S-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(O-(C=S)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(S-(C=S))-L-P 1 、V 1 -NH-(CH2CH2O)10 -CH2CH2-NH-(O-(C=O)-NH)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(S-(C=S)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-O-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-O-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-S-(C=O))-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-S-(C=O)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-O-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-O-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-S-(C=O)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-S-(C=S)-O)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-O-(C=S)-S)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-S-(C=S))-L-P 1 、V 1 -NH-(CH2CH2O) 10-CH2CH2-NH-(M-O-(C=O)-NH)-L-P 1 、V 1 -NH-(CH2CH2O) 10 -CH2CH2-NH-(M-S-(C=S)-S)-L-P 1 、P-(O-(C=O)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-((C=O)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-((C=O)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-(O-(C=O)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-(O-(C=S)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-(S-(C=O)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-(S-(C=O)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-(O-(C=S)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-(S-(C=S)-O-M)-NH(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-((C=S)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-(NH-(C=O)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P-(S-(C=S)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1-L-(O-(C=O)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-((C=O)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-((C=O)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=O)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=S)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-(S-(C=O)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-(S-(C=O)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-(O-(C=S)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-(S-(C=S)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 as well as P 1 -L-((C=S)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 、P 1 -L-(NH-(C=O)-O-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2 as well as P 1 -L-(S-(C=S)-S-M)-NH-(CH2CH2O) 10 -CH2CH2-(C=O)-V 2Among them, V, P, P 1 As defined above, and L, V 1 For the peptide of formula (8b), V 2 The peptide is of formula (8a), and preferably, V 1 V 2 P, P 1 At least one of L and M (e.g., two, three, four or more) is defined as follows:

[0557] (α) V 1 For a peptide of formula (8b'), V 2 The peptide of formula (8a');

[0558] (γ) P or P 1 It is derived from the following parts:

[0559] (γ1) NOTA, DOTA, NODAGA, DTPA, each of which may optionally chelate radionuclides selected from the following: 89 Zr、 111 In、 64 Cu、 177 Lu、 68 Ga and 99m Tc, preferably selected from 89 Zr、 111 In、 64 Cu,

[0560] (γ2) N3, TZ, TCO, DBCO, BCN,

[0561] (γ3) Auristatin (e.g., MMAE) or PNU-159582;

[0562] (δ) L is a connector selected from the following:

[0563] (a1) Alkylene groups having 2 to 6 carbon atoms (-(CH2)) 2-6 -),

[0564] (b1) Formula -NH-(CH2CH2O) n1 -CH2CH2- polyalkylene groups, n1 being an integer from 0 to 35, and

[0565] (c1) A peptide linker comprising 2 to 12 amino acids, wherein the peptide linker is optionally cleavable, preferably a cleavable peptide linker comprising a Val-Cit unit, a Val-Ala unit, a Val-Cit-PABC unit, or a Val-Cit-PABC-DMEA unit; and

[0566] (ε) M is a group of formula (5a) or (5e), ​​preferably a group of formula (5a).

[0567] According to the preferred implementation scheme, in the above formula, V 1 V 2 M is defined as follows:

[0568] (a) V 1 For a peptide of formula (8b'), V 2 A peptide of formula (8a'); and

[0569] (ε) M is a group of formula (5a).

[0570] If (γ)P 1 For a derivative derived from auristatin (e.g., MMAE), (δ)L preferably represents a cleavable linker comprising a Val-Cit unit, a Val-Ala unit, or a Val-Cit-PABC unit, more preferably a cleavable linker comprising a Val-Cit-PABC unit. If (γ)P 1 If it is a part derived from PNU-159582, then (δ)L preferably represents a pyrolytic connector containing a Val-Cit-PABC-DMEA unit.

[0571] In one embodiment, the compound of formula (1) is selected from:

[0572] ,

[0573] ,

[0574] ,

[0575] ,

[0576] ,

[0577] ,

[0578] ,

[0579] and,

[0580] ;

[0581] Wherein P represents the effective load as defined above, preferably a chelating agent that optionally chelates radionuclides, more preferably a portion derived from DTPA, DOTA, DFO, NOOTA, PCTA, CH-X-DTPA, NODAGA, or DOTAGA; Y' represents a portion derived from a compound containing a binding group, which is preferably selected from biotin, DBCO, TCO, BCN, alkynes, azides, bromoacetamide, maleimide, and thiols, more preferably from biotin, DBCO, BCN, and azides. The number of repeats of the polyethylene oxide portion of the spacer in the above compounds (i.e., 9) can be replaced by any one of 5 to 35, preferably any one of 7 to 19, with a spacer having 9 polyethylene oxide repeating units being the most preferred choice.

[0582] In one embodiment, the compound of formula (1) is selected from:

[0583] ,

[0584] ,

[0585] ,

[0586] ,

[0587] ,

[0588] ,

[0589] ,

[0590] ,

[0591] ,

[0592] ,

[0593] ,

[0594] ,

[0595] ,

[0596] ,

[0597] ,

[0598] ,

[0599] ,

[0600] 、

[0601] 、

[0602] 、

[0603] 、

[0604] 、

[0605] 、

[0606] 、

[0607] 、

[0608] 、

[0609] 、

[0610] 、

[0611] 、

[0612] 、

[0613] 、

[0614] 、

[0615] 、

[0616] 、

[0617] 、

[0618] 、

[0619] 、

[0620] 、

[0621] 、

[0622] 、

[0623] 、

[0624] 、

[0625] 、

[0626] 、

[0627] 、

[0628] 、

[0629] 、

[0630] 、

[0631] 、

[0632] 、

[0633] 、

[0634] 、

[0635] 、

[0636] 、

[0637] 、

[0638] 、

[0639] 、

[0640] 、

[0641] 、

[0642]

[0643]

[0644]

[0645]

[0646] 、

[0647] 、

[0648] 、

[0649] 、

[0650] 、

[0651] 、

[0652] 、

[0653] 、

[0654] 、

[0655] 、

[0656] 、

[0657] 、

[0658] 、

[0659] 、

[0660] 、

[0661] 、

[0662] 、

[0663] 、

[0664] 、

[0665] 、

[0666] 、

[0667] 、

[0668] 、

[0669] 、

[0670] 、

[0671] 、

[0672] 、

[0673] 、

[0674] 、

[0675] 、

[0676] 、

[0677] 、

[0678] 、

[0679] 、

[0680] 、

[0681] 、

[0682] 、

[0683] 、

[0684] 、

[0685] 、

[0686] 、

[0687] 、

[0688] 、

[0689] 、

[0690] 、

[0691] 、

[0692] 、

[0693] 、

[0694] 、

[0695] 、

[0696] 、

[0697] 、

[0698] 、

[0699] ,

[0700] ,

[0701] ,

[0702] ,

[0703] ,

[0704] ,

[0705]

[0706] as well as,

[0707] .

[0708] In one embodiment, the number of repeats of the polyethylene oxide portion of the spacer in the above-mentioned compound (i.e., 9) can be replaced with any one of 5 to 35, preferably any one of 7 to 19, with a spacer having 9 polyethylene oxide repeating units being the most preferred option.

[0709] In a preferred embodiment, the compound of formula (1) is selected from...

[0710] ,

[0711] ,

[0712] ,

[0713] ,

[0714] ,

[0715] ,

[0716] ,

[0717] ,

[0718] ,

[0719] ,

[0720] ,

[0721] ,

[0722] ,

[0723] ,

[0724] ,

[0725] ,

[0726] ,

[0727] ,

[0728] ,

[0729] ,

[0730] ,

[0731] ,

[0732] ,

[0733] ,

[0734] ,

[0735] ,

[0736] ,

[0737] ,

[0738] ,

[0739] ,

[0740] ,

[0741] ,

[0742] ,

[0743] ,

[0744] ,

[0745] ,

[0746] ,

[0747] as well as,

[0748] .

[0749] The number of repeats of the polyethylene oxide portion of the spacer in the above compounds (i.e., 9) can be replaced by any one of 5 to 35, preferably any one of 7 to 19, with a spacer having 9 polyethylene oxide repeating units being the most preferred choice.

[0750] In a more preferred embodiment, the compound of formula (1) is selected from:

[0751] ,

[0752] ,

[0753] ,

[0754] ,

[0755] ,

[0756] ,

[0757] ,

[0758] ,

[0759] ,

[0760]

[0761] as well as,

[0762] .

[0763] In the above compounds, DFO represents a deferoxamine group, which is attached to the residues of the molecule via its amino group to form a thiourea group together with the thiocarbonyl group to which it is attached. The number of repeating polyoxyethylene moieties in the spacer in the above compounds (i.e., 9) can be replaced with any one of 5 to 35, preferably any one of 7 to 19, with a spacer having 9 repeating polyoxyethylene units being the most preferred choice.

[0764] 4. Kits for site-specific modification of antibodies or antibody fragments

[0765] In some aspects, the present invention relates to a kit comprising the compounds described above and a buffer, which can be used for the region-selective modification (e.g., for labeling) of antibodies or fragments thereof, wherein the antibody fragments are optionally incorporated into Fc-fusion proteins, and the kit is particularly for the region-selective modification of therapeutic antibodies.

[0766] The compounds and buffers of the present invention (together forming a kit) can exist separately, for example in a single primary container (which can be shipped to the customer in a single box), which can be stored for extended periods without degradation. The compounds and buffers can be formulated and proportioned to a given amount of the antibody or fragment thereof to be modified. In some aspects, the compounds of the present invention are present as solids (e.g., as lyophilized powders, or non-covalently adsorbed or covalently bound to a solid matrix further described below) or as solutions in suitable solvents, such as water-soluble, polar aprotic solvents (e.g., DMF, DMSO), which can be mixed with buffers shortly before antibody or antibody fragment modification.

[0767] The buffer solution used in the kit of the present invention is not particularly limited. Preferably, the pH of the buffer solution is 5.5 to 11, more preferably 7.5 to 9.5. The buffer can be selected from, for example, 2-bis(2-hydroxyethyl)aminoacetic acid (Bicine), carbonate-bicarbonate, tris(hydroxymethyl)methylaminopropane sulfonic acid (TAPS), and 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES). Preferably, the buffer is a carbonate-bicarbonate or bicine buffer with a pH of 7.5 to 9.5 (e.g., about 9.0).

[0768] According to one embodiment, the compounds of the present invention are immobilized on a solid matrix (solid support), such as on beads. The compounds can be immobilized using methods known in the art, such as high-affinity (e.g., biotin-streptavidin, biotin-neutral avidin) binding, "click" chemistry (as defined by Kolb Kolb et al. in "Click Chemistry: Diverse Chemical Function from a Few Good Reactions," Angewandte Chemie Int. Ed. 2001, 40(11), 2004-2021), hydrazone linkage reactions, etc. Preferably, the solid matrix is ​​an inert matrix, such as a polymer gel, comprising a three-dimensional structure, lattice, or network of materials. More preferably, the solid matrix is ​​a material used for affinity chromatography, such as a dry gel. Such a gel shrinks upon drying into a dense solid containing only a gel matrix. When the dried dry gel is resuspended in a liquid, the gel matrix absorbs the liquid, swells, and returns to the gel state. Examples of dry gels suitable for use in the present invention include polymer gels such as cellulose, cross-linked dextran gels (e.g., Sephadex). ® ), agarose, cross-linked agarose, polyacrylamide gel, polyacrylamide-agarose gel.

[0769] In one embodiment, the compound is immobilized on a solid matrix via a binding group Y' in formula (8a), for example by high-affinity binding (such as biotin-streptavidin or biotin-neutral avidin binding) (in which case Y' in formula (8a) represents, for example, a biotin-containing group), by click chemistry (in which case Y' represents, for example, a group containing DBCO-, azide or alkynyl), by tetrazine linkage reaction (in which case Y' in formula (8a) represents a group containing TCO or TZ), by reaction between thiols and maleimide or between thiols and acetamide (in which case Y' in formula (8a) represents, for example, a group containing maleimide or (chloro)acetamide).

[0770] 5. Use of reactive conjugates in methods for the regioselective modification of antibodies or antibody fragments

[0771] The compounds of the present invention can be used in methods of regionally selectively modifying antibodies or fragments thereof, wherein the antibody fragments are optionally incorporated into Fc-fusion proteins. This method produces modified antibodies or modified antibody fragments (e.g., ADCs) that can be used in methods for diagnosing, monitoring (e.g., monitoring the effectiveness of treatment (e.g., over time), imaging, or treating diseases, as further described below.

[0772] In one embodiment, the method includes the step of reacting (contacting) an antibody or a fragment thereof with a compound, which may be included in the kit described above. The reaction mixture can be purified using techniques known in the art, such as gel permeation chromatography using a suitable solvent.

[0773] When the compounds of the present invention are immobilized on a solid matrix, the immobilized compounds are contacted with a sample containing the antibody or antibody fragment to be modified, and then the solid matrix is ​​washed with a suitable solvent that will substantially remove all substances from the sample except for the antibody bound to the solid matrix. Finally, the solid matrix is ​​washed with another suitable solvent, such as a glycine buffer at pH 2.5, which will release the modified antibody / antibody fragment (e.g., ADC) from the solid matrix.

[0774] The method of this invention can be applied to any antibody (e.g., IgG protein), antibody fragment, or Fc fusion protein, provided that the antibody contains an Fc region for interacting with ligand V. In one embodiment, the antibody to be modified is a monoclonal antibody (mAb), preferably selected from the group consisting of: adalimumab, adunarumab, alenmab, pentiazemab, atezolizumab, anneltuzumab, avelumab, bapizumab, baliximab, betumab, bemizumab, bemiscizumab, bevacizumab, belotuszumab, bentuximab, blotuszumab, brentuximab, brodamab, bonnetumab, caputuzumab, cimiprimab, cetuximab. Monoclonal antibodies, simpanelumab, cristatumab, Titan-cristatumab, Titan-kranezine, dazumab, daratumumab, denosumab, denutoxicum, durvalumab, ezorol, erlotinib, epavalizumab, envertumab, vetin-envertumab, epazolizumab, epazolizumab-SN-38, edazolizumab, gemutuzumab, gemutuzumab oxozamicin, gemutuzumab, gonemab, tiimozumab, inbirizumab, infliximab Monoclonal antibodies, intuzumab, olibutuzumab, ipilimumab, esatuzumab, isibetatumab, J591, PSMA antibody, labetuzumab, leicatumab, moglizazumab, nexitozumab, nitozumab, natalizumab, nivolumab, olibutuzumab, olfamotuzumab, olaramuzumab, ozovuzumab, panitumab, pembrolizumab, pertuzumab, polotozumab, verbotozumab, pronitozumab, ramucirumab, rituximab, storutuzumab Rituximab, saxituzumab, gosatuzumab, cirrusazumab, storuximab, solanineumab, taluzumab, tetrumumab, telanelumab, tocilizumab, tosimoumab, trastuzumab, dextromethorphan trastuzumab, emtansine trastuzumab, TS23, ustekinumab, vedoluzumab, votumumab, zegetene, zalumab, zalumab, fragments and derivatives thereof; more preferably atezolizumab, durvalumab, pembrolizumab, rituximab or trastuzumab.

[0775] In one embodiment, the antibody to be modified, or a fragment thereof, is a commercially formulated antibody, preferably an antibody with marketing authorization from the U.S. EMA or the Food and Drug Administration (FDA). According to one embodiment, the commercially formulated antibody is selected from Humira. ® Lemtrada ® Camppath ® Tecentriq ® Bavencio ® Simulect® LymphoScan ® Xilonix ® Scintimun ® Avastin ® Zinplava ® Blincyto ® Libtayo ® Erbitux ® hPAM4-Cide ® Zenapax ® Darzalex ® Prolia ® Unituxin ® Imfinzi ® Panorex ® Empliciti ® Gamifant ® Rencarex ® Remicade ® Besponsa ® Yervoy ® CEA-Cide ® Poteligeo ® Tysabri ® Portrazza ® Theracim ® Opdivo ® Arzerra ® Lartruvo ® Omnitarg ® Vaxira ® Cyramza ® MabThera ® Rituxan ® Sylvant ® , Bexxar ® Herceptin ® Kadcyla ® Stelara ® HuMax-EGFr ® HuMax-CD4 ®and its bioanalytes; preferably selected from MabThera ® and Herceptin ® .

[0776] Commercially available antibodies are typically formulated with histidine to maintain stability. When commercially available antibodies are mixed with reactive conjugates, histidine is expected to competitively act on the reaction site, thereby degrading the reactive conjugate and leading to a decrease in ADC yield. However, the inventors were surprised to find that the yield was not affected, or was significantly affected, when using the compounds of the present invention. Not wishing to be bound by theory, the inventors believe this is due to an increased reaction rate between the compounds of the present invention and amino acids on the side chains of antibodies or antibody fragments (e.g., lysine or cysteine). This favorable kinetics may be related to the sharp increase in local concentration at the reaction site near the target amino acid after the carrier binds to the Fc fragment.

[0777] In one embodiment, the antibody fragment to be modified is incorporated into an Fc-fusion protein, which is preferably selected from berazip, aflibercept, ziv-aflibercept, dulaglutide, linasip, romilastine, abatacept, and alfacilitation.

[0778] 6. Modified antibodies or modified antibody fragments

[0779] Modified antibodies and modified antibody fragments (optionally incorporated into Fc-fusion proteins) obtained by reacting the compounds of the present invention with antibodies or antibody fragments include one or more payloads attached to the antibody or fragments thereof by means of a divalent group, which is a group derived from the reactive part Y in formula (1) (i.e., it corresponds to the reactive part Y in formula (1) which has been reacted with amino acid side chains exposed on the surface of the antibody or fragments thereof).

[0780] According to one implementation scheme, the modified antibody or modified antibody fragment is represented by the following formula (10):

[0781] (PW) p -A (10)

[0782] in,

[0783] P is the payload as described above, preferably the portion specified in items (i) to (iii) above;

[0784] W is F1-RC', where F1 is attached to P and RC' is a portion derived from the reaction center (RC) attached to A, and F1 and RC are defined as in equations (3a) and (3b);

[0785] A is a portion derived from an antibody or antibody fragment optionally incorporated into the Fc-fusion protein, said antibody or antibody fragment as defined above; and

[0786] p is an integer from 1 to 4. Preferably, p is from 1 to 2.

[0787] In those examples where the reactive portion reacts with the Lys side chain, the attachment of the active load to the antibody or antibody fragment occurs via a nitrogen-containing group (such as an amide group, ethyl carbamate group, ethyl thiocarbamate group, ethyl dithiocarbamate group, etc.). For example, if the compound of the present invention includes a reactive portion of formula (4a) or (4d) (or formula (4a') or (4d'), then the divalent group W in formula (10) is an ethyl carbamate group, wherein the nitrogen atom forms part of the Lys side chain. If the compound includes a reactive portion of, for example, formula (4e), (4f), or (4j) (or formula (4e'), (4f'), or (4j')), then the divalent group W is an ethyl thiocarbamate group.

[0788] p represents the degree of conjugation (DoC; sometimes referred to as the drug-antibody-ratio (DAR)) of the modified antibody or modified antibody fragment.

[0789] According to one implementation scheme, the modified antibody or modified antibody fragment is represented by the following formula (11):

[0790] (P1-LW) p -A(11)

[0791] in,

[0792] P 1 L, W, A, and p are defined as above.

[0793] 7. Use of modified antibodies or modified antibody fragments for diagnostic and / or therapeutic purposes.

[0794] Modified antibodies and modified antibody fragments obtained (or available) by reacting the compounds of the present invention with antibodies or antibody fragments (the antibody fragments optionally incorporated into Fc-fusion proteins) can be used for the diagnosis and / or treatment of diseases, particularly cancer. This treatment can be therapeutic and / or preventative, aimed at preventing, reducing, or stopping undesirable physiological changes or disorders. In some cases, treatment can prolong the survival of a subject compared to the expected survival without treatment.

[0795] The disease treated by modifying antibodies or modified antibody fragments (e.g., ADCs) can be any disease that benefits from the treatment, including chronic and acute disorders or diseases, and pathological conditions predisposing one to such disorders. In some cases, the disease is an oncological disease, such as cancer, which can be treated by targeting and destroying tumor cells. Non-limiting examples of treatable cancers include benign and malignant tumors, whether solid or liquid-filled. Leukemia and lymphoid malignancies, as well as breast cancer, ovarian cancer, gastric cancer, endometrial cancer, salivary gland cancer, lung cancer, kidney cancer, colon cancer, thyroid cancer, pancreatic cancer, prostate cancer, or bladder cancer. The disease can be a neuronal disease, glial disease, astrocyte disease, hypothalamic disease or other glandular disease, macrophage disease, epithelial disease, stromal disease, and blastocystic disease; or an inflammatory disease, angiogenic disease, or immune disease. An exemplary disease is a solid, malignant tumor.

[0796] According to one implementation scheme, the disease or its treatment is selected from the group consisting of: Alzheimer's disease, amyotrophic lateral sclerosis (ALS), cerebral arteriosclerosis, encephalopathy, Huntington's disease, multiple sclerosis, Parkinson's disease, progressive multifocal leukoencephalopathy, systemic lupus erythematosus, systemic sclerosis, angina pectoris (including unstable angina), aortic aneurysm, atherosclerosis, heart transplantation, diagnosis of cardiotoxicity, coronary artery bypass grafting, heart failure (including systolic heart failure terminated by atrial fibrillation), and hypercholesterolemia. Cholesterolemia, local ischemia, myocardial infarction, thromboembolism, thrombosis, ankylosing spondylitis, autoimmune cytopenia, autoimmune myocarditis, Crohn's disease, graft-versus-host disease, granulomatous polyangiitis, idiopathic thrombocytopenic purpura, juvenile arthritis, juvenile diabetes (type 1 diabetes), lupus, microscopic polyangiitis, multiple sclerosis, plaque psoriasis, psoriasis, psoriatic arthritis, rheumatoid arthritis, ulcerative colitis (UC), uveitis, and vasculitis.

[0797] According to one implementation scheme, the disease to be treated involves cells selected from: lymphoma cells, myeloma cells, renal cell carcinoma cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cell carcinoma cells, small cell lung cancer cells, testicular cancer cells, pancreatic cancer cells, liver cancer cells, melanoma, head and neck cancer cells, and any cells that grow and divide at an unregulated and accelerated rate, resulting in cancer; preferably selected from breast cancer cells, small cell lung cancer cells, lymphoma cells, colorectal cancer cells, and head and neck cancer cells.

[0798] According to one embodiment, the modified antibody or modified antibody fragment is used in methods for diagnosing, monitoring (e.g., monitoring the effectiveness of treatment over time), imaging, and / or treating a disease (e.g., cancer) by administering the modified antibody or modified antibody fragment to a subject (e.g., a patient).

[0799] This molecule can be administered to a subject in a single dose or as part of a series of treatments. Depending on the type and severity of the disease, and / or the payload, and / or the antibody or antibody fragment, in a first-in-human trial, approximately 0.1 µg / kg to 1 mg / kg of the drug may be used as the initial candidate dose for the first administration, for example, by a single or multiple administrations alone or by continuous infusion. Typical daily dose ranges from approximately 0.1 mg / kg to 50 mg / kg or more, or from approximately 0.5 mg / kg to approximately 30 mg / kg, for example, from 0.5 mg / kg to approximately 25 mg / kg of the patient's body weight. However, typical doses will depend on a number of factors, including the specific payload (active agent), age, weight, general health, sex, and the subject's diet; whether the administration is for imaging, monitoring, or therapeutic purposes; and other factors well-known in the medical field.

[0800] In cancer treatment, observed therapeutic effects may include a reduction in the number of cancer cells; a reduction in the size of small tumors; inhibition or delay of cancer cell infiltration into surrounding organs; inhibition of tumor growth; and / or relief of one or more cancer-related symptoms.

[0801] According to a preferred embodiment, the modified antibody or modified antibody fragment is administered by injection (e.g., parenteral, intravenous, subcutaneous, intramuscular).

[0802] According to another embodiment, the modified antibody or modified antibody fragment is used in methods for diagnosing, monitoring (e.g., monitoring the effectiveness of treatment (e.g., over time)), imaging, and / or treating cancer, and the modified antibody or modified antibody fragment is administered concurrently with one or more other therapeutic agents, such as chemotherapeutic agents, radiotherapy agents, immunotherapy agents, agents for autoimmune diseases, anti-infective agents, or one or more other modified antibodies or modified antibody fragments. Other therapeutic agents may also be administered before or after the modified antibody or modified antibody fragment.

[0803] 8. Preparation of the compounds of the present invention

[0804] The following provides methods for preparing ligands, spacers, payload-connectors, and compounds (reactive conjugates), and their use in the regioselective modification of therapeutic antibodies or therapeutic proteins (e.g., Fc-fusion proteins). The compounds of the present invention can be synthesized using standard chemical methods and Fmoc-based solid-phase peptide synthesis (SPPS), including resin-based peptide conjugation and polymerization strategies. Examples of the introduction of various payloads and the immobilization of compounds on solid-phase matrices are also illustrated below. These are known to those skilled in the art and in practice... Figure 2 , Figure 5 and Figure 9 The document describes general strategies and methods that can be used to prepare the compounds of this invention.

[0805] 9. Examples

[0806] 9.1 List of abbreviations used in the embodiments:

[0807] ACN: Acetonitrile

[0808] DCM: Dichloromethane

[0809] DIC: Diisopropylcarbodiimide

[0810] DIEA: Diisopropylethylamine

[0811] DMF: Dimethylformamide

[0812] DMSO: Dimethyl sulfoxide

[0813] FL or FITC: Fluorescein

[0814] HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine-3-oxafluorophosphate

[0815] HPLC: High Performance Liquid Chromatography

[0816] HRMS: High Resolution Mass Spectrometry

[0817] PBS: Phosphate-buffered saline

[0818] SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

[0819] SPPS: Solid-phase peptide synthesis

[0820] TFA: Trifluoroacetic acid

[0821] TIS: Triisopropylsilane

[0822] UPLC: Ultra-high performance liquid chromatography

[0823] WLC: Worm-like chain

[0824] 9.2 Raw materials and chemicals:

[0825] The main raw materials and chemicals used in the following examples are listed below:

[0826] Resins used for solid-phase peptide synthesis (Fmoc-Rink amide AM resin, 4-Fmoc-hydrazinobenzoyl AMNovagel) TM ) and protected amino acids, N,N-diisopropylcarbodiimide (DIC), and piperazine from Novabiochem (Switzerland), unless otherwise stated;

[0827] Solvents, deprotecting agents, and cleavage reagents for synthesis from Merck or Fischer Scientific AG (Switzerland);

[0828] Maleimide propionic acid, 4-nitrophenyl chloroformate, TFA, TIS and DIEA from Sigma-Aldrich (Switzerland);

[0829] Amino acids from Bachem AG (Switzerland), Novartis, and Aapptec (USA);

[0830] Solvents and chemicals from Macherey-Nagel (Switzerland) for high performance liquid chromatography (HPLC) and ultra performance liquid chromatography-mass spectrometry (UPLC-MS);

[0831] >Fc-III-FAM, a fluorescently labeled peptide from Genscript (USA);

[0832] Gingis Khan from Genovis (Sweden) ® ,Fabalactica ® and Fabricator ® Protease;

[0833] IdeS from Promega (Switzerland) ® Protease;

[0834] EndoS from BioConcept (Switzerland) ® Protease;

[0835] Biotin-PEG4-amine and PEG linkers from BroadPharm (USA);

[0836] Herceptin from Roche (Switzerland) ® (Commercial trastuzumab);

[0837] p-SCN-Bn-CHX-A''-DTPA.3HCl and p-SCN-Bn-PCTA.3HCl from Macrocyclics (USA);

[0838] p-NCS-Bz-DFO from Chematek (France).

[0839] Biosimilar monoclonal IgG1 antibodies (trastuzumab, alenzab, bevacizumab, rituximab) were prepared in the laboratory of Dr. G. Hagens at the University of Applied Sciences (HES-SO, Valais / Wallis, Switzerland) by culturing recombinant CHO cell lines.

[0840] GingisKhan and Fabalactica are cysteine ​​proteases that can perform site-specific cleavage of IgG1 above the hinge, producing two Fab fragments and one Fc fragment. Fabricator is a cysteine ​​protease that can perform site-specific digestion of antibodies below the hinge, producing F(ab')2 and Fc / 2 fragments.

[0841] 9.3 Method:

[0842] The following methods are used to evaluate the compounds and conjugates of the present invention:

[0843] 9.3.1 Determining the Length of Spacers

[0844] The length of the spacer (part S in Equation (1)) introduced at the N-terminus of the Fc binding vector was calculated using the worm-like chain (WLC) model, which treats the spacer as a continuous flexible rod and has proven to be a suitable model for biopolymers (Rubinstein and Colby (2003), Polymer Physics, Oxford University Press):

[0845] <R 2 >= 2×L p ×L

[0846] Where L pL is the persistence length (the length relevant to the chain direction), and L is the stretch length (the length of the fully stretched chain). The persistence length using polyethylene glycol spacers is 3.8 Å (Kienberger et al., Single Molecules 2000, 1(2), 123-128). The 20.8 Å length of the SGGPPPPPP spacers was estimated based on the steps described in the literature (Mahoney et al., Nature Chemical Biology 1997, 4(12), 953-960; Garbuio et al. Chemistry: A European Journal 2015, 21(30), 10747-10753).

[0847] 9.3.2 Saturated FP binding test

[0848] Saturated fluorescence polarization (FP) measurements were performed in flat-bottomed 384-well Corning microplates (Merck) on a SpectraMax Paradigm multi-module detection platform (Molecular Devices) using excitation and emission wavelengths of 485 nm and 535 nm, respectively. The acquisition time was 700 ms, and the readout height was 1 mm. All reagents used in the assays were diluted in PBS containing 0.05% Tween 20.

[0849] The fluorescently labeled peptide Fc-III-FAM (structure shown below) was mixed with a series of IgG1 dilutions in PBS containing 0.05% Tween to obtain a final peptide concentration of 5 nM. The sample was incubated at 27°C for 15 minutes, and fluorescence anisotropy was measured in triplicate.

[0850]

[0851] Fc-III is a 13-mer cyclic peptide, and 13-mer cyclic peptides are known to have high affinity for the Fc region of IgG antibodies (DeLano et al., Science 2000, 287, 1279-1283; Nilsson et al., Protein Eng. 1987, 1, 107-113). Using standard SPPS technology and polymerization strategies, via GenScript... ® Prepare fluorescently labeled peptide Fc-III-FAM.

[0852] 9.3.3 Competitive FP Binding Test

[0853] Competitive FP measurements were performed in flat-bottomed 384-well Corning microplates (Merck) on a SpectraMax Paradigm multi-module detection platform (MegMolecule), using excitation and emission wavelengths of 485 nm and 535 nm, respectively. The acquisition time was 700 ms, and the readout height was 1 mm. All reagents used in the assays were diluted in PBS containing 0.05% Tween 20.

[0854] The increased concentration of the test peptide was mixed with Fc-III-FAM peptide and added to IgG1 in a total volume of 80 μL. The final concentration of Fc-III-FAM was kept constant at 5 nM, and the final concentration of IgG1 was 10–30 nM. The mixture was incubated at 27 °C for 15 min, and the fluorescence signal was red on Spectramax Paradigm. All sample preparations were performed in PBS at pH 7.4 or 7.0 containing 0.05% Tween. Each experiment was performed in triplicate.

[0855] 9.3.4 Determination of peptide and conjugate concentrations

[0856] Peptide samples were prepared by dissolving purified peptides or reactive conjugates in DMSO. Trp (ε=5500 M) was applied at 280 nm in 1xPBS pH 7.4. -1 cm -1 ) residues, p-SCN-Bn-CHX-A"-DTPA (ε=13000 M) -1 cm -1 p-SCN-Bn-PCTA (ε=13000 M) -1 cm -1 p-NCS-Bz-DFO (ε=21000 M) -1 cm -1 The absorbance of 496 nm or FITC at 496 nm (ε=73000 M) -1 cm -1 The concentration was determined by the absorbance of the sample.

[0857] 9.3.5 High-resolution mass spectrometry

[0858] Prior to HRMS analysis, the antibody-load conjugate was desalted using four concentration / dilution cycles on a microconcentrator (Vivaspin, 30kD cutoff, Sartorius, Germany) against a 50mM ammonium acetate solution buffered at pH 7.0. Deglycosylation of the conjugate was achieved by incubating 1 unit of Endo S per μg of conjugate in preparation buffer (37°C – 1 hour or overnight).

[0859] Direct injection HRMS for peptide / conjugate analysis was performed on a QExactive HFOrbitrap-FT-MS (Thermo Fisher Scientific, Germany) coupled to an automated chip-based nanoelectrospray device (Triversa Nanomate, Advion, USA). Electrospray ionization was performed at a capillary voltage of 1.4 kV and a nitrogen nanoflow of 0.15 psi. MS experiments were performed at a nominal resolution of 45,000 and in positive ion mode. Data deconvolution was performed using the Xtract algorithm with a 90% fit factor and Protein Deconvolution (Thermo Fischer Scientific, USA).

[0860] For both integral mass measurement (LC-MS) and middle-down analysis (LC-HCDMS / MS), a Dionex Ultimate 3000 analytical RSLC system (Dionex, Germany) coupled with a HESI source (Thermo Fisher Scientific, Germany) was used. Samples were separated on an Acquity UPLC protein column BEHC4 (300 Å, 1.7 μm, 1 × 150 mm, Waters, USA). Separation was performed by applying a gradient of solvent B from 15% to 45% over 2 min, followed by a gradient from 45% to 60% over 10 min, at a flow rate of 90 µL / min, followed by column washing and reequilibration. Solvent A consisted of water and 0.1% formic acid, while solvent B consisted of acetonitrile and 0.1% TFA.

[0861] Eluted protein forms were analyzed on a high-resolution QExactive HF-HT-Orbitrap-FTMS benchtop instrument (Thermo Fisher Scientific, Germany). For intact mass measurements (MS1), scans were performed in protein mode at a resolution of 15,000, and the average of 10 µs scans was taken. For Fc / 2-mod, moderate-to-moderate analysis of binding site localization was performed in PRM mode at 1356 m / z, with a 300 Th isolation window at a resolution of 240,000, and the average of 10 µs scans was taken. High-energy collision-induced dissociation (HCD) was used as the fragmentation method, with normalized collision energies of 12%, 15%, and 18%, respectively.

[0862] The complete quality measurement data was analyzed using Protein Deconvolution (Thermo Fisher Scientific, USA) with the Respect algorithm, which boasts a 99% noise suppression confidence level and an average quality identification accuracy of 20 ppm. Moderately low quality data were deconvolved using MASH Suite software (Ge research group, University of Wisconsin). Data obtained with three different NCE values ​​were combined using ProSight Lite software (Kelleher research group, Northwestern University) to create fragment plots of specified b- and y-fragments with a quality tolerance of 15 ppm.

[0863] 9.3.6 Determining Coupling Degree through HRMS Analysis

[0864] The average degree of conjugation (DoC) values ​​were calculated using HRMS data and Equation 1 (Eq. 1) below. These results are derived from the relative peak intensities in the deconvolution mass spectra.

[0865] Among them, I(DoC) k () represents the relative peak intensity of a conjugate with k additional molecules / antibodies.

[0866] 9.3.7 SDS-PAGE

[0867] Reducing or non-reducing Bis-Tris SDS-PAGE was performed on Bolt 4-12% Bis-Tris Plus Gels (Thermo Fisher Scientific, Germany). Loading buffer was added to the antibody conjugate (non-reducing Bolt sample buffer, Thermo Fisher Scientific), and the sample was heated at 70°C for 10 minutes. To reduce SDS-PAGE, reduction buffer was added to the sample before loading buffer. Electrophoresis was performed on the gel using Bolt MES electrophoresis buffer at a constant voltage (200 V) for 25–30 minutes. Fluorescence was visualized on a FluoroM bioimaging system (Syngene, UK) before Coomassie blue staining.

[0868] Example 1: Preparation and characteristics of Fc binding support

[0869] The Fc-binding support and support spacer constructs (partial V or SV of Formula (1)) as described herein were prepared using standard Fmoc / tBu type SPPS (including resin-on-resin coupling and polymerization strategies). The ligands prepared in Example 1 are shown in Table 1 below (bold underlines indicate disulfide bonds between the side chains of each Cys residue). As described above, the spacer length was calculated using a WLC model.

[0870]

[0871] Table 1: Fc-binding vectors (partial V / SV according to equation (1))

[0872] Peptides were prepared using standard Fmoc / tBu SPPS, Rink amide AM resin (loading: 0.57 mmol / g), and a Liberty Blue™ automated microwave peptide synthesizer (purchased from CEM Corp., Germany).

[0873] At room temperature, using 0.2M Fmoc amino acids pre-activated with 0.5M DIC and 1M OxymaPure ® The coupling reaction for amide bond formation was performed using DMF for more than 4 minutes. Fmoc deprotection was performed using 10% piperazine in DMF (v / v).

[0874] After synthesis, the peptides were manually lysed from the resin for 1.5 hours with gentle stirring at room temperature using a TFA / TIS / water (90 / 5 / 5, v / v / v) solution. After filtration and evaporation of the lysis mixture with a nitrogen stream, the crude peptides were precipitated with cold diethyl ether, centrifuged, and washed with cold diethyl ether. The peptides were dried, dissolved in ultrapure water (ACN), frozen, and lyophilized.

[0875] For disulfide bond formation, the crude lyophilized peptides were resuspended in a mixture of DMSO / ACN / water (2 / 3 / 3, v / v / v), and water was added until the peptides became soluble (approximately 35-50 mL). The resulting solution was adjusted to pH 8.5 using NH4HCO3 or NaHCO3 (concentration: 0.1-0.5 mM). The oxidation process was monitored by analytical UPLC-MS. After the reaction was complete, salts were removed using Sep-PakC18 Plus Long Cartridge (820 mg adsorbent per cartridge, particle size: 55-105 μm, purchased from Waters, Switzerland), and the peptides were lyophilized.

[0876] Using solvent systems A (0.1% TFA in water) and B (0.1% TFA in ACN), at a flow rate of 35 mL / min and a gradient of 15-55% B over 25 min, in Kinetex ® The peptide was purified by preparative reversed-phase HPLC on an XB-C18 column (100 Å, 5 µm, 100 × 21.2 mm; Phenomenex Helvetia). Peptide elution was monitored at 214 nm. Appropriate fractions were analyzed by UPLC-MS prior to concentration and lyophilization.

[0877] For the synthesis of compounds 3-12 and 15-16, a DMF solution of Fmoc-NH-(CH2-CH2-O)n-CH2-CH2-COOH (n=2, 4, 6, 8, 10, 12, 15, 20, 24 or 36; 1.3 equivalents, 4.7 µmol) and HATU (1.2 equivalents, 4.33 µmol) was stirred for 1 min, and DIEA (2 equivalents, 7.16 µmol) was added. After pre-activation for 3 min, DMF (1 equivalent, 3.58 μmol) of the Fc-binding peptide (compound 1, 13 or 14) was added to the reaction mixture and stirred at room temperature for 1–2 h. The reaction was monitored for completion by ULPC-MS. The peptide was then precipitated with cold diethyl ether. Fmoc deprotection was performed for 30 min at room temperature with 20% piperidine (v / v) in DMF, followed by precipitation of the peptide with cold diethyl ether. Figure 2 A). The peptide was isolated after HPLC purification (as described in the previous paragraph).

[0878] The purity of the peptides was determined using a solvent system on a Waters Acquity UPLC system equipped with a Kinetex. ® Coupled with a Micromass Quattro micro API mass spectrometer on an XB-C18 column (100 Å, 1.7 µm, 50 × 2.1 mm; Phenomenex Helvetia), the solvent system used solvent systems A (0.1% TFA in water) and B (0.1% TFA in ACN) at a flow rate of 0.6 mL / min and a B gradient of 2–98% over 4 min. Elution of the conjugates was monitored at 214 nm. The results are shown in the table below.

[0879]

[0880] Table 2: Characteristics of compounds 1-16

[0881] Example 2: Saturated FP binding test

[0882] In the above-described saturated FP binding assay, the tendency of the Fc-binding ligand Fc-III-FAM (structure shown above) to bind to the Fc-region of IgG1 antibodies (i.e., trastuzumab, alemtuzumab, bevacizumab, and rituximab) was evaluated. Results are as follows... Figure 3 As shown, Fc binds to the ligand Fc-III-FAM and binds to the corresponding antibodies (trastuzumab: 14 nM, alemtuzumab: 13 nM, bevacizumab: 7 nM, rituximab: 11 nM) with high affinity.

[0883] Example 3: Competitive FP Binding Experiment

[0884] In the competitive FP binding assays described above, the tendency of the Fc-binding ligands (compounds 1, 2, 9-11, 13, 15, and 16) prepared in Example 1 to bind to the Fc region of trastuzumab against Fc-III-FAM was evaluated. The results are shown in Table 3 below. Figure 4 As shown.

[0885]

[0886] Table 3: IC50 of Fc-binding ligand in competitive FP binding assay of trastuzumab against Fc-III-FAM 50 value

[0887] These results confirm that the Fc-binding ligands (compounds 1, 2, 9, 10, and 11) of Example 1 and Fc-III-FAM compete for the same binding site on the Fc region of trastuzumab. Furthermore, the results indicate that N-terminal modification of the Fc-III peptide (compound 1) with a spacer moiety (e.g., a peptide spacer such as Ser-(Gly)2-(Pro)6 or a polyethylene glycol spacer) does not affect the binding of the modified peptide to the Fc region of the antibody. In particular, compounds 2, 9, 10, and 11 resist Fc-III-FAM (… Figure 4 It showed high affinity for trastuzumab in competitive FP binding assays.

[0888] On the other hand, modifications to the C-terminal sequence of Fc-III in compounds 15 and 16 (i.e., replacing Val-Trp-Cys-Thr (VWCT) with Trp-Ala-Cys-Thr (WACT) or Val-Trp-Ala-Thr (VWAT)) affected peptide binding to the antibody. In the following description, compounds or conjugates with the modified C-terminal sequence (WACT or VWAT) are used as negative controls.

[0889] Example 4: Preparation of DOTA-, FL-, and DBCO-carbonate derivatives and FL-thioester derivatives—compounds 17, 18, 19, 20, 21, 22, and 23

[0890] Compounds 17, 18, 19, 20, 21, 22 and 23 (part of formula (1), PY) were prepared according to the following steps and shown to be... Figure 5 The structures of compounds 17-23 are shown in the table below.

[0891]

[0892] Table 4(1): DOTA-, FL-, DBCO- carbonate derivatives and FL-carbonate-naphthalene, FL-carbonate-isoquinoline, FL-thioester-CH 2 CH 2 The structure of the derivative (based on part of formula (1) PY)

[0893] Preparation of compound 17 :

[0894]

[0895] To a 70 mL acetonitrile solution of 2.0 g of 2-(2-Boc-aminoethoxy)ethanol (9.6 mmol), 5.2 g of N,N'-disuccinimidyl carbonate (19 mmol, 2.0 equivalent) was added, followed by 2.7 mL of triethylamine (19 mmol, 2.0 equivalent). The suspension was stirred at 40 °C for 1 hour and 30 minutes. The solvent was removed under vacuum. The residue was dissolved in DCM and filtered through a silica gel column, eluted with dichloromethane / ethyl acetate 80 / 20, to give crude 2-[2-(tert-butoxycarbonylamino)ethoxy]ethyl(2,5-dioxopyrrolidine-1-yl) carbonate (purity >80%, yield: 99%). LCMS: m / z = 247 [M-BOC+H] + 369[M+Na] + . 1 H NMR (CDCl3): δ4.52–4.40(m, 2H), 3.77–3.68(m, 2H), 3.55(t, 2H), 3.32(dd, 2H), 2.84(s, 4H), 1.44(s, 9H).

[0896] A 12 mL solution of 1.5 g of 2-[2-(tert-butoxycarbonylamino)ethoxy]ethyl(2,5-dioxopyrrolidone-1-yl) carbonate (3.4 mmol, 2.0 equivalents) was treated with 0.35 g of tert-butyl 4-hydroxybenzoate (1.7 mmol), followed by treatment with 0.43 g of 4-(dimethylamino)pyridine (3.4 mmol, 2 equivalents). The reaction mixture was stirred at room temperature for 30 minutes. 50 mL of water was added and the mixture was extracted with 3 × 10 mL of dichloromethane. The organic layer was concentrated under vacuum. The residue was purified by rapid chromatography (cyclohexane / ethyl acetate, 90 / 10 to 60 / 40) to give 0.64 g of tert-butyl-4-[2-[2-(tertbutoxycarbonylamino)ethoxy]ethoxycarbonyloxy]benzoate, a colorless oil (purity >98%, yield: 88%). LCMS: m / z = 326 [M-BOC+H]+, 448 [M+Na]+. 1H NMR (DMSO) δ 7.96 (d, 2H), 7.38(d, 2H), 6.83(s, 1H), 4.41–4.28(m, 2H), 3.75–3.60(m, 2H), 3.43(t, J=6.0 Hz, 2H), 3.09(q, 2H), 1.54(s, 9H), 1.37(s, 9H).

[0897] 2.1 mL of TFA (27 mmol, 17 equivalents) was added at 0 °C to 6.3 mL of 0.67 g of the above compound (1.5 mmol) in DCM solution, and the reaction mixture was stirred at room temperature for 3 h. The mixture was concentrated under vacuum to give 0.73 g of compound 4-[2-(2-aminoethoxy)ethoxycarbonyloxy]benzoic acid; 2,2,2-trifluoroacetic acid, a white solid (purity >80%, yield: 97%). LCMS: m / z = 270 [M+H]+. 1H NMR (DMSO) δ 8.01 (d, 1H), 7.87 (s, 2H), 7.37 (d, 2H), 4.38 (dd, 2H), 3.75 (dd, 2H), 3.65 (t, 2H), 3.06–2.97 (m, 2H).

[0898] Add 0.88 mL of DIEA (5.0 mmol, 6.0 eq) to 3.5 mL of a solution of 0.70 g of DOTA-tris(tBu) ester NHS ester (0.83 mmol), then add 0.44 g of 4-[2-(2-aminoethoxy)ethoxycarbonyl]benzoic acid (0.92 mmol, 1.1 equivalences), and stir the reaction mixture at room temperature for 10 minutes (Note: dissolved solids appear immediately after sonication). Dilute the solution in 3.5 mL of water and purify by rapid C18 column chromatography (water / ACN, 90 / 10 to 0 / 100). The fraction was collected, concentrated under vacuum, and lyophilized to give 0.66 g of compound 17 (4-[2-[2-[2-[4,7,10-tris(2-tert-butoxy-2-oxo-ethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethoxy]ethoxycarbonyloxy]benzoic acid), a white solid (purity >95%, yield: 93%). LCMS: m / z = 824 [M+H]+, 413 [M / 2+H]+. 1H NMR(DMSO) δ 8.56(s,1H), 7.95(d,2H), 7.27(d,2H), 4.31(s,2H), 3.66(s,2H), 3.48–3.42(m,2H), 3.35–3.25(m,8H), 3.00(s,2H), 2.75(s,8H), 2.63(s,4H), 1.37(s,27H).

[0899] Preparation of compound 18:

[0900]

[0901] 7.6 mL of 2-methylpropane-2-ol (80 mmol, 15 equivalents) was added to 14 mL of a toluene solution of 1.0 g of 5-hydroxy-2-nitrobenzoic acid (5.4 mmol), and the reaction mixture was heated at 85 °C. 4.5 mL of N,N-dimethylformamide dinepentyl acetal (16 mmol, 3.0 equivalents) was slowly added, and the reaction mixture was stirred at 85 °C for 3 h. The reaction was cooled, and then 10 mL of a saturated aqueous solution of NaHCO3 was added, followed by extraction of the aqueous layer with 3 × 5 mL of ethyl acetate. The combined organic layers were washed with 10 mL of water and concentrated under vacuum to give 1.1 g of crude tert-butyl 5-hydroxy-2-nitrobenzoate, a yellow oil (purity: 89%, yield: 73%). LCMS: m / z = 238 [MH]-. 1H NMR (DMSO): δ8.00(d,1H), 7.00(dd,1H), 6.92(d,1H), 1.50(s,9H).

[0902] A 5.0 mL solution of 0.35 g of crude tert-butyl 5-hydroxy-2-nitrobenzoate (1.3 mmol) in DCM was added to 0.90 g of 2-[2-(tert-butoxycarbonylamino)ethoxy]ethyl(2,5-dioxopyrrolidine-1-yl) carbonate (2.6 mmol, 2.0 equivalent; prepared as described above), followed by the addition of 0.46 mL of DIEA (2.6 mmol, 2.0 equivalent). The reaction mixture was stirred at room temperature for 30 minutes. The mixture was purified by rapid chromatography (cyclohexane / ethyl acetate, 90 / 10 to 40 / 60) to give 0.18 g of 5-((11,11-dimethyl-9-oxo-2,5,10-trioxa-8-azadodecanoyl)oxy)-2-nitrobenzoic acid, a yellow oil (purity: 99%, yield: 29%). LCMS: m / z = 315 [M-Boc-(t-Bu)+H]+, 371 [M-Boc+H]+, 493 [M+Na]+. 1H NMR (DMSO): δ8.15(d,1H), 7.77(d,1H), 7.69(dd,1H), 6.84(s,1H), 4.39–4.32(m,2H), 3.71–3.65(m,2H), 3.47–3.39(m,2H), 3.14–3.05(m,2H), 1.50(s,9H), 1.37(s,9H).

[0903] 0.17 g of 5-((11,11-dimethyl-9-oxo-2,5,10-trioxo-8-azadodecanoyl)oxy)-2-nitrobenzoic acid compound (0.37 mmol) was added to 1.8 mL of 0.60 mL of TFA (7.8 mmol, 21 equivalents), and the reaction mixture was stirred at room temperature for 2 hours. 0.30 mL of TFA was added, and the mixture was stirred at room temperature for 30 minutes. The solvent was evaporated under vacuum to give 0.23 g of crude 5-[2-(2-aminoethoxy)ethoxycarbonyloxy]-2-nitrobenzoic acid; 2,2,2-trifluoroacetic acid, a yellow oil (purity: 67%, yield: quantitative). LCMS: m / z = 315 [M+H]+. 1H NMR(DMSO): δ8.13(d,1H), 7.78(d,1H), 7.67(dd,1H), 4.46–4.35(m,2H), 3.01(q,2H).

[0904] Add 0.25 g of 2,2',2'-(2-(2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-tetraazacyclododecane-1,4,7,10-tri-tert-butyl 2,2',2''-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate to 1.3 mL of acetonitrile solution containing 0.22 g of 5-(11,11-dimethyl-9-oxo-2,5,10-trioxo-8-azacyclododecane-1,4,7-triyl)triacetate. (0.30 mmol), then add 0.31 mL of N,N-diisopropylethylamine (1.8 mmol, 6.0 equivalents), and stir the reaction mixture for 10 min at room temperature. Add 1.5 mL of water and purify the solution by C18 rapid chromatography (water / acetonitrile 95 / 5 to 0 / 1) to obtain 85 mg of compound 18 (2-nitro-5-[2-[2-[2-[4,7,10-tris(2-tert-butoxy-2-oxo-ethyl)-1,4,7,10-tetrazacyclododec-1-yl]acetyl]amino]ethoxy]ethoxycarbonyloxy]benzoic acid), a clear yellow solid (purity >80%, yield: 26%). LCMS: m / z=701[M-3(t-Bu)+H]+, 757[M-2(t-Bu)+H]+, 813[M-(t-Bu)+H]+, 869[M+H]+.1H NMR (DMSO): δ8.62(s,1H), 7.69(d,1H), 7.39(d,1H), 7.23(dd,1H), 4.38–4.31(m,2H), 3.70–3.67(m,2H), 1.42(s,6H), 1.41(s,27H).

[0905] Preparation of compound 19:

[0906]

[0907] Add 0.40 g of fluorescein isothiocyanate isomer (1.0 mmol) and 4.0 mL of dimethylformamide to 4.0 mL of a solution of 0.71 g of 4-[2-(2-aminoethoxy)ethoxycarbonyloxy]benzoic acid in ACN; 2,2,2-trifluoroacetic acid (1.2 mmol, 1.2 equivalents), then add 1.1 mL of N,N-diisopropylethylamine (6.0 mmol, 6.0 equivalents). Stir the mixture at room temperature for 10 minutes. Evaporate the solvent under vacuum. The residue was purified by C18 rapid chromatography (water / acetonitrile 95 / 5 to 0 / 1) to give 0.38 g of compound 19 (4-[2-[2-[(3',6'-dihydroxy-3-oxo-spiro[isobenzofuran-1,9'-xanthene]-5-yl)carbamothioylamino]ethoxy]ethoxycarbonyloxy]benzoic acid (4-[2-[2-[(3',6'-dihydroxy-3-oxo-spiro[isobenzofuran-1,9'-xanthene]-5-yl)carbamothioylamino]ethoxy]ethoxycarbonyloxy), orange solid) (purity: 98%, yield: 56%). LCMS: m / z = 657[MH]-, 659[M+H]+. 1H NMR (DMSO): δ13.07(s,1H), 10.24–9.95(m, 3H), 8.26(s,1H), 8.16(s,1H), 7.98(d,2H), 7.74(d,1H), 7.35(d,2H), 7.18(d,1H), 6.67(d,2H), 6.61–6.53(m,H), 4.42–4.37(m,2H), 3.80–3.66(m,6H).

[0908] Preparation of compound 20:

[0909]

[0910] At 0 °C, 100 mg (0.350 mmol, 1.0 equivalent) of 3-amino-1-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-1-propanone (100 mg, 0.350 mmol, 1.0 equivalent) was added dropwise to a dry DCM (1.20 mL) solution of 194 mg tri(ethylene glycol) bis(chloroformate) (0.690 mmol, 2.0 equivalent) and 0.070 mL DIEA (0.420 mmol, 1.2 equivalent) for more than 10 minutes. The reaction was stirred at room temperature. Ten minutes later, 0.3 mL of a dry DCM solution (1.20 mL) containing DIEA (1.73 mmol, 5.0 equivalent) and 336 mg of tert-butyl 4-hydroxybenzoate (1.73 mmol, 5.0 equivalent) were added to the reaction mixture. The reaction was stirred at room temperature for 30 minutes. Then, a saturated aqueous solution of ammonium chloride was added to the reaction mixture, and the mixture was extracted with DCM (2 × 5 mL). The combined organic extracts were dried over MgSO4. After filtration, the solvent was removed under vacuum, and the residue was purified by rapid chromatography (cyclohexane / ethyl acetate, 40 / 60 to 10 / 90) to give 67.6 mg of tert-butyl 4-[2-[2-[2-[3-(2-azatricyclo[10.4.0.04,9]hexadeca-1(12),4(9),5,7,13,15-hexen-10-yn-2-yl)-3-oxopropyl]carbamoyloxy]ethoxy]ethoxy]ethoxycarbonyloxy]tert-butyl benzoate. 1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl)-3-oxo-propyl]carbamoyloxy]ethoxy]ethoxy]ethoxycarbonyloxy]benzoate). (Purity: 80%, Yield: 23%). LCMS: m / z = 673.3 [M+H] + .

[0911] A 1:1 DCM / TFA solution of 67.6 mg of tert-butyl ester was stirred at room temperature for 5 hours. The solution was concentrated under reduced pressure, and the residue was treated with C... 18Purification by rapid chromatography (water / acetonitrile, modified with 0.1% TFA 80 / 20 to 20 / 80) yielded 40.3 mg of compound 20 (purity: 80%, yield: 59%). LCMS: m / z = 615 [MH]-, 617 [M+H]+. ¹H NMR (CDCl₃): δ 8.18–8.07 (m, 2H), 8.04–7.95 (m, 2H), 7.70–7.25 (m, 8H), 5.16 (s, 2H), 4.47–4.37 (m, 2H), 4.31–4.21 (m, 2H), 3.94–3.62 (m, 8H), 3.44–3.32 (m, 2H).

[0912] Preparation of compound 21:

[0913]

[0914] To a solution of 6-hydroxy-2-naphthoic acid (941 mg, 5.00 mmol) in 2-methyltetrahydrofuran (20.0 mL), 2-tert-butyl-1,3-diisopropylisourea (4.00 mL, 15.0 mmol) in 2-methyltetrahydrofuran (5.00 mL) was added. The reaction mixture was filtered through a silica stopper rinsed with ethyl acetate. The filtrate was washed with saturated aqueous NaHCO3 solution and brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by normal-phase chromatography (Biotage Isolera, 40 g, Silicyclesiliasep column) using heptane with 0–40% ethyl acetate to give the desired compound (715 mg, 59% yield, 99% purity) as an orange oil. ESI: m / z = 243 (MH) - . 1 H NMR (400 MHz, DMSO-d6)δ[ppm]= 1.59(s,9H), 7.14-7.21(m,2H), 7.75(d,1H), 7.82(dd,1H), 7.95(d,1H), 8.40(s,1H), 10.15(br s,1H).

[0915] 4-Dimethylaminopyridine (200 mg, 1.64 mmol) was added to dichloromethane (15.0 mL) containing tert-butyl 6-hydroxy-2-naphthoate (200 mg, 0.82 mmol) and (2-(2-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)ethoxy)ethyl)carbamate (567 mg, 1.64 mmol). The reaction mixture was stirred at ambient temperature for 2 h. The reaction mixture was washed with water, and the aqueous layer was washed with dichloromethane. The combined organic layers were concentrated under reduced pressure. The residue was purified by normal-phase chromatography (Biotage Isolera, 60 g, Silicycle siliasep column) using heptane with 10-90% ethyl acetate to give the title compound (190 mg, 49% yield, 98% purity) as a colorless solid. ESI: m / z = 498 (M+Na) + . 1 HNMR(400 MHz, DMSO-d6)δ[ppm]=1.38(s,9H), 1.61(s,9H), 3.11(q,2H), 3.46(t,2H),3.68-3.72(m,2H), 4.34-4.39(m,2H), 6.81-6.86(m,1H), 7.53(dd,1H), 7.90(d,1H),7.96-8.05(m,2H), 8.22(d,1H), 8.60(br s,1H).

[0916] tert-butyl 6-((11,11-dimethyl-9-oxo-2,5,10-trioxo-8-azadodecanoyl)oxy)-2-naphthoic acid ( tertA solution of butyl 6-((11,11-dimethyl-9-oxo-2,5,10-trioxa-8-azadodecanoyl)oxy)-2-naphthoate (190 mg, 0.400 mmol) in dichloromethane (10.0 mL) was added to trifluoroacetic acid (1.00 mL), and the reaction mixture was stirred at ambient temperature for 22 hours. The mixture was concentrated under reduced pressure. The residue was dissolved in N,N-dimethylformamide (2.00 mL) and acetonitrile (2.00 mL), followed by the addition of fluorescein isothiocyanate isomer 1 (204 mg, 0.520 mmol), and then DIPEA (343 μL, 1.97 mmol). The reaction mixture was stirred at room temperature for 90 minutes. The material was purified by reversed-phase chromatography (Biotage Isolera, 60 g, C18 SNAP Ultra Biotage column) using water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid (90:10 to 0:100). The fraction containing the product was lyophilized to give the desired compound (180 mg, yield 64%, purity 71%). ESI: m / z = 707 (MH) - . 1 H NMR (400 MHz, DMSO-d6)δ[ppm]=3.68-3.83(m,6H), 4.41-4.46(m,2H), 6.52-6.70(m,6H), 7.19(d,1H), 7.50(dd,1H), 7.75(d,1H), 7.88(d,1H), 8.01(s,2H), 8.14-8.24(m,2H), 8.28(d,1H),8.65(s,1H), 10.06(brs,1H), 10.11(br s,2H), 13.13(brs,1H).

[0917] N-hydroxysuccinimide (34.0 mg, 0.300 mmol) was added to a solution of 6-(((2-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-oxanthracene]-5-yl)thiourea)ethoxy)ethoxy)carbonyl)oxy)-2-naphthoic acid (70.0 mg, 0.099 mmol) in N,N-dimethylformamide (1.00 mL), followed by the addition of EDCI.HCl (57.0 mg, 0.300 mmol). The mixture was stirred at ambient temperature for 4 hours, and then purified on a 60 g C18 column using 5-95% acetonitrile (0.1% formic acid) and water (0.1% formic acid) as the elution buffer. The desired fractions were combined and lyophilized to give the title compound (55.0 mg, 69% yield, 95% purity). ESI: m / z = 806 (M+H) + . 1 H NMR (400 MHz, DMSO-d6)δ[ppm] = 2.94(s,4H), 3.68-3.84(m,6H), 4.42-4.48(m,2H), 6.51-6.63(m,4H), 6.66(d,2H), 7.19(d,1H), 7.61(dd,1H), 7.75(d,1H), 7.99(d,1H), 8.08(dd,1H), 8.16(d,2H), 8.28(d,1H), 8.34(d,1H), 8.91(s,1H), 10.00-10.15(m,3H).

[0918] Preparation of compound 22:

[0919]

[0920] 2-tert-butyl-1,3-diisopropylisourea (3.20 mL, 11.9 mmol) in tert-butanol (5.00 mL) was added to a solution of 6-hydroxyquinoline-2-carboxylic acid (750 mg, 3.96 mmol) in tert-butanol (40.0 mL). The reaction mixture was stirred at room temperature for 3 days. The reaction mixture was concentrated under reduced pressure, and the residue was suspended in ethyl acetate and filtered through a silica stopper rinsed with ethyl acetate. The filtrate was concentrated under reduced pressure and then purified by normal-phase chromatography (Biotage Isolera, 40 g, Silicyclesiliasep column) using heptane with 5-50% ethyl acetate to give the desired compound (378 mg, 39% yield, 95% purity) as an orange oil. ESI: m / z = 244 (MH) - . 1H NMR (400 MHz, DMSO-d6)δ[ppm]=1.60(s,9H),7.21(d,1H), 7.40(dd,1H), 7.94(d,1H), 7.99(d,1H), 8.27(d,1H), 10.42(br s1H).

[0921] To a solution of tert-butyl 6-hydroxyquinoline-2-carboxylate (200 mg, 0.820 mmol) and (2-(2-(2-(((((((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)ethoxy)ethyl)carbamate (706 mg, 2.04 mmol) in dichloromethane (15.0 mL), 4-dimethylaminopyridine (199 mg, 1.64 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was washed with water, and the aqueous layer was washed with dichloromethane. The combined organic layers were concentrated under reduced pressure. The residue was purified by normal-phase chromatography (Biotage Isolera, 40 g, Silicycle siliasep column) using heptane with 10-90% ethyl acetate to give the desired compound (278 mg, 71% yield, 94% purity) as a colorless oil. ESI: m / z = 499 (M+Na) + . 1 H NMR(400 MHz, DMSO-d6)δ[ppm]=1.38(s,9H), 1.62(s,9H), 3.07-3.16(m,2H),3.46(t,2H), 3.68-3.74(m,2H), 4.35-4.41(m,2H), 6.80-6.87(m,1H), 7.79(dd,1H),8.00(d,1H), 8.10(d,1H), 8.22(d,1H), 8.56(d,1H).

[0922] Trifluoroacetic acid (1.50 mL) was added to a solution of 6-((11,11-dimethyl-9-oxo-2,5,10-trioxo-8-azadodecanoyl)oxy)quinoline-2-carboxylic acid tert-butyl ester (290 mg, 0.61 mmol) in dichloromethane (5.00 mL), and the reaction mixture was stirred at room temperature for 26 hours. The mixture was concentrated under reduced pressure. The residue was dissolved in N,N-dimethylformamide (2.00 mL) and acetonitrile (2.00 mL), and then fluorescein isothiocyanate isomer 1 (237 mg, 0.610 mmol) was added, followed by DIPEA (530 µL, 3.04 mmol). The reaction mixture was stirred at room temperature for 2 hours. The material was purified by reversed-phase chromatography (Biotage Isolera, 60 g, C18 SNAP Ultra Biotage column) using water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid (90:10 to 100:0). The fraction containing the product was lyophilized to give the title compound (150 mg, yield 35%, purity 91%). ESI: m / z = 710 (M+H) + . 1 H NMR(400 MHz, DMSO-d6)δ[ppm]=3.68-3.83(m,6H), 4.41-4.46(m,2H), 6.52-6.69(m,6H), 7.18(d,1H), 7.72-7.80(m,2H),7.97(d,1H), 8.12-8.22(m,3H), 8.27(d,1H), 8.53(d,1H), 10.00-10.20(m,3H), 13.49(br s,1H).

[0923] N-hydroxysuccinimide (34.0 mg, 0.300 mmol) was added to a solution of 6-(((2-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-oxanthracene]-5-yl)thiourea)ethoxy)ethoxy)carbonyl)oxy)quinoline-2-carboxylic acid (70.0 mg, 0.099 mmol) in N,N-dimethylformamide (1.00 mL), followed by the addition of EDCI.HCl (57.0 mg, 0.300 mmol). The mixture was stirred at ambient temperature for 2 hours and then purified on a 60 g C18 column using 5-95% acetonitrile (0.1% formic acid) and water (0.1% formic acid) as the elution buffer. The desired fractions were combined and lyophilized to give the title compound (55.0 mg, 69% yield, 92% purity). ESI: m / z = 807 (M+H) + . 1H NMR(400 MHz, DMSO-d6)δ[ppm]=2.94(s,4H), 3.68-3.85(m,6H), 4.43-4.48(m,2H), 6.52-6.63(m,4H), 6.66(d,2H), 7.19(d,1H), 7.75(d,1H), 7.87(dd,1H), 8.09(d,1H), 8.13-8.21(m,1H), 8.25-8.33(m,3H), 8.72(d,1H), 10.00-10.13(m,3H).

[0924] Preparation of compound 23 :

[0925]

[0926] EDCI·HCl (296 mg, 1.54 mmol) was added to a solution of 3-(2-((tert-butoxycarbonyl)amino)ethoxy)propionic acid (300 mg, 1.29 mmol) in dichloromethane (3.00 mL), followed by the addition of 1-hydroxypyrrolidine-2,5-dione (177 mg, 1.54 mmol). The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with dichloromethane and washed with water. The organic layer was passed through a 15.0 mL Telos phase separation column and concentrated to give the desired product (313 mg, 79% purity), a colorless oil. It was used for the next step without further purification. ESI: m / z = 353 (M+Na) + , 231(M-Boc+H)+. 1 ¹H NMR (400MHz, DMSO-d⁶) δ[ppm] = 1.38(s, 9H), 2.82(s, 4H), 2.92(t, 2H), 3.04–3.09(m, 2H), 3.40(t, 2H), 3.69(t, 2H), 6.71–6.75(m, 1H). The NMR spectrum contains unknown impurities: 2.50 (t), 3.50 (t).

[0927] To a suspension of 2,5-dioxopyrrolidone-1-yl 3-(2-((tert-butoxycarbonyl)amino)ethoxy)propionate (200 mg, 0.606 mmol) in dichloromethane (4.00 mL), 4-mercaptohydrocinnamic acid (88.4 mg, 0.485 mmol) was added, followed by 4-dimethylaminopyridine (148 mg, 1.21 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was washed with 10% aqueous citric acid solution, followed by water. The organic layer was passed through a 15.0 mL Telos phase separation column, and the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase chromatography (Biotage Isolera, 30 g, C18 SNAP UltraBiotage column) using water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid (80:20 to 20:80). The appropriate fractions were freeze-dried to give the desired product (94.0 mg, 29% yield in two steps, 97% purity), a white solid. ESI: m / z = 298(M-Boc+H) + . 1 ¹H NMR (400 MHz, CDCl₃) δ [ppm] = 1.44 (s, 9H), 2.70 (t, 2H), 2.85–2.91 (m, 2H), 2.99 (t, 2H), 3.27–3.32 (m, 2H), 3.49 (t, 2H), 3.76 (t, 2H), 4.94 (br s, 1H), 7.27 (d, 2H) (overlapping with CHCl₃ peak), 7.36 (d, 2H).

[0928] Trifluoroacetic acid (0.59 mL, 7.70 mmol) was added to a solution of 3-(4-((3-(2-((tert-butoxycarbonyl)amino)ethoxy)propionyl)thio)phenyl)propionic acid (180 mg, 0.453 mmol) in dichloromethane (2.25 mL), and the reaction mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to give the desired product (205 mg, 80% purity), a pale yellow oil. It was used for the next step without purification. ESI: m / z = 298 (M+H) + . 1 H NMR(400 MHz, DMSO-d6)δ[ppm]=2.57(t,2H), 2.87(t,2H), 2.96-3.01(m,4H), 3.58(t,2H), 3.74(t,2H), 7.31-7.36(m,4H), 7.76(br s,3H).

[0929] To a solution of 3-(4-((3-(2-aminoethoxy)propionyl)thio)phenyl)propionate trifluoroacetate (maximum 0.453 mmol) in N,N-dimethylformamide (3.70 mL) and acetonitrile (3.70 mL), fluorescein isothiocyanate isomer 1 (176 mg, 0.453 mmol) was added, followed by DIPEA (0.12 mL, 0.680 mmol). The reaction mixture was stirred at room temperature for 1 hour, then concentrated under reduced pressure. The material was purified by reversed-phase chromatography (Biotage Isolera, 60 g, C18 SNAP UltraBiotage column) using water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid (95:5 to 20:80). The fraction containing the product was lyophilized to give the desired compound (145 mg, 51% yield in two steps, 68% purity) as an orange solid. ESI: m / z = 687 (M+H) + . 1 ¹H NMR (400 MHz, DMSO-d⁶) δ[ppm] = 2.55 (t, 2H) (overlapping with DMSO peak), 2.85 (t, 2H), 2.99 (t, 2H), 3.61 (t, 2H), 3.67-3.72 (m, 2H), 3.76 (t, 2H), 6.55-6.69 (m, 6H), 7.18 (d, 1H), 7.29-7.34 (m, 4H), 7.74 (d, 1H), 8.10 (br s, 1H), 8.26 (s, 1H), 10.05 (br s, 1H), 10.13 (br s, 2H), 12.16 (br s, 1H).

[0930] N-hydroxysuccinimide (117 mg, 1.02 mmol) was added to a solution of 3-(4-((3-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-oxanthracene]-5-yl)thiourea)ethoxy)propionyl)thio)phenyl)propionic acid (140 mg, 0.204 mmol) in N,N-dimethylformamide (4.70 mL), followed by the addition of EDCI.HCl (196 mg (1.02 mmol)). Stirring was continued for 1 hour at room temperature. The reaction mixture was purified directly by reversed-phase chromatography (Biotage Isolera, 60 g, C18 SNAP Ultra Biotage column) using water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid (80:20 to 30:70). The fraction containing the product was lyophilized to give the desired compound (25.3 g). mg (yield 16%, purity 81%), orange solid. ESI: m / z = 784 (M+H) +. 1 H NMR(400 MHz,DMSO-d6)δ[ppm]=2.81(s,4H), 2.96-3.06(m,6H), 3.62(t,2H), 3.68-3.71(m,2H), 3.76(t,2H), 6.56(dd,2H) ), 6.61(d,2H), 6.68(d,2H), 7.18(d,1H), 7.33(d,2H), 7.39(d,2H), 7.74(d,1H), 8.08(br s,1H), 8.26 (d,1H), 10.03(br s,1H), 10.13(br s,2H).

[0931] Example 5: Preparation of DOTA-containing reactive conjugates

[0932] By coupling compound 17 (or compound 19) to the N-terminus of each Fc-binding support, the Fc-binding supports prepared in Example 1 are converted into reactive conjugates of formula (1). Figure 2 B). The structures of the DOTA-containing reactive conjugates prepared in Example 5 are shown in the table below.

[0933]

[0934] Table 4(2): Structures of DOTA-containing reactive conjugates of formula (1)

[0935] To prepare the reactive conjugate, a DMF solution of the carbonate (ester) derivative (1.2 equivalents; compound 17) was added to HATU (1.1 equivalents) and stirred for 1 minute, followed by the addition of DIEA (2 equivalents). After 3 minutes, the pre-activated carbonate (ester) derivative was added to the Fc binding support, and the reaction mixture was stirred at room temperature for 2 to 4 hours. The completion of the reaction was monitored by UPLC-MS. If the reaction was not completed, an additional amount of the pre-activated carbonate (ester) derivative (approximately 1 to 3 equivalents) was added, and the mixture was stirred further for 1 to 2 hours. The reactive conjugate was precipitated with cold diethyl ether and purified by HPLC (as described above).

[0936] Subsequently, the tert-butyl protecting group of the DOTA moiety was removed by treatment with TFA / TIS / water (95 / 2.5 / 2.5, v / v / v) for more than 2.5 hours at room temperature, followed by precipitation with cold diethyl ether and purification by HPLC (as described above).

[0937] The purity of the reactive conjugates was determined using a solvent system on a Waters Acquity UPLC system equipped with Kinetex. ®Coupling was performed on a Micromass Quattro micro API mass spectrometer using an XB-C18 column (100 Å, 1.7 µm, 50 × 2.1 mm; Phenomenex Helvetia) with solvent systems A (0.1% TFA in water) and B (0.1% TFA in ACN), at a flow rate of 0.6 mL / min and a B gradient of 2–98% over 4 min. Elution of the couplings was monitored at 214 nm. The results are shown in the table below.

[0938]

[0939] Table 5: Characteristics of Reactive Couplings 24-34

[0940] Indium chelation in the DOTA fraction was performed by dissolving InCl3 in ultrapure water (1.5 equivalents, 14.2 nmol, 2 μL), mixing it with the above reactive conjugate (9.45 nmol, 5 μL) in 50 mM sodium acetate buffer (pH 5 (3 μL)), and incubating at 37°C for 5–30 minutes. In-chelation activity was monitored and analyzed by UPLC-MS.

[0941] Example 6: Preparation of trastuzumab-DOTA conjugate

[0942] Trastuzumab was used as a model system to evaluate the tendency of the reactive conjugate from Example 5 to react with the antibody. To prepare the trastuzumab-DOTA conjugate, DMF of two equivalents of the reactive conjugate prepared in Example 5 (compounds 24-33; 1.62 nmol, 0.86 μL) was added to trastuzumab (one equivalent, 0.81 nmol; commercially available trastuzumab Herceptin from Roche) diluted in 50 mM, pH 9.0 NaHCO3. ® Prior to coupling, the reaction mixture (24 μL) was buffered in phosphate-buffered saline (PBS) solution and stirred at room temperature for 2 hours.

[0943] After DOTA coupling, dilute the reaction buffer with 0.1 M glycine at pH 2.5 or use 30 kDa MWCOVivaspin. ® A 500-mesh centrifuge was used to exchange the elution solution for 0.1 M glycine at pH 2.5. The antibody-drug conjugate (ADC) was then purified by gel filtration chromatography using a pre-equilibrated Bio-spin P-30 column (bed height: 3.7 cm, total length: 5 cm; Bio-Rad, USA), followed by elution with 0.1 M glycine at pH 2.5. The purified ADC fraction was neutralized with 1 M PBS at pH 8.5.

[0944] The conjugation of DOTA moiety with trastuzumab was evaluated by HRMS analysis (as described above). An exemplary HRMS spectrum of the trastuzumab-DOTA conjugate prepared by reacting compound 31 with trastuzumab is shown in [image / data]. Figure 6 As shown in the image. The sample shows +517 Da adducts (D1-D3), which is characteristic of DOTA incorporation.

[0945] The effective loading ratio (selectivity) between Fc and F(ab)2 was assessed by digestion of the conjugate with Gingis Khan protease (1 unit per μg of antibody conjugate at 37°C for 1 hour in the presence of 2 mM cysteine, 0.1 M Tris, pH 8.0) and subsequent HRMS analysis (as described above). Exemplary HRMS spectra of the digested conjugate are shown. Figure 7 As shown in the figure, peaks D0-D2 correspond to the number of coupled DOTA portions, while G0F / G0F, G0F / G1F, and G1F / G1F correspond to different glycans in the Fc region.

[0946] The degree of conjugation (DoC) of the trastuzumab-DOTA conjugate was assessed based on the results of HRMS analysis (as described above). The HRMS analysis results are shown in Table 6 below.

[0947]

[0948] These results indicate that compounds (reactive conjugates) 27 to 33 can produce trastuzumab-DOTA conjugates with excellent selectivity for the Fc region of the antibody. In particular, compounds 27, 30, and 31 produced trastuzumab-DOTA conjugates with excellent selectivity and yield.

[0949] HRMS was used to analyze trastuzumab-DOTA conjugates via peptide mapping to determine the conjugation sites of the DOTA moiety on the antibody (data not shown). It was found that Lys317 in the Fc region was almost quantitatively labeled in most conjugates, while Lys326 labeling was additionally observed in conjugates with higher DOC, containing three DOTA moieties in each Fc region.

[0950] Example 7: Affinity of trastuzumab-DOTA conjugate and trastuzumab to SK-BR-3 (HER2+) and MD-MB-231 (HER2-) cells

[0951] The tendency of the trastuzumab-DOTA conjugate to bind to adenocarcinoma cells was assessed by measuring the affinity of the conjugate to SKBR-3 (HER2+) and MDA-MB-231 (HER2-) breast cancer cell lines. Specifically, the affinity of the trastuzumab-DOTA conjugate, prepared in the same manner as in Example 6 (similar to conjugate 8; DoC = 0.89), was measured by flow cytometry by incubating the trastuzumab-DOTA conjugate and (unlabeled) trastuzumab together with SKBR-3 or MDA-MB-231 cells. Subsequently, a trastuzumab-specific fluorescent secondary antibody was added to measure binding by fluorescence. Results were obtained in... Figure 8 As shown in the image.

[0952] like Figure 8 As shown, the median fluorescence intensity (MFI) increased in a dose-responsive manner when using unlabeled trastuzumab and the trastuzumab-DOTA conjugate, confirming that the DOTA conjugate did not affect antibody binding to SKBR-3 cells. The decreased mean fluorescence intensity of trastuzumab and its conjugate (SKBR-3 cells) at a concentration of 30 µg / mL could be explained by the high concentration of the primary antibody. No binding to MDA-MB-231 was observed in either sample (negative control).

[0953] Example 8: Preparation of FL-containing reactive conjugates

[0954] Following the same steps described in Example 5 above, the Fc-binding support prepared in Example 1 was converted into reactive conjugates (compounds 35-42) of formula (1) by coupling compound 19 to the N-terminus of each Fc-binding ligand. Figure 9 B). The FL-containing reactive conjugates prepared in Example 8 are shown in the table below.

[0955]

[0956] Table 7: Structures of FL-containing reactive conjugates of formula (1)

[0957] The purity of the reactive conjugates was determined by UPLC-MS (as described above). The results are shown in the table below.

[0958]

[0959] Table 8: Characteristics of reactive conjugates 35-42

[0960] Example 9: Preparation of trastuzumab-FL conjugate

[0961] Trastuzumab was used as a model system to evaluate the tendency of the reactive conjugate of Example 8 to react with the antibody. Trastuzumab-FL complexes were prepared using compounds 35-41 according to the same steps described in Example 6 above. The trastuzumab-FL conjugates were analyzed by SDS-PAGE. Figure 10 ).

[0962] Compounds 36-39 were found to induce effective trastuzumab labeling and good selectivity for the Fc region. Figure 10 Lanes 2-5 in the diagram). When compounds 40 and 41 were used (negative control; lanes 6 and 7), no trastuzumab labeling was observed.

[0963] To prepare trastuzumab-FITC (random) conjugates, 10 equivalents of FITC (0.47 μmol, 25.5 μL) of DMSO solution were added to trastuzumab (1 equivalent, 47 nmol; commercially available trastuzumab Herceptin purchased from Roche) diluted in 50 mM, pH 9.0 NaHCO3. ® The trastuzumab buffer was exchanged into phosphate-buffered saline (PBS) solution before conjugation, and the reaction mixture (1.4 mL) was stirred at room temperature for 16 hours.

[0964] Following FITC conjugation, the reaction buffer was diluted with 0.1 M glycine at pH 2.5. The antibody-conjugate was then purified by gel filtration chromatography using a pre-equilibrated column manually packed with Bio-spin P-30 beads (bed height: 5.0 cm), followed by elution with 0.1 M glycine at pH 2.5. The purified antibody-conjugate fraction was neutralized with 1 M phosphate buffer at pH 8.5.

[0965] The conjugation of this fraction with trastuzumab was assessed by HRMS analysis (as described above). The HRMS analysis results for trastuzumab-FITC and trastuzumab-FI are shown in Table 9 below.

[0966]

[0967] Table 9: Characteristics of the trastuzumab-FI conjugate and trastuzumab-FITC conjugate prepared in Example 9

[0968] Example 10: Affinity of trastuzumab-FL conjugate and trastuzumab-FITC conjugate (11, 12) to BT-474 (HER2+) and MDA-MB33 (HER2-) cells.

[0969] The binding affinity of trastuzumab-FL and trastuzumab-FITC conjugates to adenocarcinoma cells was assessed by measuring the affinity of the conjugates for BT-474 (HER2+) and MDA-MB33 (HER2-) breast cancer cell lines. Specifically, the affinity of the trastuzumab-FL conjugate, trastuzumab-FITC conjugate, and (unlabeled) trastuzumab, prepared as described in Example 9, was measured by flow cytometry using trastuzumab-FITC conjugates prepared as described in Example 9 (MS data are shown in Table 9).

[0970] like Figure 11 As shown, the mean fluorescence index (MFI) of both FITC-conjugated and FL-conjugated antibodies decreased in a dose-response manner after the addition of unlabeled trastuzumab (competitive antibody). A nearly 50% decrease in MFI was observed for conjugate 11, while for conjugate 12, the MFI decreased by nearly 70% at equimolar concentrations of labeled and unlabeled antibody (10 μg / ml). These results indicate that the fluorescein conjugates do not affect antibody binding to HER2 cells, while the random labeling of conjugate 12 affects antibody affinity. No binding to MDA-MB33 was observed in either sample (negative control, data not shown).

[0971] Example 11: Preparation of antibody-FL conjugates using trastuzumab, commercial trastuzumab, alemtuzumab, bevacizumab, and rituximab

[0972] Using trastuzumab, the commercial trastuzumab (Herceptin) ® The reactive conjugates of the present invention were evaluated for their tendency to react with different antibodies, including alenmab, bevacizumab, and rituximab. Antibody-FL conjugates were prepared using compound 38 and the aforementioned antibodies according to the same steps described in Example 6 above. The conjugates were analyzed by SDS-PAGE. Figure 12 ).

[0973] Compound 38 was found to induce effective antibody labeling. Figure 12 Lanes 1, 3, 5, 7, and 9 in the diagram). Trastuzumab markers were not observed when compound 40 was used (lanes 2, 4, 6, 8, and 10).

[0974] Example 12: Preparation of reactive conjugates containing DBCO and trastuzumab-DBCO conjugates

[0975] By coupling compound 20 to the N-terminus of each Fc-binding ligand, the Fc-binding ligand (compound 10) prepared in Example 1 was converted into the corresponding reactive conjugate of formula (1). The structures of the DBCO-containing reactive conjugates prepared in Example 12 are shown in the table below.

[0976]

[0977] Table 10: Structures of DBCO-containing reactive conjugates of formula (1)

[0978] The purity of the reactive conjugates was determined by UPLC-M (as described above). The results are shown in the table below.

[0979]

[0980] Table 11: Characteristics of reactive conjugate 43

[0981] Trastuzumab-DBCO conjugates were prepared using compound 43 and commercial trastuzumab (Herceptin®) following the same steps described in Example 6 above. The conjugates were digested with Gingis Khan and analyzed by HRMS as described above. The results are shown in the table below.

[0982]

[0983] Table 12: Characteristics of the trastuzumab-DBCO conjugate prepared in Example 12

[0984] Example 13: Preparation of immobilized FL-containing reactive conjugates and solid-phase modification of trastuzumab

[0985] A reactive conjugate immobilized on a solid support was prepared, and the tendency of the reactive conjugate to react with trastuzumab was evaluated.

[0986] Using 4-Fmoc-hydrazinobenzoyl AM NovaGel™ (0.61 mmol / g loading) and Liberty Blue TM An automated microwave peptide synthesizer (purchased from CEM GmbH, Germany) was used to prepare biotinylated Fc-binding carriers via standard Fmoc / tBu-type SPPS. At room temperature, 0.2 M Fmoc amino acids pre-activated with 0.5 M DIC and 1 M OxymaPure were used. ® The coupling reaction for amide bond formation was performed using DMF for more than 4 minutes. Fmoc deprotection was performed using 10% piperazine in DMF (v / v).

[0987] After synthesis, the peptide was manually cleaved from the resin by resuspending it in DMF and mixing it with 1.4 equivalents of CuII(AcO)2*H2O, 3.5 equivalents of biotin-PEG4-NH2, and 3 equivalents of pyridine. The reaction was stirred at room temperature for 4 hours. The cleavage mixture was filtered, and the peptide was precipitated with water and filtered. The precipitate was dissolved in the cleavage mixture (TFA / TIS / water 90:5:5), and the side chains of the peptide were deprotected by stirring at room temperature for 2 hours. The mixture was concentrated, and the crude peptide (compound 41) was precipitated with cold diethyl ether, centrifuged, washed with cold diethyl ether, dried, dissolved in ultrapure water / acetonitrile, lyophilized, and purified by HPLC.

[0988] Fmoc-NH-(PEG) 20 A DMF solution of -COOH (1.3 equivalents, 4.7 µmol) and HATU (1.2 equivalents, 4.33 µmol) was stirred for 1 min, and DIEA (10 equivalents, 35.8 µmol) was added. After pre-activation for 3 min, a DMF solution of biotinylated Fc-binding peptide (compound 44) ​​(1 equivalent, 3.58 µmol) was added to the reaction mixture and stirred at room temperature for 1–2 h to prepare compound 45. The reaction was monitored for completion by ULPC-MS. The peptide was then precipitated with cold diethyl ether. Fmoc deprotection was performed with 20% piperidine in DMF (v / v) at room temperature for 30 min, followed by precipitation of the peptide with cold diethyl ether and purification by HPLC.

[0989] Following the same steps described in Example 5 above, the biotinylated Fc-binding ligand was converted into a reactive conjugate (compound 46) by coupling compound 19 to the N-terminus of compound 45. The structures of the compounds prepared in Example 13 are shown in the table below.

[0990]

[0991] Table 13: Structures of the compounds prepared in Example 13

[0992] To immobilize the biotinylated reaction conjugate on a solid support, NeutrAvidin agarose resin (Thermo Fisher Scientific) was packed into a column (Thermo Fisher Scientific) and washed with binding buffer (0.1 M phosphate buffer, 0.15 M sodium chloride, pH 7.2). Compound 46 (2.1 nmol) was incubated with washed NeutrAvidin agarose beads (40 μl beads: 7.5 μg peptide) at room temperature for 30 min. Figure 13 ).

[0993] The beads were washed four times with binding buffer, and then 50 mM Bicine (pH 9.0) was added to increase the pH. Trastuzumab in PBS (2.1 nmol) at pH 7.0 was added to the beads, and the mixture was stirred at room temperature for 2 hours, followed by washing 3–4 times with binding buffer. Labeled trastuzumab (100 μl, 0.1 M glycine, pH 2.5) was eluted at a 1:10 volume ratio into a collection tube containing neutralization buffer (1 M, pH 8.5 phosphate buffer). The elution steps were repeated, and the fractions were combined. The beads were then treated with 30 kDa MWCO Vivaspin. ® A 500-centrifuge concentrator was used to buffer the eluted labeled trastuzumab with PBS at pH 7.0.

[0994] The antibody was then analyzed by SDS-PAGE. The gel showed a fluorescent band, indicating that the FL moiety was successfully conjugated with trastuzumab.

[0995] Example 14: Preparation of other reactive conjugates containing effectively loaded carbonates (esters)

[0996] By coupling different effective loads (DTPA, PCTA, DFO) to NH2-carbonate (ester)-PEG 10 -Fc-III converts the Fc-binding support prepared in Example 1 into reactive conjugates of formula (1) (compounds 47-49). The structures of these reactive conjugates containing the effective load are shown in the table below.

[0997]

[0998] Table 14: Structures of other reactive conjugates of formula (1) containing an effective loaded carbonate (ester).

[0999] The purity of the reactive conjugates was determined by UPLC-MS (as described above). The results are shown in the table below.

[1000]

[1001] Table 15: Characteristics of peptide reactive conjugates

[1002] NH2-carbonate (ester)-PEG 10 Preparation of -Fc-III:

[1003]

[1004] Step 1. At room temperature, DIEA was added to a DMF (0.65 mL) solution of 4-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxycarbonyloxy]benzoic acid (2.35 mg, 6.4 mol, 1.3 equivalents). After stirring for 1 minute at room temperature, HATU.HPF6 (2.81 mg, 5.4 mol, 1.1 equivalents) was added to the reaction mixture. After stirring for 3 minutes at room temperature, a DMF (0.65 mL) solution of compound 7 (10.0 mg, 4.9 μmol, 1.0 equivalents) was added to the reaction mixture. After stirring for 18 hours at room temperature, 2 drops of an aqueous solution of 0.1% TFA were added. The mixture was purified on a C18 plate (12 g, 30-70% ACN + 0.1% TFA in water + 12 CV + 0.1% TFA), and lyophilized to give BocHN-carbonate (ester)-PEG. 10 -Fc-III (2.4 mg, 1.0 μmol, UV purity 95%, 20% yield), white powder. UPLC-MS: Rt = 2.78 min, m / z = 1147 [M-Boc+2H] 2+ 1195 [M-2H] 2- .

[1005] Step 2. Add TFA to BocHN-carb-PEG 10 FcIII (23.9 mg, 8.3 μmol, 1.0 equivalent) was added to a DCM solution (0.5 mL). The reaction mixture was stirred at room temperature for 1.5 h, and then concentrated under vacuum. A mixture of ACN / water (1:1, 5 mL) was added, and the mixture was freeze-dried to give H2N-carbonate (ester)-PEG. 10 -Fc-III (23.7 mg, 8.3 μmol, UV purity 99%, quantitative yield), white powder. UPLC-MS: Rt = 2.20 min, m / z = 1147 [M + 2H] 2+ 1145 [M-2H] 2- .

[1006] DTPA-carbonate (ester)-PEG 10 Preparation of -Fc-III:

[1007] p-SCN-Bn-CHX-A”-DTPA.3HCl (4.47 mg, 6.0 μmol, 1.0 equivalent) was added to NH2-carbonate (ester)-PEG at room temperature. 10-Fc-III (14.35 mg, 6.0 μmol, 1.0 equivalent) was added to a DMF (0.3 mL) solution. The reaction mixture was stirred at room temperature for 5 min, and then triethylamine (4.0 μL, 30.0 μmol, 5.0 equivalent) was added. After stirring at room temperature for 36 h, p-SCN-Bn-CHX-A”-DTPA.3HCl (0.90 mg, 1.2 μmol, 0.2 equivalent) and triethylamine (0.5 μL, 3.6 μmol, 0.6 equivalent) were added, and the reaction mixture was stirred at room temperature for 18 h. The solution was purified by preparative HPLC (30-60% ACN + 0.1% FA in water + 0.1% FA) and lyophilized to obtain DTPA-carbonate (ester)-PEG. 10 -Fc-III (1.4 mg, 0.52 μmol, 8.7% yield), white powder.

[1008] PCTA-carbonate (ester)-PEG 10 Preparation of -Fc-III:

[1009] p-SCN-Bn-PCTA.3HCl (4.15 mg, 6.5 μmol, 1.05 equivalents) was added to NH2-carbonate (ester)-PEG at room temperature. 10 -Fc-III (14.9 mg, 6.2 μmol, 1.0 equivalent) was added to a DMF (0.1 mL) solution. The reaction mixture was stirred at room temperature for 5 minutes, and then triethylamine (4.2 μL, 30.0 μmol, 5.0 equivalent) was added. After stirring at room temperature for 3 hours, 1p-SCN-Bn-PCTA.3HCl (4.15 mg, 6.5 μmol, 1.05 equivalent) was added to the reaction mixture at room temperature. After stirring at room temperature for 16 hours, the mixture was purified by preparative HPLC (28-37% ACN + 0.1% TFA in water + 0.1% TFA), and lyophilized to obtain PCTA-carbonate (ester)-PEG. 10 -Fc-III (2.53 mg, 8.97 μmol, 14% yield), white powder.

[1010] DFO-carbonate (ester)-PEG 10 Preparation of -Fc-III:

[1011] At room temperature, DIEA (10 μL, 80.0 mol, 16.0 equivalents) was added to NH2-carbonate (ester)-PEG. 10A DMF (0.4 mL) solution of -Fc-III (12.42 mg, 4.9 μmol, 1.0 equivalent) and DFO-NHS (8.2 mg, 5.9 μmol, 1.2 equivalent) was added. After stirring at room temperature for 3.5 h, ACN / water / TFA (1:1:0.5%, 0.2 mL) was added, and the reaction mixture was stirred at room temperature for 5 min. The mixture was purified by preparative HPLC (25-60% ACN + 0.1% FA in water + 0.1% FA), and then lyophilized to obtain DFO-carbonate (ester)-PEG. 10 -Fc-III (1.6 mg, 0.45 μmol, UV purity 86%, 9% yield), white powder.

[1012] Example 15: Preparation of trastuzumab-DTPA / PCTA / DFO conjugate

[1013] The tendency of the reactive conjugate from Example 14 to react with the antibody was evaluated using trastuzumab. The trastuzumab DTPA / PCTA / DFO conjugate was prepared using compounds 47-49 according to the same steps described in Example 6 above. The trastuzumab DTPA / PCTA / DFO conjugate was analyzed by HRMS (Table 16).

[1014]

[1015] Table 16: Characteristics of Trastuzumab-DTPA / PCTA / DFO Conjugate

[1016] Will* Numerical extrapolation: Selectivity Fc / F(ab)2 = (DoC mAb – DoC F(ab)2) / Doc(Fab)2

[1017] Example 16: Preparation of FL-containing reactive conjugates with different chemical properties or reactivity modifiers

[1018] The Fc-binding support prepared in Example 1 was converted into a reactive conjugate by coupling FL-naphthalene carbonate / -isoquinoline carbonate / -CH2CH2 thioester (compounds 21-23) to the N-terminus of Fc-binding ligand 7 according to the following steps. The structures of these reactive conjugates containing the effective load prepared in Example 16 are shown in the table below.

[1019]

[1020] Table 17: Structures of Fl-containing reactive conjugates with different chemical properties or reactivity modifiers

[1021] Preparation of compound 50 (naphthalene):

[1022] At ambient temperature, DIPEA (10.0 μL, 0.057 mmol) was added to a solution of 2,5-dioxopyrrolidone-1-yl-6-(((2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-oxanthracene]-5-yl)thiourea)ethoxy)ethoxy)carbonyl)oxy)-2-naphthol (19.0 mg, 0.023 mmol) and compound 7 (40.0 mg, 0.019 mmol) in N,N-dimethylformamide (1.00 mL). The mixture was stirred for 3 hours and then purified on a 60 g C18 column using water (0.1% formic acid) elution buffer in 5-95% acetonitrile (0.1% formic acid). The desired fractions were combined and lyophilized. The obtained material was mixed with 2,5-dioxopyrrolidone-1-yl6-(((2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-oxanthracene]-5-yl)thiourea)ethoxy)ethoxy)carbonyl)oxy)-2-naphthol (11.0 mg, 0.014 mmol) and TFA.PEG. 10 Similar batches obtained from the reaction of -FcIII (30.0 mg, 0.014 mmol) and DIPEA (7.00 μL, 0.042 mmol) in N,N-dimethylformamide (1.00 mL) were combined and further purified on a 60 g C18 column with 20-60% acetonitrile (0.1% formic acid) and water (0.1% formic acid) as eluent. The desired fractions were combined and lyophilized to give the title compound (11.9 mg, 13% combined yield, 95% purity). UPLC4-MS: Rt = 1.95 min., 94.5%. ESI: m / z = 911.8 [M+3H] / 3 + .

[1023] Preparation of compound 51 (isoquinoline) :

[1024] To 2,5-dioxopyrrolidone-1-yl 6-(((2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-oxanthracene]-5-yl)thiourea)ethoxy)ethoxy)carbonyl)oxy)quinoline-2-carboxylate (ester) (11.0 mg, 0.014 mmol) and TFA.PEG 10-FcIII (30.0 mg, 0.014 mmol) was added to a solution of N,N-dimethylformamide (1.00 mL) with DIPEA (7.00 μL, 0.042 mmol). The mixture was stirred for 2 hours and then purified on a 60 g C18 column using 20-60% acetonitrile (0.1% formic acid) and water (0.1% formic acid) as eluent. The desired fractions were combined and lyophilized to give the title compound (8.70 mg, 23% yield, 93% purity) as a yellow powder. UPLC-MS: Rt = 1.93 min. ESI: m / z = 912.0 [M+3H] / 3+ .

[1025] Preparation of compound 52 (thioester) :

[1026] DIPEA (8.52 μL, 0.0489 mmol) was added to a solution of 2,5-dioxopyrrolidone-1-yl 3-(4-((3-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-oxanthracene]-5-yl)thiourea)ethoxy)propionyl)thio)phenyl)propionate (ester) (12.8 mg, 0.0163 mmol) and compound 7 (21.1 mg, 0.00980 mmol) in N,N-dimethylformamide (1.00 mL). The mixture was stirred for 2 hours at room temperature. The reaction was purified directly by reversed-phase chromatography (Biotage Isola, 60 g, C18 SNAP Ultra Biotage cartridge) using water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid (80:20 to 30:70). The fraction containing the product was freeze-dried to give the desired compound (4.88 mg, yield 4%, purity 87%) as a yellow solid. UPLC-MS: Rt = 1.67 min. ESI: m / z = 904[M+3H] / 3 + .

[1027] Example 17: Preparation of trastuzumab-FL conjugate

[1028] The tendency of the reactive conjugate from Example 16 to react with the antibody was evaluated using trastuzumab. Trastuzumab-FL conjugates were prepared using compounds 50-52, following the same steps described in Example 6 above. The obtained trastuzumab-FL conjugates were analyzed by HRMS (Table 18).

[1029]

[1030] Table 18: Characteristics of trastuzumab-Fl conjugates. sequence list <110> Debiao Pharmaceutical International Co., Ltd. <120> Reactive conjugates <130> 228970 <160> 3 <170> BiSSAP 1.3.6 <210> 1 <211> 14 <212> PRT <213> Artificial sequence <220> <223> The peptide portion of the compound as shown in formula (8a) of this application <220> <221> MOD_RES <222> (1)..(1) <223> As defined in Project 1, P-Y-S <220> <221> variants <222> (1)..(1) <223> This refers to an amino acid, a dicarboxylic acid, or a peptide portion represented by formula (9a) on page 16 of this application. <220> <221> disulfides <222> (2)..(12) <223> <220> <221> variants <222> (4) (8) <223> Each represents an amino acid independently. <220> <221> variants <222> (13)..(13) <223> This refers to an amino acid or a peptide portion represented by formula (9b) on page 17 of this application. <220> <221> variants <222> (14)..(14) <223> This refers to a single covalent bond or a trifunctional amino acid, such as diaminocarboxylic acid. <220> <221> MOD_RES <222> (14)..(14) <223> Other groups or atoms, Z1 and Y' as described on pages 17-18 of this application <400> 1 Xaa Cys Ala Xaa Xaa Xaa Xaa Xaa Leu Val Trp Cys Xaa Xaa 1 5 10 <210> 2 <211> 14 <212> PRT <213> Artificial sequence <220> <223> peptide portion of the compound <220> <221> variants <222> (1)..(1) <223> This refers to a single covalent bond or a trifunctional amino acid, such as diaminocarboxylic acid. <220> <221> MOD_RES <222> (1)..(1) <223> Other groups or atoms, Z2 and Y' as described on pages 17-18 of this application <220> <221> variants <222> (2)..(2) <223> This refers to an amino acid, a dicarboxylic acid, or a peptide moiety represented by formula (9a) on page 17 of this application. <220> <221> disulfides <222> (3)..(13) <223> <220> <221> variants <222> (5)..(9) <223> Each represents an amino acid independently. <220> <221> variants <222> (14)..(14) <223> This refers to an amino acid or a peptide portion represented by formula (9b) on page 17 of this application. <220> <221> MOD_RES <222> (14)..(14) <223> As defined in Project 1, P-Y-S <400> 2 Xaa Xaa Cys Ala Xaa Xaa Xaa Xaa Xaa Leu Val Trp Cys Xaa 1 5 10 <210> 3 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Fc-III-FAM as shown on page 128 of this application <220> <221> disulfides <222> (2)..(12) <223> <220> <221> MOD_RES <222> (13)..(13) <223> The FAM mark is shown on page 128 of this application. <400> 3 Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10

Claims

1. A compound represented by the following formula (1): PYSV (1) in, P represents the payload; Y represents the reactive portion that can react with the side chain of an amino acid; V is a carrier that can interact with the Fc region of a crystallizable fragment of an antibody or antibody fragment; S is a spacer of length Z, where Z is the length such that when the carrier V interacts with the crystallizable fragment Fc region of the antibody or antibody fragment, the reactive portion Y is able to react with the side chain of amino acid residues on the antibody or antibody fragment. The reactive component is represented by one of the following formulas: (6a) (6b) (6c) (6d) (6e) (6j) (6l) (6m) (6n) (6t) (6v) (6y) (6a') (6j') (6k') (6l') in, Refers to covalent attachment to spacer S, and Refers to covalent attachment to the payload P; and Wherein, the spacer is (a2) Groups represented by the following formula (7): –X 1 –(CH2CH2O) n2 –CH2CH2–X 2 –(7) in, X 1 It is NH, O, or S; X 2 It is NH or C=O; and n2 is an integer between 6 and 28; or (b2) A peptide group with 6 to 25 amino acids in the main chain, each amino acid being selected from the group consisting of: Pro, Gly, and Ser; The carrier is a peptide represented by one of the following formulas (8a) and (8b): Axx represents Asp; Bxx represents Trp; Cxx stands for His; Dxx stands for Leu; Exx is Gly; Fxx stands for Glu; Gxx represents Thr; Hxx represents a single covalent bond or a trifunctional amino acid; the trifunctional amino acid is selected from the group consisting of: 2,3-diamino-propionic acid Dap, 2,4-diamino-butyric acid Dab, Lys, guanylic acid Orn, and high lysine. Z1 indicates If Hxx is a single covalent bond, then Z1 represents a group covalently bonded to the C-terminus of Gxx, the group being selected from: -N(H)(R), where R represents a hydrogen atom, an alkyl or cycloalkyl group, or a portion of a compound containing a bonding group, the portion of the compound containing the bonding group being selected from biotin, dibenzocyclooctyne DBCO, trans-cyclooctene TCO, bicyclo[6.1.0]nonyne BCN, alkynes, azides, bromoacetamide, maleimide, and thiols; If Hxx is a trifunctional amino acid and Y' is bound to the side chain of Hxx, then Z1 represents a group covalently bound to the C-terminus of Hxx, wherein the group is selected from -N(H)(R), where R represents a hydrogen atom, an alkyl group, or a cycloalkyl group; or If Hxx is a trifunctional amino acid and Y' is bound to the C-terminus of Hxx, then Z1 represents the hydrogen atom bound to the side chain of Hxx; Z2 indicates If Hxx is a single covalent bond, then Z2 represents a group covalently bonded to the N-terminus of Axx, the group being selected from hydrogen atoms, carbonyl groups, or portions of compounds containing bonding groups, the portions of compounds containing bonding groups being selected from biotin, DBCO, TCO, BCN, alkynes, azides, bromoacetamide, maleimide, and thiols. If Hxx is a trifunctional amino acid and Y' is bound to the side chain of Hxx, then Z2 represents a group covalently bound to the N-terminus of Hxx, said group being selected from hydrogen atoms or carbonyl groups; or If Hxx is a trifunctional amino acid and Y' is bound to the N-terminus of Hxx, then Z2 represents a hydrogen atom bound to the side chain of Hxx; If Hxx is a trifunctional amino acid, then only Y' exists; and If Z1 binds to the C-terminus of Hxx, or if Z2 binds to the N-terminus of Hxx, then Y' represents the portion covalently bound to the side chain of Hxx. If Z1 is bound to the side chain of Hxx, then Y' represents the portion covalently bound to the C-terminus of Hxx, or If Z2 is bound to the side chain of Hxx, then Y' represents the portion covalently bound to the N-terminus of Hxx; Y' is selected from compounds containing a binding group, wherein the binding group is selected from biotin, DBCO, TCO, BCN, alkynes, azides, bromoacetamide, maleimide or thiols; X 3 It indicates a single covalent bond or a divalent group containing one or more atoms selected from carbon, nitrogen, and oxygen; Refers to covalent attachment to spacer S.

2. The compound according to claim 1, wherein, The antibody fragment is incorporated into the Fc-fusion protein.

3. The compound according to claim 1 or 2, wherein, The payload includes one of the following: (i) The marking portion includes a radioactive nuclide. Color clusters; or Fluorescein; (ii) A portion comprising a bonding group, said portion comprising a conjugated diene, tetraazine (TZ), alkyne or azide, DBCO, TCO, BCN; or (iii) Selected from the following groups: Antitumor agents; RNA polymerase II inhibitor; Antimetabolites; kinase inhibitors; Immunomodulators; or Anti-infective agents.

4. The compound according to claim 3, wherein, The antitumor agents include topoisomerase inhibitors, DNA cleavage agents, or antimitotic agents.

5. The compound according to claim 1 or 2, wherein, The effective load is a chelating agent.

6. The compound according to claim 5, wherein, The chelating agent chelates radionuclides.

7. The compound according to claim 5, wherein, The chelating agent is selected from the group consisting of: diethylenetriaminepentaacetic acid (DTPA), cyclohexyldiethylenetriaminepentaacetic acid (CH-X-DTPA), deferoxamine (DFO), 1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-tetraacetic acid (NODAGA), 1,4,7,10-tetraazacyclododecane-1-pentanoic acid-4,7,10-triacetic acid (DOTAGA), and 2,2'-(1,4,7-triazacyclononane) 1,4-Diyl)diacetate NO2A, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid DOTA, 1,4,7-triazacyclononane-1,4,7-triacetic acid NOTA, ethylenediaminetetraacetic acid EDTA, ethylenediaminediacetic acid, triethylenetetraminehexaacetic acid TTHA, 1,4,8,11-tetraazacyclotetradecane CYCLAM, 1,4,8,11-tetraazacyclotetradecane-1,4 ,8,11-Tetraacetic acid TETA, 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid CB-TE2A, 2,2',2''-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetamide DO3AM, 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid DO2A, 1,5,9-triazacyclododecane TACD, (3a1 s,5a1s)-decahydro-3a,5a,8a,10a-tetraazapyrene, 1,4,7-triazacyclononane TACN, 1,4,7,10-tetraazacyclododecane, tris(hydroxypyridinone)THP, 3-(((4,7-bis((hydroxy(hydroxymethyl)phosphoryl)methyl)-1,4,7-triazolidine-1-yl)methyl)(hydroxy)phosphoryl)propionic acid NOPO, 3,6,9,15-tetraazabicyclo[9.3].[1] Pentadecane-1(15),11,13-trien-3,6,9-triacetic acid PCTA, 2,2',2'',2'''-(1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetraacetic acid TRITA, 2,2',2'',2'''-(1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetraacetamide TRITAM, 2,2',2''-(1,4,7,10-tetraazacyclotridecane-1,4,7-triyl)triacetamide TRITRAM, trans-N- Dimethylcyclopamide, 2,2',2''-(1,4,7-triazacyclononane-1,4,7-triyl)triacetamide (NOTAM), oxocyclopamide, dioxocyclopamide, 1,7-dioxa-4,10-diazacyclododecane, cross-linked bridged cyclopamide, triazacyclononane phosphonite (TRAP), bispyridoxine diphosphate (DPDP), meso-tetra(4-sulfonylphenyl)porphyrin (TPPS4), ethylidene dihydroxyphenylglycine (EHPG), hexamethylenediaminetetraacetic acid, dimethylphosphomethane (DMPE), methylene diphosphate, or dimercaptosuccinic acid (DMPA).

8. The compound according to claim 6, wherein, The radionuclides are selected from the group consisting of: 124 I, 131 I, 86 Y、 90 Y、 177 Lu、 111 In、 188 Re、 55 Co、 64 Cu、 67 Cu、 68 Ga、 89 Zr、 203 Pb, 212 Pb, 212 Bi、 213 Bi、 72 As、 211 At、 225 Ac、 223 Ra、 97 Ru、 149 Tb, 152 Tb, 161 Tb, 99m Tc, 226 Th、 227 Th、 201 Tl、 89 Sr、 44 Sc、 43 Sc、 47 Sc、 153 Sm、 133 Xe or Al 18 F.

9. The compound according to claim 7, wherein, The chelating agent chelates the radionuclide, and the radionuclide is selected from the group consisting of: 124 I, 131 I, 86 Y、 90 Y、 177 Lu、 111 In、 188 Re、 55 Co、 64 Cu、 67 Cu、 68 Ga、 89 Zr、 203 Pb, 212 Pb, 212 Bi、 213 Bi、 72 As、 211 At、 225 Ac、 223 Ra、 97 Ru、 149 Tb, 152 Tb, 161 Tb, 99m Tc, 226 Th、 227 Th、 201 Tl、 89 Sr、 44 Sc、 43 Sc、 47 Sc、 153 Sm、 133 Xe or Al 18 F.

10. The compound according to claim 1 or 2, wherein, The effective payload is selected from the group consisting of: eczema, PNU-159682, muscarinic acid, docamycin, auristatin, maytansine, tubulolysin, calcitrazine, SN-38, paclitaxel, doxorubicin, vinca alkaloid, doxorubicin, methotrexate, pyrrolobenzodiazepines, pyrrole-based spindle kinesin KSP inhibitors, or indoline-benzodiazepine dimers.

11. The compound according to claim 1 or 2, wherein, P is represented by the following equation (2): P 1 -L-- (2) in, P 1 The payload as defined in any one of claims 3 to 10; L stands for connector; Refers to the covalent attachment to the reactive portion Y.

12. The compound according to claim 11, wherein, The connector is one of the following: (a1) An alkylene group having 1 to 12 carbon atoms; (b1) A polyepoxyalkylene compound having 2 or 3 carbon atoms and having 1 to 36 repeating units; or (c1) A peptide group having 2 to 12 amino acids.

13. The compound according to claim 1 or 2, wherein the compound is one of the following: 、 、 、 、 、 、 、 or, ； in, P is as defined in any one of claims 3 to 10, and Y' is as defined in claim 1.

14. The compound according to claim 1 or 2, wherein the compound is one of the following: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or, 。 15. A kit for site-specific modification of antibodies or antibody fragments, said kit comprising a compound and a buffer according to any one of claims 1 to 14.

16. The kit according to claim 15, wherein the kit is used for regioselectively modifying antibodies or antibody fragments, wherein, The compound is immobilized on a solid matrix.

17. A method for regioselectively modifying an antibody or antibody fragment, the method comprising reacting the antibody or a fragment thereof with a compound according to any one of claims 1 to 14.

18. The method of claim 17, wherein, The antibody is a monoclonal antibody; or The antibody fragment was incorporated into the Fc-fusion protein.

19. The method of claim 18, wherein The antibodies mentioned are adalimumab, adoranamumab, alenumab, atezolizumab, annatuzumab, avelumumab, bavizumab, baliximab, betumomab, bemexazol, bexixabumab, bevacizumab, belotuszumab, bentusizumab, brentuximab, verbentuximab, brodatumab, bonnetumab, caputuzumab, cimiprimab, cetuximab, simppaneumab, crimetuzumab, Titan-crimetuzumab, Titan-crimetuzumab, Titan-crimetuzumab. Ranocidumab, Dazumab, Daratonumab, Denosumab, Denutoximab, Dvalumab, Ezotrozumab, Erotoximab, Epatavamab, Envertumab, Vitin-Entertoximab, Epatavamab, Idazumab, Gelatinumab, Gelatinximab, Gonemab, Teimozumab, Inblilizumab, Infliximab, Intoximab, Osintuzumab, Ipilimumab, Esatuzumab, Isisibemab, J591 PSMA antibody, Labezizumab, Lecarbazil, Mogliflozil, Nectuizumab, Nitotuzumab, Natalizumab, Nivolumab, Orizumab, Ofamumab, Olarumab, Ogovomab, Panitumumab, Pembrolizumab, Pertuzumab, Polotuzumab, Verbotuzumab, Pronizumab, Rituximab, Ramucirumab, Rituximab, Si Toxicitummab, Saxitozumab, Gosatutuzumab, Cirrenole, Solanine, Tacillinumab, Tiltromumab, Tilanelumab, Tocillinumab, Tosimo, Trastuzumab, Dexamethasone, Trastuzumab emtansine, TS23, Ustekinumab, Vedolizumab, Votumumab, Zegettena, Zalumab, Zalumab or fragments thereof; or The antibody fragment is incorporated into an Fc-fusion protein, wherein the Fc-fusion protein is berazip, aflibercept, ziv-aflibercept, dulaglutide, linasip, romilastine, abatacept, or afasicept.

20. A modified antibody or modified antibody fragment, said modified antibody or modified antibody fragment being obtained by reacting the antibody or antibody fragment with a compound according to any one of claims 1 to 14.

21. The modified antibody or modified antibody fragment of claim 20, wherein the modified antibody or modified antibody fragment is used for diagnosing, monitoring, imaging or treating a disease, the method comprising administering the modified antibody or the modified antibody fragment to a subject.

22. The modified antibody or modified antibody fragment according to claim 21, wherein, The diseases mentioned are neurological diseases, cardiovascular diseases, autoimmune diseases, or cancer.

23. The modified antibody or modified antibody fragment according to claim 21 or 22, wherein, The diseases selected are from the group consisting of: encephalopathy, systemic sclerosis, angina pectoris, aortic aneurysm, atherosclerosis, heart failure, hypercholesterolemia, ischemia, myocardial infarction, thromboembolism, thrombosis, ankylosing spondylitis, autoimmune cytopenia, autoimmune myocarditis, Crohn's disease, graft-versus-host disease, granulomatous polyangiitis, idiopathic thrombocytopenic purpura, juvenile arthritis, juvenile diabetes, lupus, psoriasis, psoriatic arthritis, rheumatoid arthritis, ulcerative colitis (UC), uveitis, and vasculitis, wherein juvenile diabetes is type 1 diabetes.

24. The modified antibody or modified antibody fragment according to claim 23, wherein, The brain diseases mentioned are Alzheimer's disease, amyotrophic lateral sclerosis, cerebral arteriosclerosis, Huntington's disease, multiple sclerosis, Parkinson's disease, and progressive multifocal leukoencephalopathy.

25. The modified antibody or modified antibody fragment according to claim 23, wherein, The psoriasis mentioned is plaque psoriasis.

26. The modified antibody or modified antibody fragment according to claim 23, wherein, The lupus mentioned is systemic lupus erythematosus.

27. The modified antibody or modified antibody fragment according to claim 21 or 22, wherein, The diseases mentioned involve lymphoma cells, myeloma cells, kidney cancer cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cell carcinoma cells, lung cancer cells, testicular cancer cells, pancreatic cancer cells, liver cancer cells, melanoma, or head and neck cancer cells.

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