Cleavable Linker and Its Use

Abstract

The present application relates to a cleavable Linker and its uses, and particularly to a compound of formula (I) or a pharmaceutically acceptable salt thereof, X-L-Y-R1 (I) wherein X is a chelating agent; L is a linker and L may be absent; Y is a polypeptide that can be cleaved by enkephalinase; R1 is a group that binds to the amino or carboxyl group of the side chain of Y and has a functional group capable of binding to a target molecule recognition unit or its linking group, or is a hydrogen atom of the amino or carboxyl group of the side chain of Y.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and particularly to a cleavable Linker and its uses. Background Art

[0002] Fluorescence resonance energy transfer (FRET) is a non-radiative energy transition process that transfers the energy of the donor excited state to the acceptor excited state through the intermolecular electric dipole interaction. FRET peptides are convenient tools for studying peptidase specificity. Since their reaction process can be continuously monitored, they provide a convenient method for detecting enzyme activity. The fluorescence generated after the peptide bond hydrolysis of the donor / acceptor pair can measure the enzyme activity at the nanomolar concentration level. When the FRET peptide is intact, internal fluorescence quenching is exhibited, but when any peptide bond of the donor / acceptor pair is broken, fluorescence is released, and this fluorescence can be continuously detected, thereby enabling quantitative analysis of the enzyme activity. FRET peptides can be used as suitable substrates for various enzyme studies, such as the kinetic and functional characteristics of peptidases, proteases, kinases, and phosphatases; the screening and detection of new proteolytic enzymes.

[0003] NEP (Neutral endopeptidase, also known as Neprilysin) is a type II transmembrane glycoprotein of the M13 zinc-dependent metalloprotease family, also known as enkephalinase, and the zinc atom carried on it is located at the active site of the enzyme. The molecular weight of NEP is about 97 kDa, consisting of 750 amino acids, including a signal peptide and two hydrophilic domains. The intracellular fragment has 26 amino acids, and the extracellular fragment has 700 amino acids. NEP has obvious specificity and can cleave the peptide bond on the amino group with a large N-terminal aromatic and hydrophobic group, causing the polypeptide chain to hydrolyze. Summary of the Invention

[0004] The present invention screens out a polypeptide sequence cleavable by NEP through FRET, and then designs a compound structure containing a ligand molecule for tumor targeting, a tripeptide (tetrapeptide) sequence cleavable by renal brush border NEP, and a bifunctional linker for chelating radionuclides. It can significantly reduce the radioactive accumulation and retention in the kidney while maintaining the same tumor uptake, improving the clinical diagnosis and treatment value of the radioligand, and having good clinical application prospects.

[0005] On the one hand, the present application provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0006] X-L-Y-R1

[0007] Formula (I)

[0008] Wherein, X is a chelating agent;

[0009] L is a linker, and L can be absent;

[0010] Y is a polypeptide that can be cleaved by neutral endopeptidase (NEP);

[0011] R1 is a group that binds to the amino or carboxyl group of the side chain of Y and has a functional group capable of binding to the target molecule recognition unit or its linking group, or is a hydrogen atom of the amino or carboxyl group of the side chain of Y.

[0012] In certain embodiments, wherein said Y is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide or decapeptide.

[0013] In certain embodiments, wherein said Y is: -(A1)m-A2-A3-, where m is 0, 1 or 2; A1 and A2 are amino acid residues, A3 is an amino acid residue having an amino or carboxyl group on the side chain, and R1 is a group that binds to the amino or carboxyl group of the side chain of A3 and has a functional group capable of binding to the target molecule recognition unit or its linking group, or is a hydrogen atom of the amino or carboxyl group of the side chain of A3.

[0014] In certain embodiments, wherein said Y is selected from: -Ser-leu-Lys-(SLK), -Ser-Phe-Lys-(SFK), -Asp-Phe-Lys-(DFK), -Val-Met-Lys-(VMK), -Met-Val(Dap)-(MV(Dap)), -Met-Val-

[0015] (MV), -(D-Arg)-Ser-Phe-Lys-(rSFK), -Gly-Trp-Lys-(GWK), -Met-Asn-Lys-(MNK), -Gln-Leu-Lys-(QLK) and -Thr-ILe-Lys-(TIK).

[0016] In certain embodiments, wherein said R1 is composed of one or more of the following components: -C1-C 18 alkylene, -(CH2CH2O) n -, -CO(CH2) n -, -NH(CH2) n-, arylthio (PYS), p - aminobenzyloxycarbonyl (PAB), aminobenzylthio, oxybenzylthio, alkoxyamino (AOA), dioxobenzylthio, diaminobenzylthio, aminooxybenzylthio, alkoxyamino (AOA), 4 - methyl - 4 - dithiovaleryl (MPDP), triazole, dithio, sulfonyl, phosphonyl, (4 - acetyl) aminobenzoyl (SIAB), 4 - thiobutyryl, 4 - thio - 2 - sulfonatobutyryl (2 - SO3 - -SPDB), 4 - thiopropionyl (SPDP), hydrazone, aminoethylamine, hydrazine, oxime, thioaminooxobut - 2 - enoic acid, thioaminooxobutyric acid, a peptide containing 1 to 20 amino acid residues, and where n is any integer between 1 and 1000, for example n can be 1 to 900, 1 to 800, 1 to 700, 1 to 600, 1 to 500, 1 to 400, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 90, 1 to 80, - 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 10 to 900, 10 to 800, 10 to 700, 10 to 600, 10 to 500, 10 to 400, 10 to 300, 10 to 250, 10 to 200, 10 to 150, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 70, 10 to 60, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 19, 10 to 18, 10 to 17, 10 to 16 or 10 to 15.

[0017] In certain embodiments, wherein said R1 is selected from the following structures: -(CH2) n -Mal, -(CH2CH2O) n- Mal, -CO(CH2)n - Mal, -NH(CH2) n -Mal, -NH(CH2CH2O) n- Mal, where n is any integer between 1 and 1000.

[0018] In certain embodiments, wherein said R1 is selected from the following structures: 6-Maleimidohexanoyl (MC), maleimidopropionyl (MP), maleimidoethylamine (ME), 6-maleimidohexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-VC-PAB), 4-(N-maleimidomethyl)cyclohexane-1-carbonyl (SMCC), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP).

[0019] In certain embodiments, the chelator is selected from one or more of DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG3, MAG2, HYNIC, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA, HEHAA, and derivatives thereof.

[0020] In certain embodiments, X is selected from the following structures:

[0021]

[0022] and derivatives thereof; wherein each R2 is independently a hydrogen atom or an optionally substituted C1-C8 hydrocarbon group.

[0023] In certain embodiments, X is selected from the following structures:

[0024]

[0025] and derivatives thereof.

[0026] In certain embodiments, L is absent or L is selected from the following structures:

[0027] where n is any integer from 0 to 10, for example, n can be from 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0028] In certain embodiments, L is absent or L is selected from the following structures:

[0029]

[0030] In certain embodiments, X-L is selected from the following structures:

[0031]

[0032] wherein each R2 is independently a hydrogen atom or an optionally substituted C1-C8 hydrocarbon group, and n is any integer between 1 and 1000.

[0033] In certain embodiments, X-L is selected from the following structures:

[0034]

[0035] In certain embodiments, the compound represented by formula (I) is selected from: X-L-SLK(Mal), X-L-SFK-(CH2)2-Mal, X-L-SFK-(CH2)2-Mal, X-SFK-(CH2)2-Mal, X-L-DFK-(CH2)2-Mal, X-L-VMK-(CH2)2-Mal, X-L-VMK-PEG4-Mal, X-L-VMK-PEG4-Mal, X-VMK-(CH2)2-Mal, X-VMK-PEG4-Mal, X-L-MV(Dap)-PEG4-Mal, X-L-MV(Dap)-PEG4-Mal, X-L-MV(Dap)-PEG4-Mal, X-MV(Dap)-PEG4-Mal, X-MV(Dap)-PEG4-Mal, X-MV-(CH2)2-Mal, X-L-MV-PEG4-Mal, X-L-MV-PEG4-Mal, X-L-rSFK-PEG4-Mal, X-L-GWK-PEG4-Mal, X-L-MNK-PEG4-Mal, X-L-QLK-PEG4-Mal and X-L-TIK-PEG4-Mal;

[0036] wherein X is selected from:

[0037]

[0038]

[0039] X-L is selected from:

[0040]

[0041] In certain embodiments, the compound represented by formula (I) is selected from: NOTA-Bn-SLK(Mal), NOTA-Bn-SFK-(CH2)2-Mal, DOTA-Bn-SFK-(CH2)2-Mal, NODAGA-SFK-(CH2)2-Mal, NOTA-Bn-DFK-(CH2)2-Mal, NOTA-Bn-VMK-(CH2)2-Mal, DOTA-Bn-VMK-PEG4-Mal, DOTA-PEG4-VMK-PEG4-Mal, DOTA-VMK-(CH2)2-Mal, DOTA-VMK-PEG4-Mal, NOTA-Bn-MV(Dap)-PEG4-Mal, DOTA-Bn-MV(Dap)-PEG4-Mal, DOTA-PEG4-MV(Dap)-PEG4-Mal, DOTA-MV(Dap)-PEG4-Mal, NODAGA-MV(Dap)-PEG4-Mal, DOTA-MV-(CH2)2-Mal, DOTA-PEG4-MV-PEG4-Mal, DOTA-Bn-MV-PEG4-Mal, DOTA-Bn-rSFK-PEG4-Mal, DOTA-Bn-GWK-PEG4-Mal, DOTA-Bn-MNK-PEG4-Mal, DOTA-Bn-QLK-PEG4-Mal, and DOTA-Bn-TIK-PEG4-Mal.

[0042] On the other hand, the present application provides an immunoconjugate comprising i) the compound represented by formula (I) as described above or a pharmaceutically acceptable salt thereof and ii) a target molecule recognition unit.

[0043] In certain embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is bound to the target molecule recognition unit or a linking group of the target molecule recognition unit through R1.

[0044] In certain embodiments, the immunoconjugate further comprises an active moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a viral coat protein, a VLP, or a combination thereof, wherein the active moiety is linked to the chelator.

[0045] In certain embodiments, the detectable label is selected from one or more reagents of the group consisting of: a radionuclide, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, and an enzyme.

[0046] In certain embodiments, the radionuclide is suitable for medical imaging and / or therapy.

[0047] In certain embodiments, the radionuclide includes110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu,

[0048] 67 Cu, 67 Ga, 68 Ga, 68 Ge, 86 Y, 90 Y, 89 Zr, 94m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd,

[0049] 32 P, 11 C, 13 N, 15 O, 186 Re, 188 Re, 51 Mn, 52m Mn, 72 As, 75 Br, 76 Br, 82m Rb, 83 Sr, 225 Ac,

[0050] 211 At or other α, γ, β-, or positron emitters.

[0051] In certain embodiments, the immunoconjugate has the structure shown in formula (II):

[0052]

[0053] Wherein, X is a chelating agent, and X is connected or not connected to the active moiety;

[0054] T is a target molecule recognition unit;

[0055] L is a linker, and L may be absent;

[0056] A1 and A2 are amino acid residues, and m is 0, 1 or 2;

[0057] A3 is an amino acid residue having an amino group or a carboxyl group on the side chain, and R1 is a group that binds to the amino group or carboxyl group on the side chain of A3 and has a functional group capable of binding to the target molecule recognition unit or its linking group;

[0058] -(A1) m -A2-A3- can be cleaved by neprilysin.

[0059] In certain embodiments, wherein -(A1) m -A2-A3- is selected from: -Ser-leu-Lys- (SLK), -Ser-Phe-Lys- (SFK), -Asp-Phe-Lys- (DFK), -Val-Met-Lys- (VMK), -Met-Val(Dap)- (MV(Dap)), -Met-Val- (MV), -(D-Arg)-Ser-Phe-Lys- (rSFK), -Gly-Trp-Lys- (GWK), -Met-Asn-Lys- (MNK), -Gln-Leu-Lys- (QLK) and -Thr-ILe-Lys- (TIK).

[0060] In certain embodiments, wherein said X is selected from the following structures:

[0061]

[0062] and their derivatives; wherein, R2 is independently a hydrogen atom or an optionally substituted C1-C8 hydrocarbon group.

[0063] In certain embodiments, wherein said X is selected from the following structures:

[0064]

[0065] and their derivatives.

[0066] In certain embodiments, wherein said L is absent or L is selected from the following structures:

[0067] where n is any integer from 0 to 10, for example n can be from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3 or from 1 to 2.

[0068] In certain embodiments, wherein said L is absent or L is selected from the following structures:

[0069]

[0070] In certain embodiments, wherein X-L is selected from the following structures:

[0071]

[0072] Where n is any integer between 1 and 1000. For example, n can be between 1 and 900, 1 and 800, 1 and 700, 1 and 600, 1 and 500, 1 and 400, 1 and 300, 1 and 250, 1 and 200, 1 and 150, 1 and 100, 1 and 90, 1 and 80, 1 and 70, 1 and 60, 1 and 50, 1 and 45, 1 and 40, 1 and 35, 1 and 30, 1 and 25, 1 and 20, 1 and 19, 1 and 18, 1 and 17, 1 and 16, 1 and 15, 1 and 14, 1 and 13, 1 and 12, 1 and 11, 1 and 10, 1 and 9, 1 and 8, 1 and 7, 1 and 6, 1 and 5, 1 and 4, 1 and 3, 1 and 2, between 10 and 900, between 10 and 800, between 10 and 700, between 10 and 600, between 10 and 500, between 10 and 400, between 10 and 300, between 10 and 250, between 10 and 200, between 10 and 150, between 10 and 100, between 10 and 90, between 10 and 80, between 10 and 70, between 10 and 70, between 10 and 60, between 10 and 50, between 10 and 45, between 10 and 40, between 10 and 35, between 10 and 30, between 10 and 25, between 10 and 20, between 10 and 19, between 10 and 18, between 10 and 17, between 10 and 16 or between 10 and 15.

[0073] In certain embodiments, X-L is selected from the following structures:

[0074]

[0075] In certain embodiments, it is selected from the following structures:

[0076]

[0077]

[0078] Wherein, T is a targeting molecular unit;

[0079] -(A1) m -A2-A3- is selected from: -Ser-leu-Lys-(SLK), -Ser-Phe-Lys-(SFK), -Asp-Phe-Lys-(DFK), -Val-Met-Lys-(VMK), -Met-Val(Dap)-(MV(Dap)), -Met-Val-(MV), -(D-Arg)-Ser-Phe-Lys-(rSFK), -Gly-Trp-Lys-(GWK), -Met-Asn-Lys-(MNK), -Gln-Leu-Lys-(QLK) and -Thr-ILe-Lys-(TIK);

[0080] The said R1 is selected from the following structures: -(CH2) n-Mal, -(CH2CH2O) n- Mal, -CO(CH2)n-Mal, -NH(CH2) n -Mal;

[0081] n is each independently any integer between 1 and 1000, for example, n can be 1 to 900, 1 to 800, 1 to 700, 1 to 600, 1 to 500, 1 to 400, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 10 to 900, 10 to 800, 10 to 700, 10 to 600, 10 to 500, 10 to 400, 10 to 300, 10 to 250, 10 to 200, 10 to 150, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 70, 10 to 60, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 19, 10 to 18, 10 to 17, 10 to 16 or 10 to 15;

[0082] Each R2 is independently a hydrogen atom or an optionally substituted C1-C8 hydrocarbon group.

[0083] In certain embodiments, the immunoconjugate is selected from the following structures:

[0084] NOTA-Bn-SLK(Mal)-T,

[0085] NOTA-Bn-SFK-(CH2)2-Mal-T,

[0086] DOTA-Bn-SFK-(CH2)2-Mal-T,

[0087] NODAGA-SFK-(CH2)2-Mal-T,

[0088] NOTA-Bn-DFK-(CH2)2-Mal-T,

[0089] NOTA-Bn-VMK-(CH2)2-Mal-T,

[0090] DOTA-Bn-VMK-PEG4-Mal-T,

[0091] DOTA-PEG4-VMK-PEG4-Mal-T,

[0092] DOTA-VMK-(CH2)2-Mal-T,

[0093] DOTA-VMK-PEG4-Mal-T,

[0094] NOTA-Bn-MV(Dap)-PEG4-Mal-T,

[0095] DOTA-Bn-MV(Dap)-PEG4-Mal-T,

[0096] DOTA-PEG4-MV(Dap)-PEG4-Mal-T,

[0097] DOTA-MV(Dap)-PEG4-Mal-T,

[0098] NODAGA-MV(Dap)-PEG4-Mal-T,

[0099] DOTA-MV-(CH2)2-Mal-T,

[0100] DOTA-PEG4-MV-PEG4-Mal-T,

[0101] DOTA-Bn-MV-PEG4-Mal-T,

[0102] DOTA-Bn-rSFK-PEG4-Mal-T,

[0103] DOTA-Bn-GWK-PEG4-Mal-T,

[0104] DOTA-Bn-MNK-PEG4-Mal-T,

[0105] DOTA-Bn-QLK-PEG4-Mal-T and

[0106] DOTA-Bn-TIK-PEG4-Mal-T;

[0107] wherein T is a target molecule recognition unit, and the target molecule recognition unit includes an antigen-binding protein. In certain embodiments, it is selected from the following structures:

[0108] A-NOTA-Bn-SLK(Mal)-T,

[0109] A-NOTA-Bn-SFK-(CH2)2-Mal-T,

[0110] A-DOTA-Bn-SFK-(CH2)2-Mal-T,

[0111] A-NODAGA-SFK-(CH2)2-Mal-T,

[0112] A-NOTA-Bn-DFK-(CH2)2-Mal-T,

[0113] A-NOTA-Bn-VMK-(CH2)2-Mal-T,

[0114] A-DOTA-Bn-VMK-PEG4-Mal-T,

[0115] A-DOTA-PEG4-VMK-PEG4-Mal-T,

[0116] A-DOTA-VMK-(CH2)2-Mal-T,

[0117] A-DOTA-VMK-PEG4-Mal-T,

[0118] A-NOTA-Bn-MV(Dap)-PEG4-Mal-T,

[0119] A-DOTA-Bn-MV(Dap)-PEG4-Mal-T,

[0120] A-DOTA-PEG4-MV(Dap)-PEG4-Mal-T,

[0121] A-DOTA-MV(Dap)-PEG4-Mal-T,

[0122] A-NODAGA-MV(Dap)-PEG4-Mal-T,

[0123] A-DOTA-MV-(CH2)2-Mal-T,

[0124] A-DOTA-PEG4-MV-PEG4-Mal-T,

[0125] A-DOTA-Bn-MV-PEG4-Mal-T,

[0126] A-DOTA-Bn-rSFK-PEG4-Mal-T,

[0127] A-DOTA-Bn-GWK-PEG4-Mal-T,

[0128] A-DOTA-Bn-MNK-PEG4-Mal-T,

[0129] A-DOTA-Bn-QLK-PEG4-Mal-T and

[0130] A-DOTA-Bn-TIK-PEG4-Mal-T;

[0131] wherein T is a target molecule recognition unit and A is an active moiety.

[0132] In certain embodiments, the target molecule recognition unit comprises an antigen-binding protein, a scaffold protein, or a ligand.

[0133] In certain embodiments, the antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

[0134] In certain embodiments, the antibody comprises a monoclonal antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

[0135] In certain embodiments, the antigen-binding fragment comprises a Fab, Fab’, Fv fragment, F(ab’)2, scFv, VHH, and / or dAb.

[0136] In certain embodiments, the target molecule recognition unit comprises a VHH.

[0137] In certain embodiments, the VHH is camelid, chimeric, human, partially humanized, or fully humanized.

[0138] In certain embodiments, the target molecule recognition unit targets a tumor antigen and / or a non-tumor antigen.

[0139] In certain embodiments, the target molecule recognition unit specifically binds to an antigen selected from the group consisting of: AXL, BAFFR, BCMA, BDCA2, BDCA4, BTLA, BTNL2, BTNL3, BTNL8, BTNL9, C10orf54, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CD11c, CD137, CD138, CD14, CD163, CD168, CD 177, CD19, CD20, CD209, CD209L, CD22, CD226, CD248, CD25, CD27, CD274, CD276, CD28, CD30, CD300A, CD32b, CD33, CD37, CD38, CD4, cluster of differentiation 40 (CD40), CD44, CD45, CD46, CD47, CD48, CD5, CD52, CD55, CD56, CD59, CD62E, CD68, CD69, CD70, CD74, CD79a, CD79b, CD8, CD80, CD86, CD90.2, CD96, OX40 (CD134), CD123, CD97, CD179a, CLEC12A, CLEC12B, CLEC7A, CLEC9A, CR1, CR3, CRTAM, CSF1R, CTLA4, CXCR1 / 2, CXCR4, CXCR5, DDR1, DDR2, DEC-205, DLL4, DR6, FAP, FCamR, FCMR, FcR’s, Fire, GITR, HHLA2, HLA class II, HVEM, ICOSLG, IFNAR, IFNAR1, IFNLR1, IL10R1, IL10R2, IL12R, IL13RA1, IL13RA2, IL15R, IL17RA, IL17RB, IL17RC, IL17RE, IL20R1, IL20R2, IL21R, IL22R1, IL22RA, IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R, IL3RA, IL4R, IL6R, IL5R, IL7R, IL9R, Integrins, LAG3, LIFR, MAG / Siglec-4, MMR, MSR1, NCR3LG1, NKG2D, NKp30, NKp46, PDCD1, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, Siglec-1, Siglec-10, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, SIRPA, SLAMF7, TACI, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2, TGFBR3, TIGIT, TLR2, TLR4, TNFα, TROY, TSLPR, TYRO, VLDLR, VSIG4, IL2R-y, VTCN1, TSHR, CD171, CS-1, CLL-1, GD3, Tn Ag, FLT3, B7H3, B7H4, KIT, IL-13Ra2, IL-11Ra, PSCA, PSMA, PRSS21, EGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, FOLR1, FOLR2, TEM7R, CLDN6, CLDN18.2, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TAARP, WT1, ETV6-AML, SPA17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, FOSL1, hTERT, ML-IAP, ERG, NA17, PAX3, AR, cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, CD72, LAIR1, FCAR, LILRA2, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, HER2, ROR1, TAAG72, GD2, gp100Tn, FAP, tyrosinase, EPCAM, CEA, IGF-1R, EphB2, mesothelin, cadherin 17, EGFRvIII, GPNMB, GPR64, HER3, LRP6, LYPD8, NKG2D, SLC34A2, SLC39A6, SLITRK6, GUCY2C, 5T4 and / or TACSTD2.

[0140] In certain embodiments, the target molecule recognition unit comprises an anti-PD-L1 VHH antibody, an anti-HER2 VHH antibody, a scaffold protein targeting HER2, and / or an anti-CD8α VHH antibody.

[0141] In certain embodiments, the anti-CD8α VHH antibody comprises: CDR1 having the amino acid sequence shown in SEQ ID NO:1, CDR2 having the amino acid sequence shown in SEQ ID NO:2, and CDR3 having the amino acid sequence shown in SEQ ID NO:3.

[0142] In certain embodiments, the anti-CD8α VHH antibody comprises the amino acid sequence shown in SEQ ID NO:4.

[0143] In certain embodiments, the anti-HER2 VHH antibody comprises: CDR1 having the amino acid sequence shown in SEQ ID NO:5, CDR2 having the amino acid sequence shown in SEQ ID NO:6, and CDR3 having the amino acid sequence shown in SEQ ID NO:7.

[0144] In certain embodiments, the anti-HER2 VHH antibody comprises the amino acid sequence shown in SEQ ID NO:8.

[0145] In certain embodiments, the HER2-targeting scaffold protein comprises the amino acid sequence shown in SEQ ID NO:9.

[0146] In certain embodiments, the immunoconjugate is selected from the following structures:

[0147]

[0148]

[0149] wherein,

[0150] -(A1) m -A2-A3- is selected from: -Ser-leu-Lys-(SLK), -Ser-Phe-Lys-(SFK), -Asp-Phe-Lys-(DFK), -Val-Met-Lys-(VMK), -Met-Val(Dap)-(MV(Dap)), -Met-Val-(MV), -(D-Arg)-Ser-Phe-Lys-(rSFK), -Gly-Trp-Lys-(GWK), -Met-Asn-Lys-(MNK), -Gln-Leu-Lys-(QLK) and -Thr-ILe-Lys-(TIK); and the R1 is conjugated to an antigen-binding protein, a scaffold protein or a ligand.

[0151] In certain embodiments, the R1 is selected from the following structures: -(CH2) n -Mal, -(CH2CH2O) n- Mal, -CO(CH2)n-Mal, -NH(CH2) n -Mal, where n is any integer between 1 and 10.

[0152] In certain embodiments, the immunoconjugate is selected from the following structures:

[0153] NOTA-Bn-SLK(Mal)-VHH,

[0154] NOTA-Bn-SFK-(CH2)2-Mal-VHH,

[0155] DOTA-Bn-SFK-(CH2)2-Mal-VHH,

[0156] NODAGA-SFK-(CH2)2-Mal-VHH,

[0157] NOTA-Bn-DFK-(CH2)2-Mal-VHH,

[0158] NOTA-Bn-VMK-(CH2)2-Mal-VHH,

[0159] DOTA-Bn-VMK-PEG4-Mal-VHH,

[0160] DOTA-PEG4-VMK-PEG4-Mal-VHH,

[0161] DOTA-VMK-(CH2)2-Mal-VHH,

[0162] DOTA-VMK-PEG4-Mal-VHH,

[0163] NOTA-Bn-MV(Dap)-PEG4-Mal-VHH,

[0164] DOTA-Bn-MV(Dap)-PEG4-Mal-VHH,

[0165] DOTA-PEG4-MV(Dap)-PEG4-Mal-VHH,

[0166] DOTA-MV(Dap)-PEG4-Mal-VHH,

[0167] NODAGA-MV(Dap)-PEG4-Mal-VHH,

[0168] DOTA-MV-(CH2)2-Mal-VHH,

[0169] DOTA-PEG4-MV-PEG4-Mal-VHH,

[0170] DOTA-Bn-MV-PEG4-Mal-VHH,

[0171] DOTA-Bn-rSFK-PEG4-Mal-VHH,

[0172] DOTA-Bn-GWK-PEG4-Mal-VHH,

[0173] DOTA-Bn-MNK-PEG4-Mal-VHH,

[0174] DOTA-Bn-QLK-PEG4-Mal-VHH and

[0175] DOTA-Bn-TIK-PEG4-Mal-VHH.

[0176] For another example, the immunoconjugate is selected from the following structures:

[0177] A-NOTA-Bn-SLK(Mal)-VHH,

[0178] A-NOTA-Bn-SFK-(CH2)2-Mal-VHH,

[0179] A-DOTA-Bn-SFK-(CH2)2-Mal-VHH,

[0180] A-NODAGA-SFK-(CH2)2-Mal-VHH,

[0181] A-NOTA-Bn-DFK-(CH2)2-Mal-VHH,

[0182] A-NOTA-Bn-VMK-(CH2)2-Mal-VHH,

[0183] A-DOTA-Bn-VMK-PEG4-Mal-VHH,

[0184] A-DOTA-PEG4-VMK-PEG4-Mal-VHH,

[0185] A-DOTA-VMK-(CH2)2-Mal-VHH,

[0186] A-DOTA-VMK-PEG4-Mal-VHH,

[0187] A-NOTA-Bn-MV(Dap)-PEG4-Mal-VHH,

[0188] A-DOTA-Bn-MV(Dap)-PEG4-Mal-VHH,

[0189] A-DOTA-PEG4-MV(Dap)-PEG4-Mal-VHH,

[0190] A-DOTA-MV(Dap)-PEG4-Mal-VHH,

[0191] A-NODAGA-MV(Dap)-PEG4-Mal-VHH,

[0192] A-DOTA-MV-(CH2)2-Mal-VHH,

[0193] A-DOTA-PEG4-MV-PEG4-Mal-VHH,

[0194] A-DOTA-Bn-MV-PEG4-Mal-VHH,

[0195] A-DOTA-Bn-rSFK-PEG4-Mal-VHH,

[0196] A-DOTA-Bn-GWK-PEG4-Mal-VHH,

[0197] A-DOTA-Bn-MNK-PEG4-Mal-VHH,

[0198] A-DOTA-Bn-QLK-PEG4-Mal-VHH and

[0199] A-DOTA-Bn-TIK-PEG4-Mal-VHH;

[0200] wherein A is the active moiety.

[0201] In certain embodiments, wherein A is a detectable label, the detectable label is selected from one or more reagents of the group consisting of: radionuclides, fluorophores, chemiluminescent agents, bioluminescent agents, paramagnetic ions, and enzymes.

[0202] In certain embodiments, wherein A is a radionuclide, the radionuclide includes 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 67 Cu, 67 Ga, 68 Ga, 68 Ge, 86 Y, 90 Y, 89 Zr, 94m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C, 13 N, 15 O, 186 Re, 188 Re, 51 Mn, 52m Mn, 72 As, 75 Br, 76 Br, 82m Rb, 83 Sr, 225 Ac, 211 At or other α, γ, β-, or positron emitters.

[0203] On the other hand, the present application provides a radionuclide complex, which comprises i) the compound represented by the foregoing formula (I) or a pharmaceutically acceptable salt thereof and ii) a radionuclide.

[0204] In certain embodiments, the radionuclide is linked to X in the compound represented by the formula (I).

[0205] On the other hand, the present application provides a composition, which comprises the compound represented by the foregoing formula (I) or a pharmaceutically acceptable salt thereof, the foregoing immunoconjugate, and optionally a pharmaceutically acceptable carrier.

[0206] In certain embodiments, the composition is a detection agent or a therapeutic agent.

[0207] In certain embodiments, the detection agent is a reagent for detecting an antigen.

[0208] In certain embodiments, the detection agent is a contrast agent.

[0209] In certain embodiments, the contrast agent is a contrast agent for detecting an antigen.

[0210] In certain embodiments, the antigen is a tumor antigen.

[0211] In certain embodiments, the therapeutic agent is used for treating tumors.

[0212] On the other hand, the present application provides the use of the compound represented by the foregoing formula (I) or a pharmaceutically acceptable salt thereof, the foregoing immunoconjugate, the foregoing radionuclide complex, and / or the foregoing composition in the preparation of a reagent, a test plate, or a kit; wherein the reagent, test plate, or kit is used for detecting an antigen in a sample.

[0213] On the other hand, the present application provides the use of the compound represented by the foregoing formula (I) or a pharmaceutically acceptable salt thereof, the foregoing immunoconjugate, the foregoing radionuclide complex, and / or the foregoing composition in the preparation of a drug; wherein the drug is used for treating tumors.

[0214] On the other hand, the present application provides the use of the compound represented by the foregoing formula (I) or a pharmaceutically acceptable salt thereof, the foregoing immunoconjugate, the foregoing radionuclide complex, and / or the foregoing composition in the preparation of a radiolabeled drug.

[0215] On the other hand, the present application provides a radiolabeled drug, which comprises the compound represented by the foregoing formula (I) or a pharmaceutically acceptable salt thereof, the foregoing immunoconjugate, the foregoing radionuclide complex, and / or the foregoing composition.

[0216] On the other hand, the present application provides a radiological imaging diagnostic drug, which comprises the compound represented by the foregoing formula (I) or a pharmaceutically acceptable salt thereof, the foregoing immunoconjugate, the foregoing radionuclide complex, and / or the foregoing composition.

[0217] On the other hand, the present application provides a method for detecting the presence and / or amount of an antigen in a biological sample, comprising: contacting the biological sample with the compound represented by the foregoing formula (I) or a pharmaceutically acceptable salt thereof, the foregoing immunoconjugate, the foregoing radionuclide complex, and / or the foregoing composition.

[0218] In certain embodiments, the contacting is carried out in vitro or ex vivo.

[0219] In certain embodiments, the biological sample is a tissue.

[0220] In certain embodiments, the tissue is selected from blood tissue, lymphoid tissue, and tumor tissue.

[0221] In certain embodiments, the method comprises detecting the presence and / or amount of tumor antigen-positive cells in a biological sample.

[0222] In certain embodiments, the presence and / or amount of tumor antigen-positive cells in the biological sample is determined by imaging.

[0223] In certain embodiments, the presence and / or amount of tumor antigen-positive cells in the biological sample is determined by flow cytometry.

[0224] On the other hand, the present application provides a method for detecting and / or diagnosing tumors, comprising administering to a subject in need thereof the compound represented by the foregoing formula (I) or a pharmaceutically acceptable salt thereof, the foregoing immunoconjugate, the foregoing radionuclide complex, and / or the foregoing composition.

[0225] In certain embodiments, the method further comprises imaging the subject.

[0226] In certain embodiments, the imaging includes ECT imaging.

[0227] In certain embodiments, the ECT imaging includes SPECT imaging or PET imaging.

[0228] On the other hand, the present application provides a method for treating and / or preventing tumors, the method comprising administering to a subject in need thereof the compound represented by the foregoing formula (I) or a pharmaceutically acceptable salt thereof, the foregoing immunoconjugate, the foregoing radionuclide complex, and / or the foregoing composition.

[0229] On the other hand, the present application provides a method for monitoring the efficacy of an anti-tumor therapy in a subject, the method comprising:

[0230] (i) administering to a subject having a tumor and being treated with an anti-tumor therapy the compound of formula (I) as described above or a pharmaceutically acceptable salt thereof, the immunoconjugate as described above, the radionuclide complex as described above, and / or the composition as described above; and

[0231] (ii) determining the amount of tumor antigen-positive cells in the tumor of the subject.

[0232] In certain embodiments, the presence and / or amount of SNA004-positive cells in the tumor of the subject is determined by imaging.

[0233] In certain embodiments, the tumor comprises a solid tumor.

[0234] In certain embodiments, the tumor is selected from at least one of tumors such as breast cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, ovarian cancer, pancreatic cancer, endometrial cancer, cervical squamous cell carcinoma, salivary gland tumor, bladder cancer, lung cancer, colorectal cancer, head and neck cancer, prostate cancer, osteosarcoma, childhood medulloblastoma, etc.

[0235] On the other hand, the present application provides a kit comprising the compound of formula (I) as described above or a pharmaceutically acceptable salt thereof, the immunoconjugate as described above, the radionuclide complex as described above, and / or the composition as described above.

[0236] Those skilled in the art can easily insight into other aspects and advantages of the present application from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the content of the present application enables those skilled in the art to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention involved in the present application. Accordingly, the descriptions in the drawings and the specification of the present application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0237] The specific features of the invention involved in the present application are shown as in the appended claims. The features and advantages of the invention involved in the present application can be better understood by referring to the exemplary embodiments and the drawings described in detail below. A brief description of the drawings is as follows:

[0238] Figure 1 Shows 68 In vitro human serum stability results of Ga-NOTA-Bn-MV(Dap)-PEG4-006.

[0239] Figure 2 Shows68 In vitro human serum stability results of Ga-NOTA-Bn-VMK-(CH2)2-006.

[0240] Figure 3 Shown is 68 In vivo urine stability results of Ga-NOTA-Bn-MV(Dap)-PEG4-006.

[0241] Figure 4 Shown is 68 In vivo urine stability results of Ga-NOTA-Bn-VMK-(CH2)2-006.

[0242] Figure 5 Shown are the distribution results of the radionuclide-labeled precursor described in this application in mice.

[0243] Figure 6 Shown are the kinetic results of the radionuclide-labeled precursor described in this application in mice.

[0244] Figure 7 Shown are the SPECT / CT images at each time point after administration of the radionuclide-labeled precursor described in this application to tumor model mice.

[0245] Figure 8 Shown is 177 ROI of SPECT / CT images of the radiolabeled drug Lu-DOTA-Bn-VMK-PEG4-SNA004. Detailed implementation mode

[0246] The following specific examples illustrate the implementation mode of the invention of this application. Those familiar with this technology can easily understand other advantages and effects of the invention of this application from the content disclosed in this specification.

[0247] Term definition

[0248] In this application, the term "hydrocarbyl", alone or as part of another substituent, means - unless otherwise specified - a straight-chain, branched-chain or cyclic hydrocarbon group or a combination thereof, which can be fully saturated, mono- or poly-unsaturated, and can include di- and poly-valent groups, having the specified number of carbon atoms (that is, C1-C 10(Means from one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers such as n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated hydrocarbon radicals are those having one or more double or triple bonds. Examples of unsaturated hydrocarbon radicals include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. The term "hydrocarbon radical" also means, unless otherwise noted, those hydrocarbon radical derivatives defined in detail below, such as "heterohydrocarbon radical".

[0249] In the present application, the term "alkyl" generally refers to a branched or unbranched saturated hydrocarbon radical. Suitably, the alkyl group contains from 1 to 100, preferably 3 to 30 carbon atoms, more preferably 5 to 25 carbon atoms. Exemplary alkyls include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl or hexyl. "Alkenyl" generally refers to a branched or unbranched hydrocarbon radical containing one or more carbon-carbon double bonds. Suitably, the alkenyl group contains from 2 to 30 carbon atoms, preferably 5 to about 25 carbon atoms. "Alkynyl" generally refers to a branched or unbranched hydrocarbon radical containing one or more carbon-carbon triple bonds. Suitably, the alkynyl group contains from about 3 to about 30 carbon atoms, such as from about 5 to about 25 carbon atoms.

[0250] In the present application, the term "halogen" generally refers to fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.

[0251] In the present application, the term "cycloalkyl" generally refers to an alicyclic moiety, suitably having 3, 4, 5, 6, 7 or 8 carbon atoms. The group may be a bridged or polycyclic system. More common cycloalkyls are monocyclic. The term includes references to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo[2.2.2]octyl, etc.

[0252] In the present application, the term "aryl" refers to an aromatic carbocyclic system, suitably containing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring carbon atoms. An aryl may be a polycyclic system having two or more rings, at least one of which is aromatic. The term includes references to groups such as phenyl, naphthyl, fluorenyl, azulyl, indenyl, anthracenyl, etc.

[0253] The prefix "(hetero)" in this text indicates that one or more carbon atoms of the group can be replaced by nitrogen, oxygen, phosphorus, silicon or sulfur. Heteroalkyl groups include, for example, alkoxy and alkylthio groups. The heterocycloalkyl or heteroaryl groups in this text can have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, at least one of which is selected from nitrogen, oxygen, phosphorus, silicon and sulfur. In particular, 3- to 10-membered rings or ring systems, more particularly 5- or 6-membered rings, which can be saturated or unsaturated. For example, selected from oxiranyl, aziridinyl, 1,2-oxathiolanyl, imidazolyl, thienyl, furyl, tetrahydrofuryl, pyranyl, thienyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chromenyl, 2H-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidinyl, piperazinyl, pyridazinyl, morpholinyl, thiomorpholinyl, especially thiomorpholinyl, indolizinyl, 1,3-dioxo-1,3-dihydroisoindolyl, 3H-indolyl, indolyl, benzimidazolyl, coumarinyl, indazolyl, triazolyl, tetrazolyl, purinyl, 4H-quinolyl, isoquinolyl, quinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl, benzofuranyl, dibenzofuranyl, benzothienyl, dibenzothienyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, quinazolyl, cinnamyl, pteridinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, piperidinyl, phenanthrolinyl, furyl, phenazinyl, phenothiazinyl, phenoxazinyl, chromenyl, isochromanyl, chromanyl, 3,4-dihydro-2H-isoquinolin-1-one, 3,4-dihydro-2H-isoquinolinyl, etc.

[0254] Unless otherwise defined in the claims, the term "optionally" as used herein means that the subsequent described event can occur or can not occur, and includes both the occurrence and non-occurrence of the event.

[0255] In the present application, the term "substituted" means that one or more, especially up to 5, more especially 1, 2 or 3 hydrogen atoms in the said moiety are independently substituted by the corresponding number of substituents. As used herein, the term "optionally substituted" includes substituted or unsubstituted. Of course, it should be understood that the substituents are only at positions where they are chemically possible, and those skilled in the art can determine (experimentally or theoretically) whether a particular substitution is possible without undue effort. For example, an amino or hydroxyl group with a free hydrogen may be unstable if bonded to a carbon atom with an unsaturated (e.g., alkene) bond. Preferably, the term "substituted" means that one or more, especially up to 5, more especially 1, 2 or 3 hydrogen atoms in the said moiety are independently substituted by the corresponding number of substituents selected from OH, SH, NH2, halogen, cyano, carboxyl, alkyl, cycloalkyl, aryl and heteroaryl. In addition, the substituents described herein can themselves be substituted by any substituent, subject to the above limitations on suitable substitutions recognized by those skilled in the art. Preferably, any of the above substituents can be further substituted by any of the above substituents, and each substituent can be further substituted by any of the above substituents.

[0256] Suitable substituents can include halogen atoms and haloalkyl groups, such as CF3 and CCl3; oxygen-containing groups, such as oxo, hydroxyl, carboxyl, carboxyalkyl, alkoxy, alkanoyl, alkanoyloxy, aryloxy, aroyl and aroyloxy; nitrogen-containing groups, such as amino, alkylamino, dialkylamino, cyano, azide and nitro; sulfur-containing groups, such as mercapto, alkylthiol, sulfonyl and sulfoxide; heterocyclic groups which can themselves be substituted; alkyl groups which can themselves be substituted; and aryl groups which can themselves be substituted, such as phenyl and substituted phenyl. Alkyl includes substituted and unsubstituted benzyl.

[0257] When two or more moieties are described as being "each independently" selected from a list of atoms or groups, this means that the moieties can be the same or different. Thus the identity of each moiety is independent of the identity of one or more of the other moieties.

[0258] In the present application, the term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861), i.e., bind to CD8α (such as human CD8α, murine CD8α, cynomolgus CD8α or rhesus CD8α). The antibody can be murine, human, humanized, chimeric, or derived from other species.

[0259] A full-length antibody typically refers to an antibody composed of two "full-length antibody heavy chains" and two "full-length antibody light chains". A "full-length antibody heavy chain" is usually a polypeptide that, in the N-terminal to C-terminal direction, consists of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CH1), an antibody hinge region (HR), an antibody constant heavy chain domain 2 (CH2), and an antibody constant heavy chain domain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3; and in the case of antibodies of the IgE subclass, optionally also includes an antibody constant heavy chain domain 4 (CH4). In some embodiments, a "full-length antibody heavy chain" is a polypeptide consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length antibody light chain" is usually a polypeptide that, in the N-terminal to C-terminal direction, consists of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), abbreviated as VL-CL. The antibody light chain constant domain (CL) can be kappa (κ) or lambda (λ). The two full-length antibody chains are linked together by an inter-polypeptide disulfide bond between the CL domain and the CH1 domain and an inter-polypeptide disulfide bond between the hinge region of the full-length antibody heavy chain. Examples of typical full-length antibodies are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE).

[0260] In the present application, the term "antigen-binding fragment" generally refers to a part of an antibody molecule that contains the amino acids responsible for the specific binding between the antibody and the antigen. The part of the antigen that is specifically recognized and bound by the antibody is called the "epitope" as described above. The antigen-binding domain typically can include the variable region of the antibody light chain (VL) and the variable region of the antibody heavy chain (VH); however, it does not necessarily have to include both. The Fd fragment, for example, has two VH regions and generally retains some antigen-binding function of the intact antigen-binding domain. Examples of antigen-binding fragments of an antibody include (1) Fab fragment, a monovalent fragment having VL, VH, constant light chain (CL), and CH1 domains; (2) F(ab’)2 fragment, a divalent fragment having two Fab fragments linked by a disulfide bridge in the hinge region; (3) Fd fragment having two VH and CH1 domains; (4) Fv fragment having VL and VH domains of a single arm of the antibody, (5) dAb fragment (Ward et al., "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli," Nature 341:544-546 (1989), which is incorporated herein by reference in its entirety), which has a VH domain; (6) isolated complementarity-determining regions (CDRs); (7) single-chain Fv (scFv), for example, derived from an scFV-library.Although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by recombinant methods using a synthetic linker, which allows them to be prepared as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv)) (see, for example, Huston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli," Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)); and (8) VHH, "VHH" refers to the variable antigen-binding domain from heavy-chain antibodies of the Camelidae family (camels, dromedaries, llamas, alpacas, etc.) (see Nguyen V.K. et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and review Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH can also be referred to as nanobody (Nb) and / or single-domain antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the function of the fragments is evaluated in the same manner as that of intact antibodies.

[0261] In the present application, the term "variable domain" generally refers to the variable domain of an antibody capable of specifically binding to an epitope. For example, the antibody variable domains VH and VL (VH domain and VL domain). Another example of a variable domain is the "VHH domain" (or simply "VHH"). The "VHH domain", also known as a heavy-chain single-domain antibody, VHH, V HThe H domain, VHH antibody fragment, and VHH antibody are variable domains of antigen-binding immunoglobulins known as "heavy-chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" is used to distinguish the variable domain from the heavy-chain variable domain present in conventional 4-chain antibodies (referred to herein as the "VH domain") and the light-chain variable domain present in conventional 4-chain antibodies (referred to herein as the "VL domain"). The VHH domain specifically binds to an epitope without the need for other antigen-binding domains (in contrast to the VH or VL domains in conventional 4-chain antibodies, where the epitope is recognized by the VL domain together with the VH domain).

[0262] In the present application, a "variable domain" generally has the same general structure, each domain containing four framework (FR) regions that are highly conserved in sequence, where the FR regions include four "framework regions" of "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", and are connected by three "complementary determining regions" or "CDRs" of "complementary determining region 1" or "CDR1", "complementary determining region 2" or "CDR2", and "complementary determining region 3" or "CDR3". The general structure or sequence of a variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The antibody variable domain confers antibody specificity for an antigen due to the presence of an antigen-binding site.

[0263] In the present application, the term "CDR" generally refers to the complementarity determining regions within the variable sequences of an antibody. There are three CDRs present in each variable region of the heavy and light chains, and for each variable region, they are designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991))) not only provides a clear residue numbering system applicable to any variable region of an antibody, but also provides the precise residue boundaries defining these three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and his colleagues (Chothia & Lesk, J. Mol. Biol. 196:901 - 917 (1987) and Chothia et al., Nature 342:877 - 883 (1989)) found that some sub - portions within the Kabat CDRs adopt nearly identical peptide backbone conformations, despite large differences at the amino acid sequence level. These sub - portions are designated L1, L2, and L3, or H1, H2, and H3, where "L" and "H" refer to the light chain and heavy chain regions, respectively. These regions may be called Chothia CDRs, which have boundaries overlapping with the Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described in Padlan (FASEB J. 9:133 - 139 (1995)) and MacCallum (J. Mol. Biol. 262(5):732 - 45 (1996)). Other CDR boundaries may not strictly follow one of the above - mentioned systems, but still overlap with the Kabat CDRs, and although they may be shortened or lengthened according to the following predictions or experimental findings, specific residues or groups of residues or even entire CDRs do not significantly affect antigen binding. Unless otherwise clearly stated in the specification, as used in the present application, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2", and "LCDR3" include the CDRs defined by any of the methods (Kabat, Chothia, or IMGT) described above.

[0264] In the present application, the term "sequence identity" generally refers to the nucleic acid or amino acid sequences that are the same when two or more aligned sequences are aligned using a sequence alignment program. The term "% sequence identity" herein generally refers to the level of nucleic acid or amino acid sequence identity between two or more aligned sequences when aligned using a sequence alignment program. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity. For the determination of sequence identity, reference can be made, for example, to: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0265] In the present application, amino acid residues will be represented according to the standard three-letter or one-letter amino acid codes well-known and agreed upon in the art. When comparing two amino acid sequences, the term "amino acid difference" generally refers to the insertion, deletion, or substitution of a specified number of amino acid residues at a particular position in a reference sequence compared to another sequence. In some embodiments, the substitution is a conservative amino acid substitution, where a conservative amino acid means that an amino acid residue is replaced by another amino acid residue with a similar chemical structure and has little or no effect on the function, activity, or other biological properties of the polypeptide. Conservative amino acid substitutions are well-known in the art. For example, a conservative amino acid substitution is the replacement of one amino acid within one of the following groups (i)-(v) by another amino acid residue within the same group: (i) small aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (iii) polar positively charged residues: His, Arg, and Lys; (iv) large aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (v) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative amino acid substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln, or Glu; Met is replaced by Leu, Tyr, or Ile; Phe is replaced by Met, Leu, or Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; Val is replaced by Ile or Leu. In some embodiments, the substitution is a non-conservative amino acid substitution. For example, Ala is replaced by Asp, Asn, Glu, or Gln.

[0266] In the present application, the term "affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a polypeptide or an antibody) and its binding partner (e.g., a target or an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, such as surface plasmon resonance, and also includes those methods reported in the present application. A higher affinity of molecule X for its binding partner Y is seen in lower Kd values and / or EC50 values.

[0267] In the present application, the term "isolated" generally refers to a molecule (such as an antibody, nucleic acid, etc.) that is at least partially separated from other molecules that are normally associated with it in its natural state. An "isolated polypeptide" is substantially free of other biomolecules such as nucleic acids, proteins, lipids, carbohydrates, cell debris, and growth media. An "isolated nucleic acid" is typically present in a form or context different from that in which it is naturally found.

[0268] In the present application, the term "immunoconjugate" generally refers to a conjugate formed by linking an antibody or an antibody fragment thereof to other active agents, such as chemotherapeutic agents, toxins, immunotherapeutic agents, radioactive elements, imaging probes, spectroscopic probes, and the like. The linkage can be a covalent bond or a non-covalent interaction, such as by electrostatic forces. A variety of linkers known in the art can be used to form the immunoconjugate. The conjugate can specifically bind to an antigen on a target cell (e.g., a tumor cell) through the antibody or its antigen-binding fragment, thereby delivering the other reagent to the target cell. In addition, the immunoconjugate can be provided in the form of a fusion protein, which can be expressed from a polynucleotide encoding the immunoconjugate.

[0269] In the present application, the term "chelating agent" generally refers to an organic molecule capable of forming a complex with a metal ion. Chelating agents are often used to label proteins or peptides. The end products of metal ion conjugates are used in radioimmunoassay, radioimmunotherapy, magnetic resonance imaging, photodynamic therapy, or other similar modalities. Non-limiting examples of chelating agents or complexing agents are DTPA (diethylenetriaminepentaacetic anhydride) and its derivatives, NOTA (1,4,7-triazacyclononane-N,N’,N”-triacetic acid) and its derivatives such as NODA-GA (NODAGA), Maleimide-NODAGA, DOTA (1,4,7,10-tetraazacyclododecane-N,N’,N”,N”’-tetraacetic acid) (for binding radioactive metal ions) and its derivatives, TETA (1,4,8,11-tetraazacyclotetradecane-N,N’,N”,N”’-tetraacetic acid) and its derivatives, DTTA (N-(p-isothiocyanatobenzyl)-diethylenetriamine-N,N’,N”,N”’-tetraacetic acid). These and other chelating agents are readily available from commercial sources.

[0270] In the present application, the term "detectable label" generally refers to a moiety having a detectable physical or chemical property, and the label can generate a signal that can be detected by visual or instrumental means. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotin groups (which can be detected by labeled avidin (e.g., a molecule containing a streptavidin moiety) and a fluorescent label or enzymatic activity detectable by optical or calorimetric means), and predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal-binding domains, epitope tags).

[0271] In the present application, the term "pharmaceutically acceptable salt" generally refers to salts including those commonly used to form alkali metal salts and addition salts of free acids or free bases. The nature of the salt is not critical as long as it is pharmaceutically acceptable. Suitable pharmaceutically acceptable acid addition salts of the compounds can be prepared from inorganic acids or from organic acids. Examples of such inorganic acids are hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid and phosphoric acid. Suitable organic acids can be selected from organic acids of the aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic or sulfonic acid categories, examples of which are formic acid, acetic acid, fatty acids, butyric acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, methanesulfonic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, pamoic acid, methanesulfonic acid, ethanesulfonic acid, ethanedisulfonic acid, benzenesulfonic acid, pantothenic acid, 2-hydroxyethanesulfonic acid, toluenesulfonic acid, p-aminobenzenesulfonic acid, cyclohexylaminosulfonic acid, camphoric acid, camphorsulfonic acid, digluconic acid, cyclopentanepropionic acid, dodecylsulfonic acid, glucoheptanoic acid, glycerophosphoric acid, heptanoic acid, hexanoic acid, 2-hydroxyethanesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid, oxalic acid, palmitic acid, pectinic acid, persulfuric acid, 2-phenylpropionic acid, picric acid, pivalic propionic acid, succinic acid, tartaric acid, thiocyanic acid, methanesulfonic acid, undecanoic acid, stearic acid, alginic acid, β-hydroxybutyric acid, salicylic acid, galactaric acid and galacturonic acid. Suitable pharmaceutically acceptable base addition salts of the compounds include metal salts such as those prepared from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc, or salts prepared from organic bases including primary amines, secondary amines and tertiary amines, substituted amines including cyclic amines such as caffeine, arginine, diethylamine, N-ethylpiperidine, histidine, glucosamine, isopropylamine, lysine, morpholine, N-ethylmorpholine, piperazine, piperidine, triethylamine, trimethylamine. All of these salts can be prepared from the compounds by conventional methods, for example by reacting a suitable acid or base with the corresponding compound of the present invention. Once the pharmaceutical composition is formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid or dehydrated or lyophilized powder. Such formulations can be stored in a ready-to-use form or in a form that requires reconstitution prior to administration (e.g., a lyophilized form).

[0272] In the present application, the term "pharmaceutically acceptable carrier" generally refers to one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient. Such formulations can routinely contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations can also contain compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans. Other contemplated carriers, excipients, and / or additives that can be used in the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweetening agents, antioxidants, antistatic agents, lipids, protein excipients (such as serum albumin, gelatin, casein), salt-forming balancing ions (such as sodium), and the like. These and other known pharmaceutical carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art, for example, as listed in "Remington: The Science & Practice of Pharmacy", 21st Edition, Lippincott Williams & Wilkins (2005) and "Physician's Desk Reference", 60th Edition, Medical Economics, Montvale, New Jersey (2005). Pharmaceutically acceptable carriers can be routinely selected that are suitable for the desired or required mode of administration, solubility, and / or stability.

[0273] In the present application, the terms "administer" and like terms generally are not limited to bodily administration, and suitable methods include ex vivo, ex vivo then in vivo, or in vivo methods. For example, any administration method known to those skilled in the art for contacting cells, organs, or tissues with a composition can be employed. For example, the compound can be introduced into the body of a subject in need of treatment by any introduction or delivery route. In some embodiments, the compositions of the present application can be administered orally, topically, intranasally, intramuscularly, subcutaneously, intradermally, intrathecally, intraperitoneally, or transdermally.

[0274] In the present application, the terms "ex vivo" and "in vitro" are interchangeable and generally refer to activities carried out in cells, tissues, and / or organs that have been removed from a subject's body in a controlled environment.

[0275] In the present application, the term "diagnosis" generally refers to detecting a disease or disorder, or determining the state or degree of a disease or disorder. The term "diagnosis" can also include detecting the cause of a disease or disorder, determining the therapeutic effect of a drug treatment, or predicting the response pattern to a drug treatment.

[0276] In the present application, the term "treatment" generally refers to: (i) preventing a disease, disorder, and / or condition from occurring in a patient who may be predisposed to the disease, disorder, and / or condition but has not been diagnosed with the disease; (ii) inhibiting the disease, disorder, and / or condition, i.e., arresting its development; and (iii) alleviating the disease, disorder, and / or condition, i.e., causing the disease, disorder, and / or condition and / or symptoms associated with the disease, disorder, and / or condition to subside.

[0277] In the present application, the terms "tumor" and "cancer" are used interchangeably and generally refer to neoplastic or malignant cell growth. The tumors of the present application may be benign or malignant. The tumors of the present application may be solid or non-solid.

[0278] In the present application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys, etc.

[0279] In the present application, the term "about" generally refers to a variation within a range of 0.5% - 10% above or below a specified value, for example, within a range of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% above or below the specified value.

[0280] In the present application, the term "comprising" and its variants, including other forms such as "containing" and "including", generally mean including other components, elements, values, steps, etc. Detailed Description of the Invention

[0282] Compound represented by formula (I)

[0283] On the one hand, the present application provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0284] X-L-Y-R1

[0285] Formula (I)

[0286] Wherein, X is a chelating agent;

[0287] L is a linker, and L may be absent;

[0288] Y is a polypeptide that can be cleaved by neutral endopeptidase (NEP); for example, Y is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide that can be cleaved by peptidase

[0289] R1 is a group that binds to the amino or carboxyl group of the side chain of Y and has a functional group capable of binding to a target molecule recognition unit or its linking group, or is a hydrogen atom of the amino or carboxyl group of the side chain of Y.

[0290] For example, where Y can be selected from: -Ser-leu-Lys-(SLK), -Ser-Phe-Lys-(SFK), -Asp-Phe-Lys-(DFK), -Val-Met-Lys-(VMK), -Met-Val(Dap)-(MV(Dap)), -Met-Val-(MV), -(D-Arg)-Ser-Phe-Lys-(rSFK), -Gly-Trp-Lys-(GWK), -Met-Asn-Lys-(MNK), -Gln-Leu-Lys-(QLK) and -Thr-ILe-Lys-(TIK).

[0291]

[0292]

[0293] In certain embodiments, R1 can independently consist of one or more of the following components:

[0294]

[0295]

[0296]

[0297] In certain embodiments, one or more selected from DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG3, MAG2, HYNIC, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA, HEHAA and their derivatives, analogs. The term "derivative" includes chemical modifications of a compound. Examples of such modifications include replacing hydrogen with a halogen group, an alkyl group, an acyl group or an amino group, etc. The modification can increase or decrease one or more hydrogen bond interactions, charge interactions, hydrophobic interactions, van der Waals interactions and / or dipole interactions. The term "analog" includes any enantiomers, racemates and stereoisomers of such compounds, as well as all pharmaceutically acceptable salts and hydrates.

[0298] For example, where X can be selected from the following structures:

[0299]

[0300] and their derivatives; wherein each R2 is independently a hydrogen atom or an optionally substituted C1-C8 hydrocarbyl group.

[0301] For example, X-L is selected from the following structures:

[0302]

[0303] wherein each R2 is independently a hydrogen atom or an optionally substituted C1-C8 hydrocarbyl group; n is any integer between 1 and 1000.

[0304] Again for example, the present application provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0305] X-L-Y-R1

[0306] Formula (I)

[0307] wherein, X may be selected from the following structures (L is absent):

[0308]

[0309] and their derivatives; wherein each R2 is independently a hydrogen atom or an optionally substituted C1-C8 hydrocarbyl group;

[0310] X-L is selected from the following structures:

[0311]

[0312] Wherein n is any integer between 1 and 1000. For example, n can be from 1 to 900, from 1 to 800, from 1 to 700, from 1 to 600, from 1 to 500, from 1 to 400, from 1 to 300, from 1 to 250, from 1 to 200, from 1 to 150, from 1 to 100, from 1 to 90, from 1 to 80, from 1 to 70, from 1 to 70, from 1 to 60, from 1 to 50, from 1 to 45, from 1 to 40, from 1 to 35, from 1 to 30, from 1 to 25, from 1 to 20, from 1 to 19, from 1 to 18, from 1 to 17, from 1 to 16, from 1 to 15, from 1 to 14, from 1 to 13, from 1 to 12, from 1 to 11, from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 10 to 900, from 10 to 800, from 10 to 700, from 10 to 600, from 10 to 500, from 10 to 400, from 10 to 300, from 10 to 250, from 10 to 200, from 10 to 150, from 10 to 100, from 10 to 90, from 10 to 80, from 10 to 70, from 10 to 70, from 10 to 60, from 10 to 50, from 10 to 45, from 10 to 40, from 10 to 35, from 10 to 30, from 10 to 25, from 10 to 20, from 10 to 19, from 10 to 18, from 10 to 17, from 10 to 16 or from 10 to 15;

[0313] Y can be selected from: -Ser-leu-Lys-(SLK), -Ser-Phe-Lys-(SFK), -Asp-Phe-Lys-(DFK), -Val-Met-Lys-(VMK), -Met-Val(Dap)-(MV(Dap)), -Met-Val-(MV), -(D-Arg)-Ser-Phe-Lys-(rSFK), -Gly-Trp-Lys-(GWK), -Met-Asn-Lys-(MNK), -Gln-Leu-Lys-(QLK) and -Thr-ILe-Lys-(TIK);

[0314] R1 can be selected from the following structures: 6-maleimidohexanoyl (MC), maleimidopropionyl (MP), maleimidoethylamine (ME), 6-maleimidohexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-VC-PAB), 4-(N-maleimidomethyl)cyclohexane-1-carbonyl (SMCC), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP).

[0315] Target molecule recognition unit

[0316] The immune conjugates of the present application and the like are compounds or their pharmacologically acceptable salts obtained by binding a target molecule recognition unit to a compound represented by formula (I) or its pharmaceutically acceptable salt. The target molecule recognition unit can be bound to the compound represented by formula (I) or its pharmaceutically acceptable salt through a linking group, or can be directly bound thereto.

[0317] In the present application, the term "target molecule recognition unit" generally refers to a molecule, substituent, functional group or atomic group that can bind to a target molecule in vivo and can recognize the target molecule. As the target molecule recognition unit, a polypeptide or other ligand that binds to the target molecule can be cited. The polypeptide is usually a polypeptide that binds to the target molecule, and is preferably a polypeptide that specifically binds to the target molecule. Specific binding means binding to the target molecule, but not binding or weakly binding to molecules other than the target molecule. The term "target molecule" generally refers to a target site that becomes a diagnostic object by radiolabeled drugs, such as a molecule present in tissues or cells, preferably a specifically expressed molecule. The term "specific expression" generally means expression at the target site, but not expression or low expression at sites other than the target site. As the target molecule recognition unit, for example, a ligand that binds to a protein highly expressed in tissue construction accompanied by inflammation, tumor cell infiltration, etc., or a protein specifically expressed in tumor cells, and an antibody and an antigen-binding region fragment of the antibody can be cited.

[0318] As the antibody, for example, monoclonal antibodies such as anti-CD8α antibody and anti-HER2 antibody can be cited. As the antigen-binding region fragment of the antibody, for example, Fab fragment (hereinafter also simply referred to as "Fab"), F(ab')2 fragment, F(ab)2 fragment, variable region fragment (hereinafter also referred to as "Fv fragment") can be cited.

[0319] As other target molecule recognition units, for example, a cyclic pentapeptide having an affinity for integrin highly expressed in tumor neovascularization, such as cyclo-Arg-Gly-Asp-D-Phe-Lys (hereinafter also referred to as "c(RGDfK)") can be cited. In addition, bisphosphonic acid, oligomeric aspartic acid, oligomeric glutamic acid having an affinity for hydroxyapatite abundantly present in osteocarcinoma (bone metastasis), a peptide having an affinity for the scavenger receptor present on the surface of macrophages, i.e., fMet-Leu-Phe (fMLP), folic acid and its derivatives that bind to the folate receptor expressed in tumor cells, etc. can be cited.

[0320] It should be noted that the target molecule recognition unit is not limited to these exemplified polypeptides, and any polypeptide that binds to the target molecule can be optionally used.

[0321] Regarding the target molecule recognition unit, for example, a thiolation reagent such as 2-iminothiolane can be used to introduce a linking group that reacts with the functional group of the compound and bind it. The introduction of the linking group into the Fab fragment can be achieved by reacting the thiolation reagent under the conditions of pH 7-9 to attach a mercapto group to the amino group on the Fab cross-section.

[0322] Immunoconjugate

[0323] On the other hand, the present application provides an immunoconjugate comprising i) the compound represented by the foregoing formula (I) or a pharmaceutically acceptable salt thereof and ii) a target molecule recognition unit.

[0324] In certain embodiments, the immunoconjugate has the structure represented by formula (II):

[0325]

[0326] Wherein, X is a chelating agent, and X is connected or not connected to an active moiety;

[0327] T is a target molecule recognition unit;

[0328] L is a linker, and L may be absent;

[0329] A1 and A2 are amino acid residues, and m is 0, 1 or 2;

[0330] A3 is an amino acid residue having an amino group or a carboxyl group on its side chain, and R1 is a group that binds to the amino group or carboxyl group on the side chain of A3 and has a functional group capable of binding to the target molecule recognition unit or its linking group;

[0331] -(A1) m -A2-A3- can be cleaved by enkephalinase. For example, wherein-(A1) m -A2-A3- can be selected from: -Ser-leu-Lys-(SLK), -Ser-Phe-Lys-(SFK), -Asp-Phe-Lys-(DFK), -Val-Met-Lys-(VMK), -Met-Val(Dap)-(MV(Dap)), -Met-Val-(MV), -(D-Arg)-Ser-Phe-Lys-(rSFK), -Gly-Trp-Lys-(GWK), -Met-Asn-Lys-(MNK), -Gln-Leu-Lys-(QLK) and -Thr-ILe-Lys-(TIK).

[0332] In certain embodiments, wherein X is selected from the following structures:

[0333]

[0334] and their derivatives; wherein, R2 is independently a hydrogen atom or an optionally substituted C1-C8 hydrocarbon group.

[0335] In certain embodiments, X-L is selected from the following structures:

[0336]

[0337] wherein n is any integer between 1 and 1000, for example, n can be 1 to 900, 1 to 800, 1 to 700, 1 to 600, 1 to 500, 1 to 400, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 10 to 900, 10 to 800, 10 to 700, 10 to 600, 10 to 500, 10 to 400, 10 to 300, 10 to 250, 10 to 200, 10 to 150, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 70, 10 to 60, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 19, 10 to 18, 10 to 17, 10 to 16 or 10 to 15.

[0338] In certain embodiments, R1 is selected from the following structures: -(CH2) n -Mal, -(CH2CH2O) n- Mal, -CO(CH2)n-Mal, -NH(CH2) n -Mal;

[0339] For example, the immunoconjugate may have the structure shown in formula (II):

[0340]

[0341] wherein, X can be selected from the following structures (L is absent):

[0342]

[0343] and their derivatives; wherein, R2 is independently a hydrogen atom or an optionally substituted C1-C8 hydrocarbon group;

[0344] Wherein X-L is selected from the following structures:

[0345]

[0346] Wherein n is any integer between 1 and 1000, for example, n can be 1 to 900, 1 to 800, 1 to 700, 1 to 600, 1 to 500, 1 to 400, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 10 to 900, 10 to 800, 10 to 700, 10 to 600, 10 to 500, 10 to 400, 10 to 300, 10 to 250, 10 to 200, 10 to 150, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 70, 10 to 60, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 19, 10 to 18, 10 to 17, 10 to 16 or 10 to 15;

[0347] T is a target molecule recognition unit;

[0348] -(A1) m -A2-A3- can be selected from: -Ser-leu-Lys-(SLK), -Ser-Phe-Lys-(SFK), -Asp-Phe-Lys-(DFK), -Val-Met-Lys-(VMK), -Met-Val(Dap)-(MV(Dap)), -Met-Val-(MV), -(D-Arg)-Ser-Phe-Lys-(rSFK), -Gly-Trp-Lys-(GWK), -Met-Asn-Lys-(MNK), -Gln-Leu-Lys-(QLK) and -Thr-ILe-Lys-(TIK);

[0349] The said R1 is selected from the following structures: -(CH2) n -Mal, -(CH2CH2O) n- Mal, -CO(CH2)n-Mal, -NH(CH2) n -Mal.

[0350] For example, the immunoconjugate may be selected from the following structures:

[0351] NOTA-Bn-SLK(Mal)-T,

[0352] NOTA-Bn-SFK-(CH2)2-Mal-T,

[0353] DOTA-Bn-SFK-(CH2)2-Mal-T,

[0354] NODAGA-SFK-(CH2)2-Mal-T,

[0355] NOTA-Bn-DFK-(CH2)2-Mal-T,

[0356] NOTA-Bn-VMK-(CH2)2-Mal-T,

[0357] DOTA-Bn-VMK-PEG4-Mal-T,

[0358] DOTA-PEG4-VMK-PEG4-Mal-T,

[0359] DOTA-VMK-(CH2)2-Mal-T,

[0360] DOTA-VMK-PEG4-Mal-T,

[0361] NOTA-Bn-MV(Dap)-PEG4-Mal-T,

[0362] DOTA-Bn-MV(Dap)-PEG4-Mal-T,

[0363] DOTA-PEG4-MV(Dap)-PEG4-Mal-T,

[0364] DOTA-MV(Dap)-PEG4-Mal-T,

[0365] NODAGA-MV(Dap)-PEG4-Mal-T,

[0366] DOTA-MV-(CH2)2-Mal-T,

[0367] DOTA-PEG4-MV-PEG4-Mal-T,

[0368] DOTA-Bn-MV-PEG4-Mal-T,

[0369] DOTA-Bn-rSFK-PEG4-Mal-T,

[0370] DOTA-Bn-GWK-PEG4-Mal-T,

[0371] DOTA-Bn-MNK-PEG4-Mal-T,

[0372] DOTA-Bn-QLK-PEG4-Mal-T and

[0373] DOTA-Bn-TIK-PEG4-Mal-T;

[0374] wherein T is a target molecule recognition unit, and the target molecule recognition unit includes an antigen-binding protein.

[0375] Also, for example, the chelator can be conjugated with the active moiety A, and the immunoconjugate can be selected from the following structures: A-NOTA-Bn-SLK(Mal)-T,

[0376] A-NOTA-Bn-SFK-(CH2)2-Mal-T,

[0377] A-DOTA-Bn-SFK-(CH2)2-Mal-T,

[0378] A-NODAGA-SFK-(CH2)2-Mal-T,

[0379] A-NOTA-Bn-DFK-(CH2)2-Mal-T,

[0380] A-NOTA-Bn-VMK-(CH2)2-Mal-T,

[0381] A-DOTA-Bn-VMK-PEG4-Mal-T,

[0382] A-DOTA-PEG4-VMK-PEG4-Mal-T,

[0383] A-DOTA-VMK-(CH2)2-Mal-T,

[0384] A-DOTA-VMK-PEG4-Mal-T,

[0385] A-NOTA-Bn-MV(Dap)-PEG4-Mal-T,

[0386] A-DOTA-Bn-MV(Dap)-PEG4-Mal-T,

[0387] A-DOTA-PEG4-MV(Dap)-PEG4-Mal-T,

[0388] A-DOTA-MV(Dap)-PEG4-Mal-T,

[0389] A-NODAGA-MV(Dap)-PEG4-Mal-T,

[0390] A-DOTA-MV-(CH2)2-Mal-T,

[0391] A-DOTA-PEG4-MV-PEG4-Mal-T,

[0392] A-DOTA-Bn-MV-PEG4-Mal-T,

[0393] A-DOTA-Bn-rSFK-PEG4-Mal-T,

[0394] A-DOTA-Bn-GWK-PEG4-Mal-T,

[0395] A-DOTA-Bn-MNK-PEG4-Mal-T,

[0396] A-DOTA-Bn-QLK-PEG4-Mal-T and

[0397] A-DOTA-Bn-TIK-PEG4-Mal-T;

[0398] wherein T is a target molecule recognition unit and A is an active moiety.

[0399] For example, the target molecule recognition unit may be an antibody or an antigen-binding fragment thereof. Alternatively, the target molecule recognition unit T may be a VHH antibody.

[0400] In certain embodiments, the immunoconjugate is selected from the following structures:

[0401] NOTA-Bn-SLK(Mal)-VHH,

[0402] NOTA-Bn-SFK-(CH2)2-Mal-VHH,

[0403] DOTA-Bn-SFK-(CH2)2-Mal-VHH,

[0404] NODAGA-SFK-(CH2)2-Mal-VHH,

[0405] NOTA-Bn-DFK-(CH2)2-Mal-VHH,

[0406] NOTA-Bn-VMK-(CH2)2-Mal-VHH,

[0407] DOTA-Bn-VMK-PEG4-Mal-VHH,

[0408] DOTA-PEG4-VMK-PEG4-Mal-VHH,

[0409] DOTA-VMK-(CH2)2-Mal-VHH,

[0410] DOTA-VMK-PEG4-Mal-VHH,

[0411] NOTA-Bn-MV(Dap)-PEG4-Mal-VHH,

[0412] DOTA-Bn-MV(Dap)-PEG4-Mal-VHH,

[0413] DOTA-PEG4-MV(Dap)-PEG4-Mal-VHH,

[0414] DOTA-MV(Dap)-PEG4-Mal-VHH,

[0415] NODAGA-MV(Dap)-PEG4-Mal-VHH,

[0416] DOTA-MV-(CH2)2-Mal-VHH,

[0417] DOTA-PEG4-MV-PEG4-Mal-VHH,

[0418] DOTA-Bn-MV-PEG4-Mal-VHH,

[0419] DOTA-Bn-rSFK-PEG4-Mal-VHH,

[0420] DOTA-Bn-GWK-PEG4-Mal-VHH,

[0421] DOTA-Bn-MNK-PEG4-Mal-VHH,

[0422] DOTA-Bn-QLK-PEG4-Mal-VHH and DOTA-Bn-TIK-PEG4-Mal-VHH.

[0423] For example, the immunoconjugate may be selected from the following structures: A-NOTA-Bn-SLK(Mal)-VHH, A-NOTA-Bn-SFK-(CH2)2-Mal-VHH, A-DOTA-Bn-SFK-(CH2)2-Mal-VHH, A-NODAGA-SFK-(CH2)2-Mal-VHH, A-NOTA-Bn-DFK-(CH2)2-Mal-VHH, A-NOTA-Bn-VMK-(CH2)2-Mal-VHH, A-DOTA-Bn-VMK-PEG4-Mal-VHH, A-DOTA-PEG4-VMK-PEG4-Mal-VHH, A-DOTA-VMK-(CH2)2-Mal-VHH, A-DOTA-VMK-PEG4-Mal-VHH, A-NOTA-Bn-MV(Dap)-PEG4-Mal-VHH, A-DOTA-Bn-MV(Dap)-PEG4-Mal-VHH, A-DOTA-PEG4-MV(Dap)-PEG4-Mal-VHH, A-DOTA-MV(Dap)-PEG4-Mal-VHH, A-NODAGA-MV(Dap)-PEG4-Mal-VHH, A-DOTA-MV-(CH2)2-Mal-VHH, A-DOTA-PEG4-MV-PEG4-Mal-VHH,

[0424] A-DOTA-Bn-MV-PEG4-Mal-VHH,

[0425] A-DOTA-Bn-rSFK-PEG4-Mal-VHH,

[0426] A-DOTA-Bn-GWK-PEG4-Mal-VHH,

[0427] A-DOTA-Bn-MNK-PEG4-Mal-VHH,

[0428] A-DOTA-Bn-QLK-PEG4-Mal-VHH and

[0429] A-DOTA-Bn-TIK-PEG4-Mal-VHH;

[0430] wherein A is the active moiety.

[0431] For example, the active moiety may be a detectable label, such as a radionuclide, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, and an enzyme. For another example, the active moiety may be a radionuclide.

[0432] For another example, the immunoconjugate may be selected from the following structures:

[0433] A-NOTA-Bn-SLK(Mal)-VHH,

[0434] A-NOTA-Bn-SFK-(CH2)2-Mal-VHH,

[0435] A-DOTA-Bn-SFK-(CH2)2-Mal-VHH,

[0436] A-NODAGA-SFK-(CH2)2-Mal-VHH,

[0437] A-NOTA-Bn-DFK-(CH2)2-Mal-VHH,

[0438] A-NOTA-Bn-VMK-(CH2)2-Mal-VHH,

[0439] A-DOTA-Bn-VMK-PEG4-Mal-VHH,

[0440] A-DOTA-PEG4-VMK-PEG4-Mal-VHH,

[0441] A-DOTA-VMK-(CH2)2-Mal-VHH,

[0442] A-DOTA-VMK-PEG4-Mal-VHH,

[0443] A-NOTA-Bn-MV(Dap)-PEG4-Mal-VHH,

[0444] A-DOTA-Bn-MV(Dap)-PEG4-Mal-VHH,

[0445] A-DOTA-PEG4-MV(Dap)-PEG4-Mal-VHH,

[0446] A-DOTA-MV(Dap)-PEG4-Mal-VHH,

[0447] A-NODAGA-MV(Dap)-PEG4-Mal-VHH,

[0448] A-DOTA-MV-(CH2)2-Mal-VHH,

[0449] A-DOTA-PEG4-MV-PEG4-Mal-VHH,

[0450] A-DOTA-Bn-MV-PEG4-Mal-VHH,

[0451] A-DOTA-Bn-rSFK-PEG4-Mal-VHH,

[0452] A-DOTA-Bn-GWK-PEG4-Mal-VHH,

[0453] A-DOTA-Bn-MNK-PEG4-Mal-VHH,

[0454] A-DOTA-Bn-QLK-PEG4-Mal-VHH and

[0455] A-DOTA-Bn-TIK-PEG4-Mal-VHH;

[0456] wherein A may include 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 67 Cu, 67 Ga, 68 Ga, 68 Ge, 86 Y, 90 Y, 89 Zr, 94m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C, 13 N, 15 O, 186 Re, 188 Re, 51 Mn, 52m Mn, 72 As, 75 Br, 76 Br, 82m Rb, 83 Sr, 225 Ac, 211 At or other α, γ, β-, or positron emitters.

[0457] Radioactive complex

[0458] In the present application, the term "radioactive complex" generally refers to any complex that includes a radioisotope or radionuclide, such as any radioisotope or radionuclide described herein. The term "complex" generally refers to a substance obtained by coordination of a ligand with an atom or ion of a radionuclide and radionuclide-like element, also known as a coordination compound. Coordination refers to the formation of a coordination bond between a ligand and a central metal and the arrangement of the ligand around the central metal. A complex is formed by a coordination bond between a ligand and a metal. The formation of a complex by a ligand and a metal is also referred to as complexation. A coordination bond refers to a bond in which the two valence electrons participating in a bond are provided by only one atom.

[0459] As used herein, the term "radionuclide" refers to an atom that is capable of undergoing radioactive decay (e.g., 3 H, 14 C, 15 N, 18 F, 35 S, 47 Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 75 Br, 76 Br, 77 Br, 89 Zr, 86 Y, 87 Y,

[0460] 90 Y, 97 Ru, 99 Tc, 99m Tc 105 Rh, 109 Pd, 111 In, 123 I, 124 I, 125 I, 131 I, 149 Pm, 149 Tb, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 198 Au, 199 Au, 203 Pb, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra,

[0461] 225 Ac, 227 Th, 229Th , 66 Ga, 67 Ga, 68 Ga, 82 Rb, 117m Sn, 201 Tl). The terms radionuclide, radioactive isotope or radioisotope may also be used to describe a radionuclide. A radionuclide can be used as a detection agent as described above. In some embodiments, the radionuclide can be an α, γ, β−, or positron-emitting radionuclide.

[0462] The radionuclide is not limited to these specific examples, and can be optionally used as long as it has radiation, radiation dose, and half-life suitable for diagnosis using a radiolabeled drug, etc. From the viewpoint of reducing the influence on normal tissues and cells in radiographic imaging diagnosis, it is preferable to use a short half-life metal radioisotope.

[0463] The preparation of the radioactive complex can be achieved by using the compound bound to the target molecule recognition unit as a ligand and complexing it with a metal radioisotope in vitro. The complexation can be achieved by a simple operation using a conventionally known complexation reaction.

[0464] Pharmaceutical composition

[0465] The compounds described in the present application may be in the form of a pharmaceutical composition, which can be used in humans and animals in human and veterinary medicine and generally contains any one or more pharmaceutically acceptable diluents, carriers or excipients. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Company (edited by A.R. Gennaro 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected according to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may contain, as a carrier, excipient or diluent or any other suitable binder, lubricant, suspending agent, coating agent, solubilizing agent.

[0466] Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and parabens. Antioxidants and suspending agents can also be used.

[0467] Depending on the different delivery systems, there may be different composition / formulation requirements. For example, a pharmaceutical composition can be formulated for administration using a micropump or via a mucosal route, e.g., as a nasal spray or aerosol for inhalation or an ingestible solution, or parenterally, where the composition is formulated in an injectable form for delivery, e.g., via intravenous, intramuscular or subcutaneous routes. Optionally, the formulation can be designed for administration via multiple routes.

[0468] If the agent is to be administered via the gastrointestinal mucosa, it should be able to remain stable during passage through the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acidic pH, and resistant to the detergency of bile.

[0469] When appropriate, the pharmaceutical composition can be administered by inhalation, in the form of a suppository or vaginal suppository, topically in the form of a lotion, solution, cream, ointment or powder, by using a transdermal patch, orally in the form of a tablet containing excipients (such as starch or lactose), or in the form of a capsule or ovule, alone or mixed with excipients, or in the form of an elixir, a solution or suspension containing flavoring or coloring agents, or the pharmaceutical composition can be injected parenterally, e.g., intravenously, intramuscularly or subcutaneously. For parenteral administration, the composition is preferably used in the form of a sterile aqueous solution, which may contain other substances, e.g., sufficient salts or monosaccharides to render the solution isotonic with blood. For oral or sublingual administration, the composition can be administered in the form of a tablet or lozenge, which can be formulated in a conventional manner.

[0470] The compounds of the present invention can be administered in the form of pharmaceutically acceptable salts or active salts. Pharmaceutically acceptable salts are well known to those skilled in the art and include, for example, those mentioned by Berge et al., Journal of Pharmaceutical Sciences (J. Pharm. Sci.), 66, 1-19 (1977). The salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, mesylate, esylate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts.

[0471] The route of administration (delivery) can include, but is not limited to, one or more of the following: oral (e.g., as tablets, capsules or as an ingestible solution), topical, mucosal (e.g., as a nasal spray or aerosol for inhalation), nasal, parenteral (e.g., by injection), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intraventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, oral, vaginal, epidural, sublingual.

[0472] Generally, a physician will determine the actual dose most suitable for an individual subject. The specific dosage level and frequency of administration for any particular patient may vary and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and duration of action of that compound, age, body weight, general health status, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular disorder, and the individual undergoing therapy.

[0473] The formulations can be packaged in unit dose or multi-dose containers, such as sealed ampoules and vials, and can be stored under lyophilized (freeze-dried) conditions, requiring only the addition of a sterile liquid carrier, such as water, for administration. Ready-to-use injection solutions and suspensions are prepared from sterile powders, granules and tablets of the foregoing types. Exemplary unit dose formulations contain the daily dose or the daily unit sub-dose of the active ingredient or an appropriate fraction thereof.

[0474] The pharmaceutical composition can be provided in lyophilized form. The lyophilized pharmaceutical composition is preferably reconstituted in a suitable buffer, advantageously based on an aqueous carrier, prior to administration.

[0475] The pharmaceutical composition of the present invention also provides a medicament for preparing a medicament for reducing the nephrotoxic side effects of radiolabeled and non-radiolabeled therapeutic and diagnostic compounds.

[0476] The pharmaceutical composition or medicament is preferably used for reducing the nephrotoxic side effects of radiolabeled therapeutic and diagnostic compounds for imaging or treating diseases, particularly tumor diseases, such as neuroendocrine tumors, prostate cancer, pancreatic cancer, renal cancer, bladder cancer, brain cancer, gastrointestinal cancer, medullary thyroid cancer, small cell or non-small cell lung cancer, and stromal ovarian cancer, pancreatic ductal adenocarcinoma, insulinoma, gastrinoma, breast cancer, or sarcoma.

[0477] Kit

[0478] On the other hand, the present application also provides a kit, which includes pharmaceutical ingredients used according to the present application, such as the compounds represented by formula (I) as described in the present application or pharmaceutically acceptable salts thereof, radiolabeled or non-radiolabeled therapeutic or diagnostic compounds represented by formula (I), the immunoconjugates described in the present application, the radionuclide complexes described in the present application, and / or the pharmaceutical compositions described in the present application.

[0479] Optionally, the kit may include at least one other reagent as defined herein in the context of pharmaceutical compositions, including amino acids such as lysine and arginine and mixtures thereof, gelatin, amifostine, albumin-derived peptides, PSMA-binding molecules (such as PMPA), vitamins, radionuclides, antimicrobials, solubilizing agents, etc.

[0480] The kit can be a two-part or multi-part kit containing any of the above-listed components in a suitable container. For example, each container can be in the form of a vial, bottle, squeeze bottle, jar, sealed sleeve, envelope or sac, tube, or blister pack or any other suitable form, provided that the container preferably prevents premature mixing of the components. Each different component can be provided separately, or some different components can be provided together (i.e., in the same container).

[0481] The container can also be a vial, tube, jar, or envelope, or sleeve, or blister pack or a compartment or chamber within a bottle, provided that the contents of one compartment cannot be physically associated with the contents of another compartment until intentionally mixed by a pharmacist or doctor.

[0482] The kit or part of the kit may also contain a technical manual, which contains information about the administration and dosage of any of its components.

[0483] Therapeutic and diagnostic methods and uses

[0484] The compounds described in the present application can be used to treat diseases. The treatment can be therapeutic and / or prophylactic treatment, aiming to prevent, reduce or stop undesired physiological changes or conditions. Compared with the expected survival period of those not receiving treatment, the treatment can extend the survival period.

[0485] The diseases treated by the compound can be any diseases that may benefit from the treatment. This includes chronic and acute conditions or diseases, including those pathological conditions prone to the condition.

[0486] The terms "cancer" and "cancerous" are used in their broadest sense, meaning a physiological condition in mammals that is typically characterized by unregulated cell growth. A tumor contains one or more cancer cells.

[0487] In the treatment of cancer, the observed therapeutic effects can be a decrease in the number of cancer cells; a reduction in tumor size; inhibition or retardation of cancer cell invasion into peripheral organs; inhibition of tumor growth; and / or alleviation of one or more symptoms associated with cancer.

[0488] In animal models, efficacy can be evaluated by physical measurement of the tumor during treatment and / or by determining partial and complete remission of cancer. For cancer therapies, efficacy can be measured, for example, by evaluating time to progression (TTP) and / or determining response rate (RR).

[0489] Particularly preferred embodiments of the treatment methods according to the invention are shown in the appended claims.

[0490] This application also discloses methods of treating a human or animal body, such as by surgery or therapy, or diagnostic methods practiced on a human or animal body, which methods involve the step of administering a therapeutically or diagnostically effective amount of a compound or pharmaceutical composition described herein to a subject in need thereof. More specifically, methods for treating (e.g., by treating or preventing) a subject having or at risk of having a disease or disorder are disclosed herein; or guiding surgery practiced on a subject having or at risk of having a disease or disorder; methods for diagnosing a disease or disorder, e.g., diagnostic methods practiced on a human or animal body and / or involving nuclear medicine imaging techniques (such as positron emission tomography (PET) or single photon emission computed tomography (SPECT)); methods for targeted delivery of a therapeutic or diagnostic agent to a subject having or at risk of having a disease or disorder. In the foregoing methods, the disease or disorder can independently be selected from cancer, inflammation, atherosclerosis, fibrosis, tissue remodeling, and keloid disorders, preferably wherein the cancer is selected from the group consisting of: breast cancer, pancreatic cancer, small intestine cancer, colon cancer, multi-drug resistant colon cancer, rectal cancer, colorectal cancer, metastatic colorectal cancer, lung cancer, non-small cell lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocarcinoma, renal clear cell carcinoma, neuroendocrine tumors, oncogenic osteomalacia, sarcoma, CUP (cancer of unknown primary), thymic cancer, desmoid tumors, glioma, astrocytoma, cervical cancer, skin cancer, renal cancer, and prostate cancer. When used in the methods disclosed herein, the compound has an extended residence time at the disease site at therapeutically or diagnostically relevant levels, preferably more than 1 h after injection, more preferably more than 6 h.

[0491] On the other hand, the present invention relates to the use of a compound of formula (I) as described above or a pharmaceutically acceptable salt thereof, the immunoconjugate as described above, the radionuclide complex as described above, and / or the composition as described above for use as a medicament for reducing the nephrotoxic side effects of radiolabeled and non-radiolabeled therapeutic and diagnostic compounds in a subject.

[0492] Also relates to a pharmaceutical composition or a kit as described above for use in a method of reducing the nephrotoxic side effects of radiolabeled and unradiolabeled therapeutic and diagnostic compounds in a subject.

[0493] In a further aspect, the present application also provides a method of reducing the nephrotoxic side effects of radiolabeled and unradiolabeled therapeutic and diagnostic compounds in a subject, the method comprising administering to the subject the pharmaceutical composition or the kit as described above during imaging or therapy with a radiolabeled and / or unradiolabeled compound.

[0494] In a further aspect, the present application also provides a method of reducing the nephrotoxic side effects of radiolabeled and unradiolabeled therapeutic and diagnostic compounds in a subject, the method comprising administering to the subject the compound represented by the foregoing formula (I) or a pharmaceutically acceptable salt thereof, the foregoing immunoconjugate, the foregoing radionuclide complex and / or the foregoing composition, wherein the administration is before and / or during and / or after the administration of a radiolabeled or unradiolabeled therapeutic or diagnostic compound.

[0495] Method for preparing a conjugate

[0496] The present application also discloses a method for preparing a conjugate, the method comprising the step of conjugating a compound represented by the foregoing formula (I) with a target molecule recognition unit. Non-limitingly, the compound represented by formula (I) is conjugated with the target molecule recognition unit by reacting therewith to form a covalent bond.

[0497] The conjugate can be a therapeutic agent and / or a diagnostic agent and can correspond to the payload portion as detailed above with respect to the conjugates according to the invention. The method further comprises formulating the conjugate into a pharmaceutical composition or a diagnostic composition. The pharmaceutical or diagnostic composition can be used in humans and veterinary medicine for humans or animals and generally comprises any one or more pharmaceutically acceptable diluents, carriers or excipients. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Company (edited by A.R. Gennaro 1985). The choice of carrier, excipient or diluent can be selected according to the intended route of administration and standard pharmaceutical practice. The pharmaceutical or diagnostic composition can comprise, as a carrier, excipient or diluent or in addition any suitable binder, lubricant, suspending agent, coating agent, solubilizing agent. All of the formulation details and aspects disclosed in the above "pharmaceutical composition" section also apply fully herein.

[0498] Unless otherwise indicated, the practice of the present invention employs conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology, and pharmacology, which are known to those of ordinary skill in the art. These techniques are well explained in the literature. See, for example, Gennaro, A.R., ed. (1990) Remington: The Science and Practice of Pharmacy, 18th ed., Mack Publishing Co.; Hardman, J.G., Limbird, L.E., and Gilman, A.G., eds. (2001) The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill Co.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press; Weir, D.M. and Blackwell, C.C., eds. (1986) Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications; Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al., eds. (1999) Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press; Newton, C.R. and Graham, A., eds. (1997) PCR (Introduction to Biotechniques Series), 2nd ed., Springer Verlag.

[0499] This application also includes the following embodiments:

[0500] 1. A compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0501] X-L-Y-R1

[0502] Formula (I)

[0503] wherein X is a chelating agent;

[0504] L is a linker, and L can be absent;

[0505] Y is a polypeptide that can be cleaved by neutral endopeptidase (NEP);

[0506] R1 is a group that binds to the amino or carboxyl group of the side chain of Y and has a functional group capable of binding to the target molecule recognition unit or its linking group, or is a hydrogen atom of the amino or carboxyl group of the side chain of Y.

[0507] 2. The compound of formula (I) according to Embodiment 1 or a pharmaceutically acceptable salt thereof, wherein Y is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide or decapeptide.

[0508] 3. The compound of formula (I) according to any one of Embodiments 1-2 or a pharmaceutically acceptable salt thereof, wherein Y is: -(A1)m-A2-A3-, where m is 0, 1 or 2; A1 and A2 are amino acid residues, A3 is an amino acid residue having an amino or carboxyl group on the side chain, and R1 is a group that binds to the amino or carboxyl group of the side chain of A3 and has a functional group capable of binding to the target molecule recognition unit or its linking group, or is a hydrogen atom of the amino or carboxyl group of the side chain of A3.

[0509] 4. The compound of formula (I) according to any one of Embodiments 1-3 or a pharmaceutically acceptable salt thereof, wherein Y is selected from: -Ser-leu-Lys-(SLK), -Ser-Phe-Lys-(SFK), -Asp-Phe-Lys-(DFK), -Val-Met-Lys-(VMK), -Met-Val(Dap)-(MV(Dap)), -Met-Val-(MV), -(D-Arg)-Ser-Phe-Lys-(rSFK), -Gly-Trp-Lys-(GWK), -Met-Asn-Lys-(MNK), -Gln-Leu-Lys-(QLK) and -Thr-ILe-Lys-(TIK).

[0510] 5. The compound of formula (I) according to any one of Embodiments 1-4 or a pharmaceutically acceptable salt thereof, wherein R1 is composed of one or more of the following components: -C1-C 18 alkylene, -(CH2CH2O) n -, -CO(CH2) n -, -

[0511] NH(CH2) n-, arylthio (PYS), p-aminobenzyloxycarbonyl (PAB), aminobenzylthio, oxybenzylthio, alkoxyamino (AOA), dioxobenzylthio, diaminobenzylthio, aminooxybenzylthio, alkoxyamino

[0512] (AOA), 4-methyl-4-dithiovaleryl (MPDP), triazole, dithio, sulfonyl, phosphonyl, (4-acetyl) aminobenzoyl (SIAB), 4-thiobutyryl, 4-thio-2-sulfonatobutyryl (2-SO3 - -SPDB), 4-thiopropionyl (SPDP), hydrazone, aminoethylamine, hydrazine, oxime, thioaminoxycrotonic acid, thioaminoxybutyric acid, a peptide containing 1 to 20 amino acid residues, and where n is any integer between 1 and 1000.

[0513] 6. The compound of formula (I) according to any one of embodiments 1-5 or a pharmaceutically acceptable salt thereof, wherein said R1 is selected from the following structures: -(CH2) n -Mal, -(CH2CH2O) n- Mal, -CO(CH2)n-Mal, -NH(CH2) n -Mal, where n is any integer between 1 and 1000.

[0514] 7. The compound of formula (I) according to any one of embodiments 1-6 or a pharmaceutically acceptable salt thereof, wherein said R1 is selected from the following structures: 6-maleimidohexanoyl (MC), maleimidopropionyl (MP), maleimidoethylamine (ME), 6-maleimidohexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-VC-PAB), 4-(N-maleimidomethyl) cyclohexane-1-carbonyl (SMCC), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP).

[0515] 8. The compound of formula (I) according to embodiment 6 or a pharmaceutically acceptable salt thereof, wherein said chelating agent includes one or more selected from DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG3, MAG2, HYNIC, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA, HEHAA and their derivatives.

[0516] 9. The compound of formula (I) according to any one of Embodiments 1-8 or a pharmaceutically acceptable salt thereof, wherein X is selected from the following structures:

[0517]

[0518] and their derivatives; wherein each R2 is independently a hydrogen atom or an optionally substituted C1-

[0519] C8 hydrocarbyl.

[0520] 10. The compound of formula (I) according to any one of Embodiments 1-9 or a pharmaceutically acceptable salt thereof, wherein X is selected from the following structures:

[0521]

[0522] and their derivatives.

[0523] 11. The compound of formula (I) according to any one of Embodiments 1-10 or a pharmaceutically acceptable salt thereof, wherein L is absent or L is selected from the following structures:

[0524] wherein n is any integer from 0 to 10.

[0525] 12. The compound of formula (I) according to any one of Embodiments 1-11 or a pharmaceutically acceptable salt thereof, wherein L is absent or L is selected from the following structures:

[0526]

[0527] 13. The compound of formula (I) according to any one of Embodiments 1-12 or a pharmaceutically acceptable salt thereof, wherein

[0528] X-L is selected from the following structures:

[0529]

[0530] wherein each R2 is independently a hydrogen atom or an optionally substituted C1-C8 hydrocarbyl, and n is any integer between 1 and 1000.

[0531] 14. The compound of formula (I) according to any one of Embodiments 1-13 or a pharmaceutically acceptable salt thereof, wherein

[0532] X-L is selected from the following structures:

[0533]

[0534] 15. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of Embodiments 1-14, wherein the compound of formula (I) is selected from: X-L-SLK(Mal), X-L-SFK-(CH2)2-Mal, X-L-SFK-(CH2)2-Mal, X-SFK-(CH2)2-Mal, X-L-DFK-(CH2)2-Mal, X-L-VMK-(CH2)2-Mal, X-L-VMK-PEG4-Mal, X-L-VMK-PEG4-Mal, X-VMK-(CH2)2-Mal, X-VMK-PEG4-Mal, X-L-MV(Dap)-PEG4-Mal, X-L-MV(Dap)-PEG4-Mal, X-L-MV(Dap)-PEG4-Mal, X-MV(Dap)-PEG4-Mal, X-MV(Dap)-PEG4-Mal, X-MV-(CH2)2-Mal, X-L-MV-PEG4-Mal, X-L-MV-PEG4-Mal, X-L-rSFK-PEG4-Mal, X-L-GWK-PEG4-Mal, X-L-MNK-PEG4-Mal, X-L-QLK-PEG4-Mal and X-L-TIK-PEG4-Mal;

[0535] Wherein, X is selected from:

[0536]

[0537] X-L is selected from:

[0538]

[0539]

[0540] 16. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of Embodiments 1-15, wherein

[0541] The compound of formula (I) is selected from: NOTA-Bn-SLK(Mal), NOTA-Bn-SFK-(CH2)2-Mal,

[0542] DOTA-Bn-SFK-(CH2)2-Mal, NODAGA-SFK-(CH2)2-Mal, NOTA-Bn-DFK-(CH2)2-Mal,

[0543] NOTA-Bn-VMK-(CH2)2-Mal, DOTA-Bn-VMK-PEG4-Mal, DOTA-PEG4-VMK-PEG4-

[0544] Mal, DOTA-VMK-(CH2)2-Mal, DOTA-VMK-PEG4-Mal, NOTA-Bn-MV(Dap)-PEG4-

[0545] Mal, DOTA-Bn-MV(Dap)-PEG4-Mal, DOTA-PEG4-MV(Dap)-PEG4-Mal, DOTA-

[0546] MV(Dap)-PEG4-Mal, NODAGA-MV(Dap)-PEG4-Mal, DOTA-MV-(CH2)2-Mal, DOTA-

[0547] PEG4-MV-PEG4-Mal, DOTA-Bn-MV-PEG4-Mal, DOTA-Bn-rSFK-PEG4-Mal, DOTA-Bn-

[0548] GWK-PEG4-Mal, DOTA-Bn-MNK-PEG4-Mal, DOTA-Bn-QLK-PEG4-Mal, and DOTA-Bn-TIK-PEG4-Mal.

[0549] 17. An immunoconjugate comprising i) a compound of formula (I) as described in any one of embodiments 1-16 or a pharmaceutically acceptable salt thereof and ii) a target molecule recognition unit.

[0550] 18. The immunoconjugate according to embodiment 17, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is bound to the target molecule recognition unit or a linking group of the target molecule recognition unit through R1.

[0551] 19. The immunoconjugate according to any one of embodiments 17-18, the immunoconjugate further comprising an active moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a viral coat protein, a VLP, or a combination thereof, the active moiety being linked to a chelator.

[0552] 20. The immunoconjugate according to any one of embodiments 17-19, wherein the detectable label is selected from one or more reagents of the group consisting of: a radionuclide, a fluorophore, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, and an enzyme.

[0553] 21. The immunoconjugate according to any one of embodiment 20, wherein the radionuclide is suitable for medical imaging and / or therapy.

[0554] 22. The immunoconjugate according to any one of embodiments 20-21, wherein the radionuclide comprises 110 In,

[0555] 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 67 Cu, 67 Ga, 68 Ga, 68 Ge, 86 Y, 90 Y, 89 Zr, 94m Tc, 120 I,

[0556] 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C, 13 N, 15 O, 186 Re, 188 Re, 51 Mn, 52m Mn,

[0557] 72 As, 75 Br, 76 Br, 82m Rb, 83 Sr, 225 Ac, 211 At or other α, γ, β-, or positron emitters.

[0558] 23. The immunoconjugate according to any one of embodiments 17-22, which has the structure shown in formula (II):

[0559]

[0560] Wherein, X is a chelating agent, and X is connected or not connected to the active moiety;

[0561] T is a target molecule recognition unit;

[0562] L is a linker, and L may be absent;

[0563] A1 and A2 are amino acid residues, and m is 0, 1 or 2;

[0564] A3 is an amino acid residue having an amino group or a carboxyl group on the side chain, and R1 is a group that binds to the amino group or carboxyl group of the side chain of A3 and has a functional group capable of binding to a target molecule recognition unit or its linking group;

[0565] -(A1) m -A2-A3- can be cleaved by enkephalinase.

[0566] 24. The immunoconjugate according to embodiment 23, wherein -(A1) m -A2-A3- is selected from: -Ser-leu-Lys-(SLK), -Ser-Phe-Lys-(SFK), -Asp-Phe-Lys-(DFK), -Val-Met-Lys-(VMK), -Met-Val(Dap)-(MV(Dap)),

[0567] -Met-Val-(MV), -(D-Arg)-Ser-Phe-Lys-(rSFK), -Gly-Trp-Lys-(GWK), -Met-Asn-Lys-

[0568] (MNK), -Gln-Leu-Lys-(QLK) and -Thr-ILe-Lys-(TIK).

[0569] 25. The immunoconjugate according to any one of embodiments 23-24, wherein the X is selected from the following structures:

[0570]

[0571] and their derivatives; wherein, R2 is independently a hydrogen atom or an optionally substituted C1-C8

[0572] hydrocarbyl group.

[0573] 26. The compound of formula (I) according to any one of embodiments 23-25 or a pharmaceutically acceptable salt thereof, wherein the X is selected from the following structures:

[0574]

[0575] and their derivatives.

[0576] 27. The immunoconjugate according to any one of embodiments 23-26, wherein the L is absent or L is selected from the following structures:

[0577] wherein n is any integer from 0 to 10.

[0578] 28. The immunoconjugate according to any one of embodiments 23-27, wherein L is absent or L is selected from the following structures:

[0579]

[0580] 29. The immunoconjugate according to any one of embodiments 23-28, wherein X-L is selected from the following structures:

[0581]

[0582] wherein each R2 is independently a hydrogen atom or an optionally substituted C1-C8 hydrocarbon group, and n is any integer between 1 and 1000.

[0583] 30. The immunoconjugate according to any one of embodiments 23-29, wherein X-L is selected from the following structures:

[0584]

[0585] 31. The immunoconjugate according to any one of embodiments 23-30, which is selected from the following structures:

[0586]

[0587]

[0588] wherein T is a targeting molecular unit;

[0589] -(A1) m -A2-A3- is selected from: -Ser-leu-Lys-(SLK), -Ser-Phe-Lys-(SFK), -Asp-Phe-Lys-(DFK),

[0590] -Val-Met-Lys-(VMK), -Met-Val(Dap)-(MV(Dap)), -Met-Val-(MV), -(D-Arg)-Ser-Phe-

[0591] Lys-(rSFK), -Gly-Trp-Lys-(GWK), -Met-Asn-Lys-(MNK), -Gln-Leu-Lys-(QLK) and

[0592] -Thr-ILe-Lys-(TIK);

[0593] The R1 is selected from the following structures: -(CH2) n -Mal, -(CH2CH2O) n-Mal, -CO(CH2)n-Mal,

[0594] -NH(CH2) n -Mal;

[0595] n is each independently any integer between 1 and 1000;

[0596] Each R2 is independently a hydrogen atom or an optionally substituted C1-C8 hydrocarbon group.

[0597] 32. The immunoconjugate according to any one of Embodiments 23-31, which is selected from the following structures:

[0598] NOTA-Bn-SLK(Mal)-T,

[0599] NOTA-Bn-SFK-(CH2)2-Mal-T,

[0600] DOTA-Bn-SFK-(CH2)2-Mal-T,

[0601] NODAGA-SFK-(CH2)2-Mal-T,

[0602] NOTA-Bn-DFK-(CH2)2-Mal-T,

[0603] NOTA-Bn-VMK-(CH2)2-Mal-T,

[0604] DOTA-Bn-VMK-PEG4-Mal-T,

[0605] DOTA-PEG4-VMK-PEG4-Mal-T,

[0606] DOTA-VMK-(CH2)2-Mal-T,

[0607] DOTA-VMK-PEG4-Mal-T,

[0608] NOTA-Bn-MV(Dap)-PEG4-Mal-T,

[0609] DOTA-Bn-MV(Dap)-PEG4-Mal-T,

[0610] DOTA-PEG4-MV(Dap)-PEG4-Mal-T,

[0611] DOTA-MV(Dap)-PEG4-Mal-T,

[0612] NODAGA-MV(Dap)-PEG4-Mal-T,

[0613] DOTA-MV-(CH2)2-Mal-T,

[0614] DOTA-PEG4-MV-PEG4-Mal-T,

[0615] DOTA-Bn-MV-PEG4-Mal-T,

[0616] DOTA-Bn-rSFK-PEG4-Mal-T,

[0617] DOTA-Bn-GWK-PEG4-Mal-T,

[0618] DOTA-Bn-MNK-PEG4-Mal-T,

[0619] DOTA-Bn-QLK-PEG4-Mal-T and

[0620] DOTA-Bn-TIK-PEG4-Mal-T;

[0621] wherein T is a target molecule recognition unit, and the target molecule recognition unit comprises an antigen-binding protein.

[0622] 33. The immunoconjugate according to any one of embodiments 23-32, which is selected from the following structures:

[0623] A-NOTA-Bn-SLK(Mal)-T,

[0624] A-NOTA-Bn-SFK-(CH2)2-Mal-T,

[0625] A-DOTA-Bn-SFK-(CH2)2-Mal-T,

[0626] A-NODAGA-SFK-(CH2)2-Mal-T,

[0627] A-NOTA-Bn-DFK-(CH2)2-Mal-T,

[0628] A-NOTA-Bn-VMK-(CH2)2-Mal-T,

[0629] A-DOTA-Bn-VMK-PEG4-Mal-T,

[0630] A-DOTA-PEG4-VMK-PEG4-Mal-T,

[0631] A-DOTA-VMK-(CH2)2-Mal-T,

[0632] A-DOTA-VMK-PEG4-Mal-T,

[0633] A-NOTA-Bn-MV(Dap)-PEG4-Mal-T,

[0634] A-DOTA-Bn-MV(Dap)-PEG4-Mal-T,

[0635] A-DOTA-PEG4-MV(Dap)-PEG4-Mal-T,

[0636] A-DOTA-MV(Dap)-PEG4-Mal-T,

[0637] A-NODAGA-MV(Dap)-PEG4-Mal-T,

[0638] A-DOTA-MV-(CH2)2-Mal-T,

[0639] A-DOTA-PEG4-MV-PEG4-Mal-T,

[0640] A-DOTA-Bn-MV-PEG4-Mal-T,

[0641] A-DOTA-Bn-rSFK-PEG4-Mal-T,

[0642] A-DOTA-Bn-GWK-PEG4-Mal-T,

[0643] A-DOTA-Bn-MNK-PEG4-Mal-T,

[0644] A-DOTA-Bn-QLK-PEG4-Mal-T and

[0645] A-DOTA-Bn-TIK-PEG4-Mal-T;

[0646] wherein T is a target molecule recognition unit and A is an active moiety.

[0647] 34. The immunoconjugate according to any one of embodiments 23-33, wherein the target molecule recognition unit comprises an antigen-binding protein, a scaffold protein, or a ligand.

[0648] 35. The immunoconjugate according to embodiment 34, wherein the antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

[0649] 36. The immunoconjugate according to embodiment 35, wherein the antibody comprises a monoclonal antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

[0650] 37. An immunoconjugate according to any one of embodiments 34 - 36, wherein the antigen-binding fragment comprises a Fab, Fab’, Fv fragment, F(ab’)2, scFv, VHH, and / or dAb.

[0651] 38. An immunoconjugate according to any one of embodiments 23 - 37, wherein the target molecule recognition unit comprises a VHH.

[0652] 39. An immunoconjugate according to embodiment 38, wherein the VHH is camelid, chimeric, human, partially humanized, or fully humanized.

[0653] 40. An immunoconjugate according to any one of embodiments 23 - 39, wherein the target molecule recognition unit targets a tumor antigen and / or a non-tumor antigen.

[0654] 41. The immunoconjugate according to any one of embodiments 23-40, wherein the target molecule recognition unit specifically binds to an antigen selected from the group consisting of: AXL, BAFFR, BCMA, BDCA2, BDCA4, BTLA, BTNL2, BTNL3, BTNL8, BTNL9, C10orf54, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CD11c, CD137, CD138, CD14, CD163, CD168, CD 177, CD19, CD20, CD209, CD209L, CD22, CD226, CD248, CD25, CD27, CD274, CD276, CD28, CD30, CD300A, CD32b, CD33, CD37, CD38, CD4, cluster of differentiation 40 (CD40), CD44, CD45, CD46, CD47, CD48, CD5, CD52, CD55, CD56, CD59, CD62E, CD68, CD69, CD70, CD74, CD79a, CD79b, CD8, CD80, CD86, CD90.2, CD96, OX40 (CD134), CD123, CD97, CD179a, CLEC12A, CLEC12B, CLEC7A, CLEC9A, CR1, CR3, CRTAM, CSF1R, CTLA4, CXCR1 / 2, CXCR4, CXCR5, DDR1, DDR2, DEC-205, DLL4, DR6, FAP, FCamR, FCMR, FcR’s, Fire, GITR, HHLA2, HLA class II, HVEM, Icoslg, IFNAR, IFNAR1, IFNLR1, IL10R1, IL10R2, IL12R, IL13RA1, IL13RA2, IL15R, IL17RA, IL17RB, IL17RC, IL17RE, IL20R1, IL20R2, IL21R, IL22R1, IL22RA, IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R, IL3RA, IL4R, IL6R, IL5R, IL7R, IL9R, Integrins, LAG3, LIFR, MAG / Siglec-4, MMR, MSR1, NCR3LG1, NKG2D, NKp30, NKp46, PDCD1, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, Siglec-1, Siglec-10, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, SIRPA,

[0655] SLAMF7, TACI, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2,

[0656] TGFBR3, TIGIT, TLR2, TLR4, Tumor necrosis factor α (TNFα), TROY, TSLPR, TYRO,

[0657] VLDLR, VSIG4, IL2R-y, VTCN1, TSHR, CD171, CS-1, CLL-1, GD3, Tn Ag,

[0658] FLT3, B7H3, B7H4, KIT, IL-13Ra2, IL-11Ra, PSCA, PSMA, PRSS21, EGFR2,

[0659] LewisY, CD24, PDGFR-beta, SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2,

[0660] EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, FOLR1, FOLR2, TEM7R,

[0661] CLDN6, CLDN18.2, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, GloboH,

[0662] NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TAARP,

[0663] WT1, ETV6-AML, SPA17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, FOSL1,

[0664] hTERT, ML-IAP, ERG, NA17, PAX3, AR, cyclin B1, MYCN, RhoC,

[0665] CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, CD72,

[0666] LAIR1, FCAR, LILRA2, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5,

[0667] IGLL1, HER2, ROR1, TAAG72, GD2, gp100Tn, FAP, tyrosinase, EPCAM, CEA, IGF-1R, EphB2, mesothelin, cadherin 17, EGFRvIII, GPNMB, GPR64, HER3,

[0668] LRP6, LYPD8, NKG2D, SLC34A2, SLC39A6, SLITRK6, GUCY2C, 5T4 and / or

[0669] TACSTD2.

[0670] 42. The immunoconjugate according to any one of embodiments 23 - 41, wherein the target molecule recognition unit comprises an anti-PD-

[0671] L1 VHH antibody, anti-HER2 VHH antibody, a scaffold protein targeting HER2 and / or anti-CD8α VHH antibody.

[0672] 43. The immunoconjugate according to embodiment 42, wherein the anti-CD8α VHH antibody comprises: CDR1 having the amino acid sequence as shown in SEQ ID NO:

[0673] 1, CDR2 having the amino acid sequence as shown in SEQ ID NO: 2, and CDR3 having the amino acid sequence as shown in SEQ ID NO: 3.

[0674] 44. The immunoconjugate according to any one of embodiments 42-43, wherein the anti-CD8α VHH antibody comprises the amino acid sequence as shown in SEQ ID NO: 4.

[0675] 45. The immunoconjugate according to any one of embodiments 42-44, wherein the anti-HER2 VHH antibody comprises:

[0676] CDR1 having the amino acid sequence as shown in SEQ ID NO: 5, CDR2 having the amino acid sequence as shown in SEQ ID NO: 6,

[0677] and CDR3 having the amino acid sequence as shown in SEQ ID NO: 7.

[0678] 46. The immunoconjugate according to any one of embodiments 42-45, wherein the anti-HER2 VHH antibody comprises the amino acid sequence as shown in SEQ ID NO: 8.

[0679] 47. The immunoconjugate according to any one of embodiments 42-46, wherein the scaffold protein targeting HER2 comprises the amino acid sequence as shown in SEQ ID NO: 9.

[0680] 48. The immunoconjugate according to any one of embodiments 23-47, having a structure selected from the following:

[0681]

[0682]

[0683] wherein,

[0684] -(A1) m-A2-A3-Selected from: -Ser-leu-Lys- (SLK), -Ser-Phe-Lys- (SFK), -Asp-Phe-Lys- (DFK), -Val-Met-Lys- (VMK), -Met-Val(Dap)- (MV(Dap)), -Met-Val- (MV), -(D-Arg)-Ser-Phe-Lys- (rSFK), -Gly-Trp-Lys- (GWK), -Met-Asn-Lys- (MNK), -Gln-Leu-Lys- (QLK) and -Thr-ILe-Lys- (TIK); said R1 conjugated antigen-binding protein, scaffold protein or ligand.

[0685] 49. The immunoconjugate according to embodiment 48, wherein said R1 is selected from the following structures: -(CH2) n -Mal, -(CH2CH2O) n- Mal, -CO(CH2)n-Mal, -NH(CH2) n -Mal, where n is any integer between 1 and 10.

[0686] 50. The immunoconjugate according to any one of embodiments 23-49, selected from the following structures:

[0687] NOTA-Bn-SLK(Mal)-VHH,

[0688] NOTA-Bn-SFK-(CH2)2-Mal-VHH,

[0689] DOTA-Bn-SFK-(CH2)2-Mal-VHH,

[0690] NODAGA-SFK-(CH2)2-Mal-VHH,

[0691] NOTA-Bn-DFK-(CH2)2-Mal-VHH,

[0692] NOTA-Bn-VMK-(CH2)2-Mal-VHH,

[0693] DOTA-Bn-VMK-PEG4-Mal-VHH,

[0694] DOTA-PEG4-VMK-PEG4-Mal-VHH,

[0695] DOTA-VMK-(CH2)2-Mal-VHH,

[0696] DOTA-VMK-PEG4-Mal-VHH,

[0697] NOTA-Bn-MV(Dap)-PEG4-Mal-VHH,

[0698] DOTA-Bn-MV(Dap)-PEG4-Mal-VHH,

[0699] DOTA-PEG4-MV(Dap)-PEG4-Mal-VHH,

[0700] DOTA-MV(Dap)-PEG4-Mal-VHH,

[0701] NODAGA-MV(Dap)-PEG4-Mal-VHH,

[0702] DOTA-MV-(CH2)2-Mal-VHH,

[0703] DOTA-PEG4-MV-PEG4-Mal-VHH,

[0704] DOTA-Bn-MV-PEG4-Mal-VHH,

[0705] DOTA-Bn-rSFK-PEG4-Mal-VHH,

[0706] DOTA-Bn-GWK-PEG4-Mal-VHH,

[0707] DOTA-Bn-MNK-PEG4-Mal-VHH,

[0708] DOTA-Bn-QLK-PEG4-Mal-VHH and

[0709] DOTA-Bn-TIK-PEG4-Mal-VHH.

[0710] 51. An immunoconjugate according to any one of embodiments 23 - 50, selected from the following structures:

[0711] A-NOTA-Bn-SLK(Mal)-VHH,

[0712] A-NOTA-Bn-SFK-(CH2)2-Mal-VHH,

[0713] A-DOTA-Bn-SFK-(CH2)2-Mal-VHH,

[0714] A-NODAGA-SFK-(CH2)2-Mal-VHH,

[0715] A-NOTA-Bn-DFK-(CH2)2-Mal-VHH,

[0716] A-NOTA-Bn-VMK-(CH2)2-Mal-VHH,

[0717] A-DOTA-Bn-VMK-PEG4-Mal-VHH,

[0718] A-DOTA-PEG4-VMK-PEG4-Mal-VHH,

[0719] A-DOTA-VMK-(CH2)2-Mal-VHH,

[0720] A-DOTA-VMK-PEG4-Mal-VHH,

[0721] A-NOTA-Bn-MV(Dap)-PEG4-Mal-VHH,

[0722] A-DOTA-Bn-MV(Dap)-PEG4-Mal-VHH,

[0723] A-DOTA-PEG4-MV(Dap)-PEG4-Mal-VHH,

[0724] A-DOTA-MV(Dap)-PEG4-Mal-VHH,

[0725] A-NODAGA-MV(Dap)-PEG4-Mal-VHH,

[0726] A-DOTA-MV-(CH2)2-Mal-VHH,

[0727] A-DOTA-PEG4-MV-PEG4-Mal-VHH,

[0728] A-DOTA-Bn-MV-PEG4-Mal-VHH,

[0729] A-DOTA-Bn-rSFK-PEG4-Mal-VHH,

[0730] A-DOTA-Bn-GWK-PEG4-Mal-VHH,

[0731] A-DOTA-Bn-MNK-PEG4-Mal-VHH,

[0732] A-DOTA-Bn-QLK-PEG4-Mal-VHH and

[0733] A-DOTA-Bn-TIK-PEG4-Mal-VHH;

[0734] Wherein A is the active moiety.

[0735] 52. The immunoconjugate according to embodiment 51, wherein A is a detectable label selected from one or more reagents of the following group: radionuclides, fluorophores, chemiluminescent agents, bioluminescent agents, paramagnetic ions, and enzymes.

[0736] group of one or more reagents: radionuclides, fluorophores, chemiluminescent agents, bioluminescent agents, paramagnetic ions, and enzymes.

[0737] 53. The immunoconjugate according to any one of embodiments 51-52, wherein A is a radionuclide, and the radionuclide includes 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 67 Cu, 67 Ga, 68 Ga, 68 Ge, 86 Y, 90 Y, 89 Zr, 94m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C, 13 N, 15 O, 186 Re, 188 Re, 51 Mn, 52m Mn, 72 As,

[0738] 75 Br, 76 Br, 82m Rb, 83 Sr, 225 Ac, 211 At or other α, γ, β-, or positron emitters.

[0739] 54. A radionuclide complex comprising i) a compound of formula (I) according to any one of embodiments 1-16 or a pharmaceutically acceptable salt thereof and ii) a radionuclide.

[0740] 55. The radionuclide complex according to embodiment 54, wherein the radionuclide is linked to X in the compound of formula (I).

[0741] 56. A composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1-16, the immunoconjugate as described in any one of Embodiments 17-53, and optionally a pharmaceutically acceptable carrier.

[0742] 57. The composition according to Embodiment 56, wherein the composition is a detection agent or a therapeutic agent.

[0743] 58. The composition according to Embodiment 57, wherein the detection agent is a reagent for detecting an antigen.

[0744] 59. The composition according to Embodiment 56, wherein the detection agent is a contrast agent.

[0745] 60. The composition according to Embodiment 59, wherein the contrast agent is a contrast agent for detecting an antigen.

[0746] 61. The composition according to Embodiment 60, wherein the antigen is a tumor antigen.

[0747] 62. The composition according to Embodiment 61, wherein the therapeutic agent is for treating tumors.

[0748] 63. Use of the compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1-16, the immunoconjugate as described in any one of Embodiments 17-53, the radionuclide complex as described in any one of Embodiments 54-55, and / or the composition as described in any one of Embodiments 56-62 in the preparation of a reagent, a test plate or a kit; wherein the reagent, the test plate or the kit is for detecting an antigen in a sample.

[0749] 64. Use of the compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1-16, the immunoconjugate as described in any one of Embodiments 17-53, and / or the composition as described in any one of Embodiments 56-62 in the preparation of a drug;

[0750] wherein the drug is for treating tumors.

[0751] 65. Use of the compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1-16, the immunoconjugate as described in any one of Embodiments 17-53, the radionuclide complex as described in any one of Embodiments 54-55, and / or the composition as described in any one of Embodiments 56-62 in the preparation of a radiolabeled drug.

[0752] 66. A radiolabeled drug, which comprises the compound represented by formula (I) according to any one of Embodiments 1-16 or a pharmaceutically acceptable salt thereof, the immunoconjugate according to any one of Embodiments 17-53, the radionuclide complex according to any one of Embodiments 54-55, and / or the composition according to any one of Embodiments 56-62.

[0753] 67. A radiological imaging diagnostic drug, which comprises the compound represented by formula (I) according to any one of Embodiments 1-16 or a pharmaceutically acceptable salt thereof, the immunoconjugate according to any one of Embodiments 17-53, the radionuclide complex according to any one of Embodiments 54-55, and / or the composition according to any one of Embodiments 56-62.

[0754] 68. A method for detecting the presence and / or amount of an antigen in a biological sample, comprising: contacting the biological sample with the compound represented by formula (I) according to any one of Embodiments 1-16 or a pharmaceutically acceptable salt thereof, the immunoconjugate according to any one of Embodiments 17-53, and / or the composition according to any one of Embodiments 56-62.

[0755] 69. The method according to Embodiment 68, wherein the contacting is carried out in vitro or ex vivo.

[0756] 70. The method according to any one of Embodiments 68-69, wherein the biological sample is a tissue.

[0757] 71. The method according to Embodiment 70, wherein the tissue is selected from blood tissue, lymphoid tissue, and tumor tissue.

[0758] 72. The method according to any one of Embodiments 68-71, the method comprising detecting the presence and / or amount of tumor antigen-positive cells in a biological sample.

[0759] 73. The method according to Embodiment 72, wherein the presence and / or amount of tumor antigen-positive cells in the biological sample is determined by imaging.

[0760] 74. The method according to Embodiment 72 or 73, wherein the presence and / or amount of tumor antigen-positive cells in the biological sample is determined by flow cytometry.

[0761] 75. A method for detecting and / or diagnosing a tumor, comprising administering to a subject in need thereof the compound represented by formula (I) according to any one of Embodiments 1-16 or a pharmaceutically acceptable salt thereof, the immunoconjugate according to any one of Embodiments 17-53, and / or the composition according to any one of Embodiments 56-62.

[0762] 76. The method according to embodiment 75, wherein the method further comprises imaging the subject.

[0763] 77. The method according to embodiment 76, wherein the imaging comprises ECT imaging.

[0764] 78. The method according to embodiment 77, wherein the ECT imaging comprises SPECT imaging or PET imaging.

[0765] 79. A method of treating and / or preventing a tumor, the method comprising administering to a subject in need thereof a compound of formula (I) as described in any one of embodiments 1-16 or a pharmaceutically acceptable salt thereof, an immunoconjugate as described in any one of embodiments 17-53, and / or a composition as described in any one of embodiments 56-62.

[0766] 80. A method for monitoring the efficacy of an anti-tumor therapy in a subject, the method comprising:

[0767] (i) administering to a subject having a tumor and being treated with an anti-tumor therapy a compound of formula (I) as described in any one of embodiments 1-16 or a pharmaceutically acceptable salt thereof, an immunoconjugate as described in any one of embodiments 17-53, and / or a composition as described in any one of embodiments 56-62; and

[0768] (ii) determining the amount of tumor antigen-positive cells in the tumor of the subject.

[0769] 81. The method according to embodiment 80, wherein the presence and / or amount of SNA004-positive cells in the tumor of the subject is determined by imaging.

[0770] 82. The method according to any one of embodiments 80-81, wherein the tumor comprises a solid tumor.

[0771] 83. The method according to any one of embodiments 80-82, wherein the tumor is selected from at least one of tumors such as breast cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, ovarian cancer, pancreatic cancer, endometrial cancer, cervical squamous cell carcinoma, salivary gland adenoma, bladder cancer, lung cancer, colorectal cancer, head and neck cancer, prostate cancer, osteosarcoma, childhood medulloblastoma, etc.

[0772] 84. A kit comprising a compound of formula (I) as described in any one of embodiments 1-16 or a pharmaceutically acceptable salt thereof, an immunoconjugate as described in any one of embodiments 17-53, and / or a composition as described in any one of embodiments 56-62.

[0773] Without being bound by any theory, the examples below are merely for the purpose of illustrating the compounds, preparation methods, uses, etc. of the present application, and are not intended to limit the scope of the invention of the present application.

[0774] Examples

[0775] In the following examples and comparative examples, the following abbreviations are used for substituents, compounds, and organic solvents.

[0776] Boc: tert-Butoxycarbonyl

[0777] p-SCN-Bn-NOTA: 2-S-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid

[0778] p-SCN-Bn-DOTA: 2-[(4-Isothiocyanatophenyl)methyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid

[0779] NODAGA-NHS: NODAGA-Succinimide ester

[0780] DOTA-PEG4-TFP ester: Tetraethylene glycol-(2,3,5,6-tetrafluorophenoxy)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

[0781] DOTA-tris( t Bu ester): Tris(tert-butyl) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate

[0782] NHS-(CH2)2-Mal: 3-Maleimidopropionic acid hydroxysuccinimide ester

[0783] NH2-PEG4-Mal: Maleimide-tetraethylene glycol-amine trifluoroacetate

[0784] NHS-PEG4-Mal: Maleimide-tetraethylene glycol-acrylic acid succinimide ester

[0785] TFA: Trifluoroacetic acid

[0786] MeCN: Acetonitrile

[0787] DCM: Dichloromethane

[0788] Et3N: Triethylamine

[0789] DIPEA: N,N-Diisopropylethylamine

[0790] DMF: N,N-Dimethylformamide

[0791] HATU: 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0792] DIPEA: N,N-diisopropylethylamine

[0793] In the following Examples and Comparative Examples, various physical properties, etc. were separated and / or measured by the following methods.

[0794] For separation by semi-preparative HPLC, an X-Bridge BEH C18 OBD Prep Column (Waters, 30 mm × 150 mm) was used. 0.1% (v / v) TFA / H2O (phase A) and 0.1% (v / v) TFA / MeCN (phase B) were used as the mobile phases, and by the linear gradient method, it was changed from 90% (v / v) of phase A and 10% (v / v) of phase B to 20%

[0795] (v / v) of phase A and 80% (v / v) of phase B within 0 - 30 min, and from 20% (v / v) of phase A and 80% (v / v) of phase B to 90% (v / v) of phase A and 10% (v / v) of phase B within 30 - 31 min, and eluted at a flow rate of 8.0 mL / min.

[0796] For LC-MS analysis, an LC-MS-2020 triple quadrupole liquid chromatography - mass spectrometry instrument (SHIMADZU) was used.

[0797] For MS analysis, a Q Exactive mass spectrometer (Thermo) was used

[0798] Synthesis of the compound of Example 1

[0799] 1.1 Compound 1-1 NODAGA-Mal

[0800]

[0801] Compound 1-1: LC-MS calcd. for: C 21 H 31 N5O9 497.51; found 498.64 [M + H] +

[0802] 1.2 Compound 1-2 NOTA-Bn-MVK(Mal)(L1a)

[0803] NOTA-Bn-MVK(Mal) was synthesized according to the following scheme:

[0804]

[0805] 1(a): p-SCN-Bn-NOTA

[0806] (Synthesis Example 1(a): Synthesis of the compound NOTA-Bn-Met-Val-Lys(Mal)-OH)

[0807] Dissolve the compound MVK(Mal) (2.0 mg, 4.38 μmol) and p-SCN-Bn-NOTA (3.7 mg, 6.57 μmol) in 0.5 mL of DMF, and add Et3N (8 μL). Replace with N2 and stir at room temperature for 2.0 h. Monitor the reaction by LC-MS. After completion, evaporate the solvent and purify by semi-preparative HPLC to obtain a white solid of compound 1-2 (also referred to as "NOTA-Bn-MVK(Mal)" hereinafter).

[0808] Compound 1-2: LC-MS calcd. for: C 40 H 58 N8O 12 S2 907.07; found: 907.37 [M] + 。

[0809] 1.3 Compound 1-3 NOTA-Bn-GFK(Mal) (L2a)

[0810] Synthesize NOTA-Bn-GFK(Mal) according to the following scheme:

[0811]

[0812] 2(a): p-SCN-Bn-NOTA

[0813] (Synthesis Example 2(a): Compound 1-2 NOTA-Bn-Gly-Phe-Lys(Mal)-OH)

[0814] Dissolve the compound GFK(Mal) (2.0 mg, 4.65 μmol) and p-SCN-Bn-NOTA (3.9 mg, 6.97 μmol) in 0.5 mL of DMF, and add Et3N (8 μL). Replace with N2 and stir at room temperature for 2.0 h. Monitor the reaction by LC-MS. After completion, evaporate the solvent, purify by semi-preparative HPLC, and lyophilize to obtain a white solid of compound 1-3 (also referred to as "NOTA-Bn-GFK(Mal)" hereinafter).

[0815] Compound 1-3: LC-MS calcd. for: C 41 H 52 N8O 12 S 880.97; found: 881.40 [M + H]+ .

[0816] 1.4 Compound 1-4 NOTA-Bn-PEG4-Mal (L3a)

[0817] Synthesize NOTA-Bn-PEG4-Mal according to the following scheme

[0818]

[0819] 3(a): NH2-PEG4-Mal

[0820] (Synthesis Example 3(a): Synthesis of Compound 1-4 (NOTA-Bn-PEG4-Mal))

[0821] Dissolve p-SCN-Bn-NOTA (3.3 mg, 5.81 μmol) and NH2-PEG4-Mal (2.0 mg, 4.84 μmol) in 0.5 mL of DMF, add Et3N (4 μL). Stir at room temperature for 2.0 h. Detect the reaction by LC-MS. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of Compound 1-4 (also referred to as "NOTA-Bn-PEG4-Mal" hereinafter). Compound 1-4: LC-MS calcd. for: C 34 H 50 N6O 12 S 766.86; found: 767.10 [M+H] + .

[0822] The following compounds are examples:

[0823] 1.5 Compound 1-5: NOTA-Bn-SLK(Mal) (L10a)

[0824] Synthesize the following shown NOTA-Bn-SLK(Mal) in the same manner as in Reference Example 1.3, except that SLK(Mal) is used instead of GFK(Mal) in Example 1.3.

[0825]

[0826] Compound 1-5: LC-MS calcd. for: C 39 H 56 N8O 13 S 876.98; Found: 952.40 [M+75] + .

[0827] 1.5.1 Compound 1-5a: NOTA-Bn-SLK-(CH2)2-Mal (L10d)

[0828] Except for using SLK-(CH2)2-Mal to replace GFK(Mal) in Example 1.3, NOTA-Bn-SLK-(CH2)2-Mal shown below was synthesized in the same manner as in Reference Example 1.3.

[0829]

[0830] Compound 1-5a: C 42 H 61 N9O 14 S LC-MS calculated value: 947.41; measured value: 948.65 [M+H] + .

[0831] 1.5.1 Compound 1-5d: DOTA-Bn-SLK(Mal)(L10b)

[0832] Except for using DOTA to replace NOTA in Example 1.5, DOTA-Bn-SLK(Mal) shown below was synthesized in the same manner as in Reference Example 1.5.

[0833]

[0834] Compound 1-5d: C 43 H 63 N9O 15 S LC-MS calculated value: 977.08; not characterized by LC-MS, correctly characterized after coupling.

[0835] 1.6 Compound 1-6: NOTA-Bn-SFK-(CH2)2-Mal(L8b)

[0836] NOTA-Bn-SFK-(CH2)2-Mal was synthesized according to the following scheme

[0837]

[0838] 4(a): NHS-(CH2)2-Mal; 4(b): TFA, DCM; 4(c): p-SCN-Bn-NOTA

[0839] (Synthesis Example 4(a): Synthesis of Intermediate 1-1 (Boc-Ser-Phe-Lys(CH2)2-Mal))

[0840] Dissolve Boc-SFK (2.0 mg, 4.16 μmol) and NHS-(CH2)2-Mal (1.3 mg, 5.00 μmol) in 0.5 mL of DMF, add Et3N (8 μL), and stir at room temperature for 2.0 h. Detect the reaction by LC-MS. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of intermediate 1-1 (also referred to as "Boc-SFK-(CH2)2-Mal" hereinafter).

[0841] Intermediate 1-1: LC-MS calcd. for: C 30 H 41 N5O 10 631.68; found: 632.80 [M+H] + .

[0842] (Synthesis Example 4(b): Synthesis of intermediate 1-2 (NH2-Ser-Phe-Lys(CH2)2-Mal))

[0843] Dissolve intermediate 1-1 (2.6 mg, 4.16 μmol) in 1.0 mL of DCM, add 0.5 mL of TFA, and stir at room temperature for 2.0 h. After the reaction is completed, concentrate and dry by suction to obtain a white solid of crude intermediate 1-2 (also referred to as "NH2-SFK-(CH2)2-Mal" hereinafter).

[0844] Intermediate 1-2: LC-MS calcd. for: C 25 H 33 N5O8 531.57; found: 532.35 [M+H] + .

[0845] (Synthesis Example 4(c): Synthesis of compound 1-6 (NOTA-Bn-Ser-Phe-Lys(CH2)2-Mal))

[0846] Dissolve the above-mentioned crude intermediate 1-2 in 0.5 mL of DMF, add p-SCN-Bn-NOTA (3.5 mg, 6.24 μmol), and finally add Et3N (8 μL). Stir at room temperature for 2 h. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of the compound (also referred to as "NOTA-Bn-SFK-(CH2)2-Mal" hereinafter).

[0847] Compound 1-6: LC-MS calcd. for: C 45 H 59 N9O 14 S 982.08; found: 983.15 [M+H] +.

[0848] 1.6.1 Compound 1-6-1: DOTA-Bn-SFK-(CH2)2-Mal (L8ac)

[0849]

[0850] 5(a): p-SCN-Bn-DOTA

[0851] (Synthesis Example 5(a): Synthesis of Compound 1-6-1 (DOTA-Bn-Ser-Phe-Lys(CH2)2-Mal))

[0852] Dissolve NH2-SFK-(CH2)2-Mal (2.0 mg, 3.76 μmol) in 0.5 mL of DMF, add p-SCN-Bn-DOTA (3.1 mg, 4.51 μmol), and finally add Et3N (4 μL). Stir at room temperature for 3 h. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of the compound (also referred to as "DOTA-Bn-SFK-(CH2)2-Mal" hereinafter). Compound 1-6-1: LC-MS calcd. for: C 49 H 66 N 10 O 16 S 1082.44; found: 1083.45 [M+H] + .

[0853] 1.6.2 Compound 1-6-2: NODAGA-SFK-(CH2)2-Mal (L8ad)

[0854]

[0855] 6(a) NODAGA-NHS

[0856] (Synthesis Example 6(a): Synthesis of Compound 1-6-2 (NODAGA-Ser-Phe-Lys(CH2)2-Mal))

[0857] Dissolve NH2-SFK-(CH2)2-Mal (2.0 mg, 3.76 μmol) in 0.5 mL of DMF, add NODAGA-NHS (3.3 mg, 4.50 μmol), and finally add Et3N (4 μL). Stir at room temperature for 3 h. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of the compound (also referred to as "NODAGA-SFK-(CH2)2-Mal" hereinafter).

[0858] Compound 1-6-2: LC-MS calcd. for: C 40 H 56 N8O 15 888.39; found: 889.35 [M+H] + .

[0859] 1.7 Compound 1-7 NOTA-Bn-DFK-(CH2)2-Mal (L9b)

[0860] The following shown NOTA-Bn-DFK-(CH2)2-Mal was synthesized in the same manner as in Example 1.6, except that Boc-DFK was used instead of Boc-SFK in Compound 1.6.

[0861]

[0862] Compound 1-7: LC-MS calcd. for: C 46 H 59 N9O 15 S 1010.09; Found: 1010.90 [M] + .

[0863] 1.8 Compound 1-8: NOTA-Bn-VMK-(CH2)2-Mal (L13)

[0864] The following shown NOTA-Bn-DFK-(CH2)2-Mal was synthesized in the same manner as in Example 1.6, except that Boc-VMK was used instead of Boc-SFK in Example 1.6.

[0865]

[0866] Compound 1-8: LC-MS calcd. for: C 43 H 63 N9O 13 S2 978.15; Found: 978.15 [M] +

[0867] 1.8.1 Compound 1-8-1: DOTA-Bn-VMK-PEG4-Mal (L13a)

[0868]

[0869] 7(a): NHS-PEG4-Mal; 7(b): TFA; 7(c): p-SCN-Bn-DOTA

[0870] (Synthesis Example 7(a): Synthesis of Intermediate 2-1 (Boc-Val-Met-Lys(PEG)4-Mal))

[0871] Dissolve Boc-VMK (10.0 mg, 20.98 μmol) and NHS-PEG4-Mal (11.2 mg, 25.18 μmol) in 0.5 mL of DMF, add Et3N (10 μL), and stir at room temperature for 3.0 h. Detect the reaction by LC-MS. After the reaction is completed, purify by semi-preparative HPLC and freeze-dry again. To obtain a white solid of Intermediate 2-1 (hereinafter also referred to as "Boc-VMK-PEG4-Mal"). Intermediate 2-1: LC-MS calcd. for: C 36 H 61 N5O 13 S 803.97; found: 804.40 [M+H] + .

[0872] (Synthesis Example 7(b): Synthesis of Intermediate 2-2 (NH2-Val-Met-Lys(PEG)4-Mal))

[0873] Dissolve Intermediate 2-1 (2.4 mg, 3.00 μmol) in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is completed, concentrate and dry by suction to obtain a white solid of crude Intermediate 2-2 (hereinafter also referred to as "NH2-VMK-PEG4-Mal").

[0874] Intermediate 2-2: LC-MS calcd. for: C 31 H 53 N5O 11 S 703.85; found: 704.20 [M+H] + .

[0875] (Synthesis Example 7(c): Synthesis of Compound 1-6 (DOTA-Bn-Val-Met-Lys(PEG)4-Mal))

[0876] Dissolve the above-mentioned crude Intermediate 2-2 in 0.5 mL of DMF, add p-SCN-Bn-DOTA (3.1 mg, 4.51 μmol), and finally add Et3N (8 μL). Stir at room temperature for 3 h. After the reaction is completed, purify by semi-preparative HPLC and freeze-dry again to obtain a white solid of the compound (hereinafter also referred to as "DOTA-Bn-VMK-PEG4-Mal").

[0877] Compound 1-8-1: LC-MS calcd. for: C 55 H 86 N10 O 19 S2 1255.46; found: 628.75 [M / 2 + H] + .

[0878] 1.8.2 Compound 1-8-2: DOTA-PEG4-VMK-PEG4-Mal (L13b)

[0879]

[0880] 8(a): NHS-PEG4-Mal; 8(b): TFA; 8(c): DOTA-PEG4-TFP ester

[0881] (Synthesis Example 8(a): Synthesis of Intermediate 3-1 (Boc-Val-Met-Lys(PEG)4-Mal))

[0882] Dissolve Boc-VMK (10.0 mg, 20.98 μmol) and NHS-PEG4-Mal (11.2 mg, 25.18 μmol) in 0.5 mL of DMF, add Et3N (10 μL), and stir at room temperature for 2.0 h. Monitor the reaction by LC-MS. After the reaction is complete, purify by semi-preparative HPLC and lyophilize again. To obtain a white solid of Intermediate 3-1 (also referred to as "Boc-VMK-PEG4-Mal" hereinafter). Intermediate 3-1: LC-MS calcd. for: C 36 H 61 N5O 13 S 803.97; found: 805.35 [M + H] + .

[0883] (Synthesis Example 8(b): Synthesis of Intermediate 3-2 (NH2-Val-Met-Lys(PEG)4-Mal))

[0884] Dissolve Intermediate 3-1 (2.4 mg, 3.00 μmol) in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is complete, concentrate and dry by suction to obtain a white solid of crude Intermediate 3-2 (also referred to as "NH2-VMK-PEG4-Mal" hereinafter).

[0885] Intermediate 3-2: LC-MS calcd. for: C 31 H 53 N5O 11 S 703.85; found: 704.20 [M + H] + .

[0886] (Synthesis Example 8(c): Synthesis of Compound 1-6 (DOTA-PEG4-Val-Met-Lys(PEG)4-Mal))

[0887] Dissolve the above crude intermediate 3-2 in 0.5 mL of DMF, add DOTA-PEG4-TFP ester (4.6 mg, 4.51 μmol), and finally add Et3N (8 μL). Stir at room temperature for 3 h. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of the compound (also referred to as "DOTA-PEG4-VMK-PEG4-Mal" hereinafter).

[0888] Compound 1-8-2: LC-MS calcd. for: C 58 H 100 N 10 O 23 S 1336.67; found: 665.95 [M / 2 - 2H] + .

[0889] 1.8.3 Compound 1-8-3: DOTA-VMK-(CH2)2-Mal (L13d)

[0890]

[0891] 8(d): NHS-(CH2)2-Mal; 8(e): TFA; 8(f): DOTA-tris( t Bu ester); 8(g): TFA

[0892] (Synthesis Example 8(d): Synthesis of Intermediate 3-1-1 (Boc-Val-Met-Lys(CH2)2-Mal))

[0893] Dissolve Boc-VMK (30.0 mg, 62.94 μmol) and NHS-(CH2)2-Mal (20.11 mg, 75.53 μmol) in 0.5 mL of DMF, add Et3N (38 μL), and stir at room temperature for 3.0 h. Detect the reaction by LC-MS. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again. To obtain a white solid of intermediate 3-1-1 (also referred to as "Boc-VMK-(CH2)2-Mal" hereinafter).

[0894] Intermediate 3-1-1: LC-MS calcd. for: C 28 H 45 N5O9S 627.75; found: 628.45 [M + H] + .

[0895] (Synthesis Example 8(e): Synthesis of Intermediate 3-2-1 (NH2-Val-Met-Lys(CH2)2-Mal))

[0896] Dissolve Intermediate 3-1-1 (20.0 mg, 24.88 μmol) in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is completed, concentrate and dry by suction to obtain a white solid of crude Intermediate 3-2-1 (hereinafter also referred to as "NH2-VMK-(CH2)2-Mal"). It is directly used for the next step.

[0897] (Synthesis Example 8(f): Synthesis of Intermediate 3-3-1 (DOTA-tris( t Bu ester)-Val-Met-Lys(CH2)2-Mal))

[0898] Dissolve DOTA-tris( t Bu ester) (18.52 mg, 32.34 μmol) in 1.0 mL of DMF, add HATU (26.65 mg, 70.09 μmol), and stir at room temperature for 30 min. Add the above-mentioned crude Intermediate 3-2-1 NH2-VMK-(CH2)2-Mal to the reaction system, and finally add DIPEA (27.7 μL). Stir at room temperature for 3 h. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of the compound (hereinafter also referred to as "DOTA-tris( t Bu ester)-VMK-(CH2)2-Mal").

[0899] Intermediate 3-3-1: LC-MS calcd. for: C 51 H 87 N9O 14 S 1082.37; found: 1065.05 [M-H3O] + .

[0900] (Synthesis Example 8(g): Synthesis of Compound 1-8-3 (DOTA-Val-Met-Lys(CH2)2-Mal))

[0901] Dissolve Intermediate 3-3-1 (8.0 mg, 7.39 μmol) in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is completed, concentrate and purify by semi-preparative HPLC and lyophilize again to obtain a white solid of the compound (hereinafter also referred to as "DOTA-VMK-(CH2)2-Mal").

[0902] Compound 1-8-3: LC-MS calcd. for: C 39 H 63N9O 14 S 914.04; found: [M-H2O] + = 896.42

[0903] 1.8.4 Compound 1-8-4: DOTA-VMK-PEG4-Mal (L13c)

[0904]

[0905] 8(h): NHS-PEG4-Mal; 8(i): TFA; 8(j): DOTA-tris( t Bu ester); 8(k): TFA

[0906] (Synthesis Example 8(h): Synthesis of Intermediate 3-1-2 (Boc-Val-Met-Lys(PEG)4-Mal))

[0907] Dissolve Boc-VMK (30.0 mg, 62.94 μmol) and NHS-PEG4-Mal (33.6 mg, 75.53 μmol) in 0.5 mL of DMF, add Et3N (38 μL), and stir at room temperature for 3.0 h. Detect the reaction by LC-MS. After the reaction is completed, purify by semi-preparative HPLC and freeze-dry again. To obtain a white solid of Intermediate 3-1-2 (also hereinafter referred to as "Boc-VMK-PEG4-Mal").

[0908] Intermediate 3-1-2: LC-MS calcd. for: C 36 H 61 N5O 13 S 803.97; found: 805.00 [M+H] + .

[0909] (Synthesis Example 8(i): Synthesis of Intermediate 3-2-2 (NH2-Val-Met-Lys(PEG)4-Mal))

[0910] Dissolve Intermediate 3-1-2 (20.0 mg, 24.88 μmol) in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is completed, concentrate and dry by suction to obtain a white solid of crude Intermediate 3-2-2 (also hereinafter referred to as "NH2-VMK-PEG4-Mal"). Directly used for the next step.

[0911] (Synthesis Example 8(j): Synthesis of Intermediate 3-3-2 (DOTA-tris( t Bu ester)-Val-Met-Lys(PEG)4-Mal))

[0912] Dissolve DOTA-tris( t Bu ester) (18.52 mg, 32.34 μmol) in 1.0 mL of DMF, add HATU (20.81 mg, 54.73 μmol), stir at room temperature for 30 min, add the above-mentioned crude intermediate 3-2-2NH2-VMK-PEG4-Mal to the reaction system, and finally add DIPEA (22 μL), stir at room temperature for 3 h. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of the compound (also referred to as "DOTA-tris( t Bu ester)-VMK-PEG4-Mal") hereinafter. Intermediate 3-3-2: LC-MS calcd. for: C 59 H 103 N9O 18 S 1258.58; found: 1240.85 [M-H2O] + .

[0913] (Synthesis Example 8(k): Synthesis of Compound 1-8-3 (DOTA-Val-Met-Lys(PEG)4-Mal))

[0914] Dissolve intermediate 3-3-2 (8.0 mg, 6.36 μmol) in 0.5 mL of TFA, stir at room temperature for 30 min. After the reaction is completed, concentrate, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of the compound (also referred to as "DOTA-VMK-PEG4-Mal") hereinafter.

[0915] Compound 1-8-4: LC-MS calcd. for: C 47 H 79 N9O 18 S 1090.25; found: 1072.53 [M-H2O] + .

[0916] 1.9 Compound 1-9: NOTA-Bn-MV(Dap)-PEG4-Mal (L14f)

[0917]

[0918] 9(a): NHS-PEG4-Mal; 9(b): TFA; 9(c): p-SCN-Bn-NOTA

[0919] (Synthesis Example 9(a): Synthesis of Intermediate 4-1 (Boc-Met-Val(Dap)-PEG4-Mal))

[0920] Dissolve Boc-MV(Dap) (2.0 mg, 4.60 μmol) and NHS-PEG4-Mal (2.4 mg, 5.52 μmol) in 0.5 mL of DMF, add Et3N (4 μL), and stir at room temperature for 2.0 h. Detect the reaction by LC-MS. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again. To obtain a white solid of intermediate 4-1 (also referred to as "Boc-MV(Dap)-PEG4-Mal" hereinafter).

[0921] Intermediate 4-1: LC-MS calcd. for: C 33 H 55 N5O 13 S 761.89; found: 762.45 [M+H] + .

[0922] (Synthesis Example 9(b): Synthesis of intermediate 4-2 (H-Met-Val(Dap)-PEG4-Mal))

[0923] Dissolve intermediate 4-1 (3.5 mg, 4.60 μmol) in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is completed, concentrate and dry by suction to obtain a white solid of crude intermediate 4-2 (also referred to as "NH2-MV(Dap)-PEG4-Mal" hereinafter).

[0924] Intermediate 4-2: LC-MS calcd. for: C 28 H 47 N5O 11 S 661.77; found: 662.05 [M+H] + .

[0925] (Synthesis Example 9(c): Synthesis of compound 1-9 (NOTA-Bn-Met-Val(Dap)-PEG4-Mal))

[0926] Dissolve the above crude intermediate 4-2 in 0.5 mL of DMF, add p-SCN-Bn-NOTA (3.2 mg, 5.72 μmol), and finally add Et3N (8 μL). Stir at room temperature for 2 h. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of the compound (also referred to as "NOTA-Bn-MV(Dap)-PEG4-Mal" hereinafter).

[0927] Compound 1-9: LC-MS calcd. for: C 48 H 73 N9O 17 S2 1112.28; found: 1112.50 [M]+ .

[0928] 1.9.1 Compound 1-9-1: DOTA-Bn-MV(Dap)-PEG4-Mal (L14b)

[0929]

[0930] 10(a): NHS-PEG4-Mal; 10(b): TFA; 10(c): p-SCN-Bn-DOTA

[0931] (Synthesis Example 10(a): Synthesis of Intermediate 5-1 (Boc-Met-Val(Dap)-PEG4-Mal))

[0932] Dissolve Boc-MV(Dap) (10.0 mg, 23.01 μmol) and NHS-PEG4-Mal (12.2 mg, 28.06 μmol) in 0.5 mL of DMF, add Et3N (4 μL), and stir at room temperature for 2.0 h. Monitor the reaction by LC-MS. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of Intermediate 5-1 (also hereinafter referred to as "Boc-MV(Dap)-PEG4-Mal").

[0933] Intermediate 5-1: LC-MS calcd. for: C 33 H 55 N5O 13 S 761.89; found: 762.45 [M+H] + .

[0934] (Synthesis Example 10(b): Synthesis of Intermediate 5-2 (NH2-Met-Val(Dap)-PEG4-Mal))

[0935] Dissolve Intermediate 5-1 (5.0 mg, 6.56 μmol) in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is completed, concentrate and dry by suction to obtain a white solid of crude Intermediate 5-2 (also hereinafter referred to as "NH2-MV(Dap)-PEG4-Mal").

[0936] Intermediate 5-2: LC-MS calcd. for: C 28 H 47 N5O 11 S 661.77; found: 662.05 [M+H] + .

[0937] (Synthesis Example 10(c): Synthesis of Compound 1-9-1 (DOTA-Bn-Met-Val(Dap)-PEG4-Mal))

[0938] Dissolve the above crude intermediate 5-2 in 0.5 mL of DMF, add p-SCN-Bn-DOTA (5.4 mg, 7.85 μmol), and finally add Et3N (8 μL). Stir at room temperature for 2 h. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of the compound (also referred to as "DOTA-Bn-MV(Dap)-PEG4-Mal" hereinafter).

[0939] Compound 1-9-1: LC-MS calcd. for: C 52 H 80 N 10 O 19 S2 1212.50; found: 1213.65 [M+H] + .

[0940] 1.9.2 Compound 1-9-2: DOTA-PEG4-MV(Dap)-PEG4-Mal (L14a)

[0941]

[0942] 10(a): NHS-PEG4-Mal; 10(b): TFA; 11(c): DOTA-PEG4-TFP ester

[0943] (Synthesis Example 10(a): Synthesis of Intermediate 5-1 (Boc-Met-Val(Dap)-PEG4-Mal))

[0944] Dissolve Boc-MV(Dap) (10.0 mg, 23.01 μmol) and NHS-PEG4-Mal (12.2 mg, 28.06 μmol) in 0.5 mL of DMF, add Et3N (4 μL), and stir at room temperature for 2.0 h. Detect the reaction by LC-MS. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again. To obtain a white solid of intermediate 5-1 (also referred to as "Boc-MV(Dap)-PEG4-Mal" hereinafter).

[0945] Intermediate 5-1: LC-MS calcd. for: C 33 H 55 N5O 13 S 761.89; found: 762.45 [M+H] + .

[0946] (Synthesis Example 10(b): Synthesis of Intermediate 5-2 (NH2-Met-Val(Dap)-PEG4-Mal))

[0947] Dissolve Intermediate 5-1 (5.0 mg, 6.56 μmol) in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is completed, concentrate and dry by suction to obtain a white solid of crude Intermediate 5-2 (hereinafter also referred to as "NH2-MV(Dap)-PEG4-Mal").

[0948] Intermediate 5-2: LC-MS calcd. for: C 28 H 47 N5O 11 S 661.77; found: 662.05 [M+H] + .

[0949] (Synthesis Example 11(c): Synthesis of Compound 1-9 -1 (DOTA-PEG4-Met-Val(Dap)-PEG4-Mal))

[0950] Dissolve the above-mentioned crude Intermediate 5-2 in 0.5 mL of DMF, add DOTA-PEG4-TFP ester (8.0 mg, 7.88 μmol), and finally add Et3N (8 μL). Stir at room temperature for 2 h. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of the compound (hereinafter also referred to as "DOTA-PEG4-MV(Dap)-PEG4-Mal").

[0951] Compound 1-9-2: LC-MS calcd. for: C 55 H 94 N 10 O 23 S 1295.46; found: 1296.70 [M+H] + .

[0952] 1.9.3 Compound 1-9-3: DOTA-MV(Dap)-PEG4-Mal (L14e)

[0953]

[0954] 10(a): NHS-PEG4-Mal; 10(b): TFA; 12(c): DOTA-tris( t Bu ester); 12(d) TFA

[0955] (Synthesis Example 10(a): Synthesis of Intermediate 5-1 (Boc-Met-Val(Dap)-PEG4-Mal))

[0956] Dissolve Boc-MV(Dap) (10.0 mg, 23.01 μmol) and NHS-PEG4-Mal (12.2 mg, 28.06 μmol) in 0.5 mL of DMF, add Et3N (4 μL), and stir at room temperature for 2.0 h. Detect the reaction by LC-MS. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again. To obtain a white solid of intermediate 5-1 (also referred to as "Boc-MV(Dap)-PEG4-Mal" hereinafter).

[0957] Intermediate 5-1: LC-MS calcd. for: C 33 H 55 N5O 13 S 761.89; found: 762.45 [M+H] + .

[0958] (Synthesis Example 10(b): Synthesis of intermediate 5-2 (NH2-Met-Val(Dap)-PEG4-Mal))

[0959] Dissolve intermediate 5-1 (5.0 mg, 6.56 μmol) in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is completed, concentrate and dry by suction to obtain a white solid of crude intermediate 5-2 (also referred to as "NH2-MV(Dap)-PEG4-Mal" hereinafter). Intermediate 5-2: LC-MS calcd. for: C 28 H 47 N5O 11 S 661.77; found: 662.05 [M+H] + .

[0960] (Synthesis Example 12(c): Synthesis of intermediate 6-1 (DOTA-tris( t Bu ester)-Met-Val(Dap)-PEG4-Mal))

[0961] Dissolve DOTA-tris( t(5.4 mg, 7.88 μmol) of [[Bu ester]] was dissolved in 0.5 mL of anhydrous DMF, and then HATU (5.5 mg, 14.44 μmol) was added. Under a N₂ atmosphere, the mixture was stirred at room temperature for 1 h. The crude intermediate 5-2NH₂-MV(Dap)-PEG4-Mal was dissolved in 1.0 mL of anhydrous DMF and added to the above system. Then DIPEA (5.7 μL, 32.8 μmol) was added, and the mixture was stirred at room temperature for 4 h under a N₂ atmosphere. The reaction was monitored by LC-MS. After the reaction was completed, it was lyophilized. Then it was purified by semi-preparative HPLC (CH₃CN-H₂O: 0.1% TFA) and lyophilized to obtain the white solid of intermediate 6-1 (also referred to as “DOTA-tris( t Bu ester)-MV(Dap)-PEG4-Mal”) below).

[0962] Intermediate 6-1: LC-MS calcd. for: C 56 H 97 N9O 18 S 1215.67; found: 1214.20 [M-H] - .

[0963] (Synthesis Example 12(d): Synthesis of Compound 1-9-3 (DOTA-Met-Val(Dap)-PEG4-Mal))

[0964] The above crude intermediate 6-1 was dissolved in 0.5 mL of TFA and stirred at room temperature for 5 h. After the reaction was completed, it was purified by semi-preparative HPLC and lyophilized again to obtain the white solid of the compound (also referred to as “DOTA-MV(Dap)-PEG4-Mal”) below. Compound 1-9-3: LC-MS calcd. for: C 44 H 73 N9O 18 S 1047.48; found: 1046.15 [M-H] - .

[0965] 1.9.4 Compound 1-9-4: NODAGA-MV(Dap)-PEG4-Mal (L14c)

[0966]

[0967] 10(a): NHS-PEG4-Mal; 10(b): TFA; 13(c): NODA-GA-NHS

[0968] (Synthesis Example 10(a): Synthesis of Intermediate 5-1 (Boc-Met-Val(Dap)-PEG4-Mal))

[0969] Dissolve Boc-MV(Dap) (10.0 mg, 23.01 μmol) and NHS-PEG4-Mal (12.2 mg, 28.06 μmol) in 0.5 mL of DMF, add Et3N (4 μL), and stir at room temperature for 2.0 h. Detect the reaction by LC-MS. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of intermediate 5-1 (also hereinafter referred to as "Boc-MV(Dap)-PEG4-Mal").

[0970] Intermediate 5-1: LC-MS calcd. for: C 33 H 55 N5O 13 S 761.89; found: 762.45 [M+H] + .

[0971] (Synthesis Example 10(b): Synthesis of Intermediate 5-2 (NH2-Met-Val(Dap)-PEG4-Mal))

[0972] Dissolve Intermediate 5-1 (5.0 mg, 6.56 μmol) in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is completed, concentrate and dry by suction to obtain a white solid of crude Intermediate 5-2 (also hereinafter referred to as "NH2-MV(Dap)-PEG4-Mal"). Intermediate 5-2: LC-MS calcd. for: C 28 H 47 N5O 11 S 661.77; found: 662.05 [M+H] + .

[0973] (Synthesis Example 13(c): Synthesis of Compound 1-9-4 (NODAGA-Met-Val(Dap)-PEG4-Mal))

[0974] Dissolve the above crude Intermediate 5-2 in 0.5 mL of DMF, add NODAGA-NHS (5.8 mg, 7.88 μmol), and finally add Et3N (6 μL). Stir at room temperature for 2 h. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of the compound (also hereinafter referred to as "NODAGA-MV(Dap)-PEG4-Mal").

[0975] Compound 1-9-4: LC-MS calcd. for: C 43 H 70 N8O 18 S 1019.13; found: 1020.15 [M+H]+ .

[0976] 1.10 Compounds 1 - 10: DOTA - MV-(CH2)2 - Mal (L15a)

[0977]

[0978] 14(a): Mal-(CH2)2 - NH2; 14(b): TFA; 14(c): DOTA - tris( t Bu ester); 14(d): TFA

[0979] (Synthesis Example 14(a): Synthesis of Intermediate 7 - 1 (Boc - Met - Val-(CH2)2 - Mal))

[0980] Dissolve Boc - MV(Dap) (2.7 mg, 15.29 μmol) in 0.5 mL of anhydrous DMF, then add HATU (12.6 mg, 33.14 μmol). Stir at room temperature for 1 h under a N2 atmosphere. Dissolve Mal-(CH2)2 - NH2 in 1.0 mL of anhydrous DMF, add it to the above system, then add DIPEA (13.3 μL, 76.44 μmol). Stir at room temperature for 4 h under a N2 atmosphere. Detect the reaction by LC - MS. After the reaction is completed, freeze - dry. Then purify using semi - preparative HPLC (CH3CN - H2O: 0.1% TFA) and freeze - dry to obtain Intermediate 7 - 1 (also referred to as "Boc - MV-(CH2)2 - Mal" hereinafter)

[0981] Intermediate 7 - 1: LC - MS calcd. for: C 21 H 34 N4O6S 470.59; found: 471.50[M + H] + .

[0982] (Synthesis Example 14(b): Synthesis of Intermediate 7 - 2 (NH2 - Met - Val-(CH2)2 - Mal))

[0983] Dissolve Intermediate 7 - 1 in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is completed, concentrate and dry by suction to obtain a white solid of crude Intermediate 7 - 2 (also referred to as "NH2 - MV-(CH2)2 - Mal" hereinafter).

[0984] Synthesis 14(c): Synthesis of Intermediate 7 - 3 (DOTA - tris( t Bu ester)-Met - Val-(CH2)2 - Mal))

[0985] Dissolve DOTA - tris(t (3.2 mg, 5.52 μmol) of [[Bu ester]] was dissolved in 0.5 mL of anhydrous DMF. Then HATU (3.6 mg, 9.35 μmol) was added, and the mixture was stirred at room temperature for 1 h under a nitrogen atmosphere. The crude intermediate 7-2NH2-MV-(CH2)2-Mal was dissolved in 1.0 mL of anhydrous DMF and added to the above system. Then DIPEA (3.7 μL, 21.25 μmol) was added, and the mixture was stirred at room temperature for 4 h under a nitrogen atmosphere. The reaction was monitored by LC-MS. After the reaction was completed, the mixture was lyophilized. Then it was purified by semi-preparative HPLC (CH3CN-H2O: 0.1% TFA) and lyophilized to obtain a white solid of intermediate 7-3 (hereinafter also referred to as “DOTA-tris( t Bu ester)-MV-(CH2)2-Mal”).

[0986] Intermediate 7-3: LC-MS calcd. for: C 44 H 76 N8O 11 S 925.20; found: 926.55 [M+H] + .

[0987] (Synthesis Example 14(d): Synthesis of Compound 1-10 (DOTA-Met-Val-(CH2)2-Mal))

[0988] Intermediate 7-3 (3.9 mg, 4.25 μmol) was dissolved in 0.5 mL of TFA and stirred at room temperature for 5 h. After the reaction was completed, it was purified by semi-preparative HPLC and lyophilized again to obtain a white solid of the compound (hereinafter also referred to as “DOTA-MV-(CH2)2-Mal”).

[0989] Compound 1-10: LC-MS calcd. for: C 32 H 52 N8O 11 S 756.87; found: 757.60 [M+H] + .

[0990] 1.10.1 Compound 1-10-1: DOTA-PEG4-MV-PEG4-Mal (L15b)

[0991]

[0992] 14(e): Mal-PEG4-NH2; 14(f): TFA; 14(g): DOTA-PEG4-TFP ester

[0993] (Synthesis Example 14(e): Synthesis of Intermediate 7-1-1 (Boc-Met-Val-PEG4-Mal))

[0994] Dissolve Boc-MV (30.0 mg, 86.12 μmol) in 0.5 mL of anhydrous DMF, then add HATU (43.5 mg, 114.40 μmol), and stir at room temperature for 1 h under a nitrogen atmosphere. Dissolve Mal-PEG4-NH2 in 0.5 mL of anhydrous DMF, add it to the above system, then add DIPEA (51.8 μL, 297.55 μmol), and stir at room temperature for 3 h under a nitrogen atmosphere. Detect the reaction by LC-MS. After the reaction is completed, freeze-dry. Then purify by semi-preparative HPLC (CH3CN-H2O: 0.1% TFA) and freeze-dry to obtain Intermediate 7-1-1 (also hereinafter referred to as "Boc-MV-PEG4-Mal").

[0995] Intermediate 7-1-1: LC-MS calcd. for: C 29 H 50 N4O 10 S 646.80; found: 647.33 [M+H] + .

[0996] (Synthesis Example 14(f): Synthesis of Intermediate 7-2-1 (NH2-Met-Val-PEG4-Mal))

[0997] Dissolve Intermediate 7-1-1 in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is completed, concentrate and dry by suction to obtain a white solid of crude Intermediate 7-2-1 (also hereinafter referred to as "NH2-MV-PEG4-Mal").

[0998] (Synthesis Example 14(g): Synthesis of Compound 1-10-1 (DOTA-PEG4-Met-Val-PEG4-Mal))

[0999] Dissolve the above crude Intermediate 7-2-1 in 0.5 mL of DMF, add DOTA-PEG4-TFP ester (11.92 mg, 11.60 μmol), and finally add Et3N (8.0 μL), and stir at room temperature for 2 h. After the reaction is completed, purify by semi-preparative HPLC and freeze-dry again to obtain a white solid of the compound (also hereinafter referred to as "DOTA-PEG4-MV-PEG4-Mal").

[1000] Compound 1-10-1: LC-MS calcd. for: C 51 H 89 N9O 20S 1180.38; found: 1181.25 [M+H] + .

[1001] 1.10.2 Compound 1-10-2: DOTA-Bn-MV-PEG4-Mal (L15c)

[1002]

[1003] 14(h): Mal-PEG4-NH2; 14(i): TFA; 14(j): p-SCN-Bn-DOTA

[1004] (Synthesis Example 14(h): Synthesis of Intermediate 7-1-2 (Boc-Met-Val-PEG4-Mal))

[1005] Dissolve Boc-MV (30.0 mg, 86.12 μmol) in 0.5 mL of anhydrous DMF, then add HATU (43.5 mg, 114.40 μmol). Stir at room temperature for 1 h under a N2 atmosphere. Dissolve Mal-PEG4-NH2 in 0.5 mL of anhydrous DMF, add it to the above system, then add DIPEA (51.8 μL, 297.55 μmol). Stir at room temperature for 3 h under a N2 atmosphere. Detect the reaction by LC-MS. After the reaction is completed, lyophilize. Then purify using semi-preparative HPLC (CH3CN-H2O: 0.1% TFA) and lyophilize to obtain Intermediate 7-1-2 (also referred to as "Boc-MV-PEG4-Mal" hereinafter)

[1006] Intermediate 7-1-2: LC-MS calcd. for: C 29 H 50 N4O 10 S 646.80; found: 647.33 [M+H] + .

[1007] (Synthesis Example 14(i): Synthesis of Intermediate 7-2-2 (NH2-Met-Val-PEG4-Mal))

[1008] Dissolve Intermediate 7-1-2 in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is completed, concentrate and dry to obtain a white solid of crude Intermediate 7-2-2 (also referred to as "NH2-MV-PEG4-Mal" hereinafter).

[1009] (Synthesis Example 14(j): Synthesis of Compound 1-10-2 (DOTA-Bn-Met-Val-PEG4-Mal))

[1010] Dissolve the above crude intermediate 7-2-2 in 0.5 mL of DMF, add p-SCN-Bn-DOTA (15.93 mg, 23.19 μmol), and finally add Et3N (32.0 μL). Stir at room temperature for 3 h. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of the compound (also referred to as "DOTA-Bn-MV-PEG4-Mal" hereinafter).

[1011] Compound 1-10-2: LC-MS calcd. for: C 48 H 75 N9O 16 S2 1098.30; found: 1098.49 [M] + .

[1012] 1.11 Compound 1-11: DOTA-Bn-rSFK-PEG4-Mal (L12)

[1013]

[1014] 15(a): Mal-PEG4-NHS; 15(b): TFA; 15(c): p-SCN-Bn-DOTA

[1015] (Synthesis Example 15(a): Synthesis of Intermediate 8-1 (Boc-(D-Arg)-Ser-Phe-Lys(PEG4)-Mal))

[1016] Dissolve Boc-rSFK (10.0 mg, 15.70 μmol) and NHS-PEG4-Mal (7.6 mg, 17.28 μmol) in 0.5 mL of DMF, add Et3N (4 μL), and stir at room temperature for 3.0 h. Detect the reaction by LC-MS. After the reaction is completed, purify by semi-preparative HPLC and lyophilize. To obtain a white solid of Intermediate 8-1 (also referred to as "Boc-rSFK-PEG4-Mal" hereinafter).

[1017] Intermediate 8-1: LC-MS calcd. for: C 44 H 69 N9O 15 964.08; found: 965.15 [M+H] + .

[1018] (Synthesis Example 15(b): Synthesis of Intermediate 8-2 (NH2-(D-Arg)-Ser-Phe-Lys(PEG4)-Mal))

[1019] Dissolve intermediate 8-1 in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is completed, concentrate and dry by suction to obtain a white solid of crude intermediate 8-2 (also referred to as "NH2-rSFK-PEG4-Mal" hereinafter).

[1020] (Synthesis Example 15(c): Synthesis of Compound 1-11 (DOTA-Bn-(D-Arg)-Ser-Phe-Lys(PEG4)-Mal)) Dissolve the above-mentioned crude intermediate 8-2 in 0.5 mL of DMF, add p-SCN-Bn-DOTA (2.8 mg, 4.05 μmol), and finally add Et3N (8.0 μL). Stir at room temperature for 3 h. After the reaction is completed, purify by semi-preparative HPLC and freeze-dry again to obtain a white solid of the compound (also referred to as "DOTA-Bn-rSFK-PEG4-Mal" hereinafter).

[1021] Compound 1-11: LC-MS calcd. for: C 63 H 94 N 14 O 21 S 1415.58; found: 1416.15 [M+H] + .

[1022] 1.12 Compound 1-12: DOTA-Bn-GWK-PEG4-Mal (L20)

[1023]

[1024] 16(a): Mal-PEG4-NHS; 16(b): TFA; 16(c): p-SCN-Bn-DOTA

[1025] (Synthesis Example 16(a): Synthesis of Intermediate 9-1 (Boc-Gly-Trp-Lys(PEG4)-Mal))

[1026] Dissolve Boc-GWK (3.0 mg, 6.12 μmol) and NHS-PEG4-Mal (3.3 mg, 7.35 μmol) in 0.5 mL of DMF, add Et3N (4 μL), and stir at room temperature for 3.0 h. Detect the reaction by LC-MS. After the reaction is completed, purify by semi-preparative HPLC and freeze-dry. To obtain a white solid of intermediate 9-1 (also referred to as "Boc-GWK-PEG4-Mal" hereinafter).

[1027] Intermediate 9-1: LC-MS calcd. for: C 39 H 56 N6O 13816.91; found: 817.55 [M+H] + .

[1028] (Synthesis Example 16(b): Synthesis of Intermediate 9-2 (NH2-Gly-Trp-Lys(PEG4)-Mal))

[1029] Dissolve Intermediate 9-1 in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is completed, concentrate and dry by suction to obtain a white solid of crude Intermediate 9-2 (hereinafter also referred to as "NH2-GWK-PEG4-Mal").

[1030] (Synthesis Example 16(c): Synthesis of Compound 1-12 (DOTA-Bn-Gly-Trp-Lys(PEG4)-Mal))

[1031] Dissolve the above-mentioned crude Intermediate 9-2 in 0.5 mL of DMF, add p-SCN-Bn-DOTA (4.6 mg, 6.74 μmol), and finally add Et3N (8.0 μL). Stir at room temperature for 3 h. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of the compound (hereinafter also referred to as "DOTA-Bn-GWK-PEG4-Mal").

[1032] Compound 1-12: LC-MS calcd. for: C 58 H 81 N 11 O 19 S 1268.40; found: 1268.55 [M] + .

[1033] 1.13 Compound 1-13: DOTA-Bn-MNK-PEG4-Mal (L22)

[1034]

[1035] 17(a): Mal-PEG4-NHS; 17(b): TFA; 17(c): p-SCN-Bn-DOTA

[1036] (Synthesis Example 17(a): Synthesis of Intermediate 10-1 (Boc-Met-Asn-Lys(PEG4)-Mal))

[1037] Dissolve Boc-MNK (4.8 mg, 9.78 μmol) and NHS-PEG4-Mal (5.2 mg, 11.74 μmol) in 0.5 mL of DMF, add Et3N (4 μL), and stir at room temperature for 3.0 h. Detect the reaction by LC-MS. After the reaction is completed, purify by semi-preparative HPLC and lyophilize to obtain a white solid of intermediate 10-1 (also referred to as "Boc-MNK-PEG4-Mal" hereinafter).

[1038] Intermediate 10-1: LC-MS calcd. for: C 35 H 58 N6O 14 S 818.94; found: 819.65 [M+H] + .

[1039] (Synthesis Example 17(b): Synthesis of Intermediate 10-2 (NH2-Met-Asn-Lys(PEG4)-Mal))

[1040] Dissolve Intermediate 10-1 in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is completed, concentrate and dry by suction to obtain a white solid of crude Intermediate 10-2 (also referred to as "NH2-MNK-PEG4-Mal" hereinafter).

[1041] (Synthesis Example 17(c): Synthesis of Compound 1-13 (DOTA-Bn-Met-Asn-Lys(PEG4)-Mal))

[1042] Dissolve the above crude Intermediate 10-2 in 0.5 mL of DMF, add p-SCN-Bn-DOTA (4.0 mg, 5.86 μmol), and finally add Et3N (8.0 μL). Stir at room temperature for 3 h. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of the compound (also referred to as "DOTA-Bn-MNK-PEG4-Mal" hereinafter).

[1043] Compound 1-13: LC-MS calcd. for: C 54 H 83 N 11 O 20 S2 1270.44; found: 1270.50 [M] + .

[1044] 1.14 Compound 1-14: DOTA-Bn-QLK-PEG4-Mal (L23)

[1045]

[1046] 18(a): Mal-PEG4-NHS; 18(b): TFA; 18(c): p-SCN-Bn-DOTA

[1047] (Synthesis Example 18(a): Synthesis of Intermediate 11-1 (Boc-Gln-Leu-Lys(PEG4)-Mal))

[1048] Dissolve Boc-QLK (4.8 mg, 9.78 μmol) and NHS-PEG4-Mal (4.8 mg, 9.84 μmol) in 0.5 mL of DMF, add Et3N (4 μL), and stir at room temperature for 3.0 h. Monitor the reaction by LC-MS. After the reaction is completed, purify by semi-preparative HPLC and lyophilize to obtain a white solid of Intermediate 11-1 (hereinafter also referred to as "Boc-QLK-PEG4-Mal").

[1049] Intermediate 11-1: LC-MS calcd. for: C 37 H 62 N6O 14 814.93; found: 815.90 [M+H] + .

[1050] (Synthesis Example 18(b): Synthesis of Intermediate 11-2 (NH2-Gln-Leu-Lys(PEG4)-Mal))

[1051] Dissolve Intermediate 11-1 in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is completed, concentrate and dry to obtain a white solid of crude Intermediate 11-2 (hereinafter also referred to as "NH2-QLK-PEG4-Mal").

[1052] (Synthesis Example 18(c): Synthesis of Compound 1-14 (DOTA-Bn-Gln-Leu-Lys(PEG4)-Mal))

[1053] Dissolve the above crude Intermediate 11-2 in 0.5 mL of DMF, add p-SCN-Bn-DOTA (4.0 mg, 5.82 μmol), and finally add Et3N (8.0 μL). Stir at room temperature for 3 h. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of the compound (hereinafter also referred to as "DOTA-Bn-QLK-PEG4-Mal").

[1054] Compound 1-14: LC-MS calcd. for: C 56 H 87 N 11 O 20S 1266.43; found: 1266.45 [M] + .

[1055] 1.15 Compound 1-15: DOTA-Bn-TIK-PEG4-Mal (L21)

[1056]

[1057] 19(a): Mal-PEG4-NHS; 19(b): TFA; 19(c): p-SCN-Bn-DOTA

[1058] (Synthesis Example 19(a): Synthesis of Intermediate 12-1 (Boc-Thr-ILe-Lys(PEG4)-Mal))

[1059] Dissolve Boc-TIK (4.7 mg, 10.30 μmol) and NHS-PEG4-Mal (5.5 mg, 12.36 μmol) in 0.5 mL of DMF, add Et3N (4 μL), and stir at room temperature for 3.0 h. Detect the reaction by LC-MS. After the reaction is completed, purify by semi-preparative HPLC and freeze-dry to obtain Intermediate 12-1, a white solid (also hereinafter referred to as "Boc-TIK-PEG4-Mal").

[1060] Intermediate 12-1: LC-MS calcd. for: C 36 H 61 N5O 14 787.91; found: 788.25 [M+H] + .

[1061] (Synthesis Example 19(b): Synthesis of Intermediate 12-2 (NH2-Thr-ILe-Lys(PEG4)-Mal))

[1062] Dissolve Intermediate 12-1 in 0.5 mL of TFA and stir at room temperature for 30 min. After the reaction is completed, concentrate and dry by suction to obtain a crude product of Intermediate 12-2, a white solid (also hereinafter referred to as "NH2-TIK-PEG4-Mal").

[1063] (Synthesis Example 19(c): Synthesis of Compound 1-15 (DOTA-Bn-Thr-ILe-Lys(PEG4)-Mal))

[1064] Dissolve the above crude intermediate 12-2 in 0.5 mL of DMF, add p-SCN-Bn-DOTA (4.0 mg, 5.82 μmol), and finally add Et3N (8.0 μL). Stir at room temperature for 3 h. After the reaction is completed, purify by semi-preparative HPLC and lyophilize again to obtain a white solid of the compound (also referred to as "DOTA-Bn-TIK-PEG4-Mal" hereinafter).

[1065] Compound 1-15: LC-MS calcd. for: C 55 H 86 N 10 O 20 S 1239.40; found: 1247.15 [M+8] + .

[1066] Compound 1-16: DOTA-Mal (commercially available)

[1067]

[1068] Example 2 Cleavable Linker and Protein Conjugation as Precursors

[1069] Protein Sequence

[1070] SNA006 is a single-domain antibody targeting CD8+ T cells (anti-CD8α VHH), and its sequence is:

[1071] QVQLVESGGGLVQPGGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTY YADSVKGRFTISRDNSKNHVYLQMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKG TLVTVSSGSC (SEQ ID NO: 4)

[1072] SNA004 is a single-domain antibody targeting the HER2 target (anti-HER2 VHH), and its sequence is:

[1073] QLQLVESGGGLVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTN YKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQ VTVSSGSC (SEQ ID NO: 7)

[1074] SNA018 is a scaffold protein drug targeting the HER2 target, and its sequence is:

[1075] GHEHEHEDANSLAAAKETALYHLDRLGVADAYKDLIDKAKTVEGVKARYFEILHALP GSSC (SEQ ID NO:8)

[1076] Coupling method of precursor

[1077]

[1078]

[1079] Example 3 Radionuclide labeling radioactive labeling

[1080]

[1081] Example 4 Analysis of the stability of radionuclide-labeled precursor in human serum

[1082] The radionuclide-labeled precursor (100 μL) prepared in Example 3 was added to human serum and incubated at 37 °C. Samples at each time point were analyzed by Radio-HPLC at 0 h, 1 h, 2 h, and 4 h to calculate the radioactive ratio of the unchanged precursor.

[1083] 68 Human serum stability results of Ga-NOTA-Bn-MV(Dap)-PEG4-SNA006( Figure 1 ), more than 85% of the radioactivity still remained on the NOTA-Bn-MV(Dap)-PEG4-SNA006 precursor after 4 h;

[1084] 68 Human serum stability results of Ga-NOTA-Bn-VMK-(CH2)2-SNA006( Figure 2 ), more than 80% of the radioactivity still remained on the NOTA-Bn-VMK-(CH2)2-SNA006 precursor after 2 h.

[1085] Example 5 Analysis of the radioactivity of radionuclide-labeled precursor in mouse urine

[1086] The radionuclide-labeled precursor prepared in Example 3, solutions with precursor concentrations adjusted to 1 μg and ~20 μCi each, were administered via the tail vein of mice with subcutaneous tumor models. Mouse urine was collected 1.5 h after administration, and then analyzed by Radio-HPLC. The analysis results of the radioactivity in the urine excreted in the examples are as shown in( Figure 3 and Figure 4 ). 68Ga-NOTA-Bn-MV(Dap)-PEG4-SNA006( Figure 3 ) and 68 Ga-NOTA-Bn-VMK-(CH2)2-SNA006( Figure 4 ) showed that 60%-70% of the radioactivity was mainly retained in the precursor with an elution time of 17.6 minutes.

[1087] Example 6 Diagnostic Radionuclide 68 Analysis of the Distribution of Ga-Labeled Precursors in Mice

[1088] In vivo studies were performed using female nude mice aged 6-8 weeks. The nude mice were housed in a SPF environment with free access to food and water and a standard 12-hour light-dark cycle. For xenograft transplantation, animals were injected with 100 μL of cells / PBS. Cells were implanted subcutaneously in the left and right front legs, respectively. The cell seeding density was approximately 5-6×10 6 cells / mouse. Implantation was performed under isoflurane anesthesia. Under these conditions, a viable tumor model was obtained in more than 80% of the injected animals after 2 weeks.

[1089] The radionuclide-labeled precursors prepared in Example 3, with precursor concentrations adjusted to 1 μg and solutions of ~20 μCi each, were administered via the tail vein of subcutaneous tumor model mice. The mice were sacrificed 1.5 h after administration, and blood, urine, and vital organs were collected and weighed. Then, cpm was measured using a gamma counting system, and the radioactive %ID / g in each organ tissue was calculated.

[1090] After 68 Ga-NODAGA-SNA006, 68 Ga-NOTA-Bn-SFK-(CH2)2-SNA006, 68 Ga-NOTA-Bn-DFK-(CH2)2-SNA006, 68 Ga-NOTA-Bn-SLK-SNA006, 68 Ga-NOTA-Bn-VMK-(CH2)2-SNA006 and 68 Ga-NOTA-Bn-MV(Dap)-PEG4-SNA006 were administered to subcutaneous tumor model mice, respectively, the in vivo radioactive distributions at 1.5 h are shown in Table 1. The tumor and kidney distributions are as Figure 5 shown. After 1.5 h of administration, 68 Ga-NOTA-Bn-VMK-(CH2)2-SNA006, 68 Ga-NOTA-Bn-MV(Dap)-PEG4-SNA006 showed high tumor-to-kidney ratios of 0.27 and 0.23, respectively.

[1091] Table 1 Radioisotope-labeled precursor, radioactive distribution in mice within 1.5 h

[1092]

[1093]

[1094] 6.1 Diagnostic radioisotopes 68 Kinetic study of Ga-labeled precursor in mice

[1095] The radioisotope-labeled precursors prepared in Example 3 were adjusted to solutions with precursor concentrations of 1 μg and ~20 μCi, and administered via the tail vein of subcutaneous tumor model mice. After administration via the tail vein of subcutaneous tumor model mice, blood was collected from the mouse orbital cavity at 2 min, 5 min, 10 min, 15 min, 30 min, 60 min, 90 min, and 120 min after administration, weighed, the cpm was measured by a gamma counting system, and %ID / g in the blood was calculated.

[1096] Results are shown ( Figure 6 ): There were no significant changes in the in vivo kinetics of each sample in Example within 2 h after administration compared with the reference example. It was revealed that after applying the cleavable Linker in the radiopharmaceutical, the kidney retention became lower while not affecting the stability of the prototype drug, and the universality of the application of the cleavable Linker was confirmed.

[1097] 6.2 Therapeutic radioisotopes 177 Application of Lu-labeled precursor

[1098] The radioisotope-labeled 177 Lu-DOTA-SNA004, 177 Lu-DOTA-Bn-VMK-PEG4-SNA004 prepared according to the above method was diluted with PBS buffer. Each radioisotope-labeled solution with the radioactive precursor concentration adjusted to 10 μg / 100 μL and ~300 μCi was administered via the tail vein of the above-mentioned subcutaneous tumor model mice. Using MicroSPECT / CT, SPECT / CT scans of the two administered mice were imaged at time points of 0 h, 1 h, 2 h, 4 h, 24 h, 48 h, and 72 h after administration.

[1099] The 177 Lu-DOTA-SNA004, 177 SPECT / CT images of Lu-DOTA-Bn-VMK-PEG4-SNA004 at each time point after administration to tumor model mice are as shown in ( Figure 7 ). After administration for 1 - 24 h, the examples77 Lu-DOTA-Bn-VMK-PEG4-SNA004 accumulates less in the kidneys and clearly images tumors. On the other hand, in the reference example 177 Lu-DOTA-SNA004, although it also images tumors, higher radioactivity is also observed in the kidneys.

[1100] According to the ROI shown in the SPECT / CT image ( Figure 8 ), after administration for 1 - 72 h, 177 Lu-DOTA-SNA004 and 177 the tumor uptake rates of Lu-DOTA-Bn-VMK-PEG4-SNA004 are close at each time point, but 177 higher radioactivity is observed in the kidneys of Lu-DOTA-SNA004.

[1101] Above, 177 the accumulation of the Lu-DOTA-Bn-VMK-PEG4-SNA004 radiolabeled drug in the kidneys becomes lower and still maintains a high tumor targeting property. The potential of the cleavable linker applied to therapeutic radiopharmaceuticals is revealed.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, X-L-VMK-R1 Formula (I) Among them, X is a chelating agent; L is a linker, and L may be absent; -VMK- has the following structural formula ； R1 is a group that binds to the amino or carboxyl group of the side chain of -VMK- and has a functional group capable of binding to a target molecule recognition unit or its linking group, or is a hydrogen atom of the amino or carboxyl group of the side chain of -VMK-.

2. The compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R1 is composed of one or more of the following components: -C1-C 18 alkylene, -(CH2CH2O) n -, -CO(CH2) n -, -NH(CH2) n -, arylthio (PYS), p-aminobenzyloxycarbonyl (PAB), aminobenzylthio, oxybenzylthio, alkoxyamino (AOA), dioxobenzylthio, diaminobenzylthio, aminooxybenzylthio, alkoxyamino (AOA), 4-methyl-4-dithiovaleryl (MPDP), triazole, dithio, sulfonyl, phosphonyl, (4-acetyl)aminobenzoyl (SIAB), 4-thiobutyryl, 4-thio-2-sulfobutyryl (2- SPDB), 4-thiopropionyl (SPDP), hydrazone, aminoethylamine, hydrazine, oxime, thioaminooxobutenoic acid, thioaminooxobutyric acid, a peptide containing 1 to 20 amino acid residues, and (Mal); where n is any integer between 1 and 1000.

3. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R1 is selected from the following structures: (Mal), 6-maleimidohexanoyl (MC), maleimidopropionyl (MP), maleimidoethylamine (ME), 6-maleimidohexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-VC-PAB), 4-(N-maleimidomethyl)cyclohexane-1-carbonyl (SMCC), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP).

4. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of formula (I) is selected from: X-L-VMK-(CH2)2-Mal, X-L-VMK-PEG4-Mal, X-L-VMK-PEG4-Mal, X-VMK-(CH2)2-Mal, X-VMK-PEG4-Mal; Among them, X is selected from: ， ， ， ， ； X-L is selected from: , and , .

5. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of formula (I) is selected from: NOTA-Bn-VMK-(CH2)2-Mal, DOTA-Bn-VMK-PEG4-Mal, DOTA-PEG4-VMK-PEG4-Mal, DOTA-VMK-(CH2)2-Mal, DOTA-VMK-PEG4-Mal.

6. An immunoconjugate comprising i) a compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1-5 and ii) a target molecule recognition unit.

7. The immunoconjugate according to claim 6, which has the structure of formula (II): Formula (II) Among them, X is a chelating agent, and X is connected or not connected to an active moiety; T is a target molecule recognition unit; L is a linker, and L may be absent; wherein-(A1) m -A2-A3- is -Val-Met-Lys-, and the structural formula of -Val-Met-Lys- is shown below 。 8. The immunoconjugate according to claim 6, which is selected from the following structures: NOTA-Bn-VMK-(CH2)2-Mal-VHH, DOTA-Bn-VMK-PEG4-Mal-VHH, DOTA-PEG4-VMK-PEG4-Mal-VHH, DOTA-VMK-(CH2)2-Mal-VHH, DOTA-VMK-PEG4-Mal-VHH.

9. The immunoconjugate according to claim 6, which is selected from the following structures: A-NOTA-Bn-VMK-(CH2)2-Mal-VHH, A-DOTA-Bn-VMK-PEG4-Mal-VHH, A-DOTA-PEG4-VMK-PEG4-Mal-VHH, A-DOTA-VMK-(CH2)2-Mal-VHH, A-DOTA-VMK-PEG4-Mal-VHH wherein A is an active moiety.

10. A radionuclide complex comprising i) a compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1-5 and ii) a radionuclide.

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