CN116077686A - Enzyme-cleavable Linker and application thereof - Google Patents

Enzyme-cleavable Linker and application thereof Download PDF

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CN116077686A
CN116077686A CN202211620156.0A CN202211620156A CN116077686A CN 116077686 A CN116077686 A CN 116077686A CN 202211620156 A CN202211620156 A CN 202211620156A CN 116077686 A CN116077686 A CN 116077686A
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mal
peg
dota
vhh
dap
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须涛
王超
韩涛
司东阳
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Suzhou Zhihe Biomedical Technology Co ltd
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Abstract

The application relates to an enzyme-cleavable Linker and application thereof, in particular to a compound shown in a formula (I) or pharmaceutically acceptable salt thereof, X-L-Y-R 1 Formula (I)) Wherein X is a chelating agent; l is a linker, and L may be absent; y is a polypeptide which is capable of being cleaved by enkephalinase; r is R 1 Is a group that binds to an amino group or a carboxyl group of a side chain of Y and has a functional group capable of binding to a target molecule recognition unit or a linking group thereof, or is a hydrogen atom of an amino group or a carboxyl group of a side chain of Y.

Description

Enzyme-cleavable Linker and application thereof
Technical Field
The application relates to the field of biological medicine, in particular to an enzyme-cleavable Linker and application thereof.
Background
Fluorescence resonance energy transfer (Fluorescence resonance energy transfer, FRET) is a non-radiative energy transfer process that transfers energy from a donor excited state to an acceptor excited state through intermolecular electric dipole interactions. FRET peptides are convenient tools for studying peptidase specificity, and as the reaction process can be continuously monitored, a convenient method is provided for detecting enzyme activity, and fluorescence generated after hydrolysis of peptide bonds of donor/acceptor pairs can measure nanomolar concentration of enzyme activity. When the FRET peptide is intact, internal fluorescence quenching is exhibited, but fluorescence is released when any peptide bond of the donor/acceptor pair is cleaved, which fluorescence can be continuously detected, allowing quantitative analysis of the enzyme activity. FRET peptides can be suitable substrates for various enzyme studies, such as: dynamic and functional characteristics of peptidases, proteases, kinases, phosphatases; screening and detection of novel proteolytic enzymes.
NEP (Neutral endopeptidase, also known as Neprilysin) is a type II transmembrane glycoprotein of the M13 zinc-dependent metalloprotease family, also known as enkephalinase, with the zinc atom carried on the enzyme at the active site. NEP has a molecular weight of about 97kDa and consists of 750 amino acids, comprises a signal peptide and two hydrophilic domains, and has 26 amino acids in the intracellular fragment and 700 amino acids in the extracellular fragment. NEP has obvious specificity, and can cleave peptide bond on amino with great N-terminal aromatic and hydrophobicity to hydrolyze polypeptide chain.
Disclosure of Invention
According to the invention, the polypeptide sequence which can be digested by NEP is screened out through FRET, then the designed compound structure contains ligand molecules for targeting tumor, tri (tetra) peptide sequences which can be digested by NEP at the renal brush border, and a bifunctional linker for chelating radionuclides, so that the concentration and retention of radioactivity in the kidney can be obviously reduced under the condition of keeping the tumor uptake unchanged, the clinical diagnosis and treatment value of the radioligand is improved, and the preparation method has good clinical application prospect.
In one aspect, the present application provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,
X-L-Y-R 1
formula (I)
Wherein X is a chelating agent;
l is a linker, and L may be absent;
Y is a polypeptide which is capable of being cleaved by enkephalinase (Neutral endopeptidase, NEP);
R 1 is a group that binds to an amino group or a carboxyl group of a side chain of Y and has a functional group capable of binding to a target molecule recognition unit or a linking group thereof, or is a hydrogen atom of an amino group or a carboxyl group of a side chain of Y.
In certain embodiments, wherein the Y is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide.
In certain embodiments, wherein Y is: - (A) 1 )m-A 2 -A 3 -, wherein m is 0, 1 or 2; a is that 1 And A 2 Is an amino acid residue, A 3 Is an amino acid residue having an amino group or a carboxyl group in a side chain, R 1 Is with A 3 A group having an amino group or a carboxyl group of a side chain thereof and having a functional group capable of binding to a target molecule recognition unit or a linking group thereof, or A 3 Hydrogen atoms of amino groups or carboxyl groups of the side chains of (a).
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-
(MV), - (D-Arg) -Ser-Phe-Lys- (rSFK), -Gly-Trp-Lys- (GWK), -Met-Asn-Lys- (MNK), -Gln-Leu-Lys- (QLK) and-Thr-ILe-Lys- (TIK).
In certain embodiments, wherein said R 1 Is composed of one or more of the following components: -C 1 -C 18 Alkylene, - (CH) 2 CH 2 O) n -，-CO(CH 2 ) n -，-NH(CH 2 ) n Arylthio (PYS), p-aminobenzyloxycarbonyl (PAB), aminobenzylthio, oxybenzylthio, alkoxyamino (AOA), dioxybenzylthio, diaminobenzylthio, aminooxybenzylthio, alkoxyamino (AOA), 4-methyl-4-dithiopentanoyl (MPDP), triazole, dithio, sulfonyl, phosphonyl, (4-acetyl) aminobenzoyl (SIAB), 4-thiobutyryl, 4-thio-2-sulfonylbutyryl (2-SO) 3 - -SPDB), 4-thiopropionyl (SPDP), hydrazone, aminoethylamine, hydrazine, oxime, thioamino oxybutyric acid, peptide containing 1 to 20 amino acid residues, and
Figure BDA0004001699340000021
wherein n is any integer between 1 and 1000, for example n may 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 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.
In certain embodiments, wherein said R 1 Selected from the following structures:
Figure BDA0004001699340000031
-(CH 2 ) n -Mal，-(CH 2 CH 2 O) n- Mal，-CO(CH 2 )n-Mal，-NH(CH 2 ) n -Mal，-NH(CH 2 CH 2 O) n- mal, where n is any integer between 1 and 1000.
In some embodimentsWherein said R is 1 Selected from the following structures:
Figure BDA0004001699340000032
6-Maleimidocaproyl (MC), maleimidopropionyl (MP), maleimidoethyl (ME), 6-maleimidocaproyl-valine-citrulline-p-aminobenzoic acid oxycarbonyl (MC-VC-PAB), 4- (N-maleimidomethyl) cyclohexane-1-acyl (SMCC), 4- (2-pyridylthio) pentanoic acid N-succinimidyl ester (SPP).
In certain embodiments, wherein the chelator comprises 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.
In certain embodiments, wherein X is selected from the following structures:
Figure BDA0004001699340000033
Figure BDA0004001699340000034
and their derivatives; wherein R is 2 Each independently is a hydrogen atom or an optionally substituted C 1 -C 8 A hydrocarbon group.
In certain embodiments, wherein X is selected from the following structures:
Figure BDA0004001699340000041
Figure BDA0004001699340000042
and their derivatives.
In certain embodiments, wherein either L default or L is selected from the following structures:
Figure BDA0004001699340000043
where n is any integer from 0 to 10, for example n may 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.
In certain embodiments, wherein either L default or L is selected from the following structures:
Figure BDA0004001699340000044
in certain embodiments, wherein X-L is selected from the following structures:
Figure BDA0004001699340000045
Figure BDA0004001699340000046
wherein R is 2 Each independently is a hydrogen atom or an optionally substituted C 1 -C 8 Hydrocarbyl, n is any integer between 1 and 1000.
In certain embodiments, wherein X-L is selected from the following structures:
Figure BDA0004001699340000051
in certain embodiments, wherein the compound of formula (I) is selected from the group consisting of: X-L-SLK (Mal), X-L-SFK- (CH) 2 ) 2 -Mal，X-L-SFK-(CH 2 ) 2 -Mal，X-SFK-(CH 2 ) 2 -Mal，X-L-DFK-(CH 2 ) 2 -Mal，X-L-VMK-(CH 2 ) 2 -Mal，X-L-VMK-PEG 4 -Mal，X-L-VMK-PEG 4 -Mal，X-VMK-(CH 2 ) 2 -Mal，X-VMK-PEG 4 -Mal，X-L-MV(Dap)-PEG 4 -Mal，X-L-MV(Dap)-PEG 4 -Mal，X-L-MV(Dap)-PEG 4 -Mal，X-MV(Dap)-PEG 4 -Mal，X-MV(Dap)-PEG 4 -Mal，X-MV-(CH 2 ) 2 -Mal，X-L-MV-PEG 4 -Mal，X-L-MV-PEG 4 -Mal，X-L-rSFK-PEG 4 -Mal，X-L-GWK-PEG 4 -Mal，X-L-MNK-PEG 4 -Mal，X-L-QLK-PEG 4 -Mal and X-L-TIK-PEG 4 -Mal；
Wherein X is selected from:
Figure BDA0004001699340000052
Figure BDA0004001699340000061
X-L is selected from:
Figure BDA0004001699340000062
in certain embodiments, wherein the compound of formula (I) is selected from the group consisting of: NOTA-Bn-SLK (Mal), NOTA-Bn-SFK- (CH) 2 ) 2 -Mal，DOTA-Bn-SFK-(CH 2 ) 2 -Mal，NODAGA-SFK-(CH 2 ) 2 -Mal，NOTA-Bn-DFK-(CH 2 ) 2 -Mal，NOTA-Bn-VMK-(CH 2 ) 2 -Mal，DOTA-Bn-VMK-PEG 4 -Mal，DOTA-PEG 4 -VMK-PEG 4 -Mal，DOTA-VMK-(CH 2 ) 2 -Mal，DOTA-VMK-PEG 4 -Mal，NOTA-Bn-MV(Dap)-PEG 4 -Mal，DOTA-Bn-MV(Dap)-PEG 4 -Mal，DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal，DOTA-MV(Dap)-PEG 4 -Mal，NODAGA-MV(Dap)-PEG 4 -Mal，DOTA-MV-(CH 2 ) 2 -Mal，DOTA-PEG 4 -MV-PEG 4 -Mal，DOTA-Bn-MV-PEG 4 -Mal，DOTA-Bn-rSFK-PEG 4 -Mal，DOTA-Bn-GWK-PEG 4 -Mal，DOTA-Bn-MNK-PEG 4 -Mal，DOTA-Bn-QLK-PEG 4 -Mal and DOTA-Bn-TIK-PEG 4 -Mal。
In another aspect, the present application provides an immunoconjugate comprising I) a compound represented by formula (I) as described above or a pharmaceutically acceptable salt thereof, and ii) a target molecule recognition unit.
In certain embodiments, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof passes through R 1 To the target recognition unit or to a linking group of the target recognition unit.
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, the active moiety being linked to a chelator.
In certain embodiments, wherein the detectable label is selected from one or more of the reagents of the group consisting of: radionuclides, fluorescers, chemiluminescent agents, bioluminescent agents, paramagnetic ions and enzymes.
In certain embodiments, wherein the radionuclide is suitable for medical imaging and/or therapy.
In certain embodiments, wherein the radionuclide comprises 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 alpha, gamma, beta-, or positron emitters.
In certain embodiments, the immunoconjugate has a structure represented by formula (II):
Figure BDA0004001699340000071
wherein X is a chelator, X is attached or not attached to the active moiety;
t is a target molecule recognition unit;
l is a linker, L being defaulting;
A 1 and A 2 Is an amino acid residue, m is 0,1 or 2;
A 3 is an amino acid residue having an amino group or a carboxyl group in a side chain, R 1 Is with A 3 A group which is bound to an amino group or a carboxyl group of a side chain of (a) and has a functional group capable of binding to a target molecule recognition unit or a linking group thereof;
-(A 1 ) m -A 2 -A 3 -being capable of being cleaved by enkephalinase.
In certain embodiments, wherein- (A) 1 ) m -A 2 -A 3 -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), -gin-Leu-Lys- (QLK) and-Thr-ILe-Lys- (TIK).
In certain embodiments, wherein X is selected from the following structures:
Figure BDA0004001699340000081
/>
Figure BDA0004001699340000082
and their derivatives; wherein R is 2 Independently a hydrogen atom or optionally substituted C 1 -C 8 A hydrocarbon group.
In certain embodiments, wherein X is selected from the following structures:
Figure BDA0004001699340000083
Figure BDA0004001699340000084
and their derivatives.
In certain embodiments, wherein either L default or L is selected from the following structures:
Figure BDA0004001699340000085
where n is any integer from 0 to 10, for example n may 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.
In certain embodiments, wherein either L default or L is selected from the following structures:
Figure BDA0004001699340000086
in certain embodiments, wherein X-L is selected from the following structures:
Figure BDA0004001699340000091
/>
Figure BDA0004001699340000092
where n is any integer between 1 and 1000, for example n may 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 60, 10 to 50, 10 to 45 to 10, 10 to 40, 10 to 35 0,10 to 25, 10 to 20, 10 to 19, 10 to 18, 10 to 17, 10 to 16 or 10 to 15.
In certain embodiments, wherein X-L is selected from the following structures:
Figure BDA0004001699340000093
in certain embodiments, it is selected from the following structures:
Figure BDA0004001699340000101
/>
Figure BDA0004001699340000111
wherein T is a targeting molecule unit;
-(A 1 ) m -A 2 -A 3 -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), -gin-Leu-Lys- (QLK) and-Thr-ILe-Lys- (TIK);
the R is 1 Selected from the following structures:
Figure BDA0004001699340000112
-(CH 2 ) n -Mal，-(CH 2 CH 2 O) n- Mal，-CO(CH 2 )n-Mal，-NH(CH 2 ) n -Mal；
n is each independently any integer between 1 and 1000, for example n may 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 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;
R 2 Each independently is a hydrogen atom or an optionally substituted C 1 -C 8 A hydrocarbon group.
In certain embodiments, the immunoconjugate is selected from the following structures:
NOTA-Bn-SLK(Mal)-T，
NOTA-Bn-SFK-(CH 2 ) 2 -Mal-T，
DOTA-Bn-SFK-(CH 2 ) 2 -Mal-T，
NODAGA-SFK-(CH 2 ) 2 -Mal-T，
NOTA-Bn-DFK-(CH 2 ) 2 -Mal-T，
NOTA-Bn-VMK-(CH 2 ) 2 -Mal-T，
DOTA-Bn-VMK-PEG 4 -Mal-T，
DOTA-PEG 4 -VMK-PEG 4 -Mal-T，
DOTA-VMK-(CH 2 ) 2 -Mal-T，
DOTA-VMK-PEG 4 -Mal-T，
NOTA-Bn-MV(Dap)-PEG 4 -Mal-T，
DOTA-Bn-MV(Dap)-PEG 4 -Mal-T，
DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal-T，
DOTA-MV(Dap)-PEG 4 -Mal-T，
NODAGA-MV(Dap)-PEG 4 -Mal-T，
DOTA-MV-(CH 2 ) 2 -Mal-T，
DOTA-PEG 4 -MV-PEG 4 -Mal-T，
DOTA-Bn-MV-PEG 4 -Mal-T，
DOTA-Bn-rSFK-PEG 4 -Mal-T，
DOTA-Bn-GWK-PEG 4 -Mal-T，
DOTA-Bn-MNK-PEG 4 -Mal-T，
DOTA-Bn-QLK-PEG 4 Mal-T and
DOTA-Bn-TIK-PEG 4 -Mal-T；
wherein T is a target molecule recognition unit comprising an antigen binding protein. In certain embodiments, it is selected from the following structures:
A-NOTA-Bn-SLK(Mal)-T，
A-NOTA-Bn-SFK-(CH 2 ) 2 -Mal-T，
A-DOTA-Bn-SFK-(CH 2 ) 2 -Mal-T，
A-NODAGA-SFK-(CH 2 ) 2 -Mal-T，
A-NOTA-Bn-DFK-(CH 2 ) 2 -Mal-T，
A-NOTA-Bn-VMK-(CH 2 ) 2 -Mal-T，
A-DOTA-Bn-VMK-PEG 4 -Mal-T，
A-DOTA-PEG 4 -VMK-PEG 4 -Mal-T，
A-DOTA-VMK-(CH 2 ) 2 -Mal-T，
A-DOTA-VMK-PEG 4 -Mal-T，
A-NOTA-Bn-MV(Dap)-PEG 4 -Mal-T，
A-DOTA-Bn-MV(Dap)-PEG 4 -Mal-T，
A-DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal-T，
A-DOTA-MV(Dap)-PEG 4 -Mal-T，
A-NODAGA-MV(Dap)-PEG 4 -Mal-T，
A-DOTA-MV-(CH 2 ) 2 -Mal-T，
A-DOTA-PEG 4 -MV-PEG 4 -Mal-T，
A-DOTA-Bn-MV-PEG 4 -Mal-T，
A-DOTA-Bn-rSFK-PEG 4 -Mal-T，
A-DOTA-Bn-GWK-PEG 4 -Mal-T，
A-DOTA-Bn-MNK-PEG 4 -Mal-T，
A-DOTA-Bn-QLK-PEG 4 Mal-T and
A-DOTA-Bn-TIK-PEG 4 -Mal-T；
wherein T is a target molecule recognition unit and A is an active moiety.
In certain embodiments, wherein the target molecule recognition unit comprises an antigen binding protein, a scaffold protein, or a ligand.
In certain embodiments, wherein the antigen binding protein comprises an antibody or antigen binding fragment thereof.
In certain embodiments, the antibodies comprise monoclonal antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, and/or fully human antibodies.
In certain embodiments, the antigen binding fragment comprises Fab, fab ', fv fragment, F (ab') 2 scFv, VHH and/or dAb.
In certain embodiments, wherein the target molecule recognition unit comprises a VHH.
In certain embodiments, wherein the VHH is camelid, chimeric, human, partially humanized or fully humanized.
In certain embodiments, wherein the target molecule recognition unit targets a tumor antigen and/or a non-tumor antigen.
In certain embodiments, wherein the target molecule recognition unit specifically binds to an antigen selected from the group consisting of: AXL, BAFFR, BCMA, BDCA, 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, CD 2098, CD22, CD226, CD248, CD25, CD27, CD274, CD276, CD28, CD30, CD300A, CD32B, CD33, CD37, CD38, CD4, differentiation cluster 40 (CD 40), CD44, CD45, CD46, CD47, CD48, CD5, CD52, CD55, CD56, CD59, CD62E, CD68, CD69, CD70, CD74, CD79A, CD79B, CD8, CD80, CD86, CD90.2, CD96, OX40 (CD 134), 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, HHH La2, type II HLA (HLA class II), HVEM, ICOSLG, IFNAR, type I interferon receptor subunit (IFNAR 1), IFNLR1, 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 (Integrins), LAG3, LIFR, MAG/Siglec-4 (sialic acid binding immunoglobulin-like lectin-4), MMR, MSR1, NCR3LG1, NKG2D, NKp30, NKp46, PDCD1, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, siglec-1 (sialic acid binding immunoglobulin-like lectin-1), siglec-10, siglec-5, siglec-6, siglec-7, siglec-8, siglec-9, SIRPA, SLAMF7, TACI, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2, TGR 3, TIGIT, TLR2, TLR4, tumor necrosis factor alpha (TNF alpha), TROY, TSLPR, TYRO, VLDLR, VSIG4, IL2R-y, VTCN1, TSHR, CD171, CS-1, cll-1, gd3, tn ag, flt3, B7H4, KIT, IL-13ra2, IL-11ra, psca, psma, prss21, egfr2, lewis y, CD24, pdgfr-beta, SSEA-4, muc1, egfr, ncam, caix, lmp2, epha2, fucosyl GM1, sLe, GM3, TGS5, hmw maa, FOLR1, FOLR2, TEM7R, CLDN6, CLDN18.2, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, upk2, havcr1, adrb3, panx3, gpr20, ly6k, or51e2, taw 6, etv6, SPA17, XAGE1, tie 2, MAD-CT-1, MAD-CT-2, FOSL1, hTERT, ML-IAP, ERG, NA17, PAX3, AR, cyclin B1, MYCN, rhoC, CYP B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, CD72, LAIR1, FCAR, LILRA2, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGFL 1, HER2, ROR1, TAAG72, GD2, gp100Tn, FAP, tyrosinase, EPCAM, CEA, IGF-1R, ephB2, mesothelin, cadherin 17, EGFRvIII, GPB, GPR64, HER3, LRP6, LYPD8, NKG2D, SLC34A2, C39A6, SLRK6, GURKC 4 and/or TACST 2.
In certain embodiments, wherein the target molecule recognition unit comprises an anti-PD-L1 VHH antibody, an anti-HER 2 VHH antibody, a HER 2-targeting scaffold protein, and or an anti-CD 8 a VHH antibody.
In certain embodiments, wherein the anti-CD 8 a VHH antibody comprises: CDR1 of the amino acid sequence shown as SEQ ID NO. 1, CDR2 of the amino acid sequence shown as SEQ ID NO. 2 and CDR3 of the amino acid sequence shown as SEQ ID NO. 3.
In certain embodiments, the anti-CD 8. Alpha. VHH antibody comprises the amino acid sequence shown in SEQ ID NO. 4.
In certain embodiments, wherein the anti-HER 2 VHH antibody comprises: CDR1 of the amino acid sequence shown as SEQ ID NO. 5, CDR2 of the amino acid sequence shown as SEQ ID NO. 6 and CDR3 of the amino acid sequence shown as SEQ ID NO. 7.
In certain embodiments, the anti-HER 2 VHH antibody comprises an amino acid sequence shown in SEQ ID No. 8.
In certain embodiments, the HER 2-targeting scaffold protein comprises the amino acid sequence shown in SEQ ID No. 9.
In certain embodiments, the immunoconjugate is selected from the following structures:
Figure BDA0004001699340000151
/>
Figure BDA0004001699340000161
wherein, the liquid crystal display device comprises a liquid crystal display device,
-(A 1 ) m -A 2 -A 3 -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), -gin-Leu-Lys- (QLK) and-Thr-ILe-Lys- (TIK); the R is 1 Conjugated antigen binding proteins, scaffold proteins or ligands.
In certain embodiments, a subjectThe R is 1 Selected from the following structures:
Figure BDA0004001699340000171
-(CH 2 ) n -Mal，-(CH 2 CH 2 O) n- Mal，-CO(CH 2 )n-Mal，-NH(CH 2 ) n mal, where n is any integer between 1 and 10.
In certain embodiments, the immunoconjugate is selected from the following structures:
NOTA-Bn-SLK(Mal)-VHH，
NOTA-Bn-SFK-(CH 2 ) 2 -Mal-VHH，
DOTA-Bn-SFK-(CH 2 ) 2 -Mal-VHH，
NODAGA-SFK-(CH 2 ) 2 -Mal-VHH，
NOTA-Bn-DFK-(CH 2 ) 2 -Mal-VHH，
NOTA-Bn-VMK-(CH 2 ) 2 -Mal-VHH，
DOTA-Bn-VMK-PEG 4 -Mal-VHH，
DOTA-PEG 4 -VMK-PEG 4 -Mal-VHH，
DOTA-VMK-(CH 2 ) 2 -Mal-VHH，
DOTA-VMK-PEG 4 -Mal-VHH，
NOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，
DOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，
DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal-VHH，
DOTA-MV(Dap)-PEG 4 -Mal-VHH，
NODAGA-MV(Dap)-PEG 4 -Mal-VHH，
DOTA-MV-(CH 2 ) 2 -Mal-VHH，
DOTA-PEG 4 -MV-PEG 4 -Mal-VHH，
DOTA-Bn-MV-PEG 4 -Mal-VHH，
DOTA-Bn-rSFK-PEG 4 -Mal-VHH，
DOTA-Bn-GWK-PEG 4 -Mal-VHH，
DOTA-Bn-MNK-PEG 4 -Mal-VHH，
DOTA-Bn-QLK-PEG 4 Mal-VHH
DOTA-Bn-TIK-PEG 4 -Mal-VHH。
For another example, the immunoconjugate is selected from the following structures:
A-NOTA-Bn-SLK(Mal)-VHH，
A-NOTA-Bn-SFK-(CH 2 ) 2 -Mal-VHH，
A-DOTA-Bn-SFK-(CH 2 ) 2 -Mal-VHH，
A-NODAGA-SFK-(CH 2 ) 2 -Mal-VHH，
A-NOTA-Bn-DFK-(CH 2 ) 2 -Mal-VHH，
A-NOTA-Bn-VMK-(CH 2 ) 2 -Mal-VHH，
A-DOTA-Bn-VMK-PEG 4 -Mal-VHH，
A-DOTA-PEG 4 -VMK-PEG 4 -Mal-VHH，
A-DOTA-VMK-(CH 2 ) 2 -Mal-VHH，
A-DOTA-VMK-PEG 4 -Mal-VHH，
A-NOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，
A-DOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，
A-DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal-VHH，
A-DOTA-MV(Dap)-PEG 4 -Mal-VHH，
A-NODAGA-MV(Dap)-PEG 4 -Mal-VHH，
A-DOTA-MV-(CH 2 ) 2 -Mal-VHH，
A-DOTA-PEG 4 -MV-PEG 4 -Mal-VHH，
A-DOTA-Bn-MV-PEG 4 -Mal-VHH，
A-DOTA-Bn-rSFK-PEG 4 -Mal-VHH，
A-DOTA-Bn-GWK-PEG 4 -Mal-VHH，
A-DOTA-Bn-MNK-PEG 4 -Mal-VHH，
A-DOTA-Bn-QLK-PEG 4 Mal-VHH
A-DOTA-Bn-TIK-PEG 4 -Mal-VHH；
Wherein A is an active moiety.
In certain embodiments, wherein a is a detectable label selected from one or more of the reagents of the group consisting of: radionuclides, fluorescers, chemiluminescent agents, bioluminescent agents, paramagnetic ions and enzymes.
In certain embodiments, wherein A is a radionuclide comprising 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 alpha, gamma, beta-, or positron emitters.
In another aspect, the present application provides a radionuclide complex comprising I) a compound of formula (I) as described above or a pharmaceutically acceptable salt thereof, and ii) a radionuclide.
In certain embodiments, wherein the radionuclide is attached to X in the compound of formula (I).
In another aspect, the present application provides a composition comprising a compound of formula (I) as described above or a pharmaceutically acceptable salt thereof, an immunoconjugate as described above, and optionally a pharmaceutically acceptable carrier.
In certain embodiments, the composition is a detection agent or a therapeutic agent.
In certain embodiments, wherein the detection agent is an agent for detecting an antigen.
In certain embodiments, wherein the detection agent is a contrast agent.
In certain embodiments, wherein the contrast agent is a contrast agent that detects an antigen.
In certain embodiments, wherein the antigen is a tumor antigen.
In certain embodiments, wherein the therapeutic agent is used to treat a tumor.
In another aspect, the present application provides the use of a compound of formula (I) as described above or a pharmaceutically acceptable salt thereof, an immunoconjugate as described above, a radionuclide complex as described above, and/or a composition as described above, in the preparation of a reagent, assay plate, or kit; wherein the reagent, assay plate or kit is used to detect an antigen in a sample.
In another aspect, the present application provides the use of a compound of formula (I) as described above or a pharmaceutically acceptable salt thereof, an immunoconjugate as described above, a radionuclide complex as described above, and/or a composition as described above, in the preparation of a medicament; wherein the medicament is for the treatment of a tumor.
In another aspect, the present application provides the use of a compound of formula (I) as described above, or a pharmaceutically acceptable salt thereof, an immunoconjugate as described above, a radionuclide complex as described above, and/or a composition as described above, in the preparation of a radiolabeled drug.
In another aspect, the present application provides a radiolabeled drug comprising a compound of formula (I) as described above or a pharmaceutically acceptable salt thereof, an immunoconjugate as described above, a radionuclide complex as described above, and/or a composition as described above.
In another aspect, the present application provides a radiographic diagnostic drug comprising a compound represented by the aforementioned formula (I) or a pharmaceutically acceptable salt thereof, the aforementioned immunoconjugate, the aforementioned radionuclide complex, and/or the aforementioned composition.
In another aspect, the present application provides a method of detecting the presence and/or amount of an antigen in a biological sample, comprising: contacting the biological sample with a compound of formula (I) or a pharmaceutically acceptable salt thereof, an immunoconjugate, a radionuclide complex, and/or a composition.
In certain embodiments, wherein the contacting is performed in vitro or ex vivo.
In certain embodiments, wherein the biological sample is tissue.
In certain embodiments, wherein the tissue is selected from the group consisting of blood tissue, lymphoid tissue, and tumor tissue.
In certain embodiments, the method comprises detecting the presence and/or amount of tumor antigen positive cells in the biological sample.
In certain embodiments, wherein the presence and/or amount of tumor antigen positive cells in the biological sample is determined by imaging.
In certain embodiments, wherein the presence and/or amount of tumor antigen positive cells in the biological sample is determined by flow cytometry.
In another aspect, the present application provides a method of detecting and/or diagnosing and oncologically diagnosing comprising administering to a subject in need thereof a compound of formula (I) as described above or a pharmaceutically acceptable salt thereof, an immunoconjugate as described above, a radionuclide complex as described above, and/or a composition as described above.
In certain embodiments, wherein the method further comprises imaging the subject.
In certain embodiments, wherein the imaging comprises ECT imaging.
In certain embodiments, wherein the ECT imaging comprises SPECT imaging or PET imaging.
In another aspect, the present application provides 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 above or a pharmaceutically acceptable salt thereof, an immunoconjugate as described above, a radionuclide complex as described above, and/or a composition as described above.
In another aspect, the present application provides a method for monitoring the efficacy of an anti-tumor therapy in a subject, the method comprising:
(i) Administering to a subject suffering from a tumor and treated with an anti-tumor therapy a compound represented by the aforementioned formula (I) or a pharmaceutically acceptable salt thereof, the aforementioned immunoconjugate, the aforementioned radionuclide complex, and/or the aforementioned composition; and
(ii) Determining the amount of tumor antigen positive cells in the tumor of the subject.
In certain embodiments, the presence and/or amount of SNA004 positive cells in the tumor of the subject is determined by imaging.
In certain embodiments, wherein the tumor comprises a solid tumor.
In certain embodiments, wherein the tumor is selected from at least one of 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, and the like.
In another aspect, the present application provides a kit comprising a compound of formula (I) as described above or a pharmaceutically acceptable salt thereof, an immunoconjugate as described above, a radionuclide complex as described above, and/or a composition as described above.
Other aspects and advantages of the present application will become readily apparent to those skilled in the art 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 present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the invention as described herein. Accordingly, the drawings and descriptions herein are to be regarded as illustrative in nature and not as restrictive.
Drawings
The specific features of the invention related to this application are set forth in the appended claims. The features and advantages of the invention that are related to the present application will be better understood by reference to the exemplary embodiments and the drawings that are described in detail below. The drawings are briefly described as follows:
FIG. 1 shows that 68 Ga-NOTA-Bn-MV(Dap)-PEG 4 -006 in vitro human serum stability results.
FIG. 2 shows 68 Ga-NOTA-Bn-VMK-(CH 2 ) 2 -006 in vitro human serum stability results.
FIG. 3 shows 68 Ga-NOTA-Bn-MV(Dap)-PEG 4 -006 in vivo urine stability results.
FIG. 4 shows 68 Ga-NOTA-Bn-VMK-(CH 2 ) 2 -006 in vivo urine stability results.
Figure 5 shows the distribution of radionuclide-labeled precursors described in this application in mice.
Figure 6 shows the in vivo kinetics of radionuclide-labeled precursors described herein in mice.
Figure 7 shows SPECT/CT images at various time points after administration of radionuclide-labeled precursors described in this application to tumor model mice.
FIG. 8 shows 177 Lu-DOTA-Bn-VMK-PEG 4 -SPECT/CT image ROI of SNA004 radiolabeled drug.
Detailed Description
Further advantages and effects of the invention of the present application will become apparent to those skilled in the art from the disclosure of the present application, from the following description of specific embodiments.
Definition of terms
In the present application, the term "hydrocarbyl", alone or as part of another substituent, means-unless otherwise specified-a straight or branched or cyclic hydrocarbon radical or combination thereof, which may be fully saturated, mono-or poly-unsaturated, may include di-and poly-valent radicals having the indicated number of carbon atoms (that is, C 1 -C 10 Representing one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl,Such as n-pentyl, n-hexyl, n-heptyl, n-octyl homologs and isomers, and the like. Unsaturated hydrocarbon radicals are those having one or more double or triple bonds. Examples of unsaturated hydrocarbyl groups 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 "hydrocarbyl" is also meant to include those hydrocarbyl derivatives, such as "heterohydrocarbyl", which are defined in detail below, unless otherwise noted.
In this application, the term "alkyl" generally refers to a branched or unbranched saturated hydrocarbon group. Suitably, the alkyl group comprises from 1 to 100, preferably from 3 to 30 carbon atoms, more preferably from 5 to 25 carbon atoms. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, or hexyl. "alkenyl" generally refers to a branched or unbranched hydrocarbon group containing one or more carbon-carbon double bonds. Suitably, the alkenyl group comprises from 2 to 30 carbon atoms, preferably from 5 to about 25 carbon atoms. "alkynyl" generally refers to a branched or unbranched hydrocarbon group containing one or more carbon-carbon triple bonds. Suitably, the alkynyl group contains from about 3 to about 30 carbon atoms, for example from about 5 to about 25 carbon atoms.
In the present application, the term "halogen" generally refers to fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.
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 ring system. More commonly cycloalkyl groups are monocyclic. The term includes references to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo [2.2.2] octyl, and the like.
In the present application, the term "aryl" refers to an aromatic carbocyclic ring system, suitably comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring carbon atoms. Aryl groups may be polycyclic systems having two or more rings, at least one of which is aromatic. The term includes references to groups such as phenyl, naphthyl fluorenyl, azulenyl, indenyl, anthracenyl and the like.
The prefix (hetero) herein means that one or more carbon atoms of the group may be replaced by nitrogen, oxygen, phosphorus, silicon or sulfur. Heteroalkyl groups include, for example, alkoxy and alkylthio groups. The heterocycloalkyl or heteroaryl groups herein may 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 may be saturated or unsaturated. For example, selected from the group consisting of oxiranyl, aziridinyl, 1, 2-oxathiolanyl, imidazolyl, thienyl, furyl, tetrahydrofuranyl, pyranyl, thienyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chroenyl, 2H-pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidinyl, piperazinyl, pyridazinyl, morpholinyl, thiomorpholinyl, in particular thiomorpholinyl, indolazinyl, 1, 3-dioxo-1, 3-dihydro-isoindolyl, 3H-indolyl, benzimidazolyl, coumaryl, indazolyl, triazolyl, tetrazolyl, purinyl, 4H-quinolinyl, isoquinolyl, quinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, octahydroquinolinyl, benzofuranyl, dihydroquinolinyl, benzofuranyl, 3-quinolinyl, 3-naphthyridinyl, 4H-quinolinyl, 2-quinolinyl, phenanthridinyl, 4-quinolinyl, 4-naphthyridinyl, 4-quinolinyl, and the like.
The term "optionally" as used herein means that the subsequently described event may or may not occur, and includes both event occurrence and event non-occurrence, unless otherwise defined in the claims.
In the present application, the term "substituted" means one of the moietiesOr a plurality, in particular up to 5, more in particular 1, 2 or 3, hydrogen atoms are substituted independently of one another by a corresponding number of substituents. As used herein, the term "optionally substituted" includes substituted or unsubstituted. Of course, it is to be understood that substituents are only in their chemically possible positions, and that one skilled in the art will be able to determine (experimentally or theoretically) whether a particular substitution is possible without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable if bound to carbon atoms having unsaturated (e.g., olefinic) bonds. Preferably, the term "substituted" means that one or more, in particular up to 5, more in particular 1, 2 or 3 hydrogen atoms of said moiety are each independently substituted by a corresponding number of substituents selected from OH, SH, NH 2 Halogen, cyano, carboxyl, alkyl, cycloalkyl, aryl and heteroaryl. Furthermore, the substituents described herein may themselves be substituted with any substituent, but are limited by the appropriate substitution identified above for those skilled in the art. Preferably, any of the above substituents may be further substituted with any of the above substituents, and each substituent may be further substituted with any of the above substituents.
Substituents may suitably include halogen atoms and halomethyl groups, e.g. CF 3 And CCl 3 The method comprises the steps of carrying out a first treatment on the surface of the Oxygen-containing groups such as oxo, hydroxy, carboxy, 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 mercaptans, alkanethiols, sulfonyl groups and sulfoxides; heterocyclic groups which may themselves be substituted; alkyl groups which may themselves be substituted; and aryl groups which may themselves be substituted, such as phenyl and substituted phenyl. Alkyl groups include substituted and unsubstituted benzyl groups.
When two or more moieties are described as "each independently" selected from a list of atoms or groups, this means that the moieties may be the same or different. The identity of each portion is thus independent of the identity of one or more other portions.
In this application, the term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity (Milleretal (2003) journal. Ofimmunology 170:4854-4861), i.e., bind to CD8 a (such as human CD8 a, murine CD8 a, cynomolgus monkey CD8 a, or rhesus monkey CD8 a). The antibody may be murine, human, humanized, chimeric, or derived from other species.
Full length antibodies typically refer to antibodies that consist of two "full length antibody heavy chains" and two "full length antibody light chains. A "full length antibody heavy chain" is generally a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CH 1), an antibody Hinge Region (HR), an antibody heavy chain constant domain 2 (CH 2), and an antibody heavy chain constant domain 3 (CH 3), abbreviated as VH-CH1-HR-CH2-CH3, in the N-terminal to C-terminal direction; and optionally also antibody heavy chain constant domain 4 (CH 4) in the case of antibodies of the IgE subclass. In some embodiments, a "full length antibody heavy chain" is a polypeptide consisting of VH, CH1, HR, CH2, and CH3 in the N-to C-terminal direction. A "full length antibody light chain" is generally a polypeptide consisting of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), abbreviated VL-CL, in the N-to C-terminal direction. The antibody light chain constant domain (CL) may be kappa (kappa) or lambda (lambda). 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 regions of the full length antibody heavy chains. Examples of typical full length antibodies are natural antibodies such as IgG (e.g., igG1 and IgG 2), igM, igA, igD, and IgE.
In the present application, the term "antigen binding fragment" generally refers to a portion of an antibody molecule that comprises amino acids responsible for specific binding between the antibody and the antigen. The portion of the antigen specifically recognized and bound by an antibody is referred to as an "epitope" as described above. The antigen binding domain may typically comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily include both. Fd fragments have, for example, two VH regions and are generally conservedLeaving some of the antigen binding function of the antigen binding domain intact. Examples of antigen-binding fragments of antibodies include (1) Fab fragments, monovalent fragments having VL, VH, constant light Chain (CL), and CH1 domains; (2) F (ab') 2 A fragment, a bivalent fragment having two Fab fragments linked by a disulfide bridge of a hinge region; (3) Fd fragment with two VH and CH1 domains; (4) Fv fragments with VL and VH domains of an antibody single arm, (5) dAb fragments (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), with VH domains; (6) an isolated Complementarity Determining Region (CDR); (7) Single chain Fv (scFv), e.g., derived from a scFV-library. Although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that allows them to be prepared as a Single protein chain in which the VL and VH regions pair to form a monovalent molecule, known as a Single chain Fv (scFv) (see, e.g., 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 variable antigen binding domains from heavy chain antibodies of the family camelidae (camel, dromedary, llama, alpaca, etc.) (see, nguyen v.k. Et al, 2000,The EMBO Journal,19, 921-930;Muyldermans S, 2001,J Biotechnol, 74, 277-302 and reviewed vanland choot p. Et al, 2011,Antiviral Research 92, 389-407). VHH may also be referred to as Nanobody (Nb) and/or single domain antibodies. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the function of the fragments is assessed in the same manner as for the whole antibody.
In the present application, the term "variable domain" generally refers to the variable domain of an antibody capable of specifically binding to an epitope of an antigen. For example, antibody variable domains VH and VL (VH domain and VL domain). Another example of a variable domain is a "VHH domain" (or simply "VHH"). "VHH domain", also known as heavy chainSingle domain antibodies, VHHs, V H The H domain, VHH antibody fragment and VHH antibody are the 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 (which is referred to herein as a "VH domain") present in conventional 4-chain antibodies, and the light chain variable domain (which is referred to herein as a "VL domain") present in conventional 4-chain antibodies. The VHH domain specifically binds to the epitope without the need for additional antigen binding domains (this is in contrast to VH or VL domains in conventional 4-chain antibodies, in which case the epitope is recognized by the VL domain along with the VH domain).
In this application, the "variable domains" generally have the same general structure, each domain comprising 4 Framework (FR) regions that are highly conserved in sequence, wherein the FR regions comprise "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and four "framework regions" of "framework region 4" or "FR4", the FR regions "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and three "complementarity determining regions" or "CDRs" junctions of "complementarity determining region 3" or "CDR 3". The general structure or sequence of the variable domain can be expressed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The antibody variable domains confer specificity to the antigen to the antibody by having an antigen binding site.
In this application, the term "CDR" generally refers to complementarity determining regions within an antibody variable sequence. There are 3 CDRs in each of the variable regions of the heavy and light chains, which are referred to as CDR1, CDR2, and CDR3 for each variable region. The exact boundaries of these CDRs have been defined differently depending on the system. The system described by Kabat (Kabat et al, sequences of Proteins of Immunological Interest (National Institutes ofHealth, bethesda, md. (1987) and (1991)) not only provides a well-defined residue numbering system for any variable region of an antibody, but also provides precise residue boundaries defining these three CDRs; lesk, j.moi.biol.196:901-917 (1987) and Chothia et al, nature 342:877-883 (1989)) found that some of the sub-portions within the Kabat CDR taken almost the same peptide framework image, although with great differences at the amino acid sequence level, these sub-portions were designated L1, L2 and L3, or H1, H2 and H3, respectively, where "L" and "H" refer to the light and heavy chain regions, respectively, these regions could be referred to as Chothia CDRs with boundaries overlapping Kabat CDRs.
In this application, the term "sequence identity" generally refers to nucleic acid or amino acid sequences that are identical in two or more aligned sequences when aligned using a sequence alignment program. The term "% sequence identity" as used 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 assessing 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 may be used to determine identity. For a 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,20von heinje, g., academic Press,1987 and Sequence Analysis Primer, gribskov, m.and deveeux, j., eds., M Stockton Press, new York,1991.
In this application, amino acid residues will be represented according to standard three-letter or one-letter amino acid codes as known and agreed upon in the art. When comparing two amino acid sequences, the term "amino acid difference" generally refers to an insertion, deletion or substitution of a specified number of amino acid residues at a position in a reference sequence as compared to the other sequence. In some embodiments, the substitution is a conservative amino acid substitution, which refers to the replacement of an amino acid residue with another amino acid residue of similar chemical structure, with little or no effect on the function, activity, or other biological property of the polypeptide. Such conservative amino acid substitutions are well known in the art, e.g. conservative amino acid substitutions are substitutions of one amino acid within the following groups (i) - (v) by another amino acid residue within the same group: (i) smaller aliphatic nonpolar or low polar residues: ala, ser, thr, pro and Gly; (ii) a polar negatively charged residue and (uncharged) amide: asp, asn, glu and Gln; (iii) a polar positively charged residue: his, arg and Lys; (iv) a larger aliphatic nonpolar residue: met, leu, ile, val and Cys; (v) aromatic residues: phe, tyr and Trp. Particularly preferred conservative amino acid substitutions are as follows: substitution of Ala with Gly or Ser; arg is replaced by Lys; asn is substituted with Gln or His; asp is substituted with Glu; cys is replaced by Ser; gln is substituted with Asn; glu is substituted with Asp; substitution of Gly with Ala or Pro; his is substituted with Asn or Gln; lie is substituted with Leu or Val; leu is substituted with Ile or Val; lys is substituted with Arg, gin or Glu; met is substituted with Leu, tyr or Ile; phe is substituted with Met, leu or Tyr; ser is substituted by Thr; thr is replaced by Ser; trp is substituted with Tyr; tyr is substituted by Trp or Phe; val is replaced by Ile or Leu. In some embodiments, the substitution is a non-conservative amino acid substitution, e.g., an Ala substitution with Asp, asn, glu or Gin.
In this 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 antibody) and its binding partner (e.g., a target or antigen). The affinity of a molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, such as surface plasmon resonance, and also include those reported herein. The higher affinity of molecule X for its binding partner Y can be seen in lower Kd values and/or EC50 values.
In this application, the term "isolated" generally refers to a molecule (e.g., antibody, nucleic acid, etc.) that is at least partially separated from other molecules to which it is normally bound 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 background that is different from that which it is found in nature.
In this application, the term "immunoconjugate" generally refers to a conjugate formed by an antibody or antibody fragment thereof linked to other active agents, such as chemotherapeutic agents, toxins, immunotherapeutic agents, radioactive elements, imaging probes, spectroscopic probes, and the like. The linkage may be a covalent bond, or a non-covalent interaction, for example by electrostatic forces. A variety of linkers known in the art may be used to form immunoconjugates. The conjugate may be capable of delivering the additional agent to a target cell (e.g., a tumor cell) by specific binding of the antibody or antigen binding fragment thereof to an antigen on the target cell. In addition, the immunoconjugate may be provided in the form of a fusion protein that may be expressed from a polynucleotide encoding the immunoconjugate.
In this application, the term "chelator" 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 product of the metal ion conjugate is used in radioimmunoassay, radioimmunotherapy, magnetic resonance imaging, photodynamic therapy or other similar modes. Non-limiting examples of chelating or complexing agents are DTPA (diethylenetriamine pentaacetic anhydride) and its derivatives, NOTA (1, 4, 7-triazacyclononane-N, N ', N "-triacetic acid) and its derivatives such as NODA-GA (nodga), maleimido-nodga, DOTA (1, 4,7, 10-tetraazacyclododecane-N, N ', N", N ' "-tetraacetic acid) (binding a radioactive metal ion) and its derivatives, TETA (1, 4,8, 11-tetraazacyclotetradecane-N, N ', N", N ' "-tetraacetic acid) and its derivatives, DTTA (N- (p-isothiocyanate) -diethylenetriamine-N, N ', N", N ' "-tetraacetic acid). These and other chelators are readily available from commercial sources.
In this application, the term "detectable label" generally refers to a moiety having a detectable physical or chemical property, which can produce 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 (rhodomine), 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., molecules containing streptavidin moieties) and fluorescent labels or enzymatic activity detectable by optical or calorimetric methods), and predetermined polypeptide epitopes (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags) recognized by secondary reporter.
In this application, the term "pharmaceutically acceptable salt" is generally meant to include salts commonly used to form alkali metal salts and to form 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 may 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 may be selected from the group of aliphatic, cycloaliphatic, aromatic aliphatic, heterocyclic, carboxylic or sulfonic acids, examples of which are formic acid, acetic acid, fatty acid, 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, sulfanilic acid, cyclohexylsulfamic acid, camphoric acid, camphorsulfonic acid, digluconic acid, cyclopentanepropionic acid, dodecylsulfonic acid, glucoheptonic acid, glycerophosphonic acid, heptanoic acid, caproic acid, 2-hydroxy-ethanesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid, oxalic acid, palmitic acid, pectic acid, persulfuric acid, 2-phenylpropionic acid, picric acid, pivalic acid, succinic acid, tartaric acid, thiocyanic acid, methanesulfonic acid, undecanoic acid, stearic acid, alginic acid, β -hydroxybutyric acid, galactonic acid, and galactonic acid. Suitable pharmaceutically acceptable base addition salts of the compounds include metallic salts, such as those prepared from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc, or salts prepared from organic bases including primary, secondary and tertiary amines, substituted amines including cyclic amines such as caffeine, arginine, diethylamine, N-ethylpiperidine, aistine, glucosamine, isopropylamine, lysine, morpholine, N-ethylmorpholine, piperazine, piperidine, triethylamine, trimethylamine. All of these salts can be prepared from the corresponding compounds of the present invention by conventional methods, for example, by reacting the appropriate acid or base with the compounds. Once the pharmaceutical composition is formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations may be stored as ready-made forms or forms that require reconstitution prior to administration (e.g., lyophilized forms).
In this 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 may conveniently contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations may also contain compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a human. Other contemplated carriers, excipients, and/or additives that may be used in the formulations described herein include: for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients (e.g., serum albumin, gelatin, casein), salt forming counterions (e.g., 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's pharmaceutical Science and practice (Remington: the Science & # x26; practice of Pharmacy)", 21 st edition, lipping williams & Wilkins publishing company (Lippincott Williams & # x26; wilkins) (2005) and "Physician's desk Reference" (60 th edition, medical economic publishing company (Medical Economics), montvale (Montvale), new jersey (2005). Pharmaceutically acceptable carriers can be routinely selected that are appropriate for the desired or required mode of administration, solubility, and/or stability.
In this application, the term "administration" and similar terms are generally not limited to bodily administration, and suitable methods include in vitro, ex vivo, or in vivo methods. For example, any method of administration known to those skilled in the art for contacting cells, organs or tissues with a composition may be employed. For example, the compound may be introduced into the body of a subject in need of treatment by any route of introduction or delivery. In some embodiments, the compositions of the present application may be administered orally, topically, intranasally, intramuscularly, subcutaneously, intradermally, intrathecally, intraperitoneally, or transdermally.
In this application, the term "ex vivo" is interchangeable with "in vitro" and generally refers to an activity performed in a cell, tissue and/or organ that has been removed from a subject in a controlled environment.
In this application, the term "diagnosis" generally refers to detecting a disease or disorder, or determining the status or extent of a disease or disorder. The term "diagnosis" may also include detecting the cause of a disease or disorder, determining the therapeutic effect of a drug therapy, or predicting the response pattern to a drug therapy.
In this application, the term "treatment" generally refers to: (i) Preventing the occurrence of a disease, disorder, or condition in a patient who may be susceptible to the disease, disorder, and/or condition, but has not been diagnosed with the disease; (ii) Inhibiting the disease, disorder or condition, i.e., inhibiting its development; and (iii) alleviating the disease, disorder or condition, i.e., causing regression of the disease, disorder and/or condition and/or symptoms associated with the disease, disorder and/or condition.
In this 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.
In this 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, monkeys, etc.
In this application, the term "about" generally means ranging from 0.5% to 10% above or below the specified value, e.g., ranging from 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.
In this application, the terms "comprises," "comprising," and variations thereof, including "includes," "including," and the like, are used to specify the presence of stated features, elements, values, steps, etc.
Detailed Description
A compound of formula (I)
In one aspect, the present application provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,
X-L-Y-R 1
Formula (I)
Wherein X is a chelating agent;
l is a linker, and L may be absent;
y is a polypeptide which is capable of being cleaved by enkephalinase (Neutral endopeptidase, NEP); for example, Y is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide or decapeptide capable of being cleaved by a rphin enzyme
R 1 Is a group that binds to an amino group or a carboxyl group of a side chain of Y and has a functional group capable of binding to a target molecule recognition unit or a linking group thereof, or is a hydrogen atom of an amino group or a carboxyl group of a side chain of Y.
For example, wherein said Y may 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), -gin-Leu-Lys- (QLK) and-Thr-ILe-Lys- (TIK).
Figure BDA0004001699340000301
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Figure BDA0004001699340000311
In certain embodiments, R 1 May independently be composed of one or more of the following components:
Figure BDA0004001699340000312
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Figure BDA0004001699340000321
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Figure BDA0004001699340000331
in certain embodiments, 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 and analogs thereof. The term "derivative" includes chemical modification of a compound. Examples of such modifications include substitution of hydrogen with halogen groups, alkyl groups, acyl groups, or amino groups, and the like. The modification may increase or decrease one or more of hydrogen bond interactions, charge interactions, hydrophobic interactions, van der waals interactions, and/or dipole interactions. The term "analog" includes any enantiomer, racemate, and stereoisomer of such compounds, as well as all pharmaceutically acceptable salts and hydrates.
For example, wherein X may be selected from the following structures:
Figure BDA0004001699340000332
Figure BDA0004001699340000333
and their derivatives; wherein R is 2 Each independently is a hydrogen atom or an optionally substituted C 1 -C 8 A hydrocarbon group.
For example, wherein X-L is selected from the following structures:
Figure BDA0004001699340000334
Figure BDA0004001699340000341
wherein R is 2 Each independently is a hydrogen atom or an optionally substituted C 1 -C 8 A hydrocarbon group; n is any integer between 1 and 1000.
For another example, the present application provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,
X-L-Y-R 1
formula (I)
Wherein, the X may be selected from the following structures (ldefault):
Figure BDA0004001699340000342
Figure BDA0004001699340000343
and their derivatives; wherein R is 2 Each independently is a hydrogen atom or an optionally substituted C 1 -C 8 A hydrocarbon group;
X-L is selected from the following structures:
Figure BDA0004001699340000344
Figure BDA0004001699340000351
where n is any integer between 1 and 1000, for example n may 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 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;
The Y may 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), -gin-Leu-Lys- (QLK) and-Thr-ILe-Lys- (TIK);
the R is 1 May be selected from the following structures:
Figure BDA0004001699340000352
6-Maleimidohexanoyl (MC), maleimidopropionyl (MP), maleimidoethyl (M)E) 6-maleimidocaproyl-valine-citrulline-p-aminobenzoic acid oxycarbonyl (MC-VC-PAB), 4- (N-maleimidomethyl) cyclohexane-1-acyl (SMCC), 4- (2-pyridylthio) pentanoic acid N-succinimidyl ester (SPP).
Target molecule recognition unit
The immunoconjugate and the like of the present application are compounds obtained by binding a target molecule recognizing unit to a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a pharmacologically acceptable salt thereof. The target molecule recognition unit may be bound to the compound represented by formula (I) or a pharmaceutically acceptable salt thereof via a linking group, or may be directly bound thereto.
In the present application, the term "target molecule recognition unit" generally refers to a molecule, substituent, functional group or atomic group capable of binding to a target molecule or the like in vivo and recognizing the target molecule. The target molecule recognition unit may be a polypeptide or other ligand that binds to the target molecule. The polypeptide is typically a polypeptide that binds to a target molecule, and preferably a polypeptide that specifically binds to a target molecule. Specific binding refers to binding to a 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 is the subject of diagnosis by radiolabeling a drug, e.g., a molecule present in a tissue or cell, preferably a specifically expressed molecule. The term "specifically expressed" generally refers to expression at a target site, but not at sites other than the target site, or under-expressed. Examples of the target molecule recognizing unit include: ligands that bind to proteins found to be highly expressed in tissue constructs accompanied by inflammation, tumor cell infiltration, and the like, or proteins specifically expressed in tumor cells, and antibodies, antigen-binding region fragments of antibodies, and the like.
Examples of the antibody include monoclonal antibodies such as an anti-CD 8 a antibody and an anti-HER 2 antibody. Examples of the antigen-binding region fragment of the antibody include Fab fragments (hereinafter also abbreviated as "Fab"), F (ab') 2 Fragments, F (ab) 2 Fragments, variable region fragments (hereinafter also referred to as "Fv fragments").
Examples of the other target molecule recognition unit include: cyclic pentapeptides having affinity for integrin highly expressed in tumor neovascular tissue, such as cyclo-Arg-Gly-Asp-D-Phe-Lys (hereinafter also referred to as "c (RGDfK)"). Further, there may be mentioned: bisphosphonic acid, oligoaspartic acid, oligoglutamic acid, which have affinity for hydroxyapatite, which is frequently present in osteogenic cancer (bone metastasis), fMet-Leu-Phe (fMLP), which is a peptide having affinity for a scanning factor receptor present on the surface of macrophages, folic acid, which binds to a folic acid receptor expressed in tumor cells, and derivatives thereof, and the like.
It is to be noted that the target molecule recognizing unit is not limited to these exemplified polypeptides, and may be optionally used as long as it is a polypeptide that binds to a target molecule.
For the target molecule recognizing unit, for example, a thiolating agent such as 2-iminothiolane may be used to introduce and bind a linking group that reacts with a functional group of the compound. The introduction of the linking group into the Fab fragment can be achieved by reacting the thiolating reagent at a pH of 7 to 9 to add a thiol group to the amino group on the Fab cross-section.
Immunoconjugates
In another aspect, the present application provides an immunoconjugate comprising I) a compound represented by formula (I) as described above or a pharmaceutically acceptable salt thereof, and ii) a target molecule recognition unit.
In certain embodiments, the immunoconjugate has a structure represented by formula (II):
Figure BDA0004001699340000361
wherein X is a chelator, X is attached or not attached to the active moiety;
t is a target molecule recognition unit;
l is a linker, L being defaulting;
A 1 and A 2 Is an amino acid residue, m is 0,1 or 2;
A 3 is a side chainAmino acid residues having amino or carboxyl groups thereon, R 1 Is with A 3 A group which is bound to an amino group or a carboxyl group of a side chain of (a) and has a functional group capable of binding to a target molecule recognition unit or a linking group thereof;
-(A 1 ) m -A 2 -A 3 is capable of being cleaved by enkephalinase, e.g. wherein- (A) 1 ) m -A 2 -A 3 -may 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), -gin-Leu-Lys- (QLK) and-Thr-ILe-Lys- (TIK).
In certain embodiments, wherein X is selected from the following structures:
Figure BDA0004001699340000371
Figure BDA0004001699340000372
and their derivatives; wherein R is 2 Independently a hydrogen atom or optionally substituted C 1 -C 8 A hydrocarbon group.
In certain embodiments, wherein X-L is selected from the following structures:
Figure BDA0004001699340000373
/>
Figure BDA0004001699340000374
where n is any integer between 1 and 1000, for example n may 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 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.
In certain embodiments, the R 1 Selected from the following structures:
Figure BDA0004001699340000381
-(CH 2 ) n -Mal，-(CH 2 CH 2 O) n- Mal，-CO(CH 2 )n-Mal，-NH(CH 2 ) n -Mal；
for example, the immunoconjugate may have a structure represented by formula (II):
Figure BDA0004001699340000382
wherein, the X may be selected from the following structures (ldefault):
Figure BDA0004001699340000383
Figure BDA0004001699340000384
and their derivatives; wherein R is 2 Independently a hydrogen atom or optionally substituted C 1 -C 8 A hydrocarbon group;
wherein X-L is selected from the following structures:
Figure BDA0004001699340000391
Figure BDA0004001699340000392
where n is any integer between 1 and 1000, for example n may 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 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;
t is a target molecule recognition unit;
-(A 1 ) m -A 2 -A 3 -may 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), -gin-Leu-Lys- (QLK) and-Thr-ILe-Lys- (TIK);
The R is 1 Selected from the following structures:
Figure BDA0004001699340000393
-(CH 2 ) n -Mal，-(CH 2 CH 2 O) n- Mal，-CO(CH 2 )n-Mal，-NH(CH 2 ) n -Mal。
for example, the immunoconjugate may be selected from the following structures:
NOTA-Bn-SLK(Mal)-T，
NOTA-Bn-SFK-(CH 2 ) 2 -Mal-T，
DOTA-Bn-SFK-(CH 2 ) 2 -Mal-T，
NODAGA-SFK-(CH 2 ) 2 -Mal-T，
NOTA-Bn-DFK-(CH 2 ) 2 -Mal-T，
NOTA-Bn-VMK-(CH 2 ) 2 -Mal-T，
DOTA-Bn-VMK-PEG 4 -Mal-T，
DOTA-PEG 4 -VMK-PEG 4 -Mal-T，
DOTA-VMK-(CH 2 ) 2 -Mal-T，
DOTA-VMK-PEG 4 -Mal-T，
NOTA-Bn-MV(Dap)-PEG 4 -Mal-T，
DOTA-Bn-MV(Dap)-PEG 4 -Mal-T，
DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal-T，
DOTA-MV(Dap)-PEG 4 -Mal-T，
NODAGA-MV(Dap)-PEG 4 -Mal-T，
DOTA-MV-(CH 2 ) 2 -Mal-T，
DOTA-PEG 4 -MV-PEG 4 -Mal-T，
DOTA-Bn-MV-PEG 4 -Mal-T，
DOTA-Bn-rSFK-PEG 4 -Mal-T，
DOTA-Bn-GWK-PEG 4 -Mal-T，
DOTA-Bn-MNK-PEG 4 -Mal-T，
DOTA-Bn-QLK-PEG 4 Mal-T and
DOTA-Bn-TIK-PEG 4 -Mal-T；
wherein T is a target molecule recognition unit comprising an antigen binding protein.
For another example, the chelator may be conjugated to active moiety a, and the immunoconjugate may be selected from the structures: A-NOTA-Bn-SLK (Mal) -T,
A-NOTA-Bn-SFK-(CH 2 ) 2 -Mal-T，
A-DOTA-Bn-SFK-(CH 2 ) 2 -Mal-T，
A-NODAGA-SFK-(CH 2 ) 2 -Mal-T，
A-NOTA-Bn-DFK-(CH 2 ) 2 -Mal-T，
A-NOTA-Bn-VMK-(CH 2 ) 2 -Mal-T，
A-DOTA-Bn-VMK-PEG 4 -Mal-T，
A-DOTA-PEG 4 -VMK-PEG 4 -Mal-T，
A-DOTA-VMK-(CH 2 ) 2 -Mal-T，
A-DOTA-VMK-PEG 4 -Mal-T，
A-NOTA-Bn-MV(Dap)-PEG 4 -Mal-T，
A-DOTA-Bn-MV(Dap)-PEG 4 -Mal-T，
A-DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal-T，
A-DOTA-MV(Dap)-PEG 4 -Mal-T，
A-NODAGA-MV(Dap)-PEG 4 -Mal-T，
A-DOTA-MV-(CH 2 ) 2 -Mal-T，
A-DOTA-PEG 4 -MV-PEG 4 -Mal-T，
A-DOTA-Bn-MV-PEG 4 -Mal-T，
A-DOTA-Bn-rSFK-PEG 4 -Mal-T，
A-DOTA-Bn-GWK-PEG 4 -Mal-T，
A-DOTA-Bn-MNK-PEG 4 -Mal-T，
A-DOTA-Bn-QLK-PEG 4 Mal-T and
A-DOTA-Bn-TIK-PEG 4 -Mal-T；
wherein T is a target molecule recognition unit and A is an active moiety.
For example, the target molecule recognition unit may be an antibody or antigen binding fragment thereof, and for example, the T target molecule recognition unit may be a VHH antibody.
In certain embodiments, the immunoconjugate is selected from the following structures:
NOTA-Bn-SLK(Mal)-VHH，
NOTA-Bn-SFK-(CH 2 ) 2 -Mal-VHH，
DOTA-Bn-SFK-(CH 2 ) 2 -Mal-VHH，
NODAGA-SFK-(CH 2 ) 2 -Mal-VHH，
NOTA-Bn-DFK-(CH 2 ) 2 -Mal-VHH，
NOTA-Bn-VMK-(CH 2 ) 2 -Mal-VHH，
DOTA-Bn-VMK-PEG 4 -Mal-VHH，
DOTA-PEG 4 -VMK-PEG 4 -Mal-VHH，
DOTA-VMK-(CH 2 ) 2 -Mal-VHH，
DOTA-VMK-PEG 4 -Mal-VHH，
NOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，
DOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，
DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal-VHH，
DOTA-MV(Dap)-PEG 4 -Mal-VHH，
NODAGA-MV(Dap)-PEG 4 -Mal-VHH，
DOTA-MV-(CH 2 ) 2 -Mal-VHH，
DOTA-PEG 4 -MV-PEG 4 -Mal-VHH，
DOTA-Bn-MV-PEG 4 -Mal-VHH，
DOTA-Bn-rSFK-PEG 4 -Mal-VHH，
DOTA-Bn-GWK-PEG 4 -Mal-VHH，
DOTA-Bn-MNK-PEG 4 -Mal-VHH，
DOTA-Bn-QLK-PEG 4 -Mal-VHH and DOTA-Bn-TIK-PEG 4 -Mal-VHH。
For another example, the immunoconjugate may be selected from the following structures: A-NOTA-Bn-SLK (Mal) -VHH, A-NOTA-Bn-SFK- (CH) 2 ) 2 -Mal-VHH，A-DOTA-Bn-SFK-(CH 2 ) 2 -Mal-VHH，A-NODAGA-SFK-(CH 2 ) 2 -Mal-VHH，A-NOTA-Bn-DFK-(CH 2 ) 2 -Mal-VHH，A-NOTA-Bn-VMK-(CH 2 ) 2 -Mal-VHH，A-DOTA-Bn-VMK-PEG 4 -Mal-VHH，A-DOTA-PEG 4 -VMK-PEG 4 -Mal-VHH，A-DOTA-VMK-(CH 2 ) 2 -Mal-VHH，A-DOTA-VMK-PEG 4 -Mal-VHH，A-NOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，A-DOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，A-DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal-VHH，A-DOTA-MV(Dap)-PEG 4 -Mal-VHH，A-NODAGA-MV(Dap)-PEG 4 -Mal-VHH，A-DOTA-MV-(CH 2 ) 2 -Mal-VHH，A-DOTA-PEG 4 -MV-PEG 4 -Mal-VHH，
A-DOTA-Bn-MV-PEG 4 -Mal-VHH，
A-DOTA-Bn-rSFK-PEG 4 -Mal-VHH，
A-DOTA-Bn-GWK-PEG 4 -Mal-VHH，
A-DOTA-Bn-MNK-PEG 4 -Mal-VHH，
A-DOTA-Bn-QLK-PEG 4 Mal-VHH
A-DOTA-Bn-TIK-PEG 4 -Mal-VHH；
Wherein A is an active moiety.
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.
For another example, the immunoconjugate may be selected from the following structures:
A-NOTA-Bn-SLK(Mal)-VHH，
A-NOTA-Bn-SFK-(CH 2 ) 2 -Mal-VHH，
A-DOTA-Bn-SFK-(CH 2 ) 2 -Mal-VHH，
A-NODAGA-SFK-(CH 2 ) 2 -Mal-VHH，
A-NOTA-Bn-DFK-(CH 2 ) 2 -Mal-VHH，
A-NOTA-Bn-VMK-(CH 2 ) 2 -Mal-VHH，
A-DOTA-Bn-VMK-PEG 4 -Mal-VHH，
A-DOTA-PEG 4 -VMK-PEG 4 -Mal-VHH，
A-DOTA-VMK-(CH 2 ) 2 -Mal-VHH，
A-DOTA-VMK-PEG 4 -Mal-VHH，
A-NOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，
A-DOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，
A-DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal-VHH，
A-DOTA-MV(Dap)-PEG 4 -Mal-VHH，
A-NODAGA-MV(Dap)-PEG 4 -Mal-VHH，
A-DOTA-MV-(CH 2 ) 2 -Mal-VHH，
A-DOTA-PEG 4 -MV-PEG 4 -Mal-VHH，
A-DOTA-Bn-MV-PEG 4 -Mal-VHH，
A-DOTA-Bn-rSFK-PEG 4 -Mal-VHH，
A-DOTA-Bn-GWK-PEG 4 -Mal-VHH，
A-DOTA-Bn-MNK-PEG 4 -Mal-VHH，
A-DOTA-Bn-QLK-PEG 4 Mal-VHH
A-DOTA-Bn-TIK-PEG 4 -Mal-VHH；
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 alpha, gamma, beta-, or positron emitters.
Radioactive complexes
In this application, the term "radioactive complex" generally refers to any complex including a radioisotope or radionuclide, such as any radioisotope or radionuclide described herein. The term "complex" generally refers to a substance in which ligands are coordinated about atoms or ions of radionuclides and radionuclide-like elements, also referred to as coordination compounds. Coordination refers to the ligand forming a coordination bond with the central metal and being arranged around the central metal. The complex is formed by a coordination bond between the ligand and the metal. The formation of a complex from a ligand and a metal is also referred to as complexation. Coordination bond refers to a bond in which two valence electrons participating in a bond are provided by only one atom.
As used herein, the term "radionuclide" refers to an atom capable of undergoing radioactive decay (e.g., 3 H、 14 C、 15 N、 18 F、 35 S、 47 Sc、 55 Co、 60 Cu、 61 Cu、 62 Cu、 64 Cu、 67 Cu、 75 Br、 76 Br、 77 Br、 89 Zr、 86 Y、 87 Y、
90 Y、 97 Ru、 99 Tc、 99m Tc 105 Rh、 109 Pd、 111 In、 123 I、 124 I、 125 I、 131 I、 149 Pm、 149 Tb、 153 Sm、 166 Ho、 177 Lu、 186 Re、 188 Re、 198 Au、 199 Au、 203 Pb、 211 At、 212 Pb、 212 Bi、 213 Bi、 223 Ra、
225 Ac、 227 Th、 229Th 、 66 Ga、 67 Ga、 68 Ga、 82 Rb、 117m Sn、 201 tl). The term radionuclide, radioisotope, or radioisotope may also be used to describe radionuclides. Radionuclides may be used as detection agents, as described above. In some embodiments, the radionuclide may be an alpha, gamma, beta-, or positron emitting radionuclide.
The radionuclide is not limited to these specific examples, and may be optionally used as long as it has radiation, radiation amount, half-life, etc. suitable for diagnosis using a radiolabeled drug. From the viewpoint of reducing the influence on normal tissues and cells in radiographic diagnosis, it is preferable to use a short half-life metal radioisotope.
The preparation of the radioactive complex can be achieved by using said compound bound to the target molecule recognition unit as ligand and complexing with the metallic radioisotope in vitro. Complexation can be achieved by simple manipulations using previously known complexation reactions.
Pharmaceutical composition
The compounds described herein may be in the form of pharmaceutical compositions that may be used in human and veterinary medicine for humans or animals, and typically comprise any one or more pharmaceutically acceptable diluents, carriers or excipients. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical arts and are described, for example, in the Mack publishing company (a.r. gennaro editors 1985) in the pharmaceutical sciences of ramington. The choice of pharmaceutically acceptable carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may comprise as carrier, excipient or diluent or in addition any suitable combination, lubricant, suspension, coating, solubilising agent.
Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and parabens. Antioxidants and suspensions may also be used.
Depending on the delivery system, there may be different composition/formulation requirements. For example, the pharmaceutical composition may be formulated for administration using a micropump or by a mucosal route, e.g., as a nasal spray or aerosol for inhalation or ingestion of a solution, or parenterally, wherein the composition is formulated for delivery in an injectable form, e.g., by an intravenous, intramuscular, or subcutaneous route. Alternatively, the formulation may be designed to be administered by a variety of routes.
If the agent is to be administered through the mucosa of the gastrointestinal tract, 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 decontamination of bile.
Where appropriate, the pharmaceutical composition may be administered by: inhalation, external use in the form of suppositories or pessaries, external use in the form of lotions, solutions, creams, ointments or dusting powders, oral administration in the form of tablets containing excipients such as starch or lactose, or capsule or bead (ovile) alone or in admixture with excipients, or elixirs, solutions or suspensions containing flavoring or coloring agents, or pharmaceutical compositions may be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions are preferably used in the form of sterile aqueous solutions which may contain other substances, for example, sufficient salts or monosaccharides to render the solution isotonic with blood. For buccal or sublingual administration, the compositions may be administered in the form of tablets or lozenges, which may be formulated in conventional manner.
The compounds of the invention may 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 science (j.pharm.sci.), 66,1-19 (1977). 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, ascorbic acid, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1' -methylene-bis- (2-hydroxy-3-naphthoate)) salts.
The route of administration (delivery) may include, but is not limited to, one or more of the following: oral (e.g., as a tablet, capsule, 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, buccal, vaginal, epidural, sublingual.
Typically, the physician will determine the actual dosage that best suits the individual subject. The specific dosage level and frequency of administration for any particular patient can vary and will depend upon a variety of factors including the activity of the particular compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.
The formulations may be packaged in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for administration. Ready-to-use injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Exemplary unit dose formulations contain a daily dose or unit daily sub-dose of the active ingredient or an appropriate fraction thereof.
The pharmaceutical composition may be provided in lyophilized form. The lyophilized pharmaceutical composition is preferably reconstituted in a suitable buffer prior to administration, advantageously based on an aqueous carrier.
The pharmaceutical compositions of the present invention also provide for the preparation of medicaments for reducing nephrotoxic side effects of radiolabeled and non-radiolabeled therapeutic and diagnostic compounds.
The pharmaceutical composition or medicament is preferably used to reduce the nephrotoxic side effects of radiolabeled therapeutic and diagnostic compounds for imaging or treating diseases, particularly neoplastic diseases, such neuroendocrine tumors, prostate, pancreatic, renal, bladder, brain, gastrointestinal, medullary thyroid, small or non-small cell lung cancer, and interstitial ovarian, pancreatic ductal adenocarcinoma, insulinoma, gastrinoma, breast cancer, or sarcomas.
Kit for detecting a substance in a sample
In another aspect, the present application also provides a kit comprising a pharmaceutical composition for use according to the present application, a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, a radiolabeled or non-radiolabeled therapeutic or diagnostic compound of formula (I), an immunoconjugate as described herein, a radionuclide complex as described herein and/or a pharmaceutical composition as described herein.
Optionally, the kit may comprise at least one additional agent as defined herein in the context of pharmaceutical compositions, including, for example, amino acids such as lysine and arginine and mixtures thereof, gelatin, amifostine, albumin-derived peptides, PSMA binding molecules (e.g., PMPA), vitamins, radionuclides, antimicrobial agents, solubilizing agents, and the like.
The kit may be a two-part or more kit comprising any of the components exemplified above in a suitable container. For example, each container may be in the form of a vial, bottle, squeeze bottle, jar, sealed sleeve, envelope or pouch, tube or blister pack, or any other suitable form, provided that the container preferably prevents premature mixing of the components. Each of the different components may be provided separately, or some of the different components may be provided together (i.e., in the same container).
The container may also be a compartment or chamber within a vial, tube, jar, or envelope, or sleeve, or blister package or bottle, provided that the contents of one compartment cannot be physically associated with the contents of another compartment before the pharmacist or doctor intentionally mixes.
The kit or part of the kit may also comprise instructions containing information about the administration and dosage of any of its components.
Therapeutic and diagnostic methods and uses
The compounds described herein are useful in the treatment of diseases. The treatment may be a therapeutic and/or prophylactic treatment with the aim of preventing, reducing or halting an undesired physiological change or disorder. Treatment may extend survival compared to the expected survival without treatment.
The disease treated by the compound may be any disease that may benefit from treatment. This includes chronic and acute disorders or diseases, including those pathological conditions susceptible to such disorders.
The terms "cancer" and "cancerous" are used in their broadest sense to mean physiological conditions in mammals that are typically characterized by unregulated cell growth. The tumor comprises one or more cancer cells.
In treating cancer, the observed therapeutic effect may be a reduction in the number of cancer cells; tumor size is reduced; inhibit or delay infiltration of cancer cells into peripheral organs; inhibiting tumor growth; and/or alleviating one or more symptoms associated with cancer.
In animal models, efficacy can be assessed by physical measurements of tumors during treatment and/or by determining partial and complete remission of cancer. For cancer therapy, efficacy may be measured, for example, by assessing time to disease progression (TTP) and/or determining Rate of Remission (RR).
Particularly preferred embodiments of the treatment methods related to the invention are shown in the appended claims.
Also disclosed are methods of treating, or performing a diagnostic method on, a human or animal body, for example, by surgery or therapy, involving the step of administering to a subject in need thereof a therapeutically or diagnostically effective amount of a compound or pharmaceutical composition described herein. More specifically, disclosed herein are methods for treating (e.g., by treating or preventing) a subject suffering from or at risk of suffering from a disease or disorder; or by guided surgery performed on a subject suffering from or at risk of suffering from a disease or disorder; methods for diagnosing diseases or disorders, e.g., performing and/or involving nuclear medicine imaging techniques, such as Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT), on the human or animal body; methods for targeted delivery of a therapeutic or diagnostic agent to a subject suffering from or at risk of suffering from a disease or disorder. In the foregoing method, the disease or condition may be independently selected from the group consisting of 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, hypopharynx cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocarcinoma, renal clear cell carcinoma, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, CUP (primary focus unknown cancer), thymus cancer, hard fibromas, glioma, astrocytomas, cervical cancer, skin cancer, renal cancer, and prostate cancer. When used in the methods disclosed herein, the compounds have an extended residence time at the disease site at therapeutically or diagnostically relevant levels, preferably exceeding 1h, more preferably exceeding 6h after injection.
In another aspect, the present invention relates to the use of a compound of formula (I) as described above or a pharmaceutically acceptable salt thereof, an immunoconjugate as described above, a radionuclide complex as described above and/or a composition as described above, for reducing the nephrotoxic side effects of radiolabeled and non-radiolabeled therapeutic and diagnostic compounds in a subject.
Also relates to a pharmaceutical composition as described above or a kit as described above for use in a method of reducing nephrotoxic side effects of radiolabeled and non-radiolabeled therapeutic and diagnostic compounds in a subject.
In a further aspect, the present application also provides a method of reducing nephrotoxic side effects of radiolabeled and non-radiolabeled therapeutic and diagnostic compounds in a subject, the method comprising administering to the subject a pharmaceutical composition as described above or a kit as described above during imaging or therapy with radiolabeled and/or non-radiolabeled compounds.
In a further aspect, the present application also provides a method of reducing nephrotoxic side effects of radiolabeled and non-radiolabeled therapeutic or diagnostic compounds in a subject, the method comprising administering in the subject a compound of formula (I) as hereinbefore described or a pharmaceutically acceptable salt thereof, an immunoconjugate as hereinbefore described, a radionuclide complex as hereinbefore described and/or a composition as hereinbefore described, before and/or during and/or after administration of the radiolabeled or non-radiolabeled therapeutic or diagnostic compound.
Method for preparing conjugates
Also disclosed is a method for preparing a conjugate comprising the step of conjugating a compound of formula (I) as described above to a target molecule recognition unit. Without limitation, the compound of formula (I) is conjugated to the target molecule recognition unit by reacting with it to form a covalent bond.
The conjugate may be a therapeutic and/or diagnostic agent and may correspond to the payload portion already described in detail above in relation to the conjugate according to the invention. The method further comprises formulating the conjugate into a pharmaceutical composition or a diagnostic composition. The pharmaceutical or diagnostic composition may be used in human 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 arts and are described, for example, in the Mack publishing company (a.r. gennaro editors 1985) in the pharmaceutical sciences of ramington. The choice of carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical or diagnostic composition may comprise as carrier, excipient or diluent or in addition any suitable combination, lubricant, suspension, coating, solubilising agent. All of the formulation details and aspects disclosed in the "pharmaceutical composition" section above are also fully applicable thereto.
The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and pharmacology, which are known to those skilled in the art. These techniques are well explained in the literature. See, e.g., gennaro, a.r., editors (1990) leimington pharmaceutical science, 18 th edition, mack publishing company; hardman, J.G., limbird, L.E., and Gilman, A.G., editors (2001) pharmacological foundation of therapeutics (ThePharmacological Basis of Therapeutics), 10 th edition, mcGraw-Hill Co.; colowick, S. et al, editorial, methods of enzymology (Methods In Enzymology), academic publishing company; weir, D.M. and Blackwell, C.C., editions (1986) laboratory immunology handbook (Handbook of Experimental Immunology), volumes I-IV, blackwell science publishers; maniatis, T.et al, edited (1989) molecular cloning: a laboratory manual (molecular cloning: ALaboratory Manual), version 2, volumes I-III, cold spring harbor laboratory Press; ausubel, f.m. et al, edit (1999) short protocol for molecular biology (Short Protocols in Molecular Biology), 4 th edition, john Wiley & Sons; ream et al, edit (1998) molecular biology techniques: strengthening laboratory courses (MolecularBiology Techniques: an Intensive Laboratory Course), academic press; newton, C.R. and Graham, A., editions (1997) PCR (guide on biotechnology series) (PCR (Introduction to BiotechniquesSeries)), 2 nd edition, springer Verlag.
The present application also includes the following embodiments:
1. a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof,
X-L-Y-R 1
formula (I)
Wherein X is a chelating agent;
l is a linker, and L may be absent;
y is a polypeptide which is capable of being cleaved by enkephalinase (Neutral endopeptidase, NEP);
R 1 is a group that binds to an amino group or a carboxyl group of a side chain of Y and has a functional group capable of binding to a target molecule recognition unit or a linking group thereof, or is a hydrogen atom of an amino group or a carboxyl group of a side chain of Y.
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.
3. The compound of formula (I) according to any one of embodiments 1-2, or a pharmaceutically acceptable salt thereof, wherein Y is: - (A) 1 )m-A 2 -A 3 -, wherein m is 0, 1 or 2; a is that 1 And A 2 Is an amino groupAcid residue, A 3 Is an amino acid residue having an amino group or a carboxyl group in a side chain, R 1 Is with A 3 A group having an amino group or a carboxyl group of a side chain thereof and having a functional group capable of binding to a target molecule recognition unit or a linking group thereof, or A 3 Hydrogen atoms of amino groups or carboxyl groups of the side chains of (a).
4. A 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), -gin-Leu-Lys- (QLK) and-Thr-ILe-Lys- (TIK).
5. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4, wherein R 1 Is composed of one or more of the following components: -C 1 -C 18 Alkylene, - (CH) 2 CH 2 O) n -，-CO(CH 2 ) n -，-
NH(CH 2 ) n Arylthio (PYS), p-aminobenzyloxycarbonyl (PAB), aminobenzylthio, oxybenzylthio, alkoxyamino (AOA), dioxybenzylthio, diaminobenzylthio, aminooxybenzylthio, alkoxyamino
(AOA), 4-methyl-4-dithiopentanoyl (MPDP), triazole, dithio, sulfonyl, phosphonyl, (4-acetyl) aminobenzoyl (SIAB), 4-thiobutyryl, 4-thio-2-sulfobutyryl (2-SO) 3 - -SPDB), 4-thiopropionyl (SPDP), hydrazone, aminoethylamine, hydrazine, oxime, thioamino oxybutyric acid, peptide containing 1 to 20 amino acid residues, and
Figure BDA0004001699340000501
Wherein n is any integer between 1 and 1000.
6. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-5, wherein R 1 Selected from the followingThe structure is as follows:
Figure BDA0004001699340000502
-(CH 2 ) n -Mal，-(CH 2 CH 2 O) n- Mal，-CO(CH 2 )n-Mal，-NH(CH 2 ) n mal, where n is any integer between 1 and 1000.
7. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-6, wherein R 1 Selected from the following structures:
Figure BDA0004001699340000503
6-Maleimidocaproyl (MC), maleimidopropionyl (MP), maleimidoethyl (ME), 6-maleimidocaproyl-valine-citrulline-p-aminobenzoic acid oxycarbonyl (MC-VC-PAB), 4- (N-maleimidomethyl) cyclohexane-1-acyl (SMCC), 4- (2-pyridylthio) pentanoic acid N-succinimidyl ester (SPP).
8. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to embodiment 6, wherein the chelator comprises one or more of the chelators 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 derivatives thereof.
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:
Figure BDA0004001699340000511
Figure BDA0004001699340000512
and their derivatives; wherein R is 2 Each independently is a hydrogen atom or an optionally substituted C 1 -
C 8 A hydrocarbon group.
10. A 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:
Figure BDA0004001699340000513
Figure BDA0004001699340000514
and their derivatives.
11. A compound of formula (I) according to any one of embodiments 1-10, or a pharmaceutically acceptable salt thereof, wherein L default or L is selected from the following structures:
Figure BDA0004001699340000515
wherein n is any integer from 0 to 10.
12. A compound of formula (I) according to any one of embodiments 1-11, or a pharmaceutically acceptable salt thereof, wherein L default or L is selected from the following structures:
Figure BDA0004001699340000521
13. the compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-12, wherein
X-L is selected from the following structures:
Figure BDA0004001699340000522
Figure BDA0004001699340000523
wherein R is 2 Each independently is a hydrogen atom or an optionally substituted C 1 -C 8 Hydrocarbyl radicals n is from 1 to 1000Any integer therebetween.
14. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-13, wherein
X-L is selected from the following structures:
Figure BDA0004001699340000524
15. the 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- (CH) 2 ) 2 -Mal，X-L-SFK-(CH 2 ) 2 -Mal，X-SFK-(CH 2 ) 2 -Mal，X-L-DFK-(CH 2 ) 2 -Mal，X-L-VMK-(CH 2 ) 2 -Mal，X-L-VMK-PEG 4 -Mal，X-L-VMK-PEG 4 -Mal，X-VMK-(CH 2 ) 2 -Mal，X-VMK-PEG 4 -Mal，X-L-MV(Dap)-PEG 4 -Mal，X-L-MV(Dap)-PEG 4 -Mal，X-L-MV(Dap)-PEG 4 -Mal，X-MV(Dap)-PEG 4 -Mal，X-MV(Dap)-PEG 4 -Mal，X-MV-(CH 2 ) 2 -Mal，X-L-MV-PEG 4 -Mal，X-L-MV-PEG 4 -Mal，X-L-rSFK-PEG 4 -Mal，X-L-GWK-PEG 4 -Mal，X-L-MNK-PEG 4 -Mal，X-L-QLK-PEG 4 -Mal and X-L-TIK-PEG 4 -Mal；
Wherein X is selected from:
Figure BDA0004001699340000531
X-L is selected from:
Figure BDA0004001699340000532
Figure BDA0004001699340000541
16. the compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-15, wherein
The compound shown in the formula (I) is selected from the following compounds: NOTA-Bn-SLK (Mal), NOTA-Bn-SFK- (CH) 2 ) 2 -Mal，
DOTA-Bn-SFK-(CH 2 ) 2 -Mal，NODAGA-SFK-(CH 2 ) 2 -Mal，NOTA-Bn-DFK-(CH 2 ) 2 -Mal，
NOTA-Bn-VMK-(CH 2 ) 2 -Mal，DOTA-Bn-VMK-PEG 4 -Mal，DOTA-PEG 4 -VMK-PEG 4 -
Mal，DOTA-VMK-(CH 2 ) 2 -Mal，DOTA-VMK-PEG 4 -Mal，NOTA-Bn-MV(Dap)-PEG 4 -
Mal，DOTA-Bn-MV(Dap)-PEG 4 -Mal，DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal，DOTA-
MV(Dap)-PEG 4 -Mal，NODAGA-MV(Dap)-PEG 4 -Mal，DOTA-MV-(CH 2 ) 2 -Mal，DOTA-
PEG 4 -MV-PEG 4 -Mal，DOTA-Bn-MV-PEG 4 -Mal，DOTA-Bn-rSFK-PEG 4 -Mal，DOTA-Bn-
GWK-PEG 4 -Mal，DOTA-Bn-MNK-PEG 4 -Mal，DOTA-Bn-QLK-PEG 4 -Mal and DOTA-Bn-TIK-PEG 4 -Mal。
17. An immunoconjugate comprising I) a compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to any one of embodiments 1 to 16, and ii) a target molecule recognition unit.
18. The immunoconjugate according to embodiment 17, wherein the compound represented by formula (I) or a pharmaceutically acceptable salt thereof passes through R 1 To the target recognition unit or to a linking group of the target recognition unit.
19. The immunoconjugate according to any one of embodiments 17-18, 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.
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: radionuclides, fluorescers, chemiluminescent agents, bioluminescent agents, paramagnetic ions and enzymes.
21. The immunoconjugate according to any one of embodiments 20, wherein the radionuclide is suitable for medical imaging and/or therapy.
22. The immunoconjugate according to any one of embodiments 20-21, wherein the radionuclide comprises 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 alpha, gamma, beta-, or positron emitters.
23. The immunoconjugate according to any one of embodiments 17-22, having a structure represented by formula (II):
Figure BDA0004001699340000551
wherein X is a chelator, X is attached or not attached to the active moiety;
t is a target molecule recognition unit;
l is a linker, L being defaulting;
A 1 and A 2 Is an amino acidResidue, m is 0,1 or 2;
A 3 is an amino acid residue having an amino group or a carboxyl group in a side chain, R 1 Is with A 3 A group which is bound to an amino group or a carboxyl group of a side chain of (a) and has a functional group capable of binding to a target molecule recognition unit or a linking group thereof;
-(A 1 ) m -A 2 -A 3 -being capable of being cleaved by enkephalinase.
24. The immunoconjugate according to embodiment 23, wherein- (a) 1 ) m -A 2 -A 3 -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).
25. The immunoconjugate according to any one of embodiments 23-24, wherein the X is selected from the following structures:
Figure BDA0004001699340000552
Figure BDA0004001699340000553
and their derivatives; wherein R is 2 Independently a hydrogen atom or optionally substituted C 1 -C 8
A hydrocarbon group.
26. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of embodiments 23-25, wherein X is selected from the following structures:
Figure BDA0004001699340000561
Figure BDA0004001699340000562
and their derivatives.
27. The immunoconjugate according to any one of embodiments 23-26, wherein the defaults or L is selected from the following structures:
Figure BDA0004001699340000563
wherein n is any integer from 0 to 10.
28. The immunoconjugate according to any one of embodiments 23-27, wherein the defaults or L is selected from the following structures:
Figure BDA0004001699340000564
29. the immunoconjugate according to any one of embodiments 23-28, wherein X-L is selected from the following structures:
Figure BDA0004001699340000565
Figure BDA0004001699340000566
wherein R is 2 Each independently is a hydrogen atom or an optionally substituted C 1 -C 8 Hydrocarbyl, n is any integer between 1 and 1000.
30. The immunoconjugate according to any one of embodiments 23-29, wherein X-L is selected from the following structures:
Figure BDA0004001699340000571
31. The immunoconjugate according to any one of embodiments 23-30, selected from the structures:
Figure BDA0004001699340000572
/>
Figure BDA0004001699340000581
wherein T is a targeting molecule unit;
-(A 1 ) m -A 2 -A 3 -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)；
the R is 1 Selected from the following structures:
Figure BDA0004001699340000591
-(CH 2 ) n -Mal，-(CH 2 CH 2 O) n- Mal，-CO(CH 2 )n-Mal，
-NH(CH 2 ) n -Mal；
n is each independently any integer between 1 and 1000;
R 2 each independently is a hydrogen atom or an optionally substituted C 1 -C 8 A hydrocarbon group.
32. The immunoconjugate according to any one of embodiments 23-31, selected from the structures:
NOTA-Bn-SLK(Mal)-T，
NOTA-Bn-SFK-(CH 2 ) 2 -Mal-T，
DOTA-Bn-SFK-(CH 2 ) 2 -Mal-T，
NODAGA-SFK-(CH 2 ) 2 -Mal-T，
NOTA-Bn-DFK-(CH 2 ) 2 -Mal-T，
NOTA-Bn-VMK-(CH 2 ) 2 -Mal-T，
DOTA-Bn-VMK-PEG 4 -Mal-T，
DOTA-PEG 4 -VMK-PEG 4 -Mal-T，
DOTA-VMK-(CH 2 ) 2 -Mal-T，
DOTA-VMK-PEG 4 -Mal-T，
NOTA-Bn-MV(Dap)-PEG 4 -Mal-T，
DOTA-Bn-MV(Dap)-PEG 4 -Mal-T，
DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal-T，
DOTA-MV(Dap)-PEG 4 -Mal-T，
NODAGA-MV(Dap)-PEG 4 -Mal-T，
DOTA-MV-(CH 2 ) 2 -Mal-T，
DOTA-PEG 4 -MV-PEG 4 -Mal-T，
DOTA-Bn-MV-PEG 4 -Mal-T，
DOTA-Bn-rSFK-PEG 4 -Mal-T，
DOTA-Bn-GWK-PEG 4 -Mal-T，
DOTA-Bn-MNK-PEG 4 -Mal-T，
DOTA-Bn-QLK-PEG 4 Mal-T and
DOTA-Bn-TIK-PEG 4 -Mal-T；
wherein T is a target molecule recognition unit comprising an antigen binding protein.
33. The immunoconjugate according to any one of embodiments 23-32, selected from the structures:
A-NOTA-Bn-SLK(Mal)-T，
A-NOTA-Bn-SFK-(CH 2 ) 2 -Mal-T，
A-DOTA-Bn-SFK-(CH 2 ) 2 -Mal-T，
A-NODAGA-SFK-(CH 2 ) 2 -Mal-T，
A-NOTA-Bn-DFK-(CH 2 ) 2 -Mal-T，
A-NOTA-Bn-VMK-(CH 2 ) 2 -Mal-T，
A-DOTA-Bn-VMK-PEG 4 -Mal-T，
A-DOTA-PEG 4 -VMK-PEG 4 -Mal-T，
A-DOTA-VMK-(CH 2 ) 2 -Mal-T，
A-DOTA-VMK-PEG 4 -Mal-T，
A-NOTA-Bn-MV(Dap)-PEG 4 -Mal-T，
A-DOTA-Bn-MV(Dap)-PEG 4 -Mal-T，
A-DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal-T，
A-DOTA-MV(Dap)-PEG 4 -Mal-T，
A-NODAGA-MV(Dap)-PEG 4 -Mal-T，
A-DOTA-MV-(CH 2 ) 2 -Mal-T，
A-DOTA-PEG 4 -MV-PEG 4 -Mal-T，
A-DOTA-Bn-MV-PEG 4 -Mal-T，
A-DOTA-Bn-rSFK-PEG 4 -Mal-T，
A-DOTA-Bn-GWK-PEG 4 -Mal-T，
A-DOTA-Bn-MNK-PEG 4 -Mal-T，
A-DOTA-Bn-QLK-PEG 4 Mal-T and
A-DOTA-Bn-TIK-PEG 4 -Mal-T；
wherein T is a target molecule recognition unit and A is an active moiety.
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.
35. The immunoconjugate of embodiment 34, wherein the antigen binding protein comprises an antibody or antigen binding fragment thereof.
36. The immunoconjugate of embodiment 35, wherein the antibody comprises a monoclonal antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, and/or a fully human antibody.
37. The immunoconjugate according to any one of embodiments 34-36, said antigen binding fragment comprising Fab, fab ', fv fragment, F (ab') 2 scFv, VHH and/or dAb.
38. The immunoconjugate of any one of embodiments 23-37, wherein the target molecule recognition unit comprises a VHH.
39. The immunoconjugate of embodiment 38, wherein the VHH is camelid, chimeric, human, partially humanized, or fully humanized.
40. The 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.
41. The immunoconjugate according to any one of embodiments 23-40, wherein the target molecule recognition unit specifically binds an antigen selected from the group consisting of: AXL, BAFFR, BCMA, BDCA, 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, CD209L, CD22, CD226, CD248, CD25, CD27, CD274, CD276, CD28, CD30, CD300A, CD32B, CD33, CD37, CD38, CD4, differentiation cluster 40 (CD 40), CD44, CD45, CD46, CD47, CD48, CD5, CD52, CD55, CD56, CD59, CD62E, CD68, CD69, CD70, CD74, CD79A, CD79B, CD8, CD80, CD86, CD90.2, CD96, OX40 (CD 134), 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, HHHHA 2, type II HLA (HLA class II), HVEM, ICOSLG, IFNAR, type I interferon receptor subunit (IFNAR 1), 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 (Intercins), LAG3, LIFR, MAG/Siglec-4 (sialic acid binding immunoglobulin-like lectin-4), MMR, MSR1, NCR3LG1, NKG2D, NKp30, NKp46, PDCD1, PROKR1, PVR, PVRIG, PVRL, PVRL3, RELT, SIGIRR, siglec-1 (sialic acid binding immunoglobulin-like lectin-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, tumor necrosis factor alpha (tnfa), 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, egfrvlll, gpnmb, gpr64, her3,
LRP6, LYPD8, NKG2D, SLC34A2, SLC39A6, SLITRK6, GUCY2C,5T4 and/or
TACSTD2。
42. The immunoconjugate according to any one of embodiments 23-41, wherein the target molecule recognition unit comprises an anti-PD-
L1 VHH antibodies, anti-HER 2 VHH antibodies, HER 2-targeting scaffold proteins, or anti-CD 8 a VHH antibodies.
43. The immunoconjugate of embodiment 42, wherein the anti-CD 8 a VHH antibody comprises: as set forth in SEQ ID NO:
1, a CDR2 of the amino acid sequence shown as SEQ ID NO. 2 and a CDR3 of the amino acid sequence shown as SEQ ID NO. 3.
44. The immunoconjugate of any one of embodiments 42-43, wherein the anti-CD 8 a VHH antibody comprises the amino acid sequence shown in SEQ id No. 4.
45. The immunoconjugate of any one of embodiments 42-44, wherein the anti-HER 2 VHH antibody comprises:
CDR1 of the amino acid sequence shown as SEQ ID NO. 5 and CDR1 of the amino acid sequence shown as SEQ ID NO. 6
CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 7.
46. The immunoconjugate of any one of embodiments 42-45, wherein the anti-HER 2 VHH antibody comprises the amino acid sequence shown in SEQ id No. 8.
47. The immunoconjugate according to any one of embodiments 42 to 46, wherein the HER 2-targeting scaffold protein comprises the amino acid sequence shown in seq id No. 9.
48. The immunoconjugate according to any one of embodiments 23-47, selected from the structures:
Figure BDA0004001699340000631
/>
Figure BDA0004001699340000641
wherein, the liquid crystal display device comprises a liquid crystal display device,
-(A 1 ) m -A 2 -A 3 -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), -gin-Leu-Lys- (QLK) and-Thr-ILe-Lys- (TIK); the R is 1 Conjugated antigen binding proteins, scaffold proteinsOr a ligand.
49. The immunoconjugate according to embodiment 48, wherein the R 1 Selected from the following structures:
Figure BDA0004001699340000642
-(CH 2 ) n -Mal，-(CH 2 CH 2 O) n- Mal，-CO(CH 2 )n-Mal，-NH(CH 2 ) n Mal, where n is any integer between 1 and 10.
50. The immunoconjugate according to any one of embodiments 23-49, selected from the structures:
NOTA-Bn-SLK(Mal)-VHH，
NOTA-Bn-SFK-(CH 2 ) 2 -Mal-VHH，
DOTA-Bn-SFK-(CH 2 ) 2 -Mal-VHH，
NODAGA-SFK-(CH 2 ) 2 -Mal-VHH，
NOTA-Bn-DFK-(CH 2 ) 2 -Mal-VHH，
NOTA-Bn-VMK-(CH 2 ) 2 -Mal-VHH，
DOTA-Bn-VMK-PEG 4 -Mal-VHH，
DOTA-PEG 4 -VMK-PEG 4 -Mal-VHH，
DOTA-VMK-(CH 2 ) 2 -Mal-VHH，
DOTA-VMK-PEG 4 -Mal-VHH，
NOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，
DOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，
DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal-VHH，
DOTA-MV(Dap)-PEG 4 -Mal-VHH，
NODAGA-MV(Dap)-PEG 4 -Mal-VHH，
DOTA-MV-(CH 2 ) 2 -Mal-VHH，
DOTA-PEG 4 -MV-PEG 4 -Mal-VHH，
DOTA-Bn-MV-PEG 4 -Mal-VHH，
DOTA-Bn-rSFK-PEG 4 -Mal-VHH，
DOTA-Bn-GWK-PEG 4 -Mal-VHH，
DOTA-Bn-MNK-PEG 4 -Mal-VHH，
DOTA-Bn-QLK-PEG 4 Mal-VHH
DOTA-Bn-TIK-PEG 4 -Mal-VHH。
51. The immunoconjugate according to any one of embodiments 23-50, selected from the structures:
A-NOTA-Bn-SLK(Mal)-VHH，
A-NOTA-Bn-SFK-(CH 2 ) 2 -Mal-VHH，
A-DOTA-Bn-SFK-(CH 2 ) 2 -Mal-VHH，
A-NODAGA-SFK-(CH 2 ) 2 -Mal-VHH，
A-NOTA-Bn-DFK-(CH 2 ) 2 -Mal-VHH，
A-NOTA-Bn-VMK-(CH 2 ) 2 -Mal-VHH，
A-DOTA-Bn-VMK-PEG 4 -Mal-VHH，
A-DOTA-PEG 4 -VMK-PEG 4 -Mal-VHH，
A-DOTA-VMK-(CH 2 ) 2 -Mal-VHH，
A-DOTA-VMK-PEG 4 -Mal-VHH，
A-NOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，
A-DOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，
A-DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal-VHH，
A-DOTA-MV(Dap)-PEG 4 -Mal-VHH，
A-NODAGA-MV(Dap)-PEG 4 -Mal-VHH，
A-DOTA-MV-(CH 2 ) 2 -Mal-VHH，
A-DOTA-PEG 4 -MV-PEG 4 -Mal-VHH，
A-DOTA-Bn-MV-PEG 4 -Mal-VHH，
A-DOTA-Bn-rSFK-PEG 4 -Mal-VHH，
A-DOTA-Bn-GWK-PEG 4 -Mal-VHH，
A-DOTA-Bn-MNK-PEG 4 -Mal-VHH，
A-DOTA-Bn-QLK-PEG 4 Mal-VHH
A-DOTA-Bn-TIK-PEG 4 -Mal-VHH；
Wherein A is an active moiety.
52. The immunoconjugate according to embodiment 51, wherein a is a detectable label selected from the group consisting of
One or more reagents of the group: radionuclides, fluorescers, chemiluminescent agents, bioluminescent agents, paramagnetic ions and enzymes.
53. The immunoconjugate according to any one of embodiments 51-52, wherein a is a radionuclide comprising 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 alpha, gamma, beta-, or positron emitters.
54. A radionuclide complex comprising I) a compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to any one of embodiments 1 to 16, and ii) a radionuclide.
55. The radionuclide complex according to embodiment 54, wherein the radionuclide is attached to X in the compound of formula (I).
56. A composition comprising a compound of formula (I) as set forth in any one of embodiments 1-16, or a pharmaceutically acceptable salt thereof, an immunoconjugate of any one of embodiments 17-53, and optionally a pharmaceutically acceptable carrier.
57. The composition of embodiment 56, which is a detection agent or therapeutic agent.
58. The composition of embodiment 57, wherein the detection agent is an agent for detecting an antigen.
59. The composition of embodiment 56, wherein the detection agent is a contrast agent.
60. The composition of embodiment 59, wherein the contrast agent is a contrast agent that detects an antigen.
61. The composition of embodiment 60, wherein the antigen is a tumor antigen.
62. The composition of embodiment 61, wherein the therapeutic agent is for treating a tumor.
63. Use of a compound of formula (I) according to any one of embodiments 1 to 16 or a pharmaceutically acceptable salt thereof, an immunoconjugate according to any one of embodiments 17 to 53, a radionuclide complex according to any one of embodiments 54 to 55, and/or a composition according to any one of embodiments 56 to 62, in the preparation of a reagent, assay plate, or kit; wherein the reagent, assay plate or kit is used to detect an antigen in a sample.
64. Use of a compound of formula (I) according to any one of embodiments 1 to 16 or a pharmaceutically acceptable salt thereof, an immunoconjugate according to any one of embodiments 17 to 53 and/or a composition according to any one of embodiments 56 to 62 for the preparation of a medicament;
Wherein the medicament is for the treatment of a tumor.
65. Use of a compound of formula (I) according to any one of embodiments 1 to 16 or a pharmaceutically acceptable salt thereof, an immunoconjugate according to any one of embodiments 17 to 53, a radionuclide complex according to any one of embodiments 54 to 55, and/or a composition according to any one of embodiments 56 to 62, for the preparation of a radiolabeled drug.
66. A radiolabeled drug comprising a compound of formula (I) as defined in any one of embodiments 1 to 16 or a pharmaceutically acceptable salt thereof, an immunoconjugate of any one of embodiments 17 to 53, a radionuclide complex of any one of embodiments 54 to 55, and/or a composition of any one of embodiments 56 to 62.
67. A radiographic diagnostic drug comprising a compound of formula (I) as described in any one of embodiments 1-16 or a pharmaceutically acceptable salt thereof, an immunoconjugate of any one of embodiments 17-53, a radionuclide complex of any one of embodiments 54-55, and/or a composition of any one of embodiments 56-62.
68. A method of detecting the presence and/or amount of an antigen in a biological sample, comprising: contacting the biological sample with a compound of formula (I) as described in any one of embodiments 1-16 or a pharmaceutically acceptable salt thereof, an immunoconjugate of any one of embodiments 17-53, and/or a composition of any one of embodiments 56-62.
69. The method of embodiment 68, wherein the contacting is performed in vitro or ex vivo.
70. The method of any one of embodiments 68-69, wherein the biological sample is tissue.
71. The method of embodiment 70, wherein the tissue is selected from the group consisting of blood tissue, lymphoid tissue, and tumor tissue.
72. The method of any one of embodiments 68-71, comprising detecting the presence and/or amount of tumor antigen positive cells in a biological sample.
73. The method of embodiment 72, wherein the presence and/or amount of tumor antigen positive cells in the biological sample is determined by imaging.
74. The method of embodiment 72 or 73, wherein the presence and/or amount of tumor antigen positive cells in the biological sample is determined by flow cytometry.
75. A method of detecting and/or diagnosing and oncologically 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 of any one of embodiments 17-53 and/or a composition of any one of embodiments 56-62.
76. The method of embodiment 75, wherein the method further comprises imaging the subject.
77. The method of embodiment 76 wherein the imaging comprises ECT imaging.
78. The method of embodiment 77, wherein the ECT imaging comprises SPECT imaging or PET imaging.
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 of any one of embodiments 17-53, and/or a composition of any one of embodiments 56-62.
80. A method for monitoring the efficacy of an anti-tumor therapy in a subject, the method comprising:
(i) Administering to a subject suffering from a tumor and 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 of any one of embodiments 17-53, and/or a composition of any one of embodiments 56-62; and
(ii) Determining the amount of tumor antigen positive cells in the tumor of the subject.
81. The method of embodiment 80, determining the presence and/or amount of SNA004 positive cells in the tumor of the subject by imaging.
82. The method of any one of embodiments 80-81, wherein the tumor comprises a solid tumor.
83. The method of any one of embodiments 80-82, wherein the tumor is selected from at least one of breast cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, ovarian cancer, pancreatic cancer, endometrial cancer, cervical squamous cell carcinoma, salivary gland carcinoma, bladder cancer, lung cancer, colorectal cancer, head and neck cancer, prostate cancer, osteosarcoma, childhood medulloblastoma, and the like.
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 of any one of embodiments 17-53, and/or a composition of any one of embodiments 56-62.
Without intending to be limited by any theory, the following examples are meant to illustrate the compounds, methods of preparation, uses, etc. of the present application and are not intended to limit the scope of the invention of the present application.
Examples
In the following examples and comparative examples, the following abbreviations are used for substituents, compounds, and organic solvents.
Boc: t-Butoxycarbonyl group
p-SCN-Bn-NOTA:2-S- (4-isothiocyanatobenzyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetic acid
p-SCN-Bn-DOTA:2- [ (4-Isothiocyanophenyl) methyl ] -1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid
nodga-NHS: NODAGA-succinimidyl ester
DOTA-PEG 4 TFP counter: tetrapolyethylene glycol- (2, 3,5, 6-tetrafluorophenoxy) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triyl) triacetic acid
DOTA-tris( t Bu ester): tri-tert-butyl 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid
NHS-(CH 2 ) 2 Mal: 3-Maleimidopropionic acid hydroxysuccinimide ester
NH 2 -PEG 4 Mal: maleimide-tetra polyethylene glycol-amine trifluoroacetate salt
NHS-PEG 4 Mal: maleimide-tetra polyethylene glycol-acrylic acid succinimidyl ester
TFA: trifluoroacetic acid
MeCN: acetonitrile
DCM: dichloromethane (dichloromethane)
Et 3 N: triethylamine
DIPEA N, N-diisopropylethylamine
DMF: dimethylformamide
HATU:2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate
DIPEA: n, N-diisopropylethylamine
In the following examples and comparative examples, various physical properties and the like were isolated and/or measured by the following methods.
For isolation by semi-preparative HPLC, X-Bridge BEH C18 OBD Prep Column (Waters, 30 mm. Times.150 mm) attached was used. Using 0.1% (v/v) TFA/H 2 O (A phase) and 0.1% (v/v) TFA/MeCN (B phase) as mobile phases, and changing from 90% (v/v) of A phase to 10% (v/v) of B phase to 20% of A phase by linear gradient method in 0-30 min
(v/v) and 80% (v/v) of phase B, and the elution was performed at a flow rate of 8.0mL/min 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 in 30 to 31 min.
For LC-MS analysis, a triple quadrupole liquid chromatography-mass spectrometer (SHIMADZU/Shimadzu) was used for LC-MS-2020.
For MS analysis, Q exact mass spectrometer (Thermo) was used
Example 1 Synthesis of Compounds
1.1 Compounds 1-1NODAGA-Mal
Figure BDA0004001699340000691
Compounds 1-1 LC-MS calcd.for: C 21 H 31 N 5 O 9 497.51；found 498.64[M+H] +
1.2 Compounds 1-2NOTA-Bn-MVK (Mal) (L1 a)
NOTA-Bn-MVK (Mal) was synthesized according to the following protocol:
Figure BDA0004001699340000701
1(a):p-SCN-Bn-NOTA
( Synthesis example 1 (a): synthesis of NOTA-Bn-Met-Val-Lys (Mal) -OH )
Compound MVK (Mal) (2.0 mg,4.38. Mu. Mol) and p-SCN-Bn-NOTA (3.7 mg, 6.57. Mu. Mol) were dissolved in 0.5mL DMF and Et was added 3 N (8. Mu.L). Draw and change N 2 Stirring at room temperature for 2.0h. After completion of the LC-MS detection reaction, the solvent was drained and purified by semi-preparative HPLC to give compound 1-2 (hereinafter also referred to as "NOTA-Bn-MVK (Mal)") as a white solid.
Compounds 1-2 LC-MS calcd.for: C 40 H 58 N 8 O 12 S 2 907.07；found:907.37[M] + 。
1.3 Compounds 1-3NOTA-Bn-GFK (Mal) (L2 a)
NOTA-Bn-GFK (Mal) was synthesized according to the following protocol:
Figure BDA0004001699340000702
2(a):p-SCN-Bn-NOTA
( Synthesis example 2 (a): compound 1-2NOTA-Bn-Gly-Phe-Lys (Mal) -OH )
The compound GFK (Mal) (2.0 mg, 4.65. Mu. Mol) and p-SCN-Bn-NOTA (3.9 mg, 6.97. Mu. Mol) were dissolved in 0.5mL of DMF and Et was added 3 N (8. Mu.L). Draw and change N 2 Stirring at room temperature for 2.0h. After completion of the LC-MS detection reaction, the solvent was drained, purified by semi-preparative HPLC, and lyophilized to give compounds 1-3 (hereinafter also referred to as "NOTA-Bn-GFK (Mal)") as white solids.
Compounds 1-3 LC-MS calcd.for: C 41 H 52 N 8 O 12 S 880.97；found:881.40[M+H] + .
1.4 Compounds 1-4NOTA-Bn-PEG 4 -Mal(L3a)
NOTA-Bn-PEG was synthesized according to the following protocol 4 -Mal
Figure BDA0004001699340000711
3(a):NH 2 -PEG 4 -Mal
Synthesis example 3 (a) Compound 1-4 (NOTA-Bn-PEG 4 -Mal) synthesis
P-SCN-Bn-NOTA (3.3 mg, 5.81. Mu. Mol) and NH 2 -PEG 4 Mal (2.0 mg, 4.84. Mu. Mol) in 0.5mL DMF was taken up in Et 3 N (4. Mu.L). Stirring at room temperature for 2.0h. LC-MS detection reaction, after completion of the reaction, semi-preparative HPLC purification, and lyophilization again to give compounds 1-4 (hereinafter also referred to as "NOTA-Bn-PEG 4 Mal ") white solid. Compounds 1-4 LC-MS calcd.for: C 34 H 50 N 6 O 12 S 766.86；found:767.10[M+H] + .
The following compounds are examples:
1.5 Compounds 1-5 NOTA-Bn-SLK (Mal) (L10 a)
NOTA-Bn-SLK (Mal) shown below was synthesized in the same manner as in reference example 1.3 except that GFK (Mal) in example 1.3 was replaced with SLK (Mal).
Figure BDA0004001699340000712
Compounds 1-5 LC-MS calcd.for: C 39 H 56 N 8 O 13 S 876.98；Found:952.40[M+75] + .
1.5.1 Compounds 1-5a NOTA-Bn-SLK- (CH) 2 ) 2 -Mal(L10d)
Except for SLK- (CH 2) 2 NOTA-Bn-SLK- (CH 2) shown below was synthesized in the same manner as in reference example 1.3 except that Mal was used instead of GFK (Mal) in example 1.3 2 -Mal。
Figure BDA0004001699340000721
Compounds 1-5a: C 42 H 61 N 9 O 14 S LC-MS calculated 947.41; found 948.65[ M+H ]] + .
1.5.1 Compounds 1-5d DOTA-Bn-SLK (Mal) (L10 b)
DOTA-Bn-SLK (Mal) shown below was synthesized in the same manner as in reference example 1.5, except that DOTA was used instead of NOTA in example 1.5.
Figure BDA0004001699340000722
Compounds 1-5d: C 43 H 63 N 9 O 15 S LC-MS calculated 977.08; LC-MS was not characterized, and characterization was correct after coupling.
1.6 Compounds 1-6 NOTA-Bn-SFK- (CH) 2 ) 2 -Mal(L8b)
NOTA-Bn-SFK- (CH) was synthesized according to the following scheme 2 ) 2 -Mal
Figure BDA0004001699340000731
4(a):NHS-(CH 2 ) 2 -Mal；4(b):TFA,DCM；4(c):p-SCN-Bn-NOTA
Synthesis example 4 (a) intermediate 1-1 (Boc-Ser-Phe-Lys (CH) 2 ) 2 -Mal) synthesis
Boc-SFK (2.0 mg, 4.16. Mu. Mol) and NHS- (CH) 2 ) 2 Mal (1.3 mg, 5.00. Mu. Mol) was dissolved in 0.5mL DMF and Et was added 3 N (8. Mu.L) was stirred at room temperature for 2.0h. LC-MS detection reaction, after the reaction, semi-preparative HPLC purification, freeze-drying again. To obtain intermediate 1-1 (hereinafter also referred to as "Boc-SFK- (CH) 2 ) 2 Mal ") white solid.
Intermediate 1-1 LC-MS calcd.for: C 30 H 41 N 5 O 10 631.68；found:632.80[M+H] + .
Synthesis example 4 (b) intermediate 1-2 (NH) 2 -Ser-Phe-Lys(CH 2 ) 2 -Mal) synthesis
Intermediate 1-1 (2.6 mg, 4.16. Mu. Mol) was dissolved in 1.0mL of DCM, 0.5mL of TFA was added and stirred at room temperature for 2.0h. After the completion of the reaction, it was concentrated and pumped to dryness to give crude intermediate 1-2 (hereinafter also referred to as "NH") 2 -SFK-(CH 2 ) 2 Mal ") white solid.
Intermediate 1-2 LC-MS calcd.for: C 25 H 33 N 5 O 8 531.57；found:532.35[M+H] + .
Synthesis example 4 (c) Compounds 1-6 (NOTA-Bn-Ser-Phe-Lys (CH) 2 ) 2 -Mal) synthesis
The crude intermediate 1-2 was dissolved in 0.5mL DMF and p-SCN-Bn-NOTA (3.5 mg, 6.24. Mu. Mol) was added, followed by Et 3 N (8. Mu.L), stirred at room temperature for 2 hours, after completion of the reaction, purified by semi-preparative HPLC, and lyophilized again to give the compound (hereinafter also referred to as "NOTA-Bn-SFK- (CH) 2 ) 2 Mal ") white solid.
Compounds 1-6 LC-MS calcd.for: C 45 H 59 N 9 O 14 S 982.08；found:983.15[M+H] + .
1.6.1 Compounds 1-6-1:DOTA-Bn-SFK- (CH) 2 ) 2 -Mal(L8ac)
Figure BDA0004001699340000741
5(a):p-SCN-Bn-DOTA
Synthesis example 5 (a) Compound 1-6-1 (DOTA-Bn-Ser-Phe-Lys (CH) 2 ) 2 -Mal) synthesis
NH is added to 2 -SFK-(CH 2 ) 2 Mal (2.0 mg, 3.76. Mu. Mol) was dissolved in 0.5mL DMF and p-SCN-Bn-DOTA (3.1 mg, 4.51. Mu. Mol) was added, followed by Et 3 N (4. Mu.L), stirred at room temperature for 3 hours, after completion of the reaction, purified by semi-preparative HPLC, and lyophilized again to give the compound (hereinafter also referred to as "DOTA-Bn-SFK- (CH) 2 ) 2 Mal ") white solid. Compounds 1-6-1 LC-MS calcd.for: C 49 H 66 N 10 O 16 S 1082.44；found:1083.45[M+H] + .
1.6.2 Compounds 1-6-2 NODAGA-SFK- (CH) 2 ) 2 -Mal(L8ad)
Figure BDA0004001699340000742
6(a)NODAGA-NHS
Synthesis example 6 (a) Compound 1-6-2 (NODAGA-Ser-Phe-Lys (CH) 2 ) 2 -Mal) synthesis
NH is added to 2 -SFK-(CH 2 ) 2 Mal (2.0 mg, 3.76. Mu. Mol) was dissolved in 0.5mL DMF and NODAGA-NHS (3.3 mg, 4.50. Mu. Mol) was added, followed by Et 3 N (4. Mu.L), stirred at room temperature for 3 hours, after completion of the reaction, purified by semi-preparative HPLC, and lyophilized again to give the compound (hereinafter also referred to as "NODAGA-SFK- (CH) 2 ) 2 Mal ") white solid.
Compounds 1-6-2 LC-MS calcd.for: C 40 H 56 N 8 O 15 888.39；found:889.35[M+H] + .
1.7 Compounds 1-7NOTA-Bn-DFK- (CH) 2 ) 2 -Mal(L9b)
NOTA-Bn-DFK- (CH) shown below 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 2 ) 2 -Mal。
Figure BDA0004001699340000751
Compounds 1-7 LC-MS calcd.for: C 46 H 59 N 9 O 15 S 1010.09；Found:1010.90[M] + .
1.8 Compounds 1-8: NOTA-Bn-VMK- (CH) 2 ) 2 -Mal(L13)
NOTA-Bn-DFK- (CH) shown below 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 2 ) 2 -Mal。
Figure BDA0004001699340000752
Compounds 1-8 LC-MS calcd.for: C 43 H 63 N 9 O 13 S 2 978.15；Found:978.15[M] +
1.8.1 Compounds 1-8-1: DOTA-Bn-VMK-PEG 4 -Mal(L13a)
Figure BDA0004001699340000761
7(a):NHS-PEG 4 -Mal；7(b):TFA；7(c):p-SCN-Bn-DOTA
Synthesis example 7 (a) intermediate 2-1 (Boc-Val-Met-Lys (PEG) 4 -Mal) synthesis
Boc-VMK (10.0 mg, 20.98. Mu. Mol) and NHS-PEG 4 Mal (11.2 mg, 25.18. Mu. Mol) was dissolved in 0.5mL DMF and Et was added 3 N (10. Mu.L), stirring at room temperature for 3.0h. LC-MS detection reaction, after the reaction, semi-preparative HPLC purification, freeze-drying again. To give intermediate 2-1 (hereinafter also referred to as "Boc-VMK-PEG 4 Mal ") white solid. Intermediate 2-1 LC-MS calcd.for: C 36 H 61 N 5 O 13 S 803.97；found:804.40[M+H] + .
Synthesis example 7 (b) intermediate 2-2 (NH) 2 -Val-Met-Lys(PEG) 4 -Mal) synthesis
Intermediate 2-1 (2.4 mg, 3.00. Mu. Mol) was dissolved in 0.5mL of TFA and stirred at room temperature for 30min. After the reaction, it is concentrated and pumped to dryness to give crude intermediate 2-2 (hereinafter also referred to as "NH") 2 -VMK-PEG 4 Mal ") white solid.
Intermediate 2-2 LC-MS calcd.for: C 31 H 53 N 5 O 11 S 703.85；found:704.20[M+H] + .
Synthesis example 7 (c) Compounds 1-6 (DOTA-Bn-Val-Met-Lys (PEG) 4 -Mal) synthesis
The crude intermediate 2-2 was dissolved in 0.5mL DMF and p-SCN-Bn-DOTA (3.1 mg, 4.51. Mu. Mol) was added, followed by Et 3 N (8. Mu.L), stirred at room temperature for 3 hours, after completion of the reaction, purified by semi-preparative HPLC, and lyophilized again to give the compound (hereinafter also referred to as "DOTA-Bn-VMK-PEG) 4 Mal ") white solid.
Compounds 1-8-1 LC-MS calcd.for: C 55 H 86 N 10 O 19 S 2 1255.46；found:628.75[M/2+H] + .
1.8.2 Compounds 1-8-2: DOTA-PEG 4 -VMK-PEG 4 -Mal(L13b)
Figure BDA0004001699340000771
8(a):NHS-PEG 4 -Mal；8(b):TFA；8(c):DOTA-PEG 4 -TFP ester
Synthesis example 8 (a) intermediate 3-1 (Boc-Val-Met-Lys (PEG) 4 -Mal) synthesis
Boc-VMK (10.0 mg, 20.98. Mu. Mol) and NHS-PEG 4 Mal (11.2 mg, 25.18. Mu. Mol) was dissolved in 0.5mL DMF and Et was added 3 N (10. Mu.L), stirring at room temperature for 2.0h. LC-MS detection reaction, after the reaction, semi-preparative HPLC purification, freeze-drying again. To obtain intermediate 3-1 (hereinafter also referred to as "Boc-VMK-PEG 4 Mal ") white solid. Intermediate 3-1 LC-MS calcd.for: C 36 H 61 N 5 O 13 S 803.97；found:805.35[M+H] + .
Synthesis example 8 (b) intermediate 3-2 (NH) 2 -Val-Met-Lys(PEG) 4 -Mal) synthesis
Intermediate 3-1 (2.4 mg, 3.00. Mu. Mol) was dissolved in 0.5mL of TFA and stirred at room temperature for 30min. After the completion of the reaction, it was concentrated and dried to give crude intermediate 3-2 (hereinafter also referred to as "NH") 2 -VMK-PEG 4 Mal ") white solid.
Intermediate 3-2 LC-MS calcd.for: C 31 H 53 N 5 O 11 S 703.85；found:704.20[M+H] + .
Synthesis example 8 (c) Compounds 1-6 (DOTA-PEG 4 -Val-Met-Lys(PEG) 4 -Mal) synthesis
Dissolving the crude intermediate 3-2 in 0.5mL DMF, adding DOTA-PEG 4 TFP ester (4.6 mg, 4.51. Mu. Mol) and Et were added last 3 N (8. Mu.L), stirred at room temperature for 3 hours, after completion of the reaction, purified by semi-preparative HPLC, and lyophilized again to give the compound (hereinafter also referred to as "DOTA-PEG 4 -VMK-PEG 4 Mal ") white solid.
Compounds 1-8-2 LC-MS calcd.for:C 58 H 100 N 10 O 23 S 1336.67；found:665.95[M/2-2H] + .
1.8.3 Compounds 1-8-3: DOTA-VMK- (CH) 2 ) 2 -Mal(L13d)
Figure BDA0004001699340000781
8(d):NHS-(CH 2 ) 2 -Mal；8(e):TFA；8(f):DOTA-tris( t Bu ester)；8(g):TFA
Synthesis example 8 (d) intermediate 3-1-1 (Boc-Val-Met-Lys (CH) 2 ) 2 -Mal) synthesis
Boc-VMK (30.0 mg, 62.94. Mu. Mol) and NHS- (CH) 2 ) 2 Mal (20.11 mg, 75.53. Mu. Mol) was dissolved in 0.5mL DMF and Et was added 3 N (38. Mu.L) was stirred at room temperature for 3.0h. LC-MS detection reaction, after the reaction, semi-preparative HPLC purification, freeze-drying again. To obtain intermediate 3-1-1 (hereinafter also referred to as "Boc-VMK- (CH) 2 ) 2 Mal ") white solid.
Intermediate 3-1 LC-MS calcd.for: C 28 H 45 N 5 O 9 S 627.75；found:628.45[M+H] + .
Synthesis example 8 (e) intermediate 3-2-1 (NH) 2 -Val-Met-Lys(CH 2 ) 2 -Mal) synthesis
Intermediate 3-1-1 (20.0 mg, 24.88. Mu. Mol) was dissolved in 0.5mL of TFA and stirred at room temperature for 30min. After the reaction, it is concentrated and pumped to dryness to give crude intermediate 3-2-1 (hereinafter also referred to as "NH") 2 -VMK-(CH 2 ) 2 Mal ") white solid. Directly used in the next step.
Synthesis example 8 (f) intermediate 3-3-1 (DOTA-tris ] t Bu ester)-Val-Met-Lys(CH 2 ) 2 -Mal) synthesis
DOTA-tris @ t Bu ester) (18.52 mg, 32.34. Mu. Mol) was dissolved in 1.0mL of DMF, HATU (26.65 mg, 70.09. Mu. Mol) was added and stirred at room temperature for 30min to give the crude intermediate 3-2-1NH 2 -VMK-(CH 2 ) 2 Mal additionTo the reaction system, DIPEA (27.7 μl) was finally added, stirred at room temperature for 3 hours, after completion of the reaction, semi-preparative HPLC was purified, and lyophilized again to give a compound (hereinafter also referred to as "DOTA-tris t Bu ester)-VMK-(CH 2 ) 2 Mal ") white solid.
Intermediate 3-3-1 LC-MS calcd.for: C 51 H 87 N 9 O 14 S 1082.37；found:1065.05[M-H 3 O] + .
Synthesis example 8 (g) Compounds 1-8-3 (DOTA-Val-Met-Lys (CH) 2 ) 2 -Mal) synthesis
Intermediate 3-3-1 (8.0 mg, 7.39. Mu. Mol) was dissolved in 0.5mL of TFA and stirred at room temperature for 30min. After the reaction, concentration, semi-preparative HPLC purification and lyophilization again to give the compound (hereinafter also referred to as "DOTA-VMK- (CH) 2 ) 2 Mal ") white solid.
Compounds 1-8-3 LC-MS calcd.for: C 39 H 63 N 9 O 14 S 914.04；found:[M-H 2 O] + ＝896.42
1.8.4 Compounds 1-8-4: DOTA-VMK-PEG 4 -Mal(L13c)
Figure BDA0004001699340000791
8(h):NHS-PEG 4 -Mal；8(i):TFA；8(j):DOTA-tris( t Bu ester)；8(k):TFA
Synthesis example 8 (h) intermediate 3-1-2 (Boc-Val-Met-Lys (PEG) 4 -Mal) synthesis
Boc-VMK (30.0 mg, 62.94. Mu. Mol) and NHS-PEG 4 Mal (33.6 mg, 75.53. Mu. Mol) was dissolved in 0.5mL DMF and Et was added 3 N (38. Mu.L) was stirred at room temperature for 3.0h. LC-MS detection reaction, after the reaction, semi-preparative HPLC purification, freeze-drying again. To obtain intermediate 3-1-2 (hereinafter also referred to as "Boc-VMK-PEG 4 Mal ") white solid.
Intermediate 3-1-2 LC-MS calcd.for: C 36 H 61 N 5 O 13 S 803.97；found:805.00[M+H] + .
Synthesis example 8 (i) intermediate 3-2-2 (NH) 2 -Val-Met-Lys(PEG) 4 -Mal) synthesis
Intermediate 3-1-2 (20.0 mg, 24.88. Mu. Mol) was dissolved in 0.5mL of TFA and stirred at room temperature for 30min. After the reaction, it is concentrated and pumped to dryness to give crude intermediate 3-2-2 (hereinafter also referred to as "NH") 2 -VMK-PEG 4 Mal ") white solid. Directly used in the next step.
Synthesis example 8 (j) intermediate 3-3-2 (DOTA-tris ] t Bu ester)-Val-Met-Lys(PEG) 4 -Mal) synthesis
DOTA-tris @ t Bu ester) (18.52 mg, 32.34. Mu. Mol) was dissolved in 1.0mL of DMF, HATU (20.81 mg, 54.73. Mu. Mol) was added and stirred at room temperature for 30min to give the crude intermediate 3-2-2NH 2 -VMK-PEG 4 Mal was added to the reaction system, DIPEA (22. Mu.L) was added last, stirred at room temperature for 3 hours, after completion of the reaction, semi-preparative HPLC was purified, and lyophilized again to give a compound (hereinafter also referred to as "DOTA-tris t Bu ester)-VMK-PEG 4 Mal ") white solid. Intermediate 3-3-2 LC-MS calcd.for: C 59 H 103 N 9 O 18 S 1258.58；found:1240.85[M-H 2 O] + .
Synthesis example 8 (k) Compounds 1-8-3 (DOTA-Val-Met-Lys (PEG) 4 -Mal) synthesis
Intermediate 3-3-2 (8.0 mg, 6.36. Mu. Mol) was dissolved in 0.5mL of TFA and stirred at room temperature for 30min. After the reaction, concentration, semi-preparative HPLC purification and lyophilization again to give the compound (hereinafter also referred to as "DOTA-VMK-PEG 4 Mal ") white solid.
Compounds 1-8-4 LC-MS calcd.for: C 47 H 79 N 9 O 18 S 1090.25；found:1072.53[M-H 2 O] + .
1.9 Compounds 1-9: NOTA-Bn-MV (Dap) -PEG 4 -Mal(L14f)
Figure BDA0004001699340000801
9(a):NHS-PEG 4 -Mal；9(b):TFA；9(c):p-SCN-Bn-NOTA
Synthesis example 9 (a) intermediate 4-1 (Boc-Met-Val (Dap) -PEG 4 -Mal) synthesis
Boc-MV (Dap) (2.0 mg, 4.60. Mu. Mol) and NHS-PEG were combined 4 Mal (2.4 mg, 5.52. Mu. Mol) was dissolved in 0.5mL DMF and Et was added 3 N (4. Mu.L) was stirred at room temperature for 2.0h. LC-MS detection reaction, after the reaction, semi-preparative HPLC purification, freeze-drying again. To obtain intermediate 4-1 (hereinafter also referred to as "Boc-MV (Dap) -PEG) 4 Mal ") white solid.
Intermediate 4-1: LC-MS calcd.for: C 33 H 55 N 5 O 13 S 761.89；found:762.45[M+H] + .
Synthesis example 9 (b) intermediate 4-2 (H-Met-Val (Dap) -PEG 4 -Mal) synthesis
Intermediate 4-1 (3.5 mg, 4.60. Mu. Mol) was dissolved in 0.5mL TFA and stirred at room temperature for 30min. After the completion of the reaction, it was concentrated and dried to give crude intermediate 4-2 (hereinafter also referred to as "NH") 2 -MV(Dap)-PEG 4 Mal ") white solid.
Intermediate 4-2: LC-MS calcd.for: C 28 H 47 N 5 O 11 S 661.77；found:662.05[M+H] + .
Synthesis example 9 (c) Compounds 1-9 (NOTA-Bn-Met-Val (Dap) -PEG 4 -Mal) synthesis
The crude intermediate 4-2 was dissolved in 0.5mL DMF and p-SCN-Bn-NOTA (3.2 mg, 5.72. Mu. Mol) was added, followed by Et 3 N (8. Mu.L), stirred at room temperature for 2 hours, after completion of the reaction, purified by semi-preparative HPLC, and lyophilized again to give the compound (hereinafter also referred to as "NOTA-Bn-MV (Dap) -PEG) 4 Mal ") white solid.
Compounds 1-9: LC-MS calcd.for: C 48 H 73 N 9 O 17 S 2 1112.28；found:1112.50[M] + .
1.9.1 Compound 1-9-1: DOTA-Bn-MV (Dap) -PEG 4 -Mal(L14b)
Figure BDA0004001699340000811
10(a):NHS-PEG 4 -Mal；10(b):TFA；10(c):p-SCN-Bn-DOTA
Synthesis example 10 (a) intermediate 5-1 (Boc-Met-Val (Dap) -PEG 4 -Mal) synthesis
Boc-MV (Dap) (10.0 mg, 23.01. Mu. Mol) and NHS-PEG 4 Mal (12.2 mg, 28.06. Mu. Mol) was dissolved in 0.5mL DMF and Et was added 3 N (4. Mu.L) was stirred at room temperature for 2.0h. LC-MS detection reaction, after the reaction, semi-preparative HPLC purification, freeze-drying again. To obtain intermediate 5-1 (hereinafter also referred to as "Boc-MV (Dap) -PEG) 4 Mal ") white solid.
Intermediate 5-1 LC-MS calcd.for: C 33 H 55 N 5 O 13 S 761.89；found:762.45[M+H] + .
Synthesis example 10 (b) intermediate 5-2 (NH) 2 -Met-Val(Dap)-PEG 4 -Mal) synthesis
Intermediate 5-1 (5.0 mg, 6.56. Mu. Mol) was dissolved in 0.5mL of TFA and stirred at room temperature for 30min. After the completion of the reaction, it was concentrated and pumped to dryness to give crude intermediate 5-2 (hereinafter also referred to as "NH") 2 -MV(Dap)-PEG 4 Mal ") white solid.
Intermediate 5-2 LC-MS calcd.for: C 28 H 47 N 5 O 11 S 661.77；found:662.05[M+H] + .
Synthesis example 10 (c) Compound 1-9-1 (DOTA-Bn-Met-Val (Dap) -PEG 4 -Mal) synthesis
The crude intermediate 5-2 was dissolved in 0.5mL DMF and p-SCN-Bn-DOTA (5.4 mg, 7.85. Mu. Mol) was added, followed by Et 3 N (8. Mu.L), stirred at room temperature for 2h, after completion of the reaction, purified by semi-preparative HPLC, and lyophilized again to give the compound (hereinafter also referred to as "DOTA-Bn-MV (Dap) -PEG) 4 Mal ") white solid.
Compounds 1-9-1 LC-MS calcd.for: C 52 H 80 N 10 O 19 S 2 1212.50；found:1213.65[M+H] + .
1.9.2 Compounds 1-9-2: DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal(L14a)
Figure BDA0004001699340000821
10(a):NHS-PEG 4 -Mal；10(b):TFA；11(c):DOTA-PEG 4 -TFP ester
Synthesis example 10 (a) intermediate 5-1 (Boc-Met-Val (Dap) -PEG 4 -Mal) synthesis
Boc-MV (Dap) (10.0 mg, 23.01. Mu. Mol) and NHS-PEG 4 Mal (12.2 mg, 28.06. Mu. Mol) was dissolved in 0.5mL DMF and Et was added 3 N (4. Mu.L) was stirred at room temperature for 2.0h. LC-MS detection reaction, after the reaction, semi-preparative HPLC purification, freeze-drying again. To obtain intermediate 5-1 (hereinafter also referred to as "Boc-MV (Dap) -PEG) 4 Mal ") white solid.
Intermediate 5-1: LC-MS calcd.for: C 33 H 55 N 5 O 13 S 761.89；found:762.45[M+H] + .
Synthesis example 10 (b) intermediate 5-2 (NH) 2 -Met-Val(Dap)-PEG 4 -Mal) synthesis
Intermediate 5-1 (5.0 mg, 6.56. Mu. Mol) was dissolved in 0.5mL of TFA and stirred at room temperature for 30min. After the completion of the reaction, it was concentrated and pumped to dryness to give crude intermediate 5-2 (hereinafter also referred to as "NH") 2 -MV(Dap)-PEG 4 Mal ") white solid.
Intermediate 5-2: LC-MS calcd.for: C 28 H 47 N 5 O 11 S 661.77；found:662.05[M+H] + .
Synthesis example 11 (c) Compound 1-9-1 (DOTA-PEG) 4 -Met-Val(Dap)-PEG 4 -Mal) synthesis
Dissolving the crude intermediate 5-2 in 0.5mL DMF, adding DOTA-PEG 4 TFP ester (8.0 mg, 7.88. Mu. Mol) and Et were added last 3 N (8. Mu.L), stirred at room temperature for 2 hours, after completion of the reaction, purified by semi-preparative HPLC, and lyophilized again to give the compound (hereinafter also referred to as "DOTA-PEG 4 -MV(Dap)-PEG 4 Mal ") white solid.
Compounds 1 to 9-2：LC-MS calcd.for:C 55 H 94 N 10 O 23 S 1295.46；found:1296.70[M+H] + .
1.9.3 Compounds 1-9-3: DOTA-MV (Dap) -PEG 4 -Mal(L14e)
Figure BDA0004001699340000831
10(a):NHS-PEG 4 -Mal；10(b):TFA；12(c):DOTA-tris( t Bu ester)；12(d)TFA
Synthesis example 10 (a) intermediate 5-1 (Boc-Met-Val (Dap) -PEG 4 -Mal) synthesis
Boc-MV (Dap) (10.0 mg, 23.01. Mu. Mol) and NHS-PEG 4 Mal (12.2 mg, 28.06. Mu. Mol) was dissolved in 0.5mL DMF and Et was added 3 N (4. Mu.L) was stirred at room temperature for 2.0h. LC-MS detection reaction, after the reaction, semi-preparative HPLC purification, freeze-drying again. To obtain intermediate 5-1 (hereinafter also referred to as "Boc-MV (Dap) -PEG) 4 Mal ") white solid.
Intermediate 5-1 LC-MS calcd.for: C 33 H 55 N 5 O 13 S 761.89；found:762.45[M+H] + .
Synthesis example 10 (b) intermediate 5-2 (NH) 2 -Met-Val(Dap)-PEG 4 -Mal) synthesis
Intermediate 5-1 (5.0 mg, 6.56. Mu. Mol) was dissolved in 0.5mL of TFA and stirred at room temperature for 30min. After the completion of the reaction, it was concentrated and pumped to dryness to give crude intermediate 5-2 (hereinafter also referred to as "NH") 2 -MV(Dap)-PEG 4 Mal ") white solid. Intermediate 5-2 LC-MS calcd.for: C 28 H 47 N 5 O 11 S 661.77；found:662.05[M+H] + .
Synthesis example 12 (c) intermediate 6-1 (DOTA-tris ] t Bu ester)-Met-Val(Dap)-PEG 4 -Mal) synthesis
DOTA-tris @ t Bu ester) (5.4 mg, 7.88. Mu. Mol) was dissolved in 0.5mL anhydrous DMF and HATU (5.5 mg, 14.44. Mu. Mol), N was added 2 Stirring for 1h at room temperature under an atmosphere. Crude intermediate 5-2NH 2 -MV(Dap)-PEG4-Mal was dissolved in 1.0mL of anhydrous DMF, added to the above system, followed by DIPEA (5.7. Mu.L, 32.8. Mu. Mol), N 2 Stirring for 4h at room temperature under atmosphere. LC-MS detection reaction, after the reaction, freeze drying. After this time semi-preparative HPLC (CH 3 CN-H 2 O0.1% TFA) and freeze-drying to give intermediate 6-1 (hereinafter also referred to as "DOTA-tris t Bu ester)-MV(Dap)-PEG 4 Mal ") white solid.
Intermediate 6-1 LC-MS calcd.for: C 56 H 97 N 9 O 18 S 1215.67；found:1214.20[M-H] - .
Synthesis example 12 (d) Compounds 1-9-3 (DOTA-Met-Val (Dap) -PEG 4 -Mal) synthesis
The above crude intermediate 6-1 was dissolved in 0.5mL of TFA, stirred at room temperature for 5 hours, and after completion of the reaction, semi-preparative HPLC was purified, and lyophilized again to give the compound (hereinafter also referred to as "DOTA-MV (Dap) -PEG) 4 Mal ") white solid. Compounds 1-9-3 LC-MS calcd.for: C 44 H 73 N 9 O 18 S 1047.48；found:1046.15[M-H] - .
1.9.4 Compounds 1-9-4: NODAGA-MV (Dap) -PEG 4 -Mal(L14c)
Figure BDA0004001699340000841
10(a):NHS-PEG 4 -Mal；10(b):TFA；13(c):NODA-GA-NHS
Synthesis example 10 (a) intermediate 5-1 (Boc-Met-Val (Dap) -PEG 4 -Mal) synthesis
Boc-MV (Dap) (10.0 mg, 23.01. Mu. Mol) and NHS-PEG 4 Mal (12.2 mg, 28.06. Mu. Mol) was dissolved in 0.5mL DMF and Et was added 3 N (4. Mu.L) was stirred at room temperature for 2.0h. LC-MS detection reaction, after the reaction, semi-preparative HPLC purification, freeze-drying again. To obtain intermediate 5-1 (hereinafter also referred to as "Boc-MV (Dap) -PEG) 4 Mal ") white solid.
Intermediate 5-1 LC-MS calcd.for: C 33 H 55 N 5 O 13 S 761.89；found:762.45[M+H] + .
Synthesis example 10 (b) intermediate 5-2 (NH) 2 -Met-Val(Dap)-PEG 4 -Mal) synthesis
Intermediate 5-1 (5.0 mg, 6.56. Mu. Mol) was dissolved in 0.5mL of TFA and stirred at room temperature for 30min. After the completion of the reaction, it was concentrated and pumped to dryness to give crude intermediate 5-2 (hereinafter also referred to as "NH") 2 -MV(Dap)-PEG 4 Mal ") white solid. Intermediate 5-2 LC-MS calcd.for: C 28 H 47 N 5 O 11 S 661.77；found:662.05[M+H] + .
Synthesis example 13 (c) Compounds 1-9-4 (NODAGA-Met-Val (Dap) -PEG 4 -Mal) synthesis
The crude intermediate 5-2 was dissolved in 0.5mL DMF and NODAGA-NHS (5.8 mg, 7.88. Mu. Mol) was added followed by Et 3 N (6. Mu.L), stirred at room temperature for 2h, after completion of the reaction, purified by semi-preparative HPLC, and lyophilized again to give the compound (hereinafter also referred to as "NODAGA-MV (Dap) -PEG) 4 Mal ") white solid.
Compounds 1-9-4 LC-MS calcd.for: C 43 H 70 N 8 O 18 S 1019.13；found:1020.15[M+H] + .
1.10 Compounds 1-10: DOTA-MV- (CH) 2 ) 2 -Mal(L15a)
Figure BDA0004001699340000851
14(a):Mal-(CH 2 ) 2 -NH 2 ；14(b):TFA；14(c):DOTA-tris( t Bu ester)；14(d):TFA
Synthesis example 14 (a) intermediate 7-1 (Boc-Met-Val- (CH) 2 ) 2 -Mal) synthesis
Boc-MV (Dap) (2.7 mg, 15.29. Mu. Mol) was dissolved in 0.5mL anhydrous DMF and HATU (12.6 mg, 33.14. Mu. Mol), N was added 2 Stirring for 1h at room temperature under an atmosphere. Mal- (CH) 2 ) 2 -NH 2 Dissolving in 1.0mL anhydrous DMF, adding to the above system, adding DIPEA (13.3 μL,76.44 μmol), N 2 Stirring for 4h at room temperature under atmosphere. LC (liquid Crystal) device-MS detection reaction, after completion of the reaction, lyophilization. After this time semi-preparative HPLC (CH 3 CN-H 2 O0.1% TFA) purification, lyophilization to give intermediate 7-1 (hereinafter also referred to as "Boc-MV- (CH) 2 ) 2 -Mal”)
Intermediate 7-1: LC-MS calcd.for: C 21 H 34 N 4 O 6 S 470.59；found:471.50[M+H] + .
Synthesis example 14 (b) intermediate 7-2 (NH) 2 -Met-Val-(CH 2 ) 2 -Mal) synthesis
Intermediate 7-1 was dissolved in 0.5mL TFA and stirred at room temperature for 30min. After the completion of the reaction, it was concentrated and dried to give crude intermediate 7-2 (hereinafter also referred to as "NH") 2 -MV-(CH 2 ) 2 Mal ") white solid.
Synthesis 14 (c): intermediate 7-3 (DOTA-tris t Bu ester)-Met-Val-(CH 2 ) 2 -Mal) synthesis
DOTA-tris @ t Bu ester) (3.2 mg, 5.52. Mu. Mol) was dissolved in 0.5mL anhydrous DMF and HATU (3.6 mg, 9.35. Mu. Mol), N was added 2 Stirring for 1h at room temperature under an atmosphere. Crude intermediate 7-2NH 2 -MV-(CH 2 ) 2 Mal was dissolved in 1.0mL of anhydrous DMF, added to the above system, followed by DIPEA (3.7. Mu.L, 21.25. Mu. Mol), N 2 Stirring for 4h at room temperature under atmosphere. LC-MS detection reaction, after the reaction, freeze drying. After this time semi-preparative HPLC (CH 3 CN-H 2 O0.1% TFA) and freeze-drying to give intermediate 7-3 (hereinafter also referred to as "DOTA-tris t Bu ester)-MV-(CH 2 ) 2 Mal ") white solid.
Intermediate 7-3 LC-MS calcd.for: C 44 H 76 N 8 O 11 S 925.20；found:926.55[M+H] + .
Synthesis example 14 (d) Compounds 1-10 (DOTA-Met-Val- (CH) 2 ) 2 -Mal) synthesis
Intermediate 7-3 (3.9 mg, 4.25. Mu. Mol) was dissolved in 0.5mL of TFA, stirred at room temperature for 5 hours, and after completion of the reaction, semi-preparative HPLC was purified and lyophilized again to give the compound (hereinafter also referred to as "DOTA-MV- (CH) 2 ) 2 Mal ") white solid.
Compounds 1-10 LC-MS calcd.for: C 32 H 52 N 8 O 11 S 756.87；found:757.60[M+H] + .
1.10.1 Compound 1-10-1: DOTA-PEG 4 -MV-PEG 4 -Mal(L15b)
Figure BDA0004001699340000861
14(e):Mal-PEG 4 -NH 2 ；14(f):TFA；14(g):DOTA-PEG 4 -TFP ester
Synthesis example 14 (e) intermediate 7-1-1 (Boc-Met-Val-PEG 4 -Mal) synthesis
Boc-MV (30.0 mg, 86.12. Mu. Mol) was dissolved in 0.5mL anhydrous DMF and HATU (43.5 mg, 114.40. Mu. Mol) N was added 2 Stirring for 1h at room temperature under an atmosphere. Mal-PEG 4 -NH 2 Dissolving in 0.5mL anhydrous DMF, adding into the above system, adding DIPEA (51.8 μL,297.55 μmol), and adding N 2 Stirring for 3h at room temperature under atmosphere. LC-MS detection reaction, after the reaction, freeze drying. After this time semi-preparative HPLC (CH 3 CN-H 2 O0.1% TFA) purification, lyophilization to give intermediate 7-1-1 (hereinafter also referred to as "Boc-MV-PEG 4 -Mal”)
Intermediate 7-1-1: LC-MS calcd.for: C 29 H 50 N 4 O 10 S 646.80；found:647.33[M+H] + .
Synthesis example 14 (f) intermediate 7-2-1 (NH) 2 -Met-Val-PEG 4 -Mal) synthesis
Intermediate 7-1-1 was dissolved in 0.5mL TFA and stirred at room temperature for 30min. After the reaction was completed, it was concentrated and dried to give crude intermediate 7-2-1 (hereinafter also referred to as "NH") 2 -MV-PEG 4 Mal ") white solid.
Synthesis example 14 (g) Compound 1-10-1 (DOTA-PEG) 4 -Met-Val-PEG 4 -Mal) synthesis
Dissolving the crude intermediate 7-2-1 in 0.5mL DMF, adding DOTA-PEG 4 TFP ester (11.92 mg, 11.60. Mu. Mol) and Et added last 3 N (8.0. Mu.L), stirred at room temperature for 2h, after completion of the reaction, purified by semi-preparative HPLC, and lyophilized again to give the compound (hereinafter also referred to as "DOTA-PEG) 4 -MV-PEG 4 Mal ") white solid.
Compounds 1-10-1 LC-MS calcd.for: C 51 H 89 N 9 O 20 S 1180.38；found:1181.25[M+H] + .
1.10.2 Compounds 1-10-2: DOTA-Bn-MV-PEG 4 -Mal(L15c)
Figure BDA0004001699340000871
14(h):Mal-PEG 4 -NH 2 ；14(i):TFA；14(j):p-SCN-Bn-DOTA
Synthesis example 14 (h) intermediate 7-1-2 (Boc-Met-Val-PEG 4 -Mal) synthesis
Boc-MV (30.0 mg, 86.12. Mu. Mol) was dissolved in 0.5mL anhydrous DMF and HATU (43.5 mg, 114.40. Mu. Mol) N was added 2 Stirring for 1h at room temperature under an atmosphere. Mal-PEG 4 -NH 2 Dissolving in 0.5mL anhydrous DMF, adding into the above system, adding DIPEA (51.8 μL,297.55 μmol), and adding N 2 Stirring for 3h at room temperature under atmosphere. LC-MS detection reaction, after the reaction, freeze drying. After this time semi-preparative HPLC (CH 3 CN-H 2 O0.1% TFA) purification, lyophilization to give intermediate 7-1-2 (hereinafter also referred to as "Boc-MV-PEG 4 -Mal”)
Intermediate 7-1-2: LC-MS calcd.for: C 29 H 50 N 4 O 10 S 646.80；found:647.33[M+H] + .
Synthesis example 14 (i) intermediate 7-2-2 (NH) 2 -Met-Val-PEG 4 -Mal) synthesis
Intermediate 7-1-2 was dissolved in 0.5mL TFA and stirred at room temperature for 30min. After the reaction, it is concentrated and pumped to dryness to give crude intermediate 7-2-2 (hereinafter also referred to as "NH") 2 -MV-PEG 4 Mal ") white solid.
Synthesis example 14 (j) Compound1-10-2 (DOTA-Bn-Met-Val-PEG) 4 -Mal) synthesis
The crude intermediate 7-2-2 was dissolved in 0.5mL DMF and p-SCN-Bn-DOTA (15.93 mg, 23.19. Mu. Mol) was added, followed by Et 3 N (32.0. Mu.L), stirred at room temperature for 3 hours, after completion of the reaction, purified by semi-preparative HPLC, and lyophilized again to give the compound (hereinafter also referred to as "DOTA-Bn-MV-PEG) 4 Mal ") white solid.
Compounds 1-10-2: LC-MS calcd.for: C 48 H 75 N 9 O 16 S 2 1098.30；found:1098.49[M] + .
1.11 Compounds 1-11: DOTA-Bn-rSFK-PEG 4 -Mal(L12)
Figure BDA0004001699340000881
15(a):Mal-PEG 4 -NHS；15(b):TFA；15(c):p-SCN-Bn-DOTA
Synthesis example 15 (a) intermediate 8-1 (Boc- (D-Arg) -Ser-Phe-Lys (PEG) 4 ) -Mal) synthesis
Boc-rSFK (10.0 mg, 15.70. Mu. Mol) and NHS-PEG 4 Mal (7.6 mg, 17.28. Mu. Mol) was dissolved in 0.5mL DMF and Et was added 3 N (4. Mu.L) was stirred at room temperature for 3.0h. LC-MS detection reaction, semi-preparative HPLC purification after reaction, freeze-drying. To obtain intermediate 8-1 (hereinafter also referred to as "Boc-rSFK-PEG 4 Mal ") white solid.
Intermediate 8-1: LC-MS calcd.for: C 44 H 69 N 9 O 15 964.08；found:965.15[M+H] + .
Synthesis example 15 (b) intermediate 8-2 (NH) 2 -(D-Arg)-Ser-Phe-Lys(PEG 4 ) -Mal) synthesis
Intermediate 8-1 was dissolved in 0.5mL TFA and stirred at room temperature for 30min. After the completion of the reaction, it was concentrated and dried to give crude intermediate 8-2 (hereinafter also referred to as "NH") 2 -rSFK-PEG 4 Mal ") white solid.
Synthesis example 15 (c) Compound 1-11 (DOTA-Bn- (D-Arg) -Ser-Phe-Lys (PEG) 4 ) -Mal) Synthesis) the crude intermediate 8-2 was dissolved in 0.5mL DMF and p-SCN-Bn-DOTA (2.8 mg, 4.05. Mu. Mol) was added, followed by Et 3 N (8.0. Mu.L), stirred at room temperature for 3 hours, after completion of the reaction, purified by semi-preparative HPLC, and lyophilized again to give the compound (hereinafter also referred to as "DOTA-Bn-rSFK-PEG) 4 Mal ") white solid.
Compounds 1-11: LC-MS calcd.for: C 63 H 94 N 14 O 21 S 1415.58；found:1416.15[M+H] + .
1.12 Compounds 1-12: DOTA-Bn-GWK-PEG 4 -Mal(L20)
Figure BDA0004001699340000891
16(a):Mal-PEG 4 -NHS；16(b):TFA；16(c):p-SCN-Bn-DOTA
Synthesis example 16 (a) intermediate 9-1 (Boc-Gly-Trp-Lys (PEG) 4 ) -Mal) synthesis
Boc-GWK (3.0 mg, 6.12. Mu. Mol) and NHS-PEG 4 Mal (3.3 mg, 7.35. Mu. Mol) was dissolved in 0.5mL DMF and Et was added 3 N (4. Mu.L) was stirred at room temperature for 3.0h. LC-MS detection reaction, semi-preparative HPLC purification after reaction, freeze-drying. To obtain intermediate 9-1 (hereinafter also referred to as "Boc-GWK-PEG 4 Mal ") white solid.
Intermediate 9-1: LC-MS calcd.for: C 39 H 56 N 6 O 13 816.91；found:817.55[M+H] + .
Synthesis example 16 (b) intermediate 9-2 (NH) 2 -Gly-Trp-Lys(PEG 4 ) -Mal) synthesis
Intermediate 9-1 was dissolved in 0.5mL TFA and stirred at room temperature for 30min. After the completion of the reaction, it was concentrated and dried to give crude intermediate 9-2 (hereinafter also referred to as "NH") 2 -GWK-PEG 4 Mal ") white solid.
Synthesis example 16 (c) Compounds 1-12 (DOTA-Bn-Gly-Trp-Lys (PEG) 4 ) -Mal) synthesis
The crude intermediate 9-2 was dissolved in 0.5mL DMF and p-SCN-Bn-D was addedOTA (4.6 mg, 6.74. Mu. Mol) and final Et addition 3 N (8.0. Mu.L), stirred at room temperature for 3 hours, after completion of the reaction, purified by semi-preparative HPLC, and lyophilized again to give the compound (hereinafter also referred to as "DOTA-Bn-GWK-PEG) 4 Mal ") white solid.
Compounds 1-12 LC-MS calcd.for: C 58 H 81 N 11 O 19 S 1268.40；found:1268.55[M] + .
1.13 Compounds 1-13: DOTA-Bn-MNK-PEG 4 -Mal(L22)
Figure BDA0004001699340000901
17(a):Mal-PEG 4 -NHS；17(b):TFA；17(c):p-SCN-Bn-DOTA
Synthesis example 17 (a) intermediate 10-1 (Boc-Met-Asn-Lys (PEG) 4 ) -Mal) synthesis
Boc-MNK (4.8 mg, 9.78. Mu. Mol) and NHS-PEG 4 Mal (5.2 mg, 11.74. Mu. Mol) was dissolved in 0.5mL DMF and Et was added 3 N (4. Mu.L) was stirred at room temperature for 3.0h. LC-MS detection reaction, semi-preparative HPLC purification after reaction, freeze-drying. To obtain intermediate 10-1 (hereinafter also referred to as "Boc-MNK-PEG 4 Mal ") white solid.
Intermediate 10-1: LC-MS calcd.for: C 35 H 58 N 6 O 14 S 818.94；found:819.65[M+H] + .
Synthesis example 17 (b) intermediate 10-2 (NH) 2 -Met-Asn-Lys(PEG 4 ) -Mal) synthesis
Intermediate 10-1 was dissolved in 0.5mL TFA and stirred at room temperature for 30min. After the completion of the reaction, it was concentrated and dried to give crude intermediate 10-2 (hereinafter also referred to as "NH") 2 -MNK-PEG 4 Mal ") white solid.
Synthesis example 17 (c) Compounds 1-13 (DOTA-Bn-Met-Asn-Lys (PEG) 4 ) -Mal) synthesis
The crude intermediate 10-2 was dissolved in 0.5mL DMF and p-SCN-Bn-DOTA (4.0 mg, 5.86. Mu. Mol) was added, followed by Et 3 N (8.0. Mu.L), stirred at room temperature for 3h, and invertedAfter completion of this, semi-preparative HPLC purification and lyophilization again were carried out to give the compound (hereinafter also referred to as "DOTA-Bn-MNK-PEG 4 Mal ") white solid.
Compounds 1-13 LC-MS calcd.for: C 54 H 83 N 11 O 20 S 2 1270.44；found:1270.50[M] + .
1.14 Compounds 1-14: DOTA-Bn-QLK-PEG 4 -Mal(L23)
Figure BDA0004001699340000911
18(a):Mal-PEG 4 -NHS；18(b):TFA；18(c):p-SCN-Bn-DOTA
Synthesis example 18 (a) intermediate 11-1 (Boc-Gln-Leu-Lys (PEG) 4 ) -Mal) synthesis
Boc-QLK (4.8 mg, 9.78. Mu. Mol) and NHS-PEG 4 Mal (4.8 mg, 9.84. Mu. Mol) was dissolved in 0.5mL DMF and Et was added 3 N (4. Mu.L) was stirred at room temperature for 3.0h. LC-MS detection reaction, semi-preparative HPLC purification after reaction, freeze-drying. To obtain intermediate 11-1 (hereinafter also referred to as "Boc-QLK-PEG 4 Mal ") white solid.
Intermediate 11-1: LC-MS calcd.for: C 37 H 62 N 6 O 14 814.93；found:815.90[M+H] + .
Synthesis example 18 (b) intermediate 11-2 (NH) 2 -Gln-Leu-Lys(PEG 4 ) -Mal) synthesis
Intermediate 11-1 was dissolved in 0.5mL TFA and stirred at room temperature for 30min. After the completion of the reaction, it was concentrated and dried to give crude intermediate 11-2 (hereinafter also referred to as "NH") 2 -QLK-PEG 4 Mal ") white solid.
Synthesis example 18 (c) Compounds 1-14 (DOTA-Bn-Gln-Leu-Lys (PEG) 4 ) -Mal) synthesis
The crude intermediate 11-2 was dissolved in 0.5mL DMF and p-SCN-Bn-DOTA (4.0 mg, 5.82. Mu. Mol) was added, followed by Et 3 N (8.0. Mu.L), stirred at room temperature for 3h, after completion of the reaction, purified by semi-preparative HPLC, and lyophilized again to give the compound (belowAlso referred to herein as "DOTA-Bn-QLK-PEG 4 Mal ") white solid.
Compounds 1-14 LC-MS calcd.for: C 56 H 87 N 11 O 20 S 1266.43；found:1266.45[M] + .
1.15 Compounds 1-15: DOTA-Bn-TIK-PEG 4 -Mal(L21)
Figure BDA0004001699340000921
19(a):Mal-PEG 4 -NHS；19(b):TFA；19(c):p-SCN-Bn-DOTA
Synthesis example 19 (a) intermediate 12-1 (Boc-Thr-ILe-Lys (PEG) 4 ) -Mal) synthesis
Boc-TIK (4.7 mg, 10.30. Mu. Mol) and NHS-PEG 4 Mal (5.5 mg, 12.36. Mu. Mol) was dissolved in 0.5mL DMF and Et was added 3 N (4. Mu.L) was stirred at room temperature for 3.0h. LC-MS detection reaction, semi-preparative HPLC purification after reaction, freeze-drying. To obtain intermediate 12-1 (hereinafter also referred to as "Boc-TIK-PEG 4 Mal "white solid.
Intermediate 12-1: LC-MS calcd.for: C 36 H 61 N 5 O 14 787.91；found:788.25[M+H] + .
Synthesis example 19 (b) intermediate 12-2 (NH) 2 -Thr-ILe-Lys(PEG 4 ) -Mal) synthesis
Intermediate 12-1 was dissolved in 0.5mL TFA and stirred at room temperature for 30min. After the completion of the reaction, it was concentrated and dried to give crude intermediate 12-2 (hereinafter also referred to as "NH") 2 -TIK-PEG 4 Mal ") white solid.
Synthesis example 19 (c) Compound 1-15 (DOTA-Bn-Thr-ILe-Lys (PEG) 4 ) -Mal) synthesis
The crude intermediate 12-2 was dissolved in 0.5mL DMF and p-SCN-Bn-DOTA (4.0 mg, 5.82. Mu. Mol) was added, followed by Et 3 N (8.0. Mu.L), stirred at room temperature for 3 hours, after completion of the reaction, purified by semi-preparative HPLC, and lyophilized again to give the compound (hereinafter also referred to as "DOTA-Bn-TIK-PEG) 4 Mal ") white solid.
Compounds 1-15 LC-MS calcd.for: C 55 H 86 N 10 O 20 S 1239.40；found:1247.15[M+8] + .
1-16 compounds 1-16: DOTA-Mal (commercially available)
Figure BDA0004001699340000931
Example 2 enzyme-cleavable Linker coupled to protein as precursor
Protein sequence
SNA006 is a single domain antibody (anti-CD 8 a VHH) targeting cd8+ T cells, with the sequence:
QVQLVESGGGLVQPGGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTY YADSVKGRFTISRDNSKNHVYLQMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKG TLVTVSSGSC(SEQ ID NO:4)
SNA004 is a single domain antibody (anti-HER 2 VHH) targeting HER2 target, the sequence of which is:
QLQLVESGGGLVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTN YKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQ VTVSSGSC(SEQ ID NO:7)
SNA018 species of scaffold protein drugs targeting HER2 target, the sequence of which is:
GHEHEHEDANSLAAAKETALYHLDRLGVADAYKDLIDKAKTVEGVKARYFEILHALP GSSC(SEQ ID NO:8)
method for coupling precursors
Figure BDA0004001699340000932
Figure BDA0004001699340000941
EXAMPLE 3 radionuclide-labeled radiolabel
Figure BDA0004001699340000942
/>
Figure BDA0004001699340000951
EXAMPLE 4 analysis of the stability of radionuclide-labeled precursors in human serum
The radionuclide-labeled precursor (100. Mu.L) prepared in example 3 was added to human serum and incubated at 37℃to calculate the radioactivity ratio of the unchanged precursor by analyzing samples at various time points by Radio-HPLC after 0h, 1h, 2h, 4 h.
68 Ga-NOTA-Bn-MV(Dap)-PEG 4 The results of human serum stability of SNA006 indicate (FIG. 1) that more than 85% of the radioactivity remains in NOTA-Bn-MV (Dap) -PEG after 4 hours 4 -on SNA006 precursor;
68 Ga-NOTA-Bn-VMK-(CH 2 ) 2 the SNA006 human serum stability results indicate (FIG. 2) that more than 80% of the radioactivity remains in NOTA-Bn-VMK- (CH) after 2h 2 ) 2 -SNA006 precursor.
EXAMPLE 5 analysis of radionuclide-labeled precursor radioactivity in mouse urine
The radionuclide-labeled precursor prepared in example 3 was administered by adjusting the precursor concentration to 1. Mu.g, 20. Mu. Ci of each solution through the tail vein of the subcutaneous tumor model mouse. And urine from mice 1.5 hours after administration was collected and then analyzed by Radio-HPLC, and the analysis results of radioactivity in urine excreted in the examples are shown (fig. 3 and 4). 68 Ga-NOTA-Bn-MV(Dap)-PEG 4 -SNA006 (FIG. 3) and 68 Ga-NOTA-Bn-VMK-(CH 2 ) 2 both 60% -70% of radioactivity remained predominantly on the 17.6 minute off-peak precursor for SNA006 (fig. 4).
EXAMPLE 6 diagnosis of radionuclides 68 Analysis of Ga-labeled precursor distribution in mice
In vivo studies were performed with female nude mice ranging in age from 6 to 8 weeks. Raising nude mice in SPF environment, food and water can be obtained at will, and standard day-night time is 12 hoursAnd (3) an illumination cycle. For xenografts, animals were injected with 100 μl of cell/PBS. Cells were subcutaneously implanted in the left and right forelegs, respectively. The cell inoculation density is about 5 to 6X 10 6 Individual cells/mice. Implantation was performed under isoflurane anesthesia. Under these conditions, in more than 80% of the injected animals, a usable tumor model was obtained after 2 weeks.
The radionuclide-labeled precursor prepared in example 3 was administered by adjusting the precursor concentration to 1. Mu.g, 20. Mu. Ci of each solution through the tail vein of the subcutaneous tumor model mouse. Mice were sacrificed 1.5h after dosing, blood, urine and vital organs were collected and weights were determined, cpm was determined by a gamma counting system, and the% radioactivity ID/g in each organ tissue was calculated.
Will be 68 Ga-NODAGA-SNA006， 68 Ga-NOTA-Bn-SFK-(CH 2 ) 2 -SNA006， 68 Ga-NOTA-Bn-DFK-(CH 2 ) 2 -SNA006， 68 Ga-NOTA-Bn-SLK-SNA006， 68 Ga-NOTA-Bn-VMK-(CH 2 ) 2 -SNA006 68 Ga-NOTA-Bn-MV(Dap)-PEG 4 The in vivo radioactivity distribution of SNA006 after 1.5h following separate administration to subcutaneous tumor model mice is shown in table 1. The tumor and kidney distribution is shown in fig. 5. After the administration of the drug for 1.5 hours, 68 Ga-NOTA-Bn-VMK-(CH 2 ) 2 -SNA006、 68 Ga-NOTA-Bn-MV(Dap)-PEG 4 SNA006 showed a high tumor kidney ratio of 0.27, 0.23.
TABLE 1 in vivo distribution of radionuclide-labeled precursor 1.5h in mice
Figure BDA0004001699340000961
/>
Figure BDA0004001699340000971
6.1 diagnosis of radionuclides 68 In vivo kinetics study of Ga-labeled precursor in mice
The radionuclide-labeled precursor prepared in example 3 was administered by adjusting the precursor concentration to 1. Mu.g, 20. Mu. Ci of each solution through the tail vein of the subcutaneous tumor model mouse. Administration was performed through the tail vein of a subcutaneous tumor model mouse, blood was collected from the orbit of the mouse 2min, 5min, 10min, 15min, 30min, 60min, 90min, 120min after administration, the weight was measured, cpm was measured by a gamma counting system, and the% ID/g in the blood was calculated.
Results represent (fig. 6): the in vivo kinetics of each sample of the examples within 2 hours after administration were not significantly altered from that of the reference examples. After the cleavable Linker is applied to the radiopharmaceuticals, the kidney retention is lower, the stability of the proto-drug is not affected, and the application universality of the cleavable Linker is confirmed.
6.2 treatment of radionuclides 177 Application of Lu labeling precursor
Dilution of radionuclide-labeled prepared according to the above method with PBS buffer 177 Lu-DOTA-SNA004、 177 Lu-DOTA-Bn-VMK-PEG 4 -SNA004. The radionuclide label solutions, whose concentration of radioactive precursors was adjusted to 10. Mu.g/100. Mu.L, 300. Mu. Ci, were administered from the tail vein of the subcutaneous tumor model mice described above. Two mice SPECT/CT scans were imaged at time points 0h, 1h, 2h, 4h, 24h, 48h, 72h post-dose using Micro SPECT/CT.
Will be 177 Lu-DOTA-SNA004、 177 Lu-DOTA-Bn-VMK-PEG 4 SPECT/CT images at various time points after administration of SNA004 to tumor model mice are shown (fig. 7). Examples 1 to 24 hours after administration 77 Lu-DOTA-Bn-VMK-PEG 4 SNA004 accumulated less to the kidneys and clearly imaged tumors. Reference example on the other hand 177 Lu-DOTA-SNA004, although tumors were also imaged, higher radioactivity was also observed in the kidneys.
Based on SPECT/CT image ROI display (fig. 8), 1-72 hours after administration, 177 Lu-DOTA-SNA004 and 177 Lu-DOTA-Bn-VMK-PEG 4 tumor uptake rates were close at each time point of SNA004, but at 177 The Lu-DOTA-SNA004 kidney has higher radioactivity.
Above mentioned， 177 Lu-DOTA-Bn-VMK-PEG 4 The accumulation of SNA004 radiolabeled drug to the kidneys became lower and still able to maintain high tumor targeting. The potential of cleavable Linker for therapeutic radiopharmaceuticals is disclosed.

Claims (10)

1. A compound represented by the formula (I) or a pharmaceutically acceptable salt thereof,
X-L-Y-R 1
formula (I)
Wherein X is a chelating agent;
l is a linker, and L may be absent;
y is a polypeptide which is capable of being cleaved by enkephalinase (Neutral endopeptidase, NEP);
R 1 is a group that binds to an amino group or a carboxyl group of a side chain of Y and has a functional group capable of binding to a target molecule recognition unit or a linking group thereof, or is a hydrogen atom of an amino group or a carboxyl group of a side chain of Y.
2. A compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R 1 Is composed of one or more of the following components: -C 1 -C 18 Alkylene, - (CH) 2 CH 2 O) n -，-CO(CH 2 ) n -，-NH(CH 2 ) n Arylthio (PYS), p-aminobenzyloxycarbonyl (PAB), aminobenzylthio, oxybenzylthio, alkoxyamino (AOA), dioxybenzylthio, diaminobenzylthio, aminooxybenzylthio, alkoxyamino (AOA), 4-methyl-4-dithiopentanoyl (MPDP), triazole, dithio, sulfonyl, phosphonyl, (4-acetyl) aminobenzoyl (SIAB), 4-thiobutyryl, 4-thio-2-sulfonylbutyryl (2-SO) 3 - -SPDB), 4-thiopropionyl (SPDP), hydrazone, aminoethylamine, hydrazine, oxime, thioamino oxybutyric acid, peptide containing 1 to 20 amino acid residues, and
Figure FDA0004001699330000011
wherein n isIs any integer between 1 and 1000.
3. A compound of formula (I) according to any one of claims 1-2, or a pharmaceutically acceptable salt thereof, wherein R 1 Selected from the following structures:
Figure FDA0004001699330000012
6-Maleimidocaproyl (MC), maleimidopropionyl (MP), maleimidoethyl (ME), 6-maleimidocaproyl-valine-citrulline-p-aminobenzoic acid oxycarbonyl (MC-VC-PAB), 4- (N-maleimidomethyl) cyclohexane-1-acyl (SMCC), 4- (2-pyridylthio) pentanoic acid N-succinimidyl ester (SPP).
4. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein the compound of formula (I) is selected from: X-L-SLK (Mal), X-L-SFK- (CH) 2 ) 2 -Mal，X-L-SFK-
(CH 2 ) 2 -Mal，X-SFK-(CH 2 ) 2 -Mal，X-L-DFK-(CH 2 ) 2 -Mal，X-L-VMK-(CH 2 ) 2 -Mal，X-L-
VMK-PEG 4 -Mal，X-L-VMK-PEG 4 -Mal，X-VMK-(CH 2 ) 2 -Mal，X-VMK-PEG 4 -Mal，X-L-MV(Dap)-PEG 4 -Mal，X-L-MV(Dap)-PEG 4 -Mal，X-L-MV(Dap)-PEG 4 -Mal，X-
MV(Dap)-PEG 4 -Mal，X-MV(Dap)-PEG 4 -Mal，X-MV-(CH 2 ) 2 -Mal，X-L-MV-PEG 4 -Mal，X-L-MV-PEG 4 -Mal，X-L-rSFK-PEG 4 -Mal，X-L-GWK-PEG 4 -Mal，X-L-MNK-PEG 4 -
Mal，X-L-QLK-PEG 4 -Mal and X-L-TIK-PEG 4 -Mal；
Wherein X is selected from:
Figure FDA0004001699330000021
X-L is selected from
Figure FDA0004001699330000022
5. A compound of formula (I) according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is selected from: NOTA-Bn-SLK (Mal), NOTA-Bn-SFK- (CH) 2 ) 2 -Mal，DOTA-Bn-SFK-(CH2)2-Mal，NODAGA-SFK-(CH2)2-Mal，NOTA-Bn-DFK-(CH2)2-Mal，NOTA-Bn-VMK-(CH 2 ) 2 -Mal，DOTA-Bn-VMK-PEG 4 -Mal，DOTA-PEG 4 -VMK-PEG 4 -
Mal，DOTA-VMK-(CH 2 ) 2 -Mal，DOTA-VMK-PEG 4 -Mal，NOTA-Bn-MV(Dap)-PEG 4 -
Mal，DOTA-Bn-MV(Dap)-PEG 4 -Mal，DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal，DOTA-
MV(Dap)-PEG 4 -Mal，NODAGA-MV(Dap)-PEG 4 -Mal，DOTA-MV-(CH 2 ) 2 -Mal，DOTA-
PEG 4 -MV-PEG 4 -Mal，DOTA-Bn-MV-PEG 4 -Mal，DOTA-Bn-rSFK-PEG 4 -Mal，DOTA-Bn-GWK-PEG 4 -Mal，DOTA-Bn-MNK-PEG 4 -Mal，DOTA-Bn-QLK-PEG 4 -Mal and DOTA-Bn-TIK-PEG 4 -Mal。
6. An immunoconjugate comprising I) a compound of formula (I) or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 5 and ii) a target molecule recognition unit.
7. The immunoconjugate according to claim 6, having a structure represented by formula (II):
Figure FDA0004001699330000031
wherein X is a chelator, X is attached or not attached to the active moiety;
t is a target molecule recognition unit;
l is a linker, L being defaulting;
A 1 and A 2 Is an amino acid residue, m is 0,1 or 2;
A 3 is an amino acid residue having an amino group or a carboxyl group in a side chain, R 1 Is with A 3 A group which is bound to an amino group or a carboxyl group of a side chain of (a) and has a functional group capable of binding to a target molecule recognition unit or a linking group thereof;
-(A 1 ) m -A 2 -A 3 -being capable of being cleaved by enkephalinase.
8. The immunoconjugate according to any one of claims 6 to 7, selected from the structures:
NOTA-Bn-SLK(Mal)-VHH，
NOTA-Bn-SFK-(CH 2 ) 2 -Mal-VHH，
DOTA-Bn-SFK-(CH 2 ) 2 -Mal-VHH，
NODAGA-SFK-(CH 2 ) 2 -Mal-VHH，
NOTA-Bn-DFK-(CH 2 ) 2 -Mal-VHH，
NOTA-Bn-VMK-(CH 2 ) 2 -Mal-VHH，
DOTA-Bn-VMK-PEG 4 -Mal-VHH，
DOTA-PEG 4 -VMK-PEG 4 -Mal-VHH，
DOTA-VMK-(CH 2 ) 2 -Mal-VHH，
DOTA-VMK-PEG 4 -Mal-VHH，
NOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，
DOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，
DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal-VHH，
DOTA-MV(Dap)-PEG 4 -Mal-VHH，
NODAGA-MV(Dap)-PEG 4 -Mal-VHH，
DOTA-MV-(CH 2 ) 2 -Mal-VHH，
DOTA-PEG 4 -MV-PEG 4 -Mal-VHH，
DOTA-Bn-MV-PEG 4 -Mal-VHH，
DOTA-Bn-rSFK-PEG 4 -Mal-VHH，
DOTA-Bn-GWK-PEG 4 -Mal-VHH，
DOTA-Bn-MNK-PEG 4 -Mal-VHH，
DOTA-Bn-QLK-PEG 4 Mal-VHH
DOTA-Bn-TIK-PEG 4 -Mal-VHH。
9. The immunoconjugate according to any one of claims 6-8, selected from the structures: A-NOTA-Bn-SLK (Mal) -VHH,
A-NOTA-Bn-SFK-(CH 2 ) 2 -Mal-VHH，
A-DOTA-Bn-SFK-(CH 2 ) 2 -Mal-VHH，
A-NODAGA-SFK-(CH 2 ) 2 -Mal-VHH，
A-NOTA-Bn-DFK-(CH 2 ) 2 -Mal-VHH，
A-NOTA-Bn-VMK-(CH 2 ) 2 -Mal-VHH，
A-DOTA-Bn-VMK-PEG 4 -Mal-VHH，
A-DOTA-PEG 4 -VMK-PEG 4 -Mal-VHH，
A-DOTA-VMK-(CH 2 ) 2 -Mal-VHH，
A-DOTA-VMK-PEG 4 -Mal-VHH，
A-NOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，
A-DOTA-Bn-MV(Dap)-PEG 4 -Mal-VHH，
A-DOTA-PEG 4 -MV(Dap)-PEG 4 -Mal-VHH，
A-DOTA-MV(Dap)-PEG 4 -Mal-VHH，
A-NODAGA-MV(Dap)-PEG 4 -Mal-VHH，
A-DOTA-MV-(CH 2 ) 2 -Mal-VHH，
A-DOTA-PEG 4 -MV-PEG 4 -Mal-VHH，
A-DOTA-Bn-MV-PEG 4 -Mal-VHH，
A-DOTA-Bn-rSFK-PEG 4 -Mal-VHH，
A-DOTA-Bn-GWK-PEG 4 -Mal-VHH，
A-DOTA-Bn-MNK-PEG 4 -Mal-VHH，
A-DOTA-Bn-QLK-PEG 4 Mal-VHH
A-DOTA-Bn-TIK-PEG 4 -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 as claimed in any one of claims 1 to 5 and ii) a radionuclide.
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