WO2024051794A1 - Radionuclide-drug conjugate and pharmaceutical composition and use thereof - Google Patents

Abstract

Provided are a radionuclide-drug conjugate and a pharmaceutical composition and use thereof. Specifically, a precursor of the radionuclide-drug conjugate has a structure represented by formula I, wherein LG represents a targeting ligand; S represents a spacer; L represents a linker; and C represents a chelating agent capable of containing a radionuclide. The radionuclide-drug conjugate can specifically deliver the radionuclide to a target cell. When containing one LG, the drug conjugate can specifically deliver the radionuclide to the target cell. When containing two LGs, the drug conjugate can function in various ways to enhance therapeutic effects and reduce toxic and side effects. Meanwhile, the presence of two LGs also enhances the affinity of the drug conjugate for the target cell, reducing off-target toxicity.

Description

Radionuclide conjugated drugs and pharmaceutical compositions and applications thereof

Cross-references to related applications

The present invention claims the priority of the invention patent application titled "Radionuclide Conjugated Drugs and Pharmaceutical Compositions and Applications thereof" and application number 202211105422.6, submitted in China on September 9, 2022, which is incorporated by reference. The entire content of the patent application is incorporated herein.

Technical field

The invention belongs to the field of precision diagnosis and treatment, and specifically relates to a radionuclide conjugated drug, a pharmaceutical composition based on the coupled drug, and its application in the medical field.

Background technique

Radiopharmaceuticals refer to a type of special medicine that uses particles or rays released by radionuclides for medical diagnosis and treatment. Radionuclides and their labeled compounds are used to study human physiology, pathology and drug processes in the body. Belongs to the category of radiopharmaceuticals. Radiopharmaceuticals are widely used in tumor diagnosis and treatment, myocardial imaging, early detection of neurodegenerative diseases and inflammatory tissue imaging diagnosis, etc., to achieve fast, non-destructive real-time imaging of physiological and pathological processes, which is an important part of molecular imaging and precision medicine. medicine), providing new means and approaches for early diagnosis and timely treatment in the true sense.

Under the trend of precision medicine, nuclear medicine has also begun to move closer to precise targeting. Some physical methods or the specificity of tissue absorption can achieve partial precise targeting. For example, thyroid cancer and its metastases will specifically absorb iodine, so it can be treated with iodine-131 (I-131); or through minimally invasive methods Radioembolization can be achieved by inserting yttrium-90 (Y-90) microspheres. However, these targeted effects do not have the possibility of large-scale promotion and can only be applied in specific scenarios.

In recent years, with the widespread clinical application of ligands with targeting effects (e.g., antibody drug conjugates/ADCs, peptide drug conjugates/PDCs, small molecule drug conjugates/SMDC, etc.), these ligands are associated with The combination of radioactive chelates has also formed a new track - radionuclide drug conjugates/RDC. Similar to ADC, RDC uses specific targeting mediated by antibodies, small molecules or peptides to deliver radionuclides used as radiation/imaging factors to the target location, thereby focusing the rays generated by the radionuclides. Targeted at local tissues, it provides efficient and precise diagnosis and treatment while reducing damage to other tissues caused by systemic exposure. Different from ADC, the radionuclides loaded by RDC can be used for both diagnosis and treatment. In addition, RDC is also slightly different from ADC in structure, requiring the introduction of specific structural fragments for chelating radionuclides. ——Chelates.

Specifically, in terms of drug structure, RDC mainly consists of ligands (such as antibodies, polypeptides or small molecules, etc.), linkers, chelators and radioactive isotopes that mediate the targeting effect. (Radioisotope) composition. Depending on the type of ligand, RDC can be divided into antibody-targeted radionuclide antibody conjugates (RAC), radionuclide conjugates based on small molecules (including peptides), etc. Depending on the type of radionuclide, RDC can perform different functions in imaging or treatment, and some nuclides even have both functions.

Currently, relatively few therapeutic RDCs have been approved for marketing, only Novartis’s Lutathera (Lu-177dotatate, or 177Lu-dotatate, used to treat somatostatin receptor-positive gastrointestinal neuroendocrine tumors) and Pluvicto (Lu -177 vipivotide tetraxetan, or 177Lu-PSMA-617, for the treatment of prostate-specific membrane antigen (PSMA)-positive metastatic castration-resistant prostate cancer) and Biogen's Zevalin (Y-90 ibritumomab tiuxetan, or 90Y -ibritumomab tiuxetan, used to treat non-Hodgkin's lymphoma) and a few other RDCs, there is an urgent need to develop more therapeutic or dual-purpose RDCs to meet the growing demand in the field of precision diagnosis and treatment.

Contents of the invention

Invent the problem to be solved

In view of the current problems of few types of therapeutic RDCs and single targeting ligands, the present invention discovered a series of brand-new RDCs, including a variety of dual-ligand RDCs constructed based on dual-targeting technology, which further enriched the range of therapeutic RDCs. Or the type of RDC that is used for both diagnosis and treatment.

solutions to problems

<First aspect>

The invention provides a radionuclide-conjugated drug precursor, which includes a targeting ligand, a chelating agent, an optional linker and an optional spacer.

In the present invention, the above-mentioned radionuclide conjugated drug precursor may have a structure shown in formula I,

in,

LG represents targeting ligand;

S represents spacer;

L represents the linker;

C represents chelating agent;

m is 1 or 2;

n is 0 or 1;

p is 0 or 1; and

When p is 0, C is directly connected to S or LG;

When n is 0, LG is directly connected to L or C;

When m is 2, the two LGs are the same or different from each other, and the two LGs are connected to S, L, or C at the same time, or the two LGs are connected to each other and then to S, L, or C.

In one embodiment of the present invention, the above-mentioned radionuclide-conjugated drug precursor having a structure shown in Formula I may have a structure shown in Formula I-1,
LG-LC
I-1

Wherein, LG, L and C are as defined in Formula I.

In another embodiment of the present invention, the above-mentioned radionuclide-conjugated drug precursor having a structure shown in Formula I may have a structure shown in Formula I-2,
LG-C
I-2

Where, LG and C are as defined in Formula I.

In yet another embodiment of the present invention, the above-mentioned radionuclide-conjugated drug precursor having a structure shown in Formula I may have a structure shown in Formula I-3,

Wherein, LG, S, L and C are as defined in Formula I.

In yet another embodiment of the present invention, the above-mentioned radionuclide-conjugated drug precursor having a structure shown in Formula I may have a structure shown in Formula I-4,

Wherein, LG, S and C are as defined in Formula I.

In yet another embodiment of the present invention, the above-mentioned radionuclide-conjugated drug precursor having a structure shown in Formula I may have a structure shown in Formula I-5,

Wherein, LG, L and C are as defined in Formula I.

In yet another embodiment of the present invention, the above-mentioned radionuclide-conjugated drug precursor having a structure shown in Formula I may have a structure shown in Formula I-6,

Wherein, LG, L and C are as defined in Formula I.

In one embodiment of the invention, the above-mentioned targeting ligand (ie, LG) can bind to the following cell surface proteins: PSMA, FORL1, TRPV6, FAPI, C-MET, CAIX, RGD, Hepsin or Sigma.

When containing only one targeting ligand, the conjugated drug prodrug including it (for example, the conjugated prodrug as shown in Formula I-1 or Formula I-2) can bind to the following cell surface proteins: Any one (single target single match), especially PSMA, TRPV6, FAPI, C-MET or CAIX.

When two targeting ligands are contained, the conjugated drug prodrugs containing them (for example, the conjugated prodrugs represented by Formula I-3, Formula I-4, Formula I-5 or Formula I-6 substance) can bind to any one of the following cell surface protein combinations (single-target double-matching or double-target double-matching), especially PSMA/FORL1, FORL1/TRPV6, PSMA/TRPV6, PSMA/FAPI, PSMA/RGD, PSMA/Hepsin , FAPI/RGD, FAPI/FAPI, FAPI/Hespin, PSMA/Sigma or FAPI/CAIX.

In one embodiment of the invention, the above-mentioned targeting ligand can be formed from a polypeptide, an antibody or a small molecule.

In one embodiment of the present invention, the above-mentioned conjugated drug represented by Formula I, Formula I-1, Formula I-2, Formula I-3, Formula I-4, Formula I-5 or Formula I-6 Each LG in the precursor can be independently represented by formulas LG-I, LG-II, LG-III, LG-IV, LG-V, LG-VI, LG-VII, LG-VIII, and LG-IX. Any kind of ligand compound is formed,

preferred

In formula LG-I (or LG-I’),

R ^LG1 is amino,

in

R ₁ is hydrogen or optionally substituted cycloalkylformyl (e.g., 4-(aminomethyl)cyclohexylformyl, i.e. preferred Or, 4-(6-aminocaproylaminomethyl)cyclohexylformyl, that is preferred

R ₂ and R ₃ are each independently hydrogen or optionally substituted C ₆ -C ₁₀ aryl (e.g., phenyl, naphth-1-yl, naphth-2-yl, etc.);

R ₄ is hydroxyl, optionally substituted C ₁ -C ₆ alkylamino (e.g., 6-aminohexylamino, i.e. ), an amino acid derivative group (for example, -NH-Asp-COOH) or an oligopeptide derivative group consisting of 2-6 amino acids (for example, -NH-Asp-Asp-Lys-COOH);

R ₅ is optionally substituted C ₁ -C ₆ alkyl; when substituted, the substituent is amino-substituted C ₁ -C ₆ alkanoyl, amino-substituted C ₆ -C ₁₀ aroyl, or both through amide The acyl group obtained by the reaction (for example, 4-(6-aminocaproylaminomethyl)benzoyl);

Formula LG-II is pteroic acid or folic acid or an analog thereof; preferably, the analog of folic acid is selected from the group consisting of 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, 10-formylfolate, and methotrexate. rosin, 5,10-methylenetetrahydrofolate, aminopterin, and raltitrexed;

Formula LG-III includes all or part of the amino acids in the polypeptide EGKLSSNDTEGGLCKEFLHPSKVDLPR; preferably, formula LG-III includes 9 to 27 amino acids in the above polypeptide; more preferably, formula LG-III has at least 70% and 75% of the amino acids in the polypeptide KEFLHPSKVDLPR. , 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity;

preferred

In formula LG-IV (or LG-IV’),

R ^LG2 is halogen (e.g., bromine), amino,

in

R ₈ is hydrogen, in

R _8' is carboxyl-substituted C ₁ -C ₆ alkylamino (for example, 5-amino-5-carboxypentylamino), carboxyl-substituted C ₃ -C ₈ cycloalkylmethylamino (for example, (trans- 4-carboxycyclohexyl)methylamino) or carboxyl-substituted C ₁ -C ₂ alkoxy C ₁ -C ₂ alkoxy C ₁ -C ₂ alkylamino (for example, 2-(2-(carboxymethoxy )ethoxy)ethylamino);

R _8″ is hydrogen, amino-substituted C ₁ -C ₆ alkanoyl (for example, 6-aminocaproyl), amino-substituted C ₁ -C ₂ alkoxy C ₁ -C ₂ alkoxy C ₁ -C ₂ Alkanoyl (e.g., 2-(2-(2-aminoethoxy)ethoxy)acetyl), carboxyl-substituted C ₁ -C ₆ alkanoyl (e.g., 3-carboxypropionyl) or carboxyl-substituted C ₁ -C ₂ alkoxy C ₁ -C ₂ alkoxy C ₁ -C ₂ alkanoyl (e.g., 2-(2-(carboxymethoxy)ethoxy)acetyl);

R ₉ is in

R _9' is carbonyl or methylene;

In the formula LG-V,

X is a single bond or -(CH ₂ CH ₂ O) _n CH ₂ CONH-, where

n is 0, 1, 2 or 3;

R ^LG3 is hydrogen or in

R ₁₀ is a hydroxyl or carboxyl substituted C ₁ -C ₆ alkylamino group (for example, 5-amino-5-carboxypentylamino);

In the formula LG-VI,

B ₁ to B ₅ are each independently an amino acid fragment formed from any one of the following amino acids: phenylalanine (Phe), glutamic acid (Glu), arginine (Arg), glycine (Gly), and aspartate Amino acid (Asp); preferably, the formula LG-VI is and

Optionally introduce into the residue of at least one of B ₁ to B ₅ an optionally substituted alkanoyl group (for example, 3-amino-3-carboxypropionyl) and/or an optionally substituted amino group (for example, 5-amino -5-carboxypentylamino);

In formula LG-VII,

C ₁ to C ₄ each independently represent an amino acid fragment, wherein the C terminal of C ₁ is connected to a benzothiazolyl group, and the N terminal of C ₄ is connected to an acetyl group, and

optionally introducing an optionally substituted alkanoyl group (e.g., 3-carboxypropionyl) into at least one residue of C ₁ to C ₄ ;

In formula LG-VIII,

Y is One end marked with * is connected to the carboxyl group (COOH), and the other end is connected to the ring Z;

Ring Z is a C ₆ -C ₁₀ aromatic ring (for example, benzene ring, naphthalene ring, etc.) or a 5-9 membered heteroaromatic ring (for example, furan ring, benzofuran ring, etc.);

R ^LG4 is hydrogen, C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy;

Formula LG-IX contains the polypeptide Cys ^a -X1-Cys ^c -X2-Gly-Pro-Pro-X3-Phe-Glu-Cys ^d -Trp-Cys ^b -Tyr-X4-X5-X6, where: X1 is Asn, His or Tyr; X2 is Gly, Ser, Thr or Asn; X3 is Thr or Arg; X4 is Ala, Asp, Glu, Gly or Ser; X5 is Ser or Thr ^; Cysteine residue; Preferably, residues Cys ^a and Cys ^b and Cys ^c and Cys ^d are cyclized respectively to form two independent disulfide bonds; further, the formula LG-IX includes the polypeptide Ala-Gly-Ser- Cys ^a -Tyr-Cys ^c -Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys ^d -Trp-Cys ^b -Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys, Wherein: Cys ^ad is each independently a cysteine residue; preferably, the residues Cys ^a and Cys ^b and Cys ^c and Cys ^d are cyclized respectively to form two independent disulfide bonds; more preferably, the formula IX The carboxyl group of the terminal lysine is amidated; alternatively, Formula IX has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the polypeptide, wherein: Cys ^ad is each independently a cysteine residue; preferably, residues Cys ^a and Cys ^b and Cys ^c and Cys ^d are cyclized respectively to form two independent disulfide bonds.

In one embodiment of the present invention, the above-mentioned conjugated drug represented by Formula I, Formula I-1, Formula I-2, Formula I-3, Formula I-4, Formula I-5 or Formula I-6 Each LG in the precursor can independently be a targeting ligand formed from a ligand compound of the formula LG-I or its optical isomer and bound to the cell surface protein PSMA; a ligand compound of the formula LG-II or its optical isomer. The optical isomer forms and binds to the targeting ligand of the cell surface protein FORL1; the targeting ligand formed from the ligand compound of formula LG-III or its optical isomer and binds to the cell surface protein TRPV6; the targeting ligand of the formula LG-IV The body compound or its optical isomer forms and binds the targeting ligand of the cell surface protein FAPI; the formula LG-V ligand compound or its optical isomer forms and binds the targeting ligand of the cell surface protein CAIX; by the formula The LG-VI ligand compound or its optical isomer forms a targeting ligand that binds to the cell surface protein RGD; the LG-VII ligand compound or its optical isomer forms a targeting ligand that binds to the cell surface protein Hepsin. Body; formed from a ligand compound of formula LG-VIII or its optical isomer and bound to the target of the cell surface protein Sigma Ligand; or a targeting ligand formed from a ligand compound of the formula LG-IX or an optical isomer thereof and binding to the cell surface protein C-MET.

In one embodiment of the present invention, the above-mentioned conjugated drug represented by Formula I, Formula I-1, Formula I-2, Formula I-3, Formula I-4, Formula I-5 or Formula I-6 Each LG in the precursor can be independently formed from any of the following ligand compounds.

In some embodiments of the present invention, the LG in the above-mentioned conjugated drug prodrug (monocoupled drug prodrug) represented by Formula I-1 or Formula I-2 can be represented by the formulas LG-I, LG- III, LG-IV, LG-V, LG-IX are formed from any ligand compound (or its optical isomer); preferably, LG can be formed by the formula LG-1, LG-1A, LG-1B, LG-1C, LG-1D, LG-1E, LG-1G, LG-1H, LG-1K, LG-1L, LG-3, LG-4, LG-4A, LG-4B, LG-4C, LG- 4E, LG-4F, LG-11, LG-12, LG-12A, LG-12B, LG-12C, LG-12D, LG-12E, LG-14, LG-5, LG-6, LG-6A, Either ligand compound (or optical isomer thereof) of LG-6B and LG-6C is formed.

In some embodiments of the present invention, each LG in the above-mentioned conjugated drug prodrugs (branched double-chain conjugated drug prodrugs) represented by Formula I-3, Formula I-4 or Formula I-6 Can be independently composed of formulas LG-I, LG-II, LG-III, LG-IV, LG-V, LG-VI, LG-VII, Any one of the ligand compounds (or optical isomers thereof) in LG-VIII and LG-IX is formed; preferably, each LG can be independently formed by the formulas LG-1, LG-1A, LG-1B, LG -1C, LG-1D, LG-1E, LG-1G, LG-1H, LG-1K, LG-1L, LG-2, LG-3, LG-4, LG-4A, LG-4B, LG-4C , LG-4E, LG-4F, LG-11, LG-12, LG-12A, LG-12B, LG-12C, LG-12D, LG-12E, LG-14, LG-5, LG-6, LG - Any ligand compound (or its optical isomer); more preferably, the two LGs can be respectively formed by the formulas LG-1 and LG-2, LG-1 and LG-11, LG-1B and LG-4, LG-1B and LG-7, LG -1B and LG-8A, LG-1B and LG-10, LG-1E and LG-3, LG-1L and LG-2, LG-2 and LG-3, LG-4A and LG-4B, LG-4A and LG-6C, LG-4A and LG-7A or LG-4A and LG-8B ligand compounds (or optical isomers thereof); further preferably, the two LGs can be formed by the formulas LG-1B and LG- 4. Formation of LG-1B and LG-10 or LG-4A and LG-4B ligand compounds (or optical isomers thereof).

In some embodiments of the present invention, each LG in the above-mentioned conjugated drug prodrugs (linear dual-coupled drug prodrugs) represented by formula I-5 can be independently composed of formulas LG-I, Any ligand compound in LG-VIII (or its optical isomer) is formed; preferably, each LG can be independently formed by the formula LG-1, LG-1A, LG-1B, LG-1C, LG - Formation of any ligand compound (or its optical isomer) among 1D, LG-1E, LG-1G, LG-1H, LG-1K, LG-1L, LG-9, and LG-10; more preferably Specifically, the two LGs can be formed by ligand compounds of formulas LG-1 and LG-9 (or optical isomers thereof) respectively; further preferably, the two LGs can be formed of ligand compounds of formulas LG-1 and LG-9 respectively. (or its optical isomer) is formed, and LG formed by the formula LG-9 ligand compound (or its optical isomer) is connected to L.

In the present invention, the LG formed from the above-mentioned ligand compound can be of various types, and the present invention does not impose any specific limitation on this. For example, it can be a monovalent group (i.e., a secondary amino group) obtained by losing a hydrogen atom from the primary amino group in the structure (preferably the primary amino group located at the end of the main chain), which can be coupled with other structural fragments in the drug precursor. The carboxyl group is connected to form an amide group), or it can be a monovalent group obtained after the carboxyl group in the structure (preferably the carboxyl group located at the end of the main chain) loses the hydroxyl group (i.e., the carboxylic group, which can be coupled with the drug prodrug It can also be a monovalent group (such as an alkoxy group or aromatic group) obtained by losing a hydrogen atom from a hydroxyl group in the structure (preferably the hydroxyl group located at the end of the main chain). Oxygen group, which can be linked to the carboxylic group of other structural segments coupled to the prodrug to form an ester group).

In the present invention, the above-mentioned conjugated drug precursors such as Formula I-3, Formula I-4, Formula I-5 or Formula I-6 are relatively preferred, because the structures of these precursors all contain The two targeting ligands can work in a variety of ways to improve the therapeutic effect and reduce toxic side effects; at the same time, the two targeting molecules enhance the affinity of the coupled compound to target cells and reduce off-target toxicity.

Further, the radionuclide chelating agent (i.e., C) contained in the radionuclide conjugated drug precursor of the present invention can complex diagnostic and/or therapeutic radioisotopes, especially therapeutic or dual-purpose radioisotopes. . When the radionuclide-conjugated drug precursor is chelated with the radioisotope (for example, chelated in an equimolar ratio), the radionuclide-conjugated drug can be obtained. Therefore, the radionuclide-conjugated drug of the present invention includes the radionuclide-conjugated drug precursor of the present invention and the radioactive isotope chelated thereto.

In the present invention, examples of radioactive isotopes that can be used for diagnosis (imaging) include, but are not limited to, ⁴⁵ Ti, ⁵² Fe, ⁵⁹ Fe, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶³ Zn, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁹ Zr, ^99m Tc, ^{111 In} , ^117m Sn, ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ^191m Pt, 193m Pt, ^195m Pt, ¹⁹⁸ Au, ¹⁹⁹ Au, etc.

In the present invention, examples of radioactive isotopes that can be used for treatment include, but are not limited to, ^58m Co, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁸⁹ Sr, ⁹⁰ Y, ^103m Rh, ¹⁰³ Pd, ¹¹¹ In, ^117m Sn, ¹¹⁹ Sb, ¹⁵³ Sm, ¹⁵³ Gd, ¹⁶¹ Tb, ¹⁶¹ Ho, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ^193m Pt, 195m Pt, ¹⁹⁷ Pt, ¹⁹⁸ Au, ¹⁹⁹ Au, ^{201 Tl} , ²⁰³ Pb, ²¹² Bi, ²¹³ Bi, ²¹¹ At, ²²³ Ra, ²²⁴ Ra, ²²⁵ Ac, ²²⁷ Th, etc.

In one embodiment of the present invention, the above-mentioned conjugated drug represented by Formula I, Formula I-1, Formula I-2, Formula I-3, Formula I-4, Formula I-5 or Formula I-6 C in the precursor can chelate any of the following radionuclides: ⁴⁷ Sc, ⁴⁸ Sc, ⁵¹ Cr, ⁵⁵ Fe, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁹ Zn, ⁶⁷ Ga, ⁶⁸ Ga, ⁷² Ga, ⁷² As, ⁷² Se, ⁸⁹ Sr, ⁸⁸ Y, ⁹⁰ Y, ⁹⁹ Tc, ^99m Tc, ⁹⁷ Ru, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ In, ¹¹⁹ Sb, ¹²⁸ Ba, ¹³⁹ La, ¹⁴⁰ La, ¹⁴² Pr, ¹⁴⁹ Pm, ¹⁵³ Sm , ¹⁵⁹ Gd, ¹⁶⁵ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁷⁵ Yb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au, ¹⁹⁹ Au, ¹⁹⁷ Hg, ²⁰¹ Tl, ²⁰² Pb, ²⁰³ Pb, ²¹² Pb, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac and ²²⁷ Th.

In one embodiment of the present invention, the above-mentioned conjugated drug represented by Formula I, Formula I-1, Formula I-2, Formula I-3, Formula I-4, Formula I-5 or Formula I-6 C in the precursor can chelate any of the following radionuclides: ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁹ Sr, ⁹⁰ Y, ^99m Tc, ¹¹¹ In, ¹¹⁹ Sb, ¹⁵³ Sm, ¹⁶⁶ Ho, ^{177 Lu} , ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au, 199 Au, ²⁰¹ Tl, ²⁰³ Pb, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac and ²²⁷ Th, preferably any of the following radionuclides : ⁶⁴ Cu, ⁶⁷ Cu, ¹¹¹ In, ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au and ¹⁹⁹ Au.

In one embodiment of the present invention, the above-mentioned conjugated drug represented by Formula I, Formula I-1, Formula I-2, Formula I-3, Formula I-4, Formula I-5 or Formula I-6 C in the precursor can chelate ¹⁷⁷ Lu.

In the present invention, the chelating agent may contain a variable number of heteroatoms (usually N, O, S or P atoms, especially N, O or S atoms, preferably N or O atoms) to complex radionuclides , and may be cyclic or non-cyclic (especially cyclic).

Examples of cyclic chelating agents include, but are not limited to, 1,4,7-triazacyclononane; 1,4,7-triazacyclononane-triacetic acid; 1,4,7,10- Tetraazacyclododecane; 1,4,7,10-tetraazacyclotridecane; 1,4,7,11-tetraazacyclotetradecane; 1,4,7,10-tetraaza Heterocyclododecane-1,4,7,10-tetraacetic acid; 2-(1,4,7,10-tetraazacyclododecane-1-yl)acetic acid; 2,2'-(1, 4,7,10-tetraaacyclododecane-1,7-diyl)diacetic acid; 2,2',2″-(1,4,7,10-tetraaacyclododecane-1 ,4,7-triyl)triacetic acid; 1,4,8,11-tetraaacyclotetradecane; 1,4,8,12-tetraaacyclopentadecane; 1,5,9,13 -Tetraazacyclohexadecane; 2-(1,4,8,11-tetraazacyclotetradecan-1-yl)acetic acid; 2,2'-(1,4,8,11-tetraaza Heterocyclotetradecane-1,8-diyl)diacetic acid, etc.

Examples of acyclic chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (i.e., 2,2',2",2"'-(ethane-1,2-diylbis(nitrilo))) Tetraacetic acid); Diethylenetriaminepentaacetic acid (i.e. 2,2',2",2"'-(((carboxymethyl)nitrilo)bis(ethane-2,1-diyl))bis (nitrilo))tetraacetic acid); 2,2'-((2-((carboxymethyl)(2-hydroxyethyl)amino)ethyl)nitrilo)diacetic acid; 2,3-bis( 2-Mercaptoacetamido)propionic acid; 2,2',2",2"'-(((4'-(3-amino-4-methoxyphenyl)-[2,2':6', 2"-terpyridine]-6,6"-diyl)bis(methylene))bis(nitrilo))tetraacetic acid; 2,2'-((1-carboxyethyl)nitrilo)bis Acetic acid; 2,2'-(ethane-1,2-diylbis(nitrogen))bis(2-(2-hydroxyphenyl)acetic acid); 2,2',2",2"'- ((cyclohexane-1,2-diyl)bis(nitrilo))tetraacetic acid, etc.

In one embodiment of the present invention, the above-mentioned conjugated drug represented by Formula I, Formula I-1, Formula I-2, Formula I-3, Formula I-4, Formula I-5 or Formula I-6 C in the precursor can be formed from any of the following chelating compounds.

In one embodiment of the present invention, the above-mentioned conjugated drug represented by Formula I, Formula I-1, Formula I-2, Formula I-3, Formula I-4, Formula I-5 or Formula I-6 C in the precursor may be formed from a chelating agent compound of formula C-1.

In the present invention, C formed by the above-mentioned chelating agent compound can be various, and the present invention does not impose any specific limitation on this. For example, it can be a carboxylic acid group obtained by losing the hydroxyl group from the carboxyl group in the structure (preferably the carboxyl group located at the end of the main chain) (which can be connected to the secondary amino group of other structural fragments in the coupled drug precursor to form an amide group).

In one embodiment of the present invention, LG and C can be directly connected to form a radionuclide conjugated drug prodrug (i.e., a conjugated drug prodrug as shown in Formula I-2), for example, a targeted formulation The body is connected to the carboxylic acid group (-C(=O)-) of the chelating agent through its secondary amino group (-NH-) to form an amide group (-NH-(C=O)-).

Further, the linker (i.e., L) in the radionuclide-conjugated drug prodrug of the present invention can be used as an intermediate fragment to connect the chelating agent (C) and the targeting ligand (LG); or, when conjugating the drug before When there is a spacer (S) in the body structure, L can also be used as an intermediate segment to connect C and S. In the present invention, the linker can be either cleavable (or degradable) or non-cleavable (or non-degradable), and the latter is preferred.

In one embodiment of the present invention, L in the above-mentioned conjugated drug prodrugs represented by Formula I, Formula I-1, Formula I-3, Formula I-5 or Formula I-6 can be Formula L-I , any one of L-II, L-III, L-IV, L-V,

preferred

In the formula L-I (or L-I'), the end marked with * is connected to LG in the conjugated drug precursor or is terminated with a hydroxyl or amino group, and the end marked with ** is connected to LG in the conjugated drug precursor. LG or C is either terminated with hydrogen or an acetyl group; R is absent or is a bivalent structural fragment formed of amino acids; preferably, R is a bivalent structural fragment formed of lysine, tryptophan or valine;

Preferably, in formula LI (or L-I'), the end marked with * is connected to the LG in the conjugated drug precursor or is terminated with a hydroxyl or amino group, and the end marked with ** on the left side is connected to the conjugated drug. C in the precursor, the end marked with ** on the right side is connected to LG in the conjugated drug precursor or is terminated with hydrogen or acetyl group; R does not exist or is a bivalent formed by lysine (Lys) structural fragment;

In formula L-II, the end marked by *** is connected to S or LG in the conjugated drug precursor, and the end marked by ** is connected to C in the conjugated drug precursor; X is NH or O, Preferably NH; n is any integer from 2 to 8, preferably 5;

preferred

In the formula L-III (or L-III'), the end marked with *** is connected to S or LG in the conjugated drug precursor, and the end marked with ** is connected to C in the conjugated drug precursor. ; X does not exist or is NH or O, preferably NH; n is any integer from 1 to 4, preferably 1;

preferred

In the formula L-IV (or L-IV'), the end marked with * is connected to the LG in the conjugated drug precursor or is terminated with a hydroxyl or amino group, and the end marked with ** is connected to the conjugated drug precursor. LG or C in is either terminated with hydrogen or acetyl group; n is any integer from 1 to 6;

Preferably, in the formula L-IV (or L-IV'), the end marked by * is terminated with a hydroxyl group, the end marked by ** on the left is connected to C in the conjugated drug precursor, and the end marked by * on the right *The marked end is connected to LG in the conjugated drug precursor; n is 4;

In the formula LV, the end marked with * is connected to LG in the conjugated drug precursor, and the end marked with ** is connected to C in the conjugated drug precursor; n is any integer from 1 to 5; R ₁ does not exist or is -NH-CH ₂ -CH ₂ -NH-; R ₂ does not exist or is One end marked with * is connected to NH in LV, and m is any integer from 1 to 7;

Preferably, in the formula LV, the end marked with * is connected to LG in the conjugated drug precursor, and the end marked with ** is connected to C in the conjugated drug precursor; n is 2 or 3; when R ₁ does not exist, R ₂ does not exist or is One end marked with * is connected to NH in formula LV, and m is 5; or, when R ₁ is -NH-CH ₂ -CH ₂ -NH-, R ₂ does not exist.

In one embodiment of the present invention, L in the above-mentioned conjugated drug prodrugs represented by Formula I, Formula I-1, Formula I-3, Formula I-5 or Formula I-6 can be the following structure Any of the fragments, in which the end marked by * is connected to LG in the conjugated drug precursor, the end marked by ** is connected to LG or C in the conjugated drug precursor, and the end marked by *** is connected Conjugate S or LG in the prodrug.

In one embodiment of the present invention, L in the above-mentioned conjugated drug prodrugs represented by Formula I, Formula I-1, Formula I-3, Formula I-5 or Formula I-6 can be Formula L -1, one of L-2, L-3, L-10, L-11, L-12, L-13, L-14, L-15.

In some embodiments of the present invention, L in the above-mentioned conjugated drug prodrugs represented by Formula I-1 or Formula 1-5 can be one of Formulas L-I, L-III, L-IV, and L-V; preferably Land, L can be one of the formulas L-3, L-3A, L-3B, L-10, L-11, L-12, L-13, L-14, and L-15.

In some embodiments of the present invention, L in the above-mentioned conjugated drug prodrug as shown in formula I-3 can be formula L-II, L-III; preferably, L can be formula L-2, L -3. One of L-3A and L-3B; more preferably, L can be formula L-2.

In some embodiments of the present invention, L in the above-mentioned conjugated drug prodrug represented by formula I-6 can be one of formulas L-1 and L-1A.

In the present invention, the above-mentioned L can be connected with LG, C and optional S. For example, they are connected to each other through a condensation reaction between amino groups and carboxyl groups; specifically, it can be a condensation reaction between the primary amino group in the linker compound (preferably the primary amino group located at the end of the main chain) and the carboxyl group in other compounds to form an amide. The group can also be a carboxyl group in the linker compound (preferably the carboxyl group located at the end of the main chain) and a primary amino group in other compounds to form an amide group through a condensation reaction.

In one embodiment of the present invention, when only one LG is contained, L can connect LG and C to form a radionuclide conjugated drug prodrug (i.e., a conjugated drug prodrug as shown in Formula I-1 ), for example, the targeting ligand is connected to the carboxyl group of the linker through its secondary amino group, and at the same time, the linker is connected to the carboxyl group of the chelating agent through its secondary amino group to form an amide group respectively.

In another embodiment of the invention, when two LGs are contained, L can connect one of the LGs (which is also connected to the other LG) with C to form a radionuclide conjugated drug precursor (i.e., as Coupled drug prodrugs represented by formula I-5), for example, the first targeting ligand is connected to the carboxylic group of the second targeting ligand through its secondary amino group, and the second targeting ligand is connected to the carboxylic acid group of the second targeting ligand through its secondary amino group. The carboxyl group of the linker is connected, and at the same time, the linker is connected to the carboxyl group of the chelating agent through its secondary amino group to form an amide group respectively.

In yet another embodiment of the present invention, when two LGs are contained, L can simultaneously connect two LGs (which are not connected to each other) and C to form a radionuclide conjugated drug precursor (i.e., as shown in Formula I The coupled drug precursor shown in -6), for example, the first targeting ligand and the second targeting ligand are each independently connected to the carboxylic acid group or secondary amino group of the linker through its secondary amino group or carboxylic acid group, At the same time, the linker is connected to the carboxylic acid group of the chelating agent through its secondary amino group to form an amide group.

Further, when the radionuclide-conjugated drug precursor of the present invention contains two targeting ligands (LG), a spacer (i.e., S) can be used as an intermediate segment to connect the chelating agent (C) and the targeting ligand. body (LG); alternatively, when there is a linker (L) in the conjugate structure, S can also be used as an intermediate segment to connect L and LG. In the present invention, the spacer may be either cleavable (or degradable) or non-cleavable (or non-degradable), and the latter is preferred.

In one embodiment of the present invention, S in the above-mentioned conjugated drug prodrug as shown in Formula I, Formula I-3 or Formula I-4 can be in Formula S-I, S-II or S-III. any kind,

preferred

In the formula SI (or S-I'), one end marked by * is connected to one LG in the conjugated drug precursor, one end marked by ** is connected to the other LG in the conjugated drug precursor, and **The other end marked is connected to L or C in the conjugated drug prodrug; A ₁ to A ₃ are each independently a bivalent structural fragment formed of amino acids; preferably, A ₁ to A ₃ are each independently a bivalent structural fragment. Asp, Lys, Glu, His or Trp formation A bivalent structural fragment; n and m are each independently any integer from 2 to 8;

Preferably, in formula SI (or S-I'), the end marked with * is connected to one LG of the conjugated drug precursor, and the end marked with ** above is connected to the other end of the conjugated drug precursor. LG, the end marked with ** below is connected to L or C in the conjugated drug precursor; A ₁ -A ₂ -A ₃ is -Asp-Asp-Lys; m is 4, n is 4;

preferred

In the formula S-II (or S-II'), one end marked with * is connected to one LG in the conjugated drug precursor, and one end marked with ** is connected to the other LG in the conjugated drug precursor. , the other end marked by ** is connected to L or C in the conjugated drug precursor; n is any integer from 2 to 6, preferably n is 4;

preferred

In formula S-III (or S-III'), the end marked with * is connected to LG in the conjugated drug precursor, and the end marked with ** is connected to L or C in the conjugated drug precursor; n is any integer from 1 to 5, preferably n is 1 or 2.

In one embodiment of the present invention, S in the above-mentioned conjugated drug prodrug as shown in Formula I, Formula I-3 or Formula I-4 can be any one of the following structural fragments, wherein * The end marked by *** is connected to one LG in the conjugated drug precursor, the end marked by ** is connected to the other LG in the conjugated drug precursor, and the end marked by *** is connected to the LG in the conjugated drug precursor. L or C.

In one embodiment of the present invention, S in the above-mentioned conjugated drug prodrug as shown in Formula I, Formula I-3 or Formula I-4 can be Formula S-6, S-9, S-13 , one of S-18.

In one embodiment of the present invention, S in the above-mentioned conjugated drug prodrug as shown in formula I-3 can be one of the formulas S-II and S-III; preferably, S can be the formula S- 13. One of S-18; more preferably, S can be formula S-13.

In one embodiment of the present invention, S in the above-mentioned conjugated drug prodrug as shown in formula I-4 can be one of formulas S-I and S-II; preferably, S can be formula S-6, One of S-9 and S-13.

In the present invention, the above-mentioned S can be connected with LG, C and optional L. For example, they are connected to each other through a condensation reaction between amino groups and carboxyl groups; specifically, it can be a condensation reaction between a primary amino group in a spacer compound (preferably a primary amino group located at the end of the main chain) and a carboxyl group in other compounds to form an amide. The group can also be a carboxyl group in the spacer compound (preferably the carboxyl group located at the end of the main chain) and a primary amino group in other compounds to form an amide group through a condensation reaction.

In one embodiment of the present invention, S can simultaneously connect two LGs (which are not connected to each other) and C to form a radionuclide conjugated drug precursor (i.e., a conjugated drug as shown in Formula I-4 Precursor), for example, the first targeting ligand and the second targeting ligand are each independently connected to the carboxyl group or secondary amino group of the spacer through its secondary amino group or carboxyl group, and at the same time, the spacer is connected through its secondary amino group Connected to the carboxylic acid group of the chelating agent to form an amide group respectively.

In another embodiment of the present invention, S can simultaneously connect two LG (which are not connected to each other) and L, and then connect C through L to form a radionuclide conjugated drug precursor (ie, as shown in Formula I- The coupled drug prodrug shown in 3), for example, the first targeting ligand and the second targeting ligand are each independently connected to the carboxyl group or secondary amino group of the spacer through its secondary amino group or carboxyl group, and the spacer The linker is connected to the carboxyl group of the linker through its secondary amino group, and at the same time, the linker is connected to the carboxyl group of the chelating agent through its secondary amino group to form an amide group respectively.

In one embodiment of the invention, in formula I-1,

LG can be formed from any of the ligand compounds (or optical isomers thereof) in the formulas LG-I, LG-IV, and LG-V; preferably, it can be formed from the formulas LG-1B, LG-1D, and LG-1E. , any one of the ligand compounds (or optical isomers thereof) in LG-4 and LG-6 is formed;

L can be one of the formulas L-I, L-IV, L-V (or its optical isomer); preferably, it can be L-10, L-11, L-12, L-13, L-14, L-15 One (or its optical isomer);

C can be formed from a chelating compound of formula C-1.

In one embodiment of the invention, in Formula I-2,

LG can be formed by any ligand compound (or its optical isomer) in the formula LG-I, LG-III or LG-5; preferably, it can be formed by the formula LG-1D, LG-3, LG-5 Any one of the ligand compounds (or its optical isomers) is formed;

C can be formed from a chelating compound of formula C-1.

In one embodiment of the invention, in formula I-3,

The two LGs can be respectively formed by any one of the ligand compounds (or optical isomers thereof) in the formulas LG-I, LG-IV, LG-V, LG-VI, LG-VII, LG-VIII; preferably Ground, can be represented by the formula LG-I and LG-IV, LG-I and LG-VI, LG-I and LG-VII, LG-I and LG-VIII, LG-IV and LG-IV, LG-IV and LG -V, LG-IV and LG-VI or LG-IV and LG-VII ligand compounds (or optical isomers thereof) are formed; more preferably, they can be formed by formulas LG-1 and LG-11, LG-1B and LG-4, LG-1B and LG-7, LG-1B and LG-8A, LG-1B and LG-10, LG-4A and LG-4B, LG-4A and LG-6C, LG-4A and LG-7A or LG-4A and LG-8B ligand compounds (or optical isomers thereof) Formed; further preferably, can be formed by formulas LG-1B and LG-4, LG-1B and LG-10 or LG-4A and LG-4B ligand compounds (or optical isomers thereof);

S can be one of the formulas S-II and S-III (or its optical isomer); preferably, it can be one of the formulas S-13 and S-18 (or its optical isomer); more preferably, It can be formula S-13 (or its optical isomer);

L can be one of the formulas L-II and L-III (or its optical isomer); preferably, it can be one of the formulas L-2 and L-3; more preferably, L can be the formula L-2;

C can be formed from a chelating compound of formula C-1.

In one embodiment of the invention, in formula I-4,

The two LGs can be formed by any ligand compound (or its optical isomer) in the formulas LG-I, LG-II, and LG-III respectively; preferably, they can be formed by the formulas LG-I and LG-II, LG-I and LG-III or LG-II and LG-III ligand compounds (or optical isomers thereof) are formed; more preferably, they can be formed by formulas LG-1 and LG-2, LG-1E and LG-3 or the formation of LG-2 and LG-3 ligand compounds (or optical isomers thereof);

S can be one of the formulas S-I and S-II (or its optical isomer); preferably, it can be one of the formulas S-6, S-9 and S-13 (or its optical isomer);

C can be formed from a chelating compound of formula C-1.

In one embodiment of the invention, in Formula I-5,

The two LGs can be formed by ligand compounds of formulas LG-I and LG-VIII (or optical isomers thereof) respectively; preferably, they can be formed by ligand compounds of formulas LG-1 and LG-9 (or optical isomers thereof). ) is formed, and LG formed by the ligand compound of formula LG-9 (or its optical isomer) is connected to L;

L can be formula L-III (or its optical isomer); preferably, it can be formula L-3 (or its optical isomer);

C can be formed from a chelating compound of formula C-1.

In one embodiment of the invention, in Formula I-6,

The two LGs can be formed by any ligand compound (or its optical isomer) in the formulas LG-I, LG-II, and LG-III respectively; preferably, they can be formed by the formulas LG-I and LG-II, LG-I and LG-III or LG-II and LG-III ligand compounds (or optical isomers thereof) are formed; more preferably, they can be formed by formulas LG-1E and LG-3, LG-1L and LG-2 or the formation of LG-2 and LG-3 ligand compounds (or optical isomers thereof);

L can be formula L-I (or its optical isomer); preferably, it can be one of formula L-1 and L-1A (or its optical isomer);

C can be formed from a chelating compound of formula C-1.

In the present invention, different structural fragments used to construct radionuclide-conjugated drug prodrugs can be connected to each other in series through amide groups, and the amide groups can in turn be connected through primary amino groups (for example, to form linkers, spacers or targeting It is formed by the condensation reaction of the terminal amino group in the compound of the ligand) and the carboxyl group (for example, multiple carboxyl groups in the compound that forms the chelating agent). Usually, the above-mentioned condensation reaction can be carried out in the presence of a coupling reagent in order to activate the carboxylic acid into a better electrophile, thereby promoting the forward progress of the reaction. Exemplary coupling reagents include, but are not limited to, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), DCC (dicyclohexylcarbodiimide), HOBt (1 -hydroxybenzotriazole), HATU (O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate), etc. Alternatively, amide bond formation can also be achieved by activating carboxylic acids with N-hydroxysuccinimide (NHS) to form succinimide esters, which can be combined with amines without any other coupling reagents. further reaction.

<Second aspect>

The present invention provides the following specific presentation forms of radionuclide conjugated drug precursors.

Preferably, the present invention provides the following specific presentation forms of radionuclide-conjugated drug precursors.

At the same time, the present invention also provides a dual-ligand compound, which can be directly connected or indirectly connected to a chelating agent through a connecting fragment, thereby obtaining the radionuclide-conjugated drug precursor of the present invention.

In the present invention, the above-mentioned dual-ligand compound may be in the following specific presentation forms.

<Third aspect>

The present invention provides a radionuclide-conjugated drug, which includes the radionuclide-conjugated drug precursor described in the <first aspect> or the <second aspect>, and a radioactive isotope chelated thereto.

In one embodiment of the present invention, the radioactive isotope in the above-mentioned radionuclide conjugated drug is selected from ⁴⁷ Sc, ⁴⁸ Sc, ⁵¹ Cr, ⁵⁵ Fe, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁹ Zn, ⁶⁷ Ga, ⁶⁸ Ga, ⁷² Ga, ⁷² As, ⁷² Se, ⁸⁹ Sr, ⁸⁸ Y, ⁹⁰ Y, ⁹⁹ Tc, ^99m Tc, ⁹⁷ Ru, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ In, ¹¹⁹ Sb, 128 Ba, ¹³⁹ La, ^{140 La, 142 Pr} , ¹⁴⁹ Pm, ¹⁵³ Sm, ¹⁵⁹ Gd, ¹⁶⁵ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁷⁵ Yb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au, ¹⁹⁹ Au, ¹⁹⁷ Hg, ²⁰¹ Tl, ²⁰² Pb, ²⁰³ Pb, ²¹² Any of Pb, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac and ²²⁷ Th.

In one embodiment of the present invention, the radioactive isotope in the above-mentioned radionuclide conjugated drug is selected from ⁶⁴ Cu, ⁶⁷ Cu ^{, 67 Ga, 68 Ga, 89 Sr, 90 Y, 99m Tc , 111 In , 119} Sb, Any one of ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au, ¹⁹⁹ Au, ²⁰¹ Tl, ²⁰³ Pb, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac and ²²⁷ Th, preferably ⁶⁴ Cu, ⁶⁷ Cu , any one of ¹¹¹ In, ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au and ¹⁹⁹ Au.

In one embodiment of the present invention, the radioisotope in the above-mentioned radionuclide conjugated drug is ¹⁷⁷ Lu.

<The fourth aspect>

The present invention provides a pharmaceutical composition, which contains the radionuclide conjugated drug precursor described in the <first aspect> or the <second aspect> or the radionuclide conjugated drug precursor described in the <third aspect>. combined drugs.

Preferably, the pharmaceutical composition further contains at least one pharmaceutically acceptable excipient.

<Fifth aspect>

The present invention provides the radionuclide-conjugated drug precursor described in the <first aspect> or the <second aspect> or the radionuclide-conjugated drug described in the <third aspect> or the <fourth aspect> Use of the pharmaceutical composition described in for the preparation of medicaments for the prevention and/or treatment of diseases or conditions.

<Sixth aspect>

The present invention provides the radionuclide-conjugated drug precursor described in the <first aspect> or the <second aspect> or the radionuclide-conjugated drug described in the <third aspect> or the <fourth aspect> The pharmaceutical composition described in is used to prevent and/or treat diseases or conditions.

<Seventh aspect>

The present invention provides a method for preventing and/or treating a disease or condition, comprising:

A prophylactically and/or therapeutically effective amount of the radionuclide-conjugated drug precursor described in the <first aspect> or the <second aspect> or the radionuclide-conjugated drug described in the <third aspect> or The pharmaceutical composition described in <Fourth Aspect> is administered to an individual in need thereof.

Preferably, in the <fifth aspect>, <sixth aspect> and/or <seventh aspect>, the disease or condition is cancer, preferably prostate cancer, breast cancer, liver cancer, pancreatic cancer, ovarian cancer, gastric cancer or Lung cancer.

Effect of the invention

The radionuclide conjugated drug of the present invention (or its pharmaceutically acceptable derivative form, such as an addition salt) can specifically deliver radionuclides to target cells. When the conjugated drug contains one targeting ligand (LG), it can specifically deliver radionuclides to target cells; and when the conjugated drug contains two targeting ligands (LG), it can deliver radionuclides to target cells in multiple ways. It works in this way to improve the therapeutic effect and reduce toxic and side effects; at the same time, the existence of two targeting ligands (LG) also enhances the affinity of the conjugated drug to the target cells and reduces off-target toxicity.

Description of the drawings

Figure 1 is the HPLC spectrum of compound 1’.

Figure 2 is the LC-MS spectrum of compound 1’.

Figure 3 is the HPLC spectrum of compound 2’.

Figure 4 is the LC-MS spectrum of compound 2'.

Figure 5 is the HPLC spectrum of compound 4'.

Figure 6 is the LC-MS spectrum of compound 4'.

Figure 7 is the HPLC spectrum of compound 17'.

Figure 8 is the LC-MS spectrum of compound 17'.

Figure 9 is the HPLC spectrum of compound 21'.

Figure 10 is the LC-MS spectrum of compound 21'.

Figure 11 is the HPLC spectrum of compound 24'.

Figure 12 is the LC-MS spectrum of compound 24'.

Figure 13 is a SPECT dynamic image of a double-formed radionuclide conjugated drug prepared from compound 17'.

Figure 14 is a quantitative organ enrichment diagram of a double-formed radionuclide conjugated drug prepared from compound 17'.

Figure 15 is a SPECT dynamic image of a double-formed radionuclide conjugated drug prepared from compound 21'.

Figure 16 is a quantitative organ enrichment diagram of a double-formed radionuclide conjugated drug prepared from compound 21'.

Figure 17 is a SPECT dynamic image of a single radionuclide conjugated drug prepared from compound 1'.

Figure 18 is a quantitative organ enrichment diagram of a monoparticulate radionuclide conjugated drug prepared from compound 1'.

Figure 19 is a SPECT dynamic image of a single radionuclide conjugated drug prepared from compound 2'.

Figure 20 is a quantitative diagram of organ enrichment of monoparticulate radionuclide conjugated drugs prepared from compound 2'.

Figure 21 is a SPECT dynamic image of a single radionuclide conjugated drug prepared from compound 4'.

Figure 22 is a quantitative graph of organ enrichment of monoparticulate radionuclide conjugated drugs prepared from compound 4'.

Detailed ways

General terms and definitions

Unless stated to the contrary, the terms used in the present invention have the following meanings.

In the present invention, "target" or "drug target" refers to the binding site between drugs and biological macromolecules in the body, mainly involving receptors, enzymes, ion channels, transporters, immune systems, genes, etc. Among existing drugs, more than 50% of drugs target receptors, and receptors have become the main and most important targets; more than 20% of drugs target enzymes, especially enzyme inhibitors. It has a special status in clinical application; about 6% of drugs target ion channels; 3% of drugs target nucleic acids; the targets of 20% of drugs still need further research. Specifically, when certain medical treatments (such as radiotherapy) are performed on a subject, radiation is irradiated from different directions and concentrated on the lesion, and the lesion is also a target.

In the present invention, "radioactive nuclide" or "radioactive isotope" refers to an unstable element that can release rays (or radiation) during the decay process. Includes both naturally occurring elements and elements primarily produced during nuclear fission or fusion processes. According to the different rays released during the decay process of radionuclides, they can be roughly divided into three categories: α, β, and γ.

In the present invention, "conjugated drug" or "drug conjugate" refers to fragments with different functions connected to each other through optional connecting fragments. Pharmaceutical compounds formed and able to exert corresponding activities, such as antibody drug conjugates (ADC), peptide drug conjugates (PDC), small molecule drug conjugates (SMDC), radionuclide drug conjugates (RDC), etc.

In the present invention, "radionuclide conjugated drug" or "radionuclide drug conjugate" refers to fragments such as chelating agents containing radionuclides and targeting ligands (Ligand) for at least one target through A pharmaceutically acceptable compound formed by connecting optional linkers and spacers to each other and capable of exerting targeted radiotherapy activity. The chelator part is used to complex radioisotopes and targeting ligands. The linker part and the spacer part are used to connect the chelator part and the targeting ligand part to each other to form a radionuclide couple. The complete structure of the combined drug. In the present invention, "radionuclide conjugate drug precursor" or "radionuclide drug conjugate precursor" means that the chelating agent part in the radionuclide conjugate drug has not yet complexed with the radionuclide. precursor form.

In the present invention, "targeting ligand" refers to any molecule or part capable of targeting to a target site, target tissue, target organ, target cell, or region within a target cell. In some embodiments, the targeting ligand causes the moiety linked to the targeting ligand to be present in the target site, target tissue, or in the non-target tissue as compared to the non-target site, non-target tissue, non-target organ, non-target cell, or non-target intracellular region. , the target organ, the target cell or the area within the target cell is allocated more, for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more. higher. In some embodiments, a conjugate compound or agent with a targeting ligand distributes more to the target site, target tissue, target organ, target cell, or region within the target cell than without the targeting ligand. , for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more. In some embodiments, the targeting ligand can trigger or promote the specific binding of the conjugate compound containing such targeting ligand to the target molecule, trigger or promote the endocytosis of the conjugate compound by the target cell, trigger or Promote the concentration of the conjugate compound around the target cells and/or enter the target cells.

In the present invention, "ligand" may include a variety of chemical molecules or polypeptides that have specific binding affinity for a selected target, which may be a cell surface protein ( For example, cell surface receptors or cell surface antigens), specific proteins, cells, tissues, organs, etc. In some embodiments, a ligand can specifically bind to a cell surface receptor. In some embodiments, a ligand can specifically bind to a cell surface antigen. In some embodiments, a ligand may specifically bind to a specific protein that may be responsible for a disease. In some embodiments, overexpression of the particular protein causes disease or the particular protein is a mutant protein that causes disease. In some embodiments, the ligands of the present application bind to the target with an affinity of 10 ^-6 to 10 ^-11 M (K _d value). In some embodiments, ligands of the present application bind to the target with an affinity of at least 10 ^-6 , at least 10 ^-7 , at least 10 ^-8 or at least 10 ^-9 M (K _d value). In some embodiments, the ligands of the present application bind to the target with a certain affinity, which refers to binding to a non-target (for example, other cell surface receptors, cell surface antigens or specific Compared with the affinity of protein, etc.), the affinity of the ligand for the target is at least two times, three times, four times, five times, six times, eight times, ten times, twenty times, fifty times, A hundred times or more. In some embodiments, the expression of cell surface receptors, cell surface antigens, and specific proteins of the present application on the surface of target cells (for example, cancer cells or cells with physiological abnormality) or within target cells is significantly higher than the expression in normal cells. . The term "significant" as used in this application refers to a statistically significant difference, or a significant difference that can be recognized by a person skilled in the art.

In the present invention, "FOLR1", folate receptor 1, is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein that binds folate with nanomolar affinity, thereby promoting receptor-mediated endocytosis. effect. Rapidly growing solid malignancies, including ovarian and lung cancers, depend on folate for metabolism and nucleic acid synthesis.

In the present invention, "TRPV6" is transient receptor potential cation channel subfamily V member 6 (transient receptor potential cation channel subfamily V member 6), which is a highly selective calcium ion transmembrane transport channel that mediates calcium ions from cells. Active transport from the outside into the cell. TRPV6 is expressed in normal human kidneys, gastrointestinal tract, pancreas, mammary gland, salivary glands, etc., but is mainly expressed in intestinal epithelial cells, where it is involved in the transport of calcium ions into cells. Therefore, when the number or function of TRPV6 channels changes, , can cause changes in calcium ion regulation, further leading to structural or functional abnormalities in related tissues and organs. Compared with normal tissues, the expression of TRPV6 is significantly higher in malignant tumors such as breast cancer, cholangiocarcinoma, ovarian cancer, lung squamous cell carcinoma, and prostate cancer. Its abnormal expression may be related to the formation and progression of tumors.

In the present invention, "PSMA" is prostate-specific membrane antigen, which refers to a type II transmembrane glycoprotein present in the prostate epithelial cell membrane. It consists of 750 amino acids, which has 19 intracellular amino acids, 24 transmembrane amino acids in the membrane region and 707 amino acids in the extracellular region. Prostate-specific membrane antigen is expressed in normal prostate epithelial cells, but its expression levels are much higher in prostate cancer cells. Mutually For prostate-specific antigen, which is traditionally used for clinical detection, prostate-specific membrane antigen is a more sensitive and specific prostate cancer tumor marker. It is especially highly expressed in hormone-refractory prostate cancer and prostate cancer metastases. High sensitivity and specificity in distinguishing prostate cancer from other types of malignancies. At the same time, in a variety of solid tumors of non-prostate origin (such as lung cancer, bladder cancer, gastric cancer, pancreatic cancer, renal cancer, and colorectal cancer, etc.), prostate-specific membrane antigen is also highly specifically expressed on tumor vascular endothelial cells.

In the present invention, "C-MET" is a protein product encoded by the C-MET proto-oncogene. It is a receptor for hepatocyte growth factor (HGF), has tyrosine kinase activity, and interacts with various oncogene products. Related to regulatory proteins, it participates in the regulation of cell information transmission and cytoskeleton rearrangement, and is an important factor in cell proliferation, differentiation and movement. C-MET is closely related to the occurrence and metastasis of various cancers. Studies have shown that many tumor patients have C-MET overexpression and gene amplification during the occurrence and metastasis of their tumors. Because C-MET acts on different substrates in different cells and at different stages of differentiation, it can exhibit multiple functions under specific conditions, such as promoting the division of hepatocytes, endothelial cells and melanocytes, and causing the dispersion and induction of epithelial cells. Cell morphological changes.

In the present invention, "pharmaceutical composition" refers to a pharmaceutically acceptable composition, which contains small molecule drugs as active pharmaceutical ingredients (API), polypeptides, antibodies (or antibody ligands) or conjugates thereof, and other components (such as pharmaceutically acceptable excipients). Pharmaceutical compositions may be prepared using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding and/or lyophilizing processes.

In the present invention, "pharmaceutically acceptable auxiliary materials" refer to auxiliary materials widely used in the field of pharmaceutical production. The main purpose of using excipients is to provide a pharmaceutical composition that is safe to use, stable in nature and/or has specific functionality, and also to provide a method so that after the drug is administered to the subject, the active ingredient can be used in the desired manner. rate dissolution, or promote effective absorption of the active ingredient in the subject to whom it is administered. Pharmaceutically acceptable excipients may be inert fillers or functional ingredients that provide a certain function for the pharmaceutical composition (such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredients in the composition). Examples of "pharmaceutically acceptable excipients" include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, Glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, sweeteners, etc.

The technical solutions of the present invention will be described below with reference to specific examples. The following examples are provided to further illustrate the present invention, but are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the invention without departing from the spirit and scope of the invention.

The starting materials used in the present invention can be synthesized by methods known in the art, or purchased through conventional commercial means. The isolation and purification of the compounds of the present invention can be achieved by methods well known to those skilled in the art, including but not limited to column chromatography (CC), high-performance liquid chromatography (HPLC), ultra-high performance liquid chromatography (UPLC), etc. . The structural identification of the compounds of the present invention can be achieved by methods well known to those skilled in the art, including but not limited to nuclear magnetic resonance (NMR), mass spectrometry (MS), etc.

abbreviation list

Example 1: Preparation of compound 1’

step 1:

Under nitrogen protection, add DMAP (1.8g) and CDI (50g) to a three-necked flask, use an ice bath to control the reaction temperature between -5°C and -10°C, then add DCM (300ml) to the flask, stir and dissolve. Weigh SM1 (40g) and dissolve it in DCM (200ml). Add the dissolved SM1 to a constant pressure separatory funnel. Control the flow rate of the funnel according to the reaction temperature. Slowly drop SM1 into CDI and stir for 5 hours. After the reaction is completed, extract with 10% citric acid aqueous solution (2L) and EA (2L), add silica gel (50.0g) to the organic phase and mix the sample through the column. Under DCM/MeOH (30:1, v/v) conditions, The distillate was monitored by TLC, collected and concentrated to obtain 46.7g of product, namely PSMA-001.

Step 2:

Under nitrogen protection, add SM2 (43.81g) and PSMA-001 (42g) to a single-necked flask, then add DMSO (500ml) and stir to dissolve. Then add DIEA (46.5g) and stir magnetically for 3 hours. After the reaction is completed, extract with water (2L) and EA (2L). Add silica gel (50g) to the organic phase to mix the sample and pass through the column. The product is eluted with ethyl acetate, collected and concentrated to obtain 32.2g of product, namely PSMA-002. Purity 71%.

Step 3:

PSMA-002 (6.00g, 3.0eq) reacted with wang resin (1.0eq), HOBt (3.0eq), DIC (3.8eq) and DMAP (0.6eq) in DMF (7V) system for 1h, sampling, ninhydrin Color development was performed to confirm that the reaction was complete and the product PSMA-003 was obtained.

Step 4:

Use 20% piperidine/DMF to remove Fmoc from PSMA-002, react for 30 minutes, take a sample, and develop color with ninhydrin to confirm that the reaction is complete, and the product PSMA-004 is obtained.

Step 5:

PSMA-004 (1.0eq), Fmoc-2-Nal-OH (3.0eq), HOBt (3.0eq) and DIC (3.8eq) reacted in DMF (7V) system for 1 hour, took samples, developed color with ninhydrin, and confirmed The reaction is complete and product CB6-001 is obtained.

Step 6:

Use 20% piperidine/DMF to remove Fmoc from CB6-001, react for 30 minutes, take a sample, and develop color with ninhydrin to confirm that the reaction is complete. Then react with Fmoc-tranexamic acid (3.0eq), HOBt (3.0eq) and DIC (3.8eq) in a DMF (7V) system for 1 hour, take a sample, develop color with ninhydrin, confirm that the reaction is complete, and obtain the product CB6- 002.

Step 7:

Use 20% piperidine/DMF to remove Fmoc from CB6-002, react for 30 minutes, take a sample, and develop color with ninhydrin to confirm that the reaction is complete. Then react with Fmoc-Cys(Trt)-OH (3.0eq), HOBt (3.0eq) and DIC (3.8eq) in a DMF (7V) system for 1 hour, take a sample, and develop color with ninhydrin to confirm that the reaction is complete. Finally, react with acetic anhydride (10.0eq) and pyridine (10.0eq) in a DMF (7V) system for 2 hours, take a sample, and develop color with ninhydrin to confirm that the reaction is complete, and the product CB6-003 is obtained.

Step 8:

Prepare a lysis solution equivalent to 7V of crude resin (i.e. CB6-003) weight (formula: 90% TFA, 2.5% purified water, 2.5% phenol, 2.5% p-cresolthiophenol, 2.5% TIPS), pre-cooled at 0-5°C Then pour the crude resin into the lysis solution, raise it to 25°C, protect it with nitrogen, stir and cleave for 3 hours, and after the reaction is complete, remove the resin by suction filtration to obtain the lysis solution. Slowly drop into 0°C pre-cooled MTBE (the volume of MTBE is 10V of the volume of the lysis solution), stir to precipitate the solid, centrifuge and settle to obtain the crude product, and prepare and purify (acetonitrile/water: 5% ~ 80%, V/V) to obtain CB6 -004 pure product 100.2mg, off-white powder, purity 87.6%.

Step 9:

2-CTC resin (1.0eq) was swollen with DMF for 2h, then reacted with Fmoc-Lys(Dde)-OH (3.0eq) and DIEA (4.0eq) in DMF (7V) system for 3h, and finally with MeOH (10.0eq) ) and DIEA (10.0eq) reacted for 2 hours to obtain intermediate 1-1.

Step 10:

Use 20% piperidine/DMF to remove Fmoc in intermediate 1-1, react for 30 minutes, take a sample, and develop color with ninhydrin to confirm that the reaction is complete. Then react with Fmoc-Val-OH (3.0eq), HOBt (3.0eq) and DIC (3.8eq) in DMF (7V) system for 1 hour, take a sample, develop color with ninhydrin, confirm that the reaction is complete, and obtain intermediate 1- 2.

Step 11:

Use 20% piperidine/DMF to remove Fmoc in intermediate 1-2, react for 30 minutes, take a sample, and develop color with ninhydrin to confirm that the reaction is complete. Then react with Boc-Met-OH (3.0eq), HOBt (3.0eq) and DIC (3.8eq) in DMF (7V) system for 1 hour, take a sample, develop color with ninhydrin, confirm that the reaction is complete, and obtain intermediate 1- 3.

Step 12:

Use 2% hydrazine/DMF (7V) to remove Dde from intermediate 1-3, react for 30 minutes, take a sample, and develop color with ninhydrin to confirm that the reaction is complete to obtain intermediate 1-4.

Step 13:

Prepare a lysis solution (formula: 30% TFE/DCM) equivalent to 7V of the crude resin (i.e., intermediate 1-4), pre-cool the lysis solution at 0-5°C, add the crude resin to the lysis solution, and raise it to 25°C. Under nitrogen protection, stir and lyse for 3 hours. After the control reaction is complete, the resin is removed by suction filtration to obtain a lysis solution. Concentrate under reduced pressure at 30°C to remove DCM in the lysate. Pre-cool the lysate concentrate in MTBE with a volume of 10V to 0°C, slowly drop the lysate concentrate into it, stir to precipitate the solid, and centrifuge to settle to obtain crude intermediate 1-5.

Step 14:

Add intermediate 1-5 (4.4710mM, 1.0eq) and saturated NaHCO ₃ aqueous solution (40ml) to a 100ml single-neck jacketed bottle, stir evenly, protect with nitrogen, cool the circulating bath to 4.0°C, add NMCM (4.4710mM, 1.0 eq), the reaction was stirred for 2 hours, and HPLC sampling showed that the reaction was complete. At 4°C, add DCM (40ml) to the reaction solution, drop in saturated citric acid aqueous solution, adjust the pH to acidic, separate the liquids, re-extract the water phase with DCM, separate the liquids, combine the DCM phases, and concentrate under reduced pressure to obtain an off-white color. 1.80g of solid was purified by column (DCM/MeOH=10/1, V/V) to obtain 1.10g of off-white solid, which is intermediate 1-6.

Step 15:

Under ice-water bath conditions, add 4M HCl/dioxane (11ml) to the reaction bottle, then add intermediate 1-6 (1.10g, 1.9760mM), under nitrogen protection, stir the reaction for 2 hours, and control the reaction to be complete by HPLC. Concentrate under reduced pressure at 30°C, then add MTBE to beat, and centrifuge to settle to obtain crude intermediate 1-7.

Step 16:

Add DOTA-COOH (10.00g, 1.01eq), 4-aminomethyl-N-Boc-aniline (3.80g, 1.0eq), HATU (9.80g, 1.5eq), DIEA (6.60g, 3.0eq) and DMF (100ml), stir at 25-30°C for 1-2h. After the TLC monitoring reaction is completed, add EA (10ml) and water (25ml), stir for 2 minutes, let stand and separate the layers, separate the organic phase, extract the aqueous layer once with EA, combine the organic phases, dry over anhydrous sodium sulfate, concentrate, and column Purify (DCM/MeOH=10/1, V/V) to obtain 13.0 g of white solid, which is intermediate 2-1.

Step 17:

Add intermediate 2-1 (10.00g, 1.0eq), DCM (60mL) and TES (18mL) to a 500mL three-necked flask. Cool the temperature to 0-5°C, add TFA (120 mL) dropwise, bring it to room temperature after the addition, and stir for 3-5 hours. After the reaction was monitored by HPLC, the solvent was concentrated and removed. The oil obtained by concentration was added to MTBE to beat, filtered, and washed with MTBE to obtain 5.2 g of white solid, namely intermediate 2-2.

Step 18:

Add intermediate 2-2 (4.00g, 1.0eq) and water (60ml) to a 250mL three-necked flask, and add dropwise a solution of CSCl ₂ (13.50g, 15.0eq) in chloroform (60ml) with stirring. After the dropwise addition is completed, stir at room temperature for 1-2 hours. After the HPLC control reaction was completed, the mixture was allowed to stand for liquid separation. The aqueous phase was washed twice with methylene chloride, combined and concentrated to obtain 2.88g of yellow solid, which is intermediate 2-3.

Step 19:

Add intermediate 2-3 (1.7g, 1.6eq), intermediate 1-7 (1.0g, 1.0eq) and DMF (20ml) to a 100mL single-neck bottle, cool down in an ice bath, add TEA (1.2ml), and raise to room temperature Stir for 1.0h. After the reaction is completed, the reaction solution is added dropwise to MTBE (200 ml) to make a slurry, and filtered to obtain 2.7 g of white solid, namely DOTA-Met-Val-Lys(Mal)-OH, with a purity of 92%.

Step 20:

Add CB6-004 (58mg, 1.0eq) and DMF (2ml) to the 25mL jacketed bottle, stir and dissolve, then add DIEA (2ml), control the pH to 8-9, then add DOTA-Met-Val-Lys ( Mal)-OH (218.9 mg, 3.0 eq), after nitrogen replacement three times, the reaction was stirred at 25°C for 1 hour. After the reaction was monitored by HPLC, the reaction solution was purified by preparative chromatography (acetonitrile/water: 5% to 80%, V/V) to obtain 77.8 mg of white solid, namely compound 1', with a purity of 97.07%.

LC-MS(ESI ⁺ ): m/z 1808.1[M+H] ⁺ ,904.9[M+2H] ⁺ /2.

Example 2: Preparation of compound 2’

A similar method to Example 1 was adopted, but in step 5, Fmoc-L-3,3-diphenylalanine was used instead of Fmoc-2-Nal-OH. The reaction solution was purified by preparative chromatography to obtain a white solid 172.9 mg, namely compound 2', purity 90.94%.

LC-MS(ESI ⁺ ): m/z 1834.2[M+H] ⁺ ,918.1[M+2H] ⁺ /2.

Example 3: Preparation of compound 3’

Steps 1-11 are similar to steps 9-19 in Example 1 to obtain DOTA-Met-Val-Lys(Mal)-OH.

Step 12:

2-CTC-Resin (1.0eq) was reacted with Fmoc-Cys(Trt)-OH (3.0eq) and DIEA (4.0eq) in DMF (7V) system for 3 hours, and then with MeOH (10.0eq) and DIEA (10.0 eq) reacted for 2 hours to obtain intermediate 3-1.

Step 13:

Use 20% piperidine/DMF (7V) to remove Fmoc in intermediate 3-1, react for 30 minutes, take a sample, develop color with ninhydrin, and confirm the reaction. should be completely. Then react with intermediate 3-2 (3.0eq), HOBt (3.0eq) and DIC (3.8eq) in DMF (7V) system for 1 hour and take samples. Ninhydrin develops color to confirm that the reaction is complete, and intermediate 3-3 is obtained. .

Step 14:

Prepare a lysis solution equivalent to 7V of crude resin (i.e., intermediate 3-3) weight (formula: 90% TFA, 2.5% purified water, 2.5% phenol, 2.5% p-cresolthiophenol, 2.5% TIPS), 0-5°C Pre-cool the pyrolysis solution, then pour the crude resin into the pyrolysis solution, raise it to 25°C, protect it with nitrogen, stir and pyrolyze it for 3 hours, and after the reaction is complete, remove the resin by suction filtration to obtain the pyrolysis solution. Slowly drop it into 0°C pre-cooled MTBE (the volume of MTBE is 10V of the volume of the lysis solution), stir to precipitate the solid, and centrifuge to settle to obtain 0.9g of Cys-108 crude product.

Step 15 is similar to step 20 in Example 1, but Cys-108 is used instead of CB6-004. The reaction solution is purified by preparative chromatography to obtain 137 mg of white solid, namely compound 3', with a purity of 96.62%.

LC-MS(ESI ^- ): m/z 1618.8[MH] ^- ,809.7[M-2H] ^- /2.

Example 4: Preparation of compound 4’

step 1:

Swell the wang resin (3g, 1.0eq) with DMF, and then mix it with Fmoc-Lys(Dde)-OH (3.0eq), HOBt (3.0eq), DIC (3.8eq) and DMAP (0.6eq) in DMF (7V) React in the system for 3 hours, wash with DMF, then react with acetic anhydride (10.0eq) and pyridine (10.0eq) in a DMF (7V) system for 2 hours, wash with DMF, and obtain intermediate 4-1.

Step 2:

Use 20% piperidine/DMF to remove Fmoc in intermediate 4-1, react for 30 minutes, take a sample, and develop color with ninhydrin to confirm that the reaction is complete. Then react with DOTA-COOH (3.0eq), HBTU (3.0eq), HOBt (1.5eq) and DIEA (4.5eq) in a DMF (7V) system for 1 hour, take a sample, develop color with ninhydrin, and confirm that the reaction is complete, we get Intermediate 4-2.

Step 3:

Use 2% hydrazine/DMF to remove Dde from intermediate 4-2, react for 30 minutes, take a sample, and develop color with ninhydrin to confirm that the reaction is complete. Then react with intermediate 3-2 (3.0eq), HOBt (3.0eq) and DIC (3.8eq) in DMF (7V) system for 1 hour and take samples. Ninhydrin develops color to confirm that the reaction is complete, and intermediate 4-3 is obtained. .

Step 4:

Prepare a lysis solution equivalent to 7V of crude resin (i.e. intermediate 4-3) weight (formula: 90% TFA, 2.5% purified water, 2.5% phenol, 2.5% p-cresolthiophenol, 2.5% TIPS), 0-5°C Pre-cool the pyrolysis solution, then pour the crude resin into the pyrolysis solution, raise it to 25°C, protect it with nitrogen, stir and pyrolyze it for 3 hours, and after the reaction is complete, remove the resin by suction filtration to obtain the pyrolysis solution. Slowly drop into 0°C pre-cooled MTBE (the volume of MTBE is 10V of the volume of the lysate), stir to precipitate the solid, centrifuge and settle to obtain the crude product, which is purified by preparative chromatography to obtain 1109.4 mg of white solid, namely compound 4', with a purity of 99.38%.

LC-MS(ESI ⁺ ): m/z 1025.8[M+H] ⁺ ,513.3[M+2H] ⁺ /2.

Example 5: Preparation of compound 5’

Steps 1-6 were similar to steps 1-6 in Example 2 to obtain CB2-002.

Step 7:

Use 20% piperidine/DMF to remove Fmoc from CB2-002, react for 30 minutes, take a sample, and develop color with ninhydrin to confirm that the reaction is complete. Then react with DOTA-(COOH) ₄ (3.0eq), HBTU (3.0eq), HOBt (1.5eq) and DIEA (4.5eq) in DMF (7V) system for 1 hour, take a sample, develop color with ninhydrin, and confirm the reaction Completely, get CB2-003.

Step 8:

Prepare a lysis solution equivalent to 7V of crude resin (i.e. CB2-003) weight (formula: 90% TFA, 2.5% purified water, 2.5% phenol, 2.5% p-cresolthiophenol, 2.5% TIPS), pre-cooled at 0-5°C Then pour the crude resin into the lysis solution, raise it to 25°C, protect it with nitrogen, stir and cleave for 3 hours, and after the reaction is complete, remove the resin by suction filtration to obtain the lysis solution. Slowly drop into 0°C pre-cooled MTBE (the volume of MTBE is 10V of the volume of the lysate), stir to precipitate the solid, centrifuge and settle to obtain the crude product, which is purified by preparative chromatography to obtain 240.7 mg of pure product, namely compound 5’, with a purity of 99.14%.

LC-MS(ESI ⁺ ): m/z 1068.8[M+H] ⁺ ,535.0[M+2H] ⁺ /2.

Example 6: Preparation of compound 7’

Steps 1-11 are similar to steps 9-19 in Example 1 to obtain DOTA-Met-Val-Lys(Mal)-OH.

Step 12:

Add CB9-001 (200mg, 1.0eq, obtained by a method similar to the preparation method of CB10-001 in Example 8) and DMF (2ml) into a 25mL jacketed bottle, stir and dissolve, then add DIEA (2ml), control The pH was 8-9, then DOTA-Met-Val-Lys(Mal)-OH (185.19 mg, 1.4eq) was added, and after nitrogen replacement three times, the reaction was stirred at 25°C for 1 hour. After the HPLC monitoring reaction was completed, the reaction solution was purified by preparative chromatography to obtain 128.4 mg of yellow solid, namely compound 7', with a purity of 95.95%.

LC-MS(ESI ⁺ ): m/z 2529.7[M+H] ⁺ ,1265.3[M+2H] ⁺ /2.

Example 7: Preparation of compound 8’

step 1:

Use 20% piperidine/DMF to remove TRPV6 resin (the resin is sequentially connected to the resin with Arg, Pro, Leu, Asp, Val, Lys, Ser, Pro, His, Leu, Phe, Fmoc in Glu and Lys and introducing Fmoc at the N-terminus), react for 30 minutes, take a sample, and develop color with ninhydrin to confirm that the reaction is complete. Then react with DOTA-COOH (3.0eq), HBTU (3.0eq), HOBt (1.5eq) and DIEA (4.5eq) in a DMF (7V) system for 1 hour, take a sample, develop color with ninhydrin, and confirm that the reaction is complete, we get Intermediate 8-1.

Step 2:

Prepare a lysis solution equivalent to 7V of crude resin (i.e., intermediate 8-1) weight (formula: 90% TFA, 2.5% purified water, 2.5% phenol, 2.5% p-cresolthiophenol, 2.5% TIPS), 0-5°C Pre-cool the pyrolysis solution, then pour the crude resin into the pyrolysis solution, raise it to 25°C, protect it with nitrogen, stir and pyrolyze it for 3 hours, and after the reaction is complete, remove the resin by suction filtration to obtain the pyrolysis solution. Slowly drop into 0°C pre-cooled MTBE (the volume of MTBE is 10V of the volume of the lysate), stir to precipitate the solid, centrifuge and settle to obtain the crude product, which is purified by preparative chromatography to obtain 51.6 mg of pure product, namely compound 8', with a purity of 98.27%.

LC-MS(ESI ⁺ ): m/z 1952.3[M+H] ⁺ ,977.2[M+2H] ⁺ /2.

Example 8: Preparation of compound 9’

Steps 1-11 are similar to steps 9-19 in Example 1 to obtain DOTA-Met-Val-Lys(Mal)-OH.

Step 12:

Load the resin into the solid-phase reaction column, add DMF, bubble nitrogen into the solvent, and swell the resin for 30 minutes; add Fmoc-Arg-OH, DCC (dicyclohexylcarbodiimide) and DMAP at 25°C. React for 3 hours, then cap it with acetic anhydride and pyridine for 1 hour, then wash with DMF 3 times, use DBLK to remove the Fmoc protecting group on the resin, then wash with DMF 5 times, weigh Fmoc-Pro-OH, HOBt, and use Dissolve DMF, add DIC to the above solution in an ice-water bath at 0°C, mix and activate for 5 minutes, add the solution to the above reaction column, and after 3 hours of reaction, drain the solvent and wash the resin in the reaction column three times. Then use DBLK to remove the Fmoc protecting group. Repeat the above operation and couple Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)OH, Fmoc- in sequence according to the structure. Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Glu-OtBu and Pteroic acid. After the resin is lysed with lysis solution, it is filtered, and the lysis solution is poured into MTBE to precipitate the solid, which is washed with MTBE to obtain a crude product. The crude product is prepared by Pre-HPLC to obtain CB10-001.

Add CB10-001 (200mg, 1.0eq) and DMF (2ml) into a 25mL jacketed bottle, stir and dissolve, then add DIEA (2ml), control the pH to 8-9, then add DOTA-Met-Val-Lys ( Mal)-OH (134.76 mg, 1.4 eq), after nitrogen replacement three times, the reaction was stirred at 25°C for 1 hour. After the HPLC monitoring reaction was completed, the reaction solution was purified by preparative chromatography to obtain 142.2 mg of yellow solid, namely compound 9', with a purity of 97.38%.

LC-MS(ESI ⁺ ): m/z 3099.5[M+H] ⁺ ,1034.4[M+3H] ⁺ /3.

Example 9: Preparation of compound 10'

step 1:

Use 20% piperidine/DMF to remove Fmoc from TRPV6 resin, take samples after 30 minutes of reaction, and develop color with ninhydrin to confirm that the reaction is complete. Then react with Cys-108 (3.0eq), HOBt (3.0eq) and DIC (3.8eq) in a DMF (7V) system for 1 hour, take a sample, develop color with ninhydrin, confirm that the reaction is complete, and obtain intermediate 10-1.

Step 2:

Prepare a lysis solution equivalent to 7V of crude resin (i.e. intermediate 10-1) weight (formula: 92.5% TFA, 2.5% purified water, 2.5% phenol, 2.5% p-cresolthiophenol, 2.5% TIPS), 0-5°C Pre-cool the pyrolysis solution, then pour the crude resin into the pyrolysis solution, raise it to 25°C, protect it with nitrogen, stir and pyrolyze it for 3 hours, and after the reaction is complete, remove the resin by suction filtration to obtain the pyrolysis solution. Slowly drop it into 0°C pre-cooled MTBE (the volume of MTBE is 10V of the volume of the lysis solution), stir to precipitate the solid, centrifuge and settle to obtain the crude product, which is preparative and purified to obtain CB11-001.

Steps 3-13 are similar to steps 9-19 in Example 1 to obtain DOTA-Met-Val-Lys(Mal)-OH.

Step 14:

Add CB11-001 (200mg, 1.0eq) and phosphate buffer (2ml) with pH=7.4 into a 25mL jacketed bottle. After stirring to dissolve, add 0.15M disodium hydrogen phosphate aqueous solution to control the pH to 8-9. , then add DOTA-Met-Val-Lys(Mal)-OH (186.35 mg, 2.0 eq), replace with nitrogen 3 times, and stir the reaction at 25°C for 1 hour. After the HPLC monitoring reaction was completed, the reaction solution was purified by preparative chromatography to obtain 111.5 mg of white solid, namely compound 10', with a purity of 95.00%.

LC-MS(ESI ⁺ ): 3168.7[M+H] ⁺ ,793.6[M+4H] ⁺ /4.

Example 10: Preparation of compound 11'

step 1:

Use 20% piperidine/DMF to remove Fmoc from TRPV6 resin, react for 30 minutes, take a sample, and develop color with ninhydrin to confirm that the reaction is complete. Then react with Fmoc-Lys(Dde)-OH (3.0eq), HOBt (3.0eq) and DIC (3.8eq) in DMF (7V) system for 1 hour, take a sample, ninhydrin develops color, confirm that the reaction is complete, and get the intermediate Body 11-1.

Step 2:

Use 20% piperidine/DMF to remove Fmoc in intermediate 11-1, take a sample after 30 minutes of reaction, and develop color with ninhydrin to confirm that the reaction is complete. Then react with 108 (3.0eq), HOBt (3.0eq) and DIC (3.8eq) in a DMF (7V) system for 1 hour, take a sample, and develop color with ninhydrin to confirm that the reaction is complete, and intermediate 11-2 is obtained.

Step 3:

Use 2% hydrazine/DMF to remove Dde from intermediate 11-2, react for 30 minutes, take a sample, and develop color with ninhydrin to confirm that the reaction is complete. Then react with DOTA-COOH (3.0eq), HBTU (3.0eq), HOBt (1.5eq) and DIEA (4.5eq) in a DMF (7V) system for 1 hour, take a sample, develop color with ninhydrin, and confirm that the reaction is complete, we get Intermediate 11-3.

Step 4:

Prepare a lysis solution equivalent to 7V of crude resin (i.e., intermediate 11-3) weight (formula: 90% TFA, 2.5% purified water, 2.5% phenol, 2.5% p-cresolthiophenol, 2.5% TIPS), 0-5°C Pre-cool the pyrolysis solution, then pour the crude resin into the pyrolysis solution, raise it to 25°C, protect it with nitrogen, stir and pyrolyze it for 3 hours, and after the reaction is complete, remove the resin by suction filtration to obtain the pyrolysis solution. Slowly drop into 0°C pre-cooled MTBE (the volume of MTBE is 10V of the volume of the lysis solution), stir to precipitate the solid, and centrifuge to settle to obtain the crude product. After purification by preparative chromatography, 222.2 mg of the pure product is obtained, namely compound 11', with a purity of 93.975%.

LC-MS(ESI ⁺ ): m/z 2572.91[M+H] ⁺ ,644.06[M+4H] ⁺ /4.

Example 11: Preparation of compound 12'

step 1:

Add DOTA-COOH (1g, 1.0eq) and acetonitrile (20mL) to a 100mL jacketed bottle, stir to dissolve, then add N-hydroxysuccinimide (0.3014g, 1.5eq) and HBTU (0.9936g, 1.5eq) ), after nitrogen replacement three times, the reaction was stirred at 25°C for 2 hours. After the TLC monitoring reaction is completed, add 10% citric acid aqueous solution (10ml) and water (100ml) to the reaction solution, then extract with DCM (100mL*3), combine the organic phases, wash with saturated brine, and dry over anhydrous sodium sulfate. Filter, and the filtrate is concentrated under reduced pressure in a 35°C water bath to obtain 1.34 g of white solid, which is intermediate 12-1.

Step 2:

Add 2-(2-(2-aminoethoxy)ethoxy)acetic acid (0.3917g, 1.2eq) and saturated _NaHCO water to a 25mL jacketed flask solution (3.5ml), control the pH to 8-9, then add a solution of intermediate 12-1 (1.34g, 1.0eq) in DME (3.5ml) and THF (3.5ml), after nitrogen replacement 3 times, The reaction was stirred at 25°C for 2 h. After the reaction is monitored by TLC, pour the reaction solution into water (50ml), then extract with DCM (50mL*3), combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and store the filtrate in a 35°C water bath Concentrate under reduced pressure to obtain 1.06 g of oily solid, namely intermediate 12-2.

Step 3:

To the 25mL jacketed bottle, add intermediate 12-2 (1.06g, 1.0eq), N-Boc-ethylenediamine (0.2990g, 1.5eq), DMF (10ml), HATU (0.7095g, 1.5eq) and DIEA (0.4823g, 3.0eq), after nitrogen replacement three times, the reaction was stirred at 25°C for 2 hours. After the reaction is monitored by HPLC, pour the reaction solution into water (100ml), and then extract with DCM (100mL*3). Combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and store the filtrate in a 35°C water bath Concentrate under reduced pressure, stir the sample, and purify by column chromatography (gradient elution, DCM:MeOH=50:1 to 10:1) to obtain 310 mg of white solid, which is intermediate 12-3.

Step 4:

Add intermediate 12-3 (310 mg, 1.0 eq) and DCM (10 ml) to a 100 mL single-neck bottle. After stirring to dissolve, add TFA (20 ml), TES (3 ml) and purified water (3 ml), and stir at 25°C. Reaction 2h. After the reaction is monitored by HPLC, the reaction solution is concentrated under reduced pressure in a 25°C water bath to nearly dryness, then methanol (0.5ml) is added to dissolve, and added dropwise to MTBE (20ml) and stirred for 0.5h. The precipitated solid is filtered and dried under reduced pressure. , 200 mg of white solid was obtained, namely intermediate 12-4.

Step 5:

Add intermediate 12-5-1 (200mg), N-hydroxysuccinimide (155mg) and DMF (2mL) in sequence to a 25mL jacketed bottle. After stirring and dissolving, add EDCI (342mg) and replace with nitrogen for 3 seconds. After several times, the reaction was stirred at 25°C for 2 h. After the reaction is monitored by TLC, pour the reaction solution into water (30mL), then extract with DCM (30mL*3), combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and store the filtrate in a 35°C water bath Concentrate under reduced pressure to obtain 400 mg of yellow oily solid, namely intermediate 12-5.

Add intermediate 12-4 (200mg, 1.0eq) and saturated NaHCO ₃ aqueous solution (3ml) into a 50mL single-neck bottle, control the pH to 8-9, then add intermediate 12-5 (275.07mg, 1.5eq) in DME (3 ml) and THF (3 ml), after nitrogen replacement three times, the reaction was stirred at 25°C for 2 h. After the reaction was monitored by HPLC, the reaction solution was purified by preparative chromatography to obtain 114 mg of oily solid, namely compound 12', with a purity of 95.39%.

LC-MS(ESI ⁺ ): m/z 1019.6[M+H] ⁺ ,510.7[M+2H] ⁺ /2.

Example 12: Preparation of compound 13’

Steps 1-11 are similar to steps 9-19 in Example 1 to obtain DOTA-Met-Val-Lys(Mal)-OH.

Step 12:

Add S-trityl-L-cysteine (200mg, 1.0eq) and saturated NaHCO ₃ aqueous solution (2mL) to a 25mL jacketed bottle, control the pH to 8-9, and then add intermediate 12-5 (448.19mg, 1.5eq) in DME (2ml) and THF (2ml), after nitrogen replacement three times, the reaction was stirred at 25°C for 2h. After the reaction is monitored by HPLC, pour the reaction solution into water (30ml), then extract with DCM (30mL*3), combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and store the filtrate in a 35°C water bath Concentrate under reduced pressure to obtain 580 mg of crude intermediate 13-1.

Step 13:

Add intermediate 13-1 (580 mg, 1.0 eq) and DCM (15 ml) to a 100 mL single-neck bottle, stir and dissolve, then add TFA (15 ml) and TES (283.50 mg, 3.33 eq), and stir for 2 hours at 25°C. . After the reaction was monitored by HPLC, the reaction solution was concentrated under reduced pressure in a 25°C water bath to nearly dryness. MTBE (20 ml) was added and stirred for 0.5 h. The precipitated solid was filtered and concentrated under reduced pressure to obtain 306 mg of crude intermediate 13-2.

Step 14:

Add intermediate 13-2 (300mg, 1.0eq) and DMF (2ml) to a 25mL jacketed bottle, stir and dissolve, add DIEA, control the pH to 8-9, and then add DOTA-Met-Val-Lys (Mal) -OH (991 mg, 1.8 eq), after nitrogen replacement three times, the reaction was stirred at 25°C for 1 hour. After the HPLC monitoring reaction was completed, the reaction solution was purified by preparative chromatography to obtain 140.5 mg of orange-red solid, namely compound 13', with a purity of 97.00%.

LC-MS (ESI ⁺ ): m/z 1554.6[M+H] ⁺ ,779.3[M+2H] ⁺ /2.

Example 13: Preparation of compound 14'

Take CR211D6 (50.0mg, 1.0eq, for the preparation method, see Nature Medicine., 2015, vol.21, pp.955-961), dissolve it in DMF (1ml), and add DOTA-OSu-(COOH) ₃ (18.0mg, 2.0 eq), then add DIEA, control the pH to 9, and stir the reaction at room temperature for 3 hours. HPLC detected that the reaction was complete, and after preparative liquid phase purification, 42 mg of the product, namely compound 14', was obtained with a purity of 87.73%.

LC-MS(ESI ⁺ ): m/z 3167.3[M+H] ⁺ ,1584.2[M+2H] ⁺ /2.

Example 14: Preparation of compound 15’

step 1:

Add DOTA-COOH (1000.0mg, 1.0eq) into a 100ml single-mouth bottle, then add N-hydroxysuccinimide (226.0mg, 1.1eq), HBTU (740.0mg, 1.1eq) and acetonitrile (20ml), Stir at room temperature overnight. Add 5% citric acid aqueous solution to quench reaction, and then added DCM for extraction. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 0.87g of white waxy substance, namely DOTA-OSu.

Step 2:

Add intermediate 15-1 (100.0 mg, 1.0 eq) into a 100 ml single-neck bottle, then add DOTA-OSu (276.0 mg, 1.5 eq) and acetonitrile (10 ml). After stirring and dissolving, add DIEA (150 μl) dropwise in an ice bath. ), control the pH to 9, remove the ice bath, and stir at room temperature overnight. Add 5% citric acid aqueous solution to quench the reaction, and then add DCM for extraction. The organic phase is dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 0.272 mg of a transparent oil, which is intermediate 15-2.

Step 3:

Add intermediate 15-2 (100.0 mg, 1.0 eq) into a 100 ml single-neck bottle, then add DCM (5 ml) and TFA (5 ml), add TES (0.2 ml), and stir at room temperature overnight. After HPLC detects that the reaction is complete, the reaction solution is concentrated under reduced pressure to nearly dryness, and is added dropwise to MTBE. The supernatant is removed by centrifugation. The solid is purified by preparative chromatography to obtain 30 mg of product, namely compound 15', with a purity of 95.60%.

LC-MS(ESI ⁺ ): m/z 756.28[M+H] ⁺ .

Example 15: Preparation of compound 16'

Steps 1-3 are similar to steps 16-18 in Example 1 to obtain intermediate 2-3.

Step 4:

Add intermediate 15-1 (1.00g, 1.0eq), 6-Fmoc aminocaproic acid (0.96g, 1.05eq), HOBT (0.52g, 1.5eq), and EDCI (0.74g, 1.5eq) to the 50mL three-necked flask in sequence. ), DMAP (0.032g, 0.1eq), DIEA (1.00g, 3.0eq) and DMF (10V), stir at room temperature for 2-3h. After the HPLC monitoring reaction was completed, the reaction solution was purified by preparative chromatography to obtain 1.01g of white solid, namely CB3-001.

Step 5:

Add CB3-001 (1.00g, 1.0eq), DMF (3.2ml) and piperidine (0.8ml) to a 50mL three-necked flask, and stir at room temperature for 30min. After the reaction was monitored by HPLC, the reaction solution was dropped into MTBE (40 ml), and centrifuged to obtain 600 mg of white solid, namely CB3-002.

Step 6:

Add CB3-002 (393mg, 1.0eq), intermediate 2-3 (717mg, 1.6eq), DIEA (632mg, 6.0eq) and DMF (5ml) to a 50mL three-necked flask, and stir at room temperature for 2-3h. After the HPLC monitoring reaction was completed, the reaction solution was purified by preparative chromatography to obtain 458.6 mg of white solid, namely compound 16', with a purity of 95.88%.

LC-MS(ESI ⁺ ): m/z 1033.7[M+H] ⁺ .

Example 16: Preparation of compound 17'

Steps 1-6 are similar to steps 1-6 in Example 1 to obtain CB6-002.

Step 7:

Use 20% piperidine/DMF to remove Fmoc from CB6-002, react for 30 minutes, take a sample, and develop color with ninhydrin to confirm that the reaction is complete. Then react with Fmoc-Lys(Boc)-OH (3.0eq), HOBt (3.0eq) and DIC (3.8eq) in DMF (10V) system for 1 hour, take a sample, ninhydrin develops color, confirm that the reaction is complete, and get the intermediate Body 17-1.

Step 8:

Use 20% piperidine/DMF to remove Fmoc in intermediate 17-1, react for 30 minutes, take a sample, and develop color with ninhydrin to confirm that the reaction is complete. Then react with Fmoc-aminocaproic acid (3.0eq), HOBt (3.0eq) and DIC (3.8eq) in a DMF (10V) system for 1 hour, take a sample, develop color with ninhydrin, confirm that the reaction is complete, and obtain intermediate 17- 2.

Step 9:

Use 20% piperidine/DMF to remove Fmoc in intermediate 17-2, react for 30 minutes, take a sample, and develop color with ninhydrin to confirm that the reaction is complete. Then react with DOTA-COOH (3.0eq), HBTU (3.0eq), HOBt (3.0eq) and DIEA (6.0eq) in a DMF (10V) system for 2 hours, take a sample, develop color with ninhydrin, and confirm that the reaction is complete, we get Intermediate 17-3.

Step 10:

Prepare a pyrolysis solution (formula: 90% TFA/DCM) equivalent to 10V of the weight of the crude resin (i.e., intermediate 17-3), pre-cool the pyrolysis solution at 0-5°C, add the crude resin to the pyrolysis solution, and raise it to 25°C. Under nitrogen protection, stir and lyse for 3 hours. After the control reaction is complete, the resin is removed by suction filtration to obtain a lysis solution. Concentrate under reduced pressure at 30°C to remove DCM in the lysate. Pre-cool MTBE with 10 times the volume of the cleavage concentrate to 0°C, slowly drop the cleavage concentrate into it, stir to precipitate the solid, centrifuge and settle to obtain the crude product, which is purified by preparative chromatography to obtain 903 mg of the product, which is intermediate 17-4.

Step 11:

Take intermediate 12-5 (100 mg, 1.0 eq) and intermediate 17-4 (237 mg, 1.0 eq), add DIEA to adjust the pH of the reaction solution to 9, and stir the reaction with magnetic stirring at room temperature for 2 hours. After the HPLC monitoring reaction was completed, the reaction solution was purified by preparative chromatography to obtain 70.9 mg of product, namely compound 17', with a purity of 98.87%.

LC-MS(ESI ⁺ ): m/z 1710.83[M+H] ⁺ ,427.3[M+4H] ⁺ /4.

Example 17: Preparation of compound 18'

step 1:

Add intermediate 18-1 (202.0mg, 1.0eq) into a 25ml single-neck bottle, add DMF (3ml) to dissolve, then add EDCI (234.0mg, 4.0eq), stir magnetically, and then add N-hydroxysuccinyl Imine (50.0mg, 1.2eq), react at room temperature for 2h. After HPLC monitors that the reaction is complete, the reaction solution is added dropwise to MTBE. After the solid precipitates, the supernatant is removed by centrifugation, and the solid is filtered and dried to obtain 243.1 mg of product, which is intermediate 18-2.

Step 2:

Add intermediate 18-2 (100.0mg, 1.0eq) and intermediate 17-4 (169.3mg, 1.0eq) to a 25ml single-neck bottle, then add DMF (5ml) to dissolve, stir magnetically at room temperature, add DIEA dropwise to complete the reaction The pH of the liquid was adjusted to 9, and the reaction was carried out overnight. HPLC monitors that the reaction is complete, and the reaction solution is added dropwise to MTBE. After the solid is precipitated, the supernatant is removed by centrifugation, and the solid is dried to obtain 245.3 mg of the product, which is intermediate 18-3.

Step 3:

Add intermediate 18-3 (245.3 mg) to a 25 ml single-neck bottle, then add 50% TFA/DCM mixed solvent (5 ml), and react with magnetic stirring at room temperature for 1 hour. HPLC controlled the reaction to be complete. The reaction solution was concentrated under reduced pressure and purified by preparative chromatography to obtain 130 mg of product, namely compound 18', with a purity of 98.09%.

LC-MS(ESI ⁺ ): m/z 1869.93[M+H] ⁺ ,936.0[M+2H] ⁺ /2.

Example 18: Preparation of compound 19’

step 1:

Intermediate 19-1 was synthesized according to the method described in the literature (DOI: 10.1002/cmdc.201500600), and 1.23 g of light yellow solid was obtained.

Step 2:

Add intermediate 19-1 (279.1mg, 2.0eq) and intermediate 17-4 (169.3mg, 1.0eq) to a 25ml single-neck bottle, then add DMF (5ml) to dissolve, stir magnetically at room temperature, and then add DIEA dropwise. The pH of the reaction solution was adjusted to 9, and the reaction was carried out overnight. LCMS was used to control the complete reaction of the raw materials. The reaction solution was added dropwise to MTBE. After the solid precipitated, the supernatant was removed by centrifugation and the solid was dried to obtain 273 mg of the product, namely intermediate 19-2.

Step 3:

Add intermediate 19-2 (273 mg) to a 25 ml single-neck bottle, then add 5 ml of TFA, and react with magnetic stirring at room temperature for 3 hours. HPLC controlled the reaction to be complete. The reaction solution was concentrated under reduced pressure and purified by preparative chromatography to obtain 140 mg of product, compound 19', with a purity of 82.29%.

LC-MS(ESI ⁺ ): m/z 2096.10[M+H] ⁺ ,524.86[M+4H] ⁺ /4.

Example 19: Preparation of compound 20'

step 1:

Add Cl-CTC-resin (1.09mmol/g, 10.0g, 1.0eq) to DCM (250ml), stir mechanically for 2h to fully swell, and add Fmoc-Lys(Boc)-OH (16.4g, 3.0eq) , then add DIEA (4.23g, 3.0eq), react at room temperature for 3 hours, transfer to the solid-phase synthesis reaction kettle, drain the solvent, add DMF (100ml) and wash 2 times, drain the solvent, and add 20% piperidine/ DMF solution (100ml), nitrogen bubbling reaction for 0.5h, ninhydrin develops color, the reaction is complete, then add DMF (100ml) and wash 5 times, then add the next amino acid (2.0eq), HATU (3.0eq) and DIEA (3.0eq), after 1 hour of reaction at room temperature by bubbling nitrogen, ninhydrin developed color and the reaction was complete. According to the above method, Fmoc-Lys(Dde)-OH, Fmoc-aminocaproic acid, DOTA-COOH and Fmoc-tranexamic acid were sequentially connected to Fmoc-Lys(Boc)-OH.

After completion of amino acid condensation and docking, the resin was washed three times with DCM (100 ml) to remove residual DMF, washed twice with methanol (100 ml), and dried. Soak the dried resin in 20% trifluoroethanol/DCM solution (200 ml), react with magnetic stirring at room temperature for 3 hours, and remove the condensed amino acid fragments from the resin. HPLC monitored that the reaction was complete. The reaction solution was filtered to remove the resin. The organic phase was concentrated under reduced pressure to nearly dryness and purified by preparative chromatography to obtain 7.47g of a white solid, which was intermediate 20-1.

Step 2:

Add intermediate 20-1 (445mg, 1.0eq) and HBTU (455mg, 1.2eq) to a 50ml single-neck bottle, dissolve with DMF (10ml), add DIEA (388mg, 3.0eq), stir magnetically and react at room temperature for 0.5h. Intermediate 12-5-1 (1182 mg, 1.0 eq) was added, and the reaction was continued to stir for 2 h. HPLC detects that the reaction is complete. Add 5% citric acid aqueous solution (10ml) to quench the reaction, add DCM (100ml) for extraction, wash twice with 5% citric acid aqueous solution (100ml), add saturated brine for washing, and anhydrous sulfuric acid. Dry the organic phase over sodium, Concentrate under reduced pressure to obtain 1.63g of oil, namely intermediate 20-2.

Step 3:

Add intermediate 20-2 (1.63g) to a 50ml single-neck bottle, add an equal volume of mixed TFA/DCM solution (20ml), and add TES (0.2ml), and react at room temperature for 2h. After LC-MS monitors that the reaction is complete, the reaction solution is concentrated under reduced pressure to nearly dryness and added dropwise to MTBE. After solid precipitation, the supernatant is removed. The solid is purified by preparative chromatography to obtain 545 mg of the product, which is intermediate 20-3.

Step 4:

Add intermediate 20-4 (202.0mg, 1.0eq) into a 25ml single-neck bottle, add DMF (3ml) to dissolve, add EDCI (234.0mg, 4.0eq), stir magnetically, and then add N-hydroxysuccinimide (50.0mg, 1.2eq), react at room temperature for 2h. After HPLC monitors that the reaction is complete, the reaction solution is added dropwise to MTBE. After the solid is precipitated, the supernatant is removed by centrifugation. The solid is filtered and dried to obtain 243.1 mg of product, which is intermediate 20-5.

Step 5:

Add intermediate 20-5 (100.0mg, 1.0eq) and intermediate 20-4 (134.0mg, 1.0eq) to a 25ml single-neck bottle, then add DMF (5ml) to dissolve, stir magnetically at room temperature, add DIEA dropwise to complete the reaction The pH of the liquid was adjusted to 9, and the reaction was carried out overnight. HPLC monitors the completeness of the reaction. The reaction solution is added dropwise to MTBE. After the solid is precipitated, the supernatant is removed by centrifugation and the solid is dried to obtain 206.7 mg of the product, which is intermediate 20-6.

Step 6:

Add intermediate 20-6 (206.7 mg) to a 25 ml single-neck bottle, then add 50% TFA/DCM mixed solvent (5 ml), and react with magnetic stirring at room temperature for 1 hour. HPLC controlled the reaction to be complete. The reaction solution was concentrated under reduced pressure and purified by preparative chromatography to obtain 9.6 mg of product, namely compound 20', with a purity of 86.49%.

LC-MS(ESI ⁺ ): m/z 1926.96[M+H] ⁺ ,643.4[M+3H] ⁺ /3.

Example 20: Preparation of compound 21'

Step 1-3 was similar to step 1-3 in Example 19, and 545 mg of white solid, namely intermediate 20-3, was obtained.

Step 4:

Add intermediate 20-3 (100.0mg, 1.0eq) and intermediate 12-5 (134.1mg, 1.0eq) to a 25ml single-neck bottle, then add DMF (5ml) to dissolve, stir magnetically at room temperature, add DIEA dropwise to complete the reaction The pH of the liquid was adjusted to 9, and the reaction was carried out overnight. HPLC was used to control the reaction to be complete. The reaction solution was added dropwise to MTBE. After solid precipitation, the supernatant was removed by centrifugation. The solid was purified by preparative chromatography to obtain 6.4 mg of pure product, namely compound 21', with a purity of 90.78%.

LC-MS(ESI ⁺ ): m/z 1767.86[M+H] ⁺ ,884.6[M+2H] ⁺ /2.

Example 21: Preparation of compound 22’

step 1:

Add intermediate 19-1 (279.1mg, 2.0eq) and intermediate 20-3 (134.0mg, 1.0eq) to a 25ml single-neck bottle, then add DMF (5ml) to dissolve, stir magnetically at room temperature, add DIEA dropwise to complete the reaction The pH of the liquid was adjusted to 9, and the reaction was carried out overnight. LC-MS monitors that the reaction is complete. The reaction solution is added dropwise to MTBE. After the solid is precipitated, the supernatant is removed by centrifugation and the solid is dried to obtain 216 mg of product, namely intermediate 22-1.

Step 2:

Add intermediate 22-1 (216 mg) to a 25 ml single-neck bottle, then add TFA (5 ml), and react with magnetic stirring at room temperature for 3 hours. HPLC monitored that the reaction was complete, and the reaction solution was concentrated under reduced pressure and purified by preparative chromatography to obtain 105 mg of product, namely compound 22', with a purity of 83.66%.

LC-MS(ESI ⁺ ): m/z 2153.21[M+H] ⁺ ,539.17[M+4H] ⁺ /4.

Example 22: Preparation of compound 23’

step 1:

Add intermediate 23-1 (200mg, 1.0eq) and EDCI (114mg, 1.5eq) into a 25ml single-neck bottle, then add DCM (5ml) to dissolve, stir magnetically for 5 minutes, add N-hydroxysuccinimide ( 92mg, 2.0eq), stirred at room temperature for 2h. After HPLC monitors that the reaction is complete, add DCM to dilute, then add saturated brine to wash, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 235 mg of yellow solid, which is intermediate 23-2.

Step 2:

Take intermediate 23-3 (580 mg, 1.0 eq), add DMF (5 ml) to dissolve, stir magnetically, then add DBU (153 mg, 1.0 eq), and react at room temperature for 1 hour. After HPLC monitors that the reaction is complete, add the reaction solution dropwise to MTBE. Oily matter precipitates. Remove the supernatant. The oily matter is transferred to a one-neck bottle and concentrated under reduced pressure to obtain 281 mg of solid, which is intermediate 23-4.

Add intermediate 23-2 (213mg, 1.0eq) and intermediate 23-4 (140mg, 1.0eq) into a 25ml single-neck bottle, then add THF (5ml) to dissolve, stir magnetically for 5 minutes, add DIEA (138mg, 3.0eq) ), stir the reaction at room temperature for 2 h. HPLC monitored the reaction to be complete, and the reaction solution was purified by preparative chromatography to obtain 79 mg of product, namely intermediate 23-5.

Step 3:

Add intermediate 23-5 (78 mg, 1.0 eq) into a 25 ml single-neck bottle, add DCM (5 ml) to dissolve, then add TFA (2 ml) for magnetic stirring, and stir for 2 hours at room temperature. HPLC monitored that the reaction was complete, and the reaction solution was concentrated under reduced pressure to obtain 53 mg of a glassy solid, namely intermediate 23-6.

Step 4 and Step 5:

Add intermediate 23-7 (300 mg, 1.0 eq) into a 25 ml single-neck bottle, then add DCC (174 mg, 2.0 eq). After DMF (5 ml) is dissolved, stir magnetically for 5 minutes. Then add N-hydroxysuccinimide (146mg, 3.0eq) and react at room temperature for 2h. LC-MS monitors that the reaction is complete. Add 5% citric acid aqueous solution (100ml) to quench the reaction. Add DCM (100ml) for extraction. Wash with saturated brine twice. The organic phase is dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a white solid. 473 mg, which is intermediate 23-8.

Add intermediate 23-6 (53 mg, 1.0 eq) into a 25 ml single-neck bottle, add DMF (3 ml) to dissolve, then add intermediate 23-8 (120 mg, 2.0 eq), stir magnetically, and dropwise add DIEA (29 mg, 3.0 eq), stir the reaction at room temperature for 2 h. HPLC monitors the completeness of the reaction. The reaction solution is added dropwise to MTBE. After the solid is precipitated, the supernatant is removed by centrifugation. Transfer the solid (i.e., crude intermediate 23-9) to a 25 ml single-neck bottle, add TFA (5 ml), and stir overnight. LC-MS monitors that the reaction is complete. Concentrate under reduced pressure and purify through preparative chromatography to obtain 18.3 mg of the product, which is the compound. 23', purity 99.71%.

LC-MS(ESI ⁺ ): m/z 1230.64[M+H] ⁺ ,616.03[M+2H] ⁺ /2.

Example 23: Preparation of compound 24'

Add intermediate 24-1 (50mg, 1.0eq) into a 25m single-neck bottle, add DMF (2ml) to dissolve, then add HBTU (65mg, 1.5eq) and EDCI (22mg, 1.0eq), stir for 5 minutes, and then Add N-hydroxysuccinimide (29 mg, 2.0 eq) and stir magnetically at room temperature for 1 h. HPLC monitors the completion of the reaction. The reaction solution is added dropwise to MTBE. After the white solid precipitates, the supernatant is removed by centrifugation, filtered, and dried to obtain 63 mg of solid, which is intermediate 24-2.

Take intermediate 24-2 63mg (1.0eq) and intermediate 17-4 (226mg, 1.5eq), add TEA (36mg, 3.0eq), adjust the pH of the reaction solution to alkaline, and stir the reaction with magnetic stirring at room temperature for 2 hours. HPLC monitored that the reaction was complete, and the reaction solution was purified by preparative chromatography to obtain 20.6 mg of product, namely compound 24', with a purity of 91.60%.

LC-MS(ESI ⁺ ): m/z 1703.89[M+H] ⁺ ,852.28[M+2H] ⁺ /2.

Example 24: Preparation of compound 26’

step 1:

Add intermediate 26-1 (400 mg, 1.0 eq) into a 25 ml single-neck bottle, add DCM (10 ml) to dissolve, then add TFA (2 ml), stir magnetically, and stir for 2 hours at room temperature. HPLC monitored that the reaction was complete, and the reaction solution was concentrated under reduced pressure to obtain 313 mg of a light yellow solid. Add the solid (313mg, 1.0eq) into a 25ml single-neck bottle, add DMF (5ml) to dissolve, then add Fmoc-Asp-OtBu (451mg, 1.2eq) and HATU (497mg, 1.5eq), stir magnetically, and then Add DIEA (338 mg, 3.0 eq) and stir overnight. HPLC monitored that the reaction was complete, diluted with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 670 mg of orange-red oil, which was intermediate 26-2.

Step 2:

Add intermediate 26-2 (670 mg, 1.0 eq) into a 50 ml single-neck bottle, add DCM (10 ml) to dissolve, then add TFA (2 ml), stir magnetically, and stir for 2 hours at room temperature. HPLC monitored the reaction to be complete. The reaction solution was concentrated under reduced pressure and purified by preparative chromatography to obtain 436 mg of yellow solid, which was intermediate 26-3.

Step 3:

Add intermediate 26-3 (200mg, 1.0eq) into a 25ml single-neck bottle, add DMF (5ml) to dissolve, then add EDCI (111mg, 2.0eq) and N-hydroxysuccinimide (100mg, 3.0eq) ), magnetically stirred, and the reaction was stirred at room temperature for 3 h. HPLC monitored that the reaction was complete, diluted with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 231 mg of yellow solid, which was intermediate 26-4.

Step 4:

Add intermediate 26-4 (200mg, 1.0eq) and intermediate 23-4 (181mg, 2.0eq) into a 25ml single-neck bottle, add DMF (5ml) to dissolve, stir magnetically for 5 minutes, then add DIEA (98mg, 3.0eq) ), stir the reaction at room temperature for 2 h. HPLC monitored the reaction to be complete, and the reaction solution was purified by preparative chromatography to obtain 162 mg of product, namely intermediate 26-5.

Step 5 and Step 6:

Take intermediate 26-5 (100 mg, 1.0 eq), add DMF (5 ml) to dissolve, stir magnetically, then add DBU (16 mg, 1.0 eq), and react at room temperature for 1 hour. HPLC monitored that the reaction was complete (intermediate 26-6 was produced), added intermediate 23-8 (162 mg, 2.0 eq), then added DIEA to adjust the pH of the reaction solution to above 9, and stirred the reaction at room temperature for 3 hours. HPLC monitored that the reaction was complete, diluted with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 121 mg of yellow solid, which was intermediate 26-7.

Step 7:

Intermediate 26-7 (121 mg) was added to a 25 ml single-neck bottle, TFA (5 ml) was added, and the mixture was stirred at room temperature overnight. LC-MS monitored that the reaction was complete. The reaction solution was concentrated under reduced pressure and purified by preparative chromatography to obtain 29.6 mg of product, compound 26', with a purity of 98.71%.

LC-MS(ESI ⁺ ): m/z 1301.43[M+H] ⁺ .

Example 25: Preparation of compound 27’

Steps 1-3 were similar to steps 1-3 in Example 19, and 545 mg of product, namely intermediate 20-3, was obtained.

Step 4:

Add intermediate 15-1 (369mg, 1.0eq) into a 25ml single-neck bottle, add DMF (2ml) to dissolve, add succinic anhydride (120mg, 1.2eq), then add DIEA (388mg, 3.0eq), and react at room temperature 2h. LC-MS monitors that the reaction is complete. The reaction solution is added dropwise to MTBE. After solid precipitation, it is filtered and dried to obtain 470 mg of white solid, which is intermediate 27-1. Add intermediate 27-1 (470mg, 1.0eq) into a 25ml single-neck bottle, add DMF (2ml) to dissolve, then add EDCI (287mg, 1.5eq) and N-hydroxysuccinimide (230mg, 2.0eq) , stir the reaction at room temperature for 2h. LC-MS detected that the reaction was complete. The reaction solution was added dropwise to MTBE. After solid precipitation, the supernatant was removed and the solid was dried to obtain 435 mg of product, namely intermediate 27-2.

Step 5:

Add intermediate 27-2 (57 mg, 1.0 eq) and intermediate 20-3 (134.1 mg, 1.0 eq) to a 25 ml single-neck bottle, add DMF (5 ml) to dissolve, stir magnetically at room temperature, and then add DIEA dropwise to mix the reaction solution The pH was adjusted to 9 and the reaction was carried out overnight. HPLC monitored that the reaction was complete, and the reaction solution was added dropwise to MTBE. After the solid precipitated, the supernatant was removed by centrifugation. The solid was purified by preparative chromatography to obtain 11.3 mg of pure product, namely compound 27', with a purity of 93.78%.

LC-MS(ESI ⁺ ): m/z 1791.81[M+H] ⁺ .

Experimental Example 1: Imaging study of radionuclide conjugated drugs in CDX model

1. Sample preparation

Take the radionuclide coupled drug precursor of the present invention (the actual dosage of the precursor is about one thousandth of its theoretical molecular weight), add it to physiological saline (1ml), take 100μl and add to 900μl acetic acid solution (pH=4.5, 0.4M), take 10 μl and add 0.5 mCi of ¹⁷⁷ LuCl ₃ solution, then add 30 μl of acetic acid solution, heat at 60°C for 30 minutes, cool to room temperature after the reaction is completed, and obtain radionuclide coupling drug. Taking compound 7' as an example, its actual dosage is 2.5 mg, and the chelation is completed in ¹⁷⁷ LuCl ₃ solution Finally, the corresponding ¹⁷⁷ Lu conjugated drug was obtained.

2.Construction of CDX mouse model

293T-F/P (high expression of FOLR1 receptor and PSMA receptor) cell preparation: transfect FOLR receptor and PSMA receptor plasmids into 293T cells to obtain 293T cells with high expression of FOLR1 receptor and PSMA receptor, that is, 293T-F/P cells. Relevant tests have shown that the cells can also highly express FAPI receptors and can therefore be used to screen radionuclide-conjugated drugs targeting FAPI receptors. This experiment uses the above-mentioned 293T-F/P cells to test compound activity.

Model construction: Inoculate the prepared 293T-F/P cells into the subcutaneous skin of the right limb of BALB/c nude mice (5 weeks old, 18-20g, female). When the tumor grows and expands to ^300-500mm3 , the model The build is complete.

3. Group drug administration observation

Grouping: After the tumors grow to an average length of about ^300-500mm , they are divided into groups, and a model control group (BALB/c nude mice inoculated with 293T cells) and different drug administration groups are set up.

Experimental observation and measurement: Turn on the computer according to the Micro-SPECT/CT operating procedures and open the scanning software; after anesthetizing the tumor-bearing mice with isoflurane, inject imaging agent into the tail vein at a dose of 1 nmol/500 μCi/0.2 mL/mouse; give Data collection was performed at 30min, 1h, 2h, 4h, 8h, 24h, 48h and 72h after taking the drug. The collection methods were static 5min SPECT and medium-resolution whole-body CT.

4. Experimental results and analysis

It can be seen from Figure 13 and Figure 14 that the conjugate prepared from precursor 17' is highly enriched in tumors, enriched quickly, and has a long retention time (the drug is still enriched in tumors after 72 hours); renal metabolism is rapid ( Basic metabolism is completed in 8 hours), and the enrichment in other organs is low. The above results show that the conjugate and its precursor have good specificity for binding to PSMA/FAP, low toxicity to kidneys and other organs, high safety, and can be used for the treatment or diagnosis of tumors.

It can be seen from Figures 15 and 16 that the conjugate prepared from precursor 21' is highly enriched in tumors, and the enrichment is rapid, with a long retention time (a certain amount of drug is still enriched in tumors after 72 hours), and rapid renal metabolism (2 hours Basic metabolism is completed), other organs have low enrichment, and metabolism is fast. The above results show that the conjugate and its precursor have good specificity for binding to FAP/FAP, have low toxicity to other organs and are highly safe, and can be used for the treatment or diagnosis of tumors.

It can be seen from Figures 17 and 18 that the conjugate prepared from precursor 1' is highly enriched in tumors, and the enrichment is rapid, the retention time is long (a certain amount of drug is still enriched in tumors after 72 hours), and the kidney metabolism is rapid ( Basic metabolism is completed in 24 hours), other organs have low enrichment and fast metabolism. The above results show that the conjugate and its precursor have good specificity for binding to PSMA, low toxicity, and high safety, and can be used for the treatment or diagnosis of tumors.

It can be seen from Figures 19 and 20 that the conjugate prepared from precursor 2' is highly enriched in tumors, and the enrichment is rapid, the retention time is long (more than 8 hours), and the kidney metabolism is rapid (basic metabolism is completed in 8 hours). Organ enrichment is low. The above results show that the conjugate and its precursor have good specificity for binding to PSMA, low nephrotoxicity, high safety, and can be used for the treatment or diagnosis of tumors.

It can be seen from Figures 21 and 22 that the conjugate prepared from precursor 4' is highly enriched in tumors, and the enrichment is rapid, the residence time is long (more than 8 hours), and the kidney metabolism is rapid (basic metabolism is completed in 8 hours). Organ enrichment is low. The above results show that the conjugate and its precursor have good specificity for binding to PSMA, low nephrotoxicity, high safety, and can be used for the treatment or diagnosis of tumors.

The above results show that the precursor of the present invention has good tumor targeting properties, and the radionuclide conjugated drug prepared therefrom is rapidly enriched in tumors and has a long treatment time, while it is enriched in other organs (especially kidneys). It has low concentration, fast metabolism and low toxicity, and can be used for the treatment or diagnosis of tumors.

Although the embodiments of the present invention have been shown and described above, it should be understood that the above-described embodiments are illustrative and should not be construed as limitations of the present invention. Without departing from the principles and purposes of the present invention, those of ordinary skill in the art can make changes, modifications, substitutions and modifications to the above embodiments within the scope of the present invention, and these changes, modifications, substitutions and modifications are all covered by within the scope of the present invention.

Claims (44)

A radionuclide conjugated drug precursor having a structure shown in formula I,
in, LG represents targeting ligand; S represents spacer; L represents the linker; C represents chelating agent; m is 1 or 2; n is 0 or 1; p is 0 or 1; and When p is 0, C is directly connected to S or LG; When n is 0, LG is directly connected to L or C; When m is 2, the two LGs are the same or different from each other, and the two LGs are connected to S, L, or C at the same time, or the two LGs are connected to each other and then to S, L, or C. The radionuclide conjugated drug precursor according to claim 1, characterized in that it has a structure shown in formula I-1,
LG-LC
I-1 Wherein, LG, L and C are as defined in claim 1. The radionuclide conjugated drug precursor according to claim 1, characterized in that it has a structure shown in formula I-2,
LG-C
I-2 Wherein, LG and C are as defined in claim 1. The radionuclide conjugated drug precursor according to claim 1, characterized in that it has a structure shown in formula I-3,
Wherein, LG, S, L and C are as defined in claim 1. The radionuclide conjugated drug precursor according to claim 1, characterized in that it has a structure shown in formula I-4,
Wherein, LG, S and C are as defined in claim 1. The radionuclide conjugated drug precursor according to claim 1, characterized in that it has a structure shown in formula I-5,
Wherein, LG, L and C are as defined in claim 1. The radionuclide conjugated drug precursor according to claim 1, characterized in that it has a structure shown in formula I-6,
Wherein, LG, L and C are as defined in claim 1. The radionuclide conjugated drug precursor according to any one of claims 1-7, characterized in that, The targeting ligand binds to the following cell surface proteins: PSMA, FORL1, TRPV6, FAPI, C-MET, CAIX, RGD, Hepsin or Sigma. The radionuclide conjugated drug precursor according to claim 2 or 3, characterized in that, The targeting ligand binds to the following cell surface proteins: PSMA, TRPV6, FAPI, C-MET or CAIX. The radionuclide conjugated drug precursor according to any one of claims 4 to 7, characterized in that, The targeting ligand binds to the following cell surface proteins: PSMA/FORL1, FORL1/TRPV6, PSMA/TRPV6, PSMA/FAPI, PSMA/RGD, PSMA/Hepsin, FAPI/RGD, FAPI/FAPI, FAPI/Hespin, PSMA/ Sigma or FAPI/CAIX. The radionuclide conjugated drug precursor according to any one of claims 1 to 10, characterized in that, The targeting ligand is formed from polypeptides, antibodies or small molecules. The radionuclide conjugated drug precursor according to any one of claims 1-7, characterized in that, Each of the targeting ligands is independently composed of the formulas LG-I, LG-II, LG-III, LG-IV, LG-V, LG-VI, LG-VII, LG-VIII, and LG-IX. Any ligand compound is formed,
In formula LG-I, R ^LG1 is amino, in R ₁ is hydrogen or optionally substituted cycloalkylformyl; R ₂ and R ₃ are each independently hydrogen or optionally substituted C ₆ -C ₁₀ aryl; R ₄ is a hydroxyl group, an optionally substituted C ₁ -C ₆ alkylamino group, an amino acid derivative group or an oligopeptide derivative group composed of 2-6 amino acids; R ₅ is optionally substituted C ₁ -C ₆ alkyl; when substituted, the substituent is amino-substituted C ₁ -C ₆ alkanoyl, amino-substituted C ₆ -C ₁₀ aroyl, or both through amide The acyl group obtained by the chemical reaction; Formula LG-II is pteroic acid or folic acid or its analogues; Preferably, the folic acid analog is selected from the group consisting of 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, 10-formyltetrahydrofolate, methotrexate, 5,10-methylenetetrahydrofolate, amino pterin and raltitrexed; Formula LG-III includes all or part of the amino acids in the polypeptide EGKLSSNDTEGGLCKEFLHPSKVDLPR; Preferably, formula LG-III comprises 9 to 27 amino acids in said polypeptide; Alternatively, Formula LG-III has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the polypeptide KEFLHPSKVDLPR;
In formula LG-IV, R ^LG2 is halogen, amino, in R ₈ is hydrogen, in R _8' is carboxyl-substituted C ₁ -C ₆ alkylamino, carboxyl-substituted C ₃ -C ₈ cycloalkylmethylamino or carboxyl-substituted C ₁ -C ₂ alkoxy C ₁ -C ₂ alkoxy C ₁ -C _2alkylamino ; R _8″ is hydrogen, amino-substituted C ₁ -C ₆ alkanoyl, amino-substituted C ₁ -C ₂ alkoxy, C ₁ -C ₂ alkoxy C ₁ -C ₂ alkanoyl, carboxyl substituted C ₁ -C ₆ alkanoyl or carboxyl substituted C ₁ -C ₂ alkoxy C ₁ -C ₂ alkoxy C ₁ -C ₂ alkanoyl; R ₉ is in R _9' is carbonyl or methylene;
In the formula LG-V, X is a single bond or -(CH ₂ CH ₂ O) _n CH ₂ CONH-, where n is 0, 1, 2 or 3; R ^LG3 is hydrogen or in R ₁₀ is a hydroxyl- or carboxyl-substituted C ₁ -C ₆ alkylamino group;
In the formula LG-VI, B ₁ to B ₅ are each independently an amino acid fragment formed from any one of the following amino acids: phenylalanine, glutamic acid, arginine, glycine and aspartic acid; and optionally introducing an optionally substituted alkanoyl group and/or an optionally substituted amino group into at least one residue of B ₁ to B ₅ ;
In formula LG-VII, C ₁ to C ₄ each independently represent an amino acid fragment, wherein the C terminal of C ₁ is connected to a benzothiazolyl group, and the N terminal of C ₄ is connected to an acetyl group, and optionally introducing an optionally substituted alkanoyl group into at least one residue of C ₁ to C ₄ ;
In formula LG-VIII, Y is One end marked with * is connected to the carboxyl group, and the other end is connected to the ring Z; Ring Z is a C ₆ -C ₁₀ aromatic ring or a 5-9 membered heteroaromatic ring; R ^LG4 is hydrogen, C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy; Formula LG-IX contains the polypeptide Cys ^a -X1-Cys ^c -X2-Gly-Pro-Pro-X3-Phe-Glu-Cys ^d -Trp-Cys ^b -Tyr-X4-X5-X6, where: X1 is Asn, His or Tyr; X2 is Gly, Ser, Thr or Asn; X3 is Thr or Arg; X4 is Ala, Asp, Glu, Gly or Ser; X5 is Ser or Thr ^; Cysteine residue; preferably, residues Cys ^a and Cys ^b and Cys ^c and Cys ^d are cyclized respectively to form two independent disulfide bonds; Further, the formula LG-IX includes the polypeptide Ala-Gly-Ser-Cys ^a -Tyr-Cys ^c -Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys ^d -Trp-Cys ^b -Tyr-Glu-Thr -Glu-Gly-Thr-Gly-Gly-Gly-Lys, wherein: Cys ^ad is each independently a cysteine residue; preferably, the residues Cys ^a and Cys ^b and Cys ^c and Cys ^d are respectively cyclized to form Two independent disulfide bonds; more preferably, the carboxyl group of the terminal lysine of Formula IX is amidated; or, Formula IX has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, wherein: Cys ^ad is each independently a cysteine residue; preferably, the residues Cys ^a and Cys ^b and Cys ^c and Cys ^d are respectively cyclized to form two independent disulfide bonds. The radionuclide conjugated drug precursor according to claim 2 or 3, characterized in that, The targeting ligand is formed from any ligand compound of formula LG-I, LG-III, LG-IV, LG-V, LG-IX or its optical isomer. The radionuclide conjugated drug precursor according to any one of claims 4 to 7, characterized in that, Each of the targeting ligands is independently formulated by any one of the formulas LG-I, LG-II, LG-III, LG-IV, LG-V, LG-VI, LG-VII, and LG-VIII. Formation of bulk compounds or optical isomers thereof; Preferably, the two targeting ligands are respectively composed of the formulas LG-I/LG-II, LG-I/LG-IV, LG-I/LG-VI, LG-I/LG-VII, LG-I/ LG-VIII, LG-I/LG-III, LG-II/LG-III, LG-IV/LG-IV, LG-IV/LG-V, LG-IV/LG-VI or LG-IV/LG- VII ligand compound or its optical isomer is formed. The radioactive conjugated drug precursor according to claim 8, characterized in that, The targeting ligand is any one of the following: A targeting ligand formed from a ligand compound of formula LG-I or an optical isomer thereof and binding to the cell surface protein PSMA; A targeting ligand formed from a ligand compound of formula LG-II or an optical isomer thereof and binding to the cell surface protein FORL1; A targeting ligand formed from a ligand compound of formula LG-III or an optical isomer thereof and binding to the cell surface protein TRPV6; A targeting ligand formed from a ligand compound of formula LG-IV or an optical isomer thereof and binding to the cell surface protein FAPI; A targeting ligand formed from a ligand compound of formula LG-V or an optical isomer thereof and binding to the cell surface protein CAIX; A targeting ligand formed from a ligand compound of formula LG-VI or an optical isomer thereof and binding to the cell surface protein RGD; A targeting ligand formed from a ligand compound of formula LG-VII or an optical isomer thereof and bound to the cell surface protein Hepsin; A targeting ligand formed from a ligand compound of formula LG-VIII or an optical isomer thereof and binding to the cell surface protein Sigma; and A targeting ligand formed from a ligand compound of formula LG-IX or an optical isomer thereof and binding to the cell surface protein C-MET. The radionuclide conjugated drug precursor according to any one of claims 1 to 7, characterized in that, Each of the targeting ligands is independently formed from any one of the following ligand compounds:




The radionuclide conjugated drug precursor according to any one of claims 1-3, characterized in that, The targeting ligand is formed from any one of the following ligand compounds:

The radionuclide conjugated drug precursor according to any one of claims 1 and 4-7, characterized in that, Each of the targeting ligands is independently formed from any one of the following ligand compounds:


The radionuclide conjugated drug precursor according to any one of claims 1-18, characterized in that, The chelating agent contains a variable number of heteroatoms to complex radionuclides, and is cyclic or acyclic, wherein: the heteroatoms are N, O, S or P atoms, preferably N, O or S atom, more preferably N or O atom; Preferably, the cyclic chelating agent is 1,4,7-triazacyclononane, 1,4,7-triazacyclononane-triacetic acid, 1,4,7,10-tetraaza Heterocyclododecane, 1,4,7,10-tetraazacyclotridecane, 1,4,7,11-tetraazacyclotetradecane, 1,4,7,10-tetraazacyclotetradecane Dodecane-1,4,7,10-tetraacetic acid, 2-(1,4,7,10-tetraazacyclododecan-1-yl)acetic acid, 2,2'-(1,4, 7,10-tetraazacyclododecane-1,7-diyl)diacetic acid, 2,2',2″-(1,4,7,10-tetraazacyclododecane-1,4 ,7-triyl)triacetic acid, 1,4,8,11-tetraazacyclotetradecane, 1,4,8,12-tetraazacyclopentadecane, 1,5,9,13-tetraazacyclotetradecane Azacyclohexadecane, 2-(1,4,8,11-tetraazacyclotetradecan-1-yl)acetic acid or 2,2'-(1,4,8,11-tetraazacyclohexadecane Tetradecan-1,8-diyl)diacetic acid; Preferably, the acyclic chelating agent is 2,2',2",2"'-(ethane-1,2-diylbis(nitrilo))tetraacetic acid, 2,2',2 ”,2”'-(((carboxymethyl)nitrilo)bis(ethane-2,1-diyl))bis(nitrilo))tetraacetic acid, 2,2'-((2- ((carboxymethyl)(2-hydroxyethyl)amino)ethyl)nitrilo)diacetic acid, 2,3-bis(2-mercaptoacetamido)propionic acid, 2,2',2”,2” '-(((4'-(3-amino-4-methoxyphenyl)-[2,2':6',2"-terpyridine]-6,6"-diyl)bis(methylene base))bis(nitrilo))tetraacetic acid, 2,2'-((1-carboxyethyl)nitrilo)diacetic acid, 2,2'-(ethane-1,2-diylbis( Nitrogen))bis(2-(2-hydroxyphenyl)acetic acid) or 2,2',2",2"'-((cyclohexane-1,2-diyl)bis(nitrilo) ) tetraacetic acid. The radionuclide conjugated drug precursor according to any one of claims 1-18, characterized in that, The chelating agent is formed from any one of the following chelating agent compounds:

The radionuclide conjugated drug precursor according to any one of claims 1-20, characterized in that, The linker is cleavable or non-cleavable, preferably non-cleavable. The radionuclide-coupled drug precursor according to any one of claims 1 to 20, characterized in that the linker is in the formulas LI, L-II, L-III, L-IV, and LV. any kind,
In formula LI, the end marked with * is connected to the targeting ligand in the conjugated drug precursor or is terminated with a hydroxyl or amino group, and the end marked with ** is connected to the targeting ligand in the conjugated drug precursor. Or a chelating agent or terminated with hydrogen or an acetyl group; R does not exist or is a bivalent structural fragment formed by amino acids; preferably, R is a bivalent structural fragment formed by lysine, tryptophan or valine;
In formula L-II, the end marked with *** is connected to the spacer or targeting ligand in the conjugated drug precursor, and the end marked with ** is connected to the chelating agent in the conjugated drug precursor; X is NH or O; n is any integer from 2 to 8;
In formula L-III, the end marked by *** is connected to the spacer or targeting ligand in the conjugated drug precursor, and the end marked by ** is connected to the chelating agent in the conjugated drug precursor; X Does not exist or is NH or O; n is any integer from 1 to 4;
In formula L-IV, the end marked with * is connected to the targeting ligand in the conjugated drug prodrug or is terminated with a hydroxyl or amino group, and the end marked with ** is connected to the targeting ligand in the conjugated drug prodrug. The ligand or chelating agent may be terminated with hydrogen or acetyl group; n is any integer from 1 to 6;
In formula LV, the end marked with * is connected to the targeting ligand in the conjugated drug precursor, and the end marked with ** is connected to the chelating agent in the conjugated drug precursor; n is any one from 1 to 5. Integer; R ₁ is absent or -NH-CH ₂ -CH ₂ -NH-; R ₂ is absent or is One end marked with * is connected to NH in LV, and m is any integer from 1 to 7. The radionuclide conjugated drug precursor according to any one of claims 1-20, characterized in that, The linker is any one of the formulas L-I, L-III, L-IV or L-V. The radionuclide conjugated drug precursor according to any one of claims 1-20, characterized in that, The linker is of formula L-II or L-III. The radionuclide conjugated drug precursor according to any one of claims 1-20, characterized in that, The linker is of formula L-I. The radionuclide conjugated drug precursor according to any one of claims 1-22, characterized in that, The linker is any one of the following structural fragments, in which the end marked with * is connected to the targeting ligand in the conjugated drug precursor, and the end marked with ** is connected to the target in the conjugated drug precursor. Connect the spacer or targeting ligand in the conjugated drug precursor to the ligand or chelating agent at the end marked with ***:

The radionuclide conjugated drug precursor according to claim 1, 2 or 6, characterized in that, The linker is any one of the following structural fragments, in which the end marked with * is connected to the targeting ligand in the conjugated drug precursor, and the end marked with ** is connected to the chelate in the conjugated drug precursor. The mixture consists of one end marked with *** connected to the targeting ligand in the conjugated drug precursor:

The radionuclide conjugated drug precursor according to claim 1 or 4, characterized in that, The linker is any one of the following structural fragments, in which the end marked by ** is connected to the chelating agent in the conjugated drug precursor, and the end marked by *** is connected to the spacer in the conjugated drug precursor. son:
The radionuclide conjugated drug precursor according to claim 1 or 7, characterized in that, The linker is any one of the following structural fragments, in which the end marked with * is connected to the targeting ligand in the conjugated drug precursor, and the end marked with ** is connected to the target in the conjugated drug precursor. To ligand or chelating agent:
The radionuclide conjugated drug precursor according to any one of claims 1-29, characterized in that, The spacer is cleavable or non-cleavable, preferably non-cleavable. The radionuclide conjugated drug precursor according to any one of claims 1-29, characterized in that, The spacer is any one of formulas SI, S-II and S-III,
In formula SI, one end marked by * is connected to one targeting ligand in the conjugated drug precursor, one end marked by ** is connected to another targeting ligand in the conjugated drug precursor, and * *The other end marked is connected to the linker or chelator in the conjugated drug prodrug; A ₁ to A ₃ are each independently a bivalent structural fragment formed of amino acids; preferably, A ₁ to A ₃ are each independently A bivalent structural fragment formed by Asp, Lys, Glu, His or Trp; n and m are each independently any integer from 2 to 8;
In formula S-II, one end marked with * is connected to one targeting ligand in the conjugated drug precursor, and one end marked with ** is connected to another targeting ligand in the conjugated drug precursor. The other end marked by ** is connected to the linker or chelator in the conjugated drug precursor; n is any integer from 2 to 6;
In formula S-III, the end marked with * is connected to the targeting ligand in the conjugated drug precursor, and the end marked with ** is connected to the linker or chelator in the conjugated drug precursor; n is 1 Any integer within -5. The radionuclide conjugated drug precursor according to any one of claims 1-29, characterized in that, The spacer is of formula S-II or S-III. The radionuclide conjugated drug precursor according to any one of claims 1-29, characterized in that, The spacer is of formula S-I or S-II. The radionuclide conjugated drug precursor according to any one of claims 1-31, characterized in that, The spacer is any one of the following structural fragments, in which the end marked with * is connected to a targeting ligand in the conjugated drug precursor, and the end marked with ** is connected to a targeting ligand in the conjugated drug precursor. Another targeting ligand is connected to the linker or chelator in the conjugated drug prodrug by the ***-marked end:

The radionuclide conjugated drug precursor according to claim 1 or 4, characterized in that, The spacer is any one of the following structural fragments, in which the end marked with * is connected to a targeting ligand in the conjugated drug precursor, and the end marked with ** is connected to a targeting ligand in the conjugated drug precursor. Another targeting ligand is connected to the linker or chelator in the conjugated drug prodrug by the ***-marked end:
The radionuclide conjugated drug precursor according to claim 1 or 5, characterized in that, The spacer is any one of the following structural fragments, in which the end marked with * is connected to a targeting ligand in the conjugated drug precursor, and the end marked with ** is connected to a targeting ligand in the conjugated drug precursor. Another targeting ligand, with the ***-marked end connected to the chelating agent in the conjugated drug prodrug:
The following radionuclide conjugated drug precursors:





A radionuclide-conjugated drug, which contains the radionuclide-conjugated drug precursor according to any one of claims 1 to 37, and a radioactive isotope chelated thereto. The radionuclide conjugated drug according to claim 38, characterized in that the radioactive isotope is selected from ⁴⁷ Sc, ⁴⁸ Sc, ⁵¹ Cr, ⁵⁵ Fe, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁹ Zn, ⁶⁷ Ga, ⁶⁸ Ga , ⁷² Ga, ⁷² As, ⁷² Se, ⁸⁹ Sr, ⁸⁸ Y, ⁹⁰ Y, ⁹⁹ Tc, ^99m Tc, ⁹⁷ Ru, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ In, ¹¹⁹ Sb, ¹²⁸ Ba, ¹³⁹ La, ¹⁴⁰ La, ¹⁴² Pr, ¹⁴⁹ Pm, ¹⁵³ Sm, ¹⁵⁹ Gd, ¹⁶⁵ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁷⁵ Yb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au, ¹⁹⁹ Au, ¹⁹⁷ Hg, ²⁰¹ Tl, ²⁰² Pb, ²⁰³ Pb, Any one of ²¹² Pb, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac and ²²⁷ Th; Preferably, the radioactive isotope is selected from ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁹ Sr, ⁹⁰ Y, ^99m Tc, 111 In, ¹¹⁹ Sb, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ^{186 Re} , ¹⁸⁸ Re , any one of ¹⁹⁸ Au, ¹⁹⁹ Au, ²⁰¹ Tl, ²⁰³ Pb, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac and ²²⁷ Th; More preferably, the radioactive isotope is selected from any one of ⁶⁴ Cu, ⁶⁷ Cu, ¹¹¹ In, ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au and ¹⁹⁹ Au; Further preferably, the radioactive isotope is ¹⁷⁷ Lu. A pharmaceutical composition comprising a radionuclide-conjugated drug precursor according to any one of claims 1-37 or a radionuclide-conjugated drug according to claims 38 or 39; Preferably, the pharmaceutical composition further contains at least one pharmaceutically acceptable excipient. The radionuclide-conjugated drug precursor according to any one of claims 1 to 37 or the radionuclide-conjugated drug according to claim 38 or 39 or the pharmaceutical composition according to claim 40 in Use in the preparation of medicaments for the prevention and/or treatment of diseases or conditions; Preferably, the disease or condition is cancer, preferably prostate cancer, breast cancer, liver cancer, pancreatic cancer, ovarian cancer, gastric cancer or lung cancer. The radionuclide-conjugated drug precursor according to any one of claims 1-37 or the radionuclide-conjugated drug according to claim 38 or 39 or the pharmaceutical composition according to claim 40, its use to prevent and/or treat diseases or conditions; Preferably, the disease or condition is cancer, preferably prostate cancer, breast cancer, liver cancer, pancreatic cancer, ovarian cancer, gastric cancer or lung cancer. A method for preventing and/or treating diseases or conditions, which includes: combining a preventive and/or therapeutically effective amount of a radionuclide-conjugated drug precursor according to any one of claims 1-37 or The radionuclide conjugated drug according to claim 38 or 39 or the pharmaceutical composition according to claim 40 is administered to an individual in need thereof; Preferably, the disease or condition is cancer, preferably prostate cancer, breast cancer, liver cancer, pancreatic cancer, ovarian cancer, gastric cancer or lung cancer. The following two-ligand compounds: