Conjugate using succinyl as linker, and preparation method therefor and use thereof

Abstract

Disclosed in the present invention are a conjugate using succinyl as a linker, and a preparation method therefor and the use thereof. Specifically, the conjugate using succinyl as the linker of the present invention is as represented by formula (I), wherein the succinyl is used as a linker, which is linked at the α-position to a ligand with a linker for specific tissue-targeted recognition, and is linked via an amide bond to an active ligand containing a peptide chain that has therapeutic and / or diagnostic effects on a disease. The use of the succinyl linker can improve the plasma stability and targeting property of the conjugate, and the conjugate containing the linker can be used for treating, diagnosing and / or preventing a targeted disease.

Description

Couplings with succinyl groups as linking chains, their preparation methods and uses Technical Field

[0001] This invention relates to conjugates, using succinyl groups as the linking chain, that link tissue-targeting recognition ligands with active substances / labeled substances related to disease treatment, diagnosis, and / or prevention, as well as their preparation methods and uses. Specifically, this invention relates to a class of conjugates, using succinyl groups as the linking chain, that link a ligand targeting a liver-targeting desialyl glycoprotein receptor with an active substance / labeled substance for disease treatment, as well as their preparation methods and uses. Background Technology

[0002] Peptide-containing drug conjugates are widely used and researched in disease treatment and diagnosis. These conjugates target specific tissues, releasing the active substance or marker at the site of disease. However, various esterases in plasma can rapidly hydrolyze these conjugates, causing the active substance to be released before reaching the target tissue. This results in non-tissue specificity of the active substance or marker molecule, and the associated toxic side effects limit their use. For example, carboxylesterase 1c (Ces1c), highly expressed in plasma, can hydrolyze peptide chains, leading to premature release of the active substance.

[0003] The liver is one of the most important organs in the human body, and desialized glycoprotein receptors (ASGPRs) are highly expressed on the cell membranes of hepatocytes. ASGPRs, through a 15-minute cycle, mediate the recognition and endocytosis of glycoproteins terminally modified with β-D-galactose (Gal) and N-acetylgalactosamine (GalNAc) residues, followed by lysosomal digestion and degradation, thus maintaining endogenous glycoprotein homeostasis. Among all the natural ligands of ASGPRs, GalNAc has the highest affinity.

[0004] The applicant of this invention previously developed a conjugate of a GalNAc fragment and a histone B-linked bioactive molecule fragment linked by a cyclic succinimide (cyclic succinamide) (Patent Application No.: 202310079838.3, Application Date: January 17, 2023), which improved plasma stability and liver targeting of the released active substance to a certain extent. Summary of the Invention

[0005] The inventors of this application discovered in later research that conjugates linked by cyclic succinimide still exhibit some instability in animal and human plasma, resulting in poor tissue targeting. Based on these issues, this invention uses a succinyl group as a linker to connect different ligands targeting specific tissues with a release active substance containing a peptide chain that has therapeutic, diagnostic, and / or preventative effects against diseases. Using conjugates with this type of linker chain improves the stability of the conjugate in human and mouse plasma and significantly enhances the distribution of the active substance in target tissues. In vivo pharmacodynamic experiments show that conjugates containing this linker chain significantly improve in vivo activity and reduce toxic side effects, making them suitable for the treatment, diagnosis, and / or prevention of targeted diseases.

[0006] Based on this, the first object of the present invention is to provide conjugates of Formula I, which use succinyl groups as linking chains to connect tissue-targeting recognition ligands and ligands related to disease treatment, diagnosis and / or prevention, or pharmaceutically acceptable salts or solvates thereof; in particular, the present invention provides a class of conjugates using succinyl groups as linking chains to connect ligands of liver-targeting desialyl glycoprotein receptors with bioactive substances or markers.

[0007] A second object of the present invention is to provide the use of the conjugate of Formula I, which is linked by a succinyl group as a linking chain, in a pharmaceutical.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a conjugate of Formula I with a succinyl group as the linking chain, or a pharmaceutically acceptable salt or solvate thereof.

[0010] in,

[0011] A is a ligand containing a connecting chain that specifically targets and recognizes tissues;

[0012] B is an active ligand containing a peptide chain that has therapeutic and / or diagnostic effects on diseases;

[0013] X1 is either S or NR; where R is selected from H, C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group, C6-C 20 Aryl, C6-C 20 Aryl C1-C 10 Alkyl groups, 5-20 membered heteroaryl groups containing one or more (e.g., 2, 3, 4) heteroatoms selected from N, O, and S, and 5-20 membered heteroaryl C1-C 10 Alkyl group; preferably, X1 is S;

[0014] X2 is O, S, or NR; where R is selected from H, Cl-C.20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group, C6-C 20 Aryl, C6-C 20 Aryl C1-C 10 Alkyl groups, 5-20 membered heteroaryl groups containing one or more (e.g., 2, 3, 4) heteroatoms selected from N, O, and S, and 5-20 membered heteroaryl C1-C 10 Alkyl; preferably, R is H, C1-C4 alkyl, C6-C 20 Aryl, C6-C 12 Aryl C1-C4 alkyl; preferably, X2 is O, NH, or NCH3;

[0015] P is selected from H, C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group, C6-C 20 Aryl, C6-C 20 Aryl C1-C 10 Alkyl groups, 5-20 membered heteroaryl groups containing one or more (e.g., 2, 3, 4) heteroatoms selected from N, O, and S, and 5-20 membered heteroaryl C1-C 10 Alkyl; preferably, P is selected from H, C1-C4 alkyl, C6-C 20 Aryl, C6-C 12 Aryl C1-C4 alkyl; more preferably, P is H or -CH S -CH2CH S or -benzyl;

[0016] L1 is selected from -(CH2) k1 -Z1-(CH2) k2 -、-(CH2) k3 -(O-CH2-CH2-O) k4 -(CH2) k5 -、-(CH2) k6 -NH-C(O)-(CH2) k7 -Z1-(CH2) k8 -C(O)NH-(CH2) k9 - and -(CH2) k10 -NH-C(O)-(CH2) k11 -(O-CH2-CH2-O) k12 -C(O)NH-(CH2) k13- where k1 to k13 are each independently an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.); Z1 is independently NH, O, or S; preferably, L1 is -(CH2). k1 , where k1 is an integer from 1 to 10, especially -(CH2)6-.

[0017] In a specific embodiment, the conjugate with succinyl group as the linking chain shown in Formula I has the structure shown in Formula II.

[0018] in,

[0019] X1 is S or NH; preferably, X1 is S;

[0020] X2 is O, S, or NR; where R is H, C1-C4 alkyl, or C6-C4 alkyl. 20 Aryl, C6-C 12 Aryl C1-C4 alkyl; preferably, X2 is O, NH, or NCH3;

[0021] P is selected from H, C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group, C6-C 20 Aryl, C6-C 20 Aryl C1-C 10 Alkyl groups, 5-20 membered heteroaryl groups containing one or more (e.g., 2, 3, 4) heteroatoms selected from N, O, and S, and 5-20 membered heteroaryl C1-C 10 Alkyl groups; preferably selected from H, C1-C4 alkyl groups, C6-C4 alkyl groups. 20 Aryl, C6-C 12 Aryl C1-C4 alkyl; preferably, P is H, -CH3, -CH2CH3 or -benzyl;

[0022] L1 is selected from -(CH2) k1 -Z1-(CH2) k2 -、-(CH2) k3 -(O-CH2-CH2-O) k4 -(CH2) k5 -、-(CH2) k6 -NH-C(O)-(CH2) k7 -Z1-(CH2) k8 -C(O)NH-(CH2) k9 - and -(CH2) k10 -NH-C(O)-(CH2) k11 -(O-CH2-CH2-O) k12-C(O)NH-(CH2) k13 - where k1 to k13 are each independently an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.); Z1 is independently NH, O, or S; preferably, L1 is -(CH2). k1 , where k1 is an integer from 1 to 10, especially -(CH2)6-;

[0023] In Part A:

[0024] G1, G2, and G3 are each independently an N-acetylgalactosamine group or its analogues, which can bind to the ASGPR receptor highly expressed in hepatocytes or liver cancer cells.

[0025] L2, L3, and L4 are each independently selected from -(CH2). n1 -(O-CH2-CH2) n2 -(CH2) n3 -、-(CH2) n4 -(O-CH2-CH2) n5 -NH-C(O)-(CH2) n6 -, where n1 to n6 are each an independent integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.); preferably, L1, L2, and L3 are each independently -(CH2). n1 , where n1 is an integer from 1 to 10, specifically selected from -(CH2)6- and -(CH2)4-;

[0026] Y1, Y2, and Y3 are each independently selected from -Z2-(CH2). m1 - and -NHC(O)-(CH2) m2 -Z2-(CH2) m3 - where m1 to m3 are each independently an integer from 0 to 10 (e.g., 1, 2, 3, etc.), and Z2 is independently NH or O; preferably, Y1, Y2, and Y3 are each independently selected from -O-CH2- and -NH-C(O)-(CH2)2-O-CH2-;

[0027] L5 is selected from -(CH2) j1 -Z3-(CH2) j2 -、-(CH2) j3 -(O-CH2-CH2-O) j4 -(CH2) j5 -、-(CH2) j6 -NH-C(O)-(CH2) j7 -、-(CH2) j8 -NH-C(O)-(CH2) j9-Z3-(CH2) j10 -C(O)NH-(CH2) j11 - and -(CH2) j12 -NH-C(O)-(CH2) j13 -(O-CH2-CH2-O) j14 -C(O)NH-(CH2) j15 - where j1 to j15 are each an independent integer from 0 to 10 (e.g., 1, 2, 3, 4, 5, 6, etc.), and Z3 is independently NH, O, or S; preferably, L5 is -(CH2). j6 NH-C(O)-(CH2) j7 -, where j6 to j7 are each an independent integer from 0 to 6, especially -NH-C(O)-(CH2)3-;

[0028] In Part B:

[0029] L6 is -(CH2) q1 -C(O)-, where q1 is an integer from 0 to 10 (e.g., 1, 2, 3, 4, 5); preferably, L6 is -(CH2)5-C(O)-;

[0030] Q represents a peptide fragment that can be hydrolyzed and cleaved in target tissues of the liver to release T;

[0031] T refers to a small molecule, nucleic acid, polypeptide, or protein that is bioactive or labeled for the treatment, diagnosis, and / or prevention of diseases.

[0032] In a specific implementation, in Formula II, G1, G2, and G3 are all N-acetylgalactosamine groups.

[0033] In a specific implementation, in Formula II, L2, L3, and L4 are each independently selected from -(CH2)6- and -(CH2)4-.

[0034] In a specific implementation, in Formula II, Y1, Y2 and Y3 are each independently selected from -O-CH2- and -NH-C(O)-(CH2)2-O-CH2-.

[0035] In a specific implementation, X1 in Formula II is S.

[0036] In a specific implementation, X2 in Formula II is -O, -NH, or -NCH3.

[0037] In a specific implementation, in Formula II, P is -H, -CH3, -CH2CH3, or -benzyl.

[0038] In a specific embodiment, in Formula II, Q represents a dipeptide or polypeptide fragment structure that can be cleaved by cathepsins in lysosomes; preferably, Q is a group of valine-citrulline-p-aminobenzylcarbamate (PAB) as shown in the following formula:

[0039] In a specific embodiment, in Formula II, T represents a small molecule compound for treating liver diseases, such as a cytotoxin, a chemotherapeutic agent (including inhibitors or agonists targeting related disease therapeutic targets), an immunomodulator, etc.; and a labeling molecule for treating liver diseases, such as a radiolabeled, fluorescently labeled, diagnostic agent, or detectable modified enzyme that is a catalytic substrate. Preferably, the cytotoxin is methylaurestatin E (MMAE) or a derivative thereof.

[0040] In some implementations, T is The wavy line indicates the junction of toxin molecules via amide bonds.

[0041] In a specific embodiment, the compound represented by Formula I or Formula II may be selected from the following compounds II-1 to II-4:

[0042] Secondly, the present invention provides a method for preparing the conjugate of Formula I with succinyl as the linking chain, the reaction route of which is as follows:

[0043] Where A, B, L1, X1, X2 and P are defined as previously, and when X2 is 0, P is not simultaneously H;

[0044] (1) Compound III is hydrolyzed under alkaline conditions to give compound I-1;

[0045] (2) Compound I-1 undergoes a condensation reaction with compound HX2P to obtain compound I-2.

[0046] In a specific embodiment, step (1) is carried out under solvent conditions, wherein the solvent is an organic solvent, such as methanol; the base includes inorganic bases and organic bases, preferably selected from one or more of sodium methoxide, sodium ethoxide, potassium tert-butoxide, etc.; the reaction temperature is 0 degrees to room temperature; the reaction time is 0.5-24 hours.

[0047] In a specific embodiment, in step (2), the condensing agent used in the condensation reaction is selected from one or more of benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate (HBTU), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), 1-hydroxybenzotriazole (HOBt), 1,8-diazabicycloundec-7-ene (DBU), N,N-diisopropylethylamine, triethylamine, and pyridine. Preferably, the condensing agent used is a combination of benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate (HBTU) and N,N-diisopropylethylamine.

[0048] In a specific implementation, step (2) is carried out under solvent conditions, wherein the solvent is an organic solvent, such as dichloromethane or dimethylformamide; the reaction temperature is 0°C to 60°C; and the reaction time is 0.5 to 24 hours.

[0049] In specific embodiments, compound III can be obtained by conventional methods in the art, or similarly by methods described in Chinese patent application No. 202310079838.3.

[0050] Thirdly, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the succinyl-linked conjugate or a pharmaceutically acceptable salt or solvate thereof as described in the first aspect, and optionally a pharmaceutically acceptable excipient.

[0051] Fourthly, the present invention also provides the use of the conjugate of the first aspect with succinyl as the linking chain or a pharmaceutically acceptable salt or solvate thereof, and the use of the pharmaceutical composition of the third aspect in the preparation of a medicament for treating liver diseases.

[0052] In a specific implementation, the liver disease is liver cancer.

[0053] Terminology Explanation

[0054] As used herein, "alkyl" refers to a fully saturated straight-chain or branched monovalent hydrocarbon group. Alkyl groups preferably contain 1-20 carbon atoms, more preferably 1-16 carbon atoms, 1-10 carbon atoms, 1-8 carbon atoms, 1-6 carbon atoms, 1-4 carbon atoms, or 1-3 carbon atoms. The number preceding the alkyl group indicates the number of carbon atoms. For example, "C1-C6 alkyl" indicates an alkyl group having 1-6 carbon atoms. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc.

[0055] As used herein, "alkenyl" refers to a straight-chain or branched monovalent hydrocarbon group containing at least one double bond. Alkenyl groups preferably contain 2-20 carbon atoms, more preferably 2-10 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Representative examples of alkenyl groups include, but are not limited to, vinyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, isopentenyl, hexenyl, heptenyl, octenyl, etc.

[0056] As used herein, "alkynyl" refers to a straight-chain or branched monovalent hydrocarbon group containing at least one triple bond. The alkynyl group preferably contains 2-20 carbon atoms, more preferably 2-10 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Representative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, isopropynyl, butynyl, isobutynyl, pentyynyl, isopentenynyl, hexynyl, heptyynyl, and octyynyl.

[0057] As used herein, "aryl" refers to an aryl group consisting of one or more fused rings having 6-20, preferably 6-14, more preferably 6-12, and most preferably 6-10 rings. An aryl group having 6-10 ring carbon atoms is C6-C... 10 Aryl groups include: monocyclic aryl groups (e.g., phenyl); or fused bicyclic systems, wherein one ring is an aromatic ring and the other ring is an aromatic ring (e.g., in naphthalene, biphenyl) or a non-aromatic ring (e.g., in dihydroindene, tetrahydronaphthalene). Non-limiting examples of aryl groups include phenyl, biphenyl, naphthyl, tetrahydronaphthyl, indene, dihydroindene, or anthracene.

[0058] As used herein, "heteroaryl" refers to a 5-20 membered, preferably 5-10 membered, more preferably 5-7 membered or 5-6 membered aromatic ring system containing 1-4, preferably 1-3, cyclic heteroatoms selected from N, O or S, including monocyclic, bicyclic or fused polycyclic rings, with the remaining ring atoms being carbon atoms. Examples of heteroaryl groups include, but are not limited to: pyrrole, furanyl, thiophene, pyrazolyl, imidazolyl, triazolyl, thiazolyl, isothiazolyl, oxazolyl, pyridinyl, pyranyl, pyrazinyl, pyridazinyl, pyrimidinyl, oxazinyl, oxadiazinyl, quinolinyl, isoquinolinyl, borazinyl, quinazolinyl, quinoxalinyl, benzoxazinyl, 2H-chromene, benzopyranyl, benzothiophene, indole, inzolyl, benzene Pyrazole, benzimidazolyl, imidazopyridyl, benzoxazolyl, benzothiazolyl, 7-azaindolyl, 6-azaindolyl, 5-azaindolyl, 4-azaindolyl, 1H-benzo[d][1,2,3]triazolyl, [1,2,4]triazolo[1,5-a]pyridyl, [1,2,4]triazolo[4,3-a]pyridine, pyrazolo[1,5-a]pyridine, etc.

[0059] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Beneficial effects

[0060] Compared with previous conjugates using cyclic succinamide as the linking chain, the conjugate of the present invention using succinyl as the linking chain significantly improves plasma stability and enhances the liver tissue targeting of the released active or labeled substances, making it more suitable for the treatment and diagnosis of liver diseases. Attached Figure Description

[0061] Figure 1 shows the hydrolysis efficiency of compounds II-0 to II-4 under the action of cathepsin B.

[0062] Figure 2 shows the tissue distribution of MMAE (bottom) and MMAE released by hydrolysis of compounds II-0 (middle) and II-1 (top) in a mouse orthotopic liver cancer model.

[0063] Figure 3 shows the inhibitory effects of MMAE and compound II-1 on the growth of human hepatocellular carcinoma PLC / PRF / 5-Luciferase in nude mice. Figure A shows the luminescence intensity of mouse liver tumors under different doses of II-1, Figure B shows the changes in mouse body weight under different doses of II-1, and Figure C shows the imaging photographs taken by a small animal in vivo imaging system on days 0, 7, 14, 21, and 28 of the dosing cycle. Detailed Implementation

[0064] These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0065] The following examples are provided to illustrate the preparation process of the GalNAc-peptide-cytotoxin conjugate with succinyl as the linker chain provided by the present invention, and its application in liver diseases, but the present invention is not limited to these examples.

[0066] In the following examples, 1H NMR spectra were recorded using a Bruker AMX-400 NMR spectrometer. Chemical shifts δ are expressed in ppm. NMR calibrations were: δH 7.26 ppm (CDCl3), 2.50 ppm (DMSO-d6), 3.15 ppm (CD3OD). Unless otherwise specified, all reaction solvents were purified using conventional methods. Silica gel (200-300 mesh) used for column chromatography was manufactured by Qingdao Marine Chemical Plant. Unless otherwise specified, all solvents were analytical grade reagents, purchased from Sinopharm Chemical Reagent Co., Ltd. Ultraviolet fluorescence was used for color development. Removal of organic solvents under reduced pressure was performed in a rotary evaporator. In the pharmacological examples, SRB was purchased from Sigma-Aldrich; RPMI-1640 and DMEM culture media were purchased from Corning Cellgro; FBS fetal bovine serum was purchased from Gibco; mass spectrometry was performed by Xevo TQ-S (Waters); ultra-high performance liquid chromatography was performed by UPLC I-Class (Waters); centrifuge was performed by Automatic System; acetonitrile was purchased from Merck; DMSO was purchased from Sigma-Aldrich; and water was purchased from Millipore.

[0067] Compound II-0 can be synthesized according to the method of II-3 in Example II-3 of Chinese Patent Application No. 202310079838.3, the relevant content of which is incorporated herein by reference.

[0068] The specific preparation process of II-0 is as follows:

[0069] II-c: Compound II-b (52 mg, 0.04 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 ml), and 6-amino-1-mercaptohexane (5.3 mg, 0.04 mmol) was added. The mixture was stirred at room temperature for 1 hour, and the reaction was monitored by LC-MS until complete. Then, compound II-a (76.1 mg, 0.04 mmol) and N,N-diisopropylethylamine (10 mg, 0.08 mmol) were added, and the mixture was stirred at room temperature overnight. The reaction was monitored by LC-MS until complete. The mixture was separated by direct C-18 reverse-phase column chromatography, 40%–60% acetonitrile / water (0.1% trifluoroacetic acid). The collected liquid was freeze-dried to give intermediate II-c, a white solid powder (55 mg, 0.017 mmol, 43%). MS (ESI): Calculated value C 148 H 235 N 19 O 50 S is 3166.7, m / z shows [M / 2+H] + =1584.3, [M / 3+H] + =1056.6.

[0070] II-0: Intermediate II-c (55 mg, 0.017 mmol) was dissolved in 2 mL of methanol and stirred at 0 °C for 5 min. 0.27 M sodium methoxide / methanol solution was added dropwise to adjust the pH to 10–11. The reaction was allowed to proceed at room temperature for 2 h. LC-MS was used to monitor the reaction until complete. Preparative separation was performed using a reverse-phase C-18 column. The solution was thawed in 30%–40% acetonitrile / water (0.1% trifluoroacetic acid). The collected liquid was then lyophilized to give II-0, a white solid powder (45 mg, 0.016 mmol, 95%). MS (ESI): Calculated value C 134 H 225 N 19 O 41 S is 2790.43, m / z shows [M / 2+H] + =1396.2, [M / 3+H] + =931.1.

[0071] Example 1: Synthesis of Compound II-1

[0072] II-1: Compound II-0 (30 mg, 0.011 mmol) was dissolved in 2 mL of methanol and stirred at 0 °C for 5 min. A 0.27 M sodium methoxide / methanol solution was added dropwise to adjust the pH to 11–12. The reaction was carried out at 40 °C for 4 h. LC-MS was used to monitor the completeness of the reaction. Preparative separation was performed using a reverse-phase C-18 column. The solution was 30%–40% acetonitrile / water (0.1% trifluoroacetic acid). The collected liquid was freeze-dried to give II-1, a white solid powder (27 mg, 0.010 mmol, 90%). MS (ESI): Calculated value C 134 H 227 N 19 O 42 S is 2808.44, m / z shows [M / 2+H] + =1405.2, [M / 3+H] + =937.1.

[0073] Examples 2-4: Synthesis of compounds II-2 to II-4

[0074] II-2: Compound II-1 (16 mg, 0.0057 mmol), methylamine hydrochloride (2 mg, 0.017 mmol), benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate (HBTU) (4 mg, 0.012 mmol), and N,N-diisopropylethylamine (DIPEA) (5 mg, 0.034 mmol) were dissolved in 2 mL of anhydrous N,N-dimethylformamide (DMF) under argon protection. The reaction was carried out overnight at room temperature. The reaction was monitored by LC-MS until complete. Preparative separation was performed on a reverse-phase C-18 column. The solution was thawed in 30%–40% acetonitrile / water (0.1% trifluoroacetic acid). II-2 was obtained as a white solid powder (9 mg, 0.0032 mmol, 56%). MS (ESI): Calculated value C 135 H 230 N 20 O 41 S is 2821.48, m / z shows [M / 2+H] + =1411.7, [M / 3+H] + =941.5.

[0075] II-3: Compound II-1 (16 mg, 0.0057 mmol), dimethylamine (2 mg, 0.044 mmol), HBTU (4 mg, 0.012 mmol), and DIPEA (5 mg, 0.034 mmol) were dissolved in 2 mL of anhydrous DMF under argon protection and reacted overnight at room temperature. The reaction was monitored by LC-MS until complete. Preparative separation was performed using a reverse-phase C-18 column. The solution was thawed in 30%–40% acetonitrile / water (0.1% trifluoroacetic acid), and the collected liquid was thawed to give II-3 as a white solid powder (10 mg, 0.0035 mmol, 62%). MS (ESI): Calculated value C 135 H 230 N 20 O 41 S is 2835.51, m / z shows [M / 2+H] + =1418.8, [M / 3+H] + =946.2.

[0076] II-4: Compound II-1 (16 mg, 0.0057 mmol), benzylamine (5 mg, 0.044 mmol), HBTU (4 mg, 0.012 mmol), and DIPEA (5 mg, 0.034 mmol) were dissolved in 2 mL of anhydrous DMF under argon protection and reacted overnight at room temperature. The reaction was monitored by LC-MS until complete. Preparative separation was performed using a reverse-phase C-18 column. The collected liquid was lyophilized in 40%-50% acetonitrile / water (0.1% trifluoroacetic acid) to give II-4 as a white solid powder (5 mg, 0.0018 mmol, 31%). MS (ESI): Calculated value C 135 H 230 N 20 O 41 S is 2897.58, m / z shows [M / 2+H] + =1449.8, [M / 3+H] + =966.9.

[0077] Pharmacological test

[0078] Pharmacological test example 1: Stability test of compounds II-0 to II-4 in human and mouse plasma

[0079] The specific steps are as follows: The experiment was conducted in 96-well plates, with a final incubation volume of 320 μL per well. Each well contained 156 μL of plasma, 8 μL (40 μM) of the test compound, and 156 μL of PBS (pH 7.4). 50 μL of culture medium was collected at 0, 0.5, 1, 2, and 5 h of incubation and prepared into 96-well plates containing 200 μL of acetonitrile. After the reaction, the plates were centrifuged, and the supernatant was analyzed by LC-MS / MS to calculate the half-life (T) of each compound in plasma. 1 / 2 The results are shown in Table 1.

[0080] Table 1. Stability tests of compounds II-0 to II-4 in human and mouse plasma

[0081] The results showed that compounds II-1 to II-4 of the present invention exhibited superior stability in human plasma compared to conjugate II-0, which uses a conventional cyclic succinimide linkage. Furthermore, compounds II-1 to II-4 showed improved stability in mouse plasma compared to conjugate II-0. Specifically, compound II-1, with a carboxyl group as its ring-opening component, demonstrated significantly improved stability in mouse plasma. This indicates that the compounds of the present invention can overcome the hydrolytic effects of highly expressed carboxylesterases in plasma.

[0082] Pharmacological test example 2: Hydrolysis efficiency test of compounds II-0 to II-4 in cathepsin B (CTSB)

[0083] Cathepsin B was purchased from Merk (Merk, C0150, derived from human placenta).

[0084] The specific steps are as follows: A stock solution of CTSB (Merk, C0150, derived from human placenta) was prepared in an activation buffer containing 352 mM KH₂PO₄, 48 mM Na₂HPO₄, 4 mM EDTA, and 8 mM L-cysteine ​​HCl (pH 5.0). The CTSB stock solution was then diluted with the activation buffer to obtain a final concentration of 3 μM / mL. The test compound was added to a final concentration of 1 μM, and DMSO (for solubilization) was added to a final concentration of 0.002%. The mixture was incubated at 37°C. A control group was prepared in a buffer without CTSB. Samples were collected at different time points and analyzed by UPLC-MS / MS. The results are shown in Figure 1.

[0085] The results showed that both compound II-1-II-4 of the present invention and conjugate II-0, which uses a conventional cyclic succinimide linkage, were hydrolyzed by cathepsin B to release approximately 60% of MMAE. This demonstrates that the compounds of the present invention possess the ability to release bioactive molecules through hydrolysis.

[0086] Pharmacological Experiment Example 3: Evaluation of tissue distribution of compound MMAE and MMAE released by II-0 and II-1 in an orthotopic hepatocellular carcinoma mouse model.

[0087] Five-week-old male BALB / cA nude mice (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.) were used for this experiment. Under aseptic conditions, PLC-PRF5-Luciferase cells were orally inoculated into the livers of male nude mice. (The psPAX2 plasmid, pMD2.G plasmid, and pLX304Luciferase-V5 blast plasmid (Addgene plasmid, #12259, #12260, and #98580) were transfected into logarithmically growing HEK-293T cells using Lipofectamine 3000. The virus was harvested 48 h post-transfection, filtered through a 0.45 μm filter, and stored at 4°C for later use. PLC / PRF5 cells were infected with lentivirus by incubating with 8 μg / mL polybrene. After 72 h of transfection, 2.5 μg / mL cymoxanil was added for selection. The bioluminescent signal of the PLC / PRF5-Luciferase cell line was detected using a glow-type firefly luciferase reporter gene assay kit (Yeasen, #11404ES60). The cell seeding density was 2.5 × 10⁶ cells / year. 6 / mouse (day d0). Specific steps are as follows: Under aseptic conditions, mice were intraperitoneally injected with 50 mg / kg of Shutai 50 (telatamine hydrochloride for injection, 5ml: 250 mg, batch number: BN 93LWA). After anesthesia, the mouse peritoneum was opened, and the cell suspension (20uL) was directly inoculated into the nude mouse liver in situ, followed by suturing with aseptic sutures. On day 35, all surgical mice were injected with 150 mg / kg of substrate D-luciferin (Cas: 115144-35-9, Lot.: N1102D) according to body weight. Within 10-15 minutes of injection, the mice were anesthetized using a gas anesthesia machine XGI-8qitimazuixitong (anesthesia agent: Isoflurane, batch number: 2023110302). After anesthesia, the mice were photographed and observed using a small animal in vivo imaging system (IVIS Lumina II) (photography parameters: Exposure Time: 5s; Binning: 8; F / Stop: 1.2). After imaging, mice with in situ tumor growth were selected based on luminescence values. Taking into account tumor size, they were randomly divided into a solvent control group (n=6, Vehicle) and different drug administration groups (n=3, Liver tumor-bearing mice at each time point). Compounds II-0, II-1, and the positive control drug MMAE were completely dissolved in a 1:1 ratio of dimethylacetamide (DMAC):polyethylene glycol 15-hydroxystearate (HS15) and stored at -80°C. Before use, they were diluted with physiological saline to the required concentration. On day 42 (d42), compounds II-0 and II-1 were administered via tail vein injection at a dose of 2.5 mg / kg (approximately twice the molar amount of 0.3 mg / kg MMAE), and the positive control drug MMAE was administered at a dose of 0.3 mg / kg. Blood samples were collected at 0.5 h, 1 h, and 4 h after injection, and an esterase inhibitor (Pefabloc in PBS solution, with a final concentration of 1 mM) was immediately added. Simultaneously, after perfusion of mice, the heart, liver, tumor, spleen, lung, and kidney were removed, weighed, and homogenized with a homogenizing reagent containing 1 mM esterase inhibitor at a concentration of 3 mL / g homogenizing factor (4 mL / g homogenizing factor for spleen). The obtained tissue homogenates were stored at -80℃. Samples were then analyzed using UPLC-MS / MS, and the results are shown in Figure 2.

[0088] Tissue distribution results in a mouse orthotopic liver cancer model showed that, after intravenous administration, the content of active molecules released by compounds II-0 and II-1 in liver-related tissues was higher than that of the unconjugated active molecule MMAE, indicating that the conjugates of the present invention have stronger liver-targeting ability, and the tissue distribution of MMAE released by the hydrolysis of compound II-1 of the present invention is significantly better than that of II-0.

[0089] Pharmacological Experiment Example 4: Evaluation of the anti-hepatocellular carcinoma efficacy of the compound and MMAE in a human hepatocellular carcinoma PLC / PRF / 5-Luciferase nude mouse model of hepatoma in situ.

[0090] Five-week-old male BALB / cA nude mice (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.) were used for the experiment.

[0091] Human hepatocellular carcinoma PLC / PRF5-Luciferase cells were orally inoculated into the livers of male nude mice at a seeding density of 2.5 × 10⁻⁶ cells / mL. 6 / mouse. The specific steps are as follows: Under aseptic conditions, mice were intraperitoneally injected with 50 mg / kg of Shutai 50 (telatamine hydrochloride for injection, 5 ml: 250 mg, batch number: BN 93LWA). After anesthesia, the peritoneal cavity of the mice was opened, and the cell suspension (20 uL) was directly inoculated into the liver of nude mice in situ, followed by suturing with aseptic sutures. On the 7th day after inoculation (d0), all surgical mice were injected with 150 mg / kg of substrate D-fluorescein (Cas: 115144-35-9, Lot.: N1 102D) according to their body weight. Within 10-15 minutes after injection, the mice were anesthetized with a gas anesthesia machine XGI-8qitimazuixitong (anesthesia agent is Isoflurane, batch number: 2023110302), and then photographed and observed using a small animal in vivo imaging system (IVIS Lumina II) (photography parameters: Exposure Time: 5s; Binning: 8; F / Stop: 1.2). After imaging, mice with in situ tumor growth were selected based on luminescence values. Taking into account tumor size, they were randomly divided into a solvent control group and different drug administration groups, with 9 mice in each group. The II-15 mg / kg, 2.5 mg / kg, and 1 mg / kg groups received weekly tail vein injections for 4 weeks; the MMAE 0.3 mg / kg group received tail vein injections every four days for 4 weeks (a total of 7 administrations). The solvent control group received an equal volume of physiological saline. Throughout the experiment, mice were anesthetized weekly, and images were taken using a small animal in vivo imaging system (Exposure Time: 5s; Binning: 8; F / Stop: 1.2), while mouse weight was measured. The results are shown in Figure 3.

[0092] The results showed that MMAE 0.3 mg / kg had no inhibitory effect on the growth of human hepatocellular carcinoma PLC / PRF5-Luciferase xenografts implanted in situ in mouse livers. The conjugates of this invention exhibited significant dose-dependent tumor growth inhibition; the high-dose group (5 mg / kg) showed significant tumor inhibition and complete tumor regression, without significant weight loss in mice. Therefore, the compounds of this invention are superior to direct administration of the active molecule, achieving targeted and effective treatment of diseases, reducing the toxic side effects caused by tissue distribution of the active molecule, and improving the safety of this type of linker conjugate.

[0093] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The conjugate of Formula I with succinyl as the linking chain, or a pharmaceutically acceptable salt or solvate thereof, in, A is a ligand containing a connecting chain that specifically targets and recognizes tissues; B is an active ligand containing a peptide chain that has therapeutic and / or diagnostic effects on diseases; X1 is either S or NR; where R is selected from H, C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group, C6-C 20 Aryl, C6-C 20 Aryl C1-C 10 Alkyl groups, 5-20 membered heteroaryl groups containing one or more heteroatoms selected from N, O, and S, and 5-20 membered heteroaryl C1-C 10 alkyl; X2 is O, S, or NR; where R is selected from H, Cl-C. 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group, C6-C 20 Aryl, C6-C 20 Aryl C1-C 10 Alkyl groups, 5-20 membered heteroaryl groups containing one or more heteroatoms selected from N, O, and S, and 5-20 membered heteroaryl C1-C 10 alkyl; P is selected from H, C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group, C6-C 20 Aryl, C6-C 20 Aryl C1-C 10 Alkyl groups, 5-20 membered heteroaryl groups containing one or more heteroatoms selected from N, O, and S, and 5-20 membered heteroaryl C1-C 10 alkyl; L1 is selected from -(CH2) k1 -Z1-(CH2) k2 -、-(CH2) k3 -(O-CH2-CH2-O) k4 -(CH2) k5 -、-(CH2) k6 -NH-C(O)-(CH2) k7 -Z1-(CH2) k8 -C(O)NH-(CH2) k9 - and -(CH2) k10 -NH-C(O)-(CH2) k11 -(O-CH2-CH2-O) k12 -C(O)NH-(CH2) k13 - where k1 to k13 are each an integer from 0 to 10; Z1 is independently NH, O or S.

2. The conjugate of Formula I according to claim 1, with succinyl as the linking chain, or a pharmaceutically acceptable salt or solvate thereof, characterized in that, The conjugate shown in Formula I, with succinyl as the linking chain, has the structure shown in Formula II. in, X1 is S or NH; preferably, X1 is S; X2 is O, S, or NR; where R is H, C1-C4 alkyl, or C6-C4 alkyl. 20 Aryl, C6-C 12 Aryl C1-C4 alkyl; preferably, X2 is O, NH, or NCH3; P is selected from H, C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group, C6-C 20 Aryl, C6-C 20 Aryl C1-C 10 Alkyl groups, 5-20 membered heteroaryl groups containing one or more heteroatoms selected from N, O, and S, and 5-20 membered heteroaryl C1-C 10 Alkyl groups; preferably selected from H, C1-C4 alkyl groups, C6-C4 alkyl groups. 20 Aryl, C6-C 12 Aryl C1-C4 alkyl; preferably, P is H, -CH3, -CH2CH3 or -benzyl; L1 is selected from -(CH2) k1 -Z1-(CH2) k2 -、-(CH2) k3 -(O-CH2-CH2-O) k4 -(CH2) k5 -、-(CH2) k6 -NH-C(O)-(CH2) k7 -Z1-(CH2) k8 -C(O)NH-(CH2) k9 - and -(CH2) k10 -NH-C(O)-(CH2) k11 -(O-CH2-CH2-O) k12 -C(O)NH-(CH2) k13 - where k1 to k13 are each an independent integer from 0 to 10; Z1 is independently NH, O, or S; preferably, L1 is -(CH2). k1 , where k1 is an integer from 1 to 10, especially -(CH2)6-; In Part A: G1, G2, and G3 are each independently an N-acetylgalactosamine group or an analogue thereof; preferably, G1, G2, and G3 are all N-acetylgalactosamine groups. L2, L3, and L4 are each independently selected from -(CH2). n1 -(O-CH2-CH2) n2 -(CH2) n3 -、-(CH2) n4 -(O-CH2-CH2) n5 -NH-C(O)-(CH2) n6 - where n1 to n6 are each an independent integer from 0 to 10; preferably, L1, L2, and L3 are each independently -(CH2). n1 , where n1 is an integer from 1 to 10, specifically selected from -(CH2)6- and -(CH2)4-; Y1, Y2, and Y3 are each independently selected from -Z2-(CH2). m1 - and -NHC(O)-(CH2) m2 -Z2-(CH2) m3 - where m1 to m3 are each an integer from 0 to 10, and Z2 is NH or O independently; preferably, Y1, Y2, and Y3 are each independently selected from -O-CH2- and -NH-C(O)-(CH2)2-O-CH2-; L5 is selected from -(CH2) j1 -Z3-(CH2) j2 -、-(CH2) j3 -(O-CH2-CH2-O) j4 -(CH2) j5 -、-(CH2) j6 -NH-C(O)-(CH2) j7 -、-(CH2) j8 -NH-C(O)-(CH2) j9 -Z3-(CH2) j10 -C(O)NH-(CH2) j11 - and -(CH2) j12 -NH-C(O)-(CH2) j13 -(O-CH2-CH2-O) j14 -C(O)NH-(CH2) j15 - where j1 to j15 are each an independent integer from 0 to 10 (e.g., 1, 2, 3, 4, 5, 6, etc.), and Z3 is independently NH, O, or S; preferably, L5 is -(CH2). j6 NH-C(O)-(CH2) j7 -, where j6 to j7 are each an independent integer from 0 to 6, especially -NH-C(O)-(CH2)3-; In Part B: L6 is -(CH2) q1 -C(O)-, where q1 is an integer from 0 to 10; preferably, L6 is -(CH2)5-C(O)-; Q represents a peptide fragment that can be hydrolyzed and cleaved in target tissues of the liver to release T; T refers to a small molecule, nucleic acid, polypeptide, or protein that is bioactive or labeled for the treatment, diagnosis, and / or prevention of diseases.

3. The conjugate of Formula I according to claim 2, with succinyl as the linking chain, or a pharmaceutically acceptable salt or solvate thereof, characterized in that, In formula II, Q is a dipeptide or polypeptide fragment structure that can be cleaved by cathepsins in lysosomes; preferably, Q is a valine-citrulline-p-aminobenzylcarbamate group as shown in the following formula:

4. The conjugate of Formula I according to claim 2, with succinyl as the linking chain, or a pharmaceutically acceptable salt or solvate thereof, characterized in that, In Formula II, T represents a small molecule compound for treating liver diseases and a marker molecule for treating liver diseases; The small molecule compounds include cytotoxins, chemotherapeutic agents (including inhibitors or agonists of targets for treatment of related diseases), and immunomodulators; The labeling molecules for treating liver diseases include radiolabels, fluorescent labels, diagnostic agents, or detectable modified enzymes of catalytic substrates; Preferably, the cytotoxin is methylauratestatin E or a derivative thereof; More preferably, T is The wavy line indicates the junction of toxin molecules via amide bonds.

5. The conjugate of Formula I according to claim 1, with succinyl as the linking chain, or a pharmaceutically acceptable salt or solvate thereof, characterized in that, The compound represented by Formula I is selected from the following compounds II-1 to II-4:

6. The method for preparing the coupling compound with succinyl group as the linking chain according to any one of claims 1-5, wherein the reaction route is as follows: in, A, B, L1, X1, X2 and P are respectively defined as in claims 1-5, and when X2 is 0, P is not simultaneously H; Its preparation methods include: (1) Compound III is hydrolyzed under alkaline conditions to give compound I-1; (2) Compound I-1 undergoes a condensation reaction with compound HX2P to obtain compound I-2.

7. The preparation method according to claim 6, characterized in that, Step (1) is carried out under solvent conditions, wherein the solvent is an organic solvent, such as methanol; the base includes inorganic bases and organic bases, preferably one or more selected from sodium methoxide, sodium ethoxide, and potassium tert-butoxide; the reaction temperature is 0°C to room temperature; the reaction time is 0.5-24 hours; and / or In step (2), the condensing agent used in the condensation reaction is selected from one or more of benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate, dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole, 1,8-diazabicycloundec-7-ene, N,N-diisopropylethylamine, triethylamine, and pyridine; the preferred condensing agent is a combination of benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine; and / or Step (2) is carried out under solvent conditions, wherein the solvent is an organic solvent, such as dichloromethane or dimethylformamide; the reaction temperature is 0°C to 60°C; and the reaction time is 0.5 to 24 hours.

8. A pharmaceutical composition comprising a therapeutically effective amount of the succinyl-linked conjugate or its pharmaceutically acceptable salt or solvate as described in any one of claims 1-5, and optionally a pharmaceutically acceptable excipient.

9. The use of the conjugate with succinyl as the linking chain as claimed in any one of claims 1-5, or a pharmaceutically acceptable salt or solvate thereof, and the use of the pharmaceutical composition of claim 8 in the preparation of a medicament for treating liver diseases.

10. The use according to claim 9, characterized in that, The liver disease mentioned is liver cancer.

Images