Compound taking nitrogen-containing heterocyclic ring as mother nucleus, preparation method of compound, conjugate and medicine

Abstract

The invention provides a compound taking a nitrogen-containing heterocyclic ring as a parent nucleus, a preparation method of the compound, a conjugate and a medicine, and belongs to the technical field of gene engineering. The compound taking the nitrogen-containing heterocyclic ring as the parent nucleus provided by the invention contains a 1, 4, 7, 10-tetraazacyclododecane structure, so that the stability of the whole compound in a living body can be improved, the compound containing a cell-targeting ligand is not easy to degrade in the living body, and further, the effect of improving the stability of the compound and biomolecules (such as a cell-targeting ligand) is facilitated. The stability of the conjugate formed by connecting the nucleic acid molecules in an organism can be improved, the stability of the biomolecules can also be improved, and the decomposition rate of the biomolecules can be reduced; when the conjugate is used for cell targeted delivery, the effectiveness of a delivery system can be improved, the delivery effect is improved, and the effect of biomolecules is further improved.

Description

[0001] This application claims the priority right to the patent application filed on July 12, 2023, with application number 2023108548155 and invention name “Compounds with nitrogen-containing heterocycle as parent core, preparation methods, conjugates and drugs thereof”. Technical Field

[0002] The present application relates to the field of genetic engineering technology, and in particular to a compound with a nitrogen-containing heterocycle as a parent core, a preparation method thereof, a conjugate and a drug. Background Art

[0003] The delivery system connects cell targeting ligands to compounds such as oligonucleotide drugs and delivers the compounds to specific cells to achieve therapeutic or diagnostic effects.

[0004] However, existing delivery systems have poor stability in vivo and are easily degraded in vivo, resulting in the expected amount of oligonucleotide drugs and other compounds in the delivery system reaching specific cells, or even the inability of oligonucleotide drugs and other compounds in the delivery system to effectively reach specific cells, thereby making it impossible to effectively carry out treatment or diagnosis. Summary of the invention

[0005] The purpose of the present application is to provide a compound with a nitrogen-containing heterocycle as a parent core and its salt, salt hydrate, enantiomer or optical isomer and its preparation method, conjugate and drug, which can improve the stability of the compound containing cell targeting ligand in the organism, thereby improving the delivery effect of the delivery system.

[0006] In a first aspect, the present application provides a compound with a nitrogen-containing heterocycle as a parent core and its salt, hydrate of the salt, enantiomer or optical isomer. The structural formula of the compound with a nitrogen-containing heterocycle as a parent core is shown in Formula I.

[0007] Formula I is as follows:

[0008]

[0009] Wherein, L1, L2, L3 and each independently are linking groups; X1, X2, X3 and X4 are each independently methylene, imino, O atom or S atom; at least one of S1, S2 and S3 is a cell targeting ligand; A1 is a linker for connecting biological molecules; B1 is a solid phase carrier, and z is 0 or 1.

[0010] The compound with a nitrogen-containing heterocycle as a parent core provided in the present application contains a 1,4,7,10-tetraazacyclododecane structure, which can improve the stability of the entire compound and its salts, salt hydrates, enantiomers or optical isomers in the body, so that the compound containing a cell targeting ligand and its salts, salt hydrates, enantiomers or optical isomers are not easily degraded in the body, which is beneficial to improve the stability of the conjugate formed by connecting the compound and its salts, salt hydrates, enantiomers or optical isomers with biological molecules (for example, nucleic acid molecules) in the body, and can also improve the stability of the biological molecules and reduce the decomposition rate of the biological molecules; the conjugate is used for cell-targeted delivery, which can improve the effectiveness of the delivery system, improve the delivery effect, and thus enhance the effect of the biological molecule in the organ (for example, cancer cells in the liver).

[0011] In the second aspect, the present application provides a method for preparing a compound with a nitrogen-containing heterocycle as a parent core and its salt, hydrate of the salt, enantiomer or optical isomer as provided in the first aspect above; z in formula I is 0, and the preparation method comprises: deprotecting G1 in the substance shown in formula II, and coupling it with a group containing a linker for connecting biological molecules. Or, z in formula I is 1, and the preparation method comprises: deprotecting G1 in the substance shown in formula II, and coupling it with a group containing a linker for connecting biological molecules to obtain a substance shown in formula III; and coupling the substance shown in formula III with a solid phase carrier. Or, z in formula I is 0, and the preparation method comprises: replacing Q in formula IV with a linker containing a linker for connecting biological molecules. Or, z in formula I is 1, and the preparation method comprises: replacing Q in formula IV with a linker containing a linker for connecting biological molecules to obtain a substance shown in formula III; and coupling the substance shown in formula III with a solid phase carrier.

[0012] Wherein, formula II is as follows:

[0013]

[0014] L1, L2, L3, L4, X1, X2, X3 and X4 are each independently a linker; at least one of S1, S2 and S3 is a cell targeting ligand; and G1 is a protecting group.

[0015] Formula III is as follows:

[0016]

[0017] L1, L2, L3, L4, X1, X2, X3 and X4 are each independently a linker; at least one of S1, S2 and S3 is a cell targeting ligand; A1 is a linker for connecting biological molecules.

[0018] Formula IV is as follows:

[0019]

[0020] L1, L2, L3 and L4 are each independently a linker; X1, X2, X3 and X4 are each independently a methylene group, an imino group, an O atom or an S atom; at least one of S1, S2 and S3 is a cell targeting ligand; Q is a hydrogen atom.

[0021] By using the above-mentioned preparation method provided in the present application, the compound with nitrogen-containing heterocycle as the parent core and having good stability in the organism provided in the first aspect and its salt, hydrate of the salt, enantiomer or optical isomer can be prepared.

[0022] In a third aspect, the present application provides a conjugate, which includes a covalently linked biological molecule and a compound with a nitrogen-containing heterocycle as a parent core as provided in the first aspect above, and its salt, hydrate of the salt, enantiomer or optical isomer; wherein the biological molecule is covalently linked to A1 in formula I.

[0023] The conjugate provided in the present application is connected with the compound with a nitrogen-containing heterocycle as a parent core provided in the first aspect above and its salt, hydrate of the salt, enantiomer or optical isomer, so the stability of the conjugate in the organism can be improved. The conjugate is used for cell-targeted delivery, which can improve the effectiveness of the delivery system, improve the delivery effect, and thus enhance the effect of the biological molecule in the organ (for example, cancer cells in the liver).

[0024] In a fourth aspect, the present application provides a drug, which includes the conjugate provided in the third aspect above. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 This is the H NMR spectrum of compound 1a-7 prepared in step (5) of Example 1 of the present application.

[0027] Figure 2 This is the H NMR spectrum of compound 1a prepared in Example 1 of the present application.

[0028] Figure 3 This is the H NMR spectrum of compound 1a' prepared in Example 2 of the present application.

[0029] Figure 4 This is a comparison chart of the delivery effects of compound 1a' prepared in Example 2 of the present application and the compound provided in the comparative example. DETAILED DESCRIPTION

[0030] The present application provides a compound with a nitrogen-containing heterocycle as a parent core and its salt, hydrate of the salt, enantiomer or optical isomer. The structural formula of the compound with a nitrogen-containing heterocycle as a parent core is shown in Formula I.

[0031] Formula I is as follows:

[0032]

[0033] Wherein, L1, L2, L3 and L4 are each independently a linking group; X1, X2, X3 and X4 are each independently selected from a methylene group, an imino group, an O atom or an S atom; at least one of S1, S2 and S3 is a cell targeting ligand; A1 is a linker for connecting biological molecules; B1 is a solid phase carrier, and z is 0 or 1.

[0034] It should be noted that, in the present application, a cell targeting ligand refers to a ligand that binds to a cell surface receptor. Biomolecules include nucleic acid molecules (e.g., siRNA), amino acids or proteins, etc. A salt of a "compound with a nitrogen-containing heterocycle as a parent nucleus" refers to a chemically acceptable salt of a compound with a nitrogen-containing heterocycle as a parent nucleus. A hydrate of a salt of a "compound with a nitrogen-containing heterocycle as a parent nucleus" refers to a substance formed by a chemically acceptable salt of a compound with a nitrogen-containing heterocycle as a parent nucleus combined with water molecules.

[0035] It can be understood that “at least one of S1, S2 and S3 is a cell targeting ligand” means: only one of S1, S2 and S3 can be a cell targeting ligand, any two of S1, S2 and S3 can be cell targeting ligands, or all of S1, S2 and S3 can be cell targeting ligands.

[0036] The compound with nitrogen-containing heterocycle as the parent core provided by the present application contains 1,4,7,10-tetraazacyclododecane structure, which can improve the stability of the whole compound and its salt, salt hydrate, enantiomer or optical isomer in the organism, so that the compound containing cell targeting ligand and its salt, salt hydrate, enantiomer or optical isomer (that is, the compound with nitrogen-containing heterocycle as the parent core of the present application and its salt, salt hydrate, enantiomer or optical isomer) is not easy to degrade in the organism, which is conducive to improving the stability of the conjugate formed by the compound and its salt, salt hydrate, enantiomer or optical isomer and biological molecules (for example, nucleic acid molecules) in the organism, and can also improve the stability of biological molecules and reduce the decomposition rate of biological molecules. The conjugate is used for cell-targeted delivery, which can improve the effectiveness of the delivery system, improve the delivery effect, and thus enhance the effect of biological molecules in organs (for example, cancer cells in the liver).

[0037] The inventors found that compared with the group -CO- between the N atom and X1, X2, X3 and X4 in the tetraazacyclic ring (i.e., the structure is as shown in formula a), the group between the N atom and X1, X2, X3 and X4 in the tetraazacyclic ring is -CO-CH2- (i.e., the compound with a nitrogen-containing heterocycle as the parent core of the present application and its salt, hydrate of the salt, enantiomer or optical isomer, the structure is as shown in formula b), which can improve the stability of the compound containing the cell targeting ligand and its salt, hydrate of the salt, enantiomer or optical isomer in the organism; the inventors analyzed that the reason may be: in the tetraazacyclic parent core as shown in formula b, the methylene group between the N atom and -CO- is beneficial to reduce the risk of degradation of the tetraazacyclic parent core by amide hydrolases in the body, thereby improving the stability of the compound containing the cell targeting ligand and its salt, hydrate of the salt, enantiomer or optical isomer in the organism.

[0038] Wherein, the structural formula of formula a is as follows:

[0039]

[0040] The structural formula of formula b is as follows:

[0041]

[0042] If the compound with a nitrogen-containing heterocycle as the parent core provided in the present application and its salt, salt hydrate, enantiomer or optical isomer A1 are connected to siRNA to form a conjugate, the structural stability of the conjugate in the organism can be better. When the conjugate is used to interfere with the translation of RNA protein, the RNA interference ability of the conjugate can be improved, the translation of the target protein can be inhibited, and the production of the target protein can be reduced.

[0043] In some feasible embodiments of the present application, L1, L2, L3 and L4 are each independently selected from at least one of a C2-C30 alkyl group and a C2-C30 alkoxy group, and the C2-C30 alkyl group and the C2-C30 alkoxy group are each independently connected or not connected to at least one of an imino group (-NH-), a carbonyl group (-CO-) and an amide group (-CONH-). L1, L2, L3 and L4 are selected from the above groups, so that the compound containing the cell targeting ligand and its salt, salt hydrate, enantiomer or optical isomer (i.e., the compound with a nitrogen-containing heterocycle as the parent core and its salt, salt hydrate, enantiomer or optical isomer of the present application) can have a higher stability in the body and is not easily degraded.

[0044] It should be noted that, in the present application, "C2-C30 alkyl" refers to: an alkyl group with 2 to 30 carbon atoms; "C2-C30 alkoxy" refers to: an alkoxy group with 2 to 30 carbon atoms; by way of example, the number of carbon atoms can be 2, 3, 5, 8, 10, 12, 15, 20, 25 or 30, etc.

[0045] Further, L1, L2, L3 and L4 are each independently selected from -(CH2-CH2-O) m2 -CH2-, -(CH2) m3 -O-(CH2) m4 -、-(CH2) m5 -O-, -(CH2-CH2-O) m2 -(CH2) m6 -、-(CH2) m7 -O-(CH2) m8 -O- and -(CH2) m9 -O-(CH2) m10 -O-(CH2) m11 -at least one of, m2, m3, m4, m5, m7, m8, m9, m10 and m11 are each independently a natural number of 1 to 5, and m6 is a natural number of 2 to 5. L1, L2, L3 and L4 are each independently selected from the above groups, which can improve the hydrophilicity of the compound containing the cell targeting ligand and its salt, hydrate of the salt, enantiomer or optical isomer (i.e., the compound with a nitrogen-containing heterocycle as the parent core and its salt, hydrate of the salt, enantiomer or optical isomer of the present application), and is more suitable for the in vivo environment.

[0046] Illustratively, m2 is 2, 3 or 4; m3 and m4 are each independently 3, 4 or 5; m5 to m11 are each independently 2, 3, 4 or 5.

[0047] Further, L1, L2, L3 and L4 are each independently selected from -(CH2)6-CO-, -(CH2) 12-NH-CO-, -(CH2)5-CO-NH-(CH2)5-, -(CH2)4-CO-NH-(CH2)3-, -(CH2)4-CO-NH-(CH2)4-, -(CH2)5-NH-(CH2)3-, -(CH2)5-NH-(CH2)4-CO-, -(CH2)6-NH-CH2-NH-CO-, -(CH2CH2O)2-(CH2)2-NH-CO-. L1, L2, L3 and L4 are each independently selected from the above groups, which can improve the hydrophilicity of the compound containing the cell targeting ligand and its salt, salt hydrate, enantiomer or optical isomer (i.e., the compound with nitrogen-containing heterocycle as the parent core and its salt, salt hydrate, enantiomer or optical isomer of the present application) and be more suitable for the in vivo environment.

[0048] It should be noted that, in other feasible embodiments, L1, L2, L3 and L4 can also be independently selected from -(CH2) m1 -; wherein m1 is a natural number of 2-10; as an example, L1, L2, L3 and L4 are each independently selected from -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2) 11 -wait.

[0049] In some feasible embodiments of the present application, L1, L2, L3 and L4 are each independently selected from -(CH2)6-, -(CH2)6-O-, -(CH2)2-, -(CH2)2-O-, -(CH2)3-, -(CH2)3-O-, -(CH2)4-, -(CH2)4-O-, -(CH2)3-O-(CH2)2-, -(CH2)3-O-(CH2)2-O-, -(CH2)3-O-(CH2-CH2-O)2- or -(CH2)3-O-CH2-CH2-O-CH2-CH2-.

[0050] It should be noted that in other feasible embodiments of the present application, L1, L2, L3 and L4 may also be -CH2- independently.

[0051] In some feasible implementations of the present application, L4 may also be represented by the following structural formula:

[0052]

[0053] Wherein, L5, L6 and L7 are each independently a linking group; Q1 is selected from a methine group or a N atom; and M1 is a protecting group.

[0054] L4 is selected from the structure shown in the above formula, which can make the compound containing the cell targeting ligand and its salt, salt hydrate, enantiomer or optical isomer (that is, the compound with a nitrogen-containing heterocycle as the parent core and its salt, salt hydrate, enantiomer or optical isomer in the present application) have higher stability in the organism and are not easy to degrade.

[0055] Further, L5, L6 and L7 are each independently selected from at least one of a C1-C30 alkyl group and a C1-C30 alkoxy group, and the C1-C30 alkyl group and the C1-C30 alkoxy group are each independently connected to or not connected to at least one of an imino group (-NH-), a carbonyl group (-CO-) and an amide group (-CONH-). For example, L5, L6 and L7 are each independently -CH2-O- or -CH2-.

[0056] It should be noted that, in the present application, "C1-C30 alkyl" refers to: an alkyl group with 1 to 30 carbon atoms; "C1-C30 alkoxy" refers to: an alkoxy group with 1 to 30 carbon atoms; by way of example, the number of carbon atoms can be 1, 2, 3, 5, 8, 10, 12, 15, 20, 25 or 30, etc.

[0057] In some feasible embodiments, L5, L6 and L7 are each independently selected from an imino group (-NH-), a carbonyl group (-CO-) and an amide group (-CONH-), which can improve the hydrophilicity of the compound containing the cell targeting ligand and its salt, salt hydrate, enantiomer or optical isomer (i.e., the compound with a nitrogen-containing heterocycle as the parent core and its salt, salt hydrate, enantiomer or optical isomer) and is more suitable for the in vivo environment. For example, L5, L6 and L7 are each independently

[0058] Further, M1 is selected from any one of acetyl, benzoyl, benzyl, dimethoxytrityl, methoxytrityl, trityl, methoxymethyl ether, p-methoxybenzyl ether, pivaloyl and tetrahydropyranyl. For example, M1 is dimethoxytrityl.

[0059] As an example, L4 is shown in the following structural formula:

[0060]

[0061] In some feasible embodiments of the present application, X1, X2, X3 and X4 are all imino groups, which can make the compound containing the cell targeting ligand and its salt, salt hydrate, enantiomer or optical isomer more stable in the body and not easy to degrade.

[0062] In some feasible embodiments of the present application, A1 is a linker containing a phosphoramidite group or a succinate group, which can make A1 easy to connect with biological molecules, and can also make the conjugate formed by connecting A1 with biological molecules in the compound with a nitrogen-containing heterocycle as a parent core, its salt, salt hydrate, enantiomer or optical isomer better in vivo.

[0063] In some feasible embodiments of the present application, the structural formula of A1 is as follows:

[0064]

[0065] Wherein, R1 and R2 are independently selected from C1-C20 alkyl groups; or, -R1R2 is a cyclic carbon chain containing or not containing heteroatoms, and the heteroatoms are selected from at least one of N atoms and O atoms, and the cyclic carbon chain is a five-membered carbon chain or a six-membered carbon chain. X5 is selected from methylene, O atoms or S atoms; n is 0 or 1. R3 is selected from a C1-C20 alkyl group substituted with a thio group, a thio group, an oxo group, a halogen or a cyano group (-CN).

[0066] Further, -NR1R2 is selected from any one of dimethylamino, diethylamino, di-n-propylamino, diisopropylamino, dibutylamino, pyrrolidinyl, piperidinyl, 2,6-dimethylpiperidinyl, morpholinyl, imidazolyl and 4-methylimidazolyl. -NR1R2 is selected from the above groups to improve the bonding stability of P-NR1R2 and the reactivity of P-NR1R2 in coupling reactions with biomolecules.

[0067] -R3 is selected from any one of 2-cyanoethyloxy, 2-cyanoethylthio, methoxy, ethoxy, S-isobutyryl-2-(2-mercaptoethoxy)ethoxy, S-pivaloyl-2-(2-mercaptoethoxy)ethoxy and S-pivaloyl-2-mercaptoethoxy.

[0068] Further, -NR1R2 is diisopropylamino, and -R3 is 2-cyanoethoxy. The above technical scheme can make the compound containing the cell targeting ligand and its salt, salt hydrate, enantiomer or optical isomer (i.e. the compound with nitrogen-containing heterocycle as the parent core and its salt, salt hydrate, enantiomer or optical isomer of the present application) more stable in vivo, and A1 has higher reactivity and is easier to connect with biological molecules (e.g., nucleic acid molecules, etc.). For example, -NR1R2 is diisopropylamino, n is 0, and -R3 is 2-cyanoethoxy.

[0069] As an example, the structural formula of A1 is as follows:

[0070]

[0071] In some feasible embodiments of the present application, B1 is a porous glass (CPG) support or a polystyrene (PS) support.

[0072] It should be noted that in other feasible embodiments, B1 can be other solid phase carriers, for example, polypropylene supports, polyethylene and cycloolefin copolymer supports, etc.

[0073] In some feasible embodiments of the present application, -A1-(B1) z Select from any of the following structures:

[0074] Wherein, Y is a porous glass (CPG) support or a polystyrene (PS) support. -A1-(B1) z By selecting any of the above structures, the compound containing the cell targeting ligand and its salt, salt hydrate, enantiomer or optical isomer (i.e., the compound with a nitrogen-containing heterocycle as the parent core and its salt, salt hydrate, enantiomer or optical isomer in the present application) can have stronger structural stability in the body, and A1 has higher reactivity and is easier to connect with biological molecules (for example, nucleic acid molecules, etc.).

[0075] In some feasible embodiments of the present application, the active groups in S1, S2 and S3 are protected by protecting groups.

[0076] Furthermore, the protecting groups in S1, S2 and S3 are selected from any one of acetyl, benzoyl, benzyl, dimethoxytrityl, methoxytrityl, trityl, methoxymethyl ether, p-methoxybenzyl ether, pivaloyl and tetrahydropyranyl.

[0077] In some feasible embodiments of the present application, S1, S2 and S3 are all cell-targeting ligands; and S1, S2 and S3 are each independently a liver-targeting specific ligand or a thyroid hormone receptor agonist.

[0078] In some feasible embodiments of the present application, S1, S2 and S3 are each independently a targeting polydentate saccharide ligand.

[0079] Further, S1, S2 and S3 are each independently selected from N-acetylgalactosamine, D-pyranose, L-pyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-pyranose, β-D-mannopyranose, α-D-glucose, β-D-glucose, α-D-furanose, At least one of glucopyranose, β-D-glucopyranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, ribose, D-ribose, D-4-thioribose, L-ribose and L-4-thioribose.

[0080] Furthermore, S1, S2 and S3 are all N-acetylgalactosamine (ie, GalNAc), which can tightly bind to hepatocyte proteins (hepatic acylglycoprotein receptor, ASGPR) to achieve targeting of liver ASGPR.

[0081] In some feasible embodiments of the present application, any active group in S1, S2 and S3 is protected by a protecting group, and the protecting group is selected from any one of acetyl, benzoyl, benzyl, dimethoxytrityl, methoxytrityl, trityl, methoxymethyl ether, p-methoxybenzyl ether, pivaloyl and tetrahydropyranyl.

[0082] Furthermore, the active group is a hydroxyl group and the protecting group is an acetyl group.

[0083] As an example, S1, S2 and S3 can each independently be the following structure:

[0084]

[0085] It should be noted that, in other feasible embodiments, S1, S2 and S3 may also be selected from other cell targeting ligands.

[0086] In some feasible embodiments of the present application, Formula I is as follows:

[0087]

[0088] Wherein, L1, L2, L3 and L4 are each independently a linking group; X1, X2, X3 and X4 are each independently a methylene group, an imino group, an O atom or an S atom; at least one of S1, S2 and S3 is a cell targeting ligand; and W1 is a protecting group.

[0089] The present application obtains the structure shown in the above formula by designing the structure of the compound with nitrogen-containing heterocycle as the parent core and its salt, hydrate of salt, enantiomer or optical isomer, so that when the compound with nitrogen-containing heterocycle as the parent core and its salt, hydrate of salt, enantiomer or optical isomer is connected with the nucleic acid molecule to form a conjugate, the compound with nitrogen-containing heterocycle as the parent core and its salt, hydrate of salt, enantiomer or optical isomer can be connected to the 3' end of the nucleic acid molecule.

[0090] Further, W1 is selected from any one of acetyl, benzoyl, benzyl, dimethoxytrityl, methoxytrityl, trityl, methoxymethyl ether, p-methoxybenzyl ether, pivaloyl and tetrahydropyranyl. For example, W1 is dimethoxytrityl.

[0091] In some feasible embodiments of the present application, Formula I is as follows: Formula I is as follows:

[0092]

[0093] Wherein, L1, L2, L3 and L4 are each independently a linking group; X1, X2, X3 and X4 are each independently a methylene group, an imino group, an O atom or an S atom; at least one of S1, S2 and S3 is a cell targeting ligand; W2 is a protecting group; and B1 is a solid phase carrier.

[0094] The present application obtains the structure shown in the above formula by designing the structure of the compound with nitrogen-containing heterocycle as the parent core and its salt, hydrate of salt, enantiomer or optical isomer, so that when the compound with nitrogen-containing heterocycle as the parent core and its salt, hydrate of salt, enantiomer or optical isomer is connected with the nucleic acid molecule to form a conjugate, the compound with nitrogen-containing heterocycle as the parent core and its salt, hydrate of salt, enantiomer or optical isomer can be connected to the 3' end of the nucleic acid molecule.

[0095] Further, W2 is selected from any one of acetyl, benzoyl, benzyl, dimethoxytrityl, methoxytrityl, trityl, methoxymethyl ether, p-methoxybenzyl ether, pivaloyl and tetrahydropyranyl. For example, W2 is dimethoxytrityl.

[0096] In some feasible embodiments of the present application, Formula I is selected from any of the following structures:

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104] Among them, Y1 is a porous glass support, Y2 is a polystyrene support, Y3 is a porous glass support or a polystyrene support, T1 is a solid phase carrier; Ac is an acetyl group, DMTr is a dimethoxytrityl group, and CN is a cyano group.

[0105] Formula I is a substance selected from any of the above structures, which can make the entire compound and its salt, salt hydrate, enantiomer or optical isomer have better stability in the body, so that the compound containing GalNAc and its salt, salt hydrate, enantiomer or optical isomer is not easy to degrade in the body, which is beneficial to improve the stability of the conjugate formed by connecting the compound and its salt, salt hydrate, enantiomer or optical isomer with a biological molecule (for example, a nucleic acid molecule) in the body. The conjugate is used for cell-targeted delivery, which can improve the effectiveness of the delivery system, improve the delivery effect, and thus enhance the effect of the biological molecule in the organ (for example, cancer cells in the liver).

[0106] Furthermore, Formula I is selected from any of the following structures:

[0107]

[0108] Formula I is a substance selected from any of the above structures. The conjugate formed by the substance with the above structure and an oligonucleotide (eg, siRNA) is more conducive to the function of the oligonucleotide.

[0109] The present application provides a method for preparing a compound with a nitrogen-containing heterocycle as a parent core and its salt, hydrate of the salt, enantiomer or optical isomer as provided in the first aspect above. When z in formula I is 0, the method for preparing a compound with a nitrogen-containing heterocycle as a parent core and its salt, hydrate of the salt, enantiomer or optical isomer comprises: deprotecting G1 in the substance shown in formula II and coupling it with a group containing a linker for connecting biological molecules.

[0110] Or, when z in formula I is 1, the preparation method of the compound with a nitrogen-containing heterocycle as the parent core and its salt, salt hydrate, enantiomer or optical isomer comprises: deprotecting G1 in the substance shown in formula II, coupling with a group containing a linker for connecting biological molecules to obtain the substance shown in formula III; coupling the substance shown in formula III with a solid phase carrier.

[0111] Or, when z in formula I is 0, the preparation method of the compound with a nitrogen-containing heterocycle as a parent core and its salt, salt hydrate, enantiomer or optical isomer comprises: replacing Q in formula IV with a linker containing a linker for connecting biological molecules.

[0112] Or, z in formula I is 1, and the preparation method of the compound with a nitrogen-containing heterocycle as the parent core and its salt, hydrate of the salt, enantiomer or optical isomer comprises: replacing Q in formula IV with a linker containing a compound for connecting biological molecules to obtain a substance shown in formula III; coupling the substance shown in formula III with a solid phase carrier.

[0113] Wherein, formula II is as follows:

[0114]

[0115] L1, L2, L3 and L4 are each independently a linking group; X1, X2, X3 and X4 are each independently a methylene group, an imino group, an O atom or an S atom; at least one of S1, S2 and S3 is a cell targeting ligand; G1 is a protecting group.

[0116] Formula III is as follows:

[0117]

[0118] L1, L2, L3 and L4 are each independently a linker; X1, X2, X3 and X4 are each independently a methylene group, an imino group, an O atom or an S atom; at least one of S1, S2 and S3 is a cell targeting ligand; A1 is a linker for connecting biological molecules.

[0119] Formula IV is as follows:

[0120]

[0121] L1, L2, L3 and L4 are each independently a linker; X1, X2, X3 and X4 are each independently a methylene group, an imino group, an O atom or an S atom; at least one of S1, S2 and S3 is a cell targeting ligand; Q is a hydrogen atom.

[0122] By using the above-mentioned preparation method provided in the present application, the above-mentioned compound with a nitrogen-containing heterocycle as a parent core and its salt, hydrate of the salt, enantiomer or optical isomer which has good stability in the organism can be prepared, and the preparation method is simple and easy.

[0123] As an example, G1 can be selected from dimethoxytriphenylmethane chloride (DMT-Cl), trityl chloride, or 4′-methoxytrityl chloride, etc.

[0124] In some feasible embodiments of the present application, the preparation method of the substance represented by Formula II comprises: coupling the substance represented by Formula V with the substance represented by Formula VI. Wherein, Formula V is as follows:

[0125] L4 is a linking group, X4 is a methylene group, an imino group, an O atom or a S atom, and G1 is a protecting group.

[0126] Formula VI is as follows:

[0127] a is 1, 2 or 3.

[0128] It should be noted that the present application does not limit the specific steps of coupling, as long as the coupling reaction can be achieved.

[0129] It should be noted that other methods may be used to prepare the substance shown in Formula II, which is not limited in this application; other methods may be used to prepare the substance shown in Formula I, which is not limited in this application.

[0130] As an example, the present application provides the following three exemplary methods for preparing compounds with nitrogen-containing heterocycles as parent cores and their salts, hydrates of salts, enantiomers or optical isomers:

[0131] The synthesis path of Example 1 is as follows:

[0132]

[0133]

[0134] The synthesis path of Example 2 is as follows:

[0135]

[0136]

[0137] The synthesis path of Example 3 is as follows:

[0138]

[0139]

[0140]

[0141] The present application provides a conjugate, which includes a covalently linked biological molecule and a compound with a nitrogen-containing heterocycle as a parent core as provided in the first aspect above, and its salt, hydrate, enantiomer or optical isomer; wherein the biological molecule is covalently linked to A1 in formula I.

[0142] The conjugate provided in the present application is connected with the above-mentioned compound with a nitrogen-containing heterocycle as a parent core and its salt, hydrate of the salt, enantiomer or optical isomer, so that the stability of the conjugate in the organism can be improved, and the stability of the biological molecule can be improved, and the decomposition rate of the biological molecule can be reduced; the conjugate is used for cell-targeted delivery, which can improve the effectiveness of the delivery system, improve the delivery effect, and thereby enhance the effect of the biological molecule in the organ (for example, cancer cells in the liver).

[0143] In some feasible embodiments of the present application, in the conjugate, the active groups in S1, S2 and S3 may be protected by a protecting group or may not be protected by a protecting group.

[0144] Illustratively, the protecting group is selected from any one of acetyl, benzoyl, benzyl, dimethoxytrityl, methoxytrityl, trityl, methoxymethyl ether, p-methoxybenzyl ether, pivaloyl and tetrahydropyranyl.

[0145] In some feasible embodiments of the present application, the biomolecule in the conjugate is a nucleic acid molecule.

[0146] As an example, in the conjugate, a compound with a nitrogen-containing heterocycle as a core and a salt, a hydrate of the salt, an enantiomer or an optical isomer thereof is linked to the 3' end or the 5' end of the nucleic acid molecule.

[0147] As an example, in the conjugate, A1 in the compound with a nitrogen-containing heterocycle as a parent core and its salt, salt hydrate, enantiomer or optical isomer is connected to the nucleic acid molecule through a phosphate group, a thiophosphate group or a phosphonate group.

[0148] Furthermore, the biological molecule in the conjugate is an oligonucleotide, and the conjugate can be used to prepare drugs or vaccines for treating cancer. The prepared drugs or vaccines have good stability and are not easily degraded, which can make the drugs or vaccines have good therapeutic and preventive effects.

[0149] As an example, the nucleoside base in the oligonucleotide includes any one of a natural nucleoside base and a modified nucleoside base; wherein the natural nucleoside base is selected from any one of adenine, uraine, guanine, hypoxanthine, cytosine, thymine, adenine derivatives, uraine derivatives, guanine derivatives, cytosine derivatives and thymine derivatives; the modified nucleoside base refers to a compound obtained by replacing one or more hydrogen atoms of a natural nucleoside base with a modifying group, and the modified nucleoside base can be selected from N 6 -methyladenine, N 1 -methyladenine, N 6 -2'-O-dimethyladenosine, pseudouridine, N 1-methyl pseudouridine, 5-iodouridine, 4-thiouridine, 2-thiouridine, 5-methyluridine, pseudoisocytosine, 5-methoxycytosine, 2-thiocytosine, 5-hydroxycytosine, N 1 -methylcytosine, 5-hydroxymethylcytosine, hypoxanthine, N 1 -methylguanine, N 1 -Methylguanine and isoguanine.

[0150] In some feasible embodiments of the present application, the biomolecule in the conjugate is siRNA. The conjugate is not easily degraded, and when the conjugate is used for RNA interference protein translation, the production of the target protein can be reduced, that is, the RNA interference ability is strong.

[0151] The present application also provides a drug, which includes the conjugate provided above.

[0152] As an example, the drug may also include pharmaceutically acceptable diluents, solvents, carriers, salts, adjuvants, etc., which are not limited in this application and can be selected according to actual needs.

[0153] In some feasible embodiments of the present application, the biomolecule in the drug conjugate is an oligonucleotide, and the drug also includes a polynucleotide that is complementary to the oligonucleotide in the conjugate.

[0154] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0155] Example 1

[0156] This embodiment provides a compound with a nitrogen-containing heterocycle as a parent core (denoted as 1a), and its synthesis path is as follows:

[0157]

[0158]

[0159] Compound 1a with a nitrogen-containing heterocycle as the parent core is prepared by the following steps:

[0160] (1) 1,4,7,10-Tetraazacyclododecane (50 g, 290.6 mmol) and sodium acetate (78.6 g, 959.3 mmol) were dissolved in DMAc (N,N-dimethylacetamide, 600 mL) to obtain a mixed system; tert-butyl bromoacetate (141.4 mL, 959.3 mmol) was dissolved in DMAc (200 mL) and the mixture was slowly added dropwise to the mixed system at -20°C. After the addition was completed, the mixture was stirred at 25°C for 14 hours.

[0161] The reaction solution was cooled to 0°C, and a 5% by mass sodium bicarbonate aqueous solution was slowly added dropwise. After suction filtration, the solid phase was concentrated under high vacuum to obtain a white solid 1a-1.

[0162] (2) Compound 1a-1 (132 g, 256.8 mmol) and K2CO3 (92.4 g, 770.4 mmol) were dissolved in ACN (acetonitrile, 1320 mL) to obtain a mixed system; ethyl bromoacetate (48.84 mL) was added dropwise to the mixed system at -20°C, and the reaction solution was reacted at 85°C for 24 hours after the addition was completed.

[0163] The reaction solution was cooled to 0°C and filtered, the filter cake was washed twice with ACN (acetonitrile, 132 mL), the filtrates were combined and concentrated in a water bath at 30°C to obtain a crude product. The crude product was passed through a silica gel column and washed with dichloromethane / methanol 40 / 1 (v / v) to obtain product 1a-2.

[0164] (3) Compound 1a-2 (125 g, 208.3 mmol) was dissolved in dioxane (1250 mL), sodium hydroxide (416 mL) was slowly added, and the mixture was reacted at 25° C. for 16 hours.

[0165] The reaction solution was cooled to -5°C, 6N hydrochloric acid (34.3 mL) was slowly added dropwise under stirring, and the reaction was allowed to react for 4 hours. The reaction solution was concentrated in a water bath at 45°C. The concentrate was dissolved in DCM (dichloromethane, 800 mL), washed twice with 800 mL of deionized water, and once with a 10% sodium chloride aqueous solution, the organic phase was separated, anhydrous sodium sulfate (125 g) was added to the organic phase, dried, filtered, and concentrated in a water bath at 35°C to obtain a crude product 1a-3.

[0166] (4) Compound 1a-3 (68 g, 118.9 mmol) was dissolved in DMF (N,N-dimethylformamide, 680 mL), 6-aminohexanol (27.8 g, 237.8 mmol) and TEA (triethylamine, 156.3 g, 951 mmol) were added, and the mixture was stirred for 30 minutes. HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 90.2 g, 237.8 mmol) was added, and the mixture was reacted at 25°C for 8 hours.

[0167] The reaction solution was slowly added dropwise to 680 mL of deionized water and stirred for 10 minutes. Then 680 mL of DCM (dichloromethane) was added and stirred for 5 minutes. The lower organic phase was separated and washed four times with a 5% sodium chloride aqueous solution. Then 68 g of anhydrous sodium sulfate was added to the organic phase and dried. The filtrate was concentrated in a water bath at 35°C to obtain a crude product 1a-4.

[0168] (5) Compound 1a-5 (27 g) was dissolved in DMF (N,N-dimethylformamide, 360 mL), and powdered molecular sieves (20 g) were added, followed by stirring at room temperature for 0.5 hour. DIPEA (N-ethyldiisopropylamine, 83 g), 1a-6 (83 g) and HOBt (1-hydroxybenzotriazole, 3.6 g) were added to the system in sequence. After stirring at 25°C for 10 minutes, EDCI (1-ethyl-3 (3-dimethylpropylamine) carbodiimide, 40.7 g) was added, and the mixture was reacted at 25°C for 15 hours.

[0169] The reaction solution was filtered through diatomaceous earth, and the filter cake was rinsed with DMF (N,N-dimethylformamide, 30 mL). Ethyl acetate (1.8 L) was added to the filtrate, and the mixture was washed twice with 20% brine (1 L each time). After standing, the liquid phase was separated into three layers. Acetonitrile (200 mL) was added to the middle layer to dissolve it and then concentrated to dryness to obtain a yellow solid crude product. The crude product was prepared by reverse phase, 3.3 L acetonitrile was added to the prepared solution to dilute it, 2 kg of NaCl solid was added, and the mixture was stirred at room temperature for 0.5 hour, and then stood for stratification. The organic phase was concentrated and dried again with 1 L acetonitrile, and then suspended in 400 mL acetonitrile at room temperature for 0.5 hour, and then filtered. The mother liquor was concentrated to dryness, acetonitrile (60 mL) was added to dissolve it, and then filtered. After concentration, the product 1a-7 was obtained by vacuum drying.

[0170] (6) Compound 1a-7 (6 g) was concentrated to dryness with acetonitrile, and DCM (dichloromethane, 60 mL) was added. After the KF was measured to be 33 ppm, powdered molecular sieves (6 g) were added and stirred at 25°C for 0.5 hour. After the system was cooled to 5°C, TEA (triethylamine, 0.9 mL), phosphoramidite (1.2 g) and tetrazole (0.19 g) were added in sequence and stirred at 25°C for 3.5 hours.

[0171] The reaction solution was filtered through diatomaceous earth under N2 protection, and the filtrate was extracted with a 5% aqueous solution of NaHCO3 and a 10% aqueous solution of NaCl. Powdered molecular sieves (10 g) were added to the organic phase for drying and concentration to obtain a crude product. The crude product was dissolved in 12 mL of dichloromethane, and 60 mL of Hept (n-heptane) was added dropwise to the crude product, and the bottom product was separated by standing (repeated three times). The product 1a was obtained by passing through a silica gel column with a 1\1 volume of a mixed solution of dichloromethane and acetonitrile (containing 2% triethylamine).

[0172] Example 2

[0173] This embodiment provides a compound with a nitrogen-containing heterocycle as a parent core (denoted as 1a'), and its synthesis route is as follows:

[0174]

[0175]

[0176]

[0177] Compound 1a' with a nitrogen-containing heterocycle as a parent core is prepared by the following steps:

[0178] (1) Compound 1a-7 (1 g) was dissolved in acetonitrile (10 mL) at 20°C, cooled to 0°C, and powdered molecular sieves (0.6 g) were directly added. After stirring for 20 min, compound 1a-8 (0.77 g) was added, and then tetrazole (0.078 g) was added. The mixture was reacted at 27°C for 1 h to obtain a system containing compound 1a-9.

[0179] For the preparation of compound 1a-7, please refer to steps (1) to (5) in Example 1.

[0180] (2) Add tert-butyl hydroperoxide (0.1 g) to the system containing compound 1a-9 obtained in step (1), and react at 27° C. for 1 h to obtain a system containing compound 1a-10.

[0181] (3) A mixed solution (45 mL) of aqueous ammonia and ethanol in a volume ratio of 2:1 was added to the system containing compound 1a-10 obtained in step (1), and the mixture was reacted at 27°C for 1 h to obtain a reaction solution. The reaction solution was filtered with 2 g of diatomaceous earth and concentrated to dryness under reduced pressure at 30°C to obtain 1.5 g of a crude product. The crude product was prepared by reverse phase, and 1 L of the reverse phase preparation solution was concentrated to dryness under reduced pressure at 30°C to obtain 0.6 g of compound 1a-11.

[0182] (4) Compound 1a-11 (0.5 g) was dissolved in a mixed solution of 10 mL of methanol and 3 mL of dichloromethane, and dodecanethiol (0.735 g) and dichloroacetic acid (0.47 g) were added at 0°C for 15 h to obtain a reaction solution. 3 mL of 5% sodium bicarbonate aqueous solution was added to the reaction solution for quenching, and the mixture was stirred for 10 min. The reaction system was concentrated to dryness under reduced pressure at 30°C to obtain 0.7 g of a crude product. The crude product was prepared by reverse phase, and 250 mL of the reverse phase preparation solution was concentrated to dryness under reduced pressure at 30°C to obtain 0.11 g of compound 1a'.

[0183] Comparative Example

[0184] This comparative example provides a compound, which is L96 product, and its structural formula is as follows:

[0185]

[0186] Experimental Example 1

[0187] The compound obtained in step (5) of Example 1 (i.e., 1a-7), the compound with a nitrogen-containing heterocycle as the parent nucleus (i.e., 1a), and the compound 1a' obtained in Example 2 were characterized by NMR spectra. The characterization results were as follows: Figures 1 to 3 shown.

[0188] from Figure 1 It can be seen that the NMR results are consistent with the expected structure. 1 H NMR (500MHz, DMSO) δ8.49(d,J=5.5Hz,3H),8.34(d,J=9.2Hz,3H),5.64(d,J=3.3Hz,3H),5.40(dd,J=11.2,3.3H z,3H),4.93(d,J=8.4Hz,3H),4.80(t,J=5.1Hz,1H),4.43(dt,J=11.5,6.4Hz,9H),4.30(dd,J=19.9,9.0Hz,3H) ,4.12(dt,J=9.8,6.1Hz,3H),3.93-3.64(m,20H),3.41(dd,J=67.4,22.9Hz,16H),3.04-2.86(m,4H),2.65(s,7 H),2.54(d,J=10.5Hz,9H),2.42(s,9H),2.32(d,J=9.6Hz,10H),2.20(s,9H),1.99-1.74(m,17H),1.68(s,16H).

[0189] from Figure 2 It can be seen that the NMR results are consistent with the expected structure. 1H NMR (500MHz, DMSO) δ8.11(d,J=16.9Hz,4H),7.97(d,J=8.4Hz,3H),5.21(s,3H),4.97(d,J=11.2Hz,3H),4.52(d,J=8.4Hz, 3H), 4.00 (dd, J=18.2, 5.3Hz, 9H), 3.87 (dd, J=19.1, 9.3Hz, 3H), 3.71 (dd, J=26.8, 8.1Hz, 5H), 3.57 (dd, J=18.3, 11.5Hz, 4 H),3.40(d,J=8.4Hz,3H),3.34(s,2H),3.00(d,J=51.3Hz,16H),2.75(t,J=5.6Hz,3H),2.50(s,4H),2.22(d,J=18.1Hz,8H ), 2.08 (d, J = 14.3Hz, 10H), 1.99 (s, 9H), 1.90 (d, J = 24.5Hz, 9H), 1.77 (s, 9H), 1.59-1.16 (m, 33H), 1.12 (t, J = 7.0Hz, 11H).

[0190] from Figure 3 It can be seen that the NMR results are consistent with the expected structure. 1 H NMR(500MHz,DMSO)δ8.34(t,J=13.5Hz,1H),8.17-7.97(m,3H),7.97-7.74(m ,3H),7.63(dd,J=7.3,1.7Hz,1H),7.51-7.37(m,J=12.1,8.9Hz,3H),7.33-7 .14(m,2H),7.13-6.98(m,2H),6.89(dd,J=15.3,12.0Hz,1H),6.78-6.65(m, 3H),6.62(d,J=13.5Hz,1H),5.28(d,J=15.2Hz,1H),4.24-4.16(m,6H),3.81- 3.60(m,16H),3.58-3.50(m,J=8.3Hz,5H),3.49-3.39(m,7H),3.37-3.23(m, 7H),3.13-2.83(m,18H),2.59(s,9H),2.03-1.94(m,2H),1.90(d,J=8.1Hz,2 H),1.85(s,1H),1.78(s,12H),1.69(d,J=3.0Hz,8H),1.48(d,J=26.3Hz,5H) ,1.43-1.32(m,14H),1.21(d,J=21.8Hz,20H),0.93(dd,J=28.4,21.2Hz,2H).

[0191] Experimental Example 2

[0192] The compound with a nitrogen-containing heterocycle as a parent core (ie, 1a) prepared in Example 1 was subjected to a stability test. The test results are shown in Tables 1 and 2.

[0193] The stability test method is as follows: the compound with a nitrogen-containing heterocycle as the parent core (i.e., 1a) prepared in Example 1 is dissolved in acetonitrile (referred to as solution A), an acetonitrile solution containing triethylamine (the mass proportion of triethylamine in the solution is 0.1%) (referred to as solution B), tetrahydrofuran (referred to as solution C), and tetrahydrofuran containing triethylamine (the mass proportion of triethylamine in the solution is 0.1%) (referred to as solution D), and the concentration of the compound with a nitrogen-containing heterocycle as the parent core (i.e., 1a) prepared in Example in solution A, solution B, solution C, and solution D is 2M. The HPLC concentrations of the compound with a nitrogen-containing heterocycle as the parent core (i.e., 1a) dissolved for 0 days, 1 day, and 3 days are recorded respectively.

[0194] Table 1

[0195]

[0196]

[0197] Table 2

[0198]

[0199] It can be seen from Table 1 that the compound 1a prepared in Example 1 of the present application has high stability and is not easily degraded.

[0200] Experimental Example 3

[0201] The protein binding effects of compound 1a' prepared in Example 2 and the compound provided in the comparative example were compared. The results are as follows: Figure 4 As shown; Figure 4 In the examples, L96 represents the compound provided in the comparative example.

[0202] The delivery effect was tested by flow cytometry on human liver cancer cells (HepG2) to test the endocytosis effect of the two test substances. The specific method is as follows:

[0203] (1) Cell culture:

[0204] ① Thoroughly mix EMEM medium and FBS to obtain complete medium; the mass fraction of FBS in the complete medium is 15%.

[0205] ②Resuscitate HepG2 cells and select cell lines with good growth status after two generations.

[0206] ③ Collect HepG2 cells in the logarithmic growth phase, count them and detect cell viability.

[0207] ④ Resuspend the cells in complete culture medium to an appropriate cell density to obtain a cell suspension.

[0208] ⑤Inoculate the cell suspension into a 96-well plate at 100 μL / well, and place the 96-well plate in a high humidity, 37°C, 5% CO2 incubator overnight.

[0209] (2) Drug configuration:

[0210] Using the complete culture medium prepared in step (1), 1a' prepared in Example 2 and the compound provided in the comparative example were diluted to concentrations of 100 μM, 20 μM, 4 μM, 0.8 μM, 0.16 μM, 0.032 μM, 0.0064 μM, 0.00128 μM, and 0.000256 μM, respectively, to obtain drug delivery systems of different concentrations.

[0211] (3) Drug administration and incubation:

[0212] The drug delivery system prepared in step (2) was added to the 96-well plate of step (1) at 25 μL / well, and the blank control group was supplemented with complete culture medium to a liquid phase of 125 μL / well; the 96-well plate was then placed in an incubator for incubation, and the incubation time was set to 24 h.

[0213] (4) Sample collection and testing:

[0214] ① Take the 96-well plate out of the incubator, wash the cells once with PBS, add preheated trypsin for digestion, then terminate the digestion with the complete medium prepared in step (1), centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0215] ② Add PBS (100 μL / well) to the 96-well plate to resuspend the cells. Centrifuge again and resuspend the cells with PBS to obtain the sample for the machine.

[0216] ③ Use Beckman cytoflex flow cytometer to collect samples, determine the voltage parameters according to the control group, and after completing the collection, use Flowjo software to analyze the MFI value.

[0217] from Figure 4 It can be seen that compared with the compound provided in the comparative example, 1a' prepared in Example 2 of the present application has a better cell uptake effect.

[0218] In summary, the compound with a nitrogen-containing heterocycle as the parent core provided by the present application contains a 1,4,7,10-tetraazacyclododecane structure, which can improve the stability of the entire compound in the organism, so that the compound containing a cell targeting ligand is not easily degraded in the organism, which is beneficial to improve the stability of the conjugate formed by connecting the compound with a biological molecule (for example, a nucleic acid molecule) in the organism, and can also improve the stability of the biological molecule and reduce the decomposition rate of the biological molecule; using the conjugate for cell-targeted delivery can improve the effectiveness of the delivery system, improve the delivery effect, and thereby enhance the effect of the biological molecule in an organ (for example, cancer cells in the liver).

[0219] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

Claims

1. A compound with a nitrogen-containing heterocycle as a parent nucleus and its salt, hydrate, enantiomer or optical isomer, characterized in that: The structural formula of the compound with a nitrogen-containing heterocycle as a parent core is shown in Formula I; The formula I is as follows: Wherein, L1, L2, L3 and L4 are each independently a linking group; X1, X2, X3 and X4 are each independently a methylene group, an imino group, an O atom or a S atom; At least one of S1, S2 and S3 is a cell targeting ligand; A1 is a linker used to connect biomolecules; B1 is a solid support, and z is 0 or 1.

2. The compound with a nitrogen-containing heterocycle as a parent nucleus and its salt, hydrate, enantiomer or optical isomer according to claim 1, characterized in that: The L1, L2, L3 and L4 are each independently selected from at least one of a C2-C30 alkyl group and a C2-C30 alkoxy group, and the C2-C30 alkyl group and the C2-C30 alkoxy group are each independently connected to or not connected to at least one of an imino group, a carbonyl group and an amide group; Optionally, the L1, L2, L3 and L4 are each independently selected from -(CH2) m1 -、-(CH2-CH2-O) m2 -CH2-, -(CH2) m3 -O-(CH2) m4 -、-(CH2) m5 -O-, -(CH2-CH2-O) m2 -(CH2) m6 -、-(CH2) m7 -O-(CH2) m8 -O- and -(CH2) m9 -O-(CH2) m10 -O-(CH2) m11 - is at least one of, m2, m3, m4, m5, m7, m8, m9, m10 and m11 are each independently a natural number of 1-5, and m6 is a natural number of 2-5.

3. The compound with a nitrogen-containing heterocycle as a parent nucleus and its salt, hydrate, enantiomer or optical isomer according to claim 1, characterized in that: The structural formula of L4 is as follows: Wherein, L5, L6 and L7 are each independently a linking group; Q1 is selected from a methine or a N atom; M1 is a protecting group; Optionally, the L5, L6 and L7 are each independently selected from at least one of a C1-C30 alkyl group and a C1-C30 alkoxy group, and the C1-C30 alkyl group and the C1-C30 alkoxy group are each independently connected to or not connected to at least one of an imino group, a carbonyl group and an amide group; Optionally, the M1 is selected from any one of acetyl, benzoyl, benzyl, dimethoxytrityl, methoxytrityl, trityl, methoxymethyl ether, p-methoxybenzyl ether, pivaloyl and tetrahydropyranyl.

4. The compound with a nitrogen-containing heterocycle as a parent nucleus and its salt, hydrate, enantiomer or optical isomer according to any one of claims 1 to 3, characterized in that: A1 is a linker containing a phosphoramidite group or a succinate group; Optionally, the structural formula of A1 is as follows: Wherein, R1 and R2 are independently selected from C1-C20 alkyl groups; or, -R1R2 is a cyclic carbon chain containing or not containing heteroatoms, and the heteroatoms are selected from at least one of N atoms and O atoms, and the cyclic carbon chain is a five-membered carbon chain or a six-membered carbon chain; X5 is selected from a methylene group, an O atom or a S atom; n is 0 or 1; R3 is selected from a C1-C20 alkyl group substituted by thio, thio, oxo, halogen or cyano; Optionally, -NR1R2 is selected from any one of dimethylamino, diethylamino, di-n-propylamino, diisopropylamino, dibutylamino, pyrrolidinyl, piperidinyl, 2,6-dimethylpiperidinyl, morpholinyl, imidazolyl and 4-methylimidazolyl; Optionally, -R3 is selected from any one of 2-cyanoethyloxy, 2-cyanoethylthio, methoxy, ethoxy, S-isobutyryl-2-(2-mercaptoethoxy)ethoxy, S-pivaloyl-2-(2-mercaptoethoxy)ethoxy and S-pivaloyl-2-mercaptoethoxy; Optionally, the -NR1R2 is diisopropylamino, and the -R3 is 2-cyanoethoxy.

5. The compound with a nitrogen-containing heterocycle as a parent nucleus and its salt, hydrate, enantiomer or optical isomer according to any one of claims 1 to 3, characterized in that: The S1, S2 and S3 are all cell-targeting ligands; and the S1, S2 and S3 are each independently a liver-targeting specific ligand or a thyroid receptor agonist; Optionally, S1, S2 and S3 are each independently selected from N-acetylgalactosamine, D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucose, β-D-glucose, α-D - at least one of furanose, β-D-glucose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, ribose, D-ribose, D-4-thioribose, L-ribose and L-4-thioribose; Optionally, the S1, the S2 and the S3 are all N-acetylgalactosamine; Optionally, any active group in S1, S2 and S3 is protected by a protecting group, and the protecting group is selected from any one of acetyl, benzoyl, benzyl, dimethoxytrityl, methoxytrityl, trityl, methoxymethyl ether, p-methoxybenzyl ether, pivaloyl and tetrahydropyranyl; Optionally, the active group is a hydroxyl group, and the protecting group is an acetyl group.

6. The compound with a nitrogen-containing heterocycle as a parent core according to any one of claims 1 to 3, and its salt, hydrate, enantiomer or optical isomer, characterized in that: The formula I is as follows: Wherein, L1, L2, L3 and L4 are each independently a linking group; X1, X2, X3 and X4 are each independently a methylene group, an imino group, an O atom or a S atom; At least one of S1, S2 and S3 is a cell targeting ligand; W1 is a protecting group; Or the formula I is as follows: Wherein, L1, L2, L3 and L4 are each independently a linking group; X1, X2, X3 and X4 are each independently a methylene group, an imino group, an O atom or a S atom; At least one of S1, S2 and S3 is a cell targeting ligand; W2 is a protecting group; B1 is a solid phase carrier.

7. The compound with a nitrogen-containing heterocycle as a parent core according to any one of claims 1 to 3, and its salt, hydrate, enantiomer or optical isomer, characterized in that: The formula I is selected from any of the following structures: Among them, Y1 is a porous glass support, Y2 is a polystyrene support, Y3 is a porous glass support, and T1 is a solid phase carrier.

8. A method for preparing a compound with a nitrogen-containing heterocycle as a parent core and a salt, a hydrate of the salt, an enantiomer or an optical isomer thereof as claimed in any one of claims 1 to 7, characterized in that: The z in the formula I is 0, and the preparation method comprises: deprotecting G1 in the substance shown in the formula II, and coupling with a group containing a linker for connecting biological molecules; Or, the z in the formula I is 1, and the preparation method comprises: deprotecting G1 in the substance shown in formula II, coupling with a group containing a linker for connecting biological molecules to obtain a substance shown in formula III; coupling the substance shown in formula III with a solid phase carrier; Or, the z in the formula I is 0, and the preparation method comprises: replacing Q in the formula IV with a linker containing a linker for connecting biomolecules; Or, the z in the formula I is 1, and the preparation method comprises: replacing Q in the formula IV with a linker containing a linker for connecting biological molecules to obtain a substance represented by the formula III; coupling the substance represented by the formula III with a solid phase carrier; Wherein, the formula II is as follows: L1, L2, L3 and L4 are each independently a linking group; X1, X2, X3 and X4 are each independently a methylene group, an imino group, an O atom or a S atom; At least one of S1, S2 and S3 is a cell targeting ligand; G1 is a protecting group; The formula III is as follows: L1, L2, L3 and L4 are each independently a linking group; X1, X2, X3 and X4 are each independently a methylene group, an imino group, an O atom or a S atom; At least one of S1, S2 and S3 is a cell targeting ligand; A1 is the linker used to connect biomolecules; The formula IV is as follows: L1, L2, L3 and L4 are each independently a linking group; X1, X2, X3 and X4 are each independently a methylene group, an imino group, an O atom or a S atom; At least one of S1, S2 and S3 is a cell targeting ligand; Q is a hydrogen atom.

9. A conjugate, characterized in that The conjugate comprises a covalently linked biomolecule and a compound with a nitrogen-containing heterocycle as a parent core as claimed in any one of claims 1 to 7, and a salt, a hydrate of the salt, an enantiomer or an optical isomer thereof; wherein the biomolecule is covalently linked to the A1 in the formula I; Optionally, the biomolecule is a nucleic acid molecule; Optionally, the biomolecule is siRNA.

10. A drug, characterized in that The drug comprises the conjugate according to claim 9; Optionally, the biomolecule in the conjugate is a nucleic acid molecule; Optionally, the biomolecule is siRNA.

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