Conjugates and compositions and methods of making and using

Abstract

The present disclosure provides a conjugate. The conjugate comprises one or more delivery groups and one or more functional groups, each of the delivery groups being independently linked to the functional group via a covalent bond or via a linking group, and each of the functional groups being selected from small molecule therapeutic groups having a therapeutic effect on tumors. The conjugate according to the present disclosure can be efficiently targeted and delivered to tumor tissue, thereby effectively treating tumors and tumor-related diseases.

Description

[Technical field]

[0001] The present disclosure relates to conjugates and pharmaceutical compositions that include an aptamer-based delivery group and a functional group. The present disclosure also relates to methods of making and using these conjugates and pharmaceutical compositions. [Background technology]

[0002] A tumor refers to a neoplasm formed by the proliferation of local tissue cells under the action of various tumorigenic factors in a living body. A state in which tumor cells metastasize and invade surrounding tissues is called a malignant tumor. When classified according to the tissue cells from which the tumor is generated, it is generally classified into malignant tumors (carcinomas) arising from epithelial cells, malignant tumors (sarcomas) arising from mesenchymal tissue cells, malignant tumors (leukemia, etc.) arising from blood stem cells, and malignant tumors (gliomas) arising from neuroglial cells. Gliomas are the most common intracranial primary malignant tumors, accounting for approximately 40% to 50% of brain tumors, with an annual incidence rate of 3 to 8 cases per 100,000 people worldwide. According to the WHO pathological classification criteria, gliomas belong to neuroepithelial tumors and include multiple pathological types, including but not limited to pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglioma, and anaplastic oligodendroglioma.

[0003] Currently, one of the key problems in the field of treatment for tumors, especially gliomas, is how to specifically deliver therapeutic agents into tumor tissues and cells so that these therapeutic agents produce the corresponding therapeutic effects at the right time and in the right manner.

[0004] Aptamers or nucleic acid aptamers are oligonucleotide molecules that can bind to various target molecules, such as small molecular compounds, proteins, nucleic acids, and even cells, tissues, and organs. Aptamers can provide the important property of "recognizing specific molecules", and are therefore often used in biological technology and treatment, just like antibodies. Aptamers have the excellent properties of being designed in a test tube, being quickly synthesized by chemical methods, being easy to store, and being low or non-immunogenic, and therefore have attracted the attention of researchers in this field in recent years. However, aptamers suitable for tumor targeting delivery are still in need of further development and use in this field. Summary of the Invention [Problem to be solved by the invention]

[0005] The inventors of the present disclosure have unexpectedly discovered a conjugate that can specifically target tumor cells, particularly glioma cells. The conjugate exhibits high specificity to tumor cells, particularly glioma cells, and can thereby be effectively concentrated in tumor cells, particularly glioma cells, for effective targeted therapy. Thus, the inventors have made the following invention. [Means for solving the problem]

[0006] In one aspect, the disclosure provides a conjugate comprising one or more delivery groups and one or more functional groups, the delivery groups being formed by removing one or more hydrogen atoms or one or more functional groups from an aptamer, the aptamer comprising a contiguous nucleotide sequence, the groups connecting two adjacent nucleotides being independently a phosphate group or a phosphate group having a modified group, each nucleotide being selected from one of A, U, C, or G, modified or unmodified, the contiguous nucleotide sequence having the sequence shown in formula (1): 5'-T1-S1-N a -S2-N b -S3-N c -S4-T2-3' Formula (1) wherein T1 is a motif consisting of 1 to 3 nucleotides, T2 is a motif consisting of 0 to 15 nucleotides, and T2 does not contain a motif that is completely reverse complementary to T1; S1 and S4 are motifs each consisting of 3 to 7 nucleotides, S1 is the same length as S4 and is completely reverse complementary; N a and N c are motifs consisting of 1 to 4 nucleotides, respectively, and N a Each nucleotide in c is not complementary to any nucleotide in a and N c The total number of U's in a and N c accounts for more than 50% of the total number of all nucleotides in S2 and S3 are motifs each consisting of 1 to 4 nucleotides, S2 is the same length as S3 and is a perfect reverse complement; N b is a motif consisting of 3 to 6 nucleotides and N b The nucleotides at both ends of the sequence do not form an AU or GC complement. Each of the delivery groups is independently attached to a functional group via a covalent bond or via a linking group, and each of the functional groups is selected from small molecule therapeutic groups that have a therapeutic effect on tumors.

[0007] In another aspect, the present disclosure further provides a pharmaceutical composition comprising a conjugate according to the present disclosure and a pharma- ceutically acceptable carrier.

[0008] In yet another aspect, the present disclosure further provides the use of the conjugate and / or pharmaceutical composition described herein in the manufacture of a medicament for treating tumors and tumor-related diseases or conditions.

[0009] In yet another aspect, the present disclosure further provides a method of treating tumors and tumor-related diseases or conditions, comprising administering to a subject in need thereof an effective amount of a conjugate and / or pharmaceutical composition as described herein.

[0010] In yet another aspect, the present disclosure further provides kits comprising the conjugates and / or pharmaceutical compositions described herein.

[0011] INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Effect of the Invention

[0012] The conjugates and pharmaceutical compositions of the present disclosure have an excellent ability to target tumors, especially glioma tissues and cells, and can significantly treat or alleviate tumors and tumor-related diseases and / or symptoms.

[0013] In one embodiment, the delivery group in the conjugate of the present disclosure can specifically deliver various small molecule drug groups, such as small molecule toxin groups, to tumor tissues, and can show excellent tumor inhibition effects.For example, the conjugate of the present disclosure can effectively deliver MMAE to different tumor tissues, showing tumor targeting ability and reducing the toxicity risk caused by the distribution of MMAE molecules to other tissues, and various administration methods can effectively inhibit the tumor volume increase rate and tumor weight, thereby showing that the conjugate of the present disclosure can effectively inhibit tumor growth.In addition, when the dosage of the conjugate is further increased, there is almost no increase in tumor volume during the test, showing a better antitumor effect.

[0014] Furthermore, the inventors of the present disclosure unexpectedly discovered that the complex and / or pharmaceutical composition of the present disclosure can efficiently pass through the blood-brain barrier, and when administered systemically, can target gliomas in the brain, and significantly inhibit the increase in tumor volume, thereby reducing the volume to 1 / 10 or less of the initial volume, and even to 1 / 100 or less compared to the control group. This indicates that the complex of the present disclosure can effectively penetrate the blood-brain barrier and efficiently target and enter brain gliomas, has an excellent effect of inhibiting tumor growth, and shows good treatment compliance and high drug discovery potential for efficiently inhibiting tumors.

[0015] As can be seen, the conjugate of the present disclosure can remarkably and effectively inhibit tumor growth, and has good application prospects. [Brief description of the drawings]

[0016] [Figure 1A-1C] 13 is a set of photographs showing the results of fluorescent imaging in mice 1 h, 24 h, and 48 h after administration of different complexes. [Figure 1D] Photographs of fluorescent signal imaging of tumor tissues and kidneys of mice in each group after the mice were sacrificed on D5. [Figure 2A-2C] 13 is a set of photographs showing the results of fluorescent imaging in mice 1 h, 24 h, and 48 h after administration of different complexes. [Figure 2D] Photographs of fluorescent signal imaging of tumor tissues and kidneys of mice in each group after the mice were sacrificed on D6. [Diagram 3] 1 is a line graph showing the change in tumor volume over time in each group of mice after administration of a conjugate according to the present disclosure or a control compound. [Figure 4] These are photographs of fluorescent imaging of mouse brain tissues in which an in-situ tumor model of U118MG was constructed, 24 h and 48 h after administration of different complexes, respectively. [Diagram 5] 1 is a line graph showing the change over time in tumor light intensity values ​​in a U118MG in situ tumor model mouse after administration of a conjugate according to the present disclosure or a control compound. [Figure 6] 1 is a line graph showing the change in tumor volume over time in U118MG subcutaneous tumor model mice after administration of a conjugate according to the present disclosure or a control compound. [Figure 7] 1 is a line graph showing the change in tumor volume over time in U118MG subcutaneous tumor model mice after administration of different concentrations of a conjugate according to the present disclosure or a control compound. [Figure 8] 1 is a line graph showing the change in tumor volume over time in U118MG subcutaneous tumor model mice after administration of different concentrations of a conjugate according to the present disclosure or a control compound. [Figure 9] 1 is a line graph showing the change in tumor volume over time in A549 subcutaneous tumor model mice after administration of different concentrations of a conjugate according to the present disclosure or a control compound. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Specific embodiments of the present disclosure will be described in detail below. It should be understood that the specific embodiments described herein are merely for the purpose of explaining and interpreting the present disclosure, and are not intended to limit the present disclosure.

[0018] definition In this disclosure, unless otherwise stated, A, U, C, G and T refer to adenine nucleotide, uracil nucleotide, cytosine nucleotide, guanine nucleotide and thymine nucleotide, respectively, and 2-methylcytosine nucleotide refers to a nucleotide in which the hydrogen at the 2' position on the cytosine base in the cytosine nucleotide is replaced with a methyl group. The structure of these nucleotides is well known to those skilled in the art. As used in this disclosure, a "nucleic acid motif" or "motif" is a nucleic acid sequence fragment in an oligonucleotide, consisting of one or more nucleotides. In some embodiments, the motif is a nucleic acid sequence fragment having a biological function.

[0019] As used in this disclosure, "alkyl group" refers to straight and branched chain saturated hydrocarbon groups having a specified number of carbon atoms, generally 1 to 20 carbon atoms, e.g., 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms. For example, C1-C6 alkyl group refers to straight and branched chain alkyl groups containing 1 to 6 carbon atoms. When referring to an alkyl residue having a specific number of carbons, it is intended to include all branched and straight chain forms having that number of carbons. Thus, for example, "butyl group" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl groups, and "propyl" includes n-propyl and isopropyl groups. Alkylene groups are a subset of alkyl groups and refer to residues similar to alkyl groups but having two points of attachment.

[0020] As used herein, an "alkenyl group" refers to an unsaturated branched or straight chain alkyl group having one or more carbon-carbon double bonds obtained by removing one hydrogen from adjacent carbon atoms of a parent alkyl group. The group may be in the cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to, vinyl groups, propenyl groups such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl group), prop-2-en-2-yl, and butenyl groups such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, and buta-1,3-dien-2-yl. In some embodiments, alkenyl groups have from 2 to 20 carbon atoms, and in other embodiments, from 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of the alkenyl group, referring to residues similar to the alkenyl group, but that have two points of attachment.

[0021] As used herein, an "alkynyl group" refers to an unsaturated branched or straight chain alkyl group having one or more carbon-carbon triple bonds obtained by removing two hydrogens from adjacent carbon atoms of a parent alkyl group. Typical alkynyl groups include, but are not limited to, ethynyl groups, propynyl groups, such as prop-1-yn-1-yl, prop-2-yn-1-yl, and butynyl groups, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl. In some embodiments, alkynyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms. An alkynylene group is a subset of the alkynyl group and refers to a residue that is the same as the alkynyl group but has two points of attachment.

[0022] As used herein, a "heterocyclic group" refers to a stable 3- to 18-membered non-aromatic cyclic group containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise stated in the specification, a heterocyclic group is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, and may include fused or bridged ring systems. The heteroatoms in a heterocyclic group may be oxidized heteroatoms. One or more nitrogen atoms, if present, may be quaternized nitrogen atoms. A heterocyclic group is partially saturated or fully saturated. A heterocyclic group may be attached to the remainder of the molecule through any ring atom. Examples of such heterocyclic groups include dioxanyl, thiophenyl[1,3]disulfonyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindole, octahydroisoindole, 2-oxapiperazinyl, 2-oxapiperidyl, 2-oxapyrrolidinyl, oxazolidinyl, piperidyl, piperazinyl, and 4-piperidonyl. Examples of heterocyclic groups include, but are not limited to, pyrrolidinyl, pyrazolidyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Heterocyclylene is a subset of heterocycle and refers to the same residues as heterocycle but with two points of attachment.

[0023] As used herein, an "aryl group" refers to a group formed by removing a hydrogen atom from a ring carbon atom derived from an aromatic monocyclic or polycyclic hydrocarbon ring system. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon from 6 to 18 carbon atoms, and one or more rings in the ring system are fully unsaturated, i.e., contain a cyclic, delocalized (4n+2) π-electron system according to the Hückel theory. Aryl groups include, but are not limited to, groups such as phenyl, fluorenyl, and naphthyl. An arylene group is a subset of the aryl group and refers to a residue that is the same as the aryl group but has two points of attachment.

[0024] "Heteroaryl group" refers to a group derived from a 3-18 membered aromatic ring radical containing 2-17 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen and sulfur. As used in this disclosure, a heteroaryl group may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system in which one or more rings are fully unsaturated, i.e., contain a cyclic, delocalized (4n+2) π-electron system according to the Hückel theory. Heteroaryl groups include fused or bridged ring systems. Heteroatoms in a heteroaryl group may be oxidized heteroatoms. One or more nitrogen atoms (if present) may be quaternized nitrogen atoms. A heteroaryl group is bonded to the remainder of the molecule through any ring atom. Examples of heteroaryl groups include azepinyl, acridinyl, benzimidazolyl, benzoindole, 1,3-benzodioxazolyl, benzofuryl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyronyl, benzofuryl, and benzofuranonyl. group, benzothiophenyl group, benzothieno[3,2-d]pyrimidinyl group, benzotriazolyl group, benzo[4,6]imidazo[1,2-a]pyridyl group, carbazolyl group, cinnolinyl group, cyclopenta[d]pyrimidinyl group, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl group, 5,6-dihydrobenzo[h]quinazolinyl group, 5,6-dihydrobenzo[h]cinnolinyl group, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl group, dibenzofuryl group, dibenzothiophenyl group, furyl group, furanonyl group, furo[3,2-c]pyridyl group, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl group, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl group, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridyl group, isothiazolyl group, imidazolyl group, indazolyl group, indole group, isoindole group, indolinyl group, isoindolinyl group, isoquinolyl group, indolizinyl group, isoxazolyl group, 5,8-methano-5,6 ,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[H]quinazolinyl, 1-phenyl-1H-pi aryl group, phenazinyl group, phenothiazinyl group, phenoxazinyl group, phthalazinyl group, pteridinyl group, purinyl group, pyrrolyl group, pyrazolyl group, pyrazolo[3,4-d]pyrimidinyl group, pyridyl group, pyrido[3,2-d]pyrimidinyl group, pyrido[3,4-d]pyrimidinyl group, pyrazinyl group, pyrimidinyl group, pyridazinyl group, pyrrolyl group, quinazolinyl group, quinoxalinyl group, quinolyl group, tetrahydroquinolyl group, 5,6,7 ,8-tetrahydroquinazolinyl group, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl group, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl group, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl group, thiazolyl group, thiadiazolyl group, triazolyl group, tetrazolyl group, triazinyl group, thieno[2,3-d]pyrimidinyl group, thieno[3,2-d]pyrimidinyl group, thieno[2,3-c]pyridinyl group (thieno[2,Heteroarylene is a subset of the heteroaryl group and refers to residues similar to the heteroaryl group but that have two points of attachment.

[0025] The complex according to the present disclosure In one aspect, the present disclosure provides a conjugate comprising one or more delivery groups and one or more functional groups, the delivery groups being formed by removing one or more hydrogen atoms or one or more functional groups from the aptamer, each delivery group being independently attached to the functional group via a covalent bond or via a linking group, each functional group being selected from small molecule therapeutic groups having a therapeutic effect on tumors. By linking the functional groups via a covalent bond or a linking group to form a conjugate, the conjugate of the present disclosure can deliver the functional group to the tumor. In the conjugate of the present disclosure, the delivery group is formed by removing one or more hydrogen atoms or one or more functional groups from an aptamer, the aptamer comprising a contiguous nucleotide sequence, wherein the groups connecting two adjacent nucleotides are independently a phosphate group or a phosphate group having a modified group, and each nucleotide is selected from one of A, U, C, or G, modified or unmodified, and the contiguous nucleotide sequence has the sequence shown in formula (1): 5'-T1-S1-N a -S2-N b -S3-N c -S4-T2-3' Formula (1) Here, T1 is a motif consisting of 1 to 3 nucleotides. The present inventors have discovered that the presence of T1 allows the complex according to the present disclosure to exhibit an efficient tumor targeting effect. In some embodiments, T1 consists of two nucleotides, in which case the complex according to the present disclosure has a better tumor targeting ability. In some embodiments, T1 consists of two nucleotides and includes at least one C. In some embodiments, from 5' to 3', T1 is CU, UC, or AC.

[0026] T2 is a motif consisting of 0-15 nucleotides. The present inventors have found that these numbers of nucleotides and various nucleotide sequences of T2 do not significantly affect the tumor targeting ability of the complex of the present disclosure. In some embodiments, T2 consists of 0-10 nucleotides. In some embodiments, from 5' to 3', T2 consists of 1-9 nucleotides starting with U, in which case the aptamer may have better stability.

[0027] T2 does not contain a motif that is completely reverse complementary to T1. In the context of this disclosure, "reverse complementary" refers to a pair of nucleotide sequences or motifs that can form hydrogen bonds according to the rules of nucleic acid base pairing, and each nucleotide from 5' to 3' of one nucleotide sequence or motif can form base pairs with each nucleotide from 3' to 5' of the other nucleotide sequence or motif. In some embodiments, "reverse complementary" includes one or more of the complements of AU, GC, and UG.

[0028] S1 and S4 are motifs consisting of 3-7 nucleotides, respectively, and S1 is the same length as S4 and is completely reverse complementary, and the aptamer having the S1 and S4 motifs has excellent stability and can target tumor tissues and cells for a long time. In some embodiments, S1 and S4 are each consisting of 3-5 nucleotides and are the same length. In some embodiments, in the reverse complement formed by S1 and S4, GC complementation accounts for 40% or more of all complementation numbers, in which case the complex according to the present disclosure has even better stability and tumor targeting ability. In some embodiments, from 5' to 3', S1 is GCU and S4 is AGC, or S1 is GAGU and S4 is GCUC, or S1 is GGAGU and S4 is GCUCU, or S1 is UAUGG and S4 is CCAUG.

[0029] N a and N c are motifs consisting of 1 to 4 nucleotides, respectively, and N a Each nucleotide in c is not complementary to any nucleotide in a and N c The total number of U's in a and N c It accounts for more than 50% of the total number of all nucleotides in the a and N c Aptamers having the motif exhibit superior tumor tissue targeting ability. a and N c The sum of the numbers of nucleotides in N a and N c The sum of the number of nucleotides in N is 3 or 4, and a and N c The sum of the number of U's in is 2 or 3. a and / or N c is U, UU, UC, or CU.

[0030] S2 and S3 are motifs of 1-4 nucleotides each, S2 is the same length as S3, and is a perfect reverse complement. By including S2 and S3 motifs, the complex according to the present disclosure exhibits good stability and excellent tumor targeting ability. In some embodiments, S2 and S3 are each composed of 2-3 nucleotides and are the same length. In some embodiments, the reverse complement formed by S2 and S3 includes at least one GC complement, in which case the reverse complement has better stability. In some embodiments, from 5' to 3', S2 is CA and S3 is UG, or S2 is AC and S3 is GU, or S2 is GCC and S3 is GGU.

[0031] N bis a motif consisting of 3 to 6 nucleotides, N b The nucleotides between both ends of the N are not AU or GC complementary. b Aptamers having the motif can retain a specific spatial arrangement, thereby enabling the conjugates of the present disclosure to stably and efficiently target tumor tissues and cells. b In some embodiments, from 5' to 3', N b is GACG, GACGU, GACCG, UACU, GUUG, or GAUCU.

[0032] The inventors of the present disclosure have discovered that a delivery group formed by an aptamer having the sequence shown in formula (1) above can effectively target tumors, particularly glioma tissues, thereby enabling the conjugate of the present disclosure to specifically enter tumor cells and more effectively deliver the therapeutic group at the cellular level.

[0033] In some embodiments, in the conjugate according to the present disclosure, the length of the contiguous nucleotide sequence is 18 to 50 nucleotides, or 20 to 40 nucleotides, or 21 to 36 nucleotides, or 24 to 32 nucleotides. A delivery group formed by an aptamer having these contiguous nucleotide lengths and a conjugate according to the present disclosure comprising the delivery group can more easily target tumors and has a good balance between synthesis cost and targeting effect.

[0034] In some embodiments, in the complex of the present disclosure, the contiguous nucleotide sequence has the sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3: 5'-CUGCUUCAGACGUGUUAGCUU-3' (SEQ ID NO: 1) where, from 5' to 3', T1 is CU, S1 is GCU, and N a is U, S2 is CA, and N bis GACG, S3 is UG, and N c is UU, S4 is AGC, T2 is UU, 5'-CUGAGUUCAGACGUGUUGCUCU-3' (SEQ ID NO: 2) where, from 5' to 3', T1 is CU, S1 is GAGU, and N a is U, S2 is CA, and N b is GACG, S3 is UG, and N c is UU, S4 is GCUC, T2 is U, 5'-UCUAUGGCUGCCGAUCUGGUCUCCAUGUACGU-3' (SEQ ID NO: 3) where, from 5' to 3', T1 is CU, S1 is GAGU, and N a is U, S2 is CA, and N b is GACG, S3 is UG, and N c is UU, S4 is GCUC, and T2 is U.

[0035] In some embodiments, the contiguous nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO:4, 5'-N6GGAGUUCAN1N2N3N4UGN5GCUCN7-3' (SEQ ID NO: 4) Here, N1, N2 and N3 are each independently one of A, U, C and G, N4 is U, C or G, or a motif consisting of two of U, C or G, N5 is U, CU or UU, N6 is CU, UC or AC, N7 is U, UU or UUN8, and N8 is a motif consisting of 1 to 15 nucleotides.

[0036] In the above nucleotide sequence, from 5' to 3', T1 is a motif represented by N6, S1 is a motif represented by GGAGU, and N a is U, S2 is CA, and N bis the motif N1N2N3N4 consisting of N1, N2, N3 and N4, S3 is UG, and N c is the motif represented by N5, S4 is a motif consisting of GCUC and the first nucleotide in N7, and T2 is a motif consisting of the other nucleotides in N7.

[0037] An aptamer comprising the nucleotide sequence shown in SEQ ID NO:4 can more effectively target tumors, particularly gliomas, and be concentrated in tumor tissue.

[0038] Experimental verification shows that the above selection of N1, N2, N3 and N4 in the nucleotide sequence shown in SEQ ID NO: 4 does not significantly affect the tumor targeting ability of the complex of the present disclosure. In some embodiments, the motif N1N2N3N4 consisting of N1, N2, N3 and N4 is one of GACG, GACGU, GACCG, UACU, GUUG or GAUCU, and the aptamer containing these motifs has a higher tumor-specific targeting effect.

[0039] In some embodiments, in the nucleotide sequence shown in SEQ ID NO: 4, N5 is U or UU. In this case, the complex according to the present disclosure has an excellent targeting effect on tumors.

[0040] In some embodiments, the aptamer has a nucleotide sequence as shown in any one of SEQ ID NOs:5-11. 5'-CUGGAGUUCAGACGUGUUGCUCUU-3' (SEQ ID NO:5) 5'-CUGGAGUUCAGACGUUGUGCUCUU-3' (SEQ ID NO:6) 5'-CUGGAGUUCAGACCGUGUGCUCUU-3' (SEQ ID NO:7) 5'-CUGGAGUUCAGACGUGUUGCUCU-3' (SEQ ID NO:8) 5'-ACGGAGUUCAGACGUGUUGCUCUU-3' (SEQ ID NO: 9) 5'-CUGGAGUUCACUACUGUUGCUCUU-3' (SEQ ID NO: 10) 5'-UGGAGUUCAGUUGUGUUGCUCUU-3' (SEQ ID NO:11)

[0041] The complex of the present disclosure having the above nucleotide sequence exhibits high targeting effect against tumors.

[0042] In some embodiments, motif N8 consists of 1 to 15 nucleotides. In some embodiments, N8 consists of 1 to 8 nucleotides.

[0043] In some embodiments, the presence of motif N8 makes the complex of the present disclosure more stable against exonucleases in the body, thereby enabling it to exert a tumor targeting effect in the body for a longer period of time. In some embodiments, N8 can increase or maintain the tumor targeting effect of the complex of the present disclosure. Considering the balance between stability, targeting, and synthesis efficiency, in some embodiments, motif N8 consists of 8 nucleotides. In some embodiments, the nucleotide sequence of motif N8 from 5' to 3' is CCGAUCUC. In some embodiments, the consecutive nucleotide sequence has a nucleotide sequence shown in any one of SEQ ID NOs: 12 to 14. 5'-CUGGAGUUCAGACGUGUUGCUCUUCCGAUCUC-3' (SEQ ID NO: 12) 5'-CUGGAGUUCAGACGUUGUGCUCUUCCGAUCUC-3' (SEQ ID NO: 13) 5'-CUGGAGUUCAGACCGUGUGCUCUUCCGAUCUC-3' (SEQ ID NO: 14)

[0044] In the contiguous nucleotide sequence, the terminal groups of the ribose 5'-end of the 5'-terminal nucleotide and the ribose 3'-end of the 3'-terminal nucleotide are independently a hydroxyl group or a phosphate group, and the selection of these terminal groups does not change the targeting ability of the complex according to the present disclosure. In some embodiments, in the contiguous nucleotide sequence, the terminal groups of the ribose 5'-end of the 5'-terminal nucleotide and the ribose 3'-end of the 3'-terminal nucleotide are both hydroxyl groups.

[0045] In the complex of the present disclosure, each nucleotide may be modified or unmodified. In general, modification of nucleotide may change the stability and / or tumor targeting ability of the complex of the present disclosure. In some embodiments, at least one nucleotide in the complex of the present disclosure is a modified nucleotide. In some embodiments, at least one group that connects two adjacent nucleotides in the complex of the present disclosure is a phosphate group having a modified group.

[0046] Modifications of the nucleotide include, but are not limited to, modifications to the sugar, modifications to the base, and / or substitution of the nucleotide with a nucleotide analog. In some embodiments, in the complexes of the present disclosure, each of the modified nucleotides is independently one of a 2'-halogen modified nucleotide, a 2'-alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, a 2'-deoxyribonucleotide, a base modified nucleotide, and a nucleotide analog.

[0047] In the context of this disclosure, a "fluoro-modified nucleotide" refers to a nucleotide having the structure shown in formula (7) below, in which the hydroxyl at the 2' position of the ribose group of the nucleotide is replaced with fluorine. A "non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl at the 2' position of the ribose group of the nucleotide is replaced with a non-fluorine group. In some embodiments, each non-fluoro-modified nucleotide is an independently selected one from among nucleotides or nucleotide analogs in which the hydroxyl at the 2' position of the ribose group of the nucleotide is replaced with a non-fluorine group.

[0048] These nucleotides in which the 2'-hydroxyl of ribose is substituted with a non-fluorine group are well known to those skilled in the art, and these nucleotides may be one selected from 2'-alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, and 2'-deoxynucleotides.

[0049] In some embodiments, the 2'-alkoxy modified nucleotide is a methoxy modified nucleotide (2'-OMe) as shown in formula (8). In some embodiments, the 2'-amino modified nucleotide (2'-NH2) is as shown in formula (9). In some embodiments, the 2'-deoxyribonucleotide (DNA) is as shown in formula (10).

[0050] [ka]

[0051] Those skilled in the art are aware of various ways to modify the base of a nucleotide. In some embodiments, the base modification includes, but is not limited to, adding one or more methyl groups to the base. In some embodiments, thymine (T) is considered a type of base modification uracil (U). In some embodiments, 2-methylcytosine is considered a type of base modification cytosine (C).

[0052] A nucleotide analog refers to a group that can substitute for a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, a nucleotide analog can be an isonucleotide, a bridged nucleotide (abbreviated as BNA), or an acyclic nucleotide.

[0053] BNA refers to a constrained or inaccessible nucleotide. BNAs may include five-, six-, or seven-membered bridged structures with a "fixed" C3'-endo sugar puckering. Typically, the bridge is introduced at the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide. In some embodiments, the BNA may be an LNA as shown in formula (12), an ENA as shown in formula (13), a cET BNA as shown in formula (14), or the like.

[0054] [ka]

[0055] An acyclic nucleotide is a nucleotide in which the sugar ring of the nucleotide is opened. In some embodiments, the acyclic nucleotide may be an unlocked nucleic acid (UNA) as shown in formula (15), a glycerol nucleic acid (GNA) as shown in formula (16), or a peptide nucleic acid (PNA).

[0056] [ka]

[0057] In the above formula (15) and formula (16), R is selected from H, OH, or alkoxy (O-alkyl group).

[0058] Peptide nucleic acids are nucleotide analogs formed from a polypeptide backbone substituted glycoside-phosphate backbone. In some embodiments, peptide nucleic acids may be nucleotide analogs formed from, for example, 2-aminoethylglycine-linked substituted glycoside-phosphate units.

[0059] An isonucleotide refers to a compound in which the position of the base in the ribose ring in a nucleotide is changed. In some embodiments, an isonucleotide may be a compound in which the base is shifted from the 1'-position to the 2'-position or 3'-position of the ribose ring, as shown in formula (17) or (18).

[0060] In the compounds of formula (17) and formula (18) above, Base represents a nucleic acid base such as A, U, G, C or T, and R is selected from H, OH, F or the non-fluorine groups described above.

[0061] [ka]

[0062] In some embodiments, the nucleotide analog is one selected from an isonucleotide, an LNA, an ENA, a cET, an UNA, and a GNA. In some embodiments, each non-fluoro modified nucleotide is a methoxy modified nucleotide, which in this context refers to a nucleotide in which the 2'-hydroxy group of the ribose is replaced with methoxy.

[0063] In this context, "fluoro-modified nucleotide", "2'-fluoro-modified nucleotide", "nucleotide in which the 2'-hydroxy group of the ribose group is replaced with fluorine" and "nucleotide having 2'-fluoro ribose" have the same meaning and all refer to a compound having the structure shown in formula (7) in which the 2'-hydroxy group of the nucleotide is replaced with fluorine, and "methoxy-modified nucleotide", "2'-methoxy-modified nucleotide", "nucleotide in which the 2'-hydroxy group of the ribose group is replaced with methoxy" and "nucleotide having 2'-methoxy ribose" have the same meaning and all refer to a compound having the structure shown in formula (8) in which the 2'-hydroxy group of the ribose group of the nucleotide is replaced with methoxy.

[0064] In some embodiments, each cytosine nucleotide in the contiguous nucleotide sequence in the complex of the present disclosure is a fluoro-modified cytosine nucleotide, and / or each uracil nucleotide in the contiguous nucleotide sequence is a fluoro-modified uracil nucleotide. In some embodiments, each nucleotide in the contiguous nucleotide sequence in the complex of the present disclosure is a 2'-methoxy modified nucleotide. In some embodiments, one or more uracil nucleotides in the complex of the present disclosure have a modified base. In some embodiments, a thymine base (T) is considered to be a methyl-modified uracil base (U).

[0065] The group linking two adjacent nucleotides may be a phosphate group or a modified phosphate group. The modification of the phosphate group may, for example, replace at least one non-bridging oxygen atom in the phosphate group with a sulfur atom to form a thiophosphate group or a dithiophosphate group. In some embodiments, at least one group linking two adjacent nucleotides in the complex according to the present disclosure is a thiophosphate group. In some embodiments, at least one of the three groups linking two adjacent nucleotides between the first four nucleotides at the 5' end in the consecutive nucleotide sequence is a thiophosphate group. In some embodiments, at least two of the three groups linking two adjacent nucleotides between the first four nucleotides at the 5' end in the consecutive nucleotide sequence are thiophosphate groups. In some embodiments, at least one of the groups linking two adjacent nucleotides between the first four nucleotides at the 3' end in the consecutive nucleotide sequence is a thiophosphate group. In some embodiments, at least two of the three groups linking two adjacent nucleotides among the first four nucleotides at the 3' end in the contiguous nucleotide sequence are phosphorothioate groups. In some embodiments, each group linking two adjacent nucleotides in the contiguous nucleotide sequence is a phosphorothioate group.

[0066] The conjugates of the present disclosure having the above modifications are not only low in cost, but also can make the binding groups less susceptible to cleavage by ribonucleases in the body, thereby increasing the stability of the conjugates of the present disclosure and making them more resistant to nuclease hydrolysis.The conjugates of the present disclosure containing the above modified delivery groups also have high activity in targeting tumor tissues and / or cells.

[0067] In some embodiments, the contiguous nucleotide sequence has a nucleotide sequence set forth in one of SEQ ID NOs: 15 to 39, 5'-CfUfGGAGUfUfCfAGACfGUfGUfUfGCfUfCfUfUfCfCfGAUfCfUfCf-3' (SEQ ID NO: 15) 5'-CfUfGGAGUfUfCfAGACfGUfGUfUfGCfUfCfUfUf-3' (SEQ ID NO: 16) 5'-CfUfGGAGUfUfCfAGACfGUfUfGUfGCfUfCfUfUfCfCfGAUfCfUfCf-3' (SEQ ID NO: 17) 5'-CfUfGGAGUfUfCfAGACfGUfUfGUfGCfUfCfUfUf-3' (SEQ ID NO: 18) 5'-CfUfGGAGUfUfCfAGACfCfGUfGUfGCfUfCfUfUfCfCfGAUfCfUfCf-3' (SEQ ID NO: 19) 5'-CfUfGGAGUfUfCfAGACfCfGUfGUfGCfUfCfUfUf-3' (SEQ ID NO: 20) 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmUmCmCmGmAmUmCmUmCm-3' (SEQ ID NO: 21) 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUmUm-3' (SEQ ID NO: 22) 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmUmGmUmCmUmCmUmCmGmAmUmCmUmCm-3' (SEQ ID NO: 23) 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmUmGmUmGmCmUmCmUmUm-3' (SEQ ID NO: 24) 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmCmGmUmGmUmCmUmCmUmCmGmAmUmCmUmCm-3' (SEQ ID NO: 25) 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmCmGmUmGmUmGmCmUmCmUmUm-3' (SEQ ID NO: 26) 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUm-3' (SEQ ID NO: 27) 5'-CmUmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUm-3' (SEQ ID NO: 28) 5'-CmUmGmCmUmUmCmAmGmAmCmGmUmGmUmUmAmGmCmUmUm-3' (SEQ ID NO: 29) 5'-AmCmGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUmUm-3' (SEQ ID NO: 30) 5'-CmUmGmGmAmGmUmUmCmAmCmUmAmCmUmGmUmUmGmCmUmCmUmUm-3' (SEQ ID NO: 31) 5'-CmUmGmGmAmGmUmUmCmAmGmUmUmGmUmUmGmUmUmGmCmUmCmUmUm-3' (SEQ ID NO: 32) 5'-UmCmUmAmUmGmGmCmUmGmCmCmGmAmUmCmUmGmGmUmCmCmCmAmUmGmUmAmCmGmUm-3' (SEQ ID NO: 33) 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUmCmCmGmAmUmCmsUmsCm-3' (SEQ ID NO: 34) 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmsUmsUm-3' (SEQ ID NO: 35) 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmGmUmUmGmUmCmUmCmUmCmGmAmUmCmsUmsCm-3' (SEQ ID NO: 36) 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmGmUmUmGmUmGmCmUmCmsUmsUm-3' (SEQ ID NO: 37) 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmCmGmUmGmUmCmUmCmUmCmCmGmAmUmCmsUmsCm-3' (SEQ ID NO: 38) 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmCmGmUmGmUmGmCmUmCmsUmsUm-3' (SEQ ID NO: 39) Here, the capital letters C, G, U, and A represent the base composition of the nucleotide, the lower case letter m represents that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide, the lower case letter f represents that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide, and the lower case letter s represents that the two nucleotides on the left and right of the letter s are bonded by a thiophosphate group.

[0068] In some embodiments, the complex of the present disclosure has a structure shown in formula (101):

[0069] [ka] (101) In the formula, each R AP The groups are independently groups having the structure shown in formula (2).

[0070] [ka] (102) wherein each AP group is the same or different and independently represents one of the delivery groups; each A0 group is the same or different and independently represents one of the functional groups; R j , each R k Or each R i are the same or different and each independently represent a covalent bond or a linking group, and R i and R k are not both a covalent bond at the same time; m0 is an integer of 1 to 6; n0 is an integer of 1 to 6; and each n1 independently represents an integer of 0 to 4; JPEG2025512110000008.jpg5127 represents the site to which the group is covalently attached.

[0071] In some embodiments, m0 is an integer of 1 to 6, i.e., the conjugate represented by formula (101) contains 1 to 6 functional groups A0. In consideration of delivery efficiency and synthesis cost, in some embodiments, m0 is an integer of 1 to 4, i.e., the conjugate represented by formula (101) contains 1 to 4 functional groups A0. In some embodiments, m0 is 1, i.e., the conjugate represented by formula (101) contains one functional group A0.

[0072] In some embodiments, n0 is an integer from 1 to 6, i.e., the conjugate of formula (101) comprises 1 to 6 R AP From the viewpoint of delivery efficiency and cost, in some embodiments, n0 is an integer of 1 to 3, i.e., the conjugate represented by formula (101) contains 1 to 3 R AP In some embodiments, n0 is 1, i.e., the conjugate of formula (101) contains one R AP Contains a group.

[0073] In some embodiments, each n1 independently represents an integer from 0 to 4, and R i and R k are not both covalent bonds at the same time, so that each R AP The group contains 1 to 5 delivery groups AP. In some embodiments, each n1 independently represents an integer from 0 to 1, whereby each R AP The group contains 1 to 2 delivery groups AP. In some embodiments, n0 is 1 and n1 is 0, in which case the conjugate shown in formula (101) contains one delivery group AP.

[0074] R AP In the group, R k and R i The role of the delivery group AP is to j Covalently bonded to the R j The purpose of the present invention is to bind the functional group A0 via a group. Therefore, any R group that can achieve the above-mentioned binding and does not adversely affect the effectiveness of the delivery group AP and the functional group A0 can be used. k or Ri In some embodiments, any of the R k or each of the above R i are independently a straight chain alkylene group having a length of 1 to 70 carbon atoms, or one or more of the carbon atoms in the straight chain alkylene group is selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, OP(O)2, OP(O)(S), a C5-C8 glycosidic group, a C2-C 10 Alkenylene group, C2-C 10 Alkynylene group, C6-C 10 Arylene group, C3-C 18 Heterocyclylene groups and C5-C 10 heteroarylene groups, and the linear alkylene group is substituted with one or more selected from the group consisting of C1 to C 10 Alkyl groups, C6-C 10 Aryl groups, C5-C 10 Heteroaryl groups, C1-C 10 Halogenated alkyl groups, -OC1~C 10 Alkyl group, -OC1~C 10 Alkylphenyl group, -C1~C 10 Alkyl-OH, -OC1~C 10 Halogenated alkyl groups, -SC1~C 10 Alkyl groups, -SC1~C 10 Alkylphenyl group, -C1~C 10 Alkyl-SH, -SC1~C 10 Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH2, -C1~C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl group), -NH(C1-C 10 Alkyl group), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl group), -NH(C1-C 10 alkylphenyl group), cyano group, nitro group, -CO2H, -C(O)O(C1-C 10 Alkyl group), -CON(C1-C 10 Alkyl)(C1-C 10Alkyl group), -CONH(C1-C 10 Alkyl group), -CONH2, -NHC(O)(C1-C 10 alkyl group), -NHC(O)(phenyl group), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl group), -N(C1-C 10 Alkyl)C(O)(phenyl group), -C(O)C1~C 10 Alkyl group, -C(O)C1~C 10 Alkylphenyl group, -C(O)C1~C 10 Halogenated alkyl groups, -OC(O)C1~C 10 Alkyl group, -SO2(C1-C 10 Alkyl group), -SO2(phenyl group), -SO2(C1-C 10 Halogenated alkyl group), -SO2NH2, -SO2NH(C1-C 10 alkyl group), -SO2NH (phenyl group), -NHSO2 (C1-C 10 alkyl group), -NHSO2 (phenyl group) and -NHSO2 (C1-C 10 In consideration of the synthesis cost, difficulty, and tumor targeting effect of the complex, in some embodiments, each n1 is 0, and each R i are independently a covalent bond or a C1-C 20 The bond may be one or a combination of more than one of an alkylene group, a phosphate ester bond, a thiophosphate ester bond, an amide bond, an ester bond, an ether bond, a thioether bond, a disulfide bond, a 1,2,3-triazole subunit, a polyethylene glycol subunit, a pyrrolidine subunit, a 2-oxopyrrolidine subunit, a phenylene group, a cyclohexylene group, a 2-succinimide subunit, a 2-thiosuccinimide subunit, an amino acid subunit, and a nucleotide subunit.

[0075] In some embodiments, the linking group R j includes any linking group that can be used for antibody drug conjugates known to those skilled in the art.j may be degradable or non-degradable. In some embodiments, the linking group R j In this context, "degradable" refers to the fact that the linking group R can be degraded after the conjugate of the present disclosure has been targeted to the tumor. j This refers to the fact that the linking group R undergoes covalent bond cleavage in the intratumoral environment and / or within tumor cells, releasing a single therapeutic agent group to produce a therapeutic effect. j comprises one or more of an activated enzyme linking group, a sulfatase-cleavable linking group, a galactose-cleavable linking group, a lysosomal protease-sensitive linking group, a peptide linking group, a glucuronide linking group, an acid-sensitive cleavable linking group, or a glutathione-sensitive disulfide linking group. In some embodiments, the linking group R j comprises a peptide linking group. In some embodiments, the peptide linking group is one or more selected from the following: a valine-citrulline dipeptide linker (Val-Cit), an alanine-alanine dipeptide linker (Ala-Ala), a valine-alanine dipeptide linker (Val-Ala), a glycine-glycine-phenylalanine-glycine tetrapeptide linker (Gly-Gly-Phc-Gly). In some embodiments, the linking group R j is one selected from N-succinylidene 4-(2-dithiopyridine)butyrate (SPDB), N-succinimido-4-(2-thiopyridylidene)valerate (SPP), (S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanoic acid (Val-Cit-PAB-OH), N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or 2-(phosphate-(CH2)6-S-)-maleimidocaproyl-valine-citrulline-p-aminobenzyl subunit. In some embodiments, the linking group R jincludes the linking groups listed in Mckertish CM, Kayser V. Advances and Limitations of Antibody Drug Conjugates for Cancer. Biomedicines. 2021 Jul 23;9(8):872., the contents of which are incorporated by reference in their entirety into this specification.

[0076] In some embodiments, the linking group R j comprises one or more of a valine-citrulline dipeptide linker (Val-Cit), a polyethylene glycol subunit, an iminohexyl subunit, an N-succinimidyl group, or a GAU trinucleotide linking group. i is independently a combination of one or two of a covalent bond, a disulfide bond, a dodecylene group, a valine-citrulline dipeptide linker (Val-Cit), a polyethylene glycol subunit, an iminohexyl subunit, an N-succinimidyl group, or a GAU trinucleotide subunit.

[0077] R j The role of the group is R AP By attaching a group to the functional group A0, R AP The objective of the present invention is to specifically deliver the functional group A0 to tumor tissues and / or cells through the tumor targeting effect of the delivery group AP in the group. Therefore, any R that can achieve the above-mentioned binding and does not affect the tumor targeting effect of the delivery group AP and the effect of the functional group A0 can be used. j Any of the R groups can achieve the object of the present invention and solve the technical problem that the present invention is intended to solve. In some embodiments, after the conjugate of formula (1) reaches the tumor tissue and / or enters the tumor cell, the R j is decomposed to release a pharmacoactive molecule corresponding to the single functional group A0. In some embodiments, the R j is not broken down in the body, and in this case, R in the complex j Groups and R APThe presence of the group does not affect the ability of the functional group A0 to exert a therapeutic effect.

[0078] In some embodiments, R j is a covalent bond, and m is 1, in which case the complex shown in formula (101) has one functional group A and one R AP groups, each R AP A group is directly bonded to the functional group A. In some embodiments, each R AP groups are attached to the same atom of functional group A. In some embodiments, each R AP The groups are attached to different atoms of the functional group A0.

[0079] In some embodiments, R j is a bonding group, and the bonding group R j comprises a main chain portion, a side chain portion and a complex linkage portion.

[0080] The backbone moiety is attached to the complex linkage and the side chain moiety, respectively. In some embodiments, the backbone moiety is a straight chain alkylene group having a length of 1 to 70 carbon atoms, or one or more of the carbon atoms in the straight chain alkylene group is selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycosidic groups, C2-C 10 Alkenylene group, C2-C 10 Alkynylene group, C6-C 10 Arylene group, C3-C 18 Heterocyclylene groups and C5-C 10 heteroarylene groups, and the linear alkylene group is substituted with one or more selected from the group consisting of C1 to C 10 Alkyl groups, C6-C 10 Aryl groups, C5-C 10 Heteroaryl groups, C1-C 10 Halogenated alkyl groups, -OC1~C 10 Alkyl group, -OC1~C 10 Alkylphenyl group, -C1~C 10 Alkyl-OH, -OC1~C 10 Halogenated alkyl groups, -SC1~C 10Alkyl groups, -SC1~C 10 Alkylphenyl group, -C1~C 10 Alkyl-SH, -SC1~C 10 Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH2, -C1~C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl group), -NH(C1-C 10 Alkyl group), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl group), -NH(C1-C 10 alkylphenyl group), cyano group, nitro group, -CO2H, -C(O)O(C1-C 10 Alkyl group), -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl group), -CONH(C1-C 10 Alkyl group), -CONH2, -NHC(O)(C1-C 10 alkyl group), -NHC(O)(phenyl group), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl group), -N(C1-C 10 Alkyl)C(O)(phenyl group), -C(O)C1~C 10 Alkyl group, -C(O)C1~C 10 Alkylphenyl group, -C(O)C1~C 10 Halogenated alkyl groups, -OC(O)C1~C 10 Alkyl group, -SO2(C1-C 10 Alkyl group), -SO2(phenyl group), -SO2(C1-C 10 Halogenated alkyl group), -SO2NH2, -SO2NH(C1-C 10 alkyl group), -SO2NH (phenyl group), -NHSO2 (C1-C 10 alkyl group), -NHSO2 (phenyl group) and -NHSO2 (C1-C 10 halogenated alkyl groups).

[0081] The side chain part is the main chain part and RAP In some embodiments, each side chain moiety is independently a covalent bond or a straight chain alkylene group that is 1-70 carbon atoms in length, or one or more of the carbon atoms in the straight chain alkylene group is selected from C(O), NH, O, S, CH=N, S(O)2, OP(O)2, a C5-C8 glycosidic group, a C2-C 10 Alkenylene group, C2-C 10 Alkynylene group, C6-C 10 Arylene group, C3-C 18 Heterocyclylene groups and C5-C 10 heteroarylene groups, and the linear alkylene group is substituted with one or more selected from the group consisting of C1 to C 10 Alkyl groups, C6-C 10 Aryl groups, C5-C 10 Heteroaryl groups, C1-C 10 Halogenated alkyl groups, -OC1~C 10 Alkyl group, -OC1~C 10 Alkylphenyl group, -C1~C 10 Alkyl-OH, -OC1~C 10 Halogenated alkyl groups, -SC1~C 10 Alkyl groups, -SC1~C 10 Alkylphenyl group, -C1~C 10 Alkyl-SH, -SC1~C 10 Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH2, -C1~C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl group), -NH(C1-C 10 Alkyl group), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl group), -NH(C1-C 10 alkylphenyl group), cyano group, nitro group, -CO2H, -C(O)O(C1-C 10 Alkyl group), -CON(C1-C 10 Alkyl group) (C1-C 10 Alkyl group), -CONH(C1-C 10Alkyl group), -CONH2, -NHC(O)(C1-C 10 alkyl group), -NHC(O)(phenyl group), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl group), -N(C1-C 10 Alkyl)C(O)(phenyl group), -C(O)C1~C 10 Alkyl group, -C(O)C1~C 10 Alkylphenyl group, -C(O)C1~C 10 Halogenated alkyl groups, -OC(O)C1~C 10 Alkyl group, -SO2(C1-C 10 Alkyl group), -SO2(phenyl group), -SO2(C1-C 10 Halogenated alkyl group), -SO2NH2, -SO2NH(C1-C 10 alkyl group), -SO2NH (phenyl group), -NHSO2 (C1-C 10 alkyl group), -NHSO2 (phenyl group) and -NHSO2 (C1-C 10 halogenated alkyl groups), The composite linkages are attached to the main chain portion and the functional group A0, respectively. In some embodiments, each composite linkage is independently a covalent bond or a C1-C 10 The bond may be one or a combination of more than one of an alkylene group, a phosphate ester bond, a thiophosphate ester bond, an amide bond, an ester bond, an ether bond, a thioether bond, a disulfide bond, a 1,2,3-triazole subunit, a polyethylene glycol subunit, a pyrrolidine subunit, a 2-oxopyrrolidine subunit, a phenylene group, a cyclohexylene group, a 2-succinimide subunit, a 2-thiosuccinimide subunit, an amino acid subunit, and a nucleotide subunit.

[0082] In some embodiments, the linking group R jEach of the composite bonds in each of the functional groups A0 is bonded to the main chain portion, and the number of the side chain portions is n0. Each of the side chain portions is bonded to the main chain portion and one of the R AP Thus, each functional group A and R AP The groups each independently represent a linking group R j In some embodiments, all of the side chain moieties are attached to the same atom in the main chain moiety, or alternatively, each side chain moiety is attached to a different atom in the main chain moiety.

[0083] In some embodiments, m0 is 1 and the linking group R j includes the structure shown in formula (301).

[0084] [ka] Formula (301) In the formula, k is an integer of 1 to 3, and L C is the main chain portion, and L A is the side chain moiety, and L B is the composite joint, JPEG2025512110000010.jpg6136 represents the site to which the group is covalently attached.

[0085] The main chain portion L C is a covalent bond or a divalent to tetravalent, linear or branched C1 to C 25 or one or more carbon atoms in the saturated hydrocarbon group are selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycoside group, C2-C5 alkenylene group, C2-C5 alkynylene group, C6-C 10 Arylene groups, C3-C8 heterocyclylene groups and C5-C 10 heteroarylene groups, and the saturated hydrocarbon group is substituted with one or more selected from the group consisting of C1 to C5 alkyl groups, C6 to C 10 Aryl groups, C5-C 10Heteroaryl group, -O-C1-C5 alkyl group, -OC1-C5 alkylphenyl group, -C1-C5 alkyl-OH, -SC1-C5 alkyl group, nitro, -C(O)O(C1-C5 alkyl group), -CON(C1-C5 alkyl)(C1-C5 alkyl group), -CONH(C1-C5 alkyl group), -CONH2, -NHC(O)(C1-C5 alkyl group), -NHC(O)(phenyl group), -N(C1-C5 alkyl)C(O)(C1-C5 alkyl group), - It may have one or more substituents selected from the group consisting of N(C1-C5 alkyl)C(O) (phenyl group), -C(O)C1-C5 alkyl group, -C(O)C1-C5 alkylphenyl group, -OC(O)C1-C5 alkyl group, -SO2(C1-C5 alkyl group), -SO2(phenyl group), -SO2NH2, -SON2NH(C1-C5 alkyl group), -SON2NH(phenyl group), -NHSO2(C1-C5 alkyl group) and -NHSO2(phenyl group). In some embodiments, L C is divalent to tetravalent C5 to C 20 or one or more carbon atoms in the saturated hydrocarbon group are selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycoside group, C2-C5 alkenylene group, C2-C5 alkynylene group, C6-C 10 Arylene groups, C3-C8 heterocyclylene groups and C5-C 10 heteroarylene groups, and the saturated hydrocarbon group is substituted with one or more selected from the group consisting of C1 to C5 alkyl groups, C6 to C 10 Aryl groups, C5-C 10 In some embodiments, L may have one or more substituents selected from the group consisting of a heteroaryl group, an -O-C1 to C5 alkyl group, an -O-C1 to C5 alkylphenyl group, a -C1 to C5 alkyl-OH group, an -SC1 to C5 alkyl group, a nitro group, and -CONH2. C The length of L is 5 to 30 atoms. C The length of is L C L in A From an atom directly bonded to L BThe number of chain atoms in the longest chain of atoms formed up to the atom directly bonded to L. C The length ranges from 8 to 25 atoms.

[0086] The side chain moiety L A is a covalent bond or C1-C 20 or one or more carbon atoms in the alkylene group are C(O), NH, O, S, CH=N, S(O)2, OP(O)2, a C5-C8 glycoside group, a C2-C5 alkenylene group, a C2-C5 alkynylene group, a C6-C 10 Arylene groups, C3-C8 heterocyclylene groups and C5-C 10 heteroarylene groups, and the alkylene group is substituted with one or more selected from the group consisting of C1 to C5 alkyl groups, C6 to C 10 Aryl groups, C5-C 10Heteroaryl group, -O-C1-C5 alkyl group, -OC1-C5 alkylphenyl group, -C1-C5 alkyl-OH-SC1-C5 alkyl group, -SC1-C5 alkylphenyl group, -C1-C5 alkyl group, -SH, -OH, -SH, -NH2, -C1-C5 alkyl group, -NH2, -N(C1-C5 alkyl)(C1-C5 alkyl group), -NH(C1-C5 alkyl group), -N(C1-C5 alkyl)(C1-C5 alkylphenyl group), -NH(C1-C5 alkylphenyl group), nitro group, -C(O)O(C1-C5 alkyl group), -CON(C1-C5 alkyl)(C1-C5 alkyl group), -CONH(C1-C5 alkyl may have one or more substituents selected from the group consisting of -CONH2, -NHC(O)(C1-C5 alkyl group), -NHC(O)(phenyl group), -N(C1-C5 alkyl)C(O)(C1-C5 alkyl group), -N(C1-C5 alkyl)C(O)(phenyl group), -C(O)C1-C5 alkyl group, -C(O)C1-C5 alkylphenyl group, -OC(O)C1-C5 alkyl group, -SO2(C1-C5 alkyl group), -SO2(phenyl group), -SO2NH2, -SON2NH(C1-C5 alkyl group), -SON2NH(phenyl group), -NHSO2(C1-C5 alkyl group) and -NHSO2(phenyl group).

[0087] The composite joint L B is one or a combination of one to five bonds selected from the group consisting of a phosphate bond, a thiophosphate bond, an amide bond, an ester bond, an ether bond, and a disulfide bond.

[0088] In some embodiments, k is an integer from 1 to 3, and L C contains any one of the groups represented by formulae (L1) to (L3), and is connected to L via an ether bond in the group represented by formulae (L1) to (L3). A is bonded to the part.

[0089] [ka] represents the site at which the group is attached to the remainder of the molecule. In some embodiments, k=1 and L C contains a group represented by formula (L1), and the O atom in the group (L1) is L A In some embodiments, k=2 and L C contains a group represented by the formula (L2), and each of the two O atoms in the group (L1) is one L A In some embodiments, k=4 and L C contains a group represented by the formula (L3), and each of the three O atoms in the group (L3) is one L A is directly bonded to

[0090] L B is a phosphate bond or a disulfide bond, Each L A is a covalent bond, or each L A is selected from the group consisting of groups (L4) to (L23) and combinations thereof.

[0091] [ka] In the formula, each j1 is an integer from 1 to 10, Each R' is C1 to C 10 is an alkyl group, Each Ra is a hydrogen atom, C1 to C 10 An alkyl group or a group selected from the group consisting of the groups (L24) to (L37).

[0092] [ka]

[0093] In some embodiments, L A The length of L is 3 to 35 atoms. A The length of is L A L in C From an atom directly bonded to L A R in APIn some embodiments, each L A is a combination of at least two bonds among groups (L4) to (L9), (L13), (L14), and (L18). A is a combination of at least two bonds among groups (L4), (L5), (L7), (L9), (L13), (L14), and (L18).

[0094] In some embodiments, L A has a structure containing an amide bond as shown in formula (302), and L B has a structure containing an N-acylpyrrolidine represented by formula (303), contains a carbonyl group and an oxygen atom, and L C is a linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trihydroxymethylaminomethane.

[0095] [ka] In the formula, n 302 , q 302 and p 302 are each independently an integer of 2 to 6, and preferably, 302 , q 302 and p 302 are each independently 2 or 3; n 303 is an integer from 4 to 16, preferably, 303 is an integer between 8 and 12, JPEG2025512110000015.jpg6152 represents the site to which the group is covalently attached.

[0096] In some embodiments, each said side chain moiety L A Each is an R AP group via a phosphate ester bond, an ether bond or an ester bond, and C and the oxygen atom of the hydroxyl group in the main chain portion L Cand are bonded by forming an ether bond, B represents the carbonyl group in formula (303) and the main chain portion L C and the nitrogen atom of the amino group in formula (303) is bonded to the functional group A0 by forming an amide bond, and the oxygen atom in formula (303) is bonded to the functional group A0 by forming a phosphate ester bond, an ether bond, or an ester bond. C is a linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trishydroxymethylaminomethane, and the main chain portion L C The oxygen atom of the hydroxyl group is A and the nitrogen atom of the amino group is bonded to the complex bond L via an ether bond. B is bonded to the linking group R via an amide bond. j is a compound having 1 to 3 side chains bonded to the carbon atom of the same aminomethyl group and a complex bond L B via R containing the delivery group AP is bonded to the group.

[0097] In some embodiments, the complex has a structure shown in formula (305):

[0098] [ka] Formula (305)

[0099] In some embodiments, the linking group R j includes the structure shown in formula (306).

[0100] [ka] Formula (306) In the formula, n 306 is an integer from 0 to 3, and each p 306 are independently an integer from 1 to 6, JPEG2025512110000018.jpg7140 represents the site where the group is covalently attached, the combination of bonds formed by all pyrrolidine subunits and any possible phosphodiester groups constitutes the main chain moiety, the chain of atoms between the carbonyl group attached to the nitrogen atom in the pyrrolidine subunit and the oxygen atom marked with an * constitutes each side chain moiety, the oxygen atom marked with an * constitutes each side chain moiety, AP At least one of the oxygen atoms marked with # is a complex bond and forms an ether bond, ester bond or phosphate bond with the functional group A0, and the remaining oxygen atoms marked with # are bonded to a hydrogen atom to form a hydroxyl group or to a C1-C3 alkyl group to form a C1-C3 alkoxy group. Therefore, the bonded group R j The 1 to 3 side chain moieties in the AP is bonded to the group.

[0101] In some embodiments, a complex according to the present disclosure has a structure shown in formula (307a), (307b), or (307c).

[0102] [ka]

[0103] In some embodiments, the complex of the present disclosure has the structure shown in formula (308):

[0104] [ka] Formula (308) In the formula, n 308 may be an integer of 1 to 10. In some embodiments, n is an integer of 1 to 10, taking into consideration various aspects such as ease of synthesis, structure / process cost, and tumor cell specificity. 308 is an integer from 2 to 6. In some embodiments, n 308 is 3 or 4.

[0105] Each R3 is independently a functional group A0, or an R AP In some embodiments, at least one R is a functional group A and at least one R is a R AP In some embodiments, one R is a functional group A and the remaining R is R AP It is based on

[0106] In some embodiments, each m 308 is independently selected from an integer of 2 to 10, it is believed that the spatial position between the multiple delivery groups AP in the complex can be made more suitable for interaction with the corresponding receptors on the surface of the tumor cell, and in order to make the compound represented by formula (308) simpler, easier to synthesize, and / or less costly, according to some embodiments of the present disclosure, each m 308 is independently an integer from 2 to 5, and in some embodiments, each m 308 are all equal.

[0107] Those skilled in the art will recognize that each R 308 H, C1~C 10 Alkyl groups, C1-C 10 Halogenated alkyl groups and C1-C 10 It will be appreciated that when independently selected from alkoxy groups, any of them can achieve the objectives of the present disclosure without changing the properties of the conjugate shown in formula (308). 308 is independently selected from H, a methyl group, or an ethyl group. 308 are both H.

[0108] Each L1 bonded to the functional group A0 represents the composite bond, and the R AP Each L1 bonded to represents the side chain moiety. In some embodiments, one R3 is the functional group A0 and the remaining R3 is the R APIn some embodiments, one or more L1 is a group as the side chain moiety, R AP The nitrogen-containing backbone is provided with a linking group R j In the context of the present disclosure, a "nitrogen-containing backbone" refers to the chain structure in the structure shown in formula (308), and R 308 The carbon atom to which is bonded and the N atom are bonded to each other.

[0109] Considering the delivery efficiency and synthesis cost, in some embodiments, the length of each L1 is independently 3 to 25 atoms. In some embodiments, the length of each L1 is independently 4 to 15 atoms. For convenience, L1 is defined as a straight chain alkylene group, but it will be understood by those of skill in the art that the amino and alkenyl groups resulting from the above-mentioned replacement and / or substitution may not be straight chain groups or may have different names. For purposes of this disclosure, the length of L1 is the number of atoms in the chain connecting the two attachment points. For this purpose, for a ring (e.g., a heterocyclylene or heteroarylene group) obtained by replacing carbon atoms of the straight chain alkylene group, the length of the portion of the chain corresponding to the ring is calculated based on the minimum number of atoms between the attachment points on the ring.

[0110] In some embodiments, L1 is selected from the group consisting of the groups shown in formulas L4-L23 above and any bond combinations thereof. In some embodiments, each L1 is independently selected from the group consisting of a combination of at least two bonds among groups L4-L9, L13, L14, and L18. In some embodiments, each L1 is independently a combination of at least two bonds among groups L4, L5, L7, L9, L13, L14, and L18.

[0111] In some embodiments, in the conjugate of formula (308), each L1 has a binding site bonded to an N atom on the nitrogen-containing backbone and a functional group A0 or APA bond site that is bonded to a group is included together with the site that is bonded to an N atom on the nitrogen-containing backbone to form an amide bond with the N atom. In some embodiments, one or more L1 is selected from B5, B6, B5', or B6'.

[0112] [ka] During the ceremony, JPEG2025512110000022.jpg5136 represents the site to which the group is covalently attached, and q2 is an integer from 1 to 10. In some embodiments, q2 is an integer from 1 to 5.

[0113] In some embodiments, each R AP The group comprises one or more delivery groups. In some embodiments, the compound of formula (308) comprises multiple functional groups. In some embodiments, each functional group in the compound of formula (308) is the same functional group. In some embodiments, each functional group in the compound of formula (308) is a functional group for the same purpose and function. In some embodiments, the compound of formula (308) comprises different types of functional groups for different purposes and functions.

[0114] In some embodiments, the compound of formula (308) has a structure of formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), (422), (423), (424), (425), (426) or (427).

[0115] [ka] JPEG2025512110000024.jpg224143JPEG2025512110000025.jpg221139JPEG2025512110000026.jpg216131JPEG2025512110000027.jpg25114 9JPEG2025512110000028.jpg250143JPEG2025512110000029.jpg249139JPEG2025512110000030.jpg250145JPEG2025512110000031.jpg82135

[0116] In some embodiments, the linking group R jcomprises a nucleotide sequence I and a nucleotide sequence II, each of which comprises 5-25 modified or unmodified nucleotides, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse complementary, the delivery group being attached to the nucleotide sequence I, the functional group being attached to the nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II not eliciting an immune or toxic reaction in a subject. In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary or completely reverse complementary, or the nucleotide sequence I and the nucleotide sequence II are equal in length and each of which comprises 10-20 modified or unmodified nucleotides, or the nucleotide sequence I and the nucleotide sequence II each consist of 17 nucleotides and are completely reverse complementary. In some embodiments, the 3' terminus of the delivery group is linked via a phosphate bond to the 5' ribose position of the 5' terminal nucleotide of nucleotide sequence I and the functional group is linked via a phosphate bond to the 5' ribose position of the 5' terminal nucleotide of nucleotide sequence II, or the functional group comprises a nucleotide sequence, the 3' terminus of the nucleotide sequence is linked via a phosphate bond to the 5' ribose position of the 5' terminal nucleotide of nucleotide sequence I. In some embodiments, the nucleotide sequence I and the nucleotide sequence II have the sequences set forth in SEQ ID NO:40 and SEQ ID NO:41, respectively. 5'-GUACAUUCUAGAUAGCC-3' (SEQ ID NO:40) 5'-GGCUAUCUAGAAUGUAC-3' (SEQ ID NO:41)

[0117] In some embodiments, the nucleotide sequence I and the nucleotide sequence II have the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. 5'-GmUfAmCfAmUfUfCfUfAmGmAmUfAmGmCfCf-3' (SEQ ID NO: 42) 5'-GmGmCfUfAmUfCfUfAmGmAmAmUfGmUfAmCf-3' (SEQ ID NO: 43)

[0118] In some embodiments, the functional group is a small molecule therapeutic group having a therapeutic effect on tumors, each small molecule therapeutic group being independently selected from a cytotoxin group, an antibiotic group, or an angiogenesis inhibitor. By including a small molecule therapeutic group, the conjugate of the present disclosure can specifically deliver the small molecule therapeutic group to the tumor, thereby treating and / or alleviating the progression or symptoms of the tumor disease through the effect of the small molecule therapeutic group, for example, the conjugate of the present disclosure specifically delivers the cytotoxin group to the tumor, thereby specifically eliminating cancer cells in the tumor, thereby reducing side effects due to the poor targeting of the cytotoxin itself, and significantly reducing the number of cancer cells in the tumor, thereby treating the tumor.

[0119] In some embodiments, the small molecule therapeutic agent group is formed by removing one or more hydrogen atoms or one or more functional groups from the following small molecule therapeutic agents: the small molecule therapeutic agent is selected from methotrexate, doxorubicin, vinca alkaloids, auristatins (including MMAE and MMAF), calicheamicin, maytansine, camptothecin, and calicheamicin. In some embodiments, the small molecule therapeutic agent group is a group formed by removing one or more hydrogen atoms or one or more functional groups from monomethylauristatin E (MMAE).

[0120] The functional group may be included in the conjugate of the present disclosure in any suitable manner, for example, the functional group A0 and the main chain portion may be linked by the conjugate linkage described above.

[0121] In some embodiments, the conjugates according to the present disclosure further comprise one or more delivery aid groups, said delivery aid groups being 10 ~C 30The delivery aid group may be one or more selected from the group consisting of a hydrocarbon group, a cholesterol group, and a phospholipid group. By including the delivery aid group, the complex of the present disclosure may be better compatible with the internal environment in the central nervous system, may have better bioavailability, and / or may be more efficiently delivered to a tumor. In some embodiments, the delivery aid group is attached to the delivery group or the linking group via a covalent bond or a linking group. In some embodiments, the delivery aid group is attached to the functional group.

[0122] One of skill in the art can prepare the conjugates of the present disclosure by any reasonable synthetic route.

[0123] In some embodiments, the method for synthesizing a complex according to the present disclosure includes contacting a protected complex with a deprotection reagent in a solvent under deprotection reaction conditions, and isolating the complex to obtain a complex according to the present disclosure. The protected complex is a compound formed by protecting any active functional group in the complex according to the present disclosure with a protecting group. In some embodiments, the active functional group includes, but is not limited to, a hydroxyl group, an amino group, and / or a phosphate group, and the protecting group is correspondingly a hydroxyl protecting group, an amino protecting group, and / or a hydroxyl phosphate protecting group (e.g., a cyanoethyl protecting group). Depending on the type of protecting group, the solvent, deprotection reaction conditions, and deprotection reagent to be used are selected and determined. In some embodiments, the deprotection reaction conditions, solvent, and deprotection reagent are the deprotection reaction conditions, solvent, and reagent used in solid-phase synthesis of nucleic acid. In some embodiments, the method includes adding the protected complex to a mixed solution of a methylamine aqueous solution and an ammonia solution, and the deprotection reaction conditions include reacting at room temperature and normal pressure for 1 to 5 h. In some embodiments, the methylamine aqueous solution and saturated concentrated ammonia water are mixed in equal volumes to obtain the mixed solution, and the amount of the solution relative to the protected complex is 0.1-10 ml / μmol. In some embodiments, the separation includes purification by column chromatography, collection of the product eluate, and removal of the solvent. The purification conditions may be, for example, using a preparative ion chromatography purification column and eluting with a gradient eluent of an aqueous sodium chloride solution and an aqueous sodium phosphate solution. In some embodiments, a solvent of 20 mM sodium phosphate (pH 8.1), water / acetonitrile = 9:1 (volume ratio) is used as eluent A, and a solvent of 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), water / acetonitrile = 9:1 (volume ratio) is used as eluent B, and gradient elution is performed with an elution gradient of eluent A:eluent B = 100:0-50:50.

[0124] In some embodiments, a conjugate according to the present disclosure has a structure according to formula (101), and a method for synthesizing the protected conjugate comprises reacting an active group R x1and a compound containing a delivery group, the compound being treated with an active group R x2 and a compound containing a functional group to obtain the protected complex, wherein the delivery group is formed by removing one or more hydrogen atoms or one or more functional groups from the aptamer, each functional group being independently a small molecule therapeutic agent group having a therapeutic effect against a tumor, any active group among the delivery group and the functional group is protected by a protecting group, and the active group R x1 and the active group R x2 is converted into a covalent bond or a bonding group R j In some embodiments, the active group R x1 and an active group R x2 is bound to the functional group is m0:n0 molar ratio. In some embodiments, the active groups in the delivery group and functional group include, but are not limited to, one or more of a hydroxyl group, an amino group, and a phosphate group, and the protecting group is correspondingly one or more of a hydroxyl protecting group, an amino protecting group, and a phosphate hydroxyl protecting group (e.g., a cyanoethyl protecting group).

[0125] Those skilled in the art can easily identify the active group R x1 and a delivery group. In some embodiments, the active group R x1 Compounds containing the active group R and the delivery group can be obtained by nucleic acid synthesis methods well known to those skilled in the art, such as phosphoramidite solid phase synthesis or phosphodiester / phosphotriester solution phase synthesis. x1 and a delivery group can be obtained by phosphoramidite solid phase synthesis, the method comprising the steps of sequentially linking nucleoside monomers under conditions of phosphoramidite solid phase synthesis according to the order of nucleotides in a single strand of oligonucleotide, at least one nucleoside monomer carrying an active group R x1 or after all the nucleoside monomers are linked, the activating group R x1and then removing the protecting group to leave an active group R x1 The phosphoramidite solid phase synthesis method is well known to those skilled in the art, and the process and conditions thereof are disclosed in detail in Methods in Molecular Biology, vol. 288: Oligonucleotide Synthesis: Methods and Applications, pp. 17-31, the entire contents of which are incorporated herein by reference.

[0126] In some embodiments, the coupling reaction conditions are condensation reaction conditions or mercapto-disulfide bond exchange reaction conditions.

[0127] In some embodiments, the coupling reaction conditions are condensation reaction conditions, the condensation reaction conditions being acylation condensation reaction conditions, dehydration condensation reaction conditions, or click chemistry reaction conditions, and the active group R x1 and active group R x2 is a group capable of the condensation reaction. In some embodiments, the condensation reaction conditions are acylation condensation reaction conditions, and the active group R x1 and R x2 generates an acylation condensation reaction to give R I In some embodiments, the condensation reaction conditions are dehydration condensation reaction conditions, and the active group R x1 and R x2 In some embodiments, the condensation reaction conditions are click chemistry conditions, and the active group R x1 and R x2 In some embodiments, the condensation reaction conditions are Michael addition reaction conditions, and the active group R x1 and R x2In some embodiments, the condensation reaction conditions are N-hydroxysuccinimide-carbodiimide (NHS-EDC)-assisted coupling reaction conditions, and the active group R x1 and R x2 One of them is a group containing an N-hydroxysuccinimide (NHS) group, and the other is a group containing a carbodiimide group (EDC).

[0128] In some embodiments, the active group R x1 and a compound containing a delivery group, which, under the conditions of the coupling reaction, converts to an active group R x0 The active group R X0 and the acylation group form a covalent bond via a coupling reaction, thereby attaching the click chemistry active group to the aptamer of the present disclosure.

[0129] In some embodiments, the active group R x1is an active group containing 1 to 3 click chemistry active groups at the terminal, and the click chemistry active group includes a terminal alkynyl group. In some embodiments, the acylation group is an active ester group, and may be, for example, one of an NHS ester group, an imino ester group, and a pentafluorophenyl group. Those skilled in the art can obtain the crosslinker in various ways, for example, when the acylation group is a pentafluorophenyl group and the click chemistry group includes a terminal alkynyl group, the crosslinker can be prepared according to the method described in Scheme 1a(A) in φstergaard, Michael E., et al. "Efficient synthesis and biological evaluation of 5'-GalNAc conjugated antisense oligonucleotides." Bioconjugate chemistry 26.8 (2015):1451-1455, the entire contents of which are incorporated herein by reference. In some embodiments, the active group R x0 is an amino group. In some embodiments, the coupling conditions are alkaline conditions. In some embodiments, the alkaline conditions are conditions in which a weak alkaline aqueous solution is present, for example, conditions in which a sodium bicarbonate aqueous solution is present.

[0130] Those skilled in the art can easily convert the active group R x0 and in some embodiments, the active group R x0 Aptamers having the active group R are prepared by using phosphoramidite monomers containing active groups at the corresponding positions during synthesis of the aptamer. Those skilled in the art can obtain phosphoramidite monomers containing active groups in a variety of ways. In some embodiments, the active group R x0 is an amino group, and R x0 Phosphoramidite monomers containing R x0The phosphoramidite monomer containing the active group R may be 6-(trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite monomer, which is readily available commercially. x0 is an amino group, and the active group R x0 can be obtained by linking the phosphoramidite monomer to a single-stranded oligonucleotide by phosphoramidite solid-phase synthesis, followed by removal of the trifluoroacetyl protecting group by a deprotection reaction (e.g., aminolysis with concentrated aqueous ammonia) that can be easily achieved by a person skilled in the art.

[0131] In some embodiments, the coupling reaction conditions are one of a mercapto-disulfide bond exchange reaction, and the active group R x1 and R x2 One of the active groups R is a group containing a mercapto group, and the other is a group containing a leaving group linked by a disulfide bond. x1 R in the phosphoramidite monomer containing x1 is a protected R x1 ', and the preparation method includes deprotecting the protected active group R x1 The compound prepared containing the ' and delivery group is contacted with a deprotection reagent to produce an active group R x1 In some embodiments, the compound further comprises obtaining a compound comprising the R x1 wherein R′ comprises a disulfide bond leaving group, the deprotection reaction conditions are mercapto-disulfide bond exchange reaction conditions, and the deprotection reagent is a disulfide bond activator. In some embodiments, the disulfide bond activator is dithiodipyridine. Those skilled in the art will appreciate that the activator R′ can be deprotected by a variety of methods. x1 or R x1 ', and in some embodiments, the activating group R x1 or R x1Phosphoramidite monomers containing ' are commercially available, for example, the phosphoramidite monomer shown in formula (105) is commercially available.

[0132] [ka] Formula (105) In the formula, n 105 and m 105 are each independently an integer of 1 to 10.

[0133] Those skilled in the art can easily convert the active group R x2 and a functional group. In some embodiments, the coupling reaction conditions are mercapto-disulfide bond exchange reaction conditions, and the active group R x2 contains a mercapto group, and the active group R x2 Compounds containing functional groups R can be obtained by those skilled in the art in a variety of known ways, for example, they can be produced by phosphoramidite solid phase synthesis using phosphoramidite monomers containing mercapto groups, or they can be commercially available. In some embodiments, the coupling reaction conditions are phosphoramidite solid phase synthesis reaction conditions, and the active group R x2 is a phosphoramidite group, and the active group R x2 and the compound comprising the functional group may be, for example, a compound comprising a phosphoramidite group and a small molecule therapeutic group, which are readily available commercially. In some embodiments, the coupling reaction conditions are Michael addition reaction conditions, and the active group R x2 is an N-succinimide group, and the active group R x2 The compound containing the functional group may be, for example, a compound containing an N-succinimide group and a small molecule therapeutic agent group, which is readily available commercially. In some embodiments, the active group R x1 and active group R x2are nucleotide sequence I and nucleotide sequence II, respectively, the nucleotide sequence I and the nucleotide sequence II each containing 5 to 25 modified or unmodified nucleotides, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, the delivery group is bound to the nucleotide sequence I, the functional group is bound to the nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II do not induce an immune response in the body of a subject or a toxic response in the subject, and the coupling reaction conditions are reaction conditions that anneal to form a nucleic acid duplex. In some embodiments, the nucleotide sequence I and the nucleotide sequence II each consist of 17 nucleotides and are completely reverse complementary. In some embodiments, the nucleotide sequence I and the nucleotide sequence II have the sequences shown in SEQ ID NO: 40 and SEQ ID NO: 41, respectively. In some embodiments, the nucleotide sequence I and the nucleotide sequence II have the sequences shown in SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

[0134] The delivery group is formed by removing one or more hydrogen atoms or functional groups from the aptamer. In some embodiments, the ribose 5' group of the 5'-terminal nucleotide and the ribose 3' group of the 3'-terminal nucleotide of the consecutive nucleotide sequence are both hydroxyl groups, and the delivery group is formed by the aptamer removing one hydrogen atom from the 5'-hydroxyl group of the 5'-terminal nucleotide. In some embodiments, the delivery group is formed by the aptamer removing one hydrogen atom from the 3'-hydroxyl group of the 3'-terminal nucleotide. In some embodiments, the delivery group is formed by the aptamer removing the 5'-hydroxyl group from the 5'-terminal nucleotide, and in some embodiments, the delivery group is formed by the aptamer removing the 3'-hydroxyl group from the 3'-terminal nucleotide. In some embodiments, the delivery group is formed by removing the ribose 2'-hydroxyl group from the nucleotide contained in the aptamer. The aptamer can be obtained by a conventional aptamer production method in the art (e.g., nucleic acid solid phase synthesis and liquid phase synthesis methods). Solid-phase nucleic acid synthesis is already available as a commercial customization service. Modified nucleotides can be introduced into the complexes of the present disclosure by using nucleoside monomers with corresponding modifications, and methods for producing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotides into aptamers are also well known to those skilled in the art. All modified nucleoside monomers may be purchased commercially or may be produced by known methods.

[0135] In some embodiments, the complex may be used in the present disclosure in the form of its pharma- ceutically acceptable salt or precursor compound. In the context of the present disclosure, "pharma-ceutically acceptable salt" refers to forming a corresponding salt of a drug, such as potassium salt, sodium salt, carboxylate, etc., that does not have any additional side effects on the human body, in order to increase the stability, solubility, and / or bioavailability of the drug. "Precursor compound" refers to a compound that is not completely the same in structure and function as the complex itself, but can react after entering the body or in a body fluid environment to form the complex of the present disclosure, thereby exerting its effect and achieving the purpose of the present disclosure. In some cases, these precursor compounds have the effect of increasing the stability of the drug, extending the sustained release time, increasing bioavailability, etc. In some embodiments, the precursor compound includes a precursor group that can react in the human body to form all the functional groups A0 in the complex. In some embodiments, the precursor compound includes a compound in which all the active hydroxyl groups in the complex are replaced with acetoxy groups. In some embodiments, the precursor compound includes a prodrug group, which is a residue formed by a precursor compound of the therapeutic agent corresponding to the functional group in the conjugate. In some embodiments, the prodrug group may be, for example, a group in which an active hydrogen in a hydroxy or amino functional group in the functional group is replaced with an acyl group, an alkyl group, or a phosphoryl group. As will be appreciated by those of skill in the art, the use of pharma- ceutically acceptable salts and precursor compounds of these are also within the scope of the present disclosure.

[0136] Pharmaceutical Compositions In one aspect, the present disclosure further provides a pharmaceutical composition comprising a conjugate according to the present disclosure and a pharma- ceutically acceptable carrier.

[0137] The pharma- ceutically acceptable carrier may be a carrier commonly used in the art, such as water, saline, magnetic nanoparticles (e.g., nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate ester ... Examples of suitable polymerizable compounds include, but are not limited to, one or more of poly(N,N-dimethylaminoethyl methacrylate, PPEEA) and poly(2-dimethylaminoethyl methacrylate, PDMAEMA) and derivatives thereof.

[0138] In some embodiments, the pharma- ceutically acceptable carrier comprises a physiologically acceptable compound, which, for example, serves to stabilize the pharmaceutical composition or to increase or decrease the absorption of the conjugate and / or pharmaceutical composition. The physiologically acceptable compound is one or more selected from carbohydrates such as glucose, sucrose, and / or dextran, antioxidants such as ascorbic acid and / or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of any co-administered substances, excipients, stabilizers, and buffers. Detergents can also be used to stabilize the composition or to increase or decrease the absorption of the pharmaceutical composition. The physiologically acceptable compound may further comprise one or more of a wetting agent, an emulsifying agent, a dispersing agent, or a preservative, which is particularly used to prevent the growth or action of microorganisms. The physiologically acceptable compounds are known to those skilled in the art and will not be described in detail in this disclosure. As one of ordinary skill in the art can readily appreciate, the choice of pharma- ceutically acceptable carrier and physiologically acceptable compound will depend, for example, on the route of administration and the particular physiochemical properties of any co-administered substances.

[0139] In some embodiments, the pharma- ceutically acceptable carrier is sterile and generally free of unnecessary substances.The pharmaceutical composition according to the present disclosure may further include pharma- ceutically acceptable auxiliary substances such as pH adjusting and buffering agents such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, toxicity adjusting agents, etc., as necessary to approximate physiological conditions, and the concentration of the complex according to the present disclosure in the pharmaceutical composition can vary within a wide range and is selected according to the specific method of administration, mainly based on the fluid volume, viscosity, body weight, etc.

[0140] In some embodiments, there is no special requirement for the content of the complex and the pharma- ceutically acceptable carrier in the pharmaceutical composition. In some embodiments, the weight ratio of the complex to the pharma- ceutically acceptable carrier may be 1:(1-500), and in some embodiments, the weight ratio is 1:(1-50).

[0141] In some embodiments, the pharmaceutical composition may include other pharma- ceutically acceptable additives, which may be one or more of various agents or compounds commonly used in the art. For example, the other pharma-ceutically acceptable additives may include at least one of a pH buffer, a protectant, and an osmolality regulator.

[0142] The pH buffer solution may be a trishydroxymethylaminomethane hydrochloride buffer solution having a pH of 7.5 to 8.5 and / or a phosphate buffer solution having a pH of 5.5 to 8.5, for example, a phosphate buffer solution having a pH of 5.5 to 8.5.

[0143] The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The content of the protective agent may be 0.01 to 30% by weight based on the total weight of the pharmaceutical composition.

[0144] The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator is determined so that the osmotic pressure of the pharmaceutical composition is 200 to 700 milliosmoles per kilogram (mOsm / kg). Depending on the desired osmotic pressure, a person skilled in the art can easily determine the content of the osmotic pressure regulator. In some embodiments, the dosage during administration of the formulation prepared from the pharmaceutical composition is adjusted according to the administration method.

[0145] In some embodiments, the pharmaceutical composition may be a liquid formulation such as an injection solution, or a lyophilized powder injection that is mixed with a liquid additive at the time of administration to prepare a liquid formulation. The liquid formulation may be used for subcutaneous, intramuscular, or intravenous administration, but is not limited thereto, and the pharmaceutical composition may be delivered by a method such as, but not limited to, puncture injection, oral inhalation, or nasal administration. In some embodiments, the pharmaceutical composition is used for subcutaneous, intramuscular, intravenous, or intrathecal injection administration.

[0146] In some embodiments, the pharmaceutical composition may be in the form of a liposome formulation. In some embodiments, the pharma- ceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter also referred to as organic amine), an auxiliary lipid, and / or a polyethylene glycolated lipid. The organic amine, auxiliary lipid, and polyethylene glycolated lipid may be one or more selected from the amine-containing transfection compound or its pharma- ceutically acceptable salt or derivative, auxiliary lipid, and polyethylene glycolated lipid described in Chinese patent application CN103380113A (incorporated herein by reference in its entirety).

[0147] In some embodiments, the organic amine may be a compound represented by formula (201) or a pharma- ceutically acceptable salt thereof, as described in Chinese Patent Application CN103380113A.

[0148] [ka] Formula (201) During the ceremony, X 101 and X 102 are each independently O, S, NA or CA, and A is hydrogen or C1-C 20 is a hydrocarbon chain, Y 101 and Z 101 are each independently C=O, C=S, S=O, CH-OH or SO2; R 101 , R 102 , R 103 , R 104 , R 105 , R 106 and R 107are each independently hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain heteroaliphatic group, a substituted or unsubstituted, branched or straight-chain acyl group, a substituted or unsubstituted, branched or straight-chain aryl group, or a substituted or unsubstituted, branched or straight-chain heteroaryl group; x is an integer from 1 to 10; n is an integer of 1 to 3, m is an integer of 0 to 20, and p is 0 or 1, where m=p=0, R 102 is hydrogen, When at least one of n and m is 2, R 103 and the nitrogen in formula (201) form a structure represented by formula (202) or formula (203).

[0149] [ka] In the formula, g, e, and f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each * N represents a nitrogen atom in formula (201).

[0150] In some embodiments, R 103 is a polyamine. In another embodiment, R 103 In some embodiments, R in formula (201) is a ketal. 101 and R 102 are each independently an optionally substituted or unsubstituted, branched or straight chain alkyl or alkenyl group having 3 to about 20 carbon atoms, e.g., 8 to about 18 carbon atoms, and 0 to 4 double bonds, e.g., 0 to 2 double bonds.

[0151] In some embodiments, when each of n and m is independently 1 or 3, R 103 may be any one of the following formulas (204) to (213).

[0152] [ka] In formulae (204) to (213), g, e, and f are each independently an integer of 1 to 6, each "HCC" represents a hydrocarbon chain, * is R 103 and the nitrogen atom in formula (201), and * Each H on position may be substituted to provide a bond with the nitrogen atom in formula (201).

[0153] Those skilled in the art can obtain the compound of formula (201) by any reasonable method. In some embodiments, the compound of formula (201) may be prepared according to the description in Chinese patent application CN103380113A.

[0154] In some embodiments, the organic amine is an organic amine represented by formula (214) and / or an organic amine represented by formula (215).

[0155] [ka]

[0156] the co-lipid is cholesterol, a cholesterol analogue and / or a cholesterol derivative; The polyethylene glycolated lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

[0157] In some embodiments, the molar ratio of the organic amine, the colipid, and the polyethylene glycolated lipid in the pharmaceutical composition is (19.7 to 80):(19.7 to 80):(0.3 to 50), and may be, for example, (50 to 70):(20 to 40):(3 to 20).

[0158] In some embodiments, the particles of the pharmaceutical composition formed by the conjugate of the present disclosure and the amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, and more typically, the average diameter of the liposome particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm, for example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160 nm.

[0159] In some embodiments, the weight ratio (weight / weight ratio) of the complex to total lipid (e.g., organic amine, auxiliary lipid, and / or PEGylated lipid) in the pharmaceutical composition formed by the complex of the present disclosure and the amine-containing transfection reagent is within the range of about 1:1 to about 1:50, about 1:1 to about 1:30, about 1:3 to about 1:20, about 1:4 to about 1:18, about 1:5 to about 1:17, about 1:5 to about 1:15, about 1:5 to about 1:12, about 1:6 to about 1:12, or about 1:6 to about 1:10, for example, the weight ratio of the complex of the present disclosure to total lipid is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, or 1:18.

[0160] In some embodiments, the pharmaceutical composition may be marketed with each component being independent, or may be in the form of a liquid formulation when used. In some embodiments, the pharmaceutical composition formed by the conjugate according to the present disclosure and the pharma- ceutical acceptable carrier may be produced according to various known methods, and the conjugate according to the present disclosure may be used instead of a conventional aptamer or conjugate. In some embodiments, the pharmaceutical composition may be produced according to the following method.

[0161] The organic amine, auxiliary lipid and polyethylene glycolated lipid are suspended in alcohol in the above molar ratio and mixed uniformly to obtain a lipid solution. The amount of alcohol is determined so that the total mass concentration of the obtained lipid solution is 2 to 25 mg / mL, for example, 8 to 18 mg / mL. The alcohol is one or more selected from pharma- ceutically acceptable alcohols, for example, alcohols that are liquid around room temperature, such as ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, and polyethylene glycol 400, and may be, for example, ethanol.

[0162] The complex according to the present disclosure is dissolved in a buffer salt solution to obtain an aqueous complex solution. The concentration of the buffer salt solution is 0.05 to 0.5 M, and may be, for example, 0.1 to 0.2 M. The pH of the buffer salt solution is adjusted to 4.0 to 5.5, and may be, for example, 5.0 to 5.2. The amount of the buffer salt solution is determined so that the concentration of the complex is 0.6 mg / mL or less, and may be, for example, 0.2 to 0.4 mg / mL. The buffer salt is one or more selected from soluble acetates and soluble citrates, and may be, for example, sodium acetate and / or potassium acetate.

[0163] After mixing the lipid solution and the aqueous complex solution, the resulting product is incubated at 40-60°C for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome formulation. The volume ratio of the lipid solution to the aqueous complex solution is 1:(2-5), and may be, for example, 1:4.

[0164] The incubated liposome preparation is concentrated or diluted, impurities are removed, and bacteria are removed to obtain the pharmaceutical composition according to the present disclosure. Its physicochemical parameters are pH 6.5-8, encapsulation efficiency not less than 80%, particle size 40-200 nm, polydispersity index not more than 0.30, and osmotic pressure 250-400 mOsm / kg. For example, the physicochemical parameters may be pH 7.2-7.6, encapsulation efficiency not less than 90%, particle size 60-100 nm, polydispersity index not more than 0.20, and osmotic pressure 300-400 mOsm / kg.

[0165] Here, the concentration or dilution may be performed before removing impurities, after removing impurities, or simultaneously. As a method for removing impurities, various conventional methods may be used, for example, ultrafiltration may be performed under conditions of 100K Da using a tangential flow system and a hollow fiber column, and the ultrafiltration exchange solution may be a phosphate buffer solution (PBS) of pH 7.4. As a method for sterilization, various conventional methods may be used, for example, sterilization may be performed by filtering through a 0.22 μm filter.

[0166] Uses of the Conjugates of the Present Disclosure In yet another aspect, the present disclosure further provides the use of the conjugate and / or pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating tumors and tumor-related diseases or conditions.

[0167] In yet another aspect, the present disclosure further provides a method of treating tumors and tumor-related diseases or conditions, comprising administering to a subject in need thereof a conjugate and / or pharmaceutical composition as described herein.

[0168] By administering the conjugate and / or pharmaceutical composition according to the present disclosure, the method of the present disclosure can effectively treat tumors and tumor-related diseases or conditions, and the highly specific targeting effect of the conjugate according to the present disclosure can reduce the distribution of the therapeutic agent in other organs / tissues of the body that are not intended, and reduce potential side effects, which is of great significance and significant value, especially for radiotherapy and / or chemotherapy drugs that are commonly used in the field of tumor treatment and are known to have serious side effects.

[0169] The term "pharmaceutical administration / administration" as used in this disclosure refers to placing the complex and / or pharmaceutical composition into the body of a subject by a method or route that at least partially localizes the complex and / or pharmaceutical composition to a desired site to produce a desired effect. Routes of administration suitable for the methods of this disclosure include local administration and systemic administration. Generally, local administration delivers more of the complex and / or pharmaceutical composition to a particular site than to the entire body of the subject, while systemic administration delivers the complex and / or pharmaceutical composition to substantially the entire body of the subject.

[0170] Furthermore, the inventors of the present disclosure have unexpectedly discovered that the conjugates and / or pharmaceutical compositions of the present disclosure can efficiently cross the blood-brain barrier and target tumors in the brain when administered systemically, thereby further improving the delivery efficiency of functional groups, saving costs, and reducing undesirable side reactions.

[0171] Administration to a subject may be by any suitable route known in the art, including, but not limited to, oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, transdermal, intratracheal (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration. The frequency of administration may be once or more per day, week, biweekly, triweekly, monthly, or yearly.

[0172] The dose of the conjugate and / or pharmaceutical composition of the present disclosure may be a dose common in the art, and the dose may be determined according to various parameters, in particular the age, weight and sex of the subject. Toxicity and therapeutic efficacy are measured by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., LD 50 (the dose that kills 50% of the colony) and ED 50 (For a quantitative response, this refers to the dose which produces 50% of the maximum response strength; for a qualitative response, this refers to the dose at which a positive response occurs in 50% of experimental subjects) may be determined. A range of human dosages can be derived based on data obtained from cell culture assays and animal studies.

[0173] When administering the complex and / or pharmaceutical composition described in the present disclosure to, for example, a male or female, 6-12 week old, 18-25 g body weight C57BL / 6J or C3H / HeNCrlVr mouse, the dose of the complex may be 0.001-100 mg / kg body weight, in some embodiments, 0.01-50 mg / kg body weight, in further embodiments, 0.05-20 mg / kg body weight, in still further embodiments, 0.1-15 mg / kg body weight, and in still further embodiments, 0.1-10 mg / kg body weight. When administering the complex and / or pharmaceutical composition described in the present disclosure, the above doses are preferred.

[0174] kit The present disclosure provides a kit, the kit comprising a conjugate and / or pharmaceutical composition according to the present disclosure.

[0175] In some embodiments, the kits described herein may provide the conjugate and / or pharmaceutical composition in a container. In some embodiments, the kits described herein may include a container providing a pharma- ceutically acceptable excipient. In some embodiments, the kits may include other components, such as stabilizers or preservatives. In some embodiments, the kits of the present disclosure may include at least one other therapeutic agent in a container other than the container providing the conjugate and / or pharmaceutical composition described herein. In some embodiments, the kits may include instructions for mixing the conjugate and / or pharmaceutical composition with a pharma- ceutically acceptable carrier and / or excipient or other components, if present.

[0176] In the kits of the present disclosure, the conjugate and pharma- ceutically acceptable carrier and / or additive, and the pharmaceutical composition, and / or pharma- ceutically acceptable additive, can be provided in any form, such as liquid, dry, or lyophilized. In some embodiments, the conjugate and pharma- ceutically acceptable carrier and / or additive, and the pharmaceutical composition and any pharma- ceutically acceptable additive, are essentially clean and / or sterile. In some embodiments, sterile water can be provided in the kits of the present disclosure.

[0177] The present disclosure will be further described below with reference to examples, but the present disclosure is not limited thereto in any way. EXAMPLES

[0178] Unless otherwise specified, all reagents and media used in the following examples are commercially available products, and all operations such as nucleic acid electrophoresis and real-time PCR are performed with reference to the methods described in Molecular Cloning (Cold Spring Harbor Borough Press (1989)).

[0179] Production Examples 1 to 8 and 18 Synthesis of Complexes AP1 to AP8 and AP18 The conjugates numbered AP1-AP8 and AP18 in Table 1 were synthesized by solid-phase synthesis, and all nucleoside monomers were sequentially linked from 3' to 5' according to the nucleotide sequences corresponding to AP1-AP8 and AP18 in Table 1, respectively, and then Cy5 phosphoramidite monomer (purchased from Suzhou Jima Co., Ltd., lot number CY5P21H1B) was linked according to the method of linking nucleoside phosphoramidite monomers by solid-phase synthesis. Then, the nucleotide sequence was added to an equal volume mixture of methylamine aqueous solution and ammonia water, the amount of the solution for the oligonucleotide was 0.5ml / μmol, and the reaction was carried out at 25°C for 2h, filtered to remove solids, and the supernatant was vacuum concentrated and dried.

[0180] The purification of the produced complex was completed by gradient elution with NaCl aqueous solution using a production type ion chromatography purification column (Source 15Q). Specifically, 20 mM sodium phosphate (pH 8.1), water / acetonitrile = 9: 1 (volume ratio) solvent was used as eluent A, 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), water / acetonitrile = 9: 1 (volume ratio) solvent was used as eluent B, and gradient elution was performed with an elution gradient of eluent A: eluent B = 100: 0 to 50: 50. The eluent of the product was collected and then combined, and desalted by reverse phase chromatography purification column, the specific conditions were desalted by Sephadex column, the packing material was Sephadex G25, and eluted with deionized water. The resulting eluate was concentrated to remove the solvent and lyophilized to obtain complexes AP1 to AP8 and AP18, each of which has the 5'-position of ribose in the 5'-terminal nucleotide bound to the fluorescent group Cy5 via a phosphate ester group linking group.

[0181] After the synthesis of the above complexes AP1 to AP8 and AP18 was completed, the complexes obtained were washed with ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ *The mixture was diluted to a concentration of 0.2 mg / mL at 25°C (cm) and then the molecular weight was detected using a liquid chromatography mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model number: LCT Premier). As a result, the measured molecular weight value was consistent with the theoretical value, indicating that the desired complex was obtained.

[0182] The molecular weight and MS value of the complex are shown in Table 1a below.

[0183] Table 1a Mass spectrometry results of the complex [Table 1]

[0184] Comparative Preparation Examples 9 to 17 and 19 Synthesis of control complexes Comparative AP9 to Comparative AP17 and Comparative AP19 According to the method of Preparation Example 1, the conjugates numbered Comparative AP9 to Comparative AP17 and Comparative AP19 in Table 1 were each synthesized, and the molecular weights were detected to confirm the synthesized conjugates. The only difference was that the nucleoside monomers were linked in order according to the sequences corresponding to Comparative AP9 to Comparative AP17 and Comparative AP19 in Table 1, respectively. As a result, control conjugates Comparative AP9 to Comparative AP17 and Comparative AP19 were obtained, in which the 5' position of ribose in the 5'-terminal nucleotide is linked to the fluorescent group Cy5 via a phosphate ester group linking group.

[0185] Table 1 Nucleotide sequences of the complexes [Table 2]

[0186] In Table 1, capital letters C, G, U, and A represent the base composition of nucleotides, lowercase letter m represents that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide, lowercase letter f represents that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide, and Cy5 represents the binding site in the aptamer of the fluorescent dye group Cy5 (Cyanine 5) group.

[0187] Preparation Example 20: Synthesis of Complex 20 Table 2 Nucleotide sequences in the complex [Table 3]

[0188] In Table 2, capital letters C, G, U, and A represent the base composition of a nucleotide, lowercase letter m represents that one nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide, and MMAE represents the binding site in the aptamer of the small molecule drug group MMAE (monomethylauristatin E) group.

[0189] In this preparation example, conjugate 20 is prepared according to the following steps, in which the dye group in conjugate AP2 is replaced with a small molecule drug group MMAE, and the linking group is 2-(phosphate-(CH2)6-S-)-maleimidohexanoyl-valine-citrulline-p-aminobenzyl subunit.

[0190] (20-1) Preparation of oligonucleotide S1 The oligonucleotide sequence in the complex 20 in Table 2 was synthesized by solid phase synthesis. The only difference was that the nucleoside monomers were linked in order according to the oligonucleotide sequence corresponding to the complex 20 in Table 2, and during solid phase synthesis, the last nucleoside monomer at the 5' end was linked, and then a phosphoramidite monomer containing a HO-(CH2)6-SS-(CH2)6- group (purchased from Hongene Biotech) was linked according to the method of linking a nucleoside phosphoramidite monomer, and the oligonucleotide single strand was cleaved from the solid phase support to obtain the oligonucleotide single strand S1 (70.00 mg, 6.42 μmol) shown in formula (20-a).

[0191] [ka] Formula (20-a) During the ceremony, JPEG2025512110000041.jpg7135 represents the oligonucleotide sequence corresponding to complex 20.

[0192] (20-2) Synthesis of oligonucleotide S2 [ka] 70.0 mg of S1 (6.42 μmol) prepared in step (20-1) was dissolved in 10.0 ml of purified water, and then 105 mg of TCEP (tris(2-chloroethyl)phosphate, 0.37 mmol, purchased from BiDe Pharmaceutical, lot number: BD155793) was dissolved in 10.0 ml of purified water to obtain an aqueous TCEP solution. The mixture was mixed uniformly and reacted at room temperature for 2 hours. The reaction solution was diluted with 10 mL of purified water and filtered to obtain 28 mL of reaction solution. The reaction solution was transferred to a 3K standard ultrafiltration tube and centrifuged at 3900 rpm for 30 min. The ultrafiltration and centrifugation steps were repeated twice again to collect the product in the filtration membrane, and oligonucleotide S2 (67.0 mg, yield: 95.7%) was obtained.

[0193] (20-3) Synthesis of oligonucleotide 20 [ka] 24 mg of Vc MMAE (18.56 μmol, 5 eq, purchased from CSN, lot number CSN16143-005) was dissolved in 6.0 ml of DMF, and 60 μl of triethylamine was added to obtain a Vc MMAE solution. 40.0 mg of oligonucleotide S2 (3.71 μmol, 1 eq) prepared in step (20-2) was dissolved in 6.0 ml of purified water, and the above Vc MMAE solution was added to the obtained solution. After reacting at room temperature for 2 hours, a crude product of complex 20 was obtained (represented as S3 in the process diagram).

[0194] The obtained crude product of complex 20 was diluted with 0.5 ml of purified water, filtered through a 0.45 μm filter membrane, and the filtrate was purified by an Agilent semi-preparative reversed-phase column. The column used was Kromasil 100-10-C18, 100 Å, 10 um, 21.2*250 mm. Gradient elution was performed using 100 mM triethylamine acetate buffer (TEAA, pH = 7.0-7.3):acetonitrile = 95:5-35:65 as the mobile phase. The peak eluent of the product was collected and the solvent was removed by evaporation to obtain complex 20 (55 mg, yield 56.7%). The molecular weight was measured by LC-MS, and the theoretical value was 12092.73, and the actual value was 12091.39, which agreed with the theoretical value. This shows that conjugate 20 has the structure shown in S3, in which conjugate 20 replaces the dye group in conjugate AP2 with a small molecule drug group MMAE, and the linking group is 2-(phosphate-(CH2)6-S-)-maleimidohexanoyl-valine-citrulline-p-aminobenzyl subunit (2-(phosphate-(CH2)6-S-)-MC-Val-Cit-PAB).

[0195] Preparation Examples 21 to 26 Synthesis of Complexes 21 to 23, 26 and Comparative Complexes 24 to 25 According to the method of Preparation Example 20, Conjugate 21, Conjugate 22, Conjugate 23, Comparative Conjugate 24, Comparative Conjugate 25 and Conjugate 26 numbered in Table 2 were synthesized, and the molecular weights were detected to confirm the synthesized conjugates. The only difference was that the nucleoside monomers were linked in order according to the sequences corresponding to Conjugate 21, Conjugate 22, Conjugate 23, Comparative Conjugate 24, Comparative Conjugate 25 and Conjugate 2 in Table 2, respectively.

[0196] The molecular weight and MS value of the complex are shown in Table 2a below.

[0197] Table 2a: Mass spectrometry results of the complex. [Table 4]

[0198] Experimental Example 1: Targeting of the complex in mice In this experimental example, the targeting properties of the prepared complexes AP2, AP3 to 8, and comparative AP9 to comparative AP12 in mice were investigated.

[0199] U118MG human glioma cells (purchased from Guangzhou Genio Biotech Co., Ltd.) were cultured in DMEM complete medium (MACGENE, product number CM15019) supplemented with 10% fetal bovine serum (FBS, RMBIO) at 37°C in an incubator containing 5% CO2 / 95% air.

[0200] Logarithmically growing U118MG human glioma cells were taken and digested with (0.25% pancreatin), the cells were collected, centrifuged to remove the supernatant, and the cells were resuspended in DMEM medium supplemented with 10% FBS to a concentration of 1 × 10 8 A cell culture medium was prepared with 1000 cells / mL.

[0201] The experimental animals were 24 12-week-old female NOD-SCID mice (purchased from Beijing Sibaifu Biotechnology Co., Ltd.). The above cell culture medium was inoculated into the right front leg subcutaneous site of the NOD-SCID mice, with an inoculation volume of 100 μL per mouse, i.e., 1 × 10 7After injection, the mice were kept for 20 days to obtain mice inoculated with U118MG subcutaneous tumors.

[0202] Using DMEM medium, the aptamers AP2, AP3 to 8, and Comparative AP9 to Comparative AP12 produced above were each prepared into a 0.3 mg / mL solution.

[0203] Administration began 14 days after inoculation of U118MG cells, and the day of administration was designated as D1. In the experiment, the mice were administered once a day via tail vein injection for a total of three times.

[0204] Twenty-four mice inoculated with U118MG subcutaneous tumors were randomly divided into 12 groups with 2 mice in each group.

[0205] For 7 groups of mice, AP2, AP3, AP4, AP5, AP6, AP7 or AP8 was administered to each mouse in each group. The single dose volume was 10 μL / g mouse body weight, and the calculated single dose was 3 mg / kg. These were recorded as test groups 1A to 1G, respectively.

[0206] For the other four groups of mice, comparative AP9, comparative AP10, comparative AP11, or comparative AP12 was administered to each mouse in each group. The single-dose volume was 10 μL / g mouse body weight, and the calculated single-dose amount was 3 mg / kg. These were recorded as control groups 1H to 1K, respectively.

[0207] For another group of 2 mice, each mouse was administered with DMEM medium, with an administration volume of 10 μL / g mouse body weight, and recorded as the blank control group 1Y.

[0208] 1 h, 24 h, and 48 h after the first administration, each mouse was subjected to bioimaging using a small animal biooptical imaging system IVIS Lumina Series III. On D5, the mice in each group were sacrificed, and tumor tissues and kidneys were taken for fluorescence imaging.

[0209] 1A-1C are photographs showing the results of fluorescence imaging in mice after administration of different complexes for 1 h, 24 h, and 48 h, respectively. The mouse on the left side of the three mice in each photograph is a mouse in the blank control group 1Y. As can be seen from FIG. 1A, the blank control group did not show any fluorescence signal. In contrast, after administration for 1 h, the mice in each test group and the control group all showed fluorescence signals in the subcutaneous tumor. As can be seen from FIG. 1B and 1C, after administration for 24 h and 48 h, only the mice in the test groups 1A-1G showed strong fluorescence signals in the subcutaneous tumor, while the mice in the control groups 1H-1K showed little or only weak fluorescence signals. Furthermore, FIG. 1D is a photograph of the fluorescence signal imaging of the tumor tissue and kidney of the mice in each group after the mice were sacrificed on D5, and Blank represents the blank control group 15Y. As can be seen from Figure 1D, the tumor tissues of the mice in the blank control group 1Y and the control groups 1H-1K showed little or only very weak fluorescent signals, whereas in contrast, the tumor tissues of the mice in the test groups 1A-1G administered with the conjugate of the present disclosure all showed strong fluorescent signals, and only a weak fluorescent signal was shown in the metabolic organ kidney. This shows that the conjugate containing the delivery group in the conjugate of the present disclosure can stably and efficiently target tumor tissues, compared to the control conjugate.

[0210] Experimental Example 2: Targeting of the complex in mice In this experimental example, the targeting properties of the prepared complexes AP2, AP1, and comparative AP13 to comparative AP17 in mice were investigated.

[0211] U118MG human glioma cells (purchased from Guangzhou Genio Biotech Co., Ltd.) were cultured in DMEM complete medium (MACGENE, product number CM15019) supplemented with 10% fetal bovine serum (FBS, RMBIO) at 37°C in an incubator containing 5% CO2 / 95% air.

[0212] Logarithmically growing U118MG human glioma cells were taken and digested with (0.25% pancreatin), the cells were collected, centrifuged to remove the supernatant, and the cells were resuspended in DMEM medium supplemented with 10% FBS to a concentration of 1 × 10 8 A cell culture medium was prepared with 1000 cells / mL.

[0213] The experimental animals were 16 12-week-old male NOD-SCID mice (purchased from Beijing Sibaifu Biotechnology Co., Ltd.). The above cell culture medium was inoculated into the right forelimb subcutaneous site of the NOD-SCID mice, with an inoculation volume of 100 μL per mouse, i.e., 1 × 10 7 After injection, the mice were kept for 20 days to obtain mice inoculated with U118MG subcutaneous tumors.

[0214] Using DMEM medium, the aptamers AP2, AP1 and Comparative AP13 to Comparative AP17 produced above were each prepared into a 0.3 mg / mL solution.

[0215] Administration began 21 days after inoculation of U118MG cells, and the day of administration was designated as D1. In the experiment, the mice were administered once a day via tail vein injection for a total of three times.

[0216] Sixteen mice inoculated with U118MG subcutaneous tumors were randomly divided into eight groups with two mice in each group.

[0217] For the two groups of mice, AP2 or AP1 was administered to each mouse in each group. The single-dose volume was 10 μL / g mouse body weight, and the calculated single-dose amount was 3 mg / kg, which were recorded as test groups 2A to 2B, respectively.

[0218] For the other five groups of mice, comparative AP13, comparative AP14, comparative AP15, comparative AP16, or comparative AP17 was administered to each mouse in each group. The single-dose volume was 10 μL / g mouse body weight, and the calculated single-dose amount was 3 mg / kg. These were recorded as control groups 2C to 2G, respectively.

[0219] For another group of 2 mice, each mouse was administered with DMEM medium, the administration volume was 10 μL / g mouse body weight, and was recorded as the blank control group 2Y.

[0220] 1 h, 24 h, and 48 h after the first administration, each mouse was subjected to bioimaging using a small animal biooptical imaging system IVIS Lumina Series III. On D6, the mice in each group were sacrificed, and tumor tissues and kidneys were taken for fluorescence imaging.

[0221] Figures 2A to 2C are photographs showing the results of fluorescence imaging in mice after administration of different complexes for 1 h, 24 h, and 48 h, respectively. The mouse on the left side of the three mice in each photograph is a mouse in the blank control group 2Y. As can be seen from Figure 2A, the blank control group did not show any fluorescence signal, but in contrast, after administration for 1 h, the mice in each test group and the control group all showed fluorescence signals in the subcutaneous tumor. As can be seen from Figures 2B and 2C, after administration for 24 h and 48 h, only the mice in the test groups 2A and 2B showed strong fluorescence signals in the subcutaneous tumor, while the mice in the blank control group 2Y and the control groups 2C to 2G did not show any fluorescence signals. Furthermore, Figure 2D is a photograph of the fluorescence signal imaging of the tumor tissue and kidney of the mice in each group after the mice were sacrificed on D6, and Blank represents the blank control group 2Y. As can be seen from Figure 2D, the tumor tissues of the mice in the blank control group 2Y and the control groups 2C to 2G did not show any fluorescent signal, whereas in contrast, the tumor tissues of the mice in the test groups 2A and 2B administered with the complex of the present disclosure all showed strong fluorescent signals, and only a very weak fluorescent signal was shown in the metabolic organ kidney. This shows that, compared to the control aptamer, the various aptamers having the sequence shown in formula (1) can all stably and efficiently target tumor tissue, and further shows that the complexes according to the present disclosure containing the delivery groups formed by these aptamers can effectively reach tumor tissue.

[0222] Experimental Example 3 Activity of the complex of the present disclosure in mice In this experimental example, the antitumor activity of the prepared complex 20 in mice was investigated.

[0223] The mice used in this experiment were purchased from Siberia, the germ cell line was NOD-SCID, the grade was SPF, the sex was female, and the age was 6 to 8 weeks. U118MG glioma cells were purchased from Genio.

[0224] Logarithmically growing U118MG cells were digested and resuspended in DMEM medium (MACGENE, product number CM15019) containing 10% fetal bovine serum (FBS, GIBCO) to a cell density of 1 × 10 8 The U118MG cell-containing culture solution was then inoculated into the right forelimb subcutaneous site of each mouse, with an injection volume of 100 μL. Thus, each mouse received 1 × 10 7 U118MG glioma cells were inoculated.

[0225] The above-prepared conjugate AP2 was prepared into a 1.94 mg / mL solution using PBS. The conjugate 20 was prepared into 0.625 mg / mL, 1.25 mg / mL and 2.06 mg / mL solutions (all calculated based on the amount of oligonucleotide) using PBS, and MMAE (purchased from Shanghai McKin Co., Ltd., lot number C12886583) was dissolved into a 0.038 mg / mL solution using a mixture of 10% DMSO + 90% PBS (volume ratio).

[0226] The day of cell inoculation was designated as D1, and administration was performed once each on D8, D12, D16, and D20.

[0227] Thirty-six mice were randomly divided into the following six groups, with six mice in each group:

[0228] For the blank control group 3a, PBS was administered via tail vein injection, with a single administration volume of 10 μL / g.

[0229] The control group 3b was administered the above complex AP2 solution by tail vein injection, with a single dose volume of 10 μL / g and a single dose of 15.5 mg / kg.

[0230] The control group 3c was administered the above MMAE solution via tail vein injection, with a single dose volume of 10 μL / g and a single dose of 0.3 mg / kg.

[0231] For test group 3d, the above-mentioned concentration of 0.625mg / mL of the complex 20 solution was administered by tail vein injection, with a single administration volume of 10μL / g, a single administration amount of 5mg / kg (calculated by the mass of oligonucleotide), and a dose containing MMAE was equivalent to 0.3mg / kg.

[0232] For test group 3e, the above-mentioned concentration of 2.06 mg / mL of the complex 20 solution was administered by tail vein injection, with a single dose volume of 10 μL / g, a single dose amount of 16.5 mg / kg (calculated by the mass of oligonucleotide), and a dose containing MMAE was equivalent to 1 mg / kg.

[0233] For test group 3f, the above-mentioned concentration of 1.25 mg / mL of the complex 20 solution was administered by subcutaneous injection, with a single dose volume of 5 μL / g and a single dose of 5 mg / kg (calculated by the mass of oligonucleotide), which corresponds to a dose of 0.3 mg / kg containing MMAE.

[0234] [3] Detection The long and short diameters of the tumor were measured by external measurement. The tumor volume was calculated using the formula 1 / 2 (long diameter × short diameter). 2 Before the first administration on D8, the tumor volume of each group was measured and the average tumor volume was recorded, and from D16, the tumor volume of each group was measured and recorded, and was measured twice a week.

[0235] FIG. 3 is a line graph showing the change in tumor volume over time in mice of each group. As can be seen from the results of FIG. 3, the tumor volume rapidly increased in the blank control group 3a and the control group 3b in which only PBS and AP2 were administered, whereas the rate of increase in tumor volume slowed in the control group 3c in which only MMAE was administered. This indicates that MMAE itself exhibits an inhibitory effect on tumor growth. Furthermore, the tumor volumes of the test groups 3d and 3f, in which the content of MMAE corresponds to the control group 3c, were significantly smaller than those of the control group 3c during the test, and showed better antitumor activity than the control group 3c in which MMAE was administered alone. This indicates that the conjugate according to the present disclosure can effectively deliver MMAE to tumor tissue, demonstrating tumor targeting ability, while reducing the toxicity risk caused by the distribution of MMAE molecules to other tissues, and that various administration methods can effectively inhibit tumor growth. In addition, the tumor volume of the test group 3e, in which the dosage was further increased, hardly increased during the test period, indicating a better antitumor effect.

[0236] As is clear from the above results, the conjugate of the present disclosure can effectively target and deliver a small molecule drug group having an inhibitory effect on tumors to tumor tissue, and can exhibit excellent antitumor activity and dose-dependent effects.

[0237] Experimental Example 4 Distribution of the complex in the body of U118MG cell in-situ tumor model mice U118MG human glioma cells (purchased from Guangzhou Genio Biotech Co., Ltd.) were cultured in DMEM complete medium (MACGENE, product number CM15019) supplemented with 10% fetal bovine serum (FBS, RMBIO) at 37°C in an incubator containing 5% CO2 / 95% air.

[0238] Cells were harvested by digestion with 0.25 wt% pancreatin, the supernatant was aspirated, and the cells were resuspended in DMEM medium supplemented with 10% FBS to a cell density of 4 × 10 7 A cell culture medium was prepared with 1000 cells / mL.

[0239] The experimental animals were six 12-week-old male NOD-SCID mice (purchased from Beifong Biological Technology Co., Ltd.). The above cell culture fluid was inoculated into the NOD-SCID mice, and the cell culture fluid was injected into the right striatum of the mice by mouse lateral ventricle injection at AP (anteroposterior / orthoposition): 1 mm, ML (medial lateral): 1.5 mm, DV (dorsal ventral): 3.5 mm, with an injection volume of 10 μL, i.e., 4 × 10 cells were injected into each mouse. 5 After injection, the mice were kept for 14 days.

[0240] Using 1xDMEM medium, AP2 and comparative AP19 were dissolved in a complex solution of 0.3mg / mL concentration (calculated by aptamer). Four mice were taken and injected with AP2 and comparative AP19 solutions by tail vein injection, and the dosage was calculated according to the body weight of each animal, and the dosage volume was 10μL / g. The dosage per animal was 3mg / kg calculated by the amount of aptamer. Two mice were administered in each group, and recorded as test group 4a and control group 4b, respectively.

[0241] Each of the two mice was injected with 10 μL of DMEM medium and recorded as the blank control group 4Y.

[0242] 24 hours after administration, one mouse from each group was sacrificed to obtain brain tissue, and 48 hours after administration, the remaining mice were sacrificed to obtain brain tissue, and fluorescent imaging of mouse brain tissue was performed using an IVIS Lumina Series III. The results are shown in Figure 4.

[0243] FIG. 4 shows the fluorescence imaging photographs of mouse brain tissues in which an in-situ tumor model of U118MG was constructed after 24h and 48h administration of the blank control group 4Y, the test group 4a, and the control group 4b, respectively. Blank represents the blank control group, Ith represents intrathecal injection, and iv represents tail vein injection. As can be seen from the results of FIG. 4, the blank control group and the control group 4b administered with control AP19 did not show any fluorescent signal in the brain, indicating that there was no significant targeting effect on the in-situ tumor brain glioma; in contrast, the test group 4a administered with the complex according to the present disclosure showed significant fluorescent signals at the tumor inoculation position at 24h and 48h. This indicates that the AP2 complex can still reach and effectively target brain gliomas when administered via the tail vein, and that the complex according to the present disclosure is expected to penetrate the blood-brain barrier (BBB) ​​and enter brain gliomas even when administered via the system, demonstrating excellent targeting effects and potential for drug development.

[0244] Experimental Example 5: Activity of the complex in vivo in U118MG in-situ tumor model mice According to the method of Experimental Example 4, U118MG human glioma cells expressing the luciferase (Photinus pyralis) reporter gene were cultured, hereinafter referred to as U118MG-luc human glioma cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.). Logarithmically grown U118MG-luc human glioma cells were taken and digested with 0.25 wt% pancreatin to collect the cells. After centrifugation, the supernatant was sucked out and the cells were resuspended in serum-free DMEM medium until the cell density reached 4 × 10 7 A cell culture medium was prepared at 100 μg / mL.

[0245] The experimental animals were 24 12-week-old male Balb / C-nude mice (purchased from Beifong Biological Technology Co., Ltd.). The above cell culture solution was inoculated into the striatum of Balb / C-nude mice, and the cell culture solution was injected into the right striatum of the mice by mouse striatal injection at AP (anteroposterior): 1 mm, ML (medial lateral): 1.5 mm, and DV (dorsal ventral): 3.5 mm, with an injection volume of 10 μL, i.e., 4 × 10 5 After in-situ tumor inoculation, the mice were kept for 14 days.

[0246] Using 1x DMEM (purchased from Zhongke Minchen (Beijing) Technology Co., Ltd., lot number K1902200), complex 20 was dissolved in a complex solution with a concentration of 1 mg / mL (calculated based on aptamer). Complex 21 and comparative complex 24 were each dissolved in a complex solution with a concentration of 0.8 mg / mL (calculated based on aptamer).

[0247] On the 15th day after in-situ tumor inoculation, bioimaging was performed on each mouse using the small animal bio-optical imaging system IVIS Lumina Series III, and the mice were divided into groups based on brain fluorescence intensity, with 6 mice per group. The day of administration was designated as D1 (i.e., day 1 of the experiment; hereafter, D4, D8, etc. represent days 4 and 8 of the experiment, and will be inferred in this manner).

[0248] The bioimaging method is as follows. Each mouse was intraperitoneally injected with 15 mg / mL D-fluorescein potassium salt working solution (purchased from Yeasen Biotechnology (Shanghai) Co., Ltd.) at a concentration of 10 μL / g body weight, and bioimaging (IVIS® Lumina III small animal bioimaging system) was performed 10 min after injection. After imaging, the fluorescent region of interest (ROI) in the mouse brain was surrounded, and the fluorescence intensity (Radiance) was measured by software. Under this condition, the Luciferase (Photinus pyralis) reporter gene expressed in U118MG-luc human glioma cells can generate a fluorescent response, so the fluorescence intensity can reflect the proliferation number of glioma cells. The higher the fluorescence intensity, the higher the number of glioma cells.

[0249] In the experiment, the mice were administered subcutaneously on D1, D4, D8 and D12. The mice were weighed before administration and administered according to their body weight.

[0250] For test group 5a, each mouse was administered with conjugate 20. The single dose volume was 5 μL / g mouse body weight, and the calculated single dose was 5 mg / kg, which corresponds to a dose of 0.3 mg / kg containing MMAE.

[0251] For test group 5b, each mouse was administered with conjugate 21. The single dose volume was 5 μL / g mouse body weight, and the calculated single dose was 4 mg / kg, which corresponds to a dose of 0.3 mg / kg containing MMAE.

[0252] For test group 5c, each mouse was administered with comparative conjugate 24. The single dose volume was 5 μL / g mouse body weight, and the calculated single dose was 4 mg / kg, which corresponds to a dose of 0.3 mg / kg containing MMAE.

[0253] For the blank control group, each mouse was administered with DMEM medium respectively, with a single administration volume of 5 μL / g mouse body weight.

[0254] According to the above method, the mice of each group were subjected to bioimaging analysis to measure the fluorescence intensity on D1, D22, D31 and D39, respectively, and the results are shown in Figure 5.

[0255] FIG. 5 is a line graph showing the time course of tumor fluorescence intensity in U118MG in-situ tumor model mice after administration of the complex according to the present disclosure or a control compound. As can be seen from the results of FIG. 5, the tumor fluorescence intensity (Radiance) of the blank control group and the control group clearly increased with the extension of the observation time, compared to D1 after administration. This indicates that the number of U118MG human glioma cells is clearly increased, and in contrast, the tumor fluorescence intensity of test groups 5a and 5b administered with the complex according to the present disclosure is clearly decreased, with the decrease reaching a maximum of one digit, and reaching more than two digits compared to the control group. This indicates that the number of U118MG human glioma cells is clearly decreased, decreasing to 1 / 10 of the start of the experiment compared to the start of the experiment, and may even decrease to 1% or less of the control group. As described above, even by subcutaneous administration alone, the complex according to the present disclosure can effectively penetrate the blood-brain barrier and efficiently target brain gliomas, has an excellent effect of inhibiting tumor growth, and shows good treatment compliance and high drug development potential for efficiently inhibiting tumors.

[0256] Experimental Example 6 Inhibitory activity of the complex against U118MG subcutaneous tumor in mice U118MG human glioma cells (purchased from Guangzhou Geniobiotech Co., Ltd.) were cultured according to the method of Experimental Example 2. Logarithmically grown U118MG human glioma cells were taken and digested with (0.25% pancreatin), the cells were harvested, centrifuged to remove the supernatant, and the cells were resuspended in serum-free DMEM medium to a concentration of 1 × 10 8 A cell culture medium was prepared with 1000 cells / mL.

[0257] The experimental animals were 36 12-week-old male NOD-SCID mice (purchased from Beijing Sibaifu Biotechnology Co., Ltd.). The above cell culture medium was inoculated into the subcutaneous site of the right dorsum of the NOD-SCID mice, with an inoculation volume of 100 μL per mouse, i.e., 1 × 10 7 After injection, the mice were kept for 7 days.

[0258] Using serum-free DMEM medium, complex 20 was dissolved in a complex solution with a concentration of 1 mg / mL (calculated by aptamer). Complex 21, complex 22 and complex 23 were each dissolved in a complex solution with a concentration of 0.8 mg / mL (calculated by aptamer). MMAE was dissolved in a solution with a concentration of 0.06 mg / mL using 10% DMSO + 90% serum-free DMEM medium (volume ratio).

[0259] The administration was started 7 days after the inoculation of U118MG cells, and the day of administration was designated as D8. In the experiment, the mice were administered subcutaneously in the abdomen once on D8, D12, D16, and D20, for a total of four administrations.

[0260] For the blank control group 6a, mice in each group were administered DMEM respectively, with a single administration volume of 5 μL / g mouse body weight.

[0261] For the control group 6b, MMAE was administered to the mice in each group, with the single-dose volume set at 5 μL / g mouse body weight, and the calculated single-dose dose was 0.3 mg / kg.

[0262] For test group 6c, each mouse was administered with conjugate 20. The single dose volume was 5 μL / g mouse body weight, and the calculated single dose was 5 mg / kg, which corresponds to a dose of 0.3 mg / kg containing MMAE.

[0263] Mice were divided into three groups and administered with Conjugate 21, Conjugate 22 or Conjugate 23. The single dose volume was 5 μL / g mouse body weight, and the calculated single dose was 4 mg / kg, which was equivalent to a dose of 0.3 mg / kg containing MMAE, and these groups were recorded as test groups 6d, 6e and 6f, respectively.

[0264] Tumor volumes were measured on D16, D20, D25, D29, D33, D36, D41, D48, and D60. The long and short diameters of the tumor were measured by external measurement. Tumor volumes were calculated using the formula 1 / 2 (long diameter × short diameter) 2 ) was used for calculation. After the experiment was completed, tumor tissue was taken and weighed on D60. FIG. 6 is a line graph showing the time course of mouse tumor volume on different days after administration of different complexes. As can be seen from FIG. 6, the tumor volume and tumor weight of the mice in test groups 6c to 6f administered with the complex of the present disclosure were both significantly reduced, compared with the control group 6b or the blank control group. As is clear from the above results, the complex of the present disclosure can effectively reach tumor tissue and exhibit excellent antitumor activity.

[0265] Experimental Example 7: In vivo activity of U118MG subcutaneous tumor model mice by long-term administration of the complex According to the method of Experimental Example 2, 42 mice inoculated with U118MG subcutaneous tumors were obtained, and the mice were kept after injection.

[0266] Using 10% DMSO + 90% serum-free DMEM medium (volume ratio), MMAE was dissolved in solutions of 0.03 mg / mL and 0.01 mg / mL concentrations, complex 21 was dissolved in solutions of 0.5 mg / mL and 0.165 mg / mL concentrations (calculated based on aptamer), and comparative complex 25 was dissolved in solutions of 0.5 mg / mL and 0.165 mg / mL concentrations (calculated based on aptamer).

[0267] Seven days after inoculation with U118MG cells, all mice were randomly divided into 7 groups, with 6 mice in each group, and the mice in each group were administered the drug on the day of administration, which was designated as D8. In the experiment, the mice were administered subcutaneously in the abdomen once on D8, D11, D15, D29, D32 and D36, for a total of 6 doses. Before administration, the mice were weighed, and the administration volume was calculated according to their body weight.

[0268] For the blank control group, each mouse was administered with DMEM medium respectively, with a single administration volume of 10 μL / g mouse body weight.

[0269] For test group 7a, each mouse was administered MMAE at a concentration of 0.01 mg / mL. The single dose volume was 10 μL / g mouse body weight, and the calculated single dose was 0.1 mg / kg.

[0270] For test group 7b, each mouse was administered 0.165 mg / mL of conjugate 21. The single dose volume was 10 μL / g mouse body weight, and the calculated single dose was 1.65 mg / kg (corresponding MMAE dose was 0.1 mg / kg).

[0271] For the control group 7c, each mouse was administered a 0.165 mg / mL concentration of comparative conjugate 25. The single dose volume was 10 μL / g mouse body weight, and the calculated single dose was 1.65 mg / kg (corresponding MMAE dose was 0.1 mg / kg).

[0272] For test group 7d, each mouse was administered MMAE at a concentration of 0.03 mg / mL. The single dose volume was 10 μL / g mouse body weight, and the calculated single dose was 0.3 mg / kg.

[0273] For test group 7e, each mouse was administered 0.5 mg / mL of conjugate 21. The single dose volume was 10 μL / g mouse body weight, and the calculated single dose was 5 mg / kg (corresponding MMAE dose was 0.3 mg / kg).

[0274] For the control group 7f, each mouse was administered a 0.5 mg / mL concentration of comparative conjugate 25. The single dose volume was 10 μL / g mouse body weight, and the calculated single dose was 5 mg / kg (corresponding MMAE dose was 0.3 mg / kg).

[0275] Tumor volumes were measured on D1, D9, D16, D19, D21, D24, D26, D29, D32, D36, D39, D43, D47, D53, D57, D60, D64, D67, D71, D74, D78, D81, D84, D88, D92, D95 and D99. The blank control group was measured on D53, and the test groups 7a, 7b and control group 7c were measured on D60, after which the experiment was terminated.

[0276] The long and short diameters of the tumor were measured by external measurement. The tumor volume was calculated using the formula 1 / 2 (long diameter × short diameter). 2 After the experiment was stopped, tumor tissues from each group were taken and weighed, and the average value was calculated. The results are shown in Figure 7.

[0277] FIG. 7 is a line graph showing the change in tumor volume over time in U118MG subcutaneous tumor model mice after administration of different concentrations of a conjugate according to the present disclosure or a control compound.

[0278] As can be seen from the results in Figure 7, the tumor volume increased rapidly in the blank control group, and the rate of increase in tumor volume slowed in the test group administered with MMAE alone, indicating that MMAE itself has an inhibitory effect on tumor growth.

[0279] Furthermore, the tumor volume of test group 7b was significantly smaller than that of test group 7a and control group 7c, both of which correspond to the MMAE content, during the test period, and the tumor volume of test group 7e was significantly smaller than that of test group 7d and control group 7f, both of which correspond to the MMAE content, during the test period, showing superior antitumor activity to that of MMAE test groups 7a and 7d alone and that of comparative complex 25. After the experiment was stopped, the tumor weight in the mice administered with the complex of the present disclosure was also significantly lower than that of the MMAE group and the control group. As is clear from the above, the complex of the present disclosure can effectively deliver MMAE to tumor tissue, exhibiting tumor targeting ability, while reducing the toxicity risk caused by the distribution of MMAE molecules to other tissues, and exhibiting dose-related and excellent antitumor effects.

[0280] As is clear from the above results, the conjugate of the present disclosure can effectively target and deliver a small molecule drug group having an inhibitory effect on tumors to tumor tissue, and can exhibit excellent antitumor activity and dose-dependent effects.

[0281] Experimental Example 8: In vivo activity of the complex in U118MG subcutaneous tumor model mice According to the method of Experimental Example 2, 42 mice inoculated with U118MG subcutaneous tumors were obtained, and the mice were kept after injection.

[0282] MMAE was dissolved in a solution of 0.02 mg / mL concentration in 10% DMSO + 90% serum-free DMEM medium (volume ratio), complex 20 and comparative complex 25 were dissolved in a solution of 0.33 mg / mL concentration (calculated with aptamer), complex 21 and complex 23 were dissolved in a solution of 0.26 mg / mL concentration (calculated with aptamer), and complex 26 was dissolved in a solution of 0.23 mg / mL concentration (calculated with aptamer).

[0283] Seven days after inoculation of U118MG cells, the mice were divided into groups, each with 6 mice, and the mice were administered the drug on the day of administration, designated as D8. Before administration, the mice were weighed, and the administration volume was calculated assuming an average body weight of 20 g.

[0284] In the experiment, the mice were administered subcutaneously in the abdomen once on D8, D12, D15 and D19, respectively, for a total of four times.

[0285] For the blank control group, each mouse was administered with DMEM medium respectively, with a single administration volume of 100 μL / g.

[0286] For test group 8a, each mouse was administered MMAE, with a single dose volume of 100 μL and a calculated single dose of 0.1 mg / kg.

[0287] For test group 8b, each mouse was administered with Conjugate 20. The single dose volume was 100 μL, and the calculated single dose was 1.65 mg / kg.

[0288] For the control group 8c, each mouse was administered with Comparative Complex 25. The single dose volume was 100 μL, and the calculated single dose was 1.65 mg / kg.

[0289] For test group 8d, each mouse was administered with Conjugate 21. The single dose volume was 100 μL, and the calculated single dose was 1.32 mg / kg.

[0290] For test group 8e, each mouse was administered with conjugate 23. The single dose volume was 100 μL, and the calculated single dose was 1.32 mg / kg.

[0291] For test group 8f, each mouse was administered with complex 26. The single dose volume was 100 μL, and the calculated single dose was 1.17 mg / kg. The doses of the above 8b to 8f groups each correspond to 0.1 mg / kg of MMAE.

[0292] Tumor volumes were measured on D1, D9, D16, D19, D22, D26, D30, D36, D40, D43, D47, D50, D54, D57, D61, D64, D68, and D71. The blank control group was measured on D54, and group 8a (administered only MMAE) was measured on D64, after which the experiment was terminated.

[0293] The long and short diameters of the tumor were measured by external measurement. The tumor volume was calculated using the formula 1 / 2 (long diameter × short diameter). 2 After the experiment was terminated, tumor tissues were taken from each group and weighed. The results are shown in Figure 8.

[0294] Figure 8 shows a line graph of the change in tumor volume over time in mice of each group and the tumor weight at D72. As can be seen from the results of Figure 8, the tumor volume of the blank control group increased rapidly, and the tumor volume increase rate of each of the other groups decreased. However, compared with test group 8a and control group 8c, which were administered with only MMAE, the tumor volume increase rate of each of the test groups administered with the conjugate of the present disclosure was significantly further decreased when the single dose of MMAE was equivalent to 0.1 mg / kg. In addition, conjugate 20, conjugate 21, conjugate 23 and conjugate 26 showed a better antitumor effect at the end of the experiment D72, with a tumor weight reduction of at least 58% compared with test group 8a.

[0295] Experimental Example 9: In vivo activity of the complex in A549 subcutaneous tumor model mice A549 human lung adenocarcinoma cells (purchased from Guangzhou Genio Biotech Co., Ltd.) were cultured in DMEM complete medium (MACGENE, product number CM15019) containing 10% FBS (Gibco, product number 10099-141) at 37°C in a 5% CO2 / 95% air incubator. Cells were harvested by digestion with 0.25 wt% pancreatin, the supernatant was aspirated, and the cells were resuspended in serum-free DMEM medium to a cell density of 1 × 10 8 A cell culture medium was prepared with 1000 cells / mL.

[0296] According to the method of Experimental Example 2, 30 mice inoculated with A549 subcutaneous tumor were obtained, and the mice were kept after injection.

[0297] Using serum-free DMEM medium, complex 20 and complex 21 were dissolved in a complex solution with a concentration of 1 mg / mL (calculated by aptamer). Comparative complex 24 was dissolved in a complex solution with a concentration of 0.8 mg / mL (calculated by aptamer). MMAE was dissolved in a solution with a concentration of 0.06 mg / mL using 10% DMSO + 90% serum-free DMEM medium (volume ratio).

[0298] Seven days after inoculation of A549 human lung cancer cells, all mice were divided into groups, with 6 mice in each group, and each mouse was administered the drug on the day of administration, designated as D8. The mice were weighed before administration, and the administration volume was calculated according to their weight.

[0299] Mice in each group were administered once on D8, D12, D15 and D19, respectively, for a total of four doses. For the blank control group 9a, mice in each group were administered DMEM, respectively, with a single dose volume of 5 μL / g mouse body weight.

[0300] For the control group 9b, MMAE was administered to the mice in each group, with the single-dose volume being 5 μL / g mouse body weight, and the calculated single-dose amount was 0.3 mg / kg.

[0301] For test group 9c, each mouse was administered with conjugate 20. The single dose volume was 5 μL / g mouse body weight, and the calculated single dose was 5 mg / kg, which corresponds to a dose of 0.3 mg / kg containing MMAE.

[0302] For test group 9d, each mouse was administered with conjugate 21. The single dose volume was 5 μL / g mouse body weight, and the calculated single dose was 5 mg / kg, which corresponds to a dose of 0.3 mg / kg containing MMAE.

[0303] For test group 9e, each mouse was administered with comparative conjugate 24. The single dose volume was 5 μL / g mouse body weight, and the calculated single dose was 5 mg / kg, which corresponds to a dose of 0.3 mg / kg containing MMAE.

[0304] Mice in each group were administered once on D46, D50, and D54, respectively. The single-dose volume was 10 μL / g mouse body weight, and the calculated dose of MMAE contained in the single dose was equivalent to 0.6 mg / kg.

[0305] Tumor volumes were measured on D1, D9, D16, D19, D22, D26, D30, D36, D40, D43, D47, D50, D54 and D57. The blank control group was discontinued after the measurement on D50.

[0306] The long and short diameters of the tumor were measured by external measurement. The tumor volume was calculated using the formula 1 / 2 (long diameter × short diameter). 2 The results are shown in Figure 9.

[0307] 9 is a line graph showing the time course of tumor volume in A549 subcutaneous tumor model mice after administration of different concentrations of the conjugate of the present disclosure or the control compound. As can be seen from the results of FIG. 9, the tumor volume of the mice in the blank control group increased rapidly, the tumor volume increase rate of each of the remaining groups all decreased, and the tumor volumes of the mice administered with conjugates 20 and 21 at each time point were all smaller than those of the control groups 9b and 9e. As can be seen from the above results, the conjugate of the present disclosure can effectively target A549 lung cancer tumor tissue and exhibit excellent antitumor activity.

[0308] Although several embodiments of the present disclosure have been described in detail above, the present disclosure is not limited to the specific details of the above embodiments, and within the scope of the technical idea of ​​the present disclosure, several simple modifications can be made to the technical means of the present disclosure, and all of these simple modifications fall within the scope of protection of the present disclosure.

[0309] It should be noted that the specific technical features described in the above embodiments can be combined in any suitable manner unless they are contradictory, and in order to avoid unnecessary duplication, the present disclosure does not separately describe various possible combination manners.

[0310] Furthermore, the various embodiments of the present disclosure can be combined in any manner and should be considered as being disclosed in the present disclosure unless they deviate from the spirit of the present disclosure.

Claims

1. It comprises one or more delivery groups and one or more functional groups, the delivery group being formed by removing one or more hydrogen atoms or one or more functional groups from one aptamer, the aptamer comprising adjacent nucleotide sequences, the group connecting two adjacent nucleotides being independently a phosphate ester group or a phosphate ester group having a modifying group, each nucleotide being selected from one of modified or unmodified A, U, C or G, and the adjacent nucleotide sequences having the sequence shown in formula (1), 5'-T １ -S １ -N ａ -S ２ -N ｂ -S ３ -N ｃ -S ４ -T ２ -3' Formula (1) Here, T １ This is a motif consisting of 1 to 3 nucleotides, T ２ It is a motif consisting of 0 to 15 nucleotides, and T ２ T １ It does not contain any motifs that are completely inversely complementary to it. S １ and S ４ Each of these is a motif consisting of 3 to 7 nucleotides, S １ is, S ４ They have the same length and are completely inversely complementary. N ａ and N ｃ Each of these is a motif consisting of 1 to 4 nucleotides, N ａ Each nucleotide inside is N ｃ None of the nucleotides inside are complementary to each other, and N ａ and N ｃ The total number of U's inside is N ａ and N ｃ S accounts for more than 50% of the total number of nucleotides in the body. ２ and S ３ Each of these is a motif consisting of 1 to 4 nucleotides, S ２ is, S ３ They have the same length and are completely inversely complementary. N ｂ This is a motif consisting of 3 to 6 nucleotides, N ｂ The nucleotides at both ends do not form AU or GC complements. Each of the aforementioned delivery groups is independently bonded to the functional group via a covalent bond or via a binding group, and each of the aforementioned functional groups is selected from low molecular weight therapeutic groups having a therapeutic effect against tumors, forming a complex.

2. The complex according to claim 1, wherein the length of the adjacent nucleotide sequences is 18 to 50 nucleotides, or 20 to 40 nucleotides, or 21 to 36 nucleotides, or 24 to 32 nucleotides.

3. T １ It consists of two nucleotides, or T １ It consists of two nucleotides and contains at least one C, or from 5' to 3', T １ is CU, UC, or AC, or T ２ It consists of 0 to 10 nucleotides, or from 5' to 3', T ２ It consists of 1 to 9 nucleotides starting with U, or S １ and S ４ Each consists of 3 to 5 nucleotides and has the same length, or S １ and S ４ In the inverse complement formed by , GC complement accounts for at least 40% of all complements, or from 5' to 3', S １ is GCU and S ４ It is AGC, or S １ It is GAGU and S ４ GCUC, or S １ It is GGAGU and S ４ It is GCUCU, or S １ is UAUGG and S ４ is CCAUG, or The aforementioned N ａ and N ｃ The sum of the number of nucleotides inside is an integer between 2 and 4, or the aforementioned N ａ and N ｃ The sum of the number of nucleotides in the N is 3 or 4, and the N ａ and N ｃ The sum of the numbers of U inside is 2 or 3, or N from 5' to 3'. ａ or N ｃ These are independently U, UU, UC, or CU, or S ２ and S ３ Each consists of 2 to 3 nucleotides and is of the same length, or S ２ and S ３ The inverse complement formed by includes at least one GC complement, or S from 5' to 3'. ２ is CA and S ３ is UG, or S ２ AC and S ３ It is GU, or S ２ It is GCC and S ３ is GGU, or N ｂ It consists of four or five nucleotides, or from 5' to 3', N ｂ The composite according to claim 1, wherein is GACG, GACGU, GACCG, UACU, GUUG, or GAUCU.

4. The adjacent nucleotide sequence has the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or The adjacent nucleotide sequence has the nucleotide sequence shown in Sequence ID No.

4. 5'-N ６ GGAGUUCAN １ N ２ N ３ N ４ UGN ５ GCUCN ７ -3' (Sequence ID 4) Here, N １ , N ２ and N ３ Each of these is independently one of A, U, C, and G, and N ４ is a motif consisting of U, C, or G, or two of U, C, or G, N ５ is U, CU, or UU, N ６ is CU, UC, or AC, and N ７ is U, UU, or UUN ８ And N ８ The complex according to claim 1, wherein is a motif consisting of 1 to 15 nucleotides.

5. N １ 、N ２ 、N ３ and N ４ The motif N consisting of １ N ２ N ３ N ４ is one of GACG, GACGU, GACCG, UACU, GUUG or GAUCU, or The complex according to claim 4, wherein N8 is a motif consisting of 1 to 8 nucleotides, or the nucleotide sequence of N8 from 5' to 3' is CCGAUCUC, or the adjacent nucleotide sequence has the sequence shown in one of sequence numbers 12 to 14.

6. The complex according to claim 5, wherein the adjacent nucleotide sequences have a nucleotide sequence shown in any one of sequence numbers 5 to 11.

7. Each cytosine nucleotide in the adjacent nucleotide sequence is a fluoromodified cytosine nucleotide, and / or each uracil nucleotide in the adjacent nucleotide sequence is a fluoromodified uracil nucleotide, or each nucleotide in the adjacent nucleotide sequence is a 2'-methoxymodified nucleotide, or N in the adjacent nucleotide sequence ｂ and S ３ The complex according to claim 1, wherein one or more uracil nucleotides among the motifs have a modified base.

8. The complex according to claim 7, wherein the adjacent nucleotide sequence has a nucleotide sequence shown in one of sequence numbers 15 to 33.

9. The complex according to claim 1, wherein at least one group that connects two adjacent nucleotides in the adjacent nucleotide sequence is a thiophosphate ester group, or each of the groups that connect two adjacent nucleotides is a thiophosphate ester group.

10. The complex according to claim 9, wherein the adjacent nucleotide sequence has a nucleotide sequence shown in one of sequence numbers 34 to 39.

11. Having the structure shown in formula (101), 【Chemistry 1】 （１０１） In the formula, each R ＡＰ The group is independently a group having the structure shown in formula (102), 【Chemistry 2】 （１０２） In the formula, each AP group is the same or different and independently represents one of the delivery groups, R ｊ , each R ｋ or each R ｉ is the same or different and each independently represents a covalent bond or a linking group, and R ｉ and R ｋ are not both covalent bonds at the same time, and each n １ independently represents an integer from 0 to 4, Each A ０ The groups, whether the same or different, independently represent one of the aforementioned functional groups, m ０ n is an integer from 1 to 6. ０ These are integers from 1 to 6, 【change】 The composite according to claim 10, wherein is a site on which the group is covalently bonded.

12. I understand ０ is an integer from 1 to 4, and / or n ０ is an integer from 1 to 3, and / or each n １ These are independent integers between 0 and 1. Or, m ０ is 1 and / or n ０ is 1 and / or at least one or each n １ The composite according to claim 11, wherein is 0.

13. Each of the R ｋ or each of the R ｉ These are independently covalent or linear alkylene groups having a length of 1 to 70 carbon atoms, or one or more carbon atoms in the linear alkylene group are C(O), NH, O, S, CH=N, S(O) ２ , OP(O) ２ ,OP(O)(S),C ５ ~C ８ Glycoside group, C ２ ~C １０ Alkenylene group, C ２ ~C １０ Alkynylene group, C ６ ~C １０ Arylene group, C ３ ~C １８ Heterocyclylene group and C ５ ~C １０ The linear alkylene group is substituted with one or more selected from the group consisting of heteroarylene groups, and the linear alkylene group is C １ ~C １０ Alkyl alkyl group, C ６ ~C １０ Aryl group, C ５ ~C １０ heteroaryl group, C １ ~C １０ Alkyl halogenated groups, -OC １ ~C １０ Alkyl alkyl group, -OC １ ~C １０ Alkylphenyl group, -C １ ~C １０ Alkyl-OH, -OC １ ~C １０ Alkyl halogens, -SC １ ~C １０ Alkyl alkyl group, -SC １ ~C １０ Alkylphenyl group, -C １ ~C １０ Alkyl-SH, -SC １ ~C １０ Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH ２ , -C １ ~C １０ Alkyl-NH ２ , -N(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkyl alkyl group), -NH(C １ ~C １０ Alkyl alkyl group), -N(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkylphenyl group), -NH(C １ ~C １０ Alkylphenyl group, cyano group, nitro group, -CO ２ H, -C(O)O(C １ ~C １０ Alkyl(alkyl group), -CON(C １ ~C １０ (Alkyl alkyl group) (C １ ~C １０ Alkyl(alkyl group), -CONH(C １ ~C １０ Alkyl(alkyl group), -CONH ２ , -NHC(O)(C １ ~C １０ Alkyl alkyl group), -NHC(O)(phenyl group), -N(C １ ~C １０ Alkyl)C(O)(C １ ~C １０ Alkyl alkyl group), -N(C １ ~C １０ Alkyl)C(O)(phenyl group), -C(O)C １ ~C １０ Alkyl alkyl group, -C(O)C １ ~C １０ Alkylphenyl group, -C(O)C １ ~C １０ Alkyl halogenated groups, -OC(O)C １ ~C １０ Alkyl, -SO ２ (C １ ~C １０ Alkyl(alkyl group), -SO ２ (phenyl group), -SO ２ (C １ ~C １０ (Halogenated alkyl group), -SO ２ NH ２ , -SO ２ NH(C) １ ~C １０ Alkyl(alkyl group), -SO ２ NH (phenyl group), -NHSO ２ (C １ ~C １０ (Alkyl group), -NHSO ２ (phenyl group) and -NHSO ２ (C １ ~C １０ The composite according to claim 11, which may have one or more substituents selected from the group consisting of alkyl halogens.

14. each n １ All of these are 0, and each R ｉ These are, independently, covalently bonded or bonded to a C group. １ ~C ２０ A combination of one or more bonds from alkylene groups, phosphate ester bonds, thiophosphate ester bonds, amide bonds, ester bonds, ether bonds, thioether bonds, disulfide bonds, 1,2,3-triazole subunits, polyethylene glycol subunits, pyrrolidine subunits, 2-oxopyrrolidine subunits, phenylene groups, cyclohexylene groups, 2-succinimide subunits, 2-thiosuccinimide subunits, amino acid subunits, and nucleotide subunits, or Each R ｉ The complex according to claim 13, wherein is independently a combination of one or two bonds selected from a covalent bond, a disulfide bond, a propylene phosphate ester group, a 2-thiosuccinimide subunit, an amino acid subunit, or a GAU trinucleotide subunit.

15. R ｊ It is a covalent bond, m ０ is 1, or R ｊ is a bonding group, and the bonding group R ｊ It includes a main chain portion, a side chain portion and a composite bonding portion, the main chain portion is connected to the composite bonding portion and the side chain portion, and each of the side chain portions is connected to the main chain portion and the R ＡＰ The base is bonded, and each of the composite bonding portions is bonded to the main chain portion and the functional group A, respectively. ０ Combined, The main chain portion is a linear alkylene group having a length of 1 to 70 carbon atoms, or one or more carbon atoms in the linear alkylene group are C(O), NH, O, S, CH=N, S(O) ２ , OP(O) ２ , C ５ ~C ８ Glycoside group, C ２ ~C １０ Alkenylene group, C ２ ~C １０ Alkynylene group, C ６ ~C １０ Arylene group, C ３ ~C １８ Heterocyclylene group and C ５ ~C １０ The linear alkylene group is substituted with one or more selected from the group consisting of heteroarylene groups, and the linear alkylene group is C １ ~C １０ Alkyl alkyl group, C ６ ~C １０ Aryl group, C ５ ~C １０ heteroaryl group, C １ ~C １０ Alkyl halogenated groups, -OC １ ~C １０ Alkyl alkyl group, -OC １ ~C １０ Alkylphenyl group, -C １ ~C １０ Alkyl-OH, -OC １ ~C １０ Alkyl halogens, -SC １ ~C １０ Alkyl alkyl group, -SC １ ~C １０ Alkylphenyl group, -C １ ~C １０ Alkyl-SH, -SC １ ~C １０ Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH ２ , -C １ ~C １０ Alkyl-NH ２ , -N(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkyl alkyl group), -NH(C １ ~C １０ Alkyl alkyl group), -N(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkylphenyl group), -NH(C １ ~C １０ Alkylphenyl groups), cyano, nitro, -CO ２ H, -C(O)O(C １ ~C １０ Alkyl(alkyl group), -CON(C １ ~C １０ (Alkyl alkyl group) (C １ ~C １０ Alkyl(alkyl group), -CONH(C １ ~C １０ Alkyl(alkyl group), -CONH ２ , -NHC(O)(C １ ~C １０ Alkyl alkyl group), -NHC(O)(phenyl group), -N(C １ ~C １０ Alkyl)C(O)(C １ ~C １０ Alkyl alkyl group), -N(C １ ~C １０ Alkyl)C(O)(phenyl group), -C(O)C １ ~C １０ Alkyl alkyl group, -C(O)C １ ~C １０ Alkylphenyl group, -C(O)C １ ~C １０ Alkyl halogenated groups, -OC(O)C １ ~C １０ Alkyl, -SO ２ (C １ ~C １０ Alkyl(alkyl group), -SO ２ (phenyl group), -SO ２ (C １ ~C １０ (Halogenated alkyl group), -SO ２ NH ２ , -SO ２ NH(C) １ ~C １０ Alkyl(alkyl group), -SO ２ NH (phenyl group), -NHSO ２ (C １ ~C １０ (Alkyl group), -NHSO ２ (phenyl group) and -NHSO ２ (C １ ~C １０ It may have one or more substituents selected from the group consisting of halogenated alkyl groups. Each of the aforementioned side chain portions is independently covalently bonded, or is a linear alkylene group having a length of 1 to 70 carbon atoms, or one or more carbon atoms in the linear alkylene group are C(O), NH, O, S, CH=N, S(O) ２ , OP(O) ２ , C ５ ~C ８ Glycoside group, C ２ ~C １０ Alkenylene group, C ２ ~C １０ Alkynylene group, C ６ ~C １０ Arylene group, C ３ ~C １８ Heterocyclylene group and C ５ ~C １０ The linear alkylene group is substituted with one or more selected from the group consisting of heteroarylene groups, and the linear alkylene group is C １ ~C １０ Alkyl alkyl group, C ６ ~C １０ Aryl group, C ５ ~C １０ heteroaryl group, C １ ~C １０ Alkyl halogenated groups, -OC １ ~C １０ Alkyl alkyl group, -OC １ ~C １０ Alkylphenyl group, -C １ ~C １０ Alkyl-OH, -OC １ ~C １０ Alkyl halogens, -SC １ ~C １０ Alkyl alkyl group, -SC １ ~C １０ Alkylphenyl group, -C １ ~C １０ Alkyl-SH, -SC １ ~C １０ Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH ２ , -C １ ~C １０ Alkyl-NH ２ , -N(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkyl alkyl group), -NH(C １ ~C １０ Alkyl alkyl group), -N(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkylphenyl group), -NH(C １ ~C １０ Alkylphenyl group, cyano group, nitro group, -CO ２ H, -C(O)O(C １ ~C １０ Alkyl(alkyl group), -CON(C １ ~C １０ (Alkyl alkyl group) (C １ ~C １０ Alkyl(alkyl group), -CONH(C １ ~C １０ Alkyl(alkyl group), -CONH ２ , -NHC(O)(C １ ~C １０ Alkyl alkyl group), -NHC(O)(phenyl group), -N(C １ ~C １０ Alkyl)C(O)(C １ ~C １０ Alkyl alkyl group), -N(C １ ~C １０ Alkyl)C(O)(phenyl group), -C(O)C １ ~C １０ Alkyl alkyl group, -C(O)C １ ~C １０ Alkylphenyl group, -C(O)C １ ~C １０ Alkyl halogenated groups, -OC(O)C １ ~C １０ Alkyl, -SO ２ (C １ ~C １０ Alkyl(alkyl group), -SO ２ (phenyl group), -SO ２ (C １ ~C １０ (Halogenated alkyl group), -SO ２ NH ２ , -SO ２ NH(C) １ ~C １０ Alkyl(alkyl group), -SO ２ NH (phenyl group), -NHSO ２ (C １ ~C １０ (Alkyl group), -NHSO ２ (phenyl group) and -NHSO ２ (C １ ~C １０ It may have one or more substituents selected from the group consisting of halogenated alkyl groups. Each of the aforementioned composite bonding portions is independently a covalent bond or a bond structure C １ ~C １０ The complex according to claim 11, wherein the combination of bonds is one or more of the following: alkylene group, phosphate ester bond, thiophosphate ester bond, amide bond, ester bond, ether bond, thioether bond, disulfide bond, 1,2,3-triazole subunit, polyethylene glycol subunit, pyrrolidine subunit, 2-oxopyrrolidine subunit, phenylene group, cyclohexylene group, 2-succinimide subunit, 2-thiosuccinimide subunit, amino acid subunit, and nucleotide subunit.

16. The aforementioned bonding group R ｊ Each of the composite bonding portions within consists of the main chain portion and one of the functional groups A. ０ The side chain portion is connected to n ０ Each side chain portion consists of the main chain portion and one of the R ＡＰ Bonded to the base, or The composite according to claim 15, wherein all of the side chain portions are bonded to the same atom in the main chain portion, or each of the side chain portions is bonded to a different atom in the main chain portion.

17. I understand ０ is 1, and the bonding group R ｊ This includes the structure shown in formula (301), 【Transformation 3】 Formula (301) In the formula, k is an integer from 1 to 3, and L Ｃ L is the main chain portion. Ａ This is the side chain portion, L Ｂ This is the composite joint, 【change】 This represents the site where the group is covalently bonded. The main chain portion L Ｃ C is covalent or 2-4 valent, linear or branched. １ ~C ２５ A saturated hydrocarbon group, or one or more carbon atoms in the saturated hydrocarbon group are C(O), NH, O, S, CH=N, S(O) ２ , OP(O) ２ , C ５ ~C ８ Glycoside group, C ２ ~C ５ Alkenylene group, C ２ ~C ５ Alkynylene group, C ６ ~C １０ Arylene group, C ３ ~C ８ Heterocyclylene group and C ５ ~C １０ The saturated hydrocarbon group is substituted with one or more selected from the group consisting of heteroarylene groups, and the saturated hydrocarbon group is C １ ~C ５ Alkyl alkyl group, C ６ ~C １０ Aryl group, C ５ ~C １０ Heteroaryl group, -O-C １ ~C ５ Alkyl alkyl group, -OC １ ~C ５ Alkylphenyl group, -C １ ~C ５ alkyl-OH, -SC １ ~C ５ Alkyl alkyl groups, nitro, -C(O)O(C １ ~C ５ Alkyl(alkyl group), -CON(C １ ~C ５ (Alkyl) (C １ ~C ５ Alkyl(alkyl group), -CONH(C １ ~C ５ Alkyl(alkyl group), -CONH ２ , -NHC(O)(C １ ~C ５ Alkyl alkyl group), -NHC(O)(phenyl group), -N(C １ ~C ５ Alkyl)C(O)(C １ ~C ５ Alkyl alkyl group), -N(C １ ~C ５ Alkyl)C(O)(phenyl group), -C(O)C １ ~C ５ Alkyl alkyl group, -C(O)C １ ~C ５ Alkylphenyl group, -OC(O)C １ ~C ５ Alkyl, -SO ２ (C １ ~C ５ Alkyl(alkyl group), -SO ２ (phenyl group), -SO ２ NH ２ , -SO ２ NH(C) １ ~C ５ Alkyl(alkyl group), -SO ２ NH (phenyl group), -NHSO ２ (C １ ~C ５ Alkyl(alkyl group) and -NHSO ２ It may have one or more substituents selected from the group consisting of (phenyl groups), Each of the aforementioned side chain portions is independently covalently bonded, or is a linear alkylene group having a length of 1 to 70 carbon atoms, or one or more carbon atoms in the linear alkylene group are C(O), NH, O, S, CH=N, S(O) ２ , OP(O) ２ , C ５ ~C ８ Glycoside group, C ２ ~C １０ Alkenylene group, C ２ ~C １０ Alkynylene group, C ６ ~C １０ Arylene group, C ３ ~C １８ Heterocyclylene group and C ５ ~C １０ The linear alkylene group is substituted with one or more selected from the group consisting of heteroarylene groups, and the linear alkylene group is C １ ~C １０ Alkyl alkyl group, C ６ ~C １０ Aryl group, C ５ ~C １０ heteroaryl group, C １ ~C １０ Alkyl halogenated groups, -OC １ ~C １０ Alkyl alkyl group, -OC １ ~C １０ Alkylphenyl group, -C １ ~C １０ Alkyl-OH, -OC １ ~C １０ Alkyl halogens, -SC １ ~C １０ Alkyl alkyl group, -SC １ ~C １０ Alkylphenyl group, -C １ ~C １０ Alkyl-SH, -SC １ ~C １０ Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH ２ , -C １ ~C １０ Alkyl-NH ２ , -N(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkyl alkyl group), -NH(C １ ~C １０ Alkyl alkyl group), -N(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkylphenyl group), -NH(C １ ~C １０ Alkylphenyl group, cyano group, nitro group, -CO ２ H, -C(O)O(C １ ~C １０ Alkyl(alkyl group), -CON(C １ ~C １０ (Alkyl alkyl group) (C １ ~C １０ Alkyl(alkyl group), -CONH(C １ ~C １０ Alkyl(alkyl group), -CONH ２ , -NHC(O)(C １ ~C １０ Alkyl alkyl group), -NHC(O)(phenyl group), -N(C １ ~C １０ Alkyl)C(O)(C １ ~C １０ Alkyl alkyl group), -N(C １ ~C １０ Alkyl)C(O)(phenyl group), -C(O)C １ ~C １０ Alkyl alkyl group, -C(O)C １ ~C １０ Alkylphenyl group, -C(O)C １ ~C １０ Alkyl halogenated groups, -OC(O)C １ ~C １０ Alkyl, -SO ２ (C １ ~C １０ Alkyl(alkyl group), -SO ２ (phenyl group), -SO ２ (C １ ~C １０ (Halogenated alkyl group), -SO ２ NH ２ , -SO ２ NH(C) １ ~C １０ Alkyl(alkyl group), -SO ２ NH (phenyl group), -NHSO ２ (C １ ~C １０ (Alkyl group), -NHSO ２ (phenyl group) and -NHSO ２ (C １ ~C １０ It may have one or more substituents selected from the group consisting of halogenated alkyl groups. Each of the aforementioned composite bonding portions is independently a covalent bond or a bond structure C １ ~C １０ The complex according to claim 16, wherein the combination of bonds is one or more of the following: alkylene group, phosphate ester bond, thiophosphate ester bond, amide bond, ester bond, ether bond, thioether bond, disulfide bond, 1,2,3-triazole subunit, polyethylene glycol subunit, pyrrolidine subunit, 2-oxopyrrolidine subunit, phenylene group, cyclohexylene group, 2-succinimide subunit, 2-thiosuccinimide subunit, amino acid subunit, and nucleotide subunit.

18. Each of the aforementioned small molecule therapeutic groups is independently selected from cytotoxic groups, antibiotic groups, or angiogenic inhibitors, or The aforementioned low molecular weight therapeutic agent group is formed by removing one or more hydrogen atoms or one or more functional groups from one or more of the low molecular weight therapeutic agents methotrexate, doxorubicin, vinca alkaloids, auristatin, calichiamicin, meitansine, camptothecin, and calitiamycin, or The complex according to claim 1, wherein the low molecular weight therapeutic group is a group formed by removing one or more hydrogen atoms or one or more functional groups from monomethyl auristatin E (MMAE).

19. The complex is in the form of a pharmaceutically acceptable salt, or The composite according to claim 1, wherein the pharmaceutically acceptable salt is a potassium salt, a sodium salt, or a carboxylate salt.

20. A pharmaceutical composition comprising the complex described in any one of claims 1 to 19 and a pharmaceutically acceptable carrier.

21. The complex according to any one of claims 1 to 19 for treating tumors and tumor-related diseases or symptoms.

22. The complex according to claim 21, wherein the tumor is one or more of glioma, renal cancer, and lung cancer.

Images