Application of conjugate in medicine for diagnosing or treating colorectal cancer and related diseases

Abstract

The invention relates to an application of a conjugate in preparing a medicine for diagnosing or treating colorectal cancer, the conjugate comprises one or more delivery groups and one or more functional groups, each delivery group is independently connected with the functional group through a covalent bond or a linking group, the delivery group is formed by removing one or more hydrogen atoms or one or more functional groups from an aptamer, the aptamer comprises a continuous nucleotide sequence, and the continuous nucleotide sequence has a sequence as shown in a formula (1). The conjugate used in the application can effectively target tumor tissues and / or cells of the colorectal cancer, so that the function of a functional group is efficiently played, and diagnosis and / or treatment of the colorectal cancer are / is realized.

Description

Technical Field

[0001] The present disclosure relates to the use of a conjugate in the preparation of a drug for diagnosing or treating colorectal cancer and related diseases. The present disclosure also relates to a method for diagnosing or treating colorectal cancer and related diseases. Background Art

[0002] Colorectal cancer (CRC) is a common fatal disease. About 70% of newly diagnosed colorectal cancers each year are colon cancers, and most of the rest originate from the rectum. The incidence of colorectal cancer in my country is very high, with the majority of cases occurring in the middle and late stages, and there is an increasing trend year by year.

[0003] Most colorectal cancers are diagnosed after symptoms appear, through colonoscopy screening, non-invasive stool testing, or removal of tissue samples for testing (biopsy). Studies have shown that early screening can detect asymptomatic early malignancies and reduce mortality. However, although the accuracy and convenience of colorectal cancer diagnosis are relatively low, relatively few people are screened. Therefore, in the early diagnosis and treatment of colorectal cancer, it is of great significance to detect the presence of colorectal cancer earlier.

[0004] In clinical practice, colorectal cancer is generally treated with a comprehensive treatment based on surgery, as well as surgery, radiotherapy, chemotherapy and targeted therapy. Because targeted therapies have a more concentrated effect, their side effects tend to be smaller than chemotherapy. However, the actual impact and its severity depend largely on its targeting accuracy. There are also some unmet clinical needs for targeted therapy, for example: most targeted drugs are not suitable for all cancer patients, and only when genetic testing shows gene mutations can the corresponding targeted drugs be used for treatment, and some drugs will have greater side effects when used, such as dry mouth, nausea, vomiting, diarrhea, rash, etc., and different targeted drugs have different side effects.

[0005] Therefore, there is an urgent need to find new targeted treatment and diagnostic methods for colorectal cancer. Summary of the invention

[0006] The present disclosure provides a use of a conjugate in the preparation of a drug for diagnosing or treating colorectal cancer, wherein the conjugate comprises one or more delivery groups and one or more functional groups, each of the delivery groups is independently connected to at least one of the functional groups or other delivery groups through at least one linking group, and each functional group is independently connected to at least one of the delivery groups or at least one other functional group through at least one linking group;

[0007] Each of the functional groups is independently one of a diagnostic agent group, a small molecule therapeutic agent group having a therapeutic effect on colorectal cancer, a functional oligonucleotide group having a therapeutic effect on colorectal cancer, and a delivery aid group;

[0008] The delivery group is formed by removing one or more hydrogen atoms or one or more functional groups from an aptamer, wherein the aptamer comprises a continuous nucleotide sequence, wherein the group connecting two adjacent nucleotides is independently a phosphate group or a phosphate group with a modified group, and each nucleotide is selected from one of modified or unmodified A, U, C or G, and the continuous nucleotide sequence has a sequence shown in formula (1):

[0009] 5'-T1-S1-N a -S2-N b -S3-N c -S4-T2-3' formula (1)

[0010] Wherein, T1 is a motif consisting of 1-3 nucleotides, T2 is a motif consisting of 0-15 nucleotides, and T2 does not contain a motif that is completely reverse complementary to T1;

[0011] S1 and S4 are each a motif consisting of 3-7 nucleotides, S1 and S4 are of the same length and are completely reverse complementary;

[0012] N a and N c Each is a motif consisting of 1-4 nucleotides, N a Each nucleotide in c Each nucleotide in N is not complementary, and N a and N c The total number of U in N a and N c More than 50% of the total number of nucleotides in

[0013] S2 and S3 are each a motif consisting of 1-4 nucleotides, S2 and S3 are of the same length and are completely reverse complementary;

[0014] N b is a motif consisting of 3-6 nucleotides, and N b The nucleotides at both ends do not form AU or GC complementarity.

[0015] Incorporated by Reference

[0016] All publications, patents, and patent applications mentioned in this specification are incorporated by reference into the present disclosure to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference into the present disclosure.

[0017] Beneficial Effects

[0018] The conjugates and pharmaceutical compositions provided by the present disclosure have excellent ability to target colorectal cancer tumor cells. By administering the conjugates provided by the present invention, the diagnostic accuracy of colorectal cancer can be significantly improved, or colorectal cancer-related diseases and / or symptoms can be treated / alleviated.

[0019] On the one hand, the conjugates provided by the present disclosure can be targeted to colorectal cancer tumor tissues, so that the diagnostic agent groups contained in the conjugates, such as fluorescent groups, can specifically play a role in colorectal cancer tumor tissues. For example, the different conjugates provided by the present disclosure can all show strong fluorescence signals at the site of colon cancer tumor tissue inoculated in mice, indicating that the conjugates provided by the present disclosure can specifically target colorectal cancer tumor tissues; further, compared with the control group, the conjugates provided by the present disclosure still show strong fluorescence signals at 24h to 48h after administration. In addition, the conjugates provided by the present disclosure, after 96h of administration of the conjugates, the fluorescence signal intensity of the tumor tissues still remains at a high level, indicating that the conjugates provided by the present disclosure can efficiently and stably target colorectal cancer tumors for a long time, and have good application effects.

[0020] On the other hand, the conjugates provided by the present disclosure, which contain various functional oligonucleotides or various small molecule drug groups, such as small molecule cytotoxin groups, can be specifically enriched and enter tumor tissues, and show excellent tumor inhibition effects. For example, the conjugates provided by the present disclosure can effectively deliver small molecule therapeutic agent groups to tumor tissues, while showing tumor targeting capabilities, while also reducing the toxicity risks caused by the distribution of small molecule therapeutic agents in other tissues, and various modes of administration can effectively inhibit the rate of increase in tumor volume and tumor weight, indicating that the conjugates disclosed in the present disclosure can effectively inhibit tumor proliferation. In addition, further increasing the dosage of the conjugate can make the tumor volume almost unchanged during the test, showing a more excellent anti-tumor effect.

[0021] This indicates that the conjugate provided by the present disclosure has excellent ability to target colorectal cancer tissues and cells, can significantly improve the success rate of colorectal cancer diagnosis, treat / alleviate colorectal cancer-related diseases and / or symptoms, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1A and Figure 1B The in vivo imaging images at 24h and 48h after administering the conjugate provided by the present disclosure to HCT-116 human colon cancer cell subcutaneous tumor model mice are shown respectively; Figure 1CFIG. 5 is a fluorescence imaging diagram showing the tumor tissue of mice 96 hours after the conjugate provided by the present disclosure was administered to HCT-116 human colon cancer cell subcutaneous tumor model mice. DETAILED DESCRIPTION

[0023] The specific embodiments of the present disclosure are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure, and are not used to limit the present disclosure.

[0024] definition

[0025] In the present disclosure, unless otherwise specified, A, U, C, G and T refer to adenine nucleotide, uracil nucleotide, cytosine nucleotide, guanine nucleotide and thymine nucleotide, respectively, and 5-methylcytosine nucleotide refers to a nucleotide in which the hydrogen at the 5' position of the cytosine base in the cytosine nucleotide is replaced by a methyl group. The structures of these nucleotides are well known to those skilled in the art.

[0026] As used in the present disclosure, "motif" refers to a nucleic acid sequence fragment in an aptamer, consisting of one or more nucleotides. In some embodiments, the motif is a nucleic acid sequence fragment with biological function.

[0027] As used in the present disclosure, "alkyl" refers to straight and branched saturated hydrocarbon radicals having a specified number of carbon atoms, typically 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 refers to straight and branched alkyl radicals containing 1 to 6 carbon atoms. When referring to an alkyl residue having a specific number of carbons, it is intended to cover all branched and straight chain forms having that number of carbons; thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl; "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to residues that are identical to alkyl but have two points of attachment.

[0028] As used in the present disclosure, "alkenyl" refers to an unsaturated branched or straight chain hydrocarbon group having one or more carbon-carbon double bonds, which is obtained by removing a molecule of hydrogen from the adjacent carbon atoms of the parent alkyl group. The group can be in the cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to, vinyl; propenyl, such as prop-1-ene-1-yl, prop-1-ene-2-yl, prop-2-ene-1-yl (allyl), prop-2-ene-2-yl; butenyl, such as but-1-ene-1-yl, but-1-ene-2-yl, 2-methylprop-1-ene-1-yl, but-2-ene-1-yl, but-2-ene-2-yl, but-1,3-diene-1-yl, but-1,3-diene-2-yl, etc. In some embodiments, the alkenyl group has 2 to 20 carbon atoms, and in other embodiments, has 2 to 10, 2 to 8 or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl and refers to residues identical to alkenyl but with two points of attachment.

[0029] As used in the present disclosure, "alkynyl" refers to an unsaturated branched or straight chain hydrocarbon radical having one or more carbon-carbon triple bonds, which are obtained by removing two molecules of hydrogen from adjacent carbon atoms of a parent alkyl group. Typical alkynyl groups include, but are not limited to, ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc. In certain embodiments, the alkynyl group has 2 to 20 carbon atoms, and in other embodiments, has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl and refers to residues that are identical to alkynyl, but with two points of attachment.

[0030] As used in the present disclosure, "heterocyclyl" refers to a stable 3- to 18-membered non-aromatic ring radical containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen and sulfur. Unless otherwise specified in the specification, a heterocyclyl is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include a fused ring or a bridged ring system. The heteroatoms in the heterocyclyl may be oxidized heteroatoms. One or more nitrogen atoms (if present) may be quaternized nitrogen atoms. The heterocyclyl is partially saturated or fully saturated. The heterocyclyl may be connected to the rest of the molecule through any ring atom. Examples of such heterocyclic groups include, but are not limited to, dioxanyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxapyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Heterocyclylene is a subset of heterocyclyl and refers to residues identical to heterocyclyl but with two points of attachment.

[0031] As used in the present disclosure, "aryl" refers to a radical derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing hydrogen atoms from ring carbon atoms. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon of 6 to 18 carbon atoms, wherein 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 includes, but is not limited to, radicals such as phenyl, fluorenyl, and naphthyl. Arylene is a subset of aryl and refers to residues identical to aryl but having two points of attachment.

[0032] "Heteroaryl" refers to a group derived from a 3- to 18-membered aromatic ring radical, containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. As used in the present disclosure, a heteroaryl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein 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. Heteroaryl groups include fused or bridged ring systems. The heteroatoms in a heteroaryl group can be oxidized heteroatoms. One or more nitrogen atoms, if present, can be quaternized nitrogen atoms. The heteroaryl group is attached to the rest of the molecule through any ring atom. Examples of heteroaryl groups include, but are not limited to, azepine, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxazolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzo[d]thiazolyl, benzo[b][1,4]oxadiazolyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzo[d]thiazolyl, benzo[b][1,4]oxazinyl ... naphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothiophenyl, benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5 ]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanone, furano[3,2-c]pyridinyl, 5,6,7,8 ,9,10-hexahydrocyclooctan[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocyclooctan[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocyclooctan[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, isoindolyl, dihydroindole, isoindolyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl (5,8-methano-5,6,7,8-tetrahydroquinazolinyl)8-tetrahydroquinazolinyl), naphthyl, 1,6-naphthyl-onyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[H]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d ]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl / thienyl. Heteroarylene is a subset of heteroaryl and refers to residues identical to heteroaryl but with two points of attachment.

[0033] Unless otherwise specified, in the chemical formulas in the context of the present disclosure, an atom or group enclosed by brackets "()" indicates that the atom or group is connected to the atom adjacent to the left side of the bracket outside the chain in the chemical formula, that is, the atom or group is not connected in series inside the atomic chain shown in the chemical formula. For example, -C(O)- represents a carbonyl group, -CH2O- represents a methyleneoxy group, -N(R1)- represents an imino group substituted with R1, and so on.

[0034] The term "subject", as used herein, refers to any animal, such as a mammal or marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, rabbits, sheep, rats, and poultry of any kind.

[0035] As used herein, "treatment" refers to an approach to obtaining beneficial or desired results, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. In addition, a therapeutic benefit is obtained by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

[0036] Application of conjugates

[0037] In one aspect, the present disclosure provides a use of a conjugate in the preparation of a drug for diagnosing or treating colorectal cancer, wherein the conjugate comprises one or more delivery groups and one or more functional groups, each of the delivery groups is independently connected to at least one of the functional groups or other delivery groups through at least one connecting group, and each functional group is independently connected to at least one of the delivery groups or at least one other functional group through at least one connecting group; each of the functional groups is independently one of a diagnostic agent group, a small molecule therapeutic agent group having a therapeutic effect on colorectal cancer, a functional oligonucleotide group having a therapeutic effect on colorectal cancer, and a delivery aid group; the delivery group is formed by removing one or more hydrogen atoms or one or more functional groups from an aptamer, the aptamer comprises a continuous nucleotide sequence, the group connecting two adjacent nucleotides is independently a phosphate group or a phosphate group having a modified group, each nucleotide is selected from one of modified or unmodified A, U, C or G, and the continuous nucleotide sequence has a sequence shown in formula (1):

[0038] 5'-T1-S1-N a -S2-N b -S3-N c -S4-T2-3' formula (1)

[0039] Wherein, T1 is a motif consisting of 1-3 nucleotides. The inventors have found that the presence of T1 enables the conjugate provided by the present disclosure to show a high colorectal cancer tumor targeting effect. In some embodiments, T1 consists of 2 nucleotides, in which case the conjugate provided by the present disclosure has a more excellent colorectal cancer tumor targeting ability. In some embodiments, T1 consists of 2 nucleotides and contains at least one C. In some embodiments, in the 5'-3' direction, T1 is CU, UC or AC.

[0040] T2 is a motif consisting of 0-15 nucleotides. The inventors have found that T2 with these nucleotide numbers and various nucleotide sequences does not significantly affect the colorectal cancer targeting ability of the conjugate provided by the present disclosure. In some embodiments, T2 consists of 0-10 nucleotides. In some embodiments, in the 5'-3' direction, T2 consists of 1-9 nucleotides starting from U, at which time, the conjugate provided by the present disclosure may have more excellent stability.

[0041] T2 does not contain a motif that is completely reverse complementary to T1. In the context of the present disclosure, "reverse complementarity" refers to the formation of hydrogen bonds between two nucleotide sequences or motifs according to the rules of nucleic acid base pairing, and each nucleotide of a nucleotide sequence or motif in the 5'-3' direction can form base pairing with each nucleotide of the other end nucleotide sequence or motif in the 3'-5' direction. In some embodiments, "reverse complementarity" includes one or more of AU, GC and UG complementarity.

[0042] S1 and S4 are each a motif consisting of 3-7 nucleotides, and S1 and S4 are of the same length and completely reverse complementary. The conjugate provided by the present disclosure having the above-mentioned S1 and S4 motifs has excellent stability and can target colorectal cancer tumor tissues and cells for a long time. In some embodiments, S1 and S4 are each composed of 3-5 nucleotides and have the same length. In some embodiments, in the reverse complement formed by S1 and S4, GC complementation accounts for more than 50% of the total complementary number. At this time, the conjugate provided by the present disclosure has further excellent stability and colorectal cancer tumor targeting ability. In some embodiments, in the 5'-3' direction, 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.

[0043] N a and N c Each is a motif consisting of 1-4 nucleotides, N a Each nucleotide in c Each nucleotide in N is not complementary, and N a and N c The total number of U in N a and N c More than 50% of the total number of nucleotides in the a and N c In some embodiments, N a and N c The sum of the number of nucleotides in is an integer from 2 to 4. In some embodiments, N aand N c The sum of the number of nucleotides in is 3 or 4, and the N a and N c The sum of the number of U in is 2 or 3. In some embodiments, in the 5'-3' direction, N a and / or N c It is U, UU, UC or CU.

[0044] S2 and S3 are each a motif consisting of 1-4 nucleotides, and S2 and S3 are of the same length and completely reverse complementary. By comprising the S2 and S3 motifs, the conjugate provided by the present disclosure shows good stability and excellent colorectal cancer tumor targeting ability. In some embodiments, S2 and S3 are each composed of 2-3 nucleotides and have the same length. In some embodiments, the reverse complement formed by S2 and S3 includes at least one GC complement, and the reverse complement has better stability. In some embodiments, in the 5'-3' direction, S2 is CA and S3 is UG, or S2 is AC and S3 is GU, or S2 is GCC and S3 is GGU.

[0045] N b is a motif consisting of 3-6 nucleotides, and N b The nucleotides at both ends are not complementary. Without being limited by theory, the above N b The delivery group of the motif can maintain a specific configuration in space, so that the conjugate provided by the present disclosure can stably and efficiently target colorectal cancer tumor tissues and cells. b In some embodiments, according to the 5'-3' direction, N b It is GACG, GACGU, GACCG, UACU, GUUG or GAUCU.

[0046] The inventors of the present disclosure unexpectedly discovered that the aptamer comprising the sequence shown in the above formula (1) can effectively target colorectal cancer tissue, so that the conjugate comprising a functional group that can have a diagnostic / therapeutic effect on colorectal cancer provided by the present disclosure can efficiently reach and enter colorectal cancer tumor tissue and / or cells, thereby allowing the diagnostic agent / therapeutic agent group to more efficiently and accurately exert the corresponding diagnostic / therapeutic effect in triple-negative breast cancer tissue and / or cells.

[0047] In some embodiments, in the conjugate provided by the present disclosure, the length of the continuous nucleotide sequence is 18-50 nucleotides, or 20-40 nucleotides, or 21-36 nucleotides, or 24-32 nucleotides. The delivery group formed by the aptamer with these continuous nucleotide sequence lengths can be more easily targeted to the tumor, and has a good balance in terms of synthesis cost and targeting effect.

[0048] In some embodiments, in the conjugate provided by 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:

[0049] 5'-CUGCUUCAGACGUGUUAGCUU-3'(SEQ ID NO:1)

[0050] Among them, according to the 5'-3' direction, T1 is CU, S1 is GCU, N a is U, S2 is CA, N b is GACG, S3 is UG, N c is UU, S4 is AGC, T2 is UU;

[0051] 5'-CUGAGUUCAGACGUGUUGCUCU-3'(SEQ ID NO:2)

[0052] Among them, according to the 5'-3' direction, T1 is CU, S1 is GAGU, N a is U, S2 is CA, N b is GACG, S3 is UG, N c is UU, S4 is GCUC, T2 is U;

[0053] 5'-UCUAUGGCUGCCGAUCUGGUCUCCAUGUACGU-3' (SEQ ID NO: 3).

[0054] Among them, according to the 5'-3' direction, T1 is CU, S1 is GAGU, N a is U, S2 is CA, N b is GACG, S3 is UG, N c is UU, S4 is GCUC, and T2 is U.

[0055] In some embodiments, the contiguous nucleotide sequence has the nucleotide sequence shown in SEQ ID NO:4:

[0056] 5'-N6GGAGUUCAN1N2N3N4UGN5GCUCN7-3'(SEQ ID NO:4),

[0057] Among them, N1, N2 and N3 are each independently one of A, U, C and G, N4 is U, C or G or a motif composed 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 composed of 1-15 nucleotides.

[0058] In the above nucleotide sequence, in the 5'-3' direction, T1 is the motif represented by N6, S1 is the motif represented by GGAGU, N a is U, S2 is CA, N b The motif N1N2N3N4 is composed of N1, N2, N3 and N4, S3 is UG, N c is the motif represented by N5, S4 is the motif composed of GCUC and the first nucleotide in N7, and T2 is the motif composed of the remaining nucleotides in N7.

[0059] The conjugate comprising the nucleotide sequence shown in the above SEQ ID NO:4 can be more effectively enriched in colorectal cancer tissues.

[0060] Experimental verification shows that in the nucleotide sequence shown in SEQ ID NO: 4, the above selection of N1, N2, N3 and N4 will not significantly affect the colorectal cancer tumor targeting ability of the conjugate provided by the present disclosure. In some embodiments, the motif N1N2N3N4 composed of N1, N2, N3 and N4 is one of GACG, GACGU, GACCG, UACU, GUUG or GAUCU, and the conjugate provided by the present disclosure containing these motifs has a higher colorectal cancer tumor-specific targeting effect.

[0061] In some embodiments, in the nucleotide sequence shown in SEQ ID NO: 4, N5 is U or does not exist. In this case, the conjugates provided by the present disclosure have excellent targeting effects on colorectal cancer tumors.

[0062] In some embodiments, the contiguous nucleotide sequence has a nucleotide sequence as shown in any one of SEQ ID NOs: 5-11:

[0063] 5'-CUGGAGUUCAGACGUGUUGCUCUU-3'(SEQ ID NO:5)

[0064] 5'-CUGGAGUUCAGACGUUGUGCUCUU-3'(SEQ ID NO:6)

[0065] 5'-CUGGAGUUCAGACCGUGUGCUCUU-3'(SEQ ID NO:7)

[0066] 5'-CUGGAGUUCAGACGUGUUGCUCU-3'(SEQ ID NO:8)

[0067] 5'-ACGGAGUUCAGACGUGUUGCUCUU-3'(SEQ ID NO:9)

[0068] 5'-CUGGAGUUCACUACUGUUGCUCUU-3'(SEQ ID NO:10)

[0069] 5'-CUGGAGUUCAGUUGUGUUGCUCUU-3' (SEQ ID NO: 11).

[0070] The conjugate provided by the present disclosure having the above nucleotide sequence shows a high degree of targeting effect on colorectal cancer tumors.

[0071] In some embodiments, motif N8 consists of 1-15 nucleotides. In some embodiments, N8 consists of 1-8 nucleotides.

[0072] In some embodiments, the presence of motif N8 makes the conjugate provided by the present disclosure more stable to the exonuclease in vivo, thereby being able to exert the targeting effect of colorectal cancer tumors for a longer period of time in vivo. In some embodiments, N8 can increase or maintain the targeting effect of the conjugate provided by the present disclosure on colorectal cancer tumors. Considering the balance between stability, targeting and synthesis efficiency, in some embodiments, motif N8 consists of 8 nucleotides. In some embodiments, in the 5'-3' direction, the nucleotide sequence of motif N8 is CCGAUCUC.

[0073] In some embodiments, the contiguous nucleotide sequence has the nucleotide sequence shown in any one of SEQ ID NOs: 12-14:

[0074] 5'-CUGGAGUUCAGACGUGUUGCUCUUCCGAUCUC-3'

[0075] (SEQ ID NO:12)

[0076] 5'-CUGGAGUUCAGACGUUGUGCUCUUCCGAUCUC-3'

[0077] (SEQ ID NO:13)

[0078] 5'-CUGGAGUUCAGACCGUGUGCUCUUCCGAUCUC-3'

[0079] (SEQ ID NO:14)

[0080] In the continuous nucleotide sequence, the terminal groups at the 5' end of the ribose of the 5' terminal nucleotide and the 3' end of the ribose of the 3' terminal nucleotide are independently hydroxyl groups or phosphate groups, and the selection of these terminal groups will not change the colorectal cancer tumor targeting ability of the conjugate provided by the present disclosure. In some embodiments, in the continuous nucleotide sequence, the terminal groups at the 5' end of the ribose of the 5' terminal nucleotide and the 3' end of the ribose of the 3' terminal nucleotide are both hydroxyl groups.

[0081] In the conjugates provided by the present disclosure, each nucleotide may be a modified or unmodified nucleotide. In general, the modification of the nucleotide may change the stability of the conjugates provided by the present disclosure and / or the targeting ability to colorectal cancer tumors. In some embodiments, at least one nucleotide in the conjugates provided by the present disclosure is a modified nucleotide. In some embodiments, at least one phosphate group in the conjugates provided by the present disclosure is a phosphate group with a modifying group.

[0082] The modification of nucleotides includes, but is not limited to, modification of sugars, modification of bases, and / or replacement of nucleotides with nucleotide analogs. In some embodiments, in the conjugates provided by 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'-deoxynucleotide, a base-modified nucleotide, and a nucleotide analog.

[0083] In the context of the present disclosure, "fluorinated modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and has a structure shown in the following formula (7). "Non-fluorinated modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group. In some embodiments, each non-fluorinated modified nucleotide is independently selected from one of the nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group.

[0084] The nucleotides formed by replacing the hydroxyl group at the 2' position of these ribose groups with non-fluorinated groups are well known to those skilled in the art, and these nucleotides can be 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.

[0085] 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'-deoxynucleotide (DNA) is as shown in formula (10):

[0086]

[0087] Those skilled in the art know various ways to modify the bases of nucleotides. In some embodiments, 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 modified uracil (U) of the base. In some embodiments, 2-methylcytosine is considered a modified cytosine (C) of the base.

[0088] Nucleotide analogs refer to groups that can replace nucleotides in nucleic acids but have structures different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxyribonucleotides. In some embodiments, nucleotide analogs can be isonucleotides, bridged nucleic acids (BNAs) or acyclic nucleotides.

[0089] BNA refers to a constrained or inaccessible nucleotide. BNA may contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endosugar condensed bridge structure. The bridge is usually incorporated into the 2'-, 4'-position of the ribose to provide a 2', 4'-BNA nucleotide. In some embodiments, BNA may be LNA, ENA, cET BNA, etc., wherein LNA is shown in formula (12), ENA is shown in formula (13), and cET BNA is shown in formula (14):

[0090]

[0091] Acyclic nucleotides are a type of nucleotides formed by opening the sugar ring of a nucleotide. In some embodiments, the acyclic nucleotide may be an unlocked nucleic acid (UNA), a glycerol nucleic acid (GNA), or a peptide nucleic acid (PNA), wherein UNA is as shown in formula (15), and GNA is as shown in formula (16):

[0092]

[0093] In the above formulae (15) and (16), R is selected from H, OH or alkoxy (O-alkyl).

[0094] Peptide nucleic acid is a type of nucleotide analog formed by replacing the glycoside-phosphate main chain with a polypeptide backbone. In some embodiments, the peptide nucleic acid can be a nucleotide analog formed by replacing the glycoside-phosphate unit with a 2-aminoethylglycine bond.

[0095] Isonucleotides are compounds formed by a change in the position of a base on the ribose ring of a nucleotide. In some embodiments, an isonucleotide may be a compound formed by a base moving from the 1'-position to the 2'-position or the 3'-position of the ribose ring, as shown in formula (17) or (18).

[0096] In the compounds of formula (17)-(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 a non-fluorine group as described above.

[0097]

[0098] In some embodiments, the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET, UNA and GNA. In some embodiments, each non-fluorinated modified nucleotide is a methoxy-modified nucleotide, and in the above and below, the methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.

[0099] In the above and below, "fluorinated modified nucleotide", "2'-fluorinated modified nucleotide", "nucleotide in which the 2'-hydroxyl group of the ribose group is substituted by fluorine" and "nucleotide having a 2'-fluorinated ribose group" have the same meaning, all referring to compounds having a structure shown in formula (7) formed by replacing the 2'-hydroxyl group of the nucleotide with fluorine; "methoxy-modified nucleotide", "2'-methoxy-modified nucleotide", "nucleotide in which the 2'-hydroxyl group of the ribose group is substituted by methoxy" and "nucleotide having a 2'-methoxyribose group" have the same meaning, all referring to compounds having a structure shown in formula (8) formed by replacing the 2'-hydroxyl group of the ribose group of the nucleotide with methoxy.

[0100] In some embodiments, each cytosine nucleotide in the continuous nucleotide sequence in the delivery group of the conjugate used in the application provided by the present disclosure is a fluorinated modified cytosine nucleotide, and / or each uracil nucleotide in the continuous nucleotide sequence is a fluorinated modified uracil nucleotide. In some embodiments, each nucleotide in the continuous nucleotide sequence in the delivery group of the conjugate used in the application provided by the present disclosure is a 2'-methoxy modified nucleotide. In some embodiments, one or more uracil nucleotides in the delivery group of the conjugate used in the application provided by the present disclosure have a modified base. .

[0101] The group connecting two adjacent nucleotides can be a phosphate group or a modified phosphate group. The modification of the phosphate group, for example, replaces 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 connecting two adjacent nucleotides in the delivery group used in the application provided by the present disclosure is a thiophosphate group. In some embodiments, at least one of the three groups connecting two adjacent nucleotides between the first four nucleotides at the 5' end of the delivery group used in the application provided by the present disclosure is a thiophosphate group. In some embodiments, at least two of the three groups connecting two adjacent nucleotides between the first four nucleotides at the 5' end of the delivery group used in the application provided by the present disclosure are thiophosphate groups. In some embodiments, at least one of the groups connecting two adjacent nucleotides between the first four nucleotides at the 3' end of the delivery group used in the application provided by the present disclosure is a thiophosphate group. In some embodiments, at least two of the three groups connecting two adjacent nucleotides between the first four nucleotides at the 5' end of the delivery group used in the application provided by the present disclosure are thiophosphate groups. In some embodiments, each group connecting two adjacent nucleotides in the delivery group in the conjugate used in the applications provided by the present disclosure is a phosphorothioate group.

[0102] The conjugate provided by the present disclosure with the above modification is not only low in cost, but also can make it difficult for ribonucleases in the body to cut the delivery group, thereby increasing the stability of the conjugate provided by the present disclosure, making it more resistant to nuclease hydrolysis. At the same time, the conjugate provided by the present disclosure with the above modification has a higher activity of targeting tumor tissues and / or cells.

[0103] In some embodiments, the conjugate provided by the present disclosure has a nucleotide sequence shown in one of SEQ ID NOs: 15-39: 5'-CfUfGGAGUfUfCfAGACfGUfGUfUfGCfUfCfUfUfCfCfGAUfCfUfCf-3'

[0104] (SEQ ID NO:15)

[0105] 5'-CfUfGGAGUfUfCfAGACfGUfGUfUfGCfUfCfUfUf-3' (SEQ ID NO: 16) 5'-CfUfGGAGUfUfCfAGACfGUfUfGUfGCfUfCfUfUfCfCfGAUfCfUfCf-3'

[0106] (SEQ ID NO:17)

[0107] 5'-CfUfGGAGUfUfCfAGACfGUfUfGUfGCfUfCfUfUf-3'(SEQ ID NO:18)5'-CfUfGGAGUfUfCfAGACfCfGUfGUfGCfUfCfUfUfCfCfGAUfCfUfCf-3'

[0108] (SEQ ID NO:19)

[0109] 5'-CfUfGGAGUfUfCfAGACfCfGUfGUfGCfUfCfUfUf-3'(SEQ ID NO:20)5'-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUmUmCmCmGmAmUmCmUmCm-3'(SEQ ID NO:21)

[0110] 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUmUm-3'(SEQ ID NO:22)

[0111] 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmUmGmUmGmCmUmCmUmUmCmCmGmAmUmCmUmCm-3'(SEQ ID NO:23)

[0112] 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmUmGmUmGmCmUmCmUmUm-3'(SEQ ID NO:24)

[0113] 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmCmGmUmGmUmGmCmUmCmUmUmCmCmGmAmUmCmUmCm-3'(SEQ ID NO:25)

[0114] 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmCmGmUmGmUmGmCmUmCmUmUm-3'(SEQ ID NO:26)

[0115] 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUm-3'(SEQ ID NO:27)

[0116] 5'-CmUmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUm-3'(SEQ ID NO:28)

[0117] 5'-CmUmGmCmUmUmCmAmGmAmCmGmUmGmUmUmAmGmCmUmUm-3'(SEQ ID NO:29)

[0118] 5'-AmCmGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUmUm-3'(SEQ ID NO:30)

[0119] 5'-CmUmGmGmAmGmUmUmCmAmCmUmAmCmUmGmUmUmGmCmUmCmUmUm-3'(SEQ ID NO:31)

[0120] 5'-CmUmGmGmAmGmUmUmCmAmGmUmUmGmUmGmUmUmGmCmUmCm UmUm-3'(SEQ ID NO:32)

[0121] 5'-UmCmUmAmUmGmGmCmUmGmCmCmGmAmUmCmUmGmGmUmCmUm CmCmAmUmGmUmAmCmGmUm-3'(SEQ ID NO:33)

[0122] 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUmUmCmCmGmAmUmCmsUmsCm-3'(SEQ ID NO:34)

[0123] 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmC msUmsUm-3'(SEQ IDNO:35)

[0124] 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmGmUmUmGmUmGmCmUmCmUmUmCmCmGmAmUmCmsUmsCm-3'(SEQ ID NO:36)

[0125] 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmGmUmUmGmUmGmCmUmC msUmsUm-3'(SEQ IDNO:37)

[0126] 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmCmGmUmGmUmGmCmUmCmUmUmCmCmGmAmUmCmsUmsCm-3' (SEQ ID NO: 38)

[0127] 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmCmGmUmGmUmGmCmUmC msUmsUm-3' (SEQ IDNO: 39)

[0128] Among them, capital letters C, G, U, and A represent the base composition of the nucleotide; the lowercase letter m indicates that the nucleotide represented by the capital letter adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide represented by the capital letter adjacent to the left of the letter f is a fluorine-modified nucleotide; the lowercase letter s indicates that the nucleotides represented by the two capital letters on the left and right of the letter s are connected by thiophosphate groups.

[0129] The aptamers described in the present disclosure can be obtained by conventional oligonucleotide preparation methods in the art (such as solid phase synthesis and liquid phase synthesis methods). Among them, solid phase synthesis already has commercial customization services. The modified nucleotide group can be introduced into the delivery group in the conjugate used in the application provided by the present disclosure by using a nucleoside monomer with a corresponding modification. The method for preparing a nucleoside monomer with a corresponding modification and the method for introducing the modified nucleotide group into the aptamer are also well known to those skilled in the art. All modified nucleoside monomers can be purchased commercially or prepared by known methods. In some embodiments, the conjugate provided by the present disclosure has a structure shown in formula (2):

[0130]

[0131] Among them, each R AP The groups are independently groups having a structure as shown in formula (3):

[0132]

[0133] In the formula, each AP group is the same or different and independently represents a delivery group; each A0 group is the same or different and independently represents a delivery group or a functional group, and at least one A0 is a delivery group and at least one A0 is a functional group; R j , each R k or each R i are the same or different, and each independently represents a covalent bond or a linking group, and R i and R kThe two are not covalent bonds at the same time; m0 is an integer of 1-6; n0 is an integer of 1-6, and each n1 independently represents an integer of 0-4; Indicates the site to which a group is covalently attached.

[0134] In some embodiments, m0 is an integer of 1-6, that is, the conjugate shown in formula (2) contains 1-6 A0 groups. From the perspective of delivery efficiency and synthesis cost, in some embodiments, m0 is an integer of 1-4, that is, the conjugate shown in formula (2) contains 1-4 A0 groups. In some embodiments, m0 is 1, that is, the conjugate shown in formula (2) contains 1 A0 group.

[0135] In some embodiments, n0 is an integer of 1-6, that is, the conjugate represented by formula (2) contains 1-6 R AP From the perspective of delivery efficiency and synthesis cost, in some embodiments, n0 is an integer of 1-3, that is, the conjugate shown in formula (2) contains 1-3 R AP In some embodiments, n0 is 1, that is, the conjugate represented by formula (2) contains 1 R AP Group.

[0136] In some embodiments, each n1 independently represents an integer from 0 to 4, and R i and R k The two are not covalent bonds at the same time, so each R AP The group contains 1-5 delivery groups AP obtained from the aptamer disclosed in the present invention. In some embodiments, each n1 independently represents an integer from 0 to 1, so that each R AP The group contains 1-2 delivery groups AP. In some embodiments, n0 is 1 and n1 is 0. In this case, the conjugate represented by formula (2) contains 1 delivery group AP.

[0137] R AP In the group, R k and R i The role of AP is to covalently link the delivery group to R j group, and through R j Therefore, as long as the above connection can be achieved and the delivery group A P and the effect of the A0 group negatively affects any R k and / or R i In some embodiments, each of the R k and / or each of the R iThe straight chain alkylene group is independently a straight chain alkylene group with a length of 1 to 70 carbon atoms, or one or more carbon atoms in the straight chain alkylene group are replaced by one or more selected from the group consisting of the following groups: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, OP(O)(S), C5-C8 glycoside group, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 wherein the linear alkylene group may have any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl).

[0138] Considering the cost and difficulty of synthesis and the tumor targeting effect of the conjugate, in some embodiments, each n1 is independently 0 or 1, and each R i is independently a combination of one or more of the following linking groups: C1-C 20 alkylene, -OP(O)2-group, thiophosphate group (-OP(O)(S)-), -CONH group, -O-group, -S-group, -C(O)-O-group, -SS-group, 1,2,3-triazole subunit, polyethylene glycol subunit, pyrrolidine subunit, 2-oxopyrrolidine subunit, phenylene, cyclohexylene, 2-succinimide subunit, 2-thiosuccinimide subunit, amino acid subunit, nucleotide subunit. In some embodiments, each R i The linkages are independently one or a combination of two of a covalent bond, a disulfide bond, a dodecylene bond or a GAU trinucleotide subunit.

[0139] R j The role of the group is to AP The group is connected to the A0 group, thereby AP The tumor targeting effect of the delivery group AP in the group specifically delivers the A0 group A0 to tumor tissues and / or cells. Therefore, any R that can achieve the above connection and does not affect the tumor targeting effect of the delivery group AP and the effect of the A0 group j In some embodiments, after the conjugate represented by formula (2) reaches the tumor tissue and / or enters the tumor cell, the R j The cleavage occurs to release the functional molecule corresponding to the separate A0 group. j No cleavage occurs in vivo. j Group and R AP The presence of the group does not affect the diagnostic and / or therapeutic effects of the functional group.

[0140] In some embodiments, R j is a covalent bond, m0 is 1, and in this case, the conjugate shown in formula (2) contains 1 A0 group A0 and n0 R AP Group, each R AP The group is directly connected to the A0 group. AP The group is attached to the same atom of the A0 group. AP The groups are attached to different atoms of the A0 group.

[0141] In some embodiments, R j is a connecting group, the connecting group R j It comprises a main chain part, a side chain part and a conjugated linker part.

[0142] The main chain part is connected to the conjugated linker and the side chain part respectively. In some embodiments, the main chain part is a straight chain alkylene with a length of 1-70 carbon atoms, or one or more carbon atoms in the straight chain alkylene are replaced by one or more selected from the group consisting of the following groups: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycoside group, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 wherein the linear alkylene group may have any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl).

[0143] The side chain parts are respectively connected to the main chain part and R AP In some embodiments, each side chain portion is independently a covalent bond, or a straight chain alkylene group with a length of 1-70 carbon atoms, or one or more carbon atoms in the straight chain alkylene group are replaced by one or more selected from the group consisting of the following groups: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycoside group, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 and the linear alkylene group may have any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl);

[0144] The conjugated linker is connected to the main chain part and the A0 group respectively. In some embodiments, each conjugated linker is independently a covalent bond or a combination of one or more of the following connection structures: C1-C10 Straight-chain alkylene group, phosphate group, thiophosphate group, -CONH group, -O- group, -S- group, -C(O)-O- group, -SS- group, 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, nucleotide subunit.

[0145] In some embodiments, the linking group R j The present invention comprises a linking group known to those skilled in the art that can be used for antibody-drug coupling. j Can be cleavable or non-cleavable. In some embodiments, the linking group R j In this context, "cleavable" means that after the conjugate of the present disclosure is targeted to the tumor, the linker R j In the tumor environment and / or in tumor cells, the covalent bond breaks, releasing the individual functional groups to produce diagnostic and / or therapeutic effects. j The invention comprises one or more of an activating enzyme linking group, a sulfatase-cleavable linking group, a galactose-cleavable linking group, a linking group sensitive to lysosomal proteases, a peptidyl linking group, a glucuronide linking group, an acid-sensitive cleavable linking group, or a disulfide linking group sensitive to glutathione. j In some embodiments, the peptidyl linking group is selected from one or more of a valine-citrulline dipeptide linker (Val-Cit), an alanine-alanine dipeptide linker (Ala-Ala), a valine-alanine dipeptide linker (Val-Ala), and a glycine-glycine-phenylalanine-glycine tetrapeptide linker (Gly-Gly-Phc-Gly). In some embodiments, the linking group R j is selected from N-succinimidyl 4-(2-dithiopyridinyl)butyrate (SPDB), N-succinimidyl-4-(2-thiopyridinyl)valerate (SPP), (S)-2-((S)-2-amino-3-methylbutyramide)-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 jThe linking groups include those listed in Mclertish CM, Kayser V. Advances and Limitations of Antibody Drug Conjugates for Cancer. Biomedicines. 2021 Jul 23; 9 (8): 872., the entire contents of which are incorporated herein by reference. In some embodiments, the linking group R j It is not cleavable.

[0146] In some embodiments, the linking group R j The invention comprises one or more of a valine-citrulline dipeptide linker (Val-Cit), a polyethylene glycol subunit, an iminohexyl subunit, an N-succinimidyl group, and a GAU trinucleotide linker. In some embodiments, each R i The linkages are independently one or a combination of two of a covalent bond, a disulfide bond, a dodecylene group, a valine-citrulline dipeptide linker (Val-Cit), a polyethylene glycol subunit, an imidohexyl subunit, an N-succinimide group, or a GAU trinucleotide subunit.

[0147] In some embodiments, the linking group R j Each of the conjugated linking parts is respectively connected to the main chain part and one of the A0 groups; the side chain parts are n0, each of which is respectively connected to the main chain part and one of the R AP Thus, each A0 group and R AP The groups are each independently connected to the linking group R j In some embodiments, all of the side chain moieties are attached to the same atom in the backbone moiety; alternatively, each side chain moiety is attached to a different atom in the backbone moiety.

[0148] In some embodiments, m0 is 1, the linking group R j Contains the structure shown in formula (301):

[0149]

[0150] Wherein, k is an integer from 1 to 3; L C is the main chain part, L A is the side chain part, L B is the conjugated linker, Indicates the site to which a group is covalently attached.

[0151] The main chain part L C It is a covalent bond or a 2-4 valent, straight or branched C1-C 25A saturated hydrocarbon group, or one or more carbon atoms in the saturated hydrocarbon group are replaced by one or more selected from the group consisting of the following groups: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycosidyl, C2-C5 alkenyl, C2-C5 alkynyl, C6-C 10 Arylene, C3-C8 heterocyclylene and C5-C 10 wherein the saturated hydrocarbon group may have any one or more substituents selected from the group consisting of: C1-C5 alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl, -O-C1-C5 alkyl, -OC1-C5 alkylphenyl, -C1-C5 alkyl-OH, -SC1-C5 alkyl, nitro, -C(O)O(C1-C5 alkyl), -CON(C1-C5 alkyl)(C1-C5 alkyl), -CONH(C1-C5 alkyl), -CONH2, -NHC(O)(C1-C5 alkyl), -NHC(O)(phenyl), -N(C1-C5 alkyl)C(O) (C1-C5 alkyl), -N(C1-C5 alkyl)C(O)(phenyl), -C(O)C1-C5 alkyl, -C(O)C1-C5 alkylphenyl, -OC(O)C1-C5 alkyl, -SO2(C1-C5 alkyl), -SO2(phenyl), -SO2NH2, -SO2NH(C1-C5 alkyl), -SO2NH(phenyl), -NHSO2(C1-C5 alkyl) and -NHSO2(phenyl).

[0152] In some embodiments, L C C5-C 20 A saturated hydrocarbon group, or one or more carbon atoms in the saturated hydrocarbon group are replaced by one or more selected from the group consisting of the following groups: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycosidyl, C2-C5 alkenyl, C2-C5 alkynyl, C6-C 10 Arylene, C3-C8 heterocyclylene and C5-C 10 wherein the saturated hydrocarbon group may have any one or more substituents selected from the group consisting of: C1-C5 alkyl, C6-C 10 Aryl, C5-C 10 In some embodiments, L C The length of L is 5-30 atoms, wherein the L C The length is L C Zhong and LA Directly connected to the atom with L B The number of atoms in the longest chain of atoms formed by directly connected atoms. C The length of the molecule is 8-25 atoms.

[0153] The side chain part L A is a covalent bond, or C1-C 20 Alkylene, or one or more carbon atoms in the alkylene are replaced by one or more selected from the group consisting of the following groups: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycosidylene, C2-C5 alkenylene, C2-C5 alkynylene, C6-C 10 Arylene, C3-C8 heterocyclylene and C5-C 10 wherein the alkylene group may have any one or more substituents selected from the group consisting of: C1-C5 alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl, -O-C1-C5 alkyl, -OC1-C5 alkylphenyl, -C1-C5 alkyl-OH-SC1-C5 alkyl, -SC1-C5 alkylphenyl, -C1-C5 alkyl-SH, -OH, -SH, -NH2, -C1-C5 alkyl-NH2, -N(C1-C5 alkyl)(C1-C5 alkyl), -NH(C1-C5 alkyl), -N(C1-C5 alkyl)(C1-C5 alkylphenyl), -NH(C1-C5 alkylphenyl), nitro, -C(O)O(C1-C5 alkyl), -CON(C1-C5 alkyl)(C1-C5 alkyl), -CONH (C1-C5 alkyl), -CONH2, -NHC(O)(C1-C5 alkyl), -NHC(O)(phenyl), -N(C1-C5 alkyl)C(O)(C1-C5 alkyl), -N(C1-C5 alkyl)C(O)(phenyl), -C(O)C1-C5 alkyl, -C(O)C1-C5 alkylphenyl, -OC(O)C1-C5 alkyl, -SO2(C1-C5 alkyl), -SO2(phenyl), -SO2NH2, -SO2NH(C1-C5 alkyl), -SO2NH(phenyl), -NHSO2(C1-C5 alkyl) and -NHSO2(phenyl).

[0154] The conjugated linker L B It is a combination of one or more of 1-5 of the following connecting bonds: phosphate bond, phosphorothioate bond, amide bond, ester bond, ether bond, and disulfide bond.

[0155] In some embodiments, k is an integer from 1 to 3; L CContains any one of the groups shown in formula (L1)-(L3), and L is bonded to the group shown in formula (L1)-(L3) through an ether bond. A Partial connection:

[0156]

[0157] It indicates the site at which a group is attached to the rest of the molecule;

[0158] In some embodiments, k=1, L C Contains a group as shown in formula (L1), the O atom and L in the group (L1) A In some embodiments, k=2, L C Containing a group as shown in formula (L2), the two O atoms in the group (L1) each have one L A In some embodiments, k=4, L C Contains a group as shown in formula (L3), wherein the three O atoms in the group (L3) are each A Directly connected.

[0159] L B is a phosphate bond or a disulfide bond;

[0160] Each L A is a covalent bond, or each L A Selected from the group consisting of groups (L4)-(L23) and their connected combinations:

[0161]

[0162] In the formula, each j1 is an integer from 1 to 10;

[0163] Each R' is C1-C 10 alkyl;

[0164] Each Ra is a hydrogen atom, C1-C 10 Alkyl, or selected from the group consisting of radicals (L24)-(L37):

[0165]

[0166] In some embodiments, L A The length of L is 3-35 atoms, wherein the L A The length is L A Zhong and L C Directly connected to the atom with L A Middle and R AP The number of atoms in the longest chain of atoms formed by directly connected atoms.A is a combination of at least two of the groups (L4)-(L9), (L13), (L14), and (L18). A It is a combination of at least two of the groups (L4), (L5), (L7), (L9), (L13), (L14), and (L18).

[0167] In some embodiments, L A has a structure including an amide bond as shown in formula (302), L B It has a structure containing N-acylpyrrolidine as shown in formula (303), containing a carbonyl group and an oxygen atom, L C is a linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trishydroxymethylaminomethane:

[0168]

[0169] Among them, n 302 ,q 302 and p 302 Each independently represents an integer from 2 to 6, optionally, n 302 ,q 302 and p 302 Each independently is 2 or 3; n 303 An integer between 4 and 16. Optionally, n 303 is an integer between 8 and 12, Indicates the site to which a group is covalently attached.

[0170] In some embodiments, each of the side chain moieties L A With an R AP The groups are connected by phosphate bonds, ether bonds or ester bonds and by the main chain part L C The oxygen atom of the hydroxyl group in the main chain L C The conjugated linker L B Through the carbonyl group in formula (303) and the main chain part L C The nitrogen atom of the amino group in the formula (303) forms an amide bond to connect, and the oxygen atom in the formula (303) forms a phosphate bond, an ether bond or an ester bond to connect with the A0 group. In some embodiments, the main chain portion L C is a linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trihydroxymethylaminomethane, the main chain part L C Through the oxygen atom of the hydroxyl group and each of the side chain parts L A The conjugated linker L is connected via an ether bond and via the nitrogen atom of the amino group. B The connecting group R j1-3 side chains are connected to the carbon atom of the same aminomethyl group and are conjugated to the linker L B Connected to R containing a delivery group AP Group.

[0171] In some embodiments, the conjugate has a structure as shown in formula (305):

[0172]

[0173] In some embodiments, the linking group R j Contains the structure shown in formula (306):

[0174]

[0175] Among them, n 306 is an integer from 0 to 3, each p 306 are independently an integer from 1 to 6, Indicates the site of covalent attachment of a group; the structure formed by alternating pyrrolidine subunits and phosphodiester groups constitutes the main chain part, and each side chain part is composed of an atomic chain between a carbonyl group connected to a nitrogen atom on the pyrrolidine subunit and an oxygen atom marked by *, and the side chain part is connected to R through an oxygen atom marked by *. AP The groups form an ether bond connection; at least one of the oxygen atoms marked by # is a conjugated linker and forms an ether bond, ester bond or phosphate bond with the A0 group, and the remaining oxygen atoms marked by # are connected to hydrogen atoms to form a hydroxyl group, or are connected to a C1-C3 alkyl group to form a C1-C3 alkoxy group. j 1-3 side chain moieties are attached to different carbon atoms in the main chain moiety and are connected to the R containing the delivery group through an oxygen atom. AP Group.

[0176] In some embodiments, the compound has a structure as shown in Formula (307a), (307b) or (307c):

[0177]

[0178] In some embodiments, the compound has a structure as shown in formula (308):

[0179]

[0180] Among them, n 308 It can be an integer of 1-10. In some embodiments, considering the ease of synthesis, structure / process cost and tumor cell specificity, n 308 is an integer from 2 to 6. In some embodiments, n 308 Is 3 or 4.

[0181] Each R3 is independently an A0 group, or an R containing a delivery group AP AP In some embodiments, at least one R3 is an A0 group, and at least one R3 is R AP In some embodiments, one R3 is an A0 group, and the remaining R3 is R AP Group.

[0182] In some embodiments, when each m 308 When independently selected from integers of 2-10, it is believed that the spatial positions between the multiple delivery groups AP in the compound may be more suitable for interacting with the corresponding receptors on the surface of tumor cells. In order to make the compound represented by formula (308) simpler, easier to synthesize and / or reduce the cost, according to some embodiments of the present disclosure, each m 308 is independently an integer of 2-5. In some embodiments, each m 308 Are equal.

[0183] Those skilled in the art will appreciate that when each R 308 Independently selected from H, C1-C 10 Alkyl, C1-C 10 Haloalkyl and C1-C 10 When each R is alkoxy, the properties of the compound represented by formula (308) are not changed, and the purpose of the present disclosure can be achieved. 308 is independently selected from H, methyl or ethyl. In some embodiments, each R 308 Both are H.

[0184] Each L1 connected to the A0 group represents the conjugated linker and is connected to the R AP Each L1 represents the side chain portion. In some embodiments, one R3 is the A0 group, and the remaining R3 is the R AP In some embodiments, one or more L1 is used as the side chain part, and R AP The group is connected to the N atom on the nitrogen-containing skeleton; and the other one or more L1s serve as the conjugated linker to connect the A0 group to the N atom on the nitrogen-containing skeleton. The nitrogen-containing skeleton together constitutes the connecting group R j In the context of the present disclosure, "nitrogen-containing skeleton" refers to the chain structure in the structure shown in formula (308), in which R 308 The carbon atoms and N atoms are connected to each other.

[0185] From the perspective of delivery efficiency and synthesis cost, in some embodiments, the length of each L1 is independently 3-25 atoms. In some embodiments, the length of each L1 is independently 4-15 atoms. It will be understood by those skilled in the art that although L1 is defined as a linear alkylene group for convenience, it may not be a linear group or the name is different, such as an amine or alkenyl group produced by the above-mentioned replacement and / or substitution. For the purpose of this disclosure, the length of L1 is the number of atoms in the chain connecting the two connection points. For this purpose, the ring (such as a heterocyclyl or heteroaryl group) obtained by replacing the carbon atoms of the straight-chain alkylene group is calculated according to the minimum number of atoms between the connection points on the ring.

[0186] In some embodiments, L1 is selected from the group consisting of the groups represented by the above formulas L4-L23 and any combination thereof. In some embodiments, each L1 is independently selected from the group consisting of a combination of at least two of the groups (L4)-(L9), (L13), (L14), (L18). In some embodiments, each L1 is independently a combination of at least two of the groups (L4), (L5), (L7), (L9), (L13), (L14), (L18).

[0187] In some embodiments, in the conjugate of formula (308), each L1 contains both a connection site for connecting to the N atom on the nitrogen-containing skeleton and a connection site for connecting to the A0 group or the R AP In some embodiments, one or more L1s are 4-15 atoms in length. In some embodiments, one or more L1s are selected from B5, B6, B5' or B6':

[0188]

[0189] in, 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.

[0190] In some embodiments, each R AP The group contains one or more delivery groups. In some embodiments, the compound represented by formula (308) contains 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 used for the same purpose and function. In some embodiments, the compound of formula (308) contains different types of functional groups used for different purposes and functions.

[0191] In some embodiments, the compound represented by formula (308) has a structure represented by 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):

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201] In some embodiments, the linking group R jThe invention comprises a nucleotide sequence I and a nucleotide sequence II, wherein each of the nucleotide sequence I and the nucleotide sequence II comprises 5-25 modified or unmodified nucleotides, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, the delivery group is connected to the nucleotide sequence I, the functional group is connected to the nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II do not induce an immune response or a toxic response in the 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 are both 10-20 modified or unmodified nucleotides; or, the nucleotide sequence I and the nucleotide sequence II are both composed of 17 nucleotides and are completely reverse complementary. In some embodiments, the 3' end of the delivery group is linked to the 5' position of the ribose of the 5' terminal nucleotide of the nucleotide sequence I via a phosphate bond, and the functional group is linked to the 5' position of the ribose of the 5' terminal nucleotide of the nucleotide sequence II; or, the functional group comprises a nucleotide sequence, and the 3' end of the nucleotide sequence is linked to the 5' position of the ribose of the 5' terminal nucleotide of the nucleotide sequence I via a phosphate bond. 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:

[0202] 5'-GUACAUUCUAGAUAGCC-3'(SEQ ID NO:40)

[0203] 5'-GGCUAUCUAGAAUGUAC-3' (SEQ ID NO: 41).

[0204] 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:

[0205] 5'-GmUfAmCfAmUfUfCfUfAmGmAmUfAmGmCfCf-3'

[0206] (SEQ ID NO:42)

[0207] 5'-GmGmCfUfAmUfCfUfAmGmAmAmUfGmUfAmCf-3'

[0208] (SEQ ID NO:43).

[0209] The conjugates provided by the present disclosure may include one or more functional groups. In the context of the present disclosure, the functional group refers to a group containing a free radical formed by removing one or more atoms (e.g., removing a hydrogen atom) from an active agent molecule with corresponding functionality (e.g., a diagnostic agent compound, a small molecule therapeutic agent group compound with a therapeutic effect on colorectal cancer, and / or a functional oligonucleotide compound that regulates the expression level of cancer-related genes in tumor cells), and the functional group forms a covalent connection with the other parts of the molecule of the conjugate provided by the present disclosure through the free radical.

[0210] In some embodiments, at least one functional group in the conjugate provided by the present disclosure is a diagnostic agent group, and each of the functional groups is independently selected from a contrast agent group or a fluorescent label tracer group. In some embodiments, all functional groups in the conjugate provided by the present disclosure are the above-mentioned diagnostic agent groups. By including functional groups, the conjugate provided by the present disclosure can deliver the diagnostic agent group to colorectal cancer tumor cells / tissues, thereby specifically, efficiently and accurately diagnosing the presence of tumor cells / tissues and related diseases and / or symptom information such as the disease progression state. In some embodiments, the diagnostic agent group can be used to diagnose tumor cells / tissues in samples to be tested in in vitro experiments. In some embodiments, the diagnostic agent group can be used to diagnose the presence and / or properties of colorectal cancer tumor cells / tissues in a subject.

[0211] Contrast agent groups suitable for different imaging techniques can be independently selected from groups formed by one or more of the following contrast agents known in the art: computed tomography (CT) contrast agents, magnetic resonance tomography (MRI) contrast agents, magnetic particle imaging contrast agents, and ultrasound (US) contrast agents. In some embodiments, at least one contrast agent group in the specific contrast agent compound of the present disclosure is a contrast agent group formed by an MRI contrast agent. In some embodiments, all contrast agent groups in the conjugates provided by the present disclosure are contrast agent groups formed by MRI contrast agents.

[0212] The contrast agent groups of different types of compounds can be, for example, any iodine-containing group known in the art, a chelating group and a metal ion chelated with the chelating group, a surface modification group of surface-modified iron particles, or a group formed by a perfluorinated compound.

[0213] In some embodiments, the computed tomography (CT) contrast agent can be selected from iodine titanate, iohexol, diatrizoate, iopamidol, ethiodized oil, or iopanoate.

[0214] In some embodiments, the magnetic resonance tomography (MRI) contrast agent or magnetic particle imaging contrast agent can be selected from gadolinium chelates, manganese chelates, chromium chelates or iron particles.

[0215] In some embodiments, the ultrasound (US) contrast agent may be selected from perfluorinated compounds or perfluorinated compound analogs.

[0216] In some embodiments, the fluorescently labeled tracer group (also referred to as a fluorescent imaging group or fluorescent group in the art) can be a group formed by a fluorescently labeled tracer, and the fluorescently labeled tracer can be selected from fluorescein, fluorescein derivatives, indocyanine green, Oregon green, Oregon green derivatives, rhodamine green, rhodamine green derivatives, eosin, erythrosine, Texas red, Texas red derivatives, malachite green, nano-gold sulfosuccinimidyl esters, cascade blue, coumarin derivatives, naphthalene, pyridyl oxazole derivatives, cascade yellow dyes or dapoxyl dyes. In some embodiments, the fluorescently labeled tracer group is a Cy5 (Cyanine 5) fluorescent dye group or a Cy3 (Cyanine 3) fluorescent dye group.

[0217] The diagnostic agent group can be included in the conjugate provided by the present disclosure in any suitable manner. For example, the diagnostic agent group A0 can be connected to the main chain part through the aforementioned conjugation linker.

[0218] In some embodiments, the conjugate provided by the present disclosure further comprises at least one delivery aid group, wherein the delivery aid group is selected from C 10 -C 30 One or more of a hydrocarbon group, a cholesterol group, and a phospholipid group. By including the delivery aid group, the conjugate provided by the present disclosure can be better compatible with the in vivo environment in the central nervous system, may have better bioavailability, and / or enable the conjugate provided by the present disclosure to be more effectively delivered to a tumor. In some embodiments, the delivery aid group is connected to the delivery group or the connecting group via a covalent bond or a connecting group. In some embodiments, the delivery aid group is connected to the functional group.

[0219] In some embodiments, at least one functional group in the conjugate provided by the present disclosure is a small molecule therapeutic agent group that has a therapeutic effect on colorectal cancer, and each of the small molecule therapeutic agent groups is independently selected from a cytotoxic group, an antibiotic group, an angiogenesis inhibitor or an antibody drug group. In some embodiments, at least one or all of the small molecule therapeutic agent groups are cytotoxic groups formed by removing one or more atoms from a cytotoxic molecule, and the cytotoxic molecule is selected from one or more of the following: microtubule inhibitors (such as auristatins, maytansinoids), DNA damaging agents (such as calicheamicin, duocarmycins, anthracyclines, pyrrolobenzodiazepinedimers) and DNA transcription inhibitors (Amatoxin and Quinolinalkaloid (SN-38)). In some embodiments, all functional groups in the conjugate provided by the present disclosure are the above-mentioned small molecule therapeutic agent groups. By including the above-mentioned small molecule therapeutic agent group, the conjugate provided by the present disclosure can specifically deliver the small molecule therapeutic agent group to colorectal cancer tissue, thereby treating and / or alleviating the disease process or symptoms of colorectal cancer through the action of the small molecule therapeutic agent group. For example, the cytotoxin group is specifically delivered to colorectal cancer tissue through the conjugate provided by the present disclosure, so that the cancer cells are specifically eliminated, thereby reducing the side effects caused by the low targeting of the cytotoxin itself, and significantly reducing the number of cancer cells, thereby treating colorectal cancer. In some embodiments, at least one functional group in the conjugate provided by the present disclosure is a functional oligonucleotide group, and the functional oligonucleotide group can regulate the expression level of cancer-related genes in tumor cells. In some embodiments, all functional groups in the conjugate provided by the present disclosure are the above-mentioned functional oligonucleotide groups. By including the functional oligonucleotide group, the conjugate provided by the present disclosure can specifically deliver the functional oligonucleotide group to the tumor, thereby regulating the expression level of cancer-related genes in tumor cells through the action of the functional oligonucleotide group, such as the RNAi effect, such as inhibiting the expression of oncogenes, thereby treating and / or alleviating the disease process or symptoms related to the tumor. In some embodiments, the functional oligonucleotide is siRNA. In some embodiments, the functional oligonucleotide is siRNA targeting STAT3mRNA, RRM2mRNA or PLK1.In some embodiments, the functional oligonucleotide is one or more of the siRNAs having a sense chain as shown in SEQ ID NO:44 and an antisense chain as shown in SEQ ID NO:45, or a siRNA having a sense chain as shown in SEQ ID NO:46 and an antisense chain as shown in SEQ ID NO:47, or a siRNA having a sense chain as shown in SEQ ID NO:48 and an antisense chain as shown in SEQ ID NO:49: 5'-CmsUmsAmGmAmAmAfAfCfUmGmGmAmUmAmAmCmGmUm-3'.

[0220] (SEQ ID NO:44)

[0221] 5'-AmsCfsGmUmUmAfUmCmCmAmGmUmUmUfUmCfUmAmGmsCmsCm-3'

[0222] (SEQ ID NO:45)

[0223] 5'-CUUCUUAUUGACACUUACAdT-S-dT-3'(SEQ ID NO:46)

[0224] 5'-UGUAAGUGUCAUAAGAAGdT-S-dT-3'(SEQ ID NO:47)

[0225] 5'-CAAGAAGAAUGAAUACAGUdT-S-dT-3'(SEQ ID NO:48)

[0226] 5'-ACUGUAUUCAUUCUUCUUGdT-S-dT-3'(SEQ ID NO:49)

[0227] The functional group can be included in the conjugate provided by the present disclosure in any suitable manner. For example, the A0 group can be connected to the main chain part through the aforementioned conjugation linker.

[0228] Those skilled in the art can use any reasonable synthetic route to prepare the conjugates provided in the present disclosure.

[0229] In some embodiments, the synthetic method of the conjugate provided by the present disclosure includes contacting the protected conjugate with a deprotecting agent in a solvent under deprotection reaction conditions, and separating and obtaining the conjugate provided by the present disclosure. The protected conjugate is a compound formed by protecting any active functional group in the conjugate provided by the present disclosure. In some embodiments, the active functional group includes but is not limited to hydroxyl, amino and / or phosphate, and the protecting group is correspondingly a hydroxyl protecting group, an amino protecting group and / or a phosphohydroxyl protecting group (e.g., a cyanoethyl protecting group). Depending on the protecting group, the solvent, deprotection reaction conditions and deprotection reagent used are selected and determined. In some embodiments, the deprotection reaction conditions, solvent and deprotection reagent are the deprotection reaction conditions, solvents and reagents used in nucleic acid solid phase synthesis. In some embodiments, the method includes adding the protected conjugate to a mixed solution of methylamine aqueous solution and ammonia water, and the deprotection reaction conditions include reacting at room temperature and pressure for 1-5h. 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 conjugate is 0.1-10 ml / μmol. In some embodiments, the separation includes purification by column chromatography, collecting the product eluent and removing the solvent. The purification conditions can be, for example, using a preparative ion chromatography purification column, eluting with a gradient eluent of an aqueous sodium chloride solution and an aqueous sodium phosphate solution. In some embodiments, 20 mM sodium phosphate (pH 8.1), the solvent is water / acetonitrile = 9: 1 (volume ratio); eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), the solvent is water / acetonitrile = 9: 1 (volume ratio); elution gradient: eluent A: eluent B = 100: 0-50: 50 gradient elution.

[0230] In some embodiments, the conjugate provided by the present disclosure has a structure shown in formula (101), and the synthesis method of the protected conjugate comprises: in an organic solvent, under coupling reaction conditions, x1 and a compound containing a delivery group and an active group R x2 and a compound containing a functional group, and reacting to obtain the protected conjugate, wherein the delivery group is formed by removing one or more hydrogen atoms or one or more functional groups from the delivery group in the conjugate used in the application provided by the present disclosure, wherein any active group in 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 capable of reacting to form a covalent bond or a linking group R j In some embodiments, the active group R x1 The delivery group and the active group Rx2 The molar ratio of the functional groups is m0:n0. In some embodiments, the active groups in the delivery group and the functional group include, but are not limited to, one or more of hydroxyl, amino, and phosphate groups, and the protecting group is correspondingly one or more of hydroxyl protecting groups, amino protecting groups, and phosphate hydroxyl protecting groups (e.g., cyanoethyl protecting groups).

[0231] Those skilled in the art can obtain the active group R by various methods. x1 In some embodiments, the compound comprising the active group R x1 The compound containing the active group R 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 liquid phase synthesis. x1 The compound containing the delivery group is obtained by phosphoramidite solid phase synthesis, which comprises connecting nucleoside monomers in sequence according to the order of nucleotides in the oligonucleotide single chain under phosphoramidite solid phase synthesis conditions, wherein at least one nucleoside monomer is a nucleoside monomer having an active group R x1 Alternatively, after connecting all the nucleoside monomers, a nucleoside monomer having an active group R is connected according to the phosphoramidite solid phase synthesis method. x1 The phosphoramidite monomer or protected phosphoramidite monomer is then removed to form an active group R x1 The phosphoramidite solid phase synthesis method is well known to those skilled in the art, and its process and conditions are disclosed in detail in Methods in Molecular Biology, vol. 288: Oligonucleotide Synthesis: Methods and Applications, P17-P31, the entire contents of which are incorporated herein by reference.

[0232] In some embodiments, the coupling reaction conditions are condensation reaction conditions or thiol-disulfide exchange reaction conditions.

[0233] In some embodiments, the coupling reaction conditions are condensation reaction conditions, and the condensation reaction conditions are acylation condensation reaction conditions, dehydration condensation reaction conditions, or click chemistry reaction conditions, and the active group R x1 With active group R x2 In some embodiments, the condensation reaction conditions are conditions for an acylation condensation reaction, and the active group R x1 and R x2 It is possible to undergo acylation condensation reaction to form R IIn some embodiments, the condensation reaction conditions are dehydration condensation reaction conditions, and the active group R x1 and R x2 One of them is a group containing an acyl halide group or a carboxyl group, and the other is a group containing an amino group or a hydroxyl group. 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 the conditions of a Michael addition reaction, and the active group R x1 and R x2 One of the groups is a thiol group, and the other is a succinimide group. In some embodiments, the condensation reaction conditions are N-hydroxysuccinimide-carbodiimide (NHS-EDC) coupling reaction conditions, and the active group R x1 and R x2 One of them is a group containing N-hydroxysuccinimide (NHS), and the other is a group containing a carbodiimide group (EDC).

[0234] In some embodiments, the active group R x1 and a delivery group is prepared by reacting a reactive group R x0 The delivery group in the conjugate used in the application provided by the present disclosure is prepared by contacting a cross-linking agent, wherein the cross-linking agent contains a click chemistry active group and an acylation group. X0 The click chemistry active group is connected to the aptamer of the present invention by forming a covalent connection with the acylated group through a coupling reaction.

[0235] In some embodiments, the active group R x1 The active group contains 1-3 click chemistry active groups at the terminal, and the click chemistry active group contains a terminal alkynyl group. In some embodiments, the acylated group is an active ester group, for example, it can be one of an NHS ester group, an imidate ester group, and a pentafluorophenyl ester group. Those skilled in the art can obtain the cross-linking agent by various methods. For example, when the acylated group is a pentafluorophenyl ester group and the click chemistry group contains a terminal alkynyl group, the cross-linking agent can be prepared as follows: 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 In some embodiments, the coupling condition is a basic condition. In some embodiments, the basic condition is a condition in which a weak base aqueous solution exists, such as a condition in which a sodium bicarbonate aqueous solution exists.

[0236] Those skilled in the art can obtain the active group R in various ways. x0 In some embodiments, the aptamer having an active group R x0 The aptamer is prepared by using a phosphoramidite monomer containing an active group at the corresponding position during the synthesis of the aptamer. Those skilled in the art can obtain the phosphoramidite monomer containing an active group in various ways. In some embodiments, the active group R x0 It is amino, containing R x0 The phosphoramidite monomers can be commercially obtained or prepared by methods well known to those skilled in the art, for example, x0 The phosphoramidite monomer can be a commercially available 6-(trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite monomer, wherein the active group R x0 is an amino group, the active group R x0 It can be obtained by connecting the phosphoramidite monomer to a single-stranded oligonucleotide by a phosphoramidite solid phase synthesis method, and then removing the trifluoroacetyl protecting group by a deprotection reaction (such as aminolysis with concentrated ammonia water) that can be easily achieved by a person skilled in the art. In these cases, the active group R connected by the phosphoramidite solid phase synthesis method x0 Linked to the ribose 5' end of the 5' terminal nucleotide of the aptamer via a phosphodiester bond.

[0237] In some embodiments, the coupling reaction conditions are one of the sulfhydryl-disulfide exchange reactions, and the active group R x1 and R x2 One of the groups is a thiol group, and the other is a leaving group connected via a disulfide bond. In order to avoid side reactions, in some embodiments, the above-mentioned active group R x1 R in the phosphoramidite monomerx1 With the protected R x1 ', the preparation method further comprises the step of: x1 ' and the delivery group compound is contacted with a deprotecting agent to separate the active group R x1 and a compound of a delivery group. In some embodiments, the R x1 ' contains a disulfide bond leaving group, the deprotection reaction conditions are sulfhydryl-disulfide bond exchange reaction conditions, and the deprotection reagent is a disulfide bond activator. In some embodiments, the disulfide bond activator is disulfide dipyridine. Those skilled in the art can obtain the above-mentioned containing active group R by various methods. x1 or R x1 'phosphoramidite monomer, in some embodiments, the phosphoramidite monomer containing the active group R x1 or R x1 The phosphoramidite monomer of ' is commercially available, for example, the phosphoramidite monomer shown in formula (105) can be obtained commercially.

[0238]

[0239] Where n 105 and m 105 Each independently represents an integer from 1 to 10.

[0240] Those skilled in the art can obtain the active group R in various ways. x2 In some embodiments, the coupling reaction conditions are sulfhydryl-disulfide exchange reaction conditions, and the active group R x2 Contains a thiol group, the active group R x2 The compound containing the functional group can be obtained by a person skilled in the art in various known ways, for example, by using a phosphoramidite monomer containing a thiol group to prepare it by a phosphoramidite solid phase synthesis method, or 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, the active group R x2 The compound containing a phosphoramidite group and a functional group can be, for example, a compound that is easily obtained by commercial order or obtained by a nucleic acid solid phase synthesis method. In some embodiments, in some embodiments, the coupling reaction conditions are Michael addition reaction conditions, and the active group R x2 is an N-succinimide group, the active group R x2The compound containing an N-succinimide group and a functional group can be, for example, a compound containing an N-succinimide group and a functional group that is easily commercially available or obtained by a nucleic acid solid phase synthesis method. Among them, the N-succinimide group can be connected to the functional group by, for example, using a phosphoramidite monomer of an N-succinimide group that is easily commercially available, and by a phosphoramidite solid phase synthesis method. In some embodiments, the active group R x1 With active group R x2 They are nucleotide sequence I and nucleotide sequence II, respectively, and each of the nucleotide sequence I and the nucleotide sequence II comprises 5-25 modified or unmodified nucleotides, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, the delivery group is connected to the nucleotide sequence I, the functional group is connected to the nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II do not induce an immune response or a toxic response in the subject, and the coupling reaction conditions are reaction conditions for annealing to form a double-stranded nucleic acid. In some embodiments, the nucleotide sequence I and the nucleotide sequence II are both composed 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.

[0241] 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 continuous nucleotide sequence are both hydroxyl groups, and the delivery group is formed by removing a hydrogen atom from the 5' hydroxyl group of the 5' terminal nucleotide by the aforementioned aptamer; in some embodiments, the delivery group is formed by removing a hydrogen atom from the 3' hydroxyl group of the 3' terminal nucleotide by the aforementioned aptamer; in some embodiments, the delivery group is formed by removing the 5' hydroxyl group from the 5' terminal nucleotide by the aptamer described in the present disclosure; in some embodiments, the delivery group is formed by removing the 3' hydroxyl group from the 3' terminal nucleotide by the aptamer described in the present disclosure. In some embodiments, the delivery group is formed by removing the ribose 2'-hydroxyl group from the nucleotides contained in the aptamer described in the present disclosure. The aptamer can be obtained by conventional aptamer preparation methods in the art (such as nucleic acid solid phase synthesis and liquid phase synthesis methods). Among them, nucleic acid solid phase synthesis already has commercial customization services. The modified nucleotide groups can be introduced into the aptamers described in the present disclosure by using nucleoside monomers with corresponding modifications, and the methods for preparing nucleoside monomers with corresponding modifications and the methods for introducing modified nucleotide groups into aptamers are also well known to those skilled in the art. All modified nucleoside monomers can be purchased commercially or prepared by known methods.

[0242] In some embodiments, the conjugate can also be used in the present disclosure in the form of its pharmaceutically acceptable salt or precursor compound. In the context of the present disclosure, "pharmaceutically acceptable salt" refers to a salt that forms a drug into a corresponding drug that does not produce additional side effects on the human body in order to increase the stability, solubility and / or bioavailability of the drug, such as potassium salt, sodium salt, carboxylate, etc. In some embodiments, the delivery group or conjugate used in the application provided by the present disclosure may exist at least partially in the form of a salt. In some embodiments, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or thiophosphorothioate diester bond in the delivery group or conjugate used in the application provided by the present disclosure is at least partially combined with a metal ion (such as a sodium ion) that can form a water-soluble salt, and the delivery group or conjugate in the application provided by the present disclosure is a pharmaceutically acceptable salt of at least part of the water-soluble salt. In some embodiments, the delivery group or conjugate in the application provided by the present disclosure is a sodium salt or a partial sodium salt.

[0243] In some embodiments, in the conjugates disclosed herein, each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate diester bond, and the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or the phosphorothioate diester bond carries a negative charge, which may exist in the form of a hydroxyl group or a thiol group, and the hydrogen ions in the hydroxyl group or the thiol group may also be partially or completely replaced by cations. The cations may be any cations, such as metal cations, ammonium ions NH4 + , one of the organic ammonium cations. In order to improve solubility and / or improve bioavailability, in some embodiments, the pharmaceutically acceptable salt is a water-soluble salt of part or all of the conjugate. In some embodiments, the water-soluble salt may be an amine salt, an alkali metal salt or an alkaline earth metal salt. In some embodiments, the amine salt is selected from one or more of ammonium salts, methylamine salts, tertiary amine salts, and quaternary ammonium salts, the alkali metal salt is selected from potassium salts or sodium salts, and the alkaline earth metal salt is selected from calcium salts or magnesium salts. In some embodiments, the tertiary amine salt is a triethylamine salt, a triisopropylamine salt, or a N,N-diisopropylethylamine salt. In some embodiments, the pharmaceutically acceptable salt of the conjugate is a mixture of a methylamine salt and an ammonium salt of the conjugate. In some embodiments, the pharmaceutically acceptable salt of the conjugate is a sodium salt or a partial sodium salt of the conjugate.

[0244] "Precursor compound" refers to a compound that, although it is not completely identical to the conjugate in structure and function, can react after entering the body or in the body fluid environment to form the conjugate provided by the present disclosure, thereby exerting an effect and achieving the purpose of the present disclosure. In some cases, these precursor compounds can increase the stability of the drug, prolong the sustained release time, increase the bioavailability, and the like. In some embodiments, the precursor compound includes a precursor group that can react in the human body to form all functional groups in the conjugate. In some embodiments, the precursor compound includes a compound formed by replacing all active hydroxyl groups in the conjugate with acetoxy groups. In some embodiments, the precursor compound includes a prodrug group, which is a residue formed by a precursor compound of the functional group in the conjugate. In some embodiments, the prodrug group can be, for example, a group formed by replacing the active hydrogen in the hydroxyl or amino functional group in the functional group with an acyl group, an alkyl group, or a phosphoryl group. It can be understood by those skilled in the art that the use of these pharmaceutically acceptable precursor compounds is also within the scope of the present disclosure.

[0245] Pharmaceutically acceptable excipients

[0246] In some embodiments, the conjugate used for the application of the present disclosure can also be provided in the form of a pharmaceutical composition, hereinafter referred to as the pharmaceutical composition of the present disclosure. In some embodiments, the pharmaceutical composition also contains a pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient is one or more of the various ingredients conventionally used in the art, such as a solvent, a protective agent, an osmotic pressure regulator, and one or more of other pharmaceutically acceptable carriers.

[0247] For example, when the conjugate is an injection, the pharmaceutically acceptable excipient is a solvent, such as deionized water, water for injection, ethanol or a pH buffer. The pH buffer may be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH value of 7.5-8.5 and / or a phosphate buffer with a pH value of 5.5-8.5, for example, a phosphate buffer with a pH value of 5.5-8.5.

[0248] The amount of the solvent is adjusted according to the required solution concentration. Based on the functional groups in the conjugate, the concentration of the conjugate in the injection can be 0.01 mg / mL-5 mg / mL siRNA, 0.1 mg / mL-5 mg / mL, or 0.5 mg / mL-3 mg / mL.

[0249] The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.

[0250] The osmotic pressure regulator can be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 milliosmole / kilogram (mOsm / kg). According to the required osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator. In some embodiments, the dosage of the preparation made of the pharmaceutical composition during administration can be adjusted due to different administration methods.

[0251] The other pharmaceutically acceptable carriers may be carriers conventionally used in the art, such as but not limited to water, physiological saline, magnetic nanoparticles (such as nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly (D&L-lactic / glycolic acid) copolymer (PLGA), poly (2-aminoethyl ethylene phosphate ... Phosphate), PPEEA) and poly(methacrylate-N,N-dimethylaminoethyl ester) (poly(2-dimethylaminoethylmethacrylate), PDMAEMA) and one or more of their derivatives.

[0252] In some embodiments, the other pharmaceutically acceptable carrier contains a physiologically acceptable compound that acts, for example, to stabilize the pharmaceutical composition or to increase or decrease the absorption of the conjugate and / or the pharmaceutical composition. The physiologically acceptable compound is selected from one or more of the following compounds: 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 may also be used to stabilize the composition or increase or decrease the absorption of the pharmaceutical composition. The physiologically acceptable compound may also include one or more of a wetting agent, an emulsifier, a dispersant, or a preservative particularly for preventing microbial growth or action. The physiologically acceptable compound is known to those skilled in the art and will not be described in detail in this disclosure. It is readily understood by those skilled in the art that the selection of pharmaceutically acceptable carriers and physiologically acceptable compounds depends, for example, on the route of administration and the specific physiochemical properties of any co-administered substance.

[0253] In some embodiments, the pharmaceutically acceptable excipients are sterile and generally free of undesirable substances. The pharmaceutical compositions of the present disclosure may further contain pharmaceutically acceptable auxiliary substances as needed to approach physiological conditions, such as pH adjusters and buffers, toxicity regulators, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the conjugate provided by the present disclosure in the pharmaceutical composition may vary within a large range and is mainly selected according to a specific administration method based on fluid volume, viscosity, body weight, etc.

[0254] In some embodiments, in the pharmaceutical composition, there is no special requirement for the content of the conjugate and the pharmaceutically acceptable carrier. In some embodiments, the weight ratio of the conjugate to the pharmaceutically acceptable carrier can be 1:(1-500). In some embodiments, the above weight ratio is 1:(1-50).

[0255] In some embodiments, the pharmaceutical composition may be a liquid preparation, such as an injection; or a lyophilized powder injection, which is mixed with a liquid excipient during administration to prepare a liquid preparation. The liquid preparation may be, but is not limited to, used for subcutaneous, intramuscular or intravenous administration, and may be, but is not limited to, delivered by puncture injection, or by oropharyngeal inhalation, or nasal administration. In some embodiments, the pharmaceutical composition is used for subcutaneous, intramuscular or intravenous administration.

[0256] In some embodiments, the pharmaceutical composition can be in the form of a liposome preparation. In some embodiments, the pharmaceutically acceptable carrier used in the liposome preparation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), an auxiliary lipid and / or a pegylated lipid. Wherein, the organic amine, auxiliary lipid and pegylated lipid can be selected from one or more of the amine-containing transfection compound or its pharmaceutically acceptable salt or derivative, auxiliary lipid and pegylated lipid described in Chinese patent application CN103380113A (incorporated herein by reference in its entirety).

[0257] In some embodiments, the organic amine may be a compound described in Chinese patent application CN103380113A as shown in formula (201) or a pharmaceutically acceptable salt thereof:

[0258]

[0259] in:

[0260] X 101 and X 102 are each independently O, S, NA or CA, wherein A is hydrogen or C1-C 20 hydrocarbon chain;

[0261] Y 101 and Z 101 Each independently is C=O, C=S, S=O, CH-OH or SO2;

[0262] R 101 , R 102 , R 103 , R 104 , R 105 , R 106 and R 107 each independently represents 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, a substituted or unsubstituted, branched or straight-chain heteroaryl group;

[0263] x is an integer from 1 to 10;

[0264] n is an integer of 1-3, m is an integer of 0-20, and p is 0 or 1; wherein, if m=p=0, then R 102 It is hydrogen;

[0265] And, if at least one of n or m is 2, then R 103 and the nitrogen in formula (201) to form a structure as shown in formula (202) or formula (203):

[0266]

[0267] wherein 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).

[0268] In some embodiments, R 103 is a polyamine. In other embodiments, R 103 In some embodiments, R in formula (201) 101 and R 102 Each of is independently an optionally substituted or unsubstituted, branched or straight chain alkyl or alkenyl group having 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.

[0269] In some embodiments, if each of n and m independently has a value of 1 or 3, then R 103 It can be any one of the following formulas (204) to (213):

[0270]

[0271] Wherein, in formula (204) to formula (213), g, e and f are each independently an integer of 1 to 6, each "HCC" represents a hydrocarbon chain, and each * indicates R 103 Possible points of attachment to the nitrogen atom in formula (201), wherein each H at any * position can be replaced to achieve attachment to the nitrogen atom in formula (201).

[0272] Those skilled in the art can obtain the compound represented by formula (201) by any reasonable method. In some embodiments, the compound represented by formula (201) can be prepared according to the description in Chinese patent application CN103380113A.

[0273] In some embodiments, the organic amine is an organic amine as shown in formula (214) and / or an organic amine as shown in formula (215):

[0274]

[0275] The auxiliary lipid is cholesterol, a cholesterol analog and / or a cholesterol derivative;

[0276] The PEGylated lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

[0277] In some embodiments, in the pharmaceutical composition, the molar ratio of the organic amine, the auxiliary lipid and the pegylated lipid is (19.7-80):(19.7-80):(0.3-50), for example, it can be (50-70):(20-40):(3-20).

[0278] In some embodiments, the pharmaceutical composition particles formed by the conjugate provided by the present disclosure and the above-mentioned amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, 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.

[0279] In some embodiments, in the pharmaceutical composition formed by the conjugate provided by the present disclosure and the above-mentioned amine-containing transfection reagent, the weight ratio (weight / weight ratio) of the conjugate to all lipids (e.g., organic amines, helper lipids and / or pegylated lipids) is from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10, for example, the weight ratio of the conjugate provided by the present disclosure to all lipids 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.

[0280] In some embodiments, each component of the pharmaceutical composition can exist independently when it is sold, and can exist in the form of a liquid preparation when it is used. In some embodiments, the pharmaceutical composition formed by the conjugate provided by the present disclosure and the above-mentioned pharmaceutically acceptable carrier can be prepared according to various known methods, except that the conjugate provided by the present disclosure is used to replace the existing conjugate; in some embodiments, it can be prepared according to the following method:

[0281] The organic amine, the auxiliary lipid and the PEGylated lipid are suspended in alcohol according to the above molar ratio and mixed to obtain a lipid solution; the amount of alcohol used is such that the total mass concentration of the obtained lipid solution is 2-25 mg / mL, for example, 8-18 mg / mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid at around room temperature, for example, ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, one or more, for example, ethanol.

[0282] The conjugate provided by the present disclosure is dissolved in a buffered saline solution to obtain a conjugate aqueous solution. The concentration of the buffered saline solution is 0.05-0.5M, for example, 0.1-0.2M, and the pH of the buffered saline solution is adjusted to 4.0-5.5, for example, 5.0-5.2, and the amount of the buffered saline solution is such that the concentration of the conjugate does not exceed 0.6mg / mL, for example, 0.2-0.4mg / mL. The buffer salt is selected from one or more of soluble acetate and soluble citrate, for example, sodium acetate and / or potassium acetate.

[0283] The lipid solution and the conjugate aqueous solution are mixed, and the mixed product is incubated at 40-60° C. for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome preparation. The volume ratio of the lipid solution to the conjugate aqueous solution is 1:(2-5), for example, 1:4.

[0284] The incubated liposome preparation is concentrated or diluted, impurities are removed, and sterilization is performed to obtain the pharmaceutical composition provided by the present disclosure, wherein the physicochemical parameters are a pH value of 6.5-8, an encapsulation rate of not less than 80%, a particle size of 40-200 nm, a polydispersity index of not more than 0.30, and an osmotic pressure of 250-400 mOsm / kg; for example, the physicochemical parameters may be a pH value of 7.2-7.6, an encapsulation rate of not less than 90%, a particle size of 60-100 nm, a polydispersity index of not more than 0.20, and an osmotic pressure of 300-400 mOsm / kg.

[0285] The concentration or dilution can be performed before, after or simultaneously with the removal of impurities. The impurity removal method can adopt various existing methods, such as using a tangential flow system, a hollow fiber column, ultrafiltration under 100K Da conditions, and the ultrafiltration exchange solution is a phosphate buffered saline (PBS) with a pH of 7.4. The sterilization method can adopt various existing methods, such as filtration sterilization on a 0.22 μm filter.

[0286] The term "administration / administration" as used in the present disclosure refers to placing a conjugate and / or pharmaceutical composition into a subject's body by a method or route that allows the conjugate and / or pharmaceutical composition to be at least partially localized at a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include local administration and systemic administration. In general, local administration results in more conjugates and / or pharmaceutical compositions being delivered to a specific site compared to the entire body of the subject; whereas systemic administration results in the conjugate and / or pharmaceutical composition being delivered to substantially the entire body of the subject.

[0287] The drug may be administered to a subject by any suitable route known in the art, including but not limited to oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and topical administration (including oral administration and sublingual administration). The frequency of administration may be once or more per day, per week, every two weeks, every three weeks, every month, or every year.

[0288] The dosage of the conjugate and / or pharmaceutical composition disclosed herein can be a conventional dosage in the art, which can be determined according to various parameters, especially the age, weight and sex of the subject. Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as determining LD50 (a dose that causes 50% of the population to die) and ED50 (a dose that causes 50% of the maximum response intensity in quantitative reactions, and a dose that causes 50% of the experimental subjects to have a positive reaction in qualitative reactions). The range of human dosage can be derived based on the data obtained from cell culture analysis and animal studies.

[0289] When administering the conjugate and / or pharmaceutical composition of the present disclosure, for example, for male or female C57BL / 6J or C3H / HeNCrlVr mice of 6-12 weeks of age and weighing 18-25 g, the amount of the conjugate in the conjugate and / or pharmaceutical composition is calculated as follows: for the conjugate formed by the conjugate and the pharmaceutically acceptable conjugate molecule, the amount of the conjugate can 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 further embodiments 0.1-15 mg / kg body weight, and in yet further embodiments 0.1-10 mg / kg body weight. When administering the conjugate and / or pharmaceutical composition of the present disclosure, the above amount may be preferred.

[0290] The present disclosure will be further described below by way of examples, but the present disclosure is not limited thereby.

[0291] Example

[0292] Unless otherwise specified, the reagents and culture media used in the following examples are commercially available products, and the nucleic acid electrophoresis, real-time PCR and other operations used are all performed according to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

[0293] For the conjugates synthesized and used in the following examples, unless otherwise specified, all obtained were sodium salts of the conjugates in which the hydroxyl hydrogen ions in all the phosphate groups in the conjugates were replaced by sodium ions.

[0294] Preparation Example 1 Synthesis of the conjugate provided by the present disclosure

[0295] The conjugates 1 in Table 1 were synthesized by a solid phase synthesis method, and after all the nucleoside monomers were sequentially connected from the 3'-5' direction according to the nucleotide sequence corresponding to the conjugate 1 in Table 1, the Cy5 phosphoramidite monomer (purchased from Suzhou Jima Company, batch number CY5P21H1B) was connected in the same manner as the nucleoside phosphoramidite monomer was connected by the solid phase synthesis method. Subsequently, the nucleotide sequence was added to a mixed solution of equal volumes of methylamine aqueous solution and ammonia water, the amount of the solution relative to the conjugate 1 was 0.5 ml / μmol, the reaction was carried out at 25° C. for 2 h, the solid was filtered out, and the supernatant was concentrated to dryness in vacuo.

[0296] The Cy5 phosphoramidite monomer has a structure as shown in formula (106):

[0297]

[0298] The prepared conjugate was purified by gradient elution of NaCl aqueous solution using a preparative ion chromatography purification column (Source 15Q). Specifically, eluent A: 20 mM sodium phosphate (pH 8.1), solvent water / acetonitrile = 9:1 (volume ratio); eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), solvent water / acetonitrile = 9:1 (volume ratio); elution gradient: eluent A: eluent B = 100:0-50:50 gradient elution. The product eluate was collected and combined, and desalted using a reverse phase chromatography purification column. The specific conditions included desalting using a dextran gel column, the filler was dextran gel G25, and eluted with deionized water. The obtained eluate was concentrated to remove the solvent and lyophilized to obtain the conjugate 1 with the fluorescent group Cy5 connected to the 5' end. The fluorescent group Cy5 is connected to the ribose 5' end of the 5' terminal nucleotide of the continuous nucleotide sequence through a phosphodiester bond.

[0299] The conjugate 1 is a sodium salt of a compound having the following structure:

[0300]

[0301] In the formula, It represents the nucleotide sequence corresponding to conjugate 1, and a Cy5 fluorescent group is connected to the 5' end.

[0302] After the synthesis of conjugate 1 was completed, ultrapure water (Milli-Q ultrapure water instrument, resistivity 18.2MΩ*cm (25°C)) was used to dilute the prepared conjugate to a concentration of 0.2 mg / mL, and then the molecular weight was detected by liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier). Among them, the theoretical molecular weight: 8493.809; the measured molecular weight: [M+H+] = 8491.52. As a result, the measured molecular weight value was consistent with the theoretical value, indicating that the target conjugate was obtained.

[0303] Table 1 Structure of the conjugate

[0304]

[0305] In Table 1, capital letters C, G, U, and A represent the base composition of the nucleotide; lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; Cy5 represents the fluorescent dye group Cy5 (Cyanine 5) group.

[0306] Experimental Example 1 Distribution of the conjugate in HCT-116 human colon cancer cell subcutaneous tumor model mice

[0307] HCT-116 human colon cancer cells (purchased from Guangzhou Yuanjing Biotechnology Co., Ltd.) were cultured in DMEM complete medium (MACGENE, catalog number K1902200) supplemented with 10% fetal bovine serum (FBS, RMBIO) at 37°C in an incubator containing 5% CO2 / 95% air.

[0308] HCT-116 human colon cancer cells in logarithmic phase were digested with 0.25% trypsin, the cells were collected, the supernatant was removed by centrifugation, and the cells were resuspended in DMEM complete medium supplemented with 10% FBS to a concentration of 5×10 7 cells / mL of cell culture medium.

[0309] Experimental animals: 3 Balb / c-nu mice (purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.), female, 12 weeks old. The above cell culture solution was inoculated subcutaneously in the right forelimb of the mouse, with an inoculation volume of 100 μL per mouse, that is, 5×10 6 The mice were kept for 30 days after injection.

[0310] Conjugate 1 and control conjugate 1 were dissolved into solutions with a concentration of 0.3 mg / mL using 1×DMEM (purchased from Beijing Zhongke Maichen Technology Co., Ltd., batch number K1902200) culture medium.

[0311] Three mice inoculated with HCT-116 human colon cancer cells were injected into the tail vein, and the dosage volume was calculated as 10 μL / g body weight. One group of mice was given PBS, and the other two groups of mice were given conjugate 1, which were recorded as blank control group and test group, respectively. Thus, the dosage of each mouse was 3 mg / kg (calculated as aptamer).

[0312] 24h and 48h after administration, mice in each group were placed in the small animal in vivo optical imaging system IVIS Lumina Series III (PerkinElmer) in turn, and the mice were anesthetized with isoflurane gas. The anesthetized mice were placed with their abdomen facing upward in the small animal in vivo optical imaging system for in vivo imaging. The results are shown in Figure 1A and 1B 96 hours after administration, one mouse in the blank control group and one mouse in the test group were killed, and the tumor tissues were taken for fluorescence imaging. The results are shown in Figure 1C shown.

[0313] Figure 1A and Figure 1BThe figures respectively show the in vivo imaging at 24h and 48h after the conjugate provided by the present disclosure is administered to HCT-116 human colon cancer cell subcutaneous tumor model mice, wherein blank represents blank control group 1. Figure 1C Figure 4 shows fluorescence imaging of tumor tissues when the conjugate provided by the present disclosure was administered to HCT-116 human colon cancer cell subcutaneous tumor model mice for 96 hours, wherein blank represents a blank control group. Figure 1A and Figure 1B It can be seen that the blank control group did not show any fluorescent signal in the mouse body; in contrast, the conjugate 1 provided by the present disclosure showed a strong fluorescent signal at the tumor inoculation site in the mouse body, indicating that the conjugate provided by the present disclosure can target human colon cancer cells and produce fluorescent signals through the diagnostic agent group contained in the conjugate to play a diagnostic role, and can still show a strong fluorescent signal 24h to 48h after administration, which is helpful for the detection, diagnosis and monitoring of the treatment process of colorectal cancer tumor tissue. In addition, from Figure 1C It can be seen that 96 hours after the conjugate provided by the present disclosure was administered to subcutaneous tumor model mice, the fluorescence signal intensity of the tumor tissue still remained at a high level, indicating that the conjugate provided by the present disclosure can stably target to colon cancer tumor tissues and cells for a long time.

[0314] Some embodiments of the present disclosure are described in detail above; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0315] It should also be noted that the various specific technical features described in some of the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0316] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. Use of a conjugate or a pharmaceutically acceptable salt thereof in the preparation of a drug for diagnosing or treating colorectal cancer, wherein: The conjugate comprises one or more delivery groups and one or more functional groups, each of the delivery groups is independently linked to at least one of the functional groups or other delivery groups via at least one linking group, and each functional group is independently linked to at least one of the delivery groups or at least one additional functional group via at least one linking group; Each of the functional groups is independently one of a diagnostic agent group, a small molecule therapeutic agent group having a therapeutic effect on colorectal cancer, a functional oligonucleotide group having a therapeutic effect on colorectal cancer, and a delivery aid group; The delivery group is formed by removing one or more hydrogen atoms or one or more functional groups from an aptamer, the aptamer comprises a continuous nucleotide sequence, the group connecting two adjacent nucleotides is independently a phosphate group or a phosphate group with a modified group, each nucleotide is selected from one of modified or unmodified nucleotides, and the continuous nucleotide sequence has a 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 1-3 nucleotides, T2 is a motif consisting of 0-15 nucleotides, and T2 does not contain a motif that is completely reverse complementary to T1; S1 and S4 are each a motif consisting of 3-7 nucleotides, S1 and S4 are of the same length and are completely reverse complementary; N a and N c Each is a motif consisting of 1-4 nucleotides, N a Each nucleotide in c Each nucleotide in N is not complementary, and N a and N c The total number of U in N a and N c More than 50% of the total number of nucleotides in S2 and S3 are each a motif consisting of 1-4 nucleotides, S2 and S3 are of the same length and are completely reverse complementary; N b is a motif consisting of 3-6 nucleotides, and N b The nucleotides at both ends do not form AU or GC complementarity.

2. The use according to claim 1, wherein: The length of the continuous nucleotide sequence is 18-50 nucleotides, or 20-40 nucleotides, or 21-36 nucleotides, or 24-32 nucleotides.

3. The use according to claim 2, wherein: T1 consists of 2 nucleotides; or, T1 consists of 2 nucleotides and contains at least one C; or, in the 5'-3' direction, T1 is CU, UC or AC; Alternatively, T2 consists of 0-10 nucleotides; alternatively, in the 5'-3' direction, T2 consists of 1-9 nucleotides starting with U; Alternatively, S1 and S4 each consist of 3-5 nucleotides and have the same length; alternatively, in the reverse complement formed by S1 and S4, GC complementation accounts for at least 40% of the total complementation number; alternatively, in the 5'-3' direction, 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; Alternatively, the N a and N c The sum of the number of nucleotides in is an integer of 2-4; or, a and N c The sum of the number of nucleotides in is 3 or 4, and the N a and N c The sum of the number of U in is 2 or 3; or, in the 5'-3' direction, N a or N c independently U, UU, UC or CU; Alternatively, S2 and S3 each consist of 2-3 nucleotides and have the same length; alternatively, the reverse complement formed by S2 and S3 contains at least one GC complement; alternatively, in the 5'-3' direction, S2 is CA and S3 is UG, or S2 is AC and S3 is GU, or S2 is GCC and S3 is GGU; Or, N b Composed of 4 or 5 nucleotides; or, in the 5'-3' direction, N b It is GACG, GACGU, GACCG, UACU, GUUG or GAUCU.

4. The use according to claim 3, wherein: The continuous nucleotide sequence has the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; Alternatively, the continuous nucleotide sequence has the nucleotide sequence shown in SEQ ID NO:4: 5'-N6GGAGUUCAN1N2N3N4UGN5GCUCN7-3' (SEQ ID NO:4), Among them, N1, N2, and N3 are each independently one of A, U, C and G; N4 is U, C or G or a motif composed 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 composed of 1-15 nucleotides.

5. The use according to claim 4, wherein: The motif N1N2N3N4 composed of N1, N2, N3 and N4 is one of GACG, GACGU, GACCG, UACU, GUUG or GAUCU.

6. The use according to claim 4 or 5, wherein: The continuous nucleotide sequence has the nucleotide sequence shown in any one of SEQ ID NOs: 5-11; Alternatively, N8 is a motif consisting of 1-8 nucleotides; alternatively, in the 5'-3' direction, the nucleotide sequence of N8 is CCGAUCUC; alternatively, the continuous nucleotide sequence has a sequence shown in one of SEQ ID NOs: 12-14.

7. The use according to any one of claims 2 to 6, wherein: Each cytosine nucleotide in the continuous nucleotide sequence is a fluorinated modified cytosine nucleotide, and / or each uracil nucleotide in the continuous nucleotide sequence is a fluorinated modified uracil nucleotide; or, each nucleotide in the continuous nucleotide sequence is a 2'-methoxy modified nucleotide; or, N b and one or more uracil nucleotides in the S3 motif have a modified base.

8. The use according to claim 7, wherein: The continuous nucleotide sequence has a nucleotide sequence shown in one of SEQ ID NOs: 15-33.

9. The use according to claim 2, wherein: At least one group connecting two adjacent nucleotides in the continuous nucleotide sequence is a phosphorothioate group, or each group connecting two adjacent nucleotides is a phosphorothioate group.

10. The use according to claim 9, wherein: The continuous nucleotide sequence has a nucleotide sequence shown in one of SEQ ID NOs: 34-39.

11. The use according to claim 2, wherein: The conjugate has a structure as shown in formula (2): Among them, each R AP The groups are independently groups having a structure as shown in formula (3): In the formula, each AP group is the same or different and independently represents a delivery group; each A0 group is the same or different and independently represents a delivery group or a functional group, and at least one A0 is a delivery group and at least one A0 is a functional group; R j , each R k or each R i are the same or different, and each independently represents a covalent bond or a linking group, and R i and R k The two are not covalent bonds at the same time; each n1 independently represents an integer from 0 to 4; m0 is an integer from 1 to 6; n0 is an integer from 1 to 6, Indicates the site to which a group is covalently attached.

12. The use according to claim 11, wherein: m0 is an integer from 1 to 4, and / or n0 is an integer from 1 to 3, and / or each n1 is independently an integer from 0 to 1; Alternatively, m0 is 1, and / or n0 is 1, and / or at least one or each n1 is 0; Alternatively, each of the R k and / or each of the R i It is independently a covalent bond or a straight chain alkylene group with a length of 1-70 carbon atoms, or one or more carbon atoms in the straight chain alkylene group are replaced by one or more selected from the group consisting of the following groups: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, OP(O)(S), C5-C8 glycoside group, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 wherein the linear alkylene group may have any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl); Alternatively, each n1 is independently 0 or 1, and each R i are independently covalent bonds, or are a combination of one or more of the following linking groups: C1-C 20 Alkylene group, phosphate group, thiophosphate group, -CONH- group, -O- group, -S- group, -C(O)-O- group, -SS- group, 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, nucleotide subunit; Or, each R i Independently one or a combination of two of a covalent bond, a disulfide bond, a propylene phosphate group, a 2-thiosuccinimide subunit, an amino acid subunit, or a GAU trinucleotide subunit; Or, R j For a covalent bond, m0 is 1.

13. The use according to any one of claims 11 to 12, wherein: R j is a connecting group, the connecting group R j The invention comprises a main chain part, a side chain part and a conjugated connecting part, wherein the main chain part is connected to the conjugated connecting part and the side chain part, and each of the side chain parts is connected to the main chain part and the R AP Each of the conjugated linking parts is connected to the main chain part and the A0 group, wherein The main chain part is a straight chain alkylene group with a length of 1-70 carbon atoms, or one or more carbon atoms in the straight chain alkylene group are replaced by one or more selected from the group consisting of the following groups: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycoside group, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 wherein the linear alkylene group may have any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl); Each of the side chain moieties is independently a covalent bond, or a straight chain alkylene group having a length of 1 to 70 carbon atoms, or one or more carbon atoms in the straight chain alkylene group are replaced by one or more selected from the group consisting of the following groups: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycoside group, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 and the linear alkylene group may have any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl); Each of the conjugated linkers is independently a covalent bond or a combination of one or more of the following linking structures: C1-C 10 Straight chain alkylene group, -OP(O)2- group, thiophosphate group (-OP(O)(S)-), -CONH group, -O- group, -S- group, -C(O)-O- group, -SS- group, 1,2,3-triazole subunit, polyethylene glycol subunit, pyrrolidine subunit, 2-oxopyrrolidine subunit, phenylene, cyclohexylene, 2-succinimide subunit, 2-thiosuccinimide subunit, amino acid subunit, nucleotide subunit; Alternatively, the linking group R j Each of the conjugated linking parts is respectively connected to the main chain part and one of the A0 groups; the side chain parts are n0, and each of the side chain parts is respectively connected to the main chain part and one of the R AP Group connection; Alternatively, all of the side chain moieties are attached to the same atom in the main chain moiety; alternatively, each of the side chain moieties is attached to a different atom in the main chain moiety.

14. The use according to claim 13, wherein: m0 is 1, the connecting group R j Contains the structure shown in formula (301): Wherein, k is an integer from 1 to 3; L C is the main chain part, L A is the side chain part, L B is the conjugated linker, represents the site of covalent attachment of a group; The main chain part L C It is a covalent bond or a 2-4 valent, straight or branched C1-C 25 A saturated hydrocarbon group, or one or more carbon atoms in the saturated hydrocarbon group are replaced by one or more selected from the group consisting of the following groups: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycosidyl, C2-C5 alkenyl, C2-C5 alkynyl, C6-C 10 Arylene, C3-C8 heterocyclylene and C5-C 10 wherein the saturated hydrocarbon group may have any one or more substituents selected from the group consisting of: C1-C5 alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl, -O-C1-C5 alkyl, -OC1-C5 alkylphenyl, -C1-C5 alkyl-OH, -SC1-C5 alkyl, nitro, -C(O)O(C1-C5 alkyl), -CON(C1-C5 alkyl)(C1-C5 alkyl), -CONH(C1-C5 alkyl), -CONH2, -NHC(O)(C1-C5 alkyl), -NHC(O)(phenyl), -N(C1-C5 alkyl)C(O) (C1-C5 alkyl), -N(C1-C5 alkyl)C(O)(phenyl), -C(O)C1-C5 alkyl, -C(O)C1-C5 alkylphenyl, -OC(O)C1-C5 alkyl, -SO2(C1-C5 alkyl), -SO2(phenyl), -SO2NH2, -SO2NH(C1-C5 alkyl), -SO2NH(phenyl), -NHSO2(C1-C5 alkyl) and -NHSO2(phenyl); Each of the side chain moieties is independently a covalent bond, or a straight chain alkylene group having a length of 1 to 70 carbon atoms, or one or more carbon atoms in the straight chain alkylene group are replaced by one or more selected from the group consisting of the following groups: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycoside group, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 and the linear alkylene group may have any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl); Each of the conjugated linkers is independently a covalent bond or a combination of one or more of the following linking structures: C1-C 10 Straight-chain alkylene group, -OP(O)2- group, thiophosphate group (-OP(O)(S)-), -CONH group, -O- group, -S- group, -C(O)-O- group, -SS- group, 1,2,3-triazole subunit, polyethylene glycol subunit, pyrrolidine subunit, 2-oxopyrrolidine subunit, phenylene, cyclohexylene, 2-succinimide subunit, 2-thiosuccinimide subunit, amino acid subunit, nucleotide subunit.

15. The use according to any one of claims 2 to 14, wherein: At least one or all of the functional groups in the conjugate are diagnostic agent groups, and each of the diagnostic agent groups is independently selected from a contrast agent group or a fluorescent imaging group.

16. The use according to any one of claims 2 to 14, wherein: At least one or all of the functional groups in the conjugate are small molecule therapeutic agent groups that have a therapeutic effect on tumors, and each of the small molecule therapeutic agent groups is independently selected from a cytotoxin group, an antibiotic group, an angiogenesis inhibitor or an antibody drug group.

17. The use according to any one of claims 2 to 14, wherein: At least one or all of the functional groups in the conjugate are functional oligonucleotide groups, and the functional oligonucleotide groups can regulate the expression level of cancer-related genes in tumor cells.

18. The use according to any one of claims 2 to 14, wherein: At least one of the functional groups in the conjugate is a delivery aid group, and the delivery aid group is one of the lipophilic groups; or, the delivery aid group is selected from C 10 -C 30 One or more of hydrocarbon groups, cholesterol groups, and phospholipid groups.

19. The use according to claim 1, wherein: The pharmaceutically acceptable salt is a water-soluble salt of part or all of the conjugate; Alternatively, the water-soluble salt is an amine salt or an alkali metal salt; Alternatively, the amine salt is selected from one or more of ammonium salts, methylamine salts, tertiary amine salts, and quaternary ammonium salts, and the alkali metal salt is selected from potassium salts or sodium salts; Alternatively, the tertiary amine salt is a triethylamine salt, a triisopropylamine salt or an N,N-diisopropylethylamine salt; Alternatively, the pharmaceutically acceptable salt is a sodium salt or a partial sodium salt of the conjugate.

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