WO2023198200A9 - 缀合物与组合物及制备方法和用途 - Google Patents

缀合物与组合物及制备方法和用途 Download PDF

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WO2023198200A9
WO2023198200A9 PCT/CN2023/088459 CN2023088459W WO2023198200A9 WO 2023198200 A9 WO2023198200 A9 WO 2023198200A9 CN 2023088459 W CN2023088459 W CN 2023088459W WO 2023198200 A9 WO2023198200 A9 WO 2023198200A9
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group
alkyl
conjugate
nucleotide sequence
nucleotides
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WO2023198200A1 (zh
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梁子才
张鸿雁
高山
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苏州瑞博生物技术股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B8/00—Diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/08—Detecting organic movements or changes, e.g. tumours, cysts, swellings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/24—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having more than one carboxyl group bound to the carbon skeleton, e.g. aspartic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/06—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present disclosure relates to conjugates and pharmaceutical compositions comprising aptamer-based delivery groups and functional groups.
  • the present disclosure also relates to methods of preparation and uses of these conjugates and pharmaceutical compositions.
  • Tumors refer to new organisms formed by the proliferation of local tissue cells in the body under the action of various tumorigenic factors. Among them, the situation in which tumor cells metastasize and invade surrounding tissues is called malignant tumor. According to the classification of tissue cells from which tumors originate, they are generally divided into malignant tumors (cancer) produced by epithelial cells, malignant tumors produced by mesenchymal cells (sarcoma), malignant tumors produced by blood stem cells (leukemia, etc.), and glial cells. Malignant tumors (glioma) produced, etc. Among them, glioma is the most common primary intracranial malignant tumor, accounting for approximately 40%-50% of brain tumors. The global annual incidence rate is 3-8 cases per 100,000 people.
  • gliomas are neuroepithelial tumors, including a variety of pathological types, including but not limited to pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma tumors, oligodendroglioma, anaplastic oligodendroglioma, etc.
  • Aptamers, or nucleic acid aptamers are oligonucleotide molecules that can bind to a variety of target molecules, such as small molecule compounds, proteins, nucleic acids, and even cells, tissues, and organs. Aptamers can provide the important property of "recognition of specific molecules" and are therefore often used in biotechnology and therapy, similar to antibodies. Aptamers can be designed in test tubes and can be quickly synthesized using chemical methods. They also have the excellent properties of being easy to preserve and having low or no immunogenicity. Therefore, they have gradually attracted the attention of researchers in this field in recent years. However, aptamers suitable for tumor-targeted delivery still require further development and application in this field.
  • the inventors of the present disclosure unexpectedly discovered a conjugate that can specifically target tumor cells, especially glioma cells, and the conjugate shows high specificity for tumor cells, especially glioma cells, thereby It can be effectively enriched in tumor cells, especially glioma cells, and can effectively target tumors. Therefore, the inventor made the following invention:
  • the present disclosure provides a conjugate comprising one or more delivery groups and one or more functional groups; the delivery group consists of an aptamer removing one or more hydrogen atoms Or one or more functional groups are formed, the aptamer includes a continuous nucleotide sequence, and 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 A, U, C or G, and the continuous nucleotide sequence has a sequence represented by formula (1):
  • T 1 is a motif composed of 1-3 nucleotides
  • T 2 is a motif composed of 0-15 nucleotides
  • T 2 does not contain a completely reverse complementary motif to T 1 ;
  • S 1 and S 4 are each a motif composed of 3-7 nucleotides. S 1 and S 4 are the same length and completely reverse complementary;
  • Na and Nc are each a motif consisting of 1-4 nucleotides, each nucleotide in Na is not complementary to every nucleotide in Nc , and in Na and Nc The total number of U accounts for more than 50% of the total number of nucleotides in N a and N c ;
  • S 2 and S 3 are each a motif composed of 1-4 nucleotides. S 2 and S 3 are the same length and completely reverse complementary;
  • N b is a motif composed of 3-6 nucleotides, and the nucleotides at both ends of N b do not form AU or GC complementarity;
  • Each of the delivery groups is independently connected to the functional group via a covalent bond or through a linking group; each of the functional groups is selected from the group consisting of small molecule therapeutic agents that have a therapeutic effect on tumors. group.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the conjugate of the present disclosure, and a pharmaceutically acceptable carrier.
  • the present disclosure also provides use of the conjugates and/or pharmaceutical compositions of the present disclosure in the preparation of medicaments for the treatment of tumors and tumor-related diseases or symptoms.
  • the present disclosure also provides a method for treating tumors and tumor-related diseases or symptoms, the method comprising administering to a subject in need an effective amount of the conjugate of the present disclosure and/or or pharmaceutical compositions.
  • the present disclosure also provides a kit comprising the conjugates and/or pharmaceutical compositions of the present disclosure.
  • the conjugates and pharmaceutical compositions provided by the present disclosure have excellent ability to target tumors, especially glioma tissues and cells, and can significantly treat or alleviate tumors and tumor-related diseases and/or symptoms.
  • the delivery group in the conjugate provided by the present disclosure can specifically deliver various small molecule drug groups, such as small molecule toxin groups, to tumor tissues and show excellent tumor inhibitory effects.
  • the conjugate of the present disclosure can effectively deliver MMAE to different tumor tissues. While showing tumor targeting ability, it also reduces the risk of toxicity caused by the distribution of MMAE molecules in other tissues, and various administration All methods can effectively inhibit the increase rate of tumor volume and tumor weight, indicating that the conjugate of the present disclosure can effectively inhibit tumor proliferation.
  • further increasing the dosage of the conjugate resulted in almost no increase in tumor volume during the test period, showing a more excellent anti-tumor effect.
  • the inventors of the present disclosure unexpectedly discovered that the conjugates and/or pharmaceutical compositions of the present disclosure can efficiently pass through the blood-brain barrier and can be targeted to gliomas in the brain under systemic administration. , and significantly inhibited the increase in tumor volume and even reduced it to less than 1/10 of the initial volume, and even reduced it to less than 1/100 compared to the control group, indicating that the conjugate of the present disclosure can effectively penetrate the blood-brain barrier and efficiently It targets brain glioma and has a good effect on inhibiting tumor growth, showing good treatment compliance and high drug-making ability to effectively inhibit tumors. .
  • Figures 1A-1C are graphs respectively showing the fluorescence imaging results in mice at 1 hour, 24 hours and 48 hours after administration of different conjugates.
  • Figure 1D is a diagram showing fluorescence signal imaging of tumor tissues and kidneys of mice in each group after the mice were sacrificed on D5.
  • Figures 2A-2C are graphs respectively showing the fluorescence imaging results in mice at 1 hour, 24 hours and 48 hours after administration of different conjugates.
  • Figure 2D is a diagram showing fluorescence signal imaging of tumor tissues and kidneys of mice in each group after the mice were sacrificed on D6.
  • Figure 3 is a line graph showing changes in tumor volume over time in each group of mice after administration of conjugates or control compounds provided by the present disclosure.
  • Figure 4 is a fluorescence imaging diagram showing the brain tissue of mice establishing U118MG orthotopic tumor model after administration of different conjugates at 24h and 48h after administration.
  • Figure 5 is a line graph showing the changes in tumor light intensity values in U118MG orthotopic tumor model mice over time after administration of the conjugates or control compounds provided by the present disclosure.
  • Figure 6 is a line graph showing the changes in tumor volume over time in U118MG subcutaneous tumor model mice after administration of the conjugates or control compounds provided by the present disclosure.
  • Figure 7 is a line graph showing the changes in tumor volume over time in U118MG subcutaneous tumor model mice after administration of different concentrations of conjugates or control compounds provided by the present disclosure.
  • Figure 8 is a line graph showing the changes in tumor volume over time in U118MG subcutaneous tumor model mice after administration of different concentrations of conjugates or control compounds provided by the present disclosure.
  • Figure 9 is a line graph showing the changes in tumor volume over time in A549 subcutaneous tumor model mice after administration of different concentrations of conjugates or control compounds provided by the present disclosure.
  • A, U, C, G and T refer to adenine nucleotides, uracil nucleotides, cytosine nucleotides, guanine nucleotides and thymine nucleotides, respectively.
  • 2-Methylcytosine nucleotide refers to a nucleotide in which the hydrogen at the 2' position of the cytosine base in the cytosine nucleotide is replaced by a methyl group. The structure of these nucleotides is well known to those skilled in the art.
  • nucleic acid motif refers to a fragment of a nucleic acid sequence in an oligonucleotide, consisting of 1 or more nucleotides.
  • a motif is a fragment of a nucleic acid sequence that has a biological function.
  • alkyl refers to straight and branched chain saturated hydrocarbon radicals having a specified number of carbon atoms, typically 1 to 20 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms.
  • C 1 -C 6 alkyl refers to straight and branched chain alkyl groups containing 1 to 6 carbon atoms.
  • alkyl residue having a particular number of carbons it is intended to encompass 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 the same residue as alkyl but with two points of attachment.
  • alkenyl refers to an unsaturated branched or linear alkyl group having one or more carbon-carbon double bonds formed by adjacent atoms from the parent alkyl group. Obtained by removing a molecule of hydrogen from a carbon atom. The group can be in the cis or trans configuration of the double bond.
  • alkenyl groups include, but are not limited to: vinyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl base), prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl base, but-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl, etc.
  • alkenyl groups have 2 to 20 carbon atoms, and in other embodiments 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkenylene is a subset of alkenyl and refers to the same residue as alkenyl but with two points of attachment.
  • alkynyl refers to an unsaturated branched or linear alkyl group having one or more carbon-carbon triple bonds formed by adjacent atoms from the parent alkyl group. Obtained by removing two molecules of hydrogen from a carbon atom.
  • 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.
  • alkynyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkynylene is a subset of alkynyl and refers to the same residue as alkynyl but with two points of attachment.
  • heterocyclyl refers to a stable 3- to 18-membered non-aromatic cyclic group containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen , oxygen and sulfur. Unless otherwise stated in the specification, heterocyclyl is a monocyclic, bicyclic, tricyclic or tetracyclic ring system and may include fused or bridged ring systems. The heteroatoms in the heterocyclyl group may be oxidized heteroatoms. One or more nitrogen atoms, if present, may be quaternized nitrogen atoms. Heterocyclyl groups are partially or fully saturated. A heterocyclyl group can be attached to the rest of the molecule through any ring atom.
  • heterocyclyl groups include, but are not limited to: dioxanyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidine base, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxa Pyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithianyl ), tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-o-
  • aryl refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removal of a hydrogen atom from a ring carbon atom.
  • the aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and 6 to 18 carbon atoms of carbon, wherein one or more rings in the ring system are completely unsaturated, i.e., contain according to Hückel's theory Cyclic, delocalized (4n+2) ⁇ -electron system.
  • Aryl groups include, but are not limited to, phenyl, fluorenyl, and naphthyl groups.
  • Arylene is a subset of aryl and refers to the same residue as aryl but with two points of attachment.
  • Heteroaryl refers to a group derived from a 3- to 18-membered aromatic ring free radical, containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur.
  • a heteroaryl group may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, wherein one or more rings in the ring system are fully unsaturated, i.e., contain cyclic ionization according to Hückel's theory. Domain (4n+2) ⁇ -electron system.
  • Heteroaryl groups include fused or bridged ring systems. The heteroatoms in the heteroaryl group may be oxidized heteroatoms.
  • heteroaryl group is attached to the rest of the molecule through any ring atom.
  • heteroaryl groups include, but are not limited to: azepantrienyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzobisoxazolyl, benzofuranyl, benzene Oxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[ b][1,4]oxazinyl (benzo[b][1,4]oxazinyl), 1,4-benzodioxanyl (1,4-benzodioxanyl), benzonaphthofuranyl, benzo Oxazolyl, benzodioxolyl (benzodioxolyl), benzodioxin
  • the present disclosure provides a conjugate comprising one or more delivery groups and one or more functional groups; the delivery group is formed by the aforementioned aptamer removing one or more hydrogen atoms or One or more functional groups are formed; each of the delivery groups is independently connected to the functional group via a covalent bond, or connected through a linking group; each of the functional groups is selected from the group that has the ability to treat tumors The acting small molecule therapeutic agent group.
  • the conjugates of the present disclosure are capable of delivering functional groups to tumors by linking them via covalent bonds or linking groups to form conjugates.
  • the delivery group is formed by removing one or more hydrogen atoms or one or more functional groups from an aptamer.
  • the aptamer includes a continuous nucleotide sequence connecting two adjacent ones.
  • the groups of each nucleotide are 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
  • the nucleotide sequence has the sequence shown in formula (1):
  • T 1 is a motif consisting of 1-3 nucleotides. The inventors found that the presence of T 1 allows the conjugates provided by the present disclosure to display efficient tumor targeting effects.
  • T 1 consists of 2 nucleotides, in which case the conjugate provided by the present disclosure has more excellent tumor targeting ability.
  • T1 consists of 2 nucleotides and contains at least one C.
  • T 1 in the 5'-3' direction, T 1 is CU, UC, or AC.
  • T 2 is a motif consisting of 0-15 nucleotides. The inventors found that T with these nucleotide numbers and various nucleotide sequences did not significantly affect the tumor targeting ability of the conjugates provided by the present disclosure.
  • T2 consists of 0-10 nucleotides. In some embodiments, according to the 5'-3' direction, T2 consists of 1-9 nucleotides starting from U. In this case, the aptamer may have better stability.
  • T 2 does not contain a motif that is completely reverse complementary to T 1 .
  • 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 a nucleotide sequence or motif according to the 5' Each nucleotide in the -3' direction can form base pairing with each nucleotide in the 3'-5' direction of the nucleotide sequence or motif at the other end.
  • "reverse complement” includes one or more of AU, GC, and UG complement.
  • S 1 and S 4 are each a motif composed of 3-7 nucleotides, and S 1 and S 4 are the same length and completely reverse complementary. Aptamers with the above S 1 and S 4 motifs have better stability and can target tumor tissues and cells for a longer period of time.
  • S 1 and S 4 each consist of 3-5 nucleotides and are the same length.
  • GC complementation accounts for more than 40% of the total complementation amount. At this time, the conjugate provided by the present disclosure has further better stability and tumor targeting. ability.
  • S 1 is GCU and S 4 is AGC, or S 1 is GAGU and S 4 is GCUC, or S 1 is GGAGU and S 4 is GCUCU, or S 1 is UAUGG and S 4 is CCAUG.
  • Na and Nc are each a motif consisting of 1-4 nucleotides, each nucleotide in Na is not complementary to every nucleotide in Nc , and in Na and Nc
  • the total number of U accounts for more than 50% of the total number of nucleotides in Na and Nc .
  • Aptamers with the above Na and Nc motifs show excellent tumor tissue targeting ability.
  • the sum of the numbers of nucleotides in Na and Nc is an integer from 2 to 4.
  • the sum of the number of nucleotides in Na and Nc is 3 or 4, and the sum of the number of Us in Na and Nc is 2 or 3.
  • Na and/or Nc are U, UU, UC, or CU in the 5'-3' direction.
  • S 2 and S 3 are each a motif consisting of 1-4 nucleotides. S 2 and S 3 are the same length and are completely reverse complementary. By including S2 and S3 motifs, the conjugates provided by the present disclosure show good stability and excellent tumor targeting ability. In some embodiments, S2 and S3 each consist of 2-3 nucleotides and are the same length. In some embodiments, the reverse complement formed by S 2 and S 3 includes at least one GC complement, in which case 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.
  • N b is a motif consisting of 3-6 nucleotides, and the nucleotides at both ends of N b are not AU or GC complementary. Without being limited by theory, an aptamer with the above N b motif can maintain a specific configuration in space, thereby enabling the conjugate provided by the present disclosure to stably and efficiently target tumor tissues and cells.
  • Nb consists of 4-5 nucleotides.
  • N b in the 5'-3' direction, N b is GACG, GACGU, GACCG, UACU, GUUG, or GAUCU.
  • the inventors of the present disclosure unexpectedly discovered that the delivery group formed by the aptamer having the sequence represented by the above formula (1) can effectively target tumors, especially glioma tissue, thereby allowing the conjugation provided by the present disclosure
  • the drug can specifically enter tumor cells, thereby delivering therapeutic groups more effectively at the cellular level.
  • the length of the continuous nucleotide sequence is 18-50 nucleotides, or 20-40 nucleotides, or 21-36 nucleosides. acid, or 24-32 nucleotides.
  • the delivery group formed by aptamers with these continuous nucleotide lengths and the conjugates provided by the present disclosure including the delivery group can be targeted to tumors more easily, and have advantages in terms of synthesis cost and targeting effect. Good balance.
  • the continuous nucleotide sequence has the sequence shown in the following SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3:
  • T 1 is CU
  • S 1 is GCU
  • Na is U
  • S 2 is CA
  • N b GACG
  • S 3 is UG
  • N c is UU
  • S 4 is AGC
  • T 2 is UU;
  • T 1 is CU
  • S 1 is GAGU
  • Na is U
  • S 2 is CA
  • N b is GACG
  • S 3 is UG
  • N c is UU
  • S 4 is GCUC
  • T 2 is U;
  • T 1 is CU
  • S 1 is GAGU
  • Na is U
  • S 2 is CA
  • N b is GACG
  • S 3 is UG
  • N c is UU
  • S 4 is GCUC
  • T 2 is U.
  • the continuous nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 4:
  • N 1 , N 2 and N 3 are each independently one of A, U, C and G, and N 4 is U, C or G or a motif composed of two of U, C or G; N 5 is U, CU or UU; N 6 is CU, UC or AC; N 7 is U, UU or UUN 8 , and N 8 is a motif composed of 1-15 nucleotides.
  • T 1 is the motif represented by N 6
  • S 1 is the motif represented by GGAGU
  • Na is U
  • S 2 is CA
  • Nb is N 1
  • the motif N 1 N 2 N 3 N 4 composed of N 2 , N 3 and N 4
  • S 3 is UG
  • N c is the motif represented by N 5
  • S 4 is GCUC and the first nucleoside in N 7 Motif composed of acid
  • T 2 is the motif composed of the remaining nucleotides in N 7 .
  • the aptamer containing the nucleotide sequence shown in SEQ ID NO: 4 above can more effectively target tumors, especially gliomas, and be enriched in tumor tissues.
  • the motif N 1 N 2 N 3 N 4 composed of N 1 , N 2 , N 3 and N 4 is one of GACG, GACGU, GACCG, UACU, GUUG or GAUCU, including these motifs
  • the aptamer has higher tumor-specific targeting effect.
  • N 5 is U or UU.
  • the conjugates provided by the present disclosure all have excellent targeting effects on tumors.
  • the aptamer has the nucleotide sequence shown in any one of SEQ ID NOs: 5-11:
  • conjugates provided by the present disclosure having the above nucleotide sequence show a high targeting effect on tumors.
  • motif N8 consists of 1-15 nucleotides. In some embodiments, N8 consists of 1-8 nucleotides.
  • the presence of the motif N 8 makes the conjugates provided by the present disclosure more stable to exonucleases in the body, thereby enabling the tumor-targeting effect to be exerted for a longer period of time in the body.
  • N8 can increase or maintain the tumor targeting effect of the conjugates provided by the present disclosure.
  • the motif N 8 consists of 8 nucleotides.
  • the nucleotide sequence of motif N 8 is CCGAUCUC in the 5'-3' direction.
  • the contiguous nucleotide sequence has the nucleotide sequence shown in any one of SEQ ID NOs: 12-14:
  • the end groups of the ribose 5' end of the 5' end nucleotide and the ribose 3' end of the 3' end nucleotide are independently hydroxyl or phosphate groups, and the selection of these end groups
  • the targeting capabilities of the conjugates provided by the present disclosure are not altered.
  • the terminal groups at the 5' end of the ribose sugar of the 5' terminal nucleotide and the 3' end of the ribose sugar of the 3' terminal nucleotide are both hydroxyl groups.
  • each nucleotide may be a modified or unmodified nucleotide.
  • modification of nucleotides may alter the stability and/or tumor targeting capabilities of the conjugates provided by the present disclosure.
  • at least one nucleotide in the conjugates provided by this disclosure is a modified nucleotide.
  • at least one of the groups connecting two adjacent nucleotides in the conjugates provided by the present disclosure has a phosphate group of a modifying group.
  • each of the modified nucleotides is independently a 2′-halogen modified nucleotide, a 2′-alkoxy modified nucleotide, 2 '-alkyl modified nucleotide, 2'-substituted alkyl modified nucleotide, 2'-amino modified nucleotide, 2'-substituted amino modified nucleotide, 2'- One of deoxynucleotides, nucleotides with modified bases, and nucleotide analogs.
  • fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is substituted with fluorine and has a structure represented by the following formula (7).
  • Non-fluorinated modified nucleotides refers to nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by a non-fluorinated group.
  • each non-fluorinated modified nucleotide is independently selected from nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosyl group of the nucleotide with a non-fluorinated group. A sort of.
  • Nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorine group are well known to those skilled in the art. 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, 2′-deoxynucleoside One of the glycosides.
  • the 2'-alkoxy modified nucleotide is a methoxy-modified nucleotide (2'-OMe), as shown in formula (8).
  • the 2'-amino modified nucleotide (2'- NH2 ) is represented by formula (9).
  • the 2'-deoxynucleotide (DNA) is represented by formula (10):
  • base modifications include, but are not limited to, adding one or more methyl groups to a base.
  • thymine (T) is considered a type of base-modified uracil (U).
  • 2-methylcytosine is considered a type of base-modified cytosine (C).
  • Nucleotide analogues are those that can replace nucleotides in nucleic acids, but are structurally different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxyribonucleotides Ribonucleotide group.
  • nucleotide analogs may be isonucleotides, bridged nucleic acid (BNA), or acyclic nucleotides.
  • BNA refers to constrained or inaccessible nucleotides.
  • BNA may contain a five-membered ring, a six-membered ring, or a seven-membered ring bridged structure with a "fixed"C3'-endoglycocondensation. The bridge is typically incorporated into the 2',4'-position of the ribose sugar to provide a 2',4'-BNA nucleotide.
  • BNA can be LNA, ENA, cET BNA, etc., wherein LNA is represented by formula (12), ENA is represented by formula (13), and cET BNA is represented by formula (14):
  • Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of the nucleotide.
  • the acyclic nucleotide can be unlocked nucleic acid (UNA), glycerol nucleic acid (GNA) or peptide nucleic acid (PNA), wherein UNA is represented by formula (15) and GNA is represented by formula (16) :
  • R is selected from H, OH or alkoxy (O-alkyl).
  • Peptide nucleic acids are a class of nucleotide analogs formed by replacing the glycoside-phosphate backbone with a polypeptide backbone.
  • the peptide nucleic acid may be, for example, a nucleotide analog formed by substituting a 2- aminoethylglycine linkage for a glycoside-phosphate unit.
  • Isonucleotides are compounds formed by changing the positions of the bases on the ribose ring in nucleotides.
  • the isonucleotide can be a compound formed by moving the base from the 1′-position of the ribose ring to the 2′-position or the 3′-position, as shown in formula (17) or (18).
  • Base represents a nucleic acid base, such as A, U, G, C or T; R is selected from H, OH, F or a non-fluorine group as described above.
  • the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET, UNA, and GNA.
  • each non-fluoro-modified nucleotide is a methoxy-modified nucleotide, above and below, the methoxy-modified nucleotide refers to the 2' of the ribosyl group -Nucleotides formed by replacing the hydroxyl group with a methoxy group.
  • each cytosine nucleotide in the contiguous nucleotide sequence in the conjugates provided by the present disclosure is a fluoro-modified cytosine nucleotide, and/or the Each uracil nucleotide in the contiguous nucleotide sequence is a fluorinated modified uracil nucleotide.
  • each nucleotide in the contiguous sequence of nucleotides in the conjugates provided by the present disclosure is a 2'-methoxy modified nucleotide.
  • the present disclosure provides conjugates in which one or more uracil nucleotides have modified bases.
  • a thymine base (T) is considered a uracil base (U) with a methyl modification.
  • the group connecting two adjacent nucleotides can be a phosphate group or a modified phosphate group.
  • the modification of the phosphate group is, for example, replacing at least one non-bridging oxygen atom in the phosphate group with a sulfur atom to form a phosphorothioate group or a phosphorodithioate group.
  • at least one group connecting two adjacent nucleotides in the conjugates provided by the present disclosure is a phosphorothioate group.
  • at least one of the three groups connecting two adjacent nucleotides between the first four nucleotides at the 5' end of the continuous nucleotide sequence is a phosphorothioate Ester group.
  • At least 2 of the 3 groups connecting two adjacent nucleotides between the first four nucleotides at the 5' end of the continuous nucleotide sequence are phosphorothioates. Ester group. In some embodiments, at least one of the groups connecting two adjacent nucleotides between the first four nucleotides at the 3' end of the continuous nucleotide sequence is a phosphorothioate group. In some embodiments, at least 2 of the 3 groups connecting two adjacent nucleotides between the first four nucleotides at the 3' end of the continuous nucleotide sequence are phosphorothioates. Ester group. In some embodiments, each group connecting two adjacent nucleotides in the contiguous nucleotide sequence is a phosphorothioate group.
  • the conjugate provided by the present disclosure with the above modifications is not only low in cost, but also makes it difficult for ribonucleases in the body to cleave the linking group, thereby increasing the stability of the conjugate provided by the present disclosure and making it more resistant. Nuclease hydrolysis performance.
  • the conjugate provided by the present disclosure including the above-mentioned modified delivery group has higher activity of targeting tumor tissue and/or cells.
  • the contiguous nucleotide sequence has a nucleotide sequence set forth in one of SEQ ID NOs: 15-39:
  • the capital letters C, G, U, and A represent the base composition of nucleotides;
  • the small letter m means that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide;
  • the small letter f means The adjacent nucleotide to the left of the letter f is a fluorinated modified nucleotide;
  • the lowercase letter s indicates that the two nucleotides to the left and right of the letter s are connected by a phosphorothioate group.
  • the present disclosure provides a conjugate having a structure represented by formula (101):
  • each R AP group is independently a group having a structure shown in formula (2):
  • each AP group is the same or different and independently represents one of the delivery groups; each A 0 group is the same or different and independently represents one of the functional groups; R j , each R k Or each R i is the same or different, each independently represents a covalent bond or a connecting group, and both R i and R k are different at the same time as a covalent bond; m 0 is an integer from 1 to 6; n 0 is 1- An integer of 6, each n 1 independently represents an integer of 0-4; Indicates the site at which a group is covalently attached.
  • m 0 is an integer of 1-6, that is, the conjugate represented by formula (101) contains 1-6 functional groups A 0 .
  • m 0 is an integer of 1-4, that is, the conjugate represented by formula (101) contains 1-4 functional groups A 0 .
  • m 0 is 1, that is, the conjugate represented by formula (101) contains 1 functional group A 0 .
  • n 0 is an integer from 1 to 6, that is, the conjugate represented by formula (101) contains 1 to 6 R AP groups. From the perspective of delivery efficiency and cost, in some embodiments, n 0 is an integer of 1-3, that is, the conjugate represented by formula (101) contains 1-3 R AP groups. In some embodiments, n 0 is 1, that is, the conjugate represented by formula (101) contains 1 R AP group.
  • each n 1 independently represents an integer from 0 to 4, and both Ri and Rk are not covalently bonded at the same time, thus each R AP group contains 1 to 5 delivery groups. AP. In some embodiments, each n 1 independently represents an integer from 0 to 1, such that each R AP group contains 1 to 2 delivery groups AP. In some embodiments, n 0 is 1 and n 1 is 0. In this case, the conjugate represented by formula (101) contains 1 delivery group AP.
  • the function of R k and R i is to covalently connect the delivery group AP to the R j group and to the functional group A 0 via the R j group. Therefore, any R k or R i that can achieve the above connection without negatively affecting the effects of the delivery group AP and the functional group A 0 can be used in the present invention.
  • each n 1 is 0, and each R i is independently a covalent bond, or one of the following linking groups
  • One or more connection combinations C 1 -C 20 alkylene, phosphate bond, phosphorothioate bond, amide bond, ester bond, ether bond, thioether bond, disulfide bond, 1, 2, 3- Triazole subunit, polyethylene glycol subunit, pyrrolidine subunit, 2-oxopyrrolidine subunit, phenylene, cyclohexylene, 2-succinimide subunit, 2-thiobutanediyl Imide subunit, amino acid subunit, nucleotide subunit.
  • the linking group Rj includes linking groups known to those skilled in the art to be useful in antibody drug conjugates.
  • the linking group R j may be cleavable or non-cleavable.
  • linking group Rj may be cleavable.
  • "cleavable" means that upon targeting of the conjugates of the present disclosure to a tumor, the linker group Rj undergoes covalent cleavage in the intratumoral environment and/or within the tumor cells, releasing the individual therapeutic The agent group produces the therapeutic effect.
  • the linker R j comprises an activating enzyme linker, a sulfatase-cleavable linker, a galactose-cleavable linker, a lysosomal protease-sensitive linker, a peptidyl linker group, a glucuronide linking group, an acid-sensitive cleavable linking group, or a glutathione-sensitive disulfide linking group.
  • linking group R j comprises a peptidyl linking group.
  • the peptidyl linking group is selected from the group consisting of valine-citrulline dipeptide linker (Val-Cit), alanine-alanine dipeptide linker (Ala-Ala), valine - One or more of the alanine dipeptide linker (Val-Ala) and the glycine-glycine-phenylalanine-glycine tetrapeptide linker (Gly-Gly-Phc-Gly).
  • linking group R j is selected from N-succinimide 4-(2-dithiopyridine)butyrate (SPDB), N-succinimide-4-(2-thiopyridine) Subunit) valerate (SPP), (S)-2-((S)-2-amino-3-methylbutanamide)-5-ureidopentanoic acid (Val-Cit-PAB-OH), N -Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or 2-(phosphate-(CH 2 ) 6 -S-)- One of the maleimidocaproyl-valine-citrulline-p-aminobenzyl subunits.
  • SPDB N-succinimide 4-(2-dithiopyridine)butyrate
  • SPP N-succinimide-4-(2-thiopyridine) Subunit) valerate
  • SPP S)-2-((S)-2-amino
  • the linking group R j comprises a linking group enumerated in McKertish CM, Kayser V. Advances and Limitations of Antibody Drug Conjugates for Cancer. Biomedicines. 2021 Jul 23;9(8):872., as cited The entire content of this document is incorporated into this article in its entirety.
  • the linking group R j includes a valine-citrulline dipeptide linker (Val-Cit), a polyethylene glycol subunit, an iminohexyl subunit, an N-succinimidyl group, One or more of the GAU trinucleotide linking groups.
  • each R i is independently a covalent bond, a disulfide bond, a dodecylene group, a valine-citrulline dipeptide linker (Val-Cit), a polyethylene glycol subunit , iminohexyl subunit, N-succinimidyl group or GAU trinucleotide subunit or a connection combination of two.
  • the function of the R j group is to connect the R AP group with the functional group A 0 , thereby specifically delivering the functional group A 0 to the tumor through the tumor targeting effect of the delivery group AP in the R AP group.
  • Tumor tissue and/or cells Tumor tissue and/or cells. Therefore, any R j group that can achieve the above connection without affecting the tumor targeting effect of the delivery group AP and the effect of the functional group A 0 can achieve the purpose of the present invention and solve the technology to be solved by the present invention. question.
  • the conjugate represented by formula (1) after the conjugate represented by formula (1) reaches the tumor tissue and/or enters the tumor cell, the R j is cleaved, releasing the pharmaceutically active molecule corresponding to the separate functional group A 0 .
  • the R j does not undergo cleavage in vivo, and the presence of the R j group and the R AP group in the conjugate will not affect the therapeutic effect of the functional group A 0 .
  • R j is a covalent bond
  • m 0 is 1.
  • the conjugate represented by formula (101) contains 1 functional group A 0 and 1 R AP group, each R AP groups are directly linked to the functional group A 0 .
  • each R AP group is linked to the same atom of functional group A0 .
  • each R AP group is attached to a different atom of functional group A0 .
  • R j is a linking group comprising a backbone moiety, a side chain moiety , and a conjugation linkage.
  • each side chain moiety is independently a covalent bond, or a linear alkylene group of 1 to 70 carbon atoms in length, or one or more of the linear alkylene groups
  • each conjugation linkage is independently a covalent bond or a combination of one or more of the following linkage structures: C 1 -C 10 linear alkylene, phosphate ester bond, phosphorothioate Ester bond, amide bond, ester bond, ether bond, disulfide bond, 1,2,3-triazole subunit, polyethylene glycol subunit, pyrrolidine subunit, 2-oxopyrrolidine subunit, phenylene base, cyclohexylene group, 2-succinimide subunit, 2-thiosuccinimide subunit, amino acid subunit, nucleotide subunit.
  • each of the conjugated linking parts in the linking group R j is respectively connected to the main chain part and one of the functional groups A 0 ; the number of side chain parts is n 0 , Each side chain moiety is respectively linked to the backbone moiety and one of the R AP groups.
  • each functional group A 0 and R AP group is each independently linked to the linking group R j .
  • all side chain moieties are connected to the same atom in the backbone moiety; alternatively, each side chain moiety is connected to a different atom in the backbone moiety.
  • m 0 is 1, and the linking group R j includes a structure shown in formula (301):
  • k is an integer from 1 to 3; LC is the main chain part, L A is the side chain part, LB is the conjugation connection part, Indicates the site at which a group is covalently attached.
  • the length of LC is 5-30 atoms, wherein the length of LC refers to the longest atomic chain in LC formed from the atoms directly connected to LA to the atoms directly connected to LB The number of atoms in the chain. To simplify the structure, in some embodiments, the length of LC is 8-25 atoms.
  • the conjugated linker LB is a connection combination of one or more of the following 1-5 linkages: phosphate bond, phosphorothioate bond, amide bond, ester bond, ether bond, and disulfide bond.
  • k is an integer of 1-3;
  • L C contains any one of the groups represented by formula (L1)-(L3), through the group represented by formula (L1)-(L3) The ether bond in the group is connected to the L A part:
  • L C contains a group represented by formula (L1), and the O atom in group (L1) is directly connected to L A.
  • L B is a phosphate bond or disulfide bond
  • Each L A is a covalent bond, or each L A is selected from the group consisting of groups (L4)-(L23) and their connecting combinations:
  • each j1 is an integer from 1 to 10;
  • Each R' is C 1 -C 10 alkyl
  • Each Ra is a hydrogen atom, a C 1 -C 10 alkyl group, or selected from the group consisting of groups (L24) - (L37):
  • each LA is a connected combination of at least 2 of the groups (L4)-(L9), (L13), (L14), (L18). In some embodiments, each LA is a connected combination of at least 2 of the groups (L4), (L5), (L7), (L9), (L13), (L14), (L18).
  • L A has an amide bond-containing structure as shown in Formula (302)
  • L B has an N-acylpyrrolidine-containing structure as shown in Formula (303), containing a carbonyl group and an oxygen atom
  • L C is a linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trimethylolaminomethane:
  • n 302 , q 302 and p 302 are each independently an integer from 2 to 6.
  • n 302 , q 302 and p 302 are each independently an integer from 2 or 3;
  • n 303 is an integer from 4 to 16.
  • Select the location, n 303 is an integer from 8 to 12, Indicates the site at which a group is covalently attached.
  • each of the side chain portions L A is connected to one R AP group through a phosphate bond, an ether bond or an ester bond, and is connected to the oxygen atom of the hydroxyl group in the main chain portion L C through a phosphate bond, an ether bond or an ester bond.
  • the main chain part LC is connected by forming an ether bond;
  • the conjugated connection part LB is connected by forming an amide bond between the carbonyl group in the formula (303) and the nitrogen atom of the amino group in the main chain part LC , and is connected by
  • the oxygen atom in the formula (303) is connected to the functional group A 0 by forming a phosphate bond, an ether bond or an ester bond.
  • the backbone moiety LC is a linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trimethylolaminomethane via the oxygen atom of the hydroxyl group.
  • Each of the side chain moieties L A is connected by an ether bond and is connected to the conjugate linker L B by an amide bond via the nitrogen atom of the amino group.
  • 1-3 side chains in the linking group Rj are connected to the carbon atoms of the same aminomethyl group and are connected to the R AP group containing the delivery group through the conjugated linker LB.
  • the conjugate has the structure shown in formula (305):
  • the linking group R j comprises a structure shown in formula (306):
  • n 306 is an integer from 0 to 3
  • each p 306 is independently an integer from 1 to 6,
  • the linkage combination formed by all pyrrolidin subunits and any possible phosphodiester groups constitutes the backbone part, consisting of the carbonyl group attached to the nitrogen atom of the pyrrolidin subunit and the one marked by *
  • a chain of atoms between the oxygen atoms shown constitutes each side chain moiety, and the side chain part is connected to the R AP group by an ether bond formed by the oxygen atom marked by *;
  • at least one of the oxygen atoms marked by # To conjugate the linking part and form an ether bond, ester bond or phosphate bond with the functional group A 0 , and the remaining oxygen atoms marked by # are connected to hydrogen atoms to form hydroxyl groups, or with C 1 -C 3 alkyl groups The connection forms a C 1 -C 3 alkoxy group.
  • the present disclosure provides conjugates having a structure shown in formula (307a), (307b) or (307c):
  • the present disclosure provides conjugates having a structure shown in Formula (308):
  • n 308 can be an integer from 1 to 10; in some embodiments, n 308 can be an integer from 2 to 6, taking into account various aspects such as ease of synthesis, structure/process cost, and tumor cell specificity. In some embodiments, n 308 is 3 or 4.
  • Each R 3 is independently a functional group A 0 , or a R AP group including a delivery group AP;. In some embodiments, at least one R3 is the functional group A0 , and at least one R3 is RAP . In some embodiments, one R 3 is a functional group A 0 and the remaining R 3 are R AP groups.
  • each m 308 when each m 308 is independently selected from an integer of 2-10, it is considered possible to make the spatial positions between the multiple delivery groups AP in the conjugate more suitable for contact with the surface of tumor cells. The corresponding receptors interact.
  • each m 308 is independently an integer of 2-5, In some embodiments, each m 308 is equal.
  • each R 308 is independently selected from H, C 1 -C 10 alkyl, C 1 -C 10 haloalkyl, and C 1 -C 10 alkoxy, formula (308) does not change The properties of the conjugates indicated are suitable for achieving the purposes of this disclosure.
  • each R 308 is independently selected from H, methyl, or ethyl.
  • each R 308 is H.
  • Each L 1 connected to the functional group A 0 represents the conjugation linkage, and each L 1 connected to the R AP represents the side chain moiety.
  • one R 3 is the functional group A 0 and the remaining R 3 are the R AP group.
  • one or more L 1 serves as the side chain moiety connecting the R AP group to an N atom on the nitrogen-containing backbone; and an additional one or more L 1 serves as the conjugation linker , connect the functional group A 0 to the N atom on the nitrogen-containing skeleton.
  • the nitrogen-containing skeleton together constitutes the main chain part of the connecting group Rj .
  • nitrogen-containing backbone refers to the chain structure in the structure represented by formula (308), in which the carbon atom to which R 308 is attached and the N atom are interconnected.
  • each L 1 is independently 3-25 atoms.
  • each L1 is independently 4-15 atoms in length.
  • L1 is defined as a linear alkylene group for convenience, it may not be a linear group or have a different name, such as an amine or alkenyl group resulting from the above substitutions and/or substitutions.
  • the length of L is the number of atoms in the chain connecting two junction points.
  • the ring obtained by replacing the carbon atoms of the linear alkylene group (such as heterocyclylene or heteroarylene) is calculated according to the minimum number of atoms between the connection points on the ring corresponding to the ring in the chain. The length of the section.
  • L 1 is selected from the group consisting of the groups represented by the above formulas L4-L23 and any connected combinations thereof. In some embodiments, each L 1 is independently selected from the group consisting of linked combinations of at least 2 of groups L4-L9, L13, L14, L18. In some embodiments, each L 1 is independently a connected combination of at least 2 of the groups L4, L5, L7, L9, L13, L14, and L18.
  • each L 1 simultaneously contains a connection site connected to an N atom on the nitrogen-containing skeleton and a functional group A 0 or the R
  • the connection site where the AP group is connected to the N atom on the nitrogen-containing skeleton forms an amide bond with the N atom.
  • one or more L1 is selected from B5, B6, B5' or B6':
  • q 2 is an integer from 1 to 10. In some embodiments, q2 is an integer from 1 to 5.
  • each R AP group contains multiple functional groups.
  • each functional group in the compound of formula (308) is the same functional group.
  • each functional group in the compound of formula (308) is a functional group serving the same purpose and function.
  • the compound of formula (308) contains different types of functional groups for different purposes and functions.
  • the compound represented by formula (308) has 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):
  • linking group R j comprises nucleotide sequence I and nucleotide sequence II, each of said nucleotide sequence I and said nucleotide sequence II comprising 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, and the functional group is connected to the Nucleotide sequence II, and said nucleotide sequence I and said nucleotide sequence II do not trigger an immune response or toxic reaction in a subject.
  • 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 The lengths are equal and both are 10-20 modified or unmodified nucleotides; alternatively, the nucleotide sequence I and the nucleotide sequence II are both composed of 17 nucleotides and are completely reverse complementary.
  • the 3' end of the delivery group is connected to the ribose 5' position of the 5' terminal nucleotide of the nucleotide sequence I via a phosphate bond
  • the functional group is connected to the The ribose 5' position of the 5' terminal nucleotide of the nucleotide sequence II;
  • the functional group includes a nucleotide sequence, the 3' end of the nucleotide sequence is connected to the The ribose sugar 5' position of the 5' terminal nucleotide of the nucleotide sequence I.
  • the nucleotide sequence I and the nucleotide sequence II have the sequences shown in SEQ ID NO: 40 and SEQ ID NO: 41 respectively:
  • nucleotide sequence I and the nucleotide sequence II have the sequences shown in SEQ ID NO: 42 and SEQ ID NO: 43 respectively:
  • the functional group is a small molecule therapeutic agent group that has a therapeutic effect on tumors, and each small molecule therapeutic agent group is independently selected from a cytotoxic group, an antibiotic group, or an angiogenesis inhibitory group. agent.
  • the conjugates of the present disclosure can specifically deliver the small molecule therapeutic agent group to the tumor, thereby affecting the disease process or symptoms of the tumor through the action of the small molecule therapeutic agent group.
  • cytotoxic groups for example, by specifically delivering cytotoxic groups to tumors through the conjugates of the present disclosure, causing specific death of cancer cells in the tumors, thereby reducing the low-targeting band of the cytotoxin itself. While causing side effects, it can significantly reduce the number of cancer cells in the tumor, thereby treating the tumor.
  • the small molecule therapeutic agent group is formed by removing one or more hydrogen atoms or one or more functional groups from the following small molecule therapeutic agent: the small molecule therapeutic agent is selected from the group consisting of methotrexate, Mycins, vinca alkaloids, auristatin (including MMAE and MMAF), calicheamicin, maytansine, camptothecin, and calicheamicin.
  • the small molecule therapeutic agent group is a group formed by removing one or more hydrogen atoms or one or more functional groups from monomethyl auristatin E (MMAE).
  • MMAE monomethyl auristatin E
  • the functional groups may be included in the conjugates of the present disclosure by any suitable means.
  • the functional group A 0 can be connected to the main chain moiety through the aforementioned conjugation linker.
  • the conjugates provided by the present disclosure further comprise one or more delivery aid groups selected from the group consisting of C 10 -C 30 hydrocarbon groups, cholesterol groups, and phospholipid groups. one or more.
  • the conjugates provided by the present disclosure are better compatible with the in vivo environment in the central nervous system, may have better bioavailability, and/or make the conjugates provided by the present disclosure more compatible with the in vivo environment in the central nervous system. Deliver drugs to tumors more efficiently.
  • the delivery aid group is linked to the delivery group or the linking group via a covalent bond or linking group. In some embodiments, the delivery aid group is linked to the functional group.
  • the synthesis method of the conjugate provided by the present disclosure includes contacting the protected conjugate with a deprotection reagent in a solvent under deprotection reaction conditions, and isolating to obtain the conjugate provided by the present disclosure.
  • a protected conjugate is a compound in which any active functional group in the conjugate provided by the present disclosure is protected by a protecting group.
  • the reactive functional groups include, but are not limited to, hydroxyl, amino and/or phosphate groups, and the protecting groups are, respectively, hydroxyl protecting groups, amino protecting groups and/or phosphate hydroxyl protecting groups (e.g., cyanoethyl base protecting group).
  • the solvent, deprotection reaction conditions and deprotection reagents used are selected and determined.
  • the deprotection reaction conditions, solvents and deprotection reagents are those used in solid phase synthesis of nucleic acids.
  • the method includes adding the protected conjugate into a mixed solution of methylamine aqueous solution and ammonia water, and the deprotection reaction conditions include reaction at normal temperature and pressure for 1-5 h.
  • the methylamine aqueous solution and the saturated concentrated ammonia solution are mixed in equal volumes to obtain the mixed solution, and the dosage of the solution relative to the protected conjugate is 0.1-10 ml/ ⁇ mol.
  • the separation includes purification by column chromatography, collecting the product eluate and removing the solvent.
  • the purification conditions may be, for example, using a preparative ion chromatography purification column and eluting with a gradient eluent of sodium chloride aqueous solution and sodium phosphate aqueous solution.
  • the conjugate provided by the present disclosure has a structure represented by formula (101), and the synthesis method of the protected conjugate includes in an organic solvent, under coupling reaction conditions, will contain active
  • the compound of the group R One or more hydrogen atoms or one or more functional groups are formed, and each of the functional groups is independently a small molecule therapeutic agent group that has a therapeutic effect on tumors, wherein the delivery group and the functional group Any reactive group of is protected by a protecting group, and the reactive group R x1 and the reactive group R x2 are groups capable of forming a covalent bond or connecting group R j through reaction.
  • the molar ratio of the delivery group connected to the active group R x1 and the functional group connected to the active group R x2 is m 0 :n 0 .
  • the active groups in the delivery group and functional group include, but are not limited to, one or more of hydroxyl, amino, and phosphate groups, and the protective group is correspondingly a hydroxyl protective group. , one or more of amino protecting group, phosphate hydroxyl protecting group (for example, cyanoethyl protecting group).
  • the compound containing the reactive group R x1 and the delivery group can obtain compounds containing the reactive group R x1 and the delivery group through various methods.
  • the compound containing the reactive group R Obtained through synthesis.
  • the compound containing the reactive group R The sequence of nucleotides in the chain is used to connect the nucleoside monomers in sequence, where at least one nucleoside monomer is a nucleoside monomer with an active group R
  • the phosphoramide solid-phase synthesis method connects a phosphoramidite monomer or a protected phosphoramidite monomer with an active group R x1 , and then removes the protecting group to form an active group R x1 .
  • the coupling reaction conditions are condensation reaction conditions or thiol-disulfide exchange reaction conditions.
  • the coupling reaction conditions are condensation reaction conditions, and the condensation reaction conditions are acylation condensation reaction conditions, dehydration condensation reaction conditions or click chemical reaction conditions, and the reactive group R x1 and the reactive group R x2 is a group capable of undergoing the aforementioned condensation reaction.
  • the condensation reaction conditions are conditions for an acylation condensation reaction, and the active groups R x1 and R x2 are groups capable of undergoing an acylation condensation reaction to form R I .
  • the condensation reaction conditions are conditions for dehydration condensation reaction, one of the active groups R x1 and R x2 is a group containing an acid halide group or a carboxyl group, and the other is a group containing an amino group or a hydroxyl group. group.
  • the condensation reaction conditions are click chemistry conditions, one of the reactive groups R x1 and R x2 is a group containing an alkynyl group, and the other is a group containing an azide group.
  • the condensation reaction conditions are conditions of a Michael addition reaction, one of the active groups R x1 and R x2 is a group containing a thiol group, and the other is a group containing a succinimide group. group.
  • the condensation reaction conditions are N-hydroxysuccinimide-carbodiimide (NHS-EDC) coupling reaction conditions, and one of the active groups R x1 and R x2 One is a group containing N-hydroxysuccinimide (NHS), and the other is a group containing a carbodiimide group (EDC).
  • the compound containing the reactive group R x1 and the delivery group is prepared by contacting an aptamer with the reactive group R x0 and a cross-linking agent under the conditions of a coupling reaction,
  • the cross-linking agent contains click chemical active groups and acylation groups.
  • the reactive group R is an active ester group, for example, it can be one of an NHS ester group, an imide ester group, and a pentafluorophenyl ester group.
  • the cross-linking agent can be done as follows: Michael E., et al.
  • the reactive group RxO is amino.
  • the coupling conditions are basic conditions.
  • the alkaline condition is a condition in which a weak alkali aqueous solution is present, such as a sodium bicarbonate aqueous solution.
  • the aptamer with the active group R x0 is synthesized by synthesizing the aptamer. It is prepared by using phosphoramidite monomers containing active groups at corresponding positions in the process. Those skilled in the art can obtain phosphoramidite monomers containing reactive groups in various ways.
  • the reactive group R The monomer may be 6-(trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite monomer which is easily commercially available, wherein the reactive group R x0 is an amino group, and the reactive group R x0 can be a deprotection reaction that can be easily realized by those skilled in the art after connecting the phosphoramidite monomer to the single chain of the oligonucleotide through the phosphoramidite solid phase synthesis method. It is obtained by removing the trifluoroacetyl protecting group (such as ammonolysis of concentrated ammonia).
  • the coupling reaction condition is one of sulfhydryl-disulfide bond exchange reactions, one of the reactive groups R x1 and R Sulfur bonded leaving group.
  • R x1 in the above-mentioned phosphoramidite monomer containing reactive group R x1 exists in the form of protected R x1 ', and the preparation method also includes deprotection reaction conditions Next, the step of isolating the compound containing the active group R x1 and the delivery group by contacting the prepared compound containing the protected active group R x1 ' and the delivery group with a deprotecting reagent.
  • the disulfide bond activator is a disulfide pyridine.
  • phosphorus acid amide monomer containing the reactive group R Amide monomers are commercially available, for example, phosphoramidite monomers represented by formula (105) can be obtained commercially.
  • n 105 and m 105 are each independently an integer from 1 to 10.
  • the coupling reaction conditions are thiol- disulfide bond exchange reaction conditions, the reactive group R
  • the skilled person can obtain it in various known ways, for example, by using a phosphoramidite monomer containing a thiol group, prepared by a phosphoramidite solid phase synthesis method, or obtained commercially.
  • the coupling reaction conditions are phosphoramidite solid phase synthesis reaction conditions
  • the reactive group R x2 is a phosphoramidite group
  • the reactive group R x2 and a functional group include
  • the compound may be, for example, a readily commercially available compound containing a phosphoramidite group and a small molecule therapeutic agent group.
  • the coupling reaction conditions are Michael addition reaction conditions
  • the reactive group R x2 is an N-succinimide group
  • the reactive group R x2 is included
  • the compound with the functional group may be, for example, a readily commercially available compound containing an N-succinimide group and a small molecule therapeutic agent group.
  • the reactive group R x1 and the reactive group R x2 are nucleotide sequence I and nucleotide sequence II respectively, and the nucleotide sequence I and the nucleotide sequence II each comprise 5-25 modified or unmodified nucleotides, said nucleotide sequence I and said nucleotide sequence II being at least partially reverse complementary, said delivery group being linked to said nucleotide sequence I,
  • the functional group is connected to the nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II do not trigger an immune response or toxic reaction in the subject, and the coupling reaction conditions It is the reaction condition for annealing to form nucleic acid double strands.
  • nucleotide sequence I and the nucleotide sequence II each consist of 17 nucleotides and are completely reverse complementary. In some embodiments, the nucleotide sequence I and the nucleotide sequence II have the sequences shown in SEQ ID NO: 40 and SEQ ID NO: 41, respectively. In some embodiments, the nucleotide sequence I and the nucleotide sequence II have the sequences shown in SEQ ID NO: 42 and SEQ ID NO: 43, respectively.
  • the delivery group is formed by the removal of one or more hydrogen atoms or functional groups by the aptamer.
  • 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 an aptamer at The delivery group is formed by removing a hydrogen atom from the 5' hydroxyl group of the 5' terminal nucleotide; in some embodiments, the delivery group is formed by the aptamer removing a hydrogen atom from the 3' hydroxyl group of the 3' terminal nucleotide; in some In embodiments, the delivery group is formed by an aptamer removing the 5' hydroxyl group from the 5' terminal nucleotide; in some embodiments, the delivery group is formed by an aptamer removing the 3' hydroxyl group from the 3' terminal nucleotide.
  • the delivery group is formed by the aptamer removing the ribose 2'-hydroxyl group from the nucleotide it contains.
  • 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.
  • Modified nucleotide groups can be introduced into the conjugates provided by the present disclosure by using nucleoside monomers with corresponding modifications, methods of preparing nucleoside monomers with corresponding modifications, and modifying nucleotide groups. Methods for introducing aptamers are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or prepared using known methods.
  • the conjugates may also be used in the present disclosure in the form of pharmaceutically acceptable salts or precursor compounds thereof.
  • pharmaceutically acceptable salts refer to the formation of corresponding salts of the drug in order to increase the stability, solubility and/or bioavailability of the drug without causing additional pharmaceutical side effects to the human body, For example, potassium salt, sodium salt, carboxylate, etc.
  • Precursor compound means that although it is structurally and functionally identical to the conjugate, it can react to form the conjugate provided by the present disclosure after entering the body or in a body fluid environment. , thereby exerting effects and achieving the purpose provided by the present disclosure.
  • these precursor compounds can increase the stability of the drug, extend the sustained release time, increase bioavailability and other effects.
  • the precursor compound includes a precursor group that reacts in humans to form all functional groups A0 in the conjugate.
  • the precursor compound includes a compound in which all active hydroxyl groups in the conjugate are replaced with acetoxy groups.
  • the precursor compound includes a prodrug group that is a residue formed from a precursor compound of the therapeutic agent corresponding to the functional group in the conjugate.
  • the prodrug group may 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.
  • the present disclosure also provides a pharmaceutical composition comprising the conjugate provided by the present disclosure and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can be a carrier commonly used in the art, such as but not limited to water, physiological saline, magnetic nanoparticles (such as nanoparticles based on Fe 3 O 4 or Fe 2 O 3 ), carbon Nanotubes (carbon nanotubes), mesoporous silicon (mesoporous silicon), calcium phosphate nanoparticles (calcium phosphate nanoparticles), polyethylenimine (PEI), polyamide dendrimer (polyamidoamine (PAMAM) dendrimer), Poly(L-lysine), PLL, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) , poly(D&L-lactic/glycolic acid)copolymer (PLGA), poly(2-aminoethyl ethylene phosphate) (poly(2-aminoethyl ethylene phosphate), PPEEA) and one or more of poly(2-dimethylaminoethy
  • the 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; Molecular weight proteins; compositions that reduce clearance or hydrolysis of any co-administered substances; excipients; stabilizers and buffers. Detergents may also be used to stabilize the composition or to increase or decrease absorption of the pharmaceutical composition.
  • the physiologically acceptable compound may also include one or more wetting agents, emulsifiers, dispersants or preservatives, especially to prevent the growth or action of microorganisms.
  • the physiologically acceptable compounds are known to those skilled in the art and will not be described in detail in this disclosure. As will be readily understood by those skilled in the art, 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 substances.
  • pharmaceutically acceptable carriers are sterile and generally free of undesirable materials.
  • the pharmaceutical composition provided by the present disclosure may further contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjusters and buffers, toxicity adjusters, etc., such as sodium acetate, sodium chloride, potassium chloride, chloride Calcium, sodium lactate, etc., the concentration of the conjugate provided by the present disclosure in the pharmaceutical composition can vary within a wide range, and is mainly selected according to a specific administration method based on fluid volume, viscosity, body weight, etc.
  • the pharmaceutical composition has no special requirements on the content of the conjugate and the pharmaceutically acceptable carrier.
  • the weight ratio of the conjugate to the pharmaceutically acceptable carrier can be It is 1: (1-500). In some embodiments, the above weight ratio is 1: (1-50).
  • the pharmaceutical composition may also contain other pharmaceutically acceptable auxiliary materials, which may be one or more of various preparations or compounds commonly used in the art.
  • the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.
  • the pH buffer can be a trishydroxymethylaminomethane hydrochloride buffer with a pH of 7.5-8.5 and/or a phosphate buffer with a pH of 5.5-8.5, for example, it can be a phosphate with a pH of 5.5-8.5. Buffer.
  • the protective agent may be at least one of myo-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.
  • the osmotic pressure regulator may be sodium chloride and/or potassium chloride.
  • the content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliosmole/kg (mOsm/kg).
  • the content of the osmotic pressure regulator can be easily determined by those skilled in the art based on the desired osmotic pressure.
  • the dosage of the preparation made from the pharmaceutical composition during administration will be adjusted due to different administration methods.
  • the pharmaceutical composition can be a liquid preparation, such as an injection; it can also be a freeze-dried powder injection, which is mixed with liquid excipients during administration to prepare a liquid preparation.
  • the liquid preparation can be used for, but is not limited to, subcutaneous, intramuscular or intravenous injection, and the pharmaceutical composition can also be delivered by, but not limited to, puncture injection, oropharyngeal inhalation, or nasal administration.
  • the pharmaceutical composition is for subcutaneous, intramuscular, intravenous or intrathecal administration.
  • the pharmaceutical composition may be in the form of a liposome formulation.
  • the pharmaceutically acceptable carrier used in the liposome formulation includes an amine-containing transfection compound (hereinafter also referred to as an organic amine), a helper lipid, and/or a pegylated Lipids.
  • an organic amine, auxiliary lipid and pegylated lipid can be respectively selected from the amine-containing transfection compounds described in Chinese patent application CN103380113A (which is incorporated by reference into this disclosure in its entirety) or One or more of its pharmaceutically acceptable salts or derivatives, auxiliary lipids and pegylated lipids.
  • the organic amine can be a compound represented by formula (201) described in Chinese patent application CN103380113A or a pharmaceutically acceptable salt thereof:
  • X 101 and X 102 are each independently O, S, NA or CA, where A is hydrogen or a C 1 -C 20 hydrocarbon chain;
  • R 101 , R 102 , R 103 , R 104 , R 105 , R 106 and R 107 are each independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or linear aliphatic group Group, cyclic or acyclic, substituted or unsubstituted, branched or linear heteroaliphatic group, substituted or unsubstituted, branched or linear acyl group, substituted or unsubstituted Substituted, branched or linear aryl, substituted or unsubstituted, branched or linear heteroaryl;
  • x is an integer from 1 to 10;
  • n is an integer from 1 to 3
  • m is an integer from 0 to 20
  • g, e and f are each independently an integer from 1 to 6
  • HCC represents a hydrocarbon chain
  • each *N represents a nitrogen atom in formula (201).
  • R 103 is a polyamine. In other embodiments, R 103 is a ketal. In some embodiments, each of R 101 and R 102 in formula (201) is independently any substituted or unsubstituted, branched or linear alkyl or alkenyl group, the alkyl The radical or alkenyl group has 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.
  • R 103 may be any of the following formulas (204) to (213):
  • g, e and f are each independently an integer from 1 to 6
  • each "HCC” represents a hydrocarbon chain
  • each * shows that R 103 is the same as in formula (201)
  • Possible attachment points to the nitrogen atoms in where each H at any * position can be replaced to effect attachment to the nitrogen atom in formula (201).
  • the compound represented by formula (201) can be prepared according to the description in Chinese patent application CN103380113A.
  • the organic amine is an organic amine represented by formula (214) and/or an organic amine represented by formula (215):
  • the auxiliary lipid is cholesterol, cholesterol analogs and/or cholesterol derivatives
  • the pegylated lipid is 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy (polyethylene glycol)]-2000.
  • the molar ratio between 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).
  • composition particles formed from the conjugates provided by the present disclosure and the above-described amine-containing transfection reagents have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, and more typically, the 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.
  • 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 160nm.
  • the conjugate in a pharmaceutical composition formed from a conjugate provided by the present disclosure and the above-mentioned amine-containing transfection reagent, is PEGylated with all lipids (e.g., organic amines, auxiliary lipids, and/or
  • the weight ratio (weight/weight ratio) of 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 in the range 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.
  • the weight ratio of the conjugate provided by the present disclosure to all lipids is
  • each component of the pharmaceutical composition may exist independently when sold, and may exist in the form of a liquid preparation when used.
  • 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 existing adapter is replaced by the conjugate provided by the present disclosure.
  • the body or conjugate is sufficient; in some embodiments, it can be prepared as follows:
  • the organic amine, auxiliary lipid and PEGylated lipid are suspended in alcohol according to the above molar ratio and mixed to obtain a lipid solution; the amount of alcohol is such that the total mass concentration of the obtained lipid solution is 2-25 mg/mL, For example, it can be 8-18 mg/mL.
  • the alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid near room temperature, for example, ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 One or more, for example, it can be ethanol.
  • the conjugate provided by the present disclosure is dissolved in a buffered salt solution to obtain an aqueous conjugate solution.
  • concentration of the buffer salt solution is 0.05-0.5M, for example, it can be 0.1-0.2M.
  • the pH of the buffer salt solution is adjusted to 4.0-5.5, for example, it can be 5.0-5.2.
  • the amount of the buffer salt solution is such that the concentration of the conjugate does not exceed 0.6mg/mL, for example, it can be 0.2-0.4mg/mL.
  • the buffer salt is selected from one or more of soluble acetate and soluble citrate, for example, it can be sodium acetate and/or potassium acetate.
  • the volume ratio of the lipid solution and the aqueous conjugate solution is 1:(2-5), for example, it can be 1:4.
  • 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.
  • Its physical and chemical parameters are a pH value of 6.5-8, an encapsulation rate of not less than 80%, and a particle size of 40-200nm, polydispersity index not higher than 0.30, osmotic pressure 250-400mOsm/kg; for example, the physical and chemical parameters can be pH value 7.2-7.6, encapsulation rate not less than 90%, particle size 60-100nm, and more
  • the dispersion index is not higher than 0.20, and the osmotic pressure is 300-400mOsm/kg.
  • concentration or dilution can be performed before, after or at the same time as impurities are removed.
  • Various existing methods can be used to remove impurities, such as using a tangential flow system, a hollow fiber column, ultrafiltration at 100K Da, and the ultrafiltration exchange solution is phosphate buffer saline (PBS) with pH 7.4.
  • PBS phosphate buffer saline
  • the sterilization method can adopt various existing methods, for example, filtration sterilization can be performed on a 0.22 ⁇ m filter.
  • the present disclosure also provides use of the conjugates and/or pharmaceutical compositions provided by the present disclosure in the preparation of medicaments for the treatment of tumors and tumor-related diseases or symptoms.
  • the present disclosure also provides a method of treating tumors and tumor-related diseases or symptoms, the method comprising administering to a subject in need thereof a conjugate and/or pharmaceutical combination described in the present disclosure. things.
  • the methods of the present disclosure can effectively treat tumors and tumor-related diseases or symptoms; and, under the highly specific targeting effect of the conjugates provided by the present disclosure, Distribution of therapeutic agents to undesirable other body organs/tissues can be reduced, reducing potential side effects.
  • radiotherapy and/or chemotherapy drugs that are commonly used in the field of tumor treatment and are known to have significant side effects, it is of great significance and significant value.
  • administering/administering refers to the administration of a conjugate and/or pharmaceutical composition by a method or pathway that at least partially localizes the conjugate and/or pharmaceutical composition to a desired site to produce the desired effect. or the pharmaceutical composition is placed into a subject.
  • Routes of administration suitable for the methods of the present disclosure include local administration and systemic administration. Generally speaking, local administration results in the delivery of more conjugates and/or pharmaceutical compositions to a specific site compared to the subject's entire body; whereas systemic administration results in the delivery of the conjugates and/or pharmaceutical compositions in combination with The substance is delivered to substantially the entire body of the subject.
  • the inventors of the present disclosure unexpectedly discovered that the conjugates and/or pharmaceutical compositions of the present disclosure can efficiently pass through the blood-brain barrier and can be targeted to tumors in the brain under systemic administration. This further improves the delivery efficiency of functional groups, saves costs and reduces undesirable side reactions.
  • Administration to the subject may be by any suitable route known in the art, including, but not limited to, oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, or transdermal. medicine, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration and topical administration (including buccal administration and sublingual administration).
  • the frequency of administration may be once or more daily, weekly, every two weeks, every three weeks, monthly or yearly.
  • the dosage of the conjugates and/or pharmaceutical compositions described in the present disclosure can be conventional dosages in the art, and the dosage can be determined according to various parameters, especially the age, weight and gender of the subject. Toxicity and efficacy can be measured in cell culture or experimental animals by standard pharmaceutical procedures, such as determining the LD50 (the dose that causes 50% of the population to be lethal) and the ED50 (the dose that causes 50% of the maximum response intensity in a quantitative response, and in a qualitative response Medium refers to the dose that causes a positive reaction in 50% of the experimental subjects). Dosage ranges for humans can be derived based on data from cell culture assays and animal studies.
  • the conjugates are administered to The amount of the conjugate in the conjugate and/or pharmaceutical composition: 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 It is 0.05-20 mg/kg body weight, in a further embodiment it is 0.1-15 mg/kg body weight, and in still further embodiment it is 0.1-10 mg/kg body weight.
  • the above amounts may be preferred when administering the conjugates and/or pharmaceutical compositions of the present disclosure.
  • the present disclosure provides a kit comprising a conjugate and/or pharmaceutical composition provided by the present disclosure.
  • kits described in the present disclosure may provide the conjugate and/or pharmaceutical composition in one container.
  • a kit of the present disclosure may include a container providing a pharmaceutically acceptable excipient.
  • the kit may also include other ingredients, such as stabilizers or preservatives.
  • a kit of the present disclosure can include at least one additional therapeutic agent in a container other than the container in which the conjugates and/or pharmaceutical compositions of the present disclosure are provided.
  • the kit may contain instructions for mixing the conjugate and/or pharmaceutical composition with pharmaceutically acceptable carriers and/or excipients or other ingredients, if any.
  • the conjugate and pharmaceutically acceptable carrier and/or excipients and the pharmaceutical composition, and/or pharmaceutically acceptable excipients can be provided in any form, such as liquid form, dry form form or lyophilized form.
  • the conjugate and pharmaceutically acceptable carrier and/or excipients and the pharmaceutical composition and optional pharmaceutically acceptable excipients are substantially pure and/or sterile.
  • sterile water can be provided in the kits of the present disclosure.
  • the reagents and culture media used in the following examples are all commercially available products.
  • the nucleic acid electrophoresis, real-time PCR and other operations used are all described in Molecular Cloning (Cold Spring Harbor LBboratory Press (1989)). method to proceed.
  • the conjugates numbered AP1-AP8 and AP18 in Table 1 were synthesized by solid-phase synthesis method, and the nucleotide sequences corresponding to AP1-AP8 and AP18 in Table 1 were followed from the 3′-5′ direction. After connecting all the nucleoside monomers, connect the Cy5 phosphoramidite monomer (purchased from Suzhou Jima Company, batch number CY5P21H1B) according to the solid-phase synthesis method to connect the nucleoside phosphoramidite monomers. Subsequently, the nucleotide sequence was added to a mixed solution of equal volumes of methylamine aqueous solution and ammonia water.
  • the dosage of the solution relative to the oligonucleotide was 0.5 ml/ ⁇ mol.
  • the reaction was carried out at 25°C for 2 hours.
  • the solid was removed by filtration, and the supernatant was vacuumed. Concentrate to dryness.
  • the specific conditions include using a Sephadex column for desalting.
  • the filler is Sephadex G25 and eluted with deionized water.
  • the obtained eluate was concentrated to remove the solvent and lyophilized to obtain conjugates AP1-AP8 and AP18 in which the 5' position of the ribose sugar of the 5' terminal nucleotide was connected to the fluorescent group Cy5 through a phosphate linking group, respectively.
  • the prepared conjugates are diluted to a concentration of 0.2 using ultrapure water (made by Milli-Q ultrapure water instrument, resistivity 18.2M ⁇ *cm (25)) mg/mL, use liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters Company, model: LCT Premier) for molecular weight detection.
  • ultrapure water made by Milli-Q ultrapure water instrument, resistivity 18.2M ⁇ *cm (25)
  • LC-MS liquid chromatography-mass spectrometry
  • the capital letters C, G, U, and A indicate the base composition of nucleotides; the small letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the small letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; f indicates that the nucleotide adjacent to the left side of the letter f is a fluorinated modified nucleotide; CY5 indicates the connection site of the fluorescent dye group Cy5 (Cyanine 5) group on the aptamer.
  • the capital letters C, G, U, and A represent the base composition of nucleotides; the small letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; MMAE represents The connection site of the small molecule drug group MMAE (monomethyl auristatin E) group on the aptamer.
  • conjugate 20 was prepared according to the following steps.
  • the dye group in conjugate AP2 was replaced by the small molecule drug group MMAE, and the linking group was 2-(phosphate Ester-(CH 2 ) 6 -S-)-maleimidocaproyl-valine-citrulline-p-aminobenzyl subunit.
  • Vc MMAE Dissolve 24 mg Vc MMAE (18.56 ⁇ mol, 5eq, purchased from CSN Company, batch number CSN16143-005) in 6.0 ml DMF, and add 60 ⁇ l triethylamine to obtain a Vc MMAE solution.
  • the obtained crude conjugate 20 was diluted with 0.5 ml of purified water and filtered with a 0.45 ⁇ m membrane.
  • the filtrate was purified using Agilent semi-preparative reversed-phase column chromatography.
  • the product peak eluate was collected, and the solvent was evaporated to remove conjugate 20 (55 mg, yield 56.7%).
  • the molecular weight was detected by LC-MS, the theoretical value: 12092.73, the measured value: 12091.39, the measured value is consistent with the theoretical value, indicating that the conjugate 20 has the structure shown in S3, and the conjugate 20 combines the dye in the conjugate AP2
  • the group is replaced with the small molecule drug group MMAE, and the linking group is 2-(phosphate-(CH 2 ) 6 -S-)-maleimidocaproyl-valine-citrulline-p Aminobenzyl subunit (2-(phosphate-(CH 2 ) 6 -S-)-MC-Val-Cit-PAB).
  • This experimental example examines the targeting properties of the prepared conjugates AP2, AP3-8 and comparison AP9-contrast AP12 in mice.
  • U118MG human colloid was cultured in DMEM complete medium (MACGENE Company, Cat. No. CM15019) supplemented with 10% fetal bovine serum (FBS, RMBIO Company) at 37 in an incubator containing 5% CO 2 /95% air.
  • Tumor cells purchased from Guangzhou Genio Biotechnology Co., Ltd.).
  • mice 24 NOD-SCID mice (purchased from Spefford (Beijing) Biotechnology Co., Ltd.), female, 12 weeks old.
  • the above cell culture medium was inoculated into the subcutaneous position of the right forelimb of NOD-SCID mice.
  • the inoculation volume was 100 ⁇ L per mouse, that is, 1 ⁇ 10 7 cells were inoculated into each mouse. After injection, the mice were continued to be raised for 20 days to obtain mice inoculated with U118MG subcutaneous tumors.
  • the aptamers AP2, AP3-8 and comparison AP9-comparison AP12 prepared above were prepared into 0.3 mg/mL solutions using DMEM medium.
  • Administration began 14 days after U118MG cells were inoculated, and the day of administration was recorded as D1.
  • mice inoculated with U118MG subcutaneous tumors were randomly divided into 12 groups, with 2 mice in each group:
  • mice For 7 groups of mice, AP2, AP3, AP4, AP5, AP6, AP7 or AP8 were administered to each mouse in each group respectively.
  • the volume of single administration was 10 ⁇ L/g mouse body weight. Calculation shows that single administration The dose is 3mg/kg, which are recorded as test groups 1A-1G respectively;
  • each mouse in each group was given contrast AP9, contrast AP10, contrast AP11 or contrast AP12 respectively.
  • the single administration volume was 10 ⁇ L/g mouse body weight, and the single administration dose was calculated. 3mg/kg, respectively recorded as control group 1H-1K;
  • each mouse was given DMEM culture medium with a dosage volume of 10 ⁇ L/g mouse body weight, which was recorded as the blank control group 1Y.
  • each mouse was imaged in vivo using the small animal in vivo optical imaging system IVIS Lumina Series III. On D5, mice in each group were sacrificed and tumor tissues and kidneys were harvested for fluorescence imaging.
  • FIGs 1A-1C are respectively diagrams showing the fluorescence imaging results in mice at 1h, 24h and 48h after administration of different conjugates, in which the leftmost one of the three mice in each small picture is It is the blank control group of 1Y mice.
  • the blank control group did not show any fluorescence signal; unlike this, 1 hour after administration, the mice in each test group and the control group showed fluorescence signals at the subcutaneous tumors; as can be seen from Figures 1B and 1C, At 24h and 48h after administration, only the mice in the test group 1A-1G showed strong fluorescence signals at the subcutaneous tumors, while the mice in the control group 1H-1K showed almost no fluorescence signals or only very weak ones. Fluorescent signal.
  • Figure 1D is a diagram showing the fluorescence signal imaging of tumor tissues and kidneys of mice in each group after the mice were sacrificed on D5, where Blank represents the blank control group 15Y.
  • Blank represents the blank control group 15Y.
  • the tumor tissues of the mice in the blank control group 1Y and the control group 1H-1K showed almost no fluorescence signal or only a very weak fluorescence signal; in contrast, the test group 1A given the conjugate of the present disclosure
  • the tumor tissues of the -1G mice all showed strong fluorescence signals, while only weak fluorescence signals were shown at the metabolic organ kidney, indicating that compared with the control conjugate, the delivery group in the conjugate provided by the present disclosure is included.
  • the conjugates of the group can target tumor tissues more stably and efficiently.
  • This experimental example examines the targeting properties of the prepared conjugates AP2, AP1 and comparison AP13-contrast AP17 in mice.
  • U118MG human colloid was cultured in DMEM complete medium (MACGENE Company, Cat. No. CM15019) supplemented with 10% fetal bovine serum (FBS, RMBIO Company) at 37 in an incubator containing 5% CO 2 /95% air.
  • Tumor cells purchased from Guangzhou Genio Biotechnology Co., Ltd.).
  • mice 16 NOD-SCID mice (purchased from Spefford (Beijing) Biotechnology Co., Ltd.), male, 12 weeks old.
  • the above cell culture medium was inoculated into the subcutaneous position of the right forelimb of NOD-SCID mice.
  • the inoculation volume was 100 ⁇ L per mouse, that is, 1 ⁇ 10 7 cells were inoculated into each mouse. After injection, the mice were continued to be raised for 20 days to obtain mice inoculated with U118MG subcutaneous tumors.
  • the aptamers AP2, AP1 and comparison AP13-contrast AP17 prepared above were prepared into 0.3 mg/mL solutions using DMEM medium.
  • Administration began 21 days after U118MG cells were inoculated, and the day of administration was recorded as D1.
  • mice inoculated with U118MG subcutaneous tumors were randomly divided into 8 groups, with 2 mice in each group:
  • mice For the two groups of mice, AP2 or AP1 was administered to each mouse in each group, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 3 mg/kg, which were recorded in sequence as Test Group 2A-2B;
  • each mouse in each group was given contrast AP13, contrast AP14, contrast AP15, contrast AP16, or contrast AP17.
  • the volume of a single dose was 10 ⁇ L/g mouse body weight. The calculation showed that a single dose The dosage was 3 mg/kg, which were recorded as control group 2C-2G respectively;
  • each mouse was given DMEM culture medium with a dosage volume of 10 ⁇ L/g mouse body weight, which was recorded as the blank control group 2Y.
  • each mouse was imaged in vivo using the small animal in vivo optical imaging system IVIS Lumina Series III. On D6, mice in each group were sacrificed and tumor tissues and kidneys were harvested for fluorescence imaging.
  • FIGs 2A-2C are respectively diagrams showing the fluorescence imaging results in mice at 1h, 24h and 48h after administration of different conjugates, in which the leftmost one of the three mice in each small picture is It is the blank control group of 2Y mice.
  • the blank control group did not show any fluorescence signal; unlike this, 1 hour after administration, the mice in each test group and the control group showed fluorescence signals at the subcutaneous tumors; as can be seen from Figures 2B and 2C, At 24h and 48h after administration, only the mice in test groups 2A and 2B showed strong fluorescence signals at the subcutaneous tumors, while the mice in the blank control group 2Y and the control groups 2C-2G showed no fluorescence signals at all.
  • Figure 2D is a diagram showing the fluorescence signal imaging of tumor tissues and kidneys of mice in each group after the mice were sacrificed on D6, where Blank represents the blank control group 2Y.
  • Blank represents the blank control group 2Y.
  • the tumor tissues of the mice in the blank control group 2Y and the control group 2C-2G showed no fluorescent signal at all; in contrast, the tumor tissues of the mice in the test group 2A or 2B were administered with the conjugate of the present disclosure.
  • aptamers with various sequences shown in formula (1) can stably and efficiently Targeting tumor tissue further demonstrates that the conjugates provided by the present disclosure containing delivery groups formed by these aptamers can effectively reach tumor tissue.
  • This experimental example examined the anti-tumor activity of the prepared conjugate 20 in mice.
  • mice in this experiment were purchased from Spefford Company.
  • the germ line is NOD-SCID, the grade is SPF, both genders are female, and the age is 6-8 weeks.
  • U118MG glioma cells were purchased from Genio.
  • U118MG cells growing in the logarithmic phase were digested and resuspended in DMEM complete medium (MACGENE Company, Cat. No. CM15019) supplemented with 10% fetal bovine serum (FBS, GIBCO Company) and cultured until the cell density was 1 ⁇ 10 8 cells/mL to obtain a culture medium containing U118MG cells.
  • the above culture medium containing U118MG cells was inoculated into the subcutaneous position of the right forelimb of each mouse, with an injection volume of 100 ⁇ L. Thus, each mouse was inoculated with 1 ⁇ 10 7 U118MG glioma cells.
  • the conjugate AP2 prepared above was prepared into a 1.94 mg/mL solution using PBS.
  • the day of cell inoculation was recorded as D1, and the cells were administered once each on D8, D12, D16 and D20.
  • mice 36 mice were randomly divided into the following 6 groups, with 6 mice in each group:
  • PBS was administered by tail vein injection, with a single administration volume of 10 ⁇ L/g;
  • control group 3b the above-mentioned conjugate AP2 solution was administered by tail vein injection, with a single administration volume of 10 ⁇ L/g and a single administration dose of 15.5 mg/kg;
  • MMAE solution was administered by tail vein injection, with a single administration volume of 10 ⁇ L/g and a single administration dose of 0.3 mg/kg;
  • the conjugate 20 solution with the above concentration of 0.625 mg/mL was administered by tail vein injection, with a single administration volume of 10 ⁇ L/g and a single administration dose of 5 mg/kg (based on oligonucleotide mass basis) containing MMAE at a dose equivalent to 0.3 mg/kg;
  • the conjugate 20 solution with the above concentration of 2.06 mg/mL was administered by tail vein injection, with a single administration volume of 10 ⁇ L/g and a single administration dose of 16.5 mg/kg (based on oligonucleotide acid mass) containing MMAE at a dose equivalent to 1 mg/kg;
  • the conjugate 20 solution with the above concentration of 1.25 mg/mL was administered by subcutaneous injection, with a single administration volume of 5 ⁇ L/g and a single administration dose of 5 mg/kg (based on the mass of the oligonucleotide). ) containing MMAE at a dose equivalent to 0.3 mg/kg.
  • the long diameter and short diameter of the tumor were determined by in vitro measurement. Tumor volume was calculated according to the formula 1/2 (long diameter ⁇ short diameter 2 ). Before the first administration on D8, the tumor volume of each group was measured and the average tumor volume was recorded. The tumor volume of each group was measured and recorded on D16, twice a week.
  • Figure 3 is a line graph showing changes in tumor volume over time in each group of mice. It can be seen from the results in Figure 3 that in the blank control group 3a and control group 3b that were only given PBS and AP2, the tumor volume increased rapidly; in the control group 3c that was only given MMAE, the tumor volume increase rate was reduced, indicating that MMAE itself has an effect on tumor proliferation. Shows inhibitory effect. Furthermore, test groups 3d and 3f, whose MMAE content was equivalent to that of the control group 3c, had significantly smaller tumor volumes during the test period than the control group 3c, showing more excellent anti-tumor activity than the control group 3c given MMAE alone. This shows that the conjugate provided by the present disclosure can effectively deliver MMAE to tumor tissue.
  • tumor targeting ability While showing tumor targeting ability, it also reduces the risk of toxicity caused by the distribution of MMAE molecules in other tissues, and various administration All methods can effectively inhibit tumor proliferation.
  • the tumor volume of test group 3e which further increased the dosage, hardly increased during the test period, showing a more excellent anti-tumor effect.
  • U118MG human colloid was cultured in DMEM complete medium (MACGENE Company, Cat. No. CM15019) supplemented with 10% fetal bovine serum (FBS, RMBIO Company) at 37 in an incubator containing 5% CO 2 /95% air.
  • Tumor cells purchased from Guangzhou Genio Biotechnology Co., Ltd.).
  • the cells were digested with 0.25wt% trypsin and collected. The supernatant was aspirated and the cells were resuspended in DMEM medium supplemented with 10% FBS to prepare a cell culture medium with a cell density of 4 ⁇ 10 7 cells/mL.
  • NOD-SCID mice purchased from Spefford (Beijing) Biotechnology Co., Ltd.
  • the above cell culture fluid was inoculated into NOD-SCID mice, and the cell culture fluid was injected into the right striatum of the mouse using the lateral cerebral ventricle injection method.
  • the position was AP (anteroposterior anteroposterior/anterior position): 1 mm
  • ML medial lateral
  • DV diorsal ventral
  • injection volume 10 ⁇ L that is, each mouse is inoculated with 4 ⁇ 10 5 cells.
  • the mice were kept for 14 days after injection.
  • AP2 and comparative AP19 were dissolved into conjugate solutions with a concentration of 0.3 mg/mL (based on aptamer) using 1 ⁇ DMEM medium. Take 4 of the aforementioned mice and administer them through tail vein injection. Inject AP2 and comparative AP19 solutions respectively. The dosage of all animals is calculated according to their body weight. The dosage volume is 10 ⁇ L/g. Based on the amount of aptamer, each The animal dosage was 3 mg/kg, and each group was administered to 2 mice, which were recorded as test group 4a and control group 4b respectively.
  • mice in each group were killed and the brain tissue was taken.
  • mice were killed and the brain tissue was taken.
  • the mouse brain tissue was analyzed in IVIS Lumina Series III. Fluorescence imaging. The results are shown in Figure 4.
  • Figure 4 is a fluorescence imaging diagram showing the brain tissue of mice establishing U118MG orthotopic tumor model after administration of blank control group 4Y, test group 4a and control group 4b at 24h and 48h after administration.
  • Blank represents the blank control group
  • Ith represents intrathecal injection
  • iv represents tail vein injection.
  • U118MG human glioma cells expressing the Luciferase (Photinus pyralis) reporter gene were cultured according to the method of Experimental Example 4. Take the U118MG-luc human glioma cells growing in the logarithmic phase, digest them with 0.25wt% trypsin and collect the cells. After centrifugation, aspirate the supernatant and resuspend the cells in serum-free DMEM medium to make the cell density: Cell culture medium of 4 ⁇ 10 7 cells/mL.
  • mice 24 Balb/C-nude nude mice (purchased from Spefford (Beijing) Biotechnology Co., Ltd.), male, 12 weeks old.
  • the above cell culture fluid was inoculated into the striatum of Balb/C-nude nude mice, and the mouse striatum injection method was used to inject the cell culture fluid into the right striatum of the mouse at the position of AP (anteroposterior/anterior/anterior). position): 1 mm, ML (medial lateral): 1.5 mm, DV (dorsal ventral): 3.5 mm, injection volume 10 ⁇ L, that is, each mouse is inoculated with 4 ⁇ 10 5 cells. After in situ tumor inoculation, the mice were continued to be fed for 14 days.
  • Conjugate 20 was dissolved into a conjugate solution with a concentration of 1 mg/mL (based on aptamer) using 1 ⁇ DMEM (purchased from Zhongke Maichen (Beijing) Technology Co., Ltd., batch number: K1902200) medium.
  • Conjugate 21 and comparative conjugate 24 were respectively dissolved into conjugate solutions with a concentration of 0.8 mg/mL (based on aptamer).
  • the small animal in vivo optical imaging system IVIS Lumina Series III was used to conduct in vivo imaging of each mouse. According to the brain fluorescence intensity, there were 6 mice in each group. The day of administration was recorded as D1 (i.e. , the first day of the experiment, the following D4, D8, etc. correspond to the fourth day, the eighth day of the experiment, and so on).
  • In vivo imaging method Each mouse was injected intraperitoneally with 10 ⁇ L/g body weight concentration of 15 mg/mL D-luciferin potassium salt working solution (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.), and in vivo imaging was performed 10 min after injection ( Lumina III small animal in vivo imaging system). After imaging, circle the fluorescence region (ROI) of the mouse brain, and the software measures the fluorescence intensity (Radiance). Under these conditions, the Luciferase (Photinus pyralis) reporter gene expressed in U118MG-luc human glioma cells can produce a fluorescent response, so the fluorescence intensity can reflect the proliferation number of glioma cells. The higher the fluorescence intensity, the greater the number of glioma cells.
  • mice in each group were administered drugs on D1, D4, D8 and D12 respectively. Weigh before administration and administer according to weight.
  • conjugate 20 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg, and the dose containing MMAE was equivalent to 0.3 mg/kg.
  • conjugate 21 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 4 mg/kg, and the dose containing MMAE was equivalent to 0.3 mg/kg.
  • control conjugate 24 was administered to each mouse respectively, with a single administration volume of 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 4 mg/kg, and the dose containing MMAE was equivalent to 0.3mg/kg.
  • DMEM culture medium was given to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight.
  • Figure 5 shows a line chart showing the changes in tumor fluorescence intensity over time in U118MG orthotopic tumor model mice after administration of the conjugates or control compounds provided by the present disclosure.
  • the tumor fluorescence intensity (Radiance) of the blank control group and the control group increased significantly, indicating that the number of U118MG human glioma cells increased significantly.
  • the fluorescence intensity of the test groups 5a and 5b administered with the conjugate provided by the present disclosure was significantly reduced, and the reduction was up to 1 order of magnitude, and compared with the control group, it could reach more than 2 orders of magnitude, indicating that U118MG human glue
  • the number of tumor cells is significantly reduced, and may be reduced to 1/10 of that at the beginning of the experiment, and may even be reduced to less than 1% of that of the control group. It can be seen that even if it is only administered subcutaneously, the conjugate provided by the present disclosure can effectively penetrate the blood-brain barrier and efficiently target into brain glioma, and has a good tumor growth inhibition effect, showing good treatment compliance. It has high drug potential and high efficacy in inhibiting tumors.
  • U118MG human glioma cells purchased from Guangzhou Genio Biotechnology Co., Ltd. were cultured according to the method of Experimental Example 2. Take the U118MG human glioma cells growing in the logarithmic phase, digest them with (0.25% trypsin), collect the cells, centrifuge to remove the supernatant, and resuspend the cells in serum-free DMEM medium to make a concentration of 1 ⁇ 10 8 cells/mL of cell culture medium.
  • mice 36 NOD-SCID mice (purchased from Spefford (Beijing) Biotechnology Co., Ltd.), male, 12 weeks old.
  • the above cell culture medium was inoculated subcutaneously on the right back of NOD-SCID mice, and the inoculation volume was 100 ⁇ L per mouse, that is, 1 ⁇ 10 7 cells were inoculated into each mouse.
  • the mice were kept for 7 days after injection.
  • Conjugate 20 was dissolved in serum-free DMEM medium to a conjugate solution with a concentration of 1 mg/mL (based on aptamer).
  • Conjugate 21, conjugate 22 and conjugate 23 were respectively dissolved into conjugate solutions with a concentration of 0.8 mg/mL (based on aptamer).
  • Administration began 7 days after U118MG cells were inoculated, and the day of administration was recorded as D8.
  • the experiment adopted abdominal subcutaneous administration, with administration once each on D8, D12, D16 and D20, for a total of 4 administrations.
  • DMEM was administered to each group of mice respectively, and the single administration volume was 5 ⁇ L/g mouse body weight;
  • MMAE was administered to each group of mice respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. Calculation showed that the single administration dose was 0.3 mg/kg;
  • conjugate 20 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg, and the dose containing MMAE was equivalent to 0.3 mg/kg;
  • conjugate 21, conjugate 22 or conjugate 23 were administered to each group of mice respectively.
  • the single administration volume was 5 ⁇ L/g mouse body weight.
  • the calculation showed that the single administration dose was 4mg/kg, which contains the dose of MMAE equivalent to 0.3mg/kg; respectively recorded as test groups 6d, 6e and 6f;
  • FIG. 6 is a line graph showing the change in tumor volume over time in mice at different days after administration of different conjugates. As can be seen from Figure 6, compared with the control group 6b or the blank control group, the tumor volume and tumor weight of the mice in the test groups 6c-6f administered the conjugate of the present disclosure were significantly reduced. The above results indicate that the conjugate of the present disclosure can effectively reach tumor tissues and display good anti-tumor activity.
  • mice inoculated with U118MG subcutaneous tumors were obtained, and the mice were continued to be raised after injection.
  • mice Seven days after inoculation of U118MG cells, all mice were randomly divided into 7 groups, with 6 mice in each group, and the mice in each group were administered drugs. The day of administration was recorded as D8. The experiment adopted abdominal subcutaneous administration, once each on D8, D11, D15, D29, D32 and D36, for a total of 6 administrations. Weigh before administration and calculate administration volume based on weight.
  • DMEM culture medium was given to each mouse respectively, and the single administration volume was 10 ⁇ L/g mouse body weight.
  • each mouse was given MMAE at a concentration of 0.01 mg/mL, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 0.1 mg/kg.
  • each mouse was administered conjugate 21 at a concentration of 0.165 mg/mL, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 1.65 mg/kg (corresponding to MMAE dose is 0.1mg/kg).
  • each mouse was administered a concentration of 0.165 mg/mL of comparative conjugate 25, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 1.65 mg/kg ( The corresponding MMAE dose is 0.1 mg/kg).
  • each mouse was given MMAE at a concentration of 0.03 mg/mL.
  • the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 0.3 mg/kg.
  • each mouse was administered conjugate 21 at a concentration of 0.5 mg/mL, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg (corresponding to MMAE The dose is 0.3mg/kg).
  • each mouse was given a concentration of 0.5 mg/mL of comparative conjugate 25, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg (corresponding to MMAE dose is 0.3mg/kg).
  • D9, D16, D19, D21, D24, D26, D29, D32, D36, D39, D43, D47, D53, D57, D60, D64, D67, D71, D74, D78, D81, D84, D88, D92 , D95 and D99 were used to measure the tumor volume.
  • the blank control group was measured at D53
  • the test groups 7a, 7b and the control group 7c were measured at D60 and the experiment was terminated.
  • the long diameter and short diameter of the tumor were determined by in vitro measurement. Tumor volume was calculated according to the formula 1/2 (long diameter ⁇ short diameter 2 ). After the experiment was terminated, tumor tissues from each group were collected, weighed, and the average value was determined. The results are shown in Figure 7.
  • Figure 7 is a line graph showing the changes in tumor volume over time in U118MG subcutaneous tumor model mice after administration of different concentrations of conjugates or control compounds provided by the present disclosure.
  • the tumor volume of test group 7b was significantly smaller than that of test group 7a and control group 7c, which were equivalent to MMAE content; during the test period, the tumor volume of test group 7e was significantly smaller than that of test group 7d and control group 7f, which were equivalent to MMAE content. , showing better anti-tumor activity than the administration of MMAE test groups 7a and 7d alone, as well as the administration of comparison conjugate 25.
  • the tumor weight in the mice administered the conjugate of the present disclosure was also significantly lower than that in the MMAE group and the control group. The above shows that the conjugate provided by the present disclosure can effectively deliver MMAE to tumor tissue. While showing tumor targeting ability, it also reduces the risk of toxicity caused by the distribution of MMAE molecules in other tissues, showing dose-related and Excellent anti-tumor effect.
  • mice inoculated with U118MG subcutaneous tumors were obtained, and the mice were continued to be raised after injection.
  • MMAE Dissolve MMAE into a solution with a concentration of 0.02 mg/mL using 10% DMSO + 90% serum-free DMEM medium (volume ratio); dissolve conjugate 20 and comparison conjugate 25 into a concentration of 0.33 mg/mL (to adapt Conjugate 21 and conjugate 23 were dissolved into a solution with a concentration of 0.26 mg/mL (based on aptamer); Conjugate 26 was dissolved into a solution with a concentration of 0.23 mg/mL (based on aptamer) (calculated) solution.
  • mice inoculated above Seven days after inoculation of U118MG cells, the mice inoculated above were divided into groups, with 6 mice in each group. The mice in each group were administered drugs, and the day of administration was recorded as D8. Weigh before administration, and calculate the administration volume based on the average weight of 20g per animal.
  • the experiment adopted abdominal subcutaneous administration, once each on D8, D12, D15 and D19, for a total of 4 administrations.
  • DMEM culture medium was given to each mouse respectively, with a single administration volume of 100 ⁇ L.
  • MMAE was administered to each mouse respectively, with a single administration volume of 100 ⁇ L. Calculation showed that the single administration dose was 0.1 mg/kg.
  • conjugate 20 was administered to each mouse respectively, with a single administration volume of 100 ⁇ L, and the calculation showed that the single administration dose was 1.65 mg/kg.
  • the comparison conjugate 25 was administered to each mouse respectively, with a single administration volume of 100 ⁇ L, and the calculation showed that the single administration dose was 1.65 mg/kg.
  • conjugate 21 was administered to each mouse respectively, with a single administration volume of 100 ⁇ L, and the calculation showed that the single administration dose was 1.32 mg/kg.
  • conjugate 23 was administered to each mouse respectively, with a single administration volume of 100 ⁇ L, and the calculation showed that the single administration dose was 1.32 mg/kg.
  • test group 8f conjugate 26 was administered to each mouse respectively, with a single administration volume of 100 ⁇ L, and the calculation showed that the single administration dose was 1.17 mg/kg.
  • the dosages of the above-mentioned groups 8b-8f are equivalent to containing MMAE at 0.1 mg/kg.
  • Tumor volume was measured on D1, D9, D16, D19, D22, D26, D30, D36, D40, D43, D47, D50, D54, D57, D61, D64, D68 and D71.
  • the blank control group was measured on D54, and the 8a group (only given MMAE) was measured on D64 and the experiment was terminated.
  • the long diameter and short diameter of the tumor were determined by in vitro measurement. Tumor volume was calculated according to the formula 1/2 (long diameter ⁇ short diameter 2 ). After the experiment, tumor tissues were collected from each group and weighed. The results are shown in Figure 8.
  • Figure 8 is a line graph showing changes in tumor volume over time and D72 tumor weight in each group of mice. It can be seen from the results of Figure 8 that the tumor volume of the blank control group increased rapidly, and the tumor volume increase rate of the other groups decreased; and compared with the test group 8a and the control group 8c that were only given MMAE, the conjugate of the present disclosure was given In each test group, the tumor volume increase rate was significantly further reduced when the equivalent single dose of MMAE was 0.1 mg/kg.
  • Conjugate 20, Conjugate 21, Conjugate 23 and Conjugate 26 reduced the tumor weight by at least 58% compared with test group 8a at the experimental end point D72, showing more excellent anti-tumor effects.
  • the culture conditions of A549 human lung adenocarcinoma cells were DMEM complete medium (MACGENE Company, catalog number CM15019) containing 10% FBS (Gibco, catalog number 10099-141), at 37.5% Culture in a CO 2 /95% air incubator. Digest and collect the cells with 0.25wt% trypsin, aspirate the supernatant and resuspend the cells in serum-free DMEM medium to prepare a cell culture medium with a cell density of 1 ⁇ 10 8 cells/mL.
  • DMEM complete medium (MACGENE Company, catalog number CM15019) containing 10% FBS (Gibco, catalog number 10099-141)
  • FBS Gibco, catalog number 10099-141
  • mice inoculated with A549 subcutaneous tumors were obtained according to the method of Experimental Example 2, and the mice were continued to be raised after injection.
  • Conjugate 20 and conjugate 21 were dissolved in serum-free DMEM medium to a conjugate solution with a concentration of 1 mg/mL (based on aptamer).
  • Comparative conjugate 24 was dissolved into a conjugate solution with a concentration of 0.8 mg/mL (based on aptamer).
  • 10% DMSO + 90% serum-free DMEM medium (volume ratio) MMAE was dissolved into a solution with a concentration of 0.06 mg/mL.
  • mice Seven days after inoculation of A549 human lung cancer cells, all mice were divided into groups, with 6 mice in each group, and each mouse was administered a drug. The day of administration was recorded as D8. Weigh before administration, and calculate the administration volume based on mouse body weight.
  • mice in each group were administered once each on D8, D12, D15 and D19, for a total of 4 times.
  • DMEM was administered to each group of mice respectively, and the single administration volume was 5 ⁇ L/g mouse body weight;
  • MMAE was administered to each group of mice respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. Calculation showed that the single administration dose was 0.3 mg/kg;
  • conjugate 20 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg, and the dose containing MMAE was equivalent to 0.3 mg/kg;
  • conjugate 21 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg, and the dose containing MMAE was equivalent to 0.3 mg/kg;
  • control conjugate 24 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg, and the dose containing MMAE was equivalent to 0.3mg/kg;
  • mice in each group were administered once each on D46, D50 and D54.
  • the volume of single administration was 10 ⁇ L/g mouse body weight. Calculation showed that the dose containing MMAE in a single administration was equivalent to 0.6 mg/kg.
  • Tumor volume was measured on D1, D9, D16, D19, D22, D26, D30, D36, D40, D43, D47, D50, D54 and D57.
  • the experiment was terminated after D50 measurement.
  • Figure 9 is a line graph showing the changes in tumor volume over time in A549 subcutaneous tumor model mice after administration of different concentrations of conjugates or control compounds provided by the present disclosure. It can be seen from the results in Figure 9 that the tumor volume of the mice in the blank control group increased rapidly, and the increase rate of tumor volume in the other groups decreased; at each time period, the tumor volumes of mice given conjugates 20 and 21 were all the same. smaller than controls 9b and 9e. The above results show that the conjugate of the present disclosure can effectively target and reach A549 lung cancer tumor tissue and show good anti-tumor activity.
  • any combination of various embodiments of the present disclosure can also be carried out, and as long as they do not violate the idea of the present disclosure, they should also be regarded as the contents disclosed in the present disclosure.

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Abstract

本公开提供了一种缀合物，缀合物包含一个或多个递送基团和一个或多个功能性基团；每个所述递送基团独立地与所述功能性基团经共价键连接，或通过连接基团连接；每个所述功能性基团选自对肿瘤具有治疗作用的小分子治疗剂基团。本公开提供的缀合物可以高效地靶向递送至肿瘤组织，从而有效地对肿瘤及肿瘤相关疾病进行治疗。

Description

缀合物与组合物及制备方法和用途 技术领域
本公开涉及一种包含基于适配体的递送基团和功能性基团的缀合物与药物组合物。本公开还涉及这些缀合物与药物组合物的制备方法和用途。
 背景技术
肿瘤是指机体在各种致瘤因子作用下，局部组织细胞增生所形成的新生物。其中，肿瘤细胞发生转移与侵入周遭组织的情形，称为恶性肿瘤。按照生成肿瘤的来源组织细胞分类，一般分为上皮细胞产生的恶性肿瘤(癌症)、间叶组织细胞产生的恶性肿瘤(肉瘤)、血液干细胞产生的恶性肿瘤(白血病等)、以及神经胶质细胞产生的恶性肿瘤(胶质瘤)等。其中，胶质瘤是最常见的颅内原发性恶性肿瘤，约占脑肿瘤的40％-50％，全球年发病率为每10万人3-8例。按照WHO病理分型标准，胶质瘤属于神经上皮肿瘤，包括多种病理类型，包括但不限于毛细胞性星形细胞瘤、弥漫星形细胞瘤、间变星形细胞瘤、胶质母细胞瘤、少突胶质细胞瘤、间变型少突胶质细胞瘤等。
目前，本领域中对肿瘤、特别是胶质瘤的治疗的重要问题之一是如何将治疗剂特异性地递送至肿瘤组织和细胞内，并在合适的时间、以合适的方式使这些治疗剂产生相应的治疗作用。
适配体或称核酸适体，是一种能够与多种的目标分子，像小分子化合物、蛋白质、核酸，甚至细胞、组织与器官等等结合的寡核苷酸分子。适配体能够提供“对特定分子辨识”这一重要特性，因此与抗体类似地常应用于生物技术与治疗等方面。适配体可以在试管中设计、并能够快速的利用化学方法合成、同时具有易于保存、免疫原性较低或无免疫原性的优异性质，因此近年来逐渐得到本领域研究人员的重视。然而，适合用于肿瘤靶向递送的适配体在本领域中仍需要进一步开发与应用。
发明内容
本公开的发明人意外发现了一种能够特异性靶向肿瘤细胞、特别是胶质瘤细胞的缀合物，该缀合物对肿瘤细胞、特别是胶质瘤细胞显示出高度特异性，从而可以在肿瘤细胞、特别是胶质瘤细胞中有效富集并对肿瘤进行有效靶向治疗。从而，发明人作出如下发明：
在一方面，本公开提供了一种缀合物，包含一个或多个递送基团和一个或多个功能性基团；所述递送基团由一种适配体去除一个或多个氢原子或一个或多个官能团形成，所述适配体包含一段连续的核苷酸序列，连接相邻的两个核苷酸的基团独立地为磷酸酯基或者具有修饰基团的磷酸酯基，每个核苷酸选自修饰或未修饰A、U、C或G中的一种，所述连续的核苷酸序列具有式(1)所示的序列：
5′-T1-S1-Na-S2-Nb-S3-Nc-S4-T2-3′      式(1)
其中，T1是由1-3个核苷酸组成的基序，T2是由0-15个核苷酸组成的基序，并且T2中不包含与T1完全反向互补的基序；
S1和S4各自是由3-7个核苷酸组成的基序，S1与S4长度相同并且完全反向互补；
Na和Nc各自是由1-4个核苷酸组成的基序，Na中的每个核苷酸与Nc中的每个核苷酸均不互补，并且Na和Nc中U的总个数占Na和Nc中全部核苷酸总个数的50％以上；
S2和S3各自是由1-4个核苷酸组成的基序，S2与S3长度相同并且完全反向互补；
Nb是由3-6个核苷酸组成的基序，并且Nb两端的核苷酸之间不形成AU或GC互补；
每个所述递送基团独立地与所述功能性基团经共价键连接，或通过连接基团连接；每个所述功能性基团选自对肿瘤具有治疗作用的小分子治疗剂基团。
在另一方面，本公开还提供了一种药物组合物，包含本公开所述的缀合物，以及药学上可接受的载体。
在又一方面，本公开还提供了本公开所述的缀合物和/或药物组合物在制备用于对肿瘤及肿瘤相关疾病或症状进行治疗的药物中的应用。
在又一方面，本公开还提供了一种对肿瘤及肿瘤相关疾病或症状进行治疗的方法，所述方法包括向有需要的受试者给予有效量的本公开所述的缀合物和/或药物组合物。
在又一方面，本公开还提供了一种试剂盒，包括本公开所述的缀合物和/或药物组合物。
以引用的方式并入
本说明书中提及的所有出版物、专利以及专利申请均以引用的方式并入本公开，其程度与每一单独的出版物、专利或专利申请均专门并且单独地以引用的方式并入本公开的程度相同。
 有益效果
本公开提供的缀合物和药物组合物具有优异的靶向肿瘤、特别是胶质瘤组织和细胞的能力，并且能够显著治疗或缓解肿瘤及肿瘤相关的疾病和/或症状。
在一方面，本公开提供的缀合物中的递送基团能够将各种小分子药物基团，如小分子毒素基团特异性地递送至肿瘤组织，并显示出优异的肿瘤抑制效果。例如，本公开的缀合物能够有效地将MMAE递送至不同的肿瘤组织，在显示出肿瘤靶向能力的同时，还降低了MMAE分子在其它组织分布带来的毒性风险，且各种给药方式均能够有效抑制肿瘤体积的增加速度和肿瘤重量，表明本公开的缀合物能够有效抑制肿瘤增殖。此外，进一步提高给药剂量的缀合物可使肿瘤体积在测试期间几乎不增加，显示出更加优异的抗肿瘤效果。
进一步地，本公开的发明人意外地发现，本公开的缀合物和/或药物组合物能够高效地通过血脑屏障，在全身给药的情况下就能够靶向至脑内的胶质瘤中，并显著抑制肿瘤体积的增加甚至降低至初始体积的1/10以下、甚至相比对对照组可降低至1/100以下，表明本公开的缀合物能够有效穿透血脑屏障并高效靶向进入脑胶质瘤，并具有良好的抑制肿瘤生长效果，显示出良好的治疗依从性和高效抑制肿瘤的高成药能力。。
由此说明，本公开的缀合物能够显著有效抑制肿瘤增殖，具有良好的应用前景。
 附图说明
图1A-1C依次分别是示出了给予不同缀合物后1h、24h和48h时，小鼠体内的荧光成像结果的图。图1D是示出了D5时处死小鼠后，各组小鼠肿瘤组织和肾脏的荧光信号成像的图。
图2A-2C依次分别是示出了给予不同缀合物后1h、24h和48h时，小鼠体内的荧光成像结果的图。图2D是示出了D6时处死小鼠后，各组小鼠肿瘤组织和肾脏的荧光信号成像的图。
图3是示出了给予本公开提供的缀合物或对照化合物后，各组小鼠中肿瘤体积随时间变化的折线图。
图4是示出了给药后24h和48h时，给予不同缀合物后建立U118MG原位瘤模型小鼠脑组织的荧光成像图。
图5是示出了给予本公开提供的缀合物或对照化合物后，U118MG原位瘤模型小鼠肿瘤光强数值随时间变化的折线图。
图6是示出了给予本公开提供的缀合物或对照化合物后，U118MG皮下瘤模型小鼠肿瘤体积随时间变化的折线图。
图7是示出了给予不同浓度本公开提供的缀合物或对照化合物后，U118MG皮下瘤模型小鼠肿瘤体积随时间变化的折线图。
图8是示出了给予不同浓度本公开提供的缀合物或对照化合物后，U118MG皮下瘤模型小鼠肿瘤体积随时间变化的折线图。
图9是示出了给予不同浓度本公开提供的缀合物或对照化合物后，A549皮下瘤模型小鼠肿瘤体积随时间变化的折线图。具体实施方式
以下对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。
定义
本公开中，如无特别说明，A、U、C、G和T分别指腺嘌呤核苷酸、尿嘧啶核苷酸、胞嘧啶核苷酸、鸟嘌呤核苷酸和胸腺嘧啶核苷酸，2-甲基胞嘧啶核苷酸指胞嘧啶核苷酸中的胞嘧啶碱基上2′位的氢被甲基取代得到的核苷酸。这些核苷酸的结构为本领域技术人员所公知。如本公开所使用的，“核酸基序”或“基序”是指寡核苷酸中的核酸序列片段，由1个或多个核苷酸组成。在一些实施方式中，基序是具有生物学功能的核酸序列片段。
如本公开所使用的，“烷基”是指具有指定数量的碳原子的直链和支链饱和烃基，所述数量通常为1至20个碳原子，例如1至10个碳原子，如1至8个或1至6个碳原子。例如，C1-C6烷基指包含1至6个碳原子的直链和支链烷基。当提及具有特定数量的碳的烷基残基时，旨在涵盖具有该数量的碳的所有支链和直链形式；因此，例如，“丁基”意味着包括正丁基、仲丁基、异丁基和叔丁基；“丙基”包括正丙基和异丙基。亚烷基是烷基的子集，指与烷基相同、但具有两个连接点的残基。
如本公开所使用的，“烯基”是指具有一个或多个碳-碳双键的不饱和支链或直链烷基，所述碳-碳双键是通过从母体烷基的相邻碳原子中除去一分子氢而获得的。该基团可以处于双键的顺式或反式构型。典型的烯基基团包括但不限于：乙烯基；丙烯基，如丙-1-烯-1-基、丙-1-烯-2-基、丙-2-烯-1-基(烯丙基)、丙-2-烯-2-基；丁烯基，例如丁-1-烯-1-基、丁-1-烯-2-基、2-甲基丙-1-烯-1-基、丁-2-烯-1-基、丁-2-烯-2-基、丁-1，3-二烯-1-基、丁-1，3-二烯-2-基等等。在某些实施方式中，烯基基团具有2到20个碳原子，而在其他实施方式中，具有2至10个、2至8个或2至6个碳原子。亚烯基是烯基的一个子集，指与烯基相同、但具有两个连接点的残基。
如本公开所使用的，“炔基”是指具有一个或多个碳-碳三键的不饱和支链或直链烷基，所述碳-碳三键是通过从母体烷基的相邻碳原子中除去两分子氢而获得的。典型的炔基基团包括但不限于：乙炔基；丙炔基，如丙-1-炔-1-基，丙-2-炔-1-基；丁炔基，例如丁-1-炔-1-基，丁-1-炔-3-基，丁-3-炔-1-基等。在某些实施方式中，炔基具有2到20个碳原子，而在其他实施方式中，具有2至10、2至8或2至6个碳原子。亚炔基是炔基的一个子集，指的是与炔基相同、但有两个连接点的残基。
如本公开所使用的，“杂环基”是指稳定的3-至18-元非芳香族环基，包含2-12个碳原子和1-6个杂原子，所述杂原子选自氮、氧和硫。除非说明书中另有说明，杂环基是单环、双环、三环或四环系统，可包括稠环或桥环系统。杂环基中的杂原子可以是被氧化的杂原子。一个或多个氮原子(如果存在的话)可以是季铵化的氮原子。杂环基是部分饱和或完全饱和的。杂环基可以通过任何环原子连接至分子的其余部分。此类杂环基的实例包括但不限于：二噁烷基、噻吩基[1，3]二硫酰基(thienyl[1，3]dithianyl)、十氢异喹啉基、咪唑啉基、咪唑烷基、异噻唑烷基、异噁唑烷基、吗啉基、八氢吲哚基、八氢异吲哚基、2-氧杂哌嗪基、2-氧杂哌啶基、2-氧杂吡咯烷基、噁唑烷基、哌啶基、哌嗪基、4-哌啶酮基、吡咯烷基、吡唑烷基、奎宁环基、噻唑烷基、四氢呋喃基、三硫酰基(trithianyl)、四氢吡喃基、硫代吗啉基(thiomorpholinyl)、硫杂吗啉基(thiamorpholinyl)、1-氧代硫吗啉基(1-oxo-thiomorpholinyl)和1，1-二氧代硫吗啉基(1，1-dioxo-thiomorpholinyl)。亚杂环基是杂环基的一个子集，指与杂环基相同、但具有两个附着点的残基。
如本公开所使用的，“芳基”是指通过从环碳原子中除去氢原子而衍生自芳香族单环或多环烃环系统形成的基团。所述芳香族单环或多环烃环系统仅含有氢和6至18个碳原子的碳，其中所述环系统中的一个或多个环是完全不饱和的，即，包含根据Hückel理论的环状、离域的(4n+2)π-电子体系。芳基包括但不限于苯基、芴基和萘基等基团。亚芳基是芳基的子集，指与芳基相同、但具有两个连接点的残基。
“杂芳基”指由3-至18-元芳香环自由基衍生而成的基团，包含2个至17个碳原子和选自氮、氧和硫的1至6个杂原子。如本公开所使用的，杂芳基可以是单环、双环、三环或四环系统，其中环系统中的一个或多个环是完全不饱和的，即，包含根据Hückel理论的环状离域(4n+2)π-电子体系。杂芳基包括稠环或桥环系统。杂芳基中的杂原子可以是被氧化的杂原子。一个或多个氮原子(如果存在的话)可以是季铵化的氮原子。杂芳基通过任何环原子附着至分子的其余部分。杂芳基的实例包括但不限于：氮杂环庚三烯基、吖啶基、苯并咪唑基、苯并吲哚基、1，3-苯并二噁唑基、苯并呋喃基、苯并噁唑基、苯并[d]噻唑基、苯并噻二唑基、苯并[b][1，4]二噁庚英基(benzo[b][1，4]dioxepinyl)、苯并[b][1，4]噁嗪基(benzo[b][1，4]oxazinyl)、1，4-苯并二噁烷基(1，4-benzodioxanyl)、苯并萘并呋喃基、苯并噁唑基、苯并间二氧杂环戊烯基(benzodioxolyl)、苯并二噁英基(benzodioxinyl)、苯并吡喃基、苯并吡喃酮基、苯并呋喃基、苯并呋喃酮基、苯并噻吩基、苯并噻吩并[3，2-d]嘧啶基、苯并三唑基、苯并[4，6]咪唑并[1，2-a]吡啶基、咔唑基、噌啉基(cinnolinyl)、环戊烷并[d]嘧啶基、6，7-二氢-5H-环戊烷并[4，5]噻吩并[2，3-d]嘧啶基、5，6-二氢苯并[h]喹唑啉基(5，6-dihydrobenzo[h]quinazolinyl)、5，6-二氢苯并[h]噌啉基(5，6dihydrobenzo[h]cinnolinyl)、6，7-二氢-5H-苯并[6，7]环庚烷并[1，2-c]哒嗪基、二苯并呋喃基、二苯并噻吩基、呋喃基、呋喃酮基、呋喃并[3，2-c]吡啶基、5，6，7，8，9，10-六氢环辛烷并[d]嘧啶基、5，6，7，8，9，10-六氢环辛烷并[d]哒嗪基、5，6，7，8，9，10-六氢环辛烷并[d]吡啶基、异噻唑基、咪唑基、吲唑基(indazolyl)、吲哚基、异吲哚基、二氢吲哚基、异二氢吲哚基、异喹啉基、吲哚嗪基(indolizinyl)、异噁唑基、5，8-甲醇-5，6，7，8-四氢喹唑啉基(5，8-methano-5，6，7，8-tetrahydroquinazolinyl)、萘啶基(naphthyridinyl)、1，6-萘啶酮基(1，6-naphthyridinonyl)、噁二唑基、2-氧杂吖庚因基(2-oxoazepinyl)、噁唑基、氧杂环丙烷基(oxiranyl)、5，6，6a，7，8，9，10，10a-八氢苯并[H]喹唑啉基、1-苯基-1H-吡咯基、吩嗪基、吩噻嗪基、吩噁嗪基、酞嗪基(phthalazinyl)、蝶啶基(pteridinyl)、嘌呤基、吡咯基、吡唑基、吡唑并[3，4-d]嘧啶基、吡啶基、吡啶并[3，2-d]嘧啶基、吡啶并[3，4-d]嘧啶基、吡嗪基、嘧啶基、哒嗪基、吡咯基、喹唑啉基、喹喔啉基(quinoxalinyl)、喹啉基、四氢喹啉基、5，6，7，8-四氢喹唑啉基、5，6，7，8-四氢苯并[4，5]噻吩并[2，3-d]嘧啶基、6，7，8，9-四氢-5H-环庚烷并[4，5]噻吩并[2，3-d]嘧啶基、5，6，7，8-四氢吡啶并[4，5-c]哒嗪基、噻唑基、噻二唑基、三唑基、四唑基、三嗪基、噻吩并[2，3-d]嘧啶基、噻吩并[3，2-d]嘧啶基、噻吩并[2，3-c]吡啶基(thieno[2，3-c]pridinyl)和噻吩基(thiophenyl/thienyl)。亚杂芳基是杂芳基的子集，指与杂芳基相同、但具有两个连接点的残基。
本公开提供的缀合物
在一方面，本公开提供了一种缀合物，包含一个或多个递送基团和一个或多个功能性基团；所述递送基团由前述适配体去除一个或多个氢原子或一个或多个官能团形成；每个所述递送基团独立地与所述功能性基团经共价键连接，或通过连接基团连接；每个所述功能性基团选自对肿瘤具有治疗作用的小分子治疗剂基团。通过经共价键或连接基团连接功能性基团来形成缀合物，本公开的缀合物能够将功能性基团递送至肿瘤。本公开提供的缀合物中，递送基团由适配体去除一个或多个氢原子或一个或多个官能团形成，所述适配体包含一段连续的核苷酸序列，连接相邻的两个核苷酸的基团独立地为磷酸酯基或者具有修饰基团的磷酸酯基，每个核苷酸选自修饰或未修饰A、U、C或G中的一种，所述连续的核苷酸序列具有式(1)所示的序列：
5′-T1-S1-Na-S2-Nb-S3-Nc-S4-T2-3′      式(1)
其中，T1是由1-3个核苷酸组成的基序。发明人发现，T1的存在可使得本公开提供的缀合物显示出高效的肿瘤靶向作用。在一些实施方式中，T1由2个核苷酸组成，此时，本公开提供的缀合物具有更优异的肿瘤靶向能力。在一些实施方式中，T1由2个核苷酸组成并且含有至少一个C。在一些实施方式中，按照5′-3′方向，T1为CU、UC或AC。
T2是由0-15个核苷酸组成的基序。发明人发现，具有这些核苷酸数量和各种核苷酸序列的T2均不会显著影响本公开提供的缀合物的肿瘤靶向能力。在一些实施方式中，T2由0-10个核苷酸组成。在一些实施方式中，按照5′-3′方向，T2由U起始的1-9个核苷酸组成，此时，适配体可能具有更优异的稳定性。
T2中不包含与T1完全反向互补的基序。在本公开的上下文中，“反向互补”是指两段核苷酸序列或基序之间，按照核酸碱基配对的规律形成氢键连接，并且一段核苷酸序列或基序按照5′-3′方向的每一个核苷酸依次与另一端核苷酸序列或基序按照3′-5′方向的每一个核苷酸能够形成碱基配对。在一些实施方式中，“反向互补”包括AU、GC和UG互补中的一种或多种。
S1和S4各自是由3-7个核苷酸组成的基序，且S1与S4长度相同并且完全反向互补。具有上述S1和S4基序的适配体具有较优的稳定性，能够较长时间地靶向肿瘤组织和细胞。在一些实施方式中，S1和S4各自由3-5个核苷酸组成且长度相同。在一些实施方式中，S1和S4形成的反向互补中，GC互补占全部互补数量的40％以上，此时，本公开提供的缀合物具有进一步更优的稳定性和肿瘤靶向能力。在一些实施方式中，按照5′-3′方向，S1为GCU且S4为AGC，或S1为GAGU且S4为GCUC，或S1为GGAGU且S4为GCUCU，或S1为UAUGG且S4为CCAUG。
Na和Nc各自是由1-4个核苷酸组成的基序，Na中的每个核苷酸与Nc中的每个核苷酸均不互补，并且Na和Nc中U的总个数占Na和Nc中全部核苷酸总个数的50％以上。具有上述Na和Nc基序的适配体显示出优异的肿瘤组织靶向能力。在一些实施方式中，Na和Nc中核苷酸的数量之和为2-4的整数。在一些实施方式中，Na和Nc中核苷酸的数量之和为3或4，并且所述Na和Nc中U的数量之和为2或3。在一些实施方式中，按照5′-3′方向，Na和/或Nc为U、UU、UC或CU。
S2和S3各自是由1-4个核苷酸组成的基序，S2与S3长度相同并且完全反向互补。通过包含S2和S3基序，本公开提供的缀合物显示出好的稳定性和优异的肿瘤靶向能力。在一些实施方式中，S2和S3各自由2-3个核苷酸组成且长度相同。在一些实施方式中，S2和S3形成的反向互补中至少包含一个GC互补，此时该反向互补具有更好的稳定性。在一些实施方式中，按照5′-3′方向，S2为CA且S3为UG，或S2为AC且S3为GU，或S2为GCC且S3为GGU。
Nb是由3-6个核苷酸组成的基序，并且Nb两端的核苷酸不是AU或GC互补。不受理论限制地，具有上述Nb基序的适配体能够在空间方面保持特定构型，从而使本公开提供的缀合物稳定、高效地靶向肿瘤组织和细胞。在一些实施方式中，Nb由4-5个核苷酸组成。在一些实施方式中，按照5′-3′方向，Nb为GACG、GACGU、GACCG、UACU、GUUG或GAUCU。
本公开的发明人意外地发现，由具有上述式(1)所示序列的适配体形成的递送基团能够有效地靶向肿瘤、特别是胶质瘤组织，从而允许本公开提供的缀合物能够特异性地进入肿瘤细胞，从而从细胞水平上更有效地递送治疗剂基团。
在一些实施方式中，本公开提供的缀合物中，所述连续的核苷酸序列的长度为18-50个核苷酸，或者20-40个核苷酸，或者21-36个核苷酸，或者24-32个核苷酸。由具有这些连续核苷酸长度的适配体形成的递送基团以及包含该递送基团的本公开提供的缀合物能够更容易地靶向至肿瘤，并且在合成成本和靶向效果方面具有良好的平衡。
在一些实施方式中，本公开提供的缀合物中，连续的核苷酸序列具有以下SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3所示的序列：
5′-CUGCUUCAGACGUGUUAGCUU-3′(SEQ ID NO：1)
其中，按照5′-3′方向，T1为CU，S1为GCU，Na为U，S2为CA，Nb为GACG，S3为UG，Nc为UU，S4为AGC，T2为UU；
5′-CUGAGUUCAGACGUGUUGCUCU-3′(SEQ ID NO：2)
其中，按照5′-3′方向，T1为CU，S1为GAGU，Na为U，S2为CA，Nb为GACG，S3为UG，Nc为UU，S4为GCUC，T2为U；
5′-UCUAUGGCUGCCGAUCUGGUCUCCAUGUACGU-3′(SEQ ID NO：3)，
其中，按照5′-3′方向，T1为CU，S1为GAGU，Na为U，S2为CA，Nb为GACG，S3为UG，Nc为UU，S4为GCUC，T2为U。
在一些实施方式中，所述连续的核苷酸序列具有SEQ ID NO：4所示的核苷酸序列：
5′-N6GGAGUUCAN1N2N3N4UGN5GCUCN7-3′(SEQ ID NO：4)，
其中，N1、N2和N3各自独立地为A、U、C和G中的一种，N4为U、C或G或者由U、C或G中的两个组成的基序；N5为U、CU或UU；N6为CU、UC或AC；N7为U、UU或UUN8，N8为由1-15个核苷酸组成的基序。
上述核苷酸序列中，按照5′-3′方向，T1为N6所示的基序，S1为GGAGU表示的基序，Na为U，S2为CA，Nb为N1、N2、N3和N4组成的基序N1N2N3N4，S3为UG，Nc为N5表示的基序，S4为GCUC以及N7中的第一个核苷酸组成的基序，T2为N7中的其余核苷酸组成的基序。
包含上述SEQ ID NO：4所示的核苷酸序列的适配体能够更有效地靶向肿瘤、特别是胶质瘤并在肿瘤组织中富集。
实验验证表明，SEQ ID NO：4所示的核苷酸序列中，N1、N2、N3和N4的上述选择不会显著影响本公开提供的缀合物的肿瘤靶向能力。在一些实施方式中，N1、N2、N3和N4组成的基序N1N2N3N4为GACG、GACGU、GACCG、UACU、GUUG或GAUCU中的一种，包含这些基序的适配体具有更高的肿瘤特异性靶向效果。
在一些实施方式中，SEQ ID NO：4所示的核苷酸序列中，N5为U或UU。此时，本公开提供的缀合物对肿瘤均具有优异的靶向作用。
在一些实施方式中，所述适配体具有SEQ ID NO：5-11中任意一项所示的核苷酸序列：
具有上述核苷酸序列的本公开提供的缀合物显示出对肿瘤的高度靶向作用。
在一些实施方式中，基序N8由1-15个核苷酸组成。在一些实施方式中，N8由1-8个核苷酸组成。
在一些实施方式中，基序N8的存在使得本公开提供的缀合物对体内的核酸外切酶更加稳定，从而在体内能够更长时间地发挥肿瘤的靶向作用。在一些实施方式中，N8能够增加或维持本公开提供的缀合物对肿瘤的靶向作用。从稳定性、靶向性和合成效率的平衡出发考虑，在一些实施方式中，基序N8由8个核苷酸组成。在一些实施方式中，按照5′-3′方向，基序N8的核苷酸序列为CCGAUCUC。在一些实施方式中，连续的核苷酸序列具有SEQ ID NO：12-14中任意一项所示的核苷酸序列：
所述连续的核苷酸序列中，5′末端核苷酸的核糖5′端以及3′末端核苷酸的核糖3′端的末端基团独立地是羟基或磷酸基，这些末端基团的选择不会改变本公开提供的缀合物的靶向能力。在一些实施方式中，连续的核苷酸中，5′末端核苷酸的核糖5′端以及3′末端核苷酸的核糖3′端的末端基团均为羟基。
本公开提供的缀合物中，每个核苷酸均可以是修饰或未修饰的核苷酸。一般而言，核苷酸的修饰可能会改变本公开提供的缀合物的稳定性和/或对肿瘤的靶向能力。在一些实施方式中，本公开提供的缀合物中的至少一个核苷酸为修饰的核苷酸。在一些实施方式中，本公开提供的缀合物中的至少一个连接相邻的两个核苷酸的基团具有修饰基团的磷酸酯基。
核苷酸的修饰包括但不限于对糖的修饰、对碱基的修饰和/或以核苷酸类似物代替核苷酸。在一些实施方式中，本公开提供的缀合物中，每个所述修饰的核苷酸独立地为2′-卤素修饰的核苷酸、2′-烷氧基修饰的核苷酸、2′-烷基修饰的核苷酸、2′-经取代的烷基修饰的核苷酸、2′-氨基修饰的核苷酸、2′-经取代的氨基修饰的核苷酸、2′-脱氧核苷酸、碱基经修饰的核苷酸和核苷酸类似物中的一种。
在本公开的上下文中，“氟代修饰的核苷酸”指核苷酸的核糖基2′位的羟基被氟取代形成的核苷酸，其具有以下式(7)所示的结构。“非氟代修饰的核苷酸”指核苷酸的核糖基2′位的羟基被非氟基团取代形成的核苷酸、或核苷酸类似物。在一些实施方式中，每一个非氟代修饰的核苷酸独立地选自核苷酸的核糖基2′位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物中的一种。
这些核糖基2′位的羟基被非氟基团取代形成的核苷酸是本领域技术人员所公知的，这些核苷酸可以选自2′-烷氧基修饰的核苷酸、2′-烷基修饰的核苷酸、2′-经取代的烷基修饰的核苷酸、2′-氨基修饰的核苷酸、2′-经取代的氨基修饰的核苷酸、2′-脱氧核苷酸中的一种。
在一些实施方式中，2′-烷氧基修饰的核苷酸为甲氧基修饰的核苷酸(2′-OMe)，如式(8)所示。在一些实施方式中，2′-氨基修饰的核苷酸(2′-NH2)如式(9)所示。在一些实施方式中，2′-脱氧核苷酸(DNA)如式(10)所示：
本领域技术人员知晓各种对核苷酸的碱基进行修饰的方式。在一些实施方式中，碱基修饰包括但不限于在碱基上增加一个或多个甲基。在一些实施方式中，将胸腺嘧啶(T)视为碱基经修饰的尿嘧啶(U)的一种。在一些实施方式中，将2-甲基胞嘧啶视为碱基经修饰的胞嘧啶(C)的一种。
核苷酸类似物指能够在核酸中代替核苷酸，但结构不同于腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶脱氧核糖核苷酸的基团。在一些实施方式中，核苷酸类似物可以是异核苷酸、桥联的核苷酸(bridged nucleic acid，简称BNA)或无环核苷酸。
BNA是指受约束的或不能接近的核苷酸。BNA可以含有五元环、六元环、或七元环的具有“固定的”C3′-内切糖缩拢的桥联结构。通常将该桥掺入到该核糖的2′-、4′-位处以提供一个2′，4′-BNA核苷酸。在一些实施方式中，BNA可以是LNA、ENA、cET BNA等，其中，LNA如式(12)所示，ENA如式(13)所示，cET BNA如式(14)所示：
无环核苷酸是核苷酸的糖环被打开形成的一类核苷酸。在一些实施方式中，无环核苷酸可以是解锁核酸(UNA)、甘油核酸(GNA)或肽核酸(PNA)，其中，UNA如式(15)所示，GNA如式(16)所示：
上述式(15)和式(16)中，R选自H、OH或烷氧基(O-烷基)。
肽核酸是多肽骨架取代糖苷-磷酸主链形成的一类核苷酸类似物。在一些实施方式中，肽核酸可以是例如以2-氨基乙基甘氨酸键取代糖苷-磷酸单元形成的核苷酸类似物。
异核苷酸是指核苷酸中碱基在核糖环上的位置发生改变而形成的化合物。在一些实施方式中，异核苷酸可以是碱基从核糖环的1′-位移动至2′-位或3′-位而形成的化合物，如式(17)或(18)所示。
上述式(17)-式(18)化合物中，Base表示核酸碱基，例如A、U、G、C或T；R选自H、OH、F或者如上所述的非氟基团。
在一些实施方式中，核苷酸类似物选自异核苷酸、LNA、ENA、cET、UNA和GNA中的一种。在一些实施方式中，每一个非氟代修饰的核苷酸均为甲氧基修饰的核苷酸，在上文和下文中，所述甲氧基修饰的核苷酸指核糖基的2′-羟基被甲氧基取代而形成的核苷酸。
在上文及下文中，“氟代修饰的核苷酸”、“2′-氟修饰的核苷酸”、“核糖基团的2′-羟基被氟取代的核苷酸”和“具有2′-氟代核糖基的核苷酸”意义相同，均指核苷酸的2′-羟基被氟取代，而形成的具有如式(7)所示结构的化合物；“甲氧基修饰的核苷酸”、“2′-甲氧基修饰的核苷酸”、“核糖基团的2′-羟基被甲氧基取代的核苷酸”和“具有2′-甲氧基核糖基的核苷酸”意义相同，均指核苷酸核糖基团的2′-羟基被甲氧基取代而形成的具有如式(8)所示结构的化合物。
在一些实施方式中，本公开提供的缀合物中的所述连续的核苷酸序列中的每个胞嘧啶核苷酸为氟代修饰的胞嘧啶核苷酸，和/或所述所述连续的核苷酸序列中的每个尿嘧啶核苷酸为氟代修饰的尿嘧啶核苷酸。在一些实施方式中，本公开提供的缀合物中的所述连续的核苷酸序列中的每个核苷酸均为2′-甲氧基修饰的核苷酸。在一些实施方式中，本公开提供的缀合物中的一个或多个尿嘧啶核苷酸具有修饰的碱基。在一些实施方式中，将胸腺嘧啶碱基(T)视为具有甲基修饰的尿嘧啶碱基(U)。
连接相邻的两个核苷酸的基团可以是磷酸酯基或修饰的磷酸酯基。磷酸酯基的修饰例如将磷酸酯基中的至少一个非桥接氧原子替换为硫原子，形成硫代磷酸酯基或二硫代磷酸酯基。在一些实施方式中，本公开提供的缀合物中的至少一个连接相邻的两个核苷酸的基团为硫代磷酸酯基。在一些实施方式中，所述连续的核苷酸序列中的5′末端前四个核苷酸之间的3个连接相邻的两个核苷酸的基团中至少1个为硫代磷酸酯基。在一些实施方式中，所述连续的核苷酸序列中的5′末端前四个核苷酸之间的3个连接相邻的两个核苷酸的基团中至少2个为硫代磷酸酯基。在一些实施方式中，所述连续的核苷酸序列中的3′末端前四个核苷酸之间连接相邻的两个核苷酸的基团中至少1个为硫代磷酸酯基。在一些实施方式中，所述连续的核苷酸序列中的3′末端前四个核苷酸之间的3个连接相邻的两个核苷酸的基团中至少2个为硫代磷酸酯基。在一些实施方式中，所述连续的核苷酸序列中的每个连接相邻的两个核苷酸的基团均为硫代磷酸酯基。
具有上述修饰的本公开提供的缀合物不仅成本低，而且可使体内的核糖核酸酶不易切割连接基团，由此增加本公开提供的缀合物的稳定性，使其具有更强的抵抗核酸酶水解的性能。同时，包含上述修饰的递送基团的本公开提供的缀合物具有较高的靶向肿瘤组织和/或细胞的活性。
在一些实施方式中，所述连续的核苷酸序列具有SEQ ID NO：15-39中的一种所示的核苷酸序列：
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母s左右两个核苷酸之间为硫代磷酸酯基连接。
在一些实施方式中，本公开提供的缀合物具有式(101)所示的结构：
其中，每个RAP基团独立地为具有如式(2)所示的结构的基团：
式中，每个AP基团相同或不同，独立地表示一个所述递送基团；每个A0基团相同或不同，独立地表示一个所述功能性基团；Rj、每个Rk或每个Ri相同或不同，分别独立地表示共价键或者连接基团，且Ri和Rk二者不同时为共价键；m0为1-6的整数；n0为1-6的整数，每个n1各自独立地表示0-4的整数；表示基团共价连接的位点。
在一些实施方式中，m0为1-6的整数，即，式(101)所示的缀合物中包含1-6个所述功能性基团A0。从递送效率和合成成本的角度考虑，在一些实施方式中，m0为1-4的整数，即，式(101)所示的缀合物中包含1-4个所述功能性基团A0。在一些实施方式中，m0为1，即，式(101)所示的缀合物中包含1个所述功能性基团A0。
在一些实施方式中，n0为1-6的整数，即，式(101)所示的缀合物中包含1-6个RAP基团。从递送效率和成本的角度考虑，在一些实施方式中，n0为1-3的整数，即，式(101)所示的缀合物中包含1-3个RAP基团。在一些实施方式中，n0为1，即，式(101)所示的缀合物中包含1个RAP基团。
在一些实施方式中，每个n1各自独立地表示0-4的整数，且Ri和Rk二者不同时为共价键，从而，每个RAP基团中包含1-5个递送基团AP。在一些实施方式中，每个n1各自独立地表示0-1的整数，从而，每个RAP基团中包含1-2个递送基团AP。在一些实施方式中，n0为1、且n1为0，此时，式(101)所示的缀合物中包含1个递送基团AP。
RAP基团中，Rk和Ri的作用是将递送基团AP共价连接至Rj基团，并经由Rj基团连接至功能性基团A0。因此，只要是能够实现上述连接、并且不对递送基团AP和功能性基团A0的作用产生负面影响的任何Rk或Ri均可用于本发明。在一些实施方式中，每个所述Rk或每个所述Ri独立地为长度为1-70个碳原子的直链亚烷基，或者，所述直链亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、OP(O)(S)、C5-C8亚糖苷基、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中，所述直链亚烷基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)。从合成成本和难度以及缀合物的肿瘤靶向效果出发考虑，在一些实施方式中，每个n1均为0，每个Ri独立地为共价键，或者为以下连接基团的一种或多种的连接组合：C1-C20亚烷基、磷酸酯键、硫代磷酸酯键、酰胺键、酯键、醚键、硫醚键、二硫键、1，2，3-三唑亚基、聚乙二醇亚基、吡咯烷亚基、2-氧代吡咯烷亚基、亚苯基、亚环己基、2-丁二酰亚胺亚基、2-硫代丁二酰亚胺亚基、氨基酸亚基、核苷酸亚基。
在一些实施方式中，连接基团Rj包含本领域技术人员知晓能够用于抗体偶联药物的连接基团。连接基团Rj可以是可裂解的或不可裂解的。在一些实施方式中，连接基团Rj可以是可裂解的。在上下文中，“可裂解”是指在本公开的缀合物靶向至肿瘤后，连接基团Rj在肿瘤内环境下和/或肿瘤细胞内发生共价键断裂，释放出单独的治疗剂基团产生治疗效果。在一些实施方式中，连接基团Rj包含活化酶连接基团、硫酸酯酶-可切割连接基团、半乳糖可切割连接基团、对溶酶体蛋白酶敏感的连接基团、肽基连接基团、葡糖苷酸连接基团、酸敏可切割连接基团、或对谷胱甘肽敏感的二硫化物连接基团的一种或多种。在一些实施方式中，连接基团Rj包含肽基连接基团。在一些实施方式中，肽基连接基团选自缬氨酸-瓜氨酸二肽连接子(Val-Cit)、丙氨酸-丙氨酸二肽连接子(Ala-Ala)、缬氨酸-丙氨酸二肽连接子(Val-Ala)、甘氨酸-甘氨酸-苯丙氨酸-甘氨酸的四肽连接子(Gly-Gly-Phc-Gly)中的一种或多种。在一些实施方式中，连接基团Rj选自N-琥珀酰亚基4-(2-二硫吡啶)丁酸盐(SPDB)、N-琥珀酰亚胺-4-(2-硫代吡啶亚基)戊酸盐(SPP)、(S)-2-((S)-2-氨基-3-甲基丁酰胺)-5-脲基戊酸(Val-Cit-PAB-OH)、N-琥珀酰亚胺基-4-(N-马来酰亚胺甲基)环己烷-1-羧酸盐(SMCC)或者2-(磷酸酯基-(CH2)6-S-)-马来酰亚胺己酰基-缬氨酸-瓜氨酸-对氨基苯甲基亚基中的一种。在一些实施方式中，连接基团Rj包含Mckertish CM，Kayser V.Advances and Limitations of Antibody Drug Conjugates for Cancer.Biomedicines.2021Jul 23；9(8)：872.中列举的连接基团，以引用的方式将该文献的全部内容整体并入本文。
在一些实施方式中，连接基团Rj包含缬氨酸-瓜氨酸二肽连接子(Val-Cit)、聚乙二醇亚基、亚氨基己基亚基、N-琥珀酰亚胺基、GAU三核苷酸连接基团中的一种或多种。在一些实施方式中，每个Ri独立地为共价键、二硫键、亚十二烷基、缬氨酸-瓜氨酸二肽连接子(Val-Cit)、聚乙二醇亚基、亚氨基己基亚基、N-琥珀酰亚胺基或者GAU三核苷酸亚基中的一种或2种的连接组合。
Rj基团的作用是将RAP基团与功能性基团A0相连接，从而通过RAP基团中递送基团AP的肿瘤靶向作用将功能性基团A0特异性地递送至肿瘤组织和/或细胞。因此，任何能够实现上述连接、且不会影响递送基团AP的肿瘤靶向作用和功能性基团A0的效果的Rj基团都能够实现本发明的目的、解决本发明所要解决的技术问题。在一些实施方式中，在式(1)所示的缀合物到达肿瘤组织和/或进入肿瘤细胞后，所述Rj发生裂解，释放出单独的功能性基团A0对应的药物活性分子。在一些实施方式中，所述Rj在体内不发生裂解，此时缀合物中Rj基团和RAP基团的存在不会影响功能性基团A0发挥治疗作用。
在一些实施方式中，Rj为共价键，m0为1，此时，式(101)所示的缀合物中包含1个功能性基团A0和1个RAP基团，每个RAP基团直接连接至该功能性基团A0。在一些实施方式中，每个RAP基团连接至功能性基团A0的同一原子。在一些实施方式中，每个RAP基团连接至功能性基团A0的不同原子。
在一些实施方式中，Rj为连接基团，所述连接基团Rj包含主链部分、侧链部分和缀合连接部。
主链部分分别与缀合连接部和侧链部分连接。在一些实施方式中，主链部分为长度为1-70个碳原子的直链亚烷基，或者，所述直链亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中，所述直链亚烷基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)。
侧链部分分别与主链部分和RAP基团连接。在一些实施方式中，每个侧链部分独立地是共价键，或者是长度为1-70个碳原子的直链亚烷基，或者，所述直链亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且，所述直链亚烷基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)；
缀合连接部分别与主链部分和功能性基团A0连接。在一些实施方式中，每个缀合连接部独立地为共价键或者以下连接结构的一种或多种的连接组合：C1-C10直链亚烷基、磷酸酯键、硫代磷酸酯键、酰胺键、酯键、醚键、二硫键、1，2，3-三唑亚基、聚乙二醇亚基、吡咯烷亚基、2-氧代吡咯烷亚基、亚苯基、亚环己基、2-丁二酰亚胺亚基、2-硫代丁二酰亚胺亚基、氨基酸亚基、核苷酸亚基。
在一些实施方式中，连接基团Rj中的每个所述缀合连接部分别与所述主链部分和一个所述功能性基团A0连接；所述侧链部分为n0个，每个侧链部分分别与所述主链部分和一个所述RAP基团连接。从而，每个功能性基团A0和RAP基团各自独立地连接至连接基团Rj。在一些实施方式中，全部侧链部分连接至主链部分中的同一原子；或者，每个侧链部分连接至主链部分中的不同原子。
在一些实施方式中，m0为1，所述连接基团Rj包含如式(301)所示的结构：
其中，k为1-3的整数；LC为所述主链部分，LA为所述侧链部分，LB为所述缀合连接部，表示基团共价连接的位点。
所述主链部分LC为共价键或2-4价、直链或支链的C1-C25饱和烃基，或者，所述饱和烃基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C5亚烯基、C2-C5亚炔基、C6-C10亚芳基、C3-C8亚杂环基和C5-C10亚杂芳基；其中，所述饱和烃基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C5烷基、C6-C10芳基、C5-C10杂芳基、-O-C1-C5烷基、-OC1-C5烷基苯基、-C1-C5烷基-OH、-SC1-C5烷基、硝基、-C(O)O(C1-C5烷基)、-CON(C1-C5烷基)(C1-C5烷基)、-CONH(C1-C5烷基)、-CONH2，-NHC(O)(C1-C5烷基)、-NHC(O)(苯基)、-N(C1-C5烷基)C(O)(C1-C5烷基)、-N(C1-C5烷基)C(O)(苯基)、-C(O)C1-C5烷基、-C(O)C1-C5烷基苯基、-OC(O)C1-C5烷基、-SO2(C1-C5烷基)、-SO2(苯基)、-SO2NH2、-SO2NH(C1-C5烷基)、-SO2NH(苯基)、-NHSO2(C1-C5烷基)和-NHSO2(苯基)。在一些实施方式中，LC为2-4价的C5-C20饱和烃基，或者，所述饱和烃基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C5亚烯基、C2-C5亚炔基、C6-C10亚芳基、C3-C8亚杂环基和C5-C10亚杂芳基；其中，所述饱和烃基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C5烷基、C6-C10芳基、C5-C10杂芳基、-O-C1-C5烷基、-OC1-C5烷基苯基、-C1-C5烷基-OH、-SC1-C5烷基、硝基、-CONH2。在一些实施方式中，LC的长度为5-30个原子，其中所述LC的长度指LC中与LA直接连接的原子到与LB直接连接的原子形成的最长的原子链上的成链原子的个数。为了简化结构，在一些实施方式中，LC的长度为8-25个原子。
所述侧链部分LA为共价键，或者C1-C20亚烷基，或者，所述亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C5亚烯基、C2-C5亚炔基、C6-C10亚芳基、C3-C8亚杂环基和C5-C10亚杂芳基；其中，所述亚烷基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C5烷基、C6-C10芳基、C5-C10杂芳基、-O-C1-C5烷基、-OC1-C5烷基苯基、-C1-C5烷基-OH-SC1-C5烷基、-SC1-C5烷基苯基、-C1-C5烷基-SH、-OH、-SH、-NH2、-C1-C5烷基-NH2、-N(C1-C5烷基)(C1-C5烷基)、-NH(C1-C5烷基)、-N(C1-C5烷基)(C1-C5烷基苯基)、-NH(C1-C5烷基苯基)、硝基、-C(O)O(C1-C5烷基)、-CON(C1-C5烷基)(C1-C5烷基)、-CONH(C1-C5烷基)、-CONH2，-NHC(O)(C1-C5烷基)、-NHC(O)(苯基)、-N(C1-C5烷基)C(O)(C1-C5烷基)、-N(C1-C5烷基)C(O)(苯基)、-C(O)C1-C5烷基、-C(O)C1-C5烷基苯基、-OC(O)C1-C5烷基、-8O2(C1-C5烷基)、-8O2(苯基)、-SO2NH2、-SO2NH(C1-C5烷基)、-SO2NH(苯基)、-NHSO2(C1-C5烷基)和-NHSO2(苯基)。
所述缀合连接部LB为1-5个以下连接键中的一种或多种的连接组合：磷酸酯键、硫代磷酸酯键、酰胺键、酯键、醚键、二硫键。
在一些实施方式中，k为1-3的整数；LC含有如式(L1)-(L3)所示的基团中的任意一个，通过如式(L1)-(L3)所示的基团中的醚键与LA部分连接：
表示基团连接至分子其余部分的位点；
在一些实施方式中，k＝1，LC含有如式(L1)所示的基团，基团(L1)中的O原子和LA直接相连。在一些实施方式中，k＝2，LC含有如式(L2)所示的基团，基团(L1)中的2个O原子各自和1个LA直接相连。在一些实施方式中，k＝4，LC含有如式(L3)所示的基团，基团(L3)中的3个O原子各自和1个LA直接相连。
LB为磷酸酯键或二硫键；
每个LA为共价键，或者每个LA选自于由基团(L4)-(L23)及其连接组合所组成的组：
式中，每个j1为1-10的整数；
每个R′为C1-C10烷基；
每个Ra为氢原子，C1-C10烷基，或者选自由基团(L24)-(L37)组成的组：
在一些实施方式中，LA的长度为3-35个原子，其中所述LA的长度指LA中与LC直接连接的原子到与LA中与RAP直接连接的原子形成的最长的原子链上的成链原子的个数。在一些实施方式中，每个LA为基团(L4)-(L9)、(L13)、(L14)、(L18)中至少2个的连接组合。在一些实施方式中，每个LA为基团(L4)、(L5)、(L7)、(L9)、(L13)、(L14)、(L18)中至少2个的连接组合。
在一些实施方式中，LA具有如式(302)所示的包含酰胺键的结构，LB具有如式(303)所示的包含N-酰基吡咯烷的结构，含有羰基和氧原子，LC为基于羟甲基氨基甲烷、二羟甲基氨基甲烷或三羟甲基氨基甲烷的连接基团：
其中，n302、q302和p302各自独立地为2-6的整数，可选地，n302、q302和p302各自独立地为2或3；n303为4-16的整数，可选地，n303为8-12的整数，表示基团共价连接的位点。
在一些实施方式中，每个所述侧链部分LA分别与一个RAP基团通过磷酸酯键、醚键或酯键而连接，并通过所述主链部分LC中羟基的氧原子与所述主链部分LC形成醚键而连接；所述缀合连接部LB通过式(303)中的羰基与所述主链部分LC中氨基的氮原子形成酰胺键而连接，并通过式(303)中的氧原子与所述功能性基团A0形成磷酸酯键、醚键或酯键而连接。在一些实施方式中，主链部分LC是基于羟甲基氨基甲烷、二羟甲基氨基甲烷或三羟甲基氨基甲烷的连接基团，所述主链部分LC经由羟基的氧原子与各个所述侧链部分LA通过醚键相连接，并且经由氨基的氮原子与所述缀合连接部LB通过酰胺键相连接。从而，该连接基团Rj中1-3个侧链连接至同一氨甲基的碳原子上，并通过缀合连接部LB连接至包含递送基团的RAP基团。
在一些实施方式中，所述缀合物具有如式(305)所示的结构：
在一些实施方式中，所述连接基团Rj包含如式(306)所示的结构：
其中，n306为0-3的整数，每个p306独立地为1-6的整数，表示基团共价连接的位点；由全部吡咯烷亚基和任何可能的磷酸二酯基团形成的连接组合构成主链部分，由连接至吡咯烷亚基上氮原子的羰基与由*标出的氧原子之间的原子链构成每个侧链部分，所述侧链部分通过由*标出的氧原子与RAP基团形成醚键连接；由#标出的氧原子中的至少一个为缀合链接部并与功能性基团A0形成醚键、酯键或磷酸酯键而连接，其余由#标出的氧原子与氢原子连接形成羟基，或者与C1-C3烷基连接形成C1-C3烷氧基。从而，该连接基团Rj中1-3个侧链部分连接至主链部分中不同的碳原子上，并通过氧原子连接至包含递送基团的RAP基团。
在一些实施方式中，本公开提供的缀合物具有如式(307a)、(307b)或(307c)所示的结构：
在一些实施方式中，本公开提供的缀合物具有如式(308)所示的结构：
其中，n308可为1-10的整数；在一些实施方式中，从合成容易程度、结构/工艺成本和肿瘤细胞特异性等多方面综合考虑，n308为2-6的整数。在一些实施方式中，n308为3或4。
每个R3独立地为功能性基团A0，或者包含递送基团AP的RAP基团；。在一些实施方式中，至少一个R3为功能性基团A0，且至少一个R3为RAP。在一些实施方式中，一个R3为功能性基团A0，其余R3为RAP基团。
在一些实施方式中，当每个m308独立地选自2-10的整数时，认为可能使得该缀合物中，多个递送基团AP之间的空间位置更适合于与肿瘤细胞表面的相应受体发生相互作用，为了使式(308)表示的化合物更为简单，更容易合成和/或降低成本，根据本公开的一些实施方式，每个m308独立地为2-5的整数，在一些实施方式中，每个m308均相等。
本领域技术人员可以理解，当每个R308独立地选自H、C1-C10烷基、C1-C10卤代烷基和C1-C10烷氧基时，不改变式(308)表示的缀合物的性质，均可实现本公开的目的。在一些实施方式中，每个R308独立地选自H、甲基或乙基。在一些实施方式中，每个R308均为H。
连接至所述功能性基团A0的每个L1表示所述缀合连接部，并且连接至所述RAP的每个L1表示所述侧链部分。在一些实施方式中，一个R3为所述功能性基团A0，其余R3为所述RAP基团。在一些实施方式中，一个或多个L1作为所述侧链部分，将RAP基团与含氮骨架上的N原子连接；并且另外的一个或多个L1作为所述缀合连接部，将功能性基团A0与含氮骨架上的N原子连接。所述含氮骨架共同组成连接基团Rj的主链部分。在本公开的上下文中，“含氮骨架”是指式(308)所示结构中的链结构，其中连接有R308的碳原子与N原子互相连接。
从递送效率和合成成本的角度出发考虑，在一些实施方式中，每个L1的长度独立地为3-25个原子。在一些实施方式中，每个L1的长度独立地为4-15个原子。本领域技术人员会理解，尽管为了方便起见，L1被定义为线性亚烷基，但是它可能不是线性基团或者名称不同，例如由于上述替换和/或取代而产生的胺或烯基。为了本公开的目的，L1的长度是连接两个连接点的链中的原子数。为此目的，将替换所述直链亚烷基的碳原子而得到的环(如亚杂环基或亚杂芳基)按照环上连接点之间的最小原子数计算链中与该环相应部分的长度。
在一些实施方式中，L1选自上述式L4-L23所示基团及其任意连接组合所组成的组。在一些实施方式中，每个L1独立地选自于由基团L4-L9、L13、L14、L18中至少2个的连接组合所组成的组。在一些实施方式中，每个L1独立地为基团L4、L5、L7、L9、L13、L14、L18中至少2个的连接组合。
在一些实施方式中，式(308)所示的缀合物中，每个L1上同时含有与含氮骨架上的N原子连接的连接位点和与功能性基团A0或所述RAP基团连接的连接位点，与含氮骨架上的N原子连接的位点与该N原子形成酰胺键。在一些实施方式中，一个或多个L1选自B5、B6、B5′或B6′：
其中，表示基团共价连接的位点，q2为1-10的整数。在一些实施方式中，q2为1-5的整数。
在一些实施方式中，每个RAP基团中包含一个或多个递送基团。在一些实施方式中，式(308)所示的化合物中包含多个功能性基团。在一些实施方式中，式(308)化合物中每个功能性基团是相同的功能性基团。在一些实施方式中，式(308)化合物中每个功能性基团是用于同一目的和功能的功能性基团。在一些实施方式中，式(308)化合物中含有不同种类的用于不同目的和功能的功能性基团。
在一些实施方式中，所述式(308)所示的化合物具有式(403)、(404)、(405)、(406)、(407)、(408)、(409)、(410)、(411)、(412)、(413)、(414)、(415)、(416)、(417)、(418)、(419)、(420)、(421)、(422)、(423)、(424)、(425)、(426)或(427)所示的结构：
在一些实施方式中，连接基团Rj包含核苷酸序列I和核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II各自包含5-25个修饰或未修饰的核苷酸，所述苷酸序列I和所述核苷酸序列II至少部分地反向互补，所述递送基团连接至所述核苷酸序列I，所述功能性基团连接至所述核苷酸序列II，并且所述核苷酸序列I和所述核苷酸序列II在受试者体内不引发免疫反应或毒性反应。在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II实质上反向互补或者完全反向互补；或者，所述核苷酸序列I和所述核苷酸序列II的长度相等并且均为10-20个修饰或未修饰的核苷酸；或者，所述核苷酸序列I和所述核苷酸序列II均由17个核苷酸组成并且完全反向互补。在一些实施方式中，所述递送基团3′末端经磷酸酯键连接至所述核苷酸序列I的5′末端核苷酸的核糖5′位，所述功能性基团连接至所述核苷酸序列II的5′末端核苷酸的核糖5′位；或者，所述功能性基团包含一段核苷酸序列，所述核苷酸序列的3′末端经磷酸酯键连接至所述核苷酸序列I的5′末端核苷酸的核糖5′位。在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II分别具有SEQ ID NO：40和SEQ ID NO：41所示的序列：
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II分别具有SEQ ID NO：42和SEQ ID NO：43所示的序列：
在一些实施方式中，所述功能性基团为对肿瘤具有治疗作用的小分子治疗剂基团，每个小分子治疗剂基团独立地选自细胞毒素基团、抗生素基团或血管生成抑制剂。通过包含小分子治疗剂基团，本公开的缀合物能够特异性地将该小分子治疗剂基团递送至肿瘤，从而通过该小分子治疗剂基团的作用，对肿瘤的疾病进程或症状进行治疗和/或缓解，例如，通过本公开的缀合物将细胞毒素基团特异性地递送至肿瘤，使肿瘤中的癌细胞特异性地消亡，从而在减少细胞毒素本身低靶向性带来的副作用的同时，显著减少肿瘤中的癌细胞数量，从而对肿瘤进行治疗。
在一些实施方式中，所述小分子治疗剂基团由下述小分子治疗剂去除一个或多个氢原子或一个或多个官能团形成：所述小分子治疗剂选自甲氨蝶呤、阿霉素、长春花生物碱、澳瑞他汀(包括MMAE和MMAF)、卡利奇霉素、美登素、喜树碱、以及加利车霉素。在一些实施方式中，所述小分子治疗剂基团是一甲基澳瑞他汀E(MMAE)去除一个或多个氢原子或一个或多个官能团形成的基团。
所述功能性基团可通过任何合适的方式包含于本公开的缀合物中。例如，可以通过前述的缀合连接部将功能性基团A0与所述主链部分相连接。
在一些实施方式中，本公开提供的缀合物还包含一个或多个递送助剂基团，所述递送助剂基团选自于C10-C30烃基、胆固醇基、磷脂基团中的一种或多种。通过包含该递送助剂基团，本公开提供的缀合物能够更好地与中枢神经系统中的体内环境相容，可能具有更好的生物利用度，和/或使本公开提供的缀合物更有效地递送至肿瘤。在一些实施方式中，所述递送助剂基团通过共价键或连接基团连接至所述递送基团或者所述连接基团。在一些实施方式中，所述递送助剂基团连接至所述功能性基团。
本领域技术人员可以采用任意合理的合成路线制备本公开提供的缀合物。
在一些实施方式中，本公开提供的缀合物的合成方法包括在脱保护反应条件下，在溶剂中，将经保护的缀合物与脱保护试剂接触，分离获得本公开提供的缀合物。经保护的缀合物是本公开提供的缀合物中任意活性官能团均被保护基团保护而形成的化合物。在一些实施方式中，所述活性官能团包括但不限于羟基、氨基和/或磷酸基，所述保护基团相应地为羟基保护基、氨基保护基和/或磷酸羟基保护基(例如，氰乙基保护基)。根据保护基团的不同，对所使用的溶剂、脱保护反应条件和脱保护试剂进行选择和确定。在一些实施方式中，所述脱保护反应条件、溶剂和脱保护试剂是核酸固相合成中使用的脱保护反应条件、溶剂和试剂。在一些实施方式中，所述方法包括将经保护的缀合物加入甲胺水溶液和氨水的混合溶液中，所述脱保护反应条件包括在常温常压下反应1-5h。在一些实施方式中，以等体积混合所述甲胺水溶液与饱和浓氨水得到所述混合溶液，所述溶液相对于经保护的缀合物的用量为0.1-10ml/μmol。在一些实施方式中，所述分离包括通过柱色谱分离进行纯化，收集产品洗脱液并除去溶剂。纯化条件可以是例如使用制备型离子色谱纯化柱，以氯化钠水溶液与磷酸钠水溶液的梯度洗脱剂进行洗脱。在一些实施方式中，以20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9∶1(体积比)；洗脱剂B：1.5M氯化钠，20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9∶1(体积比)；洗脱梯度∶洗脱剂A∶洗脱剂B＝100∶0-50∶50梯度洗脱。
在一些实施方式中，本公开提供的缀合物具有式(101)所示的结构，所述经保护的缀合物的合成方法包括在有机溶剂中，在偶联反应条件下，将包含活性基团Rx1和递送基团的化合物与包含活性基团Rx2和功能性基团的化合物接触，反应获得所述经保护的缀合物，其中，所述递送基团由前述适配体去除一个或多个氢原子或一个或多个官能团形成，每个所述功能性基团独立地为对肿瘤具有治疗作用的小分子治疗剂基团，其中所述递送基团和功能性基团中的任何活性基团均被保护基团保护，所述活性基团Rx1和所述活性基团Rx2是能够经反应形成共价键或者连接基团Rj的基团。在一些实施方式中，连接有活性基团Rx1的递送基团和连接有活性基团Rx2的功能性基团的摩尔比为m0∶n0。在一些实施方式中，所述递送基团和功能性基团中的活性基团包括但不限于羟基、氨基、磷酸基中的一种或多种，所述保护基团相应地为羟基保护基、氨基保护基、磷酸羟基保护基(例如，氰乙基保护基)中的一种或多种。
本领域的技术人员可以通过各种方法获得包含活性基团Rx1和递送基团的化合物。在一些实施方式中，所述包含活性基团Rx1和递送基团的化合物可以通过本领域技术人员熟知的核酸合成方法，例如亚磷酰胺固相合成或者磷酸二酯法/磷酸三酯法液相合成获得。在一些实施方式中，所述包含活性基团Rx1和递送基团的化合物采用亚磷酰胺固相合成法得到，该方法包括，在亚磷酰胺固相合成条件下，按照寡核苷酸单链中核苷酸的顺序，将核苷单体依次连接，其中，至少一个核苷单体为具有活性基团Rx1的核苷单体，或者，在连接全部核苷单体后，再按照亚磷酰胺固相合成法连接一个具有活性基团Rx1的亚磷酰胺单体或经保护的亚磷酰胺单体，随后再脱除该保护基团形成活性基团Rx1。亚磷酰胺固相合成法为本领域技术人员所公知，其过程和条件在Methods in Molecular Biology，vol.288：Oligonucleotide Synthesis：Methods and Applications，P17-P31中详细公开，以引用的方式将其全部内容整体并入本文。
在一些实施方式中，所述偶联反应条件是缩合反应条件或者巯基-二硫键交换反应的条件。
在一些实施方式中，所述偶联反应条件是缩合反应条件，所述缩合反应条件是酰基化缩合反应条件、脱水缩合反应条件或者点击化学反应的条件，活性基团Rx1与活性基团Rx2是能够发生前述缩合反应的基团。在一些实施方式中，所述缩合反应条件是酰基化缩合反应的条件，所述活性基团Rx1和Rx2是能够发生酰基化缩合反应形成RI的基团。在一些实施方式中，所述缩合反应条件是脱水缩合反应的条件，所述活性基团Rx1和Rx2中的一个是包含酰卤基团或羧基的基团，另一个是包含氨基或羟基的基团。在一些实施方式中，所述缩合反应条件是点击化学的条件，所述活性基团Rx1和Rx2中的一个是包含炔基的基团，另一个是包含叠氮基团的基团。在一些实施方式中，所述缩合反应条件是Michael加成反应的条件，所述活性基团Rx1和Rx2中的一个是包含巯基的基团，另一个是包含琥珀酰亚胺基的基团。在一些实施方式中，所述缩合反应条件是N-羟基琥珀酰亚胺-碳二亚胺(NHS-EDC)联用偶联反应的条件，所述活性基团Rx1和Rx2中的一个是包含N-羟基琥珀酰亚胺(NHS)的基团，另一个是包含碳二亚胺基(EDC)的基团。
在一些实施方式中，所述包含活性基团Rx1和递送基团的化合物是通过在偶联反应的条件下，将具有活性基团Rx0的适配体和交联剂接触制备得到的，所述交联剂含有点击化学活性基团和酰基化基团。所述活性基团RX0与所述酰基化基团通过发生偶联反应而形成共价连接，使所述点击化学活性基团连接至本公开的适配体。
在一些实施方式中，活性基团Rx1是末端含有1-3个点击化学活性基团的活性基团，所述点击化学活性基团包含末端炔基。在一些实施方式中，所述酰基化基团是活性酯基团，例如可以是NHS酯基、亚氨酸酯基以及五氟苯酯基中的一种。本领域的技术人员可以通过各种方法获得所述交联剂，例如，当所述酰基化基团是五氟苯酯基，所述点击化学基团包含末端炔基时，所述交联剂可以按照如Michael E.，et al.″Efficient synthesis and biological evaluation of 5′-GalNAc conjugated antisense oligonucleotides.″Bioconjugate chemistry 26.8(2015)：1451-1455中Scheme 1a(A)中描述的方法制备获得，以引用的方式将其全部内容整体并入本文。在一些实施方式中，所述活性基团Rx0是氨基。在一些实施方式中，所述偶联条件是碱性条件。在一些实施方式中，所述碱性条件是有弱碱水溶液存在的条件，例如有碳酸氢钠水溶液存在的条件。
本领域的技术人员可以通过各种方式获得所述具有活性基团Rx0的适配体，在一些实施方式中，所述具有活性基团Rx0的适配体是通过在合成适配体的过程中在相应位置使用含有活性基团的亚磷酰胺单体制备得到的。本领域的技术人员可以通过各种方式获得含有活性基团的亚磷酰胺单体。在一些实施方式中，所述活性基团Rx0是氨基，含有Rx0的亚磷酰胺单体可以通过本领域技术人员熟知的方法商购获得或制备获得，例如，含有Rx0的亚磷酰胺单体可以是容易商购获得的6-(三氟乙酰氨基)-己基-(2-氰乙基)-(N，N-二异丙基)-亚磷酰胺单体，其中，活性基团Rx0为氨基，该活性基团Rx0可以是通过亚磷酰胺固相合成法将所述亚磷酰胺单体连接至寡核苷酸单链后，经本领域技术人员容易实现的脱保护反应(如浓氨水氨解)脱除三氟乙酰基保护基而获得。
在一些实施方式中，所述偶联反应条件是巯基-二硫键交换反应中的一种，所述活性基团Rx1和Rx2中的一个是包含巯基的基团，另一个包含经二硫键连接的离去基团。为了避免副反应发生，在一些实施方式中，上述含有活性基团Rx1的亚磷酰胺单体中的Rx1以被保护的Rx1’形式存在，所述制备方法还包含在脱保护反应条件下，通过将制备得到的包含被保护的活性基团Rx1’和递送基团的化合物与脱保护试剂接触，分离得到包含活性基团Rx1和递送基团的化合物的步骤。在一些实施方式中，所述Rx1’含有二硫键离去基团，所述脱保护反应条件是巯基-二硫键交换反应条件，所述脱保护试剂是二硫键活化剂。在一些实施方式中，所述二硫键活化剂是二硫二吡啶。本领域的技术人员可以通过各种方法获得上述含有活性基团Rx1或Rx1’的亚磷酰胺单体，在一些实施方式中，所述含有活性基团Rx1或Rx1’的亚磷酰胺单体是商购得到的，例如可以通过商购得到如式(105)所示的亚磷酰胺单体。
其中n105和m105各自独立地是1-10的整数。
本领域的技术人员可以通过各种方式获得所述包含活性基团Rx2和功能性基团的化合物。在一些实施方式中，所述偶联反应条件是巯基-二硫键交换反应条件，所述活性基团Rx2包含巯基，所述包含活性基团Rx2和功能性基团的化合物可以由本领域技术人员按照各种已知的方式获得，例如通过使用包含巯基的亚磷酰胺单体通过亚磷酰胺固相合成方法制备获得，或者商购获得。在一些实施方式中，所述偶联反应条件是亚磷酰胺固相合成反应条件，所述活性基团Rx2是亚磷酰胺基团，所述包含活性基团Rx2和功能性基团的化合物可以是例如容易商购获得的包含亚磷酰胺基团和小分子治疗剂基团的化合物。在一些实施方式中，在一些实施方式中，所述偶联反应条件是Michael加成反应条件，所述活性基团Rx2是N-琥珀酰亚胺基团，所述包含活性基团Rx2和功能性基团的化合物可以是例如容易商购获得的包含N-琥珀酰亚胺基团和小分子治疗剂基团的化合物。在一些实施方式中，所述活性基团Rx1与活性基团Rx2分别是核苷酸序列I和核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II各自包含5-25个修饰或未修饰的核苷酸，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补，所述递送基团连接至所述核苷酸序列I，所述功能性基团连接至所述核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II在受试者体内不引发免疫反应或毒性反应，所述偶联反应条件是退火形成核酸双链的反应条件。在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II均由17个核苷酸组成并且完全反向互补。在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II分别具有SEQ ID NO：40和SEQ ID NO：41所示的序列。在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II分别具有SEQ ID NO：42和SEQ ID NO：43所示的序列。
递送基团由适配体去除一个或多个氢原子或官能团形成。在一些实施方式中，连续的核苷酸序列的5′末端核苷酸的核糖5′基团以及3′末端核苷酸的核糖3′基团均为羟基，递送基团由适配体在5′末端核苷酸的5′羟基中去除一个氢原子形成；在一些实施方式中，递送基团由适配体在3′末端核苷酸的3′羟基中去除一个氢原子形成；在一些实施方式中，递送基团由适配体在5′末端核苷酸中去除5′羟基形成；在一些实施方式中，递送基团由适配体在3′末端核苷酸中去除3′羟基形成。在一些实施方式中，递送基团由适配体在其包含的核苷酸中去除核糖2′-羟基形成。所述适配体可以通过本领域常规的适配体制备方法(例如核酸固相合成和液相合成的方法)得到。其中，核酸固相合成已经有商业化订制服务。可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开提供的缀合物中，制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入适配体的方法也是本领域技术人员所熟知的。所有修饰的核苷单体均可以商购得到或者采用已知方法制备得到。
在一些实施方式中，所述缀合物还可以其药学上可接受的盐或前体化合物的形式用于本公开。在本公开提供的上下文中，“药学上可接受的盐”是指为了增加药物的稳定性、溶解性和/或生物利用度而将药物形成相应的在药学上不对人体产生额外副作用的盐，例如钾盐、钠盐、羧酸盐等。“前体化合物”是指尽管其本身与所述缀合物结构和功能上都不完全相同，但在进入体内之后或在体液环境下能够发生反应、形成所述包含本公开提供的缀合物、从而发挥效果、实现本公开提供的目的的化合物。某些情况下，这些前体化合物能够增加药物的稳定性、延长缓释时间、增加生物利用度等作用。在一些实施方式中，所述前体化合物包括可在人体内反应形成缀合物中全部功能性基团A0的前体基团。在一些实施方式中，所述前体化合物包括缀合物中全部活性羟基被乙酰氧基取代形成的化合物。在一些实施方式中，所述前体化合物包括药物前体基团，所述药物前体基团是由缀合物中所述功能性基团对应的治疗剂的前体化合物形成的残基。在一些实施方式中，所述药物前体基团可以是例如所述功能性基团中羟基或氨基官能团中的活性氢被酰基、烷基、或者磷酰基取代而形成的基团。本领域技术人员可以理解，这些药学上可接受的盐以及前体化合物的应用同样处于本公开提供的范围之内。
药物组合物
在一方面，本公开还提供一种药物组合物，所述药物组合物包含本公开提供的缀合物以及药学上可接受的载体。
所述药学上可接受的载体可以是本领域常规使用的载体，例如但不限于水、生理盐水、磁性纳米粒(magnetic nanoparticles，如基于Fe3O4或Fe2O3的纳米粒)、碳纳米管(carbon nanotubes)、介孔硅(mesoporous silicon)、磷酸钙纳米粒(calcium phosphate nanoparticles)、聚乙烯亚胺(polyethylenimine，PEI)、聚酰胺型树形高分子(polyamidoamine(PAMAM)dendrimer)、聚赖氨酸(poly(L-lysine)，PLL)、壳聚糖(chitosan)、1，2-二油酰基-3-三甲铵丙烷(1，2-dioleoyl-3-trimethylammonium-propane，DOTAP)、聚D型或L型乳酸/羟基乙酸共聚物(poly(D&L-lactic/glycolic acid)copolymer，PLGA)、聚(氨乙基乙撑磷酸酯)(poly(2-aminoethyl ethylene phosphate)，PPEEA)和聚(甲基丙烯酸-N，N-二甲氨基乙酯)(poly(2-dimethylaminoethyl methacrylate)，PDMAEMA)以及它们的衍生物中的一种或多种。
在一些实施方式中，所述药学上可接受的载体含有生理学可接受的化合物，所述化合物起到例如稳定药物组合物或增加或减少缀合物和/或药物组合物的吸收的作用。所述生理学可接受的化合物选自如下化合物中的一种或多种：碳水化合物，例如葡萄糖、蔗糖和/或葡聚糖；抗氧化剂，例如抗坏血酸和/或谷胱甘肽；螯合剂；低分子量蛋白；降低任何共同给药的物质的清除或水解的组合物；赋形剂；稳定剂和缓冲剂。也可以将清洁剂用于稳定组合物或增加或减少药物组合物的吸收。所述生理学可接受的化合物还可以包括湿润剂、乳化剂、分散剂或特别用于防止微生物生长或作用的防腐剂中的一种或多种。所述生理学可接受的化合物是本领域技术人员已知的，本公开不再赘述。本领域技术人员容易理解的是，药学上可接受的载体以及生理学可接受的化合物的选择取决于例如给药途径和任何共同给药的物质的特定生理化学特性。
在一些实施方式中，药学上可接受的载体是无菌的并且通常不含不合需要的物质。本公开提供的药物组合物可根据需要进一步包含可药用的辅助物质以接近生理学条件，例如pH调节剂和缓冲剂，毒性调节剂等，例如乙酸钠、氯化钠、氯化钾、氯化钙、乳酸钠等，药物组合物中的本公开提供的缀合物的浓度可以在大的区间内变化，并且主要依据流体体积、粘度、体重等按照特定的给药方式加以选择。
在一些实施方式中，所述药物组合物中，对缀合物和药学上可接受的载体的含量没有特别要求，在一些实施方式中，缀合物与药学上可接受的载体的重量比可以为1∶(1-500)，在一些的实施方式中，上述重量比为1∶(1-50)。
在一些实施方式中，所述药物组合物中，还可以包含药学上可接受的其它辅料，该辅料可以为本领域常规采用的各种制剂或化合物的一种或多种。例如，所述药学上可接受的其它辅料可以包括pH缓冲液、保护剂和渗透压调节剂中的至少一种。
所述pH缓冲液可以为pH值7.5-8.5的三羟甲基胺基甲烷盐酸盐缓冲液和/或pH值5.5-8.5的磷酸盐缓冲液，例如可以为pH值5.5-8.5的磷酸盐缓冲液。
所述保护剂可以为肌醇、山梨醇、蔗糖、海藻糖、甘露糖、麦芽糖、乳糖和葡萄糖中的至少一种。以所述药物组合物的总重量为基准，所述保护剂的含量可以为0.01-30重量％。
所述渗透压调节剂可以为氯化钠和/或氯化钾。所述渗透压调节剂的含量使所述药物组合物的渗透压为200-700毫渗摩尔/千克(mOsm/kg)。根据所需渗透压，本领域技术人员可以容易地确定所述渗透压调节剂的含量。在一些实施方式中，所述药物组合物所制成的制剂在给药过程中的剂量会因给药方式的不同而发生调整。
在一些实施方式中，所述药物组合物可以为液体制剂，例如注射液；也可以为冻干粉针剂，实施给药时与液体辅料混合，配制成液体制剂。所述液体制剂可以但不限于用于皮下、肌肉或静脉注射给药，也可以但不限于通过穿刺注射、或通过口咽吸入、或鼻腔给药等方式递送所述药物组合物。在一些实施方式中，所述药物组合物用于皮下、肌肉、静脉或鞘内注射给药。
在一些实施方式中，所述药物组合物可以为脂质体制剂的形式。在一些实施方式中，所述脂质体制剂中使用的药学上可接受的载体包含含胺的转染化合物(下文也可将其称为有机胺)、辅助脂质和/或聚乙二醇化脂质。其中，所述有机胺、辅助脂质和聚乙二醇化脂质可分别选自于中国专利申请CN103380113A(通过引用的方式将其整体并入本公开)中所描述的含胺的转染化合物或其药学上可接受的盐或衍生物、辅助脂质和聚乙二醇化脂质中的一种或多种。
[根据细则26改正 07.12.2023]
在一些实施方式中，所述有机胺可为中国专利申请CN103380113A中描述的如式(201)所示的化合物或其药学上可接受的盐：
其中：
X101和X102各自独立地是O、S、N-A或C-A，其中A是氢或C1-C20烃链；
Y101和Z101各自独立地是C＝O、C＝S、S＝O、CH-OH或SO2；
R101、R102、R103、R104、R105、R106和R107各自独立地是氢，环状或无环的、被取代的或未被取代的、支链或直链脂族基团，环状或无环的、被取代的或未被取代的、支链或直链杂脂族基团，被取代的或未被取代的、支链或直链酰基，被取代的或未被取代的、支链或直链芳基，被取代的或未被取代的、支链或直链杂芳基；
x是1-10的整数；
n是1-3的整数，m是0-20的整数，p是0或1；其中，如果m＝p＝0，则R102是氢；
[根据细则26改正 07.12.2023]
并且，如果n或m中的至少一个是2，那么R103和在式(201)中的氮形成如式(202)或式(203)所示的结构：
其中，g、e和f各自独立地是1-6的整数，“HCC”代表烃链，且每个*N代表式(201)中的氮原子。
在一些实施方式中，R103是多胺。在其它实施方式中，R103是缩酮。在一些实施方式中，在式(201)中的R101和R102中的每一个独立地是任意的被取代的或未被取代的、支链或直链烷基或烯基，所述烷基或烯基具有3至约20个碳原子，诸如8至约18个碳原子，和0至4个双键，诸如0至2个双键。
[根据细则26改正 07.12.2023]
在一些实施方式中，如果n和m中的每一个独立地具有1或3的值，那么R103可以是下述式(204)-式(213)中的任一个：
其中，式(204)-式(213)中，g、e和f各自独立地是1-6的整数，每个“HCC”代表烃链，且每个*显示R103与在式(201)中的氮原子的可能连接点，其中在任意*位置上的每个H可以被替换以实现与在式(201)中的氮原子的连接。
本领域技术人员可以通过任何合理的方法获得式(201)所示的化合物。在一些实施方式中，式(201)所示化合物可以根据中国专利申请CN103380113A中的描述制备。
在一些实施方式中，所述有机胺为如式(214)所示的有机胺和/或如式(215)所示的有机胺：
所述辅助脂质为胆固醇、胆固醇的类似物和/或胆固醇的衍生物；
所述聚乙二醇化脂质为1，2-二棕榈酰-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)]-2000。
在一些实施方式中，所述药物组合物中，所述有机胺、所述辅助脂质和所述聚乙二醇化脂质三者之间的摩尔比为(19.7-80)∶(19.7-80)∶(0.3-50)，例如可以为(50-70)∶(20-40)∶(3-20)。
在一些实施方式中，由本公开提供的缀合物与上述含胺的转染试剂形成的药物组合物颗粒具有约30nm至约200nm的平均直径，通常为约40nm至约135nm，更通常地，该脂质体颗粒的平均直径是约50nm至约120nm、约50nm至约100nm、约60nm至约90nm或约70nm至约90nm，例如，该脂质体颗粒的平均直径是约30、40、50、60、70、75、80、85、90、100、110、120、130、140、150或160nm。
在一些实施方式中，由本公开提供的缀合物与上述含胺的转染试剂形成的药物组合物中，缀合物与全部脂质(例如有机胺、辅助脂质和/或聚乙二醇化脂质)的重量比(重量/重量比)在从约1∶1至约1∶50、从约1∶1至约1∶30、从约1∶3至约1∶20、从约1∶4至约1∶18、从约1∶5至约1∶17、从约1∶5至约1∶15、从约1∶5至约1∶12、从约1∶6至约1∶12或从约1∶6至约1∶10的范围内，例如，本公开提供的缀合物与全部脂质的重量比为约 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或1∶18。
在一些实施方式中，所述药物组合物在销售时各组分可以独立存在，在使用时可以液体制剂的形式存在。在一些实施方式中，本公开提供的缀合物与上述药学上可接受的载体形成的药物组合物可以按照已知的各种方法制备，只是用本公开提供的缀合物替代现有适配体或缀合物即可；在一些实施方式中，可以按照如下方法制备：
将有机胺、辅助脂质和聚乙二醇化脂质按照上述摩尔比悬浮于醇中并混匀得到脂质溶液；醇的用量使得到的脂质溶液的总质量浓度为2-25mg/mL，例如可以为8-18mg/mL。所述醇选自药学上可接受的醇，诸如在室温附近为液体的醇，例如，乙醇、丙二醇、苯甲醇、甘油、聚乙二醇200，聚乙二醇300，聚乙二醇400中的一种或多种，例如可以为乙醇。
将本公开提供的缀合物溶解于缓冲盐溶液中，得到缀合物水溶液。缓冲盐溶液的浓度为0.05-0.5M，例如可以为0.1-0.2M，调节缓冲盐溶液的pH至4.0-5.5，例如可以为5.0-5.2，缓冲盐溶液的用量使缀合物的浓度不超过0.6mg/mL，例如可以为0.2-0.4mg/mL。所述缓冲盐选自可溶性醋酸盐、可溶性柠檬酸盐中的一种或多种，例如可以为醋酸钠和/或醋酸钾。
将脂质溶液和缀合物水溶液混合，将混合后得到的产物在40-60孵育至少2分钟，例如可以为5-30分钟，得到孵育后的脂质体制剂。脂质溶液和缀合物水溶液的体积比为1∶(2-5)，例如可以为1∶4。
将孵育后的脂质体制剂浓缩或稀释，去除杂质，除菌，得到本公开提供的药物组合物，其理化参数为pH值为6.5-8，包封率不低于80％，粒径为40-200nm，多分散指数不高于0.30，渗透压为250-400mOsm/kg；例如理化参数可以为pH值为7.2-7.6，包封率不低于90％，粒径为60-100nm，多分散指数不高于0.20，渗透压为300-400mOsm/kg。
其中，浓缩或稀释可以在去除杂质之前、之后或同时进行。去除杂质的方法可以采用现有各种方法，例如可以使用切相流系统、中空纤维柱，在100K Da条件下超滤，超滤交换溶液为pH7.4的磷酸盐缓冲液(PBS)。除菌的方法可以采用现有各种方法，例如可以在0.22μm滤器上过滤除菌。
本公开的缀合物的应用
在又一方面，本公开还提供了本公开提供的缀合物和/或药物组合物在制备用于对肿瘤及肿瘤相关疾病或症状进行治疗的药物中的应用。
在又一方面，本公开还提供了一种对肿瘤及肿瘤相关疾病或症状进行治疗的方法，所述方法包括向有需要的受试者给予本公开所述的缀合物和/或药物组合物。
通过给予本公开提供的缀合物和/或药物组合物，本公开的方法能够有效治疗肿瘤及肿瘤相关疾病或症状；并且，在本公开提供的缀合物的高特异性靶向作用下，可以减少治疗剂在不期望的身体其它器官/组织处的分布，降低潜在的副反应。特别是对于肿瘤治疗领域中常用的、已知副反应明显的放疗和/或化疗药物而言，具有重要意义和显著价值。
本公开所使用的术语“给药/给予”是指通过使得至少部分地将缀合物和/或药物组合物定位于期望的位点以产生期望效果的方法或途径，将缀合物和/或药物组合物放置入受试者体内。适于本公开方法的给药途径包括局部给药和全身给药。一般而言，局部给药导致与受试者整个身体相比将更多缀合物和/或药物组合物递送至特定位点；而全身给药导致将所述缀合物和/或药物组合物递送至受试者的基本整个身体。
进一步地，本公开的发明人意外地发现，本公开的缀合物和/或药物组合物能够高效地通过血脑屏障，在全身给药的情况下就能够靶向至脑内的肿瘤中，从而进一步提高功能性基团的递送效率、节省成本并降低不期望的副反应。
可通过本领域已知的任何合适途径向受试者给药，所述途径包括但不仅限于：口服或胃肠外途径，如静脉内给药、肌肉内给药、皮下给药、经皮给药、气道给药(气雾剂)、肺部给药、鼻部给药、直肠给药和局部给药(包括口腔含化给药和舌下给药)。给药频率可以是每天、每周、每两周、每三周、每个月或每年1次或多次。
本公开所述的缀合物和/或药物组合物的使用剂量可为本领域常规的剂量，所述剂量可以根据各种参数、尤其是受试者的年龄、体重和性别来确定。可在细胞培养或实验动物中通过标准药学程序测定毒性和疗效，例如测定LD50(使50％的群体致死的剂量)和ED50(在量反应中指能引起50％最大反应强度的剂量，在质反应中，指引起50％实验对象出现阳性反应时的剂量)。可基于由细胞培养分析和动物研究得到的数据得出人用剂量的范围。
在给予本公开所述的缀合物和/或药物组合物时，例如，对于雄性或雌性、6-12周龄、体重18-25g的C57BL/6J或C3H/HeNCrlVr小鼠，以所述缀合物和/或药物组合物中的缀合物的量计：其缀合物用量可以为0.001-100mg/kg体重，在一些实施方式中为0.01-50mg/kg体重，在进一步的实施方式中为0.05-20mg/kg体重，在更进一步的实施方式中为0.1-15mg/kg体重，在又进一步的实施方式中为0.1-10mg/kg体重。在给予本公开所述的缀合物和/或药物组合物时，可优选上述用量。
试剂盒
本公开提供了一种试剂盒，所述试剂盒包含本公开提供的缀合物和/或药物组合物。
在一些实施方式中，本公开所述的试剂盒可在一个容器中提供缀合物和/或药物组合物。在一些实施方式中，本公开所述的试剂盒可包含一个提供药学上可接受的赋形剂的容器。在一些实施方式中，所述试剂盒中还可包含其它成分，如稳定剂或防腐剂等。在一些实施方式中，本公开所述的试剂盒可在不同于提供本公开所述缀合物和/或药物组合物的容器以外的其它容器中包含至少一种其它治疗剂。在一些实施方式中，所述试剂盒可包含用于将缀合物和/或药物组合物与药学上可接受的载体和/或辅料或其它成分(若有的话)进行混合的说明书。
在本公开的试剂盒中，所述缀合物和药学上可接受的载体和/或辅料以及所述药物组合物，和/或药学上可接受的辅料可以任何形式提供，例如液体形式、干燥形式或冻干形式。在一些实施方式中，所述缀合物和药学上可接受的载体和/或辅料以及所述药物组合物和任选的药学上可接受的辅料基本上纯净和/或无菌。在一些实施方式中，可在本公开的试剂盒中提供无菌水。
下面将通过实施例来进一步说明本公开，但是本公开并不因此而受到任何限制。
 实施例
除非特别说明，以下实施例中所用到的试剂、培养基均为市售商品，所用到的核酸电泳、real-time PCR等操作均参照Molecular Cloning(Cold Spring Harbor LBboratory Press(1989))所记载的方法进行。
制备例1-8和18缀合物AP1-AP8、AP18的合成
通过固相合成方法分别合成了表1中编号为AP1-AP8和AP18的缀合物，并且，在分别按照表1中AP1-AP8和AP18对应的核苷酸序列从3′-5′方向依次连接全部核苷单体后，再按照固相合成方法连接核苷亚磷酰胺单体的方式连接Cy5亚磷酰胺单体(购自苏州吉玛公司，批号CY5P21H1B)。随后，将核苷酸序列加入甲胺水溶液与氨水等体积混合溶液中，溶液相对于寡核苷酸的用量为0.5ml/μmol，在25℃下反应2h，过滤除去固体，将上清液真空浓缩至干。
利用制备型离子色谱纯化柱(Source 15Q)，通过NaCl水溶液的梯度洗脱，完成制备的缀合物的纯化。具体而言为：洗脱剂A：20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9∶1(体积比)；洗脱剂B：1.5M氯化钠，20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9∶1(体积比)；洗脱梯度：洗脱剂A：洗脱剂B＝100∶0-50∶50梯度洗脱。收集产品洗脱液后合并，采用反相色谱纯化柱进行脱盐，具体条件包括采用葡聚糖凝胶柱进行脱盐，填料为葡聚糖凝胶G25，以去离子水洗脱。将获得的洗脱液浓缩除去溶剂并冻干，分别获得了5′末端核苷酸的核糖5′位通过磷酸酯基连接基团连接荧光基团Cy5的缀合物AP1-AP8和AP18。
在上述缀合物AP1-AP8和AP18合成完成后，使用超纯水(Milli-Q超纯水仪自制，电阻率18.2MΩ*cm(25))将制备获得的缀合物稀释至浓度为0.2mg/mL后，利用液质联用仪(LC-MS，Liquid Chromatography-Mass Spectrometry，购于Waters公司，型号：LCT Premier)进行分子量检测。其结果，分子量实测值与理论值一致，表明获得了目标缀合物。
缀合物的分子量和MS值如下表1a：
表1a缀合物的质谱表征结果
对比制备例9-17和19对照缀合物对比AP9-对比AP17、对比AP19的合成
按照制备例1的方法，分别合成了表1中编号为对比AP9-对比AP17、对比AP19的缀合物并检测分子量以对合成的缀合物进行确认，区别仅在于，分别按照表1中对应于对比AP9-对比AP17、对比AP19的序列，依次连接核苷单体。从而，分别获得了5′末端核苷酸的核糖5′位通过磷酸酯基连接基团连接荧光基团Cy5的对照缀合物对比AP9-对比AP17、对比AP19。
表1缀合物的核苷酸序列
表1中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；CY5表示荧光染料基团Cy5(Cyanine 5)基团在适配体上的连接位点。
制备例20缀合物20-的合成
表2缀合物中的核苷酸序列
表2中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；MMAE表示小分子药物基团MMAE(一甲基澳瑞他汀E)基团在适配体上的连接位点。
本制备例中，按照以下步骤制备获得了缀合物20，该缀合物20将缀合物AP2中的染料基团替换为小分子药物基团MMAE，所述连接基团为2-(磷酸酯基-(CH2)6-S-)-马来酰亚胺己酰基-缬氨酸-瓜氨酸-对氨基苯甲基亚基。
(20-1)寡核苷酸S1的制备
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式中，代表缀合物20对应的寡核苷酸序列。
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(20-2)寡核苷酸S2的合成：
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用10.0ml纯化水溶解70.0mg步骤(20-1)中制备得到的S1(6.42μmol)后，向所得溶液中加入105mg TCEP(三(2-氯乙基)磷酸酯，0.37mmol，购自毕得医药，批号：BD155793)溶于10.0ml纯化水得到的TCEP水溶液。混匀，在室温下反应2小时，将反应液用10mL纯化水稀释并过滤，得到反应液28mL，将反应液转移至3K规格的超滤管中，在3900rpm的条件下离心30min。再重复超滤和离心的步骤2次，收集滤膜内产品，获得寡核苷酸S2(67.0mg，产率：95.7％)。
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(20-3)寡核苷酸20的合成：
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将24mg Vc MMAE(18.56μmol，5eq，购自CSN公司，批号CSN16143-005)溶于6.0ml DMF中，并加入60μl三乙胺，获得Vc MMAE溶液。用6.0ml纯化水溶解40.0mg步骤(20-2)中制备得到的寡核苷酸S2(3.71μmol，1eq)后，向所得溶液中加入上述Vc MMAE溶液，在室温下反应2小时后，获得缀合物20粗品(工艺图中表示为S3)。
将所得到的缀合物20粗品加入0.5 ml纯化水稀释，并以0.45μm滤膜过滤，滤液使用安捷伦半制备反相柱色谱纯化，所使用的色谱柱为Kromasil 100-10-C18，10um，21.2*250mm；以100mM三乙胺乙酸盐缓冲液(TEAA，PH＝7.0-7.3)∶乙腈＝95∶5-35∶65作为流动相进行梯度洗脱。收集产物峰洗脱液，蒸发除去溶剂，获得缀合物20(55 mg，收率56.7％)。经LC-MS检测分子量，理论值：12092.73，实测值：12091.39，实测值与理论值一致，表明该缀合物20具有S3所示的结构，该缀合物20将缀合物AP2中的染料基团替换为小分子药物基团MMAE，并且连接基团为2-(磷酸酯基-(CH2)6-S-)-马来酰亚胺己酰基-缬氨酸-瓜氨酸-对氨基苯甲基亚基(2-(磷酸酯基-(CH2)6-S-)-MC-Val-Cit-PAB)。
制备例21-26缀合物21-23，26和对比缀合物24-25的合成
按照制备例20的方法，分别合成了表2中编号为缀合物21、缀合物22、缀合物23、对比缀合物24、对比缀合物25和缀合物26并检测分子量以对合成的缀合物进行确认，区别仅在于，分别按照表2中对应于的缀合物21、缀合物22、缀合物23、对比缀合物24、对比缀合物25和缀合物2序列，依次连接核苷单体。
缀合物的分子量和MS值如下表2a：
表2a：缀合物的质谱表征结果
实验例1缀合物在小鼠体内的靶向性
本实验例考察了制备得到的缀合物AP2、AP3-8以及对比AP9-对比AP12在小鼠体内的靶向性。
在添加有10％的胎牛血清(FBS，RMBIO公司)的DMEM完全培养基(MACGENE公司，货号CM15019)中，于37在含5％CO2/95％空气的培养箱中培养U118MG人胶质瘤细胞(购自广州吉妮欧生物科技有限公司)。
取对数期生长的U118MG人胶质瘤细胞，以(0.25％的胰酶)消化，收集细胞，离心去上清，将细胞重悬于添加10％FBS的DMEM培养基中制成浓度为1×108cells/mL的细胞培养液。
实验动物NOD-SCID小鼠24只(购自斯贝福(北京)生物技术有限公司)，雌性，12周龄。将上述细胞培养液接种于NOD-SCID小鼠右前肢皮下位置，接种体积为每只100μL，即，每只小鼠接种1×107个细胞。注射后继续饲养小鼠20天，获得接种U118MG皮下瘤的小鼠。
用DMEM培养基将上述制备的适配体AP2、AP3-8以及对比AP9-对比AP12分别配制为0.3mg/mL的溶液。
U118MG细胞接种后14天开始给药，给药当天记为D1。实验采用尾静脉注射给药方式，每天给药一次，共三次给药。
将接种U118MG皮下瘤的24只小鼠随机分为12组，每组2只小鼠：
对于7组小鼠，分别向每组中的每只小鼠给予AP2、AP3、AP4、AP5、AP6、AP7或AP8，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为3mg/kg，依次分别记为测试组1A-1G；
对于另外4组小鼠，分别向每组中的每只小鼠给予对比AP9、对比AP10、对比AP11或对比AP12，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为3mg/kg，依次分别记为对照组1H-1K；
对于另外1组中的2只小鼠，向每只小鼠给予DMEM培养基，给药容积为10μL/g小鼠体重，记为空白对照组1Y。
于首次给药后1h、24h和48h时，使用小动物活体光学成像系统IVIS Lumina Series III对各小鼠进行活体成像。在D5时，处死各组小鼠并取肿瘤组织和肾脏进行荧光成像。
图1A-1C依次分别是示出了给予不同缀合物后1h、24h和48h时，小鼠体内的荧光成像结果的图，其中每一小图内3只小鼠中最左侧的1只为空白对照组1Y的小鼠。由图1A可知，空白对照组不显示任何荧光信号；与其不同，在给药后1h时，各测试组和对照组的小鼠在皮下肿瘤处均显示出荧光信号；由图1B和1C可知，在给药后24h和48h时，仅测试组1A-1G的小鼠在皮下肿瘤处显示出较强的荧光信号，而对照组1H-1K的小鼠几乎不显示荧光信号或仅显示很弱的荧光信号。进一步地，图1D是示出了D5时处死小鼠后，各组小鼠肿瘤组织和肾脏的荧光信号成像的图，其中，Blank表示空白对照组15Y。由图1D可知，空白对照组1Y和对照组1H-1K的小鼠的肿瘤组织几乎不显示荧光信号或仅显示很弱的荧光信号；与此相对，给予本公开的缀合物的测试组1A-1G的小鼠的肿瘤组织均显示出强烈的荧光信号，同时在代谢器官肾脏处仅显示微弱的荧光信号，表明与对照缀合物相比，包含本公开提供的缀合物中的递送基团的缀合物能够更稳定、高效地靶向肿瘤组织。
实验例2缀合物在小鼠体内的靶向性
本实验例考察了制备得到的缀合物AP2、AP1以及对比AP13-对比AP17在小鼠体内的靶向性。
在添加有10％的胎牛血清(FBS，RMBIO公司)的DMEM完全培养基(MACGENE公司，货号CM15019)中，于37在含5％CO2/95％空气的培养箱中培养U118MG人胶质瘤细胞(购自广州吉妮欧生物科技有限公司)。
取对数期生长的U118MG人胶质瘤细胞，以(0.25％的胰酶)消化，收集细胞，离心去上清，将细胞重悬于添加10％FBS的DMEM培养基中制成浓度为1×108cells/mL的细胞培养液。
实验动物NOD-SCID小鼠16只(购自斯贝福(北京)生物技术有限公司)，雄性，12周龄。将上述细胞培养液接种于NOD-SCID小鼠右前肢皮下位置，接种体积为每只100μL，即，每只小鼠接种1×107个细胞。注射后继续饲养小鼠20天，获得接种U118MG皮下瘤的小鼠。
用DMEM培养基将上述制备的适配体AP2、AP1以及对比AP13-对比AP17分别配制为0.3mg/mL的溶液。
U118MG细胞接种后21天开始给药，给药当天记为D1。实验采用尾静脉注射给药方式，每天给药一次，共三次给药。
将接种U118MG皮下瘤的16只小鼠随机分为8组，每组2只小鼠：
对于2组小鼠，分别向每组中的每只小鼠给予AP2或AP1，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为3mg/kg，依次分别记为测试组2A-2B；
对于另外5组小鼠，分别向每组中的每只小鼠给予对比AP13、对比AP14、对比AP15、对比AP16或对比AP17，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为3mg/kg，依次分别记为对照组2C-2G；
对于另外1组中的2只小鼠，向每只小鼠给予DMEM培养基，给药容积为10μL/g小鼠体重，记为空白对照组2Y。
于首次给药后1h、24h和48h时，使用小动物活体光学成像系统IVIS Lumina Series III对各小鼠进行活体成像。在D6时，处死各组小鼠并取肿瘤组织和肾脏进行荧光成像。
图2A-2C依次分别是示出了给予不同缀合物后1h、24h和48h时，小鼠体内的荧光成像结果的图，其中每一小图内3只小鼠中最左侧的1只为空白对照组2Y的小鼠。由图2A可知，空白对照组不显示任何荧光信号；与其不同，在给药后1h时，各测试组和对照组的小鼠在皮下肿瘤处均显示出荧光信号；由图2B和2C可知，在给药后24h和48h时，仅测试组2A和2B的小鼠在皮下肿瘤处显示出较强的荧光信号，而空白对照组2Y及对照组2C-2G的小鼠完全不显示荧光信号。进一步地，图2D是示出了D6时处死小鼠后，各组小鼠肿瘤组织和肾脏的荧光信号成像的图，其中，Blank表示空白对照组2Y。由图2D可知，空白对照组2Y和对照组2C-2G的小鼠的肿瘤组织完全不显示荧光信号；与此相对，给予本公开的缀合物的测试组2A或2B的小鼠的肿瘤组织均显示出强烈的荧光信号，同时在代谢器官肾脏处仅显示微弱的荧光信号，表明与对照适配体相比，具有各种式(1)所示序列的适配体均能够稳定、高效地靶向肿瘤组织，进而表明，包含由这些适配体形成的递送基团的本公开提供的缀合物能够有效地到达肿瘤组织。
实验例3本公开的缀合物在小鼠体内的活性
本实验例考察了制备得到的缀合物20在小鼠体内的抗肿瘤活性。
本实验的小鼠购自斯贝福公司，种系为NOD-SCID，等级为SPF，性别均为雌性，周龄为6-8周龄；U118MG神经胶质瘤细胞购自吉尼欧。
取对数期生长的U118MG细胞，消化重悬于添加有10％的胎牛血清(FBS，GIBCO公司)的DMEM完全培养基(MACGENE公司，货号CM15019)中进行培养，直至细胞密度为1×108细胞/mL，获得含有U118MG细胞的培养液。将上述含有U118MG细胞的培养液接种于各小鼠右前肢皮下位置，注射体积100μL。从而，每只小鼠接种1×107个U118MG胶质瘤细胞。
用PBS将上述制备的缀合物AP2配制为1.94mg/mL的溶液。用PBS将缀合物20分别配制为0.625mg/mL、1.25mg/mL以及2.06mg/mL的溶液(均以寡核苷酸的量计算)；用10％DMSO+90％PBS(体积比)的混合溶液将MMAE(购自上海麦克林公司，批号C12886583)溶解为0.038mg/mL的溶液。
将细胞接种当天记为D1，分别于D8、D12、D16和D20各给药一次。
将36只小鼠随机分为以下6组，每组6只小鼠：
对于空白对照组3a，采用尾静脉注射方式给予PBS，单次给药容积为10μL/g；
对于对照组3b，采用尾静脉注射方式给予上述缀合物AP2溶液，单次给药容积为10μL/g，单次给药剂量为15.5mg/kg；
对于对照组3c，采用尾静脉注射方式给予上述MMAE溶液，单次给药容积为10μL/g，单次给药剂量为0.3mg/kg；
对于测试组3d，采用尾静脉注射方式给予上述浓度为0.625mg/mL的缀合物20溶液，单次给药容积为10μL/g，单次给药剂量为5mg/kg(以寡核苷酸质量计)，其中包含MMAE的剂量相当于0.3mg/kg；
对于测试组3e，采用尾静脉注射方式给予上述浓度为2.06mg/mL的缀合物20溶液，单次给药容积为10μL/g，单次给药剂量为16.5mg/kg(以寡核苷酸质量计)，其中包含MMAE的剂量相当于1mg/kg；
对于测试组3f，采用皮下注射方式给予上述浓度为1.25mg/mL的缀合物20溶液，单次给药容积为5μL/g，单次给药剂量为5mg/kg(以寡核苷酸质量计)，其中包含MMAE的剂量相当于0.3mg/kg。
[3]检测
通过体外测量的方式测定测量肿瘤的长径和短径。肿瘤体积根据公式1/2(长径×短径2)进行计算。在D8首次给药前，测定各组肿瘤体积并记录平均肿瘤体积，D16开始分别测量并记录各组肿瘤体积，每周测量两次。
图3是示出了各组小鼠中肿瘤体积随时间变化的折线图。由图3结果可知，在仅给予PBS与AP2的空白对照组3a和对照组3b中，肿瘤体积迅速增加；仅给予MMAE的对照组3c中肿瘤体积增加速度有所降低，表明MMAE本身对肿瘤增殖显示出抑制效果。进一步地，MMAE含量与对照组3c相当的测试组3d和3f在测试期间肿瘤体积均显著小于对照组3c，显示出比单独给予MMAE的对照组3c更加优异的抗肿瘤活性。这表明，本公开提供的缀合物能够有效地将MMAE递送至肿瘤组织，在显示出肿瘤靶向能力的同时，还降低了MMAE分子在其它组织分布带来的毒性风险，且各种给药方式均能够有效抑制肿瘤增殖。此外，进一步提高给药剂量的测试组3e的肿瘤体积在测试期间几乎不增加，显示出更加优异的抗肿瘤效果。
上述结果表明，本公开提供的缀合物能够有效地将对肿瘤具有抑制作用的小分子药物基团靶向递送至肿瘤组织，显示出良好的抗肿瘤活性和剂量依赖效应。
实验例4缀合物在U118MG细胞原位瘤模型小鼠体内分布
在添加有10％的胎牛血清(FBS，RMBIO公司)的DMEM完全培养基(MACGENE公司，货号CM15019)中，于37在含5％CO2/95％空气的培养箱中培养U118MG人胶质瘤细胞(购自广州吉妮欧生物科技有限公司)。
以0.25wt％胰酶消化并收集细胞，吸去上清并将细胞重悬于添加10％FBS的DMEM培养基中制成细胞密度为4×107cells/mL的细胞培养液。
实验动物NOD-SCID小鼠6只(购自斯贝福(北京)生物技术有限公司)，雄性，12周龄。将上述细胞培养液接种于NOD-SCID小鼠中，采用小鼠侧脑室注射方式，将细胞培养液注射于小鼠右侧纹状体，位置为AP(anteroposterior前后/正位)：1mm，ML(medial lateral内侧)：1.5mm，DV(dorsal ventral背腹侧)：3.5mm，注射体积10μL，即，每只小鼠接种4×105个细胞。注射后继续饲养小鼠14天。
用1×DMEM培养基将AP2和对比AP19分别溶解成0.3mg/mL浓度(以适配体计)的缀合物溶液。取4只前述小鼠，通过尾静脉注射给药，分别注射AP2和对比AP19溶液，所有动物根据体重计算给药剂量，给药体积均为10μL/g，以适配体的量计，每只动物给药量为3mg/kg，每组给予2只小鼠，分别记为测试组4a和对照组4b。
向2只小鼠中的每一只分别注射10μL的DMEM培养基，记为空白对照组4Y。
在给药后24h时，分别杀死各组小鼠中的一只并取脑组织，给药后48h时杀死剩余小鼠并取脑组织，在IVIS Lumina Series III中对小鼠脑组织进行荧光成像。结果参见图4所示。
图4是示出了给药后24h和48h时，给予空白对照组4Y、测试组4a和对照组4b后建立U118MG原位瘤模型小鼠脑组织的荧光成像图。其中，Blank表示空白对照组，Ith表示鞘内注射，iv表示尾静脉注射。从图4的结果可以看出，空白对照组和给予对比AP19的对照组4b在脑内均未显示出任何荧光信号，表明对原位脑胶质瘤没有显著靶向效果；与此不同，给予本公开提供的缀合物的测试组4a在24h和48h时在接种肿瘤位置均显示出明显的荧光信号，表明缀合物AP2在尾静脉给药时仍能到达并有效靶向脑胶质瘤，说明本公开提供的缀合物即使是系统给药的情况下，也有望穿透血脑屏障(Blood-Brain-Barrier，BBB)进入脑胶质瘤，显示出优异的靶向作用和成药能力。
实验例5缀合物对U118MG原位瘤模型小鼠体内活性
按照实验例4的方法培养表达Luciferase(Photinus pyralis)报告基因的U118MG人胶质瘤细胞，以下简称U118MG-luc人胶质瘤细胞(购自南京科佰生物科技有限公司)。取对数期生长的U118MG-luc人胶质瘤细胞，以0.25wt％胰酶消化并收集细胞，离心后吸去上清液并将细胞重悬于无血清DMEM培养基中制成细胞密度为4×107cells/mL的细胞培养液。
实验动物Balb/C-nude裸鼠24只(购自斯贝福(北京)生物技术有限公司)，雄性，12周龄。将上述细胞培养液接种于Balb/C-nude裸鼠纹状体中，采用小鼠纹状体注射方式，将细胞培养液注射于小鼠右侧纹状体，位置为AP(anteroposterior前后/正位)：1mm，ML(medial lateral内侧)：1.5mm，DV(dorsal ventral背腹侧)：3.5mm，注射体积10μL，即，每只小鼠接种4×105个细胞。原位瘤接种后继续饲养小鼠14天。
用1×DMEM(购自中科迈晨(北京)科技有限公司，批号为K1902200)培养基将缀合物20溶解成1mg/mL浓度(以适配体计)的缀合物溶液。缀合物21和对比缀合物24分别溶解成0.8mg/mL浓度(以适配体计)的缀合物溶液。
接种原位瘤后第15天，使用小动物活体光学成像系统IVIS Lumina Series III对各小鼠进行活体成像，根据脑部荧光强度分组，每组6只小鼠，给药当天记为D1(即，实验第1天，以下D4、D8等相应表示实验第4天、第8天，以此类推)。
活体成像方法：每只小鼠腹腔注射10μL/g体重浓度15mg/mL D-荧光素钾盐工作液(购自翌圣生物科技(上海)股份有限公司)，注射后10min活体成像(Lumina III小动物活体成像系统)。成像后圈选小鼠脑部荧光区域(ROI)，软件测定荧光强度(Radiance)。在此条件下，U118MG-luc人胶质瘤细胞中表达的Luciferase(Photinus pyralis)报告基因能够产生荧光响应，因此荧光强度能够反映胶质瘤细胞的增殖数量。荧光强度越高，表明胶质瘤细胞的数量越大。
实验通过皮下注射给药方式，每组小鼠分别于D1、D4、D8和D12给药。于给药前称重，按重量给药。
对于测试组5a，分别向每只小鼠给予缀合物20，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为5mg/kg，其中包含MMAE的剂量相当于0.3mg/kg。
对于测试组5b，分别向每只小鼠给予缀合物21，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为4mg/kg，其中包含MMAE的剂量相当于0.3mg/kg。
对于对照组5c，分别向每只小鼠给予对比缀合物24，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为4mg/kg，其中包含MMAE的剂量相当于0.3mg/kg。
对于空白对照组，分别向每只小鼠给予DMEM培养基，单次给药容积为5μL/g小鼠体重。
按照上述方法，分别于D1、D22、D31和D39对每组小鼠进行活体成像分析，测定荧光强度。结果参见图5所示。
图5示出了给予本公开提供的缀合物或对照化合物后，U118MG原位瘤模型小鼠肿瘤肿瘤荧光强度随时间变化的折线图。从图5的结果可以看出，与给药后D1相比较，随着观察时间的延长，空白对照组和对照组肿瘤荧光强度(Radiance)明显升高，表明U118MG人胶质瘤细胞数量明显增加；与此不同，给予本公开提供的缀合物的测试组5a和5b肿瘤荧光强度显著降低，降低幅度最高可达1个数量级，与对照组相比可达2个数量级以上，表明U118MG人胶质瘤细胞数量明显减少，与实验开始时相比可能降至开始时的1/10、甚至可能减少至对照组的1％以下。可见，即使是仅通过皮下给药，本公开提供的缀合物也能够有效穿透血脑屏障并高效靶向进入脑胶质瘤，并具有良好的抑制肿瘤生长效果，显示出良好的治疗依从性和高效抑制肿瘤的高成药能力。
实验例6缀合物在小鼠体内对U118MG皮下瘤的抑制活性
按照实验例2的方法培养U118MG人胶质瘤细胞(购自广州吉妮欧生物科技有限公司)。取对数期生长的U118MG人胶质瘤细胞，以(0.25％的胰酶)消化，收集细胞，离心去上清，将细胞重悬于无血清DMEM培养基中制成浓度为1×108cells/mL的细胞培养液。
实验动物NOD-SCID小鼠36只(购自斯贝福(北京)生物技术有限公司)，雄性，12周龄。将上述细胞培养液接种于NOD-SCID小鼠右侧背部皮下位置，接种体积为每只100μL，即，每只小鼠接种1×107个细胞。注射后继续饲养小鼠7天。
用无血清DMEM培养基将缀合物20溶解成1mg/mL浓度(以适配体计)的缀合物溶液。缀合物21、缀合物22和缀合物23分别溶解成0.8mg/mL浓度(以适配体计)的缀合物溶液。用10％DMSO+90％无血清DMEM培养基(体积比)将MMAE溶解成0.06mg/mL浓度的溶液。
U118MG细胞接种后7天开始给药，给药当天记为D8。实验采用腹部皮下给药方式，分别于D8、D12、D16和D20各给药一次，共4次给药。
对于空白对照组6a，分别向每组小鼠给予DMEM，单次给药容积为5μL/g小鼠体重；
对于对照组6b，分别向每组小鼠给予MMAE，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为0.3mg/kg；
对于测试组6c，分别向每只小鼠给予缀合物20，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为5mg/kg，其中包含MMAE的剂量相当于0.3mg/kg；
对于3组小鼠，分别向每组小鼠给予缀合物21、缀合物22或者缀合物23，单次给药容积均为5μL/g小鼠体重，计算可知单次给药剂量为4mg/kg，其中包含MMAE的剂量相当于0.3mg/kg；依次分别记为测试组6d、6e和6f；
于D16、D20、D25、D29、D33、D36、D41、D48、D60测量肿瘤体积。通过体外测量的方式测定测量肿瘤的长径和短径。肿瘤体积根据公式1/2(长径×短径2)进行计算。实验结束D60取肿瘤组织，称重。图6显示给予不同缀合物后，不同天数时小鼠肿瘤体积随时间变化的折线图的折线图。由图6可知，与对照组6b或者空白对照组相比，给予本公开缀合物的测试组6c-6f的小鼠的肿瘤体积和肿瘤重量均明显降低。上述结果表明，本公开的缀合物能够有效地到达肿瘤组织，显示出良好的抗肿瘤活性。
实验例7缀合物长间隔给药对U118MG皮下瘤模型小鼠体内活性
按照实验例2的方法获得接种U118MG皮下瘤的小鼠42只，注射后继续饲养小鼠。
用10％DMSO+90％无血清DMEM培养基(体积比)将MMAE溶解成0.03mg/mL和浓度0.01mg/mL的溶液；将缀合物21溶解成0.5mg/mL浓度和0.165mg/mL浓度(以适配体计)的溶液；对比缀合物25溶解成0.5mg/mL浓度和0.165mg/mL浓度(以适配体计)的溶液。
在接种U118MG细胞后7天，将全部小鼠随机分为7组，每组6只，并向各组小鼠给药，给药当天记为D8。实验采用腹部皮下给药方式，于D8、D11、D15、D29、D32和D36各给药一次，共6次给药。于给药前称重，按重量计算给药容积。
对于空白对照组，分别向每只小鼠给予DMEM培养基，单次给药容积为10μL/g小鼠体重。
对于测试组7a，分别向每只小鼠给予浓度0.01mg/mL的MMAE，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为0.1mg/kg。
对于测试组7b，分别向每只小鼠给予浓度0.165mg/mL的缀合物21，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为1.65mg/kg(对应MMAE剂量为0.1mg/kg)。
对于对照组7c，分别向每只小鼠给予浓度0.165mg/mL的对比缀合物25，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为1.65mg/kg(对应MMAE剂量为0.1mg/kg)。
对于测试组7d，分别向每只小鼠给予浓度0.03mg/mL的MMAE，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为0.3mg/kg。
对于测试组7e，分别向每只小鼠给予浓度0.5mg/mL的缀合物21，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为5mg/kg(对应MMAE剂量为0.3mg/kg)。
对于对照组7f，分别向每只小鼠给予浓度0.5mg/mL的对比缀合物25，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量为5mg/kg(对应MMAE剂量为0.3mg/kg)。
于D1、D9、D16、D19、D21、D24、D26、D29、D32、D36、D39、D43、D47、D53、D57、D60、D64、D67、D71、D74、D78、D81、D84、D88、D92、D95和D99测量肿瘤体积，其中，空白对照组在D53时、测试组7a、7b和对照组7c在D60时测量后中止实验。
通过体外测量的方式测定测量肿瘤的长径和短径。肿瘤体积根据公式1/2(长径×短径2)进行计算。实验中止后取各组肿瘤组织，称重并测定平均值。结果参见图7所示。
图7是示出了给予不同浓度本公开提供的缀合物或对照化合物后，U118MG皮下瘤模型小鼠肿瘤体积随时间变化的折线图。
由图7结果可知，在空白对照组中，肿瘤体积迅速增加；仅给予MMAE的测试组肿瘤体积增加速度有所降低，表明MMAE本身对肿瘤增殖显示出抑制效果。
进一步地，测试组7b在测试期间肿瘤体积均显著小于与MMAE含量相当的测试组7a和对照组7c；测试组7e在测试期间肿瘤体积均显著小于与MMAE含量相当的测试组7d和对照组7f，显示出比单独给予MMAE测试组7a和7d，以及给予对比缀合物25均更为优异的抗肿瘤活性。实验中止后，给予本公开的缀合物的小鼠中的肿瘤重量也显著低于MMAE组和对照组。上述表明本公开提供的缀合物能够有效地将MMAE递送至肿瘤组织，在显示出肿瘤靶向能力的同时，还降低了MMAE分子在其它组织分布带来的毒性风险，显示出剂量相关性和优异的抗肿瘤效果。
上述结果表明，本公开提供的缀合物能够有效地将对肿瘤具有抑制作用的小分子药物基团靶向递送至肿瘤组织，显示出良好的抗肿瘤活性和剂量依赖效应。
实验例8缀合物对U118MG皮下瘤模型小鼠体内活性
按照实验例2的方法获得接种U118MG皮下瘤的小鼠42只，注射后继续饲养小鼠。
用10％DMSO+90％无血清DMEM培养基(体积比)将MMAE溶解成0.02mg/mL浓度的溶液；将缀合物20和对比缀合物25溶解成0.33mg/mL浓度(以适配体计)的溶液；将缀合物21和缀合物23溶解成0.26mg/mL浓度(以适配体计)的溶液；将缀合物26溶解成0.23mg/mL浓度(以适配体计)的溶液。
U118MG细胞接种后7天，对上述接种的小鼠进行分组，每组6只，向各组小鼠给药，给药当天记为D8。于给药前称重，按平均重量2每只20g计算给药容积。
实验采用腹部皮下给药方式，分别于D8、D12、D15和D19各给药一次，共4次给药。
对于空白对照组，分别向每只小鼠给予DMEM培养基，单次给药容积为100μL。
对于测试组8a，分别向每只小鼠给予MMAE，单次给药容积为100μL，计算可知单次给药剂量为0.1mg/kg。
对于测试组8b，分别向每只小鼠给予缀合物20，单次给药容积为100μL，计算可知单次给药剂量为1.65mg/kg。
对于对照组8c，分别向每只小鼠给予对比缀合物25，单次给药容积为100μL，计算可知单次给药剂量为1.65mg/kg。
对于测试组8d，分别向每只小鼠给予缀合物21，单次给药容积为100μL，计算可知单次给药剂量为1.32mg/kg。
对于测试组8e，分别向每只小鼠给予缀合物23，单次给药容积为100μL，计算可知单次给药剂量为1.32mg/kg。
对于测试组8f，分别向每只小鼠给予缀合物26，单次给药容积为100μL，计算可知单次给药剂量为1.17mg/kg。上述8b-8f组的给药剂量均相当于包含0.1mg/kg的MMAE。
于D1、D9、D16、D19、D22、D26、D30、D36、D40、D43、D47、D50、D54、D57、D61、D64、D68和D71测量肿瘤体积。其中，空白对照组在D54时，8a组(仅给予MMAE)在D64时测量后中止实验。
通过体外测量的方式测定测量肿瘤的长径和短径。肿瘤体积根据公式1/2(长径×短径2)进行计算。中止实验后对各组取肿瘤组织，称重。结果参见图8所示。
图8是示出了各组小鼠中肿瘤体积随时间变化的折线图和D72肿瘤重量。由图8结果可知，空白对照组的肿瘤体积迅速增加，其它各组的肿瘤体积增加速度均有降低；而与仅给予MMAE的测试组8a和对照组8c相比，给予本公开的缀合物的各测试组在相当于MMAE单次给药剂量为0.1mg/kg的情况下，肿瘤体积增加速度均显著进一步更为降低。此外，缀合物20、缀合物21、缀合物23和缀合物26在实验终点D72时，较测试组8a肿瘤重量至少减少了58％，显示出更加优异的抗肿瘤效果。
实验例9缀合物对A549皮下瘤模型小鼠体内活性
A549人肺腺癌细胞(购自广州吉尼欧生物有限公司)培养条件为含10％FBS(Gibco，货号10099-141)的DMEM完全培养基(MACGENE公司，货号CM15019)，于37，5％CO2/95％空气的培养箱中培养。以0.25wt％胰酶消化并收集细胞，吸去上清并将细胞重悬于无血清DMEM培养基中制成细胞密度为1×108cells/mL的细胞培养液。
按照实验例2的方法获得接种A549皮下瘤的小鼠30只，注射后继续饲养小鼠。
用无血清DMEM培养基将缀合物20和缀合物21溶解成1mg/mL浓度(以适配体计)的缀合物溶液。对比缀合物24溶解成0.8mg/mL浓度(以适配体计)的缀合物溶液。10％DMSO+90％无血清DMEM培养基(体积比)MMAE溶解成0.06mg/mL浓度的溶液。
A549人肺癌细胞接种后7天，对全部小鼠进行分组，每组6只，向每只小鼠给药，给药当天记为D8。于给药前称重，按小鼠体重计算给药容积。
对于各组小鼠于D8、D12、D15和D19各给药一次，共给药4次。
对于空白对照组9a，分别向每组小鼠给予DMEM，单次给药容积为5μL/g小鼠体重；
对于对照组9b，分别向每组小鼠给予MMAE，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为0.3mg/kg；
对于测试组9c，分别向每只小鼠给予缀合物20，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为5mg/kg，其中包含MMAE的剂量相当于0.3mg/kg；
对于测试组9d，分别向每只小鼠给予缀合物21，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为5mg/kg，其中包含MMAE的剂量相当于0.3mg/kg；
对于测试组9e，分别向每只小鼠给予对比缀合物24，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为5mg/kg，其中包含MMAE的剂量相当于0.3mg/kg；
对于各组小鼠于D46、D50和D54各给药一次，单次给药容积为10μL/g小鼠体重，计算可知单次给药剂量包含MMAE的剂量相当于0.6mg/kg。
于D1、D9、D16、D19、D22、D26、D30、D36、D40、D43、D47、D50、D54和D57测量肿瘤体积。空白对照组于D50测量后中止实验
通过体外测量的方式测定测量肿瘤的长径和短径。肿瘤体积根据公式1/2(长径×短径2)进行计算。结果参见图9所示。
图9是示出了给予不同浓度本公开提供的缀合物或对照化合物后，A549皮下瘤模型小鼠肿瘤体积随时间变化的折线图。从图9的结果可以看出，空白对照组小鼠肿瘤体积迅速增加，其余各组肿瘤体积增加速度均有所降低；在各个时间段，给予缀合物20和21的小鼠的肿瘤体积均小于对照组9b和9e。上述结果表明，本公开的缀合物能够有效地靶向到达A549肺癌肿瘤组织，并显示出良好的抗肿瘤活性。
以上详细描述了本公开的一些实施方式，但是，本公开并不限于上述实施方式中的具体细节，在本公开的技术构思范围内，可以对本公开的技术方案进行多种简单变型，这些简单变型均属于本公开的保护范围。
另外需要说明的是，在上述一些实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本公开对各种可能的组合方式不再另行说明。
此外，本公开的各种不同的实施方式之间也可以进行任意组合，只要其不违背本公开的思想，其同样应当视为本公开所公开的内容。

Claims (53)

  1. 一种缀合物，包含一个或多个递送基团和一个或多个功能性基团；所述递送基团由一种适配体去除一个或多个氢原子或一个或多个官能团形成，所述适配体包含一段连续的核苷酸序列，连接相邻的两个核苷酸的基团独立地为磷酸酯基或者具有修饰基团的磷酸酯基，每个核苷酸选自修饰或未修饰A、U、C或G中的一种，所述连续的核苷酸序列具有式(1)所示的序列：
    5′-T1-S1-Na-S2-Nb-S3-Nc-S4-T2-3′   式(1)
    其中，T1是由1-3个核苷酸组成的基序，T2是由0-15个核苷酸组成的基序，并且T2中不包含与T1完全反向互补的基序；
    S1和S4各自是由3-7个核苷酸组成的基序，S1与S4长度相同并且完全反向互补；
    Na和Nc各自是由1-4个核苷酸组成的基序，Na中的每个核苷酸与Nc中的每个核苷酸均不互补，并且Na和Nc中U的总个数占Na和Nc中全部核苷酸总个数的50％以上；
    S2和S3各自是由1-4个核苷酸组成的基序，S2与S3长度相同并且完全反向互补；
    Nb是由3-6个核苷酸组成的基序，并且Nb两端的核苷酸之间不形成AU或GC互补；
    每个所述递送基团独立地与所述功能性基团经共价键连接，或通过连接基团连接；每个所述功能性基团选自对肿瘤具有治疗作用的小分子治疗剂基团。
  2. 如权利要求1所述的缀合物，其中，所述连续的核苷酸序列的长度为18-50个核苷酸，或者20-40个核苷酸，或者21-36个核苷酸，或者24-32个核苷酸。
  3. 如权利要求1或2所述的缀合物，其中，T1由2个核苷酸组成；或者，T1由2个核苷酸组成并且含有至少一个C；或者，按照5′-3′方向，T1为CU、UC或AC。
  4. 如权利要求1或2所述的缀合物，其中，T2由0-10个核苷酸组成；或者，按照5′-3′方向，T2由U起始的1-9个核苷酸组成。
  5. 如权利要求1所述的缀合物，其中，S1和S4各自由3-5个核苷酸组成且长度相同；或者，S1和S4形成的反向互补中，GC互补占全部互补数量的至少40％；或者，按照5′-3′方向，S1为GCU且S4为AGC，或S1为GAGU且S4为GCUC，或S1为GGAGU且S4为GCUCU，或S1为UAUGG且S4为CCAUG。
  6. 如权利要求1所述的缀合物，其中，所述Na和Nc中核苷酸的数量之和为2-4的整数；或者，所述Na和Nc中核苷酸的数量之和为3或4，并且所述Na和Nc中U的数量之和为2或3；或者，按照5′-3′方向，Na或Nc独立地为U、UU、UC或CU。
  7. 如权利要求1所述的缀合物，其中，S2和S3各自由2-3个核苷酸组成且长度相同；或者，S2和S3形成的反向互补中至少包含一个GC互补；或者，按照5′-3′方向，S2为CA且S3为UG，或S2为AC且S3为GU，或S2为GCC且S3为GGU。
  8. 如权利要求1所述的缀合物，其中，Nb由4个或5个核苷酸组成；或者，按照5′-3′方向，Nb为GACG、GACGU、GACCG、UACU、GUUG或GAUCU。
  9. 如权利要求1所述的缀合物，其中，所述连续的核苷酸序列具有SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3所示的序列。
  10. 如权利要求1所述的缀合物，所述连续的核苷酸序列具有SEQ ID NO：4所示的核苷酸序列：
    5′-N6GGAGUUCAN1N2N3N4UGN5GCUCN7-3′(SEQ ID NO：4)，
    其中，N1、N2、N3各自独立地为A、U、C和G中的一种；N4为U、C或G或者由U、C或G中的两个组成的基序；N5为U、CU或UU；N6为CU、UC或AC；N7为U、UU或UUN8，N8为由1-15个核苷酸组成的基序。
  11. 如权利要求10所述的缀合物，其中，N1、N2、N3和N4组成的基序N1N2N3N4为GACG、GACGU、GACCG、UACU、GUUG或GAUCU中的一种。
  12. 如权利要求10或11所述的缀合物，其中，所述连续的核苷酸序列具有SEQ ID NO：5-11中任意一项所示的核苷酸序列。
  13. 如权利要求10所述的缀合物，其中，N8是由1-8个核苷酸组成的基序；或者，按照5′-3′方向，N8的核苷酸序列为CCGAUCUC；或者，所述连续的核苷酸序列具有SEQ ID NO：12-14中的一种所示的序列。
  14. 如权利要求1所述的缀合物，其中，所述连续的核苷酸序列中的每个胞嘧啶核苷酸为氟代修饰的胞嘧啶核苷酸，和/或所述连续的核苷酸序列中的每个尿嘧啶核苷酸为氟代修饰的尿嘧啶核苷酸；或者，所述连续的核苷酸序列中的每个核苷酸均为2′-甲氧基修饰的核苷酸；或者，所述连续的核苷酸序列中Nb和S3基序中的一个或多个尿嘧啶核苷酸具有修饰的碱基。
  15. 如权利要求14所述的缀合物，其中，所述连续的核苷酸序列具有SEQ ID NO：15-33中的一种所示的核苷酸序列。
  16. 如权利要求1所述的缀合物，所述连续的核苷酸序列中的至少一个连接相邻的两个核苷酸的基团为硫代磷酸酯基，或者每个连接相邻的两个核苷酸的基团均为硫代磷酸酯基。
  17. 如权利要求16所述的缀合物，其中，所述连续的核苷酸序列具有SEQ ID NO：34-39中的一种所示的核苷酸序列。
  18. 如权利要求17所述的缀合物，具有如式(101)所示的结构：
    其中，每个RAP基团独立地为具有如式(102)所示的结构的基团：
    式中，每个AP基团相同或不同，独立地表示一个所述递送基团；Rj、每个Rk或每个Ri相同或不同，分别独立地表示共价键或者连接基团，且Ri和Rk二者不同时为共价键；每个n1各自独立地表示0-4的整数；
    每个A0基团相同或不同，独立地表示一个所述功能性基团；m0为1-6的整数；n0为1-6的整数，表示基团共价连接的位点。
  19. 如权利要求18所述的缀合物，其中，m0为1-4的整数，和/或n0为1-3的整数，和/或每个n1独立地为0-1的整数；
    或者，m0为1，和/或n0为1，和/或至少一个或者每个n1为0。
  20. 如权利要求18或19所述的缀合物，其中，每个所述Rk或每个所述Ri独立地为共价键或者长度为1-70个碳原子的直链亚烷基，或者，所述直链亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、OP(O)(S)、C5-C8亚糖苷基、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中，所述直链亚烷基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)。
  21. 如权利要求20所述的缀合物，其中，每个n1均为0，每个Rj独立地为共价键，或者为以下连接基团的一种或多种的连接组合：C1-C20亚烷基、磷酸酯键、硫代磷酸酯键、酰胺键、酯键、醚键、硫醚键、二硫键、1，2，3-三唑亚基、聚乙二醇亚基、吡咯烷亚基、2-氧代吡咯烷亚基、亚苯基、亚环己基、2-丁二酰亚胺亚基、2-硫代丁二酰亚胺亚基、氨基酸亚基、核苷酸亚基。
  22. 如权利要求21所述的缀合物，其中，每个Ri独立地为共价键、二硫键、亚丙基磷酸酯基、2-硫代丁二酰亚胺亚基、氨基酸亚基、或者GAU三核苷酸亚基中的一种或2种的连接组合。
  23. 如权利要求18-22中任意一项所述的缀合物，其中，Rj为共价键，m0为1。
  24. 如权利要求18-22中任意一项所述的缀合物，其中，Rj为连接基团，所述连接基团Rj包含主链部分、侧链部分和缀合连接部，所述主链部分分别与所述缀合连接部和所述侧链部分连接，每个所述侧链部分分别与所述主链部分和所述RAP基团连接，每个所述缀合连接部分别与所述主链部分和所述功能性基团A0连接，其中，
    所述主链部分为长度为1-70个碳原子的直链亚烷基，或者，所述直链亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中，所述直链亚烷基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)；
    每个所述侧链部分独立地是共价键，或者是长度为1-70个碳原子的直链亚烷基，或者，所述直链亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且，所述直链亚烷基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)；
    每个所述缀合连接部独立地为共价键或者以下连接结构的一种或多种的连接组合：C1-C10直链亚烷基、磷酸酯键、硫代磷酸酯键、酰胺键、酯键、醚键、二硫键、1，2，3-三唑亚基、聚乙二醇亚基、吡咯烷亚基、2-氧代吡咯烷亚基、亚苯基、亚环己基、2-丁二酰亚胺亚基、2-硫代丁二酰亚胺亚基、氨基酸亚基、核苷酸亚基。
  25. 如权利要求24所述的缀合物，其中，所述连接基团Rj中的每个所述缀合连接部分别与所述主链部分和一个所述功能性基团A0连接；所述侧链部分为n0个，每个侧链部分分别与所述主链部分和一个所述RAP基团连接。
  26. 如权利要求24或25所述的缀合物，其中，全部所述侧链部分连接至所述主链部分中的同一原子；或者，每个所述侧链部分连接至所述主链部分中的不同原子。
  27. 如权利要求26所述的缀合物，其中，m0为1，所述连接基团Rj包含如式(301)所示的结构：
    其中，k为1-3的整数；LC为所述主链部分，LA为所述侧链部分，LB为所述缀合连接部，表示基团共价连接的位点；
    所述主链部分LC为共价键或2-4价、直链或支链的C1-C25饱和烃基，或者，所述饱和烃基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C5亚烯基、C2-C5亚炔基、C6-C10亚芳基、C3-C8亚杂环基和C5-C10亚杂芳基；其中，所述饱和烃基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C5烷基、C6-C10芳基、C5-C10杂芳基、-O-C1-C5烷基、-OC1-C5烷基苯基、-C1-C5烷基-OH、-SC1-C5烷基、硝基、-C(O)O(C1-C5烷基)、-CON(C1-C5烷基)(C1-C5烷基)、-CONH(C1-C5烷基)、-CONH2，-NHC(O)(C1-C5烷基)、-NHC(O)(苯基)、-N(C1-C5烷基)C(O)(C1-C5烷基)、-N(C1-C5烷基)C(O)(苯基)、-C(O)C1-C5烷基、-C(O)C1-C5烷基苯基、-OC(O)C1-C5烷基、-SO2(C1-C5烷基)、-SO2(苯基)、-SO2NH2、-SO2NH(C1-C5烷基)、-SO2NH(苯基)、-NHSO2(C1-C5烷基)和-NHSO2(苯基)；
    每个所述侧链部分独立地是共价键，或者是长度为1-70个碳原子的直链亚烷基，或者，所述直链亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且，所述直链亚烷基可具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)；
    每个所述缀合连接部独立地为共价键或者以下连接结构的一种或多种的连接组合：C1-C10直链亚烷基、磷酸酯键、硫代磷酸酯键、酰胺键、酯键、醚键、二硫键、1，2，3-三唑亚基、聚乙二醇亚基、吡咯烷亚基、2-氧代吡咯烷亚基、亚苯基、亚环己基、2-丁二酰亚胺亚基、2-硫代丁二酰亚胺亚基、氨基酸亚基、核苷酸亚基。
  28. 如权利要求27所述的缀合物，其中，所述缀合物具有如式(305)所示的结构：
  29. 如权利要求27所述的缀合物，其中，所述连接基团Rj具有式(306)所示的结构：
    其中，n306为0-3的整数，每个p306独立地为1-6的整数，表示基团共价连接的位点；由*标出的氧原子与所述RAP基团形成磷酸酯键、醚键或酯键连接；由#标出的氧原子中的至少一个与所述功能性基团A0形成醚键、酯键或磷酸酯键而连接，其余由#标出的氧原子与氢原子连接形成羟基，或者与C1-C3烷基连接形成C1-C3烷氧基。
  30. 如权利要求29所述的缀合物，其中，所述缀合物具有如式(307a)、(307b)或(307c)所示的结构：
  31. 如权利要求27所述的缀合物，其中，所述缀合物具有式(308)所示的结构：
    其中，
    n308为选自1-10的整数；
    每个m308独立地为选自2-10的整数；
    每个R308独立地为H、C1-C10烷基、C1-C10卤代烷基或C1-C10烷氧基；
    每个R3独立地为所述功能性基团A0，或者为所述RAP基团，并且至少一个R3为所述功能性基团A0，且至少一个R3为所述RAP基团；或者，一个R3为所述功能性基团A0，其余R3为所述RAP基团；
    连接至所述功能性基团A0的每个L1表示所述缀合连接部，并且连接至所述RAP的每个L1表示所述侧链部分。
  32. 如权利要求31所述的缀合物，其中，每个L1独立地选自于由基团L4-L23及其任意连接组合所组成的组。
  33. 如权利要求32所述的缀合物，其中，每个L1独立地选自于由基团L4-L9、L13、L14、L18中至少2个的连接组合所组成的组；或者，每个L1独立地为基团L4、L5、L7、L9、L13、L14、L18中至少2个的连接组合。
  34. 如权利要求31-33中任意一项所述的缀合物，其中，每个L1的长度独立地为3-25个原子；或者，每个L1的长度独立地为4-15个原子。
  35. 如权利要求31-34中任意一项所述的缀合物，其中，n308为2-6的整数，2-4个R3为所述RAP基团，其余的R3为所述功能性基团。
  36. 如权利要求31-35中任意一项所述的缀合物，其中，每个m308各自独立地为2-5的整数，和/或每个m308均相等。
  37. 如权利要求31-36中任意一项所述的缀合物，其中，n308为选自2-4的整数；每个m308独立地为选自2-4的整数；每个R308均为H。
  38. 如权利要求37所述的缀合物，其中，一个R3为所述功能性基团A0，其余R3为所述RAP基团。
  39. 如权利要求37或38所述的缀合物，其中，每个L1上同时含有与含氮骨架上的N原子连接的连接位点和与所述功能性基团A0或所述RAP基团连接的连接位点，所述与含氮骨架上的N原子连接的位点与该N原子形成酰胺键；或者，一个或多个L1选自B5、B6、B5′或B6′：

    其中，表示基团共价连接的位点，q2为1-10的整数；或者，q2为1-5的整数。
  40. 如权利要求31-39中任一项所述的缀合物，具有式(403)、(404)、(405)、(406)、(407)、(408)、(409)、(410)、(411)、(412)、(413)、(414)、(415)、(416)、(417)、(418)、(419)、(420)、(421)、(422)、(423)、(424)、(425)、(426)或(427)所示的结构：








  41. 如权利要求18-22中任意一项所述的缀合物，其中，Rj包含核苷酸序列I和核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II各自包含5-25个修饰或未修饰的核苷酸，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补，所述递送基团连接至所述核苷酸序列I，所述功能性基团连接至所述核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II在受试者体内不引发免疫反应或毒性反应。
  42. 如权利要求41所述的缀合物，其中，所述递送基团的3′末端经磷酸酯键连接至所述核苷酸序列I的5′末端核苷酸的核糖5′位，所述功能性基团连接至所述核苷酸序列II的5′末端核苷酸的核糖5′位；或者，所述功能性基团包含一段核苷酸序列，所述核苷酸序列的3′末端经磷酸酯键连接至所述核苷酸序列II的5′末端核苷酸的核糖5′位。
  43. 如权利要求42所述的缀合物，其中，所述核苷酸序列I和所述核苷酸序列II实质上反向互补或者完全反向互补；或者，所述核苷酸序列I和所述核苷酸序列II的长度相等并且均为10-20个修饰或未修饰的核苷酸；或者，所述核苷酸序列I和所述核苷酸序列II均由17个核苷酸组成并且完全反向互补；或者，所述核苷酸序列I和所述核苷酸序列II分别具有SEQ ID NO：40和SEQ ID NO：41所示的序列：
    5′-GUACAUUCUAGAUAGCC-3′(SEQ ID NO：40)
    5′-GGCUAUCUAGAAUGUAC-3′(SEQ ID NO：41)，
    或者，所述核苷酸序列I和所述核苷酸序列II分别具有SEQ ID NO：42和SEQ ID NO：43所示的序列：
    5′-GmUfAmCfAmUfUfCfUfAmGmAmUfAmGmCfCf-3′
    (SEQ ID NO：42)
    5’-GmGmCfUfAmUfCfUfAmGmAmAmUfGmUfAmCf-3’
    (SEQ ID NO：43)。
  44. 如权利要求18-43中任意一项所述的缀合物，其中，Rj为可裂解的。
  45. 如权利要求1-44中任意一项所述的缀合物，其中，每个所述小分子治疗剂基团独立地选自细胞毒素基团、抗生素基团或血管生成抑制剂。
  46. 如权利要求45所述的缀合物，其中，所述小分子治疗剂基团由下述小分子治疗剂中的一种去除一个或多个氢原子或一个或多个官能团形成：甲氨蝶呤、阿霉素、长春花生物碱、澳瑞他汀、卡利奇霉素、美登素、喜树碱、以及加利车霉素。
  47. 如权利要求46所述的缀合物，其中，所述小分子治疗剂基团是一甲基澳瑞他汀E(MMAE)去除一个或多个氢原子或一个或多个官能团形成的基团。
  48. 一种药物组合物，包含权利要求1-47中任意一项所述的缀合物以及药学上可接受的载体。
  49. 权利要求1-47中任意一项所述的缀合物和/或权利要求48所述的药物组合物在制备用于治疗肿瘤及肿瘤相关疾病或症状的药物中的应用。
  50. 如权利要求49所述的应用，其中，所述肿瘤是胶质瘤、肾癌、肺癌中的一种或多种。
  51. 一种肿瘤及肿瘤相关疾病或症状的治疗方法，包括向有需要的受试者给予有效量的权利要求1-47中任意一项所述的缀合物和/或权利要求48所述的药物组合物。
  52. 如权利要求51所述的方法，其中，所述肿瘤是胶质瘤、肾癌、肺癌中的一种或多种。
  53. 一种试剂盒，包括权利要求1-47中任意一项所述的缀合物和/或权利要求48所述的药物组合物。
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