WO2022222993A1 - 缀合物及其制备方法和用途 - Google Patents

缀合物及其制备方法和用途 Download PDF

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WO2022222993A1
WO2022222993A1 PCT/CN2022/088173 CN2022088173W WO2022222993A1 WO 2022222993 A1 WO2022222993 A1 WO 2022222993A1 CN 2022088173 W CN2022088173 W CN 2022088173W WO 2022222993 A1 WO2022222993 A1 WO 2022222993A1
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propargyl
conjugate
nucleic acid
modified
small nucleic
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席真
杨超
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Nankai University
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Nankai University
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Priority to CN202280028989.XA priority Critical patent/CN117177773A/zh
Priority to JP2023565281A priority patent/JP7668378B2/ja
Priority to US18/556,303 priority patent/US20250319193A1/en
Priority to EP22791099.9A priority patent/EP4316525A4/en
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    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • 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
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • 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
    • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the field of biotechnology, in particular to a conjugate and its preparation method and use.
  • PSMA Prostate-specific membrane antigen
  • FOLH1 folate hydrolase 1
  • GCP II glutamate carboxypeptidase II
  • NAALAD1 N-acetylated- ⁇ -linked acid dipeptidase I
  • PSMA belongs to The M28B subfamily of the M28 family of peptidases.
  • PSMA is a type II transmembrane protein with a homodimeric structure, and each subunit can bind two zinc ions.
  • PSMA has both folate hydrolase and N-acetylated- ⁇ -linked acid dipeptidase (NAALADase) activities, and its role is to regulate the hydrolysis of N-acyl poly ⁇ -glutamic acid and its derivatives.
  • PSMA is highly expressed in the nervous system, prostate, pancreas, kidney and small intestine, and is much higher in the epithelial layer of most prostate cancers and in the neovascularization of other solid tumors than in normal tissues. This tissue specificity makes PSMA one of the most attractive targets for prostate cancer delivery.
  • RNA interference is a novel technology that uses therapeutic oligonucleotides such as siRNA to treat various diseases including cancer. Encouraged by the U.S.
  • RNAi Food and Drug Administration (FDA) approvals of Patisiran and Givosiran, RNAi is also in the spotlight for the treatment of prostate cancer. Moreover, the approval of Givosiran makes the conjugates formed by the direct linking of ligands and siRNAs a simple and effective new way of targeting tumors. However, how to effectively deliver therapeutic siRNA to prostate cancer tissues/cells for cancer treatment remains a great challenge. In addition, how to break through the limitations of the solid-phase synthesis method used by Givosiran (such as poor flexibility, cumbersome operation, unsatisfactory synthesis efficiency, and narrow scope of application) is also a key problem that needs to be solved urgently for RNAi therapy.
  • FDA Food and Drug Administration
  • the purpose of the present invention is to provide a conjugate and a preparation method thereof in order to overcome the technical problems of the prior art such as poor flexibility in preparing nucleic acid conjugates, cumbersome operations, and small production scale, which have both targeting properties The dual characteristics of high and high activity.
  • the method provided by the present invention has the advantages of rapidity, high efficiency, simplicity and wide application range.
  • the present invention also provides the application of the nucleic acid conjugate prepared by the present invention in the preparation of a medicament for the treatment of any one of prostate cancer, colon cancer, pancreatic cancer, breast cancer, kidney disease and nervous system-related diseases, especially It is the application in the medicine for the treatment of prostate cancer.
  • the first aspect of the present invention provides a conjugate, which is formed by covalently linking an azide-modified targeting ligand and a propargyl-modified small nucleic acid sequence.
  • a second aspect of the present invention provides a method for preparing the aforementioned conjugate, the method comprising: contacting an azide-modified targeting ligand with a propargyl-modified small nucleic acid sequence in the presence of a monovalent copper catalyst.
  • the third aspect of the present invention provides the use of the aforementioned conjugate or the conjugate prepared by the aforementioned method in the preparation of a medicament for treating a related disease caused by abnormality in a tissue expressing PSMA, and the disease preferably occurs in glandular tissue , colon, kidney and nervous system; wherein, the glandular tissue is selected from one of prostate, pancreas, breast and thymus.
  • the present invention is accomplished through the ingenious design of propargyl-modified oligonucleotides (especially at the 3'-terminus) and azide-modified targeting ligands, and utilizing the methods of the present invention (ie, post-synthetic modification strategies (copper-catalyzed clicks) chemistry)) to efficiently and rapidly construct the nucleic acid conjugates of the present invention.
  • the conjugate nucleic acid conjugate
  • the conjugate provided by the present invention has high specific recognition performance, gene silencing performance and tumor cell generation inhibition performance.
  • the method for preparing conjugates (nucleic acid conjugates) provided by the present invention has broad application prospects in the targeted delivery and treatment of RNAi therapeutic agents.
  • the preparation method provided by the present invention only involves simple chemical reactions, can realize flexible and efficient synthesis of nucleic acid conjugates, and is also suitable for constructing other various ligand-targeted nucleic acid conjugates, which has good practicability.
  • FIG. 1 is a schematic diagram of a small nucleic acid conjugated with KUE in the present invention
  • Fig. 2 is the high performance liquid chromatogram and mass spectrogram of oligonucleotide 1 (ON1) modified by 3'-terminal propargyl in the present invention
  • Fig. 3 is the high performance liquid chromatogram and mass spectrogram of oligonucleotide 2 (ON2) modified by 3'-terminal propargyl in the present invention
  • Fig. 4 is the high performance liquid chromatogram and mass spectrogram of oligonucleotide 3 (ON3) modified by 3'-terminal propargyl in the present invention
  • Fig. 5 is the polyacrylamide gel electrophoresis characterization diagram of different KUE-PEG-siRNA conjugates in the present invention.
  • Figure 6 is a polyacrylamide gel electrophoresis characterization diagram of different DUPA-PEG 5 -siRNA conjugates in the present invention.
  • Example 7 is a mass spectrum of nucleic acid conjugate KUE-PEG 2 -siRNA in Example 1 of the present invention.
  • Figure 8 is a mass spectrum of nucleic acid conjugate KUE-PEG 5 -siRNA in Example 21 of the present invention.
  • Figure 9 is a mass spectrum of nucleic acid conjugate KUE-PEG 12 -siRNA in Example 22 of the present invention.
  • Figure 10 is the mass spectrum of nucleic acid conjugate DUPA-PEG 5 -siRNA in Example 23 of the present invention.
  • Figure 11 is the mass spectrum of nucleic acid conjugate DUPA-PEG 5 -siRNA in Example 24 of the present invention.
  • Figure 12 is a cell imaging effect diagram of different nucleic acid conjugates in a specific embodiment of the present invention.
  • Figure 13 is a statistical graph of the intracellular mean fluorescence intensity of different nucleic acid conjugates in a specific embodiment of the present invention.
  • Fig. 14 is a graph showing the evaluation effect of different nucleic acid conjugates in the cell internalization mode according to a specific embodiment of the present invention.
  • Figure 15 is a graph showing the evaluation of the uptake efficiency of different nucleic acid conjugates in prostate cancer cells according to a specific embodiment of the present invention.
  • Figure 16 is a graph of the mRNA silencing effect of different nucleic acid conjugates in a specific embodiment of the present invention.
  • Figure 17 is a graph of the protein expression inhibition effect of different nucleic acid conjugates in a specific embodiment of the present invention.
  • Figure 18 is a graph showing the effect of different nucleic acid conjugates in the present invention on promoting apoptosis.
  • the inventors have found through extensive research that targeting ligands modified by azide (such as azide-modified KUE ligands, azide-modified DUPA ligands, etc.) and 3'-terminal propargyl-modified small nucleic acids ( oligonucleotides) can flexibly and efficiently construct antigen-targeted siRNA conjugates such as PSMA.
  • azide such as azide-modified KUE ligands, azide-modified DUPA ligands, etc.
  • 3'-terminal propargyl-modified small nucleic acids oligonucleotides
  • the first aspect of the present invention provides a conjugate formed by covalently linking an azide-modified targeting ligand and a propargyl-modified small nucleic acid sequence.
  • the azide group in the azide-modified targeting ligand is covalently linked to the targeting ligand through at least one fragment of polyethylene glycol.
  • the number of polyethylene glycol monomers in the polyethylene glycol segment is not particularly limited, for example, it can be 1-100.
  • the fragment of the polyethylene glycol mainly plays the role of linking.
  • the targeting ligand is a dipeptide formed by two amino acids whose at least one amino acid is glutamic acid; preferably, the amino acid is selected from lysine and glutamic acid, glutamic acid Acid and glutamic acid and glutamic acid and glutamic acid analogs; wherein, the targeting ligand can recognize and bind to the antigen expressed on the surface of the target cell through glutamate; the antigen is PSMA.
  • the terminal of the targeting ligand contains a carboxyl group; the targeting ligand is linked to the antigen on the surface of the target cell through the carboxyl group.
  • the target cells may be selected from at least one of prostate cancer cells, neuronal cells, renal cancer cells and colon cancer cells.
  • the azide-modified targeting ligand has the structure shown below:
  • n is an integer from 1-100.
  • n is an integer of 1-50, more preferably an integer of 1-20, still more preferably an integer of 1-15.
  • it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15.
  • the azide-modified targeting ligand can be prepared by the following method:
  • tert-butyl ester, benzyl ester-protected lysine and tert-butyl ester-protected glutamic acid are firstly connected to synthesize KUE through triphosgene; then ethylene glycol is acrylated and azide to obtain azido PEG propionic acid; Finally, both (KUE and azidoPEG propionic acid) can be condensed to give the targeting ligand.
  • the reaction temperature of the first reaction may be 20-40° C., and the reaction time may be 5-20 hours.
  • the amount of sodium metal may be 0.05-2 mmol, and the amount of tert-butyl acrylate may be 0.3-0.8 mmol.
  • the first reaction is carried out in the presence of a first solvent; wherein, the amount of the first solvent can be 1-2 mL per mmol of polyethylene glycol; the first solvent can be tetrahydrofuran.
  • the reaction temperature of the second reaction may be -20°C to 10°C, and the reaction time may be 5-20 hours.
  • the amount of triethylamine is 0.5-1 mL
  • the amount of p-toluenesulfonyl chloride can be 1.2-1.8 mmol.
  • the second reaction is carried out in the presence of a second solvent; wherein, the amount of the second solvent may be 1-3 mL per mmol of the compound represented by formula 2a-c.
  • the second solvent may be dichloromethane.
  • the second reaction is preferably carried out under an inert atmosphere, which may be provided by argon.
  • the reaction temperature of the third reaction may be 60-90° C., and the reaction time may be 5-24 hours.
  • the amount of sodium azide used may be 1.2-1.8 mmol per mmol of the compound represented by formula 3a-c.
  • the amount of the third solvent may be 2-3 mL per mmol of the compound represented by formula 3a-c.
  • the third solvent may be DMF (N,N dimethylformamide).
  • the reaction temperature of the fourth reaction may be 20-40° C., and the reaction time may be 5-24 hours.
  • the amount of KUE can be 0.9-1.5 mmol per mmol of the compound represented by formula 4a-c.
  • the trifluoroacetic acid before the compound shown in 4a-c is subjected to the fourth reaction with KUE, the trifluoroacetic acid (with respect to 1 mol of the compound shown in 4a-c, the amount of trifluoroacetic acid is 2-3 mL) can be carried out in the presence of trifluoroacetic acid.
  • HATU and DIPEA can be used to deprotect the compounds shown in 4a-c
  • the product is subjected to activation treatment; wherein, the amount of HATU and DIPEA can be 1.2-1.8 mmol and 2-4 mmol, respectively, per mmol of the compound represented by formula 4a-c.
  • the fourth reaction is performed in the presence of a fourth solvent; wherein, the amount of the fourth solvent may be 0.8-1.5 mL per mmol of the compound represented by formula 4a-c.
  • the fourth solvent may be DMF (N,N dimethylformamide).
  • the reaction temperature of the fifth reaction may be 20-40° C., and the reaction time may be 5-24 hours.
  • the amount of trifluoroacetic acid can be 20-30 mL per mmol of the compound represented by formula 5a-c.
  • the post-treatment of the reaction in steps (1)-(5) is not particularly limited, and purified products can be obtained by separation by column chromatography.
  • the small nucleic acid sequence may be selected from at least one of small nucleic acids targeting STAT3, PHB1, Notch1, PLK1 and BRD4.
  • the propargyl modification is preferably a 3'-terminal propargyl modification.
  • the propargyl-modified small nucleic acid sequence adopts the phosphoramidite method to modify the propargyl compound to the 3'-end of the small nucleic acid sequence by solid-phase synthesis technology; wherein, the propargyl The base compound contains at least one reactive hydroxyl group.
  • the propargyl modification may be multiple propargyl modifications (1, 2, 3).
  • the method of modifying the propargyl compound to the 3'-end of the small nucleic acid sequence is not particularly limited, and it can be carried out in the following manner: First, based on the prior art (Bioorg.Med.Chem., 2013, 5583–5588), the 1-O-propargyl-2-deoxy-D-furanose phosphoramidite monomer was synthesized through a 4-step reaction with commercialized hofchlorose as raw material; then, the obtained phosphoramidite mono The 3'-end of the propargyl monomer X oligonucleotide is connected to the solid phase carrier (CPG) by an automatic nucleic acid synthesizer; finally, the CPG and the protective group are removed to obtain the 3'-end propargyl modified small nucleic acids.
  • CPG solid phase carrier
  • the propargyl compound has the following structure:
  • a second aspect of the present invention provides a method for preparing the aforementioned conjugate, the method comprising: contacting an azide-modified targeting ligand with a propargyl-modified small nucleic acid sequence in the presence of a monovalent copper catalyst.
  • the molar ratio of the azide-modified targeting ligand to the propargyl-modified small nucleic acid sequence may be (1.05-10):1, preferably (2-4):1.
  • the monovalent copper catalyst may be selected from at least one of Cu(I)-TBTA, CuBr and CuCl, preferably Cu(I)-TBTA.
  • the amount of the monovalent copper catalyst may be 2-10 mol, preferably 3-6 mol.
  • the contact conditions include: the temperature is 35.5-38.5°C, preferably 36.5-37.5°C; the time is 1-5h, preferably 2-4h.
  • the rotational speed in the contact, there is no particular limitation on the rotational speed, as long as it can meet the requirements of the present invention, for example, the rotational speed can be 600-1000 r/min, preferably 700-900 r/min.
  • the contact is preferably performed under the condition of shaking, and the shaking condition may be: shaking once every 0.4-0.6 h.
  • the contacting is performed in the presence of an organic solvent selected from at least one of N,N-dimethylformamide (DMF), methanol, ethanol and acetone.
  • an organic solvent selected from at least one of N,N-dimethylformamide (DMF), methanol, ethanol and acetone.
  • the amount of the organic solvent is such that the concentration of the small nucleic acid sequence is maintained at 20-200 ⁇ M, preferably 80-120 ⁇ M.
  • the conjugate may refer to a single-stranded nucleic acid conjugate or a double-stranded nucleic acid conjugate; when the conjugate is a double-stranded nucleic acid conjugate, the sense strand can be separated from the sense strand with reference to a conventional method in the art Nucleic acid and antisense strand nucleic acid are annealed to form double-stranded nucleic acid conjugate, for example, sense strand nucleic acid and antisense strand nucleic acid are formed in equimolar ratio in annealing buffer (containing magnesium ions) at 90-100°C.
  • annealing buffer containing magnesium ions
  • the small nucleic acid sequence is an siRNA sequence comprising a sense strand and an antisense strand, and the method includes:
  • the annealing buffer is selected from the annealing buffer selected from 1.5-2.5 mM magnesium acetate solution.
  • the third aspect of the present invention provides the use of the aforementioned conjugate or the conjugate prepared by the aforementioned method in the preparation of a medicament for treating a related disease caused by abnormality in a tissue expressing PSMA, and the disease preferably occurs in glandular tissue , colon, kidney and nervous system; wherein, the glandular tissue is selected from one of prostate, pancreas, breast and thymus.
  • the disease is preferably cancer.
  • conjugate refers to "KUE-PEG-siRNA conjugate” or "DUPA-PEG-siRNA conjugate”.
  • This preparation example is used to illustrate the preparation of azide-modified KUE ligands 6a-c
  • n 2, 5, 12.
  • This preparation example is used to illustrate the preparation of azide-modified DUPA ligands
  • This preparation example is used to illustrate the synthesis of 3'-terminal propargyl modified small nucleic acid
  • X 1-O-Propargyl-2-deoxy-D-furanose
  • oligo small nucleic acid
  • Example 2 The procedure was carried out as in Example 1, except that 6a was replaced with an equimolar amount of azide-modified targeting ligand 6b. The final yield was 96%.
  • the single-stranded conjugate obtained in Example 1-22 and the Cy5-labeled complementary strand small nucleic acid were incubated in annealing buffer (2mM magnesium acetate containing magnesium ions) at a molar ratio of 1:1 at 95°C for 3 minutes.
  • annealing buffer (2mM magnesium acetate containing magnesium ions) at a molar ratio of 1:1 at 95°C for 3 minutes.
  • Nucleic acid conjugate KUE-PEG 2 -siRNA, nucleic acid conjugate KUE-PEG 5 -siRNA, nucleic acid conjugate KUE- The polyacrylamide gel electrophoresis characterization charts of PEG 12 -siRNA and DUPA-PEG 5 -siRNA are shown in Figure 5 and Figure 6; the mass spectra are shown in Figure 7, Figure 8, Figure 9, Figure 10 (Example 23) and Figure 11, respectively (Example 24).
  • LNCaP cells PSMA positive, purchased from Chinese Academy of Sciences
  • PC3 cells PSMA negative, purchased from Chinese Academy of Sciences
  • Cy5-labeled KUE-PEG-siRNA conjugate and siRNA 50 nM were co-incubated with two prostate cancer cells, and the cells were subjected to fluorescence imaging under 649 nm excitation light 6 hours after transfection ( Figure 12A (fluorescence imaging of LNCaP cells). ) and Figure 12B (fluorescence imaging of PC3 cells)).
  • the average fluorescence intensity of the cell fluorescence imaging in Figure 12A was calculated (see Figure 13). From the statistical results in Figure 13, it was found that the average fluorescence intensity of the KUE-PEG-siRNA conjugate group in LNCaP cells was 95%, while lipofectamine 2000 The mean fluorescence intensity of the transfected siRNA group was 82%; the mean fluorescence intensity of KUE-PEG-siRNA conjugates in PC-3 cells was less than 5%. These results above indicate that siRNA can hardly enter any cells without the aid of transfection reagents, while KUE-PEG-siRNA conjugates can selectively enter LNCaP cells without the aid of transfection reagents.
  • KUE-PEG-siRNA conjugate into LNCaP cells was further confirmed by adding a PSMA-specific inhibitor (ZJ43, prepared with reference to Eur. J. Neurosci., 2004, 483-494). Among them, LNCaP cells were incubated with ZJ43 in advance, and then Cy5-labeled KUE-PEG-siRNA conjugate was added. At 6 hours, LNCaP cells were subjected to fluorescence imaging under excitation light at 640 nm (see Figure 14). The results in FIG.
  • ZJ43 PSMA-specific inhibitor
  • PSMA-positive LNCaP cells purchased from Chinese Academy of Sciences
  • PSMA-positive LNCaP cells purchased from Chinese Academy of Sciences
  • Cy5-labeled KUE-PEG-siRNA conjugate (50 nM) and siRNA (50 nM) were co-incubated with LNCaP cells, wherein siRNA was co-transfected with lipofectamine 2000 (brand: Invitrogen, manufacturer: Thermo Fisher Scientific) as a control group. After 4 hours, the cells were processed according to the operation of AnnexinV-FITC/PI cell apoptosis double staining kit (brand: 556547, manufacturer: BD Company, USA).
  • the prepared cell samples were analyzed by FACS Calibur flow cytometer under the detection channel corresponding to Cy5.
  • the blank control group, the siRNA transfection alone group, the siRNA and lipofectamine 2000 co-transfection group, and the different KUE-PEG-siRNA conjugate transfection groups were analyzed in turn, and 10 4 cells were collected and tested in each group. .
  • the experimental results were analyzed using FlowJo software (Fig. 15). From Figure 15, it can be found that siRNA (siSTAT3) without ligand-conjugation can hardly enter LNCaP cells, which is consistent with the results of cell imaging.
  • KUE-siRNA conjugates (KUE-PEG 2 -siRNA, KUE-PEG 5 -siRNA, KUE-PEG 12 -siRNA) in LNCaP cells exhibited higher cellular uptake efficiency (>99%).
  • the obtained single-stranded conjugate and complementary small nucleic acid were incubated in annealing buffer (2 mM magnesium acetate) at a molar ratio of 1:1 at 95° C. for 3 minutes to form KUE-PEG-siSTAT3 conjugate.
  • KUE-PEG-siSTAT3 KUE-PEG-siRNA conjugates (100 nM) targeting signal transducer and activator of transcription 3 (STAT3) were co-incubated with LNCaP cells. After 48 hours, mRNA expression was detected by reverse transcription polymerase chain reaction (RT-PCR). As shown in Figure 16A, all KUE-PEG-siRNA conjugates were able to effectively reduce the mRNA expression of STAT3, wherein KUE-PEG5 - siSTAT3 had comparable silencing activity to siRNA transfected with lipofectamine 2000. In addition, from FIG. 16B , it can be found that the mRNA expression level gradually decreases as the PEG length in the conjugate increases.
  • KUE-PEG-siSTAT3 conjugates can effectively reduce the expression of target genes after being taken up by LNCaP cells. Also, the conjugate KUE-PEG 12 -siSTAT3 induced stronger mRNA down-regulation than KUE-PEG 2 -siSTAT3 and KUE-PEG 5 -siSTAT3.
  • KUE-PEG-siSTAT3 KUE-PEG-siRNA conjugate
  • prostate cancer cell LNCaP prostate cancer cell LNCaP
  • RNAi mechanism resulting in a significant decrease in the mRNA and protein expression of STAT3.
  • the effect of KUE-PEG-siSTAT3 conjugate on the growth of LNCaP cells was detected by flow cytometer (manufacturer: BD Company, USA, model: FACS Calibur) (see Figure 18).
  • the KUE-PEG-siSTAT3 conjugate constructed in the present invention can be selectively taken up by prostate cancer cells (LNCaP cells), and can trigger the RNAi mechanism in LNCaP cells to finally successfully inhibit the growth of prostate cancer cells.
  • LNCaP cells prostate cancer cells
  • conjugates of the present invention are as effective as conventional means (eg, liposomes) delivering small nucleic acids in reducing the expression of target genes (or inhibiting cancer cell growth).

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Abstract

本发明涉及生物技术领域，公开了一种缀合物及其制备方法和用途。该缀合物由叠氮修饰的靶向配体与炔丙基修饰的小核酸序列共价连接而成。本发明提供的缀合物在药物靶向递送中具有广阔的应用前景。另外本发明还提供了一种制备所述缀合物的方法和用途，该方法仅涉及简单的化学反应，能够实现灵活、高效合成核酸缀合物的目的，并且适用于构建其他多种配体靶向核酸缀合物，具有较好的实用性。

Description

缀合物及其制备方法和用途
相关申请的交叉引用
本申请要求2021年04月23日提交的中国专利申请202110442394.6的权益，该申请的内容通过引用被合并于本文。
技术领域
本发明涉及生物技术领域，具体涉及一种缀合物及其制备方法和用途。
背景技术
前列腺特异性膜抗原(PSMA)又称为叶酸水解酶1(FOLH1)、谷氨酸羧肽酶II(GCP II)、N-乙酰化-α-连接酸性二肽酶I(NAALAD1)，它属于肽酶M28家族中的M28B亚家族。PSMA是一种结构为同型二聚体的II型跨膜蛋白，每个亚基能够结合2个锌离子。PSMA既具有叶酸水解酶又具有N-乙酰化-α-连接的酸性二肽酶(NAALADase)活性，其作用是调节N-酰基聚γ-谷氨酸及其衍生物的水解。相对于其他组织，PSMA在神经系统、前列腺、胰腺、肾脏和小肠中表达较高，并且在大多数前列腺癌的上皮层以及其他实体瘤的新生血管中表达量远高出正常组织的表达量。这种组织特异性使得PSMA成为前列腺癌递送最有吸引力的靶标之一。
由于PSMA在前列腺癌细胞中的高度富集，特异性识别PSMA的抗体、适体和小分子配体被竞相研究开发。其中，由于合成的简单性以及较高的蛋白结合亲和力，PSMA的小分子配体的研究一直备受关注。作为一种PSMA的小分子配体，赖氨酸和谷氨酸通过脲基连接而形成的二肽(KUE)已被成功地用于前列腺癌的成像及靶向治疗中。另外，RNA干扰(RNAi)是一种使用siRNA等治疗性寡核苷酸治疗包括癌症在内的各种疾病的新型技术。受到美国食品药品监督管理局(FDA)对Patisiran和Givosiran的批准的鼓舞，RNAi在治疗前列腺癌上也备受瞩目。而且，Givosiran的获批使得配体与siRNA的直接连接形成的缀合物成为靶向治疗肿瘤的一种简单、有效的新途径。但是，如何将治疗性siRNA有效地递送到前列腺癌组织/细胞中继而实现癌症的治疗仍然是一个巨大的挑战。此外，如何突破Givosiran所采用的固相合成法的局限(比如灵活性较差、操作繁琐、合成效率不理想、适用范围窄等)也是RNAi疗法急需解决的关键问题。
发明内容
本发明的目的是为了克服现有技术存在的制备核酸缀合物灵活性差、操作繁琐、生产规模小等技术问题，提供一种缀合物及其制备方法，该缀合物兼具靶向性高与活性高的双重特点。并且，本发明提供的方法具有快速、高效、简便、适用范围广的优势。此外，本发明还提供本发明制备的核酸缀合物在制备用于治疗前列腺癌、结肠癌、胰腺癌、乳腺癌、肾脏疾病和神经系统相关疾病中任意一种疾病的药物中的应用，特别是用于治疗前列腺癌的药物中的应用。
为了实现上述目的，本发明第一方面提供一种缀合物，该缀合物由叠氮修饰的靶向配体与炔丙基修饰的小核酸序列共价连接而成。
本发明第二方面提供一种制备前述缀合物的方法，该方法包括：在一价铜催化剂的存在下，使叠氮修饰的靶向配体与炔丙基修饰的小核酸序列进行接触。
本发明第三方面提供一种前述缀合物或前述方法制备的缀合物在制备用于治疗表达PSMA的组织出现异常导致的相关疾病的药物中的应用，所述疾病优选发生在腺体组织、结肠、肾脏和神经系统中；其中，所述腺体组织选自前列腺、胰腺、乳腺和胸腺中的一种。
本发明通过炔丙基修饰的寡核酸(特别是在3'-末端修饰)以及叠氮修饰的靶向配体的巧妙设计，并且利用本发明的方法(即合成后修饰策略(铜催化的点击化学))高效、快速地构建本发明的核酸缀合物。通过细胞成像、基因沉默评价以及细胞凋亡实验，发现本发明提供的缀合物(核酸缀合物)具有较高的特异性识别性能、基因沉默性能、抑制肿瘤细胞生成性能。本发明提供的制备缀合物(核酸缀合物)的方法在RNAi治疗试剂的靶向递送及治疗中具有广阔的应用前景。另外，本发明提供的制备方法仅涉及简单的化学反应，能够实现灵活、高效合成核酸缀合物，并且也适用于构建其他多种配体靶向核酸缀合物，具有较好的实用性。
附图说明
图1是本发明中KUE缀合的小核酸示意图；
图2是本发明中3'-末端炔丙基修饰的寡核酸1(ON1)的高效液相色谱谱图及质谱图；
图3是本发明中3'-末端炔丙基修饰的寡核酸2(ON2)的高效液相色谱谱图及质谱图；
图4是本发明中3'-末端炔丙基修饰的寡核酸3(ON3)的高效液相色谱谱图及质谱图；
图5是本发明中不同的KUE-PEG-siRNA缀合物的聚丙烯酰胺凝胶电泳表征图；
图6是本发明中不同的DUPA-PEG 5-siRNA缀合物的聚丙烯酰胺凝胶电泳表征图；
图7是本发明实施例1中核酸缀合物KUE-PEG 2-siRNA质谱图；
图8是本发明实施例21中核酸缀合物KUE-PEG 5-siRNA质谱图；
图9是本发明实施例22中核酸缀合物KUE-PEG 12-siRNA质谱图；
图10是本发明实施例23中核酸缀合物DUPA-PEG 5-siRNA质谱图；
图11是本发明实施例24中核酸缀合物DUPA-PEG 5-siRNA质谱图；
图12是本发明一种具体实施方式中不同核酸缀合物的细胞成像效果图；
图13是本发明一种具体实施方式中不同核酸缀合物的细胞内平均荧光强度统计图；
图14是本发明一种具体实施方式中不同核酸缀合物的细胞内化方式评价效果图；
图15是本发明一种具体实施方式中不同核酸缀合物在前列腺癌细胞的摄入效率评价图；
图16是本发明一种具体实施方式中不同核酸缀合物的mRNA沉默效果图；
图17是本发明一种具体实施方式中不同核酸缀合物的蛋白表达抑制效果图；
图18是本发明中不同核酸缀合物促进细胞凋亡的效果图。
具体实施方式
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
发明人经大量的研究发现，通过叠氮修饰的靶向配体(例如叠氮修饰的KUE配体、叠氮修饰的DUPA配体等)与3'-末端的炔丙基修饰的小核酸(寡核酸)可灵活、高效地构建抗原(如PSMA)靶向的siRNA缀合物。并且，为了验证3'-末端炔丙基修饰小核酸是否能够保持热稳定性、整体构象以及RNAi活性，发明人对3'-末端炔丙基修饰小核酸的相关性质进行了研究，例如融链温度、构象以及RNAi活性评价等方面的研究，发现在siRNA的3'-末端进行炔丙基修饰的不但不会影响RNA固有性质，而且凸显了该siRNA在合成后修饰属性。
基于以上内容，本发明第一方面提供一种缀合物，该缀合物由叠氮修饰的靶向配体与炔丙基修饰的小核酸序列共价连接而成。
根据本发明的一些实施方式，所述叠氮修饰的靶向配体中叠氮基团与靶向配体通过至少一个聚乙二醇的片段共价连接。其中，聚乙二醇的片段中聚乙二醇单体数量没有特别的限定，例如可以为1-100。其中，所述聚乙二醇的片段主要起连接作用。
根据本发明的一些实施方式，所述靶向配体为至少一种氨基酸为谷氨酸的两个氨基酸形成的二肽；优选地，所述氨基酸选自赖氨酸和谷氨酸、谷氨酸和谷氨酸以及谷氨酸和谷氨酸类似物；其中，所述靶向配体能够通过谷氨酸识别并结合靶细胞表面表达的抗原；所述抗原为PSMA。
根据本发明的一些实施方式，所述靶向配体的末端含有羧基；所述靶向配体通过羧基与靶细胞表面的抗原相连。
根据本发明的一些实施方式，所述靶细胞可以选自前列腺癌细胞、神经元细胞、肾癌细胞和结肠癌细胞中的至少一种。
根据本发明的一些实施方式，所述叠氮修饰的靶向配体具有如下所示的结构：
Figure PCTCN2022088173-appb-000001
其中n为1-100的整数。优选n为1-50的整数，更优选为1-20的整数，进一步优选为1-15的整数。例如可以为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15。
本发明中，所述叠氮修饰的靶向配体可以通过如下方法制备：
首先，先将叔丁酯、苄酯保护的赖氨酸与叔丁酯保护的谷氨酸通过三光气连接合成KUE；之后乙二醇经过丙烯酸化、叠氮化得到叠氮基PEG丙酸；最后，两者(KUE和叠氮基PEG丙酸)可以通过缩合反应得到所述靶向配体。
本发明中，优选地，所述叠氮修饰的靶向配体(6a(n＝2)、6b(n＝5)、6c(n＝12))可以按照如下步骤制备：
(1)使聚乙二醇1a-c与金属钠接触，再与丙烯酸叔丁酯进行第一反应，得到2a-c所示的化合物；
Figure PCTCN2022088173-appb-000002
(2)在三乙胺的存在下，使式2a-c所示的化合物与甲苯磺酰氯进行第二反应得到式3a-c所示的化合物；
Figure PCTCN2022088173-appb-000003
(3)在第三溶剂的存在下，使式3a-c所示的化合物与叠氮化钠进行第三反应，得到式4a-c所示的化合物；
Figure PCTCN2022088173-appb-000004
(4)在HATU(2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯)与DIPEA(N,N-二异丙基乙胺)的存在下，使4a-c所示的化合物与KUE进行第四反应，得到式5a-c所示的化合物；
Figure PCTCN2022088173-appb-000005
(5)使式5a-c所示的化合物与三氟乙酸进行第五反应，得到式6a-c所示的化合物；
Figure PCTCN2022088173-appb-000006
在步骤(1)中，所述第一反应的反应温度可以20-40℃，反应时间可以为5-20小时。其中，相对于每毫摩尔的聚乙二醇(1a-c)，金属钠的用量可以为0.05-2mmol，丙烯酸叔丁酯的用量可以为0.3-0.8mmol。所述第一反应在第一溶剂的存在下进行；其中，相对于每毫摩尔的聚乙二醇，所述第一溶剂的用量可以为1-2mL；所述第一溶剂可以为四氢呋喃。
步骤(2)中，所述第二反应的反应温度可以为-20℃至10℃，反应时间可以为5-20小时。其中，相对于每毫摩尔的式2a-c所示的化合物，三乙胺的用量为0.5-1mL，对甲苯磺酰氯的用量可以为1.2-1.8mmol。所述第二反应在第二溶剂的存在下进行；其中，相对于每毫摩尔的式2a-c所示的化合物，所述第二溶剂的用量可以为1-3mL。其中，所述第二溶剂可以为二氯甲烷。所述第二反应优选在惰性气氛下进行，所述惰性气氛可以由氩气提供。
步骤(3)中，所述第三反应的反应温度可以为60-90℃，反应时间可以5-24小时。其中，相对于每毫摩尔的式3a-c所示的化合物，叠氮化钠的用量可以为1.2-1.8mmol。其中，相对于每毫摩尔的式3a-c所示的化合物，所述第三溶剂的用量可以为2-3mL。所述第三溶剂可以为DMF(N,N二甲基甲酰胺)。
步骤(4)中，所述第四反应的反应温度可以为20-40℃，反应时间可以为5-24小时。其中，相对于每毫摩尔的式4a-c所示的化合物，KUE的用量可以为0.9-1.5mmol。其中，在 4a-c所示的化合物与KUE进行第四反应之前，可以先在三氟乙酸(相对于1mol的4a-c所示的化合物，三氟乙酸的用量为2-3mL)的存在下，使4a-c所示的化合物脱保护，其中，所述脱保护的温度可以为20-30℃，时间为10-24h；之后可以用HATU与DIPEA对4a-c所示的化合物的脱保护产物进行活化处理；其中，相对于每毫摩尔的式4a-c所示的化合物，HATU和DIPEA的用量可以分别为1.2-1.8mmol和2-4mmol。所述第四反应在第四溶剂的存在下进行；其中，相对于每毫摩尔的式4a-c所示的化合物，所述第四溶剂的用量可以为0.8-1.5mL。所述第四溶剂可以为DMF(N,N二甲基甲酰胺)。
步骤(5)中，所述第五反应的反应温度可以为20-40℃，反应时间可以为5-24小时。其中，相对于每毫摩尔的式5a-c所示的化合物，三氟乙酸的用量可以为20-30mL。
其中，对步骤(1)-(5)中的反应的后处理没有特别的限定，均可以通过柱层析分离得到纯化产物。
本发明中，KUE的结构式如下所示：
Figure PCTCN2022088173-appb-000007
根据本发明的一些实施方式，所述小核酸序列可以选自靶向STAT3、PHB1、Notch1、PLK1和BRD4的小核酸中的至少一种。
根据本发明优选的实施方式，所述炔丙基修饰优选为3'-末端炔丙基修饰。
根据本发明的一些实施方式，所述炔丙基修饰的小核酸序列采用亚磷酰胺法通过固相合成技术将炔丙基化合物修饰到小核酸序列的3'-末端；其中，所述炔丙基化合物至少含有一个活性羟基。其中，所述炔丙基修饰可以为多个炔丙基修饰(1个、2个、3个……)。本发明中，对于将炔丙基化合物修饰到小核酸序列的3'-末端的方式不做特别的限定，可以通过如下方式进行：首先，基于现有技术(Bioorg.Med.Chem.，2013，5583–5588)，以商业化的霍夫氯糖为原料历经4步反应合成1-O-炔丙基-2-脱氧-D-呋喃糖亚磷酰胺单体；然后，得到的亚磷酰胺单体与固相载体(CPG)连接后通过自动核酸合成仪将炔丙基单体X寡核苷酸的3'-末端；最后，脱除CPG以及保护基得到3'-末端炔丙基修饰的小核酸。
本发明中，优选地，所述炔丙基化合物具有如下所示的结构：
Figure PCTCN2022088173-appb-000008
其中，
Figure PCTCN2022088173-appb-000009
表示小核酸连接位点。
本发明第二方面提供一种制备前述缀合物的方法，该方法包括：在一价铜催化剂的存在下，使叠氮修饰的靶向配体与炔丙基修饰的小核酸序列进行接触。
根据本发明的一些实施方式，所述叠氮修饰的靶向配体与炔丙基修饰的小核酸序列的摩尔比可以为(1.05-10)：1，优选为(2-4)：1。
根据本发明的一些实施方式，所述一价铜催化剂可以选自Cu(I)-TBTA、CuBr和CuCl中的至少一种，优选为Cu(I)-TBTA。
根据本发明的一些实施方式，相对于1mol的小核酸序列，所述一价铜催化剂的用量可以为2-10mol，优选为3-6mol。
根据本发明的一些实施方式，所述接触的条件包括：温度为35.5-38.5℃，优选为36.5-37.5℃；时间为1-5h，优选为2-4h。
本发明中，所述接触中，对转速没有特别的限定，只要能够满足本发明的需求即可，例如转速可以为600-1000r/min，优选为700-900r/min。
本发明中，所述接触优选在震荡的条件下进行，所述震荡的条件可以为：每隔0.4-0.6h震荡一次。
根据本发明的一些实施方式，所述接触在有机溶剂的存在下进行，所述有机溶剂选自N,N-二甲基甲酰胺(DMF)、甲醇、乙醇和丙酮中的至少一种。
根据本发明的一些实施方式，所述有机溶剂的用量使得所述小核酸序列的浓度维持在20-200μM，优选为80-120μM。
本发明中，所述缀合物可以指单链核酸缀合物或双链核酸缀合物；当所述缀合物为双链核酸缀合物时，可以参照本领域常规的方式由正义链核酸和反义链核酸经退火形成双链核酸缀合物，例如将正义链核酸和反义链核酸以等摩尔的比例在退火缓冲液(含镁离子)中，90-100℃下形成。
根据本发明的一些实施方式，所述小核酸序列为包括正义链和反义链的siRNA序列，所述方法包括：
(1)分别获得炔丙基修饰的正义链和炔丙基修饰的反义链；
(2)将叠氮修饰的靶向配体与炔丙基修饰的正义链或炔丙基修饰的反义链接触，得到单链缀合物；
(3)将单链缀合物与另一条链在退火缓冲液中90-100℃条件下孵育1-5分钟得到所述缀合物；
根据本发明优选的实施方式，所述退火缓冲液选自所述退火缓冲液选自1.5-2.5mM的醋酸镁溶液。
本发明第三方面提供一种前述缀合物或前述方法制备的缀合物在制备用于治疗表达PSMA的组织出现异常导致的相关疾病的药物中的应用，所述疾病优选发生在腺体组织、结肠、肾脏和神经系统中；其中，所述腺体组织选自前列腺、胰腺、乳腺和胸腺中的一种。其中，所述疾病优选为癌症。
本发明中，“缀合物”指的是“KUE-PEG-siRNA缀合物”或“DUPA-PEG-siRNA缀合物”。
以下将通过实施例对本发明进行详细描述。
以下实施例中，所有的原料为市售品。
制备例1
本制备例用于说明叠氮修饰的KUE配体6a-c的制备
Figure PCTCN2022088173-appb-000010
n＝2、5、12。
合成路线一：
Figure PCTCN2022088173-appb-000011
具体操作步骤：
Figure PCTCN2022088173-appb-000012
(i)将聚乙二醇1a-c(100mmol,1eq)溶解于干燥THF(150mL)中，向其中加入金属钠(230mg,10mmol,0.1eq)，室温下搅拌直至钠完全溶解。随后，向混合液中缓慢滴加丙烯酸叔丁酯(6.4g,50mmol,0.5eq)，并在室温下反应12小时。反应结束后，加入8mL的1M盐酸水溶液淬灭反应。搅拌10分钟后，将反应液倒进200mL饱和食盐水，用乙酸乙酯萃取3 次，收集有机相，用饱和食盐水洗涤，无水硫酸钠干燥，快速柱色谱分离纯化(PE/EA＝10:1-1:1)得到微黄色单叔丁酯产物。
Figure PCTCN2022088173-appb-000013
1H-NMR(400MHz,CDCl 3)δ3.77–3.70(m,4H),3.69–3.59(m,6H),2.78(s,1H),2.54–2.49(m,2H),1.45(s,9H)； 13C-NMR(101MHz,CDCl 3)δ170.92,80.63,72.49,70.33,70.31,66.81,61.70,36.14,28.05.HRMS:calculated for C 11H 22NaO 5[M+Na] +:257.1365,found 257.1357.
Figure PCTCN2022088173-appb-000014
1H-NMR(400MHz,CDCl 3)δ3.79–3.59(m,22H),2.91(s,1H),2.53–2.48(m,2H),1.45(s,9H)； 13C-NMR(101MHz,CDCl 3)δ170.93,80.51,72.52,70.61,70.59,70.56,70.54,70.48,70.33,66.88,61.72,36.24,28.09.HRMS:calculated for C 17H 34NaO 8[M+Na] +:389.2151,found 389.2146.
Figure PCTCN2022088173-appb-000015
1H-NMR(400MHz,CDCl 3)δ3.76–3.54(m,49H),2.77(s,2H),2.53–2.48(m,2H),1.45(d,J＝2.4Hz,9H)； 13C-NMR(101MHz,CDCl 3)δ170.94,80.53,72.58,70.61,70.57,70.51,70.37,70.29,66.90,61.71,36.26,28.10.HRMS:calculated for C 31H 62NaO 15[M+Na] +:697.3986,found 697.3981.
Figure PCTCN2022088173-appb-000016
(ii)合物2a-c(12.3mmol,1eq)溶于30mL二氯甲烷和10mL三乙胺的混合物中，并在氩气氛围下，冰浴中搅拌。然后，分批加入(3.53g,18.5mmol,1.5eq)对甲苯磺酰氯。混合物在室温搅拌过夜，然后，加入10g少量硅胶(100-200目)浓缩至干，并在硅胶(300-400目)上进行色谱分离(PE/EA＝10:1-1:3)，产物为无色油状物。
Figure PCTCN2022088173-appb-000017
1H-NMR(400MHz,CDCl 3)δ7.79(d,J＝8.4Hz,2H),7.35(d,J＝8.0Hz,2H),4.19–4.11(m,2H),3.67(dd,J＝8.0,4.8Hz,4H),3.55(q,J＝4.8Hz,4H),2.47(dd,J＝12.4,6.0Hz,5H),1.44(s,9H)； 13C-NMR(101MHz,CDCl 3)δ170.81,144.80,132.99,129.82,127.95,80.50,70.60, 70.29,69.26,68.64,66.88,36.21,28.07,21.61.HRMS:calculated for C 18H 28NaO 7S[M+Na] +:411.1453,found 411.1448.
Figure PCTCN2022088173-appb-000018
1H-NMR(400MHz,CDCl 3)δ7.85–7.74(m,2H),7.37–7.33(m,2H),4.24–4.08(m,2H),3.83–3.51(m,20H),2.59–2.36(m,5H),1.45(d,J＝5.6Hz,9H)； 13C-NMR(101MHz,CDCl 3)δ170.89,144.78,133.02,129.82,127.98,80.49,70.74,70.58,70.51,70.36,69.24,68.67,66.89,36.27,28.09,21.64.HRMS:calculated for C 24H 40NaO 10S[M+Na] +:543.2240,found 543.2246.
Figure PCTCN2022088173-appb-000019
1H-NMR(400MHz,DMSO)δ7.83–7.74(m,2H),7.49(d,J＝7.6Hz,2H),4.13–4.10(m,2H),3.64–3.29(m,48H),2.43(s,5H),1.40(s,9H)； 13C-NMR(101MHz,DMSO)δ170.87,145.34,132.88,130.59,128.10,80.15,70.44,70.25,70.17,70.14,68.35,66.69,36.29,28.20,21.55.HRMS:calculated for C 38H 68NaO 17S[M+Na] +:851.4075,found 851.4071.
Figure PCTCN2022088173-appb-000020
(iii)将化合物3a-c(2mmol,1eq)溶于5mL二甲基甲酰胺中，并加入叠氮化钠(195mg,3mmol,1.5eq)。混合物在80℃条件下反应过夜，然后，加入2g少量硅胶(100-200目)浓缩至干，并在硅胶(300-400目)上进行色谱分离(PE/EA＝20:1-1:2)，产物为无色油状物。
Figure PCTCN2022088173-appb-000021
1H-NMR(400MHz,CDCl 3)δ3.76–3.60(m,8H),3.42–3.35(m,2H),2.51(t,J＝6.4Hz,2H),1.45(s,9H)； 13C-NMR(101MHz,CDCl 3)δ170.84,80.45,70.56,70.38,70.00,66.92,50.64,36.22,28.04.HRMS:calculated for C 11H 21N 3NaO 4[M+Na] +:282.1430,found 282.1426.
Figure PCTCN2022088173-appb-000022
1H-NMR(400MHz,CDCl 3)δ3.77–3.58(m,20H),3.47–3.30(m,2H),2.50(t,J＝6.8Hz,2H),1.45(s,9H)； 13C-NMR(101MHz,CDCl 3)δ170.92,80.51,70.70,70.67,70.64,70.59,70.50, 70.37,70.04,66.90,50.68,36.26,28.09.HRMS:calculated for C 17H 33N 3NaO 7[M+Na] +:414.2216,found 414.2211.
Figure PCTCN2022088173-appb-000023
1H-NMR(400MHz,CDCl 3)δ3.75–3.57(m,48H),3.40(d,J＝4.4Hz,2H),2.53–2.48(m,2H),1.45(d,J＝2.4Hz,9H)； 13C-NMR(101MHz,CDCl 3)δ170.91,80.49,70.69,70.66,70.63,70.56,70.49,70.36,70.04,66.88,50.67,36.25,28.09.HRMS:calculated for C 31H 61N 3NaO 4[M+Na] +:722.4051,found 722.4048.
Figure PCTCN2022088173-appb-000024
(iv)将化合物4a-c(2mmol,1eq)溶于5mL三氟乙酸中，在室温下反应过夜，然后，浓缩除去剩余的三氟乙酸，加入二氯甲烷和2g硅胶(100-200目)浓缩至干，并在硅胶(300-400目)上进行色谱分离(PE/EA＝5:1-1:5)，产物为无色油状物，并溶于2mL二甲基甲酰胺中。加入HATU(1.15g,3mmol,1.5eq)以及DIPEA(775.5mg,6mmol,3eq)进行活化。15min后，加入KUE(1.17g,2.4mmol,1.2eq)。混合物在室温下反应过夜，然后，加入3g少量硅胶(100-200目)浓缩至干，并在硅胶(300-400目)上进行色谱分离(PE/EA＝10:1-1:5)，产物为无色油状物。
Figure PCTCN2022088173-appb-000025
1H-NMR(400MHz,CDCl 3)δ6.87(s,1H),5.64(dd,J＝20.0,6.4Hz,2H),4.38–4.17(m,2H),3.71(d,J＝28.0Hz,7H),3.44(s,2H),3.34–3.12(m,2H),2.52(s,2H),2.32(d,J＝5.2Hz,2H),2.06(d,J＝6.0Hz,1H),1.84(dd,J＝32.0,25.2Hz,2H),1.67–1.23(m,33H)； 13C-NMR(101MHz,CDCl 3)δ172.58,172.52,157.40,82.17,81.66,80.67,70.19,70.00,69.76,67.35,53.35,52.99,50.56,38.84,36.42,32.02,31.55,28.63,28.04,27.96,22.23.HRMS:calculated for C 31H 56N 6NaO 10[M+Na] +:695.3956,found 695.3953.
Figure PCTCN2022088173-appb-000026
1H-NMR(400MHz,CDCl 3)δ7.05(s,1H),5.78(dd,J＝16.4,8.0Hz,2H),4.38–4.17(m,2H),3.71(d,J＝29.2Hz,15H),3.41(d,J＝4.0Hz,1H),3.26(dd,J＝23.2,17.2Hz,2H),3.00(s,3H),2.50(s,1H),2.32(d,J＝6.4Hz,2H),2.05(s,1H),1.91–1.70(m,2H),1.69–1.17(m,36H)； 13C-NMR(101MHz,CDCl 3)δ172.21,161.56,157.58,157.41,81.74,81.19,80.34,70.47,70.45,70.41,70.37,70.35,70.28,70.18,70.01,69.87,67.24,53.35,52.83,50.54,45.11,39.74,38.74,36.60,32.17,31.92,31.47,29.03,28.70,28.20,27.97,27.91,22.55,22.31.HRMS:calculated for C 37H 68N 6NaO 13[M+Na] +:827.4742,found 827.4738.
Figure PCTCN2022088173-appb-000027
1H-NMR(400MHz,CDCl 3)δ6.89(s,1H),5.57(s,2H),4.26(d,J＝20.4Hz,2H),3.67(s,45H),3.41(s,2H),3.21(d,J＝16.8Hz,2H),2.99(s,1H),2.53(s,1H),2.32(s,2H),2.05(s,1H),1.80(d,J＝44.4Hz,1H),1.65–1.18(m,36H)； 13C-NMR(101MHz,CDCl 3)δ172.41,157.42,81.87,81.44,80.50,70.37,70.21,70.05,69.99,69.82,69.71,69.62,69.49,69.37,53.47,53.02,50.61,38.87,31.97,31.55,28.20,28.06,27.98,22.31.HRMS:calculated for C 51H 96N 6NaO 20[M+Na] +:1135.6577,found 1135.6569.
Figure PCTCN2022088173-appb-000028
(v)将化合物5a-c(0.2mmol,1eq)溶于5mL三氟乙酸中，在室温下反应过夜，然后，浓缩除去剩余的三氟乙酸，加入二氯甲烷和1g少量硅胶(100-200目)浓缩至干，并在硅胶(300-400目)上进行色谱分离(DCM/MeOH＝10:1-1:2)，产物为无色油状物。对6a-c所示的化合物进行高分辨质谱确认。
6a:145mg,Yield 96％,HRMS:calcd.for C 19H 32N 6O 10[M-H] -:503.2102；MALDI-TOF-MS:m/z 503.2105；
6b:179mg,Yield 94％,HRMS:calcd.for C 25H 44N 6O 13[M-H] -:635.2888；MALDI-TOF-MS:m/z 635.2892；
6c:260mg,Yield 92％,HRMS:calcd.for C 39H 72N 6O 20[M-H] -:943.4723；MALDI-TOF-MS:m/z 943.5601。
制备例2
本制备例用于说明叠氮修饰的DUPA配体的制备
Figure PCTCN2022088173-appb-000029
n＝5。
合成路线二：
Figure PCTCN2022088173-appb-000030
具体操作步骤：
Figure PCTCN2022088173-appb-000031
(i)将化合物S1(244mg,0.5mmol)和2-(7-氮杂苯并三唑-1-基)-N,N,N',N'-四甲基脲鎓六氟磷酸盐(HATU,228mg,0.6mmol)一起加入到5mL的二甲基甲酰胺中。搅拌条件下加入N,N-二异丙基乙胺(DIPEA,129mg,1mmol)后，将混合物在室温下搅拌15分钟。立即将17-叠氮基-3,6,9,12,15-五氧杂十七烷-1-胺(182mg，0.6mmol)以喷雾的形式加入混合物中，室温条件下继续反应6小时。反应结束后，将反应混合物倒入20mL水中，乙酸乙酯萃取(50mL×3)，收集有机相，饱和食盐水洗涤，无水硫酸钠干燥，减压除去溶剂得到橘黄色油状粗产物。残余物经柱色谱分离纯化(乙酸乙酯/石油醚＝1/5)，得到黄色油状化合物S2(338mg，产率：87％)。
1H NMR(400MHz,CDCl 3)δ6.93(s,1H),5.67(s,2H),4.28(d,J＝7.3Hz,2H),3.66-3.50(m,20H),3.48-3.30(m,4H),2.30-1.96(m,6H),1.89-1.72(m,2H),1.47-1.29(m,27H)； 13C NMR(101MHz,CDCl 3)δ172.81,172.56,172.28,171.95,157.49,81.89,81.66,80.44,70.64,70.62,70.55,70.52,70.48,70.43,70.42,70.05,69.98,69.49,53.02,50.63,39.15,32.54,31.63,29.19,28.18,28.05,27.98.HRMS(ESI):m/z[M+Na] +calculated for C 35H 64N 6NaO 13 +:799.4424,found  799.4428.
Figure PCTCN2022088173-appb-000032
(ii)在冰浴条件下，将化合物S2(472mg,0.5mmol)溶解在二氯甲烷和三氟乙酸的等体积混合溶液(5mL)中，反应液在室温下反应6小时。蒸发除去溶剂，所得残余物通过快速柱层析纯化(MeOH/CH2Cl2＝1/3)纯化得到油状化合物S3(238mg，98％)。
1H NMR(400MHz,DMSO-d 6)δ12.52(s,3H),7.94(d,J＝9.2Hz,2H),6.36(dd,J＝7.9,5.6Hz,2H),4.16-3.98(m,2H),3.63-3.58(m,2H),3.57-3.50(m,14H),3.42-3.35(m,5H),3.18(q,J＝5.9Hz,2H),2.29-2.19(m,2H),2.14-2.08(m,2H),1.95-1.84(m,2H),1.77-1.65(m,2H)； 13C NMR(101MHz,DMSO)δ174.77,174.67,174.27,171.83,162.78,157.73,70.28,70.24,70.16,70.14,69.72,55.40,52.58,52.15,50.43,38.97,36.25,32.00,31.23,30.42,28.71,28.09.HRMS(ESI):m/z[M-H] ˉcalculated for C 23H 39N 6O 13 ˉ:607.2581；found:607.2578.
制备例3
本制备例用于说明3'-末端炔丙基修饰小核酸的合成
1-O-炔丙基-2-脱氧-D-呋喃糖(X，即炔丙基单体)，作为一种通用的悬突修饰，通过使用ABI 394 DNA/RNA合成仪(ABI，USA)连接到小核酸(寡核酸)的3'-末端(如图1所示)。
3'-末端炔丙基修饰小核酸的合成的具体步骤如下：首先，以商业化的霍夫氯糖为原料合成1-O-炔丙基-2-脱氧-D-呋喃糖亚磷酰胺单体(Bioorg.Med.Chem.，2013，5583–5588)；然后，得到的亚磷酰胺单体与固相载体(CPG)连接后通过前述自动核酸合成仪将炔丙基单体X寡核苷酸的3'-末端；最后，脱除CPG以及保护基得到不同的3'-末端炔丙基修饰单链小核酸，本发明制备的所有炔丙基修饰的单链核酸的纯度经过高效液相色谱仪确认而且结构经过质谱仪确认(图2-4)。
表1 不同的3'-末端炔丙基修饰单链小核酸
ONs编号 序列(SS/AS)  
ON1 5’Cy5-CAUGUUGUUCAGCUGCUGCUUX-3’ SEQ ID NO：1
ON2 5’-CAUGUUGUUCAGCUGCUGCUUX-3’ SEQ ID NO：2
ON3 5’-GCAGCAGCUGAACAACAUGUUX-3’ SEQ ID NO：3
ON4 5’-GCGACGACCUUACAGAGCGUUX-3’ SEQ ID NO：4
ON5 5’-CGCUCUGUAAGGUCGUCGCUUX-3’ SEQ ID NO：5
实施例1
本实施例用于说明KUE-PEG-siRNA缀合物的高效合成
KUE-PEG 2-siRNA缀合物的合成：向200μL灭菌离心管中依次加入炔丙基修饰的小核酸序列(ON3)(10nmol)、叠氮修饰的靶向配体6a(30nmol)、Cu(I)-TBTA(50nmol)，使得RNA终浓度维持在100μM左右。向体系中加入DMF(体积占反应液体积的占比为25％)。反应体系经轻轻涡旋混匀后放置在金属反应器中，在37℃、900r/min的条件下震荡反应3h，用15％变性聚丙烯酰胺凝胶电泳对反应结果进行监测。最后，产物经寡核酸提取试剂盒分离纯化得到，产物的纯度以及浓度经过NanoDrop 2000超微量分光光度计(Thermo,USA)确认，最终收率为98％。
实施例2-12
按照实施例1的方式进行，不同的是，改变加入有机溶剂的种类和/或用量。结果见表2。
表2
编号 有机溶剂 有机溶剂体积占反应液体积的占比(％) 收率(％)
1 DMF 25 98
2 DMF 5 58
3 DMF 50 90
4 甲醇 5 50
5 甲醇 25 78
6 甲醇 50 65
7 乙醇 5 48
8 乙醇 25 82
9 乙醇 50 62
10 丙酮 5 43
11 丙酮 25 68
12 丙酮 50 52
实施例13-20
按照实施例1的方式进行，不同的是，改变催化剂的种类和/或用量。实验结果见表3。
表3
编号 铜催化剂 催化剂与小核酸序列的摩尔比 收率(％)
13 Cu(I)-TBTA 1.5 62
14 Cu(I)-TBTA 3 88
15 CuCl 1.5 40
16 CuCl 3 75
17 CuCl 5 89
18 CuBr 1.5 42
19 CuBr 3 77
20 CuBr 5 86
实施例21
按照实施例1的方式进行，不同的是，用等摩尔量的叠氮修饰的靶向配体6b代替6a。最终收率为96％。
实施例22
按照实施例1的方式进行，不同的是，用等摩尔量的叠氮修饰的靶向配体6c代替6a。最终收率为95％。
实施例23
本实施例用于说明靶向不同基因的KUE-PEG-siRNA缀合物的高效合成
按照实施例1的方式进行，不同的是，使用靶向PHB-1基因的RNA(ON5)代替靶向STAT3基因的RNA(ON3)。最终收率为97％。
实施例24
本实施例用于说明DUPA-PEG-siRNA缀合物的高效合成
按照实施例1的方式进行，不同的是，用等摩尔量的叠氮修饰的DUPA配体代替6a。最终收率为98％。
实施例25
将实施例1-22得到的单链缀合物和Cy5标记的互补链小核酸以摩尔比1:1的比例在退火缓冲液(2mM醋酸镁含镁离子)中，95℃下孵育3分钟形成不同的KUE-PEG-siRNA缀合物以及DUPA-PEG-siRNA缀合物。
实施例1、实施例21、实施例22、实施例23和实施例24制备得到的核酸缀合物KUE-PEG 2-siRNA、核酸缀合物KUE-PEG 5-siRNA、核酸缀合物KUE-PEG 12-siRNA和DUPA-PEG 5-siRNA的聚丙烯酰胺凝胶电泳表征图见图5和图6；质谱图分别见图7、图8、 图9、图10(实施例23)和图11(实施例24)。
测试实施例1
KUE-PEG-siRNA缀合物在前列腺癌细胞摄入的选择性评价
细胞成像：选择LNCaP细胞(PSMA阳性，购自中国科学院)和PC3细胞(PSMA阴性，购自中国科学院)评价KUE-PEG-siRNA缀合物的细胞摄取。使用Cy5标记的KUE-PEG-siRNA缀合物以及siRNA(50nM)与两种前列腺癌细胞共孵育，转染6小时后细胞在649nm激发光条件下进行荧光成像(图12A(LNCaP细胞的荧光成像)和图12B(PC3细胞的荧光成像))。从图12A可以发现只有在lipofectamine 2000(牌号：Invitrogen，厂家：赛默飞)存在下才能在两种细胞中观察到来自siRNA组的红色荧光信号，而在LNCaP细胞中可以观察到来自KUE-PEG-siRNA缀合物组的红色荧光信号。此外，图12B中，在PC-3细胞中几乎无法观察到来自KUE-PEG-siRNA缀合物组的红色荧光信号。以上这些结果表明，KUE-PEG-siRNA缀合物，与未缀合的siRNA不同，KUE-PEG-siRNA缀合物能够选择性地进入前列腺癌细胞，并且这种内化不借助lipofectamine 2000也能高效率地完成。将图12A的细胞荧光成像进行平均荧光强度统计(见图13)，从图13的统计结果中发现在LNCaP细胞中KUE-PEG-siRNA缀合物组的平均荧光强度为95％，而lipofectamine 2000转染的siRNA组的平均荧光强度为82％；在PC-3细胞中KUE-PEG-siRNA缀合物的平均荧光强度小于为5％。以上这些结果表明，siRNA在不借助转染试剂时几乎无法进入任何细胞，而KUE-PEG-siRNA缀合物不借助转染试剂能够选择性地进入LNCaP细胞。
细胞内化方式：通过加入PSMA特异性抑制剂(ZJ43，参照Eur.J.Neurosci.，2004，483-494制备得到)进一步确认KUE-PEG-siRNA缀合物进入LNCaP细胞的途径。其中，LNCaP细胞提前与ZJ43一起孵育，然后再加入Cy5标记的KUE-PEG-siRNA缀合物。6小时，LNCaP细胞在640nm激发光条件下进行荧光成像(见图14)。图14的结果表明相对于未加入PSMA特异性抑制剂的对照组而言，ZJ43的加入使得LNCaP细胞几乎观察不到来自KUE-PEG-siRNA缀合物的红色荧光信号。这表明相对于未加入ZJ43组，ZJ43的加入能明显地抑制LNCaP细胞对KUE-PEG-siRNA缀合物的摄入。
测试实施例2
KUE-PEG-siRNA缀合物在前列腺癌细胞的摄入效率评价
选择PSMA阳性表达的LNCaP细胞(购自中国科学院)评价KUE-PEG-siRNA缀合物的细胞摄取效率。Cy5标记的KUE-PEG-siRNA缀合物(50nM)和siRNA(50nM)与LNCaP细胞共孵育，其中siRNA与lipofectamine 2000(牌号：Invitrogen，厂家：赛默飞)共同转染 作为对照组。4小时后，按照AnnexinV-FITC/PI细胞凋亡双染试剂盒(牌号：556547，厂家：美国BD公司)的操作对细胞进行处理。制备好的细胞样品通过FACS Calibur流式细胞仪在Cy5相对应的检测通道下进行分析。实验过程中依次对空白独照组、siRNA单独转染组、siRNA与lipofectamine 2000共同转染组以及不同KUE-PEG-siRNA缀合物转染组进行分析，并且每一组收集检测10 4个细胞。所有实验组的细胞检测完成后，使用FlowJo软件对实验结果进行分析(图15)。从图15可以发现未进行配体缀合的siRNA(siSTAT3)几乎无法进入LNCaP细胞中，这与细胞成像的结果一致。此外，相对于lipofectamine 2000转染组的细胞摄入效率(74.2％)，KUE-siRNA偶联物(KUE-PEG 2-siRNA，KUE-PEG 5-siRNA，KUE-PEG 12-siRNA)在LNCaP细胞中展现出更高的细胞摄入效率(>99％)。这些实验结果意味着配体KUE的缀合对于递送siRNA进入特定的前列腺癌细胞(LNCaP细胞)是不可或缺的。
测试实施例3
为了验证KUE-PEG-siRNA缀合物的RNAi活性，本测试实施例用到靶向STAT3基因的治疗性小核酸，其序列见表4。
表4
ONs编号 序列(SS/AS)  
ON2 5’-CAUGUUGUUCAGCUGCUGCUUX-3’ SEQ ID NO：2
向200μL灭菌离心管中依次加入炔丙基修饰的小核酸序列(ON2)(10nmol)、叠氮修饰的靶向配体(30nmol)、Cu(I)-TBTA(50nmol)，使得RNA终浓度维持在100μM左右。向体系中加入DMF(体积为反应液体积的25％)。反应体系经轻轻涡旋混匀后放置在金属反应器中，在37℃、900r/min的条件下震荡反应3h。最后，用15％变性聚丙烯酰胺凝胶电泳对反应结果进行监测，经寡核酸提取试剂盒分离纯化得到单链缀合物。当使用的靶向配体为6a时，收率为97.7％；当使用靶向配体为6b时，收率为96.2％；当使用靶向配体6c时，收率为95.6％。
之后将得到的单链缀合物和互补链小核酸以摩尔比1:1的比例在退火缓冲液(2mM醋酸镁)中，95℃下孵育3分钟形成KUE-PEG-siSTAT3缀合物。
1)KUE-PEG-siSTAT3缀合物的基因沉默效果评价：
靶标基因沉默：将靶向信号转导与转录激活因子3(STAT3)的KUE-PEG-siSTAT3(KUE-PEG-siRNA)缀合物(100nM)与LNCaP细胞共孵育。48小时后，mRNA的表达通过逆转录聚合酶链反应法(RT-PCR)进行检测。如图16A所示，所有KUE-PEG-siRNA缀合物能够有效地降低STAT3的mRNA表达，其中KUE-PEG 5-siSTAT3与借助lipofectamine 2000转染的siRNA具有相当的沉默活性。此外，从图16B可以发现随着缀合物中的PEG长度增 加mRNA表达量逐渐降低。以上的结果说明KUE-PEG-siSTAT3缀合物被LNCaP细胞摄取后能够有效地降低靶基因的表达。并且，缀合物KUE-PEG 12-siSTAT3比KUE-PEG 2-siSTAT3和KUE-PEG 5-siSTAT3能诱导更强的mRNA下调效果。
蛋白质表达抑制：除了mRNA下调实验外，还通过蛋白质印迹法(Western Blot)探究了缀合物对于STAT3蛋白的表达的影响(见图17A和图17B)。图17A中的结果表明所有的KUE-PEG-siSTAT3缀合物实验组对应的条带灰度，相对于未缀合的siSTAT3组，均呈现能不同程度地降低，这说明KUE-PEG-siSTAT3缀合物可以抑制STAT3蛋白的表达。图17B中定量实验结果表明KUE-PEG 12-siSTAT3缀合物下调STAT3蛋白表达的幅度最大。
2)KUE-PEG-siRNA缀合物的前列腺癌细胞生长抑制效果评价
基于上述的生化测试评价，可以确定KUE-PEG-siSTAT3(KUE-PEG-siRNA)缀合物能够被前列腺癌细胞LNCaP特异性摄取并促发RNAi机制从而导致STAT3的mRNA以及蛋白表达量显著降低。用流式细胞仪(厂家：美国BD公司，型号：FACS Calibur)检测KUE-PEG-siSTAT3缀合物对于LNCaP细胞生长状况影响(见图18)。图18中，流式细胞结果表明靶向STAT3基因的KUE-PEG-siSTAT3缀合物均能不同程度地促进LNCaP细胞凋亡(25.01％-34.94％)，并且缀合物KUE-PEG 12-siSTAT3(KUE-PEG 12-siRNA)能促进34.94％的LNCaP细胞凋亡。
通过以上内容可知，本发明构建的KUE-PEG-siSTAT3缀合物能够被前列腺癌细胞(LNCaP细胞)选择性地摄取，并且能够触发LNCaP细胞中的RNAi机制最终成功地抑制前列腺癌细胞的生长。
而且，进一步的研究表明，本发明的缀合物与常规方式(如脂质体)递送小核酸在降低靶基因的表达(或抑制癌细胞生长)方面的效果相当。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。

Claims (11)

  1. 一种缀合物，其特征在于，该缀合物由叠氮修饰的靶向配体与炔丙基修饰的小核酸序列共价连接而成。
  2. 根据权利要求1所述的缀合物，其中，所述叠氮修饰的靶向配体中叠氮基团与靶向配体通过至少一个聚乙二醇的片段共价连接。
  3. 根据权利要求1或2所述的缀合物，其中，所述靶向配体为至少一种氨基酸为谷氨酸的两个氨基酸形成的二肽；优选地，所述氨基酸选自赖氨酸和谷氨酸、谷氨酸和谷氨酸以及谷氨酸和谷氨酸类似物；其中，所述靶向配体能够通过谷氨酸识别并结合靶细胞表面表达的抗原；所述抗原为PSMA；
    和/或，所述靶细胞选自前列腺癌细胞、神经元细胞、肾癌细胞和结肠癌细胞中的至少一种。
  4. 根据权利要求1-3中任意一项所述的缀合物，其中，所述叠氮修饰的靶向配体具有如下所示的结构：
    Figure PCTCN2022088173-appb-100001
    和/或
    Figure PCTCN2022088173-appb-100002
    其中n为1-100的整数。
  5. 根据权利要求1-4中任意一项所述的缀合物，其中，所述小核酸序列选自靶向STAT3、PHB1、Notch1、PLK1和BRD4的小核酸中的至少一种；
    和/或，所述炔丙基修饰为3'-末端炔丙基修饰；
    和/或，所述炔丙基修饰的小核酸序列采用亚磷酰胺法通过固相合成技术将炔丙基化合物修饰到小核酸序列的3'-末端；其中，所述炔丙基化合物至少含有一个活性羟基；
    优选地，所述炔丙基化合物具有如下所示的结构：
    Figure PCTCN2022088173-appb-100003
    其中，
    Figure PCTCN2022088173-appb-100004
    表示小核酸连接位点。
  6. 一种制备缀合物的方法，其特征在于，该方法包括：在一价铜催化剂的存在下，使叠氮修饰的靶向配体与炔丙基修饰的小核酸序列进行接触。
  7. 根据权利要求6所述的方法，其中，所述叠氮修饰的靶向配体与炔丙基修饰的小核酸序列的摩尔比为(1.05-10)：1，优选为(2-4)：1；
    和/或，靶向配体为至少一种氨基酸为谷氨酸的两个氨基酸形成的二肽；优选地，所述氨基酸选自赖氨酸和谷氨酸、谷氨酸和谷氨酸以及谷氨酸和谷氨酸类似物；
    和/或，所述叠氮修饰的靶向配体具有如下所示的结构：
    Figure PCTCN2022088173-appb-100005
    和/或
    Figure PCTCN2022088173-appb-100006
    其中n为1-100的整数；
    和/或，所述小核酸序列选自靶向STAT3、PHB1、Notch1、PLK1和BRD4的小核酸中的至少一种；
    和/或，所述炔丙基修饰为3'-末端炔丙基修饰；
    和/或，所述炔丙基修饰的小核酸序列采用亚磷酰胺法通过固相合成技术将炔丙基化合物修饰到小核酸序列的3'-末端；
    优选地，所述炔丙基化合物具有如下所示的结构：
    Figure PCTCN2022088173-appb-100007
    其中，
    Figure PCTCN2022088173-appb-100008
    表示小核酸连接位点。
  8. 根据权利要求6或7所述的方法，其中，所述一价铜催化剂选自Cu(I)-TBTA、CuBr和CuCl中的至少一种，优选为Cu(I)-TBTA；
    和/或，相对于1mol的小核酸序列，所述一价铜催化剂的用量为2-10mol，优选为3-6mol；
    和/或，所述接触的条件包括：温度为35.5-38.5℃，优选为36.5-37.5℃；时间为1-5h，优选为2-4h。
  9. 根据权利要求6-8中任意一项所述的方法，其中，所述接触在有机溶剂的存在下进行，所述有机溶剂选自N,N-二甲基甲酰胺、甲醇、乙醇和丙酮中的至少一种。
  10. 根据权利要求6-9中任意一项所述的方法，其中，所述小核酸序列为包括正义链和反义链的siRNA序列，所述方法包括：
    (1)分别获得炔丙基修饰的正义链和炔丙基修饰的反义链；
    (2)将叠氮修饰的靶向配体与炔丙基修饰的正义链或炔丙基修饰的反义链接触，得到单链缀合物；
    (3)将单链缀合物与另一条链在退火缓冲液中90-100℃条件下孵育1-5分钟得到所述缀合物；
    优选地，所述退火缓冲液选自1.5-2.5mM的醋酸镁溶液。
  11. 权利要求1-5中任意一项所述的缀合物或权利要求6-10中任意一项所述的方法制备的缀合物在制备用于治疗表达PSMA的组织出现异常导致的相关疾病的药物中的应用，所述疾病优选发生在腺体组织、结肠、肾脏和神经系统中；其中，所述腺体组织选自前列腺、胰腺、乳腺和胸腺中的一种。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120130045A1 (en) * 2009-06-01 2012-05-24 Ablitech, Inc. Biomolecule-polymer conjugates and methods of making same
WO2020146521A2 (en) * 2019-01-09 2020-07-16 Arrowhead Pharmaceuticals, Inc. Rnai agents for inhibiting expression of hif-2 alpha (epas1), compositions thereof, and methods of use
CN113304274A (zh) * 2021-04-23 2021-08-27 南开大学 缀合物及其制备方法和用途

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2021351A2 (en) * 2006-04-28 2009-02-11 Centre National de la Recherche Scientifique Method for the synthesis of triazole-containing oligonucleotide derivatives
EP2321414B1 (en) 2008-07-25 2018-01-10 Alnylam Pharmaceuticals, Inc. Enhancement of sirna silencing activity using universal bases or mismatches in the sense strand
US9198972B2 (en) * 2010-01-28 2015-12-01 Alnylam Pharmaceuticals, Inc. Monomers and oligonucleotides comprising cycloaddition adduct(s)
WO2012024526A2 (en) * 2010-08-20 2012-02-23 Cerulean Pharma Inc. Conjugates, particles, compositions, and related methods
WO2012030745A1 (en) * 2010-08-30 2012-03-08 Access Pharmaecuticals, Inc MULTIVITAMIN TARGETING OF RNAi THERAPEUTICS
WO2012075114A2 (en) * 2010-12-01 2012-06-07 Ablitech, Inc. Nucleic acid-polymer conjugates and uses thereof
CA3112661A1 (en) * 2013-08-19 2015-02-26 Abbott Molecular Inc. Nucleotide analogs
JP6908964B2 (ja) * 2013-10-18 2021-07-28 ピーエスエムエー ディベロップメント カンパニー，エルエルシー Ｐｓｍａリガンドコンジュゲートによる併用療法
KR101639599B1 (ko) 2015-11-09 2016-07-14 서울대학교산학협력단 펩타이드 싸이오우레아 유도체, 이를 포함하는 방사성 동위원소 표지 화합물 및 이를 유효 성분으로 함유하는 전립선암 치료 또는 진단용 약학적 조성물
JPWO2018003739A1 (ja) 2016-06-30 2019-04-18 レナセラピューティクス株式会社 機能的リガンドを含む核酸複合体
JP7492826B2 (ja) * 2016-10-21 2024-05-30 ザ ユナイテッド ステイツ オブ アメリカ， アズ リプレゼンテッド バイ ザ セクレタリー， デパートメント オブ ヘルス アンド ヒューマン サービシーズ 分子ナノタグ

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120130045A1 (en) * 2009-06-01 2012-05-24 Ablitech, Inc. Biomolecule-polymer conjugates and methods of making same
WO2020146521A2 (en) * 2019-01-09 2020-07-16 Arrowhead Pharmaceuticals, Inc. Rnai agents for inhibiting expression of hif-2 alpha (epas1), compositions thereof, and methods of use
CN113304274A (zh) * 2021-04-23 2021-08-27 南开大学 缀合物及其制备方法和用途

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BIOORG. MED. CHEM., 2013, pages 5583 - 5588
DERKS YVONNE H.W., LÖWIK DENNIS W. P. M., SEDELAAR J. P. MICHIEL, GOTTHARDT MARTIN, BOERMAN OTTO C., RIJPKEMA MARK, LÜTJE SUSANNE,: "PSMA-targeting agents for radio- and fluorescence-guided prostate cancer surgery", THERANOSTICS, vol. 9, no. 23, 1 January 2019 (2019-01-01), AU , pages 6824 - 6839, XP055977969, ISSN: 1838-7640, DOI: 10.7150/thno.36739 *
EUR. J. NEUROSCI., 2004, pages 483 - 494
See also references of EP4316525A4
TAI WANYI; LI JUNWEI; COREY EVA; GAO XIAOHU: "A ribonucleoprotein octamer for targeted siRNA delivery", NATURE BIOMEDICAL ENGINEERING, vol. 2, no. 5, 2 April 2018 (2018-04-02), London , pages 326 - 337, XP036513430, DOI: 10.1038/s41551-018-0214-1 *

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