Conjugate and preparation method and application thereof

Abstract

The invention relates to the technical field of biology, and discloses a conjugate as well as a preparation method and application thereof. The conjugate is formed by covalently linking an azide-modified targeting ligand and a propargyl-modified small nucleic acid sequence. The conjugate provided by the invention has a wide application prospect in drug targeted delivery. In addition, the invention also provides a preparation method and application of the conjugate, the method only relates to a simple chemical reaction, can realize the purpose of flexible and efficient synthesis of the nucleic acid conjugate, is suitable for construction of other multiple ligand targeted nucleic acid conjugates, and has good practicability.

Description

[0001] This invention is a divisional application of Chinese patent application 202110442394.6 filed on April 23, 2021. Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular to a conjugate and a preparation method and use thereof. Background Art

[0003] Prostate-specific membrane antigen (PSMA), also known as folate hydrolase 1 (FOLH1), glutamate carboxypeptidase II (GCPII), and N-acetylated-α-linked acid dipeptidase I (NAALAD1), belongs to the M28B subfamily of the peptidase M28 family. PSMA is a type II transmembrane protein with a homodimeric structure, and each subunit can bind 2 zinc ions. PSMA has both folate hydrolase and N-acetylated-α-linked acid dipeptidase (NAALADase) activities, and its function is to regulate the hydrolysis of N-acyl poly-γ-glutamic acid and its derivatives. Compared with other tissues, PSMA is highly expressed in the nervous system, prostate, pancreas, kidney, and small intestine, and its expression level in the epithelial layer of most prostate cancers and the new blood vessels of other solid tumors is much higher than that in normal tissues. This tissue specificity makes PSMA one of the most attractive targets for prostate cancer delivery.

[0004] Due to the high enrichment of PSMA in prostate cancer cells, antibodies, aptamers and small molecule ligands that specifically recognize PSMA have been developed. Among them, due to the simplicity of synthesis and high protein binding affinity, the research on small molecule ligands of PSMA has always attracted much attention. As a small molecule ligand of PSMA, a dipeptide (KUE) formed by the connection of lysine and glutamic acid through urea groups has been successfully used in the imaging and targeted therapy of prostate cancer. In addition, RNA interference (RNAi) is a new technology that uses therapeutic oligonucleotides such as siRNA to treat various diseases including cancer. Encouraged by the approval of Patisiran and Givosiran by the US Food and Drug Administration (FDA), RNAi has also attracted much attention in the treatment of prostate cancer. Moreover, the approval of Givosiran makes the conjugate formed by the direct connection of ligands and siRNA a simple and effective new way to target tumors. However, how to effectively deliver therapeutic siRNA to prostate cancer tissues / cells and then achieve cancer treatment remains a huge challenge. In addition, how to overcome the limitations of the solid-phase synthesis method used by Givosiran (such as poor flexibility, cumbersome operation, unsatisfactory synthesis efficiency, narrow scope of application, etc.) is also a key issue that RNAi therapy urgently needs to solve. Summary of the invention

[0005] The purpose of the present invention is to overcome the technical problems of poor flexibility, cumbersome operation, small production scale, etc. in the prior art in preparing nucleic acid conjugates, and to provide a conjugate and a preparation method thereof, wherein the conjugate has the dual characteristics of high targeting and high activity. Moreover, the method provided by the present invention has the advantages of being fast, efficient, simple, and having a wide range of applications. In addition, the present invention also provides the use of the nucleic acid conjugate prepared by the present invention in the preparation of a drug for treating any of prostate cancer, colon cancer, pancreatic cancer, breast cancer, kidney disease, and nervous system-related diseases, in particular, in a drug for treating prostate cancer.

[0006] In order to achieve the above-mentioned object, 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.

[0007] The 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.

[0008] In a third aspect, the present invention provides a use of the aforementioned conjugate or the conjugate prepared by the aforementioned method in the preparation of a drug for treating diseases caused by abnormalities in PSMA-expressing tissues, wherein the disease preferably occurs in glandular tissues, colon, kidneys and nervous system; wherein the glandular tissue is selected from one of the prostate, pancreas, breast and thymus.

[0009] The present invention efficiently and quickly constructs the nucleic acid conjugate of the present invention through the ingenious design of propargyl-modified oligonucleic acids (especially at the 3'-terminal modification) and azide-modified targeting ligands, and utilizes the method of the present invention (i.e., post-synthesis modification strategy (copper-catalyzed click chemistry)). Through cell imaging, gene silencing evaluation, and cell apoptosis experiments, it is found that the conjugate (nucleic acid conjugate) provided by the present invention has high specific recognition performance, gene silencing performance, and tumor cell generation inhibition performance. The method for preparing the conjugate (nucleic acid conjugate) provided by the present invention has broad application prospects in the targeted delivery and treatment of RNAi therapeutic agents. In addition, the preparation method provided by the present invention only involves simple chemical reactions, can achieve flexible and efficient synthesis of nucleic acid conjugates, and is also suitable for constructing a variety of other ligand-targeted nucleic acid conjugates, and has good practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is a schematic diagram of a KUE-conjugated small nucleic acid in the present invention;

[0011] Figure 2 It is the high performance liquid chromatography spectrum and mass spectrum of the oligonucleic acid 1 (ON1) modified with a 3'-terminal propargyl group in the present invention;

[0012] Figure 3 It is the high performance liquid chromatography spectrum and mass spectrum of the oligonucleotide 2 (ON2) modified with a 3'-terminal propargyl group in the present invention;

[0013] Figure 4 It is the high performance liquid chromatography spectrum and mass spectrum of the oligonucleotide 3 (ON3) modified with a 3'-terminal propargyl group in the present invention;

[0014] Figure 5 is a polyacrylamide gel electrophoresis characterization diagram of different KUE-PEG-siRNA conjugates in the present invention;

[0015] Figure 6 is a mass spectrum of the nucleic acid conjugate KUE-PEG2-siRNA in the preparation example of the present invention;

[0016] Figure 7 is a mass spectrum of the nucleic acid conjugate KUE-PEG5-siRNA in the preparation example of the present invention;

[0017] Figure 8 It is the nucleic acid conjugate KUE-PEG in the preparation example of the present invention 12 -siRNA mass spectrum;

[0018] Fig. 9 This is a cell imaging effect diagram of different nucleic acid conjugates in a specific embodiment of the present invention;

[0019] Fig.10 is a statistical graph of the intracellular average fluorescence intensity of different nucleic acid conjugates in one embodiment of the present invention;

[0020] Fig.11 This is a diagram showing the evaluation of the cellular internalization mode of different nucleic acid conjugates in a specific embodiment of the present invention;

[0021] Fig.12 is a diagram showing the mRNA silencing effect of different nucleic acid conjugates in one specific embodiment of the present invention;

[0022] Fig.13 It is a diagram showing the protein expression inhibition effect of different nucleic acid conjugates in one specific embodiment of the present invention;

[0023] Fig.14 It is a diagram showing the effect of different nucleic acid conjugates in the present invention on promoting cell apoptosis. DETAILED DESCRIPTION

[0024] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0025] After extensive research, the inventors found that antigen (such as PSMA)-targeted siRNA conjugates can be flexibly and efficiently constructed by using azide-modified targeting ligands (such as azide-modified KUE ligands) and small nucleic acids (oligonucleic acids) modified with propargyl groups at the 3'-terminus. In addition, in order to verify whether the 3'-terminal propargyl-modified small nucleic acids can maintain thermal stability, overall conformation and RNAi activity, the inventors studied the relevant properties of the 3'-terminal propargyl-modified small nucleic acids, such as melting temperature, conformation and RNAi activity evaluation, and found that the propargyl modification at the 3'-terminus of the siRNA not only does not affect the inherent properties of the RNA, but also highlights the post-synthesis modification properties of the siRNA.

[0026] Based on the above, 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.

[0027] According to some embodiments of the present invention, the azide group in the azide-modified targeting ligand is covalently linked to the targeting ligand via at least one polyethylene glycol fragment. The number of polyethylene glycol monomers in the polyethylene glycol fragment is not particularly limited, for example, it can be 1-100. The polyethylene glycol fragment mainly plays a connecting role.

[0028] According to some embodiments of the present invention, the targeting ligand is a dipeptide formed by two amino acids, at least one of which is glutamate; preferably, the amino acids are selected from lysine and glutamate, glutamate and glutamate, and glutamate and glutamate analogs; wherein the targeting ligand can recognize and bind to an antigen expressed on the surface of a target cell through glutamate; and the antigen is PSMA.

[0029] According to some embodiments of the present invention, the terminal end of the targeting ligand contains a carboxyl group; the targeting ligand is connected to the antigen on the surface of the target cell via the carboxyl group.

[0030] According to some embodiments of the present invention, the target cell may be selected from at least one of a prostate cancer cell, a neuronal cell, a renal cancer cell and a colon cancer cell.

[0031] According to some embodiments of the present invention, the azide-modified targeting ligand has the structure shown below:

[0032] n is an integer from 1 to 100.

[0033] In the present invention, the azide-modified targeting ligand can be prepared by the following method:

[0034] First, tert-butyl ester and benzyl ester protected lysine and tert-butyl ester protected glutamic acid are connected by triphosgene to synthesize KUE; then ethylene glycol is subjected to acrylate and azidation to obtain azido PEG propionic acid; finally, the two (KUE and azido PEG propionic acid) can be condensed to obtain the targeting ligand.

[0035] In the present invention, preferably, the azide-modified targeting ligands (6a (n=2), 6b (n=5), 6c (n=12)) can be prepared according to the following steps:

[0036] (1) contacting polyethylene glycol 1a-c with metallic sodium, and then reacting with tert-butyl acrylate to obtain compounds shown in 2a-c;

[0037]

[0038] (2) in the presence of triethylamine, subjecting the compound represented by Formula 2a-c to a second reaction with toluenesulfonyl chloride to obtain a compound represented by Formula 3a-c;

[0039]

[0040] (3) In the presence of a third solvent, the compound represented by formula 3a-c is reacted with sodium azide to obtain

[0041] The compound shown in formula 4a-c;

[0042]

[0043] (4) in the presence of HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate) and DIPEA (N,N-diisopropylethylamine), the compound represented by Formula 4a-c is reacted with KUE to obtain a compound represented by Formula 5a-c;

[0044]

[0045] (5) subjecting the compound represented by Formula 5a-c to a fifth reaction with trifluoroacetic acid to obtain a compound represented by Formula 6a-c;

[0046]

[0047] In step (1), the reaction temperature of the first reaction can be 20-40°C, and the reaction time can be 5-20 hours. The amount of sodium metal can be 0.05-2 mmol, and the amount of tert-butyl acrylate can be 0.3-0.8 mmol per millimole of polyethylene glycol (1a-c). The first reaction is carried out in the presence of a first solvent; the amount of the first solvent can be 1-2 mL per millimole of polyethylene glycol; and the first solvent can be tetrahydrofuran.

[0048] In step (2), the reaction temperature of the second reaction can be -20°C to 10°C, and the reaction time can be 5-20 hours. Wherein, the amount of triethylamine used is 0.5-1mL, and the amount of p-toluenesulfonyl chloride used can be 1.2-1.8mmol per millimole of the compound shown in formula 2a-c. The second reaction is carried out in the presence of a second solvent; wherein, the amount of the second solvent used can be 1-3mL per millimole of the compound shown in formula 2a-c. Wherein, the second solvent can be dichloromethane. The second reaction is preferably carried out under an inert atmosphere, and the inert atmosphere can be provided by argon.

[0049] In step (3), the reaction temperature of the third reaction can be 60-90°C, and the reaction time can be 5-24 hours. Wherein, the amount of sodium azide used can be 1.2-1.8 mmol per millimole of the compound shown in formula 3a-c. Wherein, the amount of the third solvent used can be 2-3 mL per millimole of the compound shown in formula 3a-c. The third solvent can be DMF (N,N dimethylformamide).

[0050] In step (4), the reaction temperature of the fourth reaction can be 20-40°C, and the reaction time can be 5-24 hours. Wherein, the amount of KUE can be 0.9-1.5mmol per millimole of the compound shown in formula 4a-c. Wherein, before the compound shown in 4a-c and KUE are subjected to the fourth reaction, the compound shown in 4a-c can be deprotected in the presence of trifluoroacetic acid (relative to 1 mol of the compound shown in 4a-c, the amount of trifluoroacetic acid is 2-3mL), wherein the deprotection temperature can be 20-30°C, and the time is 10-24h; then, HATU and DIPEA can be used to activate the deprotected product of the compound shown in 4a-c; wherein, the amount of HATU and DIPEA can be 1.2-1.8mmol and 2-4mmol, respectively, per millimole of the compound shown in formula 4a-c. The fourth reaction is carried out in the presence of a fourth solvent; wherein, the amount of the fourth solvent can be 0.8-1.5mL per millimole of the compound shown in formula 4a-c. The fourth solvent may be DMF (N,N-dimethylformamide).

[0051] In step (5), 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 used may be 20-30 mL per mmol of the compound represented by formula 5a-c.

[0052] There is no particular limitation on the post-treatment of the reactions in steps (1) to (5), and the products can be purified by column chromatography.

[0053] In the present invention, the structural formula of KUE is as follows:

[0054]

[0055] According to some embodiments of the present invention, the small nucleic acid sequence may be selected from at least one of small nucleic acids targeting STAT3, PHB1, Notch1, PLK1 and BRD4.

[0056] According to a preferred embodiment of the present invention, the propargyl modification is preferably a 3'-terminal propargyl modification.

[0057] According to some embodiments of the present invention, 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 through solid phase synthesis technology; wherein the propargyl compound contains at least one active hydroxyl group. wherein the propargyl modification can be multiple propargyl modifications (1, 2, 3...). in the present invention, there is no particular limitation on the method of modifying the propargyl compound to the 3'-end of the small nucleic acid sequence, and it can be carried out in the following manner: first, based on the prior art (Bioorg. Med. Chem., 2013, 5583-5588), commercialized Hov's chlorosugar is used as a raw material to synthesize 1-O-propargyl-2-deoxy-D-furanose phosphoramidite monomer through 4 steps of reaction; then, the obtained phosphoramidite monomer is connected to a solid phase carrier (CPG) and then the propargyl monomer X oligonucleotide is connected to the 3'-end of the oligonucleotide by an automatic nucleic acid synthesizer; finally, CPG and the protecting group are removed to obtain the 3'-end propargyl-modified small nucleic acid.

[0058] In the present invention, preferably, the propargyl compound has the structure shown below:

[0059]

[0060] in, Indicates the small nucleic acid ligation site.

[0061] The 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.

[0062] According to some embodiments of the present invention, the molar ratio of the azide-modified targeting ligand to the propargyl-modified small nucleic acid sequence can be (1.05-10):1, preferably (2-4):1.

[0063] According to some embodiments of the present invention, the monovalent copper catalyst may be selected from at least one of Cu(I)-TBTA, CuBr and CuCl, preferably Cu(I)-TBTA.

[0064] According to some embodiments of the present invention, the usage of the monovalent copper catalyst may be 2-10 mol, preferably 3-6 mol, relative to 1 mol of the small nucleic acid sequence.

[0065] According to some embodiments of the present invention, the contacting conditions include: a temperature of 35.5-38.5° C., preferably 36.5-37.5° C.; and a time of 1-5 h, preferably 2-4 h.

[0066] In the present invention, there is no particular limitation on the rotation speed during the contacting, as long as it can meet the requirements of the present invention. For example, the rotation speed may be 600-1000 r / min, preferably 700-900 r / min.

[0067] In the present invention, the contacting is preferably carried out under shaking conditions, and the shaking conditions may be: shaking once every 0.4-0.6 hours.

[0068] According to some embodiments of the present invention, the contacting is performed in the presence of an organic solvent, and the organic solvent is selected from at least one of N,N-dimethylformamide (DMF), methanol, ethanol and acetone.

[0069] According to some embodiments of the present invention, the amount of the organic solvent used is such that the concentration of the small nucleic acid sequence is maintained at 20-200 μM, preferably 80-120 μM.

[0070] In the present invention, 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 double-stranded nucleic acid conjugate can be formed by annealing the sense strand nucleic acid and the antisense strand nucleic acid in a conventional manner in the art, for example, the sense strand nucleic acid and the antisense strand nucleic acid are annealed in an annealing buffer (containing magnesium ions) at an equimolar ratio at 90-100°C.

[0071] According to some embodiments of the present invention, the small nucleic acid sequence is a siRNA sequence comprising a sense strand and an antisense strand, and the method comprises:

[0072] (1) obtaining a propargyl-modified sense strand and a propargyl-modified antisense strand respectively;

[0073] (2) contacting the azide-modified targeting ligand with the propargyl-modified sense strand or the propargyl-modified antisense strand to obtain a single-stranded conjugate;

[0074] (3) incubating the single-stranded conjugate with another strand in an annealing buffer at 90-100° C. for 1-5 minutes to obtain the conjugate;

[0075] According to a preferred embodiment of the present invention, the annealing buffer is selected from 1.5-2.5 mM magnesium acetate solution.

[0076] In a third aspect, the present invention provides a use of the aforementioned conjugate or the conjugate prepared by the aforementioned method in the preparation of a drug for treating a disease caused by abnormalities in PSMA-expressing tissues, wherein the disease preferably occurs in glandular tissues, colon, kidneys and nervous system; wherein the glandular tissue is selected from the group consisting of prostate, pancreas, breast and thymus. wherein the disease is preferably cancer.

[0077] In the present invention, "conjugate" refers to "KUE-PEG-siRNA conjugate".

[0078] The present invention will be described in detail below through examples.

[0079] In the following examples, all raw materials are commercially available.

[0080] Preparation Example 1

[0081] This preparation example is used to illustrate the preparation of azide-modified targeting ligands 6a-c

[0082]

[0083] Synthesis route:

[0084]

[0085] Specific steps:

[0086]

[0087] (i) Polyethylene glycol 1a-c (100 mmol, 1 eq) was dissolved in dry THF (150 mL), and sodium metal (230 mg, 10 mmol, 0.1 eq) was added thereto, and stirred at room temperature until the sodium was completely dissolved. Subsequently, tert-butyl acrylate (6.4 g, 50 mmol, 0.5 eq) was slowly added dropwise to the mixture, and the mixture was reacted at room temperature for 12 hours. After the reaction was completed, 8 mL of 1 M aqueous hydrochloric acid was added to quench the reaction. After stirring for 10 minutes, the reaction solution was poured into 200 mL of saturated brine, extracted with ethyl acetate 3 times, the organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and separated and purified by flash column chromatography (PE / EA=10:1-1:1) to obtain a slightly yellow mono-tert-butyl ester product.

[0088]

[0089] 1 H-NMR (400MHz, CDCl3) 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); 13 C-NMR (101MHz, CDCl3) δ170.92,80.63,72.49,70.33,70.31,66.81,61.70,36.14,28.05.HRMS: calculated for C 11 H 22 NaO5[M+Na] +:257.1365,found257.1357.

[0090]

[0091] 1 H-NMR(400MHz,CDCl3)δ3.79–3.59(m,22H),2.91(s,1H),2.53–2.48(m,2H),1.45(s,9H); 13 C-NMR(101MHz,CDCl3)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 17 H 34 NaO8[M+Na] + :389.2151,found389.2146.

[0092]

[0093] 1 H-NMR(400MHz,CDCl3)δ3.76–3.54(m,49H),2.77(s,2H),2.53–2.48(m,2H),1.45(d,J=2.4Hz,9H); 13 C-NMR(101MHz,CDCl3)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 31 H 62 NaO 15 [M+Na] + :697.3986,found 697.3981.

[0094]

[0095] (ii) Compound 2a-c (12.3 mmol, 1 eq) was dissolved in a mixture of 30 mL of dichloromethane and 10 mL of triethylamine and stirred in an ice bath under an argon atmosphere. Then, p-toluenesulfonyl chloride (3.53 g, 18.5 mmol, 1.5 eq) was added in batches. The mixture was stirred at room temperature overnight, then 10 g of a small amount of silica gel (100-200 mesh) was added, concentrated to dryness, and chromatographed on silica gel (300-400 mesh) (PE / EA=10:1-1:3), and the product was a colorless oil.

[0096]

[0097] 1 H-NMR (400MHz, CDCl3) 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); 13 C-NMR (101MHz, CDCl3) 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 18 H 28 NaO7S[M+Na] + :411.1453, found 411.1448.

[0098]

[0099] 1 H-NMR (400MHz, CDCl3) 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); 13 C-NMR (101MHz, CDCl3) δ170.89,144.78,133.02,129.82,127.98,80.49,70.74,7 0.58,70.51,70.36,69.24,68.67,66.89,36.27,28.09,21.64.HRMS:calculated for C 24 H40 NaO 10 S[M+Na] + :543.2240,found543.2246.

[0100]

[0101] 1 H-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); 13 C-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 38 H 68 NaO 17 S[M+Na] + :851.4075, found 851.4071.

[0102]

[0103] (iii) Compound 3a-c (2 mmol, 1 eq) was dissolved in 5 mL of dimethylformamide, and sodium azide (195 mg, 3 mmol, 1.5 eq) was added. The mixture was reacted at 80°C overnight, and then 2 g of a small amount of silica gel (100-200 mesh) was added and concentrated to dryness. The mixture was chromatographed on silica gel (300-400 mesh) (PE / EA=20:1-1:2), and the product was a colorless oil.

[0104]

[0105] 1 H-NMR (400MHz, CDCl3) δ3.76–3.60 (m, 8H), 3.42–3.35 (m, 2H), 2.51 (t, J = 6.4Hz, 2H), 1.45 (s, 9H); 13 C-NMR(101MHz, CDCl3)δ170.84,80.45,70.56,70.38,70.00,66.92,50.64,36.22,28.04.HRMS:calculated for C 11 H21 N3NaO4[M+Na] + :282.1430,found282.1426.

[0106]

[0107] 1 H-NMR(400MHz,CDCl3)δ3.77–3.58(m,20H),3.47–3.30(m,2H),2.50(t,J=6.8Hz,2H),1.45(s,9H); 13 C-NMR(101MHz,CDCl3)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 17 H 33 N3NaO7[M+Na] + :414.2216,found 414.2211.

[0108]

[0109] 1 H-NMR(400MHz,CDCl3)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); 13 C-NMR(101MHz,CDCl3)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 forC 31 H 61 N3NaO4[M+Na] + :722.4051,found 722.4048.

[0110]

[0111] (iv) Compound 4a-c (2 mmol, 1 eq) was dissolved in 5 mL of trifluoroacetic acid and reacted at room temperature overnight. Then, the remaining trifluoroacetic acid was removed by concentration, dichloromethane and 2 g of silica gel (100-200 mesh) were added, concentrated to dryness, and chromatographed on silica gel (300-400 mesh) (PE / EA=5:1-1:5). The product was a colorless oil and dissolved in 2 mL of dimethylformamide. HATU (1.15 g, 3 mmol, 1.5 eq) and DIPEA (775.5 mg, 6 mmol, 3 eq) were added for activation. After 15 min, KUE (1.17 g, 2.4 mmol, 1.2 eq) was added. The mixture was reacted at room temperature overnight, then 3 g of a small amount of silica gel (100-200 mesh) was added, concentrated to dryness, and chromatographed on silica gel (300-400 mesh) (PE / EA=10:1-1:5). The product was a colorless oil.

[0112]

[0113] 1 H-NMR (400MHz, CDCl3) 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); 13 C-NMR (101MHz, CDCl3) 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 31 H 56 N6NaO 10 [M+Na] + :695.3956,found 695.3953.

[0114]

[0115] 1H-NMR(400MHz,CDCl3)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); 13 C-NMR(101MHz,CDCl3)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 37 H 68 N6NaO 13 [M+Na] + :827.4742,found827.4738.

[0116]

[0117] 1 H-NMR(400MHz,CDCl3)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, CDCl3) 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 51 H 96 N6NaO 20 [M+Na] + :1135.6577, found 1135.6569.

[0118]

[0119] (v) Compound 5a-c (0.2 mmol, 1 eq) was dissolved in 5 mL of trifluoroacetic acid, reacted at room temperature overnight, then concentrated to remove the remaining trifluoroacetic acid, added dichloromethane and 1 g of a small amount of silica gel (100-200 mesh), concentrated to dryness, and chromatographed on silica gel (300-400 mesh) (DCM / MeOH=10:1-1:2), the product was a colorless oil. The compounds shown in 6a-c were confirmed by high-resolution mass spectrometry.

[0120] 6a:145mg,Yield 96%,HRMS:calcd.for C 19 H 32 N6O 10 [MH] - :503.2102;MALDI-TOF-MS:m / z 503.2105;

[0121] 6b:179mg,Yield 94%,HRMS:calcd.for C 25 H 44 N6O 13 [MH] - :635.2888; MALDI-TOF-MS: m / z 635.2892;

[0122] 6c:260mg,Yield 92%,HRMS:calcd.for C 39 H 72 N6O 20 [MH] - :943.4723; MALDI-TOF-MS: m / z 943.5601.

[0123] Preparation Example 2

[0124] This preparation example is used to illustrate the synthesis of 3'-terminal propargyl-modified small nucleic acids

[0125] 1-O-propargyl-2-deoxy-D-furanose (X, i.e., propargyl monomer), as a general overhang modification, was attached to the 3'-end of small nucleic acids (oligonucleotides) (e.g., Figure 1 shown).

[0126] The specific steps of synthesizing the 3'-terminal propargyl-modified small nucleic acid are as follows: first, 1-O-propargyl-2-deoxy-D-furanose phosphoramidite monomer is synthesized using commercial Hofmannose as a raw material (Bioorg. Med. Chem., 2013, 5583-5588); then, the obtained phosphoramidite monomer is connected to a solid phase carrier (CPG) and the 3'-end of the propargyl monomer X oligonucleotide is synthesized by the aforementioned automatic nucleic acid synthesizer; finally, CPG and the protecting group are removed to obtain different 3'-terminal propargyl-modified single-stranded small nucleic acids. The purity of all propargyl-modified single-stranded nucleic acids prepared by the present invention is confirmed by high performance liquid chromatography and the structure is confirmed by mass spectrometry ( Figure 2-4 ).

[0127] Table 1 Different 3'-terminal propargyl-modified single-stranded small nucleic acids

[0128] ONs No. Sequence (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

[0129] Example 1

[0130] This example is used to illustrate the efficient synthesis of KUE-PEG-siRNA conjugates

[0131] Synthesis of KUE-PEG2-siRNA conjugate: A propargyl-modified small nucleic acid sequence (ON3) (10 nmol), an azide-modified targeting ligand 6a (30 nmol), and Cu(I)-TBTA (50 nmol) were added to a 200 μL sterile centrifuge tube in sequence to maintain the final RNA concentration at about 100 μM. DMF (25% of the volume of the reaction solution) was added to the system. The reaction system was gently vortexed and placed in a metal reactor, and the reaction was shaken at 37°C and 900 r / min for 3 hours. The reaction results were monitored by 15% denaturing polyacrylamide gel electrophoresis. Finally, the product was separated and purified by an oligonucleic acid extraction kit. The purity and concentration of the product were confirmed by a NanoDrop 2000 ultra-micro spectrophotometer (Thermo, USA), and the final yield was 98%.

[0132] Example 2-12

[0133] The method of Example 1 was followed, except that the type and / or amount of the organic solvent added was changed. The experimental results are shown in Table 2.

[0134] Table 2

[0135]

[0136]

[0137] Examples 13-20

[0138] The method of Example 1 was followed, except that the type and / or amount of the catalyst was changed. The experimental results are shown in Table 3.

[0139] Table 3

[0140] serial number Copper Catalyst Molar ratio of catalyst to small nucleic acid sequence Yield (%) 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

[0141] Embodiment 21

[0142] The reaction was carried out in the same manner as in Example 1, except that an equimolar amount of azide-modified targeting ligand 6b was used instead of 6a. The final yield was 96%.

[0143] Embodiment 22

[0144] The reaction was carried out in the same manner as in Example 1, except that an equimolar amount of azide-modified targeting ligand 6c was used instead of 6a. The final yield was 95%.

[0145] Embodiment 23

[0146] The single-stranded conjugate obtained in Example 1-22 and the complementary strand small nucleic acid labeled with Cy5 were incubated at a molar ratio of 1:1 in an annealing buffer (2 mM magnesium acetate containing magnesium ions) at 95° C. for 3 minutes to form different KUE-PEG-siRNA conjugates.

[0147] The nucleic acid conjugates KUE-PEG2-siRNA, KUE-PEG5-siRNA and KUE-PEG prepared in Examples 1, 21 and 22 12 -siRNA polyacrylamide gel electrophoresis characterization diagram is shown in Figure 5 ; Mass spectra are shown in Figure 6 , Figure 7 and Figure 8 .

[0148] Test Example 1

[0149] Selective evaluation of KUE-PEG-siRNA conjugate uptake in prostate cancer cells

[0150] Cell imaging: LNCaP cells (PSMA positive, purchased from the Chinese Academy of Sciences) and PC3 cells (PSMA negative, purchased from the Chinese Academy of Sciences) were selected to evaluate the cellular uptake of KUE-PEG-siRNA conjugates. Cy5-labeled KUE-PEG-siRNA conjugates and siRNA (50 nM) were co-incubated with two prostate cancer cells. After 6 hours of transfection, the cells were imaged with fluorescence under 649 nm excitation light ( Fig. 9 A (Fluorescence imaging of LNCaP cells) and Fig. 9 B (Fluorescence imaging of PC3 cells). Fig. 9 A It can be found that only in the presence of lipofectamine 2000 (brand: Invitrogen, manufacturer: Thermo Fisher) can the red fluorescence signal from the siRNA group be observed in both cells, while the red fluorescence signal from the KUE-PEG-siRNA conjugate group can be observed in LNCaP cells. In addition, Fig. 9 In B, the red fluorescence signal from the KUE-PEG-siRNA conjugate group was almost unobservable in PC-3 cells. These results indicate that the KUE-PEG-siRNA conjugate, unlike the unconjugated siRNA, can selectively enter prostate cancer cells, and this internalization can be achieved efficiently without the aid of lipofectamine 2000. Fig. 9 The average fluorescence intensity of A cells was measured by fluorescence imaging (see Fig.10 ),from Fig.10 The statistical results show that the average fluorescence intensity of the KUE-PEG-siRNA conjugate group in LNCaP cells was 95%, while the average fluorescence intensity of the siRNA group transfected with lipofectamine 2000 was 82%; the average fluorescence intensity of the KUE-PEG-siRNA conjugate in PC-3 cells was less than 5%. The above results show that siRNA can hardly enter any cells without the help of transfection reagents, while KUE-PEG-siRNA conjugates can selectively enter LNCaP cells without the help of transfection reagents.

[0151] Cell internalization: The pathway of KUE-PEG-siRNA conjugate entering LNCaP cells was further confirmed by adding a PSMA-specific inhibitor (ZJ43, prepared by referring to Eur. J. Neurosci., 2004, 483-494). LNCaP cells were incubated with ZJ43 in advance, and then Cy5-labeled KUE-PEG-siRNA conjugate was added. After 6 hours, LNCaP cells were imaged with fluorescence under 649 nm excitation light (see Fig.11 ). Fig.11The results showed that compared with the control group without the addition of PSMA-specific inhibitor, the addition of ZJ43 made it almost impossible to observe the red fluorescence signal from the KUE-PEG-siRNA conjugate in LNCaP cells. This indicates that compared with the group without the addition of ZJ43, the addition of ZJ43 can significantly inhibit the uptake of KUE-PEG-siRNA conjugate by LNCaP cells.

[0152] Test Example 2

[0153] In order to verify the RNAi activity of the KUE-PEG-siRNA conjugate, this test example used a therapeutic small nucleic acid targeting the STAT3 gene, the sequence of which is shown in Table 4.

[0154] Table 4

[0155] ONs No. Sequence (SS / AS) ON3 5'-CAUGUUGUUCAGCUGCUGCUUX-3' SEQ ID NO: 2

[0156] To a 200 μL sterile centrifuge tube, add propargyl-modified small nucleic acid sequence (ON3) (10 nmol), azide-modified targeting ligand (30 nmol), and Cu(I)-TBTA (50 nmol) in sequence to maintain the final RNA concentration at about 100 μM. Add DMF to the system (the volume is 25% of the reaction solution volume). After gently vortexing and mixing, the reaction system is placed in a metal reactor and shaken at 37°C and 900 r / min for 3 hours. Finally, the reaction results are monitored by 15% denaturing polyacrylamide gel electrophoresis, and the single-stranded conjugate is separated and purified by an oligonucleic acid extraction kit. When the targeting ligand used is 6a, the yield is 97.7%; when the targeting ligand used is 6b, the yield is 96.2%; when the targeting ligand 6c is used, the yield is 95.6%.

[0157] The resulting single-stranded conjugate and complementary small nucleic acid were then incubated at a molar ratio of 1:1 in an annealing buffer (2 mM magnesium acetate) at 95°C for 3 minutes to form a KUE-PEG-siSTAT3 conjugate.

[0158] 1) Evaluation of gene silencing effect of KUE-PEG-siSTAT3 conjugate:

[0159] Target gene silencing: LNCaP cells were incubated with KUE-PEG-siSTAT3 (KUE-PEG-siRNA) conjugates (100 nM) targeting signal transducer and activator of transcription 3 (STAT3). After 48 hours, the expression of mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Fig.12As shown in A, all KUE-PEG-siRNA conjugates can effectively reduce the mRNA expression of STAT3, among which KUE-PEG5-siSTAT3 has comparable silencing activity with siRNA transfected by lipofectamine 2000. Fig.12 B It can be found that as the length of PEG in the conjugate increases, the mRNA expression level gradually decreases. The above results show that the KUE-PEG-siSTAT3 conjugate can effectively reduce the expression of the target gene after being taken up by LNCaP cells. 12 -siSTAT3 induced a stronger mRNA downregulation effect than KUE-PEG2-siSTAT3 and KUE-PEG5-siSTAT3.

[0160] Protein expression inhibition: In addition to the mRNA downregulation experiment, the effect of the conjugate on the expression of STAT3 protein was also investigated by Western Blot (see Fig.13 A and Fig.13 B). Fig.13 The results in A show that the grayscale of the bands corresponding to all KUE-PEG-siSTAT3 conjugate experimental groups was reduced to varying degrees compared with the unconjugated siSTAT3 group, indicating that the KUE-PEG-siSTAT3 conjugate can inhibit the expression of STAT3 protein. Fig.13 The quantitative experimental results in B show that KUE-PEG 12 -siSTAT3 conjugate downregulated STAT3 protein expression to the greatest extent.

[0161] 2) Evaluation of the inhibitory effect of KUE-PEG-siRNA conjugate on prostate cancer cell growth

[0162] Based on the above biochemical test evaluation, it can be determined that the KUE-PEG-siSTAT3 (KUE-PEG-siRNA) conjugate can be specifically taken up by prostate cancer cells LNCaP and trigger the RNAi mechanism, thereby significantly reducing the mRNA and protein expression of STAT3. The effect of the KUE-PEG-siSTAT3 conjugate on the growth of LNCaP cells was detected by flow cytometry (manufacturer: BD Company, USA, model: FACS Calibur) (see Fig.14 ). Fig.14 The flow cytometry results showed that the KUE-PEG-siSTAT3 conjugates targeting the STAT3 gene could promote the apoptosis of LNCaP cells to varying degrees (25.01%-34.94%), and the conjugate KUE-PEG 12 -siSTAT3(KUE-PEG 12-siRNA) can promote 34.94% apoptosis of LNCaP cells.

[0163] From the above, it can be seen that the KUE-PEG-siSTAT3 conjugate constructed by the present invention can be selectively taken up by prostate cancer cells (LNCaP cells), and can trigger the RNAi mechanism in LNCaP cells to ultimately successfully inhibit the growth of prostate cancer cells.

[0164] Furthermore, further studies have shown that the conjugates of the present invention are comparable to conventional methods (such as liposomes) in delivering small nucleic acids in terms of reducing the expression of target genes (or inhibiting the growth of cancer cells).

[0165] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A conjugate, characterized in that The conjugate is formed by covalently linking an azide-modified targeting ligand to a propargyl-modified small nucleic acid sequence, wherein the propargyl modification is a 3'-terminal propargyl modification.

2. The conjugate according to claim 1, wherein In the azide-modified targeting ligand, the azide group is covalently linked to the targeting ligand via at least one polyethylene glycol fragment.

3. The conjugate according to claim 1 or 2, wherein The targeting ligand is a dipeptide formed by two amino acids, at least one of which is glutamic acid; preferably, the amino acids are selected from lysine and glutamic acid, glutamic acid and glutamic acid, and glutamic acid and glutamic acid analogs; wherein the targeting ligand can recognize and bind to an antigen expressed on the surface of a target cell through glutamic acid; the antigen is PSMA; And / or, the target cell is selected from at least one of prostate cancer cells, neuronal cells, renal cancer cells and colon cancer cells.

4. The conjugate according to any one of claims 1 to 3, wherein The azide-modified targeting ligand has the structure shown below: n is an integer from 1 to 100.

5. The conjugate according to any one of claims 1 to 4, wherein The small nucleic acid sequence is selected from at least one of the small nucleic acids targeting STAT3, PHB1, Notch1, PLK1 and BRD4; And / or, the propargyl-modified small nucleic acid sequence is modified with a propargyl compound to the 3'-end of the small nucleic acid sequence by solid phase synthesis technology using the phosphoramidite method; wherein the propargyl compound contains at least one active hydroxyl group; Preferably, the propargyl compound has the structure shown below: in, Indicates the small nucleic acid ligation site.

6. A method for preparing a conjugate, characterized in that: The method comprises: contacting an azide-modified targeting ligand with a propargyl-modified small nucleic acid sequence in the presence of a monovalent copper catalyst, wherein the propargyl modification is a 3'-terminal propargyl modification.

7. The method according to claim 6, wherein: The molar ratio of the azide-modified targeting ligand to the propargyl-modified small nucleic acid sequence is (1.05-10):1, preferably (2-4):1; And / or, the targeting ligand is a dipeptide formed by two amino acids, at least one of which is glutamic acid; preferably, the amino acids are selected from lysine and glutamic acid, glutamic acid and glutamic acid, and glutamic acid and a glutamic acid analog; And / or, the azide-modified targeting ligand has the structure shown below: n is an integer from 1 to 100; and / or, the small nucleic acid sequence is selected from at least one of the small nucleic acids targeting STAT3, PHB1, Notch1, PLK1 and BRD4; And / or, the propargyl-modified small nucleic acid sequence is modified with a propargyl compound to the 3'-end of the small nucleic acid sequence by solid phase synthesis technology using the phosphoramidite method; Preferably, the propargyl compound has the structure shown below: in, Indicates the small nucleic acid ligation site.

8. The method according to claim 6 or 7, wherein: The monovalent copper catalyst is selected from at least one of Cu(I)-TBTA, CuBr and CuCl, preferably Cu(I)-TBTA; and / or, relative to 1 mol of the small nucleic acid sequence, the amount of the monovalent copper catalyst is 2-10 mol, preferably 3-6 mol; And / or, the contacting conditions include: temperature of 35.5-38.5°C, preferably 36.5-37.5°C; time of 1-5h, preferably 2-4h.

9. The method according to any one of claims 6 to 8, wherein: The contacting is performed in the presence of an organic solvent, and the organic solvent is at least one selected from N,N-dimethylformamide, methanol, ethanol and acetone.

10. The method according to any one of claims 6 to 9, wherein: The small nucleic acid sequence is a siRNA sequence including a sense strand and an antisense strand, and the method comprises: (1) obtaining a propargyl-modified sense strand and a propargyl-modified antisense strand respectively; (2) contacting the azide-modified targeting ligand with the propargyl-modified sense strand or the propargyl-modified antisense strand to obtain a single-stranded conjugate; (3) incubating the single-stranded conjugate with another strand in an annealing buffer at 90-100° C. for 1-5 minutes to obtain the conjugate; Preferably, the annealing buffer is selected from 1.5-2.5 mM magnesium acetate solution.

11. Use of the conjugate according to any one of claims 1 to 5 or the conjugate prepared by the method according to any one of claims 6 to 10 in the preparation of a drug for treating diseases related to abnormalities in tissues expressing PSMA, wherein the disease preferably occurs in glandular tissues, colon, kidneys and nervous system; wherein, The glandular tissue is selected from the group consisting of prostate, pancreas, breast and thymus.

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