Liver-targeting compounds, conjugates and uses thereof
By designing liver-targeting compounds and conjugates, and utilizing high-affinity molecular cluster structures to conjugate with nucleic acid drugs, the problem of efficient delivery and long-term inhibition of small nucleic acid drugs in liver tissue has been solved, achieving efficient liver-targeted delivery and a simple preparation process.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- YANTAI INSTITUTE OF PHARMACEUTICAL SCIENCE
- Filing Date
- 2023-10-30
- Publication Date
- 2026-07-07
Smart Images
Figure CN117466959B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, and in particular relates to a liver-targeting compound, conjugate, and its application. Background Technology
[0002] The desialyl glycoprotein receptor (ASGPR) is primarily expressed in hepatocytes and is a hepatocyte-specific receptor that mediates the desialylation and degradation of glycoproteins by hepatocytes. ASGPR participates in various physiological functions, including glycoprotein and lipid metabolism, and viral hepatitis, playing a crucial role in the diagnosis and treatment of liver-related diseases. ASGPR specifically recognizes and binds to galactose, galactosamine, and N-acetylgalactosamine; therefore, compounds or complexes containing galactose, galactosamine, and their derivatives can specifically and efficiently deliver drugs to the liver and hepatocytes. Utilizing galactose and its derivatives that target the ASGPR receptor on the hepatocyte surface enables efficient drug delivery to the liver. The use of ASGPR-specific ligands in drug delivery to hepatocytes has been extensively studied and applied.
[0003] Nucleic acid drugs include aptamers, antisense nucleic acids (ASOs), small interfering RNA (siRNA), and microRNAs (miRNAs). Due to their high versatility and ability to specifically inhibit target genes within cells, nucleic acid drugs show great promise in disease treatment. However, the challenge of in vivo delivery of nucleic acid drugs remains a key constraint on their development.
[0004] Therefore, there is a need for targeted delivery vectors that can efficiently deliver small nucleic acid drugs to liver tissue and maintain a long-lasting inhibitory effect. Summary of the Invention
[0005] To address the shortcomings of the existing technology, this invention provides a liver-targeting compound, a conjugate, and its applications. The specific technical solution is as follows:
[0006] The first objective of this invention is to provide a liver-targeting compound having the structure shown in Formula I:
[0007]
[0008] T is the target group, B is the branch structure, L1 is the linking group, L2 is the connecting part between the target group and the branch structure, and X is an integer between 2 and 6.
[0009] Wherein, L1 is selected from the following groups:
[0010]
[0011] a, c, d, and e are integers between 0 and 12, b is an integer between 0 and 3; DMTr stands for bis(4-methoxyphenyl)phenylmethyl;
[0012] L2 is selected from groups with the following structures:
[0013]
[0014] a1, a2, a3, a4, and a5 are integers between 0 and 12, preferably integers between 1 and 8.
[0015] The liver-targeting compound of the present invention can efficiently deliver drugs such as small nucleic acids to liver tissue and maintain a long-lasting inhibitory effect.
[0016] Furthermore, the structure of B is as follows:
[0017]
[0018] Furthermore, the T-targeting group is galactose, galactosamine, or a galactosamine derivative.
[0019] Furthermore, the T-targeting group is selected from galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, or N-propionylgalactosamine; preferably N-acetylgalactosamine.
[0020] Furthermore, L1 is selected from the following groups:
[0021]
[0022] Furthermore, the L2 is selected from the following groups:
[0023]
[0024] Furthermore, X is 3 or 4.
[0025] This invention links multiple galactose, galactosamine, or galactosamine derivative molecules into tri- or tetra-cluster molecules, forming a molecular cluster form. This cluster has a significantly higher affinity for ASGPR than for monosaccharides, thereby improving the liver-targeted delivery capability of drugs.
[0026] Furthermore, the structure of the targeted compound is shown in Formula H03:
[0027]
[0028] Furthermore, the structure of the targeted compound is shown in Formula H05:
[0029]
[0030] A second objective of this invention is to provide a liver-targeting conjugate, which is formed by linking a functional group to the aforementioned targeting compound.
[0031] Furthermore, the liver-targeting conjugate also includes its pharmaceutically acceptable salt.
[0032] Furthermore, the liver-targeting conjugate also includes, but is not limited to, pharmaceutically acceptable excipients, diluents, buffers, or stabilizers.
[0033] Furthermore, the functional group is connected to the linking group via a phosphate ester group, a thiophosphate ester group, a phosphonic acid group, an ester bond, or an ether bond.
[0034] Furthermore, the functional groups include small molecule drugs, peptides, nucleic acids, or proteins, which are biologically active substances.
[0035] Furthermore, the nucleic acid is selected from one or more of small interfering RNA, microRNA, antisense nucleic acid, or mRNA fragments.
[0036] Furthermore, the nucleic acid is a single-stranded oligonucleotide or a double-stranded oligonucleotide.
[0037] Furthermore, the linker group L1 is attached to the end of the nucleotide.
[0038] Furthermore, the linker L1 is attached to the 5' end of the nucleotide or to the 3' end of the nucleotide.
[0039] The liver-targeting compounds provided by this invention can be prepared through conventional substitution, esterification, and amidation reactions. Various condensing agents can be used for amidation reactions; for example, references such as Org. Process Res. Dev. 2022, 26, 1562-1689 can be used for the synthesis of such compounds. The liver-targeting conjugates can be conjugated to the 3' end of nucleic acid sequences using solid-phase synthesis methods. Solid-phase synthesis involves cyclically extending the nucleotide sequence through deprotection, coupling, oxidation, and capping processes.
[0040] A third aspect of the present invention provides the use of a compound or conjugate in the preparation of a medicament for the diagnosis, prevention, treatment or relief of liver-related diseases and symptoms.
[0041] The beneficial effects of this invention are as follows:
[0042] The liver-targeting compounds and conjugates of this invention have clearly defined structures. Multiple galactose, galactosamine, or galactosamine derivative molecules are linked into tri- or tetra-cluster molecules to form molecular clusters. The affinity for ASGPR is significantly higher than that for monosaccharides, which can improve the liver-targeting delivery capability of drugs, with high delivery efficiency and long-lasting inhibitory effect. Moreover, the synthetic route is clear and the preparation process is simple. Attached Figure Description
[0043] Figure 1 This is the mass spectrum of the H03 compound of the present invention;
[0044] Figure 2 The H03 compound of this invention, H 1 -NMR spectrum;
[0045] Figure 3 This is the mass spectrum of compound H05 of the present invention;
[0046] Figure 4 The H05 compound of this invention, H 1 -NMR spectrum;
[0047] Figure 5 This image shows the effect of the liver-targeting siRNA conjugate of the present invention on the inhibition of TTR mRNA in mice. Detailed Implementation
[0048] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.
[0049] Example 1: Synthesis of liver-targeting compound H03
[0050] 1) Synthesis of compound 2
[0051]
[0052] Compound 1 (50.0 g, 128.4 mmol) was dissolved in 300 mL of anhydrous 1,2-dichloroethane. Trimethylsilyl trifluoromethanesulfonate (TMSOTf, 34.3 g, 154.3 mmol) was added under nitrogen protection in an ice-water bath, and the reaction was carried out overnight at room temperature. 500 mL of saturated sodium bicarbonate aqueous solution was added to the reaction mixture, and the mixture was stirred until homogeneous. The organic phase was separated, and the aqueous phase was extracted three times with 200 mL of dichloromethane. The combined organic phases were washed once each with 200 mL of saturated sodium bicarbonate solution and 200 mL of saturated sodium chloride solution. The organic phase was then separated, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 38.3 g of a pale yellow, viscous, syrupy crude product. This product was directly added to the next reaction without purification.
[0053] 2) Synthesis of compound 3
[0054]
[0055] Compound 2 (38.0 g, 115.4 mmol) was dissolved in 200 mL of anhydrous 1,2-dichloroethane, and 5-hexen-1-ol (12.3 g, 122.8 mmol) was added. The mixture was stirred at room temperature for 30 min, and then trimethylsilyl trifluoromethanesulfonate (TMSOTf, 12.3 g, 55.4 mmol) was added under nitrogen protection in an ice bath. The mixture was stirred overnight at room temperature. The reaction solution was diluted with 200 mL of dichloromethane, and then washed with 400 mL of saturated sodium bicarbonate aqueous solution after stirring for 10 min. The organic phase was separated. The aqueous phase was extracted with 200 mL of dichloromethane, and the organic phase was washed once with 200 mL of saturated sodium bicarbonate aqueous solution and once with 200 mL of saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 39.6 g of a yellow syrupy product. MS m / z [M+H] + (ESI):430.05.
[0056] 3) Synthesis of compound 4
[0057]
[0058] Compound 3 (39.5 g, 92.0 mmol) was dissolved in a mixed solvent of 200 mL dichloromethane and 200 mL acetonitrile. 270 mL of deionized water and sodium periodate (59.0 g, 275.8 mmol) were added separately. The mixture was stirred in an ice-water bath for 10 min, and then ruthenium trichloride (600 mg, 2.9 mmol) was added. The reaction mixture was allowed to react overnight at room temperature. 800 mL of deionized water was added to the reaction solution and stirred. The pH was adjusted to approximately 7.5 with saturated sodium bicarbonate solution. The phases were separated. The aqueous phase was extracted three times with 500 mL dichloromethane, and the organic phase was discarded. The pH of the aqueous phase was adjusted to approximately 3.0 with citric acid solid, and then extracted three times with 500 mL dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 9.2 g of the product. MS m / z [M+H] + (ESI):448.57.
[0059] 4) Synthesis of Compound 5
[0060]
[0061] Compound 4 (3.2 g, 7.1 mmol) was dissolved in 32 mL of dichloromethane, and diisopropylethylamine (2.8 g, 21.7 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 3.3 g, 8.7 mmol) were added. The mixture was stirred overnight at room temperature. The reaction solution was washed with 100 mL of saturated sodium bicarbonate solution after stirring for 10 min. The organic phase was separated, and the aqueous phase was extracted with 100 mL of dichloromethane. The combined organic phases were washed with 100 mL of saturated sodium bicarbonate solution and 100 mL of saturated sodium chloride solution, respectively. The phases were separated, and the organic phase was dried over anhydrous sodium sulfate. The product was separated by silica gel column chromatography using eluent (dichloromethane:methanol = 2%–20%, V:V). The solvent was evaporated to dryness to give 3.0 g of a yellow syrupy product. MS m / z [M+H] + (ESI):604.48.
[0062] 5) Synthesis of Compound 6
[0063]
[0064] Compound 5 (40.0 g, 66.3 mmol) was dissolved in 160 mL of dichloromethane, and 80 mL of dioxane hydrochloride solution (4 M) was added in portions. The mixture was stirred at room temperature for 4 h. The solvent was evaporated under reduced pressure to obtain 42.0 g of a foamy, pale orange solid.
[0065] 6) Synthesis of Compound 8
[0066]
[0067] Compound 7 (2.0 g, 16.6 mmol) was dissolved in 50 mL of dichloromethane, and 4,4'-dimethoxytriphenylchloromethane (DMTrCl, 6.8 g, 20.1 mmol) and triethylamine (4.0 g, 39.5 mmol) were added. The mixture was stirred overnight at room temperature. The reaction solution was washed twice with 50 mL of saturated sodium chloride water, and the phases were separated. The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure to give 9.0 g of a dark green viscous crude product. MS m / z [M+H] + (ESI):423.23.
[0068] 7) Synthesis of Compound 9
[0069]
[0070] Compound 8 (3.0 g, 7.1 mmol) was dissolved in 30 mL of tetrahydrofuran, and an aqueous solution of lithium hydroxide monohydrate (LiOH·H2O) (0.9 g dissolved in 9 mL of water) was added. The mixture was stirred overnight at room temperature. The reaction solution was allowed to stand and the phases were separated. The organic phase was washed twice with 30 mL of saturated sodium chloride, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The crude product was washed with 80 mL of methyl tert-butyl ether, filtered, and the filter cake was dried to give 1.5 g of a pale yellow solid powder.
[0071] 8) Synthesis of Compound 10
[0072]
[0073] Compound 9 (1.0 g, 2.4 mmol) was dissolved in 10 mL of dichloromethane, and tert-butyl 4-aminobutyrate hydrochloride (0.47 g, 2.4 mmol), Carter's condensing agent (BOP, 1.28 g, 2.9 mmol), and triethylamine (0.61 g, 6.0 mmol) were added. The mixture was stirred overnight at room temperature. The reaction solution was washed three times with 10 mL of saturated sodium bicarbonate solution and once with 10 mL of saturated sodium chloride solution. The phases were separated, and the organic phase was dried over anhydrous sodium sulfate and evaporated to dryness to give 1.3 g of a pale orange-yellow semi-solid. MS m / z [M+H] + (ESI):550.38.
[0074] 9) Synthesis of Compound 11
[0075]
[0076] Compound 10 (1.2 g, 2.2 mmol) was dissolved in 18 mL of tetrahydrofuran, 6 mL of methanol was added, and an aqueous solution of lithium hydroxide monohydrate (LiOH·H₂O) (0.83 g dissolved in 12 mL of water) was added. The mixture was stirred overnight at room temperature. The reaction solution was evaporated to dryness under reduced pressure, and 100 mL of saturated sodium chloride and 50 mL of tetrahydrofuran were added. The phases were separated. The aqueous phase was extracted twice again with 50 mL of tetrahydrofuran. The organic phases were combined, washed once with 50 mL of saturated sodium chloride, and separated. The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness to give 1.1 g of a transparent viscous substance.
[0077] 10) Synthesis of Compound 13
[0078]
[0079] Compound 12 (10.0 g, 23.5 mmol) was dissolved in 100 mL of dichloromethane, and L-glutamic acid di-tert-butyl hydrochloride (7.0 g, 23.5 mmol), diisopropylethylamine (10.6 g, 81.8 mmol), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 9.8 g, 25.8 mmol) were added. The mixture was stirred overnight at room temperature. The reaction solution was washed three times with 100 mL of saturated sodium bicarbonate solution and once with 100 mL of saturated sodium chloride solution. The phases were separated, and the organic phase was dried over anhydrous sodium sulfate and evaporated to dryness to give 21 g of a viscous substance. MS m / z [M+H] + (ESI):667.79.
[0080] 11) Synthesis of compound 14
[0081]
[0082] Compound 13 (19.0 g, 28.5 mmol) was dissolved in 500 mL of dichloromethane, and 95 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature for 4 h. The reaction solution was evaporated to dryness under reduced pressure, and then slurried twice with 200 mL of dichloromethane. The mixture was filtered, and the filter cake was dried at 45 °C to give 11.7 g of a light purple solid. MS m / z [M+H] + (ESI):499.56.
[0083] 12) Synthesis of Compound 15
[0084]
[0085] Compound 14 (3.5 g, 7.0 mmol) was dissolved in 120 mL of N,N-dimethylformamide, and compound 6 (12.5 g, 23.2 mmol), diisopropylethylamine (9.5 g, 73.7 mmol), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 9.6 g, 25.3 mmol) were added. The mixture was stirred overnight at room temperature. The reaction solution was extracted twice with 800 mL of saturated sodium chloride and then 200 mL of dichloromethane. The organic phases were combined. The organic phase was washed once with 200 mL of saturated sodium chloride, separated, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. Crystallization was precipitated by adding 50 mL of dichloromethane and 50 mL of methyl tert-butyl ether, filtered, and the filter cake was reconstituted with methanol and evaporated to dryness under reduced pressure to give 15.1 g of a yellowish-white solid. MS m / z [M+H] + (ESI):1955.17.
[0086] 13) Synthesis of Compound 16
[0087]
[0088] Compound 15 (10.0 g, 5.1 mmol) was dissolved in 100 mL of N,N-dimethylformamide and 40 mL of tetrahydrofuran. 100 mL of diisopropylethylamine was added, and the mixture was stirred overnight in an oil bath at 55 °C. The reaction solution was evaporated to dryness under reduced pressure to obtain an oily substance. Ethyl acetate was stirred and refluxed, and after standing, the supernatant was poured off and evaporated to dryness under reduced pressure to give 4.0 g of a yellowish-brown foamy solid. MS m / z [M+H] + (ESI):1732.92.
[0089] 14) Synthesis of Compound 17
[0090]
[0091] Compound 16 (2.0 g, 1.15 mmol) was dissolved in 20 mL of dichloromethane. Compound 11 (0.58 g, 1.15 mmol), diisopropylethylamine (0.37 g, 2.89 mmol), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 0.48 g, 1.27 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction mixture was extracted with 200 mL of NaCl solution and 200 mL of dichloromethane. The phases were separated, and the organic phase was evaporated to dryness under reduced pressure. 200 mL of ethyl acetate was added, and the mixture was stirred to induce crystallization. The crystals were filtered, and the filter cake was reconstituted with methanol and evaporated to dryness under reduced pressure to give 1.3 g of a brown, foamy solid. MS m / z [M+H] + (ESI):2209.31.
[0092] 15) Synthesis of H03
[0093]
[0094] Compound 17 (1.2 g, 0.54 mmol) was dissolved in 12 mL of dichloromethane, and succinic anhydride (544 mg, 5.43 mmol), triethylamine (825 mg, 8.15 mmol), and 4-dimethylaminopyridine (DMAP, 664 mg, 5.43 mmol) were added. The mixture was stirred overnight at room temperature. The reaction solution was diluted with 200 mL of dichloromethane, washed once with 200 mL of NaCl solution, washed twice with 200 mL of saturated ammonium chloride aqueous solution, and washed twice with 200 mL of NaCl solution. The phases were separated, and the organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain 840 mg of crude yellow-brown solid. Further purification was performed using a preparative liquid chromatography system (HPLC) on a Gilson GX281 column (Waters X-bridge C18, 19 x 250 mm, 10 μm). Mobile phase A: ammonium bicarbonate aqueous solution, pH 8-9; mobile phase B: acetonitrile. The flow rate was 20 ml / min, the monitoring wavelength was 214 nm, gradient elution was performed, and the product was freeze-dried to obtain a pale yellow solid powder.
[0095] The mass spectrum of compound H03 is as follows: Figure 1 As shown, the molecular weight of compound H03, determined by high-resolution mass spectrometry, is HRMS m / z [M+Na]. + (ESI): 2331.00, consistent with the theoretical molecular weight. (By...) Figure 2 H03 compound H 1 The NMR spectrum showed that the major H atom peaks of the target product were detected and their structure was consistent with that of the target product. In conclusion, this confirms the successful synthesis of the compound.
[0096] Example 2: Synthesis of liver-targeting compound H05
[0097] 1) Synthesis of compound 20
[0098]
[0099] Compound 19 (100 g, 651 mmol) was dissolved in 1.3 L of acetonitrile, followed by the addition of triethylamine (161.4 g, 1595 mmol) at room temperature. Ethyl trifluoroacetate was then added in a water bath at 25 °C, and the reaction was carried out at room temperature for 24 h. The reaction mixture was filtered, the filtrate was concentrated, washed with 1 L of ethyl acetate, filtered to remove insoluble matter, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to constant weight, yielding 157 g of a brown oily crude product. This product was used directly in subsequent reactions without further purification.
[0100] 2) Synthesis of compound 21
[0101]
[0102] Compound 20 (651 mmol) was dissolved in 1.6 L of dichloromethane, and triethylamine (98.8 g, 976 mmol) was added, followed by purging with nitrogen. DMTTrCl (4.269 g, 12.6 mmol) was added to the reaction mixture under nitrogen protection, and the reaction was carried out at room temperature for 4 h. 1.5 L of saturated sodium chloride was added to the reaction mixture, and the mixture was stirred to separate the layers. The aqueous phase was extracted twice with 600 mL of dichloromethane. The organic phases were combined and concentrated under reduced pressure to constant weight, yielding 384 g of a brown oily crude product. This product was used directly in subsequent reactions without further purification.
[0103] 3) Synthesis of compound 22
[0104]
[0105] Compound 21 (651 mmol) was dissolved in 1.8 L of methanol. Then, an aqueous solution of potassium hydroxide (73.06 g, 1302 mmol dissolved in 430 mL of deionized water) was added to the reaction mixture, and the mixture was reacted at room temperature for 70 min. The reaction solution was concentrated to remove the solvent, and the mixture was washed twice with 400 mL of deionized water. The viscous solid was dissolved in ethyl acetate and washed with saturated sodium chloride. The solution was evaporated to dryness to give approximately 300 g of a syrupy substance.
[0106] 4) Synthesis of compound 23
[0107]
[0108] Monomethyl dodecanoate (7.4 g, 30.1 mmol) was placed in a 500 mL three-necked flask, compound 22 (12.7 g, 30.1 mmol) was added, followed by BOP (16.0 g, 36.2 mmol), and then 74 mL of dichloromethane was added and stirred until dissolved. The reaction flask was placed in a water bath at room temperature, and triethylamine (7.6 g, 75.4 mmol) was slowly added with stirring. The mixture was stirred overnight at room temperature. The reaction solution was washed three times with 80 mL of saturated sodium bicarbonate solution, once with 100 mL of deionized water, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to obtain 29.3 g of an orange-yellow oil.
[0109] 5) Synthesis of compound 24
[0110]
[0111] Compound 23 (29.3 g, 45.4 mmol) was placed in a 1 L single-necked flask and dissolved in 300 mL of tetrahydrofuran. While stirring, an aqueous solution of lithium hydroxide monohydrate (LiOH·H₂O) (17.2 g, 408.3 mmol dissolved in 170 mL of deionized water) was added, and the mixture was stirred overnight at room temperature. The reaction solution was allowed to stand and the phases separated. The upper organic phase was washed twice with 170 mL of saturated sodium chloride, dried, and evaporated to dryness under reduced pressure. The solution was dissolved in 260 mL of dichloromethane, and 520 mL of methyl tert-butyl ether was added dropwise. The precipitated solid was filtered, dried, and yielded 18.8 g of a white powder.
[0112] 6) Synthesis of Compound 25
[0113]
[0114] Compound 16 (2.0 g, 1.15 mmol) was dissolved in 20 mL of N,N-dimethylformamide, and compound 24 (729 mg, 1.15 mmol), diisopropylethylamine (0.37 g, 2.89 mmol), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 0.48 g, 1.27 mmol) were added. The mixture was stirred overnight at room temperature. The reaction solution was extracted three times with 300 mL of saturated sodium chloride and 200 mL of dichloromethane. The organic phases were combined, washed once with 200 mL of saturated sodium chloride, separated, dried, and evaporated to dryness under reduced pressure. The mixture was washed with 200 mL of ethyl acetate, filtered, and the filter cake was reconstituted with methanol and evaporated to dryness under reduced pressure to give 2.1 g of a brown solid.
[0115] 7) Synthesis of H05
[0116]
[0117] Compound 25 (2.0 g, 0.85 mmol) was dissolved in 40 mL of dichloromethane, and succinic anhydride (853 mg, 8.52 mmol), triethylamine (1.3 g, 12.78 mmol), and 4-dimethylaminopyridine (1.0 g, 8.52 mmol) were added. The mixture was stirred overnight at room temperature.
[0118] The reaction solution was diluted with 200 mL of dichloromethane, and 20 mL of methanol was added to increase the product solubility. The product was washed once with 200 mL of sodium chloride, twice with 200 mL of saturated ammonium chloride aqueous solution, and twice with 200 mL of sodium chloride. The phases were separated, the organic phase was dried, and then evaporated to dryness. The crude product was recrystallized from a mixed solution of methanol:dichloromethane:ethyl acetate (100:10:200, V:V:V) and filtered. The crude product was further purified by preparative liquid chromatography (HPLC) using a Gilson GX281 chromatogram and a Waters X-bridge C18 column (19*250 mm, 10 μm). Mobile phase A was ammonium bicarbonate aqueous solution, pH 8-9; mobile phase B was acetonitrile. The flow rate was 20 mL / min, the monitoring wavelength was 214 nm, gradient elution was used, and the product was lyophilized to obtain a pale yellow solid powder.
[0119] The mass spectrum of compound H05 is as follows: Figure 3 As shown, the molecular weight of compound H05, determined by high-resolution mass spectrometry, is HRMS m / z [M+Na]. + (ESI): 2469.10, consistent with the theoretical molecular weight. (From...) Figure 4 H05 compound H 1 The NMR spectrum showed that the major H atom peaks of the target product were detected and their structure was consistent with that of the target product. In conclusion, this confirms the successful synthesis of the compound.
[0120] Example 3: Preparation and Activity Assay of siRNA Conjugates
[0121] Synthesize siRNA targeting the mouse TTR gene, with a galactose molecule cluster (Formula I-x, x = 1 to 10 compounds) linked to the 3' end of the SS strand.
[0122] SS chain (5'-3'): asasc agu GuU CUu gcu cua uaa (SEQ ID No.1)
[0123] AS chain (5'-3'): usUsa uaG agc aag aAc Acu guus usu (SEQ ID No.2)
[0124] Compounds H03 and H05 were dissolved in appropriate amounts of DMF, followed by the addition of two equivalents of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIEA, 1), and then an amino-modified solid support (CPG-NH2). The reaction was carried out at 25°C with shaking for 24 h. After the reaction was completed, the mixture was washed successively with acetonitrile and dichloromethane. Then, 20% acetic anhydride / 80% acetonitrile was added, and the reaction was carried out at 25°C with shaking for 24 h. After the reaction was completed, the mixture was washed successively with acetonitrile and dichloromethane to obtain the target product H03-CPG or H05-CPG on the solid support.
[0125] The nucleoside monomers required for siRNA synthesis, such as 2'-O-methyl nucleoside phosphoramidite monomers, were purchased from Shanghai Zhaowei Technology Development Co., Ltd. 3% dichloroacetic acid was used as a deprotecting agent, 0.25M 5-ethylthio-1H-tetrazole acetonitrile solution as an activating agent, N,N-dimethyl-N'-(3-thio-3H-1,2,4-dithioazol-5-yl)formamidinium pyridine solution as a sulfiding agent, 0.05M iodine / pyridine / aqueous solution as an oxidizing agent, 20% acetic anhydride acetonitrile solution as capping agent A, and 20% acetonitrile / N-methylimidazolium / pyridine solution as capping agent B. All the above-mentioned synthetic reagents were purchased from Suzhou Kelema Biotechnology Co., Ltd. Each RNA single strand was synthesized using phosphoramidite solid-phase synthesis, starting with HO3-CPG or HO5-CPG, and the nucleoside phosphoramidite monomers were ligated using a DNA synthesizer according to the synthesis program. The linkage of each nucleoside monomer involves four steps: deprotection, coupling, oxidation or sulfidation, and capping.
[0126] After solid-phase synthesis, the oligonucleotides were ammonolyzed with 25%-28% ammonia at 62℃ for 6 h. The supernatant was concentrated and evaporated to dryness, then purified using a Resource 15Q column with gradient elution using sodium bromide solution and removal of DMTr using 3% trifluoroacetic acid solution to obtain oligonucleotide chains. The eluent was collected and desalted using a dextran G25 gel column. The obtained oligonucleotide chains were collected, lyophilized, and purity was determined by ion-pair chromatography. The molecular weight of the target product was analyzed by high-resolution mass spectrometry. The obtained single-stranded oligonucleotides were quantified by UV spectrophotometry, equimolarly complementary pairing was performed, dissolved in water, and double-stranded siRNA was formed by conventional annealing method, and adjusted to the required concentration for later use. Table 1 shows the structures of the SS and AS chains of TTR-H03 and TTR-H05.
[0127] Table 1. Structures of the SS and AS chains in TTR-H03 and TTR-H05
[0128]
[0129] Note: The lowercase letter f indicates that the nucleotide to the left of the letter f is a 2'-fluorine modified nucleotide; the lowercase letters a, g, c, and u are 2'-OMe modified nucleotides; and the lowercase letter s indicates that the two nucleotides to the left and right of the letter s are connected by a phosphothioester bond.
[0130] Inhibition of TTR mRNA expression in mice
[0131] Eight-week-old C57BL / 6 mice (Jinan Pengyue Experimental Animal Breeding Co., Ltd., SPF, female) were randomly divided into groups. On day 0, 0.9 mg / kg of each siRNA conjugate was administered to the skin of the neck and shoulder of the mice, while physiological saline was administered as a control group. Three mice from each group were euthanized at days 0, 7, 14, 21, and 28, and liver tissue samples were collected. The mRNA expression level of TTR in mouse liver tissue was detected using real-time fluorescence PCR. Total RNA was extracted from the liver using the RNAeasy Mini kit (Qiagen, catalog number 74104), and cDNA was obtained by reverse transcription using High Capacity cDNA Reverse Transcription Kits (Thermo Fisher, catalog number 4368814) for RT-PCR. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control gene, and the mRNA expression levels of mouse TTR and GAPDH were detected using the RT-PCR method shown in Table 1 and the Taqman probe primers for mouse TTR and GAPDH (Table 2). The expression level of mouse TTR mRNA was calculated using the 2^-ΔΔCt method, and the inhibitory effects of siRNA conjugates TTR-H03 and TTR-H05 on TTR mRNA were calculated.
[0132] Table 2 Real-time Quantitative PCR Conditions
[0133]
[0134] Table 3 Detection primer sequences
[0135]
[0136] Figure 5 To illustrate the inhibitory levels of liver-targeting conjugates TTR-H03 and TTR-H05 on hepatic TTR mRNA, TTR mRNA inhibition curves were plotted after normalization with a control group. Figure 5 As shown, both siRNA conjugates TTR-H03 and TTR-H05 have the ability to target oligonucleotides to the liver. Both compounds can effectively reduce the level of liver TTR mRNA and can control the TTR mRNA expression level below 40% for up to 35 days. Among them, compound H05 has better delivery efficiency than compound H03.
[0137] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A liver-targeting compound, characterized in that, The structure of the target compound is shown in formula H03 or formula H05: H03； H05。 2. A liver-targeting conjugate, characterized in that, It is formed by linking a functional group to the target compound as described in claim 1; the functional group includes small molecule drugs, peptides, nucleic acids or proteins.
3. The use of a targeting compound as described in claim 1 or a targeting conjugate as described in claim 2 in the preparation of a medicament for the diagnosis, prevention, treatment or relief of liver-related diseases and symptoms.