NOVEL COMPOUND AND APPLICATION THEREOF
20230110095 · 2023-04-13
Inventors
Cpc classification
A61K47/55
HUMAN NECESSITIES
A61K47/549
HUMAN NECESSITIES
A61P1/16
HUMAN NECESSITIES
International classification
C12N15/113
CHEMISTRY; METALLURGY
A61K47/55
HUMAN NECESSITIES
Abstract
The present invention relates to a novel compound and application thereof in the inhibition of HBV gene expression. The structure of the compound comprises an interfering nucleic acid for inhibiting HBV gene expression, transition points, and delivery chains of the interfering nucleic acid. By means of the delivery chains, two or three N-acetylgalactosamines can be introduced to an antisense strand 3′ end of such siRNA, and two or one N-acetylgalactosamine can be correspondingly introduced to a sense strand 5′ end, the total number of the introduced N-acetylgalactosamines being four. In vitro and in vivo pharmacological experiments prove that such a novel compound can continuously and efficiently inhibit HBV gene expression.
Claims
1. A compound comprising: an interfering nucleic acid having a sense strand and an antisense strand, transition points, and delivery chains of the interfering nucleic acid, wherein said delivery chains each independently comprises a linking chain D, a linker B, a branched chain L and a liver targeting specific ligand X, and wherein the compound has a structure of formula (I): ##STR00183## wherein: (1) n is an integer of 1 and m is an integer of 3; or n is an integer of 2 and m is an integer of 2; (2) R.sub.1 is —NH(CH.sub.2).sub.xCH.sub.2-, wherein x is an integer of 3-10; and R.sub.2 is —NHIICH.sub.2CH(OH)—CH-.sub.2(OH)—, or a pyrrole or piperidine ring with a primary and secondary alcohol moiety; (3) each X is independently selected from the residues of galactose, galactosamine and N-acetylgalactosamine; (4) each L is independently a C3-C18 linear chain comprising carbonyl, amido, phosphoryl, oxygen atom or a combination thereof; (5) each linker B is independently selected from the following formulae: ##STR00184## wherein, each A.sub.1 is independently C, O, S or NH; each r1 is independently an integer of 1-15, each r2 is independently an integer of 0-5; and each A.sub.2 is independently C, O, S, amino, carbonyl, arnido, phosphoryl or thiophosphoryl; and (6) D is a linking chain comprising 5 to 20 carbon atoms, which may comprise amino, carbonyl, amido, an oxygen atom, a sulfur atom, thiophosphoryl, phosphoryl, a cyclic structure, or a combination thereof.
2. The compound of claim 1, wherein R.sub.1 is —NH(CH.sub.2)5CH.sub.2-.
3. The compound of claim 1, wherein R.sub.2 is ##STR00185##
4. The compound of claim 1, wherein X is a residue of N-acetylgalactosamine.
5. The compound of claim 1, wherein X has the structure of ##STR00186## and R.sub.1 is NHCOCH.sub.3.
6. The compound of claim 1, wherein L is selected from the following structures: ##STR00187## wherein each r1 is independently an integer of 1-12, each r2 is independently an integer of 0-20, and Z is H or CH.sub.3.
7. The compound of claim 1, wherein the delivery chain at the 5′ end of the sense strand is selected from the following structures: ##STR00188## ##STR00189## ##STR00190## ##STR00191##
8. The compound of claim 1, wherein the delivery chain at the 3′ end of the antisense strand is selected from the following structures: ##STR00192## ##STR00193## ##STR00194## ##STR00195##
9. The compound of claim 1, wherein n is an integer of 1 and m is an integer of 3.
10. The compound of claim 1, wherein n is an integer of 2 and m is an integer of 2.
11. The compound of claim 1, wherein the combination of the delivery chain at the 5′ end of the sense strand and the delivery chain at the 3′ end of the antisense strand is selected from the following: TABLE-US-00019 GBL-01
12. The compound of claim 11, wherein the combination of the delivery chain at the 5′ end of the sense strand and the delivery chain at the 3′ end of the antisense strand is the combination designated as GBL-O1.
13. The compound of claim 11, wherein the combination of the delivery chain at the 5′ end of the sense strand and the delivery chain at the 3′ end of the antisense strand is the combination designated as GBL-02.
14. The compound of claim 11, wherein the combination of the delivery chain at the 5′ end of the sense strand and the delivery chain at the 3′ end of the antisense strand is the combination designated as GBL-03.
15. The compound of claim 11, wherein the combination of the delivery chain at the 5′ end of the sense strand and the delivery chain at the 3′ end of the antisense strand is the combination designated as GBL-04.
16. The compound of claim 11, wherein the combination of the delivery chain at the 5′ end of the sense strand and the delivery chain at the 3′ end of the antisense strand is the combination designated as GBL-05.
17. The compound of claim 11, wherein the combination of the delivery chain at the 5′ end of the sense strand and the delivery chain at the 3′ end of the antisense strand is the combination designated as GBL-06.
18. The compound of claim 11, wherein the combination of the delivery chain at the 5′ end of the sense strand and the delivery chain at the 3′ end of the antisense strand is the combination designated as GBL-07.
19. The compound of claim 11, wherein the combination of the delivery chain at the 5′ end of the sense strand and the delivery chain at the 3′ end of the antisense strand is the combination designated as GBL-08.
20. The compound of claim 11, wherein the combination of the delivery chain at the 5′ end of the sense strand and the delivery chain at the 3′ end of the antisense strand is the combination designated as GBL-09.
21. The compound of claim 11, wherein the combination of the delivery chain at the 5′ end of the sense strand and the delivery chain at the 3′ end of the antisense strand is the combination designated as GBL-10.
22. The compound of claim 11, wherein the combination of the delivery chain at the 5′ end of the sense strand and the delivery chain at the 3′ end of the antisense strand is the combination designated as GBL-11.
23. The compound of claim 11, wherein the combination of the delivery chain at the 5′ end of the sense strand and the delivery chain at the 3′ end of the antisense strand is the combination designated as GBL-12.
24. The compound of claim 11, wherein the combination of the delivery chain at the 5′ end of the sense strand and the delivery chain at the 3′ end of the antisense strand is the combination designated as GBL-13.
25. The compound of claim 11, wherein the combination of the delivery chain at the 5′ end of the sense strand and the delivery chain at the 3′ end of the antisense strand is the combination designated as GBL-14.
26. The compound of claim 11, wherein the combination of the delivery chain at the 5′ end of the sense strand and the delivery chain at the 3′ end of the antisense strand is the combination designated as GBL-15.
27. The compound of claim 11, wherein the combination of the delivery chain at the 5′ end of the sense strand and the delivery chain at the 3′ end of the antisense strand is the combination designated as GBL-16.
28. The compound of claim 1, wherein said interfering nucleic acid is siRNA, miRNA or Agomir.
29. The compound of claim 1, wherein said interfering nucleic acid is siRNA.
30. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable auxiliary material.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] To make the objectives, technical solutions and beneficial effects of the present invention more clear, a brief description of the attached drawings is provided as below:
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DETAILED DESCRIPTION
[0076] The following examples illustrate some embodiments disclosed in the present invention, but the present invention is not limited thereto. In addition, when providing specific embodiments, the inventors anticipated application of some specific embodiments, for example, compounds with specifically same or similar chemical structures for treatment of different liver-derived diseases.
[0077] Explanations:
[0078] DMF refers to N,N-dimethylformamide;
[0079] HBTU refers to O-benzotriazole-tetramethylurea hexafluorophosphate;
[0080] DIPEA (DIEA) refers to N,N-diisopropylethylamine;
[0081] DCM refers to dichloromethane;
[0082] DMAP refers to 4-dimethylaminopyridine;
[0083] DMT-CL refers to 4,4′-dimethoxytriphenylchloromethane;
[0084] THF refers to tetrahydrofuran;
[0085] TBTU refers to O-benzotriazol-N,N,N′,N-tetramethylurea tetrafluoroborate;
[0086] DBU refers to 1,8-diazabicycloundec-7-ene;
[0087] HOBt refers to 1-hydroxybenzotri zole;
[0088] DCC refers to dicyclohexylcarbodiimide;
[0089] Pd—C refers to palladium-carbon catalyst;
[0090] .circle-solid. refers to a solid phase carrier, such as a resin.
[0091] Example 1. Synthesis of GBL-0401
[0092] 1. Synthesis of Kys-01 [0093] 1.1. Compounds of 5′YICd-01: Synthesis of 5′YICd-01-PFP [0094] 1.1.1. Synthesis of 5′YICd-01-c1
##STR00120##
[0095] Into 2-hydroxyethyarnine (5.0 g, 81.9 mmol), were added 50 mL of dimethyl sulfoxide and 5 mL of a sodium hydroxide solution at a concentration of 1 g/mL, followed by dropwise addition of 12 mL of tert-butyl acrylate (81.9 mmol) within 1 hour. The mixture was reacted at room temperature for 24 h, and then 100 mL of petroleum ether was added, and the mixture was washed with saturated brine twice. The organic layer was dried and passed over a column to get 7.5 g of colorless oil. [0096] 1.1.2. Synthesis of 5′YICd-01-c2
##STR00121##
[0097] Into 5′YICd-01-c1 (7.5 g, 39.7 mmol), were added 50 mL of DCM and 23 mL of a sodium carbonate solution (25%), followed by dropwise addition of benzyl chloroformate (7.7 g, 45.0 mmol) at room temperature. The mixture was reacted at room temperature overnight, washed with saturated brine twice, dried over anhydrous sodium sulfate, and evaporated off the solvent. The residue was passed over a chromatographic column (ethyl acetate:petroleum ether=15%-30%) to get 11.3 g of an oil. [0098] 1.1.3 Synthesis of 5′YICd-01-c3
##STR00122##
[0099] 5′YICd-01-c2 (11.3 g, 35.0 mmol) was added with 20 mL of formic acid, and reacted at room temperature overnight. The solvent was evaporated off at reduced pressure to get 9.2 g of 5′YICd-01-c3. [0100] 1.1.4. Synthesis of 5′YICd-01-c4
##STR00123##
[0101] 1.0 g (3.73 mmol) of 5′YICd-01-c3 and 2.0 g (4.48 mmol) of dlSANC-c4 were added into 30 mL of DMF, then added with 0.38 g of HOBt and 2.30 g of HBTU, followed by slow addition of 1.0 mL of DIEA. The mixture was added with 20 mL of water and extracted with 40 mL of DCM. The organic phase was washed with 100 mL of saturated brine, dried over anhydrous sodium sulfate, and evaporated at reduced pressure to dryness. The residue was purified by chromatography on a silica gel column (Eluent: 1-15% methanol in DCM) to get 2.2 g of a white foamy solid, of which the high-resolution mass spectrum is shown in
##STR00124##
[0103] 2.2 g (3.2 mmol) of 5′YICd-01-c4 was dissolved in 30 mL of methanol, added with 1.0 g of 10% Pd-C(wet Degussa-type E101 NE/W), and hydrogenated at normal pressure overnight. The reaction mixture was filtered with diatomite, and the filtrate was evaporated at reduced pressure to dryness to get 1.70 g of white foam. [0104] 1.1.6. Synthesis of 5′YICd-01-c6
##STR00125##
[0105] 0.80 g (3.60 mmol) of monobenzyl glutarate was weighed and dissolved in 2 mL DMF, added with 1.28 g of TBTU and 2.0 mL of DIEA, reacted with stirring for 5 minutes, and then added with 1.70 g (3.0 mmol) of 5′YICd-01-c5, and reacted at room temperature with stirring overnight. The reaction solution was evaporated at reduced pressure, added with 50 mL of DCM and 50 mL of water and stirred for 5 minutes. The layers were separated, and the organic layer was dried over anhydrous sodium sulfate, passed over a chromatographic column (Eluent: DCM:methanol=1%-10%), and the solvent was evaporated at reduced pressure to get 2.1 g of a white product. [0106] 1.1.7. Synthesis of 5′YICd-01-c7
##STR00126##
[0107] Into a 100 mL single-necked flask, were added 2.1 g (2.7 mmol) of 5′YICd-01-c6 and 0.2 g of palladium-carbon. The flask was evacuated by a water pump and supplemented with hydrogen in triplicate. The reaction was conducted under pressurized hydrogen overnight. On the next day, TLC showed that the reaction was completed. Palladium-carbon was filtered with diatomite, and the filtrate was evaporated at reduced pressure to get 1.8 g of a product. [0108] 1.1.8. Synthesis of 5′YICd-01-PFP
##STR00127##
[0109] Into a 100 mL single-necked flask, were added 1.8 g (2.66 mmol) of 5′YICd-0 1-c7 and 20 mL of DCM. 1.1 g (4.0 mmol) of pentafluorophenyl trifluoromethanesulfonate was dropwise added, and reacted at room temperature for 1 hour. The reaction mixture was washed with 40 mL of water and 10 mL of saturated sodium bisulfite. The organic layer was dried over anhydrous sodium sulfate and evaporated at reduced pressure to dryness to get 2.3 g of a product. [0110] 1.2. Solid-phase synthesis of C6NH—S-O1With mG as the initiation monomer and with C6NH phosphoramidite monomer as the end monomer, different phosphoramidite monomers were introduced by coupling through a solid-phase phosphoramidite method. The solid-phase phosphoramidite method includes the following basic steps: 1) deprotection: removing the protective group (DMT) on the oxygen atom of the solid phase carrier; 2) coupling: adding a first nucleotide monomer, coupling in the direction of 3′ to 5′; 3) oxidation: oxidizing the resulting nucleoside phosphite into a more stable nucleoside phosphate (that is, oxidization of trivalent phosphorus to pentavalent phosphorus); 4) blocking: blocking 5′-OH of the nucleotide monomer unreacted in the previous step by capping to prevent it from reacting further; the above steps were repeated until the desired sequence was achieved. After being synthesized, the ester bond for linking the compound to the initial nucleoside on the solid phase carrier was cleaved with methylamine ethanol solution and aqueous ammonia, and protective groups on various bases and phosphoric acid on the oligonucleotide, including cyanoethyl (P), benzoyl (mA, fA), acetyl (mC, fC), isobutyryl (mG, fG) and 4-methoxy triphenylmethyl (C6NH), were removed. The product was purified by HPLC, filtered and sterilized, and freeze-dried. [0111] 1.3. Liquid-phase synthesis of Kys-01 [0112] 1.3.1. Synthesis of Kys-01-c1
##STR00128##
[0113] The purified and freeze-dried C6NH-S-01 (12.5 mg) was weighed and completely dissolved in a sodium borate buffer (650 μL, 0.06 mol/L). 5′YICd-01-PFP (10.3 mg) was weighed and dissolved in dimethyl sulfoxide (100 μL), and added into C6NH-S-01 and mixed uniformly, followed by addition of N-methylmorpholine (5 μL). The reaction mixture was ultrasonicated at room temperature for 3 h, and purified over a C18 column after HPLC detection showed the completion of the reaction. [0114] 1.3.2. Synthesis of Kys-01
##STR00129##
[0115] The purified Kys-01-c1 (32 mL, 5 mg) was taken into 25% hydrazine hydrate (16 mL), mixed uniformly, ultrasonicated at room temperature for 10 min, and purified through a C18 column after HPLC detection showed the completion of the reaction. The product was then freeze-dried to get Kys-01 (2 mg) as a white freeze-dried powder.
[0116] 2. Synthesis of Kyas-01 [0117] 2.1. Compounds of 3′SANCd-01: Synthesis of 3′SANCd-01 resin [0118] 2.1.1. Synthesis of 3′SANCd-01-c1
##STR00130##
3-amino-propanediol (9.114 g, 0.100 mol) was weighed and dissolved in THF (50 mL), cooled, dropwise added with ethyl trifluoroacetate (15.62 g, 0.110 mol), and reacted at room temperature for 1 h. The reaction solution was rotary evaporated to get crude 3′SANCd-01-c1 (18.871 g). [0119] 2.1.2. Synthesis of 3′SANCd-01-c2
##STR00131##
[0120] 3′SANCd-01-c1 (5.480 g, 0.030 mol) was dissolved in pyridine (30 mL) and cooled, added with DMT-CL (10.423 g, 0.031 mol) batchwise, reacted in dark overnight, and then rotary evaporated to remove pyridine. The residue was dissolved in CH.sub.2Cl2 (50 mL), and washed with saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and rotary evaporated. The residue was passed over a column to get the product 3′SANCd-01-c2 (10.805 g). [0121] 2.1.3. Synthesis of 3′SANCd-01-c3
##STR00132##
[0122] 3′SANCd-01-c2 (10.805 g, 0.022 mol) was dissolved in methanol (60 mL) and THF (30 mL), cooled, dropwise added with a solution of KOH (5.69 g) in water (24 mL), reacted at room temperature for 2 h, and rotary evaporated to remove methanol and THF. The residue was added with water (50 mL) and extracted with EtOAc (30 mL*3). The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and rotary evaporated. The residue was passed over a column with an eluent containing 1% triethylamine to get the product 3′SANCd-01-c3 (8.286 g). [0123] 2.1.4. Synthesis of 3′SANCd-01-c4
##STR00133##
[0124] 3′SANCd-01-c3(2.890 g,0.007 mol) was dissolved in CH.sub.2Cl2 (20 mL) and cooled, dropwise added with a solution of DCC (1.680 g) in CH.sub.2Cl2 (10 mL), stirred for 20 minutes, added with a solution of monomethyl suberate (1.522 g) in CH.sub.2Cl2 (10 mL), and reacted at room temperature overnight. The reaction was quenched with 5% NaHCO.sub.3(20 mL) and extracted with CH.sub.2Cl2 (20 mL*2). The organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and rotary evaporated. The residue was passed over a column with an eluent containing 1% triethylamine to get the product 3′SANCd-01-c4 (3.193 g). [0125] 2.1.5. Synthesis of 3′SANCd-01-c5
##STR00134##
[0126] 3′SANCd-01-c4 (2.193 g, 0.004 mol) was dissolved in THF (10 mL) and cooled, dropwise added with a solution of LiOH (0.645 g) in water (4.5 g) and reacted for 2 h. TLC indicated that there was no raw material. The reaction solution was rotary evaporated to remove the solvent. The residue was neutralized with saturated ammonium chloride, and extracted with CH.sub.2Cl2 (20 mL*2). The organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and rotary evaporated. The residue was passed over a column with an eluent containing 1% triethylamine to get the product 3′SANCd-01-c5 (1.979 g). [0127] 2.1.6. Synthesis of 3′SANCd-01-c6
##STR00135##
[0128] 3′SANCd-01-c5 (0.389 g, 0.004 mol) was dissolved in DMF (2 mL) and cooled, added with DIPEA (0.15 mL) and TBTU (0.183 g), stirred for 10 minutes, added with a solution of dlSANC-c12 (0.756 g, 0.0005 mol) in DMF (2 mL), and reacted at room temperature overnight. The reaction was quenched with water (20 mL) and extracted with CH.sub.2Cl2 (20 mL*2). The organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and rotary evaporated.
[0129] The residue was passed over a column with an eluent containing 5% triethylamine to get the product 3′SANCd-01-c6 (0.803 g), of which the high-resolution mass spectrum is shown in
##STR00136##
[0131] Into a reaction flask, 3′SANCd-01-c6 (2.15 g 0.001 mol) and 22 mL of DCM were added in order and dissolved with stirring at room temperature, and then added with DBU (0.156 g) and succinic anhydride (0.3 g, 0.003 mmol) in order, and reacted with stirring at room temperature. TLC analysis showed the reaction was completed. The reaction mixture was concentrated to remove DCM, and then added with water and extracted with DCM. The organic phase was further washed with saturated brine and dried over anhydrous sodium sulfate, filtered, and concentrated. Finally the residue was purified over a silica gel column to get 2.03 g of 3′SANCd-01-c7. [0132] 2.1.8. Synthesis of 3′SANCd-01 resin
##STR00137##
[0133] Into a reaction flask, 3′SANCd-01-c7 (1.13 g, 0.0005 mmol) and 12 mL of DMF were added in order and dissolved with stirring at room temperature, added with HBTU (0.11 g), DIPEA (0.104 g) and GE resin (1.80 g) in order, and shaken in a shaker at 35° C. for 24 h. The mixture was transferred into a synthesis tube and filtered. Under bubbled with nitrogen, the resin was rinsed with DMF for 4 times. Then CAP A +CAP B were added to conduct the end-capping reaction for half an hour under bubbling with nitrogen. A little amount of resin was taken for a kaiser test until the test solution appeared yellow. After completion of the end-capping, the filter cake was rinsed with methanol, DCM and methanol, respectively, and dried in vacuum to get 2.48 g of 3′SANCd-01 resin, of which the degree of substitution was 150 μmol/g. [0134] 2.2 Solid-phase synthesis of Kyas-01
[0135] With mU as the initiation monomer and with MU as the end monomer, different phosphoramidite monomers were introduced by coupling through a solid-phase phosphoramidite method. The solid-phase phosphoramidite method includes the following basic steps: 1) deprotection: removing the protective group (DMT) on the oxygen atom of 3′SANCd-01 resin; 2) coupling: adding a first nucleotide monomer, coupling in the direction of 3′ to 5′; 3) oxidation: oxidizing the resulting nucleoside phosphite into a more stable nucleoside phosphate (that is, oxidization of trivalent phosphorus to pentavalent phosphorus); 4) blocking: blocking 5′-OH of the nucleotide monomer unreacted in the previous step by capping to prevent it from reacting further; the above steps were repeated until the desired sequence was achieved. After being synthesized, the ester bond for linking the compound to the initial nucleoside on the solid phase carrier was cleaved with methylamine ethanol solution and aqueous ammonia, and protective groups on various bases and phosphoric acid on the oligonucleotide, including cyanoethyl (P), benzoyl (mA, fA), acetyl (mC, fC) and isobutyryl (mG, fG), were removed. The product was purified by HPLC, filtered and sterilized, and freeze-dried to get Kyas-01.
[0136] 3. Synthesis of GBL-0401
[0137] Kys-01 and Kyas-01 solutions were determined accurately for their concentration, mixed at equal molarity, added with 1 M PBS solution at 1/20 of the volume and mixed uniformly again. The mixed system was heated to 95° C. for 5 min, cooled naturally for 3 h to 40° C. or room temperature, and detected by HPLC. If the single-strand residue was <5%, the reaction is considered complete.
[0138] Example 2. Synthesis of GBL-0402
[0139] 1. Synthesis of Kys-02 [0140] 1.1. Compounds of 5′YICc-01: Synthesis of 5′YICc-01-PFP [0141] 1.1.1. Synthesis of 5′YICc-01-c1
##STR00138##
[0142] SANC-c8 (7.0 g, 40.0 mmol) and 5′YICd-01-c3 (9.2 g, 34.4 mmol) were dissolved in 25 mL of DMF, added with 9.0 g TBTU and cooled to 10° C., then added with 2 mL of DIEA and reacted at room temperature overnight. 30 mL of water and 50 mL of dichloromethane were added. The organic layer was washed with saturated brine for three times, dried, and evaporated at reduced pressure to dryness. The residue was passed over a chromatographic column (Eluent: dichloromethane:methanol =1%-10%) to get 10.0 g of a yellow sticky solid. [0143] 1.1.2. Synthesis of 5′YICc-01-c2
##STR00139##
[0144] 15 mL of concentrated hydrochloric acid was added into 10.0 g of 5′YICc-01-c1. The mixture was reacted at room temperature overnight, and then evaporated at reduced pressure to get 7.3 g of a product. [0145] 1.1.3. Synthesis of 5′YICc-01-c3
##STR00140##
[0146] 5′YICc-01-c2 (7.3 g, 22.6 mmol) and SANC-c4 (12.1 g, 27.1 mmol) were added into 60 mL of DMF, added with 3.8 g of HOBt and 12.4 g of HBTU, followed by slow addition of 5.0 ml of DIEA.
[0147] The reaction solution was reacted at room temperature with stirring overnight. Then 50 mL of water was added, and the reaction solution was extracted with 100 mL of dichloromethane. The organic phase was washed with 100 mL of saturated brine, dried over anhydrous Na2SO4, and evaporated at reduced pressure to dryness. The residue was purified by chromatography on a silica gel column (Eluent:3-15% MeOH in DCM) to get 8.3 g of a white foamy solid. [0148] 1.1.4. Synthesis of 5′YICc-01-c7
[0149] The synthetic steps were the same as those in 1.1.5 of Example 1, and the high-resolution mass spectrum is shown in
[0151] The synthetic steps were the same as those in 1.1.6 of Example 1. [0152] 1.1.6. Synthesis of 5′YICc-01-c9
[0153] The synthetic steps were the same as those in 1.1.7 of Example 1. [0154] 1.1.7. Synthesis of 5′YICc-01-PFP
[0155] The synthetic steps were the same as those in 1.1.8 of Example 1. [0156] 1.2. Solid-phase synthesis of C6NH-S-02
[0157] With mG as the initiation monomer and with C6NH phosphoramidite monomer as the end monomer, different phosphoramidite monomers were introduced by coupling through a solid-phase phosphoramidite method. The synthetic steps were the same as those in 1.2 solid-phase synthesis of Example 1. [0158] 1.3. Liquid-phase synthesis of Kys-02 [0159] 1.3.1. Synthesis of Kys-02-c1
##STR00141##
[0160] The synthetic steps were the same as those in 1.3.1 of Example 1. [0161] 1.3.2. Synthesis of Kys-02
##STR00142##
[0162] The synthetic steps were the same as those in 1.3.2 of Example 1.
[0163] 2. Synthesis of Kyas-02 [0164] 2.1. Compounds of 3′SANCc-01: Synthesis of 3′SANCc-01 resin
[0165] The synthetic route and process steps of 3′SANCc-01 resin were consistent with those of 3′SANCd-01 resin, except the synthesis of 3′SANCc-01-c6. [0166] 2.1.1. Synthesis of 3′SANCc-01-c1
##STR00143##
[0167] 3′SANCd-01-c5 (0.295 g) was dissolved in DMF (2 mL) and cooled, added with DIPEA (0.14 mL) and TBTU (0.177 g) and stirred for 10 minutes, then added with a solution of SANC-c12 (0.756 g) in DMF (2 mL), and reacted at room temperature overnight. The system was quenched with water (50 mL) and extracted with CH.sub.2Cl2 (20 mL*2). The organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and rotary evaporated. The residue was passed over a column with an eluent containing 5% triethylamine to get the product 3′SANCc-01-c6 (0.815 g). [0168] 2.2. Solid-phase synthesis of Kyas-02
[0169] With mU as the initiation monomer and with mU as the end monomer, different phosphoramidite monomers were introduced by coupling through a solid-phase phosphoramidite method. The synthetic steps were the same as those in 2.2 Solid-phase synthesis of Kyas-01 in Example 1.
[0170] 3. Synthesis of GBL-0402
[0171] Kys-02 and Kyas-02 solutions were determined accurately for their concentration. The synthetic steps were the same as those in 3. Synthesis of GBL-0401 in Example 1.
[0172] Example 3. Synthesis of GBL-0403
[0173] 1. Synthesis of Kys-03 [0174] 1.1. Compounds of 5′ERCd-01: Synthesis of 5′ERCd-01-PFP [0175] 1.1.1. Synthesis of 5′ERCd-01-c1
##STR00144##
[0176] 5.0 g (54.9 mmol) of 2-amino-1,3-propanediol was weighed, added with 50 mL of DMSO and 5 mL of a solution of sodium hydroxide at a concentration of 1 g/mL and cooled to 0° C., dropwise added with 20 mL (137.8 mol) of tert-butyl acrylate over 2 hours and reacted at room temperature for 48 h. The mixture was added with 100 mL petroleum ether. The organic phase was washed with saturated brine twice, dried and passed over a chromatographic column (Eluent: ethyl acetate:petroleum ether=25%-75% containing 0.05% triethylamine) to get 6.2 g of a colorless oil. [0177] 1.1.2. Synthesis of 5′ERCd-01-c2
##STR00145##
[0178] 5′ERCd-01-c1 (6.2 g, 17.9 mmol) was weighed, added with 50 mL of dichloromethane and 23 mL of a sodium carbonate solution (25%), followed by dropwise addition of 8.2 mL (57.4 mmol) of benzyl chloroformate at room temperature over 2 hours. The mixture was reacted at room temperature overnight, washed with saturated brine for three times, dried over anhydrous sodium sulfate, and evaporated off the solvent. The residue was passed over a chromatographic column (ethyl acetate:petroleum ether=5%-30%) to get 4.0 g of an oil. [0179] 1.1.3. Synthesis of 5′ERCd-01-c3
##STR00146##
[0180] 4.0 g (8.3 mmol) of 5′ERCd-01-c2 was added with 12 mL of formic acid, reacted at room temperature overnight, and evaporated off the solvent at reduced pressure to get 2.8 g of 5′ERCd-01-c3. [0181] 1.1.4. Synthesis of 5′ERCd-01-c4
##STR00147##
[0182] 5′ERCd-O1-c3 (1.11 g, 3.0 mmol) and dlSANC-c4 (3.6 g, 8.04 mmol) were added into 60 mL of DMF, added with 2.24 g of HOBt and 3.36 g of HBTU, followed by slow addition of 4.16 mL of DIEA. The reaction solution was reacted with stirring at room temperature for 3 hours. Water was then added, and the aqueous layer was extracted with dichloromethane (2×10 mL). The organic layer was combined, and then washed with 80 mL of saturated NaHCO.sub.3, water (2×60 mL), and saturated brine (60 mL) in order, dried over anhydrous Na2SO4, and evaporated at reduced pressure to dryness. The residue was purified by chromatography on a silica gel column (Eluent: .sup.3-15% MeOH in DCM), to get 3.24 g of a light yellow solid. [0183] 1.1.5. Synthesis of 5′ERCd-01-c5
##STR00148##
[0184] 3.24 g (2.6 mmol) of 5′ERCd-01-c4 was dissolved in 60 mL of methanol, added with 0.3 g of 10% Pd-C(wet Degussa-type E101 NE/W) and 2.0 mL of acetic acid, and hydrogenated at normal pressure overnight. The reaction solution was filtered with diatomite, and the filtrate was evaporated at reduced pressure to get 2.9 g of an oil. [0185] 1.1.6. Synthesis of 5′ERCd-01-c6
##STR00149##
[0186] 0.21 g (0.001 mol) of monobenzyl glutarate was weighed and dissolved in 2 mL of DMF, added with 0.36 g of TBTU and 0.4 mL of DIEA, reacted with stirring for 5 minutes, added with 0.50 g of 5′ERCd-01-c5 (dissolved in 10 ml DMF), and reacted at room temperature with stirring overnight.
[0187] The reaction solution was evaporated at reduced pressure to dryness, and 40 mL of dichloromethane and 20 mL of water were added and stirred for 5 minutes. The layers were separated. The organic layer was dried over anhydrous sodium sulfate, and passed over a chromatographic column (Eluent: dichloromethane:methanol=1%-10%). The eluate was evaporated off the solvent at reduced pressure to get 0.51 g of a white product. [0188] 1.1.7. Synthesis of 5′ERCd-01-c7
##STR00150##
[0189] Into a 100 mL single-necked flask, were added 0.51 g (0.42 mmol) of 5′ERCd-01-c6 and 127 mg of palladium-carbon. The flask was evacuated with a water pump and supplemented with hydrogen in triplicate. The mixture was reacted under pressurized hydrogen overnight. On the next day, TLC showed that the reaction was complete. Palladium-carbon was filtered with diatomite, and the filtrate was evaporated at reduced pressure to dryness to get 0.40 g of a product, of which the high-resolution mass spectrum is shown in
##STR00151##
[0191] Into a 50 mL single-necked flask, were added 0.40 g (0.33 mmol) of 5′ERCd-01-c7 and 10 mL of dichloromethane, and then dropwise added with 0.19 g (0.6 mmol) of pentafluorophenyl trifluoromethanesulfonate over 10 minutes and reacted at room temperature for 2 hours. The reaction solution was washed with 10 mL of water twice, and then with 5 mL of saturated sodium bisulfate once. The organic layer was dried over anhydrous sodium sulfate for 10 minutes and evaporated at reduced pressure to dryness to get 0.5 g of a product. [0192] 1.2. Solid-phase synthesis of C6NH-S-03
[0193] With mG as the initiation monomer and with C6NH phosphoramidite monomer as the end monomer, different phosphoramidite monomers were introduced by coupling through a solid-phase phosphoramidite method. The synthetic steps were the same as those in 1.2 Solid-phase synthesis in Example 1. [0194] 1.3. Liquid-phase synthesis of Kys-03 [0195] 1.3.1. Synthesis of Kys-03-c1
##STR00152##
[0196] The synthetic steps were the same as those in 1.3.1 of Example 1. [0197] 1.3.2. Synthesis of Kys-03
##STR00153##
[0198] The synthetic steps were the same as those in 1.3.2 of Example 1.
[0199] 2. Synthesis of Kyas-03 [0200] 2.1. Compounds of 3′ERCd-01: Synthesis of 3′ERCd-01 resin [0201] 2.1.1. Synthesis of 3′ERCd-01-cI
##STR00154##
[0202] Into a reaction flask, 3′SANCd-01-c5 (0.824 g, 0.0015 mol) and 10 mL of DMF were added in order and dissolved with stirring at room temperature, and then added with TBTU (0.563 g) and DIPEA (0.517 g) in order and dissolved with stirring at room temperature, and finally added with dlERC-c12 (1.09 g, 0.001 mol) and reacted with stirring at room temperature overnight. TLC analysis showed the reaction was complete, the reaction mixture was concentrated to remove DMF, added with water and extracted with DCM. The organic phase was further washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. Finally the residue was purified over a silica gel column to get 1.3 g of an off-white foamy solid. [0203] 2.1.2. Synthesis of 3′ERCd-01-c2
##STR00155##
[0204] The synthetic steps were the same as those in 2.1.7 of Example 1. [0205] 2.1.3. Synthesis of 3′ERCd-01 resin
##STR00156##
[0206] The synthetic steps were the same as those in 2.1.8 of Example 1. 2.2. Solid-phase synthesis of Kyas-03 With mA as the initiation monomer and with T as the end monomer, different phosphoramidite monomers were introduced by coupling through a solid-phase phosphoramidite method. The synthetic steps were the same as those in 2.2 Solid-phase synthesis of Kyas-01 in Example 1. 3. Synthesis of GBL-0403 Kys-03 and Kyas-03 solutions were determined accurately for their concentration. The synthetic steps were the same as those in 3. Synthesis of GBL-0401 in Example 1.
[0207] Example 4. Synthesis of GBL-0404 1. Synthesis of Kys-04 1.1. Compounds of 5′ERCc-01: Synthesis of 5′ERCc-01-PFP 1.1.1. Synthesis of 5′ERCc-01-c1
##STR00157##
[0208] N-tert-butoxycarbonyl-1,3-propanediamine (5.0 g, 28.7 mmol) and 5′ERCd-01-c3 (2.8 g, 7.6 mmol) were dissolved in 25 mL of DMF, added with 9.0 g of TBTU and 2 mL of DIEA and reacted at room temperature overnight. 30 mL of water and 50 mL of DCM were added. The organic layer was washed with saturated brine and evaporated at reduced pressure to dryness. The residue was passed over a chromatographic column loaded with petroleum ether and rinsed with 1 L petroleum ether (Eluent: DCM:methanol=5%-10%) to get 2.9 g of a yellow sticky solid. [0209] 1.1.2. Synthesis of 5′ERCc-01-c2
##STR00158##
[0210] 2.9 g of 5′ERCc-01-c1 was weighed, added with 9 mL of concentrated hydrochloric acid and reacted at room temperature overnight. The mixture was evaporated at reduced pressure to get 2.7 g of a product. [0211] 1.1.3. Synthesis of 5′ERCc-01-c3
##STR00159##
[0212] 5′ERCc-01-c2 (1.56 g, 2.44 mmol) and Sanc-c4 (3.6 g, 8.04 mmol) were added into 60 mL of DMF, added with 2.24 g of HOBt and 3.36 g of HBTU, followed by slow addition of 4.16 mL of DIEA. The reaction solution was reacted at room temperature with stirring for 1 hour. Water was then added, and the aqueous layer was extracted with DCM (2×10 mL). The organic layer was combined, and then washed with 80 mL of saturated sodium bicarbonate, 40 mL of water, and 60 mL of saturated brine in order, dried over anhydrous sodium sulfate, and evaporated at reduced pressure to dryness. The residue was purified by chromatography on a silica gel column (Eluent: .sup.3-15% methanol in DCM), to get 2.36 g of a light yellow solid. [0213] 1.1.4. Synthesis of 5′ERCc-01-c4
##STR00160##
[0214] 2.36 g (1.2 mmol) of 5′ERCc-01-c3 was dissolved in 120 mL of methanol, added with 1.0 g of 10% Pd-C(wet Degussa-type E101 NE/W), and hydrogenated at normal pressure overnight. The reaction solution was filtered with diatomite, and the filtrate was evaporated at reduced pressure to dryness to get 1.8 g of oil, of which the high-resolution mass spectrum is shown in
##STR00161##
[0216] 0.21 g (0.001 mol) of monobenzyl glutarate was dissolved in 2 mL of DMF, added with 0.36 g of TBTU and 0.4 mL of DIEA and reacted with stirring for 5 minutes, and added with 1.09 g of 5′ERCc-01-c4 and reacted at room temperature with stirring overnight. The reaction solution was evaporated at reduced pressure to dryness, added with 40 mL of DCM and 20 mL of water and stirred for 5 minutes. The layers were separated, and the organic layer was dried over anhydrous sodium sulfate and passed over a chromatographic column (Eluent: DCM:methanol=1%-10%), and the solvent was evaporated at reduced pressure to dryness to get 0.85 g of a white product. [0217] 1.1.6. Synthesis of 5′ERCc-01-c6
##STR00162##
[0218] Into a 100 mL single-necked flask, were added 0.85 g (0.43 mmol) of 5′ERCc-01-c5 and 127 mg of palladium-carbon. The flask was evacuated by a water pump and supplemented with hydrogen in triplicate. The reaction was conducted under pressurized hydrogen overnight. On the next day, TLC showed the reaction was complete. Palladium-carbon was filtered with diatomite, and the filtrate was evaporated at reduced pressure to dryness to get 0.76 g of a product. [0219] 1.1.7. Synthesis of 5′ERCc-01-PFP
##STR00163##
[0220] Into a 50 mL single-necked flask, were added 0.76 g (0.40 mmol) of 5′ERCc-01-c6 and 10 mL of DCM, and dropwise added with 0.19 g (0.6 mmol) of pentafluorophenyl trifluoromethanesulfonate, reacted at room temperature for 1 hour, and washed with 10 mL of water and 5 mL of saturated sodium bisulfite in order. The organic layer was dried over anhydrous sodium sulfate for 10 minutes and evaporated at reduced pressure to dryness to get 0.8 g of a product. [0221] 1.2. Solid-phase synthesis of C6NH-S-04
[0222] With mA as the initiation monomer and with C6NH phosphoramidite monomer as the end monomer, different phosphoramidite monomers were introduced by coupling through a solid-phase phosphoramidite method. The synthetic steps were the same as those in 1.2 Solid-phase synthesis in Example 1. [0223] 1.3. Liquid-phase synthesis of Kys-04 [0224] 1.3.1. Synthesis of Kys-04-c1
##STR00164##
[0225] The synthetic steps were the same as those in 1.3.1 of Example 1. [0226] 1.3.2. Synthesis of Kys-04
##STR00165##
[0227] The synthetic steps were the same as those in 1.3.2 of Example 1. [0228] 2. Synthesis of Kyas-04 [0229] 2.1. Compounds of 3′ERCc-01: Synthesis of 3′ERCc-01 resin [0230] 2.1.1. Synthesis of 3′ERCc-01-c1
##STR00166##
[0231] Into a reaction flask were added 3′SANCd-01-c5 (0.824 g, 0.0015 mol) and 10 mL of DMF in order and dissolved with stirring at room temperature, and then added with TBTU (0.563 g) and DIPEA (0.517 g) in order and dissolved with stirring at room temperature, and finally added with ERC-c12 (1.21 g, 0.001 mol) and reacted with stirring at room temperature overnight. TLC analysis showed that the reaction was complete, the reaction mixture was concentrated to remove DMF, added with water and extracted with DCM. The organic phase was further washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered and concentrated. Finally the residue was purified over a silica gel column to get 1.4 g of a white foamy solid. [0232] 2.1.2. Synthesis of 3′ERCc-01-c2
##STR00167##
[0233] The synthetic steps were the same as those in 2.1.7 of Example 1. [0234] 2.1.3. Synthesis of 3′ERCc-01 resin
##STR00168##
[0235] The synthetic steps were the same as those in 2.1.8 of Example 1. [0236] 2.2. Solid-phase synthesis of Kyas-04
[0237] With mG as the initiation monomer and with mU as the end monomer, different phosphoramidite monomers were introduced by coupling through a solid-phase phosphoramidite method. The synthetic steps were the same as those in 2.2 Solid-phase synthesis of Kyas-01 in Example 1.
[0238] 3. Synthesis of GBL-0404
[0239] Kys-04 and Kyas-04 solutions were determined accurately for their concentration. The synthetic steps were the same as those in 3. Synthesis of GBL-0401 in Example 1.
Example 5. Synthesis of GBL-0409
[0240] 1. Synthesis of Kyas-09 [0241] 1.1. Compounds of 3′qfSANCd-01: Synthesis of 3′qfSANCd-01 resin [0242] 1.1.1. Synthesis of 3′qfSANCd-01-c1
##STR00169##
[0243] Into a reaction flask were added hydroxyprolinol hydrochloride (1.53 g, 0.01 mol) and 15 mL of DMF in order and dissolved with stirring at room temperature, and then added with monomethyl suberate (1.98 g, 0.0105 mol), HBTU (4.55 g) and DIPEA (3.88 g) in order, and reacted with stirring at room temperature overnight. TLC analysis showed that the reaction was complete, and the reaction mixture was concentrated to remove DMF, added with water and extracted with DCM. The organic phase was further washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. Finally the residue was purified over a silica gel column to get 2.38 g of a yellow sticky liquid. [0244] 1.1.2. Synthesis of 3′qfSANCd-01-c2
##STR00170##
[0245] Into a reaction flask were added 3′qfSANCd-01-c1 (2.87 g 0.01 mol) and 30 ml of pyridine in order and dissolved with stirring at room temperature, and then added with DMAP (0.61 g) and DMT-CL (4.06 g, 0.012 mol) in order, and reacted with stirring at room temperature overnight. TLC analysis showed that the reaction was complete, and the reaction mixture was concentrated to remove pyridine, added with water and extracted with DCM. The organic phase was further washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. Finally the residue was purified over a silica gel column to get 4.13 g of a yellow sticky liquid (yield 70%). [0246] 1.1.3. Synthesis of 3′qfSANCd-01-c3
##STR00171##
[0247] Into a reaction flask were added 3′qfSANCd-01-c2 (5.89 g 0.01 mol) and 60 mL of a solvent (TIIF/water/methanol=1:1:4) in order and dissolved with stirring at room temperature, and then added with LiOH (1.26 g) and reacted with stirring at room temperature for 2 h. TLC analysis showed that the reaction was complete, and the reaction mixture was concentrated to remove the solvent, added with water and extracted with DCM. The organic phase was further washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. Finally the residue was purified over a silica gel column to get 4.5 g of a yellow sticky liquid. [0248] 1.1.4. Synthesis of 3′qfSANCd-O1-c4
##STR00172##
[0249] Into a reaction flask were added 3′qfSANCd-01-c3 (0.863 g, 1.5 mmol) and 10 mL of DMF in order and dissolved with stirring at room temperature, and then added with TBTU (0.963 g) and DIPEA (0.517 g) in order and dissolved with stirring at room temperature, and finally added with dlSANC-c12 (1.62 g 1 mmol) and reacted with stirring at room temperature overnight. TLC analysis showed that the reaction was complete, and the reaction mixture was concentrated to remove DMF, added with water and extracted with DCM. The organic phase was further washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated.
[0250] Finally the residue was purified over a silica gel column to get 1.743 g of a yellow sticky liquid. [0251] 1.1.5. Synthesis of 3′qfSANCd-01-c5
##STR00173##
[0252] Into a reaction flask were added 3′qfSANCd-01-c4 (2.18 g, 0.001 mol) and 10 mL of DCM in order and dissolved with stirring at room temperature, and then added with DBU (0.256 g) and succinic anhydride (0.3 g, 0.003 mmol) in order and reacted with stirring at room temperature. TLC analysis showed that the reaction was complete, and the reaction mixture was concentrated to remove DCM, added with water and extracted with DCM. The organic phase was further washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. Finally the residue was purified over a silica gel column to get 2.05 g of 3′qfSANCd-01-c5. [0253] 1.1.6. Synthesis of 3′qfSANCd-01-c6
##STR00174##
[0254] Into a reaction flask were added 3′qfSANCd-01-c5 (1.14 g, 0.0005 mmol) and 12 mL of DMVF in order and dissolved with stirring at room temperature, and then added with HBTU (0.19 g), DIPEA (0.194 g) and GE resin (1.83 g) in order, and shaken in a shaker at 35° C. for 4 h. The mixture was transferred into a synthesis tube and filtered. Under bubbling with nitrogen, the resin was rinsed with DMF for 4 times, added with CAP A +CAP B to conduct the end-capping reaction for half an hour under bubbling with nitrogen. A little amount of the resin was taken for a kaiser test until the test solution appeared yellow. After completion of the end-capping, the filter cake was rinsed with methanol and DCM respectively, and dried in vacuum to get 2.5 g of 3′qfSANCd-01, of which the degree of substitution was 140 μmol/g. [0255] 1.2 Solid-phase synthesis of Kyas-09
[0256] With mU as the initiation monomer and with mU as the end monomer, different phosphoramidite monomers were introduced by coupling through a solid-phase phosphoramidite method. The synthetic steps were the same as those in 2.2 Solid-phase synthesis of Kyas-01 in Example 1.
[0257] 2. Synthesis of GBL-0409
[0258] Kys-01 and Kyas-09 solutions were determined accurately for their concentration. The synthetic steps were the same as those in 3. Synthesis of GBL-0401 in Example 1.
Example 6. Synthesis of GBL-0410
[0259] 1. Synthesis of Kys-10 [0260] 1.1. Solid-phase synthesis of C9NH-S-01
[0261] With mU as the initiation monomer and with C9NH phosphoramidite monomer as the end monomer, different phosphoramidite monomers were introduced by coupling through a solid-phase phosphoramidite method. The synthetic steps were the same as those in 1.2 Solid-phase synthesis of C6NH-S-01 in Example 1. [0262] 1.2. Liquid-phase synthesis of Kys-10 [0263] 1.2.1. Synthesis of Kys-10-c1
##STR00175##
[0264] The synthetic steps were the same as those in 1.3.3 of Example 1. [0265] 1.2.2. Synthesis of Kys-01
##STR00176##
[0266] The synthetic steps were the same as those in 1.3.4 of Example 1.
[0267] 2. Synthesis of Kyas-10 [0268] 2.1. Compounds of 3′pdSANCd-O1: Synthesis of 3′pdSANCd-O1 resin [0269] 2.1.1. Synthesis of 3′pdSANCd-O1-c1
##STR00177##
[0270] Into a reaction flask, 4,4-piperidinediyl dimethanol (1.59 g, 0.01 mol) and 20 mL of DMF were added in order and dissolved with stirring at room temperature, and then added with monomethyl suberate (1.98 g, 0.0105 mol), HBTU (4.55 g) and DIPEA (3.88 g) in order and reacted with stirring at room temperature overnight. TLC analysis showed that the reaction was complete, and the reaction mixture was concentrated to remove DMF, added with water and extracted with DCM. The organic phase was further washed with a saturated aqueous solution of sodium chloride and dried over anhydrous sodium sulfate, filtered, and concentrated. Finally the residue was purified over a silica gel column to get 2.65 g of a yellow sticky liquid. [0271] 2.1.2. Synthesis of 3′pdSANCd-01-c2
##STR00178##
[0272] Into a reaction flask, 3′pdSANCd-01-c1 (3.29 g, 0.01 mol) and 33 mL pyridine were added in order and dissolved with stirring at room temperature, and then added with DMAP (0.61 g) and DMT-CL (4.06 g, 0.012 mol) in order and reacted with stirring at room temperature overnight. TLC analysis showed that the reaction was complete, and the reaction mixture was concentrated to remove pyridine, added with water and extracted with DCM. The organic phase was further washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. Finally the residue was purified over a silica gel column to get 4.74 g of a yellow sticky liquid. [0273] 2.1.3. Synthesis of 3′pdSANCd-01-c3
##STR00179##
[0274] Into a reaction flask, 3′pdSANCd-01-c2 (3.16 g, 5 mmol) and 32 mL of a solvent (TIIF/water /methanol=1:1:4) were added in order and dissolved with stirring at room temperature, and then added with LiOH (0.63 g) and reacted with stirring at room temperature for 2 h. TLC analysis showed that the reaction was complete, and the reaction mixture was concentrated to remove the solvent, added with water and extracted with DCM. The organic phase was further washed with a saturated aqueous solution of sodium chloride and dried over anhydrous sodium sulfate, filtered, and concentrated. Finally the residue was purified over a silica gel column to get 2.78 g of a yellow sticky liquid. [0275] 2.1.4. Synthesis of 3′pdSANCd-01-c4
##STR00180##
[0276] Into a reaction flask, 3′pdSANCd-01-c3 (0.93 g, 1.5 mmol) and 10 mL if DMF were added in order and dissolved with stirring at room temperature, and then added with TBTU (0.963 g) and DIPEA (0.517 g) in order and dissolved with stirring at room temperature, and finally added with dlSANC-c12 (0.562 g, 1 mmol) and reacted with stirring at room temperature overnight. TLC analysis showed that the reaction was complete, and the reaction mixture was concentrated to remove DMF, added with water and extracted with DCM. The organic phase was further washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. Finally the residue was purified over a silica gel column to get 1.688 g of a yellow sticky liquid. [0277] 2.1.5. Synthesis of 3′pdSANCd-01-c5
##STR00181##
[0278] Into a reaction flask, 3′pdSANCd-01-c4 (2.22 g, 0.001 mol) and 22 mL of DCM were added in order and dissolved with stirring at room temperature, and then added with DBU (0.256 g) and succinic anhydride (0.3 g, 0.003 mmol) in order and reacted with stirring at room temperature. TLC analysis showed that the reaction was complete, and the reaction mixture was concentrated to remove DCM, added with water and extracted with DCM. The organic phase was further washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. Finally the residue was purified over a silica gel column to get 2.11 g of 3′pdSANCd-01-c5. [0279] 2.1.6. Synthesis of 3′pdSANCd-01
##STR00182##
[0280] Into a reaction flask, 3′pdSANCd-01-c5 (1.16 g, 0.0005 mmol) and 12 mL of DMF were added in order and dissolved with stirring at room temperature, and then added with HBTU (0.19 g), DIPEA (0.194 g) and GE resin (1.85 g) in order, and shaken in a shaker at 35° C. for 4 h. The mixture was transferred into a synthesis tube and filtered. Under bubbling with nitrogen, the resin was rinsed with DMF for 4 times, and then added with CAP A +CAP B to conduct the end-capping reaction for half an hour under bubbling with nitrogen. A little amount of the resin was taken for a kaiser test until the test solution appeared yellow. After completion of the end-capping, the filter cake was rinsed with methanol and DCM respectively, and dried in vacuum to get 2.6 g of 3′pdSANCd-01, of which the degree of substitution was 145 μmol/g.
[0281] 2. Solid-phase synthesis of Kyas-10
[0282] With mU as the initiation monomer and with mU as the end monomer, different phosphoramidite monomers were introduced by coupling through a solid-phase phosphoramidite method. The synthetic steps were the same as those in 2.2 Solid-phase synthesis of Kyas-01 in Example 1.
[0283] 3. Synthesis of GBL-0410
[0284] Kys-10 and Kyas-10 solutions were determined accurately for their concentration. The synthetic steps were the same as those in of Example 1 3, Synthesis of GBL-0401.
Example 7. GBL0405 to GBL0408 and GBL0411 to GBL0418 were synthesized referring to GBL-0401 to GBL0404
Example 8. In Vitro Assay of the inhibition effects of the compounds against HBV genes in HepG2.2.15 cells
[0285] 1. Experimental grouping
[0286] Blank control group: Adding a DMEM medium containing 2% FBS and incubating for 72 h.
[0287] Test sample groups: A test sample dilution at a concentration of 5 nM, 0.5 nM or 0.05 nM was added respectively. Each concentration was done in triplicate. The incubation was conducted in an incubator at 37° C. and 5% CO2 for 72 h.
[0288] 2. Experimental materials
[0289] HepG2.2.15 cells
[0290] 3. Experimental Reagents
TABLE-US-00009 Name Brand Lot No. DMEM medium with high glucose Gibco 8119164 Fetal bovine serum Gibco 20190907 PBS Solarbio 20190624 Trypsin-EDTA solution Gibco 2062475 Dual antibiotic solution Gibco 2029632 (Penicillin/Streptomycin solution) HBsAg, HBeAg kit Shanghai Kehua 201812381
[0291] 4. Experimental Instruments
TABLE-US-00010 Name Brand Model No. Biosafety cabinet Haier HR40-IIA2 CO.sub.2 Incubator ASTEC SCA-165DS Ordinary optical microscope Nikon TS2-S-SM Low-speed centrifuge Flying pigeon KA-1000 Multi-door refrigerator MeiLing BCD-318WTPZM (E)
[0292] 5. Test Samples
TABLE-US-00011 No. Code of new compounds Weight Purity 1 GBL-0401 13.8 μg 92.3% 2 GBL-0402 12.9 μg 86.4% 3 GBL-0403 13.4 μg 89.3% 4 GBL-0404 14.0 μg 93.3% 5 GBL-0405 13.7 μg 91.3% 6 GBL-0406 20.5 μg 88.3% 7 GBL-0407 20.1 μg 94.4% 8 GBL-0408 20.3 μg 92.3% 9 GBL-0409 20.4 μg 93.6% 10 GBL-0410 20.2 μg 90.5% 11 GBL-0411 20.0 μg 89.5% 12 GBL-0412 15.1 μg 94.8% 13 GBL-0413 15.2 μg 92.5% 14 GBL-0414 15.5 μg 90.6% 15 GBL-0415 15.7 μg 91.5% 16 GBL-0416 16.0 μg 93.4% 17 GBL-0417 15.9 μg 91.7% 18 GBL-0418 15.5 μg 92.5%
[0293] 6. Test process
[0294] HepG2.2.15 cells were incubated in a 96-well cell culture plate, and fresh medium was replaced every three days. Drug-containing culture media with different concentrations formulated above were added on Day 6, and the incubation continued until Day 9. The supernatants were collected and the contents of HBsAg, HbeAg and HBV DNA in the cell supernatant were detected with a detection kit.
[0295] The results of OD values were compared with that of the control group without administration, and the effectiveness can be determined according to the ratio.
[0296] 7. Experimental results [0297] 7.1 Inhibition effects on HbsAg in HepG2.2.15 cells: see
[0300] Example 9. In vivo Assay on inhibitory effects of the new compounds against HBV genes in Transgenic Mice
[0301] 1. Experimental protocol
[0302] The experimental assay was performed on male HBV transgenic mice of proper age (requiring that HBsAg was significantly expressed). 90 mice weighing about 25 g were chosen and randomly divided into 18 groups, with 5 mice in each group. On Day 0, each mouse was administered by subcutaneous injection at 3 mg/kg with an administration volume of 100-200 jaL. Before administration, HBsAg in the blood of mice was determined, and the average level of HBsAg in various groups was tried to be kept consistent.
[0303] 2. Test samples and reagents
TABLE-US-00012 No. Code of new compounds Specification Purity/Content 1 GBL-0401 500 μg 92.3% 2 GBL-0402 500 μg 86.4% 3 GBL-0403 500 μg 89.3% 4 GBL-0405 500 μg 93.3% 5 GBL-0406 500 μg 91.3% 6 GBL-0407 500 μg 88.3% 7 GBL-0408 500 μg 94.4% 8 GBL-0409 500 μg 92.3% 9 GBL-0410 500 μg 93.6% 10 GBL-0411 500 μg 90.5% 11 GBL-0412 500 μg 89.5% 12 GBL-0413 500 μg 94.8% 13 GBL-0414 500 μg 92.5% 14 GBL-0414 500 μg 90.6% 15 GBL-0415 500 μg 91.5% 16 GBL-0416 500 μg 93.4% 17 GBL-0417 500 μg 91.7% 18 GBL-0418 500 μg 92.5% 19 Normal saline 500 ml/flask 0.9%
[0304] 3. Kit
TABLE-US-00013 Kit Name Lot No. Manufacturer Kit for hepatitis B virus surface 39531900 Roche Diagnostics antigen (Electrochemiluminescence) (Shanghai) Ltd. Co.
[0305] 5. Experimental Results
TABLE-US-00014 Name Model No. Manufacturer Vortex blender MIX-28 DragonLAB Centrifuge S1010E THERMO Full-automatic 602 Roche Diagnostics GmbH chemiluminescent analyzer
[0306] The inhibition effects were shown in
[0307] Example 10. In vivo Assay on inhibitory effect of GBL-0401 on expression of HBV HBsAg in Transgenic Mice
[0308] 1. Experimental protocol
[0309] The experimental assay was performed on male HBV transgenic mice of proper age (requiring that HBsAg was significantly expressed). 10 mice weighing about 25 g were chosen and randomly divided into 2 groups, a control group and an administration group respectively, with 5 mice in each group. On Day 0, each mouse was administered at 3 mg/kg by subcutaneous injection with an administration volume of 100-200 pL. Before administration, blood was taken to determine HBsAg, and the level of HBsAg in various groups was tried to be kept consistent. Whole blood was collected from orbital venous plexus of mice at the following time points: before administration (Day 0), after administration-Week 1, Week 2, Week 3, Week 4, Week 5 and Week 6, to detect HBsAg and investigate the persistence of GBL-0401 in inhibiting the expression of HBV gene.
[0310] The specific administration information was shown in the table below:
TABLE-US-00015 Administration Number of Administration No. Test drug dosage mice/group Solvent route 1 Blank — 5 Normal Subcutaneous solvent saline injection 2 GBL-0401 3 mg/kg 5 Normal Subcutaneous saline injection
[0311] 2. Samples and Reagents
TABLE-US-00016 No. Name Specification Purity/Content 1 GBL-0401 500 μg/vial*1 vial 92.3% 2 Normal saline 500 ml/bottle 0.9%
[0312] 3. Kit
TABLE-US-00017 Kit Name Lot No. Manufacturer Kit for hepatitis B virus surface 39531900 Roche Diagnostics antigen (Electrochemiluminescence) (Shanghai) Ltd. Co.
[0313] 4. Experimental instruments
TABLE-US-00018 Name Model No. Manufacturer Vortex blender MIX-28 DragonLAB Centrifuge S1010E THERMO Full-automatic 602 Roche Diagnostics GmbH chemiluminescent analyzer
[0314] 5. Test results
[0315] The results showed that, GBL-0401 reached the optimal inhibitory effect of 99.08% at Week 1, with a slightly decreasing trend from Week 2 to Week 3, but still presented a high inhibitory rate of about 90%, and a declining trend from Week 4 to Week 6, but still maintained an inhibitory effect of about 75%. GBL-0401 has a continuous inhibitory effect on the expression of HBV HBsAg, and can inhibit the expression stably for a period of about 6 weeks. The diagram showing the in vivo inhibitory effect of GBL-0401 on HBV HbsAg is shown in