Phosphoramidite cationic monomer and synthesis method thereof

Abstract

The structure of the phosphoramidite cationic monomer is shown in the following formula: # imgabs0 #, wherein B is a basic group and is selected from one of A, U, C, G and T; r1 is selected from a methoxy group, an ethyoxyl group or a fluorine atom; r2 is alkyl; a1 is O or C; n is a natural number from 1 to 20; and X is selected from one of F, Cl, Br, I, SO4 < 2-> and PO4 < 3->.

Description

Technical Field

[0001] The present invention relates to the technical field of organic synthesis, and particularly relates to a phosphoramidite cation monomer and a synthesis method thereof. Background Art

[0002] Phosphoramidite cation monomers are a new type of monomers. Due to carrying a positive charge, there are great differences in the pharmacodynamic effects of synthesized oligonucleic acids compared with ordinary oligonucleic acid drugs, and the physical properties will change significantly. Due to reasons such as the original small nucleic acid structure being easily degraded by nucleases in the body and poor pharmacokinetic properties, they generally cannot be directly used as drugs and need to undergo multi-site chemical modifications to improve their affinity, stability, metabolic properties, etc. After decades of accumulation, the above chemical modifications have developed three generations of technologies. Commonly used chemical modifications are the first-generation phosphorothioates and the second-generation methylphosphonates. Methyl phosphonate is uncharged, so it is more lipophilic than natural DNA or RNA and can penetrate cells better. Subsequently, third-generation technologies such as PNA and LNA have emerged. The present invention belongs to the technical field of chemical synthesis of nucleotides, and particularly relates to a phosphoramidite cation monomer and a synthesis method.

[0003] The unique chemical structure of traditional oligonucleotide drugs shows poor druggability: large molecular weight, strong hydrophilicity, highly negative charge, not following Lipinski's principle, and also having poor pharmacokinetic characteristics, being unable to pass through biological membranes, and having off-target effects. These disadvantages will be greatly amplified after using cationic monomers. Summary of the Invention

[0004] The effective delivery of oligonucleotide therapies to various tissues remains a major challenge. Oligonucleotides are usually large hydrophilic polyanions (single-stranded ASO is about 410 kDa, double-stranded siRNA is about 14 kDa), which means they are not easily passed through the plasma membrane. After synthesizing oligonucleic acids using cationic phosphoramidite monomers, due to carrying a positive charge, the physical properties will change significantly, and many disadvantages of traditional oligonucleic acid drugs can be overcome, such as improving permeability and delivery ability.

[0005] An embodiment of the present invention provides a phosphoramidite cation monomer, and its structure is shown in the following formula:

[0006] Wherein B is a base, selected from one of A, U, C, G, and T; R1 is selected from methoxy, ethoxy, or fluorine atom; R2 is an alkyl group; n is a natural number from 1 to 20; X is selected from one of F, Cl, Br, I, SO4 2- , PO4 3- one of them.

[0007] An embodiment of the present invention also provides a method for preparing the phosphoramidite cation monomer as described above, and the route of the preparation method is as follows:

[0008]

[0009] Another embodiment of the present invention also provides a method for preparing the phosphoramidite cation monomer as described above, and the route of the preparation method is as follows:

[0010]

[0011] The excellent technical effects of the present invention include:

[0012] 1. The synthesis method provided in the embodiment of the present invention has mild reaction conditions, is low-energy and environmentally friendly, uses a small amount of solvents and buffers required for the purification technology, and is suitable for commercial production.

[0013] 2. After synthesizing oligonucleic acids using the cationic phosphoramidite monomer, since the cationic phosphoramidite monomer is positively charged, the physical properties will change significantly, and many disadvantages of traditional oligonucleic acid drugs can be overcome, such as improving permeability and delivery ability. After introducing a structure with such characteristics into the oligonucleotide fragment, it is expected to achieve breakthrough results in related research fields (especially in the field of siRNA drug research and development).

[0014] 3. The purification time is short, not exceeding 24 hours, and the rotation cycle is small. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the linear graph of the half-life of the compound prepared in the embodiment of the present invention;

[0016] Figure 2 is the trend graph of liver microsome stability. DETAILED DESCRIPTION OF THE INVENTION

[0017] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and the accompanying drawings. However, those skilled in the art know that the present invention is not limited to the drawings and the following embodiments.

[0018] Technical Solution 1:

[0019]

[0020] Technical Solution 2:

[0021]

[0022] Example 1:

[0023]

[0024] Step 1:

[0025] Synthesis of N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methyl-3'-O-(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphino]cytidine

[0026] Controlled at -20 °C, a solution of bis(N,N-diisopropylamino)chlorophosphine (323 mg, 1.21 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N4-benzyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methylmethylcytidine A (646 mg, 0.97 mmol) and N-ethyl-N,N-diisopropylamine (238 mg, 1.84 mmol) in anhydrous dichloromethane (4 mL). The solution was stirred at room temperature for 2 hours. 1H-Tetrazole (0.45 M in MeCN, 0.9 mL) was added to the resulting mixture, and then stirred at room temperature for 14 hours. The reaction mixture was quenched by adding an excess of 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). The extract was washed with brine, dried over anhydrous Na2SO4, and evaporated to an oil. The product was separated by column chromatography on silica gel, and the fractions were evaporated to give the title compound as a viscous oil, yield 1038 mg (81.2%). 31P NMR (diastereoisomer mixture, CD3CN): δ 149.17 148.75.

[0027] Step 2:

[0028] 1H-Tetrazole (0.4 equiv.) was added to a mixture of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methyl-3'-O-[[(N,N-diisopropylamino-diisopropyl)B and choline (1.5 eq.) and CH2Cl2 (10 mL). The resulting solution was stirred at room temperature for 2 hours. Aqueous NaHCO3 (5%, 10 mL) was added, the emulsion was diluted with brine (50 mL), and the product was extracted with ethyl acetate (3×75 mL). The extract was washed with brine (3×50 mL), dried over Na2SO4, and evaporated to dryness. The residue was dissolved in toluene (25 mL), applied to a silica gel column, and eluted with a gradient of ethyl acetate / hexane / triethylamine from 15:80:5 to 80:15:5. The collected fractions were evaporated, co-evaporated with dry MeCN (2×50 mL), and dried on an oil pump to give the target compound.

[0029] Example 2:

[0030]

[0031] Step 1:

[0032] N4-Benzyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methylmethylcytidine A (100 mg, 1.0 eq.) was dissolved in dichloromethane (20 mL). The temperature of the mixture was adjusted to 0 °C, DIEA (2.0 eq.) was added, 1-chloro-N,N,N',N'-tetraisopropylphosphorodiamidite (1.5 eq.) was added, and the reaction was carried out at 0 °C for 2 h. The reaction solution was washed with saturated aqueous sodium bicarbonate solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The product was separated by column chromatography, and the eluent was n-heptane / ethyl acetate = 50:1 - 1:1 to obtain compound B.

[0033] Step 2:

[0034] The product compound B (80 mg, 1.0 eq.) from the previous step was added to dichloromethane (50 mL), 1H-tetrazole (0.5 eq.) and choline (1.5 eq.) were added, and the reaction was carried out at room temperature for 2 h. The reaction solution was washed with saturated aqueous sodium bicarbonate solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The product was separated by column chromatography, and the eluent was n-heptane / ethyl acetate = 50:1 - 1:1 to obtain compound C0.

[0035] Step 3:

[0036] The product compound C0 (50 mg, 1.0 eq.) from the previous step was added to DMF (20 mL), the temperature was adjusted to 0 °C, cesium carbonate (2.0 eq.) was added, triisopropylamine (1.2 eq.) was added, and the reaction was carried out for 17 h. Water (100 mL) was added, ethyl acetate (150 mL) was added, the lower aqueous phase was separated, the organic phase was washed with water (20 mL), and then washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was added to a 10 mL mixed solution of MeCN:n-heptane = 1:5, stirred for 1 h and then filtered. The obtained solid was separated by column chromatography, and the eluent was ethyl acetate / n-heptane / triethylamine = 15:80:5 to obtain the target compound C.

[0037] Detection and comparison of activity data:

[0038] Six representative phosphoramidite monomers were synthesized in the examples of the present invention (B = A, R1 = MeO, R2 = Me, A1 = O, n = 2, X are F, Cl, Br, I, SO4 2- , PO4 3-) Oligonucleic acids were synthesized using Cytiva OP100 for detecting hepatic microsomal stability. The data of hepatic microsomal stability, half-life and clearance rate, as well as the activity charts, are shown in the appendix. Figure 1 and Figure 2 (where SO4 2- , PO4 3- The synthesis of oligonucleic acids from two monomers failed, possibly due to the too strong electronegativity of the anionic groups. The X of compounds 1, 2, 3, and 4 are F, Cl, Br, and I respectively)

[0039] Half-life and clearance rate data:

[0040] Compound 1

[0041]

[0042] Compound 2

[0043]

[0044]

[0045] Compound 3

[0046]

[0047] Compound 4

[0048]

[0049] Result analysis: Hepatic microsomes are one of the main sites of drug metabolism. If the hepatic microsomal stability is poor, toxic substances or the accumulation of metabolites may be produced during the metabolism of drugs in the liver, leading to adverse reactions such as liver damage. The hepatic microsomal stability of the compounds prepared in the examples of the present invention is good, which means high stability of drug metabolism in the liver, helping to reduce adverse reactions and risks during the metabolism of drugs in the body.

[0050] Secondly, a long half-life means that the drug stays in the body for a long time and can exert a more lasting therapeutic effect. In drug research and development, if a drug has a very short half-life, it needs to be administered frequently to maintain the therapeutic effect, which will bring inconvenience and pain to patients. However, the compounds prepared in the examples of the present invention have a long half-life, which can reduce the dosing frequency and improve the medication compliance and comfort of patients.

[0051] The %CV of the half-life and clearance rate data represents the coefficient of variation of these data, that is, the ratio of the standard deviation to the mean, which is used to reflect the degree of data variation. For the half-life, %CV can reflect the difference in half-life among different individuals. If the %CV is large, it indicates that there are significant differences in the half-life among different individuals, and there may be factors such as individual differences and pathological conditions. At 45 minutes, the coefficient of variation of Compound 1 increased significantly. Preliminary analysis suggests that this may be due to individual differences or pathological conditions.

[0052] From the above data and the attached Figure 1 、 Figure 2 It can be seen that in vitro, each compound was activated by human liver microsomes. Among them, 16.71% of Compound 1 remained after 60 hours. The trend graphs of the liver microsome stability and half-life of Compound 1 are also relatively gentle, and the half-life and clearance rate data are good.

Claims

1. A phosphoramidite cationic monomer, characterized in that, Its structure is shown as follows: Wherein B is a base selected from one of A, U, C, G, and T; R1 is selected from methoxy, ethoxy, or a fluorine atom; R2 is an alkyl group; A1 is O or C; n is a natural number from 1 to 20; X is selected from F, Cl, Br, I, SO4 2- , PO4 3- and one of the following.

2. The preparation method of the phosphoramidite cationic monomer according to claim 1, characterized in that, The preparation method route is as follows:

3. The preparation method according to claim 2, characterized in that, The preparation method includes: Step 1: Synthesis of N4-benzoyl-5'-O-(4,4'-dimethoxytrimethyl)-2'-O-methyl-3'-O-(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphino]cytidine Controlled at a temperature of -20 °C, a solution of bis(N,N-diisopropylamino)chlorophosphine in anhydrous dichloromethane was added dropwise to a stirred anhydrous dichloromethane solution of N4-benzyl-5'-O-(4,4'-dimethoxytrimethyl)-2'-O-methylmethylcytidine A and N-ethyl-N,N-diisopropylamine; the solution was stirred at room temperature for 2 hours; 1H-tetrazole was added to the resulting mixture, and then stirred at room temperature for 14 hours; the reaction mixture was quenched by adding an excess of 5% aqueous NaHCO3 solution; the emulsion was diluted with 5% aqueous NaHCO3 solution, and the product was extracted with ethyl acetate; the extract was washed with brine, dried over anhydrous Na2SO4, and evaporated to an oil; the product was separated by column chromatography on silica gel, and the fractions were evaporated to obtain the target compound; Step 2: 1H-tetrazole was added to a mixture of N4-benzoyl-5'-O-(4,4'-dimethoxytrimethyl)-2'-O-methyl-3'-O-[[(N,N-diisopropylamino-diisopropyl)B and choline and CH2Cl2; the resulting solution was stirred at room temperature for 2 hours; aqueous NaHCO3 solution was added, the emulsion was diluted with brine, and the product was extracted with ethyl acetate; the extract was washed with brine, dried over Na2SO4, and evaporated to dryness; the residue was dissolved in toluene, applied to a silica gel column for elution, the collected fractions were evaporated, co-evaporated with dry MeCN, and dried on an oil pump to obtain the target compound; Preferably, the eluent used for elution on the silica gel column is a gradient separation eluent of ethyl acetate / hexane / triethylamine 15:80:5 to 80:15:

5.

4. The preparation method of the phosphoramidite cationic monomer according to claim 1, characterized in that, The preparation method route is as follows:

5. The preparation method according to claim 4, characterized in that, The preparation method includes: Step 1: N4-benzyl-5'-O-(4,4'-dimethoxytrimethyl)-2'-O-methylmethylcytidine A was dissolved in dichloromethane, the temperature of the mixture was adjusted to 0 °C, DIEA was added, 1-chloro-N,N,N',N'-tetraisopropylphosphorodiamide was added, and the reaction was carried out at 0 °C for 2 h. The reaction solution was washed with saturated aqueous sodium bicarbonate solution, and the organic phase was concentrated to dryness after drying over anhydrous sodium sulfate. The product was separated by column chromatography to obtain compound B; preferably, the eluent was n-heptane / ethyl acetate = 50:1 - 1:1; Step 2: Add the product compound B from the previous step to dichloromethane, add 1H-tetrazole and choline, react at room temperature for 2 h, wash the reaction solution with saturated sodium bicarbonate aqueous solution, dry the organic phase over anhydrous sodium sulfate and concentrate to dryness, and separate the product by column chromatography to obtain compound C0; preferably, the eluent for column chromatography separation is n-heptane / ethyl acetate = 50:1 - 1:1; Step 3: Add the product compound C0 from the previous step to DMF, adjust the temperature to 0 °C, add cesium carbonate, add triisopropylamine, react for 17 h, add water, add ethyl acetate, separate the lower aqueous phase, wash the organic phase with water and then with saturated brine, dry the organic phase over anhydrous sodium sulfate and concentrate to dryness, add the residue to a mixed solution of MeCN:n-heptane = 1:5, stir for 1 h and then filter, separate the obtained solid by column chromatography to obtain the target compound C; preferably, the eluent used for the column chromatography separation is ethyl acetate / n-heptane / triethylamine = 15:80:5.

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