Preparation method of SNA phosphoramidite, intermediate of SNA phosphoramidite and preparation method of intermediate

Abstract

The invention discloses a preparation method of SNA phosphoramidite, an intermediate of the SNA phosphoramidite and a preparation method of the intermediate. Specifically disclosed is a preparation method of a compound as shown in a formula 3, which comprises the following steps: in an organic solvent, carrying out a reduction reaction on a compound as shown in a formula 2 in the presence of a reducing agent to obtain the compound as shown in the formula 3, the reducing agent is NaBH4 and / or LiBH4; when the reducing agent is NaBH4, the reduction reaction is carried out in the presence of calcium chloride and / or lithium chloride. According to the preparation method of the compound as shown in the formula 3, racemization is completely avoided in the reaction process, and the product completely keeps the chirality of raw materials. The compound shown in the formula 3 can be directly used for preparing L-serinol with single chirality and high ee value, and can be further used for preparing an SNA phosphoramidite monomer containing four basic groups. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a preparation method of SNA phosphoramidite, an intermediate thereof, and a preparation method of the intermediate. Background Art

[0002] Compared with traditional DNA or RNA sequences, oligonucleotides synthesized from acyclic nucleic acid monomers have the following advantages: 1) they can effectively form an antiparallel double helix structure; 2) due to the flexibility of the backbone, in the single-stranded state, there is no need to introduce a special pre-assembled structure; 3) the thermal stability of the double helix structure is much higher than that of ordinary DNA and RNA.

[0003] Currently, the most common acyclic oligonucleotide monomers include GNA (glycerol nucleic acid), aTNA (threonine nucleic acid), and SNA (serine nucleic acid), etc. Among these acyclic oligonucleotide monomers, SNA has its unique advantages: 1) compared with GNA, the advantage of having one more carbon makes the oligonucleotide chain synthesized therefrom have stronger stability; 2) compared with aTNA, by reducing a methyl group on the chiral carbon to eliminate chirality, the oligonucleotide chain synthesized from SNA has stronger flexibility, so that it can be complementary paired with natural DNA and RNA, enabling its unique specific recognition function to play a role, which is exactly what aTNA does not have.

[0004] The common intermediate for synthesizing SNA phosphoramidite monomers with different bases is L-serinol. Existing literature (Tetrahedron Letters, 1998, 39, 6167-6170) reported a method for synthesizing L-serinol using L-serine as a raw material. However, the applicant found through experiments that in the synthesis process, chiral amino acids are prone to racemization, and the actually obtained product is a racemate of serinol, and the actual yield of the target product L-serinol is relatively low. Moreover, the obtained target product L-serinol needs to be purified by SFC, and the cost of SFC purification is about 6000 yuan / g, and the synthesis cost is very high. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that the existing preparation methods of SNA phosphoramidite and its intermediate have defects such as difficult control of racemization by-products and the need for expensive SFC chiral separation treatment. For this reason, the present invention provides a synthesis method of SNA phosphoramidite, an intermediate thereof, and a preparation method of the intermediate. The present invention provides a preparation method of a compound shown in Formula 3, in which no racemization occurs during the reaction process, and the product completely retains the chirality of the raw material. The compound shown in Formula 3 can be directly used to prepare L-serinol with a single chirality and a high ee value, without expensive SFC chiral separation treatment, greatly reducing the synthesis cost of L-serinol. L-serinol can be further used to prepare SNA phosphoramidite monomers containing four bases.

[0006] The present invention solves the above technical problems through the following technical solutions:

[0007] The present invention provides a method for preparing a compound represented by Formula 3, which comprises the following steps: in an organic solvent, the compound represented by Formula 2 undergoes a reduction reaction in the presence of a reducing agent to obtain the compound represented by Formula 3;

[0008]

[0009] Wherein, R is methyl or ethyl;

[0010] The reducing agent is NaBH 4 and / or LiBH 4 ;

[0011] When the reducing agent contains NaBH 4 , the reduction reaction is carried out in the presence of calcium chloride and / or lithium chloride.

[0012] In one embodiment, R is methyl.

[0013] In the reduction reaction, the organic solvent is a conventional organic solvent for this type of reduction reaction in the art, such as an ether solvent and / or an alcohol solvent, preferably a mixed solvent of an ether solvent and an alcohol solvent, more preferably a mixed solvent of an ether solvent and an alcohol solvent with a volume ratio of 1:1.

[0014] In one embodiment, the ether solvent is tetrahydrofuran and / or diethyl ether.

[0015] In one embodiment, the alcohol solvent is ethanol and / or methanol.

[0016] In one embodiment, the organic solvent is a mixed solvent of tetrahydrofuran and ethanol with a volume ratio of 1:1.

[0017] In one embodiment, in the reduction reaction, the molar concentration of the compound represented by Formula 2 in the organic solvent is 0.05 mol / L - 0.5 mol / L, preferably 0.1 mol / L - 0.2 mol / L, more preferably 0.11 mol / L.

[0018] In one embodiment, the molar ratio of the reducing agent to the compound represented by Formula 2 is (1.0 - 4.0):1, preferably (1.0 - 3.0):1, more preferably (1.5 - 2.5):1, and most preferably 2.0:1.

[0019] In one embodiment, the molar ratio of the calcium chloride and / or lithium chloride to the compound represented by Formula 2 is (0.5 to 3.0):1, preferably (0.5 to 1.5):1, and more preferably 1.0:1.

[0020] In one embodiment, the raw materials for the reduction reaction consist of the organic solvent, the compound represented by Formula 2, the reducing agent, and the calcium chloride and / or lithium chloride.

[0021] In the present invention, the temperature of the reduction reaction can be the conventional temperature for this type of reduction reaction in the art, such as 10°C to 40°C, preferably 20°C to 30°C, and more preferably 25°C.

[0022] In the present invention, the reduction reaction is monitored by means such as LC-MS and TLC. Those skilled in the art can terminate the reaction based on the reaction scale, the conversion degree of the raw materials, the conversion degree of the intermediates, the reaction efficiency (i.e., the relationship between the yield and the reaction time), the generation of impurities, etc., in order to obtain a preferred yield and purity.

[0023] In one embodiment, the feeding method of the reduction reaction is to sequentially mix the calcium chloride and / or lithium chloride and the reducing agent with the compound represented by Formula 2 (or a solution of the compound represented by Formula 2). Preferably, the entire feeding process is completed at 0 ± 5°C.

[0024] In one embodiment, the post-treatment method of the reduction reaction is to mix the reaction solution with a saturated ammonium chloride aqueous solution and perform extraction. The post-treatment after extraction may further include steps such as washing, drying, filtration, concentration, and column chromatography.

[0025] In one embodiment, the washing is performed using saturated brine.

[0026] In one embodiment, the preparation method of the compound represented by Formula 3 further includes the preparation method of the compound represented by Formula 2. The preparation method of the compound represented by Formula 2 includes the following steps: In an organic solvent, the compound represented by Formula 1 undergoes a hydroxyl protection reaction in the presence of 4,4'-dimethoxytrityl chloride (DMTrCl) to obtain the compound represented by Formula 2;

[0027]

[0028] In the hydroxyl protection reaction, the organic solvent is a conventional organic solvent for this type of hydroxyl protection reaction in the art, such as a basic solvent, preferably pyridine.

[0029] In one embodiment, in the hydroxyl protection reaction, the molar concentration of the compound represented by Formula 1 in the organic solvent is 0.1 mol / L - 1 mol / L, preferably 0.5 mol / L - 0.8 mol / L, and more preferably 0.6 mol / L.

[0030] In one embodiment, the molar ratio of 4,4'-dimethoxytriphenylmethyl chloride to the compound represented by Formula 1 is (1.0 - 3.0):1, preferably (1.0 - 2.0):1, more preferably (1.0 - 1.5):1, and most preferably 1.2:1.

[0031] In one embodiment, the hydroxyl protection reaction is carried out under the protection of nitrogen and / or inert gas.

[0032] In one embodiment, the compound represented by Formula 1 is in an anhydrous state.

[0033] In the present invention, the temperature of the hydroxyl protection reaction is the conventional temperature for this type of hydroxyl protection reaction in the art, such as 10°C to 40°C, preferably 20°C to 30°C, and more preferably 25°C.

[0034] In one embodiment, the hydroxyl protection reaction is monitored by means such as LC-MS, TLC, etc. Those skilled in the art can terminate the reaction according to the reaction scale, the conversion degree of raw materials, the conversion degree of intermediates, the reaction efficiency (i.e., the relationship between yield and reaction time), the generation of impurities, etc., in order to obtain a preferred yield and purity.

[0035] In one embodiment, the feeding method of the hydroxyl protection reaction is to add 4,4'-dimethoxytriphenylmethyl chloride to the compound represented by Formula 1 (or a solution of the compound represented by Formula 1). Preferably, the feeding is carried out under the protection of nitrogen. Preferably, the compound represented by Formula 1 is dehydrated and then mixed with an organic solvent.

[0036] In the present invention, the post-treatment method of the hydroxyl protection reaction can be the conventional post-treatment method for this type of hydroxyl protection reaction in the art, such as quenching, concentration, and column chromatography.

[0037] In one embodiment, the quenching is to mix the reaction solution with methanol.

[0038] The present invention provides a method for preparing a compound represented by Formula 2, and the method for preparing the compound represented by Formula 2 is as described above;

[0039]

[0040] wherein, R is methyl or ethyl.

[0041] The present invention provides a method for preparing a compound as shown in Formula 4, which comprises the following steps:

[0042] (1) Prepare the compound as shown in Formula 3 according to the method for preparing the compound as shown in Formula 3 above;

[0043] (2) In an organic solvent, perform a de-Fmoc reaction on the compound as shown in Formula 3 with a deprotection reagent to obtain the compound as shown in Formula 4;

[0044]

[0045] In the de-Fmoc reaction, the organic solvent is a conventional organic solvent for this type of de-Fmoc reaction in the art, such as an amide solvent, preferably N,N-dimethylformamide.

[0046] In one embodiment, in the de-Fmoc reaction, the molar concentration of the compound as shown in Formula 3 in the organic solvent is 0.05 mol / L - 0.5 mol / L, preferably 0.2 mol / L - 0.3 mol / L, more preferably 0.23 mol / L.

[0047] In one embodiment, the deprotection reagent is a basic deprotection reagent, preferably piperidine.

[0048] In one embodiment, the molar ratio of the deprotection reagent to the compound as shown in Formula 3 is (1.0 - 3.0):1, preferably (1.0 - 2.0):1, more preferably (1.0 - 1.5):1, and most preferably 1.0:1.

[0049] In the present invention, the temperature of the de-Fmoc reaction can be a conventional temperature for this type of de-Fmoc reaction in the art, such as 10°C - 40°C, preferably 20°C - 30°C, more preferably 25°C.

[0050] In one embodiment, the de-Fmoc reaction is monitored by means such as LC-MS, TLC, etc., and those skilled in the art can terminate the reaction according to the reaction scale, the conversion degree of the raw materials, the conversion degree of the intermediates, the reaction efficiency (i.e., the relationship between the yield and the reaction time), the generation of impurities, etc. to obtain a preferred yield and purity.

[0051] The feeding mode of the de-Fmoc reaction is a conventional feeding mode for this type of de-Fmoc reaction in the art, such as adding the deprotection reagent to the compound as shown in Formula 3 (or a solution of the compound as shown in Formula 3).

[0052] In one embodiment, the post-treatment mode of the de-Fmoc reaction is to mix the reaction solution with saturated sodium bicarbonate aqueous solution and perform extraction. After extraction, it may also include steps such as washing, drying, filtering, concentrating, and column chromatography.

[0053] In one embodiment, the method for preparing the compound represented by Formula 4 further includes the method for preparing the compound represented by Formula 2, and the method for preparing the compound represented by Formula 2 is as described above.

[0054] The present invention provides a compound represented by Formula 2-1;

[0055]

[0056] The present invention provides a method for preparing SNA phosphoramidite, which includes the following steps:

[0057] (1) Prepare the compound represented by Formula 4 according to the method for preparing the compound represented by Formula 4 as described above;

[0058] (2) In an organic solvent, react the compound represented by Formula 4 according to the following route to obtain SNA phosphoramidite;

[0059]

[0060] Wherein,

[0061] R 1 is

[0062] R 2 is a halogen or

[0063] The coupling reaction is carried out under the protection of nitrogen and / or inert gas.

[0064] In one embodiment, in the condensation reaction, the organic solvent is a conventional organic solvent for this type of condensation reaction in the art, such as an amide solvent, preferably N,N-dimethylformamide.

[0065] In one embodiment, in the condensation reaction, the molar concentration of the compound represented by Formula I in the organic solvent is 0.1 mol / L - 1.0 mol / L, preferably 0.2 mol / L - 0.6 mol / L, and more preferably 0.40 mol / L.

[0066] In one embodiment, in the condensation reaction, the molar ratio of the compound represented by Formula 4 to the compound represented by Formula I is (1.0 - 3.0):1, preferably (1.0 - 1.5):1, and more preferably 1.2:1.

[0067] In one embodiment, the condensation reaction is carried out in the presence of N,N - diisopropylethylamine and HBTU. Preferably, the molar ratio of N,N - diisopropylethylamine to the compound shown in Formula I is (1.0 - 5.0):1, preferably (2.5 - 3.5):1, more preferably 3.0:1; the molar ratio of HBTU to the compound shown in Formula I is (1.0 - 3.0):1, preferably (1.0 - 1.5):1, more preferably 1.2:1.

[0068] In the present invention, the temperature of the condensation reaction can be the conventional temperature for this type of condensation reaction in the art, such as 0 ± 5°C.

[0069] In one embodiment, in the coupling reaction, the organic solvent is a polar aprotic solvent, preferably anhydrous dichloromethane.

[0070] In one embodiment, in the coupling reaction, the molar concentration of the compound shown in Formula II in the organic solvent is 0.1 mol / L - 1.0 mol / L, preferably 0.1 mol / L - 0.2 mol / L, more preferably 0.15 mol / L.

[0071] In one embodiment, in the coupling reaction, the molar ratio of the compound shown in Formula III to the compound shown in Formula II is (1.0 - 3.0):1, preferably (1.0 - 2.0):1, more preferably 1.5:1.

[0072] In one embodiment, the coupling reaction is carried out in the presence of 4,5 - dicyanoimidazole. Preferably, the molar ratio of 4,5 - dicyanoimidazole to the compound shown in Formula II is (0.1 - 0.8):1, preferably (0.1 - 0.5):1, more preferably 0.3:1.

[0073] In the present invention, the temperature of the coupling reaction can be the conventional temperature for this type of coupling reaction in the art, such as 10°C - 40°C, preferably 20°C - 30°C, more preferably 25°C.

[0074] On the basis of conforming to the common knowledge in the art, the above - mentioned preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0075] The reagents and raw materials used in the present invention are all commercially available.

[0076] The positive and progressive effects of the present invention are as follows: In the preparation method of the compound shown in Formula 3, no racemization occurs during the reaction process, and the product completely retains the chirality of the raw material. The compound shown in Formula 3 can be directly used to prepare L - serinol with single chirality and high ee value, without SFC chiral separation, saving costs. L - serinol can be further used to prepare SNA phosphoramidite monomers containing four bases. Description of the Drawings

[0077] Figure 1 It is the SFC chromatogram of the racemate 1 of the compound in Comparative Example 1.

[0078] Figure 2 It is the SFC chromatogram of Intermediate 1 in Preparation Example 1.

[0079] Figure 3 It is the SFC chromatogram of Compound 4C in Example 1.

[0080] Figure 4 It is the phosphorus spectrum of the final product SNA A(Bz) phosphoramidite in Example 4.

[0081] Figure 5 It is the phosphorus spectrum of the final product SNA T phosphoramidite in Example 2.

[0082] Figure 6 It is the phosphorus spectrum of the final product SNA C(Bz) phosphoramidite in Example 1.

[0083] Figure 7 It is the phosphorus spectrum of the final product SNA G(iBu) phosphoramidite in Example 3. Detailed implementation manners

[0084] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0085] Comparative Example 1

[0086] The racemate 1, an important intermediate of SNA phosphoramidite reported in the literature Tetrahedron Letters - 1998 - 39 - 6167, has the following synthesis method:

[0087]

[0088] The serine methyl ester (Compound B) protected by trifluoroacetyl group was protected with 4,4 - dimethoxytriphenylmethyl to obtain the fully protected Compound C; finally, reduction and deprotection were carried out through the sodium borohydride - Lewis acid system to obtain the 4,4 - dimethoxytriphenylmethyl - protected L - serine alcohol racemate 1. Experiments were carried out with reference to the synthesis operations in the above - mentioned literature, and the results are as follows:

[0089] Preparation of the starting compound B

[0090] Under the condition of room temperature of 20 - 25 °C, L-serine methyl ester hydrochloride (100 g, 643 mmol, 1 eq) was added to anhydrous methanol (2.00 L). Then, triethylamine TEA (163 g, 1.61 mol, 224 mL, 2.50 eq) and ethyl trifluoroacetate (183 g, 1.29 mol, 177 mL, 2.00 eq) were added to this reaction system. The reaction was stirred at room temperature of 20 - 25 °C for 12 hours and monitored by TLC (methylene chloride:methanol = 5:1) until the reaction was complete. Most of the organic solvents were concentrated under reduced pressure to obtain 200 g of the crude product of compound B. The crude product was separated and purified by column chromatography (stationary phase: silica gel of 100 - 200 mesh, eluent: methylene chloride:methanol = 15:1 - 1:1) to obtain 113 g of the pure product of compound B, which was a yellow oil (yield: 81.7%).

[0091] Step 1:

[0092] Under the condition of room temperature of 25 °C, compound B (95.0 g, 442 mmol, 1.00 eq) was added to dried pyridine Py (900 mL). The reaction system was purged with nitrogen three times, and under the atmosphere of nitrogen bubbling at 0 °C, DMTrCl (142.14 g, 419.52 mmol, 0.95 eq) was added portionwise to the reaction system. After the whole reaction system gradually returned to room temperature of 25 °C, the reaction was continued to stir for 6 hours until the reaction was detected to be complete by LCMS. A saturated aqueous sodium bicarbonate solution (1.00 L) was added to the reaction system. It was extracted 3 times with ethyl acetate (800 mL / time). Then, the combined organic phases after extraction were washed 2 times with saturated aqueous sodium chloride solution (600 mL / time). The finally obtained organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product of compound C. The crude product was separated and purified by column chromatography (stationary phase: silica gel of 100 - 200 mesh, eluent: methylene chloride:methanol = 20:1 - 1:1) to obtain 185 g of the crude product of compound C (LCMS purity: 80%), which was a yellow oil (yield: 64.7%)

[0093] Step 2:

[0094] Under the condition of room temperature of 20 - 25 °C, compound C (200 g, 386 mmol, 1.00 eq) was added to the mixed solvent of anhydrous tetrahydrofuran (1.40 L) and anhydrous ethanol (1.40 L). At this temperature, anhydrous calcium chloride (42.9 g, 386 mmol, 1.00 eq) and sodium borohydride NaBH 4 (29.2 g, 773 mmol, 2.00 eq) were successively added to this reaction system, and the stirring was continued for 16 hours until the reaction was detected to be complete by LCMS. A saturated aqueous ammonium chloride solution with ice (6.00 L) was slowly added to this reaction system to quench the residual excessive NaBH4 , and extracted 7 times with ethyl acetate (1.00 L / time). The combined organic phases after extraction were washed once with saturated sodium chloride aqueous solution (1.00 L / time). The finally obtained organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product of racemate 1. It was separated and purified by column chromatography (stationary phase: 100 - 200 mesh silica gel, eluent: dichloromethane:methanol = 50:1 - 1:1) to obtain 80 g of the pure product of racemate 1 (HPLC purity was 94%), which was a yellow oil. Analyzed by SFC [column model: Chiralcel OJ-3 (150 mm × 4.6 mm × 3 um); mobile phase: phase A was supercritical carbon dioxide, phase B was methanol (0.1% isopropylamine); gradient (B%): 10% - 50%; flow rate: 2.5 mL / min] (see Figure 1 ), it was known that by comparing with the standard product, the peak with a retention time of 2.573 min was the target S-configuration product, and ee = 35.52%. The yield of the target S-configuration product was 49.4%.

[0095] In summary, the synthetic method of the SNA common intermediate - L-serinol reported in the literature Tetrahedron Letters - 1998 - 39 - 6167 would lead to the racemization of the chiral center. Specifically, racemization would occur in the second step of this route. Therefore the yield was relatively low. And it needed expensive SFC purification to obtain which greatly increased the synthesis cost.

[0096] Synthesis of the common intermediate of SNA phosphoramidite - Intermediate 1 in Preparation Example 1

[0097]

[0098] Step 1:

[0099] Dissolve compound 1 (45.0 g, 131.8 mmol, 1.00 eq) after azeotropically removing water with anhydrous toluene in dry pyridine (225 mL) solvent. Under a nitrogen atmosphere, add 4,4'-dimethoxytrityl chloride (53.6 g, 158.2 mmol, 1.20 eq) to the above solution and stir and react at room temperature of 25 °C for 4 hours. After monitoring the reaction by LCMS and it was completed, add methanol (300 mL) to the reaction solution to quench the reaction, concentrate and remove the organic phase solvent under reduced pressure, and purify by passing through a silica gel column of 100 - 200 mesh (polarity of eluent: petroleum ether:ethyl acetate = 10:1) to obtain 57.5 g of a yellow oil, which was the pure product of compound 2 (purity was 95.8%, yield was 64.5%). Those skilled in the art knew that the above hydroxyl protection reaction would not change the chirality of the compound, and after the reaction ended, it was not necessary to carry out SFC separation treatment to obtain the intermediate 1 of a single configuration.

[0100] Structural characterization of Compound 2:

[0101] 1 H NMR (400 MHz, CDCl 3 ): δ 8.65 (s, 1H), 7.80 (d, J = 8 Hz, 1H), 7.65 - 7.60 (m, 2H), 7.43 - 7.17 (m, 14H), 6.82 (dd, J = 2.8 Hz, J = 8 Hz, 4H), 5.72 (d, J = 8.4 Hz, 1H), 4.53 - 4.50 (m, 1H), 4.40 - 4.23 (m, 3H), 3.83 - 3.76 (m, 9H), 3.59 (dd, J = 3.6 Hz, J = 2.8 Hz, 1H), 3.43 (dd, J = 2.8 Hz, J = 2.8 Hz, 1H).

[0102] Step 2:

[0103] Dissolve Compound 2 (50.0 g, 77.7 mmol, 1.00 eq) in a mixed solvent of anhydrous tetrahydrofuran (350 mL) and anhydrous ethanol (350 mL). After cooling to 0 °C in an ice - water bath, while stirring, add calcium chloride (8.62 g, 77.67 mmol, 1.00 eq) and sodium borohydride (5.88 g, 155.35 mmol, 2.00 eq) in sequence. The temperature change during the entire feeding process is controlled within ±5 °C. After the feeding is completed, restore the system to room temperature (25 °C) and continue stirring for 16 hours. Monitor the reaction by TLC (petroleum ether: ethyl acetate = 1:1). Control the temperature of the system at about 0 °C, add saturated ammonium chloride aqueous solution (1000 mL) dropwise, then extract the aqueous phase with ethyl acetate (150 mL × 2), combine the organic phases and wash them once with saturated brine (500 mL). Finally, dry over anhydrous sodium sulfate, filter, and concentrate the organic phase solvent under reduced pressure to obtain the crude product of this compound. Pass it through a silica gel column with a mesh size of 100 - 200 (eluent: petroleum ether: ethyl acetate = 2:1) to obtain 26 g of a white solid, which is the pure Compound 3 (purity is 98.0%, yield is 53.2%).

[0104] In the subsequent de - Fmoc protection step (Compound 3 → Intermediate 1), the ee of the product is 100%, and it is well - known to those skilled in the art that the de - Fmoc protection reaction does not change the chirality of the compound. Therefore, it can be inferred without doubt that the ee value of Step 2 is also 100%.

[0105] Structural characterization of Compound 3:

[0106] 1 H NMR (400 MHz, CDCl 3): δ 7.79 - 7.17 (m, 17H), 6.85 - 6.77 (m, 4H), 5.38 (dd, J = 2.4 Hz, J = 7.6 Hz, 1H), 4.46 - 4.23 (m, 3H), 3.86 - 3.77 (m, 9H), 3.52 - 3.21 (m, 1H), 2.26 - 2.21 (m, 1H).

[0107] Step 3:

[0108] Dissolve compound 3 (43.0 g, 69.8 mmol, 1.00 eq) in N,N-dimethylformamide (300 mL). Add piperidine (5.95 g, 69.8 mmol, 6.90 mL, 1.00 eq) to the above reaction solution, and stir the reaction at room temperature (25 °C) for 3 hours. Monitor the completion of the reaction by TLC (dichloromethane:methanol = 10:1). Add saturated aqueous sodium bicarbonate solution (300 mL) to the reaction solution and extract with dichloromethane (100 mL x 2). Wash the combined organic phases once with saturated brine (300 mL), dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the crude product. Purify the crude product by column chromatography on silica gel (100 - 200 mesh) (eluent polarity: dichloromethane:methanol = 20:1) to obtain 22.0 g of a white solid, which is intermediate 1 (yield 53.5%, ee = 100%).

[0109] In this route, the absolute configuration of the product is determined by the starting material, and there is no chemical operation involving chiral inversion in the synthetic route. Therefore, it can be confirmed that the obtained main product is the target chiral product, namely intermediate 1. Analyzed by SFC [column model: Chiralcel OJ-3 (150 mm × 4.6 mm × 3 μm); mobile phase: phase A is supercritical carbon dioxide, phase B is methanol (0.1% isopropylamine); gradient (B%): 10% - 50%; flow rate: 2.5 mL / min] (see Figure 2 ), it can be known that the ee of this step of the reaction is 100%, Figure 2 and the main peak of

[0110] Structural characterization of intermediate 1: 1 H NMR (400 MHz CDCl 3 ): δ 7.39 - 7.20 (m, 9H), 6.88 (d, J = 8.8 Hz, 4H), 3.72 (s, 6H), 3.42 (dd, J = 4.4 Hz, J = 4.0 Hz, 1H), 3.27 (dd, J = 5.6 Hz, J = 5.2 Hz, 1H), 2.94 - 2.84 (m, 3H).

[0111] Example 1

[0112]

[0113] Step 1:

[0114] Dissolve compound 1C (40.0 g, 185.8 mmol, 1.00 eq) in N,N-dimethylformamide (400 mL), control the temperature at 0 °C, add sodium hydride (8.62 g, 215 mmol, 60% purity, 1.16 eq) to the above reaction system, and keep stirring the system at 0 °C for 1 hour. Then, at 0 °C, dropwise add tert-butyl bromoacetate (39.8 g, 204.5 mmol, 30.2 mL, 1.10 eq) to the reaction system. After all the addition, the reaction system is restored to room temperature (25 °C) and stirred for another 1 hour. Monitor the completion of the reaction by TLC (dichloromethane:methanol = 15:1). Add ice water (200 mL) to the reaction system and stir at 0 °C for 10 minutes. White solid precipitates. Filter under reduced pressure, wash the filter cake with water, and drain the filter cake as much as possible. Dry the solid on the filter cake under vacuum to obtain the crude product. At 20 °C, homogenize and purify the crude product with a mixed solvent of ethyl acetate:petroleum ether = 1:5 (total volume 900 mL). Stir for 30 minutes, then filter under reduced pressure and drain the filter cake as much as possible. Dry the solid on the filter cake under vacuum to obtain 38.0 g of off-white solid, which is compound 2C (yield: 63.3%).

[0115] Structure characterization of compound 2C:

[0116] MS-ESI: m / z = 330.3 [M+H] + .

[0117] 1 H NMR (400 MHz, DMSO-d 6 ): δ 8.11 (d, J = 7.2 Hz, 1H), 8.01 (d, J = 7.6 Hz, 2H), 7.63 (t, J = 7.2 Hz, 1H), 7.51 (t, J = 8.0 Hz, 2H), 7.34 (d, J = 7.2 Hz, 1H), 4.55 (s, 2H), 1.42 (s, 9H).

[0118] Step 2:

[0119] Compound 2C (38.0 g, 115.3 mmol, 1.00 eq) was dissolved in anhydrous dichloromethane (190 mL). At 20 - 25 °C, hydrochloric acid / ethyl acetate solution (4.00 M, 1140 mL, 39.5 eq) was added to the above reaction solution, and the reaction was continuously stirred at this temperature for 16 hours. The reaction was monitored by LCMS until completion. The reaction solution was concentrated under reduced pressure to obtain the crude product. At 25 °C, this crude product was suspended in ethyl acetate (200 mL), homogenized for 1 hour and filtered under reduced pressure. This operation was repeated twice, and the obtained solid cake was dried under vacuum to finally obtain 34.0 g of a white solid product, which was the crude product of compound 3C.

[0120] Structural characterization of compound 3C:

[0121] MS-ESI: m / z = 274.3 [M+H] + .

[0122] 1 H NMR (400 MHz DMSO-d 6 ): δ 8.19 (d, J = 7.6 Hz, 1H), 8.03 (d, J = 7.6 Hz, 2H), 7.64 (t, J = 7.2 Hz, 1H), 7.53 (t, J = 8.0 Hz, 2H), 7.35 (d, J = 7.6 Hz, 1H), 4.62 (s, 2H).

[0123] Step 3:

[0124] At 25 °C, intermediate 1 (4.32 g, 10.9 mmol, 1.20 eq), N,N-diisopropylethylamine (3.55 g, 27.4 mmol, 4.78 mL, 3 eq), and compound 3C (2.50 g, 9.15 mmol, 1.00 eq) were successively added to the solvent N,N-dimethylformamide (22.5 mL) to obtain a homogeneous reaction solution S1. At 25 °C, HBTU (4.16 g, 10.9 mmol, 1.20 eq) was dissolved in N,N-dimethylformamide (20 mL) to obtain a homogeneous solution S2. At 0 °C, solution S2 was slowly added dropwise to reaction solution S1, and after complete addition, the reaction was continued to stir at 0 °C for 1 hour. After monitoring the reaction to completion by LCMS, water (100 mL) was added to the reaction system, and then the aqueous phase was extracted with ethyl acetate (50 mL x 4). The organic phases were combined and washed three times with saturated brine (100 mL x 3). After drying over anhydrous sodium sulfate and filtration, the organic phase solvent was concentrated under reduced pressure to obtain the crude product of this product. The crude product was purified by passing through a silica gel column with a mesh size of 100-200 (eluent polarity: dichloromethane:methanol = 1:0 to 30:1) to obtain 3.5 g of a yellow solid, which was compound 4C (yield 59.0%). Analysis by SFC detection [column model: (S,S)-WHELK-O1 (50 mm × 4.6 mm × 3.5 um; mobile phase: phase A was supercritical carbon dioxide, phase B was methanol (0.1% isopropylamine); gradient (B%): 50%; flow rate: 4 mL / min] (see Figure 3 ) showed that the target product was at a retention time of 1.161 min and the chiral purity was 99.29%.

[0125] Structural characterization of compound 4C:

[0126] MS-ESI: m / z = 647.2 [M-H] - .

[0127] 1 H NMR (400 MHz CDCl 3): δ11.2 (s, 1H), 8.18 (d, J = 8.4 Hz, 1H), 8.02 (t, J = 7.6 Hz, 3H), 7.62 (t, J = 7.2 Hz, 3H), 7.51 (t, J = 7.6 Hz, 2H), 7.39 (d, J = 7.2 Hz, 2H), 7.33 (t, J = 3.6 Hz, 3H), 7.26 - 7.21 (m, 5H), 6.89 (dd, J = 1.6 Hz, J = 1.2 Hz, 4H), 4.68 (t, J = 5.2 Hz, 1H), 4.56 (d, J = 6.4 Hz, 2H), 4.05 - 3.95 (m, 1H), 3.73 (s, 6H), 3.55 - 3.46 (m, 2H), 3.06 - 2.93 (m, 2H).

[0128] Step 4:

[0129] Dissolve compound 4C (1.50 g, 2.31 mmol, 1.00 eq) in anhydrous dichloromethane (15.0 mL). Under a nitrogen atmosphere at 0 °C, add 4,5 - dicyanoimidazole (81.9 mg, 696.7 mmol, 0.30 eq) and bis - (diisopropylamino)(2 - cyanoethoxy)phosphine (1.05 g, 3.47 mmol, 1.10 mL, 1.50 eq) to this solution successively. After complete addition, displace the nitrogen in the reaction system three times. Gradually warm the reaction to 25 °C and continue stirring for 2 hours. Monitor the reaction by LCMS until it is complete. Add saturated sodium bicarbonate solution (30 mL) to the reaction solution, extract the aqueous phase with dichloromethane (20 mL × 4), combine the organic phases, wash with saturated sodium bicarbonate solution (30 mL × 3), and then wash once with saturated brine (30 mL). Dry over anhydrous sodium sulfate, filter, and concentrate the organic phase solvent under reduced pressure to obtain the crude product. Purify this crude product by passing it through a basic silica gel column (100 - 200 mesh, eluent polarity: ethyl acetate:acetone:triethylamine = 20:1:1% - 1:1:1%). Add the product purified by column chromatography to anhydrous dichloromethane (3 mL), slowly dropwise add the poor solvent n - hexane (30 mL), stir for 10 minutes, an oily substance will precipitate. Let it stand for 5 minutes, pour off the upper clear liquid, repeat this operation ten times, and concentrate under reduced pressure to remove all the organic solvents coating the oily product to obtain 2.3 g of a yellow solid, which is the pure product of SNA C(Bz) phosphoramidite (yield: 58.6%).

[0130] Structural characterization of SNA C(Bz) phosphoramidite:

[0131] 1 H NMR (400 MHz CD 3CN): δ 9.46 (s, 1H), 7.96 (d, J = 7.2 Hz, 2H), 7.77 - 7.74 (m, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.59 - 7.177 (m, 12H), 7.05 (t, J = 9.6 Hz, 1H), 6.84 (d, J = 8.8 Hz, 4H), 4.63 - 4.42 (m, 2H), 4.27 - 4.18 (m, 1H), 3.81 - 3.46 (m, 12H), 3.20 - 3.11 (m, 2H), 2.59 - 2.54 (m, 2H), 1.14 - 1.05 (m, 12H).

[0132] The phosphorus spectrum of the final product SNAC(Bz) phosphoramidite is shown in Figure 6 .

[0133] Examples 2 - 3

[0134] Referring to the steps of Example 1 around the key common intermediate Intermediate 1, Example 2: SNAT phosphoramidite and Example 3: SNA G(iBu) phosphoramidite were synthesized, with the difference that the starting materials were the corresponding thymine and guanine.

[0135]

[0136] Example 4

[0137]

[0138] Step 1:

[0139] Dissolve compound 1A (50.0 g, 209.0 mmol, 1.00 eq) in N,N-dimethylformamide (250 mL). While controlling the temperature at 0 - 5 °C, add sodium hydride (8.36 g, 215 mmol, 60% purity, 1.00 eq) to the above reaction solution, stir continuously for 30 minutes, then restore to room temperature and continue stirring for 1 hour. Cool the system to 0 °C again, and control the temperature change not to exceed ±5 °C. Slowly add ethyl bromoacetate (34.9 g, 209.00 mmol, 23.1 mL, 1.00 eq) dropwise to the reaction system. After complete addition, restore to room temperature (25 °C) and continue stirring the reaction for 2 hours. Monitor the reaction by LCMS until completion. Add ice water (200 mL) to the reaction system and stir at 0 °C for 10 minutes. White solid precipitates. Filter under reduced pressure, wash the filter cake with water, and drain the filter cake as much as possible. Dry the solid on the filter cake under vacuum to obtain the crude product of this product. Separate by preparative HPLC {column: Phenomenex luna C18 (250x150mm x 15um); mobile phase: [water (0.1% trifluoroacetic acid) - acetonitrile]; acetonitrile gradient: 20% - 65%, 20.0 minutes} to separate the two isomers attached to adenine N 7 and N 9 positions to obtain 31.0 g of white solid, which is the target product compound 2A attached to the N 9 position (yield 45.6%).

[0140] Structural characterization of compound 2A:

[0141] MS-ESI: m / z = 326.0 [M+H] + .

[0142] Step 2:

[0143] At 0 °C, dissolve sodium hydroxide (4.18 g, 104.5 mmol, 2.00 eq) in water (187 mL) (there will be an obvious exothermic reaction). Restore the prepared solution to room temperature and at 20 - 25 °C, add compound 2A (17.0 g, 52.26 mmol, 1.00 eq) to it. After complete addition, continue stirring the reaction solution for 2 hours and monitor the reaction by LCMS until completion. Add hydrochloric acid (12.0 M) to the reaction system until the pH value of the reaction solution reaches 2.5. At this time, white solid precipitates. Filter under reduced pressure and wash the filter cake with deionized water. After drying the solid on the filter cake under vacuum, obtain 14.0 g of white solid, which is the crude product of compound 3A (yield approximately 89.8%).

[0144] Structural characterization of compound 3A:

[0145] MS-ESI: m / z = 298.0 [M+H] +.

[0146] 1 H NMR (400 MHz, DMSO-d 6 ): δ 11.2 (s, 1H), 8.73 (s, 1H), 8.46 (s, 1H), 8.06 (d, J = 7.2 Hz, 2H), 7.64 (t, J = 6 Hz, 1H), 7.56 (t, J = 7.6 Hz, 2H), 5.12 (s, 1H).

[0147] Step 3:

[0148] At 20 °C, compound 3A (1.50 g, 5.05 mmol, 1.00 eq) and HBTU (2.30 g, 6.06 mmol, 1.20 eq) were dissolved in N,N-dimethylformamide (12.0 mL) to obtain a clear reaction solution S1. The common intermediate intermediate 1 (2.18 g, 5.55 mmol, 1.10 eq) and N,N-diisopropylethylamine (1.96 g, 15.1 mmol, 2.64 mL, 3.00 eq) were dissolved in N,N-dimethylformamide (15.0 mL) to obtain a clear solution S2. At 20 °C, solution S2 was added dropwise to reaction solution S1, and after complete addition, the reaction was stirred for an additional 1 hour. After monitoring the reaction to completion by LCMS, saturated aqueous sodium bicarbonate (50 mL) was added to the reaction system, and then the aqueous phase was extracted with ethyl acetate (40 mL x 3). The organic phases were combined and washed three times with saturated brine (100 mL x 3). After drying over anhydrous sodium sulfate and filtration, the organic phase solvent was concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by preparative HPLC {column: Phenomenex luna C18 (250 x 150 mm x 15 um); mobile phase: [water - acetonitrile]; acetonitrile gradient: 10% - 45%, 20.0 minutes}, to obtain 2.50 g of a white solid, which is compound 4A, (yield 73.6%).

[0149] Step 4:

[0150] Dissolve compound 4A (2.0 g, 2.97 mmol, 1.00 eq) in anhydrous dichloromethane (14.0 mL). At 0 °C under a nitrogen atmosphere, add 4,5-dicyanoimidazole (105.3 mg, 891.9 μmol, 0.30 eq) and phosphine reagent bis-(diisopropylamino)(2-cyanoethoxy)phosphine (1.34 g, 4.46 mmol, 1.42 mL, 1.50 eq) to this solution in sequence. After complete addition, displace nitrogen in this reaction system three times. Gradually warm up to 25 °C and continue stirring the reaction for 2 hours. Monitor the reaction by LCMS until completion. Add saturated sodium bicarbonate solution (20 mL) to the reaction system, extract the aqueous phase with dichloromethane (10 mL × 2), combine the organic phases, wash with saturated sodium bicarbonate solution (20 mL × 3), and then wash once with saturated brine (20 mL). Dry over anhydrous sodium sulfate, filter, and concentrate the organic phase solvent under reduced pressure to obtain the crude product of this product. Purify through a basic silica gel column (100 - 200 mesh, eluent polarity: ethyl acetate:acetone:triethylamine = 20:1:1% - 15:1:1%) to obtain 1.4 g of a pale yellow solid, which is the pure product of the final product SNA A(Bz) phosphoramidite (yield: 53.9%).

[0151] Structural characterization of compound SNA A(Bz) phosphoramidite:

[0152] MS-ESI: m / z = 871.3 [M-H] - .

[0153] 1 H NMR (400 MHz CD 3 CN): δ 9.50 (s, 1H), 8.58 (s, 1H), 8.00 (d, J = 8.15 Hz, 1H), 8.00 (d, J = 7.6 Hz, 2H), 7.54 (t, J = 7.6 Hz, 1H), 7.40 (t, J = 7.6 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 7.27 - 7.21 (m, 7H), 7.22 (d, J = 7.6 Hz, 1H), 6.84 (d, J = 8.8 Hz, 4H), 4.94 (d, J = 8.4 Hz, 2H), 4.26 - 4.24 (m, 1H), 3.75 - 3.53 (m, 12H), 3.16 - 3.12 (m, 2H), 2.60 - 2.56 (m, 2H), 1.15 - 1.0 (m, 12H).

[0154] The phosphorus spectrum of the final product SNA A(Bz) phosphoramidite is shown in Figure 4 .

Claims

1. A method for preparing a compound represented by Formula 3, which comprises the following steps: In an organic solvent, the compound represented by Formula 2 undergoes a reduction reaction in the presence of a reducing agent to obtain the compound represented by Formula 3; wherein, R is methyl or ethyl; The reducing agent is NaBH 4 and / or LiBH 4 ; When the reducing agent contains NaBH 4 the reduction reaction is carried out in the presence of calcium chloride and / or lithium chloride.

2. The preparation method according to claim 1, characterized in that it satisfies one or more of the following conditions: (1) R is methyl; (2) In the reduction reaction, the organic solvent is an ether solvent and / or an alcohol solvent, preferably a mixed solvent of an ether solvent and an alcohol solvent, more preferably a mixed solvent of an ether solvent and an alcohol solvent with a volume ratio of 1:1; the ether solvent is preferably tetrahydrofuran and / or diethyl ether; The alcohol solvent is preferably ethanol and / or methanol; the organic solvent is more preferably a mixed solvent of tetrahydrofuran and ethanol with a volume ratio of 1:1; (3) In the reduction reaction, the molar concentration of the compound represented by Formula 2 in the organic solvent is 0.05 mol / L - 0.5 mol / L, preferably 0.1 mol / L - 0.2 mol / L, more preferably 0.11 mol / L; (4) The molar ratio of the reducing agent to the compound represented by Formula 2 is (1.0 - 4.0): 1, preferably (1.0 - 3.0):1, more preferably (1.5 - 2.5):1, most preferably 2.0:1; (5) When the reducing agent is NaBH 4 4, the reduction reaction is carried out in the presence of calcium chloride and / or lithium chloride; preferably, the molar ratio of the calcium chloride and / or lithium chloride to the compound shown in Formula 2 is (0.5 to 3.0):1, preferably (0.5 to 1.5):1, more preferably 1.0:1; (6) The temperature of the reduction reaction is 10°C - 40°C, preferably 20°C - 30°C, more preferably 25°C; (7) The feeding mode of the reduction reaction is to mix calcium chloride and / or lithium chloride, and the reducing agent with the compound represented by Formula 2 or the solution of the compound represented by Formula 2 in sequence; preferably, the whole feeding process is completed at 0 ± 5°C; (8) The post-treatment mode of the reduction reaction is to mix the reaction solution with saturated ammonium chloride aqueous solution and extract; preferably, the extraction is followed by washing, drying, filtering, concentrating, and column chromatography steps; the washing is preferably carried out using saturated brine.

3. The preparation method according to claim 1, characterized in that The method for preparing the compound represented by Formula 3 further includes the method for preparing the compound represented by Formula 2. The method for preparing the compound represented by Formula 2 includes the following steps: In an organic solvent, the compound represented by Formula 1 undergoes a hydroxyl protection reaction in the presence of 4,4'-dimethoxytrityl chloride to obtain the compound represented by Formula 2; 4. The preparation method according to claim 3, characterized in that The method for preparing the compound represented by Formula 2 satisfies one or more of the following conditions: (1) In the hydroxyl protection reaction, the organic solvent is a basic solvent, preferably pyridine; (2) In the hydroxyl protection reaction, the molar concentration of the compound represented by Formula 1 in the organic solvent is 0.1 mol / L - 1 mol / L, preferably 0.5 mol / L - 0.8 mol / L, more preferably 0.6 mol / L; (3) The molar ratio of the 4,4'-dimethoxytriphenylmethyl chloride to the compound represented by Formula 1 is (1.0 - 3.0):1, preferably (1.0 - 2.0):1, more preferably (1.0 - 1.5):1, and most preferably 1.2:1; (4) The hydroxy protection reaction is carried out under the protection of nitrogen and / or inert gas; (5) The compound represented by Formula 1 is in an anhydrous state; (6) The temperature of the hydroxy protection reaction is 10°C - 40°C, preferably 20°C - 30°C, and more preferably 25°C; (7) The feeding method of the hydroxy protection reaction is to add the 4,4'-dimethoxytriphenylmethyl chloride to the compound represented by Formula 1 or a solution of the compound represented by Formula 1; preferably, the feeding is carried out under the protection of nitrogen; preferably, the compound represented by Formula 1 is dehydrated and then mixed with an organic solvent; (8) The post-treatment method of the hydroxy protection reaction is quenching, concentration, and column chromatography; the quenching is preferably to mix the reaction solution with methanol.

5. A method for preparing a compound represented by Formula 2, wherein the method for preparing the compound represented by Formula 2 is as described in claim 3 or 4; 6. A method for preparing a compound represented by Formula 4, which comprises the following steps: (1) According to the method for preparing the compound represented by Formula 3 as described in any one of claims 1 - 4, the compound represented by Formula 3 is prepared; (2) In an organic solvent, the compound represented by Formula 3 is subjected to a de-Fmoc reaction with a deprotection reagent to obtain the compound represented by Formula 4; 7. The preparation method according to claim 6, characterized in that the preparation method of the compound represented by Formula 4 satisfies one or more of the following conditions: (1) In the de-Fmoc reaction, the organic solvent is an amide solvent, preferably N,N-dimethylformamide; (2) In the de-Fmoc reaction, the molar concentration of the compound represented by Formula 3 in the organic solvent is 0.05 mol / L - 0.5 mol / L, preferably 0.2 mol / L - 0.3 mol / L, and more preferably 0.23 mol / L; (3) The deprotection reagent is a basic deprotection reagent, preferably piperidine; (4) The molar ratio of the deprotection reagent to the compound represented by Formula 3 is (1.0 - 3.0):1, preferably (1.0 - 2.0):1, more preferably (1.0 - 1.5):1, and most preferably 1.0:1; (5) The temperature of the de-Fmoc reaction is 10°C - 40°C, preferably 20°C - 30°C, and more preferably 25°C; (6) The feeding method of the de-Fmoc reaction is to add the deprotection reagent to the compound represented by Formula 3 or a solution of the compound represented by Formula 3; (7) The post-treatment method of the de-Fmoc reaction is to mix the reaction solution with a saturated aqueous sodium bicarbonate solution and extract; preferably, after extraction, it may further include washing, drying, filtering, concentrating, column chromatography steps.

8. The preparation method according to claim 6, characterized in that The preparation method of the compound shown in Formula 4 further includes the preparation method of the compound shown in Formula 2, and the preparation method of the compound shown in Formula 2 is as described in Claim 3 or 4.

9. A compound shown in Formula 2-1; 10. A preparation method of SNA phosphoramidite, which comprises the following steps: (1) Prepare the compound shown in Formula 4 according to the preparation method of the compound shown in Formula 4 as described in Claim 6; (2) In an organic solvent, react the compound shown in Formula 4 according to the following route to obtain SNA phosphoramidite; R 1 For R 2 is a halogen or The coupling reaction is carried out under the protection of nitrogen and / or inert gas.

11. The preparation method as described in Claim 10, characterized in that the preparation method of the SNA phosphoramidite satisfies one or more of the following conditions: (1) In the condensation reaction, the organic solvent is an amide solvent, preferably N,N-dimethylformamide; (2) In the condensation reaction, the molar concentration of the compound shown in Formula I in the organic solvent is 0.1 mol / L - 1.0 mol / L, preferably 0.2 mol / L - 0.6 mol / L, more preferably 0.40 mol / L; (3) In the condensation reaction, the molar ratio of the compound shown in Formula 4 to the compound shown in Formula I is (1.0 - 3.0):1, preferably (1.0 - 1.5):1, more preferably 1.2:1; (4) The condensation reaction is carried out in the presence of N,N-diisopropylethylamine and HBTU; Preferably, the molar ratio of N,N-diisopropylethylamine to the compound shown in Formula I is (1.0 - 5.0):1, preferably (2.5 - 3.5):1, more preferably 3.0:1; the molar ratio of HBTU to the compound shown in Formula I is (1.0 - 3.0):1, preferably (1.0 - 1.5):1, more preferably 1.2:1; (5) The temperature of the condensation reaction is 0 ± 5 °C; (6) In the coupling reaction, the organic solvent is a polar aprotic solvent, preferably anhydrous dichloromethane; (7) In the coupling reaction, the molar concentration of the compound shown in Formula II in the organic solvent is 0.1 mol / L - 1.0 mol / L, preferably 0.1 mol / L - 0.2 mol / L, more preferably 0.15 mol / L; (8) In the coupling reaction, the molar ratio of the compound shown in Formula III to the compound shown in Formula II is (1.0 - 3.0):1, preferably (1.0 - 2.0):1, more preferably 1.5:1; (9) The coupling reaction is carried out in the presence of 4,5-dicyanoimidazole; preferably, the molar ratio of 4,5-dicyanoimidazole to the compound shown in Formula II is (0.1 - 0.8):1, preferably (0.1 - 0.5):1, more preferably 0.3:1; (10) The temperature of the coupling reaction is 10 °C to 40 °C, preferably 20 °C to 30 °C, more preferably 25 °C.

Images