Preparation method of TLR8 agonist intermediate

Abstract

The invention provides a preparation method of a TLR8 agonist intermediate. Specifically, 2-butyl-2-diethyl methylmalonate is used as a raw material and is subjected to resolution and desymmetry by adopting outsourced esterase E2 (expressed by escherichia coli), the yield of the obtained (S)-2-(ethoxycarbonyl)-2-methylhexanoic acid product can reach 95% or above, the chiral purity reaches 99.9% or above, and the yield of the (S)-2-(ethoxycarbonyl)-2-methylhexanoic acid product can reach 95% or above. The preparation method comprises the following steps: carrying out esterification reaction on an (S)-2-(ethoxycarbonyl)-2-methylhexanoic acid product to obtain tert-butyl ester, then selectively hydrolyzing ethyl ester, carrying out amidation reaction, carrying out Hofmann degradation reaction, and finally hydrolyzing the tert-butyl ester to obtain the TLR8 agonist intermediate (R)-2-amino-2-methylhexanoic acid, and the total yield of six steps can reach 81%. Compared with the prior art, the preparation method disclosed by the invention has the advantages of cheap and easily available raw materials, simple reaction operation, high chiral purity, safety, mild reaction conditions, high yield, high chiral purity of the product, environmental friendliness and convenience in industrial production.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry. Specifically, the present invention relates to a preparation method of a novel vericiguat and a new intermediate for preparing 4,6-diamino-pyrido[3,2-d]pyrimidine compounds, and the 4,6-diamino-pyrido[3,2-d]pyrimidine compounds are TLR8 agonists. Background Art

[0002] Toll-like receptors (TLRs) are an important pattern recognition receptor for innate immune responses and are widely distributed in mammalian myeloid dendritic cells, monocytes, and mononuclear macrophages. On the one hand, TLRs can recognize specific microbial PAMPs (such as lipopolysaccharide, flagellin, single / double-stranded RNA, etc.), thereby activating the body's innate immunity; on the other hand, different TLRs can induce the expression of genes in specific functional regions, thereby activating the body's antigen-specific acquired immune response.

[0003] In mammals, 13 TLR members have been discovered. Among them, TLR1-TLR9 and TLR11 are common to humans and mice, and TLR10, TLR12, and TLR13 are unique to mice. TLR8 is a member of the TLRs subgroup (TLRs 3, 7, 8, and 9) and is confined to the endosomal compartment of cells specialized in recognizing non-self nucleic acids. TLR8 is mainly expressed in humans by monocytes, NK cells, and myeloid dendritic cells (mDC). TLR8 agonists can cause the release of various pro-inflammatory cytokines, such as IL-6, IL-12, TNF-α, and IFN-γ.

[0004] After activation, TLR8 mediates inflammatory immunity to promote the clearance of virus-infected cells and tumor cells in the body. Its agonists can be used as independent immunotherapeutic drugs or immunoadjuvants and show important clinical application prospects in immunotherapy. TLR8 activation is closely related to the natural immune response against infection and can mediate the occurrence and development of viral infectious diseases such as HBV, HCV, HIV, herpes virus, tumors, autoimmune diseases, and metabolic diseases.

[0005] CN107108615B discloses a series of 4,6-diamino-pyrido[3,2-d]pyrimidine compounds, such as Example 98, etc., which have good agonist activity for TLR8 activation and are thus used for the treatment of HBV and proliferative diseases. Among them, (R)-2-amino-2-methylhexanoic acid (i.e., corresponding to compound (VII) of the present application) is an important synthetic raw material for synthesizing such compounds, but this raw material is expensive. Currently, the prior art (Organic Process Research And Development, 2023, 27(11): 2111-2122) also discloses that the preparation method of (R)-2-amino-2-methylhexanoic acid is mainly a chiral resolution method, but the yield is low, and its route is as follows:

[0006]

[0007] Therefore, there is a need to develop a preparation method of (R)-2-amino-2-methylhexanoic acid that is safe, efficient, environmentally friendly, economical, simple to operate, high in yield, high in purity, and high in optical purity. Summary of the Invention

[0008] The present invention uses diethyl 2-butyl-2-methylmalonate as a raw material, and uses esterase E2 (expressed in Escherichia coli) to resolve and desymmetrize it. The yield of the obtained (S)-2-(ethoxycarbonyl)-2-methylhexanoic acid product can reach more than 95%, and the chiral purity is as high as more than 99.9%. The (S)-2-(ethoxycarbonyl)-2-methylhexanoic acid product is esterified to obtain a tert-butyl ester, then the ethyl ester is selectively hydrolyzed, followed by an amidation reaction, and then a Hofmann degradation reaction. Finally, after hydrolysis of the tert-butyl ester, the TLR8 agonist intermediate (R)-2-amino-2-methylhexanoic acid is obtained, and the total yield of the six steps can reach 81%. Compared with the prior art process, the preparation method disclosed in the present invention uses inexpensive and easily available raw materials, has high chiral purity, simple reaction operation, is safe, has mild reaction conditions, is environmentally friendly, and is convenient for industrial production.

[0009] On the one hand, the present invention provides a preparation method of a compound shown in formula (VII), characterized in that the method includes step 1),

[0010]

[0011] Step 1): The compound shown in formula (I) undergoes a desymmetrization reaction under the action of esterase E2 to obtain a compound shown in formula (II);

[0012] wherein, R 1 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, phenyl-CH2- or phenyl-(CH2)2-.

[0013] In some embodiments of the preparation method of the compound represented by formula (VII) according to the present invention, the desymmetrization reaction in step 1) is carried out at a temperature of 35°C to 70°C. In some embodiments of the present invention, the desymmetrization reaction in step 1) is carried out at a temperature of 40°C to 70°C. In some embodiments of the present invention, the desymmetrization reaction in step 1) is carried out at a temperature of 50°C to 70°C.

[0014] In some embodiments of the preparation method of the compound represented by formula (VII) according to the present invention, the reaction system of the symmetrization reaction in step 1) is carried out under alkaline conditions. In some embodiments of the present invention, the reaction system of the symmetrization reaction in step 1) is carried out under the condition of pH value = 8 - 10. In some embodiments of the present invention, the reaction system of the symmetrization reaction in step 1) is carried out under the condition of pH value = 8.5 - 9.5.

[0015] In some embodiments of the preparation method of the compound represented by formula (VII) according to the present invention, the reaction solvent for the symmetrization reaction in step 1) is phosphate buffer solution.

[0016] In some embodiments of the preparation method of the compound represented by formula (VII) according to the present invention, the concentration of the phosphate buffer solution is 0.1 - 1 mol / L.

[0017] In some embodiments of the preparation method of the compound represented by formula (VII) according to the present invention, the concentration of the phosphate buffer solution is 0.2 mol / L.

[0018] In some embodiments of the preparation method of the compound represented by formula (VII) according to the present invention, the phosphate is sodium dihydrogen phosphate, sodium hydrogen phosphate or a combination thereof.

[0019] In some embodiments of the preparation method of the compound represented by formula (VII) according to the present invention, the solvent dosage for the reaction in step 1) is 5 - 20 mL of solvent per 1 g of the compound represented by formula (I). In some embodiments of the present invention, the solvent dosage for the reaction in step 1) is 10 - 15 mL of solvent per 1 g of the compound represented by formula (I).

[0020] In some embodiments of the preparation method of the compound represented by formula (VII) according to the present invention, the feeding sequence in step 1) is that esterase E2 is dissolved in phosphate buffer solution, and then the compound represented by formula (I) is added;

[0021] In some embodiments of the preparation method of the compound represented by formula (VII) of the present invention, step 1) includes post-treatment, and the post-treatment is as follows: first, acidify the reaction solution after the reaction with 1M hydrochloric acid to a pH value of 2-3, then extract with an organic solvent, and finally concentrate the organic solvent.

[0022] In some embodiments of the preparation method of the compound represented by formula (VII) of the present invention, the method further includes step 2).

[0023]

[0024] Step 2): The compound represented by formula (II) undergoes an esterification reaction in the presence of tert-butanol to obtain the compound represented by formula (III).

[0025] In some embodiments of the preparation method of the compound represented by formula (VII) of the present invention, the reaction in step 2) further includes that the reaction is carried out in the presence of Boc anhydride.

[0026] In some embodiments of the preparation method of the compound represented by formula (VII) of the present invention, step 2) further includes carrying out the reaction under the condition of a catalyst.

[0027] In some embodiments of the preparation method of the compound represented by formula (VII) of the present invention, the catalyst in step 2) is DMAP.

[0028] In some embodiments of the preparation method of the compound represented by formula (VII) of the present invention, the esterification reaction in step 2) further includes carrying out the reaction in reaction solvent A.

[0029] In some embodiments of the preparation method of the compound represented by formula (VII) of the present invention, the reaction solvent A is DCM, DMF, DMAc, THF, acetonitrile, tert-butanol or a mixed solvent thereof.

[0030] In some embodiments of the preparation method of the compound represented by formula (VII) of the present invention, the method further includes step 3).

[0031]

[0032] Step 3): The compound of formula (III) undergoes a selective ester hydrolysis reaction in the presence of a base to obtain compound (IV).

[0033] In some embodiments of the preparation method of the compound represented by formula (VII) of the present invention, the base in step 3) is sodium hydroxide, potassium hydroxide, lithium hydroxide or their hydrates.

[0034] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, the base in step 3) is lithium hydroxide or its hydrate (such as lithium hydroxide monohydrate).

[0035] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, step 3) further includes carrying out in reaction solvent B.

[0036] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, the reaction solvent B in step 3) is methanol, ethanol, propanol, n-butanol, DCM, DMF, DMAc, THF, water or a mixed solvent thereof.

[0037] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, the method further includes step 4).

[0038]

[0039] Step 4): The compound represented by formula (IV) undergoes an amidation reaction in the presence of an ammonia source to obtain the compound represented by formula (V).

[0040] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, the ammonia source in step 4) is ammonium chloride or ammonia water.

[0041] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, the amidation reaction in step 4) is carried out in the presence of a base.

[0042] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, the base in step 4) is ammonia water.

[0043] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, the amidation reaction in step 4) is carried out in the presence of a condensing agent.

[0044] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, the condensing agent in step 4) is selected from HATU, EDCI, CDI or HBTU.

[0045] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, the amidation reaction in step 4) is carried out in reaction solvent C.

[0046] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, the reaction solvent C in step 4) is ethanol, DCM, DMF, DMAc, THF, water or a mixed solvent thereof.

[0047] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, the reaction solvent in step 4) is DCM solvent.

[0048] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, the method further comprises step 5),

[0049]

[0050] Step 5): Compound (V) undergoes Hofmann degradation reaction in the presence of a base to obtain compound (VI).

[0051] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, the base in step 5) is sodium hydroxide, potassium hydroxide, magnesium hydroxide, sodium hypochlorite, sodium hypobromite, or a combination thereof.

[0052] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, step 5) is carried out in reaction solvent D.

[0053] Optionally, in some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, the reaction solvent D in step 5) is methanol, ethanol, propanol, n-butanol, DCM, DMF, DMAc, THF, water, or a mixed solvent thereof.

[0054] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, the method further comprises step 6),

[0055]

[0056] Step 6): Compound (VI) undergoes ester hydrolysis reaction in the presence of an acid to obtain compound (VII).

[0057] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, the acid in step 6) is trifluoroacetic acid or concentrated hydrochloric acid.

[0058] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, step 6) is carried out in reaction solvent E.

[0059] In some embodiments of the method for preparing the compound represented by formula (VII) of the present invention, the reaction solvent E in step 6) is DCM, 1,4-dioxane, THF, or a mixed solvent thereof.

[0060] On the other hand, the present invention also relates to a compound, which is one of the following compounds or a salt thereof,

[0061] The compounds (3), (IV) and (V) of the present invention can be used for the preparation of compound (VII) ((R)-2-amino-2-methylhexanoic acid), and thus can be used for the preparation of 4,6-diamino-pyrido[3,2-d]pyrimidine compounds.

[0062] General synthetic method of the compounds of the present invention

[0063] Generally, the compounds of the present invention can be prepared by the methods described in the present invention. The following reaction schemes and examples are used to further illustrate the content of the present invention.

[0064] In the examples described below, unless otherwise indicated, all temperatures are in degrees Celsius (°C). Reagents were purchased from commercial suppliers such as Aldrich Chemical Company, Arco Chemical Company and Alfa Chemical Company and were used without further purification, unless otherwise indicated.

[0065] The chromatographic column used was a silica gel column, and the silica gel (200 - 300 mesh) was purchased from Qingdao Marine Chemical Factory. Nuclear magnetic resonance spectra were recorded in CDC13, DMSO-d6, CD3OD or acetone-d6 as solvents (reported in ppm), using TMS (0 ppm) or chloroform (7.25 ppm) as the reference standard. When multiplets appeared, the following abbreviations were used: s (singlet), d (doublet), t (triplet), m (multiplet), br (broadened), dd (doublet of doublets), dt (doublet of triplets), br.s (broadened singlet), q (quartet). The coupling constant J is expressed in Hertz (Hz).

[0066] Low-resolution mass spectrometry (MS) data were determined using a spectrometer of the Agilent 6320 series LC-MS equipped with a G1312A binary pump and a G1316A TCC (column temperature maintained at 30 °C), with a G1329A autosampler and a G1315B DAD detector for analysis, and an ESI source for the LC-MS spectrometer.

[0067] Low-resolution mass spectrometry (MS) data was determined using a spectrometer of the Agilent 6120 series LC-MS equipped with a G1311A quaternary pump and a G1316A TCC (column temperature maintained at 30 °C). A G1329A autosampler and a G1315D DAD detector were applied for analysis, and an ESI source was applied to the LC-MS spectrometer.

[0068] Both of the above spectrometers were equipped with an Agilent Zorbax SB-C18 column with a specification of 2.1×30 mm, 5 μm. The injection volume was determined by the sample concentration; the flow rate was 0.6 mL / min; the HPLC peaks were recorded and read at UV-Vis wavelengths of 210 nm and 254 nm. The mobile phase was a 0.1% formic acid acetonitrile solution (phase A) and a 0.1% formic acid ultrapure water solution (phase B). The gradient elution conditions are shown in Table 1:

[0069] Table 1: Gradient elution conditions

[0070] Time (min) <![CDATA[A(CH3CN, 0.1% HCOOH)]]> <![CDATA[B (H2O, 0.1% HCOOH)]]> 0-3 5-100 95-0 3-6 100 0 6-6.1 100-5 0-95 6.1-8 5 95

[0071] Compound purity was evaluated by an Agilent 1100 series high performance liquid chromatography (HPLC), where UV detection was at 230 nm, an IC chromatographic column with a specification of 4.6×250 mm, 5 μm, for 15 minutes, the flow rate was 0.8 mL / min, n-hexane - isopropanol - trifluoroacetic acid (100:3:0.1, v / v / v), and the column temperature was maintained at 30 °C.

[0072] Abbreviations:

[0073] DMAP: 4-dimethylaminopyridine

[0074] CDI: N,N'-carbonyldiimidazole

[0075] DMF: N,N-dimethylformamide

[0076] DCM: Dichloromethane

[0077] DMAc: N,N-dimethylacetamide

[0078] EDCI: 1-ethyl-3(3-dimethylpropylamine)carbodiimide

[0079] HBTU: Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0080] MeOH: Methanol

[0081] h: Hour

[0082] RT, rt: Room temperature

[0083] THF: Tetrahydrofuran

[0084] min: minute;

[0085] Room temperature refers to 10 - 35 °C, preferably 20 - 30 °C;

[0086] HATU: 2-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0087] TEA: Triethylamine

[0088] Synthesis route of the TLR8 agonist intermediate of the present invention

[0089]

[0090] Example 1: Synthesis of (S)-2-(Ethoxycarbonyl)-2-methylhexanoic acid (Compound (22))

[0091]

[0092] Dissolve esterase E2 (25 mg, wt 5%) in sodium dihydrogen phosphate buffer (5 mL, 0.2 M, pH 8.0), then add diethyl 2-butyl-2-methylmalonate (500 mg, 2.17 mmol) to the buffer. The reaction mixture is stirred at 60 °C for 24 h. During the reaction, the pH value of the reaction system is maintained at 8.5 - 9.5 by adding 15% sodium carbonate solution, and HPLC is used to detect until the reaction ends. Adjust the pH to 2.0 - 3.0 with 1 M hydrochloric acid, extract with ethyl acetate (5 mL), and concentrate the organic phase under reduced pressure at 40 °C to obtain Compound (2), which is a pale yellow liquid, 420 mg, with an ee value > 99.9% and a yield of 95.65%.

[0093] 1 1H NMR (400 MHz, CDCl3) δ 4.24 (q, J = 7.1 Hz, 2H), 1.97–1.82 (m, 2H), 1.46 (s, 3H), 1.39–1.20 (m, 7H), 0.92 (t, J = 7.2 Hz, 3H).

[0094] In addition, the inventors changed the reaction temperature, carried out experimental operations according to the method of Example 1, and investigated the influence of the reaction temperature on the experiment. The experimental results are shown in Table A below.

[0095] Table A: Influence results of changing the reaction temperature on the experiment

[0096]

[0097]

[0098] Comparative Example 1

[0099] According to the method of Example 1, the inventors tried to conduct reactions and condition tests on various hydrolases, such as: 1) Lipases: Lipase AK, Lipase G, Lipase PPL, Lipase AY10, Lipase AS, Lipase M, Lipase AYS, Lipase PS, Lipase CRL, and Lipase CAL; 2) Esterases: Esterase E1, Esterase E3, Esterase E4, Esterase L1, Esterase L2, Esterase L3, Esterase L4, Esterase P1, Esterase P2, Esterase P3, Esterase P4, and Porcine liver esterase PLE; 3) Neutral protease, alkaline protease, acidic protease, bromelain, papain, etc. None of these enzymes could convert compound (1) into the target compound (2), or the yield and / or ee value of the target compound (2) was extremely low. For example, among these enzymes, the best ee effect of porcine liver esterase PLE was only 17.06% for the ee value. Again, among these enzymes, the best conversion rate of esterase E3 that could convert compound (1) into the target compound (2) was only 1.86%. The inventors also tried to change the reaction conditions, such as changing the temperature from 20 °C to 70 °C, but it did not improve the yield and / or ee value of the target product.

[0100] Comparative Example 2

[0101] According to the method of Example 1, the inventors also tried the situation of lipase E2 catalyzing different substrates, and the results are shown in Table B below:

[0102]

[0103] Table B: ee results of products with different substrates

[0104] <![CDATA[R 1 > <![CDATA[R 2 > ee value of product Ethyl n-Propyl 96.94％ Ethyl n-Amyl 91.56％ Ethyl n-Hexyl 91.44％ Ethyl Vinyl 83.20％ Ethyl 2-Methylpropenyl 90.14％ Ethyl Ethyl 85.92％ Ethyl <![CDATA[Cyanoethyl (CN(CH2)2-)]]> 53.22％

[0105] The experimental results show that: R 2 is different, and the ee value of the product (II-a) is different, indicating that the catalytic effect of lipase E2 is greatly affected by the substrate.

[0106] Example 2: 1-(tert-butyl)-3-ethyl-(S)-2-butyl-2-methylmalonate (Compound (3))

[0107]

[0108] Compound (2) (400 mg, 1.74 mmol), DMAP (424.4 mg, 3.47 mmol) and acetonitrile (2.5 mL) were added to a reaction flask. Boc2O (758.1 mg, 3.47 mmol) was added, and the reaction was stirred at 30 °C for 2 h. tert-Butanol (4 mL) was added dropwise, and the reaction was stirred at room temperature for 12 h. After the reaction was complete, the solvent was removed by distillation under reduced pressure. Water (2 mL) was added, and 1 M dilute hydrochloric acid was added to adjust the pH to acidic. The mixture was extracted with ethyl acetate (10 mL) three times, and the organic phases were combined, washed twice with saturated sodium bicarbonate solution (5 mL), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain compound (3), a yellow oil (441.1 mg, yield: 98.3%).

[0109] Example 3: (S)-2-(tert-Butoxycarbonyl)-2-methylhexanoic acid (Compound (IV))

[0110]

[0111] Compound (3) (400 mg, 1.55 mmol) and ethanol (4 mL) were added to a reaction flask. An aqueous solution (4 mL) of lithium hydroxide monohydrate (194.9 mg, 4.64 mmol) was added dropwise, and the reaction was stirred at 30 °C for 12 h. After the reaction was complete, the organic solvent was removed by distillation under reduced pressure. 1 M dilute hydrochloric acid solution was added to adjust the pH to acidic. The mixture was extracted with ethyl acetate (10 mL) four times, and the organic phases were combined. The aqueous phase was adjusted to acidic again with dilute hydrochloric acid, and the mixture was extracted with ethyl acetate (10 mL) twice. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure from the filtrate to obtain compound (IV), a yellow oil (356.6 mg, yield: 100%).

[0112] Example 4: tert-Butyl (S)-2-carbamoyl-2-methylhexanoate (Compound (V))

[0113]

[0114] Compound (IV) (300 mg, 1.3 mmol), CDI (253.5 mg, 1.56 mmol) and dichloromethane (5 mL) were added to a reaction flask. The reaction was cooled to 0 °C, and ammonia water (wt 25%, 730 mg, 5.21 mmol) was added. The reaction was warmed to 30 °C and reacted for 12 h. After the reaction was complete, the reaction system was washed successively with water (10 mL), saturated sodium bicarbonate solution (15 mL), and saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure from the filtrate to obtain compound (V), a colorless oil (274.8 mg, yield: 92.1%).

[0115] Example 5: tert-Butyl (R)-2-amino-2-methylhexanoate (Compound (VI))

[0116]

[0117] Compound (V) (250 mg, 1.09 mmol), methanol solution (3 mL), water (1.5 mL) and sodium hydroxide (130.8 mg) were added to a reaction flask. The reaction was cooled to 0 °C, and a sodium hypochlorite solution (wt 10%, 1.22 g, 1.64 mmol) was added dropwise. After the addition was complete, the reaction was transferred to 30 °C and reacted for 12 h. After the reaction was complete, the organic solvent was removed by concentration under reduced pressure. The residue was added with ethyl acetate (20 ml) and water (5 mL), stirred, and allowed to stand for liquid separation. The aqueous phase was extracted twice with ethyl acetate (10 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was distilled under reduced pressure to obtain compound (VI), a colorless oil (205.2 mg, yield: 93.5%).

[0118] Example 6: (R)-2-Amino-2-methylhexanoic acid (Compound (VII))

[0119]

[0120] Compound (VI) (200 mg, 0.993 mmol), DCM (3 mL) and trifluoroacetic acid (339.8 mg, 2.92 mmol) were added to a reaction flask. The reaction was carried out at 30 °C for 3 h. After the reaction was complete, the solvent was removed by distillation under reduced pressure to obtain compound (VII), a white solid (144.26 mg, yield: 100%. Ee value: 99.1%) MS (ESI, pos.ion) m / z: 146.2 [M+1] + ; 1 H NMR (400 MHz, D2O) δ 2.02–1.89 (m, 1H), 1.90–1.77 (m, 1H), 1.56 (s, 3H), 1.45–1.29 (m, 3H), 1.28–1.17 (m, 1H), 0.88 (t, J = 7.1 Hz, 3H).

[0121] In the description of this specification, the description with reference to the terms "one embodiment", "an embodiment", "example", "specific example", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0122] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing compound (VII), characterized in that: The method comprises the steps of 1), Step 1): the compound represented by formula (I) undergoes a desymmetric reaction under the action of esterase E2 to obtain a compound represented by formula (II); Among them, R 1 It is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, phenyl-CH2- or phenyl-(CH2)2-.

2. The preparation method according to claim 1, characterized in that: The desymmetrization reaction in step 1) is carried out at a temperature of 35°C-70°C; preferably at a temperature of 40°C-70°C; more preferably at a temperature of 50°C-70°C.

3. The preparation method according to claim 1 or 2, characterized in that: The reaction system of the symmetrization reaction in step 1) is carried out under alkaline conditions, more preferably under the condition of pH value = 8-10; more preferably under the condition of pH value = 8.5-9.

5.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The reaction solvent of the symmetrization reaction in step 1) is phosphate buffer; Optionally, the concentration of the phosphate buffer is 0.1 to 1 mol / L, preferably 0.2 mol / L; Optionally, the amount of solvent used in step 1) is 5 to 20 mL per 1 g of the compound represented by formula (I); preferably 10 to 15 mL; Optionally, the order of adding materials in step 1) is to dissolve esterase E2 in phosphate buffer and then add the compound represented by formula (I); Optionally, the step 1) includes post-treatment, which includes: first acidifying the reaction solution after the reaction to a pH value of 2-3 with 1M hydrochloric acid, then extracting with an organic solvent, and finally concentrating the organic solvent.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The method further comprises step 2), Step 2): the compound represented by formula (II) undergoes esterification reaction in the presence of tert-butyl alcohol to obtain the compound represented by formula (III); Optionally, the step 2) reaction further comprises being carried out in the presence of Boc anhydride; Optionally, the step 2) further comprises reacting in the presence of a catalyst, preferably, the catalyst is DMAP; optionally, the esterification reaction in the step 2) further comprises reacting in a reaction solvent A, preferably, the reaction solvent A is DCM, DMF, DMAc, THF, acetonitrile, tert-butanol or a mixed solvent thereof.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The method further comprises step 3), Step 3) The compound of formula (III) undergoes selective ester hydrolysis in the presence of a base to obtain compound (IV); Preferably, the base in step 3) is sodium hydroxide, potassium hydroxide, lithium hydroxide or a hydrate thereof, preferably lithium hydroxide or a hydrate thereof; optionally, step 3) also includes being carried out in a reaction solvent B, preferably, the reaction solvent B is methanol, ethanol, propanol, n-butanol, DCM, DMF, DMAc, THF, water or a mixed solvent thereof.

7. The preparation method according to any one of claims 1 to 6, characterized in that: The method further comprises step 4), Step 4): The compound represented by formula (IV) undergoes an amidation reaction in the presence of an ammonia source to obtain a compound represented by formula (V); Preferably, the ammonia source in step 4) is ammonium chloride or ammonia water; Optionally, the amidation reaction in step 4) is carried out in the presence of a base; preferably, the base is aqueous ammonia; Optionally, the amidation reaction in step 4) is carried out in the presence of a condensing agent; preferably, the condensing agent is selected from HATU, EDCI, CDI or HBTU; Optionally, the amidation reaction in step 4) is carried out in a reaction solvent C. Preferably, the reaction solvent C is ethanol, DCM, DMF, DMAc, THF, water or a mixed solvent thereof; more preferably, DCM solvent.

8. The preparation method according to any one of claims 1 to 7, characterized in that: The method further comprises step 5), Step 5): Compound (V) undergoes Hofmann degradation reaction in the presence of a base to obtain compound (VI); Preferably, the alkali in step 5) is sodium hydroxide, potassium hydroxide, magnesium hydroxide, sodium hypochlorite, sodium hypobromite or a combination thereof; Optionally, the step 5) is reacted in a reaction solvent D. Preferably, the reaction solvent D is methanol, ethanol, propanol, n-butanol, DCM, DMF, DMAc, THF, water or a mixed solvent thereof.

9. The preparation method according to any one of claims 1 to 8, characterized in that: The method further comprises step 6), Step 6) The compound (VI) undergoes ester hydrolysis in the presence of an acid to obtain compound (VII); Preferably, the acid is trifluoroacetic acid or concentrated hydrochloric acid; Optionally, the step 6) is carried out in a reaction solvent E. Preferably, the reaction solvent E is DCM, 1,4-dioxane, THF, or a mixed solvent thereof.

10. A compound which is one of the following compounds or a salt thereof,

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