Integrin ligand key intermediate and preparation method therefor

Abstract

The present invention relates to an integrin ligand key intermediate and a preparation method therefor, and specifically provides a new process for synthesizing target compound (7). Compared with existing synthesis processes, the new synthesis process can avoid racemic intermediate compound resolution, improve yields and reduce costs, thereby strongly supporting cost reduction and large-scale production of an αvβ6 integrin ligand.

Description

An integrin ligand key intermediate and a method of making the same

[0001] Cross-reference to Related Applications

[0002] This application is based on and claims priority to CN application No. 202410814083.1, filed on June 21, 2024, and CN application No. 202411244143.7, filed on September 5, 2024, the contents of which are incorporated herein in their entirety. TECHNICAL FIELD

[0003] The present application relates to the field of biological medicine, and in particular to an integrin ligand key intermediate and a method of making the same. BACKGROUND

[0004] Integrin alpha-v beta-6 (avb6), which is expressed in various cell types including epithelial cells, is a receptor for the latency associated peptide (LAP) of TGF-b and the extracellular matrix (ECM) proteins fibronectin, vitronectin and tenascin. Although barely detectable in normal healthy adult epithelial cells, avb6 integrin is upregulated during wound healing and in different cancers, such as colon, ovarian, endometrial and gastric cancer, and is often associated with poor cancer prognosis. It has been shown that avb6 integrin can promote cell invasion and migration in metastasis and inhibit apoptosis. avb6 integrin can also regulate the expression of matrix metalloproteinases (MMPs) and activate TGF-b1. Increasing evidence, mainly from in vitro studies, suggests that avb6 integrin can promote cancer progression. Therefore, integrin avb6 is attractive as a tumor marker and potential therapeutic target, especially considering its role in the expression of matrix metalloproteinases (MMPs) and activation of TGF-b1.

[0005] In vivo delivery of therapeutically effective compounds, such as pharmaceutical compounds, to desired cells and / or tissues has been a universal challenge for the development of pharmaceutical products. There has been a need for stable and effective targeting ligands that can selectively target cells or tissues that can be used to facilitate the targeted delivery of transported / delivered molecules (e.g., therapeutically active compounds or ingredients) to specific cells or tissues. In fact, there has been a universal need for targeting ligands that can be conjugated to one or more selected transported molecules, such as one or more pharmaceutical products or other payloads, to facilitate the in vivo delivery of the transported molecules to desired cells or tissues. Furthermore, there has been a need for compounds that target integrin ανβ6that are suitable for conjugation to transported molecules to deliver the transported molecules to cells expressing integrin ανβ6in vivo. For particular transported molecules, such as therapeutic oligonucleotide-based compounds (e.g., antisense oligonucleotides or RNAi agents), there has been a need for targeting ligands that can target integrin ανβ6that can be conjugated to the oligonucleotide-based compounds to deliver the therapeutic agents to cells and / or tissues expressing integrin ανβ6and to facilitate the entry of the therapeutic agents into the cells by receptor-mediated endocytosis, pinocytosis, or by other means. SUMMARY

[0006] Based on the above problems in the prior art, the present inventors have conducted research and obtained a new ανβ6integrin ligand (also referred to herein as an ανβ6ligand), i.e., a compound represented by Formula (I) or Formula (II). The ανβ6integrin ligand of the present application is stable in serum and has affinity for and can specifically bind to ανβ6integrin. The ανβ6integrin ligand can be conjugated to transported / delivered molecules to facilitate the delivery of the transported / delivered molecules to desired cells or tissues expressing ανβ6integrin, such as to epithelial cells.

[0007] For the αvβ6 integrin ligand, i.e. the compound shown in formula (I), (S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-(2-(3-((S)-8-methyl-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-3-yl)propanamido)acetamido)propanoic acid, the compound shown in formula (II), (S)-3-(2-(3-((S)-8-methyl-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-3-yl)propanamido)acetamido)-3-(4-(4-(2-(prop-2-yn-1-yloxy)ethoxy)naphthalen-1-yl)phenyl)propanoic acid. A key intermediate for synthesizing the compound of formula (I) or formula (II) is compound 7. The inventors of the present application have previously provided a process for synthesizing the compound shown in formula (I), formula (II) (see the Preparations of the present application), however, the process obtains the chiral compound 7 by resolving the racemic compound 6, which is inefficient.

[0008] In order to improve the efficiency of synthesizing the key intermediate compound 7, the inventors of the present application have designed and optimized the original synthesis process, and further provided two new and different process routes. Both of the two new synthesis processes directly synthesize the target compound 7 from a chiral raw material. Compared with the original synthesis process, the two new synthesis processes can avoid resolving the racemic intermediate compound, improve the yield, and reduce the cost, thereby, can effectively support the cost reduction and large-scale production of the compound of formula (I), formula (II).

[0009] Therefore, in a first aspect of the present application, the present application provides a method for preparing the target compound 7, which comprises:

[0010] (1-1) reacting compound with compound to obtain compound

[0011] (1-2) subjecting compound to a Boc-deprotection reaction to obtain compound

[0012] (1-3) subjecting compound to an intramolecular coupling reaction in the presence of a catalyst and a ligand to obtain compound

[0013] (1-4) subjecting compound to an ester hydrolysis reaction to obtain the target compound 7

[0014] Alternatively, the method comprises:

[0015] (2-1) reacting compound with compound to obtain compound

[0016] (2-2) reacting compound under conditions of intramolecular coupling reaction in the presence of a catalyst and a ligand to obtain compound

[0017] (2-3) reacting compound under conditions of amide hydrolysis reaction to obtain target compound 7

[0018] In some embodiments, in step (1-1), the reaction is carried out at a temperature of -10-0 °C.

[0019] In some embodiments, in step (1-1), the reaction is carried out in a solvent of tetrahydrofuran.

[0020] In some embodiments, in step (1-1), the molar ratio of compound to compound is 1.5:1-1:1.5, preferably 1:1.

[0021] In some embodiments, in step (1-1), the reaction is carried out under a nitrogen atmosphere.

[0022] In some embodiments, in step (1-1), the reaction is carried out in the presence of triphenylphosphine and diisopropyl azodicarboxylate (DIAD).

[0023] In some embodiments, in step (1-1), the molar ratio of compound to the triphenylphosphine is 1:1-1:1.5.

[0024] In some embodiments, in step (1-1), the molar ratio of compound to the diisopropyl azodicarboxylate is 1:1-1:1.5.

[0025] In some embodiments, in step (1-2), the Boc removal reaction is carried out under acidic conditions.

[0026] In some embodiments, in step (1-2), the acidic conditions are provided by a hydrogen chloride dioxane solution.

[0027] In some embodiments, in step (1-2), the de-Boc reaction is carried out at room temperature.

[0028] In some embodiments, in step (1-2), the de-Boc reaction is carried out in the presence of a solvent.

[0029] In some embodiments, in step (1-3), the ligand is a chiral phosphine ligand, preferably Josiphos (CAS: 158923-11-6).

[0030] In some embodiments, in step (1-3), the compound is in a mass ratio of 5:1-10:1 (such as 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1), preferably 6:1, to the catalyst.

[0031] In some embodiments, in step (1-3), the compound is in a mass ratio of 4:1-10:1 (such as 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1), preferably 5:1, to the ligand.

[0032] In some embodiments, in step (1-3), the intramolecular coupling reaction is carried out under a nitrogen atmosphere.

[0033] In some embodiments, in step (1-3), the catalyst is Pd2(dba)3.

[0034] In some embodiments, in step (1-3), the intramolecular coupling reaction is carried out in the presence of a basic additive.

[0035] In some embodiments, in step (1-3), the basic additive is sodium tert-butoxide.

[0036] In some embodiments, in step (1-3), the intramolecular coupling reaction is carried out at a temperature of 70-90 °C (such as 80 °C).

[0037] In some embodiments, in step (1-3), the intramolecular coupling reaction is carried out in the presence of a solvent.

[0038] In some embodiments, in step (1-4), the ester hydrolysis reaction is carried out under basic conditions.

[0039] In some embodiments, in step (1-4), the basic conditions are provided by potassium hydroxide.

[0040] In some embodiments, the ester hydrolysis reaction in steps (1-4) is carried out under a nitrogen atmosphere.

[0041] In some implementations, the ester hydrolysis reaction in steps (1-4) is carried out at room temperature.

[0042] In some embodiments, in steps (1-4), the solvent for the ester hydrolysis reaction is water, isopropanol, or any mixture thereof (e.g., a mixture of water and isopropanol in a volume ratio of 2:1).

[0043] In some implementations, in step (2-1), the compound With compounds The molar ratio of the substances is 1.5:1 to 1:1.5, preferably 1:1.

[0044] In some implementations, the reaction in step (2-1) is carried out under a nitrogen atmosphere.

[0045] In some embodiments, in step (2-1), the reaction is carried out in the presence of triphenylphosphine and diisopropyl azodicarbonate (DIAD).

[0046] In some implementations, in step (2-1), the compound The molar ratio of the substance to the triphenylphosphine is 1:1 to 1:1.5.

[0047] In some implementations, in step (2-1), the compound The molar ratio of the substance to the diisopropyl azodicarbonate is 1:1 to 1:1.5.

[0048] In some implementations, the reaction in step (2-1) is carried out at room temperature.

[0049] In some implementations, step (2-1) includes a compound. compound The mixture of the triphenylphosphine and the diisopropyl azodicarbonate (DIAD) was mixed at a temperature of -10 to 0°C.

[0050] In some embodiments, in step (2-1), the reaction is carried out in a solvent of tetrahydrofuran.

[0051] In some implementations, in step (2-2), the ligand is a chiral phosphine ligand, preferably Josiphos (CAS: 158923-11-6).

[0052] In some implementations, in step (2-2), the compound with the catalyst in a mass ratio of 5: 1-10: 1 (such as 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1), preferably 6: 1.

[0053] In some embodiments, in step (2-2), the intramolecular coupling reaction is carried out under a nitrogen atmosphere. with the ligand in a mass ratio of 4: 1-10: 1 (such as 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1), preferably 5: 1.

[0054] In some embodiments, in step (2-2), the intramolecular coupling reaction is carried out under a nitrogen atmosphere.

[0055] In some embodiments, in step (2-2), the catalyst is Pd2(dba)3.

[0056] In some embodiments, in step (2-2), the intramolecular coupling reaction is carried out in the presence of a basic additive.

[0057] In some embodiments, in step (2-2), the basic additive is sodium tert-butoxide.

[0058] In some embodiments, in step (2-2), the intramolecular coupling reaction is carried out at a temperature of 70-90 °C (such as 80 °C).

[0059] In some embodiments, in step (2-2), the intramolecular coupling reaction is carried out in a solvent of toluene.

[0060] In some embodiments, in step (2-3), the amide hydrolysis reaction is carried out under basic conditions.

[0061] In some embodiments, in step (2-3), the basic conditions are provided by sodium hydroxide.

[0062] In some embodiments, in step (2-3), the amide hydrolysis reaction is carried out under a nitrogen atmosphere.

[0063] In some embodiments, in step (2-3), the amide hydrolysis reaction is carried out at a temperature of 90-110 °C (such as 100 °C).

[0064] In some embodiments, in step (2-3), the solvent for the amide hydrolysis reaction is water.

[0065] In some embodiments, the preparation method of the compound comprises:

[0066] (a) reacting a compound reacting with elemental iodine to obtain the compound

[0067] In some embodiments, in step (a), the compound with elemental iodine is in a molar ratio of 1.5:1 to 1:1.5, preferably 1:1.

[0068] In some embodiments, in step (a), the reaction is carried out under a nitrogen atmosphere.

[0069] In some embodiments, in step (a), the reaction is carried out under basic conditions.

[0070] In some embodiments, in step (a), the basic conditions are provided by sodium carbonate.

[0071] In some embodiments, in step (a), the reaction is carried out at room temperature.

[0072] In some embodiments, in step (a), the mixture comprising and sodium carbonate is mixed with the elemental iodine at a temperature of -10 to 0°C.

[0073] In some embodiments, in step (a), the solvent of the reaction is water.

[0074] In some embodiments, the method for preparing the compound comprises:

[0075] (b-1) activating the carboxyl group of the compound to obtain a carboxyl-activated

[0076] (b-2) subjecting the carboxyl-activated to a reduction reaction to obtain the compound

[0077] In some embodiments, in step (b-1), the activation is achieved using isobutyl chloroformate.

[0078] In some embodiments, in step (b-1), the activation is carried out under a nitrogen atmosphere.

[0079] In some embodiments, in step (b-1), the activation is carried out under basic conditions.

[0080] In some embodiments, in step (b-1), the basic conditions are provided by N-methylmorpholine.

[0081] In some embodiments, in step (b-1), the activation is carried out at a temperature of -10 to 0 °C.

[0082] In some embodiments, in step (b-1), the activation is carried out in a solvent of tetrahydrofuran.

[0083] In some embodiments, in step (b-2), the reduction reaction is achieved by using a reducing agent.

[0084] In some embodiments, in step (b-2), the reducing agent is sodium borohydride.

[0085] In some embodiments, in step (b-2), the reduction reaction is carried out under a nitrogen atmosphere.

[0086] In some embodiments, in step (b-2), the reduction reaction is carried out at room temperature.

[0087] In some embodiments, in step (b-2), the mixing of the carboxyl-activated with the sodium borohydride is carried out at a temperature of -10 to 0 °C.

[0088] In some embodiments, in step (b-2), the reduction reaction is carried out in a solvent of methanol.

[0089] In a second aspect of the present application, the present application provides a method for preparing a compound represented by formula (I) or formula (II), which comprises:

[0090] (c-1) preparing a target compound 7

[0091] (c-2) reacting the target compound 7 with a compound to obtain a compound

[0092] or, reacting the target compound 7 with a compound to obtain a compound

[0093] (c-3) reacting a compound to obtain a compound represented by formula (I)

[0094] or, reacting a compound to obtain a compound represented by formula (II)

[0095] In the present invention, "room temperature" refers to 20-25 °C.

[0096] In addition, after the reaction of each step above, the person skilled in the art also needs to carry out a post-treatment step, which aims to purify the reaction product, remove impurities, and obtain a high-purity target product. These methods are based on the common knowledge in the field of chemical synthesis, and the person skilled in the art will choose one or more post-treatment methods or combinations according to the specific reaction type, product characteristics, and scale. Some conventional post-treatment methods are listed below, but are not limited to these post-treatment methods.

[0097] Drying: removing water or organic solvents from the reaction system, common methods include vacuum drying, heating drying or using drying agents (such as sodium sulfate) to obtain dry products or prepare for subsequent steps.

[0098] Filtration: separating solids from liquids to remove insoluble impurities or solid byproducts, common methods include gravity filtration, vacuum filtration or centrifugal filtration, especially suitable for cases where the reaction solution contains precipitates.

[0099] Concentration: reducing the volume of the solution to enrich the product, usually achieved by rotary evaporation, vacuum distillation or inert gas blowing, commonly used to obtain concentrated solutions or solids after preliminary separation.

[0100] Column chromatography: separating different components in a mixture using a chromatography column (such as a silica gel column), based on the difference in polarity to achieve purification, a widely used purification technique in the laboratory, especially suitable for organic synthesis.

[0101] Washing: washing the product with solvents (such as water, alcohol or organic solvents) to remove soluble impurities or residual reagents, common methods include shaking washing or using a separatory funnel, commonly used for preliminary purification of crude products.

[0102] Extraction: transferring target compounds by selective solvents to separate impurities, typical methods such as liquid-liquid extraction (e.g. separation of aqueous and organic phases), especially suitable for the removal of water-soluble or insoluble impurities.

[0103] Acid / base adjustment of pH: adjusting the acidity or alkalinity of the solution by adding acid (such as hydrochloric acid) or base (such as sodium hydroxide), affecting the solubility of the compound or promoting precipitation (such as crystallization or precipitation purification).

[0104] MPLC (medium pressure liquid chromatography): an automated column chromatography technique that uses moderate pressure to improve separation efficiency and yield, especially suitable for large-scale purification or treatment of complex mixtures, and is a supplement to conventional HPLC (high pressure liquid chromatography).

[0105] Re-crystallization: Dissolve the crude product in hot solvent, and re-crystallize after cooling to purify the solid compound.

[0106] Distillation: Used to purify liquid products, such as normal pressure distillation or reduced pressure distillation, to separate components with different boiling points.

[0107] Centrifugation: High-speed rotation to separate mixtures with different densities, often used to remove precipitates in microbial or colloidal systems.

[0108] Freeze-drying: Suitable for dehydration purification of heat-sensitive products, such as in aqueous systems.

[0109] As understood by those skilled in the art, these post-treatment methods are often used in combination (e.g., first extraction, then concentration, and then purification by column chromatography, etc.) to improve product purity and yield. In actual operation, the choice of method depends on factors such as the specific conditions of the reaction, the nature of the product, and cost efficiency, and those skilled in the art have the ability to choose the appropriate post-treatment method.

[0110] In a third aspect of the present application, the present application provides intermediate compounds selected from the following: BRIEF DESCRIPTION OF DRAWINGS

[0111] Figure 1: Mass spectrum of compound 9.

[0112] Figure 2: Mass spectrum of compound 12.

[0113] Figure 3: Mass spectrum of compound 13.

[0114] Figure 4: Mass spectrum of compound 14.

[0115] Figure 5: Mass spectrum of compound 7.

[0116] Figure 6: Mass spectrum of compound 15.

[0117] Figure 7: Mass spectrum of compound 16. DETAILED DESCRIPTION

[0118] The above content of the present application is further described in detail through the specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present application to only the following examples. Any technology realized based on the above content of the present application falls within the scope of the present application.

[0119] The structure of the compounds is determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). NMR shifts (δ) are given in units of 10-6 (ppm). The determination of NMR is carried out by using (Bruker Avance NOE 400MHz and Bruker Avance NEO 600MHz) nuclear magnetic instrument, and the determination solvent is deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD), and the internal standard is tetramethylsilane (TMS).

[0120] The determination of LC-MS uses Shimadzu liquid chromatograph-mass spectrometer (Shimadzu LC-MS2020 (ESI)). The determination of HPLC uses Shimadzu high-pressure liquid chromatograph (Shimadzu LC-20A). MPLC (medium-pressure preparative chromatography) uses Gilson GX-281 reverse-phase preparative chromatograph. The thin-layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, and the specification used for the separation and purification of products by thin-layer chromatography is 0.4mm-0.5mm. Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

[0121] The known starting materials of the present application can be synthesized by using or according to the methods known in the art, or can be purchased from companies such as Angene Chemical, Chengdu Kelong Chemical, Shaoyuan Chemical Technology, and Bailingwei Technology.

[0122] In the examples, the solution refers to an aqueous solution unless otherwise specified. In the examples, the reaction temperature is room temperature unless otherwise specified. In the examples, M is moles per liter unless otherwise specified.

[0123] In the examples, the reagents are abbreviated as follows:

[0124] DMF: N,N-dimethylformamide; DCM: dichloromethane; DIPEA: N,N-diisopropylethylamine; HATU: 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; Pd2(dba)3: tris(dibenzylideneacetone)dipalladium.

[0125] Preparations

[0126] 1. Synthesis of compounds 1-7

[0127] Step 1. Synthesis of compound 1-2

[0128] To a 1 L three-necked flask was added DMF (245 mL) under ice-bath, then Boc-S-3-amino-3-(4-bromo-phenyl)-propionic acid (45.00 g, 131.20 mmol) was added into the flask and stirred to dissolve completely. 2-(7-Azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (59.83 g, 157.44 mmol) and N,N-diisopropyl ethylamine (33.85 g, 262.40 mmol) were added into the above system and stirred for 10 min. Then, methanol (90 mL) was added into the above system slowly and the system was moved to normal temperature overnight. After the raw material was completely reacted by LC-MS monitoring, the methanol in the system was concentrated, then washed with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (ethyl acetate: petroleum ether = 5: 1) to obtain compound 1-2 (45 g, 95.74% yield). LCMS (E+) m / z: 303.9 [M-56] + .

[0129] Step 2. Synthesis of compound 1-3

[0130] To a 1 L three-necked flask was added (S)-3-(4-bromophenyl)-3-((tert- butoxycarbonyl)amino)propionic acid methyl ester (25.00 g, 69.83 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthalenol (18.80 g, 69.83 mmol), sodium carbonate (14.80 g, 139.66 mmol), tetrakis(triphenylphosphine)palladium (807.23 mg, 0.70 mmol) and toluene / water = 4: 1 (250 mL) under nitrogen protection, the system was stirred at 80 °C overnight and monitored by LC-MS. After the reaction was completed, it was cooled to 0 °C, then the pH was adjusted to 5.0, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography twice (petroleum ether: ethyl acetate = 3: 1, dichloromethane) to obtain compound 1-3 (2.00 g, 6.80% yield). LCMS (E+) m / z: 420.2 [M-H] + .

[0131] Step 3. Synthesis of compound 1-4

[0132] Into a 100 mL three-necked flask, was added compound 1-3 (2.00 g, 4.75 mmol), then dissolved in DMF (20 mL), followed by the addition of compound 1-3' (2.20 g, 5.23 mmol) and potassium carbonate (1.3 g, 9.50 mmol). After the addition was complete, the reaction was stirred at 90 °C for 4 h, which was monitored by LC-MS. After the reaction was completed, the reaction solution was prepared by MPLC to obtain compound 1-4 (2.80 g, 88.38% yield). LCMS (E+) m / z: 689.2 [M+Na] + .

[0133] Step 4. Synthesis of compound 1-5

[0134] Into a 100 mL single-necked flask, was added compound 1-4 (2.80 g, 4.20 mmol), followed by the addition of hydrochloric acid dioxane (20 mL). After the addition was complete, the reaction was stirred at room temperature for 2 h, which was monitored by LC-MS. After the reaction was completed, the reaction solution was concentrated to obtain the crude product of compound 1-5 (3.00 g).

[0135] Step 5. Synthesis of compound 1-6

[0136] Into a 20 mL single-necked flask, was added the hydrochloride salt of compound 1-5 (1.50 g, 2.48 mmol), N-Boc-glycine (434.00 mg, 2.48 mmol), 2-(7-azobenzo-triazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.04 g, 2.73 mmol) and DMF (10 mL). After the system was stirred uniformly, N,N-diisopropyl ethylamine (1.38 g, 9.92 mmol) was added. The system was stirred at room temperature for 2 h, which was monitored by LC-MS. The reaction solution was directly prepared by MPLC to obtain compound 1-6 (1.30 g, 72.40% yield).

[0137] Step 6. Synthesis of compound 1-7

[0138] Into a 50 mL single-necked flask, was added compound 1-6 (1.30 g, 1.80 mmol) and hydrochloric acid dioxane. The system was stirred at room temperature for 2 h, which was monitored by LC-MS. The reaction solution was concentrated to obtain compound 1-7 (1.10 g, 92.59% yield).

[0139] 2. Synthesis of compound represented by formula (I)

[0140] Step 1. Synthesis of compound 3

[0141] Compound 1 (2.00 g, 12.98 mmol), 2 (2.98 g, 14.29 mmol), potassium carbonate (3.60 g, 25.90 mmol) and DMF (20 mL) were added under nitrogen protection. After addition, it was stirred at room temperature for 3 hours, and LC-MS was monitored. After the reaction was completed, water (500 mL) was added, and it was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and prepared by reversed phase to obtain compound 3 (2.00 g, 54.55% yield). LCMS (E+) m / z: 283.1 [M+H] + .

[0142] Step 2. Synthesis of compound 4

[0143] Compound 3 (2.00 g, 7.09 mmol) was added to a 100 mL reaction bottle under nitrogen protection and dissolved with ethanol (20 mL). Then acetic acid (5 mL) and iron powder (2.80 g, 50.00 mmol) were added. After addition, it was reacted at room temperature for 5 hours, and LC-MS was monitored. After the reaction was completed, the system was filtered with diatomite and concentrated, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and then prepared by MPLC to obtain compound 4 (1.78 g, 99.62% yield). LCMS (E+) m / z: 253.0 [M+H] + .

[0144] Step 3. Synthesis of compound 5

[0145] Compound 4 (1.78 g, 7.06 mmol) was added to a 100 mL three-necked flask under nitrogen protection and dissolved with DCE (50 mL). Then sodium triacetoxyborohydride (1.65 g, 7.81 mmol) and acetic acid (5 drops) were added. After addition, it was reacted at room temperature overnight, and LC-MS was monitored. After the reaction was completed, the pH was adjusted to 5 with hydrochloric acid, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and prepared by MPLC to obtain compound 5 (1.00 g, 60.00% yield). LCMS (E+) m / z: 238.2 [M+H] + .

[0146] Step 4. Synthesis of compound 7

[0147] Compound 5 (1.00 g, 4.24 mmol) was added to a 50 mL reaction bottle under nitrogen protection and dissolved with methanol (10 mL). Then sodium hydroxide solution (1 M, 10 mL) was added. After addition, it was reacted at room temperature for 2 hours, and LC-MS was monitored. After the reaction was completed, methanol was removed by concentration, the residue was adjusted to pH = 1 with hydrochloric acid, and prepared by MPLC to obtain hydrochloride of compound 6 (1.00 g, 91.41% yield). LCMS (E+) m / z: 223.3 [M+H]+ .

[0148] Compound 6 was chiral split by SFC to obtain compound 7 and compound 7-iso.

[0149] Step 5. Synthesis of compound 7A

[0150] Into a 4 mL reaction vial was placed compound 7 (50.00 mg, 0.19 mmol) and 1-7 (114.00 mg, 0.19 mmol) and dissolved with N,N'-dimethylformamide (2 mL). To the mixture was added 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (73.00 mg, 0.19 mmol) and N,N-diisopropylethylamine (68.00 mg, 0.60 mmol) with stirring. After addition, the reaction was stirred at room temperature for 2 hours, which was monitored by LC-MS. After the reaction was completed, the reaction solution was prepared by MPLC to obtain compound 7A (80 mg, 50.91% yield). LCMS (E+) m / z: 828.7 [M+H] + .

[0151] Step 6. Synthesis of compound of formula (I)

[0152] Into a 20 mL reaction vial was placed compound 7A (80.00 mg, 0.10 mmol) and dissolved with methanol (2 mL). Then 1M sodium hydroxide solution (2 mL) was added. After addition, the reaction was stirred at room temperature for 1 hour, which was monitored by LC-MS. After the reaction was completed, the compound of formula (I) was directly prepared by MPLC (50.00 mg, 61.50% yield). LCMS (E+) m / z: 814. [M+H] + .

[0153] 1H NMR (600 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.30 - 8.20 (m, 2H), 7.77 (d, J = 8.3 Hz, 1H), 7.51 (dt, J = 15.1, 8.1 Hz, 2H), 7.44 (d, J = 7.8 Hz, 2H), 7.41 (d, J = 4.9 Hz, 1H), 7.31 (dd, J = 23.7, 7.8 Hz, 3H), 7.03 (d, J = 8.0 Hz, 1H), 6.70 (s, 1H), 6.33 (d, J = 5.0 Hz, 1H), 5.23 (s, 1H), 4.31 (d, J = 5.2 Hz, 2H), 4.10 (dd, J = 10.6, 2.8 Hz, 1H), 3.92 (d, J = 4.7 Hz, 2H), 3.81 (dd, J = 10.7, 5.9 Hz, 1H), 3.74 (d, J = 5.8 Hz, 2H), 3.68 (dd, J = 5.8, 3.8 Hz, 2H), 3.63 - 3.48 (m, 10H), 3.42 (s, 1H), 3.38 - 3.35 (m, 2H), 2.60 (d, J = 14.0 Hz, 3H), 2.32 (t, J = 7.6 Hz, 2H), 2.03 (s, 3H), 1.72 (m, 2H).

[0154] 3. Synthesis of compound represented by formula (II)

[0155] Step 1. Synthesis of compound 1008-2

[0156] Compound 1008-1 (2.00 g, 4.75 mmol), 1008-1’ (2.20 g, 5.69 mmol) and potassium acetate (654.83 mg, 4.75 mmol) were dissolved in N,N-dimethylformamide (40 mL) under nitrogen protection. After addition, it was stirred at 90 °C for 16 hours, and LC-MS was monitored. After the reaction was completed, it was cooled to room temperature, water (50 mL) was added, and it was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and a yellow solid compound 1008-2 (2.60 g, 3.89 mmol, 81.88% yield) was obtained by reverse phase preparation. LCMS (E+) m / z: 636.31 [M+H] + .

[0157] Step 2. Synthesis of intermediate 1008-3

[0158] Compound 1008-2 (2.20 g. 3.46 mmol) was added to a mixture of dichloromethane (15 mL) and trifluoroacetic acid (5 mL) at 0 °C. After addition, it was stirred at 0 °C for 1 h. The reaction was monitored by LC-MS. After the reaction was completed, it was dried, and the crude product was purified by reverse phase to give compound intermediate 1008-3 (1.70 g, 3.01 mmol, 87.10% yield) as yellow oil. LCMS (E+) m / z: 536.26 [M+H] + .

[0159] Step 3. Synthesis of compound 1008-4

[0160] To a 25 mL single neck flask was added compound N-Boc glycine (302.63 mg, 0.53 mmol) and dissolved with DMF (5 mL). 1008-3 (110.25 mg, 0.63 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-l-yl) uronium hexafluorophosphate (220.40 mg, 0.58 mmol) and N,N-diisopropylethylamine (135.45 mg, 1.05 mmol) were added successively with stirring. After addition, the reaction was monitored by LC-MS for 30 min. After the reaction was completed, it was purified by MPLC to give compound 1008-4 (352.92 mg, 0.51 mmol, 96.23% yield). LCMS (E+) m / z: 693.6 [M+H] + .

[0161] Step 4. Synthesis of compound 1008-5

[0162] To a 25 mL single neck flask was added compound 1008-4 (352.92 mg, 0.51 mmol) and dissolved with dichloromethane (5 mL). Then trifluoroacetic acid (2 mL) was added with stirring under ice water bath at 0 °C. After addition, the reaction was monitored by LC-MS for 10 min at room temperature. After the reaction was completed, it was purified by MPLC to give intermediate 1008-5 (301.00 mg, 0.51 mmol, 99.70% yield). LCMS (E+) m / z: 593.9 [M+H] + .

[0163] Step 5. Synthesis of compound 1008-6A

[0164] Into a 4 mL reaction vial, was placed compound 7 (50.00 mg, 0.24 mmol), 1008-5 (100.00 mg, 0.17 mmol), 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (95.00 mg, 0.23 mmol) and N,N-diisopropyl ethylamine (100 μL, 0.60 mmol). After addition, the reaction mixture was stirred at room temperature for 1 h until LC-MS indicated the starting material was consumed completely. The reaction mixture was directly subjected to MPLC to give compound 1008-6A (70.00 mg, 87.84 μmol, 51.73% yield). LCMS (E+) m / z: 797.6 [M+H] + .

[0165] Step 6. Synthesis of compound of formula (II)

[0166] Into a 25 mL reaction vial, was placed compound 1008-6A (70.00 mg, 87.84 μmol), then dissolved in methanol (3 mL), and sodium hydroxide solution (2 N, 3 mL) was added dropwise. The reaction mixture was stirred at 25 °C for 1 h. After the reaction was completed, the methanol was concentrated, and the mixture was subjected to MPLC to give compound of formula (II) (50.00 mg, 63.87 μmol, 72.56% yield). LCMS (E+) m / z: 783.4 [M+H] + .

[0167] 1H NMR (400 MHz, DMSO-d6) δ 9.67 (s, 1H), 8.36 (t, J = 6.0 Hz, 1H), 8.26 (dd, J = 7.8, 1.8 Hz, 1H), 7.78 (dd, J = 7.8, 1.7 Hz, 1H), 7.50 (dddd, J = 15.3, 8.3, 6.8, 1.6 Hz, 2H), 7.45 - 7.38 (m, 3H), 7.30 (dd, J = 8.0, 3.5 Hz, 3H), 7.02 (d, J = 8.0 Hz, 1H), 6.81 (d, J = 2.5 Hz, 1H), 6.32 (d, J = 5.0 Hz, 1H), 5.09 (d, J = 6.6 Hz, 1H), 4.32 (dd, J = 5.8, 3.5 Hz, 2H), 4.12 (d, J = 2.4 Hz, 2H), 4.08 (dd, J = 10.7, 2.9 Hz, 1H), 3.92 (dd, J = 5.7, 3.5 Hz, 2H), 3.84 (dd, J = 10.7, 5.6 Hz, 1H), 3.79 - 3.63 (m, 4H), 3.59 (dd, J = 5.9, 3.7 Hz, 2H), 3.56 - 3.49 (m, 9H), 3.41 (t, J = 2.4 Hz, 2H), 2.46 (d, J = 5.7 Hz, 2H), 2.39 - 2.29 (m, 2H), 2.03 (s, 3H), 1.74 (dt, J = 12.9, 7.0 Hz, 2H).

[0168] Example 1: Method 1 for synthesis of compound 7

[0169] Step 1. Synthesis of compound 9

[0170] Under the protection of nitrogen, 5.00 g of compound 8 (Anjie Chemical, item number: B041392) was added into a 1 L three-necked flask, 500 mL of water was added, 14.57 g of sodium carbonate was added, and stirred at room temperature until the solution was clear. The system was cooled to 0-10°C, 11.63 g of elemental iodine was added in batches, after addition, it was raised to room temperature and stirred for 12 hours. LCMS detection showed that the reaction was completed, the reaction solution was filtered, the filter cake was washed with 20 mL of water, the water phase was combined, and extracted with 100 mL of ethyl acetate and 100 mL of methanol*3. The organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. 8.92 g of compound 9 was obtained with a yield of 82%. Mass spectrum (ESI, m / z): 235.87 [M+H] + As shown in Figure 1.

[0171] Step 2. Synthesis of compound 11

[0172] To a 1 L three-necked flask was added 20.00 g of compound 10 (Anatrace, Catalog No. A070484) and 9.3 g of N-methylmorpholine (NMM) under nitrogen protection, and 200 mL of tetrahydrofuran was added to stir until clear. The system was cooled to -10-0 °C, and 12.6 g of isobutyl chloroformate was added in batches, and the reaction was stirred for 0.5 h. The reaction solution was filtered, washed with 40 mL of tetrahydrofuran, and the filtrate was collected in a 1000 mL three-necked flask. The system was cooled to -10-0 °C, and 3.0 g of sodium borohydride and 200 mL of methanol were added, and the system was stirred at room temperature for 12 h. LCMS detection showed that the reaction was complete, 40 mL of acetone and water were added, and the reaction was quenched by stirring for 0.5 h. 400 mL of ethyl acetate and water were added and stirred, and after standing, the organic phase was taken, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain 13.5 g of compound 11 with a yield of 70.7%. Mass spectrum (ESI, m / z): 248.29 [M+H]+.

[0173] Step 3. Synthesis of compound 12

[0174] To a 1 L three-necked flask was added 20.00 g of compound 10 (Anatrace, Catalog No. A070484) and 9.3 g of N-methylmorpholine (NMM) under nitrogen protection, and 200 mL of tetrahydrofuran was added to stir until clear. The system was cooled to -10-0 °C, and 12.6 g of isobutyl chloroformate was added in batches, and the reaction was stirred for 0.5 h. The reaction solution was filtered, washed with 40 mL of tetrahydrofuran, and the filtrate was collected in a 1000 mL three-necked flask. The system was cooled to -10-0 °C, and 3.0 g of sodium borohydride and 200 mL of methanol were added, and the system was stirred at room temperature for 12 h. LCMS detection showed that the reaction was complete, 40 mL of acetone and water were added, and the reaction was quenched by stirring for 0.5 h. 400 mL of ethyl acetate and water were added and stirred, and after standing, the organic phase was taken, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain 13.5 g of compound 11 with a yield of 70.7%. Mass spectrum (ESI, m / z): 248.29 [M+H]+. + As shown in FIG. 2.

[0175] For this step, the inventors explored the effects of reaction temperature and solvent on yield, and the following Comparative Examples 1-1 to 1-4 are provided.

[0176] Comparative Example 1-1

[0177] To a 1 L three-necked flask was added 20.00 g of compound 10 (Anatrace, Catalog No. A070484) and 9.3 g of N-methylmorpholine (NMM) under nitrogen protection, and 200 mL of tetrahydrofuran was added to stir until clear. The system was cooled to -10-0 °C, and 12.6 g of isobutyl chloroformate was added in batches, and the reaction was stirred for 0.5 h. The reaction solution was filtered, washed with 40 mL of tetrahydrofuran, and the filtrate was collected in a 1000 mL three-necked flask. The system was cooled to -10-0 °C, and 3.0 g of sodium borohydride and 200 mL of methanol were added, and the system was stirred at room temperature for 12 h. LCMS detection showed that the reaction was complete, 40 mL of acetone and water were added, and the reaction was quenched by stirring for 0.5 h. 400 mL of ethyl acetate and water were added and stirred, and after standing, the organic phase was taken, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain 13.5 g of compound 11 with a yield of 70.7%. Mass spectrum (ESI, m / z): 248.29 [M+H]+.

[0178] Comparative Example 1-2

[0179] In a 100 mL flask, add 1.90 g of compound 9, 2.00 g of compound 11 and 2.76 g of triphenylphosphine under nitrogen protection, add 24 mL of dichloromethane and stir, and cool the system to -10-0 °C. Dissolve 2.30 g of diisopropyl azodicarboxylate (DIAD) in 8 mL of dichloromethane, slowly drop into the flask, and stir at -10-0 °C for 12 hours. Detect the completion of the reaction by LCMS, add 30 mL of n-heptane and stir with zinc bromide for 0.5 hours. Filter the reaction solution, and concentrate under reduced pressure, then prepare 2.35 g of compound 12 by MPLC with a yield of 63%.

[0180] Comparative Example 1-3

[0181] In a 100 mL flask, add 1.90 g of compound 9, 2.00 g of compound 11 and 2.76 g of triphenylphosphine under nitrogen protection, add 24 mL of dichloromethane and stir, and cool the system to -10-0 °C. Dissolve 2.30 g of diisopropyl azodicarboxylate (DIAD) in 8 mL of dichloromethane, slowly drop into the flask, and stir at -10-0 °C for 12 hours. Detect the completion of the reaction by LCMS, add 30 mL of n-heptane and stir with zinc bromide for 0.5 hours. Filter the reaction solution, and concentrate under reduced pressure, then prepare 2.35 g of compound 12 by MPLC with a yield of 63%.

[0182] Comparative Example 1-4

[0183] In a 100 mL flask, add 1.90 g of compound 9, 2.00 g of compound 11 and 2.76 g of triphenylphosphine under nitrogen protection, add 24 mL of dichloromethane and stir, and cool the system to -10-0 °C. Dissolve 2.30 g of diisopropyl azodicarboxylate (DIAD) in 8 mL of dichloromethane, slowly drop into the flask, and stir at -10-0 °C for 12 hours. Detect the completion of the reaction by LCMS, add 30 mL of n-heptane and stir with zinc bromide for 0.5 hours. Filter the reaction solution, and concentrate under reduced pressure, then prepare 2.35 g of compound 12 by MPLC with a yield of 63%.

[0184] It can be seen from the comparison that the selection of reaction temperature and solvent in this step has a significant impact on the yield.

[0185] Step 4. Synthesis of compound 13

[0186] Into a 500 mL three-necked flask was placed 20 g of compound 12, dissolved in 100 mL of dichloromethane, and then 30 mL of 4 M hydrogen chloride dioxane solution was slowly added. After stirring at room temperature for 3 hours, 150 mL of water was added and stirred, and then the water phase was taken after standing, extracted with 150 mL of ethyl acetate. The organic phase was obtained after adjusting the pH of the water phase to 9-10, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. 16.5 g of compound 13 was obtained with a yield of 99%. Mass spectrum (ESI, m / z): 364.96 [M+H] + As shown in Figure 3.

[0187] Step 5. Synthesis of compound 14

[0188] Into a 500 mL three-necked flask was placed 20 g of compound 12, dissolved in 100 mL of dichloromethane, and then 30 mL of 4 M hydrogen chloride dioxane solution was slowly added. After stirring at room temperature for 3 hours, 150 mL of water was added and stirred, and then the water phase was taken after standing, extracted with 150 mL of ethyl acetate. The organic phase was obtained after adjusting the pH of the water phase to 9-10, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. 16.5 g of compound 13 was obtained with a yield of 99%. Mass spectrum (ESI, m / z): 364.96 [M+H] + As shown in Figure 4.

[0189] For this step, the inventors explored the effect of ligands on the yield, and the following Comparative Examples 1-5~1-7 are provided.

[0190] Comparative Example 1-5

[0191] Into a 500 mL three-necked flask was placed 20 g of compound 12, dissolved in 100 mL of dichloromethane, and then 30 mL of 4 M hydrogen chloride dioxane solution was slowly added. After stirring at room temperature for 3 hours, 150 mL of water was added and stirred, and then the water phase was taken after standing, extracted with 150 mL of ethyl acetate. The organic phase was obtained after adjusting the pH of the water phase to 9-10, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. 16.5 g of compound 13 was obtained with a yield of 99%. Mass spectrum (ESI, m / z): 364.96 [M+H]

[0192] Comparative Example 1-6

[0193] Into a 500 mL three-necked flask was placed 20 g of compound 12, dissolved in 100 mL of dichloromethane, and then 30 mL of 4 M hydrogen chloride dioxane solution was slowly added. After stirring at room temperature for 3 hours, 150 mL of water was added and stirred, and then the water phase was taken after standing, extracted with 150 mL of ethyl acetate. The organic phase was obtained after adjusting the pH of the water phase to 9-10, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. 16.5 g of compound 13 was obtained with a yield of 99%. Mass spectrum (ESI, m / z): 364.96 [M+H]

[0194] Comparative Examples 1-7

[0195] In a 100 mL three-necked flask, 5 g of compound 13, 0.62 g of Pd2(dba)3, 0.79 g of Xantphos (CAS: 161265-03-8) and 2.64 g of sodium tert-butoxide were added under nitrogen protection, 50 mL of toluene was added for stirring, and after being warmed to 80°C, it was stirred for 1 hour. LCMS detection showed that the reaction was complete, the reaction solution was filtered, and after being concentrated under reduced pressure, 1.225 g of compound 14 was obtained by MPLC preparation, with a yield of 35%.

[0196] It can be seen by comparison that in this step, the selection of the ligand has a significant impact on the yield. Compared with other ligands, when the ligand is Josiphos, the yield is increased by at least 1 times or more, which is significantly advantageous.

[0197] Step 6. Synthesis of compound 7

[0198] In a 1 L three-necked flask, 20.00 g of compound 14, 100 mL of water and 50 mL of isopropyl alcohol were stirred under nitrogen protection, 110 mL of 0.11 M potassium hydroxide aqueous solution was added in batches, and stirred at room temperature for 4 hours. LCMS detection showed that the reaction was complete, 37% (v / v) hydrochloric acid aqueous solution was added in batches, and the pH value was adjusted to 1-2. The organic layer was separated, washed with saturated sodium chloride solution three times, and dried over magnesium sulfate. The reaction solution was filtered and concentrated under reduced pressure to obtain 18.2 g of compound 7 with a yield of 97.0%. Mass spectrum (ESI, m / z): 223.04 [M+H]+, as shown in Figure 5.

[0199] 1H NMR (400 MHz, DMSO-d6) δ 7.44 (d, J = 5.0 Hz, 1H), 6.66 (d, J = 2.3 Hz, 1H), 6.36 (d, J = 5.0 Hz, 1H), 4.12 (dd, J = 10.6, 2.9 Hz, 1H), 3.82 (dd, J = 10.7, 6.0 Hz, 1H), 3.55-3.40 (m, 1H), 2.48-2.27 (m, 2H), 2.08 (s, 1H), 2.06 (s, 3H), 1.82-1.58 (m, 2H).

[0200] Example 2: Synthesis method 2 of compound 7

[0201] Step 1. Synthesis of compound 9

[0202] The synthesis of compound 9 is the same as step 1 in Example 1.

[0203] Step 2. Synthesis of compound 15

[0204] In a 500 mL flask, add 9.50 g of compound 9, 4.66 g of L-pyroglutamic alcohol (CAS: 17342-08-4, Anaspec, Catalog No: E020312) and 13.8 g of triphenylphosphine under nitrogen protection, add 120 mL of tetrahydrofuran and stir, and cool the system to -10-0°C. Dissolve 11.5 g of diisopropyl azodicarboxylate (DIAD) in 40 mL of tetrahydrofuran, slowly drop into the flask, and stir at room temperature for 12 hours. Detect the completion of the reaction by LCMS, add 150 mL of n-heptane and zinc bromide and stir for 0.5 hours. Filter the reaction solution, concentrate under reduced pressure, and then use MPLC to prepare 11.7 g of compound 15 with a yield of 87.3%. Mass spectrum (ESI, m / z): 333.00 [M+H]+, as shown in Figure 6.

[0205] Step 3. Synthesis of compound 16

[0206] In a 500 mL flask, add 9.50 g of compound 9, 4.66 g of L-pyroglutamic alcohol (CAS: 17342-08-4, Anaspec, Catalog No: E020312) and 13.8 g of triphenylphosphine under nitrogen protection, add 120 mL of tetrahydrofuran and stir, and cool the system to -10-0°C. Dissolve 11.5 g of diisopropyl azodicarboxylate (DIAD) in 40 mL of tetrahydrofuran, slowly drop into the flask, and stir at room temperature for 12 hours. Detect the completion of the reaction by LCMS, add 150 mL of n-heptane and zinc bromide and stir for 0.5 hours. Filter the reaction solution, concentrate under reduced pressure, and then use MPLC to prepare 11.7 g of compound 15 with a yield of 87.3%. Mass spectrum (ESI, m / z): 333.00 [M+H]+, as shown in Figure 6.

[0207] For this step, the inventors explored the effect of ligands on the yield, and the following Comparative Examples 2-1 to 2-3 are provided.

[0208] Comparative Example 2-1

[0209] In a 500 mL flask, add 9.50 g of compound 9, 4.66 g of L-pyroglutamic alcohol (CAS: 17342-08-4, Anaspec, Catalog No: E020312) and 13.8 g of triphenylphosphine under nitrogen protection, add 120 mL of tetrahydrofuran and stir, and cool the system to -10-0°C. Dissolve 11.5 g of diisopropyl azodicarboxylate (DIAD) in 40 mL of tetrahydrofuran, slowly drop into the flask, and stir at room temperature for 12 hours. Detect the completion of the reaction by LCMS, add 150 mL of n-heptane and zinc bromide and stir for 0.5 hours. Filter the reaction solution, concentrate under reduced pressure, and then use MPLC to prepare 11.7 g of compound 15 with a yield of 87.3%. Mass spectrum (ESI, m / z): 333.00 [M+H]+, as shown in Figure 6.

[0210] Comparative Example 2-2

[0211] Under the protection of nitrogen, 5.00 g of compound 15, 0.62 g of Pd2(dba)3, 0.79 g of Xantphos (CAS: 161265-03-8) and 2.64 g of sodium tert-butoxide were added to a 100 mL three-neck flask, stirred with 50 mL of toluene, heated to 80°C and stirred for 1 hour. LCMS detection showed that the reaction was complete, the reaction solution was filtered and concentrated under reduced pressure, and 1.50 g of compound 16 was obtained by MPLC preparation, with a yield of 49%.

[0212] Comparative Example 2-3

[0213] Under the protection of nitrogen, 5.00 g of compound 15, 0.62 g of Pd2(dba)3, 0.79 g of Xantphos (CAS: 161265-03-8) and 2.64 g of sodium tert-butoxide were added to a 100 mL three-neck flask, stirred with 50 mL of toluene, heated to 80°C and stirred for 1 hour. LCMS detection showed that the reaction was complete, the reaction solution was filtered and concentrated under reduced pressure, and 1.50 g of compound 16 was obtained by MPLC preparation, with a yield of 49%.

[0214] It can be seen from the comparison that in this step, the selection of ligand has a significant impact on the yield.

[0215] Step 4. Synthesis of compound 7

[0216] Under the protection of nitrogen, 5.00 g of compound 15, 0.62 g of Pd2(dba)3, 0.79 g of Xantphos (CAS: 161265-03-8) and 2.64 g of sodium tert-butoxide were added to a 100 mL three-neck flask, stirred with 50 mL of toluene, heated to 80°C and stirred for 1 hour. LCMS detection showed that the reaction was complete, the reaction solution was filtered and concentrated under reduced pressure, and 1.50 g of compound 16 was obtained by MPLC preparation, with a yield of 49%.

[0217] [M+H]+, as shown in Figure 5.

[0218] Test Example 1: Detection of the activity of the compound in blocking the binding of integrin protein to its ligand

[0219] The activity of the compound of formula (I) in blocking the binding of integrin protein αvβ6 to its ligand was detected by ELISA experiment. The integrin and its ligand used in the experiment are as follows: αvβ6 and its ligand rhLAP. All experimental materials were provided by WuXi.

[0220] The specific detection method is as follows: TBS buffer is used to prepare ligand, and 50 μL of the solution is transferred to a 96-well plate and incubated at 4°C overnight. 150 μL of blocking solution is added, and the plate is incubated at 37°C for 1 h. The integrin protein is prepared using a buffer containing 0.1% BSA, 50 μL of the integrin protein is transferred to the 96-well plate, 1 μL of different concentrations of the compound, Ligand SM6.1 (ArrowHead, WO2023070082A2) or DMSO is added, and the plate is incubated at room temperature for 2 h, then washed with washing buffer for 3 times. The antibody is prepared using a buffer containing 0.1% BSA, wherein the αv antibody is used to detect αvβ6, 50 μL of the prepared antibody is added, and the plate is incubated at room temperature for 1 h, then washed with washing buffer for 3 times. 50 μL of Streptavidin-HRP is added, and the plate is incubated at room temperature for 20 min. 50 μL of TMB substrate is added, and the plate is incubated at room temperature for 20 min. Finally, 25 μL of stop buffer is added, and the OD value is read at 450 nm on a microplate reader. The IC 50 value is calculated using GraphPad Prism 5 software.

[0221] The results are shown in Table 1, and the activity of the compound of formula (I) in blocking the binding of αvβ6 protein to its ligand is significantly better than that of Ligand SM6.1.

[0222] Table 1 Activity of compounds in blocking the binding of αvβ6 integrin protein to its ligand

[0223] Test Example 2 Compound blocking integrin receptor-mediated cell adhesion experiment

[0224] To test the activity of the compound of formula (I) in blocking the binding of cell surface integrin protein to its ligand, a cell adhesion experiment is designed, and all experimental materials are provided by WuXi. TBS buffer is used to prepare ligand rhLAP (recognizing αvβ6) and Fibronectin (recognizing α5β1), and 50 μL of the solution is transferred to a 96-well plate and incubated at 4°C overnight. 150 μL of blocking solution is added, and the plate is incubated at 37°C for 1 h. The HT-29 (αvβ6) and α5β1-K562 cells are resuspended in buffer, respectively, and 50 μL of the cells is transferred to a 96-well plate. 1 μL of different concentrations of the compound of formula (I), Ligand SM6.1 (ArrowHead, WO2023070082A2) or DMSO is added, and the HT-29 and α5β1-K562 cells are incubated at room temperature for 2 h. The liquid in the 96-well plate is removed, 50 μL of substrate is added, and the plate is incubated at 37°C for 2 h. Finally, 90 μL of stop buffer is added, and the OD value is read at 405 nm on a microplate reader. The IC 50 value is calculated using GraphPad Prism 5 software.

[0225] Table 2 Compounds blocking integrin receptor-mediated cell adhesion

[0226] The experimental results are shown in Table 2. The results show that the compounds of formula (I) and formula (II) can effectively block αvβ6 and its ligand-mediated cell adhesion, and the activity is significantly better than Ligand SM6.1. Moreover, the activity of the compounds of formula (I) and formula (II) in blocking α5β1 / K562 cell adhesion is significantly different from the activity in blocking αvβ6 / HT-29 cell adhesion, even more than 50 times, with excellent αvβ6 selective specificity.

Claims

1. A method of preparing a target compound 7 comprising: (1-1) reacting a compound with compounds The reaction is carried out to obtain a compound (1-2) reacting a compound Boc-deprotection to obtain compound (1-3) reacting a compound The intramolecular coupling reaction occurs in the presence of a catalyst and a ligand to obtain a compound (1-4) reacting a compound ester hydrolysis reaction to obtain the target compound 7 Alternatively, the method comprises: (2-1) reacting a compound with a compound The reaction is carried out to obtain a compound (2-2) reacting a compound The intramolecular coupling reaction occurs in the presence of a catalyst and a ligand to obtain a compound (2-3) reacting a compound Amide hydrolysis reaction occurs to obtain the target compound 7 2. The method of claim 1, wherein, Step (1-1) further has one or more technical features selected from (i)-(v): ( i) In step (1-1), the reaction is carried out at a temperature of -10-0 °C; (ii) In step (1-1), the reaction is carried out in a solvent of tetrahydrofuran; (iii) in step (1-1), the compound with a compound the molar ratio of the two substances is 1.5:1-1:1.5, preferably 1:1; (iv) In step (1-1), the reaction is carried out under a nitrogen atmosphere; (v) In step (1-1), the reaction is carried out in the presence of triphenylphosphine and diisopropyl azodicarboxylate (DIAD); Preferably, the compound the molar ratio of the two substances is 1:1-1:1.5; Preferably, the compound the molar ratio of the two substances is 1:1-1:1.

5.

3. The method of claim 1 or 2, wherein, In step (1-2), the Boc removal reaction is carried out under acidic conditions; Preferably, the acidic conditions are provided by a hydrogen chloride dioxane solution.

4. The method according to any one of claims 1 to 3, wherein, Step (1-3) further has one or more technical features selected from (i)-(vi): ( i) In step (1-3), the ligand is a chiral phosphine ligand, preferably Josiphos; (ii) in step (1-3), the compound the mass ratio of the two substances is 5:1-10:1, preferably 6:1; (iii) in step (1-3), the compound the mass ratio of the two substances is 4:1-10:1, preferably 5:1; (iv) In step (1-3), the intramolecular coupling reaction is carried out under a nitrogen atmosphere; (v) In step (1-3), the catalyst is Pd2(dba)3; (vi) In step (1-3), the intramolecular coupling reaction is carried out in the presence of a basic additive; Preferably, the basic additive is sodium tert-butoxide.

5. The method according to any one of claims 1 to 4, wherein, Step (1-4) further has one or more technical features selected from (i)-(ii): ( i) In step (1-4), the ester hydrolysis reaction is carried out under basic conditions; Preferably, the basic conditions are provided by potassium hydroxide; (ii) In step (1-4), the ester hydrolysis reaction is carried out under a nitrogen atmosphere.

6. The method according to any one of claims 1 to 5, wherein, Step (2-1) further has one or more technical features selected from (i)-(iii): ( i) in step (2-1), the compound with a compound the molar ratio of the two substances is 1.5:1-1:1.5, preferably 1:1; (ii) In step (2-1), the reaction is carried out under a nitrogen atmosphere; (iii) In step (2-1), the reaction is carried out in the presence of triphenylphosphine and diisopropyl azodicarboxylate (DIAD); Preferably, the compound the molar ratio of the two substances is 1:1-1:1.5; Preferably, the compound the molar ratio of the two substances is 1:1-1:1.

5.

7. The method according to any one of claims 1 to 6, wherein, Step (2-2) further has one or more technical features selected from (i)-(vi): ( i) In step (2-2), the ligand is a chiral phosphine ligand, preferably Josiphos; (ii) in step (2-2), the compound the mass ratio of the two substances is 5:1-10:1, preferably 6:1; (iii) in step (2-2), the compound of formula (II) the mass ratio of the two substances is 4:1-10:1, preferably 5:1; (iv) in step (2-2), the intramolecular coupling reaction is carried out under a nitrogen atmosphere; (v) in step (2-2), the catalyst is Pd2(dba)3; (vi) in step (2-2), the intramolecular coupling reaction is carried out in the presence of a basic additive; Preferably, the basic additive is sodium tert-butoxide.

8. The method according to any one of claims 1 to 7, wherein, Step (2-3) further has one or more technical features selected from the following (i)-(ii): ( i) in step (2-3), the amide hydrolysis reaction is carried out under basic conditions; Preferably, the basic conditions are provided by sodium hydroxide; ii) in step (2-3), the amide hydrolysis reaction is carried out under a nitrogen atmosphere.

9. The method of claim 1, wherein, The compounds The process for the preparation of comprises: (a) contacting a compound reacting with elemental iodine to obtain the compound 10. The method of claim 9, wherein, Step (a) further has one or more technical features selected from the following (i)-(iii): ( i) in step (a), the compound the molar ratio of the substance to elemental iodine is 1.5:1-1:1.5, preferably 1:1; (ii) in step (a), the reaction is carried out under a nitrogen atmosphere; (iii) in step (a), the reaction is carried out under basic conditions; Preferably, the basic conditions are provided by sodium carbonate.

11. The method of claim 1, wherein, The compounds The process for the preparation of comprises: (b-1) the compound carboxyl group of the compound of Formula (I) to obtain a compound of Formula (I) (b-2) activating the carboxyl group reduction reaction to obtain a compound 12. The method of claim 11, wherein, Step (b-1) further has one or more technical features selected from the following (i)-(iii): ( i) in step (b-1), the activation is achieved using isobutyl chloroformate; (ii) in step (b-1), the activation is carried out under a nitrogen atmosphere; (iii) in step (b-1), the activation is carried out under basic conditions; Preferably, the basic conditions are provided by N-methylmorpholine.

13. The method of claim 11 or 12, wherein, Step (b-2) further has one or more technical features selected from the following (i)-(ii): ( i) in step (b-2), the reduction reaction is achieved using a reducing agent; Preferably, the reducing agent is sodium borohydride; ii) in step (b-2), the reduction reaction is carried out under a nitrogen atmosphere.

14. A method of preparing a compound of Formula (I) or Formula (II) comprising: (c-1) The target compound 7 is prepared by the method described in any one of claims 1-13 (c-2) subjecting the target compound 7 with compounds The reaction occurs to obtain a compound or, making target compound 7 with a compound The reaction occurs to obtain a compound (c-3) reacting a compound of formula (c-2) The ester hydrolysis reaction occurs to obtain the compound shown in formula (I) or, making a compound The ester hydrolysis reaction occurs to obtain a compound represented by formula (II) 15. An intermediate compound selected from the group consisting of:

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