Novel synthesis method of FimH antagonist

Abstract

About 1.5 billion persons suffer from urinary tract infections (UTIs) in the whole world every year, and the morbidity of the UTIs in women reaches up to 12.6%; meanwhile, along with the improvement of the living standard, the incidence rate of Crohn's disease is also increased year by year. The vast majority of urinary tract infection is caused by urinary tract pathogenic escherichia coli (UPEC), and the flagellum terminal FimH protein is a key molecule for causing inflammation by forming a biological membrane through adhesion and colonization of the bacteria. Inflammatory bowel disease (IBD) is a chronic recurrent inflammatory disease of the intestinal tract, including ulcerative colitis (UC) and Crohn's disease (CD). Related researches show that AIECs can induce related inflammations through FimH protein at the tail end of flagellum. The FimH antagonist can block immunoreactions caused by UPEC and AIEC, and then antibiotics are developed to be used for treating urinary tract infection and Crohn's disease. The GSK company publishes a FimH synthesis and application patent (WO 2020 / 012336A1), wherein GSK-3882347 is applied to clinical test research. The route in the GSK patent needs 13 steps, and the synthesis of the FimH antagonist is realized at the total yield of about 3%. According to the invention, the synthesis problem of the FimH antagonist is solved, i.e., the synthesis of the FimH antagonist is realized at the total yield of 34-48% by starting from protected mannose bromoglycoside and carrying out four-step chemical conversion of reductive cross coupling, stereoselective carbonyl reduction, configuration inversion and protecting group removal.

Description

Technical Field

[0001] This invention provides a novel method for synthesizing FimH antagonists, having structures shown in general formulas VI and VII. These FimH antagonists are carbon-mannose structures with outstanding metabolic stability and good drug-like properties. They can be used to treat urinary tract infections caused by pathogenic Escherichia coli (WO2020012336A1) and Crohn's disease caused by Adhesive Escherichia coli (WO2014100158A1), both currently in clinical trials. The new technical route includes the following steps: (1) Reduction cross-coupling: Under the action of inexpensive metals such as nickel and its ligands, the protected mannobromoglycoside and substituted benzoic acid and its derivatives undergo reduction cross-coupling in the presence of reducing agents, activators and additives to prepare compound III; (2) Reduction of carbonyl group: At low temperature, the reducing agent selectively reduces the carbonyl group to obtain S-chiral benzyl alcohol glycosides; (3) Hydroxyl configuration inversion: The S-chiral benzyl alcohol glycoside undergoes a Mitsunobu reaction with a carboxylic acid compound to invert the stereoconfiguration of the hydroxyl group; (4) Removal of protecting group: After removing the protecting group, compound VI is obtained, namely the FimH antagonist; (5) Removal of protecting group: The S-chiral benzyl alcohol glycoside obtained in (2) is removed by removing the protecting group to obtain the S-FimH antagonist. The synthetic method has the advantages of short steps, excellent stereoselectivity, high yield, simple operation, mild reaction conditions, inexpensive and readily available reagents, and easy scale-up production. This invention belongs to the field of technical invention. Background Technology

[0002] Carbon glycosides are derived from glycosides, where carbon-carbon bonds replace unstable chemical bonds such as carbon-oxygen and carbon-nitrogen bonds, significantly improving their stability, particularly in resistance to hydrolases and acids, thus greatly enhancing the drug-like properties of glycosides. Carbon glycosides possess a wide range of activities, including antitumor, antibacterial, antiviral, cell-promoting, and immunomodulatory effects, making them a potential class of drugs. Currently, most carbon glycosides are linked directly to the glycosyl group via carbon-carbon bonds around aryl, alkenyl, and unfunctionalized alkyl groups. This strategy cannot effectively mimic naturally occurring oxygen / nitrogen glycosides, resulting in significant variations in biological activity depending on the target, thus limiting the development of glycoside-based drugs. Novel carbon glycosides, by introducing methylene hydroxyl groups, can better mimic the binding of naturally occurring oxygen / nitrogen heteroatoms to specific targets. These novel carbon glycosides not only retain the stability advantages of carbon glycosides but also potentially enhance their affinity for targets; therefore, the development of this class of novel glycosides is of great significance. Urinary tract infections (UTIs), also known as urinary system infections, generally refer to inflammation of the urinary tract caused by pathogens infecting the urinary tract tissues. UTIs typically include nephritis, cystitis, and urethritis, and are common infectious diseases. Severe UTIs can spread into the bloodstream, endangering life. Globally, approximately 150 million people suffer from UTIs each year, with an incidence rate as high as 12.6% in women, creating a significant social burden. Furthermore, UTIs are highly prone to recurrence; about one-third of female patients will be reinfected within six months. With repeated infections, antibiotic resistance often develops, necessitating new treatment methods.

[0003] The vast majority of urinary tract infections are caused by pathogenic Escherichia coli (UTC). Type I flagellated FimH protein is a key molecule for the adhesion, colonization, biofilm formation, and subsequent inflammation of these bacteria. FimH can recognize the high-mannose glycosyl groups of high-mannose glycoproteins on epithelial cells, adhering to the epithelium and avoiding being carried away by the system. Furthermore, FimH protein is only present in pathogenic E. coli; therefore, FimH protein inhibitors can selectively eliminate pathogenic E. coli while avoiding the killing of beneficial bacteria, potentially becoming a new anti-infective treatment (Sarshar et al., review Antibiotics (Basel). 2020 9(7):397).

[0004] Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disease of the intestine, including ulcerative colitis (UC) and Crohn's disease (CD). The mechanisms that trigger IBD are not fully understood, but it is generally believed to be caused by immune system disorders or gut microbiota dysbiosis. Related studies have shown that adherent Escherichia coli (AIEC) can induce related inflammatory diseases. Like UPEC, AIEC has FimH protein at the end of its flagella (Miquel et al., PLoS One. 2010; 5(9):e12714. Prudent et al. Communication Biology (2021) 4:627).

[0005] The synthesis of some class VI and VII molecules is described in GlaxoSmithKline's patent (WO 2020 / 012336A1). The synthetic route is cumbersome, starting with mannosyl methylglycoside and involving 13 steps to obtain the target molecule in an overall yield of less than 3%. The synthesis process also involves flammable butyllithium, highly toxic osmium tetroxide, explosive sodium periodate, strong acid boron trichloride, three steps of the noble metal palladium, multiple oxidation / reduction steps, and protecting and deprotecting operations. The route is lengthy (13 steps), with a low overall yield of 3%, and is expensive, placing a financial burden on patients. Furthermore, the lengthy reaction steps hinder the development of structure-activity relationship studies, making it difficult to further develop more active FimH inhibitors.

[0006] This patent solves the problem of the difficulty in preparing class VI and VII compounds, and at the same time determines their hemagglutination inhibitory activity against UPEC and AIEC, providing a technical route for subsequent research on FimH inhibitors and related drugs. Summary of the Invention

[0007] This invention provides a novel method for preparing a new type of carbon glycoside, having the structure shown in general formulas VI and VII. R 1 It can be hydrogen, methyl, chloro, or trifluoromethyl; R2 is chlorine, bromine, alkyl, or aryl; In a preferred embodiment of the present invention, the compounds of formulas VI and VII are selected from...

[0008] The purpose of this invention is to provide a method for preparing compound VI-a to VI-k, which is achieved through four chemical transformations. The technical route is as follows:

[0009] The purpose of this invention is to provide a method for preparing compounds VII-a to VII-k of formula VII, which is achieved through three chemical transformations, wherein the technical route is as follows:

[0010] Nickel salt and its ligand, anhydrous magnesium dichloride, and freshly activated zinc powder were rapidly sealed and purged with argon three times. A dry tetrahydrofuran / acetonitrile mixed solution was then added. Subsequently, a tetrahydrofuran / acetonitrile solution containing a protected glycosylated bromoglycoside was added under argon protection. Immediately afterwards, an activated acid glycoside (a mixture of fatty acid or aromatic acid and dimethyl carbonate at room temperature) was added, and the reaction was allowed to proceed at room temperature. After the reaction was confirmed to be complete by TLC, the solvent was evaporated to dryness, and column chromatography was used to obtain intermediate III. In the preferred embodiment of this invention, the optimal nickel catalyst is nickel perchlorate; In the preferred embodiment of this invention, the optimal ligand is di-tert-butylbipyridine; The preferred additive in the preferred embodiment of this invention is magnesium chloride; In the preferred embodiment of this invention, the optimal reducing agent is activated zinc powder; In the preferred embodiment of the present invention, the optimal solvent is a mixture of tetrahydrofuran and acetonitrile, with an optimal ratio of 9:1. In the preferred embodiment of the present invention, the OP in the optimal I is a lipid protecting group such as OAc or OBz.

[0011] The reaction tube containing compound III was placed in a -78°C cold trap, methanol was added, followed by sodium borohydride or deuterated sodium borohydride. After reacting for 3 hours, saturated ammonium chloride solution was added to quench the reaction. After removing methanol by vacuum distillation, the mixture was extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate. The solution was then evaporated to dryness and subjected to column chromatography to obtain target compound IV. Compound IV was dissolved in methanol, a catalytic amount of sodium methoxide was added, and after 1 hour, the solution was evaporated to dryness and subjected to column chromatography to obtain target compound VII. In the preferred embodiment of this invention, the reducing agent is preferably sodium borohydride / deuterated sodium borohydride; In the preferred embodiment of the present invention, the solvent is preferably an alcohol, such as methanol or ethanol. In the preferred embodiment of this invention, the alkali is sodium methoxide, potassium carbonate, etc. In the preferred embodiment of the present invention, the OP in I, III, and IV is a lipid protecting group such as OAc or OBz.

[0012] Take IV, add p-nitrobenzoic acid and triphenylphosphine, seal the reaction and replace the argon gas three times, add tetrahydrofuran solution, then add azo reagent, and after the reaction is complete as detected by TLC, extract three times with ethyl acetate, combine the organic phases and dry with anhydrous sodium sulfate, evaporate to dryness, and column chromatography to obtain target compound V; dissolve V in methanol, add a catalytic amount of base, after the reaction is complete, evaporate to dryness, and column chromatography to obtain target compound VI. In the preferred embodiment of this invention, the carboxylic acid can be an arylic acid, fatty acid, or other carboxylic acid. In the preferred embodiment of this invention, the trivalent phosphorus can be triphenylphosphine or trialkylphosphine; In a preferred embodiment of the present invention, the alkali can be sodium methoxide, potassium carbonate, cesium carbonate, etc. The preferred technical solution of this invention is that the azo reagent can be diethyl azodicarbonate, diisopropyl azodicarbonate, etc. In the preferred embodiments of the present invention, the solvents used in IV to V can be common solvents such as tetrahydrofuran and toluene; In the preferred embodiments of the present invention, V to VI, the solvents used can be common saponification systems such as alcohols and tetrahydrofuran water. Detailed Implementation

[0013] To provide a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the following detailed description of specific embodiments is provided to further illustrate the above-mentioned content of the present invention. However, this should not be construed as limiting the scope of implementation of the present invention. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention.

[0014] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0015] In the experimental methods described in the following examples, all solvents and reagents used were analytically pure or chemically pure; anhydrous solvents were processed according to standard methods; column chromatography and silica gel plates were all standard models. Unless otherwise specified, anhydrous sodium sulfate was used as the drying agent for the extracted organic phase. LC-MS was performed using an Agilent 1260 high-performance liquid chromatography-ion trap mass spectrometer (ESI source) with a diode array detector (DAD) at detection wavelengths of 210 nm and 254 nm. Unless otherwise specified, all system models are standard methods; all reagents and materials, unless otherwise specified, are commercially available.

[0016] The preparation route of (S)-FimH antagonists is as follows:

[0017] The preparation route of FimH antagonist is as follows:

[0018] The optimal nickel catalysts are nickel perchlorate hexahydrate and nickel perchlorate.

[0019] Nickel ligands can have the following structures:

[0020] The optimal ligand for nickel perchlorate hexahydrate is L4.

[0021] Nickel perchlorate hexahydrate (5.5 mg, 0.015 mmol, 0.1 eq), ligand L4 (4.9 mg, 0.018 mmol, 0.12 eq), anhydrous magnesium dichloride (28 mg, 0.3 mmol, 2 eq), and freshly prepared zinc powder (29 mg, 0.45 mmol, 3 eq) were added to a reaction tube. The tube was quickly sealed and purged with argon three times. 0.5 mL of dry tetrahydrofuran / acetonitrile (9 / 1) solution was added. Mannobromoglycosides I and II were dissolved in 0.2 mL of dry tetrahydrofuran / acetonitrile (9 / 1) solution and added to the reaction tube. Then, dimethyl dicarbonate (50 μL, 0.45 mmol, 3 eq) was added, and the reaction was carried out at room temperature. After the reaction was confirmed to be complete by TLC, the solvent was evaporated to dryness, and column chromatography was used to obtain compound III.

[0022] The reaction tube of compound III obtained from entry 0020 was placed in a cold well at -78°C. 3 ml of methanol was added, followed by sodium borohydride (17 mg, 0.45 mmol, 3 eq). After reacting for 3 hours, saturated ammonium chloride solution was added to quench the reaction. Methanol was removed by vacuum distillation, and the mixture was extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was then evaporated to dryness and subjected to column chromatography to obtain compound IV.

[0023] Compound IV obtained from entry 0021 was added to p-nitrobenzoic acid (4 eq) and triphenylphosphine (3.8 eq). The mixture was rapidly sealed and purged with argon three times. Tetrahydrofuran solution was added, followed by diethyl azodicarbonate (4.5 eq). After the reaction was confirmed to be complete by TLC, the mixture was extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was then evaporated to dryness and subjected to column chromatography to obtain compound V.

[0024] Compound V obtained from entry 0022 was dissolved in methanol, and 0.1 eq of sodium methoxide in methanol was added. After 1 hour, the solution was evaporated to dryness and then subjected to column chromatography to obtain compound VI.

[0025] Compound IV obtained from entry 0021 was dissolved in methanol, and 0.1 eq of a methanol solution of sodium methoxide was added. After 1 hour, the solution was evaporated to dryness and then subjected to column chromatography to obtain compound VII.

[0026] Structural analysis and spectral data of type I-xx glycosides obtained in the examples

[0027] In this embodiment, the prepared mannose derivative compounds VI-a to VII-h were tested for hemagglutination inhibition (HAI) to determine their inhibitory effect on the function / activity of FimH protein.

[0028] Preparation of guinea pig red blood cell solution: Anesthetize adult guinea pigs with ether, collect blood from the heart, place the blood in an EDTA blood collection tube, centrifuge at 2000 rpm for 5 min, discard the serum, transfer to a 15 mL centrifuge tube, add sterile PBS, centrifuge at 1500 rpm for 10 min, discard the supernatant, transfer to a 50 mL centrifuge tube, add PBS, centrifuge at 500 rpm for 10 min, repeat the washing step twice, gently aspirate the red blood cells and transfer them to a new 50 mL centrifuge tube to prepare 5% guinea pig red blood cells.

[0029] Bacterial culture preparation: UPEC strain UTI89 was inoculated onto agar plates and incubated overnight at 37°C. Single colonies were picked and inoculated into sterile Erlenmeyer flasks containing LB broth, and incubated overnight at 37°C and 170 rpm. When the OD at 540 nm was approximately 0.8, the bacterial concentration was approximately 10⁻⁶. 8 -10 9 Centrifuge the bacterial culture at 2000 rpm for 10 min (cfu / mL), discard the supernatant, and prepare 0.1 mL of PBS bacterial culture per 1 mL of bacterial culture. Add 50 μL of PBS bacterial culture to the first well of a 96-well V-type culture plate. Add 25 μL of PBS to wells 2-12. After mixing in the first well, aspirate 25 μL and add it to the second well. Repeat this dilution process until the 12th well, discarding 25 μL each time. The last row is used as a negative control, containing only red blood cells and no bacteria. Shake well to mix, and incubate at 25°C for approximately 20-40 minutes. When reading the values, the well showing complete agglutination is taken as the hemagglutination titer (HAU) of the bacteria. For example, if the endpoint titer of hemagglutination is 1:256, then the dilution factor for 4HAU antigen should be 1:64 (256 divided by 4). Prepare bacterial cultures of appropriate concentrations based on the experimental results.

[0030] Activity assay: Add the test compound or standard to a 96-well microplate. The initial concentration of the drug in the first well is 200 μM, 50 μL. For subsequent wells, add 25 μL of PBS to each well for comparative dilution. After mixing in the first well, aspirate 25 μL and add it to the second well, continuing this dilution process until the last well, discarding the 25 μL solution. Add the bacterial culture to the diluted drug in the microplate, 25 μL per well, and incubate at 37°C for 40 min. Add 25 μL of 5% guinea pig red blood cell suspension to each well, mix gently, and incubate at 4°C for approximately 1 h. To determine the results, tilt the plate 60° and observe for any teardrop-like flow of red blood cells.

[0031] Guinea pig erythrocytes express mannosyl protein on their surface, which can bind to the FimH protein of UTI89, inducing a hemagglutination reaction. Macroscopically, this manifests as a thin film of erythrocyte aggregation at the bottom of the well, with a loose red dot visible in the center. The FimH inhibitor competitively binds to the FimH protein, inhibiting the hemagglutination reaction. Macroscopically, all erythrocytes sink and concentrate at the bottom of the well, forming a dense dot. The minimum concentration required to inhibit the hemagglutination reaction is the HAI value of the compound. The final concentration of the compound in the first well was 66700 nM, the concentration in the second well was half that of the first well, the concentration in the third well was half that of the second well, and so on. With a HAI value of 8 nM for the compound, the minimum concentration corresponding to the HAI value for inhibiting the hemagglutination reaction is 0.13 nM. A lower HAI value indicates higher activity of the compound.

[0032] The HAI values ​​of the compounds in the examples for UPEC are... and VI(2μM) VII (8 μM) VI-b(<2nM) VII-b(0.5 μM) VI-c(<2nM) VII-c(0.5 μM) VI-d(<2nM) VII-d(1.0 μM) VI-e(<2nM) VII-e(1.0 μM) VI-f(<2nM)) VII-f(1.0 μM) VI-g(4nM) VII-g(1.0 μM) VI-h(8nM) VII-h(2.0 μM) .

Claims

1. A novel method for synthesizing a FimH antagonist, the technical route of which is as follows: The steps include the following: (1) Reduction cross-coupling: In solvent A, under the action of inexpensive metals such as nickel and their ligands, the protected mannose bromoglycoside (I) and substituted benzoic acid and its derivatives (II) undergo reduction cross-coupling under the action of reducing agent, activator and additive to prepare compound III. (2) Reduction of carbonyl group: Compound IV is prepared by reacting compound III with a reducing agent in solvent B; (3) Hydroxyl configuration inversion: In solvent C, compound IV reacts with carboxylic acid compound via the Mitsunobu reaction, inverting the stereoconfiguration of the hydroxyl group in IV, thus achieving the preparation of compound V; (4) Removal of protecting group: In solvent D, compound V is deprotected to obtain compound VI, i.e. FimH antagonist; (5) Removal of protecting group: In solvent D, compound IV is deprotected to obtain compound VII, namely S-FimH antagonist.

2. The novel synthetic method for a FimH antagonist according to claim 1, characterized in that, Step (1) includes one or more of the following conditions: a) The solvent A is anhydrous tetrahydrofuran, acetonitrile, toluene, dimethylformamide, etc.; the optimal ratio of solvent A is tetrahydrofuran / acetonitrile = 9 / 1 (volume ratio); b) P in Formula I is a common acyl protecting group, with acetyl being the most preferred; c) In Formula II, R1 is hydrogen, methyl, chlorine, trifluoromethyl, and R2 is chlorine, bromine, alkyl, or aryl; d) The inexpensive metal catalyst is nickel or cobalt, and the preferred inexpensive metal catalyst is nickel perchlorate or nickel perchlorate hydrate. The molar ratio of the catalyst to the compound of formula II is 0.05-0.

2. e) The ligands of the low-cost metal catalyst are bipyridine, phenanthroline, terpyridine, etc.; the ratio of the low-cost metal catalyst to its ligand is 1:0.8–1:2, preferably 1:1.2; f) The inexpensive reducing agent is zinc powder, manganese powder, etc., preferably zinc powder; the molar ratio of the reducing agent to Formula II is 2:1–5:1, preferably 3:1; g) The additive is anhydrous lithium chloride, magnesium chloride, etc.; the molar ratio of the reducing agent to Formula II is 2:1–5:1, preferably 3:1; h) The activator of the compound of formula II may be the corresponding ditert-butyl dicarbonate, dimethyl dicarbonate, or the corresponding anhydride, thioester, and imide amide of formula II, preferably dimethyl dicarbonate; the molar ratio of the activator to formula II is 2:1–5:1, preferably 3:

1.

3. The novel synthetic method for a FimH antagonist according to claim 1, characterized in that, Step (2) includes one or more of the following conditions: a) Solvent B is methanol, ethanol, or anhydrous tetrahydrofuran, etc.; preferably, solvent B is methanol. b) The reducing agent can be used under common conditions such as reducing carbonyl groups to hydroxyl groups, and sodium borohydride and deuterated sodium borohydride are preferred. c) The temperature can be 0 to -78 degrees Celsius, with lower temperatures exhibiting better stereoselectivity.

4. A novel synthetic method for a FimH antagonist according to claim 1, characterized in that, Step (3a) includes one or more of the following conditions: a) The solvent C and reagents mentioned are all under the common conditions for the Mitsunobu reaction; b) The preferred carboxylic acid is p-nitrobenzoic acid.

5. A novel synthetic method for a FimH antagonist according to claim 1, characterized in that, Steps (3b) and (4) include one or more of the following conditions: a) The solvent D and reagents described herein are all under conventional acyl group removal conditions; b) This invention uses sodium methoxide for methanol removal under certain conditions.

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