9-position mannih base-containing tetracycline compounds, and preparation method and application thereof

Abstract

The application discloses a 9-position Mannich base-containing tetracycline compound and a preparation method and application thereof. The preparation method comprises the following steps: reacting a compound shown as formula I-A, a compound shown as formula SM-1 and formaldehyde in a solvent to obtain a compound shown as formula I. The method has the advantages of simple steps, mild reaction conditions, strong operability, and the like, and the product with high yield and purity can be obtained through simple purification. The compound shown as formula I can be used for treating diseases caused by bacterial, viral or microbial infections.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical pharmacy, and particularly relates to a 9-position Mannich base-containing tetracycline compound and a preparation method and application thereof. BACKGROUND

[0002] Tetracycline antibiotics are a class of oral broad-spectrum antibiotics fermented by Streptomyces, which have good pharmacological effects on many gram-positive bacteria and gram-negative bacteria, as well as rickettsia, mycoplasma, chlamydia, lymphogranuloma venereum pathogen, inclusion conjunctivitis pathogen and psittacosis pathogen.

[0003] The first tetracycline antibiotic was aureomycin isolated from Streptomyces aureus in 1948, and then oxytetracycline, tetracycline and demeclocycline were developed successively, all of which are natural products with high drug resistance and various side effects. After that, the chemical structure of these compounds was studied, and demethyl tetracycline antibiotics and dimethylamine tetracycline antibiotics were synthesized. However, due to the widespread use of tetracycline, the drug resistance of bacteria to these antibiotics is becoming more and more serious, which makes the tetracycline antibiotics be reduced in use.

[0004] Common tetracycline antibiotics containing Mannich base include two kinds: omadacycline and sarecycline. Among them, omadacycline (PTK-0796) is the first member of a new class of aminomethylcyclines developed by Paratek Pharma, and its structural formula is as follows:

[0005]

[0006] Omadacycline is a semisynthetic derivative of minocycline and the first member of a new class of aminomethylcyclines. The structural modification at C7 and C9 positions enables omadacycline to overcome two major mechanisms of bacterial resistance to tetracyclines, namely drug efflux mechanisms and ribosome protection mechanisms, and has fewer adverse reactions. It was approved by FDA in 2018 for the treatment of acute bacterial skin and skin structure in adults, and can also be used for the treatment of complicated urinary tract infections.

[0007] Comparing the structures of minocycline and omadacycline, the difference is only one Mannich base at the 9-position. The 7-position of other tetracycline antibiotics such as sarecycline is also a Mannich base. The structural formula of sarecycline is as follows:

[0008]

[0009] At present, the method for constructing Mannich base of tetracycline compounds is mainly as follows three methods taking omadacycline as an example.

[0010] Route one: first, through the reaction of minocycline and amino alkylating agent hydroxymethyl phthalimide, deprotection by methylamine to get 9-amino methyl derivative, pivaldehyde reductive amination to get omadacycline. This route does not use expensive metal catalyst and organic ligand, but 9-amino methyl minocycline is extremely stable, and boron is used in reductive amination, with many side reactions, complex post-treatment, purification difficulties, and low product yield, with a yield of 35%. Therefore, this route is difficult to adapt to industrial production.

[0011]

[0012] Route two: 2,2-dimethylpropylamine and paraformaldehyde react to get triazine compound, then react with acid anhydride to get side chain linker, connect the side chain with minocycline through Tscherniac-Einhorn reaction, and then hydrolyze chloroacetyl and hydroxymethyl to get omadacycline. This route has relatively mild reaction conditions, but has problems such as long reaction steps and low yield, with a yield of 45% and a purity of >98%.

[0013]

[0014] Route three: minocycline reacts with iodinating agent to get 9-iodo minocycline, which is then converted into 9-aldehyde derivative, and finally reacts with pivalamine and hydrogen under palladium carbon catalysis to get omadacycline. This route is simple in operation and simple in raw materials, but uses metal catalyst and expensive ligand, with a yield of 69% and a purity of only 64.91%.

[0015]

[0016] For tetracycline antibiotics containing Mannich base, the synthesis route shows that in the current synthesis method of tetracycline antibiotics, the method for connecting Mannich base all has problems such as long steps, complicated operation, low yield or poor purity, and some also need to use noble metal and ligand, which is not conducive to industrial production. If the Mannich base of tetracycline antibiotics can be connected to tetracycline in a simpler way, it will greatly promote the improvement of omadacycline synthesis process, and provide a new idea for the new drug development and process improvement of tetracycline antibiotics. SUMMARY

[0017] The technical problem to be solved by the present application is to overcome the defects in the prior art, such as long steps, complicated operation, low yield, poor purity, the need to use noble metals and ligands, and the like, in the synthesis method of tetracycline compounds, and to provide a 9-position Mannich base-containing tetracycline compound, a preparation method and application thereof. The method for connecting a Mannich base to a tetracycline compound in the present application has simple steps, mild reaction conditions, strong operability, and can obtain a product with high purity after simple purification.

[0018] The present application provides a preparation method of a 9-position Mannich base-containing tetracycline compound as shown in formula I, which comprises the following steps:

[0019] In a solvent, a compound as shown in formula I-A, a compound as shown in formula SM-1 and formaldehyde are subjected to a reaction as shown below to obtain a compound as shown in formula I;

[0020]

[0021] wherein:

[0022] X is present or absent, and when X is present, it is an inorganic acid;

[0023] R 9a and R 9b each independently is hydrogen, C1-C6 alkyl, halogen-substituted C1-C6 alkyl, C1-C6 alkoxy, 6-9 membered spirocyclyl, 6-9 membered heterospirocyclyl, 6 membered saturated fused cyclyl or 6 membered saturated heterofused cyclyl; wherein the heteroatoms in the 6-9 membered heterospirocyclyl and 6 membered saturated heterofused cyclyl are selected from N, O and S, and the number of heteroatoms is 1, 2 or 3;

[0024] or R 9a and R 9b together with the connected N atom form a 4-6 membered heterocyclyl, halogen-substituted 4-6 membered heterocyclyl, 6-9 membered heterospirocyclyl or 6 membered saturated heterofused cyclyl; wherein the heteroatoms in the 4-6 membered heterocyclyl, 6-9 membered heterospirocyclyl and 6 membered saturated heterofused cyclyl are selected from N, O and S, and the number of heteroatoms is 1, 2 or 3.

[0025] Preferably, the R 9a and R 9b each independently is C1-C6 alkyl or halogen-substituted C1-C6 alkyl; further preferably C1-C6 alkyl, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, i-pentyl, t-pentyl, most preferably methyl or ethyl, particularly preferably R 9a and R 9bsimultaneously ethyl.

[0026] Preferably, said R 9a 9b together with the N atom to which they are attached form a 4-6 membered heterocyclyl or a halogen substituted 4-6 membered heterocyclyl, more preferably a 4-6 membered heterocyclyl. Said 4-6 membered heterocyclyl is preferably a five membered nitrogen containing heterocyclyl or a six membered nitrogen containing heterocyclyl, further preferably a tetrahydropyrrolyl or a hexahydropyridinyl.

[0027] Preferably, said compound of formula I-A is a hydrochloride.

[0028] Preferably, said formaldehyde is formaldehyde aqueous solution (37 wt.%) or paraformaldehyde, preferably formaldehyde aqueous solution (37 wt.%).

[0029] Preferably, said reaction is carried out under gas protection, further preferably, said reaction is carried out under nitrogen protection.

[0030] Preferably, said solvent is a mixture of organic solvent and water. Wherein, said organic solvent is preferably one or more of alcohol solvent, amide solvent and sulfoxide organic solvent, more preferably alcohol solvent. Said alcohol solvent is preferably one or more of methanol, ethanol and propanol, more preferably methanol. Preferably, said water is deionized water. The amount of said organic solvent and water can not be specifically limited, as long as it does not affect the reaction. The volume ratio of said organic solvent and water is preferably 1:1-10:1, preferably 2:1-5:1.

[0031] Preferably, the mass volume ratio of said compound of formula I-A and said organic solvent is 1:2-1:50, further preferably 1:2-1:20 (e.g. 1:10).

[0032] Preferably, the mass volume ratio of said compound of formula I-A and said water is 1:2-1:50, further preferably 1:2-1:20 (e.g. 1:5).

[0033] Preferably, the molar ratio of said compound of formula I-A and said compound of formula SM-1 is 1:2-1:50, further preferably 1:2-1:20, most preferably 1:4-1:6.

[0034] Preferably, the molar ratio of said compound of formula I-A and formaldehyde is 1:2-1:50, further preferably 1:2-1:20, most preferably 1:4-1:6.

[0035] Preferably, the reaction temperature of said reaction is 50-120°C, preferably 60-80°C.

[0036] ​The reaction progress is generally determined by the end of the reaction. Preferably, the end of the reaction is determined by HPLC detection of the content of the compound of formula I in the reaction solution being less than 5%.

[0037] Preferably, the method for preparing the compound of formula I comprises the following steps:

[0038] (a) mixing the compound of formula I-A with a solvent to obtain a mixture;

[0039] (b) adding the compound of formula SM-1 and formaldehyde to the mixture obtained in step (a) to obtain the compound of formula I.

[0040] Preferably, the temperature of the mixture in step (a) is 10-40°C, and more preferably 25°C.

[0041] Preferably, the method for preparing the compound of formula I comprises the following steps:

[0042] After the reaction is completed, it further comprises the following steps of post-treatment:

[0043] (1) removing the solvent in the mixed solution obtained after the reaction is completed to obtain a mixture, and then mixing the mixture with water to obtain a mixed solution;

[0044] (2) adjusting the pH value of the mixed solution obtained in step (1) to 1-10 to obtain a purified solution;

[0045] (3) separating and purifying the purified solution obtained in step (2) to obtain the target compound.

[0046] Preferably, in step (1), the reaction solution obtained after the reaction is completed is concentrated under reduced pressure to obtain a mixture, and the temperature during the concentration under reduced pressure can be 10-80°C, preferably 30-50°C, and more preferably 35°C or lower, and even more preferably room temperature.

[0047] Preferably, in step (1), the water is preferably deionized water. The mass / volume ratio of the compound of formula I-A to water is 1:2-1:50, and preferably 1:10-1:20.

[0048] Preferably, in step (2), the pH value of the mixed solution obtained in step (1) is adjusted to 2-3, 4-5, 5-6, or 7-8.

[0049] Preferably, in step (2), the pH value can be adjusted by an inorganic acid or an organic acid. Preferably, the inorganic acid is hydrochloric acid, sulfuric acid, or nitric acid, and most preferably hydrochloric acid, such as concentrated hydrochloric acid. Preferably, the organic acid is p-toluenesulfonic acid.

[0050] Preferably, in step (3), the separation and purification is by column chromatography. The column chromatography filler is preferably a macroporous resin of the XAD series. The macroporous resin of the XAD series is preferably a macroporous resin of XAD4, XAD7HP, XAD16HP, XAD1600, XAD1600N, XAD1180 or XAD761, more preferably a macroporous resin of XAD1180. The column chromatography column specification is 2:1-5:1, preferably 2.5:1-3.5:1, relative to the volume of the solution to be purified.

[0051] Preferably, in step (3), the column chromatography mobile phase is mobile phase A and mobile phase B, the mobile phase A is an alcohol solvent, such as methanol and / or ethanol, and the mobile phase B is water, such as deionized water.

[0052] Preferably, in step (3), the column of the column chromatography is first washed with water, and then the solution to be purified is added to the column for separation; the water is preferably deionized water, and the volume ratio of the water to the solution to be purified is 10:1-20:1.

[0053] Preferably, in step (3), after the solution to be purified is added to the column, the column is first washed with water, the water is preferably deionized water, and the volume ratio of the water to the solution to be purified is 10:1-20:1.

[0054] Preferably, the column chromatography is gradient elution, the flow rate is 2 mL / min-5 mL / min, and the mobile phase gradient change and amount are shown in the following table:

[0055]

[0056] In step (3), the eluent containing the target compound is collected, the solvent is removed (concentrated under reduced pressure), and dried (oven dried under reduced pressure, the oven drying temperature is, for example, 0°C-100°C, preferably 20°C-50°C, more preferably 35°C).

[0057] The present application also provides a 9-position Mannich base-containing tetracycline compound as shown in I or a pharmaceutically acceptable salt thereof.

[0058]

[0059] wherein, R 9a and R 9b are as defined above, but R 9a and R 9b are not simultaneously hydrogen.

[0060] Preferably, the 9-position Mannich base-containing tetracycline compound as shown in I is any one of the following compounds:

[0061]

[0062] The present application also provides a pharmaceutical composition comprising the 9-position Mannich base-containing tetracycline compound as described above shown as I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0063] The present application also provides use of the 9-position Mannich base-containing tetracycline compound as described above shown as I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a disease caused by bacterial, viral or microbial infection.

[0064] The disease caused by bacterial, viral or microbial infection can be a disease caused by Gram-positive bacteria, Gram-negative bacteria, Rickettsia, Mycoplasma, Chlamydia, lymphogranuloma venereum pathogen, inclusion conjunctivitis pathogen or psittacosis pathogen.

[0065] The disease caused by bacterial, viral or microbial infection can be a disease caused by Staphyloccocus aureus, Enterococcus Faecium, Pseudomonas aeruginosa, Acinetobacter baumannii or Escherichia coli or drug-resistant bacteria thereof.

[0066] In the present application, the functional group to be protected can be protected if necessary, and then the protecting group is removed by a conventional method.

[0067] In the present application, the separation and purification of the macroporous resin comprises the following steps: the reaction liquid obtained from the reaction is separated and purified by a separation column of XAD series macroporous adsorption resin, so that the 9-position Mannich base-containing tetracycline compound as described above shown as I with higher purity can be obtained. The operation steps and operation conditions of the separation column of XAD series macroporous adsorption resin can be selected according to the conventional operation steps and operation conditions of the separation column of XAD series macroporous adsorption resin.

[0068] On the basis of not violating the common sense in the art, the above-mentioned preferred conditions can be combined arbitrarily, so as to obtain each preferred embodiment of the present application.

[0069] The "pharmaceutically acceptable salt" of the 9-position Mannich base-containing tetracycline compound of Formula I refers to a salt formed with a suitable inorganic or organic cation (base) when there is an acidic functional group (such as -COOH, -OH, SOH, etc.) in the 9-position Mannich base-containing tetracycline compound of Formula I, including a salt formed with an alkali metal such as sodium, potassium, lithium, etc.; a salt formed with an alkaline earth metal such as calcium, magnesium, etc.; a salt formed with other metals such as aluminum, iron, zinc, etc.; a salt formed with an inorganic base such as ammonium, etc.; a salt formed with an organic base such as tertiary octylamine, piperazine, tetramethylamine, tris(hydroxymethyl)aminomethane, etc.; and a salt formed with a suitable inorganic or organic anion (acid) when there is a basic functional group (such as -NH2, etc.) in the 9-position Mannich base-containing tetracycline compound of Formula I, including a salt formed with an inorganic acid such as nitric acid, perchloric acid, sulfuric acid, phosphoric acid, hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.; a salt formed with a sulfonic acid such as methanesulfonic acid, p-toluenesulfonic acid, etc.; a salt formed with an organic acid such as acetic acid, malic acid, fumaric acid, etc.; a salt formed with an amino acid such as glycine, trimethylglycine, aspartic acid, etc.

[0070] The reagents and raw materials used in the present application are commercially available.

[0071] The positive progress effect of the present application is that the method of the present application is simple in steps, mild in reaction conditions, strong in operability, and can obtain a product with high yield and purity after simple purification. DETAILED DESCRIPTION

[0072] The present application will be further described by way of examples, but the present application is not limited to the scope of the examples. The experimental methods in the following examples, for which no specific conditions are indicated, are selected according to conventional methods and conditions, or according to the instructions of the products.

[0073] Example 1: Preparation of Compound 1

[0074]

[0075] In a 250 mL three-necked flask, under nitrogen protection, 10 g of minocycline hydrochloride (content greater than 98%) was added, 100 mL of methanol was added, stirred and dissolved at room temperature for 1 min, 7.77 g of tetrahydropyrrole, 8.2 g of formaldehyde aqueous solution (37 wt.%) and 50 mL of water were sequentially added, and the reaction was stirred at 70°C. HPLC detection of the reaction solution showed that the minocycline was 0.22%, and the reaction was stopped.

[0076] The above reaction solution was concentrated under reduced pressure, and the concentration temperature was not higher than 35°C. The concentrated solution was dissolved in 100 mL of deionized water. After adjusting the pH to 2-3 with concentrated hydrochloric acid, a purified solution was obtained, and was filtered and purified.

[0077] A 250 mL XAD 1180 macroporous adsorption resin column was packed and pretreated (usually by soaking with methanol for 6-24 h), and then washed with 1500 mL of deionized water. The above solution to be purified was loaded onto the column, and then the column was washed with 1500 mL of deionized water, and then sequentially washed with 500 mL of 20% methanol solution, 500 mL of 40% methanol solution, 500 mL of 60% methanol solution, 500 mL of 80% methanol solution, and 500 mL of 95% methanol solution, to obtain a methanol solution containing the target component. The flow rate during elution was 2-5 mL / min.

[0078] The methanol solution containing the target component was collected and concentrated to dryness, and then transferred to a vacuum oven and dried at 35°C to obtain 9.17 g of a brownish yellow powder, which was the target compound 1, with a total yield of 85.23% and a purity of 98%.

[0079] Molecular formula: C 28 H 34 N4O7, molecular weight: 538.24, MS (m / e): 529.25 (M+H) (found).

[0080] 1 H NMR (600 MHz, D2O) δ 6.90 (s, 1H), 6.58 (s, 1H), 4.00-3.87 (m, 2H), 3.68 (d, J = 7.3 Hz, 1H), 3.35 (d, 1H), 3.23-3.13 (m, 4H), 2.96-2.92 (m, 1H), 2.82 (d, 1H), 2.75 (s, 6H), 2.43 (s, 6H), 1.83 (d, J = 13.9 Hz, 4H).

[0081] Example 2: Preparation of compound 2

[0082]

[0083] In a 250 mL three-necked flask, under nitrogen protection, 10 g of minocycline hydrochloride (content greater than 98%) was added, 100 mL of methanol was added, and stirring was performed at room temperature for 1 min. Then 9.3 g of hexahydropyridine and 8.2 g of formaldehyde aqueous solution (37 wt.%) were sequentially added, and 50 mL of water was added, and stirring was performed at 70°C. HPLC detection of the reaction solution showed that the minocycline content was 1.67%, and the reaction was stopped.

[0084] The above reaction solution was concentrated under reduced pressure, and the concentration temperature was not higher than 35°C. The concentrated solution was dissolved in 100 mL of deionized water. After adjusting the pH to 5-6 with concentrated hydrochloric acid, a solution to be purified was obtained, which was filtered and purified.

[0085] A 250 mL XAD 1180 macroporous adsorption resin column was packed and pretreated (usually by soaking with methanol for 6-24 h), and then washed with 1500 mL of deionized water. The above solution to be purified was loaded onto the column, and then the column was washed with 1500 mL of deionized water, and then sequentially washed with 500 mL of 20% methanol solution, 500 mL of 40% methanol solution, 500 mL of 60% methanol solution, 500 mL of 80% methanol solution, and 500 mL of 95% methanol solution, to obtain a methanol solution containing the target component. The flow rate during elution was 2-5 mL / min.

[0086] The methanol solution containing the target component was collected and concentrated to dryness, and then transferred to a vacuum oven and dried at 35°C to obtain 9.46 brownish yellow powder, which was the target compound 2, with a total yield of 85.61% and a purity of 98.56%.

[0087] Molecular formula: C 29 H 36 N4O7, molecular weight: 552.26, MS (m / e): 553.28 (M+H) (found).

[0088] 1 H NMR (400 MHz, DMSO) δ 7.01 (s, 1H), 6.65 (s, 1H), 4.02 (d, J = 9.4 Hz, 2H), 3.29-3.24 (m, 1H), 3.13 (d, J = 11.7 Hz, 2H), 2.97 (d, J = 5.7 Hz, 4H), 2.90 (d, 1H), 2.59 (s, 6H), 2.34 (d, J = 3.6 Hz, 6H), 1.65-1.62 (m, 4H), 1.25-1.20 (m, 2H).

[0089] Example 3: Preparation of compound 3

[0090]

[0091] In a 250 mL three-necked flask, under nitrogen protection, 10 g of minocycline hydrochloride (content greater than 98%) was added, 100 mL of ethanol was added, and stirring was performed at room temperature for 1 min. Then, 12.3 g of dimethylamine aqueous solution (40% wt.% in H2O), 8.2 g of formaldehyde aqueous solution (37 wt.%), and 20 mL of water were sequentially added, and the reaction was stirred at 70°C. HPLC detection of the reaction solution showed that the minocycline content was 1.67%, and the reaction was stopped.

[0092] The above reaction solution was concentrated under reduced pressure, and the concentration temperature was not higher than 35°C. The concentrated solution was dissolved in 100 mL of deionized water. After adjusting the pH to 4-5 with concentrated hydrochloric acid, a solution to be purified was obtained, and filtration purification was performed.

[0093] A 250 mL XAD 1180 macroporous adsorption resin column was packed and pretreated (usually by soaking with methanol for 6-24 h), and then washed with 1500 mL of deionized water. The above solution to be purified was loaded onto the column, and then the column was washed with 1500 mL of deionized water, and then sequentially washed with 500 mL of 20% methanol solution, 500 mL of 40% methanol solution, 500 mL of 60% methanol solution, 500 mL of 80% methanol solution, and 500 mL of 95% methanol solution, to obtain a methanol solution containing the target component. The flow rate during elution was 2-5 mL / min.

[0094] The methanol solution containing the target component was collected and concentrated to dryness, and then transferred to a vacuum oven and dried at 35°C to obtain 9.1 g of a brownish yellow powder, which was the target compound 3. The total yield was 88.87%, and the purity was 98.88%.

[0095] Molecular formula: C 26 H 32 N2O4, molecular weight: 512.23, MS (m / e): 511.24 (M-H) (observed).

[0096] 1 H NMR (400 MHz, DMSO) δ 7.01 (s, 1H), 6.67 (s, 1H), 4.14-4.06 (m, 2H), 3.29-3.24 (m, 1H), 3.17 (d, J = 10.5 Hz, 1H), 3.13 (s, 1H), 2.97-2.91 (m, 1H), 2.66 (s, 6H), 2.56 (s, 6H), 2.36 (s, 6H).

[0097] Example 4: Preparation of compound 4

[0098]

[0099] In a 250 mL three-necked flask, under nitrogen protection, 10 g of minocycline hydrochloride (content greater than 98%) was added, 150 mL of methanol was added, and stirring was performed at room temperature for 1 min. Then, 7.98 g of diethylamine and 8.2 g of formaldehyde aqueous solution (37 wt.%) were sequentially added, and 75 mL of water was added. The reaction was stirred at 70°C. HPLC detection of the reaction solution showed that the minocycline content was 1.67%, and the reaction was completed.

[0100] The above reaction solution was concentrated under reduced pressure, and the concentration temperature was not higher than 35°C. The concentrated solution was dissolved in 100 mL of deionized water. After adjusting the pH to 7-8 with concentrated hydrochloric acid, a solution to be purified was obtained, and filtration purification was performed.

[0101] A 250 mL XAD1180 macroporous adsorption resin column was packed and pretreated (usually soaked with methanol for 6-24 h), and then washed with 1500 mL of deionized water. After the above-mentioned solution to be purified was loaded onto the column, the column was washed with 1500 mL of deionized water, and then sequentially washed with 500 mL of 20% methanol solution, 500 mL of 40% methanol solution, 500 mL of 60% methanol solution, 500 mL of 80% methanol solution, and 500 mL of 95% methanol solution, to obtain a methanol solution containing the target component. The flow rate during elution was 2-5 mL / min.

[0102] The methanol solution containing the target component was collected and concentrated to dryness, and then transferred to a vacuum oven and dried at 35°C to obtain 9.28 g of a brownish yellow powder, which was the target compound 4, with a total yield of 85.85% and a purity of 97.24%.

[0103] Molecular formula: C 28 H 36 N4O7, molecular weight: 540.26, MS (m / e): 541.25 (M+H) (found).

[0104] 1 H NMR (600 MHz, DMSO+H2O) δ 7.02 (s, 1H), 6.70 (s, 1H), 4.14 (q, J = 13.2 Hz, 2H), 3.29-3.26 (m, 1H), 3.05 (q, J = 7.3 Hz, 4H), 2.95 (d, J = 2.4 Hz, 1H), 2.91 (d, J = 7.2 Hz, 1H), 2.88 (d, J = 7.2 Hz, 1H), 2.62 (s, 6H), 2.35 (s, 6H), 1.22 (t, J = 7.3 Hz, 6H).

[0105] Example 5: In vitro antibacterial activity test of the compound

[0106] Test strains:

[0107]

[0108]

[0109] The above strains were obtained by clinical isolation, and the selection of the strains was based on Antimicrobial activity of omadacycline in vitro against bacteria isolated from 2014 to 2017 in China, a multi-center study [J]. BMC Microbiology, 2020, 20(1).

[0110] Test substances: control drug omilancin and compounds of the present application (Examples 1-4).

[0111] Experimental method: The minimum inhibitory concentration (MIC) of the four compounds was determined by the micro-broth dilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS).

[0112] Experimental results and conclusion:

[0113]

[0114] MIC represents minimum inhibitory concentration

[0115] As shown in the table, the compounds of the present application have antibacterial activity against the above test strains, which is not inferior to that of omilancin, and the antibacterial activity against Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli is better than that of omilancin, which has good clinical potential.

Claims

1. A method for preparing a tetracycline compound containing a Mannich base at the 9-position as shown in Formula I, characterized in that, comprising the following steps: carrying out a reaction as shown below on a compound as shown in formula I-A, a compound as shown in formula SM-1 and formaldehyde in a solvent to obtain a compound as shown in formula I; ， wherein: X is present or not, when X is present, it is an inorganic acid; R 9a and R 9b each independently is C1-C6alkyl; or, R 9a and R 9b together with the attached N atom form a tetrahydropyrrolyl or hexahydropyridinyl group; the reaction temperature of the reaction is 50°C to 120°C.

2. The preparation method of claim 1, wherein, R 9a and R 9b each independently is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, t-pentyl.

3. The production method according to claim 2, wherein R 9a and R 9b each independently is methyl or ethyl.

4. The production method according to claim 1 or 2, wherein the compound as shown in formula SM-1 is dimethylamine, diethylamine, tetrahydropyrrole or hexahydropyridine; and / or, the compound as shown in formula I-A is minocycline hydrochloride; and / or, the formaldehyde is formaldehyde aqueous solution or paraformaldehyde; and / or, the reaction is carried out under gas protection; and / or, the solvent is a mixed solvent of organic solvent and water; and / or, the molar ratio of the compound as shown in formula I-A and the compound as shown in formula SM-1 is 1:2-1:50; and / or, the molar ratio of the compound as shown in formula I-A and formaldehyde is 1:2-1:50; and / or, the reaction temperature of the reaction is 60-80℃.

5. The production method according to claim 4, wherein the reaction is carried out under gas protection, and the gas is nitrogen; or, the organic solvent is one or more of alcohol solvent, amide solvent and sulfoxide organic solvent; or, the water is deionized water; or, the volume ratio of the organic solvent and water is 1:1-10:1; or, the mass-volume ratio of the compound as shown in formula I-A and the organic solvent is 1:2-1:50; or, the mass-volume ratio of the compound as shown in formula I-A and the water is 1:2-1:50; or, the molar ratio of the compound as shown in formula I-A and the compound as shown in formula SM-1 is 1:2-1:20; or, the molar ratio of the compound as shown in formula I-A and formaldehyde is 1:2-1:

20.

6. The production method according to claim 5, wherein the alcohol solvent is one or more of methanol, ethanol and propanol; or, the volume ratio of the organic solvent and water is 1:1-5:1; or, the mass-volume ratio of the compound as shown in formula I-A and the organic solvent is 1:2-1:20; or, the mass-volume ratio of the compound as shown in formula I-A and the water is 1:2-1:

20.

7. The production method according to claim 1, wherein comprising the following steps: (a) mixing a compound as shown in formula I-A with a solvent to obtain a mixture; (b) adding a compound as shown in formula SM-1 and formaldehyde to the mixture obtained in step (a) to carry out the reaction to obtain a compound as shown in formula I.

8. The preparation method of claim 7, wherein, the temperature of mixing in step (a) is 10-40℃; and / or, the (b) adding a compound as shown in formula SM-1 and formaldehyde to the mixture obtained in step (a) to carry out the reaction.

9. The preparation method of claim 8, wherein, the temperature of mixing in step (a) is 25℃.

10. The production method according to claim 1, wherein It further comprises a step of post-treatment, and the post-treatment comprises the following steps: (1) removing the solvent in the mixed solution obtained after the reaction is completed to obtain a mixture, and then mixing with water to obtain a mixed solution; (2) adjusting the pH value of the mixed solution obtained in step (1) to 1-10 to obtain a solution to be purified; (3) adding a base to the solution to be purified to obtain a purified solution. (3) separating and purifying the solution obtained in step (2) to obtain the target compound.

11. The preparation method of claim 10, wherein: In step (1), the reaction solution obtained after the reaction is concentrated under reduced pressure to obtain a mixture, the temperature during the concentration under reduced pressure is below 35°C; the water is deionized water; and the mass-volume ratio of the compound of formula I-A to the water is 1:2-1:50, based on the compound of formula I-A; Or, in step (2), the pH value of the mixture obtained in step (1) is adjusted to 2-3, 4-5, 5-6 or 7-8; and the pH value is adjusted by using an inorganic acid or an organic acid in step (2); Or, in step (3), the separation and purification is performed by column chromatography, the filler of the column chromatography is an XAD series macroporous resin; the volume ratio of the column specification of the column chromatography to the solution to be purified is 2:1-5:1; and the mobile phase of the column chromatography is a mobile phase A and a mobile phase B, the mobile phase A is an alcohol solvent, and the mobile phase B is water; Or, in step (3), the column of the column chromatography is first washed with water, and then the solution to be purified is added to the column for separation; the water is deionized water, and the volume ratio of the water to the solution to be purified is 10:1-20:1; Or, in step (3), after the solution to be purified is added to the column, the column is first washed with water, the water is deionized water, and the volume ratio of the water to the solution to be purified is 10:1-20:1; Or, the column chromatography is gradient elution, the flow rate is 2 mL / min-5 mL / min, and the gradient change and the amount of the mobile phase are shown in the following table; 。 12. The preparation method of claim 11, wherein: In step (1), the mass-volume ratio of the compound of formula I-A to the water is 1:10-1:20; Or, in step (2), the pH value is adjusted by using hydrochloric acid, sulfuric acid, nitric acid or p-toluenesulfonic acid; Or, in step (3), the filler of the column chromatography is an XAD4, XAD7HP, XAD16HP, XAD1600, XAD1600N, XAD1180 or XAD761 macroporous resin; Or, the volume ratio of the column specification of the column chromatography to the solution to be purified is 2.5:1-3.5:1; Or, the mobile phase A is methanol and / or ethanol.

13. The production method according to claim 1, wherein The compound of formula I is any one of the following compounds: ， ， ， 。

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