Preparation method of oxamacycline

Abstract

The invention belongs to the field of medicine synthesis, and relates to a preparation method of ombracin and application of the ombracin to preparation of the ombracin.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of Chinese Patent Application No. 202410586345.3, filed with the State Intellectual Property Office of the People's Republic of China on May 11, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure pertains to the field of pharmaceutical synthesis and relates to a method for preparing omalicycline and its use in the preparation of omalicycline. Background Technology

[0004] Omadacycline, also known as 9-[(2,2-dimethyl-propylamino)-methyl]-minocycline, can bind to the 30S subunit of the ribosome, preventing aminoacyl-tRNA from binding to ribonucleoproteins, thereby treating infections in adult patients caused by susceptible microorganisms, such as community-acquired bacterial pneumonia, acute bacterial skin and skin structure infections.

[0005] Existing technology CN107613988A discloses a preparation route for omalicycline:

[0006]

[0007] Compound 5 was reacted with pivalal under Pd / C and hydrogen pressure to prepare compound 1 (the free base of omacycline). This method is unsuitable for industrial production, and the synthesis process involves a large number of β-isomer impurities, making subsequent purification difficult and resulting in low yields. Therefore, there is a need to find a simpler, higher-yield, less impurity-containing, lower-cost method for the synthesis of omacycline that is more suitable for industrial production. Summary of the Invention

[0008] On one hand, this disclosure provides a method for preparing compound 1, characterized by comprising: step (i) reacting compound II with compound a to obtain compound 1.

[0009]

[0010] In some embodiments of this disclosure, in step (i), compound II reacts with compound a in the presence of an organic solvent and a reducing agent to give compound 1.

[0011] In some embodiments of this disclosure, in step (i), the reducing agent is selected from 2-methylpyridine-N-methylborane, sodium triacetylborohydride, sodium borohydride, sodium cyanoborohydride, borane, dimethyl sulfide borohydride, tetrahydrofuran borohydride, N,N-diisopropylethylamine borane complex, pyridine borohydride complex, morpholine borohydride complex, triethylamine borohydride complex, dimethylamine borohydride complex, ethylenediamine borohydride complex, or 5-ethyl-2-methylpyridine borane complex; preferably 2-methylpyridine-N-methylborane, pyridine borane complex, 5-ethyl-2-methylpyridine borane complex, or morpholine borohydride complex; preferably 2-methylpyridine-N-methylborane or pyridine borane complex; more preferably 2-methylpyridine-N-methylborane.

[0012] In some embodiments of this disclosure, in step (i), the organic solvent is selected from one or more mixed solvents selected from acetonitrile, methanol, ethanol, ethylene glycol, butanol, octanol, octyl acetate, methyl tert-butyl ether, dioxane, tetrahydrofuran, N,N-dimethylformamide, or dimethyl sulfoxide; preferably one or more mixed solvents selected from acetonitrile, methanol, ethanol, methyl tert-butyl ether, or tetrahydrofuran; more preferably methanol.

[0013] In some embodiments of this disclosure, in step (i), the molar ratio of compound II to compound a is 1:1 to 10; preferably 1:1 to 5. In a specific embodiment of this disclosure, the molar ratio of compound II to compound a is 1:1, 1:2, 1:3, 1:4 or 1:5; preferably 1:3.

[0014] In some embodiments of this disclosure, in step (i), the molar ratio of the compound of formula II to the reducing agent is 1:1 to 3; preferably 1:1 to 2. In a specific embodiment of this disclosure, the molar ratio of the compound of formula II to the reducing agent is 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2; preferably 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5 or 1:1.6; more preferably 1:1.2.

[0015] In some embodiments of this disclosure, in step (i), the weight ratio of the compound of formula II to the organic solvent is 1:1 to 10; preferably 1:2 to 5. In a specific embodiment of this disclosure, the weight ratio of the compound of formula II to the organic solvent is 1:2, 1:3, 1:4 or 1:5; preferably 1:4.

[0016] In some embodiments of this disclosure, in step (i), the compound of formula II reacts with compound a under a nitrogen atmosphere.

[0017] In some embodiments of this disclosure, in step (i), the compound of formula II is first stirred evenly in an organic solvent, then compound a is added, followed by the addition of a reducing agent.

[0018] In some embodiments of this disclosure, in step (i), the reducing agent may be added all at once or in batches. Preferably, the reducing agent is added in batches; more preferably, the reducing agent is added in 2, 3, 4 or 5 batches on average. More preferably, the reducing agent is added in 3 batches on average.

[0019] In some embodiments of this disclosure, in step (i), the reaction temperature of compound II with compound a is 0–40°C; preferably 10–30°C; more preferably 20–30°C.

[0020] In some embodiments of this disclosure, in step (i), the reaction time between the compound of formula II and compound a is 1 to 10 hours; preferably 1 to 5 hours; more preferably 1 to 3 hours.

[0021] In some embodiments of this disclosure, step (i) further includes a post-processing step.

[0022] In some embodiments of this disclosure, step (i) is post-processed to obtain a concentrated solution of compound 1.

[0023] In some embodiments of this disclosure, step (i) further includes crystallizing the concentrate of compound 1.

[0024] In some embodiments of this disclosure, step (i) crystallization of compound 1 concentrate includes: dissolving compound 1 concentrate in a first solvent, and then adding a second solvent and stirring to induce crystallization.

[0025] In some embodiments of this disclosure, the first solvent in the crystallization step of the compound 1 concentrate is selected from dichloromethane, ethyl acetate, tetrahydrofuran, 1,4-dioxane, or n-propanol; dichloromethane is preferred.

[0026] In some embodiments of this disclosure, the second solvent in the crystallization step of the concentrated compound 1 is selected from methyl tert-butyl ether, n-heptane, n-pentane, n-hexane, diethyl ether, anisole, or isopropyl ether; preferably methyl tert-butyl ether or n-heptane; more preferably n-heptane.

[0027] In some embodiments of this disclosure, the weight ratio of the first solvent and the second solvent in the crystallization step of the concentrated compound 1 solution is 1:1 to 3; preferably 1:1 to 2. In a specific embodiment of this disclosure, the weight ratio of the first solvent and the second solvent in the crystallization step of the concentrated compound 1 solution is 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2; preferably 1:1.5.

[0028] In some embodiments of this disclosure, the post-processing step (i) includes: directly adding purified water to the reaction solution of compound 1, adjusting the pH to dissolve, and washing with a first solvent. In some embodiments of this disclosure, the post-processing step (i) includes: directly adding purified water to the reaction solution of compound 1, adjusting the pH to 3.0–5.0 to dissolve, and washing with a first solvent.

[0029] In some embodiments of this disclosure, the first solvent in the post-processing step (i) is selected from dichloromethane, ethyl acetate, tetrahydrofuran, 1,4-dioxane, or n-propanol; dichloromethane is preferred.

[0030] In some embodiments of this disclosure, the weight ratio of the first solvent to the compound of formula II is 1:0.05 to 0.5. In some embodiments of this disclosure, the weight ratio of the first solvent to the compound of formula II is 1:0.05, 1:0.06, 1:0.07, 1:0.08, 1:0.09, 1:0.1, 1:0.2, 1:0.3, 1:0.4, or 1:0.5; preferably 1:0.1.

[0031] In some embodiments of this disclosure, step (i) further includes a crystallization step.

[0032] In some embodiments of this disclosure, the crystallization step includes adjusting the pH of the compound 1 solution to 8.0–14.0 and then crystallizing; preferably, adjusting the pH of the compound 1 solution to 10–12 and then crystallizing.

[0033] In some embodiments of this disclosure, in step (i), after the reaction of compound II with compound a is completed, crystallization can be performed directly in the solvent without post-treatment.

[0034] In some embodiments of this disclosure, in step (i), after the reaction of compound II with compound a is completed, purified water is added, the pH is adjusted to 9.0 to 12.0, and crystallization occurs; preferably, the pH is adjusted to 10 to 12, and crystallization occurs.

[0035] In some embodiments of this disclosure, the weight ratio of the purified water to the compound of formula II is 1:0.1 to 0.5; preferably 1:0.1 to 0.3. In a specific embodiment of this disclosure, the weight ratio of the purified water to the compound of formula II is 1:0.1, 1:0.15, 1:0.2, 1:0.25, or 1:0.3; preferably 1:0.2.

[0036] In some embodiments of this disclosure, in step (i), after the reaction of compound II with compound a is completed, a second solvent is directly added dropwise to precipitate the product.

[0037] In some embodiments of this disclosure, the second solvent is selected from methyl tert-butyl ether, n-heptane, or diethyl ether; preferably methyl tert-butyl ether or n-heptane; more preferably methyl tert-butyl ether.

[0038] In some embodiments of this disclosure, the compound 1 obtained in step (i) further includes a purification step.

[0039] In some embodiments of this disclosure, the purification step includes: dissolving the solid obtained by the above crystallization, eluting it through a preparative column, concentrating the eluent, and crystallizing it again to obtain the purified compound 1.

[0040] In some embodiments of this disclosure, in the above-mentioned refining steps, the preparation column is a reverse-phase preparation column, preferably a preparation column with reverse-phase polymer filler or C18 filler.

[0041] In some embodiments of this disclosure, the eluent used in the above purification step, specifically the reverse-phase preparative column purification, is an acetonitrile-water solution.

[0042] In some embodiments of this disclosure, in the purification step described above, the eluent used for column purification is an acidic solution of acetonitrile-water, and the acidity adjuster is hydrochloric acid, methanesulfonic acid, trifluoroacetic acid, sulfuric acid, oxalic acid, or formic acid. In some embodiments of this disclosure, in the purification step described above, the pH of the eluent is 1.0–5.0; preferably 2.0–4.0; more preferably 3.0.

[0043] In some embodiments of this disclosure, in the purification step described above, the eluent gradient used for the preparative column purification is isocratic elution with a 6% (v / v) acetonitrile-water solution.

[0044] In some embodiments of this disclosure, the re-crystallization step in the above purification step includes: concentrating the eluent and dissolving it in a first solvent, then adding a second solvent and stirring to induce crystallization.

[0045] In some embodiments of this disclosure, the re-crystallization step in the above purification step includes: concentrating the eluent and then directly adding it dropwise to a second solvent while stirring to induce crystallization.

[0046] In some embodiments of this disclosure, the re-crystallization step in the above-mentioned purification step includes: concentrating the eluent and then directly adding it dropwise to the second solvent and stirring to induce crystallization, wherein the volume ratio of the concentrated eluent to the second solvent is 1:1 to 1:10; preferably 1:2 to 1:5; and more preferably 1:2, 1:3, 1:4 or 1:5.

[0047] In some embodiments of this disclosure, in the above-mentioned purification step, the first solvent in the re-crystallization step is selected from dichloromethane, ethyl acetate, tetrahydrofuran, 1,4-dioxane, or n-propanol; dichloromethane is preferred.

[0048] In some embodiments of this disclosure, in the above-mentioned purification step, the second solvent in the re-crystallization step is selected from methyl tert-butyl ether, n-heptane, or diethyl ether; preferably methyl tert-butyl ether or n-heptane; more preferably n-heptane.

[0049] In some embodiments of this disclosure, compound 1 obtained from the above purification steps is amorphous.

[0050] In some embodiments of this disclosure, the X-ray powder diffraction spectrum of the amorphous form of compound 1 is as follows: Figure 1 As shown.

[0051] On the other hand, this disclosure provides a method for preparing omacycline methanesulfonate, characterized by comprising: step (i) reacting compound II with compound a to obtain compound 1; and step (ii) reacting compound 1 with a solution of p-toluenesulfonic acid monohydrate to crystallize and obtain omacycline methanesulfonate.

[0052]

[0053] This disclosure provides a method for preparing omacycline methanesulfonate, wherein the conditions of step (i) are as described above.

[0054] In some embodiments of this disclosure, step (ii) includes: dissolving omacycline methanesulfonate in solvent I, and then adding solvent II to react.

[0055] In some embodiments of this disclosure, in step (ii), the molar ratio of compound 1 to p-toluenesulfonic acid monohydrate is 1:1 to 5; preferably 1:1 to 2. In a specific embodiment of this disclosure, the molar ratio of compound 1 to p-toluenesulfonic acid monohydrate is 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, or any range of the above values; preferably 1:1.

[0056] In some embodiments of this disclosure, in step (ii), solvent I and solvent II are each independently selected from one or more mixed solvents selected from acetonitrile, methanol, ethanol, ethylene glycol, butanol, octanol, octyl acetate, methyl tert-butyl ether, dioxane, tetrahydrofuran, N,N-dimethylformamide, or dimethyl sulfoxide; preferably one or more mixed solvents selected from acetonitrile, methanol, ethanol, methyl tert-butyl ether, or tetrahydrofuran; further preferably one or more mixed solvents selected from methanol or methyl tert-butyl ether; even more preferably methanol or methyl tert-butyl ether.

[0057] In one specific embodiment of this disclosure, in step (ii), the solvent I is selected from methanol.

[0058] In one specific embodiment of this disclosure, in step (ii), solvent II is selected from methyl tert-butyl ether.

[0059] In some embodiments of this disclosure, in step (ii), the weight ratio of solvent I to solvent II is 1:0.1 to 3; preferably 1:0.5 to 1.5. In a specific embodiment of this disclosure, in step (ii), the weight ratio of solvent I to solvent II is 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, or a range formed by any of the above values; preferably 1:1.1 or 1:1.2.

[0060] In some embodiments of this disclosure, in step (ii), the solvent in the p-toluenesulfonic acid monohydrate solution is selected from one or more mixed solvents selected from water, acetonitrile, methanol, ethanol, ethylene glycol, butanol, octanol, octyl acetate, methyl tert-butyl ether, dioxane, tetrahydrofuran, N,N-dimethylformamide, or dimethyl sulfoxide; preferably, one or more mixed solvents selected from acetonitrile, methanol, ethanol, methyl tert-butyl ether, or tetrahydrofuran.

[0061] Solvent; preferably a mixture of methanol and methyl tert-butyl ether.

[0062] In some embodiments of this disclosure, in step (ii), compound 1 and p-toluenesulfonic acid monohydrate react under a nitrogen atmosphere.

[0063] In some embodiments of this disclosure, in step (ii), the crystallization temperature of compound 1 and p-toluenesulfonic acid monohydrate is 0–40°C; preferably 10–30°C; more preferably 15–20°C.

[0064] In some embodiments of this disclosure, in step (ii), the crystallization time of compound 1 and p-toluenesulfonic acid monohydrate is 1 to 10 hours; preferably 2 to 5 hours; more preferably 3 hours.

[0065] In some embodiments of this disclosure, the omacycline methanesulfonate is in crystalline form.

[0066] In some embodiments of this disclosure, the X-ray powder diffraction spectrum of the omacycline methanesulfonate crystal form is as follows: Figure 2 As shown.

[0067] On the other hand, this disclosure provides the use of a method for preparing compound 1 in the preparation of omacycline, the method being as follows: comprising: step (i) reacting compound II with compound a to obtain compound 1,

[0068]

[0069] In some embodiments of this disclosure, step (i) of the above-described uses is subject to the conditions described above.

[0070] On the other hand, this disclosure provides a compound 1 with a 4β-isomer impurity content of no more than 5%, wherein the 4β-isomer impurity is as shown in compound III; in some preferred embodiments, the 4β-isomer impurity content in compound 1 is no more than 2%; in some preferred embodiments, the 4β-isomer impurity content in compound 1 is no more than 1.5%.

[0071]

[0072] On the other hand, this disclosure also provides a method for preparing compound 1 with a 4β-isomer impurity content of no more than 5%, the preparation method being as follows: including step (i) reacting compound II with compound a to obtain compound 1. In some preferred embodiments, this disclosure provides a method for preparing compound 1 with a 4β-isomer impurity content of no more than 2%, the preparation method being as follows: including step (i) reacting compound II with compound a to obtain compound 1. In some preferred embodiments, this disclosure provides a method for preparing compound 1 with a 4β-isomer impurity content of no more than 1.5%, the preparation method being as follows: including step (i) reacting compound II with compound a to obtain compound 1;

[0073]

[0074] In some embodiments of this disclosure, the conditions of step (i) in the above-described method for preparing compound 1 with a 4β-isomer impurity content of not more than 5% are as described above.

[0075] In some embodiments of this disclosure, the conditions of step (i) in the above-described method for preparing compound 1 with a 4β-isomer impurity content of not more than 2% are as described above.

[0076] In some embodiments of this disclosure, the conditions of step (i) in the above-described method for preparing compound 1 with a 4β-isomer impurity content of not more than 1.5% are as described above.

[0077] As described in this application, the 4β-isomer impurity content is the area normalization ratio of HPLC, and the detection wavelength is 280 nm.

[0078] As described in this application, compound 1 is an omacycline free base.

[0079] The compound of formula II described herein can be prepared with reference to patent documents US9434680B2, WO2023047323A1 or CN114591216A, or reference Tetrahedron Letters 49(2008)6095–6100.

[0080] In this disclosure, all tautomers are included within the scope of this disclosure.

[0081] Both compound a and p-toluenesulfonic acid monohydrate in this disclosure are commercially available or can be prepared by methods in the prior art.

[0082] In this disclosure, the X-ray powder diffraction spectra of the samples were determined under the following conditions: Instrument: Rigaku MinFlex II X-ray powder diffractometer; Target: Cu: Kα; Wavelength 2θ angle range: 3~60°; scanning speed 10° / min; Cu target tube voltage and current: 30KV, 15mA.

[0083] It should be noted that in X-ray powder diffraction spectroscopy, the diffraction patterns obtained from crystalline compounds are often characteristic of a specific crystal. The relative intensities of bands (especially at low angles) can vary due to the dominant orientation effect resulting from differences in crystallization conditions, particle size, and other measurement conditions. Therefore, the relative intensity of diffraction peaks is not characteristic of the specific crystal. When determining whether a crystal is identical to a known crystal, the relative positions of the peaks should be considered more than their relative intensities. Furthermore, slight errors in peak positions are known in crystallography for any given crystal. For example, peak positions can shift due to temperature variations during sample analysis, sample movement, or instrument calibration; the measurement error for 2θ values ​​can sometimes be approximately ±0.2°. Therefore, this error should be taken into account when determining each crystalline structure. In XRD patterns, peak positions are typically represented by the 2θ angle or the interplanar distance d, with a simple conversion: d = λ / 2sinθ, where d represents the interplanar distance, λ represents the wavelength of the incident X-ray, and θ is the diffraction angle. For the same crystal of the same compound, the peak positions of their XRD spectra are generally similar, but the relative intensity errors may be relatively large. It should also be noted that in the identification of mixtures, factors such as decreased content may cause the absence of some diffraction lines. In this case, it is unnecessary to rely on all bands, or even just one band, observed in a high-purity sample.

[0084] It may also be characteristic of a given crystal.

[0085] In this disclosure, methanol includes anhydrous methanol, and ethanol includes anhydrous ethanol.

[0086] In this disclosure, TLC represents thin-layer chromatography; HPLC represents high-performance liquid chromatography.

[0087] Technical effect

[0088] The preparation method of compound 1 (omalcycline free base) provided in this disclosure has the advantages of simple operation and high yield, and significantly reduces the content of 4β-isomer, better controls product quality, and is more suitable for use in industrial production. Attached Figure Description

[0090] Figure 1 X-ray powder diffraction (XRPD) pattern of the amorphous compound 1.

[0091] Figure 2 X-ray powder diffraction (XRPD) pattern of omacycline methanesulfonate crystals. Detailed Implementation

[0092] The specific embodiments described below are intended to enable those skilled in the art to better understand and implement the present invention. They should not be considered as limitations on the scope of protection of the present invention, but are merely exemplary illustrations and typical representatives of the invention. Those skilled in the art should understand that there are other synthetic routes for forming the compounds disclosed herein, and the following are non-limiting examples.

[0093] Unless otherwise stated, temperatures are in Celsius. The solvents used in this disclosure are commercially available.

[0094] Example 1: Preparation of Compound II

[0095] Step 1: Preparation of Compound I

[0096]

[0097] Under nitrogen protection, minocycline hydrochloride (1 kg, 2.0 mol), N-hydroxymethyl phthalimide I (0.72 kg, 4.0 mol), and methanesulfonic acid (5.0 kg, 52.0 mol) were added to a glass reactor and stirred at room temperature until dissolved. Methanesulfonic anhydride (0.70 kg, 4.0 mol) was slowly added, and the reaction was stirred at room temperature. HPLC monitoring was maintained until the initial reaction was complete. After the reaction was complete, the reaction solution was added to ice water, filtered, and the filter cake was dissolved in acetone. The solution was adjusted to neutral with alkali, purified water was added, and the mixture was filtered again. The filter cake was dried to obtain approximately 1.6 kg of compound I, with a yield of approximately 90%.

[0098] HPLC detection was performed using a Waters Cortecs C18 column (4.6*150mm, 2.7um). Mobile phase A consisted of 10 mmol / L ammonium formate solution (containing 0.05% formic acid), and mobile phase B consisted of acetonitrile. Gradient elution was used, and the detection wavelength was 250 nm. The area normalization ratio of formulas I-1 and I-2 was 8:1.

[0099] MSm / z (ESI): Equation I-1: 776.00 [M+H] + Formula I-2: 935.00 [M+H] +

[0100] 1H-NMR(500M,DMSO-d6)δ14.65(brs,~1H),11.84(s,1H),9.87(s,1H),7.82-7.90(m,9H),7.23(s,1H),5.15(m,1H),4.98(m,1H),4 .76(s,2H),4.34(d,J=3.0Hz,1H),3.65(brs,1H),3.13(m,1H),2.68(s,6H),2.44(s,6H),2.36(s,1H),2.07(m,1H),1.47(m,1H).

[0101] Step 2: Preparation of Compound II

[0102]

[0103] In a glass reactor, compound I (1.5 kg, 1.75 mol) and methanol I (3.67 kg) were added. Under nitrogen protection, methylamine-ethanol solution (2.5 kg, 27-32%, w / w) was added dropwise. The reaction was stirred at room temperature, and HPLC monitoring was performed until the initial reactants reacted completely. After the reaction was complete, methyl tert-butyl ether (8.0 kg) was added dropwise to the reaction solution. The mixture was filtered, and the filter cake was slurried with methanol, filtered again, and the filter cake was dried under vacuum to obtain approximately 840 g of compound II, with a yield of approximately 90%.

[0104] HPLC detection was performed using a Waters ACQUITY UPLC BEH Shield Rp18 column (2.1*100mm, 1.7um). Mobile phase A consisted of 0.1% trifluoroacetic acid solution, and mobile phase B consisted of methanol. Gradient elution was used, and the detection wavelength was 250nm. The area normalization ratio of formula II-1 and formula II-2 was 1:1.

[0105] MSm / z (ESI): Equation II-1: 487.00 [M+H] + Formula II-2: 516.10 [M+H] +

[0106] 1 H-NMR(500M,D2O)δ6.92(s,1H),4.13(s,1H),3.78(brs,1H),2.86(m,2H),2.44(s,6H),2.28(s,8H),1.77(s,1H),1.22(m,2H).

[0107] Example 2: Preparation of the sodium bisulfite adduct of pentamalonaldehyde

[0108]

[0109] Purified water (210 mL) and NaHSO3 (101 g, 0.97 mol) were added to a reaction flask and stirred at room temperature until dissolved. After dissolution, pentylaldehyde (70 g, 0.81 mol) was added dropwise. After the addition was complete, anhydrous ethanol (210 mL) was added, the temperature was raised to 50 °C and stirred for 1 hour, then cooled to room temperature. Anhydrous ethanol (420 mL) was added, the temperature was lowered to 0–10 °C, and the mixture was stirred for 1 hour. The mixture was then filtered, the filter cake was washed with anhydrous ethanol, and dried at 50 °C to obtain 137 g of the adduct, with a yield of approximately 88%.

[0110] MSm / z (ESI): 212.9 [M+Na] +

[0111] 1 H-NMR(500M,DMSO-d6)δ4.72(d,J=4.5Hz,1H),3.64(d,J=4.5Hz,1H),0.99(s,9H).

[0112] Example 3: Preparation of Omacycline Bases

[0113]

[0114] Under nitrogen protection, anhydrous methanol (50 mL) and compound II (10 g, 20 mmol) were added to a reaction flask, followed by the sodium bisulfite adduct of pentylaldehyde (11.5 g, 60.0 mmol). After the addition was complete, the temperature was controlled below 20 °C, and 2-methylpyridine-N-methylborane (3.38 g, 31.6 mmol) was added in three portions. After the addition was complete, the temperature was raised to room temperature, and the reaction was monitored by HPLC until compound II reacted completely. Methyl tert-butyl ether (250 mL) was added dropwise, and the mixture was filtered. The filter cake was dried at 30–40 °C to obtain 15.5 g of crude compound 1 with a main peak purity of 70–80%.

[0115] The crude compound 1 was dissolved in hydrochloric acid solution, filtered, and the filtrate was loaded onto a DAC reversed-phase preparative column (reverse-phase polymer packing). The column was isocratically eluted with 6% acetonitrile aqueous solution at pH 3.0. The preparative solution was collected and concentrated by nanofiltration through a 100-200 Da nanofiltration membrane to obtain the nanofiltration concentrate. The main peak purity was >95%, and the yield was 75-85%.

[0116] MSm / z (ESI): 557.20 [M+H] +

[0117] 1H-NMR (500M, DMSO-d6) δ11.24(brs,~1H),9.14(s,1H),8.04(s,1H),7.40(s,1H),3.90(d,J=6.0Hz,1H),3.13(dd,J=15.5,4.5Hz,1H ),2.78(m,1H),2.51(s,8H),2.42(s,6H),2.36(m,1H),2.16(t,J=14.5Hz,1H),2.05(m,1H),1.58(m,1H),1.27(m,1H),0.92(s,9H).

[0118] Example 4 Synthesis of Omacycline p-Toluenesulfonate

[0119] Step 1: Preparation of Omacycline Bases

[0120]

[0121] Anhydrous methanol (2.0 kg) and compound II (500 g, 1.00 mol) were added to a glass reactor and stirred until homogeneous. Under nitrogen protection, the sodium bisulfite adduct of pentamalon (570.6 g, 3.0 mol) was slowly added. After the addition was complete, the temperature was controlled below 20 °C, and 2-methylpyridine-N-methylborane (127 g, 1.20 mol) was added in three batches. After the addition was complete, the temperature was controlled at 20–30 °C, and the reaction was monitored by HPLC until compound II had essentially completed.

[0122] After the reaction was complete, 5 times the volume of purified water was slowly added to the reaction solution, and hydrochloric acid was added to adjust the pH to 4.0–4.5. The solution was washed once with 10 times the volume of dichloromethane, separated, and the aqueous phase was adjusted to pH 10–12 with sodium hydroxide solution at room temperature. The mixture was then filtered to obtain the crude product of compound 1. HPLC analysis showed that the content of the 4β isomer was 1.84%.

[0123] The crude compound 1 was dissolved in a 0.1% aqueous methanesulfonic acid solution, and the pH was adjusted to 2-3 by adding methanesulfonic acid dropwise. The solution was filtered, and the filtrate was loaded into a DAC preparative column (reverse-phase polymer packing). The column was eluted isocratically with a 6% acetonitrile aqueous solution at pH 3.0, and the preparative eluent with a purity of ≥95% was collected. The collected solutions were combined, and sodium bisulfite (0.1‰-0.5‰ of the preparative solution volume) was added to the collected preparative solution. The solution was then concentrated by nanofiltration through a 100-200 Da membrane to obtain omacycline nanofiltrate.

[0124] The nanofiltration concentrate of compound 1 was adjusted to pH 4.0–4.5 with hydrochloric acid, washed with 10 times the amount of dichloromethane, and the aqueous phase was adjusted to pH 7.5–8.5 with sodium hydroxide solution. It was extracted four times with 40 times the amount of dichloromethane, and the organic phases were combined. The organic phase was concentrated to about 2 L at 20–25 °C, and then added dropwise to 6 L of n-heptane. The mixture was stirred and crystallized for 0.5 hours under nitrogen protection. After filtration, the filter cake was washed with n-heptane and dried to obtain about 350 g of compound 1. The content of the 4β isomer was determined to be 1.15% by HPLC.

[0125] MSm / z (ESI): 557.20 [M+H] +

[0126] 1 H-NMR (500M, DMSO-d6) δ11.24(brs,~1H),9.14(s,1H),8.04(s,1H),7.40(s,1H),3.90(d,J=6.0Hz,1H),3.13(dd,J=15.5,4.5Hz,1H ),2.78(m,1H),2.51(s,8H),2.42(s,6H),2.36(m,1H),2.16(t,J=14.5Hz,1H),2.05(m,1H),1.58(m,1H),1.27(m,1H),0.92(s,9H).

[0127] Step 2: Synthesis of Omacycline p-Toluenesulfonate

[0128]

[0129] Compound 1 (350 g, 0.63 mol) and methanol I (1.66 kg) were stirred until dissolved under nitrogen protection. Methyl tert-butyl ether I (2.09 kg) was added dropwise at 15–20 °C. After the addition was complete, the temperature of the solution was controlled at 15–20 °C, and a solution of p-toluenesulfonic acid methanol II / methyl tert-butyl ether II (119.6 g, 0.63 mol, 554 g / 518 g) was slowly added dropwise. After the addition was complete, crystallization was carried out at 15–20 °C for at least 3 hours under nitrogen protection. The solution was filtered, and the filter cake was washed with methanol III / methyl tert-butyl ether III (676 g / 1.9 kg). The filter cake was then dried under vacuum at 40–50 °C to obtain approximately 375 g of omacycline methanesulfonate, with a yield of approximately 80%.

[0130] MSm / z (ESI): 557.20 [M+H] +

[0131] 1H-NMR(500M,DMSO-d6)δ11.24(brs,~1H),9.14(s,1H),8.04(s,1H),7.40(s,1H),3.90(d,J=6.0Hz,1H),3.13(dd,J=15.5,4.5Hz,1H),2.78(m,1H),2.51(s,8H),2.42(s,6H),2.36(m,1H),2.16(t,J=14.5Hz,1H),2.05(m,1H),1.58(m,1H),1.27(m,1H),0.92(s,9H)。

Claims

1. A method for preparing compound 1, characterized in that, include: Step (i) involves reacting compound II with compound a to obtain compound 1.

2. The method for preparing compound 1 according to claim 1, wherein in step (i), compound II reacts with compound a in the presence of an organic solvent and a reducing agent to obtain compound 1.

3. The method for preparing compound 1 according to claim 2, wherein the reducing agent is selected from 2-methylpyridine-N-methylborane, sodium triacetylborohydride, sodium borohydride, sodium cyanoborohydride, borane, dimethyl sulfide borohydride, tetrahydrofuran borohydride, N,N-diisopropylethylamine borane complex, pyridine borohydride complex, morpholine borohydride complex, triethylamine borohydride complex, dimethylamine borohydride complex, ethylenediamine borohydride complex, or 5-ethyl-2-methylpyridine borane complex; preferably 2-methylpyridine-N-methylborane, pyridine borane complex, 5-ethyl-2-methylpyridine borane complex, or morpholine borohydride complex; preferably 2-methylpyridine-N-methylborane or pyridine borane complex; more preferably 2-methylpyridine-N-methylborane.

4. The method for preparing compound 1 according to claim 2, wherein the organic solvent is selected from one or more mixed solvents selected from acetonitrile, methanol, ethanol, ethylene glycol, butanol, octanol, octyl acetate, methyl tert-butyl ether, dioxane, tetrahydrofuran, N,N-dimethylformamide or dimethyl sulfoxide; preferably one or more mixed solvents selected from acetonitrile, methanol, ethanol, methyl tert-butyl ether or tetrahydrofuran; more preferably methanol.

5. The method for preparing compound 1 according to any one of claims 1-4, wherein in step (i), the molar ratio of compound II to compound a is 1:1 to 10; preferably, 1:1 to 5; more preferably, 1:1, 1:2, 1:3, 1:4 or 1:5; Optionally, in step (i), the molar ratio of the compound of formula II to the reducing agent is 1:1 to 3; preferably 1:1 to 2; more preferably 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2; even more preferably 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5 or 1:1.

6.

6. The method for preparing compound 1 according to any one of claims 1-5, wherein step (i) further includes a post-processing step; Optionally, the post-processing steps of step (i) include: Purified water was added directly to the reaction solution of compound 1 to adjust the pH and dissolve it. The solution was then washed with the first solvent. Optionally, the first solvent is selected from dichloromethane, ethyl acetate, tetrahydrofuran, 1,4-dioxane, or n-propanol; preferably dichloromethane; optionally, the weight ratio of the first solvent to the compound of formula II is 1:0.05 to 0.5; preferably, 1:0.05, 1:0.06, 1:0.07, 1:0.08, 1:0.09, 1:0.1, 1:0.2, 1:0.3, 1:0.4, or 1:0.5; Optionally, step (i) further includes a crystallization step; The crystallization step includes adjusting the pH of the compound 1 solution to 8.0–14.0, followed by crystallization; preferably, adjusting the pH of the compound 1 solution to 10–12. Crystallization; Optionally, the weight ratio of purified water to compound of formula II is 1:0.1 to 0.5; preferably 1:0.1 to 0.3; more preferably 1:0.

2.

7. The method for preparing compound 1 according to any one of claims 1-6, wherein the compound 1 obtained in step (i) further comprises a purification step; optionally, the purification step comprises: The solid obtained by crystallization was dissolved, then eluted with a preparative column, the eluent was concentrated, and crystallized again to obtain the purified compound 1. Optionally, in the purification step, the eluent used for the preparative column purification is an acidic solution of acetonitrile-water, and the acidity adjuster is hydrochloric acid, methanesulfonic acid, trifluoroacetic acid, sulfuric acid, oxalic acid, or formic acid; Optionally, the pH of the eluent is 1.0 to 5.0; preferably, the pH is 2.0 to 4.0; and more preferably, the pH is 3.

0.

8. The method for preparing compound 1 as described in claim 7, wherein the re-crystallization step comprises: The eluent was concentrated and dissolved in the first solvent, and then the second solvent was added and stirred to induce crystallization. Optionally, the re-crystallization step includes: concentrating the eluent and then adding it dropwise to a second solvent while stirring to induce crystallization; Optionally, the first solvent in the re-crystallization step is selected from dichloromethane, ethyl acetate, tetrahydrofuran, 1,4-dioxane, or n-propanol; dichloromethane is preferred. The second solvent in the re-crystallization step is selected from methyl tert-butyl ether, n-heptane, or diethyl ether; preferably methyl tert-butyl ether or n-heptane; more preferably n-heptane.

9. A method for preparing omacycline methanesulfonate, characterized in that, include: Step (i) involves reacting compound II with compound a to obtain compound 1; step (ii) involves reacting compound 1 with a solution of p-toluenesulfonic acid monohydrate, followed by crystallization to obtain omacycline methanesulfonate.

10. A method for preparing compound 1 with a 4β-isomer impurity content of not more than 5%, the preparation method comprising: Step (i) involves reacting compound II with compound a to obtain compound 1; Optionally, the content of 4β-isomer impurities is not higher than 2%; Alternatively, the content of 4β-isomer impurities shall not exceed 1.5%.

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