Macrolide compound preparation method therefor, and use thereof

Abstract

A macrolide compound, a preparation method therefor, and the use thereof. The macrolide compound has a structure as represented by formula (I). Further provided are a pharmaceutical composition or a veterinary drug composition, which comprise the macrolide compound having the structure as represented by formula (I). Further provided is a pharmaceutical preparation, which comprises the macrolide compound having the structure as represented by formula (I). The macrolide compound, the veterinary drug composition, and the pharmaceutical formulation may be used in the treatment of pathogenic infections, and particularly in the treatment of animal pathogenic infections.

Description

A macrolide compound and its preparation method and application Technical Field

[0001] The present invention relates to the technical field of heterocyclic compound synthesis and animal pharmacy, and in particular to a macrolide compound and a preparation method and application thereof. Background Art

[0002] Macrolides are an important class of antibiotics with excellent anti-infective properties and minimal side effects, making them widely used in animal and poultry veterinary clinics (Wang Xiuru, "Properties, Characteristics, and Applications of Macrolides." Veterinary Guide, 2018, 54-55; Kuang Baoxiao, "Tedirosin: A New Animal-Specific Antibiotic." Today's Swine Industry, 2022, 98-100). Based on their chemical structure, macrolide antibiotics are divided into 14-, 15-, and 16-membered macrolides. Major varieties include erythromycin, azithromycin, kitasamycin, tylosin, tilmicosin, tylosin, and gamithromycin (Yu Zhichao, "Research Progress in the Veterinary Antibiotic Gamithromycin." Contemporary Animal Husbandry, 2020, 22-23; Liu Wang; Pei Wei, "Recent Research Progress in Azithromycin." Animal Husbandry and Veterinary Science and Technology Information, 2014, 15). It is mainly used to treat bovine respiratory diseases, mastitis, arthritis and otitis media caused by Mycoplasma bovis infection, swine asthma caused by Mycoplasma hyopneumoniae, porcine proliferative enteritis caused by Lawsonia intracellularis, and chronic respiratory diseases in chickens caused by Mycoplasma gallinarum (He Chengguang; Kong Lingcong; Sun Zhe. Research progress on antibiotic resistance of bovine Mycoplasma. Jilin Animal Husbandry and Veterinary Medicine 2018, 39, 11-13. Wan Jin; Ma Nini; Wang Cong. Research progress on the efficacy of tartrate tartrate in the prevention and treatment of specific livestock and poultry diseases. China Animal Quarantine, 2021, 38, 75-78.).

[0003] Tylosin, also known as Tylosin in English, is an important macrolide antibiotic for livestock and poultry. It has a 16-membered macrolide structure and was first extracted from the culture medium of Streptomyces fradiae in 1959. Products used in livestock and veterinary clinical settings primarily include tylosin tartrate, tylosin lactate, tylosin sulfate, tylosin hydrochloride, and tylosin phosphate (Chen Dong, Zhang Xiaoqiang, Na Qi, Suo Jiawei. Optimization of Tylosin Purification Process. Contemporary Chemical Research, 2023, 170-172. Liu Jia, Hao Shengyan, Pan Faming. Research Progress on Tylosin Residue Patterns in Animal Products. Animal Husbandry and Veterinary Medicine, 2022, 54, 148-152). Tylosin has good antibacterial activity against Gram-negative bacteria, Gram-positive bacteria, mycoplasma and other pathogens. It can not only be used to treat diseases such as swine dysentery, poultry mycoplasma infection, ruminant pneumonia, etc., but also can be used as a feed additive to promote animal growth (Wang Lixia; Li Shenglong; Chen Dangtong; Wang Jun. Establishment of high-performance liquid chromatography detection method for tylosin. Anhui Agricultural Sciences 2020, 48, 206-209.).

[0004] In order to develop new macrolide antibiotics, domestic and foreign scholars have made various modifications to the structure of tylosin and have synthesized a series of tylosin derivatives (Zhao Dongfeng; Ren Xiang; Zhu Li. Research Progress of Tylosin and Its Derivatives. Pharmaceutical Industry Information, 2006, 46-48.). For example, 10,11,12,13-tetrahydro-desmycosin derivatives (Narandja, A.; Kelneric, K.; Kolacny-Babic, L.; Djokic, S. 10,11,12,13-Tetrahydro Derivatives of Tylosin. Ii. Synthesis, Antibacterial Activity and Tissue Distribution of 4'-Deoxy-10,11,12,13-Tetrahydrodesmycosin. Journal of Antibiotics. 1995, 48, 248-253. Narandja, A.; Djokic, S. Derivatives of 10,11,12,13-tetra-hydrodesmycosin, processes for preparation, and use thereof in obtaining pharmaceuticals. Patent EP0490311, 1992-06-17), 9-oxime tylosin derivatives (Wang Huanhuan; Yang Pu; Zhai Hongjin; Zhang Shuo; Cao Yaquan; Yang Yingxue; Wu Chunli. Design, synthesis and activity evaluation of new tylosin derivatives. Organic Chemistry 2022, 42, 557-571.), 12,13-epoxy tylosin (Narandja, A.; Lopotar, N. Derivatives of 12,13-Epoxy-tylosin and processes of manufacture thereof. Patent US5688924, 1997-11-18), tadilaosin (Zhang, C.; Song, M.; Qi, P.; Zhang, G.; Ge, X.; Zhao, M.; Wu, J.; Ma, J.; Wang, D.; Process for preparation of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.Patent CN 104892704 B,2017-08-08.), Tylvalosin (Research progress on the efficacy of Tylvalosin tartrate in the prevention and treatment of specific livestock and poultry diseases. China Animal Quarantine, 2021, 38, 75-78.), etc.

[0005] Summary of the Invention

[0006] The present invention aims to provide a macrolide compound and a preparation method and application thereof, wherein the compound can be used to treat or prevent animal pathogen infection with significant effect.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a macrolide compound having a structure as shown in Formula I:

[0009] Wherein: R is selected from 2-hydroxyethylamino, 3-hydroxypropylamino, bis(2-hydroxyethylamino), bis(3-hydroxypropylamino), (R)-2-hydroxymethyltetrahydropyrrolyl, (S)-2-hydroxymethyltetrahydropyrrolyl, 4-hydroxypiperidinyl, 4-hydroxymethylpiperidinyl.

[0010] Specifically, the macrolide compounds are: 20-(2-hydroxyethylamino) tylosin, 20-(3-hydroxypropylamino) tylosin, 20-(bis(2-hydroxyethylamino)) tylosin, 20-(bis(3-hydroxypropylamino)) tylosin, 20-((R)-2-hydroxymethyltetrahydropyrrolyl) tylosin, 20-((S)-2-hydroxymethyltetrahydropyrrolyl) tylosin, 20-(4-hydroxypiperidinyl) tylosin, and 20-(4-hydroxymethylpiperidinyl) tylosin.

[0011] Preferably, the macrolide compound has a structure as shown in any one of Formula Ia, Formula Ib, Formula Ic or Formula Id:

[0012] The present invention also provides pharmaceutically acceptable salts of the above-mentioned macrolide compounds.

[0013] The pharmaceutically acceptable salt refers to a salt formed by the macrolide compound and an acid.

[0014] The acid includes tartaric acid, hydrochloric acid, phosphoric acid, sulfuric acid, salicylic acid, methanesulfonic acid, lactic acid, malic acid, formic acid, acetic acid, propionic acid, fumaric acid, citric acid, oxalate, maleic acid, succinic acid, benzoic acid, ethanedisulfonic acid, and the like.

[0015] In a second aspect, the present invention further provides a method for preparing the above-mentioned macrolide compound, comprising the following steps:

[0016] S1, tylosin A reacts with amino alcohol;

[0017] S2. Add a reducing agent or acid to the system obtained by the reaction in step S1 to react to obtain the macrolide compound.

[0018] Furthermore, the preparation method includes synthetic route 1 and synthetic route 2:

[0019] The synthetic route 1 comprises the following steps:

[0020] (1) Tylosin A reacts with amino alcohol in a polar solvent to obtain an imine solution;

[0021] (2) adding a reducing agent to the imine solution to react and obtain a macrolide compound modified with a hydroxyl secondary amino group.

[0022] In step (1), the amino alcohol is 2-aminoethanol or 3-aminopropanol.

[0023] In step (1), the molar ratio of the amino alcohol to the tylosin A is 2 to 5:1, preferably 3 to 3.5:1.

[0024] In step (1), the polar solvent is one or more of methanol, ethanol, propanol, isopropanol, n-butanol and ethylene glycol.

[0025] In step (1), the reaction conditions are: temperature is room temperature, and time is 12 to 13 hours.

[0026] In step (2), the reducing agent is one or more of sodium borohydride, sodium triacetoxyborohydride and LiAlH4.

[0027] In step (2), the molar ratio of the reducing agent to the tylosin A is 1 to 4:1, preferably 2 to 2.5:1.

[0028] In step (2), the reaction conditions are: room temperature, and 2 to 3 hours.

[0029] Furthermore, the synthetic route 1 further comprises: before adding the reducing agent, performing TLC to monitor the reaction to ensure that the raw material is completely converted into imine.

[0030] Furthermore, the synthetic route 1 also includes a post-treatment step; the post-treatment is performed according to the following operation: adding an aqueous solution of an alkali to the reaction system to quench the reaction, and then concentrating under reduced pressure to remove the alcohol solvent; the remaining aqueous solution is extracted with an organic solvent, and the combined organic phase is washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure; wherein the alkali is selected from one or more of potassium carbonate, sodium carbonate, potassium hydroxide or sodium hydroxide; and the organic solvent is selected from one or more of dichloromethane, ethyl acetate or diethyl ether.

[0031] For example, the synthesis route of the above-mentioned synthesis route 1 is as follows:

[0032] The synthetic route 2 comprises the following steps:

[0033] (A) tylosin A reacts with amino alcohol in a non-polar solvent to obtain a reaction solution;

[0034] (B) adding an acid to the reaction solution obtained in step (A) to react to obtain a macrolide compound modified with a hydroxyl tertiary amino group.

[0035] In step (A), the amino alcohol is 2-aminoethanol, 3-aminopropanol, (R)-prolinol, (S)-prolinol, 4-hydroxypiperidine, or 4-hydroxymethylpiperidine.

[0036] In step (A), the molar ratio of the amino alcohol to the tylosin A is 2 to 5:1, preferably 2.5 to 3.5:1.

[0037] In step (A), the non-polar solvent is one or more of ethylene glycol dimethyl ether, benzene and toluene.

[0038] In step (B), the acid is formic acid.

[0039] In step (B), the acid is added when the temperature of the reaction system reaches 75-85°C, preferably 80°C.

[0040] In step (B), the molar ratio of the acid to the tylosin A is 3 to 6:1, preferably 5 to 6:1.

[0041] In step (B), the reaction conditions are: temperature of 78-80° C., and time of 2-2.5 h.

[0042] Furthermore, the synthetic route 2 also includes a post-treatment step; the post-treatment is carried out according to the following operation: distilled water is added to the reaction system, the pH of the aqueous phase after separation is adjusted to 9-11 with a base, the aqueous solution is extracted with an organic solvent, the combined organic phase is washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure; wherein the base is selected from one or more of potassium carbonate, sodium carbonate, potassium hydroxide or sodium hydroxide; the organic solvent is selected from one or more of dichloromethane, ethyl acetate or diethyl ether.

[0043] Furthermore, the preparation method of the macrolide compound provided by the present invention also includes a purification step: adding the obtained crude product to a silica gel chromatography column, selecting two organic solvents to form eluents of different polarities, and using gradient elution to remove impurities in the crude product, thereby obtaining a pure macrolide compound; wherein the eluent can be selected from any two of diethyl ether, ethyl acetate, methanol, isopropanol, acetone or dichloromethane.

[0044] For example, the synthesis route of the above-mentioned synthesis route 2 is as follows:

[0045] In a third aspect, the present invention further provides a veterinary drug composition, which comprises the macrolide compound having the structure shown in Formula I above.

[0046] In a fourth aspect, the present invention further provides a pharmaceutical preparation comprising the macrolide compound having the structure shown in Formula I above.

[0047] The dosage forms of the pharmaceutical preparation are powder, tablet, premix, soluble powder and injection.

[0048] In a fifth aspect, the present invention further provides the use of the above-mentioned macrolide compounds, veterinary drug compositions and pharmaceutical preparations in the preparation of anti-pathogen infection drugs. For example, the anti-pathogen infection drugs are products for clinical use in livestock and poultry veterinary medicine.

[0049] In the application, the pathogen is mycoplasma, Pasteurella, Pasteurella multocida, Histophaga, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, beta-hemolytic Streptococcus, Escherichia coli, Haemophilus influenzae, Actinobacillus pneumoniae, Salmonella, Mannheimia, and Erysipelothrix rhusiopathiae. DETAILED DESCRIPTION

[0050] The present invention will be further described below with reference to specific examples, but the present invention is not limited to the following examples.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0052] Unless otherwise specified, the reagents, materials, instruments, etc. used in the following examples can be obtained from commercial sources.

[0053] The raw materials used in the following examples are from the following sources:

[0054] Example 1

[0055] Tylosin A (3.00 g, 3.27 mmol) was added to a 50 mL Shrek bottle, methanol (18 mL) was added, and then 3-amino-1-propanol (0.74 g, 9.85 mmol) was slowly added with a syringe, and the reaction was stirred at room temperature for 12 h.

[0056] After TLC showed that the raw material was completely converted into imine, sodium triacetoxyborohydride (1.39 g, 6.56 mmol) was slowly added at room temperature, and the reaction was continued with stirring at room temperature for 2 h.

[0057] After TLC detection of the reaction completion, 1M aqueous NaOH solution (3 mL) was added to quench the reaction. The MeOH was then removed by concentration under reduced pressure, and the residue was extracted with dichloromethane (10 mL × 3). The combined extracts were washed with saturated aqueous NaCl solution (10 mL) and dried over anhydrous sodium sulfate. Filtered, the filtrate was concentrated under reduced pressure to obtain the crude product. Finally, purification by silica gel column chromatography (dichloromethane / methanol = 8:1) afforded macrolide compound Ia as a white solid (1.40 g, 44% yield).

[0058] 1 H NMR (500MHz, CDCl3) δ7.37(d,J=14.7Hz,1H),6.30(d,J=14.8Hz,1H),5.95(s,1H),5.08(d,J=9.6Hz,1H),4.96(d,J=10.6Hz,1H),4.58(t,J =10.5Hz,1H),4.33–4.25(s,4H),4.09–4.08(m,1H),4.01–3.99(m,1H ),3.80–3.75(m,4H),3.67–3.61(m,4H),3.56(s,2H),3.49–3.47(m,2H ),3.29(t,J=10.5Hz,2H),3.19(d,J=10.4Hz,1H),3.02–2.95(m,4H),2.87–2.80(m,3H),2.72–2.68(m,4H),2.51–2.44(m,8H),2.06–1.95( m,3H),1.87–1.74(m,8H),1.63–1.62(m,3H),1.53–1.48(m,2H),1.31–1.23(m,14H),1.16–1.11(m,4H),1.03(s,3H),0.93(d,J=8.2Hz,3H).

[0059] 13C NMR (126MHz, CDCl3) δ203.84,173.60,148.26,142.99,134.55,117.88,103.71,101.01,96.38 ,81.69,79.83,79.50,76.32,75.03,74.88,72.84,72.67,71.67,70.34,69.39,69.02,68.70,6 6.59,65.88,61.88,61.66,59.51,47.26,46.07,45.94,44.97,41.91,41.05,40.85,39.32,33.47,32.41,30.42,29.55,26.14,25.34,25.18,19.03,18.18,17.69,17.52,12.82,10.52,9.55.

[0060] TLC R f =0.4 (dichloromethane / methanol=8:1)

[0061] HRMS (ESI, m / z): [M+H] + calcd for C 49 H 87 N2O 17 ,975.59993; found 975.60059.

[0062] Example 2

[0063] Add tylosin A (0.50 g, 0.55 mmol) to a 50 mL three-necked flask equipped with a condenser, add toluene (6 mL), and stir to dissolve. Then add diethanolamine (0.17 g, 1.62 mmol), raise the temperature to 80°C, and add formic acid (0.14 g, 3.04 mmol). Continue stirring at 80°C for 2 h.

[0064] After TLC confirmed the reaction was complete, distilled water (5 mL) was added to quench the reaction and the layers were separated. The aqueous phase was adjusted to pH 10 with 5 M sodium hydroxide solution and then extracted with dichloromethane (15 mL x 3). The combined extracts were dried over anhydrous sodium sulfate. Filtered, the filtrate was concentrated under reduced pressure to yield the crude product. Finally, purification by silica gel column chromatography (dichloromethane / methanol = 8:1) afforded macrolide compound Ib as a white solid (0.27 g, 49% yield).

[0065] 1H NMR (500MHz, CDCl3) δ7.44(d,J=14.4Hz,1H),6.31(d,J=15.7Hz,1H),5.98(s,1H),5.08(d,J=10.2Hz,1H),4.93(d,J=9.9Hz,1H), 4.58(t,J=9.1Hz,1H),4.33–4.29(m,2H),4.11–4.08(m,1H),4.03–3.99(m,1H),3.79–3.57(m,13H),3.47–3.45(m,2H),3.32–3.2 8(m,2H),3.21–3.17(m,1H),3.05–2.95(m,3H),2.72–2.60(m,7H),2.51–2.49(m,8H),2.42–2.38(m,2H),2.28–2.26(m,1H),2.06 –2.01(m,1H),1.95–1.75(m,7H),1.66–1.59(m,3H),1.50–1.46(m,2H),1.32–1.21(m,16H),1.12–1.05(m,7H),0.96–0.91(m,3H).

[0066] 13 C NMR (126MHz, CDCl3) δ205.13,173.62,149.18,143.77,134.55,117.45,103.57,101.03,96.33,8 1.66,80.09,79.83,76.33,75.15,75.07,72.76,72.68,71.74,70.36,69.40,69.10,68.75,66.3 3,65.89,61.64,59.58,59.47,59.43,57.17,53.02,45.97,45.94,45.27,44.89,41.93,41.01,40.88,39.39,33.80,33.65,26.06,25.34,25.25,19.10,18.18,17.69,17.46,12.76,10.89,9.66.

[0067] TLC R f =0.2 (dichloromethane / methanol=8:1)

[0068] HRMS (ESI, m / z): [M+H] + calcd for C 50 H 89 N2O 18,1005.61049; found 1005.62402.

[0069] Example 3

[0070] Add tylosin A (0.50 g, 0.55 mmol) to a 50 mL three-necked flask equipped with a condenser. Add toluene (6 mL), then add (R)-prolinol (0.17 g, 1.68 mmol) and stir to dissolve. Raise the temperature to 80°C, add formic acid (0.14 g, 3.04 mmol), and continue the reaction at 80°C for 2 h.

[0071] After TLC confirmed the reaction was complete, distilled water (5 mL) was added to quench the reaction and the layers were separated. The aqueous phase was adjusted to pH 10 with 5 M sodium hydroxide solution and then extracted with dichloromethane (15 mL x 3). The combined extracts were dried over anhydrous sodium sulfate. Filtered, the filtrate was concentrated under reduced pressure to obtain the crude product. Finally, purification by silica gel column chromatography (dichloromethane / methanol = 8:1) afforded the macrolide compound Ic as a white solid (0.32 g, 58% yield).

[0072] 1 H NMR(500MHz, CDCl3)δ7.36(d,J=17.5Hz,1H),6.30(d,J=14.8Hz,1H),5.94(s,1H),5.09–5.07(m,1H),4.97–4.94(m,1H),4.58–4.55(m,1H), 4.29–4.26(m,2H),4.11–4.06(m,1H),4.02–3.98(m,1H),3.83–3.81(m ,1H),3.76–3.73(m,1H),3.62–3.53(m,7H),3.47–3.44(m,4H),3.33–3 .29(m,2H),3.19–3.17(m,2H),3.03–2.93(m,3H),2.72–2.63(m,3H), 2.59–2.54(m,6H),2.50–2.46(m,5H),2.36–2.26(m,2H),2.04–2.01(m ,2H),1.89–1.83(m,3H),1.79–1.72(m,7H),1.63–1.55(m,4H),1.31–1 .19(m,16H),1.08–1.06(m,5H),1.01–0.99(m,2H),0.93–0.91(m,3H).

[0073] 13C NMR (126MHz, CDCl3) δ203.98,173.63,162.63,161.84,148.07,143.06,134.38,117.97,103.97,101 .01,96.36,82.44,81.69,79.86,76.32,75.01,74.91,72.95,72.66,71.65,70.35,69.37,69.07,68. 80,66.48,65.89,65.12,63.03,61.64,59.50,55.00,54.69,46.01,45.11,41.93,41.63,40.87,39.36,34.95,34.22,27.64,26.75,25.34,25.17,23.48,19.13,18.18,17.69,12.76,11.16,9.60,9.33.

[0074] TLC R f =0.3 (dichloromethane / methanol=8:1)

[0075] HRMS (ESI, m / z): [M+H] + calcd for C 51 H 89 N2O 17 ,1001.61558; found 1001.61896.

[0076] Example 4

[0077] Add tylosin A (1.00 g, 1.09 mmol) to a 50 mL three-necked flask equipped with a condenser. Add toluene (8 mL), then add (S)-prolinol (0.33 g, 3.26 mmol) and stir to dissolve. Raise the temperature to 80°C, add formic acid (0.27 g, 5.86 mmol), and continue the reaction at 80°C for 2 h.

[0078] After TLC confirmed the reaction was complete, distilled water (8 mL) was added to quench the reaction and the layers were separated. The aqueous phase was adjusted to pH 10 with 5 M sodium hydroxide solution and then extracted with dichloromethane (15 mL x 3). The combined extracts were dried over anhydrous sodium sulfate. Filtered, the filtrate was concentrated under reduced pressure to yield the crude product. Finally, purification by silica gel column chromatography (dichloromethane / methanol = 8:1) afforded the macrolide compound Id (0.64 g, 59% yield) as a white solid.

[0079] 1H NMR(500MHz, CDCl3)δ7.31(d,J=15.4Hz,1H),6.29(d,J=15.5Hz,1H),5.92(s,1H),5.11–5.06(m,1H),4.97–4.91(m,1H),4 .59–4.55(m,1H),4.32–4.26(m,2H),4.11–4.07(m,1H),4.02–3.97(m,1H),3.76–3.72(m,2H),3.65–3.61(m,4H),3.57–3. 53(m,3H),3.50–3.42(m,4H),3.33–3.27(m,2H),3.21–3.17(m,2H),3.02–2.93(m,3H),2.87–2.68(m,3H),2.61–2.42(m,1 3H),2.16–2.09(m,2H),1.88–1.74(m,10H),1.65–1.56(m,4H),1.33–1.19(m,16H),1.10–1.02(m,7H),0.96–0.90(m,3H).

[0080] 13 C NMR (126MHz, CDCl3) δ203.54,172.99,162.58,161.82,147.71,142.84,134.41,117.96,103.63,10 0.97,96.28,81.63,79.82,79.45,76.29,75.01,74.77,72.76,72.64,71.71,70.29,69.33,69.12, 68.71,66.41,65.83,65.56,61.65,61.58,59.45,53.48,53.38,45.95,45.14,41.91,41.17,40.84,39.62,33.60,32.58,26.98,26.66,25.31,23.27,23.21,19.05,18.14,17.65,12.79,11.10,9.59.

[0081] TLC R f =0.3 (dichloromethane / methanol=8:1)

[0082] HRMS (ESI, m / z): [M+H] + calcd for C 51 H 89 N2O 17 ,1001.61558; found 1001.61746.

[0083] Test Example 1 Determination of antibacterial activity of the compounds of the present invention

[0084] The antibacterial activities of the compounds obtained in Examples 1-4 of the present invention were determined by the broth microdilution method using tylosin as a positive control.

[0085] The test method is as follows:

[0086] Add broth culture medium to a 96-well plate, dilute the prepared drug solution in a micro-two-fold decreasing concentration, then inoculate an appropriate amount of bacterial solution. After incubation for 24 hours, observe the minimum inhibitory concentration of the drug.

[0087] The culture medium used in the experiment was CAMHB broth and CAMHB+5% defibrinated sheep blood broth.

[0088] The preserved bacteria were inoculated into serum plate medium and cultured at 37℃ for 16-18 hours. The appropriate amount of bacteria and physiological saline after subculture were placed in a turbidimetric tube. The McFarland turbidimeter was calibrated to the McFarland turbidimetric standard. The bacterial suspension was diluted 10 times with physiological saline to prepare a certain concentration (5×10 5 ~5×10 6 cfu / mL) of the test bacterial solution for later use.

[0089] Dissolve tylosin and the compounds obtained in the examples in methanol to the desired concentration (1.0 mg / mL). Store in sterile brown vials, stopper, and seal until ready for use. The working concentration range for Gram-negative bacteria is 0.25 μg / mL to 128 μg / mL; for Gram-positive bacteria, the working concentration range is 0.098 μg / mL to 50 μg / mL.

[0090] The 96-well plate micro-dilution method was used. Broth culture medium was added to the 96-well plate, and the prepared drug solution was diluted in micro-dilution in a two-fold decreasing manner, so that the drug solution concentration in the first well to the tenth well showed a two-fold decreasing relationship. No drug solution was added to the eleventh and twelfth wells. Finally, the prepared bacterial solution (concentration of 5×10 5 ~5×10 6 cfu / mL). The twelfth well was left untreated as a blank control. The 96-well plate was placed in a 37°C incubator and incubated for 24 hours. Bacterial growth in each well was observed. The solution in wells that inhibited bacterial growth was transparent, while the solution in wells that did not inhibit bacterial growth was turbid. The concentration corresponding to the well with a transparent solution was the minimum antimicrobial concentration (MIC) for that sample.

[0091] The results are shown in the following table.

[0092] Table 1 MIC values ​​of compounds of the present invention (μg / mL)

[0093] The results showed that compared with tylosin, the compounds obtained in Examples 1-4 showed superior or comparable in vitro antibacterial activity against Streptococcus pneumoniae (a representative of Gram-positive bacteria) and Escherichia coli (a representative of Gram-negative bacteria), indicating that the compound represented by Formula I has antibacterial activity against Gram-positive bacteria, some Gram-negative bacteria, and mycoplasmas. Specifically, derivative Ia, obtained by reaction with 3-amino-1-propanol, was slightly better than tylosin against Streptococcus pneumoniae ATCC 49169 and had an antibacterial effect comparable to tylosin against Escherichia coli 8099. It is speculated that the R-position group increased binding to Streptococcus pneumoniae and enhanced antibacterial activity. Derivative Ic, obtained by reaction with (R)-prolinol, had antibacterial effects superior to tylosin against both Streptococcus pneumoniae ATCC 49169 and Escherichia coli 8099. While derivative Id, obtained by reaction with (S)-prolinol, had an N-containing pyrrole ring that further enhanced its antibacterial activity, this increase in antibacterial activity was not achieved, likely due to steric hindrance.

[0094] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

[0095] Cross-reference to related applications:

[0096] This application claims priority to the Chinese patent application (application number 202311620585.2) filed on November 30, 2023, and the entire contents of that patent application are incorporated herein by reference.

[0097] Industrial Applications

[0098] The present invention has the following technical advantages:

[0099] The present invention provides a macrolide compound, a preparation method and application thereof. The compound or a pharmaceutically acceptable salt thereof can be used to treat or prevent bacterial or mycoplasma infections, providing more selectivity for animal husbandry and veterinary clinics.

Claims

1. A macrolide compound, characterized in that: The macrolide compound has a structure as shown in Formula I: Wherein: R is selected from 2-hydroxyethylamino, 3-hydroxypropylamino, di(2-hydroxyethylamino), di(3-hydroxypropylamino), (R)-2-hydroxymethyltetrahydropyrrolyl, (S)-2-hydroxymethyltetrahydropyrrolyl, 4-hydroxypiperidinyl, 4-hydroxymethylpiperidinyl.

2. The macrolide compound according to claim 1, characterized in that: The macrolide compound has a structure as shown in any one of Formula Ia, Formula Ib, Formula Ic or Formula Id:

3. The method for preparing the macrolide compound according to claim 1 or 2, characterized in that: The steps include: S1, tylosin A reacts with amino alcohol; S2, adding a reducing agent or an acid to the system obtained by reaction in step S1 to react to obtain the macrolide compound.

4. The method for preparing a macrolide compound according to claim 3, characterized in that: The preparation method includes synthesis route 1 and synthesis route 2; The synthetic route 1 is carried out according to the following steps: (1) Tylosin A reacts with amino alcohol in a polar solvent to obtain an imine solution; (2) adding a reducing agent to the imine solution to react and obtain a hydroxyl secondary amino modified macrolide compound; The synthetic route 2 is carried out according to the following steps: (A) tylosin A reacts with amino alcohol in a non-polar solvent to obtain a reaction solution; (B) adding an acid to the reaction solution to obtain a macrolide compound modified with a hydroxyl tertiary amino group.

5. The method for preparing a macrolide compound according to claim 4, characterized in that: In the synthetic route 1: In step (1): The amino alcohol is 2-aminoethanol or 3-aminopropanol; The molar ratio of the amino alcohol to the tylosin A is 2 to 5:1; The polar solvent is one or more of methanol, ethanol, propanol, isopropanol, n-butanol and ethylene glycol; The reaction conditions are: room temperature, time 12 to 13 hours; In step (2): The reducing agent is one or more of sodium borohydride, sodium triacetoxyborohydride and LiAlH4; The molar ratio of the reducing agent to the tylosin A is 1 to 4:1; The reaction conditions are: room temperature and 2 to 3 hours.

6. The method for preparing a macrolide compound according to claim 4, characterized in that: In the synthetic route 2: In step (A): The amino alcohol is one or more of 2-aminoethanol, 3-aminopropanol, (R)-prolinol, (S)-prolinol, 4-hydroxypiperidine, and 4-hydroxymethylpiperidine; The molar ratio of the amino alcohol to the tylosin A is 2 to 5:1; The non-polar solvent is one or more of ethylene glycol dimethyl ether, benzene and toluene; In step (B): The acid is formic acid; The acid is added at a time when the temperature of the reaction system reaches 75 to 85°C; The molar ratio of the acid to the tylosin A is 3 to 6:1; The reaction conditions are: temperature of 78-80°C and time of 2-3h.

7. A veterinary drug composition, characterized in that: The veterinary composition comprises the macrolide compound according to claim 1 or 2.

8. A veterinary drug composition for use in resisting pathogen infection, characterized in that: The veterinary composition comprises the macrolide compound according to claim 1 or 2.

9. The veterinary drug composition according to claim 8, characterized in that: The pathogen is selected from one or more of the following: Mycoplasma, Pasteurella, Pasteurella multocida, Histophaga, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, beta-hemolytic Streptococcus, Escherichia coli, Haemophilus influenzae, Actinobacillus pneumoniae, Salmonella, Mannheimia, and Erysipelothrix rhusiopathiae.

10. A pharmaceutical preparation, characterized in that: The pharmaceutical preparation comprises the macrolide compound according to claim 1 or 2.

11. A pharmaceutical preparation for use in preventing pathogen infection, characterized in that: The pharmaceutical preparation comprises the macrolide compound according to claim 1 or 2.

12. The pharmaceutical preparation according to claim 11, characterized in that: The pathogen is selected from one or more of the following: Mycoplasma, Pasteurella, Pasteurella multocida, Histophaga, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, beta-hemolytic Streptococcus, Escherichia coli, Haemophilus influenzae, Actinobacillus pneumoniae, Salmonella, Mannheimia, and Erysipelothrix rhusiopathiae.

13. Use of the macrolide compound according to claim 1 or 2, the veterinary composition according to claim 7 or the pharmaceutical preparation according to claim 10 in the preparation of drugs for preventing and treating pathogenic infections.

14. The use according to claim 13, characterized in that: The pathogen is selected from one or more of the following: Mycoplasma, Pasteurella, Pasteurella multocida, Histophaga, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, beta-hemolytic Streptococcus, Escherichia coli, Haemophilus influenzae, Actinobacillus pneumoniae, Salmonella, Mannheimia, and Erysipelothrix rhusiopathiae.

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