WO2020168865A1 - Anti-infective pharmaceutical and preparation method and application thereof - Google Patents

Abstract

The present invention provides an anti-infective pharmaceutical, or stereoisomer, hydrate, deuterate, ester, solvate, crystalline form, metabolite, pharmaceutically acceptable salt, or prodrug thereof, and application in anti-infection; the structure of the anti-infective pharmaceutical is as represented by formula (I). The anti-infective pharmaceutical of the present invention, or stereoisomer, hydrate, deuterate, ester, solvate, crystalline form, metabolite, pharmaceutically acceptable salt, or prodrug thereof has stronger antibacterial activity, is less toxic, and is more suitable for the treatment of infectious diseases.

Description

Anti-infective medicine and its preparation method and application Technical field

The invention relates to an anti-infective medicine, a preparation method and application thereof, and belongs to the technical field of medicine.

Background technique

Antibiotics are currently the first choice for humans to treat bacterial infectious diseases, but the excessive use and abuse of antibiotics has made the problem of bacterial resistance increasingly serious. The rapid development of various antibiotics and antibacterial drug-resistant bacteria has seriously threatened the lives and health of patients with infectious diseases. The exploration of new drugs against drug-resistant gram-positive bacteria has become a research hotspot in the medical field at home and abroad. Oxazolidinone antibacterial drugs are a new type of chemical fully synthetic antibacterial drugs developed after sulfonamides and fluoroquinolones in the past 30 years. They can kill Gram-positive pathogens by inhibiting protein synthesis in the very early stage. The efficacy of multi-drug resistant Gram-positive bacteria. For example, Methicillin-resistant Staphylococcus aureus (MRSA), Methicillin-resistant Staphylococcus epidermidis (MRSE), Penicillin-resistant Streptococcus pneumoniae (PRSP), and Vancomycin-resistant Enterococcu (Vancomycin-resistant Enterococcu, VRE), etc. The emergence of these resistant bacteria has severely reduced the efficacy of existing drugs, resulting in a significant extension of patient treatment time and an increase in mortality.

Oxazolidinone compounds are a new class of drugs for the treatment of bacterial infections. They can inhibit the initial stage of protein synthesis and rarely cross-resistance. Because of their unique mechanism of action, they have attracted attention. The first oxazolidinone antibacterial agent Linezolid (Linezolid) was approved for marketing in the United States in April 2000 for the treatment of diseases caused by infection with multi-drug-resistant Gram-positive bacteria. Linezolid has been proven to be an important drug for the treatment of serious infections caused by gram-positive bacteria. It is currently widely used to treat hospital-acquired pneumonia and complex skin and soft tissue infections caused by MRSA.

Anaerobic bacteria are a type of bacteria that grow under anaerobic conditions, but cannot grow on the surface of solid media with air (containing 18% oxygen) and/or 10% carbon dioxide. According to their tolerance to oxygen, they can be divided into obligate anaerobes, microaerobes and facultative anaerobes. Such bacteria lack a complete metabolic enzyme system, and their energy metabolism is carried out by anaerobic fermentation. Anaerobic bacteria mainly include: Gram-positive anaerobes (Peptostreptococcus, Finegoldia, Anaerococcus, Peptoniphilus, Veillonella, etc.), Gram-negative anaerobes (Veillonella), Gram-positive anaerobic bacteria (Propionibacterium, lactobacillus, Actinomyces, Eubacterium, Eggerthella, Atopobium, Bifidobactrium, Mobiluncus, etc.), and Gram-negative anaerobic bacilli (Bacteroides, Helicobacter Helicobacter, Porphyromonas, Prevotella, Fusobacterium, etc.).

Ornidazole and Levoornidazole are used in the treatment of Bacteroides fragilis, Bacteroides diundi, Bacteroides ovalifolia, Bacteroides polymorpha, Bacteroides vulgaris, Clostridium, Eubacteria, Peptococcus and Peptostreptococcus, A variety of infectious diseases caused by sensitive anaerobic bacteria such as Helicobacter pylori, Bacteroides melanogaster, Fusobacterium, CO ₂ weaving bacteria, Bacteroides gingivalis. It has significant effects on abdominal infections, pelvic infections, oral infections, brain infections and treatment of severe amoebiasis in the digestive system.

But there are also more and more common drug-resistant bacteria production and drug side effects. Therefore, more and better antibacterial drugs are needed clinically.

Summary of the invention

In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide an anti-infective drug and its application, which can effectively anti-bacterial and has higher safety, and can treat infectious diseases.

The purpose of the present invention is achieved through the following technical solutions:

An anti-infective drug, the general structure of the anti-infective drug is shown in I:

The structure shown in formula I, wherein R ₁ represents piperazinyl, 2-methylpiperazinyl, 2,4-dimethylpiperazinyl, (S,S)-2,8-diazabicyclo [4 ,3,0]nonanyl, (R,R)-2,8-diazabicyclo[4,3,0]nonyl, 2,8-diazabicyclo[4,3,0]non alkyl;

R ₂ represents hydroxyl, phosphate ester group, phosphate ester base,

R ₆ represents an alkyl group of 1-10;

R ₃ represents halogen, hydrogen group;

R ₄ represents an amide group, a hydroxyl group, a phosphate group, a phosphate group,

R ₆ represents an alkyl group of 1-10;

R ₅ stands for methyl, methyloxy, ethoxy,

X represents halogen, hydrogen, and n2 represents 1-3.

The structure shown in formula I includes the following compounds:

The present invention also provides a method for preparing the above-mentioned anti-infective medicine, as shown in Reaction Formula II.

In reaction formula II, R ₁ , R ₃ , R ₄ and R ₅ are as defined in formula I. R ₆ is a glycidyl group or an ethanol group.

According to the present invention, the preparation method of the anti-infective drug includes: reacting levornidazole cyclization or ornidazole cyclization (A) with oxazolidinone derivative (B) to generate oxazolidinone-nitazolimidazole Compound (C) (Step 1);

The oxazolidinone-nitroimidazole compound (C) is reacted with phosphorus oxychloride and then hydrolyzed into a salt to form an anti-infective drug (D) (step 2);

The present invention also provides the above-mentioned anti-infective drugs, or their stereoisomers, hydrates, deuterated products, esters, solvates, crystal forms, metabolites, pharmaceutically acceptable salts or prodrugs, which are used in the preparation of therapeutic anaerobic bacteria Application of drugs for diseases caused by infections.

The present invention also provides an anti-infective pharmaceutical composition, the combined components of which include the above-mentioned anti-infective drugs (ie compounds of formula I), or stereoisomers, hydrates, deuterated compounds, esters, solvates, Crystal form, metabolite, pharmaceutically acceptable salt or prodrug.

The above-mentioned anti-infective drugs having hydroxyl groups react with phosphate esters to form anti-infective prodrugs. This prodrug has better solubility than the compound that has not formed a prodrug; the solubility of the prodrug is greater than 100mg/ml, the prodrug is stable in aqueous solution, and passes through the esterase and phosphatase in the blood It is converted into active ingredients, and thus a preparation for injection or oral administration is developed.

The composition of the present invention may include at least one active ingredient having functions similar to anti-infective drugs.

With regard to formulating pharmaceutical compositions, at least one compound of formula I may be mixed with at least one pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include physiological saline, sterile water, Ringer's solution, physiological saline buffer solution, glucose solution, maltodextrin solution, glycerol, ethanol, and the like. According to user needs, the pharmaceutical composition may contain conventional excipients, such as antioxidants, buffers, soil cleaners, and the like. The composition is also mixed with diluents, disintegrants, surfactants, binders, lubricants, aqueous solutions, suspensions, etc., to form injections, powders, capsules, granules, tablets, and the like. Preferably, according to the disease or component, the formulation is prepared by using the method described in Remington's Pharmaceutical Science (latest edition) (Mack Publishing Company, Easton PA, etc.).

The compounds of the present invention can be administered orally or parenterally, such as intravenously, subcutaneously, intraperitoneally, topically, and the like. The dosage of the compound may vary with the specific compound used, the mode of administration, the symptoms and severity of the condition to be treated, and various physical factors associated with the treatment of the individual. When the compound of the present invention is administered to an individual at a daily dose of about 10-25 mg, preferably 13-20 mg per kilogram of body weight, when needed, satisfactory results can be obtained according to the usage of the present invention. More preferably, the above-mentioned daily dose is divided into several administrations per day.

In the acute toxicity test, the half-lethal dose (LD ₅₀ ) of the anti-infective drug was found to be greater than 1g/kg, so the anti-infective drug was found to be safe.

The anti-infective drug of the present invention has inhibitory activity and low toxicity against a broad spectrum of bacteria. The prodrug prepared by reacting a compound having a hydroxyl group with a phosphate ester has high water solubility.

Further, the derivatives of the present invention can be shown to include Gram-positive bacteria such as Staphylococcus, Enterococcus and Streptococcus, anaerobic microorganisms such as Bacteroides and Clostridium and acid-resistant microorganisms such as Mycobacterium tuberculosis, Mycobacterium avium The potent antibacterial activity of human and animal pathogens.

Therefore, the composition containing the anti-infective drug is used in antibiotics.

detailed description

The aforementioned anti-anaerobic drug combination composition may include the anti-infective drug shown in formula I, the stereoisomers, hydrates, deuterated products, esters, solvates, crystal forms, and metabolic Combination of at least one of the product and a pharmaceutically acceptable salt or prodrug with antibacterial drugs known in the art.

The present invention also provides the application of the above-mentioned pharmaceutical composition in the preparation of a medicine for treating diseases caused by anaerobic bacteria infection in humans.

The outstanding effects of the present invention are:

The anti-infective drug of the present invention, or its stereoisomers, hydrates, deuterated products, esters, solvates, crystal forms, metabolites, pharmaceutically acceptable salts or prodrugs, can effectively resist anaerobic bacteria and then treat anaerobic bacteria Infectious diseases.

detailed description

In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solutions of the present invention are now described in detail below, but they should not be understood as limiting the scope of the present invention. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, can be obtained from commercial sources.

Example 1: Preparation of 2-methyl-5-nitro-1-(((S)-oxiran-2-yl)methyl)1H-imidazole

Weigh 100g of Levoornidazole and add it to 500ml of dichloromethane and stir to dissolve, cool to -10～-15℃; weigh 36.5g of sodium hydroxide, add 500ml of water, stir to dissolve and let cool, slowly add dropwise to the dichloromethane, Control the temperature not to exceed -10°C. After the addition, keep the reaction at 0～5°C for 2 hours, separate the dichloromethane layer, extract the water layer with 100ml dichloromethane, combine the dichloromethane layers, and wash the dichloromethane layer with 100ml water Twice, add an appropriate amount of anhydrous sodium sulfate to dry, filter, and concentrate the filtrate under reduced pressure to obtain 75.7 g of brown oil.

Example 2: Preparation of 2-methyl-5-nitro-1-(((R)-oxiran-2-yl)methyl)1H-imidazole

Weigh 100g of dexornidazole and add 500ml of dichloromethane and stir to dissolve, and cool to -10～-15℃; weigh 36.5g of sodium hydroxide, add 500ml of water, stir to dissolve and let cool, slowly add dropwise to the dichloromethane, Control the temperature not to exceed -10°C. After the addition, keep the reaction at 0～5°C for 2 hours, separate the dichloromethane layer, extract the water layer with 100ml dichloromethane, combine the dichloromethane layers, and wash the dichloromethane layer with 100ml water Twice, add an appropriate amount of anhydrous sodium sulfate to dry, filter, and concentrate the filtrate under reduced pressure to obtain 72.5 g of brown oil.

Example 3: Preparation of 2-methyl-5-nitro-1-((oxiran-2-yl)methyl)1H-imidazole

Weigh 100g of ornidazole into 500ml of dichloromethane and stir to dissolve, and cool to -10～-15℃; weigh 36.5g of sodium hydroxide, add 500ml of water, stir to dissolve and let cool, slowly add dropwise to dichloromethane, control The temperature should not exceed -10°C. After the addition, the reaction was kept at 0～5°C for 2 hours. The dichloromethane layer was separated, the water layer was extracted with 100ml dichloromethane, the dichloromethane layers were combined, and the dichloromethane layer was washed with 100ml water 2 Add an appropriate amount of anhydrous sodium sulfate for drying, filter, and concentrate the filtrate under reduced pressure to obtain 76.3 g of brown oil.

Example 4: Preparation of Compound 1

Take 1 10g of anhydrous ethanol 50ml of linezolid derivative into the reaction flask and stir the solution, warm up to 60℃ and slowly add 2-methyl-5-nitro-1-(((S)-oxirane-2 -Base) methyl) 1H-imidazole 2g, after the addition, keep the reaction for 6-8 hours, cool to room temperature, a large amount of solid precipitates, stir and crystallize for 2 hours, filter, the filter cake is washed with an appropriate amount of absolute ethanol, and dried to obtain Compound 1 solid 10.1g.

Example 5: Preparation of Compound 2

Add 1 10g of anhydrous ethanol 50ml of linezolid derivative into the reaction flask and stir the solution, warm up to 60℃ and slowly add 2g of deuterated ornidazole, after the addition, keep the reaction for 6-8 hours, cool to room temperature, there is a lot of solid After precipitation, stirring and crystallization for 2 hours, filtration, the filter cake was washed with an appropriate amount of absolute ethanol, and dried to obtain 9.8 g of compound 2 as a solid.

Example 6: Preparation of Compound 3

Take 2 10g of linezolid derivative and 50ml of absolute ethanol into the reaction flask and stir the solution, warm up to 60℃ and slowly add 2-methyl-5-nitro-1-((oxiran-2-yl) (Methyl) 1H-imidazole 2g, after the addition, keep the reaction for 6-8 hours, cool to room temperature, a large amount of solid precipitates, stir and crystallize for 2 hours, filter, the filter cake is washed with an appropriate amount of absolute ethanol, and dried to obtain compound 3 as a solid 10.8g.

Example 7: Preparation of Compound 8

Take 3 10g of linezolid derivative and 50ml of absolute ethanol into the reaction flask and stir the solution. The temperature is raised to 60℃ and slowly add 2-methyl-5-nitro-1-(((S)-ethylene oxide- 2-yl)methyl)1H-imidazole 2.8g, after the addition, keep the reaction for 6-8 hours, cool to room temperature, a large amount of solid precipitates, stir and crystallize for 2 hours, filter, wash the filter cake with an appropriate amount of absolute ethanol, and dry , 11.2 g of compound 8 was obtained as a solid.

Example 8: Preparation of compound 11

Take 20g of compound 1 and 100ml of dichloromethane into the reaction flask and stir to dissolve. Cool down to 0℃ and slowly add 4.2g of phosphorous oxychloride, keep the temperature for 6-8 hours, and slowly add 5ml of water after the reaction to complete the hydrolysis. After adjusting the pH to 4.0 with hydrochloric acid, a large amount of solids precipitated, stirred for 2 hours, filtered, and the filter cake was washed with an appropriate amount of acetone and dried to obtain 9.9 g of compound 11 as a solid.

Example 9: Preparation of compound 13

Take 3 10g of linezolid derivative and 50ml of absolute ethanol into the reaction flask and stir the solution. Heat to 60℃ and slowly add 2-trifluoromethyl-5-nitro-1-(((S)-Ethylene oxide) Alk-2-yl)methyl)1H-imidazole 2.8g, after the addition, keep the reaction for 6-8 hours, cool to room temperature, a large amount of solid precipitates, stir and crystallize for 2 hours, filter, and wash the filter cake with an appropriate amount of absolute ethanol And dried to obtain 10.5 g of compound 13 as a solid.

Example 10: Preparation of Compound 15

In the same way as compound 11, replacing compound 1 with compound 8 to obtain compound 15.

Experimental Example 1

In this example, the in vitro antibacterial activity of Compounds 1-18 and Comparative Compound 1 and Comparative Compound 2 were tested to illustrate their general antibacterial ability.

In this example, the in vitro antibacterial activity test on 8 standard strains was used first, and the samples were frozen and stored in a low-temperature refrigerator at -80°C. The use needs to be recovered 2 days in advance. Scrape a small amount of frozen bacteria with a sterile inoculating loop, streak it on a suitable solid medium plate, and place it in a suitable gas culture environment at 35±2°C for 20-48 hours. The classification numbers of the standard strains are shown in Table 1. For comparison, the following compounds were selected: linezolid, levornidazole and moxifloxacin hydrochloride. The compound was dissolved in DMSO and made into a 40-fold concentration stock solution on the day of the test.

Table l

Bacterial species	Gram stain classification	Strain number
Staphylococcus aureus	G+	ATCC29213
Enterococcus faecalis	G+	ATCC700221
Clostridium difficile	G+	ATCC43255
Clostridium perfringens	G+	ATCC13124
Pseudomonas aeruginosa	G-	ATCC27853
Escherichia coli	G-	ATCC25922
Klebsiella pneumoniae	G-	ATCC43816
Acinetobacter baumannii	G-	ATCC19606

The culture medium in this embodiment includes the following: Trypticase soy agar (TSA) (BDBBL211043), TSA+5% sheep blood (TSA II), ion-corrected Ma Yixin Ershi Broth (Cation-adjusted Mueller Hinton) broth, CAMHB) (BD BBL 212322), Brucella broth (Brucella broth, BB) (BDBBL 211088), Brucella agar (Brucella agar, BA) (BD BBL 211086), sheep blood (Quad Five 630-500) ).

The in vitro antibacterial activity test of this example determines the minimum inhibitory concentration (Minimum Inhibitory Concentration, MIC) of the compound. Methods as below:

Follow the standard methods recommended by the Clinical and Laboratory Standards Institute (CLSI). The highest tested concentration of compound 1-18, comparative compound 1, comparative compound 2, linezolid and levornidazole was 64ug/ml, which was diluted by 2 times. The highest tested concentration of moxifloxacin hydrochloride is 16ug/ml, which is diluted by 2 times.

Add 98ul of the corresponding bacterial inoculum to the test plate (except sterile control wells). For Clostridium difficile ATCC43255, first add 98ul of 50°C pre-prepared Bruce's agar (containing 5ug/ml of hemin and 10ug/ml of vitamin K1) to the test plate, mix well and wait for it to cool and solidify. Then add 5ul of Clostridium difficile ATCC 43255 bacterial inoculum on the agar surface. After the system is added, cover the test plate with a sterile cover, put it in a centrifuge at 800 rpm for 30 seconds, then shake on a plate shaker at 400 rpm for 1 minute, mix well, and put it in an ordinary incubator or anaerobic tank (Clostridium difficile ATCC 43255 and C. perfringens ATCC13124) was cultured at 35±2°C for 20 hours. The number of colonies of different dilutions of bacterial inoculum in the TSA plate was recorded by visual observation.

The test results are shown in Table 2 below:

The results showed that compound 1-18 has antibacterial effect on Gram-negative bacteria Klebsiella ATCC 43816, Acinetobacter baumannii ATCC 19606, Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 (MIC less than 32ug /mL); For Gram-positive bacteria, Anaerobic Clostridium difficile ATCC 43255 is the most sensitive to 17 test compounds, with MIC values between <0.063-0.125ug/mL; Anaerobic Clostridium perfringens ATCC 13124 is relatively sensitive, and its MIC value is between <0.063-0.25ug/mL; it has a slight inhibitory effect on Enterococcus faecalis ATCC 700221, and its MIC value is about 2-8ug/mL; compound 1-18 is against Staphylococcus aureus ATCC The antibacterial ability of 29213 is similar, and the MIC value is 4-8ug/mL. Comparative compounds 1 and 2 have a certain antibacterial effect on Gram-positive bacteria, but the antibacterial effect of the compound of the present invention is not significant.

Experimental Example 2: Acute toxicity test of the compound of the present invention on mice intravenously

In order to test the acute toxicity of the compound of the present invention and the comparative compound, the following experiment was performed.

Compound 1-18 was dissolved in water and administered to 5 ICR mice (5 weeks old, male, mice weighing 20 g ± 2 g). Intravenous administration to determine the median lethal dose (LD ₅₀ , mg/ml). Linezolid, Levoornidazole and Ornidazole were used as controls. The results are shown in Table 4.

Table 4

Compound	LD50 (LD 50 , mg/kg)
Linezolid	500
Levoornidazole	520
Ornidazole	550
Compound 1	＞1000
Compound 2	＞1000
Compound 3	＞1000
Compound 4	＞1000
Compound 5	＞1000
Compound 6	＞1000
Compound 7	＞1000
Compound 8	＞1000
Compound 9	＞1000
Compound 10	＞1000
Compound 11	＞1000
Compound 12	＞1000
Compound 13	＞1000
Compound 14	＞1000
Compound 15	＞1000
Compound 16	＞1000
Compound 17	＞1000
Compound 18	＞1000
Comparative compound 1	450
Comparative compound 2	450

According to Table 4, the toxicity of the compound of the present invention is less than that of the control drug, indicating that the compound of the present invention has excellent low toxicity and higher safety.

Example formulation: preparation of pharmaceutical composition

l. Preparation of powder

Anti-infective drugs 2 grams

Lactose 1 grams

The above materials are mixed, and then the mixture is filled into a sealed package to prepare a powder.

2. Preparation of tablets

The above materials are mixed, and then the mixture is compressed into tablets by known methods.

3. Preparation of capsules

The above materials are mixed and the mixture is filled into a gelatin capsule by a known method to make a capsule.

4. Preparation of injection

Dissolve the anti-infective drugs and glucose with water, adjust the pH to 4.0-9.0 with a pH regulator, and place in a freeze-drying oven for freeze-drying, after drying, stoppering, and capping.

Claims (6)

1. An anti-infective drug whose structure is shown in formula I:

   
2. The anti-infective drug according to claim 1, wherein the anti-infective drug comprises its stereoisomers, hydrates, deuterated products, esters, solvates, crystal forms, metabolites, pharmaceutically acceptable salts or pro- medicine.
3. The anti-infective drug according to claim 1, wherein R ₁ in the structure of the anti-infective drug represents piperazinyl, 2-methylpiperazinyl, 2,4-dimethylpiperazinyl, (S ,S)-2,8-diazabicyclo[4,3,0]nonanyl, (R,R)-2,8-diazabicyclo[4,3,0]nonyl, 2, 8-Diazabicyclo[4,3,0]nonanyl;

   R ₂ represents hydroxyl, phosphate ester group, phosphate ester base,

   

   R ₆ represents an alkyl group of 1-10;

   R ₃ represents halogen, hydrogen group;

   R ₄ represents an amide group, a hydroxyl group, a phosphate group, a phosphate group,

   

   R ₆ represents an alkyl group of 1-10;

   R ₅ stands for methyl, methyloxy, ethoxy,

   

   X represents halogen, hydrogen, and n2 represents 1-3.
4. The anti-infective drug according to claim 1, wherein the anti-infective drug comprises the following compounds:

   

   

   

   
5. An anti-infective drug composition, characterized in that: the components include the anti-infective drug according to claim 1, or its stereoisomers, hydrates, deuterated products, esters, solvates, crystal forms, metabolites, pharmaceuticals The acceptable salt or prodrug.
6. The use of the pharmaceutical composition of claim 5 in the preparation of a medicine for treating diseases caused by human infection.