Emodin mannich base, its preparation method and application

Abstract

The present application relates to the field of pharmaceutical chemistry, and specifically discloses a emodin mannich base, a preparation method and application thereof. The emodin mannich base has a structure as shown in formula I, the structure is simple, has strong in-vitro antimicrobial activity, especially high inhibitory activity to staphylococcus aureus, bacillus subtilis, micrococcus luteus, escherichia coli, proteus vulgaris, pseudomonas aeruginosa, shigella dysenteriae, salmonella typhimurium and other bacteria, and candida utilis, candida albicans, saccharomyces cerevisiae, monascus and other fungi, and can be used for preparing antibacterial and / or antifungal drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, and particularly relates to a emodin mannich base, a preparation method and application thereof. BACKGROUND

[0002] Emodin is an effective component of Chinese herbal medicine rhubarb. Research shows that emodin has various biological activities, such as vasodilation, immune inhibition, anti-tumor, anti-inflammatory, anti-oxidation, liver protection, catharsis, and diuresis, and thus has good clinical medical value. At present, there are few studies on the antimicrobial activity of emodin and its derivatives, so the present application aims to provide an emodin mannich base compound with good antimicrobial activity. SUMMARY

[0003] Therefore, the present application aims to provide an emodin mannich base with good antimicrobial activity, and a preparation method and application thereof.

[0004] To achieve the above-mentioned purpose, the present application provides the following technical solutions.

[0005] According to an embodiment of the present application, in a first aspect, an emodin mannich base is provided, having a structure as shown in formula I:

[0006]

[0007] wherein Im is a nitrogen-containing five-membered heteroaryl group.

[0008] It should be noted that the nitrogen-containing five-membered heteroaryl group, such as thiazolyl, oxazolyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, etc., has a unique aromatic structure containing at least one heteroatom and an electron-rich property, making it more likely to interact with enzymes, ion channels, carrier proteins, DNA, RNA, etc. in the body through various non-covalent bonds, and thus showing low drug resistance. By introducing a nitrogen-containing five-membered heteroaryl group into the parent structure of emodin, a new emodin mannich base with a mechanism of action different from traditional drugs can be obtained. The present application has found that the emodin mannich base having the structure shown in formula I shows excellent antibacterial and antifungal activity, and is expected to be developed into a high-efficiency antimicrobial drug.

[0009] In an alternative embodiment, Im is any one of 1,2,3-triazolyl, 1,2,4-triazolyl, oxazolyl, imidazolyl, pyrazolyl, thiazolyl, tetrazolyl, benzotriazolyl, benzimidazolyl, and carbazolyl.

[0010] According to an embodiment of the present application, in a second aspect, a method for preparing the emodin mannich base of the first aspect is provided, comprising the following steps:

[0011] Im-NH2 reacts with formaldehyde and emodin in a polar organic solvent under the action of an acidic catalyst to prepare the emodin Mannich base shown in formula I.

[0012] The preparation method provided by the application has a simple synthesis route and is easy to operate, and the target product can be prepared in one step, has high yield, causes little pollution and is suitable for industrial production.

[0013] In an alternative embodiment, Im-NH2 reacts with formaldehyde and an acidic catalyst in a polar organic solvent under reflux conditions for 0.5-1 h, and then emodin is added to continue reflux reaction for 3-6 h, so that the emodin Mannich base azole compound can be quickly prepared.

[0014] It can be understood that the chemical structure of the reaction raw material Im-NH2 can be 2-aminothiazole, 2-aminooxazole, 2-aminoimidazole, 3-amino-triazole or 5-amino-tetrazole.

[0015] In an alternative embodiment, the molar ratio of Im-NH2 to formaldehyde to emodin is 1-3: 1-5: 1-1.2, so that the electrophilic reaction can occur at a relatively high conversion rate to obtain the emodin Mannich base.

[0016] In an alternative embodiment, the polar organic solvent is at least one of n-butanol, propanol, ethanol or methanol, so that the electrophilic imine ion has a certain concentration.

[0017] In an alternative embodiment, the acidic catalyst is at least one of formic acid, glacial acetic acid or hydrochloric acid, so that the imine carbonium ion can be formed.

[0018] According to the third aspect of the embodiments of the application, the emodin Mannich base of the first aspect is used in the preparation of an antimicrobial drug.

[0019] In an alternative embodiment, the microorganism is bacteria and / or fungi.

[0020] In an alternative embodiment, the bacteria is one or more of Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Shigella dysenteriae or Salmonella typhimurium.

[0021] In an alternative embodiment, the fungi is one or more of Candida utilis, Candida albicans, Saccharomyces cerevisiae or Monascus ruber.

[0022] In an alternative embodiment, the drug is any one of a tablet, a capsule, an aerosol or an ointment.

[0023] The emodin mannich base provided by the present application has simple chemical structure, strong in-vitro antimicrobial activity, and high inhibitory activity on bacteria such as Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Shigella dysenteriae, Salmonella typhimurium, and fungi such as Candida utilis, Candida albicans, Saccharomyces cerevisiae, and Monascus, and can be used for preparing antibacterial and / or antifungal drugs, thereby providing more efficient and safe candidate drugs for clinical antimicrobial treatment, and helping to solve the increasingly serious problems of drug resistance, stubborn pathogenic microorganisms, and newly emerging harmful microorganisms in clinical treatment. DETAILED DESCRIPTION

[0024] The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application. In addition, the technical features involved in different embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0025] If the specific experimental steps or conditions are not specified in the examples and comparative examples, the operations or conditions can be performed according to the conventional experimental steps described in the literature in the art. If the reagents or instruments are not specified by the manufacturer, they are all conventional reagent products that can be obtained by purchase.

[0026] Example 1

[0027] The preparation of emodin mannich base I-1 is shown in the following reaction formula:

[0028]

[0029] The specific steps are as follows:

[0030] Into a 100 mL round-bottom flask, 0.602 g of 2-aminothiazole, 0.180 g of formaldehyde, 0.045 g of formic acid, and 70 mL of n-butanol were added, and after stirring under reflux for half an hour, 1.350 g of emodin was added and the reaction was continued under reflux for 4 hours. After the reaction was completed, n-butanol was removed by distillation under reduced pressure, the residue was extracted with dichloromethane (3 x 30 mL), the combined organic phase was dried over anhydrous sodium sulfate, and then rotary evaporation was performed. The product was purified by silica gel column chromatography using dichloromethane as the eluent, and dried to obtain 0.786 g of emodin mannich base I-1 in the form of yellow solid.

[0031] The yield of this example is 41%, the melting point is 200-202°C, and the HRMS (TOF) calcd for C 19 H 14N2O5S[M+H] + Found: 383.0706.

[0032] Example 2

[0033] Emodin Mannich Base I-2, the reaction formula is as follows:

[0034]

[0035] The specific steps are as follows:

[0036] Into a 50 mL round bottom flask, 0.251 g of 2-amino oxazole, formaldehyde 0.092 g, formic acid 0.023 g and n-butanol 30 mL were added, after stirring for half an hour under reflux, 0.676 g of emodin was added and the reaction was continued under reflux for 4 hours. After the reaction was completed, n-butanol was removed by distillation under reduced pressure, the residue was extracted with dichloromethane (3 x 20 mL), the combined organic phase was dried over anhydrous sodium sulfate and then rotary evaporated, and then purified by silica gel column chromatography using dichloromethane as the eluent, and dried to obtain 0.415 g of emodin Mannich base I-2 in the form of yellow solid.

[0037] The yield of this example was 45%; the melting point was 123-125 °C; HRMS (TOF) calcd for C 19 H 14 N2O6[M+H] + Found: 367.0930.

[0038] Example 3

[0039] Emodin Mannich Base I-3, the reaction formula is as follows:

[0040]

[0041] The specific steps are as follows:

[0042] Into a 50 mL round bottom flask, 0.250 g of 3-amino triazole, formaldehyde 0.091 g, formic acid 0.024 g and n-butanol 30 mL were added, after stirring for half an hour under reflux, 0.675 g of emodin was added and the reaction was continued under reflux for 4 hours. After the reaction was completed, n-butanol was removed by distillation under reduced pressure, the residue was extracted with dichloromethane (3 x 20 mL), the combined organic phase was dried over anhydrous sodium sulfate and then rotary evaporated, and then purified by silica gel column chromatography using dichloromethane as the eluent, and dried to obtain 0.456 g of emodin Mannich base I-3 in the form of yellow solid.

[0043] The yield of this example was 50%; the melting point was 201-203 °C; HRMS (TOF) calcd for C 18 H 14 N4O5[M+H]+ 367.1037; found, 367.1043.

[0044] Example 4

[0045] Preparation of emodin Mannich base I-4, the reaction scheme is shown below:

[0046]

[0047] The specific steps are as follows:

[0048] Into a 50 mL round bottom flask was added 0.250 g of 2-aminoimidazole, formaldehyde 0.090 g, formic acid 0.023 g and n-butanol 30 mL, after stirring for half an hour under reflux, 0.675 g of emodin was added to continue the reaction under reflux for 4 hours. After the reaction was completed, n-butanol was removed by distillation under reduced pressure, the residue was extracted with dichloromethane (3 x 20 mL), the combined organic phase was dried over anhydrous sodium sulfate, then rotary evaporation, and column chromatography on silica gel was performed using dichloromethane as the eluent, and drying, to obtain 0.405 g of emodin Mannich base I-4 in the form of yellow solid.

[0049] The yield of this example was 44%; melting point 139-140 °C; HRMS (TOF) calcd. for C 19 H 15 N3O5[M+H] + ,366.1085; found, 366.1083.

[0050] Example 5

[0051] Preparation of emodin Mannich base I-5, the reaction scheme is shown below:

[0052]

[0053] The specific steps are as follows:

[0054] Into a 50 mL round bottom flask was added 0.250 g of 2-aminoimidazole, formaldehyde 0.090 g, formic acid 0.023 g and n-butanol 30 mL, after stirring for half an hour under reflux, 0.675 g of emodin was added to continue the reaction under reflux for 4 hours. After the reaction was completed, n-butanol was removed by distillation under reduced pressure, the residue was extracted with dichloromethane (3 x 20 mL), the combined organic phase was dried over anhydrous sodium sulfate, then rotary evaporation, and column chromatography on silica gel was performed using dichloromethane as the eluent, and drying, to obtain 0.405 g of emodin Mannich base I-4 in the form of yellow solid.

[0055] The yield of this example was 46%; melting point 212-213 °C; HRMS (TOF) calcd. for C 17 H 13 N5O5[M+Na]+ Calcd, 390.0809; found, 390.0808.

[0056] Comparative Example 1

[0057]

[0058] Reference (Thidarat Chalothorn, Vatcharin Rukachaisirikul, Souwalak Phongpaichit, Sakawrat Pannara, Chittreeya Tansakul, Synthesis and antibacterial activity of emodin and its derivatives against methicillin-resistant Staphylococcus aureus, Tetrahedron Letters, 2019, 60, 151004), the specific synthesis method is as follows:

[0059] At room temperature, to a solution of emodin II (50 mg, 0.19 mmol) in dioxane (2 mL) was added dimethylamine aqueous solution (40%, 48 μL, 0.38 mmol) and benzaldehyde (38 μL, 0.38 mmol). The reaction system was heated to 65 °C for 24 hours. After the reaction was completed, dichloromethane (3 x 30 mL) was extracted, the organic phase was combined, dried over anhydrous sodium sulfate, and then rotary evaporated. Purification was performed by silica gel column chromatography using 60% ethyl acetate / dichloromethane as the eluent, and dried to obtain 0.057 g of compound III as an orange-yellow solid.

[0060] Comparative Example 2

[0061]

[0062] Reference (Thidarat Chalothorn, Vatcharin Rukachaisirikul, Souwalak Phongpaichit, Sakawrat Pannara, Chittreeya Tansakul, Synthesis of aminoderivatives of 1,6,8-trihydroxy-3-methyl-9,10-anthraquinone, Chemistry of Natural Compounds, 1999, 35(6), 619-620), the specific synthesis method is as follows:

[0063] After the morpholine (0.6 mL, 0.006 mol) was cooled to 0°C, the formaldehyde aqueous solution (40%, 0.23 g, 0.008 mol) was added dropwise, and then the emodin II (0.45 g, 0.002 mol) was added. The mixture was stirred at room temperature for one hour. After 10 mL of water was added to the reaction system, the mixture was filtered, and the filtrate was collected. The pH was adjusted to 9 by adding NaOH (10%) dropwise while stirring. The filter cake was collected, washed with water, and dried to obtain 0.35 g of the compound IV.

[0064] Experimental Example 6

[0065] The emodin Mannich bases prepared in the examples and the comparative examples were tested for in-vitro antimicrobial activity by using the 96-well microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI). The minimum inhibitory concentrations (MICs) of the compounds against Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Shigella dysenteriae, Salmonella typhimurium, Candida utilis, Candida albicans, and Saccharomyces cerevisiae were determined.

[0066] Specifically, the test compounds and the reference drugs were dissolved in dimethyl sulfoxide (DMSO) to prepare 6.4 mg·mL -1 solution. A sterile 96-well plate was prepared, and 100 μL of a bacterial solution was added to each well. 100 μL of the test drug solution diluted with sterile broth was added to well No. 1, and 100 μL of the solution obtained after mixing was added to well No. 2. The solution was diluted by a factor of two in each of the subsequent wells. The DMSO content in each well was less than 1% to exclude the influence of DMSO on the in-vitro antibacterial and antifungal activity. Well No. 12 did not contain the drug and was used as a positive control. The drug sensitivity plates were incubated at 37°C for 24 hours on a constant-temperature shaker. The results are shown in Tables 1 and 2.

[0067] Table 1. Antimicrobial activity (MIC, μmol / mL)

[0068]

[0069]

[0070] Table 2. Antifungal activity (MIC, μmol / mL)

[0071] Compound Candida utilis Aspergillus flavus Saccharomyces cerevisiae Candida albicans Candida I-1 0.021 0.042 0.042 0.010 0.010 I-2 0.022 0.022 0.022 0.011 0.044 I-3 0.011 0.044 0.011 0.022 0.011 I-4 0.022 0.044 0.011 0.011 0.022 I-5 0.022 0.011 0.022 0.022 0.022 II 0.948 0.474 0.474 0.474 0.474 III 0.159 0.317 0.635 0.635 0.635 IV 0.693 0.347 0.173 0.347 0.173 fluconazole 0.026 0.837 0.013 0.013 0.013

[0072] As shown in Table 1, the emodin Mannich bases I-1 to I-5 exhibited good inhibitory effects on the tested bacteria. Table 2 shows that the emodin Mannich bases I-1 to I-5 exhibited certain inhibitory effects on the tested fungi. Thus, the emodin Mannich bases provided by the present application have strong antimicrobial activity and can be used to prepare antibacterial and / or antifungal drugs.

[0073] Example 7

[0074] Preparation of tablets

[0075] Prescription: emodin mannich base I-1 100g, starch 40g, microcrystalline cellulose 80g, magnesium stearate 3.0g, hydroxypropyl methyl cellulose E-30 (40% solution) appropriate amount, a total of 4000 tablets.

[0076] Method: prepare a 4% hydroxypropyl methyl cellulose E-30 solution, ready for use; take 20g of starch, dry at 105°C for 5 hours to get dry starch, ready for use; take 20g of starch and the amount of emodin mannich base I-1, microcrystalline cellulose, mix evenly, crush through 80 mesh sieve, use 4% hydroxypropyl methyl cellulose E-30 solution to make soft material, 20 mesh sieve to granulate, dry at 50-60°C to about 3% moisture, pass through 20 mesh sieve to granulate, add dry starch 20g and the amount of magnesium stearate in the dry granules, mix evenly, and press into tablets.

[0077] Usage and dosage: according to the patient's condition and individual differences, it is recommended to take 3-6 tablets per day, equivalent to 0.075-0.15g emodin mannich base I-1 / 60kg body weight / day, taken after meals, drink a full cup of water when taking; or according to the doctor's advice.

[0078] Example 8

[0079] Preparation of capsules

[0080] Prescription: emodin mannich base I-2 100g, modified starch (120 mesh) 50g, microcrystalline cellulose (100 mesh) 30g, low-substituted hydroxypropyl cellulose (100 mesh) 10g, talc (100 mesh) 10g, sweetener 5g, orange flavor 1g, pigment appropriate amount, water appropriate amount, made into 4000 particles.

[0081] Method: micronize the emodin mannich base I-2 in the prescription amount into fine powder, mix with the amount of modified starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, talc, sweetener, orange flavor and pigment, use water to make soft material, 12-14 mesh sieve to granulate, dry at 40-50°C, sieve to granulate, fill into empty capsules, and get it.

[0082] Usage and dosage: according to the patient's condition and individual differences, it is recommended to take 3-6 capsules per day, equivalent to 0.075-0.15g emodin mannich base I-2 / 60kg body weight / day, taken after meals, drink a full cup of water when taking; or according to the doctor's advice.

[0083] Example 9

[0084] Preparation of aerosol

[0085] Prescription: emodin mannich base I-3 2.5g, Span20 3g, talc (100 mesh) 4g, trichloromonofluoromethane to the appropriate amount.

[0086] Preparation: emodin mannich base I-3, Span20 and talc are dried in a vacuum drying oven for several hours, cooled to room temperature in a desiccator, and then crushed into powder with an air flow crusher. Then mix them according to the prescription, pour them into airtight containers, and add trichloromonofluoromethane to the specified amount.

[0087] Usage and dosage: according to the patient's condition and individual differences, it is recommended to use 3-4 times a day.

[0088] Example 10

[0089] Preparation of ointment

[0090] Prescription: emodin mannich base I-4 2g, stearic acid 12g, vaseline 3g, monoglyceride 3g, nipagin ethyl 0.5g, distilled water 60mL, borax 1g, potassium hydroxide 0.5g, potassium sorbate 0.3g, glycerol 0.5g, liquid paraffin 0.5g, a total of 85g.

[0091] Preparation: Take the prescribed amount of stearic acid, vaseline, monoglyceride and nipagin ethyl, melt and pass through a 100 mesh sieve, keep at 80°C as oil phase; take the prescribed amount of distilled water, borax, potassium hydroxide, potassium sorbate, glycerol and liquid paraffin, heat to boiling as water phase; cool the water phase to 85°C, add the oil phase under constant stirring, emulsify, then add the prescribed amount of emodin mannich base I-4, cool and stir, and it is ready.

[0092] Usage and dosage: apply to the affected area, rub repeatedly until the skin is hot, twice a day.

[0093] Example 11

[0094] Preparation of liniment

[0095] Prescription: emodin mannich base I-5 4g, potassium soap 7.5g, camphor 5g, distilled water to 100mL.

[0096] Preparation: heat the potassium soap to liquefy, ready for use; dissolve camphor in 95% ethanol, ready for use; take the prescribed amount of emodin mannich I-5, add the potassium soap solution under constant stirring, then add the camphor ethanol solution, and gradually add water. After complete emulsification, add distilled water to the full amount.

[0097] Usage and dosage: 1-2 drops on the affected area without damage, or use a cotton swab to apply the appropriate amount of liquid to the affected area, twice a day.

[0098] Obviously, the above embodiments are merely example for clearly illustrating but not limitation to the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments need not and can not be enumerated. The obvious changes or variations derived from the above description are still within the protection scope of the present application.

Claims

1. Rhamnosine Mannich base, having the structure shown in Formula I: in, Im is a nitrogen-containing five-membered heteroaryl group.

2. The emodin Mannich base according to claim 1, characterized in that Im is any one of 1,2,3-triazolyl, 1,2,4-triazolyl, oxazolyl, imidazolyl, pyrazolyl, thiazolyl, and tetrazolyl.

3. The method for preparing the emodin Mannich base according to claim 1 or 2, characterized in that: The steps include: In a polar organic solvent, under the action of an acidic catalyst, Im-NH2 reacts with formaldehyde and emodin to prepare an emodin Mannich base having a structure shown in formula I.

4. The method for preparing the emodin Mannich base according to claim 3, wherein: In a polar organic solvent, under reflux conditions, Im-NH2 reacts with formaldehyde and an acidic catalyst for 0.5-1 h, and then emodin is added and the reflux reaction is continued for 3-6 h.

5. The method for preparing the emodin Mannich base according to claim 3 or 4, characterized in that: The molar ratio of the Im-NH2 to the formaldehyde and the emodin is 1-3:1-5:1-1.

2.

6. The method for preparing the emodin Mannich base according to claim 3 or 4, characterized in that: The polar organic solvent is at least one of n-butanol, propanol, ethanol and methanol.

7. The method for preparing the emodin Mannich base according to claim 3 or 4, characterized in that: The acidic catalyst is at least one of formic acid, glacial acetic acid and hydrochloric acid.

8. Use of the emodin Mannich base according to any one of claims 1 to 2 in the preparation of antimicrobial drugs, wherein the microorganism is a bacterium and / or a fungus.

9. The use of the emodin Mannich base in the preparation of antimicrobial drugs according to claim 8, characterized in that: The medicine is any one of tablets, capsules, aerosols and ointments.

10. Use of the emodin Mannich base according to claim 8 or 9 in the preparation of antimicrobial drugs, characterized in that: The bacteria are one or more of Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Escherichia coli, Proteus, Pseudomonas aeruginosa, Shigella dysenteriae or Salmonella typhimurium; The fungus is one or more of Candida utilis, Candida albicans, Saccharomyces cerevisiae or Monascus.

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