A cholesteric compound or its salt, disinfectant, and preparation method and application thereof

Abstract

The present invention discloses a cholesteric compound or its salt, a disinfectant, and its preparation method and application, belonging to the field of medical and health technology. The cholesteric compound has a structure as shown in Formula I, Formula II, or Formula III, and can effectively kill Escherichia coli and Pseudomonas aeruginosa. It can also achieve a satisfactory killing effect against Staphylococcus aureus and Candida albicans within 1 minute under neutral and alkaline environments. The present invention also discovers for the first time that the cholesteric compound can achieve a highly effective disinfection effect against spores in a short period of time, meeting the national technical standard for high-efficiency disinfection ("Technical Specifications for Disinfection" (2002 edition)). Therefore, the cholesteric compound and its salt of the present invention have broad application prospects in the preparation of disinfectants such as hand disinfectants and disinfectants for medical devices and supplies. #imgabs0##imgabs1##imgabs2#

Description

Technical Field

[0001] The present invention belongs to the field of medical and health technology, and specifically relates to a cholesteric compound or its salt, a disinfectant, and a preparation method and application thereof. Background Art

[0002] Disinfectants are agents used to kill pathogenic microorganisms on transmission media, rendering them harmless. Unlike antibiotics, their primary role in disease prevention is to eliminate pathogens from the human body, cutting off the transmission pathways of infectious diseases and ultimately controlling them.

[0003] Disinfectants can be categorized by their effectiveness as high-efficacy disinfectants, intermediate-efficacy disinfectants, and low-efficacy disinfectants. High-efficacy disinfectants specifically refer to preparations that can kill all bacterial vegetative forms (including mycobacteria), viruses, fungi, and their spores, and also have a certain killing effect on bacterial spores (pathogenic spore-forming bacteria), meeting high-level disinfection requirements. Intermediate-efficacy disinfectants refer to preparations that can only kill mycobacteria, fungi, viruses, and bacterial vegetative forms, meeting disinfection requirements. Low-efficacy disinfectants refer to preparations that can only kill bacterial vegetative forms and lipophilic viruses, meeting disinfection requirements.

[0004] According to the application scenario, disinfectants can be divided into hand disinfection, skin and mucous membrane disinfection, air disinfection, medical equipment and supplies disinfection, food (drinking utensils) disinfection, general object surface and fabric disinfection, drinking water and swimming pool water disinfection, fruit and vegetable disinfection, etc.

[0005] Disinfectants for these different application scenarios must be tested for efficacy in accordance with national disinfectant technical requirements. For example, hand disinfectants must be tested against at least Staphylococcus aureus (Gram-positive bacteria), Escherichia coli (Gram-negative bacteria), Pseudomonas aeruginosa (Gram-negative bacteria), and Candida albicans (fungus). High-level disinfection of medical devices and supplies requires testing against Bacillus subtilis var. niger spores.

[0006] The national disinfectant technical specifications require that qualified disinfectants demonstrate a sufficiently strong killing effect against bacteria or spores within the killing time. Killing time, also known as KT, is used in biological indicator resistance testing to define the minimum time (in minutes) required for all test indicator samples to exhibit sterile growth after exposure to a bactericidal agent. While achieving the same sterilization effect, a shorter killing time is preferred. According to the national hand disinfectant technical standard (GB27950-2020dz), qualified hand disinfectants must meet the following killing criteria within 1 minute at the intended concentration: a logarithmic killing limit of 5 or greater against Staphylococcus aureus (Gram-positive bacteria), Escherichia coli (Gram-negative bacteria), and Pseudomonas aeruginosa (Gram-negative bacteria) (determined by suspension method, the same below); and a logarithmic killing limit of 4 or greater against Candida albicans (fungus). Qualified high-efficiency disinfectants for medical devices and supplies must meet the requirement that the logarithmic killing limit for spores is greater than or equal to 5 within the declared killing time (generally no more than 1 hour).

[0007] With the increasing use of various antibiotics and disinfectants in the environment and on humans, bacteria and viruses are constantly mutating under the pressure of survival and evolution, and humanity faces increasingly serious threats. The development of new disinfectants is crucial for maintaining public health. An ideal new disinfectant should be safe for humans and environmentally friendly at low concentrations, with a shortest possible kill time and broad-spectrum, high-efficiency disinfection.

[0008] Ceragenins are a class of compounds with antibacterial activity. Their antibacterial and anti-biofilm activities have been widely reported. For example, the literature (New β-Lactam Antibiotics and Ceragenins – A Study to Assess Their Potential in Treatment of Infections Caused by Multidrug-Resistant Strains of Pseudomonas aeruginosa. Infection and Drug Resistance. 2021:14 5681–5698) investigated the minimum bactericidal concentration of CSA-13 (reference compound 8 in this invention) against Pseudomonas aeruginosa. These antibacterial and bactericidal activity tests all followed the European Committee on Antimicrobial Susceptibility Testing (EUCAST) susceptibility testing procedures. The compound's interaction time with bacteria was 18 ± 2 hours, which is fundamentally different from the shorter kill time (measured in minutes) required for disinfectants. To date, there have been no reports of ceragenins being used for disinfection with shorter kill times.

[0009] In fact, the vast majority of compounds with good antibacterial activity, including approved antibiotics, fail to meet the technical specifications for disinfectants because they cannot rapidly and broadly kill pathogens. In short, based on the potential needs of public health, it is extremely necessary to research and develop new disinfectants that are safe, effective, and fast-acting. Summary of the Invention

[0010] In order to solve the above problems existing in the prior art, the purpose of the present invention is to provide a cholesteric compound or its salt, a disinfectant, and a preparation method and application thereof.

[0011] In order to achieve the above object, the present invention adopts the following technical solutions:

[0012] The present invention provides a cholesteric compound or a salt thereof, wherein the cholesteric compound has a structure as shown in Formula I, Formula II or Formula III:

[0013]

[0014]

[0015]

[0016] Furthermore, the salt is hydrochloride, glucuronide, acetate, butyrate or 1,5-naphthalene disulfonate.

[0017] The present invention also provides a method for preparing the above-mentioned cholesteric compound, which comprises the following steps:

[0018] (1) Bile acid compounds react with halogenated olefins under alkaline conditions to obtain intermediate 1;

[0019] (2) Intermediate 1 reacts with amine to obtain intermediate 2;

[0020] (3) Intermediate 2 undergoes hydroboration-oxidation reaction to obtain intermediate 3;

[0021] (4) Intermediate 3 first reacts with a sulfonylating agent to form a sulfonate, which then reacts with a nucleophile under alkaline conditions to obtain intermediate 4;

[0022] (5) Intermediate 4 is hydrolyzed under acidic conditions and reacts with a reducing agent to obtain a cholesteric compound;

[0023] Among them, when the bile acid compound is allochenodeoxycholic acid, intermediate 1 is When the amine is octylamine, the intermediate 2 is , intermediate 3 is , intermediate 4 is , cholesteric compounds are ;

[0024] When the bile acid compound is hyodeoxycholic acid, intermediate 1 is When the amine is octylamine, the intermediate 2 is , intermediate 3 is , intermediate 4 is , cholesteric compounds are ;

[0025] When the bile acid compound is hyodeoxycholic acid, intermediate 1 is When the amine is laurylamine, intermediate 2 is , intermediate 3 is , intermediate 4 is , cholesteric compounds are .

[0026] Furthermore, the bile acid compound in step (1) is allochenodeoxycholic acid or hyodeoxycholic acid;

[0027] The amine in step (2) is R-NH2, R is C 3~12 alkyl;

[0028] The hydroboration-oxidation reagent in step (3) is 9-borabicyclo[3.3.1]nonane and H2O2;

[0029] In step (4), the sulfonylating agent is methanesulfonic acid chloride, and the nucleophilic agent is bis(tert-butyloxycarbonyl)amine;

[0030] The reducing agent in step (5) is borane.

[0031] Furthermore, the amine is octylamine or laurylamine.

[0032] The present invention also provides a disinfectant, which is a preparation prepared with the above-mentioned cholesteric compound or its salt, compound 6 or its salt, compound 8 or its salt as active ingredients, and the pH of the disinfectant is ≥7.0.

[0033] Furthermore, the pH of the disinfectant is 7.0~11.0.

[0034] Furthermore, the disinfectant also includes a solvent, a co-solvent acceptable to the disinfectant, and a pH regulator.

[0035] Furthermore, the solvent is water, the co-solvent is glycerol, and the pH adjuster is at least one of tetrasodium ethylenediaminetetraacetic acid, sodium metasilicate pentahydrate, sodium phosphate, and sodium carbonate.

[0036] Furthermore, the solvent is water, the co-solvent is glycerol, and the pH adjuster is sodium metasilicate pentahydrate.

[0037] The present invention also provides the use of the above-mentioned compound or its salt or the above-mentioned disinfectant in the preparation of disinfection products for disinfecting mucous membranes and skin, hands and feet, medical devices and supplies, fabrics, food (drinking) utensils, drinking water and swimming pool water, and fruits and vegetables.

[0038] The present invention also provides the use of the above compound or its salt or the above disinfectant in preparing a disinfection product for killing at least one of bacteria, fungi and their spores or viruses.

[0039] Furthermore, the disinfection product is a disinfection product for killing at least one of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Bacillus subtilis var. niger spores.

[0040] The present invention has achieved the following beneficial effects:

[0041] 1. The closest disclosed prior art to the present invention is the Ceragenins represented by compounds 6 and 8. Although a variety of antibacterial activities have been reported for this class of compounds, their application in disinfectants has not yet been seen. In particular, for hand disinfection, in accordance with national technical specifications, it is necessary to simultaneously meet the requirements of achieving a killing effect of 4 or 5 logarithms against Candida albicans, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa in 1 min (1 minute). Compounds 1, 2, 3, 4, 5, 6, 7, and 8 are relatively similar in structure, but have significantly different disinfecting activities. As described in Experimental Example 2, at the same concentration, compounds 3, 5, and 7 did not show a qualified killing effect against Escherichia coli and Pseudomonas aeruginosa for 1 min, among which compound 7 had the worst effect. Compounds 1, 2, 4, 6, and 8 can kill Escherichia coli and Pseudomonas aeruginosa. However, in terms of killing Candida albicans and Staphylococcus aureus in 1 minute, as described in Experimental Examples 3 and 5, under the same conditions, compounds 1 and 2 are the most effective, with significantly superior activity compared to compounds 6 and 8; compound 4 also has a strong killing effect.

[0042] 2. The present invention discovered for the first time the disinfection pH effect of this class of compounds. As described in Experimental Example 3, even if compounds 6 and 8 were increased to 20 mg / ml under weak acidity, they could not meet the qualified killing effect of Candida albicans and Staphylococcus aureus in 1 minute. As described in Experimental Examples 4 and 5, pH ≥ 7 is the key condition for meeting the qualified killing effect of Staphylococcus aureus and Candida albicans in 1 minute. The 1-minute killing effect of Escherichia coli and Pseudomonas aeruginosa is not sensitive to changes in pH, indicating that the pH effect is significantly different for different strains. Experimental Example 8 proves that changes in pH do not affect the 18±2-hour antibacterial and bactericidal effects. In short, this pH effect is the first discovery of the present invention, has never been reported in the relevant literature on Ceragenins, and cannot be inspired by the prior art, and is non-obvious.

[0043] 3. The present invention found that the 1-min killing effect of this type of compound, in addition to the significant influence of pH mentioned above, the structural differences of the compounds also affect the activity. The results of Experimental Example 5 show that although they are all cholesteric structural analogs, differences in substituent position, number, chirality, etc. will affect the activity. Under the same conditions, compounds 1 and 2 achieved complete killing of Staphylococcus aureus and Candida albicans, and the effect was significantly better than compounds 6 and 8. As mentioned in 1 above, the 1-min killing experiment on Escherichia coli and Pseudomonas aeruginosa also demonstrated the importance of structural differences, and this structure-activity relationship is unpredictable.

[0044] 4. In addition to the pH effect, the present invention also discovered for the first time that the disinfectant co-solvent system has a significant impact on the 1-min killing effect.

[0045] 5. The present invention also found that compounds 1 and 2 can achieve a highly effective disinfection effect on killing spores in a short period of time, meeting the national technical standard for high-efficiency disinfection ("Technical Specifications for Disinfection" (2002 edition)).

[0046] 6. Compounds 1 and 2 of the present invention have higher oral gavage safety than the structural analog compound 8.

[0047] In summary, the new compounds 1, 2, 4 of the present invention and the known compounds 6, 8 and their salts, especially the new compounds 1, 2, 4 and their salts, have broad application prospects in practical disinfection applications.

[0048] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0049] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The scatter plot, fitting curve and curve equation of the logarithmic value of the dose of compound 1 and the probability unit value are shown in FIG.

[0051] Figure 2 The scatter plot, fitting curve and curve equation of the dose logarithm and probability unit value of compound 2 are shown in FIG.

[0052] Figure 3 The scatter plot, fitting curve and curve equation of the dose logarithm and probability unit value of compound 8 are shown in FIG. DETAILED DESCRIPTION

[0053] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0054] The following experiments, where no temperature is specified, are reactions conducted at room temperature, which is 25±5°C.

[0055] Example 1: Preparation of Compound 1 and its salts

[0056] 1. Preparation of Compound 1

[0057] The synthetic route of compound 1 is as follows:

[0058]

[0059]

[0060]

[0061] Step 1: Add 5 g of allochenodeoxycholic acid (from Zhongshan Bailing Biotechnology Co., Ltd., cas: 15357-34-3) to 50 ml of tetrahydrofuran (THF), cool to -10-0°C, slowly add 8 g of sodium hydride, and stir for 2-3 minutes after the addition. Then add 16 g of allyl iodide (cas: 556-56-9), stir for 2-3 minutes after the addition, raise the temperature to 80°C, and reflux for 4-5 hours. Monitor the reaction for completion, cool the reaction solution, slowly pour it into water, and extract it three times with ethyl acetate (EA). The organic phases are combined, concentrated, and then purified by silica gel column chromatography to obtain 5.1 g of an oil, namely, compound B1.

[0062] Compound B1 has the following structure:

[0063]

[0064] Step 2: Add 5 g of the intermediate compound B1 obtained in the previous step, 10 g of O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), 5 g of N,N-diisopropylethylamine (DIPEA), and 2.5 g of octylamine (cas: 111-86-4) to 20 ml of DMF, stir at room temperature for 1-2 hours, add to water, and extract twice with EA. The organic layer is washed with dilute acid and saturated brine, dried and concentrated to obtain a crude product. Silica gel column chromatography is used to obtain 5.7 g of the intermediate, namely compound C1.

[0065] Compound C1 has the following structure:

[0066]

[0067] Step 3: Place 5 g of the intermediate compound C1 obtained in the previous step in a single-necked flask, followed by 25 ml of THF. At low temperature, add 35 ml of a 0.5 M solution of 9-borabicyclo[3.3.1]nonane in THF (9-BBN, cas: 280-64-8) dropwise. After nitrogen replacement, stir at room temperature for 5 hours to obtain reaction solution 1. Add 10 ml of 20% sodium hydroxide to another single-necked flask, cool to -10-0°C, and slowly add 10 ml of 30% hydrogen peroxide. Stir for 2-3 minutes after addition to obtain reaction solution 2. Slowly add reaction solution 1 dropwise to reaction solution 2, controlling the temperature not to exceed 0°C. After addition, reflux for 3-5 hours. Quench the reaction with aqueous sodium bisulfite solution, extract three times with EA, dry and concentrate, and then chromatograph on a silica gel column to obtain 4.1 g of an oil, namely, compound D1.

[0068] Compound D1 has the following structure:

[0069]

[0070] Steps 4 and 5: Dissolve 4 g of the intermediate compound D1 from the previous step in DCM, cool to 0°C, add 2 mL of triethylamine and 1.5 g of methanesulfonic acid chloride, and stir at room temperature for 1-2 hours. Monitor the reaction for completion, concentrate under reduced pressure, mix the residue with water, extract three times with EA, wash twice with aqueous sodium bicarbonate, and dry and concentrate the organic layer. Without purification, dissolve in an appropriate amount of DMF, add 6 g of cesium carbonate and 4.5 g of bis(tert-butyloxycarbonyl)amine (cas: 51779-32-9), and heat at 70-80°C for 3-5 hours. Monitor the reaction for completion, add water, extract three times with EA, wash the organic layer with saturated brine, dry, concentrate, and column chromatography to obtain 3.5 g of the intermediate, namely, compound E1.

[0071] Compound E1 has the following structure:

[0072]

[0073] Steps 6 and 7: Add 20 ml of methanol to 2 g of the intermediate compound E1, cool to 0°C, add 2 ml of acetyl chloride dropwise, stir at room temperature overnight, concentrate to dryness, add 15 ml of THF, cool to -10~0°C, and slowly add 20 ml of borane (BH3, 1 M) in THF solution. After addition, react at 80°C for 4~5 hours, add aqueous sodium bicarbonate solution, extract three times with DCM, and dry and concentrate to obtain 0.9 g of crude compound 1. The crude product was purified by preparative reverse-phase chromatography to obtain an oil with an HPLC purity of >98%, which is the target compound 1.

[0074] NMR data of compound 1:

[0075] 1H NMR (CD3OD, 400 MHz) δ: 3.62(dt, J=9.1, 5.7Hz, 1H), 3.55-3.51(m,1H), 3.48-3.44(m, 2H), 3.21(dt, J=9.1, 5.9Hz, 1H), 2.74(td, J=7.2, 2.7Hz,4H),2.58-2.54(m, 4H), 1.99-1.82((m,2H), 1.80-1.66(m, 5H), 1.64-1.43(m, 14H),1.43-1.16(m, 16H), 1.16-1.04(m,4H), 0.95(d, J=6.4Hz, 3H), 0.93-0.86(m, 4H),0.82(s, 3H), 0.67(s, 3H);

[0076] Mass spectrometry data of compound 1:

[0077] ESI-MS m / z: 604.87[M+1] + .

[0078] 2. Preparation of Compound 1 Hydrochloride

[0079] Compound 1 prepared according to the above preparation method of Compound 1 was dissolved in methyl tert-butyl ether, and hydrochloric acid gas was introduced to precipitate a white solid, which was filtered to obtain the hydrochloride of Compound 1.

[0080] NMR data of compound 1 hydrochloride:

[0081] 1 HNMR(CD3OD, 400MHz)δ: 3.70(dt, J-10.3, 5.5Hz, 1H), 3.59(brs, 1H), 3.56-3.49(m, 2H), 3.40-3.35(m, 1H), 3.29-3.22(m, 2H), 3.10-2.83(m, 8H), 2.00-1.86(m, 6H),1.81-1.64(m, 4H), 1.60-1.43(m, 12H), 1.39-1.29(m, 10H), 1.24-1.06(m, 6H), 0.97(d, J-6.4Hz, 3H), 0.95-0.86(m, 3H), 0.83(s, 3H), 0.69(s, 3H);

[0082] Mass spectrometry data of compound 1 hydrochloride:

[0083] ESI-MS m / z: 604.87[M+1]+ .

[0084] 3. Preparation of Compound 1 Naphthalene Disulfonate

[0085] Compound 1, prepared according to the preparation method of Compound 1, was dissolved in methanol and 2 equivalents of 1,5-naphthalene disulfonic acid were added. A white solid gradually precipitated and was filtered and dried to obtain its naphthalene disulfonic acid salt. NMR data showed that Compound 1 formed a salt with 1.5 molecules of 1,5-naphthalene disulfonic acid.

[0086] NMR data of compound 1 naphthalene disulfonate:

[0087] 1 H NMR (d6-DMSO, 400 MHz) δ: 8.87 (d, J=8.6Hz, 3H), 8.24 (brs, 2H), 7.96 (d, J=7.0Hz, 3H), 7.75-7.70 (m, 6H), 7.44 (dd, J=9.1, 7.0Hz, 3H),3.54-3.44(m,2H), 3.36(t, J=6.1Hz, 2H), 3.26(s,1H),3.18-3.14(m,1H), 2.88-2.78(m,8H), 1.90-1.87(m,1H), 1.82-1.28(m,17H), 1.25-0.96(m,14H),0.90-0.82(m,6H), 0.75(s,3H),0.61(s, 3H);

[0088] Mass spectrometric data of compound 1 naphthalene disulfonate:

[0089] ESI-MS m / z: 604.87[M+1] + ,286.95[M-1] - (1,5-naphthalene disulfonic acid).

[0090] 4. Preparation of Glucuronate, Acetate and Butyrate of Compound 1

[0091] Compound 1 prepared according to the above preparation method of Compound 1 is dissolved in methyl tert-butyl ether, and at least 3 equivalents of glucuronic acid are added. The solid is precipitated under stirring, and the solid is filtered and dried to obtain the glucuronic acid salt of Compound 1.

[0092] ESI-MS m / z: 604.87[M+1] + ,193.05[M-1] - (glucuronic acid).

[0093] By replacing glucuronic acid with acetic acid or butyric acid, the acetate and butyrate of compound 1 can be obtained, respectively.

[0094] Example 2. Preparation of Compound 2 and its salts

[0095] 1. Preparation of Compound 2

[0096] Compound 2 has the following structure:

[0097]

[0098] Compound 2 was prepared by referring to the preparation method of compound 1 in Example 1, except that chenodeoxycholic acid was replaced with commercially available hyodeoxycholic acid (cas: 83-49-8).

[0099] NMR data of compound 2:

[0100] 1 HNMR (CD3OD, 400MHz) δ: 3.65(dt, J=11.8, 4.6Hz, 1H), 3.59-3.50(m, 3H), 3.50-3.40(m, 1H), 3.30-3.18(m, 2H), 2.71(td, J=6.9Hz, 4H), 2.58-2.46(m, 4H),2.05-1.98(m,1H), 1.94-1.78(m, 2H),1.78-1.64((m, 5H), 1.52-1.38(m, 6H), 1.35-1.25(m, 12H), 1.25-1.09(m, 6H), 0.95(d, J=6.4Hz, 3H), 0.92(s, 3H), 0.91-0.87(m, 3H), 0.68(s,3H);

[0101] Mass spectrometry data of compound 2:

[0102] ESI-MS m / z:604.85 [M+1] + ;

[0103] 2. Preparation of Compound 2 Hydrochloride

[0104] Compound 2 prepared according to the preparation method of Compound 2 was dissolved in methyl tert-butyl ether, and hydrochloric acid gas was introduced to precipitate a white solid, which was filtered and dried to obtain the hydrochloride of Compound 2.

[0105] NMR data of compound 2 hydrochloride:

[0106] 1HNMR(CD3OD, 400MHz)δ: 3.74-3.67(m, 1H), 3.66-3.57(m, 3H), 3.54-3.44(m,1H), 3.08-3.00(m, 4H), 3.00-2.90(m, 4H), 2.06-1.99(m, 1H), 1.93-1.66((m,8H),1.50-1.27(m, 17H), 1.23-1.03(m, 6H), 0.98(d, J=6.3Hz, 3H), 0.93(s, 3H), 0.92-0.86(m, 3H), 0.69(s, 3H);

[0107] 3. Preparation of Compound 2 Naphthalene Disulfonate

[0108] Compound 2, prepared according to the preparation method of Compound 2, was dissolved in tetrahydrofuran and 2 equivalents of 1,5-naphthalenedisulfonic acid were added. A white solid gradually precipitated and was filtered and dried to obtain its naphthalenedisulfonic acid salt. NMR data indicated that Compound 2 formed a salt with 1.5 molecules of 1,5-naphthalenedisulfonic acid.

[0109] NMR data of compound 2 naphthalene disulfonate:

[0110] 1 H NMR (CD3OD, 400 MHz) δ: 9.02 (d, J=8.6Hz, 4H), 8.22 (d, J=7.2Hz, 4H), 7.60 (dd, J=8.7, 7.2Hz, 4H), 3.76-3.68(m, 1H), 3.68-3.60(m, 1H), 3.59-3.50(m,3H), 3.49-3.39(m,1H), 3.05-2.86(m, 6H), 1.95-1.75(m, 9H), 1.74-1.62(m, 8H),1.43-1.28(m, 17H), 1.20-0.96(m, 4H),1.34-0.89(m, 7H), 0.89(s, 3H), 0.61(s,3H);

[0111] Mass spectrometry data of compound 2 naphthalene disulfonate:

[0112] ESI-MS m / z: 604.85[M+1] + ,287.12[M-1] - (1,5-naphthalene disulfonic acid).

[0113] 4. Preparation of Glucuronate, Acetate and Butyrate of Compound 2

[0114] Compound 2 prepared according to the preparation method of Compound 2 is dissolved in methyl tert-butyl ether, and at least 3 equivalents of glucuronic acid are added. Solids are precipitated under stirring, and the solids are filtered and dried to obtain the glucuronic acid salt of Compound 2.

[0115] ESI-MS m / z: 604.85[M+1] + ,193.05[M-1] - (glucuronic acid).

[0116] By replacing glucuronic acid with acetic acid or butyric acid, the acetate and butyrate of compound 2 can be obtained, respectively.

[0117] Example 3. Preparation of Compound 4 and its salts

[0118] 1. Preparation of Compound 4

[0119] Compound 4 has the following structure:

[0120]

[0121] Referring to the preparation method of compound 2 in Example 2, the only difference is that octylamine is replaced by laurylamine to obtain compound 4.

[0122] 2. Preparation of Compound 4 Hydrochloride

[0123] Compound 4 prepared according to the preparation method of compound 4 is dissolved in methyl tert-butyl ether, and hydrochloric acid gas is introduced to precipitate a white solid, which is filtered and dried to obtain the hydrochloride of compound 4.

[0124] NMR data of compound 4 hydrochloride:

[0125] 1 H NMR (CD3OD, 400 MHz) δ: 3.66-3.57(m, 1H), 3.56-3.48(m, 3H), 3.44-3.36(m, 1H), 2.992.91(m, 4H), 2.89-2.75(m, 4H), 1.97-1.90(m,1H), 1.86-1.55(m,10H),1.45-1.20(m, 24H), 1.17-0.96(m, 10H), 0.98-0.88(m, 6H), 0.84(s, 3H),0.60(s, 3H);

[0126] Mass spectrometry data of compound 4 hydrochloride:

[0127] ESI-MS m / z: 660.92[M+1] +

[0128] Example 4: High-Level Disinfectant Configuration

[0129] Dissolve 50g of glycerol and 15g of sodium metasilicate pentahydrate in 800g of pure water to obtain an aqueous solution. Dissolve 50g of the compound hydrochloride in the aqueous solution and dilute to 1000ml with pure water to obtain a disinfectant with a concentration of 50mg / ml and a pH of 8.0. This disinfectant can be diluted at a ratio of 1:4 between disinfectant and pure water and can be used for high-level disinfection of medical devices and supplies.

[0130] Dissolve 50g of glycerol and 8-10g of sodium metasilicate pentahydrate in 850g of pure water to obtain an aqueous solution. Dissolve 10g of the compound hydrochloride in the aqueous solution and dilute to 1000ml with pure water to obtain a disinfectant with a concentration of 10mg / ml and a pH of 7.0-9.0. This disinfectant can be directly used for high-level disinfection of medical devices and supplies.

[0131] The hydrochloride of the compound is any one of the hydrochloride of compound 1, compound 2, and compound 4.

[0132] Example 5: Hand disinfectant preparation

[0133] Take 10g of glycerol and 0.3g of sodium metasilicate pentahydrate and dissolve them in 900g of water to obtain a solution. Take 1g of the hydrochloride of compound 1 or compound 2, dissolve it in the above aqueous solution, and dilute the volume to 1000ml with pure water to obtain a disinfectant with a concentration of 1mg / ml and a pH of 8.0. This disinfectant can be used for hand disinfection.

[0134] Example 6: Skin and mucous membrane disinfectant preparation

[0135] Take 10g of glycerol and 0.2-0.3g of sodium metasilicate pentahydrate and dissolve them in 900g of water to obtain a solution, take 0.5g of the hydrochloride of compound 1 or compound 2, dissolve it in the above aqueous solution, and dilute to 1000ml with pure water to obtain a disinfectant with a concentration of 0.5mg / ml and a pH of 7.0-8.0. The disinfectant can be used for skin and mucous membrane disinfection.

[0136] Example 7: Disinfectant preparation for tableware, drinking water, fruits and vegetables

[0137] Dissolve 10g of glycerol and 0.1g of sodium metasilicate pentahydrate in 900g of water to obtain a solution. Dissolve 0.01g of the hydrochloride of Compound 1 or Compound 2 in the above aqueous solution and dilute to 1000ml with pure water to obtain a disinfectant with a concentration of 0.01mg / ml and a pH of 7.2. This disinfectant can be used to disinfect tableware, drinking water, fruits, and vegetables. It can also be added to swimming pool water according to this ratio for disinfection.

[0138] The following is the preparation of the reference compound and its salt.

[0139] Comparative Example 1, Preparation of Compound 3 and its salt

[0140] 1. Preparation of Compound 3

[0141] Compound 3 has the following structure:

[0142]

[0143] Referring to the preparation method of compound 2 in Example 2, the only difference is that octylamine is replaced by propylamine to obtain compound 3.

[0144] 2. Preparation of Compound 3 Hydrochloride

[0145] Compound 3 prepared according to the preparation method of compound 3 is dissolved in methyl tert-butyl ether, and hydrochloric acid gas is introduced to precipitate a white solid, which is filtered and dried to obtain the hydrochloride of compound 3.

[0146] NMR data of compound 3 hydrochloride:

[0147] 1 H NMR (CD3OD, 400 MHz) δ: 3.76-3.68(m, 1H), 3.68-3.60(m, 3H), 3.56-3.48(m, 1H), 3.11-2.91(m, 8H), 2.08-2.01(m, 1H), 1.98-1.70(m, 14H), 1.52-1.43(m, 2H),1.41-1.26(m, 16H), 1.26-1.06(m, 4H), 1.00(d, J=6.4Hz, 3H), 0.96(s,3H), 0.94-0.87(m, 4H), 0.72(s, 3H);

[0148] Mass spectrometry data of compound 3 hydrochloride:

[0149] ESI-MS m / z: 535.23[M+1] +

[0150] Comparative Example 2, Preparation of Compound 5

[0151] 1. Preparation of Compound 5

[0152] Compound 5 has the following structure:

[0153]

[0154] Compound 5 was prepared by referring to the preparation method of compound 1 in Example 1, except that chenodeoxycholic acid was replaced with commercially available ursodeoxycholic acid (cas: 128-13-2).

[0155] 2. Preparation of Compound 5 Hydrochloride

[0156] Compound 5 prepared according to the preparation method of compound 5 is dissolved in methyl tert-butyl ether, and hydrochloric acid gas is introduced to precipitate a white solid, which is filtered and dried to obtain the hydrochloride of compound 5.

[0157] NMR data of compound 5 hydrochloride:

[0158] 1 HNMR (CD3OD, 400MHz) δ: 3.68-3.58(m, 3H), 3.41-3.33(m,1H), 3.27(m,1H), 3.19-3.16(m, 1H), 3.07-2.86(m, 8H), 2.08-2.00(m, 1H), 1.94-1.64(m, 14H),1.54-1.44(m, 8H), 1.44-1.16(m, 14H), 1.16-1.02(m, 2H), 0.99(d, J=6.4Hz, 3H),0.96(s, 3H), 0.94-0.86(m, 4H), 0.70(s, 3H);

[0159] Mass spectrometry data of compound 5 hydrochloride:

[0160] ESI-MS m / z: 605.10[M+1] +

[0161] Comparative Example 3, Preparation of Compound 6

[0162] 1. Preparation of Compound 6

[0163] Compound 6 has the following structure:

[0164]

[0165] Compound 6 was prepared by referring to the preparation method of compound 1 in Example 1, except that chenodeoxycholic acid was replaced with commercially available chenodeoxycholic acid (cas: 474-25-9).

[0166] 2. Preparation of Compound 6 Hydrochloride

[0167] Compound 6 prepared according to the preparation method of compound 6 is dissolved in methyl tert-butyl ether, and hydrochloric acid gas is introduced to precipitate a white solid, which is filtered and dried to obtain the hydrochloride of compound 6.

[0168] NMR data of compound 6 hydrochloride:

[0169] 1 HNMR(CD3OD, 400MHz)δ: 3.78(dt, J=9.9, 5.4Hz, 1H), 3.65-3.56(m, 2H), 3.37-3.32(m, 1H), 3.23-3.13(m, 2H), 3.11-2.88(m, 8H), 2.16-1.97(m,1H),1.97-1.64(m, 7H), 1.64-1.43(m, 3H), 1.39-1.27(m, 12H), 1.24-1.10(m, 4H), 0.98(d, J=6.4Hz, 3H), 0.94(s, 3H), 0.92-0.86(m, 4H), 0.69(s, 3H);

[0170] Mass spectrometry data of compound 6 hydrochloride:

[0171] ESI-MS m / z: 605.10[M+1] +

[0172] Comparative Example 4, Preparation of Compound 7

[0173] 1. Preparation of Compound 7

[0174] Compound 7 has the following structure:

[0175]

[0176] Compound 7 was prepared by referring to the preparation method of compound 1 in Example 1, except that chenodeoxycholic acid was replaced by commercially available lithocholic acid (cas: 434-13-9).

[0177] 2. Preparation of Compound 7 Hydrochloride

[0178] Compound 7 prepared according to the preparation method of compound 7 was dissolved in methyl tert-butyl ether, and hydrochloric acid gas was introduced to precipitate a white solid, which was filtered and dried to obtain the hydrochloride of compound 7.

[0179] NMR data of compound 7 hydrochloride:

[0180] 1HNMR(CD3OD, 400MHz)δ: 3.65-3.55(m, 3H), 3.08-3.00(m, 2H), 3.00-2.86(m,4H), 2.05-1.98(m, 1H), 1.97-1.79(m, 5H), 1.79-1.63(m, 5H), 1.63-1.55(m, 2H), 1.50-1.38(m, 6H), 1.38-1.23(m, 12H), 1.23-1.05(m, 6H), 1.03-0.99(m, 1H), 0.97(d, J=6.5Hz, 3H), 0.94(s, 3H), 0.93-0.86(m, 3H), 0.69(s, 3H);

[0181] Mass spectrometry data of compound 7 hydrochloride:

[0182] ESI-MS m / z: 532.01[M+1] +

[0183] Comparative Example 5: Preparation of Compound 8

[0184] 1. Preparation of Compound 8

[0185] Compound 8 has the following structure:

[0186]

[0187] Compound 8 was prepared by referring to the preparation method of compound 1 in Example 1, except that chenodeoxycholic acid was replaced with commercially available cholic acid (cas: 81-25-4).

[0188] 2. Preparation of Compound 8 Hydrochloride

[0189] Compound 8 prepared according to the above preparation method of Compound 8 was dissolved in methyl tert-butyl ether, and hydrochloric acid gas was introduced to precipitate a white solid, which was filtered and dried to obtain the hydrochloride of Compound 8.

[0190] Mass spectrometry data of compound 8 hydrochloride:

[0191] ESI-MS m / z: 678.03[M+1] +

[0192] HPLC analysis conditions: a HILIC column as the stationary phase, a 60% methanol / 40% 0.1% trifluoroacetic acid aqueous solution as the mobile phase, and a differential refractive index detector. HPLC analysis results showed that the purity of the above compounds was >98%.

[0193] The following experimental examples demonstrate the beneficial effects of the present invention. Similar to the Ceragenin compounds reported in the literature, the non-salt compounds of the present invention are oily, which is not conducive to practical application, while the salt compounds thereof are mostly solid. Therefore, in the experimental examples of the present invention, compounds 1-8 were uniformly prepared and implemented using solid hydrochloride salts.

[0194] Experimental Example 1: In vitro inhibitory activity of the compounds of the present invention against various bacteria (18±2 hours of culture)

[0195] 1. Test methods

[0196] After thawing, strains (Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Escherichia coli were streaked onto MH agar plates for 18-24 hours. Colonies were picked and diluted into MH broth to a McFarland concentration of 0.5. Compounds 1-8 (test compounds) prepared in Examples 1-7 and Comparative Example 1, respectively, were dissolved in appropriate amounts of DMSO and diluted into MH broth. The starting concentration of the test compound was set at 256 μg / ml, and two-fold dilutions were made across a total of 10 concentration gradients. The bacterial solution and MH broth containing the test compound were inoculated at a 1:1 ratio into a 96-well culture plate in a total volume of 200 μl and incubated at 37°C for 16-20 hours. The clear wells (i.e., the wells where the culture medium was clear) were inoculated onto MH agar plates and incubated at 37°C for 24 hours. Because the compound solution is absorbed by the agar, if the bacteria are not killed by the compound, they will grow and form colonies in the absence of compound inhibition. Therefore, the minimum concentration at which no colonies grow can be considered as the minimum bactericidal concentration (MBC) of the compound.

[0197] 2. Test results

[0198] The minimum bactericidal concentration (MBC) values ​​of compounds 1-8 against various bacterial strains are shown in Table 1 below:

[0199] The MBC value of the compound was + when >100 μg / ml, ++ when 10-100 μg / ml, and +++ when <10 μg / ml.

[0200] Table 1 Minimum bactericidal concentrations (MBC) of compounds 1-8 for various bacterial strains

[0201]

[0202] As shown in the 18 ± 2-hour antibacterial assay, compound 8 exhibited strong antibacterial and bactericidal activity, consistent with literature reports. Analysis of MBC concentrations revealed that compounds 1, 2, 4, and 6 were the most active among the structural analogs (i.e., compounds 1-7).

[0203] The present invention investigated the disinfectant activity of compounds 1-8 through subsequent experimental examples. First, the neutralizing agent was determined in accordance with the provisions of the "Technical Specifications for Disinfection (2002 Edition)." Neutralizing agents are used to neutralize residual disinfectant on the surface of microorganisms in a suspension of test microorganisms and disinfectant, thereby eliminating their inhibitory and killing effects on microorganisms. D / E Neutralizing Broth can be used as a neutralizing agent for compounds 1-8.

[0204] Experimental Example 2: 1-min quantitative killing test of the weakly acidic disinfectant compound of the present invention against Escherichia coli and Pseudomonas aeruginosa

[0205] 1. Quantitative killing test of Escherichia coli by the disinfectant of the present invention

[0206] 1. Preparation of Escherichia coli (8099) suspension:

[0207] Take a freeze-dried tube of Escherichia coli, open it under aseptic operation, add an appropriate amount of nutrient broth with a capillary pipette, and gently pipette several times to melt and disperse the bacteria. Take a test tube containing 10.0ml nutrient broth medium, drop a small amount of bacterial suspension into it, and culture it at 37℃ for 18h~24h. Use an inoculation loop to take the bacterial suspension of the first generation culture, streak it on the nutrient agar medium plate, and culture it at 37℃ for 18h~24h. Pick out the typical colonies from the above second generation culture, inoculate them on the nutrient agar slant, and culture it at 37℃ for 18h~24h, which is the third generation culture. Take fresh slant cultures of the nutrient agar medium of the 3rd to 14th generations of the strain (each generation is cultured for 18h to 24h), use a 5.0ml pipette to draw 3.0ml to 5.0ml of diluent (tryptone saline solution, the diluent described in the following dilutions and other operations is the same) and add it to the slant test tube, blow and aspirate repeatedly to wash off the bacterial moss. Subsequently, use a 5.0ml pipette to transfer the washing liquid to another sterile test tube and mix (oscillate) with an electric mixer for 20s to evenly suspend the bacteria. The preliminary bacterial suspension is first roughly measured for its bacterial concentration using the bacterial concentration turbidimetric assay, and then diluted with diluent to about 5×10 8 The concentration of cfu / ml should be kept in a refrigerator at 4℃ for future use. It should not be kept overnight for use on the same day.

[0208] 2. Experimental groups:

[0209] The experiment was divided into a test group with disinfectant added, a control group with disinfectant replaced by diluent, and a blank group without E. coli inoculation. The blank group was used to monitor whether the culture medium was contaminated by other bacteria.

[0210] 3. Preparation of disinfectant:

[0211] Accurately weigh the hydrochlorides of compounds 1-8 prepared according to the methods described in the Examples and Control Examples and directly prepare them with neutral sterile water to a concentration 1.25 times the test concentration, i.e., 1.25 mg / ml. (According to Section 2.1.1.7 of the "Technical Specifications for Disinfection 2002," since the bacterial solution and test solution must be mixed in a 1:4 ratio during the procedure, the test solution should be prepared at 1.25 times the test concentration, resulting in a final concentration of 1.0 mg / ml after dilution. This principle applies to the remaining experimental examples.) The solutions prepared with the hydrochloride salts of the above compounds all showed weak acidity, as measured by a pH meter, with a pH range of 4.5-6.5. The prepared disinfectant was placed in a water bath at 20°C ± 1°C until ready for use.

[0212] 4. Test operation:

[0213] Take the E. coli suspension obtained in step 1, the concentration of which is about 5×10 8 cfu / ml. For the disinfection test, add 0.5ml of the E. coli suspension to a sterile large test tube, followed by 0.5ml of an organic interfering substance (3% BSA solution). Mix thoroughly and place in a 20°C ± 1°C water bath for 5 minutes. Then, use a sterile pipette to pipette 4.0ml of the disinfectant obtained in step 3 into the test tube, mix rapidly, and immediately begin counting. Allow the E. coli and disinfectant to interact for 1 minute. Then, pipette 0.5ml of the E. coli-disinfectant mixture into 4.5ml of sterilized neutralizer and mix thoroughly. After the E. coli-disinfectant mixture has been incubated with the neutralizer for 10 minutes, a 1.0ml sample is drawn and the number of viable cells is determined using the viable cell culture method. Two plates are inoculated with each sample. If a large number of colonies grow on the plates, perform a 10-fold serial dilution and then perform a viable cell culture count. Simultaneously, perform a parallel experiment using the diluent instead of the disinfectant as a control group, and a parallel experiment using the diluent instead of the bacterial suspension as a blank group. All test samples were cultured in a 37°C incubator for 48 hours, and the final results were observed. The test was repeated three times, and the viable bacterial concentration (cfu / ml) of each group was calculated and converted into a logarithmic value (N). The logarithmic killing value was then calculated according to the following formula:

[0214] Killing logarithm (KL) = logarithm of the average viable bacterial concentration in the control group (No) - logarithm of the viable bacterial concentration in the test group (Nx)

[0215] Killing rate = average live bacteria concentration of the control group - live bacteria concentration of the experimental group / average live bacteria concentration of the control group * 100%

[0216] Table 2 The killing effect of compound weak acid disinfectant on Escherichia coli in 1 minute

[0217]

[0218] 2. Quantitative Killing Test of Pseudomonas aeruginosa by the Disinfectant of the Present Invention

[0219] The quantitative killing effect of Pseudomonas aeruginosa (ATCC15442) was tested by referring to the quantitative killing test method of Escherichia coli by the above disinfectant, with the only difference being that Escherichia coli was replaced by Pseudomonas aeruginosa.

[0220] Table 3 Compound weak acid disinfectant killing effect on Pseudomonas aeruginosa 1min

[0221]

[0222] In summary, the test results are shown in Table 2-3. Under the same conditions, compounds 3, 5, and 7 did not meet the acceptable limits for killing Escherichia coli and Pseudomonas aeruginosa, with compound 7 having the worst bactericidal effect. The remaining compounds (i.e., compounds 1, 2, 4, 6, and 8) all met the acceptable limits.

[0223] Experimental Example 3: 1-min quantitative killing test of the weakly acidic disinfectant compound of the present invention against Staphylococcus aureus and Candida albicans

[0224] 1. Quantitative killing test of Staphylococcus aureus

[0225] 1. Preparation of Staphylococcus aureus (ATCC6538) suspension:

[0226] The preparation method of Staphylococcus aureus suspension refers to the preparation method of Escherichia coli suspension in Experimental Example 2.

[0227] 2. Preparation of disinfectant:

[0228] Compounds 1-8 hydrochlorides were prepared directly with sterile water to a concentration 1.25 times the intended test concentration, i.e., 12.5 mg / ml (the intended test concentration, i.e., the final concentration after dilution with the bacterial solution, is 10 mg / ml). The pH range was 4.5-6.5. The prepared disinfectant was placed in a water bath at 20°C ± 1°C until ready for use.

[0229] The experimental grouping and experimental operation were the same as those described in Experimental Example 2. The results are shown in Table 4:

[0230] Table 4 Killing effect of compound weak acid disinfectant on Staphylococcus aureus in 1 min

[0231]

[0232] 2. Quantitative killing test of Candida albicans

[0233] 1. Preparation of Candida albicans (ATCC10231) suspension:

[0234] Take a tube of freeze-dried bacterial culture and aseptically open it. Use a capillary pipette to add an appropriate amount of Sandcastle liquid culture medium to the tube. Gently pipette several times to dissolve and disperse the culture. Add a small amount of the bacterial suspension to a test tube containing 10.0 ml of Sandcastle liquid culture medium and incubate at 37°C for 18–24 hours. Use an inoculating loop to take a sample of the bacterial suspension from the first-generation culture and streak it onto a Sandcastle agar plate. Incubate at 37°C for 18–24 hours. Select a representative colony from the second-generation culture and inoculate it onto a Sandcastle agar slant. Incubate at 37°C for 18–24 hours to create the third-generation culture. Seal the culture and store at 4°C for no more than 6 weeks. During testing, take the third-generation slant culture and serially passage it on Sandcastle agar slant using the same method as for the third-generation culture. Take a fresh slant culture (18-24 hours old) on a 6-passage Sabouraud agar medium and pipette 3-5 ml of diluent into the slant test tube. Repeatedly pipette and aspirate to wash off the bacterial moss. Then, pipette the wash solution into another sterile test tube using a 5-ml pipette and mix with an electric mixer for 20 seconds to evenly suspend the Candida albicans. Dilute with diluent to a bacterial count of approximately 5 × 10 8 cfu / ml, refrigerate at 4℃ for future use and do not store overnight for use on the same day.

[0235] 2. Preparation of disinfectant:

[0236] Compounds 1-8 hydrochlorides were prepared directly with neutral sterile water at a concentration 1.25 times the intended test concentration, i.e., 12.5 mg / ml (the intended test concentration, i.e., the final concentration after dilution with the bacterial solution, is 10 mg / ml). The pH range was 4.5-6.5. Compound 2 hydrochloride had a pH of 5.5. The prepared disinfectant was placed in a water bath at 20°C ± 1°C until ready for use.

[0237] 3. Test operation:

[0238] Take the Candida albicans suspension obtained in step 1, the concentration of which is about 5×10 8cfu / ml. In a sterile large test tube, add 0.5 ml of the Candida albicans suspension, followed by 0.5 ml of an organic interfering substance (3% BSA solution). Mix thoroughly. Place in a 20°C ± 1°C water bath for 5 minutes. Then, using a sterile pipette, pipette 4.0 ml of the disinfectant obtained in step 2 into the test tube, mix rapidly, and immediately begin counting. Allow the test bacteria (i.e., Candida albicans) to interact with the disinfectant for 1 minute. Then, pipette 0.5 ml of the test bacteria and disinfectant mixture into 4.5 ml of sterilized neutralizer and mix thoroughly. After the neutralizer has been added for 10 minutes, pipette 1.0 ml of each sample from each tube and determine the number of viable cells using the viable cell culture method. Inoculate two plates of each sample using Sabouraud dextrose agar. If a large number of colonies grow on the plates, perform a series of 10-fold dilutions before performing viable cell culture counts. Parallel experiments were conducted using the diluent instead of the disinfectant as a control group, and the diluent instead of the bacterial solution as a blank group. All test samples were incubated in a 37°C incubator, and propagules were cultured for 48 hours and the final results were observed. The experiment was repeated three times, and the viable bacterial concentration (cfu / ml) was calculated for each group and converted to a logarithmic value (N). The logarithmic kill value was then calculated using the following formula:

[0239] Killing logarithm (KL) = logarithm of the average viable bacterial concentration in the control group (No) - logarithm of the viable bacterial concentration in the test group (Nx)

[0240] Killing rate = average live bacteria concentration of the control group - live bacteria concentration of the experimental group / average live bacteria concentration of the control group * 100%

[0241] The average log kill and average killing rate are the average of three replicates.

[0242] Table 5 Killing effect of compound weak acid disinfectant on Candida albicans in 1 min

[0243]

[0244] The results are shown in Tables 4-5. When the pH value of the disinfectant is weakly acidic, the above-mentioned compounds 1-8, even if the concentration is prepared at 10 mg / ml, do not show a qualified killing effect against Staphylococcus aureus and Candida albicans; under the same conditions, the bactericidal activity of each compound shows great differences, among which compounds 1 and 2 have the best killing effect.

[0245] Considering the aforementioned failures, the concentration of compounds 6 and 8, previously reported in the literature, was increased from 10 mg / ml to 20 mg / ml and retested for their 1-minute killing efficacy against Candida albicans. The preparation protocol used neutral sterile water as previously described, and the disinfectants prepared with the hydrochloride salts of compounds 6 and 8 were both weakly acidic.

[0246] Table 6 Killing effect of compound 6 and 8 weak acid disinfectant on Candida albicans in 1 min at increasing concentrations

[0247]

[0248] The results, shown in Table 6, show that even at a concentration increase from 10 mg / ml to 20 mg / ml, the reported Ceragenins 6 and 8 still failed to meet the 1-minute killing efficiency against Candida albicans. Furthermore, a two-fold increase in concentration did not significantly improve the killing rate or logarithmic killing value. The 20 mg / ml concentration was already well above the aforementioned MBC values ​​(for example, the MBC values ​​for compounds 6 and 8 were both <0.1 mg / ml in the 18 ± 2 hour inhibition test described in Experimental Example 1).

[0249] The present invention first discovered that the pH condition of the disinfectant preparation is extremely critical for the 1-minute killing effect on Staphylococcus aureus and Candida albicans. The following experimental example 4 uses compound 2 as an example to systematically study the killing effect of disinfectants prepared at different pH values.

[0250] Experimental Example 4: 1-min quantitative killing test of compound 2 against Candida albicans / Staphylococcus aureus under different pH conditions

[0251] 1. The preparation of Candida albicans suspension was the same as that described in Experimental Example 3.

[0252] 2. Disinfectant Preparation: Prepare a phosphate buffer system containing 5% DMSO to a final pH of 5.0, 7.0, and 9.0. Dissolve an appropriate amount of compound 2 in each of the three phosphate buffer solutions. Monitor the pH using a pH meter and adjust the pH to the indicated values. The compound concentration is 12.5 mg / ml, 1.25 times the test concentration.

[0253] According to the operating method described in Experimental Example 3, the killing effect of the disinfectant containing Compound 2 as the active ingredient at a test concentration of 10 mg / ml on Candida albicans and Staphylococcus aureus under the above three pH conditions was tested, and the average value was taken after three repetitions.

[0254] Table 7 Killing effect of compound 2 at different pH on Candida albicans / Staphylococcus aureus in 1 min

[0255]

[0256] The results, as shown in Table 7, show that pH significantly affects the 1-minute killing effect of disinfectants containing Compound 2 of the present invention as the active ingredient against Candida albicans and Staphylococcus aureus. At a concentration of 10 mg / ml, the pH 5.0 system failed to achieve a satisfactory killing effect, but as the pH increased, the log kill rate significantly increased. At pH values ​​of 7.0 and 9.0, the disinfectant at a concentration of 10 mg / ml achieved a satisfactory killing effect, and the log kill value at pH 9.0 was significantly higher than that at pH 7.0.

[0257] The 10 mg / ml compound 2 disinfectant at different pH values ​​was used to test the killing of Escherichia coli and Pseudomonas aeruginosa in 1 minute. It was found that pH had little effect on the killing of Escherichia coli and Pseudomonas aeruginosa. At a concentration of 10 mg / ml, 100% killing effect was achieved regardless of the pH value of 5.0, 7.0, or 9.0. In addition, concentration dilution tests showed that at pH values ​​of 5.0, 7.0, or 9.0, concentrations as low as 0.02 mg / ml of compound 1 and compound 2 were able to kill Escherichia coli and Pseudomonas aeruginosa in 1 minute.

[0258] The results show that the pH of the disinfectant solution of the compound of the present invention is very important for the 1-min killing of Staphylococcus aureus and Candida albicans, but has little effect on Escherichia coli and Pseudomonas aeruginosa.

[0259] Experimental Example 5: 1-min quantitative killing test of the compounds of the present invention against Staphylococcus aureus and Candida albicans at pH 9.0

[0260] 1. The preparation of Staphylococcus aureus suspension and Candida albicans suspension was the same as that in Experimental Example 3.

[0261] 2. After studying Compound 2 and discovering its pH effect against Candida albicans in Experimental Example 4, this experiment further compared the activity of Compounds 1, 2, 4, 6, and 8 under the same pH conditions. Compounds 3, 5, and 7 exhibited significantly weaker killing activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans, failing to meet acceptable standards and therefore lacking meaningful comparison. Disinfectant preparation was the same as in Experimental Example 4.

[0262] According to the operating method described in Experimental Example 4, the killing effects of the disinfectants of the above compounds on Staphylococcus aureus and Candida albicans at pH 9.0 were compared.

[0263] Table 8 Killing effect of compounds on Staphylococcus aureus at pH 9.0 for 1 min

[0264]

[0265] Table 9 Killing effect of compounds on Candida albicans at pH 9.0 for 1 min

[0266]

[0267] The results are shown in Tables 8-9: Compounds 1, 2, and 4 all achieved acceptable killing of Candida albicans and Staphylococcus aureus within 1 minute at a concentration of 10 mg / ml and pH 9.0. Comparison of surviving colony counts and log kill rates revealed that Compounds 1 and 2 demonstrated significant killing effects against S. aureus and C. albicans, with observable colony counts reaching 0, indicating complete killing of both S. aureus and C. albicans. However, Compound 4 still exhibited a small number of colonies.

[0268] Compared to the results in Table 5 of Experimental Example 3, the 10 mg / ml concentrations of compounds 6 and 8 significantly improved their 1-min killing activity against Candida albicans by adjusting the pH from weakly acidic to 9.0. Compound 8 still showed a small number of colonies, but achieved the acceptable killing logarithm; compound 6 was close to the acceptable level.

[0269] At pH 9.0, compounds 1 and 2 were significantly more effective than compounds 6 and 8 in killing Staphylococcus aureus and Candida albicans, while compounds 4 and 8 were comparable in activity.

[0270] Experimental Example 6: 1-min quantitative killing test of disinfectants with different formulations of the present invention against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans

[0271] The above-mentioned compound 1-8 hydrochloride is easily soluble in aqueous solution, but it is easy to precipitate when the pH is adjusted to neutral or alkaline. Experimental Examples 3, 4, and 5 all used DMSO solubilization and phosphate to adjust the pH condition to study the pH effect, but DMSO solubilization is rarely used in the actual application of disinfectants. The present invention selects three co-solvent systems: hydroxypropyl-β-cyclodextrin, glycerol, and EDTA tetrasodium salt for comparison in accordance with the co-solvents listed in the national standard "GB38850-2020".

[0272] Preparation of disinfectant: Sodium metasilicate pentahydrate was used to adjust the pH to 8.0. Compounds 1 and 2 were prepared using three systems, namely 5% glycerol aqueous solution, 5% hydroxypropyl-β-cyclodextrin aqueous solution, and 0.01% tetrasodium EDTA aqueous solution, to a concentration of 0.5 mg / ml for a 1-min killing test.

[0273] The killing test was performed the same as before. For Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, an average kill logarithm of ≥5.00 over three times was considered acceptable, while a kill logarithm <5.00 was considered unacceptable. For Candida albicans, an average kill logarithm of ≥4.00 over three times was considered acceptable, while a kill logarithm <4.00 was considered unacceptable. The test results are shown in Table 10 below:

[0274] Table 10 Killing effects of compounds 1 and 2 in three different formulations at pH 8.0 for 1 min

[0275]

[0276] The results showed that the cosolvent system also affected disinfection activity. Of the three systems, compounds 1 and 2 achieved acceptable killing activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans within 1 minute, only in the glycerol / sodium metasilicate pentahydrate system at a concentration as low as 0.5 mg / ml. Similarly, at this low concentration, the hydroxypropyl-β-cyclodextrin system failed to achieve acceptable killing activity against all bacteria and fungi. The tetrasodium EDTA system achieved acceptable killing activity only against Escherichia coli, Pseudomonas aeruginosa, and Candida albicans, but not against Staphylococcus aureus. The specific mechanism by which these cosolvents lead to these differences in activity remains unclear.

[0277] Experimental Example 7: Quantitative killing test of compound 2 against Candida albicans and Staphylococcus aureus in 1 min under different concentrations and pH values

[0278] This experiment further explored the effectiveness of Compound 2 against Candida albicans and Staphylococcus aureus at different concentrations and pH values ​​using glycerol as a cosolvent and sodium metasilicate pentahydrate as a pH adjuster. The experimental procedure was the same as in Experimental Example 3. The average of three replicates was used as the standard, with a logarithmic kill value of ≥4.00 for Candida albicans and ≥5.00 for Staphylococcus aureus considered acceptable.

[0279] Table 11 Killing results of compound 2 against Candida albicans at different pH / concentrations for 1 min

[0280]

[0281] Table 12 Killing results of compound 2 against Staphylococcus aureus at different pH / concentrations for 1 min

[0282]

[0283] The results are shown in Tables 11-12, which once again prove that pH seriously affects the killing effect.

[0284] Against Candida albicans, even at a concentration of 12.5 mg / ml, Compound 2 failed to meet the acceptable standards at pH 6.5. At a neutral pH of 7.0, the lowest killing concentration was 2.5 mg / ml. At pH 7.5, 8.0, 8.5, and 9.0, the bactericidal effect was comparable, with the lowest killing concentration being 0.1 mg / ml. A pH ≥ 7 is a critical parameter for the killing system using Compound 2 as the active ingredient.

[0285] Against Staphylococcus aureus, compound 2 failed to meet the acceptable sterilization standards at a pH of 6.5, even at a concentration of 12.5 mg / ml. At a neutral pH of 7.0, the lowest sterilization concentration was 0.5 mg / ml. At pHs of 8.0, 8.5, and 9.0, the sterilization effect was comparable, with the lowest sterilization concentration being 0.1 mg / ml. The study found that further increasing the pH to 11 resulted in the compound being equally effective against Candida albicans or Staphylococcus aureus within 1 minute.

[0286] The results showed that both the concentration and pH of the compound would affect the killing effect, but pH ≥ 7 was an extremely important condition to ensure that the disinfectant could kill qualified bacteria.

[0287] Experimental Example 8: Antibacterial activity test of compound 8 under different pH conditions (18±2 hours)

[0288] This experiment uses compound 8 reported in the literature as an example to study whether pH affects the antibacterial or bactericidal effect of Ceragenin compounds for 18±2 hours.

[0289] The experimental method was similar to that of Experimental Example 1. After incubation of Candida albicans with Compound 8 at various pH conditions for 18 ± 2 hours, the minimum bactericidal concentration (MBC) was determined. Compound 8 at varying concentration gradients and pH values ​​was prepared using a glycerol / sodium metasilicate pentahydrate system (adjusting the pH to an acidic state with a small amount of dilute hydrochloric acid).

[0290] MBC values ​​>100µg / ml were considered +, 10-100µg / ml were considered ++, and <10µg / ml were considered +++.

[0291] Table 13 Candida albicans killing results of compound 8 under different pH conditions for 18±2 hours

[0292]

[0293] The results shown in Table 13 indicate that when the compound is incubated with the bacterial solution for a sufficient period of 18 ± 2 hours, the effect of pH on MBC is not significant. This suggests that pH influences the compound's short-term killing rate. Once the compound and bacterial solution are incubated for a sufficient period of time, the killing effect at different pH levels converges. However, as previously mentioned, the killing time is crucial for disinfectant applications, and the influence of pH is particularly significant.

[0294] Experimental Example 9: Bacillus subtilis var. niger spore killing test

[0295] 1. Preparation of Bacillus subtilis var. niger spore suspension (ATCC9372):

[0296] Take the freeze-dried bacterial culture tube, open it under sterile operation, add an appropriate amount of nutrient broth culture medium with a capillary pipette, and gently pipette several times to melt and disperse the bacteria. Take a test tube containing 10ml nutrient broth culture medium, drop a small amount of bacterial suspension into it, and culture it at 37℃ for 18h~24h. Use an inoculation loop to take the bacterial suspension of the first generation culture, streak it on the nutrient agar medium plate, and culture it at 37℃ for 18h~24h. Pick out the typical colonies in the above second generation culture, inoculate it into nutrient broth culture medium, and culture it at 37℃ for 18h~24h, which is the third generation culture. Use a 10.0ml pipette to draw 5.0-10.0ml of the 18-24h-old nutrient broth culture from the third to fifth passages and inoculate it onto the surface of the nutrient agar in a Roche flask. Shake the broth until the entire surface is covered. Remove any excess broth and place the flask in a 37°C incubator for 5-7 days. Use an inoculating loop to smear a small amount of the bacteria onto a glass slide. Fix the sample using a modified spore stain method and examine it under an oil immersion microscope. When the sporulation rate reaches 95% or higher, proceed to the following treatments. Otherwise, continue to stand at room temperature for a period of time until the sporulation rate reaches the specified value before proceeding to the following treatments.

[0297] Use a 10.0ml pipette to add 10.0ml of sterile distilled water to each Roche flask. Gently scrape the bacterial moss with an L-shaped rod. Aspirate the first wash of the bacterial suspension, then add 5.0ml of sterile distilled water to the flask and repeat the wash. Combine the first and second washes of the bacterial suspension in a sterile Erlenmeyer flask containing glass beads and shake for 5 minutes to break up the bacterial clumps and create a uniform spore suspension. If necessary, place the Erlenmeyer flask containing the bacterial suspension in a 45°C water bath for 24 hours to allow the bacteria to autolyze and break down into individual spores. Filter the spore suspension through sterile cotton or gauze to remove any agar clots. Place the filtered spore suspension into a sterile centrifuge tube and centrifuge at 3000 rpm for 30 minutes. Discard the supernatant and resuspend the spores by pipetting in distilled water. Repeat the centrifugation and resuspending three times. Suspend the cleaned spores in distilled water in a conical flask and add an appropriate amount of small glass beads. Place the spore solution in an 80°C water bath for 10 minutes (or 60°C for 30 minutes) to kill any remaining bacterial vegetative cells. After cooling to room temperature, store in a refrigerator at 4°C until ready for use. The effective shelf life is six months.

[0298] 2. Experimental groups:

[0299] The experiment was divided into a test group with disinfectant added, a control group with disinfectant replaced by diluent, and a blank group without inoculation of Bacillus subtilis var. niger spores.

[0300] 3. Disinfectant configuration:

[0301] The disinfectant adopts a 5% glycerol / sodium metasilicate pentahydrate aqueous solution system (i.e., a system obtained by adding sodium metasilicate pentahydrate to an aqueous solution containing 5% by mass of glycerol and adjusting it to the target pH value), with a pH value of 8.0. The compounds are compounds 1 and 2 of the present invention, and the test concentration is 1.25 times, i.e., 12.5 mg / ml. The killing effect is tested under the conditions of 15 min, 30 min, and 45 min, respectively.

[0302] 4. Experimental operation: refer to Experimental Example 2 for the operation.

[0303] 5. Test results:

[0304] Table 14 Killing effect of the compounds of the present invention on Bacillus subtilis var. niger spores within 45 minutes

[0305]

[0306] The results are shown in Table 14. The logarithmic killing values ​​of the disinfectants of compounds 1 and 2 against Bacillus subtilis var. niger spores were ≥5.00 when the disinfectants were applied for 15 min, 30 min, and 45 min, respectively, meeting the requirements of the technical specifications for efficient disinfection.

[0307] Experimental Example 10, LD of Compounds 1, 2 and 8 of the present invention 50 Detection test

[0308] The LD values ​​of compound 1, compound 2 and compound 8 were tested according to the method described in item 2.3.1 of the Disinfection Technical Specifications 2002. 50 value.

[0309] 1. Experimental Animals:

[0310] 180 Balb / c mice, half male and half female, 6 weeks old, weighing 18-22 g, were randomly divided into 18 groups after one week of adaptive feeding. These mice were given 6 doses of Compound 1, Compound 2, and Compound 8, at doses of 200 mg / kg, 250 mg / ml, 300 mg / kg, 350 mg / kg, 400 mg / kg, and 450 mg / kg, respectively.

[0311] 2. Test operation:

[0312] Before the test, each group of animals was fasted overnight but not allowed to drink water. The test compound was dissolved in 5% hydroxypropyl-β-cyclodextrin aqueous solution and prepared into 6 concentrations of 40mg / ml, 50mg / ml, 60mg / ml, 70mg / ml, 80mg / ml and 90mg / ml, corresponding to 6 dosage groups. Each mouse was given a dose of 50μl / 10g body weight by oral gavage. After the administration of the compound, the animals in each group were raised normally and observed for 14 days. During this period, the signs of animal poisoning, the number of deaths and the time of death were observed and recorded. All remaining animals were killed after the end of the test. Animals that died during the observation period and animals that were finally killed were immediately dissected and observed with the naked eye to see if there were any abnormal organs or tissues. The LD values ​​of compound 1, compound 2 and compound 8 were also calculated. 50 .

[0313] The test results are shown in Table 15 below:

[0314] Table 15 Results of each group of experimental animals after administration

[0315]

[0316] Calculation of the LD values ​​of the three compounds using the probit-log plot method 50 Based on the mortality rate of each group of animals, the probability unit of each group was found in Table 16. The probability units for mortality rates of 0% and 100% were found in Table 17. The mortality rates and corresponding probability indices for different doses of the three compounds are shown in Table 18 below.

[0317] Table 16 Percentage-Probability Unit Conversion Table

[0318]

[0319] Note: The horizontal numbers are the units digit of the mortality rate, and the vertical numbers are the tens digit of the mortality rate.

[0320] Table 17 Probability units for response rates of 0% and 100%

[0321]

[0322] Table 18 Mortality and corresponding probability index at different doses of compounds 1, 2, and 8

[0323]

[0324] Draw a scatter plot based on the results in Table 18, with the horizontal axis representing the logarithm of the dose (X) and the vertical axis representing the probability unit value (Y), and fit a straight line. Take the logarithm of the dose corresponding to the probability unit of 5 on the straight line, and its antilog is the LD 50According to the "Technical Specifications for Disinfection 2002," the mortality rate of animals in the highest dose group should generally be ≥90%, and the mortality rate in the lowest dose group should be ≤10%. Therefore, the data for the 200 mg / kg group of Compound 1, the 200 mg / kg group of Compound 2, and the 450 mg / kg group of Compound 8 in Table 18 do not meet these requirements and are not included in the scatter plot. mg / kg is equivalent to mpk.

[0325] The dose-probability unit scatter plots for compound 1, compound 2, and compound 8 are shown in Figure 2. Figure 1 、 Figure 2 、 Figure 3 As shown. The calculated LD of compound 1 50 The value was 338.4 mg / kg, and the LD of compound 2 50 The value was 363.3 mg / kg, and the LD of compound 8 50 The value is 305.1mg / kg.

[0326] The test results showed that the LD of compound 1 and compound 2 50 The value is greater than the LD of compound 8 50 Value, and from the test compound LD 50 It can also be observed in the experiment with the same dose that the toxicity of compound 1 and compound 2 is less than that of compound 8. This shows that the safety of compound 1 and compound 2 is higher than that of compound 8, especially compound 2, which has higher safety and lower toxicity.

[0327] In summary, the compounds of the present invention can achieve qualified killing of Escherichia coli and Pseudomonas aeruginosa, and can achieve qualified killing effects against Staphylococcus aureus and Candida albicans in 1 minute under neutral and alkaline environments. The present invention also discovered for the first time that compounds 1 and 2 can achieve efficient disinfection effects against spores in a short period of time, meeting the national technical standards for efficient disinfection. Compared with the prior art compound 8, compounds 1 and 2 of the present invention have higher oral gavage safety. Therefore, the compounds of the present invention and their salts have broad application prospects in practical disinfection applications.

Claims

1. A cholesteric compound or a salt thereof, characterized in that The cholesteric compound has a structure as shown in Formula I, Formula II or Formula III: （I） (II) (III).

2. The cholesteric compound or salt thereof according to claim 1, characterized in that The salt is hydrochloride, glucuronide, acetate, butyrate or 1,5-naphthalene disulfonate.

3. A method for preparing the cholesteric compound according to claim 1, characterized in that: The method comprises the following steps: (1) Bile acid compounds react with halogenated olefins under alkaline conditions to obtain intermediate 1; (2) Intermediate 1 reacts with amine to obtain intermediate 2; (3) Intermediate 2 undergoes hydroboration-oxidation reaction to obtain intermediate 3; (4) Intermediate 3 first reacts with a sulfonylating agent to form a sulfonate, which then reacts with a nucleophile under alkaline conditions to obtain intermediate 4; (5) Intermediate 4 is hydrolyzed under acidic conditions and reacts with a reducing agent to obtain a cholesteric compound; Among them, when the bile acid compound is allochenodeoxycholic acid, intermediate 1 is When the amine is octylamine, the intermediate 2 is , intermediate 3 is , intermediate 4 is , cholesteric compounds are ; When the bile acid compound is hyodeoxycholic acid, intermediate 1 is When the amine is octylamine, the intermediate 2 is , intermediate 3 is , intermediate 4 is , cholesteric compounds are ; When the bile acid compound is hyodeoxycholic acid, intermediate 1 is When the amine is laurylamine, intermediate 2 is , intermediate 3 is , intermediate 4 is , cholesteric compounds are .

4. The method according to claim 3, characterized in that The bile acid compound in step (1) is allochenodeoxycholic acid or hyodeoxycholic acid; The amine in step (2) is octylamine or laurylamine; The hydroboration-oxidation reagent in step (3) is 9-borabicyclo[3.3.1]nonane and H2O2; In step (4), the sulfonylating agent is methanesulfonic acid chloride, and the nucleophilic agent is bis(tert-butyloxycarbonyl)amine; The reducing agent in step (5) is borane.

5. A disinfectant, characterized in that The disinfectant is a preparation prepared with the cholesteric compound or its salt according to claim 1 as an active ingredient, and the pH of the disinfectant is ≥7.

0.

6. The disinfectant according to claim 5, characterized in that The disinfectant further comprises a solvent, a co-solvent acceptable to the disinfectant and a pH adjuster.

7. The disinfectant according to claim 6, characterized in that The solvent is water, the cosolvent is glycerol, and the pH regulator is at least one of tetrasodium ethylenediaminetetraacetic acid, sodium metasilicate pentahydrate, sodium phosphate, and sodium carbonate.

8. Use of the compound or salt thereof according to claim 1 or the disinfectant according to any one of claims 5 to 7 in the preparation of a disinfectant product for disinfecting mucous membranes and skin, hands, feet, medical devices and supplies, fabrics, tableware, drinking water and swimming pool water, and fruits and vegetables.

9. Use of the compound or salt thereof according to claim 1 or the disinfectant according to any one of claims 5 to 7 in the preparation of a disinfectant product for killing spores of at least one of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Bacillus subtilis var. niger.

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