Allocholic acid derivative with antibacterial activity and pharmaceutical composition thereof

Abstract

The present invention provides an allocholic acid derivative having antibacterial activity and a pharmaceutical composition thereof, belonging to the field of medical technology. The allocholic acid derivative is a compound represented by formula (I), or a stereoisomer thereof, or a salt thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof. This type of compound can effectively inhibit the growth of Clostridium difficile bacteria, has significant antibacterial activity, and has very good application prospects in the preparation of drugs for preventing and / or treating Clostridium difficile infectious diseases, recurrence of Clostridium difficile infectious diseases, or complications of Clostridium difficile infectious diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to an allocholic acid derivative with antibacterial activity and a pharmaceutical composition thereof. Background Art

[0002] Clostridium difficile is an obligate anaerobic bacterium of the genus Clostridium. It is very sensitive to oxygen and difficult to isolate and culture, hence its name. It generally parasitizes in the human intestine. Clostridium difficile infection is usually caused by excessive use of certain antibiotics, which disrupts the balance of intestinal flora, accelerates the growth rate of Clostridium difficile flora, and causes inflammation. Clostridium difficile produces exotoxins A and B, which have different effects at different times. Toxin A is an enterotoxin that first binds to mucosal cells in the early stage, causing primary damage, which can cause inflammation of the intestinal wall, cell infiltration, increased intestinal wall permeability, bleeding and necrosis. Toxin B is a cytotoxin that damages the cytoskeleton, causing cell shrinkage and necrosis, and directly damages intestinal wall cells, leading to diarrhea.

[0003] Clostridium difficile infection is a disease caused by infection with Clostridium difficile bacteria and / or Clostridium difficile spores. Pseudomembranous colitis is a common Clostridium difficile infection, with clinical manifestations of diarrhea, abdominal pain, and systemic toxicity. Symptoms begin suddenly and are accompanied by low blood pressure, often accompanied by fever and leukocytosis, and can later lead to death. It is a very serious disease. In addition, infection with Clostridium difficile bacteria and / or Clostridium difficile spores may also cause complications of Clostridium difficile infection. Common complications include pyelonephritis, meningitis, abdominal and vaginal infections, bacteremia, and gas gangrene. In recent years, Clostridium difficile has become an important pathogen causing nosocomial infectious diseases and is receiving increasing attention.

[0004] The current mainstay of treatment for Clostridium difficile infections is the use of antibacterial agents. Metronidazole and vancomycin are the two most commonly used antibacterial agents. However, patients with Clostridium difficile infections treated with metronidazole and vancomycin have a high rate of relapse. Given that bacterial resistance under evolutionary pressure will eventually become a major treatment challenge, and given the emergence of difficult-to-treat strains resistant to metronidazole and vancomycin, the development of new, highly active antibacterial agents against Clostridium difficile is urgent.

[0005] Bile acids are a class of structurally similar tetracyclic fused-ring steroidal compounds containing 23-25 ​​carbon atoms, belonging to the steroid acid class. Among them, allo-bile acids are bile acid derivatives with the 5-hydrogen in the alpha configuration. They are widely present in mammals, but due to their relatively low abundance compared to the beta stereoisomer, research on their physiological functions is limited. Summary of the Invention

[0006] The object of the present invention is to provide an allocholic acid derivative having antibacterial activity and a pharmaceutical composition thereof, wherein the allocholic acid derivative and the pharmaceutical composition thereof have high inhibitory activity against Clostridium difficile bacteria.

[0007] The present invention provides a compound represented by formula (I), or a stereoisomer thereof, or a salt thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof:

[0008]

[0009] in,

[0010] n = 0, 1, 2;

[0011] represents a single bond or a double bond, Each independently selected from -H, -OH, -NH2, =O, =NOH, C 1-5 alkyl, -OSO3H or a salt thereof, -OPO3H2 or a salt thereof.

[0012] Furthermore, when n=1, the compound has a structure represented by the following formula (II):

[0013]

[0014] in,

[0015] represents a single bond or a double bond, Each independently selected from -H, -OH, -NH2, =O, =NOH, C 1-5 alkyl, -OSO3H or a salt thereof, -OPO3H2 or a salt thereof.

[0016] Furthermore, when n=0, the compound has a structure represented by the following formula (III):

[0017]

[0018] in,

[0019] represents a single bond or a double bond, Each independently selected from -H, -OH, -NH2, =O, =NOH, C 1-5 alkyl, -OSO3H or a salt thereof, -OPO3H2 or a salt thereof.

[0020] Furthermore,

[0021] represents a single bond or a double bond, Each independently selected from -H, -OH, =O, C1-2 of alkyl.

[0022] Furthermore, the compound has a structure represented by the following formula (IV):

[0023]

[0024] in,

[0025] represents a single bond or a double bond, Each independently selected from -H, -OH, -NH2, =O, =NOH, C 1-5 Alkyl, -OSO3H or its salt, -OPO3H2 or its salt;

[0026] Preferably, Each independently selected from -H, -OH, =O, C 1-2 of alkyl.

[0027] Furthermore, the compound has a structure represented by the following formula (V):

[0028]

[0029] in,

[0030] represents a single bond or a double bond, Each independently selected from -H, -OH, -NH2, =O, =NOH, C 1-5 Alkyl, -OSO3H or its salt, -OPO3H2 or its salt;

[0031] Preferably, Each is independently selected from -H, -OH.

[0032] Furthermore, the compound has a structure represented by the following formula (VI):

[0033]

[0034] in,

[0035] represents a single bond or a double bond, Each independently selected from -H, -OH, -NH2, =O, =NOH, C 1-5 Alkyl, -OSO3H or its salt, -OPO3H2 or its salt;

[0036] Preferably, are each independently selected from -H, -OH, and Not -H or -OH at the same time.

[0037] Furthermore, the compound is:

[0038]

[0039]

[0040] The present invention also provides the use of the aforementioned compound, or its stereoisomer, or its salt, or its solvate, or its prodrug, or its metabolite in the preparation of an antibacterial agent.

[0041] Furthermore, the antibacterial agent is a drug that inhibits Clostridium difficile bacteria.

[0042] Furthermore, the drug is a drug capable of preventing and / or treating Clostridium difficile infectious diseases, recurrence of Clostridium difficile infectious diseases, or complications of Clostridium difficile infectious diseases.

[0043] Furthermore, the Clostridium difficile infectious disease, recurrence of Clostridium difficile infectious disease, or complications of Clostridium difficile infectious disease are caused by infection with Clostridium difficile bacteria;

[0044] The Clostridium difficile infectious disease complication is a digestive tract infection syndrome caused by Clostridium difficile bacterial infection.

[0045] Furthermore, the digestive tract infection syndrome is selected from pseudomembranous colitis, diverticulitis, and antibiotic-associated diarrhea.

[0046] The present invention also provides a drug for inhibiting Clostridium difficile bacteria, which is a preparation prepared with the aforementioned compound, or its stereoisomer, or its salt, or its solvate, or its prodrug, or its metabolite as the active ingredient, and pharmaceutically acceptable excipients.

[0047] Furthermore, the pharmaceutically acceptable excipient is selected from any one or more of a diluent, a filler, a colorant, a glidant, a lubricant, an adhesive, a stabilizer, a suspending agent or a buffer.

[0048] Furthermore, the preparation is an oral preparation;

[0049] Preferably, the oral preparation is selected from granules, capsules, tablets, and pills.

[0050] Furthermore, the dosage of the active ingredient contained in the pharmaceutical unit preparation is 5-2500 mg;

[0051] The unit preparation refers to one preparation unit, namely one tablet, one capsule, one bag of granules, one bag of pills, or one pill filled in a capsule.

[0052] In the present invention, "salt" refers to an acidic and / or basic salt formed by a compound or its stereoisomer with an inorganic and / or organic acid and / or base, including zwitterionic salts (inner salts), and also includes quaternary ammonium salts, such as alkylammonium salts. These salts can be directly obtained in the final separation and purification of the compound. It can also be obtained by mixing the compound, or its stereoisomer, with a certain amount of acid or base appropriately (e.g., equivalents). These salts may form a precipitate in the solution and be collected by filtration, or be recovered after solvent evaporation, or be obtained by freeze-drying after reaction in an aqueous medium. The salt described in the present invention can be a hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compound.

[0053] Prefix (C a-b ) refers to any alkyl group containing "a" to "b" carbon atoms. For example, C 1-5 The alkyl group refers to a straight-chain or branched alkyl group containing 1 to 5 carbon atoms.

[0054] The present invention provides a class of allocholic acid derivatives in which the 5-hydrogen is alpha. These compounds can effectively inhibit the growth of Clostridium difficile bacteria and have significant antibacterial activity. They have very good application prospects in the preparation of drugs for preventing and / or treating Clostridium difficile infectious diseases, recurrence of Clostridium difficile infectious diseases, or complications of Clostridium difficile infectious diseases.

[0055] 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.

[0056] 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. DETAILED DESCRIPTION

[0057] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercial products or referring to the preparation methods in public literature.

[0058] All the raw materials for the allocholic acid compounds were commercially available (HPLC purity greater than 98%), including 5α-CA (allocholic acid), 5α-CDCA (allo chenodeoxycholic acid), 5α-DCA (allo deoxycholic acid), 5α-LCA (allo lithocholic acid), 5α-UDCA (allo ursodeoxycholic acid), 5α-HCA (allo hyocholic acid), 5α-HDCA (allo hyodeoxycholic acid), 5α-OCA (alloobeticholic acid), 5α-PCA (allo phocaecholic acid), 5α-7-ketoLCA, 5α-DHCDCA, 5α-DHCA, 5α-7-ketoDCA, 5α-3-DHDCA, and 5α-3-DHCA. They correspond to the structures of the hydroxyl groups of the above-mentioned natural bile acids oxidized to carbonyl groups. The abbreviations and corresponding structures of each compound are as follows:

[0059]

[0060] The decarbonized bile acid raw materials are as follows, and the synthesis method is based on the method of Journal of Lipid Research, 1988, 29: 1387:

[0061]

[0062] Example 1. Synthesis of Compound I-1

[0063]

[0064] 1.2 g of 5α-CA, 15 ml of DMF (N,N-dimethylformamide), 1.0 g of TBTU (benzotriazoletetramethyltetrafluoroborate), 1 g of DIPEA (N,N-diisopropylethylamine) and 0.42 g of 2-amino-5-nitrothiazole were added to a single-necked flask and stirred at room temperature for 5-6 hours. The reaction progress was monitored by TLC until the reaction of 5α-CA was complete. 60 ml of water and 60 ml of ethyl acetate were added, stirred and allowed to stand, the organic phase was separated, the aqueous phase was extracted twice with ethyl acetate and combined, and the organic phase was washed successively with 10 ml of 2% dilute hydrochloric acid, 1% potassium carbonate aqueous solution, and saturated brine, dried, and concentrated in vacuo. The residue was subjected to silica gel column chromatography and rinsed with petroleum ether / ethyl acetate to obtain 0.91 g of a light yellow solid.

[0065] 1HNMR (400MHz, CD3OD) δppm: 8.37 (s, 1H), 3.97-3.91 (m, 1H), 3.77 (q, J = 3.0Hz, 1H), 3.44-3.32 (m, 1H), 2.64-2.58 (m, 1H), 2.51-2.45 (m, 1H), 2.3 6-2.21(m,2H),2.05-1.70(m,8H),1.70-1.28(m,7H),1.23(t,J=7.1Hz, 1H),1.18-0.92(m,2H),1.06(d,J=5.8Hz,3H),0.88(s,3H),0.78(s,3H);

[0066] ESI-MS m / z:534.02[M-1] - .

[0067] Example 2. Synthesis of Compound I-2

[0068]

[0069] Using 5α-CDCA as raw material, the synthesis method was similar to that in Example 1 to obtain compound I-2.

[0070] 1 HNMR(400MHz,CD3OD)δppm:8.36(s,1H),3.79-3.75(m,1H),3.42-3.30(m,1H),2.68-2.50(m,1H),2.49-2.41(m,1H),2.30-2.20(m,1H),2.0 2-1.98(m,3H),1.98-1.92(m,3H),1.92-1.37(m,8H),1.37-1.09(m,4H ),1.00(d,J=6.4Hz,3H),1.05-1.96(m,1H),0.97(s,3H),0.69(s,3H);

[0071] ESI-MS m / z:518.03[M-1] - .

[0072] Example 3. Synthesis of Compound I-3

[0073]

[0074] Using 5α-DCA as raw material, the synthesis method was similar to that of Example 1 to obtain compound I-3.

[0075] ESI-MS m / z:518.11[M-1] - .

[0076] Example 4. Synthesis of Compound I-4

[0077]

[0078] Using 5α-LCA as raw material, the synthesis method was similar to that in Example 1 to obtain compound I-4.

[0079] 1 HNMR(400MHz,d6-DMSO)δppm:12.94(s,1H),8.58(s,1H),4.41(brs,1H),2.58-2.34(m,1H),1.96-1.85(m,1H),1.81-1.74(m,3H ),1.72-1.52(m,3H),1.46-1.26(m,9H),1.25-1.02(m,7H),0.98-0.84(m,1H),0.96(d,J=6.4Hz,3H),0.84(s,3H),0.65(s,3H);

[0080] ESI-MS m / z:502.04[M-1] - .

[0081] Example 5. Synthesis of Compound I-5

[0082]

[0083] Using 5α-UDCA as raw material, the synthesis method was similar to that of Example 1 to obtain compound I-5.

[0084] 1 HNMR(400MHz,d6-DMSO)δppm:13.05(s,1H),8.60(s,1H),4.46(brs,1H),3.9 5-3.84(m,1H),3.30-3.25(m,1H),2.66-2.39(m,1H),1.98(s,1H),1.95-1.9 3(m,1H),1.91-1.59(m,4H),1.52-1.36(m,3H),1.36-1.23(m,6H),1.23-1.0 1(m,5H),1.01-0.94(m,1H),0.96(d,J=6.3Hz,3H),0.86(s,3H),0.65(s,3H);

[0085] ESI-MS m / z:518.04[M-1] - .

[0086] Example 6. Synthesis of Compound I-6

[0087]

[0088] Using 5α-7-ketoLCA as raw material, the synthesis method was similar to that in Example 1 to obtain compound I-6.

[0089] 1 HNMR(400MHz,d6-DMSO)δppm:13.01(s,1H),8.56(s,1H),4.41(brs,1H),2.88(dd,J=12.0,5.8Hz,1H),2.58-2.36(m,2H),1.96-1.84 (m,1H),1.84-1.60(m,4H),1.54-1.14(m,8H),1.12(s,3H),1.14-0.98(m,7H),0.98-0.84(m,2H),0.95(d,J=6.3Hz,3H),0.63(s,3H);

[0090] ESI-MS m / z:516.03[M-1] - .

[0091] Example 7. Synthesis of Compound I-7

[0092]

[0093] Using 5α-HCA as raw material, the synthesis method was similar to that in Example 1 to obtain compound I-7.

[0094] ESI-MS m / z:534.02[M-1] - .

[0095] Example 8. Synthesis of Compound I-8

[0096]

[0097] Using 5α-HDCA as the raw material, the synthesis method was similar to that in Example 1 to obtain compound I-8.

[0098] 1HNMR (400MHz, d6-DMSO) δppm: 13.01 (s, 1H), 8.65 (s, 1H), 4.40 (d, J = 4.2Hz, 1H), 4.22(d,J=3.9Hz,1H),3.85-3.75(m,1H),3.34-3.28(m,1H),2.61-2.39(m,2H),1 .98-1.88(m,1H),1.89-1.72(m,3H),1.68-1.60(m,1H),1.54-1.25(m,7H),1.28- 1.09(m,4H),1.08-0.91(m,2H),0.95(d,J=6.3Hz,3H),0.82(s,3H),0.64(s,3H);

[0099] ESI-MS m / z:518.01[M-1] - .

[0100] Example 9. Synthesis of Compound I-9

[0101]

[0102] Using 5α-OCA as the raw material, the synthesis method was similar to that in Example 1 to obtain compound I-9.

[0103] ESI-MS m / z:546.08[M-1] - .

[0104] Example 10. Synthesis of Compound I-10

[0105]

[0106] Using 5α-PCA as the raw material, the synthesis method was similar to that in Example 1 to obtain compound I-10.

[0107] ESI-MS m / z:534.03[M-1] - .

[0108] Example 11. Synthesis of Compound I-11

[0109]

[0110] Using 5α-DHCDCA as raw material, the synthesis method was similar to that in Example 1 to obtain compound I-11.

[0111] ESI-MS m / z:514.04[M-1] - .

[0112] Example 12. Synthesis of Compound I-12

[0113]

[0114] Using 5α-DHCA as raw material, the synthesis method was similar to that of Example 1 to obtain compound I-12.

[0115] ESI-MS m / z:528.01[M-1] - .

[0116] Example 13. Synthesis of Compound I-13

[0117]

[0118] Using 5α-7ketoDCA as raw material, the synthesis method was similar to that in Example 1 to obtain compound I-13.

[0119] ESI-MS m / z:532.00[M-1] - .

[0120] Example 14. Synthesis of Compound I-14

[0121]

[0122] Using 5α-3-DHDCA as raw material, the synthesis method was similar to that in Example 1 to obtain compound I-14.

[0123] ESI-MS m / z:516.24[M-1] -

[0124] Example 15. Synthesis of Compound I-15

[0125]

[0126] Using 5α-3-DHCA as raw material, the synthesis method was similar to that in Example 1 to obtain compound I-15.

[0127] ESI-MS m / z: 532.23 [M-1] -

[0128] Example 16. Synthesis of Compound I-16

[0129]

[0130] Using 5α-nor-CA as raw material, the synthesis method was similar to that in Example 1 to obtain compound I-16.

[0131] ESI-MS m / z:520.19[M-1] - .

[0132] Example 17. Synthesis of Compound I-17

[0133]

[0134] Using 5α-nor-CDCA as raw material, the synthesis method was similar to that in Example 1 to obtain compound I-17.

[0135] ESI-MS m / z:504.06[M-1] - .

[0136] Example 18. Synthesis of Compound I-18

[0137]

[0138] Using 5α-nor-UDCA as raw material, the synthesis method was similar to that in Example 1 to obtain compound I-18.

[0139] ESI-MS m / z:504.23[M-1] - .

[0140] Example 19. Synthesis of Compound I-19

[0141]

[0142] Using 5α-nor-7DHCA as raw material, the synthesis method was similar to that in Example 1 to obtain compound I-19.

[0143] ESI-MS m / z:518.19[M-1] - .

[0144] Example 20. Synthesis of Compound I-20

[0145]

[0146] Using 5α-7-keto-nor-LCA as raw material, the synthesis method was similar to that in Example 1 to obtain compound I-20.

[0147] ESI-MS m / z:502.05[M-1] - .

[0148] Example 21: Preparation of a pharmaceutical tablet composition of the compound of the present invention

[0149] The pharmaceutical tablet composition of compound I-1 comprises 1 part by weight of compound I-1, 0.1-0.3 parts by weight of lactose, 0.4-0.2 parts by weight of starch, 0.008-0.014 parts by weight of sodium carboxymethyl starch, an appropriate amount of povidone K30, 0.01-0.05 parts by weight of magnesium stearate, and 0.5 parts by weight of 40% ethanol; tablets are prepared according to the above proportions to obtain pharmaceutical tablets of compound I-1 of the present invention, each tablet containing 50-1500 mg of compound 1-1.

[0150] Using the same method as above, pharmaceutical tablet compositions of compounds I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-13, I-17, and I-20 were prepared respectively.

[0151] Example 22: Preparation of a Colon-Targeted Tablet Composition of the Compound of the Invention

[0152] Preparation of a colon-targeted tablet composition for Compound I-2: Weigh 1 part by weight of Compound I-2, 0.1-0.4 parts by weight of starch, 0.4-0.1 parts by weight of microcrystalline cellulose, 0.008-0.014 parts by weight of sodium carboxymethyl starch, an appropriate amount of povidone K30, 0.01-0.05 parts by weight of magnesium stearate, and 0.8 parts by weight of 40% ethanol; prepare tablet cores according to the above proportions. Weigh 0.1-0.2 parts by weight of Eudragit S100, 0.2-0.1 parts by weight of Eudragit FS30D, and 0.05 parts by weight of PEG4000; dissolve the mixture in 0.3 parts by weight of ethanol to prepare an enteric coating solution, and coat the tablet cores to produce the medicinal colon-targeted tablets for Compound I-2 of the present invention. Each tablet contains 50-1500 mg of Compound I-2.

[0153] Using the same method as above, pharmaceutical tablet compositions of compounds I-1, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-13, I-17, and I-20 were prepared respectively.

[0154] Example 23: Pharmaceutical capsule composition of the compound of the present invention

[0155] A pharmaceutical capsule composition of compound I-3 contains 300 g of compound I-3, 193 g of microcrystalline cellulose, 7 g of micropowdered silica gel, a total of 500 g, and a size 2 hollow capsule; or contains 1200 g of compound I-3, 279 g of microcrystalline cellulose, 21 g of micropowdered silica gel, a total of 1500 g, and a size 00 hollow capsule. The preparation method is as follows:

[0156] a. Mixing compound I-3, microcrystalline cellulose, and silica gel powder to obtain a mixed powder;

[0157] b. Pass the mixed powder through a 120-mesh sieve, fill it into capsules, and seal them. Make a total of 1,000 capsules.

[0158] Each capsule contains 300 mg or 1200 mg of compound I-3.

[0159] Using the same method as above, pharmaceutical capsule compositions of compounds I-1, I-2, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-13, I-17, and I-20 were prepared respectively.

[0160] Example 24: Preparation of a Colon-Targeting Micropellet Composition of the Compound of the Invention

[0161] A medicinal colon-targeted micropellet composition of compound I-1 was prepared, containing 1 part by weight of compound I-1, 0.2 parts by weight of microcrystalline cellulose, 0.01 parts by weight of povidone, 0.01 parts by weight of magnesium stearate, 0.1 parts by weight of hydroxypropyl methylcellulose, 0.1 parts by weight of Eudragit S100, 0.1 parts by weight of PEG 800, and 0.8 parts by weight of 40% ethanol.

[0162] The preparation method comprises: preparing pellet cores with microcrystalline cellulose in the aforementioned proportions; dissolving a mixture of Compound I-1 and povidone in 0.3 parts by weight of 40% ethanol to prepare a drug solution; dissolving hydroxypropyl methylcellulose and 0.05 parts by weight of PEG 800 in 0.2 parts by weight of 40% ethanol to prepare an isolation coating solution; and dissolving Eudragit S100 and 0.05 parts by weight of PEG 800 in 0.3 parts by weight of 40% ethanol to prepare an enteric coating solution. Using a bottom spray coating technique, the pellet cores are coated with a drug, an inner isolation layer, and an outer enteric layer to produce colonic micropellets. The resulting micropellets are then quality tested and filled into capsules, each containing 50-1500 mg of Compound I-1.

[0163] By using the same method as above, medicinal colon-targeted micropellet compositions of compounds I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-13, I-17, and I-20 were prepared respectively.

[0164] The beneficial effects of the present invention are demonstrated below through specific experimental examples.

[0165] Experimental Example 1: Inhibition of the in vitro activity of the compounds of the present invention on Clostridium difficile

[0166] (1) Experimental methods

[0167] The experiment selected ATCC 630 Clostridium difficile strains and tested them on supplemented Brucella agar medium. The strains were frozen in glycerol at -80℃ and inoculated onto solid agar medium. They were placed in a 37℃ incubator for anaerobically incubated for 24-48h. The test range of the compounds of the present invention was 32μg / ml-0.0156μg / ml, with a total of 11 two-fold dilution concentration gradients; the control compound was vancomycin; the compounds of the present invention were prepared into high-concentration working solutions at 100 times the test concentration and used on the same day, with the solvent being 100% DMSO. For the preparation of each dilution concentration agar plate, 20μl of the high-concentration working solution was mixed with 2ml of melted, supplemented Brucella agar (45-55℃) and added to a six-well plate and allowed to solidify. 1% DMSO was used as a growth control. On the day of the experiment, an appropriate number of single colonies were picked and suspended in physiological saline. The turbidity of the bacterial solution was adjusted to OD600=0.2 using a turbidimeter, containing approximately 1×10 8 cfu / ml. This 2μl bacterial solution was directly spotted on the compound solid dilution agar plate. Therefore, each well of the six-well plate contained about 10 5 CFU of Clostridium difficile. This is the test plate. Incubate all prepared test plates anaerobically at 35±2°C for 48 hours. After 48 hours of incubation, visually inspect the plate to determine the minimum drug concentration that completely or significantly inhibits bacterial growth, as determined by the MIC.

[0168] (2) Experimental results

[0169] The experimental results are recorded in Table 1 below. The results show that compared with the control drug, the compounds of the present invention have higher inhibitory activity against Clostridium difficile bacteria, especially compounds I-1, I-2, I-3, I-5, I-6, I-7, I-8, I-10, I-12, and I-14, which have MICs of ≤0.5 μg / ml against Clostridium difficile bacteria, showing significant antibacterial activity.

[0170] Table 1. Inhibitory activity of the compounds of the present invention against Clostridium difficile

[0171] Compound Clostridium difficile ATCC630 MIC (μg / ml) I-1 0.125 I-2 0.25 I-3 0.25 I-4 8 I-5 0.5 I-6 0.5 I-7 0.125 I-8 0.5 I-9 4 I-10 0.25 I-11 2 I-12 0.5 I-13 1 I-14 0.5 I-15 1 I-16 2 I-17 1 I-18 2 I-19 8 I-20 1 Vancomycin 1

[0172] Experimental Example 2: Recovery rate of the compound of the present invention in feces

[0173] CN110878113A discloses a series of bile acid derivatives with antibacterial activity, among which three compounds have the structures shown in the following formula:

[0174]

[0175] The above three compounds were used as control compounds.

[0176] 1. Experimental method: 18 C57BL / 6J male mice were adapted to feeding for 7 days and were fasted before the start of the experiment. The mice were divided into 6 groups, with 3 mice in each group. Each group of mice was given a single dose of compound I-1, compound I-2, compound I-5 of the present invention and the three control compounds a, b, and c mentioned above, at a dose of 150 mg / kg, by gavage, and the solvent was normal saline containing 10% Tween-80. After administration, the mice were raised normally, and the feces of each mouse were collected 24 hours and 48 hours after administration. First, the mouse fecal sample was accurately weighed, dried and homogenized at 65 Hz for 30 seconds, and then methanol was added at a mass volume ratio of 1:5. After vortex mixing, the mixture was homogenized again (40 Hz, homogenized for 60 seconds); after vortexing for 30 seconds, 800 μL was taken and centrifuged (13000 rpm for 5 minutes, 4°C), and the supernatant was the fecal sample extract. Take 75 μl of fecal sample extract, add 25 μl of internal standard and 175 μl of methanol, vortex mix for 2 min, centrifuge at 12000 rpm at 4°C for 10 min, and take the supernatant for LC-MS / MS analysis.

[0177] Calculate the total amount of each compound in the feces and the recovery rate. Recovery rate = mass of compound in feces ÷ total mass of oral compound * 100%, and record it in the following Table 2:

[0178] Table 2. Fecal recovery of the compounds of the present invention

[0179] Group Recovery rate Group Recovery rate Compound I-1 46.8±4.2％ Control compound a 26.2±2.9％ Compound I-2 31.7±3.3％ Control compound b 20.2±4.4％ Compound I-5 37.3±2.5％ Control compound c 24.4±3.7％

[0180] The results show that the compound of the present invention has a higher fecal recovery rate than the disclosed compounds, indicating that the exposure of the compound of the present invention in the intestine is higher than that of the disclosed compounds, and has more advantages in treating Clostridium difficile infection.

[0181] In summary, the present invention provides a class of allocholic acid derivatives with alpha as the 5-hydrogen group. These compounds can effectively inhibit the growth of Clostridium difficile bacteria and have significant antibacterial activity. They have very good application prospects in the preparation of drugs for preventing and / or treating Clostridium difficile infectious diseases, recurrence of Clostridium difficile infectious diseases, or complications of Clostridium difficile infectious diseases.

Claims

1. The following compounds, or their salts: 、 、 。 2. Use of the compound according to claim 1 or a salt thereof in the preparation of an antibacterial agent; the antibacterial agent is a drug that inhibits Clostridium difficile bacteria.

3. The use according to claim 2, characterized in that: The drug is a drug capable of preventing and / or treating Clostridium difficile infectious diseases, recurrence of Clostridium difficile infectious diseases, or complications of Clostridium difficile infectious diseases.

4. The use according to claim 3, characterized in that: The Clostridium difficile infection disease, recurrence of Clostridium difficile infection disease, or complication of Clostridium difficile infection disease is caused by infection with Clostridium difficile bacteria; The Clostridium difficile infectious disease complication is a digestive tract infection syndrome caused by Clostridium difficile bacterial infection.

5. The use according to claim 4, characterized in that: The digestive tract infection syndrome is selected from pseudomembranous colitis, diverticulitis, and antibiotic-associated diarrhea.

6. A drug for inhibiting Clostridium difficile bacteria, characterized in that: The drug is a preparation prepared with the compound according to claim 1 or its salt as the active ingredient and pharmaceutically acceptable excipients.

7. The drug according to claim 6, characterized in that: The pharmaceutically acceptable excipient is selected from any one or more of a diluent, a filler, a colorant, a glidant, a lubricant, a binder, a stabilizer, a suspending agent or a buffer.

8. The drug according to claim 6, characterized in that: The preparation is an oral preparation.

9. The drug according to claim 8, characterized in that: The oral preparation is selected from granules, capsules, tablets and pills.

10. The drug according to any one of claims 6 to 9, characterized in that: The amount of active ingredient contained in the pharmaceutical unit preparation is 5-2500 mg; The unit preparation refers to one preparation unit, namely one tablet, one capsule, one bag of granules, one bag of pills, or one pill filled in a capsule.

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