Bile acid-norfloxacin complex, method of preparation and use as an antibacterial agent

Abstract

The application discloses a preparation method of a bile acid-norfloxacin compound, which comprises the following steps: reacting bile acid and norfloxacin in the presence of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and diisopropylethylamine in THF solvent at 10-30 DEG C for 12-30 h, and then carrying out post-treatment to obtain the bile acid-norfloxacin compound. The bile acid-norfloxacin compound is obtained by condensation reaction between the carboxyl group of the bile acid and the piperazine group of the norfloxacin, and the compound structure is combined with the bile acid and the norfloxacin. The antibacterial activity of the norfloxacin is obviously improved, the norfloxacin has better inhibition effect on Escherichia coli, Helicobacter pylori and the like, and also has better inhibition effect on Pseudomonas aeruginosa, Staphylococcus aureus and Pneumococcus, and meanwhile, the preparation operation process is simple.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a bile acid-norfloxacin complex, its preparation method, and its application as an antibacterial agent. Background Technology

[0002] Norfloxacin, also known as norfloxacin, chemically named 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid, is a third-generation quinolone antibiotic. It inhibits the activity of DNA gyrase in pathogenic bacteria in the digestive tract, thus hindering bacterial DNA replication and exhibiting antibacterial activity. Due to its broad antibacterial spectrum, low incidence of allergic reactions, and mild toxicity, it is widely used clinically. Norfloxacin has good inhibitory effects against Gram-negative bacteria such as Escherichia coli, Proteus, Helicobacter pylori, and Enterobacter aerogenes, but its inhibitory effect on Gram-negative bacteria such as Pseudomonas aeruginosa and some Gram-positive bacteria such as Staphylococcus aureus and Streptococcus pneumoniae is relatively poor. By modifying the structure of quinolones, novel antibacterial drugs with better antibacterial activity can be obtained, such as ciprofloxacin, which has strong anti-Gram-negative bacteria activity; norfloxacin, ofloxacin, and moxifloxacin, which have strong anti-Gram-positive bacteria activity; and levofloxacin, which has good broad-spectrum antibacterial activity. Research on the structural modification of quinolones is still ongoing, but the chemical synthesis process of related derivatives generally involves many reaction steps and is becoming increasingly complex. Summary of the Invention

[0003] The purpose of this invention is to provide a bile acid-norfloxacin complex and its preparation method. This complex exhibits superior antibacterial activity compared to norfloxacin. Furthermore, the preparation process of the complex is simple, requiring only a one-step reaction and minimal post-treatment. Specifically, this invention adopts the following technical solution:

[0004] On one hand, the present invention provides a method for preparing a bile acid / norfloxacin complex, which involves reacting bile acids and norfloxacin in a THF solvent at 10-30°C for 12-30 h in the presence of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) and diisopropylethylamine (DIPEA), followed by post-treatment.

[0005] In the preparation method described above, preferably, the molar ratio of bile acid, norfloxacin, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and diisopropylethylamine is 1:1.1-1.3:1.1-1.3:1.5-3; more preferably, the molar ratio of bile acid, norfloxacin, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and diisopropylethylamine is 1:1.2:1.2:2.

[0006] In the preparation method described above, preferably, the bile acid is selected from cholic acid, chenodeoxycholic acid, or ursodeoxycholic acid; when the bile acid is selected from cholic acid, the reaction is carried out overnight at 25°C; when the bile acid is selected from ursodeoxycholic acid, the reaction is carried out in THF solvent at 15°C for 24 hours.

[0007] On the other hand, when the bile acid is selected from bile acids, the bile acid / norfloxacin complex obtained by the above preparation method of the present invention is a bile acid / norfloxacin complex with the molecular formula C. 40 H 56 FN3O7 has the following structural formula (Ⅰ):

[0008]

[0009] Furthermore, when the bile acid is selected from ursodeoxycholic acid, the bile acid / norfloxacin complex obtained by the preparation method described in this invention has the molecular formula Cursodeoxycholic acid / norfloxacin. 40 H 56 FN3O6, with the following structural formula (II):

[0010]

[0011]

[0012] This invention involves a condensation reaction between the carboxyl group of bile acids and the piperazine group of norfloxacin to obtain a complex structure combining bile acids and norfloxacin. This structure significantly enhances the antibacterial activity of norfloxacin, exhibiting better inhibitory effects not only against Escherichia coli and Helicobacter pylori, but also against Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae. Furthermore, the preparation process of this invention is simple. Detailed Implementation

[0013] The following embodiments are further illustrations of the present invention and serve as explanations of the technical content of the present invention. However, the essence of the present invention is not limited to the embodiments described below. Those skilled in the art can and should know that any simple changes or substitutions based on the spirit of the present invention should fall within the protection scope claimed by the present invention.

[0014] Example 1

[0015] The preparation method of the bile acid / norfloxacin complex is as follows:

[0016]

[0017] The feeding details are shown in Table 1 below:

[0018] Table 1: Feeding Status

[0019] name relative molecular weight Feeding amount Mole ratio source cholic acid 408.5 6g 1.0 Bailing CA2171202 Norfloxacin 319.3 5.6g 1.2eq Bid CMU114 HATU 380.24 6.7g 1.2eq McLean DIPEA 129.24 3.8g 2.0eq McLean THF / 100mL / Cologne

[0020] The specific operation process is as follows:

[0021] Add 6g of cholic acid to 100ml of THF, then add 6.7g of HATU and 3.8g of DIPEA, and finally add 5.6g of norfloxacin. React overnight at room temperature (25°C). Monitor the reaction for completeness by TLC (eluent EA, phosphomolybdic acid). Purify by silica gel column chromatography (eluent EA), evaporate to dryness, and then slurry with ethyl acetate to obtain 4g of white powder.

[0022] Structural confirmation:

[0023] 1 HNMR (400MHz, DMSO) δ: 15.33 (s, 1H), 8.94 (s, 1H), 7.88 (d, J = 12Hz, 1H), 7.17 (d, J = 6.4Hz, 1H), 4.58(d,J=6.8Hz,2H),4.04-3.79(m,6H),3.67-3.17(m,9H),2.40-0.79(m,33H),0.58(s,3H).

[0024] ESI-MS m / z: 710.60 [M+1].

[0025] Example 2

[0026] The preparation method of the ursodeoxycholic acid / norfloxacin complex is as follows:

[0027]

[0028] The feeding details are shown in Table 2 below:

[0029] Table 2: Feeding Status

[0030] name relative molecular weight Feeding amount Mole ratio source Ursodeoxycholic acid 392.58 4g 1.0 Nightingale Norfloxacin 319.3 3.9g 1.2eq Bid HATU 380.24 4.7g 1.2eq Annegi Diisopropylethylamine 129.24 2.6g 2eq McLean THF / 80mL / Cologne

[0031] The specific operation process is as follows:

[0032] Ursodeoxycholic acid, norfloxacin, HATU, diisopropylethylamine, and THF were added to a reaction flask and stirred at 15°C for 24 hours. The reaction was monitored by TLC (dichloromethane / methanol = 10:1, phosphomolybdic acid for color development). After the reaction was completed, the mixture was filtered, and the mother liquor was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10:1). The product was then added to 200 ml of ethyl acetate / methanol (20:1) and stirred for 2 hours. After filtration, the product was dried at 70°C to obtain 4.7 g.

[0033] Structural confirmation:

[0034] 1 H NMR (400MHz, DMSO) δ15.32(s,1H),8.96(s,1H),7.93(d,J=13.2Hz,1H),7.19(d,J=7.2Hz,1H),4.59(q,J=6.8Hz,2H),4.43(d,J=4.4 Hz,1H),3.86(d,J=6.7Hz,1H),3.67(s,4H),2.33(ddd,J=36.3,9.8,5.3Hz,2H),2.02-1.01(m,31H),0.99-0.83(m,8H),0.62(s,3H).

[0035] ESI-MS m / z: 694.6 [M+1] 。

[0036] Example 3

[0037] In vitro antibacterial activity experiment:

[0038] Experimental methods: Helicobacter pylori (ATCC43504) was inoculated in MH medium (microaerobic environment) and incubated at 37℃ for 20 h; Pseudomonas aeruginosa (ATCC27853) and Escherichia coli (ATCC25922) were inoculated in MH medium and incubated at 37℃ for 17-20 h; Streptococcus pneumoniae (ATCC49619) was inoculated in MH medium (containing 5% sheep blood) and incubated at 35-37℃ with 5% carbon dioxide for 24 h; Staphylococcus aureus (ATCC6538) was inoculated in MH medium (2% NaCl) and incubated at 37℃ for 24 h; each culture was diluted to contain 10% of the test bacteria. 5 The concentration of bacteria was measured at 1 / ml using a multi-point inoculation device. The minimum inhibitory concentration (MIC) of the bile acid / norfloxacin complex of the present invention was determined using the agar dilution method, with norfloxacin, bile acid, and ursodeoxycholic acid as controls. The results are shown in Table 3 below.

[0039] Table 3: In vitro antibacterial activity test

[0040]

[0041]

[0042] As can be seen from the in vitro antibacterial results in Table 3 above, the cholic acid-norfloxacin complex obtained in Example 1 and the ursodeoxycholic acid-norfloxacin complex obtained in Example 2 of this invention exhibit significantly better antibacterial activity against Escherichia coli ATCC25922, Helicobacter pylori ATCC43504, Pseudomonas aeruginosa ATCC27853, Pneumococcus ATCC49619, and Staphylococcus aureus ATCC6538 than norfloxacin, and are also significantly higher than the inhibitory activity of cholic acid or ursodeoxycholic acid against the corresponding bacteria. The cholic acid-norfloxacin complex and ursodeoxycholic acid-norfloxacin complex of this invention demonstrate excellent antibacterial properties.

[0043] It should be noted that the above-described technical content of this invention is merely an explanation and clarification to enable those skilled in the art to understand the technical essence of this invention, and therefore is not intended to limit the scope of protection of this invention. The scope of protection of this invention should be determined by the claims. Those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made based on the essential spirit of this invention should be within the scope of protection of this invention.

Claims

1. A bile acid / norfloxacin complex, with the following structural formula (Ⅰ): Equation (Ⅰ).

2. The method for preparing the bile acid / norfloxacin complex according to claim 1, characterized in that, Cholic acid and norfloxacin were reacted in THF solvent at 10-30°C for 12-30 h in the presence of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and diisopropylethylamine, followed by post-treatment to obtain the final product.

3. The preparation method according to claim 2, characterized in that, The molar ratio of cholic acid, norfloxacin, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and diisopropylethylamine is 1:1.1-1.3:1.1-1.3:1.5-3.

4. The preparation method according to claim 3, characterized in that, The molar ratio of cholic acid, norfloxacin, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and diisopropylethylamine is 1:1.2:1.2:

2.

5. The preparation method according to claim 2, characterized in that, React overnight at 25°C in THF solvent.

6. A bile acid / norfloxacin complex, with the following structural formula (II): Formula (II).

7. The method for preparing the bile acid / norfloxacin complex according to claim 6, characterized in that, Ursodeoxycholic acid and norfloxacin were reacted in THF solvent at 10-30°C for 12-30 h in the presence of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and diisopropylethylamine, followed by post-treatment to obtain the final product.

8. The preparation method according to claim 7, characterized in that, The molar ratio of ursodeoxycholic acid, norfloxacin, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and diisopropylethylamine is 1:1.1-1.3:1.1-1.3:1.5-3.

9. The preparation method according to claim 8, characterized in that, The molar ratio of ursodeoxycholic acid, norfloxacin, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and diisopropylethylamine is 1:1.2:1.2:

2.

10. The preparation method according to claim 6, characterized in that, React in THF solvent at 15°C for 24 hours.

11. The use of the bile acid / norfloxacin complex according to claim 1 or 6 in the preparation of an antibacterial agent, wherein the antibacterial agent is a drug against Escherichia coli, Helicobacter pylori, Pseudomonas aeruginosa, Staphylococcus aureus and / or Streptococcus pneumoniae.

12. The use of the bile acid / norfloxacin complex obtained by any one of claims 2-5 in the preparation of an antibacterial agent, wherein the antibacterial agent is a drug against Escherichia coli, Helicobacter pylori, Pseudomonas aeruginosa, Staphylococcus aureus and / or Streptococcus pneumoniae.

13. The use of the bile acid / norfloxacin complex obtained by any one of claims 7-10 in the preparation of an antibacterial agent, wherein the antibacterial agent is a drug against Escherichia coli, Helicobacter pylori, Pseudomonas aeruginosa, Staphylococcus aureus and / or Streptococcus pneumoniae.

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