Application of oxoalkanoyl clinafloxacin derivatives in the preparation of antibacterial drugs

Abstract

The invention discloses the use of an oxyalkanoyl clinafloxacin derivative having the following structural formula in the preparation of an antibacterial drug, wherein the antibacterial drug is a drug for resisting Gram-positive bacteria and / or Gram-negative bacteria, wherein n is 1, 2 or 3, and R is acetyl, propionyl, butyryl or H. Antibacterial activity experiments confirm that all compounds show good inhibitory activity against tested Gram-positive strains and the vast majority of Gram-negative strains, especially have strong inhibitory activity against Gram-positive bacteria, which is much higher than that of a positive control drug ciprofloxacin, and do not induce rapid development of drug resistance.

Description

Technical Field

[0001] The present invention relates to pharmaceutical use of oxyalkanoyl clinafloxacin derivatives. Background Art

[0002] In the previous research work, the inventors synthesized a class of novel oxoalkanoyl clinafloxacin derivatives and studied their anti-tuberculosis activity (ZL202110839437.0 Oxoalkanoyl clinafloxacin derivatives and their preparation methods and applications). The results showed that the synthesized compounds all showed good anti-tuberculosis activity, among which the anti-tuberculosis activity of compound TM2 was comparable to that of positive control drugs clinafloxacin (CLX) and moxifloxacin (MOX); the activity of compound TM1 was even twice that of clinafloxacin and nearly twice that of moxifloxacin (MOX is one of the "gold standards" recommended by WHO for the treatment of drug-resistant tuberculosis patients), and the target of compound TM1 was different from that of conventional fluoroquinolones. Highly active molecules have great potential for further development. Summary of the invention

[0003] In view of this, the purpose of the present invention is to further study the biological activities of the synthesized oxyalkanoyl clinafloxacin derivatives to expand their pharmaceutical uses.

[0004] After research, the present invention provides the following technical solutions:

[0005] 1. Use of an oxyalkanoyl clinafloxacin derivative in the preparation of an antibacterial drug, wherein the oxyalkanoyl clinafloxacin derivative has the following structural formula:

[0006]

[0007] In the formula, n is 1, 2 or 3, and R is acetyl, propionyl, butyryl or H;

[0008] That is, the oxoalkanoyl clinafloxacin derivative is any one of the following compounds TM1 to TM12:

[0009]

[0010]

[0011] The antibacterial drug is a drug against Gram-positive bacteria and / or Gram-negative bacteria.

[0012] Preferably, the oxoalkanoyl clinafloxacin derivatives are compounds TM8 and TM12.

[0013] Preferably, the antibacterial drug is a drug against Gram-positive bacteria.

[0014] Furthermore, the Gram-positive bacteria are one or more of Staphylococcus aureus, Salmonella enteritidis and Micrococcus luteus.

[0015] More preferably, the oxoalkanoyl clinafloxacin derivative is any one of compounds TM2 to TM12, and the antibacterial drug is a drug against drug-resistant Staphylococcus aureus. The drug-resistant Staphylococcus aureus includes but is not limited to methicillin-resistant Staphylococcus aureus.

[0016] More preferably, the oxoalkanoyl clinafloxacin derivative is compound TM2 or TM10, and the antibacterial drug is an anti-Salmonella drug.

[0017] More preferably, the oxoalkanoyl clinafloxacin derivative is any one of compounds TM2 to TM5, TM10 and TM11, and the antibacterial drug is a drug against Micrococcus luteus.

[0018] Furthermore, the Gram-negative bacteria are one or more of Escherichia coli, Klebsiella pneumonia, Acinetobacter baumannii and Pseudomonas aeruginosa.

[0019] More preferably, the oxazoacyl clinafloxacin derivative is compound TM12, and the antibacterial drug is an anti-Escherichia coli drug.

[0020] More preferably, the oxoalkanoyl clinafloxacin derivative is any one of compounds TM2, TM6 and TM8, and the antibacterial drug is a drug against Klebsiella pneumoniae.

[0021] More preferably, the oxoalkanoyl clinafloxacin derivative is any one of compounds TM7, TM10 and TM12, and the antibacterial drug is a drug against Acinetobacter baumannii.

[0022] The beneficial effects of the present invention are as follows: the present invention discloses the use of oxyalkanoyl clinafloxacin derivatives in the preparation of antibacterial drugs, and antibacterial activity experiments confirm that all compounds show good inhibitory activity against tested Gram-positive strains and most Gram-negative strains, especially have strong inhibitory activity against Gram-positive bacteria, which is much higher than that of the positive control drug ciprofloxacin, and do not induce the rapid development of drug resistance, and can be used to prepare drugs against Gram-positive bacteria and / or Gram-negative bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The effect of compounds TM8 and TM12 on intracellular bacteria of methicillin-resistant Staphylococcus aureus USA300.

[0024] Figure 2 The resistance development of compounds TM8 and TM12 against methicillin-resistant Staphylococcus aureus USA300. DETAILED DESCRIPTION

[0025] In order to make the objectives, technical solutions and beneficial effects of the present invention more clear, preferred embodiments of the present invention will be described in detail below.

[0026] The oxyalkanoyl clinafloxacin derivatives TM1 to TM12 described in the preferred embodiments are all prepared according to the method described in Chinese invention patent ZL202110839437.0 (oxyalkanoyl clinafloxacin derivatives, preparation methods and applications thereof).

[0027] The strain information used in the antibacterial activity test in the preferred embodiment is shown in Table 1.

[0028] Table 1 Test strain information

[0029]

[0030]

[0031] Example 1 Preliminary screening of the inhibitory activity of oxoalkanoyl clinafloxacin derivatives TM1-TM12 against human pathogenic bacteria

[0032] The minimum inhibitory concentration (MIC) of oxaloyl clinafloxacin derivatives against the test strain was detected by broth dilution method (Clinical and Laboratory Standards Institute, CLSI, 2017). The specific steps are as follows: the test strain was cultured in MHB broth medium at 37°C in a shaking incubator, and the bacterial solution concentration was adjusted to 1×10 6 CFU mL -1 ; Dissolve the compound in DMSO, and then dilute it in sequence according to the 2-fold dilution method to obtain a concentration gradient compound solution; Take a 96-well plate, first add the bacterial suspension with adjusted concentration (97.5 μL per well), and then add different concentrations of compound solutions (2.5 μL per well), use an equal volume of DMSO as a negative control, and use the broad-spectrum antibiotic ciprofloxacin (CIP) as a positive control; Incubate at 37°C for 18-24 hours, and observe the lowest concentration that inhibits bacterial growth in the well, i.e., MIC.

[0033] The initial screening results are shown in Table 2.

[0034] Table 2 Inhibitory activity of oxoalkanoyl clinafloxacin derivatives against human pathogenic bacteria - initial screening

[0035]

[0036] As can be seen from Table 2, the inhibitory activity of oxaloyl clinafloxacin derivatives TM1 to TM12 against 7 strains of human pathogenic bacteria is generally strong, and most of them are stronger than or equivalent to the positive control. Especially for Acinetobacter baumannii ATCC19606, the MIC of compounds TM1 to TM12 (0.0354 to 0.2923 μmol·mL -1 ) was much lower than the positive control CIP (0.7726 μmol·mL -1 ), the inhibitory activity was 21.8 to 2.6 times that of CIP, among which compound TM12 had the strongest inhibitory activity.

[0037] Example 2 Rescreening of the inhibitory activity of oxoalkanoyl clinafloxacin derivatives TM1-TM12 against human pathogenic bacteria

[0038] Because the lowest test concentration of the compound in the initial screening test was 2 μg mL -1 In order to obtain the MIC of highly active compounds, a rescreening was performed. The results of the rescreening are shown in Table 3.

[0039] Table 3 Inhibitory activity of oxoalkanoyl clinafloxacin derivatives against human pathogenic bacteria - rescreening

[0040]

[0041] Analysis of the data in Table 3 shows that, except for Escherichia coli ATCC25922, the MIC of the positive control CIP against the other five strains of bacteria is the same as the initial screening results, but the MIC of some oxyalkanoyl clinafloxacin derivatives against some strains is significantly lower than the initial screening results, showing strong inhibitory activity against these strains; in general, the MIC of oxyalkanoyl clinafloxacin derivatives TM1 to TM12 against the other five strains of bacteria are all lower than the positive control CIP, indicating that their inhibitory activity is stronger than CIP. Specifically:

[0042] (1) For Salmonella ATCC13076, except for TM3, the MICs of other compounds were 2.0839-4.5677 μmol·L -1 ) are much lower than CIP (24.1438 μmol·L -1 ), the inhibitory activity was 11 to 5 times that of CIP, among which compounds TM2 and TM10 had the strongest inhibitory activity;

[0043] (2) For Pseudomonas aeruginosa ATCC27853, the MICs of compounds TM1 to TM12 were 3.8316 to 4.7189 μmol·L -1) is also lower than CIP (6.0360 μmol·L -1 ), the inhibitory activity was 1.5 to 1.2 times that of CIP;

[0044] (3) For Micrococcus luteus, the MICs of compounds TM1 to TM12 ranged from 0.0737 to 4.4260 μmol·L -1 ) is much lower than CIP (48.2877 μmol·L -1 ), the inhibitory activity was 655-10 times that of CIP, among which compound TM10 had the strongest inhibitory activity, followed by TM2-TM5 and TM11;

[0045] (4) Inhibitory activity of compounds TM1 to TM12 against Staphylococcus aureus ATCC25129 (MIC 3.8316 to 4.7189 μmol·L -1 ) is CIP (MIC 96.5754 μmol·L -1 ) 25 to 20 times;

[0046] (5) For Staphylococcus aureus ATCC14125, except for TM11, the MICs of other compounds were significantly lower than those of the initial screening results; among them, the inhibitory activities of compounds TM4-TM7, TM9, and TM10 (MIC 0.0599-0.0737 μmol·L -1 ) is CIP (MIC 12.0719 μmol·L -1 ) by 201 to 163 times; the inhibitory activity of compounds TM1 to TM3 (MIC 0.1265 to 0.1342 μmol·L -1 ) is 95 to 89 times that of CIP; even TM11, which has the poorest activity among the compounds, has an inhibitory activity (MIC4.5677μmol·L -1 ) is also 2.6 times of CIP.

[0047] Example 3 Inhibitory activity of oxoalkanoyl clinafloxacin derivatives TM1-TM12 against other Gram-positive bacteria

[0048] As can be seen from Tables 2 and 3, for the seven human pathogens tested, the inhibitory activity of the oxoalkanoyl clinafloxacin derivatives TM1 to TM12 against Gram-positive bacteria is stronger than that against Gram-negative bacteria. In order to verify this inference, more different Gram-positive bacterial strains were further tested. The test results are shown in Table 4.

[0049] Table 4 Inhibitory activity of oxoalkanoyl clinafloxacin derivatives against other Gram-positive bacteria

[0050]

[0051] From the data in Table 4, we can see that, except for TM1, the oxoalkanoyl clinafloxacin derivatives TM2 to TM12 have no significant effect on the five G + The inhibitory activity of the strains was much stronger than that of the positive control CIP, and it had strong inhibitory activity against both the standard strain ATCC330041 and the four strains of methicillin-resistant Staphylococcus aureus. The highest activity increased by 382 times, and the lowest increased by at least 6 times, indicating that these compounds have great potential in treating Gram-positive bacterial infections. Specifically:

[0052] (1) TM5 and TM7 had the best inhibitory activity against methicillin-resistant Staphylococcus aureus USA300 (MIC 0.0158 μmol·L -1 ), CIP (MIC 1.5090 μmol·L -1 ) 95 times; TM10 was second (MIC 0.0184 μmol·L -1 ), which is 82 times that of CIP; followed by TM9, TM3, and TM4 (MIC 0.0299-0.0326 μmol·L -1 ), which is 50 to 46 times that of CIP; TM8 had the worst inhibitory activity (MIC 0.1230 μmol·L -1 ), but also reached 12 times of CIP;

[0053] (2) TM7 had the best inhibitory activity against Staphylococcus aureus ATCC330041 (MIC 0.0158 μmol·L -1 ), CIP (MIC 1.5090 μmol·L -1 ) was 95 times higher than that of TM10 (MIC 0.0184 μmol·L -1 ), which is 82 times that of CIP; followed by TM9 and TM3-TM6 (MIC 0.0299-0.0326 μmol·L -1 ), which is 50 to 46 times that of CIP; TM2 had the worst inhibitory activity (MIC 0.1302 μmol·L -1 ), but also reached 11 times the CIP;

[0054] (3) TM9 had the best inhibitory activity against methicillin-resistant Staphylococcus aureus MRSA3390 (MIC 0.0150 μmol·L -1 ), CIP (MIC 1.5090 μmol·L -1 ) 100 times; followed by TM3-TM5 and TM7 (MIC 0.0158-0.0163 μmol·L -1), which is 95-92 times that of CIP; followed by TM6 and TM10 (MIC 0.0308-0.0369 μmol·L -1 ), which is 48-40 times that of CIP; TM8 had the worst inhibitory activity (MIC 0.2461 μmol·L -1 ), but it also reached 6 times of CIP;

[0055] (4) TM3, TM5, TM7, and TM9 had the best inhibitory activity against methicillin-resistant Staphylococcus aureus MRSA3392 (MIC 0.0075-0.0079 μmol·L -1 ), CIP (MIC 0.7545 μmol·L -1 ) was 100 to 95 times higher than that of TM10 (MIC 0.0092 μmol·L -1 ), which is 82 times that of CIP; followed by TM4, TM6, TM11 and TM12 (MIC 0.0163~0.0357μmol·L -1 ), which is 46 to 21 times that of CIP; TM8 had the worst inhibitory activity (MIC 0.1230 μmol·L -1 ), but it also reached 6 times of CIP;

[0056] (5) TM7 had the best inhibitory activity against methicillin-resistant Staphylococcus aureus MRSA3393 (MIC 0.0158 μmol·L -1 ), CIP (MIC 6.0360 μmol·L -1 ) was 382 times higher than that of TM9 (MIC 0.0299 μmol·L -1 ), which is 201 times that of CIP; followed by TM3 and TM10 (MIC 0.0316 and 0.0369 μmol·L -1 ), which were 191 and 163 times that of CIP respectively; TM2 had the worst inhibitory activity (MIC 0.5210 μmol·L -1 ), but it also reached 11 times the CIP.

[0057] Example 4 Inhibitory activity of oxoalkanoyl clinafloxacin derivatives TM1-TM12 against other Gram-negative bacteria

[0058] As can be seen from Tables 2 and 3, for the seven human pathogens tested, the inhibitory activity of the oxoalkanoyl clinafloxacin derivatives TM1 to TM12 against Gram-positive bacteria is stronger than that against Gram-negative bacteria. In order to verify this inference, more different Gram-negative bacterial strains were further tested. The test results are shown in Table 5.

[0059] Table 5 Inhibitory activity of oxoalkanoyl clinafloxacin derivatives against other Gram-negative bacteria

[0060]

[0061] From the data in Table 5, it can be seen that the oxoalkanoyl clinafloxacin derivatives TM1 to TM12 have a significant effect on G - The inhibitory activity of G + However, the inhibitory activity of some compounds on some strains was comparable to that of the positive control CIP, or even stronger than that of CIP.

[0062] For E. coli, the inhibitory activity of TM12 against the three strains (S, R, ATCC11775) was stronger than that of CIP, which was 2 to 8 times that of CIP, indicating that TM12 could exert excellent antibacterial effect against E. coli; the inhibitory activity of TM7, TM8 and TM10 against strain ATCC11775 was also comparable to that of CIP;

[0063] For resistant Acinetobacter baumannii 1346, CIP had no detectable antibacterial activity, while the MICs of TM7 and TM10 were 8 and 16 μg·mL, respectively. -1 The antibacterial activity of TM7 and TM10 against Acinetobacter baumannii ATCC19606 was at least 4 to 2 times that of CIP, indicating that these two compounds are potential effective compounds against drug-resistant Acinetobacter baumannii. Combined with the results in Table 2, the MIC of TM7 and TM10 against Acinetobacter baumannii ATCC19606 was 64 μg mL -1 The inhibitory activity is 4 times that of CIP, indicating that TM7 and TM10 have good inhibitory activity against both standard strains and resistant strains of Acinetobacter baumannii.

[0064] For Klebsiella pneumoniae, TM8 had the most significant antibacterial activity. Its inhibitory activity against three strains (3617, 3024, and 3588) was comparable to or even stronger than that of CIP, and its inhibitory activity against two strains (ATCC700603 and 3026) was slightly lower than that of CIP. The inhibitory activities of TM2 and TM6 against two strains (3617 and 3588) were comparable to those of CIP. The inhibitory activity of TM12 against strain ATCC 700603 was comparable to that of CIP.

[0065] According to Table 5, it can be found that TM8 and TM12 have good antibacterial effects on most of the tested Gram-negative strains; combined with Table 4, TM1 to TM 12 have excellent antibacterial ability against Gram-positive bacteria, so TM8 and TM12 can be considered to have good broad-spectrum antibacterial effects.

[0066] Example 5 Test of the inhibitory activity of oxoalkanoyl clinafloxacin derivatives TM8 and TM12 against intracellular bacteria

[0067] Taking methicillin-resistant Staphylococcus aureus USA300 as the test strain, the broad-spectrum antibacterial oxaloyl clinafloxacin derivatives TM8 and TM12 were selected as representative compounds, and CIP was used as the control to test the intracellular bacteria inhibition activity.

[0068] The specific steps are as follows: RAW264.7 cells were diluted with DMEM complete medium containing 10% fetal bovine serum and then plated in a 96-well plate, and placed in a cell culture incubator for overnight culture at 37°C. Pick a single colony of S.aureus USA300, culture it with LB medium at 37°C for 6 hours, centrifuge the bacterial solution at 8000rpm for 5 minutes, discard the supernatant, resuspend it with an equal volume of PBS, centrifuge it again and resuspend it with an equal volume of PBS. According to the volume ratio of bacterial solution: DMEM complete medium = 1:300, prepare DMEM complete medium with bacteria (to ensure that 20-30 bacteria are engulfed in one cell), use DMEM complete medium as a blank control (to ensure that the cells are not contaminated by bacteria during the experiment), culture it at 37°C for 2 hours, discard the supernatant, and wash it 3 times with PBS; use 50μg mL -1 Compound solutions of different concentrations (0.25 μg mL -1 , 0.5 μg mL -1 , 1 μg mL -1 , 2 μg mL -1 , 4 μg mL -1 ), 100 μL of the prepared compound solution was pipetted into a 96-well plate, with at least 5 replicate wells for each concentration, and DMEM complete medium containing only 50 μg / mL gentamicin was added to one column as a negative control (to observe whether gentamicin can completely eliminate extracellular bacteria); incubated at 37°C for 6 h, the supernatant was discarded, and the cells were washed 3 times with PBS, 100 μL of 1% Triton cell lysis buffer was added, gently blown, and treated for 0.5 h, PBS was diluted and plated for counting, and 5 concentration gradients were diluted in total, and then incubated at 37°C for 24 h, and the number of colonies was counted.

[0069] Test results see Figure 1 The results of TM8 and CIP were basically the same. Under the five concentration gradients, TM8 had no clearance effect on intracellular Staphylococcus aureus USA300. However, compound TM12 had no clearance effect on intracellular Staphylococcus aureus USA300 under the lower concentration conditions (0.25, 0.5, 1, 2 μg·mL -1 ), but when its concentration increased to 4 μg mL -1 When TM12 was added to the mixture, it had a 10-fold clearance effect on intracellular bacteria, indicating that TM12 had a potential therapeutic effect on the clearance of intracellular bacteria at high concentrations.

[0070] Example 6 Bacterial resistance test of oxoalkanoyl clinafloxacin derivatives TM8 and TM12

[0071] The methicillin-resistant Staphylococcus aureus USA300 was used as the test strain, and the oxaloyl clinafloxacin derivatives TM8 and TM12 with broad-spectrum antibacterial activity were selected as representative compounds. CIP was used as the control to conduct a bacterial resistance test.

[0072] The specific steps are as follows: Methicillin-resistant Staphylococcus aureus USA300 was cultured in LB medium at 37°C for 6 h, and the bacterial solution concentration was adjusted to 1.0×10 6 CFU mL -1 , culture for a period of time under the action of 1 / 2MIC compound, observe the growth of bacteria every day, detect the changes in the MIC of bacteria to the compound, and judge its resistance.

[0073] Test results see Figure 2 The inhibitory activity of CIP against methicillin-resistant Staphylococcus aureus USA300 began to decrease significantly after the 10th generation. Compared with the 0th generation, the antibacterial ability of CIP in the 10th generation was reduced by at least 8 times, indicating that CIP has developed resistance to the USA300 strain; while the inhibitory activity of compounds TM8 and TM12 against methicillin-resistant Staphylococcus aureus USA300 remained almost unchanged within 15 generations, and the inhibitory activity began to decrease after the 15th generation, indicating that compared with CIP, compounds TM8 and TM12 did not induce the development of rapid resistance to methicillin-resistant Staphylococcus aureus USA300 in a short period of time, suggesting that these two compounds have development prospects in the field of antibacterial drugs for multidrug-resistant bacteria.

[0074] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. Use of an oxyalkanoyl clinafloxacin derivative in the preparation of an antibacterial drug, wherein the oxyalkanoyl clinafloxacin derivative has the following structural formula: In the formula, n is 3, R is propionyl or H; That is, the oxoalkanoyl clinafloxacin derivative is the following compound TM8 or TM12: The antibacterial drug is a drug against Staphylococcus aureus.

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