JPH0160010B2 - - Google Patents

Description

[Detailed description of the invention]

The present application relates to a drug comprising a cephalosporin antibiotic. Specifically, the present invention relates to a drug having cephalosporin-like activity, which loses antibacterial activity by chemically modifying a cephalosporin antibiotic, but regains antibacterial activity once absorbed into the body. Cephalosporin antibiotics are currently widely used and are drugs with excellent selective toxicity against bacteria. However, since it has an antibacterial effect on beneficial bacterial flora resident in the living body,
In particular, these drugs have the serious drawback of disturbing the bacterial flora in the intestines. This drawback is significant when antibiotics are taken orally. As a result, diseases such as bacterial alternation may occur, and in some cases, colitis, diarrhea, etc. may occur. The present inventors have intensively investigated antibiotics having cephalosporin-like activity that do not have these drawbacks, and have found that cephalosporin derivatives represented by the general formula () are effective, leading to the present invention. Therefore, an object of the present invention is to provide an antibacterial agent containing a cephalosporin antibiotic as an active ingredient. The present invention will be explained in detail below. The present invention is based on the general formula () [In the formula, R ₁ is

【formula】

[Formula] {wherein R ₃ is -O (CH ₂ ) _o H (in the formula, n represents 2 to 4), -OCH
(CH ₃ ) ₂ , -OC(CH ₃ ) ₃ ,

【formula】

[Formula] or

The present invention relates to an antibacterial agent characterized in that it contains a cephalosporin antibiotic derivative represented by the following formula as an active ingredient. The antibacterial agent of the present invention can be used particularly as an oral antibacterial agent. Furthermore, it can be used as an oral antibacterial agent that does not disturb the resident flora of living organisms. The compound represented by the above general formula () (hereinafter referred to as the present substance) can be obtained by chemically modifying a cephalosporin antibiotic.
It is a brand new type of antibiotic that is absorbed without affecting the resident flora of the body when administered, and only shows antibacterial activity once it enters the bloodstream.It is also an extremely safe substance as its acute toxicity is low. It can be said. The pharmacological effects of this substance are shown below. (a) Acute toxicity: Acute toxicity was investigated by intraperitoneal and forced oral administration using ICR-JCL mice. This substance was dispersed in physiological saline for both intraperitoneal and oral administration, and the solution was adjusted to a predetermined amount using a syringe or a stomach tube and administered. After administration, poisoning symptoms were continued to be observed, and the LD ₅₀ value was determined from the mortality rate over time up to the 7th day. Survival cases,
In both cases of death, autopsies were performed to obtain findings. _LD50 values are Litchfield-Wilcoxon (Litchfield
-Wilcoxon) Calculated using graphic calculation method. As a result, both intraperitoneal and oral administration of this substance
The _LD50 value was 10g/Kg or more. (b) Effect on intestinal bacteria: This substance was orally administered to mice at 500 mg/Kg for 2 days, and mouse feces were collected before and on the 1st day after administration. A portion of this was cultured in various media at 25° C. or 37° C. for 1 to 5 days and examined for Escherichia coli, Pseudomonas aeruginosa, Streptococcus, Lactic acid bacteria, Bifidobacterium, and Bacteroides. There was almost no difference in the number of each of the above bacteria before and after administration of this substance. Therefore, it can be seen that this substance does not substantially affect the intestinal flora. (c) Antibacterial activity: Examined in accordance with the standard method of the Japanese Society of Chemotherapy.
Esherichia coli as a test bacterium
The minimum inhibitory concentration (MIC) was determined using IFO12734Staphylococcus aureus IAM1011. (d) In order to prove that the activity changes when absorbed into the body, the following experiment was conducted using a metabolically activating enzyme [rat liver homogenate (hereinafter referred to as S-9mix)]. Prepare a preculture of Staphylcocccus aureus IAM1011 at 108/ml, then 50 times the volume of Mueller
The preculture solution was added to Hinton agar medium and plated. Place a penicillin cup with a diameter of 8 mm on a flat plate,
0.1 ml of this substance or a culture of this substance and S-9mix was added thereto, incubated at 37°C for 18 hours, and the diameter of the circle of inhibition was measured. When comparing the diameter of the inhibition circle for cephalothin sodium or penicillin as 100, it was 0 for the system containing only this substance. On the other hand, it was 0 to 1 for the system of this substance + S-9 mix. This indicates that although this substance has low antibacterial properties as it is, it is activated by enzymes after entering the body. (e) Effect on infectious diseases: In order to confirm that it is activated in vivo, we conducted therapeutic experiments on infectious diseases using this substance. Esherichia intraperitoneally in 20 mice in each group.
After inoculation and infection with E. coli IFO, 500 mg/Kg of each of the present substances was orally administered immediately after infection and 4 hours later, and the presence or absence of death due to infection was determined on the 7th day. All cases in the untreated group died on the second day, whereas all cases of this substance showed a survival rate of 35% or more, indicating that it is effective as an oral anti-infective agent. As mentioned above, this substance is safe, has no effect on intestinal flora, and can be said to be a new cephalosporin antibiotic that enters the body and becomes active. Since this substance is converted into a cephalosporin antibiotic in vivo, it can be used as an antibacterial agent in the same field as cephalosporin antibiotics. The present substance can be used as a pharmaceutical composition containing at least one cephalosporin antibiotic represented by the general formula () and a pharmaceutically acceptable carrier, diluent or auxiliary agent, and further in a unit dosage form. These can be administered orally, by injection or by rectal administration. Oral administration may be in the form of tablets, capsules, powders, granules, powders, pills, ampoules, and the like. These include fillers, extenders, binders, wetting agents,
Disintegrants, dissolution slowing agents, reabsorption promoters, adsorption carriers,
Includes lubricants, etc. Specifically, powder, mannitol, silicic acid, cellulose derivatives, gelatin, alginate, glycerin, agar, calcium carbonate, sodium bicarbonate, paraffin, quaternary ammonium compounds, glycerin monostearate, kaolin, bentonite, talc, Examples include potassium stearate, magnesium stearate, and polyethylene glycol. It may also be a pharmaceutically acceptable emulsion solution or suspension. Suppositories may include polyethylene glycol and fatty acids or esters thereof. Sills and elixirs may be used as liquid compositions containing an inert diluent such as water or paraffin and suitable for oral administration. They may also contain auxiliary agents such as wetting agents, sweetening agents and flavoring agents. Compositions for injection administration may be sterile, aqueous or non-aqueous solutions, suspensions, or emulsions, and may contain, for example, propylene glycol, polyethylene glycol, olive oil, and the like. When used as a composition, the material may contain from 0.01 to 99.5%, usually from 0.1 to 90%, as active ingredient. This substance is used in a similar way to cephalosporin antibiotics and is useful in treating infections of bacterial origin. The dosage of the drug varies depending on the degree of infection and the patient's condition, but it is generally administered per adult patient per day.
Administer 0.1-10g in several doses. Hereinafter, the present invention will be explained in more detail based on examples. However, the present invention is not limited only to the following examples. Example 1 1587 mg of 7-(thiophene-2-acetamido)cephalosporanic acid was suspended in 20 ml of benzene. After adding one drop of DMF, 1016 mg of oxalyl chloride was slowly added dropwise. The reaction was carried out at 7 to 10°C for 45 minutes with gentle stirring.
After the reaction was completed, benzene was distilled off under reduced pressure. The acid chloride of cephalosporin thus obtained was dissolved in 10 ml of methylene chloride. 4 ml of a methylene chloride solution containing 2.4 g of n-propyl alcohol and 404 mg of triethylamine was slowly added dropwise. Stir for about 1 hour at 4°C. After the reaction was completed, 20 ml of methylene chloride was added thereto, and the methylene chloride layer was washed with 10 ml of an aqueous HCl solution of PH3. Next 1
Washed with 10 ml of ₃ % NaHCO3 aqueous solution. Finally water 10
After washing with ml of methylene chloride, the methylene chloride layer was dried with MgSO ₄ .
After concentration under reduced pressure, 1210 mg of crude crystals were obtained. Recrystallization from methylene chloride-n-hexane yielded 964 mg of product. The yield was 44%. The results of elemental analysis values are shown below. C(%) H(%) N(%) Theoretical value 52.04 5.06 6.38 Measured value 51.4 4.9 6.4 Example 2 A similar reaction was carried out using iso-propyl alcohol in place of n-propyl alcohol in the reaction of Example 2 to obtain 560 mg of a crude product. Recrystallization from methylene chloride-n-hexane gave 328 mg of product. The yield was 15%, and the melting point was 139-140°C. The elemental analysis values are shown below. C(%) H(%) N(%) Theoretical value 52.04 5.06 6.38 Measured value 52.8 5.2 6.2 Example 3 A similar reaction was carried out using t-butyl alcohol instead of n-propyl alcohol in the reaction of Example 2 to obtain 516 mg of a crude product. Recrystallization from methylene chloride-n-hexane gave 330 mg of product. The yield was 15%, and the melting point was 175-176°C. The elemental analysis values are shown below. C(%) H(%) N(%) Theoretical value 53.10 5.3 6.19 Actual value 53.4 5.3 6.1 Example 4 Phenyl 7-(thiophene-2-acetamido)cephalosporanate was synthesized by the following method. 7-(thiophene-2-acetamido)cephalosporanic acid 2.0 g, phenol 0.47 g and N,
1.05 g of N'-dicyclohexylcarbodiimide was dissolved in 100 ml of tetrahydrofuran, and the solution was stirred at room temperature for 24 hours. After removing the generated N,N'-dicyclohexylurea, the solvent of the liquid was distilled off, and the residue was then dissolved in 100 ml of chloroform. Add the chloroform solution to 5% aqueous hydrochloric acid solution,
% aqueous sodium bicarbonate solution and water, and then dried over anhydrous magnesium sulfate. After evaporating the solvent, the residue was recrystallized from ethyl acetate-n-hexane to obtain 1.6 g (yield: 68%) of crystals. The melting point is
The temperature was 162-163℃. The physical property values are shown below. (a) Infrared absorption spectrum ν _nax (cm ^-1 ) (KBr) 1778; 1660; 1492; ; 1230 (b) Elemental analysis value (as C ₂₂ H ₂₀ O ₆ N ₂ S ₂ ) C(%) H( %) N(%) Calculated value 55.46 4.24 5.93 Actual value 55.40 4.23 5.88 Example 5 1.6 g of 7-(thiophene-2-acetamido)cephalosporanic acid was dissolved in 20 ml of THF solution containing 400 mg of triethylamine and cooled on ice. 4ml
A solution of 430 mg of ethyl chlorocarbonate dissolved in THF was slowly added dropwise. After reacting for 1 hour, it was further cooled for 1 hour. After evaporating the solvent under reduced pressure, the residue was dissolved in 10 ml of chloroform. The chloroform solution was thoroughly washed with 1% HCl, 2% NaHCO ₃ solution, and water in that order, and then dried with MgSO ₄ . Recrystallization from carbon tetrachloride yielded 200 mg of product.
The melting point was 96-99°C. The physical property values are shown below. (a) Infrared absorption spectrum ν _nax (cm ^-1 ) (KBr) 3301; 1777; 1733; 1655; 1535; 1238 (b) Ultraviolet absorption spectrum n _nax (nm) (CH ₃ CN) 234266 (c) Elemental analysis C(%) H(%) N(%) Calculated value 48.71 4.30 5.98 Actual value 49.4 4.4 6.0 Example 6 (

[Formula] represents a cyclohexane ring) The above compound was obtained as a by-product of the reaction of Example 5. After distilling off the solvent of the recrystallization solution in Example 5, the residue was recrystallized from n-hexane containing a small amount of ethyl acetate to obtain 0.24 g (yield: 21%) of crystals. The melting point was 151-152°C. The physical property values are shown below. (a) Infrared absorption spectrum ν _nax (cm ^- ) (KBr) 3340; 2905; 1782; 1702; 1652; 1225 (b) Elemental analysis value (as C ₂₉ H ₃₈ N ₄ C ₆ S ₂ ) C (%) H(%) N(%) Calculated value 57.78 6.35 9.29 Actual value 57.8 6.3 9.3 (c) Ultraviolet absorption spectrum λ _nax (nm) (CH ₃ CN) 236; 266 Example 7 Effect of each compound on intestinal flora Examined. Each of the above drugs was orally administered at a dose of 500 mg/Kg per day for 2 days to a group of 5 female ICR mice (6 weeks old). Before administration and on the 7th day after administration, feces from each mouse were collected, diluted with 100 times the volume of anaerobic diluent (phosphate buffer), ground, and 0.1ml of each sample shown in Table 1 below. It is applied to the culture medium of the bacteria to be measured and cultured aerobically or anaerobically (anaerobic glove box method) at 37°C or 25°C for 1 to 5 days.
The numbers of streptococci, lactic acid bacteria, bifidus bacteria, and bacteroides bacteria were measured.

【table】 The results are shown in Table 2.

【table】

[Table] As is clear from Table 2, an increase in Escherichia coli was observed in the cephalothin administration, but the numbers of each of the substances did not change much from before administration. Furthermore, while cephalothin reduces the number of lactic acid bacteria, the number of lactic acid bacteria in each of these substances remains unchanged from the number before administration. Example 8 Antibacterial activity was measured by the agar plate dilution method in accordance with the standard method of the Japanese Society of Chemotherapy. Test method: Test bacteria Esherichia coli IFO 12734 Staphylococcus aureus IAM 1011 The above strains were inoculated into Mueller-Hinton medium at 37°C.
After culturing for 18 to 48 hours, the culture was adjusted to 10 ⁶ /ml and used as a test bacterial solution. Add 1/9 volume of each sample solution at the specified temperature to Mueller-Hinton medium as a medium for drug susceptibility measurement.
An agar plate was prepared. The above test bacterial solution was smeared with a platinum loop on each plate in approximately 2 cm streaks, then cultured at 37°C for 18 to 24 hours, and the concentration at which bacterial growth was completely inhibited was defined as the minimum inhibitory concentration. . The results are shown in Table 3.

[Table] Example 9 The following method was adopted as a model experiment to prove that it is activated in the body. Rat liver homogenate (S-9, manufactured by Oriental Yeast Co., Ltd.) was used as a metabolic activating enzyme with the following composition (hereinafter referred to as S-9mix).
It was used in Composition in 1ml S-9 0.5ml KCl 3.3μmol MgCl ₂・6H ₂ O 8μmol Glucose-6-phosphate 5μmol NADH 4μmol NADPA 4μmol 0.2M phosphate buffer 0.5ml (PH7.4) Sample solution 0.1ml and S-9mix0 Mix .9ml or 0.9ml of 0.1M phosphate buffer as a control and incubate at 37℃ for 20 minutes.
The cells were cultured in separate spindles and subjected to susceptibility tests.
Mueller Staphylococcus aureus IAM1011
After inoculating Hinton medium and culturing at 37℃ for 18 hours,
A 50-fold amount of Mueller-Hinton agar medium was adjusted to 10 ⁸ /ml and plated. Place a penicillin cup (diameter 8 mm) on top of it, and place 0.1 of the above reaction solution in it.
ml and left at 4°C for 2 hours, cultured at 37°C for 18 hours, and the diameter of the growth inhibition zone was measured. The results are shown in Table 4.

[Table] Example 10 Effect on experimental infection in mice: Esherichia doli was administered to each group of 20 ddY SPF mice.
IFO 12734 1.4 × 10 ⁸ was inoculated intraperitoneally and infected, and 500 mg/Kg of this substance was orally administered twice, immediately after infection and 4 hours later, and the presence or absence of death due to infection was observed for 7 days. All of the animals in this group died on the second day after infection, but more than 20% of the animals in all groups administered with this substance were still alive on the seventh day after infection. Example 11 [1] Tablet This substance obtained in Example 1 175mg Lactose 16mg Starch 5mg Hydroxypropylcellulose 3.0mg Magnesium stearate 1.0mg 200mg/tablet This substance and lactose were mixed, and an aqueous hydroxypropylcellulose solution was added and kneaded. Then dry and crush. Magnesium stearate dispersed in starch was added and mixed in advance to this pulverized product, and the mixture was compressed into tablets using a conventional method. [2] Granules: The present substance obtained in Example 2: 176 mg Lactose: 16 mg Starch: 4 mg Hydroxypropyl cellulose: 4 mg The present substance, starch, and lactose are mixed together, an aqueous solution of hydroxypropyl cellulose is added, the mixture is dried, and pulverized. Granules were obtained by sieving in the range of 12 to 48 meshes.

Claims (1)

[Claims] 1 General formula () [In the formula, R ₁ represents [Formula], Y represents [Formula] {In the formula, R ₃ represents -O (CH ₂ ) _o H (in the formula, n represents 2 to 4) -OCH
An antibacterial agent characterized by containing as an active ingredient a cephalosporin antibiotic derivative represented by (CH ₃ ) ₂ , -OC(CH ₃ ) ₃ , [Formula] [Formula] or [Formula]]. 2. The antibacterial agent according to claim 1, wherein the antibacterial agent has an oral administration form. 3. The antibacterial agent according to claim 1 or 2, wherein the antibacterial agent has the effect of not disturbing intestinal flora.