JPS6345392B2 - - Google Patents

Description

Detailed Description of the Invention The present invention relates to a novel 7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid derivative, more specifically, the following formula () In the formula, Y represents a hydroxyl group, a lower alkoxy group, a lower alkanoyloxy group, a phenylacetylamino group, or a mono- or di-(lower alkyl)amino group; _R2 represents a hydrogen atom or an unsubstituted or substituted benzyl group The present invention relates to compounds represented by , and salts thereof, and their use as antibacterial agents. The present inventors previously calculated the following formula () We developed a new antibiotic with the structure shown in
This was named antibiotic PS-5 and published [J.
Antibiotics, 31 , 480-482 (1978) and JP-A-Sho
See No. 53-121702 and Publication No. 54-30195]. The present inventors have conducted extensive research on derivatives that have even better antibacterial activity and/or stability than the above antibiotic PS-5, and have found that the compound represented by the above formula () has excellent antibacterial activity. Heading Completing the Invention. In this specification, the term "lower" refers to a group or compound to which this term is attached having 6 or less carbon atoms;
This means that the number is preferably 4 or less. Therefore, in the above formula (), examples of the "lower alkoxy group" include methoxy, ethoxy, n -propoxy, isopropoxy, n -butoxy, etc., and the "lower alkanoyloxy group"
Examples include acetoxy, propionyloxy, n -butyryloxy, isobutyryloxy, etc., and mono- or di-(lower alkyl)
Examples of "amino group" include methylamino, ethylamino, propylamino, dimethylamino,
Included are diethylamino, methylethylamino, dipropylamino, and the like. In addition, examples of the substituent on the benzene ring in the "unsubstituted or substituted benzyl group" include a halogen atom, a lower alkyl group, a lower alkoxy group,
Examples include a lower haloalkyl group, a nitro group, a lower alkylsulfonyl group, and the benzene ring is preferably monosubstituted with such a group. Specific examples of such "unsubstituted or substituted benzyl group" include benzyl, p-nitrobenzyl, o
-Nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl, p-fluorobenzyl, p-bromobenzyl, p-methoxybenzyl, p-methylsulfonylbenzyl, p-trifluoromethylbenzyl, etc., among which p- The -nitrobenzyl group is advantageous because it can be easily removed by hydrogenolysis. Among the compounds represented by the above formula (), a preferable group of compounds includes compounds represented by the formula () in which Y represents a hydroxyl group or a di-(lower alkyl)amino group. Furthermore, in formula (), R ₂ is preferably a hydrogen atom or a p-nitrobenzyl group, particularly,
Hydrogen atoms are advantageous for antibacterial activity. The compound of the above formula () when R ₂ is a hydrogen atom can also exist in the form of a carboxylate salt, examples of such salts include alkali metal salts such as sodium salts, potassium salts, lithium salts, etc. Salts; alkaline earth metal salts such as calcium salts and magnesium salts; other metal salts such as aluminum salts; ammonium salts; primary and secondary salts such as monoethylamine, dimethylamine, trimethylamine, monoethanolamine, diethanolamine, etc. or salts with tertiary amines; salts with other organic bases such as benzathine salts and procaine salts, etc. Among them, pharmaceutically acceptable salts are preferred, and among them, alkali salts such as sodium salts and potassium salts are preferred. Metal salts are preferred. Representative examples of the compound of formula () or a salt thereof provided by the present invention are as follows. 3-(2-hydroxyethylthio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0] hept-2-ene-2-carboxylic acid-p-nitrobenzyl ester, 3-(2-hydroxyethyl thio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid sodium salt, 3-(2-hydroxyethylthio)-6-ethyl-7-oxo- 1-Azabicyclo[3.2.0] hept-2-ene-2-carboxylic acid benzyl ester, 3-(2-acetoxyethylthio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0] hepta-2 -ene-2-carboxylic acid sodium salt, 3-(2-acetoxyethylthio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid-p-nitro Benzyl ester 3-(2-ethoxyethylthio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid-p-nitrobenzyl ester, 3-(2- ethoxyethylthio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid sodium salt, 3-(2-phenoxyethylthio)-6-ethyl- 7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylic acid-benzyl ester, 3-(2-phenoxyethylthio)-6-ethyl-7-oxo-1-azabicyclo [ 3.2.0] hept-2-ene-2-carboxylic acid sodium salt, 3-(2-N,N-dimethylaminoethylthio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0] hepta- 2-ene-2-carboxylic acid-p-nitrobenzyl ester, 3-(2-N,N-dimethylaminoethylthio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0]hepta-2- En-2-carboxylic acid. 3-(2-phenylacetamidoethylthio)-
6-ethyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid-p
-Nitrobenzyl ester 3-(2-phenylacetamidoethylthio)-
6-Ethyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-carboxylic acid sodium salt, 3-(2-N-methylaminoethylthio)-6
-Ethyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid-p
-Nitrobenzyl ester, 3-(2-N-methylaminoethylthio)-6
-Ethyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, etc. () according to the present invention, (A) the following formula () In the formula, _R2 ' represents an unsubstituted or substituted benzyl group, (i) A compound represented by the following formula () Y- _CH2 - _CH2 -SH...() In the formula, Y has the above meaning or (ii) react with a reactive derivative of the thiol compound of the above formula (), (B) then obtain the following formula: (-c) In the formula, Y and R ₂ ' have the above-mentioned meanings. A compound represented by the formula can be easily produced by subjecting it to hydrogenolysis, if necessary. The reaction between a compound of formula () and a thiol compound of formula () or a reactive derivative thereof is usually performed in an N,N-
In solvents such as dimethylformamide (DMF), tetrahydrofuran (THF), dioxane, hexamethylphosphoramide (HMPA), glyme, etc., especially
In DMF, about -20°C or less, preferably about -30°~
Advantageously, it is carried out under low temperature conditions in the range of about -50°C. The reaction can be completed under such conditions, usually within 15 minutes to 60 minutes. When the thiol compound of formula () is used as it is, it is usually carried out in the presence of a base, but the reaction proceeds even in the absence of a base. Usable bases include, for example, sodium hydride, potassium hydride, sodium amide, potassium amide, sodium hydroxide, potassium hydroxide, sodium ethoxide, sodium methoxide, potassium butoxide, triethylamine, tripropylamine, and the like. These bases are advantageously used in a proportion of at least 0.9 equivalents, preferably 1.0 to 1.2 equivalents, per mole of the thiol compound. Furthermore, in the above reaction, instead of the presence of a base, a reactive derivative of the thiol compound of formula () may be used, and examples of such reactive derivatives include sodium ethanethiolate, sodium butanethiolate, potassium ethanethiolate,
Potassium butane thiolate and the like can be mentioned. Although the amount of the thiol compound of formula () or its reactive derivative used is not critical, it is generally used in a ratio of at least 1.0 mol, preferably 1.1 to 1.5 mol, per 1 mol of the compound of formula (). It's advantageous. In this way, a compound of the above formula (-c) is obtained, which can be isolated and purified by the method described below if necessary, and then subjected to hydrogenolysis to remove the group R ₂ '. With this, the following formula (-b) In the formula, Y has the above-mentioned meaning and can be changed to a compound represented by or a salt thereof. Isolation and purification of the compound of formula (-c) above can be carried out, for example, by diluting the reaction mixture with an inert solvent that is immiscible with water, such as benzene, toluene, methylene chloride, ethyl acetate, etc. liquid or sodium bicarbonate solution and then PH6.8
After washing with phosphate buffer and drying this solvent layer over anhydrous sodium sulfate, it was evaporated under reduced pressure, and the residue was dissolved in a small amount of the above inert solvent, and Biobeads S-series (styrene-divinylbenzene polymer; This can be carried out by passing the reaction mixture through a column (manufactured by BioRad) and developing it with the same solvent, or by combining methods such as passing it through a silica gel column if necessary. compounds can be isolated. Hydrogenolysis of the compound of formula (-c) can be carried out according to a method known per se, for example, in a solvent such as water or an ether such as dioxane or tetrahydrofuran in the presence of a buffer with a pH of 8 or higher. This can be carried out by treatment with hydrogen in the presence of a hydrogenation catalyst such as platinum oxide, palladium on carbon, palladium black or the like. The compound of formula (-b) thus obtained can be isolated in the form of a common salt from the reaction mixture by the method described above. The compound of formula () used as a starting material in the above method is a new compound, for example, the antibiotic PS-
5, by an esterification method similar to that used for conventionally known carboxyl group-containing antibiotics (e.g. penicillin, cephalosporin, etc.).
For example, by the esterification method described in JP-A-52-149374, pages 36-48, the following formula () In the formula, R ₂ ' has the above-mentioned meaning, and can be produced by forming an unsubstituted or substituted benzyl ester of antibiotic PS-5 represented by and then S-oxidizing the compound of formula (). can. The S-oxidation of the compound of formula () can be carried out by a method known per se, for example, the S-oxidation of sulfur-containing β-lactam antibiotics such as penicillins and cephalosporins.
The oxidation can be carried out according to methods often used for oxidation. For example, the compound of the formula () is a mild oxidizing agent that does not substantially act on the 7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid skeleton, that is, perbenzoic acid, It is reacted with phenyldichloro iodide or sodium metaperiodate. Suitable such mild oxidizing agents are organic peracids, in particular perbenzoic acid and m-chloroperbenzoic acid, most preferably substituted perbenzoic acids such as m-chloroperbenzoic acid. . The reaction between the compound of formula () and the above oxidizing agent can be carried out using an inert solvent such as methylene chloride, chloroform,
in a solvent such as carbon tetrachloride at room temperature or lower;
It is advantageous to conduct the reaction under mild temperature conditions, preferably about -30° to about 20°C. The reaction can be completed under such conditions, usually within 3 minutes to 3 hours. The amount of the oxidizing agent used to oxidize the compound of formula () can vary widely depending on the type of oxidizing agent, reaction conditions, etc.;
It is useful within the range of 0.3 to 1.8 molar equivalents, preferably 0.7 to 1.3 molar equivalents, per mole of the compound. After completion of the reaction, the compound of formula (), which is the S-oxidation product, can be isolated and purified by various methods known per se. It can be isolated from the reaction solution depending on the method utilized. The compound of the formula () obtained by the method described above, in which R ₂ is a hydrogen atom, can be prepared by methods known per se, such as sodium hydrogen carbonate, disodium phosphate, dipotassium phosphate, 2-ethyl It can be converted into a salt by treatment with an inorganic base such as potassium hexanoate or ammonia; or an organic base such as monoethylamine, dimethylamine, triethylamine, monoethanolamine, diethanolamine, benzathine, or procaine. The compound of formula () used as a starting material in the above method, for example the antibiotic PS-5, is
J. Antibiotics, 31 , 480-482 (1978) and JP-A-53-121702 and JP-A-54-30195, antibiotics obtained by fermentation are produced by fermentation. PS-5 is the following formula (-a) It has been confirmed that the compound of the formula () of the present invention derived from this antibiotic PS-5 also has the 5,6-trans configuration shown by the following formula (-a). In the formula, Y and R ₂ have the meanings given above and have a 5,6-trans configuration as shown. Compounds of the above formula () provided by the present invention, especially the following formula (-b) The compound represented by the following formula or a salt thereof, in which Y has the above-mentioned meaning, has excellent antibacterial activity. For example, the following formula The antibacterial activity of the compound represented by or its sodium salt is as shown in Table 1 below. [Table] [Table] The original language names of the test bacteria used in Table 1 above are as follows. Bacillus subtilis ATCC6633
ATCC6633) Sarcina lutea Staphylococcus aureus FDA209P (Staphy-lococcus aureus
FDA209P) Staphylococcus aureus Smith strain
Staphylococcus aureus Smith)
aureus Russell) Staphylococcus epidermidis
epidermidis) Alcaligenes fuecalis A1 (Alcaligenes epidermidis)
faecalis Al) Citrobacter freundei GN346
(Citrobacter freundii GN346) Comamonas terrigena B-996 (Comamonas
terrigena B-996) Enterobacter aerogenes E19
(Enterobacter aerogenesE19) Enterobacter cloacae45
(Enterobacter cloacae 45) Enterobacter E8 (Enterobacter sp.E8) Escherichia coli K-12 (Escherichia coli K-12) Escherichia coli RCN823 (Escherichia coli RGN823) Klebsiella pneumoniae K13
pneumoniae K-13) Proteus mirabilis P6 (Proteus mirabilis P6)
mirabillis P6) Proteus rettgeri P7 (Proteus rettgeri P7) Proteus vulgaris GN (Proteus vulgaris GN)
GN76) Proteus P22 (Proteus sp. P22) Providencia P8 (Providencia sp. P8) Pseudomonas aeruginosa IF03445
IFO3445) Pseudomonas aeruginosa NCTC10490 (Pseudomonas aeruginosa)
NCTC10490) Serratia marcescens S18 (Serratia
marcescens S18) Serratia marcescens T55 (Serratia
marcescens T55) The above antibacterial activity was measured as follows. Namely, heart infusion agar medium “Difco” [manufactured by Difco Laboratories]
The minimum inhibitory concentration of the compound of the formula (-b) against the various test bacteria in Table 1 above was determined by the agar dilution test method using the following method. The specimen was dissolved in sterile distilled water, and multiple dilutions were prepared. 1 ml of this antibiotic solution and 9 ml of Heart Infusion Agar medium (Difco), which had been dissolved and sterilized and heated to about 60° C., were mixed in a 9 cm diameter shear dish and plated. Trypto soy broth medium [manufactured by Eiken Chemical Co., Ltd.]
The inoculum of the test bacteria shown in the table above, obtained by static culture at 35°C for 18 to 20 hours, was diluted with the same trypto soy broth medium to adjust the number of bacteria to approximately 10 ⁸ cells/ml, and then Agar plates containing the antibiotic were inoculated with an inoculator. After culturing this plate at 35°C for 20 hours, the presence or absence of growth is observed, and the lowest concentration at which no growth is observed is defined as the minimum inhibitory concentration of the compound against the bacterium. The antibacterial spectrum characteristics of the compound of formula (-b) measured in this manner are as described above. PS-5 sodium salt, which is the starting material of the derived compound of formula (-b), exhibits superior antibacterial activity compared to cefazolin (CEZ), which is commonly used among known β-lactam drugs, and has been used extensively. It is known to be effective against Pseudomonas aeruginosa, Serratia sp., Proteus sp., Klebsiella sp. and Enterobacter sp.
However, the compound of formula (-b) has better antibacterial activity than PS-5 sodium salt, which has excellent antibacterial activity, and in particular, the 3-di-(lower alkyl)aminoethylthio derivative It has about 2 to 4 times more antibacterial activity than PS-5, and has the same or higher activity against Gram-negative bacteria. For example, it has about 2 to 4 times more activity than PS-5 against Pseudomonas aeruginosa. ing. In particular, 3-(2-dimethylaminoethylthio) derivative (compound 2) can be cited as an excellent antibacterial compound. In addition, 2 mg of the compound of formula (-b) was subcutaneously administered to each of 5 mice, and 5 minutes after administration, a small amount of blood was collected from the eye into a heparinized capillary tube and centrifuged. , the concentration of the compound in the supernatant (plasma, plasma) of Comamonas
The measurement was carried out by a bioassay method using Terigena B-996 as a test bacterium. Table 2 below shows the average values of 5 mice. Table: All compounds of formula (-b) tested were well absorbed and gave fairly high maximum concentrations. Among them, 3-
The di-(lower alkylamino)ethylthio-derivative gave excellent highest concentrations. Also, the expression (-
In b), compounds having a di-(lower alkyl)aminoethyl group or a hydroxyethyl group at the end of the S side chain at the 3-position had good sustained blood concentration. Next, mice were intraperitoneally infected with Staphylocotcus aureus Smith strain at 5 × 10 ⁵ cells per mouse (5 mice per group, male ddy mice, Shizuoka).
The in vivo activity of the compound of formula (-b) was measured using Two hours after infection, an injection solution containing the sodium salt or inner salt of the compound of formula (-b) was administered subcutaneously. Infection treatment test ( _CD50 )
The results are shown in Table 3 below. PS-5 of raw materials
As is clear from the results in Table 3, the sodium salt [Table] is 2-
Dimethylaminoethylthio compound (compound 2)
shows a better therapeutic effect than the starting material PS-5 sodium salt. In addition, among the compounds of formula (-b), 2-hydroxyethylthio compound (compound 1) and 2-dimethylaminoethylthio compound (compound 2) were intraperitoneally administered up to 20 mg (1 g/Kg) per mouse.
No compounds were found to cause mouse death. From the above in vitro and in vivo activity data, the following formula (-b) Among the compounds represented by or salts thereof, compounds in which Y is dimethylamino, hydroxy, etc. (such as the above-mentioned compounds 2 and 1) and salts thereof can be said to be particularly suitable as antibacterial agents. As mentioned above, the compound of formula (-b) or a salt thereof exhibits antibacterial activity and can be used as an active ingredient of an antibacterial agent for the prevention, treatment and/or treatment of infectious diseases caused by Gram-positive and Gram-negative bacteria. It can be effectively used for the prevention, treatment, treatment, etc. of bacterial infections not only for humans but also for non-human animals such as mammals, poultry, and fish. The compound of formula (-b) or a salt thereof can be administered orally, topically, or parenterally (intravenously, intramuscularly, intraperitoneally, etc.), and depending on the method of administration, it is usually administered. It can be formulated and used in various drug forms as described above. For example, the compound of formula (-b) or a salt thereof may be prepared in solid form (e.g., tablet, capsule, powder, granule, dragee, troche, powder, spray) together with a pharmaceutically acceptable carrier material, diluent, etc. , suppositories, etc.), semisolid forms (e.g., ointments, creams, semisolid capsules, etc.), or liquid forms (e.g., solutions, emulsions, suspensions, lotions, syrups, injections, liquid sprays, etc.). can do. The unit dosage form containing the compound of formula (-b) or a salt thereof, whether liquid, semi-solid or solid, generally contains 0.1 to 99% by weight, preferably 10% by weight.
May contain ~60% by weight of active ingredient.
The content of the active ingredient in the drug varies depending on the dosage form and total amount of the drug, but is usually about 10 mg to about 1000 mg.
mg, preferably in the range of about 100 mg to about 1000 mg. The unit dosage form for parenteral administration is usually a compound of formula (-b) of the present invention or a salt thereof dissolved in sterile water with a purity close to 100%, or a compound with a solubility that can be easily made into a solution. It can be made into powder. In normal oral or parenteral administration, the formula (-
The compound b) is generally 0.05 to 500 mg/day.
Kg, preferably in the range of 0.5 to 200 mg/Kg, but it goes without saying that the dosage may be higher or lower than the above, depending on the condition of the patient to be treated. It is. The compound of formula (-b) can not only be used as a drug, but also be added to animal feed directly or as a concentrate for feed formulation, and as an active ingredient in food preservatives or disinfectants. can also be used. Next, the present invention will be further explained by examples. Example 1 3-(2-acetamidoethylthio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0]
Production method of hept-2-ene-2-carboxylic acid benzyl ester (PS- 5 benzyl ester) 3-(2-acetamidoethyl Drying of 1204 mg (purity 82%) of sodium thio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0] hept-2-ene-2-carboxylate (hereinafter abbreviated as PS-5 sodium salt) 4.75 ml of triethylamine (hereinafter referred to as TEA) was added to a 200 ml solution of dimethylformamide (hereinafter referred to as DMF), and 3.68 ml of benzyl bromide was added while stirring under ice cooling in a stream of room air. After returning to room temperature and stirring for 3 hours, the reaction mixture was poured into 1000 ml of benzene, washed three times with 300 ml of 0.1M, PH6.8 phosphate buffer, and the solvent layer was dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure at 30°C or below, it was dissolved in a small amount of benzene and subjected to column chromatography (120 g, 3.2 x 75.0 cm) using Bio-Beas S-X3 using benzene as a developing agent. Each eluted fraction was confirmed by thin layer chromatography (hereinafter abbreviated as TLC), fractions containing the target compound were collected, and the solvent was distilled off under reduced pressure to obtain 1081 mg of colorless powder. [α] ²¹ _D +19.97° (c0.33, CHCl ₃ ). UVλ ^MeOH _nax nm (ε): 318 (11000). IRν ^CHCl _3nax cm ^-1 : 3430 (CO NH ), 1770 (β-lactam
CO), 1690 (ester CO), 1665 ( CO NH). NMR (CDCl ₃ ) δ: 1.04 (3H, t, J = 7Hz,
CH ₂ CH ₃ ), 1.82 (2H, m, CH ₂ CH ₃ ), 1.96
(3H, s, COCH ₃ ), 2.80~3.60 (7H, m,
3XCH ₂ , CH), 3.94 (1H, dt, J=9.0Hz, J
= 3Hz, C-5H), 5.21 (1H, d, J = 12Hz,
Ar CH・H), 5.38 (1H, d, J=12Hz,
ArCH・H ), 5.90-6.10 (1H, brs, NH ),
7.34 (5H, distorted, s, Ar H ). Mass(m/
e): 388 (M ⁺ ). Example 2 3-(2-acetamidoethylthio)-6-ethyl-7-oxo-1-azabicyclo [3.2.0]
Production of hept-2-ene-2-carboxylic acid-(p-nitrobenzyl) ester (PS-5/p-nitrobenzyl ester) 275 mg of PS-5 sodium salt was dissolved in 20 ml of dry DMF, and while stirring at room temperature. 197 μl of TEA was added. Ten minutes later, p-nitrobenzyl bromide 179
A solution prepared by dissolving 5 ml of dry DMF in 5 ml of dry DMF was added at 5° C. with stirring, and the mixture was stirred at the same temperature for 3 hours. After the reaction is complete, pour the reaction mixture into 150ml of benzene,
It was washed three times with 100 ml of 0.1M, PH6.8 phosphate buffer. After drying over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was transferred to 30 g of silica gel (1.8 x 30 cm).
The mixture was subjected to column chromatography using a benzene-acetone (3:1) elution fraction to obtain yellow crystals as a solvent distillation residue. When this is recrystallized from benzene, mp: 240mg of yellow needle crystals at 163-165℃
I got it. [α] ²¹ _D +70.7° (c1.00, CHCl ₃ ). UV λ ^CHCl _nax nm (ε): 322 (12800), 269 (12000). IR ν ^CHCl3 _nax cm ^-1 : 3460 ( NH CO), 1780 (β-lactam CO), 1710 (ester CO), 1675 (NH CO ),
1530, (NO ₂ ), 1350 (NO ₂ ). NMR (CDCl ₃ ) δ: 1.04 (3H, t, J = 7.0Hz,
CH ₂ CH ₃ ), 1.84 (2H, m, CH ₂ CH ₃ ), 1.96
(3H, s, NHCO CH ₃ ), 2.80-3.60 (7H, m,
3XCH ₂ , CH), 3.98 (1H, dt, J=9.0Hz, J
=3Hz, C- 5H ), 5.19(1H, d, J=14Hz,
ArC H・H), 5.49 (1H, d, J=14Hz,
ArCH・H ), 5.95 (1H, brs, NH ), 7.61
(2H, d, J=9Hz, Ar H ), 8.18 (2H, d,
J=9Hz, ArH ). Mass (m/e): 433 (M ⁺ ), 363 (M ⁺ −EtCH=C
=O). Anal・Calcd.forC ₂₀ H ₂₃ O ₅ N ₃ S:C, 55.43;H,
5.35; N, 9.70. Found: C, 55.29; H,
5.24; N, 9.40. Example 3 Process for producing sulfoxide of PS-5 benzyl ester PS-5 benzyl ester 50 obtained in Example 1
Dissolve 28.74 mg of m-chloroperbenzoic acid in 5 ml of dry methylene chloride, cool with ice water, and stir while stirring.
A solution of mg (1.1 times mole based on the raw material) dissolved in 1 ml of dry methylene chloride was added dropwise, and the mixture was vigorously stirred at the same temperature for 15 minutes. After the reaction is completed, dilute with 50 ml of methylene chloride and wash twice with 25 ml each of 0.1M, PH8.7 and 6.8 phosphate buffers. The solvent layer was dried over anhydrous sodium sulfate, evaporated under reduced pressure, and immediately dissolved in 1 ml of benzene. Bio-Beas S-X3 column (1.2×
75.0 cm), and after developing with benzene, fractions containing the target substance were collected, and the solvent was distilled off under reduced pressure to obtain 53.5 mg of a colorless dry solid. [α] ²¹ _D −43.96 (c1.00, MeOH). UVλ ^MeOH _nax nm (ε): 307 (4440). IRν ^CHCl3 _nax cm ^-1 : 3400 ( NH CO), 1790 (β-lactam CO), 1710 (ester CO), 1670 (NH
CO). NMR (CDCl ₃ ) δ: 1.04 (3H, t, J = 7.0Hz, J
=3Hz, CH ₂ CH ₃ ), 1.86 (2H, m, CH ₂ CH ₃ ),
1.94 (3H, s, CO CH ₃ ), 2.90-3.84 (7H, m,
_3XCH2 , CH ), 3.88-4.20 (1H, m, C-5
H), 5.26 (2H, s, ArcH ₂ ), 6.40 (1H, m,
NH), 7.36 (5H, s, ArH ). Mass (m/e): 404 (M ⁺ ). Example 4 Preparation of sulfoxide of PS-5.p-nitrobenzyl ester 240 mg of PS-5.p-nitrobenzyl ester was dissolved in 25 ml of dry methylene chloride and cooled to -30°C. 104.9 mg of m-chloroperbenzoic acid was dissolved in 12 ml of dry methylene chloride, added to the above solution with stirring, and reacted at the same temperature for 40 minutes. After the reaction was completed, an ice-cooled solution was prepared by adding 0.09 ml of TEA to 100 ml of ethyl acetate, and the reaction solution was poured into this solution. After cooling on ice and stirring for 5 minutes, add 100 ml of ethyl acetate, transfer to a separatory funnel, and add 0.1 M
Washed four times with PH6.8 phosphate buffer. After drying the solvent layer with anhydrous sodium sulfate, benzene was added and 1
It was concentrated under reduced pressure to ml. Silica gel column 18 where the concentrated solution was pre-wetted with benzene-acetone (1:1)
(42 ml, 2.0 x 13.4 cm, same as the above-mentioned silica gel) and developed with about 50 ml of the same solvent, 14.1 mg of the raw material was eluted in this section. Further, the title target compound was eluted (yield 211 mg, 89
%). The Rf value of this substance on a silica gel TLC plate (same as the TLC plate above) is benzene-
Acetone (1:2) showed 0.36. [α] ²¹ _D +18.0° (c1.00, CHCl ₃ ). UVλ ^CHCl3 _nax nm (ε): 267 (13079), 310 (7775)
. IRν ^CHCl _3nax cm ^-1 : 3460 ( NH CO ), 1785 (β-lactam CO ), 1720 (ester CO ), 1680 ( NH CO ). NMR ( _CDCl3 ) δ: 1.00 (3H, t, J=7.0Hz, _CH2CH3 ), 1.70-2.00 (2H, m _{, CH2CH3} ), 2.00 ₍ 3H, s, _NHCOCH3 ), 3.00 ~3.88 (7H, m, 3XCH ₂ , CH), 3.92 ~ 4.23 (1H, m, CH), 5.12 ~ 5.60 (2H, distorted d, ArC H・H,
ArCH.H ), 6.25-6.55 (1H, brs, NH ), 7.53-7.72 (2H, ArH ), 8.12-8.28 (2H, ArH ). Mass (m/e): 449 (M ⁺ ) (FDMass). Example 5 3-(2-hydroxyethylthio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0]
Preparation of hept-2-ene-2-carboxylic acid para-nitrobenzyl ester A solution of 200 mg of PS-5 para-nitrobenzyl ester sulfoxide in 50 ml of dry DMF was mixed with 58 μl of TEA and 2-mercaptoethanol under stirring at -45°.
Add 39 μl and stir at the same temperature. After 40 minutes, the reaction mixture was added to 200 ml of benzene, washed four times with 150 ml of 0.1 M, PH6.8 phosphate buffer, and the solvent was dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain a colorless oil. Ta. When this was subjected to column chromatography (2.6 x 12 cm) using silica gel (30 g), benzene-acetone (10:1, V/V)
149.2 mg (85%) of colorless powder was obtained. IRν ^CHCl3 _nax cm ^-1 : 3450 (OH), 1790 (β-lactam C=O), 1700 (ester C=O). UVλ ^THF _nax nm (ε): 320 (10400), 270 (10600). [α] ²² _D ⁺ 33.2° (C1.0inTHF). NMR δ ( _CDCl3 ): 1.04 (3H, t, J, 7.5Hz, _{OH2CH3 )} , 1.75-2.05 ( 2H, m , _CH2CH3 ), 2.85-3.30 ₍ 5H, m, 2XCH3) ₂ , CH), 3.70-4.10 (3H, m, C-5H, -CH2OH ) 5.18 (1H, d, J, 14Hz, ArC H H), 5.49 ₍ 1H, d, J, 14Hz, ArCH H ), 7.80 (2H, d, J, 9.0Hz, Ar H ), 8.16 (2H, d, J, 9.0Hz Ar H ), MS (m/e): 392 (M ⁺ ), 322 (M ⁺ −EtCH= C=
O), 304 (M ⁺ -EtCH=C=O- _H2O ). Example 6 3-(2-hydroxyethylthio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0]
Production of hept-2-ene-2-carboxylic acid sodium salt 3-(2-hydroxyethylthio-6-ethyl-7-oxo-1-azabicyclo[3.2.0] hept-2-ene-2-carboxylic acid -20mg of paranitrobenzyl ester in 2ml of dioxane, 0.1MPH8.4
Dissolved in 1.5 ml of phosphate buffer mixed solvent,
Add 20mg and use Pearl reduction equipment (hydrogen pressure 4Kg/
Catalytic reduction was carried out using (cm ² ) for 4 hours at room temperature.
After the reaction, the catalyst was removed using a filter aid, and the filtrate was adsorbed on a QAE-Sephadex A-25 column (1.1 x 20 cm) that had been treated with 0.01M phosphate buffer to reduce the concentration of 0 to 0.4M sodium chloride. Gradient gradient method (using 100ml of 0.01M phosphate buffer and 100ml of 0.01M phosphate buffer with 0.4M sodium chloride concentration)
It was eluted. In this eluate, we collected the sections with maximum absorption at 300 nm in the ultraviolet absorption spectrum,
After adjusting the salt concentration to 3% with sodium chloride, it was adsorbed onto a Diaion Hp-20 column (1.1 x 20 cm). This is done using the 0-30% acetone concentration gradient method (using 100ml of ion-exchanged water and 100ml of 30% acetone water).
5.6
mg (39.4%). UVλ ^H2O 〓 _nax nm (ε): 300 (7302) *0.1M phosphate buffer (PH7.0). NMR δ( _D2O ): 1.00 (3H, t, J _, 7Hz, CH2CH3 ), 1.60-2.00 (2H, m _, CH2CH3 ₎ , 2.80-3.40 ₍ 5H, m, 2XCH3 ) ₂ , C H ), 3.74 (2H, t, J, 6Hz, C H ₂ OH), 4.00 (1H, dt, J, 9Hz, J, 3Hz, O H ), Example 7 3-(2-acetoxy ethylthio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0]
Production of hept-2-ene-2-carboxylic acid paranitrobenzyl ester [Method A] 3-(2-hydroxyethylthio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0] hepta-2 13.3 μl of TEA was added to a solution of 3.75 mg of -ene-2-carboxylic acid-paranitrobenzyl ester in 2 ml of dichloromethane, and 6.8 μl of acetyl chloride was added dropwise while stirring at -10°. After stirring at the same temperature for 10 minutes, the reaction mixture was diluted with 10 ml of benzene to 0.1 M
It was washed three times with 5 ml of phosphate buffer at pH 6.8.
After drying the solvent over Na ₂ SO ₄ , the solvent was distilled off under reduced pressure to obtain a yellow oil. Dissolve this in a small amount of benzene,
Bio-beads S- using benzene as a developing solvent
Column chromatography using X3 (100g,
3.2 x 75.0 cm), then each eluted fraction was confirmed by thin layer chromatography, fractions containing the target compound were collected, and the solvent was distilled off under reduced pressure to obtain 3.2 mg (77.1%) of a colorless oil. UVλ ^THF _nax nm (ε): 317 (9600), 268 (9800). IRν ^CHCl3 _nax cm ^-1 : 1780 (β-lactam C=O),
1740 (O- CO Me), 1700 (ester C=O). _NMR δ ( _CDCl3 ): 1.08 (3H, t, J _, 7Hz, CH2CH3 ), 1.70-2.20 ( _4H , m , _{CH2CH3 )} , 2.00 (3H, s, COC H3 ), 2.80~3.50 (5H, m, 2XC H ₂ , CH), 3.98 (1H, dt, J, 9.5Hz, J, 3Hz, C ₅ -H), 4.20 (2H, t, J, 7.5Hz, CH ₂ OCO -), 5.20 (1H, d, J, 14Hz, ArCH H ), 5.50 (1H, d, J, 14Hz, ArCH H ), 7.59 (2H, d, J, 9Hz.0, ArH ), 8.15 ( 2H, d, J, 9.0Hz, ArH ). [Method B] 3 mg of 3-(2-hydroxyethylthio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0] hept-2-ene-2-carboxylic acid-paranitrobenzyl ester was dissolved in tetrahydrofuran (hereinafter referred to as , abbreviated as THF), dissolved in 2 ml of pyridine
3.0 μl and 7.2 μl of acetic anhydride were added, and the mixture was stirred at room temperature for 3 hours. After the reaction is complete, the reaction solution is cooled on ice to a pH of 6.8.
The mixture was poured into 3 ml of 0.1M phosphate buffer and stirred at the same temperature overnight. The mixture was extracted with benzene, and saturated aqueous sodium hydrogen carbonate solution (5 ml x 2), and
It was washed with 0.1M phosphate buffer (5 ml x 3) of pH 6.8. After drying the solvent over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure to obtain a yellow oil. This was treated in the same manner as in Method A to obtain 2.2 mg (66%) of the desired product. Example 8 Production of 3-(2-ethoxyethylthio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid-paranitrobenzyl ester PS-5. To a solution of 20 mg of paranitrobenzyl ester sulfoxide in 15 ml of dry DMF were added 5.4 μl of TEA and 5.9 μl of 2-ethyloxyethyl mercaptan while stirring at −45°, and the mixture was stirred at the same temperature for 20 minutes. Add the reaction mixture to 40ml of benzene and add 0.1M
After washing four times with 20 ml of PH6.8 phosphate buffer, the solvent was dried over anhydrous sodium sulfate and evaporated under reduced pressure.
A yellow oil was obtained. This was subjected to column chromatography (1.3 x 4 cm) using silica gel (5 g).
When doing this, benzene-acetone (20:1, V/
From V), 10.6 mg (56.7%) of a colorless oil was obtained. IRν ^CHCl3 _nax cm ^-1 : 1775 (β-lactam C=O),,
1700 (ester C=O). UVλ ^THF _nax nm (ε): 320 (14100), 270 (12400). [α] ²² _D : +45.7° ( c 1.0, THF). NMR δ ( _CDCl3 ): 1.06 (3H, t, J, 7.0, CH2CH3 ), 1.18 (3H _, t _, J, 7.0, CH2CH3 ), 1.70-2.00 _{( 2H} , m, C H ₂ CH ₃ ), 2.90-3.70 (9H, m, C-6H, C-4H,
3XCH ₂ ), 3.91 (1H, dt, J, 9Hz, J, 3Hz, CH _-5 ), 5.18 (1H, d, J, 13Hz, ArC H H), 5.48 (1H, d, J, 13Hz, ArCH H ), 7.60 (2H, d, J, 9Hz, Ar H ), 8.15 (2H, d, J, 9Hz, Ar H ). MS (m/e): 420 (M ⁺ ), 350 (M ⁺ −EtCH=C=
O), 304 (M ⁺ -EtCH=C=O-EtOH). Example 9 3-(2-N,N-dimethylaminoethylthio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid-paranitrobenzyl ester Production: 150 mg (0.335 mmol) of PS-5 p-nitrobenzyl ester sulfoxide in 70 ml of dry DMF
and cooled to -60°C. Add 107μl of TEA to this solution while stirring, and add N,N
-Dimethylcysteamine, hydrochloride 54.7mg
(0.387 mmol) was added and reacted for 30 minutes. The reaction solution was poured into 300 ml of benzene, and washed with 0.1 M phosphate buffer (PH8.5) (150 ml x 2) and 200 ml of saturated saline. The organic layer was dried over anhydrous sodium sulfate, and benzene was distilled off under reduced pressure to obtain a pale yellow oil. This product was swollen with acetone, applied to a packed Sephadex LH20 column (φ1.2 x 80 cm), developed and eluted with acetone. By searching for the target substance using thin layer chromatography, collecting the target substance-containing fractions, and distilling off the solvent under reduced pressure, 105mg of
(yield 75%) of the target product was obtained. IRν ^CHCl3 _nax cm ^-1 : 1779 (β-lactam C=O),
1705 (ester C=O). UVλ ^THF _nax nm (ε): 322 (12100), 270 (10700). [α] ²² _D +38.5° (c1.0, THF). NMR δ ( _CDCl3 ): 1.04 (3H, t, J=7.5Hz, -CH2CH3 ), 1.70-2.00 (2H _, m _, -CH2CH3 ), 2.28 _{( 6H} , s, -N ( CH3 ) ₂ ), 2.44-3.40 (7H _, m, C- 6H , C- 4H2 _,
N-C H ₂ C H ₂ -S), 3.98 (1H, dt, J=4Hz, J=9Hz, C-5
H), 5.24 (1H, d, J = 14Hz, -CH H Ar), 5.54 (1H, d, J = 14Hz, CH H Ar), 7.68 (2H, d, J = 9Hz, -CH ₂ Ar H ) , 8.24 (2H, d, J=9Hz, -CH ₂ ArH), Mass (m/e): 419 (M ⁺ ), 404 (M ⁺ -CH ₃ ), 349
(M ⁺ -EtCH=C=O). Example 10 Production of 3-(2-N,N-dimethylaminoethylthio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid 3-(2 -N,N-dimethylaminoethylthio)-6-ethyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid paranitrobenzyl ester 120mg to 8ml 50%
The mixture was dissolved in an aqueous dioxane solution, 120 mg of platinum oxide was added, and a reaction was carried out at room temperature for 4 hours in a Parr reduction apparatus (hydrogen pressure 4 Kg/cm ² ). The catalyst was separated, and the liquid and washing liquid were combined and concentrated under reduced pressure to 3 ml. Transfer the concentrated solution to Dowex50W (×8) Na type column (φ1.0×20cm)
The mixture was eluted with water and fractionated into 5 g portions. 297nm
The sections with ultraviolet absorption are collected and freeze-dried,
32 mg (yield 39%) of the target product was obtained. UVλ ^H2O _nax nm (ε): 297 (7026). _NMR δ( _D2O ): 1.01 (3H, t, J=7.5Hz, -CH2CH3 ), 1.60-2.00 (2H _, m , _-CH2CH3 ), 2.65 ( _6H , sN( C H ₃ ) ₂ ), 3.00-3.50 (7H, m, C-4 H ₂ , C-6・H,
-S-C H ₂ C H ₂ N), 4.04 (1H, dt, J=3Hz, J=9HzC- 5H ). Example 11 3-(2-phenylacetamidoethylthio)-
6-Ethyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid-
Production of para-nitrobenzyl ester 150 mg (0.334 mmol) of PS-5-p-nitrobenzyl ester sulfoxide was dissolved in 40 ml of DMF and cooled to -50°C. 51μl of this solution
(0.366 mmol) of TEA was added with stirring.
Furthermore, a solution of 72 mg (0.369 mmol) of N-phenylacetylcysteamine dissolved in 1 ml of DMF was added dropwise and reacted for 1 hour. Pour the reaction solution into 80ml of benzene, and add this to 0.1M phosphate buffer (PH6.8) (100ml).
After drying the organic layer over anhydrous sodium sulfate, benzene was distilled off under reduced pressure to obtain a pale yellow oil. This product was applied to a column (1.3 x 21 cm) packed with silica gel, and developed and eluted with a benzene-acetone (1:1 V/V) solvent. The target product was searched for by thin layer chromatography and the target categories were collected, yielding 134.4 mg (79%) of the target product. [α] ²² _D +50.4. (c0.80, THF) IRν ^CHCl3 _nax cm ^-1 : 1775 (β-lactam CO), 1700
(ester) UVλ ^THF _nax nm (ε): 321 (11300), 268 (9800) NMR (CDCl ₃ ) δ: 1.07 (3H, t, J = 7.2Hz - CH ₂ C H ₃ ) 1.70-2.00 (2H, m, CH ₂ CH ₃ ) 2.80-3.55 (7H, m, C-6H, C-4H,
2XCH ₂ ) 3.56 (2H, s, COCH ₂ ) 3.97 (1H, dt J=3.0Hz, J=9.0Hz, C-5H), 5.22 (1H, d, J=14.0HzArC H H) 5.51 (1H, d , J=14.0HzArCH H ) 5.82 (1H, m, NH) 7.30 (5H, s Ar H ) 7.65 (2H, d, J=9.0HzAr H ) 8.21 (2H, d, J=9.0HzAr H ) Mass (m /e): 509 (M ⁺ ) 439 (M ⁺ -EtCHCO) Example 12 3-(2-phenylacetamidoethylthio)-
Production of 6-ethyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid sodium salt 3-(2-phenylacetamidoethylthio)-
120 mg of 6-ethyl-7-oxo-1-azabicyclo[3.2.0] hept-2-ene-2-carboxylic acid paranitrobenzyl ester, 10.2 ml of THF,
Dissolved in 9.4ml of 0.1M phosphate buffer (PH7.0) mixed solvent, added 170mg of platinum oxide, and dissolved under hydrogen pressure (4.0ml).
Kg/cm ² ) The mixture was stirred at room temperature for 3.5 hours using a Pearl reduction apparatus. After the reaction, the product was treated and purified in the same manner as in Example 6 to obtain 31.8 mg (yield: 34%) of the desired product. UVλ ^H2O _nax nm (ε): 301 (4800). NMR δ(D ₂ O): 1.02 (3H, t, J7.5Hz-CH ₂ C H ₃ ), △1.80 (2H, m, -CH ₂ CH ₃ ), 2.7-3.35 (5H, m, [Formula ]C ₄ - H, C ₆ -H), 3.43 (2H, t, J=6.0Hz, -S-C H ₂ -), 3.62 (2H, s, [formula]), 3.90 (1H, m, C ₅ -H), 7.28 (5H, s, -CH ₂ ph ). Next, a method for producing a typical pharmaceutical preparation containing the compound of formula (-b) or a salt thereof of the present invention will be illustrated. In addition, the compound number has the above-mentioned meaning. [Table] Grind the above antibiotics and excipients in a mortar and mix uniformly. Approximately 200 mg per capsule is packed into a No. 8 hard gelatin capsule with an enteric coating. [Table] Thoroughly mix the antibiotic with half of the lactose and corn starch in the mixing ratio above. The mixture is granulated with 10% starch paste and sieved. Add the remainder of the corn starch and magnesium stearate to this, mix well, and make a 1cm diameter and weight
Form into 500mg tablets. This is coated with sugar and then coated with an enteric coating. [Table] Dissolve the above antibiotics in sterile water for injection,
Disinfect. The solution is dispensed into sterilized ampoules, water is removed aseptically by freeze-drying, and the opening of the ampule is sealed. Before use, open the package, add 2 ml of sterile physiological saline to the contents of the ampoule, and dissolve. [Table] Compound 3 and cephaloridine were mixed, formulated and molded in the same manner as in the formulation method of Example B, and coated with sugar coating and then coated with an enteric coating. [Table] [Table] Compound 1 and aminobenzylpenicillin are mixed and prepared in the same manner as in Example B. [Table] The above antibiotic derivative and excipients are polished and mixed uniformly. Approximately 200 mg per capsule is packed into a No. 8 hard gelatin capsule with an enteric coating. [Table] Thoroughly mix the antibiotic derivative with half of the lactose and corn starch in the above mixing ratio. mixture
Mix with 10% starch paste solution to granulate, and pass through a sieve. Add the remaining corn starch and magnesium stearate to this, mix well, and make a
cm, mold into tablets with a weight of 500 mg per tablet. This is coated with sugar and then coated with an enteric coating. [Table] Dissolve the above antibiotic derivatives in sterile water for injection and sterilize by filtration. The solution is dispensed into sterilized ampoules, water is removed aseptically by freeze-drying, and the mouth of the ampule is sealed. Before use, open the package, add 2 ml of 70% sterile N-(β-hydroxyethyl)lactamide aqueous solution to the contents of the ampoule, dissolve, and use. [Table] Mix this antibiotic derivative with cephaloridine,
Formulated and molded in the same manner as the formulation method of Example G,
After sugar coating, it is further coated with an enteric coating. [Table] Compound 2 and aminobenzylpenicillin are mixed and formulated in the same manner as for [tablets].

Claims (1)

[Claims] 1. The following formula () In the formula, Y represents a hydroxyl group, a lower alkoxy group, a lower alkanoyloxy group, a phenylacetylamino group, or a mono- or di-(lower alkyl)amino group; _R2 represents a hydrogen atom or an unsubstituted or substituted benzyl group , and its salts. 2 The compound of the formula () has the following formula (-a) The compound according to claim 1 and a salt thereof having a 5.6-trans configuration represented by: 3 The following formula (-b) In the formula, Y represents a hydroxyl group, a lower alkoxy group, a lower alkanoyloxy group, a phenylacetylamino group, or a mono- or di-(lower alkyl)amino group. An antibacterial agent containing a compound or a salt thereof as an active ingredient. .