JPH0245631B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention has the general formula () which has excellent antibacterial effect and β-lactamase inhibitory activity. (wherein R is optionally substituted ethyl and n represents 0 or 1, respectively) or a method for producing a salt thereof. More specifically, a compound represented by the general formula () or a salt thereof [hereinafter sometimes simply referred to as "compound ()"] is a compound represented by the general formula () (In the formula, R and n have the same meanings as above)
The compound represented by or its salt (hereinafter sometimes simply referred to as "compound ()") is added to an excess of 4
It can be produced by isomerizing ammonium halide. The isomerization reaction is preferably carried out in an organic solvent, and it is better to avoid using a highly polar solvent as the organic solvent. The principle of the above reaction can be considered as follows. In other words, both compounds () are water-soluble substances and cannot be extracted into the non-polar solvent layer using normal solvent extraction methods.However, when compound () and excess quaternary ammonium halide coexist, they become water-soluble. The sulfonic acid or carboxylic acid groups in the molecule form a quaternary ammonium salt, and the entire molecule becomes more fat-soluble and migrates to the non-polar solvent layer. The ammonium halide used in excess selectively acts on the double bond in the side chain portion, and the E (trans) type coordination compound () has a better yield than the Z (cis) type coordination compound (). It is converted with . Note that the addition reaction of ammonium salts to sulfonic acid groups or carboxylic acid groups occurs preferentially to sulfonic acid groups due to the difference in acidity of these groups. Quaternary ammonium halides that can be used in the present invention include reagents commonly used in ion-paired extraction methods, such as tetra n-pentylammonium, tetra n-hexylammonium,
Four substituents (e.g., carbon Quaternary ammonium chloride and bromide having a total carbon number of about 18 to 30 (alkyl (number 1 to 25), benzyl) can be used, but chloride is better in terms of extractability and reaction yield. be. These quaternary ammonium halides are used in excess amounts, and are usually used in amounts of 3 to 3 to
It is about 1000 times the mole. Preferably, the amount is about 3 to 100 times the mole for a starting compound having a sulfonic acid group, and about 100 to 1000 times the amount for a starting compound not having a sulfonic acid group. Examples of organic solvents include chloroform,
dichloromethane, 1,2-dichloroethane, 1,
1,1-trichloroethane, benzene, toluene, ethyl acetate, diisopropyl ether, etc. are used, but most preferably chloroform,
Examples include halogenated hydrocarbons such as dichloromethane and 1,2-dichloroethane. As described above, it is generally preferable to use a nonpolar solvent or a weakly polar solvent, but even if a small amount of a strongly polar solvent such as methanol, dimethylformamide, tetrahydrofuran, or water is mixed in the above organic solvent, it may interfere with the reaction. Must not be. The organic solvent is usually used so that the concentration of quaternary ammonium halide is about 0.5 to 3% with respect to the organic solvent. The reaction temperature is around the boiling point of each solvent, but the reaction can proceed even at room temperature (approximately 15°C), and 40 to 70°C is particularly convenient, and the time varies depending on the temperature and type of solvent, but the reaction can proceed at room temperature (approximately 15°C). It can be completed within 3 days. To isolate the target compound () from the reaction solution,
First, when the quaternary ammonium salt and quaternary ammonium halide of the compound () in the reaction solution are extracted with an aqueous solution of sodium iodide, potassium iodide, etc., the sodium or potassium salt of the compound () is extracted into the aqueous layer; The ammonium iodide salt is separated into an organic solvent layer. Furthermore, when quaternary ammonium chloride is used, it is also possible to use sodium bromide, potassium bromide, etc. Next, the aqueous layer containing the compound () can be separated from the starting compound () by, for example, chromatography. An example of a suitable chromatography system is high porous resin HP-20.
(manufactured by Mitsubishi Kasei Co., Ltd., Japan), basic cephadex
QAE-25 (Type C1, manufactured by Pharmacia, Sweden) and activated carbon (manufactured by Takeda Pharmaceutical, Japan) are used as carriers, and water or an aqueous solution of methanol or butanol or an aqueous solution of inorganic salts at an appropriate concentration is used as a carrier. can be used for elution and purification from the carrier. In general formulas () and (), R is, for example, general formula () [In the formula, R ₁ is hydrogen or methyl and R ₂ is hydrogen, hydroxy, R ₃ COO-[R ₃ is R ₄ or-]
NHR ₄ (R ₄ is alkyl, alkenyl, phenyl,
or phenyl or alkyl substituted with phenyloxy, provided that the phenyl group may be substituted with lower alkyl, alkoxy or halogen)] or R ₅ O ₃ SO- (R ₅ is hydrogen or lower alkyl), respectively] Examples include the "optionally substituted ethyl group" shown in
The alkyl for R ₄ is preferably an alkyl having 1 to 6 carbon atoms such as methyl, ethyl, propyl, butyl, pentyl, hexyl, etc., and R ₄ and
The lower alkyl for R ₅ is preferably methyl, ethyl, etc. having up to 3 carbon atoms. Examples of the alkenyl represented by R ₄ include vinyl, propenyl, butenyl, pentenyl, hexenyl and the like having up to 6 carbon atoms. As the alkoxy for R ₄ , methoxy, ethoxy, propoxy having up to 3 carbon atoms can be used.
The halogen for R ₄ is preferably chloro or fluoro. These raw material compounds are described in the publication of JP-A No. 54-144394 and the documents listed in Table 1, or are prepared using the compounds listed in Table 1 as raw materials.
It can be produced according to the method described in Publication No. 144394.

[Table] The structural formula of each compound () obtained from compound () by an isomerization reaction may be indicated by the "number" shown in Table 2.

【table】

【table】

[Table] Compounds () and () mentioned above were prepared using Microbondapak C ₁₈ (manufactured by Waters, USA) as a carrier and methanol - 0.02M as a mobile phase.
Phosphate buffer (PH6.3), UV (254nm) as detector
Each is quantified separately by high performance liquid chromatography (abbreviated as HPLC) using a detector.
Each Rt value is shown in the examples described below. In the present invention, examples of compounds () and salts of () include salts of alkali metals such as sodium and potassium, and salts of alkaline earth metals such as magnesium and calcium. Among the compounds obtained by the method of the present invention, general formula () The compound represented by (in the formula, R ₆ represents hydrogen or -SO ₃ H) and its salt are new compounds. The object compound () of the present invention is a valuable compound that exhibits activity against various Gram-positive and Gram-negative bacteria and exhibits potent β-lactamase inhibitory activity. In addition, these compounds have significantly improved stability in mouse kidney homogenate compared to the raw material compound, and are compounds that can be expected to have strong therapeutic effects due to increased stability in body fluids. Next, the antibacterial spectrum and inhibitory activity of the compound obtained according to the present invention are shown below.

【table】

[Table] The compound () obtained by the present invention exhibits antibacterial activity against Gram-positive bacteria and Gram-negative bacteria, as is clear from the above-mentioned antibacterial spectrum. Therefore, the compounds () can be used to treat bacterial infections in mammals (eg, mice, rats, dogs, humans) and domestic animals (eg, chickens, ducks). In order to use the compound () as a therapeutic agent for E. coli infection, for example, the compound () can be dissolved in physiological saline and administered parenterally subcutaneously or intramuscularly at 0.1 to 200 mg/Kg/day, preferably as an injection. is administered at a dose of 1 to 50 mg/Kg/day. Also, as an oral agent,
Compound () is mixed with lactose to make capsules,
The compound () is administered at 1 to 500 mg/Kg/day, preferably 5 to 200 mg/Kg/day. Moreover, the compound () obtained by the present invention
can be used as a fungicide. For example, a solvent in which the compound () is dissolved in distilled water at a concentration of 0.01 to 1.0 W/V%, or a base of petrolatum or lanolin, containing 0.5 to 50 mg of the compound () per gram;
Preferably, the ointment containing 2 to 20 mg can be used for sterilizing and disinfecting the hands, feet, eyes, ears, etc. of the above-mentioned animals. Compound () exhibits a beta-lactamase inhibitory effect and significantly enhances the susceptibility of penicillin- or cephalosporin-resistant bacteria to ampicillin and cefotiam due to their ability to produce beta-lactamase. Therefore, by combining the compound () with penicillins or cephalosporins, it is possible to treat bacteria in mammals (e.g. mice, rats, dogs, humans) and poultry (e.g. chickens, ducks), especially beta-lactam antibiotics. It can be used to treat infections caused by resistant bacteria. Compound () can be used in combination with other beta-lactam drugs, for example, to treat infections caused by beta-lactam antibiotic-resistant E. coli.
For example, the same amount of compound (2) and ampicillin are dissolved in physiological saline and administered as an injection parenterally subcutaneously or intramuscularly at 0.1 to 20 mg/Kg/day, preferably 0.5 to 5 mg/Kg/day. Further, as an oral preparation, a capsule containing the same amount of the compound (2) and cephalexin is administered at 1 to 200 mg/Kg/day, preferably 5 to 100 mg/Kg/day. As a bactericidal agent, for example, compound ()0.1~
10W/V% concentration and benzylpenicillin 0.1~
Hands and feet of the above-mentioned animals, either as a solution in an aqueous solution containing a concentration of 1.0 W/V%, or as an ointment based on petrolatum or lanolin and containing 5 to 20 mg of the compound () and 5 to 20 mg of benzylpenicillin per gram. It can be used to sterilize and disinfect the eyes, ears, etc. Compound () is also an extremely promising compound as a synthetic intermediate for new pharmaceuticals. Aqueous solutions of the compounds of the present invention are stable in the neutral pH range. Next, the present invention will be explained in more detail with reference to production examples and examples, but the present invention is not limited thereto. Production example 1 Streptomyces SB C-19393 strain (FERM-P No.4774, IFO 13886, ATCC31486)
200 ml of T-fold (2% oatmeal, 2% tomato paste,
0.2% Bovril (manufactured by Bovril, UK) and 2
% agar (PH7.0 medium) to allow spores to settle. Then, the number of viable bacteria is determined by adding the spores to sterile water.
Suspend at 1.2×10 ⁸ /ml. Dilute the spore suspension 10 times with sterile water, inoculate 1 ml of it into 40 ml of seed medium in a 200 ml Erlenmeyer flask, and incubate at 28°C for 2 hours.
The culture was cultured on a rotary shaking incubator for 1 day, and the culture solution was inoculated into 500 ml of seed medium in a 2-volume Sakaguchi shaking flask, and cultured at 28°C for 2 days on a reciprocating shaking incubator. Transplant into seed medium 100 containing 50 ml of Actocol in a steel fermenter, heat at 28°C, aerate at 70/min, and stir at 150 rpm.
The cells were cultured for 2 days. Next, the culture solution was transferred to 4 m ³ of the main medium in a 6 m ³ fermenter, and the aeration rate was 2800 at 30°C.
The cells were cultured for 3 days with stirring at 150 rpm. The seed medium contains 20 g of glucose, 30 g of soluble starch, 10 g of raw soybean flour, 10 g of corn steep liquor, 5 g of polypeptone (manufactured by Daigo Nutrient Chemical Co., Ltd.), 3 g of salt, and 5 g of precipitated calcium carbonate per seed medium.
g, and its pH was adjusted to 7.0 before sterilization. The main medium is 30g of glucose, 30g of soluble starch, 15g of defatted soybean flour, 15g of cotton seed flour, and 2g of phosphoric acid.
Potassium hydrogen 0.25g, potassium monohydrogen phosphate 0.6
g, cobalt chloride 0.002 g, and Actocol 0.5 g, and its pH was adjusted to 7.0 before sterilization. All media were steam sterilized at 120°C for 20 minutes. Hyflo Super Cell was added to the culture solution obtained as described above and filtered to obtain a solution of 4000.
After adjusting the liquid to PH6.3, Amberlite IRA-402
(Cl ^- form) through a column of 0.02M saline solution.
After washing with 200 ml of water, it was eluted with 1.5M saline solution with 1000 ml of 1.5M saline.
After passing the eluate through a column (70) packed with HP-20, the antibiotic was eluted with water 280. The eluate was passed through a column packed with activated carbon 15. water 45
After washing with water, the antibiotic was eluted with 7% isobutanol water. After concentrating the eluate to a concentration of 10%, 500 g of common salt was added and the mixture was passed through an HP-20 column (6).
Elution was carried out with 5% saline 36, and the eluate was passed through a column packed with activated carbon 3. After washing with water 7.5, 8
Eluted with 12% isobutanol in water. The eluate was concentrated under reduced pressure to 8%, and then WA-30 (acetic acid form)
column (500ml). It was washed with 2.5 ml of 0.2M acetic acid-sodium acetate buffer, and then the active ingredient was eluted with 5 ml of 1M saline in the same buffer. The eluate was passed through an activated carbon column (500 ml).
After washing with 5% saline, elution was performed with 2.5% of a 4:1 mixed solvent of 5% saline and methanol. Distill methanol under reduced pressure and reapply to activated carbon column (200ml)
passed through. After washing with 600 ml of water and 600 ml of 20% methanol water, elution was performed with 600 ml of 8% isobutanol. The eluate was concentrated under reduced pressure, acetone was added to the residue, and the powder was collected by filtration to obtain 580 mg of powder. Dissolve this powder in a small amount of water and use Amberlite.
It was passed through a column packed with 360 ml of XAD-(100-200 mesh), eluted with water, and fractionated. When the antibacterial fractions are collected, concentrated, dried, and acetone is added,
100 mg of powder was obtained. This was dissolved in a small amount of water, passed through a column packed with 40 ml of QAE-Sephadex A-25 (Cl ^- form), and fractionated by elution with 0.04 M phosphate buffer. The fractions that were found to have antibacterial properties were each subjected to the liquid chromatography analysis described above, and the fractions showing a single peak were collected and passed through a column packed with 10 ml of activated carbon. After washing with 30 ml of water, elute with 50 ml of 8% isobutanol water, concentrate the eluate to dryness, add acetone, and add C-
20 mg of sodium salt of 19393E ₅ was obtained. Production example 2 Antibiotic C-19393H _disodium salt crude powder (30% purity, 8 mg) was added to 10% methanol water (10 ml)
A mixture of 10% methanol water (10 ml) and 10% palladium-carbon (20 mg), which had been hydrogenated for 30 minutes, was added. The mixture was then reduced with hydrogen at 1 atm for 3 hours at room temperature, the catalyst was filtered off and the filtrate was concentrated in vacuo to a volume of 2 ml. This solution was poured into a column (10 ml) packed with XAD-2 (100-200 meshes) to adsorb the target compound, washed with water (50 ml), and eluted with 20% methanol-water. Fractions containing the compound were pooled. After distilling off the methanol from the effective fraction, lyophilize it to obtain 1.0 mg of [5R, 6R]-
3-[(E)-2-acetamidoethenylthio]-6
-[1-Hydroxy-1-methylethyl]-7-
A powder of sodium oxo-1-azabicyclo[3,2,0]hept-2-ene-2-carboxylate was obtained. UV: λmax (H ₂ O) 229 and 310 nm IR: νmax (KBr) 1760, 1620 cm ^-1 Thin layer chromatography [cellulose f (manufactured by Tokyo Kasei Co., Ltd.)]: Rf = 0.87 (solvent system: propanol: water = 4 :1) High performance liquid chromatography (Waters Inc., USA): Rt=8.2 minutes [Micro Bonder Pack
_C18 /14% methanol 0.02M-phosphate buffer (PH
6.3), 2 ml/min/cm (2000 psi)] However, under the same conditions, the Rt of the raw material compound was 4.3 minutes. NMR: δ (100MHz, D ₂ O, TMS): 1.33 (3H,
s, C ₈ - CH ₃ ), 1.44 (3H, s, C ₈ - CH ₃ ), 2.10
(3H, s, COCH ₃ ), 3.03 (1H, dd, J=10,
19, C ₄ - H), 3.85 (1H, dd, J = 10.5, 19, C ₄
-H), 3.72 (1H, d, J=6, C ₆ -H), 4.28
(1H, m, C ₅ - H), 6.10 (1H, d, J = 14, S
-CH=), 7.20 (1H, d, N-CH=) Production example 3 Antibiotic C-19393S ₂ Disodium salt coarse powder (30% purity, 60mg) was mixed with 10% methanol water (20ml)
This solution was added to a mixture of 10% methanol water (10 ml) and 10% palladium-carbon (20 mg) that had been hydrogenated for 30 minutes. The mixture was then reduced with hydrogen at 1 atm for 3 hours at room temperature, the catalyst was filtered off, and the filtrate was concentrated in vacuo to a volume of 2 ml. This solution was passed through a column (50 ml) of XAD-2 (100-200 mesh) to adsorb the target compound, and then eluted with water. 45ml to 150
Collect the elution fractions containing up to ml of the target compound,
Freeze-dry [5R, 6R]-3-[(E)-2-acetamidoethenylthio]-6-[1-(hydroxysulfonyloxy)-1-methylethyl]-7
7.3 mg of powder of disodium -oxo-1-azabicyclo[3,2,0]hept-2-ene-2-carboxylate was obtained. UV: λmax ( _H2O ) 228 and 309nm IR: νmax (KBr) 1760, 1620, 1240, 1050cm
^-1 Thin layer chromatography [cellulose f (manufactured by Tokyo Kasei Co., Ltd.)]: Rf = 0.65 (solvent system: propanol: water = 4:1) High performance liquid chromatography (manufactured by Waters Corporation): Rt = 4.4 minutes [ Micro Bonder Pack C _18/14
% methanol 0.02M-phosphate buffer (PH6.3), 2
ml/min/cm (2000 psi)], but under the same conditions, the Rt of the raw material compound was 2.2 minutes. NMR: δ (100MHz, _D2O , TMS): 1.63 (3H,
s, C ₈ - CH ₃ ), 1.70 (3H, s, C ₈ - CH ₃ ), 2.10
(3H, s, COCH ₃ ), 3.05 (1H, dd, J=10,
19, C ₄ - H), 3.82 (1H, dd, J = 10.5, 19, C ₄
-H), 3.88 (1H, d, J=6, C ₆ -H), 4.20
(1H, m, C ₅ - H), 6.10 (1H, d, J = 14, S
-CH=), 7.20 (1H, d, N-CH=) Production Example 4 (1) Dissolve the compound obtained in Production Example 3 (h100mg) in methanol (50ml) and add meta-chloroperbenzoic acid (78mg). added. Mixed liquid at 0-5℃
Stir for 30 minutes. The reaction solution was added to 0.02M phosphate buffer (PH6.3, 100ml) and concentrated. The concentrated solution (50 ml) was washed with ethyl acetate (50 ml), and the aqueous layer was washed with HP-20, which had been previously treated with 5% saline.
The resulting product was subjected to column chromatography (100-200 mesh, 100ml) and eluted and fractionated with a solvent system of methanol:5% saline (5:95). The fraction containing the target compound was detected by HPLC, and the effective fraction was desalted by activated carbon chromatography and lyophilized to produce [5R, 6R]-3-[(E)-2-acetamidoethenyl-(S). -Sulfinyl]-6-
Disodium salt of [1-hydroxysulfonyloxy-1-methyl-ethyl]-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid (k, 37.2 mg ) was obtained. (2) The compound obtained in Production Example 3 (h, 2 mg) was dissolved in acetonitrile (6 ml), and 12% hydrogen peroxide solution (4 ml) was added. The mixture was stirred at room temperature for 2 hours. HPLC confirmed the formation of compound (k) obtained in (1) in the reaction solution at a yield of 22%. Thin layer chromatography (hereinafter abbreviated as TLC) [Cellulose f (manufactured by Tokyo Kasei Co., Ltd.)]: Rf=
0.38 (Solvent system: propanol: water = 4:1) HPLC (manufactured by Waters): Rt = 1.6 min [Radial pack A, 2 ml/min (same below), 8%
Methanol 0.02M phosphate buffer (PH6.3, hereafter P.
B.) UV: λ ^H2O _nax nm (E1% 1cm) = 250 (298) and
288.5 (251) CD: [θ] ^H2O _on 240 (ε-56900) and 290 (+
32900) IR: ν ^KBr _nax 1760, 1700, 1255, ^{1050cm -1} PMR: δ (100MHz. D ₂ O, TMS - the same below); 1.66, 1.73 (3H × 2, s, 8 - CH ₃ ),
2.15 (3H, s, _NHCOCH3 ), 3.19 and 3.91
(1H×2, dd, H ₄ ), 3.93 (1H, d, H ₆ ), 6.44
(1H, d, S-CH=), 7.65 (1H, d, N-
CH=)ppm. Production Example 5 The compound obtained in Production Example 2 (g76 mg) was dissolved in methanol (38 ml), and meta-chloroperbenzoic acid (70 mg) was added. The mixture was stirred at 0-5°C for 30 minutes. Add water (40ml) to the reaction solution and mix with phosphate buffer.
The pH was adjusted to 6.3, concentrated, and washed with ethyl acetate. The aqueous layer was subjected to HP-20 (100 ml) column chromatography, eluted with water, and fractionated. Concentrate and freeze-dry the fraction containing the target compound [5R, 6R] -
3-[(E)-2-acetamidoethenyl-(S)-
Sodium salt of sulfinyl]-6-[1-hydroxy-1-methyl-ethyl]-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid (j, 25 mg) I got it. TLC: Rf = 0.64 HPLC: Rt = 3.8 min [8% methanol-PB] UV: λ ^H2O _nax nm (E1% 1cm) = 249 (476) and 285
(354) IR: ν ^KBr _nax 1770, 1700, 1630cm ^-1 CD: [θ] ^H2O _on 240 (ε-55000) and 287 (+
10100) PMR: 1.34, 1.45 (3H×2, s, 8-CH ₃ ),
2.15 (3H, s, NHCOCH ₃ ), 3.14, 3.83 (1H×
2, dd, H ₄ ), 3.84 (1H, d, H ₆ ), 6.31 (1H,
d, S-CH=), 7.56 (1H, d, N-CH=) Example 1 (1) Dissolve compound b (50 mg) in water (20 ml),
Extraction was performed with 1% tri n-octylmethylammonium chloride/dichloromethane solution (20 ml). After the extract was left at room temperature for 3 days, it was redissolved with 0.375% sodium iodide solution (20 ml). The aqueous layer was concentrated, and the concentrated solution was subjected to column chromatography on HP-20 (100-200 mesh, 30 ml), which had been previously treated with 5% saline, and eluted and fractionated with 5% saline and water. The resulting product was detected by HPLC, and single peak fractions were collected, concentrated, and lyophilized to produce [5R,6R]-3-[(Z)-2-acetamidoethenyl-(R)-sulfinyl]-6.
-[1-hydroxysulfonyloxy-1-
Methyl-ethyl]-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-
Disodium salt of carboxylic acid (b, 24.4mg)
I got it. HPLC: 8% methanol 0.02M phosphate buffer (PH6.3), 2ml/min (same conditions except methanol concentration, Rt = 5.9min, (Rt of b =
3.1min) UV: λ ^H2O _nax nm (E1% 1cm) = 241.5 (310) and
292.5 (215) IR: ν ^KBr _nax 1770, 1630, 1250, 720 cm ^-1 PMR (100 MHz): δ ^D2O _PPn 1.66 and 1.73 (8-
(CH ₃ ) ₂ , 3H each), 5.90 (S-CH=, 1H, d,
J=8Hz), 7.39(N-CH=,1H,d,J=8
Hz) (2) Dissolve compound b (42 mg) in water (10 ml),
Extraction was performed with 1% tri n-octylmethylammonium chloride/dichloromethane solution (20 ml). The extract was heated under reflux for 8 hours, and after cooling, 0.75
% sodium iodide (20ml). The aqueous layer was post-treated in the same manner as in (1) above to obtain the disodium salt of compound b (19.3 mg). (3) Dissolve compound b (100mg) in water (50ml),
Extraction was performed with a 1% tri-n-octylmethylammonium chloride/chloroform solution (50 ml). The extract was heated to reflux for 1.5 hours. HPLC showed a peak of 88% of the product. The reaction solution was transferred with 0.75% sodium iodide solution (25 ml). Concentrate the aqueous layer and use the concentrated liquid as HP-20 (100-
200 mesh, 125 ml) column chromatography and eluted with water. Concentrate the fraction containing the product, add acetone to the concentrated residue,
Powdered disodium salt of compound b (56.5
mg) was obtained. (4) Using compound b, as shown in Table 3, 4
The reaction was carried out by changing the combination of grade ammonium halide and solvent and the reaction conditions, and the same treatment as in (1) above was carried out, and the reaction product b was measured by HPLC.
The results are shown in Table 3. (The concentration of raw material compound b is 200μg/ml in each case)

[Table] Example 2 (1) Compound a (30 mg) was dissolved in dimethylformamide (3 ml), and a 2% tri-n-octylmethylammonium chloride/dichloromethane solution (300 ml) was added. The solution was heated to reflux for 8 hours, and after cooling, 3% sodium iodide (150 ml) was added.
It was transferred and melted. HPLC showed a peak of 82% of the product. After concentrating the aqueous layer, the concentrated liquid is HP-
20 (100-200 mesh, 30 ml) column chromatography, and eluted and fractionated with water. Concentrate and freeze-dry the fraction of the product, [5R, 6R] -
3-[(Z)-2-acetamidoethenyl-
(R)-sulfinyl]-6-[1-hydroxy-1-methyl-ethyl]-7-oxo-1-
Sodium salt of azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid (a,
19.8mg) was obtained. HPLC: 15% methanol, Rt=7.5min (a
Rt = 3.6min) UV: λ ^H2O _nax nm (E1% 1cm) 239 (367) and 295
(255) IR: ν ^KBr _nax 1765, 1630, 1260, 1000 cm ^-1 PMR: δ ^D2O _ppn 1.34 and 1.45 (8-(CH ₃ ) ₂ ,
3H each), 5.88 (S-CH=, 1H, d, J=8
Hz), 7.40 (N-CH=,1H,d,J=8Hz) (2) If methanol is used instead of dimethylformamide in (1) above, the reaction solution after 9 hours contains A peak of 85% a was observed on HPLC. Example 3 Compound d (12.8 mg) was dissolved in water (10 ml),
Extraction was performed with 1% tri-n-octylmethylammonium chloride/dichloromethane solution (10 ml).
The extract was heated under reflux for 8 hours. A peak of 88% of the product was observed by HPLC. The reaction solution was extracted three times with 0.375% sodium iodide solution (10 ml). 2
Concentrate only the second extract, and use the concentrated liquid as HP-20 (100
-200 mesh, 60ml) column chromatography, and eluted and fractionated with water. The eluted fraction was concentrated and lyophilized, [5R, 6R, 8S]-3-[(Z)
-2-acetamidoethenyl-(R)-sulfinyl]-6-[1-hydroxysulfonyloxyethyl]-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2- The disodium salt of caboric acid (d, 3.2 mg) was obtained. HPLC: 4% methanol, Rt=4.8min (d
Rt = 2.3min) UV: λ ^H2O _nax nm (E1% 1cm) = 242 (281) and 290
(196) IR: ν ^KBr _nax 1770, 1700, 1260, 1040cm ^-1 PMR: 1.55 (8-CH ₃ , d, J = 6Hz), 5.90
(S-CH=, d, J=8Hz), 7.39 (N-CH=,
d, J = 8 Hz) Example 4 Compound c (15 mg) was dissolved in water (200 ml),
% tri-n-octylmethylammonium chloride/chloroform solution (200 ml).
The extract was heated under reflux for 2 hours, and the reaction solution was dissolved with 3% sodium iodide (50 ml). 78% of the products were recognized by HPLC. Example 2 - Post-treatment of aqueous layer
Perform as in (1) [5R, 6R, 8S] -3- [(Z)
-2-acetamidoethenyl-(R)-sulfinyl]-6-[1-hydroxy-ethyl]-7-
The sodium salt of oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid (c, 6.6 mg) was obtained. HPLC: 8% methanol, Rt=5.5min (c
Rt = 2.8min) UV: λ ^H2O _nax nm (E1% 1cm) = 238 (330) and 291
(228) IR: ν ^KBr _nax 1770, 1630, 1260 cm ^-1 PMR: δ ^D2O _ppn 1.38 (8-CH ₃ , d, J = 6Hz),
5.90 (S-CH=d, J=8Hz), 7.40 (N-CH=
d, J = 8 Hz) Example 5 Compound e (64 mg) was dissolved in water (1.28),
Extraction was performed with a 2.5% tri-n-octylmethylammonium chloride/chloroform solution (1.28). The extract was heated under reflux for 1 hour, cooled, and then transferred with 7.5% sodium iodide solution (160 ml).
A peak of 95% of the product was observed by HPLC. Post-treatment of the aqueous layer was carried out as in Example 2-(1) [5R,
6R, 8S〕-3-[(Z)-2-acetamidoethenyl-thio]-6-[1-hydroxy-ethyl]
-7-oxo-1-azabicyclo(3,2,0)
-Hept-2-ene-2-carboxylic acid sodium salt (e, 33 mg) was obtained. HPLC: 6% methanol, Rt=11.6min (e
Rt = 6.8min) UV: λ ^H2O _nax nm (E1% 1cm) = 226 (320) and 306
(268) IR: ν ^KBr _nax 1755, 1630, 1265 cm ^-1 PMR: δ ^D2O _ppn 1.35 (8-CH ₃ , d, J = 6Hz),
5.74 (S-CH=, d, J=7.5Hz), 7.20 (N-CH
=, d, J = 7.5 Hz) Example 6 Compound f (20.9 mg) was dissolved in water (10 ml),
Extraction was performed with 1% tri-n-octylmethylammonium chloride/dichloromethane solution (10 ml).
The extract was heated under reflux for 9 hours, cooled, and then transferred with 0.375% sodium iodide solution (10 ml). HPLC showed 95% reaction products. The aqueous layer concentrate was subjected to HP-20 (100-200 mesh, 130 ml) column chromatography, and eluted and fractionated with water and 5% methanol water. Concentrate and freeze-dry the product categories, [5R, 6R, 8S]-3-[(Z)-2-
Acetamidoethenyl-thio]-6-[1-hydroxysulfonyloxyethyl]-7-oxo-1-azabicyclo(3,2,0)-hept-2
-en-2 carboxylic acid disodium salt (f,
8.8mg) was obtained. HPLC: 4% methanol, Rt=5.2min (f
Rt=4.1min) UV: λ ^H2O _nax nm (E1% 1cm) = 228 (291) and 307
(179) IR: ν ^KBr _nax 1750, 1620, 1260, 1035 cm ^-1 PMR: δ ^D2O _ppn 1.50 (8-CH ₃ , d, J = 6Hz),
5.74 (S-CH=, d, J=8Hz), 7.21 (N-CH
=, d, J = 8 Hz) Example 7 Compound g (10 mg) was dissolved in water (200 ml), and 2
% tri-n-octylmethylammonium chloride/dichloromethane solution (200 ml).
The extract was heated under reflux for 8 hours and then dissolved with 3% sodium iodide (50 ml). Example 2 Post-treatment of aqueous layer
- Do as in (1), [5R, 6R] -3 - [(Z) -
2-acetamidoethenyl-thio]-6-[1-
Hydroxy-1-methyl-ethyl]-7-oxo-1-azabicyclo(3,2,0)-hept-2
Sodium salt of -ene-2-carboxylic acid (g,
7 mg) was obtained. Compound g is TLC, HPLC,
The UV, IR and PMR spectra matched the standard. Example 8 Compound h (40 mg) was dissolved in water (20 ml) and 1
% tri-n-octylmethylammonium chloride/chloroform solution (20 ml). The extract was heated under reflux for 2 hours and then dissolved with 0.75% sodium iodide (10 ml). Example 1 Post-treatment of aqueous layer
- Perform as in (3), [5R, 6R] -3- [(Z)-
2-acetamidoethenyl-thio]-6-[1-
Hydroxy-sulfonyloxy-1-methyl-
ethyl]-7-oxo-1-azabicyclo(3,
The disodium salt of 2,0)-hept-2-ene-2-carboxylic acid (h, 31 mg) was obtained. The TLC, HPLC, UV, IR, and PMR spectra of compound h matched the standard. Example 9 Dissolve 130 mg of compound i in water (2.0) to give 2%
Extraction was performed with tri-n-octylmethylammonium chloride/chloroform solution (2.0).
The extract was heated under reflux for 1.5 hours, cooled, and then transferred with 6.6% sodium iodide solution (375 ml). by HPLC
A peak of 93% of deliverables was observed. Post-treatment of the aqueous layer was carried out as in Example 2-(1) [5R, 6S,
8S]-3-[(Z)-2-acetamidoethenyl-thio]-6-[1-hydroxy-ethyl]-7
-Oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid sodium salt (i, 64 mg) was obtained. HPLC: 10% methanol, Rt=14.0min (i
Rt = 6.7min) UV: λ ^H2O _nax nm (E1% 1cm) = 231 (390) and 308
(344) IR: ν ^KBr _nax 1755, 1620, 1440, 1265 cm ^-1 PMR: δ ^D2O _ppn 1.70 (8-CH ₃ , d, J = 6Hz),
5.70 (S-CH=d, J=8Hz), 7.16 (N-CH=,
d, J = 8 Hz) Example 10 The compound (j, 7 mg) obtained in Production Example 5 was dissolved in water (140 ml) and extracted with a 2% tri-n-octylmethylammonium chloride/chloroform solution (140 ml). The extract was heated under reflux for 3 hours, and after cooling, 1.5% sodium iodide solution (100ml) was added.
It was transferred and melted. A peak of 83% of the product was observed by HPLC. The aqueous layer was post-treated as in Example 2-(1) [5R, 6R]-3-[(Z)-2-acetamidoethenyl-(S)-sulfinyl]-6-
[1-hydroxy-1-methyl-ethyl]-7-
The sodium salt of oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid (j, 5 mg) was obtained. TLC: Rf = 0.64 HPLC: Rt = 4.2 min [8% methanol-PB] UV: λ ^H2O _nax nm (E1% 1cm) = 237 (408) and 290
(262) IR: ν ^KBr _nax 1765, 1705, 1635, 1385, 1270, 1020
cm ^-1 CD: [θ] ^H2O _on 235 (ε-22700) and 286 (+
32800) PMR: 1.32, 1.41 (3H×2, s, 8-CH ₃ ),
2.15 (3H, s, NHCOCH ₃ ), 3.22, 3.83 (1H×
2, dd, H ₄ ), 3.82 (1H, d, H ₆ ), 5.71 (1H,
d, S-CH=), 7.27 (1H, d, N-CH=) Example 11 The compound (k, 10 mg) obtained in Production Example 4 was dissolved in water (10 ml), and 1% tri-n-octylmethyl Extracted with ammonium chloride/chloroform solution (10ml). The extract was heated under reflux for 3 hours,
After cooling, the solution was transferred with 0.75% sodium iodide solution (10 ml). A peak of 88% of the product was observed by HPLC. The aqueous layer was previously treated with 5% saline.
It was subjected to column chromatography on HP-20 (100-200 mesh, 20 ml), eluted and fractionated with 5% saline and methanol:5% saline (3:97). Fractions containing the target substance were collected and concentrated, and the concentrated solution was subjected to column chromatography using activated carbon (5 ml). 8% iso-butanol water, N/
50 ammonia water: 8% isobutanol water (2:
98) to elute the target substance. The eluate was concentrated and lyophilized to obtain [5R,6R]-3-[(Z)-2-acetamidoethenyl-(S)-sulfinyl]-6-
Disodium salt of [1-hydroxysulfonyloxy-1-methyl-ethyl]-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid (k, 8.4 mg ) was obtained. TLC: Rf = 0.30 HPLC: Rt = 1.8 min (8% methanol-PB) UV: λ ^H2O _nax nm (E1% 1cm) = 237 (276) and 292
(188) CD: [θ] ^H2O _on 232 (ε-12900) and 282 (+
31400) IR: ν ^KBr _nax 1765, 1700, 1260, 1050 cm ^-1 NMR: δ1.65, 1.68 (3H×2, s, 8-CH ₃ ),
2.17 (3H, s, NHCOCH ₃ ), 3.26 and 3.92 (1H
×2, dd, H ₄ ), 3.98 (1H, d, H ₆ ), 5.85 (1H,
d, S-CH=), 7.30 (1H, d, N-CH=)

Claims (1)

[Claims] 1. General formula (wherein R is optionally substituted ethyl and n represents 0 or 1, respectively) or a salt thereof is allowed to act on excess quaternary ammonium halide to isomerize it. general formula (In the formula, R and n have the same meanings as above)
A method for producing a compound represented by or a salt thereof.