JPS6354715B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention relates to general formula (-1) (In the formula, R ₂ represents hydrogen or ester residue)
The present invention relates to an optically active form of a cephalosporin analog represented by, salts thereof, and a method for producing the same. In the general formula (-1), the ester group represented by COOR ₂ is a group that is used in the chemistry of penicillins and cephalosporins and is easily induced into COOH. R ₂ is a straight chain or branched alkyl group having 1 to 5 carbon atoms (e.g. methyl, ethyl, n-
propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl group, etc.), straight chain or branched lower alkoxymethyl group having 2 to 5 carbon atoms (e.g. methoxymethyl, ethoxymethyl group, etc.), carbon number 1 ~5 linear or branched halogenated alkyl groups (e.g. chloromethyl,
2,2,2-trichloromethyl, 2,2,2-trifluoroethyl, etc.), lower alkylsulfonylethyl groups (such as methylsulfonylethyl,
(ethylsulfonylethyl group, etc.), carbon number 7 or more
19 arylmethyl groups (e.g. benzyl, diphenylmethyl, trityl groups, etc.), carbon number 7-20
Substituted arylmethyl group (examples of substituents include methoxy group, nitro group, etc., and the number of substituents on the phenyl group is 1 to 5) or general formula () (In the formula, R ₄ represents a straight chain or branched lower alkyl group having 1 to 5 carbon atoms, a straight chain or branched lower alkoxy group having 1 to 5 carbon atoms, or a phenyl group, and R ₅ represents hydrogen or a straight chain or branched lower alkyl group having 1 to 5 carbon atoms. Examples include ester residues represented by 5 (representing a straight chain or branched lower alkyl group). Carbacefuem compounds in which the sulfur atom of cephalosporin is replaced with a carbon atom [Carbacefuem is named after J.Am.Chem.Soc., 96 , 7584
(1974)], compounds having a substituted methyl group at the 3-position are known [as described above and J.Med.Chem., 20 , 551 (1977), etc.]
No compounds are known that exhibit particularly strong antibacterial activity. The present inventors previously demonstrated that 4
We succeeded in synthesizing compounds having various substituents at the 5-position, 5-position, or 3-position, and these compounds
No. 34696 (Japanese Unexamined Patent Publication No. 128591/1983), No. 53-
No. 122403 (No. 55-49376), No. 53-133072 (No. 55-59186), No. 53-162005 (No. 55-59186)
No. 87788) and No. 54-8408 (No. 55-100384). Furthermore, the present inventors also succeeded in synthesizing an acyl compound having a novel carbacephem core. These acyl forms are novel antibiotics with strong antibacterial activity, and they are also used in Japanese Patent Application No. 53-34696 (Japanese Unexamined Patent Publication No. 54-128591).
Publication No. 53-122402 (Publication No. 55-49375),
53-127027 (publication number 55-53290), 53-
No. 133071 (No. 55-59185), No. 53-162006 (No. 55-87789), No. 53-162007 (No. 55-87789)
87790), 53-162008 (55-87791), 54-8409 (55-100391),
No. 54-92035 (publication No. 56-16491), September 13, 1954
Filing date: Cephalosporin analogues (patent application filed in 1982)
No. 116720; JP-A-56-40683). However, since the carbacefem nuclei described above are produced by synthetic methods using optically inactive raw materials, all of them are optically inactive DL.
body [or expressed as (±) body]. That is,
Compounds corresponding to the general formula () in which the hydrogens at the 6th and 7th positions are in a cis relationship are the formula (-1) and the formula (-2), which are in an enantiomeric relationship with each other. [In formulas (), (-1) and (-2), R ₂
is the same as defined above] is present as a mixture of equal amounts of the compound represented by the following. The present inventors have discovered that separation of one optically active enantiomer of a compound represented by the general formula () [described later],
The preparation was successful, and patent application No. 14533 (1982) was published.
108872) General formula () in the specification (In the formula, R ₇ represents the same meaning as R ₂ , R ₆ represents hydrogen or a lower alkyl group, and has an absolute structure of 6R7S). As a result of further studies, the present inventors succeeded in separating and preparing an optically active form of the compound represented by the general formula (), and completed the present invention. That is, the present inventors used a compound in which a specific acyl group was introduced into an optically inactive dl form as a raw material (substrate), and carried out an optically selective deacylation reaction using an enzyme to obtain a compound represented by the general formula (). For the first time, we succeeded in producing an optically active form of a cephalosporin analog. The optically active substance obtained by the present invention has various physical properties as described below, and its acyl form exhibits stronger antibacterial activity than the corresponding optically inactive form (dl form). Based on this knowledge, it is considered to have the absolute structure of the above general formula (-1). Therefore, the following description will be made using the structural formula of general formula (-1). The compounds of Examples are also named as having this presumed absolute structural formula. The present invention will be explained in detail below. Optically active form of cephalosporin analog represented by general formula () or estimated absolute structural formula (-1)
Among the compounds represented by R ₂ =H, the compound (-
4) [Described later] is the general formula () (In the formula, R represents a substituted or unsubstituted unsaturated 6-membered carbon ring or a 5-membered hetero ring, and X represents hydrogen, an amino group, a hydroxyl group, or a lower alkyl group) [hereinafter referred to as compound ()] Abbreviate. In addition, the compounds represented by general formula (), general formula ()... are hereinafter abbreviated as compound (), compound (),...] are produced by optically selective deacylation of . Unsaturated carbon 6-membered rings, hetero 5-membered rings include phenyl, cyclohexenyl, cyclohexadienyl,
Examples include thienyl and furyl, and examples of substituents thereof include hydroxyl group, halogen group, nitro group, methanesulfonamide group, and the like. A lower alkyl group refers to a linear or branched alkyl group having 1 to 5 carbon atoms (for example, methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, sec-butyl, t-butyl group, etc.)
It is. The optically selective deacylation reaction of compound () to optically active compound (-4) is performed by converting compound () into optically active compound (-4).
This can be carried out using a microorganism capable of optically selectively deacylating to produce optically active compound (-4) and/or a cultured product of the microorganism. Examples of microorganisms capable of carrying out the above-mentioned optical selective deacylation reaction include microorganisms belonging to the genus Kluybella. A specific example of the bacterial cell is Kluyvera citrophylla ATCC21285. In addition to using enzymes produced by the above-mentioned microorganisms for optical selective deacylation, the following methods are used: Culture solution or cultured product of the above-mentioned microorganisms Isolated bacterial bodies [separated from the culture solution by centrifugation or the like. Furthermore, the bacterial cells are soaked in saline solution (usually about 1
%), may be used after washing with buffer, etc.],
Or an isolated bacterial cell suspension, a bacterial cell disruption solution [a liquid obtained by mechanically or chemically disrupting the bacterial cells obtained above] a bacterial cell disruption solution [the bacterial cell residues were removed from the bacterial cell disruption solution obtained above] Solution] Purified enzyme solution [After adding ammonium sulfate etc. to the enzyme solution obtained above, preferably the supernatant solution to precipitate the enzyme protein, this is purified by gel filtration method, ion exchange cellulose column chromatography, or ion exchange cellulose column chromatography. An enzyme solution purified by means such as Adex column chromatography] can also be used in the same manner. Furthermore, an immobilized enzyme obtained by immobilizing bacterial cells or an extracted and purified enzyme by a conventional method can also be used. The reaction is carried out in an inert solvent that does not affect the reaction.
It is carried out at a temperature of 15 to 35°C, preferably 15 to 35°C, and a pH of 5 to 10.
The most preferred solvent is water, but organic solvents (eg, acetone, methanol, ethanol, N,N-dimethylformamide, dimethyl sulfoxide, etc.) can also be used to dissolve the cephalosporin analog substrate.
In order to control the pH during the reaction, it is also effective to add a phosphate buffer, veronal buffer, or citrate buffer. The reaction time is influenced by the type of enzyme, concentration, type of substrate, reaction temperature, reaction pH, etc., but is usually 30 minutes to 24 hours. Most preferably, the reaction is terminated when the reaction yield reaches a maximum value. The bacterial cell concentration used is dry weight, 1 ml of reaction solution
1 mg to 50 mg per serving is preferred. In addition, the substrate concentration used is that compound () is per ml of reaction solution.
0.5mg to 50mg is preferred. In addition, if the bacterial cells used contain enzyme activities that interfere with this reaction, such as β-lactamase or esterase, the bacterial cells are subjected to mutation treatment to eliminate this enzyme activity, and then the bacterial cells are removed. This bacterial cell can also be used. Furthermore, the reaction yield can be improved by adding an inhibitor of these interfering enzymes to the reaction system. After completion of the reaction, the target product is generally isolated by adsorption using various carriers, ion exchange chromatography,
Alternatively, gel filtration, liquid-liquid extraction, etc. may be used. Furthermore, among the compounds represented by general formula (-1), general formula (-3) (In the formula, _R'2 represents an ester residue) The optically active form of the cephalosporin analogue is the general formula (-4) obtained as described above. It can be obtained by esterifying an optically active form of a cephalosporin analog represented by the following by a conventional method. Optically active compound of the present invention, compound (-1)
is itself a useful compound that can be expected to have antibacterial activity, but the compound obtained by acylating the optically active compound (-1) is similar to the corresponding inactive compound ().
It is a useful compound that exhibits extremely strong antibacterial activity compared to acylated compounds. Examples of these compounds and their antibacterial activity are shown in Reference Examples. Furthermore, the general formula () The compound represented by the formula (in which R and Compounds equivalent to these (hereinafter collectively referred to as 7-amino compounds) and general formula () (In the formula, R and It is produced by removing the protecting group using a conventional method. The protecting groups and removal methods used are those commonly used in the chemistry of penicillins and cephalosporins. As for the acylation method, methods commonly used in the chemistry of penicillins and cephalosporins are also applied. In addition, the compound () is shown in the reference process diagram below []
and synthesized according to the process shown in [ ]. This method generally produces compounds in which the hydroxyl group at the 4th position is in a cis relationship with the hydrogen atoms at the 6th and 7th positions,
The isomer in which the hydroxyl group and the 6- and 7-position hydroxyl atoms are in a trans relationship can be produced using the following reference process diagram []. Compounds (4), (6), (10), and (11) are used as starting materials [compound ()]. Examples of the present invention will be shown below. Example 1 (4S,6R,7S,)-7-amino-4-hydroxy-1-azabicyclo[4.2.0]oct-2-
En-8-oso-2-carboxylic acid [(4S, 6R,
7S)-7-amino-2-carboxy-4-hydroxy-1-azabicyclo[4.2.0]octo-
2-en-8-one] Production: (1-1) Preparation of bacterial cell suspension (a) Cultivation of microorganisms having optical selective deacylation ability Kluyvera citrophila ATCC21285 [Mycology The description is J.General Applied Microbiology
3, 28-31 (1957)]. As a seed medium, an aqueous solution containing 1% polypeptone, 1% yeast extract, 0.5% meat extract, 0.5% monosodium glutamate, and 0.25% salt was used.
A solution adjusted to pH 7.0 with 5N-NaOH was used. Place 1 platinum loopful of the inoculum into a 50ml thick test tube.
ml of seed medium was inoculated and cultured with shaking at 30°C for 24 hours. The entire volume of this seed medium was placed in a pleated Erlenmeyer flask as shown in No. 2.
Inoculate into 300ml and culture with shaking at 30℃. The fermentation medium used had the same composition as the seed medium. (b) Preparation of bacterial cell suspension After 24 hours of the above main culture, the bacterial cells were taken out from the fermentation liquid by centrifugation and added to 50 ml of 0.9% saline.
Wash twice with The cells are suspended in 1/30M phosphate buffer (PH8.0). The bacterial cell concentration is
At 40 mg/ml, the bacterial weight is calculated in terms of dry bacterial cells. (1-2) Preparation of substrate solution (±)-cis-7β-phenylacetamide-4α-hydroxy-1-azabicyclo[4.2.0]oct-2- prepared in the same manner as Reference Example 4 described later.
En-8-one-2-carboxylic acid 200mg 1/30
Add to 9 ml of M phosphate buffer (PH8.0). At this time, the compound did not dissolve, but when 2N-NaOH was added little by little and the pH was adjusted to 8.0 again, it dissolved. Finally, add deionized water to bring the total volume to 10
Adjusted to ml. (1-3) Enzyme reaction Add 10ml of the above bacterial suspension to 10ml of substrate solution,
Carry out the enzymatic reaction at 40°C for 80 minutes. Table 1 shows the time course of the reaction. [Table] A certain compound.
(1-4) Isolation and purification of the target product (a) After the reaction is completed, the bacterial cells are removed from the reaction solution by centrifugation, and after concentration under reduced pressure, Diaion HP
A column filled with 43 ml of 100 to 200 meshes (column diameter 0.88 cm, height 70 cm) of -20AG (manufactured by Mitsubishi Kasei Corporation) was charged. When eluted with water, the target product was eluted in a fraction of 36 ml to 45 ml. After concentrating this again under reduced pressure, high-performance liquid chromatography [manufactured by JASCO Corporation, TRI
ROTAR]. The column was Shodex OH Pak B-804 manufactured by Showa Denko Co., Ltd., and elution was performed using water. The sample thus collected was concentrated under reduced pressure and lyophilized to obtain 37.6 mg of white powder. The physical properties of this product are as follows. IRν ^KBr _nax (cm ^-1 ): 3530, 3190, 1746, 1620,
1550 NMR ( _D2O , DSS internal standard) δ (ppm): 6.17
(1H, d, J = 5.4Hz), 4.95 (1H, d, J
=5.4Hz), 4.59 (1H, m), 4.11 (1H, m),
2.23 (1H, m), 1.80 (1H, m) These values agree well with the values of the corresponding dl body. Optical rotation [α] ³⁰ _D = +24.2° (c = 0.153, _H2O ) It is thought that dextrorotatory compounds are mixed in. Silica gel thin layer chromatography [Thin layer plate Merck Art5721 (manufactured by E. Merck), developing solvent; ethanol:acetic acid:water = 4:1:1]
According to the study, a single spot with positive ninhydrin coloring was observed at an Rf value of 0.38. this
The Rf value coincides with that of the optically active dl-body spot. (b) After the enzymatic reaction was completed in the same manner as in (1-1) to (1-3) above, the bacterial cells were removed from the reaction solution by centrifugation, concentrated under reduced pressure, and then Diaion HP-20AG ( A column filled with 43 ml of 100-200 mesh (column diameter 0.88 cm, height 70 cm) (manufactured by Mitsubishi Kasei Corporation) was charged. When eluted with water, the target product was eluted in a fraction of 36 ml to 45 ml. After concentrating this again under reduced pressure, 20 ml of Diaion WA-30-S (manufactured by Mitsubishi Chemical Corporation)
It was charged into a Rakam with an inner diameter of 0.88 cm and a height of 33 cm. The ion exchange resin used was an acetic acid type resin that had been previously passed through with 40 ml of a 0.5N acetic acid aqueous solution. When eluted with water, inorganic ions mixed in the solution containing the target substance were eluted. 20ml water
After passing through the solution, 0.5N acetic acid aqueous solution is passed through. 0.5N
After passing through the acetic acid aqueous solution, the target product is eluted in a 30-45 ml fraction. This target product was freeze-dried to obtain 32 mg of pale yellow powder. The physical properties of this product were as follows, and it was identified as the acetate salt of the title compound. IRν ^KBr _nax (cm ^-1 ): 3400, 1805, 1760, 1745
(Sh), 1600, 1560 (Sh), 1410 NMR ( _D2O , DSS internal standard) δ (ppm): 6.16
(1H, d, J = 5.4Hz), 4.89 (1H, d, J
= 5.4Hz), 4.57 (m, overlaps with H ₂ O signal), 4.08 (1H, m), 2.21 (1H, m),
1.97 (3H, s), 1.78 (1H, m) Optical rotation [α] ²⁰ _D = -62.1° (c = 0.16, 1M phosphate buffer, PH7.0) (c) (4S, 6R, 7S) - 7-amino-4-hydroxy-1-azabicyclo[4.2.0]octo-
Production of 2-en-8-one-2-carboxylic acid (alternative method) (c-1) (4S, 6R, 7S)-7-t-butoxycarbonylamino-4-hydroxy-1-azabicyclo [4.2.0 ] Octo-2-en-
Preparation of 8-one-2-carboxylic acid. After the enzymatic reaction was completed in the same manner as in (1-1) to (1-3) above, the bacterial cells were removed from the reaction solution by centrifugation and concentrated under reduced pressure.
-200 mesh diamond ion HP-20AG
(Mitsubishi Kasei Corporation) Inner diameter 0.88 filled with 43ml
cm, and charged into a column with a height of 70 cm. Elution was performed with water, and fractions of 36 to 45 ml were collected, concentrated under reduced pressure, and lyophilized to obtain 100 mg of white powder. Add 1.0ml of dioxane, 1.0ml of water,
Triethylamine 21μ and S-t-butoxycarbonyl-4,6-dimethyl-2
- After adding 40 mg of mercaptopyrimidine and stirring at room temperature for 4 days and at 40°C for 17 hours, the reaction solution was concentrated under reduced pressure to about 1/2 volume. After prepurifying the residue three times with ethyl acetate, the pH of the aqueous layer is adjusted to about 3 with a 10% aqueous citric acid solution under ice cooling. After extraction with ethyl acetate five times, the ethyl acetate layer was washed twice with saturated brine, dried over Glauber's salt, and concentrated under reduced pressure to obtain white crystals.
Get 6.5mg. The physical properties of this product were as follows, and it was identified as the title compound. IRν ^KBr _nax (cm ^-1 ): 3450, 3350, 1785, 1765,
1640, 1540 NMR (CD ₃ OD) δ (ppm): 6.42 (1H, d,
J = 5.4Hz) 5.27 (1H, d, J = 5.4Hz),
4.41 (1H, m), 3.95 (1H, m), 2.3~1.2
(2H, m), 1.46 (9H, s) Optical rotation [α] ²¹ _D = -38.2° (c = 0.11,
_CH3OH ) (c-2) (4S,6R,7S)-7-Amino-4-hydroxy-1-azabicyclo[4.2.0]Production of oct-2-en-8-one-2-carboxylic acid. (4S,6R,7S)-7-t-butoxycarbonylamino-4-hydroxy-1-azabicyclo[4.2.0]oct-2-ene-8 obtained in the same manner as the above step (c-1)
3 ml of anhydrous methylene chloride was added to 63 mg of -one-2-carboxylic acid, and 3 ml of trifluoroacetic acid was added while stirring under ice cooling, and the mixture was stirred at the same temperature for 3.5 hours. Thereafter, the reaction solution was concentrated under reduced pressure, and the resulting brown oil was powdered with ethyl ether to obtain 53 mg of the crude target product as a yellowish brown powder. This is Diaion HP-20AG
(manufactured by Mitsubishi Kasei Corporation) Charge a column packed with 50 ml, elute with water, collect the ninhydrin color positive fraction, and concentrate under reduced pressure to 31 mg.
(47.0%) of the target product, trifluoroacetate, was obtained. The physical properties of this product were as follows, and it was identified as the trifluoroacetate of the title compound. IRν ^KBr _nax (cm ^-1 ): 3400, 1800, 1780, 1680,
1620 NMR (D ₂ O) δ (ppm): 6.31 (1H, d, J
= 5.4Hz), 5.00 (1H, d, J = 5.4Hz),
4.60 (1H, m), 4.16 (1H, m), 2.37~
1.66 (2H, m) Optical rotation [α] ²¹ _D = -61.9° (c = 0.0743,
H ₂ O) The target compounds obtained in (b) and (c) exhibited the same behavior as that obtained in (a) when subjected to thin layer chromatography under the same conditions. Example 2 (4S,6R,7S)-7-amino-4-hydroxy-1-azabicyclo[4.2.0]oct-2-en-8-one-2-carboxylic acid [(4S,6R,
7S)-7-amino-2-carboxy-4-hydroxy-1-azabicyclo[4.2.0]octo-
Production of 2-en-8-one] (alternative method): (2-1) Preparation of immobilized enzyme Kluybella citrophylla cultured in 5 flasks in the same manner as in (1-1) (a)
40mg/ml of ATCC21285 bacterial cells in terms of dry weight
It was suspended in 1/30M phosphoric acid buffer (PH7.0) so that The cells were disrupted for 2 minutes at 200 watts using an ultrasonic generator model UR-200P manufactured by Tomy Seiko Co., Ltd., and the cell residue was removed by centrifugation to obtain a supernatant. 1% by weight of streptomycin sulfate was added thereto, and the mixture was allowed to stand overnight to remove nucleic acids, and then ammonium sulfate was added to 70% saturation to precipitate the enzyme protein. This precipitate was collected by centrifugation, dissolved again in 50 ml of deionized water, and desalted by dialysis. This enzyme solution was concentrated under reduced pressure to 10 ml, and 0.5 ml of 1M acetic acid-acetic acid soda buffer (PH5) was added.
On the other hand, Diaion WK-10 manufactured by Mitsubishi Kasei Corporation
10ml of M/20 acetic acid-acetic acid soda buffer (PH
5) Soak in advance. The above enzyme solution and WK−
10 were mixed and shaken at 30°C overnight. An immobilized enzyme was prepared as described above. (2-2) Reaction and isolation and purification of target product 10 ml of the above immobilized enzyme and (1-2) of Example 1
10 ml of the substrate solution prepared in the same manner as above was mixed in a large test tube and shaken at 40°C for 2 hours. Example 1 The reaction solution was separated by decantation.
Purification was carried out in the same manner as in (1-4). The obtained material showed almost the same physical property values as Example 1. Reference example 1 (4S, 6R, 7S)-7-[2-(2-amino-4-
thiazolyl)-2-syn-methoxyiminoacetamide]-4-hydroxy-1-azabicyclo[4.2.0]oct-2-en-8-one-2
-Production of carboxylic acid (Reference 1-1) 2-(2-tritylamino-4-thiazolyl)-
2-Syn-methoxyiminoacetic acid 78.8mg (0.178m
mole) in 0.8 ml of anhydrous dichloromethane,
Add 25 μ (0.178 mmole) of triethylamine at -20°C. Next, 37 mg (0.178 mmole) of phosphorus pentachloride was added and reacted at -20°C for 40 minutes with stirring, concentrated under reduced pressure, and the residue was dissolved in 40 ml of anhydrous tetrahydrofuran to prepare an acid chloride solution. On the other hand, 34 mg (0.162 mmole) of the compound obtained in the same manner as in Section (1-4) (a) of Example 1 was dissolved in a mixture of 1 ml of tetrahydrofuran and 1 ml of water.
To this add 79 μ (0.565 mmole) of triethylamine. The acid chloride solution prepared above is added dropwise to this solution while stirring on ice. Add 20μ of triethylamine and react for 2 hours and 45 minutes under ice cooling. Then adjust the pH to 2.0 with 10% hydrochloric acid and add 10ml of ethyl acetate.
After extracting twice with
After washing with 10 ml of sodium sulfate and drying with Glauber's salt, the mixture was concentrated under reduced pressure to obtain 114 mg of the crude acyl compound. 50 for this
Add and dissolve 10 ml of % acetic acid, stir at 50°C for 30 minutes, cool to room temperature, and concentrate the reaction solution. 1 for residue
Add 1 ml of methanol to dissolve, add 2 ml of ether and 20 ml of n-hexane, and obtain a precipitate by centrifugation. This is dried under vacuum to obtain 51 mg of solid. Dissolve this in methanol-water (1:1) 30
Charge a column packed with ml of Diaion HP-20AG. Elute with 50 ml of water, 40 ml of water:methanol=10:1, 30 ml of 8:1, 30 ml of 4:1,
Perform using 150 ml of 1:1. Silica gel thin layer chromatography [Plate: Merck Art.5719 (manufactured by E.Merck),
Developing solvent; butanol: acetic acid: water = 4:1:
1:], fractions corresponding to Rf=0 and 3 spots are collected and concentrated under reduced pressure. Dissolve this in 1 ml of methanol, 20 ml of ether, and n-hexane.
Precipitate with 20 ml. The solid is collected by centrifugation and vacuum dried to obtain 29.8 mg (yield 45.5%) of the desired product as white crystals. The physical properties of this product are as follows. IRν ^KBr _nax (cm ^-1 ): 3450, 1775, 1670, 1635, 1550 NMR (DMSOd ₆ − CD ₃ OD) δ (ppm): 9.28 (1H,
d, J=8.4Hz), 7.07 (2H, s), 6.75 (1H,
s), 6.27 (1H, d, J=5.4Hz), 5.56 (2H,
m), 2.77 to 2.04 (2H, m), -OCH ₃ signals overlap with the solvent-derived signals and cannot be distinguished. [α] ²⁴ _D = -32° (c = 0.5, methanol) (Reference 1-2) Obtained crystals subjected to the same procedure for further purification
Dissolve 200mg in 1ml of methanol, heat to 50℃ and add 1ml of hot water. White crystals are obtained when the solution is left at room temperature. This crystallization operation is repeated twice, and the resulting crystals are washed with 1 ml of water and dried under vacuum at 45°C for 12 hours. Yield 120mg. The physical properties of this product were as follows, and it was identified as the title compound. From the melting point of around 140℃, it turns purple and gradually turns brown.
Decomposes at 176-178℃. [α] ¹⁹ _D = −1° (c = 0.9, menol) NMR (DMSO-d ₆ , 100M) δ (ppm): 13.1 (1H,
br), 9.25 (1H, d, J = 8.3Hz), 7.20 (2H,
br), 6.76 (1H, s), 6.25 (1H, d, J=5.4
Hz), 5.48 (1H, dd, J=8.3, 5.1Hz), 5.26
(1H, br), 4.30 (1H, m), 3.84 (3H, s),
1.43-2.05 (2H, m), the proton at position 6 is -
It overlaps with the signal of OCH ₃ (δ3.84). Elemental analysis Actual value C: 43.33%, H: 4.43%, N: 17.89% Theoretical value C: 43.08%, H: 4.13%, N: 17.94% (as C ₁₄ H ₁₅ N ₅ O ₆ S1/2H ₂ O) High resolution mass spectrometry The above crystals were heated at 60°C for 5 hours in N,O-bistrimethylsilylacetamide and then subjected to analysis. Mass=669.23408 (C ₂₆ H ₄₇ O ₆ N ₅ SSi ₄ ) (Reference 1-3) Alternative method Obtained in (1-4) (b) and (1-4) (c) above (4S, 6R, 7S )-7-amino-4-hydroxy-
1-azabicyclo[4.2.0]oct-2-ene-
Reference example (Reference 1) using 8-one-2-carboxylic acid
-1) and (Reference 1-2) were performed to obtain the title compound in the same manner. Reference Example 2 The antibacterial activity of the compound obtained in Reference Example 1 is shown in the following table. Measurements were performed using the Heart Infusion Agar Dilution method (PH7.2). Cefazolin and the compounds obtained in Reference Examples 3 and 7 below were used as controls. [Table] [Table] Reference example 3 (±)-cis-7β-[2-(2-amino-4-thiazolyl)-2-syn-methoxyiminoacetamide]-4α-hydroxy-1-azabicyclo[4.2.0 ] Production of oct-2-en-8-one-2-carboxylic acid: (±)-cis-7β-amino-4α-hydroxy-1-azabicyclo[4.2.0]octo-2 synthesized in the same manner as described in JP-A-54-128591
Dissolve 60 mg (0.203 mmole) of -en-8-one-2-carboxylic acid/trifluoroacetate in 1 ml of water and 1 ml of tetrahydrofuran, and add 56 μm of triethylamine (referred to as reaction solution A). On the other hand, 2
-(2-tritylamino-4-thiazolyl)-2-
Syn-methoxyiminoacetic acid 125.7mg (0.280m
mole) in 1.2 ml of anhydrous methylene chloride, and cooled the solution in a dry ice-carbon tetrachloride bath.
After reacting with stirring for 50 minutes, the reaction solution was concentrated under reduced pressure, and 1 ml of anhydrous tetrahydrofuran was added to the residue to prepare an acid chloride solution. This acid chloride solution and 28μ of triethylamine were each divided into three portions and added alternately to the above reaction solution (A) over about 5 minutes. After reacting for an additional 35 minutes, adjust the pH to 2.5 with 10% citric acid and add sodium chloride until saturation. Then, the reaction solution was diluted with ethyl acetate for 3
Extract twice, wash the organic layer with saturated brine, dry over anhydrous sodium sulfate, filter and concentrate, and purify the residue with silica gel chromatography (15 g of silica gel, developing solvent: methanol:chloroform=1:3).
185.5 mg of crude acyl compound was obtained. Add 3 ml of 50% acetic acid to 86 mg of this crude acyl compound [compound (13)], and
After heating and stirring for 50 minutes, the precipitated triphenyl carbinol was removed by cooling and the liquid was concentrated under reduced pressure.
(manufactured by Mitsubishi Kasei Corporation), 8 ml (developing solvent; methanol:
Purification was performed using water = 1:9 to 2:1 (volume ratio, same hereinafter) to obtain 14.5 mg (44.3%) of the target product. The physical properties of this product are as follows. IRν ^KBr _nax (cm ^-1 ): 3450, 1775, 1670, 1635, 1550 NMR (DMSOd ₆ − CD ₃ OD) δ (ppm): 9.28 (1H,
d, J=8.4Hz), 7.07 (2H, s), 6.75 (1H,
s), 6.27 (1H, d, J=5.4Hz), 5.56 (2H,
m), 2.77-2.04 (2H, m) -OCH ₃ signal overlaps with the solvent-derived signal and cannot be distinguished. Reference example 4 (±)-cis-7β-phenylacetylamino-
4α-hydroxy-1-azabicyclo [4.2.0]
Preparation of oct-2-en-8-one-2-carboxylic acid. (±)-cis-7β-amino-4α-hydroxy-
1-azabicyclo[4.2.0]oct-2-ene-
8-one-2-carboxylic acid, trifluoroacetate
194.6mg (0.623mmole) in 3.1ml of water, 6.2ml of acetone
ml and add 209 mg of sodium bicarbonate to this.
(2.49mmole) is added. This was mixed with phenylacetyl chloride under ice-cooling and stirring.
Dissolve 125 mg (0.810 mmole) in 2 ml of acetone and add dropwise. 10.5 mg (0.068 mmole) of phenylacetyl chloride after 1.5 hours, and an additional 17.6 mg after 2.5 hours.
Add mg (0.114 mmole). After 2 hours and 45 minutes, concentrate under reduced pressure to remove acetone, add 10 mg of water and 1 ml of 1N hydrochloric acid, and extract three times with 20 ml of ethyl acetate. The ethyl acetate layer was washed once with saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting brown oil is tritiated by adding ether, filtered and dried to obtain the desired compound.
120 mg (60.4%) was obtained as a powder. The physical properties of this product are as follows. IRν ^KBr _nax (cm ^-1 ): 3400, 1780, 1670, 1640, NMR (CD ₃ OD) δ (ppm): 7.27 (5H, s), 6.39
(1H, d, J = 5.4Hz), 5.46 (1H, d, J = 5.1
Hz), 4.37 (1H, m), 4.01 (1H, m), 3.57 (2H,
s), 2.0 to 1.1 (2H, m) Reference example 5 (±)-cis-7β-[2-(2-amino-4-thiazolyl)-2-syn-methoxyiminoacetamide]-4β-hydroxy-1- Production of azabicyclo[4.2.0]oct-2-en-8-one-2-carboxylic acid. 2-(2-tritylamino-4-thiazolyl)-
2-Syn-methoxyiminoacetic acid 127 mg (0.286 (m
mole) in 1.3 ml of anhydrous dichloromethane, -
Cool to 20°C, stir and add 29 mg of triethylamine.
(0.286mmole) is added. Next, 60 mg (0.286 mmole) of phosphorus pentachloride is added and stirred at -18 to -20°C for 30 minutes. The reaction solution was concentrated under reduced pressure, dried in vacuo for 30 minutes, and diluted with anhydrous tetrahydrofuran 3.
ml and dissolve to prepare an acid chloride solution. On the other hand, (±)-cis-7β-amino-4β-hydroxy-1 obtained in the same manner as Reference Example 11 described later
-Azabicyclo [4.2.0] Octo-2-ene-8
-one-2-carboxylic acid, trifluoroacetate 63.8
mg (0.204 mmole) was suspended in 1.3 ml of water and 1.3 ml of tetrahydrofuran, and 103 mg of triethylamine was added.
(1.02 mmole). The acid chloride solution prepared above was added dropwise to this while stirring under ice cooling, and 22 mg (0.22 mmole) of triethylamine was further added.
After reacting for 4 hours and 45 minutes under ice-cooling and stirring, 2 ml of 1N HCl was added, and the mixture was extracted three times with 20 ml of ethyl acetate. The extracted layer was dried over sodium sulfate and concentrated under reduced pressure to obtain a crude product of the target trityl-protected product [compound (16)]. Dissolve this in 20 ml of 50% acetic acid and stir for 1 hour on a water bath at 50-60°C. After returning to room temperature, the reaction solution was filtered under reduced pressure, and the solution was concentrated under reduced pressure to obtain the desired crude product.
Get 173mg. Add this to Diaion HP-20AG (manufactured by Mitsubishi Kasei Corporation) 40ml (developing solvent; water-methanol = 10:1~
1:1). Obtain 32 mg (42%) of the desired product as a pale yellow powder. The physical properties of this product are as follows. IRν ^KBr _nax (cm ^-1 ): 3475, 1780, 1680, 1670, 1655,
1645 NMR (DMSO-d ₆ ) δ (ppm): 9.33 (1H, d, J
=8.8Hz), 7.18 (2H, s), 6.75 (1H, s), 6.06
(1H, d, J = 1.4Hz), 5.38 (2H, of which 1H is q, J = 8.8, 5.4Hz), 4.50 (1H, m), 3.98
(1H, m), 3.84 (3H, s), 2.17~1.37 (2H,
m) Reference example 6 (±)-cis-7β-[(R)-2-t-butyloxycarbonylamino-2-phenylacetamide]-4α-hydroxy-1-azabicyclo[4.2.0]octo-2- Production of en-8-one-2-carboxylic acid, tert-butyl ester. Dissolve 23.8 mg (0.095 mmole) of (R)-Nt-butyloxycarbonylphenylglycine in 1 ml of anhydrous tetrahydrofuran, and add -30
0.095 ml (0.095 mmole) of 1N-N-methylmorpholine-tetrahydrofuran and 0.095 ml of 1N-isobutyl chloroformate-tetrahydrofuran at °C.
(0.095 mmole) and stir for 30 minutes. To this, (±)-cis-7β-amino-4α-hydroxy-
1-azabicyclo[4.2.0]oct-2-ene-
8-one-2-carboxylic acid, tert-butyl ester
20 mg (0.079 mmole) was dissolved in 1 ml of anhydrous methylene chloride, added, reacted at -30°C for 45 minutes, and further reacted at 0°C for 4 hours. This reaction solution was diluted with 5 ml of methylene chloride, water, 1N HCl, 5%
Wash with NaHCO ₃ , water, and saturated saline in that order, dry with Glauber's salt, and concentrate to obtain 51 mg of the crude acyl compound. This was chromatographed on silica gel (5 g of silica gel, developing solvent;
Divided with n-hexane: ethyl acetate = 1.5:1),
A total of 22 mg (57%) of 10 mg of a highly polar isomer, 8 mg of a low polar isomer, and 4 mg of a mixture thereof were obtained. The physical properties of this product are as follows. Highly polar isomer IRν ^CHCl3 _nax (cm ^-1 ): 3430, 1780,
1725 (sh), 1717, 1697, 1630 Low polar isomer IRν ^CHCl3 _nax (cm ^-1 ): 3430, 1780,
1722(sh), 1715, 1695, 1630 Reference example 7 Cis-7β-[(R)-phenylglycinamide]
-4α-hydroxy-1-azabicyclo [4.2.0]
Preparation of oct-2-en-8-one-2-carboxylic acid. Cis-7β- obtained in the same manner as Reference Example 6
[(R)-2-t-butyloxycarbonylamino-2-phenylacetamide]-4α-hydroxy-1-azabicyclo[4.2.0]oct-2-en-8-one-2-carboxylic acid, tertiary Dissolve 56 mg (0.118 mmole) of the butyl ester low polar isomer in 1 ml of anhydrous methylene chloride and 1 ml of anisole, and add 2 ml of trifluoroacetic acid to this solution under ice cooling.
After standing under ice cooling for an hour, it was concentrated under reduced pressure. Dry benzene was added and concentrated again. Ether was added to the resulting oily substance, trituration was performed, and the resulting precipitate was removed to obtain the trifluoroacetate of the target product as a pale yellow powder.
Obtained 42.1 mg (80%). The physical properties of this product are as follows. IRν ^KBr _nax (cm ^-1 ): 3485, 1780 (sh), 1770, 1700
(sh), 1685, 1635 The obtained trifluoroacetate was dissolved in 2 ml of 1M phosphoric acid buffer (PH7.0), and Diaion HP
-20AG (50ml, developing solvent; water to water:methanol = 9:1) and lyophilized to 28mg (72%)
Obtained the desired object. The physical properties of this product are as follows. [α] ²⁴ _D = −26.0 (c = 0.53, H ₂ O) IRν ^KBr _nax (cm ^-1 ): 3480, 1780, 1770, 1680 ~ 1705,
1570-1650 NMR ( _D2O ) δ (ppm): 7.52 (5H, s), 6.03
(1H, d, J = 5.4Hz), 5.5 (1H, d, J = 5.1
Hz), 5.21 (1H, S), 4.28 (1H, m), 4.06~
3.85 (1H, m), 1.76-0.99 (2H, m) Reference example 8 (±)-cis-7-azido-1-azabicyclo[4.2.0]oct-2-ene-4,8-dione-
Production of 2-carboxylic acid, tert-butyl ester: N-chlorsuccinimide 374mg (2.760m
mole) was added to 7 ml of anhydrous toluene, cooled to -25°C under a nitrogen stream, and stirred. Next, 0.35 ml (4.766 mmole) of methyl sulfide was added, stirred for 10 minutes, and then heated to -15°C. -1-Azabicyclo [4.2.0]
oct-2-en-8-one-2-carboxylic acid,
A solution of 107 mg (0.382 mmole) of tertiary butyl ester dissolved in 25 ml of anhydrous toluene is added and stirred for 2.5 hours. Then 0.773ml of anhydrous triethylamine
A solution prepared by dissolving . This solution is washed with a mixture of 3 ml of 5% hydrochloric acid and 20 ml of saturated saline, and then washed twice with 5 ml of saturated saline. The ether layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 145.7 mg of the crude target product as an oil. This was chromatographed on silica gel (Wako gel).
C-200, 10g, developing solvent: n-hexane-ethyl acetate = 2; Purification with 1) yielded 42.5mg of the target product.
(0.153 mmole) of oil. yield
40.1% The physical properties of this product are as follows. IRν ^CHCl3 _nax (cm ^-1 ): 2125, 1802, 1740, 1698, (sh
),
1690 NMR ( _CDCl3 ) δ (ppm): 6.16 (1H, s), 5.22
(1H, d, J=5.5Hz), 4.44 (1H, m), 3.10~
2.48 (2H, m), 1.55 (9H, s) Reference example 9 (±)-cis-7β-azido-4β-hydroxy-
Production of 1-azabicyclo[4.2.0]oct-2-en-8-one-2-carboxylic acid, tert-butyl ester. (±)-cis- synthesized in the same manner as Reference Example 8
7-azido-1-azabicyclo[4.2.0]oct-2-ene-4,8-cyone-2-carboxylic acid,
Tertiary butyl ester 47mg (0.169mmole) 1%
Dissolve in 1.6 ml of tetrahydrofuran containing water, −
Stir at 40°C. Next, 3.2 mg (0.0845 mmole) of sodium borohydride is added and stirred for 15 minutes. Then, add a mixed solution of 10 ml of saturated saline and 1 ml of 10% hydrochloric acid, and extract twice with 20 ml of ether. The ether layer was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure to obtain 43.9 mg (0.157 mmole) of the desired product as an oil. Yield: 92.7% The physical properties of this product are as follows. IRν ^CHCl3 _nax (cm ^-1 ): 3450, 2118, 1789, 1784 (sh)
,
1729 (sh), 1722, 1629, 1632 (sh) NMR (CDCl ₃ ) δ (ppm): 6.20 (1H, d, J =
1.22Hz), 4.86 (1H, d, J = 5.37Hz), 4.62
(1H, m), 3.98 (1H, m), 2.45~1.65 (3H,
m), 1.53 (9H, s) Reference example 10 (±)-cis-7β-amino-4β-hydroxy-
Production of 1-azabicyclo[4.2.0]oct-2-en-8-one-2-carboxylic acid, tert-butyl ester. (±)-cis-7β- synthesized in the same manner as Reference Example 9
Azido-4β-hydroxy-1-azabicyclo[4.2.0]oct-2-en-8-one-2-carboxylic acid, tert-butyl ester 41.9 mg (0149m
mole) in 1.5ml of ethyl alcohol, 10%
Add 12 mg of palladium-carbon and stir in a hydrogen stream for 2 hours and 15 minutes under ice cooling. Then, the palladium-carbon is removed, the liquid is concentrated under reduced pressure, and the concentrated residue is dissolved in 10 ml of ether acetate. This solution was extracted four times with 5 ml of 10% citric acid aqueous solution, and the extract was extracted with 5 ml of ethyl acetate.
After washing with 30 ml of ethyl acetate, adjust the pH to 8 with sodium carbonate, saturate with common salt, and extract three times with 30 ml of ethyl acetate. The extract was washed with 37 ml of saturated saline, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain 23.4 mg (0.092 mmole) of the desired product as a pale yellow powder. Yield 61.6%. The physical properties of this product are as follows. IRν ^CHCl3 _nax (cm ^-1 ): 3430, 1774, 1734 (sh), 1724
,
1625 (sh), 1618 NMR ( _CDCl3 ) δ (ppm): 6.18 (1H, bs), 4.53
(1H, m), 3.83 (1H, m), 2.83 (3H, bs),
2.20 (1H, m), 1.51 (3H, s) Reference example 11 (±)-cis-7β-amino-4β-hydroxy-
Production of 1-azabicyclo[4.2.0]oct-2-en-8-one-2-carboxylic acid, trifluoroacetate. (±)-cis-7β obtained in the same manner as Reference Example 10
-amino-4β-hydroxy-1-azabicyclo[4.2.0]oct-2-en-8-one-2-carboxylic acid, butyl ester 172.4 mg (0.678 m
mole) in 8 ml of anhydrous methylene chloride, add 8 ml of trifluoroacetic acid while stirring on ice, and heat at the same temperature.
Stir for 1.5 hours. Thereafter, the reaction solvent was distilled off under reduced pressure, and the resulting brown oil was powdered with ethyl ether, yielding 121.3 mg of the target product as a yellowish brown powder.
(0.389 mmole) obtained. Yield 57.3%. The physical properties of this product are as follows. IRν ^KBr _nax (cm ^-1 ): 3440, 1770, 1750 (sh), 1700
(sh), 1685

Claims (1)

[Claims] 1 General formula (-1) (In the formula, _R2 represents hydrogen or an ester residue)
Cephalosporin analogs and their salts represented by 2 General formula () (In the formula, R is substituted or unsubstituted unsaturated carbon 6
represents a membered ring group, or a substituted or unsubstituted 5-membered heterocyclic group, X represents hydrogen, an amino group, a hydroxyl group, or a lower alkyl group, and hydrogen at the 6th and 7th positions are in a cis relationship The compound of general formula (-4) is obtained by optically selective deacylation of A microorganism belonging to the genus Kluybella having the ability to produce a cephalosporin analog represented by
Or the general formula (-
4) A method for producing a cephalosporin analog or a salt thereof.