JPH0520069B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to the reduction of 3-acetyl-2-azetidinone derivatives by microorganisms belonging to the genus Pichia, Satucharomyces, Trigonopsis, Crotuchera, Schizosatucharomyces, Candida, Trichosporon, Streptomyces, or Aspergillus. This invention relates to a method for producing optically active 3-(1-hydroxyethyl)-2-azetidinone derivatives. The 3-(1-hydroxyethyl)-2-azetidinone derivative obtained by the present invention is an important intermediate leading to carbapenems and penem derivatives. Optically active 3-(1-hydroxyethyl)-2-
Although various methods for producing azetidinone derivatives are known, all of them require a large number of steps and the reaction operations are complicated. Optically active 3-(1-hydroxyethyl)-2-azetidinone is efficiently produced by reduction with microorganisms belonging to the genus Crotuschella, Schizosatucharomyces, Candeida, Trichosporon, Streptomyces, or Aspergillus. The present invention was completed based on the discovery that the present invention can be obtained. general formula R ¹ is an alkyl group which may have a substituent (for example, a methyl, ethyl, propyl, or butyl group) and may have 1 to 3 of the same or different substituents shown below. The substituents include an alkyl group (for example, methyl, ethyl, propyl, butyl, isopropyl, or t-butyl), a CO ₂ R ³ group (wherein R ³ is a hydrogen atom, methyl, ethyl, propyl, butyl, isopropyl, etc.). ,
or t-butyl, a phenyl group which may have a substituent (the substituent is methyl, ethyl, propyl, methoxy, methylmercapto, nitro, cyano, acetamide, fluorine, chlorine, bromine, etc.). ), a benzyl group which may have a substituent (such as methoxy, methylmercapto, methyl, ethyl, propyl, nitro, cyano, acetamide, fluorine, chlorine or bromine), a halogen atom (for example, fluorine, (such as chlorine or bromine), -COSR ⁴ group (wherein R ⁴ is methyl, ethyl, propyl, or a phenyl group which may have a substituent (the substituent is the above-mentioned R ³ has the same meaning as the substituent of the phenyl group which may have a substituent), a benzyl group which may have a substituent (the substituent is the same as the substituent of ^the phenyl group which may have a substituent) ), -SR ³ (in the formula, R 3 has the same meaning as above), -CONR ⁵ R ⁶ (in the formula, R ³ has the same meaning as the above-mentioned substituent). , ^R5
and R ⁶ are the same or different hydrogen atoms, alkyl groups (e.g. methyl, ethyl, propyl,
(butyl, t-butyl, etc.), cyclohexyl, benzyl, etc. ), -OR ⁷ group (wherein R ⁷ is a hydrogen atom, an alkyl group (such as methyl, ethyl, or propyl) or an acyl group (such as acetyl, propionyl, etc.),
butyryl, benzoyl, etc.), or -COR ⁸ group (wherein R ⁸ is methyl,
ethyl, phenyl, etc.], an alkenyl group which may have a substituent (for example, vinyl, allyl, or butenyl, and having 1 to 3 of the same or different substituents as shown below) Good too. The substituents include ^an alkyl group (for example, methyl, ethyl, propyl, butyl, isopropyl, or t-butyl), a _-CO2R3 group (wherein ^R3 has the same meaning as defined above), -
COSR ⁴ groups (in the formula, R ⁴ has the same meaning as above), -SR ³ groups (in the formula, R ³ has the same meaning as above), -OR ⁷ groups (in the formula, R ⁷ has the same meaning as above), or a phenyl group that may have a substituent (the substituent is a substituent of the phenyl group that R ³ mentioned above may have a substituent) (having the same meaning as the group), an alkynyl group which may have a substituent (for example, an ethynyl or propargyl group, which may have 1 to 3 of the same or different substituents as shown below) . The substituent is an alkyl group (for example, methyl, ethyl, propyl, butyl, isopropyl, or t-butyl), -CO ₂ R ³ group (in the formula, R ³ has the same meaning as R ³ described above), - ⁴ COSR groups (R ⁴ in the formula is as described above)
Shows the same meaning as R ⁴ . ), -SR ³ group (in the formula, R ³ has the same meaning as R ³ described above), -OR ⁷ group (in the formula, R ⁷ is
Shows the same meaning as R ⁷ above. ), or a phenyl group which may have a substituent (the substituent has the same meaning as the substituent of the phenyl group in which R ³ described above may have a substituent), etc. A phenyl group that may have a substituent (it may have 1 to 3 of the same or different substituents as shown below).The substituent is an alkyl group (for example, methyl, ethyl,
(such as propyl or isopropyl), alkoxy groups (such as methoxy, ethoxy, propoxy, butoxy, or t-butoxy), halogens (such as fluorine, chlorine, or bromine), nitro, cyano, acetyl, acetoxy,
or hydroxyl group, etc.), alkylthio group -
SR ⁸ (wherein R ⁸ is methyl, ethyl, propyl, butyl, isopropyl, or t-butyl, etc.),
Alkylsulfonyl group -SO ₂ R ⁸ (in the formula, R ⁸ has the same meaning as R ⁸ described above), phenylthio group which may have a substituent (1 to 3 of the same or different substituents shown below) The substituent may be an alkyl group (such as methyl, ethyl, propyl, or isopropyl), an alkoxy group (such as methoxy, ethoxy, propoxy,
butoxy or t-butoxy), halogens (such as fluorine, chlorine, or bromine),
These include nitro, cyano, acetyl, acetoxy, or hydroxyl groups. ), a phenylsulfonyl group which may have a substituent (the substituent has the same meaning as the substituent of the phenylthio group which may have a substituent as described above), or an acyloxy group, -OCOR ⁹
(In the formula, R ⁹ is an optionally substituted alkyl group having 1 to 10 carbon atoms (for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl,
octyl, nonyl, or decyl), the substituent being an alkyl group having 1 to 5 carbon atoms (such as methyl, ethyl, propyl, butyl, pentyl, isopropyl, or t-butyl),
A phenyl group that may have a substituent (the substituent has the same meaning as the substituent of the phenyl group that R ³ described above may have a substituent), or a phenyl group that may have a substituent. and a benzyl group which may have a substituent (the substituent has the same meaning as the substituent of the benzyl group in which R ³ described above may have a substituent). ) etc. R ² may have a protecting group for a hydrogen atom or a nitrogen atom [for example, a silyl group (such as trimethylsilyl, triethylsilyl, triphenylsilyl, t-butyldimethylsilyl, or t-butoxydiphenylsilyl), or a substituent; Good alkyl groups {for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, or heptyl, etc., may have 1 to 3 of the same or different substituents as shown below. The substituents include alkyl groups (e.g., methyl, ethyl, propyl, isopropyl, butyl, or t-butyl),
CO ₂ R ³ group (in the formula, R ³ has the same meaning as above), -OR ¹⁰ group (in the formula, R ¹⁰ is a hydrogen atom, an alkyl group (such as methyl, ethyl, propyl, or butyl), a substituted a benzyl group that may have a group (the substituent has the same meaning as the substituent of the benzyl group that R ³ described above may have a substituent),
A phenyl group that may have a substituent (the substituent has the same meaning as the substituent of the phenyl group that R ³ described above may have a substituent), or a phenyl group that may have a substituent. a benzyl group which may have a substituent (the substituent has the same meaning as the substituent of the benzyl group where R ³ may have a substituent described above)}, an alkenyl group which may have a substituent {(For example, it is a vinyl or allyl group, and may have 1 to 3 substituents that are the same or different as shown below. The substituent is an alkyl group (for example, methyl, ethyl,
propyl, butyl, etc.), a phenyl group that may have a substituent (the substituent is as described above),
R ³ has the same meaning as the general formula of a phenyl group which may have a substituent. ), or -CO ₂ R ³ group (in the formula
R ³ has the same meaning as described above. )}, a phenyl group which may have a substituent (the substituent has the same meaning as the substituent of the phenyl group in which R ³ mentioned above may have a substituent, and is the same or different) may have 1 to 3 of these substituents), a benzyl group which may have a substituent (the substituent is
R ³ described above has the same meaning as the substituent of the benzyl group which may have a substituent, and may have 1 to 3 of the same or different substituents. ),
or a cycloalkyl group that may have a substituent (for example, cyclopentyl or cyclohexyl, where the substituent is an alkyl group that may have a substituent when R ² is a protecting group for the nitrogen atom) (showing the same meaning as a substituent of a group)], etc. Among the compounds having general formula (1), preferred compounds are
R ¹ is an alkyl group having a substituent, and the substituent is a -CO ₂ R ³ group (in the formula, R ³ has the same meaning as described above) or a -COSR ⁴ group (in the formula, R ⁴
has the same meaning as above. ), an alkynyl group which may have a substituent, and the substituent is -
SR ³ group (in the formula, R ³ has the same meaning as above) or -OR ⁷ group (in the formula, R ⁷ has the same meaning as above), an alkylsulfonyl group -
SO ₂ R ⁸ (in the formula, R ⁸ has the same meaning as described above), a phenylsulfonyl group which may have a substituent, or an acyloxy group -OCOR ⁹ (in the formula, R ⁹
has the same meaning as above. ), etc.
R ² is a hydrogen atom, an alkyl group having a substituent, and the substituent is a -CO ₂ R ³ group (in the formula, R ³ has the same meaning as above), an alkyl group, OR ¹⁰
group (in the formula, R ¹⁰ has the same meaning as defined above), an alkenyl group which may have a substituent, and the substituent is an alkyl group, or a -CO ₂ R ³ group (in the formula, R ³ has the same meaning as described above), a phenyl group which may have a substituent, or a benzyl group which may have a substituent. The genus Pichia, Satucharomyces, and Trigonopsis to be subjected to the stereospecific microbial reduction of the present invention,
Microorganisms belonging to the genus Crotucela, Schizosatucharomyces, Candeida, Trichosporon, Streptomyces, or Aspergillus can be selected with reference to many years of experience and numerous books. The microbial species that seem most promising for achieving this goal are yeast, followed by actinomycetes and filamentous fungi. For example, Pichia terricola (SANK 51684) (FERM P 8001) Saccharomyces cerevisiae (SANK 50161) (IAM 4512) Saccharomyces rosei (SANK 50268) Trigonopsis variabilis (SANK 59963) (IAM 4443) Kloeckera africana (SANK 58163) (I AM 4191) Schizosaccharomyces pombe (SANK 57362) (IAM 4863) Saccharomyces montanous (CBS 4506) Candida albicans, (YU 1200) Saccharomycopsis lipolytica (IFO 0746) Trichosporon penicillatum (FERM P 8002) Streptomyces cattleya (NRRL 8057) Candida krusei (FERM P 8000) Saccharomyces fermentati ( Microorganisms such as Aspergillus niger (SANK 13658) (IAM 4771) (ATCC 9142) can be mentioned. The experimental method when testing microorganisms belonging to the genus Pichia, Satucharomyces, Trigonopsis, Crotuchera, Schizosatucharomyces, Candeida, Trichosporon, Streptomyces, or Aspergillus is as follows: It can be broadly classified into B method. Method A - Inoculate the strain in any medium in which the test microorganism shows good growth, culture for 1 to 2 days (usually rotary shaking culture - reciprocating shaking culture is also possible), and when vigorous growth is observed. Add 30 to 200 mg% of the substrate (either directly to the medium as a fine powder or dissolve it in the range of 0.5 to 3.0% in an organic solvent that is well miscible with any water), and continue culturing under the same conditions to complete microbial reduction. This is the so-called growing bacterial cell method. For example, glucose 2%, polypeptone 1%,
Dissolve an aqueous solution of 0.1% yeast extract in 200ml of tap water and dispense 100ml each into two 500ml Erlenmeyer flasks.
High pressure sterilize at 120℃, 15lbs, 20 minutes. After cooling, inoculate 3 ml of culture solution obtained by culturing bacteria in the same medium for 3 days, and shake at 28° C. with rotation. One day later, when vigorous growth is observed, an appropriate amount of substrate dissolved in an appropriate aqueous solvent is added and culture is continued for two days. The pH is 5.6 to 5.7 at the end of the microbial reaction. The culture solution is extracted with ethyl acetate to obtain a crude product. Method B - The strain is inoculated into any medium in which the test microorganism shows good growth, cultured for 2 to 4 days (normal shaking culture - reciprocating shaking culture is also possible), centrifugal collection of bacterial cells, and physiological Wash twice with saline. 1 to 5 g of the wet bacterial cells thus obtained were suspended in a 1% to 30% sugar tap aqueous solution, acclimatized by placing them in a rotating (reciprocating) shaker for 0.5 to 2 hours, and then using the same method as in Method A. The so-called bacterial cell suspension method involves adding a substrate and completing microbial reduction under the same conditions. Here, similar results can be expected even if a buffer solution with a pH of 5.0 to 7.0, such as a phosphate buffer solution, is used instead of tap water. In addition, in place of the inoculated bacterial cells in Method A and the wet bacterial cells in Method B, easily available live bacterial cells, such as commercially available yeast for breads, yeast for sweet breads, etc., can be easily used to achieve the purpose. It is something. In method B, in the extraction operation after the completion of microbial reduction,
There are fewer contaminants from the bacterial cell suspension than in Method A, and therefore the target substance can be easily isolated and purified, and the yield is high. Furthermore, in Method A, the microbial cell suspension method tends to cause secondary reactions to occur next to the desired primary (reduction) reaction, and multiple results may be obtained, but in Method B, the microbial cell suspension method It is simplified and only desired results can be obtained efficiently. For example, 3g of commercially available bread yeast (2g of wet bacteria)
Suspend in 20ml of tap water containing sucrose and leave for 30-60 minutes.
Shake rotatably at 28℃. Next, an appropriate amount of the substrate is dissolved in an appropriate water-soluble solvent and added to carry out a microbial reaction. Check the time-course changes by TLC for 1 to 2 days after the start of the reaction, and if the substrate remains, add 1 g of sucrose.
to complete the microbial reaction. The reaction solution is extracted with ethyl acetate to obtain a crude product. Note that any medium that can be used for culturing microorganisms in Methods A and B can achieve the purpose as long as vigorous growth of microorganisms is observed. These media include natural media, semi-synthetic media, and synthetic media, but in order to obtain microbial cells with high microbial reduction activity,
Preferably, a natural medium is used. An example of a natural medium is glucose 1-5%, peptone 1-3%.
%, yeast extract 0.05-0.5% PH = 6.5, etc. In this case, depending on the microorganism species, glucose can be replaced with other sugar sources such as sucrose, maltose, or liquid sugar, and peptone and yeast extract can be replaced with other nitrogen sources such as soybean flour and firmamedea. Furthermore, in addition to carbon and nitrogen sources, inorganic salts (e.g. FeSO ₄
7H ₂ O, MgSO ₄・7H ₂ O, ZnSO ₄・7H ₂ O, etc.)
Activation of bacterial cells can be expected by adding 0.001 to 0.01%. Compounds having general formulas (2) to (wherein R ¹ and R ² have the same meanings as described above) are obtained by adding alcohol, acetone or It is dissolved in DMF or directly added to yeast or mold culture medium and cultured for 1 to 5 days according to method A or method B. During this time, the conversion of Compound 1~ to Compound 2~ is checked from time to time by thin layer chromatography or the like. After an appropriate time, extraction is performed with an appropriate solvent such as ethyl acetate, ether, benzene, hexane, or chloroform, and the extract is subjected to column chromatography, thin layer chromatography, recrystallization, etc. Optically active azetidinone derivatives 2- are separated and purified. Usually, the optically active compounds 2 to 2 are obtained as a mixture of compounds having different stereochemistry, but each can be separated and purified as a pure compound by the above-mentioned means. (2)-', which is one of the compounds having the general formula (2)- obtained by the present invention, can be converted into compound (3)-' by the following reaction.
can lead to. Compound (3) ~ is patent application No. 58-127143 (Japanese Patent Application No. 60-19763)
Compound (4) can be obtained by the method disclosed in No.
Compound (4) - Patent application No. 58-127143 (Japanese Patent Application No. 60-19763)
Carbapenem derivatives having antibacterial activity can be obtained by the method disclosed in JP-A-59-51286. Next, the present invention will be further explained with reference to reference examples and examples. The present invention is not limited thereto. Example 1 3,4-trans-1-(4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone and 3,4-cis-1
-(4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone dl3-acetoneazetidinone obtained in Reference Example 1
100mg A with Schizosaccharomyces pombe
146 mg of crude biomass extracted with ethyl acetate after shaking culture for 2 days was separated and purified using silica gel preparative TLC, and cyclohexane:ethyl acetate = 1:1
In the system, extract the parts sensitive to UV lamps with Rf = 0.32 and Rf = 0.23. Rf=0.32 part: 30 mg, 3,4-trans-1-
(4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone mp135℃ (recrystallized from ethyl acetate: ether) [α] ²²゜_D + 205゜ (c = 1, CHCl ₃ ) NMR (CDCl ₃ ) δ: 1.27 (3H, d, J = 6Hz), 2.55 (1H, d , J=2Hz), 3.38 (1H, dd, J=2 and 4Hz), 3.75 (3H, s), 4.1~4.5 (1H), 4.60 (1H, t, J=2Hz), 6.75~7.60 (4H, A ₂ B ₂ type) Elemental analysis: C ₁₄ H ₁₅ NO ₃ Calculated value: C, 68.55; H, 6.16; N, 5.71 Actual value: C, 68.34; H, 6.18; N, 5.69 Part of Rf = 0.23: 37 mg, 3,4-cis-1-(4
-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone mp153~155゜[α] ²²゜_D -180゜(c=1, _CHCl3 ) IR (Nujol) cm ^-1 : 3570, 3240, 1735, 1590 NMR ( _CDCl3 ) δ: 1.43 (3H, d, J = 6Hz), 2.64 (1H, d, J = 2Hz), ~3.05 (OH), 3.50 (1H, t, J = 6Hz), 3.77 (3H, s), 4.37 (1H, q J = 6Hz), 4.66 (1H, dd, J=2 and 6Hz), 6.8-7.6 (4H, _A2B type ₂ ) Example 2 3,4-trans-1-(4-methoxyphenyl)-3-(1-hydroxy ethyl)-4-ethynyl-2-azetidinone and 3,4-cis-1
-(4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone dl3-acetylazetidinone obtained in Reference Example 1
840mg of Trigonopsis variabilis was cultured with shaking for 2 days using Method A, and 1.008g of the crude product extracted with ethyl acetate was separated and purified using preparative TLC on silica gel, and purified using a cyclohexane:ethyl acetate = 1:1 system.
Extract the parts sensitive to UV-lamp with Rf=0.32 and Rf=0.20. Rf=0.32 part: 350 mg, 3,4-trans-1
-(4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone [α] ²² ° _D + 61° (c = 1, CHCl ₃ ), by repeating recrystallization of this sample twice from ether:cyclohexane [α] ²² ° _D + 210° (c = 1, CHCl ₃ )
Give a sample. NMR is consistent with the trans isomer of Example 1. Rf=0.20 part: 250 mg, 3,4-cis-1-(4
-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone [α] ²²゜_D -40゜ (c = 1, CHCl ₃ ) mp121 ~ 122゜ NMR (CDCl ₃ ) δ: 1.47 (3H, d, J = 6Hz), ~2.6 (OH) 2.66 (1H, d, J=2Hz), 3.43 (1H, d, d, J=6 and 9Hz), 3.77 (3H, s), 4.70 (d, d, J=2 and 6Hz), 4.45 (1H, dq, J=6 and 9Hz), 6.8-7.6 (4H, _{A2B2 type} ) Elemental analysis: _{as C14H15NO3} Calculated value: C, 68.55; H _, 6.16; N, 5.71 Actual value: C, 68.52; H, 6.04; N, 5.69 Example 3 3,4-trans-1-(4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone dl3-acetylazetidinone obtained in Reference Example 1
115mg was cultured with SaccharomyceS cerevisiae by method A, and 230mg of crude material was extracted with ethyl acetate.
was separated and purified using preparative TLC, and UV irradiated with Rf = 0.32 in a cyclohexane:ethyl acetate = 1:1 system.
- Extract the part sensitive to the lamp. Get 90mg. [α] ²² ° _D +85° (c=1, CHCl ₃ ) NMR shows 3,4-trans-1-(4-methoxyphenyl)-3-(1-hydroxyethyl)- obtained in Example 1. It was consistent with that of 4-ethynyl-2-azetidinone. Example 4 3,4-trans-1-(4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone 991 mg of dl3-acetylazetidinone obtained in Reference Example 1 was added to Kloeckera africana. 1.22g of crude product cultured with method A and extracted with ethyl acetate
was separated and purified using silica gel preparative TLC, and Rf was purified using cyclohexane:ethyl acetate = 1:1
= 0.32 UV - Extract the part sensitive to lamp 770
Get mg. [α] When the crystal showing ²² _D +52° (c=1, CHCl ₃ ) is recrystallized from 3 ml of methanol, mp126-7°C
Obtain 730 mg of crystals of [α] ²² _D +177° (c=1, CHCl ₃ ). NMR is 3,4-trans-1 obtained in Example 1.
-(4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone. Example 5 3,4-trans-1-(4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone and 3,4-cis-1
-(4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone 50 mg of dl3-acetyl azetidinone obtained in Reference Example 1 was cultured with Saccharomyces rosei for 2 days, and ethyl acetate Extract and separate and purify the extracted crude biomass using preparative silica gel TLC to obtain Rf=
0.32 (cyclohexane: ethyl acetate = 1:1)
Extract the part sensitive to UV-lamp to obtain 28 mg. [α] ²² ° _D −56° (c=1, CHCl ₃ ) NMR (CDCl ₃ ) is the dl obtained by Reference Example 12.
Acetylazetidinone NaBH ₄ reduced form Rf =
It agrees with that of the mixture of OH coordination of 0.32. Extract the part sensitive to UV-lamp with Rf = 0.23 to obtain 16 mg of 3,4-cis-1-(4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone. . [α] ²² ° _D +160.3° (c=1, CHCl ₃ ) mp160° NMR matched that of the 3,4-cis form obtained in Example 1. Example 6 3,4-trans-1-(4-methoxybenzyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone and 3,4-cis-1
-(4-Methoxybenzyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone 1-(4-methoxybenzyl)-3-acetyl-4-ethynyl-2-azetidinone obtained from Reference Example 4 Schizosaccharomyces 760mg
900mg of crude biomass was cultured with pombe using method A for 24 hours and extracted with ethyl acetate for silica gel separation.
Separation and purification using TLC, Rf = 0.43 and Rf = 0.34 in a cyclohexane: ethyl acetate = 1:3 system.
Extract the part sensitive to UV-lamp. Rf=
Part 0.43: 155 mg, 3,4-trans-1-(4
-methoxybenzyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone mp91゜ (recrystallized from ether) [α] ²²゜_D +22゜ (c=1, CHCl ₃ ) IR (Nujol) cm ^-1 : 3380, 3280, 1750, 1620 NMR (CDCl ₃ ) δ: 1.18 (3H, d, J=6Hz), 2.39 (1H, d, J=2Hz), 3.22 (1H, dd, J=5 and 2Hz), 3.70 (3H, s), 4.9-4.4 (1H, m), 3.97 (1H , d, J = 15Hz) and 4.59 (1H, d, J = 15Hz), 6.70-7.25 (4H, A ₂ B ₂ type) Elemental analysis: C ₁₅ H ₁₇ NO ₃ Calculated value: C, 69.48; H, 6.61; N, 5.40% Actual value: C, 69.65; H, 6.45; N, 5.58% Part of Rf = 0.34: 196 mg, 3,4-cis-1-(4
-methoxybenzyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone [α] ²² ° _D -57° (c=1, _CHCl3 ) NMR ( _CDCl3 ) δ: 1.49 (3H, d, J=6Hz), 2.55 (1H, d, J=2Hz), 2.90 (OH) , 3.17 (1H, dd, J = 6 and 7Hz), 3.69 (3H, s), 3.85-4.40 (2H, m), 4.0 (1H, d, J = 15Hz), and 4.6 (1H, d, J = 15Hz), 6.7-7.25 (4H, _A2B type ₂ ) Example 7 3,4-trans-1-(4-methoxybenzyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone and 3,4-cis-1
-(4-methoxybenzyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone 1-(4-methoxybenzyl)-3 obtained in Reference Example
-acetyl-4-ethynyl-2-azetidinone
300 mg was cultured with Saccharomyces fermentati for 2 days using Method A, and 514 mg of the crude biomass was extracted with ethyl acetate, separated and purified using silica gel preparative TLC, and purified using a cyclohexane:ethyl acetate = 1:3 system.
Extract the parts sensitive to UV-lamp with Rf=0.43 and Rf=0.34. Part of Rf = 0.43: 102 mg [α] ²² ° _D +5° (c = 1, CHCl ₃ ) NMR is 3,4-trans-1-(4-methoxybenzyl)-3-(1-hydroxyethyl)-4
-It is a mixture of two coordinations of the 1-hydroxy group of -ethynyl-2-azetidinone. Rf=0.34 portion: 84 mg [α] ²² ° _D +55° NMR was consistent with that of the 3,4-cis form of Example 6. Example 8 3,4-trans-1-(1-benzyloxycarbonylprop-1-enyl)-3(1-hydroxyethyl)-4-ethynyl-2-azetidinone and 3,4-cis-1-(1 -benzyloxycarbonylprop-1-enyl)-3-(1
-hydroxyethyl)-4-ethynyl-2-azetidinone 1-(1-benzyloxycarbonylprop-1-enyl)-3-acetyl of Reference Example 9 obtained from diastereomer (2) obtained in Reference Example 8 -4-ethynyl-2-azetidinone ([α] _D +18°) 60mg
2 by method A along with Saccharomyces cerevisiae
After culturing for 1 day, 91.5 mg of the ethyl acetate extract was subjected to silica gel chromatography, and cyclohexane:
Parts with Rf = 0.26 and 0.20 are isolated in a system of ethyl acetate = 1.1. Rf=0.26 part: 17 mg, 3,4-trans-1-
(1-benzyloxycarbonylprop-1-
enyl)-3-(1-hydroxyethyl)-4
-Ethynyl-2-azetidinone NMR ( _CDCl3 ) δ: 1.31 (3H, d, J = 6Hz), 1.92 (3H, d, J = 7Hz), 2.42 (1H, d, J = 2Hz), 3.37 (1H, dd. br.s.) Rf=0.20 part: 10 mg, 3,4-cis-1-(1
-benzyloxycarbonylprop-1-enyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone NMR ( _CDCl3 ) δ: 1.40 (3H, d, J = 6Hz), 1.91 (3H, d , J=7Hz), 2.52 (1H, d, J=2Hz), 3.37 (1H, t, J=5.5Hz), 4.1~4.5 (1H), 4.86 (1H, dd, J=2 and 5.5Hz) 5.15 (2H, s), 7.02 (1H, q, J = 7Hz), 7.32 (5H, br.s) Example 9 3,4-trans-1-(1-benzyloxycarbonylprop-1-enyl)-3 -(1-hydroxyethyl)-4-ethynyl-2-azetidinone 1-(1-benzyloxycarbonylprop-1-enyl)- of Reference Example 9 obtained from diastereomer (1) obtained in Reference Example 8 70 mg of 3-acetyl-4-ethynyl-2-azetidinone [α] ²² _D -18° (C = 0.5, CHCl ₃ ) was added to Saccharomyces
cerevisiae for 2 days, extracted with ethyl acetate, and 130 mg of the extract was subjected to silica gel chromatography to obtain cyclohexane:ethyl acetate = 1.1.
The parts with Rf = 0.26 and 0.20 are isolated and purified using the system.
15 mg of the portion of Rf=0.26 obtained in Example 8, Rf=
Get 0.2 portions 10mg. Example 10 3,4-trans-1-(2-hydroxy-1
-benzyloxycarbonylethyl)-3-(1
-Hydroxyethyl)-4-ethynyl-2-azetidinone 1 obtained using L-serine as a starting material in Reference Example 10
-(2-hydroxy-1-benzyloxycarbonylethyl)-3-acetyl-4-ethynyl-2
-90 mg of azetidinone (diastereomer (1))
Cultivated with Schizosaccharomyces pombe for 2 days using method A, extracted with ethyl acetate, and obtained 142 mg of extract.
was subjected to silica gel chromatography, and the portion corresponding to Rf = 0.24 was isolated and purified using a methylene chloride:ethyl acetate = 3:1 system to obtain 1-(2-hydroxy-1-benzyloxycarbonylethyl)-3. −
25 mg of (1-hydroxyethyl)-4-ethynyl-2-azetidinone are obtained. NMR (CDCl ₃ ) δ: 1.39 (3H, d, J = 6Hz),
2.58 (1H, d, J = 2Hz), 3.34 (1H, t, J =
5.6Hz), 3.9-4.3 (4H, m), 4.44 (1H, dd, J=
5.6 and 2Hz), 5.19 (2H, S), 7.30 (5H, S) Example 11 3,4-trans-1-(2-hydroxy-
1-benzyloxycarbonylethyl-3-
(1-hydroxyethyl)-4-ethynyl-2-
Azetidinone 60 mg of 1-(2-hydroxy-1-benzyloxycarbonylethyl)-3-acetyl-4- obtained from Reference Example 10 using L-serine as a starting material
Ethynyl-2-azetidinone (diastereomer)
(1)) was cultured with Saccharomyces fermentati for 2 days using Method A, and the living body was extracted with ethyl acetate. Evaporate the solvent to obtain 81 mg of crude extract. Methylene chloride:
TLC on silica gel was performed in a system of ethyl acetate = 3:1 to obtain 20 mg of the desired compound corresponding to Rf = 0.24. NMR (CDCl ₃ ) δ: 1.33 (3H, d, J = 6Hz),
2.44 (1H, d, J = 2Hz), 3.30 (1H, dd, J = 6
and 2Hz), 4.22 (1H, t, J = 2Hz), 3.8-4.4
(4H, m), 5.19 (2H, S), 7.33 (5H, S). Example 12 3,4-trans-1-(4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone dl3,4-trans-1-(4-methoxyphenyl) -100 mg of 3-acetyl-4-ethynyl-2-azetidinone was added to mature baker's yeast Saccharomyces
cerevisiae for 24 hours according to method B. During this time, add sugar. Extract with ethyl acetate to obtain 192 mg of crude product. Separate and purify using silica gel preparative TLC, and extract the portion sensitive to UV lamps with Rf = 0.5 and Rf = 0.32 using a cyclohexane:ethyl acetate = 1:1 system. Rf=0.5 part 30mg

[Formula] Crystal, recrystallized from ether-n-hexane mp97
°C [α] _D −120° (C=0.3, CHCl ₃ ) Optically active 3-
Acetyl azeticinone was obtained. NMR is dldl1-(4-methoxyphenyl)-3
-Acetyl-4-ethynyl-2-azetidinone (Reference Example 1). Rf=0.32 part 42mg crystal, ethyl acetate: recrystallized from ether mp135゜[α] ²²゜_D +210° (C=1, CHCl ₃ )

[Formula] NMR is 3,4-trans-1- of Example 1
It corresponds to that of (4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone. Similar results are obtained using mature Saccharomyces rosei. Example 13 3,4-trans-1-(4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone and 3,4-cis-1
-(4-Methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone 90 mg of dl3-acetyl azetidinone obtained in Reference Example 1 was cultured with Trichosporon penicillatum for 24 hours by method B. do. After extraction with ethyl acetate, the solvent was concentrated under reduced pressure, and 90.5 mg of the crude extract was separated and purified using silica gel preparative TLC, and Rf = 0.32 and
Extract the part sensitive to the UV-lamp with Rf=0.23. Rf=0.32 part: 34.8mg

[Formula] [α] ²² ° _D +144° (C=1, CHCl ₃ ) NMR shows 3,4-trans-1- of Example 1
It corresponds to that of (4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone. Rf=0.23 part: 25mg

[Formula] [α] ²² ° _D -197° (C=1, CHCl ₃ ) NMR shows 3,4-cis-1-(4-
methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone. Example 14 3,4-cis-1-(4-methoxyphenyl)-
3-(1-hydroxyethyl)-4-ethynyl-
2-Azetidinone The dl3-acetyl azetidinone obtained in Reference Example 1 was cultured with 100 mg of Pichia terricola according to Method B (24 hours), and the living body was extracted with ethyl acetate.
139 mg of the crude extract was subjected to Rf using silica gel preparative TLC in a system of cyclohexane:ethyl acetate = 1:1.
Separate and purify the portion with Rf = 0.32 and the portion with Rf = 0.20. Rf=0.32 part: 48.8 mg, [α] _D +184° NMR shows 3,4-trans-1 (P
-methoxyphenyl)-3-(1'-hydroxyethyl)-4-ethynyl-2-azetidinone. Rf=0.20 part: 14 mg [α] ²² _D −144° (C=1,
CHCl ₃ ) NMR is 3,4-cis-1-(4-
methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone. Example 15 3,4-trans-1-(4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone and 3,4-cis-1
-(4-Methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone 100 mg of dl-3-acetyl azetidinone obtained in Reference Example 1 was cultured with Streptomyces cattleya for 24 hours by method B. Then, extract the living body with ethyl acetate. The solvent was distilled off under reduced pressure to obtain the crude volume.
Separate and purify 125.6 mg using silica gel preparative TLC,
In a system of cyclohexane: ethyl acetate = 1:1,
The parts sensitive to UV lamps of Rf=0.32 and Rf=0.2 are isolated and extracted. Rf=0.32 part: 21.2mg [α] ²²゜_D +145゜(C=1,
CHCl ₃ ) NMR is 3,4-trans-1- of Example 1.
It corresponds to that of (4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone. Rf=0.2 part: 15.4 mg [α] ²² ° _D −170° (C=1,
CHCl ₃ ) NMR completely matches that of the 3,4-cis form of Example 2. Example 16 3,4-trans-1-(4-methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone and 3,4-cis-1
-(4-Methoxyphenyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone B
After culturing according to the method, the living body was extracted with ethyl acetate to obtain 129 mg of crude living body. For preparative silica gel
Cyclohexane:ethyl acetate =
The portions with Rf=0.32 and 0.23 are separated and purified in a 1:1 system. Rf=0.32 part: 17.3 mg [α] _D +143° NMR is 3,4-trans-1- of Example 1
It corresponds to that of (P-methoxyphenyl)-3-(1'-hydroxyethyl)-4-ethynyl-2-azetidinone. Rf = 0.23 part: 63.3 mg [α] ²² ° _D -68° (C = 1,
CHCl ₃ ) NMR completely matches that of the 3,4-cis form of Example 1. Example 17 3,4-trans-1-(4-methoxybenzyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone and 3,4-cis-1
-(4-Methoxybenzyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone 200 mg of the 3-acetyl compound obtained in Reference Example 4 was added to
30 by method A with Saccharomyces cerevisiae
After culturing for an hour and extracting ethyl acetate, crude biomass 208
Get mg. This was separated and purified using silica gel preparative TLC, and cyclohexane:ethyl acetate=
Separate the parts of Rf=0.35 and 0.26 in a 1:2 system. Rf = 0.35 part: 65 mg, [α] ²² ° _D -6° (C = 1,
CHCl ₃ ) NMR corresponds to that of the 3,4-trans form of Example 6. Rf=0.26 part: 27mg

[Formula] [α] ²² ° _D +17° (C=1, CHCl ₃ ) NMR (CDCl ₃ ) δ: 1.40 (3H, d, J=6.)
Hz), 2.55 (1H, d, J = 2Hz), 2.40 (OH), 3.22
(1H, dd, J=5.5 and 9Hz), 3.75 (3H, S),
3.9-4.4 (2H), 3.97 (1H, d, J = 15Hz) and
4.64 (1H, d, J=15Hz), 6.7~7.25 (4H, A ₂ B ₂
Example 18 3,4-trans-1-(4-methoxyphenyl)-3-(1-hydroxyethyl)-4-trimethylsilylethynyl-2-azetidinone

[Formula] 330 mg of the 3-acetyl azetidinone obtained in Reference Example 7 is cultured with Saccharomyces cerevisiae for 48 hours according to Method A. Extracted with ethyl acetate,
284 mg of crude product are obtained. Separated and purified using silica gel preparative TLC, cyclohexane: ethyl acetate =
In a 2:1 system, the part sensitive to a UV-lamp with Rf=0.24 was extracted to obtain 33 mg. [α] _D −30° (C=1, CHCl ₃ ) NMR (CDCl ₃ ) δ: 0.15 (9H, S), 1.28 (1.5H,
d, J=6Hz), 1.37 (1.5H, d, J=6Hz),
3.25-3.45 (1H), 3.72 (3H, S), 3.9-4.3 (1H),
4.38 (0.5H, d, J = 2.5Hz), 4.52 (0.5H, d, J
= 2.5Hz), 6.7 to 7.5 (4H, _A2B type ₂ ) Reference example 1 dl3,4-trans-1-(4-methoxyphenyl)-3-acetyl-4-ethynyl-2-azetidinone Propargylaldehyde 4g Dissolve in 50 ml of benzene, add 9 g of para-anisidine and 4A molecular sieve (about 4 g), and stir at 20°C for 30 minutes. The reaction solution was filtered, the solvent was distilled off (under reduced pressure), the residue was dissolved in 60 ml of anhydrous methylene chloride, 4.3 g of imidazole was added, and the entire system was heated under nitrogen at -20 °C.
℃. Slowly add 7.8g of diketene at -20° to -10°C. Raise the reaction temperature to 20℃ over about 2 hours, add 40ml of methylene chloride, wash with water, wash with dilute hydrochloric acid, wash with water,
Dry over _MgSO4 . After distilling off the solvent, the desired target compound is isolated and purified by silica gel rapid chromatography (solvent system: cyclohexane:ethyl acetate=3:1). Get 5.7g. TLC, Rf = 0.5 (cyclohexane: ethyl acetate = 1:1) mp85° (recrystallized from ethyl ether) NMR (CDCl ₃ ) δ: 2.35 (3H, S), 2.50 (1H,
d, J=2Hz), 3.75 (3H, S), 4.33 (1H, d,
J=2Hz), 4.91 (1H, t, J=2Hz), 6.7-7.5
(4H, A ₂ B ₂ type) IR (Nujol) cm ^-1 : 1760, 1720, 2100 Reference example 2 dl3,4-trans-1-benzyl-3-acetyl-4-ethynyl-2-azetidinone Propargyl aldehyde 500mg Dissolve in 10ml benzene and add 1g _MgSO4 . While stirring, 990 mg of benzylamine was further added, and the mixture was stirred at 20°C for 2 hours. Insoluble matter in the reaction solution was removed, the solvent was distilled off under reduced pressure, the residue was dissolved in 15 ml of anhydrous methylene chloride, 555 mg of imidazole was added, and the entire system was cooled to -20°C under a nitrogen atmosphere. Add 1 ml of diketene to this using a syringe.
Add at 20° to -10° and slowly raise the reaction temperature to 10°C (about 2 hours). Add 20ml methylene chloride,
Wash with water, dilute hydrochloric acid, water, and dry the extract over _MgSO4 . After distilling off the solvent, the desired target compound is isolated and purified by silica gel rapid chromatography (cyclohexane:ethyl acetate=5:1). Get 720mg. oily substance. TLC, Rf = 0.31 (cyclohexane: ethyl acetate = 2:1) NMR (CDCl ₃ ) δ: 2.30 (3H, S), 2.49 (1H,
d, J = 2Hz), 4.13 (1H, d, J = 15Hz) and
4.68 (1H, d, J = 15Hz), 4.29 (1H, d, J = 2
Hz), 4.49 (1H, t, J = 3Hz), 7.34 (5H, br.s.) Reference example 3 3,4-trans-1-(α-methylbenzyl)
-3-Acetyl-4-ethynyl-2-azetidinone To a solution of 500 mg of propargylaldehyde in 5 ml of anhydrous benzene was added 1.12 g of (+)D-phenethylamine in the presence of 1 g of 4A molecular sieve, and the mixture was heated at 20°C.
Stir for an hour. Insoluble matter in the reaction solution was removed, the solvent was distilled off under reduced pressure, the residue was dissolved in 20 ml of anhydrous methylene chloride, 0.56 g of imidazole was added, the whole system was brought to -20°C under a nitrogen atmosphere, and 1 ml was dissolved at the same temperature. of diketene was slowly added and the reaction temperature was raised to 10°C over 3 hours. Diluted with methylene chloride, washed with water, washed with dilute hydrochloric acid,
After washing with water, dry with MgSO ₄ . After evaporating the solvent, the residue was subjected to silica gel and rapid chromatography using cyclohexane:ethyl acetate=1:1 with Rf=0.70.
1.15 g of the target compound corresponding to is isolated. The product is a mixture of diastereomers in an approximately 1:1 ratio, but the two can be separated by careful separation using silica gel chromatography or a Rover column. Isomer 1 NMR (CDCl ₃ ) δ: 1.70 (3H, d, J = 7Hz),
2.26 (3H, s), 2.38 (1H, d, J=2Hz), 4.4~
4.75 (1H, m), 7.25 (5H, S.), 4.15 (1H, d, J
= 2Hz), 4.35 (1H, t, J = 2Hz) Isomer 2 NMR (CDCl ₃ ) δ: 1.66 (3H, d, J = 7Hz),
2.24 (3H, S), 2.39 (1H, d, J = 2Hz), 4.15
(1H, d, J=2Hz), 4.35 (1H, t, J=2
Hz), 4.89 (1H, qJ=7Hz) Reference example 4 dl3,4-trans-1-(4-methoxybenzyl)-3-acetyl-4-ethynyl-2-azetidinone Dissolve 1 g of propargylaldehyde in 15 ml of anhydrous benzene Then, 2.28 g of P-methoxybenzylamine is added, followed by 6 g of MgSO ₄ and stirred for 30 minutes at room temperature. Insoluble matter in the reaction solution was removed, the solvent was distilled off under reduced pressure, and 30 ml of anhydrous methylene chloride was added.
Another 1.28 g of imidazole is added and the whole system is cooled to −20° C. under N ₂ atmosphere. Slowly add 1.54 ml of diketene at the same temperature and raise the reaction temperature from -20°C to 10°C over about 1.5 hours. Most of the methylene chloride was distilled off under reduced pressure, ethyl acetate was added, and after washing with water.
Dry with _MgSO4 . After distilling off the solvent, the target compound corresponding to Rf=0.4 (cyclohexane:ethyl acetate=1:1) was separated and purified by chromatography to obtain 980 mg of an oily substance. NMR (CDCl ₃ ) δ: 2.23 (3H, S), 2.55 (1H,
d, J = 2Hz), 3.70 (3H, S), 4.0 (1H, d, J
= 15Hz) and 4.52 (1H, d, J = 15Hz), 4.22
(1H, d, J=2Hz), 4.38 (1H, t, J=2Hz)
6.7-7.3 (4H, A ₂ B ₂ type) IR (Liq) cm ^-1 : 1762, 1715, 2120 Reference example 5 dl3,4-trans-1-(2,4-dimethoxybenzyl)-3-acetyl-4 -ethynyl-
2-Azetidinone To a solution of 1 g of propargylaldehyde in 15 ml of anhydrous benzene, add 15 ml of an aqueous benzene solution of 2,4-dimethoxybenzylamine obtained from 2.48 g of 2,4-dimethoxybenzylamine hydrochloride, and add 7 g of anhydrous MgSO ₄ at room temperature. Stir for 30 minutes. Insoluble matter in the reaction solution was removed, the solvent was distilled off under reduced pressure, the solution was dissolved in 30 ml of anhydrous ethylene chloride, 1.5 g of imidazole was added, and the whole system was cooled to -20°C in a nitrogen atmosphere. Slowly add 1.8 ml of diketene at -20° to -10°C,
The reaction temperature was raised to 10°C over about 1 hour, the solvent was distilled off under reduced pressure, ethyl acetate was added, the mixture was washed with water, and dried with MgSO ₄ . After solvent distillation Rf = 0.33 (cyclohexane:
The desired compound corresponding to ethyl acetate (1:1) was isolated and purified using silica gel column chromatography to obtain 700 mg of an oily substance. IR (Liq.) cm ^-1 : 2120, 1760, 1710 NMR (CDCl ₃ ) δ: 2.25 (3H, S), 2.44 (1H,
d, J=2Hz), 3.75 (6H, S), 4.05 (1H, d,
J=15Hz) and 4.58 (1H, d, J=15Hz), 4.20
(1H, d, J=2Hz), 4.38 (1H, t, J=2
Hz), 6.3-6.6 (2H, m), 6.95-7.28 (1H, m) Reference example 6 dl3,4-trans-1-(3,4-dimethoxybenzyl)-3-acetyl-4-ethynyl-
2-Azetidinone 5 g of 3,4-dimethoxybenzylamine instead of 2,4-dimethoxybenzylamine in Reference Example 5,
Propargyl aldehyde 2g, imidazole 3g,
The reaction was carried out in exactly the same manner as in Reference Example 5 using 3.6 ml of diketene and 1.4 g of the desired title compound was obtained. Rf = 0.24 (cyclohexane: ethyl acetate = 1:
1) IR (Liq) cm ^-1 : 2120, 1762, 1710 NMR (CDCl ₃ ) δ: 2.26 (3H, S), 2.60 (1H,
d, J = 2Hz), 3.80 (6H, S), 4.0 (1H, d, J
= 15Hz) and 4.55 (1H, d, J = 15Hz), 4.28
(1H, d, J=2Hz), 4.41 (1H, t, J=2
Hz), 6.71 (2H, S), 7.40 (1H, S) Reference example 7 dl3,4-trans-1-(α,α-dimethylbenzyl)-3-acetyl-4-ethynyl-2
-Azetidinone Add 850 mg of cumylamine to a solution of 400 mg of propargyl aldehyde in 10 ml of benzene, add 2 g of MgSO ₄
Stir for 30 min in the presence of. Insoluble matters in the reaction solution were removed, the solvent was distilled off, the solution was dissolved in anhydrous methylene chloride, and 513 mg of imidazole was added. The entire system is kept at -20℃ in a nitrogen atmosphere.
0.62 ml of diketene was added and the reaction temperature was brought to 10°C for 1.5 hours. The solvent was distilled off under reduced pressure,
Add ethyl acetate and wash with water. The product was subjected to rapid chromatography in a cyclohexane:ethyl acetate ratio of 3:1 to obtain 283 mg of the desired compound. Rf = 0.51 (cyclohexane: ethyl acetate = 1:
1) NMR (CDCl ₃ ) δ: 1.78 (6H, S), 2.26 (3H,
S), 2.33 (1H, d, J=2Hz), 4.11 (1H, d,
J=2Hz), 4.54 (1H, t, J=2Hz), 7.1~7.4
(5H, S) Reference example 8 3,4-trans-1-(1-benzyloxycarbonyl-2-hydroxy)propyl-3-
Acetyl-4-ethynyl-2-azetidinone 1) SMTenneson and B.Belleau separately in a 30ml benzene solution of 600mg propargyl aldehyde,
Can, J.Chem.58 1605 (1980), 2.4g of L-threonine-0-tert-butyldimethylsilylbenzyl ester was mixed with 5g of L-threonine-0-tert-butyldimethylsilylbenzyl ester.
After adding in the presence of MgSO ₄ and stirring at room temperature for 30 minutes, the solvent was distilled off under reduced pressure. Dissolve in 30 ml of anhydrous methylene chloride, add 800 mg of imidazole and then cool the entire system to -20° C. under nitrogen atmosphere. Add 1.1 g of diketene at -20°C to -10° and bring the reaction temperature to 10°C over about 2 hours. Add 100 ml of ethyl acetate, wash with water 5 times, dry the organic layer over MgSO ₄ and evaporate the solvent under reduced pressure. The product was subjected to silica gel rapid chromatography to obtain 1.1 g of a compound corresponding to Rf=0.6 (cyclohexane:ethyl acetate=2:1) on TLC. IR (Liq) cm ^-1 : 2120, 1770, 1742, 1720 The compound is a mixture of two diastereomers and can be separated by method 2) below. 2) Dissolve 1.1 g of the compound obtained by the method in 1) in 15 ml of acetonitrile, and add 0.3 ml of BF ₃ -etherate under ice cooling. After 15 minutes, confirm that the desilylation has completely progressed by TLC, and then add ethyl acetate.
Add 40ml and wash with water. After drying with MgSO ₄ , the solvent is distilled off, and the product is subjected to rapid chromatography to separate and purify the product to obtain the target compound. Diastereomer (1) Rf=0.35 630 mg is obtained.
(Cyclohexane: ethyl acetate = 1:1) IR (Liq) cm ^-1 : ~3200, ~3300, 2130, 1750,
1740, 1720 NMR (CDCl ₃ ) δ: 1.27 (3H, d, J = 6Hz),
2.29 (3H, S), 2.50 (1H, d, J=2Hz), 3.99
(1H, d, J=4Hz), 4.25 (1H, d, J=2
Hz), 4.73 (1H, t, J=2Hz), 4.2-4.6 (1H,
m.), 5.19 (2H, S), 7.35 (5H, S) diastereomer (2) Rf = 0.26 to obtain 360 mg (cyclohexane: ethyl acetate = 1:1) IR (Liq) cm ^-1 : ~ 3200, ~3300, 2130, 1760,
1740, 1719 NMR (CDCl ₃ ) δ: 1.26 (3H, d, J = 6Hz),
2.30 (3H, S), 2.46 (1H, d, J=2Hz), 4.13
(1H, d, J=4Hz), 4.35 (1H, d, J=2
Hz), 4.89 (1H, t, J=2Hz), 5.20 (2H, S),
7.35 (5H, S) Reference Example 9 3,4-trans-1-(1-benzyloxycarbonyl-prop-1-enyl)-3-acetyl-4-ethynyl-2-azetidinone Diastereo obtained in Reference Example 8 Mar(1) 630mg 10
Dissolve in ml of anhydrous methylene chloride and add 220 mg of triethylamine. Slowly add 250 mg of mesyl chloride (methanesulfonyl chloride) to this solution and stir for 20 minutes. After confirming that diastereomer (1) has disappeared, add methylene chloride and wash with water.
After drying over MgSO ₄ , the product is separated by silica gel chromatography to obtain 300 mg of the desired compound. Rf = 0.5 (cyclohexane: ethyl acetate = 2:
1) NMR (CDCl ₃ ) δ: 1.89 (3H, d, J = 6Hz),
2.28 (3H, S), 2.44 (1H, d, J = 2Hz), 4.28
(1H, d, J=4Hz), 5.10 (1H, t, J=2
Hz), 5.18 (2H, S), 7.05 (1H, q, J = 6Hz),
7.35 (5H, S) [α] ²²゜_D -18゜ (C = 0.5, CHCl ₃ ) 360 mg of diastereomer (2) obtained in Reference Example 8 was treated in the same manner as in the above example to obtain the desired title. Obtain 150 mg of compound. Rf = 0.5 (cyclohexane: ethyl acetate = 2:
1) IR (Liq) cm ^-1 : 2110, 1765, 1720, 1645 [α] ²²゜_D +18゜ (C = 1, CHCl ₃ ) Reference examples 8 and 9 are similar even if D-threonine derivatives are used. The reactions proceed to give the respective corresponding compounds. Reference example 10 3,4-trans-1-(1-benzyloxycarbonyl-2-hydroxy)ethyl-3-acetyl-4-ethynyl-2-azetidinone 1) 10 ml of 400 mg propargyl aldehyde
Can.J.Chem58 1605 (1980) in anhydrous benzene solution
1.4 g of benzyl-L-serine-0-tert-butyldimethylsilyl synthesized according to the method of
The ester was added in the presence of 4 g _MgSO4 at room temperature.
After stirring for 20 minutes, the filtrate was distilled off under reduced pressure. Dissolve in 25 ml of anhydrous methylene chloride and put the whole system under nitrogen atmosphere.
Bring the temperature to 20℃ and slowly add 0.67ml of diketene. The reaction temperature was increased to +5° over 2 hours, 65 ml of ethyl acetate was added, and the organic layer was washed with water, diluted hydrochloric acid, water, and dried over MgSO ₄ . After evaporation, the crude product was subjected to silica gel rapid chromatography and developed in a developing system of cyclohexane:ethyl acetate=4:1 to obtain 750 mg of the desired compound. Rf = 0.5 (cyclohexane: ethyl acetate = 2:
1) IR (Liq) cm ^-1 : 2120, 1770, 1745, 1720 The compound is a mixture of two diastereomers and is separated by the method 2) below. 2) Dissolve 750 mg of the compound obtained by the method in 1) in 10 ml of acetonitrile, and add 0.3 ml of BF ₃ -etherate under ice cooling. After 15 minutes, after confirming that the desilylation had completely progressed by TLC, 40% ethyl acetate was added.
ml, washed with water, dried with MgSO ₄ , and the solvent was distilled off. The target compounds, which are two diastereomers, are separated and purified using preparative silica gel TLC. Diastereomer (1): 230 mg, Rf = 0.55 (methylene chloride: ethyl acetate = 3:1) IR (Liq) cm ^-1 : ~3440, ~3300, 2120, 1765,
1740, 1720 NMR ( _CDCl3 ) δ: 2.29 (3H, S), 2.46 (1H,
d, J=2Hz), ~4.10 (3H, br.), 4.22 (1H,
d, J=2Hz), 4.73 (1H, t, J=2Hz), 5.18
(2H, S), 7.34 (5H, S) [α] ²² ° _D +5° (C = 1, CHCl ₃ ) Diastereomer (2): 110 mg, Rf = 0.40 (methylene chloride: ethyl acetate = 3:1 ) IR (Liq) cm ^-1 : ~3440, ~3300, 2120, 1765,
1742, 1720 NMR ( _CDCl3 ) δ: 2.29 (3H, S), 2.43 (1H,
d, J=2Hz), 4.05 (2H, br.d.), 4.30 (1H, d,
J = 2Hz), 4.20-4.45 (1H), 4.85 (1H, t, J
=2Hz), 5.18 (2H, S), 7.34 (5H, S.) [α] ²² _D +21° (C = 1, CHCl ₃ ) Reference example 11 dl3,4-trans-1-(1-benzyloxycarbonyl ) ethynyl-3-acetyl-4-
Ethynyl-2-azetidinone Diastereomer (1) obtained from Reference Example 10
37mg pyridine dissolved in anhydrous methylene chloride under ice cooling
25mg, then 40mg of methanesulfonyl chloride was added, the whole system was stirred at 20℃ for 10 hours, and the portion of Rf = 0.45 (cyclohexane: ethyl acetate = 2:1) was collected using preparative silica gel TLC to obtain 10mg of the target compound.
get. [α] ²²゜_D -23゜ (C = 0.5, CHCl ₃ ) IR (Liq) cm ^-1 : 2130, 1765, 1720, 1620 NMR (CDCl ₃ ) δ: 2.30 (3H, S), 2.39 (1H,
d, J=2Hz), 4.27 (1H, d, J=2Hz), 5.23
(2H, S), ~5.24 (1H, m), 6.02 (1H, S) and 6.09 (1H, S), 7.36 (5H, S) Diastereomer (2) obtained according to Reference Example 10
Dissolve 100mg in anhydrous methylene chloride, add 100mg of triethylamine, then add methanesulfonyl chloride.
Add 75 mg under ice cooling. After 5 hours, the mixture is treated in the usual manner, and a portion with Rf = 0.45 (cyclohexane:ethyl acetate = 2:1) is fractionated using preparative silica gel TLC. Obtain 58 mg of the target compound. [α] ²² ° _D +23° (C=1, CHCl ₃ ) IR and NMR are the same as those obtained from diastereomer (1). Reference example 12 dl3,4-trans-1-(4-methoxyphenyl)-3-(α)-(1-(S ^* )-hydroxyethyl)-4-ethynyl-2-azetidinone Reference examples 12 to 15 are This was carried out to confirm the absolute coordination of the reduced product obtained by microbial reduction of azetidinone derivatives. 370 mg of dl3-acetylazetidinone obtained in Reference Example 1 was dissolved in 5 ml of methanol and heated to 0°C.
Add 30 mg of NaBH ₄ as a solid in 4 portions. Stir for 15 minutes at the same temperature. After confirming that the raw material has completely disappeared by TLC, add ethyl acetate and wash with water, dilute hydrochloric acid, water, and dry with Mg ^- SO ₄ . The solvent was distilled off under reduced pressure, and the residue was purified by chromatography to obtain 300 mg of crystals. Rf = 0.32 (cyclohexane: ethyl acetate = 1:
1) The compound is a mixture of OH coordinations, but one isomer can be separated as a crystal by recrystallizing from methanol. mp107-109゜ (methanol recrystallization) IR (Nujol) cm ^-1 : 3550, 3220, 2120, 1730 NMR (CDCl ₃ ) δ: 1.37 (3H, d, J = 6Hz),
2.55 (1H, d, J=2Hz), 3.40 (1H, dd, J=2
and 4Hz), 3.75 (3H, S), 3.9-4.4 (1H, m),
4.45 (1H, t, J=2Hz), 6.75~7.6 (4H, A ₂ B ₂
Type) Elemental analysis Calculated value as C ₁₄ H ₁₅ NO ₃ : C, 68.55,
H, 6.16; N, 5.71 Actual value: C, 68.57, H,
6.23; N, 5.76 Reference example 13 dl3,4-trans-1-(4-methoxyphenyl)-3-(α)-(1-(S ^* )-t-butyldimethylsilyloxy)ethyl-4-ethynyl −
2-Azetidinone 80 mg of one isomer isolated as a crystal in Reference Example 12 (mp107-109°C) was dissolved in 2 ml of DMF, 30 mg of imidazole and 130 mg of tert-butyldimethylsilyl chloride were added at room temperature,
Stir for a minute. Add ethyl acetate, wash with water, and dry with MgSO ₄ . The portion with Rf = 0.8 was isolated and extracted using cyclohexane:ethyl acetate = 1:1 using silica gel preparative TLC to obtain 120 mg of the target compound. NMR (CDCl ₃ ) δ: 0.08 (6H, S), 0.80 (9H,
S), 1.33 (3H, d, J=6Hz), 2.47 (1H, d,
J=2Hz), 3.43 (1H, J=2 and 3Hz), 3.75
(3H, S), 4.0-4.35 (1H, m), 4.37 (1H, t,
J=2Hz), 6.75-7.5 (4H, _A2B type ₂ ) Reference example 14 dl3,4-trans-1-(4-methoxyphenyl)-3-(α)-(1-(S ^* )- t-Butyldimethylsilyloxy)ethyl-4-(phenylthio)ethynyl-2-azetidinone 120 mg of the silyl compound obtained in Reference Example 13 was anhydrous.
Dissolve in 2 ml of THF and 0.3 ml of butyllithium solution (1
A hexane solution containing 1.6 mmol butyllithium in ml is added at -78°C. After stirring for another 30 minutes, a 1.5 ml THF solution containing 108 mg of diphenyl disulfide was added at the same temperature, and the mixture was stirred at -78° to -20°C for about 3 hours. TLC
After confirming that the upper raw material has disappeared, add ethyl acetate and wash with water 3 times. After drying the organic layer with MgSO ₄ , the product was subjected to preparative TLC in a cyclohexane:ethyl acetate=5:1 system to isolate 128 mg of the target compound. TLC Rf = 0.58 (cyclohexane: ethyl acetate = 5:1) NMR (CDCl ₃ ) δ: 0.08 (6H, S), 0.82 (9H,
S), 1.38 (3H, d, J=6Hz), 3.48 (1H, dd,
J=3 and 4Hz), 3.74 (3H, S), 4.0~4.4 (1H,
m), 4.64 (1H, d, J=3Hz), 6.75-7.55 (4H,
A ₂ B type ₂ ) Reference example 15 dl3,4-trans-3-(α)-(1-(S ^* )-
t-Butyldimethylsilyloxy)ethyl-4
-Phenylthioethynyl-2-azetidinone 108 mg of the S-phenylated compound obtained in Reference Example 14 was dissolved in 4 ml of acetonitrile, and 3 ml of an aqueous solution of 400 mg of Selic ammonium nitrite was added under ice cooling, followed by stirring for 20 minutes. Add ethyl acetate and wash with water 3
After drying with MgSO ₄ twice, the solvent was distilled off under reduced pressure. Rf=0.54 in cyclohexane:ethyl acetate=2:1 system
Isolate 50 mg of the target compound corresponding to the portion of . mp 93℃ (recrystallized from petroleum ether) IR (Nujol) cm ^-1 : 3200, 2175, 1770, 1760 NMR (CDCl ₃ ) δ: 0.08 (6H, S), 0.89 (9H,
S), 1.31 (3H, d, J=6Hz), 3.42 (1H, br,
t, J≒3), 4.1-4.5 (1H, m), 4.33 (1H, d,
J = 2.5Hz), ~6.1 (1H, br.S), 7.32 (5H, m). Reference example 16 3,4-trans-1-(4-methoxybenzyl)-3-acetyl-4-ethynyl-2-azetidinone 3,4-cis-1-(4-
Dissolve 196 mg of methoxybenzyl)-3-(1-hydroxyethyl)-4-ethynyl-2-azetidinone ([α] ²² _D -57°) in 5 ml of anhydrous methylene chloride, and dissolve pyridinium chlorochromate (PCC).
Add 500 mg under ice-cooling and stir at 25°C for an additional 4 hours.
The solvent was distilled off under reduced pressure, ethyl acetate was added, and the mixture was washed with water.
The product is separated and purified by silica gel chromatography to obtain 106 mg of the desired compound corresponding to Rr=0.4 (cyclohexane:ethyl acetate=1:1).
An optically active 3-acetyl azetidinone whose IR and NMR completely correspond to those of Reference Example 4 is obtained. [α] ²² ° _D +39° (C=1, CHCl ₃ ) Reference example 17 dl3,4-trans-1-(4-methoxyphenyl)-3-acetyl-4-trimethylsilylethynyl-2-azetidinone Trimethylsilylpropargyl aldehyde 614
Dissolve mg in 20 ml of benzene, add 500 mg of para-anisidine and 600 mg of magnesium sulfate, and add 20 mg of para-anisidine and 600 mg of magnesium sulfate.
Stir at ℃ for 30 minutes. The reaction solution was filtered, the solvent was distilled off under reduced pressure, the residue was dissolved in 20 ml of anhydrous methylene chloride, 331 mg of imidazole was added, and the whole system was brought to -20°C under nitrogen. Slowly add 0.4ml of diketene at -20° to -10°. Raise the reaction temperature to 10°C over about 1 hour. After adding methylene chloride and washing with water, MgSO ₄
Dry at. After distilling off the solvent, the desired target compound was subjected to silica gel rapid chromatography (solvent system:
cyclohexane:ethyl acetate=10:1),
Isolate and purify. Get 620mg. TLC, Rf = 0.35 (cyclohexane: ethyl acetate = 5:1) NMR (CDCl ₃ ) δ: 0.15 (9H, S), 2.31 (3H,
S), 3.70 (3H, S), 4.32 (1H, d, J = 2.5Hz),
4.91 (1H, d, J=2.5Hz), 6.7~7.5 (4H, A ₂ B ₂
Type) IR (Liq.) cm ^-1 : 1760, 1718, 2160.

Claims (1)

[Claims] 1. General formula [In the formula, R ¹ is an alkyl group that may have a substituent,
R 2 represents an alkenyl group, an alkynyl group, an aryl group, an alkylthio group, an alkylsulfonyl group, an arylthio group, an arylsulfonyl group, or an acyloxy group, and R ² represents a hydrogen atom or a protecting group for a nitrogen atom. ] of the genus Pichia,
The general formula is reduced by microorganisms belonging to the genus Satucharomyces, Trigonopsis, Crotuchela, Schizosatucharomyces, Candeida, Trichosporon, Streptomyces, or Aspergillus. [In the formula, R ¹ and R ² have the same meanings as described above. A method for producing an optically active β-lactam compound, characterized by leading to a compound having the following.