JP2813342B2 - Method for producing optically active 3-hydroxypyrrolidine derivative - Google Patents

Description

Description: BACKGROUND OF THE INVENTION [0001] The present invention relates to a compound of the general formula [I]: (Wherein X is hydrogen or carbon number 1-17)
Represents a substituted or unsubstituted alkyl group or alkenyl group. )
The mixture of (R) and (S) -N-benzyl-3-acyloxypyrrolidine represented by the formula is stereoselectively hydrolyzed to give a compound of the general formula [II]: [0005] It has a stereoselective esterase activity to generate optically active (S) -N-benzyl-3-hydroxypyrrolidine represented by Esterase enzyme represented by the general formula [I] (R)
And (S) -N-benzyl-3-acyloxypyrrolidine to act on a mixture of optically active (S) -N-benzyl-3-hydroxypyrrolidine [II] and a general formula [S] I ′] embedded image (Wherein X is the same as above), and optically resolving it into (R) -N-benzyl-3-acyloxypyrrolidine represented by the following formula. The present invention relates to a method for producing optically active (S) -N-benzyl-3-hydroxypyrrolidine and its enantiomer, optically active (R) -N-benzyl-3-acyloxypyrrolidine. The optically active (S) -N-benzyl-3-hydroxypyrrolidine and (R) -N-benzyl-3-acyloxypyrrolidine produced by the present invention are useful synthetic intermediates for pharmaceuticals, agricultural chemicals and the like. is there. With respect to a method for producing optically active (S) -N-benzyl-3-hydroxypyrrolidine, there is known a method of synthesizing L-malic acid as a raw material (JP-A-61-63652). However, because of partial racemization, it is necessary to further perform optical resolution with D-mandelic acid to improve the optical purity. A method of reducing N-benzyl-3-pyrroline using (+)-diisopiinocamphylborane as an asymmetric reducing agent is also known [Journal of American Chemical Society (J.
Am. Chem. Soc.), 108 , 2049, 1986].
In addition, (S) -3-hydroxypyrrolidine having no benzyl group at the N-position is derived from L-glutamic acid (Synthetic Communication, 16 , 181).
5, 1986), and the method of obtaining the opposite (R) form by decarboxylation of trans-4-hydroxy-L-proline (JP-A-60-23328) is known.
These can also be synthesized by benzylation. However, any of these methods is difficult to consider as an economical industrial production method because the steps are long and a special reagent is required. DISCLOSURE OF THE INVENTION The present invention relates to an optically active (S) -N-benzyl-3-hydroxypyrrolidine represented by the formula [II] useful as a raw material for synthesis of pharmaceuticals and the like; I '] is a mixture of (R) and (S) -N-benzyl-3-acyloxypyrrolidine which can easily synthesize optically active (R) -N-benzyl-3-acyloxypyrrolidine. It is intended to produce economically by optical resolution by stereoselectively hydrolyzing with a microorganism or an enzyme. Means for Solving the Problems First, the present inventors have
Racemic (R) is an equal mixture of (R) and (S)
S) -N-Benzyl-3-acyloxypyrrolidine [I]
Is a novel compound which has not been reported before, but it can be easily synthesized from inexpensive DL-malic acid or the like, with (RS) -N-benzyl-3-hydroxypyrrolidine and a fatty acid halide. They found that they can be easily synthesized by reacting, and applied for a patent. Next, the present inventors
This novel compound (R) and (S) -N-benzyl-3-acyloxypyrrolidine mixture [I] was subjected to asymmetric hydrolysis using a microorganism or an enzyme to conduct optical resolution and study. As a result, (S) of compound [I]
Optically active compound [II]
After the reaction, a microorganism and an enzyme capable of producing the compound were found, and after the reaction, the optically active (S) -N-benzyl-3-hydroxypyrrolidine [II] and the optically active (S) were chiralized by a simple separation operation. It has been found that R) -N-benzyl-3-acyloxypyrrolidine [I '] can be collected. Further, optically active (R) -N-benzyl-3-acyloxypyrrolidine [I '] can be further used for microorganisms and enzymes which act on alkali hydrolysis or act stereospecifically on the (R) form, lipases having no stereoselectivity, and the like. Can be converted into an optically active (R) -N-benzyl-3-hydroxypyrrolidine retaining the steric configuration by using a microorganism or enzyme having any stereospecificity. ) And (S) -form N-benzyl-3-hydroxypyrrolidine enantiomers can be obtained at the same time. By the present invention, both enantiomers of optically active N-benzyl-3-hydroxypyrrolidine and optically active N-benzyl-3-acyloxypyrrolidine can be produced more economically than the conventional method. Became possible. DETAILED DESCRIPTION OF THE INVENTION The general formula [I] used as a substrate of the present invention: The (R) and (S) -N-benzyl-3-acyloxypyrrolidines represented by the formula (I) can be either a racemic form, which is an equal mixture of the (R) form and the (S) form, or the (R) form. S) Mixtures with a higher content of any of the forms may also be used. As the substituent X, hydrogen or a group having 1 carbon atom
Unsubstituted ones with up to 17 linear or branched alkyl groups or alkenyl groups, or those substituted with halogen or the like can also be used. For example, a methyl group, an ethyl group,
Ethylene group, n-propyl group, isopropyl group, 1-propylene group, n-butyl group, isobutyl group, 1-butylene group, n-pentyl group, n-hexyl group, n-heptyl group, n-octyl group, n -Nonacyl group, n-decyl group,
linear or branched unsubstituted alkyl such as n-undecyl group, n-dodecyl group, n-tridecyl group, n-tetradecyl group, n-pentadecyl group, n-hexadecyl group, n-heptadecyl group and n-octadecyl group Group, alkenyl group and the like, for example, chloromethyl group, dichloromethyl group,
Examples thereof include a halogen-substituted substituted alkyl group such as a trifluoromethyl group, a 2-chloroethyl group, a 3-chloropropyl group, and a 4-chlorobutyl group. Compound [I] is hydrolyzed stereoselectively,
Microorganisms or enzymes having stereoselective esterase activity that produces optical activity [II] can be easily found by performing the following screening. For example, as a specific example, 10 ml of a nutrient medium capable of growing microorganisms is placed in a large test tube, and the inoculated bacteria are inoculated.
Incubate with shaking at 5-35 ° C for 1-3 days. Next, 2% (w / v) of (RS) -N-benzyl-3-butyryloxypyrrolidine was added to this culture solution, and further added at 20 to 35 ° C.
The reaction is performed for 1 to 3 days while adjusting the pH to 5 to 7 with NaOH. The progress of the hydrolysis is determined by adjusting the pH of the reaction solution with sulfuric acid.
2.0, butyric acid produced was extracted with twice the amount of ethyl acetate, and extracted with gas chromatography (filler, Shimadzu FA
LM6% / Simalite, φ0.3 × 100 cm column, temperature 140 ° C.). The time-dependent change of each reaction solution is taken, and a strain whose reaction is extremely slowed when about 1/2 equivalent of butyric acid is generated with respect to the added substrate is primarily selected. Next, set the reaction scale to 500
ml and perform the same culture and hydrolysis as above,
After completion, the mixture is extracted four times with 500 ml of dichloromethane, and the solvent is removed under reduced pressure. This was loaded on a silica gel column (Wakogel C-200, 250 g), and unreacted N-benzyl-3-butyryloxypyrrolidine was eluted with hexane / ethyl acetate (10/2). Further, the N-benzyl-3-hydroxypyrrolidine generated with ethyl acetate is eluted. By concentrating each fraction, an oily optically active N-benzyl-
3-butyryloxypyrrolidine and optically active N-benzyl-3-hydroxypyrrolidine are obtained. When optically active N-benzyl-3-butyryloxypyrrolidine is hydrolyzed with NaOH, it becomes optically active N-benzyl-3-hydroxypyrrolidine without impairing the optical activity, and these are purified by distillation. And
By measuring the specific rotation, it can be easily determined whether or not the microorganism has stereoselective esterase activity. In screening for enzymes of various origins, 0.5 g of the enzyme can be dissolved in 10 ml of 0.1 M phosphate buffer instead of the microorganism culture solution, and the screening can be performed in the same manner. Thus, microorganisms or enzymes capable of stereoselectively hydrolyzing N-benzyl-3-butyryloxypyrrolidine include various N-types as described above.
-Benzyl-3-acyloxypyrrolidine shows the same stereoselective hydrolysis activity. (R) and (S) -N-benzyl-3-
Microorganisms having a stereoselective esterase activity which stereoselectively hydrolyzes an acyloxypyrrolidine mixture to give [II] and [I '] include microorganisms belonging to the genus Rhizops, phycomyces and the like.・ Japonicas (Rhizopus japonicus) IFO 4
758, Rhizopus delemer I
FO 4697, Phycomyc nitens
es nitens) IFO 5694, and the like. These microorganisms can be used in the form of a culture solution itself, but can also be used by extracting and purifying enzymes from the culture solution or the cells. A commercially available enzyme can also be used directly. For example, lipase PN (derived from the genus Phycomyces, manufactured by Wako Pure Chemical Industries, Ltd.), lipase (derived from the genus Rhizops, manufactured by Seikagaku Corporation), lipase “Saiken” 10
0 (derived from Rhizopus, manufactured by Osaka Bacterial Research Laboratories, Inc.), Neurase (derived from Rhizopus, manufactured by Amano Pharmaceutical Co., Ltd.) and the like are suitable for this purpose. In the hydrolysis reaction, the substrate [I] is added to a microorganism culture solution or an enzyme extract or an aqueous solution of the enzyme in an amount of 0.5 to 75%.
% (W / v) concentration, and the mixture is stirred at a temperature of 10 to 55 ° C. Although the amount of the enzyme depends on the enzyme activity, the amount is usually in the range of equivalent to 1/1000 of [I]. The pH of the reaction is in the range of 5.0 to 8.5, but when the pH changes during the hydrolysis reaction, it is desirable to maintain the pH at an optimum value with an appropriate acid or alkali aqueous solution. In the reaction, microorganisms or enzymes can be immobilized on a water-insoluble carrier or the like and used repeatedly.
Immobilization of microorganisms, for example, acrylamide polymer, urethane polymer, ethylene glycol derivative polymer,
Methods using carrageenan, calcium alginate and the like can be mentioned. Amberlite XAD is used for enzyme immobilization.
-7, Amberlite XAD-2, Diaion HP2
0, a method using a synthetic adsorbent such as Diaion HP2MG, Octyl Sepharose CL-4B, and the like. The solubility of the substrate [I] in water is generally low,
Sufficient stirring will not hinder the reaction. However, a hydrophilic solvent such as acetone or ethanol, a surfactant, or the like may be added to the extent that the reaction is not hindered for the purpose of promptly proceeding the reaction. The reaction can be traced by analyzing the organic acid generated by the hydrolysis by gas chromatography and terminating the reaction when 0.5 equivalent of the added [II] organic acid is generated. As a method for separating the hydrolyzate [II] and the remaining [I '], a hydrophobic organic solvent such as hexane, cyclohexane, petroleum ether, methylene chloride, chloroform, carbon tetrachloride or the like can be used. 0.0 to 8.5
[I '] containing only [I'] or slightly [II] was extracted and separated from the reaction solution adjusted to the range described above, and if necessary, [I '] was purified by silica gel chromatography (separated with a hexane-acetone solvent). And [II] can be separated to obtain pure products. Further, [II] remaining in the reaction solution can be completely recovered by adjusting the pH of the reaction solution to 9 to 14 with NaOH or the like and then extracting with the above-mentioned solvent. It should be noted here that since [I '] undergoes hydrolysis under alkaline conditions, extracting [II] and [I'] under alkaline conditions from the beginning causes a reduction in the optical purity of [II]. This is a bad point. The separation of [I '] and [II] can be carried out by the above-mentioned silica gel chromatography by using other inorganic adsorbents such as florisil, alumina, zeolite or the like, or synthetic adsorbents such as Amberlite XAD-7, Amberlite. XAD-2, Diaion HP20, Diaion H
Separation can also be performed using P2MG, Octyl Sepharose CL-4B, or the like. [I '] and [I
If it is I], it can also be separated by distillation. The optically active [I '] can be used as a raw material for synthesis as it is, but NaOH, KOH, Ca
By hydrolyzing chemically with an alkali such as (OH) ₂ or using an esterase or a microorganism having different stereoselectivity, it is possible to easily lead to [II] while maintaining the optical activity. The optical purity of [II] and [I '] can be measured by the following method. [I '] is hydrolyzed by the above-mentioned method to form [II], and [II] is converted into methylene chloride in
After tosylation by reaction with an equimolar amount of p-toluenesulfonyl chloride, this was subjected to high performance liquid chromatography (H
PLC) (column: chiral cell OB (manufactured by JASCO)
0.46 × 25 cm, eluent hexane: isopropanol (20: 1), flow rate 1.5 ml / min, detection 22
1 nm, retention time (R): 36.7 minutes, (S) 5
(1.3 minutes). EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to only these Examples. Examples 1-3 Glucose 2%, yeast extract 0.5%, meat extract 0.3%, peptone 0.3%, olive oil 1% (pH
7.0) of nutrient liquid medium having a composition of
After placing in a liter mini jar and sterilizing at 120 ° C. for 20 minutes, the microorganisms shown in Table 1 were inoculated, and cultured at 30 ° C. for 40 hours with aeration at 1 vvm and stirring at 500 rpm. Thereafter, the pH was adjusted to 7.0, 5 g of (RS) -N-benzyl-3-hexanoyloxypyrrolidine was added, and the mixture was reacted at 30 ° C for 48 hours. After the reaction, the cells were removed by centrifugation to remove the cells, and the supernatant was extracted three times with an equal volume of hexane to recover unreacted N-benzyl-3-hexanoyloxypyrrolidine. Next, the extraction residue was adjusted to pH 13 with a NaOH solution and extracted three times with an equal volume of methylene chloride to obtain hydrolyzed N-benzyl-3-hydroxypyrrolidine. After removing the solvent under reduced pressure, colorless and transparent N-benzyl-3 was obtained by distillation.
-Hydroxypyrrolidine, which after tosylation,
The optical purity measured by LC was as shown in Table 1. [Table 1] Examples 4 to 7 10 g of (RS) -N-benzyl-3-hexanoyloxypyrrolidine was added to 100 ml of a 0.1 M phosphate buffer (pH 7.0). The reaction was carried out at 40 ° C. under stirring for 22 to 120 hours. The reaction solution was extracted three times with an equal volume of hexane to recover unreacted N-benzyl-3-hexanoyloxypyrrolidine.
Thereafter, the pH was adjusted to 13 with a NaOH solution, and the mixture was extracted three times with an equal volume of methylene chloride to obtain hydrolyzed N-benzyl-3-.
Hydroxypyrrolidine was extracted and separated. The solvent was removed under reduced pressure, and the residue was purified by distillation to obtain N-benzyl-3-hydroxypyrrolidine as a colorless transparent oil. After this was tosylated, the optical purity was measured by HPLC.
Shown in [Table 2] According to the present invention, optically active (S) -N
-Benzyl-3-hydroxypyrrolidine and optically active (R) -N-benzyl-3-acyloxypyrrolidine can be obtained economically.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C12P 41/00 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] General formula [I] [In the formula, X represents hydrogen or a substituted or unsubstituted alkyl group or alkenyl group having 1 to 17 carbon atoms. ], A mixture of (R) and (S) -N-benzyl-3-acyloxypyrrolidine represented by the general formula [II]: Has the stereoselective esterase activity for producing optically active (S) -N-benzyl-3-hydroxypyrrolidine represented by the formula: and has a stereoselective esterase activity derived from a microorganism belonging to the genus Rhizopus or Phycomyces or from the microorganism. The enzyme is allowed to act on a mixture of (R) and (S) -N-benzyl-3-acyloxypyrrolidine represented by the general formula [I] to give a compound of the formula [II] And an optically active (S) -N-benzyl-3-hydroxypyrrolidine represented by the general formula [I '] (Wherein X is the same as defined above).
Optically active (S) -N-benzyl-3-hydroxypyrrolidine and / or a pair thereof, wherein the optically active compound is optically resolved into -N-benzyl-3-acyloxypyrrolidine, and each optically active compound is separated and collected. Optical activity of palm body (R)
A method for producing -N-benzyl-3-acyloxypyrrolidine. 2. The method according to claim 1, wherein the enzyme is lipase. 3. 3. The method according to claim 1, wherein a microorganism or enzyme having stereoselective esterase activity is immobilized on a water-insoluble carrier. 4. (R) and (S) -N- represented by the general formula [I]
Optically active (S) -N-benzyl-3-hydroxypyrrolidine [II] and optically active (R)-obtained by stereoselectively hydrolyzing a mixture of benzyl-3-acyloxypyrrolidine with a microorganism or an enzyme. N-benzyl-3-
When separating and collecting [II] and [I '] from a mixture with acyloxypyrrolidine [I'], separate and collect each by column chromatography using an inorganic adsorbent or a synthetic adsorbent as a carrier. The method according to any one of claims 1 to 3, wherein: 5. (R) and (S) -N- represented by the general formula [I]
Optically active (S) -N-benzyl-3-hydroxypyrrolidine [II] and optically active (R)-obtained by stereoselectively hydrolyzing a mixture of benzyl-3-acyloxypyrrolidine with a microorganism or an enzyme. N-benzyl-3-
When [II] and [I '] are separated and collected from the mixture with acyloxypyrrolidine [I'], [I '] is extracted using a hydrophobic solvent and separated from [II]. The method according to any one of claims 1 to 3, wherein the sample is collected.