CN112708641A - Chemical-enzymatic synthesis method of tomoxetine - Google Patents

Chemical-enzymatic synthesis method of tomoxetine Download PDF

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CN112708641A
CN112708641A CN201911019678.3A CN201911019678A CN112708641A CN 112708641 A CN112708641 A CN 112708641A CN 201911019678 A CN201911019678 A CN 201911019678A CN 112708641 A CN112708641 A CN 112708641A
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张福利
陈少欣
倪国伟
邹杰
汤佳伟
江锣斌
陈一波
王宏毅
晏海军
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Shanghai Institute of Pharmaceutical Industry
China State Institute of Pharmaceutical Industry
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Abstract

The invention provides a chemical-enzymatic synthesis method of tomoxetine, which comprises the steps of taking 3-chloropropiophenone as a raw material, carrying out asymmetric reduction reaction under the catalysis of carbonyl reductase and coenzyme to prepare (S) -3-chloropropiophenol, wherein the ee value of chiral purity is 99.9%, carrying out a mitsunobcasting reaction and a methylaminolysis reaction, and then carrying out salification by hydrochloric acid to prepare the tomoxetine. The preparation method can obviously reduce the production cost, is green and environment-friendly, and has potential industrial application value.

Description

Chemical-enzymatic synthesis method of tomoxetine
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a chemical-enzymatic synthesis method of tomoxetine.
Background
The trade name of the tomoxetine hydrochloride is Sessida (Strettera), the Chinese cultural name is (R) -N-Methyl-3- (2-methylphenoxy) amphetamine hydrochloride, the English name is (R) -N-Methyl-3- (2-methylphenoxy) benzanepropanamine hydrochloride, and the molecular formula is C17H22ClNO, relative molecular mass 291.82, CAS registry number 82248-59-7, in the form of a capsule (10 mg; 18 mg; 25 mg; 40mg) indicated for attention deficit hyperactivity disorder.
Figure BDA0002246790490000011
Tomoxetine hydrochloride was developed by the american gift pharmaceutical company (Eli Lily) and approved for sale in the us in 2003. Tomoxetine is the first FDA-approved non-central excitatory ADHD therapeutic drug and is a highly Selective Norepinephrine Reuptake Inhibitor (SNRI). A representative synthetic route for tomoxetine hydrochloride is as follows:
route one
Using 3-chlorophenyl acetone as initial raw material, and processing with NaBH4Reduction, Mitsunobu reaction, methyl amination, mandelic acid resolution and salification by hydrochloric acid to obtain the atomoxetine hydrochloride.
Figure BDA0002246790490000012
Route two
Kumar et al uses 3-phenyl-3-ketone-ethyl propionate as initial raw material, and makes it undergo the processes of chiral reduction of carbonyl group by Baker's yeast, reaction with methylamine and LiAlH reaction4Reduction, Boc protection, Mitsunobu reaction with o-cresol, and acidification with hydrochloric acid to obtain the product tomoxetine hydrochloride.
However, the biocatalysis stereoselectivity in the step is not high, the ee value of the final product is only 70%, and the reaction route has Boc protection and deprotection processes, so that the reaction step is long.
Figure BDA0002246790490000021
Route three
Vogl et al, using 3-chlorophenyl acetone as starting material, selectively reducing carbonyl by carbonyl reductase Candida tenuis xylose reductase, Mitsunobu reaction, and methylamine reaction to obtain tomoxetine. In the route, (S) -3-chlorophenylpropanol is prepared by catalytic reduction of carbonyl reductase CtXR, but the substrate concentration is only 2mM, the chiral purity is 98%, the space-time yield of the product is low, and the method is not suitable for industrial production.
Figure BDA0002246790490000022
The existing industrial preparation method for preparing tomoxetine mainly adopts a traditional resolution method, the method is limited by the theoretical yield of only 50%, and the existing biological preparation method reports that the catalytic activity of carbonyl reductase is low, the substrate concentration is low, the chiral purity of the product is only 98%, and the requirement of industrial production is difficult to meet.
Therefore, there is a strong need in the art for a chemo-enzymatic method for the industrial efficient preparation of tomoxetine.
Disclosure of Invention
Aiming at the problems of the existing preparation method, the invention aims to provide a chemo-enzymatic method for preparing the tomoxetine, which shows a remarkable industrial application prospect.
In a first aspect of the present invention, there is provided a method for preparing (S) -3-chlorophenylpropanol, comprising the steps of:
(a) in a liquid reaction system, 3-chlorophenyl propanone (compound of formula 2) is used as a substrate, and asymmetric reduction reaction is carried out in the presence of coenzyme under the catalysis of carbonyl reductase, so as to form (S) -3-chlorophenyl propanol [ compound of formula (S) -3 ];
Figure BDA0002246790490000031
and
(b) optionally separating (S) -3 from the reaction system after the reaction of the previous step;
wherein the carbonyl reductase is selected from the group consisting of:
(i) the amino acid sequence is shown as SEQ ID NO. 2;
(ii) the amino acid sequence shown in SEQ ID NO.2 is obtained by carrying out substitution, deletion, change, insertion or addition of one or more amino acids within the range of keeping the enzyme activity.
In another preferred embodiment, in the reaction system, the concentration of the compound of formula 2 is 50-1000 g/L.
In another preferred embodiment, in the reaction system, the concentration of the compound of formula 2 is 60-700 g/L.
In another preferred embodiment, in the reaction system, the concentration of the compound of formula 2 is 80-600 g/L.
In another preferred embodiment, in the reaction system, the concentration of the compound of formula 2 is 20-500 g/L.
In another preferred embodiment, in the reaction system, the concentration of the compound of formula 2 is 50-200 g/L.
In another preferred embodiment, in the reaction system, the concentration of the compound of formula 2 is 50-150 g/L.
In another preferred embodiment, in the reaction system, the concentration of the compound of formula 2 is 100-150 g/L.
In another preferred embodiment, the coenzyme is selected from the group consisting of: a reducing coenzyme, an oxidizing coenzyme, or a combination thereof.
In another preferred embodiment, the reducing coenzyme is selected from the group consisting of: NADH, NADPH, or a combination thereof.
In another preferred embodiment, the oxidative coenzyme is selected from the group consisting of: NAD, NADP, or a combination thereof.
In another preferred embodiment, the ratio of the amount of NAD to the amount of substrate is 0.01% to 1.0% (w/w), preferably 0.01% to 0.5% (w/w).
In another preferred embodiment, the ratio of the amount of NADP to the amount of substrate is 0.01-1.0% (w/w), preferably 0.01-0.5% (w/w).
In another preferred embodiment, an enzyme for coenzyme regeneration is also present in the reaction system.
In another preferred embodiment, the enzyme for coenzyme regeneration is selected from the group consisting of: alcohol dehydrogenase (such as isopropanol dehydrogenase), formate dehydrogenase, glucose dehydrogenase, or a combination thereof.
In another preferred example, in the reaction system, an enzyme for coenzyme regeneration is further present, and the enzyme for coenzyme regeneration is isopropanol dehydrogenase, formate dehydrogenase, or glucose dehydrogenase.
In another preferred embodiment, in step (a), the temperature is from 10 ℃ to 50 ℃, preferably from 20 ℃ to 40 ℃, more preferably from 25 ℃ to 35 ℃.
In another preferred embodiment, in step (a), the time is 0.1 to 240 hours, preferably 0.5 to 120 hours, more preferably 1 to 72 hours, still more preferably 20 to 24 hours, still more preferably 3 to 10 hours.
In another preferred embodiment, in step (a), the pH is 6 to 9, preferably 6.5 to 8.5, more preferably 7.0 to 7.5.
In another preferred embodiment, in the reaction system, the carbonyl reductase is an enzyme in a free form, an immobilized enzyme, or an enzyme in the form of bacterial cells.
In another preferred embodiment, the reaction system is a phosphate buffer system.
In another preferred embodiment, the reaction system further comprises a cosolvent.
In another preferred embodiment, the cosolvent is selected from the following group: dimethyl sulfoxide, methanol, ethanol, isopropanol, acetonitrile, toluene, acetone, or a combination thereof.
In another preferred embodiment, the concentration of the cosolvent is 5-30%.
In another preferred embodiment, in step (b), the separating comprises: adding isopropanol, centrifuging, partially concentrating, extracting with methyl tert-ether, and concentrating the organic layer.
In another preferred embodiment, in step (b), the separating comprises: heating to 50-80 deg.C to inactivate protein, adding extraction solvent such as methyl tert-ether, dichloromethane, toluene, ethyl acetate, etc., centrifuging to obtain organic layer, drying, filtering, and concentrating the organic layer to obtain the product.
In another preferred embodiment, in step (b), the ee value of the compound of formula (S) -3 in the reaction system after the reaction is 90% or more, preferably 95% or more, and more preferably 99% or more.
In another preferred embodiment, in step (b), 80% or more (preferably 85% or more, more preferably 90% or more) of the compound of formula 2 is converted into the compound of formula (S) -3 in the reaction system after the reaction.
In another preferable example, in the step (b), the concentration of the compound of formula (S) -3 in the reaction system after the reaction is 50-1000 g/L.
In another preferred embodiment, the gene sequence encoding the carbonyl reductase is selected from the group consisting of:
(a) the sequence shown as SEQ ID NO.1 (accession number CP000677.1 at NCBI);
(b) a polynucleotide complementary to the sequence defined in (a); or
(c) Any polynucleotide or complementary sequence having at least 70% (preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99%) or more sequence identity to the sequence defined in (a).
In another preferred embodiment, the carbonyl reductase gene is constructed on an expression vector.
In another preferred embodiment, a co-substrate is also present in the reaction system.
In another preferred embodiment, the co-substrate is selected from the group consisting of: isopropanol, glucose, ammonium formate, or a combination thereof, preferably glucose.
In another preferred embodiment, in the reaction system, a co-substrate is further present, and the co-substrate is isopropanol, ammonium formate or glucose.
In another preferred embodiment, the concentration of the cosubstrate in the reaction system is 5-30%.
In another preferred embodiment, the amount of the co-substrate used in the reaction system is 2 to 5 equivalents of the amount of compound 2.
In a second aspect of the present invention, there is provided a process for the preparation of tomoxetine, wherein a compound of formula (S) -3 prepared by the process of the first aspect of the present invention is prepared by the following reaction steps:
Figure BDA0002246790490000051
in another preferred example, the method comprises the following steps:
(1) carrying out a light extension reaction on (S) -3-chlorophenylpropanol prepared by the method of the first aspect of the invention and o-methylphenol to prepare a compound 4;
(2) activating the compound 4 obtained in the step (1) by sodium iodide, and salifying methylamine substituted oxalic acid to prepare the oxalate of the tomoxetine;
(3) and (3) dissociating the oxalate obtained in the step (2) by using a saturated sodium bicarbonate solution, and salifying the oxalate with hydrogen chloride to obtain the atomoxetine hydrochloride.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the chiral pattern of 3-chlorophenylpropanol racemate.
FIG. 2 shows a chiral spectrum of (S) -3-chlorophenylpropanol.
Detailed Description
The present inventors have extensively and intensively studied and found that the present invention improves the key carbonyl reduction step in the existing tomoxetine production process to the production of (S) -3-chlorophenylpropanol (compound of formula (S) -3) by the catalytic reduction of 3-chlorophenylacetone (compound of formula 2) by carbonyl reductase, i.e., the stereoselective reduction of the compound of formula 2 to the compound of formula (S) -3 in the presence of carbonyl reductase and a coenzyme (even under the condition of a high concentration of a substrate, e.g., 50 to 1000g/L, the compound of formula (S) -3 can be efficiently produced, and then the subsequent reaction is further carried out using the compound of formula (S) -3 as a substrate for the production of pharmaceutical products such as tomoxetine.
Term(s) for
Carbonyl reductases
In the present invention, the "carbonyl reductase" is an enzyme capable of stereoselectively catalyzing asymmetric reduction of a prochiral ketone to give a chiral alcohol.
In the present invention, the carbonyl reductase may be wild-type or mutant. Furthermore, they may be isolated or recombinant.
The amino acid sequence of a typical carbonyl reductase is shown as SEQ ID No.2, and the coding gene is shown as SEQ ID No. 1.
Due to the degeneracy of codons, the base sequence encoding the amino acid sequence shown in SEQ ID NO.2 is not limited to only SEQ ID NO. 1. The homologues of this base sequence may be obtained by those skilled in the art by appropriately introducing substitutions, deletions, alterations, insertions or additions, and the present invention encompasses these homologues as long as the recombinant enzyme expressed therefrom retains catalytic reduction activity for the substrate compound 2. The homologue of the polynucleotide of the present invention can be produced by substituting, deleting or adding one or more bases of the base sequence SEQ ID NO.1 within a range in which the activity of the enzyme is maintained.
The carbonyl reductase of the invention also comprises an amino acid sequence obtained by replacing, deleting, changing, inserting or adding one or more amino acids of the amino acid sequence shown in SEQ ID NO.2 in the range of keeping the activity of the enzyme.
According to the general knowledge in the art, recombinant enzyme resting cells, wet cells, crude enzyme solutions, pure enzymes, crude enzyme powders, and the like constructed using the carbonyl reductase can be used in the reaction system. In order to obtain a higher conversion efficiency, it is preferable to use a crude enzyme solution. The ratio of the amount of the carbonyl reductase to the amount of the substrate is preferably 1% to 6% (w/w), or the ratio of the mass of resting cells to the mass of the substrate is 10% to 100%.
Coenzyme
In the present invention, "coenzyme" means a coenzyme capable of effecting electron transfer in a redox reaction.
Typically, the coenzyme of the invention is a reducing coenzyme NADH, NADPH or an oxidizing coenzyme NAD+、NADP+. Since the reducing coenzyme is expensive, the oxidizing coenzyme NAD is preferably selected+、NADP+。
When the oxidative coenzyme is selected, a method for realizing coenzyme regeneration needs to be selected, and the method mainly comprises three types of (1) glucose dehydrogenase and cosubstrate glucose; (2) alcohol dehydrogenase and co-substrate isopropanol; (3) formate dehydrogenase co-substrate ammonium formate.
In a preferred embodiment, the coenzyme is NADP+The coenzyme regenerating system is glucose dehydrogenase, and the glucose dehydrogenase and the cosubstrate glucose are preferred in the invention.
In another preferred embodiment of the invention, the oxidative coenzyme NAD+The concentration of (A) is 0.05-0.1g/L, and the buffer system is 0.1mol/L phosphate buffer salt.
Typically, the pH of the buffer is 7.0-7.3.
Typically, the coenzyme of the invention can be regenerated using glucose dehydrogenase with glucose.
In another preferred embodiment, the carbonyl reductase of the present invention can also achieve coenzyme regeneration by autocatalytic conversion of isopropanol to acetone by the addition of isopropanol.
Cosolvent
In the present invention, a co-solvent may be added or not added to the reaction system.
As used herein, the term "co-solvent" refers to a sparingly soluble substance that forms a soluble intermolecular complex, association, double salt, or the like with an added third substance in a solvent to increase the solubility of the sparingly soluble substance in the solvent. This third material is referred to as a co-solvent.
In the present invention, the substrate compound 2 is hardly soluble in water, and when the substrate concentration is increased, the reaction conversion rate is seriously affected. Therefore, it is necessary to improve the substrate solubility by adding a cosolvent to improve the reaction conversion. Optional cosolvent is dimethyl sulfoxide, methanol, ethanol, isopropanol, acetonitrile, toluene, acetone, concentration is preferably 5-30% (v/v), and dimethyl sulfoxide, methanol, ethanol, isopropanol are preferably selected.
Biological preparation method
The invention provides a preparation method of a compound (S) -3, which comprises the following steps:
(a) in a liquid reaction system, taking a compound shown in a formula 2 as a substrate, and carrying out asymmetric reduction reaction in the presence of coenzyme under the catalysis of carbonyl enzyme to form (S) -3-chlorophenylpropanol;
Figure BDA0002246790490000081
and
(b) optionally separating (S) -3-chlorophenylpropanol from the reaction system after the reaction in the previous step.
In the present invention, the above reaction may be coupled with or without a coenzyme regeneration system.
Preferably, the above reaction is to add (couple) coenzyme regeneration method (system) in the same system, thereby further improving production efficiency, reducing production cost and improving substrate tolerance.
Furthermore, the preferred features of the various classes or combinations thereof described in the first aspect of the invention are equally applicable to the reaction system described herein.
Application of reaction system
The present invention also provides the use of a reaction system according to the second aspect of the present invention for the preparation of a compound of formula (S) -3, comprising the steps of: using said reaction system, carrying out an enzymatic reaction under conditions suitable for enzymatic catalysis, thereby producing a compound of formula (S) -3:
Figure BDA0002246790490000082
the main advantages of the invention are:
(1) the carbonyl reductase of the invention is used for biocatalytically reducing the compound 2 to prepare (S) -3, only extraction operation is needed, the operation is simple, the cost is low, and the cost of the enzyme required for producing 1 kg of product is 30 yuan.
(2) The preparation method is green and environment-friendly, and is more suitable for industrial production of the tomoxetine key intermediate (S) -3 with high chemical purity and high optical purity.
(3) The concentration of the substrate is 50-150g/L, the conversion rate is more than 98%, and the ee value is more than 99.5%.
(4) The carbonyl reductase has high stereoselectivity, the ee value is more than 99.5%, the carbonyl reductase has good tolerance to organic solvents and substrates, the substrate concentration is obviously improved, when a reaction system is amplified to a hectogram level, the substrate concentration can reach 100g/L to the maximum, the substrate concentration is the highest in the existing biocatalysis method, and compared with the existing literature, the carbonyl reductase is the method for catalyzing 3-chloropropiophenone to prepare (S) -3 with the highest efficiency.
The following specific examples further illustrate the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for specific conditions not noted in the following examples are generally performed according to conventional conditions, for example, the conditions described in (Sambrook and Russell et al, Molecular Cloning: A Laboratory Manual (third edition) (2001CSHL Press), or according to the conditions suggested by the manufacturer.
Material
The racemate of compound 2 was purchased from Shanghai Michelin chemical reagents, Inc., and the total gene synthesis was performed by Shanghai Bailey, Inc. Carbonyl reductases 1500 and 1306 were obtained from Yihui BioLimited, Huzhou, carbonyl reductases K142, K157, K168, K195 and K201 were obtained from Shanghai Fukuang BioLimited.
The carbonyl reductase is obtained by synthesizing commercial whole gene, then constructing the coding gene into an expression vector, introducing the expression vector into host bacteria, and inducing expression.
Method
Chiral HPLC conditions: CHIRALPAK OD-3 column (4.6X 250mm,5 μm);
mobile phase: n-hexane/isopropanol 98/2;
flow rate: 1.0 mL/min;
ultraviolet detection wavelength: 224 nm;
column temperature: at 30 ℃.
Process for producing enzyme
The gene of the carbonyl reductase is constructed on the same plasmid pET28a (+) vector by the conventional technology in the field, then is introduced into an expression host escherichia coli, and is fermented and induced to express, so as to obtain the thallus containing the target enzyme. The bacteria can be obtained directly by centrifugation, or the enzyme liquid can be obtained by breaking the walls of the bacteria, and the enzyme powder is used for the subsequent biotransformation reaction.
EXAMPLE 1 preparation of (S) -3-Chlorobenzopropanol [ (S) -3]
Figure BDA0002246790490000101
Phosphate buffer (28mL, 100mM/L, pH 8.0), dimethyl sulfoxide (12mL), glucose (4.2g), ketone 2(4.0g,0.012mol), NAD+(0.02g) and wet cells (8.0g) of the carbonyl reductase of the present invention and glucose dehydrogenase (4.0g) were sequentially placed in a 250mL reaction flask and reacted for 24 hours in a 45 ℃ oil bath with mechanical stirring at 200 rpm. Sampling, and monitoring the reaction process by HPLC, wherein the raw materials are completely reacted. The reaction solution was extracted three times with dichloromethane (200mL × 3), the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give (S) -3, 3.6g as a pale yellow solid, crude yield 87.8%, HPLC purity 84.56%, ee value:>99.9％。
1HNMR(600MHz,CDCl3):δ＝7.37(d,J＝4.2Hz,4H),7.33-7.29(m,1H), 4.96-4.94(m,1H),3.77-3.73(m,1H),3.59-3.55(m,1H),2.27-2.22(m,1H), 2.12-2.07(m,1H),1.99(s,1H)；13CNMR(150MHz,CDCl3):δ＝143.69,128.67, 127.94,125.77,71.34,41.72,41.43。
FIG. 1 shows a chiral pattern of a 3-chlorophenylpropanol racemate, and FIG. 2 shows a chiral pattern of (S) -3-chlorophenylpropanol. As can be seen from a comparison of FIGS. 1 and 2, the enzymatic conversion product of the present invention has a high chiral purity with an ee value of > 99.9%.
TABLE 1 screening results of enzymes
Figure BDA0002246790490000102
Figure BDA0002246790490000111
Screening conditions are as follows: 10mL of reaction system:
(a)5 g/L2 and 2g/L GDH, 20g/L cells, 0.5g/L NAD+1-fold equivalent of glucose and phosphate buffer (100mM, pH 7.0) was reacted for 2 hours at 25 ℃ in a shaker at 220 rpm;
(b)5 g/L2 and 2g/L GDH, 20g/L thallus, 0.5g/L NADP+1-fold equivalent of glucose and phosphate buffer (100mM, pH 7.0) was reacted in a shaker at 25 ℃ and 220rpm 2h；
(c)5 g/L2, 20g/L thallus, 0.5g/L NADP+20% isopropanol and phosphate buffer (100mM, pH 7.0) were reacted for 2h at 25 ℃ in a shaker at 220 rpm;
as is clear from Table 1, there are significant differences in the reduction of the enzyme-catalyzed substrate Compound 2 of the present invention by different enzymes. For example, in the case of producing (S) -3 by the catalytic reduction of Compound 2 with the enzyme No. 1500 carbonyl reductase, the conversion rate reached 98%, but the stereoselectivity was low and was only 1.28% ee. Compared with carbonyl reductases 1500, 1306K 142, K157, K168, K195 and K201, the carbonyl reductases of the invention can simultaneously achieve significantly better conversions (98.91%) and stereoselectivities (ee > 99.9%).
Comparative example 1:
Figure BDA0002246790490000112
phosphate buffer (10mL, 100mM/L, pH 8.0), dimethyl sulfoxide (2mL), glucose (0.2g), ketone 2(0.5g,0.012mol), NAD + (0.01g), and the carbonyl reductase wet cell of the present invention (1g), and glucose dehydrogenase (0.5g) were sequentially added to a 50mL reaction flask, and reacted at 45 ℃ in an oil bath with mechanical stirring at 200rpm for 24 hours. Sampling, and monitoring the reaction process by HPLC, wherein the raw materials are completely reacted. The reaction solution was extracted three times with dichloromethane (200mL × 3), the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give a pale yellow solid (S) -50.3 g, crude yield 60%, chiral purity, ee value: 90.2 percent.
Compound 4 is structurally very close to compound 2, differing only in that one more methyl substituent is present on the phenyl ring of compound 4. However, the ee value of the product after the reaction of compound 4 is only 90.2%. Thus, the carbonyl reductase of the present invention can convert a specific substrate compound 2 with high selectivity.
EXAMPLE 2 Synthesis of (R) -1-chloro-3-phenyl-3- (2-methylphenoxy) propanol (4)
Figure BDA0002246790490000121
In a 100mL three-necked flask, (S) -3(1.7g, 0.010mol) was dissolved in 20mL of anhydrous tetrahydrofuran, and triphenylphosphine (3.4g, 0.013mol), o-cresol (1.4g, 0.013mol), and N were added in this order2Diethyl azodicarboxylate (DEAD) (2.3g, 0.013mol) was added slowly at 0 ℃ for protection. After dropping, the mixture was stirred at room temperature for 24 hours. After adding 50mL of petroleum ether and stirring for 1 hour, a large amount of white solid was generated, and the solution obtained by filtration was washed with water (50mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain colorless oily liquid 4, 72.4 g, yield 92.31%, HPLC purity: 88.63 percent.
1H NMR(600MHz,CDCl3):δ＝7.44-7.39(m,4H),7.33(t,J＝7.2Hz,1H),7.19(d, J＝7.8Hz,1H),7.04(t,J＝7.8Hz,1H),6.86(t,J＝7.5Hz,1H),6.71(d,J＝7.8Hz,1H), 5.48-5.45(m,1H),3.89-3.85(m,1H),3.71-3.67(m,1H),2.59-2.53(m,1H),2.40(s,3H), 2.34-2.28(m,1H)；13C NMR(150MHz,CDCl3) δ 155.20,140.47,130.11,128.18, 127.24,126.39,126.07,125.23,119.94,112.27,75.84,40.96,40.79, 15.96; HR-MS theoretical value (calcd for) C16H17ClNaO[M+Na]+m/z 283.0860, Experimental value (found) m/z 283.0854.
EXAMPLE 3 Synthesis of (R) -N-methyl-3- (2-methylphenoxy) -3-phenyl-1-propylamine (1. oxalate) oxalate
Figure BDA0002246790490000122
In a 100mL three-necked flask, 4(1.0g, 0.004mol) was dissolved in 10mL of acetone, and sodium iodide (0.6g, 0.004mol) was added to conduct a reaction under reflux for 12 hours. Cooled to room temperature, filtered, the filtrate concentrated under reduced pressure and dissolved in 5mL of tetrahydrofuran.
And (3) adding 10mL of 30% methylamine water solution into a 100mL three-necked bottle, dropwise adding the tetrahydrofuran solution of the product obtained in the previous step, and reacting for 8 hours at 40 ℃. After cooling to room temperature, the mixture was poured into 30mL of water and extracted with methylene chloride (30 mL. times.2). The dichloromethane layer was washed with water (30 mL. times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated.
The concentrated product from the previous step was dissolved in 10mL of acetone and oxalic acid (0.6g, 0.007mol) was added with stirring to precipitate a white solid continuously. Stirring for 6h, filtering, washing a filter cake with acetone, and drying to obtain 0.9g of white solid 1. oxalate, wherein the yield is as follows: 67.9%, HPLC purity: 98.86%, ee value: 99.00 percent.
1HNMR(600MHz,DMSO-d6):δ＝8.85(s,2H),7.39-7.34(m,4H),7.29-7.26(m, 1H),7.12-7.11(m,1H),6.98-6.96(m,1H),6.77-6.74(m,1H),6.70(d,J＝7.8Hz,1H), 5.49-5.47(m,1H),3.09-2.99(m,2H),2.57(s,3H),2.29-2.22(m,4H),2.16-2.11(m, 1H)；13CNMR(150MHz,DMSO-d6) δ 165.04,155.52,141.32,130.99,129.16, 128.30,127.06,126.76,126.30,120.83,113.38,76.18,45.78,34.76,33.01, 16.70; HR-MS theoretical value (calcd for) C17H22NO[M+H]+m/z 256.1696, Experimental value (found) m/z 256.1700.
EXAMPLE 4 Synthesis of (R) -N-methyl-3- (2-methylphenoxy) -3-phenyl-1-propylamine (6) hydrochloride
Oxalate-1 (0.5g, 0.0014mol) was dissolved in a mixed solvent of 5mL of methylene chloride and 5mL of a saturated sodium bicarbonate solution, stirred for 30min, extracted with methylene chloride (2 × 10mL), washed with water (1 × 10mL), the methylene chloride layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated.
Dissolving the oily substance obtained in the previous step in 5mL of ethyl acetate, adding 1mL of an ethyl acetate solution of 2mol/L hydrogen chloride, stirring for 5 hours at room temperature, filtering to obtain a white solid, washing with ethyl acetate, and drying to obtain 1.HCl 0.4g, yield: 94.7%, HPLC purity: 99.58%, ee value: 99.9 percent.
1HNMR(600MHz,DMSO-d6):δ＝9.15(s,2H),7.41-7.35(m,4H),7.29-7.26(m, 1H),7.12-7.10(m,1H),6.98-6.95(m,1H),6.76-6.72(m,2H),5.57-5.55(m,1H), 3.05-2.96(m,2H),2.53(s,3H),2.32-2.27(m,1H),2.26(s,3H),2.21-2.15(m,1H)；13CNMR(150MHz,DMSO-d6) δ 155.53,141.35,130.98,129.14,128.29,127.06, 126.75,126.34,120.82,113.44,76.12,45.69,34.72,32.79, 16.72; HR-MS theoretical value (calcd for) C17H22NO[M+H]+m/z 256.1696, Experimental value (found) m/z 256.1696.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence information related to the present invention:
DNA sequence SEQ ID No.1
atgacgattgctctcaacaatgtggtcgccgtcgtcaccggcgcggcgggaggcatcggccgcgaactggtcaaggc gatgaaggccgccaacgccatcgtcatcgccaccgacatggccccctcggccgatgtcgaaggcgcggaccattatc tccagcacgacgtgacgagcgaggccggctggaaggccgtcgcggcgctggcccaggaaaagtacgggcgcgtc gatgcgctggtgcacaacgcgggcatctcgatcgtcacgaagttcgaagacactccgctgtccgatttccaccgcgtg aacacggtcaacgtcgattccatcatcatcggtacgcaggtcctgctgccgctgctcaaggaaggcggcaaggcgcg cgcagggggcgcctcggtggtcaacttctccagcgtcgcgggcctgcgcggcgcggcgttcaatgcggcctattgca ccagcaaggcggcggtgaagatgctctcgaagtgcctcggcgcggaattcgcggcgctcggctacaacatccgcgtc aactccgtgcatccgggcggcatcgataccccgatgctcggctcgctcatggacaagtacgtcgaactcggcgctgcc ccctcgcgcgaggtggcccaggccgcgatggaaatgcgccacccgatcggtcgcatgggtcgccctgccgaaatgg gcggcggcgtggtctatctctgctccgacgcagcaagcttcgtcacctgcacggaattcgtgatggacggcggcttca gccaggtc
Amino acid sequence SEQ ID No.2
mtialnnvvavvtgaaggigrelvkamkaanaiviatdmapsadvegadhylqhdvtseagwkavaalaqeky grvdalvhnagisivtkfedtplsdfhrvntvnvdsiiigtqvllpllkeggkaraggasvvnfssvaglrgaafnaay ctskaavkmlskclgaefaalgynirvnsvhpggidtpmlgslmdkyvelgaapsrevaqaamemrhpigrmg rpaemgggvvylcsdaasfvtctefvmdggfsqv。
Sequence listing
<110> Shanghai institute for pharmaceutical industry
China Pharmaceutical Industry Research Institute
<120> chemo-enzymatic synthesis method of tomoxetine
<130> P2019-1036
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 777
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
atgacgattg ctctcaacaa tgtggtcgcc gtcgtcaccg gcgcggcggg aggcatcggc 60
cgcgaactgg tcaaggcgat gaaggccgcc aacgccatcg tcatcgccac cgacatggcc 120
ccctcggccg atgtcgaagg cgcggaccat tatctccagc acgacgtgac gagcgaggcc 180
ggctggaagg ccgtcgcggc gctggcccag gaaaagtacg ggcgcgtcga tgcgctggtg 240
cacaacgcgg gcatctcgat cgtcacgaag ttcgaagaca ctccgctgtc cgatttccac 300
cgcgtgaaca cggtcaacgt cgattccatc atcatcggta cgcaggtcct gctgccgctg 360
ctcaaggaag gcggcaaggc gcgcgcaggg ggcgcctcgg tggtcaactt ctccagcgtc 420
gcgggcctgc gcggcgcggc gttcaatgcg gcctattgca ccagcaaggc ggcggtgaag 480
atgctctcga agtgcctcgg cgcggaattc gcggcgctcg gctacaacat ccgcgtcaac 540
tccgtgcatc cgggcggcat cgataccccg atgctcggct cgctcatgga caagtacgtc 600
gaactcggcg ctgccccctc gcgcgaggtg gcccaggccg cgatggaaat gcgccacccg 660
atcggtcgca tgggtcgccc tgccgaaatg ggcggcggcg tggtctatct ctgctccgac 720
gcagcaagct tcgtcacctg cacggaattc gtgatggacg gcggcttcag ccaggtc 777
<210> 2
<211> 259
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 2
Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val Thr Gly Ala Ala
1 5 10 15
Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys Ala Ala Asn Ala
20 25 30
Ile Val Ile Ala Thr Asp Met Ala Pro Ser Ala Asp Val Glu Gly Ala
35 40 45
Asp His Tyr Leu Gln His Asp Val Thr Ser Glu Ala Gly Trp Lys Ala
50 55 60
Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val Asp Ala Leu Val
65 70 75 80
His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu Asp Thr Pro Leu
85 90 95
Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp Ser Ile Ile Ile
100 105 110
Gly Thr Gln Val Leu Leu Pro Leu Leu Lys Glu Gly Gly Lys Ala Arg
115 120 125
Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val Ala Gly Leu Arg
130 135 140
Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys Ala Ala Val Lys
145 150 155 160
Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala Leu Gly Tyr Asn
165 170 175
Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp Thr Pro Met Leu
180 185 190
Gly Ser Leu Met Asp Lys Tyr Val Glu Leu Gly Ala Ala Pro Ser Arg
195 200 205
Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro Ile Gly Arg Met
210 215 220
Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr Leu Cys Ser Asp
225 230 235 240
Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met Asp Gly Gly Phe
245 250 255
Ser Gln Val

Claims (10)

1. A preparation method of (S) -3-chlorophenylpropanol is characterized by comprising the following steps:
(a) in a liquid reaction system, 3-chlorophenyl propanone (compound of formula 2) is used as a substrate, and asymmetric reduction reaction is carried out in the presence of coenzyme under the catalysis of carbonyl reductase, so as to form (S) -3-chlorophenyl propanol [ compound of formula (S) -3 ];
Figure FDA0002246790480000011
and
(b) optionally separating (S) -3 from the reaction system after the reaction of the previous step;
wherein the carbonyl reductase is selected from the group consisting of:
(i) the amino acid sequence is shown as SEQ ID NO. 2;
(ii) the amino acid sequence shown in SEQ ID NO.2 is obtained by carrying out substitution, deletion, change, insertion or addition of one or more amino acids within the range of keeping the enzyme activity.
2. The process according to claim 1, wherein the carbonyl reductase in the reaction system is a free form enzyme, an immobilized enzyme, or a bacterial form enzyme.
3. The method of claim 1, wherein the coenzyme is selected from the group consisting of: a reducing coenzyme, an oxidizing coenzyme, or a combination thereof.
4. The process according to claim 1, wherein an enzyme for coenzyme regeneration is further present in the reaction system, and the enzyme for coenzyme regeneration is isopropanol dehydrogenase, formate dehydrogenase, or glucose dehydrogenase.
5. The process according to claim 1, wherein in step (b), the ee value of the compound of formula (S) -3 in the reaction system after the reaction is 90% or more, preferably 95% or more, more preferably 99% or more.
6. The method of claim 1, wherein the carbonyl reductase is encoded by a gene sequence selected from the group consisting of:
(a) the sequence shown as SEQ ID NO.1 (accession number CP000677.1 at NCBI);
(b) a polynucleotide complementary to the sequence defined in (a); or
(c) Any polynucleotide or complementary sequence having at least 70% (preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99%) or more sequence identity to the sequence defined in (a).
7. The method according to claim 1, wherein a co-substrate is further present in the reaction system, and the co-substrate is isopropanol, ammonium formate, or glucose.
8. The method according to claim 7, wherein the concentration of the cosubstrate in the reaction system is 5 to 30%.
9. A process for the preparation of tomoxetine, wherein the compound of formula (S) -3 prepared by the process of claim 1 is prepared by the following reaction steps:
Figure FDA0002246790480000021
10. the method of claim 9, wherein the method comprises the steps of:
(1) subjecting (S) -3-chlorophenylpropanol prepared by the method of claim 1 to a mitsunobu reaction with o-methylphenol to obtain compound 4;
(2) activating the compound 4 obtained in the step (1) by sodium iodide, substituting methylamine and salifying oxalic acid to prepare the oxalate of the tomoxetine;
(3) and (3) dissociating the oxalate obtained in the step (2) by using a saturated sodium bicarbonate solution, and salifying the oxalate with hydrogen chloride to obtain the atomoxetine hydrochloride.
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CN113816836A (en) * 2021-09-29 2021-12-21 山东寰酶生物制药有限公司 Enzymatic production method of (S) -1- (4-chlorphenyl) -1, 3-propylene glycol
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