A preparation method of L-penicillamine

Abstract

The invention provides a method for preparing L-penicillamine, comprising the following steps: subjecting a compound shown in formula I-1 to a nitration reaction under concentrated nitric acid conditions; subjecting a nitrated product shown in formula II-1 to a thio reagent R2-SH under the catalytic action of an alkaline catalyst in a third organic solvent to a Michael addition reaction under an inert gas atmosphere; reducing the nitro group in the thio product shown in formula III-1 to an amino group to generate a racemic penicillamine shown in formula IV-1; subjecting the racemic penicillamine shown in formula IV-1 to ester hydrolase decomposition to generate a thiol-protected L-penicillamine shown in formula V-1; removing the thiol protecting group from the thiol-protected L-penicillamine shown in formula V-1 under strong acid conditions to generate an L-penicillamine salt, and performing alkaline hydrolysis to generate the L-penicillamine; the method of the invention solves the problems of high raw material cost, complicated synthesis steps and low synthesis efficiency in the prior art.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical chemicals, and in particular to a method for preparing L-penicillamine. Background Art

[0002] Penicillamine is an optically active substance with D-type, L-type and D, L-type. At present, there are many reports on the synthesis or production of D-penicillamine at home and abroad, such as the degradation of penicillin and its derivatives, chemical synthesis of thiazole ring hydrolysis or alkylation of bromide and thio reagent and then cyano hydrolysis to synthesize D-penicillamine or racemic penicillamine. There are fewer reports on the synthesis of L-penicillamine, which is mainly obtained by synthesizing racemic penicillamine and then splitting it.

[0003] like Figure 1 As shown, Sun Risheng et al. (Applied Chemistry, 2006, 47(12), 734-736) disclosed that D-penicillamine reacts with acetyl chloride in acetic acid, and racemization occurs after acylation to obtain racemic acetylated penicillamine, which is then resolved using L-chloramine as a resolution agent to obtain L-configuration acetylated penicillamine chloramine salt, which is then freed with concentrated hydrochloric acid, and the acetyl protecting group is removed with 15% hydrochloric acid to obtain L-penicillamine, and the D-configuration acetylated penicillamine is recovered and reused. This strategy can successfully obtain L-penicillamine after racemization of D-penicillamine, but the cost of D-penicillamine raw materials and L-chloramine resolution agents is relatively high, and is not suitable for large-scale production.

[0004] like Figure 2 As shown, Chinese patent CN12500323A discloses that D-penicillamine and acetone undergo nucleophilic addition reaction to cyclize isopropyl penicillamine, and then the amino group of isopropyl penicillamine undergoes acylation reaction with ethyl formate to obtain N-formylisopropyl penicillamine, which is then racemized under acetic acid conditions to obtain a racemate of N-formylisopropyl penicillamine, the racemate is hydrolyzed under hydrochloric acid conditions to remove the formyl group and isopropyl group to obtain a racemate of penicillamine hydrochloride, triethylamine is added, the pH value is adjusted, and penicillamine hydrochloride is liberated to obtain racemic penicillamine, the racemic penicillamine is resolved under L-tartaric acid to obtain the tartrate of penicillamine, and the pH is adjusted with triethylamine to liberate the tartrate of penicillamine to obtain L-penicillamine. This strategy can successfully obtain L-penicillamine by racemization of D-penicillamine and then splitting it, but the synthesis steps are cumbersome, and a total of 7 steps are required to obtain L-penicillamine. In addition, the cost of D-penicillamine raw materials is relatively high, resulting in a long scale-up production cycle and high costs. Therefore, the existing strategies all obtain L-penicillamine by racemization of D-penicillamine and then splitting it, which has high raw material costs, and the process requires optically pure penicillamine-racemic penicillamine-optically pure penicillamine, and the synthesis steps are cumbersome and the synthesis efficiency is low. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for preparing L-penicillamine to solve the problems in the prior art, such as high raw material cost, complicated synthesis steps, low synthesis efficiency and unsuitability for large-scale production.

[0006] In order to achieve the above object, according to one aspect of the present invention, a method for preparing L-penicillamine is provided, comprising the following steps:

[0007] Step S1.1, subjecting the compound represented by formula I-1 to a nitration reaction under concentrated nitric acid conditions to generate a nitration product represented by formula II-1;

[0008] Step S1.2, under an inert gas atmosphere, the nitration product of formula II-1 and the thiolation reagent R2-SH are subjected to a Michael addition reaction in a third organic solvent under the catalytic action of an alkaline catalyst to generate a thiolation product of formula III-1;

[0009] Step S1.3, reducing the nitro group in the thio product of formula III-1 to an amino group to generate a nitro-reduced product of formula IV-1;

[0010] Step S1.4, the nitro-reduction product of formula IV-1 is split by ester hydrolase to generate thiol-protected L-penicillamine of formula V-1;

[0011] Step S1.5, removing the thiol protecting group of the thiol-protected L-penicillamine shown in formula V-1 under strong acid conditions to generate L-penicillamine salt, and then alkaline hydrolyzing to generate L-penicillamine;

[0012]

[0013] Among them, R1 is selected from one of C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy, aryl substituted or unsubstituted by at least one substituent in halogen atom, and benzyl; R2 is selected from one of alkyl, alkoxy, phenyl substituted or unsubstituted by at least one substituent in halogen atom, diphenylmethyl, triphenylmethyl, benzyl, and 2-ethylhexyl propionate.

[0014] Furthermore, R1 is selected from one of methyl, ethyl, propyl, isopropyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, allyl, phenyl and benzyl.

[0015] Preferably, R2 is selected from phenyl, diphenylmethyl, triphenylmethyl, benzyl, 2-ethylhexyl propionate, substituted or unsubstituted with at least one substituent selected from methyl, methoxy, fluorine, chlorine, and bromine.

[0016] Furthermore, in step S1.1, the mass fraction of nitric acid in concentrated nitric acid is 68% to 98%.

[0017] Furthermore, in step S1.1, the molar ratio of the compound represented by formula I-1 to the nitric acid in concentrated nitric acid is 1:(2-10).

[0018] Furthermore, in step S1.1, the compound represented by formula I-1 is added dropwise to concentrated nitric acid at 0-5°C. After the addition is completed, stirring is continued at 0-5°C for 1-1.5h, and then the temperature is raised to room temperature and stirring is continued until the reaction is complete.

[0019] Preferably, in step S1.1, during the nitration reaction, TLC is used to monitor whether the reaction is complete.

[0020] Further, in step S1.2, the alkaline catalyst is selected from one or more of triethylamine, tri-n-butylamine, diisopropylethylamine, pyridine, 2,6-lutidine, dimethylaminopyridine, imidazole, N-methylpiperidine, N-methylmorpholine, 1,8-diazobispiro[5.4.0]undec-7-ene (DBU), 1,4-diazabicyclo[2.2.2]octane, D-proline, L-proline, quinine, quinidine, lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, potassium tert-butoxide, sodium tert-butoxide, lithium diisopropylamide, lithium bistrimethylsilylamide, lithium bistrimethylsilylamide, potassium bistrimethylsilylamide, sodium hydroxide, potassium hydroxide, and sodium hydride.

[0021] Furthermore, in step S1.2, the alkaline catalyst is selected from one or more of triethylamine (TEA), D-proline, L-proline, quinine, and quinidine.

[0022] Furthermore, in step S1.2, the molar ratio of the nitration product represented by formula II-1, the thiolation reagent and the alkaline catalyst is 1: (1.0~10.0): (0.05~5.0).

[0023] Preferably, in step S1.2, the molar ratio of the nitration product represented by formula II-1, the thiolation reagent and the alkaline catalyst is 1: (1.0-2.0): (0.5-2).

[0024] Furthermore, in step S1.2, the third organic solvent is selected from one or more of methyl tert-butyl ether, anisole, ethylene glycol dimethyl ether, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, dichloromethane, chloroform, 1,2-dichloroethane, 1,2-dibromoethane, n-heptane, n-hexane, N-methylpyrrolidone, acetonitrile, ethyl acetate, tetrahydrofuran, 2-methyltetrahydrofuran, and 1,4-dioxane.

[0025] Preferably, in step S1.2, the third organic solvent is selected from dichloromethane, ethyl acetate and toluene.

[0026] Furthermore, in step S1.2, nitrogen gas is introduced into the third solvent to form an inert gas atmosphere.

[0027] Furthermore, in step S1.2, the temperature of the Michael addition reaction is 15-30°C.

[0028] Preferably, in step S1.2, the temperature of the Michael addition reaction is room temperature.

[0029] Furthermore, in step S1.2, the Michael addition reaction time is 1 to 5 hours.

[0030] Furthermore, in step S1.2, during the Michael addition reaction, HPLC is used to monitor whether the reaction is complete.

[0031] Furthermore, in step S1.3, the nitro group in the thio product shown in formula III-1 is reduced to an amino group by a metal reducing agent; the metal reducing agent is zinc powder and / or iron powder.

[0032] Preferably, in step S1.3, the step of reducing the nitro group in the product shown in formula III-1 to an amino group by a metal element reducing agent is as follows: a metal element reducing agent is added to a mixed system comprising a thio product shown in formula III-1, a first acidic substance and a first organic solvent to carry out a reduction reaction to generate a nitro reduction product shown in formula IV-1.

[0033] More preferably, in step S1.3, the molar ratio of the thioproduct represented by formula III-1, the metal reducing agent and the first acidic substance is 1:(4-6):(10-20).

[0034] More preferably, in step S1.3, the first acidic substance is selected from one or more of hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, methanesulfonic acid, trifluoromethanesulfonic acid, sulfuric acid, nitric acid, acetic acid, trifluoroacetic acid, ammonium chloride, trimethylchlorosilane, ferrous chloride, zinc chloride, cupric chloride, aluminum chloride, boron tribromide, and boron trichloride.

[0035] More preferably, the first acidic substance is selected from one or more of hydrochloric acid, hydrobromic acid, phosphoric acid, methanesulfonic acid, sulfuric acid, nitric acid and acetic acid.

[0036] More preferably, in step S1.3, the first organic solvent is selected from one or more of methanol, ethanol, isopropanol, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, acetic acid, acetonitrile, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, dichloromethane, chloroform, 1,2-dichloroethane, and 1,2-dibromoethane.

[0037] More preferably, in step S1.3, the metal reducing agent is added in batches.

[0038] More preferably, in step S1.3, before the metal reducing agent is completely added, the reaction temperature is controlled to be 0-5°C. After the metal reducing agent is completely added, the reaction is first carried out at a temperature of 0-5°C for 0.5-1.5h, and then the temperature is raised to 15-30°C for 1.5-3h.

[0039] More preferably, in step S1.3, during the reduction reaction of the metal reducing agent, HPLC is used to monitor whether the reaction is complete.

[0040] Furthermore, in step S1.3, the nitro group in the thio product represented by formula III-1 is reduced to an amino group by catalytic hydrogenation using a precious metal catalyst, and the precious metal catalyst is selected from one or more of palladium carbon, platinum carbon, palladium hydroxide carbon, and Raney nickel.

[0041] Preferably, in step S1.3, the step of reducing the nitro group in the thio product shown in formula III-1 to an amino group by catalytic hydrogenation with a precious metal catalyst is as follows: hydrogen is introduced into a reactor containing the thio product shown in formula III-1, a second organic solvent, a third acidic substance and a precious metal catalyst for replacement, and the pressure in the reactor is controlled to be 1.5~2.5Mpa, and the reaction is carried out at a temperature of 70~80°C to generate a nitro reduction product shown in formula IV-1.

[0042] More preferably, in step S1.3, the noble metal catalyst is selected from one or more of Raney nickel, palladium carbon with a palladium mass fraction of 5% to 10%, platinum carbon with a platinum mass fraction of 5% to 10%, and palladium hydroxide carbon with a palladium hydroxide mass fraction of 10% to 20%.

[0043] More preferably, in step S1.3, the reaction time of the catalytic hydrogenation with the noble metal catalyst is 12 to 36 hours.

[0044] More preferably, in step S1.3, the second organic solvent is selected from one or more of methanol, ethanol, isopropanol, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, acetic acid, acetonitrile, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, dichloromethane, chloroform, 1,2-dichloroethane, and 1,2-dibromoethane.

[0045] More preferably, in step S1.3, the third acidic substance is selected from one or more of hydrochloric acid, acetic acid, trifluoroacetic acid, and p-toluenesulfonic acid.

[0046] More preferably, the third acidic substance is selected from one or more of hydrochloric acid, trifluoroacetic acid and p-toluenesulfonic acid.

[0047] More preferably, in step S1.3, the molar ratio of the thioaddition product to the third acidic substance is 1:(1-1.5).

[0048] More preferably, in step S1.3, the mass ratio of the noble metal catalyst to the thioproduct represented by formula III-1 is (0.05~0.3):1.

[0049] Furthermore, in step S1.4, the ester hydrolase is selected from one or more of acetylcholinesterase, gluconolactonase, phosphomonoester hydrolase, and serine protease.

[0050] Furthermore, in step S1.4, the step of splitting the nitro-reduction product represented by formula IV-1 by ester hydrolase is as follows: a solution of the nitro-reduction product represented by formula IV-1 is added dropwise to a buffer solution containing an organic reducing agent and an ester hydrolase, and then reacted at 35-45° C. to generate the thiol-protected L-penicillamine represented by formula V-1; the pH of the buffer solution is 6.8-7.2, and the organic reducing agent is selected from dithiothreitol, tris(2-carbonylethyl)phosphine hydrochloride, or a mixture of the two.

[0051] Preferably, in step S1.4, the solution of the nitro reduction product represented by formula IV-1 is obtained by dissolving the nitro reduction product represented by formula IV-1 in one or more of water, acetonitrile, methanol, ethanol, propanol, isopropanol and butanol.

[0052] Preferably, in step S1.4, the buffer is a potassium phosphate buffer, and the concentration of the buffer is 0.1-5 mol / L.

[0053] Preferably, in step S1.4, the mass ratio of the organic reducing agent, the ester hydrolase and the nitro reduction product represented by formula IV-1 is (0.03~0.08):(0.3~0.8):1.

[0054] Furthermore, the pH of the system after the splitting reaction is completed is adjusted to 1-2, and the mixture is stirred for 10-30 minutes and then filtered. The filter cake is washed with water, the filtrates are combined and the pH of the combined filtrates is adjusted to 6-7, and the combined filtrates are extracted with isopropyl acetate for multiple times to remove the remaining substrate. The aqueous phase is concentrated, and the pH of the aqueous phase is adjusted to 1-2 after concentration, and then ice-bathed and filtered to obtain the thiol-protected L-penicillamine solid shown in Formula V-1.

[0055] Furthermore, in step S1.5, the step of removing the thiol protecting group of the thiol-protected L-penicillamine shown in formula V-1 under strong acid conditions to generate L-penicillamine salt is as follows: adding acid solution to the thiol-protected L-penicillamine shown in formula V-1 at 15~30°C, and then stirring for 6~24h under heating conditions at 30~60°C to remove the thiol protecting group to generate L-penicillamine salt.

[0056] Preferably, in step S1.5, the acid solution is selected from trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, or is selected from a second acidic substance solution.

[0057] More preferably, in step S1.5, the second acidic substance in the second acidic substance solution is selected from one or more of hydrochloric acid, hydrobromic acid, hydroiodic acid, trifluoroacetic acid, methanesulfonic acid, and trifluoromethanesulfonic acid, and the solvent in the second acidic substance solution is selected from one or more of water, acetic acid, methanol, ethanol, isopropanol, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, acetic acid, acetonitrile, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, dichloromethane, chloroform, 1,2-dichloroethane, and 1,2-dibromoethane.

[0058] More preferably, in step S1.5, the mass fraction of the second acidic substance in the second acidic substance solution is 10-90%.

[0059] More preferably, in step S1.5, the molar ratio of the thiol-protected L-penicillamine represented by formula V-1 to the second acidic substance is 1:(1-30).

[0060] Furthermore, in step S1.5, the alkaline hydrolysis step is as follows: the system after the reaction of removing the thiol protecting group is completed is concentrated to remove the second acidic substance, dichloromethane is added to dissolve the residue, and then an alkaline substance is added to adjust the pH to 4-5, and concentrated to obtain a crude product of L-penicillamine.

[0061] Preferably, in step S1.5, the alkaline substance is selected from an inorganic base aqueous solution or an organic base, the inorganic base is selected from one or more of NaOH, Na2CO3, KOH, K2CO3, or the organic base is selected from one or more of TEA, DBU, DIPEA, DABCO, DBN, DMAP, pyridine, N-methylmorpholine, and tetramethylethylenediamine.

[0062] Preferably, in step S1.5, the crude L-penicillamine is slurried with dichloromethane, stirred for 6 to 24 hours, and filtered to obtain solid L-penicillamine.

[0063] According to another aspect of the present invention, there is also provided a method for preparing L-penicillamine, comprising the following steps:

[0064] Step S2.1, subjecting the compound represented by formula I-2 to a nitration reaction under concentrated nitric acid conditions to generate a nitration product represented by formula II-2;

[0065] Step S2.2, under an inert gas atmosphere, the nitration product of formula II-2 and the thiolation reagent R2-SH are subjected to a Michael addition reaction in a third organic solvent under the catalytic action of an alkaline catalyst to generate a thiolation product of formula III-2;

[0066] Step S2.3, reducing the nitro group in the thio product of formula III-2 to an amino group to generate a nitro-reduced product of formula IV-2;

[0067] Step S2.4, splitting the nitro-reduction product of Formula IV-2 by ester hydrolase to generate amino-protected L-penicillamine of Formula V-2;

[0068] Step S2.5, removing the amino protecting group of the amino-protected L-penicillamine shown in V-2 under strong acid conditions to generate L-penicillamine salt, and then alkaline hydrolyzing to generate L-penicillamine;

[0069]

[0070] Wherein, R3 is selected from one of C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy, aryl substituted or unsubstituted with at least one substituent in the halogen atom, and benzyl;

[0071] R4 is selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, benzoyl or acetyl which is substituted or unsubstituted with at least one substituent in the halogen atom.

[0072] Preferably, h and R3 are selected from one of methyl, ethyl, propyl, isopropyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, allyl, phenyl and benzyl.

[0073] Preferably, R4 is selected from methyl, methoxy, fluorine, chlorine, bromine, substituted or unsubstituted benzoyl, acetyl.

[0074] Preferably, in step S2.5, the ester hydrolase is selected from one or more of acetylcholinesterase, gluconolactonase, phosphomonoester hydrolase and serine protease.

[0075] According to another aspect of the present invention, there is also provided a method for preparing L-penicillamine, comprising the following steps:

[0076] Step S4.1, subjecting the compound represented by formula I-4 to a nitration reaction under concentrated nitric acid conditions to generate a nitration product represented by formula II-4;

[0077] Step S4.2, under an inert gas atmosphere, the nitration product of formula II-4 and the thiolation reagent R2-SH are subjected to Michael addition reaction in a third organic solvent under the catalytic action of an alkaline catalyst to generate the thiolation product of formula III-4;

[0078] Step S4.3, reducing the nitro group in the thio product of formula III-4 to an amino group to generate a nitro-reduced product of formula IV-4;

[0079] Step S4.4, splitting the nitro-reduction product of formula IV-4 by acyl hydrolase to generate L-penicillamine;

[0080]

[0081] Wherein, R6 is selected from one of C1-C6 alkyl, C1-C6 alkoxy, benzoyl and acetyl which are substituted or unsubstituted with at least one substituent in the halogen atom.

[0082] Preferably, R6 is selected from methyl, methoxy, fluorine, chlorine, bromine, substituted or unsubstituted benzoyl, acetyl.

[0083] Preferably, in step S4.4, the acyl hydrolase is selected from one or more of acylcarnitine hydrolase, acetyl-CoA hydrolase and acyl-CoA hydrolase.

[0084] The technical scheme of the present invention is applied, commercially available enoic acid or enoic acid ester is used as a starting material, a nitration reaction is carried out under concentrated nitric acid conditions to generate a nitration product, and a nitro group is introduced into the raw material; then, under an inert gas atmosphere, the nitration product and a thio reagent R-SH are subjected to Michael addition, thereby introducing RS into the raw material and generating a racemic thio product; then, the nitro group in the racemic thio product is reduced to an amino group to generate a racemic penicillamine with a protective group; finally, the racemic penicillamine is split by a hydrolase to obtain L-penicillamine.

[0085] The target product L-penicillamine was synthesized by the above method. The synthesis strategy is simple, efficient and easy to operate. Racemic penicillamine is first synthesized in three steps, and then an optically pure product L-penicillamine is obtained by an efficient and highly selective enzyme resolution technology with a high yield. Furthermore, the starting raw materials are cheap and easily available, and the material cost is low, which provides a more economical and practical method for the synthesis of L-penicillamine. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0087] Figure 1 A synthetic route of L-penicillamine in the prior art is shown;

[0088] Figure 2 Another synthesis route of L-penicillamine in the prior art is shown;

[0089] Figure 3 A synthetic route diagram of L-penicillamine according to one embodiment of the present invention is shown. DETAILED DESCRIPTION

[0090] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0091] As described in the background art, the prior art has the problems of high raw material cost, complicated synthesis steps, low synthesis efficiency, and is not suitable for large-scale production. In order to solve the above problems, according to one aspect of the present invention, a method for preparing L-penicillamine is provided, such as Figure 3 As shown, the following steps are included:

[0092] Step S1.1, subjecting the compound represented by formula I-1 to a nitration reaction under concentrated nitric acid conditions to generate a nitration product represented by formula II-1;

[0093] Step S1.2, under an inert gas atmosphere, the nitration product of formula II-1 and the thiolation reagent R2-SH are subjected to a Michael addition reaction in a third organic solvent under the catalytic action of an alkaline catalyst to generate a thiolation product of formula III-1;

[0094] Step S1.3, reducing the nitro group in the thio product of formula III-1 to an amino group to generate a nitro-reduced product of formula IV-1;

[0095] Step S1.4, the nitro-reduction product of formula IV-1 is decomposed by ester hydrolase to generate thiol-protected L-penicillamine of formula V-1;

[0096] Step S1.5, removing the thiol protecting group of the thiol-protected L-penicillamine shown in formula V-1 under strong acid conditions to generate L-penicillamine salt, and then alkaline hydrolyzing to generate L-penicillamine;

[0097]

[0098] Wherein, R1 is selected from one of C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy, aryl substituted or unsubstituted by at least one substituent in halogen atom, and benzyl; R2 is selected from one of C1-C6 alkyl, C1-C6 alkoxy, phenyl substituted or unsubstituted by at least one substituent in halogen atom, diphenylmethyl, triphenylmethyl, benzyl, and 2-ethylhexyl propionate.

[0099] In order to reduce production costs and scale up production, the present invention provides a method for preparing L-penicillamine. In the first step, commercially available enoic acid ester is used as a starting material, and a nitration reaction is carried out under concentrated nitric acid conditions to generate a nitration product shown in formula II-1, thereby introducing a nitro group into the raw material; in the second step, under an inert gas atmosphere, the nitration product shown in formula II-1 is subjected to Michael addition with a thio reagent R2-SH with different substituents, thereby introducing R2S into the raw material and generating a racemic thio product shown in formula III-1; in the third step, the nitro group in the thio product shown in formula III-1 is reduced to an amino group to generate a nitro reduction product shown in formula IV-1 with a protecting group; in the fourth step, the nitro reduction product shown in formula IV-1 with a protecting group is split by an ester hydrolase to obtain a thiol-protected L-penicillamine shown in formula V-1; in the fifth step, the thiol protecting group is removed from the thiol-protected L-penicillamine of formula V-1 under strong acid conditions to generate an L-penicillamine salt, and the salt is hydrolyzed with an organic base to obtain a free L-penicillamine. The target product L-penicillamine is synthesized through five steps by adopting the above method. The synthesis strategy is simple, efficient and simple to operate. The racemate is first synthesized through three steps, and then an optically pure product is obtained through efficient and highly selective enzyme resolution technology. Finally, the protecting group is removed to obtain L-penicillamine with a high yield. Furthermore, the starting raw materials are cheap and easy to obtain, and the material cost is low, which provides a new strategy for synthesizing L-penicillamine that is simple, efficient and low-cost and suitable for large-scale production.

[0100] In a preferred embodiment, R1 is selected from one of methyl, ethyl, propyl, isopropyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, allyl, phenyl and benzyl. The raw material cost is low and it is simple and easy to obtain; R2 mainly affects the yield of the thio product shown in formula III-1 during the Michael addition reaction and the yield of L-penicillamine salt during the removal of the thiol protecting group, thereby affecting the yield of L-penicillamine. In order to obtain a higher yield of L-penicillamine, R2 is preferably selected from one of phenyl, diphenylmethyl, triphenylmethyl, benzyl and 2-ethylhexyl propionate substituted or unsubstituted with at least one substituent selected from methyl, methoxy, fluorine, chlorine and bromine. When R2 is a structure such as phenyl or benzyl, the stability and yield of the thio product shown in formula III-1 can be improved.

[0101] In order to obtain a higher nitration efficiency and yield, in a preferred embodiment, the mass fraction of nitric acid in concentrated nitric acid is 68%~98%; preferably, the molar ratio of the compound represented by formula I-1 to the nitric acid in concentrated nitric acid is 1:(2~10), so that the compound represented by formula I-1 is fully nitrated; preferably, the compound represented by formula I-1 is added dropwise to concentrated nitric acid at 0~5°C, and after the addition is completed, stirring is continued at 0~5°C for 1~1.5h, and then the temperature is raised to room temperature and stirring is continued until the reaction is complete, and temperature control is used to prevent temperature surge and spraying; more preferably, during the nitration reaction, TLC is used to monitor whether the reaction is complete.

[0102] In order to obtain a higher thio product represented by formula III-1, in a preferred embodiment, in step S1.2, the alkaline catalyst is selected from triethylamine, tri-n-butylamine, diisopropylethylamine, pyridine, 2,6-lutidine, dimethylaminopyridine, imidazole, N-methylpiperidine, N-methylmorpholine, 1,8-diazobispiro[5.4.0]undec-7-ene (DBU), 1,4-diazabicyclo[2.2.2]octane, D-proline, L-proline, quinine, quinidine, lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, potassium tert-butoxide , sodium tert-butoxide, lithium tert-butoxide, lithium diisopropylamide, lithium bistrimethylsilylamide, lithium bistrimethylsilylamide, potassium bistrimethylsilylamide, sodium hydroxide, potassium hydroxide, sodium hydride; preferably, the alkaline catalyst is selected from one or more of triethylamine (TEA), D-proline, L-proline, quinine, and quinidine; preferably, the molar ratio of the nitration product represented by formula II-1, the thiolation reagent, and the alkaline catalyst is 1: (1.0-10.0): (0.05-5.0), so that the nitration product represented by the raw material formula II-1 is fully thiolated. Conversion; more preferably, the molar ratio of the nitration product represented by formula II-1, the thiolation reagent and the alkaline catalyst is 1: (1.0~2.0): (0.5~2); preferably, the third organic solvent is selected from methyl tert-butyl ether, anisole, ethylene glycol dimethyl ether, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, dichloromethane, chloroform, 1,2-dichloroethane, 1,2-dibromoethane, n-heptane, n-hexane, N-methylpyrrolidone, acetonitrile, ethyl acetate, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane and one or more thereof; more ... The three organic solvents are selected from dichloromethane, ethyl acetate, and toluene to obtain a higher product yield; preferably, nitrogen is introduced into the third solvent to form an inert gas atmosphere; in order to obtain a higher yield, the temperature of the Michael addition reaction is preferably 15-30°C; more preferably, the temperature of the Michael addition reaction is room temperature, which is more economical; preferably, the time of the Michael addition reaction is 1-5h, under which the raw materials are basically reacted; preferably, during the Michael addition reaction, HPLC is used to monitor whether the reaction is complete.

[0103] In a preferred embodiment, in step S1.3, the nitro group in the thioproduct represented by formula III-1 is reduced to an amino group by a metal element reducing agent; the metal element reducing agent is zinc powder and / or iron powder, and the reduction by zinc powder and / or iron powder is efficient and low in cost, economical and practical; preferably, the step of reducing the nitro group in the product represented by formula III-1 to an amino group by a metal element reducing agent is as follows: in a mixed system comprising the thioproduct represented by formula III-1, a first acidic substance and a first organic solvent, a metal element reducing agent is added The reduction reaction is carried out with an agent to generate a nitro reduction product shown in formula IV-1; more preferably, the molar ratio of the thio product shown in formula III-1, the metal element reducing agent and the first acidic substance is 1: (4-6): (10-20) to obtain a higher reduction yield; more preferably, the first acidic substance is selected from hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, methanesulfonic acid, trifluoromethanesulfonic acid, sulfuric acid, nitric acid, acetic acid, trifluoroacetic acid, ammonium chloride, trimethylsilyl chloride, ferrous chloride, zinc chloride, cupric chloride, aluminum chloride, boron tribromide, boron trichloride. The first acidic substance is preferably one or more selected from hydrochloric acid, hydrobromic acid, phosphoric acid, methanesulfonic acid, sulfuric acid, nitric acid, and acetic acid; the first acidic substance is preferably added in the form of an aqueous solution to prevent volatilization or to facilitate rapid dissolution and dispersion; the first organic solvent is more preferably selected from methanol, ethanol, isopropanol, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, acetic acid, acetonitrile, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, dichloromethane, chloroform, 1,2-dichloroethane one or more of alkane, 1,2-dibromoethane; more preferably, the metal reducing agent is added in batches; in order to avoid too fast temperature rise in the initial stage of the reaction to reduce the occurrence of side reactions, it is further preferred that the reaction temperature is controlled to be 0-5°C before the metal reducing agent is completely added, and after the metal reducing agent is completely added, the reaction is first carried out at a temperature of 0-5°C for 0.5-1.5h, and then the temperature is raised to 15-30°C for 1.5-3h; more preferably, during the reduction reaction of the metal reducing agent, HPLC is used to monitor whether the reaction is complete.

[0104] Optionally, in addition to using the above-mentioned metal element to reduce the nitro group, in step S1.3, the nitro group in the thioproduct shown in formula III-1 can also be reduced to an amino group by catalytic hydrogenation using a noble metal catalyst, and the noble metal catalyst is selected from one or more of palladium carbon, platinum carbon, palladium hydroxide carbon, and Raney nickel; preferably, the step of reducing the nitro group in the thioproduct shown in formula III-1 to an amino group by catalytic hydrogenation using a noble metal catalyst is as follows: hydrogen is introduced into a reactor containing the thioproduct shown in formula III-1, a second organic solvent, a third acidic substance, and a noble metal catalyst, and the pressure in the reactor is controlled to be 1.5~2.5Mpa, and the nitro reduction product shown in formula IV-1 is generated at a temperature of 70~80°C; in order to obtain a higher catalytic efficiency, it is more preferred that the noble metal catalyst is selected from Raney nickel, palladium carbon with a palladium mass fraction of 5%~10%, platinum carbon with a platinum mass fraction of 5%~10%, palladium hydroxide mass fraction of 1%~10%, and palladium carbon with a platinum mass fraction of 1%~10%. More preferably, the reaction time of the noble metal catalyst catalytic hydrogenation is 12 to 36 hours; more preferably, the second organic solvent is selected from methanol, ethanol, isopropanol, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, acetic acid, acetonitrile, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, dichloromethane, chloroform, 1,2-dichloroethane, 1,2-dibromoethane, and more preferably, the third acidic substance is selected from one or more of hydrochloric acid, acetic acid, trifluoroacetic acid, and p-toluenesulfonic acid, and further preferably, the third acidic substance can be added in the form of an aqueous solution to prevent volatilization or facilitate rapid dissolution and dispersion; more preferably, the molar ratio of the thio-addition product to the third acidic substance is 1: (1 to 1.5); more preferably, the mass ratio of the noble metal catalyst to the thio-product shown in formula III-1 is (0.05 to 0.3): 1.

[0105] In the splitting reaction, the ester hydrolase plays two main roles. One is to hydrolyze the ester group to generate a carboxyl group (the nucleophilic amino acid residue in the active site forms a nucleophilic addition with the carbonyl carbon atom in the ester molecule to form a transition state. In the transition state, the hydroxyl oxygen of the ester group forms a hydrogen bond with the ketone oxygen atom in the enzyme. The ketone oxygen atom in the enzyme grabs the proton of the ester carbonyl carbon, and the ester group breaks to form a carboxyl group). The other is to control the generation of a hydrolysis product of a specific configuration (i.e., thiol-protected L-penicillamine shown in Formula V-1) in the process of hydrolyzing the ester group to generate a carboxyl group. In order to obtain a higher splitting efficiency, in a preferred embodiment, the ester group is selected from one or more of acetylcholinesterase, gluconolactonase, phosphomonoester hydrolase, and serine protease; in the preferred step S1.4, the step of splitting the nitro-reduced product of formula IV-1 by ester hydrolase is as follows: in a buffer solution containing an organic reductant and an ester hydrolase, a solution of the nitro-reduced product of formula IV-1 is added dropwise, and then reacted at 35-45°C to generate the thiol-protected L-penicillamine of formula V-1; in order to obtain a higher enzyme activity , the pH of the buffer is 6.8-7.2, the organic reducing agent is selected from one of dithiothreitol and tris(2-carbonylethyl)phosphine hydrochloride or a mixture of the two, which is used to reduce and resolve the disulfide bonds in the hydrolase; the solution of the nitro-reduced product of formula IV-1 is preferably obtained by dissolving the nitro-reduced product of formula IV-1 in one or more of water, acetonitrile, methanol, ethanol, propanol, isopropanol and butanol; the buffer is preferably a potassium phosphate buffer, and the concentration of the buffer is 0.1-5 mol / L; the organic reducing agent, the ester hydrolase and the formula The mass ratio of the nitro reduction product shown in IV-1 is (0.03~0.08): (0.3~0.8): 1; preferably, the pH of the system after the splitting reaction is completed is adjusted to 1~2, stirred for 10~30 minutes and then filtered, the filter cake is washed with water, the filtrate is combined and the pH of the combined filtrate is adjusted to 6~7, the combined filtrate is extracted with isopropyl acetate multiple times to remove the remaining substrate, the aqueous phase is concentrated, and the pH of the aqueous phase is adjusted to 1~2 after concentration, and then ice-bathed and filtered to obtain the thiol-protected L-penicillamine solid shown in formula V-1.

[0106] In order to fully remove the protecting group structure, in a preferred embodiment, in step S1.5, the step of removing the thiol protecting group of the thiol-protected L-penicillamine shown in formula V-1 under strong acid conditions to generate L-penicillamine salt is as follows: adding acid solution to the thiol-protected L-penicillamine shown in formula V-1 at 15-30°C, and then stirring for 6-24h under heating conditions at 30-60°C to remove the thiol protecting group to generate L-penicillamine salt; preferably, the acid solution is selected from trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, and pure acid is used to quickly dissolve the raw material and remove the protecting group, or is selected from a second acidic substance solution; more preferably, the second acidic substance in the second acidic substance solution is selected from hydrochloric acid, hydrobromic acid, The second acidic substance solution is selected from one or more of iodine, hydroiodic acid, trifluoroacetic acid, methanesulfonic acid, and trifluoromethanesulfonic acid; the solvent in the second acidic substance solution is selected from one or more of water, acetic acid, methanol, ethanol, isopropanol, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, acetic acid, acetonitrile, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, dichloromethane, chloroform, 1,2-dichloroethane, and 1,2-dibromoethane; it is further preferred that the mass fraction of the second acidic substance in the second acidic substance solution is 10-90% so as to efficiently remove the protecting group; it is further preferred that the molar ratio of the thiol-protected L-penicillamine shown in formula V-1 to the second acidic substance is 1: (1~30).

[0107] In a preferred embodiment, in step S1.5, the alkaline hydrolysis step is as follows: the system after the reaction of removing the thiol protecting group is completed is concentrated to remove the second acidic substance, dichloromethane is added to dissolve the residue, and then an alkaline substance is added to adjust the pH to 4-5, and concentrated to obtain a crude product of L-penicillamine; preferably, the alkaline substance is selected from an inorganic base aqueous solution or an organic base, and the inorganic base is selected from one or more of NaOH, Na2CO3, KOH, and K2CO3, or the organic base is selected from one or more of TEA, DBU, DIPEA, DABCO, DBN, DMAP, pyridine, N-methylmorpholine, and tetramethylethylenediamine; preferably, the crude L-penicillamine is slurried with dichloromethane, stirred for 6-24 hours, and filtered to obtain a solid L-penicillamine.

[0108] According to another aspect of the present invention, there is also provided a method for preparing L-penicillamine, comprising the following steps:

[0109] Step S2.1, subjecting the compound represented by formula I-2 to a nitration reaction under concentrated nitric acid conditions to generate a nitration product represented by formula II-2;

[0110] Step S2.2, under an inert gas atmosphere, the nitration product of formula II-2 and the thiolation reagent R2-SH are subjected to a Michael addition reaction in a third organic solvent under the catalytic action of an alkaline catalyst to generate a thiolation product of formula III-2;

[0111] Step S2.3, reducing the nitro group in the thio product of formula III-2 to an amino group to generate a nitro-reduced product of formula IV-2;

[0112] Step S2.4, splitting the nitro-reduction product of Formula IV-2 by ester hydrolase to generate amino-protected L-penicillamine of Formula V-2;

[0113] Step S2.5, removing the amino protecting group of the amino-protected L-penicillamine shown in V-2 under strong acid conditions to generate L-penicillamine salt, and then alkaline hydrolyzing to generate L-penicillamine;

[0114]

[0115] Among them, R3 is selected from one of C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy, aryl substituted or unsubstituted by at least one substituent in halogen atom, and benzyl; R4 is selected from one of C1-C6 alkyl, C1-C6 alkoxy, benzoyl substituted or unsubstituted by at least one substituent in halogen atom, and acetyl.

[0116] In this scheme, the first step is to use commercially available enoate as the starting material, and to undergo a nitration reaction under concentrated nitric acid conditions to generate a nitration product, thereby introducing a nitro group into the raw material; the second step is to react the nitration product with a thio reagent R4-SH under an inert gas atmosphere to undergo Michael addition, thereby introducing R4S (the main structure of R4 is an acetyl group or a benzoyl group) into the raw material and generating a racemic thio product; the third step is to reduce the nitro group in the racemic thio product to an amino group to generate a racemate with a protecting group (amino group with a protecting group), and when the main structure of R4 is an acetyl group or a benzoyl group, the acetyl group or the benzoyl group migrates to the amino group, and the final product is the racemic penicillamine shown in formula IV-2; the fourth step is to split the racemate with a protecting group by an ester hydrolase; the fifth step is to remove the protecting group and perform alkaline hydrolysis to obtain the free L-penicillamine.

[0117] In a preferred embodiment, R3 is selected from one of methyl, ethyl, propyl, isopropyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, allyl, phenyl and benzyl. The raw material cost is low and it is simple and easy to obtain. In order to obtain a higher yield of L-penicillamine, R4 is preferably selected from one of methyl, methoxy, fluorine, chlorine, bromine, substituted or unsubstituted benzoyl and acetyl.

[0118] In order to obtain a higher nitration efficiency and yield, in a preferred embodiment, in step S2.1, the mass fraction of nitric acid in concentrated nitric acid is 68%~98%; preferably, the molar ratio of the compound represented by formula I-2 to the nitric acid in concentrated nitric acid is 1:(2~10), so that the compound represented by formula I-2 is fully nitrated; preferably, the compound represented by formula I-2 is added dropwise to concentrated nitric acid at 0~5°C, and after the addition is completed, stirring is continued at 0~5°C for 1~1.5h, and then the temperature is raised to room temperature and stirring is continued until the reaction is complete. The above-mentioned temperature control can prevent temperature surge and material spraying; more preferably, during the nitration reaction, TLC is used to monitor whether the reaction is complete.

[0119] In order to obtain a higher yield of the thioproduct represented by formula III-2, in a preferred embodiment, in step S2.2, the basic catalyst is selected from triethylamine, tri-n-butylamine, diisopropylethylamine, pyridine, 2,6-lutidine, dimethylaminopyridine, imidazole, N-methylpiperidine, N-methylmorpholine, 1,8-diazobispiro[5.4.0]undec-7-ene (DBU), 1,4-diazabicyclo[2.2.2]octane, D-proline, L-proline, quinine, quinidine, lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, potassium tert-butoxide, sodium tert-butoxide, One or more of lithium tert-butoxide, lithium diisopropylamide, lithium bistrimethylsilylamide, lithium bistrimethylsilylamide, potassium bistrimethylsilylamide, sodium hydroxide, potassium hydroxide, and sodium hydride; preferably, the alkaline catalyst is selected from one or more of triethylamine (TEA), D-proline, L-proline, quinine, and quinidine; preferably, the molar ratio of the nitration product represented by formula II-2, the thiolation reagent, and the alkaline catalyst is 1: (1.0-10.0): (0.05-5.0), so that the nitration product represented by the raw material formula II-2 is fully thiolated; more preferably, the nitration product represented by formula II-2 The molar ratio of the sulfide product, the thiolation reagent and the alkaline catalyst is 1: (1.0-2.0): (0.5-2); preferably, the third organic solvent is selected from one or more of methyl tert-butyl ether, anisole, ethylene glycol dimethyl ether, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, dichloromethane, chloroform, 1,2-dichloroethane, 1,2-dibromoethane, n-heptane, n-hexane, N-methylpyrrolidone, acetonitrile, ethyl acetate, tetrahydrofuran, 2-methyltetrahydrofuran and 1,4-dioxane; more preferably, the third organic solvent is selected from dichloromethane, ethyl acetate and toluene to obtain a relatively High product yield; preferably, nitrogen is introduced into the third solvent to form an inert gas atmosphere; in order to obtain a higher yield, the temperature of the Michael addition reaction is preferably 15-30°C; more preferably, the temperature of the Michael addition reaction is room temperature, and room temperature reaction is more economical; preferably, the time of the Michael addition reaction is 1-5h; preferably, during the Michael addition reaction, HPLC is used to monitor whether the reaction is complete; preferably, after the Michael addition reaction is complete, the third organic solvent in the reaction system is concentrated and dried to obtain the thio product shown in formula III-2.

[0120] In a preferred embodiment, in step S2.3, the nitro group in the thioproduct shown in formula III-2 is reduced to an amino group by a metal element reducing agent; the metal element reducing agent is zinc powder and / or iron powder, and the reduction by zinc powder and / or iron powder is efficient and low in cost, and economical and practical; preferably, the step of reducing the nitro group in the product shown in formula III-2 to an amino group by a metal element reducing agent is as follows: in a mixed system comprising the thioproduct shown in formula III-2, a first acidic substance and a first organic solvent, a metal element reducing agent is added to carry out a reduction reaction to generate a nitro reduction product shown in formula IV-2; More preferably, the molar ratio of the thioproduct represented by formula III-2, the metal element reducing agent and the first acidic substance is 1: (4-6): (10-20) to obtain a higher reduction yield; more preferably, the first acidic substance is selected from one or more of hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, methanesulfonic acid, trifluoromethanesulfonic acid, sulfuric acid, nitric acid, acetic acid, trifluoroacetic acid, ammonium chloride, trimethylchlorosilane, ferrous chloride, zinc chloride, cupric chloride, aluminum chloride, boron tribromide, and boron trichloride; further preferably, the first acidic substance is selected from one or more of hydrochloric acid, hydrobromic acid, phosphoric acid, methanesulfonic acid, sulfuric acid, nitric acid, and acetic acid. ; It is further preferred that the first acidic substance can be added in the form of an aqueous solution to prevent volatilization or facilitate rapid dissolution and dispersion; It is more preferred that the first organic solvent is selected from one or more of methanol, ethanol, isopropanol, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, acetic acid, acetonitrile, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, dichloromethane, chloroform, 1,2-dichloroethane, and 1,2-dibromoethane; It is more preferred that the metal reducing agent is added in batches; It is further preferred that before the metal reducing agent is completely added, the reaction temperature is controlled to be 0~5°C, and the metal reducing agent is After the addition of the agent is complete, the reaction is first carried out at a temperature of 0-5°C for 0.5-1.5h, and then the temperature is raised to 15-30°C for 1.5-3h; more preferably, during the reduction reaction of the metal reducing agent, the reaction is monitored by HPLC to see whether it is complete; more preferably, after the reduction reaction of the metal reducing agent is completed, the system after the reaction is completed is filtered to obtain a filtrate, the filtrate is concentrated and diluted with water, the diluted system is extracted multiple times with dichloromethane and the organic phases are combined, the combined organic phases are dried over anhydrous Na2SO4, and the organic phases are concentrated to obtain a crude nitro reduction product shown in Formula IV-2.

[0121] Optionally, in addition to using the above-mentioned metal element to reduce the nitro group, in step S2.3, the nitro group in the thioproduct shown in formula III-2 can also be reduced to an amino group by catalytic hydrogenation with a precious metal catalyst, and the precious metal catalyst is selected from one or more of palladium carbon, platinum carbon, palladium hydroxide carbon, and Raney nickel; preferably, the step of reducing the nitro group in the thioproduct shown in formula III-2 to an amino group by catalytic hydrogenation with a precious metal catalyst is as follows: hydrogen is introduced into a reactor containing the thioproduct shown in formula III-2, a second organic solvent, a third acidic substance and a precious metal catalyst, and the pressure in the reactor is controlled to be 1.5~2.5Mpa, and the nitro reduction product shown in formula IV-2 is generated at a temperature of 70~80°C; in order to obtain a higher catalytic efficiency, it is more preferred that the precious metal catalyst is selected from Raney nickel, palladium carbon with a palladium mass fraction of 5%~10%, platinum carbon with a platinum mass fraction of 5%~10%, palladium hydroxide mass fraction of 10%~20 % of palladium hydroxide on carbon; more preferably, the reaction time of the noble metal catalyst catalytic hydrogenation is 12 to 36 hours; more preferably, the second organic solvent is selected from methanol, ethanol, isopropanol, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, acetic acid, acetonitrile, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, dichloromethane, chloroform, 1,2-dichloroethane, 1,2-dibromoethane, one or more; more preferably, the third acidic substance is selected from hydrochloric acid, acetic acid, trifluoroacetic acid, p-toluenesulfonic acid, one or more; more preferably, the molar ratio of the thio addition product to the third acidic substance is 1: (1 to 1.5); more preferably, the mass ratio of the noble metal catalyst to the thio product shown in formula III-2 is (0.05 to 0.3): 1; more preferably, the system after the reaction of the noble metal catalyst catalytic hydrogenation is completed is cooled, filtered after cooling, and the filtrate is concentrated to obtain the crude nitro reduction product shown in formula IV-2.

[0122] In order to obtain a higher resolution efficiency, in a preferred embodiment, in step S2.4, the ester hydrolase is selected from one or more of acetylcholinesterase, gluconolactonase, phosphomonoester hydrolase, and serine protease; preferably, in step S2.4, the step of resolving the nitro-reduced product shown in formula IV-2 by ester hydrolase is as follows: in a buffer solution containing an organic reducing agent and an ester hydrolase, a solution of the nitro-reduced product shown in formula IV-2 is added dropwise, and then reacted at 35-45°C to generate amino-protected L-penicillamine shown in formula V-2; in order to obtain a higher enzyme activity, the pH of the buffer solution is 6.8-7.2, and the organic reducing agent is selected from one of dithiothreitol and tris(2-carbonylethyl)phosphine hydrochloride or a mixture of the two, which is used to reduce the disulfide bonds of the hydrolase to improve the efficiency of enzyme recognition and resolution; preferably, the nitro-reduced product shown in formula IV-2 is added dropwise to a buffer solution containing an organic reducing agent and an ester hydrolase. The solution of the nitro reduction product is obtained by dissolving the nitro reduction product shown in formula IV-2 in one or more of water, acetonitrile, methanol, ethanol, propanol, isopropanol, and butanol; the preferred buffer is potassium phosphate buffer, and the concentration of the buffer is 0.1~5 mol / L; the preferred mass ratio of the organic reducing agent, the ester hydrolase and the nitro reduction product shown in formula IV-2 is (0.03~0.08): (0.3~0.8): 1; the pH of the system after the splitting reaction is preferably adjusted to 1~2, stirred for 10~30 minutes and then filtered, the filter cake is washed with water, the filtrate is combined and the pH of the combined filtrate is adjusted to 6~7, the combined filtrate is extracted with isopropyl acetate for multiple times to remove the remaining substrate, the aqueous phase is concentrated, and the pH of the aqueous phase is adjusted to 1~2 after concentration, and then ice-bathed and filtered to obtain the amino-protected L-penicillamine solid shown in formula V-2.

[0123] In order to fully remove the protecting group structure, in a preferred embodiment, in step S2.5, the step of removing the amino protecting group of the amino-protected L-penicillamine shown in formula V-2 under strong acid conditions to generate L-penicillamine salt is as follows: at 15-30°C, add acid solution to the amino-protected L-penicillamine shown in formula V-2, and then stir under heating conditions at 30-60°C for 6-24h to remove the amino protecting group to generate L-penicillamine salt; preferably, the acid solution is selected from trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, and pure acid is used to quickly dissolve the raw material and remove the protecting group, or is selected from a second acidic substance solution; more preferably, the second acidic substance in the second acidic substance solution is selected from hydrochloric acid, hydrobromic acid, The solvent in the second acidic substance solution is selected from one or more of water, acetic acid, methanol, ethanol, isopropanol, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, acetic acid, acetonitrile, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, dichloromethane, chloroform, 1,2-dichloroethane, 1,2-dibromoethane; it is further preferred that the mass fraction of the second acidic substance in the second acidic substance solution is 10-90%, so as to efficiently remove the protecting group; it is further preferred that the molar ratio of the amino-protected L-penicillamine shown in formula V-2 to the second acidic substance is 1: (1~30).

[0124] In a preferred embodiment, in step S2.5, the alkaline hydrolysis step is as follows: the system after the removal of the amino protecting group is completed is concentrated to remove the second acidic substance, dichloromethane is added to dissolve the residue, and then an alkaline substance is added to adjust the pH to 4-5, and concentrated to obtain a crude product of L-penicillamine; preferably, the alkaline substance is selected from an inorganic alkali aqueous solution or an organic base, and the inorganic base is selected from one or more of NaOH, Na2CO3, KOH, and K2CO3, or the organic base is selected from one or more of TEA, DBU, DIPEA, DABCO, DBN, DMAP, pyridine, N-methylmorpholine, and tetramethylethylenediamine; preferably, the crude L-penicillamine is slurried with dichloromethane, stirred for 6-24 hours, and filtered to obtain a solid L-penicillamine.

[0125] According to another aspect of the present invention, there is also provided a method for preparing L-penicillamine, comprising the following steps:

[0126] Step S4.1, subjecting the compound represented by formula I-4 to a nitration reaction under concentrated nitric acid conditions to generate a nitration product represented by formula II-4;

[0127] Step S4.2, under an inert gas atmosphere, the nitration product of formula II-4 and the thiolation reagent R2-SH are subjected to Michael addition reaction in a third organic solvent under the catalytic action of an alkaline catalyst to generate the thiolation product of formula III-4;

[0128] Step S4.3, reducing the nitro group in the thio product of formula III-4 to an amino group to generate a nitro-reduced product of formula IV-4;

[0129] Step S4.4, splitting the nitro-reduction product of formula IV-4 by acyl hydrolase to generate L-penicillamine;

[0130]

[0131] Wherein, R6 is selected from one of C1-C6 alkyl, C1-C6 alkoxy, benzoyl and acetyl which are substituted or unsubstituted with at least one substituent in the halogen atom.

[0132] In this scheme, the first step is to use commercially available enoic acid as the starting material, and to undergo a nitration reaction under concentrated nitric acid conditions to generate a nitration product, thereby introducing a nitro group into the raw material; the second step is to subject the nitration product to Michael addition with a thio reagent R6-SH with different substituents under an inert gas atmosphere, thereby introducing R6S (the main structure of R6 is acetyl or benzoyl) into the raw material and generating a racemic thio product; the third step is to reduce the nitro group in the racemic thio product to an amino group to generate a racemic penicillamine with a protecting group (amino with a protecting group), and when the main structure of R6 is an acetyl group or a benzoyl group, the acetyl group or the benzoyl group migrates to the amino group, and the final product is the racemic penicillamine shown in formula IV-4; the fourth step is to directly split the racemic form with a protected group by an acyl hydrolase to obtain L-penicillamine.

[0133] In a preferred embodiment, in order to obtain a higher yield of L-penicillamine, it is more preferred that R6 is selected from methyl, methoxy, fluorine, chlorine, bromine, substituted or unsubstituted benzoyl, acetyl.

[0134] In order to obtain a higher nitration efficiency and yield, in a preferred embodiment, in step S4.1, the mass fraction of nitric acid in concentrated nitric acid is 68%~98%; preferably, the molar ratio of the compound represented by formula I-1 to the nitric acid in concentrated nitric acid is 1:(2~10) to fully nitrate the raw material; preferably, the compound represented by formula I-4 is added dropwise to the concentrated nitric acid at 0~5°C, and after the addition is completed, stirring is continued at 0~5°C for 1~1.5h, and then the temperature is raised to room temperature and stirring is continued until the reaction is complete. The above-mentioned temperature control can prevent temperature surge and material spraying; in order to obtain a nitration product of higher purity, it is more preferred to monitor whether the reaction is complete by TLC during the nitration reaction.

[0135] In order to obtain a higher yield of thioproducts, in a preferred embodiment, in step S4.2, the basic catalyst is selected from triethylamine, tri-n-butylamine, diisopropylethylamine, pyridine, 2,6-lutidine, dimethylaminopyridine, imidazole, N-methylpiperidine, N-methylmorpholine, 1,8-diazobispiro[5.4.0]undec-7-ene (DBU), 1,4-diazabicyclo[2.2.2]octane, D-proline, L-proline, quinine, quinidine, lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, potassium tert-butoxide, sodium tert-butoxide, lithium tert-butoxide , lithium diisopropylamide, lithium bistrimethylsilylamide, lithium bistrimethylsilylamide, potassium bistrimethylsilylamide, sodium hydroxide, potassium hydroxide, sodium hydride or one or more thereof; preferably, the alkaline catalyst is selected from one or more of triethylamine (TEA), D-proline, L-proline, quinine, and quinidine; preferably, the molar ratio of the nitration product shown in formula II-4, the thiolation agent and the alkaline catalyst is 1: (1.0 to 10.0): (0.05 to 5.0) so that the nitration product is fully thiolated; more preferably, the nitration product shown in formula II-4, the thiolation agent and the base catalyst are The molar ratio of the active catalyst is 1: (1.0-2.0): (0.5-2); preferably, the third organic solvent is selected from one or more of methyl tert-butyl ether, anisole, ethylene glycol dimethyl ether, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, dichloromethane, chloroform, 1,2-dichloroethane, 1,2-dibromoethane, n-heptane, n-hexane, N-methylpyrrolidone, acetonitrile, ethyl acetate, tetrahydrofuran, 2-methyltetrahydrofuran, and 1,4-dioxane; more preferably, the third organic solvent is selected from dichloromethane, ethyl acetate, and toluene to obtain a higher product yield. ; Preferably, nitrogen is introduced into the third solvent to form an inert gas atmosphere; in order to obtain a higher yield, the temperature of the Michael addition reaction is preferably 15~30°C; more preferably, the temperature of the Michael addition reaction is room temperature, which is more economical; preferably, the time of the Michael addition reaction is 1~5h; preferably, during the Michael addition reaction, HPLC is used to monitor whether the reaction is complete; preferably, after the Michael addition reaction is complete, the third organic solvent in the reaction system is concentrated and dried to obtain the thio product shown in formula III-4.

[0136] In a preferred embodiment, in step S4.3, the nitro group in the thioproduct shown in formula III-4 is reduced to an amino group by a metal element reducing agent; the metal element reducing agent is zinc powder and / or iron powder, and the reduction is carried out by zinc powder and / or iron powder, which is efficient and low in cost; preferably, the step of reducing the nitro group in the product shown in formula III-4 to an amino group by a metal element reducing agent is as follows: in a mixed system comprising the thioproduct shown in formula III-4, a first acidic substance and a first organic solvent, a metal element reducing agent is added to carry out a reduction reaction to generate a nitro reduction product shown in formula IV-4; more preferably, the molar ratio of the thioproduct shown in formula III-4, the metal element reducing agent and the first acidic substance is 1: (4~6): (10~20) to obtain a higher reduction yield; more preferably, the first acidic substance is selected from hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, methanesulfonic acid, trifluoromethanesulfonic acid, sulfuric acid, nitric acid, acetic acid, trifluoroacetic acid, ammonium chloride, trimethylsilyl chloride, ferrous chloride, zinc chloride, cupric chloride, aluminum chloride, tribromide boron, boron trichloride; further preferably, the first acidic substance is selected from one or more of hydrochloric acid, hydrobromic acid, phosphoric acid, methanesulfonic acid, sulfuric acid, nitric acid, and acetic acid; further preferably, the first acidic substance can be added in the form of an aqueous solution to prevent volatilization or facilitate rapid dissolution and dispersion; more preferably, the first organic solvent is selected from one or more of methanol, ethanol, isopropanol, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, acetic acid, acetonitrile, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, dichloromethane, chloroform, 1,2-dichloroethane, and 1,2-dibromoethane; more preferably, the metal reducing agent is added in batches; further preferably, before the metal reducing agent is completely added, the reaction temperature is controlled to 0-5°C, and after the metal reducing agent is completely added, the reaction is first reacted at a temperature of 0-5°C for 0.5-1.5h, and then the temperature is raised to 15-30°C for 1.5-3h; more preferably, during the reduction reaction of the metal reducing agent, HPLC is used to monitor whether the reaction is complete.

[0137] Optionally, in addition to using the above-mentioned metal element to reduce the nitro group, in a preferred embodiment, in step S4.3, the nitro group in the thio product shown in formula III-4 is reduced to an amino group by catalytic hydrogenation with a noble metal catalyst, and the noble metal catalyst is selected from one or more of palladium carbon, platinum carbon, palladium hydroxide carbon, and Raney nickel; preferably, the step of reducing the nitro group in the thio product shown in formula III-4 to an amino group by catalytic hydrogenation with a noble metal catalyst is as follows: hydrogen is introduced into a reactor containing the thio product shown in formula III-4, a second organic solvent, a third acidic substance and a noble metal catalyst, and the pressure in the reactor is controlled to be 1.5~2.5Mpa, and the nitro reduction product shown in formula IV-4 is generated at a temperature of 70~80°C; in order to obtain a higher catalytic efficiency, it is more preferred that the noble metal catalyst is selected from Raney nickel, palladium carbon with a palladium mass fraction of 5%~10%, platinum carbon with a platinum mass fraction of 5%~10%, and hydrogen peroxide. More preferably, the second organic solvent is selected from one or more of methanol, ethanol, isopropanol, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, acetic acid, acetonitrile, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, dichloromethane, chloroform, 1,2-dichloroethane, 1,2-dibromoethane; more preferably, the third acidic substance is selected from one or more of hydrochloric acid, acetic acid, trifluoroacetic acid, and p-toluenesulfonic acid; further preferably, the first acidic substance can be added in the form of an aqueous solution to prevent volatilization or facilitate rapid dissolution and dispersion; more preferably, the molar ratio of the thio-addition product to the third acidic substance is 1: (1-1.5); more preferably, the mass ratio of the noble metal catalyst to the thio-product shown in formula III-4 is (0.05-0.3): 1.

[0138] In the resolution reaction, in order to obtain a higher resolution efficiency, in a preferred embodiment, in step S4.4, the acyl hydrolase is selected from one or more of acylcarnitine hydrolase, acetyl-CoA hydrolase, and acyl-CoA hydrolase; preferably in step S4.4, the step of resolving the nitro-reduction product shown in formula IV-4 by acyl hydrolase is as follows: in a buffer solution containing an organic reducing agent and an acyl hydrolase, a solution of the nitro-reduction product shown in formula IV-4 is added dropwise, and then reacted at 35 to 45°C to generate L-penicillamine; the pH of the buffer solution is 6.8 to 7.2, and the organic reducing agent is selected from one of dithiothreitol and tris(2-carbonylethyl)phosphine hydrochloride or a mixture of the two, which is used to reduce the disulfide bonds of the hydrolase to improve the efficiency of enzyme recognition and resolution; preferably the nitro-reduction product shown in formula IV-4 is The solution of the original product is obtained by dissolving the nitro reduction product shown in formula IV-4 in one or more of water, acetonitrile, methanol, ethanol, propanol, isopropanol, and butanol; the preferred buffer is potassium phosphate buffer, and the concentration of the buffer is 0.1~5 mol / L; the preferred mass ratio of the organic reducing agent, the acyl hydrolase and the nitro reduction product shown in formula IV-4 is (0.03~0.08): (0.3~0.8): 1; the pH of the system after the splitting reaction is preferably adjusted to 1~2, stirred for 10~30 minutes and then filtered, the filter cake is washed with water, the filtrate is combined and the pH of the combined filtrate is adjusted to 6~7, the combined filtrate is extracted with isopropyl acetate multiple times to remove the remaining substrate, the aqueous phase is concentrated, and the pH of the aqueous phase is adjusted to 1~2 after concentration, and then ice-bathed and filtered to obtain L-penicillamine.

[0139] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0140] Example 1

[0141]

[0142] Step 1: Add pure water (20 mL) to a 500 mL four-necked reaction bottle, cool to 0°C, and under stirring, add 90% fuming nitric acid (85.0 mL, 5.1 eq) by mass into the reaction bottle through a constant pressure titration funnel and control the temperature at 0~5°C. After the addition is complete, slowly add the enoate (50.1 g, 54.3 mL, 1.0 eq) shown in formula I-1-1 into the system through another constant pressure titration funnel and control the temperature at 0~5°C. After the addition is complete, keep stirring at 0°C for 1.25 hours, and then warm to room temperature and continue stirring after 1.25 hours. When TLC monitoring shows that the raw materials in the mixed system have been completely converted, stop stirring. ; Pour the mixed system into 500ml ice water to quench, extract with 500mL dichloromethane after quenching and extract four times continuously under the same conditions, combine the organic phases, then wash the organic phase with 300mL purified water and wash twice continuously under the same conditions, then wash the organic phase with 300mL saturated sodium bicarbonate solution and wash four times continuously under the same conditions, then wash the organic phase with 300mL saturated brine and wash twice continuously under the same conditions, then dry the organic phase with anhydrous Na2SO4, take the organic phase after filtering and concentrate the organic phase to obtain a light yellow liquid nitration product shown in formula II-1-1, and the yield of the nitration product shown in formula II-1-1 is 91%. Structural characterization data of the product: 1 H NMR (400MHz, CDCl3) δ 4.26 (q, J = 7.1 Hz, 2H), 2.24 (s, 3H), 1.96 (s, 3H), 1.28 (s, 3H).

[0143] Step 2: Add 400 mL of dichloromethane to a 1L reaction bottle, blow nitrogen into the reaction bottle to deoxygenate, blow until the dichloromethane remains to about 364 mL, add the nitration product (36.7 g, 1.0 eq) and triethylamine (TEA, 60.0 mL, 2.0 eq) of formula II-1-1 obtained in step 1 to the reaction bottle, stir at room temperature, add the thiolation reagent p-methoxythiophenol (31.0 mL, 1.05 eq) dropwise to the reaction system, continue stirring at room temperature for 3.0 h, HPLC monitors the complete conversion of the raw materials, stop stirring, directly concentrate and dry the solvent in the reaction system to obtain the crude thiolation product of formula III-1-1, which is a dark brown oil with a yield of 93%. Structural characterization data of the product: 1H NMR (400MHz, CDCl3) δ 7.22 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 5.25 (s, 1H), 3.82 (dd, J = 13.6, 2.0 Hz, 1H), 3.78 (s,2H), 1.62 (d, J = 6.1 Hz, 6H), 1.32 (t, J = 7.1 Hz, 3H).

[0144] Step 3: Reduce the nitro group in the thioproduct represented by formula III-1-1 to an amino group by zinc powder. The specific steps are as follows: add the thioproduct represented by formula III-1-1 (5.0 g, 1.0 eq) and ethanol (50 mL) obtained in step 2 to a 100 mL reaction bottle, control the temperature at 0-5 ° C and stir, add concentrated hydrochloric acid (37%, 20.0 mL, 15.7 eq) dropwise to the reaction system at 0-5 ° C, keep the temperature at 0-5 ° C and continue stirring, add zinc powder (5.0 g, 1.0 eq) to the reaction system in four batches at 0 ° C and stirring. , 5.0eq), after the zinc powder is added, keep stirring at 0~5℃ for 1.0h, then warm to room temperature and continue stirring for 2.0h. HPLC monitors that the raw material has been completely converted and stops stirring; filter the excess zinc powder, concentrate the system, and after the ethanol is dried, add 50mL of pure water to dilute the concentrated hydrochloric acid system, then extract with 50mL of dichloromethane and extract twice under the same conditions, combine the organic phases, and then dry with Na2SO4, concentrate the organic phases to obtain the crude nitro reduction product, that is, the crude nitro reduction product shown in formula IV-1-1, with a yield of 88%. Structural characterization data of the product: 1 H NMR (400MHz, DMSO-d6) δ 7.22 (d, J = 8.6 Hz, 2H), 6.85 (d, J =8.6 Hz, 2H), 4.11 (qd, J = 7.1, 1.8 Hz, 2H), 3.80 – 3.70 (m, 2H), 3.72 (s,3H), 3.38 (s, 1H), 1.30 (s, 3H), 1.29 (s, 3H), 1.22 (t, J = 7.1 Hz, 3H).

[0145] Step 4: Add KPB (54mL, 0.3M), DTT (0.1g) and serine protease (1.0g) to a 250mL conical flask and mix well. Dissolve the crude nitro-reduction product (2.0g) of formula IV-1-1 obtained in step 3 in acetonitrile (3mL) and slowly add it to the system. Then place the system on a 40°C shaker for 16h. Adjust the pH of the system to 1~2 after the reaction, stir for 20min and filter, wash the filter cake with 10mL pure water, combine the filtrates and adjust the pH of the combined filtrates to 6~7, extract with 40mL isopropyl acetate and extract three times under the same conditions to remove the remaining substrate. After the aqueous phase is concentrated to 10mL, adjust the pH to 1~2 and crystallize in an ice bath for 1.0h. Filter to obtain 1.2g of light yellow solid thiol-protected L-penicillamine of formula V-1-1, with an ee value of 98.7% and a yield of 36%. Structural characterization data of the product: 1 H NMR (400MHz, DMSO-d6) δ 10.41 (s, 1H), 8.36 (s,3H), 7.24 (d, J = 8.5 Hz, 2H), 6.81 (d, J = 8.6 Hz, 2H), 3.90 (s, 1H), 3.82 –3.69 (m, 3H), 1.47 (s, 3H), 1.31 (s, 3H).

[0146] Step 5: Add the thiol-protected L-penicillamine (1.0 g) and trifluoroacetic acid (5.0 mL) shown in formula V obtained in step 4 to a 20 mL reaction bottle, stir at room temperature, add trifluoromethanesulfonic acid (0.32 mL) dropwise to the reaction system at room temperature, heat to 40 ° C and stir overnight after the addition is complete; after the reaction is completed, concentrate to remove trifluoroacetic acid and trifluoromethanesulfonic acid, add dichloromethane to dissolve the residue, add TEA to adjust the pH to 4-5, concentrate to obtain a crude product, slurry the crude product with 5 mL of dichloromethane, stir overnight, filter and discard the filtrate to obtain L-penicillamine, with a yield of 70%. Structural characterization data of the product: 1 H NMR (400MHz, DMSO-d6) δ 13.89 (s, 1H), 8.34 (s, 3H), 3.93 (s, 1H), 3.08 (s, 1H), 1.48 (s,3H), 1.42 (s, 3H).

[0147] Embodiment 2-5

[0148] The difference from Example 1 is that the type and amount of the alkaline catalyst and the type of the third organic solvent in step 2 are different, as shown in Table 1 for details.

[0149] Table 1. Type and amount of alkaline catalyst, type of third organic solvent and yield of thioproduct in Examples 2-5

[0150]

[0151] Example 6

[0152]

[0153] Step 1: The compound represented by formula I-1-1 is subjected to nitration reaction under concentrated nitric acid conditions to generate a nitration product represented by formula II-1-1. The specific steps are the same as step 1 in Example 1.

[0154] Step 2: Add the nitration product (50.00 g, 288.74 mmol, 1.0 eq), thioacetic acid (109.89 g, 1.44 mol, 5.0 eq) and toluene (150 mL) obtained in step 1 to a 500 mL four-necked bottle, cool to 0 ° C, and then add TEA (58.3 g, 577.48 mmol, 2.0 eq) in batches. After the addition is completed, the reaction system is warmed to room temperature and stirred for 4.0 hours. After HPLC monitoring shows that the raw material is basically completely converted, wash it twice with a 2% HCl aqueous solution, extract it twice with toluene after washing, combine the organic phases, wash it three times with a saturated NaHCO3 aqueous solution, and then wash it once with a saturated NaCl aqueous solution. The organic phase is dried and concentrated to obtain the thio product shown in the oily formula III-1-2, with a yield of 63%. Structural characterization data of the product: 1 H NMR(400 MHz, CDCl3) δ 6.16 (s, 1H), 4.28 (q, J = 7.2 Hz, 2H), 2.27 (s, 3H), 1.74(s, 3H), 1.66 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H).

[0155] Step 3: Reducing the nitro group in the thio product shown in formula III-2-1 to an amino group by zinc powder to obtain a nitro reduction product shown in formula IV-2-1. The specific steps are the same as step 3 in Example 1; wherein the structural characterization data of product IV-2-1 are: 1H NMR (400 MHz, CDCl3) δ 6.42 (d, J = 9.4 Hz, 1H), 4.61 (d, J = 9.4 Hz, 1H), 4.29 – 4.06 (m, 2H), 2.04 (s, 3H), 1.96 (d, J = 1.3 Hz, 1H), 1.48 (s,3H), 1.33 (d, J = 1.4 Hz, 3H), 1.27 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 170.6, 170.0, 61.5, 60.4, 46.7, 31.2, 29.6, 23.3, 14.3.

[0156] Step 4: The nitro-reduction product of formula IV-2-1 is split by ester hydrolase to obtain amino-protected L-penicillamine of formula V-2-1. The specific steps are the same as step 4 in Example 1.

[0157] Step 5: Remove the amino protecting group from the thiol-protected L-penicillamine shown in formula V-2-1 under strong acid conditions to generate L-penicillamine salt, and perform alkaline hydrolysis to generate L-penicillamine. The specific steps are the same as step 5 in Example 1.

[0158] Embodiment 7-8

[0159] The difference from Example 6 is that the type and amount of the alkaline catalyst in step 2 and the type of the third organic solvent are different, as shown in Table 2.

[0160] Table 2. Type and amount of alkaline catalyst, type of third organic solvent and yield of thioproduct in Examples 7-8

[0161]

[0162] Examples 9-13

[0163] The difference from Example 1 is that the type of reducing agent, the first acidic substance and its amount, and the type of the first organic solvent in step 3 are different, as shown in Table 3 for details.

[0164] Table 3. Type of reducing agent, acidic substance and its dosage, type of first organic solvent and yield of nitro reduction product in Examples 9-13

[0165]

[0166] Embodiment 14

[0167] The only difference from Example 1 is that palladium-carbon catalyst is used in step 3 to catalyze hydrogenation and reduce the nitro group. The specific steps of step 3 are as follows:

[0168] To a 250mL reactor, add the thio product (5.3g, 30.6mmol, 1.0eq) of formula III-1-1 and methanol (53mL). After the thio product (5.3g, 30.6mmol, 1.0eq) of formula III-1-1 is dissolved, add p-toluenesulfonic acid monohydrate (2.9g, 1.05eq) and palladium carbon (1.1g) with a palladium mass fraction of 5%. Replace with hydrogen three times and pressurize to 2.0Mpa, then heat to 75°C, react for 24h, cool down, filter out palladium carbon, add 25mL methanol to wash the filter cake, and concentrate the filtrate to obtain the crude nitro reduction product of formula IV-1-1 with a yield of 84%.

[0169] Embodiment 15

[0170] The only difference from Example 1 is that step 3 uses a palladium-carbon catalyst to catalyze hydrogenation to reduce the nitro group. The specific steps of step 3 are as follows:

[0171] To a 250mL reactor, add the thio product (5.3g, 30.6mmol, 1.0eq) of formula III-1-1 and methanol (53mL). After the thio product (36.7mmol, 1.2eq) of formula III-1-1 is dissolved, add 3.0ml (36.7mmol, 1.2eq) of 37% aqueous hydrochloric acid solution and 1.1g of palladium carbon with a palladium mass fraction of 5%. Replace with hydrogen three times and pressurize to 2.0Mpa, then raise the temperature to 75°C, react for 24h, cool down and filter out palladium carbon, add 25mL of methanol to wash the filter cake, and concentrate the filtrate to obtain the crude nitro reduction product of formula IV-1-1 with a yield of 70%.

[0172] Example 16

[0173] To a 250mL reactor, add the thio product (5.3g, 30.6mmol, 1.0eq) of formula III-1-1 and methanol (53mL). After the thio product (36.7mmol, 1.2eq) of formula III-1-1 is dissolved, add acetic acid 2.1ml (36.7mmol, 1.2eq) and palladium carbon (1.1g) with a palladium mass fraction of 5%. Replace with hydrogen three times and pressurize to 2.0Mpa, then heat to 75°C, react for 24h, cool and filter out palladium carbon, add 25mL methanol to wash the filter cake, and concentrate the filtrate to obtain the crude nitro reduction product of formula IV-1-1 with a yield of 39%.

[0174] Embodiment 17

[0175]

[0176] Step 1: Add pure water (20 mL) to a 500 mL four-necked reaction bottle, cool to 0°C, and under stirring, add 90% fuming nitric acid (85.0 mL, 5.1 eq) to the reaction bottle through a constant pressure titration funnel and control the temperature to 0~5°C. After the addition is complete, slowly add the olefinic acid (35.0 g, 1.0 eq) shown in formula I-4-1 to the system through another constant pressure titration funnel and control the temperature to 0~5°C. After the addition is complete, keep stirring at 0°C for 1.25 hours, and then warm to room temperature after 1.25 hours and continue stirring. When TLC After monitoring shows that the raw materials in the mixed system have been completely converted, stop stirring; pour the mixed system into 500ml of ice water to quench, extract with 500mL of dichloromethane after quenching and extract four times continuously under the same conditions, combine the organic phases, and then wash the organic phase with 300mL of purified water and wash it twice continuously under the same conditions, then wash the organic phase with 300mL of saturated brine and wash it twice continuously under the same conditions, then dry the organic phase with anhydrous Na2SO4, take the organic phase after filtration and concentrate the organic phase to obtain the nitration product shown in formula II-4-1, with a yield of 63.1%.

[0177] Step 2: Add 400 mL of dichloromethane to a 1L reaction flask, blow nitrogen into the reaction flask to deoxygenate, blow until the remaining dichloromethane is about 364 mL, add the nitration product (31.3 g, 0.22 mol, 1.0 eq) and triethylamine (TEA, 60.0 mL, 2.0 eq) shown in formula II-4-1 obtained in step 1 to the reaction flask, stir at room temperature, add the thiolation reagent thioacetic acid (1.05 eq) dropwise to the reaction system, continue stirring at room temperature for 3.0 hours, monitor the complete conversion of the raw material by HPLC, stop stirring, directly concentrate and dry the solvent in the reaction system to obtain the crude thiolation product shown in formula III-4-1, with a yield of 75.8%.

[0178] Step 3: Reduce the nitro group in the thioproduct to an amino group by zinc powder. The specific steps are as follows: add the thioproduct (5.0 g, 1.0 eq) and ethanol (50 mL) shown in formula III-4-1 obtained in step 2 to a 100 mL reaction bottle, control the temperature at 0-5 °C and stir, add concentrated hydrochloric acid (37%, 20.0 mL, 10.6 eq) dropwise to the reaction system at 0-5 °C, keep the temperature at 0-5 °C and continue stirring, add zinc powder (7.3 g, 1.0 eq) to the reaction system in four batches at 0 °C and stirring. , 5.0eq), after the zinc powder is added, keep stirring at 0~5℃ for 1.0h, then warm to room temperature and continue stirring for 2.0h. HPLC monitors that the raw material has been completely converted and stops stirring; filter the excess zinc powder, concentrate the system, and after the ethanol is dried, add 50mL of pure water to dilute the concentrated hydrochloric acid system, then extract with 50mL of dichloromethane and extract twice under the same conditions, combine the organic phases, and then dry with Na2SO4, concentrate the organic phase to obtain the crude nitro reduction product shown in formula IV-4-1, with a yield of 42%. Structural characterization data of the product: 1 H NMR (400 MHz, CDCl3) δ6.42 (d, J = 9.4 Hz, 1H), 4.61 (d, J = 9.4 Hz, 1H), 2.04 (s, 3H), 1.96 (d, J= 1.3 Hz, 1H), 1.48 (s, 3H), 1.33 (d, J = 1.4 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 170.6, 170.0, 61.5, 46.7, 31.2, 29.6, 23.3.

[0179] Step 4: Add KPB (54 mL, 0.3 M), DTT (0.1 g) and acyl-CoA hydrolase (1.0 g) to a 250 mL conical flask and mix well. Dissolve the crude nitro-reduction product (2.0 g) of formula IV-4-1 obtained in step 3 in acetonitrile (3 mL) and slowly add it dropwise to the system. Then place the system on a shaker at 40 °C to react for 16 h. Adjust the pH of the system after the reaction to 1-2, stir for 20 min and filter, wash the filter cake with 10 mL of pure water, combine the filtrates and adjust the pH of the combined filtrates to 6-7, extract with 40 mL of isopropyl acetate and extract three times under the same conditions to remove the remaining substrate. After the aqueous phase is concentrated to 10 mL, adjust the pH to 1-2 and crystallize in an ice bath for 1.0 h. Filter and discard the filtrate to obtain L-penicillamine with a yield of 20.5%.

[0180] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing L-penicillamine, characterized in that: The steps include: Step S2.1, subjecting the compound represented by formula I-2 to a nitration reaction under concentrated nitric acid conditions to generate a nitration product represented by formula II-2; Step S2.2, under an inert gas atmosphere, the nitration product of formula II-2 and the thiolation reagent R4-SH are subjected to a Michael addition reaction in a third organic solvent under the catalytic action of an alkaline catalyst to generate a thiolation product of formula III-2; Step S2.3, reducing the nitro group in the thio product represented by formula III-2 to an amino group to generate a nitro-reduced product represented by formula IV-2; Step S2.4, splitting the nitro-reduced product of Formula IV-2 by ester hydrolase to generate amino-protected L-penicillamine of Formula V-2; Step S2.5, removing the amino protecting group of the amino-protected L-penicillamine shown in V-2 under strong acid conditions to generate L-penicillamine salt, and alkaline hydrolysis to generate L-penicillamine; ； Wherein, R3 is selected from one of C1-C6 alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, C1-C6 alkyl, C1-C6 alkoxy, aryl substituted or unsubstituted with at least one substituent in the halogen atom, and benzyl; R4 is selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, benzoyl or acetyl substituted or unsubstituted with at least one substituent in the halogen atom; In the step S2.2, the alkaline catalyst is selected from one or more of triethylamine, tri-n-butylamine, diisopropylethylamine, pyridine, 2,6-lutidine, dimethylaminopyridine, imidazole, N-methylpiperidine, N-methylmorpholine, 1,8-diazobispiro[5.4.0]undec-7-ene, 1,4-diazabicyclo[2.2.2]octane, quinine, quinidine, lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, potassium tert-butoxide, sodium tert-butoxide, lithium diisopropylamide, lithium bistrimethylsilylamide, potassium bistrimethylsilylamide, sodium hydroxide, potassium hydroxide, and sodium hydride; In the step S2.2, the molar ratio of the nitration product represented by formula II-2, the thiolation reagent and the alkaline catalyst is 1: (1.0-10.0): (0.05-5.0); In step S2.2, the third organic solvent is selected from one or more of methyl tert-butyl ether, anisole, ethylene glycol dimethyl ether, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, dichloromethane, chloroform, 1,2-dichloroethane, 1,2-dibromoethane, n-heptane, n-hexane, N-methylpyrrolidone, acetonitrile, ethyl acetate, tetrahydrofuran, 2-methyltetrahydrofuran, and 1,4-dioxane.

2. The method for preparing L-penicillamine according to claim 1, characterized in that: The R3 is selected from one of methyl, ethyl, propyl, tert-butyl, phenyl and benzyl; and / or, The R4 is selected from methyl, methoxy, fluorine, chlorine, bromine, substituted or unsubstituted benzoyl, acetyl.

3. The method for preparing L-penicillamine according to claim 1, characterized in that: In the step S2.1, the mass fraction of nitric acid in the concentrated nitric acid is 68% to 98%; and / or, In the step S2.1, the molar ratio of the compound represented by formula I-2 to the nitric acid in the concentrated nitric acid is 1:(2-10); and / or, In the step S2.1, the compound of formula I-2 is added dropwise to the concentrated nitric acid at 0-5°C, and after the addition is completed, the mixture is stirred at 0-5°C for 1-1.5 hours, and then the mixture is heated to room temperature and stirred until the reaction is complete; and / or, In the step S2.2, nitrogen is introduced into the third organic solvent to form an inert gas atmosphere; and / or, In step S2.2, the temperature of the Michael addition reaction is 15-30° C.; and / or, In step S2.2, the Michael addition reaction time is 1 to 5 hours; and / or, In the step S2.2, during the Michael addition reaction, HPLC is used to monitor whether the reaction is complete.

4. The method for preparing L-penicillamine according to claim 1, characterized in that: In the step S2.3, the nitro group in the thioproduct represented by the formula III-2 is reduced to an amino group by a metal reducing agent; the metal reducing agent is zinc powder and / or iron powder.

5. The method for preparing L-penicillamine according to claim 4, characterized in that: In the step S2.3, the step of reducing the nitro group in the product shown in formula III-2 to an amino group by a metal element reducing agent is as follows: a metal element reducing agent is added to a mixed system comprising a thio product shown in formula III-2, a first acidic substance and a first organic solvent to carry out a reduction reaction to generate a nitro reduction product shown in formula IV-2.

6. The method for preparing L-penicillamine according to claim 5, characterized in that: In the step S2.3, the molar ratio of the thioproduct represented by formula III-2, the metal reducing agent and the first acidic substance is 1: (4-6): (10-20); and / or, In step S2.3, the first acidic substance is selected from one or more of hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, methanesulfonic acid, trifluoromethanesulfonic acid, sulfuric acid, nitric acid, acetic acid, trifluoroacetic acid, ammonium chloride, trimethylchlorosilane, ferrous chloride, zinc chloride, cupric chloride, aluminum chloride, boron tribromide, and boron trichloride; and / or, In step S2.3, the first organic solvent is selected from one or more of methanol, ethanol, isopropanol, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, acetic acid, acetonitrile, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, dichloromethane, chloroform, 1,2-dichloroethane, and 1,2-dibromoethane; and / or, In the step S2.3, the metal reducing agent is added in batches; and / or, In the step S2.3, during the reduction reaction of the metal reducing agent, HPLC is used to monitor whether the reaction is complete.

7. The method for preparing L-penicillamine according to claim 1, characterized in that: In the step S2.3, the nitro group in the thio product represented by formula III-2 is reduced to an amino group by catalytic hydrogenation using a precious metal catalyst, wherein the precious metal catalyst is selected from one or more of palladium carbon, platinum carbon, palladium hydroxide carbon, and Raney nickel.

8. The method for preparing L-penicillamine according to claim 1, characterized in that: In step S2.4, the ester hydrolase is selected from one or more of acetylcholinesterase, gluconolactonase, phosphomonoester hydrolase, and serine protease; and / or, In the step S2.4, the step of splitting the nitro-reduction product represented by the formula IV-2 by an ester hydrolase is as follows: a solution of the nitro-reduction product represented by the formula IV-2 is added dropwise to a buffer solution containing an organic reducing agent and the ester hydrolase, and then reacted at 35-45° C. to generate the amino-protected L-penicillamine represented by the formula V-2; the pH of the buffer solution is 6.8-7.2, and the organic reducing agent is selected from dithiothreitol, tris(2-carbonylethyl)phosphine hydrochloride or a mixture of the two; and / or, The pH of the system after the splitting reaction is completed is adjusted to 1-2, and the mixture is stirred for 10-30 minutes and then filtered. The filter cake is washed with water, the filtrates are combined and the pH of the combined filtrates is adjusted to 6-7, and the combined filtrates are extracted with isopropyl acetate for multiple times to remove the remaining substrate. The aqueous phase is concentrated, and the pH of the aqueous phase is adjusted to 1-2 after concentration, and then ice-bathed and filtered to obtain the amino-protected L-penicillamine solid represented by the formula V-2.

9. The method for preparing L-penicillamine according to claim 1, characterized in that: In the step S2.5, the step of removing the amino protecting group of the amino-protected L-penicillamine shown in the formula V-2 under strong acid conditions to generate L-penicillamine salt is as follows: adding acid solution to the amino-protected L-penicillamine shown in the formula V-2 at 15-30°C, and then stirring for 6-24 hours under heating conditions at 30-60°C to remove the amino protecting group to generate the L-penicillamine salt.

10. The method for preparing L-penicillamine according to claim 1, characterized in that: In the step S2.5, the alkaline hydrolysis step is as follows: the system after the removal of the amino protecting group reaction is completed is concentrated to remove the acid, dichloromethane is added to dissolve the residue, and then an alkaline substance is added to adjust the pH to 4-5, and the crude L-penicillamine is concentrated.

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