BR102015012995A2 - DEVELOPMENT OF A BIOCATALYTIC PROCESS FOR THE PRODUCTION OF PINDOLOL PHARMACEUTICAL (S) - Google Patents

Abstract

The present invention relates to the development of a biocatalytic process for the synthesis of drug (s) -pindolol, a beta blocker used in the treatment of hypertension and cardiac arrhythmia. furthermore, said drug is a 5-htla auto receptor antagonist, favoring combination with drugs from the group of selective serotonin reuptake inhibitors (isrs) and may accelerate or increase the therapeutic efficacy of antidepressants. The principal strategy in the development of said process includes enzymatic kinetic resolution of a racemic mixture of 1- (chloromethyl) -2- (1 h -indol-4-yloxy) ethyl acetate, an intermediate in the synthesis of -indolol (s). , in the presence of lipases. After screening with nine commercial lipases it was found that the free-form pseudomonas fluorescens lipase was the most efficient in the resolution of 1- (chloromethyl) -2- (lh-indol-4-yloxy) ethyl racacetate in phosphate buffer (ph 7.0), 24 hour reaction time and 40 ° C temperature, with and 115 ° C. This enzyme was immobilized on nanosilica and can be reused in up to ten reaction cycles, maintaining efficiency in selectivity and enzymatic activity. This biocatalytic process is in line with green chemistry as it makes use of a biodegradable catalyst that can be reused for several reaction cycles. Therefore, the reuse of the biocatalyst suggests an economically attractive and environmentally friendly process.

Description

(54) Title: DEVELOPMENT OF A BIOCATALYTIC PROCESS FOR THE PRODUCTION OF DRUG (S) -PINDOLOL (51) Int. Cl .: C12P 41/00; C07D 209/32; A61K 31/404; A61P 9/12 (73) Holder (s): UNIVERSIDADE FEDERAL DO CEARA (72) Inventor (s): MARCOS CARLOS DE MATTOS; GLEDSON VIEIRA LIMA; THIAGO DE SOUSA FONSECA; MARIA DA CONCEIÇÃO FERREIRA DE OLIVEIRA;

TELMA LEDA GOMES DE LEMOS; LEANDRO BEZERRA DE LIMA; LUIZ ARTHUR ZAMPIERI (57) Abstract: The present invention relates to the development of a biocatalytic process for the synthesis of the drug (S) -Pindolol, a beta-blocker used in the treatment of hypertension and cardiac arrhythmia. In addition, this drug is an antagonist of the 5-HTIA auto receptor, favoring the combination with drugs from the group of selective serotonin reuptake inhibitors (SSRIs), which may accelerate or increase the therapeutic efficacy of antidepressants. The main strategy in the development of this process includes the enzymatic kinetic resolution of a racemic mixture of l- (chloromethyl) -2- (l Hindol-4-yloxy) ethyl acetate, an intermediate in the synthesis of (S) -Pindolol, in presence of lipases. After screening with nine commercial lipases, it was possible to verify that Pseudomonas fluorescens lipase in free form was the most efficient in the resolution of ethyl l- (chloromethyl) -2- (IHindol-4-yloxy) ethyl acetate, in phosphate buffer (pH 7.0), reaction time of 24 hours and temperature of 40 ° C, with an E of 115. This enzyme was immobilized in nanosilica and can be reused in up to ten cycles (...)

1/7 “DEVELOPMENT OF A BIOCATALYTIC PROCESS FOR THE PRODUCTION OF DRUG (5) -PINDOLOL” [001] The present invention relates to the development of a biocatalytic process for the synthesis of (5) -Pindolol (A), a drug used as a beta-blocker in the treatment of hypertension and cardiac arrhythmia. In addition, said drug is an antagonist of the 5-HT1A auto receptor, favoring the combination with drugs from the group of selective serotonin reuptake inhibitors (SSRIs), which may accelerate or increase the therapeutic efficacy of antidepressants. It should be noted that despite being an old drug, new applications have been reported for (5) -Pindolol as in the treatment of fibromyalgia and chronic fatigue syndrome (WO 2003065970 A2), premenstrual syndrome / premenstrual dysphoric disorder (WO 2006030306 A2) and treatment of cachexia and sarcopenia (WO 2014016585 Al). The main strategy in the development of this process includes the enzymatic kinetic resolution of a racemic mixture of l- (chloromethyl) -2- (l // - indol-4-yloxy) ethyl (B), an intermediate in the synthesis of ( S) -Pindolol (A), in the presence of lipases.

[002] The synthesis of racemic Pindolol, rac-Pindolol, was proposed by Tejani-Butt and Brunswick (J. Med. Chem. V. 29, p. 1524-1527, 1986), using 4-hydroxindol as starting material in the presence of (±) -epichlorohydrin with the formation of epoxide, 4 (oxiran-2-yl-methoxy) -indole. The epoxide was opened in the presence of ethanol and excess isopropylamine. The earliest account of a method of synthesizing (S) -Pindolol refers to the use of (-) - epichlorohydrin as a chiral intermediate [F. Troxler and A. Hofmann (Sandoz Ltd.), Ger. Offen. 1905881, 1969], but this method has the disadvantage of presenting a decrease in the optical purity of the desired product, due to a competitive nucleophilic attack of the aryloxy anion on the α carbon to the chlorine atom (Tetrahedron Letters v. 34, p. 489- 492, 1993). Ishibashi et al. (Tetrahedron Letters v. 34, p. 489-492, 1993) performed the synthesis of (S) -Pindolol (A) using (/?) - (-) 3-chloro-1,2 propanediol as a chiral derivatization reagent , in the presence of p-toluenesulfonic acid (TsOH) and copper (II) chloride (CuCb), as a catalyst. The reaction product, 4-alkoxindole, was refluxed in the presence of sodium hydroxide (NaOH) and excess isopropylamine in aqueous ethanol, leading to the enantioselective production of (5) -Pindolol (A). Sasai et al. (Tetrahedron v. 50, p. 1231312318, 1994) reported the enantioselective synthesis of (S) -Pindolol using as

2. Chiral derivatization agent, Lanthanum-Lithium (/?) - BINOL ((/ Q-LLB). Nitromethane (CH3NO2, -50 ° C) was used in the presence of /? - LLB. 5-Pindolol ( A) after a reductive alkylation of the nitro group in the presence of PtO2, in acetone and methanol.

[003] The objective of the present invention is to solve the problem of using chemical catalysts not degraded in nature, using lipases in the enzymatic kinetic resolution of a chemical intermediate of (5) -Pindolol, the racemic mixture of l- (chloromethyl) acetate -2- (1 // - indol-4-yloxy) ethyl (B). Such biocatalysts are environmentally acceptable as they are completely biodegradable. In addition, the advantages of using mild reaction conditions such as temperatures between 20 and 40 ° C, reaction media with p 1 between 5 and 8 are added, providing a decrease in the formation of by-products from decomposition, racemization, competition of elimination reactions or isomerization, of the main product. In addition, lipase can be immobilized and reused in several reaction cycles, making the process economically attractive.

[004] The invention can be better understood with a detailed description, in line with the attached figures, namely:

[005] FIGURE 1 represents (S) -Pindolol (A).

[006] FIGURE 2 represents the kinetic resolution of 1- (chloromethyl) -2- (1 // indol-4-yloxy) ethyl acetate (B) in the presence of lipases in phosphate buffer solution (pH 7.0), leading to (1 /?) - (chloromethyl) -2- (17 / -indol-4-yloxy) ethyl (C) and (2.V) -l-chloro-3 (17 / -indol-4 -yloxy) -2-propanol (D).

[007] FIGURE 3 represents the synthesis of rac-1-chloro-3- (1 // - indol-4-yloxy) -2propanol (F) and (1 // - indol-4-yloxy) (2- oxiranyl) methane (G), from 4-hydroxyindole (E).

[008] FIGURE 4 represents the synthesis of raol-chloro-3- (l // - indol-4-yloxy) -2propanol (F) from (l // - indol-4-yloxy) (2-oxiranyl ) methane (G).

[009] FIGURE 5 represents the synthesis of l- (chloromethyl) -2- (l // - indol4-yloxy) ethyl rucetate (B) from rac-1-chloro-3- (1 Z / -indol-4-yloxy) -2-propanol (F).

[010] FIGURE 6 represents the synthesis of (2 /?) - 1-chloro-3- (1/7-indol-4-yloxy) -2propanol (H) from (l /?) - ( chloromethyl) -2- (1 // - indol-4-yloxy) ethyl (C).

3/7 [011] FIGURE 7 represents the synthesis of (S) -Pindolol (A) from (2 /?) - 1-chloro-3 (1 // - indol-4-yloxy) -2-propanol (H).

[012] The most practical way to find an ideal lipase for kinetic resolution is to perform a screening of lipases in a hydrolysis reaction of a racemic (rac) mixture of l- (chloromethyl) -2- (l // - indole-4-yloxy) ethyl (B), in buffer solution. The synthesis of l- (chloromethyl) -2- (17 / -indol-4-yloxy) ethyl (B) rac-acetate is described in figures 3- 5. In the first step, 4-hydroxyindole (E) reacted with epichlorohydrin, in a basic medium, producing halohydrin ra6-l-chloro-3- (l // - indole-4-yloxy) -2-propanol (F) and o (lf / -indol-4-yloxy) ( 2-oxiranyl) methane (G). As the objective is to obtain the halohydrin rac-1-chloro-3- (1 // - indol-4-yloxy) -2-propanol (F), a conversion step of (1/7-indole- 4-yloxy) (2-oxiranyl) methane (G) in the corresponding halohydrin, in an acidic medium. Finally, raco-1-chloro-3- (1f / -indol-4-yloxy) -2-propanol (F) halohydrin was acetylated in the presence of acetic anhydride in the corresponding 1 (chloromethyl 1) -2- (1 H- \ ndol-4-ylox i) ethyl (B).

[013] Initially, a screening was carried out with nine commercial lipases using the hydrolysis reaction of l- (chloromethyl) -2- (l / Y-indol-4-yloxy) ethyl (B) rac-acetate, in buffer phosphate (pH 7.0), using THF as a co-solvent, in 24 hours of reaction and temperature of 40 ° C.

[014] It was possible to verify that the most effective enzyme in hydrolysis of rc / c-acetate of 1 (chloromethyl) -2- (l // - indol-4-yloxy) ethyl (B) was Amano lipase AK from Pseudomonas fluorescens providing excellent results of enantiomeric excesses of (1 /?) - (chloromethyl) -2- (1 // - indol-4-yloxy) ethyl (C) (94%) and (2S) -l- chlorine-3- (1 // - indole-4-yloxy) -2-propane (D) (94%), 50% conversion and enantiomeric ratio (E) value of 115.

[015] Amano lipase AK from commercial Pseudomonas fluorescens is in free form, making it impossible to reuse. Immobilized enzymes have, as compared to free-form enzymes, greater stability, easier separation from the reaction medium and the possibility of reusing the biocatalyst, providing significant savings in the overall cost of the process. Thus, the immobilization of said enzyme in modified nanosilica was carried out. In this case, the 7 nm nanosilica functionalized in the presence of APTES (aminopropyltriethoxysilane) in phosphate buffer (pH 7.0) was used. The P. fluorescens lipase was

It is added to the functionalized support, followed by the addition of the homofunctional coupling agent glutaraldehyde. Finally, after successive centrifugation steps, it was possible to obtain the immobilized biocatalyst. Subsequently, the efficiency of the lipase of P. fluorescens immobilized in nanosilica in the kinetic resolution of the 1- (chloromethyl) -2- (1A / -indol-4-yloxy) ethyl (B) racacetate was analyzed in phosphate buffer solution ( pH 7.0) and a temperature of 40 ° C. After 12 h of reaction, (1 /?) (Chloromethyl) -2- (lf / -indol-4-yloxy) ethyl acetate (C) and (2. ^) - 1-chloro-3- ( 1/7-indol-4-yloxy) -2propanol (D) with values of enantiomeric excess of 97 and 70%, respectively, conversion of 59% and value of enantiomeric ratio (E) of 23. Then, a study of the reuse of the biocatalyst, providing the obtaining of (1 /?) (chloromethyl) -2- (l // - indol-4-yloxy) ethyl acetate (C) with 97% enantiomeric excess in up to ten reaction cycles. Subsequently, (l /?) - (chloromethyl) -2- (l // - indol-4yloxy) ethyl acetate (C) was hydrolyzed in the presence of the nanosilica immobilized Candida rugosa lipase leading to (2 / () - 1-chloro-3- (1 // - indol-4-yloxy) -2-propanol (H) in quantitative yield. It should be noted that the commercial C. rugosa lipase was immobilized under the same conditions used for the immobilization of the lipase of P. fluorescens The study of the reuse of the immobilized C. rugosa lipase in nanosilica revealed that the referred enzyme can be used in up to ten reaction cycles keeping the enzyme activity intact, and finally (2 / () - 1-chloro-3 - (1 // - indol-4-yloxy) -2propanol (H) reacted with isopropylamine to produce (5) -Pindolol (A).

[016] It is demonstrated that the present invention is an excellent alternative for the introduction of chirality in a key intermediate for the synthesis of (, V) -Pindolol (A). This biocatalytic process is in line with the aspects of Green Chemistry, since it makes use of non-toxic, biodegradable chiral biocatalysts that can be reused in up to ten reaction cycles, maintaining efficiency in selectivity and enzymatic activity.

[017] Therefore, the reuse of biocatalysts in up to ten cycles suggests an elegant and attractive process from an economic and environmentally friendly point of view.

[018] Example 1:

Synthesis of r-c-1-chloro-3- (1 // - indol-4-yloxy) -2-propanol (F):

In a round-bottom flask, 500 mg (3.75 mmol) of 4-hydroxyindole (E) and 5 mL of a 0.75 M aqueous solution of sodium hydroxide (NaOII) were added.

5/7 to the mixture were added 2.5 ml (32 mmol) of (±) epichlorohydrin and 2 ml of dioxane. The reaction was stirred for 6 hours at room temperature under an atmosphere of N2. The evolution of the reaction was monitored by thin layer chromatography using dichloromethane as eluent. After the end of the reaction, a dark green oil was obtained and the crude product was purified using a chromatographic column with flash silica, using dichloromethane as the eluent. Two products were separated, a yellowish oil characterized as / '<. · -L-chloro-3- (1 // - indol-4-yloxy) -2-propanol (F) with 65.7% yield and an oil brown characterized as (1 // - indole-4-yloxy) (2-oxiranyl) methane (G) in 21.5% yield. (1 // - indol-4-yloxy) (2-oxiranyl) methane (G) was converted to / -ac-1-chloro-3- (1/7-indol-4-yloxy) -2-propanol ( F) under the following conditions: 152.4 mg (0.81 mmol) of (177-indol-4-ioxy) (2-oxiranyl) methane (G) was dissolved in 5 ml of dichloromethane with subsequent addition of 0.5 ml of concentrated HCl. The reaction mixture was stirred for 1 hour. Then, the product was extracted with dichloromethane (3x5 ml) and dried with Na2SO.i. After filtration and evaporation of the solvent, under reduced pressure, the crude product was obtained, which was purified by chromatography using silica gel and dichloromethane as eluent. Finally, rüf-1-chloro-3- (1H-indol-4-yloxy) -2-propanol (F) can be obtained in an overall yield of 85.4%.

[019] Synthesis of 1- (chloromethyl) -2- (17 / -indol-4-yloxy) ethyl rac-acetate (B):

To a round-bottom flask, 50 mg (0.38 mmol) of rttc-1-chloro-3- (177-indol-4-yloxy) -2-propanol (F), 4 ml of dichloromethane, 22 mg (0, 19 mmol) of 4-dimethylaminopyridine (DMAP) and 360 pL (3.8 mmol) of acetic anhydride. The reaction mixture was kept under stirring at room temperature. The evolution of the reaction was monitored using CCD, completing after 1 hour, with the obtaining of a dark green oil. The crude product was purified using a chromatographic column with flash silica and dichloromethane as eluent, obtaining a yellowish oil characterized as 1- (chloromethyl) -2- (l / 7-indol-4-yloxy) cγ / c-acetate ) ethyl (B) with 93.4% yield.

[020] Kinetic enzymatic resolution of l- (chloromethyl) -2- (177indol-4-yloxy) ethyl rr / c-acetate (B) via hydrolysis reaction using commercial lipases:

The hydrolysis reactions were conducted in eppendorf tubes. 5 mg (0.019 mmol) of 1- (chloromethyl) -2- (l / 7-indol-4-yloxy) ethyl rac-acetate (B) were dissolved in 38 pL of tetrahydrofuran (THF), used as a co-solvent, followed by the addition of βπ

152 pL of a pH 7.0 phosphate buffer and 5 mg of lipase. The reaction mixture was stirred at 30 ° C at predetermined times of 2, 4, 6, 8, 12, 20 and 24 hours. After each period, liquid-liquid extraction was performed using ethyl acetate. The solvent was evaporated under reduced pressure and the hydrolysis product was dissolved in isopropanol (HPLC grade) and analyzed in HPLC using a Shimadzu LC-20AD device with a Shimadzu SPD-20A UV / vis detector. A Chiralcel® OD-H normal phase chiral column (150 x 4.6 mm x 5 pm) was used. The mobile phase employed was hexane: isopropanol (80:20) with UV-HPLC purity. The oven temperature was maintained at 35 ° C and the analyzes were performed at a wavelength of 265 nm. The solvents were filtered through nylon membranes with 0.45 pm pores (Phenomenex). In addition, the samples were filtered in a manual system through teflon membranes with 0.45 pm pores (Waters).

[021] (enzymatic mobilizations:

In an eppendorf, 10 mg of 7 nm nanosilica and 1 ml of Milli-Q water were added. The system was subjected to ultrasound for a period of 30 minutes, followed by the addition of 20 pL of APTES (aminopropyltriethoxysilane) with subsequent agitation on a shaking table (180 rpm) for 12 h at 30 ° C. Then, centrifugation was performed with removal of the supernatant. To the eppendorf content, 1 ml of Milli-Q water was added followed by centrifugation (3,500 rpm) for 10 minutes. This procedure was repeated six times. Then, 5 mg of the lipase was added with homogenization of the system on a shaking table (250 rpm) for 3 hours. Subsequently, 25 pL of glutaraldehyde was added, with stirring (250 rpm) on a shaking table for 17 hours. After this period, successive washes were performed with Milli-Q water (six cycles with 1 mL of water). With this procedure, the lipases of P. fluorescens and C. rugosa immobilized in nanosilica were obtained.

[022] Obtaining (l / f) - (chloromethyl) -2- (l / -indol-4-yloxy) ethyl (C) via hydrolysis of l- (chloromethyl) -2- (l // - indole-4-yloxy) ethyl (B) in the presence of P. fluorescens immobilized on nanosilica:

To an eppendorf, 5 mg of 1- (chloromethyl) -2- (1Hindol-4-yloxy) ethyl (B) mc-acetate (0.019 mmol), 5 mg of P. fluorescens immobilized in 10 mg of functionalized nanosilica in 200 pL of phosphate buffer (pH 7.0). The reaction system was stirred (250 rpm) on a shaking table, at 40 ° C, for 12 hours. Then, a

7/7 centrifugation with removal of the supernatant and extraction with ethyl acetate (3 x 1 mL). The solvent was evaporated under reduced pressure and the crude product was purified by flash silica chromatography, using dichloromethane as the eluent, obtaining (1fl) - (chloromethyl) -2- (1 // - indoI-4-yloxy) acetate ethyl (C) with 98% yield.

[023] Synthesis of (2 /?) - 1-chloro-3- (1 // - indol-4-yloxy) -2-propanol (H):

In an eppendorf, 5 mg (0.019 mmol) of (1 /?) (Chloromethyl) -2- (1 // - indol-4-yloxy) ethyl acetate (C) and 5 mg of C. rugosa immobilized in 10 mg of functionalized nanosilica, in 200 pL of phosphate buffer (pll 7.0). The reaction system was stirred (250 rpm) on a shaking table, at 40 ° C, for 12 hours. Then, a centrifugation was performed with removal of the supernatant and extraction with ethyl acetate (3 x 1 mL). The solvent was evaporated under reduced pressure to obtain (2 /?) - 1-chloro-3- (1 // indol-4-yloxy) -2-propanol (H) in quantitative yield.

[024] Synthesis of (S) -Pindolol (A):

In a round-bottom flask, 100 mg (0.44 mmol) of (2 /?) - chloro-3- (1 // - indol-4-yloxy) -2-propanol (H), 5 ml of ethanol and excess isopropylamine. The reaction system was kept under reflux for 24 hours. After that period, the solvent and excess amine were evaporated to obtain a white solid, characterized as (Sj-Pindolol (A), with a 66% yield.

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Claims (4)

1. “DEVELOPMENT OF A BIOCATALYTIC PROCESS FOR THE DRUG (S) -PINDOLOL” characterized by being the enzymatic kinetic resolution of the racemic mixture of the acetate of 1 - (chloromethi 1) -2- (1 // indol-4-yloxy ) ethyl, precursor of the drug fS) -Pindolol, using Amano lipase AK as a biocatalyst from Pseudomonas jluorescens, in free form and immobilized in modified nanosilica, in phosphate buffer (pl I 7.0), enzyme: substrate ratio 1: 1 and temperature of 40 ° C. 2. “DEVELOPMENT OF A BIOCATALYTIC PROCESS FOR PHARMACEUTICAL (5) -PINDOLOL” characterized by using the commercial Candida rugosa lipase immobilized in modified nanosilica in the hydrolysis of (l /?) - (chloromethyl) -2- (l / Y-indol-4-yloxy) ethyl. 3. “DEVELOPMENT OF A BIOCATALYTIC PROCESS FOR PHARMACEUTICAL (5) -PINDOLOL” characterized by immobilizing commercial Amano lipase AK from Pseudomonas jluorescens in modified nanosilica in the presence of APTES and glutaraldehyde. 4. “DEVELOPMENT OF A BIOCATALYTIC PROCESS FOR DRUG (S) -PINDOLOL” characterized by immobilizing the commercial Candida rugosa lipase in modified nanosilica in the presence of APTES and glutaraldehyde. / 1/2