Synthesis, Isolation and Analysis of Stereoisomers of Sacubitril

Subscriber access provided by YORK UNIV

Communication

Synthesis, Isolation and Analysis of Stereoisomers of Sacubitril. Aleš Halama, and Michal Zapadlo Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.8b00350 • Publication Date (Web): 13 Dec 2018 Downloaded from http://pubs.acs.org on December 13, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Organic Process Research & Development

Synthesis, Isolation and Analysis of Stereoisomers of Sacubitril. Aleš Halama,* Michal Zapadlo Department of Chemical Synthesis, Zentiva k.s., U kabelovny 130, Prague 102 01, Czech Republic

ACS Paragon Plus Environment

Organic Process Research & Development 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC

O O NH3

H N O

(2S,4R)-1 CHA sacubitril reference standard

O

O OEt

H N

O

NH3

O

O (R) (S)

OEt

(2R,4S)-1 CHA sacubitril enantiomer reference standard


Page 2 of 11

Page 3 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ABSTRACT: An efficient industrial synthetic process for sacubitril has been developed. Stereoisomers derived from sacubitril and its crucial intermediate have been synthesized, isolated and characterized for quality control. These stereoisomers were characterized by spectral data (MS and NMR) and used as reference standards for development of HPLC methods. KEYWORDS: sacubitril, AHU-377, valsartan, LCZ696, stereoisomers, development of HPLC methods

INTRODUCTION Sacubitril (2S,4R)-1 (Figure 1), known also as AHU-377, is an active pharmaceutical substance which is used in fixed-dose combination with valsartan 2 in the treatment of heart failure. This combination, known also as supramolecular complex LCZ696, is marketed by Novartis under the brand name Entresto. It is indicated to reduce the risk of cardiovascular death and hospitalization for heart failure in patients with chronic heart failure and reduced ejection fraction. It was approved as the first dual inhibitor of neutral endopeptidase (NEP) and AT1 receptors for angiotensin II by the FDA in 2015.1,2

O

H N

HO

O

O OEt

O

OH

HN N N N

N O

(2S,4R)-1 sacubitril

2 valsartan

Figure 1. Molecular structures of sacubitril (2S,4R)-1 and valsartan 2.

The first reported synthesis of sacubitril (2S,4R)-1 consists of 11 steps starting from N-Boc-D-tyrosine methyl ester, six of them to the significant intermediate ethyl ester 6 followed by another five steps to the final product, see Scheme 1.3 This process is not suitable for large scale because include big number of synthetic steps and also some problematic operations from an industrial point of view, e.g., separation of diastereoisomers by column chromatography purification of tert-butyl ester 3 in the end of the synthetic route. Some alternative processes are described for advanced chiral intermediates, e.g., diastereoselective reduction of carbon-carbon double bond in sodium carboxylate (R)-4a to carboxylic acid (2R,4S)-5 (Scheme 2).4,5 It is mandatory for a manufacturer to identify and characterize all significant impurities that are present in an active pharmaceutical ingredient (API).6 Impurities are generally the products of incomplete reactions, by-products which come from nonselective reactions and instability of intermediates or final compound, e.g., hydrolytic impurities. In the case of sacubitril a potential impurities are represented also by its stereoisomers. For that reason, it is necessary to synthesize, isolate and characterize all its stereoisomers, especially for development of analytical methods. Results related to this issue are summarized in this communication.



Page 4 of 11

Scheme 1. The First Reported Synthetic Route of Sacubitril (2S,4R)-1.

H N

Boc

OCH3

Boc

(a) (b) (c)

H

H N

O OEt

O

Boc

(g)

O

H N

OEt

(d) (e) (f) OH Boc-D-Tyr-OMe

(R)-4

(2R,4S)-5 major diastereoisomer

(h)

O

H N

t-BuO

O

O (j)

OEt

H N

MO

O

O

O

O OEt

H 2N

OEt

(i)

(k) after step (i) M=H (2S,4R)-1 after step (k) M=Na (2S,4R)-1 Na salt

3

6

(a) triflic anhydride; (b) phenylboronic acid, tetrakis(triphenylphosphine)palladium(0); (c) NaOH; (d) CH3NHOCH3, Et3N, EDC, HOBT; CH2Cl2; (e) LiAIH4; ether, KHSO4, HCl; (f) (carbethoxyethylidene)triphenylphosphorane; CH2Cl2, (g) H2/Pd/C; EtOH; (h) HCl; (i) succinic anhydride; methylenechloride, pyridine, mixture of diastereoisomers; (j) N,N-dimethylfomamide ditert-butyl acetal; toluene; separation of diastereoisomers by chromatography (SiO2/ toluene, EtOAc); (k) HCl, CH2Cl2, NaOH, THF. Scheme 2 The Industrial Diastereoselective Synthesis of the Chiral Precursor (2R,4S)-5 of Sacubitril (2S,4R)-1. Boc

H N

H

O ONa

(a)

Boc

H N

O OH

(b)

(R)-4a

(2R,4S)-5

(a) H2, Ru-Mandyphos, biphasic system [EMIM]/[NTf2]/NaOH(aq). (b) HCl (aq).

RESULTS AND DISCUSSION The short description of the industrial process for synthesis of sacubitril. The developed industrial synthetic process employs commercially available carboxylic acid (2R,4S)-5 as a starting material, consists of three synthetic steps, is scalable and no special operational techniques or equipment are required, see Scheme 3.7,8 The overall yield is approximately 87% of the sodium salt (2R,4S)-1. The product meets with all regulatory specifications. The first step combines two simple chemical transformations together. The Boc protecting group is removed by reaction with HCl (generated by reaction of thionyl chloride with ethanol) and esterification of carboxyl group is carried out simultaneously. The yield of the first step was 96% with chemical purity ≥ 99.6% by HPLC. In the second step the four carbon building block is introduced to the molecule by reaction of amine (2R,4S)-6 with succinic anhydride. The product is isolated in the form of the cyclohexyl amine salt (2S,4R)-1 CHA and it is isolated in high chemical purity (≥99.9% by HPLC and 93% yield) after simple crystallization from ethyl acetate. The purification via the cyclohexyl amine salt is crucial from point of view chemical purity. In the third step the cyclohexyl amine salt (2S,4R)-1 CHA is converted to sacubitril sodium salt with 97% yield.


Page 5 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 3. The Developed Short Industrial Process for Sacubitril Sodium Salt Synthesis Including Purification via Cyclohexyl Amine Salt. Boc

H N

O OH

O

HCl H 2N

(a)

O OEt

(2R,4S)-6

(2R,4S)-5 O Na

H N

O

(b)

(c)

O NH3

H N

O OEt

O

(2S,4R)-1 CHA salt

O OEt

O

(2S,4R)-1 Na salt

(a) SOCl2, EtOH, cyclopentylmethyl ether. (b) succinic anhydride, triethyl amine, CH2Cl2; cyclohexyl amine (CHA), EtOAc. (c) EtOAc, toluene, water, HCl; NaOH, EtOH, EtOAc.

Synthesis and isolation of stereoisomers of the starting material for industrial synthesis of sacubitril. The developed industrial process starts from carboxylic acid (2R,4S)-5. For that reason, its optical isomers are needed as reference standards of the process control. These standards have been synthesized and isolated by the conventional procedure, see Scheme 4. The mixtures of diastereoisomeric pairs have been prepared by catalytic hydrogenation from commercially available carboxylic acids (R)-4b and (S)-4b. One of these diastereoisomers is always significantly predominant (ratio approximately 75:25). The major isomers (2R,4S)-5 and (2S,4R)-5 have been isolated in pure form by crystallization from mixtures isopropyl acetate and heptane. The isolation of the minor isomers (2S,4S)-5 and (2R,4R)-5 was much more challenging. The diastereoisomers remaining in the mother liquor after separation of major isomers were converted to the corresponding cyclohexyl amine salts and originally minor diastereoisomers were isolated by repetitive fractional crystallizations from isopropyl acetate and mixtures isopropyl acetate-heptane in different ratios of the solvents used. Synthesis and isolation of stereoisomers of sacubitril. The stereoisomers of sacubitril (2S,4R)-1 CHA salt have been synthesized according to Scheme 5. These include enantiomer (2R,4S)-1 CHA salt, diastereoisomers (2S,4S)-1 CHA salt and (2S,4S)-1 CHA salt. Previously isolated carboxylic acids (2R,4S)-5, (2S,4R)-5, (2S,4S)-5 and (2R,4R)-5 or their cyclohexyl amine salts were used as starting materials. The procedure consists of two steps. The first step represents almost quantitative Boc deprotection and simultaneous esterification. The resulting amino intermediates (2R,4S)-6, (2S,4R)-6, (2S,4S)-6 and (2R,4R)-6 were not collected but used in the next synthetic step. In the second step these intermediates are reacted with succinic anhydride. The products were isolated in the form of well crystallizing cyclohexyl amine salts. All stereoisomers showed high chemical (98.0-99.9% by HPLC) as well as optical purities (all different optical isomers less than 2.5 %, usually less than 0.05%). HPLC methods for analysis of stereoisomers of sacubitril. Two robust isocratic HPLC methods have been developed for checking optical purity in the industrial synthetic process of sacubitril. The first method was developed for the chiral purity test of carboxylic acid (2R,4S)-5 and the second for the chiral purity test of sacubitril (2S,4R)-1. The first HPLC method use Chiracel OJ-H (5 μm) as the stationary phase and mixture hexane/ethanol (8:2) as the mobile phase. The retention times (RT) or the relative retentions times (RRT) are as follows: (2R,4S)-5 (RT: 23.0 min), (2S,4R)-5 (RRT 0.55), (2S,4S)-5 (RRT 0.49) and (2R,4R)-5 (RRT 0.79). The second HPLC method use Chiracel AY-H (5 μm) as the stationary phase and mixture hexane/ethanol (97:3) with addition small amount of trifluoroacetic acid as the mobile phase. The retention times (RT) or the relative retentions times (RRT) are as follows: (2S,4R)-1 CHA salt (RT: 7.6 min), (2R,4S)-1 CHA salt (RRT 1.25), (2S,4S)-1 CHA salt (RRT 0.89) and (2R,4R)-1 CHA salt (RRT 1.64). The experimental conditions of both methods are described in experimental section in detail. The results are illustrated in Figure 2 and Figure 3.



Page 6 of 11

Scheme 4. Syntheses and Molecular Structures of Stereoisomers of the Staring Material in Sacubitril Industrial Process, in Blue is Intermediate for Sacubitril and in Red are Intermediates for Optical Isomers of Sacubitril. NH2

Boc

O

H

H N

OH

H2/Pd, ethanol

Boc

H N

O (S)

Boc

OH

(R)

H N

O (S)

Boc

OH

(S)

+

Boc

H

H N

O OH

H2/Pd, ethanol

Boc

H N

(S)

Boc

OH

H 3N

H N

(2S,4S)-5 CHA salt isolated yield 10 % NH2

O (R) (R)

Boc

OH

major (2S,4R)-5 isolated yield 53 %

H N

O (R) (R)

O H 3N

+

(S)-4b

O

(S)

minor (2S,4S)-5

O (R)

O (S)

after isomers pre-separation

major (2R,4S)-5 isolated yield 58 %

(R)-4b

H N

after isomers pre-separation minor (2R,4R)-5

(2R,4R)-5 CHA salt isolated yield 12 %

Scheme 5. Syntheses and Molecular Structures of Stereoisomers of Sacubitril, in Blue is Sacubitril and in Red Are Sacubitril Isomers. Boc

H N

O (S)

HCl H 2N

OH

(R)

H N

O (S)

HCl H 2N

O

(S)

OH

HCl H 2N (a)

Boc

HCl H 2N

O (R)

NH3

O H 3N

(2R,4R)-5 CHA salt

(a)

(b)

H N

O

NH3

O (R)

OEt

(S)

O

(2R,4S)-1 CHA salt sacubitril enantiomer reference standard isolated yield 88 %

O

O

(2R,4R)-6

OEt

(S)

O

O

OEt

O (S)

(2S,4S)-1 CHA salt sacubitril diastereoisomer reference standard isolated yield 73 %

(2S,4R)-6

(2S,4R)-5

(R)

(b)

H N

O

O OEt

OEt

(R)

O

O OEt

O (S)

(2S,4R)-1 CHA salt sacubitril reference standard isolated yield 89 %

(2S,4S)-6

O (R) (S)

H N

NH3

O

(a)

(2S,4S)-5 CHA salt

H N

(b)

H N

O

(2R,4S)-6

H 3N

Boc

O OEt

(a)

(2R,4S)-5

Boc

O

(b)

H N

O

NH3

O

O (R) (R)

OEt

(2R,4R)-1 CHA salt sacubitril diastereoisomer reference standard isolated yield 59 %

(a) SOCl2, EtOH, cyclopentylmethyl ether, heptane. (b) Succinic anhydride, triethyl amine, CH2Cl2; cyclohexyl amine (CHA), EtOAc.


Page 7 of 11

(2S,4S)-5

0.008

0.006

(2S,4R)-5 (2R,4R)-5

AU

0.004

0.002

(2R,4S)-5

0.000 -0.002

0.00

4.00

8.00

12.00

16.00

20.00

24.00

28.00

Minutes

Figure 2. HPLC chromatogram of the sacubitril intermediate (2R,4S)-5 with addition of its stereoisomers (2S,4S)-5, (2S,4R)-5 and (2R,4R)-5, each stereoisomer at level 0.15 %.

(2S,4S)-1 1.50

(2S,4R)-1 1.00

(2R,4S)-1

AU

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(2R,4R)-1 0.50

0.00 4.00

6.00

7.00

8.00

9.00

10.00 11.00 12.00 13.00 14.00 15.00 Minutes

Figure 3. HPLC chromatogram of the equimolar mixture of sacubitril (2S,4R)-1 and its stereoisomers (2S,4S)-1, (2R,4S)-1 and (2R,4R)-1.

CONCLUSION The reference standards of sacubitril (2S,4R)-1 stereoisomers and its crucial intermediate (2R,4S)-5 have been synthesized, isolated and characterized by spectral methods. The syntheses were completed in analogy to the first and second steps of the industrial synthetic process but isolations of stereoisomers were different, especially isolations of minor diastereoisomers. All stereoisomers of (2S,4R)-1 were isolated in the form of well crystallizing cyclohexyl amine salts. A robust HPLC method has been developed to analyze the optical purity of sacubitril based on prepared reference standards.

EXPERIMENTAL SECTION Analytical Methods. NMR spectroscopy. 1H and 13C NMR spectra were measured with a Bruker Avance 500 spectrometer at measuring frequencies of 500.131 and 125.762 MHz, respectively. The spectra were measured in DMSO-d6. 1H chemical shifts are referenced to TMS (δ = 0.00 ppm) and 13C chemical shifts to DMSOd6 (δ = 39.5 ppm). Mass Spectrometry. The mass spectra were measured using a Sciex API 3000 Mass Spectrometer (Sciex, Canada) with positive atmospheric pressure ionization (TurboIonspray) or using LTQ Orbitrap Hybrid Mass Spectrometer (Thermo Finnigan, USA) with direct injection into APCI



source in positive mode. High Performance Liquid Chromatography (HPLC). HPLC chromatograms were measured with the WATERS Alliance with UV or PDA detector. HPLC method for optical purity (2R,4S)-5. Stationary phase: Chiralpak AY-H (5 μm); column temperature was 40 °C. Mobile phase: hexane / ethanol (8:2, V/V). The flow rate of the mobile phase was 1.5 ml/min. Ethanol was used as the solvent for preparation of the samples (25 mg to 5 ml); 25 μL of the solution was used for the injection. Time of analysis was 30 minutes. Detection at 254 nm. HPLC method for optical purity (2S,4R)-1. Stationary phase: Chiracel OJ-H (5 μm); column temperature was 40 °C. Mobile phase: hexane / ethanol (8:2, V/V) adjusted with 0.01 % of TFA. The flow rate of the mobile phase was 1.5 ml/min. Ethanol was used as the solvent for preparation of the samples (25 mg to 5 ml); 25 μl of the solution was used for the injection. Time of analysis was 15 minutes. Detection at 254 nm. (2R,4S)-5-Biphenyl-4-yl-4-tert-butoxycarbonylamino-2-methylpentanoic acid (2R,4S)-5 and (2S,4S)-5-Biphenyl-4-yl-4-tert-butoxycarbonylamino-2-methylpentanoic acid (2S,4S)-5. Carboxylic acid (R)-4b (52.9 g) was mixed with ethanol (550 ml) and the slurry of 10% palladium on carbon (1.6 g) in water (5 ml) was added. The reaction vessel was filled up with hydrogen gas (0.5 MPa) and stirred reaction mixture was heated to 40°C for approximately 3 hours to the constant pressure in the reaction vessel. After hydrogen consumption the catalyst was filtered off and ethanol was evaporated under reduced pressure. The isolated material (55 g) was a mixture of the pair of diastereioisomers, ratio (2R,4S)-5 to (2S,4S)-5 was approximately 75:25. The residue was dissolved in isopropyl acetate (110 ml) and heated to boiling point. Heptane (220 ml) was added to the refluxing solution. The mixture was gradually cooled to 40°C. The precipitated product was filtered off, washed with the solution of isopropyl acetate (30 ml) in heptane (60 ml) and dried in a vacuum oven at 50°C overnight. The first target product (2R,4S)-5 in the form of free acid was isolated as of white crystals (30.7 g, yield 58%, HPLC over 99%, content of (2S,4S)-isomer less than 1%). [M+H]+ m/z=384.2169 (C23H30NO4). 1H NMR (DMSO-d6), δ(ppm):1.05 and 1.08 (d, J = 7.2, 3H); 1.36 (m, 1H); 1.74 (m, 1H); 2.42 (m, 1H); 2.68 (d, 2H); 6.30 and 6.72 (d, J = 9, 1H); 7.24 (d, J = 8.4, 2H); 7.33 (t, J = 7.4, 2H); 7.44 (t, J = 7.4, 2H); 7.56 (d, J = 8.4, 2H); 7.63 (d, J = 7.3 Hz, 2H); 12.00 (s, 1H).13C NMR (DMSO-d6), δ(ppm):18.1, 27.8, 28.3, 35.9, 37.9, 40.7, 50.0, 77.4, 126.3, 126.5, 127.2, 128.9, 129.8, 137.7, 138.2, 140.1, 155.2, 177.2. Isolation of (2S,4S)-5 cyclohexylamine salt from mother liquors. The mother liquors containing a mixture of the two diastereoisomers were collected from several batches of preparing pure compound (2R,4S)-5. The solvents were distilled off from collected mother liquors under reduced pressure and a paste-like rest was obtained. This material was mixture of the pair of diastereioisomers (ratio (2R,4S)-5 to (2S,4S)-5 was approximately 45:55) and other minor impurities. This material was dissolved in isopropyl acetate at 70°C and cyclohexyl amine added to support of formation better crystallizing salt. Diastereoisomer (2S,4S)-5 was isolated from this mixture as a crystalline salt with cyclohexyl amine by means of repetitive fractional crystallizations from isopropyl acetate and mixtures isopropyl acetateheptane in different ratios of the solvents used. The (2S,4S)-5 cyclohexyl amine salt (6.3 g) was isolated as of white crystals (HPLC over 99%, content of (2R,4S)-isomer less than 1%). [M+H]+ m/z=384.2171 (C23H30NO4). 1H NMR (DMSO-d6), δ(ppm):0.97 (d, J = 7.2, 3H); 1.07 (m, 3H); 1.20 (m, 4H); 1.32 (m, 9H); 1.66 (m, 1H); 1.77 (d, 5H); 2.22 (m, 1H); 2.64 (m, 1H); 2.70 (d, J = 8.6, 2H); 3.67 (m, 1H); 6.42 and 6.82 (d, J = 9, 1H); 7.24 (d, J = 8.4, 2H); 7.33 (t, J = 7.4, 2H); 7.44 (t, J = 7.4, 2H); 7.56 (d, J = 8.4, 2H); 7.63 (d, J = 7.3 Hz, 2H).13C NMR (DMSO-d6), δ(ppm):17.0, 24.3, 25.2, 27.8, 28.2, 34.2, 36.9, 38.2, 39.3, 40.8, 49.5, 49.6, 77.2, 126.3, 126.4, 127.1, 128.9, 129.7, 137.7, 138.6, 140.1, 155.2, 178.4. (2S,4R)-5-Biphenyl-4-yl-4-tert-butoxycarbonylamino-2-methylpentanoic acid (2S,4R)-5 and (2R,4R)-5-Biphenyl-4-yl-4-tert-butoxycarbonylamino-2-methylpentanoic acid (2R,4R)-5. Carboxylic acid (S)-4b (4.5 g) was mixed with ethanol (110 ml) and the slurry of 5% palladium on carbon (0.45 g) in water (0.5 ml) was added. The reaction vessel was filled up with hydrogen gas (0.5 MPa) and stirred reaction mixture was heated to 40°C for approximately 3 hours to the constant pressure in the reaction vessel. After hydrogen consumption the catalyst was filtered off and ethanol was evaporated under reduced pressure. The isolated paste-like material was a mixture of the pair of diastereioisomers, ratio (2S,4R)-5 to (2R,4R)-5 was approximately 75:25. The residue was dissolved in isopropyl acetate (8 ml) and the mixture heated to boiling point. Heptane (16 ml) was added to the refluxing solution. The mixture was gradually cooled to room temperature and the precipitated solid collected by suction. The raw product, contaminated by 15% of (2R,4R)-isomer, was dissolved in isopropyl acetate (6 ml) at 70 °C, heptane (12 ml) added and the mixture gradually cooled to 40°C. The precipitated product was filtered off, washed with the solution of isopropyl acetate (2 ml) in heptane (3 ml) and dried in a vacuum


Page 8 of 11

Page 9 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


oven at 50°C overnight. The first target product (2S,4R)-7 was isolated in the form of free acid as of white crystals (2.4 g, yield 53%, HPLC over 99%, content of (2R,4R)-isomer less than 1%). [M+H]+ m/z=384.2169 (C23H30NO4). 1H NMR (DMSO-d6), δ(ppm):1.05 and 1.08 (d, J = 7.2, 3H); 1.36 (m, 1H); 1.74 (m, 1H); 2.42 (m, 1H); 2.68 (d, 2H); 6.30 and 6.73 (d, J = 9, 1H); 7.24 (d, J = 8.4, 2H); 7.33 (t, J = 7.4, 2H); 7.44 (t, J = 7.4, 2H); 7.56 (d, J = 8.4, 2H); 7.63 (d, J = 7.3 Hz, 2H); 12.00 (s, 1H).13C NMR (DMSO-d6), δ(ppm):18.1, 27.8, 28.3, 35.9, 37.9, 40.7, 50.0, 77.3, 126.3, 126.4, 127.1, 128.9, 129.8, 137.7, 138.2, 140.1, 155.2, 177.1. Isolation of (2R,4R)-5 cyclohexyl amine salt from mother liquors. The mother liquors containing a mixture of the two diastereoisomers were collected from several batches of preparing pure compound (2S,4R)-5. The solvents were distilled off from collected mother liquors under reduced pressure and a paste-like rest was obtained. This material was mixture of the pair of diastereioisomers (ratio (2S,4R)-5 to (2R,4R)-5 was approximately 45:55) and other minor impurities. This material was dissolved in isopropyl acetate at 70°C and cyclohexyl amine added to support of formation better crystallizing salt. Diastereoisomer (2R,4R)-5 was isolated from this mixture as a crystalline salt with cyclohexyl amine by means of repetitive fractional crystallizations from isopropyl acetate and mixtures isopropyl acetate-heptane in different ratios of the solvents used. It was isolated 665 mg of target (2R,4R)-5 as of white crystals (HPLC over 99%, content of (2S,4R)-isomer less than 1%). [M+H]+ m/z=384.2167 (C23H30NO4). 1H NMR (DMSO-d6), δ(ppm):0.97 (d, J = 7.2, 3H); 1.07 (m, 3H); 1.20 (m, 4H); 1.32 (m, 9H); 1.66 (m, 1H); 1.77 (d, 5H); 2.22 (m, 1H); 2.64 (m, 1H); 2.70 (d, J = 8.6, 2H); 3.67 (m, 1H); 6.42 and 6.82 (d, J = 9, 1H); 7.24 (d, J = 8.4, 2H); 7.33 (t, J = 7.4, 2H); 7.44 (t, J = 7.4, 2H); 7.56 (d, J = 8.4, 2H); 7.63 (d, J = 7.3 Hz, 2H).13C NMR (DMSO-d6), δ(ppm):16.9, 24.5, 25.3, 27.8, 28.2, 34.9, 36.5, 38.0, 39.0, 40.8, 49.6, 49.8, 77.2, 126.3, 126.5, 127.1, 128.9, 129.8, 137.7, 138.5, 140.1, 155.3, 178.0, 179.4, 179.6. Ethyl-(2R,4S)-4-amino-5-[1,1'-biphenyl]-2-methylpentanoate, hydrochloride (2R,4S)-6 Ethanol (130 ml) was stirred, cooled to -5 °C and thionyl chloride (20 ml) added dropwise during 20 minutes. The mixture was stirred for approximately 10 minutes and then carboxylic acid (2R,4S)-5 (35 g) and ethanol (80 ml) added. The mixture was intensively stirred at room temperature for a while, then gradually heated to 50°C and held at 50°C for 120 minutes. The solution was filtered and concentrated by means of rotary vacuum evaporator (70°C / 50 mbar) to the paste-like residue. This residue was dissolved in a mixture of cyclopentyl methyl ether (100 ml) and ethanol (30 ml) at 70°C. The resulting mixture was partially concentrated under vacuum (approximately 1/3 of the original volume was evaporated) and the residue solution was stirred under slow cooling. Another cyclopentyl methyl ether (100 ml) and ethanol (20 ml) were added for better homogenization of the mixture. The precipitated solid was collected by suction. The cake was washed by cyclopentyl methyl ether (50 ml) and heptane (50 ml). The isolated solid was dried in the open air at room temperature for several hours and then in a vacuum dryer at 60°C for 4 hours to give product as a white powder (30.5 g, yield 96 %). 1H NMR (DMSO-d6), δ(ppm): 1.07 (d, J = 7.1 Hz, 3H); 1,09 (t, J = 7.2, 3H); 1.27 (ddd, J = 14.2 / 8.2 / 5.2 Hz, 1H); 1.86 (ddd, J = 14.2, 8.2, 5.2, 1H); 2.81 (dd, J = 14.0 / 8.2, 1H); 2.74 (m, 1H); 3.05 (dd, J = 14.0 / 5.3, 1H); 3.38 (m, 1H); 3.99 (q, J = 7.1 Hz, 2H); 7.36 (m, 3H); 7.46 (t, J = 7.7 Hz, 2H); 7.66 (m, 4H); 8.19 (bs, 3H). 13C NMR (DMSO-d6), δ(ppm): 14.0, 17.5, 33.5, 38.1, 39.0, 50.4, 60.1, 126.5, 126.8, 127.4, 129.0, 130.0, 135.5, 138.7, 139.8, 174.7. Ethyl-(2S,4S)-4-amino-5-[1,1'-biphenyl]-2-methylpentanoate, hydrochloride (2S,4S)-6 The target compound (1.7g) was prepared from (2R,4R)-5 according to procedure described for (2R,4S)-6. The product was directly used to the next step. Ethyl-(2S,4R)-4-amino-5-[1,1'-biphenyl]-2-methylpentanoate, hydrochloride (2S,4R)-6 The target compound (0.9 g) was prepared from (2S,4R)-5 according to procedure described for (2R,4S)-6. The product was directly used to the next step. Ethyl-(2R,4R)-4-amino-5-[1,1'-biphenyl]-2-methylpentanoate, hydrochloride (2R,4R)-6 The target compound (676 mg) was prepared from (2R,4R)-5 according to procedure described for (2R,4S)-6. The product was directly used to the next step. 4-{[(2S,4R)-1-(1,1'-biphenyl)-5-ethoxy-4-methyl-5-oxo-2-pentanyl]amino}-4-oxobutanoic acid, cyclohehyl amine salt (2S,4R)-1 CHA. (2R,4S)-6 (21 g) was dissolved in 145 ml of dichloromethane, 17.7 ml of triethyl amine added and the mixture stirred at room temperature for 10 minutes. Succinic anhydride (6.15 g) and dichloromethane (30 ml) were added. The reaction mixture was stirred for 18



hours at room temperature. Hydrochloric acid (2M, 35 ml) was added to the reaction mixture. The mixture was stirred for 10 minutes, layers were separated, the aqueous phase was washed with a small amount of dichloromethane (20 ml) and the organic phases were combined. The combined dichloromethane layers were washed with water (2x50 ml), brine (50 ml) and dried over sodium sulfate. The drying agent was filtered off and washed with small amount of dichloromethane (20 ml). The combined dichloromethane solutions were concentrated in vacuum evaporator to yield a transparent honey (free carboxylic acid). The honey-like residue was dissolved in 300 ml of ethyl acetate at 65 °C, then cyclohexyl amine (6.99 ml) and ethyl acetate (50 ml) added. The mixture was stirred under slow cooling to 40-45°C. A very thick suspension was obtained. The mixture was repeatedly heated up to 65°C and cooled to 40-45°C for better consistence. The precipitated product was isolated by vacuum filtration. The cake was washed with mother liquor and ethyl acetate (50 ml).The crystalline solid was dried in the open air at room temperature overnight and then in a vacuum dryer at 65°C for 3.5 hours to give product as a white powder (28.7 g, yield 93.1%). Purity by HPLC 99.93%. [M+H]+ m/z=412.2114 (C24H30NO5), other optical isomers less than 0.05 %. 1H NMR (DMSO-d6), δ(ppm):1.12-1.22 (m, 12H); 1.36 (m, 1H); 1.54 (m, 1H); 1.65 (m, 2H); 1.74 (1H); 1.81 (m, 2H); 2.20 (s, 4H); 2.69 (m, 3H); 3.90 (m, 1H); 3.99 (m, 2H); 7.25 (d, J = 8.1 Hz, 2H); 7.33 (t, J = 7.4 Hz, 1H); 7.44 (t, J = 7.7 Hz, 2H); 7.56 (d, J = 8.1 Hz, 2H); 7.64 (d, J = 8.2 Hz, 2H); 8.01 (t, J = 8.3 Hz, 1H). 13C NMR (DMSO-d6), δ(ppm):14.1, 17.9, 24.2, 25.0, 32.2, 32.9, 35.9, 37.6, 39.0, 40.6, 48.0, 49.3, 59.7, 126.3, 126.5, 127.2, 128.9, 129.8, 137.8, 138.1, 140.0, 171.7, 175.0, 175.4. 4-{[(2S,4S)-1-(1,1'-biphenyl)-5-ethoxy-4-methyl-5-oxo-2-pentanyl]amino}-4-oxobutanoic acid, cyclohexyl amine salt (2S,4S)-1 CHA. The target compound (2S,4S)-1 CHA (1.95 g, yield 78%) was prepared from (2S,4S)-6 (1.7 g) according to procedure described for (2S,4R)-1 CHA salt. Purity by HPLC 99.34%. [M+H]+ m/z=412.2114 (C24H30NO5), the product contains 0.42% (2S,4R)-1, other optical isomers less than 0.03 %. 1H NMR (DMSO-d6), δ(ppm): 1.06 (m, 3H); 1.07-1.21(m, 8H); 1.46 (m, 1H); 1.65 (m, 1H); 1.82 (m, 5H); 2.18 (m, 4H); 2.43 (m, 1H); 2.69 (m, 3H); 4.02 (m, 3H); 7.26 (d, J = 8.1 Hz, 2H); 7.33 (t, J = 7.4 Hz, 1H); 7.44 (t, J = 7.6 Hz, 1H); 7.57 (d, J = 8.2 Hz, 2H); 7.64 (d, J = 8.1Hz, 2H); 8.01 (t, J = 9.0 Hz, 1H). 13C NMR (DMSO-d6), δ(ppm): 14.1, 16.1, 24.2, 25.0, 32.0, 33.1, 35.7, 37.6, 39.0, 40.7, 47.2, 49.3, 59.8, 126.3, 126.5, 127.2, 128.9, 129.8, 137.8, 138.2, 140.0, 171.7, 174.9, 175.9. 4-{[(2R,4S)-1-(1,1'-biphenyl)-5-ethoxy-4-methyl-5-oxo-2-pentanyl]amino}-4-oxobutanoic acid, cyclohexyl amine salt (enantiomer) (2R,4S)-1 CHA. The target compound (2R,4S)-1 CHA (1.20 g, yield 90%) was prepared from (2S,4R)-6 (0.9 g) according to procedure described for (2S,4R)-1 CHA salt. Purity by HPLC 99.45 %. [M+H]+ m/z=412.2112 (C24H30NO5), the product contains 0.22% (2R,4R)1, other optical isomers less than 0.03 %. 1H NMR (DMSO-d6), δ(ppm):1.12-1.21 (m, 12H); 1.36 (m, 1H); 1.53 (m, 1H); 1.65 (m, 2H); 1.74 (1H); 1.81 (m, 2H); 2.20 (s, 4H); 2.69 (m, 3H); 3.90 (m, 1H); 3.99 (m, 2H); 7.25 (d, J = 8.1 Hz, 2H); 7.34 (t, J = 7.4 Hz, 1H); 7.44 (t, J = 7.7 Hz, 2H); 7.57 (d, J = 8.1 Hz, 2H); 7.64 (d, J = 8.2Hz, 2H); 8.02 (t, J = 8.3 Hz, 1H). 13C NMR (DMSO-d6), δ(ppm): 14.0, 17.9, 24.2, 25.0, 32.3, 32.9, 35.9, 37.6, 39.0, 40.5, 48.0, 49.3, 59.7, 126.3, 126.5, 127.2, 128.9, 129.8, 137.8, 138.1, 140.0, 171.7, 175.1, 175.4. 4-{[(2R,4R)-1-(1,1'-biphenyl)-5-ethoxy-4-methyl-5-oxo-2-pentanyl]amino}-4-oxobutanoic acid, cyclohexyl amine salt (2R,4R)-1 CHA. The target compound (2R,4R)-1 CHA (624 mg, yield 63%) was prepared from (2R,4R)-6 (676 mg) according to procedure described for (2S,4R)-1 CHA salt. Purity by HPLC 98.0 %. [M+H]+ m/z=412.2112 (C24H30NO5), the product contains 2.4 % (2R,4S)-1, other optical isomers less than 0.03 %. 1H NMR (DMSO-d6), δ(ppm): 1.06-1.22 (m, 12H); 1.46 (m, 1H); 1.54 (dt, J = 12.9 / 3.1 Hz, 1H); 1.66 (m, 3H); 1.82 (m, 2H); 2.18 (m, 4H); 2.43 (m, 1H); 2.70 (m, 3H); 4.03 (m, 2H); 7.26 (d, J = 8.1 Hz, 2H); 7.33 (t, J = 7.4 Hz, 1H); 7.44 (t, J = 7.6 Hz, 1H); 7.57 (d, J = 8.2 Hz, 2H); 7.64 (d, J = 8.1Hz, 2H); 7.99 (t, J = 9.0 Hz, 1H). 13C NMR (DMSO-d6), δ(ppm): 14.1, 16.2, 24.2, 25.0, 32.3, 32.4, 32.5, 35.7, 37.6, 39.0, 40.7, 47.2, 49.2, 59.8, 126.3, 126.5, 127.2, 128.9, 129.8, 137.8, 138.3, 140.0, 171.9, 175.3, 175.9. AUTHOR INFORMATION Corresponding Author e-mail: [email protected]


Page 10 of 11

Page 11 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ACKNOWLEDGMENT We have no words to express our deepest appreciation to the management of Zentiva k.s. for versatile support of this work especially for excellent leadership of our department. The corresponding author dedicates this paper to his dear colleagues Mrs. Lucie Halamová, Mrs. Ludmila Hejtmánková, Mr. Petr Lustig, Mr. Jindřich Richter and Mr. Josef Jirman for their patience, advice and friendship over the past 21 years. REFERENCES (1) McMurray, J. J. V.; Packer, M.; Desai, A. S.; Gong, J.; Lefkowitz, M. P.; Rizkala, A. R.; Rouleau, J. L.; Shi, V. C.; Solomon, S. D.; Swedberg, K.; Zile, M. R. Investigators and Committees (August 30, 2014). Angiotensin–Neprilysin Inhibition versus Enalapril in Heart Failure. N. Engl. J. Med. 371: 993– 1004. (2) US Food and Drug Administration. https://www.fda.gov/Drugs/DevelopmentApprovalProcess/DrugInnovation/ucm474696.htm. Novel Drugs Summary 2015. (3) Ksander, G. M.; Ghai, R. D.; de Jesus, R.; Diefenbacher, C.; Yuan, A.; Berry, C.; Sakane, Y.; Trapani, A. Dicarboxylic Acid Dipeptide Neutral Endopeptidase Inhibitors. J. Med. Chem., 1995, 38 (10), 1689–1700. (4) Hook, D.; Wietfeld, B.; Lotz, M. Process for preparing biaryl substituted 4-amino-butyric acid or derivatives thereof and their use in the production of NEP inhibitors. WO2008031567 A1, 2008. (5) Piscopo, C. G.; Gallou, F.; Leitner, W.; Franciò, G. Diastereoselective Synthesis of an Industrially Relevant 4-Aminopentanoic Acid by Asymmetric Catalytic Hydrogenation in a Biphasic System Using Aqueous Sodium Hydroxide as Substrate Phase. Synthesis 2017; 49(02): 353-357. (6) ICH Q3A Impurities in New Drug Substances, R2; International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH): Geneva, Switzerland, October 2006; http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3A_R2/Step4/Q3A_ R2__Guideline.pdf. (7) Halama, A.; Zvatora, P.; Dammer, O.; Stach, J.; Zapadlo, M.; Krejcik, L.; Voslar, M. A method for the preparation, isolation and purification of pharmaceutically applicable forms of AHU-377. WO2016074651 A1, 2016. (8) Halama, A.; Zvatora, P.; Voslar, M.; Stach, J.; Zapadlo, M.; Dammer, O.; Krejcik, L.; Dvorakova, L.; Rezankova, M.; Vyslouzil, R. The solid forms of the ethyl ester of (2R,4S)-5-(biphenyl-4-yl)-4-[3carboxy-propionyl)amino]-2-methylpentanoic acid, its salts and the method of its preparation. WO2017097275 A1, 2017.


Synthesis, Isolation and Analysis of Stereoisomers of Sacubitril

Recommend Documents