Synthesis, Isolation, and Analysis of Stereoisomers of Sacubitril

Article abstract of DOI:10.1021/acs.oprd.8b00350

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.

Full text of DOI:10.1021/acs.oprd.8b00350

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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
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Synthesis, Isolation and Analysis of Stereoisomers of
Sacubitril.
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Aleš Halama,* Michal Zapadlo
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Department of Chemical Synthesis, Zentiva k.s., U kabelovny 130, Prague 102 01, Czech Republic
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TOC
5
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O
O
O
O
H
H
(R)
N
N
O
NH3
OEt
O
OEt
(S)
NH3
O
O
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(2S,4R)-1 CHA
(2R,4S)-1 CHA
sacubitril enantiomer
reference standard
sacubitril
reference standard
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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
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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
OH
N
O
O
HN
N
N
H
N
N
HO
OEt
O
O
2
(2S,4R)-1
sacubitril
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.
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Scheme 1. The First Reported Synthetic Route of Sacubitril (2S,4R)-1.
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H
O
O
H
N
H
OCH3
O
N
H
(a) (b) (c)
(d)
Boc
OEt
Boc
OEt
(g)
N
Boc
(e)
(f)
9
OH
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Boc-D-Tyr-OMe
(R)-4
(2R,4S)-5
major
diastereoisomer
(h)
O
O
O
O
O
H
H
N
(j)
N
H2N
t-BuO
OEt
MO
OEt
OEt
(i)
O
O
(k)
after step (i) M=H (2S,4R)-1
after step (k) M=Na (2S,4R)-1 Na salt
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3
(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.
H
O
O
H
H
N
N
Boc
ONa
Boc
OH
(a)
(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.
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Scheme 3. The Developed Short Industrial Process for Sacubitril Sodium Salt Synthesis Including
Purification via Cyclohexyl Amine Salt.
O
O
O
O
HCl
H2N
H
H
N
(a)
N
(b)
Boc
OH
OEt
O
OEt
NH3
O
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(2S,4R)-1 CHA salt
(2R,4S)-6
(2R,4S)-5
(c)
O
O
H
N
O
OEt
Na
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.
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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
O
O
O
H
O
H2/Pd,
ethanol
H
N
H
N
H
H
N
(S)
(S)
(S)
N
Boc
O
Boc
OH
Boc
OH
Boc
OH
(S)
(S)
(R)
H3N
+
after isomers
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pre-separation
major
(2R,4S)-5
isolated yield 58 %
minor
(2S,4S)-5
(2S,4S)-5 CHA salt
isolated yield 10 %
(R)-4b
NH2
O
H
N
O
O
H
O
(R)
H
N
H
H
N
(R)
(R)
Boc
O
(R)
N
H2/Pd,
ethanol
(R)
Boc
OH
Boc
OH
Boc
OH
(S)
H3N
+
after isomers
pre-separation
major
(2S,4R)-5
isolated yield 53 %
minor
(2R,4R)-5
(S)-4b
R R
(2 ,4 )-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.
O
O
O
O
HCl
H2N
H
H
(S)
(S)
N
N
Boc
OH
OEt
O
OEt
(R)
(R)
NH3
(b)
(a)
O
(2S,4R)-1 CHA salt
sacubitril reference standard
isolated yield 89 %
(2R,4S)-5
(2R,4S)-6
O
O
O
O
HCl
H2N
H
H
(S)
(S)
N
N
Boc
O
O
NH3
OEt
OEt
(S)
(S)
(b)
(a)
O
H3N
(2S,4S)-1 CHA salt
sacubitril diastereoisomer
reference standard
(2S,4S)-6
(2S,4S)-5 CHA salt
isolated yield 73 %
O
O
O
O
HCl
H2N
H
N
H
(R)
(R)
N
Boc
OH
OEt
O
NH3
OEt
(S)
(S)
(b)
(a)
O
(2R,4S)-1 CHA salt
sacubitril enantiomer
reference standard
isolated yield 88 %
(2S,4R)-5
(2S,4R)-6
O
HCl
H2N
O
O
O
H
N
H
(R)
(R)
N
OEt
Boc
O
O
OEt
(R)
(R)
(b)
(a)
NH3
O
H3N
(2R,4R)-1 CHA salt
sacubitril diastereoisomer
reference standard
isolated yield 59 %
(2R,4R)-5 CHA salt
(2R,4R)-6
(a) SOCl2, EtOH, cyclopentylmethyl ether, heptane. (b) Succinic anhydride, triethyl amine, CH2Cl2;
cyclohexyl amine (CHA), EtOAc.
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(2S,4S)-
0.008
0.006
0.004
5
(2S,4R)-
5
(2R,4R)-
8
9
0.002
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0.000
5
(2R,4S)-
-0.002
0.00
16.00
28.00
4.00
8.00
12.00
20.00
24.00
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 %.
1
(2S,4S)-
1.50
1.00
0.50
0.00
1
(2S,4R)-
1
(2R,4S)-
1
(2R,4R)-
4.00
6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
Minutes
15.00
14.00
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 DMSO-
d6 (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
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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.
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(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 acetate-
heptane 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
1
(C23H30NO4). H 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
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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
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1%). [M+H]+ m/z=384.2167 (C23H30NO4). H 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
1
vacuum dryer at 60°C for 4 hours to give product as a white powder (30.5 g, yield 96 %). H 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
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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.
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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
1, other optical isomers less than 0.03 %. H 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: ales.halama@centrum.cz
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ACKNOWLEDGMENT
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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.
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method
of
its
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