Chemoenzymatic synthesis of the potential antihypertensive agent (2R,2′S)-β-hydroxyhomometoprolol

Article abstract of DOI:10.1016/j.tetasy.2008.11.003

The kinetic resolution of 1-chloro-3-[4-(2-methoxyethyl)phenoxy]-2-propanol rac-4 with Novozym 435 and vinyl stearate, a key step in the gram-scale synthesis of (2S)-2-[[(2R)-2-hydroxy-3-[4-(2-methoxyethyl)phenoxy]propyl]amino]-1-butanol (R,S)-1 a potent antihypertensive agent currently under investigation, is reported here. Our approach differs from the previously reported synthesis, which involves a tedious and poorly effective fractional crystallization of (R,S)-1. This novel approach incorporates an enzymatic resolution for the efficient preparation of the oxirane precursor (R)-3. The two main advantages arising from this strategy are the high enantioselectivity of the enzymatic process and the facilitated recovery of the hydrophobic stearate intermediate (S)-5.

Full text of DOI:10.1016/j.tetasy.2008.11.003

Tetrahedron: Asymmetry 19 (2008) 2439–2442
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Chemoenzymatic synthesis of the potential antihypertensive agent
(2R,20S)-b-hydroxyhomometoprolol
a
a
b
c
Ignacio Regla a, , Axel Luviano-Jardón , Patricia Demare , Enrique Hong , Alejandro Torres-Gavilán ,
*
Agustin López-Munguía c, Edmundo Castillo c,
*
a Facultad de Estudios Superiores Zaragoza, Universidad Nacional Autónoma de México, Batalla del 5 de mayo y Fuerte de Loreto, Iztapalapa 09230, Mexico
b Departamento de Farmacobiología, Centro de Investigación y Estudios Avanzados del IPN, Apartado Postal 14-740, 07000, México, D.F., Mexico
c Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Mor. 62271, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 June 2008
Accepted 5 November 2008
Available online 29 November 2008
The kinetic resolution of 1-chloro-3-[4-(2-methoxyethyl)phenoxy]-2-propanol rac-4 with Novozym 435
and vinyl stearate, a key step in the gram-scale synthesis of (2S)-2-[[(2R)-2-hydroxy-3-[4-(2-methoxy-
ethyl)phenoxy]propyl]amino]-1-butanol (R,S)-1 a potent antihypertensive agent currently under investi-
gation, is reported here. Our approach differs from the previously reported synthesis, which involves a
tedious and poorly effective fractional crystallization of (R,S)-1. This novel approach incorporates an
enzymatic resolution for the efficient preparation of the oxirane precursor (R)-3. The two main advanta-
ges arising from this strategy are the high enantioselectivity of the enzymatic process and the facilitated
recovery of the hydrophobic stearate intermediate (S)-5.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
ary alcohol center and an (S)-configuration at C-2 of the nitrogen
chain (R,S)-1 showed significant, but short-lasting hypotension as
In the search for new antihypertensive drugs with better thera-
peutic potential and fewer side effects, we recently reported the
synthesis and pharmacological evaluation of four new analogues
of Metoprolol with an additional stereogenic center (Fig. 1).1
Although these analogues did not cause b1-adrenergic receptor
inhibition, the diastereomer with (R)-configuration at the second-
well as a modest and short-lasting bradychardia, suggesting anti-
arrhythmic activity. This compound, (2S)-2-[[(2R)-2-hydroxy-
3-[4-(2-methoxyethyl)phenoxy]propyl]amino]-1-butanol (R,S)-1,
was tentatively named (2R,20S)-b-hydroxyhomometoprolol (Fig. 1).
The first step in the previously reported synthesis of the new
compounds (R,S)-1 and (S,S)-1 involves the reaction between the
O
O
O
N
H
O
N
H
OH
OH
OH
OH
O
1
(S,R)-
(R,S)-1
O
N
H
OH
( )-Metoprolol
O
O
O
N
H
O
N
H
OH
OH
OH
OH
(S,S)-1
1
(R,R)-
Figure 1. Structure of metoprolol and b-hydroxyhomometoprolol diastereomers.
* Corresponding authors. Tel.: +52 55 5623 0795; fax: +52 73 172388.
E-mail addresses: regla@unam.mx (I. Regla), edmundo@ibt.unam.mx (E. Castillo).
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.11.003

2440
I. Regla et al. / Tetrahedron: Asymmetry 19 (2008) 2439–2442
O
O
N
H
(S)
OH
OH
H2N
OH
O
1
(R,S)-
O
MeOH
O
rac-3
O
O
N
H
OH
OH
(S,S)-1
Scheme 1. Previously reported chemical synthesis of (R,S)-1 and (S,S)-1.1
racemic epoxide ( )2-((4-(2-methoxyethyl)phenoxy)methyl)oxi-
rane rac-3 and (S)-2-amino-1-butanol (Scheme 1).
tional crystallization step required for the separation of diastereo-
mers (R,S)-1 and (S,S)-1. As an alternative, we introduced a
chemoenzymatic step that would lead to the preparation of the
oxirane enantiomer (R)-3 in good enantiomeric excess. From this
enriched precursor, the addition of (S)-2-amino-1-butanol directly
provides diastereomer (R,S)-1 (Scheme 2).
While the preparation of these compounds seems straightfor-
ward, it requires the separation of the diastereomeric products
(R,S)-1 and (S,S)-1 via a tedious fractional crystallization. Further-
more, the desired diastereomer (R,S)-1 remains in the mother li-
quors, and is isolated in reduced yields (21% yield from rac-3).
Over the last few years, enzymes in organic media have become
a useful tool in organic synthesis due to their chemo-, regio-, and
enantioselective properties. Due to their high stability and catalytic
promiscuity, particular attention has been paid to lipases (triacyl-
glycerol hydrolases, EC 3.1.1.3), since they have been shown to
be suitable enzymes for the catalysis of a wide variety of chemical
reactions.2 Furthermore, because of the high enantioselectivity of
the enzymatic process, they have been used extensively in the res-
olution of alcohols, esters, carboxylic acids, amino acids, amines,
and amides.3,4 One of the most commonly used lipases in organic
syntheses is lipase B from Candida antarctica (CaLB), which has
an operational limit of up to 80 °C in its immobilized form (Nov-
ozymÒ 435) and works efficiently in different organic solvents, as
it is highly resistant to denaturalization.5–7 According to Kazlaus-
kas’ rules,8 the steric effect of the substrate is a decisive factor in
the degree of enantioselectivity in acylation reactions of chiral sec-
ondary alcohols; under this consideration, CaLB should have a pref-
erence for the (R) enantiomer.9
2. Results and discussion
In the first step, the synthesis of 2-[4-(20-methoxyethyl)-phen-
oxymethyl]-oxirane rac-3 was carried out by treatment of the
potassium salt of 4-(2-methoxyethyl)phenol 2 with an excess of
( )-epichlorohydrin (90% yield). The racemic chlorohydrin rac-4
was then obtained by the regioselective ring-opening of rac-3 with
lithium chloride and acetic acid, in a nearly quantitative yield
(99%).10
In this step, we introduced the enzymatic resolution of rac-4, by
taking advantage of the reported (R)-specificity of lipase Nov-
ozymÒ 435 during alcoholysis reactions.9 An additional advantage
was introduced using a hydrophobic acyl moiety that allows a
facilitated separation of the hydrophobic enantiomer (S)-5 from
the non-reacted polar (R)-4. Accordingly, rac-4 was reacted at
37 °C with NovozymÒ 435, and the vinyl esters of acetic, lauric,
oleic, and stearic acids were used as acyl donors in 95:5 hexane–
acetone as solvent. All reactions were stopped after 28 h, in a com-
promise between yield and ee, as lower conversions resulted in
better ee but modest yields of (S)-5. After these reaction times,
the desired (S)-chlorohydrin esters were recovered and separated
In order to carry out detailed pharmacologic and pharmacoki-
netic studies of (R,S)-1, a more efficient gram-scale chemoenzy-
matic synthesis of this compound was developed. This new
strategy is based on elimination of the previously reported frac-
O
O
O
LiCl / AcOH
1) KOH/MeOH
2) ( )-Epichlorohydrin
90 %
THF
O
O
Cl
OH
O
OH
99 %
rac-3
2
rac-4
Novozym 435
O
(CH2)16CH3
n-hexane-acetone 95:5
24 h, 37o
C
O
(S)
H2N
1M NaOH
35 %
O
O
OH
95 %
MeOH
(R,S)-1
96 %
O
O
Cl
(CH2)16CH3
O
O
O
3
(R)-
5
(S)-
Scheme 2. Chemoenzymatic synthesis of (2R,20S)-b-hydroxyhomometoprolol, (R,S)-1.

I. Regla et al. / Tetrahedron: Asymmetry 19 (2008) 2439–2442
2441
by column chromatography. It is important to note that in contrast
to other acyl residues traditionally tested in this type of resolu-
tion,11 the introduction of a stearyl residue was more advanta-
geous. Actually, the hydrophobic stearate (S)-5 was easily
separated after column chromatography due to its high solubility
in n-hexane–acetone (35.4% yield; theoretical maximum yield of
50%). Compound (S)-5 was easily recovered as a white crystalline
product, which was efficiently transesterified by treatment with
1 equiv of NaOH in MeOH to provide the desired (R)-3 in 96% yield
and 94.7% ee, along with the corresponding methyl stearate. Final-
ly, the nucleophilic opening of this epoxide with (S)-2-amino-1-
butanol led directly to the desired diastereomeric amino alcohol
(R,S)-1 in 95% yield.
4-(2-methoxyethyl)phenol 2 in 200 mL MeOH was placed. To this
solution, 45 g (683 mmol) of 85% KOH was added, and the mixture
was stirred for 1 h at 40–45 °C. Afterwards, 150 mL of MeOH was
distilled off at atmospheric pressure, and 1 L epichlorohydrin was
added to the semisolid residue. After 24 h of stirring at 40 °C and
TLC monitoring (CH2Cl2–AcOEt 95:5), the mixture was washed
with water (3 ꢀ 100 mL, pH 7.5), dried over anhydrous Na2SO4,
and vacuum distilled in order to recover excess epichlorohydrin
(190 mmHg/70 °C). Toluene (120 mL) was added to the residue,
and the mixture was distilled again at 75 mmHg/40 °C until con-
stant weight to obtain 147.11 g of a mixture containing rac-3
(90%) and rac-4 (10%).1,12
4.2.2. ( )-1-[4-(2-Methoxyethyl)phenoxy]-3-chloro-2-propanol
rac-4
3. Conclusion
To a solution of rac-3 (30 g (144.1 mmol)) in 250 mL THF,
19.78 mL (432.46 mmol, 3 equiv mol) acetic acid and 9.78 g
(230.4 mmol, 1.6 equiv mol) LiCl were added. The reaction mixture
was stirred at room temperature for 48 h. After TLC verification of
the end of the reaction (CH2Cl2–AcOEt 95:5), the reaction mixture
was concentrated under reduced pressure and partitioned in
300 mL water and 150 mL AcOEt. The organic layer was washed
with saturated NaHCO3 until free from acid, dried with anhydrous
Na2SO4, and concentrated in a rotary evaporator to yield 34.88 g of
pure product as a colorless oil in 99% yield. 1H NMR (CDCl3,
300 MHz) d: 2.82 (t, J = 6.9 Hz, 2H), 3.34 (s, 3H), 3.56 (t, J = 7.2 Hz,
2H), 3.66 (dd, J1 = 11.4, J2 = 6 Hz, 1H), 3.73 (dd, J1 = 11.4,
J2 = 5.1 Hz, 1H), 3.99 (dd, J1 = 6.3, J2 = 1.2 Hz, 1H), 4.05 (dd, J1 = 6,
J2 = 1.2 Hz, 1H), 4.17 (quintet, J = 10.5 Hz, 1H), 6.81 (d, J = 9 Hz,
2H), 7.11 (d, J = 9 Hz, 2H). 13C NMR (CDCl3, 75 MHz) d: 35.1, 45.8,
58.5, 68.5, 69.7, 73.6, 114.4, 129.7, 131.7, 156.6. Lit.13,14
The global process involving the chemoenzymatic synthesis of
target compound (R,S)-1 resulted in a 31% overall yield from the
racemic oxirane precursor rac-3. For the most part, this strategy
was successful because of the high enantioselectivity exhibited
by NovozymÒ 435 lipase and the facile recovery of (S)-5 which,
after transesterification and reaction with (S)-2-aminobutanol,
provided an easily recrystallized (R,S)-1, leaving only a small
amount of unwanted diastereomer (S,S)-1 in the mother liquors.
This chemoenzymatic approach completely avoids the use of the
inefficient fractional crystallization for (R,S)-1 involved in the
chemical syntheses previously reported.
4. Experimental
4.1. Materials and instruments
4.2.3. (2S)-1-Chloro-3-[4-(2-methoxyethyl)phenoxy]-2-
propanol-2-stearate (S)-5
In a tightly closed vessel, 12 g (49.1 mmol) of rac-4 was dis-
solved in a mixture of 360 mL hexane and 20 mL acetone. To this
solution, 4.5 g of 4 Å molecular sieves and 8 g (25.7 mmol,
Lipase B from Candida antarctica was used in its immobilized
form NovozymÒ 435. The reagents were purchased from Aldrich
Chemical Company. Reactions were monitored by TLC using silica
gel as the stationary phase and visualized using UV irradiation
254/366 nm and iodine vapors. In the chromatographic columns,
silica gel was used as the stationary phase 70–230 mesh. The enzy-
matic resolution was conducted in a Shaker bath orbital incubator.
1H and 13C NRM were determined in a JEOL Eclipse instrument
at 300 and 400 MHz, using tetramethylsilane as an internal stan-
dard and CDCl3 as a solvent. The infrared spectra were obtained
in a Bruker spectrometer model Tensor 27. Optical rotations were
determined in a Perkin Elmer polarimeter model 341 using a 1 dm
cell length. Measurements were done using the sodium D-line
(589 nm) at a sample compartment temperature of 20 oC. The mass
spectra were determined in a JEOL instrument model JMS-SX102A.
Elemental analyses were obtained with a Exeter Analytical CE-440
apparatus.
0.5 equiv mol) of vinyl stearate were added (a 60 lL aliquot was
saved as a reference) together with 3.8 g of the biocatalyst Nov-
ozym 435. This mixture was incubated for 28 h a 37 °C with orbital
stirring at 200 rpm. Afterwards, the mixture was filtered through
Celite and concentrated under reduced pressure to yield 19.5 g of
a crude product, which after separation in a chromatographic col-
umn in 370 g of silica gel (hex–AcOEt 95:5) rendered 8.85 g (35.4%)
of (S)-5 as white crystals with a mp 42–43 °C and ½a D20
¼ þ13:6 (c
ꢂ
1, CHCl3).
1H NMR (CDCl3, 300 MHz) d: 0.87 (t, J = 6.9 Hz, 3H), 1.25 (s,
14H), 1.63 (quintet, J = 7.2 Hz, 2H), 2.30–2.38 (m, 2H), 2.82 (t,
J = 6.9 Hz, 2H), 3.34 (s, 3H), 3.56 (t, J = 7.2 Hz, 2H), 3.73–3.87 (m,
2H), 4.09–4.18 (m, 2H), 5.32 (quintet, J = 10.2 Hz, 1H), 6.82 (d,
J = 9 Hz, 2H), 7.12 (d, J = 9 Hz, 2H). 13C NMR (CDCl3, 75 MHz) d:
14.0, 22.6, 24.7, 24.8, 29.0, 29.2, 29.3, 29.4, 29.5, 29.6, 31.8, 31.9,
33.7, 34.2, 35.2, 42.4, 42.5, 58.6, 66.0, 70.8, 73.7, 114.5, 129.8,
131.8, 156.7, 173.0. IR tmax (KBr) cmꢁ1: 2928, 2850, 1738, 1702,
1467, 1246, 1168, 944. EM (IE) m/z (%) M+: 511 (2.5), 359 (100),
331 (3), 244 (4), 147 (7.5), 107 (7), 71 (7.5), 57 (12), 43 (11). Ele-
mental Anal. Calcd for C30H51ClO4 (511.18): C, 70.49; H, 10.06.
Found: C, 70.50; H, 9.84.
4.2. Chiral HPLC analysis
Enantiomers of 2-{[4-(2-methoxyethyl)phenoxy] methyl}oxi-
rane were quantified in
a
CHIRALCEL OB-H column (4.6 ꢀ
250 mm) (Chiral Technologies Inc., West Chester PA, USA), using
a Waters 600E system controller and a Waters 996 photodiode ar-
ray detector at 224 nm (Waters Copr. Milford, MA, SA). The effluent
was n-hexane–ethanol 90:10 (v/v) at a flow rate of 1 ml minꢁ1. The
retention time for the (R)-3 isomer is 16.2 min and 19.5 min for the
(S)-3 enantiomer.
4.2.4. (R)-2-{[4-(2-Methoxyethyl)phenoxy]methyl}oxirane
(R)-3
16.2 mL of 1 M NaOH (1 equiv mol) was added to a solution
containing 8.3 g (16.26 mmol) of (S)-5 in 400 mL MeOH (ultra-
sound-assisted solution) and stirred for 24 h at room temperature,
while monitoring the reaction progress by TLC (hex–AcOEt 85:15).
After completion, the reaction medium was cooled to 0–5 °C for
4.2.1. ( )-2-{[4-(2-Methoxyethyl)phenoxy]methyl}oxirane
rac-3
In a 2 L three-necked flask, fitted with a mechanical stirrer, a
condenser, and a thermometer, a solution of 100 g (657 mmol) of

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I. Regla et al. / Tetrahedron: Asymmetry 19 (2008) 2439–2442
1 h, and the solids were vacuum filtered and washed with 75 mL of
cold MeOH. The filtrate was evaporated under reduced pressure,
and the remaining oily liquid (5.5 g) was purified by column chro-
matography in silica gel (1. hex–AcOEt 95:5, 2. hex–AcOEt 85:15)
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to yield 3.23 g of (R)-3 in 96% yield and 94.7% ee. ½a D20
¼ ꢁ11:3 (c
ꢂ
1, MeOH).
4.2.5. (2R,20S)-b-Hydroxyhomometoprolol (R,S)-1
To a solution of (R)-3 (2 g; 9.6 mmol) in 14 mL MeOH, 5.81 g
(65.17 mmol, 6.7 M equiv) of (S)-2-amino-1-butanol was added.
After 24 h of stirring and TLC monitoring (CH2Cl2–AcOEt 95:5),
the reaction mixture was evaporated under reduced pressure and
partitioned in 20 mL CHCl3 and 20 mL of water. After drying the or-
ganic extract and evaporating the solvent, the remaining solid
(3.1 g) was recrystallized from toluene to yield 2.66 g (95%) of
(R,S)-1, mp 74–75 °C, ½a D20
ꢂ
¼ þ10:3 (c 1, MeOH). Lit1 mp 74–
75 °C, ½a 2D0
¼ þ10:3 (c 1, MeOH).
ꢂ
Acknowledgments
The authors acknowledge DGAPA-UNAM for Ignacio Regla’s
sabbatical fellowship at IBT-UNAM. Authors are also grateful to
María de los Angeles Peña, Elizabeth Huerta, Erendira García Rios,
Javier Pérez-Flores and Rocío Patiño-Maya from Instituto de Quí-
mica, UNAM for spectroscopic analysis and to Novozymes-México
for the generous gift of CaLB.
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