Chemoenzymatic route to S-betaxolol

Article abstract of DOI:10.1080/00397911.2010.505699

An efficient chemoenzymatic route to S-betaxolol is reported. A strain (Rhodotorula mucilaginosa DQ832198) screened from soil was used as biocatalyst for the kinetic resolution of the key acetylated intermediates. Excellent enantiomeric excess (ee99%) was

Full text of DOI:10.1080/00397911.2010.505699

Synthetic Communications1, 41: 2468–2474, 2011
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2010.505699
CHEMOENZYMATIC ROUTE TO S-BETAXOLOL
Yong-Hong Li,1,2 Li-Hua Huang,1,2 and Hong-Min Liu1,3
1New Drug Research and Development Center, Zhengzhou University,
Zhengzhou, China
2Department of Chemistry, Zhengzhou University, Zhengzhou, China
3School of Pharmaceutical Sciences, Zhengzhou University,
Zhengzhou, China
GRAPHICAL ABSTRACT
Abstract An efficient chemoenzymatic route to S-betaxolol is reported. A strain (Rhodo-
torula mucilaginosa DQ832198) screened from soil was used as biocatalyst for the kinetic
resolution of the key acetylated intermediates. Excellent enantiomeric excess (ee > 99%)
was obtained under very mild conditions. The biocatalyst is quite stable and could be used
several times with little decrease of the resolving ability.
Keywords Chemoenzymatic route; kinetic resolution; Rhodotorula mucilaginosa;
S-betaxolol
INTRODUCTION
Pharmacological studies have shown that an organism often reacts in a differ-
ent way when it interacts with each enantiomer of the same molecule, and this pro-
moted chirality studies in pharmaceutical research. Recently one of the most studied
classes of drug has been the b-blockers, because of the increase in cardiovascular dis-
eases. Studies on these drugs showed that for compounds such as 1-alkylamino-3-
aryloxypropan-2-ols, the cardiovascular activity resides mainly in their (S)-isomer,
whereas the (R)-isomer is usually responsible for the side effects.[1] Our studies are
related to betaxolol, a very interesting b1-selective blocker, whose (S)-isomer has
much stronger activity in reducing intraocular pressure and is the most widely used
antiglaucoma agent.[2]
Received November 3, 2009.
Address correspondence to Hong-Min Liu, New Drug Research and Development Center,
Zhengzhou University, Zhengzhou 450001, China. E-mail: liuhm@zzu.edu.cn
2468

CHEMOENZYMATIC ROUTE TO S-BETAXOLOL
2469
Scheme 1. Synthetic outlines of S-betaxolol. (a) epichlorohydrin, K2CO3, CH3CN, reflux, 6 h; (b)
i-PrNH2, CH3OH, rt, 7 h; (c) Ac2O, pyridine, Et3N, rt; (d) Rhodotorula mucilaginosa, 28 ꢀC, 12 h; (e)
NaOH, CH3OH, rt; (f) benzaldehyde, toluene, paratoluenesulfonic acid, reflux; (g) C4H7Br, DMF,
NaH, ꢁ15 ꢀC 3 h, rt, 2 h; and (h) HCl, rt.
The synthesis of drugs in their enantiomerically pure form becomes very
important for the pharmaceutical industries, because of increased demand for more
effective and safer single isomers. Both asymmetric synthesis and chemoenzymatic
approaches have been described in the literature[3–7] for the preparation of
(S)-betaxolol. The asymmetric synthetic methods need rigorous reaction conditions,
expensive chiral catalyst, or optical voidness material. The growing need to find new
strategies with lower output cost pushes the research toward the biotransformation
field, in which the ability of microorganisms, and their cheap enzymes (especially
lipases), as enantioselective catalysts could be used for such a purpose. Lipases, with
their capacity to work in organic solvents unaffectedly and their high enantioselec-
tivity, offer an attractive route for industrial exploitation, because they could lower
output costs in a significant way. In the chemoenzymatic method reported by
Di Bono et al.[1] the enantiomeric excess of (S)-betaxolol was 82%, which was further
improved to 91% after crystallizing its hydrochloride salt. However, the catalyst used
in the method is pure enzyme, which is quite expensive, especially when a large
quantity is needed.
Herein we report a process for the chemoenzymatic synthesis of (S)-betaxolol
(Scheme 1). A strain (Rhodotorula mucilaginosa DQ832198) screened from soil was
used as biocatalyst for the kinetic resolution of the key acetylated intermediates. This
approach provides high enantioselectivity, and the catalyst is very cheap compared
with the approach reported before.
RESULTS AND DISCUSSION
Because of the interesting results reported[1] in the lipase-catalyzed resolution
of b-amino alcohols, we decided to try an enantioselective hydrolysis of the
N,O-diacetyl derivative 10 of betaxolol (9). With this target in mind, we projected

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Y.-H. LI, L.-H. HUANG, AND H.-M. LIU
the synthesis of the betaxolol in racemic form, according to the patented method-
ology.[8] After purification, betaxolol was N,O-bisacetylated in quantitative yield
by acetic anhydride in pyridine, and microbe screening was carried out on this acety-
lated product 10 to differentiate which strains were able to catalyze the hydrolysis of
either the ester or amide functionality. In this screening, 52 strains that can live on
the substrate as the only carbon source and have the ability to produce transparent
circles were used. The screening was analyzed by thin-layer chromatography (TLC)
and optical rotations, and the results showed that only two strains, 1 and 2 were able
to catalyze the hydrolysis of the substrate.
The scale-up of these reactions allowed the determination of the final products
R- and S-betaxolol via high-performance liquid chromatography (HPLC) using a
Chiralcel OJ column. Results showed that both strains exhibit poor selectivity with
E values of 1.5 and 3.5 respectively.
Thus, a new substrate compound 4 was synthesized.[1,8,9] Compound 4 (14.86 g,
0.041 mol) was prepared from the reaction of compound 3 3-[4-(2-hydroxyethyl-
pheoxy-1-(N-isopropyl) amino-propan-2-ol (12.19 g, 0.051 mol) and Ac2O[1] with
the yield of 92%, and compound 3 was prepared via two steps from compound 1
according to the patented methodology.[8]
According to a previous screening, it was noticed that only 1 and 2 strains led
to significant amount of kinetic resolution after 24 h; hence a scale-up of these reac-
tions allowed the determination of enantiomeric excesses. After 24 h, 1 strain gave
intermediate 6 with eep of 45% and an enantioselectivity factor E of 5.3, while 2
strain gave intermediate 6 with eep of 95% and E of 10.6.
These results show that strain 2 is more suitable for use in kinetic resolution. It
has been identified as Rhodotorula mucilaginosa DQ832198 and was deposited in
China General Microbiological Culture Collection Center (GGMCC2257). It was
used as a biocatalyst for kinetic resolution in further investigations.
The steroselectivity of lipase in the strain is not perfect. A product with better
optical purity was obtained with lower yield: the better the optical purity, the lower
the yield. So a two-resolution procedure was adopted. Product with lower ee value
was obtained with greater yield first, and the product obtained was acetylated and
used as resolution substrate again. Product with greater ee value can be obtained
with greater yield after the substrate resolved twice.
After kinetic resolution twice catalyzed by 2 strain for 12 h, substrate 10 com-
pletely transferred into compound 11 with the molar ratio 65% and compound 12
with the molar ratio 35%. Compound 12 was the S-isomer, the one we need, and
0.96 g compound 12 was obtained from 3 g substrate 10 with the yield of 31%. Sub-
strate 4 completely transferred into compound 5 with the molar ratio 52%, and com-
pound 6 with the molar ratio 48%. Compound 6 was the S-isomer, the one we need,
and 0.59 g compound 5 (ee 95%) was obtained from the same substrate, with the
yield of 51.2%, and 0.68 g compound 6 was obtained from 4 g substrate 4, with
the yield of 47.5%.
Optically active intermediates (ꢁ)-6 and (ꢁ)-12 were easily converted in good
yields into S-betaxolol following the known procedure.[8] Compound 12 (1 g,
0.0026 mol) could be transferred into S-betaxolol (13) (0.71 g, 0.0023 mol) directly,
with the yield of 91% and ee value of 95%. Compound 6 (5 g, 0.018 mol) was trans-
ferred into compound 7 (4.22 g, 0.017 mol) with the yield of 98% first, and then

CHEMOENZYMATIC ROUTE TO S-BETAXOLOL
2471
turned into S-betaxolol (95%) with the yield of 98.1% according to the reported
methodology.[8,9]
S-Betaxolol was transferred into its hydrochloride (14) (ee 95%), according to
the reported methodology,[8,9] and its ee value was improved to 99% after recrystal-
lization from diethyl ether (Et2O).
CONCLUSION
In summary, we have established a new chemoenzymatic route to S-betaxolol
from cheap and easily obtained material 4-(2-hydroxyethyl)-phenol (1) with good
optical purity (ee > 99%) and moderate chemical yield (32.4% overall), including a
lipase-catalyzed kinetic resolution. We envisage that our route could find application
in the synthesis of b-amino alcohols with acyl or hydroxy functionality in stereogenic
carbon such as some b-adrenergic receptor blockers (propranolol, alptenolol, oxprena-
lol, pindolol, nadolol, timolol, esmolol, and flestolol), ephedrine, epinephrine, dropro-
pizine, and salbutamol sulfate. Further investigations to improve the stereoselectivity
through screening new substrates with longer acyl chains in stereogenic carbon and
performing the kinetic resolution in organic–aqueous double systems are under way.
EXPERIMENTAL
Reagent-grade solvents were distilled prior to use. All reported NMR spectra
were collected on a Bruker DPX 400 NMR spectrometer with tetramethylsilane
(TMS) as the internal reference. Infrared (IR) spectra were recorded on Nicolet
IR2001 instrument using KBr disks in the 400- to 4000-cm regions. High-resolution
mass spectra (HRMS) were obtained on a TM Waters Micromass Q-Tof Micro
instrument using the electrospray ionization (ESI) technique. Optical rotations were
determined on a Perkin-Elmer 341 polarimeter. Enantiomeric excess was determined
by chiral HPLC at room temperature using Syltech 500 pump equipped with a UV
500 version 4.1 ultraviolet detector with a Chiralcel OJ (4.6 mm ꢂ 250 mm) column.
Screening of the Microbes
Microbe screening proceeded according the method reported.[10] A strain with
steroselectivity lipase activity (Rhodotorula mucilaginosa DQ832198) was screened
from soil and used for kinetic resolution of betaxolol intermediates. The culture
medium of the strain contained corn paste 1%, peptone 0.5%, and glycerol 2% with
an initial pH of 6.0. The culture was carried out in 500-ml shaking flasks containing
110 ml medium at 32 ꢀC and 220 rpm for 16 h.
Enzyme activity of the strains was evaluated by the substrate conversion rate
after conversion for the same period catalyzed by different strains.
Preparation and Characterization of Kinetic Resolution Substrates
Preparation and characterization of compound 4. Compound 4 was pre-
pared from the reaction of compound 3 with Ac2O,[1] and compound 3 was prepared
from compound 1 according to the patented methodology.[8]

2472
Y.-H. LI, L.-H. HUANG, AND H.-M. LIU
1H NMR: 7.14, 6.80 (each 2H, d, J ¼ 8.5 Hz, Ar-H); 3.80 (2H, t, J ¼ 6.6 Hz,
H-1); 2.88 (2H, t, J ¼ 6.6 Hz, H-2); 3.60 (1H, dd, J ¼ 1.44 Hz, J ¼ 14.0 Hz, H-31);
3.4 (1H, dd, J ¼ 7.5 Hz, J ¼ 14.0 Hz, H-32); 4.46 (1H, s, H-4); 4.10 (1H, dd, H-51);
4.03 (1H, dd, H-52); 3.70 (1H, m, H-6); 1.26, 1.25 (each 3H, d, J ¼ 6.76 Hz,
ꢃ
J ¼ 6.56 Hz, CH3 2); 2.24 (9H, s, -COCH3).
ESI-MS m=z: calcd. for C20H29NO6 379.1995. Found: 380.2067 [M þ H]þ;
402.1888 [M þ Na]þ.
Preparation of compound 10. Compound 10 was prepared from the reac-
tion of racemic betaxolol (9) with Ac2O1, and racemic betaxolol was prepared
according to the patented methodology.[8]
Kinetics Resolution
The culture fluid of Rhodotorula mucilaginosa was filtered, and 0.1 M pH 6.0
Na2HPO4–citric acid buffer and 3 g=L substrate were added to a proper amount
of the obtained cells, and the mixture was incubated at 28 ꢀC for 12 h. The resulting
broth was extracted three times with an equal volume of ethyl acetate. The organic
layer was first washed three times with saturated NaHCO3, then three times with
saturated NaCl, and last with water. The resulting saturated NaHCO3 and NaCl
layer was extracted with acetic ether. The organic phase was combined and dried
over Na2SO4.
The resolution products were separated by flash chromatograph (eluent
acetone=petroleum ether 1:2). The intermediates resolved by 2 strain and 1 strain
are mostly the same except for the optical rotations, so the date of intermediates
resolved by 2 strain are reported as the following.
Data of Compound 5 and Compound 6
Compound 5. 1H NMR: 7.14, 6.80 (each 2H, d, J ¼ 8.5 Hz, Ar-H); 3.81 (2H,
t, J ¼ 6.6 Hz, H-1); 2.81 (2H, t, J ¼ 6.6 Hz, H-2); 3.60 (1H, dd, J ¼ 1.44 Hz,
J ¼ 14.0 Hz, H-3a); 3.43 (1H, dd, J ¼ 7.5 Hz, J ¼ 14.0 Hz, H-3b); 4.46 (1H, Ws,
H-4); 4.10 (1H, dd, H-5a); 4.03 (1H, dd, H-5b); 3.60 (1H, m, H-6); 1.26, 1.21 (each
3H, d, J ¼ 6.76 Hz, J ¼ 6.56 Hz, CHꢃ32); 2.20 (3H, s, -COCH3).
ESI-MS m=z: calcd. for C16H25NO4 295.1784. Found: 296.1876 [M þ H]þ;
20
20
318.1689 [M þ Na]þ. ½aꢄD2 ¼ þ285 (C ¼ 1, CH3OH), ½aꢄD1 ¼ þ116 (C ¼ 1, CH3OH).
Compound 6. 1H NMR: 7.14, 6.8 (each 2H, d, J ¼ 8.5 Hz, Ar-H); 3.82 (2H, t,
J ¼ 6.6 Hz, H-1); 2.82 (2H, t, J ¼ 6.6 Hz, H-2); 3.60 (1H, dd, J ¼ 1.44 Hz, J ¼ 14.0 Hz,
H-3a); 3.35 (1H, dd, J ¼ 7.5 Hz, J ¼ 14.0 Hz, H-3b); 4.46 (1H, Ws, H-4); 4.10 (1H,
dd, H-5a); 4.03 (1H, dd, H-5b); 3.70 (1H, m, H-6); 1.26, 1.22 (each 3H, d,
ꢃ
J ¼ 6.76 Hz, J ¼ 6.56 Hz, CH3 2); 2.24 (6H, s, -COCH3).
ESI-MS m=z: calcd. for C18H27NO5 337.1889. Found: 338.1958 [M þ H]þ;
20
360.1774 [M þ Na]þ. ½aꢄD2 ¼ ꢁ2700 (C ¼ 1, CH3OH), [a]D201# ¼ ꢁ711 (C ¼ 1,
CH3OH), ee ¼ 95%.
The data of compound 12 are the same as those of compound 10, except
ee ¼ 85%.

CHEMOENZYMATIC ROUTE TO S-BETAXOLOL
2473
Preparation and Characterization of S-Betaxolol
Preparation of S-betaxolol. Compound 6 and compound 12 were dissolved
in methanol and then stirred with excessive NaOH together, until the completion of
the reaction (TLC control). After excess methanol was removed, the residue was dis-
solved in ethyl acetate. The organic layer was washed first three times with saturated
NaHCO3, then three times with saturated NaCl, and last with water. The organic
layer obtained was then dried over Na2SO4 and concentrated under vacuum pressure.
1
Characterization of S-betaxolol. The IR and H NMR data of S-betaxolol
were identical with those described by Di Bono et al. ESI-MS m=z: calcd. for
20
C18H29NO3 307.2147; found: 308.2229 [M þ H]þ. ½aꢄD ¼ ꢁ13.0 (c 1.0, CHCl3), and
ꢁ19.6 (C ¼ 1, CH3OH), ee > 99%, which are identical to those described in the
literature.[1,5,7]
HPLC Conditions for Resolved Betaxolol and Related Intermediates
The HPLC conditions for intermediates 5 and 6 are as follows: n-hexane–
isopropanol ¼ 85:15, OJ, 1.0 mL minꢁ1, 273 nm. T5R ¼ 21.7 min, t5S ¼ 23.5 min,
t6R ¼ 28.1 min, t6S ¼ 32.0.[11]
The HPLC conditions for resolved betaxolol are as follows: n-hexane=
isopropanol=diethylamine 85:15:0.05, OJ, 1.0 mL minꢁ1, 273 nm. tR ¼ 7.3 min,
tS ¼ 9.3 min. The results are identical to those described in the literature.[5,7]
ACKNOWLEDGMENT
We acknowledge the National Natural Science Foundation of China for finan-
cial support of this work (20572103).
REFERENCES
1. Di Bono, G.; Scilimati, A. chemoenzymatic route to both enantiomers of betaxolol.
Synthesis 1995, 6, 699.
2. (a) Osborne, N. N.; Wood, J. P.; Chidlow, G.; Casson, R.; De Santis, L.; Schmidt, K. G.
Effectiveness of levobetaxolol and timolol at blunting retinal ischaemia is related to their
calcium and sodium blocking activities: Relevance to glaucoma. Brain Res. Bull. 2004, 62,
525; (b) Aoyama, Y.; Motoki, M.; Hashimoto, M.; Aoyama, Y.; Motoki, M.; Hashimoto,
M. Nippon Ganka. Effect of various antiglaucoma eyedrops on human corneal epithelial
cells. Gakkai Zasshi 2004, 108(2), 75.
3. Binet, J.; Manoury, P. S. S-Isomer of betaxolo: Its preparation and its application in
therapy. Patent GB2130585, 1985.
4. Muthukrishnan, M.; Garud, D. R.; Joshi, R. R.; Joshi, R. A. Concise synthesis of
b-blockers (S)-metoprolol and (S)-betaxolol using hydrolytic kinetic resolution.
Tetrahedron 2007, 6, 1872.
5. Joshi, R. A.; Garud, D. R.; Muthukrishnan, M.; Joshi, R. R.; Gurjar, M. K. A convenient
synthesis of the enantiomerically pure b-blocker (S)-betaxolol using hydrolytic kinetic
resolution. Tetrahedron: Asymmetry 2005, 16, 3802.
6. Joshi, R. A.; Murugan, M.; Garud, D. R.; Borikar, S. P.; Gurjar, M. K. S-(ꢁ)-1-f4-[2-
(allyloxy)-ethyl]phenoxyg-3-isopropylamino propan-2-ol, and process for preparation
thereof and process for preparation of S-(ꢁ)betaxolol. U.S. Patent 6,989,465, 2006.

2474
Y.-H. LI, L.-H. HUANG, AND H.-M. LIU
7. Joshi, R. A.; Murugan, M.; Garud, D.; Borikar, S. P.; Gurjar, M. K. Process for
preparation of S-(ꢁ)-betaxolol and salts thereof. U.S. Patent 2006004109, 2006; U.S.
Patent 7019172, 2006.
8. Ippolito, R. M.; Vigmond, S. Process for the preparation of betaxolol and related 1-[4-[2-
(cycloalkylalkoxy)ethyl]phenoxy]-3-(alkylamino)-2-propanol pharmaceuticals. GB Patent
2187190, 1987; Process for the preparation of phenoxyisopropanolamine derivatives use-
ful as a-adrenergic blocking agents. ES Patent 544803A1, 1986.
9. Jung, S. H.; Linh, P. T.; Lim, H. K.; Kim, H. J.; Kim, K. H.; Kang, J. S. Enantioselective
preparation of metoprolol and its major metabolites. Arch. Pharmacal Res. 2000, 23, 226.
10. Li, Y. H.; Huang, L. H.; Liu, H. M.; Huang, K. X. Screening and identification of micro-
organism for enantioselective hydrolysis of betaxolol intermediate. Chin. Ind. Microb.
2009, 39, 17.
11. Li, Y. H.; Yu, M. Y.; Liu, H. M. Determination of the ee values and content of b-amino
alcohol chiral drug intermediates by HPLC. Chin. J. Z. Zhou Univ. (Med. Sci.) 2009,
44, 427.