New chemo-enzymatic approaches for the synthesis of (R)- and (S)-bufuralol

Article abstract of DOI:10.1016/j.tetasy.2014.08.002

Both enantiomers of bufuralol are pharmaceutically important molecules. While the (S)-isomer with a higher β-blocking activity is recommended for hypertension treatment, the (R)-enantiomer can be used as marker of hepatic activity. In this paper two new a

Full text of DOI:10.1016/j.tetasy.2014.08.002

Tetrahedron: Asymmetry 25 (2014) 1316–1322
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
New chemo-enzymatic approaches for the synthesis
of (R)- and (S)-bufuralol
⇑
Botond Nagy, Norbert Dima, Csaba Paizs, Jürgen Brem, Florin Dan Irimie, Monica Ioana Tosßa
Biocatalysis and Biotransformation Research Group, Babeßs-Bolyai University, Arany János 11, Cluj-Napoca Ro-400028, Romania
a r t i c l e i n f o
a b s t r a c t
Article history:
Both enantiomers of bufuralol are pharmaceutically important molecules. While the (S)-isomer with a
higher b-blocking activity is recommended for hypertension treatment, the (R)-enantiomer can be used
as marker of hepatic activity. In this paper two new alternative approaches are described for their chemo-
enzymatic synthesis, providing both highly enantiomerically enriched stereoisomers of the target mole-
cule (ee 96–98%). One route is based on the baker’s yeast mediated stereoselective biotransformation of
Received 12 July 2014
Accepted 5 August 2014
Available online 10 September 2014
a
-substituted ketones, and the other one on the lipase mediated kinetic resolution of the racemic
bromoethanol.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
step.1a Lee et al. have described a similar method involving the
asymmetric transfer hydrogenation of 1-(7-ethylbenzofuran-
2-yl)-2-mesyloxyethanol mediated by Cp⁄RhCl[S,S-TsDPEN]
using an azeotropic mixture of formic acid/triethylamine.7 The
(R)-enantiomer was successfully obtained from the corresponding
chloro-ketone8 or keto-imine9 using a sequential transfer hydroge-
nation–substitution procedure or through a combined ruthenium-
enzyme-catalyzed dynamic kinetic resolution of chlorohydrin.10
Recently, the stereoselective synthesis of (S)-bufuralol (95%
yield, ee 98%) via the corresponding (S)-cyanohydrin, was
reported.11 The latest compound was prepared by a homochiral
metal–organic catalyst mediated enantiotope selective cyanation
of the corresponding aldehyde.
Due to their generally large substrate tolerance, high stereose-
lectivity, effective catalytic activity without cofactors, and good
commercial availability, lipases (EC 3.1.1.3) are especially attrac-
tive biocatalysts for synthetic purposes. A highly enantioselective
lipase-catalyzed kinetic resolution affords both enantiomers
simultaneously from a racemic mixture (one as the unreacted
enantiomer and the other as a new reaction product).
The preparation of enantiopure compounds has gained great
importance since the enantiomers of pharmaceuticals often display
different physiological properties or metabolic behaviors and may
have their own effects on drug–ligand interactions.
Amino alcohol moieties with a certain stereochemistry can lead
to important biological activities showing antiarrhythmic, enzy-
matic inhibitory, antihypertensive, and b-adrenoacceptor blocking
activity.1
Bufuralol, developed by Hoffman-La Roche, is a widely studied
potent, nonselective, b-adrenergic receptor antagonist.2 It is effi-
cient in hypertension treatment,3 acts as inhibitor of testosterone
6b-hydroxylase,4 and it was also used in studies of cytochrome P450.
Bufuralol is a chiral molecule with an asymmetric carbon in the
ethanol amine side chain and its oxidative degradation takes
place enantioselectively and regioselectively in the liver.5 While
the b-blocking potency resides mainly in (S)-bufuralol (100 times
greater than that of the (R)-enantiomer), (R)-bufuralol is
a
commonly used marker of hepatic CYP 2D6 activity.6
Various stereoselective procedures for the synthesis of both
enantiomers of bufuralol have been already described in the
literature.
(S)-Bufuralol has been obtained with acceptable enantiomeric
excess (ee 87%) from 3-ethyl-2-hydroxy-benzaldehyde, via the
stereoselective reduction of 2-bromo-1-(7-ethylbenzofuran-2-yl)
ethanone with (À)-B-chlorodiisopino-campheylborane as the key
Consequently Turner et al. employed an enzymatic enantiomer
selective acylation of rac-7-ethylbenzofuran-2-yl-chloro-alcohol.12
It was shown that lipases from Pseudomonas and Candida species
can lead to successful resolution of the racemic chloro-alcohol pro-
viding (R)-bufuralol.
Herein we present two new chemo-enzymatic approaches for
the stereoselective synthesis of both enantiomers of bufuralol.
The first route is based on a baker’s yeast catalyzed biotransforma-
tion of 2-(7-ethylbenzofuran-2-yl)-2-oxoethyl acetate
1-(7-ethylbenzofuran-2-yl)-2-hydroxyethanone 6, affording both
5 and
⇑
Corresponding author. Tel.: +40 264 593 833; fax: +40 264 590 818.
E-mail address: mtosa@chem.ubbcluj.ro (M.I. Tosßa).
http://dx.doi.org/10.1016/j.tetasy.2014.08.002
0957-4166/Ó 2014 Elsevier Ltd. All rights reserved.

B. Nagy et al. / Tetrahedron: Asymmetry 25 (2014) 1316–1322
1317
enantiomers of the corresponding 1,2-diols. The other procedure is
based on the enantiomer selective lipase-mediated acylation of
rac-2-bromo-1-(7-ethylbenzofuran-2-yl)ethanol rac-8, affording
at 50% conversion one enantiomer as an ester and the other (slowly
reacting) enantiomer as untransformed alcohol. All of the enantio-
merically enriched intermediates obtained were further chemically
transformed into the both desired enantiomers of bufuralol with
good yields and without significant alteration of the enantiopurity.
in excellent yield. Finally the latest compound was reduced with
sodium borohydride to yield the rac-1,2-ethanediol rac-7 (84% over-
all yield starting from 4) used to set up the chiral analytical chro-
matographic separation of the racemic diol, allowing to
investigate the stereochemical outcome of the baker’s yeast-cata-
lyzed biotransformations.
The baker’s yeast mediated procedure has gained popularity for
several reasons such as mild reaction conditions, low cost, and easy
availability of the whole-cell system. Besides these facts the main
advantage of this cellular biotransformation consists in the large
substrate acceptability and stereoselectivity of the hydrolases
and oxido-reductases from Saccharomyces cerevisiae cells.
One of the most reliable biocatalytic procedures for forming
enantiopure aryl-ethanediols is the cellular biotransformation of
2. Results and discussion
2.1. Chemical synthesis
The chemical synthesis of the starting materials uses the com-
mercially available 2-ethylphenol 1 which was ortho-formylated
with paraformaldehyde affording 3-ethyl-salicylaldehyde 2 in
good yield (Scheme 1). The aldehyde 2 was transformed with
chloroacetone into 7-ethyl-benzofuran-2yl-ethanone 3 which
was further brominated with pyridinium tribromide providing
the bromo-ketone 4.
various
reduction produces one enantiomer with 100% theoretical yield.
It is known that -hydroxy and -acetoxymethyl ketones can
a-substituted ketones, since their enantiotope selective
a
a
be transformed by Saccharomyces cerevisiae with opposite stereo-
preference into both enantiomers of the corresponding diols.14
Moreover, the baker’s yeast mediated bioreduction of the
a-acet-
oxymethyl ketones usually is followed by a subsequent enzymatic
hydrolysis of the reduction product. Thus the hydroxy-monoace-
tate obtained by bioreduction is further hydrolyzed into 1,2-eth-
anediol by the hydrolases also present in baker’s yeast. Our
previous results15 have demonstrated that the enantioselective
bioreduction of 1-(benzofuran-2-yl)-2-hydroxyethanones and 2-
acetoxy-1-(benzofuran-2-yl)ethanones provided the opposite
enantiomeric forms of the diols with high enantiomeric purity
(ee 84–93%). Ketones with the relatively small and hydrophilic
hydroxymethyl group were all reduced from the same face,
whereas baker’s yeast has an opposite enantiotopic face preference
for the acetoxymethyl ketones (Scheme 3).
O
O
O
II.
I.
III.
OH
O
O
OH
Br
2
3
4
1
Scheme 1. Chemical synthesis of the a-bromo-ketone. (I) Paraformaldehyde, NEt3,
MgCl2, CH3CN; (II) chloroacetone, K2CO3, CH3CN: (III) C5H6NBr3, CH3COOH.
These observations proved to be valid also for the biotransfor-
mations of ketones 5 and 6 (Scheme 3), which were performed
under fermenting and non-fermenting conditions. In order to
increase the enantiopurity of the products, the influence of various
additives upon the stereoselectivity of the reactions was also
tested (Table 1).
As was expected, the enantiomeric excess of the products was
significantly influenced by the nature of the additives used.
Generally it was observed that, while the use of additives increases
the selectivity in the biotransformation of acetoxy-ketone 5, in the
case of hydroxy-ketone 6 most of the additives decreased the
selectivity of the bioreduction. Higher selectivity was obtained in
2.2. Synthesis of (R)- and (S)-bufuralol via the corresponding
ethane diols obtained by Baker’s yeast-mediated
biotransformations
Prochiral ketones 5 and 6, used as substrates for the baker’s yeast
mediated biotransformation, were obtained by starting from 2-
bromo-1-(7-ethylbenzofuran-2-yl)-ethanone 4, using a procedure
previously developed in our laboratories (Scheme 2).13 Under anhy-
drous conditions bromo-ethanone 4 was transformed with sodium
acetate (in the presence of crown ether 18-C6 as a phase-transfer
catalyst) into the corresponding
a-acetoxymethyl-ketone 5. The
Candida antarctica lipase B (Novozym 435) mediated ethanolysis
the fermenting system only in the presence of L-cysteine (Table 1,
of the ketoester 5 allowed us to obtain
a
-hydroxymethyl-ketone 6
O
O
O
OH
OH
I.
II.
III.
O
O
O
O
Br
OAc
OH
6
7
rac-
5
4
Scheme 2. Chemical synthesis of the
a
-substituted ketones 5 and 6 and of the racemic diol 7. (I) CH3COOÀNa+, 18-C6, 1,4-dioxane; (II) Novozym 435, EtOH; (III) NaBH4,
MeOH, rt.
OH
OH
OH
OH
OH
OAc
O
S. cerevisiae
I.
S. cerevisiae
NHtBu
YADHs
hydrolases
II.
O
O
O
O
OAc
7
(R)-
(R)-10
5
OH
OH
S. cerevisiae
O
I.
NHtBu
II.
O
O
O
OH
10
(S)-
7
(S)-
6
Scheme 3. Chemo-enzymatic transformation of a-substituted ketones 5 and 6 into (R)- and (S)-bufuralol. (I) TsCl, Et3N, Bu2SnO, CH2Cl2, rt; (II) tert-butylamine, EtOH, reflux.

1318
B. Nagy et al. / Tetrahedron: Asymmetry 25 (2014) 1316–1322
Table 1
Fermenting and non-fermenting cellular biotransformation of 5 and 6
a
b
Entry
Additives (0.5% w/w)
ee(R)-7
ee(S)-7
Fermenting
Non-fermenting
Fermenting
Non-fermenting
1
2
3
4
Without additive
Allyl alcohol
n-Hexane
92
96
98
96
90
96
95
95
86
80
50
96
87
80
78
76
L-Cysteine
5
6
7
Ethyl bromoacetate
MgCl2
DMSO
—
96
95
—
92
94
—
62
76
—
50
77
a
From prochiral acetoxy-ketone 5 as the substrate.
From prochiral hydroxy-ketone 6 as the substrate.
b
entry 4). While the use of ethyl bromoacetate inhibits the biotrans-
formation of both ketones 5 and 6, the best result for the transfor-
mation of 5 was obtained in the fermenting system using n-hexane
as an additive (Table 1, entry 3).
from Candida antarctica displayed excellent reactivity but poor
selectivity, whereas L-AK, CRL, and LPS showed good selectivity
but reduced activity in all solvents tested in the presence of both
acyl donors.
The isolated and purified (R)- and (S)-heteroaryl-1,2-diols (R)-
and (S)-7 served as valuable precursors for the chemical synthesis
of both enantiomers of bufuralol. In the first step the regioselective
tosylation of the primary alcohol group of (R)- and (S)-7 with para-
toluenesulfonyl chloride in the presence of a catalytic amount of
dibutyltin(IV) oxide16 was performed. In the second step the tosyl
group was substituted with tert-butylamine (Scheme 3) yielding
the end-product with a small decrease of the enantiopurity (ee
96% for (R)-10 and 93% for (S)-10) compared with those of (R)-
and (S)-heteroaryl-1,2-diols (ee 98% for (R)-7 and 96% for (S)-7)
(40% overall yield starting from the diols).
CaL-B proved to be the best biocatalyst. The reaction selectivity
was high in all solvents tested with both vinyl esters (Table 2).
Excellent enantiomeric excesses of the products and also high reac-
tion rates were obtained in diisopropyl ether and n-octane using
vinyl acetate as an acyl donor (Table 2, entries 2 and 9) and in tol-
uene using vinyl laurate, respectively, (Table 2, entry 8).
Furthermore a scale-up of the previously mentioned optimal
analytical scale reactions was set up, using the same substrate–
biocatalyst ratio. The reactions were stopped at an approximately
50% conversion (checked by TLC and HPLC). While a small decrease
of the ee (ꢀ97%) of the resolution products was detected when
vinyl acetate was used as the acyl-donor, the ee values were the
same as those found for small scale reactions with vinyl dodecan-
2.3. Synthesis of (R)- and (S)-bufuralol from the lipase-catalyzed
enantiopure resolution products of 2-bromo-1-(7-ethylbenzo-
furan-2-yl)-ethanol
Table 2
CaL-B catalyzed enantioselective acylation of rac-8 with vinyl acetate and vinyl
dodecanoate after 16 h
The other approach for the synthesis of (R)- and (S)-bufuralol
involved the use of the lipase-catalyzed stereoselective O-acylation
of racemic 2-bromo-1-(7-ethylbenzofuran-2-yl)ethanol rac-8
(Scheme 4), which was previously prepared by reduction of 4 with
NaBH4.
Commercially available immobilized or lyophilized lipases,
such as lipases A and B from Candida antarctica (CaL-A on Celite
and CaL-B immobilized on Eupergite commercialized as Novozyme
435), lipase from Pseudomonas fluorescens (L-AK), Candida rugosa
(CRL), and lipase from Pseudomonas sp. (LPS) were tested for the
analytical scale enantioselective acylation of rac-8 with vinyl ace-
tate and vinyl laurate as acyl donors in various solvents. Lipase A
Entry
Solvent
eep (%)
ees (%)
c (%)
E
1
2
3
4
5
6
7
8
9
MTBEa
99
99
49
41
>99
69
93
42
71
99
99
33
29
50
41
48
30
41
50
50
»200
>200
»200
>200
»200
>200
»200
»200
»200
MTBEb
DIPEa
>99
>99
>99
>99
>99
>99
>99
n-Hexanea
n-Hexaneb
Acetonitrilea
Toluenea
Tolueneb
n-Octanea
a
Vinyl acetate.
Vinyl dodecanoate.
b
O
O
R
OH
NHBut
I.
II.
O
O
O
(S)-
Br
O
9a,b
(R)-12
(R)-10
OH
Br
KR
O
O
R
OH
Br
O
Si
IV. a,b
OH
NHBut
III.
I.
8
rac-
O
O
O
Br
O
(R)-8
(R)-13
(S)-10
V.
R
a. CH3
O
b.
O
C
11H23
II.
O
R
O
12
(S)-
O
Br
9b
(R)-
Scheme 4. KR. Enzyme, acyl donor, rt, 800 rpm; (I) LiOH, EtOH, rt; (II) tert-butylamine, reflux; (III) DMCTMS, Et2O, rt; (IV) (a) tert-butylamine , MeOH, rt; (b) HF, MeOH; (V)
vinyl dodecanoate, CaL-A, DIPE.

B. Nagy et al. / Tetrahedron: Asymmetry 25 (2014) 1316–1322
1319
oate. Both enantiopure compounds were isolated in excellent
yields and used for further chemical transformations.
In order to prevent significant racemization and undesired sec-
ondary reactions, two particular chemical paths were developed
for the efficient transformation of the resolution products into
(R)- and (S)-bufuralol.
enantiomeric purities of the bufuralol enantiomers were deter-
mined using a Chirobiotic V2 column in polar ionic mode with
methanol–acetic acid–triethylamine 100:0.02:0.015 (v/v/v) as elu-
ent (tr(S)-12 19.6 min; tr(R)-12 22.4 min). Optical rotations were mea-
25
sured with a Perkin–Elmer 201 polarimeter and [
a]
values are
D
given in units 10À1 deg cm2 gÀ1
.
In our first attempt the reaction of tert-butylamine12 with enan-
tiomerically pure bromohydrin (R)-8 was investigated in various
conditions (data not given). Due to the instability of bromo-ethanol
(R)-8 under basic conditions, in each case almost racemic bufuralol
was isolated with poor yields. The mild protection of the hydroxy
group from (R)-8 with trimethylsilyl-N,N-dimethylcarbamate
(DMCTMS, Scheme 4) allowed the substitution of bromine with
tert-butyl amine on O-silylated bromohydrin (R)-13, avoiding race-
mization. Since the deprotection of (R)-13 with mild acids failed,
the HF catalyzed deprotection of the hydroxy group occurred with
partial racemization, yielding the desired (S)-bufuralol (S)-10 with
reduced enantiopurity (ee 90%) and 35% overall yield.
4.2. Reagents, solvents, and biocatalysts
2-Ethylphenol, all reagents used, and solvents were products of
Sigma–Aldrich and Alfa Aesar. Lipase from Pseudomonas fluorescens
(L-AK) and lipase from Pseudomonas sp. (LPS) were obtained from
Amano, England, while lipase from Candida rugosa (CRL) was pur-
chased from Fluka. Immobilized lipase B from Candida antarctica
(CaL-B) was purchased from Novozyme, Denmark, while lipase A
from Candida antarctica immobilized by adsorption on Celite
(CaL-A) was a gift from Professor Liisa T. Kanerva, University of
Turku.
For the synthesis of (R)-bufuralol (R)-10 the enantiomerically
pure acylated bromohydrins (S)-9a,b were transformed into the
enantiopure epoxide (R)-12 in the presence of LiOH. The latest
compound was transformed in neat tert-butylamine into (R)-10
with 53% overall yield and with excellent enantiomeric excess
(ee 98%).
Based on this result, with the aim to improve the enantiopurity
of (S)-bufuralol, we decided to reinvestigate the chemical transfor-
mation of (R)-8 applying the strategy described earlier. Since CaL-A
proved to be a highly active but unselective biocatalyst for the
acylation of rac-8, the acylation of (R)-8 with vinyl laurate in DIPE
in the presence of this lipase was performed and the isolated ester
(R)-9b (ee 99%) was further transformed into the target (S)-10 (ee
98%) as shown in Scheme 4.
4.3. Chemical transformations
4.3.1. Synthesis of 3-ethyl-2-hydroxybenzaldehyde 2
Into the mixture of 2-ethylphenol 1 (9.77 g, 80 mmol), magne-
sium chloride (11.42 g, 120 mmol) and triethylamine (30.36 g,
300 mmol) in acetonitrile (100 mL) paraformaldehyde (16.21 g,
540 mmol) was added in small portions at rt. The mixture was
refluxed for 5 h, stirred for another 12 h at rt and finally acidified
with a dilute HCl solution (5%, 200 mL). The mixture was extracted
with dichloromethane (2 Â 100 mL) and then the organic layer was
dried over Na2SO4. The solvent was removed in vacuo and the
product was isolated by vacuum distillation with 98% yield. Spec-
tral data were in accordance with those reported in the literature.1a
4.3.2. Synthesis of 1-(7-ethylbenzofuran-2-yl)ethanone 3
Chloroacetone (7.32 g, 79.2 mmol) was added dropwise to a
mixture of 2 (10.8 g, 72 mmol) and anhydrous potassium carbon-
ate (9.95 g, 72 mmol) in acetonitrile (60 mL) at 35 °C. The mixture
was refluxed for 5 h and cooled to rt. The precipitated solid was fil-
tered off and washed with acetonitrile (2 Â 10 mL). After removing
the solvent the crude product was purified by distillation followed
by recrystallization from n-hexane affording the pure desired prod-
uct 3 (9.9 g, 73% yield). Spectral data were in accordance with
those reported in the literature.1a
3. Conclusions
Using two alternative biocatalytic approaches based on baker’s
yeast mediated reactions (through the biotransformation of pro-
chiral a-substituted 7-ethyl-benzofuran-2-yl-ethanones) and on a
lipase catalyzed kinetic resolution of (7-ethyl-benzofuran-2-yl)-
bromohydrin (by enantiomer selective O-acylation), highly enanti-
omerically enriched compounds were obtained. These compounds
serve as building blocks for the synthesis of both enantiomers of
bufuralol. Due to partial racemization occurring during the chem-
ical transformation of the chiral intermediates, the enantiomeric
excesses of the target (R)- and (S)-bufuralol were decreased to
96–98%.
4.3.3. Synthesis of 2-bromo-1-(7-ethylbenzofuran-2-yl)ethan-
one 4
Into a solution of 3 (16 g, 0.1 mol) in acetic acid (100 mL) pyrid-
inium tribromide (28.7 g, 0.9 equiv.) was added in small portions at
35 °C. The reaction was completed at 50 °C (controlled by TLC with
dichloromethane as eluent, approx. 2 h). The reaction mixture was
poured on ice-cooled water; the precipitate formed was filtered
and redissolved in dichloromethane. The organic solution was
dried over Na2SO4, the solvent was further removed in vacuo,
and the crude product was purified by column chromatography
using n-hexane–dichloromethane 1:4 (v/v) as eluent.
4. Experimental
4.1. Analytical methods
The 1H and 13C NMR spectra were recorded on Bruker spectro-
meters operating at 400 MHz, 101 MHz and 600 MHz, 151 MHz,
respectively. Spectra were recorded at 21 °C and were referenced
internally to the solvent signal. Mass spectra were recorded on a
GC–MS Shimadzu QP 2010 Plus spectrometer using direct injection
and EI+ ionization at 70 eV. IR spectra were recorded on a Bruker
Vector 22 spectrometer. High performance liquid chromatography
(HPLC) analyses were conducted with an Agilent 1200 instrument
using a Chiralpak IB column and a mixture of n-hexane–2-propanol
95:5 (v/v) as eluent for the enantiomeric separation of rac-7 (tr(S)-7
19.2 min; tr(R)-7 21.5 min), and a (R,R)-Welk-O1 column and a mix-
ture of n-hexane–2-propanol 99:1 (v/v) as eluent for the separation
of rac-8 and rac-9a,b respectively, (tr(S)-8 10.6 min; tr(R)-8 12.2 min;
tr(R)-9a 3.8 min; tr(S)-9a 5.5 min; tr(R)-9b 2.9 min; tr(S)-9b 4.1 min). The
4.3.3.1. 2-Bromo-1-(7-ethylbenzofuran-2-yl)ethanone 4. Yield
85%, 19.4 g; 1H NMR (400 MHz, CDCl3) d 7.65 (s, 1H), 7.56 (d,
J = 7.8 Hz, 1H), 7.34 (d, J = 7.2 Hz, 1H), 7.27 (dd, J = 7.8, 7.2 Hz,
1H), 4.46 (s, 2H), 2.99 (q, J = 7.6 Hz, 2H), 1.38 (t, J = 7.6 Hz, 3H);
13C NMR (101 MHz, CDCl3) d 182.33, 154.65, 149.95, 129.08,
127.89, 126.62, 124.47, 120.92, 115.08, 30.30, 22.80, 13.99; MS
m/z 268 (M+2, 25.74), 267 (M+, 8.72), 266 (MÀ1, 26.75), 174
(12.86), 173 (100), 159 (17.26), 144 (12.36), 117 (12.25), 115
(29.8), 91 (9.74); IR (KBr, cmÀ1): 1693, 1558, 1376, 1313, 1138,
1001.

1320
B. Nagy et al. / Tetrahedron: Asymmetry 25 (2014) 1316–1322
4.3.4. Synthesis of 2-(7-ethylbenzofuran-2-yl)-2-oxoethyl acetate 5
Into a solution of 4 (900 mg, 3.4 mmol) in 1,4-dioxane (10 mL)
anhydrous sodium acetate (830 mg, 10.1 mmol) and phase transfer
catalyst 18-C6 (0.1 equiv, 89 mg) were added. The resulting
mixture was heated at reflux until the reaction was completed
(controlled by TLC with dichloromethane as eluent, approx. 5 h).
After cooling, the deposited solid was filtered off and washed with
dioxane (3 Â 10 mL). The solvent was removed in vacuo and the
crude product was recrystallized from ethanol.
(40 mL) at 0 °C under an argon atmosphere. The reduction was
completed under stirring at room temperature (controlled by TLC
with dichloromethane as eluent, approx. 30 min). After distillation
of methanol in vacuo at room temperature, the crude product was
resuspended in water (150 mL), acidified with 10% HCl, and
extracted with dichloromethane (3 Â 150 mL). The organic layer
was dried over anhydrous Na2SO4 and concentrated in vacuo,
affording the product as a yellow semisolid.
4.3.7.1. rac-2-Bromo-1-(7-ethylbenzofuran-2-yl)ethanol rac-8.
Yield 97%, 5.48 g; 1H NMR (400 MHz, CDCl3) d 7.42 (d, J = 7.6 Hz,
1H), 7.21 (dd, J = 7.6, 6.9 Hz, 1H), 7.16 (d, J = 6.9 Hz, 1H), 6.76 (s,
1H), 5.11 (dd, J = 6.7, 4.4 Hz, 1H), 3.87 (dd, J = 10.5, 4.3 Hz, 1H),
3.80 (dd, J = 10.5, 7.0 Hz, 1H), 2.96 (q, J = 7.6 Hz, 3H), 1.38 (t,
J = 7.6 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 155.17, 153.49,
127.84, 127.50, 123.84, 123.26, 118.84, 104.46, 68.25, 36.57,
22.89, 14.20. MS m/z 271 (M+2, 11.97), 270 (M+1, 68.24), 269
(M+, 15.05), 268 (64.69), 253 (50), 251 (50.53), 175 (100), 119
(22.59), 91 (40); IR (KBr, cmÀ1): 3400, 1602, 1426, 1300, 1274,
4.3.4.1. 2-(7-Ethylbenzofuran-2-yl)-2-oxoethyl acetate 5. Yield
90%, 735 mg; 1H NMR (400 MHz, CDCl3) d 7.57 (s, 1H), 7.52 (d,
J = 7.7 Hz, 1H), 7.30 (d, J = 6.9 Hz, 1H), 7.24 (dd, J = 7.7, 6.9 Hz, 1H),
5.32 (s, 2H), 2.96 (q, J = 7.6 Hz, 2H), 2.23 (s, 3H), 1.35 (t, J = 7.6 Hz,
3H); 13C NMR (101 MHz, CDCl3) d 183.72, 170.34, 154.35, 150.16,
128.92, 127.63, 126.37, 124.39, 120.85, 113.67, 65.82, 22.71,
20.52, 13.96; MS m/z 246 (M+, 5.96), 173 (16.37), 115 (9.21), 43
(100), 29 (8.06); IR (KBr, cmÀ1): 1745, 1563, 1378, 1230, 1160, 1008.
1184, 1073, 988; [
a]
D
25 = À43.7 (c 1.0, CHCl3) for (R)-8.
4.3.5. Synthesis of 1-(7-ethylbenzofuran-2-yl)-2-hydroxyeth-
anone 6
4.4. Cellular biotransformation of 2-(7-ethylbenzo-furan-2-yl)-
2-oxoethyl acetate 5 and 1-(7-ethylbenzofuran-2-yl)-2-hydro-
xyethanone 6 with baker’s yeast cells
The mixture of 5 (300 mg, 1.2 mmol) and CaL-B (300 mg) in eth-
anol (23 mL) was shaken at 300 rpm and rt until the reaction was
completed (controlled by TLC with dichloromethane–methanol
95:5 (v/v) as eluent, approx. 4 h). The enzyme was filtered off
and washed with ethanol (3 Â 10 mL). The solvent was removed
in vacuo from the filtrate and the crude product was recrystallized
from ethanol.
4.4.1. Non-fermenting biotransformation
Baker’s yeast (2 g) was suspended in 15 mL water. After stirring
for 15 min, the corresponding additive (see Table 1) and the ketone
5 or 6 (10 mg) dissolved in ethanol (0.5 mL) were added into the
resulting cell suspension under stirring. Samples (1 mL) were taken
after 18 h and 40 h, respectively, extracted with ethyl acetate and
dried over anhydrous Na2SO4. The solvent was evaporated, the
crude solid was redissolved in n-hexane–isopropanol 9:1 (v/v)
and analyzed by HPLC.
4.3.5.1. 1-(7-Ethylbenzofuran-2-yl)-2-hydroxyethanone 6. Yield
98%, 244 mg; 1H NMR (600 MHz, Acetone-d6) d 7.76 (s, 1H), 7.63 (d,
J = 7.8 Hz, 1H), 7.36 (d, J = 7.3 Hz, 1H), 7.28 (dd, J = 7.8, 7.3 Hz, 1H),
4.86 (s, 2H), 2.95 (q, J = 7.6 Hz, 2H), 1.33 (t, J = 7.6 Hz, 3H). 13C NMR
(151 MHz, Acetone-d6) d 190.55, 154.88, 151.19, 129.41, 128.15,
127.58, 125.11, 121.87, 114.31, 66.39, 23.28, 14.34. MS m/z 204
(M+, 16.58), 173 (100), 115 (21.9), 91 (10.24); IR (KBr, cmÀ1): 3444,
1679, 1562, 1388, 1223, 1154, 1097, 996.
4.4.2. Fermenting biotransformation
A fresh wet cake of baker’s yeast (2 g) and sucrose (1 g) were
added in water (15 mL). The resulting suspension was stirred for
30 min, the corresponding additive (see Table 1) and the ketone
5 or 6 (10 mg) dissolved in ethanol (0.5 mL) were added. Further
experiments were performed as described in the previous section.
4.3.6. Synthesis of rac-1-(7-ethylbenzofuran-2-yl)ethane-1,2-diol
rac-7
Sodium borohydride (2 equiv, 1.11 g) was added in small por-
tions into a stirred mixture of 6 (2 g, 9.8 mmol) in methanol
(40 mL), until all of the ketone was transformed (controlled by
TLC with (dichloromethane–methanol 9:1 (v/v) as eluent, approx.
1 h). The methanol was removed in vacuo and the mixture was
acidified with 5% HCl, followed by extraction with dichlorometh-
ane (3 Â 50 mL) of the crude product. The organic layer was dried
over anhydrous Na2SO4, concentrated in vacuo, and the crude
product was recrystallized from n-hexane.
4.4.3. Synthesis of (R)- and (S)-2-tert-butylamino-1-(7-ethyl-
benzofuran-2-yl)ethanol (R)- and (S)-10
To a solution of the enantiomerically pure alcohol (R)- or (S)-7
(200 mg, 1 mmol) in dichloromethane (20 mL) Bu2SnO (50 mg,
0.2 mmol), p-TsCl (190 mg, 1 mmol), and Et3N (100 mg, 1 mmol)
were added. The reaction mixture was stirred until TLC indicated
the complete consumption of the starting material (dichlorometh-
ane–methanol 9:1 (v/v) as eluent, approx. 30 min). The mixture
was filtered and the filtrate was concentrated in vacuo. The residue
was purified by column chromatography using dichloromethane as
eluent.
4.3.6.1. rac-1-(7-Ethylbenzofuran-2-yl)ethane-1,2-diol rac-7. Yield
95%, 1.9 g; 1H NMR (400 MHz, CDCl3) d 7.38 (d, J = 7.5 Hz, 1H), 7.16
(dd, J = 7.5, 7.1 Hz, 1H), 7.11 (d, J = 7.1 Hz, 1H), 6.71 (s, 1H), 4.98 (t,
J = 4.7 Hz, 1H), 4.00 (d, J = 4.9 Hz, 2H), 2.92 (q, J = 7.6 Hz, 2H), 1.34 (t,
J = 7.6 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 155.79, 153.42, 127.74,
127.54, 123.61, 123.12, 118.62, 104.07, 68.96, 65.17, 22.80, 14.10.
MS m/z 206 (M+ 38.53), 189 (23.39), 175 (100), 131 (11.67), 119
(17.11), 91 (43.98), 77 (12.29), 31 (16.25); IR (KBr, cmÀ1): 3283,
4.4.3.1. 2-Tosyloxy-1-(7-ethylbenzofuran-2-yl)ethanol. Yield
74%, 260 mg; 1H NMR (400 MHz, CDCl3) d 7.71 (d, J = 8.3 Hz, 2H),
7.35 (d, J = 7.6 Hz, 1H), 7.20 (d, J = 8.1 Hz, 2H), 7.16 (t, J = 7.5 Hz,
1H), 7.10 (d, J = 7.1 Hz, 1H), 6.67 (s, 1H), 2.84 (q, J = 7.6 Hz, 2H),
2.37 (s, 3H), 1.30 (t, J = 7.6 Hz, 3H); 13C NMR (101 MHz, CDCl3) d
153.79, 153.39, 145.13, 132.24, 129.86, 127.87, 127.68, 127.39,
123.70, 123.14, 118.75, 104.87, 71.40, 66.52, 22.69, 21.62, 14.03.
MS m/z 360 (M+ 5.38), 343 (10.40), 188 (79.32), 175 (100), 159
(24.84), 91 (60.21), 65 (34.00); IR (KBr, cmÀ1): 3536, 1599, 1360,
1180, 1097, 983.
3262; 1463, 1427, 1272, 1180, 1142, 1032; [a]D
25 = +38.3 (c 1.0, CHCl3)
for (R)-7, [a D
]
25 = À37.4 (c 1.0, CHCl3) for (S)-7.
4.3.7. Synthesis of rac-2-bromo-1-(7-ethylbenzofuran-2-yl)-
ethanol rac-8
Sodium borohydride (1 equiv, 0.8 g) was added in small por-
tions to a stirred solution of 4 (5.61 g, 21.1 mmol) in methanol
Further, into the solution of the tosylated diol (145 mg,
0.5 mmol) in absolute ethanol (10 mL) tert-butylamine (110 mg,

B. Nagy et al. / Tetrahedron: Asymmetry 25 (2014) 1316–1322
1321
1.5 mmol) was added and the mixture was heated to reflux over-
night. The volatile materials were removed in vacuo and the crude
product was purified by column chromatography using dichloro-
methane–methanol 8:2 (v/v) as eluent. The product was obtained
as a light-yellow solid.
with dichloromethane, approx. 20 min). The product was obtained
after removing the volatile materials in vacuo (589 mg, yield 93%).
tert-Butylamine (300 lL, 3 mmol) was added to a solution of
(R)-13 (500 mg, 1.46 mmol) in methanol (15 mL) and then stirred
at rt until the reaction was finished (approx. 30 min). In order to
remove the protecting trimethyl silyl group hydrofluoric acid
(1 M, 1.5 mL) was added dropwise into the reaction mixture. The
methanol was evaporated in vacuo, the residue was dissolved in
ethyl acetate (40 mL) and washed with water (2 Â 40 mL). The
organic layer was dried over anhydrous Na2SO4, the solvent was
removed in vacuo affording pure (S)-bufuralol in 35% yield (based
on (R)-8).
4.4.3.2. 2-(tert-Butylamino)-1-(7-ethylbenzofuran-2-yl)ethanol
rac-10.
Yield 54%, 56 mg; 1H NMR (400 MHz, CDCl3) d 7.37
(dd, J = 7.6, 1.1 Hz, 1H), 7.15 (t, J = 7.5 Hz, 1H), 7.09 (d, J = 7.3 Hz,
1H), 6.69 (s, 1H), 5.00 (dd, J = 6.9, 4.9 Hz, 1H), 4.02 (br s, 2H),
3.11–3.02 (m, 2H), 2.94 (q, J = 7.6 Hz, 2H), 1.35 (t, J = 7.6 Hz, 3H),
1.17 (s, 9H). 13C NMR (101 MHz, CDCl3) d 158.21, 153.40, 127.83,
127.63, 123.18, 122.90, 118.51, 103.23, 66.24, 51.31, 46.53, 28.60,
22.83, 14.18; MS m/z 262 (M+1, 23.9), 261 (M+, 2.0), 228 (14.7),
175 (13.6), 131 (11.5), 115 (11.2), 91 (16.5), 86 (100.0), 57
(18.2); IR: (KBr, cmÀ1): 3285, 3130, 1601, 1477, 1426, 1364,
1320, 1270, 1262, 1094, 1019.
4.5.4. Synthesis of (R)-bufuralol from the corresponding acylated
bromohydrins (S)-9a,b
Into the solution of the acylated bromohydrin (350 mg) in EtOH
(10 mL), LiOH Â H2O (1.2 equiv) was added and the mixture was
stirred at room temperature. After 20 min no starting material
was observed (controlled by TLC with dichloromethane as eluent).
The reaction was quenched with sat. NaHCO3 solution (2 mL) and
the EtOH was removed in vacuo. Brine (2 mL) was added to the res-
idue and the mixture was extracted with diethyl ether (3 Â 10 mL).
The combined organic phases were dried over anhydrous Na2SO4,
filtered, and concentrated.
The mixture of crude epoxide (R)-12 (approx. 200 mg) and tert-
butylamine (300 lL, 3 mmol) was stirred overnight at rt. The
excess of tert-butylamine was removed under reduced pressure.
Chromatographic purification (dichloromethane–methanol, 8:2
(v/v) as eluent) afforded 158 mg (R)-bufuralol (54% overall yield).
4.5. Kinetic resolution of 2-bromo-1-(7-ethylbenzofuran-2-yl)
ethanol rac-8
4.5.1. Analytical scale enzymatic acylation of rac-8
One of the lipases (30 mg/mL) and an acyl donor (vinyl acetate
or vinyl dodecanoate, 2 equiv) were added into the solution of rac-
8 (30 mg) in an organic solvent (1 mL). The reaction mixture was
shaken (1350 rpm) at room temperature. For HPLC analysis, the
samples (25
lL) were concentrated, diluted to 700 lL with 2-pro-
panol and n-hexane, and filtered before injection.
4.5.2. Preparative scale enzymatic acylation of rac-8
4.5.5. Synthesis of (S)-bufuralol from (R)-8 via the epoxide inter-
mediate (S)-12
Into a solution of (R)-8 (200 mg) in DIPE (5 mL), CaL-A on Celite
(50 mg) and vinyl dodecanoate (330 lL, 2 equiv) were added. The
reaction was completed at room temperature under shaking
(1350 rpm), for approx. 16 h. The enzyme was filtered off and
washed with DIPE (2 Â 0.5 mL). The solvent was removed in vacuo
and the crude product obtained (R)-9b (325 mg, 97%) was used fur-
ther without purification in the synthesis of epoxide (S)-12 and of
(S)-bufuralol, as described for the (R) enantiomer in Section 4.5.4.
CaL-B (1 g) was added into the solution of 8 (1 g), vinyl acetate,
or dodecanoate (2 equiv) in the appropriate solvent (33 mL). The
reaction mixture was shaken for 24 h at room temperature. The
solvent was evaporated in vacuo and the crude product was puri-
fied by column chromatography, using dichloromethane as eluent.
4.5.2.1. (S)-2-Bromo-1-(7-ethylbenzofuran-2-yl)ethyl acetate
(S)-9a.
1H NMR (400 MHz, CDCl3) d 7.44 (d, J = 7.4 Hz, 1H),
7.24–7.16 (m, 2H), 6.83 (s, 1H), 6.25 (t, J = 6.5 Hz, 1H), 3.87 (d,
J = 6.5 Hz, 2H), 2.99 (q, J = 7.6 Hz, 2H), 2.19 (s, 3H), 1.40 (t,
J = 7.6 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 169.66, 153.54,
151.61, 127.94, 127.20, 124.21, 123.35, 118.99, 106.57, 68.64,
30.57, 22.89, 20.87, 14.11; MS (70 eV) m/z 312 (M+2, 1.67), 310
(M+, 1.73), 230 (6.68), 188 (46.26), 175 (13.44), 157 (11.37), 115
(13.11), 87 (23.16), 74 (30.95), 43 (100); IR (KBr, cmÀ1): 1749,
Acknowledgments
This work was supported by a Grant of the Romanian National
Authority for Scientific Research, CNDI-UEFISCDI, Project PN-II-PT-
PCCA-2011-3.1-1268. B.N. thanks the Collegium Talentum Research
Program for the support.
1462, 1428, 1372, 1272, 1176; [a]
25 = +92.8 (c 1.0, CHCl3).
D
4.5.2.2. (S)-2-Bromo-1-(7-ethylbenzofuran-2-yl)ethyl dodecan-
oate (S)-9b.
1H NMR (400 MHz, CDCl3) d 7.40 (d, J = 7.3 Hz,
References
1H), 7.20–7.11 (m, 2H), 6.77 (s, 1H), 6.21 (t, J = 6.5 Hz, 1H), 3.83
(d, J = 6.5 Hz, 2H), 2.93 (q, J = 7.5 Hz, 2H), 2.47–2.31 (m, 2H),
1.71–1.62 (m, 2H), 1.34 (t, J = 7.6 Hz, 3H) overloaded with 1.2–
1.34 (m, 16H) 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 172.68, 153.62, 151.88, 128.06, 127.33, 124.24, 123.40, 119.05,
106.47, 68.50, 34.34, 32.05, 30.82, 29.73, 29.57, 29.47, 29.37,
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43 (29.03); IR (KBr, cmÀ1): 1748, 1709, 1463, 1426, 1377, 1148,
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