Chemoenzymatic Synthesis of Luliconazole Mediated by Lipases

Article abstract of DOI:10.1002/ejoc.201800250

A straightforward chemoenzymatic synthesis of luliconazole has been developed. The key step involved the preparation of the enantiomerically pure β-halohydrin (1S)-2-chloro-1-(2,4-dichlorophenyl)-1-ethanol through kinetic resolution of the corresponding racemic acetate. This was achieved by a hydrolytic approach, mediated by the lipase from Thermomyces lanuginosus or Novozym 435. The latter enzyme proved to be a robust biocatalyst for the kinetic resolution, and the (S)-β-halohydrin was obtained with high selectivity (ee > 99 %, E > 200) after just 15 min, at 45 °C. It could be reused five times with maintenance of high values of both conversion and enantioselectivity. Subsequently, the (S)-β-halohydrin was subjected to a mesylation reaction; the mesylated derivative reacted with 1-cyanomethylimidazole in the presence of CS₂ to give luliconazole in 43 % yield with >99 % ee.

Full text of DOI:10.1002/ejoc.201800250

A Journal of
Accepted Article
Title: Chemoenzymatic synthesis of luliconazole mediated by lipases
Authors: Marcos Carlos de Mattos, Thiago de Sousa Fonseca, Lara
Dias Lima, Maria da Conceição Ferreira de Oliveira, Telma
Leda Gomes de Lemos, Davila Zampieri, and Francesco
Molinari
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201800250
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10.1002/ejoc.201800250
European Journal of Organic Chemistry
FULL PAPER
Chemoenzymatic synthesis of luliconazole mediated by lipases
Thiago de S. Fonseca,[a] Lara D. Lima,[a] Maria da C. F. de Oliveira,[a] Telma L. G. de Lemos,[a] Davila
Zampieri,[a] Francesco Molinari[b] and Marcos C. de Mattos*[a]
fungi including dermatophytes.[4] Luliconazole was developed in
A straightforward chemoenzymatic synthesis of luliconazole has been
Japan and approved by Japanese health agencies in 2005.[4]
More recently, in 2013, the US FDA approved the (R)-1 for the
developed. The key step involved the preparation of the
enantiomerically
pure
β-halohydrin
(1S)-2-chloro-1-(2,4-
treatment of tinea pedis interdigital, tinea cruris and tinea corporis
caused by the microorganisms Trichophyton rubrum and
dichlorophenyl)-1-ethanol through kinetic resolution of the
corresponding racemic acetate, via hydrolytic approach, mediated by
lipase from Thermomyces lanuginosus or Novozym 435®. This latter
enzyme proved to be a robust biocatalyst in the kinetic resolution,
leading to the (S)-β-halohydrin with high selectivity (e.e. > 99%, E >
200) in just 15 min, at 45 °C, and being reused for five-times with
maintenance high values of both conversion and enantioselectivity.
Subsequently, the (S)-β-halohydrin was subjected to a mesylation
reaction and the mesylated derivative reacted with 1-
cyanomethylimidazole, in the presence of CS2, leading to
luliconazole in 43% yield and enantiomeric excess > 99%.
Epidermophyton floccosum.[5]
It is noteworthy that the
stereoisomers of luliconazole, (S,E) and/or Z-isomers, are
considered impurities and should be removed to obtain the
enantiomerically pure active pharmaceutical ingredient (R)-1.[1]
The antifungal action exhibited by (R)-1 resides in the fact that
this substance inhibits the enzyme sterol 14α-demethylase
(CYP51), which participates in the key step of ergosterol
biosynthesis.[6] The mechanism of inhibiton includes the
coordination of one of the nitrogen atoms of the imidazole ring
with the iron atom of the heme group of the enzyme.[6]
The syntheses of luliconazol have been reported involving
some chiral strategies, such as asymmetric induction in the
presence of oxazaborolidines,[1,7] dihydroxylation of Sharpless[7]
and chiral catalysts based on ruthenium, iridium or rhodium.[8]
Herein, we report an unprecedented approach to obtain
luliconazole, consisting of a lipase-catalyzed chemoenzymatic
synthesis, which has the preparation of a chiral β-halohydrin
intermediate as key step. The importance of the preparation of
chiral β-halohydrins lies in the fact that they are intermediates in
the synthesis of substances with high added value.[9,10]
Introduction
Luliconazole (R-1), Fig. 1, also chemically known as (-)-(2E)-
[(4R)-4-(2,4-dichlorophenyl)-1,3-dithiolan-2-ylidene](1H-
imidazol-1-yl)acetonitrile, is a pharmaceutically active substance
marketed under the brand names Luzu® (Valeant
Pharmaceuticals North America) and Lulicon® (Pola Pharma),
among others.[1,2]
One of the methods used to obtain chiral β-halohydrins is the
enzymatic kinetic resolution of the corresponding racemate
mediated by lipases.[11] There are some reports of the kinetic
resolution of β-halohydrins containing aromatic ring moiety via the
acylation reaction, as well as from their corresponding esters, via
hydrolytic process. Several lipases have been used in both
processes
such
as
Pseudomonas
fluorescens,[12,13,14]
Burkholderia cepacia,[15,16,17] Candida antarctica type B (Novozym
435®),[13,18,19] P. cepacia, [20,21,22,23,24] C. rugosa,[21] Pseudomonas
sp.[25] and P. aeruginosa.[26] In some of these examples, the
kinetic resolution was performed in the presence of ionic
liquids[17,23] or by dynamic kinetic resolution in the presence of
ruthenium as racemization agent.[20,26]
Figure 1. Structural formula of luliconazole (R) -1
Lipases (EC 3.1.1.3) are highlighted as one of the most used
enzymes in synthesis of enantiomerically pure drugs since no
cofactors are required and regio-, chemo- and enantioselectivities
are observed in the resolution process of racemates. Since most
of the drugs are chiral, it is important to know which of the
stereoisomers has the desired biological activity. Thus, the
patient receives only one dose of the active stereoisomer,
avoiding the side effects caused by the undesired stereoisomer.
Therefore, biocatalysis represents an alternative tool for the
production of enantiomerically pure chiral drugs over
conventional chemical processes.[27]
This drug has antifungal action, being used for the treatment
of tinea pedis, popularly known as athlete's foot, in addition to the
treatment against of both candidiasis and pityriasis.[3] In vitro, (R)-
1 is one of the most potent antifungal agents against filamentous
[a]
T. S. Fonseca, Dr. L. D. Lima, M. C. F. de Oliveira, Prof. Dr. T. L. G.
de Lemos, Prof. Dr. D. Zampieri, Prof. Dr. M. C. de Mattos, Prof. Dr.
Department of Organic and Inorganic Chemistry
Laboratory of Biotechnlogy and Organic Synthesis (LABS)
Federal University of Ceara
In this paper, we describe the chemoenzymatic synthesis of
luliconazole (R)-1, which had as key step the preparation of the
Campus do Pici, Postal Box 6044, 60455-970, Fortaleza, CE. Brazil
E-mail: mcdmatto@ufc.br
Homepage: www.labs.ufc.br
chiral
β-halohydrin
(1S)-2-chloro-1-(2,4-dichlorophenyl)-1-
ethanol (S)-3, via kinetic resolution of acetate rac-4, mediated by
a lipase, Scheme 1. Our focus was to perform an optimization of
the reactional conditions, including the enzymes recycling
outcome.
[b]
Prof. Dr. F. Molinari
Department of Food
Environmental and Nutritional Sciences (DEFENS)
University Of Milan
Via Mangigalli 25, 20133, Milan, Italy
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10.1002/ejoc.201800250
European Journal of Organic Chemistry
FULL PAPER
Results and Discussion
Table 1. Kinetic resolution of rac-4, via hydrolysis using lipases.a
The chemoenzymatic synthesis of luliconazole is depicted in
Scheme 1. The key step was the kinetic enzymatic resolution of
the racemic 2-chloro-1-(2,4-dichlorophenyl)ethyl acetate (rac-4),
via a hydrolytic approach, mediated by a lipase.
Ed
Time
(h)
e.e.s
(%)b
e.e.p
(%)b
c
Entry
Lipase
(%)c
1
2
3
P. fluorescens
P. camemberti
19
24
14
77
3
70
3
52
50
51
13
1
R. miehei
immobilized on
60
57
6
anionic resin
Amano lipase
PS from B.
cepacia
immobilized on
diatomaceous
earth
4
5
19
19
73
80
66
74
52
52
10
16
Amano lipase
PS from B.
cepacia
6
7
8
TLLe
24
24
24
>99
97
92
95
59
52
50
16
128
185
4
Novozym 435®
Amano lipase
11
from
M.
javanicus
9
C. rugosa
24
24
9
3
48
3
16
50
3
1
Scheme 1. Chemoenzymatic synthesis of luliconazole.
Porcine
10
pancreas
Synthesis of racemic halohydrin (rac-3) and its acetate (rac-
11
12
R. niveus
R. oryzae
24
19
1
64
57
2
5
6
4)
53
48
immobilized on
immobead-150
As first step, the chemical reduction of the α-chloroketone 2
was carried out by using sodium borohydride (0.5 eq.) in MeOH[28]
to yield rac-3 in 90% yield. Next, the chemical acetylation of rac-
3 was performed using Ac2O, DMAP and Et3N at room
temperature for 2 h[29] and allowed the preparation of rac-4 with
91% isolated yield, Scheme 1. Adequate chiral GC analyses were
developed for both rac-3 and rac-4 in order to achieve a reliable
method to measure the enantiomeric excesses of both remaining
substrate and the final product from the lipase-catalyzed
resolution.
[a] Conditions: 30 °C, lipase:rac-4 (2:1) at 250 r.p.m. [b]
Determined by GC. [c] Conversion, c = e.e.s/(e.e.s + e.e.p).
[d] Enantiomeric ratio, E = ln[1 − c(1 + e.e.p)]/ln[1 − c(1 −
e.e.p)]. [e] TLL: T. lanuginosus immobilized on immobead-
150.
Among the lipases evaluated in the hydrolysis reaction of rac-
4, Thermomyces lanuginosus immobilized on immobead-150
(TLL) and CAL-B (Novozym 435®) were the enzymes that
provided better results. For these enzymes, conversions of 52 and
50%, and E values of 128 and 185 were observed, respectively,
in 24 h of reaction (Table 1, entries 6 and 7). On the other hand,
lipase from Rhizomucor miehei immobilized on anionic resin was
the enzyme with the highest activity in the kinetic resolution of rac-
4, with a conversion of 51% in only 14 h, but with low selectivity
(E value of 6), Table 1 entry 3. Lipase from Rhizopus niveus was
the less active enzyme, leading to a conversion of only 2% in 24
h of reaction, (Table 1, entry 11).
The other lipases tested, as P. fluorescens, Penicillium
camemberti, Amano PS from B. cepacia immobilized on
diatomaceous earth, Amano PS from B. cepacia, Amano lipase
from Mucor javanicus, C. rugosa, porcine pancreas and Rhizopus
oryzae immobilized on immobead-150, led to conversions of
about 50%, but with low enantioselectivity (E values ranging from
1 to 16; Table 1 entries 1, 2, 4, 5, 8, 9, 10 and 12, respectively).
Screening of lipase-mediated kinetic resolution of acetate
rac-4 via hydrolysis
For initial screening of lipases, twelve commercially available
enzymes (ratio 2:1 in weight respect to rac-4) were tried in the
resolution of rac-4 at 30 °C, 250 rpm, phosphate buffer pH 7
(0.1M). Reactions were monitored for a maximum of 24 h and
stopped before this time if the conversion had reached a value
close to 50%. The product β-halohydrin (S)-3 and the remaining
acetate (R)-4 were obtained, and the results are summarized in
Table 1.
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10.1002/ejoc.201800250
European Journal of Organic Chemistry
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Improving the kinetic resolution of rac-4 through
temperature variation
Once the ideal temperature (45 °C) was determined, at
which the kinetic resolution of rac-4 occurred in the shortest
possible time (15 min), we decided to evaluate the decrease of
the loaded enzyme on the reaction.
In order to improve enzymatic resolution of rac-4, we
performed a detailed study of the reaction condition, varying some
parameters, such as temperature, reaction time and the ratio
enzyme/substrate (w/w). For this study, we selected the enzymes
TLL immobilized on immobead-150 and Novozym 435® that were
most promising in the kinetic resolution of rac-4 (Table 1, entries
6 and 7). In addition, since these two lipases are commercially
immobilized, reuse studies have been performed.
In order to reduce the reaction time, the first variable to be
changed was the temperature. With additions of 5 °C, the kinetic
resolution of rac-4 was evaluated at 35, 40 and 45 °C. The results
were summarized in Table 2.
Effect of enzyme load in the kinetic resolution of rac-4
For Novozym 435® the ratio enzyme:substrate was
,
gradually decreased (from 2:1 to 0.25:1) and the kinetic resolution
of rac-4 remained efficient up to 0.5:1 (w:w). Enantiomeric excess
> 99%, 50% conversion and E value > 200 was observed for both
product and substrate, Table 2, entry 4. However, with an
enzyme:substrate ratio of 0.25:1, the conversion had a slight
decrease (43%) leading to an enantiomeric excess of substrate
remaining of 76%, Table 2, entry 5. For TLL to maintain an optimal
kinetic resolution of rac-4, the lowest enzyme:substrate ratio was
1.5:1, Table 2, entry 9. When we decreased the ratio to 1:1, the
conversion showed a decrease (30%) and the enantiomeric
excess of the substrate was only 41%, Table 2, entry 10.
Finally, since the most efficient enzymes (Novozym 435®
and TLL) in the kinetic resolution of rac-4 are commercially
immobilized, we decided to carry out a study of their reuse in ten
reaction cycles.
Table 2. Results of kinetic resolution of rac-4, via hydrolysis, mediated by
lipases, varying temperature and enzyme/substrate ratio (w/w).
Entry
1d
Lipase
T(°C)
35
Ec
Time
(min)
e.e.s
(%)a
e.e.p
(%)a
c
(%)b
Novozym
435®
960
120
15
>99
>99
>99
>99
76
>99
>99
>99
>99
>99
50
50
50
50
43
>200
>200
>200
>200
>200
2d
40
Novozym
435®
Reuse of the immobilized lipases
3d
45
Novozym
435®
This study was carried out in order to investigate the
efficiency of Novozym 435® and TLL in maintaining satisfactory
conversion and enantioselectivity values up to ten cycles of reuse
(Fig. 2 and 3).
4e
45
15
Novozym
435®
5f
45
15
Novozym
435®
e.e.p (%)
conversion
100
50
0
6d
7d
8d
9h
10i
TLLg
TLLg
TLLg
TLLg
TLLg
35
40
45
45
45
960
120
15
97
96
50
50
50
50
30
>200
>200
>200
>200
>200
>99
>99
>99
41
>99
>99
>99
>99
(%)
15
1
2
3
4
5
6
7
8
9
10
Cycles of reuse
15
aConditions: phosphate buffer pH 7 (0.1M), 45 ºC, lipase:rac-4 (0.5:1) and 15
min at 250 rpm.
[a] Determined by GC. [b] Conversion, c = e.e.s/(e.e.s + e.e.p). [c]
Enantiomeric ratio, E = ln[1 − c(1 + e.e.p)]/ln[1 − c(1 − e.e.p)]. [d]
lipase:rac-4 (2:1). [e] lipase:rac-4 (0.5:1). [f] lipase:rac-4 (0.25:1). [g]
TLL: T. lanuginosus immobilized on immobead-150. [h] lipase:rac-4
(1.5:1). [i] lipase:rac-4 (1:1).
Figure 2. Results from the reuse of Novozym 435®.a
As we increased the temperature by 5 °C (from 30 to 35 °C),
there was a decrease in reaction time from 24 to 16 h for both
Novozym 435® and TLL enzymes, Table 2, entries 1 and 6.
However, with Novozym 435® we observed an increase in the
enantiomeric excess values for both product and remaining
substrate (> 99%), Table 2, entry 1. No significant changes in the
enantiomeric excess values of product and remaining substrate
were observed for TLL, Table 2, entry 6. On the other hand, the
increment of 5 °C in the temperature (from 35 to 40 °C) led to a
significant decrease in reaction time from 16 to 2 h (Table 2,
entries 2 and 7). In this case, both lipases yielded the product and
the remaining substrate with enantiomeric excess > 99%. A
surprising result was obtained when the kinetic resolution was
performed at 45 °C. At this temperature, the reaction time for an
ideal kinetic resolution was only 15 min and both lipases led to the
product and remaining substrate with enantiomeric excess > 99%,
conversion of 50% and E value > 200 (Table 2, entries 4 and 8).
e.e.p (%)
conversion
100
50
0
(%)
1
2
3
4
5
6
7
8
9
10
Cycles of reuse
aConditions: phosphate buffer pH 7 (0.1M), 45 ºC, lipase:rac-4 (1.5:1) and 15
min at 250 rpm.
Figure 3. Results from the reuse of TLL immobilized on immobead-150.a
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10.1002/ejoc.201800250
European Journal of Organic Chemistry
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Conversion values close to 50% were observed for Novozym
435® during ten reused cycles. Additionally, it was obtained
enantiomeric excess values of product greater than 90% up to the
fifth reaction cycle. From the sixth reaction cycle, the enzyme lost
some of its selectivity, with enantiomeric excess values of product
varying between 81 and 89% (Fig. 2).
When the reuse study was performed with TLL, it was verified
that in the second reaction cycle the activity decreased to 37%, a
value that remained constant until the fifth reaction cycle. In the
next reaction cycles, there was a slight decrease in the conversion
values, culminating in a conversion of 30% in the last cycle.
Concerning the selectivity, it was observed that the enantiomeric
excess of the product decreased to 90% in the second reaction
cycle and continued progressively decreasing until the tenth cycle
at a value of 65% (Fig. 3).
Once the enzymatic loading and reuse studies were
performed, it was possible to verify that Novozym 435® presented
more advantages compared to TLL. In fact, Novozym 435®
required a lower enzyme:substrate ratio (0.5:1) in detriment of
1.5:1 to TLL. Moreover, Novozym 435® can be reused in up to five
reaction cycles maintaining high values of selectivity and
conversion. On the other hand, in the presence of TLL, the
conversion showed a considerable decrease already in the
second reaction cycle, followed by a progressive decrease in the
selectivity in the subsequent reaction cycles.
stood out as the best lipase because it required a lower enzymatic
load enzyme:substrate (0.5:1) compared to TLL (1.5:1). Moreover,
Novozym 435® proved to be a robust biocatalyst, since it could be
reused in five reaction cycles, maintaining high values of both
conversion and enantioselectivity. Finally, this unprecedented
approach to obtain luliconazole can be considered eco-friendly,
since a stable, low cost, reusable, commercially available and
highly enantioselective biocatalyst was used. Investigation of the
kinetic resolution of β-halohydrin (rac-3) via lipase-catalyzed
acetylation will be another subject of forthcoming studies.
Experimental Section
Enzymes
(i) Immobilized lipases: C. antarctica lipase type
B
immobilized on acrylic resin (CAL-B, Novozym 435, 7,300.0 U/g)
and R. miehei lipase immobilized on anionic resin (RML, 150.0
U/g) were purchased from Novozymes®. B. cepacia lipase
immobilized on diatomaceous earth (Amano PS-IM, ≥500 U/g), T.
lanuginosus lipase immobilized on immobead-150 (TLL, 250.0
U/g) and R. oryzae lipase immobilized on immobead-150 (ROL,
340.0 U/g) were acquired from Sigma-Aldrich®. (ii) Crude lipase
preparations: M. javanicus lipase (Amano M, ≥10,000 U/g), R.
niveus lipase (RNL, 1,5 U/mg), B. cepacia lipase (Amano PS,
≥30,000 U/g), P. fluorescens lipase (AK, 22,100.0 U/g), P.
camemberti lipase (Amano G, 50.0 U/g) were acquired from
Sigma-Aldrich®. Porcine pancreas lipase (PPL, 46.0 U/g solid),
and C. rugosa lipase (CRL, 1.4 U/g) were obtained from Sigma-
Aldrich®.
After optimizing the enzymatic kinetic resolution of rac-4, we
proceed with the following steps to obtain luliconazole (R)-1.
Synthesis of luliconazole (R)-1
The kinetic resolution of rac-4 was performed under the
optimized conditions (Scheme 1), phosphate buffer (pH 7),
Novozym 435®, enzyme:substrate (0.5:1), 45 °C and 15 min of
reaction, and provided the β-halohydrin (S)-3 and the remaining
acetate (R)-4 with enantiomeric excess values > 99%, conversion
of 50% and enantioselectivity (E) > 200.
Chemical Materials
Chemical reagents were purchased from different commercial
sources and used without further purification. Methanol, DMF,
chloroform, DMSO, ethyl acetate and dichloromethane were
acquired from Synth®. Tetrahydrofuran and carbon disulfide were
acquired from Sigma-Aldrich®. Solvents were distilled over an
adequate desiccant under nitrogen. Analytical TLC analyses were
performed on aluminum sheets pre-coated with silica gel 60 F254
(0.2 mm thick) from Merck®. Flash chromatographies were
performed using silica gel 60 (230-240 mesh).
Posteriorly, the (S)-3 was subjected to a mesylation reaction
with MsCl, DMAP, Et3N in CH2Cl2 for 7 h to give the mesylated
product (S)-5 in 89% yield and enantiomeric excess >99%,
Scheme 1.[30]
Concomitantly, 1-cyanomethylimidazole 7 was obtained in
70% yield from the reaction of imidazole 6 with bromoacetonitrile,
NaH in dry THF for 4 h, Scheme 1.[31]
Finally, in the last step, the mesylate (S)-5 was subjected to
the reaction with 1-cyanomethylimidazole 7 in the presence of
CS2, K2CO3 in DMF, at room temperature and 24 h of reaction.[32]
However, under such conditions the substrate (S)-5 was
recovered unchanged from the reaction medium. In search of
better reaction conditions, we decided to use KOH as base and
DMSO as the solvent. In this case, we obtained a mixture of the
E (R-1) and Z (R-8) isomers in a ratio of 2:1. After purification, the
luliconazole (E-isomer) was obtained in 43% yield and
enantiomeric excess > 99%, Scheme 1.[6]
Analysis
Melting points were determined in open capillary tube
1
Microquímica model MQAPF-302 and are uncorrected. H and
13C NMR were obtained using Spectrometer Bruker model
Avance DPX 300, operating at frequency of 300 MHz for
hydrogen and frequency of 75 MHz for carbon. The chemical
shifts are given in delta (δ) values and the coupling constants (J)
in Hertz (Hz). Measurement of the optical rotation was done in a
Jasco P-2000 polarimeter. Gas chromatograph (GC) analyses
were carried out in a Shimadzu chromatograph model GC 2010
with a flame ionization detector using a chiral column CP-chirasil-
dex (25 m x 0.25 mm x 0.25 µm, 0.5 bar N2). For the following of
Conclusions
In summary, we prepared luliconazole via an original
approach involving the chemoenzymatic process using lipases.
Both lipases TLL immobilized on immobead-150 and Novozym
435® were able to resolve the racemic 2-chloro-1-(2,4-
dichlorophenyl)ethyl acetate (rac-4) via a hydrolytic process. The
enantiomerically pure key intermediate β-halohydrin (1S)-2-
chloro-1-(2,4-dichlorophenyl)-1-ethanol (S)-3 was obtained with
conversion of 50% in only 15 min of reaction. Novozym 435®
the
reaction
time
courses:
rac-2-chloro-1-(2,4-
dichlorophenyl)ethyl acetate (rac-4): 185 ºC; 1 ºC/min. 190 ºC
(hold 5 min.); 0.5 ºC/min. 200 ºC (hold 15 min.). Retention times
were: (R)-acetate= 8.33 min.; (S)-acetate= 8.82 min. Haloydrin
rac-3 was analyzed using a chiral column Rt®bDEXcst (30 m x
0.39 mm x 0.25 µm, 0.5 bar N2). For the following of the reaction
time courses: rac-2-chloro-1-(2,4-dichlorophenyl)ethanol (rac-3):
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Synthesis of luliconazole (R-1)
185 ºC; 1 ºC/min. 190 ºC (hold 5 min.); 0.5 ºC/min. 200 ºC (hold
15 min.). Retention times were: (S)-halohydrin= 17.90 min.; (R)-
halohydrin= 18.50 min.
A solution of nitrile 7 (0.1 g, 0.9 mmol) and carbon disulfide
(56 µL, 0.9 mmol) in of DMSO (3 mL) at 4 ºC was added to a
solution of KOH (0.210 mg, 3.7 mmol) in DMSO (3 mL). The
reaction was stirred for 2 h at room temperature to form the
dithiolate salt. Then, (S)-5 (0.560 g, 1.9 mmol) was added slowly
at 4 °C and the reaction was stirred for 24 h at room temperature.
After this time, cold water (30 mL) was added and the reaction
mixture was stirred for 10 min. Then, the product was extracted
with EtOAc (3 x 40 mL) and the organic phase were washed with
ice water (2 x 20 mL), dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure. The crude product was
purified using column chromatography with silica gel
(CHCl3/CH3CN 9:1) to afford the (R,Z)-isomer (R)-8 with 19%
yield (0.062 g, 0.18 mmol) and (R)-1 as yellow solid in 43% yield
(0.141 g, 0.4 mmol).
Synthesis of rac-β-halohydrin (rac-3)
NaBH4 (0.085 g, 2.2 mmol) was slowly added to a solution
of 2-chloro-1-(2,4-dichlorophenyl)ethanone (2) (1 g, 4.5 mmol) in
MeOH (40 mL) at 0 ºC. The reaction was stirred for 30 min at room
temperature. Upon completion, the MeOH was evaporated under
reduced pressure. Then, 1 M HCl (10 mL) of was added followed
by extraction with EtOAc (4 x 60 mL). Organic phases were
combined and dried over anhydrous Na2SO4, filtered and solvent
was evaporated under reduced pressure. Then, the crude product
was purified by performing column chromatography, employing
silica gel and CHCl3 to afford rac-β-halohydrin rac-3 in 90% yield
(0.913 g, 4.0 mmol).
General procedure for the lipase-catalyzed hydrolysis of rac-
Synthesis of rac-acetate (rac-4)
4
A suspension of DMAP (0.054 g, 0.4 mmol) and acetic
anhydride (1.25 mL, 13.3 mmol) was dissolved in CH2Cl2 (40 mL).
Then, rac-3 (1 g, 4.4 mmol) and triethylamine (616.3 µL, 4.4
mmol) were added. The reaction was stirred for 2 h at room
temperature. After this time, distilled water (10 mL) was added
followed by extraction with CH2Cl2 (3 x 50 mL). The organic
phases were combined and dried over anhydrous Na2SO4. Then,
after filtration, the solvent was evaporated under reduced
pressure and the crude product was purified using a silica gel
column chromatography and CHCl3 to afford rac-acetate rac-4 in
91% yield (1.071 g, 4.0 mmol).
A suspension of rac-4 (0.030 g, 0.1 mmol) and lipase (ratio
2:1 in weight respect to the rac-4) in phosphate buffer 100 mM
pH 7.0 (1.0 mL) was shaken in their proper temperature and time
reaction at 250 rpm. After the conversion reaches a value close
to 50%, the products were extracted with EtOAc (3 x 5 mL). The
organic phases were combined and dried over Na2SO4, filtered
and the solvent evaporated under reduced pressure. The reaction
crude was purified by flash chromatography on silica gel (100%
CHCl3), yielding (R)-acetate (R-4) and (S)-halohydrin (S-3) being
their enantiomeric excess determined by GC.
Synthesis of (R)-4 and (S)-3 mediated by Novozym 435®
Synthesis
of
(1S)-2-chloro-1-(2,4-dichlorophenyl)-2-
methanesulfonate (S-5)
A suspension of rac-4 (0.5 g, 1.9 mmol) and 0.25 g of
Novozym 435® in phosphate buffer 100 mM pH 7.0 (19 mL) was
shaken in 45 ºC and 15 min at 250 rpm. After the conversion has
reached a value close to or 50%, the products were extracted with
EtOAc (3 x 20 mL). The organic phases were combined and dried
over Na2SO4, filtered and the solvent evaporated under reduced
pressure. The reaction crude was purified by flash
chromatography on silica gel and CHCl3, yielding 46.5% of (R)-
acetate (R-4) (0.454 g, 1.7 mmol) with [α]D23=-52.3 (c=1.0 in ethyl
acetate) for 99% e.e. and 44.5% of (S)-halohydrin (S-3) (0.383 g,
1.7 mmol) with [α]D20=+51.1 (c=2.50 in chloroform) for 99% e.e.;
Lit [α]D25=-52.8 (c=2.55 in chloroform) for 99% e.e. of the (R)-
enantiomer.[33]
Triethylamine (307 µL, 2.2 mmol), DMAP (0.027 g, 0.2
mmol) and MsCl (855 µL, 11.0 mmol) were added to a solution of
(S)-3 (0.5 g, 2.2 mmol) in CH2Cl2 (25 mL) at 0 ºC for 1 h. Then,
the reaction mixture was stirred at room temperature for 6 h. After
this time, 20 mL of distilled water was added followed by
extraction with CH2Cl2 (3 x 40 mL). Then, the organic phase were
combined and treated with saturated aqueous NaHCO3 solution
(20 mL) and the organic phase was collected and dried with
anhydrous Na2SO4. Finally, after filtration, the solvent was
evaporated under reduced pressure and the crude product was
purified using column chromatography with silica gel
(CHCl3/hexane 7:3) to afford (S)-5 as yellow solid in 89% yield
(0.607 g, 2.0 mmol).
rac-2-chloro-1-(2,4-dichlorophenyl)ethanol (rac-3): 90%
yield. Orange solid. Rf=0.45 (CHCl3); m.p. 47-49 C; Lit m.p. 46-
47 C;[15] 1H NMR (300 MHz, CDCl3, TMS): δ=2.60 (br s, OH, 1H),
3.52 (dd, J=11.3, 8.4 Hz, 1H), 3.87 (dd, J=11.3, 2.8 Hz, 1H), 5.26
(dd, J=8.4, 2.8 Hz 1H), 7.31 (dd, J=8.4, 2.0 Hz 1H), 7.38 (d, J=2.0
Hz, 1H), 7.58 ppm (d, J=8.4 Hz, 1H) ; 13C NMR (75 MHz, CDCl3,
TMS): δ=49.4 (CH2), 70.5 (CH), 127.8 (CH), 128.7 (CH), 129.5
(CH), 132.7 (C), 134.8 (C), 136.1 ppm (C).
Synthesis of 1-cyanomethylimidazole (7)
To a solution of imidazole (6) (0.5 g, 7.3 mmol) in dry THF
(50 mL) under nitrogen atmosphere, NaH (0.176 g, 7.3 mmol) was
added until complete release of
hydrogen. After,
bromoacetonitrile (510 µL, 7.3 mmol) was added dropwise, with
the appearance of a yellow color in the reaction. The reaction
mixture was stirred for 4 h at room temperature. Then, the solvent
was evaporated under reduced pressure and distilled water (10
mL) was added followed by extraction with CH2Cl2 (3 x 50 mL).
The organic phase was dried over anhydrous Na2SO4. After
filtration, the solvent was evaporated under reduced pressure.
Then, the crude product was purified by performing column
chromatography on silica gel (CHCl3/MeOH 9.5:0.5) to afford (7)
as a brown solid in 70% yield (0.546 g, 5.1 mmol).
rac-2-chloro-1-(2,4-dichlorophenyl)ethyl acetate (rac-4):
1
91% yield. Yellow liquid. Rf=0.61 (CHCl3); H NMR (300 MHz,
CDCl3, TMS): δ=2.17 (s, 3H), 3.72 (dd, J=11.8, 7.0 Hz, 1H), 3.84
(dd, J=11.8, 3.7 Hz, 1H), 6.31 (dd, J=7.0, 3.7 Hz, 1H), 7.28 (dd,
J=8.5, 2.0 Hz, 1H), 7.39 (d, J=8.5 Hz, 1H), 7.40 ppm (d, J=2.0 Hz
1H); 13C NMR (75 MHz, CDCl3, TMS): δ=21.0 (CH3), 45.2 (CH2),
71.4 (CH), 127.7 (CH), 128.9 (CH), 129.8 (CH), 133.2 (C), 133.7
(C), 135.3 (C), 169.6 ppm (C).
(S)-2-chloro-1-(2,4-dichlorophenyl)-2-methanesulfonate (S-
5): 89% yield. Yellow solid. Rf=0.45 (CHCl3/hexane 7:3); m.p. 73-
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800250
European Journal of Organic Chemistry
FULL PAPER
1
76 C; [α]D20=+64.0 (c=1.0 in chloroform) for 99% e.e.; H NMR
(300 MHz, CDCl3 TMS): δ=3.10 (s, 3H), 3.75 (dd, J=12.2, 7.9 Hz,
1H), 3.87 (dd, J=12.2, 3.6 Hz, 1H), 6.06 (dd, J=7.9, 3.6 Hz, 1H),
7.36 (dd, J=8.4, 1.9 Hz, 1H), 7.45 (d, J=1.9 Hz, 1H), 7.53 ppm (d,
J=8.4 Hz, 1H); 13C NMR (75 MHz, CDCl3, TMS): δ=39.0 (CH3),
45.4 (CH2), 78.8 (CH), 128.2 (CH), 129.2 (CH), 130.0 (CH), 132.4
(C), 132.7 (C), 136.3 ppm (C).
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1-cyanomethylimidazole (7): 70% yield. Brown solid.
Rf=0.50 (CHCl3/MeOH 9.5:0.5); m.p. 141-142 C; Lit m.p. 138 C;[34]
1H NMR (300 MHz, CDCl3, TMS): δ=4.92 (s, 1H), 7.03 (s, 1H),
7.10 (s, 1H) 7.55 ppm (s, 1H); 13C NMR (75 MHz, CDCl3, TMS):
δ=34.5 (CH2), 113.9 (C), 119.1 (CH), 130.9 (CH), 137.2 ppm
(CH).
(R,Z)-Isomer (R-8): 19% yield. Yellow solid. Rf=0.46
(CHCl3/CH3CN 9:1); m.p. 113-115 C; 1H NMR (300 MHz, CDCl3,
TMS): δ=3.74 (dd, J=12.0, 7.6 Hz, 1H), 3.97 (dd, J=12.0, 5.2 Hz,
1H), 5.63 (dd, J=7.6, 5.2 Hz, 1H), 7.06 (s, 1H), 7.17 (s, 1H), 7.32
(dd, =8.4, 2.0 Hz 1H), 7.46 (d, J=2.0 Hz, 1H), 7.52 (d, J=8.4 Hz,
1H), 7.65 ppm (s, 1H); 13C NMR (75 MHz, CDCl3, TMS) δ=44.2
(CH2), 55.6 (CH), 94.2 (C), 114.6 (C), 119.3 (CH), 128.1 (CH),
129.2 (CH), 130.4 (2CH),131.4 (C), 134.4 (C), 136.1 (C), 137.2
(CH), 164.7 ppm (C).
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Luliconazole (R-1): 43% yield. Yellow solid. Rf=0.45
(CHCl3/CH3CN 9:1); m.p. 151-152 C; Lit m.p. 151.1 C;[35] [α]D20=-
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1
50.7 (c=1.0 in dimethylformamide) for 99% e.e.; H NMR (300
MHz, CDCl3, TMS): δ=3.65 (dd, J=12.0, 7.4 Hz, 1H), 3.89 (dd,
J=12.0, 5.2 Hz, 1H), 5.69 (dd, J=7.4, 5.2 Hz, 1H), 7.06 (s, 1H),
7.18 (s, 1H), 7.34 (dd, J=8.4, 1.9 Hz, 1H), 7.49 (d, J=1.9 Hz, 1H),
7.60 (d, J=8.4 Hz, 1H), 7.65 ppm (s, 1H); 13C NMR (75 MHz,
CDCl3, TMS) δ=444 (CH2), 55.3 (CH), 94.0 (C), 114.8 (C), 119.4
(CH), 128.2 (CH), 129.2 (CH), 130.4 (2CH), 131.5 (C), 134.4 (C),
136.1 (C), 137.2 (CH), 164.8 ppm (C).
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The authors thank to the Fundação Cearense de Apoio ao
Desenvolvimento Científico e Tecnológico (FUNCAP), Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
and Coordenação de Aperfeiçoamento de Ensino Superior
(CAPES) for fellowships and financial support. The authors thank
the Brazilian funding agency Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) for providing
the Special Visiting Researcher fellowship (process
400171/2014-7) under the Brazilian Scientific Program “Ciência
sem Fronteira” and the research sponsorships of M. C. de Mattos
(Process: 308034/2015-5) and M. C. F. de Oliveira (Process:
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Conflict of interest
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The authors declare no conflict of interest.
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Keywords: biocatalysis • chemoenzymatic synthesis • kinetic
resolution • luliconazole • Novozym 435®
[1]
S. B. Bhirud, K. H. Bhushan, S. S. Zhope, S. G. Ghadigaonkar, P. Singh,
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2014041708, 2014.
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Thiago de S. Fonseca, Lara D. Lima,
Maria da C. F. de Oliveira, Telma L. G. de
Lemos, Davila Zampieri, Francesco
Molinari, Marcos C. de Mattos*
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Luliconazole via biocatalytic process
A newapproach based on biocatalysis was developed for the production of the potent
antifungal luliconazole. The lipase Novozym 435® proved to be a robust biocatalyst
in the key step involving the preparation of an enantiomerically pure β-halohydrin in
only 15 min of reaction. The enzyme was reused in up to 5 reaction cycles with
maintenance of high values of activity and selectivity. After a few more steps,
luliconazole was obtained in good yield and enantiomeric excess.
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