Lipase-catalyzed kinetic resolutions of racemic 1-(10-ethyl-10H- phenothiazin-1,2, and 4-yl)ethanols and their acetates

Article abstract of DOI:10.1016/j.tetasy.2011.05.009

The synthesis of both enantiomers of 1-(10-ethyl-10H-phenothiazin-1,2, and 4-yl)ethanols 1a-c and their acetates via enantioselective methanolysis of the corresponding racemic esters rac 2a-c with lipase B from Candida antarctica (CaL-B) or/and by acylation of the racemic alcohols with the lipase A or lipase B from C. antarctica (CaL-A and CaL-B) is described. The absolute configuration of enantiopure 1-(10-ethyl-10H-phenothiazin-1-yl)ethyl acetate 2a was assigned as (R) by using QM/MM(hf/3-21g:uff) calculations within the CaL-B (1LBT crystal structure) enzymic environment.

Full text of DOI:10.1016/j.tetasy.2011.05.009

Tetrahedron: Asymmetry 22 (2011) 916–923
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Lipase-catalyzed kinetic resolutions of racemic 1-(10-ethyl-10H-
phenothiazin-1,2, and 4-yl)ethanols and their acetates
Jürgen Brem a, Sarolta Pilbák b, Csaba Paizs a, Gergely Bánóczi b, Florin-Dan Irimie a,
b,
Monica-Ioana Toßsa a, , László Poppe
⇑
⇑
a Department of Biochemistry and Biochemical Engineering, Babeßs-Bolyai University of Cluj-Napoca, Ro-400028 Cluj-Napoca, Arany János 11, Romania
b Department of Organic Chemistry and Technology and Research Group for Alkaloid Chemistry HAS, Budapest University of Technology and Economics,
}
H-1111 Budapest, Muegyetem rkp. 3, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of both enantiomers of 1-(10-ethyl-10H-phenothiazin-1,2, and 4-yl)ethanols 1a–c and
their acetates via enantioselective methanolysis of the corresponding racemic esters rac 2a–c with lipase
B from Candida antarctica (CaL-B) or/and by acylation of the racemic alcohols with the lipase A or lipase B
from C. antarctica (CaL-A and CaL-B) is described. The absolute configuration of enantiopure 1-(10-ethyl-
10H-phenothiazin-1-yl)ethyl acetate 2a was assigned as (R) by using QM/MM(hf/3–21g:uff) calculations
within the CaL-B (1LBT crystal structure) enzymic environment.
Received 27 April 2011
Accepted 17 May 2011
Available online 15 June 2011
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Candida antarctica were found to be efficient catalysts for KR
and DKR,5 and also for the enantioselective resolution of various
Phenothiazine is one of the most valuable templates for a large
number of organic compounds, exhibiting a wide variety of biolog-
ical activities and which are used as antihistaminic, antipsychotic,
anticholinergic, antiemetic, anti-inflammatory, sedative, or cito-
static drugs.1 Recently the synthesis of 2-substituted N-acylpheno-
thiazines and their antibacterial and antifungal activities was
reported.2
N-alkylated phenothiazines, such as phenothiazin-3-yl-cyano-
hydrins,6 -3-hydroxy-propanoic acids,7 or -1-ethanols.8 In order
to investigate how the position of the asymmetric moiety at-
tached to the phenothiazine could influence the activity and
selectivity of lipases, the kinetic resolution of 1-(10-ethyl-10H-
phenothiazin-1-,2- and 4-yl)ethanols rac-1a–c and of their acet-
ylated derivatives rac-2a–c was studied. The resulting optimum
conditions for the analytical scale reactions were successfully
applied for the preparative scale synthesis of the highly
enantiomerically enriched (R)- and (S)-1-(10-ethyl-10H-
phenothiazinyl)ethanols (R)- and (S)-1a–c.
Moreover, the absolute configurations of the enantiopure prod-
ucts obtained were assigned using the earlier published data for
enantiomerically pure enantiomers of 1d8 and non alkylated 1b9
and by QM/MM calculations within the CaL-B10 structure for 2a
and 2d.
Especially in the case of central nervous system drugs,3 a close
relationship between the chemical structure and the therapeutic
effects of phenothiazine derivatives has been demonstrated. The
interaction of the drug-pharmacological receptors strongly de-
pends on the nature, position, and number of functional groups
present in the drug molecule.4
Due to the chiral nature of biomacromolecules which are built
by chiral units such as amino acids and carbohydrates, most of
the biologically active compounds controlling the physiological
functions of living organisms are chiral. Moreover, the stereocom-
plementary recognition between chiral drugs and drug-receptor
has been demonstrated.
The increased interest in the understanding of how phenothia-
zine-based drugs act, explains the necessity of the development of
novel synthetic methods for the preparation of variously substi-
tuted phenothiazines in enantiopure form.
Hydrolases are frequently used as biocatalysts for the kinetic
resolution of chiral organic compounds. Lipases A and B from
2. Results and discussion
2.1. Chemical synthesis
As shown in Scheme 1 racemic 1-(10-ethyl-10H-phenothiazi-
nyl)ethanols rac-1a–c were prepared using specific methods for
each individual compound in accordance with the literature.
As starting compounds for the syntheses of alcohols rac-1a–c,
the corresponding 10-ethyl-10H-phenothiazinyl-carbaldehydes
(for 1a,c) or the commercially available 1-(10H-phenothiazin-2-
yl)ethanone (for 1b) were used (Scheme 1).
⇑
Corresponding authors. Tel.: +36 1 463 3299; fax: +36 1 463 3697 (L.P.).
E-mail address: poppe@mail.bme.hu (L. Poppe).
0957-4166/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2011.05.009

J. Brem et al. / Tetrahedron: Asymmetry 22 (2011) 916–923
917
S
S
N
S
N
S
I.
II.
III.
N
H
N
H
O
O
OH
1a
S
S
S
N
IV.
II.
O
O
OH
N
N
H
1b
OH
O
S
N
S
N
S
N
II.
V.
III.
1c
Scheme 1. Synthesis of the racemic 1-(10-ethyl-10H-phenothiazinyl)ethanols rac-1a–c. Reagents and conditions: I. (a) 2.5 equiv n-BuLi, À78 °C, 1 h, rt, 48 h; (b) DMF, À78 °C,
rt, 2 h; II. NaH, EtI, DMF; III. MeMgI, Et2O; IV. NaBH4, methanol; V. (a) BuLi, TMEDA, À78 °C, rt 1 h; (b) DMF, À78 °C, rt, 1 h.
The direct lithiation and subsequent formylation with DMF is a
facile procedure to regioselectively prepare phenothiazine deriva-
tives with the formyl group adjacent to the heteroatom.11 The di-
rect metallation of phenothiazine with 2.5 equiv of n-BuLi
followed by treatment with DMF resulted in selective formylation
at the 1-position. The N-alkylation of the resulting aldehyde with
ethyl iodide followed by Grignard reaction with methyl iodide
yielded almost quantitatively racemic 1-(10-ethyl-10H-phenothia-
zin-1-yl)ethanol rac–1a.
The 1-(10-ethyl-10H-phenothiazin-2-yl)ethanol rac-1b was ob-
tained in high overall yield by N-alkylation of the commercially
available 2-acetylphenothiazine with ethyl iodide followed by sub-
sequent reduction with NaBH4 in methanol.
For the synthesis of racemic 1-(10-ethyl-10H-phenothiazin-4-
yl)ethanol rac-1c, lithiation of N-ethyl phenotiazine was performed
in the presence of a stoichiometric amount of tetramethylethyl-
ene-diamine (TMEDA) followed by formylation with DMF.12 The
forming aldehyde was transformed into the desired alcohol rac-
1c via Grignard reaction with methyl iodide.
Chemical acetylation of alcohols rac-1a–c by acetyl chloride in
the presence of triethylamine and a catalytic amount of DMAP gave
the racemic acetates rac-2a–c (Scheme 2).
their acetates rac-2a–c, the chromatographic separation of the
enantiomers was first established (Table 7). The base-line separa-
tion of the enantiomers of all rac-1,2a–c was performed using var-
ious enantioselective HPLC columns.
Efficient enzymatic procedures for the synthesis of enantiomeri-
cally pure N-alkyl-phenothiazin-3-yl-ethanols (R)-1d and (S)-1d
and their butanoates has been already described, using L-AK and
CaL-B mediated acylations of racemic 1-(10-alkyl-10H-phenothia-
zin-3-yl)ethanol rac-1d and by CaL-B catalyzed methanolysis of the
racemic butanoate of 1d.8 Since the natureof the alkyl group attached
to the heterocyclic nitrogen exhibited no influence on the selectivity
of the enzymatic reactions, we decided to investigate only the lipase
catalyzed kinetic resolution of the racemic N-ethyl substituted
1-(phenothiazinyl)ethanols rac-1a–c and their acetates rac-2a–c.
2.2.1. Analytical scale biotransformations
2.2.1.1. Analytical scale enzymatic acetylation of rac-1a–c. First,
the analytical scale enantioselective lipase catalyzed acetylation of
racemic 1-(10-ethyl-10H-phenothiazinyl)-ethanols rac-1a–c was
studied in neat vinyl acetate using 14 free and immobilized com-
mercial lipases (listed in the Experimental parts) (Scheme 2). Most
of them exhibited low enantioselectivities and activities, or were
active in a nonselective manner. The best results were obtained
with lipases A and B from C. antarctica (CaL-A and CaL-B) and with
the lipase from Pseudomonas fluorescens (L-AK), which were active
and selective biocatalysts for the kinetic resolutions of rac-1a–c as
shown in Table 1.
2.2. Enzymatic synthesis
To investigate the stereoselectivity of the reactions involving
racemic 1-(10-alkyl-10H-phenothiazinyl)ethanols rac-1a–c and
MeOH
OAc
OAc
OH
OAc
OH
OH
OAc
CH3-COCl
lipase
lipase
+
+
R
R
Et3N, DMAP,
CH2Cl2
R
R
R
R
solvent
solvent
rac-1a-c
rac-2a-c
(S)-2a-c
(R)-1a-c
(S)-1a-c
(R)-2a-c
N
S
R=
Scheme 2. Biotransformation of the racemic 1-(10-ethyl-10H-phenothiazinyl)ethanols rac-1a–c and their acetates rac-2a–c.

918
J. Brem et al. / Tetrahedron: Asymmetry 22 (2011) 916–923
Table 1
Acylation of rac-1a–c (0.1 M) with lipases CaL-A, CaL-B or L-AK (50 mg/mL) in neat vinyl acetate at room temperature
Substrate
Enzyme
Time (h)
c (%)
ee(R)-2a–c (%)
ee(S)-1a–c (%)
E
rac-1a
CaL-A
CaL-B
L-AK
14
85
85
24
11
7
97
94
67
30
12
6
77
36
5
rac-1b
rac-1c
CaL-A
CaL-B
L-AK
24
24
24
50
49
50
97
99
99
99
99
97
>200
ꢀ200
>200
CaL-A
CaL-B
L-AK
15
24
40
46
31
40
91
95
94
79
42
32
47
61
46
The most efficient lipase for the acetylation of the racemic alco-
hols rac-1b,c in terms of selectivity proved to be CaL-B. In the case
of rac-1a, which is the most sterically hindered secondary alcohol,
lipase A from C. antarctica cross-linked with glutaraldehyde (CaL-
A-CLEA) was the most active and selective (c = 24% after 14 h,
E = 77, Table 1.).
Based on the crystal structure of CaL-A and molecular modeling,
it has been proposed that upon substrate binding, CaL-A undergoes
a conformational change whereby the active-site ‘flap’ (Gly426-
Gly440) moves around the active site, widening the available space
essentially infinite.13 This can explain why CaL-A is active and
shows enantioselectivity in the O-acylation of highly sterically hin-
dered secondary alcohols.
poor solubility of all the ester substrates in water, various water–
organic solvent mixtures were used as reaction media. For all sub-
strates in all of the reaction media used, the appearance of large
amounts of side products, which decreased the global yield for
(R)-1a–c, was observed. In this way the use of water as nucleophile
for the enzymatic resolution of rac-2a–c practically was ruled out.
Using the aforementioned optimal conditions for the resolution
of 1-(10-alkyl-10H-phenothiazin-3-yl)-ethyl butanoates,8 the
methanolysis of acetates rac-2a–c in the presence of CaL-B in ace-
tonitrile was further investigated. The rate of methanolysis was
found to be higher than those of other alcoholyses. Moreover 1-
(10-alkyl-10H-phenothiazin-3-yl)-ethanol displayed a higher sta-
bility in the presence of methanol than in the presence of other
alcohols and the reaction rate was highest when methanol was
used as the nucleophile. Finally, methanol enhanced the solubility
of all substrates and products. While methanolysis occured effi-
ciently for rac-2b,c under these conditions, CaL-B was totally inac-
tive toward rac-2a in acetonitrile. The enzyme catalyzed
methanolysis of racemic 1-(10-ethyl-10H-phenothiazin-1-yl)ethyl
acetate rac-2a was studied in several organic solvents. The reaction
only took place in non-polar solvents and the best results were ob-
tained in n-hexane when at 50% conversion the enantiopurity of
both products was high (ee >99%) (Table 3).
It is known that the nature of the solvent can significantly influ-
ence the selectivity of this reaction.8 Therefore, the acylation of
rac-1a–c with vinyl acetate in the presence of the most appropriate
enzymes (CaL-B for rac-1b,c and CaL-A for 1a) was tested in several
organic solvents (Table 2).
While the CaL-A (CLEA form) catalyzed acylation of rac-1a with
vinyl acetate as the acyl donor in polar solvents, such as dichloro-
methane, chloroform, THF, or dioxane proceeded with low activity
(c <5% after 110 h), the reaction was efficient in ethers such as DIIP-
E, MTBE, or diethyl ether and other non-polar solvents (n-hexane
and toluene). Acetonitrile was the only polar solvent in which
CaL-A showed good activity but low stereoselectivity, is in agree-
ment with our previously reported results.14 As presented in
Table 2, n-hexane was found to be an appropriate reaction media
(E = 55, c = 22% after 110 h) for the CaL-A mediated acetylation of
rac-1a. For rac-1b,c CaL-B was the most active and selective when
using chloroform as the solvent (E >200 at approx 50% conversion).
As previously found for phenotiazin-3-yl ethanols,8 during the
enzymatic reactions and especially during the work-up of the reac-
tion mixture, a moderate to strong instability of the ethanolic sub-
strates was observed. The stability decrease in the series was as
follows: rac-1a > rac-1b > rac-1d > rac-1c. This is probably the rea-
son as to why the chemical hydrolysis of the obtained (R)-2a–c was
affected when the chemical hydrolysis of these compounds was
tested. Consequently, for the synthesis (R)-1a–c the lipase cata-
lyzed kinetic resolution of rac-2a–c was investigated further.
With the aim of developing a general methodology, the meth-
anolysis in the same solvent was also tested for rac-1b,c. While
the selectivities remained high (E >200), the reaction times de-
creased significantly.
2.2.2. Preparative scale synthesis of both (R)- and (S)-1,2a–c
Using the optimal conditions found for the analytical scale
enzymatic reactions, the preparative scale enzymatic synthesis of
both (S)- and (R)-1,2a–c was performed (Table 4). To demonstrate
the usefulness of these enzymatic procedures 1 g of the substrate
was used for each enzymatic kinetic resolution.
The opposite enantiomeric forms of products (S)-2a–c and (R)-
1a–c can be prepared by methanolysis of the corresponding race-
mic acetates rac-2a–c assisted by CaL-B. The ee for both (S)-2a–c
and (R)-1a–c were >99, c = 50% and E ꢀ200 when the reaction oc-
curs in hexane in the presence of 10 equiv of methanol.
2.2.1.2. Analytical scale enzymatic alcoholysis of rac-2a–c. It is
known that lipases usually retain their enantiomeric preference
in hydrolysis or alcoholysis.11 Consequently, such reactions should
result in the opposite enantiomeric forms of the reaction counter-
parts. For obtaining the opposite enantiomers of 1,2a–c, the kinetic
resolution of racemic acetates rac-a–c was performed in the next
step.
First the enzyme mediated hydrolysis of racemic acetates rac-
2a–c was tested using CaL-B, which was found to be the appropri-
ate biocatalyst for the enzymatic kinetic resolution of racemic
1-(10-alkyl-10H-phenothiazin-3-yl)-ethyl butanoates.8 Due to the
2.3. The absolute configurations of (R)-1b,d
The absolute configurations of the (R)-(+)-1-(10H-phenothia-
zin-2-yl)ethanol [non-ethylated analogue of (R)-1b] obtained by
reduction of 2-acetyl-phenothiazine in the presence of a chiral spi-
roborate ester catalyst has already been described.15 Since the sign
of the specific rotation proved to be independent of the nature of
the N-substitution within a series of (10-substituted-10H-pheno-
thiazin-3-yl)ethanols,8 the absolute configuration of the N-ethylat-
ed 1-(10H-phenothiazin-2-yl)ethanol (+)-1b could be assigned as
(R). On the other hand, in the case of (R)-(+)-1-(10-ethyl-10H-phe-

J. Brem et al. / Tetrahedron: Asymmetry 22 (2011) 916–923
919
Table 2
The effect of solvents on the CaL-A and CaL-B catalyzed (50 mg/mL) acylation of rac-1a–c (0.1 M) with vinyl acetate (0.4 M) at room temperature
Substrate
Solvent
Time(h)
c (%)
ee(R)-2a–c (%)
ee(S)-1a–d (%)
E
rac-1aa
DIIPE
MTBE
Diethyl-ether
n-Hexane
Acetonitrile
110
110
110
110
110
15
14
20
22
21
96
93
95
99
93
16
15
23
27
25
55
29
48
>200
33
rac-1bb
CHCl3
18
24
42
24
42
50
44
50
47
49
99
98
99
99
99
>99
77
99
87
95
ꢀ200
>200
>200
>200
>200
n-Hexane
Toluene
THF
CH2Cl2
rac-1cb
CHCl3
40
47
98
95
>200
A
b
Cal-A CLEA.
CaL-B.
Table 3
Alcoholysis of rac-2a–c (0.1 M) with methanol (1 M) and lipase CaL-B (50 mg/mL) in n-hexane or acetonitrile
Racemic compound
Solvent
Time (h)
c (%)
ee(R)-1 (%)
ee(S)-2 (%)
E
2a
2b
n-Hexane
n-Hexane
Acetonitrile
n-Hexane
Acetonitrile
420
24
20
24
20
50
50
50
50
50
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
ꢀ200
ꢀ200
ꢀ200
ꢀ200
ꢀ200
2c
Table 4
Preparative scale synthesis of enantiomerically pure ethanols (S)- and (R)-1a–c and their acetates (S)- and (R)-2a–c
Acylation of rac-1
Time (h)
(R)-2a–c
(S)-1a–c
b
b
Yielda (%)
ee (%)
[a]
D
Yielda (%)
ee (%)
[a]
D
a
b
c
360
24
48
95
92
91
97
>99
97
12.1
79
3.1
93
90
92
96
>99
99
À13.7
À47.2
À144.5
Methanolysis of rac-2
Time (h)
(R)-1a–c
(J)-2a–c
a
b
c
360
24
48
97
96
97
>99
>99
>99
14.8
46.4
145.3
98
98
97
>99
>99
>99
À13
À80
À3.5
a
Isolated yields based on the maximum theoretical 50%.
b
10 mg  mLÀ1, CHCl3, t = 25 °C.
nothiazin-3-yl)ethanol (R)-(+)-1d the absolute configuration was
conformations were refined by two-layer ONIOM calculations
(hf/3–21g:uff; over 955 atoms).
unambiguously determined by using X-ray crystallography.8
The reaction of the (R)- or (S)-enantiomers of ordinary second-
ary alcohols with the acyl enzyme resulted in the formation of four
possible tetrahedral intermediates.16 The crystal structure of (R)-
1d, however, revealed two independent forms due to the asym-
metric nature of the tertiary N atom.8 Therefore, to explain the ste-
reoselectivity of the acetylation reactions of racemic 1-(10-ethyl-
10H-phenothiazinyl)ethanols 1a and 1d, eight tetrahedral inter-
mediates of each alcohol were taken into account. Thus, the lowest
energy ONIOM models for each of the eight possible tetrahedral
intermediates were compared (Table 5). The calculations deter-
mined that for both phenothiazinyl alcohols 1a and 1d, the (R)-
alcohol-(S)- tetrahedral intermediate structure is the most favored
(Table 5). Accordingly, the faster forming acetates 2a and 2d from
the two racemic alcohols have an (R)-configuration. The configura-
tions of the asymmetric N-center for the lowest energy tetrahedral
intermediate states were (R)- for (R)-1a-(S)-tetrahedral intermedi-
ate, whereas (S)- for (R)-1d-(S)-tetrahedral intermediate. The sta-
bilization effects due to several important hydrogen bonds were
similar in the two most favored intermediate states (R)-N-(R)-1a-
2.4. The absolute configuration of (R)- and (S)-1,2a and -1,2c
The assignment of the absolute configuration by QM/MM calcu-
lations within the CaL-B enzymic environment was applied earlier
to explain the stereoselectivity of reactions of racemic 1-heteroar-
ylethanols.16 Since the absolute configuration for (R)-(+)-1d was
unambiguously determined by X-ray crystallography,8 we first
confirmed the accuracy of the QM/MM calculations on the stere-
oselectivity of the CaL-B catalyzed acetylation of the enantiomers
of rac-1d. Next, we examined the acetylation of the enantiomers
of racemic 1-(10-ethyl-10H-phenothiazin-1-yl)ethanol rac-1a by
similar QM/MM calculations. For the detailed calculations, the tet-
rahedral intermediates formed by the reaction of the (R)- or (S)-
alcohol with the acyl enzyme were used to model the transition
states. First, the tetrahedral intermediates were constructed within
the CaL-B structure and systematic conformation search was per-
formed for each tetrahedral intermediate model by molecular
mechanics within a rigid enzyme environment. Next, the best

920
J. Brem et al. / Tetrahedron: Asymmetry 22 (2011) 916–923
Table 5
Comparison of the tetrahedral intermediate states of acylation of rac-1a and rac-1d within the active site of CaL-B determined by QM/MM (hf/3–21g:uff) method
OH
OH
N
S
N
rac-1a:
rac-1d:
S
Substrate
E (kcal/mol)
(R)-1-(R)-Tetrahedral intermediate
(R)-1-(S)-Tetrahedral intermediates
(S)-1-(R)-Tetrahedral intermediate
(S)-1-(S)-Tetrahedral intermediate
D
(S)-N
(R)-N
(S)-N
(R)-N
(S)-N
(R)-N
(S)-N
(R)-N
rac-1a
rac-1d
28.6
58.8
33.3
40.0
—
0.0
0.0
—
—
35.3
15.3
43.0
31.9
7.9
14.5a
a
The N-atom of the 1-(10-ethyl-10H-phenothiazin-3-yl)ethanol moiety was nearly planar in the optimized structure.
(S)-tetrahedral intermediate and (S)-N-(R)-1d-(S)-tetrahedral
intermediate (Table 6 and Fig. 1A). Irrespective of the position of
the substituents on the phenothiazine ring in (R)-1a or (R)-1d,
the alcohol moieties occupied approximately the same space of
the active site in the most favored intermediate states (Fig. 1B).
Based on these data and on the consistent (+)-signs of the spe-
cific rotations for (R)-1a,b and (R)-1d,8 the (R)-(+)-absolute config-
uration was assigned to be the faster reacting enantiomer of 1-(10-
ethyl-10H-phenothiazin-4-yl)ethanol 1c as well.
of the tetrahedral intermediates for the racemic alcohol rac-1a
within the active site of C. antarctica lipase B structure.
4. Experimental
4.1. Analytical methods
The 1H and 13C-NMR spectra were recorded in CDCl3 solution on
a Brucker Avance DPX-300 spectrometer operating at 300 and
75 MHz, respectively. Chemical shifts on the d scale are expressed
in ppm values from TMS as the internal standard. IR spectra were
recorded in KBr or CsBr plates on a Jasco FT-IR spectrometer and
the wave numbers are reported in cmÀ1. EI mass spectra were ta-
ken on a VG 7070E mass spectrometer operating at 70 eV and are
characterized by m/z (relative intensity %) values. Optical rotations
were determined on a Bellingham-Stanley ADP 220 polarimeter.
Thin Layer Chromatography (TLC) was carried out using Merck
Kieselgel 60F254 sheets. Spots were visualized by treatment with
5% ethanolic phosphomolybdic acid solution and heating. Prepara-
tive vacuum-chromatography was performed on Merck Kieselgel
3. Conclusion
Efficient enzymatic procedures for the synthesis of enantiome-
rically pure 1-(10-ethyl-10H-phenothiazinyl)ethanols 1a–c and
their acetates 2a–c has been described. Due to the low stability
of these compounds, the preparative scale biotransformations
were followed by careful isolation and purification.
While the lipase mediated acetylation afforded the enantiopure
(S)-ethanols (S)-1a–c and (R)-acetates (R)-2a–c, the CaL-B cata-
lyzed methanolysis of the racemic acetates rac-2a–c yielded the
opposite (R)-1a–c and (S)-2a–c enantiomeric forms of the target
compounds.
60 (0.063–0.200 lm). Determination of E was based using the
equation E = ln[(1 À c)(1 À eeS)]/ln[(1 À c)(1 + eeS)].17
The absolute configuration of the faster reacting enantiomer of
2a was determined by QM/MM calculations on all possible forms
High performance liquid chromatography analyses were
conducted with an Agilent 1200 instrument. For enantiomeric
Table 6
Comparison of the H–O distances between Gln106, Thr40 and the oxoanion (d1, d2, d3), His224-N and tetrahedral intermediate oxygens (d4: His224-N -Ser105-O3; d5: His224-
s
s
N -ester-O) in the two lower energy states of (R)-1-(S)-tetrahedral intermediates
s
Tetrahedral intermediate structure
d1 (Å)
d2 (Å)
d3 (Å)
d4 (Å)
d5 (Å)
d6 (Å)
d7(Å)
(R)-N-(R)-1a-(S)-Tetrahedral intermediate
(S)-N-(R)-1d-(S)-Tetrahedral intermediate
2.16
1.93
2.77
2.00
1.83
1.59
2.15
2.26
1.93
1.75
2.00
2.00
2.44
2.44
Figure 1. Comparison of the two lower energy tetrahedral intermediate states within the active site of CaL-B determined by QM/MM (hf/3–21g:uff) calculations for
acetylation of racemic 1-(10-ethyl-10H phenothiazin-1-yl)ethanol rac-1a and 1-(10-ethyl-10H-phenothiazin-3-yl)ethanol rac-1d [A, (R)-N-(R)-1-(S)- tetrahedral interme-
diate from rac-1a; B, overlay of (R)-N-(R)-1-(S)- tetrahedral intermediate from rac-1a and (S)-N-(R)-1-(S)- tetrahedral intermediate from rac-1d].

J. Brem et al. / Tetrahedron: Asymmetry 22 (2011) 916–923
921
Table 7
J = 6.4 Hz, 1H), 6.79–6.84 (m, 1H), 6.87–6.95 (m, 2H), 7.15–7.24(m,
4H); 13C NMR: (75 MHz, CDCl3): d = 13.1, 23.5, 41.8, 67.0, 114.3,
115.1, 118.8, 122.1, 122.7, 124.3, 126.9, 127.3, 127.5, 143.4, 145.1,
145.7; IR (KBr): 3353, 3063, 2969, 2857, 1593, 1564, 1480, 1459,
1443, 1367, 1325, 1249, 1110, 889, 785, 746; HRMS: M+ found (M+
calculated for C16H17NOS): 271.2 (271.1030); MS: m/z (%) = 273.2
(4, M+2), 272.2 (14, M+1), 271.2 (86, M), 243 (17), 242 (100), 226
(7), 198 (19), 191 (24), 83 (14), 43 (18).
Retention times for the enantiomers of 1a–c and 2a–c
Compound
tR (min)
Compound
tR (min)
(R)-1a
(S)-1a
(R)-1b
(S)-1b
(R)-1c
(S)-1c
6.5
7.1
7.9
7.2
6.8
7.9
(R)-2a
(S)-2a
(R)-2b
(S)-2b
(R)-2c
(S)-2c
4.1
3.5
5.5
5.3
12.0
9.4
4.3.2. Synthesis of racemic 1-(10-ethyl-10H-phenothiazin-2-
yl)ethanol rac-1b
separation of rac-1,2a
a LiChroCART (R,R)-Whelk-O1 column
Into a stirred solution of 1-(10H-phenothiazin-2-yl)ethanone
(1 mmol, 241 mg) in anhydrous DMF (20 mL), NaH (1.1 mmol,
26.5 mg) was added in small portions at 0–5 °C. After 10 min, ethyl
(4.6 Â 250 mm); for rac-1,2b Chiralpak IB column (4.6 Â 250 mm)
and for rac-1,2c Chiralpak IC column (4.6 Â 250 mm) and a mixture
of hexane and 2-propanol, 98:2, 90:10 and 90:10, respectively, (v/
v) as eluent was used, all at 1 mL/min flow rate (Table 7).
iodide (1.25 mmol, 125 lL) was added and the resulting mixture
was stirred at 50 °C. After completion of the reaction (checked by
TLC), the solvent from the mixture was distillated in vacuo, the res-
idue was dissolved in CH2Cl2 (20 mL), filtered, and concentrated.
The 1-(10-ethyl-10H-phenothiazin-2-yl)ethanone was crystallized
from ethanol as yellow semisolid and used in the next step without
further purification.
To a stirred solution of the 1-(10-ethyl-10H-phenothiazin-2-
yl)ethanone (0.8 mmol, 215 mg) in dry methanol (5 mL), NaBH4
(1 mmol, 38 mg) was added in small portions at room temperature
and the resulting mixture was stirred. After the reduction was
complete (checked by TLC), the mixture was quenched by the
dropwise addition of a 2 M HCl solution (1 mL) and evaporated
to a final volume of approximately 1 mL. To this residue, water
(3 mL) and CH2Cl2 (6 mL) were added. After separating the two lay-
ers, the aqueous layer was extracted with CH2Cl2 (6 mL). The com-
bined organic layers were dried over anhydrous MgSO4 and the
solvent was removed. The residue was purified by column chroma-
tography on silica gel using CH2Cl2 as eluent yielding the desired
product as a colorless semisolid.
4.2. Reagents and solvents
All reagents were purchased from Aldrich or Fluka and used as
received. Solvents and acyl donors for enzymatic reactions were
stored over molecular sieves unless otherwise stated. Lipases from
Aspergillus niger, P. fluorescens (L-AK), Burkholderia cepacia (L-PS),
Rhizopus oryzae (L-F), Rhizopus arrhizus, Penicillium camemberti
and Mucor javanicus were products of Amano. Lipases from Candida
rugosa (CrL), Candida lipolytica, Mucor miehei and porcine pancreas
(PPL) were purchased from Fluka. CAL-A CLEA was from Fluka. Li-
pase B from C. antarctica (CAL-B, Novozym 435) and lipase from
Thermomyces lanuginosus (Lipozyme TL IM) were purchased from
Novozymes, Denmark.
4.3. Synthesis of racemic alcohols rac-1,2a–c
4.3.1. Synthesis of racemic 1-(10-ethyl-10H-phenothiazin-yl)-
ethanols rac-1a,c
Into a stirred solution of methyl magnesium iodide, prepared
from magnesium (7.4 mmol, 0.177 g) and iodomethane (7.4 mmol,
1.05 g, 0.460 mL) in Et2O (5 mL), a solution of a 10-ethyl-10H-phe-
nothiazinyl-carbaldehyde 1a,c (6.17 mmol, 1.57 g) dissolved in
Et2O (3 mL) was added slowly at 0 °C under argon. The resulting
mixture was stirred at room temperature for 1.5–2 h. After
quenching the reaction by the slow addition of saturated ammo-
nium chloride solution (8 mL) and ethanol (2 mL) at t <0 °C to pro-
tect the crude products, the organic layer was isolated and the
aqueous layer was extracted with Et2O (2 Â 10 mL). The combined
organic layer was dried over anhydrous sodium sulfate and con-
centrated in vacuo at low temperature (t <10 °C). The solid residue
was purified by vacuum-chromatography on neutral aluminum
oxide (Brockmann IV) using dichloromethane, resulting in the
racemic alcohol rac-1a,c as a colorless semisolid.
4.3.2.1. 1-(10-Ethyl-10H-phenothiazin-2-yl)ethanol rac-1b. Yield:
94%; semisolid; 1H NMR: (300 MHz, CDCl3): d = 1.39–1.47 (m, 6H),
3.93 (q, J = 6.9 Hz, 2H), 4.81 (q, J = 6.4 Hz, 1H), 6.85–6.93 (m, 4H),
7.06–7.24 (m, 3H); 13C NMR : (75 MHz, CDCl3): d = 12.9, 25.1, 41.5,
70.1, 112.1, 115.0, 119.2, 122.2, 123.2, 124.3, 127.1, 127.2, 144.8,
145.1, 145.2; IR (KBr): 3356, 3060, 2969, 2925, 2853, 1595, 1585,
1463, 1442, 1423, 1284, 1235, 1133, 1110, 887, 815, 748; HRMS:
M+ found (M+ calculated for C16H17NOS): 271.1 (271.1030); MS: m/
z (%) = 271 (37, M), 242 (36), 196 (19), 135 (28), 128 (43), 119 (58),
105 (25), 91 (23), 58 (39), 57 (28), 43 (100).
4.3.3. Chemical acetylation of the racemic 1-(10-ethyl-10H-
phenothiazin-yl)-ethanols rac-1a–c
Into
yl)ethanol rac-1a–c (5.61 mmol) in dry CH2Cl2 (15 mL), Et3N
(6.16 mmol, 624 mg, 860 L), acetyl chloride (6.17 mmol,
484.3 mg, 438.7 L) and DMAP (0.16 mmol, 20 mg) were added.
a solution of racemic 1-(10-ethyl-10H-phenothiazin-
l
4.3.1.1. 1-(10-Ethyl-10H-phenothiazin-1-yl)ethanol rac-1a. Yield:
91%; semisolid; 1H NMR: (300 MHz, CDCl3); d = 1.15 (t, J = 7.0 Hz,
3H), 1.55 (d, J = 6.4 Hz, 3H), 3.75 (q, J = 7.0 Hz, 2H), 5.31 (q,
J = 6.4 Hz, 1H), 6.97–7.20 (m, 7H); 13C NMR: (75 MHz, CDCl3):
d = 14.2, 23.6, 50.8, 65.7, 114.3, 122.7; 125.1; 125.3; 125.4; 126.3,
127.0, 127.2; 132.8; 141.1; 142.5, 144,1; IR (KBr): 3357, 3060,
2971, 2926, 2864, 1590, 1566, 1473, 1433, 1375, 1263, 1227, 1096,
1077, 787, 758, 727; HRMS: M+ found (M+ calculated for
l
The mixture was stirred at room temperature overnight and then
quenched with water (15 mL). The isolated organic layer was dried
over anhydrous sodium sulfate and the solvent was distilled off by
rotatory evaporation. The crude product was purified by vacuum-
chromatography on neutral aluminum oxide (Brockmann IV) using
CH2Cl2 as eluent to give rac-2a–c as a semisolid.
C
16H17NOS): 271.1 (271.1030); MS: m/z (%) = 272 (7, M+1), 271
4.3.3.1. 1-(10-Ethyl-10H-phenothiazin-1-yl)ethyl acetate rac-
2a. Yield: 93%; semisolid; 1H NMR: (300 MHz, CDCl3): d = 1.07
(t, J = 6.9 Hz, 3H), 1.35 (d, J = 6.6 Hz, 3H), 2.07 (s, 3H), 3.54 (q,
J = 6.9 Hz, 2H), 6.34 (q, J = 6.9 Hz, 1H), 6.94–7.22 (m, 7H); 13C
NMR: (75 MHz, CDCl3): d = 14.6, 21.5, 22.3, 51.2, 69.2, 124.4,
124.8, 125.2, 126.0, 126.6, 127.1, 127.2, 133.0, 134.2, 138.2,
142.2, 145.2, 170.5; IR (KBr): 3061, 2975, 2929, 2865, 1732,
(31, M), 242 (37), 200 (17), 199 (24), 133 (12), 91 (13), 58 (21), 56
(12), 43 (100).
4.3.1.2. 1-(10-Ethyl-10H-phenothiazin-4-yl)ethanol rac-1c. Yield:
90%; semisolid; 1H NMR: (300 MHz, CDCl3): d = 1.41 (t, J = 6.9 Hz,
3H), 1.49 (d, J = 6.4 Hz, 3H), 3.92 (q, J = 6.9 Hz, 2H), 5.27 (q,

922
J. Brem et al. / Tetrahedron: Asymmetry 22 (2011) 916–923
1591, 1568, 1474, 1435, 1368, 1239, 1073, 1022, 788, 759, 728;
HRMS: M+ found (M+ calculated for C18H19NO2S): 313.1
(313.1136); MS: m/z (%) = 315 (5, M+2), 314 (18, M+1), 313 (100,
M), 284 (100), 270 (15), 242 (35), 241 (46), 225 (26), 224 (38),
223 (27), 200 (11), 199 (18), 198 (16), 43 (30).
300 rpm at room temperature. Samples were analyzed by HPLC
as described in Section 4.3.1. Data on conversion and enantiomeric
composition of the products are presented in Table 3.
4.5. Enzyme mediated biotransformation of racemic 1-(10-
ethyl-10H-phenothiazinyl)ethanols, rac-1a–c and their acetates
rac-2a–c on a preparative scale
4.3.3.2. 1-(10-Ethyl-10H-phenothiazin-2-yl)ethyl acetate rac-
2b. Yield: 87%; semisolid; 1H NMR: (300 MHz, CDCl3): d = 1.41
(t, J = 6.9 Hz, 3H), 1.51 (d, J = 6.6 Hz, 3H), 2.06 (s, 3H), 3.94 (q,
J = 6.9 Hz, 2H), 5.80 (q, J = 6.9 Hz, 1H), 6.83–6.92 (m, 4H), 6.83–
6.92 (m, 3H); 13C NMR: (75 MHz, CDCl3): d = 13.1, 21.5, 22.2,
41.8, 72.3, 113.4, 115.3, 120.1, 122.5, 124.3, 124.4, 127.2, 127.4,
127.5, 141.1, 144.9, 145.2, 170.4; IR (KBr): 3057, 2970, 2931,
2869, 1732, 1579, 1465, 1443, 1326, 1245, 1237, 1179, 1062,
816, 749; 745 HRMS: M+ found (M+ calculated for C18H19NO2S):
313.2 (313.1136); MS: m/z (%) = 313 (7, M), 206 (11), 192 (6),
191 (41), 149 (8), 86 (12), 84 (70), 82 (100), 71 (100), 57 (13), 47
(18); 43 (20).
Into the solution of racemic 1-(10-ethyl-10H-phenothiazi-
nyl)ethanols rac-2a–c (3.69 mmol, 1 g) in n-hexane or chloroform
depending on the enzyme used (36.9 mL), vinyl acetate
(14.7 mmol, 1.27 g, 1.36 mL) and lipase (1.84 g, CaL-A or CaL-B)
were added. The reaction mixture was shaken at 300 rpm at room
temperature. The reactions were monitored by TLC and stopped at
approx. 50% conversion by removing the enzyme by filtration. Sol-
vents were removed in vacuo at low temperature (t <10 °C) and the
crude product was purified by vacuum-chromatography on neutral
aluminum oxide (Brockmann IV) using dichloromethane as eluent,
resulting in virtually enantiopure (S)-1-(10-ethyl-10H-phenothia-
zin-yl)ethanols (S)-1a–c and (R)-1-(10-ethyl-10H-phenothiazin-
yl)ethyl acetate (R)-2a–c as semisolids.
4.3.3.3. 1-(10-Ethyl-10H-phenothiazin-4-yl)ethyl acetate rac-
2c. Yield: 81%; semisolid; 1H NMR: (300 MHz, CDCl3): d = 1.41 (t,
J = 6.9 Hz, 3H), 1.55 (d, J = 6.4 Hz, 3H), 2.10 (s, 3H), 3.93 (q,
J = 6.9 Hz, 2H), 6.21 (q, J = 6.4 Hz, 1H), 6.83–6.95 (m, 3H), 7.06–
7.09 (m, 1H), 7.14–7.21 (m, 3H); 13C NMR: (75 MHz, CDCl3):
d = 13.2, 20.9, 21.3, 42.3, 69.8, 114.9, 115.3, 119.5, 122.5, 122.6,
123.8, 124.5, 127.0, 127.5, 127.8, 139.5, 145.7, 170.2; IR (KBr):
3064, 2972, 2927, 2868, 1733, 1593, 1566, 1481, 1462, 1443,
1370, 1240, 1072, 1058, 783, 748, 729; HRMS: M+ found (M+ calcu-
lated for C18H19NO2S): 313.2 (313.1136); MS: m/z (%) = 315.2 (5,
M+2), 314.2 (18, M+1), 313.2 (100, M), 285 (10), 284 (66), 226
(10), 225 (15), 224 (28), 191 (8), 58 (26), 43 (72).
Into
a solution of racemic 1-(10-ethyl-10H-phenothiazi-
nyl)ethyl acetates rac-2a–c (3.19 mmol, 1 g) in acetonitrile
(31.9 mL), methanol (31.9 mmol, 1 g, 1.29 mL) and CaL-B (1.59 g)
were added. The reaction mixture was shaken at 300 rpm at room
temperature. The reactions were monitored by TLC and stopped at
an approx. 50% conversion by removing the enzyme by filtration.
Solvents were removed in vacuo at <10 °C and the crude product
was purified by vacuum-chromatography on neutral aluminum
oxide (Brockmann IV) using dichloromethane as eluent, resulting
in virtually enantiopure (S)-1-(10-ethyl-10H-phenothiazin-
yl)ethyl acetates (S)-2a–c and (R)-1-(10-alkyl-10H-phenothiazin-
yl)ethanols (R)-1a–c as semisolids.
4.4. Enzyme mediated biotransformation of racemic 1-(10-
ethyl-10H-phenothiazin-yl)ethanols, rac-1a–c and their
acetates racemic 1-(10-ethyl-10H-phenothiazin-yl)ethyl
acetates, rac-2a–c on an analytical scale
The desired compounds were isolated in good yields, without
altering their enantiomeric purity (Table 7).
4.6. Computational Methods
4.4.1. Enzymatic kinetic resolution of racemic 1-(10-ethyl-10H-
phenothiazinyl)ethanols rac-1a–c with vinyl acetate and
different lipases
4.6.1. Systematic conformational search within the active site of
CaLB at MM level
Into a solution of racemic 1-(10-ethyl-10H-phenothiazinyletha-
nol rac-1a–c (0.1 mmol, 27.1 mg) in vinyl acetate (1 mL), lipase
(50 mg) was added. The reaction mixture was shaken at 300 rpm
at room temperature. For HPLC analysis, samples were taken from
The calculations were performed within the active site of the
crystal structure CaL-B containing Tween80 (T80) as the substrate
mimic (PDB code: 1LBT).10 From this CaL-B structure, a part con-
taining the residues within 10 Å spheres around the Ser105 O ,
c
the reaction mixture (10
l
L), diluted to 500
l
L with 2-propanol
T80 C10 and T80 C20 atoms was cut off as the active site model
for CS and later QM/MM calculations. Next, T80 was removed
and by the Hyperchem18 standard procedure, hydrogens were
added to the amino acid residues and water oxygens. The C- and
N-termini at cutting were completed to neutral aldehyde and ami-
no moieties. During our calculations, His224 was protonated and
Asp187 was deprotonated.
and filtered before injection. Data on the conversion and enantio-
meric composition of the products for the solvents tested are pre-
sented in Table 1.
4.4.2. Enzymatic kinetic resolution of racemic 1-(10-ethyl-10H-
phenothiazin-yl)ethanols rac-1a–c with vinyl acetate in
different solvents with CaL-A/CaL-B
The tetrahedral intermediate states were built in the active site
Into
yl)ethanol rac-1a–c (0.1 mmol, 27.1 mg) in different solvents
(1 mL), vinyl acetate (0.4 mmol, 34.4 mg, 36.8 L), and lipase
(50 mg, CaL-A or CaL-B) were added. The reaction mixture was sha-
ken at 300 rpm at room temperature. Samples were analyzed by
HPLC similarly as described in Section 4.4.1. Data on the conver-
sion and enantiomeric composition of the products are presented
in Table 2.
a
solution of racemic 1-(10-ethyl-10H-phenothiazin-
model by constructing a covalent bond between the O atom of
c
Ser105 and the carbonyl carbon of the (R)- and (S)-enantiomers
of 2a and 2d acetates. In this way, starting structures were con-
structed for the eight tetrahedral intermediate states [(R)-N-(R)-
1-(R)-tetrahedral intermediates, (S)-N-(R)-1-(R)-tetrahedral inter-
mediate, (R)-N-(R)-1-(S)-tetrahedral intermediate, (S)-N-(R)-1-(S)-
tetrahedral intermediate, (R)-N-(S)-1-(R)-tetrahedral intermediate,
(S)-N-(S)-1-(R)-tetrahedral intermediate, (R)-N-(S)-1-(S)-tetrahe-
dral intermediate, (S)-N-(S)-1-(S)-tetrahedral intermediate] for
acetylation of each racemic alcohols rac-1a and rac-1d. The initial
l
4.4.3. Enzymatic alcoholysis of racemic 1-(10-ethyl-10H-
phenothiazinyl)ethyl acetates rac-2a–c
tetrahedral intermediates including C and Cb (with their hydro-
a
Into a mixture of rac-2a–c (0.1 mmol, 31.3 mg) in n-hexane/ace-
tonitrile (1 mL), CaL-B (50 mg) and methanol (1 mmol, 32 mg,
40.4 lL) were added. The reaction mixture was shaken at
gens) of Ser105 (47 atoms for 2a and 2d acetates) were optimized
by the MM+ method of Hyperchem18 within the rigid enzymic
environment (including no waters).

J. Brem et al. / Tetrahedron: Asymmetry 22 (2011) 916–923
923
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settings (MM+ forcefield; gradient: 0.1 kcal/mol; Polak-Ribiere
method until 0.05 kcal/mol RMS gradient or maximum 600 cycles;
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a
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optimized using a two-layer ONIOM method (hf/3–21g:uff) over
955 atoms. The high layer included the atoms of the alcohols 1a
and 1d, the acetate part of tetrahedral intermediate, the side chains
of the catalytic triad (Ser105, His224, Asp187 + one water), the rel-
evant parts of the residues forming the so-called oxoanion hole
(Gln106, Thr40). The high layer was relaxed with Hartree-Fock
(hf/3–21g) method. Residues from the low layer, except Trp104
(+ one water), Asp134, Gln157, Ile189, Ala281, Ala282 and Ile285,
were kept fixed during the molecular mechanics (UFF)
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Acknowledgments
J.B. gives thanks for the financial support provided from pro-
grams co financed by The Sectoral operational program human re-
sources development, Contract POSDRU 6/1.5/S/3 – ‘Doctoral
studies: through science towards society’. This work is also related
to the scientific program of ‘Development of quality-oriented and
harmonized R+D+I strategy and functional model at BME’ (TÁMOP-
4.2.1/B-09/1/KMR-2010-0002), supported by the New Hungary
Development Plan.
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