Lipase-catalysed resolution of 2-dialkylaminomethyicyclohexanols

Article abstract of DOI:10.1016/S0957-4166(98)00019-6

The lipase PS- and Novozym 435-catalysed resolutions of 2- dialkylaminomethylcyclohexanols (A-F) with various vinyl esters were studied in different organic media. High enantioselectivity (E>200) was observed when vinyl acetate was used as an acylating ag

Full text of DOI:10.1016/S0957-4166(98)00019-6

Pergamon
Tetrahedron: Asymmetry 9 (1998) 513–520
Lipase-catalysed resolution of
2-dialkylaminomethylcyclohexanols
Enikõ Forró,a,b Liisa T. Kanerva a,∗ and Ferenc Fülöp b
aDepartments of Chemistry and Biomedicine, University of Turku, FIN-20014 Turku, Finland
bInstitute of Pharmaceutical Chemistry, Albert Szent-Györgyi Medical University, Szeged, Hungary
Received 11 December 1997; accepted 15 January 1998
Abstract
The lipase PS- and Novozym 435-catalysed resolutions of 2-dialkylaminomethylcyclohexanols (A–F) with
various vinyl esters were studied in different organic media. High enantioselectivity (E>200) was observed when
vinyl acetate was used as an acylating agent and diethyl ether as a solvent. The (1R,2R) enantiomers react
preferentially in the case of the cis isomers, whereas the (1R,2S) enantiomers do so in the case of the trans
counterparts. Reaction rates were markedly affected by the solvent and by the quantity of the enzyme. © 1998
Elsevier Science Ltd. All rights reserved.
The 1,2- and 1,3-amino alcohols are compounds of considerable biological and chemical impor-
tance. The widespread investigations on alicyclic 1,2- and 1,3-amino alcohols1–4 prompted us to study
the resolution of racemic cis- and trans-2-dialkylaminomethylcyclohexanols (A–F) by using lipase-
catalysed acylation in organic media as a tool to attain highly enantiopure products (Scheme 1). The
racemates of these compounds5–14 and some of their derivatives have been examined thoroughly in
various pharmacological tests.5–9,14 The local anaesthetic activities of all four stereoisomers of 2-
dialkylaminomethylcyclohexyl benzoate are comparable with that of lidocaine.5 The cis- and trans-2-
dialkylaminomethylcyclohexyl carbanilates and carbamates, synthesized by phenyl isocyanate addition
to the corresponding cyclohexanols, display a strong anaesthetic effect, often accompanied by antiarhyth-
mic activity.6–8 2-Dialkylaminomethylcyclohexanols can occur as intermediates in the synthesis of semi-
rigid analogues of prothiadene and dithiadene, potential antidepressant and antihistamine agents.9
The investigated compounds are also of chemical importance, e.g., hydrogenolysis of the dibenzy-
lamino group of compound F provides a 1,3-amino alcohol with a primary amino group. The two
functional groups (OH and NH2) are subject to a wide variety of chemical transformations.5
∗
Corresponding author.
0957-4166/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00019-6

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E. Forró et al. / Tetrahedron: Asymmetry 9 (1998) 513–520
Table 1
Effects of various R–CO2–CH_CH2 (0.2 M) on the acylation of B (0.1 M) in the presence of Novozym
435 (30 mg ml−1) in diethyl ether at room temperature
1. Results and discussion
In previous studies, lipase PS from Pseudomonas cepacia proved applicable for the resolution of
secondary alcohols in general, and for that of cycloalkanols in particular.15–21 Some doubts arose
about the possibilities for the resolution of A–F, because N-isopropyl-2-amino-1-phenylethanol was
shown to be a nonsubstrate for lipase PS-catalysed acylation in toluene.17 The size of the N-alkyl
substituent occupying the pocket Rsmall is clearly critical according to the proposed dimensions for the
two hydrophobic pockets at the active site of the lipase.22,23 Extensive lipase screening in diethyl ether
indicated that, besides lipase PS, Novozym 435 (lipase from Candida antarctica B) is also a promising
catalyst for the asymmetric acylation of the present cycloalkanols with N-substituents at the position β
to the stereocentre. The two catalysts lead to excellent enantioselectivity (enantiomeric ratio, E, usually
over 200).24 The resolution conditions were subsequently optimized for lipase PS- and Novozym 435-
catalysed acylations by using B as a model compound.
The enzymatic reactions of R1OH (originating from RCO2R1) with the products A1–F1, which can
accompany the asymmetric acylation of cycloalkanols A–F (Scheme 1), yielded A2–F2 in decreasing
enantiopurity with time. For this reason and on the basis of previous experience with lipase PS catalysis,15
vinyl esters were used as irreversible acyl transfer reagents (vinyl alcohol as R1OH irreversibly tau-
tomerizes to acetaldehyde) throughout the work. Moreover, acetate was shown to be a better acyl donor
than esters with longer carbon chains.15,25 Vinyl acetate is also a choice for the Novozym 435-catalysed
acylation of B (Table 1). As a drawback, the Schiff base formation of the liberated acetaldehyde and the
free amino groups of a lipase may lead to partial deactivation and a loss of selectivity. In this respect, some
caution was necessary in the present work. It turned out that the acetylation of B (0.1 M) in diethyl ether in
the presence of Novozym 435 (30 mg ml-1) proceeded somewhat more slowly and less enantioselectively
with vinyl acetate (0.4 M; conversion 40% after 120 h; eealcohol 64%, eeester 96%) than when vinyl acetate
(0.2 M; conversion 46% after 120 h; eealcohol 84%, eeester ∼99%, C) was used. Acetaldehyde is produced
by enzymatic acylation with vinyl acetate and possibly by enzymatic hydrolysis of the ester with the
water present in the enzyme preparation.
The quantitative basis of solvent effects on reactivity (the time needed to reach a certain conversion)
and enantioselectivity is unclear and often unpredictable. Ether solutions are generally preferable for
work with lipase PS.25 In the present work, selected solvent screening for the Novozym 435-catalysed
resolution of B was performed (Table 2). The enzyme was practically inactive in acetone and tetrahydro-
furan. Diethyl ether was chosen as the most favourable solvent for further studies.

E. Forró et al. / Tetrahedron: Asymmetry 9 (1998) 513–520
515
Scheme 1.
Table 2
The Novozym 435-catalysed (30 mg ml−1) acetylation of B (0.1 M) with vinyl acetate (0.2 M) in
different organic solvents at room temperature
The enzymatic reactions can also be controlled via the amount of enzyme. Even though the enantiose-
lectivity in the lipase PS- and Novozym 435-catalysed acetylations of alcohols A–F is generally excellent
(usually E>200), the reactivity for the acetylation of alcohol B clearly passes through a maximum at an
enzyme content of ca 30 mg ml−1, the effect being less pronounced in the case of lipase PS (Table 3).
One of the benefits of biocatalytic reactions over conventional chemical ones is that elevated temper-
atures are not needed. Rather, the reactions proceed effectively at room temperature. Additionally, the
enantioselectivity can often be enhanced at lower temperatures.26 Enhancement of the enantioselectivity
is not necessary in the present work. On the other hand, at room temperature the time needed to reach
the theoretical 50% conversion is relatively long in the case of Novozyme 435 (e.g. a conversion of 21%
after 62 h; Table 3, row 5). In order to decrease the reaction time, the Novozym 435-catalysed acetylation
of B was performed at 40°C. Surprisingly, a slower reaction (conversion 19% after 108 h) was observed
without any effect on the enantioselectivity.
The above optimizations were mostly based on previous work with lipase PS15–21,25 and on the
Novozym 435-catalysed acetylation of B (Tables 1–3). The applicability of the optimal conditions was
tested for the acetylation of alcohols A–F. Although E>200 throughout the substrate screening, only
lipase PS effectively catalyses the asymmetric acetylation of the cis compounds A and C (Table 4).
Successful enantioselective acetylation of the trans compounds B, D and F is possible (with different
reaction rates) when either lipase PS or Novozym 435 is used. Since the real protein contents and

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Table 3
Effects of the quantity of Novozym 435 (i) and lipase PS preparationa (ii) on the acetylation of B (0.1
M) with vinyl acetate (0.2 M) in diethyl ether at room temperature
Table 4
Activity of Novozym 435 (i) 30 mg ml−1 and lipase PS (ii) 50 mg ml−1 in the acetylation of A–F (0.1
M) with vinyl acetate (0.2 M) in diethyl ether at room temperature
specific activities of the two enzyme preparations are unknown, a straightforward comparison between
their reactivities is impossible. It can be concluded, however, that lipase PS is the more favourable of
the two because, as stated above (Table 3), it is not possible to enhance the reactivity of Novozyme 435
to the level shown for lipase PS in Table 4 by increasing the enzyme content under the present reaction
conditions.
Gram-scale resolutions of compounds A–F were performed in diethyl ether in the presence of lipase
PS or Novozym 435 and the results are summarized in Table 5. The esters (A1–F1) produced by the (R)-
selective acetylation of cyclohexanols underwent spontaneous deacylation to the corresponding alcohols
(A3–F3) in methanol at room temperature, without loss of enantiopurity.
It is interesting that recrystallization from diisopropyl ether was accompanied by the spontaneous res-

E. Forró et al. / Tetrahedron: Asymmetry 9 (1998) 513–520
517
Table 5
The enzyme-catalysed resolution of A–F in the presence of Novozym 435 (i) 30 mg ml−1 or lipase PS
preparationa (ii) 50 mg ml−1 and vinyl acetate (0.2 M) in diethyl ether at room temperature
olution of racemic trans-2-dibenzylaminomethylcyclohexanol (F), yielding different crystal structures.
Well-developed crystals with high enantiopurities (ee>90%) were sorted for the two enantiomers.
1.1. Determination of absolute configurations
Chiral chromatography indicated that the corresponding enantiomers of compounds A–F react prefer-
entially with lipase PS or Novozym 435 catalysts. According to the Kazlauskas model for the active site
of lipases, the (1R) configuration was predicted for these enantiomers. The validity of this was proved in
the case of 2-dibenzylaminomethylcyclohexanol (F). For this purpose, the enantiomer F2 was reduced
catalytically to the corresponding aminoalcohol. The value of [α]D20=+31.9 (c=1.77, MeOH) for the
reduced product is in good agreement with the literature value ([α]D20=+33.9 (c=2, MeOH).27 Thus, the
absolute configuration of the unreactive cyclohexanols A2–F2 is (1S,2S) for the cis and (1S,2R) for the
trans isomers.
2. Experimental
2.1. Materials and methods
Racemic cis- and trans-2-dialkylaminomethylcyclohexanols A–F were obtained by means of the Man-
nich reaction and subsequent reduction. The Mannich reaction was carried out in excess cyclohexanone

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with paraformaldehyde and the corresponding amine. The resulting ketones were reduced with NaBH4.
2-Dimethylaminomethyl- and 2-(1-piperidinylmethyl)cyclohexanols were obtained as isomeric mixtures
(A:B=2:3; C:D=1:2), which were separated by column chromatography [A: mp 38–41°C (lit.11 mp
40–41°C); B: oil; C: mp 48–51°C; D: oil], elution being performed with ethyl acetate for the trans and
with methanol for the cis isomers. The reduction of 2-dibenzylaminomethylcyclohexanone hydrochloride
under the same conditions resulted exclusively in F with the trans configuration (mp 93–94°C).
Vinyl acetate and vinyl decanoate were purchased from Aldrich Co., and vinyl butyrate from Tokyo
Kasei Kogyo Co. Lipase PS was obtained from Amano Pharmaceuticals, and Novozym 435 as an
immobilized preparation from Novo Nordisk. Before use, lipase PS was dissolved in Tris-HCl buffer
(0.02 M; pH 7.8) in the presence of sucrose (3 g), followed by adsorption on Celite (17 g) (Sigma). The
lipase preparation thus obtained contained 20% (w/w) of lipase.
The solvents were of the best analytical grade and were dried over molecular sieves (3 Å). For gram-
scale resolution, diethyl ether was distilled just before use.
In a typical small-scale experiment, one of the 2-dialkylaminomethylcyclohexanols A–F (0.1 M
solution) in diethyl ether (3 ml) was added to the lipase PS preparation (50 mg ml−1) or Novozym
435 (30 mg ml−1). A vinyl ester (0.2 M in the reaction mixture) was added. The mixture was shaken
at room temperature (22–24°C). The progress of the reaction was followed by taking samples (0.1
ml) from the reaction mixture at intervals. In some cases, the unreacted alcohol in the sample was
derivatized with propionic anhydride in the presence of 4-dimethylaminopyridine and pyridine before
the gas chromatographic analysis. The ee values of the unreacted alcohol (A2–D2) and the produced
ester (A1–D1) enantiomers were determined by gas chromatography on a Chrompack CP-Cyclodextrin-
β-2,3,6-M-9 column (25 m). The ee values for F1 and F2 were determined by using high-pressure liquid
chromatography with a Chiralcel OG column (25 cm).
2.2. Gram-scale resolution of cis-2-dimethylaminomethylcyclohexanol (A)
Racemic A (2.35 g; 15 mmol) and vinyl acetate (2.81 ml; 30 mmol) in diethyl ether (150 ml) were
added to the lipase PS preparation (4.50 g, 50 mg ml−1). The mixture was stirred at room temperature
for 162 h. The reaction stopped at 50% conversion with 96% ee for unreacted (1S,2S)-A2 and 99% ee
for the produced (1R,2R)-A1. The enzyme was filtered off and the solvent was evaporated. The resolved
products were separated on silica gel, by elution with ethyl acetate for A2 (1.14 g oil, 7.28 mmol, ee 96%)
and with methanol for A1 (1.32 g oil, 6.6 mmol, ee 99%). Within 2 to 3 days, the ester enantiomer A1
underwent quantitative deacylation to the corresponding alcohol A3 (ee 99%) on standing in methanol at
room temperature. 1H NMR (400 MHz, CDCl3) δ (ppm) for A1: 1.2–1.9 (9H, m, 4×CH2 and remaining
CH), 2.0 (3H, s, CH3), 2.1–2.2 (2H, dd, CH2N), 2.18 (6H, s, 2×CH3), 5.0 (H, m, CHOCOCH3). Analysis:
calculated for C11H21NO2: C, 66.29; H, 10.62; N, 7.03; found: C, 65.55; H, 11.23; N, 6.94.
1H NMR (400 MHz, CDCl3) δ (ppm) for A2 and A3: 1.25–2.1 (9H, m, 4×CH2 and remaining CH),
2.2 (1H, dd, J=8.8, 3.9 Hz, CH2N), 2.38 (6H, s, 2×CH3), 3.0 (1H, dd, J=10.8, 1.7 Hz, CH2N), 3.9 (1H,
m, CHOH), 7.2 (1H, brs, OH). MS analysis for A2: M=157. Analysis: calculated for C9H19NO: C, 68.74;
H, 12.18; N, 8.91; found for A3: C, 68.73; H, 12.27; N, 8.71.
2.3. Gram-scale resolution of trans-2-dimethylaminomethylcyclohexanol (B)
With the procedure described above and in the presence of Novozym 435 (30 mg ml−1), racemic B
(1.88 g; 12 mmol) afforded the unreacted (1S,2R)-B2 (0.60 g oil, 3.83 mmol, ee 96.0%) and the ester
(1R,2S)-B1 (1.00 g oil, 5.02 mmol, ee 97.9%) in 214 h.

E. Forró et al. / Tetrahedron: Asymmetry 9 (1998) 513–520
519
1H NMR (400 MHz, CDCl3) δ (ppm) for B1: 0.9–2.01 (9H, m, 4×CH2 and remaining CH), 2.03 (3H,
s, CH3), 2.10 (1H, dd, J=12.1, 4.4 Hz, CH2N), 2. 17 (6H, s, 2×CH3), 2.19 (1H, dd, J=12.1, 8.9 Hz,
CH2N), 4.5 (1H, m, CHOCOCH3). Analysis: calculated for C11H21NO2: C, 66.29; H, 10.62; N, 7.03;
found: C, 65.50; H, 10.41; N, 7.19). MS analysis for B2: M=157. B3 (ee 96.6%). Analysis: calculated
1
for C9H19NO: C, 68.74; H, 12.18; N, 8.91; found: C, 67.98; H, 12.56; N, 8.60. H NMR (400 MHz,
CDCl3) δ (ppm) for B2 and B3: 0.8–2.0 (9H, m, 4×CH2 and remaining CH), 2.2 (1H, dd, J=9.52, 2.68
Hz, CH2N), 2.4 (1H, dd, CH2N), 3.38 (1H, m, CHOH), 5.83 (1H, brs, OH).
2.4. Gram-scale resolution of cis-2-(1-piperidinylmethyl)cyclohexanol (C)
With the procedure described above and in the presence of lipase PS (50 mg ml−1), racemic C (1.35 g;
6.8 mmol) afforded the unreacted (1S,2R)-C2 (0.61 g oil, 3.00 mmol, ee 97%) and the ester (1R,2S)-C1
(0.79 g oil, 3.30 mmol, ee 98%) in 46 h.
1H NMR (400 MHz, CDCl3) δ (ppm) for C1: 1.0–2.3 (19H, m, 9×CH2 and remaining CH), 2.05 (3H,
s, CH3), 2.3 (2H, dd, CH2N), 5.0 (1H, m, CHOCOCH3). Analysis: calculated for C14H25NO2: C, 70.25;
H, 10.53; N, 5.85; found: C, 70.31; H, 11.21; N, 5.81.
C2: Analysis: calculated for C12H23NO: C, 73.04; H, 11.75; N, 7.10; found: C, 72.67; H, 12.05; N,
6.97.
C3 (ee 98%). Analysis: calculated for C12H23NO: C, 73.04; H, 11.75; N, 7.10; found: C, 72.49; H,
11.89; N, 7.16.
1H NMR (400 MHz, CDCl3) δ (ppm) for C2 and C3: 1.2–2.8 (19H, m, 9×CH2 and remaining CH),
2.2 (1H, dd, J=9.37, 3.6 Hz, CH2N), 3.0 (1H, dd, J=10.9, 1.9 Hz, CH2N), 3.9 (1H, m, CHOH), 7.1 (1H,
brs, OH).
2.5. Gram-scale resolution of trans-2-(1-piperidinylmethyl)cyclohexanol (D)
With the procedure described above and in the presence of Novozym 435 (30 mg ml−1), racemic D
(2.35 g; 12 mmol) afforded the unreacted (1S,2R)-D2 (1.15 g oil, 5.83 mmol, ee 97%) and the ester
(1R,2S)-D1 (1.29 g oil, 5.38 mmol, ee 99%) in 255 h.
1H NMR (400 MHz, CDCl3) δ (ppm) for D1: 0.9–2.4 (19H, m, 9×CH2 and remaining CH), 2.0 (3H,
s, CH3), 2.28–2.35 (2H, dd, CH2N), 4.55 (1H, m, CHOCOCH3). Analysis: calculated for C14H25NO2:
C, 70.25; H, 10.53; N, 5.85; found: C, 70.29; H, 10.46; N, 5.85.
D2: Analysis: calculated for C12H23NO: C, 73.04; H, 11.75; N, 7.10; found: C, 72.64; H, 12.17; N,
7.05.
D3 (ee 97.5%). Analysis: calculated for C12H23NO: C, 73.04; H, 11.75; N, 7.10; found: C, 72.56; H,
11.45; N, 6.81.
1H NMR (400 MHz, CDCl3) δ (ppm) for D2 and D3: 0.8–2.6 (19H, m, 9×CH2 and remaining CH),
2.29 (1H, dd, J=9.28, 3.2 Hz, CH2N), 2.38 (1H, dd, J=12.4, 11.48 Hz, CH2N), 3.38 (1H, m, CHOH).
2.6. Gram-scale resolution of trans-2-dibenzylaminomethylcyclohexanol (F)
With the procedure described above and in the presence of lipase PS (50 mg ml−1), racemic F (1.84
g, 5.9 mmol) afforded the unreacted (1S,2R)-F2 (0.91 g crystalline product, 2.94 mmol, mp 134–136°C,
ee 99%) and the ester (1R,2S)-F1 (0.85 g oil, 2.4 mmol, ee>99%) in 34 h. Separation of the resolved
products occured on elution with diethyl ether.
1H NMR (400 MHz, CDCl3) δ (ppm) for F1: 0.7–2.2 (9H, m, 4×CH2 and remaining CH), 2.0 (3H,
s, CH3), 2.1 (1H, dd, J=10.0, 2.32 Hz, CH2N), 2.4 (1H, dd, J=8.92, 3.6, Hz, CH2N), 3.1–3.3 (4H, d,

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E. Forró et al. / Tetrahedron: Asymmetry 9 (1998) 513–520
2×CH2), 4.45 (1H, m, CHOCOCH3), 7.2–7.4 (10H, m, 10×CH). Analysis: calculated for C23H29NO2:
C, 78.60; H, 8.32; N, 3.98; found: C, 78.66; H, 8.88; N, 3.93.
F2: Analysis: calculated for C21H27NO: C, 81.51; H, 8.79; N, 4.53; found: C, 81.60; H, 9.23; N, 4.51.
F3 (Crystalline product, mp 132–134°C, ee 99%). Analysis: calculated for C21H27NO: C, 81.51; H,
8.79; N, 4.53; found: C, 81.58; H, 9.11; N, 4.49.
1H NMR (400 MHz, CDCl3) δ (ppm): 0.7–2.0 (9H, m, 4×CH2 and remaining CH), 2.2–2.6 (2H, dd,
CH2N), 3.0 (1H, m, CHOH), 3.1–4.2 (4H, d, 2×CH2), 6.9 (1H, brs, OH), 7.2–7.4 (10H, m, 10×CH).
Acknowledgements
One of us (E. F.) is grateful for a grant from the Centre for International Mobility (CIMO). Support
from MKM (FKFP 0910/1997) Hungary is also gratefully acknowledged.
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