Chemoenzymatic preparation of 1-heteroarylethanamines of low solubility

Article abstract of DOI:10.1002/ejoc.201200330

Both enantiomers of biologically and pharmaceutically interesting benzofuran-, benzothiophen-, and phenylfuran-based 1-heteroarylethanamines were prepared at close to theoretical yields by using Candida antarctica lipase B (Novozym 435) catalyzed (R)-selective N-acylation with isopropyl butanoate (enantiomeric ratio E > 200). The use of N-methyl-2-pyrrolidinone (NMP) as a cosolvent (1:30) in isopropyl butanoate solved the problem of low solubility of the compounds. Instability of the heterocyclic ring systems against traditional acid- and base-catalyzed hydrolysis was solved by using Candida antarctica lipase A as a commercial CAL-A-CLEA preparation for deprotection of the N-acylated (R) enantiomers in water. The slow, highly enantioselective (E > 200) hydrolyses of racemic butanamides was also observed in the presence of Novozym 435. Copyright

Full text of DOI:10.1002/ejoc.201200330

Job/Unit: O20330
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FULL PAPER
DOI: 10.1002/ejoc.201200330
Chemoenzymatic Preparation of 1-Heteroarylethanamines of Low Solubility
Jürgen Brem,[a,b] László-Csaba Bencze,[a] Arto Liljeblad,[b] Mihaela C. Turcu,[b]
Csaba Paizs,[a] Florin-Dan Irimie,[a] and Liisa T. Kanerva*[b]
Keywords: Asymmetric catalysis / Acylation / Amides / Enantioselectivity / Hydrolysis / Kinetic resolution
Both enantiomers of biologically and pharmaceutically inter-
esting benzofuran-, benzothiophen-, and phenylfuran-based
1-heteroarylethanamines were prepared at close to theoreti-
cal yields by using Candida antarctica lipase B (Novozym
435) catalyzed (R)-selective N-acylation with isopropyl but-
anoate (enantiomeric ratio E Ͼ 200). The use of N-methyl-2-
pyrrolidinone (NMP) as a cosolvent (1:30) in isopropyl but-
anoate solved the problem of low solubility of the com-
pounds. Instability of the heterocyclic ring systems against
traditional acid- and base-catalyzed hydrolysis was solved by
using Candida antarctica lipase A as a commercial CAL-A-
CLEA preparation for deprotection of the N-acylated (R)
enantiomers in water. The slow, highly enantioselective (E Ͼ
200) hydrolyses of racemic butanamides was also observed
in the presence of Novozym 435.
amine enantiomers are prepared by lipases on a ton-
scale.[10]
Introduction
Demand for enantiopure arylethanamines is continu-
ously increasing, especially in the pharmaceutical and fine
chemical industries. The activity of 1-arylethanamines 3a–g
(Scheme 1) have been tested for instance against varicella
zoster virus[1] and as kinase inhibitors,[2] calcium-sensing re-
ceptor regulating agents,[3,4] pesticides,[5] and as compounds
for the prevention and treatment of pain.[6] The nature of a
benzofuran, benzothiophene, and phenylfuran ring in these
and related compounds requires handling under mild reac-
tion conditions to prevent ring degradation. Therefore, bio-
catalytic rather than traditional chemocatalytic preparative
methods are favored. Lipases (E.C. 3.1.1.3) have been com-
monly used for the kinetic resolution of compounds with
primary[7,8] and – although more rarely – secondary[9]
amino groups by enantioselective N-acylation in organic
solvents. N-Acylations of amines are typically irreversible,
because most amide bonds are stable against lipases in or-
ganic media. Additional benefits of lipases include high sta-
bility, wide substrate scope, chemo-, enantio-, and regiose-
lectivity, possibilities for modifications by genetic engineer-
ing, and easy recycling, affording applications in which
Various chemocatalytic methods are known to produce
enantiomerically enriched 1-(benzofuran-2-yl)- and 1-
(benzothien-2-yl)alkanamines. One of the methods de-
scribes the formation of (S)-1-(benzofuran-2-yl)ethanamine
from N-protected α-l-amino acids by utilizing a microwave
technique.[11] Another synthesis has been reported that in-
volves rhodium-catalyzed hydrogenation of N-sulfonyl ket-
imine.[12] A similar compound with the amine frame, (R)-
N-[1-(benzo[b]thiophen-2-yl)ethyl]-O-benzylhydroxylamine,
has been prepared by using an alternative, expensive cou-
pling and an intramolecular Wittig reaction.[13] (S)-N-Aryl-
1-(benzofuran-2-yl)ethamine has been synthesized by Siga-
mide-catalyzed enantioselective reduction of ketimines with
trichlorosilane.[14]
Our aim has been to prepare the enantiomers of seven
benzofuran-, benzothiophen-, and phenylfuran-based 1-
heteroarylethanamines by a chemoenzymatic pathway, the
enantiopurity being introduced by the enantioselective N-
acylation of rac-3a–g (Scheme 1). The emphasis on the en-
zymatic kinetic resolution step focused on solving two prob-
lems associated with 3a–g and N-acylated 4a–g. The first
problem to be addressed was the extremely low solubility
of the compounds in organic solvents commonly used in
enzymatic N-acylation reactions. The second problem con-
cerned the instability of heteroaryl rings and the deprotec-
tion of amides (R)-4a–g to release the corresponding (R)-
3a–g, which is traditionally performed under acidic or basic
conditions at high temperature. The use of polar cosolvents
in enzymatic N-acylation and lipase-catalyzed hydrolysis of
amides 4a–g addressed these issues.
[a] Department of Biochemistry and Biochemical Engineering,
Babes-Bolyai University of Cluj-Napoca,
¸
Arany János 11, 400028 Cluj-Napoca, Romania
[b] Institute of Biomedicine/Department of Pharmacology, Drug
Development and Therapeutics/Laboratory of Synthetic Drug
Chemistry and Department of Chemistry, University of Turku,
20014 Turku, Finland
Fax: +358-2-333-7955
E-mail: lkanerva@utu.fi
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201200330.
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Scheme 1. Reagents and conditions: (I) (a) (PhO)2PON3, toluene; (b) Ph3P, H2O/THF. (II) (a) NH3, [Ti(OiPr)4], MeOH; (b) NaBH4,
MeOH. (III) PrCOCl, DMAP, pyridine, CH2Cl2. (IV) CAL-B, PrCO2iPr, NMP. (V) CAL-A, H2O. (VI) CAL-B, H2O.
B as a Novozym 435 preparation gave both high enantio-
selectivity (measured as enantiomeric ratio E) and reactivity
Results and Discussion
(measured as conversion after a certain time; data not
shown).
Synthesis of Amines rac-3a–g and Amides rac-4a–g
Optimization was continued with Novozym 435 as a cat-
alyst in anhydrous ethyl and isopropyl acetates, which are
common solvents for the acylation of 1-phenylethan-
amine,[7f,8,16] in the presence of molecular sieves [4 Å; in a
1:1 (w/w) ratio to the catalyst]. Molecular sieves were neces-
sary, because the so-called residual water in the seemingly
dry enzyme catalyst or traces of water in the mixture may
cause lipase-catalyzed hydrolysis of hydrolyzable com-
pounds. Under the present N-acylation conditions, the hy-
drolysis of an acyl donor may lead to ammonium salt for-
mation between an amine and the acid liberated, lowering
the enantioselectivity of the kinetic resolution.[7d,17] The ef-
fect of water was clear when the N-acylation of rac-3a was
performed in ethyl acetate saturated with water (E = 50)
compared to the reaction in anhydrous ethyl or isopropyl
acetate (E Ͼ 100; Table 1, Entries 1–3). The effect of water
is often pointed out, especially in connection with Novozym
435 (CAL-B on hydrophobic macroporous divinylbenzene-
crosslinked carrier), which readily releases adsorbed water
into the anhydrous reaction medium.[18] Additionally, water
tunnels, leading to the active site of CAL-B, were previously
shown to allow access for water to the active site.[19]
The preparation of rac-3a–g was accomplished from the
corresponding alcohols rac-1a–g and ketones 2a–g by ap-
plying literature methods (routes I and II, Scheme 1).[15]
With alcohols rac-1a–g as starting materials, low yields (10–
55%) were obtained, because rac-1a–g appeared to be un-
stable during azide formation with diphenyl phosphorazid-
ate under basic conditions (route I, step a) in which elimi-
nation of water was detected.[15a,15b] In step b, the transfor-
mation of azides into amines under basic conditions at high
temperature (40–85 °C) led to amine dimerization. Hydro-
genation of azides in the presence of Pd/C in EtOH/MeOH
before the Staudinger reaction (route I, step b) gave similar
low yields (45–55%), as did the Mitsunobu reaction of rac-
1a–g in the presence of phthalimide (35–45% yields).[15c,15d]
Finally, amines rac-3a–g were synthesized from the corre-
sponding ketones 2a–g by reductive amination with 52–
84% isolated yields (route II, Scheme 1).[15e] The reactions
were complete within 6–12 h at room temperature.
Amides rac-4a–g were obtained by chemical acylation
with an acyl chloride or acyl anhydride in dichloromethane
in the presence of pyridine and a catalytic amount of 4-
(dimethylamino)pyridine (DMAP) (route III, Scheme 1).
One clear problem, especially with amines 3c–f, concerns
their low solubility. For instance, rac-3c was soluble in ethyl
acetate only as a 1 mm solution at room temperature. Solu-
bility also remained low in acetonitrile, dioxane, toluene,
Enzymatic N-Acylation of rac-3a–f
Frequently used commercial lipase preparations tert-amyl alcohol, 2-methyltetrahydrofuran, dimethoxye-
[50 mgmL–1; lipases from Candida rugosa, Pseudomonas thane, diglyme, and tetraline. Common dipolar aprotic sol-
fluorescens, Burkholderia cepacia, Thermomyces lanuginosus, vents such as tetraglyme, dimethyl sulfoxide (DMSO) and
and Candida antarctica (CAL-A and CAL-B)] and alcalase N-methylpyrrolidinone dissolved the substrate better, but
(protease from Bacillus licheniformis) were first screened for enzymatic reactivity was then negligible. N-Methyl-2-pyr-
the N-acylation of rac-3a (0.01 m) in ethyl acetate at room rolidinone (NMP), a suitable building block for ionic li-
temperature (route IV, Scheme 1). Surprisingly, only CAL- quids[20] and pharmaceuticals such as Cefepime,[21] was se-
2
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1-Heteroarylethanamines of Low Solubility
Table 1. N-Acylation of rac-3a (0.010 m) and rac-3c (0.020 m) with CAL-B (10 mgmL–1 for rac-3a and 20 mgmL–1 for rac-3c) at room
temp. for rac-3a and 45 °C for rac-3c, with different solvents and acyl donors (t = 4 h).
Entry
Amine
Solvent/Acyl donor
Conv. [%]
(S)-3 ee [%]
(R)-4 ee [%]
E[a]
1
2
3
4
5
6
7
8
9
3a
3a
3a
3c
EtOAc (wet)
EtOAc
iPrOAc
43
43
50
6
37
51
49
51
52
51
48
51
70
73
96
5
53
97
94
98
97
94
89
Ͼ 99
92
98
96
82
92
91
94
94
91
91
98
97
50
Ͼ 100
Ͼ 100
10
40
90
Ͼ 100
Ͼ 100
90
75
Ͼ 200
Ͼ 200
NMP/iPrOAc (30:1)
NMP/iPrOAc (1:1)
NMP/iPrOAc (1:5)
NMP/iPrOAc (1:30)
NMP/iPrOAc (1:30)
NMP/EtOAc (1:30)
NMP/MeOCH2CO2Et (1:30)
NMP/PrCO2Et (1:30)
NMP/PrCO2iPr (1:30)
3c
3c
3c
3c[b]
3c
10
11
12
3c
3c
3c
[a] Enantiomeric ratio. [b] The results with Novozym 435 reused in five cycles.
Table 2. Preparative-scale N-acylation of rac-3a–f (0.020 m) with CAL-B (20 mgmL–1) at 45 °C in a mixture of NMP/isopropyl butanoate
(1:30) and in the presence of molecular sieves (4 Å; 3 mgmL–1). Reaction time 4 h.
Entry
Amine
Conv. [%]
(S)-3 ee [%]
Yield [%][a]
(R)-4 ee [%]
Yield [%][a]
E[b]
1
2
3
4
5
6
3a
3b
3c
3d
3e
3f
50
50
50
51
51
51
Ͼ 99
Ͼ 99
Ͼ 99
Ͼ 99
Ͼ 99
Ͼ 99
94
95
93
94
95
93
98
99
97
98
97
97
97
95
93
95
95
95
Ͼ 200
Ͼ 200
Ͼ 200
Ͼ 200
Ͼ 200
Ͼ 200
[a] Isolated yield. [b] Enantiomeric ratio.
lected as a cosolvent to increase the solubility of the amine.
The kinetic-resolution method developed for rac-3c was
Thus, the N-acylation of rac-3c (0.020 m) was carried out at then used for the preparative-scale kinetic resolution of rac-
45 °C in various solvent mixtures of NMP and acetate, 2- 3a–f (0.020 m) with isopropyl butanoate and Novozym 435
methoxyacetate, and butanoate esters (Table 1, Entries 4– (20 mgmL–1; Table 2). The highly enantioselective N-acyla-
12). Enantioselectivity E (10 Ǟ Ͼ 100) and conversion after tions of the reactive (R)-3a–f (E Ͼ 200) were all complete
4 h (6 Ǟ 49%) both increased considerably when the pro- within 4 h. The resolution products [the unreacted (S)-3a–f
portion of NMP was reduced from 30:1 to 1:30 with respect and the obtained (R)-4a–f] were all separated at close to
to the acyl donor (Table 1, Entries 4–7). When the N-acyl- 50% theoretical yields (93–97% from the theoretical
ation was repeated by reusing the same Novozym 435 cata- amounts at 50% conversion) in highly enantiopure form (ee
lyst, there was practically no change in the enzymatic effi- 97–99%). In conclusion, the results clearly show NMP to
ciency even after five reuse cycles (compare Table 1, En- be a usable cosolvent for the lipase-catalyzed N-acylation
tries 7 and 8), indicating that the amine and the small of amines of limited solubility.
amount of NMP (1:30) were not harmful to the lipase. The
enantioselectivity of the N-acylations with various esters as
solvents and acyl donors varied slightly, with butanoate es-
ters being clearly the best candidates (compare Table 1, En-
tries 7–10, 11, and 12). Disappointingly, the N-acylation of
Deprotection and Kinetic Resolution of 4a–g by Lipase-
Catalyzed Hydrolysis
rac-3c with acyl-activated ethyl 2-methoxyacetate (Table 1,
The instability of especially (R)-4e and -4f during base-
Entry 10), an excellent acyl donor under the solvent-free catalyzed hydrolysis, and that of (R)-4a–g in aqueous HCl,
kinetic resolution conditions for arylamines,[8] proceeded prevented their deprotection under traditional chemocata-
even worse than with the other acyl donors (Table 1, En- lyzed hydrolysis conditions. A small improvement was made
tries 7–9, 11, and 12). Thus, marked rate enhancements, when (R)-4a–g were heated to reflux overnight in dioxane
caused by favorable hydrogen-bond formation between the containing HCl (8 equiv.), leading to acceptable yields (70–
β-oxygen atom of the methoxyacetate moiety and the amine 80%). Because environmentally more acceptable methods
hydrogen atom of the reactive amine counterpart during the were desired, the studies were directed to the lipase-cata-
progress of catalysis,[22] were not detected at this time. Ac- lyzed amide hydrolysis of rac-4c (Table 3). The most impor-
cording to the results presented in Table 1, the N-acylation tant feature enabling amide hydrolysis by serine proteases
of rac-3c is the most enantioselective one in the NMP/iso- was recently attributed to the hydrogen bond from the sub-
propyl butanoate mixture (1:30; Table 1, Entry 12).
strate amide nitrogen atom to the enzyme or substrate, facil-
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Table 3. CAL-A and CAL-B-catalyzed (10 mgmL–1) hydrolyses of rac-4c (RЈ = Pr; 3.5 mm) in phosphate buffer (pH = 7.5, 0.100 m) or
water at 45 °C (t = 3 d).
Entry
Enzyme
Solvent
Conv. [%]
(S)-4c ee [%]
(R)-3c ee [%]
E[a]
1
2
3
4
5
6
7
8
9
Novozym 435
Novozym 435
buffer
water
water
water
water
water
water
buffer
water
26
33
27
10
25
3
17
60
76
35
48
38
10
33
3
21
30
32
98
Ͼ 100
Ͼ 200
Ͼ 200
Ͼ 200
Ͼ 200
Ͼ 200
Ͼ 200
4
Ͼ 99
Ͼ 99
Ͼ 99
Ͼ 99
Ͼ 99
Ͼ 99
12
CAL-B (ICR-110)
CAL-B (LCAHN)
CAL-B-T1-1500
CAL-B-T2-150
CAL-B (IMB-111)
CAL-A-CLEA
CAL-A-CLEA
16
9
[a] Enantiomeric ratio.
itating the nitrogen inversion during the catalytic process.[23] tween reactivities are based simply on the amounts of cata-
Even though lipases have the same catalytic triad, they lack lysts weighed (10 mgmL–1).
this particular hydrogen bond and, consequently, the ability
Finally, (R)-4a–f (3.5 mm; RЈ = Pr) obtained in the N-
to efficiently cleave amides. However, from a synthetic point acylation were subjected to preparative-scale hydrolysis in
of view, lipases are of particular interest because of their water in the presence of CAL-A-CLEA (Table 4). Accord-
stability and other favorable properties and – as an encour- ingly, amines (R)-3a–f were obtained at approximately
aging result – CAL-A was previously found to hydrolyze 100% conversions in 4–6 d. Additionally, preparative-scale
amides in water.[24] The ability to hydrolyze amides has also hydrolysis of rac-4a–g with Novozym 435 proceeded with
been observed with CAL-B.[25]
excellent enantioselectivities (E Ͼ 200; Table 4), albeit
Before CAL-A and CAL-B could be used for the depro- slowly (5–7 d to reach full 50% conversions). Thus, the No-
tection of (R)-butanamides by hydrolysis, it was important vozym 435 catalyzed hydrolysis allowed the preparation of
to know that the lipase catalyzes hydrolysis of the particular the enantiomers of rac-4g as produced (R)-3g and unre-
enantiomer. Thus, the hydrolysis of butanamide rac-4c (RЈ acted (S)-4g (RЈ = Me; Table 4, Entry 7) although the N-
= Pr in Scheme 1; 3.5 mmol/L) served as a model substrate acylation method totally failed due to the extremely low
in an aqueous medium (water or phosphate buffer, pH = solubility of the compounds in ester solvents even in the
7.5, 0.100 m) in the presence of CAL-A-CLEA (crosslinked presence of NMP. The N-acylation was also unsuccessful
enzyme aggregate of CAL-A) and various CAL-B-prepara- when performed in ionic liquids [1-ethyl-3-methylimid-
tions (Table 3). Because the amide hydrolyses tended to be azolium trifluoromethansulfonate, 1-hexyl-3-methylimid-
slow, elevated temperature was used. At 60 °C, the thermal azolium tetrafluoroborate, 1-butyl-3-methylimidazolium
deactivation of CAL-A-CLEA was significant; therefore, hexafluorophosphate, 1-ethyl-3-methylimidazolium bis(tri-
the hydrolyses were performed at 45 °C. In general, hydro- fluoromethanesulfonyl)imide, and tetrafluoroborate] in
lyses in water were preferable to reactions in phosphate which the amine was soluble; in these cases low conversions
buffer (compare Table 3, Entries 1 to 2 and 8 to 9). CAL- or enantioselectivities were observed (data not shown).
A-CLEA gave good reactivity (conversions 60–76% in 3 d)
but almost no enantioselectivity (Table 3, Entries 8 and 9),
Table 4. Use of CAL-A and CAL-B (10 mgmL–1) on the prepara-
whereas various CAL-B catalysts gave excellent enantio-
tive-scale hydrolyses of rac- or (R)-4a–f (RЈ = Pr; 3.5 mm) and 4g
selectivity (E Ͼ 200) with slow reactions (conversions 3–
48% in 3 d; Table 3, Entries 1–7), with the highest reactivity
(RЈ = Me; 3.5 mm) in water at 45 °C.
Entry Substrate Time
[d]
CAL-B
c [%]
Substrate Time CAL-A
being achieved with Novozym 435 (Table 3, Entry 2). Sim-
ilar reactivities also appeared with CAL-B-T1-1500
(Table 3, Entry 5) and with free lyophilized CAL-B powder,
ICR-110 (Table 3, Entry 3). CAL-B immobilized either
through adsorption on a highly hydrophobic polymer
(LCAHN; Table 3, Entry 4) or covalently (T2-150 and
IMB-111; Table 3, Entries 6 and 7, respectively) gave the
lowest reactivity. As the amounts of protein in the commer-
E[a]
[d]
c [%]
1
2
3
4
5
6
7
rac-4a
rac-4b
rac-4c
rac-4d
rac-4e
rac-4f
6
6
7
6
5
50
50
50
50
50
50
49
ϾϾ 200 (R)-4a
ϾϾ 200 (R)-4b
ϾϾ 200 (R)-4c
5
6
6
4
4
5
6
97
99
96
98
99
98
97
Ͼ 200
Ͼ 200
Ͼ 200
(R)-4d
(R)-4e
(R)-4f
5
13
rac-4g[b]
ϾϾ 200 (R)-4g
cial lipase preparations are not known, comparison be- [a] Enantiomeric ratio. [b] Enzyme content 30 mgmL–1.
4
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1-Heteroarylethanamines of Low Solubility
Scheme 2. Chemoenzymatic synthesis of (R)- and (S)-3g.
The results clearly show that Novozym 435 can be used were addressed by utilizing CAL-A as CLEA and CAL-B
to produce (R)-3a–g from the corresponding butanamide as as Novozym 435 preparations for the hydrolysis of butan-
well as to resolve rac-4a–g, whereas the deprotection of (S)- amides (R)-4a–g and rac-4a–g in water. Hydrolysis by CAL-
4a–g was unsuccessful. On the other hand, the low enantio- A-CLEA proceeded much faster than with Novozym 435,
selectivity of CAL-A-CLEA allowed the deprotection of allowing the deprotection of (R)-4a–g to yield (R)-3a–g in
either (R)- or (S)-amides.
96–99% isolated yields. The enzyme is fascinating, because
amides of the opposite enantiomers can also be depro-
tected. On the other hand, the excellent enantioselectivity
(E ϾϾ 200) of Novozym 435 made the kinetic resolution
of rac-4a–g in water possible, although not attractive, due to
long reaction times compared to N-acylation. The method,
although slow, made the preparation of (R)-3g (ee 99%)
possible by hydrolyzing rac-4g in water, because the low sol-
ubility of rac-3g prevented its kinetic resolution through N-
acylation; (R) enantioselectivity in Novozym 435 and CAL-
A-CLEA catalyses was shown by preparing the correspond-
ing (S)-alcohol and transforming it chemically into (R)-3g.
Confirmation of Absolute Configurations
The absolute configuration of (R)-3g was determined by
first preparing the corresponding alcohol according to the
previous method and resolving it with Novozym 435 in neat
vinyl acetate to yield (S)-5g with 95% enantiomeric excess
(Scheme 2).[26] Compound (S)-5g was converted into the
azide (R)-6g with diphenyl phosphorazidate [(PhO)2PON3]
in anhydrous toluene under argon (Scheme 1, route I,
step a) followed by reduction to amine (R)-3g by the Staud-
inger reaction with triphenylphosphane (step b).[15a,15b] De-
tails of the synthesis are provided in the Supporting Infor-
mation. The enantiopurity was not affected by the transfor-
mation, giving [α]2D5 = +16.8 (c = 1.0, CH3OH) at 95% ee,
which is in accordance with the value {[α]2D5 = +17.3 (c =
1.0, CH3OH) at 99% ee} obtained for the Novozym 435
catalyzed hydrolysis of rac-4g in water (Table 4). It is rea-
sonable to assume that Novozym 435 also has the same
enantiopreference with other substrates in the N-acylation
and deacylation reactions. The (R) enantiopreference of
Novozym 435 shown herein is in accordance with the en-
antiopreference observed previously for the enzyme in N-
acylations of arylethanamines.[8]
Experimental Section
Materials and Methods: Toluene was distilled from Na and stored
under argon. Solvents and acyl donors for enzymatic reactions
were stored over molecular sieves unless otherwise stated. All other
reagents were purchased from Aldrich or Fluka and used as re-
ceived. Lipases from P. fluorescens and B. cepacia were products of
Amano Europe. Lipase A from C. antarctica as cross-linked en-
zyme aggregates (CAL-A-CLEA), alcalase (protease from B.
licheniformis) and lipase from C. rugosa were obtained from Fluka,
and lipase B from C. antarctica (CAL-B, Novozym 435) and T.
lanuginosus lipase (Lipozyme TL IM) from Novozymes. CAL-B as
IMMOZYME CAL-B-T2-150 (covalently immobilized) and IM-
MOZYME CAL-B-T1-1500 (immobilized by adsorbtion) were
products of ChiralVision. CAL-B as IMB-111 (covalently immobi-
lized) and ICR-110 (lyophilized) were from Biocatalytics and as
LCAHN (on highly hydrophobic styrene and divinylbenzene copol-
ymer) from Sprin technologies. The enzymatic reactions were per-
Conclusions
Biologically and pharmaceutically interesting enantio-
mers of racemic benzofuran-, benzothiophen-, and phen-
ylfuran-based 1-heteroarylethanamines (rac-3a–f) were sep-
arated by Novozym 435 catalyzed N-acylation with iso-
propyl butanoate (t = 3–4 h; E Ͼ 200) at close to theoretical
1
formed at 23 °C (room temperature) and 45 °C. H and 13C NMR
spectra were recorded with a Bruker spectrometer operating at
500 MHz with tetramethylsilane (TMS) as an internal standard.
HRMS data were measured in ESI+ mode with a Bruker Avance
50% yields and ee Ͼ 97%. The use of NMP as a cosolvent microOTOF-Q quadrupole-TOF spectrometer. Melting points
were recorded with a Gallenkamp apparatus. Optical rotations
were determined with a PerkinElmer 241 or 341 polarimeter, and
[α]D values are given in units of 10–1 degcm2 g–1. Determination of
in isopropyl butanoate (1:30) solved the problem of low sol-
ubility of the amines 3a–f and the corresponding butan-
amides. However, the kinetic resolution of rac-3g was not
possible by this method, because the compound was not
soluble under the reaction conditions.
The instability of especially (R)-4e and (R)-4f during
base-catalyzed hydrolysis and that of (R)-4a–g in aqueous
HCl prevented the deprotection of butanamide products
under traditional hydrolysis conditions. This problem, and
the enantiomeric ratio E was based on the equation E
=
ln[(1 – c)(1 – eeS)]/ln[(1 – c)(1 + eeS)], with c = eeS/(eeS + eeP) by
using linear regression {E as the slope of the line ln[(1 – c)(1 – eeS)]
vs. ln[(1 – c)(1 + eeS)]} (subscripts S and P refer to substrate and
product, respectively).[27] Enantiomeric excesses for (R)- and (S)-
3a–g were determined as the corresponding acet-, propan- or
butanamides (amines derivatized with the corresponding anhydride
the insolubility of rac-3g under N-acylation conditions, in the presence of a catalytic amount of DMAP in pyridine) with
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L. T. Kanerva et al.
FULL PAPER
Agilent 1090 or 1050 HPLC instruments equipped with Daicel Chi-
racel OD-H or Chiralpak IA chiral columns. Enantiomeric excess
values of (S)-5g and (R)-6g were determined with an Agilent 6850
gas chromatograph equipped with a Cyclosil-B chiral column. Thin
layer chromatography (TLC) was carried out by using Merck Kie-
selgel 60F254 sheets. Spots were visualized by treatment with 5%
ethanolic phosphomolybdic acid solution and heating.
version. The enzyme was filtered off, washed with CH2Cl2, and the
combined organic phases were concentrated in vacuo. The crude
product was purified by silica gel chromatography [ethanol/
CH2Cl2, 5–30% (v/v)].
Enzymatic Deprotection: CAL-A-CLEA (500 mg, 10 mg/mL) was
weighed in a reaction vial before the butanamide (R)-4a–g (RЈ =
Pr; 50 mg) and water (50 mL) were added. The mixture was shaken
at 45 °C. At close to 100% conversion (reaction monitored by
TLC) the reaction was stopped by freezing the reaction mixture
(–20 °C) and evaporating the water by lyophilization. The product
was dissolved in CH2Cl2, and the enzyme was filtered off. The solu-
tion was concentrated in vacuo and the crude product was purified
by silica gel chromatography [ethanol/CH2Cl2, 5–30% (v/v)] to
yield (R)-3a–f. The NMR and MS data of the amines were identical
to those of rac-3a–f above. The kinetic resolution of rac-4a–g
(0.4 mmol) with Novozym 435 (1.25 g) was performed in water
(125 mL) at 45 °C by otherwise applying the deprotection pro-
cedure with CAL-A-CLEA.
Synthesis of rac-3a–g: Amines rac-3a–g were synthesized from
ketones 2a–g according to the literature.[15e,26a] In a Schlenk tube
flushed with argon, a mixture of the corresponding ketone 2a–g
(20.0 mmol) was dissolved in a solution of ammonia in methanol
(2 m, 50 mL, 100 mmol) and titanium(IV) isopropoxide (12.0 mL,
40 mmol). The mixture was stirred under argon at room tempera-
ture for 2–3 h and then cooled in an ice bath before sodium borohy-
dride (1.2 g, 30 mmol) was added in small portions. The mixture
was stirred at room temperature for 3 h and then quenched by
pouring it into ammonium hydroxide (2 m, 100 mL). The inorganic
precipitate was filtered off, the phases were separated, and the
aqueous phase was extracted several times with ethyl acetate. The
combined organic phases were washed with HCl solution (1 m,
150 mL) to separate the neutral materials. The acidic aqueous ex-
tracts were washed with ethyl acetate (250 mL), treated with aque-
ous sodium hydroxide (2 m) to obtain pH = 12 and then extracted
with ethyl acetate (250 mL). The combined organic extracts were
washed with brine (150 mL), dried with sodium sulfate, and con-
centrated in vacuo to give the corresponding crude amine. The
amines were purified by vacuum chromatography on silica gel
(dichloromethane/methanol, 7:3) to afford rac-3a–g as semisolids
with 52–78% yields.
Enantiomerically Enriched Products
(S)-3a: Yield: 130 mg (0.80 mmol, 94%); yellow semisolid; ee Ͼ
99%; [α]2D5 = –12.6 (c = 1.0, CHCl3).
(R)-4a: Yield: 191 mg (0.82 mmol, 97%); yellow solid; m.p.
(78Ϯ1) °C; ee = 98%; [α]2D5 = +148 (c = 1.0, CHCl3).
(R)-3a: CAL-A-CLEA-catalyzed hydrolysis of (R)-4a in water pro-
duced (R)-3a. Yield: 33 mg (0.20 mmol, 95%); yellow semisolid; ee
Ͼ 99%; [α]2D5 = +12.4 (c = 1.0, CHCl3).
(S)-3b: Yield: 144 mg (0.81 mmol, 95%); yellow semisolid; ee Ͼ
99%; [α]2D5 = –17.5 (c = 1.0, CHCl3).
Synthesis of rac-4a–g: DMAP (1%) in pyridine (1.11 mmol,
87.8 mg, 89.4 μL) and the acylating agent (1.11 mmol) were added
to a solution of rac-3a–g (1.00 mmol) in anhydrous CH2Cl2
(1.5 mL). The mixture was stirred at room temperature for 3 h and
then quenched with water (15 mL). The isolated organic phase was
dried with anhydrous sodium sulfate, the solvent was evaporated,
and the crude product was purified by vacuum chromatography on
silica gel (dichloromethane/methanol, 9.5:0.5) to give rac-4a–g as
semisolids with 92–96% yields. NMR and MS data were indistin-
guishable from those of their (R) enantiomers.
(R)-4b: Yield: 200 mg (0.81 mmol, 95%); white solid; m.p.
(115Ϯ1) °C; ee = 99%; [α]2D5 = +147 (c = 1.0, CHCl3).
(R)-3b: CAL-A-CLEA-catalyzed hydrolysis of (R)-4b in water pro-
duced (R)-3b. Yield: 36 mg (0.20 mmol, 98%); yellow semisolid; ee
Ͼ 99%; [α]2D5 = +17.8 (c = 1.0, CHCl3).
(S)-3c: Yield: 162 mg (0.79 mmol, 93%); white solid; m.p.
(159Ϯ1)°C; ee Ͼ 99%; [α]2D5 = –2.5 (c = 1.0, CH3OH).
(R)-4c: Yield: 242 mg (0.83 mmol, 93%); white oil; ee = 97%;
[α]2D5 = +28.5 (c = 0.5, CH3OH)].
Enzymatic Reactions
(R)-3c: CAL-A-CLEA-catalyzed hydrolysis of (R)-4c in water pro-
duced (R)-3c. Yield: 35 mg (0.16 mmol, 92%); white semisolid; ee
Ͼ 99%; [α]2D5 = +2.5 (c = 1.0, CH3OH).
Analytical-Scale N-Acylation: Novozym 435 and 3–4 beads of mo-
lecular sieves (4 Å) were weighed in a reaction vial before rac-3a or
rac-3c (10–100 mm) in anhydrous organic solvent (1 mL) and an
acyl donor were added. Reactions proceeded under shaking
(170 rpm) at 23 °C or (80 rpm) at 45 °C. Samples were taken to
monitor the reaction by filtering off the enzyme and derivatizing
the free amine with an appropriate acyl anhydride in the presence
of DMAP in pyridine. The samples were diluted with acetonitrile
and analyzed by HPLC.
(S)-3d: Yield: 178 mg (0.80 mmol, 94%); white solid; m.p.
(182Ϯ1) °C; ee Ͼ 99%; [α]2D5 = +9.6 (c = 1.0, CH3OH).
(R)-4d: Yield: 237 mg (0.81 mmol, 95%); white solid; m.p.
(136Ϯ1) °C; ee = 97.8%; [α]2D5 = 50.6 (c = 0.5, CH3OH).
(R)-3d: CAL-A-CLEA-catalyzed hydrolysis of (R)-4d in water pro-
duced (R)-3d. Yield: 36 mg (0.16 mmol, 96%); white solid; m.p.
(191Ϯ1) °C; ee Ͼ 99%; [α]2D5 = –9.0 (c = 1.0, CH3OH).
Analytical-Scale Hydrolysis: Either CAL-B or CAL-A-CLEA
(10 mg/mL) was weighed in a reaction vial before one of the amides
rac-4a–g (3.5 mmol/L) and water or phosphate buffer (10 mL, pH
= 7.5, 0.100 m) were added. The mixture was shaken at 45 °C. Sam-
ples were taken to monitor the reaction as above by freezing the
sample (–20 °C) and removing the water by freeze drying. After
derivatization, analysis by HPLC was performed as described
above.
(S)-3e: Yield: 188 mg (0.81 mmol, 95%); yellow semisolid; ee Ͼ
99%; [α]2D5 = –40.2 (c = 1.0, CH3OH).
(R)-4e: Yield: 245 mg (0.81 mmol, 95%); yellow oil; ee = 97%;
[α]2D5 = 53.0 (c = 0.5, CH3OH).
(R)-3e: CAL-A-CLEA-catalyzed hydrolysis of (R)-4e in water pro-
duced (R)-3e. Yield: 37 mg (0.16 mmol, 96%); yellow semisolid; ee
Ͼ 99%; [α]2D5 = +41.3 (c = 1.0, CH3OH).
Preparative-Scale N-Acylation: Novozym 435 (860 mg, 20 mg/mL)
and molecular sieves (4 Å, 120 mg) were weighed in a reaction vial
before rac-3a–f (0.020 mm) in NMP/isopropyl butanoate (1:30) was
added. The mixture was shaken at 45 °C for 4 h to reach 50% con-
(S)-3f: Yield: 184 mg (0.79 mmol, 93%); orange solid; m.p.
(104Ϯ1) °C; ee Ͼ 99%; [α]2D5 = +18.9 (c = 1.0, CH3OH).
6
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1-Heteroarylethanamines of Low Solubility
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Received: March 16, 2012
Published Online: ᭿
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Job/Unit: O20330
/KAP1
Date: 08-05-12 09:51:33
Pages: 8
L. T. Kanerva et al.
FULL PAPER
Kinetic Resolution
J. Brem, L.-C. Bencze, A. Liljeblad,
M. C. Turcu, C. Paizs, F.-D. Irimie,
L. T. Kanerva* .................................. 1–8
Chemoenzymatic Preparation of 1-Het-
eroarylethanamines of Low Solubility
Both enantiomers of seven 1-heteroaryl-
ethanamines were prepared by starting
from the ketones. Enantiopurity was intro-
duced by Candida antarctica lipase B cata-
lyzed (R)-selective N-acylation. Low solu-
bility issues were solved by the use of N-
methylpyrrolidine as a cosolvent. (R)-But-
anamides were deprotected by Candida ant-
arctica lipase A catalyzed hydrolysis in
water, to yield the (R)-amines.
Keywords: Asymmetric catalysis / Acyl-
ation / Amides / Enantioselectivity / Hy-
drolysis / Kinetic resolution
8
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