C. Sanfilippo et al. / Journal of Molecular Catalysis B: Enzymatic 104 (2014) 82–86
83
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and it is marketed as racemic hydrochloride salt (Ixel , Savella )
flow rate 0.5 ml/min and UV-detection at ꢁ 220 nm. Pre-column
derivatization with propionic anhydride was applied to all the sam-
ples from the enzymatic reactions, with the exception of samples
from enzymatic reaction with ethyl propionate as acyl donor that
were treated with acetic anhydride. Reference samples of racemic
amides (± )-2–(± )-5 were obtained by reaction of (± )-1 with the
suitable anhydride while (± )-7 was obtained by reaction of (± )-1
with allyl chloroformate in CH2Cl2 in the presence of pyridine.
Retention times of the racemic reference compounds: N-acetyl
milnacipran (± )-2: tR 21.2 min (1S,2R)-2 and 37.5 min (1R,2S)-2; N-
propanoyl milnacipran (± )-3: tR 16.6 min (1S,2R)-3 and 23.0 min
(1R,2S)-3; N-iso-butanoyl milnacipran (± )-4: tR 12.1 min (1S,2R)-
4 and 14.2 min (1R,2S)-4; N-(2-methoxy)-ethanoyl milnacipran
(Scheme 1).
Pharmacokinetic studies on the separate enantiomers of 1
showed higher activity for levomilnacipran, (1S, 2R)-(−)-1 [37,38]
and, in order to meet FDA and EMA guidelines, there is a growing
interest in the preparation of enantioenriched (−)-1 for novel drug
formulations [39,40]. Although some synthetic methodologies for
the preparation of optically active 1 have been reported [41,42], the
availability of a biocatalytic protocol for kinetic resolution of (± )-
1
could be valuable in terms of costs and operational simplicity.
Here we report the obtained results in lipase-catalysed N-acylation
of (± )-1 and the preparation of both enantiomers of the drug in
optically active form.
(
7
± )-5: tR 31.4 min (unresolved); N-allylcarbamoyl milnacipran (± )-
: tR 15.1 min (1S,2R)-7 and 21.9 min (1R,2S)-7. Enantiomeric
2
. Experimental
excesses for products (eep) and milnacipran (ees), the latter
converted into a suitable amide derivative, were determined
from peak areas of the corresponding enantiomers following
ee = (A> − A<)/(A> + A<) × 100. Conversion (C) values in kinetic res-
2.1. General information
Immobilized lipases PS-C
I
(from Pseudomonas cepacia),
®
s s p
olution experiments were then derived from C = ee /(ee + ee ).
Novozyme 435 (from Candida antarctica, CAL-B) and crude Lipase
AK (from Pseudomonas fluorescens) were purchased from Aldrich,
Lipozyme (immobilized lipase from Mucor miehei) was obtained
from Fluka, PPL (crude lipase from Porcine pancreas) and cross-
linked enzyme aggregate CLEA (from Candida antarctica) were from
Sigma, Chirazyme L-9 (immobilized lipase from Rhizomucor miehei)
was purchased from Boehringer Mannheim. Analytical grade chem-
Since amide 5 eluted as a single peak, the conversion value of entry
4 in Table 1 was obtained by direct comparison of peak areas after
correction of area of 5 with respect to 3 by a relative response factor
RRF = 1.90.
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2.4. General procedure for the lipase-catalyzed resolution of
1
13
(± )-1
icals were used as received. H and C NMR spectra were recorded
in CDCl3 on a Bruker Avance
TM
400 instrument at 400.13 and
In a general enzymatic reaction, 10 mg (0.04 mmol) of (± )-1
1
00.03 MHz, respectively. Chemicals shifts (ı) are reported in ppm
were dissolved in 2 ml of solvent, then 30 mg of lipase and acyl
donor (0.43 mmol) were added and the resulting suspension was
relative to the residual solvent and coupling constants (J) are given
in Hz. Optical rotations were recorded on a DIP 370 JASCO instru-
ment using a ꢀ 3.5 × 100 mm cell.
◦
shaken at 300 rpm and 40 C. Aliquots were withdrawn at vari-
ous intervals and subjected to chiral HPLC analysis to determine
both conversions and enantiomeric excesses. In the experiment in
presence of molecular sieves, they were added (40 mg) together
with the lipase to the reaction mixture. The same experimental
conditions were also applied to enzymatic reactions at variable
temperature.
2.2. Extraction of (± )-1 from the pharmaceutical preparation
Milnacipran free base (± )-1, was recovered from commercial
pharmaceutical preparation according the following procedure:
the tablets were grinded in a mortar and suspended in methanol.
The suspension was stirred at room temperature for 1 h, then fil-
tered and the solution evaporated to dryness. The residue was
suspended in aq. 2 N NaOH and the solution stirred for 30 min at
room temperature, then the aqueous phase was extracted with
tert-butyl methyl ether (t-BME). The organic solution was dried on
Na SO and taken to dryness to give (± )-1 as a pale yellow oil, with
2
.5. Semi-preparative resolution of (± )-1 by lipase catalyzed
reaction
In a semi-preparative run (± )-1 (200 mg, 0.81 mmol) was dis-
solved in a mixture of t-BME and methyl iso-butyrate (MiBu) as
acylating agent in 3:2 v/v ratio (20 ml) and the reaction was started
by adding Novozyme 435 lipase (600 mg). The suspension was
2
4
analytical purity 98% and spectral properties in agreement with
literature data [41,42].
◦
shaken at 300 rpm and 50 C until the HPLC analysis showed about
5
0% of substrate conversion (1.5 h). The enzyme was then filtered
2
.3. Chiral HPLC analyses
off and the solution evaporated to dryness. The residue was dis-
solved in 1 N HCl (5 ml) and the solution extracted with t-BME.
The organic phase was dried on anhydrous Na2SO4 and evaporated
under vacuum to give the amide product (−)-4 as a white solid in
The enantiomeric excesses were determined by chiral HPLC
◦
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at 23 C on Phenomenex Lux Cellulose-4 (250 × 4.60 mm) col-
umn using isocratic elution with n-hexane/2-propanol 70:30 (v/v),
5
1% yield (128 mg, 0.41 mmol) and 71% ee.
The acidic aqueous phase was alkalinized by addition of 2 N
NaOH and then extracted with t-BME. The organic phase was dried
on anhydrous Na SO4 and taken to dryness to afford unreacted
2
(
−)-1 as yellow pale oil in 41% yield (81 mg, 0.33 mmol) and 76% ee.
Crystallization of (−)-4 from t-BME gave white crystals in
enantiopure form (>98% ee) and 68% crystallization yield (88 mg,
◦
0
.28 mmol). Data for (−)-4: [˛]D = −148.9 (c 0.67, CHCl ), mp 138 C;
3
1
H NMR: ı 0.84 (3H, t, J = 7.1 Hz, CH CH −), 1.13 (10H, m, CH CH−,
3
2
3
CH CH − and −CH−), 1.45 (1H, m, −CHCH NH−), 1.54 (1H, dd,
3
2
2
J = 8.8 and 5.2 Hz, −CH−), 2.39 (1H, heptet, J = 6.8 Hz, CH CH−), 2.59
3
(
3
1H, m, −CH NH−), 3.33 (2H, m, CH CH −), 3.41 (1H, m, CH CH −),
2
3
2
3
2
.49 (1H, m, CH CH −), 4.10 (1H, m, −CH NH−), 7.18 (3H, m, Ph),
3
2
2
Scheme 1. Chemical structures of milnacipran enantiomers and related amide
derivatives.
13
7
.27 (2H, m, Ph); C NMR: ı 12.4, 13.0, 17.4, 19.5, 19.7, 26.9,