P. Csuka et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
5
temperature: 25 °C, k = 220 nm. Retention times, tr (min): 4.6 [rac-1],
42.9 [(R)-3], 48.8 [(S)-3].
(R)-3: 29% (entry 3 in Table 1); colorless crystals; mp: 142 °C,
lit.16 mp: 120–122 °C, [ 28 = +80.1 (c 1.0, CH2Cl2), ee = 99.9% (by
a]
D
Conversion of reactions (c) were calculated by the equation
c = (ee(S)-1)/[ee(S)-1 + ee(R)-3] using the enantiomeric excess values
of the residual substrate (S)-1 and the produced (R)-3.
The NMR spectra were recorded in CDCl3 on a Bruker DRX-300
spectrometer operating at 300 MHz for 1H and 75 MHz for 13C, and
signals are given in ppm on the d scale.
HPLC). The IR and NMR spectra were in agreement with those
obtained for the racemic amide rac-3.
4.3.2. Comparison of ethyl and isopropyl 2-cyanoacetates 2a
and 2b as acylating agents
1-Phenylethanamine rac-1 (50 mg, 0.41 mmol), CaLB N435
(100 mg), and alkyl 2-cyanoacetate (ethyl 2-cyanoacetate 2a,
23 mg, 0.5 equiv; or isopropyl 2-cyanoacetate 2b, 26 mg, 0.5 equiv)
were added to a mixture of toluene–THF 1:1 (2 mL) and the result-
ing mixture was shaken (400 rpm) at room temperature for 4 h
(sampling at 1, 2, 3, and 4 h was performed: see Fig. 2). The reac-
tion mixtures were then processed as described in Section 4.3.1.
A blank test was performed with rac-1 (50 mg, 0.41 mmol) and
alkyl 2-cyanoacetate 2a (23 mg, 0.5 equiv) or 2b (26 mg, 0.5 equiv)
in a mixture of toluene–THF 1:1 (2 mL) shaken (400 rpm) at room
temperature for 4 h. The conversion was checked by GC (0.2% with
2a, 0.3% with 2b).
Infrared spectra were recorded on a Bruker ALPHA FT-IR spec-
trometer and wavenumbers of bands are listed in cmÀ1
.
Optical rotation was measured on Perkin–Elmer 241 polarime-
ter at the -line of sodium. The polarimeter was calibrated with
D
measurements of both enantiomers of menthol.
The proliferation assay was made on 384 well plates (Perkin
Elmer, Boston, MA). The antiproliferative activity of the
compounds was determined by CellTiter-GloÒLuminescent Cell
Viability Assay (Promega, Madison, WI) according to the manufac-
turer’s instructions. The luminescent signal was measured by
AnalystÒGT Multimode Reader (Molecular Devices, Sunnyvale,
CA). XLfit (IDBS) software was used to generate the dose-effect
curves for EC50 determination.
4.3.3. Mode of ethyl 2-cyanoacetate 2a addition
To a mixture of toluene–THF 1:1 (2 mL) were added 1-pheny-
lethanamine rac-1 (50 mg, 0.41 mmol), CaLB N435 (100 mg), and
ethyl 2-cyanoacetate 2a (Method A: 23 mg, 0.5 equiv; Method B:
46 mg, 1 equiv; Method C: 23 mg, 0.5 equiv, further 11.5 mg,
0.25 equiv after 2 h and further 11.5 mg, 0.25 equiv after 4 h) and
the resulting mixture was shaken (400 rpm) at room temperature
for 24 h (sampling at 2, 4 6, and 24 h was performed: see
Table 2). The reaction mixtures were then processed as described
in Section 4.3.1. (S)-1: 41% (Method C in Table 2); pale yellow liq-
4.2. Chemical acylation of 1-phenylethanamine 1, (S)-1 by ethyl
2-cyanoacetate 2a
Amides rac-3 or (S)-3 were prepared from the corresponding
amine [racemic amine 1 or (S)-1 obtained from the lipase-catalyzed
kinetic resolution, see Section 4.3] by a modified method of Kumar
et al.16 The reaction mixture of ethyl 2-cyanoacetate 2a (113 mg,
0.01 mol) and 1-phenylethanamine rac-3 or (S)-3 (121 mg,
0.01 mol) was added to toluene (2 mL) by stirring at room
temperature for 72 h. The solvent was removed under reduced
pressure. The product was washed with cooled diethyl ether
(2 Â 3 mL).
uid; [
a]
D
28 = À31.1 (c 10, ethanol), {lit.: [
a]
28 = À40.3 (neat)25};
D
ee = 99.3% (by GC).
4.4. Preparation of Tyrphostins
rac-3: 61% (precipitated from the reaction mixture and washed
by Et2O); colorless crystals; mp: 113 °C, lit.24 mp 100–101 °C, 1H
NMR (CDCl3): 1.54 (3H, d, J = 7.2 Hz, CH3), 3.31 (2H, s, CH2), 5.10
(1H, dq, J = 7.2 Hz and J = 7.3 Hz, CH), 6.53 (1H, br, NH), 7.28–7.40
(5H, m, ArH); 13C NMR (CDCl3): 21.7 (CH3), 26.1 (CH2), 50.2 (CH),
114.9 (CN), 126.3 (2xCH), 128.0 (CH), 129.1 (2xCH), 142.2 (C)
160.3 (C@O); IR (KBr): 3279, 3068, 2975, 2921, 2258, 1648, 1554,
669. The IR and NMR spectra were in agreement with the data in
literature.24
An aldehyde 4a–f (1 mmol), and an amide rac-3, (S)-3 or (R)-3
(1 mmol) were heated at reflux in ethanol (8 mL) containing piper-
idine (1 drop) for 4 h. The mixture was cooled and stored at 4 °C for
24 h. The precipitated crystalline product was filtered off and
washed with cold diethyl ether. In the case of no precipitate form-
ing, the product was isolated by column chromatography (silica
gel, with a mixture of hexane–acetone 10:4) from the residue after
evaporation of the solvent. The results with rac-3 are compiled in
Table 3.
rac-5a: 73% (after silica gel chromatography), colorless crystals;
mp: 123 °C; 1H NMR (CDCl3): 1.62 (3H, d, J = 7.2 Hz, CH3), 5.27 (1H,
m, CH), 6.56 (1H, d, J = 5.7 Hz, NH), 7.27–7.55 (8H, m, ArH), 7.93
(2H, d, J = 6.6 Hz, ArH), 8.35 (1H, s, CH@); 13C NMR (CDCl3): 22.0
(CH3), 50.4 (CH), 106.0 (C@), 117.0 (CN), 126.4 (2 Â CH), 128.0
(CH), 129.1 (2 Â CH), 129.5 (2 Â CH), 130.9 (2 Â CH), 132.0 (C),
133.0 (CH), 143.0 (C), 153.5 (CH@), 159.0 (C@O); IR (KBr): 3295,
3062, 3032, 29702, 2939, 2215, 1666, 1654, 1597, 1541, 1523,
701, 687.
(S)-3: 59% (precipitated from the reaction mixture, washed by
Et2O); colorless crystals; mp: 141 °C, [
ee = 99.3% (by HPLC). The IR and NMR spectra were in agreement
a
]
28 = À78.6 (c 1.0, CH2Cl2),
D
with those obtained for the racemic amide rac-3.
4.3. CaLB-catalyzed kinetic resolution of 1-phenylethanamine 1
with alkyl 2-cyanoacetates 2a, b
4.3.1. Selection of the biocatalyst
1-Phenylethanamine rac-1 (100 mg, 0.825 mmol), ethyl 2-
cyanoacetate 2a (46 mg, 0.5 equiv), and 200 mg of the biocatalyst
(CaLB G250P, CaLB T2-150 or CaLB N435: see Table 1) were added
to a mixture of toluene–THF 1:1 (10 mL) and the resulting mixture
was shaken (400 rpm) at room temperature for 24 h. At the end of
the reaction, the biocatalyst was filtered off and the filtrate was
evaporated in vacuum. The residue was dissolved in 5% HCl
(7.5 mL) and the solution extracted with dichloromethane
(3 Â 12 mL). The organic phase was dried and evaporated to give
pure (R)-3. To the aqueous phase was added 40% NH3 solution
(5 mL) and then extracted with dichloromethane (3 Â 12 mL).
The organic phase was dried and evaporated to give unreacted
(S)-1-phenylethanamine (S)-1.
(R)-5a: 71% (after silica gel chromatography), colorless crystals;
mp: 73 °C; [
a]
D
28 = À4.1 (c 1.0, CH2Cl2), ee = 99.9% [by HPLC of the
starting amide (R)-3].
(S)-5a: 69%; (after silica gel chromatography), colorless crystals;
mp: 72 °C, [a]
28 = +4.0 (c 1.0, CH2Cl2), ee = 99.3% [by HPLC of the
D
starting amide (S)-3].
rac-5b: 95% (precipitated from the reaction mixture, washed by
Et2O), yellow crystals; mp: 228 °C, 1H NMR (CDCl3): 1.62 (3H, d,
J = 6.6 Hz, CH3), 5.30 (1H, m, CH), 5.43 (2H, s, CH2), 6.39 (1H, d,
J = 7.5 Hz, NH), 7.16–7.19 (2H, m, ArH), 7.28–7.41 (11H, m, ArH),
7.86–7.88 (1H, m, ArH), 8.45 (1H, s, CH@), 8.70 (1H, s, ArH); 13C
NMR (CDCl3): 22.1 (CH3), 50.0 (CH), 51.5 (CH2), 95.9 (C@), 110.8
(C), 111.1 (CN), 119.1 (CH), 119.5 (CH), 122.7 (CH), 124.1 (CH),