C. Rusconi et al. / Bioorg. Med. Chem. 20 (2012) 5980–5985
5983
other. They were equally active against CQ-R (range 19–53 nM)
5.3. Enzymatic resolution: general method
and CQ-S (range 16–28 nM) strains of P. falciparum, with no evi-
dence of cross-resistance with CQ.
Racemic AM1 and the two enantiomers demonstrated low tox-
icity against human normal cells with a selectivity index generally
>1000.
These results show an equivalent in vitro behaviour of the two
enantiomers of AM1 and make reasonable the development of the
cheaper racemate as a potential antimalarial drug. In vivo studies
are required to confirm these first results.
Step 1: (+)-Lupinyl ester formation. The lipase (10 mg/mmol of
lupinine) was added to a solution of racemic lupinine (1.77–
5.9 mmol) in toluene (1.8 ml/mmol of lupinine) and the mixture
was stirred for 19–24 h in the open flask put into a desiccators over
anh. CaCl2. Acyl donor (3.7 equiv) was added to the reaction flask,
which was immediately sealed with a septum. The mixture was
stirred for 5–120 h at rt. The reaction was quenched by filtration
and the filtrate was concentrated under reduced pressure. The res-
idue was chromatographed on a silica gel column using ethyl ace-
tate–methanol–NH3 in gradient as eluent to give the (+)-lupinine
ester as white solid.
4. Conclusions
Step 2: Ester hydrolysis. The ester (1 equiv) was dissolved in 1 N
NaOH in MeOH (3 equiv) at 0 °C and the reaction mixture was stir-
red at room temperature for 1 h. When the reaction was com-
pleted, the solution was concentrated; water was added and the
aqueous phase was extracted with ethyl acetate. The combined or-
ganic extracts were dried over anhydrous Na2SO4, evaporated to
dryness and the residue was washed with hexane to give (+)-lup-
inine as a white solid.
We have identified the best conditions to synthesize ( )-AM1
and (+)-AM1 starting, respectively, from ( )-lupinine and (+)-lupi-
nine, which was obtained through the enzymatic resolution of the
former. The racemic AM1 and the (+)-enantiomer of AM1 display
the same activity against P. falciparum and lack of toxicity as de-
scribed previously for the (ꢀ)-enantiomer. These data indicate that
racemic AM1 could be considered as a potential new antimalarial
lead with decrease costs of synthesis compared to (ꢀ)-AM1 and
broad spectrum of activity against different Plasmodium strains.
Experimental conditions and results of enzymatic resolution are
collected in Table 1.
5. Experimental
5.1. General
5.3.1. Experiment 1
Amano PS (282 mg) was added to a solution of racemic
lupinine (560 mg, 3.32 mmol) in 34 ml of toluene in the reaction
flask containing a magnetic stirring bar. The mixture in the open
flask was stirred for 24 h in a sealed container over saturated
CaCl2. 4-Nitrophenyl 3-[4-(trifluoromethyl)phenyl]propanoate
(1.127 g, 3.32 mmol) was added to the reaction flask, which was
immediately sealed with a septum. The mixture was stirred for
24 h at rt. The reaction was quenched by filtration and the filtrate
was concentrated under reduced pressure. The residue was
chromatographed on a silica gel column using ethyl acetate–
methanol–-NH3 in gradient as the eluent, to give 128 mg of ester
Amano Lipases PS (Pseudomonas cepacia, P30,000 U/g) and AK
(Pseudomonas fluorescens, P20,000 U/g) were supplied by Aldrich
and were employed without any previous treatment. All commer-
cially available solvents and reagents were used without further
purification, unless otherwise stated. Purification of products and
separations of esters and alcohols after enzymatic conversions
were performed on silica gel 60 Merck. Melting points were deter-
mined with a Büchi apparatus and are uncorrected. 1H NMR spec-
tra were recorded on Varian Mercury 300VX spectrometer, using
CDCl3 as solvent; high-resolution mass spectra (HRMS) on a APEX
II ICR-FTMS Bruker Daltonics mass spectrometer in positive electro
spray ionization (ESI). Optical rotations were measured on a
polarimeter Jasco P-1010 in EtOH at 20 °C.
as
a
white solid. 1H NMR (300 MHz, CDCl3): d = 7.57 (d,
J = 8.05 Hz, 2H), 7.34 (d, J = 7.91 Hz, 1H), 4.47–4.16 (m, 2H), 3.04
(t, J = 7.52 Hz, 2H), 2.97–2.81 (m, 2H), 2.68 (t, J = 7.59 Hz, 2H),
2.21–1.15 (m, 15H).
The produced ester was hydrolyzed (1 N NaOH in MeOH,
3.5 ml) to give (+)-lupinine (73.3 mg, 0.18 mmol, ½a D20
ꢁ
+14.9 (c = 1
5.2. (1RS, 9aRS)-Octahydro-2H-quinolizine-1-methanol; ( )-
Lupinine
in EtOH); 1H NMR (300 MHz, CDCl3): d = 5.25 (br s, 1H), 4.17–
4.11 (m, 1H), 3.68 (d, J = 10.45 Hz, 1H), 2.85–2.77 (m, 2H), 2.19–
1.97 (m, 3H), 1.95–1.67 (m, 4H), 1.63–1.45 (m, 6H), 1.31–1.20
(m, 1H).
Sodium borohydride (2.23 g; 59.13 mmol) was added in small
portions, during 2 h, to a solution of 3,4,6,7,8,9-hexahydro-2H-
quinolizine-1-carboxylic acid ethyl ester, prepared following the
method described by Cai et al.12 (2.6 g; 12.42 mmol) in anh. MeOH
(70 ml), kept between 0 and 5 °C and stirred under N2. The reaction
mixture was then diluted with water (300 ml) and extracted with
ether (twice). The combined organic extracts were dried over anh.
Na2SO4 and evaporated to dryness. A slight yellow oil was obtained
(2.3 g; 10.89 mmol; 88.5%), which was dissolved in anh. ether
(60 ml) and added dropwise to a suspension of LiAlH4 (3.5 g;
94.52 mmol). The mixture was stirred at rt, under N2, for 1.5 h.
After cooling in an ice bath, wet ether was added dropwise to the
mixture, followed by water. After filtration, the two layers were
separated and the aqueous phase was saturated with Na2CO3 and
extracted with ether. The combined ether extracts were dried over
anhydrous Na2SO4 and evaporated to dryness. A slight yellow oil
was obtained which crystallizes after treatment with hexane. Yield
81%; mp 56.5–57.8 °C. 1H NMR (300 MHz, CDCl3): d = 5.25 (br s,
1H), 4.14 (dd, J = 4.13, 10.45 Hz, 1H), 3.68 (d, J = 10.72 Hz, 1H),
2.85–2.75 (m, 2H), 2.25–1.95 (m, 3H), 1.90–1.67 (m, 4H), 1.61–
1.42 (m, 6H), 1.35–1.18 (m, 1H).
5.3.2. Experiment 2
Amano PS (60 mg) was added to a solution of racemic lupinine
(1 g, 5.9 mmol) in 10.6 ml of toluene and the mixture was stirred
for 24 h. Vinyl acetate (2 ml, 21.7 mmol) was added and the mix-
ture was stirred for 120 h at rt. After purification, were obtained
300 mg of ester (1.41 mmol) as a white solid. 1H NMR (300 MHz,
CDCl3): d = 4.38–4.33 (m, 1H), 4.22–4.16 (m, 1H), 2.84–2.80 (d,
J = 10.8 Hz, 2H), 2.24–1.73 (m, 10H), 1.69–1.30 (m, 6H), 1.30–1.24
(m, 1H).
The ester was hydrolyzed, according to general procedure (step
2), with 5 ml of 1 N NaOH in MeOH, to give (+)-lupinine (160 mg,
66%). ½a 2D0
ꢁ
= +17.67 (c = 1 in EtOH).
5.3.3. Experiment 3
Amano AK (30 mg) was added to a solution of racemic lupinine
(500 mg, 2.95 mmol) in 5 ml of toluene and the mixture was stir-
red for 24 h. Vinyl acetate (1 ml, 10.9 mmol) was added and the
mixture was stirred for 24 h at rt. After purification, were obtained
360 mg of ester (1,41 mmol) as a white solid.