Synthesis and comparison of antiplasmodial activity of (+), (-) and racemic 7-chloro-4-(N-lupinyl)aminoquinoline

Article abstract of DOI:10.1016/j.bmc.2012.07.041

Recently the N-(-)-lupinyl-derivative of 7-chloro-4-aminoquinoline ((-)-AM-1; 7-chloro-4-{N-[(1S,9aR)(octahydro-2H-quinolizin-1-yl)methyl]amino} quinoline) showed potent in vitro and in vivo activity against both Chloroquine susceptible and resistant strains of Plasmodium falciparum. However, (-)-AM-1 is synthesized starting from (-)-lupinine, an expensive alkaloid isolated from Lupinus luteus whose worldwide production is not sufficient, at present, for large market purposes. To overcome this issue, the corresponding racemic compound, derived from synthetic (±)-lupinine was considered a cheaper alternative for the development of a novel antimalarial agent. Therefore, the racemic and the 7-chloro-4-(N-(+)-lupinyl)aminoquinoline ((±)-AM-1; (+)-AM-1) were synthesized and their in vitro antimalarial activity and cytotoxicity compared with those of (-)-AM-1. The (+)-lupinine required for the synthesis of (+)-AM-1 was obtained through a not previously described lipase catalyzed kinetic resolution of (±)-lupinine. In terms of antimalarial activity, (±)-AM1 and (+)-AM1 demonstrated very good activity in vitro against both CQ-R and CQ-S strains of P. falciparum (range IC₅₀ 16-35 nM), and low toxicity against human normal cell lines (therapeutic index >1000), comparable with that of (-)-AM1. These results confirm that the racemate (±)-AM1 could be considered as a potential antimalarial agent, ensuring a decrease of costs of synthesis compared to (-)-AM1.

Full text of DOI:10.1016/j.bmc.2012.07.041

Bioorganic & Medicinal Chemistry 20 (2012) 5980–5985
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
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Synthesis and comparison of antiplasmodial activity of (+), (ꢀ) and racemic
7-chloro-4-(N-lupinyl)aminoquinoline
Chiara Rusconi a, , Nadia Vaiana a, , Manolo Casagrande a, Nicoletta Basilico b, Silvia Parapini c,
Donatella Taramelli c, Sergio Romeo a, Anna Sparatore a,
⇑
a Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milan, Italy
b Dipartimento di Scienze Biomediche, Chirurgiche e Odontoiatriche, Università degli Studi di Milano, Via Pascal 36, 20133 Milan, Italy
c Dipartimento di Scienze Farmacologiche e Biomolecolari, Università di Milano, Via Balzaretti 9, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Recently the N-(ꢀ)-lupinyl-derivative of 7-chloro-4-aminoquinoline ((ꢀ)-AM-1; 7-chloro-4-{N-
[(1S,9aR)(octahydro-2H-quinolizin-1-yl)methyl]amino}quinoline) showed potent in vitro and in vivo
activity against both Chloroquine susceptible and resistant strains of Plasmodium falciparum. However,
(ꢀ)-AM-1 is synthesized starting from (ꢀ)-lupinine, an expensive alkaloid isolated from Lupinus luteus
whose worldwide production is not sufficient, at present, for large market purposes. To overcome this
issue, the corresponding racemic compound, derived from synthetic ( )-lupinine was considered a
cheaper alternative for the development of a novel antimalarial agent. Therefore, the racemic and the
7-chloro-4-(N-(+)-lupinyl)aminoquinoline (( )-AM-1; (+)-AM-1) were synthesized and their in vitro anti-
malarial activity and cytotoxicity compared with those of (ꢀ)-AM-1. The (+)-lupinine required for the
synthesis of (+)-AM-1 was obtained through a not previously described lipase catalyzed kinetic resolution
of ( )-lupinine. In terms of antimalarial activity, ( )-AM1 and (+)-AM1 demonstrated very good activity
in vitro against both CQ-R and CQ-S strains of P. falciparum (range IC50 16–35 nM), and low toxicity
against human normal cell lines (therapeutic index >1000), comparable with that of (ꢀ)-AM1. These
results confirm that the racemate ( )-AM1 could be considered as a potential antimalarial agent, ensuring
a decrease of costs of synthesis compared to (ꢀ)-AM1.
Received 24 May 2012
Revised 20 July 2012
Accepted 22 July 2012
Available online 31 July 2012
Keywords:
Enzymatic resolution
Lipase
Lupinine
Malaria
Quinoline
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
has emerged very slowly over time. Recently, several new 4-ami-
noquinoline derivatives have been synthesized and studied.
Malaria is an infectious disease causing more than 500 million
cases and approximately 700 thousands deaths every year. The
majority of these cases are due to Plasmodium falciparum and occur
mainly in sub-Saharan Africa. Plasmodium vivax, the second most
frequent human malaria species causes more than 80 million clin-
ical episodes particularly in Asia, where it accounts for at least half
of the malaria burden.1
Chloroquine (CQ), one of the most efficacious, safe and cheap
antimalarial drugs, has been effectively used for decades against
both Plasmodium species. Unfortunately now it is largely ineffec-
tive against P. falciparum because of widespread parasite resis-
tance.2 New drugs for combating parasite resistant strains are
needed, even more now that the efforts are directed toward elim-
ination and eradication of malaria by 2050.3 Quinoline-type anti-
malarials remain an attractive class of compounds because their
mechanism of action and resistance are unrelated and resistance
Among them, AQ-13 and ferroquine are under development as
novel antimalarial drugs and entered into Phase 2 clinical trials.4–6
In the last few years, the research of our group has been direc-
ted to the study of new quinolizidinyl and quinolizidinyl-alkyl
derivatives of 7-chloro-4-aminoquinoline.7,8 Compounds highly
effective in vitro against both the D-10 (CQ-sensitive, CQ-S) and
W-2 (CQ-resistant, CQ-R) strains of P. falciparum7 and in vivo in
the murine P. berghei model,9 have been obtained. One of these
compounds, (ꢀ)-AM1 has been selected for further characteriza-
tion: (ꢀ)-AM1 is a semisynthetic product that can be obtained in
4 steps with good yield (52.5%) as a pure enantiomer, starting from
(ꢀ)-lupinine, which is transformed into (ꢀ)-aminolupinane ((1S,
9aR)-octahydro-2H-quinolizine-1-methanamine) and coupled
with 4,7-dichloroquinoline to give the final compound, as previ-
ously reported by Boido Canu et al.10 (Scheme 1).
Lupinine is an alkaloid present in several Leguminosae (Lupinus
luteus, L. hispanicus) and in Chenopadiaceae (Anabasis aphylla). It is
the major alkaloid of bitter Lupinus luteus that was cultivated in
central Europe for fixing nitrogen in soil. Unfortunately, the exten-
sion of this cultivation is presently very limited because ‘sweet’
⇑
Corresponding author. Tel.: +39 02 50319365; fax: +39 02 50319359.
E-mail address: anna.sparatore@unimi.it (A. Sparatore).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.041

C. Rusconi et al. / Bioorg. Med. Chem. 20 (2012) 5980–5985
5981
O
NH2
OH
Cl
HN
N
N
H
N
H
H
N
H
N
H
a
O
b
d
c
N
N
Cl
(_)-AM1
(_)-Lupinine
Scheme 1. Reagents and conditions: (a) SOCl2, CHCl3, reflux; (b) potassium phthalimide, DMSO, N2, 145 °C; (c) 6 N HCl, reflux; (d) 4,7-dichloroquinoline, phenol, 180 °C.
I
Cl
Cl
OH
COOEt
COOEt
O
a
e
b
c
d
+
Cl
OEt
N
N
N
( )-lupinine
4
5
COOEt
COOEt
1
2
3
Scheme 2. Reagents and conditions: (a) n-BuLi, Et2O, ꢀ30 °C; (b) NaI, CH3COCH3, reflux; (c) 3-chloropropylamine.HCl, MeCN, K2CO3, 4 Å MS, reflux; (d) NaBH4, CH3OH, 0–
5 °C; (e) LiAlH4, Et2O, rt.
varieties of Lupinus (L. albus, L. luteus), without alkaloids, are now
preferred for human consumption or for animal feeding. Lupinine
from Anabasis is available commercially, however it is quite expen-
sive and not sufficient for drug production. Therefore, although the
synthesis of (ꢀ)-AM1 from lupinine is quite straightforward, the
limited availability and the cost of the starting material represent
unsuited features for a potential antimalarial agent.
Key intermediate in the synthesis of ( )-lupinine is the unsatu-
rated quinolizidine ester 4 (Scheme 2). This compound has been
obtained by reaction of the alkyliodide 3 with 3-chloropropyl-
amine in MeCN assisted with K2CO3, which undergoes a sequential
SN2/Michael addition/SN2/SN2 reaction process.12
Selective reduction of the double bond by NaBH4,13 followed by
LiAlH4 reduction of the ester afforded rac-lupinine with an overall
yield of 30%.
Since the activity of 4-aminoquinoline antimalarials does not
seem to be correlated with the chirality of the drug, the presence
of a specific absolute configuration on the basic head should not
be necessary. Therefore, we synthesized and investigated the activ-
ity of the racemate of AM1 (( )-AM1), thus ensuring a production
independent from the natural source and, consequently, cheaper.
However, since a racemic mixture can be used in therapy only if
both enantiomers display comparable biological and toxicological
profiles, the synthesis and the pharmacological characterization
of racemic AM1 and (+)-AM1 were also planned in comparison
with (ꢀ)-AM1.
( )-AM1 was obtained from ( )-lupinine by applying the four
steps synthetic scheme shown in Scheme 1 for the synthesis of
(ꢀ)-AM1.
To obtain (+)-AM1 by resolution, two different routes were pos-
sible: either the resolution of ( )-AM1 or the resolution of ( )-lup-
inine, followed by conversion of (+)-lupinine into (+)-AM1. We
decided to attempt the second alternative considering that: (a)
the presence of an alcoholic functional group allows the use of sev-
eral chiral auxiliaries; (b) in a synthetic process, it is always pref-
erable to have the lowest yield step as early as possible.
Although the resolution of racemic primary alcohols is well
known, resolution of lupinine could be a difficult task, mainly
due to the lack of aromatic rings. An interesting approach is the ki-
netic resolution of racemic alcohols, through lipase-catalyzed
asymmetric transesterification in organic solvents. However, this
method is more efficient when applied to secondary alcohols,
while kinetic resolution of racemates of primary alcohols is more
difficult to achieve, due to the lower enantioselectivity of lipases
towards chiral primary alcohols.14–16
In this paper, we report the synthesis and the in vitro antiplas-
modial activity against different strains of P. falciparum of the race-
mate of AM1 and the enantiomers. The cytotoxicity against human
cells is also reported.
2. Chemistry
According to the synthetic pathway followed for (ꢀ)-AM1, the
synthesis of ( )-AM1 and (+)-AM1 required as starting material
( )-lupinine and (+)-lupinine, respectively, which are not commer-
cially available.
In order to obtain pure (+)-lupinine, two different synthetic
strategies could be adopted: the stereoselective synthesis of (+)-
lupinine or the total synthesis of racemic lupinine, followed by
the resolution of the two enantiomers.
It has been reported that (+)-lupinine can be obtained in seven
steps starting from (ꢀ)-pipecolic acid.11 Following this procedure
(+)-lupinine was obtained with an enantiomeric excess of
50–60%, which increased up to 80% by repeated fractional crystal-
lizations; thus, this synthetic route resulted low yielding and not
economically advantageous and it was, therefore, abandoned.
The total synthesis of racemic lupinine, followed by the resolu-
tion of the two enantiomers was then attempted.
In lipase catalyzed reactions, the type of acyl donor plays an
important role in the reaction kinetics (or in the selectivity reac-
tion).17–20 We have studied three different acyl donors, 4-nitro-
phenyl 3-[4-(trifluoromethyl)phenyl]propanoate,17 benzyl acetate
and vinyl acetate,21 using toluene as solvent. During the transeste-
rification reaction, the lipases (PS or AK) are initially acylated by
the acyl donor, and the acylated lipase then reacts enantioselec-
tively with an alcohol (Scheme 3).
As shown in Table 1, first we used 4-nitrophenyl 3-[4-(trifluoro-
methyl)phenyl]propanoate as acyl donor, but we obtained (+)-lup-
inine with only 71% of ee22
Using vinyl acetate as acyl donor, we performed enzymatic res-
olution yielding different ee depending by different incubation/
reaction times.

5982
C. Rusconi et al. / Bioorg. Med. Chem. 20 (2012) 5980–5985
O
R
H
N
O
OH
OH
H
H
N
O
a
+
+
OH
R
OR'
H
N
N
(_)-lupinine
(+)-lupinine
(_)-lupinine
R= CH3;
R'= vinyl; benzyl
R= pCF3-C6H4-CH2CH2 R'=p-nitrophenyl
OH
O
R
H
N
H
N
O
b
(+)-lupinine
Scheme 3. Reagents and conditions: (a) Amano Lipase, toluene; (b) 1 N NaOH in MeOH.
Table 1
Enzymatic resolution conditionsa
Lupinine ½a 2D0
ꢁ
Entry
Lipase
Acyl donor
Incubation time
Reaction time
ee%b
1
2
3
4
5
6
7
8
PS
PS
4-Nitrophenyl 3-[4-(trifluoromethyl)phenyl] propanoate
24 h
24 h
24 h
19 h
19 h
19 h
24 h
24 h
24 h
120 h
24 h
15 h
10 h
5 h
(+)14,9
(+)17,7
(+)13,3
(+)18,6
(+)18,6
(+)19.1
—
71%
82%
62%
87%
87%
89%
—
Vinyl acetate
Vinyl acetate
Vinyl acetate
Vinyl acetate
Vinyl acetate
Benzyl acetate
Benzyl acetate
AK
AK
AK
AK
PS
120 h
120 h
AK
—
—
a
a
For details see the Experimental Section.
Ee was calculated assuming the value (+)21.5 as 100%. A pure sample of natural (ꢀ)lupinine has ½a D20
ꢁ
ꢀ21.5 (C = 1, EtOH).22
Table 2
In vitro antimalarial and cytotoxic activities of AM1 racemate and its enantiomers
P. falciparum IC50 SDa (nM)
Cytotoxicity IC50 (nM)
b
Compound
D-10
3D7
W-2
Pf-Kenyac
HMECd
HDFe
( )-AM1
(ꢀ)-AM1
(+)-AM1
CQ
16.15 5.18
23.98 8.83
17.49 8.85
23.72 13.99
18.55 7.70
28.14 17.00
21.91 0.29
16.92 2.11
35.44 8.34
19.43 8.64
29.20 3.77
18.92 8.82
241.30 41.13
>60,000
>49,000
>60,000
>38,000
24,584
22,616
26,918
>38,000
53.67 22.72
35.77 16.94
437.62 93.47
a
b
c
pLDH method, mean of three different experiments.
MTT method.
P.falciparum strain isolated in Milan from a patient coming from Kenya, adapted to in vitro growth.
Human endothelium.
Human fibroblasts.
d
e
Esterification did not occur with benzylacetate as acyl donor, in
3. Results and discussion
presence of both lipases (PS and AK).
The best results (89% ee) were obtained after 19 h of incubation
of Lipase AK with vinyl acetate followed by 5 h of reaction with ( )-
lupinine.
Racemic AM1, (ꢀ)-AM1 and (+)-AM1 (ee >80%) were tested
in vitro against two CQ-R (W-2 and the Pf Kenia isolate) and two
CQ-S (D10–3D7) strains of P. falciparum. Their antimalarial activity
was quantified as inhibition of parasite growth23 and was com-
pared to the cytotoxicity24 on mammalian cells of human origin
(Table 2).
As shown, the antiplasmodial activities of ( )-AM1 and of the
two enantiomers against the four strains, expressed as IC50, were
in the range of 16–53 nM and were not significantly different each
To be noted that higher enantiomeric eccess (98.6%; ½a D20
ꢁ
+21.17) was obtained by submitting a second time the enriched
(+)-lupinine (½a D20
ꢁ
+13,4) to the resolution with lipase AK and vinyl
acetate (19 h of incubation and 7 h of reaction).
Finally (+)-AM1 was obtained starting from (+)-lupinine,
according to the same procedure used for (ꢀ)-AM1.10

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.

5984
C. Rusconi et al. / Bioorg. Med. Chem. 20 (2012) 5980–5985
The ester was hydrolyzed, with 6 ml of 1 N NaOH in MeOH, to
7.99 (d, J = 1.92 Hz, 1H); 7.68 (d, J = 9.08 Hz, 1H); 7.35 (dd,
J = 9.08, 2.20 Hz, 1H); 6.25 (d, J = 5.77 Hz, 1H); 3.63–3.57 (m, 1H);
give (+)-lupinine (150 mg, 63%). ½a D20
ꢁ
+13.4 (c = 1 in EtOH).
3.38–3.32 (m, 1H); 3.07–3.03 (m, 2H); 2.25–1.15 (m, 14H). HRMS
5.3.4. Experiment 4
(ESI) m/z calcd for C
19H25N3Cl [M+H]+: 330.17315; found:
Amano AK (18 mg) was added to a solution of racemic lupinine
(300 mg, 1.77 mmol) in 3 ml of toluene and the mixture was stir-
red for 19 h. Vinyl acetate (0.6 ml, 6.5 mmol) was added and the
mixture was stirred for 5 h at rt. After purification, were obtained
104 mg of ester (0.49 mmol) as a white solid.
330.17268; calcd for C19H24N3ClNa [M+Na]+: 352.15510; found:
352.15521.
5.4.2. (+)-7-Chloro-4-{N-[(1R,9aS)-(octahydro-2H-quinolizin-1-
yl)methyl]amino}-quinoline (+)-AM1
The ester was hydrolyzed with 1.6 ml of 1 N NaOH in MeOH, to
Crystallized from CH2Cl2. Yield 48%; mp 147–149 °C. ½a D20
ꢁ
+22.5
give (+)-lupinine (50 mg, 60%); ½a D20
ꢁ
+19.1 (c = 1 in EtOH).
(c = 0.98 in EtOH). 1H NMR (CDCl3): d 8.83 (s, 1H); 8.47 (d,
J = 5.51 Hz, 1H); 7.97 (d, J = 1.93 Hz, 1H); 7.67 (d, J = 9.08 Hz, 1H);
7.34 (dd, J = 9.08, 2.20 Hz, 1H); 6.24 (d, J = 5.78 Hz, 1H); 3.65–
3.50 (m, 1H); 3.40–3.20 (m, 1H); 3.10–2.95 (m, 2H); 2.30–1.00
(m, 14H); superimposable with a 1H NMR spectrum of pure sample
5.3.5. Experiment 5
Amano AK (20 mg) was added to a solution of racemic lupinine
(340 mg, 2 mmol) in 3 ml of toluene and the mixture was stirred
for 19 h. Vinyl acetate (0.68 ml, 7.4 mmol) was added and the mix-
ture was stirred for 15 h at rt. After purification, were obtained
224 mg of ester (1.06 mmol) as a white solid.
of (ꢀ)-AM1 (mp 142–144 °C; ½a D20
ꢀ27.8; c = 1 in EtOH). HRMS
ꢁ
(ESI) m/z calcd for C19H25N3Cl [M+H]+: 330.17315; found:
330.17264; calcd for C19H24N3ClNa [M+Na]+: 352.15510; found:
352.15544.
The ester was hydrolyzed with 5.3 ml of 1 N NaOH in MeOH, to
give (+)-lupinine (110 mg, 61%). ½a D20
ꢁ
+18.6 (c = 1 in EtOH).
5.5. Parasite cultures and drug susceptibility assay
5.3.6. Experiment 6
Amano AK (18 mg) was added to a solution of racemic lupinine
(300 mg, 2 mmol) in 3 ml of toluene and the mixture was stirred
for 19 h. Vinyl acetate (0.6 ml, 6.5 mmol) was added and the mix-
ture was stirred for 10 h at rt. After purification, were obtained
183 mg of ester (0.87 mmol) as a white solid.
Plasmodium falciparum cultures were carried out according to
Trager and Jensen with slight modifications.26 The CQ-sensitive,
strains D-10 and 3D7 and the CQ-resistant, strain W-2 and the
Pf-Kenia isolate27 were maintained at 5% hematocrit (human type
A-positive red blood cells) in RPMI 1640 (EuroClone, Celbio) med-
ium with the addition of 1% AlbuMax (Invitrogen, Milan, Italy),
0.01% hypoxantine, 20 mM Hepes, and 2 mM glutammine. The Pf
Kenia isolate derives from a patient with malaria returning from
Kenia to Milan and adapted to grow in culture. All the cultures
were maintained at 37 °C in a standard gas mixture consisting of
1% O2, 5% CO2, and 94% N2. Compounds were dissolved in either
water or DMSO and then diluted with medium to achieve the re-
quired concentrations (final DMSO concentration <1%, which is
non-toxic to the parasite). Drugs were placed in 96-well flat-bot-
tomed microplates (COSTAR) and serial dilutions made. Asynchro-
nous cultures with parasitaemia of 1–1.5% and 1% final hematocrit
were aliquoted into the plates and incubated for 72 h at 37 °C. Par-
asite growth was determined spectrophotometrically (OD650) by
measuring the activity of the parasite lactate dehydrogenase
(pLDH), according to a modified version of the method of Makler
in control and drug-treated cultures.23 The antimalarial activity is
expressed as 50% inhibitory concentrations (IC50); each IC50 value
is the mean and standard deviation of at least three separate exper-
iments performed in duplicate.7
The ester was hydrolyzed with 4.3 ml of 1 N NaOH in MeOH, to
give (+)-lupinine (95 mg, 60%). ½a D20
ꢁ
+18.6 (c = 1 in EtOH).
5.3.7. Experiment 7
Amano PS (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. Benzyl acetate (1.6 ml, 10.9 mmol) was added and
the mixture was stirred for 96 h at rt but the transesterification
did not occur.
5.3.8. Experiment 8
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. Benzyl acetate (1.6 ml, 10.9 mmol) was added and
the mixture was stirred for 96 h at rt but the transesterification
did not occur.
5.4. 7-Chloro-4-{N-[(1RS,9aRS)-octahydro-2H-quinolizin-1-
yl)methyl]amino}-quinoline (( )-AM1) and (+)-7-Chloro-4-{N-
[(1R,9aS)-(octahydro-2H-quinolizin-1-yl)methyl]amino}-
quinoline (+)-AM1. General method.
5.6. Cell toxicity assays
A
mixture of ( )-aminolupinane [(1RS,9aRS)-octahydro-2H-
The long-term human microvascular endothelial cell line
(HMEC-1) immortalized by SV 40 large T antigen28 was maintained
in MCDB 131 medium (Invitrogen, Milan, Italy) supplemented with
10% fetal calf serum (HyClone, Celbio, Milan, Italy), 10 ng/ml of epi-
quinolizine-1-methanamine], or (+)-aminolupinane ((1R, 9aS)-
octahydro-2H-quinolizine-1-methanamine), prepared, in accord-
ing to the method described by Sparatore F. et al,25 starting from
( )-lupinine or (+)-lupinine (0.60 g, 3.57 mmol), 4,7-dichloroquin-
oline (0.71 g, 3.58 mmol) and phenol (2.3 g) was heated for 4 h at
180 °C under N2. After cooling, the mixture was treated with 2 M
NaOH and the mixture was extracted with DCM (3 times). The
combined organic phases were dried and the solvent evaporated.
The residue was purified by silica gel chromatography (CH2Cl2/
CH3OH/conc.NH4OH; 90:9.7:0.3) and the solid crystallized from
the indicated solvent.
dermal growth factor (Chemicon), 1
2 mM glutamine, 100 U/ml of penicillin, 100 l
l
g/ml of hydrocortisone,
g/ml of streptomy-
l
cin, and 20 mM Hepes buffer (EuroClone). Human dermal fibro-
blasts (HDF) were maintained in DMEM medium (EuroClone)
supplemented with 10% fetal calf serum, 2 mM glutamine, 100
units/ml of penicillin, 100 lg/ml of streptomycin. Unless stated
otherwise, all reagents were from Sigma Italia, Milan, Italy. For
the cytotoxicity assays, cells were treated with serial dilutions of
test compounds and cell proliferation evaluated using the MTT as-
say already described.24 Plates were incubated for 72 h at 37 °C in
5.4.1. 7-Chloro-4-{N-[(1RS,9aRS)-octahydro-2H-quinolizin-1-
yl)methyl]amino}-quinoline (( )-AM1)
5% CO2, then 20 lL of a 5 mg/ml solution of 3-(4,5-dimethylthia-
Crystallized from abs. EtOH/Et2O. Yield 55%; mp 168.5–
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (M-2128 Sigma)
in PBS was added for an additional 3 h at 37 °C. The plates were
170.6 °C. 1H NMR (CDCl3): d 8.90 (s, 1H); 8.46 (d, J = 5.51 Hz, 1H);

C. Rusconi et al. / Bioorg. Med. Chem. 20 (2012) 5980–5985
5985
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2011, 18, 207.
then centrifuged, the supernatants discarded and the dark blue for-
mazan crystals dissolved using 100 L of lysing buffer consisting of
l
20% (w/v) of a solution of SDS (Sigma), 40% of N,N-dimethylform-
amide (Merck) in H2O, at pH 4.7 adjusted with 80% acetic acid.
The plates were then read on a microplate reader (Molecular De-
vices Co., Menlo Park, CA, USA) at a test wavelength of 550 nm
and a reference wavelength of 650 nm. The results are expressed
as IC50, which is the dose of compound necessary to inhibit cell
growth by 50%. All the tests were performed in triplicate at least
three times.
7. Sparatore, A.; Basilico, N.; Parapini, S.; Romeo, S.; Novelli, F.; Sparatore, F.;
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Part of this manuscript was generated in the context of the
AntiMal project, funded under the 6th Framework Programme of
the European Community (Contract No. IP-018834). The authors
are solely responsible for its content, it does not represent the
opinion of the European Community, and the Community is not
responsible for any use that might be made of the information con-
tained therein. The financial support from the University of Milan
(PUR 2008) and from MIUR (PRIN2008, 20084MMXN , Leads ad
Attività Antimalarica di Origine Naturale, Isolamento, Ottimizzazi-
one e Valutazione Biologica) is also acknowledged.
22. Enantiomeric eccess of (+)-lupinine was calculated assuming the value
+21.5 = 100% ee. A pure sample of natural (ꢀ)-lupinine has ½a D20
ꢀ21.5 (C = 1,
ꢁ
We thank the Associazione Volontari Italiani Sangue (AVIS
Comunale Milano) for providing fresh red blood cells for P. falcipa-
rum growth And Dr. R. Grande (IRCCS, Policlinico, Milano) for pro-
viding the fresh isolate of P. falciparum.
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