Short synthesis and antimalarial activity of fagaronine

Article abstract of DOI:10.1016/j.bmc.2012.05.061

Herein, we report a new synthesis of fagaronine 1, inspired by the synthesis reported by Luo for nornitidine. The in vitro biological activity of fagaronine against malaria on several chloroquine-sensitive and resistant Plasmodium falciparum strains was c

Full text of DOI:10.1016/j.bmc.2012.05.061

Bioorganic & Medicinal Chemistry 20 (2012) 4856–4861
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Short synthesis and antimalarial activity of fagaronine
M. Rivaud a,b, A. Mendoza a,b,c, M. Sauvain a,d, A. Valentin a,b, V. Jullian a,b,
⇑
a Université de Toulouse, UPS, UMR 152 (Laboratoire Pharmadev), Faculté de Pharmacie, 35, chemin des maraîchers, F-31062 Toulouse cedex 9, France
b Institut de Recherche pour le Développement (IRD), UMR 152 (Laboratoire Pharmadev), 118, rte de Narbonne, F-31062 Toulouse cedex 9, France
c Unidad en Investigación y Desarrollo de Medicamentos, Centro de Investigación en Farmacobiologia Aplicada (CIFA), Universidad de Navarra, c/Irunlarrea s/n, 31080 Pamplona, Spain
d Institut de Recherche pour le Développement (IRD), UMR 152, Mission IRD Casilla 18-1209, Lima, Peru
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we report a new synthesis of fagaronine 1, inspired by the synthesis reported by Luo for norni-
tidine. The in vitro biological activity of fagaronine against malaria on several chloroquine-sensitive
and resistant Plasmodium falciparum strains was confirmed, and the selectivity index compared to mam-
malian cells was calculated. Fagaronine was found to have very good antimalarial activity in vivo, com-
parable to the activity of the reference compound chloroquine. Therefore, fagaronine appears to be a good
potential lead for the design of new antimalarial molecules.
Received 8 February 2012
Revised 22 May 2012
Accepted 25 May 2012
Available online 5 June 2012
Keywords:
Fagaronine
Ó 2012 Elsevier Ltd. All rights reserved.
Benzo[c]phenanthridine
Synthesis
Malaria
In vivo experiment
1. Introduction
OH
Malaria is one of the world’s most widespread and deadliest
diseases, having caused 655,000 deaths in 2010.1 For many years,
chloroquine and its derivatives have been used successfully to cure
people, but the rapid development of resistant strains has led
scientists to search for new and more efficient treatments. Tradi-
tionally, plants have been used to treat malaria, and ethnopharma-
cological studies followed by phytochemical investigations have
led to the discovery of the in vitro antiplasmodial activity of many
molecules. Unfortunately, these molecules are often not further
investigated because there is a lack of in vivo experiments to assess
their potential as antimalarial drugs.
Fagaronine 1 (Fig. 1) was first isolated from Fagara zanthoxy-
loïdes2 and found to have in vitro antiplasmodial activity
against a chloroquine-sensitive strain of Plasmodium falciparum.3
Other benzo[c]phenanthridines showed antiplasmodial activity
in vitro.4–6 To the best of our knowledge, these studies are the only
literature reports on the antiplasmodial activity of benzo[c]phe-
nanthridine. Many other biological activities for fagaronine have
been reported: antileukaemic activity in vivo on L1210 and P-
388 mouse leukaemia7,8 and in vitro on K-562 human leukaemia
cells,9,10 as well as topoisomerase 1 and 2 inhibition.11 Because
fagaronine showed an activity on P. falciparum in the nanomolar
H3CO
H3CO
OCH3
I
N
CH3
1
Figure 1. Structure of fagaronine iodide 1.
range, we decided to evaluate its potential as an antimalarial drug
by performing in vivo experiments.
Many total syntheses or hemisyntheses of fagaronine have been
published,12–18 but a concise route to this benzo[c]phenanthridine
is still needed. A short synthesis of nornitidine from commercially
available reagents was published by Luo et al.19 We decided to
adapt it to this synthetic route for the synthesis of fagaronine.
2. Results and discussion
2.1. Chemistry
The method employed for the synthesis of fagaronine 1 is out-
lined in Scheme 1.
First, 2-bromobenzaldimine 3 was prepared in quantitative
yield by treatment of the commercially available aldehyde 2 with
tert-butylamine. The alkynes 5a and 5b were prepared by applying
acetate or isopropyl protecting groups, respectively, to the com-
mercially available bromide 4 followed by a Sonogashira reaction
⇑
Corresponding author. Tel.: +33 (0)5 62 25 68 23; fax: +33 (0)5 62 25 98 02.
E-mail address: jullian@cict.fr (V. Jullian).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.061

M. Rivaud et al. / Bioorg. Med. Chem. 20 (2012) 4856–4861
4857
OR
H3CO
H3CO
H3CO
H3CO
H3CO
OH
N
O
NtBu
(a)
OH
OR
Br
Br
H3CO
H3CO
3
H3CO
H3CO
2
(d)
OCH3
+
N
6a-1
6a-2
OH
R = Ac
6b-1 R= iPr
R = Ac
H3CO
HO
Br
6b-2 R= iPr
H3CO
RO
(b),(c)
(e)
4
5a
R= Ac
5b R=iPr
O
N
OR
OCH3
OH
OCH3
OH
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
OCH3
(g)
N
X
N
CH3
(f)
1
7a
R = Ac
8
7b R= iPr
O
OR
OCH3
(h)
(i)
H3CO
H3CO
I
N
CH3
9a
R = Ac
9b R= iPr
Scheme 1. Synthesis of fagaronine 1. (a) tBuNH2, rt; (b) AcCl, CH2Cl2, NEt3, 0 °C or iPrBr, K2CO3, DMF, 80 °C; (c) butyn-1-ol, Pd(PPh3)2Cl2, CuI, DIPA, reflux; (d) Ni(dppe)Br2, Zn
powder, ACN, reflux; (e) oxalyl chloride, DMSO, DIPEA, CH2Cl2, À78 °C; (f) HBr 40%, CH3COOH; (g) methylmethanesulfonate, sealed tube, 180 °C; (h) CH3I, CH2Cl2; (i) HBr 40%,
CH3COOH, reflux.
with overall 70% yield for 5a and 76% yield for 5b. We also showed
that protection of the phenol during the Sonogashira reaction was
necessary: the best yield with unprotected phenol 4 was 23%.
The key step of this synthesis is a nickel-catalysed annulation
reaction between alkyne 5a or 5b and the bromobenzaldimine 3.
The annulation of 2-bromobenzaldimine 3 with alkyne 5a using
NiBr2(dppe)/Zn as the catalyst afforded isoquinoline 6a-1 and its
regioisomer 6a-2 with a global yield of 92% and a 6a-1/6a-2 ratio
of 5/4. The same reaction with alkyne 5b proceeded with a global
yield of 70% and a 6b-1/6b-2 ratio of 75/25. The separation of the
regioisomers from each other proved to be difficult and required
successive chromatographies on silica gel to obtain isolated 6a-1
and 6b-1 with sufficient purity. After optimisation, a more efficient
purification process was finally found for compound 6b-1, result-
ing in 73% yield using medium pressure column chromatography
on silica gel.
ratio of 6/4, which was not in agreement with Luo’s yield of 73%
for 6c-1 alone. We have no explanation for this discrepancy. The
regioselectivity of this annulation reaction is discussed by Larock20
who described an isoquinoline synthesis via a palladium-catalysed
reaction between an imine and an alkyne; high regioselectivity
was achieved with unsymmetrical alkynes when the bulkiest sub-
stituent was in the a-position relative to the nitrogen atom on the
quinoline nucleus. However, they report a lower regioselectivity
when the reaction involved an electron-rich imine-like imine 3.
Korivi and Cheng21 described this reaction with nickel catalysis
and reported the same regiochemistry when phenyl substituted
alkynes are used.
The oxidation of 6a-1 and 6b-1 under Swern conditions, and
the cyclisation of the aldehydes to produce norfagaronine,
proceeded in good yields. Optimisation of the methylation of nor-
fagaronine was undertaken using methyl iodide, dimethylsulfate
and methylmethanesulfonate. Conditions with methylmethane-
sulfonate gave the best results: In a sealed tube at 180 °C, methyl-
ation occurred with 49% yield, and subsequent purification of
As Luo did not mention the regioselectivity of this annulation
reaction, we tried to reproduce the reaction described in his paper
(Scheme 2). We obtained a global yield of 86% with a 6c-1/6c-2
O
O
H3CO
NtBu
OH
OH
O
O
H3CO
Br
H3CO
H3CO
H3CO
H3CO
3
(d)
+
N
N
+
6c-1
6c-2
OH
O
O
5c
Scheme 2. Annulation reaction described by Luo.19

4858
M. Rivaud et al. / Bioorg. Med. Chem. 20 (2012) 4856–4861
Table 2
fagaronine from the reaction mixture was tedious. As N-methyla-
tion of the quinoline nucleus with methyl iodide was widely re-
ported in the literature, we decided first to methylate quinolines
7a and 7b, and then to allow the cyclisation of the methylated
aldehydes 9a and 9b to offer fagaronine. Methylation of 7a and
7b with methyl iodide in dichloromethane at room temperature
was quantitative. During the cyclisation of 9a and 9b into fagaro-
nine with HBr in refluxing acetic acid, fagaronine iodide 1 precip-
itated from the reaction mixture and could be easily isolated and
purified by filtration and precipitation from methanol with 40%
yield from 9a and 34% yield from 9b. These yields are rather low,
but the filtrates and mother liquors were also essentially fagaro-
nine as a mixture of bromide and iodide salts as shown by 1H
NMR and mass spectrometry analyses. Fagaronine obtained from
9a showed purity higher than 95% (1H NMR). Fagaronine obtained
from 9b showed purity higher than 80% (1H NMR and HPLC).
Therefore, the use of acetate as a protective group afforded fagaro-
nine of higher purity.
We successfully adapted Luo’s nornitine synthesis to achieve
the synthesis of fagaronine in a few steps. We noted that the use
of an isopropyl protecting group on the phenol moiety allowed
for both a better regioselectivity in the annulation step and an eas-
ier purification of the desired regioisomer. The use of an even bulk-
ier protecting group (tertbutyl or cyclohexyl) should potentially
improve the regioselectivity of the annulation reaction, which is
the limiting step of this short synthesis.
In vivo activity of fagaronine iodide (F) in
evaluation of the survival time
a 4 day suppressive test (ED50) and
Treatment
n
Mean
parasitaemia SD
Inhibition (%)
Mean
survival (day)
Control
5
5
5
5
5
5
5
37.0 8.6
1.9 2.5
35.8 7.1
2.3 3.3
16.7 10.1
27.0 13.7
35.2 7.8
9.5
>13
9.5
CQ 10 mg/kg/d
CQ 1 mg/kg/d
F 10 mg/kg/d
F 5 mg/kg/d
F 2.5 mg/kg/d
F 1 mg/kg/d
94.8
3.2
91.5
38.1
27.0
4.8
>13
10.7
11.5
8.6
Purity of fagaronine: >80%.
was evaluated and compared to the control. The doses that inhib-
ited 50% of the parasitaemia (ED50) were 6 mg/kg/day for fagaro-
nine and 5.5 mg/kg/day the positive control chloroquine. The
survival of the mice was also followed for 13 days after infection.
Our studies have shown that fagaronine iodide is as potent as chlo-
roquine for the inhibition of parasitaemia. We have also shown
that the effect on the survival of mice was the same for chloroquine
and fagaronine at 10 mg/kg/day (Table 2). Furthermore, no sign of
acute toxicity was observed for fagaronine at the highest dose of
10 mg/kg/day. In previous studies of fagaronine and its in vivo
activity against P388 leukaemia in mice, no toxicity was detected
at a dose as high as 50 mg/kg (single injection).8
3. Conclusions
2.2. Biology
In this study, we showed that fagaronine, a known antiplasmo-
dial molecule, was as active as chloroquine against murine malaria
in vivo. These results confirm the high potential of fagaronine as an
antimalarial drug. Further studies are underway in the laboratory
to understand the mechanism of action. Furthermore, we showed
that the synthesis proposed by Luo et al. for nornitidine could be
adapted to a short synthesis of fagaronine.
The antiplasmodial activity of fagaronine iodide was evaluated
for three strains of P. falciparum with different sensitivities to chlo-
roquine (Table 1). The IC50 values for fagaronine ranged from 7.4 to
14.9 nM and were better than the values of the reference com-
pound, chloroquine. These values were independent of the sensi-
tivity of these strains to chloroquine. Our findings were in
agreement with the first report of antiplasmodial activity of fagar-
onine extracted from Fagara zanthoxyloides by Kassim, which de-
fines an IC50 of 18 ng/ml against P. falciparum 3D7.3 Nyangulu,
however, reports a weaker activity of fagaronine mesylate, with
4. Experimental part
4.1. Chemistry
an IC50 of 2.3 and 1.8 lM against P. falciparum K39 and V1/S strains,
respectively.4 This discrepancy with our results may be due to the
counter ion, mesylate, which may alter the solubility of fagaronine.
As fagaronine is known for its cytotoxic activity, we also tested
the compound on Vero cells, a non-cancerous mammalian cell line,
to determine its antiplasmodial selectivity. As fagaronine showed
no toxicity on these healthy mammalian cells, we performed an
in vivo antimalarial assay on mice infected with P. vinckei petteri,
following the classic Peter’s four days suppressive test protocol.22
Mice were infected at day 0, and then treated intraperitoneally
each day from day 0 to 3. At day 4, the parasitaemia of each mouse
The solvents used were of analytical grade (Fisher Scientific, Ill-
kirch, France). Acetonitrile was distiled over CaH2. Flash column
chromatography was carried out on 40–63 lm silica (Geduran,
Merck). Medium pressure chromatography was carried out using
a Buchi 688 isocratic pump and a Büchi C-690 glass column packed
with 6–35 lm silica (Merck). Thin layer chromatographs (TLC)
were carried out on silica gel 60F254 (Merck), and the compounds
were detected with UV light at 254 nm. Mass spectra were re-
corded on a Finnigan LCQ MS ion trap spectrometer (Thermo-
Fisher) with ESI ionisation. High resolution mass spectra were
recorded on a spectrometer Xevo G2 Q-TOF (Waters) with ESI
ionisation or on a GCT Premier CAB109 with DCI/CH4 ionisation.
NMR spectra were recorded on a Bruker Avance 300 or a Bruker
400 AMX spectrometer. Infra-red spectra were recorded on a
Paragon 1000 FT-IR (Perkin-Elmer) with compounds deposed on
a NaCl plate. HPLC analysis was performed using a C18 Luna
Table 1
In vitro activity (IC50 in nM) of tested drugs on P. falciparum and vero cells (mean
values of two independent experiments performed in triplicate)
Cell lines
Fagaronine iodide
Chloroquined
Doxorubicin
3D7
FcB1
FcM29
RIa
7.6 1.8
14.9 0.2
7.4 0.9
1.0
32c
NT
NT
NT
170 14c
240 21c
3 lm, 100A column (Phenomenex), a Dionex P680 pump and a
Dionex UVD170U UV-detector.
Vero
>21,000
>1400
>50 000
>194
6400 900
SIb
4.1.1. 4-(4-Acetoxy-3-methoxyphenyl)-3-butyn-1-ol (5a)
Acetyl chloride (1 ml, 13.97 mmol) was added dropwise to a
solution of 4-bromoguaiacol 4 (2.58 g, 12.7 mmol) into CH2Cl2
(15 ml) at 0 °C under argon. Et3N (2 ml) was added, and the mix-
ture was allowed to warm to room temperature under stirring over
one hour. The reaction was stopped with the addition of distiled
Purity of fagaronine: >95%.
a
Resistance index: 3D7/FcM29.
Selectivity index: Vero/FcB1.
3D7, CQsensitive, FcB1 and FcM29: CQ resistant.
Evaluated every two months.
b
c
d

M. Rivaud et al. / Bioorg. Med. Chem. 20 (2012) 4856–4861
4859
water (10 ml). The organic phase was then washed with HCl 0.01 N
(10 ml), saturated NaHCO3 (10 ml) and distiled water (10 ml). The
organic layer was dried over MgSO4, filtered and concentrated to
give 2.97 g of acetyl-bromoguaiacol in quantitative yield.
m/z Calcd for C22H24NO6 [MH+]: 398.1604. Found: 398.1604 IR:
1199, 1219; 1258; 1581; 1622; 1759.4; 2900–3554 cmÀ1
.
Compound 6a-2 1H NMR (300 MHz, CDCl3): d = 2.39 (s, 3H); 3.01
(t, J = 7.2 Hz, 2H); 3.80 (s, 3H); 3.84 (s, 3H); 3.97 (t, J = 7.3 Hz, 2H);
4.05 (s, 3H); 6.67 (se, 1H); 6.88 (dd, J = 7.4, 1.8 Hz, 1H); 6.90 (bs,
1H); 7.21 (d, J = 7.4 Hz, 1H); 7.28 (s, 1H), 9.08 (s, 1H). 13C NMR
(75 MHz, CDCl3): d = 20.7 (CH3); 35.8 (CH2); 56.0 (CH3); 56.1
(CH3); 56.2 (CH3); 62.3 (CH2); 103.4 (CH); 105.4 (CH); 114.0
(CH); 122.2 (CH); 123.0 (C); 123.3 (CH); 130.2 (C); 133.1 (C);
135.6 (C); 139.4 (C); 148.4 (CH); 149.0 (C); 150.2 (C); 151.4 (C);
153.8 (C); 168.9 (C). HRMS (DCI/CH4): m/z Calcd for C22H24NO6
[MH+]: 398.1604. Found: 398.1592. IR: 1257; 1368; 1427; 1468;
1504; 1582; 1621; 1736; 1765; 2039; 2852; 2933; 3006;
Acetyl-bromoguaiacol (856 mg, 3.5 mmol) was reacted with
and 3-butyn-1-ol following a previously published protocol.19
The crude mixture was purified by silica gel column chromatogra-
phy (ethyl acetate/cyclohexane:4/6) to afford 579 mg (69% yield)
of compound 5a (oil). 1H NMR (300 MHz, CDCl3): d = 2.32 (s, 3H);
2.69 (t, J = 6.3 Hz, 2H); 3.80–3.84 (m, 5H); 6.96 (d, J = 9 Hz, 1H);
7.01–7.04 (m, 2H). 13C NMR (75 MHz, CD3OD): d = 19.1 (CH3);
22.8 (CH2); 55.0 (CH3); 60.3 (CH2); 80.6 (C); 86.6 (C); 115.3 (CH);
122.4 (C); 122.5 (CH); 123.8 (CH); 139.6 (C); 150.9 (C); 169.2 (C).
MS (EI) m/z: 234 [M+]. HRMS (+ESI): m/z Calcd for C13H15O4
[MH+]: 235.0907. Found: 235.09071. IR: 1015; 1034; 1165; 1211;
1266; 1405; 1598; 1765; 2235; 2940; 3412.
3410 cmÀ1
.
4.1.4. 6,7-Dimethoxy-4-(2-hydroxyethyl)-3-(4-isopropyloxy-3-
methoxyphenyl)-isoquinoline (6b-1) and 6,7-dimethoxy-3-(2-
hydroxyethyl)-4-(4-isopropyloxy-3-methoxyphenyl)
isoquinoline (6b-2)
4.1.2. 4-(4-Isopropyloxy-3-methoxyphenyl)-3-butyn-1-ol (5b)
Isopropyl bromide (1.39 ml, 14.77 mmol) and anhydrous K2CO3
(2.04 g, 14.77 mmol) were added to a solution of 4-bromoguaiacol
4 (2.00 g, 9.85 mmol) in anhydrous DMF (10 ml). The mixture was
stirred at 90 °C for 7 h, and then stirred at room temperature over-
night, after which 100 ml of ethyl acetate and 100 ml of water were
added. The two phases were separated and the aqueous phase was
extracted again with 50 ml of ethyl acetate. The organic phases
were pooled together and washed with 100 ml of HCl (0.05 M)
and 100 ml of water. The organic phase was dried over anhydrous
MgSO4, and the solvent was evaporated under reduced pressure.
The crude compound was purified by flash column chromatogra-
phy on silica gel (elution with 90/10 cyclohexane/ethyl acetate)
to afford 2.43 g of isopropyl-bromoguaiacol in quantitative yield.
Isopropyl-bromoguaiacol (2.43 g) was reacted with and 3-
butyn-1-ol following a previously published protocol.19 The crude
mixture was purified by flash column chromatography on silica gel
(ethyl acetate/cyclohexane:25/75 then 50/50) to afford 1.76 g (76%
yield) of compound 6b (oil). RMN 1H (300 MHz, CDCl3): d = 1.39 (d,
J = 6 Hz, 6H); 2.70 (t, 2H, J = 6.3 Hz); 3.83 (m, 2H); 3.86 (s, 3H); 4.56
(sept, J = 6 Hz, 1H); 6.82 (d, 1H, J = 8.2 Hz); 6.95 (d, 1H, J = 1.8 Hz);
7.00 (dd, 1H, J = 8.2 Hz, J = 1.8 Hz). 13C NMR (75 MHz, MeOD):
d = 21.0 (CH3); 22.9 (CH2); 55.0 (CH3); 60.8 (CH2); 71.5 (CH); 81.5
(C); 84.9 (C); 115.3 (CH); 115.7 (CH); 116.8 (C); 124.7 (CH);
146.9 (C); 150.3 (C). MS (EI): m/z = 234 [M+]. HRMS (+ESI): m/z
Calcd for C14H18O3 [MH+]: 235.1336. Found: 235.1334. IR: 1037;
Imine
3 (885 mg, 2.73 mmol) and alkyne 5b (830 mg,
3.55 mmol) were reacted according to a previously published pro-
tocol.19 The crude mixture was purified by flash column chroma-
tography on silica gel (ethyl acetate/dichlomethane:5/5 and 7/3)
to afford 185 mg of compound 6b-1 (amorphous solid), and
562 mg of a mixture of 6b-1 and 6b-2. The analysis by 1H NMR
spectroscopy of the mixture showed a 6b-1/6b-2 ratio of 67/33
(70% total yield, 6b-1/6b-2 ratio 75/25). From this mixture, 6b-2
(amorphous solid) could be purified by precipitation with dichloro-
methane for analytical purpose.
An optimisation of the purification method for compound 6b-1
was achieved. The crude mixture of the reaction of imine 3 (1.90 g;
6.31 mmol) with alkyne 5b (1.92 g, 8.20 mmol) was submitted to
medium pressure column chromatography on silica gel eluted with
acetone/cyclohexane 60/40 to give 1.84 g (73% yield) of 6b-1.
Compound 6b-1 1H NMR (400 MHz, CDCl3): d = 1.38 (t, J = 6.0 Hz,
6H); 3.31 (t, J = 7.1 Hz, 2H); 3.86 (s, 3H); 3.87 (t, J = 7.1 Hz, 2H);
4.04 (s, 3H); 4.05 (s, 3H); 4.55 (sept, J = 6.0 Hz, 1H); 6.93 (d,
J = 8.1 Hz, 1H); 6.99 (dd, J = 8.1, 1.8 Hz, 1H); 7.06 (d, J = 1.8 Hz,
1H); 7.21 (s, 1H); 7.36 (s, 1H); 8.93 (s, 1H). 13C NMR (100 MHz,
CDCl3): d = 22.1 (CH3); 32.2 (CH2); 56.0 (CH3); 56.1 (CH3); 56.2
(CH3); 62.7 (CH2); 71.5 (CH); 102.3 (CH); 105.9 (CH); 113.5 (CH);
115.4 (CH); 121.7 (CH); 123.7 (C); 132.6 (C); 134.2 (C); 146.9 (C);
147.7 (CH); 150.0 (C); 150.1 (C); 151.7 (C); 153.3 (C). HRMS
(+ESI): m/z Calcd for
C
23H28NO5 [MH+]: 398.1967. Found:
1136; 1172; 1109; 1264; 1412; 1596; 2935; 2976; 3421 cmÀ1
.
398.1962. IR: 1138; 1167; 1259; 1581; 1622.8; 2900–3554 cmÀ1
.
Compound 6b-2 1H NMR (400 MHz, CDCl3): d = 1.45 (t, J = 6.1 Hz,
6H); 3.25–3.30 (m, 2H); 3.79 (s, 3H); 3.84 (s, 3H); 4.03 (s, 3H);
4.19–4.24 (m, 2H); 4.64 (sept, J = 6.1 Hz, 1H); 6.70 (s, 1H); 6.76
(d, J = 1.9 Hz, 1H); 6.77 (dd, J = 8.8, 1.9 Hz, 1H); 7.03 (d, J = 8.8 Hz,
1H), 7.36 (s, 1H), 9.13 (s, 1H). 13C NMR (75 MHz, CDCl3): d = 22.1
(CH3); 22.2 (CH3); 33.3 (CH2); 56.2 (CH3); 56.5 (CH3); 64.2 (CH2);
71.5 (CH); 103.9 (CH); 106.1 (CH); 113.2 (CH); 115.4 (CH); 122.2
(CH); 123.3 (C); 127.9 (C); 133.0 (C); 135.2 (C); 146.4 (C); 147.4
(C); 148.8 (CH); 150.7 (C); 151.2 (C); 155.6 (C). MS (+ESI) m/z:
398 [MH+]. HRMS (DCI/CH4): m/z Calcd for C23H28NO5 [MH+]:
398.1967. Found: 398.1977. IR: 1275; 1315; 1366; 1424; 1471;
1502; 1579; 1545; 1620; 1734; 1879; 2016; 2928; 2948; 2971;
4.1.3. 3-(4-Acetoxy-3-methoxyphenyl)-6,7-dimethoxy-4-(2-
hydroxyethyl)isoquinoline (6a-1) and 4-(4-acetoxy-3-methoxy-
phenyl)-6,7-dimethoxy-3-(2-hydroxyethyl) isoquinoline (6a-2)
Imine
3 (207 mg, 0.69 mmol) and alkyne 5a (210 mg,
0.89 mmol) were reacted according to a previously published pro-
tocol.19 The crude mixture was purified by flash column chroma-
tography on silica gel (ethyl acetate/dichloromethane:7/3) to
afford 60 mg of compound 6a-1 (amorphous solid), 136 mg of a
mixture of 6a-1 and 6a-2, and 60 mg of 6a-2 (oil). The mixture of
6a-1 and 6a-2 was then further purified to afford 75 mg of 6a-1
and 60 mg of 6a-2 (92% total yield, 6a-1/6a-2 ratio 5/4).
Compound 6a-1 1H NMR (300 MHz, CDCl3): d = 2.33 (s, 3H); 2.43
(s br, 1H); 3.27 (t, J = 7.2 Hz, 2H); 3.82 (t, J = 7.3 Hz, 2H); 3.83 (s,
3H); 4.04 (s, 3H); 4.05 (s, 3H); 7.02–7.08 (m, 2H); 7.15 (d,
J = 0.9 Hz, 1H); 7.21 (s, 1H); 7.35 (s, 1H); 8.92 (s, 1H). 13C NMR
(75 MHz, CDCl3): d = 20.7 (CH3); 32.2 (CH2); 55.9 (CH3); 56.0
(CH3); 56.1 (CH3); 62.5 (CH2); 102.3 (CH); 105.9 (CH); 113.9
(CH); 121.7 (CH); 122.3 (CH); 123.9 (C); 132.3 (C); 139.2 (C);
140.3 (C); 147.9 (CH); 150.0 (C); 150.7 (C); 151.3 (C); 153.2 (C);
169.1 (C). MS (+ESI) m/z: 398 [MH+]; 356 [MH+-Ac]. HRMS (+ESI):
3430 cmÀ1
.
4.1.5. 3-(4-Acetoxy-3-methoxyphenyl)-6,7-dimethoxy-4-(2-
oxoethyl)isoquinoline (7a)
Alcohol 6a-1 (320 mg, 0.805 mmol) was reacted under Swern
conditions.19 The crude compound was purified by flash column
chromatography on silica gel (ethyle acetate/cyclohexane:7/3) to
produce 251 mg (77% yield) of compound 7a (amorphous solid).
1H NMR (300 MHz, CDCl3): d = 2.33 (s, 3H); 3.86 (s, 3H); 4.04 (s,

4860
M. Rivaud et al. / Bioorg. Med. Chem. 20 (2012) 4856–4861
3H); 4.07 (s, 3H); 4.15 (d, J = 1.7 Hz, 2H); 7.00 (s, 1H); 7.02 (dd,
J = 8.0, 1.8 Hz, 1H); 7.12 (d, J = 8.0 Hz, 1H); 7.14 (d, J = 1.8 Hz,
1H); 7.28 (s, 1H); 9.06 (s, 1H); 9.84 (t, J = 1.68 Hz, 1H). 13C NMR
(75 MHz, CDCl3): d = 20.7 (CH3); 45.2 (CH2); 55.9 (CH3); 56.1
(CH3); 56.1 (CH3); 101.9 (CH); 106.0 (CH); 113.8 (CH); 118.2 (C);
121.6 (CH); 122.6 (CH); 123.8 (C); 132.5 (C); 139.5 (C); 139.7 (C);
149.2 (CH); 150.3 (C); 151.1 (C); 152.1 (C); 153.7 (C); 168.9
(C); 199.2 (C). MS (ESI) m/z: 396 [MH+]; 354 [MH+-Ac]. HRMS
4.1.9. 6,7-Dimethoxy-3-(4-isopropyloxy-3-methoxyphenyl)-2-
methyl-4-(2-oxoethyl)isoquinolinium iodide (9b)
A solution of compound 7b (100 mg, 0.253 mmol) in CH2Cl2
(10 mL) was stirred for 24 h at room temperature with 160 ll of
methyl iodide (360 mg, 2.53 mmol). Then, methyl iodide and
CH2Cl2 were both removed under vacuum to afford 135 mg of pure
compound 9b in quantitative yield (orange oil). 1H NMR (300 MHz,
CDCl3): d = 1.45 (d, J = 6.0 Hz, 6H); 3.87 (s, 3H); 4.08 (s, 3H); 4.15 (s,
3H); 4.17 (s, 3H); 4.25 (bs, 2H); 4.65 (m, 1H); 6.86 (dd, J = 8.1,
2.0 Hz, 1H); 6.95 (d, J = 2.0 Hz, 1H); 7.03 (d, J = 8.1 Hz, 1H); 7.13
(s, 1H); 8.07 (s, 1H); 9.84 (s, 1H); 10.37 (s, 1H). 13C NMR
(75 MHz, CDCl3): d = 22.1 (CH3); 45.3 (CH2); 47.6 (CH3); 56.5
(CH3); 57.4 (CH3); 57.9 (CH3); 71.5 (CH); 102.9 (CH); 108.3 (CH);
112.5 (C); 114.7 (CH); 121.6 (CH); 122.7 (C); 123.5 (C); 127.2 (C);
135.7 (C); 144.2 (C); 146.1 (CH); 149.5 (C); 150.9 (C); 152.7 (C);
158.7 (C); 197.1 (CH). MS (+ESI) m/z: 350 [M-Ac]+, 410 [M]+. HRMS
(+ESI): m/z Calcd for C24H28NO5 [M] +: 410.1967. Found: 410.1965.
(+ESI): m/z Calcd for
396.1452. IR: 1199; 1215; 1259; 1580; 1621; 1719; 1764;
2938 cmÀ1
C
22H22NO6 [MH+]: 396.1447. Found:
.
4.1.6. 6,7-Dimethoxy-3-(4-isopropyloxy-3-methoxyphenyl)-4-
(2-oxoethyl)isoquinoline (7b)
Alcohol 6b-1 (400 mg, 1.04 mmol) was reacted under Swern
conditions.19 The crude compound was purified by flash column
chromatography on silica gel (acetone/dichloromethane: 3/7) to
afford 290 mg (72% yield) of compound 7b (amorphous solid). 1H
NMR (300 MHz, CDCl3): d = 1.41 (d, J = 6.0 Hz, 6H); 3.88 (s, 3H);
4.02 (s, 3H); 4.05 (s, 3H); 4.14 (d, J = 2.1 Hz, 2H); 4.60 (sept,
J = 6.0 Hz, 1H); 6.97 (m, 2H); 7.00 (m, 1H); 7.06 (m, 1H); 7.28 (s,
1H); 9.07 (s, 1H); 9.82 (t, J = 2.1 Hz, 1H). 13C NMR (75 MHz, CDCl3):
d = 21.8 (CH3); 45.5 (CH2); 56.0 (CH3); 56.1 (CH3); 56.1 (CH3); 71.6
(CH); 101.8 (CH); 106.1 (CH); 113.4 (CH); 115.4 (CH); 118.1 (C);
121.8 (CH); 123.6 (C); 132.7 (C); 133.6 (C); 147.3 (C); 148.9 (CH);
150.2 (C); 150.3 (C); 152.6 (C); 153.8 (C); 199.7 (C). MS (+ESI) m/
z: 396 [MH+]; 428 [MH+ + MeOH]. HRMS (+ESI): m/z Calcd for
IR 1141; 1175; 1267; 1508; 1615; 1718; 2835; 2977 cmÀ1
.
4.1.10. Fagaronine iodide (1)
To a solution of compound 9a (95 mg, 0.177 mmol) in AcOH
(3 ml) was added 0.77 ml of HBr (40%), and the mixture was stirred
under reflux for 1 h. The mixture was allowed to cool to room tem-
perature. The precipitate was collected by filtration and washed
with water. To the filtrate was added 20 ml of water to promote
further precipitation. The solid is collected by filtration and washed
with water. The two solids are pooled together and dried under
vacuum to afford 38.1 mg (45% yield) of a brown solid. The solvents
are removed from the filtrate under vacuum to give 33 mg of a res-
idue, which can be purified by precipitation with methanol to gen-
erate 13 mg of a solid that is a mixture of fagaronine iodide and
fagaronine bromide.
To a solution of compound 9b (410 mg, 0.76 mmol) in 8 ml of
acetic acid was added 3.1 ml of HBr (40%), and the mixture was
stirred under reflux for 6 h. The mixture was allowed to cool, and
the precipitate was collected by filtration. This precipitate was
washed with water and dried under vacuum to afford 263 mg of
a brown solid. This solid was precipitated from methanol to gener-
ate 164 mg (34% yield) of a brown solid.
C
23H26NO5 [MH+]: 396.1811. Found: 396.1815. IR: 1137; 1164;
1256; 1581; 1621; 1719; 2974 cmÀ1
.
4.1.7. Norfagaronine (8)
To a solution of compound 7a (40 mg, 0.101 mmol) in AcOH
(1 ml) was added 1 ml of HBr (40%), and the mixture was stirred
under reflux for 0.5 h. The mixture was allowed to cool to room
temperature and was then neutralised to pH 8 with a concentrated
solution of sodium hydroxide. This neutralised reaction mixture
was extracted with CHCl3. The organic phase was isolated and then
dried over anhydrous MgSO4, and the solvent was evaporated un-
der reduced pressure to afford 30 mg (90% yield) of a yellow solid.
1H NMR (300 MHz, CDCl3/CD3OD 2/1): d = 4.03 (s, 3H), 4.10 (s,
3H), 4.12 (s, 3H), 7.31 (s, 1H), 7.34 (s, 1H), 7.74 (d, J = 8.9 Hz, 1H),
7.81 (s, 1H), 8.18 (d, J = 8.9 Hz, 1H), 8.58 (s, 1H), 9.13 (s, 1H). 13C
NMR (75 MHz, CDCl3/CD3OD 2/1): d = 55.9 (CH3), 55.9 (CH3), 56.0
(CH3), 101.5 (CH), 103.7 (CH), 107.2 (CH), 110.5 (CH), 117.8 (CH),
119.6 (C), 121.9 (C), 126.2 (C), 126.6 (CH), 128.8 (C), 129.2 (C),
139.9 (C), 146.8 (C), 148.5 (C), 149.2 (CH), 149.6 (C), 153.1 (C).
MS (+ESI) m/z: 336 [MH]+.
From 9a, fagaronine was obtained with purity higher than 95%
(single compound on 1H NMR spectra). From 9b fagaronine was
obtained with a purity of 85% according to the 1H NMR spectra,
and a purity of 81% according to the HPLC chromatogram recorded
at 300 nm.
1H NMR (300 MHz, CDCl3/CD3OD): d = 4.14 (s, 3H), 4.16 (s, 3H),
4.28 (s, 3H), 5,03 (s, 3H), 7.52 (s, 1H), 7.80 (s, 1H), 8.04 (s, 1H), 8.12
(d, J = 9.0 Hz,1H), 8.13 (s, 1H), 8.56 (d, J = 9.0 Hz, 1H), 9.68 (s, 1H).
MS (+ESI) m/z: 350 [M]+, 335 [M-CH3]+. MS (-ESI) m/z: 127 [IÀ].
+:
4.1.8. 3-(4-Acetoxy-3-methoxyphenyl)-6,7-dimethoxy-2-
methyl-4-(2-oxoethyl)isoquinolinium iodide (9a)
HRMS (+ESI): m/z Calcd for C21H20NO4 [M]
350.1392. Found:
350.1395.
A solution of compound 7a (40 mg, 0.101 mmol) in CH2Cl2 (1 mL)
was stirred for 24 h at room temperature with 70 ll of methyl iodide
As fagaronine iodide was not enough soluble in deuterated sol-
vents to obtain a carbon in a reasonable amount of time, it has been
converted into chloride: 40 mg of fagaronine iodide were dissolved
in 50/50 methanol/dichloromethane and eluted with methanol
through an anion exchanger column (10 g, DowexÒ 1 Â 4–200,
chloride form). 20 mg of a yellow solid were obtained. 13C NMR
(75 MHz, CDCl3/CD3OD): d = 50.9 (CH3), 55.9 (CH3), 56.3 (CH3),
56.8 (CH3), 102.0 (CH), 107,0 (CH), 108.1 (CH), 112.1 (CH), 117.7
(CH), 117.9 (C), 119.9 (C), 124.0 (C), 130.3 (C), 131.7 (CH), 132.5
(C), 132.9 (C), 149.1 (C), 149.2 (C), 149.4 (CH), 152.2 (C), 159.0 (C).
(145 mg, 1.01 mmol). Then, methyl iodide and CH2Cl2 were both re-
moved under vacuum to produce 55 mg of compound 9a in quanti-
tative yield (orange oil). 1H NMR (300 MHz, CDCl3): d = 2.35 (s, 3H);
3.84 (s, 3H); 4.04 (s, 3H); 4.12 (s, 3H); 4.14 (s, 3H); 4.24 (bs, 2H); 6.92
(dd, J = 8.0, 1.9 Hz, 1H); 7.15 (s, 1H); 7.18 (d, J = 1.8 Hz, 1H); 7.22 (d,
J = 8.0 Hz, 1H); 8.00 (s, 1H); 9.83 (s, 1H); 10.20 (s, 1H). 13C NMR
(75 MHz, CDCl3): d = 20.6 (CH3); 45.2 (CH2); 47.5 (CH3); 56.7
(CH3); 57.1 (CH3); 57.9 (CH3); 103.1 (CH); 108.3 (CH); 113.5 (C);
121.3 (CH); 123.7 (C); 124.3 (CH); 127.2 (C); 129.4 (C); 135.6 (CH);
141.7 (C); 143.2 (C); 146.3 (C); 152.3 (C); 152.7 (C); 158.7 (C);
168.4 (C); 197.2 (CH). MS (+ESI) m/z: 350 [M-Ac]+, 410 [M]+, 442
4.2. Biology
+:
[M+MeOH]+. HRMS (+ESI): m/z Calcd for C23H24NO6 [M]
4.2.1. In vitro antiplasmodial activity
410.1598. Found: 410.1601. IR: 1175; 1217; 1260; 1508; 1613;
P. falciparum 3D7, FcM29 and FcB1-Columbia strains were cul-
1781; 2879; 2981 cmÀ1
.
tured as described by Trager and Jensen,23 with modifications.24

M. Rivaud et al. / Bioorg. Med. Chem. 20 (2012) 4856–4861
4861
The cultures were synchronised with a combination of magnetic
concentration and 5% -sorbitol lysis (Merck, Darmstadt,
Supplementary data
D
Germany).25,26 The cultures, mostly at ring stage (synchronization
period: 8 h) were plated in 96-well plates and the drugs were
deposited at growing dilutions in triplicates. The incubation time
between drugs and Plasmodium was 48 h.The 3D7 strain was con-
sidered to be chloroquine-sensitive (chloroquine IC50: 32 nM); the
FcM29 and FcB1-Columbia strains were considered to be chloro-
quine-resistant (chloroquine IC50: 170 14 nM and 240 21 nM,
respectively). Antiplasmodial activity was determined by the
[3H]-hypoxanthine (Amersham–France) incorporation method.27
The resistance index was calculated as follows: IC50 3D7/IC50
FcM29.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.061.
References and notes
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Acknowledgements
M.R. thanks the French Ministère de la Recherche et de
l’Enseignement Supérieur for a Grant. A.M. thanks ADA (Asociación
de Amigos) of the University of Navarra for a Grant.