Synthesis and biological activity of fatty acid derivatives of quinine

Article abstract of DOI:10.1271/bbb.69.2250

Derivatives of quinine with fatty acids including polyunsaturated fatty acids were prepared. They showed moderate antimalarial activity as compared with quinine itself using Plasmodium falciparum. The activities were not dependent on whether the fatty acyl group was saturated or unsaturated. On the other hand, the derivatives showed significantly higher cytotoxicity against a mammary tumor cell line FM3A than quinine itself. Calculating from these data, an acetyl derivative of quinine with the shortest acyl group was found to give the highest selectivity.

Full text of DOI:10.1271/bbb.69.2250

Biosci. Biotechnol. Biochem., 69 (11), 2250–2253, 2005
Note
Synthesis and Biological Activity of Fatty Acid Derivatives of Quinine
Naokazu KUMURA,1 Minoru IZUMI,1 Shuhei NAKAJIMA,1 Sakayu SHIMIZU,2
y
y
1;
Hye-Sook KIM,3 Yusuke WATAYA,3; and Naomichi BABA
1Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology,
Okayama University, Tsushimanaka, Okayama 700-8530, Japan
2Department of Applied Bioscience, Graduate School of Agriculture, Kyoto University,
Sakyo-ku, Kyoto 606-8502, Japan
3Faculty of Pharmaceutical Sciences, Okayama University, Tsushimanaka, Okayama 700-8530, Japan
Received June 29, 2005; Accepted August 1, 2005
Derivatives of quinine with fatty acids including
polyunsaturated fatty acids were prepared. They
showed moderate antimalarial activity as compared
with quinine itself using Plasmodium falciparum. The
activities were not dependent on whether the fatty acyl
group was saturated or unsaturated. On the other hand,
the derivatives showed significantly higher cytotoxicity
against a mammary tumor cell line FM3A than quinine
itself. Calculating from these data, an acetyl derivative
of quinine with the shortest acyl group was found to give
the highest selectivity.
was synthesized, and their in vitro antimalarial activity
was evaluated using Plasmodium falciparum. Their
cytotoxicity against mouse mammary tumor FM3A cells
was also examined to determine their selectivity toward
the parasite.
The hydroxy group in quinine was esterified in etha-
nol-free chloroform with fatty acid activated by diiso-
propylcarbodimide in the presence of N,N-dimethyl-
aminopyridine and traces of butylated hydroxytoluene
as an antioxidant in a nitrogen atmosphere (Scheme 1).
The product esters, (2)–(8), were purified by silica gel
column eluted with hexane/ethyl acetate (4:1), and their
structural integrity was confirmed by 1H-NMR and
ESI-MS, indicating that no oxidative damage occurred
for the non-conjugated all-cis olefinic structure.
Key words: quinine; fatty acid; antimalarial activity;
Plasmodium falciparum
Quinine (1) has been used as a natural antimalarial
medicine. Its side effects and the development of
resistance against malaria parasites stopped its use once,
and the medicine has been staged again in recent years
because of the resistance problem of most of the
antimalarial medicines sythesized so far Development
of new drugs is an urgent subject nowadays. As major
components of polyunsaturated fatty acids (PUFA) in
marine fish oil, docosahexaenoic acid (DHA) and
icosapentaenoic acid (EPA) have a non-conjugated all
cis-polyunsaturated olefinic structure and are known to
exhibit a variety of biochemical, physiological func-
tions, including enhancement of cell membrane perme-
ability,1) growth regulation or apoptosis-inducing capa-
bility to cancer cells,2,3) cytotoxicity enhancement for
some anti-cancer drugs against cancer cells,4,5) and
potential antimalarial activity.6–9) If we assume that
these physiological functions are mainly responsible for
their characteristic olefinic structure, the same functions
are also expected to play their roles even when these
acids are linked to their other bioactive compounds.
Here, in the present study, a series of saturated fatty
acid- or PUFA-derivatives (2)–(8) of quinine (Fig. 1)
The antimalarial activity and cytotoxicities of fatty
acid derivatives (2)–(8) were tested in vitro against
P. falciparum and a mammary tumor cell line FM3A
cells, respectively (Table 1). The antimalarial activity of
lipid derivatives (2)–(7) [EC50 ¼ 2 ꢀ 10ꢁ5{7 ꢀ 10ꢁ6 M]
with a long chain fatty acyl group were found to be
similar to each other and lower than quinine (1)
irrespective of whether the acyl group was saturated or
unsaturated. The activity of acetyl derivative (8) having
the shortest acyl group, however, was significantly
higher [7:5 ꢀ 10ꢁ7 M] and was closed to quinine. On the
other hand, cytotoxicity against FM3A cells [1:7 ꢀ
10ꢁ5{5:2 ꢀ 10ꢁ6 M] was found to be significantly higher
than quinine [1 ꢀ 10ꢁ4 M]. Here again, the activity
appeared to be independent of whether the acyl chain
was saturated or unsaturated. When we compared the
selectivity calculated by [(FM3A EC50)]/(P. falciparum
EC50)], as shown in Table 1, the derivatives (2)–(7)
having a long-chain acyl group showed much lower
selectivity (1–8) than quinine (1000), whereas, in the
case of acetyl derivative (8), the value (36) was
significantly higher than (2)–(7).
Combining these results, it was suggested that the
y
To whom correspondence should be addressed. Yusuke WATAYA, Tel: +81-86-251-7976; Fax: +81-86-251-7926; E-mail: wataya@cc.
okayama-u.ac.jp; Naomichi BABA, Tel: +81-86-251-8292; Fax: +81-86-251-8388; E-mail: babanaom@cc.okayama-u.ac.jp

Synthesis and Biological Activity of Quinine Derivatives
2251
H
H
N
R O
1: R = H (Quinine)
MeO
N
CO
CO
CO
2: R =
4: R =
3: R =
CO
5: R =
6: R =
7: R =
CO
CO
8: R = Ac
Fig. 1. Structure of Fatty Acid Derivatives of Qunine.
H
H
H
H
Fatty acid, DMAP, DIC
N
N
HO
R
O
CHCl3,r.t., 12 h
MeO
MeO
N
N
Quinine (1)
2-8
Ac2O, Et3N
CHCl3, r.t., 12 h
Scheme 1. Esterification of Quinine with Fatty Acids or Anhydride.
parasite used in the present study, they might exhibit
rather different antimalarial activity. Also, since the
present modification of the hydroxy group in the quinine
molecule by the acyl group did not depress the activity
completely, modification with different functional
groups and different types of chemical bond other than
the ester linkage might be possible, so that higher
activity and selectivity are expected. Overcoming the
problem of the resistance developing in malaria para-
sites is an urgent subject worldwide, and molecular
conjugation of active components is considered to be
one of the strategies in the struggle.10)
Table 1. In Vitro Antimalarial Activities of Acylated Qunines
against P. Falciparum and of Cytotoxicites against FM3A Cells
EC50 (M)
Compound
R=
Selectibility
P. falciparum
FM3A
2
3
4
5
6
7
8
lauryl
stearyl
>2:0 ꢀ 10ꢁ5 >2:0 ꢀ 10ꢁ5
2:2 ꢀ 10ꢁ6 >1:7 ꢀ 10ꢁ5
2:2 ꢀ 10ꢁ6 5:2 ꢀ 10ꢁ6
7:0 ꢀ 10ꢁ6 5:0 ꢀ 10ꢁ6
1
>8
octadecadienyl
linolenyl
0.74
3.3
eicosapentaenyl 4:5 ꢀ 10ꢁ6 4:0 ꢀ 10ꢁ5
0.89
0.91
docosahexaenyl 4:6 ꢀ 10ꢁ6 4:2 ꢀ 10ꢁ5
acetyl
H
>2:7 ꢀ 10ꢁ5 2:7 ꢀ 10ꢁ5 >36
1:0 ꢀ 10ꢁ5 1:0 ꢀ 10ꢁ5 1000
Quinine (1)
Experimental
Preparation of quinoline esters (2)–(8). ESI-MS was
taken on a Perkin Elmer Model API III by positive ion
mode and direct infusion. 1H-NMR (500 MHz) was
taken on a Varian VXR 500.
Typically, a solution of quinine (1.92 g, 5.81 mmol),
docosahexaenoic acid (0.80 g, 2.44 mmol), N,N-diiso-
propylcarbodiimide (0.90 ml, 5.8 mmol), dimethylami-
nopyridine (0.15 g, 1.0 mmol), and a trace of butylated
introduction of a hydrophobic long-chain fatty acyl
group into the quinine structure appears to lower the
toxic effect against the P. falciparum parasite grown in
red cells in spite of their possibly more efficient
interaction with red cell membranes due to the lipophilic
acyl chain. Although the lipid derivatives of quinine did
not show promising activity to the quinine-sensitive

2252
N. KUMURA et al.
hydroxytolune as an antioxidant was stirred at room
temperature in a nitrogen atmosphere for 12 h. After this
period, the solvent was removed under reduced pressure
and the residue was purified by silica gel column
chromatography, eluted with hexane/ethyl acetate (9:1),
affording the desired ester (7). 93% yield. ESI-MS m=z:
description is not repeated.
(6). 91% yield. ESI-MS m=z: calcd. for C40H54N2O3,
608.4; found, 608.3. 1H-NMR (CDCl3) ꢀ(ppm): 0.95
(3H, t, J ¼ 7:63 Hz), 2.00–2.10 (4H, m, CH3CH2–,
–CH2CH2COO–), 2.55–2.70 (2H, m, –CH2–COO–),
2.75–2.87 (8H, m, 4ꢀ –C=C–CH2–C=C–), 3.95 (3H,
s, CH3O–), 5.30–5.45 (10H, m, olefinic protons of the
icosapentaenoyl group). Since the signals for the protons
on the quinoline nucleus were almost the same as those
of (1), their description is not repeated.
(8). 95% yield. ESI-MS m=z: calcd. for C22H26N2O3,
366.2; found, 366.2. 1H-NMR (CDCl3) ꢀ(ppm): 2.12
(3H, s, CH3COO–), 3.97 (3H, s, CH3O–). Since the
signals for the protons on the quinoline nucleus were
almost the same as those of (1), their description is not
repeated.
Biological activity measurements of quinoline esters
(2)–(8). In vitro antimalarial activity of (1)–(8) against
P. falciparum was conducted according to the reported
method.11) Briefly, 10 microliters of each solution of
(1)–(8) in dimethyl sulfoxide was added to individual
well of a 24-well plate. Erythrocytes with 0.3% para-
sitemia containing 980 ml of culture medium were added
to each well to give a final hematocrit level of 3%. The
plates were incubated at 37 ꢂC for 72 h in a CO2–O2–N2
incubator (5% CO2, 5% O2, and 90% N2 atmosphere).
To evaluate the antimalarial activity of the test com-
pounds, thin blood films from each culture were
prepared and stained with Giemsa (Merck, Germany).
A total of 1 ꢀ 104 erythrocytes/1 thin blood films were
examined under microscopy. All of the test compounds
were assayed in duplicate. The EC50 value refers to the
concentration of the compound (1)–(8) necessary to
inhibit the increase in parasite density at 72 h by 50% of
the control.
In vitro toxicity against mammalian cell line was
tested using FM3A according to the reported method.11)
Briefly, prior to exposure to the derivatives, cell density
was adjusted to 5 ꢀ 104 cells/ml. A cell suspension of
995 ml was dispersed to the test plate, and derivatives
(1)–(8) were suspended in dimethyl sulfoxide (10 ml),
which was added to individual wells of 24-well plate.
The plates were incubated at 37 ꢂC in 5% CO2 for 48 h.
All the test compounds were assayed in duplicate. Cell
numbers were measured using a microcell counter CC-
130 (Toa Medical Electric Co., Japan). All data points
represent the mean of three experiments. The EC50 value
refers to the concentration of the compound necessary to
inhibit the increase in cell density at 48 h by 50% of the
control. Selectivity refers to the mean of the EC50 value
for FM3A cells per the mean of the EC50 value for
P. falciparum.
1
calcd. for C42H54N2O3, 634.4; found, 634.4. H NMR
(CDCl3) ꢀ(ppm): 0.96 (3H, t, J ¼ 7:63 Hz, –CH2CH3),
2.05–2.10 (4H, m, CH3CH2–, –CH2CH2COO–), 2.57–
2.72 (2H, m, –CH2–COO–), 2.75–2.87 (12H, m, 6 ꢀ
–C=C–CH2–C=C–), 3.97 (3H, s, CH3O–), 5.30–5.42
(10H, m, olefinic protons of the docosahexaenoyl
group), 5.80–5.87 (1H, m, H2C=CH–), 7.34 (1H, d, J ¼
4:28 Hz, a proton adjacent to CH3O– at the 5-position
from quinoline nitrogen), 7.38 (1H, d, J ¼ 16:48 Hz, a
proton adjacent to CH3O– at the 3-position from
quinoline nitrogen), 7.42 (1H, d, J ¼ 9:15 Hz, a proton
on the carbon next to the side chain on the quinoline
ring), 8.00 (1H, d, J ¼ 9:15 Hz, a proton at the 3-
position from quinoline nitrogen), 8.72 (1H, d, J ¼ 4:58
Hz, a proton next to quinoline nitrogen).
(2). 86% yield. ESI-MS m=z: calcd. for C32H46N2O3,
506.4; found, 506.4. 1H-NMR (CDCl3) ꢀ(ppm): 0.96
(3H, t, J ¼ 7:63 Hz), 2.05–2.10 (4H, m, CH3CH2–,
–CH2CH2COO–), 2.57–2.72 (2H, m, –CH2–COO–),
2.75–2.87 (10H, m, 5 ꢀ –C=C–CH2–C=C–), 3.97
(3H, s, CH3O–), 5.30–5.42 (10H, m, olefinic protons of
docosahexaenoyl group). Since the signals for the
protons on the quinoline nucleus were almost the same
as those of (1), their description is not repeated.
(3). 81% yield. ESI-MS m=z: calcd. for C38H58N2O3,
590.4; found, 590.4. 1H-NMR (CDCl3) ꢀ(ppm): 0.96
(3H, t, J ¼ 7:63 Hz), 2.05–2.10 (4H, m, CH3CH2–,
–CH2CH2COO–), 2.57–2.72 (2H, m, –CH2–COO–),
2.75–2.87 (10H, m, 5 ꢀ –C=C–CH2–C=C–), 3.97
(3H, s, CH3O–), 5.30–5.42 (10H, m, olefinic protons of
the docosahexaenoyl group). Since the signals for the
protons on the quinoline nucleus were almost the same
as those of (1), their description was not repeated.
(4). 95% yield. ESI-MS m=z: calcd. for C38H54N2O3,
586.4; found, 586.4. 1H-NMR (CDCl3) ꢀ(ppm): 0.90
(3H, m, –CH3), 1.70–1.81 (2H, m, –CH2CH2COO–),
2.00–2.10 (4H, m, –CH2–C=C, C=C–CH2–), 2.35–2.40
(2H, m, –CH2–COO–), 2.77 (2H, t, J ¼ 6:41 Hz,
–C=C–CH2–C=C–), 3.98 (3H, s, CH3O–), 5.30–5.45
(4H, m, olefinic protons of the linoleoyl group). Since
the signals for the protons on the quinoline nucleus were
almost the same as those of (1), their description is not
repeated.
(5). 71% yield. ESI-MS m=z: calcd. for C38H52N2O3,
584.4; found, 584.4. 1H-NMR (CDCl3) ꢀ(ppm):
0.98 (3H, t, J ¼ 7:63 Hz, –CH3), 1.70–1.81 (2H, m,
–CH2CH2COO–), 2.00–2.10 (4H, m, –CH2–C=C,
C=C–CH2–), 2.39 (2H, m, –CH2COO), 2.77 (2H, t,
J ¼ 6:41 Hz, –C=C–CH2–C=C–), 3.95 (3H, s, CH3O–),
5.30–5.45 (6H, m, olefinic protons of the linolenyl
group). Since signals for the protons on the quinoline
nucleus were almost the same as those of (1), their
Acknowledgments
We are obliged to the Laboratory of SC-NMR of
Okayama University and to Ikeda Toka Industries Co.,
Ltd. of Hiroshima, Japan for the gift of DHA and EPA.

Synthesis and Biological Activity of Quinine Derivatives
2253
and Phuong, Oral bioavailability of dihydroartemisinin
in Vietnamese volunteers and in patients with Falcipa-
rum malaria. British J. Clin. Pharmacol., 51, 541–546
(2001).
References
1) Stillwell, W., Ehringer, W., and Jenski, L. J., Docosa-
hexaenoic acid increases permeability of lipid vesicles
and tumor cells. Lipids, 28, 103–108 (1993).
2) Begin, M. E., Ellis, G., and Horrobin, D. F., Polyunsa-
turated fatty acid-induced cytotoxicity against tumor
cells and its relationship to lipid peroxidation. J. Natl.
Cancer Inst., 80, 188–194 (1988).
7) Kumar, C. A., and Das, U. N., Lipid peroxides, nitric
oxide and essential fatty acids in patients with Plasmo-
dium falciparum malaria. Prostaglandins, Leukotrienes
and Biochemistry, 61, 255–258 (1999).
8) Fujikawa, M., Kamitani, T., Tunru, I., Yamazaki, K.,
and Hamazaki, T., Antimalarial effects of purified and
ꢁ-tochopherol-fortified n-3 polyunsaturated fatty acids.
J. Nutr. Biochem., 4, 153–157 (1993).
9) Kumaratilake, L. M., Robinson, B. S., Ferrante, A., and
Poulos, A., Antimalarial properties of n-3 and n-6
polyunsaturated fatty acids: in vitro effects on Plasmo-
dium falciparum and in vivo effects on P. berghei. J.
Clinical. Investigation, 89, 961–967 (1992).
10) Zerouga, M., Stillwell, W., and Jenski, L. J., Synthesis of
a novel phosphatidylcholine conjugated to docosahex-
aenoic acid and methotrexate that inhihibits cell pro-
liferation. Anti-Cancer Drugs, 13, 301–311 (2002).
11) Kim, H.-S., Shibata, Y., Wataya, Y., Tsuchiya, K.,
Masuyama, A., and Nojima, M., Synthesis and antima-
larial activity of cyclic peroxides, 1,2,4,5,7-pentoxo-
canes and 1,2,4,5-tetroxanes. J. Med. Chem., 42, 2604–
2609 (1999).
3) Noding, R., Schonberg, S. A., Krokan, H. E., and Bjerve,
¨
K. S., Effects of polyunsaturated fatty acids and their n-6
hydroperoxides on growth of five malignant cell lines
and the significance of culture media. Lipids, 33, 285–
292 (1998).
4) Das, U. N., Madhavi, N., Kumar, G. S., Padma, M., and
Sangeetha, P., Can tumor cell drug resistance be
reversed by essential fatty acids and their metabolite?
Prostaglandins, Leukotrienes and Essential Fatty Acids,
58, 39–54 (1998).
5) Germain, E., Chajes, V., Cognault, S., Lhuillery, C., and
Bougnoux, P., Enhancement of doxorubicin cytotoxicity
by polyunsaturated fatty acids in the human breast tumor
cell line MDA-MB-231. Int. J. Cancer, 75, 578–583
(1998).
6) Binh, T. Q., Ilett, K. F., Davis, T. M., Hung, N. C.,
Powell, S. M., Thu, L. T., Thien, H. V., Phuong, H. L.,